Animal Cloning: 

A Draft 

Risk Assessment 

Center for Veterinary Medicine 

U. S. Food and Drug Administration 

Department of Health and Human Services 

7500 Standish Place 

Rockville, MD 20855 

12/28/2006 

 

Table of Contents 

Preface...........................................................................................................iii 

Chapter I: Executive Summary................................................................... 3

A. Overview................................................................................................................. 3 

B. Technology Overview (Chapter II)......................................................................... 4 

C. Risk Assessment Methodology (Chapter III) ......................................................... 5 

D. The Implications of Epigenetic Reprogramming for Clones and their Progeny 

 (Chapter IV)............................................................................................................ 7 

E. Risks to Animals Involved in Cloning (Chapter V)................................................ 8 

F. Food Consumption Risks (Chapter VI) .................................................................. 9 

1. Two-Pronged Approach to Identifying and Characterizing Food Consumption 

 Risks.................................................................................................................... 9 

2. Conclusions Regarding Potential Food Consumption Risks ............................ 10 

a. Cattle Clones................................................................................................. 10 

b. Swine Clones ................................................................................................ 12 

c. Sheep Clones................................................................................................. 13 

d. Goat Clones................................................................................................... 13 

e. Clone Progeny............................................................................................... 13 

G. Concluding Statements (Chapter VII) .............................................................. 14 

Chapter II: Technology Overview: Somatic Cell Nuclear Transfer and 

Other Assisted Reproductive Technologies.............................................. 19

A. What is Cloning?................................................................................................... 20 

B. Continuum of Reproductive Technologies ........................................................... 21 

1. Natural Service.................................................................................................. 21 

2. Artificial Insemination and Synchronized Estrus ............................................. 22 

3. Embryo Transfer ............................................................................................... 24 

4. In vitro Fertilization .......................................................................................... 26 

5. Embryo splitting................................................................................................ 27 

6. Blastomere Nuclear Transfer ............................................................................ 28 

7. Somatic Cell Nuclear Transfer (SCNT)............................................................ 29 

a. Donor cell...................................................................................................... 30 

b. Oöcyte........................................................................................................... 31 

c. Fusion............................................................................................................ 31 

d. Transfer to recipient...................................................................................... 32 

C. Critical Biological Events in SCNT...................................................................... 32 

D. Outcomes Observed in ARTs ............................................................................... 34 

E. Future of Reproductive Technologies in Modern Agricultural Practice .............. 36 
Chapter III: Developing the Risk Assessment Methodology................. 41

A. Charge................................................................................................................... 41 

B. General Discussion of Risk/Safety Analyses........................................................ 41 

1. Risk and Safety ................................................................................................. 41 

2. Risk Assessment vs. Risk Management............................................................ 42 

C. Risk/Safety Assessment of Cloning...................................................................... 42 

D. Transgenic Animal Clones.................................................................................... 44 

E. Methodology Development .................................................................................. 45 

1. Hazard Characterization.................................................................................... 46 

2. Potential Risks .................................................................................................. 48 

3. Proposed Approaches........................................................................................ 49 

a. Animal Health Risks..................................................................................... 49 

b. Food Consumption Risks.............................................................................. 49 

F. Two-Pronged Approach to Assessing Food Consumption Risks......................... 50 

1. Critical Biological Systems Approach.............................................................. 51 

a. Overview....................................................................................................... 51 

b. Evaluation Nodes.......................................................................................... 52 

G. Limitations of the Risk Assessment.................................................................. 54 

Chapter IV: Epigenetic Reprogramming: Implications for Clones and 

their Progeny ............................................................................................... 59

A. Overview of Epigenetic Reprogramming in Early Embryonic Development...... 61 

1. Preimplantation Reprogramming...................................................................... 63 

a. Fusion and Cleavage..................................................................................... 63 

b. Demethylation and Remethylation in Early Embryos .................................. 66 

c. Epigenetic Reprogramming in Later Development ...................................... 70 

d. Studies of Gene Expression and Development in Clones and Other ARTs . 72 

2. Gametogenic Reprogramming.......................................................................... 77 

3. Conclusions from Studies of Epigenetic Reprogramming................................ 79 

B. Phenotypic Evidence for Gametogenic Reprogramming ..................................... 80 

1. Phenotypic Anomalies Observed in Mouse Clones.......................................... 80 

a. Utility of Mouse Model ................................................................................ 80 

b. Pregnancy (Developmental Node 1)............................................................. 81 

c. Perinatal Period (Developmental Node 2) .................................................... 83 

d. Juvenile Period to Reproductive Maturity (Developmental Nodes 3 and 4) 84 

e. Maturity and Aging (Developmental Node 5).............................................. 86 

2. Conclusions from Phenotypic Studies of Gametogenic Reprogramming in 

Mouse Clones and their Progeny for Reprogramming in Domestic Livestock Clones 

and their Progeny ...................................................................................................... 87 

C. Implications of Epigenetic Reprogramming for Animal Health and Food 

Consumption Risks ....................................................................................................... 89 
Chapter V: Animal Health Risks .............................................................. 93

A. Potential Hazards and Risks to Animals Involved in Cloning.............................. 93 

B. The Critical Biological Systems Approach to the Analysis of Clone Animal 

Health: Cattle, Swine, Sheep, and Goats ...................................................................... 94 

1. Pregnancy and Parturition (Developmental Node 1)........................................ 94 

2. Perinatal Period (Developmental Node 2) ........................................................ 96 

3. Juvenile Developmental Node (Developmental Node 3) ................................. 97 

4. Reproductive Development and Function Node (Developmental Node 4)...... 98 

5. Post-Pubertal Maturation and Aging (Developmental Node 5)........................ 99 

a. Telomere Length as an Indicator of Aging................................................. 100 

C. Data on Animal Health by Species ..................................................................... 104 

1. Cattle............................................................................................................... 104 

a. Developmental Node 1: Pregnancy and Parturition.................................... 107 

i. Pregnancy................................................................................................ 107 

ii. Parturition ............................................................................................... 111 

(a) Hydrops........................................................................................... 111 

(b) Dystocia .......................................................................................... 114 

(c) Large Offspring Syndrome ............................................................. 115 

(d) Other complications........................................................................ 116 

b. Developmental Node 2: Perinatal Period.................................................... 116 

(a) Large Offspring Syndrome ............................................................. 117 

(b) Other complications........................................................................ 122 

i. Cyagra Data: Perinatal Cohort................................................................ 124 

ii. Unpublished data ................................................................................ 125 

c. Developmental Node 3: Juvenile Development ......................................... 127 

i. Peer-Reviewed Publications ................................................................... 127 

ii. Cyagra Data: 1-6 Month Cohort ............................................................. 133 

iii. Unpublished data ................................................................................ 135 

d. Developmental Node 4: Reproductive Development and Function ........... 140 

i. Peer-Reviewed Publications ................................................................... 140 

ii. Unpublished Data.................................................................................... 145 

e. Developmental Node V: Post-Pubertal Maturation and Aging .................. 147 

i. Peer-reviewed literature.......................................................................... 147 

ii. Cyagra Data: 6-18 Month Cohort ........................................................... 149 

iii. Unpublished data ................................................................................ 149 

f. Progeny of Bovine Clones .......................................................................... 151 

g. Summary for Health of Bovine Clones and Their Progeny........................ 152 

2. Swine............................................................................................................... 153 

a. Developmental Node 1: Pregnancy and Parturition.................................... 154 

i. Pregnancy................................................................................................ 154 

ii. Parturition ............................................................................................... 154 

b. Developmental Node 2: Perinatal Period.................................................... 155 

i. Peer-Reviewed Publications ................................................................... 155 

ii. Viagen dataset......................................................................................... 157 

iii. Unpublished data ................................................................................ 157 

c. Developmental Node 3: Juvenile Development ......................................... 159 
i. Peer-reviewed literature.......................................................................... 159 

ii. Viagen dataset......................................................................................... 161 

d. Developmental Node 4: Reproductive Development and Function ........... 162 

e. Developmental Node V: Post-Pubertal Maturation and Aging .................. 163 

f. Progeny of Swine Clones............................................................................ 163 

g. Summary for Health of Swine Clones and Their Progeny ......................... 164 

3. Sheep............................................................................................................... 164 

a. Developmental Node 1: Pregnancy and Parturition.................................... 165 

i. Pregnancy................................................................................................ 165 

ii. Parturition ............................................................................................... 166 

(a) Dystocia .......................................................................................... 166 

(b) Other complications........................................................................ 166 

b. Developmental Node 2: Perinatal Period.................................................... 167 

c. Developmental Node 3: Juvenile Development ......................................... 168 

d. Developmental Node 4: Reproductive Development and Function ........... 168 

e. Developmental Node V: Post-Pubertal Maturation and Aging .................. 168 

f. Progeny of Sheep Clones............................................................................ 168 

g. Summary for Health of Sheep Clones ........................................................ 168 

4. Goats ............................................................................................................... 169 

a. Developmental Node 1: Pregnancy and Parturition.................................... 169 

i. Pregnancy................................................................................................ 169 

ii. Parturition ............................................................................................... 170 

b. Developmental Node 2: Perinatal Period.................................................... 170 

c. Developmental Node 3: Juvenile Development ......................................... 171 

d. Development Node IV: Reproductive Development and Function............ 172 

e. Developmental Node V: Post-Pubertal Maturation and Aging .................. 172 

f. Progeny of Goat Clones.............................................................................. 172 

g. Summary for Health of Goat Clones .......................................................... 173 

D. Conclusions......................................................................................................... 173 

Chapter VI: Food Consumption Risks ................................................... 179

A. Potential Hazards and Food Consumption Risks Associated with Food Products 

 from Animal Clones and their Progeny .............................................................. 179 

1. Assumptions.................................................................................................... 179 

2. Critical Biological Systems Approach to Animal Clones of Cattle, Swine, 

 Sheep, and Goats............................................................................................. 179 

a. Bovine Clones............................................................................................. 180 

 i. Cell Fusion, Nuclear Reprogramming, and Embryonic and Fetal 

 Development in Bovine Clones .................................................................. 181 

(a) Peer-reviewed Publications............................................................. 182 

(b) Summary for the Embryonic/Fetal Developmental Node in Bovine 

 Clones (Developmental Node 1)..................................................... 189 

ii. Perinatal Development in Bovine Clones (Developmental Node 2) ...... 189 

(a) Peer-reviewed Publications............................................................. 189 

(b) Cyagra Dataset: Perinatal Cohort.................................................... 201 

(c) Unpublished data ............................................................................ 203 
(d) Summary for Perinatal Developmental Node in Bovine Clones 

 (Developmental Node 2)................................................................. 203 

iii. Juvenile Development in Bovine Clones (Developmental Node 3) ... 204 

(a) Peer-reviewed Publications............................................................. 204 

(b) Cyagra Data: 1-6 Month Age Cohort.............................................. 211 

(c) Unpublished data ............................................................................ 214 

(d) Summary for Juvenile Developmental Node in Bovine Clones 

 (Developmental Node 3)................................................................. 214 

iv. Reproductive Development and Function in Bovine Clones 

 (Developmental Node 4)..................................................................... 215 

(a) Peer-reviewed Publications............................................................. 215 

(b) Unpublished data ............................................................................ 224 

(c) Summary Statement for Reproductive Development and Function in 

 Bovine Clones (Developmental Node 4) ........................................ 224 

v. Post-Pubertal Maturation in Bovine Clones (Developmental Node 5)... 225 

(a) Peer-reviewed Publications............................................................. 225 

(b) Cyagra Dataset: 6-18 Month Cohort............................................... 227 

(c) Unpublished data ............................................................................ 228 

(d) Summary Statement for Post-Pubertal Maturation in Bovine Clones 

 (Developmental Node 5)................................................................. 228 

b. Swine Clones .............................................................................................. 229 

i. Cell Fusion, Nuclear Reprogramming, Embryonic and Fetal Development 

 Through the Perinatal Developmental Period in Swine Clones 

 (Developmental Nodes 1 and 2) ............................................................. 230 

(a) Peer-reviewed Publications............................................................. 230 

(b) Summary Statement on the Embryo/Fetal to Perinatal Developmental 

 in Swine Clones (Developmental Nodes 1 and 2) .......................... 234 

ii. Juvenile Development and Function in Swine Clones (Developmental 

 Node 3).................................................................................................... 234 

(a) Peer-reviewed Publications............................................................. 234 

(b) Viagen Dataset................................................................................ 237 

(c) Summary Statement for Juvenile Development and Function in 

 Swine Clones (Developmental Node 3).......................................... 239 

iii. Reproductive Development and Function in Swine Clones 

 (Developmental Node 4)..................................................................... 239 

(a) Peer-reviewed Publications............................................................. 239 

iv. Post-Pubertal Maturation in Swine Clones (Developmental Node 5) 240 

v. Conclusions Regarding the Food Safety of Swine Clones ................. 240 

c. Sheep Clones............................................................................................... 241 

i. Peer-reviewed Publications..................................................................... 241 

ii. Conclusions Regarding the Food Safety of Sheep Clones ..................... 243 

d. Goat Clones................................................................................................. 243 

i. Perinatal Development and Function in Goat Clones (Developmental 

 Node 2).................................................................................................... 244 

ii. Juvenile Development in Goat Clones (Developmental Node 3)........... 245 
iii. Reproductive Development and Function in Goat Clones 

 (Developmental Node 4)..................................................................... 246 

iv. Post-Pubertal Maturation in Goat Clones (Developmental Node 5) .. 247 

v. Summary Statement on Health Status of Goat Clones ....................... 247 

vi. Conclusions for Food Consumption Risks from Goat Clones............ 247 

3. Compositional Analysis Method..................................................................... 248 

a. Overview..................................................................................................... 248 

b. Nutritional Risk........................................................................................... 249 

i. Milk......................................................................................................... 251 

ii. Meat ........................................................................................................ 254 

c. Characterization of Milk from Cow Clones................................................ 258 

i. Peer-reviewed Reports............................................................................ 258 

ii. The Report of the Japanese Research Institute for Animal Science in 

 Biochemistry and Toxicology................................................................. 264 

iii. Additional Data................................................................................... 266 

iv. Summary Statement on Composition of Milk from Clones ............... 270 

d. Characterization of Meat from Clones and Their Progeny......................... 271 

i. Cattle....................................................................................................... 271 

ii. Swine....................................................................................................... 278 

(a) Clones ............................................................................................. 278 

(b) Swine Clone Progeny...................................................................... 282 

iii. Conclusions from Studies Evaluating the Composition of Meat and 

 Milk from Clones and Their Progeny ................................................. 285 

B. Drawing Conclusions Regarding Risks Associated with Consumption of Food 

 Products from Animal Clones............................................................................. 286 

1. Approaches for Decreasing Uncertainties ...................................................... 286 

2. Bounding the Risk Space................................................................................ 287 

3. Developing Conclusions Regarding Food Consumption Risks...................... 288 

4. Weight of Evidence Conclusions Regarding Food Consumption Risks for 

 Clones and their Progeny................................................................................ 289 

a. Cattle Clones............................................................................................... 289 

i. Risk Hypothesis Statement for Perinatal Bovine Clones........................ 290 

ii. Risk Hypothesis Statement for Juvenile Bovine Clones......................... 291 

iii. Risk Hypothesis Statement for “Adult” Bovine Clones ..................... 292 

b. Swine Clones .............................................................................................. 293 

i. Risk Hypothesis Statement for Swine Clones ........................................ 294 

c. Sheep Clones............................................................................................... 294 

i. Risk Hypothesis Statement for Sheep Clones......................................... 295 

d. Goat Clones................................................................................................. 295 

i. Risk Hypothesis Statement for Goat Clones........................................... 295 

e. Clone Progeny............................................................................................. 296 

5. Summary of Risk Hypotheses......................................................................... 296 

a. Additional Issues......................................................................................... 297 

i. Potential Allergenicity ............................................................................ 297 

ii. Microbiological Effects .......................................................................... 298 

iii. Unanticipated Effects.......................................................................... 299 
iv. Technology Changes........................................................................... 300 

5. How Much (Information) Is Enough?............................................................. 300 

Chapter VII Summary and Conclusions ................................................ 305 

Glossary ..................................................................................................... 311 

References.................................................................................................. 327 

Appendix A: Risk and Safety Assessment Primer for Animal Cloning A3

A. How has Risk Assessment Evolved? ................................................................... A3 

B. Thinking About Risk............................................................................................ A4 

C. How Do We Think About Safety?....................................................................... A6 

Appendix B :Overall Reproductive Efficiency and Health Statistics for 

US Animal Agriculture...............................................................................B3

A. Dairy cattle............................................................................................................B3 

B. Beef Cattle ............................................................................................................B6 

C. Swine.....................................................................................................................B8

D. Sheep.....................................................................................................................B9

E. Goats ...................................................................................................................B11 

Appendix C: Comparisons of Outcomes Among Assisted Reproductive 

Technologies (ARTs)...................................................................................C3

A. Successes and Failures of AI, IVP, and ET ..........................................................C8 

B. Outcomes for BNT, Fetal- and Adult-Cell SCNT ..............................................C12 

C. Conclusions regarding outcomes for ARTs........................................................C13 

Appendix D Transgenic Clones ................................................................ D3

A. Issues.................................................................................................................... D3

B. Cattle.................................................................................................................... D4

C. Swine.................................................................................................................... D6 

D. Sheep.................................................................................................................... D8 

E. Goats .................................................................................................................... D9 

F. Conclusions Regarding Transgenic Clones ....................................................... D11 

Appendix E: The Cyagra Dataset .............................................................E3

A. Response to CVM Data Requests.........................................................................E3 

B. Cyagra Dataset......................................................................................................E4 

1. Description of Clones .......................................................................................E6 
2. Evaluations Performed......................................................................................E8 

3. CVM’s Analysis of Cyagra Data: Method........................................................E9 

4. CVM’s Analysis of Cyagra Data: Results ......................................................E11 

a. Comprehensive Veterinary Examinations ..................................................E11 

b. Conclusions from Veterinary Examinations...............................................E13 

c. Laboratory Values: Selection of Most Appropriate Comparator................E13 

d. Conclusions Regarding Clone and Comparator Population Cohorts in 

Aggregate............................................................................................................E15 

e. Animal and Analyte Specific Analyses ......................................................E16 

i. Growth-Related Phenomena ...................................................................E18 

f. Animals with Measurements at Different Developmental Nodes ..............E20 

i. Age Range within 24 hours of birth (Charts E101 and E111)................E21 

(a) Analyte Evaluation..........................................................................E22 

(b) Animal Evaluation ..........................................................................E24 

(c) Conclusions for Perinatal Period: Animal Safety ...........................E25 

(d) Food Safety .....................................................................................E25 

ii. Age Range 1 to 6 months (Charts E201 and E211)................................E26 

(a) Analyte Review...............................................................................E26 

(b) Animal Evaluation (1 to 6 months Age Group)..............................E27 

(c) Conclusion for 1 to 6 month old group: Animal Safety..................E29 

(d) Conclusion for 1 to 6 month old group: Food Safety .....................E29 

iii. Age Range: 6-18 Months (Charts E301 and E311) ............................E30 

(a) Analyte Analysis.............................................................................E30 

(b) Animal Analysis..............................................................................E32 

(c) Conclusions for 6-18 Month Group: Animal Safety.......................E33 

(d) Conclusions for 6-18 Month Group: Food Safety ..........................E33 

5. Charts and Tables............................................................................................E34 

Appendix F: The Viagen Dataset ..............................................................F3

A. Background........................................................................................................... F3 

B. Experimental Design............................................................................................. F3 

1. Study 1: Clones vs. Comparators...................................................................... F5 

a. Animal Health, Hematology, Clinical Chemistry, and Urinalysis................ F6 

b. Boar Semen Evaluation................................................................................. F6 

c. Farrowing Rate.............................................................................................. F6 

2. Study 2: Progeny of Clone Boars vs. Progeny of Conventional Boars ............ F7 

a. Statistical Analysis........................................................................................ F7 

i. Methodology............................................................................................. F7 

ii. Utility of Statistical Analysis as a Tool for Assessment of Outcomes ..... F9 

b. Specific Methods for the Analysis of Animal Health, Hematology, Clinical 

 Chemistry, and Urinalysis Data .................................................................. F10 

c. Meat Composition, Carcass Characteristics, and Meat Quality Assessments 

 for Clones, Comparators, and Progeny....................................................... F10 

C. Results................................................................................................................. F11 

1. Study 1 ............................................................................................................ F11 

a. Survival....................................................................................................... F11 
b. Animal Health, Growth, Blood Clinical Chemistry, Hematology, and 

 Urinalysis .................................................................................................... F12 

c. Carcass Characteristics ............................................................................... F20 

d. Meat Composition Analysis for Clones and Comparators ......................... F21 

e. Semen and Breeding Evaluation................................................................. F23 

f. Farrowing Rate............................................................................................ F24 

2. Study 2: Progeny of Clones vs. Comparators ................................................. F26 

a. Survival....................................................................................................... F26 

b. Growth, Hematology, Clinical Chemistry, and Urinalysis......................... F32 

i. Growth .................................................................................................... F32 

ii. Hematology, Clinical Chemistry, and Urinalysis ................................... F34 

c. Carcass Characteristics ............................................................................... F40 

d. Meat Composition from the Progeny of Clones and Comparators............. F41 

D. Conclusions from the Viagen Dataset................................................................. F46 

1. Study 1: Clones vs. Comparators.................................................................... F46 

a. Animal Health............................................................................................. F46 

b. Food Safety ................................................................................................. F47 

2. Progeny of Clone Boars vs. Progeny of Comparator Boars ........................... F47 

a. Animal Health............................................................................................. F47 

b. Food Safety ................................................................................................. F48 

E. Addendum........................................................................................................... F49 

Appendix G: Investigation on Attribues of Cloned Bovine Products: 

A Study Performed by the Ministry of Health of Japan........................ G3 

A. Project Report of an Investigation of the Properties of Products from Cloned 

 Cattle.................................................................................................................... G7 

B. English language summary of the report “Investigation on the Attributes of 

 Cloned Bovine Products”................................................................................... G11 

C. Results of a 28-day feeding study in rats fed diets containing freeze dried 

 milk or beef from ordinary cattle and clone cattle............................................. G21 

Appendix H: The Comprehensive Veterinary Examination................. H3

A. Introduction to the Comprehensive Veterinary Examination .............................. H3 

B. The Importance of Species-Specific Standards ................................................... H4 

C. How a Veterinarian Performs a Comprehensive Veterinary Examination.......... H4 

1. Specific Considerations for Neonatal CVEs.................................................... H6 

2. Specific Considerations for Juvenile CVEs..................................................... H7 

3. Specific Considerations for Mature Animal CVEs.......................................... H7 

4. Specific Consideration for Reproductive Stage Examinations........................ H7 

a. Males............................................................................................................ H7 

b. Females ........................................................................................................ H8 

D. Interpreting the Comprehensive Veterinary Examination for Animal Clones .... H9 

E. Parameters Evaluated in the Comprehensive Veterinary Exam for a Risk 

 Assessment........................................................................................................... H9 

Animal Cloning: A Risk Assessment DRAFT 

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Preface 
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Preface 

The following Draft Risk Assessment is the result of a multi-year effort by staff from the 

US Food and Drug Administration’s (FDA’s) Center for Veterinary Medicine (CVM or 

the Center). Since the late 1990s, CVM has been meeting with clone producers and other 

stakeholders interested in cloning to discuss the safety and regulatory implications of 

somatic cell nuclear transfer (SCNT), the process most commonly used to generate 

animal clones during this time period. In the fall of 2000, CVM tasked the National 

Academy of Sciences (NAS) to perform an independent, scientific review of the available 

data on the safety of cloning, including holding a public meeting to identify sciencebased concerns and elicit data and information on clones and their food products from the 

scientific community. In July of 2001, the Center issued a CVM Update requesting that 

clone producers not introduce meat or milk from clones or their progeny into food or feed 

until the NAS report had been completed, and the agency had had a chance to complete 

its own review of the safety of those food products.1 

In October of 2002, NAS issued its report “Animal Biotechnology: Science-Based 

Concerns.” Following an overview of the available data on animal clones, the report 

indicated that the most likely mechanism for generating hazards to clones would stem 

from reprogramming the donor cell genome, and that any harms that might result from 

that reprogramming would be observed early in a clone’s development. They further 

noted that there were no published data comparing the composition of meat or milk from 

clones with conventional animals. Nonetheless, the report concluded that there is “no 

evidence that food products derived from adult somatic cell clones or their progeny pose 

a hazard (i.e., there is no evidence that they present a food safety concern)” (page 65). 

This Draft Risk Assessment is CVM’s subsequent independent analysis of all of the data 

relevant to assessing the health of clones and their progeny (and other animals involved 

in the cloning process) or food consumption risks resulting from edible products from 

these animals. In order to make the Risk Assessment as transparent as possible, all of this 

information is available to the public, either by virtue of its publication in peer-reviewed 

journals, or by “publication” in this risk assessment. We are actively seeking independent 

peer-review of these data by providing all of the data in raw form (not summaries) either 

in the text of the risk assessment or in appendices. In addition, we have also described the 

means by which the methodology was developed to facilitate peer-review by risk 

assessors. 

 

1 http://www.fda.gov/cvm/CVM_Updates/clones.htm
Preface iv 

CVM has attempted to be as comprehensive as possible about identifying and using all of 

the data relevant to assessing the health of clones and their progeny or food consumption 

risks resulting from their edible products. We have performed extensive literature 

reviews, engaged in conversations with scientists involved in cloning animals, and 

requested data on animal health and food composition from scientists, breeders, and food 

producers. Unpublished data were provided to us in raw, unanalyzed form, which we 

subsequently analyzed. CVM determined whether a particular publication or dataset was 

relevant to the analysis. These judgments were framed by the two overarching objectives 

of the Draft Risk Assessment: determining whether cloning poses any health risks to the 

animals involved in the cloning process, and whether any hazards arise during the 

development of clones or their progeny that may pose food consumption risks. 

Data incorporation for this version of the Draft Risk Assessment ceased in early 2006, 

when we made the final revisions to the scientific analysis in this draft. Any additional 

data, and other relevant information submitted during the public comment period, will be 

thoroughly reviewed, and revisions necessary will be made in the final Risk Assessment. 

In addition to understanding the Risk Assessment’s goals, it is equally important to 

understand what it does not consider. It does not attempt to address the question of 

whether clones are “normal;” rather it concentrates on identifying the risks that cloning 

poses to animal health or to humans and animals consuming food derived from clones 

and their progeny. It also does not attempt to explore issues such as the influence of 

different donor cell types or cell cycle stages in the “success rate” for producing clones, 

or the degree to which clones are more or less identical at the phenotypic level. Studies 

addressing these questions have been used, however, when they provided data useful to 

the identification of hazards or risks. Similarly, the Draft Risk Assessment does not 

attempt to parse out the relative effectiveness of different cloning techniques or different 

laboratories in generating live animals. Results of cloning in species not commonly used 

for food have been employed only as they have utility as model systems (e.g., mice as 

models for livestock). Uncertainties associated with those models have been identified. 

Finally, it is important to note that this Draft Risk Assessment is a framework by which 

science-based questions regarding animal health and food consumption risks are 

evaluated. It does not provide any recommendations for managing those risks, the 

circumstances under which we might recommend that food from clones or their progeny 

may be released for commercial use, or ethical concerns that may be raised by cloning. 

These issues are addressed in the accompanying Proposed Risk Management Plan and 

Draft Guidance for Industry. 
 

Chapter I: 

Executive Summary
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Chapter I: Executive Summary 

Cloning is the colloquial term used to describe the process of somatic cell nuclear transfer 

(SCNT) that falls on a continuum of assisted reproductive technologies (ARTs) currently 

used in agriculture. In this Draft Risk Assessment, the Center for Veterinary Medicine 

(CVM or the Center) at the US Food and Drug Administration (FDA) presents a sciencebased review of the available information on cloning in species traditionally used for 

food (i.e., cattle, swine, sheep, and goats). 

A. Overview 

This Draft Risk Assessment addresses SCNT technology, its impact on the health of 

animals involved in that process, and food consumption hazards that may arise in animal 

clones and their progeny2 in the context of the use of ARTs in conventional animal 

agriculture. Chapter II is a summary of ARTs currently used in food animal breeding and 

a detailed explanation of SCNT. Chapter III describes the process of risk assessment, its 

application to animal cloning, and the nature of the hazards that may arise as the result of 

cloning. A synopsis of the processes involved in epigenetic reprogramming and their 

relevance to adverse outcomes noted in animals derived via SCNT and other ARTs is 

found in Chapter IV. Chapter V addresses potential health risks to animals involved in 

the process of cloning, including surrogate dams, clones, and their progeny. Chapter VI 

addresses potential food consumption risks that may result from edible products derived 

from animal clones or their progeny. Each chapter contains conclusions relevant to that 

subject; the Risk Assessment is summarized in Chapter VII, and our overall conclusions 

are presented there. In order to make this process as transparent as possible, all of our 

methodologies are presented in the text of the risk assessment; the information and data 

that CVM evaluated are publicly available, either in peer-reviewed publications, or in 

Appendices to this document. The process by which CVM drew its conclusions is 

presented in the Risk Assessment, along with explicit statements of potential bias and 

uncertainty. The document concludes with a complete bibliography, a glossary of terms, 

and appendices containing data and background information. 

The Draft Risk Assessment is the result of a qualitative analysis that identifies and 

characterizes the nature of hazards that may be introduced into animals as a result of 

 2

 For the purposes of this analysis, an animal clone is one arising directly from a somatic cell nuclear 

transfer event. A progeny animal is one derived from sexual reproduction that has at least one animal 

clone as a parent (but could also result from two animal clones mating). Clones of clones would be 

considered as clones (i.e., directly arising from an SCNT process). 
Chapter I: Executive Summary 4 

cloning, and puts them in the context of other assisted reproductive technologies 

currently practiced in the United States. The strongest conclusions that can be drawn 

regarding positive outcomes in risk assessments of this type are “no additional risk” 

because outcomes are weighed against known comparators. If a finding of “no additional 

risk” were to be applied to the health of animal clones, it would mean that the cloning 

process would not pose any greater risk to the health of the animals involved than other 

ARTs. Applied to the safety of edible products derived from clones, a finding of “no 

additional risk” would mean that food products derived from animal clones or their 

progeny would not pose any additional risk relative to corresponding products from 

conventional animals, or that they are as safe as foods that we eat every day. As with all 

risk assessments, some uncertainty is inherent either in the approach we have used or in 

the data themselves. Where uncertainties exist, CVM has attempted to identify the degree 

of uncertainty and the reasons for its existence. 

B. Technology Overview (Chapter II) 

Assisted reproductive technologies (ARTs) have been employed extensively in animal 

agriculture for over a century, and at least one (artificial insemination) has been practiced 

for several hundred years. These technologies form a continuum that ranges from the 

fairly minimal assistance provided to animals engaged in natural service through the 

more recent development of SCNT. ARTs have aided in the genetic improvement of 

domestic livestock species by the selection and propagation of desirable phenotypes, and 

accelerating the rate at which those characteristics have been incorporated into national 

herds. Artificial insemination, for example, permitted the propagation of valuable 

genomes without the sire being physically present, thereby allowing superior genetics to 

be spread beyond relatively small geographical areas. 

Most commonly used ARTs rely on fertilization as a first step. This joining of egg and 

sperm is accompanied by the recombination of the genetic material from the sire and 

dam, and is often referred to as “shuffling the genetic deck.” From a breeder’s 

perspective, phenotypes resulting from sexual reproduction cannot be predicted—that is, 

the characteristics of the offspring from a mating may be estimated, but not predicted 

with certainty. Nuclear transfer, the most advanced of these technologies, does not 

require fertilization and allows for the propagation of known genotypes and phenotypes 

without the risk of genetic reshuffling. Thus, SCNT’s greatest immediate impact on 

animal breeding may be that it allows the propagation of genomes whose phenotypes are 

proven. It also allows the propagation of animals whose reproductive function may be 

impaired, or of very valuable animals that have died. SCNT, like the other newer forms 
Chapter I: Executive Summary 5 

of ARTs (e.g., in vitro fertilization, embryo splitting) results in some known adverse 

outcomes to the animals and possibly the dams bearing those pregnancies. 

C. Risk Assessment Methodology (Chapter III) 

Risk assessment is a science-based process used to identify hazards that may be present 

in predefined exposure scenarios, and to estimate the severity and chances of the 

outcome(s) occurring once that exposure occurs. Because many, if not all, of the 

individual steps that comprise a risk assessment contain various degrees of uncertainty, 

risk assessors should explicitly describe the sources of uncertainty and the effect(s) that 

the uncertainties may have on any judgment of risk. Risk assessment serves as the 

scientific underpinning from which risk managers may choose different options based on 

their understanding of, and responsibilities to, the broader contexts within which they 

operate. 

Qualitatively, risk may be thought of as some function of the combination of exposure 

and the intrinsic properties of the substance or process under consideration by linking an 

exposure to the likelihood of an outcome. When performing a risk analysis, it is critically 

important to distinguish between a hazard and the potential risk(s) that may result from 

exposure. A hazard can be defined as an act or phenomenon that has the potential to 

produce an adverse outcome, injury, or some sort of loss or detriment. These are 

sometimes referred to as harms, and are often identified under laboratory conditions 

designed to maximize the opportunity to detect adverse outcomes. Thus, such 

observational summaries are often referred to as “hazard identification” or “hazard 

characterization.” Risk, then, is the conditional probability that estimates the probability 

of harm given that exposure has occurred. In a qualitative assessment such as this, 

however, risks can be discussed only within a qualitative context, and no quantitative 

interpretations should be made. 

In order to address the hazards and risks to animals involved in cloning and the food 

products derived from them four issues must be addressed: identifying hazards and risks; 

determining the degree to which existing data address the question of risk; characterizing 

residual uncertainties; and selecting the most appropriate definition of risk for the risk 

assessment. 

This Risk Assessment explicitly excludes transgenic clones from the identification of 

hazards or risks experienced by “just clones” because of the inability to determine 

whether the transgenic event or cloning was causally associated with an adverse outcome. 
Chapter I: Executive Summary 6 

In addition, the Risk Assessment has assumed that, at minimum, animal clones, their 

progeny, and food products derived from them would be subject to the same laws and 

regulations as conventional animals and their food products. Because no exogenous genes 

have been introduced into animals derived via SCNT, the underlying assumption 

regarding potential hazards that could arise is that anomalies observed in animal clones 

are due to incomplete or inappropriate reprogramming of the donor cell nucleus. 

Therefore, any remaining hazards leading to food consumption risks that would result 

from inappropriate or incomplete reprogramming would be subtle. These subtle hazards 

would allow an animal clone to develop with apparently normal functions, but with subclinical physiological anomalies. These could include alterations in the expression of key 

proteins affecting the nutritional content of food and possibly lead to dietary imbalances. 

Similar hazards arise in animals generated via other ARTs. The goal of this draft risk 

assessment is to determine whether any unique hazards arise that are not noted in 

comparators, or have not been identified in cattle, swine, sheep, or goats produced via 

other ARTs. 

 

Both the animal health and food consumption risk assessments evaluated information 

within a framework developed by CVM called the Critical Biological Systems Approach

(CBSA), which divides the life cycle of an animal clone into five functional 

developmental nodes. Developmental Node 1 incorporates the initial technical steps 

involved in SCNT, from cell fusion through fetal development. Developmental Node 2 

encompasses the perinatal period, including late gestation, labor induction in the dam, 

delivery, and the critical few days after birth. The third developmental node, Juvenile 

Development and Function, covers the period of rapid growth between birth and the onset 

of puberty. The Reproductive Development and Function Node (Developmental Node 4) 

includes puberty and reproductive function throughout the reproductive life of clones. 

The Post-Pubertal Maturation Node (Developmental Node 5) consists of all nonreproductive functions of sexually maturing or mature clones, including growth, weight 

gain, disease frequency, aging, and, where available, lifespan. 

The nature of each component of the risk assessment (i.e., animal health or food 

consumption) shaped the manner in which the available data were evaluated. For 

example, identification of adverse outcomes for animal health included both the animal 

clone and the surrogate dam carrying the pregnancy. Emphasis was placed on the clones’ 

development and probability of normal development, compared with other ARTs such as 

artificial insemination (AI), in vitro fertilization (IVF), and blastomere nuclear transfer 

(BNT). For food consumption risks, however, animal clones bearing gross anomalies 

were excluded from the analysis, and emphasis was placed on identifying unique subtle 
Chapter I: Executive Summary 7 

hazards that could have arisen as the result of the SCNT process. The rationale for this 

approach is found in Chapter IV, which provides the molecular evidence for the role of 

epigenetic reprogramming as the source of these subtle hazards. Because of the 

assumption that hazards would be subtle, datasets were evaluated on as fine a level of 

resolution as possible, including individual animals or even individual analytes per 

animal in order to have as sensitive a screen as possible for adverse outcomes (and thus 

potential food consumption risks). In this risk assessment, the most detailed level of 

resolution used for evaluating animal health has been physiological and biochemical 

measures of individual animals. It is likely, as technologies mature, that molecular 

techniques such as genomics, proteomics, and their integrated metabolomic measures will 

assist in such determinations, but to date, these methods have not been standardized or 

validated (NAS 2004). 

D. The Implications of Epigenetic Reprogramming for Clones and their 

Progeny (Chapter IV) 

Epigenetics has been defined as the study of stable alterations in gene expression 

potentials that arise during development and cell proliferation. In sexual reproduction, a 

new diploid genome is created by the fusion of two haploid genomes. The subsequent 

expression of that genome into a functional organism is governed by a “program.” There 

are several examples of epigenetic control of gene expression, of which DNA 

methylation is likely the best characterized. 

Mammalian embryos experience major epigenetic reprogramming primarily at two times 

in their development, both of which have significant implications for cloning. One of 

these takes place soon after fertilization, and is referred to as preimplantation 

reprogramming; the other occurs during gametogenesis (the development of cells that 

ultimately become the sperm and egg). Because preimplantation reprogramming occurs 

after fertilization, and in the case of nuclear transfer, after fusion of the donor nucleus 

with the oöplast, it is the most immediately affected by the cloning process, and may be 

most directly implicated in the development of clones with defects. Gametogenic 

reprogramming may also be involved in the abnormalities noted in clones, but it likely 

has more far-reaching implications for progeny, because it generates the gametes used for 

the sexual reproduction of clones. 

When cloning, the donor nucleus must be coaxed to direct embryonic development as if it 

were a fertilization-derived zygote. Most of the time this is not successful. Anomalous 

epigenetic reprogramming is observed at the global genomic and individual gene level in 
Chapter I: Executive Summary 8 

clone embryos and fetuses, and in similar developmental stages of animals produced 

using ARTs with significant in vitro culturing components. Many of these are lethal, as 

demonstrated by the low success rate of IVF and the even lower success rate of SCNT. In 

the small number of successful cases that ultimately result in normal-appearing and 

functioning animals, SCNT-derived embryos appear to be able to carry out 

reprogramming just about as well as fertilization-derived embryos. Live and apparently 

healthy clones may exhibit some level of epigenetic differences relative to fertilizationderived animals. 

The Center assumes that if clones were to pose food consumption risks, the only 

mechanism by which those risks could arise would be from inappropriate epigenetic 

reprogramming, similar to those observed for other ARTs. It is important to note that the 

genes that are being dysregulated are the “normal,” naturally present genes that comprise 

the animal’s genome, and have not been introduced via recombinant DNA techniques 

from other sources (i.e., these are not transgenic or genetically engineered animals). 

Progeny of animal clones, on the other hand, are not anticipated to pose food safety 

concerns, as natural mating resulting from the production of new gametes by the clones is 

expected to reset even those residual epigenetic reprogramming errors that could persist 

in healthy, reproducing clones. 

E. Risks to Animals Involved in Cloning (Chapter V) 

This chapter compares SCNT with other ARTs with respect to effects on animal health 

and concludes that some animals involved in the cloning process (i.e., cattle and sheep 

surrogate dams, and some clones) are at increased risk of adverse health outcomes 

relative to conventional animals. None of these adverse outcomes, however, are unique to 

cloning. 

Cows and ewes used as surrogate dams for SCNT-derived pregnancies appear to be at 

increased risk of late gestational complications such as hydrops, as well as dystocia at 

parturition, that occur at a lower frequency with other ARTs that have a significant in 

vitro culturing component. Surrogate swine and goat dams bearing clones do not appear 

to be at increased risk. 

 

There is an increased risk of mortality and morbidity in perinatal calf and lamb clones 

compared with calves and lambs produced using other ARTs. In cattle and sheep, the 

increased risk appears to be related to large offspring syndrome. Survival of these clones 
Chapter I: Executive Summary 9 

appears to be a function of both the severity of the clinical signs and neonatal 

management. Morbidity and mortality do not appear to be increased in perinatal swine 

and goat clones. 

After the perinatal developmental node, no new health risks have been identified in 

clones of any of the species considered in this risk assessment. Clones in the juvenile to 

prepubertal age cohort do not appear to be at an increased risk of morbidity or mortality 

compared to animals produced by natural service or ARTs. Most animals surviving the 

neonatal period appear to grow and develop normally. No increased risk of adverse health 

effects have been reported in clones approaching reproductive maturity. Finally, the 

available information indicates that there are no increased risks to the health of maturing 

clones relative to conventional animals. Currently, it is not possible to draw any 

conclusions regarding the longevity of livestock clones due to the relatively short time 

that the technology has existed. 

Based on the biological assumptions and molecular data reviewed in Chapter IV, progeny 

of clones are expected to be normal. Consistent with these predictions, the data on the 

health status of clone progeny indicate that there is no increased risk of health problems 

in these animals compared with conventional animals. 

F. Food Consumption Risks (Chapter VI) 

1. Two-Pronged Approach to Identifying and Characterizing Food 

Consumption Risks 

In order to determine whether epigenetically-caused subtle hazards pose food 

consumption risks, CVM has developed a two-pronged approach. The first component, 

the Critical Biological Systems Approach (CBSA), incorporates a systematic review of 

the health of the animal clone or its progeny. Its role in the evaluation of food 

consumption risk analysis is premised on the hypothesis that a healthy animal is likely to 

produce safe food products. It accepts that at this time, SCNT is a biologically imprecise 

and inefficient process, but recognizes that animals are capable of biological repair or 

adaptation. The cumulative nature of the CBSA allows for the incorporation of both 

favorable and unfavorable outcomes. The former, provided that all other measures appear 

to be normal, will result in the finding that the clone is likely to produce edible products 

that pose no food consumption risks; the latter implies that clones with anomalies are 

likely to be considered unsuitable for food. The second component, the Compositional 

Analysis Method, assumes that food products from healthy animal clones and their 

progeny that are not materially different from corresponding products from conventional 
Chapter I: Executive Summary 10 

animals pose no additional risks. It relies on the comparison of individual components of 

edible products, and the identification of the appropriate comparators. 

Assessing the safety of food products from animal clones and their progeny is best 

accomplished by using both approaches: prospectively drawing on our knowledge of 

biological systems in development and maturation, and in retrograde, from an analysis of 

food products. Subtle hazards and potential risks that may be posed by animal clones 

must, however, be considered in the context of other mutations and epigenetic changes 

that occur in all food animal populations. No adverse outcomes have been noted in clones 

that have not also been observed in animals derived via other ARTs or natural mating that 

enter the food supply unimpeded. 

Because the value of clones lies in their genetics, CVM anticipates that animal clones 

might enter the food supply as meat if removed from the herd due to injury or 

senescence, but these would likely be animals near the end of their reproductive lives. 

Milk from clones, however, might enter the food supply. Progeny of clones are more 

liked to be reared as animals intended primarily for food use. 

2. Conclusions Regarding Potential Food Consumption Risks 

Based on this review of the body of data on the health of animal clones, the composition 

of meat and milk from those animals and corresponding information on clone progeny, 

CVM has drawn the following conclusions: 

a. Cattle Clones 

Edible products from perinatal bovine clones may pose some very limited human food 

consumption risk. 

The underlying biological assumption in place for this age cohort is that perinatal clones 

may be fragile at birth due to residual incomplete or inappropriate reprogramming of the 

donor nucleus. The data are consistent with that assumption; some perinatal clones do not 

survive for several reasons, including poor placentation, LOS, and in some cases, frank 

malformations. Although surviving clones can be fragile for a period of time, survivors 

tend to adjust to life outside the womb within a relatively short period, either on their 

own or with assistance from caregivers. A significant proportion of perinatal clones 

survives gestation and is born without significant health problems. Laboratory measures 

of key physiological functions do not indicate that surviving animals are very different 

from conventional newborns. It is therefore unlikely that food consumption risks have 

been introduced into these animals or that rendering these clones will pose risks in animal 

feed or to humans consuming animals fed material derived from the clones. 
Chapter I: Executive Summary 11 

Edible products from juvenile bovine clones pose no additional food consumption 

risk(s) relative to corresponding products from contemporary conventional 

comparators. 

The underlying biological assumption for this developmental node is that if any 

anomalies were to be found in the youngest clones and those animals were to survive to 

be healthy adults, the juvenile developmental node would be a period of equilibration and 

normalization. The data are consistent with such a hypothesis.

Juvenile bovine clones are largely healthy and normal. Although some clones in this 

developmental node are more physiologically unstable than their conventional 

counterparts, they are in the process of normalizing their physiological functions on the 

way to adulthood. This normalization has been observed consistently and is further 

demonstrated by the analysis of clinical chemistry and hematology data demonstrating 

that clones show the appropriate physiological responses to developmental signals. None 

of the physiological measures taken, including both clinical chemistry and hematology, 

indicate any food consumption hazards. 

Edible products derived from adult bovine clones pose no additional risk(s) 

relative to corresponding products from contemporary conventional 

comparators. 

This conclusion is based on application of both prongs (CBSA and Compositional 

Analysis) of the risk assessment approach. The body of data comprising the CBSA 

approach is consistent with the biological prediction that there are no underlying 

biological reasons to suspect that healthy animal clones pose more of a food safety 

concern than conventional animals of similar age and species. 

The data show that healthy adult clones are virtually indistinguishable from their 

comparators even at the level of clinical chemistry and hematology. These data also 

confirm the observation that physiological instabilities noted earlier in the lives of the 

clones are resolved in the juvenile developmental node (see previous conclusions 

regarding other developmental nodes), and do not reappear as the clones age. There are 

some reports of early deaths of clones; as these animals would not enter the food supply, 

they do not pose a food consumption risk. Data on reproductive function in cows or bulls 

of this age cohort indicates that healthy bovine clones surviving to reproductive maturity 

function normally and produce healthy offspring. These data are consistent across 

studies. Given that reproduction is the most difficult “biological hurdle” placed on an 

organism, the observation of normal reproductive function provides an additional degree 

of confidence to the conclusion of the appropriate development of these animals. 
Chapter I: Executive Summary 12 

All of the reports on the composition analysis of meat or milk from bovine clones show 

that there are no biologically significant differences in the composition of milk derived 

from clone and non-clone cattle. Additionally, data from one report show no difference in 

allergenic potential for meat or milk derived from clone cattle compared to meat or milk 

from non-clone comparators. Similarly, neither meat nor milk from clone or non-clone 

cattle induced mutations in a mutagenicity assay. Finally, none of the reports identified 

an endpoint that would pose a hazard for human consumption. 

b. Swine Clones 

Edible products from adult swine clones pose no additional risk(s) relative to 

corresponding products from contemporary conventional comparators. 

This conclusion is based on the same underlying biological assumption as cited for adult 

bovine clones. Because the data are more heavily weighted towards adult, market sized 

animals, judgments regarding the safety of food products from swine clones are provided 

in one aggregate set of comments.

Once piglet clones are born, they appear to be healthy. The most compelling argument for 

the normal health status of swine clones results from the evaluation of the behavior and 

physiological status of a small cohort of relatively young (15 weeks), and approximately 

market age (27 weeks) swine clones relative to closely related conventional pigs. No 

significant differences were observed in either behavior, epigenetic, or physiological 

measurements, indicating that these animals were not materially different from the 

comparators. Another small dataset on swine clones reared in very unusual settings (i.e., 

deprivation of colostrums, initial husbandry in pathogen-free conditions, switching to 

commercial settings) is confounded with respect to outcome. Nonetheless, these clones 

were able to respond appropriately to this stress ̧ and their carcass characteristics, 

reproductive performance, including semen quality, farrowing rates and litter sizes were 

within national averages. No biologically relevant differences were observed in the 

composition of meat from these clones or their comparators. 
Chapter I: Executive Summary 13 

c. Sheep Clones 

Except by relying on underlying biological assumptions, and by inference from other 

species, there is insufficient information on the health status of sheep clones to draw 

conclusions with respect to potential risks that could be posed from the consumption of 

food products. 

With the exception of reports on Dolly, CVM was unable to find any publicly available 

reports on the health status of live sheep clones. There are several studies addressing 

methodological issues for optimizing the generation of clones, but these do not address 

post-natal health. There are reports of anomalies noted in fetal sheep clones that have 

died or been terminated, and reports on the pathology associated with animals that do not 

survive. Although these are instructive for understanding the molecular and 

developmental pathways that may be perturbed during the process of SCNT, these studies 

have limited relevance to addressing food safety because the deceased animals would not 

have been allowed to enter the food supply. CVM was not able to find any reports on the 

composition of milk or meat from sheep clones. 

 

d. Goat Clones 

Edible products from goat clones pose no additional food consumption risk(s) relative 

to corresponding products from contemporary conventional comparators. 

This conclusion is based on the same underlying biological assumption cited for the other 

livestock species, and a relatively small but compelling dataset. Once clone embryos are 

transferred to surrogate dams and pregnancies are confirmed, the “success rate” for live 

births is quite high. The animals appear to have developed well through reproductive age, 

and the available data indicate their physiological responses are appropriate for age and 

breed. The reproductive development and function of male Nigerian Dwarf goat clones 

demonstrate that those animals functioned appropriately relative to age- and breedmatched comparators. One male progeny goat was derived from the buck clones; this 

animal also appeared to function in an age- and breed-appropriate manner. No meat or 

milk composition data were identified for goat clones. 

e. Clone Progeny 

Edible products derived from the progeny of clones pose no additional food 

consumption risk(s) relative to corresponding products from other animals.

Progeny of clones will likely provide the overwhelming majority of clone-derived food 

products (both meat and dairy) in the US. The underlying biological assumption for 

health of progeny animals is that passage through the process of creating the cells that 
Chapter I: Executive Summary 14 

ultimately become ova and sperm naturally resets epigenetic signals for gene expression, 

and effectively “clears” the genome of incomplete or inappropriate signals. The rationale 

for this assumption has been developed in Chapter IV, and dominates the conclusion that 

edible products from any clone progeny pose no additional food consumption risk(s) 

relative to those from any other sexually reproduced animals. It has been supported by 

detailed empirical3 evidence both in the mouse model system, which clearly indicates that 

phenotypic alterations noted in the parent clones are not passed to their sexually-derived 

progeny. Observations on the health and meat composition of progeny of livestock 

clones, with one extensive dataset on the progeny of swine clones in particular, provide 

direct data on the health of these animals and on the composition of their meat. The swine 

data support the underlying biological assumption that the progeny of clone animals are 

essentially indistinguishable from the comparable progeny of non-clone animals. 

We therefore concur with the high degree of confidence that the outside scientific 

community (NAS 2002 a,b) places in the underlying biological assumption, and conclude 

that consumption of edible products from clone progeny would not pose any additional 

food consumption risk(s) relative to consumption of similar products from sexuallyderived animals. 

G. Concluding Statements (Chapter VII) 

For Animal Health: SCNT results in an increased frequency of health risks to animals 

involved in the cloning process, but these do not differ qualitatively from those observed 

in other ARTs or natural breeding. The frequency of live normal births appears to be low, 

although the situation appears to be improving as the technology matures. Cattle and 

sheep exhibit a set of clinical signs collectively referred to as LOS that do not appear to 

be present in swine or goats. Surrogate dams are at risk of complications from birth if the 

fetus suffers from LOS, or from accumulation of fluid in the cavities of the placenta 

(hydrops). Clones exhibiting LOS may require additional supportive care at birth, but can 

recover and mature into normal, healthy animals. Most clones that survive the perinatal 

period are normal and healthy as determined by physiological measurements, behavior, 

and veterinary examinations. Progeny of animal clones also have been reported as normal 

and healthy. 

For Food Consumption Risks: Extensive evaluation of the available data has not 

identified any food consumption risks or subtle hazards in healthy clones of cattle, swine, 

 

3

 Empirical refers to that which can be seen or observed alone, often without reliance on theory. In the 

context of this risk assessment, conclusions drawn on empirical evidence are those that are drawn strictly 

based on the data. These conclusions may later be put in the context of underlying biological assumptions. 
Chapter I: Executive Summary 15 

or goats. Thus, edible products from healthy clones that meet existing requirements for 

meat and milk in commerce pose no increased food consumption risk(s) relative to 

comparable products from sexually-derived animals. The uncertainties associated with 

this judgment are a function of the empirical observations and underlying biological 

processes contributing to the production of clones. There is less uncertainty about the 

health of clones as they age and have more time to exhibit the full range of functionality 

expected of breeding stock. 

Edible products derived from the progeny of clones pose no additional food consumption 

risk(s) relative to corresponding products from other animals based on underlying 

biological assumptions, evidence from model systems, and consistent empirical 

observations. 

The results of this comprehensive risk assessment agree with the preliminary findings of 

the NAS (2002a) conclusions that “The products of offspring of clone[s] ... were 

regarded as posing no food safety concern because they are the result of natural matings,” 

and “In summary there is no current evidence that food products derived from adult 

somatic cell clones or their progeny present a food safety concern.” 
 

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Chapter II: 

Technology Overview 

Somatic Cell Nuclear Transfer 

and Other 

Assisted Reproductive Technologies
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Chapter II: 

Technology Overview: Somatic Cell 

Nuclear Transfer and Other Assisted 

Reproductive Technologies 

Since the beginnings of livestock agriculture, selection criteria have been applied to 

foster the propagation of animals with traits more desirable to humans. The expansion of 

herds with desirable traits has been limited, however, by the reproductive capacity of the 

species or breed and the prevalence of particular versions of genes (or sets of genes) 

responsible for those traits in the available gene pool. (The gene pool can be considered 

all of the animals available for breeding.) The female contribution to reproductive 

success, for example, is limited by species-specific characteristics such as average litter 

size, frequency of estrus, and gestation length. In natural breeding, male contributions are 

restricted by the degree of proximity to fertile females and the ability to inseminate 

females with a sufficient number of normal sperm. Finally, individuals of both sexes are 

limited to the extent that they may carry the desired versions of genes or combination of 

genes. 

To help overcome some of these complications, various forms of assisted reproductive 

technologies (ARTs) have been adopted in animal agriculture for over a century, and at 

least one (artificial insemination) has been used for several hundred years. These 

technologies form a continuum that ranges from the fairly minimal assistance provided to 

animals engaged in natural service through those containing components of significant in 

vitro manipulation such as in vitro fertilization and embryo splitting, to the more recent 

development of somatic cell nuclear transfer (SCNT), or what is colloquially referred to 

as “cloning”4

 (Faber et al. 2004; Sakai 2005). Beginning with the development and 

application of modern artificial insemination (AI) methodologies in the first half of the 

20th century, ARTs have aided in the genetic improvement of domestic species, including 

selection of phenotypessuch as behavioral and production traits in domesticated animals 

(Youngquist 1997, Faber and Ferre 2004). By accelerating the rate at which selective 

breeding goals can be met, improved genotypes have expanded rapidly into national 

herds in the United States and other countries (Faber et al. 2004; Wells 2005). In turn, 

this has resulted in lower costs for livestock producers and retail consumers, while 

 

4

 The term "clone" originated before the late 1990s. The British biologist J.B.S. Haldane, in a speech 

entitled "Biological Possibilities for the Human Species of the Next Ten-Thousand Years," used the 

term in 1963. The Merriam-Webster dictionary, however, dates its use in a biological context to 1903. 
Chapter II: Technology Overview 20 

simultaneously maintaining or improving the quality and consistency of foods of animal 

origin. 

Reproductive technology advances have also proven to be powerful tools in curbing the 

spread of vertically transmitted diseases (i.e., those that are passed from the dam to her 

offspring during the period immediately before and after birth, either across the placenta 

or in the dam’s milk) (Youngquist 1997). For example, embryo transfer (ET) (see 

subsequent discussion for a description of this ART) has been used to prevent vertical 

transmission of Neospora caninum in cattle (Landmann et al. 2002; Ballargeon et al. 

2001), scrapie in sheep (Wang et al. 2001), Bovine Virus Diarrhea (BVD) in cattle 

(Smith and Grimmer 2000), and Brucella abortus in an American bison (Robison et al. 

1998). Embryo transfer is commonly used in laboratory animal research to re-derive 

valuable strains of gnotobiotic (i.e., animals in which all of the bacterial species are 

known) or specific pathogen-free research animals when colonies become infected with 

undesirable disease agents that cannot be controlled through more conservative means. 

The following chapter begins with a brief overview of what cloning is, followed by an 

overview of the continuum of other ARTs commonly in use in current US agricultural 

practice, placing nuclear transfer technology into context of these breeding practices. 

Appendix B provides additional details on overall reproductive efficiency observed in 

current agricultural practice in the US, and Appendix C provides a comprehensive 

summary of the outcomes observed in ARTs, with particular emphasis on those 

technologies that contain a significant in vitro culturing component. Although all of these 

technologies are currently in practice, all are continually undergoing development and 

refinements with the goal of improving efficiencies. A reasonable expectation then, is 

that success rates (defined as the rate of production of healthy animals) will improve as 

expertise increases. 

A. What is Cloning? 

Cloning, or somatic cell nuclear transfer, is a process by which animals are reproduced 

asexually (embryo splitting and blastomere nuclear transfer are other ways of 

reproducing animals asexually and are discussed later in this chapter). In cloning, a 

differentiated somatic cell (a non-germ line cell from an existing animal) is introduced to 

an oöcyte (a cell that is the immediate precursor of a mature egg) that has had its nucleus 

(and thus its genome) removed, and then, following some manipulations, is induced to 

start replicating. If all goes well, the dividing cell is implanted into a female animal 

(dam), continues to develop normally, and is delivered just as any newborn. 
Chapter II: Technology Overview 21 

Since the first report of a clone produced by SCNT (Wilmut et al. 1997), several other 

species have been cloned (Table II.1), although in some cases (e.g., companion animals) 

only a limited number of animals have been generated. The reasons for this are multifold, but are largely driven by the relative difficulty in producing clones, and the various 

drivers, economic and technical, that affect the expansion of the technology. For 

example, the use of clones in expanding elite breeding stock in domestic livestock is 

perceived to have benefit for breeders and consumers. This risk assessment does not 

attempt to address those issues, however, and instead concentrates on those domestic 

livestock clones commonly consumed as food (e.g., cattle, swine, sheep, and goats). 

Table II.1. Species of Animals that Have Been Cloned 

Species First Citation 

Sheep Wilmut et al. (1997) 

Mouse Wakayama et al. (1998) 

Cow Forsberg et al. (2002) 

Goat Keefer et al. (2002) 

Mule Woods et al. (2003) 

Horse Galli et al. (2003) 

Rabbit Chesne et al. (2002) 

Cat Shin et al. (2002) 

Pig Polejaeva et al. (2000) 

Dog Lee et al. (2005) 

Rat Zhou et al. (2003) 

Deer Texas A&M announcement (2003) 

B. Continuum of Reproductive Technologies 

1. Natural Service 

Although many people who are not involved in intensive animal agriculture assume that 

most breeding occurs “naturally” 5 (e.g., a male animal mates with receptive female), in 

fact, human intervention is the industry standard for many livestock operations 

(Youngquist 1997). In the US dairy industry, for example, most reproduction involves 

some technological component, and swine producers rarely use natural mating for their 

production of offspring. Conversely, in the beef industry most reproduction occurs by 

 

5

 The process of non-assisted mating is referred to as natural “mating,” “coverage” or “service.” 
Chapter II: Technology Overview 22 

natural service, and most of the world’s sheep and goat production occurs under free 

range conditions and depends on natural mating. 

Humans have assisted animals in natural mating by monitoring the reproductive status of 

females, introducing receptive females to the same location (e.g., field, corral, or pen) as 

the male, and allowing nature to take its course. When this process does not result in 

sufficient offspring of the desired phenotype, or is otherwise compromised, assisted 

reproductive technologies can be called into play. 

2. Artificial Insemination and Synchronized Estrus 

The first ART developed was artificial insemination (AI), which in its simplest form 

involves the collection of semen from males and its subsequent human-assisted 

introduction into a physiologically receptive female. It is an important technique for the 

genetic improvement of animals, as a few select males can produce sufficient sperm to 

inseminate thousands of females per year, while natural service would provide for the 

insemination of only a fraction of those animals. 

Reports of AI in horses as part of breeding programs have been traced to the Arabian 

Peninsula in the 14th century (Bearden and Fuquay 2000). AI of a beagle dog was first 

described by Spallanzani in 1780 (Hafez and Hafez 2000). In 1899, the Russian Czar 

Nicholas II commissioned I.I. Ivanov to develop an AI program for horses, and by 1933 

Ivanov had developed methods for collecting semen and inseminating horses, cows, 

sheep, and pigs (Foote 2001). In 1931, 19,800 cows were bred by AI in Russia. By 1936, 

Denmark had established an AI cooperative association, and by 1939, the use of AI had 

spread to the United States. In 1970, it was estimated that 7,344,420 dairy cows were 

bred using AI (Webb 2003). 

Although there are several methods for collecting semen, most involve training males to 

ejaculate into an artificial vagina. Semen is then diluted to maximize the number of 

services that one male can provide. A normal ejaculate from a dairy bull usually contains 

between 5 and 10 billion sperm; good conception rates generally require about 12-20 

million sperm to be introduced. The diluting solution contains factors that help to 

stabilize and preserve the sperm, as well as antibiotics to inhibit bacterial growth and 

reduce the danger of spreading any potential disease or contamination. Most collected 

semen is stored in glass ampoules or plastic straws, and is generally stored either in dry 

ice and alcohol (-100o

F) or liquid nitrogen (-320oF). To date, there appears to be no limit 

on the amount of time that bovine sperm can remain frozen and regain viability upon 

appropriate thawing. Since 1997, use of AI in swine breeding has increased 
Chapter II: Technology Overview 23 

dramatically. A survey of swine producers conducted by the National Pork Board in 2003 

indicated that even among small producers (1,000 to 3,000 swine marketed annually) as 

many as 60 percent of litters were sired by AI in 2003, while for large producers (> 

50,000 swine marketed annually) 98 to 100 percent of litters were sired by AI.6 Rams 

(male sheep) and bucks (male goats) can also be donors for artificial insemination. 

In the US, AI of the female is usually performed either by trained technicians employed 

by breeding companies or large farms or by the producers themselves. The most common 

technique employed today for dairy cows involves the use of sterile, disposable catheters 

that are inserted vaginally and extended through the cervix into the body of the uterus of 

the recipient cow (whose estrous cycle has been documented). Thawed semen is warmed 

to the appropriate temperature, and sperm are deposited in the uterine/cervical regions. 

The primary advantages of AI to farmers include the ability to use semen from bulls 

anywhere in the world rather than those that are more geographically proximate, and thus 

to have desirable genetics available for propagation. It also allows the farmer to use 

multiple sires in a herd without the attendant costs of maintaining animals that are often 

difficult to handle and in multiple breeding pastures. AI tends to be less expensive than 

natural service (a straw of semen generally costs less than transporting a female to the 

sire and the stud fee) and avoids the potential physical risks to either sire or dam as part 

of the mating process. The disadvantages of AI include the need to train personnel 

engaged in the breeding operations on how to detect estrus in females (see subsequent 

discussion of estrous synchronization), and training or retaining individuals to perform 

the insemination. Further, care needs to be taken not to rely excessively on a few 

apparently superior sires so as not to reduce the genetic diversity of the resulting herds. 

Sperm collection and AI were further improved by the advent of sperm sexing, or 

selection of sperm carrying an X (female) or Y (male) chromosome.7

 Development of an 

effective and simple method for producing animals of the desired sex is economically 

desirable for livestock producers; sperm sexing is currently being used when available 

and economically feasible (Foote 2001; Faber and Ferre 2004). For example, in the dairy 

industry, females are desired because males do not produce milk; and excess males often 

become veal. In the beef industry, however, males are desired because they grow faster. 

Females can be the desired sex in the swine industry where leaner animals generally 

receive higher prices; young female pigs (gilts) tend to be leaner than castrated male pigs 

(barrows) when they arrive at market. 

 

 6 http://www.pork.org/Producers/EconomicsMarketInfo/Production%20and%20Marketing2003.doc 

7

 In normal mammalian sexual reproduction, the female always donates an “X” chromosome, and the 

male can donate either an “X” or a “Y” chromosome. XX yields a female animal; XY produces a male. 
Chapter II: Technology Overview 24 

One method that shows the most promise for predetermining the sex of offspring is 

sexing semen using flow cytometry. This technique is based on the observation that in 

livestock species, sperm with X chromosomes have about 3 percent more DNA than 

those with Y chromosomes. Collected semen is diluted, and single sperm are passed 

through a laser beam that allows for the determination of the amount of DNA in each 

individual sperm. Based on their relative DNA content, sperm are sorted into “heavier” 

(female producing) and “lighter” (male producing) fractions. Another method sexes early 

embryos by removing one or two of the cells from the early embryo, arresting the further 

growth of the embryo by freezing, and identifying genes found only on the Y 

chromosome using the polymerase chain reaction (PCR) in the selected cells (Youngquist 

1997). Semen sexing is more rapid, less invasive, and more economical, while embryo 

sexing is impractical at this time, as it is invasive, time intensive, and quite expensive. 

Further, the potential to damage the embryo by piercing the protective layer around it 

(zona pellucida), removing cells, and freezing the remaining cells in the embryo is quite 

high. 

During the breeding season, estrous synchronization further permits the efficient use of 

artificial insemination (Hafez and Hafez 2000). Estrous synchronization, or the timed 

induction of heat, is typically achieved by hormone therapy, allowing for the 

insemination of large groups of animals, and was first practiced in the US in the 1960s. 

The alternative is the time-consuming method of observing females’ behavior to gauge 

estrous initiation, and then arranging insemination for the appropriate time interval 

following initiation of estrus. Labor, as well, can be synchronized (or closely grouped) by 

the use of hormones. The advantage of linking AI to estrous synchronization lays in the 

ability of contained agricultural practices to operate on a more predictable schedule. For 

example, cattle breeders can avoid the reduced conception rates that occur during 

summer’s heat by breeding animals during the cooler spring season. Predictability can 

benefit farmers by allowing them to allocate resources (e.g., farm labor, veterinary visits) 

more efficiently, thus lowering production costs. 

3. Embryo Transfer 

It is impossible for a fertile female mammal to bear all of her potential offspring. Litter 

size, gestation time, and post-partum decreases in fertility all limit the potential number 

of progeny that she can produce. When the female animal reaches the end of her 

reproductive period, any remaining unfertilized eggs represent potential offspring that 

have been lost. One solution to this dilemma is to transfer embryos of genetically 

superior female animals to multiple surrogate dams. This technique, called “embryo 
Chapter II: Technology Overview 25 

transfer,” is particularly useful in species in which a low number of progeny are produced 

per gestation.8 In concept, then, embryo transfer (ET) is analogous to AI in that the total 

yield of offspring from a genetically superior, in this case, female animal can be 

increased (Youngquist 1997). 

In 1890, rabbit embryos were first transferred from a donor female to surrogate rabbits. 

The experiment demonstrated that the surrogate’s genetics would not influence the 

transferred embryo’s genetics or development. In 1951, a successful live bovine ET was 

accomplished, but non-surgical methods of embryo collection did not succeed until the 

late 1960s (Hafez and Hafez 2000). 

Currently, it is possible to flush large numbers of viable embryos from a superovulated 

cow with minimal stress to the animal (Hafez and Hafez 2000). Superovulation of the 

donor animal is generally accomplished by injecting the animal with follicle stimulating 

hormone or other exogenous gonadotropins before she enters estrus. The hormones 

induce production of a large quantity of ovarian follicles containing mature, preovulatory 

oöcytes. Insemination is performed at appropriate times relative to ovulation depending 

on the species and breed. Recipient surrogate mothers are synchronized in parallel with 

the donor to be ready to accept embryos for implantation and gestation. When embryos 

are about a week old, they are flushed out of the donor dam’s uterus, isolated from the 

flushing solution, and examined microscopically to determine whether they are of 

sufficiently quality to implant. If they meet the criteria for further use, embryos can be 

transferred immediately to a waiting synchronized recipient animal, frozen for later use, 

or split into halves (see embryo splitting discussion below). Fresh or thawed embryos are 

inserted into surrogate mothers, where they attach to the lining of the uterus, and progress 

through the normal course of pregnancy. 

This technique, referred to as MOET (multiple ovulation and embryo transfer), is often 

used in relatively intensive cattle breeding programs, but is less developed in other 

livestock species. Similar to fertilizing many females with sperm from one superior male, 

MOET provides the breeder the ability to expand genetic traits exhibited in superior 

females. Further, the ability to freeze embryos allows for the preservation of “genetic 

stock” to be used at a later time. Its prevalence in livestock breeding, however, is much 

lower than AI, as it is considerably more expensive (Wilmut et al. 2002).

The International Embryo Transfer Society (IETS), a professional society whose 

membership includes breeders and researchers, estimates that a total of approximately 

 

8

 Cattle, for example, usually produce one offspring, and occasionally two per gestation; sheep and goats 

generally produce one or two offspring, with an occasional triplet delivery. Swine, on the other hand, 

usually bear multiple piglets in a litter, and require multiple fetuses to maintain the pregnancy. 
Chapter II: Technology Overview 26 

550,000 in vivo derived bovine embryos were transferred worldwide in 2004 (Thibier 

2005). Most of those transfers occurred in North America (39.5 percent), with the rest 

taking place in Asia (~21.6 percent), South America (~21.1 percent), and Europe (~15.9 

percent). The numbers of embryo transfers for other species (sheep and goats) were 

considerably lower, with approximately 68,000 sheep embryos transferred, mostly in 

Australia, New Zealand and South Africa, and fewer than 1,000 goat embryos 

transferred, mostly in South Africa and Asia. According to IETS statistics, approximately 

16,016 swine embryos, most of which were either transgenic or embryo clones, were 

transferred in 2004, with almost all occurring in Korea and Canada. 

4. In vitro Fertilization 

The first in vitro fertilized (IVF) offspring was a rabbit born in 1959 (Chang 1959). Since 

that time, IVF offspring have been born to mice, rats, hamsters, cats, guinea pigs, 

squirrels, pigs, cows, monkeys, and humans (Bearden and Fuquay 2000). IVF allows for 

the production of offspring from animals where other ART methods fail due to 

difficulties with either the female (blocked oviducts, non-responsive ovaries) or male 

(marginal semen quality and/or quantity), or where disease is present. In cattle, it is also 

used for the production of embryos from sexed semen because of the low sperm counts 

resulting from current sexing protocols, and for the further extension of the semen of 

superior sires due to the relatively low level of sperm required for in vitro fertilization. 

(IVF procedures are also used to assist human couples with limited fertility.) 

The overall technique for IVF is similar among species, and involves significant 

manipulations in vitro, or outside the body of animals. In livestock species, oöcytes are 

collected from the ovaries of either living or deceased animals whose genetic potential is 

desirable (Goodhand et al. 1999). Ovaries can be obtained by transvaginal aspiration 

from live animals, or from a deceased animal at time of slaughter. Slaughterhouse ovaries 

are cross-sectioned and the contents of all of the follicles are collected; mature oöcytes 

are collected, evaluated for quality, and used for fertilization. Immature oöcytes must be 

allowed to continue to develop in a maturation medium. 

Either fresh or frozen-thawed semen can be used for fertilization. Sperm need to be 

capacitated in vitro in order to penetrate the zona pellucida and fuse with the ovum or to 

undergo the same maturation process that they would normally undergo in the female 

reproductive tract. Capacitation involves a series of cellular changes to the sperm 

including increased motility, calcium uptake and protein binding (binding to proteins 

produced by the female reproductive tract). In vitro capacitation is accomplished by 

creating a medium designed to simulate the female reproductive tract and allowing the 
Chapter II: Technology Overview 27 

sperm to incubate in it for a period of time. Sperm are then added to ova, incubated in 

culture medium for approximately 8-22 hours, and the resulting fertilized ova, called 

zygotes, are washed, examined for appropriate development, and allowed to continue to 

divide for up to seven days, again in culture. At that time, if embryos appear normal, they 

may either be frozen for future use or inserted into the uterus of a reproductively 

competent female. 

The IETS reported that 239,813 in vitro produced cattle embryos were transferred in 

2004. Over half of those transfers were performed in Asia (62.6 percent), and most of the 

rest taking place in South America (33.7 percent), Europe (2.8 percent), and North 

America (0.8 percent) (Thibier 2005). 

5. Embryo splitting 

Genetically identical individuals derived from a sole embryonic source can arise 

naturally, as in the case of spontaneous monozygotic twinning, or in vitro via the manual 

separation (splitting) of early stage embryos. Embryo splitting may be considered the first 

true “cloning” procedure involving human intervention, and was first described by 

Willadsen and Polge in 1981, when monozygotic twin calves were produced. 

Embryo splitting, or the mechanical separation of cells,9

 can be used in very early 

embryos. Briefly, two-cell embryos derived from either in vitro fertilization, or embryo 

rescue following in vivo fertilization (as described for embryo transfer) are held in place 

with micropipettes under a microscope. The zona pellucida (the clear layer of protein 

surrounding the oöcyte and fertilized ovum) of these embryos is opened, and the twocelled embryo is then split into individual cells with a finely drawn needle or pipette. One 

of the cells is left in the original zona pellucida and the other is either placed into an 

empty zona pellucida or allowed to develop without a zona pellucida. These so-called 

demi-embryos can be cultured in vitro for a few days, inspected for appropriate growth 

and then transferred directly to synchronized recipient dams or frozen for future use. 

 

9

 Common nomenclature for the early stages of development following fertilization include the zygote, 

which includes the fertilized egg contained in the zona pellucida, through about the 8 cell stage of 

development (3 days in the cow, and 3-4 days in the sow). The morula refers to the time period 

(between about 4-7 days in cows, and 4-5 days in sows) following fertilization in which cells continue 

to divide within the zona pellucida, but there is no discernable migration of cells into any particular 

region. At about 7-12 days in cattle and 5 days in swine, a group of cells migrates to a portion of the 

spherical mass, forming an inner cell mass, with the remainder forming a ring of cells around a central 

hollow core (blastocoele). This is referred to as the blastocyst. The inner cell mass continues to develop 

into most of the body mass that will constitute the fetus and the ring of cells around the perimeter, 

which is referred to as the trophoblast, will eventually make up the placenta. 
Chapter II: Technology Overview 28 

Similar procedures can be used to multiply embryos that have developed beyond the 2-

cell stage (Willadsen 1980). Each of the individual cells or blastomeres from a single 

early embryo is totipotent. That is, each cell retains the ability to generate a fully 

functional individual identical to the other individual(s) derived from the other cells of 

the original embryo. 

Although commercial applications of embryo splitting have been tracked by breeders’ 

associations, the technology has never gained significant market penetration for several 

reasons. It is a very expensive and time consuming procedure that has not provided the 

yield initially anticipated for the technology. For example, actual calf yield from 

blastomere splits is approximately 105 calves per 100 embryos, while direct transfer of 

intact embryos yields approximately 60 calves per 100 embryos (Wilmut et al. 2000). 

Unless embryos are sexed at the time of splitting, however, breeders may end up with 

half of their animals being of the undesired gender, thus incurring twice the cost for the 

desired offspring. In addition, even if the resulting calves are of the desired gender, their 

production potential is not known, making the procedure an expensive gamble. 

 

6. Blastomere Nuclear Transfer 

The next evolution of ART evolved from additional manipulations of the blastomere cell, 

and involved its fusion with an enucleated oöcyte. This method expands on the relatively 

simple early stage embryo cell separation procedure described previously by allowing the 

use of cells from later stage embryos. In this case, embryos of the eight to sixteen-cell 

stage, compact morulae, and the inner cell mass from blastocysts can be used as donor 

nuclei (First and Prather 1991). Fusion of these later stage blastomere cells, which have 

lost their totipotency, with enucleated oöcytes, reprograms the blastomere nuclei to allow 

them to develop as zygotes. Blastomeres from bovine embryos up to the 64-cell stage can 

be fused with enucleated freshly fertilized oöcytes and cultured to develop into 

genetically identical individuals (Keefer et al. 1994). Cell nuclei derived from the inner 

cell mass of expanded blastocysts transferred into enucleated host cells are also capable 

of development resulting in offspring (Sims and First 1994). 

This technology, which may be considered the true antecedent of somatic cell nuclear 

transfer, had limited commercial applicability for the same reasons as embryo splitting: 

high cost, high loss rate, and the inability to predict phenotypic performance or the 

gender of the resulting offspring. 
Chapter II: Technology Overview 29 

7. Somatic Cell Nuclear Transfer (SCNT) 

In 1962, biologist John Gurdon of Oxford University pioneered the method of the two 

step “nuclear transfer” process in frogs: the enucleation of a recipient oöcyte and the 

subsequent transfer of a differentiated somatic cell nucleus to that oöcyte. Gurdon’s 

experiments showed that despite the differentiated status of the donor nucleus, 

reconstituted cells appeared to reprogram, or dedifferentiate, the nucleus and enable it to 

function much as a naturally produced zygote. These zygotes successfully developed into 

viable embryos that hatched and grew into tadpoles. Because the tadpoles had all come 

from the gut cells of the same adult frog, they all had the same genetic material and thus 

were all clones. However, Gurdon’s nuclear transfer tadpoles clones failed to 

metamorphose into frogs. When scientists attempted to apply this technology to other 

species such as mice, cattle, or other mammals, the developmental program could not be 

reset (Gurdon and Uehlinger 1966; Byrne et al. 2002). 

Scientists continued to tackle the problem and in 1986, Randall Prather and colleagues, 

then working in Neal First’s laboratory at the University of Wisconsin-Madison, cloned a 

cow from early embryonic cells using nuclear transfer (Prather et al. 1987). Although this 

was an example of blastomere nuclear transfer, it effectively set the stage for Dolly’s 

birth a decade later, on July 5, 1996. Dolly the sheep, the first organism ever to be cloned 

from adult cells, was created by Ian Wilmut and Keith Campbell using a technique 

similar to that used to create the first sheep from differentiated embryo cells (i.e., a 

blastomere clone) in 1995 (Wilmut et al. 1997). 

In July 1998, Ryuzo Yanagimachi, Toni Perry, and Teruhiko Wakayama of the 

University of Hawaii announced that they had cloned fifty mice from adult cells using the 

“Honolulu technique” (Wakayama et al. 1998). This was particularly significant because 

mouse embryos begin to divide almost immediately after the ovum is fertilized, and 

scientists had believed that this would not allow sufficient time for reprogramming to 

occur. Sheep, on the other hand, because their ova do not divide for several hours after 

fertilization, were thought to be an “easier” species to clone, as the natural delay between 

fertilization and division might be replicated in SCNT, possibly giving the oöcyte time to 

reprogram its new nucleus. 

SCNT is a relatively new technology described by many as complex, technically 

demanding and inefficient, that continues to be developed and improved. As such, there 

is no set “method” that is universally employed, although the basic steps outlined below 

are common to most SCNT procedures at the time that this overview was written. 
Chapter II: Technology Overview 30 

a. Donor cell 

For species in which the cloning process has been relatively well developed, the first step 

is identifying the animal to use as a nuclear donor. Animals to be used for breeding 

purposes are selected because they have been shown to be genetically superior to herd 

mates for the trait(s) to be propagated. Somatic cells can be collected from the ear (hole 

punch) or skin (surgical incision or needle aspiration), although many other cell sources 

have been used. Multiple factors may influence success or failure of the nuclear transfer 

process. Coordination of the cell-cycle stage of the donor nucleus and the recipient egg 

cytoplasm appears to be important for successful development of embryos. In general, the 

selection of a cell type for commercial cloning from an adult animal has evolved to 

choosing a collection method that is relatively noninvasive and minimizes stress to the 

live animal donor. 

Several characteristics have been identified as contributors to the degree to which any 

given donor cell or type of cell will likely result in a successful cloning event. One 

example is the “replicative state” of the donor cell. In general, cells in culture accumulate 

nutrients, grow, and when they reach certain conditions, divide. Cells that adhere to a 

solid substrate, such as the bottom of a tissue culture dish tend to grow until there are so 

many of them that they begin to touch each other. Once that happens, they generally stop 

dividing, and go into a “resting state” with respect to replication (referred to as G0). Cells 

can also be directed into G0 by depleting the nutrients in their growth medium. Some 

laboratories have concluded that cells in G0 are the most effective donors (Wilmut and 

Campbell 1998, De Sousa et al. 2002). Conversely, other laboratories have found that 

actively dividing cells make good donors (Cibelli et al. 1998, Lanza et al. 2001). Some 

laboratories find that cells from embryos or fetuses are the best donors (Batchelder 2005), 

while others are successful at cloning cells from aged or even deceased animals (Hill et 

al. 2000a, Tian et al. 2001). Another characteristic that has been shown to influence the 

degree to which cells make good donors is how “inbred” the donor animal is (Rideout et 

al. 2000). These researchers have determined that “hybrid vigor” is important for the 

success rate of animal cloning and the more inbred the donor animal, the less likely it is 

that cloning will occur successfully. Further, some species appear to be more amenable to 

cloning than others (e.g., goats compared with cattle, see Chapter V), and some species 

have not been cloned at all. At this time, the best conclusion that can be drawn with 

respect to the degree to which a cell (or animal) will serve as a “good” donor is that the 

technology is not sufficiently mature to predict with certainty which set of conditions will 

optimize cloning efficiency. 
Chapter II: Technology Overview 31 

Once a cell has been isolated from culture, depending on the laboratory, either the entire 

cell or just its nucleus is transferred under the zona pellucida of the enucleated oöcyte 

using a very thin glass micropipette (Solter 2000) to await fusion. 

b. Oöcyte 

The cell type used as the recipient for the donor cell to be cloned is the mature oöcyte, the 

version of the ovum that participates in fertilization during sexual reproduction. The 

oöcyte contains all of the non-nuclear cellular components required for the early 

development of an embryo. Oöcytes can be obtained from ovaries collected at 

slaughterhouses or from live animals using aspiration techniques (see previous discussion 

of in vitro fertilization). Because the oöcyte donates only its cytoplasm (the oöplast), it 

must be enucleated prior to fusion with the donor. The nucleus is generally removed by 

microaspiration, using a finely honed needle (PIFB 200310). 

c. Fusion 

In order to begin the development process, the membranes separating the oöplast and the 

donor nucleus (or cell) must be fused. This can be accomplished in two ways: (1) by the 

administration of a brief electrical pulse, or (2) chemical fusion. Electrical stimulation 

appears to be the more commonly used technique and involves the application of one to 

several microbursts of a mild electrical current in the vicinity of the cells. This induces 

the formation of pores between the somatic donor cell and oöplast which functionally 

makes the two cells one. This process also stimulates embryonic development, which if 

successful, results in the development of blastocysts that are transplanted into surrogate 

mothers (Cervera et al. 2002). 

Technical modifications aimed at increasing the success rate of cloning by improving the 

efficiency of the enucleation and fusion approaches are steadily evolving. For example, 

Oback et al. (2003) have developed a method that removes the zona pellucida from the 

oöcyte, aligns the donor cells with enucleated oöplasts, and uses electrofusion and 

chemicals to activate the cells to begin dividing. The results of this technique seem to 

show similar success rates for generating cattle clones as the cloning techniques more 

commonly used, with the advantage of being faster to perform (in the authors’ hands), 

and requiring less expensive equipment. Peura (2003) has also described a modified 

technique for preparing fused donor/oöplasts in which sheep oöcytes whose zona 

pellucidae had been removed were enucleated after fusion with donor cells, reversing the 

 

10 http://pewagbiotech.org/events /0924/proceedings2.pdf 
Chapter II: Technology Overview 32 

order in which those steps are usually performed. This technique appears to provide a 

higher rate of development of the blastocyst stage, implying that some factors present 

near the oöcyte chromosomes may be of assistance. 

Over the next few years other technical refinements may be developed, some based on 

improved technical practice, and others on increased knowledge about basic molecular 

mechanisms involved in the developmental process. These should increase the success 

rate of cloning, and decrease the potential for adverse events to occur. 

d. Transfer to recipient 

Just as the case for other ARTs with an in vitro phase, the developing clone is transferred 

into a synchronized surrogate female at the blastocyst stage. In cloning’s earliest days, 

the surrogate mother was often chosen to be distinctively different from the donor animal 

with respect to some clearly visible trait. For example, Dolly’s donor animal was a Finn 

Dorset sheep, a breed with white faces. Dolly’s surrogate mother, however, was chosen 

to be a black-faced sheep, so that if a white-faced sheep were born, it would be clear that 

it was not a genetic relative of the surrogate mother. In addition to choosing a 

distinctively different embryo recipient, Dolly’s identity was also confirmed by DNA 

fingerprint analysis of the donor cell line from which she was derived (Wilmut et al. 

1997). DNA fingerprint analysis enables definitive confirmation that an animal clone was 

indeed derived from a specific cell type, and is now the method of choice for confirming 

genetic parentage of animal clones (First et al. 1994). 

C. Critical Biological Events in SCNT 

Although it is often said that SCNT is a highly inefficient process with a relatively low 

success rate, the extraordinary nature of the technology and the demands that it places on 

the biological system being manipulated should not be overlooked. Unlike the fertilized 

egg or early embryonic cells that may be considered totipotent (capable of becoming any 

cell in an organism) or pluripotent (capable of become many cells in an organism) 

“generalists,” donor cells tend to be specialists. That is, they have differentiated to such a 

degree that their genomes have been “reconfigured” in ways that are, as yet, not fully 

understood in order to carry out the particular function for which they have been destined 

by their particular developmental fate. Kidney cells, therefore, do not transcribe the milk 

producing instructions of the mammary gland, yet they continue to carry those genes. The 

question then, is how to “reprogram” the full set of instructions contained in the genome 

such that “normal” development can occur. The following is a general overview of the 

events that are thought to occur during the SCNT process. There are several excellent 
Chapter II: Technology Overview 33 

reviews of the overall process or individual components that interested readers can 

reference for more details, and Chapter IV deals with some of these issues in more detail 

(Kikyo and Wolffe 2000; Sinclair et al. 2000; Solter 2000; Young and Fairburn 2000; 

Fulka et al. 2001; Rideout et al. 2001; Novak and Sirard 2002; NAS 2002a,b; Colman 

2002; Dean et al. 2003, Santos et al. 2003; Morgan et al. 2005). 

In principle, SCNT has demonstrated that cell differentiation can be reversed. Genetic 

reprogramming, the process of altering the gene expression pattern associated with the 

differentiated cell to one that is appropriate or early embryonic development, is normally 

carried out at two stages in the development of fertilization-derived embryos: after 

fertilization, and during the development of gametes (the sperm and ovum). The actual 

molecular events involved in reprogramming are not fully understood, although they may 

be categorized into a few overall steps. These include altering the way in which the 

chromosomes are packaged by changing the chemical nature of the proteins involved, 

and changing the chemical structure of the DNA in portions of the molecule that are not 

responsible for base-pairing (NAS 2002 a,b). A more complete description of these 

processes is found in Chapter IV. 

The nucleus of a cell contains a complete copy of all of the genes required for life. This 

information is encoded in genes. Physically, genes are the linked nucleotides that 

comprise DNA, or the “master molecule” of biology. The total genetic material of an 

organism is referred to as its “genome,” and consists of long strands of DNA packaged in 

chromosomes, which come in pairs except for those specifying the sex of the resulting 

organism. The number of pairs of chromosomes differs among species. Cattle, for 

example, have 30 pairs, pigs have 19, sheep 27, and goats 30. (Humans have 23 pairs of 

chromosomes.) 

Chromosomes can exist in different “conformations” depending on the stage of the cell 

cycle. When DNA needs to be moved, as in when a cell divides, or when a sperm needs 

to deliver the male genome, chromosomes are tightly condensed. During the rest of the 

cell’s life cycle, chromosomes tend to exist in less tightly coiled conformations so the 

information encoded in the DNA is more accessible for processing. Specific proteins are 

responsible for holding chromosomes in different conformations. In all cells but sperm, 

these proteins are called histones; in sperm chromosomes are packaged by proteins 

referred to as protamines. 

When an ovum is fertilized by a sperm, a complex series of molecular events ensues that 

is referred to as “chromatin remodeling” (chromatin is another term for the protein:DNA 

complexes that make up chromosomes.) Although the exact steps are not known, the 
Chapter II: Technology Overview 34 

overall process involves stripping away the protamines packaging the paternal DNA, 

removing histones from the ovum’s DNA, and allowing the newly associated DNA 

molecules to reform chromatin in a way that allows the fertilized ovum and early 

embryonic cells to replicate and be “totipotent”(capable of developing into a complete 

organism). Many proteins are involved in this, only a few of which have been identified, 

and it is likely that there are chemical markers on the DNA bases that are altered (such as 

methylation). Chromatin remodeling is likely very different in SCNT. Disassembly of the 

tightly condensed sperm chromatids and the subsequent removal of protamines do not 

occur because there is no sperm present. Instead the oöplast must decondense and 

repackage the chromosomes of the donor somatic nucleus. 

In order to perform the functions of life, cells have to convert the information in the DNA 

to ribonucleic acid (RNA) (a process referred to as transcription), and then to translate 

that RNA into proteins, which are the molecules that carry out life’s functions. This 

coordinated set of activities is referred to as gene expression. Alterations in the 

expression of a given set of genes are often referred to as “epigenetic effects” (or “around 

gene effects”) because they do not require changes in the base-pairing properties of the 

DNA that comprise genes. Instead, they reflect changes in the structure of the 

chromosome around the gene (such as control regions), or on the nucleotides, but outside 

the portion of the molecule involved in coding. (See Chapter IV for a more complete 

discussion). A classic example of the manifestations of epigenetic effects is the different 

fingerprint or freckle patterns observed in human twins. These individuals have exactly 

the same coding regions, but small changes in the non-coding regions of the DNA result 

in different phenotypes. Other examples of epigenetic control of gene expression include 

the coat color or color patterns of many mammals. 

D. Outcomes Observed in ARTs 

As this risk assessment is being prepared, biologists are just beginning to understand the 

highly complex interactions that must occur to choreograph the millions of molecular 

interactions that signal the expression or silencing of genes in a particular cell or at any 

point in its life cycle. Although the exact mechanisms by which these effects occur are 

not fully understood, in all forms of reproduction, ranging from natural mating to SCNT, 

these processes may go awry in early development. Although most of the animals born 

following ARTs with significant in vitro components appear to be completely “normal,” 

some of the outcomes are not so successful. In particular, some of the adverse outcomes 

noted in these “high in vitro component ARTs” appear to have common defects in gene 

expression, particularly in the overgrowth outcomes (Humpherys et al. 2002). 
Chapter II: Technology Overview 35 

Published studies involving cattle, sheep, and mice demonstrate that embryos produced 

using in vitro systems may differ in morphology and developmental potential compared 

to embryos produced in vivo (Kruip and den Daas 1997; Young et al. 1998; Farin et al. 

2003; Farin et al. 2006; see Appendix C for a more detailed discussion and additional 

references). For example, common abnormalities have been noted in fetuses (Farin and 

Farin 1995) and calves (Behboodi et al.1995; Sinclair 1999) associated with the transfer 

of bovine embryos produced using in vitro maturation (IVM), in vitro fertilization (IVF), 

in vitro culture (IVC) systems, and SCNT (Hill et al. 2000b). One set of reported adverse 

outcomes following transfer of embryos from cloning or in vitro production systems is 

often referred to as Large Offspring Syndrome (LOS). These include lowered pregnancy 

rates, increased rates of abortion, production of oversized calves, musculoskeletal 

deformities and disproportionalities, as well as hydroallantois (abnormal accumulation of 

fluid in the placenta) and other abnormalities of placental development. 

The phenomenon of “large calves” was first described by Willadsen et al. (1991). The 

syndrome has also been identified in fetal and newborn lambs and in mice where the 

embryos were cultured in vitro (Eggan et al. 2001). Offspring with LOS tend to exhibit 

difficulties with placentation (Farin et al. 2003; Bertolini et al. 2004; Lee et al. 2004; 

Batchelder 2005). In cattle and sheep, the placentae of developing fetuses with LOS are 

unusually large for their species, and tend to have abnormal development of placentomes 

(the sites of attachment between fetal and maternally derived tissues of the placenta). 

LOS fetuses tend to have longer than usual gestation lengths, and often labor in the dams 

must be induced followed by Caesarian section deliveries. The newborns tend to be large 

for their breeds, and often have abnormal or poorly developed lungs, hearts, or other 

affected internal organs (liver and kidney), which makes it difficult for them to breathe or 

maintain normal circulation and metabolism. LOS newborns may appear to be edematous 

(fluid filled), and if they are to survive, often require significant veterinary intervention. 

Problems have also been noted in muscle and skeletal development of animals with LOS. 

These animals also often have difficulty regulating body temperature. (For a more 

detailed discussion of LOS, see Chapter V). 

Although the cause of LOS is not known with certainty, it is likely be related to changes 

in gene expression (i.e., epigenetic changes) that result from the in vitro manipulation and 

culturing of embryos. A review by Young et al. (1998) suggests that in vitro culture alone 

is adequate to perturb the embryo. This hypothesis is supported by data from Sinclair et 

al. (2000) where in vivo matured and fertilized eggs recovered from superovulated sheep 

donors, cultured in vitro for 6 days, showed an 18-36 percent increase in mean birth 

weight at day 125 of gestation, depending on the culture system used (Sinclair et al. 2000, 
Chapter II: Technology Overview 36 

Young et al. 1998). Table C-1 (Appendix C) provides a more comprehensive summary of 

adverse outcomes noted in different ARTs. 

This is an area of extensive research in the cloning and developmental biology 

communities. It is likely that advances in the understanding of these mechanisms will 

lead to significant improvements in the rates of successful outcomes of all ARTs that 

include a significant in vitro component, including cloning. 

E. Future of Reproductive Technologies in Modern Agricultural Practice 

Modern agricultural practices will likely continue to employ all of the reproductive 

modalities described in this overview. The factors that may influence which practices are 

used will likely be a function of the breeder/farmer/ranchers’ needs and opportunities. 

Seidel (2006), in a foreword to a symposium on ARTs, emphasized that current 

differences in the reproductive management of cattle in different parts of the world are 

driven by multiple considerations. Some of these have to do with the nature of the 

differences in the husbandry of beef (mostly pasture based) and dairy (mostly intensively 

housed in the US, more pasture based in Australia and New Zealand) cattle. Some are 

economic (ARTs are much more expensive than natural matings), some are practical (the 

fertility of dairy cattle has declined significantly in the last 20 years, making ARTs more 

attractive; using ARTs in beef cattle is not practical for ranchers who look for 

replacement by natural coverage), and some are technological (the ability to choose 

genetics more precisely versus the developmental problems associated with ARTs with 

significant in vitro components). 

Technological issues will be addressed by continued research and development in this 

field. To that end, several professional and scientific societies (e.g., the International 

Embryo Transfer Society, various animal science organizations and breeding 

associations) have been actively involved as clearing-houses for information and 

interaction. 

SCNT has the potential to impact animal breeding in as fundamental a manner as 

artificial insemination. Given its current high costs (approximately $20,000 for a live 

calf) and relatively low success rates (< 10 percent), SCNT will likely be used to improve 

production characteristics of food producing animals by providing breeding animals, just 

as any breeding program would select the most elite animals for breeding, and not as 

production animals. In this way, cloning does not differ from any of the other ARTs that 

have been described in this chapter. Cloning has the relative advantage of allowing for 
Chapter II: Technology Overview 37 

the propagation of animals with known phenotypes to serve as additional breeding 

animals. This is critically important in breeding programs, especially when it may take 

years to “prove” the merit of a sire or dam. Second, it allows the propagation of animals 

whose reproductive function may be impaired. It has already been used to increase the 

available genotype of a particular dairy cow with low fertility; her clones appear to be 

exhibiting normal fertility (PIFB 2003). Third, it allows the propagation of valuable 

deceased animals from which tissue samples have been appropriately collected or 

preserved, which may have profound implications for species or breeds nearing 

extinction. Finally, for the first time, cloning allows for the careful study of the “naturenurture” interactions that influence breeding programs by allowing a large enough sample 

of genetically identical animals to be raised in different environments, or with different 

diets. Such studies have been impossible to perform prior to the advent of SCNT and are 

likely to yield important information for developing livestock species to live in areas that 

have, until this time, been marginal for food animal production. This is of particular 

importance to the developing world, where even slightly increased wealth generally 

favors the incorporation of animal-based agriculture. 

Regardless of the degree to which cloning may be adopted in animal breeding programs, 

FDA’s role in performing this risk assessment is clear: the agency’s responsibility is to 

determine whether cloning poses any risk to animals involved in the cloning process, and 

whether the consumption of food products from clones or their progeny poses any 

additional risk compared with food from conventionally produced animals. This Risk 

Assessment presents the method by which we evaluated data on clones and their progeny, 

the data themselves, and the agency’s conclusions, including discussions of uncertainty. 
 

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Chapter III: 

Developing the 

Risk Assessment Methodology
 

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Chapter III: 

Developing the 

Risk Assessment Methodology 

A. Charge 

In July of 2001, the Food and Drug Administration’s (FDA or the Agency) Center for 

Veterinary Medicine (CVM or the Center) issued an open letter (CVM Update 2001) to 

producers of animal clones to ask them to refrain from putting edible products from those 

animals into the food supply until the Center evaluated the safety of those foods.11 This 

request had already been made to companies engaged in cloning food-producing animals 

during the previous year. The overall strategy chosen by the Center was to perform a risk 

assessment in order to determine what hazards might be introduced into animals as the 

result of the cloning process, to characterize the resulting potential risks, and to develop 

risk management proposals commensurate with the identified risks. 

B. General Discussion of Risk/Safety Analyses 

1. Risk and Safety 

Risk and safety can be thought of as two sides of the same coin. In general, the answer to 

the question of “Is it safe?” is addressed scientifically by determining the conditions 

under which the substance or action in question is not safe, and then limiting exposures to 

conditions outside those limits. Because knowledge is always incomplete, and not every 

circumstance can be controlled, there is no such thing as “absolute safety” or “zero risk.” 

Risk assessors attempt to identify conditions under which risks are estimated to be as low 

as possible, and risk managers use that information in developing policies to protect 

human or animal health. The methodology used to characterize potential risk is referred 

to as risk assessment.12 One of the real values in performing a risk assessment is that in 

addition to arriving at an outcome, the process of arriving at an answer provides a 

framework by which data can be organized, analyzed, and interpreted. By dividing the 

risk assessment process into discrete steps, and then reintegrating them into an overall 

 11 http://www.fda.gov/cvm/CVM_Updates/clones.htm 

12 Appendix A provides an overview of risk and safety assessments, especially as they have evolved to 

address issues relevant to cloning, and may be useful background reading for individuals not familiar 

with the processes. 
Chapter III: Developing the Risk Assessment Methodology 42

characterization of potential risk, risk assessment allows both the details and the “big 

picture” to be addressed for complex problems. 

Discussion of uncertainty must accompany every risk assessment. Uncertainties may 

stem from a lack of fundamental understanding of biological processes and/or from data 

gaps that may be filled with the appropriate empirical studies; they may be exacerbated 

by intrinsic variability in datasets. Given that the process of risk assessment identifies 

data gaps and helps direct the acquisition of data that decrease uncertainties, it should not 

be thought of as a process that is performed just once, but rather a recursive process in the 

responsible development of research programs, new products, and science-based 

regulatory strategies. 

2. Risk Assessment vs. Risk Management 

Risk management can be defined as the set of activities of identifying and evaluating 

alternative strategies (often regulatory) to deal with the risks characterized in the risk 

assessment, and then selecting among them based on social, economic, ethical, and 

political conditions or criteria (NAS 1996a). Risk managers choose among different 

options based on the risk assessment, which is generally regarded to be relatively valuefree,13 and their understanding of and responsibilities to the broader social or economic 

constructs within which they operate. Risk-benefit or risk-risk decisions are risk 

management, as they involve an active choice between two or more possible courses of 

action. A proposed risk management plan based on this Risk Assessment is presented in 

the accompanying document. 

C. Risk/Safety Assessment of Cloning 

In order to address the hazards and risks to animals involved in cloning and the food 

products derived from them (and their progeny14) four issues must be addressed: 

identifying hazards and risks; determining the degree to which existing data address 

 

13Although risk assessment is based on science and relatively value-free, it generally contains a few 

policy-based judgments such as the selection of health protective (conservative) defaults when data are 

incomplete or when choosing among datasets of equal quality. The selection of policy-driven 

alternatives should be explicitly discussed in the risk characterization, and the implications of such 

choices should be described in a risk assessment. 14 An animal clone is one arising directly from a somatic cell nuclear transfer event. A progeny animal is 

one derived from sexual reproduction that has at least one animal clone as a parent (but could result 

from two animal clones mating). 
Chapter III: Developing the Risk Assessment Methodology 43

questions of safety; characterizing residual uncertainties; and selecting the most 

appropriate risk metric for the Risk Assessment: 

(1) Identifying hazards and risks. As there are no existing risk paradigms for 

animal clones and the food products that may be derived from them, this 

assessment attempts to identify hazards and risks based on the available data and 

consideration of the biological processes affected by cloning. 

(2) Determining the degree to which existing data address questions of animal 

health or food consumption risk. At the time of this writing most of the peerreviewed publications report on the ability to generate live animal clones from 

various donor cell sources and culture conditions; the frequency of successful 

outcomes (where success is defined as a surviving dam and a live offspring with 

no apparent abnormalities); and the nature and frequency of developmental errors. 

The nature of the published reports, with some exceptions, reflects the institutions 

producing them: academic laboratories tend to report the development of new 

technologies and the observation of abnormalities, while corporate entities tend to 

report successful implementation of the technology, including summaries of the 

health status of animal clones. These studies are useful in identifying hazards to 

the health of animals involved in the cloning process and characterizing the 

potential risks that may stem from those hazards. Despite the extensive literature 

search performed, and the large number of papers that were reviewed for animal 

health, few reports directly addressed food safety. 

Reports from the peer-reviewed literature likely suffer from “publication bias,” an 

artifact of the criteria used to determine the “attractiveness” of publication in 

leading peer-reviewed journals. In general, investigators tend to submit to 

journals, and journals tend to publish, novel findings or hypothesis-testing results 

rather than surveys of the health of cohorts of animals. With a few notable 

exceptions, the literature on animal clones tends to consist of reports of studies of 

the role of various technical manipulations on the success of cloning procedures, 

descriptions of initial successes of cloning in species that have not yet been 

cloned, or descriptions of adverse outcomes. Much of the work in which cloning 

has been refined (and is therefore more likely to be successful) is being performed 

by the private sector. Given the competitive nature of the breeding and 

biotechnology industries, as well as the need to maintain business confidential 

information, much of the important information on more recent cloning outcomes 

has not been published or made publicly available. In order to keep the current 

analysis transparent to the public, however, this assessment only cites information 
Chapter III: Developing the Risk Assessment Methodology 44

that has been published in peer-reviewed journals, or otherwise made available to 

the agency by companies engaged in cloning, with explicit permission for release 

to the public. 

 

(3) Characterizing residual uncertainties persisting following a review of the 

existing data. Due in large part to the novelty of the technology, the 

concentration of data at the earliest stages of clone development, and limited data 

directly addressing food safety, uncertainty will persist in any estimates of risk 

associated with animal cloning. As with all science-based uncertainties, additional 

data may increase the confidence with which judgments are made. The decision 

as to “how much is enough,” however, is a function of the nature of the risk(s) 

(i.e., its severity), the quality and consistency of the data (i.e., the weight of the 

evidence), and the tolerance of the risk management policies for uncertainty. 

(4) Selecting the most appropriate risk metric for this risk assessment. The most 

appropriate standard to apply to the potential risk(s) associated with the 

consumption of foods derived from animal clones and their progeny is whether 

such food poses any additional risk relative to that derived from sexually-derived 

animals. For the purposes of this risk assessment, conventional animals are 

defined as those animals derived by any reproductive means other than SCNT. 

 

D. Transgenic Animal Clones 

This risk assessment addresses “just clones,” that is, animals derived via SCNT whose 

donor genomes have not intentionally been modified by molecular biology techniques. 

Transgenic clones, on the other hand, are clones whose donor cells contain exogenous 

heritable DNA inserted by molecular biology techniques. They are considered to occupy 

a different “risk space” from “just clones” because the transgenic event (the insertion of a 

heritable DNA sequence) is intrinsically accompanied by a series of potential risks. These 

include those associated with the DNA construct and those associated with the product of 

the gene (if there is a gene product). Organisms derived from transgenic cells will have 

risks specific to the inserted construct, its insertion site, and its subsequent expression. 

Although it is entirely possible for transgenic clones (or any transgenic organism) to be 

produced safely and to be a safe source of edible products, the risks associated with each 

animal must be determined separately on a case-by-case basis, because of the added 

genetic material. 
Chapter III: Developing the Risk Assessment Methodology 45

Nonetheless, much of the literature on animal clones reports on experiences with 

transgenic clones. In some cases, the transgenic nature of the animals is explicit (e.g., Hill 

et al. 1999), but in many others, only careful reading or tracing back references cited in 

the methods section of the papers allows the reader to learn the transgenic status of the 

clones (i.e., Lanza et al. 2001, Cibelli et al. 2002). The question is whether any 

information from the transgenic clone reports can inform the identification of hazards and 

characterization of risks associated with “just clones.” 

After a careful review of the key papers addressing transgenic clones, CVM has decided 

that it is not possible to determine with certainty whether any particular adverse outcome 

is due to the process of cloning, the transgenic nature of the donor cell, or some 

combination of the two. Clearly, the insertion of exogenous DNA introduces a set of 

hazards not present in non-transgenic clones, and by inference, the creation of a different 

set of risks. If transgenic animals appear to be normal, the logical inference is that neither 

cloning, nor transgenesis (or the combination of cloning and transgenesis) has perturbed 

the animals’ development. This is the case of transgenesis and cloning posing no 

significant (or apparent) risk. In either case, this risk assessment puts greatest weight on 

reports of outcomes from non-transgenic animal clones, and uses studies of transgenic 

clones for secondary or corroborative purposes. Nonetheless, given the large proportion 

of the peer-reviewed literature that reports on transgenic clones comprise, these studies 

have been cited with the preceding caveats. A more complete discussion of this topic is 

found in Appendix D: Transgenic Clones. 

E. Methodology Development 

When considering how to develop a risk assessment methodology for animal cloning, it 

became apparent the need to develop a framework that could be applied to both animal 

health and food consumption risks. Because the initial review of the data indicated that 

there were no studies explicitly evaluating the safety of food products from animal 

clones, the health status of the animals producing food would have to contribute to both 

the animal health and food safety components. 

Interpreting hazard and risk from the same dataset but for different sets of receptors 

(animal health - the animals involved in producing clones; food safety - the consumers of 

the food products) requires shaping the manner in which the data are evaluated to suit the 

ultimate outcome of the assessment. For example, identification of adverse outcomes for 

animal health requires evaluating data on both surrogate dams carrying pregnancies and 

resulting clones. For food consumption, however, animal clones that would be 
Chapter III: Developing the Risk Assessment Methodology 46

condemned at slaughter, as currently practiced with conventional food animals were 

excluded from the analysis, an emphasis is placed on the identification of unique hazards 

to food consumers that could arise as the result of the cloning process. As described in 

the following section, and Chapter VI, this requires evaluating the dataset at a finer level 

of resolution than for animal health outcomes. 

The net effect of the different ultimate outcomes of the animal health and food 

consumption risk assessments is that although the datasets considered by both 

assessments may overlap considerably, the manner in which they are evaluated differ, 

and the conclusions generated from the same (or largely overlapping) datasets vary with 

respect to the amount of risk present. 

1. Hazard Characterization 

Identifying and characterizing potential hazards is the first step in characterizing the 

nature of risks due to cloning (see Appendix A). CVM therefore sought to develop a 

framework in which adverse outcomes associated with cloning could be presented in a 

systematic manner that would facilitate interspecies comparisons of outcomes. 

For food safety purposes, the scientific and regulatory communities have traditionally 

operated under the principle that domestic animals (i.e., cattle, swine, sheep, and goats) 

commonly consumed for food have not developed specialized organs producing toxicants 

to kill prey or avoid predation (e.g., venom producing glands). Further, because the 

components of animal tissues are necessary for life, and closely resemble the processes in 

humans, it is highly unlikely that “silent” pathways to produce intrinsic toxicants exist. 

Thus, “it is convention that animal metabolites are not considered to be natural toxicants” 

(Watson 1998). 

In order to generate a viable clone, the differentiated genome of the donor cell or nucleus 

must be reprogrammed by the recipient oöplasm. Because no additional genes are being 

added, and the presumption is that there are no silent pathways to produce intrinsic 

toxicants, the only method by which hazards may arise in animal clones is from the 

incomplete or inappropriate reprogramming of the genetic information from the donor 

somatic nucleus (i.e., epigenetic effects). These phenomena are described in more detail 

in Chapter IV. 

Where, then, would the potential hazards in clones arise? As outlined in Chapter II, 

during the development of an embryo, a complex series of molecular events are 
Chapter III: Developing the Risk Assessment Methodology 47

responsible for balancing gene expression from the maternal and paternal genomes, and 

directing the appropriate expression of genes in the developing embryo and mature 

mammal. This process is referred to as “reprogramming.” Alterations in gene expression 

due to those changes are referred to as “epigenetic” variability, and are present normally 

in conventional animals, including humans. 

The most severe errors in reprogramming will result in death, obvious malformations, or 

metabolic derangements, and are reflected in the low “success rate” of cloning, the 

perinatal difficulties observed in some newborn clones, and occasional examples of 

altered metabolic pathways in very young animals (see Chapters V). These are clearly the 

subject of the animal health risk assessment. Because animals found to have a disease or 

condition that would render them adulterated (e.g., unfit for consumption, unhealthful, 

unwholesome) are prohibited from entering the human food supply, however, the only 

remaining food consumption hazards arising from gene dysregulation would be those that 

allow an animal clone to develop with apparently normal functions, but with sub-clinical 

physiological anomalies. 

These subtle hazards are outside the conventional range of hazards commonly the subject 

of food safety analyses, and can be divided into three overall classes: 

(1) Alterations in gene expression that lead to phenotypic variability such as coat 

color, size, behavior, longevity; 

(2) Disruption of immune function; and 

(3) Alterations in metabolism leading to changes in physiological “set-points” such 

that the animal has apparently compensated and appears to be normal on gross 

inspection, but whose physiology may be aberrant. 

It is important to note, however, that changes in gene expression in individuals sharing 

identical genotypes have been observed in conventional animals and in humans. This 

phenomenon is often referred to as phenotypic variability, and can be seen at the human 

level in the different fingerprint and freckle patterns that identical (monozygotic) twins 

possess. Non-clone mice of identical genotypes fed different levels of certain nutrients 

can have different coat colors, and exhibit significant differences in body weight and 

lifespan (Cooney et al. 2002). 
Chapter III: Developing the Risk Assessment Methodology 48

2. Potential Risks 

Risk is defined as the probability of an adverse outcome given that exposure has 

occurred. This concept is often presented in the format of the “risk equation” that may be 

expressed as 

Risk ∝ foutcome (exposure, hazard)

or, stated more simply, risk is some function of exposure and hazard. 

The “risk equation” can be run in the forward or reverse direction. Characterizing risks 

from a set of hypothetical hazards is a case of running the equation in the forward 

direction: it allows the estimation of the probability that adverse outcomes might occur 

once changes that create hazards have occurred. Such approaches are useful when there is 

some understanding of the underlying biological processes being evaluated. For example, 

if incomplete genetic reprogramming (a change that may result in a hazard) were to result 

in animals with altered calcium transport mechanisms, a possible animal health risk could 

be bone weakness or malformation, and a possible food consumption risk (a probability 

of an adverse outcome) could be compromised human nutrition resulting from a diet of 

milk containing lower than expected calcium levels (the adverse outcome). In the case of 

the animal health risk, the degree of risk could vary from insignificant, in which no 

physical symptoms were present, to severe, in which the animal could experience 

misshapen or fragile bones leading to difficulties in walking. Because animals found to 

have a disease or condition that would render them adulterated (e.g., unfit for 

consumption, unhealthful, unwholesome), only the animals without obvious visible 

anomalies (and therefore less severe calcium transport anomalies) would be sources of 

edible products. The food consumption risks then could possibly arise from a lower 

available calcium pool accessible to milk production, and thus a potential nutritional risk 

to individuals consuming milk from such animals. 

Analysis of end products such as milk constituents is an example of running the risk 

equation in the reverse direction: it captures the potential outcome(s) of the biological 

changes, and allows for the identification of exposures and hazards responsible for the 

risk(s). The nutritional hazard identified in the preceding example might be detected 

more efficiently by a compositional analysis of milk. Compositional analyses, however, 

are limited by available analytical methods and comparators. As far as CVM is aware, no 

complex food (e.g., bacon, beef steak, milk, cheese) has been fully characterized with 
Chapter III: Developing the Risk Assessment Methodology 49

respect to its chemical composition.15 The organisms that are or make up foods are 

comprised of hundreds of thousands of chemical substances that can be influenced 

qualitatively and quantitatively by diet, environmental conditions, and genetics. Attempts 

to characterize all of the chemical constituents of “milk” or “meat,” then, are neither 

practicable nor desirable (NAS 2004). Instead, milk and meat analyses have tended to be 

limited to characterizing proximates (e.g., water content, proteins, fats, carbohydrates, 

minerals, ash), or, when necessary or desired, to profiles of particular nutrients, antinutrients, or individual components of interest (e.g., vitamin content, fatty acid profiles, 

or protein composition). 

3. Proposed Approaches 

a. Animal Health Risks 

The Center determined that at this point in the development of the technology, risks to 

animal health are best characterized using a retrospective approach. In other words, CVM 

approached this issue by recording and cataloguing adverse outcomes in a biological 

context, rather than by elucidating specific examples of gene dysregulation and searching 

for their physiological sequellae. The Critical Biological Systems Approach (CBSA), 

described below, provides a framework in which this may be accomplished (Figure III-2). 

In general, the Center has relied on integrated physiological measurements to survey 

animal health, although it is likely that genomics, proteomics and metabolomics will see 

increased use for such purposes in the future. At the time that this risk assessment was 

prepared, however, these methods had not been sufficiently developed and validated to 

allow them to be used as survey tools. 

b. Food Consumption Risks 

Determining the safety of food products from animal clones and their progeny, at least in 

its earliest stages, is likely best accomplished by using both approaches: prospectively 

drawing on knowledge of biological systems in development and maturation, and in 

retrograde, from an analysis of food products. An intrinsic and valuable part of this 

analysis is cataloging the available information, and identifying data gaps and 

uncertainties that may in turn suggest research that could serve to decrease the identified 

uncertainties. The following sections describe the methodology CVM has proposed to 

 

15The International Life Science Institute (ILSI) is currently coordinating an effort to generate a database 

of the known chemical constituents of major food crops (e.g., corn, soy, wheat). 
Chapter III: Developing the Risk Assessment Methodology 50

accomplish a rigorous, science-based analysis of potential hazards and risks associated 

with the consumption of food products derived from animal clones and their progeny. 

Prior to undertaking such an analysis, however, subtle hazards and potential risks that 

may be posed by animal clones must be considered in the context of other mutations and 

epigenetic changes that occur in all food animal populations. Some are considered 

beneficial, and have been selected for by animal breeders when a desirable phenotype is 

obtained. For example, not-so-subtle genetic mutations that have occurred at least twice 

in nature are the development of double-muscled beef breeds such as the Belgian Blue 

and Piedmontese, which arose from different mutations in the myostatin gene 

(McPherron and Lee 1997). These animals appear to be healthy, although sexual maturity 

appears to be delayed relative to other breeds, and female fertility appears to be 

somewhat lower. Nonetheless, these animals are used in selective beef breeding programs 

in several countries as they have 20-30 percent more muscle mass than cattle with the 

wild-type myostatin gene, feed efficiency is increased, and the meat is considered to be 

more tender although lower in fat content. Meat from these animals is presumed to be 

food, and as such enters the food supply with no additional regulatory scrutiny. 

Epigenetic changes that occur on a regular basis include variations in pigmentation 

patterns (e.g., coloration patterns on Holsteins) and are perhaps most easily thought of as 

those differences observed in identical twins, such as different fingerprints and freckle 

patterns. 

Finally, it is important to remember that any discussion of subtle hazards and potential 

risks associated with the products of animal clones is not conducted in a regulatory 

vacuum. All food, including that from animal clones, must meet existing regulatory 

requirements in order to be marketed lawfully in the United States. 

F. Two-Pronged Approach to Assessing Food Consumption Risks 

Given the assumption that food derived from clones will be in compliance with existing 

regulatory requirements for food products from conventional animals, CVM proposes a 

two-pronged approach for evaluating the potential risks associated with the food products 

of animal clones and their progeny (AC/P) (Figure III-1). The first component, the 

Critical Biological Systems Approach (CBSA) is based on the hypothesis that a healthy 

animal is likely to produce safe food products, and incorporates a systematic review of 

the health of the animal clone or its progeny. The second component, or the 

Compositional Analysis Method, is based on the operating hypothesis that food products 

from healthy animal clones and their progeny that are not materially different from 

corresponding products from conventional animals are as safe to consume as their 
Chapter III: Developing the Risk Assessment Methodology 51

conventional counterparts. It relies on the comparison of individual components of edible 

products, and the identification of the appropriate comparators. 

Figure III-1: 

4/22/2003

Two-Pronged Approach to Evaluating Food 

Safety

Critical Biological Systems

a healthy animal is likely to produce safe food

Compositional Analysis

food products from healthy AC/P are not 

materially different from those derived from 

conventional animals

Entry into 

food supply

+

1. Critical Biological Systems Approach 

a. Overview 

The CBSA (Figure III-2) is based on a cumulative evaluation of health status indicators 

of animal clones. Mechanistically derived, it considers SCNT and the subsequent 

development of the animal clone from a biological “systems analysis” perspective, and 

thus may be thought of as being “HACCP16-like.” It accepts that at this time SCNT is a 

biologically imprecise and inefficient process resulting in few live births relative to the 

number of implanted embryos, and that some animals are born with obvious defects or 

subtle anomalies. It also assumes that biological systems are capable of repair or 

correction, either intrinsically or following human intervention. For example, animals 

that may have difficulty surviving on their own immediately after birth may develop into 

healthy, reproducing individuals if provided support in the form of respiratory assistance 

 

16HACCP is the Hazard Analysis Critical Control Point approach adopted by USDA and FDA for 

assuring the safety of certain food products undergoing some degree of processing. 

http://www.fsis.usda.gov/Science/HACCP_Models/index.asp 
Chapter III: Developing the Risk Assessment Methodology 52

and warmth during the period immediately after birth. Alternatively, these animals may 

not recover, and may remain “sickly” or unthrifty until they are culled. 

The cumulative nature of the CBSA allows for the incorporation of both favorable and 

unfavorable outcomes. The former, provided that all other measures appear to be normal, 

will result in the judgment that the animal will produce food that is safe for consumption; 

the latter implies that animals with anomalies may be unsuitable for food. 

Figure III-2: Critical Biological Systems Approach 

Perinatal

Development 

and Function

Reproductive Development

and Function

Post-Pubertal

Maturation

Selection of donor and recipient cells, oöcyte 

manipulation, nuclear transfer, embryo culture/transfer,

implantation, placentation, pregnancy maintenance

Cell Fusion Through 

Fetal Development

Induction of labor, parturition, 

post-natal survival

Gametogenesis, secondary sex 

characteristics, estrus, fertility, 

mating behavior, ease of delivery

Growth and development measures

Physical and physiological 

functions Progeny 

Juvenile Development

And Function

b. Evaluation Nodes 

The CBSA selects five key developmental stages of an animal clone’s life, analogous to 

the “critical control points” of the HACCP analysis. These stages provide biologicallybased developmental “collection nodes” (Developmental Nodes) (indicated in 

Figure III-2 by periwinkle-colored boxes) that also serve as agronomically appropriate 
Chapter III: Developing the Risk Assessment Methodology 53

points at which to collect data. Examples of the types of data that could be collected are 

illustrated in Figure III-2 as yellow boxes. It is important to note that these 

Developmental Nodes address functionality and not necessarily discrete time points, as 

the latter will vary among species and breeds. 

Developmental Node 1 incorporates the initial technical steps involved in SCNT, 

including cell fusion through implantation, and subsequent embryo and fetal 

development. Chapter IV covers many of the early common molecular events that occur 

during this time period common to mammals; Chapter V reviews these issues as they 

impact on the health of clones and their surrogate dams; and Chapter VI reviews these 

steps from the perspective of identifying food consumption risks. 

 

Developmental Node 2 encompasses the Perinatal period, including late gestation, labor 

induction in the dam, delivery, and the critical time period of approximately 0-72 hours 

after birth. This developmental node allows for the analysis of animal health data relevant 

to both the surrogate dam and the clone, although few food consumption risks are 

anticipated to occur at Developmental Node 2 because clones of that age would not be 

consumed as food. 

The third developmental node (Developmental Node 3), Juvenile Development and 

Function, encompasses the period of rapid growth between birth and the onset of puberty, 

and may vary in duration among the species considered. 

The Reproductive Development and Function Node (Developmental Node 4) 

encompasses puberty and reproductive function throughout the reproductive period of the 

animal. Food consumption risks arising from milk production may first be encountered at 

this point of the animal’s life. Because of the complex integration events that must occur 

for effective reproduction to take place, this developmental node is critically important 

for evaluating the health and functionality of animal clones. Proper reproductive function 

indicates that the complex and inter-related physiological systems required for the 

development and delivery of functional germ cells (and, in the case of females, viable 

offspring) have occurred appropriately. 

The Post-Pubertal Maturation Node (Developmental Node 5) encompasses all nonreproductive functions of sexually maturing or mature animals, including growth, weight 

gain, disease frequency, aging, and lifespan, where available. 

Because the value of clones lies in their use as breeding stock (and is reflected in their 

relatively high cost), “founder” animal clones are not likely to be slaughtered initially for 
Chapter III: Developing the Risk Assessment Methodology 54

meat. It is anticipated that most of the food products, especially meat, from clone lineages 

will enter the food chain as the progeny of animal clones, or their subsequent offspring. 

Milk from dairy clones could enter the food supply, following breeding and delivery of 

offspring. Meat from clones could enter the food supply if, for instance, conditions 

outside the producer’s control forced herd culling (e.g., loss of funding), or when older 

animals reach the end of their functional utility (e.g., loss of fertility in breeders). Table 

III-1 summarizes the Developmental Nodes, the types of data likely to be collected at 

each node, and the potential for the entry of clones into the food supply. 

Table III-1: Summary of Developmental Nodes and Implications for Food Consumption Risks 

Developmental Node Types of Observations/Data 

Potential for Entry 

into Food Supply 

1: Cell fusion through 

implantation, embryo and 

fetal development 

Selection of donor and recipient cells, oöcyte 

maturation and activation, fusion method, days in 

culture, culture conditions, number of fusions, 

number of blastocysts formed (if measured). Number 

of implantations, early and late gestation losses, 

placentation, pregnancy maintenance, morphological 

anomalies. 

None 

2: Perinatal period including 

immediate pre-partum, 

delivery, and up to 72 hours 

post-partum 

Number of animals delivered with/without assistance, 

survival, morphological abnormalities, postparturition survival, physiological/biochemical 

characterizations of surviving/dead animals. 

Minimal, due to low 

likelihood of entry into 

food supply as meat, 

except for injured 

animals. 

3: Juvenile Development 

(cattle: pre-weaning; swine, 

sheep, goats: post-weaning 

period) 

Survival rate, measures of growth, physiological and 

biochemical markers of health status. 

Relatively low, but 

possibly as meat 

(e.g., veal, lamb, 

suckling pig). 

4: Reproductive Development 

and Function 

Development of secondary sex characteristics, 

spermatogenesis, oögenesis, gender appropriate 

behavior, age of pubertal onset. Fertility measures for 

males and females. For females, mothering behavior, 

milk production. 

Milk 

5: Post-Pubertal Maturation Growth, weight gain, muscle/fat ratios, milk 

production. Meat, Milk 

G. Limitations of the Risk Assessment 

This is a qualitative, comparative risk assessment that does not attempt to assign a 

quantitative value to estimates of risk or safety. The strongest conclusions that can be 

drawn regarding positive outcomes in risk assessments of this type are “no additional 

risk” because outcomes are weighed against known comparators. If a finding of “no 

additional risk” were to be applied to the health of animal clones, it would mean that the 
Chapter III: Developing the Risk Assessment Methodology 55

cloning process would not pose any greater risk to the health of the animals involved than 

other ARTs. Applied to the safety of edible products derived from clones, a finding of 

“no additional risk” would mean that food products derived from animal clones or their 

progeny would not pose any additional risk relative to corresponding products from nonclones, or that they are as safe as foods that we eat every day. As with all risk 

assessments, some uncertainty is inherent either in the approach we have used or in the 

data themselves (for a more complete discussion of the uncertainties in this Risk 

Assessment, see Chapter VII). 
 

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Chapter IV: 

The Implications of 

Epigenetic Reprogramming for 

 Clones and their Progeny
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Chapter IV: Epigenetic Reprogramming: 

Implications for Clones and their Progeny 

The previous Chapters of this Risk Assessment have introduced the concept that 

incomplete or inappropriate epigenetic reprogramming appears to be one of the primary 

underlying causes for the relatively low success rate of cloning, and the source of 

potential subtle hazards for the consumption of food from animal clones. Although a 

complete discussion of the rapidly emerging field of epigenetics is beyond the scope of 

this risk assessment, readers are directed to a series of excellent reviews for more details 

(Reik and Walter 2001; Surani 2001; Bird 2002; Li 2002; Davidson et al. 2003; Kelly and 

Trasler 2004; Santos and Dean 2004; Tian 2004; Allegrucci et al. 2005; Holliday 2005; 

Morgan et al. 2005). An overview of the topic, however, is useful to put the issue of the 

source of potential subtle hazards in clones into context. 

Briefly, epigenetics has been defined as the study of stable alterations in gene expression 

potentials that arise during development and cell proliferation (Jaenisch and Bird 2003), 

or alterations in DNA function without alterations in DNA sequence (Jones and Takai 

2001). The central idea behind the concept of epigenetics is that although the DNA 

sequence of almost all the nucleated somatic cells in the body of an adult mammal is 

identical (except some very specialized cells whose development requires DNA 

rearrangements), the phenotypes of those cells can be quite different because alternate 

subsets of genes are expressed at different times in development and during cellular 

differentiation. In other words, each cell type in an organism has its own epigenetic 

profile or signature (Morgan et al. 2005). 

Epigenetic changes have been implicated as the source of the anomalies noted in clones 

and other ARTs. The primary biological assumption is that as no exogenous genes are 

being introduced into the genome being copied and expressed (as in the case of clones) or 

being expressed following the union of two gametes (as in the case of the other ARTs), 

alterations in gene expression are responsible for the adverse outcomes noted in the 

resulting animals. Although much of the focus of the ongoing research in this rapidly 

expanding field is directed towards gathering and understanding observations of 

epigenetic changes in early development, epigenetic changes also occur later in life. They 

are part of the normal and necessary way that organisms adapt to their environments. 

For example, Fraga et al. (2005) have demonstrated that monozygotic or “identical” 

human twins begin life with very similar epigenetic patterns. Over time, however, they 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 60 

accumulate epigenetic differences so that their epigenetic profiles become quite different. 

Smoking, diet, and other life experiences are proposed as exerting influence of the 

epigenetic differences observed between genetically identical twins, with more 

differences in life experiences correlated with more different epigenetic profiles later in 

life. Epigenetic changes have also been associated with various disease states that arise 

from the dysregulation of normal genes (reviewed in Egger et al. 2004, Jiang et al. 2004). 

Epigenetic differences are also noted in conventional animals, and may reflect the status 

of the uterine environment. Cooney et al. (2002) investigated the effects of maternal 

methyl food supplements17 prior to and during pregnancy on the epigenetic control of 

various health outcomes using an experimental system based on the expression of an 

epigenetically-regulated mouse coat color. The genome of mouse strain employed in this 

study includes an endogenous retrovirus containing viral genes and promoter enhancer 

sequences referred to as long terminal repeats (LTRs), which can drive the expression of 

retroviral genes and murine genes in their vicinity. When the LTR is active (relatively 

demethylated), it overpowers the endogenous mouse promoters, and allows the constant 

transcription of the genes giving rise to the “yellow” phenotype. This phenotype exhibits 

a solid yellow coat color, obesity, predisposition to cancer, diabetes, and a relatively short 

lifespan. When the LTR is suppressed (relatively methylated), the agouti18 gene locus is 

regulated by its own promoters, and is expressed cyclically and only in hair follicles. The 

phenotype of these mice is lean, healthy animals of normal lifespan and agouti-patterned 

coats. In this study, pregnant dams were fed diets containing three levels of dietary 1-

carbon sources or cofactors. The lowest consisted of typical laboratory mouse chow; the 

intermediate level was supplemented with choline, betaine, folic acid and vitamin B-12, 

while the highest supplementation included three times the supplement level of the 

intermediate diet plus methionine and zinc. In addition to evaluating coat color, Cooney 

et al. (2002) also determined the relative degree of methylation of the LTRs driving the 

agouti gene locus. They observed that as the level of methyl donors in the diet increased, 

phenotypes of progeny animals shifted towards the agouti phenotype. Corresponding 

changes were observed in the methylation status of the LTR, with increasing methylation 

in the animals whose dams received higher levels of methyl donors in their diets. 

Because the field is relatively new, and the scientific community has not identified all of 

the mechanisms involved in epigenetic remodeling, with few exceptions (e.g., X-

 

17 Methylation of DNA is performed by specific enzymes (methylases) that obtain methyl sources from 

either the diet (as folates or folic acid) or from endogenous one-carbon metabolism. The latter requires 

essential dietary components such as methionine, zinc, and vitamin B-12 to act as cofactors in the synthesis 

of intermediates that give rise to 1-carbon donors 

18 The agouti coat color is a continuous spectrum of variegated coat color patterns on a yellow background. 

In cats, this coat color pattern is referred to as “tortoiseshell.” 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 61 

chromosome inactivation), the direct links between any one mechanism (or a series of 

mechanisms) and the health outcomes in live animals are not clear. Animals produced by 

non-SCNT ARTs, including natural mating, may have different epigenetic profiles, and 

even exhibit developmental abnormalities, but are not considered to pose unique food 

consumption risks. 

A. Overview of Epigenetic Reprogramming in Early Embryonic Development 

In conventional breeding, a new diploid genome is created by the fusion of two haploid 

genomes; one each from the sperm and the egg. The subsequent expression of that newly 

formed diploid genome to generate a functional multicellular organism is governed by a 

“program.” This term was first used by the genetic pioneers Jacob and Monod, who in 

1961 proposed that “...the genome contains not only a series of blueprints, but a 

coordinated program...and a means of controlling its execution.” More than a halfcentury later, researchers are still trying to understand how that control is exerted. 

Multiple mechanisms respond to the cell’s developmental stage or its environment by 

acting as positive (more transcription19) or negative (less transcription) control elements. 

Transcriptionally active regions of DNA (or heterochromatin) may be considered to be 

“open” so that various molecules involved in DNA processing can gain access to certain 

regions, whereas “euchromatin” is physically tightly condensed, or “closed” with respect 

to access by other molecules, and transcriptionally silent. The picture emerging through 

current research (see citations above) suggests that the overall system is extremely 

complex, with many degrees of “openness” existing. 

One of the examples of this complexity is manifested via the extent and variety of 

modifications that can occur to DNA itself and its associated histones (positively charged 

proteins that are responsible for maintaining chromosome structure). These modifications 

include DNA and histone methylation at a number of positions, acetylation, 

phosphorylation, and ubiquitination of histones (Kanka 2003; Quivy et al. 2004; Cheung 

and Lau 2005; Fuks 2005; Verschure et al. 2005). Although histone modification seems 

to be important for fully appreciating the complete range and stability of regulation 

possible as well as the subtleties of the system, the methylation state of the DNA is 

central to the epigenetic regulation of gene expression. DNA methylation has been the 

subject of considerable research (reviewed by Holliday 2005, Scarano et al. 2005), as 

 

19 Information encoded in DNA is converted into RNA by a process referred to as transcription. Those 

RNA molecules (messenger RNA) that encode information for protein synthesis are converted to proteins 

by the process of translation. 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 62 

scientists begin to understand its role in gene regulation. The bulk of the discussion of 

epigenetics in this chapter centers on DNA methylation primarily because, to date, most 

of the studies of epigenetic changes in animal clones examine changes in methylation 

states. 

DNA methylation refers to the addition of methyl groups to the 5 position of cytosine, a 

non-coding portion of the nucleoside, to regulate the appropriate expression of genes (see 

Figure IV-1). Methylation tends to occur in areas of the chromosome that are rich in 

sequences that contain stretches of repeating cytosine-guanosine residues (CpG islands), 

which tend to be positioned at the 5’ ends of genes.20 Most of these regions are 

unmethylated regardless of developmental stage, tissue type, or gene expression level. 

DNA methylation in somatic cells is generally faithfully restored at each replication cycle 

(for dividing cells), although changes in methylation levels are often associated with 

aging, or occur in abnormal cells (Bird 2002, Jaensich 2004). Methylation may affect 

gene transcription by physically impeding the access of cellular transcriptional machinery 

to coding regions, or by attracting proteins that bind specifically to the modified CpG 

sequences, thus impeding the transcriptional machinery (Cezar 2003). 

Figure IV-1: Cytosine and 5-methyl cytosine Addition of a methyl group at the 5 

position of the base is shown. 

N

O N

NH2

 

N

N

CH3

NH2

O

Mammalian embryos experience major epigenetic reprogramming primarily at two times 

in their development, both of which have significant implications for cloning. One of 

these takes place soon after fertilization, and is referred to as preimplantation 

reprogramming; the other occurs during gametogenesis (the development of cells that 

ultimately become the sperm and egg). Because preimplantation reprogramming occurs 

after fertilization, and in the case of nuclear transfer, after fusion of the donor nucleus 

with the oöplast, it is the most immediately impacted by the cloning process, and may be 

most directly implicated in the anomalous development of clones with defects. 

Gametogenic reprogramming may also be involved in the anomalies noted in clones, but 

 

20 The 5’ end of a gene is often considered to be at the start of the coding sequence on the DNA molecule. 

The nomenclature is derived from the position of a hydroxyl group in the deoxyribose sugar ring at the 

beginning of the strand of the DNA. 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 63 

it likely has more far-reaching implications for progeny, because it generates the gametes 

used for the sexual reproduction of clones, (although, by definition, the absence of 

gametogenic reprogramming in the somatic cell donors used for SCNT poses a high 

biological burden for the preimplantation reprogramming (Jaenisch et al 2004)). Most of 

the literature on epigenetic dysregulation in clones and animals produced using other 

ARTs addresses preimplantation reprogramming; the literature on gametogenic 

reprogramming often evaluates endpoints related to the sexual reproduction of clones 

(Yamazaki et al. 2003). 

1. Preimplantation Reprogramming 

a. Fusion and Cleavage 

In sexual reproduction, mammals use cells of highly different morphology and function 

to deliver haploid genomes. Sperm are small relative to the oöcyte, and package their 

highly condensed DNA by tightly coiling the DNA around a set of proteins called 

protamines. The oöcyte’s genome is packaged more loosely around a different set of 

proteins called histones, also found in somatic cells (Cezar 2003). In order for the embryo 

to form a unique genome, the two chromatin structures must be resolved into one that is 

capable of coordinated gene expression. A number of factors present in the oöplasm of 

the ovum, only a few of which have been identified, are thought to aid in this remodeling 

(Kang et al. 2003). In the first hour after fertilization, the sperm head swells, the nuclear 

envelope of the sperm breaks down, and protamines are replaced with histones (Santos 

and Dean 2004). The chromatin then decondenses, and the male pronucleus21 forms 

(Mann and Bartolomei 2002). The female genome completes its second meiotic division, 

expels the resulting polar body, and then forms the maternal pronucleus. Both the male 

and female pronuclei begin to replicate DNA, and depending on the species, some 

transcription may ensue. In mice, transcription occurs in the male pronucleus in the first 

cell cycle, followed by a larger burst in the second cell cycle (Aoki et al. 1997), while in 

bovine embryos, transcription is delayed (Mann and Bartolomei 2002). 

During SCNT, however, different initial events must take place. SCNT begins with the 

removal of the nucleus of the oöcyte that contains the meiotic metaphase II chromosomespindle complex, followed by microinjecting or fusion of the donor cell or nucleus into 

the enucleated oöplast. The presence of oöcyte factors probably causes the breakdown of 

the nuclear envelope of the donor cell (similar to swelling and breakdown of the sperm 

 

21 The pronucleus is the structure that contains the haploid genome of the sperm or ovum after fertilization 

occurs, but before they fuse to make the nucleus of the zygote, or the single-celled diploid organism. 

Once the zygote has undergone the first division (or cleavage), it is referred to as an embryo. 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 64 

head). Following oöcyte activation (usually by electrical stimulation), the chromatin from 

the donor nucleus decondenses, and a pseudopronucleus is formed. If successful, DNA 

replication and cellular division follow. 

Figure IV-2: Epigenetic Reprogramming and Embryonic Development 

Legend to Figure IV-2. The top of the diagram illustrates the terminally differentiated sperm and egg 

(oöcyte). These gametes fuse to form the fertilized egg or zygote and begin preimplantation reprogramming 

(emphasized by the bracket at the left). Following the steps counter-clockwise around the figure sequential 

cell divisions are illustrated with corresponding differentiation from totipotent through pluripotent to 

differentiated. The right half of the figure represents gametogenic reprogramming (emphasized by the 

bracket at the right), the epigenetic marking of the primordial germ cells that will become the sperm or 

eggs of this new individual at sexual maturity and setting up another cycle. 

Genital 

ridge

oöcyte sperm

fertilized egg

zygote

2-cell stage

8-16 cell stage

primordial 

germ cells

primary oöcyte

spermatogonium

Imprints erased by 

demethylation

Imprints established by 

methylation

blastocyst

inner cell mass

trophectoderm

~ 12 d embryo (mouse)

TOTIPOTENT

TERMINALLY 

DIFFERENTIATED

PLURIPOTENT

DIFFERENTIATED

preimplantation reprogramming

gametogenic reprogramming

(clones)

(progeny)
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 65 

Information from either terminally differentiated gametes (fertilization-derived zygotes) 

or a terminally differentiated somatic cell (in SCNT) must be reprogrammed so that the 

resulting zygote is totipotent (capable of developing into any cell type). Totipotency 

appears to be lost early in development and almost certainly after the blastocyst is 

formed, when the trophectoderm and inner cell mass begin to separate (see Figure IV-2). 

At this point in development, the cells are pluripotent—no longer capable of being any

cell type, but retaining the ability to become many cell types. The end process is referred 

to as “terminal differentiation,” in which cells acquire a set of characteristics that allows 

them to perform a specific function (e.g., muscle cells contract, neurons transmit 

electrical pulses, and gametes serve as genome donors for subsequent generations). One 

of the ways that this overall process is accomplished is by resetting the epigenetic marks 

of the gametes. At this time, the signals that determine “lineage allocation” are not clear. 

Fujimori et al. (2003) have noted that each of the two cells in the early blastomere is 

completely totipotent; some lineage bias is observed when the developmental potency of 

four-cell stage blastomeres is evaluated. Cells inside the eight and 16 cell stage of the 

morula appear to be more likely to become committed to the inner cell mass lineage 

(which becomes the embryo), while those outside appear to be directed to the 

trophectoderm and the development of placental tissues (Morgan et al. 2005). 

The next sections provide an overview of events as they are understood in the 

development of fertilization-derived embryos, followed by examples of observations 

noted in clones and, when available, other ARTs with significant in vitro culturing 

components. The examples are intended to be illustrative and not comprehensive, as an 

encyclopedic review is beyond the scope of this discussion. The important points to be 

made are that 

• Mechanisms of epigenetic reprogramming are complex and not fully understood, 

even in “normal” fertilization-derived embryos; 

• SCNT-derived embryos often do not develop normally, and all the available 

evidence indicates that this is due to incomplete or inappropriate epigenetic 

reprogramming; 

• Genomes are “plastic” and can accommodate some errors in epigenetic 

reprogramming, regardless of whether those embryos are derived via fertilization 

or nuclear transfer; 

• Some SCNT-derived embryos go onto full gestation and delivery; clones that are 

born can range from those exhibiting some epigenetic dysregulation to no 

detectable differences. 

Some have suggested (Wilmut 2002; Jaenisch 2004) that no clone is completely “normal” 

with respect to its epigenetic profile. Although this is an important point for assessing the 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 66 

overall safety of the cloning process for any particular species, and for determining risk 

to animals involved in the cloning process, the relevance of “epigenetic normality” to 

food consumption risks is unclear. This is particularly true when considering the degree 

to which epigenetic changes are observed in other ARTs with a significant in vitro

culturing component, and the accumulation of epigenetic changes expected during the 

aging process. The most compelling conclusions that can be made about food 

consumption risks are drawn from assessments of the health status of the animals and the 

composition of food products derived from them, and not from gene expression studies. 

 

b. Demethylation and Remethylation in Early Embryos 

Dean et al. (2001) and Morgan et al. (2005) have outlined how the process of 

demethylation and epigenetic resetting occur in various mammals. Hours after 

fertilization, but prior to DNA replication and cleavage, the paternal genome of mice, 

rats, pigs, cattle, and humans, but not sheep, is actively stripped of the epigenetic 

methylation markers by mechanisms not fully understood, but that likely require the 

activity of a demethylase enzyme present in the oöcyte (Morgan et al. 2005). This 

genome-wide methylation erasure appears to be conserved among cattle, swine, and rats, 

but is not observed in sheep (Wilmut et al. 2002; Beaujean et al. 2004; Young and 

Beaujean 2004). In mice and cattle, the maternal genome retains its methylation markers 

during this period, and does not undergo demethylation until the zygote undergoes the 

first cleavage to yield the two-celled embryo. Demethylation of the maternal genome is 

thought to be passive, that is, diluted by the lack of remethylation on newly replicated 

DNA (Cezar 2003). In two to eight cell bovine embryos, Dean et al. (2001) observed a 

further reduction in methylation, consistent with the passive demethylation occurring 

during DNA replication seen in the mouse. In contrast, mouse 16-cell embryos continued 

to remain demethylated, and genome-wide de novo methylation did not occur until 

approximately four cell divisions later, and appeared to occur preferentially in the inner 

cell mass (ICM). Thus, although the overall process of demethylation and de novo

methylation appears to be conserved in the species evaluated, the timing of these 

phenomena may differ among species (Morgan et al. 2005). The more important 

observation, however, is that the first differentiation event in mammalian embryos (the 

differentiation of the trophectoderm and ICM and the resulting loss of totipotency of the 

ICM cells) is accompanied by genome-wide de novo methylation. 

Fertilization-derived bovine embryos begin to demonstrate global genomic de novo 

methylation in the eight- to 16-cell stage, what is often referred to as the maternal to 

embryonic transition (MET). During this time, the developmental program that is initially 

directed by components within the egg (maternal) is replaced by a new program directed 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 67 

by the expression of new genes (Wrenzycki et al. 2005), and is accompanied by different 

rates of demethylation of maternally and paternally derived genes to give rise to a new 

methylation pattern for the embryo. 

Although the early demethylation described above is global (occurring over the entire 

genome in general), methylation marks on imprinted single copy genes tend to be 

protected from demethylation so that parental imprints are preserved in the resulting 

somatic cells of the developing mammal (Li 2002). It is unknown whether the extensive 

global demethylation of the genome during pre-implantation development is essential for 

normal development. 

DNA-methylation patterns unique to the developing mammal are established in the 

embryo after its implantation in the uterus through lineage-specific de novo methylation 

that begins in the inner cell mass. DNA methylation increases rapidly in the primitive 

ectoderm, which gives rise to the entire embryo. Conversely, methylation is either 

inhibited or not maintained in the trophoblast and the primitive endoderm, from which 

the placenta and yolk-sac membranes develop, respectively (Li 2002). The net effect is 

that extra-embryonic tissues appear to have a lower methylation state than embryonic 

tissues. These global differences in methylation status between the embryonic and extraembryonic tissues appear to be conserved in mice, cattle, sheep, and rabbits (Morgan et 

al. 2005). 

Reprogramming the donor nucleus in SCNT or the nucleus of the early fertilized embryo 

has been the subject of considerable investigation over the past few years. Much of this 

research has been summarized in reviews by Rideout et al. 2001; Jaenisch et al. 2002; 

Mann and Bartolomei 2002; Cezar 2003; Han et al. 2003; Jouneau and Renard 2003; 

Smith and Murphy 2004; Young and Beaujean 2004; Wrenzycki et al. 2005). 

These reviews and the studies contained in them have come to approximately the same 

conclusions: although some clones may develop into healthy animals, the low success 

rate of SCNT is likely associated with the inability of clones to reprogram the somatic 

nucleus of the donor to the state of a fertilized zygote. Similarly, the rates of successful 

embryo formation resulting in term gestation and live births in ATRs that have a high 

degree of in vitro culturing are likely also due to difficulties in reprogramming (Gardner 

and Lane 2005, Wrenzycki et al. 2005). The sources of the stresses on the embryos that 

cause these difficulties may be a reflection of the intrinsic biological differences between 

fertilization- and nuclear transfer-derived embryos (e.g., the need to fully reprogram a 

differentiated nuclear donor), or technological (e.g., the in vitro environment in which 

early embryos are cultured prior to introduction into the uterus). The following discussion 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 68 

briefly outlines the current state of knowledge of how this is accomplished in 

fertilization- or nuclear transfer-derived embryos. 

In embryos derived via nuclear transfer, epigenetic modification, such as the waves of 

demethylation and de novo methylation observed following fertilization must also occur, 

but may be hampered by both the nature of the donor DNA and the partially depleted 

oöplasm. There are reports of both aberrant and “normal” demethylation and 

remethylation in clones and fertilization-derived embryos. Differences may be reflections 

of different methodologies, source cells, species differences, or may reflect unexplained 

phenomena. The following discussion summarizes the key observations that contribute to 

the body of knowledge regarding epigenetic remodeling in SCNT- and other ARTderived embryos. 

Some species-specific responses in the degree of methylation reprogramming have been 

observed, although in general, the overall processes appear to be relatively conserved 

among the clones of different species. Dean et al. (2001) found that somatic nuclei of 

mouse, rat, pig, and bovine embryos undergo the genome-wide reprogramming described 

previously, but that reprogramming occurred aberrantly in many cloned preimplantation 

embryos. Bourc’his et al. (2001), using a similar method, did not observe active 

demethylation in bovine SCNT zygotes, although they did observe that the somatic 

pattern of methylation from donor nuclei was preserved through the four cell stage. 

Ohgane et al. (2001) compared the methylation status of CpG islands (CG-rich sequences 

located at promoter regions) in placenta and skin cells of sexually reproduced mice to 

similar regions in normal-appearing mouse clones. Most of the methylated regions in 

fetal clones (99.5 percent in the placenta and 99.8 percent in the skin) were identical to 

those of the controls, but different methylation patterns were observed in the two 

different tissues. The sites of discordant methylation were located in regions responsible 

for expression of tissue-specific genes, despite the absence of grossly observable 

abnormalities. In bovine preimplantation embryos, however, Kang et al. (2001a) noted 

that bovine clone embryos failed to demethylate satellite regions of the genome (certain 

repetitive sequences), and instead maintained methylation levels similar to the donor cell. 

In a subsequent study, however, Kang et al. (2001b) were able to “rescue” the inefficient 

demethylation of bovine embryos by providing an additional “dose” of oöcyte factors to 

the early embryo. This work confirms the presence of an active element in the oöcyte for 

erasure of paternal epigenetic methylation, and implies that this component, which may 

be removed or diluted during the process of preparing an enucleated oöplast, is involved 

in the appropriate epigenetic modeling observed in zygotes and early embryos derived 

from fertilization. In a third study, Kang et al. (2001c) investigated demethylation in 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 69 

swine clone embryos relative to those derived by in vitro fertilization (IVF). They 

observed that, unlike mice and cattle, the sequences investigated (centromeric satellite 

DNA) were negligibly methylated in swine oöcytes, and hypermethylated in swine 

sperm. (Sperm satellite DNA sequences in cattle and mouse tend to be undermethylated.) 

The satellite sequences of the donor pig fibroblast cells were hypermethylated, and 

retained that status until the 4-8 cell stage. Demethylation began at that time, and the 

methylation status of the clone embryos decreased significantly in the blastocyst, just as it 

did in the blastocysts of in vitro or in vivo fertilization-derived embryos. Their finding 

thus indicated that satellite sequences of SCNT-derived pig embryos undergo 

preimplantation demethylation in a manner similar to fertilization-derived embryos. 

Analogous results were observed when another sequence, PRE-1 (from the euchromatin) 

was evaluated. These results are comparable to the pattern observed in mouse embryos by 

Dean et al. 2001. 

Whether the results obtained from these two DNA sequences studied by Kang et al. can 

be extrapolated to global DNA methylation or other single-gene sequences in the pig 

remains unknown. Additionally, the reasons for the interspecies differences between mice 

and pigs on one hand, and cattle on the other, also remain unknown. Nonetheless, one of 

the key implications of these observations is that global demethylation soon after 

fertilization appears to be a prerequisite for successful reprogramming later in embryonic 

development, and possibly for successful SCNT outcomes. 

Kang et al. (2003) have also demonstrated that at least some SCNT-derived bovine 

embryos are capable of normal remethylation during early embryogenesis. They 

evaluated the methylation status of a 170 base pair fragment of single copy gene in IVF 

and SNCT-derived bovine embryos. This sequence is negligibly methylated in both 

sperm and oöcyte DNA, and moderately (approximately 37 percent) methylated in the 

fibroblasts that served as nuclear donors for SCNT. In single celled zygotes, as well as 

the 4-to 8-cell stage embryos derived via IVF, the low methylation levels of the sperm 

and oöcyte genomes were observed. No significant changes in methylation status of the 

IVF-derived embryos were observed at the 8-16 cell stage, but by the time a blastocyst 

had formed, de novo methylation appears to have taken place. In SCNT-derived embryos, 

the methylation pattern of the donor cell was nearly completely lost by the 4-8 cell stage, 

and demethylation appeared to be complete by the 4-8 cell stage. At the blastocyst stage, 

the methylation stage of the SCNT-derived embryo was exactly that of the IVF 

blastocyst, with the same CpG sites exclusively methylated in both sets of embryos. The 

authors claim that this study is “the most elaborate example of recapitulation of normal 

embryonic process[es] occurring in [SC]NT embryos.” Although this study clearly 

demonstrates the ability of somatic cells to be epigenetically reprogrammed in an 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 70 

accurate manner relative to an IVF comparator, and that these molecular results are 

consistent with observation of apparently healthy and normal animal clones being 

generated from somatic cell donors, the predictive value of this particular gene for other 

single copy genes, or the entire genome has not been demonstrated. 

 

In another study showing differences in methylation states among species, Beaujean et al. 

(2004) evaluated the global methylation status of fertilized and SCNT-derived sheep 

embryos. They observed that unlike mice and cattle, sheep oöcytes do not appear to 

demethylate the sperm-derived pronucleus after fertilization. In vivo-derived sheep 

embryos demonstrated that a partial demethylation of the global genome occurred up to 

the 8-cell stage, with similar qualitative findings in SCNT-derived embryos (fibroblast 

cell nuclear donor), but to a lesser extent. Interestingly, between the 8-cell and blastocyst 

stages, both in vivo- and SCNT-derived embryos showed comparable overall levels of 

methylation, but the distribution of methylation patterns differed among the SCNTderived embryos and between some of the SCNT-derived embryos and those derived 

from fertilization. The authors attributed these differences to differences in the overall 

high-order chromatin structure, rather than simply to changes in methylation. They 

suggested that many SCNT-derived embryos do not undergo the rapid reorganization of 

the DNA prior to first cleavage that successful in vivo-derived (and a small proportion of 

SCNT-derived) zygotes do. Further, they suggest that perturbations in methylation (and 

possibly remodeling) correlate with the lack of appropriate trophectodermal development 

and subsequent placental development in later embryos and that these alterations may 

contribute to the high observed levels of placental defects and embryonic loss during 

SCNT-pregnancies. Beaujean et al. conclude that although “DNA methylation appears to 

be marker of reprogramming in all mammalian species examined to date, it is not yet 

clear to what extent it is a determinant.” 

c. Epigenetic Reprogramming in Later Development 

This summary covers studies of epigenetic reprogramming from the fetal through adult 

developmental nodes (as described in more detail in Chapter VI). 

In a study of genome-wide epigenetic reprogramming in bovine clone embryos and 

adults, Cezar et al. (2003) measured the amount of 5-methyl cytosine in DNA from 

various tissues in fetuses and term pregnancies generated via SCNT and fertilization. 

Their results showed that the amount of methylation was lower in spontaneously aborted 

fetal clones, fetal clones sacrificed as part of the study, and tissues collected from 

pregnancies that had experienced hydroallantois relative to controls. These results are in 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 71 

contrast to others that have found hypermethylation in clones relative to fertilizationderived controls (Bourc’his et al. 2001; Dean et al. 2001; Kang et al. 2001b). Adult 

clones, however, had similar levels of DNA methylation as adults derived via 

fertilization. Cezar et al. (2003) concluded that there may be an epigenetic 

reprogramming threshold that is met by a subset of animal clones. They also proposed 

that clones surviving into adulthood have the ability to overcome epigenetic challenges 

determined by their somatic cell origin. These hypotheses are consistent with the 

observations by Chavatte-Palmer et al. (2002), Chapters V and VI, and the Cyagra 

dataset, described in Appendix E, in which early physiological instabilities appear to 

resolve as the clones mature. 

Similar to Cezar’s observations, Chen et al (2005) also noted that aberrant methylation 

likely plays a role in the poor development noted in clones and other forms of 

reproduction. In their study, the methylation status of aborted bovine clone fetuses, 

aborted fetuses generated by artificial insemination (AI), and adult cattle generated via AI 

or cloning was studied. Three genomic regions were evaluated: a repeated sequence and 

the promoter regions of two single copy genes (interleukin 3 and cytokeratin). All of the 

aborted fetuses (AI- and SCNT-derived) were females between 60 and 90 days of 

gestation; adult animals were all classified as ‘healthy” and between 18 and 24 months of 

age. The adult animals all had approximately the same level of methylation at all of the 

loci examined, regardless of method of production. The aborted AI-derived fetuses all 

had similar, but lower levels of methylation than the healthy adults, as well as different 

methylation patterns. For the single copy genes, methylation could be classified into two 

groups: one group had very low methylation patterns in the promoter regions, while the 

other group had methylation patterns similar to the aborted AI-derived fetuses. One of 

these fetuses also showed low methylation patterns in the satellite region. Although this 

study is not conclusive, it does provide evidence that at least for certain regions of the 

genome, appropriate methylation appear to be correlated with normal development. 

Dindot et al. (2004) developed a unique bovine hybrid interspecies model (Bos gaurus x 

Bos taurus) to study epigenetic markings and imprinting in gestation day 40 female 

SCNT-derived fetuses and placentae (derived from cumulus cell donor cells) that were 

genetically identical to fetuses derived by fertilization. Previously, Hill et al. (2000b) had 

shown that more than 80 percent of bovine clone pregnancies were lost between days 30-

60 of gestation, and attributed the losses to placental anomalies including a reduction in 

the number of expected cotyledons and a decrease in chorio-allantoic blood vessels. 

These observations were similar to those of Stice et al. (1996) who reported that no 

placentomes had developed in NT fetuses that died between gestation days 33-55. Mouse 

clone pregnancies have also shown increases in placental size (Tanaka et al. 2001). These 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 72 

abnormalities have been hypothesized to arise from anomalies in nuclear reprogramming 

of the trophectoderm, which gives rise to placental structures including the chorion. By 

using the hybrid Bos gaurus/Bos taurus model, Dindot et al. were able to discriminate 

between parental alleles by following single nucleotide polymorphisms (SNPs) (changes 

in the nucleotide sequence of the DNA at only one site that allow for the cleavage or the 

lack thereof by enzymes that recognize specific DNA sequences). In particular, three 

genes associated with epigenetic reprogramming were selected including IGF-2, Gene 

trap locus 2 (GTL2), and the X chromosome inactivation specific transcript (Xist). Clone 

fetuses and placental tissues were isolated from surrogate dams at gestation day 40; none 

of the clone placentae developed cotyledons, unlike the fertilization-derived fetuses, 

which had 4, 16, and 25 cotyledons per pregnancy. Although appropriate allelic 

expression of IGF2 and GTL2 relative to fertilization-derived fetuses was observed in 

both chorionic and fetal liver tissues of the clones, disruptions of genomic imprinting of 

the Xist locus was found in the chorion, but not the liver tissues of any of the clones. 

Further analysis of two other regions of the genome in the chorion of the clone fetuses, 

the satellite I repeat element and epidermal cytokeratin promoter, indicated that the 

trophectoderm-derived tissues of the clones had higher levels of methylation relative to 

fertilization-derived controls. No differences in methylation levels were observed in the 

livers of clones or fertilization-derived embryos. In this study, at least, there were 

differences in the degree of epigenetic reprogramming between ICM-derived tissues (the 

fetus proper) and those derived from the trophectoderm, consistent with the observation 

by Hill et al. (2000b), that clones with aberrant placental structures can survive gestation 

and be born alive and apparently healthy. 

d. Studies of Gene Expression and Development in Clones and Other ARTs 

The previous sections summarized studies of alterations in methylation associated with 

cloning; the following section summarizes reports of gene expression and phenotypic 

observations in similar clone populations. The overview is intended to be more 

illustrative than comprehensive as the literature on this subject is large and growing 

rapidly. The studies indicate that for non-viable clone embryos, fetuses, or neonates, key 

genes are inappropriately expressed. In some cases, viable clones have differences in 

expression compared to fertilization-derived counterparts, leading investigators to 

speculate that genomes are plastic and that a certain level of gene dysregulation can be 

tolerated. In other studies of healthy, live clones, no significant differences can be 

observed between the expression profiles of animals generated via SCNT or other 

fertilization-based ARTs. Finally, it should be noted that studies comparing embryos 

generated via various ARTs (including SCNT) with significant in vitro culturing 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 73 

components, appear to be sensitive to the culture environment, with developmental 

success often being a function of the culture medium used. 

Most of the earliest studies of gene expression in clones were performed in mice. Boiani 

et al. (2002) and Bortvin et al. (2003) evaluated patterns of gene expression in mouse 

blastocysts derived from SCNT to identify which critical genes were involved in the 

inability of most of those blastocysts to develop further. In particular, they evaluated the 

expression of Oct4 and Oct4-related genes in these embryos. (Oct4 is a transcription 

factor specifically expressed in stem and primordial germ cells, and appears to be 

required for maintaining pluripotency and the self-renewal ability of stem cells.) Boiani 

et al. (2002) compared Oct4 expression in blastocysts cloned from somatic cell nuclei and 

germ cell nuclei to that observed in synchronous blastocysts produced by IVF and 

intracytoplasmic sperm injection (as the control groups independent of cloning but 

involving micromanipulation). Their results demonstrated that mouse blastocysts derived 

from clones had abnormal Oct4 expression, and that the failure of mouse clones embryos 

to develop beyond the blastocyst stage was related to incorrect lineage determination by 

the inappropriate expression of Oct4. Bortvin et al. (2003) identified 10 candidate genes 

with expression patterns similar to Oct4 and compared their expression in 

preimplantation embryos derived from fertilization to embryos whose SCNT donors were 

somatic cumulus and pluripotent embryonic stem cells. They demonstrated that 

successful reactivation of the full set of 10 genes correlated with the development of 

embryo clones, but also noted that almost 40 percent of the cumulus cell-derived 

blastocysts failed to reactivate these genes faithfully, even though the blastocysts were 

morphologically normal. Thus, some other factors were required to maintain the 

pluripotency of the inner cell mast cells. Marikawa et al. (2005) found that the DNA 

methylation status of the Oct4 regulatory element in mouse embryos directly influences 

the level of gene expression. They further noted that the methylation status of the Oct4

regulatory element was highly heterogeneous among alleles in a population of adult 

somatic cells, and hypothesized that that the degree to which Oct4 can be reactivated in 

SCNT may be a function of the methylation status of the donor cell(s). 

Boiani et al. (2005) further evaluated Oct4 expression in early post-activation SCNTderived zygotes, fertilization-derived early embryos and parthenotes in six different 

culture media. (Lack of expression of Oct4 precludes further development beyond the 

blastocyst). Among their first observations was that similar to fertilization-derived 

embryos, progression to blastocyst did not ensure further development of the embryos, 

and that some of the primary influences on whether such development occurred could be 

environmental in origin. They also noted that nuclear transfer embryos appeared to be 

more sensitive to environmental conditions than the other two types of embryos. They 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 74 

concluded that not only was the ability of mouse clone embryos to progress through 

development contingent on the nature of the donor nucleus and recipient oöplasm, but 

that culture conditions could have a significant impact on the expression of key genes 

required for reprogramming (and subsequent development), and the ability of the 

blastocyst to continue to develop successfully. 

To study the correlation between gene expression, survival, and fetal overgrowth 

(e.g., LOS-type symptoms), Humpherys et al. (2001) examined imprinted gene 

expression in mice cloned by nuclear transfer and in the embryonic stem cell donor 

population from which they were derived. They determined that transcript levels of 

selected imprinted genes varied widely in placentae from animal clones relative to nonclones, although alterations in the expression of one imprinted gene did not correlate with 

abnormal expression of other imprinted genes. They also observed that changes in DNA 

methylation levels at one imprinted locus did not necessarily predict changes at other 

loci. Certain genes (e.g., H19 and Igf2) were largely silenced in the heart and kidney, and 

their expression reduced in the livers of animal clones relative to conventional animals. 

No correlations were observed between changes in gene expression and birth weights, 

placental weights, or neonatal mortality. Culturing the embryonic stem cells in vitro

resulted in highly variable levels of gene expression; gene expression in the animals 

resulting from those cells was even more variable than in the cells in culture, implying 

that culturing early embryos may contribute to the degree of embryonic gene 

dysregulation. Furthermore, mice derived from the cells of the same cellular lineage 

differed in their expression of imprinted genes. Given that viable animals were generated 

with variable expression of imprinted genes, the authors concluded that “mammalian 

development may be rather tolerant to epigenetic abnormalities and that lethality may 

only result from the cumulative effects of a stochastic loss of normal gene regulation at 

multiple loci....even apparently healthy animal clones can have gene expression 

abnormalities that are not severe enough to impede development to birth but that may 

cause subtle physiological abnormalities which could be difficult to detect.” The degree 

to which such subtle abnormalities could exist within conventional populations of 

animals is not discussed. 

Humpherys et al. (2002) the evaluated gene expression in the placenta and liver tissues of 

mouse clones derived from embryonic stem cells and cumulus cells using microarray 

analyses. More than 10,000 genes were examined, with the expression of 286 found to be 

altered in clones derived from cumulus cells compared to the fertilization controls, with a 

similar level of altered expression detected in the embryonic stem cell-derived clones. 

The general concordance in the expression differences between the mouse clones from 

different donor cell types suggested to the investigators that most of the expression 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 75 

abnormalities were common to all placentae of mouse clones rather than specific to 

animals derived from one particular cell type. Consistent with their previous summary 

(2000), the authors concluded that differences in gene expression, even those that are 

highly variable, may be tolerated during differentiation and even in clones that survive. 

Sebastiano et al. (2005) noted that in single cells derived from early preimplantation 

embryos of mice developed via SCNT and in vitro fertilization, a series of genes 

important to appropriate embryonic development began transcription at approximately 

the same time in both types of embryos. Different levels of expression, however, were 

found in the nuclear transfer-derived embryos, particularly as the embryos progressed 

through development. They concluded that reprogramming was initially quickly shifted 

towards embryonic development, but that reprogramming was incomplete and inaccurate, 

particularly in the latest stages of preimplantation. 

Several studies have attempted to determine whether the expression of any particular 

gene(s) could be used as a marker to determine the developmental success of embryos 

produced via SCNT or other ARTs. Camargo et al. (2005) evaluated differences in gene 

expression in individual preimplantation bovine embryos produced via SCNT (same 

donor cell line), in vitro fertilization (IVF), or in vivo derived embryos obtained 

following superovulation, artificial insemination, and harvested, and cultured in vitro to 

reach the same degree of development as the nuclear transfer or IVF embryos. Using real 

time PCR, they studied a panel of 11 genes (including Oct4) preferentially activated at 

the maternal-embryo transition (~ the 8-12 cell stage in bovine embryos), during which 

demethylation of parental genes (or donor cell genes) largely has been accomplished and 

de novo methylation, in which transcription of embryonic genes becomes predominant. 

Also evaluated was the expression of a fibroblast gene expressed in the donor cells to 

determine whether cessation of expression of donor genes was also appropriate. The 

results indicated that the expression patterns of the 11 genes common to the IVF and 

SCNT-derived embryos were virtually indistinguishable. Further, the expression of the 

donor cell gene was appropriately turned off in the SCNT-derived embryos. Compared to 

expression levels in the in vivo derived embryos, however, all transcripts except one, 

lactate dehydrogenase, in both the IVF and SCNT-derived embryos were found at lower 

levels. They attributed the differences in expression between the in vivo- and in vitroproduced embryos to differences in culture conditions. To support this hypothesis, the 

investigators noted that the IVF and SCNT embryos exhibited similar variability in 

expression among individual embryos, but different from their in vivo counterparts. 

Miyazaki et al. (2005) compared the expression of a different set of genes from SCNTand intracytoplasmic sperm injection (ICSI)-derived 2-4 cell and blastocyst stage porcine 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 76 

embryos. The genes selected have previously been suggested as candidates as markers for 

identifying embryos that would successfully develop (Daniels et al. 2000) included two 

genes from the fibroblast growth factor family, Xist (important in X-chromosome 

inactivation), genes encoding interleukin-6 and its receptor, and c-kit ligand (another 

gene important in early embryonic development). Donor cells for the SCNT-derived 

embryos came from two different cell lines, with different degrees of success at 

developing blastocysts. Additionally, SCNT-derived embryos were developed using two 

different activation protocols. Although the percentage of embryos in which expression 

of these genes was similar between the SCNT- and ICSI-derived embryos, actual levels 

of transcripts of two of the genes (FGFr72IIIb, one of the fibroblast growth factor genes, 

and interleukin 6 receptor gene) were lower and higher, respectively, in SCNT-derived 

versus ICSI-derived embryos in one of the SCNT-activation protocols, while FGFr72IIIb 

and Xist transcripts were lower than ICSI-derived embryos when evaluating the other 

method of SCNT activation. No significant differences in gene expression were noted at 

these early developmental stages between the two different SCNT donor cell sources. No 

comparisons were made to in vivo derived embryos. It is not clear whether the differences 

between the results observed by Miyazaki et al. and Camargo et al. are due to 

experimental design, species, or the genes assayed. 

Both appropriate and inappropriate gene expression have been observed later in the 

development of fetuses, neonates, or more mature clones. Yang et al. (2005) used realtime PCR22 to compare the expression levels of three imprinted genes associated with 

growth regulation (Igf2r and Igf2) or imprinting regulation (H19) in eight tissues from 

deceased newborn calf clones, three tissue sources from apparently healthy, genetically 

identical adult bovine clones, and cattle obtained from a slaughterhouse. The deceased 

clones all exhibited signs of LOS, and exhibited abnormal and highly variable expression 

of the genes, despite being produced from one nuclear donor. The decreased levels of 

expression of Igfr2 (which inhibit fetal growth) in the deceased clones compared to 

controls were consistent with the decreased expression of the same gene noted by Young 

et al. (2001), in LOS sheep clones, but interestingly, these levels were not correlated with 

increased birth weights of the deceased clones. Expression of the three genes in the 

healthy clones was largely normal, except for Igf2 in the muscle tissue of adult clones, 

which was found to be highly variable, although lower than the reported levels for the 

newborn controls. These results are consistent with the idea that significant dysregulation 

of imprinted genes results in embryonic or neonatal death, but that those animal clones 

surviving to adulthood can be epigenetically similar to control animals. 

 

22 Real-time PCR is a technique that allows for the rapid and precise identification and quantification of 

genetic material (in this case, RNA) during the actual time that the reaction is running. 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 77 

Li et al. 2005b also used real time PCR to compare expression levels of eight 

developmentally important genes in six organs from bovine clones that within 48 hours 

of birth relative to control animals produced by artificial insemination and also 

slaughtered within 48 hours of birth. Organs that were evaluated included the heart, liver, 

kidney, spleen, lung, and brain. Aberrant and highly variable gene expression in the 

clones occurred in a tissue-specific pattern, with the heart most (five of eight genes), and 

the kidney, least (two of eight genes) again indicating the role of gene expression in the 

ability of particular tissues and organs to develop appropriately in clones. They also noted 

that organ systems could be affected independently of others, implying a stochastic 

process at work. No mention was made of whether a similar study had been performed on 

live, healthy clone births in this report. 

Finally, Archer et al. (2003a) have performed the most comprehensive study of the 

correlation between epigenetic reprogramming and live clone outcomes in a cohort of 

female swine clones. (More detailed discussions of the results of this study are found in 

Chapters V and VI). In addition to evaluating methylation in two different regions of the 

genomes of these animals and half-sibling comparators, the investigators studied the 

growth, clinical chemistry, and behavior (Archer et al. 2003b) of these animals. The 

overall degree of methylation between clones and their half-siblings was the same, with a 

small random variability in the PRE-1 SINE regions, and one CpG site in the centromeric 

satellite region. Further, the clones exhibited two patterns in specific phenotypic traits: 

one set of traits exhibited variability similar to the comparators, and another set showed 

less variability than the comparators. CpG methylation was measured in PRE-1 SINE 

(repeat sequence in a euchromatic region) and centromeric DNA (repeat sequence in a 

heterochromatic region) obtained from skin punch samples. Finally, the clones appeared 

to have grown and developed normally: no differences were observed between clones and 

their comparators with respect to growth rates, physiological measures of health, or 

behavior. 

2. Gametogenic Reprogramming 

The development mechanisms involved in gametogenic reprogramming were initially 

studied most extensively in the mouse; conservation of mechanisms involved in sexual 

reproduction are similar in all species examined to date, although the timing of events 

differs depending on the length of gestation. 

Germ cells (those developmentally destined to become gametes) are first detected as 

founder population cells at about embryonic day (E) 6.5 in the mouse. By E 7.2, 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 78 

approximately 45 primordial germ cells can be counted in the mouse embryo (Hajkova et 

al. 2002). These cells begin migration into the genital ridge (the portion of the embryo 

destined to become the reproductive organs) about 10 days after embryo formation 

(Hajkova et al. 2002, Yamazaki et al. 2003) (See Figure IV-2). Their epigenetic 

methylation status at this point resembles that of the rest of the embryo: they contain 

genomic imprints from the maternal and paternal genomes, and one of the two X 

chromosomes in female gametes has been inactivated in the somatic tissues (Surani 

2001). Once the primordial germ cells migrate into the genital ridge (the thickening near 

the kidneys of the embryo that gives rise to the ovaries and testes), however, profound 

changes in their methylation status occur. A period of rapid demethylation ensues, in 

effect “erasing” all of the epigenetic modifications that were present on the cells prior to 

their migration (Yamazaki et al. 2003, 2005). This demethylation appears to be selective 

by affecting single copy imprinted and non-imprinted genes (e.g., coding sequences), 

whereas the reprogramming of repetitive elements (whose function in the cell is not fully 

understood but is thought to be structural and regulatory) is more protected and 

incomplete. 

In describing this phenomenon, Surani (2001) states that this “mechanism also erases any 

aberrant epigenetic modifications, so preventing the inheritance of epimutations, which 

consequently occurs very rarely.” The mechanism by which erasure of the epigenetic 

markings, including demethylation, in primordial germ cells is not yet understood. Other 

“resetting” mechanisms also occur in primordial germ cells, including the restoration of 

telomere length, and repair of lesions to the coding regions of the DNA (Surani 2001). 

Random X inactivation in XX (female) germ cells also occurs during the migration phase 

of PGCs, coinciding with the timing of X inactivation in somatic tissues (reviewed by 

Avner and Heard 2001; Heard 2004). Inactivation of one X chromosome in female 

mammals is absolutely essential to compensate for the potential doubling of the “gene 

dosage” that a XX genotype would present. Although not fully understood, the process 

by which this occurs involves coating one of the X chromosomes by an RNA molecule 

itself encoded by a gene (Xist) on the X chromosome, followed by DNA methylation, and 

covalent modifications of the histones associated with the inactive chromosome. In mice, 

X inactivation first occurs in the placental trophoblast cells, where the paternal X tends to 

be inactivated by a mechanism thought to involve the expression of a maternal gene at 

the blastocyst stages that exclusively inactivates the paternal X chromosomes in the 

trophoblast cells. The end result is that the structure of the chromosome is altered from an 

active, relatively loosely coiled state to a highly condensed and transcriptionally silent 

DNA molecule (Avner and Heard 2001). 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 79 

Restoration of epigenetic modification in primordial germ cells in mice appears to take 

place several days later when the male germ line appears to acquire methylation at 15-16 

days after conception. Remethylation of the female germ line in mice does not appear to 

occur until after birth during the growth of the oöcytes, and probably continues until the 

first meiotic division (a stage in the maturation of the cells destined to become gametes in 

which the chromosome number is reduced from 2n to n) (Davis et al. 2000; Surani 2001). 

This overall process appears to be conserved in other mammals, although the exact 

timing may differ according to species. 

Although the preceding discussion has focused on methylation as the primary marker of 

imprinting, it is important to remember that there are other modifications that may 

contribute to the retention of “epigenetic memory” in germ cells whose identity and 

mechanism remain to be characterized (Davis et al. 2000; Fazzari and Greally 2004). 

3. Conclusions from Studies of Epigenetic Reprogramming 

• Inappropriate or incomplete epigenetic reprogramming is the source of the frank 

adverse outcomes and subtle anomalies that pose animal health risks in animals 

developed by SCNT or other ARTs. 

• SCNT-derived embryos must demethylate the differentiated and generally 

relatively highly methylated nuclear donors to restore totipotency. The high rate 

of failure to progress beyond the early stages of cleavage of SCNT-embryos may 

be a function of the inability to carry out that demethylation, and likely involves 

other mechanisms, some of which may involve higher-order chromatin 

remodeling. 

• In studies evaluating the differential reprogramming of trophectoderm- and ICMderived tissues, more dysregulation is observed in the trophectodermally-derived 

tissues (placental tissues) than in the somatic tissues derived from the ICM. 

Whether this disparity is a function of the more stringent requirement of 

appropriate reprogramming of the ICM-derived tissues for survival (embryos and 

fetuses with significantly altered epigenetic reprogramming simply do not 

survive) is not known. 

• Live and apparently healthy clones can exhibit some level of epigenetic 

differences relative to fertilization-derived comparators. Many of these 

differences appear to resolve as the animals age, consistent with the adaptation 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 80 

observed in clone populations studied for physiological and growth parameters. It 

is not known whether these animals are tolerant of these differences, or whether a 

“threshold” of epigenetic differences exists that has not been exceeded in the live 

and apparently healthy animals. 

B. Phenotypic Evidence for Gametogenic Reprogramming 

The initial observations confirming the biological assumption that phenotypic expression 

of underlying inaccurate epigenetic reprogramming observed in clones disappear in the 

progeny due to gametogenic reprogramming come from the studies of Shimozawa et al. 

2002 and Tamashiro et al. 2003, who demonstrated that a phenotype observed in mouse 

clones was not transmitted to their progeny. These studies have led to the conclusion that 

“Progeny of animal clones, on the other hand, are not anticipated to pose food safety 

concerns, as natural mating resulting from the production of new gametes by the clones is 

expected to reset epigenetic reprogramming errors that could persist in healthy, 

reproducing clones” (NAS 2002a). Or stating a similar conclusion “. . . epigenetic rather 

than genetic aberrations are the cause; epigenetic changes, in contrast to genetic changes, 

are reversible modifications of DNA or chromatin that are usually erased in the germ 

line” (Hochedlinger and Jaenisch 2002). This postulate can be further summarized as: 

“All epigenetic problems in the parents seem to be erased when cell nuclei go through the 

germ line” Yanagamichi (2002), and, “. . .the progeny of cloned animals will be normal” 

Fulka et al. (2004). 

In the following section, the studies that have led to these conclusions, as part of a 

summary of the utility of the mouse model for estimating risks in livestock clones are 

reviewed. It is organized by Developmental Nodes, as in the Critical Biological Systems 

Approach to evaluating the health status of livestock clones, although several nodes are 

combined to better reflect the existing mouse dataset. 

1. Phenotypic Anomalies Observed in Mouse Clones 

a. Utility of Mouse Model 

Although the subject animals of this assessment are domestic livestock clones, the use of 

the mouse as a model system provides some insights into the underlying biology of the 

cloning process and its implications for food safety, particularly for understanding the 

role of sexual reproduction in resetting residual epigenetic reprogramming errors. SCNT 

in mice was first reported by Wakayama et al. (1998) using the “Honolulu technique” at 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 81 

approximately the same time as publication of the “Dolly” paper (Wilmut et al. 1997). 

Since that time, mice have been cloned from a range of cells from embryonic and adult 

sources (reviewed by Yanagimachi 2002). The mouse model is useful because of its wellcharacterized genotypes, small size, short generation period, and shorter life span than 

larger animals. 

b. Pregnancy (Developmental Node 1) 

The key measure of the success of SCNT is the normal development, maturation, and 

reproduction of the animal clones. As with livestock, the efficiency of this process in 

mice is very low, and in the same range as livestock: approximately 0.2-3.4 percent when 

calculated from the total number of reconstructed embryos resulting in live offspring 

(Yanagimachi 2002). In mice, the rate of embryo survival is most reduced early in 

development, particularly in the days immediately before and after implantation 

(Yanagimachi 2002). Yanagimachi (2002) also found that more than 90 percent of mouse 

embryos cloned with cumulus cells had normal chromosomal constitutions, indicating 

that the poor survival rates are not due to chromosomal problems, again pointing to 

epigenetic reprogramming as the determining factor in cloning efficiency. 

Placental enlargement has been observed in almost all of the studies of mouse clones 

reported to date (Wakayama and Yanagamichi 1999; Humphreys et al. 2001; Ono et al. 

2001; Tanaka et al. 2001; Ogura et al. 2002; Yanagimachi 2002). Tanaka et al. (2001) 

performed histological examination of term placentas from mouse clones and evaluated 

the expression of a number of genes relevant to fetal development. Placentas from these 

animals were larger than from conventional controls, and exhibited histological changes 

in all three layers of the placenta (i.e., the trophoblastic giant cell, spongio-trophoblast, 

and labyrinth layers). Most of the anomalies appeared to be related to the expansion of 

the spongio-trophoblast layer, which exhibited an increased number of glycogen cells and 

enlarged spongio-trophoblast cells. Despite these morphological changes, there were no 

critical disturbances in regulation of gene expression in the placentae associated with 

term clone placentas. Unlike cattle and sheep, in which clone fetuses tended to be larger 

than comparators, the average weight of the mouse clone fetuses appeared to be lower 

than that of comparators, suggesting that a “latent negative effect from somatic cell 

cloning may occur on fetal growth, potentially due to incomplete placental function” 

(Tanaka et al. 2001). Despite the morphological changes observed in their study, Tanaka 

et al. (2001) noted that the placentas “could support full development of the fetus, 

suggesting that their functions are adequate for apparently normal fetal development” 

similar to the observation of Hill et al. (2000b) for cattle clones. 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 82 

Both Ono et al. (2001) and Ogura et al. (2002) reported morphological changes in the 

placenta of mouse clones similar to those observed by Tanaka et al. (2001). Ono et al. 

(2001) observed that increased placental size was caused by proliferation of the 

trophoblastic cells, endometrial glycogen cells, and unusually large giant cells. They also 

found limited distribution of maternal blood vessels in the spongio-trophoblast layer and 

suppressed development of the labyrinth layer. They suggested that these abnormalities 

would greatly reduce the functional capacity of the placenta and could contribute to high 

rates of neonatal death in mouse pup clones derived from somatic cells. Ogura et al. 

(2002) compared the histological findings for mouse clone placentae with those of 

embryos derived from other micromanipulation techniques, such as microinsemination, 

aggregation chimera, and pronuclear exchange. Disruption of labyrinth layer morphology 

was common to placentae from cloning and other micromanipulation techniques, whereas 

disruption of the basal layer with marked proliferation of glycogen cells was the only 

phenotype unique to cloning. 

The underlying mechanisms responsible for the observed placentomegaly are unknown, 

but Tanaka et al. (2001) cite their previous findings (Ohgane et al. 2001) of aberrant 

methylated genomic regions in placental tissues and suggest that slight disturbances in 

the expression of a number of genes, rather than a drastic change in the expression of a 

single gene, may impact on placental growth and function. Humpherys et al. (2002) 

reported that approximately 4 percent of the expressed genes in placentas from nuclear 

transfer-derived mouse clones differed dramatically in expression levels from those in 

controls. Placental size was not correlated with abnormal gene expression, indicating that 

the changes in cellular composition observed in Tanaka et al. (2001) are unlikely to 

account for the observed expression changes (i.e., changes in placental gene expression 

did not reflect changes in relative abundance of certain cell types). Ono et al. (2001) and 

Wakayama and Yanagimachi (2001) speculated that the observed placental abnormalities 

may be a function of disrupted patterns of expression of imprinted genes important for 

placental development. However, Inoue et al. (2002), using donor cells from a number of 

different sources, found that placentae of mouse clones at term were two to three times 

larger than those of controls, despite the developmentally appropriate expression of 

imprinted genes in both the placentae and fetuses of mouse clones. They concluded that 

placental genes were thus regulated by some upstream function that is independent of 

imprinting and is either dysregulated by nuclear transfer cloning itself, or by some other 

aspect of nuclear transfer. 

More recently, Ohgane et al. (2004) investigated whether placental overgrowth was 

related to the existence of aberrant DNA methylation at certain loci (and subsequent 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 83 

abnormal gene expression) in mouse clones. They identified a tissue-dependent 

differentially methylated region within the Sall3 locus that is hypermethylated in the 

placenta of all mouse clones examined. Ohgane et al. concluded, given that the 

methylation rate of the Sall3 locus correlated with the occurrence of placentomegaly in 

mouse clones, this was an example of “a genomic locus highly susceptible to epigenetic 

error caused by nuclear transfer.” 

c. Perinatal Period (Developmental Node 2) 

As in the pregnancy and parturition developmental node, mouse clones have 

demonstrated some of the same abnormalities observed in the perinatal periods of larger 

mammalian clones, including reports of perinatal deaths from respiratory problems 

similar to that observed in cattle clones (Wakayama and Yanagimachi 1999; Eggan et al. 

2001; Yanagimachi 2002). Interestingly, LOS, a relatively high frequency event in cattle 

cloning, was only evident in one mouse study (Eggan et al. 2001). 

Eggan et al. (2001) investigated whether the phenotypic abnormalities noted in mouse 

clones, such as loss of neonatal growth control, respiratory failure, and high neonatal 

mortality, were due to the effects of nuclear transfer, or instead reflected some 

fundamental characteristic of the cell(s) used as donors. Using mouse embryonic stem 

cells with either inbred or hybrid (F1) genetic backgrounds, they compared the 

phenotypes of animals created by either tetraploid embryo complementation or nuclear 

cloning. After evaluating four endpoints (embryos transferred to surrogate dam, pups 

alive at term, pups respiring after Caesarian section, and pups surviving to adulthood) the 

authors concluded that genetic heterozygosity (i.e., hybrid vigor) was crucial for 

influencing the survival of mouse clones. They further concluded that difficulties with 

neonatal mouse clone survival and respiratory competence were a function of the genetic 

makeup of the donor cell nucleus, whereas neonatal overgrowth was more likely to be a 

consequence of the nuclear transfer procedure. 

Ogura et al. (2002) reported that more than 90 percent of mouse clone fetuses that 

developed to term were mostly normal. Birth weights were not significantly different 

from controls (produced by IVF or spermatid injection), and fetal overgrowth was not 

observed. This is in contrast to the high incidence of placental enlargement observed in 

these studies (as discussed earlier in this Chapter). Of the 159 term pups, 12 had 

abnormalities: umbilical hernia (two cases), respiratory failure (six), developmental 

retardation (one), severe anemia (one), and intrauterine death shortly before birth (two). 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 84 

d. Juvenile Period to Reproductive Maturity (Developmental Nodes 3 and 4) 

The amount of information on the health status of mouse clones from postnatal 

development to reproductive maturity is limited. The finding of note within this period 

was postpubertal obesity in mouse clones reported by a single research group. 

Tamashiro et al. (2000) evaluated the postnatal growth and behavioral development of 

mice cloned from adult cumulus cells relative to control mice specifically generated to 

eliminate confounding factors associated with the effects of embryo micromanipulation, 

in vitro embryo culture, embryo transfer, litter sizes, Caesarean delivery, and pup 

placement with lactating foster mothers. No physical abnormalities were noted at birth or 

through the course of the study. Body weight at birth was not statistically significantly 

different between clones and controls. Beginning at approximately 8-10 weeks, however, 

the body weights of the clone group were significantly higher than that of controls. The 

late onset of increased body weight in clones was distinguished by the authors from the 

LOS observed at birth in many mammalian clones. Although preweaning development of 

these mouse clones was similar to that of controls, there was a delay in first appearance 

of eye opening, ear twitch, and negative geotaxis (the ability of mice placed on a 

downward slope to turn and climb upwards). Subsequent tests of spatial learning, 

memory, and motor abilities in the same subjects did not show any deficits or long-term 

behavioral alterations. There was no significant difference in activity levels of clones 

compared to controls up to 180 days of age. The authors concluded that the cloning 

procedure did not adversely affect the overall postnatal behavior of mice. 

Tamashiro et al. (2002) further investigated the obesity phenotype in mouse clones of two 

different background strains (B6C3F1 and B6D2F1). Comparisons were made relative to 

two groups: animals manipulated in vitro similar to SCNT-derived animals (in vitro

embryo manipulated, or IVEM, mice), and stock (conventional) control mice. At birth, 

animals derived from in vitro manipulation (mouse clones and IVEM mice) were both 

heavier than stock control mice. Clones and IVEM mice gained about the same amount 

of weight over the next eight weeks, after which time the clones became significantly 

heavier than either IVEM or stock mice. Clones continued to weigh more than controls 

throughout their lives, unlike control animals whose body weight peaked at 

approximately 18 months of age. The increased body weight was independent of the 

strain of mouse used as the nuclear donor. Although mouse clones ate more than the 

IVEM mice, they consumed approximately the same amount of food as the stock mice. 

All animals lost the same percentage of baseline body weight when deprived of food, and 

all animals compensated by increasing consumption when it was returned. Carcass 

analysis showed that clones had more body fat than either the IVEM or stock mice. 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 85 

Mouse clones had increased plasma levels of leptin and insulin than either control group, 

whereas plasma corticosterone levels in mouse clones did not differ significantly from the 

control groups. 

The authors concluded that mouse clones are truly obese and are not simply larger than 

controls. The process of in vitro culture appeared to be a factor in body weight, given that 

both the IVEM mice and clones were significantly heavier than controls. Further, the 

clones had more carcass fat than the IVEM mice, suggesting that some aspect of the 

somatic donor cell or the nuclear transfer technique may be a causative factor in the 

development of obesity. Faulty epigenetic programming was proposed as a possible 

mechanism responsible for the obesity phenotype observed in these clones. 

Further study by Tamashiro et al. (2002) to determine whether a malfunctioning leptinmelanocortin system was involved in the observed obesity, involved administering 

melanocortin 4 receptor (MTII) and leptin, known suppressors of intake, to mouse clones 

and examining food intake. Inui (2003), who analyzed the results of Tamashiro et al. 

(2002) in context of knowledge gained of the leptin-melanocortin system from studies in 

rodent models of obesity and human obesity, agreed that the phenotype observed in 

mouse clones is unique, is not due to defects in the leptin-melanocortin system, and may 

be attributable in part to cloning procedures. Tamashiro et al. (2003) provides a more 

thorough discussion of the role body weight regulatory systems may play in this 

phenotype, but concludes that the mechanisms for the observed obesity remain to be 

elucidated. Inui (2003) proposed that inappropriate placentation may be at least partially 

responsible for the obese phenotype. This opinion is based on observations in other 

species, including humans, indicating that decreased intra-uterine nutrient levels can have 

significant repercussions on later human health. For example, diabetic human mothers 

have been observed to have births that result in large placentae, altered birth weights, 

respiratory distress syndromes, and subsequent obesity and diabetes in offspring (the 

“thrifty-phenotype” hypothesis) (Hales and Barker 2001). 

To determine whether the obese phenotype was likely due to events in the cloning 

process, or the result of a genetic mutation, Tamashiro et al. (2002) mated male and 

female mouse clones and found that the offspring did not appear to be obese, nor did they 

have the enlarged placentas commonly found in mouse clones. Obesity was, therefore, 

not transmitted through the germline, indicating to the authors “that epigenetic 

modifications that occur during the cloning procedure are eliminated, or ‘corrected’ 

during gametogenesis.” The authors further proposed that “reproduction by natural 

mating may be recommended as soon as offspring with specific desired traits are 

produced by cloning.” 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 86 

With respect to other possible health outcomes in this developmental period, Ogura et al. 

(2002) reported that more than 90 percent of mouse clones reached puberty when nursed 

by good foster mothers, a rate not significantly different from that of microinseminationderived mice. In this study, cloning did not appear to have any adverse effects on 

reproductive performance. Of the 25 animals studied, no cases of complete sterility were 

observed; two female clones delivered only one litter and then became sterile for 

unknown reasons. No further details were provided. 

e. Maturity and Aging (Developmental Node 5)23

Given that one of the advantages of using the mouse model to study SCNT is the 

relatively short life span of mice compared to livestock animals, the impact of SCNT 

cloning on maturity and aging of animal clones has been examined in several reports. 

Ogonuki et al. (2002) followed weight gain, serum biochemical values, and lifespan in a 

group of 12 male mouse clones derived using immature Sertoli cells as donors, and 

compared them to the same values from male mice with the same genetic background 

derived from natural mating or spermatid injection. At one year after birth, weight gain of 

mouse clones did not differ from that of natural mating controls. Of the 16 serum 

biochemical values measured at 3 and 14 months of age, only lactate dehydrogenase 

(LDH), and ammonia (NH3) were significantly higher in clones than in control mice. 

Clone survival rate, however, was significantly different from the two control groups. 

The first death in the clone cohort occurred 311 days after birth, and 10 of the 12 animals 

died before day 800. Histopathological examination of necropsy samples of six of the 

mouse clones revealed severe pneumonia (6/6), extensive liver necrosis (4/6) and tumors 

(leukemia and lung cancer, 1/6 each). Elevated serum LDH and ammonium levels were 

consistent with liver damage. 

Immune function also was investigated by Ogonuki et al. (2002) in a different group of 

animals derived from Sertoli cells. In mouse clones as early as 4-5 months of age, 

antibody production following injection of live bacteria was significantly reduced relative 

to age- and genotype-matched controls. Phagocytic activity was also lower than controls, 

although the difference did not reach the level of statistical significance. 

Ogura et al. (2002) provided additional information on the same mouse clones. The 

longest surviving clone died at 857 days of age, with the 50 percent survival point of the 

mouse clones at 550 days, relative to the 1,028 days for the naturally mated control 

 

23 For a discussion of telomeres and their possible role in aging, see Chapter V. 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 87 

animals. The average lifespans of the two control groups (natural mating vs. spermatid 

injection) were not significantly different. The authors suggested that the major cause of 

early death was related to dysfunction of the respiratory system. Necropsy results showed 

that all six examined clones had severe pneumonia that resulted in destruction of alveolar 

structures throughout the entire lobes. Given that the animals were maintained in a 

pathogen-free environment, and the observed reduced immunocompetence, the authors 

suggested that the respiratory effects were caused by chronic infection by opportunistic 

organisms that are usually asymptomatic in immunocompetent mice. Interestingly, the 

early pneumonia-associated death of mouse clones was restricted to mice of a specific 

genetic background (B6D2F1). Clones of other genotypes exhibited neither early death 

nor severe pneumonia. 

In his overview of mouse cloning, Yanagimachi (2002) reported that in his laboratory’s 

experience, mice cloned with adult cumulus cells, tail-tip cells, and embryonic neural 

cells generally had normal life spans with no serious health problems before death except 

for the postpubertal obesity as described by Tamashiro et al. (2000, 2002). In reviewing 

the Ogonuki et al. (2002) data, Tamashiro et al. (2003) stressed the importance of 

considering the age and type of donor cell used in the animal clones, as they may 

influence the health status of the animal clone later in life. This is especially important in 

attempting to extrapolate data to other mouse clones, or other animal clones. Tamashiro 

et al. (2003) cite the immature Sertoli cells used by Ogonuki et al. (2002) as possibly 

harboring defects that would result in adverse effects such as the observed hepatic failure 

and immune incompetence. Tamashiro et al. (2003) summarized their own experience 

with mouse clones, observing that histopathology at the time of death of their cumulus 

cell clones indicated that most died of conditions associated with normal aging, and that 

the lifespan of their clones was comparable to animals followed by the National Institute 

of Aging. 

2. Conclusions from Phenotypic Studies of Gametogenic Reprogramming in 

Mouse Clones and their Progeny for Reprogramming in Domestic Livestock 

Clones and their Progeny 

• Mouse clones offer insights into physiological mechanisms that may be 

perturbed in animal clones, and provide evidence that certain epigenetic 

changes may lead to common anomalies in livestock clones. Placental 

enlargement, an outcome observed in cattle and sheep clone pregnancies, also 

has been observed in mouse clone pregnancies and appears to be linked to 

dysfunctional reprogramming of cells of trophectodermal origin. Fetal size, on 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 88 

the other hand, does not appear to be increased in the animals with placental 

enlargement, and in fact, appears to be decreased in mouse clones. 

• The mouse literature also confirms that the genetic make-up of the donor cells 

is critical in the development and growth of the animal clone, and that cloning 

methodology (e.g., in vitro culture conditions, effects of micromanipulation, 

methods of oöcyte activation, technical skill) may also have a significant 

effect on cloning outcomes (see also Chapters V and VI). 

• At this time, it is not possible to say whether the life span shortening observed 

in one strain of mouse clones will be observed in other species of clones. The 

shortened life-spans of mouse clones appear to be due to chronic alterations in 

metabolism, while the only observed early deaths of livestock clones appear to 

be due to more acute phenomena. Nonetheless, it is too early to make a 

definitive judgment on longevity, as most domestic livestock clones have not 

yet begun to approach even the midpoint of their natural life-spans (See 

Chapters V and VI). 

• Clones are not the only animals that exhibit differences in epigenetic 

programming relative to their genetic antecedents. There are examples of 

fertilization-derived embryos responding to dietary levels of methyl donors in 

their dam’s diets resulting in offspring whose phenotypes differ significantly 

from their parents. Although the cited case provides a clear molecular 

correlation between the exposure and outcome, it is important to remember 

that epigenetic markers are reversible by “nature’s design,” and are intended 

to help provide organisms with multiple, interactive mechanisms with which 

they may adapt to environmental challenges. 

The most important implication of the mouse clone literature for domestic livestock 

clones is the observation that anomalies noted in clones are not transmitted to their 

progeny. The obese phenotype, for example, is not transmitted to progeny of those 

clones, and progeny of mouse clones appear to be normal and healthy. This observation is 

consistent with the biological assumption that gametogenesis effectively “re-sets” 

epigenetic markings, and allows for the appropriate development of normal organisms 

(i.e., sexual reproduction). It is also consistent with the limited but consistent 

observations of healthy, fully functional progeny born to domestic livestock clones. Thus, 

the empirical evidence supports the assertion that “Progeny of animal clones, on the other 

hand, are not anticipated to pose food safety concerns, as natural mating resulting from 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 89 

the production of new gametes by the clones is expected to reset epigenetic 

reprogramming errors that could persist in healthy, reproducing clones” (NAS 2002a). 

C. Implications of Epigenetic Reprogramming for Animal Health and Food 

Consumption Risks 

The Center assumes that if clones were to pose food consumption risks, the only 

mechanism by which those risks could arise would be from inappropriate epigenetic 

reprogramming, similar to those observed for other ARTs. It is important to note that the 

genes that are being dysregulated are the “normal,” naturally present genes that comprise 

the animal’s genome, and have not been introduced via recombinant DNA techniques 

from other sources (i.e., these are not transgenic or genetically engineered animals). 

• Anomalous epigenetic reprogramming is observed at the global genomic and 

individual gene level in clone embryos and fetuses, and in similar developmental 

stages of animals produced using ARTs with significant in vitro culturing 

components. Various factors influence the success rate of SCNT and these other 

ARTs, including the source of the donor cells and oöcytes, culture medium, and 

factors that have not yet been identified. Many of these anomalies are lethal, as 

demonstrated by the low success rate of IVF and the even lower success rate of 

SCNT. 

• Because abnormalities arise from the dysregulation of intrinsic genes, adverse 

outcomes that would likely be expected in clones and animals derived via other 

ARTs are those that result from the inappropriate development of tissues and 

organs. For example, it would be reasonable to expect both overgrowth 

phenomena, and the poor development (aplasia or hypoplasia) of tissues and 

organs. Examples of outcomes that affect the health status of animal clones are 

presented in detail in Chapters V (Animal Health) and Appendix C, and those that 

may have an impact on food consumption risks are described in Chapter VI. 

 

• The studies that have evaluated epigenetic reprogramming of live, healthy clones 

indicate that although there is some variability between clones and their 

fertilization-derived counterparts, clones are capable of carrying out sufficient 

methylation-based reprogramming (and other coordinated functions) to allow for 

survival. Molecular analyses reveal relatively small methylation differences, and 

either the animals are tolerant of such differences, or that the epigenetic 
Chapter IV: Epigenetic Reprogramming: Implications for Clones and Progeny 90 

differences are below the threshold that poses observable adverse health 

outcomes. 

• It may be, as many have suggested (Wilmut 2002; Jaensich et al. 2004), that no 

clone is completely “normal” with respect to its epigenetic profile. Although this 

is an important point for assessing the overall safety of the cloning process for any 

particular species, the relevance of “epigenetic normality” to food consumption 

risks is unclear. Further, because similar abnormalities have been noted in animals 

produced using other ARTs, the issue of defining normality becomes significantly 

more complex. It may be that normality encompasses a range on a continuum, and 

that animals that are healthy, meet appropriate developmental and behavioral 

milestones, and reproduce to bear healthy young are “normal,” regardless of their 

epigenetic status. The most compelling conclusions that can be made about food 

consumption risks, then, are drawn from assessments of the health status of the 

animals and the composition of food products derived from them, and not from 

gene expression studies. 

• Progeny of animal clones, on the other hand, are not anticipated to pose food 

safety concerns, as natural mating resulting from the production of new gametes 

by the clones is expected to reset even those residual epigenetic reprogramming 

errors that could persist in healthy, reproducing clones (Tamashiro et al. 2002; 

Yanagimachi 2002; NAS 2003, Fulka et al. 2004). 
 

Chapter V: 

Animal Health Risks
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Animal Cloning: A Risk Assessment DRAFT

Chapter V: Animal Health Risks 

A. Potential Hazards and Risks to Animals Involved in Cloning 

This analysis identifies hazards and characterizes risks to animals involved in the somatic 

cell nuclear transfer (SCNT) procedure in the context of other assisted reproductive 

technologies (ARTs) in use in current US agricultural practice. Although hazards have 

been identified in the literature, a systematic assessment of potential risks is difficult, due 

to the relative newness of the technology, and the variability in outcomes among 

laboratories and species cloned. This section reviews the publicly available information 

and applies existing knowledge of animal biology and agricultural practices to cast that 

information in a risk context. This chapter also identifies information gaps that when 

filled may provide a more complete understanding of the risks to animals associated with 

SCNT technology. 

In the course of developing this overall assessment of risks associated with SCNT, CVM 

decided to rely on information that is publicly available. While increasing the 

transparency of the risk assessment, this limits the analyses to reports in peer-reviewed 

journals, or data that have been made available to the Center with express permission of 

the submitter for data to become available to the public with the release of this risk 

assessment. 

 

Because of the diversity of approaches in the peer-reviewed studies, CVM has relied on 

various ARTs including an earlier type of “cloning” called blastomere nuclear transfer 

(BNT) for context. Current agricultural statistics also are used to provide readers with a 

frame of reference for these technologies (see Appendix B). Outcomes for various ARTs 

are located in Appendix C. Peer-reviewed reports of primary findings were used as 

references for SCNT, while some recent reviews of artificial insemination (AI), embryo 

transfer (ET), and in vitro produced embryos (IVP), as well as primary data reports, were 

employed as references for the older ARTs. 

Most of the studies on SCNT and other ARTs that are of utility for identifying and 

assessing risk to animals, and that make up the subject of this Risk Assessment are in 

ruminants.24 Cattle studies are the most abundant, followed by sheep, swine (a nonruminant species) and goats. Peer-reviewed research reports on these four species, with 

supplemental data from studies in mice, primarily have been used as the basis for this 

 

24 Ruminants are animals with a complex or compartmental stomach, such as cattle, sheep, and goats. 
Chapter V: Animal Health Risks 94 

Animal Cloning: A Risk Assessment DRAFT

assessment. Additionally, CVM evaluated veterinary records, blood clinical chemistry 

and hematology, and urinalysis provided by two private firms: (1) Cyagra, Inc. provided 

data on 134 individual cattle clones ranging from birth to approximately one and a half 

years of age (Appendix E); and (2) Viagen, Inc. provided data on 11 swine clones and 

402 progeny of swine clones through slaughter age (Appendix F). Additional unpublished 

data were provided by several sources, in the form of veterinary records, blood chemistry 

and hematology, and reproductive performance on small groups of cattle and swine 

clones. These data are reproduced in their entirety in this chapter. 

Publications from peer-reviewed journals were searched for information relating to health 

of surrogate dams, animal clones, and clone progeny. Whenever possible, data on 

contemporary comparators have been used to provide reference rates for purposes of 

comparison. Where comparisons were not made within a study, the historical literature 

and other available databases (e.g., USDA National Agricultural Statistics Service 

(NASS25) or National Animal Health Monitoring Service (NAHMS26)) were searched for 

applicable comparative information. For example, Table V-1 (Survival Rates of LiveBorn Bovine Clones and Comparators) presents data on survival rates of clones and 

comparators, drawn from both contemporaneous comparators and historical datasets. 

Descriptions of how other data were analyzed are described in Appendix E (Cyagra 

Data), Appendix F (Viagen Data), and Appendix H (Comprehensive Veterinary Exam 

and Its Interpretation). 

B. The Critical Biological Systems Approach to the Analysis of Clone Animal 

Health: Cattle, Swine, Sheep, and Goats 

1. Pregnancy and Parturition (Developmental Node 1) 

Pregnancy is a remarkable time in mammalian development. A carefully orchestrated and 

incompletely understood sequence of changes in both the pregnant female and 

developing embryo/fetus must occur to produce a successful outcome: a healthy newborn 

and mother. Despite this complexity, most pregnancies in domestic livestock proceed 

normally and result in healthy offspring. 

Criticisms of cloning point to the “inefficiency” of the process, which is often translated 

to mean that successful outcomes are relatively uncommon (Wilmut 2002). Reports of 

early pregnancy loss or later-term spontaneous abortion of embryonic and fetal clones are 

 

25 http://www.nass.usda.gov/census/census02/volume1/us/index1.htm 

26 http://www.aphis.usda.gov/vs/ceah/ncahs/nahms/index.htm 
Chapter V: Animal Health Risks 95 

Animal Cloning: A Risk Assessment DRAFT

frequently cited in the literature (Le Bouhris et al. 1998; Kishi et al. 2000; ChavattePalmer et al. 2002; Lee et al. 2004). Loss due to defects in the embryo or failure to 

implant in the uterus of the surrogate dam does not pose a hazard to the dam at this early 

stage. Rather, the female simply resorbs any embryonic tissues and returns to cycling 

(Merck Veterinary Manual Online 2005). Mid- and late-term spontaneous abortions may 

be hazardous to surrogates if they are unable to expel the fetus and its associated 

membranes, possibly resulting in metritis (uterine infection), retained fetal membranes (in 

which the placenta is not expelled), or a mummified (dead, desiccated) fetus. Other 

complications can occur during pregnancy and labor that may pose a risk to both the 

pregnant female and the fetus. Developmental Node 1 examines the causes and frequency 

of pregnancy complications, and the relative risks to both the female and fetus, using 

other ARTs for comparison where such data are available. 

It is important to note that there are a number of external factors (management, 

environment) that can influence pregnancy outcomes, which are not related to breeding 

method. In evaluating any ART, including cloning, the potential impact of external 

influences should be considered before assigning the cause of pregnancy loss to the 

technology itself. For example, stress is an important risk factor in the loss of any 

pregnancy, particularly in the preimplantation phase (before the embryo attaches to the 

uterine lining). Disease, under-nutrition, and severe environmental conditions (e.g., high 

ambient temperature) are stressors known to interfere with animal fertility and embryo 

survival (Lucy 2001; Merck Veterinary Manual Online 2005). In these cases, the risk to 

the pregnancy is directly related to those stress factors, not the technology used, and must 

be mitigated in order for normal reproduction to resume. 

Another factor to consider is the methodology used in the SCNT process. A review of the 

literature suggests limiting in vitro manipulation of the embryo may improve the chances 

for successful pregnancy outcomes. Many of the abnormalities reported in cattle and 

sheep pregnancies have not been noted in goats or swine carrying SCNT clones. Of the 

reports reviewed for this assessment, goat embryos were only cultured through the first or 

second cleavage stage (less than one day in culture) before transfer to the recipients 

(Keefer et al. 2002), compared with sheep and cattle, whose embryos were generally 

cultured to the blastocyst stage (seven to eight days in culture) prior to transfer. Walker et 

al. (2002) reported success after only brief in vitro culture of swine embryos (1-3 hours 

after activation) before transferring to recipients. Onishi et al. (2000) also reported the 

successful birth of SCNT pigs following culture to the 2 to 8 cell stage (one or two days 

in culture), while none of the embryos cultured to the blastocyst stage developed to term. 

In contrast, Viagen, Inc. has indicated that they have had greater success recently 
Chapter V: Animal Health Risks 96 

Animal Cloning: A Risk Assessment DRAFT

transferring swine clone blastocysts (5 days in vitro culture) into surrogate dams (see 

CVM Memorandum II at www.fda.gov/cvm/cloning.htm). 

Abnormalities in cattle and sheep clones may result from incomplete reprogramming of 

the donor nucleus. As noted in Chapter IV, epigenetic reprogramming occurs at different 

times in embryos in different species, possibly in relation to gestation length. Despite that 

observation, it is interesting to note that although goats and sheep have the same gestation 

length (about five months), abnormal pregnancy outcomes are frequently reported with 

SCNT sheep, whereas SCNT goats have had relatively few problems reported (Wells et 

al. 1998b; Young et al. 1998; Ptak et al. 2002; Baguisi et al. 1999; Keefer et al. 2002; 

Reggio et al. 2001). It is important to note that epigenetic remodeling has been studied 

primarily in mice, swine, and cattle, and that very little is known about the timing and 

extent of reprogramming in small ruminants. 

The biology of placental attachment also may account for differences among pregnancy 

outcomes in the species evaluated in this risk assessment. In contrast to ruminants with a 

“cotyledonary” (cotyledon27) type attachment via placentomes (see discussion below on 

this type of fetal attachment to the uterine lining), swine have what is classified as a 

“diffuse” type of placenta where fetal attachment occurs over the entire surface of the 

placenta and uterine lining (Hafez and Hafez 2000). This gross morphologic difference in 

fetal attachment may influence outcomes of clone pregnancies in the ruminant vs. swine 

species. 

2. Perinatal Period (Developmental Node 2) 

The perinatal period (from initiation of labor through approximately one week post 

partum) is one of the most critical times in the lives of all young animals. Several studies 

(reviewed by Moore et al. 2002) noted that 75 percent of mortality from all causes for 

naturally produced and AI beef calves occurred within the first seven days of life. 

The process of labor and birth can be as stressful on the neonate as it is on the dam, 

particularly if complications arise during the process. The newborn must begin breathing 

almost immediately after birth, either spontaneously or with stimulation from the mother 

or human attendant. For ruminant animals, as for other herbivores, it is instinctive for the 

 27 Cotyledons are the structures in ruminant placentae that form contact points between the fetal-derived 

placental tissues with the maternal caruncles (attachment points) of the uterus to form the functional 

units called placentomes. Placentomes allow for the passage of gases and nutrients from the dam to the 

developing fetus, as well as the removal of waste products from the fetus to the dam’s blood stream, 

for final elimination. 
Chapter V: Animal Health Risks 97 

Animal Cloning: A Risk Assessment DRAFT

newborn to attempt to stand within the first 5-15 minutes after birth, and to suckle shortly 

thereafter. Swine are less mature at birth than most other farm livestock, and although 

they are able to walk and nurse almost immediately after birth, they are not able to 

control their body temperature (known as thermoregulation) for the first 10 to 14 days of 

life, and generally require supplemental heat. 

In mammals, neonates have little endogenous immune protection from disease during the 

first few weeks to months of life. Young mammals are dependent on antibodies 

transmitted from their dams either through the placenta or by consumption of colostrum 

(the antibody- and nutrient-rich first fluid secreted by the mammary glands after birth 

preceding the production of true milk). The process of providing immunity to the 

offspring in this manner is called passive transfer of immunity. In ruminants and swine, 

the principal means of this transfer is through colostrum. In species where this form of 

transfer predominates, the neonate must consume colostrum as soon after birth as 

possible to insure intestinal absorption of functional immunoglobulins, large proteins 

which contain antibodies (Merck Veterinary Manual Online 2005). Within approximately 

48 hours after birth (although this may vary among species), the neonatal intestine loses 

the ability to absorb large, functional proteins, and the opportunity for this method of 

immune transfer is lost (Donovan 1992). 

3. Juvenile Developmental Node (Developmental Node 3) 

Another critical period in the lives of young mammals is immediately post-weaning to 

approximately six months of age. In general, health and survival of any young animal 

post-weaning is dependent on management conditions. Relatively little information has 

been published in the peer-reviewed literature on health and survival of animal clones 

during this developmental node. As previously discussed, one clone producer has 

supplied data (Cyagra, Inc.), including health records and laboratory measurements that 

have been evaluated along with the published literature; these may be found in 

Appendix E. 

Age at weaning varies among species, breeds, and individual farm management. Swine 

are typically weaned at about 21 days of age, but may be weaned as early as 10 to 

14 days. Sheep and goats may be weaned between 8 and 12 weeks of age. Dairy calves 

typically receive milk replacer (after colostrum consumption is complete) until 28 to 60 

days, when they are weaned to solid feed. Beef calves may remain with their dams and 

continue to nurse for four months or longer. 
Chapter V: Animal Health Risks 98 

Animal Cloning: A Risk Assessment DRAFT

Weaning is a period of stress for all developing animals. Weight loss is common during 

weaning as the young animal must compensate for the loss of a primary source of 

nutrition and adapt to what previously may only have been offered as a supplement. 

Changing diet can induce scouring, particularly if it is done abruptly. Diarrhea is a 

common ailment in all young mammals, and can be serious, resulting in dehydration and 

death if not treated in a timely manner (Merck Veterinary Manual Online 2005). In 

addition, between two and six months of age in ruminants, or as early as 21 days in 

swine, maternally derived immunity wanes, and the young animal must depend on its 

own immune system. In some animals, such as beef cattle, this may occur concurrently 

with transportation stress when they are sold to feedlots or stocker operations, resulting in 

relatively high losses. 

4. Reproductive Development and Function Node (Developmental Node 4) 

Due to the complexity of the reproductive system, careful attention was directed to 

reports of puberty and reproductive function in clones in order to determine whether 

cloning had perturbed this delicately balanced system. Data from this stage of 

development in animal clones are sparse, however. 

In conventional cattle, inappropriate intrinsic, nutritional, and environmental factors have 

been shown to adversely influence reproduction in both male and female conventional 

animals. Under- and over-nutrition can influence the age at puberty and, particularly in 

the case of under-nutrition, can disrupt the normal estrous cycle. Environmental stressors 

such as extreme heat or cold can also suppress normal cycling and estrous behavior in 

females and reduce fertility and libido in males (Lucy 2001). Derangements in metabolic 

pathways, such as hypothyroidism, genomic disorders manifesting as freemartins28 and 

hermaphrodites,29 as well as congenital anomalies such as hypospadius30 can also result 

in reproductive failure (Merck Veterinary Manual Online 2005). 

Considerable differences exist among species and even among breeds within a species for 

age at puberty. In cattle, puberty is related to body weight, and a heifer will achieve her 

first estrus when she reaches approximately 65 percent of her adult body weight. 

Depending on management, then, heifers will typically begin cycling between 10 and 13 

months of age. Goats and sheep mature at a younger age, with first estrus typically 

 28 Freemartin -- reproductive tract hypoplasia (infantile uterus, not developing appropriately with growth 

of the rest of the calf, failure to respond to puberty). 29 A hermaphrodite is an animal with ambiguous genitalia, typically a penis with ovaries or a vulva with 

testicles. Sometimes this abnormality is not obvious. 30 Hypospadius is a condition where the urethra exits the penis on the ventral aspect of the glans penis 

and not at the tip of the penis where it is supposed to exit. 
Chapter V: Animal Health Risks 99 

Animal Cloning: A Risk Assessment DRAFT

occurring between seven and eight months. Dwarf goat and sheep varieties may mature at 

a much younger age. Nigerian Dwarf goats, such as those used in the Keefer et al. 

(2001a) study, mature as early as 4 months. Swine also mature sexually at a relatively 

young age, and gilts typically begin cycling between 6 and 8 months of age. Male 

animals generally reach sexual maturity at similar ages to females of the same breed and 

species. 

In female animals of agricultural species, the estrous cycle is typically 21 days in length, 

although some variation exists among species. For example, the estrous cycle in sheep is 

only 17 days. In cattle, both males and females are fertile year round, although fertility 

may be decreased during parts of the year in regions with hot, humid climates. Sheep and 

goats originating in temperate zones are seasonal breeders, becoming fertile in response 

to decreasing day length. Breeds of sheep and goats that originated in the tropics are less 

sensitive to day length, and some are fertile year round. Swine, like cattle, are year-round 

breeders. A cow’s gestation is approximately nine months, with some breeds having 

slightly shorter and others having slightly longer gestations. Sheep and goats have 

gestations lasting approximately five months, with less variation among breeds. In swine, 

gestation is approximately four months. 

With the exception of parturition, the reproductive period is characterized as low risk for 

the general population of healthy, properly managed agricultural animals. By this point in 

the animals’ growth the immune system is fully developed, and typically assisted by 

vaccination and parasite control practices. As previously noted, however, heifers are at 

greater risk of dystocia compared to older cows, largely because they are less than mature 

size at the time of their first calving. Although it is common practice to select sires with 

records of producing low birth weight calves (“calving ease”), dystocia continues to be a 

hazard for heifers. Dystocia is less of a concern in animals that typically bear multiple 

young, such as swine, as individual fetuses in multiple-fetus pregnancies are usually 

small compared to single births. 

5. Post-Pubertal Maturation and Aging (Developmental Node 5) 

Maturity and aging in food animal clones have not been studied extensively due to the 

relatively short time that cloning has been practiced. Common practice among 

conventional animals kept for breeding stock indicates that males may be kept to a later 

age than females, as they generally continue to be fertile for a longer period. Thus, highly 

valued males would continue in the herd as long as adequate quality semen was still 

being collected. When fertility of females declines, they are typically sold for slaughter, 
Chapter V: Animal Health Risks 100 

Animal Cloning: A Risk Assessment DRAFT

regardless of age. This decline in fertility generally occurs well before the animal shows 

other signs of aging or age-related disease. 

a. Telomere Length as an Indicator of Aging 

Studies have suggested that telomeres, long strands of repetitive DNA that “cap” the ends 

of chromosomes, are the “biological clock” that controls aging (Lanza et al. 2000, Betts 

et al. 2001). In all eukaryotic31 cells, the terminal ends of chromosomes are capped by 

short, repetitive sequences of noncoding DNA that are repeated up to many kilobases in 

length, in conjunction with specific binding proteins. Telomeres play a role in 

chromosome stability, protecting DNA from digestion by exonucleases (enzymes that 

attack the ends of chromosomes), facilitating attachment of chromosome ends to the 

nuclear envelope, ensuring proper segregation of chromosomes during replication, and 

ensuring the full replication of coding DNA during cellular divisions (Kuhholzer-Cabot 

and Brem 2002). 

Although the DNA in chromosomes is generally double stranded along its length, the end 

of the chromosome, or the telomere, differs in that it consists of a single-stranded 

overhang (called a lagging strand) of variable length that forms a loop. Conventional 

DNA polymerases (enzymes that replicate DNA) cannot replicate the extreme 5′ ends of 

chromosomes. Instead, these lagging strands are replicated in a series of fragments, rather 

than as a continuous strand. Each fragment is “primed” by a short sequence of RNA and 

the gaps between fragments are filled in by DNA polymerase. However, when the RNA 

primer at the furthest end of the lagging strand is removed, a small gap of un-copied 

DNA if left that is not filled in by the DNA polymerase. This leads to the loss of 50 to 

200 base pairs each time the cell divides. For this reason, telomeres have been proposed 

to act as “mitotic clocks” that limit the capacity of cells to replicate through the single 

stranded region, which is interpreted as a DNA damage signal. The net effect is that at 

some critical telomere length, cell cycle progression is halted, and the cell becomes 

“replicatively senescent” or incapable of further division. Senescent cells remain viable 

and metabolically active for very long periods of time with minimal cell death 

(Schaetzlein and Rudolph 2005). 

Telomeres appear to be longest in the nuclei of early stage embryos, and begin to 

decrease in length starting in the embryonic period. Early stage embryos and 

immortalized cells in culture appear to have the capacity to rebuild telomeres through the 

 

31 In contrast to bacteria, which are classified as “prokaryotes” and have a simple structure, eukaryotic cells 

have a clearly defined nucleus containing true chromosomes surrounded by a membrane. Eukaryotes also 

contain other organelles such as mitochondria. 
Chapter V: Animal Health Risks 101 

Animal Cloning: A Risk Assessment DRAFT

action of an enzyme known as telomerase (Betts et al. 2001; Xu and Yang 2001). 

Telomerase, the enzyme responsible for telomere replication and elongation, is active 

during embryogenesis, suppressed postnatally in most somatic tissues, but remains active 

in germ cells, tumor cells, and in a subset of stem/progenitor cells (as reviewed by Xu 

and Yang 2003; Schaetzlein and Rudolph 2005). The activation of telomerase appears to 

occur about the time when the genome becomes activated in the embryo: at 

approximately the 2-cell stage in mice, or the 8 to 16 cell stage in cattle (Betts and King 

2001). The ability of SCNT embryos to rebuild telomeres may depend on species, the 

source of the donor nucleus, and culture conditions for early stage embryos (Betts and 

King 2001; Miyashita et al. 2002). 

Concerns over genetic age and potential longevity of SCNT-derived animal clones were 

first raised after a report by Shiels and coworkers (1999) who noted that telomeres from 

the first SCNT clone “Dolly,” were 10-20 percent shorter than age-matched naturally 

bred sheep (Shiels et al. 1999). Since that report, studies in animal clones have examined 

the effects of the nuclear transfer process on telomere length and telomerase activity to 

determine whether the SCNT process “resets” telomere length. Some early studies in 

cattle suggested that the SCNT process may influence cellular age and senescence. For 

example, Betts et al. (2001) noted reprogramming abnormalities affected telomerase 

activity in some early bovine SCNT embryos. In contrast, Cibelli et al. (1998) cloned 

from a late-passage cell line (after 30 passages in vitro; the lifespan of cells in vitro is 

approximately 31-33 passages). At 40 days gestation, the fetus was harvested and a 

fibroblast cell line established. These fibroblasts appeared to have an extended lifespan 

compared to the original donor cells, and underwent another 31-33 passages in vitro. 

Other studies suggest that reduction in telomere length may be more related to animal 

species, type of cells used to derive the donor cell line, or duration of time in culture 

(Shiels et al. 1999; Kuhholzer-Cabot and Brem 2002; Miyashita et al. 2002; Betts et al. 

2005). Although telomere shortening may have led to a premature aging phenotype in 

telomerase-knockout mice (Blasco et al. 1997; Rudolph et al. 1999), convincing data on 

clones addressing the issue of premature aging are not currently available. 

Telomere length variation has not been observed consistently across cloning studies or 

species. The group that produced “Dolly” stated that her telomeres were of the same 

length as the cultured mammary gland cells (from a six year old ewe) from which she 

was generated (Betts et al. 2001). Betts et al. (2001) also noted that SCNT sheep 

generated from cultured embryonic or fetal cells had telomeres 10 -15 percent shorter 

than age-matched controls. Studies in cattle clones indicated that telomere lengths differ 
Chapter V: Animal Health Risks 102 

Animal Cloning: A Risk Assessment DRAFT

among tissues within an animal, and that DNA from some tissues were more amenable to 

telomere rebuilding, while DNA of nuclei from other tissues yielded clones with 

substantially shorter telomeres. For example, Miyashita et al. (2002) have reported that 

although clones derived from epithelial cells of a 13-year-old cow and clones derived 

from the oviductal epithelial cells of a six-year-old cow had telomeres shorter than agematched controls, clones derived from muscle cells of a 12-year-old bull were similar to 

age-matched controls. Similarly, Kato et al. (2000) noted that telomere lengths in ear 

fibroblasts of a calf clone were similar to that of the 10-year-old nuclear donor bull, but 

telomeres in white blood cells of the same clone were similar to those of an age-matched 

control. 

The telomere length of goat clones derived from fetal fibroblast donor cells were shorter 

than in those from age-matched control animals (Betts et al. 2005). These authors also 

noted that progeny from goat clones were found to have shorter telomere length in 

testicular biopsies compared to conventionally derived animals, and the telomere lengths 

were intermediate to the values obtained for their clone fathers’ and age-matched control 

testes (Betts et al. 2005). This suggests that there was incomplete telomere elongation in 

the offspring of clones, although as mentioned above it is uncertain whether telomere 

length is a predictor of longevity. 

By contrast, the telomere length of sheep clones (Clark et al. 2003) and cattle derived 

from adult or fetal fibroblasts were comparable to naturally bred cattle (Tian et al. 2000; 

Betts et al. 2001; Jiang et al. 2004) or even slightly increased when near senescent bovine 

fibroblasts were used for cloning (Lanza et al. 2000). 

Using a slightly different technique for measuring telomere length, Meerdo et al. (2005) 

found no significant difference between blastocysts derived from adult bovine fibroblast 

cell lines and in vitro fertilization-produced blastocysts, but the clone blastocysts had 

longer telomeres than the two donor cell lines. They also noted detectable telomerase 

activity in oöcytes and a dramatic increase in telomerase activity at the morula stage. A 

second study in cattle and one in mice also demonstrated telomere elongation during the 

transition from morula to blastocyst in clone embryos (Schaetzlein et al. 2004). Cellular 

aging in tissue culture is also reflected by telomere shortening, and its reversal during 

SCNT was evident in a study by Clark and coworkers by the partial restoration of 

telomere length after nuclear transfer from late-passage cells (Clark et al. 2003). This and 

several other studies suggest that gametes have telomerase activity sufficient to lengthen 

the telomeres through the maturation process (Xu and Yang 2000; Betts et al. 2001; 

Meerdo et al. 2005). 
Chapter V: Animal Health Risks 103 

Animal Cloning: A Risk Assessment DRAFT

Wakayama et al. (2000) evaluated successive generations of mouse clones for signs of 

premature aging and changes in telomere length in chromosomes from peripheral blood 

lymphocytes. Female mice were reiteratively recloned to six generations (i.e., Mouse G1 

was derived from a somatic cell, Mouse G2 was cloned from a cell from Mouse G1, etc. 

for 6 generations) and four generations in two independent lines. The mouse clones (n = 

35) showed no physical signs of increased aging, and behaved normally relative to agematched controls as measured by tests of learning ability, strength, and agility. There also 

was no evidence of shortening of telomeres, as had been reported in some studies of 

livestock animal clones. In contrast, telomere length increased with successive cloning, 

although this finding may be confounded by age-related contributions or by 

characteristics of the donor cells (the cumulus cells used to produce the clones were 

found to express telomerase, suggesting that these cells may have long telomeres at the 

outset). They concluded that “telomere shortening is not a necessary outcome of the 

cloning process,” and suggested the possibility that the differences among the results 

observed in various species may be due to the selection of cells of longer or shorter 

telomere length in the different SCNT protocols. Clark et al. (2003) noted that fibroblast 

cell lines derived from fetal sheep clones had the same capacity to proliferate and the 

same rate of telomere shortening as the donor cell line from which the fetuses were 

cloned. This observation led King et al. (2006) to hypothesize that replicative senescence 

was under genetic control, and not triggered by a pre-determined telomere length. 

Recently, Yonai et al. (2005) reported on the growth and production characteristics of six 

Holstein and 4 Jersey clones (described in detail in Chapter VI). These clones were 

derived from oviduct epithelial cells and had shorter telomeres than those observed in 

naturally bred old cows (Miyashita et al. 2002). The overall success rate in terms of calf 

survival beyond the perinatal period was 4.8 percent for the Holstein group and 10.8 

percent for the Jersey group. At the time of publication of their article all of these 

remaining clones had produced two calves and were artificially inseminated and had 

conceived for a third time. The authors concluded that “reduced telomere length did not 

influence productivity between birth and 3 years of age.” 

Thus, although there have been reports of different telomere length outcomes in clones, at 

this time it is not possible to determine what the exact mechanism for telomere shortening 

is in clones, as studies have demonstrated that clones do have sufficient telomerase 

activity to return the shorter telomere lengths of the donor cells to lengths appropriate for 

normally developing embryos. Further, although some studies indicate that clones have 

shorter telomere lengths than would be expected, other clones have age-appropriate 
Chapter V: Animal Health Risks 104 

Animal Cloning: A Risk Assessment DRAFT

telomere lengths, and some appear to have longer telomeres. The most detailed study of 

clones with shortened telomeres indicates that the animals appear to be healthy and 

function normally. Finally, at this time, because most clones have not been alive for the 

full “natural” lifespan of their species, it is not possible to predict whether clones with 

shortened telomeres will exhibit premature aging. 

C. Data on Animal Health by Species 

1. Cattle 

As mentioned above, the majority of available data on health of animal clones and their 

surrogate dams is derived from studies in cattle. Survival of live-born bovine clones from 

various studies is summarized in Table V-1. Because relatively few studies included 

contemporary comparators, historical data from various references and data bases were 

also incorporated into the table to provide context. 

Table V-1: Survival Rates of Live-Born Bovine Clones and Comparators 

Reference Transgenic Status 

Surviving/Total 

Live-Born Clones 

(fraction)1

Surviving /Total 

Live- Born 

Comparators 

(fraction) 

Comments 

Batchelder 2005 None 2/8 

(0.25) 

6/6 ET 

3/3 AI 

(1.00) 

Chavatte-Palmer 

et al. 2002 None 21/21 

(1.00) 

20/20 IVF 

176/176 AI 

(1.00) 

Described in Chapter 

VI 

Chavatte-Palmer 

et al. 2004 None 36/58 (0.62) NP 

Update on animals 

generated since 1998, 

includes some 

animals from the 

2002 publication 

Cyagra 2003 None 104/134 

(0.78) NP2 

Data from complete 

comparator birth 

cohort (animals 

surviving vs. animals 

born) not available 

Gibbons et al. 2002 None 8/9 

(0.89) NP 

Gong et al. 2004 None 12/27 

(0.44) NP Gong et al. 2004 

Heyman et al. 2002 None 11/15 

(0.73) 

20/25 

(0.80) 

IVF derived 

contemporary 

comparators 
Chapter V: Animal Health Risks 105 

Animal Cloning: A Risk Assessment DRAFT

Heyman et al. 2004 None 35/50 

(0.70) 

65/68 

(0.93) 

AI derived 

contemporary 

comparators 

Hill et al. 1999 All 6/8 

(0.75) NP 

Hill et al. 2000a, 

2001a All 1⁄2 (0.50) NP 

Ideta et al. 2005 None 0/1 

(0.00) NP 

Kato et al. (1998, 

2000) None 13/24 (0.54) NP 

An additional clone 

died between the 

perinatal period and 

117 days of age 

(12/24 or 0.50 overall 

survival) 

Kishi et al. 2000 None 3/4 

(0.75) NP 

Kubota et al. 2000 None 4/6 

(0.67) NP 

Lanza et al. 2000 All 6/6 

(1.00) 

5/5 

(1.00)

IVF and ET derived 

comparators 

Lanza et al. 2001 All 24/30 

(0.80) NP 

Matsuzaki and 

Shiga 2002 None 8/13 (0.62) 

7/7 

(1.00) 

IVF and AI derived 

comparators 

Meirelles et al. 

2001 None 1/1 (1.00) NP 

Pace et al. 2002 Some 82/106 

(0.78) NP 

Powell et al. 2004 All 5/8 

(0.63) NP 

Renard et al. 1999 None 0/1 

(0.00) NP Case study on a clone of clone 

Shiga et al. 2005 None 4/8 

(0.50) NP One death associated 

with Akabane virus 

Urakawa et al. 

2004 None 8/9 (0.89) NP 

Wells et al. 2004 None 104/133 

(0.78) 

37/52 

(0.71) 

Table reflects 

survival to 3 months, 

due to unexplained 

differences in 

numbers at the 

beginning of later 

periods. Reports on 

number of calves 

delivered; unclear 

how many were 

stillborn. 

Comparators are 

progeny of clones 
Chapter V: Animal Health Risks 106 

Animal Cloning: A Risk Assessment DRAFT

Wells et al. 2003 Some 22/31 (0.71) 

11/24 (0.46) NP 

Non-transgenic (31 

calves born alive) and 

transgenic (24 calves 

born alive) listed 

separately 

Zakhartchenko et 

al. 1999a None 1/2 

(0.50) NP 

USDA/NAHMS 

19973 

(12/96 – 2/97) 

NA6 NA 0.97 

Historical data from 

beef cattle produced 

through AI and natural 

mating in commercial 

operations 

USDA/NAHMS 

2002 

(1/02 – 12/02) 

NA NA 0.98 

Historical data from 

dairy cattle produced 

through AI and natural 

mating in commercial 

operations 

Nix et al. 1998 NA NA 0.96 

Historical comparison 

from a university herd 

of beef cattle using AI 

Hasler et al. 1995 NA NA 361/428 

(0.84) 

Historical data on IVF 

derived beef calves in 

a commercial 

operation 

Schmidt et al. 1996 NA NA 13/18 

(0.72) 

Calves produced by 

IVF. Two embryos 

transferred to each 

recipient, yielding 11 

live-born twins and 7 

singles; 4 twins dead 

by 14 days. One 

singleton dead by 14 

days. 

1 Survivors through the Juvenile Period/Live births 

2 NP = not provided; data not available 

3

 Beef calves; 4 Dairy heifers 5

 NA = not applicable 

Transgenic Status: All = All of the clones cited in the publication are derived from transgenic donor cells, Some = 

Some of the clones cited in the publication are derived from transgenic donor cells, None = None of the clones cited 

in the publication were derived from transgenic donor cells. 

IVF = in vitro fertilization 

AI = artificial insemination 

ET = embryo transfer 
Chapter V: Animal Health Risks 107 

Animal Cloning: A Risk Assessment DRAFT

a. Developmental Node 1: Pregnancy and Parturition 

i. Pregnancy 

Most abortions in natural service and AI pregnancies in cattle remain undiagnosed due to 

the expense of laboratory work and the low profit margin in both the beef and dairy 

industry. Producers and veterinarians become concerned when the rate of abortion 

exceeds 3-5 percent in a herd. Many causative factors, both infectious (e.g., bacterial, 

protozoal, viral, fungal) and non-infectious (e.g., genetics, nutrition, stress, toxicity), have 

been identified (Merck Veterinary Manual Online 2005). Fetal losses later in pregnancy 

may be more common in goats and swine compared to cattle (Engeland et al. 1997; van 

der Lende and van Rens 2003; Vonnahme et al. 2002), and are not necessarily associated 

with disease (Engeland et al. 1997). 

Farin et al. (2001) stated that up to 40 percent of pregnancy losses in cattle occur between 

days 8 and 18 of gestation. A recent study (Silke et al. 2002) indicated that most 

pregnancies are lost during the same period in dairy cattle, while a smaller percentage of 

pregnancies are lost between days 16 and 42 of pregnancy (late embryonic period). Total 

pregnancy loss in moderate to high yielding dairy cattle may be as high as 40 percent 

(Silke et al. 2002). Losses at later stages of pregnancy in cattle bred by AI are estimated 

to be less than 5 percent (Thompson et al. 1998). 

Early embryo loss in other forms of ARTs may be related to in vitro culture conditions 

that may cause abnormal development and early embryo/fetal death. In a review of 

studies of in vitro produced (IVP) and clone bovine embryos, Farin et al. (2004) reported 

lowered pregnancy rates and increased rates of abortion associated with in vitro 

production. Farin and Farin (1995) compared bovine IVP embryos cultured in mixed 

media containing 10 percent serum from cows in estrus and other hormones for seven to 

eight days with embryos fertilized in vivo and collected and transferred on the same day 

via embryo transfer (ET). Pregnancy rates 53 days after transfer were higher for heifers (a 

cow that has not yet produced her first calf) receiving ET (15/19 embryos transferred; 79 

percent) compared with IVP embryos (7/19 embryos transferred; 37 percent). A study of 

beef heifers indicated that losses in the first days following embryo transfer are the most 

common (Dunne et al. 2000), with similar pregnancy rates at days 14, 30, and at term (68 

percent, 76 percent, and 71.8 percent, respectively). 

Similar to other ARTs, by far the greatest loss of pregnancies resulting from SCNT 

embryos occurs prior to 60 days gestation in cattle (Le Bouhris et al. 1998; Hill et al. 
Chapter V: Animal Health Risks 108 

Animal Cloning: A Risk Assessment DRAFT

1999 with transgenic clones; Kishi et al. 2000; Lanza et al. 2000 with transgenic clones; 

Chavatte-Palmer et al. 2002; Pace et al. 2002 using mixed transgenic and non-transgenic 

clones). High pregnancy losses during the time of placental formation suggest that 

embryonic death may be a consequence of faulty placentation, possibly due to a delay in 

chorioallantoic development, as proposed by Hill et al. (2000b) and Bertolini et al. 

(2004). Abnormal placentation may lead to a build up of wastes in the fetus and 

associated membranes, or inadequate transfer of nutrients and oxygen from the dam to 

the fetus. 

Unlike other forms of ARTs, however, SCNT pregnancy losses occur at all stages of 

gestation in cattle. Clone pregnancies have been lost during the second and third 

trimesters and have been accompanied by reports of hydrops (discussed in more detail in 

section 1.a.ii.), enlarged umbilicus, and abnormal placentae (Batchelder, 2005). Indeed, a 

major factor contributing to mid- and late-term spontaneous abortion of clones of both 

embryonic and somatic cell origin is abnormal development of the placenta (Wells et al. 

1999; Farin et al. 2001; Chavatte-Palmer et al. 2002). Normal placental development is 

essential to ensure proper exchange of nutrients and gases between mother and fetus 

(Farin et al. 2001; Bertolini et al. 2004). Placental insufficiency has been cited as a 

possible cause of fetal loss in cattle, goats and swine bred by AI or natural mating (Lucy 

2001; Engeland et al. 1997; Vonnahme et al. 2002). Studies have reported too few and/or 

abnormal cotyledons present in the placentae of sheep and cattle clones (Farin et al. 2001; 

Chavatte-Palmer et al. 2002; Heyman et al. 2002; Lee et al. 2004; Batchelder 2005). 

Although fewer in number, these abnormal placentomes are found to be larger, weigh 

more, and comprise a greater surface area for exchange than “normal” placentomes. 

Enlarged placental surface area in IVP suggests an increase in substrate uptake and 

transport capacity (Bertolini et al. 2004). Failure of epigenetic reprogramming has been 

cited in numerous studies as a likely cause of early embryo failure and abnormal 

placental development for SCNT (see Chapter IV). These early losses do not pose a 

hazard to the surrogate dam, and the net result is typically a longer than normal estrous 

cycle (Merck Veterinary Manual Online 2005). 

Lee et al. (2004) noted pregnancy rates were similar between NT, AI, and IVP at 50 days 

gestation (65 vs. 67 and 58 percent, respectively), but from that point onward NT 

pregnancies were continually lost. By day 150, only 40 percent of NT embryo recipients 

were still pregnant. There were no losses during this time period for either AI or IVP 

pregnancies. Mean fetal weights at 100 days gestation were not different between the 

three groups; however, the authors noted that more NT fetuses were two standard 

deviations above the mean weight of AI fetuses (283 ± 2 g) compared to IVP fetuses (5/6 
Chapter V: Animal Health Risks 109 

Animal Cloning: A Risk Assessment DRAFT

vs. 1/4). A similar trend was noted among fetuses examined at day 150. Fetal livers and 

kidneys were larger among NT fetuses compared to AI or IVP fetuses, and one liver and 

the kidneys from three NT fetuses were noted to have fatty infiltrations. Fatty liver was 

also diagnosed on post-mortem of one neonatal calf in the recent study by ChavattePalmer et al. (2004). 

Few detailed descriptions of placentae of cattle clones exist. Lee et al. (2004) examined 

placentae of developing SCNT fetuses at 50, 100 and 150 days of gestation. These time 

periods roughly correspond to the periods before placentome formation is complete (50 

days), shortly after complete placentome formation (100 days), and the period when 

hydrops may first be detected (150 days). The authors noted that at day 50, fetal 

cotyledon formation and vascularization initiated normally in NT fetuses, but fewer 

cotyledons successfully formed placentomes compared to AI and IVP control 

pregnancies. At day 50, 5/10 NT fetuses were noted to have very good vascularization of 

the cotyledons, compared to 2/5 AI and none of the IVP fetuses, which were said to have 

pale cotyledons. However, at day 100, the mean number of caruncles among NT 

pregnancies was lower than for either AI or IVP groups (58 ± 9 vs. 103 ± 15 and 99 ± 16, 

respectively). Although numbers of cotyledons were reduced in the NT group, total 

weight of caruncles was significantly higher in NT fetuses compared to the other groups 

at day 100, suggesting an attempt to compensate for lower numbers. The authors 

described NT placentomes as larger than AI or IVP placentomes, and having thicker, fistshaped structures compared to AI or IVP placentomes, which were typically flat and 

discoid in shape. 

Batchelder (2005) conducted a systematic histological exam of placentae collected at 

birth from seven cattle clones. She noted all clone placentae exhibited one or more 

abnormalities of varying severity: moderate to severe edema, enlarged vessels, 

adventitious placentation, and large areas devoid of placentomes. No abnormalities were 

described for the comparator placentae collected (n=9). In general, clones had fewer 

(67.4 vs. 98.3) and larger placentomes (6.05 vs. 3.84 kg) compared to the pooled means 

for AI and ET comparators, and surface area of placentomes was greater and more 

variable in placentae of clones vs. comparators. The placenta of one clone contained two 

masses comprised of fatty and connective tissue with hair, but exhibiting no bone or 

organ development. These may have derived from embryos that failed to undergo 

complete differentiation, likely due to failure to completely reprogram the donor nucleus 

to a totipotent (able to become any tissue type) state (See Chapter IV). These may pose a 

potential hazard (metritis) to the dam if the fetal membranes are not completely expelled 
Chapter V: Animal Health Risks 110 

Animal Cloning: A Risk Assessment DRAFT

at termination of the pregnancy. In this study, all clones were delivered by planned Csection, and the placentae were manually removed. 

The underlying cause(s) of the higher rate of pregnancy failure and placental 

abnormalities in SCNT compared to IVP may be related to the selection of the donor cell 

for nuclear transfer. Wells et al. (2003) noted that survival rates to term differed 

depending on cell cycle of the nuclear donor cells. Putative G0 cells (cells that apparently 

were not dividing) used for the nuclear transfer had high early pregnancy losses, but no 

losses after 120 days of gestation, and no reported hydrops. Cells that had begun to divide 

(G1 phase) had higher losses to term (21/43 pregnancies lost after 120 days gestation) and 

higher incidence of hydrops (18/43 (42 percent) of pregnancies), but higher post natal 

survival than clones from G0 cells. 

In contrast to the Wells et al. study, Urakawa et al. (2004) reported success using fetal 

fibroblast donor cells in the G1 phase. Two cell lines were used, derived from fetuses 

with the same dam but two different bulls. All embryos that survived to ≥ 6 cells (day 3) 

continued to develop to the morula/blastocyst stage by day 6. Ten of these blastocysts 

were transferred into ten recipients, resulting in nine live calves. According to the 

authors, calving was “uneventful.” Differences were noted between cell lines, in that 

three calves resulting from one of the lines tended to be heavier at birth than the six 

calves of the other cell line used (actual birth weights not provided). One of these three 

heavy-weight calves died after two days without standing. The authors do not report on 

the health or survival of the remaining eight calves beyond the first six days of life. 

Similarly, Ideta et al. (2005) compared development of embryos constructed with G1 or 

M phase (the period in the cell cycle when cell division takes place) fetal fibroblasts, and 

noted that G1 SCNT embryos had higher rates of development to blastocyst than M phase 

cells (31 vs. 16 percent). Although these results are considerably lower than those noted 

in the Urakawa et al. study, the numbers are calculated based on total number of embryos 

cultured prior to first cleavage, whereas the Urakawa et al. study calculated development 

based on embryos surviving the first three days in culture. Only five surrogate cows 

received embryos in the Ideta et al. study, of which three were diagnosed pregnant on day 

30 of gestation, and one live calf was delivered. All of the transferred embryos were 

developed from G1-phase somatic cells. The single calf died two days after birth. Health 

of the surrogate dams, method of delivery, and birth weight of the single calf was not 

reported in this study. 
Chapter V: Animal Health Risks 111 

Animal Cloning: A Risk Assessment DRAFT

ii. Parturition 

(a) Hydrops 

The set of conditions generally termed hydrops refers to abnormal fluid accumulation 

(edema) in one or more compartments of the placenta and/or the fetus itself, and are 

variously referred to as hydroallantois, hydramnios or hydrops fetalis, depending on 

where the edema occurs (Heyman et al. 2002; Merck Veterinary Manual Online 2005; 

Pace et al. 2002 (including transgenic clones)). Hydrops is estimated to occur in 1 in 

7,500 pregnancies in the general population of cattle (Hasler et al.1995). The incidence is 

higher in cattle and sheep recipients of IVP embryos, with one study estimating a rate of 

approximately 1 in 200 in IVP pregnancies in cattle (Hasler et al. 1995). 

Table V-2 presents a summary of reports of hydrops in cattle from the peer-reviewed 

literature for clone, IVP, ET, and AI pregnancies. Survival rates of dams developing 

hydrops generally were not reported. Most studies that discussed outcomes indicated that 

dams developing hydrops were euthanized. 

Not all cases of hydrops in clone-bearing pregnancies develop into a significant 

complication or threat. In an interview with CVM staff (see CVM Memorandum I at 

www.fda.gov/cvm/cloning.htm), clone producers indicated that many pregnancies result 

in some excess fluid accumulation in the fetal membranes and tissues. In most cases this 

accumulation is mild or moderate, and does not threaten the surrogate dam or calf. The 

producers interviewed for this assessment indicated that they monitor surrogate dams 

closely, beginning as early as 150 days of gestation, for any signs of developing hydrops. 

They indicated that if the veterinarian determines that hydrops is sufficiently severe to 

threaten the surrogate, the pregnancy is terminated. 

A few studies have directly compared cloning procedures with other ARTs under the 

same conditions. These studies are limited, with few clones and often fewer comparators 

from alternative technologies (Heyman et al. 2002, Matsuzaki and Shiga 2002, Lee et al. 

2004, Batchelder 2005). In one such study, Matsuzaki and Shiga (2002) compared 13 

SCNT clones with five AI and two IVP-derived calves used as controls. Five of the 13 

clones required delivery by Caesarian section (C-section), while all seven controls were 

delivered without assistance. Two cows carrying clones had to be induced at 250 days 

gestation due to rapidly expanding hydroallantois, and the calves were delivered by Csection. 
Chapter V: Animal Health Risks 112 

Animal Cloning: A Risk Assessment DRAFT

Batchelder (2005) indicated that the largest clone in that study (weighing 71.0 kg at birth) 

exhibited edema at birth, particularly in the head and neck, suggesting that it suffered 

from mild hydrops fetalis. This calf was successfully delivered at term by planned Csection, although it died three days after birth. This calf’s surrogate dam apparently was 

unharmed by the complication, although another surrogate dam was euthanized at 211 

days gestation due to severe hydrops. 

In one of the largest cattle cloning studies reported, Pace et al. (2002) estimated that 

approximately 6 percent (30/535) of all pregnancies established with SCNT embryos 

resulted in hydrops, but among pregnancies with clones that lasted beyond 60 days, the 

incidence of hydrops was 17 percent (30/178). An important consideration in interpreting 

Table V-2: Incidence of Hydrops in Cattle Surrogate Dams 

Study Transgenic 

Status 

Incidence (fraction) 

in clone 

pregnancies 

Incidence (fraction) 

in comparator 

pregnancies 

Comments 

Batchelder 2005 None 1/8 

(0.13) 

0/6 

(0.00) 

Comparators were ET (n=6) 

and AI (n=3) 

Hasler et al. 1995 NA NA 1/200 

(0.005) 

Study based on commercial 

IVP operation 

Heyman et al. 2002 None 3/20 (0.15) 

5/21 (0.24) 

0/24 

(0.00) IVP comparators 

Hill et al. 1999 All 2/8 

(0.25) NP 

Lee et al. 2004 None 2/8 

(0.25) 

0/9 

(0.00) 

4 IVP and 5 AI 

comparators. A third clone 

fetus was suspected of 

developing hydrops. All 

pregnancies terminated at 

gd 150. 

Matsuzaki and 

Shiga 2002 None 2/13 (0.15) 

0/7 

(0.00) 2 IVP and 5 AI comparators 

Pace et al. 2002 Some 30/178 

(0.17) NP Pregnancies lasting beyond 60 days 

Wells et al. 2003 Some 18/43 (0.42) 

1/6 (0.17) NP 

Pregnancies lasting beyond 

120 days. Non-transgenic 

(n=43) and transgenic (n=6) 

listed separately 

Zahkartchenko et al. 

1999a None 2/5 (0.40) NP 

NA = not applicable NP = not provided; data not available Gd = gestation day or day of pregnancy 
Chapter V: Animal Health Risks 113 

Animal Cloning: A Risk Assessment DRAFT

these outcomes, however, is that approximately 75 percent of the embryo clones in this 

study were transgenic. Heyman et al. (2002) observed that 3 of 20 (15 percent) recipients 

of fetal and adult SCNT embryos (non-transgenic) developed severe hydroallantois 

during the time from approximately six months of gestation to term. In another trial 

reported in the same paper, five cases of late abnormal pregnancies were detected among 

21 SCNT recipients (24 percent) by repeated ultrasonography, and the recipients were 

euthanized between day 155 and 233 of gestation. Severe hydroallantois was confirmed 

at necropsy and the size of the placentomes from these pregnancies was measured (142.3 

± 61.7 g vs. 46.7 ± 22.7 g for controls). No abnormalities were reported among the IVFderived pregnancies in the Heyman et al. 2002 study. 

Similarly, a recent study by Wells et al. (2003) reported a high rate of pregnancy loss of 

non-transgenic bovine fetal fibroblast clones after 120 days gestation, with hydrops cited 

as the cause of pregnancy loss in 86 percent (18/21 losses) of the cases. 

Lee et al. (2004) examined survival and development of AI, IVP and SCNT fetuses at 50, 

100 and 150 days of gestation. Although there were no significant differences in fluid 

volume of fetal membranes at day 50 or 100, total fetal membrane fluid volume was 

significantly higher in SCNT (n = 8) fetuses compared to IVP (n = 4) fetuses (8033 ± 

1800 ml vs. 5088 ± 698 ml) at 150 days gestation. For AI fetuses, mean fetal membrane 

fluid volume was 6500 ± 444 ml. The study noted the high variability in membrane 

weights and fluid volume among clone fetuses, and stated that 2/8 SCNT fetuses 

examined had particularly high allantoic fluid volumes (20 and 12 L), which were largely 

responsible for the high mean fluid volume among clones. The authors stated that these 

two cases indicated developing hydrops. The authors suspected a third SCNT fetus was 

developing hydrops, but did not provide data on this case. Fluid volumes were less 

variable among membranes of AI and IVF fetuses. 

In contrast, hydrops has only been detected in one or two cows out of 250 to 300 

transgenic clone-bearing surrogate cows, as reported in discussions with clone producers, 

suggesting that these results vary considerably among labs performing animal cloning 

(see CVM Memorandum I at www.fda.gov/cvm/cloning.htm). The producers also noted 

that hydrops occurred in IVP-derived pregnancies, but less frequently than with clonebearing pregnancies, although no actual numbers were available. The causes of hydrops 

in conventional animals are unclear. Although it is possibly related to placental 

insufficiency, not all abnormal placentas develop hydrops. In SCNT, incomplete or 

improper epigenetic reprogramming and subsequent inappropriate gene expression may 

be an important factor in placental development and hydrops (see Chapter IV). 
Chapter V: Animal Health Risks 114 

Animal Cloning: A Risk Assessment DRAFT

Lee et al. (2004) suggested that the association between excessive fetal fluid 

accumulation and renal and placental growth deregulation may indicate impairment of 

renal and placental function. “Although the placenta is the major organ regulating the 

fetal environment, the fetal kidney also plays an important role in the regulation of fetal 

arterial pressure, fluid and electrolyte homeostasis, acid base balance, and hormone 

synthesis. In ruminants, fetal urine contributes to the allantoic and amniotic fluid. 

Reports have appeared of kidney defects and impaired renal function in cloned offspring 

as well as impaired liver function in cloned mice...” 

(b) Dystocia 

Dystocia, or difficult labor, is an identified hazard for any pregnancy that goes to term. A 

common cause of dystocia is incompatibility between the size of the fetus and the pelvic 

opening through which it must pass. Although oversized offspring occur in all species, it 

is more common in animals that typically produce only one or two offspring per 

pregnancy. Other causes of parturition difficulty include malpresentation of an individual 

fetus (e.g., breech birth, head or leg out of position), or simultaneous presentation of 

multiple fetuses in the birth canal. Severe dystocia may increase the risk of retained fetal 

membranes and metritis (uterine infection), and cause damage to the reproductive tract, 

including uterine adhesions, uterine rupture and uterine prolapse, and nerve and musculoskeletal damage (Merck Veterinary Manual Online 2005). Such complications could 

compromise future reproductive capability and result in culling of the dam. Another risk 

is that dystocia may lead to an emergency C-section. Complications of emergency Csection surgery may include uterine tearing, peritonitis, infected suture line, incisional 

hernia, and respiratory and circulatory compromise from anesthesia and recumbancy. 

Stress of labor is also a complicating factor in the case of emergency C-section. 

Estimates of dystocia in natural and AI-derived bovine pregnancies range between 4 and 

6 percent. Nix et al. (1998), in a large study of 2,191 births of natural and AI bred beef 

cattle at Clemson University reported that 6 percent of births required assistance. Calf 

birth weight and parity of dam (number of times she had given birth) were the major 

factors in the incidence of dystocia. Calves heavier than 40 kg were associated with 

greater calving difficulty. Heifers were more likely to experience dystocia, despite the 

common practice of selecting sires known to produce smaller calves. Dystocia 

contributed to the increased neonatal mortality of the calves and decreased reproductive 

performance of the dams in this study. In another large study that evaluated dairy cattle, 

6.3 percent (1,749/27,713) of pregnant cows experienced dystocia (Lucy 2001). USDA 

estimates the mean dystocia risk in the general cattle population at 4 percent of 

pregnancies (USDA/NAHMS 1997). 
Chapter V: Animal Health Risks 115 

Animal Cloning: A Risk Assessment DRAFT

Rates of dystocia in surrogate dams carrying clone pregnancies are difficult to determine 

as clone producers have often elected to deliver clones via planned C-section as part of 

their animal care protocol (Wells et al. 1999; Lanza et al. 2000 using transgenic clones; 

Gibbons et al. 2002; Batchelder 2005). Planned C-section deliveries are associated with 

decreased parturition risk, and in most cases the surrogate dam recovers without ill 

effects. Although this does not eliminate the risk associated with giving birth, particularly 

in the event of hydrops, very few surrogate dams are lost, and most recover normally. 

(c) Large Offspring Syndrome 

Large Offspring Syndrome (LOS) (Table V-3) has been described as occurring at a 

relatively high frequency in clone-bearing pregnancies, and at a lower frequency in cattle 

derived from IVP and ET pregnancies, and in some cases may be related to the 

development of hydrops (Kruip and den Daas 1997; Chavatte-Palmer et al. 2002). This 

syndrome will be discussed in greater detail in a later section, as it also has implications 

for the health and survival of the newborn animal. For the surrogate dam, LOS increases 

the incidence of dystocia, frequently requiring human intervention to remove the calf 

vaginally, or by C-section, due to the inability of the dam to expel the calf without 

assistance. Reported incidences of LOS in peer-reviewed publications on cattle clones 

have ranged from as low as 1/12 (8.3 percent) (Miyashita et al. 2002) to as high as 12/24 

(50 percent) (Kato et al. 2000). Average birth weight of clones (some transgenic) of 

various cattle breeds (Holstein, Brown Swiss, Angus and Holstein*Jersey crossbreds) in 

the Pace et al. (2002) study was 51 ± 11 kg, with 54/106 (51 percent) live-born calves 

weighing more than 50 kg at birth. Given the inability to distinguish between transgenic 

and “just clone” pregnancies in the Pace et al. study, it is difficult to put these numbers 

into context with other studies of non-transgenic clones. Average birth weight of calves 

produced by AI or natural service varies depending on breed, and may range from 30 kg 

in small breed cattle to 45 kg or more in large breed cattle (NAS 1996b). 

Table V-3: Clinical Signs Associated with Calves Displaying Large Offspring Syndrome LOS) 

Fetal size > 20% above average for species/breed 

Slow to stand 

Inability to thermoregulate 

Weak or absent suckle reflex 

Large umbilicus with patent blood vessels 

Deformities of limbs (tendon contracture) and /or head 

Disproportionate or immature organ development 

Increased susceptibility to infection 

Respiratory signs: insufficient lung surfactant, failure of lungs to inflate 

Cardiovascular signs: patent ductus arteriosis, enlarged heart /ventricle, septal defects 

Hydrops 
Chapter V: Animal Health Risks 116 

Animal Cloning: A Risk Assessment DRAFT

(d) Other complications 

Although other complications associated with SCNT pregnancies have been noted, 

potential interactions with transgenic manipulation of the donor cell and predisposing 

conditions in the surrogate dam make it difficult to ascribe the complications exclusively 

to the cloning process. For example, the ketonuria32 and fatty liver associated with ketosis 

and “fat cow syndrome”33 described by Hill et al. (1999) are not only confounded by the 

existing obesity of the surrogate dams at the time of diagnosis, but also by the transgenic 

nature of the fetal clones. Cows that are obese at calving are most likely to develop fatty 

liver, and cows that develop fatty liver at calving are most susceptible to ketosis. Fatty 

liver can occur whenever there is a decrease in feed intake and may be secondary to the 

onset of another disorder. Obesity in late-gestation cattle is a commonly reported problem 

resulting in anorexia (due to reduced gut capacity), ketosis, fatty liver deposits, and 

hepatic insufficiency in pregnant cattle (Merck Veterinary Manual Online 2005). 

Wells et al. (1999) noted weak or non-existent uterine contractions, poor mammary 

development and failure to lactate in cattle carrying fetal clones. Hammer et al. (2001) 

also noted similar outcomes, but the clone was of a different species (Bos gaurus) from 

the surrogate dam (Bos taurus). Actual incidence of these complications is not known, 

but all have been reported in sheep (Ptak et al. 2002) and failure to lactate was noted in 

swine surrogate dams (see CVM Memorandum I at www.fda.gov/cvm/cloning.htm). 

b. Developmental Node 2: Perinatal Period 

i. Peer-Reviewed Publications 

In the general population of cattle and sheep, neonatal death rates are typically low. 

Overall, the estimated death rate of beef calves within 24 hours of birth (including 

stillbirths) is 3.4 percent (USDA/NAHMS, 1997). Nix et al. (1998) found that dystocia 

affected calf mortality within the first 24 hours, with mortality rates increasing with 

increasing severity of dystocia. Overall calf mortality attributed to dystocia was 4.5 

percent of all calvings in this study (2,191 births). Dystocia was the most influential 

factor on calf mortality, due to trauma of difficult labor and emergency C-section. 

 

32A metabolic disorder related to energy metabolism, where breakdown products of body fat spillover 

into the urine. 

33 Pregnant cows that are obese often reduce energy intake near the time of calving, leading to rapid 

mobilization of body fat which predisposes them to metabolic disorders such as fatty liver and ketosis, 

an inability to clear the blood stream of breakdown products of fat, known as ketone bodies. 
Chapter V: Animal Health Risks 117 

Animal Cloning: A Risk Assessment DRAFT

Dystocia was also associated with high calf morbidity (illness) in a study of 2,490 beef 

cattle herds (Sanderson and Dargatz 2000). 

Among dairy replacement heifers, the highest losses occur during the first week of life 

(1.8 ± 0.3 percent deaths for all heifer calves born alive). In dairy replacement heifers, the 

most commonly reported illnesses were due to respiratory problems and scours 

(diarrhea), with incidence of these illnesses peaking during the first two weeks of life 

(USDA/NAHMS 1994). 

Because the number of animal clones available to study is small, it is difficult to draw 

conclusions on rates of morbidity and mortality of live-born clones. However, some 

trends appear to be common across most of the studies reviewed. Early reports, beginning 

in 1998, of clone mortality rates were 50 to 80 percent (reviewed by Solter 2000). 

Survival rates have improved in some recent studies, with mortality during the first 

month of life of approximately 18 percent (21/117; Pace et al. 2002 for a cohort of mixed 

transgenic and non-transgenic clones) and 20 percent (6/30; Lanza et al. 2001 for a cohort 

of transgenic cattle), with most of the deaths occurring during the first 48 hours 

postpartum. Similarly, data supplied by Cyagra, Inc. indicate 22 percent mortality in the 

first 48 hours (30/134) among non-transgenic clone calves born between 2001 and 2003. 

(For a summary of survival rates among live-born bovine clones, see Table V-1.) 

(a) Large Offspring Syndrome 

Large Offspring Syndrome (LOS) has been described in calves and lambs produced by 

ET, IVP, BNT, and SCNT, and references describing this syndrome in the following 

section include descriptions of abnormalities noted for any of these ARTs. As the name 

indicates, the most readily recognized sign is oversized fetus or newborn, characterized as 

having a birth weight greater than 20 percent above the average birth weight for that 

species, breed, and sex. Dystocia and related morbidity and mortality of the young 

animals are common in cases of LOS when C-sections are not planned. Mortality rates 

for LOS calves can be high (Behboodi et al. 1995; Farin et al. 2001; Farin et al. 2004; 

Lee et al. 2004). A summary of incidence and survival rates of calves born with LOS and 

related clinical signs are in Table V-4. Survival of LOS calves is highly variable, and 

appears to depend on severity of the clinical signs and neonatal management practices. 

Studies that included such data indicated that survival ranged from 0 to 88 percent of 

calves diagnosed with LOS. 

Stress associated with dystocia, prolonged labor and emergency C-section birth is a risk 

factor for large calves (Kato et al. 1998; Kubota et al. 2000). Matsuzaki and Shiga (2002) 
Chapter V: Animal Health Risks 118 

Animal Cloning: A Risk Assessment DRAFT

reported that SCNT clone calves born by emergency C-section had a higher mortality rate 

(4/5) compared to clone calves that were delivered vaginally (1/8). It is not clear whether 

the higher mortality is entirely due to the emergency surgery or whether adverse factors 

in the clones themselves contributed to the mortality. 

Congenital abnormalities that may be related to fetal oversize include deformities of 

limbs and head, and may be a function of crowding in the uterus (Meirelles et al. 2001; 

Zakhartchenko et al. 1999a; Hill et al. 1999 with transgenic clones; Garry et al. 1996, 

with BNT clones). Intrauterine infections may also be responsible for some of these 

abnormalities (Kato et al. 2000; Kubota et al. 2000). LOS includes a large number of 

abnormalities, only some of which may be directly related to dystocia and congenital 

effects of unusually large size. Other abnormalities reported to coincide with LOS 

include respiratory, cardiac, hepatic, renal, umbilical, and immunologic problems, and 

may occur even among animals with birth weights within the normal range for their 

breed. These abnormalities may result from dysregulation of developmentally important 

genes rather than the uterine environment (see Chapter IV). Systemic abnormalities 

including organ dysfunction result in morbidity and often result in high mortality. 

Pulmonary abnormalities include immature lung development, insufficient lung 

surfactant, and failure of the lungs to inflate. Cardiovascular abnormalities include patent 

ductus arteriosus and ventricular defects (Table V-3). 

In vitro culture conditions are suspected to contribute to development of LOS in IVPderived embryos (Farin and Farin 1995; Farin et al. 2001). Various culture systems used 

in different laboratories often use slightly different media ingredients,34 such as fetal calf 

serum, and may expose developing embryos to hormones and growth factors that may not 

be in appropriate concentrations for the stage of development, possibly contributing to 

gene dysregulation (Sinclair et al. 1999). Behboodi et al. (1995) reported that birth 

weights were not significantly different between calves produced by AI and IVP-derived 

calves when embryos were cultured to the blastocyst stage in sheep oviducts; however, 

birth weights of calves born from embryos that developed into blastocysts in vitro were 

higher than those for calves from embryos that developed in the sheep oviduct or from 

AI. In this study, 7/8 calves produced from embryos cultured in vitro died within 48 

hours of birth, compared to 1/8 calves from embryos cultured in the sheep oviduct after 

fertilization. Hasler et al. (1995) noted that approximately 7 percent of clients purchasing 

 

34 Cells in culture require media that provide the essential nutrients and other chemical components that 

allow them to grow. Scientists have attempted to simulate the growth environment of the intact 

organism in culture media by adding certain blood components, usually serum (the portion of whole 

blood that remains after clotting has occurred). 
Chapter V: Animal Health Risks 119 

Animal Cloning: A Risk Assessment DRAFT

cows carrying IVP-derived calves reported high birth weights. In this study, of 428 IVP 

calves born, 67 died at birth (15.6 percent). 

Sire selection may also contribute to the large calves resulting from ET and IVP. Knight 

et al. (2001) indicated that one of the sires used in a two year study had a tendency to 

produce large ET calves. High birth weights in this study may have contributed to low 

survival rates in a previous study in the same herd. In cattle, sires may be selected based 

on their IVP and ET calf birth weight records (Knight et al. 2001). 

In a large study comparing birth weights, dystocia incidence, and neonatal death rates in 

AI, ET, IVP, and BNT produced calves of various beef and dairy breeds from labs in 

several countries, Kruip and den Daas (1997) noted that on average 31.7 percent of IVP 

calves (n=308) weighed more than 50 kg at birth, compared to 10 percent for AI (based 

on 495,000 calf records from the Netherlands). Interestingly, only 15 percent (n=126) of 

calves produced by BNT had birth weights greater than 50 kg in this study. For one breed 

(Holstein-Friesian), perinatal losses were similar between AI (n=1,160) and ET (n=45) 

calves (6.1 ± 0.6 and 6.6 ± 0.6 percent), but loss was higher for IVP calves (14.4 ± 2.3 

percent; n=251). Perinatal death loss was higher (11.6 vs. 2.3 percent) for IVP (n=308) 

compared with BNT calves (n=126) for the six breeds studied (Holstein-Friesian, Belgian 

Blue, Simmental/Fleckvieh, Limosin, Piedmontese, and Alentejano). 

Table V-4: Incidence of LOS and related clinical signs and survival rates of calves produced with ARTs 1 

Study Transgeni

c Status 

Clone 

LOS 

incidence 

Survival 

of LOS 

clones 

Comparator 

LOS 

incidence 

Survival of 

comparators Comments 

Batchelder 

2005 None 8/8 

(1.00) 

2/8 

(0.25) 

2/9 

(0.22) 

9/9 

(1.00) 

Comparators were ET (n=6) 

and AI (n=3). See Table V-5 

for clinical signs. 

Behboodi et al. 

1995 Some NP NP 4/8 (0.50) 

0/72 (0.00) NP 8 IVF calves compared to 72 

AI calves 

Cyagra 2003 None 73/1232

(0.59)

56/733 

(0.77) NP NA 

Clinical signs: contracture; 

septicemia; nephritis; failure to 

thrive; umbilical, 

gastrointestinal, cardiaccirculatory anomalies 

Garry et al. 

1996 None 34/40 (0.85) 

26/34 

(0.77) 

0/26 

(0.00) NA 

BNT clones, AI comparators. 

Clinical signs: respiratory and 

musculo-skeletal 

Gibbons et al. 

2002 None 8/9 

(0.88) 

7/8 

(0.88) NP NA 

Clinical signs: respiratory, 

umbilical, septicemia, 

hydrocephalus, GI problems 
Chapter V: Animal Health Risks 120 

Animal Cloning: A Risk Assessment DRAFT

Gong et al. 

2004 None 7/27 (0.26) 

0/27 

(0.00) NP NA 

Hasler et al. 

1995 NA NP NA 23/343 (0.07) NP Data gathered from owners of IVF-pregnant cows 

Hill et al. 1999 All 4/8 

(0.50) 

2/4 

(0.50) NP NA 

Clinical signs: respiratory, 

umbilical, cardiac, hepatic 

anomalies; contracture, 

acidosis, weak suckling reflex 

Heyman et al. 

2004 None 7/50 

(0.14) NP NP NP 

Birth weights of AI 

comparators used to set range 

for determining LOS in clones 

Kato et al. 2000 None 6/17 

(0.35) 

3/6 

(0.50) NP NA 

Clinical signs (may be result of 

Akabane virus): musculoskeletal, kidney abnormalities 

Kubota et al. 

2000 None 6/6 (1.00) 

4/6 

(0.67) NP NA 

Clinical signs: respiratory, 

polyuria and polydypsia 

Akabane virus 

Lanza et al. 

2001 Some 14/30 (0.46) 

8/14 

(0.57) NP NA 

Miyashita et al. 

2002 None 1/12 (0.08) 

0/1 

(0.00) NP NA 

Pace et al. 2002 Some 70/106 

(0.66) 

59/70 

(0.84) NP NA 

Clinical signs: umbilical, 

respiratory, cardiac, musculoskeletal, GI; hydrocephalus, 

bacterial infection 

Zakhartchenko 

et al. 1999a None 1/2 

(0.50) 

0/1 

(0.00) NP NA 

Clinical signs: musculoskeletal and hepatic 

abnormalities 

1

 Data on live-born calves 

2

 Of 134 calves born, 123 were born alive. 

3

 Denominator is number of calves identified with LOS and/or related clinical signs 

NA = not applicable 

NP = not provided; data not available 

More recent studies in which IVP and SCNT embryos were produced under the same 

culture conditions reported considerably higher incidences of LOS in fetal and adult cell 

SCNT-derived calves compared to IVP (Heyman et al. 2002; Chavatte-Palmer et al. 

2002; Matsuzaki and Shiga 2002), indicating that culture conditions may not be the only 

factor influencing the development of LOS in cattle clones. Average birth weight of 

adult-cell SCNT clones was significantly higher than IVP-derived calves (53.1 ± 2.0 kg 

vs. 44.5 ± 2.1 kg) in the Heyman et al. (2002) study. Chavatte-Palmer et al. (2002) found 

considerable variability in organ development among calf clones, and reported that one 

apparently normal clone fetus had small kidneys for its size and stage of development. 

Also in this study, Chavatte-Palmer et al. noted differences in body temperature, plasma 

leptin, thyroxine (T4) and insulin-like growth factor-II (IGF-II) in surviving clones 

compared to IVP and AI controls during the first week to 15 days after birth, although the 
Chapter V: Animal Health Risks 121 

Animal Cloning: A Risk Assessment DRAFT

clones appeared normal and healthy. Differences between clones and controls resolved by 

50 days of age (see Chapter VI for a more complete discussion of this study). The 

differences in outcomes between SCNT and IVP pregnancies observed in these studies 

suggest that some additional factor(s) may be at least partially responsible for the higher 

rate of abnormalities in animal clones compared to IVP calves, and not solely due to 

culture conditions. One possible explanation for this increase in abnormalities is 

incomplete epigenetic reprogramming (see Chapter IV). 

In a later study by this same group (Chavatte-Palmer et al. 2004), an additional cohort of 

58 live-born calves were followed through maturity. Clone survival after the first week 

following birth was 76 percent (44/58). Clinical signs and necropsy findings for nine 

clones that died during the perinatal period included hyperthermia, umbilical hernia, 

respiratory problems, ascites (abnormal fluid accumulation) in the chest and abdomen, 

fatty liver, limb deformities, various digestive tract problems, and abnormal or 

degenerating kidneys. 

Alternatively, culture media requirements may differ between SCNT and IVP embryos. 

Mastromonaco et al. (2004) compared development to blastocyst for IVP and SCNT 

embryos using different media ingredients at different stages of the in vitro process 

(oöcytes maturation and embryo culture stages). Although IVP embryos had similar rates 

of development to blastocyst and hatched blastocyst regardless of culture media used, 

development to blastocyst was greater among SCNT embryos cultured in synthetic 

oviductal fluid with 2 percent steer serum. Unfortunately, this study only looked at 

development through day 9 of embryo culture, and did not examine in vivo embryo 

development or subsequent calving outcomes. It is possible, however, that culture 

conditions impact epigenetic reprogramming, and this may be related to differences in 

outcomes observed in the Heyman et al., Chavatte-Palmer et al., and Matsuzaki and 

Shiga studies. 

In a recent study comparing SCNT (n=8) to ET (n=6) and AI (n=3), Batchelder (2005) 

noted large birth weights among three Hereford clones (n=3), ranging from 50.0 to 71.0 

kg. By comparison, ET comparator Hereford calves ranged from 31.5 to 48.0 kg (n=3). 

Curiously, the mean weight for Holstein clones (n=5) was similar to contemporary ET 

comparators (n=3) (37.1 vs. 39.4 kg), and within the average range for Holstein heifer 

calves. Neonatal clones in this study had lower RBC (6.8 x 106

 vs. 8.6 x 106 cells/μl) and 

hematocrit at birth than their comparators, and remained low for the first hour after birth, 

but were similar to comparators thereafter. White blood cell counts (WBC) and 

differential patterns were similar between clones and comparators. Clones exhibited 
Chapter V: Animal Health Risks 122 

Animal Cloning: A Risk Assessment DRAFT

lower blood glucose and lactate levels during the first 24 hours after birth than 

comparators, but were similar to comparators by 48 hours. 

Batchelder (2005) also noted several clinical signs often associated with LOS in both 

Holstein and Hereford clones, including delayed time to suckle and stand, hypoglycemia, 

forelimb flexor tendon contracture, enlarged umbilicus, patent urachus, and respiratory 

distress. These clinical signs were not always associated with high birth weight. 

Interestingly, a small number of comparators exhibited some of these same clinical signs. 

Table V-5 is partly reproduced from Batchelder 2005. 

Table V-5: Clinical signs observed in neonatal clones and comparators for Batchelder 2005. 

Clinical Sign Clones Comparators 

Time to nurse (> 3hrs) 5/8 0/9 

Time to stand (>3 hrs) 5/8 1/9 

Hypoglycemia (≤ 50mg/dl) 3/8 2/9 

Respiratory distress 3/8 1/9 

Flexor tendon contracture 4/8 0/9 

Enlarged umbilical vessels 8/8 2/9 

Patent urachus 5/8 1/9 

All calves in this study survived the first 48 hours; however, one clone died at 72 hours 

after birth, and another at six days after birth. The first clone, a Hereford heifer, was the 

largest at birth (71.0 kg), and at necropsy was diagnosed with pulmonary hypertension 

and multiple severe organ abnormalities including diffuse fibrosis of the liver, dysplasia 

of the biliary system, right ventricle hypertrophy, and patent ductus arteriosus. The 

second clone died at six days of age after suffering bloat and various other clinical signs 

involving the heart and lungs. 

(b) Other complications 

In discussing health and mortality among clones it is often difficult to distinguish 

between defects resulting from the uterine environment, placentation, and/or difficulties 

during delivery, and epigenetic factors intrinsic to the clone that impede normal 

development of the fetus and adaptation following birth. Dystocia, for example, can 

result in premature separation of the placenta, causing inhalation of amniotic fluid prior 

to birth, predisposing the neonate to pneumonia in both conventional calves (Moore et al. 

2002) and clones (Kato et al. 1998). Respiratory failure is one of the most commonly 

reported clinical signs in neonatal clones (Table V-3), and appears to result from 

numerous causes, including inadequate surfactant and failure of the lungs to inflate, as 
Chapter V: Animal Health Risks 123 

Animal Cloning: A Risk Assessment DRAFT

well as pneumonia arising from various causes (Garry et al. 1996; Hill et al. 1999; 

Chavatte-Palmer et al. 2002). Pneumonia may result from dystocia in natural pregnancies 

as well as those derived by ARTs (Moore et al. 2002). However, many of the respiratory 

conditions reported to occur in association with LOS (failure to inflate, lack of surfactant) 

have not been reported for calves from natural service or AI, and may be peculiar to 

ARTs that involve more extensive in vitro manipulation of the embryo (i.e., IVF and 

cloning), or may be related to labor-induction protocols (Batchelder 2005). 

Calves exhibiting LOS may also show prolonged time to stand and poor or latedeveloping suckling behavior (Chavatte-Palmer et al. 2002; Pace et al. 2002 (mixed 

transgenic and non-transgenic clones); Batchelder 2005). Poor suckling may preclude 

immune transfer in colostrum-dependent species, resulting in decreased ability to respond 

to immune challenge. Most of these studies, however, indicate that colostrum was 

administered by tube-feeding if the animal failed to suckle within one to two hours 

postpartum (Garry et al. 1996 (BNT clones); Hill et al. 1999 (transgenic clones); Gibbons 

et al. 2002; Batchelder 2005). Poor immune response in such cases may be due to a 

number of causes: inability of the neonate to absorb immunoglobulins; colostrum that is 

inadequate in immunoglobulin content; excessive or overwhelming stress; or high levels 

of pathogens in the neonatal environment. Clone producers have indicated that some 

calves are born with large umbilici, often with patent (open) blood vessels. This factor 

may increase the risk of bacterial infection, and clone producers indicated that surgery 

was generally performed on the enlarged umbilici of calves to reduce the risk of infection 

(see CVM Memorandum I at www.fda.gov/cvm/cloning.htm; also Appendix E and 

Batchelder 2005). 

Most studies that reported supplemental colostrum feeding did not indicate the source of 

the colostrum or whether tests of its adequacy (gravimetric density or IgG concentration) 

had been performed. Two studies reported testing colostrum of surrogate dams for 

adequacy or blood tests of neonates to determine immunoglobulin status (Hill et al. 1999; 

Pace et al. 2002). In the study of transgenic clones by Hill et al. several of the surrogate 

dams were judged to have adequate colostrum. Transgenic calf clones that failed to 

suckle were administered colostrum by tube and fostered to other cows as needed. Pace 

et al. (2002) reported testing plasma IgG of calves 12 hours after birth, followed by 

plasma infusion if plasma IgG concentrations were less than 1,200 mg/dL. Calf clones in 

this study were reported to have normal serum IgG levels 24 hours after birth. As noted 

throughout this report, the data derived from clones that are transgenic are extremely 

difficult to extrapolate to “just clones” (the only subject of this risk assessment) because 
Chapter V: Animal Health Risks 124 

Animal Cloning: A Risk Assessment DRAFT

of the inability to determine the relative contributions of the transgenic modification and 

the cloning process to the observations. 

In Batchelder 2005, clones were provided 2 liters colostrum (either by bottle or 

esophageal tube) within three hours of birth as well as supplemental plasma by I.V. over 

40 minutes. At 24 hours after birth, clones and comparators had similar levels of serum 

IgG. However, one clone had sub-normal IgG (435 mg/dl IgG), and was classified as 

having failure of passive transfer of immunity, and a second clone was classified as 

marginally protected (1500 mg/dl IgG). Batchelder related failure of passive immune 

transfer to poor metabolic status and respiratory distress. 

i. Cyagra Data: Perinatal Cohort 

A complete discussion of the Cyagra dataset including how it was analyzed and the 

context in which results should be interpreted is presented in detail in Appendix E. 

Briefly, the Cyagra dataset provided information on the overall health status and 

laboratory tests (clinical chemistry and blood cell parameters (hemograms)) for a group 

of SCNT-derived cattle clones and their approximately age- and breed-matched 

comparators. Among 10 neonates, four liver-related analytes were lower in clones than 

comparators: AST, GGT, cholesterol, and bile acids. Except for the values from one calf 

that did not survive, all red blood cell analytes were within the comparator group range. 

Three calves, all of which were infected with rotavirus, had low lymphocyte counts 

(lymphopenia). 

Of the 134 clone calves in the Cyagra cohort, 11 were stillborn. Birth weights were 

available for 34 of the 123 live-born clones, and ranged from 19.5 kg (a twin calf) to 

76.8 kg. Eighteen of the 34 (53 percent) birth weights were at least 20 percent above the 

average for their breed. Most oversized calves (13/18 (72 percent)) survived the critical 

first 48 hours after calving. Six of the oversize calves did not exhibit any other clinical 

signs associated with LOS. Fifty-five additional calves that were not oversized at birth, or 

for which birth weights were not available, showed clinical signs often associated with 

LOS; 43 of these animals survived the first 48 hours after calving. The most common 

clinical sign was umbilical problems (41 cases), followed by tendon contracture (15 

cases), ranging from mild to severe. There were also four animals with respiratory signs, 

five with cardio-vascular signs, three with thermoregulatory problems, two with renal or 

nephric signs, and five animals listed as having “abnormal development.” Some of the 

calves exhibited more than one sign, often umbilical problems with contracture, cardiac 

or respiratory signs. 
Chapter V: Animal Health Risks 125 

Animal Cloning: A Risk Assessment DRAFT

ii. Unpublished data 

Body temperature, pulse and respiration rate data were submitted covering the first 72 

hours of life for 19 clone calves of unknown breed(s) from a commercial cloning 

company (Table V-6). 

Table V-6: Pulse, Respiration, and Body Temperature of Nineteen Cattle Clones of Unknown Breeds During the 

First 72 After Birth 

Calf Temperature (F) Pulse Respiration 

ID 

Calving 

Date Birth 24h 48h 72h Birth 24h 48h 72h Birth 24h 48h 72h 

1 2/12/2001 104.8 103 102.2 102.6 54 132 144 116 48 44 48 84 

2 3/28/2001 101.2 101.7 101.7 101.8 120 114 138 138 36 42 42 36 

3 4/10/2001 103 101.6 101.4 103.6 100 120 126 140 46 44 44 48 

4 4/12/2001 104.3 101.2 101.6 102.6 64 120 132 140 30 48 44 36 

5 4/13/2001 102.6 101.1 102 103.6 100 120 120 140 44 56 48 48 

6 4/10/2001 102.9 101.5 102.3 102.5 116 144 128 126 68 54 56 39 

7 4/11/2001 100.5 101.1 101.7 102.9 112 120 160 152 54 48 40 40 

8 1/15/2002 102.4 102 101.8 102 60 144 140 128 60 48 28 20 

9 1/30/2002 103.2 101.3 102 101.1 128 136 140 132 98 56 52 44 

10 1/31/2002 105.2 101.5 101.6 102.2 150 140 140 115 36 32 24 40 

11 1/29/2002 102.4 102 102.5 102.6 66 132 126 138 66 108 102 78 

12 3/27/2002 102.5 102.5 102 102.4 60 192 104 156 60 54 40 56 

13 3/21/2002 103.4 100.8 101.3 103 108 108 132 156 72 36 30 24 

14 4/9/2002 103.9 101.3 102.1 102.9 40 114 120 120 24 80 84 72 

15 4/4/2002 104 102.2 102.2 90 132 180 24 56 72 

16 5/1/2002 103.4 101.3 102.4 103 90 120 120 102 50 48 68 68 

17 4/30/2002 101.3 103.2 101.5 102 120 140 120 180 78 88 64 66 

18 11/11/2002 103.3 103.1 103.2 103.4 100 150 150 160 70 100 120 60 

19 11/12/2002 102 101.6 101.8 103.8 130 150 160 156 60 48 60 96 

Mean ± SD for body temperature, pulse and respiration at birth for the 19 calves were 

103.0 ± 1.2  ̊ F, 95.2 ± 30.34 beats/min, and 53.9 ± 19.4 breaths/min, respectively. At 24 

hours, means were 101.8 ± 0.7  ̊ F, 133.1 ± 19.2 beats/min, and 57.4 ± 21.1 breaths/min. 

At 48 hours, means were 102.0 ± 0.5  ̊ F, 135.8 ± 17.9 beats/min, and 56.1 ± 24.8 

breaths/min. Values for one calf were not available for the 72 hour measurements, such 

that means and standard deviations represent 18 calves. Those values were 102.7 ± 0.7  ̊

F, 138.6 ± 19.3 beats/min, and 53.1 ± 20.9 breaths/min. Heart and respiration rates vary 
Chapter V: Animal Health Risks 126 

Animal Cloning: A Risk Assessment DRAFT

with age. Respiration rates in growing steers (age not specified) were noted to be 79 ± 3.2 

breaths/min in one study (Nihsen et al. 2004). Breukelman et al. (2004) noted basal fetal 

heart rates in late gestation AI pregnancies to be 111.6 ± 1.4 beats/min. By comparison, 

basal heart rate of three week old heifer calves averaged 88.1 ± 4.04 beats/min (Van 

Reenen et al. 2005). 

The birth records of two Holstein heifer clones were submitted by a private cloning firm. 

Both heifers were delivered by C-section. One calf was a breech position (posterior 

presentation with hind limbs under body); the other calf was in a normal posterior 

position, with hind limbs extended. The placentae of these calves were described as 

normal, with some large and some small placentomes described for one placenta. Calves 

were described as normal, weighing 45 and 47.7 kg each at time of delivery. Both calves 

had normal umbilici. Some fluid was noted in the lungs of both calves, but they were 

described as breathing normally, and although some meconium staining was noted, there 

was no indication that meconium had been inhaled. Body temperatures were 100 and 

102.6 ̊F, slightly below and above the average temperature for cattle. Blood glucose, 

packed cell volume (PCV), blood total protein, and IgG concentrations were monitored 

for the first 23 to 27 hours after birth (Table V-7). Blood glucose was low for both calves 

prior to first feeding, then increased to normal levels by the second feeding. Total protein 

also increased steadily following feeding, and IgG levels were listed as “> 10” after the 

first colostrum feeding. The units for IgG measurements were not provided, and PCV 

values were the only hematology data provided, so these data are difficult to interpret. 

Total protein and serum glucose values are comparable to age-matched non-clone cattle 

in the Cyagra dataset by the second post-feeding blood sample. 

Table V-7: Reported blood values for Two Holstein Heifer Clones 

 Calf 1 Calf 2 

 Pre-feeding 1st feeding 2nd feeding Pre-feeding 1st feeding 2nd feeding 

Glucose 20 35 93 21 29 83 

PCV 29 28 25 30 33 29 

Total 

Protein 5 5.3 7.5 4.8 5.0 6.0 

IgG N/A >10 >10 N/A >10 >10 
Chapter V: Animal Health Risks 127 

Animal Cloning: A Risk Assessment DRAFT

c. Developmental Node 3: Juvenile Development 

i. Peer-Reviewed Publications 

Mortality for AI-produced and naturally bred dairy replacement heifers from weaning to 

calving was 2.4 percent according to USDA statistics (USDA/NAHMS 1996). As a 

reference for morbidity rates, in the general population of beef replacement heifers, the 

rate of illness from weaning to puberty is very low, with the most common illness 

reported as pinkeye (1.9 percent), followed by scours (diarrhea) (1.0 percent) 

(USDA/NAHMS 1997). 

Less detailed information has been published on the health of bovine clones following 

weaning than on the perinatal period. Most studies merely report that animals surviving 

the first 30 to 60 days postpartum are “healthy and normal” (Campbell et al. 1996; Lanza 

et al. 2000 with transgenic clones; Heyman et al. 2002). Kubota et al. (2000) reported that 

veterinary exams, growth curves and blood clinical chemistry were used to determine the 

health of six clone calves, and that no differences were noted between clones and age 

matched controls. 

Shiga et al. (2005) reported on growth rates of four clones (two steers and two intact 

bulls) of a 12 year old Japanese Black bull. Although the average birth weight of the 

clones was greater than that of AI-derived comparators (43.1 ± 4.1 vs. 31.3 ± 4.0 kg), 

post-natal growth rates were similar between groups, and by two years of age, body 

weight and shoulder height were similar between clones and comparators. 

In a long term study of health and survival of clones and their offspring, Wells et al. 

(2004) stated that the most common cause of mortality (either by natural death or 

euthanasia) of young clones at their facility was musculoskeletal abnormalities (severe 

tendon contracture and chronic lameness). They also reported two cases of death due to 

bloat, and an unspecified number of clones dying due to endophyte toxicity. Gastrointestinal problems, including bloat, have been reported in other studies (Cyagra 2003; 

Batchelder 2005), but can also result from poor feeding/grazing management in 

conventional cattle. Endophyte toxicity results from grazing fungus-infected grass by 

cattle sensitive to the toxin. Wells et al. acknowledge that this trait is inherited in certain 

lines of cattle, and likely was related to the genetics of the nuclear donor. (The clones 

affected by this toxicity were derived from the same donor.) Other causes of death among 

clones (besides those attributed to accident or management problems) included anemia, 

chronic heart failure, and degenerative nephrosis, problems which have been noted in 
Chapter V: Animal Health Risks 128 

Animal Cloning: A Risk Assessment DRAFT

other studies (Cyagra 2003; Chavatte-Palmer et al. 2004). Growth rates of heifer clones 

were within the range for conventional heifers raised under typical management 

conditions in New Zealand (0.677 ± 0.066 kg/day). Heyman et al. (2004) also reported 

growth rates of 23 clones were within expected limits for Holsteins (0.7 – 0.8 kg/day). 

Growth rate was not influenced by birth weight in these studies. 

Chavatte-Palmer et al. (2002) monitored the growth and development of clones (n=21) 

compared to IVP (n=20) and AI (n=176) controls. For each variable measured, numbers 

of clones and controls varied (see Table VI-2 in Chapter VI). For the first week after 

birth, the mean rectal body temperature was higher in clones (n=10) than AI controls 

(n=10), and some temperature spikes (up to 41o C for periods lasting 24 – 36 hours35) 

were observed. Body temperatures of clones were reported as remaining elevated for the 

first 50 days, although data were only provided for the first week. The investigators were 

unable to determine the cause of the elevated body temperatures in clones: no bacterial 

infections were detected, and animals did not respond to anti-inflammatory drugs 

commonly used to lower body temperature. Levels of thyroxine (T4), a hormone that 

controls metabolic rate in most tissues, were tested to determine if they could explain the 

temperature difference between clones and controls. Plasma T4 levels were lower in 

clones than controls during the first two weeks of life, and were similar to controls 

thereafter. Chavatte-Palmer et al. (2002) noted that lower plasma T4 levels coupled with 

elevated body temperatures in young calves was consistent with the findings of Carstens 

et al. (1997). 

Carstens et al. (1997) measured metabolic rates and increases in other blood parameters 

related to stress in different breeds of neonatal calves before and after stimulation with 

norepinephrine. The Carstens study focused on the regulation of brown adipose tissue by 

norepinephrine. Brown adipose tissue (BAT) is found in neonates of many mammalian 

species, and, while it contains fat, its primary function is to generate heat (unlike white 

adipose, which is primarily a fat depot) to keep the newborn warm during cold stress. 

Brown adipose cells contain large concentrations of mitochondria (which is what makes 

it brown in appearance). Mitochondria are often referred to as the “power houses” of 

cells, because they generate energy from nutrients through a process known as oxidative 

phosphorylation to produce adenosine triphosphate (ATP), the ultimate (short-term) form 

of energy storage immediately prior to use by the cell to carry out functions that require 

energy. This process is relatively inefficient, resulting in some energy loss from the 

system as heat. This heat loss is the primary source of body temperature, which is 

relatively constant in warm-blooded animals (Blaxter 1989). 

 35 For dairy cattle, normal temperature is approximately 38.5 oC. 
Chapter V: Animal Health Risks 129 

Animal Cloning: A Risk Assessment DRAFT

Brown adipose tissue metabolism is stimulated by norepinephrine (Blaxter 1989), which 

is consistent with the norepinephrine release observed in response to stressful stimuli 

such as cold (Voet and Voet 1995). Unlike other tissues, BAT cell mitochondria contain 

an extra protein, controlled through the action of norepinephrine, which allows the 

oxidative phosphorylation pathway to become “uncoupled” from the production of ATP. 

Although oxidative phosphorylation continues, ATP is not produced. This interruption in 

the pathway to ATP results in the release of large amounts of energy as heat (Voet and 

Voet 1995). In some species, BAT persists into adulthood, but in cattle and some other 

cloven-hoofed species, BAT usually disappears (is broken down and metabolized) 

following the neonatal period (Blaxter 1989). 

In most tissues of the body, metabolic rate is controlled by T4. In animals that do not 

possess BAT, body temperature is a function of metabolic rate (Voet and Voet 1995). 

According to Carstens et al. (1997) T4 appears to have differential effects on BAT 

compared to other tissues, in that elevated T4 suppresses thermogenesis (heat formation 

and release) in BAT. It may do this by reducing metabolic rate in this tissue, or by 

blocking the activity of the protein that uncouples oxidative phosphorylation, allowing 

energy to be captured as ATP, as it is in other cells, and reducing the amount of energy 

that is lost as heat. In the Carstens et al. (1997) study, T4 was not affected by 

norepinephrine challenge, but metabolic rate and body temperature increased, which the 

authors attributed to increased heat production in the calves’ BAT. 

Because the higher body temperatures of clones observed in the Chavatte-Palmer et al. 

study were independent of T4 levels, it is possible that the hyperthermia experienced by 

the clones resulted from increased BAT metabolism. However, norepinephrine was not 

measured in this study (Chavatte-Palmer et al. 2002), so it is not possible to determine 

whether that was the cause of the elevated temperature levels in these clones. 

In a follow-up study, Chavatte-Palmer et al. (2004) reported that 38/44 clones surviving 

the perinatal period lived to six months of age. The authors reported an additional four 

clones with thymic aplasia or atrophy (underdeveloped or degenerating thymus gland) 

since the first report of a clone with this condition (Renard et al. 1999). It is not clear 

from the current study whether these four clones were also the result of multiple rounds 

of cloning as in the Renard et al. report. To our knowledge, this is the only laboratory 

reporting thymic aplasia as a clinical problem in clones. On necropsy, the thymus glands 

of these calves exhibited abnormal tissue organization, suggesting epigenetic errors (see 

Chapter IV). Three calves in this group died suddenly with few or no clinical signs: two 
Chapter V: Animal Health Risks 130 

Animal Cloning: A Risk Assessment DRAFT

died following the onset of diarrhea and one calf died without any apparent cause. 

Another calf was diagnosed with diabetes insipidus. The only post-mortem finding on the 

diabetic calf was an enlarged pituitary, suggesting abnormal hormonal regulation. 

Chavatte-Palmer et al. also noted that, although hematological values for clones were 

within the normal range, hemoglobin levels of 25 clones were lower than those of 19 AI 

contemporary comparators for the first 65 days after birth. This finding appears to 

reinforce this group’s earlier conclusion that clones cannot be considered physiologically 

normal until approximately two months of age. 

Batchelder (2005) also noted periodic moderate to severe hyperthermia in young 

Hereford and Holstein clones (n=8) until approximately 60 days of age. As in ChavattePalmer et al. (2002), hyperthermia was unresponsive to treatment with either antiinflammatory drugs or mechanical attempts at cooling (fans, alcohol baths), and 

hyperthermic calves demonstrated no changes in behavior or signs of illness. Respiratory 

rates in clones followed a similar pattern to body temperature in this study, increasing 

during temperature spikes. This is expected, as increased respiration rate (including 

panting) is a means of dissipating body heat for cattle, or could be related to increased 

oxygen demand by BAT (Blaxter 1989). 

Additional endocrine measures evaluated in the Chavatte-Palmer et al. (2002) study 

included cortisol, insulin-like growth factor-I (IGF-I), IGF-II, IGF binding protein, leptin, 

and growth hormone. Blood samples for these assays were collected from all 21 clones 

and 8 AI calves (described above). Cortisol levels were decreased in both clone (n=11) 

and non-clone calves (n=2) born by C-section relative to calves born vaginally (10 clones 

and 6 non-clones). By seven days of age, clones and AI controls exhibited similar cortisol 

levels following an ACTH (adrenocorticotropic hormone) challenge (ACTH induces the 

production of cortisol). 

No differences in levels of growth hormone, IGF-I, or IGF binding protein were observed 

between clones and AI controls. Levels of IGF-II were relatively high at birth among 

clones, but rapidly decreased within 15 days, until clones had slightly lower IGF-II levels 

compared to AI controls. Leptin levels were higher in clones than controls during the first 

week of life, but were similar to controls thereafter. The role of leptin in ruminant 

metabolism is not well understood, and the relevance of this measurement to an 

assessment of animal health cannot be determined. Insulin and glucose response after 

eating were not different between clones and AI controls in this study (Chavatte-Palmer 

et al. 2002). 
Chapter V: Animal Health Risks 131 

Animal Cloning: A Risk Assessment DRAFT

Govoni et al. (2002) also published one of the few studies of postnatal growth and 

development of cattle clones. Although this study was performed on only four animals 

generated from the same donor cell, the report is fairly detailed. Holstein heifer clones 

were paired with age, sex and breed matched controls produced by AI. All calves were 

pre-pubertal at the beginning of the study (approximately 5 months of age). Control 

calves were housed in adjacent pens in the same barn as clones. Differences were noted 

over time between clones and controls in growth hormone (GH) and IGF-I levels. Over 

the course of the six month study, GH levels declined in controls, but in clones GH levels 

began to increase beginning at about nine months of age. Average plasma concentration 

of IGF-I was generally lower in clones compared to controls. Although IGF-I increased 

in both groups over the course of the study, clones continued to have lower IGF-I 

concentrations compared to age matched controls (203.7 ±13.8 vs. 306.3 ± 13.1 mg/mL). 

Growth hormone has been reported as a major modulator of systemic concentrations of 

IGF-I (Le Roith 2001), and has been demonstrated to stimulate hepatic production of 

IGF-I after its release from the hypothalamus. Somatostatin, which is stimulated by high 

levels of IGF-I, suppresses GH synthesis, which in turn causes a reduction in IGF-I 

synthesis in the liver. Mice lacking functional hepatic synthesis of IGF-I grow normally, 

perhaps because IGF-I is also synthesized in muscle, but GH levels in these mice are 

elevated. If cattle and mice were to exhibit similar control mechanisms, the expectation 

would be that the increased GH levels in clones after 9 months of age would have 

resulted in a concomitant increase in circulating IGF-I. This was not observed by Govoni 

et al. (2002). Clones were more responsive than controls to factors controlling GH 

release, but showed a similar response as controls to inhibiting factors. For example, the 

magnitude of response to injected Growth Hormone Releasing Hormone (GHRH) was 

five times higher in clones than controls, although GH returned to basal levels 40-50 

minutes post stimulation. Conversely, injecting animals with Somatotropin Release 

Inhibiting Factor (SRIF) was successful in equally inhibiting response to GHRH in both 

clones and controls. IGF Binding Protein 2 (IGFBP2) levels were not different between 

growing clones and controls in the Govoni et al. study. IGFBP3 (another binding protein 

for IGF-I) levels were lower in clones compared to controls. The altered levels may be 

related to the lower IGF-I levels in these animals, possibly resulting in down-regulation 

(reduced synthesis) of this binding protein. The Savage et al. (2003) study, discussed 

below, noted no abnormalities in growth or behavior of these clones. Neither of these 

studies noted health problems in the clones, suggesting that these differences were 

insufficient to cause any metabolic perturbation. 
Chapter V: Animal Health Risks 132 

Animal Cloning: A Risk Assessment DRAFT

Savage et al. (2003) performed behavioral studies with four Holstein heifer clones and 

age- and breed-matched control heifers (the same group of animals as reported in the 

Govoni et al. study). Animals were studied beginning between 32 and 36 weeks of age, at 

which point there were no differences in weight or height between the clones (205.5 ± 9.9 

kg; 117.0 ± 1.8 cm) and controls (211.4 ± 7.4 kg; 119.5 ± 1.4 cm). All calves were raised 

together under the same management conditions. Based on a series of studies evaluating 

approach to other animals and novel objects, clones exhibited age-appropriate behaviors, 

but were reported to be more aggressive and inquisitive than controls, and spent more 

time grooming and socializing. Clones tended to spend less time in playful behavior than 

controls. Review of records on the cow that served as the nuclear donor for the clones 

indicated that she had displayed similarly aggressive and inquisitive behavior as a young 

animal, suggesting that at least some of these behavioral traits may be genetically 

controlled. Clones spent more time in proximity to adult animals in an adjacent pen 

(which also housed the nuclear donor), and in proximity to the feed bunk compared to 

control animals. In general, clones were reported to spend more time with each other 

rather than socializing with control animals. The authors speculated as to whether clones 

exhibit genetic kinship recognition. 

Batchelder (2005) reported aggressive feeding behavior and “insatiable” appetites among 

eight juvenile clones, as well as increased water consumption vs. ET and AI comparator 

calves. The clones’ increased demand for water and milk replacer may be related to the 

higher body temperatures experienced by clones in this study during the first 60 days, and 

may represent a compensatory response to maintain hydration. Blood values for these 

older calves were not reported, so it cannot be determined whether some other underlying 

metabolic disturbance (such as differences in energy metabolism) might have contributed 

to the increased appetite and thirst exhibited by clones. Overall, no differences were 

observed in weight gain between clones and comparators in this study; however, when 

data were analyzed by breed, Hereford clones gained more rapidly than Hereford 

comparators, while Holstein clones gained less than Holstein comparators during the first 

four weeks after birth. 

The potential for long term effects of embryo manipulation on the resulting animal is a 

question that has arisen in the past. McEvoy et al. (2000) noted that IVP calves exhibiting 

LOS at birth (greater than 60 kg) were not different in body weight compared with in vivo

produced, normal birth weight controls when slaughtered at 13 months of age. LOS cattle 

had abnormally large hearts when necropsied at slaughter, although this study did not 

discuss whether the enlarged hearts showed any other anomalies which might indicate 

functional abnormalities. Wilson et al. (1995) compared birth weight and growth of 
Chapter V: Animal Health Risks 133 

Animal Cloning: A Risk Assessment DRAFT

calves derived by AI and natural mating (NM) to half- and full-sibling calves from ET or 

BNT. Male and female calves resulting from BNT had higher birth weights (49.5 vs. 39.9 

and 36.8 kg for male BNT, ET, and AI/NM; 47. vs. 37.1 and 34.6 kg for female BNT, 

ET, and AI/NM, respectively). BNT calves were also heavier than ET and AI/NM calves 

at 1 year of age (519.0 vs. 497.4 and 497.0 kg for male BNT, ET and AI/NM calves; 

429.1 vs. 356.3 and 352.9 for female BNT, ET, and AI/NM calves, respectively). No 

other physiological measurements were taken in the Wilson et al. study, and health of 

calves was not discussed. In contrast to the Wilson et al. study, Pace et al. (2002) reported 

that 52 SCNT clones (some transgenic) raised at the same facility had similar weight 

gains for the first 120 days, regardless of birth weight. No comparisons were made with 

contemporary controls in this study, and no other physiological measurements for this 

age were reported. 

Of the six calves surviving the neonatal period in the Batchelder (2005) study, three more 

calves died or were euthanized during the juvenile period. Two calves died due to 

complications involving a non-healing umbilical stalk and patent urachus. Another calf 

died of apparent pneumonia, and was diagnosed with cardiac abnormalities and 

pulmonary hypertension upon necropsy. Two of the calves exhibited neurological signs, 

including head twitching and seizures. Three clones (Holstein breed) and all nine 

comparators survived the juvenile period. 

ii. Cyagra Data: 1-6 Month Cohort 

According to the data provided by Cyagra (Appendix E), nine calves out of 104 died after 

the critical 48 hour period after birth. Of these, three died or were euthanized during the 

juvenile or weaning period. Causes of death for these three animals varied. One clone 

died at 47 days after it failed to respond to therapy to treat severe contracture of the 

limbs. Another clone was diagnosed with “failure to thrive,” indicating that it failed to 

gain weight or grow properly, or was unthrifty. The third clone died at 149 days as the 

result of gastrointestinal tract problems such as bloating and poor rumen motility.36 One 

clone that survived to weaning was culled for poor conformation. This type of culling is 

often seen in conventional breeding programs. 

Health issues observed in some of the Cyagra clones (see Appendix E) included an 

increased incidence of umbilical problems (enlargements, excessive bleeding, navel 

infection), contracted tendons, and cryptorchidism (a condition in which one or both 

testicles are retained in the body cavity). All of these conditions are seen in sexually-

 36 In ruminants, the rumen or largest compartment of the stomach contracts on average once per minute. 

This is necessary to aid digestion and normal passage of nutrients through the gastro-intestinal tract. 
Chapter V: Animal Health Risks 134 

Animal Cloning: A Risk Assessment DRAFT

derived animals, but at lower frequencies than in clones. For example, calf clones had 

umbilical surgery at a much higher rate than non-clone calves. Contracted tendons 

appeared to occur at a higher incidence in clones relative to sexually-derived animals. 

Cryptorchidism is quite uncommon in sexually-reproduced bulls, but does appear to be 

an inherited trait (Blood and Radostits 1989); cryptorchid bulls tend to sire cryptorchid 

calves, are not recommended for breeding stock, and are thus directed to food production. 

The health risk posed to the animal by cryptorchidism is the tendency for the retained 

tissues to become neoplastic (tumor-forming) in long-lived animals. 

There was no evidence, based on hemoglobin and hematocrit, of anemia in clones 

(Appendix E). One clone calf had a hemoglobin count which was above the range for the 

comparator group, but that observation does not appear to have any health consequences. 

Other red blood cell measures, such as low mean cell hemoglobin concentration (MCHC) 

and red cell distribution width (RDW) do not indicate a disease process in the absence of 

indicators of anemia such as low hemoglobin or hematocrit (these measures were within 

the normal range). White cell measures were generally within the normal range, and there 

were no differences that could indicate infection or abnormalities in immune function. 

As discussed in Appendix E, physiological indicators of growth such as calcium, 

phosphorus, and alkaline phosphatase were appropriately elevated relative to expected 

adult levels in both clones and comparators. A few clones exhibited elevated levels of 

these growth indicators relative to comparators. Although these differences may be 

attributed to differences in management, it is important to note that the animals in which 

these elevations occurred were among the youngest in this age cohort. Further, these 

clones are likely the product of superior genetics bred for improved growth and 

production characteristics. 

Although six of the 42 valid glucose values (some measurements were considered 

artifactual (see Appendix E)) were higher for clones than comparators, none of the 

urinalyses were positive for glucose. The elevated glucose is not considered to be 

clinically relevant as the absence of urinary hyperglycemia indicates that the elevated 

blood glucose levels were transient, and most likely a short-lived response to stress. 

Gamma glutamyl transferase (GGT) levels in three clones were lower than in 

comparators, and sorbitol dehydrogenase (SDH) levels were lower in three other clones 

than comparators. Although high levels of these enzymes are indicative of liver damage, 

the clinical significance of low levels is not known. 
Chapter V: Animal Health Risks 135 

Animal Cloning: A Risk Assessment DRAFT

iii. Unpublished data 

In response to CVM’s request for additional data on clones, a private veterinary clinic 

submitted hematology and clinical chemistry data on three bull clones, ranging in age 

from 5 to 7 months at the time of the first sampling. Animals were sampled a total of 

three times within a six week period. Most variables measured were within the reference 

range used by the diagnostic lab conducting the tests (Marshfield Laboratories, 

Marshfield, WI). Values outside the reference range for the testing laboratory are listed in 

Tables V-8 a, b, and c. 

CVM contacted Marshfield Laboratories on September 21, 2005, regarding the source of 

their reference range. According to the laboratory, the reference range for hematology 

and clinical chemistry was established on blood samples taken from female dairy animals 

between 1 and 8 years of age. As discussed in Appendix E, it is important that the 

selected reference range is appropriate to the animals being tested. In this case, the use of 

a reference range established using post-pubertal, near-adult and adult females may not 

provide an appropriate comparison for pre-pubertal, rapidly growing males. As no 

contemporary comparator animals were sampled, other published reference ranges 

(Meyer and Harvey 2004; Revoir 1998; Duncan and Prasse 2003) were used for crosscomparison. Results indicated that one clone (Clone #3) on the second sampling date had 

one analyte that was outside any of the reference ranges used. For Clone #3, cholesterol 

was low on October 13. However, all of this animal’s hematology and clinical chemistry 

values were considered within published ranges on the third sampling date, 

approximately two weeks later. Because serum cholesterol can be affected by diet and 

time since the last meal, the single low value for this animal was judged not to be 

biologically relevant. 

Table V-8a: Hematology and Clinical Chemistry for Three Holstein Bulls (Bull #1) 

 RMI

N RMAX Units 

Date Section 1 Collected 9/22/03 10/13/03 10/27/03 

Hemogram-Vet (VCLT) 

 Red Blood Cell Count . 7.03 5 10 x 10^6/uL 

 Hemoglobin . 9.10 8 15 g/dL 

 Hematocrit . 29.00 24 46 % 

Mean Corpuscular 

Volume . 41.20 40 60 fL 

Mean Corpuscular 

Hemoglobin . 12.90 11 17 pg 
Chapter V: Animal Health Risks 136 

Animal Cloning: A Risk Assessment DRAFT

Mean Corpuscular Hgb 

Conc. . 31.40 30 36 g/dL 

Red Cell Distribution 

Width L 21.60 26 30 % 

 Platelet Count H 720.00 230 690 x 10^3/uL 

 White Blood Cell Count . 5.30 4 12 x 10^3/uL 

Seg. Neutrophil 

Absolute # . 1.64 0.6 4 x 10^3/uL 

Banded Neutrophil 

Absolute # . 0.00 0 0.12 x 10^3/uL 

 Lymphocyte Absolute # . 3.39 2.5 7.5 x 10^3/uL 

Act Lymphocyte 

Absolute # . 0.00 x 10^3/uL 

 Monocyte Absolute # . 0.16 0.03 0.84 x 10^3/uL 

 Eosinophil Absolute # . 0.11 0 2.4 x 10^3/uL 

 Basophil Absolute # . 0.00 0 0.2 x 10^3/uL 

 Other Absolute # . 0.00 0 0 x 10^3/uL 

 Blast Absolute # 0.00 x 10^3/uL 

Promyelocyte Absolute 

# 0.00 x 10^3/uL 

 Myelocyte Absolute # 0.00 x 10^3/uL 

Metamyelocyte 

Absolute # 0.00 x 10^3/uL 

Differential, Vet. (VDIF) 

 Segmented Neutrophils 31 % 

 Lymphocytes 64 % 

 Monocytes 3 % 

 Eosinophils 2 % 

 Basophils % 

Poikilocytosis / 

polychromasia? no 

 Glucose L 47.0 . 78.0 H 87.0 55 79 mg/dL 

 AST (GOT) . 79.0 L 56.0 . 69.0 57 108 U/L 

 SDH . 12.8 . 13.3 . 15.8 12.2 46 U/L 

 Total Bilirubin . 0.1 . 0.1 . 0.1 0.1 0.4 mg/dL 

 Cholesterol L 95.0 L 90.0 L 95.0 112 331 mg/dL 

 Total Protein . 6.8 . 7.6 . 7.2 6.3 8.5 g/dL 

 Albumin . 3.4 . 3.5 . 3.5 3.2 4.3 g/dL 

 Urea N . 12.0 L 7.0 . 8.0 8 22 mg/dL 

 Creatinine L 0.5 . 0.6 L 0.5 0.6 1.4 mg/dL 

 Phosphorous . 9.0 . 8.3 . 8.6 4.4 9.2 mg/dL 

 Calcium . 10.5 . 10.1 . 10.5 7.9 10.5 mg/dL 
Chapter V: Animal Health Risks 137 

Animal Cloning: A Risk Assessment DRAFT

 Sodium . 143.0 . 150.0 . 145.0 140 151 mmol/L 

 Potassium . 5.2 . 4.8 . 5.1 3.7 5.6 mmol/L 

 Chloride . 100.0 H 110.0 . 102.0 100 109 mmol/L 

 Bicarbonate . 25.0 . 25.0 . 29.0 22 29 mmol/L 

 CK . 221.0 . 190.0 . 157.0 50 271 U/L 

 GGT . 19.0 . 14.0 . 13.0 12 30 U/L 

 Anion Gap H 23.0 . 20.0 . 19.0 13.6 21.6 mmol/L 

 Hemolysis/lipemia? no no no 

Table V-8b: Hematology and Clinical Chemistry for Three Holstein Bulls (Bull #1) 

 RMI

N RMAX Units 

Date Section 1 Collected 9/22/03 10/13/03 10/27/03 

Hemogram-Vet (VCLT) 

 Red Blood Cell Count . 7.83 5 10 x 10^6/uL 

 Hemoglobin . 9.90 8 15 g/dL 

 Hematocrit . 31.20 24 46 % 

Mean Corpuscular 

Volume L 39.80 40 60 fL 

Mean Corpuscular 

Hemoglobin . 12.60 11 17 pg 

Mean Corpuscular Hgb 

Conc. . 31.70 30 36 g/dL 

Red Cell Distribution 

Width L 21.60 26 30 % 

 Platelet Count H 769.00 230 690 x 10^3/uL 

 White Blood Cell Count . 10.10 4 12 x 10^3/uL 

Seg. Neutrophil 

Absolute # . 2.63 0.6 4 x 10^3/uL 

Banded Neutrophil 

Absolute # . 0.00 0 0.12 x 10^3/uL 

 Lymphocyte Absolute # . 6.87 2.5 7.5 x 10^3/uL 

Act Lymphocyte 

Absolute # . 0.00 x 10^3/uL 

 Monocyte Absolute # . 0.20 0.03 0.84 x 10^3/uL 

 Eosinophil Absolute # . 0.10 0 2.4 x 10^3/uL 

 Basophil Absolute # H 0.30 0 0.2 x 10^3/uL 

 Other Absolute # . 0.00 0 0 x 10^3/uL 

 Blast Absolute # 0.00 x 10^3/uL 

 Promyelocyte Abs. # 0.00 x 10^3/uL 

 Myelocyte Absolute # 0.00 x 10^3/uL 
Chapter V: Animal Health Risks 138 

Animal Cloning: A Risk Assessment DRAFT

 Metamyelocyte Abs. # 0.00 x 10^3/uL 

Differential, Vet. (VDIF) 

 Segmented Neutrophils 26 % 

 Lymphocytes 68 % 

 Monocytes 2 % 

 Eosinophils 1 % 

 Basophils 3 % 

Poikilocytosis / 

polychromasia? no 

 Glucose L 4.0 . 76.0 H 88.0 55 79 mg/dL 

 AST (GOT) . 71.0 L 46.0 . 69.0 57 108 U/L 

 SDH L 11.2 L 7.3 . 16.7 12.2 46 U/L 

 Total Bilirubin . 0.2 . 0.1 . 0.1 0.1 0.4 mg/dL 

 Cholesterol L 88.0 L 83.0 L 86.0 112 331 mg/dL 

 Total Protein . 7.5 . 7.8 . 7.6 6.3 8.5 g/dL 

 Albumin . 3.7 . 3.4 . 3.6 3.2 4.3 g/dL 

 Urea N . 10.0 . 8.0 . 13.0 8 22 mg/dL 

 Creatinine L 0.5 L 0.5 . 0.6 0.6 1.4 mg/dL 

 Phosphorous . 7.8 . 7.3 . 8.4 4.4 9.2 mg/dL 

 Calcium H 10.6 . 9.7 . 9.5 7.9 10.5 mg/dL 

 Sodium . 142.0 . 144.0 . 145.0 140 151 mmol/L 

 Potassium . 5.3 . 4.6 . 4.4 3.7 5.6 mmol/L 

 Chloride . 101.0 . 104.0 . 103.0 100 109 mmol/L 

 Bicarbonate . 24.0 . 27.0 . 28.0 22 29 mmol/L 

 CK . 234.0 . 172.0 . 179.0 50 271 U/L 

 GGT . 14.0 . 13.0 . 15.0 12 30 U/L 

 Anion Gap H 22.0 . 18.0 . 18.0 13.6 21.6 mmol/L 

 Hemolysis / lipemia? no no no 

Table V-8c: Hematology and Clinical Chemistry for Three Holstein Bulls (Bull #1) 

 RMI

N RMAX Units 

Date Section 1 Collected 9/22/03 10/13/03 10/27/03 

Hemogram-Vet (VCLT) 

 Red Blood Cell Count . 7.41 5 10 x 10^6/uL 

 Hemoglobin . 10.10 8 15 g/dL 

 Hematocrit . 31.10 24 46 % 

Mean Corpuscular 

Volume . 41.90 40 60 fL 

 Mean Corpuscular . 13.70 11 17 pg 
Chapter V: Animal Health Risks 139 

Animal Cloning: A Risk Assessment DRAFT

Hemoglobin 

Mean Corpuscular Hgb 

Conc. . 32.60 30 36 g/dL 

Red Cell Distribution 

Width L 21.90 26 30 % 

 Platelet Count . 461.00 230 690 x 10^3/uL 

 White Blood Cell Count . 4.80 4 12 x 10^3/uL 

Seg. Neutrophil 

Absolute # . 1.87 0.6 4 x 10^3/uL 

Banded Neutrophil 

Absolute # . 0.00 0 0.12 x 10^3/uL 

 Lymphocyte Absolute # . 2.59 2.5 7.5 x 10^3/uL 

 Act Lymphocyte Abs. # . 0.00 x 10^3/uL 

 Monocyte Absolute # . 0.19 0.03 0.84 x 10^3/uL 

 Eosinophil Absolute # . 0.10 0 2.4 x 10^3/uL 

 Basophil Absolute # . 0.05 0 0.2 x 10^3/uL 

 Other Absolute # . 0.00 0 0 x 10^3/uL 

 Blast Absolute # 0.00 x 10^3/uL 

 Promyelocyte Abs. # 0.00 x 10^3/uL 

 Myelocyte Absolute # 0.00 x 10^3/uL 

 Metamyelocyte Abs. # 0.00 x 10^3/uL 

Differential, Vet. (VDIF) 

 Segmented Neutrophils 39 % 

 Lymphocytes 54 % 

 Monocytes 4 % 

 Eosinophils 2 % 

 Basophils 1 % 

Poikilocytosis / 

polychromasia? no 

 Glucose L 52.0 . 76.0 H 84.0 55 79 mg/dL 

 AST (GOT) . 72.0 L 53.0 . 81.0 57 108 U/L 

 SDH L 9.7 L 10.1 . 18.6 12.2 46 U/L 

 Total Bilirubin . 0.2 . 0.1 . 0.2 0.1 0.4 mg/dL 

 Cholesterol L 85.0 L 80.0 L 87.0 112 331 mg/dL 

 Total Protein . 6.6 . 7.4 . 7.4 6.3 8.5 g/dL 

 Albumin . 3.6 . 3.4 . 3.3 3.2 4.3 g/dL 

 Urea N . 10.0 . 8.0 . 8.0 8 22 mg/dL 

 Creatinine L 0.5 . 0.6 L 0.5 0.6 1.4 mg/dL 

 Phosphorous . 8.8 . 8.1 . 9.0 4.4 9.2 mg/dL 

 Calcium H 10.7 . 9.7 . 10.1 7.9 10.5 mg/dL 

 Sodium . 143.0 . 145.0 . 144.0 140 151 mmol/L 
Chapter V: Animal Health Risks 140 

Animal Cloning: A Risk Assessment DRAFT

 Potassium . 4.9 . 4.9 . 5.1 3.7 5.6 mmol/L 

 Chloride L 99.0 . 105.0 . 101.0 100 109 mmol/L 

 Bicarbonate . 26.0 . 26.0 . 29.0 22 29 mmol/L 

 CK . 179.0 . 134.0 . 172.0 50 271 U/L 

 GGT . 15.0 . 16.0 . 17.0 12 30 U/L 

 Anion Gap H 23.0 . 19.0 . 19.0 13.6 21.6 mmol/L 

 Hemolysis / lipemia? no no no 

d. Developmental Node 4: Reproductive Development and Function 

i. Peer-Reviewed Publications 

Pace et al. (2002) reported that clone Holstein heifers (some transgenic) reached puberty 

at approximately 10 to 11 months of age, within the normal range for their breed. They 

also reported that all 22 clone heifers were inseminated and diagnosed pregnant 

(Table V-9). The heifers calved between 23 and 25 months of age, within the 

recommended range for first parity Holsteins; no details were reported in this study. The 

study would be more useful to address the safety of cloning to the animal clone if some 

of the following questions were addressed: 

• Which of the animals were transgenic? 

• Were cycles of normal length? 

• Did heifers display typical estrous behavior? 

• How many inseminations were needed before pregnancy was confirmed? 

• Did all the heifers calve normally? 

• Did any require assistance at calving? 

• Did lactation initiate normally? and 

• Did they produce adequate quality colostrum? 

Answers to some of the questions listed above were addressed in a study by Enright et al. 

(2002), which reported on the same set of animals as Govoni et al. (2002) and Savage 

et al. (2003). Four non-transgenic Holstein heifer clones of a single donor animal were 

compared to age and breed matched heifers derived from AI. The heifer clones reached 

puberty at a later age than controls (314.7 ± 9.6 days vs. 272 ± 4.4 days), and had higher 

body weights at first estrus (336.7 ± 13 vs. 302.8 ± 4.5 kg). No differences were noted 

between clones and controls in estrous cycle length, development of ovarian follicles, or 

profiles of hormonal changes. Three of the four clones and all four control heifers 

became pregnant following AI. Daily hormone profiles of lutenizing hormone (LH), 

follicle stimulating hormone (FSH), estradiol-17β, and progesterone were similar 
Chapter V: Animal Health Risks 141 

Animal Cloning: A Risk Assessment DRAFT

between clones and controls. The cause of reproductive failure in the one clone could not 

be determined. Its reproductive hormone profiles were similar to the other animals in the 

study, and no physical abnormalities could be found upon veterinary examination, 

although this heifer showed poor signs of estrus. The later age and higher weight of 

clones at time of puberty relative to the controls may have been under genetic control as 

all four animals were derived from the same cow, although no records of age at puberty 

were kept for the source cow. Average age and weight were higher in clones compared to 

the comparator animals in this study, although they were similar to ranges previously 

reported for conventional Holstein heifers (Murphy et al. 1991; Radcliff et al.1997). 

Table V-9: Pregnancy Rates for Clone and Comparator Cattle (Females) 

Study Transgenic 

Status 

Clone Pregnancy Rate 

(fraction) 

Comparator 

Pregnancy Rate 

(fraction) 

Comments 

Pace et al. 2002 Some 22/22 

(1.00) NP 

Enright et al. 2002 None 3/4 

(0.75) 

4/4 

(1.00) 

Cause of pregnancy 

failure in one clone 

not determined 

Lanza et al. 2001 All 24/24 

(1.00) NP 

Conception rate to 

first AI 87.5%, 

remaining pregnant 

after second AI 

Knobil and Neill 

1998 NA NA 0.90 beef 0.95 dairy 

Based on average 

pregnancy rates for 

replacement heifers 

Wells et al. 2004 None 25/30 

(0.83) 

9/10 

(0.90) 

Conception rates to 

two AI services 

NP = not provided 

NA = not applicable 

Table V-9 indicates pregnancy rates37 for heifer clones and comparators. Average 

historical pregnancy rates for conventional beef and dairy replacement heifers are 90 and 

95 percent, respectively (Knobil and Neill 1998). 

 37 Pregnancy rate is defined as the fraction of animals confirmed pregnant per total number of animals in 

the breeding group (i.e., animals available for breeding). Because so many dairy cattle are bred by AI, 

it is also possible to speak in terms of conceptions per insemination, such that conception rate is 

defined as the fraction of animals conceiving per number of animals inseminated. Because most beef 

cattle are bred naturally, the number of times a cow is bred is seldom recorded, so the more general 

term “pregnancy rate” is used. In this risk assessment, “pregnancy rate” is used for both dairy and beef 

cattle, regardless of method of breeding. 
Chapter V: Animal Health Risks 142 

Animal Cloning: A Risk Assessment DRAFT

Forsberg et al. (2002) reported that a bull clone had matured into a “healthy, fertile bull 

that has sired calves by artificial insemination and in vitro fertilization,” although data 

were not provided. Kato et al. (2000) report that one of the clones derived from a 

Holstein cumulus cell was artificially inseminated, conceived, and gave birth to a normal 

calf. 

In a study of a cohort of transgenic cattle clones, Lanza et al. (2001) reported that 24 

heifer clones of various breeds reached puberty between 10 and 12 months of age, with 

body weights ranging from 318 to 365 kg. Twenty-one of the 24 heifers conceived with 

the first insemination, and the remaining three conceived at the second insemination. At 

the time of publication, two calves had been born, and were reported as healthy. 

In a recent study, Shiga et al. (2005) reported on the semen quality of two clones of a 12 

year old Japanese Black bull. Semen was collected beginning when the bulls were 12 

months old, and the study followed these animals through 16 months of age. Ejaculate 

volumes were similar between the two bulls (2.34 and 2.76 mL) but were lower than the 

range for conventional Black bulls (5-8 mL). However, sperm concentration (1202 and 

834 x 106

/mL), pH, and pre-freezing motility (71.4 and 66 percent) were within 

established ranges for conventional bulls of the same breed. Fertility testing using in vitro

fertilization was performed, comparing the two clones to the donor bull. Development of 

embryos to blastocyst was not different between the clones and their donor (23.4 and 28.4 

vs. 30.9 percent). Because few cows were available for breeding, only one clone was 

used to compare pregnancy rates to AI with the donor bull. Only 22 cows were 

inseminated by the clone, compared to 102 cows inseminated by the donor. Nonetheless, 

pregnancy rates were similar between the clone and the donor bull (54.5 vs. 62.7 

percent). Two of the twelve resulting pregnancies to the clone ended in spontaneous 

abortion in mid-pregnancy, compared to five spontaneous abortions (5/64) for cows 

pregnant by the nuclear donor. 

Similarly, Wells et al. (2004) reported that rates of development to blastocyst for 

embryos fertilized in vitro by sperm from six bull clones were similar to blastocyst rates 

for four non-clone bulls (range 10-25 percent for clones vs. 13-30 percent for 

comparators). In the same report, Wells et al. stated that pregnancy and calving rates of 

heifer clones following two rounds of AI were 83 percent (25/30), and were only slightly 

less than for a small group of conventional heifers (9/10, 90 percent). Gestation length 

was slightly longer for a group of 16 clones compared to nine comparators (287 ± 3 vs. 

281 ± 3 days), but still within the range for the breed (Friesian). The heifer clones calved 
Chapter V: Animal Health Risks 143 

Animal Cloning: A Risk Assessment DRAFT

spontaneously with only minor assistance. Clones exhibited normal maternal behavior, 

and bonded with their calves. 

Tecirlioglu et al. (2005) also evaluated semen quality and fertility of three clones and 

their Holstein-Fresian donors. All three clones were derived from skin fibroblasts. Two of 

the clones (A-1 and A-2) were derived from a six-year-old bull (donor A) that was 

euthanized shortly afterward due to a spinal injury. The third clone was derived from a 

second donor (donor B, an eight-year-old bull) by SCNT. The embryos were frozen and 

thawed before transfer to recipient heifers. The donors and their clones were housed 

under similar conditions. Semen collection and freezing was performed at the same 

commercial facility for all five bulls according to routine procedures established by the 

facility. Semen was evaluated for volume, concentration, total and progressive motility, 

total, primary and secondary abnormalities. Semen was also used to evaluate in vitro 

fertilization (cleavage rate, blastocyst rate, inner cell mass, trophectoderm38 cell count, 

and total cell count). Differences in semen volume were inconsistent, with Donor A and 

Clone B-1 having lower volumes than Clones A-1, A-2 and Donor B. However, sperm 

concentrations and motility measures were similar among clones and donors. Sperm 

morphology (measures of abnormalities) was within acceptable limits for all five bulls (> 

80 percent morphologically normal sperm). In vitro fertilization rates (expressed as 

cleavage rate by 48 h) were higher for clones than for their donors; however, there were 

no differences in rates of blastocyst formation between clones and donors, except Clone 

A-2, which had a higher development to blastocyst than the other four bulls. There were 

no differences in embryo quality (as measured by cell counts of inner cell mass (ICM), 

trophectoderm (TE) and total cell number) between embryos of clones and donors. 

Pregnancy rates and pregnancy losses were compared only between Donor B and Clone 

B-1. Individual IVF embryos from Donor B (n=40) and Clone B-1 (n=37) were 

transferred to synchronized heifers. Heifers were examined for pregnancy at 30, 120 and 

240 days of gestation. Number of live calves and calf survival per sire were also reported. 

Pregnancy rates at day 30 were similar between donor (16/40) and clone (17/37), as were 

pregnancy losses measured from 30 to 240 days of gestation (2/40 vs. 5/37). All heifers 

pregnant at day 240 delivered live calves, and there was no postnatal mortality. The 

authors noted that there were no phenotypic abnormalities among the progeny, even 

though all the calves were produced via IVF and delivered by C-section. 

In a companion study to Chavatte-Palmer et al. (2004), Heyman et al. (2004) reported on 

reproduction and lactation of 17 Holstein clones compared to age-matched, AI-derived 

 

38 In the blastocyst stage of development, embryonic cells segregate into two types: the inner cell mass is 

destined to become the fetus, while the trophectoderm cells become the placenta. 
Chapter V: Animal Health Risks 144 

Animal Cloning: A Risk Assessment DRAFT

half-siblings maintained under the same conditions. Only preliminary data from three 

females were available at the time of publication. The authors reported that milk yields 

were similar for the three clones compared to the non-clone comparators (9341 ± 304 vs. 

8319 ± 1800 kg for 305 day lactation). Somatic cell counts (SCC, an indicator of 

mammary gland health) were assessed monthly throughout the first lactation in clones 

and comparators. Mean SCC for clones and comparators were 116 ± 103 x 103 vs. 113 ± 

50 x 103 cells/mL, and were not statistically different. These values are well below the 

point at which animals would be judged to have subclinical mastitis (1,000 x 103

cells/mL). 

Tian et al. (2005) also reported first lactation milk yields and SCC for four clones and 

their non-clone comparators, indicating that lactation curves were similar for both groups. 

Total milk production for the first lactation was not different between clones and 

comparators (8646 ± 743.8 vs. 9507.8 ± 743.8 kg). One clone gave birth prematurely to a 

stillborn calf, did not have complete udder development, and produced approximately 30 

percent less milk during her first lactation compared to her clone mates. Typically, the 

udder develops during the last month of pregnancy, so lack of complete development 

following premature delivery is not unusual. Overall, SCC was low for both clones and 

comparators (based on Figure 2b of the paper: ~40 x 103

 vs. 35 x 103 cells/mL). 

Heyman et al. (2004) also reported on the fertility and reproduction of male and female 

clones. Three clones of an eight year old bull were enrolled in an AI center at 

approximately 12 months of age, and semen collection was initiated when the clones 

were between 13 and 15 months of age. Clones and their semen were handled the same as 

other bulls at the center. Sperm from the three clones and the nuclear donor were 

compared. Percentages of normal sperm were not statistically different between the 

nuclear donor and the three clones (See Table V-10). Preliminary IVF trials using the 

sperm of these four animals indicated that a lower concentration of sperm from the clones 

was needed to achieve a similar fertilization rate to that of the nuclear donor. Given the 

age of the nuclear donor (approximately 9 years old at the time of this phase of the 

study), this is not surprising. Cleavage rate and development to blastocyst were not 

statistically different between the nuclear donor and the clones (see Table V-10). Only 

one of the clones (Clone #2) was used for comparison of AI with the nuclear donor. 

Overall pregnancy rate for the clone was 65 percent (41/63 inseminated). Two 

pregnancies were lost by day 90 of gestation, representing a 5 percent loss, similar to that 

reported by Thompson et al. 1998 for IVF embryos. Only 26 of the pregnancies were 

allowed to go to term, resulting in 25 live calves, and one premature stillborn. Average 

birth weight of the progeny was 36 ± 2 kg, below average for the breed. The authors 
Chapter V: Animal Health Risks 145 

Animal Cloning: A Risk Assessment DRAFT

attributed this to the fact that the nuclear donor was considered an “easy calving” bull, 

indicating that he was selected because of his ability to produce smaller calves. 

 

Table V-10: Results of IVF for nuclear donor and three clone bulls. 

Animal Normal sperm (%) Cleavage rate1 Blastocyst rate 

Donor 86.5 273/363 (75.2) 108/273 (39.6) 

Clone 1 85.0 244/386 (63.2) 93/ 244 (38.1) 

Clone 2 77.5 207/381 (54.3) 72/207 (34.8) 

Clone 3 67.0 196/359 (54.6) 80/196 (40.8) 

1

 Cleavage rate and blastocyst rate are expressed as number of embryos cleaved or forming blastocysts/ 

 total number of embryos. Percentages are in parentheses. 

Results of studies on reproduction in female clones is still preliminary; however, Heyman 

et al. (2004) reported that female clones began cycling at about 10 months of age, and 

exhibited estrous behavior by 12 months of age, within the normal range for Holstein 

heifers. Ten female clones were bred by AI to a non-clone bull, and all conceived and 

produced live, apparently normal progeny. Birth weight of progeny was 43.9 ± 4.1 kg, 

and gestation length was 281.1 ± 3.9 days, within the normal range for Holstein cattle. 

ii. Unpublished Data 

In response to CVM’s request for additional data on reproductive maturity of clones, 

results were submitted on semen evaluations of four post-pubertal bull clones. Semen 

was collected by a commercial reproduction service, from May 15, 2003 through June 19, 

2003. Age of the bulls at time of sampling was not recorded. Bulls were collected three 

times daily, approximately every three days during the observation period. Data consists 

of hand-written notes provided by the technician, and includes information on semen 

volume, concentration, and percentage of normal sperm in samples. Sperm motility was 

not presented in these reports. Percent normal sperm was not assessed in all samples. A 

summary of the results (means, minimum and maximums) is in Table V-11. The 

complete table (Table V-20) is presented at the end of this chapter. Because the original 

data was sent as a fax, some of the hand written notes may not be accurately transcribed. 

CVM attempted but was unable to establish contact with the service to clarify these 

notes. 
Chapter V: Animal Health Risks 146 

Animal Cloning: A Risk Assessment DRAFT

Table V-11: Summary results of semen evaluation of four bull clones. 

Clone # Volume (ml) Concentration (x106) Normal Sperm (%) 

 mean max min mean max min mean max min 

1 4.1 4.9 3.0 169.5 100 276 5.0 8.0 2.0 

2 3.8 6.5 3.0 686.7 1870 307 51.0 71.0 25.0 

3 5.0 7.9 3.5 712.1 1581 396 69.5 76.0 62.0 

4 4.6 6.9 3.0 730 1649 73 63.9 80.0 54.0 

Reference ranges differ somewhat, but in general normal ranges for ejaculate volume are 

from 4 to 15 mL, sperm concentrations from 800 to 1200 x 106

 sperm/mL, and percent 

normal sperm range from 65 to 95 percent for bulls (Sorenson 1979; Beardon and Fuquay 

1980; Hafez and Hafez 2000). Based on these data, unless clone #1 was very young, he 

likely would have failed a breeding soundness exam, due to the very low concentration 

and percentage of normal sperm in the samples. Clone #2 might be considered marginal, 

and depending on other, unrecorded variables such as motility, and the perceived value of 

his genetics might have been judged acceptable. The other two clones appear to have 

acceptable semen, based on the limited data presented. 

Galli et al (2003 unpublished data) also presented data to CVM on three clones of a 

Holstein bull as a follow-up to their 2002 study on cloning (Table V-12). Scrotal 

circumferences of two of the clones (clones 1 and 2) were similar to the expected range 

for bulls 18-24 months old (31 and 33 cm, respectively, at 22 months old vs. 32-33 cm 

for 18 to 24 month old bulls). Semen quality measurements on two of the clones (clones 

2 and 3) were also considered within the normal range for young bulls, although only 

data on volume (5.27 and 3.35 ml) and sperm concentration (691 and 736 million/ml) 

were presented. 

Table V-12: Scrotal circumference and semen production of clones from Galli et al. 2003 

unpublished data. 

Scrotal 

Circumference 

(cm) 

Collections 

(#)1 

Ejaculates 

(#) 

Average 

volume (ml) 

Concentration 

(x106

/ml) 

Clone 1 31 

Clone 2 33 17 32 5.27 691 

Clone 3 11 19 3.35 736 

1

 Semen was collected at irregular intervals 
Chapter V: Animal Health Risks 147 

Animal Cloning: A Risk Assessment DRAFT

Semen with ≥ 50 percent motility were frozen and thawed. Post thaw motility averaged > 

40 percent. Semen from clone # 2 resulted in a 75 percent in vitro fertilization rate. 

Semen from this clone was also used to test AI pregnancy rates on four farms. The total 

number of cows bred (n = 63) was small, and no contemporaneous comparator was used, 

so the value of this data is limited, and effects of individual farm management cannot be 

assessed. With these caveats in mind, the results of these tests by farm are presented in 

Table V-13. 

Table V-13: Artificial insemination results for one clone bull (from Galli et al. 2003 unpublished 

data). 

Farm Cows bred Cows pregnant Pregnancies lost Pregnancy rate % Loss rate % 

1 30* 22 2 73 9 

2 20 10 0 50 0 

3 3 1 0 33 0 

4 10 8 0 80 0 

total 63 41 2 65 5 

*Results for two rounds of insemination were presented. It is not clear whether any of the individual cows 

were bred twice. 

Pregnancy rates to AI in cattle vary considerably, and are affected by multiple factors, 

such as the ability of farm personnel to detect cows in heat, appropriate timing of 

insemination relative to the onset of heat, and environmental, production and nutritional 

factors. Studies in U.S. dairy cattle indicate that overall pregnancy rates to first AI are 40 

percent or less (Lucy 2001; El-Zarkouny et al. 2004). Given the small number of cows in 

this study, and the lack of a contemporaneous comparator to assess the influence of farm, 

definitive conclusions are not possible. However, overall pregnancy rates to this bull 

clone do not appear worse than the U.S. average. 

e. Developmental Node V: Post-Pubertal Maturation and Aging 

i. Peer-reviewed literature 

There are limited data on concerns related to aging and longevity of conventionallyderived cattle. As mentioned previously, McEvoy et al. (2000) noted that IVP-derived 

calves diagnosed as suffering from LOS at birth had abnormally large hearts compared to 

in vivo derived calves when slaughtered at 13 months of age, although the LOS calves 

appeared to grow normally and were not larger than in vivo controls. This group 

questioned whether the in vitro process could affect long-term animal health and 
Chapter V: Animal Health Risks 148 

Animal Cloning: A Risk Assessment DRAFT

longevity. No further details were available to indicate whether the enlarged hearts were 

functionally abnormal, nor were any follow-up studies identified. 

Wells et al. (2004) conducted a retrospective analysis of cattle clones that were generated 

through SCNT at AgResearch in New Zealand to determine their long-term survival. 

They found that 133 (13 percent) calves were born from 988 SCNT embryos transferred 

into recipient cows. Sixty seven percent of these calves (89 animal clones) survived to 

weaning (3 months of age) and 81 percent of the calves (72 animal clones) survived postweaning, with the oldest animal being 4 years of age at the time of publication of their 

article. They estimated the annual mortality rate in cattle cloned from somatic cells to be 

at least 8 percent. The reasons for death were variable, including euthanasia due to 

musculoskeletal abnormalities (4 animals), bloat (2 animals), ryegrass staggers (2 

animals), misadventure (2 animals) and one case each of anemia, heart failure, kidney 

failure, ruminal acidosis, lungworm, clostridia, and overfeeding on grain supplement. 

Some of these deaths were preventable. The musculoskeletal abnormalities included 

animals with severely contracted flexor tendons and those displaying chronic lameness, 

particularly in milking cows. In surviving cattle clones, blood profiles and other 

indicators of general physiological function such as growth rate, reproduction, rearing of 

offspring, and milk production were all within the normal phenotypic ranges. 

Wells et al. (2004) also reported on hematology and clinical chemistry of nine heifer 

clones and their progeny at two years of age. Heifer clones were compared to nine nonclones and a published reference range. Of the 13 hematological values measured, clones 

were within the range of their contemporary comparators for nine values. White blood 

cell counts (4.57 ± 0.48 vs. 6.91 ± 1.16 x 109

/L), lymphocytes (3.13 ± 0.31 vs. 4.93 ± 

0.92 x 109

/L), and eosinophils (0.11 ± 0.09 vs. 0.32 ±0.18 x 109/L) were lower for clones 

than contemporary comparators, but within the published range. Basophil counts were 

higher for clones than comparators (0.08 ± 0.07 vs. 0.01 ± 0.03 x 109/L), but still within 

the reference range. Of the 15 clinical chemistry values analyzed, 14 of the values for 

clones were within the range of their contemporary comparators. Creatine kinase, the 

only value that was outside the comparator range (191 ± 136 vs. 112 ± 76.4 IU/L) was 

within the published reference range (0-370 IU/L). 

The Chavatte-Palmer et al. (2004) study reported that of the 38 clones surviving the 

juvenile period (past six months of age), 36 were still alive among the older clones (aged 

15 months to four years). Cause of death was reported for only one of the clones that 

died: apparent heat stress during an unusually hot summer, approximately one week after 

her second calving. Twenty of the clones are currently enrolled in a long term health 
Chapter V: Animal Health Risks 149 

Animal Cloning: A Risk Assessment DRAFT

study with 20 AI comparators. This study has not been completed, but preliminary 

reports indicate that so far the only observed clinical sign has been fungal lesions of the 

skin, which have occurred in both clones and controls. The authors state that they have 

not yet evaluated whether the lesions occur more frequently in one group or the other. 

Batchelder (2005) reported the sudden death of a Holstein heifer clone at 25 months of 

age. On necropsy, the heifer was diagnosed with severe trace mineral (selenium and 

copper) deficiency. Other cattle grazing the same pasture were clinically normal. As a 

young animal, this clone was reported with frequent, mild left-sided bloat. Clones in this 

and other studies (Wells et al. 2004; Cyagra 2003) have also been reported with bloat and 

other gastro-intestinal tract problems. Two other clones in the Batchelder (2005) study 

were reported as healthy at 19 months of age. These two surviving animals required little 

supportive care at birth; however, both animals required umbilical surgery. 

ii. Cyagra Data: 6-18 Month Cohort 

Both the veterinary examination and laboratory data indicated that clones in the 6-18 

month cohort were healthy and normal, and that in general, laboratory values for this age 

cohort were more similar to the Cornell laboratory reference range than the younger age 

groups (see Appendix E). 

iii. Unpublished data 

In response to CVM’s request for data concerning this developmental node, data on two 

heifer clones, approximately 14 months old, was submitted to CVM (see Table V-14). 

These data consist of Certificates of Veterinary Inspection, results of serological testing 

for Bovine Leucosis Virus (BLV) and Bovine Viral Diarrhea (BVD), and hematology 

from the state of Wisconsin. Both heifers tested negative for BLV and BVD. According 

to the hematology report, both heifers had red cell distribution widths (RDW) slightly 

below the reference range used by the testing laboratory (22.4 and 24.0 vs. range of 26.0-

30.0 percent). As discussed earlier in this chapter and in Appendix E for Cyagra clones, 

RDW is only indicative of a health problem (anemia) when coupled with primary 

indicators such as low red blood cell count (RBC), hemoglobin and/or hematocrit. As all 

other hematology values were within the reference range, there is no evidence to indicate 

an underlying health problem in these animals. 
Chapter V: Animal Health Risks 150 

Animal Cloning: A Risk Assessment DRAFT

Table V-14: Hematology and Clinical Chemistry for Two Holstein Heifer Clones 

 Heifer #1 Heifer #2 RMIN RMAX Units 

Date Collected 12/10/2000 12/11/2001 

Hemogram-Vet 

 Red Blood Cell Count . 7.43 . 7.81 5 10 x 10^6/uL 

 Hemoglobin . 11 . 11.7 8 15 g/dL 

 Hematocrit . 30.6 . 32.7 24 46 % 

Mean Corpuscular 

Volume . 41.2 . 41.8 40 60 fL 

Mean Corpuscular 

Hemoglobin . 14.8 . 15 11 17 pg 

Mean Corpuscular Hgb 

Conc. . 35.9 . 35.9 30 36 g/dL 

Red Cell Distribution 

Width L 22.4 L 24 26 30 % 

 Platelet Count . 449 . 322 230 690 x 10^3/uL 

 White Blood Cell Count . 7.1 . 7.2 4 12 x 10^3/uL 

Seg. Neutrophil Absolute 

# . 1.99 . 1.66 0.6 4 x 10^3/uL 

Banded Neutrophil 

Absolute # . 0 . 0 0 0.12 x 10^3/uL 

 Lymphocyte Absolute # . 3.91 . 4.39 2.5 7.5 x 10^3/uL 

Act Lymphocyte 

Absolute # . 0 . 0 x 10^3/uL 

 Monocyte Absolute # . 0.78 . 0.72 0.03 0.84 x 10^3/uL 

 Eosinophil Absolute # . 0.43 . 0.36 0 2.4 x 10^3/uL 

 Basophil Absolute # . 0 . 0.07 0 0.2 x 10^3/uL 

 Other Absolute # . 0 . 0 0 0 x 10^3/uL 

 Blast Absolute # 0 0 x 10^3/uL 

 Promyelocyte Absolute # 0 0 x 10^3/uL 

 Myelocyte Absolute # 0 0 x 10^3/uL 

Metamyelocyte Absolute 

#s 0 0 x 10^3/uL 

Differential, Vet. 

 Segmented Neutrophils 28 23 % 

 Lymphocytes 55 61 % 

 Monocytes 11 10 % 

 Eosinophils 6 5 % 

 Basophils 1 % 

 Morphology * * % 

Poikilocytosis / 

polychromasia? no no 
Chapter V: Animal Health Risks 151 

Animal Cloning: A Risk Assessment DRAFT

f. Progeny of Bovine Clones 

Many clones have been bred and have been at least reported as having given birth. Lanza 

et al. (2000) reported that a transgenic cow clone had given birth, and her offspring was 

growing normally. Both the University of Connecticut (Enright et al. 2002) and Infigen 

groups (Pace et al. 2002) have reported breeding and subsequent calving of several of 

their cow clones, but no information on the health status of these progeny has been made 

available. 

Galli et al. (2003 unpublished data) presented limited data on three progeny of bull 

clones. The three calves (two female and one male) were born following normal 

gestations (271 to 280 days) and were within the normal birth weight range for Holstein 

cattle (42 to 45 kg). The presenters stated that they observed no abnormalities in the 

progeny of the bull clones. 

Shiga et al. (2005) reported on 10 calves of a Japanese Black bull clone. The clone 

produced three female and seven male calves by AI. Female progeny weighed on average 

33.2 ± 2.0 kg, and male progeny weighed 32.3 ± 4.1 kg at birth, within the normal range 

for Japanese Black cattle, and similar to the birth weights of calves of the nuclear donor 

(30.7 ± 3.5 kg for females and 34.0 ± 4.9 kg for males). No additional information was 

available on the health and development of the progeny in this study. 

Wells et al. (2004) produced 52 progeny of clones via natural mating or AI with 

conventionally bred bulls. According to their report, 85 percent of these calves were alive 

at 24 hours after birth, compared to 84 percent (27/32) for contemporary comparators. All 

progeny calves were described as “phenotypically normal.” Wells noted that this was 

similar to survival rates of clones within 24 hours after birth, but the progeny required 

less care than clones. Only one progeny animal was lost after the first 24 hours. 

Hematology and clinical chemistry of 15 progeny between the ages of one and three 

years were compared to conventionally derived age-matched cattle and the same 

published range that was used for comparing two-year-old clones. One limitation of these 

data is that only three contemporary comparator animals were used, thus possibly failing 

to capture the natural variability among cattle. Nine of 13 hematology values for progeny 

of clones were within the range of the three comparators. The four values that fell outside 

the range were MCV (44.6 ± 5.39 vs. 49.7 ± 1.53 fL), MCH (16.1 ± 1.92 vs. 17.7 ± 0.58 

pg), MCHC (363 ±11.5 vs. 354 ± 2.00 g/L), and eosinophils (0.22 ± 0.23 vs. 0.31 ± 0.13 

x 109

/L), however, these differences were small. All hematology values for progeny of 
Chapter V: Animal Health Risks 152 

Animal Cloning: A Risk Assessment DRAFT

clones fell within the published range. For a discussion of the diagnostic value of these 

measurements, see Appendices E and G. Eleven of 15 clinical chemistry values for 

progeny fell within the range of contemporary comparator values. Creatine kinase (136 ± 

98.1 vs. 168 ± 133 IU/L), AST (45.7 ± 7.49 vs. 36.7 ± 6.43 IU/L), and GDH (8.40 ± 6.99 

vs. 3.33 ± 0.58 IU/L) were higher in progeny of clones, and creatinine (101 ± 37.3 vs. 

138 ± 11.5 μmol) was lower in progeny than contemporary comparators. All clinical 

chemistry values for progeny of clones were within the published range. Interestingly, the 

average creatinine value for comparators was slightly higher than the published range 

(138 ± 11.5 vs. 55-130 μmol). 

Similar to Wells et al. (2004), Heyman et al. (2004) reported that 25 live-born progeny of 

a clone bull were physiologically normal at birth. Progeny calves and their placentae did 

not exhibit any of the phenotypic defects sometimes noted for clones. Progeny of 10 

female clones bred by AI to a non-clone bull weighed on average 43.9 ± 4.1 kg at birth, 

within the range of birth weights for their breed. All progeny calves were alive and 

appeared normal. Additionally, three female clones were bred by natural service to a 

male clone and also produced three apparently normal calves. 

g. Summary for Health of Bovine Clones and Their Progeny 

Based on a review of the literature, the SCNT process in cattle is associated with 

increased incidences of early pregnancy loss or later-term spontaneous abortion of clone 

embryos and fetuses. Other identified hazards for surrogate dams of bovine clones are 

hydrops and dystocia. The risk of developing either of these complications appears to be 

both species- and laboratory-dependent. Not all cases of hydrops in clone-bearing 

pregnancies develop into a significant complication or threat, but severe hydrops 

conditions, when not diagnosed early, may result in the death of the surrogate dam and 

the clone. Large Offspring Syndrome increases the risk of dystocia, and may be related to 

the development of hydrops. Neonatal death rates for cattle clones currently average 

approximately 20 percent. Dystocia may be the most influential factor on calf mortality, 

due to trauma of difficult labor and emergency C-section; however, abnormal organ and 

musculo-skeletal development also appear to play in important roles. Three calves 

generated by Cyagra, although surviving the early neonatal period, died during the 

juvenile period due to either congenital abnormalities or failure to thrive. The limited data 

suggests that there are no adverse effects on the reproductive health of cattle clones, 

although this tentative conclusion must be tempered by the small number of available 

studies. Only one report of apparent reproductive failure in a female Holstein heifer clone 

has been published. Data on post-pubertal maturation and aging indicate that as surviving 
Chapter V: Animal Health Risks 153 

Animal Cloning: A Risk Assessment DRAFT

clones approach maturity, they experience fewer health problems and are physiologically 

similar to non-clone comparators. Among older clones that die or are euthanized, health 

problems appear to be related to pre-existing conditions (musculo-skeletal defects, GI 

tract problems) already identified during the perinatal and juvenile periods. Progeny of 

cattle clones do not exhibit LOS, and appear to grow and develop normally. 

2. Swine 

Survival of live-born swine clones from various studies is summarized in Table V-15. As 

with cattle, relatively few studies included contemporary comparators, historical data 

from various references and data bases were also incorporated into the table to provide 

context. 

Table V-15: Survival Rates of Live-Born Swine Clones and Comparators 

Reference Transgenic 

Status 

Surviving/Total 

Live-Born Clones 

(fraction)1

Surviving /Total 

Live- Born 

Comparators 

(fraction) 

Comments 

Betthauser et al. 

2000 None 4/4 (1.00) NP 

Bondioli et al. 2001 All 2/2 

(1.00) NP Described in Chapter VI 

Boquest et al. 2002 None 1/2 

(0.50) NP 

De Sousa et al. 2002 None 1/1 

(1.00) NP 

Onishi et al. 2000 None 1/1 

(1.00) NP 

Polejaeva et al. 2000 None 5/5 

(1.00) NP 

Walker et al. 2002 None 27/28 

(0.96) NP 

Yin et al. 2002 None 8/8 

(1.00) NP Described in Chapter VI 

USDA/NAHMS 

2001 

(6/00 – 7/00) 

NA NA 0.89 

Historical data from 

animals produced by AI 

and natural service in 

commercial operations 

1 Survivors through the Juvenile Period/Live births 

2 NP = not provided; data not available 

Transgenic Status: All = All of the clones cited in the publication are derived from transgenic donor cells, Some = 

Some of the clones cited in the publication are derived from transgenic donor cells, None = None of the clones cited 

in the publication were derived from transgenic donor cells. 
Chapter V: Animal Health Risks 154 

Animal Cloning: A Risk Assessment DRAFT

a. Developmental Node 1: Pregnancy and Parturition 

i. Pregnancy 

In swine, a litter-bearing species, at least four viable embryos are needed during early 

gestation for the sow to carry a pregnancy to term (Polge et al. 1966). Fetal death in 

conventionally bred swine was reported in one study as occurring between days 35 of 

gestation and term (van der Lende and van Rens 2003). Peaks in swine fetal mortality 

appeared to coincide with changes in placental growth around day 35, from days 55-75, 

and again around day 100 of pregnancy (van der Lende and van Rens 2003). Overall fetal 

mortality in this study was 9.2 percent, with 46.9 percent of gilts (102/192) having some 

evidence of dead or mummified fetuses at farrowing (birth or parturition). Some fetal loss 

is expected in swine, and may be a function of uterine capacity (the available room in the 

sow’s uterus) (Vonnahme et al. 2002). No health problems were reported for sows in 

these studies. Typically the sow is able to resorb or expel non-viable embryos and fetuses 

without ill-effects. 

A study of a commercial swine herd found that uterine capacity becomes limiting at 

approximately day 36 of pregnancy (Vonnahme et al. 2002). Uterine capacity and fetal 

survival to term were more dependent on placental size and efficiency than the size of the 

fetus (Vonnahme et al. 2002). In this study, smaller placentae were associated with larger 

numbers of viable piglets born per litter, while individual fetuses with large, less efficient 

placentae generally did not survive. 

It is difficult, then, to draw conclusions regarding fetal loss in clone-bearing swine 

pregnancies. Betthauser et al. (2000) reported the birth of four live pigs from two sows 

(two pigs per sow) following transfer of 100 to 300 clone embryos plus 100 IVF embryos 

per sow. Similarly, Onishi et al. (2000) reported the birth of a single clone pig after 

transfer of 110 clone embryos per recipient, and Polejaeva et al. (2000) reported the birth 

of 5 clone pigs in one litter following transfer of 100 clone embryos per recipient. None 

of the studies reviewed indicated health problems in the surrogate dams. 

ii. Parturition 

Studies in non-transgenic swine clones did not report any complications with delivery 

(Betthauser et al. 2000; Onishi et al. 2000; Polejaeva et al. 2000; King et al. 2002; Walker 

et al. 2002). During discussions with CVM, clone producers indicated that agalactia 

(failure to lactate) was noted in sows giving birth to piglet clones (see CVM 

Memorandum I at www.fda.gov/cvm/cloning.htm). 
Chapter V: Animal Health Risks 155 

Animal Cloning: A Risk Assessment DRAFT

b. Developmental Node 2: Perinatal Period 

i. Peer-Reviewed Publications 

Incidence of illnesses from all causes for conventional pigs was greatest during the first 3 

days after birth (45.2 ± 2.4 percent of all pigs in this age group), with scours (diarrhea) 

being the most prevalent cause of illness (52.7 ± 4.9 percent of all reported illnesses) 

(USDA/NAHMS 1992). Preweaning mortality rates among pigs averaged 11.0 ± 0.3 

percent of herds observed (USDA/NAHMS 2001). The principal cause of preweaning pig 

deaths was due to being laid on by the sow (52.1 ± 2.0 percent of all deaths). Causes of 

death that might be attributed to infection, such as scours (9.3 ± 1.4 percent of all deaths) 

and respiratory illness (3.0 ± 0.5 percent of all deaths) were less prevalent, possibly due 

to biosecurity measures employed at most swine operations (USDA/NAHMS 2001). 

Because swine are a litter-bearing species, dystocia is less common, and was not cited as 

a cause of pig death in the USDA study (USDA/NAHMS 2001). 

Swine are the most recent of the livestock animal species considered in this assessment to 

be cloned. In general, success rates from the studies evaluated (as measured by number of 

viable offspring) are low even when compared to reports of cloning in other species. 

Most pregnancies fail to reach term, despite efforts to support surrogate sows hormonally 

or with co-transfer of IVP or parthenogenic39 embryos. There are few detailed 

descriptions on health and vitality of neonatal non-transgenic swine clones available in 

the literature, although the few studies that report successful births claim that pigs are 

typically normal and healthy. 

Betthauser et al. (2000) reported the birth of four male pigs in two litters cloned from 

cultured fetal fibroblasts. In this study, 100-300 SCNT embryos and up to an additional 

100 IVP embryos were co-transferred to each sow. The confirmed pig clones were 

reported as healthy, but no details were provided on what, if any, measurements were 

taken. Onishi et al. (2000) reported the birth of one confirmed pig clone after two 

separate attempts. None of the embryo clones cultured to blastocyst stage developed to 

term in this study. The single surviving clone pig was born after the second experiment, 

when two- to eight-cell stage embryos were transferred to sows. In both experiments, 

clone embryos were co-transferred with non-clone “helper” embryos. The single clone 

pig was reported as apparently healthy and normal, and weighed 1.2 kg at birth, within 

 39 Parthenogenesis is a form of reproduction in which an unfertilized egg develops into a new individual, 

occurring commonly among insects and certain other arthropods. Parthenotes typically do not develop 

into a viable fetus in mammals. 
Chapter V: Animal Health Risks 156 

Animal Cloning: A Risk Assessment DRAFT

the normal range for its breed. The placenta for this pig weighed 0.3 kg and was reported 

to be anatomically normal. 

Polejaeva et al. (2000) reported the birth of five pigs in a single litter following double 

SCNT (re-cloning)40 and hormonal support of pregnancy. Average birth weight of the 

clones was 1.24 kg, which was 25 percent lower than average birth weight for non-clone 

pigs of this same line (1.64 kg). Clone pigs were delivered by C-section, although no 

explanation was given for choosing this method of delivery. 

Walker et al. (2002) reported greater success with hormone supplemented pregnancies 

and large numbers of transferred SCNT embryos per gilt. All transferred embryos were 

SCNT clones, as opposed to previous studies which co-transferred so-called “helper 

embryos.” In this study, four of five recipients carried pregnancies to term, producing 

litters of five to nine pigs each, for a total of 28 pig clones. Only one of these was 

stillborn, and one was reported to be born with anal atresia (absence of an anal opening), 

necessitating euthanasia. Actual birth weights of surviving pigs were not reported, but the 

authors mentioned that they were small at birth. The authors commented that variability 

in birth weight could be attributed to uterine effects, and that none of the pigs displayed 

signs of LOS. They also stated that no placental abnormalities were noted. Similar reports 

of low birth-weight pigs have been recorded by other researchers (Boquest et al. 2002), 

although actual birth weights were not presented. 

Park et al. (2004a and 2005) reported the death of 22 of 35 live born SCNT clones within 

the first week of life. Several health problems were noted including cerebromeningitis,41

diarrhea, leg abnormalities, Leydig cell hypoplasia 42 and unknown factors. Gestation 

length was similar for the clones (117.82 + 1.94 days) and the comparators (115 + 2.4 

days). However, the authors noted low birth weights for the clones (0.80 + 0.29 kg) 

relative to the comparator pigs (1.27 + 0.30 kg). The authors attempted to characterize the 

causes of death of pig clones and noted evidence of problems with blood flow and 

cerebromeningitis. Many bacterial diseases already established in the swine industry can 

result in similar clinical signs. Although the investigators tested for the presence of 12 

types of microorganisms, they did not detect any infections. However, these clinical signs 

are commonly noted with bacterial infections which were not tested in this study 

including Actinobacillus suis, Escherichia coli septicemia, Haemophilus parasuis, 

 40 Re-cloning, double SCNT, and serial SCNT, are synonyms for the process of generating a clone of a 

clone. 41 Inflammation of the membrane covering the cerebrum or anterior portion of the brain. 

42 The Leydig cell is a type of cell found in the testes and is the primary source of testosterone. Leydig cell 

hypoplasia results in failure to generate sufficient testosterone for development of secondary sex 

characteristics, and affects spermatogenesis resulting in infertility. 
Chapter V: Animal Health Risks 157 

Animal Cloning: A Risk Assessment DRAFT

Salmonella spp., and Streptococcus suis. Therefore, it is not possible to rule out the 

presence of these organisms as possible sources of the clinical observations noted in this 

study. Also, the authors noted that some of the dead pigs were born weak, which may 

have predisposed them to bacterial or viral diseases which may have contributed to the 

clinical signs observed in the study. Finally, the authors identified low birth weight as a 

possible contributor to the neonatal morbidity. This study identified several clinical 

observations in neonatal swine clones. However, all of the clinical observations noted 

have been associated with diseases commonly reported in the swine industry (Straw 

1999). 

ii. Viagen dataset 

A complete discussion of the Viagen dataset is provided in Appendix F. For neonatal 

swine clones (n=7) only birth weights were available. Swine clones in this dataset were 

smaller at birth than AI comparators (n=16) (1.12 vs. 1.73 kg). It should be noted that 

these swine clones were delivered by C-section following induced labor on or the day 

before expected parturition, while comparator pigs were farrowed following natural onset 

of labor. Low birth weight of swine clones has also been noted in previous studies 

(Polejaeva et al. 2000; Walker et al. 2002; Boquest et al. 2002). 

iii. Unpublished data 

Additional data was submitted by a commercial cloning company on birth weight and 

average daily gain (ADG) during the first three months of life as well as body 

temperature and heart rate of pigs during the first 2 days after birth. Breed of pigs was not 

identified. Data on body temperature and heart rate was available for five pigs, while 

birth weight and ADG were available for three of the five animals. According to the 

information submitted, two of the five piglets, both from the same litter and weighing 1.0 

kg at birth, died within 48 hours of birth. The cause of death was not provided. The five 

piglets were born in two litters. The data are presented in Tables V-16 and V-17. 
Chapter V: Animal Health Risks 158 

Animal Cloning: A Risk Assessment DRAFT

Table V-16: Birth Weight, Weaning Weight and Average Daily Gain for Three 

Swine Clones 

Litter 

# 

Piglet 

ID # Birth date BW 

(kg) 

Age @ 

weighing Period Weight 

(Kg) 

ADG2 

(KG/day) 

1 1 5/24/2002 1.2 18 17 2.7 0.06 

1 1 5/24/2002 1.2 40 22 10 0.33 

1 1 5/24/2002 1.2 60 20 23.3 0.67 

1 1 5/24/2002 1.2 74 14 31.6 0.59 

1 1 5/24/2002 1.2 94 20 50 0.92 

1 2 5/24/2002 1.4 18 17 2.8 0.08 

1 2 5/24/2002 1.4 40 22 8.6 0.26 

1 2 5/24/2002 1.4 60 20 18.9 0.52 

1 2 5/24/2002 1.4 74 14 25.8 0.49 

1 2 5/24/2002 1.4 94 20 44.2 0.92 

2 4 5/27/2002 1.1 15 17 3.2 0.15 

2 4 5/27/2002 1.1 37 22 10.9 0.35 

2 4 5/27/2002 1.1 57 20 24.6 0.69 

2 4 5/27/2002 1.1 71 14 33.8 0.66 

2 4 5/27/2002 1.1 91 20 51.5 0.89 
Chapter V: Animal Health Risks 159 

Animal Cloning: A Risk Assessment DRAFT

Table V-17: Pulse, Respiration and Body Temperature for Five Swine Clones During First Five Days After Birth 

Respiration Rate Heart Rate Blood Temperature 

Litter # Pig ID # 

D1 D2 D3 D4 D5 D1 D2 D3 D4 D5 D1 D2 D3 D4 D5 

1 1 

48.3 

+/- 

3.8 

36 

+/- 

2.3 

37.9 

+/- 

2.1 

38.9 

+/- 

1.7 

41.3 

+/- 

2.8 

188.6 

+/- 

14.4 

159.8 

+/- 

13.8 

167.4 

+/- 

8.1 

160 

+/- 

6.1 

176.0 

+/- 

5.6 

99.3 

+/- 

1.2 

101.5 

+/ - 

0.2 

101.

9 +/- 

0.3 

101.2 

+/- 

0.4 

101.3 

+/- 

0.3 

1 2 

51.8 

+/- 

5.7 

39.1 

+/- 

2.2 

37.3 

+/- 

1.7 

43.7 

+/- 

3.7 

39.5 

+/- 

1.8 

202.3 

+/- 

7.3 

148.6 

+/- 

14.6 

162.3 

+/- 

5.9 

176.7 

+/- 

6.7 

157.4 

+/- 

4.8 

99.2 

+/- 

1.0 

101.8 

+/- 

0.4 

102.

4 +/-

0.2 

100.8 

+/- 

0.3 

101.3 

+/- 

0.3 

2 3 

64.3 

+/- 

9.8 

36.9 

+/- 

8.3 

NA NA NA 

162.3 

+/- 

10.6 

173.5 

+/- 

2.3 

NA NA NA 

100.6 

+/- 

0.5 

101.1 

+/- 

0.2 

NA NA NA 

2 4 

43 

+/- 

3.0 

34.9 

+/- 

1.2 

NA NA NA 

206.4 

+/- 

13.8 

169.0 

+/- 

5.8 

NA NA NA 

101.0 

+/- 

0.5 

101.1 

+/- 

0.3 

NA NA NA 

2 5 

59.8 

+/- 

5.9 

42.2 

+/- 

3.2 

NA NA NA 

172.6 

+/- 

13.9 

180.4 

+/- 

6.1 

NA NA NA 

98.8 

+/- 

0.5 

101.5 

+/-

0.3 

NA NA NA 

Note: Pigs 3, 4, and 5 were from the same litter. Pigs 1 and 2 were from another litter. Pigs 3 and 5 died shortly after birth. The 

absence of data for pig 4 was not explained. 

Birth weight and ADG vary depending on breed of swine. The breed of swine in this 

dataset was not reported, making interpretation of these data difficult. Likewise, 

interpreting respiration and heart rates in animals not typically handled is problematic, 

since the stress of handling tends to increase respiration and heart rates. Body 

temperatures of the five clones during the first two days are somewhat low; however, as 

noted earlier in this chapter, neonatal swine generally need supplemental heat because 

they lack the ability to thermoregulate. For growth, the available reference values for 

non-clone comparator swine and their progeny presented in the Viagen dataset (Appendix 

F) is instructive. Growth rates in this dataset and the Viagen dataset for non-clone 

comparators are similar. Average heart rate of day old conventional pigs was reported as 

190.75 ± 36.45 bpm in one study (Foster et al. 2001) 

c. Developmental Node 3: Juvenile Development 

i. Peer-reviewed literature 

Among conventional weaned pigs (greater than 21 days old), the total number of illnesses 

reported was 1,721/213,910 pigs weaned in the observation group (0.8 percent). The most 
Chapter V: Animal Health Risks 160 

Animal Cloning: A Risk Assessment DRAFT

commonly reported cause of illness among weaned pigs was nervous system disorders 

(12 percent of illnesses), followed by respiratory problems (10.4 percent). The number of 

weaned pigs dying was reported as 1,906/213,910 pigs weaned (0.9 percent), with most 

common causes of death also attributed to nervous disorders (13.4 percent) and 

respiratory problems (16.6 percent) (USDA/NAHMS 1992). 

Archer et al. (2003a) studied physiological and clinical chemistry markers of swine 

clones. Clones and age- and breed-matched comparators were evaluated at 15 and 27 

weeks of age. Body weights of the animal clones at 27 weeks of age did not differ 

significantly when relative coefficients of variation in body weights were compared. 

Body weights of all the animals overlapped and were within the normal range for the age 

and breed, with the exception of a single clone that was small at birth, and never attained 

the size of its littermates. Teat number was the same for all (6, 6 distribution) except one 

clone piglet (6, 7 distribution). One of the clones also exhibited an unusual hair growth 

pattern (e.g., longer and sparser), which the authors state prompted an examination of the 

histology of the skin. Results of that investigation indicated that with one exception, skin 

morphology showed no unusual variations among the pigs. The exception was a clone 

that exhibited morphology indicative of hyperkeratosis. Whether this was the same pig as 

the one exhibiting the unusual hair pattern is not specified. 

Hyperkeratosis, also referred to as parakeratosis, occurs in naturally bred and AI pigs 

between the ages of 6 and 16 weeks, and is generally associated with zinc and essential 

fatty acid deficiency or excess dietary calcium or phytate (naturally occurring compounds 

in grain that bind certain minerals). Gastrointestinal disorders may also affect zinc 

absorption, and contribute to the development of this condition (Cameron 1999). Other 

possible causes of hyperkeratosis include heredity, and other, non-specific causes of skin 

inflammation (Blood and Radostits 1989). Dermatitis vegetans is the inherited form of 

this disease in swine, and is a semi-lethal recessive gene (Blood and Radostits 1989). The 

inherited form of the disease generally presents before the pig is three weeks old. 

Results of blood clinical chemistries for clones were similar to those of age-matched 

controls. In addition, changes in alkaline phosphatase, globulin and A/G ratio between 15 

and 27 weeks were also similar among clones and controls, and are age appropriate. 

Cortisol levels are more variable among clones and controls, and across time periods 

compared to other measurements, but no consistent trend could be identified. Cortisol is a 

hormone produced in response to stress, and differences may result from individual 

variation in response to handling. 
Chapter V: Animal Health Risks 161 

Animal Cloning: A Risk Assessment DRAFT

For this set of animals, with one exception, no anomalies are present that would appear to 

have any direct impact on animal health. Nutrition-related parakeratosis has been known 

to result in reduced growth and appetite, diarrhea, and vomiting when severe. Mortality 

from this disease is uncommon. Dermatitis vegetans, the hereditary form of this disease 

in swine, may result in death of the pig, or the pig may recover completely (Blood and 

Radostits 1989). This is the only known report of hyperkeratosis occurring in clones. The 

apparently normal status of the clinical measurements indicates that the clones in this 

study possess the same physiological functions as their sexually-derived counterparts. 

In a companion study, Archer et al. (2003b) evaluated behavioral characteristics 

including food preference (to apples, bananas, saltine crackers, and carrots), temperament 

(as judged by time to remove a towel placed on the pig’s head and attempts to escape 

mild restraints (being placed on their backs and being lifted off the ground)), and time 

budgets (the amount of time spent engaged in a particular activity in their pens). The 

results of this study indicated that the behaviors of pig clones were no more homogenous 

than the behaviors of their comparators. The relevance of the study to an evaluation of the 

health of swine clones is that the animals behaved in much the same manner as 

conventional animals, and displayed no behavioral anomalies at the times tested (15-16 

weeks of age for the food trials, 8-9 weeks and 14-15 weeks for the towel test, 7 weeks 

for the restraint tests, and 13-15 weeks for the time budget tests). 

ii. Viagen dataset 

Clones weighed less at slaughter and took 27 days longer to reach slaughter weight than 

their contemporary comparators. This may be due to the fact that clones spent the first 50 

days of life in highly biosecure conditions before being moved to a conventional swine 

facility for the start of the experiment. This would have presented swine clones with a 

significant immune challenge that likely would have slowed growth as they adapted to 

their new environment. 

Three clones were described as “poor-doers:” animals that exhibited slow growth rates 

and other health problems. All three of these animals suffered from periodic or chronic 

scouring along with other health problems (see Appendix F). On average, organ weights 

as a percentage of body weight were lighter for clones than for comparators. Overall, 

swine clones had lower IGF-I and estradiol-17β levels at slaughter compared to nonclone comparators. Other blood values were variable among animals, and did not indicate 

any consistent trends. One clone was diagnosed with a lung adhesion at slaughter. 
Chapter V: Animal Health Risks 162 

Animal Cloning: A Risk Assessment DRAFT

d. Developmental Node 4: Reproductive Development and Function 

Semen collected once a week from a boar clone between 10 months to 14 months of age 

and evaluated in a study conducted by Martin et al. (2004) was reported as having 

motility, sperm concentration and ejaculate volume similar to those of non-clone boars. 

Clone gilts in the same study were reported to show first estrus at 215 + 4 days and 200 +

0.6 days for the two genetic lines in the study. Five clone gilts (four transgenic, one nontransgenic) were inseminated with the semen from the clone boar and all became 

pregnant and farrowed without incident. Gestation length, litter size, proportion of pigs 

born live and birth weights were similar between litters from the clone pigs and litters 

from non-clone pigs. Sixty-five pigs were born as a result of the matings, three of which 

were stillborns (4.5 percent). By way of comparison, the mating of five non-clone 

females to a non-clone boar resulted in 60 pigs born with four stillborns (6.7 percent). All 

live-born pigs born to the clone parents were normal except one pig which had 

contracture of the flexor tendon (arthrogryposis) of both hind limbs. The authors reported 

that the frequency of arthrogryposis was similar to reported estimates for commercial 

swine in Australia. Survival to weaning was similar for both groups with 58 of the 62 live 

born clone offspring pigs surviving (94 percent) and 53 of the 55 non-clone offspring 

pigs surviving (96 percent). 

No other peer-reviewed reports have been identified to date on puberty and reproduction 

in male or female swine clones. However, as part of a large dataset submitted to CVM by 

Viagen, Inc., four clone boars and three comparator boars (one nuclear donor and two AIderived sons of a nuclear donor) were examined for semen characteristics and fertility 

(see Appendix F for the full report). There were no differences between clones and 

comparators in sperm concentration, total sperm count, percent total motility, percent 

progressive motility, or number of sperm abnormalities. Farrowing rates were higher for 

clones than for comparators (73.5 vs. 62.5 percent), although this difference may be due 

to the age of one of the comparators, a five-year old Hamline43 boar used as the nuclear 

donor for three of the clones in this study. Litter size was more variable for clone sires 

than for comparators, and mean litter size for litters sired by clones was slightly smaller 

than for comparator boars (10.94 vs. 11.76 pigs/litter), but were similar to the average 

cited for U.S. commercial swine production (10.66 pigs/litter). 

 

43 “Hamline” refers to a specific crossbred line of swine used by Viagen, Inc. This line was developed by 

crossing various breeds, including Duroc, European Landrace, Pietran, and European Large White swine. 
Chapter V: Animal Health Risks 163 

Animal Cloning: A Risk Assessment DRAFT

e. Developmental Node V: Post-Pubertal Maturation and Aging 

No reports on aging and maturity in swine clones were identified. 

f. Progeny of Swine Clones 

Martin et al. (2004) reported that progeny of male and female clone pigs were born with 

comparable birth weights to non-clones. One offspring of the mating was reported to 

have contracture of the flexor tendon in both hind limbs. The frequency of this 

abnormality was reported by the authors as similar to reported estimates for the 

Australian swine industry. Survival rates to weaning were similar between the offspring 

of the clones and the non-clone offspring (94 percent and 96 percent respectively). 

In a follow-up to the study reported by Archer et al. (2003a) for swine clones, Mir et al. 

(2005) reported on the body weight and blood profiles of female swine clones and 

progeny of swine clones up to 27 weeks of age. To produce progeny for this study, nine 

clone and five comparator gilts were bred to the same non-clone boar. All gilts gave birth 

naturally (spontaneously, vaginally). All pigs were housed under the same conditions, 

and groups were penned together according to age. Blood samples were collected at 15 

and 27 weeks, and pigs were weighed at 27 weeks of age. Although litter sizes for clones 

and comparators were small compared to industry standards (7.78 ± 2.6 and 7.40 ± 3.0 

pigs/litter for clones and comparators, respectively), there were no differences between 

clones and contemporary comparator gilts in this study. There were no differences in 

body weight at 27 weeks between clones, comparators, or progeny of clones and 

comparators. As with the Archer et al. (2003a) study, the ranges in blood values between 

clones and contemporary comparators overlapped for the variables measured. Significant 

differences in blood urea nitrogen (BUN) at 15 weeks and alkaline phosphatase (ALP) at 

27 weeks were noted between clones and comparators as well as clone progeny and their 

comparators. The authors note that other blood variables found to be different between 

clones and comparators (creatinine, phosphorus, and calcium) were not different between 

progeny of clones and their comparators. 

No other peer-reviewed reports have been published to date on progeny of swine clones. 

However, a large study of progeny of swine clones was submitted by Viagen, Inc. The 

study included data from 402 progeny of swine clones and 300 age-matched, geneticallyrelated comparator pigs. For a full description of this study, see Appendix F. All progeny 

in this study were farrowed and raised to slaughter under similar conditions. The 

percentage of animals reaching slaughter age was lower for progeny of clones than for 

comparators (295/402, 73.4 percent vs. 243/300, 81 percent); however, much of this 
Chapter V: Animal Health Risks 164 

Animal Cloning: A Risk Assessment DRAFT

difference can be attributed to the loss of a single litter of clone progeny. When data from 

this litter is excluded, the percentage of neonatal deaths was similar for progeny of clones 

and comparators, and was similar to the averages for commercially raised U.S. swine. 

Abnormalities noted among pigs in this study (e.g., anal atresia and spraddle legs) have 

been documented in the commercial U.S. swine population at similar rates. There were 

no consistent differences between progeny of clones and comparators for blood clinical 

chemistry or hematology, and the few minor differences noted did not indicate any health 

concerns. Growth rates were similar between groups in this study, also. 

g. Summary for Health of Swine Clones and Their Progeny 

Swine carrying clone pregnancies do not appear to experience hydrops and dystocia. 

With the exception of one pig clone born with anal atresia, no other reports of frank 

deformities have been noted for this time period in non-transgenic swine clones, although 

birth weights may be lower in swine clones relative to non-clone comparators. The single 

study reporting high mortality rates in non-transgenic swine clones reported clinical signs 

that may be related to various causes, including infectious disease, which cannot be ruled 

out based on the available data. Swine clones grew more slowly and weighed less at 

slaughter than sexually-derived comparators, although this difference may have been the 

result of immune challenge when clones were transitioned from a biosecure environment 

to a more conventional rearing facility (Viagen 2005). Three clones in the Viagen study 

were described as “poor doers,” with periodic or chronic scouring and other health 

problems that resulted in poor growth. One clone was diagnosed with a lung adhesion at 

slaughter. Reports from Martin (2004) and Viagen, Inc. (Appendix H) indicate normal 

fertility in boar and gilt clones. No reports on post-pubertal maturation and aging of 

swine clones are currently available. Available reports from the literature and the Viagen 

Inc. dataset suggest that progeny of swine clones are not different from pigs derived 

through conventional breeding. The few reports of health problems in progeny of swine 

clones indicate they are not different either in quality or frequency from conventionally 

bred swine. 

3. Sheep 

Compared to other food animal species which have been cloned, data on sheep clones 

and their surrogates are sparse. Table V-18 presents a summary of survival of live-born 

sheep clones from the available literature. 
Chapter V: Animal Health Risks 165 

Animal Cloning: A Risk Assessment DRAFT

Table V-18: Survival Rates of Live-Born Clones and Comparators 

Reference Transgenic 

Status 

Surviving/Total 

Live-Born 

Clones(fraction)1

Surviving /Total 

Live- Born 

Comparators 

(fraction) 

Comments 

Peura et al. 2003 None 1/8 

(0.13) NP 

Wells et al. 1998b None 3/10 

(0.30) NP 

USDA/NAHMS 

2002 

(2/01 – 4/01) 

NA3 NA 0.98 

Historical data 

from animals 

(mostly natural 

mating) in 

commercial 

operations 

1 Survivors through the Juvenile Period/Live births 

2 NP = not provided; data not available 

3

 NA = not applicable 

Transgenic Status: All = All of the clones cited in the publication are derived from transgenic donor cells, 

Some = Some of the clones cited in the publication are derived from transgenic donor cells, None = None 

of the clones cited in the publication were derived from transgenic donor cells. 

a. Developmental Node 1: Pregnancy and Parturition 

i. Pregnancy 

Little information is available on embryo or fetal loss in sheep following natural service 

or ART pregnancies. As noted for cattle, abnormal development of the placenta in clones 

of both embryonic and somatic cell origin is one cited cause of mid- and late-term 

spontaneous abortion in sheep (Wells et al. 1998). Further, Wells et al. (1998) cite too 

few and/or abnormal cotyledons in placentae of sheep clones. Increased fetal weight was 

not associated with increased placental weight in studies of sheep IVP fetuses by Sinclair 

et al. (1999), although these investigators did not examine placental morphology in their 

study. They hypothesized that fetal overgrowth during the last trimester of pregnancy in 

sheep, with associated hypoxia (lack of oxygen) and accumulation of lactic acid, was the 

cause of hydrops in IVP pregnancies. 
Chapter V: Animal Health Risks 166 

Animal Cloning: A Risk Assessment DRAFT

ii. Parturition 

(a) Dystocia 

A similar relationship between dystocia, birth weight, and parity of conventional sheep 

dams was reported by Dwyer (2003) as for cattle (Nix et al. 1998). Ewes (female sheep), 

however, tend to carry twin pregnancies more often than cows, and ewes bearing single 

lambs were more likely to experience dystocia and require assistance during labor than 

twin-bearing ewes. Overall, the incidence of dystocia requiring assistance in Suffolk and 

Scottish Blackface ewes in the Dwyer study was 10.2 percent for twin-bearing ewes 

requiring assistance, and 31.0 percent among single-bearing ewes requiring assistance. 

LOS has been described in sheep derived from IVP pregnancies as well as in SCNTderived pregnancies (reviewed by Young et al. 1998). The incidence of LOS in lambs is 

difficult to estimate, due to the few studies of cloning in this species, the small numbers 

of animals in individual studies, and the variability among breeds for birth weight. In a 

study by Peura et al. (2003), 8/11 clone lambs were more than 20 percent above the 

average birth weight for their breed at time of delivery, and 5/8 large lambs were 

delivered by emergency C-section. Only one of the eight live-born lambs survived (Table 

V-18). This study did not record whether the surviving lamb was delivered vaginally or 

by C-section. 

In another study comparing cloning procedures with other ARTs, an increase in assisted 

deliveries was observed for ewes carrying clone and IVP-derived pregnancies compared 

to AI or natural service pregnancies (Ptak et al. 2002). Delivery was assisted because of a 

lack of adequate uterine contractions and general lack of preparedness for delivery in the 

ewes carrying clone and IVP-derived lambs. 

(b) Other complications 

Ptak et al. (2002) reported that normal maternal behavior was impaired in ewes carrying 

both IVP and clone-derived pregnancies. Ewes carrying IVP or clone embryos did not 

show common signs of labor (increased activity, bleating, contractions), and delayed 

licking neonatal lambs (to bond with lambs, and to stimulate lambs to breathe, stand and 

nurse). Ptak et al. (2002) also reported a lack of expected prepartum changes such as 

cervical dilation and swelling of the vulva in ewes carrying clone pregnancies. In such 

cases, delivery was assisted by administering hormones to induce more typical labor, or 

by C-section. 
Chapter V: Animal Health Risks 167 

Animal Cloning: A Risk Assessment DRAFT

b. Developmental Node 2: Perinatal Period 

Among lambs, the mortality rate for all causes was 2.2 ± 0.2 percent (USDA/NAHMS 

2002), although the report did not indicate the age at which losses were most prevalent. 

According to the USDA report, the principal cause of lamb death was predation (killed 

by predators) (44.1 ± 1.1 percent for all operations). However, in one large study 

comprising 4,511 lambs and their dams of various breeds (Christley et al. 2003), factors 

that had the greatest effect on neonatal mortality were birth weight and blood 

immunoglobulin concentrations. In this study, the mortality rate among neonatal 

singleton lambs was increased in both high and low-birth weight lambs, with the lowest 

death rate associated with a birth weight of about 5.5 kg. The authors suggested that the 

increased mortality rate with increasing birth weight may be attributed to the increased 

risk of dystocia. The relationship appeared to be breed-dependent in this study, with 

single lambs of Suffolk sheep at greater risk of dystocia than multiple lambs, while 

multiple lambs of Dorset sheep were at increased risk of dystocia compared to single 

lambs of this breed. Also in this study, increased serum immunoglobulin levels in lambs 

were associated with reduced risk of death in lambs 2 to 14 days old. 

Studies involving IVP and cloning in sheep report lambs born with many of the same 

clinical signs as noted for cattle clones, including LOS (reviewed by Young et al. 1998). 

Mortality rates were elevated relative to lambs produced by natural service in IVP- , 

BNT-, and SCNT-derived lambs (Campbell et al. 1996; Ptak et al. 2002). Unlike cattle, 

however, there were no differences noted in mortality of lambs produced by IVP and 

nuclear transfer (NT) (Ptak et al. 2002). Mortality for IVP and NT produced lambs was 

significantly higher compared to lambs produced by AI and natural mating in the Ptak et 

al. study. Ptak et al. (2002) compared different glucocorticoid treatments in perinatal 

lambs in an attempt to improve survival. Although mortality of untreated NT-derived 

lambs in this study was higher (~30 percent), mortality of lamb clones treated with 

glucocorticoid (betamethasone) was around 20 percent (based on Figure 3 of Ptak et al. 

2002). Actual numbers of lamb clones in each treatment group was not provided, 

although a total of 22 SCNT-derived lambs were born alive. Another recent study (Peura 

et al. 2003) looked at nutritional status of the oöcyte donor to determine if it had an effect 

on embryo development and lamb survival. Although SCNT embryos from donors on a 

high plane of nutrition had a higher rate of pregnancy initiation, pregnancy loss and 

neonatal lamb mortality was high in this study for both treatment groups. Pregnancy rate 

to term was 17.6 percent (9/51) for the high nutrition group and 5.4 percent (2/37) for the 

low nutrition group. Of the 11 pregnancies that went to term, eight lambs were born alive. 

Four of these lambs died within the first 24 hours. Three more lambs died or were 
Chapter V: Animal Health Risks 168 

Animal Cloning: A Risk Assessment DRAFT

euthanized prior to 30 days. The single surviving lamb, originating from the “high” 

nutrition group, was reported as “thriving” at 15 months of age. 

c. Developmental Node 3: Juvenile Development 

Very little information is available on either conventional sheep or sheep clones for this 

age group. Wells et al. (1998) noted that BNT lambs that survived the neonatal period 

and were raised under varying conditions (indoors or outdoors, winter and spring 

lambing) were apparently healthy, based on blood urea levels (an indicator of kidney 

function, protein metabolism, and hydration) and daily live weight gains. 

d. Developmental Node 4: Reproductive Development and Function 

In the Wells et al. (1998) study, two BNT ram clones were allowed to mate naturally with 

an unspecified number of ewes for proof of fertility. Eight ewes became pregnant and 

produced a total of 15 lambs, which were delivered without assistance. Male lambs were 

reported to weigh 5.2 ± 0.5 kg and females weighed 4.6 ± 0.5 kg, and were not different 

from lambs sired by a non-clone control ram of the same breed mix as the BNT-derived 

rams. No similar data on SCNT-derived sheep were identified. 

e. Developmental Node V: Post-Pubertal Maturation and Aging 

It has been reported in the popular press and elsewhere that “Dolly,” the first adult SCNT 

sheep, showed signs of premature arthritis (Dyer 2002), but no other reports of age 

related illnesses in sheep clones were found. Dolly was subsequently reported to have 

died of complications from a respiratory infection (Powell 2003b). Recent reports in the 

popular press have recorded the death of a relatively young sheep clone in Australia 

(Arlington 2003), although the cause of death for this animal has not been reported. 

Under ideal conditions, sheep may live to 15 years of age. 

f. Progeny of Sheep Clones 

Wells et al. (1998) reported that progeny of a male ram clone (BNT) were born healthy 

and within the expected weight range for their breed mix. 

g. Summary for Health of Sheep Clones 

Data on sheep SCNT clones is scarce and, except for anecdotal reports, do not extend 

beyond the perinatal period. Existing data for Developmental Nodes I and II suggest that 

surrogate ewes and neonatal lamb clones experience similar problems as cattle clones and 
Chapter V: Animal Health Risks 169 

Animal Cloning: A Risk Assessment DRAFT

their surrogates (hydrops, dystocia, LOS). However, given the very few studies that have 

been conducted and the few animals involved, it cannot be determined whether the 

frequency of these abnormalities are elevated compared to other ART in sheep. One 

study (Ptak et al. 2002) indicated that the incidence of LOS in lamb clones was not 

different from IVP lambs, although actual numbers of lambs with LOS for each ART 

method was not reported in this study. Data for Developmental Nodes III and IV and 

progeny are only available for BNT clones, and only from one study. The only 

information available for Developmental Node V is from the death of Dolly and another 

sheep clone of unknown age. 

4. Goats 

As with sheep, relatively few studies have been conducted with goat clones, and many of 

these have used transgenic clones. Unlike sheep, however, several of the goat studies are 

fairly detailed, and provide a more complete picture of the health of the animals involved 

at most developmental nodes. Table V-19 provides survival data for live-born goat clones 

from the four studies which reported this information. Similar data for conventionally 

bred goats is not currently available. 

Table V-19: Survival Rates of Live-Born Clones and Comparators 

Reference Transgenic 

Status 

Surviving/Total LiveBorn Clones (fraction)1

Surviving /Total Live- Born 

Comparators (fraction) 

Baguisi et al. 1999 All 3/3 (1.00) NP2 

Keefer et al. 2001 Some 1⁄4 (0.25) NP 

Keefer et al. 2002 None 7/9 (0.78) NP 

Reggio et al. 2001 All 5/5 (1.00) NP 

1 Survivors through the Juvenile Period/Live births 

2 NP = not provided; data not available 

Transgenic Status: All = All of the clones cited in the publication are derived from transgenic donor cells, 

Some = Some of the clones cited in the publication are derived from transgenic donor cells, None = None 

of the clones cited in the publication were derived from transgenic donor cells. 

a. Developmental Node 1: Pregnancy and Parturition 

i. Pregnancy 

A study of 515 healthy, conventionally bred dairy goats (Engeland et al. 1997) noted that 

the does that spontaneously aborted or delivered stillborn kids did not show any signs of 

clinical illness. Age of the doe, number of fetuses/doe (twins or other multiples), social 
Chapter V: Animal Health Risks 170 

Animal Cloning: A Risk Assessment DRAFT

status (position in the herd hierarchy) and previous history of pregnancy loss were the 

factors most closely associated with spontaneous abortion in dairy goats in this study. 

Does more than three years of age and those which had previously lost pregnancies were 

more likely to lose a pregnancy during the study compared with younger does and does 

with a history of successful births. Does carrying three or more fetuses were more likely 

to lose a fetus than does carrying only one or two fetuses, possibly due to limitations in 

uterine capacity. Does with a low status in the herd were more likely to lose their 

pregnancy than does with moderate or high status. The authors suggested that this last 

factor may be related to stress. 

In general, cloning-related problems similar to those noted for sheep and cattle have not 

been reported for goats. Because there are relatively few reports of goats bearing clone 

pregnancies (Keefer et al. 2001; Reggio et al. 2001; Baguisi et al. 1999, the latter two 

reporting on transgenic clones), and the number of animals involved in individual studies 

is small, CVM could not determine whether the lack of complications reported in this 

species was the result of differences in methodology, species-specific differences, or 

simply an artifact of the small numbers of animals involved and small number of 

published papers. 

ii. Parturition 

Data on effects on surrogate dams are not currently available. 

b. Developmental Node 2: Perinatal Period 

Although few reports on goat clones appear to have been published, the results of these 

trials contrast with those of sheep and cattle. None of the studies reported cases of LOS 

or related perinatal clinical signs in goat clones. 

Keefer et al. (2002) reported deaths of two goat clones during delivery of two twin 

pregnancies, but causes of these deaths were not reported. Keefer et al. (2001a) reported 

normal birth weights in transgenic male Nigerian Dwarf goat clones compared to 

historical records for the same breed at the same facility (average 2.35 kg), and noted no 

placental abnormalities. In this study, three young goat clones died from respiratory 

infections of bacterial origin, one at one day of age, the other two at later times (one 

month old and three months old). As mentioned previously, respiratory problems of 

various causes are the most commonly reported clinical sign in ruminant clones (Table 

V-3). As respiratory ailments, including pneumonia, are common in the general goat 

population (Merck Veterinary Manual Online 2005), it is not possible to tell from this 
Chapter V: Animal Health Risks 171 

Animal Cloning: A Risk Assessment DRAFT

study whether the infections in these clones were potentiated by the SCNT process. Also, 

as noted earlier, young ruminants are dependent on passive immunity transferred through 

colostrum. The Keefer et al. studies (2001a, 2002) provided no details on source or 

quality of colostrum provided to the goat clones after birth, or on IgG levels in kid serum. 

Other papers on cloning in goats employed transgenic cells as donors (Baguisi et al. 

1999; Reggio et al. 2001). Although transgenesis may have increased complications in 

studies of SCNT in cattle and swine (Hill et al.1999; Carter et al. 2002; Lai et al. 2002), 

studies in transgenic goat clones noted no perinatal morbidity or mortality (Baguisi et al. 

1999; Keefer et al. 2001a; Reggio et al. 2001). Birth weights of transgenic goat clones 

were within the expected range for their breed in these three studies. 

c. Developmental Node 3: Juvenile Development 

Agricultural statistics for conventional goats of this age range were not available. As 

presented in more detail in Chapter VI, Keefer and her colleagues (Keefer et al. 2001a; 

Gauthier et al. 2001; Keefer et al. 2002) reported on the life history, with particular 

emphasis on reproductive function, in a small cohort of goat clones. No adverse 

outcomes were noted in this group, and development appeared to parallel non-clone 

comparators. In a study of transgenic prepubertal goats, Reggio et al. (2001) reported that 

transgenic goat clones weighed on average 20.9 kg when weaned at 90 days of age 

(normal weight and age for weaning dairy goats), and were apparently healthy at 12 

months of age. 

In the only study encountered to date that included data on hematology and clinical 

chemistry of goat clones (Behboodi et al. 2005), a group of seven transgenic clones were 

compared to age-matched comparators and to published values (See Appendix D). 

Hematology values were similar between clones and comparators, and all hematology 

values fell within the published range (Pugh 2002). For clinical chemistry, 18/24 values 

were not significantly different between clones and their age-matched comparators. Of 

the 19 clinical chemistry values for which published ranges were available, 18 of the 

values for clones and comparators fell within the published range. The one value out of 

the published range was creatine kinase (CK) (244.6 vs. 204.4 IU/L for clones and 

comparators). However, values between clones and comparators were not statistically 

different. It is unclear whether or not the comparators in this study were also transgenic, 

whether they were the same breed as the clones, or how they were generated (AI, natural 

mating, IVP, or ET). The study also does not specify the age of the goats at time of blood 

sampling, so it is difficult to interpret the high values for CK in these animals compared 

to the published range. 
Chapter V: Animal Health Risks 172 

Animal Cloning: A Risk Assessment DRAFT

d. Development Node IV: Reproductive Development and Function 

Gauthier et al. (2001) studied sexual maturation and fertility of Nigerian Dwarf goat 

clones. Four bucks produced by AI were used as controls in this study. Average age at 

first semen collection for controls was 141 ± 22 days (approximately 20 weeks of age), 

with the earliest age at first collection 103 days. In buck clones, the earliest age at first 

collection was 125 days, but average age at first collection for three SCNT-derived bucks 

was not different from the mean of the control bucks (142 ± 8 days). First semen 

collection volume for all bucks was small (<0.1 ml). Subsequent collections were made at 

different ages for clones and controls, and thus are not appropriate to compare. Sperm 

motility did not appear to be different between clones and controls. The study did not 

mention whether there were any differences in sperm quality or morphology between 

clones and controls. Semen from two of the SCNT-derived bucks was used to inseminate 

six does. Five of the six does were determined to be pregnant, and all five delivered a 

total of nine healthy kids. Birth weights of progeny of the clone bucks ranged from 1.25 

to 2.30 kg, and were not different from non-clone Nigerian Dwarf kids born at the same 

facility. 

Reggio et al. (2001) reported only that five female transgenic SCNT-derived goats 

demonstrated estrus, were bred by natural mating, and produced kids. Age at puberty, 

number of services to conception, and details of the parturition and lactation were not 

reported in this study. 

e. Developmental Node V: Post-Pubertal Maturation and Aging 

No reports on aging and maturity in goat clones were identified. 

f. Progeny of Goat Clones 

Gauthier et al. (2001) reported that progeny of male goat clones were born healthy and 

within expected weight ranges for their breed. Reggio et al. (2001) similarly remarked 

that five transgenic goat doe clones were bred and produced kids, and that the kids were 

continuing to grow as expected. 

In one study, progeny from goat clones were found to have shorter telomere length in 

testicular biopsies compared to conventionally derived animals and the telomere lengths 

were intermediate to the values obtained for their clone fathers’ and age-matched control 

testes (Betts et al. 2005). This suggests that there was incomplete telomere elongation in 

the offspring of clones, although as mentioned above it is uncertain whether telomere 

length is a predictor of longevity. 
Chapter V: Animal Health Risks 173 

Animal Cloning: A Risk Assessment DRAFT

g. Summary for Health of Goat Clones 

Although few studies have been performed on goat clones, some data is available for four 

of the five developmental nodes, and some limited information on progeny is also 

available. Unlike cattle and sheep, goat clones do not appear to develop LOS. Likewise, 

there have been no adverse reports of pregnancy in surrogate goat does (i.e., hydrops and 

dystocia). Although three goat clones were reported to develop respiratory problems, it 

could not be determined from the study (Keefer et al. 2001a) whether this was related to 

cloning or not. Goats appear to grow and mature normally and produce normal progeny. 

The potential effect of shortened telomeres in one report on progeny of goat clones 

cannot be estimated at this time. No data on post-pubertal maturation are available for 

goats at this time. 

D. Conclusions 

Studies performed to date indicate that health problems observed in pregnancies carrying 

animal clones are not unique; similar problems are well documented in pregnancies 

produced by IVP and ET, and the same birth defects are sometimes seen in animals that 

are naturally bred. 

Early embryo loss seems to be related to in vitro culture conditions, which may cause 

abnormal development and early embryo/fetal death in both SCNT and IVP pregnancies. 

Failure of epigenetic reprogramming may also play a role in these losses for SCNT 

embryos (see Chapter IV). The impact of such events on the health of the dam is 

dependent on the stage of pregnancy when loss occurs. Losses due to defects in the 

embryo or failure to implant do not pose a hazard to the dam in early stages of pregnancy, 

whereas mid- and late-term spontaneous abortions may pose a health hazard to individual 

females if they are unable to completely expel the fetus and its associated membranes. 

As with pregnancy data, information from the perinatal period indicates that cattle clones 

are at the greatest risk of morbidity and mortality, compared with goats and swine. Also 

as observed in the pregnancy data, the abnormalities noted in animal clones are not 

unique to animals derived by SCNT; similar outcomes have been observed in other 

ARTs, albeit at lower rates. Most of the information on neonatal mortality comes from 

cattle. 

The major clinical finding associated with these observed outcomes appears to be a 

complex of clinical signs collectively known as LOS, which has been described in calves 

and lambs produced by ET, IVP, BNT, and SCNT. Some of the clinical signs reported 
Chapter V: Animal Health Risks 174 

Animal Cloning: A Risk Assessment DRAFT

may be directly related to fetal oversize, constraints of the surrogate’s uterine capacity, 

and dystocia during labor. Other signs, such as respiratory, cardiovascular, hepatic and 

renal (kidney) abnormalities do not appear to be related to intrauterine effects, and may 

occur even among calves within the normal range of birth weights for their breed, but are 

considered part of the syndrome due to the frequency of co-occurrence. Hence, LOS may 

be a misnomer, but the term has become familiar to scientists working in the ART field. 

The causes of LOS remain unclear, but may be related to in vitro culture conditions and 

other factors, such as incomplete reprogramming of the somatic cell nucleus (see Chapter 

IV). 

Most prepubertal cattle, swine and goat clones appear to grow and develop normally 

following the early neonatal period as demonstrated by reports on health status and 

laboratory measurements presented in the available published data and other reports on 

health status supplied by private companies. 

Based on the biological assumptions and molecular data reviewed in Chapter IV, progeny 

of clones are expected to be normal. Based on empirical observations, data regarding the 

health status of the progeny of animal clones indicate no increased risk of health 

problems compared to conventional animals. 

Two traits that may be genetically caused were identified (cryptorchidism in three calves 

derived from the same cell line and parakeratosis in one swine clone). These may pose 

health risks to the animals, and are certainly economically undesirable. Healthy clones 

appear to behave similarly to sexually-derived comparators or, where the information was 

available, to their genetic donor. 

Insufficient time has elapsed since the first domestic livestock clones were born to make 

any reliable observations on maturity, aging, or the lifespan of these animals. Reports on 

telomere lengths in animal clones are highly variable (see Box V-1), appear to be tissue 

dependent, and may not be reliable predictors of lifespan. As most female food animals 

are not maintained to old age, the risk of increased health problems or decreased 

longevity, if any exist, would be primarily to male animals kept as breeding stock. 

This component of the Risk Assessment has compared SCNT with other ARTs with 

respect to effects on animal health. It has not been possible to perform a strict quantitative 

analysis of the risk of SCNT to the health of animals involved in cloning for two 

fundamental reasons: the number of animals that have been studied and reported upon is 

small, and the rates of adverse outcomes are variable. Therefore, rather than evaluating 
Chapter V: Animal Health Risks 175 

Animal Cloning: A Risk Assessment DRAFT

relative rates of adverse outcomes between studies of cloning and other ARTs, the 

outcomes from cloning studies should be considered within the context of the actual 

number of animals involved within a study. Second, rates of adverse outcomes observed 

in surrogate dams and clones may be decreasing (Table V-1) as clone producers develop 

more expertise with the technology. Therefore, although the nature of the risk (the 

qualitative adverse outcome) stays the same, the risk itself (the probability of adverse 

outcome given that cloning has occurred) may decrease. Thus, it is not appropriate to 

perform a quantitative analysis of “relative risk” until these rates have stabilized. 

The conclusions from this assessment of the risks of cloning to animal health may be 

summarized as follows: 

• Cows and ewes used as surrogates for SCNT-derived pregnancies appear to be at 

increased risk (e.g., incidence) of late gestational complications such as hydrops, 

as well as dystocia at parturition, that occur, but at a lower frequency, with other 

ARTs such as IVP. The risk to surrogate swine and goats bearing clones does not 

appear to be increased compared to the general population; however, the limited 

dataset in these species increases the uncertainty associated with this conclusion. 

 

• There is an increased risk (e.g., incidence) of mortality and morbidity in perinatal 

calf and lamb clones compared with calves and lambs produced using other 

ARTs. In cattle and sheep, the increased risk appears to be a function of LOS. 

Survival of these clones appears to be a function of both the severity of the 

clinical signs and neonatal management. The available information suggests that 

morbidity and mortality is not increased in perinatal swine and goat clones; 

however, the limited dataset in these species increases the uncertainty associated 

with this conclusion. 

• Animal clones of all of the species considered in the juvenile to prepubertal age 

cohort do not appear to be at an increased risk of morbidity or mortality compared 

to animals produced by natural service or ARTs. Most animals surviving the 

neonatal period appear to grow and develop normally. 

• No increased risk of adverse health effects is apparent in bovine clones 

approaching reproductive maturity. This conclusion should be tempered by the 

relatively small dataset available for analysis. There are insufficient data to assess 

the risk in this developmental node for swine, sheep, or goat clones. 

• Insufficient data exist to assess the risk of adverse health effects to mature and 

aging animal clones. The available information indicates that there are no 
Chapter V: Animal Health Risks 176 

Animal Cloning: A Risk Assessment DRAFT

apparent risks to the health of maturing animals from cloning. Drawing empirical 

conclusions regarding longevity in domestic livestock clones is difficult due to the 

relatively short time that the technology has existed. 
 

Animal Cloning: A Risk Assessment DRAFT

Chapter VI: 

Food Consumption Risks
 

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Animal Cloning: A Risk Assessment DRAFT

Chapter VI: Food Consumption Risks 

A. Potential Hazards and Food Consumption Risks Associated with Food 

Products from Animal Clones and their Progeny 

1. Assumptions 

This Chapter of the Risk Assessment is focused on food safety concerns, and assumes 

that any clones or their products would be subject to the same local, state, and federal 

laws and regulations as conventional food animals or their products. These assumptions 

exclude animals with gross anomalies that would not enter the human food supply 

(although they might be rendered). It also assumes that any hazards arising from the 

consumption of products derived from animal clones would result from epigenetic 

dysregulation of the genome of the developing animal, as described in Chapter IV. 

Because much of the focus of this analysis is the identification of subtle hazards in 

otherwise healthy-appearing animals, the Critical Biological Systems Approach (CBSA) 

evaluates animal health data on as fine a level of resolution as possible. This includes 

individual animals or even individual analytes per animal in order to have a sensitive 

screen for adverse outcomes (and thus food consumption risks). Thus, although some of 

the data in this chapter reprises information previously addressed in Chapters IV and V, 

the methods by which the data were evaluated differed. Because the emphasis in the 

Chapter is on subtle hazards, the focus of Chapter VI is to evaluate adverse outcomes 

observed in animals to see if they can provide insight into identifying food consumption 

hazards, and not the actual risks to the animals themselves, which have been discussed in 

Chapter V. Chapter VI also includes all of the information that we could identify on the 

composition of meat or milk from clones or their progeny. Much of this information has 

been published or made available in 2005, and tends to evaluate very similar 

compositional components; much of it is on animals for which physiological data are also 

available. 

2. Critical Biological Systems Approach to Animal Clones of Cattle, Swine, 

Sheep, and Goats 

Chapter V and VI review the health outcomes reported for clones of cattle, swine, sheep, 

and goats. Over 1,700 references were identified in our literature searches; closer 

examination revealed that approximately 350-400 papers were useful to the 

understanding of the subject, and a smaller fraction of those actually cited papers were 
Chapter VI: Food Consumption Risks 180 

Animal Cloning: A Risk Assessment DRAFT

cited for information on the health of clones or the composition of their food products. 

Many of these reports are on the same cohorts of animals, but concentrate on different 

measurements or life stages. Several are reviews of adverse outcomes that have been 

observed in individual animals or cohorts of animals, but do not provide new data. As 

indicated previously within the Risk Assessment and detailed in Appendix D, some of the 

animals on which reports are provided are somatic cell nuclear transfers of transgenic 

cells, thereby actually being reports on transgenic animal clones. These have been 

included in the food consumption risk assessment when they provide corroborative 

information, and the transgenic status of the animals has been indicated when that 

information is available. 

The following section reviews the available information on animal cloning by species, 

sorting the information into developmental node-specific groupings. This approach was 

most applicable to bovine clones, where there is significantly more information compared 

to other species. For those species where information is very limited, such as sheep, the 

available information is presented as a single unit. 

a. Bovine Clones 

The largest number of publicly available publications and data sources address clones of 

dairy and beef cattle. Many reports on effects noted in the Cell Fusion/Reprogramming, 

Embryo/Fetal, and Perinatal periods tend to come from the early cloning experiments. 

Others test hypotheses regarding some component of the SCNT process (e.g., cell cycle, 

cell source, culture conditions, epigenetic reprogramming (see Chapter IV)), and either 

do not result in live births, or result in very few live births. Very few systematically 

evaluate the health of the animals, many simply state that “animals appear normal and 

healthy” or that “no differences were observed between clones and controls.” CVM has 

extracted as much information as possible from these studies, and has incorporated its 

findings into the appropriate Developmental Nodes. 

During the course of preparing this risk assessment, clone producers shared information 

on various cloning outcomes with CVM. The most comprehensive dataset was generated 

in response to preliminary presentations of the risk assessment methodology by FDA 

staff at various scientific meetings. In particular, one clone producer, Cyagra, Inc., has 

attempted to gather information on all of the cattle clones that it has produced, including 

animals that did not survive or that were culled for various reasons. In some cases, this 

has proved impracticable due to the dispersal of clones to their ultimate owners. The 

Cyagra dataset is the most comprehensive survey of the health status of cattle clones that 

has been assembled, and this information has been incorporated into this Risk 
Chapter VI: Food Consumption Risks 181 

Animal Cloning: A Risk Assessment DRAFT

Assessment. Details on the animals, the methods used to collect and interpret the data, 

and the actual data themselves can be found in Appendix E. Cyagra also collected data on 

the composition of meat from several clones; these data are also in the Appendix.44 

The information provided by Cyagra differs from that presented in the peer-reviewed 

literature for several reasons: 

• The data were collected specifically to address issues raised in this risk 

assessment, and thus are not part of a hypothesis-testing study, or written to 

provide examples of novel or unusual events; 

• They have not been peer-reviewed outside CVM (to the Center’s knowledge); 

• They include individual animal data; and 

• They are far more extensive with respect to the number of clearly non-transgenic 

animals evaluated (n=78 surviving and tracked animals), and the number of 

observations on individual animals than any other study or series of studies from a 

particular laboratory. 

i. Cell Fusion, Nuclear Reprogramming, and Embryonic and Fetal 

Development in Bovine Clones45 (Developmental Node 1) 

SCNT is a relatively inefficient process. “Successful” event estimates can be based on the 

number of fused cells, implanted blastocysts, or pregnancies confirmed at some day of 

gestation, estimates range from one in one thousand (usually based on fused cells) to one 

in four (confirmed pregnancy at gestation day 60). The former estimates include the 

earliest reports of SCNT, as well as studies testing various methodological variables, and 

reflect the “technology development” nature of the reports. When measured from the 

detection of an established pregnancy in the surrogate dam, the success rate can be 

considerably higher, and can range from 1-2 percent (as reviewed in NAS 2002b) to 

approximately 20-25 percent as related to CVM by commercial cloning ventures. 

Lack of success at the cell fusion stage is likely due to several factors, the most 

significant of which are technological (e.g., damage to the oöcyte or donor cells) or 

biological (e.g., incorrect reprogramming of the genome of the donor cells (Chapter IV) 

or possible lack of synchrony between donor cell and oöcyte). An alternative justification 

 

44 Viagen, Inc. has also developed an extensive dataset on the health and composition of swine clones and 

their progeny. This is the most comprehensive dataset on the health of swine clone progeny and the 

composition of their meat. Similar to the Cyagra dataset, these data and their detailed analyses are found in 

Appendix G, and are summarized within the text of this Chapter. 

45 This Chapter emphasizes the morphological changes observed in this Developmental Node, unlike 

Chapter IV, that summarized molecular findings. 
Chapter VI: Food Consumption Risks 182 

Animal Cloning: A Risk Assessment DRAFT

proposed by Hochedlinger and Jaenisch (2002) among others, is that the extremely low 

success frequency is a reflection of the inability of all but “stem cells” of various degrees 

of pluripotency to be reprogrammed, and the serendipitous outgrowth of such cells 

selected at random for use as donor cells. Regardless of the explanation, few fused 

donor/oöcyte pairs survive to divide or to become established as pregnancies in surrogate 

dams. 

The following overview of methods that may affect success rates of SCNT are included 

to allow the reader to understand that there are many different components that may 

influence cloning efficiency. It is important to remember, however, that the goal of this 

chapter of the risk assessment is to identify and characterize potential subtle hazards in 

clones and to determine whether they pose food consumption risks. 

(a) Peer-reviewed Publications 

The following section provides summaries of studies that contribute to identifying some 

of the factors that may contribute to successful nuclear transfer at the earliest 

developmental node. It is intended to be illustrative, and not comprehensive. 

Effect of the Zona Pellucida. The importance of the zona pellucida in embryo 

development is not clear, and there are conflicting outcomes in different studies 

evaluating its role. Dinnyes et al. (2000) compared developmental rates of cattle oöcytes 

subjected to SCNT, parthenogenetic activation, or in vitro fertilization. For the oöcytes 

undergoing SCNT (n=106), 74 percent fused, 90 percent of fused embryos cleaved by 

Day 2, and 29 percent of cleaved embryos developed to blastocysts. Eighty-one percent 

of parthenotes46 (early embryos arising from parthenogenetic activation) (n=47) 

incubated in 5 percent CO2 in air cleaved by Day 2 of the experiment, but only 17 percent 

developed into blastocysts. Parthenotes (n=98) incubated in 5 percent O2, 5 percent CO2

and 90 percent N2 had a 79 percent cleavage rate on Day 2, and a 32 percent survival to 

blastocyst stage. By comparison, in vitro fertilized oöcytes (n=98) had a 69 percent 

cleavage rate by Day 2, and 35 percent developed to blastocysts. Because parthenotes are 

“clones” that have not undergone nuclear transfer, the zona pellucida of the embryo is not 

disrupted. This disruption has been hypothesized to be a possible cause of early embryo 

failure in nuclear transfer (NT) embryos. The lack of difference in development to 

blastocyst between SCNT, parthenotes and IVF embryos cultured under the same 

 46 A form of reproduction in which an unfertilized egg develops into a new individual, which occurs 

among crustaceans and certain other arthropods. Parthenotes, unlike somatic cells, do not need to be 

reprogrammed, as they are already in an undifferentiated state. 
Chapter VI: Food Consumption Risks 183 

Animal Cloning: A Risk Assessment DRAFT

conditions suggests that disruption of the zona pellucida may not be an important factor 

in early loss of SCNT embryos. Conversely, Ribas et al. (2006) noted no difference in 

development to blastocyst in zona-free vs. zona-intact IVF mouse embryos, although the 

authors stated that zona-free blastocysts were smaller and more irregular than zona-intact 

embryos. None of the embryos in this study were transferred to recipients for gestation, 

however, so further development could not be assessed. In another study involving IVFderived sheep embryos, Ritchie et al. (2005) transferred eight zona-free embryos to four 

surrogate ewes. One of these pregnancies progressed to term and resulted in a live lamb. 

Cell Culture Conditions. Several laboratories have attempted to optimize culture 

conditions to improve cloning efficiency (Kubota et al. 2000; Li et al. 2004; Park et al. 

2004b; Du et al. 2005). These manipulations have included addition of various 

compounds to culture media, co-culture with “feeder cells,” and serum starvation. Results 

of these studies have been mixed, as described below. 

In order to study the influence of culture conditions of donor cells used for SCNT, 

Kubota et al. (2000) used fibroblasts derived from a skin biopsy obtained from a 17 year 

old Japanese Black beef bull. Donor cells for nuclear transfer were obtained from cultures 

that had undergone 5 (n=570), 10 (n = 269), or 15 (n = 264) passages.47 All cultures were 

serum starved prior to nuclear transfer, except that cells from passage number five were 

divided into two groups, one of which was serum-starved (n=288), and the other was not 

(n=282). There were no differences among groups for fusion or cleavage rates, but 

development to blastocyst stage was lower in cells from Passage 5, relative to cells from 

the higher passage rates, regardless of whether or not the cells were serum starved. A 

total of 54 blastocysts were transferred to 36 recipient cows. Fifteen cows were diagnosed 

pregnant, of which nine spontaneously aborted between 39 and 123 days of pregnancy. 

All three of the pregnancies derived from Passage 5 cell cultures spontaneously aborted. 

Six calves derived from the two more extensively passaged cultures were delivered at 

term, two from cultures that had undergone 15 passages, the other four from cells that 

had undergone 10 passages. Two calves derived from Passage 10 donor cells died shortly 

after birth. In this study it appears that cells that have been more extensively passaged 

make better donors than less extensively passaged cells. The biological basis for this is 

not clear, unless cells that have been passaged more extensively in culture somehow 

become more amenable to epigenetic reprogramming. 

 47 A passage is a cell culture process in which culture vessels that are full of cells are diluted to lower cell 

densities. This allows the cells to overcome the growth inhibition that comes with limited space. Each 

dilution is referred to as a passage, so that a culture that has been passaged five times has started with 

low cell density, grown up to high cell density, been diluted, and had that process repeated four more 

times. 
Chapter VI: Food Consumption Risks 184 

Animal Cloning: A Risk Assessment DRAFT

In another study of culture conditions, Li et al. (2004) compared development of SCNT 

embryos co-cultured with bovine cumulus cells or with one of two different types of 

serum (fetal calf serum (FCS) or bovine serum albumin (BSA)) for seven days. The rates 

of cleavage, morula and blastocyst formation were similar across treatment groups. 

Fewer blastocysts in the FCS group exhibited normal chromosomal ploidy compared to 

the BSA group (24/41 or 58.5 percent vs. 24/35 or 68.6 percent), but both of the serum 

supplemented groups performed poorly compared to the cumulus cell co-culture group, in 

which 34/42 (80.9 percent) of blastocysts had normal ploidy. 

Park et al. (2004b) noted that although not effective in improving embryo development 

alone, the combination of β-mercaptoethanol (ME) and hemoglobin (Hb) enhanced the 

rate of development of NT embryos to the morula stage compared to unsupplemented 

media (19/57 vs. 55/85). Development to blastocyst, however, was similar between 

untreated controls and either the combined treatments or ME or Hb supplementation 

alone (16/57 vs. 18/99, 15/95, and 40/104 for control, Hb, Me and Hb + ME, 

respectively). Similarly, Du et al. (2005) found no beneficial effect of adding 

phytohemagglutinin-L (PHA) to culture media for survival, cleavage or blastocyst 

formation of NT embryos. From a total of 324 fused embryos, three live calves were 

born: two from the PHA group and one from the untreated group. 

Heterogeneity of Fusion Components. Hiendleder et al (2004) studied how differences 

between nuclear and oöplasm sources can influence SCNT outcomes by using three 

breeds of cattle (Brown Swiss, Dwarf Zebu, and two varieties of Simmental) as oöcyte 

sources and granulosa cells from a Brown Swiss cow as the source for somatic cells. Four 

groups of SCNT embryos were produced. All pregnancies were terminated at 80 days 

gestation and uterine contents collected to determine the number of viable fetuses. Details 

on individual fetuses were not discussed, but the authors noted that SCNT fetuses in 

general were heavier, had a larger thorax circumference, and a reduced crown rump 

length: thorax ratio (a standard measure of body size) compared to AI fetuses. The 

proportion of viable fetuses was significantly affected by source of oöplasm, and was 

higher for fetuses produced using Dwarf Zebu oöplasts than the other three sources. The 

lowest viability was noted for one, but not both, of the Simmental sources. Interestingly, 

the difference between the two Simmental sources for viability was significantly 

different. No details regarding the oöcyte donors, other than breed, were provided, so 

there is no way to determine if other factors (e.g., age of the oöcyte donor cows, 

nutritional status, health history, or size of follicles collected) might have influenced fetal 

viability. The authors also compared mitochondrial DNA sequences between the two 

Simmental oöcyte sources, and noted extensive polymorphism in coding and non-coding 
Chapter VI: Food Consumption Risks 185 

Animal Cloning: A Risk Assessment DRAFT

regions of the two mitochondrial genomes. Although there has been speculation that 

mitochondrial dimorphism may affect development of SCNT embryos, only one study 

was identified that looked specifically at mitochondrial effects on embryo development 

(Takeda et al. 2005). Also of interest, when fetal morphology was compared in the 

Hiendleder et al. study, hybrid fetuses (reconstructed using either Zebu or Simmental 

oöplasm) were not significantly different in size compared to AI fetuses of the same 

gestational age; however, fetuses produced using the same breed as source of both 

oöplasm and nucleus (Brown Swiss) exhibited fetal overgrowth. The Brown Swiss cows 

that were used as sources of oöcytes were different individuals from the Brown Swiss 

donor of the nuclear DNA. The authors do not report whether they compared 

mitochondrial DNA of the nuclear donor with that of any of the Brown Swiss oöcyte 

donors. 

Source of Donor or Recipient Cells. Tissue source of nuclear donor cells can also affect 

development and survival of NT embryos. Galli et al. (1999) used bovine blood 

lymphocytes as nuclear donors. Lymphocytes, involved in the immune system, must 

undergo rearrangement of their DNA in order to produce immunoglobulins. Panelli et al. 

(2004) examined tissues of four aborted NT fetuses and the chondrocytes of the single 

surviving clone from the Galli et al. experiments. The results were compared to 

chondrocytes from three non-clone bulls (how the comparator bulls were generated is not 

described). The aborted fetuses exhibited DNA rearrangement in brain cells that was 

typical of terminally differentiated lymphocytes, but the surviving clone showed no 

rearrangement in chondrocytes isolated from his sperm, similar to chondrocytes collected 

from non-clone bulls. Based on this small dataset, the authors suggested that although 

terminally differentiated cells can sustain development through the late fetal stage, cells 

more amenable to reprogramming (dedifferentiation), such as stem cells, were more 

likely to result in live clones. 

Xue et al. (2002) reported on the relative success rates associated with generating clone 

embryos from three different tissues collected from a 13 year old Holstein cow. In their 

hands, ovarian cumulus cells had the highest rate of development to blastocyst (57 

percent, n=92), compared to skin fibroblast cells (34 percent, n=110) and mammary 

epithelial cells (23 percent, n=96). Six term pregnancies resulted following transfer of 

ovarian cumulus nuclear transfer (NT) embryos to recipient cows (5.5 percent, n=109), 

and four (7 percent, n=57) term pregnancies resulted from skin fibroblast NT embryos. 

None of the embryos generated from mammary epithelial cells resulted in a term 

pregnancy when transferred to recipient cows (n=34). The expression of X-chromosome 

linked genes in various tissues from deceased animals and conventional controls, and 

from the placentae of surviving clones was also investigated. Results indicated that X-
Chapter VI: Food Consumption Risks 186 

Animal Cloning: A Risk Assessment DRAFT

chromosome inactivation occurred normally in the surviving female clones, but was 

incomplete in the clones that died. Embryo samples were taken to determine if there were 

differences in cell counts in embryos from parthenotes and SCNT-derived embryos at the 

same stage of development. Cell numbers for NT embryos were lower compared to 

parthenotes at all stages examined (Day 5 morula: 35.1 ± 1.1, n=48 for NT vs. 43.5 ± 1.5, 

n=58 for parthenotes; Day 7 blastocyst: 81.0 ± 3.7, n=46 for NT vs. 93.8 ± 5.6, n=48 for 

parthenotes). The importance of differences in cell numbers is not clear from this study, 

as mammalian parthenotes generally do not develop to term. Cell counts of IVF embryos, 

which would have been a more informative comparison, were not provided. 

Gong et al. (2004) compared granulosa cells from adult cattle of two different breeds 

(Holstein and Chinese red-breed yellow cattle), skin fibroblasts from two individual 

Holsteins and a Holstein fetus, and oviductal cells from a Holstein fetus for development 

and survival through the birth of clones. The rate of blastocyst formation was lowest for 

one of the two adult skin fibroblast sources (253/906 blastocysts/fused couplets or 27.9 

percent), although the other adult fibroblast cell line was comparable to the fetal 

fibroblast cell line (52/132 or 39.4 percent vs. 1294/3412 or 37.9 percent). Fetal oviductal 

cells had the highest rate of blastocyst formation in this experiment (456/1098 or 41.5 

percent). A total of 346 Day 7 blastocysts were transferred to 171 recipients. Pregnancy 

rate at day 60 was 34.5 percent (59/171), with 25 surrogates carrying 27 calves to term. 

Because of the small numbers of calves delivered at term, no differences could be 

detected among donor cell sources for live birth. Of the 27 calves born, eight died during 

the perinatal period, and another seven died at later stages. Seven of the calves died of 

causes associated with LOS (hepatic, cardiac, or gastro-intestinal defects, respiratory 

distress), and eight animals apparently died due to management errors. It is not clear what 

portion of the perinatal deaths were due to birth defects/respiratory failure or 

management errors. Birth weights of calves were not reported. 

Some authors have suggested that the stage of the cell cycle may also influence cloning 

outcomes. However, results in different laboratories (Wells et al. 2003; Urakawa et al. 

2004; Ideta et al. 2005) using cells in different stages have been mixed. Wells et al. 

(2003) compared putative G0 cells (cells that apparently were not dividing) to G1 phase 

(cells that had begun dividing) cells for SCNT. They noted high early pregnancy losses, 

but no losses after 120 days of gestation, and no reported hydrops in the G0 group. In 

contrast, G1 phase cells had higher losses to term (21/43 pregnancies lost after 120 days 

gestation) and higher incidence of hydrops (18/43 (42 percent) of pregnancies), but 

higher post natal survival than clones from G0 cells. In contrast to the Wells et al. study, 

Urakawa et al. (2004) reported success using fetal fibroblast donor cells in the G1 phase. 

However, it should be noted that Urakawa et al. used only G1 phase cells, and did not 
Chapter VI: Food Consumption Risks 187 

Animal Cloning: A Risk Assessment DRAFT

compare to other stages of development. Two cell lines were used, derived from fetuses 

with the same dam but two different bulls. Ten blastocysts were transferred into ten 

recipients, resulting in nine live calves. According to the authors, calving was 

“uneventful.” Differences were noted between cell lines, in that three calves resulting 

from one of the lines tended to be heavier at birth than the six calves of the other cell line 

used (actual birth weights not provided). One of these three heavy-weight calves died 

after two days without standing. The authors do not report on the health or survival of the 

remaining eight calves beyond the first six days of life. Ideta et al. (2005) compared 

development of embryos constructed with G1 or M phase fetal fibroblasts, and noted that 

G1 SCNT embryos had higher rates of development to blastocyst than M phase cells (31 

vs. 16 percent). Only five surrogate cows received embryos in the Ideta et al. study, of 

which three were diagnosed pregnant on day 30 of gestation, and one live calf was 

delivered. All of the transferred embryos were developed from G1-phase somatic cells. 

The single calf died two days after birth. Health of the surrogate dams, method of 

delivery, and birth weight of the single calf was not reported in this study. 

Based on these studies, two of which used only embryos developed from G1 phase cells, 

at this time it is not possible to determine the influence of the stage of the donor cell cycle 

on subsequent development of the embryo/fetus. 

Embryo and Fetal Development. Early pregnancy failures in bovine clones are thought 

to be a function of incorrect reprogramming of the donor cell that manifest as lethal 

developmental defects (see Chapter IV). Some of those developmental defects may 

manifest as difficulties in placentation. For example, Hill et al. (2000b) noted that 

placentae from gestation day 40-50 clone embryos were hypoplastic (low cell density), 

and had poorly developed cotyledons (Hill et al. 2000b). (In ruminants, the cotyledon is 

the fetal part of the junction between the maternal and fetal sides of the placenta where 

nutrients and wastes are exchanged.) Additional placental anomalies in first trimester 

aborted fetal clones may include decreased numbers of placentomes (the junction of 

maternal and fetal components of the ruminant placenta that serve to transport nutrients 

into and waste out of the fetal environment), and poor formation of blood vessels in the 

placenta. In contrast, Lee et al. (2004) noted that although fewer cotyledons were present 

in SCNT placentae compared to AI and IVF placentae at day 50 of gestation, 

vascularization was very good, and appeared more developed in SCNT compared to AI 

or IVF placentae. Edwards et al. (2003) also studied this phenomenon in transgenic and 

non-transgenic bovine clones and observed that approximately 50 percent of transferred 

embryo clones established a pregnancy when measured by the presence of a heart beat 

between gestational days 29-32. This rate was compared favorably to that observed for 

non-clone IVF embryos. Edwards et al. (2003) noted that 50-100 percent of embryo 
Chapter VI: Food Consumption Risks 188 

Animal Cloning: A Risk Assessment DRAFT

clones spontaneously aborted between 30–60 days of pregnancy. Dindot et al. (2004) 

have noted more than 80 percent of hybrid bovine clone pregnancies (Bos gaurus X Bos 

taurus) were lost between gestational days 30 and 60. Evaluation of the early placental 

structures at gestational day 40 indicated an absence of cotyledons in each clone 

pregnancy, while the control (AI) fetuses had between 4 and 25 cotyledons per 

pregnancy). Pace et al. (2002), in a study that included transgenic clones, noted that the 

fetal abortion rate prior to gestational day 60 was 67 percent. A comparison of the crownrump length of calved and aborted clone fetuses with AI-generated fetuses from 

gestational day 25 to gestational day 70 indicated that prior to abortion fetuses grew at 

the same rate. 

Later pregnancy failures are thought to be a function of developmental defects, including 

placentation abnormalities. Heyman et al. (2002), for example, compared pregnancy loss 

between gestation day 90 and calving among clones derived from adult somatic cells, 

fetal somatic cells, blastomere nuclear transfer (BNT), and in vitro fertilization (IVF) 

animals. They noted that the somatic cell clones showed a pregnancy loss incidence of 

approximately 44 percent and 33 percent, while BNT clones were lost in only 4 percent 

of the pregnancies, and the IVF control group lost no pregnancies. 

Abnormal placentation can, however, result in the birth of a viable clone (Hill et al. 

2000b). In this case, one of six transgenic fetal clones detected at 40 days of gestation 

continued to develop to term, and when delivered vaginally weighed 37.7 kg, within the 

normal weight range for Holstein calves (35 to 45 kg). The calf was considered normal 

based on physical examination at birth. It suckled normally, and at the time of 

publication, was two years old and considered to be normal. The placenta was similar in 

weight for term Holsteins (4.3 kg vs. mean expected weight of 5.6 kg). Its structure, 

however, was highly abnormal, with only 26 cotyledons present, of which only 12 were 

judged to have been functional. These were enlarged, and the authors hypothesized that 

this increased size allowed the normal development of the calf. The authors also note that 

pregnancies resulting from IVF have also been reported to contain fewer placentomes48

than those of conventional cattle. As discussed in Chapter III, the role of transgenesis in 

the development of this pregnancy cannot be determined. Batchelder (2005), however, 

working with non-transgenic clones, also noted fewer and larger placentomes in 

placentae of eight live-born clones compared to AI and ET comparators. 

 48 The structures involved in connecting the fetal and maternal tissues consisting of a cotyledon and a 

caruncle in the cotyledonary placenta. The cotyledons or chorionic villi are of fetal origin and "plug 

into" the caruncles or receptacles in the maternal uterine wall. 
Chapter VI: Food Consumption Risks 189 

Animal Cloning: A Risk Assessment DRAFT

(b) Summary for the Embryonic/Fetal Developmental Node in Bovine Clones 

(Developmental Node 1) 

This period manifests the highest degree of risk for the developing clone. The probability 

of an SCNT-embryo implanting, and the subsequent likelihood of an implanted clone 

embryo surviving and continuing to develop are low. Various investigators have 

attempted to understand the role of various components of the donor/recipient/cell culture 

system that comprises the “cloning unit” to improve efficiency with different sources of 

nuclear or oöcyte donors or by manipulating the culture conditions. These studies have 

been met with mixed results. Lack of success can be attributed to failure of the genome to 

be reprogrammed (Chapter IV), including failure of the embryo to begin dividing and 

implant in the uterus, and failure of development in the first trimester (likely due to 

defects in reprogramming that manifest as poor placentation or other defects that do not 

allow the fetus to develop), or physical damage to the early embryo. Difficulties that may 

persist in later pregnancy are largely associated with placentation anomalies that may codevelop with Large Offspring Syndrome (LOS) (see Chapter V). Nonetheless, some of 

these early embryos do divide, implant, develop, and give rise to live animals, as 

discussed in the subsequent Developmental Nodes. 

ii. Perinatal Development in Bovine Clones (Developmental Node 2) 

In the early studies of the technology, relatively high perinatal losses were reported. 

Deaths generally resulted from phenomena associated with LOS, including poor 

development of the respiratory and cardiovascular systems. (For a more complete 

description, refer to Chapter V.) In general, animals with LOS tend to have high birth 

weights (ranging from 20-50 percent greater than breed averages), poorly developed and 

sometimes edematous (fluid-filled) lungs and other tissues, and heart malformations and 

malfunctions. These animals may also have kidney and liver anomalies, and may initially 

exhibit difficulties in maintaining homeostatic functions such as body temperature and 

glucose metabolism. The latter are discussed in more detail later in this section. As the 

expertise develops, however, more animals are either born with no apparent defects, or 

have supportive care perinatally and survive to grow into healthy cattle. 

(a) Peer-reviewed Publications 

Most of the adverse outcomes that have been reported result in loss of the fetus before 

birth, although there is another period of loss after delivery, usually within the first few 

days of life. Reproducible sets of adverse outcomes have been observed, including LOS 

and gross morphological abnormalities that may result in pregnancy loss either early in 

gestation or late in gestation. For example, contracture of tendons has been noted in some 
Chapter VI: Food Consumption Risks 190 

Animal Cloning: A Risk Assessment DRAFT

clones. None of the abnormalities noted in animal clones are unique to animals derived 

by SCNT; all have been observed in natural reproduction, as well as in ARTs such as AI 

and IVF (reviewed by Cibelli et al. 2002 and Pace et al. 2002, and in Chapter V). 

Despite the initial frequency of publications describing adverse outcomes of SCNT, two 

classes of successful outcomes actually predominate at birth. The first includes animals 

that may require assistance with delivery and immediate post-natal support in 

maintaining oxygenation and body temperature. Among others, Cibelli et al. (2002) noted 

that adverse effects associated with abnormal placental functions in the birth of a group 

of transgenic clones can be mitigated by intensive veterinary care immediately following 

birth. One bull clone described by Hill et al. (2000a) required considerable veterinary 

support immediately after birth due to respiratory problems (immature lungs and 

pulmonary hypertension), lack of suckling reflex, apparent Type I diabetes, and other 

health problems. According to this report, the calf improved rapidly, and the diabetes 

resolved (the calf was able to maintain normal blood glucose and insulin levels) by two 

months of age. This animal has fully recovered, and is reported to be a vigorous and 

healthy bull (PIFB 2003). 

The second set of successful outcomes consists of those animals born with relatively little 

assistance (due to the high cost of developing animal clones, most are delivered via 

planned C-section, and may require more supportive care than animals derived from 

more conventional breeding techniques), and appear to be normal and healthy (see 

especially the Cyagra database (Appendix E)). Although many reviews attribute the 

difference in birth weight to various degrees of LOS, higher birth weights may also be 

due to the greater care afforded surrogate dams carrying animal clones relative to 

standard husbandry of conventional animals. Alternatively, birth weight may be related to 

genetics of the nuclear donor. No data were found on birth weights of nuclear donors, but 

studies indicate that birth weight is heritable (Knight et al. 2001; Chapter V). 

Forsberg et al. (2002) reported the production of 103 cattle clones, of which 47 were 

produced from non-transgenic cells and 56 from transgenic cells, including a Holstein 

bull calf generated by recloning an embryo derived from genital ridge cells. Of five 

pregnancies initiated from that recloning, two aborted prior to gestational day 30, one 

pregnancy was terminated at gestational day 203 due to hydrops, one set of twins died at 

birth due to the surrogate dam’s ketosis, and the fifth gave rise to “Gene,” the first cattle 

clone not produced from an embryonic cell line.49 Little further information on Gene’s 

 49 The first publication describing the production of cattle SCNT clones appeared in Science in 1998 

(Cibelli et al. 1998). Gene’s gestation overlapped with Dolly’s and due to species differences in length 

of pregnancy, Dolly became the first SCNT clone born alive. 
Chapter VI: Food Consumption Risks 191 

Animal Cloning: A Risk Assessment DRAFT

birth status, growth, or development is found in the peer-reviewed literature, except that 

as of the end of 2001, when the Forsberg et al. manuscript was accepted for publication, 

Gene had matured into “a healthy, fertile bull.” In a separate recloning trial described in 

this report, fibroblast cell lines derived from another fetal clone were used as donors to 

generate 28 blastocysts that were then transferred into 14 surrogate dams. Nine 

pregnancies were initiated. Four of those pregnancies went to term, and five calves (three 

singletons and one set of twins) were produced. 

Forsberg et al. (2002) also used cells from adult animals as donors for SCNT. Ear cells 

from a bull (age and breed not specified) were used to generate 32 embryo clones that 

were transferred into 17 surrogate dams, of which 10 became pregnant. Five pregnancies 

were lost prior to gestational day 60, and two more were terminated due to hydramnios or 

hydroallantois (these conditions are also referred to as hydrops). Three live animals were 

born, but one was euthanized at 11 days of age due to a heart defect. In a separate trial 

described in the same paper, cumulus cells from an in vivo matured oöcyte from a 17 year 

old cow were used to initiate 11 pregnancies, from which three calves were born. 

Although information on the health status of many of these animals is not available, 15 of 

these animals were bred, gave birth, and their milk studied by Walsh et al. (2003) (See 

Compositional Analysis Method - Section 3). 

In addition, Pace et al. (2002) of the same group reported on the development of 117 

cattle clones from the reconstructed embryo stage through to lactation. These animals 

were born between January 1998 and February of 2000. Some of the cell lines from 

which these animals were developed were transgenic (Forsberg et al. 2002), and 75 

percent of the resulting clones were transgenic. Because this report does not distinguish 

individual animals by cell source, it is not possible to determine which of the animals are 

transgenic. Interpretation of adverse outcomes should therefore be considered within the 

context of the discussion of transgenic animals in Appendix D. Of the 117 clone births, 

106 were born alive, and 82 remained alive at the time of publication. Birth weights of 

the surviving clones ranged from 11-72 kg, with an average birth weight of 51 ± 14 kg. 

The distribution of birth weights was skewed in excess of birth weight ranges for 

conventional Holsteins. 

Pace and his colleagues (2002) divided the calf clone deaths into preventable and nonpreventable causes (summarized in Table VI-1). Of the 24 animals that did not survive, 

12 died between post partum days 1-5, nine died between days 6-122, and three died at 

more than 123 days of age. Many of the animals appear to have experienced 

complications resulting from enlarged umbilici, and three of the deaths were directly 

related to this condition. For subsequent births, this condition was managed by 
Chapter VI: Food Consumption Risks 192 

Animal Cloning: A Risk Assessment DRAFT

prophylactically tying or clamping off the umbilical arteries. Difficulties with the 

umbilicus were also observed at levels apparently higher than in conventional animals by 

Kishi et al. (2000); Gibbons et al. (2002); Cyagra (Appendix E); Edwards et al. (2003); 

and Batchelder (2005). Nonetheless, 77 percent of the clones reported on by Pace et al. 

(2002) in this study were alive and apparently healthy at the time of the study publication 

(2004). 

Table VI-1: Summary of Causes of Death of Calf Clones 

(adapted from Pace et al. 2002)

Non- Preventable Deaths 

Physiological System 

Involved 

Calves 

(n) 

Age at 

death 

(days) 

Birth 

Weight 

(kg) 

Observations 

Multiple dysfunctions 3 1-2 11-63 Failure of most systemic functions 

Placental 2 1 50-59 Apparent premature separation of placenta 

Respiratory 1 3 62 Lung immaturity, mecomium aspiration at birth 

Digestive 2 78-122 52-60 

Chronic diarrhea (n=1); 

Intussusception of small intestine with 

obstruction (n=1)1

Circulatory 1 42 52 Congenital heart defect 

Nervous 1 154 51 Hydrocephalus 

Musculoskeletal 1 298 44 Developmental orthopedic disease 

Preventable Deaths

Physiological System 

Involved 

Calves 

(n) 

Age at 

death 

(days) 

Birth 

Weight 

(kg) 

Observations 

Placental 3 1 53-69 Extensive internal bleeding from enlarged 

umbilicus 

Respiratory 3 1-5 48-66 

Developed pneumonia (n=2); 

Premature induction of labor 16 days early, 

immature lungs (n=1) 

Digestive 5 5-90 59-72 

Clostridial infection (n=1); 

Developed abomasal ulcers2

 from eating wood 

chips (n=2); 

Bloat (n=2) 

Musculoskeletal 1 328 42 Injury, dislocation of patella 
Chapter VI: Food Consumption Risks 193 

Animal Cloning: A Risk Assessment DRAFT

Urinary 1 112 59 Pyelonephritis3

 probably secondary to umbilical 

infection 

1

 Intestinal intussusception is the collapse of one portion of the intestine into another, like a telescope, often resulting in 

the obstruction of the intestine. 

2

 The abomasum is the fourth compartment of the stomach of cattle, similar to the human stomach in function. 

3 Pyelonephritis is an inflammation of the kidney brought on by bacterial infection. 

In another example of the successful production of clones, Chavatte-Palmer et al. (2002) 

reported on clinical, hematological, and endocrine characteristics of 21 apparently normal 

cattle clones and 16 abnormal SCNT-produced fetuses compared with similar outcomes 

in animals derived by AI (and summarized in Table VI-2). Initial measurements such as 

pregnancy outcome (e.g., abnormal development, stillbirth, live birth) and birth weight 

were also compared with IVF-derived animals. (Data were presented as summaries, and 

individual animal data were not presented.) Detailed discussion of the health outcomes of 

these clones are in the section describing the next developmental node (Juvenile – 

Developmental Node 3), as they extend from the perinatal period to approximately 50 

days after birth. 

In a follow-up study by this same group, including animals from the 2002 study 

(Chavatte-Palmer et al. 2004), the authors noted a 76 percent survival rate (44/58) among 

clones following the first week after birth. Causes of death during the neonatal period 

included hyperthermia, umbilical hernia, respiratory problems, ascites (abnormal fluid 

accumulation) in the chest and abdomen, fatty liver, limb deformities, various digestive 

tract problems, and abnormal or degenerating kidneys. 

Reports from research groups noting no differences between clones and naturally bred 

animals provide very few details about the health status of the clones. For example, 

Kubota et al. 2000 reported that although 30 blood measurements were taken on four 

clone calves, and that they observed no differences between the clones and their agematched peers, neither the nature nor the numerical values of the measurements were 

provided. 
Chapter VI: Food Consumption Risks 194 

Animal Cloning: A Risk Assessment DRAFT

Table VI-2: Summary of Outcomes Measured in SCNT Clones and AI Controls 

(adapted from Chavatte-Palmer et al. 2002) 

Outcome AI Controls SCNT Comment 

Pregnancy 

Outcome: 

Stillborns or 

Abnormal 

Fetuses 

NR 

11/12 exhibit “pathological gestation;” 

1 animal sacrificed for control. 

5 term stillborn (gd* 274.4 ± 2.6). 

Abdominal ascites and edema. 

7 fetal membranes show large edematous 

cotyledons, and lower mean number of 

placentomes. 

Mean and median weight of placentomes 

higher than for normal pregnancies and 

controls. 

Kidney defects: 

Fetus: 1 enlarged 

Stillborn: both autolyzed. 

1 apparently normal fetus had “seemingly 

small kidneys.” 

1 large fatty liver in fetus; seemingly large 

amount of fat surrounding abdominal organs 

in “several” fetuses (number not specified). 

No other gross morphologic abnormalities in 

other organs. 

Live Births 

 Total 

 Caesarian 

 Vaginal 

n=176 

not specified 

not specified 

n=21; 7 fetal origin; 13 adult origin 

20 (18 at term, 2 were 1 week before term). 

1 

Clones delivered via 

C-section when natural 

calving had not 

occurred by gd 282. 

All calves survived to 

at least 2 mo of age. 

Body Weight 

(kg) 

43.7± 2.7 

n=176 

55.1± 2.7; n= 26 

Difference between Clone and AI and IVP 

statistically significant at P<0.01. 

No significant 

difference between AI 

and IVF. 

Body 

Temperature 

(BT) at birth 

Lower than 

SCNT 

(approximately 

38 to 39.5oC) 

Mean rectal BT higher in SCNT than 

controls in 1st week, and until 50 days. 

Data provided for only 1st week. 

Peak temperature spike approximately 41o

 C. 

No accompanying clinical signs. 

Comparison between 

n=10 NT and n=10 

combined AI (8) and 

IVF (2). 

Not sensitive to 

NSAID; regulated by 

using wet towels and 

ventilation. 
Chapter VI: Food Consumption Risks 195 

Animal Cloning: A Risk Assessment DRAFT

Hematologic 

Parameters 

 RBC, 

 HC, 

 Hb, 

 WBC, 

 Differentials 

 Mean cell 

 Parameters 

n=8 

Mean cell 

volume (43.59± 

0.60 fl). 

Neutrophil: 

lymphocyte ratio 

at birth 3.14 ± 

1.1; higher than 

SCNT. 

n=21 live clones. 

Mean cell volume (50.07 ± 1.29 fl) higher 

than AI. 

Neutrophil: lymphocyte ratio at birth 6.28 ± 

0.9; higher than AI. 

1 animal with lymphoid aplasia (Renard et 

al. 1999), sudden decrease in lymphocyte 

and RBC counts. 

No measurements 

reported after birth. 

Clinical 

Chemistry 

 Urea 

 Creatinine 

 AST 

 ALT 

NR All values within normal limits; individual 

data not provided. 

No measurements 

reported after birth. 

Thyroxine (T4) n=4 

n=7; Lower than AI controls for days 1-15. 

Approximate kinetics the same as AI (rapid 

decrease from birth to d 4, then constant low 

level (~15-25 pmol/l) to day 15. 

Measured for 2 months 

to determine whether 

associated with 

hyperthermia. 

IGF-1 

IGF-II 

IGFBP 

n=5; No diff. 

from SCNT. 

Lower than 

SCNT at birth 

and d 15. 

No difference 

from SCNT 

n=7; no difference from AI. 

Higher than AI at birth and day 15. 

No difference from AI. 

Measured from day of 

birth until age 80 days. 

Leptin 

n=5; Lower than 

SCNT animals, 

and less interanimal 

variability. 

n=6; higher in clones than controls during 

first week after birth. More inter-animal 

variability and changes in absolute response 

in SCNT animals. 

Levels revert to normal in amount and 

amplitude after one week. 

Measured from day of 

birth until age 28 days. 

Growth 

Hormone 

n=6; 

no difference 

from SCNT 

n=5; 

no difference Same as leptin assay. 

Insulin & PostPrandial 

Glucose 

Response 

n=6; 

No significant 

difference in 

either response 

between AI and 

clones 1 to 8 

days old. 

Some clones presented with hypoglycemia 

and hypothermia during first 24 h post 

partum. 

No significant difference in either response 

between clones and AI after the first 24 

hours. 

Cortisol 

(ACTH 

n=2; C-sect, 

n=6; natural 

n=11; C-section. 

n=1; natural birth. 

Increase in plasma 

cortisol in response to 
Chapter VI: Food Consumption Risks 196 

Animal Cloning: A Risk Assessment DRAFT

Induction) birth. Basal 

levels in Csection births 

lower than 

natural birth. 

No significant differences between clones 

and controls. 

Basal levels in C-section births lower than 

natural birth. 

ACTH stimulation 

reflects appropriate 

adrenal maturation and 

function. 

Lower basal cortisol 

values probably due to 

C-section and not NT 

or IVF. 

AI = artificial insemination 

NR = Not reported 

gd = gestational day 

NSAID = Non-steroidal anti-inflammatory drug

Matsuzaki and Shiga (2002) evaluated the potential link between endocrine status and 

perinatal difficulties in Japanese Black clone calves delivered via C-section (selected by 

the investigators on the basis of a comparison of fetal size and maternal pelvic diameter, 

or rapidly expanding hydroallantois) relative to clones delivered vaginally, or Japanese 

Black calves produced via AI, and IVF calves born via spontaneous vaginal delivery. 

Birth weight, plasma cortisol levels, Adreno Cortico Tropic Hormone (ACTH), and 

components of the insulin-like growth factor signal transduction pathway (IGF) were 

evaluated. Average birth weights of clones delivered by C-section were heavier than AI 

controls; average birth weights of vaginally delivered clones and IVF animals were 

intermediate compared with C-section clones and AI control animals. Clones delivered 

by C-section had lower cortisol and IGF-I levels than AI and in vitro produced controls, 

similar ACTH levels, and had more IGF binding protein-2 (IGFBP2) relative to controls. 

The authors concluded that in C-section delivered clones the expected prepartum rise in 

plasma cortisol did not occur, and that these animals failed to initiate the switch to extrauterine IGF-I system during late gestation. Four of five C-section delivered clones died 

within the first week following birth; one of the eight vaginally delivered clones died in 

that same time period of unspecified causes. 

In their first study, Kato et al. (1998) reported that eight of 10 blastocysts derived by 

SCNT from a Japanese Black beef cow completed gestation and were born. Seven were 

delivered vaginally, while one was delivered by emergency C-section due to dystocia. 

Two of the calves were born prematurely. Four of the eight calves died. No abnormalities 

were noted, and the authors attributed the deaths to “environmental factors” as described 

in Table VI-3. 
Chapter VI: Food Consumption Risks 197 

Animal Cloning: A Risk Assessment DRAFT

Table VI-3: Summary of Clone Outcomes 

(source Kato et al. 1998) 

Calf 

Gestation 

Length 

(days)1

Vaginal (V)/ 

Cesarean (C) 

Delivery

Birth 

Weight 

(kg)2 

Status at 

Publication Cause of Death 

1 242 V 18.2 Alive NA3 

2 242 V 17.3 Alive NA 

3 266 V 32.0 Dead (day 3) Pneumonia apostematosa 

from heatstroke 

4 267 V 17.3 Dead (day 0) Inhalation of amniotic fluid 

5 267 V 34.8 Dead (day 0) Inhalation of amniotic fluid 

6 276 V 23.0 Alive NA 

7 276 V 27.5 Alive NA 

8 287 C 30.1 Dead (day 0) Dystocia and delayed delivery 

1 Average gestation length for Japanese Black cattle: 286.6 ± 0.9 days 

2

 Average weight of Japanese Black calf at birth: 27.0 ± 0.8 kg 

3 NA = not applicable

In a second publication, Kato et al. (2000) reported the production of 13 surviving clones 

of 24 deliveries of Japanese Black and Holstein donor cells. Pregnancy duration was 

approximately equivalent to that of the donor cell breed, except that “a few” recipient 

cows had shorter gestations. Calves were either born vaginally or delivered via C-section; 

no criteria were given for the decision to perform C-section. Seven animals were either 

stillborn or died at delivery. Two clones died during C-section due to dystocia, but 

presented no gross abnormalities. One clone born appeared normal at birth but died 19 

days later from septicemia. Six dead clones had significant morphological abnormalities 

of the kidney or outer extremities, including severe tendon contracture. One clone was 

born disemboweled, and another had a “warped” face. All of these abnormal births were 

attributed to infection with Akabane virus, a known teratogen (birth defect inducer), as 

antibodies to the virus were detected in the serum of afflicted animals. Mean body 

weights of clones were higher than those of controls,50 with nine clones exceeding the 

mean body weight of controls by >40 percent. Interestingly, Kato et al. report on the 

unusual appearance of some male clones derived from a bull that was 10 years of age 

when cells were taken for donors in the SCNT process. At birth, the bull calves were 

reported to exhibit “an adult appearance, displayed as many wrinkles in the skin, thick 

bone structure and rough hairs resembling those of adult males.” They speculate that 

 50 Mean body weights of Holstein calves at term were 40 kg for females and 47 kg for males; for 

Japanese Black cattle, mean female calf birth weight at term was provided as 27 kg, and male at 38 kg. 
Chapter VI: Food Consumption Risks 198 

Animal Cloning: A Risk Assessment DRAFT

these might result from mutations in the donor cells that increase with age or to telomere 

length. 

In the Kubota et al. (2000) study of clones from the 17 year old Japanese Black bull 

described in the Cell Fusion/Fetal Developmental Node (Developmental Node 1), two 

calves died shortly after birth, one of which was diagnosed as having Akabane Virus. The 

other died due to complications following a difficult delivery (dystocia). Four others 

survived, and were reported to be healthy and normal. The average gestation periods for 

the clone pregnancies was 294 days (range of 291-299 days), which was nine days longer 

than the breed average of 285 days. Average birth weight of the clones was 36 kg (range 

of 30.7 to 42.5 kg), approximately 20 percent heavier than the breed average of 30 kg. 

Kishi et al. (2000) used fibroblast cells from ear punches of Holstein or Japanese Black 

cattle, and somatic cells isolated from the colostrum of mammary gland epithelial (MGE) 

cells from Holstein cows as SCNT donors. Of the 45 embryos implanted into 31 

recipients, three pregnancies were confirmed on gestation day 60, and two calves were 

born from colostrum derived MGE cells. One clone was delivered at 279 days of 

pregnancy by C-section and weighed 44 kg; the other was vaginally delivered after 

induction of parturition at 280 days of gestation and weighed 45 kg. For the fibroblastderived clones, 43 embryos were implanted into 37 recipients. Five pregnancies were 

confirmed on gestational day 60, and 2 calves were born (one Holstein and one Japanese 

Black). The clone derived from the Japanese Black fibroblast died six hours after birth 

due to internal hemorrhage of the umbilical artery. Two of the Holstein clones (the origin 

of the cells is unclear) received blood transfusions due to anemia at some unspecified 

time after birth. The three remaining Holsteins (presumably including the posttransfusion clones) were reported as “normal and healthy.”

A series of papers (Taneja et al. (2000); Tian et al. (2000); Xu and Yang (2001); Enright 

et al. (2002); Govoni et al. (2002); Xue et al. (2002); Savage et al. (2003)) has been 

published on a group of female Holsteins cloned from a 13 year old cow by the 

laboratory of X. Yang at the University of Connecticut. Most of these studies report on 

the birth and later development of these calves, and are discussed in the sections 

appropriate to those developmental nodes. 

 

In a meeting abstract, Taneja et al. (2000) described the premature delivery of 10 

Holstein clones and the supportive care that they required. Normal gestation length for a 

Holstein averages 282 days (range 280 to 285 days). All the calves born in this study 

were premature (average gestation length 266.6 ± 2.0 days), regardless of whether labor 

was induced or occurred naturally. Three cows initiated labor spontaneously at 263.0 ± 
Chapter VI: Food Consumption Risks 199 

Animal Cloning: A Risk Assessment DRAFT

3.8 days gestation. Twin calves born to one surrogate dam were stillborn, with one 

requiring manual delivery. One of the calves in the spontaneous labor group was 

delivered by C-section, showing signs of stress and hypothermia (body temperature 

<100oF). This calf was hospitalized after 36 hours, when it began running a fever. A chest 

x-ray revealed immature lung development, and blood gas measurements indicated low 

blood oxygen concentration. The calf also underwent surgery for an umbilical abscess 

and for patent urachus (the canal connecting the bladder with the umbilicus) on day 6, 

after which it recovered and survived. The last calf born in the spontaneous labor group 

was delivered vaginally with some assistance, was diagnosed with immature lung 

development and low blood oxygen concentration; it died within 12 hours of birth. 

Necropsy of this calf indicated bacterial infection and septicemia, as well as immature 

lung development. The remaining five surrogate dams were treated with dexamethasone 

17 hours prior to planned C-sections. Four single calves and a pair of twins were born in 

the induced labor group. Two calves were delivered vaginally without assistance at 8 and 

15 hours post induction treatment. The first calf (born after eight hours) was healthy and 

did not require supportive care. The second calf (born after 15 hours) died three hours 

after birth; necropsy revealed that it had died of hypoxia and immature lungs. A set of 

twin calves and another single calf were delivered by C-section. One of the twin calves 

and the singleton survived, while the other twin and another single calf died soon after 

birth. Necropsy revealed that they had inhaled mecomium (the first intestinal discharge 

that normally occurs after birth that can appear in the amniotic fluid if the fetus is 

distressed) and the lungs failed to inflate completely. All but one of the surviving calves 

required supportive care ranging from supplemental oxygen to surgery. The four 

surviving clones were the subject of additional studies by this lab, including Enright et al. 

(2002) and Govoni et al. (2002). In the study by Xue et al. (2002) comparing the relative 

effectiveness of different cell types as donors for SCNT, four of the six calves from the 

ovarian cumulus group survived the perinatal period; all four of the calves born from 

donor skin fibroblast cells died. All deaths occurred within 24 hours of birth due to 

respiratory distress. 

Batchelder (2005) reported on the birth of eight clones (three Hereford and five Holstein) 

and nine comparators produced by AI (n=3) or ET (n=6). She noted an interaction 

between cloning and cattle breed, such that Hereford clones were heavier (range 50.0 to 

71.0 kg; n=3) than their breed-matched ET comparators (range 31.5 to 48.0 kg; n=3), 

while Holstein clones had similar birth weights to their breed-matched ET comparators 

(37.1 vs. 39.4 kg). Neonatal clones had lower RBC and hematocrit at birth and for the 

first hour, but were similar to comparators thereafter. Clones also exhibited lower blood 

glucose and lactate levels than comparators during the first 24 hours, but were similar to 

comparators by 48 hours. No differences were noted between clones and comparators for 
Chapter VI: Food Consumption Risks 200 

Animal Cloning: A Risk Assessment DRAFT

WBC and differential patterns. Although Batchelder noted several clinical signs often 

associated with LOS in both Holstein and Hereford clones (delayed time to suckle and 

stand, hypoglycemia, forelimb flexor tendon contracture, enlarged umbilicus, patent 

urachus, and respiratory distress), many of the same signs were noted in the AI-derived 

comparator group in this study (see Chapter V for more details). In this study all clones 

survived the first 48 hours after birth, but two clones were lost between 72 hours and six 

days of age. All comparator calves survived. 

Wells et al. (2004) reported that a total of 133 clone calves were delivered as a result of 

988 embryo transfers of somatic cell nuclear transfers (SCNT) using adult and fetal donor 

cells. Embryonic cloning resulted in 27 delivered clone cattle from 210 embryos derived 

from embryonic blastomeres (ENT). Both techniques were reported to result in a live 

birth success rate of 13 percent. Approximately two thirds of these calves survived to 

weaning (3 months of age). 

Yonai et al. (2005) reported on the growth, reproduction, and lactation of clones whose 

nuclear donors were a high milk performance 13 year old Holstein and a six year old 

Jersey that had previously been used for embryo transfer. These animals had previously 

been characterized as having shortened telomeres, but are otherwise indistinguishable 

from cattle of presumably normal telomere length (Miyashita et al. 2002). (Discussions of 

growth and reproductive and lactational performance of these clones are found in 

Developmental Nodes 3, 4, and Compositional Analysis, respectively). Table VI-4 

summarizes the success rates for the two breeds of dairy clones. All embryos, regardless 

of the breed of the donor cows, were implanted into multiparous Holstein surrogate dams. 

One of the recipients of Holstein embryos had twin calves. The overall success rates, as 

measured by surviving calves as a function of embryos implanted were approximately 5 

and 10 percent for the Holsteins and Jerseys, respectively. 

The authors state that although there is an approximately two-fold difference in the 

production rates between breeds, this difference is not statistically significant due to the 

low numbers in the study. The abortion rate in the surrogate dams carrying Holstein 

clones was approximately two times higher than the Jersey group (68.4 percent v 31.8 

percent). No dystocia was noted in surrogates carrying Jersey clones; incidence of 

dystocia in the surrogates carrying Holsteins was not reported. The authors attribute the 

differences in outcomes to the smaller size of the Jersey fetuses relative to the Holstein 

fetuses. Gestational periods and birth weights were reported as being within normal 

ranges for dairy cows of these breeds. Although there was more variability in birth 

weights of the Holstein clones than the Jerseys, no symptoms of LOS were noted in these 

two clone cohorts. The authors note that although cell culture conditions have been 
Chapter VI: Food Consumption Risks 201 

Animal Cloning: A Risk Assessment DRAFT

implicated as a potential source of large calves, the two cell lines used for nuclear transfer 

were cultured under identical conditions, implying that differences between the cell lines 

(i.e., heredity) was likely responsible. 

Table VI-4: Success Rates for Implantation Through Delivery for Holstein and Jersey Clones 

(source Yonai et al. 2005) 

Jersey Embryos Holstein Embryos 

Recipients 22 63 

Embryos Transferred 37 124 

Pregnancy Detected at 40 - 60 days 7 (31.8%) 18 (28.6%) 

Failure to Reach Term 1 (14.3%) 11 (61.1%) 

Calves Delivered 6/22 (27.3%) 8/63 (11.1%) 

Surviving Calves from Transferred 

Embryos 4/37 (10.8%) 6/124 (4.8%) 

Production Rate from Recipients 4/22 (18.2%) 6/63 (9.5%) 

Average Birth Weights ± SD kg (ranges) 29.4 ± 1.5 (27.5-31.0) 36.2 ± 7.7 (27.0-47.0) 

In summary, the survival rate of clones appears to be in the range of 5-18 percent, 

depending on how it is calculated. Many of the perinatal clones die of complications or 

sequellae of LOS. Newborn cattle clones may be more physiologically fragile than their 

comparators, and differences between clones and comparators include body weight, body 

temperature, alterations in the amounts of circulating IGF-II, leptin, growth hormone, T4, 

and differences in mean erythrocyte volume either on the day of birth or shortly 

thereafter. None of the differences between clones and AI- or IVF-derived controls 

persisted through the longest observation period (up to three months) (Chavatte-Palmer et 

al. 2002; 2004), and most resolved within a week or two of birth (Hill et al. 1999 (for 

transgenic clones); Enright et al. (2002); Govoni et al. (2002); and Tian et al. (2001)) 

(See subsequent discussions in the sections on the appropriate developmental nodes). 

(b) Cyagra Dataset: Perinatal Cohort51

Of the 134 clones in the Cyagra dataset that were born or delivered, 103 animals (or 77 

percent) were alive three days after birth. The remaining 31 were stillborn, died, or were 

euthanized within three days of birth. Details on health and survival of conventional, agematched comparators (comparators) are not available. At the time that data were 

collected on these animals (late March 2003), 67 were alive (64 percent of those 

 51 Data from Cyagra and the Center’s detailed analyses of the data are found in Appendix E: Cyagra 

Dataset. Summaries of the analyses are presented in the narrative of the Risk Assessment. Readers 

wishing to have the best understanding of the Cyagra Dataset are urged to read the entire Appendix 

prior to continuing with the summaries. 
Chapter VI: Food Consumption Risks 202 

Animal Cloning: A Risk Assessment DRAFT

surviving to 48 hours, or 50 percent of those born or delivered). Eight animals died 

between 4 and 149 days of birth. The problems noted at the time of birth and the causes 

of death for those clones not surviving are summarized in Table E-2 of Appendix E: The 

Cyagra Dataset. Some animals required supportive care immediately after birth (e.g.,

glucose, warming, or supplemental oxygen), and many (n=26) received umbilical surgery 

after birth. 

Blood was drawn for clinical chemistry and hematology for 10 clones within a few hours 

(or in some cases, minutes) of birth. The actual measurements provided by the Cornell 

Animal Health Diagnostic Laboratory are found in Appendix E, Tables E-100a (clinical 

chemistry), and E100b (hematology). Charts E-100, E-101, E-102, E-110, E-111, and E112 compare these values with the comparator population reared on the same farms and 

the Cornell Reference Values and are also found in Appendix E, along with all of the data 

from which they were generated. 

Ninety percent of the total clinical chemistry values of the clones were within the range 

of values exhibited by the comparators, and 90 percent of the hematology values were 

within the comparator range. Twenty-seven of the 33 analytes (substances that were 

measured, such as sodium, cholesterol, or liver enzyme activity) had either no differences 

or one difference relative to the comparators (Chart E-101). The remaining six analytes 

tended to be more variable between clones and comparators. Liver values (AST, GGT, 

cholesterol, bile acids (hBA)) were lower in several clones, for reasons likely related to 

the placental/umbilical abnormalities, or transitions from fetal to adult circulation. GGT 

levels were also low relative to the comparators, probably related to blood sampling prior 

to colostrum intake, whereas comparators were administered colostrum prior to blood 

draw. None of the out-of-range values of these analytes poses any particular concern for 

food safety, as they are relatively close to the comparator range. 

Blood cell parameters in the neonatal clones were also very similar to those of the 

comparators. Fifteen of the 17 analytes had either no differences or just one difference 

between the two groups (Chart E-111). With the exception of one clone that was infected 

with rotavirus and subsequently died, all red blood cell parameters were within the range 

of the comparator group. Three clones had white blood cell counts that were lower than 

the comparator range. One clone was infected with rotavirus but survived, indicating that 

at least in that animal, the immune system was functioning appropriately. There did not 

appear to be an increased incidence of infection in these animals, except where infection 

was associated with umbilical difficulties, also indicating that the immune systems were 

functioning appropriately. 
Chapter VI: Food Consumption Risks 203 

Animal Cloning: A Risk Assessment DRAFT

(c) Unpublished data 

In response to requests by CVM, various groups involved in cloning submitted 

unpublished data. One such group, a commercial cloning company, submitted body 

temperature, pulse and respiration rates on 19 cattle clones (breed(s) and gender not 

identified) during the first 72 hours of life. These data has been discussed in greater detail 

in Chapter V. Body temperatures were elevated during the observation period (mean 

103 ̊F at birth; 102.7 ̊F at 72 hours); heart rates appeared to increase (95.2 beats/min at 

birth; 138.6 beats/min at 72 hours); while respiration rates remained fairly constant (53.9 

breaths/min at birth; 53.1 breaths/min at 72 hours). It is often difficult to evaluate data on 

heart rate and respiration in livestock, since the stress of handling tends to increase these 

rates. Body temperature in neonatal clones appears to be quite variable, with some studies 

reporting hyperthermia (Chavatte-Palmer et al 2002; Batchelder 2005) which may persist 

through the first 50 to 60 days of life and then appears to normalize. 

Another cloning firm presented birth records on two Holstein heifer clones delivered by 

C-section. The calves weighed 45 and 47.7 kg at time of delivery, within the normal 

range for Holstein cattle; body temperatures were 100 and 102.6 ̊F at birth, slightly below 

and above normal (101.5 ̊F) for cattle. These two calves were otherwise normal, 

according to the veterinarian’s notes and limited blood chemistry (See Chapter V for 

details). 

 

(d) Summary for Perinatal Developmental Node in Bovine Clones 

(Developmental Node 2) 

The combined information from the peer-reviewed literature and the Cyagra dataset 

indicates that newborn clones tend to be more fragile than their comparators, with a 

higher incidence of perinatal death. Abnormalities noted among both dead and surviving 

clones include respiratory distress, organ malformations, flexor tendon contracture, and 

umbilical difficulties. None of the adverse outcomes observed are qualitatively different 

from adverse outcomes that have been observed in natural breeding or other assisted 

reproductive technologies. Some animals succumbed to infection, but there does not 

appear to be a decrease in immune function in the population of clones at the perinatal 

stage. Despite the perinatal deaths and noted anomalies, most clones that survive 

parturition, either with or without assistance, appear to stabilize. 

“Sentinel” markers were sought that might predict a successful outcome for perinatal 

clone calves. Based on the literature and the Cyagra data, it does not appear that any one 

analyte or analyte profile is predictive of whether a particular animal, or indeed, the entire 
Chapter VI: Food Consumption Risks 204 

Animal Cloning: A Risk Assessment DRAFT

cohort of animals will develop into normal, fully functioning, healthy animals. The 

laboratory data are consistent with the hypothesis that animals that look and behave 

normally are normal with respect to laboratory values, implying that consideration of the 

complete dataset on an individual animal is the best predictor of the health of that animal. 

Further, the seven surviving Cyagra clones that were sampled twice (# 71, 72, 73, 78, 79, 

119, and 132) provide the baseline data for a small subcohort of animals for which there 

are laboratory measurements at two different time points, as described more fully in the 

following section. 

iii. Juvenile Development in Bovine Clones (Developmental Node 3) 

Most of the information on this developmental node has been extracted from publications 

that primarily address the perinatal period. 

(a) Peer-reviewed Publications 

For purposes of following the cohorts of animals, these reviews have been grouped by 

institution. 

The Institut National de la Recherche Agronomique (INRA) Studies: Renard et al. 1999 

and Chavatte-Palmer 2002 

Renard et al. (1999) reported one case of lymphoid hypoplasia in a clone generated from 

cells in an ear biopsy of an animal that had herself been the product of blastomere (or 

embryo) nuclear transfer (BNT). An echocardiogram performed on the animal 

immediately after birth revealed an enlarged right ventricle of the heart. The animal was 

treated with an angiotensin converting enzyme (ACE) inhibitor and given diuretics for 

one month, at which time the condition was reported to be resolved. Blood samples taken 

every two days after birth revealed relatively high reticulocyte counts and immature 

blood cells in the blood during the first three weeks of life. Lymphocyte (white blood 

cell) counts were also reported as normal for about a month after birth, but counts fell 

rapidly after that time. Hemoglobin levels in the animal also decreased at about day 40. 

On day 51, the animal died from severe anemia. Histological examination of the calf 

revealed hypoplasia (lack of development) of the thymus, spleen, and lymph nodes or 

global lymphoid aplasia (absence of lymphoid cells in all organs in which they would 

likely be found) that likely began at birth. No evidence for the endogenous synthesis of 

immunoglobulin G was detected. Bovine Viral Diarrhea virus, which has been known to 

induce thymic atrophy, was ruled out. SCNT was implicated as the cause of the lymphoid 
Chapter VI: Food Consumption Risks 205 

Animal Cloning: A Risk Assessment DRAFT

aplasia, possibly due to the selection of a cell with a mutation responsible for the 

expression of the portion of the genome governing lymphoid development, or lack of 

appropriate reprogramming of the somatic cell nucleus. In a follow-up study by this 

group (Chavatte-Palmer et al. 2004) an additional four clones were diagnosed with 

thymic aplasia. Histological examination of the thymus glands of these calves indicated 

abnormal tissue organization, suggesting the aplasia was the result of epigenetic errors. It 

is not clear from the late report whether these four clones were also the result of serial 

cloning. To our knowledge, this is the only laboratory reporting thymic aplasia in clones. 

Three other calves in this cohort died suddenly with few or no clinical signs: two died of 

diarrhea, and one died without any apparent cause. 

In a separate report of the larger cohort of clones produced by the same laboratory (see 

Perinatal Developmental Node), Chavatte-Palmer (2002) monitored the growth and 

development of 21 clones. For the first week after birth, the mean rectal body temperature 

was higher in clones than AI controls, and some temperature spikes (up to 41o C; normal 

temperature is considered to be approximately 38.5 oC in dairy cows) were observed. 

Elevated temperatures in the clones persisted for 24-36 hours, and were not sensitive to 

pharmacological intervention. Animals were cooled by wrapping in wet towels and 

providing ventilation, although they did not appear to be distressed during the 

temperature spikes. No bacterial infection was detected, and no changes in hematology or 

clinical chemistry were observed. The authors state that the mean temperature remained 

elevated for 50 days, although data are only provided for the first week. Thyroxine (T4) 

levels were tested to determine if they could help explain the temperature difference 

between clones and controls. Plasma thyroxine levels were lower in clones than controls 

during the first two weeks of life, and then reverted to normal levels. Chavatte-Palmer et 

al. (2002) noted that lower plasma T4 levels coupled with elevated body temperatures in 

young calves was consistent with the findings of Carstens et al. (1997). (See discussion in 

Chapter V on metabolism and body temperature.) 

In the Chavatte-Palmer et al. (2002) study, the higher body temperatures of clone calves 

were independent of T4 levels, suggesting that the hyperthermia experienced by the 

clones may have resulted from increased brown adipose tissue (BAT) metabolism (see 

discussion in Chapter V). Chavatte-Palmer et al. did not measure norepinephrine, but did 

measure cortisol, another hormone that may be stress-induced. They observed that 

cortisol levels were decreased in both clone and non-clone calves born by C-section 

relative to calves born vaginally. By seven days of age, all of the calves exhibited similar 

cortisol levels following an ACTH challenge (AdrenoCorticoTropic Hormone induces 

the production of cortisol). In the Carstens et al (1997) study, the response to 

norepinephrine infusion tended to be breed specific: Bos indicus (breeds originating in 
Chapter VI: Food Consumption Risks 206 

Animal Cloning: A Risk Assessment DRAFT

the tropics and subtropics) calves tended to produce more basal and norepinephrineinduced cortisol than calves with more Bos taurus breeding (originating from cooler 

climates). All of Chavatte-Palmer’s calves were Holstein, or of Bos taurus origin. 

Therefore, without knowing what the norepinephrine levels were in the Chavatte-Palmer 

calves, it cannot be determined if the hyperthermia observed in clone calves was related 

to stimulation of BAT by norepinephrine, though it is plausible. 

Blood parameters evaluated by Chavatte-Palmer et al. (2002) included red blood cell 

count (RBC), hematocrit (HC), hemoglobin (Hb), and counts of white blood cells 

(WBC), including differentials (counts of the distributions of populations within the 

overall category of white cells). Mean cell volume was higher in clones than AI controls, 

and the neutrophil:lymphocyte ratio was higher in clones at birth than in AI controls. As 

previously mentioned, one clone presented with lymphoid aplasia (Renard et al. 1999), 

with decreased lymphocyte and RBC counts. All other blood parameters in clones were 

reported as not statistically different from AI controls. Clinical chemistry values were 

reported as within normal limits. With the exception of the aplastic clone in Renard et al. 

(1999), no clinically relevant findings accompanied these measurements over the time 

period of the study. (For a discussion of the nature and relevance of these tests, refer to 

Appendix F). 

In addition to thyroxine, endocrine measures that were evaluated included IGF-I, IGF-II, 

IGF binding protein, leptin, and growth hormone. No differences in levels of growth 

hormone, IGF-I, or IGF binding protein were observed between clones and AI controls, 

although levels of IGF-II were relatively high at birth but then rapidly decreased within 

15 days. Leptin levels were higher in clones than controls during the first week of life, 

but reverted to normal after that. Both insulin and post-prandial glucose response were 

measured in clones and AI controls, with no differences between the two groups 

(Chavatte-Palmer et al. 2002). 

Thus, even for physiological measures in which differences were detected between clones 

and controls, most resolved soon after birth in apparently healthy animals. Of those 

measured, even the most persistent, abnormal body temperature, resolved after 50 days. 

The study authors caution that, based on their data, apparently healthy clones should not 

be considered “physiologically normal animals until at least 50 days of age.” 

The 2004 follow-up study by Chavatte-Palmer et al. noted that clones (n=25) had slightly 

lower hemoglobin levels than AI comparators (n=19), although the hemoglobin levels of 

the clones were still considered within the normal range. The lower levels persisted for 

the first 65 days after birth before reaching the same levels as the AI comparators. This 
Chapter VI: Food Consumption Risks 207 

Animal Cloning: A Risk Assessment DRAFT

finding reinforced the group’s opinion that clones could not be considered 

physiologically normal for the first two months of life. 

The University of Connecticut Studies: Govoni et al. 2002; Enright et al. 2002; and 

Savage et al. 2003.

Govoni et al. (2002) investigated the degree to which the somatotropic axis52 in Holstein 

clones developed normally compared to AI-produced age-, gender- and breed-matched 

controls. All calves were prepubertal at the beginning of the study. Differences were 

noted over time between clones and controls in growth hormone (GH) and insulin-like 

growth factor-1 (IGF-I) levels. Over the course of the six month study, GH levels 

declined in controls, but began to increase beginning at about nine months of age in the 

clones. Although IGF-I increased in both groups over time, clones continued to have 

lower IGF-I concentrations compared to age matched controls. In a review of this issue, 

Le Roith (2001) indicates that GH is a major modulator of systemic concentrations of 

IGF-I. Growth hormone, produced in the hypothalamus, binds to liver cells and 

stimulates production of IGF-I. Somatostatin, which is stimulated by high levels of IGF-I, 

suppresses GH synthesis, which in turn causes a reduction in IGF-I synthesis in the liver. 

Clones in this study were more responsive to certain factors promoting GH release, but 

showed a similar response to controls when exposed to inhibiting factors. Response to 

Growth Hormone Releasing Hormone (GHRH) was five times higher in clones compared 

to controls, and returned to basal levels 40-50 minutes post stimulation. Somatotropin 

Release Inhibiting Factor (SRIF) was successful in inhibiting response to GHRH in both 

clones and controls. IGF Binding Protein 2 (IGFBP2) levels were not different between 

growing clones and controls in the relatively older animals of this study. Levels of 

IGFBP3, another IGF-I binding protein, however, were lower in clones compared to 

controls. Although the reasons for this are not entirely clear, this may be due to the lower 

IGF-I levels in these animals, which may down-regulate this binding protein. 

Although lower circulating IGF-I levels may be partially responsible for the later onset at 

puberty observed in this group of clones (Enright et al. 2002) (as IGF-I is involved in 

development of ovarian follicles and uterine growth (Le Roith et al. 2001)), the 

concentration of IGF-I required for normal sexual development is not known. Despite the 

reported differences in these protein levels, the clones appeared otherwise healthy and 

grew normally. Appendix F: Comprehensive Veterinary Examination discusses the 

relative weight that individual clinical chemistry values should have in the overall 

 52 The somatotropic axis governs the growth and development of the body. 
Chapter VI: Food Consumption Risks 208 

Animal Cloning: A Risk Assessment DRAFT

evaluation of the health of cattle. Interpretation of these results should occur within the 

context of that discussion. 

Savage et al. (2003) evaluated the behavior of the clones and age-matched controls 

described in the Govoni et al. (2002) study. Between 32 and 36 weeks of age, there were 

no differences in weight or height between the clones (205.5 ± 9.9 kg; 117.0 ± 1.8 cm) 

and controls (211.4 ± 7.4 kg; 119.5 ± 1.4 cm). All calves were raised together under the 

same management conditions. Based on a series of studies evaluating approach to other 

animals and novel objects, clones exhibited age-appropriate behaviors, but were reported 

to be more aggressive and inquisitive than controls, and spent more time grooming and 

socializing. Clones tended to spend less time in playful behavior than controls. Review of 

records on the cow that served as the donor for the clones indicated that she had 

displayed similarly aggressive and inquisitive behavior as a young animal, suggesting 

that at least some of these behavioral traits may be genetically controlled. Clones spent 

more time in proximity to adult animals in an adjacent pen (which also housed the 

nuclear donor), and in proximity to the feed bunk compared to control animals. In 

general, clones were reported to spend more time with each other rather than socializing 

with control animals, with the authors speculating as to whether clones exhibit some form 

of genetic kinship recognition. Nonetheless, the overall conclusion of this study was that 

the clones behaved normally. 

Other Studies 

Wells et al. (2004) and Wells (2005) followed the growth and maturity of cattle clones 

generated at their facility in New Zealand through approximately four years of age. 

Approximately 80 percent of the clones delivered alive at term survived the first 24 hours 

of live. They reported that two-thirds of the 20 percent that died was due to spinal 

fractures syndrome or to deaths from dystocia, associated with LOS (Wells 2005). 

Another 15 clones died in the time period before weaning, most commonly of 

musculoskeletal abnormalities, including tendon contracture and chronic lameness, and 

umbilical infections, attributable to complications of LOS. They also reported two clones 

dying as the result of bloat, and an unspecified number of clones dying due to endophyte 

toxicity after eating fungus-infected ryegrass. Bloat and other gastrointestinal disorders 

have been reported by others (Cyagra 2003; Batchelder 2005), but also may result from 

feeding or grazing management problems. Wells et al. use the phrase “clonal family” to 

refer to clones derived from a particular donor, and note that the bloat and susceptibility 

to endophyte toxicity was restricted to one clone family, and likely due to their genetics. 

Another clone family consisting of three clones (and five half-siblings produced by AI) 

survived with no health anomalies and at the time of reporting was 18 months old. Other 
Chapter VI: Food Consumption Risks 209 

Animal Cloning: A Risk Assessment DRAFT

health problems observed during the juvenile period included anemia, chronic heart 

failure, and degenerative nephrosis, problems that have also been noted by other 

researchers (Chavatte-Palmer et al. 2004). Additional deaths were categorized as being 

due to misadventure and accidental deaths due to clostridial disease, parasitism, and over 

feeding. Surviving animals from this group were characterized with respect to general 

health and physiological measurements; these are found in the discussion of 

Developmental Node 5 (Post-Pubertal Maturation). 

Similar to Chavatte-Palmer et al. (2002), Batchelder (2005) also noted periodic moderate 

to severe hyperthermia in Holstein and Hereford clones up to 60 days of age. As with the 

Chavatte-Palmer clones, the Batchelder clones also showed no indication of infection, 

were unresponsive to anti-inflammatory drugs, and their behavior was unchanged; the 

hyperthermia also resolved spontaneously. 

In their study of Japanese Black beef cattle clones described in the section on the 

Perinatal Developmental Node, Kato et al. (1998) reported that all of the clones that 

survived the perinatal period were alive and healthy at 85 and 120 days of age. In the 

subsequent study (Kato et al. 2000) of 13 clones that survived the perinatal period, 12 

clones were alive and healthy at 117-350 days, and one clone died at three months “for 

no clear reason.” 

Kubota et al. (2000), in their study of four surviving clones of a 17 year old Japanese 

Black bull, reported that the clones were 10-12 months of age at the time of publication. 

Based on veterinary examinations, growth curves, and 30 blood parameters no 

differences were found between the clones and their age-matched peers. No data were 

provided in the publication. Other groups have also reported normal growth rates for 

cattle clones (Wells et al. 2004; Heyman et al. 2004). 

Yonai et al. (2005) (previously mentioned in the Perinatal Developmental Node) studied 

the growth of Holstein (n= 6) and Jersey (n=4) clones with shortened telomeres. Clones 

were given at least two liters of warmed colostrum immediately after birth, fed colostrum 

twice a day for the first five days of life, and monitored for physiological functions until 

they stabilized. Clones were fed according to the guidelines presented by the US National 

Research Council Nutrient Requirements of Dairy Cattle (1989). From Day 5 through 

Day 45, calves were given milk replacer twice daily, and offered calf starter pellets, hay 

and water during this time. After Day 45, all calves (clones and comparators) were 

weaned from milk replacer, and their feed gradually changed from calf starter pellet to 

formula feed over a two week period. Calves were fed 2-3 kg/day of formula feed, hay 

and water from Day 60 until one year of age. For the first 45 days after birth, the clones 
Chapter VI: Food Consumption Risks 210 

Animal Cloning: A Risk Assessment DRAFT

were reared in individual calf huts, after which they were reared together with other 

calves produced by AI or embryo transfer. Calves were held in a large pen in mixed 

groups of clones and age-matched comparators during the weaning period. After 

weaning, groups of 10-20 animals were moved into pens, and after one year of age, all 

animals were moved to a free-stall barn for heifers. Table VI-5 summarizes the average 

daily body weight gain of the clones from birth to two years of age. Body weights were 

collected monthly from birth to one year of age, and every three months between 15 and 

24 months. 

Table VI-5: Average Daily Gain (kg/day) for Holstein and Jersey Clones 

(source Yonai et al. 2005) 

months of age 

0-3 3-6 6-9 9-12 12-15 15-18 18-21 21-24 

Jersey Clones (n=4) 

Mean 0.49 0.73 0.67 0.53 0.49 0.56 0.51 0.40 

SD 0.02 0.02 0.11 0.06 0.05 0.17 0.16 0.18 

Holstein Clones (n = 6) 

Mean 0.72 1.17 0.82 0.85 0.90 0.97 0.68 0.58 

SD 0.14 0.12 0.08 0.11 0.10 0.26 0.11 0.27 

SD = Standard Deviation 

The authors report that the average daily gain for the clones was greater than that of the 

standard of each breed. For the Holstein clones, the average bodyweights conformed to 

the standard during the first three months of age, but exceeded the standard after five 

months, while the Jersey clones exceeded the body weight of the Japanese Feeding 

Standard for Dairy Cows throughout the measured time period. The Holstein clones’ 

body weights were approximately equivalent to that of the donor animal until 18 months 

of age, but exceeded it thereafter. The Jersey clones exceeded the body weights of the 

donor from birth to two years of age. The animals were reported as healthy with normal 

growth throughout this time period. No deaths were reported after the perinatal period. 

There are other reports of clones that appear to be healthy at birth but unexpectedly die 

some time later. Gibbons et al. (2002), for example, reported a clone dying at 60 days of 

age due to respiratory and digestive problems. As mentioned above, Kato et al. (2000) 

also reported the death of a clone at 3 months. Chavatte-Palmer et al. 2004; Wells et al. 

2004; Batchelder 2005 have also noted early deaths, but their cause(s) have not been 

clearly linked to cloning. The degree to which these unexpected deaths in cattle are 

related to cloning, or some disease process that is independent of cloning, is not clear. 

Ogunuki et al. (2002) have noted shorter life spans in some of their mouse clones; the 
Chapter VI: Food Consumption Risks 211 

Animal Cloning: A Risk Assessment DRAFT

cause of death appears to be due to liver damage, pneumonia, or neoplasia. The relevance 

of mouse models to domestic livestock has been discussed in Chapter IV. 

(b) Cyagra Data: 1-6 Month Age Cohort 

The calves from the Cyagra dataset most closely correlating to the Juvenile 

Developmental Node are the 46 clones and 47 comparators found in the 1-6 month of age 

group. Tables E-200a and E-200b, and Charts E-200, E-201, E-202, E-210, E-211, and E212 describe CVM’s analyses of the information. 

In general, these clones appeared normal, although some anomalies were noted on 

physical examination. These may be related either to cloning or to the genetics of the 

animal that was being propagated. None are unique to clones, although their frequency 

appears to be higher in clones than in calves produced using other forms of reproduction 

(see Chapter V). One of the clones was culled for poor conformation (the physical 

appearance of the animal), a matter of potential business importance to the producer, but 

likely having no impact on either food or animal health. Conventional animals with poor 

conformation are generally not used in selective breeding programs, and may be culled; it 

is likely that breeders will put similar limitations on clones as well. Several of the clones 

experienced serious problems resulting from umbilical abnormalities, including 

enlargement, excessive bleeding, and infection of the navel. These were resolved 

surgically. In addition, three cases of cryptorchidism (undescended testicle) were 

identified in calves from the same cell line. Although this condition is relatively 

uncommon in conventional animals, it is observed with some frequency, and is thought to 

be hereditary. 

Interestingly, three clones derived from the same Jersey cow cell line presented with very 

different phenotypes. Clones # 87, 88, and 89 were within 10 days of age of each other 

when they were weighed and blood samples drawn (131-141 days old). All three required 

umbilical surgery. The oldest, clone #87, weighed 282 pounds. Clone #88, who was a day 

younger, weighed 197 pounds, and the youngest (at 131 days of age) weighed 215 

pounds. Otherwise, the animals were healthy on physical examination. A fourth clone 

from this cell line died at birth from LOS-related complications. 

Measurements of analyte levels in the entire 1-6 month old cohort were generally very 

close to those measured in the comparators (Chart E-201). In aggregate, 96 percent of the 

total analyte values for clones were within the range of the comparators. A few were out 

of range: glucose values were above the range of the comparators in six of the 42 likely 

valid measurements (four were considered artifactual). In order to determine whether the 
Chapter VI: Food Consumption Risks 212 

Animal Cloning: A Risk Assessment DRAFT

hyperglycemia was transient or sustained, urinalysis results were checked for the clones 

with elevated blood glucose levels. As none of those tests were positive for glucose (the 

renal threshold for glucose in cattle is approximately 100 mg/dl: i.e., if blood levels of 

glucose exceed 100 mg/dl for any appreciable time, glucose spills over into the urine), it 

is unlikely that the higher blood glucose levels (88-123) had been sustained long enough 

to allow for spillover into the urine. Most likely, these were transient elevations resulting 

from proximity to a meal or as a short-lived response to stress (as in being restrained for 

blood draws). 

The hemograms for the cohort did not reveal any significant health concerns. None of the 

clones were anemic, and there was no depression of cellular immune function. Some of 

the clones had individual values that were outside the range of the comparators, but these 

were not judged to pose either an animal health or food consumption risk (see Appendix 

E for a more complete discussion). 

It is important to note that although this time period appears to be relatively short, it 

spans an important developmental transition period for ruminants. Calves that are closer 

to one month of age are still primarily milk-fed, while those closer to six months of age 

have mostly transitioned to a more adult diet, and function as ruminants. The youngest 

animals are in a very rapid growth phase, while the older animals in the range, although 

still growing, are doing so at a slower rate. Because young animals are growing rapidly, 

measures of bone growth such as calcium, phosphate, and alkaline phosphatase might be 

expected to be higher in younger compared to older animals. Comparison of both the 

clone and comparator laboratory values to the Cornell Reference Range (which is derived 

from adult cattle) (Charts E-200 and E-202) indicates that many of the clones and 

comparators exhibit calcium, phosphate, and alkaline phosphatase levels that exceed the 

Cornell Reference Range. This finding is consistent with higher rates of growth in young 

calves relative to adults, and provides confidence that clones and comparators are 

exhibiting similar, normal physiological responses to growth stimuli. Review of Chart E201 reveals that clone alkaline phosphatase values are almost entirely within the range of 

the comparators (38 of 46 values). Most of the clones whose alkaline phosphatase levels 

exceeded the comparator range were the youngest animals. 

Another set of physiological parameters that varies with age can be seen in total protein, 

globulin, and albumin levels. These measurements reflect, among other things, the 

immune status of the animal. Immediately after birth, globulin levels, which are largely 

comprised of immunoglobulins, are derived almost entirely from colostrum (the 

antibody-rich first “milk” to be secreted by mammals). “Passive immunity” is conferred 

by the ingestion and intestinal absorption of immunoglobulin-rich maternal colostrum. In 
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Animal Cloning: A Risk Assessment DRAFT

the two to four months after birth, the calf’s own immune system begins to develop its 

production of immunoglobulins, as the circulating supply of maternally-derived 

immunoglobulins in the calf’s blood wanes. This phenomenon can be observed in Charts 

E-200 and E-202 (Clones: Reference Range (1 to 6 months) and Comparator Population: 

Reference Range). Clone and comparator globulin values are low relative to the Cornell 

lab reference range because that reference range is derived from adult animals with fully 

functional endogenous immunoglobulin production. The clone and comparator calves in 

this cohort have not fully started to produce their own antibodies from their own Blymphocytes. Review of Chart E-201 (Comparison of Clones to Comparator Population), 

however, indicates that there were few differences between the clones and the comparator 

population, reflecting the appropriate age-related lag accompanying the decrease in 

passive acquired immunity and endogenous immunoglobulin production. The globulin 

levels that are different between clones and comparators reflect this age-related 

physiological phenomenon. Clones #72 and 73 were among the youngest in the one to six 

month old group, and thus would be expected to have lower globulin levels. Comparison 

of the globulin value for clone #100 (174 days of age, globulin of 4.6g/dL) with clone 

#72 (48 days of age and globulin level of 1.6 g/dL) clearly demonstrates the age-related 

changes in the analyte, and appropriately reflects the normal developmental increase in 

endogenous globulin production. 

Sub-Cohort Analysis 

Examination of the subcohort of seven clones (# 71, 72, 73, 78, 79, 119, 132) at two time 

frames (birth and the 1-6 months of age) allows the determination that appropriate agerelated physiological changes are occurring in the clones on an individual animal basis, 

rather than on a population basis. For example, gamma glutamyl transferase (GGT) 

values appear low relative to comparators in “within 24 hours of birth” time period for 

four of these seven clones. This likely reflects the difference in timing between when the 

blood samples were drawn for clones and comparators (Clones had their blood samples 

drawn prior to colostrum administration, while comparators had their blood drawn some 

time after being fed colostrum). As colostrum has high intrinsic GGT activity, the 

difference between the two groups may be due to its effective absorption of GGT by the 

comparators. GGT values normalized by the time of the second blood draw for three of 

these animals, and were only slightly lower (4U/L vs. the comparator range of 5-32 U/L) 

in the remaining clone at Day 48. 

At birth, some of the clones in this sub-cohort had measures of liver function out of the 

comparator range (lower AST, and low bile acid or cholesterol levels). Low cholesterol is 

associated with retained fetal circulation in the livers of young animals. Were these low 
Chapter VI: Food Consumption Risks 214 

Animal Cloning: A Risk Assessment DRAFT

cholesterol levels to continue into the next developmental node, there might be cause for 

concern, but given that they normalized at the time of the second blood draw, there is 

little reason to expect that the lower values in these very young clones pose a health risk. 

The low levels at birth are more likely a reflection of the changeover from fetal to 

neonatal circulation, possibly exacerbated by the clones’ unusually large umbilical 

vessels, which often required surgical correction. The lower bile acid and AST values 

observed would also be related to the transition from fetal to neonatal circulation, and are 

not indicative of any disease state. All of these values normalized by the second 

measurement, as did additional analyte levels that were out of range for individual clones 

perinatally (low CK, TIBC, and iron). These measurements reflect normal adaptive 

physiological processes and not pathologic or disease states, and instead provide evidence 

of the “normalization” of the clones as they matured. 

A few laboratory measurements appeared outside the range of the comparators in some of 

the clones at the time of the second measurement, but these do not appear to have clinical 

relevance. Complete blood count information is only available for four of the seven 

clones measured at both time points, and do not appear to be reflective of clinical 

problems. For a more complete discussion of these data, see Appendix E. 

(c) Unpublished data 

Full hematology and clinical chemistry screens on three pre-pubertal bull clones (aged 5 

to 7 months old) were shared with CVM by a private veterinary clinic (Chapter 5 Table 

V-10). The clones were described as being clinically, physically and behaviorally normal, 

with normal growth rates and size. Blood samples were taken three times over a six week 

period. All of the clinical chemistry data, with the exception of one, were within normal 

published ranges or within the comparator range for the testing laboratory. Just as for the 

physiological data shared by Cyagra, the reference range for the testing laboratory was 

for an older cohort of animals (that were also female), and were not age-appropriate. The 

one analyte that fell outside a reference ranges occurred in a single sample in one bull 

clone, and was a low cholesterol value. All measurements in the subsequent sample from 

this bull clone were within normal ranges. 

(d) Summary for Juvenile Developmental Node in Bovine Clones 

(Developmental Node 3) 

With the exception of visible physical anomalies that were detected, individual animal 

and analyte review of the data indicated no differences between clones and conventional 

animals that reflect any food consumption hazards in clones. Clones that may be 
Chapter VI: Food Consumption Risks 215 

Animal Cloning: A Risk Assessment DRAFT

physiologically “unstable” at birth appear to normalize all of the measured variables 

within two months of birth (Chavatte-Palmer et al. 2002; Cyagra 2003). Some juvenile 

clones succumb to the sequellae of LOS. Surviving clones appear to grow normally, and 

careful evaluation of the laboratory results indicates that the clones’ physiology reflects 

normal, appropriate responses to ongoing growth and developmental signals, and that 

they are functionally indistinguishable from non-clones. 

iv. Reproductive Development and Function in Bovine Clones (Developmental 

Node 4) 

(a) Peer-reviewed Publications 

The number of studies that explicitly address the reproductive function of bovine clones 

is smaller than studies of other endpoints. Puberty onset has been reported as either 

“within normal limits” or somewhat (days) later in clones than controls. The Cyagra data 

received do not explicitly address the question of puberty onset or reproductive 

capability. 

Reproductive Function of Female Clones 

In a study of reproductive function in bovine clones, Enright et al. (2002) at the 

University of Connecticut evaluated the same clones and controls previously reported on 

by Xue et al. (2002) and Govoni et al. (2002). They reported that heifer clones reached 

puberty at a later age than controls (314.7 ± 9.6 days vs. 272 ± 4.4 days), and were 

reported as having higher body weights at first estrus (336.7 ± 13 vs. 302.8 ± 4.5 kg). No 

differences were noted between clones and controls in estrous cycle length, development 

of ovarian follicles, or profiles of hormonal changes. Three of the four clones and all four 

control heifers became pregnant following AI, although number of inseminations was not 

reported. Daily hormone profiles of lutenizing hormone (LH), follicle stimulating 

hormone (FSH), estradiol, and progesterone were similar between clones and controls. 

The cause of reproductive failure in one clone could not be determined; although this 

animal had reproductive hormone profiles similar to the other animals in the study, and 

no physical abnormalities could be found upon veterinary examination, poor signs of 

estrus were observed. This heifer did eventually conceive and produce a calf (Tian et al. 

2005) further discussed below). The cause for the later age and higher weight of clones at 

time of puberty is difficult to explain. The authors speculated that as the later onset of 

puberty can be genetically controlled in some cattle breeds, these clones may be 

expressing the genetics of the donor animal. Given that no records of age at puberty were 

kept for the donor cow, it is not possible to draw any conclusions regarding that 

hypothesis. 
Chapter VI: Food Consumption Risks 216 

Animal Cloning: A Risk Assessment DRAFT

Heyman et al. (2002) reported that from a group of clones derived from adult cells, five 

remaining animals were healthy and normal (one clone died of severe anemia (Renard et 

al. 1999, as previously discussed in the Perinatal section)). They noted that some of the 

females were more than one year old at the time of publication and were cycling 

normally, but no data were provided. In a follow-up study (Heyman et al. 2004) the 

authors stated that female clones at the INRA facility generally began cycling at 10 

months of age, and demonstrated estrous behavior by 12 months of age, within the 

normal range for their breed (Holstein). Ten female clones were bred by AI to the same 

non-clone bull. All 10 heifers conceived and produced live, apparently normal calves. 

Birth weight of progeny was 43.9 ± 4.1 kg, and gestation length was 281 ± 3.9 days, 

within the normal range for Holstein cattle. 

Wells et al. (2004) reported conception rate to two AI was 83 percent (25/30) for Holstein 

heifer clones, compared to 90 percent (9/10) for as small group of heifers produced by 

AI. Gestation length was slightly longer for clones (n=16) than for nine comparators (287 

± 3 vs. 281 ± 3 days), but within the normal range for Holsteins. Wells (2005) notes that 

despite variations in gestation length, only conventional levels of animal management 

and husbandry are required for the calving of heifer clones, indicating that the signals for 

induction of parturition and actual birth are functioning appropriately. Although most of 

the clones were separated from their offspring soon after birth, as is conventional in dairy 

practice, those dams that were not separated from their progeny exhibited normal 

maternal behavior and successfully reared their young. 

Forsberg et al. (2002) reported that Gene, the bull calf described previously, matured into 

a “healthy, fertile bull that has sired calves by artificial insemination and in vitro

fertilization.” Specific data on measures of reproductive function were not provided. 

Kato et al. (2000) report that one of the clones derived from a Holstein cumulus cell was 

artificially inseminated, conceived, and gave birth to a normal calf. 

The University of Connecticut (Tian et al. 2005) also reported first lactation milk yields 

and SCC for four clones and their non-clone comparators, indicating that lactation curves 

were similar for both groups. Total milk production for the first lactation was not 

different between clones and comparators (8,646 ± 743.8 kg vs. 9,507.8 ± 743.8 kg). One 

clone gave birth prematurely to a stillborn calf, did not have complete udder 

development, and produced approximately 30 percent less milk during her first lactation 

compared to her clone mates. Overall, SCC was low for both clones and comparators 

(based on Figure 2b of the paper: ~ 40 x 103 vs. 35 x 103 cells/mL), indicating a 
Chapter VI: Food Consumption Risks 217 

Animal Cloning: A Risk Assessment DRAFT

functional immune system, mammary gland, and low disease incidence. The role of good 

husbandry can also not be ruled out in this observation. 

Yonai et al. 2005 

Study overview: In the most comprehensive study of reproductive function in cattle 

clones, Yonai et al. (2005) (previously mentioned in other Developmental Nodes) 

performed an extensive analysis of reproductive performance in Holstein and Jersey 

clones with shortened telomeres, including puberty onset, estrus behavior, hormone 

cycling, the appearance of follicular waves, fertility and birthing for three estrus cycles. 

Once puberty onset had been determined, ovulation and formation of corpora lutea were 

monitored thrice weekly, with plasma samples to monitor progesterone levels collected 

every three days. After puberty, the estrus behavior of the clones was monitored twice 

daily until the animals became pregnant, with the length of the estrous periods and 

occurrence of standing behavior recorded. Plasma samples and ultrasonography were 

used to identify follicular waves and monitor progesterone and 17-β estradiol 

concentrations between day 18 of estrus and the day of ovulation over 17 estrus cycles in 

the Jersey clones and 28 estrus cycles in the Holstein clones. All clones were bred by 

artificial insemination using semen from the same lot of one bull (breed unspecified). 

Pregnancies were diagnosed by ultrasonography at 40 days after AI. For the first and 

second postpartum cycles, all clones were artificially inseminated at first estrus, which 

usually occurred 90 days after parturition. The length of gestation and resulting calves’ 

birth weights were recorded. Table VI-6 summarizes the data collected in this very 

detailed study. 

Table VI-6: Reproductive Parameters Evaluated for Jersey and Holstein Clones 

(adapted from Yonai et al. 2005)

Parameter Mean ± Standard Deviation 

Jerseys (n = 4) 

Age at puberty - 

Reproductive records from puberty to first parturition 

Length of estrous cycle 1 (days) 20.2 ± 1.4 

Follicle waves per cycle 1 (number) 2.3 ± 0.8 

Plasma estradiol-17 β concentration on estrous day 2

Detectable (17/17 cycles; pg/ml) 8.12 ± 2.40 

Not detectable (0/17 cycles; pg/ml) - 

Plasma progesterone under the curve 3 (ng/ml per cycle) 190.6 ± 59.4 

Number of AI for first conception 2.3 ± 1.9 
Chapter VI: Food Consumption Risks 218 

Animal Cloning: A Risk Assessment DRAFT

Age at first conception (days) 503 ± 54.9 

Gestation period (days) 279 ± 2.5 

Calf weight (first parturition) (kg) 22.0 ± 2.1 

Reproductive records after first parturition 

Interval from parturition to first ovulation (days) 51.3 ± 42.8 

Interval from parturition to first estrus (days) 85.0 ± 52.7 

Number of AI for second conception 1.3 ± 0.5 

Interval from parturition to second conception (days) 115 ± 16.8 

Age of second conception (days) 897 ± 44.8 

Calf weight (second parturition) (kg) 26.4 ± 1.1 

Reproductive records after second parturition 

Interval from parturition to first ovulation (days) 32.5 ± 19.3 

Interval from parturition to first estrus (days) 50.0 ± 27.8 

Number of AI for third conception 1.5 ± 1.0 

Interval from parturition to third conception (days) 129 ± 49.9 

Age of third conception (days) 1,304 ± 46.6 

Holsteins (n = 6) 

Age at puberty 323 ± 0.6 

Reproductive records from puberty to first parturition 

Length of estrous cycle 4 (days) 20.3 ± 1.5 

Follicle waves per cycle 4 (number) 2.3 ± 0.7 

Plasma estradiol-17 β concentration on estrous day 5

Detectable (19/28 cycles; pg/ml) 6.94 ± 2.64 

Not detectable (9/28 cycles; pg/ml) 3.95 ± 1.74 

Plasma progesterone under the curve 6 (ng/ml per cycle) 154.0 ± 58.0 

Number of AI for first conception 2.0 ± 2.0 

Age at first conception (days) 481 ± 35.0 

Gestation period (days) 277 ± 5.8 

Calf weight (first parturition) (kg) 37.8 ± 5.0 

Reproductive records after first parturition 

Interval from parturition to first ovulation (days) 56.0 ± 41.5 

Interval from parturition to first estrus (days) 86.0 ± 33.0 

Number of AI for second conception 1.2 ± 0.4 

Interval from parturition to second conception (days) 126 ± 41.7 

Age of second conception (days) 881 ± 61.7 

Calf weight (second parturition) (kg) 44.2 ± 1.9 

Reproductive records after second parturition 

Interval from parturition to first ovulation (days) 79.3 ± 18.9 

Interval from parturition to first estrus (days) 92.3 ± 19.2 

Number of AI for third conception 1.3 ± 0.5 

Interval from parturition to third conception (days) 138 ± 34.9 
Chapter VI: Food Consumption Risks 219 

Animal Cloning: A Risk Assessment DRAFT

Age of third conception (days) 1,297 ± 75.0 

1

 Twenty-six estrous cycles in four cloned heifers were included. 2

 Plasma samples were collected from 17 estrous cycles in four cloned heifers. 3

 Plasma samples were collected every three days during the 26 estrous cycles. 4

 Thirty-three estrous samples in five cloned heifers were included. 

5

 Plasma samples were collected from 28 estrous cycles in five cloned heifers. 6

 Plasma samples were collected every three days during the 33 estrous cycles. 

Reproductive function: First Estrus: Yonai et al. grouped their analysis of reproductive 

function into three stages: pubertal, post-pubertal conception and gestation, and postparturition, including rebreeding. Although some of the clones entered puberty prior to 

the initiation of this stage of the study, Yonai et al. reported that changes in plasma 

progesterone were consistent with previous reports on puberty in conventional cows. 

They also reported that corpus luteum formation was consistent with that reported in 

conventionally bred cows, and that the clones exhibited appropriate estrous behavior at 

puberty. Overall, the observations at puberty indicated that these clones exhibited normal 

early reproductive development. With respect to post-pubertal maturation of the heifer 

clones, Yonai et al. noted that there was some difficulty detecting estrus by behavior in 

the Holstein heifer clones, and that there were differences in their estradiol levels, these 

were consistent with similar observations in conventionally bred Holstein heifers. There 

were no difficulties in observing estrus in the Jersey clones. Estrus cycles lengths in both 

clone lines were comparable those observed in conventionally bred cattle. Additionally, 

the levels of progesterone secretion per cycle were reported as similar to those of 

conventionally bred heifers, which the authors interpreted as normal post-pubertal corpus 

luteum function. They conclude that the estrus cycles of the heifer clones were normal. 

All of the heifers conceived upon artificial insemination, although one heifer clone and 

one comparator needed multiple cycles of insemination; the remaining clones and 

comparators all conceived after no more than two rounds of AI. All of the clones but one 

Holstein delivered healthy, live calves. The exception delivered a stillborn calf two weeks 

before expected parturition. No obvious abnormalities were observed in the stillborn. 

Two of the Holstein clones required limited assistance for delivery; the remaining 

Holsteins and all the Jersey clones did not require any assistance in delivery. The average 

gestational periods were normal for the clones and all of the resulting calves were within 

normal body weight ranges for their breeds. All the live-born calves were reported as 

being normal. 

Second and Third Estrus. Yonai et al. noted a wide variation in the interval between 

parturition and first post-partum ovulation and estrus. The first postpartum ovulation in 
Chapter VI: Food Consumption Risks 220 

Animal Cloning: A Risk Assessment DRAFT

the Holstein clones occurred between 14 and 188 day (Table VI-6), and between 11 and 

108 days in Jersey clones; the interval between parturition to first estrus was between 62 

and 149 days for the Holstein clones, and 30 and 135 days for the Jersey clones. All 

clones had confirmed follicular waves, and pregnancy ensued in all of the clones 

following an average of 1.2 and 1.3 rounds AI for the Holstein and Jersey clones, 

respectively. The second parturition was largely uneventful for all of the clones, with one 

Holstein requiring minimal assistance calving. Gestation times for the all of the clones 

fell within normal ranges for the breeds; all of the calves had normal body weights, 

appeared to be normal at birth, and survived. Similar responses were noted for the third 

conception. 

Milk Production. Table VI-7 summarizes the yield of milk produced by the clones and 

their half-siblings and donor for the two lactation cycles following the first and second 

calvings. Data on the composition of this milk are addressed in the Food Composition 

portion of this chapter. Milk yield, although varying among the clones, was within the 

normal range for each breed for each lactation cycle. Interestingly, the Holstein clones 

produced less milk on average than their nuclear donor animal, while the Jersey clones 

produced more milk on average than their nuclear donor. The authors reported that 

mastitis was observed in the Holstein group of clones in two animals towards the end of 

the lactation cycle, and bloat was observed in two clones (not specified if the same 

animals) at approximately 130 days post-parturition. Neither was observed in the Jersey 

clones. Although not specified, the affected animals were most likely treated, and appear 

to have recovered as the number of animals did not change between cycles. 

This study, which is the first to study multiple cycles of reproductive function in any 

species of clone provides detailed information on both the individual physiological 

parameters measuring growth and reproduction (including lactation), as well as integrated 

measures of those functions. The authors conclude that despite the observation that all of 

these clones had shortened telomeres, these Holstein and Jersey clones exhibited normal 

growth, reproductive and lactation characteristics. 
Chapter VI: Food Consumption Risks 221 

Animal Cloning: A Risk Assessment DRAFT

Table VI-7: Results of Milk Yield in First and Second Lactations of Jersey and Holstein Clones 

(adapted from Yonai et al. 2005) 

Animal Milk Yield 

Jerseys (n = 4) 

First Lactation 

Clone 1 5,637.4 

Clone 2 6,077.9 

Clone 3 6,272.6 

Clone 4 5,597.7 

Mean ± Standard Deviation 5,896.4 ± 332.0 

Donor Animal 5,064.0 

Second Lactation 

Clone 1 7,006.8 

Clone 2 7,539.2 

Clone 3 7,309.6 

Clone 4 7,195.6 

Mean ± Standard Deviation 7,262.8 ± 222.6 

Donor Animal 6,087.0 

Holsteins (n = 6) 

First Lactation 

Clone 1 8,591.2 

Clone 2 9,219.5 

Clone 3 9,586.5 

Clone 4 9,836.0 

Clone 5 9,029.1 

Clone 6 9,735.6 

Mean ± Standard Deviation 9,333.0 ± 476.4 

Donor Animal 10,968.0 

Second Lactation 

Clone 1 10,678.6 

Clone 2 12,402.6 

Clone 3 11,341.4 

Clone 4 10,376.0 

Clone 5 10,110.2 

Clone 6 12,719.4 

Mean ± Standard Deviation 11,271.4 ± 1084.7 

Donor Animal 11,442.0 
Chapter VI: Food Consumption Risks 222 

Animal Cloning: A Risk Assessment DRAFT

Other Studies 

Although Lanza et al. (2001) reported on transgenic clones, conception rates for female 

clones after AI were high, with 87.5 percent of the animals conceiving on the first 

insemination and the remainder conceiving on the second insemination attempt. The two 

transgenic clones that had given birth, as of the publication date, were reported to have 

delivered calves that appeared normal in all respects, although no specific data are 

provided. 

Pace et al. (2002) reported that heifers began to display signs of reaching puberty at 10-

11 months of age, within the normal age range of conventional Holstein heifers (9 to 12 

months). They further report that all of the heifer clones that were inseminated (n=22) 

became pregnant, and calved at the age of 23-25 months, similar to non-clone cattle 

(approximately 75 percent of the cattle in Pace (2002) were transgenic). No specific 

information on gestation length or health of the progeny was provided. Analysis of the 

milk from non-transgenic clones of this cohort (Walsh et al. 2003) is presented within 

Section 3 of this Chapter. 

In an abstract, Aoki et al. (2003) present a preliminary report on the milk and milking 

behavior of two first-lactation Holstein clones derived from somatic cells isolated from 

the colostrum of mammary gland epithelial (MGE) cells described by Kishi et al (2000), 

previously discussed in the Perinatal section. These two clones were housed near the 

same automatic milking system as eight second-lactation control cows produced by AI. 

Comparisons were made between first lactation clones and second lactation controls. 

These cow clones were apparently followed for at least two calvings, and results were 

reported for the first through third post-partum ovulation and follicular development per 

estrous cycle. First postpartum ovulation was delayed in both of the clones, as well as the 

interval between the first to second postpartum ovulation. Clones were reported to have 

had two waves of follicular development per cycle. Both clones and comparator cattle 

were reported to calve normally, and did not appear to have different body weights and 

body condition scores, although no data were provided. The authors did not report 

differences between gestation length and duration of estrous cycle. They concluded that 

the clones were “normal in regard to delivery, lactation, and growth, and were similar in 

regard to the functions of their reproductive physiology.” Differences were observed, 

however, in the milking behavior, including the number of times that they voluntarily 

entered the automatic milking system relative to controls. In general, first lactation 

animals lack experience with milking equipment, and produce less milk than second and 

later parity cows, which likely contributed to differences in milking behavior between the 

two groups (Vasconcelos et al. 2004; Flis and Wattiaux 2005). Given that this is an 
Chapter VI: Food Consumption Risks 223 

Animal Cloning: A Risk Assessment DRAFT

abstract, the number of animals is very small, and the difference in the total number of 

lactation cycles the cows had experienced, the significance of the observation is unclear. 

Presentation of these data in a complete publication would aid this risk assessment and 

other analyses of clones. 

Heyman et al. (2004) reported that first lactation milk yields (9,341 ± 304 kg vs. 8,319 ± 

1,800 kg for a 305 day lactation) and somatic cell counts (SCC), which are a measure of 

mammary gland health) for three female Holstein clones were similar to those of three 

age-matched non-clone comparators. Somatic cell counts for both clones and 

comparators (116 ± 103 x 103 vs. 113 ± 50 x 103) were well below the level indicative of 

subclinical mastitis (1,000 x 103), and the SCC limit cited by the Pasteurized Milk 

Ordinance for fluid milk entering commerce. 

Reproductive Function of Male Clones 

The reproductive function of male bovine clones has also been studied. Wells (2005) 

reported on the reproductive function of six bulls cloned from the same steer. The rates of 

in vitro embryo development following fertilization of abattoir-derived oöcytes using 

sperm from these sires varied among the sires, but the development of blastocysts to 

quality grades suitable for embryo transfer were similar to that for four comparator bulls 

(10-25 percent for the clones and 13-30 percent for the comparators). Likewise, Heyman 

et al. (2004) reported that three clones of an eight year old bull were enrolled in an AI 

center, and semen was collected when the clones were between 13 and 15 months of age. 

Percentages of normal sperm, cleavage rate and blastocyst rate following IVF were not 

different between the clones and their nuclear donor. Results of AI trials were only 

presented for one clone (no comparator). Forty-one cows became pregnant out of 63 

animals inseminated, yielding a 65 percent pregnancy rate. Two pregnancies were lost by 

day 90 (5 percent loss). Only 26 pregnancies were allowed to go to term, yielding 25 live, 

healthy calves and one stillborn. 

Shiga et al. 2005 reported on the semen quality of two clones of a 12 year old Japanese 

Black bull. Semen was collected over a four month period beginning when the clones 

were approximately 12 months old. Comparisons were made using frozen semen from 

the nuclear donor and using averages for the breed. Although ejaculate volumes of the 

two bulls were lower than the range for the breed (2.34 and 2.76 mL vs. 5-8 mL), sperm 

concentration, pH, and pre-freezing motility were within established ranges for Japanese 

Black bulls. Development of IVF embryos to the blastocyst stage was not different 

between clones and their nuclear donor (23.4 and 28.4 vs. 30.9 percent). Semen from one 

of the clones was used to inseminate 22 cows, compared to 102 cows inseminated by the 
Chapter VI: Food Consumption Risks 224 

Animal Cloning: A Risk Assessment DRAFT

nuclear donor. Pregnancy rates were similar between the clone semen and semen from 

the nuclear donor (54.5 vs. 62.7 percent). Two of the 12 (17 percent) resulting 

pregnancies from the clone aborted spontaneously in mid-pregnancy, compared to 5/64 (8 

percent) abortions among the cows bred by the nuclear donor. 

(b) Unpublished data 

Semen evaluations on four healthy post-pubertal clones derived from an Angus-Chianina 

nuclear donor cross were shared with CVM (Chapter 5, Table V-17). Semen was 

collected by a commercial reproduction service from May through June 2003, three times 

daily, as is usual for industry practice. The age of the bulls at the time of collection was 

not recorded. Semen evaluation showed that one clone had a low sperm concentration 

(average 169.5 x 106 cells/ml vs. the normal range 800-1,200 x 106 sperm/mL (Sorenson 

1979; Beardon and Fuquay 1980; Hafez and Hafez 2000)) and low percentage of normal 

sperm (between 2 and 8 percent) during the observation period. This bull likely would 

have failed a breeding soundness exam, and if it had been a conventional animal, it would 

most likely have been sold to a feedlot for eventual slaughter. A second bull clone had 

marginal semen quality, and might have been retained depending on the perceived value 

of his genetics. The remaining two clones exhibited acceptable semen characteristics, and 

would likely have been retained for breeding. 

Galli et al. (unpublished data 2003) also presented data on breeding soundness and 

performance of three clones of a Holstein bull (Chapter V, Table V-10). Breeding 

soundness exams indicated that clones were acceptable for breeding. Artificial 

insemination trials using semen from one of the clones on four farms resulted in 

pregnancy rates ranging from 33 to 80 percent; however, few cows were actually bred 

(n=63 for all farms combined), there were no contemporary comparators used, and no 

details regarding farm management were provided, making these data difficult to 

interpret. Pregnancy rates to AI for this clone were within the range of the U.S. average 

for Holstein cattle. 

(c) Summary Statement for Reproductive Development and Function in Bovine 

Clones (Developmental Node 4) 

Although specific animals are rarely cited, all reports of reproductive function in bovine 

clones appear to indicate that the animals respond normally to developmental signals 

governing puberty onset and that they subsequently reproduce effectively. The results of 

the study by Yonai et al. (2005) provide further confidence by reporting on detailed 

physiological parameters required for successful reproduction, and demonstrate that the 
Chapter VI: Food Consumption Risks 225 

Animal Cloning: A Risk Assessment DRAFT

clones continued to cycle and function normally after the first pregnancy. The studies of 

lactation and milk yield indicate a consistent response demonstrating that these animals 

function normally post-partum and during subsequent reproductive cycles. Reproductive 

failure is a common phenomenon in conventional cattle, and among one of the most 

frequent causes for culling. Although cases of reproductive failure have been reported 

among clones, they are not unusual among conventional cattle, and do not raise food 

safety concerns. Reproductive function is among the most tightly regulated functions that 

a mammal performs; the demonstration that clones can reproduce normally appears to 

indicate that those clones are functioning normally for this biological criterion. 

v. Post-Pubertal Maturation in Bovine Clones (Developmental Node 5) 

(a) Peer-reviewed Publications 

Post-pubertal maturation includes the very long period of time between the development 

of reproductive capacity and the natural end of the animal’s life. Most cattle in US 

agriculture never reach the end of their “natural” life-spans for economic reasons. In 

commercial dairy establishments, dairy cows are sent to slaughter some time between the 

end of their third to fifth lactations, or sooner, depending on their health and productivity. 

Beef cattle that are not being used for breeding are generally sent to slaughter when they 

reach about 1,000 to 1,400 lbs, or at approximately 18 to 24 months of age (depending on 

breed, season, environmental conditions, etc.). Most of the possible food consumption 

risks arising from edible products of clones (e.g., milk or meat) would occur during this 

Developmental Node. 

We have not conducted a survey of clone producers or the investigators who have 

published on the health status of clones earlier in the clones’ lives to determine their vital 

or health status. At this time, there are economic disadvantages to maintaining healthy 

clones without being able to realize financial investments, so many otherwise healthy 

clones have been euthanized. The following discussion therefore summaries reports that 

have been obtained from the literature, and tends to focus on anomalies that have been 

noted. 

Kato et al. (2000) reported that as of September 1, 1999, all of the surviving clones from 

their Holstein and Japanese Black cumulus cell and fibroblast donors were healthy and 

aged 117-350 days. No further publications were found regarding the fate of these 

animals. 
Chapter VI: Food Consumption Risks 226 

Animal Cloning: A Risk Assessment DRAFT

Because of the relatively short time that cloning has been practiced, (Gene, the first 

bovine SCNT clone was born in 1997 (Cibelli et al. 1998)), little information is available 

on animals during this developmental phase, and much of that information comes in the 

form of single sentences or short mentions in journal articles that address some other 

issue. Abnormalities that have been noted in mature cattle clones appear to be sequellae 

of anomalies or defects noted earlier in life, and may be related to LOS or other earlier 

diseases. For example, Batchelder (2005) reported that one clone died suddenly at 25 

months of age. Necropsy results indicated severe trace mineral deficiency (selenium and 

copper) as the cause of death. None of the non-clone cattle grazing the same pasture 

developed signs of mineral deficiencies. Nonetheless, this particular clone was reported 

to have exhibited frequent but mild signs of bloat as a juvenile, and it is possible that its 

subsequent death may have been the result of gastro-intestinal tract problems resulting in 

reduced ability to absorb micro-nutrients. The two surviving clones were reported as 

healthy at 19 months of age. 

Second Chance, the Brahman bull clone described by Hill et al. (2000a), has been 

outlined in detail in the preceding section. The researchers speculate that the early 

diabetes had resolved at eight months of age and the calf was clinically normal. At a 

conference in September of 2002, the bull was reported to be 3 years of age, with normal 

weight, growth, behavior, and normal semen production. The investigator presenting this 

information also reported that the bull’s glucose level was elevated, although they could 

not rule out the role of stress resulting from medical procedures as a cause (Westhusin in 

PIFB 200353). In a subsequent conversation, Dr. Westhusin indicated that the blood 

glucose has remained within normal limits since the previous report. 

Lanza et al. (2000) reported on 24 sexually mature transgenic bovine clones. Physical 

examinations were reported as normal including temperature, pulse, respiratory rate, 

general appearance, lymph nodes, and abdominal palpation. Blood and urinalysis 

indicated that in general, those variables were within normal ranges although six animals 

had total urine protein levels slightly below the comparator average. Studies with 

adaptive T-cell responses indicated that these transgenic clones had functional immune 

systems, and that the animals responded to periodic infection in the same manner as 

conventional cattle. 

Pace et al. (2002) measured weight gain in their transgenic clones until the age of 540 

days. Although comparison of the overall cohort with any comparator group is difficult 

because the clones were raised at different facilities, 52 of the clones raised at the same 

 

53 http://pewagbiotech.org/events/0924/presentations/Westhusin.pdf 
Chapter VI: Food Consumption Risks 227 

Animal Cloning: A Risk Assessment DRAFT

facility had similar weight gain over the first 120 days of life (approximately 1.15 

kg/day). Weight gain of 17 clones from the same genetic line declined to 1.09 ± 0.14 and 

0.92 ± 0.10 kg/day at 365 and 540 days, respectively, entirely consistent with weight gain 

profiles of conventional animals. 

Yonai et al. (2005) reported on the growth characteristics of six Holstein and four Jersey 

clones with shortened telomeres from birth through two years of age. Those data have 

been summarized in Table VI-5. Evaluation for clones aged 12-24 months indicates that 

animals had normal weight gain for their breeds, indicating their overall health. With the 

exception of brief mentions of bloat and mastitis, no other illnesses were reported in this 

study. All of the animals that entered the study were alive at the time the manuscript was 

submitted for publication. 

Wells et al. (2005) have reported that clones produced at AgResearch have an overall 

annual mortality of eight percent over four years. Most of the mortality observed appears 

to be due to the sequellae of LOS or accidents or mishaps; no contemporaneous 

comparator exists. They also note that one clonal family and their half-siblings were all 

alive and healthy at 18 months of age, implying that there may be an association between 

the cell line used, susceptibility to LOS and its sequellae. 

(b) Cyagra Dataset: 6-18 Month Cohort 

The oldest cohort of Cyagra animals spans 6-18 months of age, and actually overlaps the 

Juvenile and Maturity developmental nodes. Clearly, the younger clones in this cohort 

have more in common with the older, but still juvenile, animals of the preceding cohort, 

while the older clones are more appropriately considered as nearing “adulthood.” 

The 6-18 month Cyagra clones were virtually indistinguishable from the comparators. 

None of the animals had any visible anomalies on physical examination (See Appendix E 

for details). The laboratory values derived from blood samples drawn from the clones are 

virtually superimposable on those of the comparators. Only three of the 294 

hematological values and six of the 592 clinical chemistry measurements were outside 

the clinically relevant range. In aggregate, 99 percent of the laboratory measurements 

were within the clinically relevant range established by the comparators.

Review of Chart E-301 indicates that only two analytes initially appeared to marginally 

exceed the range characterized by the comparators: estradiol-17β (E2), and insulin-like 

growth factor-1 (IGF-I). Neither of these findings was judged to pose clinical 

significance for the animals or any food consumption risk. Although the E2 levels of five 
Chapter VI: Food Consumption Risks 228 

Animal Cloning: A Risk Assessment DRAFT

animals exceeded the comparator range, none exceeded the Cornell Reference range, 

which as previously discussed, is derived from adult cattle. By comparison, 14 of the 20 

comparators had measurements that were lower than the Cornell Reference Range (Chart 

E-302). For a more complete discussion of the normal fluctuation of E2 levels in cattle, 

see Appendix E. IGF-I levels in the Cyagra cohort were slightly higher in males than in 

females, and in three of the bull calves (# 24, 33, and 35) were slightly increased (less 

than 10 percent) relative to the comparator Group. Review of the literature on IGF-I 

levels in cattle indicated that basal circulating levels of IGF-I vary with a range of factors 

and fluctuate dramatically among individual animals in herds (Vega et al. 1991). Plasma 

concentrations of IGF-I are strongly influenced by a number of factors including gender, 

age, and diet (Plouzek and Trenkle 1991 a,b). The primary nutritional determinants of 

basal IGF-I levels appear to be crude protein and the number of calories absorbed by the 

animal (Elsasser et al. 1989). Given that most non-transgenic clones are derived from 

animals of superior genetic merits for traits such as growth and development, 10 percent 

elevations in IGF-I levels are likely of no clinical significance for the animal, and pose no 

food consumption risk. 

No remarkable dissimilarities were noted in the blood variables of clones and 

comparators. There were no indications of problems with respect to red or white blood 

cell measurements. One animal (Clone #98) exhibited higher basophil counts than the 

comparator range, but there appeared to be no clinical correlate to that value, and as a 

result it was judged insignificant to the health of the animal or food safety. 

(c) Unpublished data 

Hematology data for two Holstein heifer clones aged 14 months old were submitted to 

CVM by a private veterinary firm. They consisted of a Veterinary Certificate of 

Inspection, results of serological testing showing the animals were free of Bovine 

leucosis virus and Bovine viral diarrhea, and standard clinical chemistry and hematology 

panels. All hematology and clinical chemistry results were within the range of the 

laboratory’s reference values except red cell distribution width, which was slightly below 

the reference range used by the testing laboratory (see Chapter V). As discussed in 

Chapter V and Appendix E, RDW is a secondary indicator, and does not on its own 

suggest a health problem. Certificates of Veterinary Inspection accompanying the 

hematology data indicate that both heifers were healthy. 

(d) Summary Statement for Post-Pubertal Maturation in Bovine Clones 

(Developmental Node 5) 
Chapter VI: Food Consumption Risks 229 

Animal Cloning: A Risk Assessment DRAFT

Clones in this age group exhibited no remarkable differences from non-clones with 

respect to their overall health. The Cyagra clones were indistinguishable from the 

comparator group on the basis of clinical and laboratory tests. The study of Yonai et al. 

indicates that clones continued to grow well for the duration of the study (two years). No 

residual health problems were noted in any of the clones in this Developmental Node that 

had not been identified in earlier developmental nodes. Some clones died prematurely for 

different reasons, including the sequellae of earlier disease. Individual animal reviews 

indicated no health problems, or changes in physiological parameters that would indicate 

a food consumption risk that would not be detected in existing food safety regulations 

(e.g., mastitis in milking cows). 

b. Swine Clones 

There are approximately twenty papers, including some reviews, within the peerreviewed literature that address cloning of swine; many of these report on the generation 

of transgenic swine by SCNT. Unlike cattle, where improvement of breeding stock has 

been a major driving force for advances in reproductive technologies, many of the earlier 

studies of SCNT in swine have focused on transgenic animals for use as xenotransplant 

organ sources (reviewed by Prather et al. 1999; Westhusin and Piedrahita 2000; Wheeler 

and Walters 2001; Carter et al. 2002; Machaty et al. 2002; and Prather et al. 2003). 

Nonetheless, cloning swine for agricultural purposes has become the focus of at least one 

large commercial venture (Viagen, Inc.), and others (Archer et al. 2003 a, b) have also 

reported extensively on the health and physiological status of non-transgenic swine 

clones. 

The cloning of swine was first described in 2000 by Polejaeva and her colleagues at what 

was then PPL Therapeutics in Blacksburg, Virginia and Roslin, UK. Several laboratories 

followed that publication with their own reports of swine cloning using different 

approaches to cell fusion, oöcyte maturation, or other technical issues (Betthauser et al. 

2000; Onishi et al. 2000; and Bondioli et al. 2001). In the subsequent years, additional 

studies have reported on the difficulties of overcoming the early stage failures (Boquest 

et al. 2002, and Yin et al. 2002; Lee et al. 2005; Zhu et al. 2004). 

 

Another issue contributing to the difficulty of cloning swine is that unlike cattle, sheep, 

and goats, swine require a minimum number of viable embryos, thought to be 

approximately four, to initiate and sustain pregnancy (Polge et al. 1966; Dzuik 1985). 

This has posed a technical limitation for the development of cloning in this species 

because the high loss of embryo clones throughout the pregnancy necessitates the transfer 

of a very large number of clone embryos into the surrogate dam (between 150 and 500) to 
Chapter VI: Food Consumption Risks 230 

Animal Cloning: A Risk Assessment DRAFT

ensure that the minimum number of embryos is maintained. A recent paper by King et al. 

(2002) explored hormonal treatments to sustain limited numbers of viable embryos to 

term, and demonstrated that pregnancies can be established with a mixture of fertilized 

and parthenote embryos and that small numbers of fertilized embryos can develop to term 

successfully with hormonal support. 

Because of these difficulties, most of the available reports describe only the implantation 

and early perinatal phase. Two publications by Archer et al. (2003 a, b) describe the 

behavior and clinical chemistry of juvenile swine clones. 

i. Cell Fusion, Nuclear Reprogramming, Embryonic and Fetal Development 

Through the Perinatal Developmental Period in Swine Clones 

(Developmental Nodes 1 and 2) 

(a) Peer-reviewed Publications 

In the first published report of swine clones by Polejaeva et al. (2000), two rounds of 

nuclear transfer were employed, with in vivo matured oöcytes as recipients and cultured 

granulosa cells as donors, to produce five live female piglet clones. Piglets were 

delivered by C-section on day 116 of the pregnancy. The only data on the health of these 

piglets indicated that the average birth weight of the clones of 2.72 pounds (range 2.28-

3.08 pounds) was approximately 25 percent lower than in piglets produced using natural 

mating in the same population as the donor cells (average birth weight of 3.6 pounds, 

range 3.3-3.9 pounds in an average litter size of 10.9 piglets). 

In the second report of swine cloning, after several unsuccessful attempts, Onishi et al. 

(2000) produced a single female piglet named “Xena” from cultured embryo fibroblast 

cells. The clone’s birth and placental weights were 1.2 kg and 0.3 kg, respectively, which 

the authors state were in the normal range for conventional offspring of that breed 

(Meishan). Xena was described as a “healthy female” but, with the exception of a 

photograph, no data were provided to confirm that observation. 

Betthauser et al. (2000) also describe multiple attempts at establishing successful 

pregnancies in surrogate dams receiving swine embryos resulting from SCNT. Of the 

seven pregnancies that were established, three were with non-transgenic embryo clones. 

Four live births resulted from two pregnancies, out of 427 embryos implanted into 

surrogate dams. The first litter yielded two male piglets born alive by vaginal delivery, 

weighing 2.0 and 3.0 pounds each. The second litter also produced two live vaginally 

delivered male clone piglets and one mummified fetus. The live piglets in this litter 
Chapter VI: Food Consumption Risks 231 

Animal Cloning: A Risk Assessment DRAFT

weighed 2.2 and 3.5 pounds. The third pregnancy was aborted at 40 days of gestation. No 

further information was provided on the health status of the clones at birth. Subsequently, 

the senior author on this report wrote a Letter to the Editor of the publication (Bishop 

2000) to inform that the piglets from the second litter had died one week after their birth 

due to the aggressive behavior of the first-time surrogate mother. This behavior limited 

the amount of time the piglets were able to nurse, and the consequent lack of adequate 

nutrition proved to be fatal to the piglets (Bishop 2000). CVM is unaware of any 

publications providing additional information on the health status of the first litter. 

An Australian group (Boquest et al. 2002) described the birth of live piglets from 

cultured fetal fibroblast cells that were frozen for two years, employing a novel cell 

fusion method in which donor nuclei were exposed to inactivated oöplasm for a period of 

time prior to chemical activation (to begin the process of cell replication). They believe 

that the lag time between fusion and activation allows for the more efficient 

reprogramming of the donor cell nuclei. The investigators transferred between 40 and 

107 embryos to 10 surrogate dams, resulting in five pregnancies. Three of those 

pregnancies were aborted, and each of the two remaining pregnancies yielded one live 

piglet. No information is provided about the health status of the clones. 

Yin et al. (2002) also developed a novel method for the production of pig clones by 

treating oöcytes to be used as recipients with demecolcine such that the condensed 

chromosomes produce a protrusion at the cell membrane that can easily be removed by 

micro-aspiration. Donor cells were obtained from an adult female four year old Landrace 

pig, and included cultured heart and kidney cells. Six surrogate dams were implanted 

with between 137 and 341 embryos. Three of the recipients never became pregnant, and 

one aborted the pregnancy on day 62. The remaining two pregnancies, both with embryos 

of heart tissue origin, resulted in live births. The first litter included four live female 

clones, and one dead fetus. The second resulted in another four live female clones, and 

two dead fetuses. None of the clones, live or dead, exhibited any morphological 

anomalies. The authors reported that the eight surviving clones were eight months old at 

the time of publication, and “appear quite healthy.” No further information is provided. 

Lee et al. (2005) found that supplementing culture media with epidermal growth factor 

(EGF) improved cleavage rate of NT embryos, but not the rate of blastocyst formation 

compared to unsupplemented media, although total cell numbers in surviving blastocysts 

were higher in EGF supplemented media. Adding EGF after morula formation did not 

affect blastocyst formation rate or cell numbers. Zhu et al. (2005) found embryos 

produced with stem cells isolated from fetal porcine skin cultures had higher 

preimplantation development rates than embryos produced using fetal fibroblast cells. 
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Animal Cloning: A Risk Assessment DRAFT

Karyotypic analysis of the two donor cell cultures indicated that porcine stem cells 

accumulated fewer abnormalities and were more stable through multiple passages 

compared to fibroblast cells. Porcine stem cells also yielded more blastocysts than 

fibroblast cells. Because neither of these groups attempted to transfer embryos to 

recipients, there is no way to know whether these improvements in early embryo 

developmental efficiency would have resulted in a higher proportion of live clones. 

Bondioli et al. (2001) reported on the generation of transgenic pig clones from cultured 

skin fibroblasts derived from an α-1,2-fucosyltransferase (H-transferase) transgenic boar. 

(H-transferase is involved in producing the sugars on the surface of a pig cell that are 

partially responsible for the acute phase of rejection observed when non-human tissues 

are transplanted into humans.) Of the 217 embryos transferred into five surrogate dams, 

two pregnancies resulted. One of the surrogate dams was euthanized at 90 days of 

gestation for health reasons that the authors state were unrelated to embryo transfer. One 

mummified fetus and one apparently viable fetus were recovered. The other pregnancy 

yielded two live piglets that were delivered by C-section at 116 days of gestation. The 

piglets were reported as “healthy,” and a photograph of two apparently normal piglets at 

two months of age is provided in the paper. 

Walker et al. (2002) have reported on the largest litters of piglets produced by SCNT. 

Donor cells were derived from Duroc fetal fibroblasts, and fused with in vitro matured 

oöcytes. A total of 511 embryos were transferred into five surrogate dams, with between 

59 and 128 embryos per recipient. All five recipients were confirmed pregnant by 

ultrasound between days 28 and 40 post-implantation. Four of the five pregnancies went 

to term, and litters containing between 5 and 9 piglet clones (total of 28) were delivered. 

Three of the four surrogate dams were induced and delivered on gestational day 115. The 

fourth was allowed to deliver naturally, and produced her litter on gestational day 117. 

One of the 28 clones was stillborn, but no abnormalities were noted on necropsy. One of 

the live born clones presented with anal atresia (no anus or tail), and was the smallest of 

all of the clones (birth weight of 0.72 kg, and crown rump length of 23.5 cm). The 

authors noted that anal atresia is a developmental abnormality seen at a natural low 

frequency in conventional piglets. The question of whether this is a random event due to 

genetic or inappropriate reprogramming cannot be answered from this dataset. 
Chapter VI: Food Consumption Risks 233 

Animal Cloning: A Risk Assessment DRAFT

Table VI-8: Summary of Birth Characteristics of Piglet Clones 

(source: Walker et al. 2002) 

Litter size Mean Birth Weight (kg)1 Mean Placental Weight (kg)1 Crown-Rump Length (cm)1 

9 1.15 ± 0.17 0.29 ± 0.09 68.8 ± 2.1 

5 1.06 ± 0.23 0.23 ±0.02 71.6 ± 7.6 

7 1.35 ± 0.13 0.29 ± 0.07 74.9 ± 1.8 

7 1.29 ± 0.26 NR NR 

Control2 1.37 ± 0.12 NR NR 

1

 All values presented as means ± SD. 

2

 The control birth weight was derived from the average weight ± SD from 10 litters of piglets from naturally 

bred Duroc pigs. 

NR = Not reported. 

The remaining piglets had birth weights that appear to be a little lower than conventional 

piglets of the same breed. The authors noted with explicit surprise that there was little 

correlation between litter size, placental weights, and fetal weights (Table VI-8). They 

predicted a correlation of 0.639 between placental and fetal weight, but noted that the 

lowest mean birth weights occurred in the litters with the smallest number of piglets. The 

authors asserted that without the appropriate controls for litter size, in vitro oöcyte 

maturation and other manipulations, it is inappropriate to assign the SCNT process as the 

cause of the difference in birth weights. Two of these litters subsequently served as the 

source of the clinical and behavioral studies of Archer et al. (2003 a, b). 

Viagen Inc. provided birth weights of seven male swine clones as part of the data 

package presented to CVM. Clones were smaller at birth than AI comparators of similar 

genetic background (See Appendix G: Viagen Dataset). No detailed health data were 

available on these clones for this developmental node. All clones survived the neonatal 

period. 

Additional data submitted to CVM included birth weight, average daily weight gain 

(ADG), body temperature, and pulse rates on another cohort of neonatal swine clones 

(see Chapter V). Birth weights for three clones ranged from 1.1 to 1.4 kg, and ADG 

ranged from 0.46 to 0.55 kg; however, because the breed of swine was not identified, it is 

not possible to determine whether these data are within normal ranges. The report 

indicated that two of the five piglets, both from the same litter and weighing 1.0 kg at 

birth, died within the first 48 hours. The cause of death was not reported, and no other 

details were provided. Body temperatures of the piglets were low (range 98.8 to 101.8 ̊F) 

during the first 48 hours compared to reference body temperature for adult swine (102-
Chapter VI: Food Consumption Risks 234 

Animal Cloning: A Risk Assessment DRAFT

103 ̊F). This finding is not unusual, however, as neonatal swine generally have difficulty 

regulating body temperature, and require supplemental heat after birth (see Chapter V). 

(b) Summary Statement on the Embryo/Fetal to Perinatal Developmental in 

Swine Clones (Developmental Nodes 1 and 2) 

The production of swine clones differs from the other livestock species discussed in this 

risk assessment because of the requirement for a minimum number of viable fetuses to 

maintain the pregnancy. The gestational losses observed are a function of the combined 

low “success rate” for embryonic and fetal development for the individual clone and the 

requirement for a minimum number of growing fetuses to implant. Clone piglets do not 

appear to exhibit the overgrowth phenomena observed in cattle, and if anything, newborn 

swine clones may be smaller than their non-clone counterparts. Although there is one 

report of an anomaly at birth (e.g., anal atresia), piglet clones appear to be normal and 

healthy. 

ii. Juvenile Development and Function in Swine Clones (Developmental Node 3) 

(a) Peer-reviewed Publications 

Archer et al. (2003 a,b) have investigated the degree of behavioral and physiological 

variability exhibited among litters of swine clones and their closely related conventional 

siblings. The derivation of these clones has been described in Walker et al. (2002). The 

clone cohorts consisted of two litters of 5 and 4 female swine derived from the same cell 

line born 6 weeks apart. The control groups consisted of a litter of four female full 

siblings (both parents in common) and a litter of four female half-siblings taken from 

three sows mated to the same boar. All animals were farrowed (born) in conventional 

farrowing crates, and weaned at 5-6 weeks of age when they were placed in adjacent 

identical pens and given continuous access to identical standard rations and water. 

Results in these studies were presented as means and ranges; individual animal data were 

not provided. 

One study (Archer et al. 2003b) evaluated behavioral characteristics including food 

preference (for apples, bananas, saltine crackers, and carrots), temperament (as judged by 

time to remove a towel placed on the pig’s head, attempts to escape mild restraints, being 

placed on their backs, and being lifted off the ground), and time budgets (the amount of 

time spent engaged in a particular activity in their pens). The results of this study 

indicated that the behaviors of swine clones were no more homogenous than the 

behaviors of siblings and may be more variable than the comparator animals, although 

the statistical power to draw such a conclusion was limited. The authors conclude that 
Chapter VI: Food Consumption Risks 235 

Animal Cloning: A Risk Assessment DRAFT

“...using nuclear transfer to replicate animals to reproduce certain behavioral 

characteristics is an unrealistic expectation.” The relevance of the study to an evaluation 

of the health of swine clones, however, is that the animals behaved in much the same 

manner as conventional animals, and displayed no behavioral anomalies at the times 

tested (15-16 weeks of age for the food trials, 8-9 weeks and 14-15 weeks for the towel 

test, 7 weeks for the restraint tests, and 13-15 weeks for the time budget tests). 

Another study performed by this group (Archer et al. 2003a) evaluated whether the 

SCNT process introduced epigenetic changes into animal clones that could be manifested 

at the genomic (e.g., methylation status) (See Chapter IV), physiological (e.g., blood 

chemistry), and anatomical (e.g., weight, size, coat) levels. Body weights of all the 

animals overlapped and were within the normal range for the age and breed, with the 

exception of a single clone that was small at birth, and never attained the size of its 

littermates. This is likely a case of “runting,” which is observed in conventional animals 

as well. Teat number was the same for all animals (6,6 distribution) except for one clone 

(6,7 distribution), within the normal variability in conventional pigs. 

One of the clones also exhibited an unusual hair growth pattern (e.g., longer and sparser), 

which the authors state prompted an examination of the histology of the skin. Results of 

that investigation indicated that with one exception, skin morphology showed no unusual 

variations among the pigs. The exception was a clone that exhibited morphology 

indicative of hyperkeratosis.54 Hyperkeratosis, also referred to as parakeratosis, also 

occurs in naturally bred and AI pigs between the ages of 6 and 16 weeks, and is generally 

associated with zinc and essential fatty acid deficiency or excess dietary calcium or 

phytates. Gastrointestinal disorders may also affect zinc absorption, and contribute to the 

development of this condition (Cameron 1999). Other possible causes of hyperkeratosis 

include heredity, and other non-specific causes of skin inflammation (Blood and 

Radostits 1989). Dermatitis vegetans is the inherited form of this disease in swine, and is 

a semi-lethal recessive gene (Blood and Radostits 1989). Although the phenotypic 

variation is interesting, it is of limited concern for food safety, as pork skin that exhibits 

severe hyperkeratosis would be condemned at slaughter. 

Blood samples were taken from the animals for analysis at 15 and 27 weeks of age (Table 

VI-9) (Archer et al. 2003a). Although the hypothesis being testing in this study addressed 

the degree of variability among clones relative to the degree of variability among 

controls, these data are very instructive in that they provide the most extensive analysis of 

 54 Hyperkeratosis, generally referred to as parakeratosis in swine, is characterized by lesions of the 

superficial layers of the epidermis. These lesions rapidly become covered with scales, and then 

develop hard, dry crusts with deep fissures. 
Chapter VI: Food Consumption Risks 236 

Animal Cloning: A Risk Assessment DRAFT

the physiological status of swine clones at two different times in development. (See 

previous discussion of Cyagra dataset). Unlike the Cyagra dataset, however, very few 

Table VI-9: Clinical Chemistry Results from Swine Clones at Two Ages 

(adapted from Archer et al. 2003a)

 Clones Controls 

Measurement Merck1 Week 15 Week 27 Week 15 Week 27 

Creatinine 

(mg/dl) 0.8-2.3 1.02 ± 0.223 

(0.7-1.4) 

1.11 ± 0.14 

(0.9-1.3) 

1.58±0.95 

(0.8-3.6) 

1.25 ± 0.32 

(0.9-1.8) 

Alkaline 

Phosphatase (U/l) 41.0-176.1 208.67 ±11.60 (192-226) 

100.78 ±17.89 

(80-128) 

235.25 ±33.12 

(201-294) 

117.88 ± 49.54 

(56-196) 

BUN2 (mg/dl) 8.2-24.6 9.69 ± 1.45 

(7.7-11.9) 

10.09 1.29 

(8.9-11.7) 

9.58 ± 2.84 

(6.3-11.9) 

7.85 ±2.04 

(5.8-11.7) 

ALT (SGPT) 

(U/l) 21.7-46.5 46.78 ± 4.24 (46-56) 

38.44 ± 2.55 

(34-42) 

53.25 ± 9.16 

(41-70) 

38.88 ± 8.32 

(22-48) 

Albumin (g/dl) 2.3-4.0 4.21 ± 0.13 

(4.0-4.3) 

4.12 ± 0.26 

(3.6-4.3) 

4.40 ± 0.21 

(4.1-4.7) 

4.15 ± 0.55 

(3.0 -4.7) 

Phosphorus 

(mg/dl) 5.5-9.3 10.29 ± 0.42 (9.6-10.6) 

7.87 ± 0.60 

(7.0-8.8) 

10.75 ± 0.82 

(9.5-11.8) 

7.75 ± 0.85 

(6.1-8.9) 

Calcium (mg/dl) 9.3-11.5 11.30 ± 0.24 

(11.0-11.7) 

11.50 ± 0.84 

(10.7-12.5) 

11.49 ± 0.57 

(10.4 -12.2) 

11.35 ± 1.12 

(9.5-12.7) 

Serum Protein 

(mg/dl) 58.3-83.2 6.34 ± 0.35 (5.7-6.8) 

6.96 ± 0.44 

(6.2 - 7.7) 

6.09 ± 0.32 

(5.9-6.5) 

7.00 ± 0.60 

(6.4-8.2) 

Glucose (mg/dl) 66.4-116.1 100.56 ± 10.03 

(101-113) 

86.89 ± 7.03 

(70-94) 

115.88 ± 14.89 

(105-151) 

99.13 ± 7.40 

(87-107) 

Globulins (g/dl) 3.9-6.0 2.13 ± 0.33 

(1.6 - 2.8) 

2.83 ± 0.57 

(2.2 -3.6) 

1.69 ± 0.22 

(1.3 - 2.0) 

2.85 ± 0.89 

(1.9-4.0) 

A/G ratio na 2.01 ± 0.30 

(1.43-2.56) 

1.52 ± 0.39 

(1.03-2.10) 

2.65 ± 0.36 

(2.15-3.23) 

1.62 ± 0.62 

(0.75-2.47) 

Total T3 (ng/dl) na 70.95 ± 10.05 

(60.09 -92.99) 

48.60 ± 9.37 

(36.71-54.63) 

95.48 ± 17.85 

(74.12- 120.07) 

43.99 ± 19.41 

(15.00-66.87) 

Cortisol (g/dl) na 5.56 ± 2.52 

(1.2-8.9) 

4.58 ± 1.76 

(3.2-8.9) 

6.56 ± 2.39 

(3.1-10.9) 

4.66 ± 3.55 

(0.9-10.0) 

1

 Merck Veterinary Manual, 

http://www.merckvetmanual.com/mvm/index.jsp?cfile=htm/bc/230100.htm, References, Table 07. 

2

 For abbreviations, see Appendix F: The Comprehensive Veterinary Examination and Its Interpretation 

3

 Values presented are means ± SD, range in parenthesis. 

animals were evaluated (nine clones and eight controls). Nonetheless, the data are 

compelling in that they demonstrate that the physiological parameters investigated do not 

indicate any material differences between the clones and controls. In addition, they 

provide confidence that these clones are responding appropriately to age-specific signals. 
Chapter VI: Food Consumption Risks 237 

Animal Cloning: A Risk Assessment DRAFT

Just as the Cyagra cattle clones, the piglet clones initially exhibit relatively high alkaline 

phosphatase levels: at week 15 both clones and controls have mean levels of 209 and 235 

U/L, respectively, while 8 weeks later (at 27 weeks of age), the mean alkaline 

phosphatase levels have decreased to 101 and 118 U/L, respectively. (Alkaline 

phosphatase provides a measure of bone growth in young animals.) Phosphorus levels, 

also an indicator of bone growth, show similar age-related changes, as does T3. 

Genomic methylation levels (also discussed in Chapter IV) were evaluated in two 

repeated sequences, one found at the centromere55 and the other in the euchromatin56

regions of the chromosomes (Archer et al. 2003a). The investigators discovered that one 

euchromatin region of clones had a different degree of methylation from controls. They 

further observed that another region had an increase in the variability of the degree of 

methylation in clones relative to controls. The investigators stated that it was not possible 

“to prove cause and effect” between alteration in methylation patterns and any of the 

measurements that they had taken on these animals. Additionally, because all of the 

animals in this study (clones and controls) appear to be healthy, with the exception of the 

pig with hyperkeratosis/parakeratosis, the developmental relevance of these methylation 

changes are not clear. It may be that animals that do not survive have higher degrees of 

variability or derangement of methylation patterns, and that what is being observed in this 

study is a set of animals that has adapted to or compensated for differences in 

methylation, or the inherent tolerance of biological systems for changes in methylation 

status of genes. 

The authors concluded that “while cloning creates animals within the normal phenotypic 

range, it does affect some traits by increasing variability associated with that phenotype.” 

Their final conclusion with respect to phenotypic variability among clones was that they 

were not necessarily less variable than their closely related, sexually reproduced halfsiblings. For pigs, at least, this implies that although genetics may have a strong 

influence, various environmental influences, including intra-uterine environments, may 

play a significant role in eventual phenotype of the animal. 

(b) Viagen Dataset 

The data on which the following discussion is based are found in Appendix F, along with 

a more detailed description of the results of the study. 

 55 The constricted region of a mitotic chromosome that holds sister chromatids together—the crossing 

point in the “X” often used to depict chromosomes. 56 The region of an interphase chromosome that likely to be transcriptionally active. 
Chapter VI: Food Consumption Risks 238 

Animal Cloning: A Risk Assessment DRAFT

Two groups of swine clones were used for Viagen Study 1. In the first group, seven 

clones (1 Duroc and 6 Hamline) were evaluated for survival, health, growth, meat and 

carcass characteristics. Fifteen conventional barrows (young males) (all Hampshire) were 

selected as comparators. Because the study was initiated after the birth of the clones 

(delivered by C-section), the observation period did not begin until shortly after they 

were weaned. Clones were followed from 50 days after birth through slaughter at 

approximately 6 months of age. comparators57 were selected as age-matched pigs 

selected from litters sired by the Hampshire nuclear donor boar in a conventional 

breeding (AI) program. 

Clones raised to slaughter weight (approximately 270 lbs) took on average 27 days longer 

to reach that approximate weight, and when finally slaughtered tended to weigh less than 

their comparators. These observations are likely due to the husbandry of the clones. 

Further, because these animals were delivered via C-section, they faced additional stress 

during the earliest stages of life. In addition, because of the late initiation of the study, 

these clones were raised under pathogen-free conditions until 50 days of age before being 

transferred to more conventional (pathogen containing) rearing conditions, while all of 

the comparators were raised under conventional conditions. Clones also did not receive 

colostrum, and were deprived of passively transferred maternal immunity. Combined 

with the change from pathogen-free rearing, the significant immune challenge that the 

clones experienced would have slowed growth as the animals adapted to their new 

environment, regardless of whether the animals were produced by sexual reproduction or 

nuclear transfer. 

Four of the clones exhibited appropriate responses to the immune challenge and were 

able to adapt and grow, albeit at a lower rate than animals which had been raised under 

more conventional conditions from birth. Three of the clones were considered “poordoers:” animals that exhibit slow growth rates and other health problems, such as chronic 

scouring. At slaughter, organ weights as a percentage of body weight were smaller for 

clones than for their comparators. The clones also had lower blood IGF-I and estradiol 

levels in their blood than comparators. It is unknown how the change from a pathogen 

free environment to a more conventional one may have impacted organ weights or 

hormone status, as none of the comparators were subjected to similar immune challenges 

because they were all raised under conventional conditions from time of birth (Appendix 

F). However, given the physiological and immunological stress that the animals 

 

57 Although this study is a more controlled experiment than the retrospective review of the Cyagra clones, 

the word “comparator” is used for consistency with the discussion of Cyagra data, rather than the more 

common term “control.” 
Chapter VI: Food Consumption Risks 239 

Animal Cloning: A Risk Assessment DRAFT

experienced, in the opinion of CVM’s veterinarians, these animals performed as well as 

could possibly be expected. 

The second group of swine clones was used to study reproductive function; that 

discussion is found in Section iii. 

(c) Summary Statement for Juvenile Development and Function in Swine Clones 

(Developmental Node 3) 

The dataset reported by Archer et al. (2003 a,b) in which both behavior and physiological 

variables were measured on an individual animal basis is the larger and more tightly 

controlled study of the two studying juvenile swine clones. Those studies indicate that 

swine clones overlap their conventional counterparts in behavior and health, and that 

there are no significant differences between the two groups. Measures of age-appropriate 

physiological responses (e.g., alkaline phosphatase, phosphorus, and serum protein 

indicative of increased globulins) indicate that clones are responding normally to growth 

signals. One case of parakeratosis was observed in this clone cohort. It is not possible to 

determine whether its incidence was due to SCNT, as it is a condition that is also present 

in conventional pigs. The Viagen dataset is less-well controlled, and its outcome 

confounded by the unconventional shift from a pathogen free environment to more 

conventional husbandry. Based on the lack of colostrums immediately after birth, and the 

transfer from pathogen free to conventional housing, it is not possible to ascribe any of 

the differences in growth to cloning. Further, most of the animals were able to respond 

appropriately to immune challenges. None of these outcomes were observed in the 

studies of Archer or Martin, again implying that the changes in husbandry were likely 

responsible for the outcomes. Finally, none of the swine clones exhibited any adverse 

outcomes that have not been observed in conventionally bred and reared swine. 

 

iii. Reproductive Development and Function in Swine Clones (Developmental 

Node 4) 

(a) Peer-reviewed Publications 

Martin et al. (2004) described birth outcomes of clone females which were mated via 

artificial insemination to clone males as normal in duration and uneventful. The 62 live 

offspring of the clone X clone mating were reported to be normal at birth with the 

exception of one pig that had contracture of the flexor tendons of both hind limbs. The 
Chapter VI: Food Consumption Risks 240 

Animal Cloning: A Risk Assessment DRAFT

authors reported that the rate for this abnormality (1.6 percent) was similar to estimates of 

the frequency within the Australian swine industry (1.2 percent). The stillborn rate for the 

clone offspring litters was 4.5 percent while a comparator group had a stillborn rate of 8 

percent. Evaluation of the semen from the boars, showed similar ejaculate volume, sperm 

concentration and motility between the clones and comparators. These investigators 

further reported that 100 percent of gilt clones (5) became pregnant following 

insemination at second estrus. Consequently, the limited data indicate that gilts and boars 

from cloning mature similar to non-clones. 

Reproduction was measured in four boar clones in the Viagen dataset (Appendix F). The 

four clones (three Hampshire and one Duroc) were compared to three genetically related 

boars derived by AI. No differences were observed between clones and comparators in 

semen quality. Farrowing rates were higher for swine clones than comparators (73.5 vs. 

62.5 percent), although this difference was attributed to the fact that the Hampshire 

comparator was five years old, and may have been nearing the end of his reproductive 

life. Litter size was more variable for boar clones, and mean litter size was slightly 

smaller for clones vs. comparators (10.94 vs. 11.76 pigs/litter), but were similar to US 

commercial swine production (10.66 pigs/litter). 

iv. Post-Pubertal Maturation in Swine Clones (Developmental Node 5) 

CVM was not able to identify any peer-reviewed studies on non-reproductive postpubertal studies in swine clones. The Viagen dataset (Appendix F) indicated that 

No remarkable differences were observed between clones and comparators for any of the 

characteristics evaluated. The small differences in backfat thickness and marbling are 

likely due to the lighter weight of clones vs. comparators at slaughter. 

v. Conclusions Regarding the Food Safety of Swine Clones 

The conclusions regarding food consumption risks from swine clones are drawn largely 

from the animal health information presented by Walker et al. (2002), Archer et al. 

(2003b), and the Viagen dataset, supported by less detailed discussions of animal health 

in the other studies reviewed. These results indicate that there are no apparent anomalies 

present that would have a direct impact on the safety of food products derived from swine 

clones. The measurements taken at 27 weeks of age are approximately the age at which 

pigs are sent to slaughter in the US, and thus provide an appropriate age cohort for the 

evaluation of food safety. The identified abnormalities in the Archer et al. (2003a) 

(parakeratosis) and Viagen dataset (lung adhesion) are abnormalities normally seen in 
Chapter VI: Food Consumption Risks 241 

Animal Cloning: A Risk Assessment DRAFT

case noted does not pose a food consumption risk, as the affected skin from the carcass 

would be condemned at the slaughterhouse, and would not enter the food supply. The 

apparently normal status of the clinical measurements indicates that the clones in this 

study possess the same physiological functions and behaviors as their conventional 

counterparts, and thus are not likely to pose a greater food consumption risk than 

conventional swine. 

c. Sheep Clones 

i. Peer-reviewed Publications 

As sheep were the first mammal to be cloned by SCNT, the relative paucity of papers on 

the developmental success of sheep clones is somewhat surprising. The seminal paper in 

the history of animal cloning is that prepared by Wilmut et al. (1997) in which they 

describe the generation of “Dolly,” the first mammal to be born (July 5, 1996) as the 

result of SCNT. (Gene, a bull clone was being gestated at the same time, but due to 

differences in the length of pregnancy between cattle and sheep, Dolly was born first). 

Dolly was derived from the mammary epithelium of a 6-year-old Finn Dorset ewe. The 

trial from which Dolly was derived included cells from two other sources besides the 

mammary epithelium, and included fetal fibroblast cells from a 26-day-old Black Welsh 

fetus, and cells derived from a nine-day-old Poll Dorset embryo. 

Table VI-10: Summary of Outcomes from Production of Sheep Clones from Different Cell Types 

from Wilmut et al. (1997) 

Cell type 

Number of 

embryos 

transferred 

Number of 

pregnancies/ 

Number 

recipients 

(%) 

Pregnancy 

Duration 

(days) 

Number of Live 

Lambs 

Birth Weight 

(kg) 

Finn Dorset 

Mammary 

epithelium 

29 1/13 

(7.7) 148 1 6.6 

Black Welsh 

Fetal (gd29) 

fibroblast 

34 

6 

4/10 

(40.0) 

1/6 

(16.6) 

152 

149 

156 

2 

1 

5.6 

2.8 

3.1 

Poll Dorset 

9 day embryo 

72 14/27 

(51.8) 

149 

152 

148 

152 

4 

6.5 

6.2 

4.2 

5.3 
Chapter VI: Food Consumption Risks 242 

Animal Cloning: A Risk Assessment DRAFT

Table VI-10 summarizes the outcomes Wilmut et al. 1997 paper. Pregnancy rate, as 

measured by detectable pregnancy at days 50-60 post transfer, ranged between ~ 8 

percent to as high as ~50 percent. A total of 62 percent of the implanted fetuses were lost. 

Wilmut et al. (1997) reported that at approximately day 110 of the pregnancies, four dead 

fetuses derived from the embryo cell lines were detected. Their surrogate dams were 

euthanized, and post-mortem examination of the fetuses revealed two cases of abnormal 

liver development, but no other abnormalities or evidence of infection. A total of eight 

live lambs were born. One lamb, derived from fetal fibroblasts, died within a few minutes 

of birth. No abnormalities were noted at the post-mortem. Wilmut et al. cite the mortality 

rate of 12.5 percent (1 of 8) as similar to that observed in a large study of commercial 

sheep breeding, where 8 percent of the lambs died within 24 hours of birth. The birth 

weights of all of these sheep were within the range of single lambs born to the surrogate 

Blackface dams used at the Roslin farm (up to 6.6 kg), and were reported to be 

appropriate to the birth weights of the donor breeds. 

The following year, Shiels et al. (1999) compared the telomere lengths of Dolly and one 

of each of the sheep derived from the different cell sources described in Table VI-10, 

with age-matched control sheep, donor mammary gland tissue, and donor cells. As 

expected, the mean size of telomere fragments in control animals decreased with 

increasing age. Mean telomere sizes were smaller in all three sheep clones than in agematched controls. Dolly’s mean telomere size in particular, was smaller than other oneyear-old age-matched sheep, and more consistent with the telomere fragment sizes 

derived from a 6-year-old sheep (the age of the animal from which the donor cells were 

derived). These observations led to speculation that clones would reflect the age of the 

donor cell, rather than effectively “resetting the biological clock” to their chronological 

age. 

Dolly’s health was scrupulously observed over the course of her life. She developed 

arthritis at an early age, and was reported to have been overweight. Dolly was euthanized 

in early 2003 at approximately six and one half years of age having contracted a virulent 

form of lung disease that was endemic at the facility where she had been housed. It is not 

clear whether any of the abnormalities that were observed with Dolly were the result of 

SCNT, the conditions under which she was housed, or some combination of the two. 

Most of the other papers in the literature refer to sheep clones generated from transgenic 

somatic cells to propagate animals with pharmaceutical potential, and data in those 

papers deal with expression of transgenes, molecular mechanisms that may be involved 

with fetal overgrowth syndromes (Young et al. 2001), or techniques to increase survival 

of nuclear transfer (Papadopoulos et al. 2002; Ptak et al. 2002) or other in vitro produced 
Chapter VI: Food Consumption Risks 243 

Animal Cloning: A Risk Assessment DRAFT

embryos. McCreath et al. (2000) reported on the post-mortem examination of transgenic 

lambs that died in utero or in the perinatal phase of development. These animals revealed 

a range of abnormalities including a high incidence of kidney defects, liver and brain 

pathology. This research group did not discuss the health of the transgenic lambs that 

survived. 

Recently, Rhind et al. (2003) published a commentary on pathology findings from both 

transgenic (n=5) and non-transgenic (n=3) sheep clones that were not viable after birth. 

(The transgenes were intended to be targeted deletions of the α-1,3 galactosyl transferase 

or prion protein genes). Of the eight animals evaluated, seven were euthanized at birth or 

shortly thereafter, the eighth survived but was euthanized after 14 days. The authors 

concluded that many of the defects (e.g., hepatobiliary changes, kidney structure changes, 

and pulmonary hyptertension) may not be contained within the “large offspring 

syndrome” (LOS) classification that may be common to other animal clones. Pulmonary 

hypertension has been observed in transgenic cattle clones (Hill et al. 1999), and in swine 

clones derived from transgenic “knock-out” piglets (Lai et al. 2002), suggesting that this 

syndrome may be common to a many species of animal clones, and that a common defect 

may be responsible. The authors call for additional research into the developmental 

mechanisms that may be responsible for the common defects in clones, although it is 

important to note that most of the animals with defects were derived from transgenic 

donor cells. The relevance of these observations to food consumption risks are limited, as 

clones that have died would not be used for food consumption purposes. 

ii. Conclusions Regarding the Food Safety of Sheep Clones 

Very few conclusions can be drawn about the health of sheep clones, due to the small 

database available for evaluation, as despite Dolly’s high public visibility, there are very 

few other reports of non-transgenic sheep clones. In the absence of specific information 

regarding the health of sheep clones, the only inferences that can be made would be 

drawn from interspecies extrapolation from other ruminant clones, i.e., cattle and goats. 

d. Goat Clones 

Relative to cattle, the database on goat clones is relatively small, but quite rich for its 

size. Much of the work that has been reported on non-transgenic goat cloning arises from 

data collected in an attempt to perfect systems by which SCNT can be harnessed to 

develop transgenic goats for commercial applications, and are effectively limited to 

publications from one group. 
Chapter VI: Food Consumption Risks 244 

Animal Cloning: A Risk Assessment DRAFT

i. Perinatal Development and Function in Goat Clones (Developmental Node 2) 

In 2002, Keefer et al. (2002) published a report on the birth of nine goat clones derived 

from two lines of adult granulosa cells and one line of fetal fibroblasts. Ninety-one 

female granulosa cell-derived embryo clones were transferred into eight surrogate dams. 

Four of those dams became pregnant, as confirmed by ultrasound on gestational day 30 

and 60. All of these pregnancies went to term, and seven clones were born. Table VI-11 

summarizes the outcomes of Keefer et al. 2002. One of the recipients delivered a single 

kid; the remaining three surrogates gestating granulosa cell-derived clones delivered 

twins. One of the female twins died at birth, but appeared to be normal. 

In addition, 54 male fetal fibroblast-derived embryo clones were implanted into six 

surrogate dams. Only one of those dams had a confirmed pregnancy and delivered two 

male kids, one of which died during delivery. The authors state that this kid also appeared 

normal. 

Table VI-11: Summary of Outcomes from Goat Cloning Study 

(Keefer et al. 2002) 

Surrogate Donor 

Cell Type 

Gestation 

(days) 

Birth 

weight 

(kg) 

Gender Status Suckling 

Response 

1 Granulosa Line 1 144 1.8 Female Live Good 

2 Granulosa Line 2 

Granulosa Line 2 150 2 1.2 

Female 

Female 

Live 

Dead 

None 

NA1 

3 Granulosa Line 2 

Granulosa Line 2 145 1.6 

1.4 

Female 

Female 

Live 

Live 

Poor/None 

Poor/None 

4 Granulosa Line 2 

Granulosa Line 2 145 1.5 2.2 

Female 

Female 

Live 

Live 

Good 

Good 

5 Fibroblast Line 1 

Fibroblast Line 1 148 1.5 1.2 

Male 

Male 

Dead 

Live 

NA 

None 

1

 NA = not applicable 

The birth weights of all of the kids were cited as being within the normal range for this 

breed (Nigerian Dwarf) at an average of 1.7 ± 0.13 kg versus 1.3 ± 0.06 kg for females 

resulting from natural breeding. The authors reported that the placentae of these kids 

appeared normal, and had cotyledon numbers that were comparable to those from the 

placentae of naturally bred Nigerian Dwarf goats. Suckling response was delayed in half 

of the granulosa cell-derived kids, and in the fibroblast-derived kid. These animals were 
Chapter VI: Food Consumption Risks 245 

Animal Cloning: A Risk Assessment DRAFT

fed colostrum by intubation, and good suckling was reported to occur by Day 2. The 

clones were otherwise reported as healthy, and having no apparent abnormalities. 

ii. Juvenile Development in Goat Clones (Developmental Node 3) 

In the Keefer et al. (2001a) study, the authors reported that blood profiles of the clones 

were monitored for one year, and showed no anomalous results. No data addressing this 

statement were presented in the Keefer et al. (2002) publication. There is, however, an 

abstract that was published in 2001 (Keefer et al. 2001b) in which some blood parameters 

are provided (Table VI-12: Selected Laboratory Parameters for Goat Clones). In this brief 

account, only a few measurements were reported; the duration of monitoring was six 

months. Alkaline phosphatase levels show the appropriate age-related changes to be 

expected for rapidly growing infants and very young animals, dropping to lower levels as 

the animals aged. Given the adult range for alkaline phosphatase in Nigerian Dwarf goats 

is reported as 16-33 U/L, these animals likely were still growing at 6 months of age. 

Table VI-12: Selected Laboratory Parameters for Goat Clones 

(from Keefer et al. 2001b) 

Measurement 1 week 3 months 6 months 

Lymphocyte counts (cells x 109) 

 Clones 

 Control 1

2.34 

2.64 

4.94 

5.94 

9.84 

7.14 

Glucose (mmol/L) 

 Clone 

 Control 

5.64 ± 0.3 

4.84 ± 0.3 

4.14 ± 0.2 

4.54 ± 0.1 

3.54 ± 0.1 

3.54 ± 0.1 

Alkaline phosphatase (U/L) 

 Clone 

 Control 

7,434 ± 84 

Not provided 

5,554 ± 73 

Not provided 

374 ± 33 

Not provided 

1

 Controls were reported as taken from Mbassa et al. 1991 (Zentralbl Veterinarmed [A] 38: 510-522). It 

is likely that these values are from adult animals, as the control values for the alkaline phosphatase levels 

were not explicitly provided. Instead, the adult range of 16-33 U/L was cited. 

Behboodi et al. (2005) compared hematology and blood clinical chemistry of four 

transgenic goat clones with four age-matched comparators and a published range for goat 

blood values (Pugh 2002). Hematology values were similar between clones and 

comparators, and all hematology values fell within the published range (Pugh 2002). For 

clinical chemistry, 18/24 values were not significantly different between clones and their 

age-matched comparators. Of the 19 clinical chemistry values for which published ranges 

were available, 18 of the values for clones and comparators fell within the published 

range. The one value out of the published range, creatine kinase (244.6 vs. 204.4 IU/L for 

clones and comparators), was not different between clones and comparators. 
Chapter VI: Food Consumption Risks 246 

Animal Cloning: A Risk Assessment DRAFT

iii. Reproductive Development and Function in Goat Clones (Developmental 

Node 4) 

One paper compares the sexual maturation and fertility of male Nigerian Dwarf clones to 

conventional bucks (Gauthier et al. 2001). Three clones (Stewart, Clint, and Danny) and 

four conventional animals that served as controls (Blue, Star, Banzai, and Ed) were 

trained to serve an artificial vagina beginning at the age of one month. Average age at 

first semen collection for both clones and controls was approximately 20 weeks, although 

volumes were small at the initial collection (<0.1 ml). Subsequent collections showed 

increased volume and increased sperm count (see Table VI-13 for a summary of the 

reproductive function in goat clones, comparators, and clone progeny). 

Semen collected from two goat clones, Clint and Danny, at seven months was used to 

impregnate six Nigerian Dwarf does (three does for each buck). Although not explicitly 

stated, the implication is that the does were not clones. Five of the six does became 

pregnant. Two does impregnated by Clint gave birth vaginally to two sets of twins. Two 

does impregnated by Danny gave birth to singletons, and one doe gave birth to triplets. 

Nine kids were produced, and all appeared to be normal and healthy. Average birth 

weights for the male and female clone progeny were 1.7 ± 0.2 kg and 1.66 ± 0.1 kg, 

respectively, which do not differ significantly from average birth weights for 

conventional animals of this breed (1.7 ± 0.07 kg (n = 41) for males and 1.3 ± 0.31 kg (n 

= 79) for females). Semen was first collected from one of the progeny males at 28.4 

weeks (Table VI-13). 

Table VI-13: Reproductive Function in Goat Clones, Comparators, and Clone Progeny 

(from Gauthier et al. 2001) 

Animal 

Derivation 

Number 

of bucks 

Mean age at 

collection ± 

SEM1

 (weeks) 

Mean sperm 

concentration ± 

SEM 

(sperm x 109

 /ml) 

Mean Ejaculate 

Volume ± SEM 

(ml) 

Range of 

Motility % 

Control 

bucks 4 20.2 ± 3.1 ND2 ND ND 

 3 36.5 ± 0.3 2.5 ± 0.6 0.37 ± 0.14 ~70-90 

 3 59 ± 1 2.1 ± 0.6 0.4 ± 0.06 ~45-90 

Clones 3 20.2 ±1.2 0.6 ± 0.073 0.25 ± 0.13 75-85 

 3 23 ± 0.6 1.2 ± 0.71 0.28 ± 0.11 30-98 

 2 79.5 ± 0.5 4.4 ± 0.3 0.37± 0.02 75-90 

Progeny 

buck 1 28.4 4.6 0.4 65 

1

 SEM = standard error of the mean 

2

 ND = not done 

3

 The sample from the first collection from one buck was too small to measure 
Chapter VI: Food Consumption Risks 247 

Animal Cloning: A Risk Assessment DRAFT

The authors concluded that male Nigerian Dwarf goat clones developed sexual maturity 

similarly to their conventional counterparts. Further, these goat clones are fertile, and 

their progeny appear to be fertile as well. 

In their study of goat clones generated from transgenic fibroblasts, Reggio et al. (2001) 

were able to produce a total of five healthy kids. Twenty-three surrogate dams were each 

impregnated with an average of eight embryo clones. Five of the dams that were detected 

as pregnant at day 30 completed their pregnancies, and gave birth naturally, providing a 

100 percent success rate based on detectable pregnancy. All of the kids appeared healthy 

and vigorous. Birth weights averaged 3.8 kg (normal for the Toggenberg breed that 

served as the donor cell), and weaning weights were also within normal range (19.1-24.5 

kg) for the breed. Each of the kids exhibited estrus, and has been bred to a buck. No 

reports of progeny were provided. Although this study is based on transgenic clones, it 

reiterates the high success rate that is experienced by researchers producing goat clones. 

iv. Post-Pubertal Maturation in Goat Clones (Developmental Node 5) 

CVM was not able to identify any published reports of measures of post-pubertal nonreproductive maturation in goat clones. Further, in the course of several presentations at 

scientific meetings, the Center learned that that the cohort of clones studied in Keefer et 

al. 2001a and 2002 has been terminated for business reasons. 

v. Summary Statement on Health Status of Goat Clones 

Based on these data, goat clones appear to have the least difficulty of any of the livestock 

species with respect to the SCNT process. Successful pregnancy outcome (when 

confirmed by ultrasound detection) is very high, and clones appear to be born at birth 

weights within the appropriate breed- and species- range. Suckling response was weak in 

some of the goat clones immediately after birth, but they appear to have recovered within 

one day. Available information on physiological parameters indicates that these animals 

appear to be normal. Data on reproductive function in these animals indicates that they 

enter puberty at the normal age range, produce viable semen, and normal, live offspring. 

The minimal reporting on one progeny animal also indicates that progeny are fertile. 

vi. Conclusions for Food Consumption Risks from Goat Clones 

Based on the data reviewed, there do not appear to be any anomalies present in the goat 

clones that would have a direct impact on the safety of food products derived these 

animals. Goats appear to be relatively “cloning friendly” with a high degree of successful 
Chapter VI: Food Consumption Risks 248 

Animal Cloning: A Risk Assessment DRAFT

live births following confirmation of pregnancy. All reports of health of the goat clones 

seem to indicate that they are normal and healthy. The available data on the physiological 

parameters of goat clones indicate that these animals respond as their conventional 

counterparts to internal signals for growth. The apparently normal status of the clinical 

measurements indicates that the clones in this study possess the same physiological 

functions and behaviors as their conventional counterparts. Further, unlike the other 

livestock clones, data on the reproductive behavior of male goat clones indicate that 

reproductive function is normal. Finally, although cursory in mention, it appears that 

male progeny of clone bucks also reach puberty at the appropriate time. Thus, although 

the number of animals that has been evaluated is not as large as in the case of bovine 

clones, goat clones appear to be healthy, and do not appear to be materially different from 

conventional goats. 

3. Compositional Analysis Method 

a. Overview 

The operating hypothesis of the second prong complement to the Critical Biological 

Systems Approach is that if food products from healthy animal clones and their progeny 

meet the local, state, and federal regulatory requirements set forth for those products 

(e.g., Pasteurized Milk Ordinance,58 USDA inspection criteria, absence of drug residues), 

and are not materially different from products from conventionally bred animals, then 

they would pose no more food consumption risk(s) than corresponding products derived 

from conventional animals. 

Information on the composition of meat or milk from animal clones has been limited for 

several reasons. Few of the cattle clones are old enough to have been bred, given birth, 

and begun lactating. In addition, there is uncertainty regarding the kinds of analyses that 

could or should be performed in order to determine whether milk from animal clones is 

materially different from milk from non-clone animals. The issues associated with the 

compositional analysis of meat are similar, but have additional practical and economic 

components. During the course of preparing this Risk Assessment, CVM has contacted 

several food testing laboratories to inquire about the minimum sample size that would be 

required in order to perform a compositional analysis of meat. The Center’s hope was 

that systems were sufficiently miniaturized to allow analysis of “punch biopsies” of a 

shoulder or rump, but were informed that the minimum sample size would require 

 58 The Grade “A” Pasteurized Milk Ordinance recommends for statutory adoption regulations for the 

production, collection, processing, sale, and distribution of milk and certain milk products. 
Chapter VI: Food Consumption Risks 249 

Animal Cloning: A Risk Assessment DRAFT

sacrificing an animal. Nonetheless, there are now several studies that have evaluated the 

composition of the milk and meat of both cattle and swine clones, and one large study 

that has evaluated the composition of the meat of the progeny of swine clones. 

b. Nutritional Risk 

The primary concern for milk and meat from animal clones is that inappropriate 

reprogramming of the nucleus of donor cells does not result in epigenetic changes 

creating subtle hazards that may pose food consumption risks (Chapter III). Because, as 

previously discussed, there is no a priori reason to expect that SCNT will introduce any 

new, potentially toxic substances into the milk or meat of otherwise healthy animals, the 

remaining food safety concerns addressed whether subtle changes have occurred that 

would alter the presence of important nutrients. The most likely dietary risk would then 

be the absence or significant decrease in levels of vitamins and minerals whose daily 

requirements are in large part met by milk or meat. 

The overall strategy we used to determine which milk or meat components could 

characterize their respective nutritional “footprints” involved selecting certain key 

nutrients and compositional parameters, while at the same time allowing sufficient 

flexibility in the non-essential components that vary with the genetic make-up and 

husbandry of the production animal. Finally, evaluation of the levels of the results of 

complex biochemical pathways in clones (e.g., saturated fats, vitamins) can further 

ensure that the clone is functioning appropriately, and thus indirectly support the 

hypothesis that the clones are appropriately reprogrammed and not materially different 

from their conventionally bred counterparts. 

In order to identify the nutrients in milk or meat whose alterations would most likely 

affect the overall diet, even if all of the dairy and meat products from conventional 

animals in the daily diet were replaced by counterparts produced by clones, we first 

determined which nutrients made a “major” or “moderate” contribution to the total daily 

diet of milk or meat consumers. For the purposes of this Risk Assessment, a nutrient in 

meat or milk was considered a major dietary source if it provides 50 percent or more of 

its recommended dietary allowance (RDA) in that food.59 Likewise, a nutrient in a food 

providing 10 to 50 percent of its RDA in that food is considered a moderate dietary 

source. For example, a single eight ounce serving of whole milk provides milk drinkers 

with between 10 to 50 percent of the RDA of vitamin B12, riboflavin (B2), pantothenic 

acid (B5), calcium, phosphorous and selenium. Another example, a single serving of three 

 

59 http://www.iom.edu/?id=4576&redirect=0 
Chapter VI: Food Consumption Risks 250 

Animal Cloning: A Risk Assessment DRAFT

ounces of roasted eye of round beef provides a meat consumer with a moderate source of 

zinc, niacin, vitamin B6, phosphorus, iron, and riboflavin, and a major source of vitamin 

B12 and selenium. 

In order to determine typical meat and milk consumption in the adult US population, we 

consulted the one-day food survey conducted by the National Health and Nutrition 

Examination Survey (NHANES) of 2000-2001. According to NHANES, the mean daily 

consumption of milk among adult milk drinkers was 11.5 ounces. At this level of 

consumption, milk becomes a major source of vitamin B12, and a moderate source of 

thiamin (B1), zinc, and potassium, in addition to the nutrients previously listed as 

provided in moderate amounts. The same survey showed that the 90th percentile 

consumption of milk by users was 24.1 ounces per day, making milk a major source of 

calcium, phosphorus, riboflavin, and pantothenic acid, and adding magnesium and 

vitamin B6 to the list of nutrients provided in moderate amounts. Among subjects who 

consumed meat, the mean intake of meat was 4.2 ounces or 120.2 grams. Among the 90th 

percentile of meat eaters, consumption was 8.4 ounces or 239.4 grams. In order to 

determine whether evaluating the mean or 90th percentile consumption of milk and meat, 

changed the actual number of nutrients designated as moderate or major, we found that 

there were no nutrients were added or deleted, although with increased consumption 

rates, some of the nutrients changed from moderate to major contributors to the diet. 

Proteins are of dietary importance because once they are digested, they provide the body 

with amino acids. In particular, some amino acids are of dietary concern because of the 

inability of mammals to synthesize them de novo in sufficient quantity to meet the body’s 

needs. For this reason they are designated as “essential.” Therefore, for purposes of 

assessing nutritional risk from food products from animal clones, the nature of the protein 

in its initial food matrix (e.g., casein or actin) is less important than whether it contains 

the same level of essential amino acids as its counterpart in foods derived from 

conventional animals. Finally, certain fatty acids such as linolenic (18:3) and linoleic 

(18:2) acid are essential components of the diets of mammals (including humans) and 

have also been selected as “key nutrients.” 

Table VI-14 summarizes the analytes that we believe could be used to assess the 

composition of milk and meat from clones and comparators to demonstrate that there are 

no material difference between the two groups of animals with respect to key nutrients 

and overall nutritional characteristics. Included are key essential vitamins, minerals, and 

fatty acids. Other less essential constituents (e.g., vitamin A in milk is often 

supplemented, iron is not a key nutrient in milk) are also included to illustrate that 

checking on the levels of non-essential nutrients can also provide a useful tool to 
Chapter VI: Food Consumption Risks 251 

Animal Cloning: A Risk Assessment DRAFT

demonstrate the similarity of milk and meat from clones and their contemporary 

comparators. 

Table VI-14: Compositional Analyses of Milk and Meat That May be Used for Showing No Material 

Differences Between Clone and Comparator Food Products 

Milk Composition Meat Composition 

Proximates1 Proximates 

Vitamins and minerals for which milk is a moderate to 

major source 

Vit A, C, B1,B2, B12, niacin, 

pantothenic acid, Ca, Fe, P 

Vitamins and minerals for which meat is a moderate to 

major source 

Vit A, C, B6, B12, niacin 

Ca, Fe, P, Zn 

Fatty Acid Profiles 

Saturated: 4:0, 6:0, 8:0, 10:0, 12:0, 14:0, 16:0, 

18:0 

Unsaturated: 18:1, 18:2, 18:3 

Fatty Acid Profiles 

Saturated: 10:0, 12:0, 14:0, 16:0, 18:0 

Unsaturated: 18:1, 18:2, 18:3, 20:4 

Cholesterol 

Protein characterization 

Essential amino acid profile 

Protein characterization 

Essential amino acid profile 

Carbohydrate 

1

 Most foods are comprised of water, protein, fat, ash, and carbohydrates; the sum of these values approximates a 

complete analysis, hence the term “proximates.” 

i. Milk 

For the purposes of this Risk Assessment, CVM uses the term “milk” to mean the “lacteal 

secretion, practically free from colostrum, obtained by the complete milking of one or 

more healthy cows,” and that “milk that is in final package form for beverage use shall 

have been pasteurized or ultra-pasteurized, and shall contain not less than 8 1/4 percent 

milk solids not fat and not less than 3 1/4 percent milkfat” (21 CFR 131.110(a)). 

The Grade “A” Pasteurized Milk Ordinance (a model ordinance for adoption by states, 

counties and municipalities to regulate the production, collection, processing, sale and 

distribution of milk and certain milk products) echoes this definition, replacing “cows” 

with the term “hooved animals.” Therefore, in this Risk Assessment, unless otherwise 

specified, the term “milk” will refer to the lacteal secretions of cows, goats, or sheep. 

Although most of the discussion in this Risk Assessment refers to cow’s milk, similar 

arguments may be applied to the milk of goats or sheep. Other hooved animals whose 

milk is covered by the PMO include water buffalos, although they are not covered by this 

Risk Assessment. 
Chapter VI: Food Consumption Risks 252 

Animal Cloning: A Risk Assessment DRAFT

The biological role of the milk of any mammal is to provide nutrition to its own newborn 

and young. In addition to mother’s milk, humans consume the milk of a few other 

species, principally from cows. Milk and milk products provide a considerable portion of 

the nutrition of other age groups, including growing children and adolescents, pregnant 

and lactating women, and the elderly. In 2001, per capita American consumption of milk 

among all age groups was approximately 23 gallons of fluid milk, 30 pounds of cheese, 

and 27 pounds of frozen dairy products (USDA ERS 2003).60 In particular, bovine milk 

and milk products (excluding butter) provided approximately nine percent of the energy, 

19 percent of the proteins, 12 percent of the fats, and 4.5 percent of the carbohydrates 

consumed by milk drinkers in the US in 200161. Ensuring that these dietary levels do not 

alter significantly is a key component of evaluating the potential nutritional risk from the 

milk of animal clones. 

The degree to which individuals may experience risk from the consumption of milk 

appears to be a function of individual susceptibility, rather than the intrinsic toxicity of 

milk. For example, certain individuals suffer from Cow’s Milk Protein Allergy, which 

has an incidence of 2-6 percent among young infants (Exl and Fritsché 2001). Cow’s 

Milk Protein Allergy usually presents during the first year of life, and generally resolves 

by school age (Bernstein et al. 2003). Lactose intolerance is another milk-related 

condition found in adults and children (to a lesser degree) that is also a function of 

individual physiology (i.e., decreased expression of the enzyme lactase), particularly 

among certain ethnic groups. Excess consumption of saturated fats, including those from 

dairy products, can lead to atherosclerosis and its consequent morbidities; again, these 

harms are a function of individual behavior and susceptibility and not an intrinsic hazard 

of milk itself. 

State regulatory agencies have managed the risk(s) posed by milk by adopting the PMO. 

It was first developed (1924) by what was then known as the Public Health Service, a 

precursor to today’s U.S. Food and Drug Administration. Now known as the Grade “A” 

Pasteurized Milk Ordinance, the PMO is revised biennially (most recently in 2003) by the 

Center for Food Safety and Applied Nutrition and other centers of the FDA, with input 

from industry and state regulatory agencies. 

Table VI-15 lists the PMO requirements for Grade A milk (as adopted by state and local 

governments). As milk from dairy clones would be subject to the same requirements as 

that from conventional dairy cows, potential risks associated with subtle changes in 

immune function that might result in increased rates of mastitis, for example, would be 

 

60 http://www.ers.usda.gov/Amberwaves/June03/DataFeature/ 61 http://www.ers.usda.gov/Data/FoodConsumption/spreadsheets/nutrients.xls#Foodgroups!a1 
Chapter VI: Food Consumption Risks 253 

Animal Cloning: A Risk Assessment DRAFT

controlled by the somatic cell and bacterial load requirements of the PMO. Likewise, 

even if clones suffered more bacterial infections, and required additional treatment with 

antibiotics, existing requirements restrict the presence of antibiotic residues in Grade A 

milk, thereby ensuring that milk from clones would pose no more bacteriological or drug 

residue risk than milk from non-clone cows. 

Table VI-15: Pasteurized Milk Ordinance (PMO) Requirements for Grade A Milk Compliance 

Standard Raw Milk Pasteurized Milk and BulkShipped Heat-Treated Milk 

Temperature 

Cooled to 10o

C (50oF) or less 

within four (4) hours after the 

commencement of the first 

milking, and to 7o

C (45oF) or 

less within two (2) hours after 

the completion of milking. 

Provided that the blend 

temperature after the first and 

subsequent milkings does not 

exceed 10o

C (50oF) 

Cooled immediately to 7o

C 

(45o

F) or less and maintained 

thereat 

Bacterial limits: Standard Plate 

Count 

Individual producer milk not to 

exceed 100,000 mL prior to 

commingling with other 

producer milk. Not to exceed 

300,000 mL as commingled 

milk prior to pasteurization 

20,000 mL limit 

Coliforms ... 

Not to exceed 10 mL. Provided, 

that in case of bulk milk 

transport tank shipments, shall 

not exceed 100 mL 

Somatic cell counts Individual producer milk not to 

exceed 750,000 per mL1

... 

Drugs 

No positive results on drug 

residue detection methods as 

referenced in Section 6 of the 

PMO 

Milk must not test positive for 

any drug residues as described 

in section 6 of the PMO 

No positive results on drug 

residue detection methods as 

referenced in Section 6 of the 

PMO 

Milk must not test positive for 

any drug residues as described 

in section 6 of the PMO 

Phosphatase ... 

Less than 350 milliunits per 

liter for fluid products and 

other milk products by the 

Fluorometer or Charm ALP or 

equivalent. 

1

 Goat milk somatic cell count NTE 1,000,000 cells/mL. 

 Source: 2003 Grade A Pasteurized Milk Ordinance. 
Chapter VI: Food Consumption Risks 254 

Animal Cloning: A Risk Assessment DRAFT

Is it possible to be reasonably certain that milk from animal clones and their progeny is 

indistinguishable from that now available in commerce? The complexity of milk itself is 

one of the primary difficulties in determining whether residual non-PMO managed 

hazards exist in the milk of animal clones. Milk from cows, sheep, and goats are mixtures 

that are estimated to be composed of more than 100,000 molecules (Jenness 1988), 

whose presence and proportion is a function of both the genetics of the animal and its 

environment. Not every component in milk has been identified and characterized; thus 

determining whether animal clones are producing a hazardous substance in their milk 

although theoretically possible, is highly impractical. 

As for new components or changes in currently present but unknown and uncharacterized 

components of milk, it is unlikely that the cloning process would trigger expression of a 

novel substance that would not have independently arisen through random mutations in 

cow populations. In addition, it seems unlikely that a reprogramming error would lead to 

expression of an excess of a metabolically active protein with no adverse effects on the 

producing animal itself. This is especially true if the many nutrients that are monitored by 

the comparison scheme in Table VI-14 are within the ranges of contemporary 

comparators, and the physiological and biochemical parameters monitored in assessments 

of animal health are also within the ranges exhibited by contemporary comparators. 

All milk is subject to Nutrition Labeling Requirements promulgated by FDA’s Center for 

Food Safety and Applied Nutrition under 21 CFR 101.9. These requirements provide a 

good starting point for milk characteristics that could be used as a basis of comparison. 

Additionally, the USDA Nutrient Database for Standard Reference 

(http://www.ars.usda.gov/ba/bhnrc/ndl) compiles data from a range of scientific, 

technical, food industry, and government agency sources to arrive at “composite” values 

of key nutrients in milk from cows, sheep, and goats. Table VI-14 provides a compilation 

of the key constituents and nutrients of milk from FDA’s Nutritional Labeling 

Requirements and USDA’s Nutrient Database. 

If milk from clones and conventional animals does not materially differ in these 

constituents, it is unlikely that individuals consuming milk from animal clones will face 

increased risk(s) relative to individuals consuming milk from conventionally bred 

animals. 

ii. Meat 

For purposes of this Risk Assessment, CVM uses the term “meat” to mean “(1) The part 

of the muscle of any cattle, sheep, swine, or goats, which is skeletal 
Chapter VI: Food Consumption Risks 255 

Animal Cloning: A Risk Assessment DRAFT

or...tongue,...diaphragm,...heart, or...esophagus, with or without the accompanying and 

overlying fat, and the portions of bone, skin, sinew, nerve, and blood vessels which 

normally accompany the muscle tissue... It does not include the muscle found in the lips, 

snout, or ears....” and “(2) The product derived from the mechanical separation of the 

skeletal muscle tissue from the bones of livestock using the advances in mechanical 

meat/bone separation machinery and meat recovery systems that do not crush, grind, or 

pulverize bones, and from which the bones emerge comparable to those resulting from 

hand-deboning....” (9 CFR 301.2) 

Meat comprises a large proportion of the average American’s diet, for both cultural and 

economic reasons (meat is relatively inexpensive in the US). In 2001, total annual per 

capita consumption of beef, veal, pork, lamb and mutton on a retail weight basis was 

approximately 122 pounds, and is estimated to have been about the same for 2002. The 

species-specific breakdown is approximately 69 pounds from beef and veal, 52 pounds 

from pork, and a little over a pound for lamb and mutton (USDA-NASS Statistical 

Highlights of US Agriculture 2001 and 2002). Goat consumption tends to be centered in 

various ethnic groups, but when averaged over the US population is about a half a pound 

per capita per year (USDA). 

Meats provide a substantial portion of the nutrition in a non-vegetarian American diet. 

For example, beef provides approximately 50 percent of the total protein in a 2,000 

calorie American diet, as well as approximately a third of the daily requirement of zinc 

and vitamin B12. It provides about 20 percent of the daily requirement for selenium, 

phosphorus, and niacin, and lesser although substantial amounts (i.e., 10-15 percent) of 

daily requirements for vitamins B6, riboflavin, thiamin, and iron (USDA Nutrient 

Database for Standard Reference Release 15, 2002). 

Similar to milk, consumption of meat for millennia has taught that there are no significant 

intrinsic toxicants in meat from cattle, swine, sheep, or goats. Examples of meat allergies 

are rather rare, although they do exist. Cases of human immune-mediated allergies to the 

cattle proteins bovine serum albumin and bovine gamma globulin have been reported 

(Wuthrich et al. 1995; Han et al. 2000; Fiocchi et al. 2000; Tanabe et al. 2002). Humans 

allergic to cat serum albumin may also exhibit cross-reactivity to swine serum albumin 

(Hilger et al. 1997), in a phenomenon referred to as the pork-cat syndrome (Drouet et al. 

1994). Children exhibiting positive skin prick test to bovine serum albumin may also 

cross react with sheep serum albumin (Fiocchi et al. 2000). As is the case for all immunemediated allergic response, the individual’s susceptibility is in large part the driver for the 

response, as allergies are examples of the dysfunction of the immune response. 
Chapter VI: Food Consumption Risks 256 

Animal Cloning: A Risk Assessment DRAFT

Just as for milk, there are no chemical composition schemes that “define” beef, pork, 

mutton, or goat meat. Due to the physiological function of muscles, and their need for 

rapid perfusion and oxygenation, meat also reflects the materials circulating the blood of 

the animal prior to slaughter. Myoglobin, the major storage protein for oxygen, is found 

in high concentration in muscle tissues. Unknown numbers of other large and small 

molecules are also found in meat, whose origins can be environmental, dietary, or 

endogenous. Each of these contributes to the complex profile that is responsible for the 

distinctive tastes and smells of meats. 

The muscle tissue that makes up meat is composed of two major protein types: 

myofibrillar proteins, actin and myosin, which make up the fibers in muscle bundles, and 

connective tissue, which primarily consists of collagen and elastin. Collagen is the major 

component of gelatin, which results from the melting of collagen in the presence of hot 

water. Elastin is not greatly affected by cooking. 

Tenderness, one of the primary considerations in carcass merit, is affected by the 

interplay of the myofibrillar and connective tissue proteins, and changes over the age of 

the animal and the amount of time since slaughter. The more connective tissue there is in 

a piece of meat, the tougher it tends to be; cooking, by solubilizing the collagen, 

decreases meat toughness. Collagen levels and structure tend to change in animals as they 

age, with the amount in young animals considerably lower than in older animals. With 

age, collagen undergoes more cross-linking, rendering it more insoluble and less likely to 

dissolve during the cooking process. The amount and distribution of fat in a muscle also 

influences tenderness. Marbling, or the presence of fatty deposits within muscles, also 

affects tenderness by functioning as a “lubricant” on the teeth or in the mouth, and by 

leaving “pockets” between muscle bundles as it melts during cooking. Changes in the 

amount of collagen or fat in the animal may affect meat quality with respect to tenderness 

or other qualities, but these would not pose nutritional or other food consumption risks. 

Further, it is likely that beef cattle clones will have changes in the amount or nature of 

marbling relative to average conventional beef cattle, as breeders will select animals as 

nuclear donors that have carcass qualities producing more uniformly tender and tasty 

meat. Similar selection procedures are being applied to animals used in conventional 

animal breeding programs, so the effect of cloning would be to speed the rate at which 

these desirable traits are introduced into breeding and production herds. 

When an animal is slaughtered, rigor mortis (muscle stiffening observed after death) 

causes stable cross-links to form in muscle fibers due to the free flow of calcium across 

the cell membranes. The carcass stiffens and lactic acid levels accumulate resulting in a 

decrease in pH. The net result is that muscle fibers contract, and the meat appears 
Chapter VI: Food Consumption Risks 257 

Animal Cloning: A Risk Assessment DRAFT

“tough.” As the meat ages, however, a set of enzymes called calpains (calcium activated 

proteases) break down some of the structural components of the muscle, relieving the 

contraction, and degrading the connective tissue proteins, also releasing the degree to 

which the muscles are held together. Calpains are thought to function in concert with 

their antagonistic regulators, calpastatins, such that if calpastatin levels are high, calpain 

activity will be inhibited and less post-mortem degradation will occur, resulting in 

tougher meat. Most processors age beef for a minimum of 14 days to allow sufficient 

time for the calpains to work. Changes in levels of either calpains or calpastatins may 

thus affect meat tenderness, but likely would not pose food consumption risks. 

 

As is the case for milk, the question of the appropriate comparator for meats may be 

approached from two perspectives. In order to determine whether cloning results in 

potential food consumption hazards relative to close genetic relatives, comparisons could 

be made to animals that are matched as closely as possible by age, husbandry (including 

diet), and environment. The second approach compares meat samples from animal clones 

more broadly to the national herds by using composite data sources. 

Unlike milk, however, meat consists of various cuts that although made up mostly of 

muscle, contain different minor tissues, and whose function may affect composition. For 

example, the muscle in loin cuts may differ in composition from the muscle used to make 

bacon (i.e., belly muscles). In order to provide the most useful data for purposes of 

determining similarity to conventionally bred animals, it would be useful to compare cuts 

from each species that have the following characteristics: 

• High US consumption levels (e.g., loin, rib, shoulder roasts, pork bellies, lamb or 

mutton shoulder or leg), and 

• Cuts of different muscularity that may have different overall compositions (e.g., if 

one tissue is lean, another may be fatty). 

USDA’s Nutrient Database (http://www.ars.usda.gov/ba/bhnrc/ndl) contains composite 

tables that provide chemical compositions of several cuts of beef, pork, and lamb meats. 

Goat meat composition is only available as a single source. 

There are no full chemical characterizations for meats. Moreover, as the definition of 

meat actually contains several tissue types, and each varies according to the genetics, 

breed, species, and environment of the food animal, it is unlikely that “complete” 

characterizations will ever be developed. The USDA requires nutritional labeling on 

“mixed” pork and beef products, and allows the voluntary labeling of raw products 

(9 CFR 317.300). Included in the labeling are calories, calories from fat, total fat, 
Chapter VI: Food Consumption Risks 258 

Animal Cloning: A Risk Assessment DRAFT

saturated fat, cholesterol, sodium protein and iron. Because meats are declared not to be a 

significant source of total carbohydrate, dietary fiber, sugars, vitamins A and C, and 

calcium, USDA does not require labeling information on them. 

c. Characterization of Milk from Cow Clones 

i. Peer-reviewed Reports 

Walsh et al. (2003) evaluated the milk produced from 15 dairy cow clones from five 

different donor cell lines and three different breeds. These animals were produced by 

Infigen, Inc., and they have been described by Forsberg et al. (2002), reviewed in the 

Critical Biological Systems Section earlier in this Chapter. Clones were bred by AI 

between 14 to 16 months of age; the paper does not specify whether all of the heifers 

were inseminated with semen from the same bull. Five different cell lines were used as 

donors to generate the clones, and the breeds represented by the cell lines included two 

Holsteins, and two cell lines derived from cows resulting from crossbreeding Jersey and 

Holstein cattle. 

 

Comparator cows were housed at different farms from the clones, but were 

approximately age and lactation-stage matched. They consisted of five Holsteins living 

on one farm, and one Brown Swiss cow raised at a farm different from the clones or the 

comparator Holsteins. Because of the different rearing sites, clones and their comparators 

were fed different rations, and for the clones, the ration was changed during the course of 

the lactation. Each cow was lactating for at least 30 days prior to sample collection, and 

samples were collected at approximately two month intervals over the entire lactation 

cycle. 

Cows were milked into individual buckets, the contents of the bucket mixed and 

distributed into various vessels appropriate to each analysis. Samples were coded at the 

collection site, although the coding was broken approximately half-way through the study 

for unspecified reasons. The milk components that were analyzed are found in Table VI16. 
Chapter VI: Food Consumption Risks 259 

Animal Cloning: A Risk Assessment DRAFT

Table VI-16: Milk Components Analyzed 

(by Walsh et al. 2003)

• Total fat 

• Lactose 

• pH 

• Nitrogen 

• Solids 

• Somatic Cell Count62

• Elements including: sodium, calcium, sulfur, 

potassium, zinc, iron, strontium, and phosphorus 

• Fatty acids including: C 4:0, 6:0, 8:0, 10:0, 12:0, 

14:0, 14:1, 16:0, 18:0, 18:1, 18:2, 18:3, and 20:063

• Milk proteins including: total protein, caseins (αs, β, 

and κ subtypes), β-lactoglobulin, α-lactalbumin, and 

immunoglobulin fraction, and a category entitled 

“other proteins.” 

• Acid Degree Value64

 

Mastitis is an infection of the udder, and a common problem in dairy cattle, that 

characteristically causes an increase in the number of somatic cells (cells from the 

circulation) in the milk of affected cows. The somatic cell count of the milk from both 

clones and non-clones indicated that none of the milk being sampled came from cows 

with mastitis. This implies that the immune function of the clones was sufficient to ward 

off infection under the husbandry conditions that the cows experienced. (A similar lack of 

impact on somatic cell count was reported by Heyman et al (2004) for milk from 50 clone 

cows compared to milk from 68 contemporary non-clone controls). The pH of the milk 

from the clones was within the range of healthy cows (~6.5-6.8). Acid degree values, 

which indicate rancidity or off-flavors, were also within the normal range for fresh milk. 

No significant differences were noted between clones and non-clones with respect to the 

concentrations of the individual milk proteins that were sampled. No significant 

differences (p> 0.05) were observed when the gross composition of milk from Holstein 

 62 Somatic Cell Count is a measure of milk quality, and is derived from counting the number of epithelial 

cells (normally shed cells) and leukocytes (white blood cells that fight infection). Both cell types are 

normally present in milk at low levels. High levels are likely caused by mastitis, which is an 

inflammation of the udder, usually caused by a bacterial infection. The Pasteurized Milk Ordinance 

sets a ceiling for somatic cell counts in milk of dairy animals. 63 The International Commission on Biochemical Nomenclature has accepted the following method for 

fatty acid nomenclature. The number before the colon represents the number of carbon atoms, and the 

number following the colon represents the number of double bonds in the carbon chain. For example, 

linoleic acid (or cis-9, cis-12-octadecadienoic acid) is named 18:2; it has 18 carbon atoms, 2 double 

bonds. 64 The Acid Degree Value helps to predict off-flavors in milk that arise from the breakdown of fat by an 

enzyme called lipase. High values of certain free fatty acids can make milk taste rancid. Pasteurization 

inactivates many lipases, but the acid degree value may still rise slowly during long storage. 
Chapter VI: Food Consumption Risks 260 

Animal Cloning: A Risk Assessment DRAFT

clones and Holstein non-clones was compared over the course of the entire lactation 

cycle (Table VI-17). 

No significant differences were reported between milk from clones and sexuallyreproduced cows with respect to the individual milk proteins that were assayed, although 

difference in the concentrations of αs-casein, κ-casein, and α-lactalbumin were noted 

over the course of the lactation. 

Table VI-17: Comparison of Gross Characteristics of Milk from Clones and Non-Clones 

(from Walsh et al. 2003)

Clone 

BrSw1 

NonClone 

BrSw 

BrSw 

Lit 

Value2 

Clone 

Hlstn3

1

Clone 

Hlstn 

2 

NonClone 

Hlstn 

Hlstn 

Lit 

Value 

Clone 

H X 

Jersey

5 

1 

Clone 

H X 

Jersey

5 

2 

Animals/ 

Samples 1/5 1/5 NP6 1/5 11/63 5/26 NP 1/5 1/5 

Solids (%) 13.4 

± 0.77 

13.5 

± 0.7 13.3 12.6 

± 1.0 

12.9 

± 1.1 

12.9 

± 1.4 12.3 12.9 

± 0.9 

13.5 

± 0.5 

Fat (%) 4.3 

± 0.9 

4.5 

±1.0 4.1 3.8 ± 0.9 

3.9 

± 1.3 

4.3 

± 1.2 3.6 4.1 ± 1.3 

4.7 

± 0.5 

Protein 

(%) 

3.6 

± 0.2 

3.2 

± 0.12 3.6 3.0 ± 0.1 

3.0 

± 0.1 

3.1 

± 0.2 3.3 3.2 ± 0.2 

2.9 

± 0.1 

Lactose 

(%) 

5.3 

± 0.5 

5.3 

± 0.4 5.0 5.0 

± 0.1 

5.0 

± 0.1 

4.9 

± 0.15 4.9 4.9 

± 0.02 

5.0 

± 0.1 

1 BrSw = Brown Swiss 

2 Lit Value = published literature value cited by Walsh et al. (2003) 

3 Hlstn = Holstein 

5 H X Jersey = a cross between a Holstein and Jersey, referring to the source of the animal that provided the 

donor cell for SCNT. 

6

 NP = not provided 

7 Values are presented as means ± standard deviation. 

For 12 of the 14 fatty acids analyzed, no significant differences were noted between clone 

and non-clone milk. Significant differences (p<0.05) were noted in the amount of 

palmitic acid (C16:0) and linolenic acid (C18:3) between clones and non-clones. The 

authors noted that the palmitic and linolenic acid levels for both clone and non-clone milk 

fall within published references for that substance, and speculated that difference between 

the levels in clones and non-clones could be attributed to diet. Differences were observed 

in the fatty acid profiles of the milks over the course of the lactation cycle. These were 

noted as being consistent with published accounts of lactation cycle differences, diet, and 

seasonality. The greatest variability was observed in the mineral content of milk from 

clones and non-clones, with significant differences noted for potassium, zinc, strontium, 
Chapter VI: Food Consumption Risks 261 

Animal Cloning: A Risk Assessment DRAFT

and phosphorus levels. The authors attribute these differences to the different diets that 

clones and non-clones were fed. (Clones and comparators were housed at different farms, 

and fed different rations.) The authors’ overall conclusion was that there were “no 

obvious differences between milk from clones and non-clones.”

In an abstract, Aoki et al. (2003) described the generation of two clones from cells 

derived from the colostrum of a Holstein cow, as well as providing summary comments 

regarding milk characteristics and milking behavior. According to the abstract, milk yield 

(measured in kilograms per week) was measured every four weeks over a 16 week 

period. They noted that significant differences were observed between milk yield at 

weeks 1, 9, 11, and 13, but in the other weeks, “they shared similar lactation curves.” 

Milk composition was apparently measured as milk fat, protein, lactose, solids-non-fat, 

and total solid percentages. The authors reported that there were “considerable 

resemblance[s]” between the milk of clones and non-clones. It should be noted, however, 

that the measurements in this study were made between clones in their first lactation, and 

comparators in their second lactation. There are often differences in milk yield and 

composition between successive lactations (Vasconcelos et al. 2004; Flis and Wattiaux 

2005). 

The laboratories at the University of Connecticut continued their surveillance of a set of 

Holstein clones (see CBSA section) by analyzing the composition of milk from clones 

(the composition of meat from Japanese Black clones is discussed in the Meat 

Composition section) (Tian et al. 2005). Ten dairy clones were produced through SCNT 

using skin fibroblast (n=4) or cumulus cells (n=6) of a 13 year old Holstein cow. Four of 

the surviving cumulus cell derived clones were compared with four age- and paritymatched comparator heifers. All animals were raised at the same facility from 2 months 

of age, with the same management and feeding. Both groups were bred by artificial 

insemination using semen from the same bull at 14-15 months of age. 

Milk production was monitored starting immediately after calving through 305 days of 

lactation; milk samples were collected three times daily. Two milk samples were 

collected from each of three milkings on a given day of each week throughout the entire 

first lactation. One of the collected samples was used for the analysis of total protein, 

total fat, lactose, total solids, milk urea nitrogen, and somatic cell counts as routinely 

monitored by the Dairy Herd Improvement Association (DHIA) at a DHIA designated 

laboratory. Individual fatty acids that were measured included C4:0, 6:0, 8:0, 10:0, 12:0, 

14:0, 14:1, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, and 20:0. The second collected sample was 

analyzed for protein profiles using denaturing SDS/PAGE (sodium dodecyl (lauryl) 

sulfate-polyacrylamide gel electrophoresis) stained with Comaisse blue. Relative 
Chapter VI: Food Consumption Risks 262 

Animal Cloning: A Risk Assessment DRAFT

quantities of each band were determined. Antibody concentrations (IgM, IgA, and IgG) 

were determined in colostrums from the first milking with a commercial assay. 

The investigators report that there were no significant differences between the 

composition of milk from clone and age-matched, closely related comparators, or breed 

comparators. Clones and comparators also showed comparable lactation curves, with 

milk production increasing during the first month, and decreasing thereafter during the 

course of the lactation. The exception was one clone that birthed prematurely, and 

produced 30 percent less milk, as would be expected. Analysis of key milk proteins 

indicated that there were no differences among major or minor bands as analyzed by 

SDS/PAGE. The four major bands representing α-caseins, β-caseins, κ-caseins, and βlactoglobulins were consistent in all milk samples whether from clones or their 

comparators. Similarly, there was no difference between groups for minor protein bands. 

Antibody concentrations in colostrums were also similar between clone and non-clone 

cows, and reported to be in the typical range for colostrum antibody composition. 

Yonai et al. (2005) (see previous discussions of these animals in the Developmental 

Nodes) presented milk composition data for six Holstein and four Jersey clone cows. 

Overall milk yield, fat, protein, and other solids not fat (SNF) were considered to be 

normal by the authors, with the observed inter-clone differences and differences from the 

donor animals attributed to diet and environmental conditions. The authors note that the 

heritability of milk production is approximately 30 percent and, considering the impact of 

environmental conditions on milk production, suggest that standardizing individual 

feeding conditions may be helpful for future comparisons. 
Chapter VI: Food Consumption Risks 263 

Animal Cloning: A Risk Assessment DRAFT

Table VI-18: Milk Composition (mean ± standard deviation) in First and Second Lactations 

(adapted from Yonai et al. 2005) 

 Milk Yield Fat (%) Fat (kg) Protein 

(%) 

Protein 

(kg) 

SNF*

(%) 

SNF 

(kg) 

Jersey (n=4) 

First 

lactation 5,896.4 ± 332.0 5.0 ± 0.2 300.3 ±

23.9 3.8 ± 0.2 225.5 ±20.4 9.4 ± 0.2 560.0 ±47.8 

Donor 

animal 5,064.0 4.9 242.3 4.0 197.1 9.6 477.2 

Second 

lactation 7,262.8 ± 222.6 5.13 ±

0.13 

375.3 ±

26.2 

3.78 ±

0.10 

274.8 ±

13.4 

9.35 ±

0.10 681.3 

Donor 

animal 6,087.0 4.6 280 3.67 224 9.30 566 

Holstein (n=6) 

First 

lactation 9,333.0 ± 476.4 4.7 ± 0.1 440.3 ±

36.7 3.3 ± 0.1 304.2 ±27.6 9.0 ± 0.1 835.5 

Donor 

animal 10,968.0 4.1 452.0 3.3 359.0 — — 

Second 

lactation

11,271.4 ±

1084.7 4.5 ± 0.2 510.5 ±53.4 3.1 ± 0.1 353.5 ±31.4 8.7 ±0.1 978.7 

Donor 

animal 11,442.0 3.9 446.2 2.8 320.4 — — 

* SNF is milk solids not fat 
Chapter VI: Food Consumption Risks 264 

Animal Cloning: A Risk Assessment DRAFT

ii. The Report of the Japanese Research Institute for Animal Science in 

Biochemistry and Toxicology 

The Japanese Research Institute for Animal Science in Biochemistry and Toxicology 

provided a report entitled “Investigation on the Attributes of Cloned Bovine Products” 

published by the Japan Livestock Technology Association (Japan 2002). CVM was able 

to obtain a seven page English-language summary translation of the original 489 page 

Japanese report. Only the English-language summary is reviewed in this risk assessment. 

The study investigated blood, milk, and meat constituents in blastomere nuclear transfer 

clones (BNT) and SCNT cattle clones. In addition, the results of rodent feeding studies 

conducted with edible products derived from the cattle clones are reported. The results 

for milk are discussed in this section; results for meat are discussed in the section 

Compositional Data on Meat from Clones. No information was provided on the 

production of the BNT or SCNT clone cattle in the English translation, and the 

comparator group was identified as “ordinary cattle.” 

Milk constituents were compared between ordinary cattle, BNT clones, and SCNT 

clones. The results are reported as the mean of samples obtained three and six weeks after 

parturition and provided in Table VI-17. No biologically significant differences were 

observed between any of the groups for the parameters tested. 

Table VI-19: Milk Constituents in BNT and SCNT Clones and Ordinary Cattle 

(from Japan 2002) 

Classification Cattle 

No. 

Protein Fats Sugars Ash 

content 

Water 

content Calcium Cholesterol 

 g/100 g mg/100 g 

Min. 

value 

Max 

value 

3.0 

3.4 

2.2 

3.3 

4.6 

4.6 

0.7 

0.7 

88.1 

89.7 

100 

110 

8 

10 Ordinary 

cattle 

Mean 

value 3.3 2.7 4.6 0.7 88.9 105 9 

BNT clones No.1 

No.2 

2.9 

2.9 

2.3 

3.6 

3.0 

3.5 

0.8 

0.7 

91.1 

89.3 

95 

105 

9 

9 

SCNT clones 

No 1 

No.2 

No.3 

3.1 

3.3 

3.3 

4.3 

2.6 

3.1 

4.6 

4.4 

4.5 

0.7 

0.7 

0.7 

87.4 

89.1 

88.5 

120 

115 

115 

9 

11 

10 
Chapter VI: Food Consumption Risks 265 

Animal Cloning: A Risk Assessment DRAFT

Milk from these cow clones was tested for allergenic potential. The ability to digest a 

protein is one index of potential allergenicity; a protein that is less able to be digested 

may be more likely to provide an allergenic response. The protein digestion rate of 

freeze-dried milk combined in feed consumed by rats is reported below for milk obtained 

from ordinary cattle, BNT clone cattle, and SCNT clone cattle. The authors report that 

there was no biological difference among the groups tested. 

Table VI-20: Protein Digestion of Freeze Dried Milk from BNT and SCNT Clones and 

Ordinary Cattle 

(from Japan 2002) 

Test Group Number of Animals Digestion Rate 

(mean ± standard deviation) 

Ordinary Cattle 5 83.0 ± 2.6 

BNT clone cattle 5 82.7 ± 2.0 

SCNT clone cattle 5 8.13 ± 3.4 

In a separate study, mice were sensitized by intraperitoneal injection to extracts of milk 

from clone and non-clone cows. Fourteen days later, the abdominal wall of the mice was 

surgically retracted and an allergic reaction induced by re-injection of the freeze-dried 

milk extract into the abdominal wall. Control mice did not receive the second injection of 

milk extract. Allergenic response was assessed based on vascular permeability as 

measured by the diameter of dye leakage from the site of injection. No statistically 

significant differences in allergenic activity were reported between groups. The data are 

presented in Table VI-21. 

Table VI-21: Allergenic Response to Milk from BNT and SCNT Clones and Ordinary Cattle 

(from Japan 2002) 

Test Group Mouse Group Number of 

Animals 

Diameter of dye leakage (mm) 

(mean ± standard deviation) 

Ordinary Cattle Control Group 

Test Group 

7 

10 

7.0 ± 3.7 

18.0 ± 2.9 

BNT clone cattle Control Group 

Test Group 

7 

10 

4.7 ± 3.2 

18.0 ± 3.9 

SCNT clone cattle Control Group 

Test Group 

7 

10 

4.9 ± 4.6 

17.9 ± 4.2 

Based on these two studies, the authors conclude that there were no biologically or 

statistically significant differences in the allergenic potential of milk from ordinary cattle 

or BNT or SCNT clones. 
Chapter VI: Food Consumption Risks 266 

Animal Cloning: A Risk Assessment DRAFT

In addition to the composition and allergenicity studies, the Japanese Research Institute 

for Animal Science in Biochemistry and Toxicology performed a 28-day rodent feeding 

study.65 Rats were fed diets containing freeze dried milk from clones and ordinary cattle 

at concentrations of 0, 2.5, 5, or 10 percent of the diet for 14 weeks. General signs, body 

weight, food consumption, urinalysis, sensory and reflex function, spontaneous 

movement frequency, general function, reproductive cycle, hematology at autopsy, blood 

chemistry, organ weights, pathology and histopathology were compared between groups. 

English-language summary tables were provided in the original Japanese-language 

report; the summary tables have been provided in Appendix G. No biologically 

significant differences were reported in rats fed milk from clones compared to rats fed 

milk from ordinary cattle. In addition, it is noted that 10 cattle fed clone milk powder at 

2.5, 5, or 10 percent of the diet showed no significant differences in body weight 

increase, indicating that the milk did not contain anti-nutrients or other toxicants to cattle. 

The duration of exposure is not reported. 

Finally, the potential for milk from BNT and SCNT clone cattle to cause clastogenic66

(DNA breaking) events was assessed using an in vivo mouse micronucleus assay. Mice 

were fed milk from ordinary cattle, BNT clone cattle, or SCNT clone cattle at 0, 2.5, 5, or 

10 percent of the diet for 14 days. In addition, a positive control group received a single 

intraperitoneal injection of 2 mg/kg mitomycin C, a known clastogen. The positive 

control group showed a statistically significant increase in the incidence of micronuclei 

appearance and polychromatic erythrocyte rate, and was considered a positive test. No 

milk-fed group, whether derived from ordinary or clone cattle, was positive in this assay. 

The report concludes that there were no biologically significant differences in the 

component analysis or the results of feeding milk from ordinary cattle, BNT clones, and 

SCNT clones. 

iii. Additional Data 

 

65 Animal feeding studies to examine the toxicity of specific components of materials contained in foods 

are significant elements of a toxicity assessment. It is, however, generally recognized that animal feeding 

studies to examine the toxicological effects of whole foods (i.e., feeding the whole food from a clone to the 

toxicology test animal) are of limited value due to the complex nature of the whole food, inability to 

provide sufficiently high doses of minor components of the whole food, and limited sensitivity of the assay. 

(Kessler, DA, MR Taylor, JH Maryanski, EL Flamm and LS Kahl. 1992. The Safety of Foods Developed 

by Biotechnology. Science. 256:1747-1832; Codex Alimentarius. 2003. Guideline for the Conduct of Food 

Safety Assessments of Foods Derived From Recombinant-DNA Plants. CAC/GL 45-2003. 

66 Clastogens are often referred to as mutagens, as most DNA breaks result in mutations if they do not first 

kill the cell or organism. The mouse micronucleus test examines the ability of a substance to cause the 

chromosomes of precursors of red blood cells in mice to break. Because mammalian red blood cells lose 

their nuclei as they mature, if any DNA is left in the mature blood cells, it is due to pieces of chromosomes 

breaking away from the rest of the nucleus as it is extruded from the immature blood cell during its 

maturation. 
Chapter VI: Food Consumption Risks 267 

Animal Cloning: A Risk Assessment DRAFT

In a preliminary report of the nutritional contribution of food from cattle clones, Tome et 

al. (2004) found no differences in the response of rats receiving meat and milk from 

clones or non-clone cattle. Rats were fed milk or meat from clones or controls for three 

weeks. Outcomes evaluated included food intake, body weight gain, body composition, 

and fasting insulin levels. In addition, no differences were detected for IgG, IgA, and 

IgM subtypes for rats receiving clone or non-clone derived diets. Further, no specific 

anti-milk or meat protein IgE responses were detected in rat sera. The authors conclude 

that there are no major differences in the nutritional value of milk or meat derived from 

clone or non-clone animals, and suggest that this study be confirmed in longer term 

exposure studies. 

Wells et al (2004) briefly reported on the composition of milk from six 2-year old 

Friesian cow clones in their first lactation. The milk composition was compared to that of 

the single donor cow in her third lactation. All animals were managed together as part of 

a single dairy herd. Variables measured are presented in Table VI-22. 

Table VI-22: Milk composition variables measured in clone Friesian cows and the non-clone donor 

(from Wells et al. 2004) 

Fat Fatty acids C18:0 

Protein C4:0 C18:1 

Lactose C6:0 C18:2 

αs-casein C8:0 C18:2 conjugated linoleic acid 

β-casein C10:0 C18:3 

κ-casein C12:0 Solid Fat Content 

α-lactalbumin C14:0 Magnesium 

β-lactalbumin C16:0 Calcium 

BSA C16:1 Sodium 

IgG C17:0 Potassium 

The comparison was made based on a single milk sample take at mid lactation. Although 

one of the protein levels (bovine serum albumin (BSA (4.52 ± 0.10 vs. 4.48)) and two of 

the fatty acids in the clones (C18:2 (3.76 ± 0.06 vs. 3.00), C18:3 (1.18 ± 0.07 vs. 0.90) 

were found to be statistically different (p>0.05) from the donor cow’s milk, they were 

reported to be within normal limits for this breed of cow, and not considered by the 

authors to be biologically significant. The authors conclude that overall milk composition 

of the clones was what might be expected for healthy cows. 

In a presentation at the January 2005 31st IETS Annual Meeting, the same group 

(Reproductive Technologies Group, AgResearch Ltd, New Zealand (Lee and Wells 2005) 
Chapter VI: Food Consumption Risks 268 

Animal Cloning: A Risk Assessment DRAFT

presented data on AgResearch’s experience with cattle cloning from 1997 onwards. Milk 

composition was evaluated in three clone cows each from three clonal families (a clonal 

family is derived from the same source animal). Milk from a total of six SCNT clones 

(selection not specified) was compared to milk from a single donor cow (also not 

specified) (Table VI-23). In this preliminary communication of data, the investigators 

noted statistically significant differences for only BSA (162 ± 6 vs. 105 mg/L), and two 

fatty acids C18:2 (3.76 ± 0.06 vs. 3.00 percent of total) and C18:3 (1.19 ± 0.03 vs. 0.90 

percent of total). They concluded that the composition of milk from clones was normal. 

Table VI-23: Comparison of Milk Composition in of Milk from Clones and the Single Donor Cow 

(from Lee and Wells 2005)

Component of Milk 

(g/kg milk unless otherwise stated) Clones (n=6) Donor Cow 

Fat 35.1 ± 1.00 36.2 

Protein 31.2 ± 0.36 31.5 

Lactose 50.1 ± 0.36 51.7 

αs-casein 10.8 ± 0.17 10.6 

β-casein 8.98 ± 0.15 9.13 

κ-casein 2.94 ± 0.09 2.76 

α-lactalbumin 0.99 ± 0.12 1.37 

β-lactalbumin 4.52 ± 0.10 4.48 

BSA (mg/L) 162 ± 6 105 

IgG (mg/L) 563 ± 13 536 

C4:0 4.59 ± 0.02 4.61 

C6:0 2.45 ± 0.07 2.48 

C8:0 1.32 ± 0.03 1.37 

C10:0 2.14 ± 0.22 2.36 

C12:0 2.32 ± 0.15 2.30 

C14:0 8.79 ± 0.30 8.95 

C16:0 25.9 ± 0.55 25.5 

C16:1 1.01 ± 0.07 1.38 

C17:0 1.77 ± 0.06 1.62 

C18:0 12.4 ± 0.26 11.3 

C18:1 26.5 ± 1.06 28.6 

C18:2 3.76 ± 0.06 3.00 

C18:2 CLA 1.18 ± 0.07 1.05 

Fatty acids 

(% of total) 

C18:3 1.19 ± 0.03 0.90 

SFC at 10°C (g/kg fat) 520 ± 15.8 477 

Magnesium (mg/100g) 9.3 ± 0.23 10.2 

Calcium (mg/100g) 124 ± 1.8 128 

Sodium (mg/100g) 33 ± 0.6 33 

Potassium (mg/100g) 149 ± 1.5 152 
Chapter VI: Food Consumption Risks 269 

Animal Cloning: A Risk Assessment DRAFT

In 2004 through 2005, milk vitamin composition was compared for 3 clone cows each 

from 3 clonal families (n=9) to control non-clone cows (Table VI-24). Subsequently, 

Wells published a summary of a slightly expanded version of these data (Wells 2005) in a 

scientific journal (Tables VI-25 and VI-26). No details were provided for the comparator 

animals. No differences were reported in selected vitamins in milk. Wells concluded that 

the composition from these clones was within the normal range for milk. 

Table VI-24: Vitamin Composition of Bovine Whole Milk Harvested In Spring 

(from Wells 2005) 

Vitamin Units Clone milk (n = 9) Comparator milk (n = 5) 

A IU/100 ml 128 ± 22 140 ± 29 

B2 mg/100 ml 0.27 ± 0.03 0.24 ±0.04 

B12 μg/100 g 0.40 ±0.09 0.20 ± 0.07 

 

Table VI-25: Mineral Composition of Bovine Whole Milks Harvested In Spring 

(from Wells 2005)

Mineral 

(mg/100g) 

Milk from Clones (n = 9) 

Mean ± Standard Deviation 

Milk from Comparators (n = 5) 

Mean ± Standard Deviation 

Calcium 133.0 ± 15.7 134.4 ± 10.1 

Iodine 0.0010 ±0.0005 0.0022 ±0.0009 

Magnesium 10.1 ± 1.5 10.0 ± 0.0 

Phosphorus 115.2 ± 12.5 103.6 ± 5.3 

Potassium 129.9 ± 13.9 125.8 ± 15.1 

Selenium 0.0005 ± 0.0 0.0008 ± 0.0004 

Sodium 27.0 ± 5.1 26.8 ± 5.0 

Zinc 0.495 ± 0.768 0.515 ± 0.077 

 
Chapter VI: Food Consumption Risks 270 

Animal Cloning: A Risk Assessment DRAFT

Table VI-26: Amino Acid Composition of Bovine Skim Milk Harvested In Spring 

(from Wells 2005)

Amino acid 

(mg/g) 

Milk from Clones (n = 9) 

Mean ± Standard Deviation 

Milk from Comparators (n = 5) 

Mean ± Standard Deviation 

Alanine 1.31 ± 0.17 1.31 ± 0.14 

Arginine 1.32 ± 0.20 1.33 ± 0.12 

Aspartic acid 3.07 ± 0.40 3.02 ± 0.26 

Cystine 0.36 ± 0.05 0.38 ±0.04 

Glutamic acid 8.78 ± 1.16 8.65 ±0.70 

Glycine 0.74 ± 0.11 0.75 ± 0.07 

Histidine 1.02 ±0.14 1.01 ±0.07 

Isoleucine 1.82 ±0.28 1.76 ±0.16 

Leucine 3.83 ±0.51 3.75 ±0.30 

Lysine 3.22 ± 0.45 3.16 ±0.26 

Methioine 0.89 ±0.12 0.88 ±0.09 

Phenylalanine 1.85 ±0.26 1.83 ±0.15 

Proline 3.87 ±0.53 3.80 ±0.33 

Serine 2.19 ±0.30 2.17 ±0.19 

Threonine 1.78 ±0.25 1.76 ±0.18 

Tryptophan 0.48 ±0.08 0.48 ± 0.06 

Tyrosine 1.81 ±0.27 1.80 ±0.17 

Valine 2.15 ± 0.32 2.08 ± 0.17 

Totals 40 ± 5.57 39.94 ± 3.40 

 

iv. Summary Statement on Composition of Milk from Clones 

Based on the available data, milk from cow clones does not appear to differ significantly 

in composition from milk from non-clones. Small differences have been noted between 

clones and comparators, but given the different diets and husbandry conditions of these 

animals, it is difficult to determine with certainty whether the small changes seen in some 

components were a function of the diet, handling, or related to cloning. In summary, none 

of the small reported differences in any of the studies indicate any concern for food 

safety. 
Chapter VI: Food Consumption Risks 271 

Animal Cloning: A Risk Assessment DRAFT

d. Characterization of Meat from Clones and Their Progeny 

i. Cattle 

Two linked reports on carcass merit67 (e.g., dressing percentage, fat depth, rib-eye area, 

yield and quality grade) of cattle produced via BNT have been published (Diles et al. 

1996a,b). Neither paper addresses food safety issues. Both papers evaluate the degree to 

which body measurements are heritable (Diles et al. 1996a), and the degree to which 

there is phenotypic variability among clones and closely related siblings. The studies 

conclude that animals derived from BNT provide good models for determining which 

traits have strong genetic correlations. 

As discussed in the section on milk composition, the Japanese Research Institute for 

Animal Science in Biochemistry and Toxicology provided an unpublished bound report 

“Investigation on the Attributes of Cloned Bovine Products” by the Japan Livestock 

Technology Association (Japan 2002).68 The results for meat are discussed in this section. 

Takahashi and Ito (2004) have published a summary of these data, including some 

information characterizing the clones and their comparators. SCNT and BNT clones were 

derived from Japanese Black cattle at the Para Prefectural Animal Research Center. 

comparator animals were selected as conventionally bred Japanese Black cattle. All 

animals used for compositional analysis were sacrificed between 27 and 28 months of 

age, after fattening. For the in vitro digestion test, samples were taken from a one-day old 

conventional calf and a four day old clone. 

Meat constituents were compared between ordinary cattle, BNT clone cattle, and SCNT 

clone cattle. The results are reported as the mean of analytical samples obtained from 9 

sites; shoulder, chuck loin, rib loin, loin end, brisket, round, silver side, rump, and tender 

loin, and are provided in Table VI-27: 

 67 Carcass merit programs have been initiated by a group of academics and beef producers to correlate 

bovine genetics and phenotypic markers for consumer-desired traits such as marbling, tenderness, and 

composition. Because consumers desire consistency in meat products, producers demonstrating that 

their herds have good performance and carcass data can leverage higher market prices for their beef. 

Identification of genetically determined traits can also lead to selective breeding programs that 

improve herd meat quality in a directed manner. 

68 Some of these data are presented in Takahashi and Ito 2004; however, we have cited the original report 

as the data reporting is more complete. 
Chapter VI: Food Consumption Risks 272 

Animal Cloning: A Risk Assessment DRAFT

Table VI-27: Meat Constituents in BNT and SCNT Clones and Ordinary Cattle 

(from Japan 2002) 

Classification Cattle No. Protein 

(g/100 g) 

Fats 

(g/100 g) 

Sugars 

(g/100 g) 

Ash 

content 

(g/100 g) 

Water 

content 

(g/100 g) 

Cholesterol 

(mg/100 g) 

Min. value 

Max value 

17.8 

19.6 

13.8 

22.9 

0.4 

0.8 

0.9 

1.0 

58.0 

64.8 

50 

Ordinary 68 

cattle 

Mean value 18.4 19.3 0.6 0.9 60.8 59 

BNT clones 17.4 21.2 0.4 0.9 60.2 56 

SCNT clones 16.8 23.8 0.5 0.9 57.9 68 

No biologically significant differences were observed between any of the groups of cattle 

(ordinary cattle, BNT clone cattle, and SCNT clone cattle) for the parameters tested 

Meat from clone cows was tested for allergenic potential by comparing protein digestion 

rates with artificial digestive juice and in a rat model, and by looking for an allergenic 

response following direct challenge in rats. The rates of digestion by artificial digestive 

juices (artificial gastric juice and artificial intestinal juice) were compared for freeze dried 

meat derived from ordinary cattle, BNT clone cattle, or SCNT clone cattle (Table VI-28). 

No information is provided in the translation regarding the artificial digestive material. 

The results are presented below as the rate of protein digestion. 

Table VI-28: Rates of in vitro Digestion of Beef from SCNT Clones or Ordinary Cattle 

(from Japan 2002) 

Rate of digestion after the start of incubation 

Digestive (per cent) 

juice Sample 

Course Start 0.75 hr 1.5 hr 3 hr 6 hr 12 hr 

Ordinary beef 0 68 79 - 95 90 Artificial 

gastric 

juice Somatic cloned beef 0 59 78 - 91 90 

Ordinary beef 0 - 20 40 66 67 Artificial 

intestinal 

juice Somatic cloned beef 0 - 28 38 67 63 

It was concluded that there were no biologically significant differences in the rates of 

digestion for meat from ordinary beef cattle or from clone beef cattle using artificial 

digestive juices. 
Chapter VI: Food Consumption Risks 273 

Animal Cloning: A Risk Assessment DRAFT

The protein digestion rate of freeze-dried meat combined in feed consumed by rats is in 

Table VI-29. The authors report that there was no biological difference among the groups 

tested. 

Table VI-29: Protein digestion rate in rats following consumption of freeze dried meat from clone 

cattle and non-clone cattle 

(from Japan 2002) 

Test Group Number of Animals Digestion Rate (mean±s.d.) 

Ordinary cattle beef 5 83.8 ± 6.6 

BNT clones 5 82.3 ± 4.7 

SCNT clones 5 84.9 ± 3.6 

In a separate study, mice were given sensitizing intraperitoneal injections of extracts of 

freeze-dried beef from clone and non-clone cows. Fourteen days later, the abdominal wall 

of the mice was surgically exposed and an allergic reaction induced by re-injection of the 

freeze-dried beef extract into the abdominal wall and administered a vascular dye. 

Control mice did not receive the second injection of beef extract and only were 

administered the dye. Allergenic response was assessed based on vascular permeability as 

measured by the diameter of dye leakage. No statistically significant difference in 

allergenic activity was reported between groups. The data are presented in Table VI-30. 

Table VI-30: Allergenic response by mice to intraperitoneal injection of extracts of freeze-dried beef 

from BNT and SCNT cloned cattle and ordinary cattle 

(from Japan 2002)

Test Group Mouse Group Number of 

Animals 

Diameter of dye leakage (mm) 

(mean±s.d.) 

Ordinary cattle Control group 

Test group 

7 

10 

5.3 ± 5.0 

13.0 ± 5.9 

BNT clones Control group 

Test group 

7 

10 

7.0 ± 4.9 

12.5 ± 3.5 

SCNT clones Control group 

Test group 

7 

10 

5.7 ± 4.2 

13.1 ± 5.0 

The authors conclude that there were no biologically or statistically significant 

differences in the allergenic potential of milk from ordinary cattle or BNT or SCNT clone 

cattle. 
Chapter VI: Food Consumption Risks 274 

Animal Cloning: A Risk Assessment DRAFT

An oral feeding study was conducted in rats to determine the effects of a diet containing 

meat derived from clone cattle.69 Freeze dried beef from ordinary cattle and clone cattle 

was fed to rats at concentrations of 0, 2.5, 5, or 10 percent of the diet for 14 weeks. 

General signs, body weight, food consumption, urinalysis, sensory and reflex function, 

spontaneous movement frequency, general function, reproductive cycle, hematology at 

autopsy, blood chemistry, organ weights, pathology and histopathology were compared 

between groups. English-language summary tables were provided in the original 

Japanese-language report; the summary tables have been provided in Appendix H. No 

biologically significant differences were reported compared to rats fed beef from ordinary 

cattle. In addition, it is noted that 10 ordinary cattle fed clone beef powder at 2.5, 5, or 10 

percent of the diet showed no significant differences in body weight increase. The 

duration of exposure is not reported. 

Finally, the potential for meat from BNT and SCNT clone cattle to cause mutations was 

assessed using the mouse micronucleus assay. Mice were fed freeze dried powdered beef 

from ordinary cattle, BNT clone cattle, or SCNT clone cattle at 0, 2.5, 5, or 10 percent of 

the diet for 14 days. In addition, a positive control group received a single intraperitoneal 

injection of 2 mg/kg mitomycin C. The positive control group showed a statistically 

significant increase in the incidence of micronucleus appearance and polychromatic 

erythrocyte rate, and was considered a positive test. No beef-fed group, whether derived 

from ordinary or clone cattle, caused mutations in this assay (i.e., no group fed beef 

derived from ordinary cattle or clone cattle was positive in this assay for mutagenicity or 

clastogenicity). The report concludes that there were no biologically significant 

differences in component analysis or the results of feeding meat from ordinary cattle, 

BNT clone cattle, and SCNT clone cattle. 

Cyagra, the cloning company that provided the extensive physiological data discussed 

earlier in the risk assessment, also provided meat composition data. Eleven clones (6 

female, 15 to 43 months; 5 male, 12 to 17 months) and an equal number of comparator 

cattle (over 12 months) were selected for the study. All animals were fed a standard 

ration for 30 days prior to slaughter. Samples (500 g each) were obtained of chuck arm 

roast, bottom sirloin tip roast, and short loin for analysis by an independent laboratory. 

 

69 Animal feeding studies to examine the toxicity of specific components of materials contained in foods 

are significant elements of a toxicity assessment. It is, however, generally recognized that animal feeding 

studies to examine the toxicological effects of whole foods (i.e., feeding the whole food from a clone to the 

toxicology test animal) are of limited value due to the complex nature of the whole food, inability to 

provide sufficiently high doses of minor components of the whole food, and limited sensitivity of the assay. 

(Kessler, DA, MR Taylor, JH Maryanski, EL Flamm and LS Kahl. 1992. The Safety of Foods Developed 

by Biotechnology. Science. 256:1747-1832; Codex Alimentarius. 2003. Guideline for the Conduct of Food 

Safety Assessments of Foods Derived From Recombinant-DNA Plants. CAC/GL 45-2003. 
Chapter VI: Food Consumption Risks 275 

Animal Cloning: A Risk Assessment DRAFT

No biologically significant differences are observed in the composition of meat from 

clones and comparators. The results of the compositional analysis summarized across 

gender and cuts of meat are summarized in Table VI-31. A detailed presentation of the 

results is provided in Appendix E, the Cyagra Dataset. 

Table VI-31: Meat Composition from Cyagra Clones and Comparators 

Meat Analysis Overall Comparison

Sample Number 

Marked ID Clone 

 

Comparator 

 

Analyte Units Mean Std dev Mean Std dev 

Crude Fat % 11.62 10.08 8.62 8.10 

Moisture % 66.18 7.68 68.57 5.51 

Protein – Combustion % 20.69 2.96 21.72 2.58 

Protein – Kjeldahl % 20.74 2.90 21.58 2.51 

Ash % 1.03 0.17 1.05 0.13 

Balance (protein+moist+ash+fat) % 99.56 1.72 99.82 0.89 

Amino Acid Profile (results below) 

Tryptophan % 0.25 0.03 0.26 0.03 

Aspartic Acid % 1.96 0.31 2.08 0.23 

Threonine % 0.93 0.15 1.01 0.12 

Serine % 0.79 0.14 0.86 0.12 

Glutamic Acid % 3.22 0.54 3.33 0.71 

Proline % 0.97 0.21 0.91 0.16 

Glycine % 1.08 0.27 1.08 0.21 

Alanine % 1.28 0.21 1.36 0.18 

Cystine % 0.23 0.04 0.24 0.04 

Valine % 0.89 0.21 1.07 0.14 

Methionine % 0.54 0.09 0.56 0.08 

Isoleucine % 0.81 0.20 0.98 0.12 

Leucine % 1.61 0.27 1.78 0.20 

Tyrosine % 0.69 0.11 0.74 0.08 

Phenylalanine % 0.84 0.14 0.91 0.10 

Histidine % 0.70 0.12 0.77 0.11 

Lysine, Total % 1.77 0.31 1.98 0.23 

Arginine % 1.33 0.23 1.41 0.17 

Hydroxyproline % 0.17 0.07 0.16 0.07 
Chapter VI: Food Consumption Risks 276 

Animal Cloning: A Risk Assessment DRAFT

Fatty Acid (results below) 

C14:0 Tetradecanoic (Myristic) % 0.28 0.24 0.23 0.24 

C14:1 Tetradecenoic (Myristoleic) % 0.15 0.17 0.09 0.10 

C15:0 Pentadecanoic % 0.04 0.04 0.04 0.04 

C15:1 Pentadecenoic % 0.00 0.00 0.00 0.00 

C16:0 Hexadecanoic (Palmitic) % 2.65 2.29 2.04 2.00 

C16:1 Hexadecenoic (Palmitoleic) % 0.69 0.68 0.45 0.44 

C16:2 Hexadecadienoic % 0.08 0.08 0.06 0.06 

C17:0 Heptadecanoic (Margaric) % 0.10 0.08 0.09 0.09 

C17:1 Heptadecenoic Margaroleic % 0.11 0.11 0.08 0.08 

C18:0 Octadecanoic (Stearic) % 1.18 0.93 1.05 1.10 

C18:1 Octadecenoic (Oleic) % 4.94 4.49 3.43 3.34 

C18:2 Octadecadienoic (Linoleic) % 0.31 0.24 0.22 0.18 

C18:3 Octadecatrienoic (Linolenic) % 0.05 0.05 0.05 0.05 

C18:4 Octadecatetraenoic % 0.07 0.08 0.04 0.04 

C20:1 Eicosenoic (Gadoleic) % 0.03 0.04 0.02 0.03 

C20:4 Eicosatetraenoic (Arachidonic) % 0.01 0.01 0.01 0.01 

Total Monounsat. Fatty Acids Calc. % 5.92 5.42 4.08 3.93 

Total Polyunsat. Fatty Acids Calc. % 0.54 0.44 0.40 0.33 

Saturated Fatty Acids % 4.25 3.51 3.47 3.42 

Total Fat (as triglycerides) % 11.24 9.76 8.34 7.99 

 

Calcium mg/100g 12.01 13.78 14.30 13.67 

Iron mg/100 g 2.29 0.74 2.32 0.71 

Phosphorus mg/100 g 179.09 27.31 191.21 28.12 

Zinc mg/100 g 3.86 0.67 4.14 0.64 

Cholesterol mg/100g 64.92 7.79 68.43 8.64 

Niacin mg/100 g 4.96 1.18 5.00 1.07 

Vitamin B1 - Thiamine Hydrochloride mg/100 g 0.10 0.04 0.09 0.02 

Vitamin B2 – Riboflavin mg/100 g 0.24 0.05 0.29 0.04 

Vitamin B6 mg/100 g 0.33 0.08 0.37 0.11 

Vitamin E IU/100g 0.50 0.15 0.44 0.15 

Hydroxyproline % 0.17 0.07 0.16 0.07 

The study by Tian and her colleagues (2005) discussed previously for milk composition 

also reports the results of studies on the composition of meat from bovine SCNT clones 

(Tian et al. 2005). Cultured skin fibroblasts or cumulus cells were used to clone an adult 

Japanese Black beef bull, selected as a superior breeding stud with superior marbling 
Chapter VI: Food Consumption Risks 277 

Animal Cloning: A Risk Assessment DRAFT

traits at 17 years of age. Six bull clones were produced, with four surviving and 

apparently normal. The clone bulls were raised in the same facility with eight genetically 

matched comparator non-clone animals and maintained on the same diet. The comparator 

bulls were produce by artificial insemination using semen from the son of the original 

donor bull. In addition, 20 age-matched sexually reproduced Japanese Black beef cattle 

were used as breed comparators to establish the normal range for each measured 

parameter. All bulls were castrated at 3 months of age and raised on standard growing 

ration from 8 to 26 months of age. The comparators and two of the clones were 

slaughtered and subjected to standard meat analyses. Variables measured included: 

• Organ or body part weights 

• Total proportion of meat and fat in the dressed carcass 

• Cross section of the left dressed carcass between the 6th and 7th rib 

• Moisture in 6 muscles (infraspinatus, longissimus thoracis, latissimus dorsi, 

adductor, biceps femoris, and semitendinosus) 

• Crude protein in 6 muscles (infraspinatus, longissimus thoracis, latissimus dorsi, 

adductor, biceps femoris, and semitendinosus) 

• Crude fat content in 6 muscles (infraspinatus, longissimus thoracis, latissimus 

dorsi, adductor, biceps femoris, and semitendinosus) 

• Fatty acid composition (lauric acid, myristic acid palmitic acid palmitoleic acid, 

stearic acid, oleic acid, linoleic acid and linolenic acid) of five major fat tissues 

(subcutaneous fat, inter-muscular fats, celom fat, and kidney leaf fat) 

• Amino acid composition of the longissimus thoracis muscle 

• Histopathology of all organs 

The 90 percent confidence intervals for each parameter were compared in a paired 

analysis between the clone and non-clone genetic comparators. There were 12 instances 

where the clones and genetic comparators showed differences: 

• Amount of mesentery fat 

• Proportion of longissimus thoracis muscle over body weight 

• Muscle moisture 

• Amount of crude protein in the semitendinosus muscle 

• Amount of linolenic acid in the kidney leaf fat 

• Amount of linolenic acid in the longissimus thoracis 

• Amount of linolenic acid in the semitendinosus muscles 

• Amount of oleic acid in the semitendinosus muscle 

• Amount of palmitic acid in the semitendinosus muscle 
Chapter VI: Food Consumption Risks 278 

Animal Cloning: A Risk Assessment DRAFT

• Amount of linoleic acid in the semitendinosus muscle 

All of the parameters were higher in the clones than in the genetic or breed comparators, 

except for crude protein or muscle moisture in semitendinosus muscle. The differences in 

mesentery fat and fatty acid content were attributed to the characteristics of the donor bull 

(superior marbling). It is noted that the clones had a marbling score of 8 out of 12, 

compared to an industry standard of 5.2, and genetic comparator score of 6.5. All of the 

other variables fell within normal industry standards. The researchers conclude that the 

meat from somatic animal clones falls within normal industry standards and does not 

significantly differ from those of the genetic or breed comparators. The differences 

observed were considered due to the superior genetics of the donor bull from which the 

line of clones was derived. 

No abnormalities were reported in the pathology or histopathology for clone tissue. 

ii. Swine 

(a) Clones 

Viagen, Inc., worked in consultation with CVM to designed two experiments that 

produced data comparing meat composition of clone swine vs. age-matched, genetically 

related, AI-derived comparator animals. Experimental design, raw data, and CVM’s 

analysis of the data are provided in Appendix G, The Viagen Dataset. Meat composition 

data were available for five clones (four Hamline and one Duroc) and 15 comparator 

animals (all Hamline). There were no differences between the Duroc and Hampshire 

clones, so data for clones were pooled. 

Carcass characteristics were provided on four Hamline clones and 15 comparator barrows 

and are summarized in Table VI-32. The Duroc clone barrow carcass was condemned at 

slaughter due to a lung adhesion, and thus data relating to growth and carcass 

characteristics were not included for these parameters. In some cases of lung adhesions 

due to bacterial infection, animals fail to thrive, thereby affecting their growth rate and 

carcass characteristics – this was considered to be the case for the Duroc clone. Two 

other clones were approximately 100 pounds lighter than any of the other animals in the 

experiment at the time of slaughter, and for this reason were excluded from carcass 

evaluation. Hot carcass weights averaged 189.0 and 199.5 pounds for clone and 

comparator barrows, respectively. Carcass lengths were 82.4 and 84.5 cm for clones and 

comparators, respectively. Dressing percentages were 70.1 and 70.2 percent for clones 

and comparators, and were similar across groups. Backfat thickness over the first rib, 

tenth rib, last rib, and lumbar vertebra were slightly greater for comparator barrows than 
Chapter VI: Food Consumption Risks 279 

Animal Cloning: A Risk Assessment DRAFT

for clone barrows which may, in part, be due to the heavier body weight of comparator 

barrows at the time of slaughter. 

Qualitative characteristics including USDA carcass muscle score, color, firmness, and 

marbling were similar across breeding regimens and are illustrated in Table VI-32. All 

animals received score 2 for carcass muscle. All of the clone and comparator barrows had 

marbling scores of either 1 or 2. 

Table VI-32: Comparison of the Carcass Characteristics of Barrows Derived by Somatic Cell 

Nuclear Transfer (Clones) or Conventional Breeding (Mean ± standard deviation) 

(from Viagen, Inc.) 

Clones 

(n=4) 

Conventionally Bred 

(n=15) 

Hot Carcass Weight (lbs) 189.0 ± 13.8 199.5 ± 13.7 

Carcass Length (cm) 82.4 ± 1.5 84.5 ± 2.7 

Dressing Percentage (%) 70.1 ± 0.8 70.2 ± 1.4 

Back fat Thickness (mm) 

 First Rib 35.3 ± 2.1b 38.7 ± 3.1a

 Tenth Rib 18.5 ± 3.1 22.2 ± 4.9 

 Last Rib 20.5 ± 4.7 23.3 ± 3.4 

 Last Lumbar 17.3 ± 3.2 21.0 ± 3.1 

Loin Eye Area (cm2) 44.0 ± 4.4 45.8 ± 4.0 

Measurements of pH at 24 hours post-slaughter on the longissimus muscle were similar. 

Loin eye area for meat cuts for clone and comparator barrows were only slightly different 

at 45.8±4.0 and 44.0 ± 4.4 inches, respectively. The Hunter L*, a* and b* values were 

only slightly different between the groups of animals with the meat from clones being 

slightly darker and more red than meat from comparator barrows. 
Chapter VI: Food Consumption Risks 280 

Animal Cloning: A Risk Assessment DRAFT

Table VI-33: Comparison of the Qualitative Carcass Characteristics of Barrows Derived by Somatic 

Cell Nuclear Transfer (Clones) or Conventional Breeding (Means ± standard deviation) 

(from Viagen, Inc.) 

Clones 

(n=4) 

Conventionally Bred 

(n=15) 

Longissimus pH at 24 hours 5.6 ± 0.1 5.7 ± 0.1 

Carcass Muscle Score 2.0 ± 0.0 2.2 ± 0.40 

NPPC Quality Scores 

 Color 3.0 ± 0.0 2.7 ± 0.6 

 Marbling 1.5 ± 0.6 1.9 ± 0.5 

 Firmness 3.5 ± 0.6 2.9 ± 0.9 

Hunter Color 

 L* 52.2 ± 2.0 56.3 ± 4.4 

 a* 9.5 ± 1.4 7.6 ± 1.2 

 b* 17.6 ± 0.7 16.9 ± 1.2 

Meat composition data were available for five clones (four Hamline and one Duroc) and 

15 comparator animals (all Hamline). There were no differences between the Duroc and 

Hamline clones, so data for clones were pooled. Means ± standard deviations for fatty 

acids, amino acids, cholesterol, minerals and vitamins measured are presented in Table 

VI-34. Differences in individual analytes for clones and comparators were very small and 

not biologically relevant. Values for niacin and vitamin B12 in both clones and control 

swine were above USDA values for a similar type of swine muscle (shoulder blade and 

loin). Values for cholesterol and vitamin B6 were similar to the USDA values. 
Chapter VI: Food Consumption Risks 281 

Animal Cloning: A Risk Assessment DRAFT

Table VI-34: Results of Meat Composition analysis for Experiment 11 

(from Viagen, Inc.) 

Component Clones Comparators 

Amino acids (g) 

Alanine 

Arginine 

Aspartate 

Cystine 

Glutamate 

Glycine 

Histidine 

Isoleucine 

Leucine 

Lysine 

Methionine 

Phenylalanine 

Proline 

Serine 

Threonine 

Tyrosine 

Valine 

1.26 ± 0.04 

1.41 ± 0.03 

2.55 ± 0.28 

0.25 ± 0.03 

3.41 ± 0.11 

0.98 ± 0.04 

0.99 ± 0.05 

1.04 ± 0.05 

1.74 ± 0.05 

1.91 ± 0.06 

0.54 ± 0.06 

0.86 ± 0.02 

0.85 ± 0.03 

0.90 ± 0.03 

1.11 ± 0.04 

0.77 ± 0.02 

1.10 ± 0.05 

1.30 ± 0.04 

1.47 ± 0.04 

2.43 ± 0.19 

0.26 ± 0.02 

3.46 ± 0.09 

1.02 ± 0.10 

1.03 ± 0.05 

1.05 ± 0.03 

1.79 ± 0.04 

1.96 ± 0.04 

0.58 ± 0.03 

0.89 ± 0.02 

0.90 ± 0.06 

0.92 ± 0.02 

1.14 ± 0.03 

0.79 ± 0.02 

1.12 ± 0.04 

Fatty Acids2 (g) 

14:0 

16:0 

16:1 

17:0 

17:1 

18:0 

18:1 

18:2 

18:3 

20:0 

20:1 

20:2 

22:6 

0.09 ± 0.06 

1.31 ± 0.82 

0.09 ± 0.04 

0.01 ± 0.01 

0.01 ± 0.01 

0.66 ± 0.41 

1.84 ± 0.84 

0.26 ± 0.08 

0.01 ± 0.01 

0.01 ± 0.01 

0.05 ± 0.03 

0.01 ± 0.01 

0.02 ± 0.03 

0.05 ± 0.03 

0.95 ± 0.49 

0.14 ± 0.05 

0.00 ± 0.01 

0.00 ± 0.01 

0.55 ± 0.27 

1.49 ± 0.50 

0.19 ± 0.06 

0.00 ± 0.01 

0.00 ± 0.01 

0.04 ± 0.02 

0.01 ± 0.01 

0.01 ± 0.01 

Cholesterol (mg) 55.5 ± 6.95 52.81 ± 2.69 

Minerals (g) 

Calcium 

Phosphorus 

Iron 

Zinc 

0.004 ± 0.000 

0.20 ± 0.01 

0.001 ± 0.000 

0.002 ± 0.000 

0.005 ± 0.003 

0.21 ± 0.01 

0.001 ± 0.001 

0.001 ± 0.000 

Vitamins 

Niacin (mg) 

B6 (mg) 

B12 (mcg) 

10.90 ± 0.83 

0.41 ± 0.09 

0.21 ± 0.28 

11.16 ± 1.58 

0.48 ± 0.12 

0.00 ± 0.00 

1

Data expressed as quantities per 100 g of homogenized meat. 

2

 Data presented reflect those fatty acids with detectable levels in pork. 
Chapter VI: Food Consumption Risks 282 

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Carcass qualitative characteristics were similar for clones and comparators. Differences 

in backfat thickness and marbling may be due to the lighter weight of clones at slaughter 

vs. comparators. Differences in meat nutrient composition were very small and likely not 

biologically relevant. No biologically relevant differences were observed in the food 

composition values between muscle of swine clones and comparators. 

(b) Swine Clone Progeny 

The Viagen company also provided CVM with data comparing the quality and 

composition of meat derived from the progeny of clone swine and non-clone swine. Data 

are reported for 412 swine: 242 clone progeny and 163 comparators. 

Table VI-35 provides the comparison of key nutrients between the progeny of clones and 

their comparators. Data were reported for 412 swine of which 242 were the progeny of 

clones and 163 were the progeny of comparator boars. (Details of this comparison can be 

found in Appendix F). The composition of the meat from the progeny of clones and 

comparators indicates that the meat samples were indistinguishable at the level of the key 

nutrients evaluated. Only two values (alanine and erucic acid) of 56 (0.03 percent) were 

not virtually identical, less than would be expected by chance alone. Neither of these 

differences is biologically significant. 

A comparison was also made between the meat composition of either the progeny of 

clones or the comparators to standard USDA values for similar cuts of pork (See 

Appendix G). This analysis reveals that neither the clones nor the comparators are as 

similar to the USDA dataset as they are to each other. The differences between the 

nutrient concentrations in progeny of clones and comparators compared to USDA 

database may be due to diet, swine genotype, or storage stability effects. The important 

conclusions from the two comparisons, however, are that (1) there are virtually no 

differences between the progeny of clones and comparators, and that (2) the closely 

genetically related comparators are a better reference point than the USDA database, and 

(3) none of the differences pose a food safety concern. These data suggest that there is no 

increased risk for humans to consume muscle from the progeny of swine clones. 

 
Chapter VI: Food Consumption Risks 283 

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Table VI-35: Comparison of Nutrient Concentrations of Progeny 

from Clones and Comparators 

Nutrients1 

Progeny from Clone 

Boars 

mean + std. dev. 

Progeny from 

Comparators Boars 

Mean + std. dev. 

Amino Acids

Aspartic acid 2.31+0.19 2.29+0.16 

Cystine 0.25+0.02 0.25+0.01 

Glutamic acid 3.76+0.34 3.71+0.27 

Glycine 1.14+0.15 1.12+0.13 

Histidine 0.98+0.09 0.98+0.07 

Isoleucine 1.03+0.12 1.03+0.10 

Leucine 1.90+0.14 1.89+0.12 

Lysine 2.06+0.17 2.07+0.16 

Methionine 0.61+0.05 0.62+0.04 

Phenylalanine 0.96+0.09 0.94+0.08 

Praline 1.09+0.13 1.11+0.13 

Serine 0.96+0.08 0.95+0.07 

Threonine 1.09+0.09 1.08+0.07 

Tyrosine 0.81+0.06 0.81+0.05 

Valine 1.09+0.12 1.10+0.10 

Fatty Acids and Cholesterol

8:0 (Caprylic acid) <0.012 0.01 

10:0 (Capric acid) 0.01+0.002 0.01+0.002 

11:0 <0.01 <0.01 

12:0 (Lauric acid) 0.01+0 0.01+0 

14:0 (Myristic acid) 0.08+0.027 0.08 + 0.029 

14:1 (Myristoleic acid) <0.01 <0.01 

15:0 <0.01 <0.01 

15:1 <0.01 <0.01 

16:0 (Palmitic acid) 1.39+0.38 1.40+0.49 

16:1 (Palmitoleic acid) 0.17+0.06 0.16+0.05 

17:0 (Margaric acid) 0.01+0.003 0.01 +0.002 

17:1 (Margaroleic acid) 0.01+0.003 0.01+0.002 

18:0 (Stearic acid) 0.66+0.24 0.68+0.25 

18:1 (Oleic acid) 2.26+0.76 2.20+0.72 

18:2 (Linoleic acid) 0.3+0.11 0.29+0.11 

18:3 (Linolenic acid) 0.02+0.001 0.01+0.005 

18:4 0.01+0.0001 0.01+0.004 
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Animal Cloning: A Risk Assessment DRAFT

20:0 (Arachidic acid) 0.01+0.005 0.01+0.005 

20:1 (Gadoleic acid) 0.08+0.04 0.07+0.04 

20:2 (Eicosadienoic acid) 0.02+0.01 0.02+0.005 

20:3 (Eicosatrienoic acid) 0.01+0.01 <0.01 

20:4 (Arachidonic acid) 0.01+0.003 0.01+0 

20:5 (Eicosapentaenoic acid) 0.01 + 0 0.01+0.004 

21:5 (Heneicosapentaenoic acid) 0.01+0 <0.01 

22:0 (Behenic acid) <0.01 <0.01 

22:1 (Erucic acid) 0.01+0.006 0.02+0.006 

22:2 (Docosadienoic acid) <0.01 0.01+0.01 

22:3 (Docosatrienoic acid) <0.01 <0.01 

22:4 (Docosatetraenoic acid) <0.01 <0.01 

22:5 (Docosapentaenoic acid) <0.01 <0.01 

22:6 (Docosahexaenoic acid) 0.02+0.01 0.02+0.01 

24:0 (Lignoceric acid) <0.01 <0.01 

24:1 (Nervonic acid) <0.01 <0.01 

Cholesterol (mg/100 g) 57.93+5.46 59.39+5.04 

Minerals

Calcium 0.01+0.003 0.01+0.002 

Iron 0.00+0.0005 0.000+0.003 

Phosphorus 0.18+0.082 0.16+0.082 

Zinc 0.00+0.0003 0.00+0.0001 

Vitamins

Niacin (mg/100g) 10.68+1.23 10.64+1.03 

Vitamin B6 (mg/100 g) 0.40+0.07 0.38+0.07 

Vitamin B12 (mcg/100 g) 1.01+0.25 0.97+0.28 

1 Unless otherwise specified, quantities are expressed as g/100g homogenized meat. 

2 Values marked with “<” indicate concentrations below the level of detection for the 

 instrument used in the assay. 

Carcass characteristics for the progeny of clone swine and their comparators are provided 

in Table VI-36, and discussed in more detail in Appendix F. Although some minor 

differences in backfat thickness and meat color were noted for progeny of clones vs. 

comparators, these do not affect food safety. No differences were noted that would have 

any impact on the quality of the meat or the safety of consuming the meat products. 
Chapter VI: Food Consumption Risks 285 

Animal Cloning: A Risk Assessment DRAFT

Table VI-36: Carcass Characteristics for Progeny Derived from Clones or Conventionally Bred 

Boars (Means ± standard deviation) 

Hampshire 

Comparator 

Hampshire 

Clone 

Duroc 

Comparator 

Duroc 

Clone 

Hot Carcass Weight (lbs) 176.2 ± 8.6 175.0 ± 8.7 173.9 ± 9.5 179.0 ± 9.1 

Carcass Length (cm) 82.7 ± 2.2 81.6 ± 2.1 82.3 ± 2.2 81.5 ± 2.3 

Loin Eye Area (cm2) 6.7 ± 0.8 6.8 ± 0.8 6.6 ± 0.8 7.2 ± 0.9 

Back fat Thickness (mm) 

 First rib 22.2 ± 4.2 23.4 ± 4.4 23.8 ± 4.1 25.9 ± 4.2 

 Last rib 16.0 ± 2.9 16.9 ± 3.2 17.4 ± 2.4 19.0 ± 2.8 

 Last Lumbar 16.6 ± 3.4 17.0 ± 3.2 18.1 ± 2.6 19.3 ± 2.7 

Longissimus pH at 24 hours 5.8 ± 0.2 5.7 ± 0.1 5.7 ± 0.1 5.7 ± 0.1 

Carcass muscle score 3 ± 0 3 ± 0 3 ± 0 3 ± 0 

NPPC Quality Scores 

 Color 3 ± 0.3 3 ± 0.2 3 ± 0.1 3 ± 0 

 Marbling 3 ± 0.7 3 ± 0.8 3 ± 0.8 3 ± 0.9 

 Firmness 2 ± 0 2 ± 0 2 ± 0 2 ± 0 

Hunter Color 

 L* 55.54 ± 2.1 55.88 ± 2.4 56.40 ± 2.4 57.24 ± 2.4 

 a* 7.47 ± 0.9 7.58 ± 1.0 7.21 ± 1.0 7.17 ± 1.0 

 b* 13.88 ± 0.9 14.12 ± 0.9 13.88 ± 0.8 14.35 ± 0.6 

iii. Conclusions from Studies Evaluating the Composition of Meat and Milk 

from Clones and Their Progeny 

The second prong of our Risk Assessment is based on the hypothesis that food products 

from healthy animal clones and their progeny that are not materially different from 

corresponding products from conventional animals are as safe to eat as their conventional 

counterparts. CVM has reviewed several peer-reviewed publications that have evaluated 

gross (e.g., milk yield, carcass characteristics) and fine (e.g., individual amino acid and 

fatty acid components) characteristics of meat and milk from clones, and in one study, their 

sexually-reproduced progeny. None of the characteristics that we examined differed in any 

biologically significant way between the clone and comparator, and none identified any 

potential nutritional or other hazards. Based on this review, CVM concludes that the data 

support the operating hypothesis underlying the Compositional Analysis approach, that is, 

meat and milk from clones and their progeny do not differ materially from that derived 

from conventional counterparts, and do not pose any additional food consumption risks 

relative food from conventional animals. 
Chapter VI: Food Consumption Risks 286 

Animal Cloning: A Risk Assessment DRAFT

B. Drawing Conclusions Regarding Risks Associated with Consumption of Food 

Products from Animal Clones 

1. Approaches for Decreasing Uncertainties 

The fundamental problem in determining the quantity and types of data required to 

reduce the uncertainties associated with a judgment of “no additional risk” has bedeviled 

the scientific, risk, and regulatory communities. The impracticality of proving a negative 

and, in the absence of its proof, determining the consequent activities to identify the 

conditions under which concerns have been minimized to levels considered “acceptable” 

becomes the goal of a comprehensive risk assessment/management process. 

In fact, certainty of prediction is unattainable in science. In its absence, risk assessment 

can provide risk managers with a systematic approach for bounding the “risk space” in 

which to operate by allowing assumptions and uncertainties to be clearly identified. 

Especially for new technologies in which uncertainty may be high, the “bounded 

framework” risk assessment process allows decision makers (both risk assessors and risk 

managers) to draw conclusions based on the data. Then, by explicitly addressing 

uncertainties, identifying biases, scientifically defensible (or alternatively, policy-based) 

judgments can be made about acceptable risk levels. The added benefit of such a process 

is that interested individuals are provided with a level of transparency that allows them to 

judge the quality of the science and the relative merits of decisions stemming from its 

evaluation. 

This risk assessment has provided an overview of the molecular evidence for epigenetic 

dysregulation as the basis for obvious and subtle hazards that may arise in animal clones, 

the biological reasons for why subtle changes would not persist in progeny of healthy 

clones, the existing data on the health of animal clones and their progeny, and 

information on the composition of foods derived from clones and their progeny. These 

data can be incorporated into four procedural steps leading about to conclusions 

regarding food safety: 

• Bounding the risk space, in which the “risk hypotheses” are explicitly identified 

and thereby the biases that influence the weight of the evidence evaluations 

regarding the health of the animals and the composition of food products derived 

from them; 
Chapter VI: Food Consumption Risks 287 

Animal Cloning: A Risk Assessment DRAFT

• Performing a weight of evidence evaluation of the data to characterize the risks

contained within the risk space, in which the information on food consumption 

hazards posed by cloning is summarized, and drawing conclusions based on the 

risk hypotheses presented in Step 1; 

• Characterizing the uncertainties associated with the data and their interpretation, 

including identifying important data gaps based on Critical Biological Systems 

and Compositional Analysis approaches; and 

• In subsequent versions of this Risk Assessment, reevaluating previously estimated 

risks based on new information to make new weight of evidence determinations. 

2. Bounding the Risk Space 

The two underlying risk hypotheses that explicitly bound the “risk space” in which the 

evaluations are being made are 

• Animal Clone Risk Hypothesis 1: Clones are the Same as Sexually-Derived 

Animals

Animal clones are biological copies of the donor animal, and data confirming overall 

findings of animal health and food product comparability are sufficient to indicate 

that no additional risk is posed by the consumption of such food products. 

• Animal Clone Risk Hypothesis 2: Clones are Different from Sexually-Derived 

Animals

Animal clones may appear to be faithful biological copies of the donor animal, but 

subtle hazards may have resulted from incomplete or inappropriate reprogramming of 

the genome as part of the SCNT process. In order to avoid additional risks above 

those posed by consumption of foods from sexually-derived animals under this 

hypothesis, comprehensive health and compositional data must be collected and 

analyzed to demonstrate that the animals are healthy, and that food products derived 

from them do not differ significantly from sexually-derived animals. 

• Clone Progeny Risk Hypothesis: Gametogenesis Resets Epigenetic 

Dysregulation 

Normal, healthy clones reproducing via sexual reproduction give rise to progeny 

animals that are as healthy as animals derived from any other sexual reproduction 

event. 
Chapter VI: Food Consumption Risks 288 

Animal Cloning: A Risk Assessment DRAFT

3. Developing Conclusions Regarding Food Consumption Risks 

The conclusions that can be drawn with respect to the safety of consuming food products 

from animal clones and their progeny based on the data reviewed in this Risk Assessment 

follow. Because risk assessment is best performed recursively, risk assessment 

conclusions should always be considered to apply to the dataset that was examined; each 

conclusion is based on the information that was available for consideration, but if 

additional data become available, a conclusion may change, or the degree of confidence 

placed in the conclusion may be adjusted. Nonetheless, risk managers need to make 

decisions at particular points in time, and despite the desire for recursive assessments, 

decisions often include statements about the degree of certainty that accompany them. 

Each conclusion is followed by a statement on whether the judgment comes from 

application of Hypothesis 1 (Assumes Clones are the same as Sexually-Derived 

Animals), or Hypothesis 2 (Assumes Clones are Different from Sexually-Derived 

Animals), and the reason for the selection of that hypothesis (and its implicit bias). 

As previously stated, the Risk Assessment assumes that all of the laws and regulations 

that apply to sexually-derived animals and the food products that come from them apply 

equally to animal clones, their progeny, and food products that are derived from them. 

Our weight of evidence risk assessment conclusions are presented on a species-specific 

basis, except for bovine clones, where the large dataset allows for the consideration of 

individual developmental nodes. The weight of evidence evaluations take into account: 

 

• All of the observations for that species (or developmental node); 

• The extent to which those observations are coherent with biological assumptions; 

• The consistency with which those observations are also seen across species, 

including the mouse model, where applicable; 

• Uncertainties that persist in the evaluation, including the source of those 

uncertainties; and 

• The confidence level in the conclusion based on all of the preceding 

considerations. 

Because this is a qualitative, comparative risk assessment, it does not attempt to assign 

quantitative values to estimates of risk or safety. The strongest conclusions that can be 

drawn regarding positive outcomes in risk assessments of this type are “no additional 
Chapter VI: Food Consumption Risks 289 

Animal Cloning: A Risk Assessment DRAFT

risk” because outcomes are weighed against known comparators. In the context of edible 

products derived from clones, a finding of no additional risk means that food products 

derived from animal clones will not pose any additional risks relative to corresponding 

products from non-clones, or are as safe as foods we eat every day. As with all risk 

assessments, some uncertainty is inherent either in the approach we have used or in the 

data themselves. For each conclusion, CVM has attempted to identify the sources and 

extent of these uncertainties. A more complete discussion of sources of uncertainties and 

their implications can be found in Chapter VII. 

4. Weight of Evidence Conclusions Regarding Food Consumption Risks for 

Clones and their Progeny 

Based on this review of the body of data on the health of animal clones, the composition 

of meat and milk from those animals and corresponding information on clone progeny, 

CVM has drawn the following conclusions: 

a. Cattle Clones 

Edible products from perinatal bovine clones may pose some very limited human food 

consumption risk. 

The underlying biological assumption in place for this age cohort is that perinatal clones 

may be fragile at birth due to residual incomplete or inappropriate reprogramming of the 

donor nucleus. Data from both the peer-reviewed publications and Cyagra are consistent 

with that assumption; some perinatal clones do not survive for several reasons, including 

poor placentation, LOS, and in some cases, frank malformations. Although surviving 

clones can be fragile for a period of time, survivors tend to adjust to life outside the 

womb within a relatively short period, either on their own or with assistance from 

caregivers (see Juvenile Developmental Node). The peer-reviewed literature and Cyagra 

data indicate that, depending on the laboratory, a significant proportion of perinatal 

clones survive gestation and are born without significant health problems. Laboratory 

measures of key physiological functions do not appear to indicate that surviving animals 

are very different from conventional newborns. It is therefore unlikely that food 

consumption risks have been introduced into these animals. 

The uncertainty associated with the preceding statement is relatively high, however, for 

the following reasons. First, postulated differences in epigenetic reprogramming between 

perinatal clones and comparators suggest that some subtle hazards may have been 

introduced into these animals. Second, the relatively poor condition of many of these 
Chapter VI: Food Consumption Risks 290 

Animal Cloning: A Risk Assessment DRAFT

perinatal clones also precludes the conclusion that no food consumption risks, such as 

nutritional imbalances, are present. Therefore, given that perinatal clones may differ from 

comparator animals of the same age, at this time, the Center concludes that they may 

pose a very limited nutritional risk for consumption as food. Rendering these clones will 

not pose such risks in animal feed or to humans consuming animals fed material derived 

from the clones. 

 

i. Risk Hypothesis Statement for Perinatal Bovine Clones 

At this time there is insufficient information to move from Hypothesis 2 (Clones are 

Different) to Hypothesis 1 (Clones are the Same), even though the available data neither 

identify nor predict the presence of food consumption hazards (and subsequent risks) 

from these very young clones. The uncertainties in the data are relatively high and lead 

the Center to have a relatively low degree of confidence in the safety of edible products 

from perinatal bovine clones. We note, however, that it is highly unlikely that clones of 

this age group would be consumed for food. 

Edible products from juvenile bovine clones pose no additional food consumption 

risk(s) relative to corresponding products from contemporary conventional 

comparators. 

The underlying biological assumption for this developmental node is that if any 

anomalies were to be found in the youngest clones and those animals were to survive to 

be healthy adults, the juvenile developmental node would be a period of equilibration and 

normalization. The data appear to be consistent with such a hypothesis. 

Juvenile bovine clones are largely healthy and normal. Although clones in this 

developmental node may be more physiologically unstable than their conventional 

counterparts, they are in the process of normalizing their physiological functions on the 

way to adulthood. For example, some animals at this developmental node may 

demonstrate alterations in physiological parameters such as body temperature, some 

hormone and cytokine levels (Chavatte-Palmer 2002, Govoni et al. 2002, ChavattePalmer 2004), these differences are resolved relatively rapidly. The normalization 

resulting in appropriate health status of these animals has been observed consistently in 

the reports reviewed in this Risk Assessment set, and is further demonstrated by the 

analysis of clinical chemistry and hematology data indicating that clones show the 

appropriate physiological responses to developmental signals. For example, measures of 

bone growth such as alkaline phosphatase, phosphorous, and calcium levels all show 
Chapter VI: Food Consumption Risks 291 

Animal Cloning: A Risk Assessment DRAFT

appropriate age-specific responses. None of the physiological measures taken, including 

both clinical chemistry and hematology, indicated any food consumption hazards. 

The Cyagra dataset, which is made up of 47 clones between the ages of one and six 

months, indicates the overall health of these animals is comparable to their age-matched 

comparators, with the exception of the sequellae of umbilical problems and 

cryptorchidism. Although these outcomes pose risk to the animals, if appropriately 

managed, they do not appear to pose any food safety concerns, and are also observed in 

non-clones. 

ii. Risk Hypothesis Statement for Juvenile Bovine Clones 

The assessment began at the position of Hypothesis 2, but the scientific evidence has 

moved the assessment from Hypothesis 2 to Hypothesis 1 for maturing juvenile clones. 

The weight of the evidence and the underlying biological assumptions lead the Center to 

conclude that there would not likely be any additional risk from the consumption of food 

from healthy juvenile clones relative to corresponding products from their conventional 

comparators. The consistency of these observations across all of the data for juvenile 

bovine clones makes the uncertainty associated with this judgment relatively low, and 

provides the Center with a relatively high degree of confidence in judgments regarding 

the health of (and consequent food safety of edible products derived from) this age cohort 

of bovine clones. 

Edible products derived from adult bovine clones pose no additional risk(s) relative to 

corresponding products from contemporary conventional comparators. 

This conclusion is based on application of both prongs (CBSA and Compositional 

Analysis) of the risk assessment approach. 

The body of data comprising the CBSA approach on adult domestic livestock clones is 

made up of two components: data and information extracted from peer-reviewed 

publications and the Cyagra dataset. The empirical evidence on the health of these 

animals is consistent with the biological prediction that there are no underlying biological 

reasons to suspect that healthy animal clones pose more of a food safety concern than 

conventional animals of similar age and species. 

The data from Cyagra survey indicate that healthy clones of the oldest cohort 

(6-18 months) are virtually indistinguishable from their comparators even at the level of 

clinical chemistry and hematology. These data also confirm the observation that 
Chapter VI: Food Consumption Risks 292 

Animal Cloning: A Risk Assessment DRAFT

physiological instabilities noted earlier in the lives of the clones are resolved juvenile 

developmental node (see previous conclusions regarding other developmental nodes), and 

do not reappear as the clones age. The statements regarding the health and apparent 

normality of animals of this age group from the peer-reviewed literature are consistent 

with the data evaluated by CVM. There are some reports of early deaths of clones; as 

these animals would not enter the food supply, they do not pose a food consumption risk. 

Data on reproductive function in cows or bulls of this age cohort indicates that that 

healthy bovine clones surviving to reproductive maturity function normally and produce 

healthy offspring. These data are consistent across studies. Given that reproduction is the 

most difficult “biological hurdle” placed on an organism, the observation of normal 

reproductive function provides an additional degree of confidence to the conclusion of 

the appropriate development of these animals. 

All of the reports on the composition analysis of meat or milk from bovine clones show 

that there are no biologically significant differences in the composition of milk derived 

from clone and non-clone cattle. Additionally, data from one report that show no 

difference in allergenic potential for meat or milk derived from clone cattle compared to 

meat or milk from non-clone comparators. Similarly, neither meat nor milk from clone or 

non-clone cattle induced mutations in a mutagenicity assay (Japan 2004). Finally, none of 

the reports identified an endpoint that would pose a hazard for human consumption. 

iii. Risk Hypothesis Statement for “Adult” Bovine Clones 

The assessment began at the position of Hypothesis 2: that animal clones may appear to 

be copies of the donor animal, but that the process of cloning may have introduced subtle 

hazards that could pose food consumption risks. As presented above, however, the weight 

of the evidence has moved the assessment from Hypothesis 2 to Hypothesis 1 (Clones are 

the same as their sexually-derived counterparts). Extensive and consistent empirical 

evidence, including epigenetic, physiological, and health data on individual animals and 

compositional analysis of milk and meat derived from individual animals, indicate that 

adult bovine clones are biologically equivalent to their contemporary comparators. 

Therefore, evidence confirming the health of the animals produced via similar methods, 

and evidence confirming the compositional similarity of meat and milk from clone and 

non-clone cattle indicates that there is no additional risk from the consumption of edible 

products from these animals relative to sexually-derived comparators. The consistency of 

the observations provide the Center with a high degree of confidence in judgments 

regarding the health of (and food safety of edible products derived from) this age cohort 

of bovine clones. 
Chapter VI: Food Consumption Risks 293 

Animal Cloning: A Risk Assessment DRAFT

We note that given the economic considerations involved, it is not likely that many adult 

clones would enter the food supply as meat at this stage of the technology, unless they 

had suffered a non-treatable injury or old age. Milk products from lactating female 

bovine clones, however, could be introduced into the food supply. 

b. Swine Clones 

Edible products from adult swine clones pose no additional risk(s) relative to 

corresponding products from contemporary conventional comparators. 

This conclusion is based on the same underlying biological assumption as cited for adult 

bovine clones (i.e., non-transgenic clones would not likely express toxicants, no 

exogenous genes, and diseased animals would not be slaughtered for food). Because the 

data are more heavily weighted towards adult, market sized animals, judgments regarding 

the safety of food products from swine clones are provided in one aggregate set of 

comments.

Although generating swine clones appears to pose more technical difficulties than bovine 

clones, once piglets are born, they appear to be healthy. The health status of perinatal 

animals is generally presented as “normal” or “healthy” in peer-reviewed publications. 

The most compelling argument for the normal health status of swine clones has been 

presented by Archer et al. (2003 a,b), who evaluated the behavior and physiological 

status of a small cohort of relatively young (15 weeks), and approximately market age 

(27 weeks) swine clones relative to closely related conventional pigs. No significant 

differences were observed in either behavior, epigenetic, or physiological measurements, 

indicating that these animals were not materially different from the comparators. Agerelated physiological measures appeared to be normal, as demonstrated by levels of 

measures of growth such as alkaline phosphatase, calcium, and phosphorus and measures 

of immune system maturity such as globulin. The case of parakeratosis was observed in 

the clones making up the cohort studied by Archer et al. (2003b). It is not known whether 

its appearance is related to cloning. The food consumption concerns are minimal, as the 

skin of that animal (or at least the portion with the lesion) would be condemned at the 

slaughterhouse, as it would had it come from a conventional animal. 

The data on the Viagen clones (Appendix F) are on a relatively small number of animals, 

reared in very unusual settings (i.e., deprivation of colostrums, initial husbandry in 

pathogen-free conditions, switching to commercial settings) and are therefore confounded 

with respect to outcome. Nonetheless, the data indicate that even though the clone 
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Animal Cloning: A Risk Assessment DRAFT

barrows were subjected to a significant immunological challenge after moving from 

pathogen-free conditions to more standard housing conditions, most clones were able to 

respond appropriately to this stress. Nonetheless, carcass qualitative characteristics were 

similar for clones and comparators in the Viagen Dataset. Further, reproductive 

performance for these clone boars appears normal. No differences were noted in semen 

quality between clones and comparator boars; farrowing rates and litter sizes were within 

national averages. No biologically relevant differences were observed in the composition 

of meat from clones or comparators. 

i. Risk Hypothesis Statement for Swine Clones 

Based on both underlying biological assumption and confirmatory data, CVM concludes 

that consumption of food from healthy adult swine clones would not pose an additional 

risk above consumption of their conventional counterparts. The data from Archer et al. 

(2003 a,b) is particularly compelling as it includes data on behavior, epigenetic 

reprogramming, and physiological measurements at two time points in the development 

of these clones. Likewise, data from Viagen includes information on growth, 

reproduction, carcass and meat composition, indicating that swine clones are not 

materially different from age-matched, genetically related swine. In this case, the Center 

finds itself at an intermediate Risk Hypothesis Level of “1 minus,” or relatively high 

certainty based on biological plausibility, consistency of observations among different 

and compelling datasets, and consistency with responses observed across other clone 

species. 

c. Sheep Clones 

Except by relying on underlying biological assumptions, and by inference from other 

species, there is insufficient information on the health status of sheep clones to draw 

conclusions with respect to potential risks that could be posed from the consumption of 

food products. 

With the exception of reports on Dolly, CVM was unable to find any publicly available 

reports on the health status of live sheep clones. There are several studies addressing 

methodological issues for optimizing the generation of clones, but these do not address 

post-natal health. There are reports of anomalies noted in fetal sheep clones that have 

died or been terminated, and reports on the pathology associated with animals that do not 

survive. Although these are instructive for understanding the molecular and 

developmental pathways that may be perturbed during the process of SCNT, these studies 

have limited relevance to addressing food safety because the deceased animals would not 
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Animal Cloning: A Risk Assessment DRAFT

have been allowed to enter the food supply. CVM was not able to find any reports on the 

composition of milk or meat from sheep clones. 

i. Risk Hypothesis Statement for Sheep Clones 

At this time there is insufficient information to support Hypothesis 1; Hypothesis 2 must 

be the default position with respect to potential food consumption risks from sheep 

clones. CVM was not able to find any studies providing specific evidence to show that 

sheep generated by SCNT are healthy and normal, and would therefore pose no 

additional food safety concerns beyond those of their conventional counterparts. 

d. Goat Clones 

Edible products from goat clones pose no additional food consumption risk(s) relative 

to corresponding products from contemporary conventional comparators. 

This conclusion is based on the same underlying biological assumption cited for the other 

livestock species, and a relatively small but compelling dataset. Once clone embryos are 

transferred to surrogate dams and pregnancies are confirmed, the “success rate” for live 

births is quite high. The only anomaly noted was that approximately half of the cohort of 

goats reported on by Keefer et al. (2001a) appeared to have poor suckling response 

immediately after birth, but by the second day were responding normally and nursing 

from their surrogate dams. The animals appear to have developed well through 

reproductive age. The available data indicate their physiological responses are 

appropriate for age and breed. The reproductive development and function of male 

Nigerian Dwarf goat clones demonstrate that those animals functioned appropriately 

relative to age- and breed-matched comparators. One male progeny goat was derived 

from the buck clones; this animal also appeared to function in an age- and breedappropriate manner. No meat or milk composition data were identified for goat clones. 

i. Risk Hypothesis Statement for Goat Clones 

Although the assessment began at Hypothesis 2, based on the underlying biological 

assumptions stated for the other clone species, consistency of responses with other 

species of clones, and a small but relatively rich dataset, CVM concludes that Hypothesis 

1 more appropriately represents the conclusions regarding the food safety of goat clones. 

CVM places particularly high weight on the study of reproductive function, as it is one of 

the most complex physiological pathways to coordinate. The consistency of appropriate 

reproductive function, even in a small cohort of animals, adds to the confidence that can 

be placed in the judgment that these animals are as normal and healthy as their sexuallyderived counterparts. Based on this finding, edible products from goats are not 
Chapter VI: Food Consumption Risks 296 

Animal Cloning: A Risk Assessment DRAFT

anticipated to pose more of a food consumption risk than their sexually-derived 

counterparts. Further, given the data on the normal reproductive function of these 

animals, and a preliminary report of normal reproductive function of one male offspring 

of a male goat clone, CVM has more confidence in the empirical demonstration that 

clone progeny are as healthy as other sexually-derived animals. 

e. Clone Progeny 

Edible products derived from the progeny of clones pose no additional food 

consumption risk(s) relative to corresponding products from other animals.

Progeny of clones, from the first sexual breeding of a clone through subsequent 

generations, will likely provide the overwhelming majority of clone-derived food 

products (both meat and dairy) in the US. The underlying biological assumption for 

health of progeny animals is that passage through the process of creating the cells that 

ultimately become ova and sperm naturally resets epigenetic signals for gene expression. 

This process is thought to effectively “clear” the genome of incomplete or inappropriate 

signals. The rationale for this assumption has been developed in Chapter IV, and 

dominates the conclusion that edible products from any clone progeny pose no additional 

food consumption risk(s) relative to those from any other sexually reproduced animals. It 

has been supported by detailed empirical evidence both in the mouse model system, 

which clearly indicates that phenotypic alterations noted in the parent clones are not 

passed to their sexually-derived progeny, and observations on the health and meat 

composition of progeny of livestock clones. In addition, the extensive information 

provided by Viagen on the progeny of clone swine provides direct data on the health of 

these animals and on the composition of meat derived from them. The swine data support 

the underlying biological assumption that the progeny of clone animals are essentially 

indistinguishable from the comparable progeny of non-clone animals. 

We therefore concur with the high degree of confidence that the outside scientific 

community (NAS 2002 a,b) places in the underlying biological assumption, and conclude 

that consumption of edible products from clone progeny would not pose any additional 

food consumption risk(s) relative to consumption of similar products from sexuallyderived animals. 

5. Summary of Risk Hypotheses 

The current weight of evidence suggests that there are no biological reasons, either based 

on underlying scientific assumptions or empirical studies, to indicate that consumption of 

edible products from cattle, pigs, or goat clones poses a greater risk than consumption of 
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Animal Cloning: A Risk Assessment DRAFT

those products from their non-clone counterparts. The level of certainty is highest for 

bovine clones, followed closely in degree of certainty by swine and, and then goat clones. 

The lack of species specific data for sheep clones precludes an evaluation of the risk for 

consumption of sheep clones at this time. Consumption of edible products from the 

progeny of clones poses no additional risk(s) relative to those from other sexually-derived 

animals, based on underlying biological assumptions and compelling evidence from the 

mouse model system and the Viagen dataset on the health of clone progeny and their 

meat composition. No food safety concerns were raised in the study of the composition of 

milk or meat from bovine clones. The level of confidence that may be placed in these 

overall conclusions is quite high, although additional data can always increase 

confidence. 

a. Additional Issues 

In addition to the hazards and risks described in the preceding portion of this risk 

assessment, there are a few issues that do not fit neatly into one of the categories that 

have been discussed previously. Many of these are overarching issues that may also have 

applicability to technologies other than SCNT. 

i. Potential Allergenicity 

The issue of allergenicity is one that is often cited for foods that do not have a long 

history of consumption. Although there is no reason to suspect that cloning will cause the 

synthesis of new proteins in animals that appear healthy and normal, there are two 

possible pathways that might pose an increased allergenic risk from the edible products 

of animal clones. One is an increase in the relative amount of an individual protein 

component of milk or meat that may only be present in very low or trace amounts. Cows’ 

milk has been associated with true allergies (Cows Milk Allergy or CMA) in 

approximately six percent of the US population (Bernstein 2003). Caseins, although the 

predominant proteins in milk, do not appear to be the key allergens associated with CMA. 

The other possible pathway is that processing of the proteins during their generation in 

the mammary gland or muscle cells somehow alters their antigenic presentation. The 

Center cautions that these are purely hypothetical pathways, and that there has been no 

demonstration that either of these actually occurs. 

In theory, evaluating the relative concentrations of milk proteins in clone and comparator 

milk could provide information to determine if the first risk exists. The study by Tian et 

al. (2005) provides just such a comparison using SDS/page technology. In practice, 

however, even this study highlights the difficulty in establishing the appropriate 
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Animal Cloning: A Risk Assessment DRAFT

comparator and minimizing variability. Milk from non-clone dairy animals may vary in 

relative composition due to the influences of breed, diet, number of lactations, where in 

the lactation cycle the milk is collected, etc. Further, the level of exposure (dose) required 

to elicit an allergenic response is not well understood, and has been the subject of much 

discussion in the scientific literature (Taylor 2002) and among international regulatory 

bodies (Codex Alimentarius 200370). Nonetheless, the limited studies provided (Japan 

2002) show that milk from both SCNT and BNT clone cattle showed similar digestibility 

characteristics both in vitro and in a rodent in vivo assay. In addition, a rodent bioassay 

for allergic response did not show any significant differences in response between clone 

and non-clone derived milk. Combined with the underlying biological assumptions, these 

data support the lack of a unique allergic response to milk derived from clone cattle. 

Similar risks are not likely to occur for meats, as meat allergies are so much less 

prevalent in the population that they are almost considered idiosyncratic, and individuals 

likely to suffer from meat-related allergies are likely to avoid those meats entirely. In 

addition, freeze dried meat from clone and non-clone cattle produced no difference in 

response in digestibility in both an in-vitro and rodent in-vivo assay, and there was no 

difference in difference in response in a rodent allergenicity bioassay (Japan 2002). 

Finally, it is important to remember that relative and potential allergenicity in food is an 

issue that vexes the scientific and regulatory communities. FDA supports further research 

into the overall risk factors that cause individuals to exhibit aberrant immune responses. 

The agency has been actively involved in the evaluation of predictive tests at the 

laboratory and clinical level that address changes in protein structure and presentation. 

Nonetheless, it is important to remember that efforts such as those undertaken by the ILSI 

Allergy and Immunology Institute, the International Biotechnology Council, the National 

Academy of Sciences, Food and Agriculture Organization and the World Health 

Organization and the Codex Alimentarius address the allergenicity of novel proteins. 

These proteins are either new to the food supply as the result of the introduction of new 

foods, or are present in different matrices, as may be the case with transgenic plants or 

animals. 

ii. Microbiological Effects 

One potential meat-based hazard that can be postulated is that epigenetic changes in 

animal clones could somehow alter the rumen and intestinal microflora of the ruminants 

(cattle, sheep, and goats), or the intestinal microflora of the monogastric species (swine) 

considered here. Such an alteration in intestinal flora could theoretically result in the 

 

70 ftp://ftp.fao.org/codex/Publications/Booklets/Biotech/Biotech_2003e.pdf 
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Animal Cloning: A Risk Assessment DRAFT

growth of a novel zoönotic pathogen or increased levels of an existing zoönotic pathogen 

contaminating the edible tissues derived from the food animal. The use of animal drugs 

has similarly been postulated to alter the intestinal flora of treated food animals, resulting 

in an increased load of zoönotic pathogens in the food supply. 

The potential for animal drugs to induce this change was considered at length by the 

January 2002 CVM Veterinary Medical Advisory Committee on that topic 

(www.fda.gov/cvm/index/vmac/winter2002meet.htm). Most of this independent 

scientific advisory committee found that animal drug use was unlikely to significantly 

impact pathogen load (or the prevalence of zoönotic pathogens), and that pathogen load 

has little or no impact on public health. It is likely that bacterial shedding from food 

animal clones poses no greater risk than that posed by conventional food animals. The 

complexity of the intestinal microflora makes this an extremely difficult question to 

address directly. Indirect evidence of normal intestinal microflora, however, can be 

inferred from the health status and growth characteristics of the animal clones, suggesting 

a normal microflora population. 

iii. Unanticipated Effects 

This risk assessment has attempted to identify the range of potential hazards and risks 

that could be generated as the result of SCNT in domestic livestock species. Although it 

may be possible for a healthy clone to express some proteins inappropriately, the same 

argument can just as easily be made for sexually-derived animals. At this time, there is no 

validated method for determining small differences in protein constituents in foods, and 

even if such methodologies existed, the question would still remain as to how to interpret 

them--what foods would be used as comparators, and what degree of variability would be 

considered to pose a risk (NAS 2004)? 

Finally, the issue of the hypothetical dysregulation of endogenous substances that may 

pose a hazard by virtue of increased dose should be addressed. The primary concern in 

this case is the up-regulation of small molecules that may retain bioactivity in the bodies 

of the human (or animal) food consumer, usually by virtue of the lack of degradation in 

the intestinal tract. For example, levels of endogenous substances that have posed some 

public concern in the past (e.g., estrogen and IGF-I) have been evaluated in bovine 

clones, and based on those data, there is no reason to expect that the levels of these 

substances in clones would pose any food consumption risks for humans. 
Chapter VI: Food Consumption Risks 300 

Animal Cloning: A Risk Assessment DRAFT

iv. Technology Changes 

This risk assessment has focused on the outcomes of cloning (i.e., clones and their 

progeny) rather than on the cloning process itself. As discussed in Chapter II and 

elsewhere, however, at the time this risk assessment was developed, most clone 

producers use the same overall technology to produce clones. Clearly, different producers 

and laboratories may modify the process to enhance the overall success rate of the 

cloning process. In general, however, the clones that were evaluated in this risk 

assessment were produced by very similar processes. From a risk perspective, the 

important constant in technology used to produce these clones is that donor nuclei and 

recipient oöcytes (or oöplasts) are not significantly manipulated beyond the obvious steps 

described in Chapter II. Thus, hazards other than epigenetic dysregulation are not 

introduced into clones. 

Significant changes in cloning technology, especially those accompanied by donor 

nucleus or oöcyte treatment regimens introducing new hazards into the overall process, 

would significantly increase the uncertainty associated with our judgments regarding the 

degree of risk that could accompany the resulting clones and clone food products. 

Without a careful evaluation of the animals arising from such methods, it would not be 

appropriate to speculate on the relative safety of the process from either an animal health 

or food safety perspective. 

5. How Much (Information) Is Enough? 

The question of determining when sufficient data have been collected in order to allow 

high confidence in risk-based decisions regarding edible products from animal clones is 

difficult to determine in the abstract. In practice, the answer is “it depends on what 

questions you ask, and how the data answer those questions.” 

Because the nature of the technology has generally precluded generating large datasets on 

clones with good statistical power, CVM constructed a systematic approach to frame the 

appropriate questions (hazard identification), evaluated the available data (hazard 

characterization), and attempted to characterize resulting risk (probability of harm given 

that exposure occurs). This weight of evidence approach allows for the evaluation of the 

data from the CBSA and Compositional Analysis prongs of the Risk Assessment as part 

of an overarching whole. The conclusions from this risk assessment represent the 

judgment of CVM veterinarians, animal scientists, toxicologists, and risk assessors. The 

underlying assumptions for clones and their progeny were that the animals needed to 

meet all relevant federal, state, and local laws and regulations for conventional animals, 
Chapter VI: Food Consumption Risks 301 

Animal Cloning: A Risk Assessment DRAFT

and the food products derived from clones or their progeny also had to meet relevant 

federal, state, and local laws and regulations. 

When considered across the Developmental Node spectrum, the data on the health of 

livestock clones were remarkably consistent across species, despite initial anomalies that 

appear to be species-specific. For example, although LOS may be more prevalent in 

cattle and sheep, most surviving animals normalize initial anomalies and become 

“healthy and normal.” This consistency has increased the value of even small datasets 

(e.g., goats), and has contributed significantly to the judgments regarding the health of 

these clones and their suitability as food sources. In addition, CVM evaluated a number 

of reports on the composition of meat and milk from clones and their progeny. No 

biologically important or safety-relevant differences were noted when compositions were 

compared to standard databases or contemporary comparator controls. If anything, these 

data confirm the rather wide variability in the composition of meat and milk eaten on a 

daily basis. In summary, no toxicological hazard of concern for the human consumer has 

been identified in any of the reported studies. Although additional data from other sets of 

animals, particularly in other species routinely used for food, could be useful in 

increasing the confidence that may be placed in overall judgments regarding food safety, 

the weight of the evidence at this time is sufficient for the agency to draw the conclusions 

it has made in this Risk Assessment with reasonable certainty. 
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Chapter VII: 

Summary and Conclusions
 

Animal Cloning: A Risk Assessment DRAFT

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Animal Cloning: A Risk Assessment DRAFT

Chapter VII 

Summary and Conclusions 

Somatic Cell Nuclear Transfer (SCNT) is a technology early in its development. Cloning 

has been accomplished in relatively few species, with most of our current information 

stemming from studies in cattle, swine, goats, and mice. This Risk Assessment has 

addressed the hazards and potential risks that may be experienced by domestic livestock 

(i.e., cattle, swine, sheep, and goats) involved in the cloning process (Animal Health 

Risks) and whether edible products from animal clones or their progeny pose food 

consumption risks beyond those of their conventional counterparts (Food Consumption 

Risks). 

The Risk Assessment employed a weight of evidence approach for drawing conclusions 

regarding risks to animal health and for consumption of food products from clones and 

their progeny. This approach consisted of four steps: 

(1) Evaluation of the empirical evidence (i.e., data on molecular mechanisms, 

physiological measurements, veterinary records, and observations of general 

health and behavior) for the species being considered; 

(2) Consideration of biological assumptions predicated on our growing 

understanding of the molecular mechanisms involved in mammalian 

development; 

(3) Evaluation of the coherence of the observations with predictions based on 

biological mechanisms; and 

(4) Evaluation of the consistency of observations across all of the species 

considered, including the mouse model system. 

The Risk Assessment also assumes that animal clones, their progeny, and all food 

products derived from either clones or progeny must meet the same federal, state, and 

local laws and regulations as conventional food animals or their edible products. 

Because no exogenous genes have been introduced into animals derived via SCNT, the 

underlying assumption has been that adverse outcomes observed in animal clones arise 

from epigenetic modifications due to incomplete reprogramming of the donor cell 
Chapter VII: Summary and Conclusions 306 

Animal Cloning: A Risk Assessment DRAFT

nucleus. Methodological and technological components (e.g., selection of donor cell, cell 

cycle stage, in vitro factors associated with the SCNT process) may also affect outcomes 

as they do for other ARTs. 

To assess the health of animal clones for both the animal health and food consumption 

risk portions of this risk assessment, we have developed the Critical Biological Systems 

Approach (CBSA), which divides the life cycle of clones into five distinct Developmental 

Nodes. Available data for each species has been sorted into these Developmental Nodes 

to evaluate the data systematically and to determine whether there are common 

developmental difficulties among the livestock clones or whether animals “recover” from 

initial infirmities related to cloning. 

The results of the CBSA indicate that significant adverse health outcomes have been 

reported for animal clones and their surrogate dams. These tend to result in dystocia and 

high gestational mortality. Post-natal mortality in clones tends to be concentrated in the 

perinatal period, and is higher in clones than in animals produced using other assisted 

reproductive technologies (ARTs), although as the technology matures, the rate of live 

births or deliveries appears to be increasing. 

To date, no adverse outcomes have been noted in clones that have not been observed in 

animals derived via other ARTs or natural mating. The incidence of these adverse 

outcomes appears to be higher in clones than in other forms of ARTs. Common adverse 

developmental outcomes that have been observed in cattle and sheep fall under the 

heading of Large Offspring Syndrome (LOS). Newborn animals with LOS tend to be 

heavy for their breed and species, may show edema or other abnormalities of the lungs 

and other parts of the body, and exhibit cardiovascular and respiratory problems. Other 

species (goats and swine) tend to develop without significant abnormalities. Mice, which 

may provide useful information as model systems, can exhibit different anomalies from 

those observed in domestic livestock species including obesity and decreased lifespan. 

Animal clones that survive the critical perinatal period appear to develop normally. Even 

animals with physiological perturbations, including less severe manifestations of LOS, 

seem to resolve them, usually within a period of weeks. Umbilical abnormalities that 

have been noted can be treated successfully with surgery. To date, all of the physiological 

instabilities that were observed resolve by the time the animals reach adolescence. Clones 

that reach reproductive age appear to be normal in all of the measures that have thus far 

been investigated, and appear to give rise to healthy, apparently normal progeny. 
Chapter VII: Summary and Conclusions 307 

Animal Cloning: A Risk Assessment DRAFT

Studies that have evaluated epigenetic reprogramming in live, healthy clones indicate that 

although there is some variability between clones and their sexually-derived counterparts, 

these clones have undergone sufficient epigenetic reprogramming to carry out 

coordinated functions necessary for survival and normal functioning. Molecular analyses 

reveal relatively small methylation differences, and either the animals are tolerant of such 

differences, or the epigenetic differences are below the threshold that poses observable 

adverse health outcomes. 

In order to evaluate potential food consumption risks associated with healthy-appearing 

clones, we have developed a two-pronged approach. The first part of the approach is 

based on the hypothesis that a healthy animal is likely to be safe to eat, and relies on the 

CBSA. The second component, or the Compositional Analysis Approach, assumes that if 

there are no material differences between the composition of milk and meat from animal 

clones (and their progeny) and their non-clone counterparts, then edible products derived 

from clone meat or milk would be as safe to eat as corresponding products from nonclones. This assessment assumes that animal clones and their progeny would be subject to 

all of the existing federal and state requirements for milk and meat. 

Because each clone arises from an independent event, identification and characterization 

of potential subtle hazards is best accomplished by the evaluation of individual animals, 

at as fine a level of resolution as possible. Characterization of the overall functionality of 

clones, however, is likely best considered by evaluating the animal as a whole, in 

particular assessing the degree to which highly complex functions have been integrated, 

for example by demonstrating successful reproduction. 

 

Progeny of animal clones are not anticipated to pose special animal health or food 

consumption concerns, as they are the product of sexual reproduction. The production of 

gametes by clones is expected to reset even those residual epigenetic reprogramming 

errors that could persist in healthy, reproducing clones. A large, well-controlled study on 

the health of swine clone progeny indicates that they are healthy and indistinguishable 

from other sexually-derived swine comparators. Because the value of clones lies in their 

genes, they are most likely to be used as breeding stock, and their food use would be 

incidental. Almost all of the production animals (i.e., sources of meat and milk) from the 

overall SCNT process are therefore likely to be sexually-reproduced progeny of clones. 

Most of the data on which the preceding conclusions are drawn have been generated from 

cattle, in particular, from a set of data including both health and physiological 

measurements on clones generated by Cyagra. Data on reproductive function in bovine 

clones indicates that healthy clones surviving to reproductive age have normal 
Chapter VII: Summary and Conclusions 308 

Animal Cloning: A Risk Assessment DRAFT

reproductive function and produce normal offspring. Although the database for swine 

clones is smaller than for cattle clones, physiological and health outcomes were 

consistent with normal functionality among the clones. Almost no data on the health of 

sheep clones were available for review. The dataset on goat clones contains information 

on reproductive function in males, and preliminary physiological measurements on males 

and females that are consistent with those evaluated for cattle and swine indicating that 

the clones function normally. 

Analysis of the composition of meat from bovine and swine clones and milk from bovine 

clones consistently indicates that there are no biologically relevant differences between 

the composition of food from clones, their close comparators, or food commonly 

consumed from these species on a daily basis. An extensive dataset on the progeny of 

swine clones indicates that the composition of meat from those animals does not differ 

from that of comparator animals. 

The food consumption portion of the risk assessment postulates that because the only 

hazards that may be present in clones would arise from epigenetic dysregulation, and 

because only healthy animals meeting the same standards that conventional food animals 

or their edible products meet would be permitted for use as food, the only hazards that 

could be present in these animals would be subtle. Allergenicity and mutagenicity studies 

confirm that there are no food safety hazards. 

Although the data indicate that the clones meeting the criterion described above would 

not likely pose a food consumption risk, some residual uncertainty is associated with this 

judgment. The source(s) of the uncertainty may be sorted into three categories: 

1. Uncertainties associated with empirical observations. Uncertainties are lowest for 

those individual clones whose health has been thoroughly evaluated and, by 

inference, clones produced subsequently using the same methodology. The 

uncertainties associated with the evaluation of empirical observations can be a 

function of the size, consistency, and quality of the data being evaluated. For 

example, the degree of confidence that can be placed in judgments arising from a 

well-conducted, consistent, and extensive dataset is much higher than from a 

small, poorly designed, and highly variable dataset. Further, because datasets tend 

to arise from an individual laboratory or producer, the uncertainties associated 

with that producer and method are lower than for other laboratories or producers 

for which less information is available. 

2. Uncertainty stemming from biological sources can be minimized by the 
Chapter VII: Summary and Conclusions 309 

Animal Cloning: A Risk Assessment DRAFT

evaluation of the clones themselves. The most important factor in this evaluation 

is the healthy survival and functionality of individual clones, indicating that either 

the animal has minimal epigenetic dysregulation, or that any initial epigenetic 

dysregulation has been resolved. Uncertainty would be the lowest for individual 

clones demonstrating successful reproduction. 

3. Uncertainties stemming from technological or methodological grounds 

encompass the degree to which judgments regarding clones arising from 

technologies in use when this risk assessment was conducted can be applied to 

modifications of the technology. These may only be resolved by the evaluation of 

the outcomes of those technological changes (i.e., the actual clones). 

Thus, our overall conclusions are: 

For Animal Health: SCNT results in an increased frequency of health risks to animals 

involved in the cloning process, but these do not differ qualitatively from those observed 

in other ARTs or natural breeding. The frequency of live normal births appears to be low, 

although the situation appears to be improving as the technology matures. Cattle and 

sheep exhibit a set of clinical signs collectively referred to as LOS that do not appear to 

be present in swine or goats. Surrogate dams are at risk of complications from birth if the 

fetus suffers from LOS, or from accumulation of fluid in the cavities of the placenta 

(hydrops). Clones exhibiting LOS may require additional supportive care at birth, but can 

recover and mature into normal, healthy animals. Most clones that survive the perinatal 

period are normal and healthy as determined by physiological measurements, behavior, 

and veterinary examinations. Progeny of animal clones also have been reported as normal 

and healthy. 

For Food Consumption Risks: Extensive evaluation of the available data has not 

identified any food consumption risks or subtle hazards in healthy clones of cattle, swine, 

or goats. Thus, edible products from healthy clones that meet existing requirements for 

meat and milk in commerce pose no increased food consumption risk(s) relative to 

comparable products from sexually-derived animals. The uncertainties associated with 

this judgment are a function of the empirical observations and underlying biological 

processes contributing to the production of clones. There is less uncertainty about the 

health of clones as they age and have more time to exhibit the full range of functionality 

expected of breeding stock. Edible products derived from the progeny of clones pose no 

additional food consumption risk(s) relative to corresponding products from other 

animals based on underlying biological assumptions, evidence from model systems, and 

consistent empirical observations. 
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Animal Cloning: A Risk Assessment DRAFT
 

Animal Cloning: A Risk Assessment DRAFT

Glossary 

The following terms are defined as they are used within the current risk assessment. 

Unless otherwise indicated, definitions provided are the commonly accepted use of the 

term(s) at the Center for Veterinary Medicine, and may have been derived from various 

sources.71

allele Any alternative form of a gene that can occupy a particular 

chromosomal locus. 

anal atresia Abnormally closed anal opening. 

analyte A substance undergoing analysis. 

 

aneuploid Describes a cell or organism which has an abnormal total number of 

chromosomes and where numbers of individual chromosomes are 

out of proportion with the numbers of the other chromosomes. Too 

many chromosomes is called hyperploidy; too few is called 

hypoploidy. 

 

animal clones Animals derived via somatic cell nuclear transfer techniques. The 

terminology employed in this assessment did not use “cloned 

animals.” The phrase “cloned animals” does not clearly differentiate 

between the animal serving as the source of genome being 

propagated, or the animal that has been generated from a particular 

source. For example, the sentence “That field contains several 

cloned animals” does not specify whether the animals had been 

used as a source of material for SCNT or whether they had been 

generated by that technology. 

ARTs Assisted reproductive technologies. 

biallelic Referring to expression of two alleles at the same time. 

 

71 The various sources used for these definitions include: Dorland’s Medical Dictionary, 30th Ed., W.B. 

Saunders Company, Philadelphia, 2003; Dictionary of Epidemiology. 3rd Ed. John M. Last. Oxford 

University Press, 1995; HTTP://bioethics.gov; http://biotech.icmb.utexas.edu; Large Animal Internal 

Medicine, 2nd Ed., Smith, B.P., Ed., Mosby – Year Book, Inc., St. Louis, 1996.; Veterinary Medicine: A 

Textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses. 7th Ed. Blood, D. C. and O. M. 

Radostits, Philadelphia: Bailliere Tindall Company, 1989. The Merck Veterinary Manual, 8th Ed. Online 

Version. C.M. Kanh and S Line, Ed. Merck & Co., Inc, NJ, 2003; and The American Heritage® 

Dictionary of the English Language, 4th Ed., Houghton Mifflin Company. 2002. 
Glossary 312 

Animal Cloning: A Risk Assessment DRAFT

bioengineered 

animals 

The broadest category of animals associated with molecular biology 

techniques, including animal clones and all genetically engineered 

animals. 

blastocyst An early stage in the development of mammalian embryos, when 

the embryo is a spherical body comprising an inner cell mass that 

will become the fetus and an outer ring of cells, the trophectoderm, 

that will become part of the placenta. 

blastomere Any one of the cells formed from the first few cell divisions in 

animal embryology. The embryo usually divides into two, then four, 

then eight blastomeres, and so on. 

Blastomere 

Nuclear Transfer 

(BNT) 

An assisted reproductive technique in which a blastomere is used as 

a donor for nuclear transfer into enucleated oöplasts.

capacitation The process of sperm maturation (or activation) that occurs postejaculation. Allows the spermatozoa to go through the acrosomal 

reaction in which factors in the sperm head that allow it to penetrate 

the egg are released and fertilize an oöcyte. 

caruncle The site of attachment in the maternal uterus of the ruminant for the 

placental cotyledon (see cotyledon). 

 

centromere 

(centromeric)

A specialized chromosome region to which spindle fibers attach 

during cell division (mitosis) that is genetically inactive. This is 

constricted region of a mitotic chromosome that holds sister 

chromatids together—the crossing point in the “X” often used to 

depict chromosomes. 

chimera An organism or recombinant DNA molecule created by joining 

DNA fragments from two or more different organisms. 

chondrocyte A mature cartilage cell. 

chorion The outermost membrane enclosing the fetus. It is formed from 

tissues on the outside of the embryo such as the trophoblast, and the 

part of it attached to the uterus wall eventually develops into the 

placenta. 

chromatid One of the two daughter strands of a duplicated chromosome. 

chromatin The network of fibers of DNA and protein that make up the 

chromosomes of the eukaryotic nucleus during interphase. 
Glossary 313 

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chromosome(s) A structure composed of one very long molecule of DNA and 

associated proteins (e.g. histones) that carries hereditary 

information. 

cleavage The series of mitotic divisions by which a fertilized animal ovum 

changes, without any overall change in size, into a ball of smaller 

cells constituting the primitive embryo. 

clone A group of cells or individuals that are genetically identical as a 

result of asexual reproduction including nuclear transfer. 

cloning Asexual reproduction of animals using somatic cell nuclear transfer 

(SCNT). 

coherence The extent to which a hypothesized causal association is compatible 

with preexisting theory and knowledge. 

colostrum The first fluid secreted by the mammary glands at the time of 

birthing that is rich in antibodies and nutrients, and precedes the 

production of true milk. Its ingestion confers passive maternal 

immunity on the offspring of some species. 

Comprehensive 

Veterinary Exam 

(CVE) 

Systematic approach for examining domestic livestock animals and 

making informed judgments as to their health. The CVE contains 

both objective and subjective information and is performed by a 

veterinarian. 

 

congenital Existing at, and usually before, birth; referring to conditions that are 

present at birth, regardless of their causation. 

consistency Close conformity between findings in different studies conducted 

by different methods or different investigators. 

cortisol The major natural glucocorticoid hormone synthesized in the zona 

fasciculata of the adrenal cortex; it affects the metabolism of 

glucose, protein, and fats. It also regulates the immune system and 

affects many other functions. 

cotyledon A lobule structure in ruminant placentae that form contact points 

between the fetal-derived placental tissues with the maternal 

caruncles (attachment points) of the uterus to form the functional 

units called placentomes. It consists mainly of a rounded mass of 

villi. 

cryptorchid A male animal with one or both testicles retained within the body 

cavity. 
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cull To remove unwanted members or parts from a herd. 

cytoplasm The living contents of the cell, exclusive of the nucleus, consisting 

of an aqueous protein matrix or gel, and where essential membranes 

and cellular organelles (mitochondria, plastids, etc.) reside. 

de novo Literally means “anew.” Beginning a process from its origin with 

out prior plans. 

dermatitis 

vegetans 

A hereditary disease of the skin in swine (see hyperkeratosis).

differentiation The process whereby relatively unspecialized cells, e.g. embryonic 

or regenerative cells, acquire specialized structural and/or functional 

features that characterize the cells, tissues, or organs of the mature 

organism or some other relatively stable phase of the organism’s 

life history. 

diploid Having two sets of chromosomes. 

DNA Abbreviation for deoxyribonucleic acid; one of the two types of 

nucleic acids that constitutes the genetic material of most known 

organisms; usually in double helix form. 

DNA polymerase The enzyme responsible for copying DNA. Common name for 

either of two categories of enzymes that catalyze the synthesis of 

DNA from deoxyribonucleoside triphosphates in the presence of a 

nucleic-acid primer. 

ductus arteriosus The blood vessel between the pulmonary artery (carries blood from 

the heart to the lungs for oxygenation) and the aorta (carries 

oxygenated blood to the rest of the body). During gestation the 

ductus arteriosus bypasses the fetal lungs, and is normally sealed 

after birth. 

ductus venosus The blood vessel between the umbilical vein and the caudal vena 

cava (carries oxygenated blood from the dam, bypassing the liver, 

through the vena cava to the heart of the fetus). It is normally sealed 

shortly after birth. 

dysregulate Abnormal or impaired control of gene expression. 

dystocia Abnormal or difficult labor. 
Glossary 315 

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ectoderm The outermost layer of tissue in a developing embryo that will 

eventually become the skin and/or other outer surface of the 

organism, the outermost parts of the nervous system, and various 

other outer and external organs depending on the organism. 

embryo In mammals, the term is restricted to the structure present in the 

early part of gestation that develops into a fetus. 

embryo cloning Another term for blastomere nuclear transfer. 

empirical That which can be seen or observed alone, often without reliance on 

theory. 

endoderm The innermost layer of tissue in a developing animal embryo that 

will eventually become the digestive tract, respiratory tract, and 

various other things depending on the organism. 

enucleate Removal of an organ or mass from its supporting tissues. 

epigenetic Describing any of the mechanisms regulating the expression and 

interaction of genes, particularly during the development process. 

These include changes that influence the phenotype but have arisen 

as a result of mechanisms such as inherited patterns of DNA 

methylation rather than differences in gene sequence: imprinting is 

an example of this. 

epigenetic 

reprogramming 

In the case of somatic cell nuclear transfer (SCNT), the process of 

altering the instructions governing the expression of genes in the 

chromosomal DNA of the donor cell such that embryonic or 

totipotent (able to differentiate along any line or into any type of 

cell) gene expression conditions are reestablished. 

epigenetic 

variation/effects 

Non-hereditary, phenotypic changes in the expression in a single 

gene.

estrous Pertaining to estrus. (Adjective) 

estrus The recurrent, restricted period of sexual receptivity in female 

mammals other than human females, marked by intense sexual urge. 

(Noun) 

euchromatin One of two types of chromatin seen during interphase of the cell 

cycle. It is genetically active (transcription occurs in it) and less 

condensed than heterochromatin (the other type of chromatin). 

eukaryote An organism whose cells have a true nucleus, i.e., one bounded by a 
Glossary 316 

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nuclear membrane, within which lie the chromosomes, combined 

with proteins and exhibiting mitosis; eukaryotic cells also contain 

many membrane-bound compartments (organelles) in which 

cellular functions are performed. 

F1 Abbreviation for filial generation 1 (first generation). The initial 

hybrid generation resulting from a cross between two parents. 

farrow In swine, the process of giving birth. Also used to describe a litter of 

pigs. 

fat cow syndrome A multifactorial disease condition often occurring in dairy cows 

following parturition; associated with excessive mobilization of fat 

to the liver in well-conditioned cows. This mobilization is induced 

by the negative energy balance and hormonal changes. Presenting 

signs usually include depression, anorexia, weight loss, and 

weakness that can lead to recumbency. 

fecundity The physiological ability to reproduce, as opposed to fertility. 

fertility The capacity to conceive or induce conception. 

foramen ovale A hole in the fetal heart between the right and left atria, for the 

purpose of bypassing the lungs. It is normally sealed shortly after 

birth. 

founder animal An organism that serves as the progenitor of a particular lineage. 

freemartin A sexually maldeveloped female calf born as a twin to a normal 

male calf. The reproductive tract hypoplasia results in an infantile 

uterus that does not develop appropriately with the growth of the 

rest of the calf and fails to respond to puberty. It is commonly 

sterile and intersexual as the result of male hormones reaching it 

through shared placental blood vessels. 

gamete A mature reproductive cell capable of fusing with a cell of similar 

origin but of opposite sex to form a zygote from which a new 

organism can develop. Gametes normally have haploid 

chromosome content. In animals, a gamete is a sperm or egg. 

gametogenesis The process of the formation of gametes. 

gene expression The process by which a cell transcribes the information stored in its 

genome to carry out the functions of life. 

genetic The process of rearranging the genome of the nucleus to restore a 
Glossary 317 

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reprogramming cell’s totipotency so it can differentiate into different types of cells 

and develop into a whole organism. Also known as dedifferentiation. 

genetically 

engineered animals 

A subset of animals associated with molecular biology techniques. 

Includes transgenic animals, animals subjected to gene therapy and 

mosaic animals. This subset does not include animal clones. 

genome The full set of genes in an individual, either haploid (the set derived 

from one parent) or diploid (the set derived from both parents). 

genotype The entire genetic constitution of an individual. 

germ cell A reproductive cell such as a spermatocyte or an oöcyte, or a cell 

that will develop into a reproductive cell. 

gilt A female pig that is intended for breeding but has not yet given 

birth. 

gonadotropin Any hormone that stimulates the testes or ovaries. 

haploid An individual or cell having only one member of each pair of 

homologous chromosomes. 

harm An adverse outcome. 

hazard Something that can produce harm. 

heifer A female bovine that has not yet produced a calf. 

hematology The branch of medicine that deals with the blood and blood-forming 

tissues. 

hemogram A written record or graphic representation of a detailed blood 

assessment such as the complete blood count or differential 

leukocyte count. 

hermaphrodite An individual characterized by the presence of both male and 

female sex organs. The condition is caused by an anomalous 

differentiation of the gonads: an animal with ambiguous genitalia, 

typically a penis with ovaries or a vulva with testicles 

heterochromatin The condensed and genetically inactivated portion of a 

chromosome. 

histones Chromatin proteins commonly associated with the DNA of somatic 
Glossary 318 

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cells in eukaryotes and they are involved in packaging of the DNA 

and the regulation of gene activity. 

hormone A chemical substance produced in the body by an organ, cells of an 

organ, or scattered cells, having a specific regulatory effect on the 

activity of an organ or organs. The term was originally applied to 

substances secreted by endocrine glands and transported in the 

bloodstream to distant target organs, but later it was applied to 

various substances having similar actions but not produced by 

special glands. 

hydroallantois Abnormal fluid accumulation in the allantoic cavity of the placenta. 

(See hydrops.) 

hydrops Edema. Hydrops refers to a set of conditions relating to abnormal 

fluid accumulation in one or more compartments of the placenta 

and/or the fetus itself, and are alternatively referred to as 

hydroallantois, hydramnios or hydrops fetalis, depending on where 

the edema occurs. 

hyperkeratosis Characterized by lesions of the superficial layers of the epidermis. 

These lesions rapidly become covered with scales, and then develop 

hard, dry crusts with deep fissures. Generally referred to as 

parakeratosis in swine. 

hypoplasia Incomplete development or underdevelopment of an organ or tissue. 

hypospadius A developmental anomaly in which the urethra opens inferior 

(below) to its usual location; usually seen in males with the opening 

on the underside of the penis or on the perineum. 

imprinted genes Those genes whose degree of expression is determined by their 

derivation from either the dam or the sire. 

in vitro Outside the organism, or in an artificial environment. This term 

applies, for example, to cells, tissues or organs cultured in glass or 

plastic containers. 

in vivo Literally means "in life;” a biologic or biochemical process 

occurring within a living organism. 

inner cell mass The group of cells in a blastocyst that are destined to form the fetus. 

inner cell mass 

(ICM) 

A cluster of cells within the blastocyst. The inner cell mass will 

form all of the tissues of the organism and these cells are 

pluripotent. 
Glossary 319 

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ketonuria Ketone bodies in the urine, as in diabetes mellitus; called also 

acetonuria and hyperketonuria. 

ketosis A metabolic disease of lactating dairy cows characterized by weight 

loss, decreased milk production, and neurologic abnormalities that 

usually occur during the first 6 weeks of lactation. 

Large Offspring 

Syndrome (LOS) 

A morphologic syndrome presumably expressed at the molecular 

and physiological level due to some alterations in embryonic gene 

expression. Animal clones with LOS may experience difficulties in 

developing and maintaining the placenta. An LOS fetus is unusually 

large for its species, has longer than usual gestation periods, and 

often has immature lungs or heart abnormalities. Kidneys and liver 

may also be affected. 

leukocytosis A transient increase in the number of leukocytes (white blood cells) 

in the blood. 

 

leukopenia A reduction in the number of leukocytes in the blood. 

locus The specific site of a gene on a chromosome. 

long terminal 

repeats 

A double-stranded sequence, generally several hundred base pairs 

long, at the two ends of the genetic sequence of retroviruses. 

mastitis Inflammation of the mammary gland or breast. 

meconium First stool in the intestine of a full-term fetus. 

meiosis The process in which a single diploid cell becomes four haploid 

cells in two consecutive divisions of the nucleus of an eukaryotic 

cell. In multicellular higher organisms this occurs only in the 

progenitors of sex cells and never in somatic cells. 

methylation The addition of a methyl group (-CH3) to a larger molecule (e.g. 

cytosine methylation) 

 cytosine 5-methyl cytosine 
Glossary 320 

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metritis Inflammation of the uterus. 

mitosis The division of a eukaryotic cell nucleus to produce two daughter 

nuclei that contain identical numbers of chromosomes and that are 

identical genetically to the parent nucleus except where crossing 

over or mutation has occurred. 

monozygotic twin One of a pair of twins derived from a single fertilized egg or zygote. 

Synonym: identical twin. 

morphology The form and structure of an organism, organ, or part. 

morula The solid mass of blastomeres formed from the cleavage of a 

fertilized ovum or egg. 

murine Pertaining to or affecting mice or rats. 

neoplasia Abnormal and uncontrolled cell growth that often produces a tumor 

(a neoplasm) that may or may not be cancerous (i.e., capable of 

spread or metastasis). 

nuclear transfer Transferring the nucleus with its chromosomal DNA from one 

(donor) cell to another (recipient) cell. 

nucleic acids A large molecule composed of nucleotide subunits. DNA 

(deoxyribonucleic acid) and RNA (ribonucleic acid) are examples. 

nucleoside A molecule composed of a purine or pyrimidine nitrogenous base 

attached to the five-carbon sugar. This glycosylamine is a 

component of nucleic acids. 

nucleotide A molecule composed of a purine or pyrimidine nitrogenous base 

attached to the five-carbon sugar which also has a phosphate group 

attached to it. It is the constitutional unit into which nucleic acids 

are broken down by partial hydrolysis and from which they are 

built. 

N

N

CH3

NH2

O

N

O N

NH2

CH3
Glossary 321 

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nucleus The most conspicuous organelle of a eukaryotic cell; it contains the 

chromosomes and is the site of genomic DNA replication and or 

RNA synthesis in the cell. 

oöcyte A cell of an animal ovary that undergoes meiosis to form an ovum. 

oöplasmic 

remodeling 

After nuclear transfer, the cytoplasm of the oöcyte (oöplasm) alters 

the morphology of the nucleus, so that it more closely resembles the 

nucleus of an embryo. 

oöplast The remaining portion of the oöcyte following enucleation. 

oviduct A tube from the ovary to the uterus through which ova (eggs) may 

pass. 

ovum The female reproductive cell which, after fertilization, becomes a 

zygote that develops into a new member of the same species. Also 

called an egg. 

parakeratosis A nutritional deficiency disease of 6- to 16-wk-old pigs that is 

characterized by lesions of the superficial layers of the epidermis. It 

is a metabolic disturbance resulting from a deficiency of zinc or an 

excess of calcium in the diet. 

parity The condition of having given birth. 

parthenogenesis The development of a new individual from an unfertilized female 

gamete. 

parturition The act or process of giving birth to offspring. 

patent ductus 

arteriosus 

The failure of the ductus arteriosus to close after birth resulting in 

extra blood flow to the lungs and recirculation of oxygenated blood 

to the lungs rather than the rest of the body. 

patent urachus The failure or the urachus to close during parturition, resulting in 

the inability to excrete urinary waste. 

phagocytosis The uptake of extracullular materials by the formation of a pocket 

from the cellular membrane and its subsequent pinching off. 

phenotype The totality of the observable functional and structural 

characteristics of an organism as determined by its genotype and its 

interaction with its environment. 
Glossary 322 

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phytate(s) A form of phosphorus commonly occurring in grain products, 

which is indigestible in non-ruminant species. 

placentomes Placental junctures consisting of the uterine caruncle and the 

placental cotyledon, which permits vascular transport of nutrients 

into and waste out of the fetal environment. 

ploidy Degree of repetition of the basic number of chromosomes. 

pluripotent Capable of differentiating into more than one cell type. 

polar body A small cell containing little cytoplasm that is the by-product of 

oöcyte meiosis in female animals. 

polycythemia An increase in the total red cell mass of the blood. 

polymorphism Describes a substance that can take on several different forms. Can 

refer to subtle differences in DNA sequences among individuals. It 

also may refer to a protein which can be coded by several different 

sequences; these variations do not ruin the protein's function. 

polyploidy The state of a cell having more than two times the haploid number 

of chromosomes in its nucleus. 

portal Anatomical nomenclature pertaining to an opening, especially the 

site of entrance to an organ of the blood vessels and other structures 

supplying or draining it. 

predation The capturing and consumption of prey as a means of maintaining 

life. 

pregnancy toxemia A pathologic metabolic disturbance of pregnancy that results when 

fetal carbohydrate or energy demand exceeds the maternal supply 

during the last trimester of pregnancy. Specific to sheep and goats. 

preimplantation A period very early in embryo development, before the embryo 

attaches to the uterus. 

progeny An animal derived from sexual reproduction that has at least one 

cloned animal as a parent (but could result from two cloned animals 

mating). 

 

promoter A sequence of the DNA molecule to which RNA polymerase will 

bind and initiate transcription. 
Glossary 323 

Animal Cloning: A Risk Assessment DRAFT

promoter A segment of DNA acting as a controlling element in the expression 

of a gene. 

promoterenhancer sequence 

A control element that can increase expression of a gene. 

pronucleus The pronucleus is the structure that contains the haploid genome of 

the sperm or ovum after fertilization occurs, but before they fuse to 

make the nucleus of the zygote, or the single-celled diploid 

organism. 

 

p-value A measure of the probability that a difference between groups 

during an experiment happened by chance. 

recumbancy Lying down. 

rendering Reducing, converting, or melting down animal by-products by 

heating; a cooking and drying process that yields fat of varying 

grades, both edible and inedible (depending on raw material 

source), and animal protein that is useful for animal feeds and 

fertilizer. 

risk A set of conditions that links an exposure to the likelihood of an 

adverse outcome. 

risk assessment The methodology used to characterize potential risks and the 

conditions that result in the potential to experience risk. 

risk management The set of activities applied to identify and evaluate alternative 

strategies (often regulatory), and select among them on the basis of 

economic, political, scientific, ethical and social conditions or 

criteria. 

RNA Abbreviation for ribonucleic acid that serves to carry information 

from DNA to other parts of the cell or that has other functions. The 

generation of messenger RNA is a critical step in gene expression. 

RNA polymerase An enzyme that transcribes the information in a DNA sequence into 

RNA. 

ruminant Animals having a rumen - a large digestive sac in which fibrous 

plant material is fermented by commensal microbes, prior to its 

digestion in a "true" stomach (the abomasum). Common farm 

ruminants are cattle and sheep. 

SCNT Acronym for Somatic Cell Nuclear Transfer. The process of 

generating a live organism asexually by transferring the diploid 
Glossary 324 

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nucleus of a somatic cell from a donor animal to the enucleated 

embryo of a recipient animal. 

scours Severe diarrhea in farm animals. 

senescence The process or condition of growing old in which cells, tissues, and 

organisms deteriorate and finally die. 

sequellae Morbid conditions occurring as a consequence of another condition 

or event. 

sexual 

reproduction 

The production of offspring by the fusion of male and female 

gametes (in contrast to ‘asexual reproduction’). 

somatic cell Any cell of an organism other than a germ cell. 

stem cell A totipotent or pluripotent cell that can replicate indefinitely and 

which can differentiate into other cells; stem cells serve as a 

continuous source of new cells. 

stochastic Pertaining to a random process, used particularly to refer to a time 

series of random variables. Arrived at by skillful conjecture; e.g. a 

stochastic model, a stochastic process. 

superovulate To produce numerous ova at one time. 

telomerase A DNA polymerase enzyme that maintains the structure of the 

telomere by adding the required repetitive sequences to the ends of 

eukaryotic chromosomes. 

telomere The structure that seals the end of a chromosome. 

tetraploid An organism or cell containing four haploid sets of chromosomes 

(see polyploidy). 

totipotent Capable of becoming any cell type in the body. 

transcription The process by which a single-stranded RNA with a base sequence 

complementary to one strand of a double-stranded DNA is 

synthesized. 

transgenic Contains heritable DNA from another source. A transgenic animal 

is one that has been intentionally altered using molecular biology 

techniques that result in heritable changes (insertions, deletions or 

rearrangements) in the nucleic acid sequence of the nucleus or 

mitochondria, and includes any offspring that inherit those changes. 
Glossary 325 

Animal Cloning: A Risk Assessment DRAFT

translation The second major step of gene expression in which the particular 

sequence of bases in the transcribed mRNA determines the 

sequence of amino acids in the proteins (or polypeptides) being 

synthesized (see transcription). 

transposable 

element 

A genetic element that has the ability to move (transpose) from one 

site on a chromosome to another. 

trophectoderm The group of cells in the blastocyst that form the placenta and other 

non-fetal tissues. 

trophoblast A layer of extra-embryonic ectodermal tissue on the outside of the 

blastocyst. It attaches the blastocyst to the endometrium of the 

uterine wall and supplies nutrition to the embryo. 

urachus A structure through which a fetus excretes urinary waste. In normal 

development, this structure would close at the time of parturition. 

ventricle 

(ventriculus) 

A small cavity or chamber within a body or organ, especially: (a) 

the chamber on the left side of the heart that receives oxygenated 

arterial blood from the left atrium and contracts to force it into the 

aorta; and (b) the chamber on the right side of the heart that receives 

deoxygenated venous blood from the right atrium and forces it into 

the pulmonary artery. 

villi Microscopic vascular protrusions from the surface of a membrane. 

wild type The phenotype that is characteristic of most of the members of a 

species occurring naturally and contrasting with the phenotype of a 

mutant. 

xist Enzyme that deactivates one of the two X chromosomes in female 

embryos. 

zona pellucida The thick, transparent, non-cellular outer layer surrounding an 

oöcyte and fertilized ovum. 

zygote The diploid cell that results from the union of a sperm cell and an 

egg cell.
 

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Appendix A: 

Risk and Safety Assessment Primer for 

Animal Cloning
 

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Animal Cloning: A Risk Assessment DRAFT

Appendix A: 

Risk and Safety Assessment Primer for 

Animal Cloning 

A. How has Risk Assessment Evolved? 

Although the overall process of dividing risk assessment into operational steps has been altered 

to address the nature of the substances or processes being evaluated, the fundamental 

components of the risk assessment process have remained relatively constant. Thus, for any 

particular etiologic (causative) agent or process, 

(1) the universe of potential outcomes that may be causally associated with exposure 

are identified and characterized; 

(2) the relationships between exposure and outcome are described; 

(3) estimates of potential exposure are made; and then, 

(4) the qualitative and quantitative (when available) components are integrated into 

an estimate of the likelihood of the potential outcomes to occur given that 

exposure also occurs. 

Because information for decision-making is often incomplete, risk characterization also must 

take into account the degree of uncertainty associated with any of the steps in the overall process, 

as well as the cumulative contribution(s) that such uncertainties may make to the overall risk 

estimate. 

At various times, the National Academy of Sciences (NAS) has attempted to describe risk 

analysis in different ways (Table A-1). The 1983 NAS report “Risk Assessment in the Federal 

Government,” first attempted to consolidate the risk assessment procedures practiced in the US 

regulatory agencies (primarily FDA’s Bureau of Foods, which subsequently became the Center 

for Food Safety and Applied Nutrition) into four coherent steps. At that time, these steps were 

appropriate to the nature of the substances on which risk assessments were performed e.g., 

radiation and chemical carcinogens. 

Chief among the shared characteristics of these substances was the ability to describe dose in 

discrete units, allowing for the relative precision of exposure and dose-response estimates. By 
Appendix A: Risk and Safety Assessment Primer A-4 

Animal Cloning: A Risk Assessment DRAFT

the time of the publication of the NAS’s 2002 report “Animal Biotechnology: Science-Based 

Concerns” (NAS 2002b), the description of the risk assessment process had evolved to be more 

accurately suited for the potential risks associated with animal biotechnology. The most 

important differences reflect the change of etiologic agents from radiation and chemicals to 

biological agents or processes. These differences are most obviously manifested in the hazard 

assessment and dose-response sections, where the range of potential adverse outcomes (harms) 

can differ in kind from radiation and chemical damage, and the concept of dose must 

accommodate biological potential. Biological potential can be thought of as the ability for the 

substance or organism being evaluated to either grow, replicate, die, or perform a catalytic 

function so that dose is no longer a constant (or possibly decreasing) amount. 

Table A-1: Risk Analysis Steps as Described by the National Academy of Sciences 

1983 “Red Book” 2002 Animal Biotechnology Report 

o Hazard Identification 

o Exposure Assessment 

o Dose Response Evaluation 

o Risk Characterization 

o Identify potential harms 

o Identify potential hazards that might produce 

those harms 

o Define what exposure means and the 

likelihood of exposure 

o Quantify the likelihood of harm given that 

exposure has occurred 

B. Thinking About Risk 

Qualitatively, risk may be thought of as some function of the combination of exposure and the 

intrinsic properties of the substance or process under consideration by linking an exposure to the 

likelihood of an outcome. The “risk equation” was first derived for the condition of carcinogen 

exposure and written as: 

Risk = (exposure) x (potency) 

where potency was estimated from an evaluation of the relationship between exposure and 

outcome (i.e., the dose-response evaluation). More generally, however, the risk equation is best 

thought of as some function of exposure and some function of the biological properties of the 

agent causing the outcome: 
Appendix A: Risk and Safety Assessment Primer A-5 

Animal Cloning: A Risk Assessment DRAFT

Risk ∝ foutcome (exposure, hazard)

In cancer risk assessment, the function of outcomes was often referred to as the “cancer potency” 

and was derived from the slope of the dose-response curve for tumor formation. For animal 

cloning, outcomes may be thought of as the adverse health effects resulting from cloning such as 

Large Offspring Syndrome, or for edible products of clones, a lack of expected nutritional 

content of milk from animal clones. 

Thinking about risk from the perspective of an “equation” is useful, even when performing 

qualitative analyses, because it allows the equation to be “solved” for any of the variables that 

have been defined. Often we ask the “forward” or prospective question: given that some process 

or exposure has occurred, what is the likelihood of a particular outcome (e.g., how likely is 

exposure to a particular contaminant in milk to cause gastrointestinal distress?). Alternatively, 

the question can be asked in the “backwards” or retrograde form: given that an outcome has 

occurred, what etiologic agent under which exposure conditions is responsible for that outcome 

(e.g., given gastrointestinal distress, did consumption of milk contaminated with x amount of y

substance cause that effect? or how much of x do you have to consume before gastrointestinal 

distress is experienced?). 

When performing a risk analysis, it is critically important to distinguish between a hazard and 

the potential risk(s) that may result from exposure. A hazard can be defined as an act or 

phenomenon that has the potential to produce an adverse outcome, injury, or some sort of loss or 

detriment. These are sometimes referred to as harms, and are often identified under laboratory 

conditions designed to maximize the opportunity to detect adverse outcomes. Thus, such 

observational summaries are often referred to as “hazard identification” or “hazard 

characterization.” Risk, as previously discussed, is the conditional probability that estimates the 

probability of harm given that exposure has occurred. In a qualitative assessment such as this, 

however, risks can be discussed only within a qualitative context, and no quantitative 

interpretations should be made. 

Another important question to consider is who experiences the risk. At its inception, risk 

assessment tended to be anthropomorphic; all risks were evaluated in the human sphere, and 

were expressed in units of the individual, that is, the probability of a person being exposed to a 

hazard and experiencing a harm over a lifetime. That individual was defined as the receptor. 

Human risks could also be expressed at the population level, or the probability of x individuals in 

the population experiencing the harm. For animal cloning issues, the receptor can be considered 
Appendix A: Risk and Safety Assessment Primer A-6 

Animal Cloning: A Risk Assessment DRAFT

to be the surrogate dam carrying a fetal clone, the animal clone itself, or humans or other animals 

consuming edible products of clones (e.g., milk and meat). 

C. How Do We Think About Safety? 

For purposes of the Draft Risk Assessment Safety may be best thought of as the condition under 

which risks would be considered unlikely, rather than the condition of no risk (as such conditions 

do not exist for any scenario). It implies that a risk analysis has been performed, and the “risk 

equation” is solved for the condition that Risk  0 (i.e., the conditions under which risk 

approaches zero). When considering food from animal clones, this risk assessment has 

approached the issue of safety from a comparative perspective. Because one of the basic 

questions that the food consumption portion of this risk assessment asks is whether animal clones 

are materially different from their conventional counterparts, the risk question that is asked is 

whether edible products from animal clones or their progeny pose an increased risk relative to 

the same products from conventional comparators. Likewise, for animal safety, the question that 

is asked is whether animals involved in the cloning process are at greater risk for any adverse 

outcome relative to other assisted reproductive technologies. 

One of the difficulties with any safety assessment is “proving the negative.” Because in practice 

the universe of conditions under which some risk may be encountered cannot be explored, there 

are always some conditions under which the null hypothesis (i.e., exposure to y μg/liter of 

Substance X will pose no significant risk) will not be disproved. Thus, a careful risk/safety 

assessment defines the boundaries of its investigation and expresses its conclusions within those 

particular limits (i.e., clones born after a carefully monitored pregnancy under closely supervised 

conditions are at a slightly increased risk of dying than animals derived via in vitro fertilization, 

or artificial insemination, or, for food safety, milk from dairy cow clones that meets existing 

regulatory standards and is not significantly different from Grade A bulk tank milk is as safe to 

drink as milk meeting existing regulatory standards from Grade A bulk tank milk derived from 

non-clone dairy cows). 
Animal Cloning: A Risk Assessment DRAFT

Appendix B: 

Overall Reproductive Efficiency and Health 

Statistics for US Animal Agriculture
Animal Cloning: A Risk Assessment DRAFT

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Animal Cloning: A Risk Assessment DRAFT

Appendix B :Overall Reproductive Efficiency 

and Health Statistics for US Animal 

Agriculture 

In order to gain a better understanding of the animal safety issues associated with SCNT, it is 

helpful to review statistics on animal health and reproduction under current agricultural 

practices. This section draws data from reports published by USDA/APHIS National Animal 

Health Monitoring System (NAHMS at http://www.aphis.usda.gov/vs/ceah/ncahs/nahms/index.htm) and 

the National Agricultural Statistics Service (NASS at 

http://www.nass.usda.gov/census/census02/volume1/us/index1.htm). 

A. Dairy cattle 

The Dairy 2002 Part I: Reference of Dairy Health and Management in the United States report 

(USDA/NAHMS 2002) surveyed a total of 2,461 dairy operations in the United States. 

According to the USDA/NASS census, in 2002, there were 9,109,600 milking dairy cows in the 

United States. The predominant breed of dairy cattle in the US is Holstein, comprising 93.4 

percent of the national herd. The next most popular breed is the Jersey, comprising about 3.6 

percent of dairy cattle in the US. Other “colored” breeds (Guernsey, Brown Swiss, Ayrshire, and 

others) make up the remaining minority, and numbers for these breeds are more variable. 

Individual dairies vary in size from fewer than 100 to as many as 10,000 cows. Due to the 

variability in size of dairies, the USDA/NAHMS report broke dairies down into three groups: 

fewer than 100 cows, 100-499 cows, and greater than 500 cows. The report does not supply 

statistics for individual breeds of dairy cattle. 

According to the USDA/NAHMS 2002 report, the most commonly reported causes of cow 

illness for all operations were clinical mastitis, infertility problems (failure to conceive by 150 

days postpartum) and lameness. Incidence of retained placenta was also a commonly reported 

problem (7.8 ± 0.2 percent), and may have contributed to incidence of reproductive problems. 

Incidence of clinical mastitis was similar across operations, and averaged 14.7 percent of all 

cows. Table B-1 presents data on causes and incidence rate of illness for operations responding 

to the survey. 
Appendix B: Overall Reproductive Efficiency and Health Statistics B-4 

Animal Cloning: A Risk Assessment DRAFT

Table B-1: Most commonly reported health problems contributing to morbidity, 

mortality and culling of US dairy cattle1

Cause % morbidity2 % mortality3 % culled4 

Clinical mastitis/udder 

problems 14.7 ± 0.3 17.1 ± 0.6 26.9 ± 0.5 

Lameness 11.6 ± 0.3 13.9 ± 0.6 

Reproductive 

 Infertility 

 Retained fetal membranes 

 Other (dystocia, metritis) 

11.9 ± 0.3 

7.8 ± 0.2 

3.7 ± 0.2 17.4 ± 0.75 

26.5 ± 0.56 

1

 Based on USDA statistics for 2002 population of 9,109,600 milking cows. 

2

 Expressed as percentage of all cows ± standard deviation of the mean. 

3

 Expressed as percentage of cows dying ± standard deviation of the mean. 

4

 Expressed as percentage of cows culled ± standard deviation of the mean. 

5

 Mortality attributed to dystocia 

6

 Culling for all reproductive problems 

The percent of dairy cows dying in 2002 was 4.8 ± 0.1 percent, and did not differ by size of 

operation. The most frequently reported causes of death for all dairy cows in this report were 

difficult labor, also known as dystocia (17.1 ± 0.6 percent) and mastitis (17.1 ± 0.6 percent). 

Mastitis may cause death by acute toxicity, or cows may be euthanized as a result of severe or 

persistent mastitis caused by treatment-resistant pathogens such as Staphyloccocus aureus or 

Mycoplasma species. The percent of cows culled for mastitis or other udder problems in 2002 

was 26.9 ± 0.5 percent for all cows culled, and represented one of the most common reasons for 

culling. Culling due to reproductive problems was an equally common reason given by producers 

in this report (26.5 ± 0.5 percent of all cows culled), with poor production not due to illness as 

the next most common reason (22.4 ± 0.8 percent of all cows culled). On average, 25.5 ± 0.3 

percent of dairy cows were culled in 2002, with culling rate slightly higher on large dairies (27.5 

± 0.6 percent for dairies with more than 500 cows) compared to smaller dairies (24.9 ± 0.6 

percent for herds with fewer than 100 cows and 23.9 ± 0.5 percent for operations with 100 to 499 

cows). 

Mortality for dairy cattle varied by age, with unweaned heifers having the highest death rate (8.7 

± 0.2) and weaned heifers having the lowest death rate (1.9 ± 0.1 percent). Smaller operations 

appeared to have a higher death loss among unweaned heifers compared to operations with more 

than 500 milking cows (9.1 ± 0.4 and 9.4 ± 0.3 percent for operations with less than 100 cows 
Appendix B: Overall Reproductive Efficiency and Health Statistics B-5 

Animal Cloning: A Risk Assessment DRAFT

and between 100 and 500 cows, vs. 7.7 ± 0.5 percent for operations with greater than 500 cows). 

Table B-2 presents data on causes of death and incidence rate for weaned and unweaned heifers 

for all operations responding to the survey. 

Table B-2: Major causes of mortality for unweaned and weaned dairy 

replacement heifers that died1

Cause Unweaned Weaned 

Diarrhea 62.1 ± 1.12 12.3 ± 1.0 

Respiratory 21.3 ± 0.9 50.4 ± 1.6 

Dystocia 4.1 ± 0.6 NA 

1

 Based on USDA statistics for 2002, with 88.8% of milking cows (9.1 million 

cows) producing live calves. 

2

 Percentage of deaths ± standard deviation of the mean. 

In the US, most dairy cattle are bred by AI, although many dairies still maintain bulls for cows 

that do not conceive to AI. According to the USDA/NAHMS 2002 report, 54.9 percent of dairies 

surveyed maintained one or more bulls. Embryo transfer has been promoted as a commercially 

feasible assisted reproductive technology (ART) for dairy cattle, particularly for dairies 

interested in using their best cows to improve herd genetics (Webb and Drost 1992). Embryos 

are also sold nationally and internationally to increase genetic advancement and overall herd 

production. The International Embryo Transfer Society, a professional society whose 

membership includes breeders and researchers, estimates that a total of approximately 550,000 in 

vivo derived bovine embryos were transferred worldwide in 2004 (Thibier 2005). Cows with less 

desirable genetics or production levels may be used as recipients of higher genetic merit 

embryos. However, ET is not a predominant means of reproduction in dairy cattle. In vitro

fertilization has been less successful than in vivo fertilized ET, and is not commonly practiced. 

The developmental competence of cultured bovine embryos remains low (Betts and King 2001), 

with less than half of bovine IVF embryos developing to blastocysts, and even fewer survive to 

attachment in the uterus. 

In cows bred by AI, pregnancy may be diagnosed by ultrasound 35 d after insemination or by 

palpation approximately 40 to 45 d after insemination. Average pregnancy loss following a 

positive pregnancy diagnosis for all cows across operations of different sizes was 4.0 ± 0.1 

percent. Pregnancy loss was highest on larger operations (4.9 ± 0.3 percent for operations with 

greater than 500 head; 3.6 ± 0.1 percent for operations with less than 100 head; 3.4 percent for 

operations with 100 to 499 head) (USDA/NAHMS 2002). 
Appendix B: Overall Reproductive Efficiency and Health Statistics B-6 

Animal Cloning: A Risk Assessment DRAFT

B. Beef Cattle 

Beef cattle in the US are managed under various systems, depending on the intended use of the 

animals. Beef cattle destined for slaughter may change hands several times before final 

disposition. Breeding stock and young nursing animals may be maintained on range or in 

pasture. These are generally referred to as “cow-calf” operations. Following weaning, animals 

destined for slaughter may go directly to feedlots or may be maintained for a brief period on high 

quality pasture, a stage referred to as “back-grounding” or “stocker.” In the US, most cattle are 

slaughtered between 15 to 18 months of age. 

The 1997 Beef Cow-Calf Health and Health Management Practices report (USDA/ 

NAHMS) surveyed 2,713 beef cow-calf operations throughout the United States, representing an 

estimated 34,280,000 head of cattle. According to the survey, approximately 1.5 ± 0.1 percent of 

breeding cattle, including weaned replacement heifers, cows and bulls, died or were euthanized 

due to various causes in the previous year. Mortality rate was higher on small operations with 

less than 50 cattle, compared with larger herds (2.4 ± 0.3 percent). Approximately 20 percent of 

these losses were due to unknown causes. The largest single category (27 percent) of losses for 

beef breeding cattle was “other known” causes, most of which producers attributed to old age. 

The next highest categories (after “unknown”) were weather (18.0 percent) and calving problems 

(17.0 percent). Table B-3 presents the leading known causes of death, where a specific cause was 

named, for cattle that died. 

Table B-3: Causes of death for beef breeding cattle (cows, bulls and 

weaned replacement heifers) that died 

Cause Percent ± SE 

Digestive 6.1 ± 0.1 

Respiratory 6.0 ± 1.0 

Weather 18.0 ±3.9 

Dystocia 17.0 ± 1.9 

Relatively few breeding females in cow-calf herds experienced health problems, according to the 

1997 survey. In general, replacement heifers experienced a higher percentage of illnesses 

compared to mature cows. Pinkeye was the most commonly reported illness, and occurred in 1.3 

percent of female breeding cattle. With the exception of pinkeye, illness rates for breeding 

females appeared fairly similar among herds of different sizes. Pinkeye incidence was reported 

highest in small herds (less than 50 head, 2.3 percent) than in large herds (more than 300 head 
Appendix B: Overall Reproductive Efficiency and Health Statistics B-7 

Animal Cloning: A Risk Assessment DRAFT

0.6 percent). There was no difference in incidence rate of retained placenta or uterine infections 

between small and larger operations (0.2 ± 0.0 percent for operations with less than 50 or more 

than 300 head). Incidence of pregnancy loss was also small and not significantly different 

between breeding females in different sized herds (0.2 ± 0.1 percent in herds with less than 50 

head; 0.3 ± 0.0 percent in herds with greater than 300 head). Major causes of health problems in 

breeding female beef cattle are listed in Table B-4. 

Table B-4: Causes of morbidity in breeding female beef cattle1

Conditions Replacement Heifers Cows All Females 

Respiratory Disease 0.9 ± 0.32 0.3 ± 0.0 0.4 ± 0.1 

Scours 1.0 ± 0.2 0.4 ± 0.1 0.5 ± 0.1 

Pinkeye 1.9 ± 0.4 1.2 ± 0.1 1.3 ± 0.1 

Cancer eye 0.0 ± 0.0 0.3 ± 0.0 0.2 ± 0.0 

Foot rot 0.8 ± 0.2 0.8 ± 0.1 0.8 ± 0.1 

Mastitis N/A 0.2 ± 0.0 0.2 ± 0.0 

Retained placenta/metritis N/A 0.4 ± 0.0 0.3 ± 0.0 

Spontaneous abortion 0.3 ± 0.1 0.3 ± 0.0 0.3 ± 0.0 

Neurologic problems 0.0 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 

1

 Expressed as average percentage of all breeding females in 1996 cattle inventory. 2

 Percentage of females by category ± SE. 

Average mortality rate of unweaned calves was approximately 3.4 ± 0.1 percent of all calves 

born during 1996, and there were no appreciable differences among operations of different sizes 

for calf mortality. Two of the most common causes of death, when death could be attributed to a 

cause, were respiratory problems and dystocia. The leading causes of calf mortality according to 

producers surveyed are expressed in Table B-5. 

Table B-5: Most common perceived causes of death for unweaned calves 

Cause Percent ± SE 

Digestive 14.4 ± 1.0 

Respiratory 16.3 ± 1.2 

Weather 20.2 ± 1.4 

Dystocia 13.9 ± 1.3 

Unknown 17.5 ± 1.4 
Appendix B: Overall Reproductive Efficiency and Health Statistics B-8 

Animal Cloning: A Risk Assessment DRAFT

The leading cause of morbidity in calves was scours (diarrhea) affecting 2.4 ± 0.2 percent of all 

calves three weeks old or younger. Older, but still unweaned calves had a slightly lower 

incidence of scours (1.7 ± 0.2 percent). Diarrhea, in part, may have contributed to death losses 

due to digestive problems. Causes of morbidity in unweaned calves are listed in Table B-6. 

Table B-6: Causes of morbidity in unweaned calves1

 

Cause 3 Weeks or Less Over 3 Weeks Old 

Respiratory 0.5 ± 0.12 0.8 ± 0.1 

Scours 2.4 ± 0.2 1.7 ± 0.2 

Pinkeye 0.1 ± 0.0 1.1 ± 0.1 

Foot rot N/A 0.2 ± 0.0 

1

 Based on all calves in survey. 

2

 Mean Percentage ± standard error. 

C. Swine 

The total US swine population was estimated at 59,848,000 head in 2000 (USDA/NAHMS 

2001). Most US swine operations are fully integrated. This means that swine remain on the same 

operation under the same general management throughout their lives. Animals are usually 

maintained under full confinement in highly biosecure facilities, to minimize disease 

transmission and for other economic and management reasons. Sows generally farrow (give 

birth) twice a year. Piglets remain with their dams for approximately 21 days, and then are 

weaned and moved to a nursery, where they are housed in small groups in raised pens for 6 to 8 

weeks. They progress through “grower” and “finisher” phases, depending on weight, and are 

generally maintained in the same groups throughout the process. 

The most complete survey of swine health and management practices in the United States was 

published in 2001. This section derived data from Part I: Reference of Swine Health and 

Management in the United States, 2000 and Part II: Reference of Swine Health and Health 

Management in the United States, 2000 (USDA/NAHMS 2001). A total of 2499 producers were 

surveyed for the report. In order to qualify for the report, operations must have had at least 100 

head of swine at the time of the survey. 

A total of 3.3 ± 0.1 percent of all breeding females died and 17.5 ± 0.7 percent were culled 

between December 1999 and May 2000. The most common reasons cited for culling were age, 

lameness, performance and reproductive failure. Measures of poor performance in this survey 
Appendix B: Overall Reproductive Efficiency and Health Statistics B-9 

Animal Cloning: A Risk Assessment DRAFT

included small litter size, high pre-weaning mortality and low birth rate. Other reasons for 

culling included upgrading herd genetics, poor body condition and liquidation of the breeding 

herd for financial reasons. Table B-7 presents the reasons for culling and the relative percentages 

of swine culled for those reasons. 

Table B-7: Reasons for culling swine 

Cause Percent of culled females ± SE Percent of all females ± SE 

Age 41.9 ± 1.8 7.3 ± 0.4 

Lameness 16.0 ± 1.2 2.8 ± 0.3 

Performance 12.0 ± 0.7 2.1 ± 0.1 

Reproductive failure 21.3 ± 1.3 3.7 ± 0.2 

Other 8.8 ± 1.6 1.6 ± 0.3 

The two most commonly reported health problems in breeding females were roundworms (an 

intestinal parasite) and Porcine Reproductive and Respiratory Syndrome (PRRS). Swine 

dysentery was the only health problem more commonly reported on small operations (less than 

250 swine) compared to large operations. Other diseases occurred at a higher rate on larger 

operations. Unfortunately, no data were presented to indicate number or percent of animals 

affected by disease. Problems at farrowing and other reproductive problems were not reported. 

D. Sheep 

Most sheep in the US are raised for the production of both wool and meat. In the Eastern US, 

most sheep are raised on farms in fenced pasture, and may be supplemented with grain. In the 

Western US, it is more common to maintain sheep on open range. Lambs are generally born in 

late winter or early spring. Age at slaughter is variable, depending on the price of lamb compared 

to the price of grain and other inputs. 

The National Agricultural Statistics Service (NASS), USDA reported 66,100 sheep operations 

with a total national herd of 6,965,000 head as of February 2002. The 2001 Reference of Sheep 

Management in the United States (USDA/NAHMS) reported that 23.8 ± 1.0 percent of rams and 

18.3 ± 0.5 percent of ewes in all flocks were culled in 2000, and 5.0 ± 0.1 percent of all sheep 

and lambs died. Sheep raised on farms had a marginally higher death loss compared to open or 

fenced range sheep (5.6 vs. 4.5 and 4.7 percent, respectively). Data on culling rates by type of 

operation were not available. Table B-8 presents primary reasons for culling by sex for animals 

culled in 2000. 
Appendix B: Overall Reproductive Efficiency and Health Statistics B-10 

Animal Cloning: A Risk Assessment DRAFT

Table B-8: Primary reasons to cull for all rams and ewes culled 

Reason for Culling Rams Ewes 

Age 47.7 ± 2.11 47.9 ± 1.82 

Teeth problems 0.8 ± 0.3 5.3 ± 0.5 

Poor mothering N/A 3.3 ± 0.3 

Mastitis N/A 3.3 ± 0.2 

Failure to lamb N/A 5.5 ± 0.4 

Ram breeding soundness 13.8 ± 1.4 N/A 

Other reproductive 3.6 ± 1.1 1.2 ± 0.4 

1

 Percent of all culled rams ± SE 

2

 Percent of all culled ewes ± SE 

Predators (23.5 ± 1.0 percent), dystocia (12.3 ± 0.5 percent) and old age (15.4 ± 0.8 percent) 

accounted for 51.2 percent of all adult sheep that died or were lost in 2000. Other problems 

included respiratory disease, other diseases, digestive and metabolic problems (including milk 

fever and pregnancy toxemia), poisoning/toxicity, weather, and theft. Table B-9 presents data on 

major causes of death for adult sheep and lambs that died in 2000. 

Table B-9. Causes of death for adult sheep that died in 2000 

Cause Sheep Lambs 

Predators 23.5 ± 1.01 44.1 ± 1.12 

Digestive 6.7 ± 0.6 9.9 ± 0.6 

Respiratory 7.0 ± 0.8 11.7 ± 0.7 

Metabolic 3.7 ± 0.4 1.0 ± 0.1 

Dystocia 12.3 ± 0.5 NR3 

Other disease 3.0 ± 0.2 2.0 ± 0.3 

1

 Based on percent of all sheep that died ± SE 

2

 Based on percent of all lambs that died ± SE 

3

 Not reported 

As for swine, incidence and causes of morbidity in sheep was presented as percentage of 

operations reporting the problem. Data on number or percent of animals affected by illness were 

not presented in the USDA/NAHMS report. The most commonly reported health problems were 

stomach or intestinal parasites, clostridial infection, contagious ecthyma (sore mouth), and foot 

rot. Respiratory and reproductive problems were not reported as causes of illness in sheep or 

lambs in this report. 
Appendix B: Overall Reproductive Efficiency and Health Statistics B-11 

Animal Cloning: A Risk Assessment DRAFT

E. Goats 

Statistics on goat production in the US were not available through USDA/NAHMS. According to 

the Agriculture Databases for Decision Support (ADDS), there are approximately 2 to 4 million 

goats raised in the US (http://www.adds.org/CGIBIN/om_isapi.dll?clientID=23885&infobase=National%20Goat%20Database&softpage=Browse_Frame_Pg). 

However, no reliable or comprehensive statistics on goat numbers or their production in the US 

could be found. Goats are generally divided into three distinct types for meat, dairy or fiber 

(mohair or cashmere) production. Goats grown for meat or fiber are raised predominantly in 

large herds on open range, while dairy goats are raised in smaller herds on limited acreage with 

grain feeding. Intestinal parasites and respiratory diseases appear to be the most common 

illnesses reported in goats, although actual data were not available (ADDS Goat Handbook 

1993).
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Animal Cloning: A Risk Assessment DRAFT

Appendix C: 

Comparison of Outcomes 

Among Assisted Reproductive 

Technologies (ARTs) 
Animal Cloning: A Risk Assessment DRAFT

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Animal Cloning: A Risk Assessment DRAFT

Appendix C: Comparisons of Outcomes 

Among Assisted Reproductive Technologies 

(ARTs) 

Although there have been several studies comparing the outcomes of somatic cell nuclear 

transfer (SCNT) with various other assisted reproductive technologies, it is important to note that 

most of these evaluated data once other technologies had matured and were well-integrated into 

agricultural practice. The following summary provides an overview of several studies comparing 

the outcomes of four key ARTs. Comparison of success rates from SCNT with these ARTs may 

not be entirely appropriate due to the relative newness of SCNT technology. However, a review 

of the available studies indicates a trend of increasing adverse outcomes with increasing 

technological assistance; specifically, the increased rate of pregnancy failure, late gestational 

complications and problems associated with Large Offspring Syndrome (LOS) are most 

commonly associated with in vitro manipulation of the embryo. Table C-1 presents outcomes 

noted in various studies of artificial insemination (AI), in vivo produced embryo transfer (ET), in 

vitro produced embryos (IVP), blastomere nuclear transfer (BNT), and SCNT. 

Table C-1. Outcomes noted among studies for various ART in cattle, swine and sheep. 

Developmental 

Node1

Gestational 

Period ART Outcome Reference Comments 

Node I 

Early 

conceptus, 

early embryo 

prior to 

completion of 

organogenesis 

(gd 42 in 

cattle) 

IVP, 

BNT 

Higher rate of embryonic 

death than AI or in vivo

produced embryos 

Reichenbach et al. 1992; 

Kruip and den Daas 

1997; 

Wells et al. 1998; 

Hasler 2000; 

Cattle and sheep 

 IVP 

Pregnancy loss following 

transfer of IVP or in vivo

produced embryos prior 

to gd2

 21 or within 2 

weeks of transfer 

Farin and Farin 1995 

McMillan et al. 1997 

 IVP 

Increased total length of 

conceptus from IVP 

embryos 2X that of in 

vivo produced at gd 12 

and 17 

Farin et al. 2001 

Lazzari et al. 2002 

 IVP Gd 16 IVP conceptuses 

shorter than in vivo Bertolini et al 2002 

Likely reflects 

survival status 

during critical 

time of maternal 
Appendix C: Comparisons of Outcomes Among ARTs C-4 

Animal Cloning: A Risk Assessment DRAFT

recognition 

 IVP 

19% of conceptuses 

from IVP blastocysts 

degenerated by gd 17 

Farin et al. 2001 

 IVP 

Altered embryonic disc 

morphology affected by 

culture medium for IVP 

embryos 

Fischer-Brown et al. 

2005 

 IVP 

Pregnancy rates 45% or 

higher in dams receiving 

IVP embryos 

Hasler 2000; 

van Wagtendonk-de 

Leeuw et al. 2000; 

Lane et al 2003 

Factors affecting 

outcome include 

embryo culture 

system, embryo 

quality, embryo 

evaluator, number 

of embryos 

implanted, 

synchrony with 

dam’s estrus 

cycle, fresh vs. 

frozen embryos 

 ET 

Higher embryo survival 

rate when embryos are 

transferred fresh rather 

than frozen-thawed 

Spell et al. 2001 

 AI Embryo loss ~30% by gd 

30in beef and dairy cattle 

Smith et al. 1982; 

Sreenan and Diskin 

1983; 

Dunne et al. 2000; 

Santos et al. 2004 

 AI 

Embryo loss by gd 21 

associated with high 

plasma estrogen levels 

on day of insemination 

Shore et al. 1998 

Possible 

estrogenic effect 

of legume in diet 

Node I 

Late 

embryonic/ 

early fetal 

period (days 

30-90) 

ET, 

IVP 

Embryo loss for in vivo

produced embryos < 5% 

(from 2 months – term) 

King et al. 1985 

Hasler et al. 1987 

 IVP 

Embryo loss for IVP 

embryos higher 

- 13% after gd 40 

- 10.7-13.1% 

- 24% total between 

gd 53-calving, 

with more between 

gd 50-80 

Hasler et al. 1995; 

Agca et al. 1998; 

Hasler 2000; 

Block et al. 2003 

Depending on 

medium 

 AI, ET, 

IVP 

Pregnancy rates at gd 22 

not different among 

groups. At gd 42, 

pregnancy rates similar 

between AI and ET, but 

increased embryo loss in 

IVP compared to AI and 

Drost et al. 1999 
Appendix C: Comparisons of Outcomes Among ARTs C-5 

Animal Cloning: A Risk Assessment DRAFT

ET 

 IVP 

Abnormal development 

of allantoic membranes 

and cavity in placentas 

of IVP embryos gd 30-

90 

Peterson et al. 2000 

Abnormal 

placental 

development and 

reduced placental 

blood membrane 

development 

 IVP 

Abnormal placentome 

and blood vessel 

morphology between gd 

70-222 

Miles et al. 2004 

Miles et al. 2005 

 IVP 

Gd 61 and older fetuses 

heavier than ET fetuses; 

altered fetal organ 

growth; excessive 

amniotic fluid 

Sinclair et al. 1999 

 IVP 

Gd 70 altered 

angiogenesis and 

placental morphometry; 

modified synthetic 

oviductal medium 

(mSOF) compared with 

medium with serum had 

fewer placentomes, low 

placental fluid volume 

and lower fetal weight: 

placental weight ratio; 

Placentomes (cotyledon 

tissue) had decreased 

density of blood vessels, 

decreased expression of 

angiogenic factor mRNA 

and vascular endothelial 

growth factor (VEGF) 

Miles et al. 2005 

Farin et al. 2006 

 AI 

Fetal loss by gd 44 30-

40% in swine 

pregnancies 

Vonnahme et al. 2002 

Fetal survival 

related to 

placental 

efficiency 

 AI 

Embryonic/fetal loss 

varies from 10 to 20% 

between gd 28 and 80 in 

beef and dairy cattle 

Pope and Hodgson-Jones 

1975; 

Kummerfeld et al. 1978; 

Bulman and Lamming 

1979; 

Lucy 2001 

Progesterone 

levels in dams’ 

milk may be 

normal through 

first 30 days of 

pregnancy, 

followed by 

sudden drop 

 AI 

Embryonic/fetal loss 11 

to 44% by gd 50 in beef 

cattle 

Bulman 1979 Attributed to bull 

Node I Late gestation ET, 

IVP 

Compensation in 

vascular beds of IVP 

bovine embryos; 

Compared with in vivo

Miles et al. 2004 

Theorized to 

compensate for 

increased fetal 

size and need for 
Appendix C: Comparisons of Outcomes Among ARTs C-6 

Animal Cloning: A Risk Assessment DRAFT

embryos, IVP had 

decreased fetal villi, 

binucleate cell volume 

densities in placentomes. 

Proportional volume of 

blood vessels in maternal 

caruncles increased in 

IVP group. Ratio of 

blood vessel volume 

density: placentome 

surface area increased. 

increased 

nutrients and gas 

exchanges, but 

increased vascular 

blood network at 

level of 

placentome 

 IVP 

IVP fetuses show 

increased glucose and 

fructose in fetal plasma 

levels; increased 

placental surface area 

Bertolini et al. 2004 

 IVP 

Hydroallantois 

frequency in IVP 

pregnancies (1/200) 

higher than in “normal” 

pregnancies (1/7,500) 

Hasler et al. 1995 

 IVP, 

SCNT 

Pregnancy loss higher in 

SCNT than IVP 

embryos; 50-100% loss 

gd 30-60; placentas 

hypoplastic and reduced 

cotyledonary 

development 

Hill et al. 2000; 

Chavette-Palmer et al. 

2002; 

Heyman et al. 2002; 

Edwards et al. 2003; 

Lee et al. 2004 

 BNT 

Late gestation abortions, 

stillbirths, 

underdeveloped fetuses 

for gestational age, 

edema, hydronephrosis, 

testicular hypoplasia, 

skull and heart 

malformations; lack of 

udder development in 

dams 

Wells et al. 1998 

 IVP, 

SCNT 

Broad distribution of 

fetal and neonatal body 

weights for both IVP and 

SCNT-derived embryos; 

shifted to “heavy” 

relative to in vivo 

embryos 

Wilson et al. 1995 

Kruip and de Daas 1997 

Farin et al. 2001 

Miles et al. 2005 

Two competing 

explanations: (1) 

“normal” for 

these animals may 

be heavier than 

for in vivo

produced 

embryos, or (2) a 

proportion of 

animals shifts 

weight 

distribution of 

population 

 

Adaptation to small 

changes in biochemical 

parameters and 

Sangild et al. 2000 
Appendix C: Comparisons of Outcomes Among ARTs C-7 

Animal Cloning: A Risk Assessment DRAFT

morphology 

 IVP, 

ET 

Increased gestation 

length, dystocia, 

perinatal mortality, fetal 

edema, altered organ 

development, abnormal 

limbs 

Kruip and den Daas 

1997; 

Behboodi et al. 1999; 

van Wagtendonk-de 

Leeuw et al. 1999; 

Farin et al 2001; 

Bertolini and Anderson 

2002; 

Edward et al. 2003; 

Rerat et al. 2005 

Frequency and 

severity of 

abnormalities: 

IVP>ET>AI 

 AI 55.9% of abortions due 

to infection Santos et al. 2004 

Node II Perinatal IVP, 

BNT 

Increased birth weight, 

Increased crown-rump 

length; increased 

mortality and physical 

deformities 

Behboodi et al. 1995; 

Wilson et al. 1995; 

Walker et al. 1996; 

Rerat et al. 2005 

 IVP 

Perinatal mortality in 

IVP ranges from 2.4-

17.9%, due to dystocia 

associated with large 

fetuses 

Hasler et al 1995; 

van Wagtendonk-de 

Leeuw et al. 1998; 

Block et al. 2003 

Lower in heifers 

than in cows 

 IVP, 

SCNT 

IVP and SCNT fetuses 

have cerebellar 

hypoplasia, respiratory 

distress, and heart 

enlargement 

Schmidt et al. 1996; 

van Wagtendonk-de 

Leeuw et al. 2000; 

Chavette-Palmer et al. 

2002 

 IVP 

Altered expression of 

mRNA for nonimprinted myostatin and 

glyceraldehydes-3-

phosphate in IVP fetuses 

Crosier et al. 2002 

 IVP, 

SCNT 

Altered expression of 

mRNA or protein in IVP 

and SCNT placentas for 

VEGF, peroxisome 

proliferators activated 

receptor γ, leptin, bovine 

placental lactogen, 

transforming growth 

factor (TGF) β1, 2, 3, 

TGF- β receptor, major 

histocompatibility class I 

antigens

Davies et al. 2004; 

Miles et al. 2004; 

Ravelich et al 2004; 

Miles et al 2005; 

Ravelich et al. 2005 

 IVP, 

SCNT 

Expression of 

demethylating enzymes 

DMT 1, 3a altered in 

IVP and SCNT 

preimplantation embryos 

Wrenzycki et al. 2004 

 BNT 

Birth weight range 26.4 

to 67.3 kg; slow to stand, 

poor suckling behavior, 

Garry et al. 1996 
Appendix C: Comparisons of Outcomes Among ARTs C-8 

Animal Cloning: A Risk Assessment DRAFT

A. Successes and Failures of AI, IVP, and ET 

Success of AI depends on a variety of factors, including health of the female and timing of 

insemination relative to ovulation. In dairy cattle, conception rates to AI following spontaneous 

estrus have declined from approximately 55 percent in the 1950s to 45 percent in the late 1990s. 

flexor tendon 

deformities, hypoxemia, 

hypoglycemia, acidosis, 

hypothermia; altered 

metabolic hormones 

(thyroxine, 

triiodothironine, and 

insulin) 

 BNT 

Calving rate ~50% using 

high quality embryos; 

some very large calves 

(up to 70.5 kg); 

contracture of limbs and 

spine, cardiac and skull 

deformities noted in a 

few calves; high rate of 

dystocia (52/100); 

hydroallantois observed 

in four cows 

Willadsen et al. 1991 

 IVP, 

SCNT 

Increased incidence of 

dystocia and C-section 

deliveries for IVP 

pregnancies compared to 

AI/NM; lack of 

contractility and other 

signs of labor in ewes; 

higher mortality among 

IVP and SCNT 

compared to AI/NM 

Ptak et al. 2002 Sheep 

 AI 

Heat stress reduces birth 

weight and passive 

transfer of immunity and 

results in low IgG 

concentration in calves 

Collier et al. 1982 

High levels of 

glucocorticoids 

accelerates “gut 

closure” 

Nodes II-III Postnatal IVP Increased feed intake and 

growth rate Rerat et al. 2005 

 IVP 

Altered glucose and 

electrolyte metabolism 

compared to AI 

persisting through early 

juvenile period 

Rerat et al. 2005 

1

 For the purposes of this table, Developmental Node 1 is divided into three stages of pregnancy: early embryo, late embryoearly fetal, and late gestation. 

2

 Gd= gestation day or day of pregnancy. 
Appendix C: Comparisons of Outcomes Among ARTs C-9 

Animal Cloning: A Risk Assessment DRAFT

The use of hormones to synchronize estrus for timed AI has further reduced conception rates to 

approximately 35 percent. The reasons for this apparent reduction in dairy cow fertility are not 

clear, although a number of factors have been cited as possibly contributing to the phenomenon, 

including increased milk production (resulting in increased stress and reduced availability of 

nutrients for reproductive function), increased average herd size (resulting in fewer person-hours 

spent observing cows for estrus behavior), nutrition, herd health, inbreeding, and environmental 

pollution (Lucy 2001). Embryo loss has been estimated to occur at a rate of 10 to 20 percent in 

dairy cattle (Lucy 2001) and as high as 30 percent in beef cattle (Dunne et al. 2000), and 

generally occurs prior to 30 days gestation. Fetal losses in swine pregnancies can be as high as 

40 percent following AI (Vonnahme et al. 2002). The reasons for these losses in utero are not 

always apparent. Lucy (2001) noted that embryo loss may occur even in cases where the 

developing embryo appeared normal. However, in swine, fetal loss appears to be related to the 

size and efficiency of the placenta (Vonnahme et al. 2002). 

Betts and King (2001) noted that the developmental competence (an embryo’s ability to progress 

through normal cell division and development) of IVP and cultured embryos was low. Using in 

vitro procedures (as published up to 2001), less than half of inseminated bovine oöcytes reached 

blastocyst stage, and of those that did, many did not implant or attach following transfer. Betts 

and King (2001) noted that chromosomal abnormalities such as aneuploidy and polyploidy 

played a fundamental role in most of these embryonic deaths. 

The evolution of IVP technology in cattle can be observed by comparing early studies 

(conducted prior to 2002) with more recent publications. Studies using IVP embryos during the 

mid- to late 1990s (Behboodi et al. 1995; Farin and Farin 1995; Hasler et al. 1995; Walker et al. 

1996; Drost et al. 1999; Sinclair et al. 1999) noted relatively high rates of embryo loss and LOS 

among fetuses and neonatal calves. In contrast, several more recent studies using IVP embryos 

have indicated few or no problems (Chavatte-Palmer et al. 2002; Heyman et al. 2002; Bertolini 

et al. 2004; Rerat et al. 2004). However, high embryonic mortality and placental abnormalities 

may still be observed with IVP in some labs (Miles et al. 2004; Miles et al. 2005). 

Embryo transfer, in which oöcytes are fertilized in utero then removed and transferred to 

surrogates, has become a commercially viable technology (See Chapter II), and is generally more 

successful than IVP. In a study by Drost et al. (1999), initial pregnancy rates, as determined by 

blood progesterone levels at gestation day 22, were similar among cows bred by AI, ET or IVP. 

However, by gestation day 42, embryo loss among cows receiving IVP embryos was higher than 

either AI or ET, while pregnancy rate was similar between cows bred by AI compared to those 

receiving ET embryos. The success of ET may be affected by treatment of the embryo prior to 

transfer and synchrony between the surrogate and the embryo donor (Pope 1988; Spell et al. 

2001). According to Spell et al. (2001) fresh embryos had a higher rate of survival than embryos 
Appendix C: Comparisons of Outcomes Among ARTs C-10 

Animal Cloning: A Risk Assessment DRAFT

that had been frozen then thawed prior to transfer. Embryo survival was also higher when 

surrogates had been in estrus within 12 hours of the embryo donor (Spell et al. 2001). 

In order to follow fates of client-owned pregnant cows carrying IVP-derived pregnancies in a 

commercial ET operation, Hasler et al. 1995 noted that for the first 100 transfers, 24 ended in 

pregnancy loss before 100 days of gestation. The success rate improved the subsequent year, 

however, with only 7 percent of IVP-derived pregnancies spontaneously aborting. They 

compared these results to 5.3 percent of ET pregnancies aborted between two and seven months 

of gestation in an earlier study. 

In a comparison of AI and IVP, Behboodi and coworkers (1995) noted an increased incidence of 

dystocia and Cesarean sections (C-section) for IVP derived pregnancies compared to AI in a 

small group of cattle (8/13 IVP-derived pregnancies vs. 7/71 AI pregnancies requiring Csection). Birth weights of calves derived from IVP embryos were higher than calves produced by 

AI, likely contributing to the observed increase in dystocia among dams carrying IVP-derived 

pregnancies. Sinclair et al. (1999) also observed large IVP-derived fetuses with altered 

development and excessive amounts of amniotic fluid. In that study, nine of 13 fetuses (69 

percent) derived from embryos co-cultured with granulosa cells (a type of cell found in the 

ovary) and one of six embryos (17 percent) incubated in synthetic oviductal fluid (SOF) plus 

steer serum were oversized, while embryos that had been incubated with SOF alone produced 

normal sized fetuses. Bovine embryos cultured for three to five days post-fertilization also were 

associated with increased dystocia due to oversized calves in a study by Walker et al. (1996). 

(See discussion of influence of culture conditions on success rates in Chapter IV). 

Farin and Farin (1995) collected bovine IVP and ET fetuses from beef heifers at seven months 

gestation and compared development between the two groups. Fetuses from the IVP group were 

heavier than their ET counterparts (18.6 ±1.1 vs. 15.4 ±0.8 kg), had greater heart girths 

(56.5 ± 1.2 vs. 52.4 ± 0.9 cm) and weights (139.7 ± 8.3 vs. 116.2 ± 5.8 g), and greater long bone 

lengths (23.1 ± 0.6 vs. 21.3 ± 0.4 cm).When organ and skeletal measures were compared on a 

per kilogram body weight basis, however, IVP fetuses had consistently smaller skeletal measures 

than ET fetuses. Internal organ weights per unit of body weight were not different between the 

two groups of calves. The authors concluded that IVP fetuses were undergoing abnormal and 

disproportionate development compared to ET fetuses. It should be noted that the most rapid 

period of prenatal growth in cattle is during the last two months of gestation (months 8 and 9) 

(NRC 2001), which would have occurred after these pregnancies were terminated. 

Young and Fairburn (2000) noted that both IVP and embryo culture have resulted in abnormal 

phenotypes, including up to two-fold increases in birth weight (LOS), excess amniotic fluid, 
Appendix C: Comparisons of Outcomes Among ARTs C-11 

Animal Cloning: A Risk Assessment DRAFT

hydrops fetalis72, altered allometric organ growth73, advanced fetal development, placental and 

skeletal defects, immunological defects, and increased perinatal death. 

Markette et al. (reviewed by Farin et al. 2001) observed that 54.7 percent of ET recipients were 

pregnant at 60 days gestation, with the majority of pregnancies lost prior to day 24 of gestation. 

In a large study, King et al. (2000) reported that the incidence of pregnancy loss in 1,776 embryo 

transfer recipients was 3.15 percent from 2 to 3 months of gestation, and 2.14 percent between 3 

to 7 months. These mid- and late-gestation spontaneous abortions were not influenced by embryo 

age, embryo quality, time between embryo collection and transfer, asynchrony of recipient with 

donor estrus, donor age, ovarian response to gonadotropin treatment, or whether or not the donor 

had a history of infertility, according to the authors. In most studies, pregnancy loss during the 

fetal period (day 42 to 280 of gestation in cattle) was greater following transfer of embryos 

produced in vitro than that for embryos produced in vivo. Mid- to late-gestation spontaneous 

abortion of about 7 to 13 percent has been reported for recipient cattle carrying fetuses derived 

from IVP embryos, and in some studies pregnancy loss has been considerably higher (Farin et al. 

2001). 

Conversely, Bertolini et al. (2004) compared fetal development in in vivo and IVP cattle 

pregnancies and reported no significant difference between groups for pregnancy rates (20/53 

and 36/112 for control and IVP groups respectively) and fetal losses after day 45 (2/20 and 3/36 

for control and IVP groups respectively). They did report that fetal losses between gestation days 

30 and 44 were 3.4-fold higher (P<0.05) in the IVP group (17/36) than in controls (4/20). Also in 

contrast to earlier studies, Bertolini et al. (2004) reported that their measurements of conceptus 

physical traits for both in vivo produced controls and IVP pregnancies on days 90 and 180 

demonstrated allometric proportionality between fetal body size and body weight with no 

physical deformities observed in any fetus. 

In a review of research on early embryo development, Gardner and Lane (2005) stated that the 

environment of the preimplantation embryo has a profound effect on the physiology and viability 

of the conceptus. Among the many factors that can influence development of IVP embryos, they 

cite the use of serum products as an important contributor to developmental abnormalities in 

cultured embryos. These authors state: “Mammalian embryos are never exposed to serum in 

vivo...Rather, serum is a pathological fluid, the composition of which is greatly undefined and 

 

72 Accumulation of fluid in the entire body of the newborn. 

73 Allometric growth refers to differences in the rate of growth of a particular organ or part in relation to the rest of 

the organism. An example of normal allometric growth is the legs of a newborn foal (horse) in proportion to its 

body size; the legs are long and out of proportion to the rest of the body. As the foal ages, the body grows (and 

fills out) more rapidly than the legs, so that in adult horses the legs appear proportional to the rest of the body. 

Altered allometric growth in the context of ARTs has resulted in enlarged hearts and undersized kidneys, as well 

as other organs, which are not appropriately proportioned to the rest of the body. 
Appendix C: Comparisons of Outcomes Among ARTs C-12 

Animal Cloning: A Risk Assessment DRAFT

varies enormously with source...serum induces premature blastulation in domestic animal 

embryos...affects embryo morphology...and leads to perturbations in ultrastructure...and energy 

metabolism.” Other factors that may influence development of embryos in vitro include 

ammonia, oxygen, inadequate nutrients, and freezing (Gardner and Lane 2005). 

Rerat et al. (2004) compared the perinatal health characteristics of IVP and AI cattle and 

observed no differences in post-natal mortality or viability. Calves in this study were generally 

healthy with the health status of IVP calves at birth and during the first 112 days of life similar to 

that of AI calves. Clinical traits such as heart rate, rectal temperature, and respiratory rate were 

nearly identical in both groups. At birth, measurements indicative of growth performance such as 

potassium, 3,5,3’-triiodothyronine (a metabolic hormone), and thyroxine concentrations were 

lower in IVP than in AI calves. Postnatally, IVP calves had a faster growth rate than AI calves 

under conditions of identical nutrient intake. 

Sakaguchi et al. (2002) induced twinning in Japanese beef cows by transferring one or two in 

vivo fertilized embryos into AI bred cows. Fetal dystocia occurred in 7 of 14 twin parturitions, in 

which some twin calves appeared to enter the uterine cervix at the same time, but no single 

parturition was accompanied by dystocia. The incidence of retained placenta was significantly 

higher in the twin parturitions (10/14; 71 percent) than in the single parturitions (2/22; 9 percent). 

These complications are known to occur with natural twins in cattle, however, and may not be 

directly related to ET technology. The incidence of retained placenta in healthy, single calfbearing dairy cows is approximately 5-15 percent, (slightly lower in beef cows) and is increased 

when there are twins. The expected incidence of dystocia is 10-15 percent in first-parity animals, 

and 3-5 percent in mature cows (Merck Veterinary Manual Online 2002). 

B. Outcomes for BNT, Fetal- and Adult-Cell SCNT 

Although success rates for various types of cloning have improved, they are still highly variable 

across studies. In earlier studies, generally less than 10 percent of all NT embryos transferred to 

recipients were born alive (Wells et al. 1999). Some of these early studies noted that both 

blastomere and somatic cell NT clones appeared to have the same low success rate and exhibited 

many of the same problems, such as poor or dysfunctional placentation and LOS (Stice et al. 

1996; Wells et al. 1998). Stice et al. (1996) reported that no fetuses derived from BNT survived 

beyond day 60 of gestation. Wells et al. (1998) reported a 64 to 80 percent pregnancy loss during 

the attachment phase for clone fetuses derived from an embryonic sheep cell line, while a further 

43 percent of pregnancies were lost in the last trimester, such that 11 percent of embryos 

survived to term (12/112). In contrast, Le Bourhis et al. (1998) reported 9/30 transferred male 

bovine BNT clones developed to calving, while 6/27 female BNT clones resulted in live calves. 

Heyman et al. (2002) compared development and survival of BNT, fetal and adult NT clones to 
Appendix C: Comparisons of Outcomes Among ARTs C-13 

Animal Cloning: A Risk Assessment DRAFT

IVP-derived embryos under the same culture conditions. Pregnancy loss from 90 days of 

gestation to calving were 43.7 percent for adult and 33.3 percent for fetal SCNT, compared to 4.3 

percent for BNT clones, while none of the IVP-derived pregnancies were lost. Pace et al. (2002) 

reported 75 percent pregnancy loss of adult (some transgenic) SCNT embryos throughout 

pregnancy. 

Results from these studies may reflect the evolution of NT technology over time. Embryonic or 

BNT cloning and IVP success rates appear to have improved. Although losses remain high for 

the newer SCNT technology, success rates for this technology also have improved over time. It 

remains to be seen what progress may be made in further reducing pregnancy loss and other risks 

associated with SCNT. 

C. Conclusions regarding outcomes for ARTs 

Based on the studies reviewed, there appears to be a general trend indicating that the frequency 

of embryo/fetal loss and abnormal pregnancy outcomes increases with increasing manipulation 

of the embryo and in vitro culture. This trend is evident, even when maturity of the technology is 

considered. Causes of embryo/fetal loss are not always evident, but late gestational 

complications (hydrops and dystocia) and fetal/neonatal abnormalities (skeletal and organ 

deformities, oversize, metabolic alterations) have all been noted in ET, IVP, BNT and SCNT. 

The frequency of these outcomes varies somewhat among laboratories, but has the general trend 

ET<IVP<BNT<SCNT. These data support a conclusion that SCNT falls on a continuum of 

ARTs, and that the adverse outcomes noted with SCNT are not unique, but are of concern due to 

their increased frequency. 
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Appendix D: 

Transgenic Clones
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Appendix D 

Transgenic Clones 

A. Issues 

The current risk assessment is limited to address the risk of clones from non-transgenic cells. 

Although not within the purview of this analysis, the results of a number of studies that address 

either transgenic animal clones or transgenic and nontransgenic animal clones (Hill et al. 1999; 

the “ACT series” including Cibelli et al.1998, Lanza et al. 2000, and Lanza et al. 2001 for cattle; 

Denning et al. 2001 and McCreath et al. 2000 in sheep; Baguisi et al. 1999 and Keefer et al. 

2001a in goats; Carter et al. 2002, Lai et al. 2002, and Lee at al. 2003 in swine) are presented 

here to clarify the relative utility of such studies for assessing potential risk(s) associated with 

non-transgenic somatic cell nuclear transfer (SCNT). 

 

Many reviews of SCNT outcomes cite these papers as indicative of the severity of adverse 

outcomes associated with cloning, or a demonstration of the positive outcomes that can come 

from cloning. Hill et al. (1999), in particular, is often cited as the seminal “adverse outcome” 

paper for cloning. On the other hand, if only the “final report” paper of the ACT series, Lanza et 

al. 2001, were read, it would not be possible to know that the cells from which the cattle were 

cloned were indeed transgenic. This distinction only becomes apparent when the earlier papers 

are also reviewed. Most recently, headlines were generated when Pearson (2003) reported on 

“sudden death syndrome” in pig clones; CVM reviewed the paper that describes their generation 

and noted that these pig clones carried two distinct transgenes (Lee et al. 2003). 

Because these animals are transgenic clones, it is not possible to determine whether adverse 

outcomes result from the direct effect of the expression of the transgenic construct, pleiotropic 

effects resulting from insertion of the construct, the SCNT process, or some interaction of any or 

all of these processes. For example in a comparison of 34 cells lines, Forsberg et al. (2002) 

reported that when otherwise similar cells are used as donors for SCNT, those that are transgenic 

(for a selectable marker gene) result in lower pregnancy initiation (22 percent vs. 32 percent) and 

calving (3.4 percent vs. 8.9 percent) rates. The authors hypothesize that the additional culturing 

required to generate transgenic cells, selection of transgenic lines, or the DNA construct itself, 

could be responsible for the lower rates. 

CVM thus assumes that transgenic clones occupy a different “risk space” from “just clones.” 

Conversely, an argument can be made that if no adverse outcomes are detected, then for these 
Appendix D: Transgenic Clones D-4 

Animal Cloning: A Risk Assessment DRAFT

animals, neither process sufficiently perturbed development to induce anomalies. We have 

included those studies in the overall risk assessments if such results were obtained. Nonetheless, 

because these studies occupy such a large segment of the cited literature, a few are presented 

here to illustrate the range of responses noted, with the appropriate caveats for interpretation. 

B. Cattle 

Hill et al. 1999

Hill et al. 1999 reports on a group of 13 transgenic clones of a Holstein bull. Twelve Brangus 

cows carrying 13 fetuses cloned from Holstein cells were originally included in the study, 

although three of these transgenic clone fetuses died prior to the perinatal period (defined in this 

paper as two weeks prior to anticipated delivery and a few days thereafter), and one cow aborted 

at eight months of gestation. Two cows developed hydroallantois and were delivered by Csection; four others were also delivered by this method due to subjective judgment regarding 

fetal size. The two remaining pregnancies delivered vaginally. Birth weights of the transgenic 

clones ranged from 44-58.6 kg (average Holstein male calf weight is in the range of 40-50 kg), 

and cited as within the weight range of in vitro produced embryos. Five of the eight live born 

clones were judged to be normal within four hours of birth based on clinical signs and blood gas 

measurements. Three of the eight were immediately diagnosed with neonatal respiratory distress. 

One of these calves died from pulmonary hypertension, pulmonary surfactant deficiency, and 

elevated systemic venous pressure at day 4. The other three animals recovered. Two of the five 

fetuses that did not survive to birth also exhibited signs of pulmonary hypertension and placental 

edema at necropsy. Another clone died at 6 weeks of age with signs of respiratory distress; 

subsequent field necropsy suggested dilated cardiomyopathy, although no definitive diagnosis 

could be made. 

The Advanced Cell Technology Series

The series of papers from the Advanced Cell Technology group (Cibelli et al. 1998, Lanza et al. 

2000, and Lanza et al. 2001) on the health of clones are similar to that of Hill et al. (1999) in that 

the animals presented are clones that are derived from transgenic cells. Interpretation of any 

adverse outcomes is thus also confounded by the potential role of the transgene and its insertion. 

The results of these studies are summarized in Lanza et al. (2001), in a short overview with 

accompanying supporting documentation provided by the journal in electronic form. Of 30 fetal 

transgenic clones that developed to term, 24 were reported healthy at 1-4 years of age, but five 

died within 7 days of cardiopulmonary difficulties that the authors speculated were secondary to 

placental insufficiencies. The sixth animal died at day 149 due to enteric disease, 

lymphadenopathy, and exhibited mild placental edema and high fever at birth. Problems 
Appendix D: Transgenic Clones D-5 

Animal Cloning: A Risk Assessment DRAFT

observed at birth included placental edema, including edematous cotyledons (attachment sites of 

the placenta to the uterus), labored breathing, froth and fluid in the lungs, pulmonary edema, 

pneumonia, high fever, septicemia, lethargy, abdominal distention, masses in the abdomen, liver 

damage due to hypoxia, and heart abnormalities. 

Birth weights of the survivors were reported as 45 ± 2 kg (this paper cites normal as 43 kg). An 

unspecified number exhibited pulmonary hypertension and respiratory distress at birth. 

Presumably, they received supportive care at that time. Another unspecified number were also 

reported as experiencing fever following vaccination. This is not an atypical response among 

calves receiving vaccinations, as stimulating a potent immune response is likely to produce at 

least a mild local and systemic (fever) reaction in the animals (Roth 1999). 

Physical and veterinary examination of surviving animals aged 1-3 years were reported as 

normal and included temperature, pulse, and respiratory rate. No abnormalities were detected in 

general appearance, on auscultation (listening to breathing, heart beat, and digestive sounds), and 

behavior appeared normal. Puberty onset was reported to occur at the expected time, and fertility 

appeared to be normal. At the time of publication (2001), two of the animals had delivered 

apparently normal progeny. 

Clinical chemistry parameters evaluated for these animals included electrolytes, urea, creatinine, 

glucose, bilirubin, aspartate aminotransferase (AAT), sorbitol dehydrogenase (SGT), albumin, 

globulin, and total protein. Globulin and total protein measurements were reported in the 

publication as “slightly below normal.” All other measurements were reported to be within 

normal range. Hemograms (analysis of cellular components of blood) were all reported as 

normal: hematocrit, hemoglobin, red blood cells, mean red cell volume, mean red cell 

hemoglobin concentration, and white blood cell numbers and differentials were within normal 

ranges. Blood gases were also within normal ranges. To examine immunocompetence in the 

clones, peripheral blood lymphocytes from the transgenic clones and conventional Holsteins 

were compared to determine whether the same ratio of cell surface markers were present, and if 

the transgenic clone cells responded to mitogen challenge in the same way as cells from 

conventional Holsteins. No significant differences were observed between the cell surface 

markers or cellular responses of cells from conventional animals or clones. 

In the early spring of 2003, an interview of an ACT executive reported in the lay press indicated 

that two animals from this cohort had developed significant health problems. One animal was 

reported to have developed a tumor, and the other was diagnosed as having neurological 

problems. The first animal apparently died during surgery to remove the tumor, and no further 
Appendix D: Transgenic Clones D-6 

Animal Cloning: A Risk Assessment DRAFT

information is available on the potential causes of the tumor. The second animal was later 

diagnosed as being positive for Johne’s disease (Mycobacterium paratuberculosis), an infectious, 

chronic, progressive disease that often presents with chronic diarrhea and eventual cachexia 

(general physical wasting and malnutrition). It is therefore unlikely that this animal’s symptoms 

were due to either cloning or transgenesis. We are unaware of any other adverse outcomes 

associated with these animals. 

C. Swine 

Carter et al. 2002 

Carter et al. 2002 reported on the overall health status of transgenic swine clones produced from 

cells transfected with green fluorescent protein (GFP). The 10 transgenic piglet clones from three 

litters were followed for the first six months of life. 

Five of the ten transgenic swine clones died or were euthanized during the study. Two piglets 

died of congestive heart failure at 7 and 35 days of age, two others died from bacterial infections 

at 3 and 116 days of age. The fifth animal died at 130 days of age, following a history of chronic 

diarrhea, decreased growth and vitamin E deficiency. The remaining five piglets were reported 

as healthy and growing similarly to conventional animals housed in the same facility at the 

conclusion of the study. Behavior was reported as “consistent with pigs of their age group.” 

Average birth weight of the transgenic clones (1,312 g) was similar to average birth weights of 

conventional piglets from similar genetic background (1,450 g). Average daily weight gain for 

transgenic clones through the first 16 weeks was (461 g) relative to the herd average (594 g), 

which the authors considered as within the normal range. 

Some of the piglets displayed physical defects. These included two piglets with contracture of 

the flexor tendons, another piglet with five digits on a forelimb (four digits are normal) and an 

enlarged dewclaw. Another piglet with low birth weight was described as having short legs and a 

large, round chest. 

Hematology and blood clinical chemistry data were collected beginning at 2 days of age and 

every two to four weeks until 24 weeks of age. Most hematological variables were similar to the 

comparator group, except for hemoglobin, hematocrit, and plasma total protein. Mild anemia and 

low blood protein concentration were observed for the first four weeks, but both these conditions 

resolved by eight weeks of age. The authors stated that decreased hematocrit and hemoglobin 

values are common in piglets reared in confinement, and that these symptoms are generally 

treated with iron dextran. Similarly, clinical chemistry results indicated decreased levels of 
Appendix D: Transgenic Clones D-7 

Animal Cloning: A Risk Assessment DRAFT

albumin and globulin during the first four weeks in the transgenic clones relative to comparators, 

but these values were back within the normal range by eight weeks of age. The authors attributed 

the decreased protein and globulin values to the decreased colostrum intake of the newborns as 

the surrogate sow bearing them did not initiate normal lactation, and piglets were dosed with 

colostrum at some unspecified point after birth. 

Seven of the transgenic clones were evaluated for cardiac function. Although no physical defects 

were found, one piglet had evidence of mitral insufficiency (a condition in which the mitral valve 

of the heart does not close all the way during contraction, resulting in regurgitation of some of 

the blood in the left ventricle), and dilation of the left atrium and ventricle. This piglet and two 

other clones had reduced cardiac output values compared to control piglets, but did not display 

clinical signs of cardiac disease. Although similar cardiac abnormalities have been noted in 

conventional swine, the incidence is reported to be very low (Carter citing Hsu et al. 1982). 

These developmental defects appear to be similar to those noted in cattle clones (see Critical 

Biological Systems discussions). 

Lai et al. 2002 

This study was reported in a brief communication, and a limited amount of data was presented. 

Piglets were generated from cell lines (derived from inbred miniature pigs) in which the α-1-3-

galactosyltransferase gene was interrupted by the insertion of a gene sequence in order to create 

α-1-3-galactosyltransferase “knock-outs.” The α-1-3-galactosyltransferase gene codes for a 

protein that causes hyperacute rejection of swine organs when transplanted into primates. 

“Knocking out” the expression of this gene increases the suitability of these animals to be used 

as donors of organs for human transplant patients. 

Six piglets were born from two litters. All but one of the piglets had low birth weights compared 

to the breed average (115 to 650 g vs. 860 g). One piglet from each litter died shortly after birth 

from what the authors termed “respiratory distress syndrome.” A third piglet died at 17 days of 

age during a routine blood draw, and was diagnosed at necropsy with a dilated right ventricle and 

thickening of the heart wall. Other abnormalities noted in these surviving transgenic piglets 

included flexor tendon deformities in three animals; abdominal ascites, enlarged right ventricle, 

pulmonary hypertension in one animal; and ocular defects and lack of patent ear canals in 

another animal. The authors attributed these abnormalities to failures in reprogramming during 

the SCNT process rather than the genetic engineering process, as they did not see a consistent 

phenotype across the piglets. 
Appendix D: Transgenic Clones D-8 

Animal Cloning: A Risk Assessment DRAFT

Lee et al. (2003) 

Recently, Pearson (2003) reported that the University of Connecticut laboratory that had 

generated four transgenic swine clones had announced that the three (of four) surviving piglets 

died suddenly of heart failure at less than six months of age. The fourth piglet died at three days 

due to infection and abnormal spine development (Lee et al. 2003). Because of the transgenic 

nature of the animals (they carried genes for human clotting factor IX and porcine lactoferrin, an 

iron transport protein found in blood), it is not possible to attribute the deaths solely to cloning. It 

is unknown whether any cardiac abnormalities were detected in these animals prior to their 

deaths, or if any measurements of cardiac function were made.

D. Sheep 

Denning et al. 2001

Denning et al. 2001 were unsuccessful in producing viable knock-out sheep lacking either the α-

(1,3)-galactosyl transferase (GGTA1) or the prion protein (PrP) gene using gene targeted fetal 

fibroblasts and SCNT. Reconstructed embryos were either incubated for six days (n=48) or 

overnight (n=93) in synthetic oviductal fluid with bovine serum albumin (concentration not 

specified). Embryos incubated overnight in vitro were then embedded in 1 percent agar chips in 

phosphate buffered saline and transferred to the ligated oviduct of an estrus-synchronized ewe 

for six days. A total of 120 morula or blastocyst stage embryos were transferred to 78 estrussynchronized Finn Dorset ewes as final recipients. It is not clear from this paper how many of the 

transferred embryos had been incubated in vitro. Although 39 pregnancies were diagnosed at 

gd 35, only eight were maintained to term, resulting in four live births. Three of the four liveborn lambs died shortly after birth. The fourth lamb survived 12 days before it was euthanized 

after developing dyspnea (difficulty breathing) due to pulmonary hypertension and right-sided 

heart failure. The authors attributed the abnormalities observed to the nuclear transfer procedure, 

as they were similar to results obtained with non-transgenic NT lambs. 

McCreath et al. 2000

McCreath et al. 2000 inserted a promoter-less neomycin selectable marker between the ovine 

α1(I)-procollagen translational stop and polyadenylation signal74 in male and female ovine fetal 

fibroblast cultures. Four transgenic female fibroblast cultures were selected as nuclear sources 

for SCNT, due to their vigor and normal chromosome number. A total of 80 morula and 

blastocyst stage embryos were transferred to recipient ewes. No description of post-fusion 

incubation or estrous cycle status of recipient ewes was provided in this report. Fourteen lambs 

 

74 These are regions of the DNA construct that provide instructions for the appropriate processing of information 

in order to make functional proteins. 
Appendix D: Transgenic Clones D-9 

Animal Cloning: A Risk Assessment DRAFT

were born alive; seven of these lambs died within 30 hours of birth. Four more lambs died 

between 3 days and 12 weeks of age. Three lambs survived and were described as thriving at one 

year of age. Necropsy of lambs that died in utero or after birth revealed a number of 

abnormalities including a high incidence of kidney defects (frequently renal pelvis dilation) and 

liver and brain abnormalities (not specified). The authors attribute these abnormalities to either 

cell treatment or the NT procedure, because the necropsy findings were similar to a previous 

nuclear transfer study using the same cell lines. 

E. Goats 

Baguisi et al. 1999

In this study from Genzyme Transgenics, six cell lines were established from 35- and 40-day old 

fetuses that resulted from the mating of a transgenic buck (carrying a human antithrombin III 

(hAT) gene with a goat β-casein promoter) to a non-transgenic doe. This study differs slightly 

from several other transgenic cloning studies reported here, in which the gene was inserted into 

the cell lines before the cultures were established. Clone embryos were cultured on goat oviduct 

epithelial cells for 48 hours (2-16 cell stage) before being transferred to estrus synchronized 

recipient does. Although overall cloning efficiency was low (3/112 embryos transferred resulted 

in live births), all pregnancy losses occurred prior to 60 days of pregnancy. There were no 

stillbirths and no abnormalities observed in the live-born kids. Kids weighed between 2.35 and 

3.5 kg, within the normal birth weight for dairy goats, and are reported as healthy. 

Keefer et al. 2001a

In this study from the Nexia Biotechnologies laboratory, goat fetal fibroblasts were transfected75 

with green fluorescent protein (eGFP) and neomycin resistance genes. These are commonly used 

as markers to demonstrate that transgenes have been inserted and are being expressed. Twenty 

seven NT embryos were produced with the transfected cells, and an additional 70 non-transgenic 

NT embryos were constructed and transferred into 13 estrus synchronized recipient does. The 

authors did not specify how many embryos (transgenic or non-transgenic) were transferred to 

each doe. Five non-transgenic male clones and one transgenic female clone were born alive. 

Three of the non-transgenic clones died of bacterial infections, but the single female transgenic 

clone lived and showed no signs of abnormalities. The kids were all within the normal birth 

weight range (1.5 to 3.1 kg) for goats at that facility, and no abnormalities were observed in the 

placentas. 

 

75 Transfection is the process of introducing exogenous DNA into cells without the use of viral vectors. Common 

methods include co-precipitating DNA with salts and polymers. 
Appendix D: Transgenic Clones D-10 

Animal Cloning: A Risk Assessment DRAFT

Behboodi et al. 2004

The authors compared development of embryos cultured with oviductal cells in vitro vs. embryos 

cultured in vivo. Embryos were constructed using skin fibroblasts of transgenic goats. Only 

embryos cultured in vivo resulted in pregnancies. Two of these pregnancies were lost early in 

gestation (after 30 days gestation), and four other pregnancies were carried to term. Two 

surrogate does delivered stillborn kids 2-3 days after their due dates; the other two does delivered 

healthy kids (one per each doe) at term. The two live clones weighed 3.8 and 4.1 kg at birth, and 

were within the normal birth weight range for their breed (Saanen). Clones were weaned at 8 

weeks of age, and had similar growth rates compared to age-matched AI derived Saanen kids 

born at the same facility (14.5 and 18.1 kg for clones vs. 14.88 ± 1.98 kg for AI comparators). 

Pathology on the dead fetuses indicated diffuse atelectasis (lung collapse) and the presence of 

amniotic fluid in the lungs. No bacterial or viral cause for the deaths of these clones could be 

identified. The presence of amniotic fluid in the lungs suggests that the clones attempted to 

breathe prematurely, a sign of fetal stress which sometimes occurs around the time of birth. 

Behboodi et al. 2005

The authors evaluated health, growth, reproduction and lactation in four female goat clones 

generated from two transgenic fetal cell lines (one cell line coding for glycosylated and the other 

for non-glycosylated protein). A total of seven clones were carried to term. One clone from the 

glycosylated group was still born with evidence that the umbilical vessel had ruptured. Two 

clones died at birth (one from each of the transgenic lines) after failing to breathe on their own, 

despite attempts at manual ventilation. Thus, two clones from each transgenic line survived to 

adulthood. There were no differences in birth or weaning weights among the four surviving 

clones or their age-matched comparators. Transgenic clones exhibited enlarged umbilical stumps 

(two live and one stillborn kid), “tendon laxity” (three of the four live-born clones), and minor 

generalized edema (number of clones affected not indicated). These conditions resolved without 

intervention. The four does were bred and produced nine kids, compared to five kids produced 

by comparators. Clones expressing the glycosylated version of the protein lactated only briefly, 

but the does expressing the non-glycosylated protein had normal lactation length and milk yields. 

This study is the only one we encountered that presented hematology and blood clinical 

chemistry data for four goat clones. These data are presented in comparison to four age-matched 

comparators and values from the literature (Pugh 2002). It is unclear whether or not the 

comparators in this study were also transgenic, whether they were the same breed as the clones, 

or how they were generated. Hematology values were similar between clones and comparators, 

and all hematology values fell within the published range. For clinical chemistry, 18/24 values 

were not significantly different between clones and their age-matched comparators. Of the 19 
Appendix D: Transgenic Clones D-11 

Animal Cloning: A Risk Assessment DRAFT

clinical chemistry values for which published ranges were available, 18 of the values for clones 

and comparators fell within the published range. The one value out of the published range was 

creatine kinase (244.6 vs. 204.4 IU/L for clones and comparators). However, values between 

clones and comparators were not statistically different. The study does not specify the age of the 

goats at time of blood sampling, so it is difficult to interpret the high values for CK in these 

animals compared to the published range. 

This study is unique among reports of goat clones because it is the first to indicate possible signs 

of LOS in goat clones (enlarged umbilici, failure to initiate breathing, tendon problems). It is 

interesting to note that similar signs have not been noted in non-transgenic goats. We should also 

note that clinical signs in the four surviving clones resolved, and their health, growth, 

reproduction, and hematology, clinical chemistry values indicate that even these transgenic 

clones are apparently normal. 

Landry et al. 2005

The authors reported on growth (weight gain, wither and hip height change) and endocrine 

profiles of two lines of transgenic goat clones. Group 1 consisted on five does carrying the ATIII gene with a β-casein promoter inserted into cells of a female Toggenburg (dairy breed). The 

gene inserted into the second line of goats (Group II; n=2) was not identified, but the cells used 

for cloning were from a female Saanen (also a dairy breed). Non-transgenic, non-clone 

comparators (n=7) were Boer X Spanish crossbred meat-type does (Group III). The authors did 

not report on overall health of the clones. One female in each group of clones died prior to the 

end of the study; one died due to an accident, the other due to a ruptured abomasum. Neither 

death appears to be related to cloning. Both groups of clones were within range for their breed 

for birth weight, and appeared to grow normally. Interpretation of hormone profiles (GH, IGF-I, 

T3, and T4) is difficult due to the fact that the clones and comparators were of different breed 

(purebred vs. crossbred) and type (dairy vs. meat) backgrounds. However, for most of the 

hormones assayed, the values for clones fell within the range of values for comparators. The one 

exception is insulin, which resulted in an extremely low value in blood samples of comparators, 

and may have been the result of difficulties with the assay. 

F. Conclusions Regarding Transgenic Clones 

The experience of these cohorts of transgenic clones can be summarized as follows: 

• A relatively large fraction of transgenic fetal bovine clones in cohorts surviving to 

late gestation presents with severe and often fatal difficulties. Some of these are 
Appendix D: Transgenic Clones D-12 

Animal Cloning: A Risk Assessment DRAFT

qualitatively similar to those observed in cattle and sheep clones that are not derived 

from transgenic cells. Due to the many other variables that have been altered in the 

generation of these animals, at this time it is not possible to attribute these 

abnormalities to either of the processes (cloning or transgenesis) or their combination. 

• Some animals in both cattle cohorts are born with varying degrees of initial 

respiratory or other physiological distress. Supportive care appears to allow most of 

these animals to survive to adulthood, although some animals that initially survive 

can succumb to possible sequellae up to six weeks later. 

• Animals surviving to adulthood in the ACT cohort that appear to be healthy on visual 

inspection also exhibit physiological values that generally fall within normal ranges. 

CVM is unaware of an update of the health status of the Hill et al. cohort. 

• Animals in the ACT cohort surviving to reproductive maturity appear to be capable of 

bearing normal offspring, although it is not clear whether the offsprings’ health has 

been examined in a rigorous manner. 

• Two severe adverse outcomes have been noted for the ACT cohort. Both cloning and 

transgenesis can likely be ruled out as causes for one (Johne’s disease) and no causal 

agent or process has been associated with the neoplasm found in the other. 

• The appearance, behavior, and physiological function of the animals that survive 

suggest that even the “riskiest” set of clones (i.e., transgenic clones) can develop into 

normally functioning animals. These results are consistent with the analysis of nontransgenic clones, and provide additional confidence that rigorous monitoring and 

responsible husbandry of such animals can allow for the selection of animals that are 

healthy. 

• Abnormalities for transgenic sheep clones appear similar to reports for non-transgenic 

sheep and cattle clones. 

• Goats appear to suffer fewer adverse effects compared to sheep and cattle. Of the 

reports reviewed, only one cohort exhibited clinical signs of LOS. 

• Abnormalities reported for transgenic swine clones are similar to those reported for 

transgenic and non-transgenic cattle clones. CVM is aware of only one report in non-
Appendix D: Transgenic Clones D-13 

Animal Cloning: A Risk Assessment DRAFT

transgenic swine clones (Park et al. 2005) in which clones exhibited similar health 

problems; however, in vitro methods used in this study likely influenced the outcome 

of swine clones in this study. 
Animal Cloning: A Risk Assessment DRAFT

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Animal Cloning: A Risk Assessment DRAFT

Appendix E: 

The Cyagra Dataset
Animal Cloning: A Risk Assessment DRAFT

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Animal Cloning: A Risk Assessment DRAFT

Appendix E: The Cyagra Dataset 

A. Response to CVM Data Requests 

The Center for Veterinary Medicine (CVM) has presented its proposed risk assessment approach 

at several public venues since the fall of 2002. As part of the proposed approach, we have 

requested that investigators engaged in cloning cattle, swine, sheep, or goats share data that they 

might have on the health status of these animals with the Center. The intent of the data request 

was to supplement the published data with unpublished data generated by the developers of these 

animals. We thought that data on the health status of animal clones would likely be in the hands 

of the private sector, which might have less impetus to publish than academic laboratories. The 

Center promised producers that, to the extent allowed by law, if they wished, their identities 

could be kept confidential by FDA, and that we would not publish the specific identity and 

location of the animals. 

We discovered that there are very few datasets describing the health of animal clones. In general, 

clones are monitored closely for the first few weeks of life (or through weaning). They are often 

then moved from “research/hospital” facilities to “farm-like” facilities, where they are often 

reared with conventional animals. Most producers kept fairly cursory veterinary records unless 

the animal was in distress. Further, because of technical issues associated with generating 

successful pregnancies, only a few clones tend to be delivered at one time or from one cell line. 

The result is that aggregating and analyzing data becomes difficult unless publications are 

planned in advance. 

As many of the clone producers either have primary employment as academics, or continue to 

maintain academic appointments, there may be data available that have not yet been shared with 

CVM because of the investigators’ desire not to jeopardize their ability to publish in peer 

reviewed journals. Because of CVM’s pledge to be fully transparent in this risk assessment, we 

determined that all data submitted would be made public through the risk assessment. We 

obtained the express permission from the submitters of data for the public release of this data. 

“Publishing” this information in the assessment could preclude formal publication in a peer 

reviewed journal, as most high quality peer reviewed journals have a policy of being the site of 

first publication. 

As our risk assessment methodology evolved, it was presented at public fora (the Pew Initiative 

for Agricultural Biotechnology’s September 2002 Biotech in the Barnyard conference, the April 
Appendix E: The Cyagra Dataset E-4 

Animal Cloning: A Risk Assessment DRAFT

2003 American Registry of Professional Animal Scientists meeting in Maryland, and the FDA 

Science Forum of April 2003). Subsequent discussions between clone producers and agency staff 

resulted in investigators returning to the field to try to collect existing data, or, in one exceptional 

case, to generate de novo data on the health status of clones. Without exception, every clone 

producer or investigator contacted was willing to answer questions on aspects of clone 

production, gestation, delivery, and care. Many have provided data or information that we have 

incorporated into this risk assessment or will use in future iterations. In order to issue the risk 

assessment in a timely manner, however, we have had to put off our analysis of some of the 

datasets until the next revision of the risk assessment. We are very grateful to those producers 

and owners who voluntarily expended significant time, effort, and in some cases, capital, to 

provide information to us. 

B. Cyagra Dataset 

One clone producer, Cyagra Inc.,76 has been engaged in the production of cattle clones since 

1999. In the late spring of 2003, Cyagra submitted an extensive database to CVM for use in the 

animal health component of our food consumption and the animal health risk assessments. These 

data were made available for CVM to use in our risk assessments with no restriction, except to 

protect to the extent allowed by law the identity and location of the animals, and their current 

owners. In order to accommodate this request, CVM issued each animal in the study a unique 

identification number. These numbers have been employed throughout this analysis. 

Cyagra has asserted that they have provided data on all of the clones that they can trace, 

including those that died, or were euthanized or culled. Animals were divided into three age 

cohorts by Cyagra: neonates (within 24 hours), 1-6 month age cohort (between 30 and 175 days 

of age), and 6-18 month age cohort (187-557 days of age). 

The age spread among these animal cohorts reflects key stages in physiological development of 

cattle. For example, digestion differs significantly among different age groups: a 2-month-old 

calf is just starting to use its rumen, while a 6-month-old calf is a fully developed, cud-chewing 

ruminant. In this case, these two calves have been grouped together even though they have 

substantive physiological differences, because they have more in common than, for example, a 

neonate and a six month old calf. For the sake of accuracy, we have classified this group as 1 to 6 

months old. A 6-18 month old calf is not quite old enough to be considered an adult, as it is still 

growing, and the younger animals in this group will still be pre-pubertal. We have therefore 

 

76Cyagra Inc. is a privately held biotechnology company commercializing SCNT technology for the agricultural 

sector. 
Appendix E: The Cyagra Dataset E-5 

Animal Cloning: A Risk Assessment DRAFT

decided to classify this group simply as “6 to 18 months.” The distribution of animals in the 

cohorts is found in Table E- 1: Distribution of Cattle Clones and Comparator Populations. 

Comparators were approximately age-matched animals reared on the same farms or facilities as 

clones. The comparators were not born at the same locations, and do not represent the same 

distribution among breeds as the clones. Comparator animals were not clones, but were produced 

by either artificial insemination (AI) or natural breeding, from either primiparous (heifers) or 

multiparous dams, and were all delivered vaginally. Blood samples from neonatal comparator 

animals were taken after colostrum administration, while neonatal clones were sampled prior to 

receiving colostrum. 

These animals do not provide a strict biological comparator that has experienced the same 

treatments and conditions as the clones. For example, the culturing conditions in the embryonic 

phase for cloned embryos could be more closely compared with those encountered by animals 

generated by in vitro fertilization. These comparators are not, strictly speaking, “control” 

animals. 

Further, given the approximate age- and breed-matching, this dataset should not be evaluated in 

the same manner as a tightly controlled prospective “laboratory” experiment. Rather, our opinion 

is that this dataset should be viewed as an attempt to compare health and laboratory test values 

between clones and conventional animals comprising part of national dairy and beef herds. These 

data were not generated or collected under “Good Laboratory Practices,” and we have not 

attempted to audit the data except insofar as we have detected errors or requested clarification(s) 

from Cyagra. 

Table E- 1: Distribution of Cattle Clones and Comparator Populations for Blood 

Analyses

Number of Animals 

neonates 1 to 6 

months 6 to 18 months Totals 

Clones 10 46 18 67 

Comparators 17 47 21 83 

In table E-1, 7 (of the 46) 1-6 month clones and 2 (of the 47) 1-6 month comparators were 

sampled in the neonatal group. The 1 to 6 month and 6 to 18 month cohort information was 

collected within a relatively short time frame. These data may best be thought of as a “snapshot” 

view of the animals during their development, rather than a longitudinal study in which the same 

animals are followed over some period of time. In fact, only nine animals were sampled or 
Appendix E: The Cyagra Dataset E-6 

Animal Cloning: A Risk Assessment DRAFT

examined at more than one time point (at birth and weaning), and of those, seven were clones 

and two were controls (Clone ID# 71, 72, 73, 78, 79, 119, and 132; Control ID# 135 and 162). 

1. Description of Clones 

All clones were derived from actively dividing cells from skin biopsies; recipient oöcytes were 

obtained from commercial abattoirs. After 7 or 8 days of in vitro culture, morula or blastocyst 

stage reconstructed embryos were implanted into recipient Holstein heifers. Pregnancies were 

monitored closely, and with few exceptions, clones were delivered via Caesarean section (Csection) to reduce the risk associated with birth. Blood samples were drawn from the neonates 

prior to colostrum administration. 

Table E-2 summarizes the information on samples taken from calves within the first 24 hours of 

birth. Some of the animals in the Cyagra dataset required some supportive care immediately after 

birth (e.g., glucose, warming, or supplemental oxygen), and many (n=29 out 134) received 

umbilical surgery after birth. Enlarged umbilical vessels which do not close naturally after birth 

are an identified hazard for clone calves, and many of these calves received surgery to prevent 

complications such as umbilical infections and bleeding (see subsequent discussion on veterinary 

examinations and health status). This appears to be a fairly common problem in clones, and may 

be associated with poor placentation. However, no direct causal attribution can be made at this 

time to any particular developmental pathway causing the umbilical problems. 

Health anomalies noted in surviving animals for which there are no additional follow-up data 

include diarrhea, fever, anemia, heart murmur, and slight contracture of the flexor tendons 

(referred to as “contracture”). 

Of the 134 clones in this review, 28 were stillborn, died, or were euthanized within 48 hours of 

birth, leaving 106 animals (or 79 percent) alive two days after birth. At the time that data were 

collected on these animals (late March 2003), 67 were alive (64 percent of those surviving to 48 

hours, or 50 percent of those born or delivered). Eleven (10 percent) of the animals alive at 48 

hours died within approximately one and a half years later. These data are summarized in Table 

E-2. Of the eleven deaths between 48 hours and one and a half years later, Cyagra considers two 

deaths not related to cloning, and the other nine as “related, possibly related, or questionably 

related” to cloning. Of those fitting the “related (to some degree) to cloning” category, one was 

clearly a fetal developmental anomaly: flexor tendon contracture (“contracture”); three 

experienced difficulties with the umbilicus ultimately leading to death either via infection or 
Appendix E: The Cyagra Dataset E-7 

Animal Cloning: A Risk Assessment DRAFT

adhesions; two had gastrointestinal problems with bloat or adhesions; two had circulatory 

problems; and one animal was euthanized for “failure to thrive.” 

Table E-2: Summary of Outcomes for Clones Not Surviving Birth 

Animal 

Number

 Birth Weight 

(kg) Age at Death (days) Problems 

Noted Cause of Death

3 NP1 0 Abnormal delivery Stillborn 

6 NP 16 NP Accident; hung in stall 

11 NP 0 Abnormal delivery Euthanized 

12 NP 0 Ruptured uterus in 

recipient Stillborn 

13 NP 0 NP Unknown 

14 33.2 13 Contracture2

, umbilical 

infection Septicemia 

16 50.0 2 

Slack abdomen, umbilical 

problems, breathing 

difficulties 

Failure to transition to 

neonatal circulation 

18 68.2 0 Polycystic kidneys Stillborn 

19 69.1 0 Umbilical problems, 

flaccid abdomen Stillborn 

20 NP 0 Abnormal development Euthanized 

23 45.5 0 

Abnormal development, 

internal bleeding, umbilical 

problems 

Euthanized 

28 NP 0 NP Stillborn (C-section) 

29 NP 0 Abnormal development Euthanized 

31 76.8 0 Abnormal renal 

development Euthanized 

34 NP 0 NP Stillborn (C-section) 

43 NP 1 Diarrhea Rotavirus 

47 NP 0 NP Stillborn (C-section) 

48 54.5 0 NP Stillborn (C-section) 

49 NP 0 NP Stillborn (C-section) 

51 NP 0 Flaccid abdomen, 

“bulldog” Stillborn (C-section) 

52 NP 0 NP Stillborn (Fetotomy) 

54 59.1 0 

Reverted to fetal 

circulation, cardiac, 

neurological problems 

Euthanized 

57 NP 23 Ruptured abomasum Ruptured abomasum 

63 NP 60 Loss of hair, appetite, 

muscle Euthanized/failure to thrive 

65 61.4 3 Lethargic GI transit; adhesions from 
Appendix E: The Cyagra Dataset E-8 

Animal Cloning: A Risk Assessment DRAFT

umbilical bleeding 

66 54.6 149 

Contracted tendons, 

recurring bloat, large 

umbilicus requiring 

surgery 

Bloat/GI motility problems 

68 NP 0 (2 weeks 

premature) Pericarditis Unable to determine 

77 NP 47 Umbilical problems Severe contracture, 

unresponsive to therapy 

80 NP 1 Diarrhea Rotavirus 

86 NP 0 Severe contracture, fluid 

filled belly Euthanized. 

92 NP 0 Depressed, pus in 

umbilicus Unable to determine 

95 NP 0 Severe contracture Euthanized 

97 NP 0 Severe contracture, fluid 

filled belly Euthanized 

105 45.5 0 Severe twisting of neck, 

contracture Euthanized 

107 NP 2 Hypoxemia, rapid 

deterioration Euthanized 

109 NP 0 Abnormal development Euthanized 

113 NP 22 Nephritis Pyelonephritis3

/ umbilical 

infection 

123 NP 9 Contracted front fetlocks Pyelonephritis/ umbilical 

infection 

125 NP 0 Severe contracture, rotation Euthanized 

1

NP = Not provided 

2

Contracture is a condition in which muscles have a fixed, high resistance to stretching due to fibrosis of the tissues 

supporting the muscles or joints, or from disorders of the muscle fibers. 3

Pyelonephritis is an inflammation of the kidney due to bacterial infection. 

2. Evaluations Performed 

Several types of information including veterinary records, clinical chemistry measurements, 

hemograms,77 and urinalysis are provided in this dataset. Not every collectable data point has 

been provided for each animal. Some information is unavailable because use of the data in a 

review such as this was not anticipated at the time the data were collected. In addition, dispersal 

of clones to their ultimate owners limited data collection to the degree to which owners made 

information or animals available. Nonetheless, this is the largest collection of information on the 

 

77 A hemogram is a panel of measurements characterizing the nature of the circulating blood in an animal or 

human. 
Appendix E: The Cyagra Dataset E-9 

Animal Cloning: A Risk Assessment DRAFT

health status of non-transgenic clones of which we are aware, and the most detailed with respect 

to health status and laboratory tests. 

The dataset includes information on the following: 

• Breed from which donor cells were collected 

• Gender of the donor 

• Birth date of the clone 

• Birth status (alive, stillborn) 

• Birth weight 

• Perinatal health status and veterinary/supportive care provided 

• Health status of animals between two and twelve months of age 

• Veterinary care, including treatment with drugs, surgery, or other therapeutic 

interventions 

• Standard blood chemistry assays (Large Animal Panels) 

• Assays for serum Insulin-like Growth Factor-1 (IGF-1), estradiol-17β, amylase, 

cholesterol, and bile acids 

• Complete blood counts (CBC) and differentials 

• Standard urinalysis 

Comprehensive veterinary examinations were performed by licensed cattle veterinarians. Blood 

samples were drawn within a few hours of birth, or at the time of veterinary examination. For 

CBC, blood was collected into standard EDTA-treated collection tubes; additionally, two 

unstained and unfixed air-dried smears were provided. For chemical analyses, whole blood was 

collected, allowed to clot, and the serum fraction separated by centrifugation. Laboratory 

analyses were all performed at the Cornell University’s Animal Health Diagnostic Laboratory. 

3. CVM’s Analysis of Cyagra Data: Method 

Our goal in evaluating the Cyagra dataset has been to determine whether extensive interrogation 

of the health status of the clones, including clinical chemistry and hematology, could 

 

(a) Distinguish clones from comparators; 

(b) Determine whether the health status of the clones was inferior to conventional 

animals and offer a predictor of a successful outcome; and 

(c) Determine whether any of the information indicated concerns regarding animal 

health or food safety. 
Appendix E: The Cyagra Dataset E-10 

Animal Cloning: A Risk Assessment DRAFT

We note that this was not a “blinded” analysis of the provided data. No attempt was made to 

disguise the identity of the animals, and whether they were clones or comparators. CVM 

personnel engaged in performing the evaluation included veterinarians, animal scientists, 

toxicologists, and risk assessors, with extensive training in evaluating clinical and physiological 

measurements of animals traditionally consumed as food in the US. 

 

For the overall health status of animals, the veterinary records were reviewed for notations 

indicating therapeutic interventions (including administration of colostrum, vaccines, dehorning, 

surgeries, drug therapies, etc.). Clinical and hematologic data were compared to both reference 

ranges provided by the testing laboratory and to the comparator animals. Additionally, laboratory 

values from the comparator animals were also compared to the testing laboratory to determine 

the degree to which the comparator group was represented by the testing laboratory’s reference 

range (see Results). In general, urinalysis data were only used qualitatively as confirmation of 

outcomes noted in the clinical chemistry (e.g., glucose, BUN or creatinine levels). Table E-3 

provides a summary of the analyses performed and tabulated in the Charts indicated. 

 

Outcomes were reviewed on an analyte basis across a cohort of animals (analyte evaluation), and 

on a per animal basis across analytes (animal evaluation). The questions asked for each animal 

and analyte tested were “How many of the total animals tested exhibited values outside the 

comparator/testing laboratory reference range for Analyte X?” and “How many values outside 

the comparator/testing laboratory reference range does Animal Y exhibit?” 

 

The Charts are a graphical summary of CVM’s analyses. For each chart, the unique identification 

number associated with each animal (“ID#” or “animal number”) is listed in columns 

horizontally across the top of the table; the analysis performed is listed in rows vertically down 

the side of the table. If the value being evaluated fit within the comparison range being used for 

that interrogation, a black rectangle was recorded in the cell corresponding to the animal 

column/analyte row pair (g). If the value was outside the comparator range, but judged to be not 

clinically relevant, a gray rectangle ( ) was recorded. If the value recorded was above or below 

the clinically relevant range, an arrow indicating whether the value was greater or less than the 

range was inserted (↑↓). Values that were considered to be so far out of range as to be 

physiologically incompatible with a healthy animal but unsupported by related clinical 

measurements were deemed artifact and labeled “X.” For example, a calf with a blood glucose 

level of 4 mg/dl would be comatose or dead. If the sample came from an animal that was not 

comatose or in distress, and there were no other related clinical measurements normally 

associated with abnormal blood glucose, we assumed that the measurement was an artifact. 

Missing values were represented by an asterisk (*). 
Appendix E: The Cyagra Dataset E-11 

Animal Cloning: A Risk Assessment DRAFT

Table E-3. Summary of Charts Describing Comparisons 

Clones: Reference Range Clones: Comparators Comparators: Reference 

Range 

Clinical Chemistry: 

 6 to 18 months 

 1 to 6 months 

 neonates 

Chart 300 

Chart 200 

Chart 100 

Chart 301 

Chart 201 

Chart 101 

Chart 302 

Chart 202 

Chart 102 

Hematology: 

 6 to 18 months 

 1 to 6 months 

 neonates 

Chart 310 

Chart 210 

Chart 110 

Chart 311 

Chart 211 

Chart 111 

Chart 312 

Chart 212 

Chart 112 

 

4. CVM’s Analysis of Cyagra Data: Results 

a. Comprehensive Veterinary Examinations 

Comprehensive Veterinary examinations were performed on 53 clones and 2 non-clones, and 

included explicit evaluations of the following: 

 

• Demeanor • Gastrointestinal system 

• Posture • Genitals 

• Gait • Neurological examination 

• Body Condition • Peripheral lymph nodes 

• Skin and coat • Responsiveness of pupils to light 

• Vocalization • Corneas and eyelids 

• Lungs (Auscultation) • Umbilicus 

• Nerves • Weight 

• Integument • Heart Rate 

• Musculo-skeletal system • Respiration rate 

• Cardiovascular system • Temperature 

• Oral/Pharyngeal region • Feces 

• Urine 

The calves in this study were examined by veterinarians specializing in cattle at roughly 1-6 

months of age or at 6-18 months of age. The most consistent abnormality reported for clones was 

umbilical surgery, often described as umbilical hernia surgery. In some instances, the records 
Appendix E: The Cyagra Dataset E-12 

Animal Cloning: A Risk Assessment DRAFT

stated, “umbilicus – had surgery.” Some other comments on the umbilicus were: “had umbilical 

hernia surgery,” “ventral hernia,” and “1 1⁄2” hernia,” “fluid filled mass,” “umb. stump.” In the 

initial submission of 58 animals, 26 animals had umbilical surgery. Other abnormalities reported 

included two clones with musculo-skeletal abnormalities, one with slight precocious (early) 

mammary development, two with harsh lung sounds, three cryptorchid (undescended testicles) 

bull calves, and one with premature ventricular contractions (PVCs, a form of cardiac 

arrhythmia) every 5 – 10 heartbeats. 

The two clones with musculo-skeletal abnormalities included a Holstein heifer (ID# 79) with 

thick withers, enlarged left carpus, and leg that deviated laterally, and an Angus heifer that was a 

dwarf tending to gastro-intestinal bloat (Clone #108). These are obvious abnormalities and the 

animals were culled. The calf with slight mammary development was a 41⁄2 month old Jersey 

(Clone #87). This age is young for mammary development but the phenomenon sometimes 

occurs in conventional heifers if they are overfed. There is no notation of follow up to determine 

if the calf continued to develop precociously. 

The two clones with harsh lung sounds were a Holstein heifer (Clone # 41) and an Angus heifer 

(Clone #58). Both also had umbilical surgery. A note at the bottom of the Angus heifer’s exam 

sheet stated that the heifer “may not return home due to permanent lung damage.” There is no 

indication as to whether this animal was culled. Three Holstein bull clones derived from the 

same cell line were diagnosed with a retained testicle (cryptorchid) (Clones #128, 130, 131). 

Although cryptorchidism is not common in bull calves, it is thought to be heritable and is seen 

with some regularity. Bulls exhibiting cryptorchidism would fail their breeding soundness 

exams, and would not be used for breeding,78 but would not be refused by an inspector at 

slaughter. 

A Holstein bull calf clone (Clone #126) was diagnosed with premature ventricular contractions 

from a single exam, but no subsequent follow up is provided to determine whether the animal 

outgrew the condition or whether the animal was culled. The frequency of cardiac arrhythmias in 

conventional calves is unknown. Thoracic auscultation (listening to the chest with a stethoscope) 

or more elaborate procedures are needed to detect cardiac arrhythmias. Calves are rarely 

examined with thoracic auscultation unless they show signs of illness. 

 

78 Cryptorchidism is undesirable because of its heritability, its adverse effect on fertility, and potential for the 

development of testicular cancer in animals living long enough to allow neoplasia to develop. From a veterinary 

standpoint, however, testicular neoplasia is more of an issue with companion animals, as they are generally 

longer-lived than farm animals. 
Appendix E: The Cyagra Dataset E-13 

Animal Cloning: A Risk Assessment DRAFT

b. Conclusions from Veterinary Examinations 

The adverse physical exam findings noted in this limited sample of clones do not present a food 

safety issue for several reasons. One of the precepts of this risk assessment is that animals found 

to have a disease or condition that would render them adulterated (e.g., unfit for food, 

unhealthful, unwholesome) are excluded from the food supply, as normally happens with 

conventional animals. Dwarf animals from conventional breeding would likely be culled 

depending on the extent of the physical abnormality. Pre-pubescent mammary development, lung 

sounds, cryptorchidism, and cardiac arrhythmias are not conditions that typically exclude 

animals from food use. If the disease process had progressed to an extent sufficiently severe to 

cause systemic changes (e.g., liver congestion, enlarged heart, edematous lungs), the carcass 

would be condemned on inspection at the slaughtering plant. In fact, all of these conditions occur 

in conventional animals. 

With respect to animal safety, these conditions may pose some cause for concern. Our review of 

these data indicates that the clone cohort appears to exhibit a higher incidence of abnormalities 

than might be expected in a random sample of conventional calves. There is, however, an 

absence of data on the prevalence of these outcomes in contemporary cattle. As some of these 

defects (e.g., dwarfism, cryptorchidism) likely have a hereditary component, in the absence of 

information on the donor cattle and their individual histories, we cannot determine whether the 

defects result from the cloning process, the selection of the donor nucleus, or some combination 

of those factors. The clustering of cryptorchidism in clones from one cell line, for example, 

implies that heredity may indeed be a contributing factor in the appearance of that outcome. 

Comparison with datasets on animal health from other clone producers would be instructive in 

determining whether these health problems are common among clones generated by different 

methods and multiple cell lines. 

c. Laboratory Values: Selection of Most Appropriate Comparator 

Two comparators were available for evaluating the Clones: the Cornell Animal Health 

Diagnostic Laboratory (“Reference Range”) and approximately age-matched, and breeddistributed cohort of animals contemporarily reared at the same farms as the clones 

(“Comparator Population” or “Comparators”). The Reference Range population from the Cornell 

Laboratory is described as follows: 

“We establish reference intervals by collecting blood from at least 50 adult healthy

animals. These healthy animals are obtained from a variety of sources (e.g., student- or 
Appendix E: The Cyagra Dataset E-14 

Animal Cloning: A Risk Assessment DRAFT

faculty-owned). Therefore, our reference intervals are only applicable for adult animals 

and not young animals. Results from young animals may fall outside our reference 

intervals because of age-dependent changes in their analytes. For example, phosphate 

concentrations and alkaline phosphatase activity are higher in young animals and 

decrease to within reference intervals at about one year of age.” 

(http://www.diaglab.vet.cornell.edu/clinpath/reference/)

Follow-up conversation with the laboratory indicates that the animals used to establish the 

laboratory’s reference range were exclusively dairy cows, and thus do not represent the beef 

breeds that are included in the Cyagra clone cohort or comparator cohorts, and may not include 

bulls. In addition, it is important to remember that the reference range is selected as a statistical 

distribution containing about 95 percent of the normal samples. As a result, as many as 5 percent 

of the test values will likely fall outside that range. Statistically, when numerous tests are run on 

the same animal, the chance of obtaining one or more results outside the “normal range” rises 

based on chance alone and not a disease state. 

Table E-3a: Fraction of Blood Values Within Comparison Range 

Animals Analysis Clones: Cornell 

Reference Range 

Clones: 

Comparator 

Population 

Comparator 

Population: Cornell 

Reference Range 

 Chart FCWR Chart FCWR Chart FCWR 

Peripubertals 

(6-18 months) 

Clinical 

Chemistry 300 0.75 301 0.99 302 0.73 

 Hematology 310 0.73 311 0.99 312 0.71 

Juveniles 

1-6 months) 

Clinical 

Chemistry 200 0.62 201 0.96 202 0.71 

 Hematology 210 0.59 211 0.96 212 0.61 

Neonates 

(<48 hours) 

Clinical 

Chemistry 100 0.31 101 0.90 102 0.36 

 Hematology 110 0.61 111 0.90 112 0.62 

FWCR= Fraction contained within range of comparison, calculated by determining the number of out of range 

analytes of potential clinical relevance to the total number of measurements collected in each Chart. 

Table E-3a provides a summary of the Charts evaluating the clinical chemistry and hematology 

tests performed on the Cyagra clones compared with the comparator population, and the Cornell 

Reference Range. In addition, the comparator population was compared to the Cornell Reference 

Range. First, as cautioned by the Cornell Laboratory, the Reference Range is not a good 

comparator for young animals. A number of the clones and comparators fall outside the 
Appendix E: The Cyagra Dataset E-15 

Animal Cloning: A Risk Assessment DRAFT

Reference Range79 but the similarity to the Reference Range increases with age for both clone 

and comparator populations. Approximately half the animals in the older cohort were less than 

one year of age, however, and all clones and comparators were less than two years of age. All of 

the animals in the older cohorts were still growing and thus do not match the laboratory reference 

adult cattle population well. Clearly, then the most relevant comparison for the clone cohorts in 

this review is the comparator population. 

d. Conclusions Regarding Clone and Comparator Population Cohorts in Aggregate 

Review of the degree to which the clone cohorts have laboratory values that fit within those of 

the comparator population cohorts indicates the following: 

1. Even at birth, 90 percent (107 of 119 measurements) of the hematology values, and 90 

percent (272 of 324 values) of the clinical chemistry values lie within the values of the 

comparator population (Table E-3, Charts E101 and E111). This is particularly 

instructive, considering that many of the clones required some assistance immediately 

after birth (no similar records were kept for the comparators, but we assume that no 

extraordinary measures were taken, and were informed that all comparators were born 

vaginally). Further, clones had blood samples drawn before colostrum administration, 

while the comparators had blood samples drawn after colostrum administration, but 

within 24 hours after birth. Colostrum consumption (quantity and quality) influences 

certain laboratory values (e.g., globulin, total protein, GGT). 

2. The 1 to 6 month age cohorts are even more similar to each other than the neonatal 

cohorts: both the clinical chemistry and hematology values have 96 percent and 95 

percent concordance respectively (707 of 742 of the hematology measurements and 1,404 

of 1,462 of the clinical chemistry measurements for clones are within the clinically 

relevant ranges) (Charts 201 and 211). 

3. The 6 to 18 month cohorts are almost superimposable with respect to laboratory values 

(Charts 301 and 311). Only three of the 294 hematological values and seven of the 592 

clinical chemistry measurements were outside the clinically relevant ranges, significantly 

less than would be expected by chance alone. 

 

79 It should be noted that because the reference range represents only 95% of the animals used in its derivation, 

even comparison of the animals used for the derivation will not fit exactly within the distribution. Thus, if the 

reference range were expanded to include those values outside the 95% distribution, it is likely that the clone and 

comparator populations would show a higher degree of “fit” than is observed in this analysis. 
Appendix E: The Cyagra Dataset E-16 

Animal Cloning: A Risk Assessment DRAFT

Based on clinical chemistry and hematology values, it is not possible to distinguish between 

these two cohorts (clones and comparators). The superimposability of the laboratory values and 

the absence of any significant health observations in the clones (based on the limited number of 

explicit veterinary exams) leads to the conclusion that the health of these animal clones during 

the 6-18 month period is not inferior to that of conventional animals. 

 

Because we have concluded that the comparator group is the appropriate basis for comparison 

for the clones, all subsequent discussion regarding clinical and hematological values will be 

considered in that context. 

e. Animal and Analyte Specific Analyses 

In addition to evaluating the overall status of the clone and comparator cohorts, individual 

animal and analyte data were reviewed to determine if more detailed evaluations could provide 

either confirmation of the overall health of the animals, or to serve as indicators of potential 

health problems that might be present in the animals that were not detected on the 

comprehensive veterinary examinations. For each Chart, the following two questions were 

asked: 

1. “For all of the clones in this age cohort, how many of the values for each analyte were 

out of the range established by the comparators? (i.e., looking across each row, how 

many arrows or grey rectangles were present?), and 

2. “For each clone in this cohort, how many of the analytes were out of the range 

established by the comparators?” (i.e., looking down each column of the Chart). 

There are three overall issues addressed by this evaluation: 

1. Whether the laboratory values of the clones were similar to those of the comparator 

population on an animal-by-animal level, or whether it would be possible to 

distinguish between the two populations based on the clinical chemistry and 

hematology data. A finding of similar laboratory values would provide confidence 

that there were no material differences in metabolic, immunologic, and hematopoetic 

(blood producing) functions between clones and conventional animals; 

2. Whether the clones respond to the internal (growth and maturation) and normal 

external (stressors, disease) environments appropriately (analyte based approach); and 
Appendix E: The Cyagra Dataset E-17 

Animal Cloning: A Risk Assessment DRAFT

3. Whether the individual values can be used to predict the long-term viability of that 

animal or that cohort (analyte and animal approaches combined). 

A description of the parameters that were evaluated and their relation to physiological status is 

provided in Appendix F: The Comprehensive Veterinary Examination. 

Clinical chemistry and hematology responses are best evaluated in the context of the whole 

animal, including its age, species, breed, husbandry, geographic location, reproductive status, 

and the laboratory performing the analysis. Laboratory findings complement the subjective 

physical diagnosis of the patient by providing objective information for the process of 

differential diagnosis, monitoring treatment, and formulation of a prognosis (see Appendix 

F). “Abnormal” laboratory measurements and examinations are often defined as those values 

lying outside the limits of the reference range. Determining what constitutes “normal” is more 

complex than simply comparing an individual value to a reference range derived from a sample 

of a representative population. 

Laboratory tests are designed to support a clinical diagnosis based on the patient’s history and 

clinical findings, with all of the information contributing to the final decision. Comparing 

clinically normal clone and comparator populations only on laboratory measurements, in the 

absence of disease or injury, is, at best, an exercise in attempting to identify subtle hazards that 

may be hypothesized to exist in animal clones. The statistical or biological significance of any 

value that may lie outside the comparison ranges available for this review in the absence of 

corroborating information on the health status of the individual animals or cohorts would be 

difficult to justify on scientific grounds. 

Further, it is important to remember that the evaluation is only as good as the sample provided. 

One of the terms used to group the inconsistent outcomes that result from sample mishandling or 

processing errors is “artifact.” Erroneous conclusions can result if the artifact is accepted as a 

true sample result. An artifact is suspected when the laboratory data are inconsistent with the 

clinical assessment or there is an inappropriate relationship between tests. For example, low 

blood glucose values can indicate hypoglycemia, with the accompanying clinical signs of 

lethargy or seizure. However, low blood glucose values in a blood test can also result from not 

separating the red cells from the serum in a timely fashion after drawing blood. In that case, the 

glucose level will be artifactually reduced. Other artifacts that can influence blood variables are 

lipemia (the presence of excess fats and fatty acids in the blood), hemolysis (the breakage of 
Appendix E: The Cyagra Dataset E-18 

Animal Cloning: A Risk Assessment DRAFT

blood cells releasing their contents into the sample), poor collection technique, and storage for 

too long a time at an inappropriate temperature. 

Therefore, a single value outside of a “normal range,” or even a number of values outside a 

“normal range” in one animal does not necessarily mean (or even imply) that an animal’s health 

is at risk. Rather, these values should serve as signals for the further investigation by experienced 

veterinarians and animal scientists who exercise professional judgment regarding that value in 

the context of weighing available evidence. 

In the Analyte Evaluation, similarities and differences in analytes across animals and within agematched cohorts were considered. One or two values outside the range could be considered 

normal and acceptable variation in the absence of other physiologically based evidence. Because 

this review attempts to use clinical values to identify potential hazards, even those few values 

outside the range were examined in the context of other physiological characteristics to 

determine if those values implied some anomalous trend. If more than two variables were out of 

the comparator range, we returned to the animal’s entire record in an attempt to understand those 

values in the context of the animal, and to attempt to determine if they represented a concern for 

the health of the animal and therefore the safety of edible products derived from it. 

In the Animal Evaluation, similarities and differences within individual animals were evaluated. 

For each animal, we first determined the total number of analytes that were considered 

sufficiently out of range to imply clinical relevance, thereby triggering further scrutiny. Those 

analytes were evaluated for internal consistency with other values to rule out artifacts, and then 

considered within the context of the animal’s health records for clinical consistency between the 

lab work and the clinical picture (based on available veterinary exam records) of the animal. 

In the following sections, we have divided our analyses into the three age cohorts: within 24 

hours of birth, 1-6 months, and 6-18 months. Results that are outside the comparator range are 

presented on an analyte and animal-specific basis. The exception to this format is the first subsection that discusses growth-related phenomena that span age groups. 

i. Growth-Related Phenomena 

Because young animals are growing rapidly, measures of bone growth such as calcium, 

phosphate, and alkaline phosphatase might be expected to be elevated relative to adults. 

(Alkaline phosphatase is an enzyme that reflects a number of physiological parameters, and in 

young animals represents the activity of bone growth and development). This is, in fact, 
Appendix E: The Cyagra Dataset E-19 

Animal Cloning: A Risk Assessment DRAFT

observed in the clones and comparators. Review of Charts E300 and E302 (6 to 18 months old), 

E200 and E202 (1 to 6 months old), and E100 and E102 (within 24 hours of birth) clearly 

indicates that all of the alkaline phosphatase levels, and a high proportion of calcium and 

phosphate levels, are elevated in both cohorts relative to the Cornell Reference range. Review of 

Charts E301, E201, and E101, however, reveals that clone alkaline phosphatase values are 

almost entirely within the range of the comparators (0/18 for 6 to 18 months, 8/46 for 1 to 

6months, and 0/10 (within 24 hours of birth) values out of range). For those animals in the one to 

6 month cohort, the increased levels of alkaline phosphatase occurred in the youngest animals, a 

finding consistent with higher rates of growth in younger animals relative to older animals. 

Total protein, globulin, and albumin reflect, among other things, the immune status of the 

organism, which varies with age. Immediately after birth, globulin levels, which are largely 

comprised of immunoglobulins, are derived almost entirely from colostrum (the antibody rich 

first “milk” secreted by mammals). “Passive immunity” is conferred by the ingestion and 

intestinal absorption of immunoglobulin-rich maternal colostrum. In the two to four months after 

birth, a calf’s own immune system begins to ramp up its production of immunoglobulins, as the 

circulating supply of maternally-derived immunoglobulins in milk wanes. This phenomenon can 

be observed in Charts E200 and E202 (Clones: Reference Range (1 to 6 months), and 

Comparator Population: Reference Range). Clone and comparator calf globulin values are low 

relative to the Cornell lab reference range because that reference range is derived from adult 

animals. The clone and comparator calves have not fully started to produce antibodies from their 

own B-lymphocytes. Review of Chart E201 (Comparison of Clones to Comparator Population), 

however, indicates that there were few differences between the clone and comparator 

populations, reflecting the appropriate age-related lag between the decrease in passive acquired 

immunity and endogenous immunoglobulin production. 

The globulin levels that are different between clones and comparators also reflect this age-related 

physiological phenomenon. Clones #72 and 73 were among the youngest in the 1 to 6 month old 

group, and thus would be expected to have lower globulin levels. For example, comparison of 

the globulin value for clone #100 (174 days of age, globulin of 4.6g/dL) with clone #72 (48 days 

of age and globulin level of 1.6 g/dL) clearly demonstrates the age-related changes in the 

analyte, and appropriately reflects the normal developmental increase in endogenous globulin 

production. 

In summary, clones and comparators exhibited entirely appropriate developmental stage 

responses in those laboratory values that reflect age-specific alterations. At least with respect to 
Appendix E: The Cyagra Dataset E-20 

Animal Cloning: A Risk Assessment DRAFT

this dataset, clones respond appropriately to the internal signals guiding normal development and 

maturation. 

f. Animals with Measurements at Different Developmental Nodes 

There is a small sub-cohort of seven clones for which laboratory values are available for both the 

earliest (neonatal) developmental group and early in the second developmental node group (1-6 

months). The seven calves are 71, 72, 73, 78, 79, 119, and 132. 

More variability was observed among the clinical chemistry values generated from six of seven 

of these clones relative to the comparators than in the hematology values (for one clone, #119, no 

differences were noted relative to the comparators). Compare Charts E101 vs. E201 for 

chemistry and Charts E111 vs. E211 for hematology. In particular, gamma glutamyl transferase 

(GGT) values appeared lower for four of the seven neonatal clones. This observation is entirely 

consistent with the difference in treatment that the animals received with respect to the timing of 

the blood draws. For clones, blood samples were drawn prior to the administration of colostrum, 

while the comparators had blood samples drawn following its administration. As colostrum has 

been shown to have high intrinsic GGT activity, the difference between the two groups could 

well be due to the reflection of absorbed colostrum by the comparators (Meyer and Harvey 

2004). GGT values normalized by the second time of measurement for three of the clones. Calf 

#73 continued to demonstrate slightly lower GGT activity (4U/L vs. comparator range of 

5 - 32 U/L) at Day 48, but this value is unlikely to have clinical significance. We would have 

expected the globulin level to be lower also, because of the young age of this clone. There was 

one comparator calf with a low enough globulin level (1.3 g/dl) to allow all of the clone calf 

values to fall within the range, had it been included in establishing the range. 

Six of the seven clones exhibited lower aspartate amino transferase (AST) values at birth relative 

to the comparators, but these values normalized by the time of the second blood draw (Chart 

E101 for neonates v. E201 for 1-6 months). Five of the seven clones had low bile acid or 

cholesterol levels at birth. All three of these analytes are produced by the liver, and their 

relatively low values may be a reflection of the changeover from fetal hepatic circulation (which 

bypasses much of the liver) to neonatal circulation in which the liver becomes more fully 

perfused. Bile acids normalized by the second measurement. The initial, relatively low creatine 

kinase (CK) (Clone #72), total iron binding capacity (TIBC) (Clone #73), and elevated iron 

(Clone #132) values resolved by the time of the second blood draw. The cholesterol level for 

clone #79 was low at birth, but was elevated relative to comparators in the 1-6 month blood 

sample. 
Appendix E: The Cyagra Dataset E-21 

Animal Cloning: A Risk Assessment DRAFT

A few measurements appeared elevated in five of the clones at the time of the second 

measurement. Glucose, alkaline phosphatase, phosphorus, creatinine, and the A/G ratios were 

elevated relative to the comparators, and anion gap, globulin and total protein were decreased 

relative to the comparators. These variations from the comparator range are discussed more fully 

in the Animal and Analyte Review portions of this Appendix. 

For hematological data, complete blood count information is only available for four of the seven 

neonatal clones (Chart E111). Of those, only Calf #78 exhibited any values outside the range of 

the comparators. At birth, that animal exhibited decreased lymphocytes and decreased platelets, 

which normalized by the time of the second blood draw. The second blood sample (Chart E211) 

showed increased banded (immature) neutrophils, whose only biological significance here is that 

it demonstrates that the clone’s bone marrow can produce normal immature neutrophils. The 

stimulus for the release of the banded neutrophils in this case is not known. No neonatal 

hematological values were available for clone #78. At the time of the second blood draw (Day 

84) clone #79 exhibited decreased total protein as measured by refractometer (TP-ref), increased 

mean platelet volume (MPV) and decreased red cell distribution width (RDW) relative to the 

comparators. These variations (increase in bands for clone #78 and decreased total protein (TP) 

and RDW and increased MPV) are discussed in the animal and analyte review sections. 

Clone #79 was eventually culled (see discussion in Animal and Analyte Section). 

In summary, with the exception of one clone (Clone #73, GGT), none of the values measured at 

birth that were out of range of the comparator group persisted into the second developmental 

cohort. Initial laboratory measurements taken at birth differed in clones versus comparators. 

Some of that difference was likely attributable to the difference in timing or source of colostrum 

administration between the two groups. Clones #71, 72, and 73 were derived from the same cell 

line. As discussed in the Animal and Analyte Review sections, most of the values that were out 

of range at the time of the second blood draw can either be attributed to the age of the animals or 

do not appear to have clinical significance. 

i. Age Range within 24 hours of birth (Charts E101 and E111) 

There were 10 live clones and 17 live comparators in this age cohort. 
Appendix E: The Cyagra Dataset E-22 

Animal Cloning: A Risk Assessment DRAFT

(a) Analyte Evaluation 

Despite an expectation of substantial differences in laboratory measurements between clones and 

comparators at birth, Chart E101 shows 27 of the 33 analytes were very similar between the 

groups: they had either one difference or no differences. The remaining six analytes tended to be 

more variable between clones and comparators. 

The values out of range in the four analytes related to liver function (AST, GGT, cholesterol, and 

bile acids (hBA)) were low relative to comparators. AST levels, in particular, were low in 9 of 10 

animals. Although these parameters can indicate zinc or vitamin B6 deficiencies, (Duncan and 

Prasse 2003), Meyer and Harvey (2004) have reported that “reduced AST activities (below the 

reference range) are noted with relative frequency in dogs and rats during pre-clinical drug 

trials....” We are uncertain whether similar observations have been made in cattle. Colostrum 

possesses an intrinsic high GGT activity (Meyer and Harvey 2004) that is passively transferred 

to neonates. Given that the clones did not receive colostrum until after blood samples were 

drawn, while comparators did, lower GGT levels in clones are not unexpected. 

Low cholesterol is associated with porto-systemic shunts (abnormal liver blood circulation) in 

young animals. Fetal circulation provides for the bypass of the bulk of the liver tissue through a 

vessel called the ductus venosus in a loop including the placenta, umbilical vein, and the ductus 

venosus. At birth, the ductus venosus closes off; the liver is then fully perfused with blood from 

the hepatic artery and other components of the hepatic portal circulation. Were these low 

cholesterol levels to continue into the next developmental age group, there might be cause for 

concern. Given that the cholesterol levels appear to normalize, based on information from the 

seven calves that were sampled at both the neonatal and weaning time periods, and review of the 

overall 1-6 month cohort, there is little reason to think that the lower values in these very young 

animals pose a health risk. The low levels at birth are more likely a reflection of the changeover 

from fetal to adult circulation, possibly exacerbated by the clones’ unusually large umbilical 

vessels, which often required surgical correction (see 1-6 month cohort discussion). The lower 

bile acid values observed may also be related to the transition from fetal to neonatal circulation, 

and are not likely indicative of any disease state. 
Appendix E: The Cyagra Dataset E-23 

Animal Cloning: A Risk Assessment DRAFT

Table E-4: Summary of Laboratory Values from Subcohort of Clones with Laboratory Measurements Taken 

at Two Developmental Nodes 

Calf ID# Parameters Charts E101/111: Birth 

Age at 

Second 

Blood 

Draw 

(Days) 

Chart E201/211: 1-6 

Months 

Current 

Status 

Clinical 

Chemistry ↓AST, ↓GGT (pa) ↑glucose, ↑alk phos 78, Holstein 

♀ Hemogram ↓lymphocytes, ↓platelets 

54 

↑banding 

Presumed 

healthy 

Clinical 

Chemistry ↓AST, ↓hBA 

↑creatinine, ↓globulin, 

↑A/G, ↑alk phos, 

↑chol 

79, 

Holstein 

♀

Hemogram No data 

65 

↓rdw, ↑MPV, ↓TP-ref 

Culled 

Clinical 

Chemistry 

↓AST, ↓hBA, 

↓GGT (pa) 

↑P, ↑glucose, ↑alk 

phos 

71, 

Holstein 

♀ Hemogram within range 

48 

within range 

Presumed 

healthy 

Clinical 

Chemistry 

↓AST, ↓ hBA, 

↓GGT, ↓CK 

↓anion gap, ↑P, ↓TP, 

↓glob, ↑A/G, ↑glu, 

↑alk phos, ↑ lipemia 

72, 

Holstein 

♀

Hemogram No data 

48 

↑ MPV 

Presumed 

healthy 

Clinical 

Chemistry 

↓AST, ↓GGT (pa), 

↓TIBC, ↓hBA, 

↓cholesterol 

↓anion gap, ↑P, ↓TP, 

↓glob, ↑A/G, ↑glu, 

↓GGT, 

73, 

Holstein 

♀

Hemogram within range 

48 

↑ MPV 

Presumed 

healthy 

Clinical 

Chemistry within range within range 119, red 

Holstein 

♂ Hemogram within range 

36 

within range 

Presumed 

healthy 

Clinical 

Chemistry 

↓AST, ↓chol, ↑iron, ↑ % 

saturation within range 132, 

Holstein 

♂ Hemogram No data 

Not 

available 

within range 

Presumed 

healthy 

pa = presumed artifact 

Other abbreviations as described in Appendix F: Comprehensive Veterinary Examination 

Within range = within the range of values for the comparator population 

 

The hemograms for the neonatal clones (Chart E111) also were very similar to the comparators: 

15 of the 17 analytes had either no values out of range or just one value out of the comparator 

range. With the exception of clone #43, which was infected with rotavirus and died at one day of 

age, all red blood cell analytes were within the range of the comparator group. Three clones had 

lymphocyte counts lower than the comparator range. 
Appendix E: The Cyagra Dataset E-24 

Animal Cloning: A Risk Assessment DRAFT

(b) Animal Evaluation 

In general, clones appeared to be more fragile perinatally than comparators. Review of Table E2a indicates that many of the animals did not survive parturition. Of those that did survive, three 

were infected with rotavirus, and two, clones #43 and #80, died from rotavirus-induced 

diarrhea.80 Clone #43 exhibited lower AST, cholesterol, GGT, and bile acids than the comparator 

range, and platelet counts higher than the comparator range. Clone #80 had sodium levels that 

slightly exceeded the comparator range (149 mg/dL, relative to the highest comparator value of 

146 mg/dL), elevated iron, and relatively high percent saturation. The elevated sodium level may 

have been related to electrolyte disturbances that occur with diarrhea. 

Calf #78, a Holstein heifer, also had WBC within comparator range at birth and developed 

normally through Day 54 of age. She had low lymphocytes and low platelets, but did not present 

clinically with infection, and survived. Low lymphocyte counts (lymphopenia) can result from 

severe systemic bacterial and viral infections, disruption of the lymphatic drainage (ruptured 

thoracic duct), or hereditary disorders in which the production of the lymphocytes is impaired. It 

may also be a function of suppressed immune function in calves. In the absence of additional 

information, we think that these data should not be overinterpreted. Of the three clones with 

lymphopenia, two died from infection, but the third survived, indicating that although perinatal 

lymphocyte count may be a useful parameter to monitor, it is not predictive of outcome. 

Clones #71, 72, 73, 78, 79, 119, and 132 were discussed in aggregate in the preceding section of 

the subcohort with laboratory measurements taken at two time points. Clone #79 was a Holstein 

heifer that was culled for poor posture and gait at 54 days of age. In addition to low AST and low 

bile acids, she was noted to have “very thick withers, and a general build resembling a beef 

calf. Her left carpus was enlarged, and her left leg deviated laterally.” 

 

Clone #75 was a Holstein heifer with no follow-up data at a later age. She exhibited AST, 

cholesterol, GGT, and bile acid levels that were lower than the comparator range. Clone #132 

was a Holstein heifer initially presented with AST and cholesterol levels lower than the 

comparator range and iron and TIBC above the comparator range, but that appeared to be 

thriving at 50 days. 

 

80 Rotavirus is a common enteric pathogen in cattle to which many calves are exposed; some succumb. There is a 

commercial vaccine for the dam to increase immunity to rotavirus in the colostrum to passively immunize calves 

in order to protect them. 
Appendix E: The Cyagra Dataset E-25 

Animal Cloning: A Risk Assessment DRAFT

(c) Conclusions for Perinatal Period: Animal Safety 

In general, clones appeared to be more fragile than comparators. Three of the ten calves died or 

were culled. Two of these died at one day of age of rotavirus infection, and one was culled for 

poor posture and gait. The major classes of adverse outcomes noted for neonates included 

stillbirth, umbilical bleeding/abscess/management, colostrum/passive transfer problems, and 

euthanasia for defects (renal, circulatory, tendon contracture, placental abnormalities, cardiac, 

abomasal, and ascites (increased fluid in the abdomen)). These problems also occur in 

conventional animals, although the incidence of the adverse outcomes appears to be higher in 

clones. Animals with these outcomes are readily identifiable. 

The laboratory values indicated a variety of anomalies in the clones. The liver values (AST, 

GGT, cholesterol, bile acids (hBA)) were decreased in several animals, for reasons likely related 

to the placental/umbilical abnormalities. GGT levels were also low relative to the comparators, 

likely related to blood sampling prior to colostrum intake. Although both red and white blood 

cell counts are more variable than in older cohorts, both systems appear to be functioning 

normally in response to environmental stress (rotavirus). 

Based on these data, it is not possible to use either a particular analyte or analyte profile to 

predict whether an individual animal, or indeed the entire cohort, will develop into normal, fully 

functioning healthy animals. The health and laboratory data are consistent with the hypothesis 

that animals that look and behave normally are most likely normal with respect to laboratory 

values. 

(d) Food Safety 

Healthy clones of this age are unlikely to be used for human food, given their potential value as 

breeding stock. It is also highly unlikely that clones of this age group would be fed to animals 

except through rendering of dead clones that occurred at parturition or by accident. In any event, 

the laboratory values do not appear to indicate that these animals are materially different from 

conventional newborns, but their physical condition at birth seems to indicate that they are more 

fragile than comparators, and by inference, other conventional animals. 

 
Appendix E: The Cyagra Dataset E-26 

Animal Cloning: A Risk Assessment DRAFT

ii. Age Range 1 to 6 months (Charts E201 and E211) 

There were 46 clones and 47 comparators in this age cohort. 

(a) Analyte Review 

Chart E201 shows glucose values were higher for clones than comparators in six of the 42 

accepted measurements (four of the values are so low as to be incompatible with life (< 2mg/dL) 

and thus were considered artifacts). The higher values (ranging from 88-123 mg/dL) may reflect 

stress responses caused by handling, dietary considerations (including proximity to a meal), or 

real differences in glucose metabolism. In order to determine whether the hyperglycemia was 

transient or sustained, urinalysis results were checked for these animals. The renal threshold (the 

blood level at which glucose spills over into the urine) in cattle is approximately 100 mg/dl. As 

none of the urinalyses tested positive for glucose, it is unlikely that the hyperglycemia observed 

in the blood had been sustained long enough to allow spillover from the blood into the urine. 

Further, no mention of increased water intake or urination was noted on any of the veterinary 

records of these clones, which are clinical signs of sustained hyperglycemia. Therefore, the most 

physiologically plausible interpretation of these elevated levels is transient hyperglycemia as a 

short-lived response to stress. 

Total Protein (TP) is an analyte that contains globulin and albumin. Two animals had TP values 

that were outside the comparator range (Clone #72 and clone #73 were low). Three animals 

(Clones #94, 102, 128) had decreased levels of SDH relative to the comparators; three others 

(Clones # 56, 73, 116) exhibited lower GGT levels than the comparators. Although elevated 

blood levels of SDH and GGT may be indicative of liver disease, the relevance of decreased 

levels is unclear. 

Evaluation of hemograms (Chart E211) indicated that there were no anemic animals. The one 

value out of range of the comparator group was an elevation, and not a decrease, in RBCs. 

Although other red cell indices such as mean cell hemoglobin (MCHC) concentration (4/44 

clones had elevated levels relative to comparators) and red cell distribution width (RDW) (3/44 

clones had lower levels than comparators) were out of comparator range, they are secondary 

indicators of red cell status. Hemoglobin (Hb) and hematocrit are the primary indicators of red 

cell status, and were effectively no different from comparators. No variables were consistently 

out of range for white blood cell evaluations. Four of the 44 clones had elevated basophil counts; 
Appendix E: The Cyagra Dataset E-27 

Animal Cloning: A Risk Assessment DRAFT

the significance of these measurements is unclear. Twelve of the 41 clones had mean platelet 

volume (MPV) values that were elevated. See clone #102 below for a discussion of MPV. 

(b) Animal Evaluation (1 to 6 months Age Group) 

Clone #41, a 141 day old Holstein heifer, exhibited no clinical chemistry and one minor 

hematology value out of range of the comparators. Her health records, however, stated that she 

had umbilical surgery and harsh lung sounds. No further information is available on this animal. 

Clone #58, a 161 day old Angus heifer had a normal veterinary exam, with indication of 

umbilical surgery. Four clinical chemistry values were out of the comparator range: creatinine, 

albumin, bile acids, and the A/G ratio were all low. Potential causes of low albumin levels 

include decreased production from chronic liver failure, or increased loss due to nephropathy (a 

kidney disease in which proteins are excreted), intestinal disease (enteropathy), or loss into a 

body cavity such as the thorax or abdomen. Chronic liver failure is accompanied by elevated bile 

acid levels; clone #58’s bile acids were low. There were no other analyte or health measures 

indicating enteropathy or nephropathy. The low albumin level was also accompanied by a high 

globulin level, which could well be attributed to the umbilical abscess that was surgically 

removed, as the globulin response was appropriate to antigenic stimulation. The relevance of low 

creatinine is unknown. This calf also had four hemogram variables that differed from 

comparators. Hemoglobin (Hb), TP, and RDW were high, and the lymphocyte count was 

relatively low. The elevated Hb is consistent with the high hemolysis index of the sample, and is 

therefore probably not a reflection of the biology of the animal. The elevated TP was likely 

caused by the elevated globulin in response to antigenic stimulation discussed in the above. 

Three Holstein calves (Clones #71, 72, and 73) were derived from the same cell line and were 

the same age at blood draw (48 days). All three presented with normal veterinary exams, 

elevated alkaline phosphatase levels, glucose, and phosphate levels. Clone #71 did not exhibit 

any other clinical values outside the comparator range. The elevated glucose measurements, as 

discussed above, appear to be transient and likely stress related. Clone #72, also presented with a 

normal veterinary exam. Her clinical chemistry measurements indicated low anion gap, TP, and 

globulin levels, and elevated phosphate, A/G ratio, glucose, and lipemia levels. Low anion gap is 

rare, and can be related to low albumin, which this animal did not exhibit. Its importance is 

unknown, and in this case, may simply be an anomaly. The low TP and globulin levels are likely 

age-related, as at this age calves are transitioning from maternal antibody to endogenous 

production, and there is often a lag in globulin concentration during this age. Plausible 

explanations for elevated phosphate, A/G, and glucose levels have been discussed 
Appendix E: The Cyagra Dataset E-28 

Animal Cloning: A Risk Assessment DRAFT

previously. The lipemia level for clone #72 was 30U compared to 25U. As the lipemia index is 

relatively arbitrary, in the absence of corroborative health evidence, it is likely that this value has 

no real clinical significance. Clone #73 had low anion gap, TP, globulin, and GGT levels in 

addition to the elevated phosphorus, A/G, and glucose values. As previously discussed, 

particularly considering the very young age and high genetic merit of these animals, the elevated 

phosphorus and alkaline phosphatase levels are not surprising. The influence of colostrum on 

GGT levels has been discussed previously, although its significance in this age group is not 

clear. Comparison of clone #41 to clone #73 shows that the former exhibited no laboratory 

values out of the range of the comparators, but did have health problems, while #73 exhibited 

many laboratory values outside the comparator range, but no health problems. 

Clone #79 was a 65-day-old Holstein heifer at the time of the blood sampling. She was culled for 

poor posture and gait. Clinical chemistry indicated elevated creatinine, A/G, alkaline 

phosphatase, cholesterol, and reduced globulin levels. 

Clones #87, 88, and 89 were all derived from the same Jersey cell line, and were, respectively, 

141, 140, and 131 day old heifers at the time of blood draw. Another animal derived from the 

same cell line died at birth from LOS-related complications. All three had umbilical surgery and 

were dehorned. The differences in body weight in these animals illustrate the variability seen 

among clones derived from the same cell line. Clone #87, the oldest at 141 days, weighed 282 

lbs; clone #88 (140 days) weighed 197 lbs; and the youngest (Clone #89 at 131 days) weighed 

215 lbs. 

Clone #100, a 174 day old Holstein bull, had an elevated WBC (26,500 cells), along with a 

history of umbilical abscess that was treated surgically. The elevated WBC is an appropriate 

response to an umbilical abscess. 

Clone #102, a135 day old Holstein heifer also had umbilical surgery, reduced platelets 

(241 x 103

), and an elevated MPV and MCHC relative to the comparators. By itself, the latter 

measurement has little relevance unless anemia is present. Based on the RBC counts, this calf 

did not have anemia. The relatively low platelet count also does not appear to be significant; for 

reference, the low end of the Cornell Reference Range is 232 x103

, or functionally the same 

number. Also, automated platelet counters may erroneously count platelets, as they tend to 

aggregate (clump together). The platelet smear listed platelets as adequate, corroborating that the 

platelet numbers were likely physiologically appropriate.
Appendix E: The Cyagra Dataset E-29 

Animal Cloning: A Risk Assessment DRAFT

(c) Conclusion for 1 to 6 month old group: Animal Safety 

The clones in this age cohort were mostly normal. Only one calf was culled for reasons of 

performance (poor conformation) and not animal health. Such calves are not selected for future 

breeding and their appearance (and subsequent culling) in a herd is not unique to clones. Culling 

occurs routinely in conventional breeding programs. The observation of poor conformation in a 

clone is interesting in that the animal providing the donor cell would likely have exhibited 

acceptable conformation, raising the question of whether conformation of this animal is a 

function of its uterine environment or changes in gene expression. Clones from the same cell line 

showed considerable variation in their phenotype (see clones #87, 88, and 89 above with respect 

to weight). 

 

Some of the clones had overt health problems. These included the increased incidence of 

umbilical problems (e.g., enlargements, excessive bleeding, oomphalitis (navel infection)) 

tendon contracture, and cryptorchidism. Clones had umbilical extirpation (surgical removal of 

tissue) at a much higher rate than comparators. This increase represents a real risk to clones 

related to surgery. Surgical risks include complications that may arise from anesthesia and 

recovery from surgery, sepsis from manipulating an infected umbilicus, dehiscence (suture line 

not holding or infection of the suture line), and aspiration of stomach contents into the lungs. 

Contracted tendons also seemed to occur at a higher frequency than in conventional calves. 

(Tendon contracture can generally be treated successfully.) Three cases of cryptorchidism were 

identified. This condition is thought to be heritable, and is relatively uncommon in calves. The 

risk to the animal is that retained abdominal testicles can develop neoplasia (testicular cancer) in 

later age. The life cycle of food animals is such that bulls rarely live long enough for neoplasia to 

develop. In fact, the only food animals that would likely survive to develop such a condition 

would be breeding bulls. Given that a cryptorchid bull would fail its breeding soundness exam 

and would not be used for breeding stock (i.e., would be castrated and sent to slaughter when the 

steer reached the appropriate weight), this eventuality is not likely to occur. 

 

(d) Conclusion for 1 to 6 month old group: Food Safety 

It is not likely that clones of this age group would be consumed for food, although there may be 

some circumstances in which culled clones might be sent into the food supply. When the results 

of the laboratory analyses are considered in the context of the Cyagra clones’ clinical 

presentation, there were no consistent analyte or physical observations indicating a food safety 

concern. For example, although some calcium, phosphorus, alkaline phosphatase, and glucose 

levels fell above the comparator range, all of the elevations can be explained by the clones’ stage 
Appendix E: The Cyagra Dataset E-30 

Animal Cloning: A Risk Assessment DRAFT

of life or stress level, and the increased levels observed do not represent a food consumption risk. 

Further, the laboratory work is consistent with clinical presentation: Calf #100 presented with 

both umbilical abscess and a high WBC count. In the unlikely event that this animal was sent to 

slaughter with a large abscess, it would be detected on inspection. The carcass would be 

condemned if there was evidence of systemic involvement. The abscess would otherwise be cut 

out and the carcass processed normally. Healthy clones of this age group do not appear to be 

materially different from the comparators, and would not likely pose a food consumption risk 

different from conventional animals. 

iii. Age Range: 6-18 Months (Charts E301 and E311) 

There were 18 clones and 21 comparators in this age cohort. 

(a) Analyte Analysis 

Review of Chart 301 (Clone: Comparator Population Clinical Chemistry) indicates that there 

were very few differences between these two cohorts: 33 of the 33 analytes showed no or one 

value out of the range defined by the comparator population. Two analytes, on first impression 

appeared to exceed that range: estradiol-17β (E2) and insulin-like growth factor-1 (IGF-I). On 

further scrutiny, these values were judged to be of no clinical relevance. Because hormones are 

important from a physiological and food safety standpoint, their lack of clinical significance is 

discussed below. 

 

IGF-I is a hormone produced by all mammals, whose presence is necessary for growth and 

development. Circulating levels of IGF-I have been linked to weight gain and growth rate, and 

higher levels have been used as a physiological marker for superior genetics in cattle, swine, 

sheep, and chickens (Davis and Simmen 2000). In this study, IGF-I levels tended to be higher in 

male clones than in females, and in three bull calves (Clone #24, 33, and 35) were slightly 

increased (less than 10 percent) relative to the comparator group (respective IGF-I levels of 924, 

916, and 938 ng/mL relative to the comparator range of 33-875 ng/mL). 

Basal circulating levels of IGF-I vary with a range of factors, and fluctuate dramatically among 

individual bovines in herds (Vega et al. 1991). In an analysis of 603 conventional Angus cattle 

conducted 42 days after weaning, the serum concentrations of IGF-I ranged from 17 to 883 

ng/mL (Davis and Simmen 2000). Basal IGF-I levels also vary between males and females, with 

12 month old bulls exhibiting higher concentrations of IGF-I than steers, heifers, or 

ovariectomized (animals whose ovaries have been surgically removed) heifers (Plouzek and 

Trenkle 1991 a,b). Plasma concentrations of IGF-I are also influenced by diet composition and 
Appendix E: The Cyagra Dataset E-31 

Animal Cloning: A Risk Assessment DRAFT

intake, with basal IGF-I levels significantly lower in cattle during feed restriction compared to 

cattle that are fed to meet maximum growth or production potential. The primary nutritional 

determinants of basal IGF-I levels appear to be crude protein and total metabolizable energy81

(Elsasser et al. 1989). Given that most non-transgenic clones are derived from animals of 

superior genetic merit for traits such as growth and development, the likelihood that their diets 

would be highly controlled, and the wide variability in normal IGF-I levels, the observed 10 

percent elevations in IGF-I levels are of no clinical significance. 

Of the five clones (# 24, 33, 35, 36, and 69) in the 6 to 18 month dataset that were identified as 

having plasma E2 levels above the comparators, all were bulls. These differences in E2 levels 

prompted closer scrutiny. The range in concentrations of these five bulls was 14.16 to 

24.33 pg/mL. The range for all 18 clones in this age group was 4.28 to 24.33 pg/mL. The 

laboratory reference range is 10 to 40 pg/mL, while the comparator range was 4.1 to 11.41 

pg/mL. As the laboratory reference range most likely included cycling females, we sought more 

specific information on E2 concentrations in bull plasma. We then compared the values to the 

Cornell Reference Range, derived from adult animals, and found that none were outside that 

range. 

Although male mammals produce E2, little research effort has been devoted to studying the role, 

normal concentration, and fluctuation of endogenous estrogens in the bull. Estrogens are 

produced in the Sertoli cells of the testis, as well as in adipose tissue and the brain (Henney et al. 

1990). Estradiol-17β (E2) is produced when testosterone binds to cells in the hypothalamus and 

is converted to E2 by the aromatase enzyme. Receptors for E2 have been identified in the 

urogenital tracts of growing and adult male mammals of several species, and may be necessary 

for normal structural and functional development of the male reproductive system (Nilsson et al. 

2001). The ratio of E2 to testosterone may be an important factor in male sexual behavior and 

libido (Henney et al. 1990). 

 

Henney et al. (1990) attempted to relate various hormone concentrations in plasma to libido in 18 

Holstein bulls aged 4 to 5 years. Mean concentration of E2 in plasma of these bulls was 

10.2 pg/mL, but ranged from 2.8 to 21.7 pg/mL. A more recent study by Sauerwein et al. (2000) 

measured fluctuations in plasma E2 in Simmental breeding bulls with an average age of 8.4 years 

with and without recombinant bovine somatotropin (rbST) treatment. Untreated controls 

(injected with vehicle only) and treated bulls exhibited fluctuations in E2 concentrations over the 

25 week study. Concentrations of E2 in untreated bulls ranged from approximately 5 pg/mL to 

approximately 23 pg/mL, based on Figure 2 in this paper, with a mean pre-injection 

 

81 Metabolizable energy is an estimate of the number of calories absorbed by the animal after digestion. 
Appendix E: The Cyagra Dataset E-32 

Animal Cloning: A Risk Assessment DRAFT

concentration of 12.0 ± 1.5 pg/mL. No papers were identified which discussed E2 concentration 

in young, growing bulls; however, given its possible role in normal development, increased 

levels of E2 in growing bulls of high genetic merit may be expected to have slightly elevated 

levels. Based on these ranges and those established by the reference laboratory, E2 

concentrations of 24 pg/mL are not a concern, and should be considered within normal 

fluctuations for bulls. 

 

Analysis of the hematological parameters for all of the clones and comparators (Chart E311) 

showed no remarkable findings. Sixteen of the 17 analytes measured for clones were within the 

range of the comparators, or had only one difference. No problems were identified with red 

blood cell measurements (e.g., anemia or polycythemia (increase in the total mass of red blood 

cells in the body)), or white blood cell problems (e.g. leukocytosis (increase in WBC count) or 

leukopenia (low WBC count)) were seen. Two animals (Clones #99 and #108) presented with a 

MCV below that of the comparators, but given the lack of corresponding evidence of anemia in 

these animals, these values have no clinical significance. Clone #108 was culled due to 

dwarfism. 

(b) Animal Analysis 

Unless specifically mentioned, no differences were observed between an individual clone’s 

clinical chemistry or hematology values and the range presented by the comparators. 

 

Clone #103 (Red Angus, 6.5 month old heifer) exhibited elevated potassium (K+), asparagine 

transferase (AST), and creatine kinase (CK) levels relative to the comparators. None of the 

values in the hemogram exceed the range defined by the comparators. A physical exam 

conducted on the same day as the blood draws showed no abnormalities. The sample drawn from 

this calf had the highest hemolysis index (353) of all of the samples, indicating poor sample 

handling. Potassium is an electrolyte found mostly within the cell, and its elevation could be 

caused by sample hemolysis. Asparagine transferase (AST) is an enzyme normally found in liver 

or muscle tissue that can be released when liver or muscle is damaged. Creatine kinase is a 

muscle specific enzyme that is released when there is muscle damage. Although these values are 

elevated, they are low enough to imply only minor muscle damage, similar to that observed 

when an animal is recumbent for an extended period of time, struggling in a squeeze chute, or 

may be artifactual due to blood sample handling leading to hemolysis. Given that this animal 

exhibited no clinical abnormalities, even if these analyte levels are not due to artifact or poor 

sample handling, these changes would likely have no clinical significance.

 
Appendix E: The Cyagra Dataset E-33 

Animal Cloning: A Risk Assessment DRAFT

Clone #108 was an Angus heifer that is characterized as a dwarf with frequent bloat. She was 

reported to have been severely deformed with abnormal gastro-intestinal (GI) motility. 

Interestingly, clinical chemistry results show only a decrease in TIBC compared to the 

comparator group. The hemogram indicated high RBC and slightly lower MCV relative to 

comparators. The hematocrit was within the range of the comparators. Despite the animal’s 

obvious physical abnormalities, laboratory values were not significantly out of range. 

(c) Conclusions for 6-18 Month Group: Animal Safety 

Clones in this age group exhibited no differences from comparators with respect to their overall 

health, and were indistinguishable from the comparator group on the basis of clinical and 

laboratory tests. No residual health problems were noted in this group of animals, with the 

exception of the dwarf calf that was identified visually and culled. Based on these observations, 

there are no apparent health risks in this age group of animals. 

(d) Conclusions for 6-18 Month Group: Food Safety 

No material differences were observed between the six to eighteen month old clone cohort and 

their approximately age-matched, similarly reared comparators. In fact, the clones are 

indistinguishable from the comparators, and thus would be unlikely to pose any risk for 

consumption as human food or in animal feed above conventional animals now in the food 

supply. 
Appendix E: The Cyagra Dataset E-34 

Animal Cloning: A Risk Assessment DRAFT

5. Charts and Tables 

The following Charts summarize the results of laboratory data derived from the Cyagra clones 

and corresponding non-clone comparators. In Charts 100, 102, 110, 112, 200, 202, 212, 300, 

302, 310, and 312, the clones’ laboratory data were compared to the Cornell veterinary 

laboratory data and to the approximately age- and breed- matched comparators’ data. A 

laboratory value from a clone that exceeded the range of the Cornell Reference Range or 

comparators was initially flagged regardless of how much it was out of range. After all of the 

comparisons had been made (i.e., clones to comparators, clones to reference range, and 

comparators to reference range), it became apparent that the most appropriate comparison for 

clinical relevance was clones to comparators. We then determined the clinical relevance of the 

out of range values. Clinical relevance was defined as laboratory value observed in the clones 

that was more than 10 percent out of the comparator range, or, one that based on veterinary 

clinical judgment, was likely to cause concern. The published literature was consulted for nonstandard clinical chemistry endpoints such as IGF-I and estrogen. Laboratory values determined 

to be not clinically relevant are represented in the Charts as gray boxes. Clinical relevance is 

presented in charts 101, 111, 201, 211, 301, and 311. 
Cyagra Inc.

Table E-5: Cloned Birth Outcome Summary

1

Calf # Sex Breed Birth Date Birth Wt (kg) Birth

Current 

Status

Age 03/24/03 

(mos)

Age at 

Death (d) Cause of Death

Glucose 

Req'd

Thermoreg. 

Prob. Calf #

Oxygen 

Req'd Umbilicus (Probs.)

Umbilicus 

Hernia/ 

Infection

Umbilical 

Surgery Noted Minor Problems Major Problems

1 F NP 4-Aug-01 45.5 NP alive 19.9 NA NA NP NP 1 NP NP NP NP NP NP

2 F NP 5-Aug-01 54.5 NP alive 19.9 NA NA NP NP 2 NP NP NP NP NP NP

3 F NP 29-Sep-01 NP NP dead NA 0 stillborn NP NP 3 NP NP NP NP NP abn dev

4 F NP 28-Sep-01 NP NP alive 18.1 NA NA NP NP 4 NP NP NP NP not clone of donor NP

5 M NP 7-Nov-01 NP NP alive 16.7 NA NA NP NP 5 NP NP NP NP NP NP

6 M NP 7-Nov-01 NP NP dead NA 16 accident NP NP 6 NP NP NP NP NP calf hung in stall

7 M NP 8-Nov-01 NP NP alive 16.7 NA NA NP NP 7 NP NP NP NP NP NP

8 M NP 7-Nov-01 NP NP alive 16.7 NA NA NP NP 8 NP NP NP yes scouring NP

9 M NP 7-Nov-01 NP NP alive 16.7 NA NA NP NP 9 NP NP NP NP fever NP

10 M NP 7-Nov-01 NP NP alive 16.7 NA NA NP NP 10 NP NP NP NP NP NP

11 M NP 7-Nov-01 NP NP dead NA 0 euthanized NP NP 11 NP NP NP NP NP abn dev

12 M NP 17-Nov-01 NP NP dead NA 0 stillborn NP NP 12 NP NP NP NP NP recip ruptured uterus

13 M NP 19-Nov-01 NP NP dead NA 0 unknown NP NP 13 NP NP NP NP NP appeared normal

14 NP AN 12-Jan-02 33.2 C-section dead NA 13 septicemia/umbilicus no no 14 yes no no no infection, oxygenation contracture

15 NP AN 12-Jan-02 37.3 C-section alive 14.5 NA NA no no 15 no no no no infection, heart murmur NP

16 NP AN 15-Jan-02 50 C-section dead NA 2 failure to trans to neonat circ no no 16 yes yes no no slack abdomen breathing

17 NP AN 16-Feb-02 44.5 Natural alive 13.4 NA NA no no 17 no no no no NP NP

18 NP AN 28-Feb-02 68.2 Natural dead NA 0 stillborn NP NP 18 NP NP NP NP NP polycystic kidneys

19 NP AN 28-Feb-02 69.1 Natural dead NA 0 stillborn NP NP 19 NP yes NP NP NP flaccid abdomen

20 M HO 25-Nov-01 NP C-section dead NA 0 euthanized NP NP 20 NP NP NP NP NP abn dev

21 M HO 28-Nov-01 41.8 C-section alive 16 NA NA no no 21 yes no no no NP NP

22 M HO 28-Nov-01 NP C-section alive 16 NA NA no no 22 yes no no no low PCV NP

23 M HO 30-Nov-01 45.5 C-section dead NA 0 euthanized yes NP 23 yes yes NP NP NP abn dev, int. bleeding

24 M HO 30-Nov-01 31.8 C-section alive 16 NA NA yes no 24 yes no no no NP NP

25 M HO 12-Dec-01 54.5 C-section alive 15.6 NA NA yes NP 25 NP NP no NP NP NP

26 M HO 18-Dec-01 NP C-section alive 15.4 NA NA no NP 26 NP NP no NP NP NP

27 M HO 22-Dec-01 NP C-section alive 15.2 NA NA no NP 27 NP NP no NP NP NP

28 M HO 25-Nov-01 NP C-section dead NA 0 stillborn NP NP 28 NP NP NP NP NP NP

29 M HO 27-Nov-01 NP C-section dead NA 0 euthanized NP NP 29 NP yes NP NP NP abn dev

30 M HO 4-Dec-01 72.3 C-section alive 15.8 NA NA NP yes 30 yes no no no hyperthermia, mild contracture NP

31 M HO 5-Dec-01 76.8 C-section dead NA 0 euthanized NP NP 31 NP no NP NP NP abn (renal) dev

32 M HO 11-Dec-01 54.5 C-section alive 15.6 NA NA no no 32 yes no no no mild contracture NP

33 M HO 17-Dec-01 57.7 C-section alive 15.4 NA NA yes no 33 yes no no no NP NP

34 M HO 27-Nov-01 NP C-section dead NA 0 stillborn NP NP 34 NP NP NP NP NP NP

35 M HO 7-Feb-02 41.8 C-section alive 13.7 NA NA yes no 35 yes no no no immature lungs NP

36 M HO 8-Feb-02 52.7 C-section alive 13.6 NA NA no no 36 no no no no NP NP

37 M HO NP NP NP alive NA NA NA NP NP 37 NP NP NP NP NP NP

38 M HO 19-Feb-02 67.3 C-section alive 13.3 NA NA NP NP 38 NP no no no anemia, slight forelimb contracture NP

39 F HO 19-Feb-03 NP NP NP NP NP NP NP NP 39 NP NP NP yes NP NP

40 F HO 24-Feb-03 NP NP NP NP NP NP NP NP 40 NP NP NP yes NP NP

41 F HO 3-Dec-02 NP C-section alive 3.7 NA NA yes no 41 yes yes yes yes contracture/splinting NP

42 F HO 10-Nov-02 NP C-section alive 4.5 NA NA yes no 42 yes large yes NP NP NP

43 F HO 10-Mar-02 NP NP dead NA 1 rotavirus NP NP 43 NP NP NP NP diarrhea rotavirus

44 F HO 29-Jan-02 45.5 C-section alive 14 NA NA no no 44 yes yes no no slight int. bleeding NP

45 F HO 29-Jan-02 NP C-section alive 14 NA NA no no 45 no yes no no slight int. bleeding NP

46 F HO 24-Feb-02 51.4 C-section alive 13.1 NA NA no no 46 yes no no no NP NP

47 F HO 31-Jan-02 NP C-section dead NA 0 stillborn NP NP 47 NP NP NP NP NP NP

48 F HO 1-Feb-02 54.5 C-section dead NA 0 stillborn NP NP 48 NP NP NP NP NP NP

49 F HO 1-Feb-02 NP C-section dead NA 0 stillborn NP NP 49 NP NP NP NP NP NP

50 F HO 8-Mar-02 59.5 C-section alive 12.7 NA NA no no 50 no yes no yes NP flaccid abdomen

51 F HO 15-Mar-02 NP C-section dead NA 0 stillborn NP NP 51 NP yes NP NP NP flaccid abdomen, bulldog

52 F HO 17-Mar-02 NP Fetotomy dead NA 0 stillborn NP NP 52 NP NP NP NP NP NP

53 F HO 2-Apr-02 56.8 C-section alive 11.9 NA NA no no 53 no yes NP yes NP NP

54 F HO 4-Jun-02 59.1 C-section dead NA 0 euthanized NP NP 54 NP NP NP NP reverted to fetal circulation cardian, neurological problems

55 F HO 6-Jun-02 54.5 C-section alive 9.7 NA NA no no 55 yes yes yes yes large abdomen NP

56 F AN 11-Nov-02 NP C-section alive 4.4 NA NA yes no 56 yes no no no NP NP

57 F AN 12-Nov-02 NP induced/vaginal dead NA 23 ruptured abomasum no no 57 yes no no no NP ruptured abdomen

58 F AN 13-Nov-02 NP C-section alive 4.4 NA NA no no 58 yes yes no yes lost blood NP

59 F Longhorn 21-May-02 27.3 C-section alive 10.2 NA NA no no 59 no no no no NP NP
Cyagra Inc.

Table E-5: Cloned Birth Outcome Summary

2

Calf # Sex Breed Birth Date Birth Wt (kg) Birth

Current 

Status

Age 03/24/03 

(mos)

Age at 

Death (d) Cause of Death

Glucose 

Req'd

Thermoreg. 

Prob. Calf #

Oxygen 

Req'd Umbilicus (Probs.)

Umbilicus 

Hernia/ 

Infection

Umbilical 

Surgery Noted Minor Problems Major Problems

60 F Longhorn 22-May-02 19.5 C-section alive 10.2 NA NA no no 60 no yes no yes NP NP

61 F Longhorn 22-May-02 23.6 C-section alive 10.2 NA NA no no 61 no no no no NP NP

62 F Longhorn 23-May-02 27.3 Natural alive 10.2 NA NA no no 62 no yes no yes NP NP

63 F Longhorn 7-Jul-02 NP Natural dead NA 60 euth/ failure to thrive no no 63 no no no no NP loss of hair, appetite, muscle

64 M HO 25-Jun-02 59.1 C-section alive 9.1 NA NA yes yes 64 no no no no contracted tendons NP

65 M HO 27-Jun-02 61.4 C-section dead NA 3 GI transit/adhesions from umbilical bleeding no yes 65 yes NP NP NP NP lethargic

66 M HO 30-Jun-02 54.6 C-section dead NA 149 Bloat/ GI motility problems yes no 66 no large yes yes contracted tendons recurring bloat

67 M HO 26-Jul-02 NP 2WP alive 8 NA NA no no 67 no yes yes yes leathery placenta NP

68 M HO 29-Jul-02 NP 2WP dead NA 0 unable to determine yes yes 68 yes no no no thick placenta pericarditis

69 M HO 15-Aug-02 49.5 C-section alive 7.4 NA NA yes no 69 yes proud flesh no yes oxygenation, glucose NP

70 M HO 16-Aug-02 NP C-section alive 7.3 NA NA no no 70 no small hernia yes yes NP NP

71 F HO 6-Mar-03 NP NP NP NP NP NP NP NP 71 NP NP NP NP NP NP

72 F HO 6-Mar-03 NP NP NP NP NP NP NP NP 72 NP NP NP NP NP NP

73 F HO 6-Mar-03 NP NP NP NP NP NP NP NP 73 NP NP NP NP NP NP

74 F HO NP NP NP alive NP NP NP 74 NP NP NP NP NP NP

75 F HO NP NP NP alive NA NA NP NP NP 75 NP NP NP NP NP NP

76 F HO 12-Dec-02 NP C-section alive 3.4 NA NA yes no 76 yes pus, healing yes NP NP NP

77 F HO 9-Dec-02 NP C-section dead NA 47 severe contracture/unresp to therapy yes no 77 yes yes yes yes NP severe contracture

78 F HO 28-Feb-03 NP NP NP NP NP NP NP NP 78 NP NP NP NP NP NP

79 F HO 3-Mar-03 NP NP cull NP NP NP NP NP 79 NP NP NP NP poor gait, posture NP

80 F HO 3-Mar-03 NP NP dead NA 1 rotavirus NP NP 80 NP NP NP NP diarrhea rotavirus

81 F HO 17-Dec-02 NP C-section alive 3.2 NA NA yes no 81 yes ripped vessels no NP required transfusion NP

82 F HO 17-Dec-02 NP C-section alive 3.2 NA NA yes no 82 yes proud flesh, pus yes NP NP NP

83 F HO 18-Dec-02 NP C-section alive 3.2 NA NA yes no 83 yes proud flesh, pus yes NP NP NP

84 F HO 19-Dec-02 NP C-section alive 3.2 NA NA yes no 84 yes proud flesh, pus yes yes NP NP

85 F HO 19-Dec-02 NP C-section alive 3.2 NA NA yes no 85 yes proud flesh, pus yes yes NP NP

86 F JE 2-Dec-02 NP C-section dead NA 0 euthanized NP NP 86 NP NP NP NP NP severe contracture, fluid filled belly

87 F JE 3-Dec-02 NP C-section alive 3.7 NA NA yes no 87 yes no no yes NP NP

88 F JE 4-Dec-02 NP C-section alive 3.7 NA NA yes no 88 yes proud flesh no yes NP NP

89 F JE 5-Dec-02 NP C-section alive 3.6 NA NA yes no 89 yes proud flesh no yes NP NP

90 F HO 12-Dec-02 NP C-section alive 3.4 NA NA yes no 90 yes no no no NP NP

91 F HO 12-Dec-02 NP C-section alive 3.4 NA NA yes no 91 yes moist, pus yes NP depressed NP

92 F HO 12-Dec-02 NP C-section dead NA 0 unable to determine yes no 92 yes pus, moist yes NP depressed NP

93 F HO 16-Dec-02 NP C-section alive 3.3 NA NA yes no 93 yes no no no NP NP

94 F HO 16-Dec-02 NP C-section alive 3.3 NA NA yes no 94 yes no no no NP NP

95 F HO 16-Dec-02 NP C-section dead NA 0 euthanized NP NP 95 NP NP NP NP NP severe contracture

96 F HO 16-Dec-02 NP C-section alive 3.3 NA NA yes no 96 yes large internal stump no NP slight contracture NP

97 F HO 16-Dec-02 NP C-section dead NA 0 euthanized NP NP 97 NP NP NP NP NP severe contracture, fluid filled belly

98 F HO 1-Sep-02 34.1 Natural alive 6.8 NA NA no yes 98 no no no no thermoregulation NP

99 F HO 1-Sep-02 34.1 Natural alive 6.8 NA NA no yes 99 no yes no yes thermoregulation NP

100 M HO 7-Nov-02 NP C-section alive 4.6 NA NA yes no 100 yes pus, proud flesh yes yes NP NP

101 F HO 9-Dec-02 NP C-section alive 3.5 NA NA yes no 101 yes proud flesh, swollen no NP mild contracture NP

102 F HO 9-Dec-02 NP C-section alive 3.5 NA NA yes no 102 yes large, proud flesh no yes NP NP

103 F AR 17-Oct-02 NP C-section alive 5.3 NA NA no no 103 no yes no no NP NP

104 F AR 18-Oct-02 NP C-section alive 5.2 NA NA no no 104 no no no no NP NP

105 F AR 5-Nov-02 45.5 C-section dead NA 0 euthanized NP NP 105 NP NP NP NP NP severe torticollis, arthrogryphosis

106 F BS 7-Mar-03 NP NP NP NP NP NP NP NP 106 NP NP NP yes NP NP

107 F HO 23-Oct-02 NP C-section dead NA 2 euthanized yes no 107 yes NP NP NP NP hypoxemia, rapid deterioration

108 F AN 31-Oct-02 NP Natural alive 4.8 NA NA yes no 108 yes pus, proud flesh yes yes NP diarrhea

109 F AN 5-Nov-02 NP C-section dead NA 0 euthanized NP NP 109 NP NP NP NP NP abn dev

110 F AN 18-Nov-02 NP C-section alive 4.2 NA NA yes no 110 yes some pus yes no NP NP

111 F AN 18-Nov-02 NP C-section alive 4.2 NA NA yes no 111 yes no no no NP NP

112 F AN 21-Nov-02 NP C-section alive 4.1 NA NA NP NP 112 NP NP NP NP NP NP

113 M Red HO 8-Jan-02 NP C-section dead NA 22 pyelonephritis/umbilicus no no 113 yes proud flesh, moist no no NP nephritis

114 M Red HO 9-Jan-02 NP C-section alive 2.5 NA NA no no 114 yes no no no NP NP

115 M Red HO 2-Feb-03 NP Natural alive 1.7 NA NA no no 115 yes no NP NP NP NP

116 M Red HO 6-Feb-03 NP C-section alive 1.5 NA NA yes no 116 yes large NP NP NP NP

117 M Red HO 10-Feb-03 NP C-section alive 1.4 NA NA yes no 117 yes no NP NP contracture NP

118 M Red HO 13-Feb-03 NP NP NP NP NP NP NP NP 118 NP NP NP yes NP NP

119 M Red HO 1-Apr-03 NP NP NP NP NP NP NP NP 119 NP NP NP NP NP NP

120 M HO 2-Jan-03 NP C-section alive 2.7 NA NA yes no 120 yes large and nasty NP NP abcess on side of neck (IV side) NP
Cyagra Inc.

Table E-5: Cloned Birth Outcome Summary

3

Calf # Sex Breed Birth Date Birth Wt (kg) Birth

Current 

Status

Age 03/24/03 

(mos)

Age at 

Death (d) Cause of Death

Glucose 

Req'd

Thermoreg. 

Prob. Calf #

Oxygen 

Req'd Umbilicus (Probs.)

Umbilicus 

Hernia/ 

Infection

Umbilical 

Surgery Noted Minor Problems Major Problems

121 M HO 2-Jan-03 NP C-section alive 2.7 NA NA yes no 121 yes no NP NP NP NP

122 M HO 6-Jan-03 NP C-section alive 2.6 NA NA yes no 122 yes proud flesh, moist NP NP contracture, req'd phys therapy transfusion needed

123 M HO 28-Jan-03 NP C-section dead NA 9 pyelonephritis/umbilicus yes no 123 yes NP NP NP contracted front fetlocks NP

124 M HO 28-Jan-03 NP C-section alive 1.8 NA NA yes no 124 yes large and moist no NP NP NP

125 M HO 6-Feb-03 NP C-section dead NA 0 euthanized NP NP 125 NP NP NP NP NP severe contracture, rotation

126 M HO 10-Feb-03 NP C-section alive 1.4 NA NA yes no 126 yes ripped vessels no NP large cotyledons, normal NP

127 M HO 10-Feb-03 NP C-section alive 1.4 NA NA yes no 127 yes NP NP NP NP NP

128 M HO 12-Feb-03 NP NP NP NP NP NP NP NP 128 NP NP NP yes cryptorchid NP

129 M HO 13-Feb-03 NP NP NP NP NP NP NP NP 129 NP NP NP yes NP NP

130 M HO 16-Feb-03 NP NP NP NP NP NP NP NP 130 NP NP NP NP right testicle not descended NP

131 M HO 21-Feb-03 NP NP NP NP NP NP NP NP 131 NP NP NP yes left testicle not descended NP

132 M HO 24-Feb-03 NP NP NP NP NP NP NP NP 132 NP NP NP NP NP NP

133 M HO 26-Apr-03 NP NP NP NP NP NP NP NP 133 NP NP NP NP NP NP

134 M HO 26-Apr-03 NP NP NP NP NP NP NP NP 134 NP NP NP NP NP NP

KEY

NP= Not Provided

2WP= Two Weeks Premature
Table E-6: Assay Dates (6-18 Month Calves) 1

Table E-6: Assay Dates (6-18 month calves)

Calf # Breed Sex DOB Assay Date Age (Days)

Clones 10 Holstein F 6-Jun-02 15-Apr-03 313

98 Holstein F 2-Sep-02 30-Apr-03 240

99 Holstein F 2-Sep-02 30-Apr-03 240

103 Red AngusF 17-Oct-02 7-May-03 202

104 Red AngusF 18-Oct-02 23-Apr-03 187

108 Angus F 31-Oct-02 7-May-03 188

60 Texas Longhorn F 22-May-02 23-Apr-03 336

24 Holstein M 30-Nov-01 10-Jun-03 557

25 Holstein M 12-Dec-01 10-Jun-03 545

26 Holstein M 18-Dec-01 10-Jun-03 539

27 Holstein M 21-Dec-01 10-Jun-03 536

33 Holstein M 17-Dec-01 10-Jun-03 540

35 Holstein M 7-Feb-02 10-Jun-03 488

36 Holstein M 8-Feb-02 10-Jun-03 487

38 Holstein M 20-Feb-02 10-Jun-03 475

67 Holstein M 26-Jul-02 10-Jun-03 319

69 Holstein M 15-Aug-02 10-Jun-03 299

70 Holstein M 16-Aug-02 10-Jun-03 298
Table E-100a: Individual Animal Clincial Chemistry at Birth 1

Birth Blood Chem.Sodium Potassium Chloride Bicarbonate Anion Gap Urea Creatine-rb Calcium Phosphate Magnesium-xb Total Protein Albumin-bulk Globulin A/G Glucose AST/P5P SDH Alk. Phos.

Ref Low 134 3.9 94 22 17 10 0.4 8.3 4.2 1.7 7.2 3.2 3.5 0.6 31 53 10 23

Ref High 145 5.3 105 30 24 25 1 10.4 7.7 2.2 9.4 4.2 5.8 1.2 77 162 50 78

Units mEq/L mEq/L mEq/L mEq/L mEq/L mg/dL mg/dL mg/dL mg/dL mEq/L g/dL g/dL g/dL mg/dL U/L U/L U/L

Controls 140 145 5.3 100 21 29 8 2.4 12.4 6.7 2.1 4.2 2.7 1.5 1.8 60 47 4 348

141 141 5.8 93 27 27 8 2.6 11.8 5.9 2 4.1 2.7 1.4 1.93 58 54 4 418

135 142 7.8 86 16 48 43 5 15.1 12.3 4.6 4.5 2.4 2.1 1.14 12 386 145 882

136 134 7.3 93 31 17 11 0.9 10.4 6.7 1.7 5.8 2.5 3.3 0.76 87 162 <2 229

137 141 8.5 99 15 36 25 2.5 13.9 13.3 3 4.6 3.2 1.4 2.29 182 182 211 379

138 140 6.2 96 28 22 10 1.4 11.5 6.9 2 4.7 2.5 2.2 1.14 75 109 <2 840

139 142 7.2 101 24 24 13 2.2 11.5 7.3 1.8 4 2.7 1.3 2.08 66 63 <2 126

186 138 9.6 97 28 23 8 1.5 11.6 8.4 2.3 5.4 2.6 2.8 0.93 73 123 4 628

187 138 6.5 94 28 23 10 1.7 11.2 7.8 2.3 5.7 2.4 3.3 0.73 69 137 4 1415

188 140 8.3 95 19 34 10 2.6 12.9 9.9 2.2 4.3 2.8 1.5 1.87 248 76 6 564

189 141 5.8 100 24 23 9 0.6 11.2 5.8 1.5 6.3 2.3 4 0.58 60 66 5 136

190 144 6.2 101 25 24 30 1.2 11.8 9.1 2.4 6.3 2.8 3.5 0.8 65 53 0 143

191 146 6.4 103 24 25 12 0.8 11.1 8.3 1.8 5.2 2.8 2.4 1.17 84 79 3 343

192 142 6.7 98 26 25 5 0.9 11.4 8.1 1.5 4.5 2.6 1.9 1.37 81 72 10 225

193 138 7.2 97 29 19 15 1.8 10.6 7.8 2 5.3 2.5 2.8 0.89 44 166 6 707

194 140 6.5 99 27 21 10 1.7 11.3 7.4 1.9 5 2.4 2.6 0.92 60 170 2 487

195 140 5.6 98 22 26 25 1.5 12.6 7.2 1.8 4.3 2.8 1.5 1.87 17 51 21 103

Clones 43 146 7.1 104 27 22 16 1.4 13 7.9 1.9 4.5 2.8 1.7 1.65 98 46 4 154

71 140 6 99 25 22 17 2.1 12.8 7.2 1.9 4.2 2.5 1.7 1.47 7 18 3 215

72 142 5.8 100 26 22 17 1.9 12.8 6.8 1.9 4.2 2.5 1.7 1.47 25 15 <2 214

73 142 5.7 101 24 23 20 3.1 12.5 8.2 2 4.1 2.6 1.5 1.73 12 22 3 241

75 143 5.5 97 21 31 20 2.2 13.3 8 2 4.4 2.6 1.8 1.44 5 15 4 204

78 143 5.4 100 19 29 13 2.4 13.3 7.9 2 4.4 2.6 1.8 1.44 10 15 <2 355

79 141 5.5 98 22 27 17 3.3 13.2 7.8 2.1 4.5 2.8 1.7 1.65 17 40 5 323

80 149 5.7 101 19 35 13 2.5 13.5 8.5 2 4.5 2.9 1.6 1.81 69 22 6 372

119 138 5.9 90 25 29 15 3.5 12.8 6 2.6 5.1 3 2.1 1.43 30 150 13 798

132 139 5.4 94 27 23 17 1.7 12.6 7.3 2 4.4 2.9 1.5 1.93 42 17 3 378

Individual animal clinical chemistry data at birth
Table E-100a: Individual Animal Clincial Chemistry at Birth 2

Birth Blood Chem.

Ref Low

Ref High

Units

Controls 140

141

135

136

137

138

139

186

187

188

189

190

191

192

193

194

195

Clones 43

71

72

73

75

78

79

80

119

132

GGT Total Bilirubin Dir. Bilirubin Ind. Bilirubin Amylase Cholesterol CK Iron TIBC % Saturation hBA-random Lipemia Hemolysis Icterus IGF-1 Estradiol

11 0 0 0 73 77 113 362 28 9 34.7 10

39 0.1 0 0.1 280 265 226 533 48 455 472.27 40

U/L mg/dL mg/dL mg/dL U/L mg/dL U/L ug/dL ug/dL % umol/L ng/ml pg/ml

21 0.3 0.1 0.2 53 22 331 136 421 32 16 39 0 203.57 29.3

21 0.6 0.1 0.5 48 26 551 156 375 42 12 15 43 1 137.4 52.73

691 0.8 0.2 0.6 40 20 11033 54 373 14 54.3 9 20 1 0.065 59.44

1217 1.7 0.1 1.6 31 37 731 70 450 16 18.1 22 53 2 nes 27.66

12 0.1 0.1 0 68 15 559 148 473 31 58.5 17 44 0 113.51 103.76

1413 1 0.1 0.9 29 34 501 88 365 24 10.5 21 117 1 141.01 17.33

8 1 0.1 0.9 19 28 356 104 367 28 13.7 21 75 1 87.77 25.5

1012 0.3 0.1 0.2 24 24 379 53 397 13 9.5 25 55 0 145.61 28.53

1829 0.9 0.1 0.8 39 27 768 82 392 21 12.7 5 66 1 57.12 33.99

19 0.1 0.1 0 14 25 1209 57 411 14 16.2 0 114 1 3.07 37.99

893 1.5 0.1 1.4 62 59 111 24 378 6 26.3 15 22 2 6.75 6.14

391 0.8 0.1 0.7 60 37 65 29 411 7 7.6 16 35 1 11.79 9.81

268 1.7 0.1 1.6 62 47 462 60 536 11 10 0 162 3 41.82 20.58

383 0.9 0.1 0.8 33 45 87 44 348 13 21.7 2 15 1 16.69 6.18

3213 1 0.2 0.8 48 44 513 47 354 13 15.2 21 251 1 127.94 18.79

1623 1.2 0.1 1.1 42 29 929 48 337 14 16.6 26 33 2 29.67 42.88

5 0.9 0.1 0.8 49 27 637 79 376 21 28.2 7 20 1 6.89 50.45

<3 0.2 0.1 0.1 33 10 125 48 532 9 0 0 293 0 92.06 46.04

3 0.9 0.1 0.8 20 14 209 66 314 21 5 13 24 1 106.85 61.51

3 0.7 0.1 0.6 20 14 61 65 318 20 2.4 13 29 1 44.54

<3 0.4 0.1 0.3 14 11 106 52 264 20 2 10 122 0 128.66 48.06

<3 0.2 0.1 0.1 15 12 118 33 342 10 1.1 25 7 0 71.67 23.34

<3 0.3 0.1 0.2 28 16 179 112 331 34 8.2 20 21 0 97.68 35.42

5 0.3 0.2 0.1 24 201 38 312 12 3.3 8 19 0 82.2 17.16

6 0.1 0.1 0 18 186 164 343 48 12.3 0 13 0 149.44 27.38

845 0.4 0.2 0.2 59 18 711 93 509 18 15.9 14 5 0 187.12

5 0.3 0.1 0.2 35 12 91 163 383 43 22 28 0 31.03
Table E-100b: Individual Animal Hematology at Birth 1

Hemogram Hematocrit (HCT) Hemoglobin (HB) RBC MCV MCH MCHC RDW WBC Seg. Neut. Band. Neut. Lymphocytes Monocytes Eosinophils Basophils

Ref Low 23 8.6 5.4 36 14 36 16.2 5.6 1.8 0 1.9 0 0 0

Ref High 25 13.2 8.2 49 19 40 19.7 13.7 7.7 0 7.4 1.2 1.4 0.3

Units % g /dL mill/uL fL pg g /dL % thou/uL thou/uL thou/uL thou/uL thou/uL thou/uL thou/uL

Controls 135 33 11.8 7.5 44 16 35 18.4 6 3.7 0 1.7 0.1 0.5 0

154 28 11 8.4 33 13 40 20.7 8.5 2.4 0 6.1 0 0 0

206 28 10.8 8.9 32 12 39 23.3 13.2 5 0 7.5 0.7 0 0

160 30 11.8 7.9 38 15 39 19.8 12.4 6 0 6.2 0.2 0 0

161 34 11.4 8.6 39 13 34 22.2 13.4 3.1 0 10.1 0.3 0 0

162 35 10.3 8.4 42 12 29 31.4 14.4 2.7 0 10.8 0.7 0 0.1

163 34 10.3 9.4 37 11 30 28.3 14.6 4.5 0 9.9 0.1 0 0

165 34 11.6 9.6 35 12 35 31.1 24.9 5.2 0 19.2 0.5 0 0

169 33 11.5 9.7 34 12 35 21.1 12.2 4.5 0 7 0.2 0.5 0

205 30 11.4 6.8 44 17 38 17.4 12.7 3.9 0 8.3 0.1 0.3 0.1

207 29 11 7.3 39 15 38 18.4 8.6 2.6 0 5.8 0.1 0.1 0.1

208

209 32 10.5 8.7 37 12 33 33.1 11.3 4.4 0 6.7 0.2 0 0

210 34 10.6 9 38 12 31 32.1 10 4.4 0 5.1 0.4 0.1 0

211 28 10.7 7.8 36 14 38 22.7 9.8 3.7 0 5.8 0.1 0.2 0

212 34 10.5 8.6 39 12 31 30.2 12.5 5.1 0 6.8 0.6 0 0

215 46 14.8 10.1 46 15 32 34.6 9.6 4.1 0 5.4 0 0.1 0

216 32 12.7 9 36 14 40 24.1 8.4 2.8 0 5.5 0.1 0 0

217 38 12.7 8 47 16 34 23.1 13.6 6.9 0 6.4 0.1 0.1 0

218 42 13.8 10.2 42 14 33 36 9.9 2.6 0 6.8 0.4 0 0.1

219 31 12.2 9 34 14 40 22.3 12.4 6.9 0 4.8 0.5 0 0.1

Clones 24 33 11 6.9 48 16 33 20.2 11.6 6.4 0 4.8 0.5 0 0

25 32 10.7 6.9 46 16 34 23.8 13.9 6.1 0 7.2 0.6 0 0

26 32 10.6 6.8 47 16 34 21.4 9.5 2.4 0 6.7 0.2 0.2 0

27 32 10.5 6.7 47 16 33 20.5 7.8 2.5 0 4.8 0.3 0.2 0

33 33 10.7 7.7 43 14 32 33 10.2 2.1 0 7 0.5 0.4 0.1

35 34 11.1 7.7 44 15 33 33.9 10.9 2.7 0 7.7 0.2 0.1 0.1

36 30 9.9 6.9 44 14 33 32.2 9.2 3 0 5.9 0.1 0.1 0.1

38 31 9.7 6.6 46 15 32 28.3 10.5 4.7 0 5.4 0 0.4 0

37 35 12.2 8.7 40 14 35 20.1 8.1 2.3 0 5.2 0.6 0.1 0

55 30 11.1 8.6 35 13 37 21.7 10.5 2.1 0 7.6 0.5 0.3 0

67 34 11.1 9.5 36 12 32 32.4 9.5 4.8 0 3.8 0.3 0.6 0

69 31 10.1 9.5 33 11 33 29.5 8.2 2.5 0 5.4 0.1 0.2 0

70 33 10.8 9.9 34 11 32 31.7 8.2 2.5 0 5.2 0.1 0.3 0

98 28 10.8 8.9 32 12 38 22.6 8.1 2.1 0 5.4 0.2 0.1 0.3

99 28 10.6 9.3 30 12 38 20.4 8.5 2.4 0 5.7 0.4 0 0

103 28 10.5 7.2 40 15 37 21.6 8.6 3.5 0 4.9 0.2 0 0

104 28 10.9 7.4 38 15 39 19.9 8.8 5.5 0 3 0.2 0.2 0

108 37 13.8 11.9 31 12 37 19.4 8.2 3.5 0 3.9 0.3 0.2 0.2

Notes:

NSA No Significant Abnormalities

NS None Seen

N Normal

SCM Smudged Cells-Moderate

Individual animal hematology data at adulthood
Table E-100b: Individual Animal Hematology at Birth 2

Hemogram

Ref Low

Ref High

Units

Controls 135

154

206

160

161

162

163

165

169

205

207

208

209

210

211

212

215

216

217

218

219

Clones 24

25

26

27

33

35

36

38

37

55

67

69

70

98

99

103

104

108

Platelet SmearPlatelets MPV TP-Ref RBC Morphology Acanthocytes Anisocytosis Echinocytes Poikilocytes Schistocytes Parasites WBC Exam Plasma Appearance

232 5.6 5.9

596 8.2 8.1 hemolysis

thou/uL fL g /dL

Incr. 812 16.8 8.3 NSA NS NSA Moderate

Adeq. 391 5.8 6.7 Mild NS NSA N

Incr. (*1) 601 5.5 6.8 NSA NS NSA N

Adeq. 259 6.6 7.3 NSA NS NSA N

Adeq. 391 7.4 7 Moderate NS NSA N

Adeq. 487 6.8 6.5 Moderate NS NSA N

Adeq. 515 6.3 7.2 Many NS NSA N

Adeq. 279 6.2 6.8 NSA NS Few N

Adeq.(*1) 559 5.9 7.2 NSA NS NSA N

Adeq. 309 10.7 7 NSA NS NSA Slight

Adeq.(*1) 6.6 NSA NS NSA Slight

Adeq.(*1) 7 Mild NS NSA Slight

Adeq.(*1) 117 6.4 7.3 Few NS NSA Slight

Adeq. 414 7.3 7 Mild NS NSA N

Adeq. 351 6.5 7.4 NSA NS NSA N

Adeq.(*1) 242 5.9 7.5 NSA NS NSA Slight

Incr. 671 6.3 6.7 NSA NS NSA Slight

Adeq. 468 6.3 6.5 Mild NS NSA Slight

Adeq. 422 6 6.9 NSA NS NSA Slight

Adeq. 583 6.3 7 Mild Few NS NSA Slight

Adeq. 398 7.2 7.3 NSA NS NSA Slight

Adeq. 313 6.7 6.8 NSA NS NSA N

Adeq.(*1) 7.2 NSA NS NSA Slight

Adeq.(*1) 7.2 NSA NS NSA N

Adeq. 392 5.8 7.8 NSA NS NSA N

Adeq.(*1) 7.4 NSA NS NSA Slight

Adeq. 335 6.6 7.4 NSA NS NSA N

Adeq.(*1) 7.5 NSA NS NSA Slight

Adeq. 328 12.4 7.5 NSA NS NSA Slight

Adeq.(*1) 6.8 NSA Slight

Adeq.(*1) 6.9 NSA NS NSA Slight

Incr. 658 5.9 7.7 NSA NS NSA N

Incr. 605 6 7.3 NSA NS NSA N

Adeq. (*1) 395 6.8 6.7 Mild NS NSA N

Incr.(*1) 622 5.5 7.4 Mild NS NSA N

Adeq. 337 11 6.8 Mod NS NSA N

Incr. 745 6.4 7.4 NSA NS NSA N

Adeq. 399 14.4 6.8 Few NS NSA Slight
Table E-200a: Individual Animal Clinical Chemistry Data at Weaning 1

0-6 months Blood Chem.Sodium Potassium Chloride Bicarbonate Anion Gap Urea Creatine-rb Calcium Phosphate Magnesium-xb Total ProteinAlbumin-bulkGlobulin A/G Glucose AST/P5P SDH Alk. Phos. GGT

Ref Low 134 3.9 94 22 17 10 0.4 8.3 4.2 1.7 7.2 3.2 3.5 0.6 31 53 10 23 11

Ref High 145 5.3 105 30 24 25 1 10.4 7.7 2.2 9.4 4.2 5.8 1.2 77 162 50 78 39

Units mEq/L mEq/L mEq/L mEq/L mEq/L mg/dL mg/dL mg/dL mg/dL mEq/L g/dL g/dL g/dL mg/dL U/L U/L U/L U/L

Controls 142 138 5.1 97 21 25 21 0.6 10.8 9.3 2 6.5 3.9 2.6 1.5 66 64 21 156 11

143 142 5 100 23 24 13 0.4 10.7 9.6 2.1 5.9 3.6 2.3 1.57 65 80 18 184 9

144 139 4.6 100 21 23 16 0.9 9.8 8.6 1.7 6.6 3.2 3.4 0.94 41 105 50 298 18

145 139 5 98 22 24 18 0.6 10.2 8 1.7 6.7 3.5 3.2 1.09 38 100 26 184 17

146 139 4.6 98 22 24 17 0.5 9.7 6.9 1.9 7.7 3.3 4.4 0.75 40 94 24 115 22

147 143 4.5 98 25 25 7 0.6 10.4 8.5 1.8 7 3.7 3.3 1.12 39 73 34 238 14

148 139 5.3 98 22 24 23 0.6 10.2 9.3 1.7 6.9 3.3 3.6 0.92 25 77 22 96 17

149 143 5.2 97 26 25 11 0.6 10.7 10.1 1.8 6.9 3.6 3.3 1.09 66 93 32 235 11

150 143 4.9 100 24 24 16 1.1 10.3 8.4 1.9 6.3 3.6 2.7 1.33 30 65 17 102 12

151 138 5 98 22 23 27 0.6 9.7 6.9 1.6 6.9 3.5 3.4 1.03 34 118 26 101 13

152 139 5.2 99 22 23 19 0.6 10.1 7.8 1.7 7.6 3.2 4.4 0.73 55 80 22 311 19

153 138 5.2 98 23 22 18 0.6 9.5 7.3 1.6 6.2 3.4 2.8 1.21 5 62 15 117 18

155 137 5.4 92 24 26 11 0.6 11 8.5 1.9 7.4 3.5 3.9 0.9 42 93 46 163 22

156 139 4.9 97 24 23 9 0.5 10 8.4 1.9 5.9 2.9 3 0.97 42 93 33 138 7

157 141 5.2 95 23 28 10 0.5 10.9 7.9 1.5 7.4 3.4 4 0.85 47 67 13 181 14

158 141 4.8 96 23 27 11 0.6 10.3 10.1 1.9 7 3.7 3.3 1.12 52 106 38 307 18

159 139 5.7 98 22 25 9 0.5 10.7 7.2 1.9 7.2 3.3 3.9 0.85 45 83 50 349 24

162 137 6.7 98 27 19 12 0.5 10.5 9.1 1.8 6.9 3.4 3.5 0.97 53 104 7 449 7

164 139 6.5 100 27 19 14 0.5 11.2 9.2 2 6.3 3.6 2.7 1.33 67 101 10 441 8

166 140 6.3 103 25 18 15 0.5 10.7 9.9 2 6.2 3.6 2.6 1.38 49 100 21 415 10

167 137 6.8 96 29 19 13 0.5 10.3 8.6 1.6 6.5 3 3.5 0.86 37 159 130 230 32

168 135 7 96 27 19 9 0.6 10.7 9 1.8 6.8 3.5 3.3 1.06 59 105 10 439 8

170 143 4.6 95 23 30 15 0.6 10.5 8.4 1.8 7.2 3.7 3.5 1.06 38 75 17 245 10

171 145 5.1 98 23 29 15 0.5 10.9 9.1 1.8 6.4 3.6 2.8 1.29 38 91 24 232 10

172 143 4.8 97 22 29 16 0.6 11.1 9.6 1.9 7.4 3.9 3.5 1.11 44 91 7 347 5

173 144 5.1 100 23 26 12 0.6 10.7 8.8 1.9 6.8 3.7 3.1 1.19 36 71 23 201 8

174 144 4.6 96 25 28 21 0.7 11.2 9.7 1.7 6.4 3.7 2.7 1.37 31 70 21 351 13

175 142 4.5 96 25 26 14 0.7 10.9 9 1.9 7.6 3.6 4 0.9 38 91 29 199 14

176 142 4.8 97 26 24 15 0.5 10 9 1.8 7 3 4 0.75 29 100 26 176 12

177 143 5.1 95 23 30 16 0.7 11 9.9 2.1 6.8 3.5 3.3 1.06 18 85 23 248 16

178 143 5 97 21 30 19 0.6 10.2 9.3 1.8 6.8 3.3 3.5 0.94 40 90 6 249 5

179 142 4.9 97 22 28 17 0.7 10.4 8.9 1.9 6.7 3.4 3.3 1.03 52 83 11 237 8

180 140 5.1 97 22 26 13 0.6 10.2 9.3 2 6.5 3.6 2.9 1.24 60 66 15 194 10

181 143 4.5 96 24 28 15 0.5 10 10.2 1.8 6.9 3.5 3.4 1.03 31 84 21 171 13

182 142 5.3 96 20 31 15 0.5 10.1 7.4 1.6 7.5 3.1 4.4 0.7 7 89 11 173 16

183 142 5.1 95 23 29 12 0.5 10.7 8.8 2.1 6.8 3.6 3.2 1.13 26 103 31 305 7

184 141 5.3 94 23 29 15 0.6 11.2 9.8 1.8 7.2 3.7 3.5 1.06 32 65 15 263 14

185 141 5.2 94 26 26 15 0.5 11.3 9.3 1.8 8.3 3.3 5 0.66 24 85 23 249 16

196 139 4.8 100 23 21 13 0.5 10.2 8.3 2 5.9 2.9 3 0.97 27 73 35 217 14

197 141 5.2 97 25 24 17 0.7 10.5 8.7 2.1 6.6 3.5 3.1 1.13 42 76 33 444 16

198 139 4.9 97 23 24 12 0.6 10.5 7.6 1.8 7.4 3.3 4.1 0.8 20 65 24 303 14

199 142 5.5 95 28 25 20 0.7 10.8 7.2 1.9 6.5 3.5 3 1.17 12 94 37 239 14

200 138 5.5 101 21 22 7 0.6 10.7 9 1.7 5.4 3.5 1.9 1.84 42 89 174 339 22

201 142 5 100 26 21 7 0.6 10.6 9.1 1.7 6.1 3.4 2.7 1.26 61 48 12 338 16

202 142 5.3 100 23 24 8 0.7 10.5 10.6 1.8 5.9 3.9 2 1.95 52 67 22 402 14

203 141 5.7 99 27 21 7 0.6 11 9.1 1.8 6.1 3.4 2.7 1.26 60 41 23 419 20

204 140 5.9 97 24 25 8 0.6 10.9 9.9 1.9 7.1 3.2 3.9 0.82 44 80 46 419 16

Clones 39 141 5.8 100 28 19 8 0.6 10.8 8.5 1.9 5.6 3.1 2.5 1.25 69 48 11 392 19

40 141 5.6 100 27 20 9 0.7 11.7 10.8 1.9 5.7 3.5 2.2 1.59 62 72 11 549 20

41 141 5.3 100 27 19 11 0.5 10.3 8.4 1.9 6.4 3.7 2.7 1.37 56 77 8 157 10

42 141 5.9 100 26 21 11 0.5 10.4 9.7 2.2 6.6 3.6 3 1.2 64 81 15 220 10

56 140 5.4 96 26 23 19 0.7 10.9 10.7 2.1 6.3 3.6 2.7 1.33 15 119 13 239 4
Table E-200a: Individual Animal Clinical Chemistry Data at Weaning 2

58 133 6.8 93 25 22 11 <0.1 9.2 7.4 1.6 7.9 2.2 5.7 0.39 9 118 <2 87 <3

Sodium Potassium Chloride Bicarbonate Anion Gap Urea Creatine-rb Calcium Phosphate Magnesium-xb Total ProteinAlbumin-bulkGlobulin A/G Glucose AST/P5P SDH Alk. Phos. GGT

Clones 71 140 5.7 102 21 23 9 0.6 10.7 11.2 1.9 5.5 3.6 1.9 1.89 105 64 12 579 12

72 140 5.5 102 30 14 7 0.5 10.9 11.3 1.9 5.1 3.5 1.6 2.19 121 64 14 616 10

73 142 6.2 105 28 15 9 0.6 11 11.4 1.8 5.2 3.5 1.7 2.06 123 72 7 474 4

74 139 4.7 98 29 17 10 0.7 11 10.9 1.7 5.6 3.5 2.1 1.67 113 54 16 531 12

76 140 5.9 97 24 25 12 0.6 10 9.6 2.2 6.9 3.5 3.4 1.03 22 102 6 191 12

78 140 5.7 101 27 18 14 0.6 11.2 9.8 2 6.2 3.8 2.4 1.58 119 76 31 627 16

79 142 6.2 99 25 24 11 1.2 11.1 10.2 1.5 5.4 3.6 1.8 2 51 64 12 511 13

81 141 5.7 98 25 24 8 0.5 10.9 8.6 2.1 7 3.5 3.5 1 <2 104 14 189 6

82 140 6.5 100 24 23 11 0.4 10.8 8.3 2.2 7.9 3.2 4.7 0.68 2 110 <2 163 <3

83 138 6.3 96 25 23 11 0.5 10.1 8.9 1.9 7.6 2.8 4.8 0.58 <2.1 82 9 159 11

85 142 6 101 26 21 11 0.6 11.2 9 2 6.6 3.5 3.1 1.13 25 80 9 145 8

87 141 6.1 95 27 25 14 0.4 10.6 11.1 1.9 7.4 3.3 4.1 0.8 17 139 26 222 15

88 139 6.1 94 27 24 12 0.3 10.2 10.7 1.9 7.2 3.3 3.9 0.85 4 177 15 176 8

89 137 6.5 95 27 22 11 0.2 10.4 10.1 2 7.1 3.1 4 0.78 22 142 22 212 <3

90 138 5.3 98 22 23 11 0.3 10.3 8.4 2.2 6.7 3.4 3.3 1.03 14 141 <2 226 <3

93 137 5.2 94 22 26 9 0.6 10.6 10.4 1.9 7.8 3 4.8 0.63 13 62 13 148 12

94 139 5.7 99 24 22 11 0.4 10.5 9.5 2.3 6.9 3.4 3.5 0.97 9 116 2 177 <3

96 137 5.2 95 25 22 17 0.5 10.6 10.3 2.2 7.5 3.7 3.8 0.97 51 85 6 223 <3

100 135 5.3 96 24 20 6 0.5 10.8 9.1 2.1 8 3.4 4.6 0.74 78 67 11 265 22

101 140 5.2 100 27 18 10 0.5 10 8.9 1.8 7.2 3.1 4.1 0.76 53 131 30 154 7

102 138 5.4 96 26 21 15 0.4 10.3 9.5 1.7 6.9 3.3 3.6 0.92 32 110 5 158 <3

106 138 6.2 101 26 17 10 0.7 10.9 9 1.8 5.9 3.6 2.3 1.57 88 69 11 670 10

110 140 5.3 99 23 23 16 0.8 10.5 11.3 1.8 6.1 3.5 2.6 1.35 45 103 15 170 6

111 140 5.4 99 24 22 17 1.1 10.1 11.8 1.8 6.5 3.5 3 1.17 35 94 25 124 14

114 138 5.3 97 26 20 9 0.7 10.8 9.1 2 6.2 3.7 2.5 1.48 31 95 6 242 6

115 143 5.2 102 26 20 12 0.6 11.6 9.9 1.9 5.8 3.8 2 1.9 60 79 35 336 10

116 141 5.2 99 26 21 11 0.7 10 9.6 1.9 6.7 3.5 3.2 1.09 30 131 37 246 3

117 142 5.4 101 27 19 9 0.4 11.6 9.8 1.8 6.9 3.3 3.6 0.92 38 92 84 261 11

118 138 5.8 100 23 21 10 0.5 10.5 9.1 1.9 7.1 3.3 3.8 0.87 27 96 11 202 6

119 138 5 96 28 19 9 0.8 11.3 9.6 1.6 5.7 3.5 2.2 1.59 59 59 25 379 21

120 137 5 98 24 20 11 0.5 10.5 9 2 7.4 3.3 4.1 0.8 45 80 9 186 8

121 141 5.5 102 23 22 8 0.6 10.4 9 1.9 6.1 3.8 2.3 1.65 42 81 15 277 11

124 140 5.2 98 28 19 14 0.6 11.2 9.1 1.8 6.7 3.3 3.4 0.97 43 108 26 254 12

126 141 6.3 102 24 21 8 0.3 10.9 9.6 2.2 5.9 3.5 2.4 1.46 33 86 <2 261 <3

127 137 4.7 96 25 21 10 0.6 11.5 8.5 1.8 8 3.2 4.8 0.67 40 70 27 226 12

128 140 5.5 98 24 24 8 0.4 10.9 9 1.9 6.5 3.6 2.9 1.24 13 94 2 232 10

129 139 5.5 98 27 20 8 0.5 10.9 9.4 2.1 5.9 3.6 2.3 1.57 60 53 6 251 13

130 142 5.9 102 25 21 9 0.3 11.7 9.2 2 5.4 3.5 1.9 1.84 60 73 7 693 6

131 140 5 99 25 21 7 0.6 11.2 8.2 1.8 6.9 3.4 3.5 0.97 38 60 15 270 12

132 143 5.5 100 25 24 7 0.7 10.7 8.8 2.1 5.9 3.7 2.2 1.68 23 44 9 258 20
Table E-200b: Individual Animal Clinical Chemistry Data at Weaning 3

0-6 months Blood Chem.

Ref Low

Ref High

Units

Controls 142

143

144

145

146

147

148

149

150

151

152

153

155

156

157

158

159

162

164

166

167

168

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

196

197

198

199

200

201

202

203

204

Clones 39

40

41

42

56

Total Bilirubin Dir. Bilirubin Ind. Bilirubin Amylase Cholesterol CK Iron TIBC % Saturation hBA-random Lipemia Hemolysis Icterus IGF-1 Estradiol

0 0 0 73 77 113 362 28 9 34.7 10

0.1 0 0.1 280 265 226 533 48 455 472.27 40

mg/dL mg/dL mg/dL U/L mg/dL U/L ug/dL ug/dL % umol/L ng/ml pg/ml

0.1 0 0.1 35 106 124 210 584 36 45.3 25 18 0

0 0 0 38 110 244 184 432 43 18.7 0 31 0

0.1 0 0.1 37 130 225 147 374 39 49.5 25 1 0

0.1 0 0.1 36 119 287 145 370 39 28.4 24 12 0

0 0 0 26 65 207 149 341 44 12.8 18 14 0

0.1 0 0.1 37 75 256 141 412 34 29.7 12 13 0

0.1 0 0.1 26 78 272 158 350 45 25.9 15 16 0

0 0 0 18 92 268 180 438 41 14.9 5 49 w2

0 0 0 86 88 197 156 411 38 26.2 14 8 0

0.1 0.1 0 35 68 373 85 336 25 26.2 16 23 0

0.1 0.1 0 28 66 185 138 371 37 52 15 2 0

0.2 0.1 0.1 22 90 103 45 370 12 70.2 8 9 0

0.1 0 0.1 20 72 213 120 369 33 43.4 19 6 0 114.97 >4.00

0.1 0 0.1 32 63 261 182 335 54 29.7 15 46 0 61.68 7.46

0.1 0 0.1 20 73 134 112 420 27 13.7 20 17 0 203.71 4.47

0.1 0 0.1 15 67 217 194 550 35 78.8 15 20 0 278.97 4.29

0.1 0 0.1 29 71 292 147 422 35 33.5 19 16 0 157.34 4

0.1 0 0.1 30 63 700 208 389 53 24.5 6 99 0 49.29 5.34

0.1 0 0.1 38 73 308 209 475 44 28.5 0 65 0 104.91 5.52

0.1 0 0.1 36 59 285 180 431 42 38.8 0 55 0 232.32 4.75

0 0 0 29 86 346 88 340 26 42.3 5 31 0 17.99 2.47

0.1 0 0.1 26 71 1427 135 403 33 26.6 11 28 0 96.89 6.84

0.1 0 0.1 33 78 168 209 449 47 14.2 6 16 0 220.82 7.78

0.1 0 0.1 37 84 249 211 459 46 27.8 5 23 0 282.16 4.07

0.1 0 0.1 41 81 169 187 487 38 17.5 8 93 0 255.05 3.12

0.1 0 0.1 41 78 161 184 449 41 14.1 11 12 0 127.16 8.1

0.1 0 0.1 44 74 183 215 511 42 28.2 3 7 0 408.27 10.63

0.1 0 0.1 32 76 180 186 417 45 21.1 0 13 0 136.19 7.06

0.1 0 0.1 27 83 215 187 391 48 17.6 5 21 0 146.96 7.23

0.1 0 0.1 47 96 248 186 423 44 27.1 1 8 0 283.27 4.48

0.1 0 0.1 29 81 258 142 345 41 11.7 6 78 0 125.93 6.91

0.1 0 0.1 52 60 299 202 467 43 14.6 14 55 0 369.11 7.05

0.1 0 0.1 47 68 147 176 411 43 9.7 4 32 0 205.57 12.31

0.1 0 0.1 33 91 248 195 386 51 27.1 1 8 0 82.3 4.74

0 0 0 35 53 161 129 422 31 11.6 6 19 0 478.03 2.92

0.1 0 0.1 12 82 426 199 437 46 24 15 14 0 1.76 5.29

0.1 0 0.1 40 80 168 167 424 39 12.8 2 7 0 257.93 14.87

0.1 0 0.1 29 37 183 145 395 37 17.6 0 17 0 159.15 2.89

0.1 0 0.1 30 50 213 136 316 43 79.5 18 11 0 17.29 18.48

0.1 0 0.1 26 58 373 243 442 55 43.1 10 39 0 28.12 7.03

0.2 0 0.2 34 54 126 130 337 39 40 15 15 0 25.95 4.82

0.1 0 0.1 49 38 233 139 494 28 38.9 11 17 0 8.41 4.17

0.1 0 0.1 16 123 184 27 540 5 33.5 17 0 0 108.21 4.82

0.1 0 0.1 33 109 72 90 564 16 24.1 17 19 0 204.5 6.29

0.2 0 0.2 22 93 165 29 620 5 18.2 20 6 0 216.46 8

0.1 0 0.1 24 89 96 22 600 4 21.7 6 14 0 192.22 5.39

0.1 0 0.1 32 99 16832 591 5 37.2 15 7 0 0 269.77 8.44

0.2 0.1 0.1 19 86 281 81 432 19 15.4 0 16 0 113.37 4.73

0.1 0 0.1 22 116 230 180 511 35 37.7 9 36 0 252.12 7.04

0.1 0 0.1 26 98 243 155 353 44 37 7 51 0 114.43 4.24

0.1 0 0.1 28 65 540 188 426 44 17.9 13 18 0 274.83 9.34

0.1 0 0.1 18 109 238 244 441 55 18.2 26 67 0 270.54 5.4
Table E-200b: Individual Animal Clinical Chemistry Data at Weaning 4

58

Clones 71

72

73

74

76

78

79

81

82

83

85

87

88

89

90

93

94

96

100

101

102

106

110

111

114

115

116

117

118

119

120

121

124

126

127

128

129

130

131

132

0.2 0 0.2 14 52 243 106 209 51 0 17 424 0 7.64 17.99

Total Bilirubin Dir. Bilirubin Ind. Bilirubin Amylase Cholesterol CK Iron TIBC % Saturation hBA-random Lipemia Hemolysis Icterus IGF-1 Estradiol

0.2 0 0.2 18 116 375 113 632 18 15.7 18 38 0

0.2 0 0.2 15 122 189 114 642 18 14.7 30 45 0 109.3

0.3 0 0.3 16 119 751 145 584 25 12.3 25 103 0

0.1 0 0.1 15 113 158 153 555 28 30.1 21 21 0 204.52

0.1 0 0.1 51 94 662 214 384 56 24.1 11 97 0 196.87 7.2

0.2 0 0.2 29 99 209 273 640 43 39 9 27 0

0.2 0 0.2 28 153 208 131 465 28 19.9 22 58 0 149.6 <4

0.2 0 0.2 23 66 1228 157 376 42 36.4 5 43 0 81.54 8.52

0.2 0 0.2 23 90 505 169 379 45 28.2 14 114 0 92.21 6.01

0.2 0 0.2 17 62 353 83 296 28 32.3 9 19 0 66.45 5.72

0.1 0 0.1 29 77 340 140 416 34 34.6 3 37 0 60.33 10.38

0.1 0 0.1 20 94 1215 203 391 52 17.1 8 9 0 150.05 5.21

0.1 0 0.1 20 105 1265 187 321 58 20.7 10 115 0 110.26 7.77

0.1 0 0.1 18 100 442 243 330 74 16 3 112 0 119.66 5.06

0.2 0 0.2 24 90 2642 209 403 52 14 27 156 0 248.1 7.33

0.1 0 0.1 23 74 151 159 337 47 16.6 16 5 0 124.6 5.14

0.1 0 0.1 26 80 510 193 373 52 25.5 10 111 0 191.99 6.23

0.1 0 0.1 29 74 282 190 465 41 10.5 11 125 0 294.68 5.25

0.1 0 0.1 10 99 110 170 474 36 24.6 10 42 0 436.09 <4

0.1 0 0.1 17 84 1225 164 340 48 17.3 9 102 0 67.59 6.34

0.1 0 0.1 25 98 287 152 407 37 13.1 13 137 0 135.97 4.55

0.2 0 0.2 24 121 556 118 449 26 45.2 17 88 0

0.1 0 0.1 20 98 238 168 361 47 53.9 2 56 0 256.46 6

0.1 0.1 0 12 76 515 156 320 49 28.4 5 17 0 85.33 9.94

0.1 0 0.1 29 72 234 238 429 55 11 0 70 0 213.19 5.47

0.1 0 0.1 42 95 127 295 459 64 17.8 7 30 0 435 8.37

0.1 0 0.1 35 83 1974 217 388 56 19.3 6 151 0 114.68 4.36

0.1 0 0.1 32 109 127 208 382 54 31.7 21 96 0 260.65 3.63

0.2 0 0.2 38 71 319 162 351 46 35.6 5 107 0 64.08 9.05

0.2 0 0.2 35 121 125 217 431 50 18.1 25 32 0 227.89 <4

0.1 0 0.1 8 71 217 170 358 47 43.1 7 57 0 155.03 5.88

0.1 0 0.1 9 68 685 171 476 36 24.9 4 47 0 244.45 7.11

0.1 0 0.1 31 50 841 143 384 37 103.1 22 33 0 143.7 <4

0.1 0 0.1 24 107 129 204 467 44 27.7 14 253 0 253.54 5.51

0.1 0 0.1 25 84 217 93 424 22 72.9 5 20 0 295.63 <4

0.1 0 0.1 25 135 1056 196 459 43 17.3 8 161 0 113.91 6.71

0.1 0 0.1 24 133 140 187 443 42 32.5 0 42 0 121.47 9.98

0.2 0 0.2 18 127 110 283 547 52 15.4 5 153 0 387.73 8.32

0.1 0 0.1 25 113 152 151 437 35 52.4 0 19 0 216.38 5.21

0.1 0 0.1 28 136 492 197 466 42 24.7 8 24 0 96.56 8.45
Table E-300a: Individual Animal Clinical Chemistry Data at Adulthood 1

Adult Blood Chem. Sodium Potassium Chloride Bicarbonate Anion Gap Urea Creatine-rb Calcium Phosphate Magnesium-xb Total ProteinAlbumin-bulkGlobulin A/G Glucose AST/P5P SDH

Ref Low 134 3.9 94 22 17 10 0.4 8.3 4.2 1.7 7.2 3.2 3.5 0.6 31 53 10

Ref High 145 5.3 105 30 24 25 1 10.4 7.7 2.2 9.4 4.2 5.8 1.2 77 162 50

Units mEq/L mEq/L mEq/L mEq/L mEq/L mg/dL mg/dL mg/dL mg/dL mEq/L g/dL g/dL g/dL mg/dL U/L U/L

Controls 135 140 4.5 103 21 21 11 0.6 9.6 8.6 2 6.8 3.4 3.4 1 71 120 5

154 138 4.7 98 27 18 12 0.7 10.9 8.1 2.1 6.7 3.7 3 1.23 88 68 18

206 136 4.7 97 24 20 5 0.6 10.7 9.6 1.7 6.9 3.4 3.5 0.97 90 83 17

160 147 5.9 101 23 29 21 0.9 10.1 9.3 1.9 7.3 3.9 3.4 1.15 31 84 23

161 143 4.7 97 22 29 18 0.7 10.5 9.4 2 6.8 3.7 3.1 1.19 38 88 22

162 140 4.8 98 23 24 16 0.7 9.7 8.5 1.8 6.4 3.1 3.3 0.94 8 65 13

163 144 4.8 99 15 35 15 0.7 10.5 9 1.9 7.4 3.5 3.9 0.9 44 86 16

165 146 4.9 99 18 34 16 0.7 10.6 8.7 1.8 7 3.9 3.1 1.26 22 100 18

169 143 4.7 96 22 30 13 0.5 11 8.8 1.8 7.3 3.8 3.5 1.09 32 72 25

205 139 4.5 95 26 23 15 0.8 10.7 8.3 1.8 7.8 3.3 4.5 0.73 81 88 15

207 137 4.9 95 26 21 10 0.7 10.4 7.7 2 6.6 3.6 3 1.2 83 72 7

208 139 4.5 96 27 21 10 0.4 10.2 8.9 1.9 7 3.4 3.6 0.94 95 77 4

209 139 4.8 94 27 23 12 0.6 9.9 9.2 1.8 7 3.2 3.8 0.84 87 109 34

210 140 4.8 96 26 23 13 0.8 10.1 7.7 2.1 7.1 3.5 3.6 0.97 92 98 15

211 138 4.3 93 26 23 10 0.6 10.3 8.1 2 7 3.2 3.8 0.84 92 85 19

212 142 5.5 98 26 24 13 0.6 10.7 8.1 2.1 7.5 3.4 4.1 0.83 90 79 30

215 141 5.2 100 19 27 16 0.9 10.6 8.8 2 7 3.8 3.2 1.19 49 109 8

216 143 4.6 101 21 26 18 0.8 9.5 9.5 1.8 6.5 3.5 3 1.17 80 99 25

217 143 4.6 100 20 28 12 0.7 9.4 10.1 1.8 6.2 3.4 2.8 1.21 74 98 23

218 141 4.7 99 21 26 18 0.7 10.3 9.7 1.9 6.6 3.7 2.9 1.28 58 74 23

219 141 4.6 99 21 26 14 0.9 10 9.7 1.9 6.7 3.7 3 1.23 89 88 26

Clones 24 140 4.7 96 24 25 18 1 10.4 8 1.9 7.2 3.6 3.6 1 78 86 13

25 141 4.3 96 25 24 12 1 10.4 8.1 1.8 6.8 3.6 3.2 1.13 72 95 16

26 139 4.5 95 25 24 17 1 10 8.6 1.8 7 3.4 3.6 0.94 80 97 19

27 139 5.2 94 25 25 13 0.9 10.5 8.7 1.9 7.2 3.5 3.7 0.95 78 100 14

33 143 4.7 98 25 25 14 1.2 10.4 8.3 1.9 7.9 3.6 4.3 0.84 73 68 11

35 143 4.7 98 26 24 14 0.9 10.2 8.1 2 7.6 3.4 4.2 0.81 75 73 14

36 140 4.3 96 27 21 12 0.9 10 7.7 1.9 7.6 3.5 4.1 0.85 81 67 15

38 139 4.2 96 25 22 15 1 9.7 9.1 1.9 7.1 3.4 3.7 0.92 88 75 15

37 140 5 100 26 19 6 0.5 10.3 8.4 2 6.5 3.6 2.9 1.24 88 92 3

55 140 5.4 99 25 21 13 0.6 10.8 9.9 2 6.6 3.8 2.8 1.36 45 81 7

67 145 5 99 23 28 17 0.6 10.6 10.6 1.9 6.9 3.5 3.4 1.03 70 103 15

69 144 4.8 97 26 26 14 0.7 10 9.3 1.7 7.5 3.1 4.4 0.7 73 85 22

70 147 4.9 100 27 25 15 0.6 10.7 9.9 2.1 7.2 3.7 3.5 1.06 81 82 20

98 136 4.2 96 27 17 11 0.6 10 8.5 1.8 6.8 3.7 3.1 1.19 91 59 15

99 135 4.5 96 23 21 10 0.6 9.4 8.5 1.8 7.4 3.4 4 0.85 92 62 13

103 144 6.6 100 23 28 13 0.2 10.7 10.2 2.1 7.1 3.7 3.4 1.09 12 133 7

104 143 6.3 100 27 22 17 0.5 10.8 9.9 2 6.9 3.5 3.4 1.03 32 122 5

108 137 5.5 97 24 22 18 1.1 10.6 9.2 1.9 6.7 3.7 3 1.23 24 97 15
Table E-300a: Individual Animal Clinical Chemistry Data at Adulthood 2

Adult Blood Chem.

Ref Low

Ref High

Units

Controls 135

154

206

160

161

162

163

165

169

205

207

208

209

210

211

212

215

216

217

218

219

Clones 24

25

26

27

33

35

36

38

37

55

67

69

70

98

99

103

104

108

Alk. Phos. GGT Total Bilirubin Dir. Bilirubin Ind. Bilirubin Amylase Cholesterol CK Iron TIBC % Saturation hBA-random Lipemia Hemolysis Icterus IGF-1 Estradiol

23 11 0 0 0 73 77 113 362 28 9 34.7 10

78 39 0.1 0 0.1 280 265 226 533 48 455 472.27 40

U/L U/L mg/dL mg/dL mg/dL U/L mg/dL U/L ug/dL ug/dL % umol/L ng/ml pg/ml

121 5 0.1 0 0.1 82 86 176 167 364 46 3.2 20 71 1

173 15 0.2 0 0.2 32 113 170 164 428 38 50.9 8 10 0 312.99 >4.0

231 11 0.1 0 0.1 44 102 227 212 450 47 28.4 14 45 0 278.28 4.2

182 15 0.1 0 0.1 38 113 153 183 476 38 34.2 5 32 0 324.29 11.14

292 12 0.1 0 0.1 26 95 196 205 493 42 14.6 5 4 0 332.05 8

167 9 0.1 0 0.1 23 69 141 224 343 65 20.4 0 26 0 43.75 4.1

276 11 0.1 0 0.1 36 74 217 157 406 39 27.5 13 19 0 193.53 4.48

191 14 0.1 0 0.1 48 88 207 239 494 48 28.6 12 42 0 291.66 6.93

280 10 0.1 0 0.1 43 74 243 200 508 39 17.7 24 0 0 153.69 5.77

134 16 0.1 0 0.1 45 105 176 114 411 28 15.1 25 14 0 732.75 9.15

193 11 0.1 0 0.1 43 90 117 195 435 45 21.3 13 39 0 752.54 11.41

178 <3 0.1 0 0.1 39 66 242 194 439 44 7.8 24 202 0 793.84 10.36

151 23 0.1 0 0.1 23 67 150 156 410 38 15.1 5 25 0 732.63 10.35

168 18 0.1 0 0.1 33 86 465 170 439 39 32.7 18 64 0 816.99 10.53

169 20 0.1 0 0.1 29 58 168 164 482 34 7.9 17 24 0 861.08 7.91

169 22 0.1 0 0.1 14 77 165 151 529 29 13.5 12 6 0 875.72 12.44

158 <3 0.1 0 0.1 24 172 936 167 403 41 18.9 33 119 1 155.44 >4.0

213 8 0.1 0 0.1 44 141 421 192 407 47 18 15 33 1 215.93 >4.0

197 6 0 0 0 45 181 328 182 401 45 19.5 2 23 1 253.77 4.3

127 8 0.1 0 0.1 44 160 253 180 376 48 20.9 7 41 2 218.14 5.26

254 10 0.1 0 0.1 30 155 640 161 433 37 26.8 9 27 1 338.43 >4.0

109 13 0.1 0 0.1 46 104 181 132 409 32 17.1 16 8 0 924.04 14.16

174 15 0.1 0 0.1 33 113 884 142 388 37 29.4 18 3 0 769.08 11.94

151 13 0.1 0 0.1 34 113 161 130 359 36 20.2 15 2 0 381.04 10.62

163 14 0.1 0 0.1 39 122 205 143 401 36 16.5 15 28 0 618.84 12.02

104 16 0.1 0 0.1 34 81 145 150 470 32 31.5 14 9 0 915.45 24.33

132 13 0.1 0 0.1 34 77 188 192 488 39 16.6 12 10 0 937.54 23.92

113 16 0.1 0 0.1 35 78 103 155 479 32 12.3 11 5 0 875.68 15.4

164 15 0.1 0 0.1 33 125 121 101 353 29 24.9 6 9 0 622.08 7.62

136 3 0.1 0 0.1 37 85 432 179 478 37 14.4 22 84 1

184 7 0.1 0 0.1 43 129 177 230 466 49 27.9 28 60 0 414.52 9.6

186 17 0.1 0 0.1 29 86 426 193 455 42 12.3 5 34 0 691.78 12.4

138 22 0.1 0 0.1 27 88 263 156 385 41 10.7 25 0 0 445.23 15.82

148 22 0.1 0 0.1 31 99 448 183 464 39 14.3 8 1 0 548.43 10.06

231 11 0.1 0 0.1 39 91 196 132 363 36 23.5 5 8 0 279.26 >4.0

192 11 0.1 0 0.1 37 96 217 129 331 39 20.8 9 5 0 217.88 4.28

182 <3 0.1 0 0.1 33 122 1222 165 390 42 7.7 20 353 0 169.03 6.91

179 <3 0.1 0 0.1 35 108 313 230 430 53 7.3 18 262 0 331.99 7.07

111 12 0.1 0 0.1 33 111 227 165 304 54 29.5 20 30 0 33.25 7.5
Table E-300b: Individual Animal Hematology Data at Adulthood 1

Hemogram Hematocrit (HCT) Hemoglobin (HB) RBC MCV MCH MCHC RDW WBC Seg. Neut. Band. Neut. Lymphocytes Monocytes Eosinophils Basophils

Ref Low 23 8.6 5.4 36 14 36 16.2 5.6 1.8 0 1.9 0 0 0

Ref High 25 13.2 8.2 49 19 40 19.7 13.7 7.7 0 7.4 1.2 1.4 0.3

Units % g /dL mill/uL fL pg g /dL % thou/uL thou/uL thou/uL thou/uL thou/uL thou/uL thou/uL

Controls 135 33 11.8 7.5 44 16 35 18.4 6 3.7 0 1.7 0.1 0.5 0

154 28 11 8.4 33 13 40 20.7 8.5 2.4 0 6.1 0 0 0

206 28 10.8 8.9 32 12 39 23.3 13.2 5 0 7.5 0.7 0 0

160 30 11.8 7.9 38 15 39 19.8 12.4 6 0 6.2 0.2 0 0

161 34 11.4 8.6 39 13 34 22.2 13.4 3.1 0 10.1 0.3 0 0

162 35 10.3 8.4 42 12 29 31.4 14.4 2.7 0 10.8 0.7 0 0.1

163 34 10.3 9.4 37 11 30 28.3 14.6 4.5 0 9.9 0.1 0 0

165 34 11.6 9.6 35 12 35 31.1 24.9 5.2 0 19.2 0.5 0 0

169 33 11.5 9.7 34 12 35 21.1 12.2 4.5 0 7 0.2 0.5 0

205 30 11.4 6.8 44 17 38 17.4 12.7 3.9 0 8.3 0.1 0.3 0.1

207 29 11 7.3 39 15 38 18.4 8.6 2.6 0 5.8 0.1 0.1 0.1

208

209 32 10.5 8.7 37 12 33 33.1 11.3 4.4 0 6.7 0.2 0 0

210 34 10.6 9 38 12 31 32.1 10 4.4 0 5.1 0.4 0.1 0

211 28 10.7 7.8 36 14 38 22.7 9.8 3.7 0 5.8 0.1 0.2 0

212 34 10.5 8.6 39 12 31 30.2 12.5 5.1 0 6.8 0.6 0 0

215 46 14.8 10.1 46 15 32 34.6 9.6 4.1 0 5.4 0 0.1 0

216 32 12.7 9 36 14 40 24.1 8.4 2.8 0 5.5 0.1 0 0

217 38 12.7 8 47 16 34 23.1 13.6 6.9 0 6.4 0.1 0.1 0

218 42 13.8 10.2 42 14 33 36 9.9 2.6 0 6.8 0.4 0 0.1

219 31 12.2 9 34 14 40 22.3 12.4 6.9 0 4.8 0.5 0 0.1

Clones 24 33 11 6.9 48 16 33 20.2 11.6 6.4 0 4.8 0.5 0 0

25 32 10.7 6.9 46 16 34 23.8 13.9 6.1 0 7.2 0.6 0 0

26 32 10.6 6.8 47 16 34 21.4 9.5 2.4 0 6.7 0.2 0.2 0

27 32 10.5 6.7 47 16 33 20.5 7.8 2.5 0 4.8 0.3 0.2 0

33 33 10.7 7.7 43 14 32 33 10.2 2.1 0 7 0.5 0.4 0.1

35 34 11.1 7.7 44 15 33 33.9 10.9 2.7 0 7.7 0.2 0.1 0.1

36 30 9.9 6.9 44 14 33 32.2 9.2 3 0 5.9 0.1 0.1 0.1

38 31 9.7 6.6 46 15 32 28.3 10.5 4.7 0 5.4 0 0.4 0

37 35 12.2 8.7 40 14 35 20.1 8.1 2.3 0 5.2 0.6 0.1 0

55 30 11.1 8.6 35 13 37 21.7 10.5 2.1 0 7.6 0.5 0.3 0

67 34 11.1 9.5 36 12 32 32.4 9.5 4.8 0 3.8 0.3 0.6 0

69 31 10.1 9.5 33 11 33 29.5 8.2 2.5 0 5.4 0.1 0.2 0

70 33 10.8 9.9 34 11 32 31.7 8.2 2.5 0 5.2 0.1 0.3 0

98 28 10.8 8.9 32 12 38 22.6 8.1 2.1 0 5.4 0.2 0.1 0.3

99 28 10.6 9.3 30 12 38 20.4 8.5 2.4 0 5.7 0.4 0 0

103 28 10.5 7.2 40 15 37 21.6 8.6 3.5 0 4.9 0.2 0 0

104 28 10.9 7.4 38 15 39 19.9 8.8 5.5 0 3 0.2 0.2 0

108 37 13.8 11.9 31 12 37 19.4 8.2 3.5 0 3.9 0.3 0.2 0.2

Notes:

NSA No Significant Abnormalities

NS None Seen

N Normal

SCM Smudged Cells-Moderate
Table E-300b: Individual Animal Hematology Data at Adulthood 2

Hemogram

Ref Low

Ref High

Units

Controls 135

154

206

160

161

162

163

165

169

205

207

208

209

210

211

212

215

216

217

218

219

Clones 24

25

26

27

33

35

36

38

37

55

67

69

70

98

99

103

104

108

Platelet SmearPlatelets MPV TP-Ref RBC Morphology Acanthocytes Anisocytosis Echinocytes Poikilocytes Schistocytes Parasites WBC Exam Plasma Appearance

232 5.6 5.9

596 8.2 8.1 hemolysis

thou/uL fL g /dL

Incr. 812 16.8 8.3 NSA NS NSA Moderate

Adeq. 391 5.8 6.7 Mild NS NSA N

Incr. (*1) 601 5.5 6.8 NSA NS NSA N

Adeq. 259 6.6 7.3 NSA NS NSA N

Adeq. 391 7.4 7 Moderate NS NSA N

Adeq. 487 6.8 6.5 Moderate NS NSA N

Adeq. 515 6.3 7.2 Many NS NSA N

Adeq. 279 6.2 6.8 NSA NS Few N

Adeq.(*1) 559 5.9 7.2 NSA NS NSA N

Adeq. 309 10.7 7 NSA NS NSA Slight

Adeq.(*1) 6.6 NSA NS NSA Slight

Adeq.(*1) 7 Mild NS NSA Slight

Adeq.(*1) 117 6.4 7.3 Few NS NSA Slight

Adeq. 414 7.3 7 Mild NS NSA N

Adeq. 351 6.5 7.4 NSA NS NSA N

Adeq.(*1) 242 5.9 7.5 NSA NS NSA Slight

Incr. 671 6.3 6.7 NSA NS NSA Slight

Adeq. 468 6.3 6.5 Mild NS NSA Slight

Adeq. 422 6 6.9 NSA NS NSA Slight

Adeq. 583 6.3 7 Mild Few NS NSA Slight

Adeq. 398 7.2 7.3 NSA NS NSA Slight

Adeq. 313 6.7 6.8 NSA NS NSA N

Adeq.(*1) 7.2 NSA NS NSA Slight

Adeq.(*1) 7.2 NSA NS NSA N

Adeq. 392 5.8 7.8 NSA NS NSA N

Adeq.(*1) 7.4 NSA NS NSA Slight

Adeq. 335 6.6 7.4 NSA NS NSA N

Adeq.(*1) 7.5 NSA NS NSA Slight

Adeq. 328 12.4 7.5 NSA NS NSA Slight

Adeq.(*1) 6.8 NSA Slight

Adeq.(*1) 6.9 NSA NS NSA Slight

Incr. 658 5.9 7.7 NSA NS NSA N

Incr. 605 6 7.3 NSA NS NSA N

Adeq. (*1) 395 6.8 6.7 Mild NS NSA N

Incr.(*1) 622 5.5 7.4 Mild NS NSA N

Adeq. 337 11 6.8 Mod NS NSA N

Incr. 745 6.4 7.4 NSA NS NSA N

Adeq. 399 14.4 6.8 Few NS NSA Slight
Animal Cloning: A Risk Assessment DRAFT

Appendix F: 

The Viagen Dataset
Animal Cloning: A Risk Assessment DRAFT

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Animal Cloning: A Risk Assessment DRAFT

Appendix F: The Viagen Dataset 

A. Background 

Viagen, Inc., a privately held company engaged in cloning swine and cattle for agricultural 

purposes, also responded to CVM’s request for additional data on clones and their progeny (for a 

similar discussion on cattle, see Appendix E: The Cyagra Dataset). Viagen designed two studies 

to evaluate the health, growth, and meat composition of swine clones, fertility of boar82 clones, 

and health, growth and meat composition of clone progeny vs. age-matched, genetically related, 

artificial insemination (AI)-derived comparator animals. Various scientists from CVM with 

expertise relevant to these studies provided peer-review comments to Viagen on the overall 

experimental design, but did not formally approve or disapprove the studies. All of the raw data 

that CVM received from Viagen is found at the end of this Appendix. 

As described in their report, the company’s objectives in running these studies were to 

1. Compare the health of swine clones vs. AI-derived pigs of a related genetic line; 

2. Compare the biological characteristics, including laboratory measurements and meat 

composition, of swine clones vs. AI-derived swine; 

3. Assess the reproductive characteristics of clone boars vs. AI-derived boars; and, 

4. Compare the biological characteristics, including laboratory measurements and meat 

composition, of the progeny of clone boars vs. progeny of AI-derived closely-related 

boars (nuclear donors for clones or progeny of nuclear donor). 

B. Experimental Design 

Study 1 Overview: Evaluation of Clones 

Survival, health, growth and meat characteristics of seven clones (6 “Hamline”83 and 1 Duroc (a 

common breed of swine used in U.S. commercial pork production), all barrows84) and 15 

conventional barrows (all Hamline) were followed from 50 days after birth through slaughter at 

approximately 6 months of age. Clones were assigned to this study shortly after they were 

 

82 A boar is a reproductively intact male pig. 

83 “Hamline” refers to a specific crossbred line of swine used by Viagen, Inc. This line was developed by crossing 

various breeds, including Duroc, European Landrace, Pietran, and European Large White swine. 84 A barrow is a castrated male pig. 
Appendix F: The Viagen Dataset F-4

Animal Cloning: A Risk Assessment DRAFT

weaned. Age-matched comparator85 pigs were selected from litters sired by the Hamline nuclear 

donor boar in a conventional breeding (AI) program. 

An additional group of four clones (three Hamline and one Duroc) and three AI-derived boars 

(the Hamline nuclear donor and two AI sons of the Duroc nuclear donor) were used for 

evaluation of semen quality and fertility. The progeny generated from these boars were used in 

another experiment to study whether progeny of clones were materially different in growth, 

health or meat characteristics from conventional swine. Because of the small number of clones, 

these animals were evaluated on a case-by-case basis and simple means generated when 

appropriate, rather than applying statistical methods to the data. 

Figure F-1: Design of Viagen Study 1 

Study 1: Design

Hamline Nuclear Donor Duroc Nuclear Donor

Clones Comparators 

(Hamline Genetics)

Hamline Comparator

Hamline Comparator Hamline Comparator

Hamline Comparator Hamline Comparator

Hamline Comparator Hamline Comparator

Hamline Comparator Hamline Comparator

Hamline Comparator Hamline Comparator

Hamline Comparator Hamline Comparator

Hamline Comparator Hamline Comparator

15 genetically unrelated 

cross-bred “Hamline” pigs

Hamline Clone 021

Hamline Clone 020

Hamline Clone 019

Hamline Clone 018 Duroc Clone 024

Hamline Clone 022

Hamline Clone 023

Study 2 Overview: Evaluation of Clone Progeny 

In the second experiment, survival, health, growth, and meat composition of progeny derived 

from clone boars (three Hamline and one Duroc) and either their nuclear donor (Hamline) or two 

sons of the nuclear donor Duroc boar were compared. Gilts86 sired by six different boars were 

randomly assigned according to their sire (such that each maternal grandsire was represented in 

 

85 Although this study is a more controlled experiment than the retrospective review of the Cyagra clones, the word 

“comparator” is used for consistency with the discussion of Cyagra data, rather than the more common term 

“control.” 

86 A gilt is a young female pig that has not given birth (termed “farrowing”). 
Appendix F: The Viagen Dataset F-5

Animal Cloning: A Risk Assessment DRAFT

each breeding group to control for effect of maternal grandsire) to each clone or comparator 

boar, and bred by AI. Sixty-eight gilts farrowed (gave birth) under standard commercial 

procedures, yielding 402 total progeny from clone boars and 300 total progeny from comparator 

boars. This included 284 progeny from Hamline clones, 118 progeny from the Duroc clone, 61 

progeny from the Hamline donor sire (Hamline comparator), and 239 progeny from Duroc donor 

sire sons (Duroc comparators). 

Figure F-2: Design of Viagen Study 2 

Study II: Design

Duroc

Nuclear Donor

5001

Duroc

Clone

007

64 

Hamline

Offspring

Hamline

Clone

018

Bred to 12 

Hamline

Sows

Hamline

Nuclear Donor

H498

Bred to 12 

Hamline Sows

64 

Hamline

Offspring

Hamline

Clone

018

Bred to 12 

Hamline

Sows

64 

Hamline

Offspring

Hamline

Clone

018

Bred to 12 

Hamline

Sows

64 

Hamline

Comparators

Bred to 13

Hamline

Sows

18128

Conventionallybred 

Son of 5001

Bred to 13

Hamline

Sows

112 

Duroc

Comparators

112 

Duroc

Offspring

18128

Conventionallybred 

Son of 5001

Bred to 13

Hamline

Sows

112 

Duroc

Comparators

1. Study 1: Clones vs. Comparators 

Clone pigs were obtained by Caesarean section (C-section) on the day of or the day before the 

sows’ predicted farrowing date. Clone piglets were kept under highly biosecure87 conditions 

prior to initiating the experiment. Clones were bottle fed commercial milk replacer and did not 

receive colostrum at the time of birth. Comparator pigs, however, were born naturally (i.e., 

vaginally) in a standard commercial farrowing house, and suckled their dams until weaning. By 

 

87 “Biosecure” connotes conditions that, although not completely sterile, are designed to prevent introduction of 

most disease-causing organisms. Because animals’ immune systems produce antibodies in response to specific 

stimuli (antigens), animals raised under biosecure conditions lack the full battery of antibodies needed to respond to 

common pathogens found in conventional environments (although they may have some antibodies if they have been 

vaccinated, these antibodies will be specific for the antigen used in the vaccine). It can take up to two weeks for an 

animal to develop a sufficient concentration of antibodies to a specific antigen following exposure. 
Appendix F: The Viagen Dataset F-6

Animal Cloning: A Risk Assessment DRAFT

the start of the test period (~50 days of age), the clones were similar in weight to the comparator 

pigs (16.4 vs. 15.3 kg for clones and comparators, respectively). In addition, comparator barrows 

were castrated during the first week of life, as per normal industry practice, but clone barrows 

were castrated at a later age (~30 days old). Pigs were assigned to this study shortly after they 

were weaned. Comparator barrows were selected from litters sired by one of the nuclear donor 

boars in a conventional (AI) breeding program. At the start of Study 1 clones and comparator 

animals were housed in the same facility. Target slaughter weight was approximately 122 kg.88 

a. Animal Health, Hematology, Clinical Chemistry, and Urinalysis 

Health records from date of birth were submitted for clones, and included birth weight, heart 

rate, respiration, body temperature, body weight, and daily health observations. All health 

problems and treatments were recorded. Necropsy reports were provided for clones that died or 

were euthanized. Blood samples of clones and comparators were collected at birth and again 

either approximately one week prior to slaughter, or immediately after animals were slaughtered. 

Urine samples were collected after slaughter (by bladder puncture). Blood hematology, clinical 

chemistry, and urine values were assayed as for the Cyagra dataset at the same independent 

testing laboratory (Cornell University’s Animal Health Diagnostic Laboratory, see Appendix E). 

b. Boar Semen Evaluation 

Semen samples were evaluated in two commercial boar lines (Hamline and Duroc). The Hamline 

included three clone boars and the sexually derived nuclear donor boar for the clones as a 

comparator. The Duroc line included one clone boar and two comparators that were AI-derived 

progeny of the donor (semen from the Duroc nuclear donor was unavailable). Clones and 

comparator animals were housed at separate facilities. Semen was collected at least once a week 

from each boar, was diluted with a commercial semen extender to yield 3 X 109 sperm per dose, 

and cooled to 19 ̊C per standard industry practice. Semen from clones was evaluated pre-freezing 

and post-freezing (post-shipping) to determine quality. Comparator boar semen was only 

evaluated post-shipping. Standard tests such as sperm concentration, percent total motility, 

percent progressive motility, and number of head/tail abnormalities were evaluated. 

c. Farrowing Rate 

Following semen evaluation, semen from the four clone boars and three comparator boars were 

used to evaluate pregnancy (number of females diagnosed pregnant divided by number of 

 

88 Swine are typically slaughtered in groups. Usually, all swine raised together in a pen will go to slaughter on the 

same day once the average weight of the animals within that pen has reached a pre-set target weight. The date of 

slaughter can be affected by the availability of transportation and the capacity of the slaughter facility. 
Appendix F: The Viagen Dataset F-7

Animal Cloning: A Risk Assessment DRAFT

females bred) and farrowing (number of females giving birth to a litter divided by the number of 

females bred) rates. Three of the clone boars were bred to 12 gilts each and one clone boar was 

bred to 13 gilts (total of 49 gilts bred). For the comparator boars, two were bred to 14 gilts each 

and one was bred to 12 gilts (a total of 40 gilts bred). All gilts were bred via AI twice, once on 

the day of observed estrus and approximately 24 hours later. 

2. Study 2: Progeny of Clone Boars vs. Progeny of Conventional Boars 

Breeding resulted in 36 litters (402 total progeny) from the clone boars and 25 litters (300 total 

progeny) from the comparator boars. Reproductive outcomes evaluated included litter size, birth 

weight, number of stillborns, number of dead/destroyed animals, abnormalities, and nipple 

counts (another measure of breeding fitness often taken at birth). Animals were slaughtered when 

they had reached the target weight of approximately 122 kg. Average age at slaughter was 169.3 

days for progeny of comparators and 174.9 days for progeny of clones. 

a. Statistical Analysis 

i. Methodology 

To aid CVM’s analysis of the very large sample sizes in Study 2, the Center’s biostatisticians 

produced data summaries to facilitate veterinary and scientific evaluation of the outcomes 

collected from the progeny. Two types of summaries were used: contingency tables and 

boxplots. Definitions and examples of both are given below. 

Contingency tables present data in a row and column format and are often used to summarize the 

results of discrete variables. A variable can be defined as discrete if its responses are limited to a 

few predetermined values. For example, a discrete variable from Study 2 is “Abnormality at 

birth” which is described according to four possible discrete outcomes: Normal, Atresia, 

Spraddle legs, or Other. For those progeny for which this variable was recorded, the distributions 

of “Abnormality” for the live-born progeny of animal clones and progeny of comparators are 

displayed in the following contingency table (Table F-1). 
Appendix F: The Viagen Dataset F-8

Animal Cloning: A Risk Assessment DRAFT

Table F-1: Contingency Table Data Description for Distribution of Abnormalities 

for Progeny of Clones and Progeny of Comparators 

 Progeny of Clones Progeny of Comparators 

Abnormality Number Percentage Number Percentage 

Normal 384 97.5% 291 99% 

Atresia 1 0.3% 0 0% 

Spraddle Legs 4 1.0% 3 1.0% 

Other 5 1.2% 0 0% 

Total 394 100% 294 100% 

By contrast, continuous variables are those whose responses can take on a continuum of values. 

Graphical displays are useful in summarizing the responses from continuous variables. The 

boxplot is one type of graphical display. An example of a continuous variable from Study 2 is 

“animal weight.” The boxplots of birth weights from the progeny experiment are provided as an 

example below. 

Figure F-3: Boxplot of birth weights of progeny of clones and comparators (in kgs) 

In Figure F-3, the boxplot on the far right includes all of the birth weights recorded for clone 

progeny and comparators. To create a boxplot, the birth weight data was ordered from smallest 
Appendix F: The Viagen Dataset F-9

Animal Cloning: A Risk Assessment DRAFT

to largest. The bottom edge of the box was drawn at a value of birth weight for which 25 percent 

of the observations have a lower value, which is called the “25th percentile”1 (or first quartile) of 

the sample. Similarly, the top of the box was drawn at a value of birth weight for which 75 

percent of the observations have a lower value, which is called the “75th percentile”89 of the 

sample. The value of the observation associated with the 75th percentile minus the value of the 

observation associated with the 25th percentile is called the interquartile range (IQR). Within the 

box, a plus sign is used to indicate the sample mean and the line from side to side of the box 

indicates the sample median which is the value of the observation associated with the 50th

percentile below which half the observations lie. Any observation values which lie above the 

“upper fence,” a point equal to 1.5 times IQR plus the value associated with the 75th percentile or 

below the “lower fence,” a value equal to the value of the observation associated with the 25th

percentile minus 1.5 times IQR, are individually plotted on the graph and are called extreme 

values. (Note the actual fence values are not plotted.) The whiskers stemming out from the boxes 

represent the observation just below the upper fence and the observation just above the lower 

fence. In the example above, the boxplots, from left to right, are for progeny of Hamline clones, 

progeny of Hamline comparator animals, progeny of Duroc clones, and progeny of Duroc 

comparator animals. 

 

Boxplots do not require assumptions about the form of the underlying continuous data 

distributions and are uniformly applicable across a range of different distributions. When 

boxplots are used to describe a variable sampled from a single population and having a normal 

distribution, they tend to identify about 1 percent of values above and 1 percent of values below 

the box as “extreme.” 

When the underlying distribution has more data in the tails than a normal distribution, then more 

than 1 percent of the distribution is identified as extreme. To evaluate the Viagen data, values 

from the progeny of clones were compared to the ends of the whiskers in the boxplots for the 

values from the progeny of comparators. 

ii. Utility of Statistical Analysis as a Tool for Assessment of Outcomes 

Results (numerical values) for any outcome from the clone progeny were compared to the ends 

of the whiskers for the distribution of values from progeny. If a result fell within the range 

defined by the box and whiskers, it was considered “within the same range as the comparators” 

and therefore posed no significant concern for the health of the animal or the composition of the 

food product. If a result for a clone fell outside the range identified by the whiskers for the 

comparators, or was identified because of its value in a contingency table, the appropriate CVM 

 

89 A percentile is defined as (1/(total sample size))*100 
Appendix F: The Viagen Dataset F-10 

Animal Cloning: A Risk Assessment DRAFT

scientist (e.g., veterinarian, animal scientist, chemist, toxicologist) traced back all of the data for 

that the individual animal, and conducted a thorough examination of all of the relevant clinical 

information to draw a conclusion regarding the biological or health significance of the value. 

In addition, to provide consistency with the presentation of the analysis developed for the Cyagra 

dataset (Appendix E), box charts were constructed. As described in that Appendix, black boxes 

or up or down arrows were assigned to values for each clone progeny by the reviewing 

veterinarian or scientist on the basis of this multi-step review procedure. 

b. Specific Methods for the Analysis of Animal Health, Hematology, Clinical 

Chemistry, and Urinalysis Data 

Progeny of clones and comparators were farrowed during a three week period from July 7-27, 

2004, at the Meat Animal Research Center, USDA, Clay Center, Nebraska. All available animals 

were sampled (serum and whole blood) in July, October, and January. Blood samples were 

obtained between 7:30 and 10:30 AM. After collection, samples were placed on ice until 

centrifugation at 900 x g for 10 min at 4°C. Serum was collected and stored at -20°C until 

analysis for estradiol-17β (E2), and Insulin like growth factor-I (IGF-I). These time points 

represented early, middle and late stages in the life of the test subjects, which mirrors the lifespan 

of animals used for commercial pork production in the U.S. When considered in the context of 

the CBSA approach used in this Draft Risk Assessment, these time points roughly correspond to 

the Perinatal (July) and Juvenile (October and January) developmental nodes, as most pigs used 

for food are slaughtered before they reach sexual maturity. July and October blood samples were 

collected from the cranial vena cava. In January, blood and urine samples were harvested postmortem. Pigs were individually identified by numbered ear tags, which did not reveal to 

caretakers or personnel taking samples whether the animals were the progeny of clones or 

comparators. 

c. Meat Composition, Carcass Characteristics, and Meat Quality Assessments for 

Clones, Comparators, and Progeny. 

As in standard US pork production, swine were slaughtered when they reached approximately 

122 kg in weight. Samples were collected from the latissimus dorsi muscle (the muscle that 

comprises the large, round-shaped muscle next to the vertebrae end of the ribs that makes up the 

boneless portion of the “rib eye” roast or steak), frozen, and held at -20o

C for approximately two 

weeks prior to shipping to a commercial laboratory for compositional analysis. Samples were 

identified by a nine digit code so that personnel at the analytical laboratory did not know which 

samples belonged to comparators, clones, or progeny of clones. Muscle samples (approximately 

0.45 kg, deboned) were held frozen at -20o

C for three to 20 days after receipt at the laboratory 
Appendix F: The Viagen Dataset F-11 

Animal Cloning: A Risk Assessment DRAFT

before being partially thawed (10 – 18 ̊C overnight) and homogenized using a commercial grade 

meat grinder with a #12 blade and passed through a 1/8 inch screen. Homogenized meat samples 

were analyzed for fatty acid and amino acid profiles, cholesterol, vitamins B6, B12, niacin, 

calcium, iron, phosphorus, and zinc. Analyses were carried out according to the Association of 

Analytical Communities International (AOAC) methods for amino acids (982.30), calcium, iron, 

phosphorus and zinc (965.17 and 985.01), cholesterol (994.10), fatty acid profile (996.06), niacin 

(944.13), vitamin B12 (952.20) and vitamin B6 (961.15). With the exception of vitamins B6, B12, 

niacin, and cholesterol, values were reported as percentages. Vitamin B6, niacin and cholesterol 

were reported as mg/100 g of sample, and vitamin B12 was reported as mcg/100 g of sample. 

Percentages were based on grams of nutrient per 100 grams of homogenized meat sample. 

Economically important traits for the swine carcass evaluation include animal live weight, 

dressing percentage, carcass length, loin muscle area, backfat depth, extent of muscling, and 

firmness. These are described in the following paragraphs. 

The normal range for dressing percentage90 is 68-77 percent (average of 72 percent) with a 

minimum carcass length of 76.2 cm. The minimum acceptable area for loin muscle is 11.4 cm2

and the maximum back fat thickness over the last rib (10th or 11th rib) is 3.8 cm.91 

Three degrees of pork carcass muscling are recognized in the pork grading standards or USDA92

carcass muscling score: muscle score #1 = thin (inferior), muscle score #2 = average, and muscle 

score #3 = thick (superior). Scores of either 2 or 3 are considered acceptable under USDA 

inspection standards. Muscle firmness is a subjective score determined by visual observation and 

physical handling of the meat. Muscle firmness is measured on a three-point scale: score #1 = 

soft, score #2 = firm, and #3 = very firm. As with muscling score, a firmness score of 2 or 3 is 

considered desirable. 

C. Results 

1. Study 1 

a. Survival 

Seven clones and 16 comparators were raised by Viagen Inc. The seven clones began the study 

at 50 days of age, and all seven survived until the end of the observation period (approximately 

195 days of age); however, two clones were euthanized due to chronic health problems at the end 

 

90 Dressing percentage refers to the proportion of the live animal that ends up as the carcass used for food 

production, or Carcass Weight / Live Weight X 100. 

91 http://www.animalrangeextension.montana.edu/Articles/Swine/2-Det-quant.htm 92 http://ars.sdstate.edu/AnimalEval/Swine/SwineGrade.htm 
Appendix F: The Viagen Dataset F-12 

Animal Cloning: A Risk Assessment DRAFT

of the study. Thus, five clones were slaughtered as per normal industry practice. One of these 

clones was diagnosed with a lung adhesion post mortem, which, by USDA standards would have 

led to condemnation of the carcass. Similarly, 15/16 comparators reached the end of the 

observation period (approximately 171 days of age), with one comparator euthanized 

approximately four months prior to the end of the study because of a chronic respiratory 

condition. 

Because litters of clones were born approximately six weeks apart, they were separated into two 

groups of animals and matched with comparators born at approximately the same time. In the 

first group, five clones and 11 comparators were raised and studied. In this first group, one clone 

(#21, farrowed 10/13/03) and one comparator (#025435) did not thrive. Clone #21, a Duroc, was 

euthanized at the end of the study (May 27, 2004) and was not included in the final carcass 

evaluations because of low weight. However, hematology and clinical chemistry values for this 

clone were used for analysis of health data. Non-clone comparator #025435 was euthanized on 

December 4, 2003 because of a respiratory condition that was not improving. 

Viagen raised two clones and five comparators in the second group. Clone #23, a Hamline, 

farrowed on 11/23/03, was described as a “poor doer” and was euthanized at the end of the 

study. All comparators from this group survived to slaughter. 

b. Animal Health, Growth, Blood Clinical Chemistry, Hematology, and Urinalysis 

Clones were smaller on average at birth than their conventionally bred counterparts (1.1 vs. 1.7 

kg) (Table F-2). By the start of the test period (~50 days), however, clones were similar in 

weight to their comparators (16.4 vs. 15.3 kg). 

Animal health records indicated that six of the seven clones developed scours (severe diarrhea) 

approximately two months after birth, which would have been shortly after they were moved 

from the biosecure environment. The affected clones were #18, 19, 20, 21, 22 and 23. One of 

these animals (Clone #22) was diagnosed at approximately four months of age with influenza 

and secondary bacterial septicemia, was periodically treated with antibiotics throughout the 

experimental period, and was eventually euthanized at the end of the study. One of the 

comparator animals (#025435) was diagnosed at approximately one month of age with influenza 

and secondary bacterial septicemia. This animal was periodically treated with antibiotics 

throughout the experimental period and was eventually euthanized. The clones weighed less at 

slaughter and took an average of 27 days longer to reach the target slaughter weight. As a result, 

the calculated average daily (weight) gain (ADG) for clones was lower than for comparators 

(0.63 vs. 0.93 kg/day). The clones were slaughtered later than comparators because of the greater 

length of time needed for them to achieve the target weight, and also because the slaughter 
Appendix F: The Viagen Dataset F-13 

Animal Cloning: A Risk Assessment DRAFT

facility could only accommodate them on a few specified days. This latter point was because 

clones are currently withheld from the food supply, and the facility preferred to slaughter the 

clones on days when no other swine were being processed, in order to prevent accidental mixing 

of the clones with conventional animals. 

Table F-2: Comparison of the Body Weight (kg) of Barrows Derived by Somatic Cell Nuclear Transfer 

(Clones) or Conventional Breeding (mean ± standard deviation)

Comparators Clones 

 Age Body Weight Age Body Weight 

Birth weight — 1.72 ± 0.28a 

(n=15) — 1.13 ± 0.09b (n=4) 

Post-weaning 22.4 ± 4.81 

(n=15) 

15.3 ± 5.99 

(n=15) 

21.2 ± 1.81 

(n=4) 

17.0 ± 3.40 

(n=4) 

End of Experiment 77.9 ± 5.62a 

(n=15) 

128.9 ± 7.26 

(n=15) 

88.5 ± 8.16b 

(n=4) 

122.2 ± 8.93 

(n=4) 

Note: one comparator is not included because it was euthanized at about 1 month of age. Three clones are not 

included due to low weight. 

As previously mentioned, clones and comparators were reared under different conditions prior to 

starting the experiment. Clones were delivered by C-section, did not receive colostrum, were fed 

a commercial milk replacer, were castrated at an older age, and were raised in a biosecure 

environment prior to initiation of the study. The clones were then housed along with the 

comparator animals in an environment similar to commercial swine facilities, where they were 

exposed to a typical range of pathogens. The comparators were farrowed vaginally following 

natural initiation of labor in a farrowing house similar to commercial swine operations, and the 

progeny were allowed to nurse off their dams. This disparity likely accounts for some of the 

differences in growth and health observed. The sudden change from a biosecure environment, 

with a low immune challenge, to a more conventional barn with a high immune challenge placed 

considerable stress on the immune systems of the young clones. This stress likely led to the 

higher incidence of scouring in this group, and may have resulted in decreased food consumption 

or absorption, and increased energy expenditure to combat illness. 

Body temperature, heart and respiration rates were measured in clones, but not in comparators. 

Because of the absence of comparator data, observations for these measurements are put in the 

context of common veterinary practice or reference texts or manuals. 
Appendix F: The Viagen Dataset F-14 

Animal Cloning: A Risk Assessment DRAFT

Table F-3: Average Body Temperature, Heart Rate, and Respiration Rate for Neonatal Clones During First 

Eight Days After Birth 

Day Temperature ( ̊F)1 Heart Rate (beats/min) Respiration Rate (breaths/min) 

1 100.38 ± 0.18 194.33 ± 1.42 87.56 ± 0.71 

2 100.80 ± 0.17 184.22 ± 0.96 79.61 ± 0.53 

3 101.40 ± 0.19 179.96 ± 0.92 75.72 ± 0.53 

4 101.43 ± 0.19 177.30 ± 1.03 72.52 ± 0.59 

5 101.53 ± 0.21 177.28 ± 1.23 71.81 ± 0.71 

6 101.41 ± 0.23 177.94 ± 1.23 71.06 ± 0.71 

7 101.59 ± 0.22 179.00 ± 1.19 70.29 ± 0.79 

8 101.89 ± 0.21 178.11 ± 1.18 72.34 ± 0.68 

1

 Temperatures were recorded and analyzed in Fahrenheit. Standard deviations are not convertible to Celsius. 

Body Temperature (BT). Clones #18, 19, 20, 22, and 24 were measured for BT 2-9 times per 

day for the first eight days of life (no records on BT for other clones were available). Body 

temperature increased over this eight day period from an average of 100.38 ± 0.18oF (37.99 ̊C) 

on Day 1 to 101.89 ± 0.21oF (38.83 ̊C) on Day 8. The low BT was 97.7o F (36.5 ̊C), and 

occurred in clones #22 and 24 early in the study (Days 1 and 2 post farrowing). The high 

temperatures were 104.6oF (40.33 ̊C), 103.8oF (39.89 ̊C), and 103oF (39.44 ̊C) and occurred in 

one clone (#24), twice on Day 1, 1, and 8 respectively. The Merck Manual’s Reference range for 

swine BT is 101-103°F (38.33 – 39.44 ̊C). The BT indicates an apparent appropriate response to 

autonomous BT regulation (adjustment to post-natal environment) after delivery from the uterus 

where BT is not self-regulated, but controlled by the intra-uterine environment. As mentioned in 

Chapter V, newborn pigs typically cannot regulate their own BT. Generally, it is recommended 

to maintain the environment of newborn pigs at a temperature of 86-93 ̊F (30-34 ̊C) for the first 

week to allow for this adaptation period (Merck Veterinary Manual Online). 

Heart rates (HR). The heart rates (HRs) of clones also were measured 4-12 times per day for 

the first eight days of life. Heart rate decreased over time from 194.33 ± 1.42 to 178.11 ± 1.18 

beats per minute (bpm). The highest value was 220 bpm for clone #18 on Day 1; the lowest 

value is 100 bpm for clone #22 on Day 1. This decrease is an appropriate response for neonates. 

Heart rates decrease with age as fetal circulation (ductus venosus,93 foramen ovale,94 and ductus 

arteriosus95 closure) normalizes and the neonate develops autonomic control (Medline, Medical 

 

93 The ductus venosus carries oxygenated blood from the umbilical vein to the caudal vena cava, bypassing the fetal 

liver. 

94 The foramen ovale is a hole between the right and left atria in the fetal heart, for the purpose of by-passing the 

fetal lungs in utero. 95 The ductus arteriosis carries oxygenated blood from the main pulmonary artery to the descending aorta, bypassing 

the fetal lungs. 
Appendix F: The Viagen Dataset F-15 

Animal Cloning: A Risk Assessment DRAFT

Encyclopedia96). There were no values from the non-clone group for direct comparison. These 

values should be evaluated with caution because the very act of ausculting (listening to) the 

thorax of an animal that is seldom handled can raise HR artifactually. In fact, it is possible that 

the reduction in HR over time represents the pigs becoming more accustomed to handling. Data 

on HR for non-clone neonatal swine are not routinely reported; however, one study (Foster et al. 

2001) recorded HR of day-old piglets before and after insertion of a breathing tube. Prior to 

insertion of the tube, average HR was 190.75 ± 36.45 bpm. In another study (Aaltonen et al. 

2003) examining the effects of mecomium aspiration and asphyxia in piglets aged 10 to 12 days. 

Average HR for the control (untreated) group was 203 ± 23 bpm. Given these data for 

comparison, the HR of clone pigs during the first eight days of life fell within or slightly below 

the range of non-clone pigs of similar ages. 

Respiration. The same clones as above were evaluated 5-12 times per day for the first eight 

days of life. Respiration rates decreased over time, ranging from 87.56 ± 0.71 breaths/minute on 

Day 1 to 70.29 ± 0.79 breaths/minute on Day 7. As with HR, data on swine neonatal respiration 

rates are not commonly reported. However, one study (McDeigan et al. 2003) measured 

respiratory responses in pigs from 3 to 7 days of age before and after exposure to either saline 

(control) or Escherichia coli endotoxin. Average pre-treatment respiration rate for control piglets 

was 44 ± 5 breaths/minute in this study, and thus considerably lower than the average respiration 

rates of the clones in the Viagen study. The lowest daily average respiration rate among the 

neonatal clones in the Viagen study was 69.6 breaths/min on day 7 (Clone #18). The cautions 

mentioned for interpreting HR data should also be applied to interpreting respiration rates. Also, 

animals delivered by C-section have a transient tachypnea (rapid breathing) because the 

pulmonary surfactant and fluid is not distributed and squeezed out of the lungs as it would in a 

vaginal birth (Medline, Medical Encyclopedia). 

Daily Health Records. Clones #18, 19, 20, 21, 22, and 23 all had scours (severe diarrhea) while 

on study. Clone #18 herniated a loop of bowel post-castration, which was repaired, and the pig 

responded well. This pig had an inguinal hernia, sometimes referred to as a “busted pig,” which 

is not uncommon in swine (Gatphayak et al. 2005). As previously mentioned, clone #21 was 

described as a “poor doer” (unthrifty, poor weight gain) and was euthanized because of low body 

weight. He had chronic scours and a skin abscess on his neck. Clone #22 had scours twice while 

on study. He developed sepsis (a blood infection), and had a low body weight at slaughter. Clone 

#23 had many health problems, including scours, respiratory disease, cyanotic (bluish) skin 

color, and grew slowly. He was euthanized because of his low body weight. Clone #24 had some 

respiratory difficulty toward the end of the study but responded to therapy. One comparator pig 

(#025435) was euthanized for non-responsive diarrhea and pneumonia early in the study. 

 

96 http://www.rashaduniversity.com/mrashad/neonphys.html 
Appendix F: The Viagen Dataset F-16 

Animal Cloning: A Risk Assessment DRAFT

Another comparator (#027446) had one reported health problem of a swollen dewclaw that 

responded to therapy. 

Although these clones had more health problems than comparators, it is important to note that 

differences in early rearing are likely to have contributed to these outcomes. First, the clones 

were not suckled on their dams, but were fed commercial milk replacer. This deprived them of 

the passive immunity provided by transfer of maternal antibodies in the colostrum. Secondly, 

pigs raised in a biosecure environment would not have developed antibodies to common 

environmental pathogens, and would be at increased risk of developing infections until their 

immune systems adjusted to the environmental challenges when they were moved to lessbiosecure conditions at day 50 of age. The comparators, on the other hand, were born in 

conditions equivalent to commercial swine breeding, and were allowed to suckle on their dams, 

thereby allowing their immune systems to develop along more conventional lines. We also note 

that even though some of the clones developed illness, most (5/7) were able to respond to 

treatment. Further, the hernia observed in clone #18 is not unique to clones, but is considered 

common among male pigs in the general swine population (Merck Veterinary Manual Online). 

Clinical Chemistry and Pathology. Clinical pathology is the term generally used for laboratory 

findings that includes clinical chemistry, hematology, and urinalysis among others. As described 

in Appendices E and G, clinical chemistry and hematology responses are best evaluated in the 

context of the whole animal, including its age, species, breed, husbandry, geographic location, 

reproductive status, and the laboratory performing the analysis. Laboratory findings complement 

the subjective physical diagnosis of the patient by providing objective information for the 

process of differential diagnosis, monitoring treatment, and formulation of a prognosis. 

“Abnormal” laboratory measurements and examinations are often defined as those values lying 

outside the limits of the reference range. Determining what constitutes “normal” is more 

complex than simply comparing an individual value to a reference range derived from a sample 

of a representative population. In this study, the firm provided serum chemistry data, white cell 

counts, red cell counts, and urinalysis using a rapid “dipstick” test. Because the number of pigs 

in the study was low, a statistical analysis of the comparator ranges was not appropriate. Values 

from clones were compared to those from the comparators. 

Across all three sampling periods, 89 percent (281/315) of clones’ hematology measurements 

were within the range of the comparator population. At the beginning of the study, 80 percent of 

the clones’ hematology values (84/105) were within the range of the comparators. This 

percentage improved at the next sampling period, approximately two months later, such that 88 

percent of the clones’ values (92/105) were within range. For clinical chemistry, 76 percent of 

the clones’ values (236/315) were within the comparator range across all time periods. As with 
Appendix F: The Viagen Dataset F-17 

Animal Cloning: A Risk Assessment DRAFT

hematology, the percentage of clone values within the range of comparators was lower at the 

beginning of the trial, but this ratio improved as the animals grew. At the beginning of the study, 

63 percent (66/105) of the clones’ values were within the comparator range. By the second 

sampling period, 83 percent (88/105) of the clones’ values were within the comparator range. By 

the end of the trial it appeared that clones for the most part had stabilized, with 84 percent of the 

hematology values and 98 percent of clinical chemistry values within the comparator range 

(Table F-4). 

Table F-4: Clone Hematology and Clinical Chemistry Values Identified As Outside The Comparator Range 

Sampling 

Period 

Number 

of 

Variables 

Number of 

Observations 

Number 

of 

Clones 

Number 

of 

Out of 

Range 

Values 

Percentage 

Out of 

Range 

Number 

of In 

Range 

Values 

Percentage In 

Range 

1st

hematology 21 105 5 21 20% 84 80% 

1st clinical 

chemistry 21 105 5 39 37% 66 63% 

2nd

hematology 21 105 5 13 12% 92 88% 

2nd clinical 

chemistry 21 105 5 17 16% 88 84% 

3rd

hematology 21 105 5 17 16% 88 84% 

3rd clinical 

chemistry 21 84 4 23 2% 82 98% 

Seven comparators had elevated WBC counts with no apparent health problem. Clone #20 had a 

high WBC (32,000 cells/μl) during an episode of diarrhea. This is an appropriate clinical 

response. One clone (#18) and one comparator (#025457) had low platelets at the end of the 

study. Red blood cell size for both clones and comparators indicated a microcytosis (low MCV). 

Because the clones and comparators were similar, this value is likely related to the laboratory 

and not a difference in health. 

Clone #21 had elevated BUN levels at the start and middle of the study and elevated liver and 

muscle enzymes at the start and end of study. He did not show an increase in WBC, and had a 

slight increase in urine glucose at the end of the study. These findings are consistent with a 

“poor-doing” animal. Animals in a negative energy balance (due to inadequate nutrition, food 

intake or utilization) will start to mobilize muscle protein for energy once their body energy 

stores are depleted. Blood urea can elevate with increased protein digestion or mobilization. CK 

(an intracellular enzyme for muscle) will also increase with muscle break down. The elevated 
Appendix F: The Viagen Dataset F-18 

Animal Cloning: A Risk Assessment DRAFT

liver enzymes indicate some insult to the liver. The cause of these health problems is not evident 

from the data presented. 

Clone #23 exhibited an increase in blood glucose at the start of the study (January) and bile acids 

at the mid-point of the study (March). By the end of the study (May), clone #23 had glucose and 

bile acid values within the range of the comparators. Clone #23 was also a “poor doer” (poor 

weight gain), but had no other clinical pathology abnormalities evident. Although increased 

blood glucose levels may indicate metabolic disturbances such as diabetes, the increased glucose 

levels in this study (in blood and once in urine) appear to have been transient and resolved 

spontaneously. They therefore do not appear to be indicative of an insulin deficiency or 

resistance. Bile acids are an indicator of liver function (see Appendix G). If the bile acid value is 

consistently elevated, liver insufficiency may be indicated. However, bile acid levels are also 

dependent on when the animal was fed, and increase after a meal. Because information on the 

relationship between blood draws and feeding times was not provided, no conclusion can be 

made about the single measurement which on its own does not suggest a health problem. 

Urinalysis. Urine values were determined by dipstick. There was apparently no Specific Gravity 

(SG) determination using a refractometer or sedimentation evaluation, so SG was measured only 

by the dipstick method, which is less accurate than these other methods. With these caveats in 

mind, two clones and one comparator had glucose in their urine. The percent of clones and 

comparators with blood and protein in their urine were similar. Seven out of 15 urine samples 

from clones were positive for blood (47 percent) vs. 16/ 44 (36 percent) samples from 

comparators. For protein, 6/15 samples from clones (40 percent) vs. 20/44 (45 percent) samples 

from comparators were positive. Color, turbidity, SG, bilirubin, pH, ketone, nitrite, and 

leukocytes were all similar between clones and comparators. The presence of blood in the urine 

may have been due to the fact that these samples were taken after the pigs had been slaughtered. 

Organ weights. The clones’ body weights were lighter on average than comparators at slaughter 

(117 vs. 128.8 kg). Clones #21 and 23 were so light (86.2 and 89.8 kg, respectively), they were 

not included in the organ weight analyses. Kidneys were smaller for clones vs. comparators on a 

percent of body weight basis. However, heart, liver, lungs, and spleen were similar as a percent 

of body weight for clones vs. comparators. With no clinical chemistry indicators of renal 

insufficiency in clones, the smaller kidney weights are interesting but not conclusive. These 

findings indicate no appreciable differences between clones and comparators in organ size as a 

percent of body weight. 

IGF-1 and E2. In general, the clones had lower levels of IGF-I than the comparators after birth 

and before slaughter (Table F-5). However, the values were within the range observed in the 

comparator group, except for one clone (Clone #19), that had lower levels at the beginning of the 

study. This clone had levels of IGF-I at the limit of assay sensitivity (low value). Before 
Appendix F: The Viagen Dataset F-19 

Animal Cloning: A Risk Assessment DRAFT

slaughter, clones #19 and #20 presented levels of IGF-I that were close or below the detectable 

values by the analytical method. 

Although, critical illness can modify and has an important impact on the human endocrine 

system (Sartin et al. 1998), these animals did not show health-associated conditions at the time of 

sampling that could explain the low levels of IGF-I. Additionally, the IGF-I axis (the glands, 

organs and hormones associated with IGF-I), as well as cortisol and gonadal steroids, are 

endocrine determinants of the growth potential of animals (Mauras and Haymond 2005). 

Nevertheless, the body weights of the animals with low IGF-I (clones #19 and #20), were not 

lower than the control animals. This observation suggests that the reduction in IGF-I levels seen 

before slaughter for these two clones is not biologically important and perhaps is associated with

the handling of samples or time of sampling relative to when the animals ate. In addition, limited 

information has been provided describing the handling and storage of samples in the current 

experiment, and hemolysis and storage of blood samples have been shown to reduce detectable 

concentrations of hormones using the assay technique employed in this study (Reimers et al 

1991). Therefore, the possibility that the low levels of IGF-I in clones #19 and #20 may be due to 

hemolyzed serum used for the hormone analysis cannot be excluded. 

Clones had slightly lower levels of E2 than the comparator group; this trend was observed during 

the perinatal period and before slaughter (Table F-6). However, the E2 values of clones were 

within the range of the comparators, but one comparator animal (#025461) displayed elevated 

levels of E2, as a result, the mean values of the comparator group increased. This animal also 

presented the highest rate of weight gain throughout the study. The body weight at slaughter was 

higher than the other comparators. If the data for this animal is removed from the comparator 

group, the apparent difference between animal clones and comparators is considerably 

diminished. Considering the above observations it can be concluded that the slight reduction in 

the levels of E2 in the clones was not biologically relevant. 

Table F-5: Estradiol-17β and IGF-I Levels in Swine Clones and Comparators 

Collection 

period Estradiol-17β (pg/ml) IGF-I (ng/ml) 

 Clones Comparators Clones Comparators 

Start 9.4 ± 0.4 (n=5) 16.10 ± 1.4 (n=15) 

11.48 ± 1.6 (n=14) 142 ± 23.4 235.9 ± 6.7 (n=15) 222.03 ± 27.6 (n=14) 

End 15.9 ± 0.9 (n=5) 26.7 ± 2.4 (n=13) 64.02 ± 12.8 93.9 ± 7.2 (n=13) 
Appendix F: The Viagen Dataset F-20 

Animal Cloning: A Risk Assessment DRAFT

Table F-6: Estradiol-17β and IGF-I Values for Individual Clones 

 Start of Study End of Study 

Clone ID Estradiol-17β

(pg/ml) 

IGF-I 

(mg/ml) 

Estradiol-17β

(pg/ml) 

IGF-I 

(ng/ml) 

18 6.82 45.80 17.84 91.60 

19 10.91 114.50 18.71 22.90 

20 10.21 68.70 16.06 22.90 

21 6.27 22.90 13.76 68.70 

22 9.48 137.40 11.05 137.40 

23 11.43 0 9.80 91.60 

24 9.74 338.17 12.87 160.30 

Conclusions for Health and Growth of Viagen Clones. The results of this study are limited by 

the small number of animals and the design of the experiment. However, results of this 

experiment indicate that even though the clone barrows were subjected to a significant 

immunological challenge after moving from more biosecure conditions to more standard housing 

conditions, most clones were able to respond appropriately to this stress. This immunological 

challenge potentially slowed the growth of clones early in this experiment and thus may have 

resulted in a delay in their maturation. 

c. Carcass Characteristics 

Carcass characteristics were provided on the four Hamline clones and 15 comparator barrows 

followed in Study 1; these are summarized in Table F-7. Hot carcass weights97 averaged 85.91 

and 90.68 kg for clone and comparator barrows, respectively, with the exception of the animals 

that were excluded (see discussion in next paragraph). Carcass lengths were 82.4 and 84.5 cm for 

clones and comparators, respectively. Dressing percentages were 70.1 and 70.2 percent for 

clones and comparators, and were similar across breed groups. Backfat thickness over the first 

rib, tenth rib, last rib, and lumbar vertebra were slightly greater for comparators than clones 

which may, in part, be due to the heavier body weight of comparator barrows at the time of 

slaughter. Qualitative characteristics including USDA carcass muscle score, firmness, and 

marbling were similar across groups and are illustrated in Table F-7. All animals received score 

2 for carcass muscle. All of the clone and comparator barrows had marbling scores of either 1 or 

2. 

 

97 Hot carcass weight refers to the weight of the eviscerated carcass post exsanguination, but prior to chilling. 
Appendix F: The Viagen Dataset F-21 

Animal Cloning: A Risk Assessment DRAFT

The Duroc clone barrow carcass was condemned at slaughter due to a lung adhesion, and thus 

data relating to growth and carcass characteristics from this animal were not included for these 

variables. The rationale for excluding the carcass for this analysis is that in some cases, lung 

adhesions are due to bacterial infection, and animals fail to thrive, (i.e., their growth and carcass 

characteristics are adversely affected). Two other clones (#21 and 23) were approximately 45.45 

kg lighter than any of the other animals in the experiment at the time of slaughter, and for this 

reason were excluded from carcass evaluation. 

Measurements of pH at 24 hours post-slaughter on the longissimus dorsi muscle were similar. 

Loin eye area for meat cuts for clones and comparators were quite similar at 116.33 ± 10.16 and 

11.76 ± 11.18 cm, respectively. 

Table F-7: Comparison of the Carcass Characteristics of Barrows Derived by Somatic Cell Nuclear 

Transfer (Clones) or Conventional Breeding (Mean ± standard deviation) 

Comparators 

(n=15) 

Clones 

(n=4) 

Hot Carcass Weight (kg) 90.5 ± 6.2 85.7 ± 6.26 

Carcass Length (cm) 84.5 ± 2.7 82.4 ± 1.5 

Dressing Percentage (%) 70.2 ± 1.4 70.1 ± 0.8 

Back Fat Thickness (mm) 

 First Rib 38.7 ± 3.1a 35.3 ± 2.1b

 Tenth Rib 22.2 ± 4.9 18.5 ± 3.1 

 Last Rib 23.3 ± 3.4 20.5 ± 4.7 

 Last Lumbar 21.0 ± 3.1 17.3 ± 3.2 

Loin Eye Area (cm2) 45.8 ± 4.0 44.0 ± 4.4 

Longissimus pH at 24 hours 5.7 ± 0.1 5.6 ± 0.1 

Carcass Muscle Score 2.2 ± 0.4 2.0 ± 0.0 

NPPC Quality Scores 

 Color 2.7 ± 0.6 3.0 ± 0.0 

 Marbling 1.9 ± 0.5 1.5 ± 0.6 

 Firmness 2.9 ± 0.9 3.5 ± 0.6 

a, b Values with different superscripts are significantly different (P<0.05) 

d. Meat Composition Analysis for Clones and Comparators 

Meat composition data were available for the five clones (four Hamline and one Duroc) and 15 

comparator animals (all Hamline). Because there were no differences between the Duroc and 

Hamline clones, data were pooled. All of the values examined (amino acids, fatty acids, 

cholesterol, nutritionally important vitamins and minerals (see discussion in Chapter 6) were 

remarkably similar, and no biologically relevant differences were noted (Table F-8). 
Appendix F: The Viagen Dataset F-22 

Animal Cloning: A Risk Assessment DRAFT

Table F-8: Comparison of Nutrient Concentrations in Meat from Swine Clones 

and Comparators from Experiment 11

Component Clones 

mean + std. dev. 

Comparators 

mean + std. dev. 

Amino acids (g) 

Alanine 1.26 ± 0.04 1.30 ± 0.04 

Arginine 1.41 ± 0.03 1.47 ± 0.04 

Aspartate 2.55 ± 0.28 2.43 ± 0.19 

Cystine 0.25 ± 0.03 0.26 ± 0.02 

Glutamate 3.41 ± 0.11 3.46 ± 0.09 

Glycine 0.98 ± 0.04 1.02 ± 0.10 

Histidine 0.99 ± 0.05 1.03 ± 0.05 

Isoleucine 1.04 ± 0.05 1.05 ± 0.03 

Leucine 1.74 ± 0.05 1.79 ± 0.04 

Lysine 1.91 ± 0.06 1.96 ± 0.04 

Methionine 0.54 ± 0.06 0.58 ± 0.03 

Phenylalanine 0.86 ± 0.02 0.89 ± 0.02 

Proline 0.85 ± 0.03 0.90 ± 0.06 

Serine 0.90 ± 0.03 0.92 ± 0.02 

Threonine 1.11 ± 0.04 1.14 ± 0.03 

Tyrosine 0.77 ± 0.02 0.79 ± 0.02 

Valine 1.10 ± 0.05 1.12 ± 0.04 

Fatty Acids2 (g) and Cholesterol (mg) 

14:0 (Myristic acid) 0.09 ± 0.06 0.05 ± 0.03 

16:0 (Palmitic acid) 1.31 ± 0.82 0.95 ± 0.49 

16:1 (Palmitoleic acid) 0.09 ± 0.04 0.14 ± 0.05 

17:0 (Margaric acid) 0.01 ± 0.01 0.00 ± 0.01 

17:1 (Margaroleic acid) 0.01 ± 0.01 0.00 ± 0.01 

18:0 (Stearic acid) 0.66 ± 0.41 0.55 ± 0.27 

18:1 (Oleic acid) 1.84 ± 0.84 1.49 ± 0.50 

18:2 (Linoleic acid) 0.26 ± 0.08 0.19 ± 0.06 

18:3 (Linolenic acid) 0.01 ± 0.01 0.00 ± 0.01 

20:0 (Arachidic acid) 0.01 ± 0.01 0.00 ± 0.01 

20:1 (Gadoleic acid) 0.05 ± 0.03 0.04 ± 0.02 

20:2 (Eicosadienoic acid) 0.01 ± 0.01 0.01 ± 0.01 

22:6 (Docosahexaenoic acid) 0.02 ± 0.03 0.01 ± 0.01 

Cholesterol (mg) 55.5 ± 6.95 52.81 ± 2.69 
Appendix F: The Viagen Dataset F-23 

Animal Cloning: A Risk Assessment DRAFT

Component Clones 

mean + std. dev. 

Comparators 

mean + std. dev. 

Minerals (g) 

Calcium 0.004 ± 0.0003 0.005 ± 0.003 

Phosphorus 0.20 ± 0.01 0.21 ± 0.01 

Iron 0.001 ± 0.0003 0.001 ± 0.001 

Zinc 0.002 ± 0.0003 0.001 ± 0.0003 

Vitamins 

Niacin (mg) 10.90 ± 0.83 11.16 ± 1.58 

B6 (mg) 0.41 ± 0.09 0.48 ± 0.12 

B12 (mcg) 0.21 ± 0.28 ND4 

1

Data expressed as quantities per 100 g of homogenized meat. 

2

 Data presented reflect those fatty acids with detectable levels in pork. 3

 Actual standard deviation was < 0.001 

4

 ND = not detected 

Conclusions for Carcass Characteristics and Meat Composition of Clones. No remarkable 

differences were observed between clones and comparators for any of the characteristics 

evaluated. The small differences in backfat thickness and marbling are likely due to the lighter 

weight of clones vs. comparators at slaughter. Differences in meat nutrient composition were 

very small and likely not biologically relevant. The lack of biologically relevant differences in 

the food composition values between muscle of swine clones and comparators supports the 

conclusion that there is no additional food consumption risk from the consumption of muscle 

from swine clones compared to their conventional counterparts. 

e. Semen and Breeding Evaluation 

Sperm concentration, percent total motility (percent of sperm in motion), percent progressive 

motility (evaluates forward movement), and number of head/tail abnormalities were similar for 

the four clones and the comparator boars. The clones did not appear to have a reduction in semen 

quality relative to the comparator boars. The lowest post-shipment semen concentration, percent 

motility, and percent progressive motility were observed in the Duroc line comparator boars. 
Appendix F: The Viagen Dataset F-24 

Animal Cloning: A Risk Assessment DRAFT

Table F-9: Evaluation of Semen From Individual Clones and Comparators 

Animal Concentration 

(sperm/ml x 109

) 

Total 

Motility 

Progressive 

Motility 

Head 

Abnormalities 

Tail 

Abnormalities 

Clone 2 5.13 82.34% 71.39% 58.00% 90.00% 

Clone 3 7.10 79.95% 71.92% 81.75% 91.00% 

Clone 5 5.23 86.18% 75.75% 58.50% 87.50% 

Clone 7 4.19 79.19% 62.68% 90.50% 79.50% 

Control H498 4.51 75.86% 70.45% 66.33% 87.22% 

Control 18128 2.53 53.28% 49.42% 92.50% 81.25% 

Control 25515 3.19 77.14% 57.20% 97.25% 83.75% 

f. Farrowing Rate 

The farrowing rate for the gilts inseminated with semen from clones and comparators was 73.5 

percent vs. 62.5 percent, respectively. Both these rates are below the industry average of 

approximately 80 percent. The PigCHAMP swine industry record keeping system farrowing rate 

average for the first quarter of 2005 was 79.1 percent (Olson98 2005). It should be noted that the 

PigCHAMP rate included all parities (the number of times a sow has farrowed), and the rate 

would be expected to be lower for gilts. The lower farrowing rates of the pigs in this study could 

be attributed to many factors including the feed intake of the gilts as well as the ability of 

technicians to detect gilts in heat. Farrowing rate is generally more attributable to the female as 

opposed to the male member of the breeding pair, and takes into account such factors as 

ovulation rate (number of oöcytes released per estrus), age/parity of the sow, and environmental 

factors (Hafez and Hafez 2000). Additionally, the rate for the comparator-bred group was low 

relative to the clone-bred group, which was largely attributable to the gilts bred by one boar 

whose farrowing rate was 41.7 percent. This boar (Hamline nuclear donor) was reported to be 

five years old at the time of semen collection (relatively old for a breeding boar), which may 

have contributed to the low farrowing rate for gilts inseminated by his semen. As the clone-bred 

group had a higher farrowing rate than the comparator-bred group, and the farrowing rate of the 

clone-bred group was only slightly lower than industry averages, cloning does not appear to 

negatively impact AI-based boar reproductive performance. 

 

The mean litter size for the progeny of the clone boars was 10.94 pigs and the median was 11.5 

pigs. For the pigs from the comparator boars, the mean litter size was 11.76 pigs and the median 

was 12.0 pigs. The mean litter size for the U.S. in the first quarter of 2005 according to 

 

98 www.pigchamp.com/overview5.asp 
Appendix F: The Viagen Dataset F-25 

Animal Cloning: A Risk Assessment DRAFT

PigCHAMP records was 10.66 pigs per litter (Olson 2005). Although the comparator boars had a 

higher mean litter size, both groups were near the U.S. average for litter size. Litter size appeared 

to be more variable in gilts inseminated by clones: 11 percent of the litters from the clone boars 

had more than 14 pigs, whereas none of the comparator litters had more than 14 pigs. Further 

analysis of litter size showed that the clone boars also had a higher frequency of litters with less 

than ten pigs. Approximately 27.8 percent (10/36) of the litters from the clone boars had fewer 

than ten pigs in the litter. For the comparator group, only one litter (out of 25 litters or 4 percent 

of all comparator litters) had fewer than ten pigs. Many factors can affect litter size in pigs 

including the estrus cycle in which the gilts are bred, genetics, nutrition, management, 

environment, and ovulation rate of the gilt (Aherne and Kirkwood 1985). Although many of 

these factors were controlled (nutrition, parity, management, and environment), other factors 

besides cloning cannot be ruled out as contributors to the wider distribution of litter size for the 

litters from the clones. Finally, as semen characteristics appear to be similar between the clones 

and their comparators, the differences in litter sizes were most likely due to gilt or uncontrollable 

management variation such as breeding cycle or feed intake. 

Table F-10: Results of Breeding Boar Clones and Comparators 

Boar 

Number 

of Gilts 

Bred 

Number 

of Gilts 

Pregnant 

Day 30 

Percentage 

of Gilts 

Pregnant 

Day 30 

Number 

of Gilts 

Pregnant 

Day 110 

Percentage 

of Gilts 

Pregnant 

Day 110 

Number 

of Gilts 

Farrowed 

Percentage 

of Gilts 

Farrowed 

Clone 2 12 9 75 7 58.33 7 58.33 

Clone 3 12 10 83.33 10 83.33 10 83.33 

Clone 5 12 9 75 8 66.67 8 66.67 

Clone 7 13 11 84.62 11 84.62 11 84.62 

Control 

H498 12 7 58.33 7 58.33 5 41.67 

Control 

18128 14 13 92.86 13 92.86 11 78.57 

Control 

25515 14 9 64.29 9 64.29 9 64.29 

Conclusions for Reproductive Performance of Clones. There were no apparent differences in 

semen quality between clones and comparator boars. Farrowing rate was slightly higher among 

gilts bred by semen from clone boars, although the difference between clones and comparators 

could be traced to the Hamline nuclear donor, which was older than any of the other boars used 

in this study. Average litter size was similar for clones and comparator boars, and farrowing rate 

and litter size for clones were similar to national averages. Therefore, reproductive performance 

does not appear to be affected by the cloning process in these animals. 
Appendix F: The Viagen Dataset F-26 

Animal Cloning: A Risk Assessment DRAFT

2. Study 2: Progeny of Clones vs. Comparators 

a. Survival 

A summary of the survival data from this study is presented in Table F-11. At the start of the 

study, there were 310 (295 made it to slaughter) live-born clone-derived progeny and 251 (243 

made it to slaughter) live-born comparator-derived progeny. The percentage of mummified pigs 

(dead, desiccated fetuses) at farrowing was 3.3, 2.8, 1.7, and 0 percent for the progeny from 

Hamline comparator, Hamline clones, Duroc comparators, and Duroc clone boars, respectively. 

In both breeds of pigs (Hamline and Duroc) the percentage of mummified pigs was slightly 

higher in the comparator group than in the clone-derived pregnancies. In this study, litters from 

Hamline clones and comparators and Duroc comparators had higher rates of mummies in their 

litters than the U.S. average (0.2 percent) (SwineReproNet)99; however, the percentage of 

mummies in the litters derived from the Duroc clone was similar to the U.S. average. A 

substantial number of pigs were lost around the time of birth, ranging from 17.0 percent-31.4 

percent, and in each breed, these losses were slightly higher in the group comprised of progeny 

derived from clones. Most of these losses were due to the categories of “weakness” or “unknown 

causes.” Further analysis of the data indicated that an entire litter of 13 progeny from a Duroc 

clone boar was lost on July 15, 2004, shortly after birth. Reasons provided for the loss of this 

litter included “unknown” (n=7) and “weak” (n=6). The accompanying animal health records 

note, however, that between July 13 and 15 sows were stressed due to both high temperature and 

humidity in the farrowing house. The records also indicate that C- section was performed on one 

of the heat-stressed gilts, and the gilt and her 13 pigs subsequently died. If these 13 progeny from 

the one litter that died are removed from the evaluation, the differences in survival rate for 

progeny from clones and from comparator boars are slight and inconsequential. 

 

99 http://www.traill.uiuc.edu/swinerepronet/paperDisplay.cfm?ContentID=6266 
Appendix F: The Viagen Dataset F-27 

Animal Cloning: A Risk Assessment DRAFT

Table F-11: Comparison of the Survival Data for Progeny of Clone or Comparator Boars 

Hampshire 

Comparator Hampshire 

Clone 

Duroc 

Comparator 

Duroc 

Clone Total 

Total pigs born 61 284 239 118 702 

 Mummies 2 (3.3%)1 8 (2.8%) 4 (1.7%) 0 (0%) 14 (2.0%) 

Died at Birth 11 (18.6%) 62 (22.5%) 40 (17.0%) 37 (31.4%) 150 (21.8%) 

 Stillborn 5 (8.5%) 21 (7.6%) 22 (9.4%) 15 (12.7%) 63 (9.2%) 

 Overlay 5 (8.5%) 27 (9.8%) 10 (4.3%) 6 (5.1%) 48 (7.0%) 

 Weak 0 (0%) 1 (0.4%) 0 (0%) 6 (5.1%) 7 (1.0%) 

 Unknown 0 (0%) 12 (4.3%) 8 (3.4%) 10 (8.5%) 30 (4.4%) 

 DDFR 0 (0%) 1 (0.4%) 0 (0%) 0 (0%) 1 (0.1%) 

 DINJ 1 (1.7%) 0 (0%) 0 (0%) 0 (0%) 1 (0.1%) 

Number of Animals 

Weaned 48 (81.4%) 214 

(77.5%) 195 (83.0%) 81 (68.6%) 538 (78.2%) 

Died Prior to 

Slaughter 0 (0%) 5 (1.8%) 7 (3.0%) 0 (0%) 12 (1.9%) 

Completed 

Experiment 48 (81.4%) 209 

(75.7%) 188 (80.0%) 81 (68.6%) 526 (76.5%) 

 Slaughtered 44 (74.6%) 181 

(65.6%) 119 (50.6%) 61 (51.7%) 405 (58.9%) 

 Comparator shipped 

to market 0 (0%) 0 (0%) 64 (27.2%) 0 (0%) 64 (9.3%) 

 Tanked at slaughter2 0 (0%) 21 (7.6%) 0 (0%) 14 (11.9%) 35 (5.1%) 

 Shipped to Perdue3 4 (6.8%) 7 (2.5%) 5 (2.1%) 6 (5.1%) 22 (3.2%) 

1

 Percentages are of total animals per group. 

2

These animals were sent for rendering because there were more progeny born than was needed for the experiment. 

3

 These animals were sent at the conclusion of the experiment to Perdue University to be enrolled in an immunology 

experiment. 

The percentage of stillborns among progeny of clones (36/394) and the progeny of the 

comparator boars (27/294) was the same (9 percent). This level is within the estimates of 

industry averages for stillborns (range 5 -10 percent (SwineReproNet)). Sixteen of the 36 litters 

from the clone boars had at least one stillborn pig (44 percent), while 13 of the 25 litters from the 

comparator boars had at least one stillborn pig (53 percent). Therefore, the frequency of 

stillborns in litters from the clone boars was lower than the comparator group. The litters of the 

clone Hamline boars had an average of 0.8 ± 0.3 rate of stillborn per litter, and were virtually 

identical to the comparator Hamline boars (0.8 ± 0.07). Similarly, the stillborn rate for the Duroc 

clone boar litters was 1.4 ± 0.4 per litter, while the rate for the comparator Duroc boar litters was 

1.1 ± 0.3 stillborns per litter. According to PigCHAMP, these rates are similar to the average 

stillborn rate of 0.93 pigs per litter for the U.S. swine industry records for the first quarter of 
Appendix F: The Viagen Dataset F-28 

Animal Cloning: A Risk Assessment DRAFT

2005 (Olson 2005). Therefore, cloning does not appear to affect the stillborn rate component of 

reproductive performance of pigs for either genetic line. 

One clone progeny pig was destroyed due to a deformity (DDFR). One comparator pig was 

destroyed due to an injury (DINJ). The destroyed pigs represent 0.25 percent and 0.3 percent for 

the progeny of the clone and comparator boars, respectively. The deformity of the destroyed 

clone progeny was not described and may more accurately fit into the “other abnormalities” 

category. Adding an additional pig to this category for the progeny of the clone boars does not 

increase the incidence rate to a level that warrants concern (see discussion on abnormalities). 

Injuries during nursing are usually due to sow overlays. Consequently, the injured pig reported in 

its own category may typically be included in the overlay category as discussed below. Adding 

this pig to the overlay category does not substantially change the frequency of overlays and 

therefore does not warrant changing the conclusion relative to overlays as discussed below. 

The rates of abnormalities in both clone and comparator derived progeny were similar to industry 

observed levels. Progeny of the clone boars had an abnormality rate of 2.5 percent (10/394), 

including anal atresia (lack of opening of the anus) (1/394, 0.3 percent), spraddle legs (leg 

weakness) (4/394, 1 percent), and “other” (5/394, 1.2 percent). Three of the 295 offspring of the 

comparator boars had abnormalities (1 percent), all of which were recorded as having spraddle 

legs. The incidence and cause(s) of spraddle legs is not well documented or fully understood, but 

it may involve several factors including genetics, management, slick flooring, mycotoxins, and a 

virus or combination of viruses (Goodband et al.1997). Furthermore, the incidence of spraddle 

legs was the same for both groups. Therefore, the occurrence of spraddle legs does not appear to 

be related to cloning. Of the two other classes of abnormalities observed in the progeny of 

clones, all have previously been reported in the literature on swine reproduction. The incidence 

of anal atresia in the general swine population has been reported to range from 0.1-1.0 percent 

(Wiedemann et al. 2005). Further investigation of the “other” category revealed that these pigs 

were from two litters. Three pigs from one litter were described as having “typical leg 

abnormalities possibly associated with overcrowding in the uterus.” These pigs were from a litter 

of 18 pigs, which would be considered large compared to industry average (Vonnahme et al. 

2002). The other two pigs were from another litter, and were described as having short legs. The 

frequency of miscellaneous abnormalities in newborn pigs has been reported by Spicer and 

coworkers (1986) as 1.2 percent, and included cleft palate, anal atresia, renal hypoplasia, 

hydrocephalus and accidental death. Because similar abnormalities have been reported in the 

swine industry at a similar frequency to that of this study, the rate of “other” abnormalities in this 

study is not a high concern. 
Appendix F: The Viagen Dataset F-29 

Animal Cloning: A Risk Assessment DRAFT

Seven of the 394 progeny (2 percent), all from the same litter (#339) derived from one of the 

clone boars were disposed of for unknown reasons. Furthermore, of the seven clone-progeny 

pigs that were categorized as “disposed of because they were weak” (2 percent), six were from 

the same litter (#339) as the pigs “disposed of for unknown reasons.” Only one other progeny of 

the clone boars was disposed of for being weak. No comparator pigs were disposed of for 

unknown reasons or because of weakness. The fact that 4 percent of the pigs from the clone 

boars were destroyed due to weakness and unknown reasons compared to 0 percent of 

comparators could be a matter for concern. However, this effect results primarily from 

observations from one litter, and the daily observation records indicate that heat stress was a 

problem in the farrowing house when this litter was delivered. Further, given most of the pigs in 

this litter were disposed on the day they were born, with only two pigs living for three days, it 

appears that heat stress of the dam and/or pigs may have contributed to the loss of this litter. The 

consequent disposal of this litter resulted in the relatively high rate (4 percent) of “unknown” and 

“weak pig” disposals for the progeny of the clone boars. The data do not, however, fully account 

for the removal and subsequent care of the pigs in this litter, and this interpretation should be 

considered preliminary. We also note that unknown disposals or weak pig disposals do not 

appear to be a problem across litters for the clone progeny group, which would be expected if 

cloning were a primary contributing factor to the incidence of weak pigs in the progeny of 

clones. 

The frequency of overlays (death due to the sow lying on top of the piglets) for pigs in litters 

from the clone boars and the litters from the comparator boars were 8.2 percent and 5.0 percent 

respectively. Industry estimates for pig deaths due to crushing by the sow are between 4.8 and 18 

percent (Lay et al. 1999). The frequencies of overlays for both groups in this study are near the 

low end of the estimated range of deaths due to crushing. Crushing has been reported to be 

related to several factors including the genetics, activity level of the sow and sow housing (Hay 

et al. 2002). Secondary factors that potentially influenced the number of pigs that die due to the 

dam lying on pigs included environmental conditions at the time of farrowing and the number of 

litters that were born on a single day. The data indicate that 315 of the 688 pigs were born on one 

of six days where the heat index was above 104oF (40 ̊C). The daily sow/litter observation 

records indicate that high temperatures in conjunction with poor cooling may have contributed to 

the number of crushing deaths, as the sows attempted to find a cooler and more comfortable 

position. The records do not differentiate between the two groups of gilts, and gilt housing was 

the same for both groups in this study. Therefore, any differences in the number of crushing 

deaths in this study were probably due to differences in sow activity. Also, the litter observation 

records indicate normal growth and behavior for all of the pigs and therefore, there is no 

evidence to indicate an increase in susceptibility to crushing of pigs in the litters from the clone 

boars. 
Appendix F: The Viagen Dataset F-30 

Animal Cloning: A Risk Assessment DRAFT

The total number of disposed pigs (stillborns, destroyed, overlays, unknown deaths and weak 

pigs) was 21 percent for the pigs from the clone boars and 14 percent for the pigs from the 

comparator boars. The difference in the two groups is primarily due to the unknown and weak 

pig disposals and a higher rate of overlays. These categories have been discussed previously. 

Also, there was a 3.0 percent loss of pigs post-weaning in pigs derived from Duroc comparators 

and a 1.8 percent loss of pigs obtained from Hampshire clone boars. Based on these data there is 

no evidence to suggest that progeny of clones are at increased risk for mortality compared to AIderived pigs. 

Initial plotting of the relationship between age and body weight suggested that, although the 

mean length of time to slaughter was similar for progeny within the four groups, there might be a 

broader range in the length of time that it took to reach slaughter weight. These data indicate that 

there was a similar range, from 144 to 210 days, in the length of time that it took for progeny 

from conventionally bred and clone boars to attain their slaughter weight. Furthermore, the data 

indicate that there were only small differences in ADG between the four groups of pigs between 

birth and slaughter or weaning and slaughter. When ADG was calculated over 4 week intervals 

there were no significant differences prior to 20 weeks of age. The data indicated that slightly 

less than 50 percent of the progeny from each of the four groups was slaughtered prior to week 

24 and that only 27 animals remained on the experiment at week 28. The small differences in 

percentage of animals could be attributed to the small number of animals remaining in the 

experiment at this point than to any one of the groups. 

Because of its retrospective survey nature, and its smaller size, the Cyagra dataset was evaluated 

using a slightly different procedure (see description in Appendix E). In that case, values from 

approximately breed- and age-matched comparators were used to establish a range against which 

values obtained from clones were compared (The Cyagra approach). To determine how 

outcomes would differ if the Viagen data had been analyzed by the same method used to 

evaluate the Cyagra dataset, both approaches were applied to the clinical chemistry and 

hematology variables from Viagen Study 2. The following tables (Tables F-12a, F-12b and F-13) 

summarize the proportion of values that were identified as outliers for further examination by 

both procedures. The boxplot procedure identified a slightly higher percentage of values for 

further examination than the procedure used to describe the Cyagra data. However, the boxplots 

identified similar percentages of outlier values for progeny of comparators as for progeny of 

clones. 
Appendix F: The Viagen Dataset F-31 

Animal Cloning: A Risk Assessment DRAFT

Table F-12a: Variables, Observations, and Outlier Values of Bloodwork from 

Progeny of Comparators Using the Box Plot Method 

Data 

Number 

of 

Variables 

Number of 

Observations 

Number 

of 

Progeny 

Number 

of 

Outlier 

Values 

Number 

of 

Missing 

Values 

Percentage 

of Outliers 

Percentage in 

Range 

July 2004 

Hematology 18 439 204 201 0 5.5 94.5 

July 2004 

Chemistry 35 513 226 267 0 3.4 96.6 

October 

2004 

Hematology 

18 452 187 108 2 3.2 96.8 

October 

2004 

Chemistry 

35 442 178 295 0 4.7 95.3 

January 

2004 

Hematology 

18 222 84 52 10 3.5 96.5 

January 

2004 

Chemistry 

35 405 164 156 0 2.7 97.3 

To derive percentage of outlier values in Tables F-12a, F12b, and F-13, multiply the number of variables by number of 

progeny. For example, from row 1 of Table F-12a: 18 variables x 235 clone progeny sampled = 4230 total 

observations for July hematology. Then divide the number of outliers in the appropriate column by total observations 

(167/4230 = .0394) and multiply by 100 to obtain the percentage of outlier values. 

Table F-12b: Variables, Observations, and Outlier Values of Bloodwork from Progeny of Clones 

Using the Box Plot Method 

Data 

Number 

of 

Variables 

Number of 

Observations 

Number 

of 

Progeny 

Number 

of 

Outlier 

Values 

Number 

of 

Missing 

Values 

Percentage 

of Outliers 

Percentage in 

Range 

July 2004 

Hematology 18 439 235 167 0 3.9 96.1 

July 2004 

Chemistry 35 513 287 320 0 3.2 96.8 

October 

2004 

Hematology 

18 452 265 151 28 3.2 96.8 

October 

2004 

Chemistry 

35 442 264 295 0 3.2 96.8 

January 

2004 

Hematology 

18 222 138 122 16 4.9 95.1 

January 

2004 

Chemistry 

35 405 241 365 0 4.3 95.7 
Appendix F: The Viagen Dataset F-32 

Animal Cloning: A Risk Assessment DRAFT

Table F-13: Percent of All Values Identified as Outliers Using the Procedure Used to Describe the Cyagra 

Data, for Progeny of Clones Only 

Data 

Number 

of 

Variables 

Number of 

Observations 

Number 

of 

Progeny 

Number 

of 

Outlier 

Values 

Number 

of 

Missing 

Values 

Percentage 

of Outliers 

Percentage in 

Range 

July 

Hematology 18 439 235 12 0 0.3% 99.7% 

July 

Chemistry 35 513 287 53 0 0.5% 99.5% 

October 

Hematology 18 452 265 36 28 0.8% 99.2% 

October 

Chemistry 35 442 264 62 0 0.7% 99.3% 

January 

Hematology 18 222 138 47 16 1.9% 98.1% 

January 

Chemistry 35 405 241 85 0 1.0% 99.0% 

b. Growth, Hematology, Clinical Chemistry, and Urinalysis 

i. Growth 

The birth weights of clone and comparator progeny were similar. To establish a base population 

for the comparator group of pigs (n = 267), mummified and stillborn pigs were excluded. A birth 

weight outlier analysis of the progeny of the clones and comparator boars (n=617) indicated that 

two progeny of clone boars were lightweight outliers (0.59 and 0.64 kg). One comparator pig 

was a lightweight outlier (0.41 kg) and one comparator pig was a heavyweight outlier (2.31 kg). 

The boxplots for birth weights showed a similar distribution for the progeny of the clones and 

comparator boars. Additionally, the mean birth weight for the offspring of the clone boars was 

1.5 kg and the mean for the comparator group was 1.45 kg. The median for both groups was 1.5 

kg. The similarity in birth weights, the birth weight distributions and the low frequency of 

outliers between the progeny of the clone boars and those of the comparator boars indicate that 

birth weight is not a health concern for progeny of clones. 
Appendix F: The Viagen Dataset F-33 

Animal Cloning: A Risk Assessment DRAFT

Table F-14: Average Birth Weights of Pigs by Sire 

Sire Number of pigs Birth weight (kg) 

Clone 2 78 1.55 

Clone 3 124 1.43 

Clone 5 75 1.54 

Clone 7 118 1.52 

Control H498 61 1.52 

Control 25515 105 1.48 

Control 18128 134 1.41 

The progeny from both clones and comparator boars had similar nipple counts with similar 

distributions of the counts. Nipple counts are important because they are genetically transmitted 

and indicative of the number of offspring a female can feed. Most of the pigs in this study had 

between 12 and 16 nipples (95.7 percent and 98.4 percent for the clones (389/394) and 

comparators (282/294), respectively). The industry standard is for at least six functional teats per 

side (total 12) of the underline of a gilt (Ahlschwede and Kuhlers 1992). The progeny of the 

clone boar group had no pigs with fewer than 11 nipples while the progeny of the comparator 

group had three pigs with 10 or fewer nipples. Nipple counts for the progeny of clone boars are 

therefore within normal bounds of these pig populations. 

Growth characteristics of these animals were also analyzed, with the data evaluated for potential 

outliers prior to statistical analysis or plotting. Forty-two outliers were identified in the dataset 

containing body weight measurements (n =2,966). Six of these outliers represented body weights 

taken prior to the death of 4 animals. Nine of the outliers were data points associated with 

animals derived from both clone and comparator sires that lost weight near the end of the 

experiment. No reason for the weight loss was provided, and these animals were excluded from 

the final analysis. The mean slaughter weights were, 121.7, 119.6, 120.2, and 121.9 kg for 

progeny derived from the Hampshire comparators, Hampshire clones, Duroc comparators, and 

Duroc clones, respectively. Body weights at the time of slaughter ranged from 108.8 to 134.7 kg 

for progeny from the Hampshire comparator boar, 97.4 to 135.7 kg for progeny from Hampshire 

clones, 97.0 to 136.2 kg for progeny from Duroc comparator boars, and 108.0 to 137.0 kg for 

progeny from the Duroc clone boar. The mean number of days from birth to slaughter was 173.7, 

174.7, 168.2, 175.5 days for progeny obtained from Hampshire comparator, Hampshire clones, 

Duroc comparators, and the Duroc clone, respectively. 

Tabulation of the calculated average daily gain (ADG) for progeny from clones and comparators 

at various time points throughout their lives is shown in Table F-15. Average daily gains from 
Appendix F: The Viagen Dataset F-34 

Animal Cloning: A Risk Assessment DRAFT

birth to slaughter were 0.69, 0.68, 0.71, and 0.69 kg/day for progeny derived from Hampshire 

comparator, Hampshire clones, Duroc comparators, and Duroc clone boars, respectively. Similar 

finds were observed for ADG between weaning and slaughter (0.76, 0.73, 0.77, and 0.74 kg/day, 

respectively). Body weights were also measured every 4 weeks with only small differences 

between the progeny of clones and comparators. 

Table F-15: Comparison of ADG for Progeny Derived from Clone Boars or Conventionally 

Derived Boars (means ± standard deviation) 

Hampshire 

Comparator 

Hampshire 

Clone 

Duroc 

Comparator 

Duroc 

Clone 

Birth to weaning 

0.40±0.13 

(n=48) 

0.44±0.09 

(n=208) 

0.43±0.10 

(n=186) 

0.45±0.09 

(n=81) 

Weaning to 8 wks 

0.65±0.13 

(n=48) 

0.73±0.15 

(n=208) 

0.82±0.14 

(n=186) 

0.84±0.14 

(n=81) 

8 wks to 12 wks 

1.59±0.20 

(n=48) 

1.51±0.21 

(n=208) 

1.54±0.18 

(n=186) 

1.55±0.22 

(n=81) 

12 wks to 16 wks 

2.04±0.30 

(n=48) 

1.96±0.25 

(n=208) 

2.07±0.28 

(n=186) 

2.01±0.30 

(n=81) 

16 wks to 20 wks 

2.12±0.35 

(n=48) 

2.10±0.32 

(n=208) 

2.18±0.31 

(n=186) 

1.92±0.30 

(n=81) 

20 wks to 24 wks 

2.11±0.29 

(n=24) 

2.00±0.38 

(n=109) 

2.05±0.32 

(n=73) 

1.98±0.35 

(n=54) 

24 wks to 28 wks 

1.81±0.32 

(n=4) 

1.77±0.52 

(n=11) 

0.84±1.88 

(n=5) 

1.86±0.41 

(n=7) 

Birth to slaughter 

1.53±0.11 

(n=48) 

1.50±0.11 

(n=208) 

1.57±0.10 

(n=186) 

1.52±0.10 

(n=81) 

Weaning to slaughter 

1.67±0.12 

(n=48) 

1.62±0.13 

(n=208) 

1.70±0.12 

(n=186) 

1.63±0.11 

(n=81) 

ii. Hematology, Clinical Chemistry, and Urinalysis 

First Blood Sampling100 (July). The results of blood clinical chemistry and hematology for July, 

when the progeny were between three and 30 days old, are in Charts F-1 and F-2, respectively. 

More that 94 percent of these values showed no differences between the progeny of clones and 

comparators. We identified the following variables to be of interest for clone progeny: 

 

100 Appendix H has detailed descriptions clinical chemistry values and what they measure. 
Appendix F: The Viagen Dataset F-35 

Animal Cloning: A Risk Assessment DRAFT

hemolysis, lipemia, percent saturation, ALT, AST, CK, Lipase, SDH, LUC, MCHC, and MPV. 

This is because greater than 5 percent of the clone values were outside the comparator range and 

therefore would be more that we would expect by chance. In approximately 5 percent of the 

clone progeny, values for hemolysis, lipemia, percent saturation, ALT, AST, CK, Lipase, SDH, 

LUC, MCHC, and MPV were outside the comparator range. To determine whether these values 

indicated concerns for the health of the animals, we compared the amount of variability between 

the clone progeny and the comparator progeny groups. Following that analysis, we determined 

that the values for hemolysis, percent saturation, ALT, SDH, LUC, MCHC, and MPV had a 

similar amount of variability and did not warrant any further concern. The values for lipemia, 

AST, CK, and lipase were out of range more frequently for clone progeny and required further 

consideration. 

Hemolysis and lipemia can be considered artifacts based on sample handling or drawing samples 

from animals that have been fed recently, respectively, and can have a significant adverse impact 

on the quality of other blood data values (Duncan and Prasse 2003). Hemolysis, either from poor 

collection technique, age of the sample, or poor handling of the sample once in the laboratory, 

has a serious effect on many blood chemistry tests, including dramatic effects on the enzymes 

alkaline phosphatase, GGT, and CK. In general, it is advisable to draw a new sample if gross 

hemolysis is noted. Lipemia (a measure of the amount of fats in the blood) can become elevated 

if animals have their blood drawn shortly after eating. The increased levels of lipids can falsely 

raise Na, K, and Cl levels and artifactually lower AST and ALT levels (Shanahan 2004). For that 

reason, lipemia is not considered a health related variable in food animals (Duncan and Prasse 

2003). Creatine kinase (CK) is an enzyme found in muscle tissue and to a lesser extent in liver 

cells, and elevations are often indicators of muscle injury (including muscle damage during 

venipuncture) or hemolysis (Duncan and Prasse 2003) (See Appendix E). It is unclear if the 

small elevations that were noted were due to those reasons, or injury near the time of handling. 

AST is an enzyme found in liver cells and muscle cells. It has a longer half-life than CK. 

Although eight more clone progeny (19/242 or 7.8 percent) had elevated AST compared to the 

non-clone progeny (11/163 or 6.8 percent), neither the level of increase nor the number of 

animals with increased levels were sufficiently high to indicate a real biological difference. 

Lipase is a pancreatic enzyme that breaks down fat; its elevation can indicate pancreatic 

inflammation (pancreatitis). A 2-3 fold increase, however, is considered the threshold for further 

evaluation to determine whether pancreatic inflammation is responsible. (Duncan and Prasse 

2003). The upper level is 93.5 U/l and average elevation is 132 U/l. In pancreatitis, there also is 

usually an increase in another enzyme (amylase) to corroborate the condition (Duncan and 

Prasse 2003). Amylase values in this study are within range. As values in this study represented 

less than a 2 fold increase, we did not consider them biologically relevant. We therefore 
Appendix F: The Viagen Dataset F-36 

Animal Cloning: A Risk Assessment DRAFT

concluded that there are no biologically relevant differences in blood values between clone 

progeny and comparator progeny at this point in their development. 

Second blood sampling (October). The results of blood clinical chemistry and hematology for 

October, when the animals were approximately 12 to 15 weeks old, are in Chart F-3 and F-4, 

respectively. In comparing the clone to non-clone progeny variability, the CK and basophil 

values were similar enough to conclude no difference. For the chemistry values in clones, 

indirect bilirubin/total bilirubin and bile acids had more outliers compared to non-clone progeny. 

Bilirubin is a breakdown product from the hemoglobin of senescent (old) RBCs. The liver 

processes this by conjugating the product to a salt and making it water soluble. Elevations in 

bilirubin can indicate reduced hepatic function (Duncan and Prasse 2003). The clones in this 

study had low bilirubin. There is no known cause for low bilirubin. For this reason, this finding 

was considered not clinically relevant. Bile acids were elevated in 25 clone progeny and 13 

comparator progeny. Bile acids can be artifactually elevated in response to eating. This value 

may also indicate some hepatic insufficiency (insufficient number of liver cells to perform the 

metabolic functions of the liver). If the liver were adversely affected, we would expect to find 

other corroborating analytes to confirm this possibility. There are no other analytes to confirm 

hepatic insufficiency in these animals. 

 

Hematology values for basophils for progeny of clones and comparators were similar enough not 

to warrant further discussion. More clone progeny had lower MCH (mean corpuscular 

hemoglobin) and MCV (mean corpuscular volume) values and more clone progeny had higher 

RBC values than the comparators. RBCs are elevated in 12 clone progeny and only 2 comparator 

progeny. Elevations in RBC can be from excitement (splenic contraction), hydration status 

(dehydration causes an increase in RBC), or an absolute polycythemia (true increase in 

production). The cause here is unknown but does not seem to indicate a health problem. MCH is 

a value derived by dividing hemoglobin by the RBC number. Because the RBC number is high, 

the MCH must be low. A decrease in mean MCV can mean an iron deficiency. This is usually 

accompanied by anemia. Anemia is defined as a reduced number of RBCs or decreased 

Hematocrit/ Packed Cell Volume (PCV). Because there was an increase in these values, its 

significance is minimal. 

Third blood sampling (January). The results of blood clinical chemistry and hematology for 

January, when the animals were approximately 24 weeks old, are in Charts F-5 and F-6, 

respectively. Values with similar variation between clone and comparator progeny are Mg++, 

LUC, and RDW. These values require no further discussion. 
Appendix F: The Viagen Dataset F-37 

Animal Cloning: A Risk Assessment DRAFT

Sodium:potassium (Na:K) ratio is a value derived from the sodium concentration and compared 

to the potassium concentration. Neither Na+ nor K+ were significantly different. Their ratio is 

used to determine adrenal function (to detect Addison’s Disease) in small animals. Its 

significance in pigs is not listed in clinical pathology texts as being clinically relevant (Duncan 

and Prasse 2003). ALT, AST, BA, CK, and SDH are analytes with significance for liver and 

muscle tissues. Because we have seen elevations in these enzymes before and discussed them 

above, we decided to determine if there was clinical relevance to the increase in clone progeny. 

As discussed, BA may be increased depending on when the blood was drawn in relation to a 

meal. Elevations in values for analytes with significance for liver and their effect on body weight 

are discussed in the next section. Daily health observations were not available. As stated in 

Appendix E and elsewhere, one can only evaluate lab tests in the context of a complete clinical 

picture. 

In the January hematology, hematocrit and RBCs were elevated in progeny of clones. Elevated 

hematocrit and RBC values are rarely an adverse health issue. The MCHC (mean corpuscular 

hemoglobin concentration) had as many high values as low values, which reduces its 

significance as an indicator of a health problem. The clone progeny had 21/138 (15.2 percent) 

animals with elevated segmented neutrophils (segs) vs. 4/84 (4.8 percent) for the comparator 

progeny. Segmented neutrophils are elevated in response to bacterial exposure. With no daily 

clinical health observations, it is difficult to interpret this observation. However, this may be an 

appropriate response to some challenge in the pig’s environment because no other analytes 

indicative of active infection (over all white cell count, banded neutrophils, globulin) are 

elevated. 

No differences in the levels of IGF-I (Fig.1), and E2 (Fig. 2) in progeny of swine clones versus 

comparator animals were found at slaughter. Male progeny of animal clones and their 

comparators have similar levels of E2. The levels of E2 were slightly, but not significantly, 

diminished in the female progeny of clones vs. the comparators. This minor decrease was 

considered to be part of the normal variation in blood levels that may occur depending on 

reproductive status of the female and time of day. The reproductive status of the female animals 

was not provided, however, these animals were slaughtered at approximately six months of age, 

when swine are generally still pre-pubertal. Nevertheless, the levels of E2 in the progeny of 

swine clones do not differ significantly from the comparators. 
Appendix F: The Viagen Dataset F-38 

Animal Cloning: A Risk Assessment DRAFT Serum levels of Estradiol (pg/mL) 

0

3

6

9

12

15

F Cl-F M Cl-M

8 0

116

7 7 123

Figure F-4 depicts the levels of IGF-I in the offspring of swine clones and comparators (female 

and males) at slaughter. CL=offspring clones, F=female, M=male. Values are mean ± SEM, the 

numbers above the bars = number of samples (animals) per group. 

F Cl-F M Cl-M

80

Serum levels of IGF-I (ng/mL) 

0

50

100

150

200

250

300

350

400

116

82

125

80
Appendix F: The Viagen Dataset F-39 

Animal Cloning: A Risk Assessment DRAFT

Figure F-5 depicts the levels of Estradiol-ß in offspring of swine clones and comparators 

(female and males) at slaughter. CL=offspring clones, F=female, M=male. Values are mean ± 

SEM, the numbers above the bars = number of samples (animals) per group. 

Urinalysis. Urine samples were harvested after slaughter, which may account for the presence of 

blood in some samples. Only one clone progeny had protein in its urine. This is not unrealistic by 

random chance and not a health issue. 

Table F-16: Frequency by Group of Urinary pH Values in Progeny of Swine Clones and Comparators 

pH 6 6.5 7 7.5 8 8.5 Total 

Number of Clone Progeny 10 12 16 54 146 4 242 

Percentage 4% 5% 7% 22% 60% 2% 100% 

Number of Comparator Progeny 13 11 13 36 89 1 163 

Percentage 8% 7% 8% 22% 54.5% 0.5% 100% 

Total Number of Animals 23 23 29 90 235 5 405 

The distribution of pH values is similar between the clone progeny and the comparator progeny 

and indicates normal urine variation. This is especially true for animals on an herbivorous diet 

which is typical of current swine management practice (Duncan and Prasse 2003). No animals in 

this experiment had glucose in the urine. 

Conclusions for Animal Health of Progeny of Clones. Although there was a higher death loss 

among progeny of clones in this study, most of this loss can be attributed to a single litter 

farrowed by a heat-stressed sow that did not survive. Causes of death (e.g., stillbirth, overlay, 

weakness) were similar to national statistics for commercially raised swine, and there were only 

minor differences between groups. Few animals were noted with abnormalities in either group, 

and the rates and types of abnormalities were similar to national statistics for commercially 

raised swine. Growth rates from birth to weaning for progeny of clones and comparators were 

similar. Differences were noted in both the early (neonatal) and mid-trial (early juvenile) blood 

values between progeny of clones and comparators in Study 2. The differences during the 

neonatal period were few and minor. The clone progeny values were considered to be within the 

range of variation for a normal population of neonatal animals. There are some differences 

between the clone progeny and comparator progeny during the second blood sampling (early 

juvenile period). The values for analytes with significance for liver for this second sampling 

period offered mixed results, none of which confirm liver abnormalities. The blood cell values 

for this second sampling are also inconsistent, offering no indication of blood cell abnormalities. 
Appendix F: The Viagen Dataset F-40 

Animal Cloning: A Risk Assessment DRAFT

There are increases in liver-function associated analytes in this dataset (late juvenile period). The 

other values indicate no negative health impact on progeny of clones. 

c. Carcass Characteristics 

Given the large variation in live weight at the time of slaughter, one might anticipate that many 

of the post-slaughter carcass characteristics, such as marbling and backfat thickness, would also 

vary considerably. Carcass characteristics are provided in Table F-17. Hot carcass weights were 

79.9, 79.4, 79.0, and 81.2 kg for progeny from Hamline comparator, Hamline clones, Duroc 

comparators, and Duroc clone boars, respectively. Carcass length was also similar, 82.7, 81.6, 

82.3, and 81.5 cm, respectively. The first rib values were 22.2, 23.4, 23.8, and 25.9 mm for 

progeny from Hamline comparator, Hamline clones, Duroc comparators, Duroc clone boars, 

respectively, whereas the last rib values were 16.0, 16.9, 17.4, and 19.0 mm, respectively. 

Table F-17: Carcass Characteristics for Progeny Derived from Clone or Comparator Boars 

Hampshire 

Comparator 

(Mean ± 

standard 

deviation) 

Hampshire 

Clone 

(Mean ± 

 standard 

deviation) 

Duroc 

Comparator 

(Mean ± 

standard 

deviation) 

Duroc Clone 

(Mean ± 

 standard 

deviation) 

Hot Carcass Weight (kg) 79.9 ±3.9 79.4±3.9 78.9±4.3 81.2±4.1 

Carcass Length (cm) 82.7 ±2.2 81.6±2.1 82.3±2.2 81.5±2.3 

Loin Eye Area (cm2) 6.7 ± 0.8 6.8±0.8 6.6±0.8 7.2±0.9 

Back fat Thickness (mm) 

 First rib 22.2 ± 4.2 23.4 ± 4.4 23.8 ± 4.1 25.9 ± 4.2 

 Last rib 16.0 ± 2.9 16.9 ± 3.2 17.4 ± 2.4 19.0 ± 2.8 

 Last Lumbar 16.6 ± 3.4 17.0 ± 3.2 18.1 ± 2.6 19.3 ± 2.7 

Longissimus pH at 24 

hours 5.8 ± 0.2 5.7 ± 0.1 5.7 ± 0.1 5.7 ± 0.1 

Carcass muscle score 3 ± 0 3 ± 0 3 ± 0 3 ± 0 

NPPC Quality Scores 

 Color 3 ± 0.3 3 ± 0.2 3 ± 0.1 3 ± 0 

 Marbling 3 ± 0.7 3 ± 0.8 3 ± 0.8 3 ± 0.9 

 Firmness 2 ± 0 2 ± 0 2 ± 0 2 ± 0 

All animals were given score 3 for carcass muscle score and 2 for firmness. Ninety-three percent 

of the progeny from the clones and comparator boars had marbling score within the 2 to 4 range. 

Measurements of pH at 24 hours post-slaughter on the longissimus muscle were similar. Loin 

eye area for meat cuts for progeny from Hamline comparator, Hamline clones and comparators, 

as well as Duroc comparators and clones were also very similar. In summary, all of the carcass 

characteristics evaluated were similar between the offspring of clones and comparators. 
Appendix F: The Viagen Dataset F-41 

Animal Cloning: A Risk Assessment DRAFT

d. Meat Composition from the Progeny of Clones and Comparators 

Table F-18 provides the comparison of key nutrients between the progeny of clones and their 

comparators. Data were reported for 412 swine of which 242 were the progeny of clones and 163 

were the progeny of comparator boars. The primary comparison was made between the reported 

nutrient concentrations of these two groups. A secondary comparison was made to reference 

swine muscle values currently in the food supply (USDA Food Composition Data for pork, fresh, 

composite of trimmed retail cuts (loin and shoulder blade, separable lean and fat, raw), USDA 

National Nutrient Database for Standard Reference Release 18)). The latter comparison is less 

tightly controlled than the comparison with the comparator, largely due to the differences in cuts, 

and the unknown nature of the breed(s) of swine used in the USDA dataset. 

The composition of the meat from the progeny of clones and comparators indicates that the meat 

samples were indistinguishable at the level of the key nutrients evaluated. Only two values 

(alanine and erucic acid) of 56 (0.04 percent) were not virtually identical, less than would be 

expected by chance alone. Neither of these differences is biologically significant. 

Comparing the meat composition of either the progeny of clones or the comparators to the 

USDA values reveals that neither is as closely comparable to that dataset as they are to each 

other. For example, values for niacin and vitamin B12 from the progeny of both clones and 

comparators were higher than the USDA values for a similar type of swine muscle (shoulder 

blade and loin), while virtually identical to each other. Other nutrients that differ from the USDA 

database include palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid 

(18:1), linoleic acid (18:2), arachidonic acid (20:4), and niacin. The levels of these six fatty acids 

are higher in the database than in the progeny of clones and comparators. Little variability was 

observed between the other values in the nutrient profiles of the USDA database and those 

obtained from the progeny of clones and comparators. The differences between the nutrient 

concentrations in progeny of clones and comparators compared to USDA database may be due to 

diet, swine genotype, or storage stability effects. The important conclusions from the two 

comparisons, however, are that (1) there are virtually no differences between the progeny of 

clones and comparators, (2) the closely genetically related comparators are a better reference 

point than the USDA data base, and (3) none of the differences pose a food safety concern. 

These data suggest that there is no increased risk for humans to consume muscle from the 

progeny of swine clones. 

 
Appendix F: The Viagen Dataset F-42 

Animal Cloning: A Risk Assessment DRAFT

Table F-18: Comparison of Nutrient Concentrations of Progeny from Clones and Comparators 

Nutrients1 

Progeny from Clone 

Boars 

mean + std. dev. 

Progeny from 

Comparators Boars 

Mean + std. dev. 

Amino Acids

Aspartic acid 2.31+0.19 2.29+0.16 

Cystine 0.25+0.02 0.25+0.01 

Glutamic acid 3.76+0.34 3.71+0.27 

Glycine 1.14+0.15 1.12+0.13 

Histidine 0.98+0.09 0.98+0.07 

Isoleucine 1.03+0.12 1.03+0.10 

Leucine 1.90+0.14 1.89+0.12 

Lysine 2.06+0.17 2.07+0.16 

Methionine 0.61+0.05 0.62+0.04 

Phenylalanine 0.96+0.09 0.94+0.08 

Nutrients1 

Progeny from Clone 

Boars 

Mean + std. dev. 

Progeny from 

Comparators Boars 

Mean + std. dev. 

Praline 1.09+0.13 1.11+0.13 

Serine 0.96+0.08 0.95+0.07 

Threonine 1.09+0.09 1.08+0.07 

Tyrosine 0.81+0.06 0.81+0.05 

Valine 1.09+0.12 1.10+0.10 

Fatty Acids and Cholesterol

8:0 (Caprylic acid) <0.012 0.01 

10:0 (Capric acid) 0.01+0.002 0.01+0.002 

11:0 <0.01 <0.01 

12:0 (Lauric acid) 0.01+0 0.01+0 

14:0 (Myristic acid) 0.08+0.027 0.08 + 0.029 

14:1 (Myristoleic acid) <0.01 <0.01 

15:0 <0.01 <0.01 

15:1 <0.01 <0.01 

16:0 (Palmitic acid) 1.39+0.38 1.40+0.49 

16:1 (Palmitoleic acid) 0.17+0.06 0.16+0.05 

17:0 (Margaric acid) 0.01+0.003 0.01 +0.002 

17:1 (Margaroleic acid) 0.01+0.003 0.01+0.002 

18:0 (Stearic acid) 0.66+0.24 0.68+0.25 

18:1 (Oleic acid) 2.26+0.76 2.20+0.72 

18:2 (Linoleic acid) 0.3+0.11 0.29+0.11 

18:3 (Linolenic acid) 0.02+0.001 0.01+0.005 
Appendix F: The Viagen Dataset F-43 

Animal Cloning: A Risk Assessment DRAFT

Nutrients1 

Progeny from Clone 

Boars 

Mean + std. dev. 

Progeny from 

Comparators Boars 

Mean + std. dev. 

18:4 0.01+0.0001 0.01+0.004 

20:0 (Arachidic acid) 0.01+0.005 0.01+0.005 

20:1 (Gadoleic acid) 0.08+0.04 0.07+0.04 

20:2 (Eicosadienoic acid) 0.02+0.01 0.02+0.005 

20:3 (Eicosatrienoic acid) 0.01+0.01 <0.01 

20:4 (Arachidonic acid) 0.01+0.003 0.01+0.002 

20:5 (Eicosapentaenoic acid) 0.01 + 0 0.01+0.004 

21:5 (Heneicosapentaenoic acid) 0.01+0 <0.01 

22:0 (Behenic acid) <0.01 <0.01 

22:1 (Erucic acid) 0.01+0.006 0.02+0.006 

22:2 (Docosadienoic acid) <0.01 0.01+0.01 

22:3 (Docosatrienoic acid) <0.01 <0.01 

22:4 (Docosatetraenoic acid) <0.01 <0.01 

22:5 (Docosapentaenoic acid) <0.01 <0.01 

22:6 (Docosahexaenoic acid) 0.02+0.01 0.02+0.01 

24:0 (Lignoceric acid) <0.01 <0.01 

24:1 (Nervonic acid) <0.01 <0.01 

Cholesterol (mg/100 g) 57.93+5.46 59.39+5.04 

Minerals

Calcium 0.01+0.003 0.01+0.002 

Iron 0.00+0.0005 0.000+0.003 

Phosphorus 0.18+0.082 0.16+0.082 

Zinc 0.00+0.0003 0.00+0.0001 

Vitamins

Niacin (mg/100g) 10.68+1.23 10.64+1.03 

Vitamin B6 (mg/100 g) 0.40+0.07 0.38+0.07 

Vitamin B12 (mcg/100 g) 1.01+0.25 0.97+0.28 

1 Unless otherwise specified, quantities are expressed as g/100g homogenized meat. 

2 Values marked with “<” indicate concentrations below the level of detection for the instrument used in 

the assay. 
Appendix F: The Viagen Dataset F-44 

Animal Cloning: A Risk Assessment DRAFT

Table F-19: Nutrient Concentrations for Pork, Fresh, Composite of Trimmed Retail Cuts (Loin 

And Shoulder Blade), Separable Lean and Fat, Raw as Listed in USDA National Nutrient 

Database 

Nutrients1 USDA2

Amino Acids

Aspartic acid 1.795 

Cystine 0.246 

Glutamic acid 3.011 

Glycine 1.011 

Histidine 0.761 

Isoleucine 0.900 

Leucine 1.556 

Lysine 1.748 

Methionine 0.508 

Phenylalanine 0.776 

Praline 0.830 

Serine 0.807 

Threonine 0.882 

Tyrosine 0.668 

Valine 1.052 

Fatty Acids and Cholesterol

8:0 (Caprylic acid) 0.000 

Nutrients1 USDA2

10:0 (Capric acid) 0.010 

11:0 not listed 

12:0 (Lauric acid) 0.010 

14:0 (Myristic acid) 0.160 

14:1 (Myristoleic acid) not listed 

15:0 not listed 

15:1 not listed 

16:0 (Palmitic acid) 2.79 

16:1 (Palmitoleic acid) 0.37 

17:0 (Margaric acid) not listed 

17:1 (Margaroleic acid) not listed 

18:0 (Stearic acid) 1.46 

18:1 (Oleic acid) 5.27 

18:2 (Linoleic acid) 1.140 

18:3 (Linolenic acid) 0.09 

18:4 0.00 
Appendix F: The Viagen Dataset F-45 

Animal Cloning: A Risk Assessment DRAFT

Nutrients1 USDA2

20:0 (Arachidic acid) not listed 

20:1 (Gadoleic acid) 0.100 

20:2 (Eicosadienoic acid) not listed 

20:3 (Eicosatrienoic acid) not listed 

20:4 (Arachidonic acid) 0.08 

20:5 (Eicosapentaenoic acid) 0.00 

21:5 (Heneicosapentaenoic acid) not listed 

22:0 (Behenic acid) not listed 

22:1 (Erucic acid) 0.00 

22:2 (Docosadienoic acid) not listed 

22:3 (Docosatrienoic acid) not listed 

22:4 (Docosatetraenoic acid) not listed 

22:5 (Docosapentaenoic acid) 0.000 

22:6 (Docosahexaenoic acid) 0.000 

24:0 (Lignoceric acid) not listed 

24:1 (Nervonic acid) not listed 

Cholesterol (mg/100 g) 64 

Minerals

Calcium 0.019 

Iron 0.00082 

Phosphorus 0.195 

Zinc 0.00187 

Vitamins

Niacin (mg/100g) 4.492 

Vitamin B6 (mg/100 g) 0.456 

Vitamin B12 (mcg/100 g) 0.63 

1

Data expressed as quantities per 100 g of homogenized meat. 

2

USDA means taken from the USDA National Nutrient Database for Standard Reference Release 18, 

item number 10226. 

3 Values marked with < indicate concentrations below level of detection for instrument used in assay. 

Conclusions for Carcass Characteristics and Meat Composition for Progeny of Clones and 

Comparators 

Although some minor differences in backfat thickness were noted for progeny of clones vs. 

comparators, they have no significance for food safety. The increased values for niacin and B12

and decreased values for six fatty acids compared to USDA values were similar for progeny of 

clones and comparators in this experiment, and may reflect differences in diet, genotype, or 

sample handling compared to the national average. Because these values were similar between 

the two groups involved in this study, there is no increased risk associated with meat from 
Appendix F: The Viagen Dataset F-46 

Animal Cloning: A Risk Assessment DRAFT

progeny of clones vs. contemporary comparators. All other meat composition values were 

similar between groups, indicating no increased risk associated with meat from progeny of 

clones vs. contemporary comparators. 

D. Conclusions from the Viagen Dataset 

1. Study 1: Clones vs. Comparators 

a. Animal Health 

The interpretation of the results of the study comparing conventionally bred and clone barrows is 

limited because it was not initiated until the animals were approximately 50 days of age, clones 

were raised under different conditions prior to initiating the experiment, and there were a limited 

number of clones. The conventionally derived barrows were selected based on two criteria: (1) 

their sire was one of the donor boars for SCNT; and (2) the pigs were similar in age and weight 

to the clone barrows. Retrospective evaluation of the birth weights for the seven clone barrows 

indicated that these animals were smaller at birth than their conventionally bred counterparts. 

The growth rate data would suggest that clone barrows grew as well as conventionally bred 

animals prior to weaning, as these animals reached the same body weight at around 50 days of 

age. However, after the clones were moved to the more conventional rearing facility to be raised 

with the AI-derived barrows, it took the clone barrows on average 27 days longer to reach their 

slaughter weight, and the clone barrows were on average 18.2 kg lighter when they were 

slaughtered than the conventionally bred barrows.101 Furthermore, three of the seven clone 

barrows were not processed at the end of the experiment. One of the clones was condemned at 

slaughter due to a lung adhesion and the other two animals were approximately 45 kg lighter 

than their counterparts. The health records for the clones indicate that these animals developed 

several health issues including scouring. These clones were born and maintained under highly 

biosecure conditions until the beginning of the study (at approximately 50 days of age), were 

potentially premature at delivery, and were deprived of colostrum. Thus, moving these animals 

to a conventional production system could have had a dramatic effect on their growth rate. 

However, four of the seven clone barrows responded appropriately and overcame the pathogenic 

challenge. 

 

101 As mentioned previously, because animal clones are voluntarily withheld from entering the food supply, the 

slaughter facility could only make certain dates available to slaughter these animals. Thus, the clones were 

slaughtered as a group on one of the pre-selected, available dates once their average weight approached the target 

weight. 
Appendix F: The Viagen Dataset F-47 

Animal Cloning: A Risk Assessment DRAFT

b. Food Safety 

The most significant difference between the comparator and clone barrows at slaughter was a 

trend for higher backfat thickness in the conventionally bred animals, consistent with the 

observation that the conventionally bred animals were heavier at the time of slaughter than 

clones. 

Data were presented on the key nutrient levels of latissimus dorsi muscle from swine clones and 

comparators. Fifty-six nutrients were measured in tissue samples of five clones (one animal was 

euthanized for health reasons) and 15 comparator swine. There were limitations in the usefulness 

of the data due to the study design and data reporting. A comparison of reported values to 

reference swine muscle values (USDA National Nutrient Database for Standard Reference 

Release 18) was possible for only four nutrients because the other nutrients were reported as 

percentages. Values for niacin and vitamin B12 in both clones and comparator swine were above 

USDA values for a similar type of swine muscle (shoulder blade and loin), but were similar to 

each other. Values for cholesterol and vitamin B6 were similar to the USDA values. Little 

variability between reported values was observed when the data were examined by nutrient. 

The lack of variability observed in the food composition values between muscle of swine clones 

and comparators supports a conclusion that there is no additional risk in the human consumption 

of muscle from swine clones. However, limitations in the study design, reporting of data, and the 

elevated niacin and vitamin B12 concentrations in both clone and comparator muscle compared to 

the USDA reference values diminish the confidence of this conclusion. A more definitive 

comparison between the food composition of clone and comparator swine muscle could be made 

if more of the analyses could be compared to values in reference (USDA) swine muscle. 

2. Progeny of Clone Boars vs. Progeny of Comparator Boars 

This experiment was designed to determine whether progeny from clones performed as well as 

progeny from comparator boars and if food products from progeny of clones would pose any 

additional risk relative to corresponding products derived from comparator animals. Data were 

provided on 300 and 402 progeny derived from comparator and clone boars, respectively. 

a. Animal Health 

Although there was a higher percentage loss of pigs at birth for progeny derived from clone 

boars, this difference was primarily due to the loss of an entire litter of 13 pigs. Secondary 

factors that potentially influenced the number of pigs that died due to the dam lying on pigs 

included environmental conditions at the time of farrowing and the number of litters that were 

born on a single day. Although there was a slight increase in the percentage of progeny of clones 
Appendix F: The Viagen Dataset F-48 

Animal Cloning: A Risk Assessment DRAFT

that were crushed by their dam, there was a similar number of pigs/litter crushed by their dams 

among comparators, and these values were similar to values that are commonly found within the 

swine industry. Thus, there do not appear to be any differences in the survival of progeny from 

clone boars when compared to progeny from conventionally derived boars. There was a similar 

percentage of mummified pigs presented at birth. The survival and growth rate data do not show 

any animal health concerns for progeny of clone boars when compared to progeny from 

conventionally derived boars. Hematology, clinical chemistry and urinalysis values for clone 

progeny were considered to be within the range of variation for a normal population of animals. 

b. Food Safety 

Data were presented on the food composition of latissimus dorsi muscle from the progeny of 

swine clones and controls. Fifty-eight nutrients were measured in the tissue samples of 242 AIderived comparators and 163 clone progeny. A positive aspect of the study is the large numbers 

of test swine and the numerous nutrients analyzed. Negative aspects of the study are the lack of 

method performance data in the test matrix (meat), the choice of latissimus dorsi as the test 

matrix instead of a retail pork cut and/or another edible tissue, and the lack of storage stability 

data. 

The nutrient concentrations of clone progeny and comparator swine are very similar. A few 

nutrients did have differences in the variability and distribution of values between clone progeny 

and comparator swine. We evaluated the differences, and determined they were minor and not 

biologically significant. 

Most of the nutrient concentrations were similar to USDA reference values. Six fatty acids had 

lower concentrations in both the clone progeny and comparator swine compared to the USDA 

values. One B vitamin was higher in the clone progeny and comparator swine than the USDA 

value. The difference between these nutrient concentrations in clone progeny and comparator 

progeny compared to USDA values may be due to effect of diet, swine breed, or storage stability 

effects on method performance. 

Based on the lack of difference in the nutrient concentrations between muscle of progeny from 

AI-derived comparator and clone boars, we conclude that there is no increased risk for humans to 

consume muscle from the progeny of clone swine. The current study provides no information 

regarding the food composition of other swine edible tissue (liver, kidney, fat). Therefore, food 

safety conclusions about muscle cannot be extrapolated to other edible tissues of swine. 
Appendix F: The Viagen Dataset F-49 

Animal Cloning: A Risk Assessment DRAFT

E. Addendum 

On January 5, 2006, Viagen Inc. faxed several pages of data from the re-assay of samples which 

they had identified as outliers using the criteria outlined for the Cyagra dataset (Appendix E). 

Samples from 15 clone progeny were re-assayed because the values for specific nutrients were > 

10 percent above or below the range of values for comparators. Table F-20 provides a 

comparison of the original and re-assay values for the nutrients assayed by animal. 

Table F-20. Comparison of original and re-assay values of select nutrients from 

meat of clone progeny. 

Nutrient1 Animal ID Original Value Re-assay Value 

Amino Acids 

 Aspartic Acid 200437509 3.33 2.30 

Fatty Acids 

 10:0 (Capric) 200430710 

200438107 

0.01 

0.02 

<0.01 

0.01 

 14:0 (Myristic) 

200430710 

200431409 

200430701 

200431606 

200432702 

200438107 

0.15 

0.03 

0.04 

0.12 

0.07 

0.21 

0.12 

0.05 

0.07 

0.09 

0.09 

0.20 

 16:0 (Palmitic) 

200430710 

200431409 

200430701 

200432702 

200438107 

2.40 

0.45 

0.53 

1.10 

3.62 

2.03 

0.99 

1.00 

1.52 

3.63 

 16:1 (Palmitoleic) 

200430710 

200431409 

200430701 

200431606 

200432702 

200438107 

0.27 

0.06 

0.06 

0.25 

0.12 

0.38 

0.15 

0.12 

0.15 

0.17 

0.20 

0.36 

 17:1 (Margaroleic) 

200430710 

200431606 

200438107 

0.02 

<0.012 

0.02 

0.01 

0.01 

0.01 

 18:0 (Stearic) 

200430710 

200431409 

200430701 

200431606 

200432702 

200438107 

1.17 

0.21 

0.23 

0.99 

0.48 

1.77 

1.11 

0.49 

0.42 

0.68 

0.68 

1.78 
Appendix F: The Viagen Dataset F-50 

Animal Cloning: A Risk Assessment DRAFT

Nutrient1 Animal ID Original Value Re-assay Value 

 18:1 (Oleic) 

200430710 

200431409 

200430701 

200431606 

200432702 

200438107 

4.63 

0.79 

0.69 

3.53 

1.38 

5.44 

3.30 

1.71 

1.76 

2.43 

2.45 

5.67 

 18:2 (Linoleic) 

200430710 

200431409 

200430701 

200431606 

200432702 

200438107 

0.70 

0.11 

0.07 

0.92 

0.06 

0.55 

0.46 

0.32 

0.29 

0.39 

0.33 

0.61 

 18:3 (Linolenic) 

200430710 

200431409 

200430701 

200431606 

200432702 

200438107 

0.04 

<0.01 

<0.01 

0.05 

<0.01 

0.02 

0.01 

0.02 

0.01 

0.01 

0.02 

0.03 

 18:4 200430710 0.01 <0.01 

 20:0 (Arachidic) 200431606 

200438107 

0.02 

0.03 

0.01 

0.02 

 20:1 (Gadoleic) 

200430710 

200430701 

200431606 

200432702 

200438107 

0.14 

0.05 

0.07 

0.04 

0.13 

0.07 

0.03 

0.04 

0.03 

0.09 

 20:2 (Eicosadienoic acid) 

200430710 

200431409 

200430701 

200431606 

200432702 

200438107 

0.04 

<0.01 

<0.01 

0.04 

<0.01 

0.03 

0.06 

0.03 

0.01 

0.03 

0.04 

0.03 

 20:3 (Eicosatrienoic acid) 200431606 0.01 <0.01 

Nutrient1 Animal ID Original Value Re-assay Value 

 20:4 (Arachidonic) 200430701 

200431606 

<0.01 

0.02 

0.01 

<0.01 

 22:1 (Erucic) 200438107 0.02 <0.01 

 22:6 (Docosahexaenoic) 

200430710 

200432702 

200438107 

0.01 

0.03 

0.05 

<0.01 

<0.01 

<0.01 

Minerals 

 Calcium 200430609 

200430708 

0.034 

0.021 

0.0042 

0.0045 

 Phosphorus 200433206 0.72 0.021 

 Zinc 200438104 

200431008 

0.0025 

0.0046 

0.0014 

0.0015 
Appendix F: The Viagen Dataset F-51 

Animal Cloning: A Risk Assessment DRAFT

Nutrient1 Animal ID Original Value Re-assay Value 

Vitamins 

 Niacin (mg/100g) 200433002 19.1 8.78 

 Vitamin B12 (mcg/100 g) 20045803 2.20 1.20 

1

 Unless otherwise specified, quantities are expressed as g/100g homogenized meat. 

2 Values marked with “<” indicate concentrations below the level of detection for the 

instrument used in the assay. 

CVM conducted a follow-up analysis of the data using the new values and found they had only 

very minor effects on the average nutrient values for clone progeny. The reanalyzed means are 

presented in Table F-21, and compared to the original means for progeny of clones and 

comparators. 

Table F-21: Comparison of Nutrient Concentrations of Progeny 

from Clones and Comparators 

Nutrients1 

Progeny 

from Clone 

Boars 

(Original) 

mean + std. 

dev. 

Progeny 

from Clone 

Boars 

(Reanalyzed) 

mean + std. 

dev. 

Progeny 

from 

Comparators 

Boars 

Mean + std. 

dev. 

Amino Acids

Aspartic acid 2.31+0.19 2.30 ± 0.17 2.29+0.16 

Fatty Acids 

10:0 (Capric acid) 0.01+0.002 0.002 ± 0.003 0.01+0.002 

14:0 (Myristic acid) 0.08+0.027 0.08 ± 0.027 0.08 + 0.029 

16:0 (Palmitic acid) 1.39+0.38 1.39 ± 0.47 1.40+0.49 

16:1 (Palmitoleic acid) 0.17+0.06 0.17 ± 0.06 0.16+0.05 

17:1 (Margaroleic acid) 0.01+0.003 0.00 ± 0.004 0.01+0.002 

18:0 (Stearic acid) 0.66+0.24 0.66 ± 0.23 0.68+0.25 

18:1 (Oleic acid) 2.26+0.76 2.26 ± 0.74 2.20+0.72 

18:2 (Linoleic acid) 0.3+0.11 0.3 ± 0.10 0.29+0.11 

18:3 (Linolenic acid) 0.02+0.001 0.01 ± 0.009 0.01+0.005 

18:4 0.01+0.000 0.00 ± 0.002 0.01+0.004 

20:0 (Arachidic acid) 0.01+0.005 0.01 ± 0.008 0.01+0.005 

20:1 (Gadoleic acid) 0.08+0.04 0.08 ± 0.04 0.07+0.04 

20:2 (Eicosadienoic acid) 0.02+0.01 0.01 ± 0.01 0.02+0.005 

20:3 (Eicosatrienoic acid) 0.01+0.01 <0.013 <0.01 

20:4 (Arachidonic acid) 0.01+0.003 0.00 ± 0.002 0.01+0.002 

22:6 (Docosahexaenoic acid) 0.02+0.01 <0.01 0.02+0.01 
Appendix F: The Viagen Dataset F-52 

Animal Cloning: A Risk Assessment DRAFT

Nutrients1 

Progeny 

from Clone 

Boars 

(Original) 

mean + std. 

dev. 

Progeny 

from Clone 

Boars 

(Reanalyzed) 

mean + std. 

dev. 

Progeny 

from 

Comparators 

Boars 

Mean + std. 

dev. 

Minerals

Calcium 0.01+0.003 0.01 ± 0.002 0.01+0.002 

Phosphorus 0.18+0.082 0.18 ± 0.075 0.16+0.082 

Zinc 0.00+0.0003 0.00 ± 0.0002 0.00+0.0001 

Vitamins

Niacin (mg/100g) 10.68+1.23 10.64 ± 1.11 10.64+1.03 

Vitamin B12 (mcg/100 g) 1.01+0.25 0.93 ± 0.34 0.97+0.28 

1

 Unless otherwise specified, quantities are expressed as g/100g homogenized meat. 

2 Values of 0.00 reflect means less than 0.01. 

3 Values marked with “<” indicate concentrations below the level of detection for 

the instrument used in the assay. 

Of the 22 nutrient values that were reanalyzed, 12 means were unchanged compared to the 

original values. Nine values were changed (0.01 g/100g) compared to the original values. Only 

the mean for Vitamin B12 differed by more than 0.01 g/100 g; however, the reanalyzed mean was 

similar to the mean for comparators (0.93 ± 0.34 vs. 0.97 ± 0.28 g/100 g). All 22 values were 

similar to values for comparators. None of these minor changes affect the assumptions regarding 

the safety of meat from clone progeny. 
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200430601 ■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■

200430602 ■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■200430603 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430604 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430605 ■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■ ↓ ■■■■200430606 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430607 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430608 ■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■200430609 ■■■ ↑ ■■■■■■■■■ ↑ ■ ■ ↓ ↓ ■ ↑ ■■■■■■■ ↓ ■ ■ ↓ ■200430610 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430611 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430701 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430702 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430703 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430704 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430705 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430706 ■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■200430707 ■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■200430708 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430709 ■■■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■■■■■■■200430710 ■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■200430711 ■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■200430801 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430802 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430803 ■ ■ ↑ ■■■■■ ↑ ■■■■■■■■■■■■■■■■ ↑ ■ ↓ ■■■■200430804 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430805 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-1 (Page 1): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (July 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200430901200430902 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200430903 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430904200430905 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430906 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431001 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431002 ■■■■■■■■

↑

↓

■

↑

■

↑ ■■■■■

↑ ■■■■■■■■■■■■

200431003 ■ ■

↑

↑

■

↑ ■■■■■

↑ ■■■■■■■■■■■

↓

■

↑ ■■■■■■

200431004 ■■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■■

200431005 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431006 ■■■■■■■■

↑ ■■■■■■■■■■■■■■■

↑ ■■■■■■■

200431007 ■■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■■

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↑ ■■■■■■

200431009 ■■■■■■■■

↑ ■ ■

↑ ■■■■■■■■■■■■

↑ ■■■■■■■

200431010 ■ ■

↑ ■■■■■■

↓

↓

■

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↓ ■■■

↓ ■■■■

200431011 ■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■

200431012 ■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■

200431013200431401

■

↑ ■■■■■

↑ ■■■■■■

↑ ■■■■■■■■■■■

↑ ■■■

↑ ■

200431402 ■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■

↑ ■

200431403 ■■■

↑ ■■■■■■■

↑ ■■■■■■■■■■■■■■

↑ ■■■■■

200431404 ■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■

200431405 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431406 ■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■

200431407 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431408 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-1 (Page 2): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (July 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200431409 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200431410 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431411 ■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■ ↓ ■■■200431501 ■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■200431502 ■■■■■■ ↑ ■■■■■■■ ↑ ■■■■■■ ↑ ■■■■■■■■■■ 200431503 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431504 ■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■200431505 ■■■■■■ ↑ ■ ↑ ■■■■■ ↑ ■■■■■■■■■■■■■■■■■200431506 ■■■■■■ ↑ ↑ ■■■■■■■■■■■■■ ↑ ■■■■■■ ↓ ■■■200431601 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■200431602 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431603 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431604 ■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■200431605 ■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■200431606 ■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■ ↓ ■■■200431607 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431608 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■200431609200431610 ■■■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■■■■■■■200431701 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431702 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431703 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431704 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431705200431706 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■200431707200431708 ■■■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■ ↑ ■■■■■

Chart F-1 (Page 3): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (July 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200432001 ■■■

↑ ■■■■■■■■■

↑ ■■■■■

↑ ■■■■■■■■■■■■

200432002 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432003 ■■■■■■

↑ ■■■■■■■

↑ ■■■■■■■■■■

↑ ■■■■■■

200432004 ■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■

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200432005 ■■■■■■

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200432006 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432007 ■■■■■■■■■■■■■

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200432008 ■■■■■■■■■■■■■

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200432101 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432102 ■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200432103 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432104 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432105 ■■■

↑ ■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■

200432106 ■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■

↓ ■■■

200432107200432108 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432109 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432301 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432302 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

200432303 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

200432304 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

200432305 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

200432306 ■■■

↑ ■■■■■■■

↑ ■■■■■■■■■■■■■

↑ ■ ■

↑ ■■■

200432307 ■■■■■■■■■■■■■

↑ ■■■■■

↑ ■■■■■■■■■■■■

200432308 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432309 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432310 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-1 (Page 4): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (July 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200432311 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200432312 ■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■200432401 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432402 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432403 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432404 ■■■■■■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■■■■■■■200432405 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■200432406 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432407 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■200432408200432501 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432502 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432503 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432504 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432505200432506 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432507 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432508 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432701 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432702 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432703 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432704 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432705 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432706 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433001 ■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ↑ ■■■■■200433002 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■200433003 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-1 (Page 5): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (July 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200433004 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

↓ ■

200433005 ■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■

200433006 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

200433007 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433008 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433009200433010 ■■■■■■■■■■

↑ ■■■■■■■■■■■■■■

↑

■

↓ ■■■■

200433011 ■■■

↑ ■■■■■■■■■■■■■

↓ ■■■■■■■

↑ ■■■■■■

200433201 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433202 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433203 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433204 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433205 ■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■

200433206 ■■■■■■■■

↑ ■■■■■■■■■■■■■■■

↑ ■■■■■■■

200433207 ■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■

↑ ■■

200433208 ■■■■■■■■■■■■■■

↑

↑

■

↑ ■■■■■■■■

↑ ■ ■

↑

↑ ■

200433601200433602 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■

200433603200433604 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■

200433605 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■

200433701 ■■■■■■■■■■■

↓ ■■■■■■■■■■■■■■■■■

↑ ■■

200433702 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■

200433703 ■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■

↑ ■■

200433704 ■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■

↑ ■■

200433705 ■■■■■■■■■■■■■

↑ ■■■■■

↑ ■■■■■■■■■

↑ ■■

200433706 ■■■■■■■■■■■■■

↑ ■■■■■

↑ ■■■■■■■■■

↑ ■■

Chart F-1 (Page 6): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (July 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200433707 ■■■■■■

↑ ■■■

↑ ■■■■■■■■■■■■■■■■■■

↑ ■■

200433708 ■■■

↑ ■■■■■■■

↑

■

↑ ■■■■■

↑ ■■■■■■■■■

↑ ■■

200433709 ■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■

↑ ■■

200433710

■

↓ ■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■

↑ ■■

200433711 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■

200433712 ■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■

↑ ■■

200433801 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■

200433802 ■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■

↑ ■■

200433803 ■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■

↑ ■■

200433804 ■■■■■■

↑ ■■■■■■■■■■■■■■■■■■

↑ ■■■

↑ ■■

200433805200433806 ■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■

↑ ■■

200433807200433808 ■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■

↑ ■■

200433809

■

↓ ■■■■■■■■■

↑

■

↑ ■■■■■

↑ ■■■■■■■■■

↑ ■■

200434101 ■■■■■■■■

↑ ■ ■

↑ ■■■■■■

↑ ■■■■■■

↑ ■■■■■■

200434102 ■ ■

↑ ■■■■■

↑ ■ ■

↑ ■ ■

↑ ■■■

↑ ■■■■■■

↑

■

↓ ■■■■

200434103 ■■■■■■■■■■■

↑ ■■■■■■■■■■■■■

↑ ■■■■■■

200434104 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

200434105 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

200434106 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434107 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434108 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434109 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434110200434111 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

200434112 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-1 (Page 7): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (July 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200434113 ■■■■■

↑ ■■■■■■■■■■■■■■■

↑

■

↓ ■■■■■■■■

200434114 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

200434115 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434401 ■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■

↑ ■

200434402 ■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■

200434403 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434501 ■■■■■■■■■■■■■

↑ ■■■■■

↑ ■■■■■

↑ ■■■■■■

200434502 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■

200434503 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

200434504 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434505200434506 ■■■■■■■■■■■

↑ ■■■■■■■■■■■■■

↑ ■■■■■■

200434507 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

200434508 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434509 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434510 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

200434511 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

200434512 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434601 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434602 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434603200434604 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

200434605 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

200434606 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434607 ■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■

200434608 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200435801 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■

Chart F-1 (Page 8): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (July 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200435802 ↑ ■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■ ↑ ■

200435803 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436001 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436002 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436003 ↑ ■ ■ ↑ ■■■■■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■200436004 ■■■■■ ↑ ■■■■■■■■■■ ↓ ■ ■ ↑ ■■■ ↓ ■■■■■■■■200436005 ■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■200436006 ■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■200436007 ■■■■■■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■■■■■■■200436008 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436009 ■■■ ↑ ■■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■■■■■■■200436010 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436011 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436012 ■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■200436013 ■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■200436014200436401 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■ ↑ ■■■■■■■200436402 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436403 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436404 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436405 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436601 ■■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■200436602 ■■■ ↑ ■■■■■■■ ↑ ■ ↑ ■■■■■ ↑ ■■■■■■■■■■■■200436603 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436604 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436605 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436606 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-1 (Page 9): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (July 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200436607 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200436608 ■■■■■■■■■■■■■■■■■ ↓ ■ ↑ ■■■■■■■■■■ ↓ ■200436609 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436610 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436611 ■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436612 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436901200436902 ■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■200436903 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436904 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436905 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436906 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436907 ■■■ ↑ ■■■■ ↑ ■ ■ ↑ ■ ↑ ■■■■■ ↑ ■■■■■■■■■■■■200436908 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436909 ■■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ↑ ■■■■200436910 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436911 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436912 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436913 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437401 ■■■■ ↑ ↑ ■■■■■ ↑ ■ ↑ ↑ ■ ■ ↑ ↑ ■■■■ ↓ ■ ↑ ■ ↓ ■ ■ ↑ ■ 200437402 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437403 ■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■200437404 ■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■200437405 ■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■200437406 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437407 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437408 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-1 (Page 10): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (July 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200437409 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200437410 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437411 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437501 ■■■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■ ↑ ■■■200437502 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437503200437504200437505 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437506 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437507 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437508200437509 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437510 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437801 ■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■200437802 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437803 ■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■200437804 ■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■ ■ ↓ ■■■■200438101 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438102 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438103 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438104 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438105 ■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■200438106 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■200438107 ■■■■■ ↓ ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■200438601 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438602 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438603 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-1 (Page 11): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (July 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200438604 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200438605 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438606 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438607 ■■■ ↑ ■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■■■■■■■200438608 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438609 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438610 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438611 ■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■200439501 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200439502 ■■■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■■■■■■■200439503200439504 ■■■ ↑ ■■■■■■■ ↑ ■■■■■■■ ↑ ■■■■■■■■■■■■200439505 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

2 4 4 15 1 9 21 6 15 2 11 17 1 19 14 1 3 7 6 28 0 4 3 5 11 37 7 9 7 26 10 

///////////////////////////////

287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 287 2

Summary

Chart F-1 (Page 12): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (July 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

200430601 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430602 ■■■■■■■■ ↑ ■ ■ ↓ ↓ ■■■■■ 3 / 18

200430603 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430604 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430605 ■■■■■■■■■■■■ ↓ ■■■■■ 1 / 18

200430606 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200430607 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200430608 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430609

200430610 ■■■■■■■■■■■ ↑ ■■■■■■ 1 / 18

200430611 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430701 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200430702 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430703 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430704 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430705 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430706 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430707

200430708 ■■■■■■■■ ↑ ■■■■■■■■■ 1 / 18

200430709

200430710 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430711 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430801 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430802

200430803 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200430804

200430805 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430901

200430902 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430903 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430904

200430905 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430906 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200431001 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200431002

200431003 ■■■■■ ↓ ■■■■■■ ↓ ■■■■■ 2 / 18

200431004 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431005 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200431006

200431007 ■■■■■■■■■■■■■■■■■■ 0 / 18

Summary

Chart F-2 (Page 1): Summary of Hematology Data for Viagen Clone Progeny and Comparators (July 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
200431008

200431009 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200431010 ■■■■■ ↓ ■■■■ ↑ ■■■■■ ↑ ↑ 4 / 18

200431011 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431012 ■■■■■■■■■■■ ↑ ■■■■■■ 1 / 18

200431013

200431401 ■■■■■ ↑ ■■■■■ ↑ ■■■■■■ 2 / 18

200431402

200431403

200431404 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431405 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431406

200431407

200431408 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431409 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431410 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431411 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431501 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431502 ■ ■ ↑ ■■■ ↓ ■■■■■■■■■■■ 2 / 18

200431503 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431504 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431505 ■■■■■■■■ ↑ ■■■■■■■■■ 1 / 18

200431506 ■■■■■■ ↓ ■■■■■■■■■■■ 1 / 18

200431601

200431602

200431603 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431604 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431605 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200431606 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431607 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431608

200431609

200431610

200431701 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

200431702 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431703 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

200431704

200431705

200431706 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431707

200431708

200432001

200432002 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432003 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

Chart F-2 (Page 2): Summary of Hematology Data for Viagen Clone Progeny and Comparators (July 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
200432004 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200432005 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200432006 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432007

200432008

200432101 ■■■■■ ↓ ■■■■■■■■ ↑ ■■■ 2 / 18

200432102

200432103 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432104 ■■■■ ↓ ↑ ■■■■■ ↑ ■ ■ ↑ ■■■ 4 / 18

200432105

200432106 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432107

200432108 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200432109 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432301 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432302 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432303 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432304 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432305

200432306

200432307

200432308

200432309 ■■■■■■■■■■■ ↑ ↑ ■■■■■ 2 / 18

200432310

200432311 ■■■■■■■■■■■■ ↑ ■ ↑ ■■■ 2 / 18

200432312 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200432401 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432402

200432403 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200432404 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432405 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200432406

200432407 ■■■■■■■■ ↑ ■■■■■■■■■ 1 / 18

200432408

200432501 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432502 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432503 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432504 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432505

200432506 ■ ■ ↑ ■ ■ ↓ ■■■■■■■■■■■■ 2 / 18

200432507 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432508 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432701 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432702 ■■■■■■■■■■■■■■■■■■ 0 / 18

Chart F-2 (Page 3): Summary of Hematology Data for Viagen Clone Progeny and Comparators (July 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
200432703 ■■■■■■■■■■■ ↑ ■■■■■■ 1 / 18

200432704 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200432705 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200432706 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433001 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433002 ■■■■■■■■■■■ ↑ ■■■■■■ 1 / 18

200433003 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433004 ■■■■■ ↑ ■■■■■■■ ↑ ■■■■ 2 / 18

200433005 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433006 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433007 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200433008 ■■■■■■■■■■■■■ ↑ ■■■■ 1 / 18

200433009

200433010 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433011

200433201 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433202 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200433203 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433204 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433205 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200433206 ■ ■ ↑ ■ ■ ↓ ■■■■■■■■■■■■ 2 / 18

200433207 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433208 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200433601 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433602 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200433603 ■ ■ ↑ ■ ■ ↓ ■■■■■■■■■■■■ 2 / 18

200433604 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433605 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433701 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200433702 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433703 ■■■■■■■■■■■■■■ ↑ ■■■ 1 / 18

200433704 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433705

200433706

200433707 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200433708

200433709 ■■■■■ ↓ ■■■■■■■■ ↑ ■■■ 2 / 18

200433710 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433711 ■■■■■■■■■■■ ↑ ■■■■■■ 1 / 18

200433712 ■■■■■ ↓ ■■■■■ ↑ ■■■■■■ 2 / 18

200433801

200433802 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200433803 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200433804 ■■■■■■■■■■■■■■■■■■ 0 / 18

Chart F-2 (Page 4): Summary of Hematology Data for Viagen Clone Progeny and Comparators (July 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
200433805

200433806 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200433807 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200433808 ■■■■■■■■■■■ ↑ ■■■■■■ 1 / 18

200433809

200434101 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200434102 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434103 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200434104 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200434105 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434106 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434107 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434108 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200434109 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200434110 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434111 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434112 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200434113 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434114 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200434115 ■■■■■ ↑ ■■■■■■■■■ ↑ ■ ■ 2 / 18

200434401 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434402

200434403 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200434501

200434502

200434503

200434504

200434505

200434506

200434507 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434508

200434509 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434510 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434511 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200434512 ■■■■ ↓ ↑ ↑ ■■■■■ ↑ ■ ↑ ■■■ 5 / 18

200434601 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434602 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434603 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434604 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434605 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200434606 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200434607

200434608 ■■■■■■■■■■■■■■■■■■ 0 / 18

200435801 ■ ■ ↑ ■ ■ ↓ ■■■■■■■■■■■■ 2 / 18

Chart F-2 (Page 5): Summary of Hematology Data for Viagen Clone Progeny and Comparators (July 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
200435802 ■■■■■ ↓ ■■■■■ ↑ ■■■■■■ 2 / 18

200435803 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436001 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436002 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200436003

200436004 ■■■■■■■■■■ ↑ ■■■■■■■ 1 / 18

200436005 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436006 ■■■■■■■■■■■■■ ↑ ■ ■ ↑ ■ 2 / 18

200436007 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436008

200436009

200436010 ■■■■■■■■■■■■■ ↑ ■■■■ 1 / 18

200436011 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436012 ■■■■■■■■■■■ ↑ ■■■■■■ 1 / 18

200436013 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436014

200436401 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200436402 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436403 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200436404

200436405 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436601 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436602

200436603 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436604 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436605 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436606 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436607 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200436608 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436609 ■■■ ↓ ↓ ↓ ■ ■ ↑ ■■■ ↑ ■ ↑ ■■■ 6 / 18

200436610 ■■■■ ↓ ↓ ■■■■■■ ↑ ■ ↑ ■■■ 4 / 18

200436611 ■ ■ ↑ ↓ ↓ ↓ ■ ■ ↑ ↓ ■■■ ↓ ↑ ■■■ 8 / 18

200436612 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436901 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436902 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436903 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436904 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436905

200436906 ■■■■■ ↓ ■■■■■■■■■■ ↑ ■ 2 / 18

200436907

200436908 ■■■■■ ↑ ↑ ■■■■■■■■■■ ↑ 3 / 18

200436909 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436910 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200436911

Chart F-2 (Page 6): Summary of Hematology Data for Viagen Clone Progeny and Comparators (July 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
200436912 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436913

200437401 ■■■■ ↑ ■■■■■■■■ ↑ ■■■■ 2 / 18

200437402 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437403 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437404 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

200437405 ■■■■■■■■ ↓ ■■■■■■■■■ 1 / 18

200437406 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437407 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437408

200437409 ■■■■■■■■■■■ ↑ ■■■■■■ 1 / 18

200437410 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437411

200437501 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437502 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437503

200437504 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437505 ■■■■■■■■ ↓ ■■■■■■■■■ 1 / 18

200437506 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200437507 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437508

200437509 ■■■■■ ↓ ■■■■■ ↑ ■■■■■■ 2 / 18

200437510 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200437801 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437802 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200437803 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200437804 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438101 ■■■■■■■■ ↓ ■■■■■■■■■ 1 / 18

200438102 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438103 ■■■■■■■■ ↓ ■ ↑ ■■■■■ ↑ ↑ 4 / 18

200438104

200438105 ■ ■ ↑ ■ ■ ↓ ■ ■ ↓ ■■■■■■■■■ 3 / 18

200438106 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200438107 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200438601 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200438602 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200438603 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438604 ■■■ ↓ ↓ ■■■■■■■ ↑ ↓ ↑ ■■■ 5 / 18

200438605 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438606 ■■■■■■■■ ↓ ■■■■■■■■■ 1 / 18

200438607

200438608

200438609 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438610 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

Chart F-2 (Page 7): Summary of Hematology Data for Viagen Clone Progeny and Comparators (July 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
200438611 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

200439501 ■■■■■■■■■■■■■■■■■■ 0 / 18

200439502

200439503 ■■■■■■■■■■■■■■■■■■ 0 / 18

200439504

200439505 ■■■■■ ↓ ■■■■■■■■■■■■ 1 / 18

0 0 9 3 7 76 4 0 12 1 3 15 9 7 10 1 7 3

//////// / / / / / / / / / /

235 235 235 235 235 235 235 235 235 235 235 235 235 235 235 235 235 235

Summary

Chart F-2 (Page 8): Summary of Hematology Data for Viagen Clone Progeny and Comparators (July 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200430601 ↓ ■ ↑ ■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■

200430602 ■■■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■■■■■■200430603 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430604 ■■■■■ ↓ ■■■■■■■■ ↑ ■■■■ ↑ ■■■■■■■■■■■200430605 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430606 ■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■200430607 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430608 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430609 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■200430610 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430611 ■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■ ↑ ■■■■■200430701 ■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■200430702 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200430703 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430704 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430705 ■ ■ ↑ ■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■200430706 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430707 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430708 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430709 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430710 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200430711 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430801 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430802 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430803 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430804 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430805 ■■■■■■■■■■■■■■■■ ↓ ■■■■■■■■■■■■■■

Chart F-3 (Page 1): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (October 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200430901200430902 ■■■■■ ↓ ■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■

200430903 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■ ↑ ■■■■■200430904200430905 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430906 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■200431001 ■ ■ ↑ ■■■■■■■■■■■ ↑ ■■■■■■■■■■ ↑ ■■■■■200431002 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431003 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431004 ■■■ ↑ ■■■■■■■■■■■■ ↓ ■■■■■■■■■■■■■■200431005 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431006 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431007 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431008 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431009 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431010 ■ ■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431011 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431012 ■■■■■■■■■■■■■■■■ ↓ ■■■■■■■■■■■■■■200431013200431401 ■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■200431402 ■■■■■ ↑ ■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■200431403 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200431404 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200431405 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■ ↑ ■■■■■200431406 ■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■200431407 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431408 ■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■■■■ ↑ ■■■■■■

Chart F-3 (Page 2): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (October 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200431409 ■■■■■ ↓ ■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■

200431410 ■■■■■■■■■■■■■■ ↑ ■■■■ ↑ ■■■■■■■■■■■200431411 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200431501 ■■■ ↑ ■ ↑ ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200431502 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200431503 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431504 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200431505 ■■■■■ ↓ ■■■■■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■ 200431506 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200431601 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431602 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431603 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431604 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431605 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431606 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431607 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431608 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431609200431610 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200431701 ■ ■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431702 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431703 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431704 ■■■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■■■■■■200431705200431706 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431707200431708 ■ ■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-3 (Page 3): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (October 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200432001 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200432002 ■■■ ↑ ■■■■ ↑ ■■■■■■■■■■■ ↓ ■■■■■■■■■■200432003 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432004 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432005 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432006 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200432007 ■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■200432008 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432101 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■200432102 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432103 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432104 ■■■■■■■■■■■■■ ↑ ↑ ■■■■ ↑ ■■■■■■■■■■■200432105 ■■■■■■■■■■■■■■■■ ↓ ■■■■■■■■■■■■■■200432106 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432107200432108 ■■■■■ ↓ ■■■■■■■■■■ ↓ ■■■■■■■■■■■■■■200432109 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432301 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432302 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432303 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■200432304 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432305 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432306 ■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■200432307 ■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■200432308 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432309 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200432310 ■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-3 (Page 4): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (October 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200432311 ■■■■■ ↓ ■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■

200432312 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200432401 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432402 ■■■■■■■■■■■■■■■■ ↓ ■■■■■■■■■■■■■■200432403 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432404 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432405 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432406 ■■■■■ ↓ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■200432407 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432408200432501 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432502 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432503 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432504 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432505200432506 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200432507 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200432508200432701 ■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■200432702 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432703 ■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■200432704 ■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■200432705 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432706 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433001 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200433002 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433003 ■■■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■■■■■■

Chart F-3 (Page 5): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (October 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200433004 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■

200433005 ■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■200433006 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433007 ■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■200433008 ■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200433009200433010 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200433011 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433201 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433202 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433203 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433204 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433205 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200433206 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200433207 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200433208 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200433601 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433602 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433603 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433604 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433605 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433701 ■■■ ↑ ■■■■■■■■■■■■■■■■ ↓ ■■■■■■■■■■200433702 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433703 ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433704200433705 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433706 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-3 (Page 6): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (October 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200433707 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200433708 ↓ ■ ↑ ■■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■200433709 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433710 ■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■200433711 ■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■■■■■■■■200433712 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433801 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433802 ■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■200433803 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■200433804 ■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■200433805200433806 ■■■■■■■■■■■■■■ ↑ ■ ■ ↓ ■■■■■■■■■■■■ ↓ 200433807 ■■■■■ ↓ ■■■■■ ↑ ■ ■ ↑ ■■■■■■■■■■■■■■■■200433808 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200433809 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200434101 ■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■200434102 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434103200434104 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434105 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434106200434107 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434108200434109 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200434110200434111 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434112

Chart F-3 (Page 7): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (October 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200434113 ■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■

200434114200434115200434401 ■■■ ↑ ■ ↑ ■ ■ ↑ ■■■■■■■■■■■■■■■■■■■■■■200434402 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434403 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200434501 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200434502 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434503 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434504 ↑ ■ ↓ ■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■200434505200434506200434507 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434508200434509 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434510200434511 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434512 ■■■■■■■■■■■ ↑ ■ ↑ ■■■■■ ↑ ■■■■■■■■■■■200434601 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434602 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434603 ■■■ ↑ ■ ↑ ■■■■ ↑ ■■■■■■■■ ↑ ■■■■■■■■■■■200434604 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434605 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434606 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434607 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200434608 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200435801 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-3 (Page 8): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (October 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200435802 ■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200435803 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436001 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436002 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436003 ■■■ ↑ ■■■■■■■■■■■■■■■■ ↓ ■ ↑ ■■■■■■■■200436004 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436005 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436006 ■■■■■ ↑ ■ ↓ ■■■■■■■■■■■■■■■■■■■■ ↑ ■■200436007 ■■■ ↑ ■ ↑ ■ ↓ ■■■■■ ↑ ■■■■■ ↑ ↓ ■■■■■■■ ↑ ■■200436008 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436009 ■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■■■■200436010 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■200436011 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436012 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436013 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200436014200436401 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436402 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436403 ↓ ■ ↑ ■■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■200436404 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436405 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436601 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436602 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436603 ■■■ ↑ ■ ↑ ↑ ■■■■ ↑ ■ ↑ ↑ ■■■■■■■■■■ ↑ ■■■ ↑ ■ 200436604 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436605200436606 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-3 (Page 9): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (October 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200436607200436608 ■ ↑ ■■■■■■■■■ ↓ ■ ■ ↑ ↓ ■■■■■■■ ↑ ■■■■■■■

200436609200436610 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■200436611200436612 ■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■200436901 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436902 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■200436903 ■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■200436904200436905 ■■■■■■ ↑ ■■■■■■■ ↑ ■■■■■■■■■■■■■■■■200436906200436907200436908200436909 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436910200436911 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436912 ■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■200436913 ■■■■■ ↑ ■■■■■■■ ↑ ■■■■■ ↑ ■ ■ ↑ ■■■■■■■■200437401 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437402 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437403200437404 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■200437405200437406 ■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■200437407200437408 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-3 (Page 10): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (October 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200437409200437410200437411200437501 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200437502 ■■■ ↑ ■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■200437503200437504 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437505200437506 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■200437507200437508200437509 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■200437510 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■ ↑ ■■■■■200437801 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437802 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■200437803 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437804 ■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■ ↑ ■■■■■200438101 ■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■200438102 ■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■■■■■■■■200438103200438104 ■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■200438105 ■■■ ↑ ■■■■■■■ ↑ ■ ↑ ■■■ ↑ ■ ■ ↓ ■■■■■■■■■■200438106 ■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■ ↑ ■200438107 ■■■ ↑ ■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■200438601200438602 ■■■■■ ↓ ↑ ■ ■ ↓ ■■■■■■ ↓ ↓ ■■■■■■■■■■ ↓ ■ ↓ 200438603 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-3 (Page 11): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (October 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200438604 ■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■

↑ 

200438605 ■■■■■■■■■

↑ ■■■■■■

↑

↑

↑ ■■■■

↑ ■ ■

↑

↑

↑

■

↑

200438606 ■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■

200438607 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438608 ■■■■■■■■■

↓ ■■■■■■■■■■■■■■■■■■■■■

200438609 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438610 ■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■

200438611 ■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■

200439501 ■■■■■■■■■■■■■■■■

↓

↓ ■■■■■■■■■■■■

↓ 

200439502 ■■■■■■■■■■■■■■■■

↓ ■■■■■■■■■■■■■

↓ 

200439503 ■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■

↑ 

200439504 ■■■■■■■■■■■■■■■■

↓

↓ ■■■■■■■■■■■■

↓ 

200439505 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

5 2 9 13 0 42 9 4 5 6 6 13 2 11 25 1 12 11 3 20 5 0 2 4 1 11 1 1 6 4 8 

///////////////////////////////

264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 264 

Summary

Chart F-3 (Page 12): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (October 2004)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

200430601 ■■■■■■■ ↓ ■■■■ ↑ ■■■■■ 2 / 18

200430602

200430603 ■■■■■■■ ↓ ■ ↓ ■■■■■■■■ 2 / 18

200430604 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430605 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430606

200430607 ■ ■ ↑ ■ ↑ ■■■■■■■■ ↑ ■■■■ 3 / 18

200430608 ■■■■■■■ ↓ ■ ↓ ■■■■■■■■ 2 / 18

200430609 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430610 ■■■■■■■ ↓ ■ ↓ ■■■ ↑ ■■■■ 3 / 18

200430611 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430701

200430702 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430703 ■■■■■■■■■■ ↑ ■■■■■■■ 1 / 18

200430704 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430705 ■■■■■■■■■■■■ ↑ ■■■■■ 1 / 18

200430706 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430707 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430708 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430709

200430710 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430711 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430801 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430802 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200430803 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430804 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430805 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430901

200430902 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430903 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430904

200430905 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430906

200431001 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431002 ■■■■■■■■■■■■ ↑ ■■■■■ 1 / 18

200431003 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200431004 ■■■■■■■■■■■ ↓ ↓ ■■■■■ 2 / 18

200431005 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431006 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431007 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

Summary

Chart F-4 (Page 1): Summary of Hematology Data for Viagen Clone Progeny and Comparators (October 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

Summary

200431008 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200431009 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431010 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431011 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431012 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431401 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431403 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431404 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431406

200431407 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431408 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431409 ↑ ■■■■■■■■■■■■■■ ↑ ■ ■ 2 / 18

200431410 ■■■■■■■ ↓ ■ ↓ ■■■ ↑ ■■■■ 3 / 18

200431411 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431501

200431502 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431503 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431504 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431505 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431506 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431601 ■■■■■■■ ↓ ■ ↓ ■■■■■■■■ 2 / 18

200431602 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431603 ■■■■■■■ ↓ ■ ↓ ■■■■■■■■ 2 / 18

200431604 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200431605 ↑ ■■■■■■■■■■■■■■ ↑ ■ ■ 2 / 18

200431606 ■■■ ↓ ↓ ■■■■■■■■■■■■■ 2 / 18

200431607

200431608 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431609

200431610 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431701 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431702 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431703 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431704 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431705

200431706 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431707

200431708 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432001 ■ ↑ ↑ ■ ■ ↑ ↑ ■■■■■■■■■■ ↑ 5 / 18

200432002 ■■■■■■■■■■■■■■■■■■ 0 / 18

Chart F-4 (Page 2): Summary of Hematology Data for Viagen Clone Progeny and Comparators (October 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

Summary

200432003 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432004 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432005 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432006 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432007 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432008 ■■■■■■■ ↑ ■■■■■■■■■■ 1 / 18

200432101 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432102

200432103

200432104

200432105 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432106 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200432107

200432108 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432109 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432301

200432302 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432303

200432304 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432305 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432306 ■■■■■■■■■■■■ ↑ ■■■■■ 1 / 18

200432307 ■■■■■■■ ↓ ■ ↓ ■■■■ ↑ ■■■ 3 / 18

200432308 ■ ↑ ■■■■ ↑ ■■■■■■■■■■■ 2 / 18

200432309 ■■■■■■■ ↓ ■ ↓ ■■■■ ↑ ■■■ 3 / 18

200432310 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432311 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432312 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432401 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432402 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432403 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432404 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432405 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432406 ■■■ ↓ ↓ ■■■■■■■■ ↓ ■■■■ 3 / 18

200432407 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432408

200432501 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432502 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432503 ■ ■ ↑ ■■■■■■■■■■■■■ ↑ ■ 2 / 18

200432504 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432505

Chart F-4 (Page 3): Summary of Hematology Data for Viagen Clone Progeny and Comparators (October 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

Summary

200432506 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432507 ■ ↑ ■■■■■■■■■■■■■■■■ 1 / 18

200432508

200432701 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432702 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432703 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200432704 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432705 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432706 ■■■■■■■ ↑ ■ ↑ ■■■■■■■■ 2 / 18

200433001 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433002 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200433003 ■ ↑ ■■■■■■■■■■■■■■■■ 1 / 18

200433004 ■ ↑ ■■■■■■■■■ ↑ ■■■■■■ 2 / 18

200433005 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433006 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433007 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433008 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433009

200433010 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

200433011 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200433201 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433202 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200433203 ■ ↑ ↑ ■■■ ↑ ■■■■■■■■■■ ↑ 4 / 18

200433204 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433205 ■ ↑ ■■■■■■■■■■■■■■■■ 1 / 18

200433206 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433207 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433208 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433601 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433602 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200433603 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433604 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433605 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200433701 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433702 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433703 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433704 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433705 ■■■■ ↑ ■■■■■■■■■■■■■ 1 / 18

200433706 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433707 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

Chart F-4 (Page 4): Summary of Hematology Data for Viagen Clone Progeny and Comparators (October 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

Summary

200433708 ■■■■■ ↑ ■ ↓ ■ ↓ ■■■■■■■■ 3 / 18

200433709 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433710 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433711 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433712 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433801 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433802 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433803 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433804 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433805

200433806 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433807 ■■■■■■■ ↓ ■ ↓ ■■■■ ↑ ■■■ 3 / 18

200433808 ■■■■■■■ ↓ ■ ↓ ■■■ ↑ ↑ ■■■ 4 / 18

200433809 ■■■■■ ↑ ■■■■■■■■ ↑ ■■■ 2 / 18

200434101 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434102 ■ ↑ ■■■■ ↑ ■■■■■■■■■■ ↑ 3 / 18

200434103 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434104 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434105 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434106 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434107 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434108 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434109 ■ ↑ ■■■■■■■■ ↑ ■■■■■■■ 2 / 18

200434110 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434111 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434112 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434113 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200434114

200434115

200434401 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434402 ■ ↑ ■■■■ ↑ ■■■■■■■■■■ ↑ 3 / 18

200434403 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434501 ■ ■ ↑ ■ ■ ↑ ■■■■■■■■■■■■ 2 / 18

200434502 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200434503 ■ ↑ ■■■■■■■■■■■■■■■■ 1 / 18

200434504 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434505

200434506 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434507 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200434508 ■■■■■■■■■■■■■■■■■■ 0 / 18

Chart F-4 (Page 5): Summary of Hematology Data for Viagen Clone Progeny and Comparators (October 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

Summary

200434509 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434510 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434511 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434512 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434601 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434602 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434603 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200434604 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200434605 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434606 ■ ↑ ■■■■■■■■■■■■■■■■ 1 / 18

200434607 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434608 ■■■■■■■■■■■■■■■■■■ 0 / 18

200435801 ■■■■■■■■■■■■■■■■■■ 0 / 18

200435802 ■■■ ↑ ↑ ■■■■■■ ↑ ■ ↑ ■■■■ 4 / 18

200435803 ■■■ ↑ ↑ ■■■■■■■■ ↑ ■■■■ 3 / 18

200436001 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436002 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436003 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436004 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436005 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436006 ■■■■■■■■■■■■■ ↑ ■■■■ 1 / 18

200436007 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436008 ■■■■■■■■■■■■■ ↑ ■■■■ 1 / 18

200436009 ■■■ ↑ ↑ ■■■■■■■■ ↑ ■■■■ 3 / 18

200436010 ■■■■■■■■■■■■■ ↑ ■■■■ 1 / 18

200436011 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436012 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436013 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436014

200436401 ■ ↑ ■■■■■■■■■■■■■■■■ 1 / 18

200436402 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436403 ■ ↑ ■■■■■■■■■■■■■■■■ 1 / 18

200436404 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436405 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436601 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436602 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436603 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436604 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436605 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436606 ■■■■■■■■■■■■■■■■■■ 0 / 18

Chart F-4 (Page 6): Summary of Hematology Data for Viagen Clone Progeny and Comparators (October 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

Summary

200436607 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436608 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436609 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200436610 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436611 ■ ↑ ■■■■■■■■ ↑ ■■■■ ↓ ■ ■ 3 / 18

200436612 ■■■■ ↑ ■■■■■■■■ ↑ ■■■■ 2 / 18

200436901 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436902 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436903 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200436904 ■ ↑ ■■■■ ↑ ■■■■■■■■■■ ↑ 3 / 18

200436905

200436906 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436907 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436908 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436909 ■■■■ ↑ ■■■■■■■■■■■■■ 1 / 18

200436910 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436911 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436912 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436913 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437401 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437402 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437403 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437404 ■ ↑ ■■■■■■■■ ↑ ■■■■■■■ 2 / 18

200437405

200437406 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437407

200437408 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437409

200437410

200437411

200437501 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437502 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431013

200431402

200431405

200437503

200437504 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437505

200437506 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437507

Chart F-4 (Page 7): Summary of Hematology Data for Viagen Clone Progeny and Comparators (October 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

Summary

200437508

200437509 ■■■■ ↑ ■■■■■■■■ ↑ ■■■■ 2 / 18

200437510 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437801 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437802 ■■■■■■■■■■■■■■ ↑ ■■■ 1 / 18

200437803 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437804 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438101 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438102

200438103

200438104

200438105 ■■■■■■■■■■■■ ↓ ■■■■■ 1 / 18

200438106 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438107 ■■■■■■■■■■■■ ↑ ■■■■■ 1 / 18

200438601

200438602 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438603 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438604 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438605 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

200438606 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438607 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438608 ■■■ ↓ ■■■ ↓ ■ ↓ ■■■■■■ ↑ ■ 4 / 18

200438609 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438610 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200438611 ■■■■■■■ ↓ ■ ↓ ■■■■■■■■ 2 / 18

200439501 ■■■■■■■■■■■■■■ ↑ ■■■ 1 / 18

200439502 ■■■ ↓ ↓ ■■■■■■■■ ↓ ■■■■ 3 / 18

200439503 ■■■■■■■■■■■■■■■■■■ 0 / 18

200439504 ■■■■■■■■■■■■■■■■■■ 0 / 18

200439505 ■■■■■■■■■■■■■■ ↑ ■■■ 1 / 18

2 17 20 7 11 9 6 16 0 14 4 3 7 14 8 3 5 5

//////////////////

265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265

Summary

Chart F-4 (Page 8): Summary of Hematology Data for Viagen Clone Progeny and Comparators (October 2004) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200430601200430602

■

↑ ■■■■■■■■■■■

↑ ■■■■■■■■■■■■

↑ ■■■■

200430603 ■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■

↑ ■

200430604 ■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■■

200430605 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430606 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430607 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430608 ■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■

↑ ■

200430609 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430610 ■ ■

↑ ■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■■

200430611 ■■■■■■■■■■

↑ ■■■■■■■■■■■

↑ ■■■■■■■■

200430701 ■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■

↑ ■

200430702 ■■■■■■■■■■■■■

↑ ■■■■■

↑ ■■■■■■■■■

↑ ■

200430703 ■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■

↑ ■

200430704 ■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■

↑ ■ ■

↑ ■

200430705 ■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■

↑ ■

200430706 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430707 ■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■■■

200430708

■

↑ ■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■

200430709 ■■■

↑ ■■■■■■■■■■■■■■■■

↓ ■■■■■■■■■■

200430710 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430711 ■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■

↓

■

↓ ■■

200430801

↓

■

↑

↑ ■■■■■

↓

↓ ■■■■■

↓ ■■■■■■■■

↑ ■■■■

↓

200430802 ■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■

↑ ■

200430803 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430804 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430805

■

↓ ■■■■■■■■■■■■■■■■■■■■■■■■

↓ ■■■■

Chart F-5 (Page 1): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (January 2005)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200430901200430902 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200430903 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200430904200430905 ■■■■■■■■■■■ ↑ ■ ■ ↑ ■■■■■■■■ ↑ ■■■■■■■200430906 ■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■200431001200431002 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431003 ■■■■■■■■■■■■■■■■■■ ↓ ■■■■■■■■■■■■200431004 ■■■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■■■■■■200431005 ■■■■■■■■■■■ ↑ ■ ↑ ■■■■■■■■■■■■■■■ ↑ ■200431006 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431007 ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■200431008 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■ ↑ ■■■■■■■200431009 ■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■ ↓ ■■200431010 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431011 ■■■■■■■■■■■■■ ↑ ↑ ■■■■■■■■■■■■■■ ↑ ■200431012 ■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■200431013200431401 ■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■200431402 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■200431403 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■ 200431404 ■■■■■■■■■■■■■■ ↑ ■■■■■■ ↑ ■■■■■■■■■200431405 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431406 ■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■200431407 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431408 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-5 (Page 2): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (January 2005)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200431409 ■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■■

200431410 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431411 ■■■

↑ ■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■

200431501 ■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■

200431502 ■■■■■■■

↑ ■■■■■

↑ ■■■■■■■■■■■■■■■■■

200431503 ■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■■

200431504 ■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■

200431505 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431506 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431601 ■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■■

200431602 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431603 ■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■

↓ ■■

200431604 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■

200431605 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431606 ■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■

↓ ■■

200431607 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■

200431608 ■■■■■■■■■■■

↑

■

↑

↑ ■■■■■■■■

↑ ■■■■■■■

200431609200431610 ■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■■

200431701 ■■■■■■■■■■■■■

↑ ■■■■■

↑ ■■■■■■■■■

↑ ■

200431702

■

↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200431703 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200431704 ■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■■

200431705200431706 ■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■

↑ 

200431707200431708 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-5 (Page 3): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (January 2005)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200432001 ■■■■■■■■■■ ↑ ■ ■ ↑ ■■■■■■■■■■■■■■■■■

200432002 ■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■200432003 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■200432004 ■■■■■■■■■■ ↑ ↑ ■ ↑ ■■■■■■■■■■■■■■■ ↑ ■200432005 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432006 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200432007 ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■200432008 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432101 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432102 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200432103 ■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■200432104 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■200432105 ↓ ■ ↑ ■■■■■■ ↓ ■■■■■■■■■■■■■■ ↓ ■ ↓ ■■■■ 200432106200432107200432108 ■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■■■ ↑ ■■■■■200432109200432301200432302 ■ ■ ↑ ■■■■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■200432303200432304 ■ ↑ ■■■■■■■■■■■■ ↑ ↓ ■■■■■■■ ↑ ■ ■ ↑ ■ ■ ↑ ■200432305200432306 ↓ ■ ↑ ■■■■■■ ↓ ■ ↓ ■■■■■■■■■■■■ ↓ ■■■■■■ 200432307200432308 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432309200432310 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-5 (Page 4): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (January 2005)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200432311200432312 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200432401 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■200432402 ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■200432403 ■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■ ↓ ■ ↓ ■■200432404 ■■■■■■■■■■■■■ ↑ ↑ ■■■■■■■■■■■■■■ ↑ ■200432405 ■■■■■■■■ ↑ ■■■■ ↑ ■■■■■■■■■■■■■■■■■200432406 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432407 ■■■■■■■■■■■ ↑ ■ ↑ ■■■■■■■■■■■■■■■ ↑ ■200432408200432501 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432502200432503 ■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■ ↓ ■■200432504 ■■■■■■ ↑ ↑ ■■■■■■■■■■■■■■■■■■■■ ↓ ■■200432505200432506 ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■200432507 ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■200432508200432701200432702 ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432703200432704 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200432705 ■■■■■■■■■■■■■■■■■■■ ↑ ■ ↑ ■■■■■■■■■200432706 ■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■ ↑ 200433001 ■■■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■ ↑ ■ ■ ↑ ■200433002 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433003 ■■■■■■■■■■■■■ ↑ ↑ ■■■■■■■■■■■■■■■■

Chart F-5 (Page 5): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (January 2005)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200433004200433005200433006200433007 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■

200433008200433009200433010 ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■200433011 ■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■200433201 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433202200433203 ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■200433204 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433205 ■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■ ↓ ■■200433206 ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■200433207 ■ ↑ ■■■■■■■■ ↑ ■■■ ↑ ↓ ■■■■■■■ ↑ ■ ■ ↑ ■■■■200433208 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433601 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433602 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433603 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433604 ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■200433605 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433701200433702200433703200433704200433705200433706

Chart F-5 (Page 6): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (January 2005)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200433707200433708 ↓ ■ ↑ ■■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■

200433709 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433710 ■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■200433711 ■■■■■■■ ↑ ■ ■ ↑ ■■■■■■■■■■■■■■■■■■■■200433712 ■■■■■■■ ↑ ■ ■ ↑ ■■■■■■■■■■■■■■■■■■■■200433801 ■■■■■■■■■■■■■ ↑ ■■■■■■ ↑ ■■■■■■■■■■200433802 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433803 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200433804 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433805200433806 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200433807 ■■■■■■■ ↑ ■■■■■■ ↑ ■■■■■■■■■■■■■ ↓ ■■200433808 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200433809 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434101 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434102 ■■■■■■■ ↑ ■■■■■■■■■ ↑ ■■■■■■■■■■■■■200434103200434104 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200434105 ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■200434106200434107 ■■■■■■■■■■■■■■■■ ↓ ■ ■ ↑ ■■■■■■■■■■■200434108200434109 ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■200434110200434111 ■■■■■■■■■■■■■■■■■■ ↓ ■■■■■■■■■■■■200434112

Chart F-5 (Page 7): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (January 2005)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200434113 ■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■

↑ ■

200434114200434115200434401 ■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■

↑ ■

200434402 ■■■■■■■

↑ ■■■■■■■■■■■■■

↑ ■■■■■■■■■

200434403 ■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■■

200434501 ■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■

↑ ■

200434502 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434503

■

↑ ■■■■■■■■■■■

↑ ■■■■■■■■■■■■

↑ ■ ■

↑ ■

200434504 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434505200434506200434507 ■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■

↑ ■

200434508200434509 ■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■■■■■

200434510200434511 ■■■■■■■■■■■■■

↑ ■■■■■

↑ ■■■■■■■■■

↑ ■

200434512 ■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■

200434601 ■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■

200434602 ■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■

200434603

■

↑ ■■■■■■■■■■■

↑ ■■■

↓

■

↑ ■■■■■■■

↑

■

↑ ■

200434604 ■■■■■■■■■■■■■■■■■■■

↑ ■■■■■

↑ ■■■■■

200434605 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■

200434606 ■■■■■■■■■■■

↓ ■■■■■■■

↑ ■■■■■■■■■■■

200434607 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200434608 ■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■

↑ ■

200435801 ■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■

Chart F-5 (Page 8): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (January 2005)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200435802 ■■■■■■■■■■■■■■■■■

↑

■

↑ ■■■■■■■■■■■

200435803 ■■■■■■■

↓ ■■■■■■■■■■■■■■■■■■■■■■■

200436001 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■

200436002 ■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■

200436003 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■

200436004 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436005 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436006 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436007 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436008

↑ ■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■

↑ ■

200436009 ■■■

↑ ■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■

200436010 ■■■■■■■

↑ ■■■■■■■■■■■

↑ ■■■■■■■■■■■

200436011 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

↑ ■

200436012 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436013 ■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■■

200436014200436401 ■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■

↑ ■

200436402 ■■■■■■■■■■■■■■

↑ ■■■■

↑ ■■■■■■■■■■■

200436403 ■ ■

↑ ■■■■

↑ ■■■■■■■■■■■■■■■■■■

↓ ■■■■

200436404

■

↑ ■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■■

200436405 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436601 ■■■■■■■

↓ ■■■■■

↑ ■■■■■■■■■■■■■■■■■

200436602 ■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■

200436603 ■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■

200436604 ■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■

↑ ■ ■

↑ ■

200436605200436606 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

Chart F-5 (Page 9): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (January 2005)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200436607 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200436608 ■ ↑ ■■■■■■■■■■■■■ ↓ ■■■■■■■■■■■■■■■200436609200436610 ■■■■■■■■ ↓ ■■■■■■■■■■■■■■■■■■■■■■200436611200436612 ■ ■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436901 ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■200436902 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436903 ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■200436904200436905 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■200436906200436907200436908200436909 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■200436910200436911 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436912 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200436913 ■■■■■■■■■■■■■■■■■■■■■■■■■■ ↓ ■■■■200437401 ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■200437402 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■ ↑ ■200437403200437404 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437405200437406 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437407200437408 ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■

Chart F-5 (Page 10): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (January 2005)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200437409200437410200437411200437501 ■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■

200437502 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437503200437504 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437505200437506 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200437507200437508200437509 ■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■ ↑ ■200437510 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200437801 ■■■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■■■■ ↑ ■200437802 ■■■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■ ↑ ■■■■■■■ 200437803 ■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■ ↓ ■■■■200437804 ■■■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■200438101 ■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■■■■■■■■200438102 ■■■■■■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■200438103200438104 ■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■■■■■■200438105 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438106 ■■■■■■■■■■■ ↑ ■■■■■ ↑ ■■■■■■■■■■■■■200438107 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438601200438602 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■200438603 ■■■■■■■■■■■■ ↑ ■■■■■■■■■■■■■■■■■■

Chart F-5 (Page 11): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (January 2005)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

A/G

Anion Gap

Globulin

Hemolysis

Icterus

Bilirubin-i

Lipemia

NaK

% Sat

Albumin-bulk

Alk Phos

ALT/P5P

Amylase

AST/P5P

hBA

Bicarbonate

Calcium

Chloride

Cholesterol

CK

Creatinine

Bilirubin-d

GGT

Glucose

Iron

Lipase

MG - XB

Phosphate

Potassium

SDH

Sodium

200438604

■

↑ ■■■■■■■■■■■

↑

■

↓ ■■■■■■■■■■■■■

↑ ■

200438605 ■■■■■■■■■■■

↑

■

↑ ■■■■■■■

↑ ■■■

↑

↑

↑

■

↑ ■

200438606 ■■■■■■■

↑ ■■■■■■■■■■■■■■■■■■■■■■■

200438607 ■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■

200438608 ■ ■

↑ ■■■■■■■■■■■■■■■■■■■■■■■■■■■■

200438609 ■■■■■■■■■■■■■■■■■■■

↑ ■■■■■■■■■■■

200438610 ■■■■■■■■■■■

↑

↑

↑ ■■■■■

↑ ■■■■■■■■■■■

200438611 ■■■■■■■■

↓

↓ ■■■■■■

↓ ■■■■■■■

↓

■

↓ ■■■■

200439501 ■■■■■■■■■■■

↑

■

↑ ■■■■■■■■■■■■■■■■■

200439502 ■■■■■■■■■■■■■

↑

↑ ■■■■■■■■■■■■■■■■

200439503 ■■■■■■■

↓ ■■■

↑

■

↑ ■■■■■■■■■■■■

↑

■

↑

↑ ■

200439504 ■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■

↑ ■ ■

↑ ■

200439505 ■■■■■■■■■■■■■

↑ ■■■■■■■■■■■■■■■■■

7 11 9 5 0 0 1 34 3 5 11 14 2 59 17 4 5 10 5 29 2 5 2 8 3 6 18 2 10 53 3 

///////////////////////////////

241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 241 

Summary

Chart F-5 (Page 12): Summary of Clinical Chemistry Data for Viagen Clone Progeny and Comparators (January 2005)■ In Comparison Range↑/↓ Animal is above/below reference rangeBlank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

200430601

200430602 ■■■■ ↑ ↑ ■■■■■■■■■■■■ 2 / 18

200430603 ■■■■■■■■■■■■■■■■■■ 0 / 18

200430604

200430605

200430606

200430607

200430608 ■■■■■■■■■■■■■ ↑ ↑ ■■■ 2 / 18

200430609

200430610 ■■■■■■■■■■■■ ↑ ■ ↑ ■ ↑ ↑ 4 / 18

200430611 ■■■■■■■■ ↓ ■■■■■ ↑ ■■■ 2 / 18

200430701 ■■■ ↑ ↑ ■■■■■■■■ ↑ ■■■■ 3 / 18

200430702 ■■■■■■■■■■■■■■ ↑ ■■■ 1 / 18

200430703

200430704

200430705

200430706

200430707

200430708

200430709

200430710

200430711

200430801 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

200430802

200430803

200430804

200430805

200430901

200430902

200430903

200430904

200430905

200430906

200431001

200431002 ↑ ■■■■■■■■■■■■■■■■■ 1 / 18

200431003

200431004 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200431005 ■■■ ↑ ↑ ■■■■■■■■■■■■■ 2 / 18

200431006

200431007 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

Summary

Chart F-6 (Page 1): Summary of Hematology Data for Viagen Clone Progeny and Comparators (January 2005) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

Summary

200431008

200431009

200431010 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431011

200431012 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431013

200431401

200431402 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200431403

200431404

200431405

200431406

200431407

200431408

200431409 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431410 ■■■■■■■■■■■■■ ↑ ■■■■ 1 / 18

200431411

200431501

200431502

200431503 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431504

200431505

200431506 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431601 ■■■■■■■■■■■■■■■■■■ 0 / 18

200431602 ■■■■■ ↑ ■ ■ ↓ ■■■■■■■ ↑ ■ 3 / 18

200431603 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200431604

200431605 ↑ ■■■■ ↑ ■■■■■■■■■■■■ 2 / 18

200431606

200431607

200431608

200431609

200431610

200431701 ↑ ■■■■ ↑ ■■■■■■■■■ ↑ ■ ■ 3 / 18

200431702 ■ ■ ↑ ■ ■ ↑ ■■■■■■■■■■■■ 2 / 18

200431703

200431704

200431705

200431706

200431707

Chart F-6 (Page 2): Summary of Hematology Data for Viagen Clone Progeny and Comparators (January 2005) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

Summary

200431708 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432001 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432002

200432003 ↑ ■■■■■■■■■■■■■■ ↑ ■ ■ 2 / 18

200432004

200432005 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432006

200432007 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200432008 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432101

200432102 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432103

200432104 ■■■■■■■■■■■■■■■■ ↑ ↑ 2 / 18

200432105

200432106

200432107

200432108

200432109

200432301

200432302

200432303

200432304 ■■■■■■■■ ↓ ■■■ ↑ ■ ↓ ■■■ 3 / 18

200432305

200432306 ■■■■■■■■ ↑ ↓ ■ ■ ↑ ■ ↓ ■■■ 4 / 18

200432307

200432308

200432309

200432310

200432311

200432312 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432401 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200432402

200432403 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432404

200432405

200432406

200432407 ■■■■■■■■■■■■■■■■■■ 0 / 18

200432408

200432501

200432502

Chart F-6 (Page 3): Summary of Hematology Data for Viagen Clone Progeny and Comparators (January 2005) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

Summary

200432503

200432504 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

200432505

200432506 ↑ ■■■■■■■■■■■■■■ ↑ ■ ■ 2 / 18

200432507 ■■■■ ↑ ■■■■■■■■ ↑ ■■■■ 2 / 18

200432508

200432701

200432702

200432703

200432704

200432705

200432706

200433001 ■■■■ ↑ ■■■■■■■■ ↑ ■■■■ 2 / 18

200433002 ■■■■■■■■■■■■■ ↑ ■■■■ 1 / 18

200433003 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200433004

200433005

200433006

200433007 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433008

200433009

200433010 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

200433011 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433201

200433202 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433203 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433204 ■■■■■■■■■■■■■ ↑ ■■■■ 1 / 18

200433205

200433206

200433207

200433208 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433601

200433602

200433603

200433604

200433605

200433701

200433702

200433703

200433704

Chart F-6 (Page 4): Summary of Hematology Data for Viagen Clone Progeny and Comparators (January 2005) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

Summary

200433705

200433706

200433707

200433708 ■■■■■■■■ ↑ ↓ ■■■■■■■■ 2 / 18

200433709 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433710

200433711

200433712 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433801 ■■■■■■■■■■■■■ ↑ ↑ ■■■ 2 / 18

200433802 ■■■■■■■■■■■■■■■■■■ 0 / 18

200433803

200433804

200433805

200433806 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

200433807

200433808

200433809

200434101 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434102 ■■■■■■■■ ↑ ■■■■■■■■■ 1 / 18

200434103

200434104 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434105 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434106

200434107 ■■■■ ↑ ■■■■■■■■■■■ ↑ ↑ 3 / 18

200434108

200434109 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434110

200434111 ■■■■■■■■■■ ↑ ■■■■■■■ 1 / 18

200434112

200434113 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434114

200434115

200434401

200434402

200434403

200434501 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434502 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200434503 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434504 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434505

Chart F-6 (Page 5): Summary of Hematology Data for Viagen Clone Progeny and Comparators (January 2005) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

Summary

200434506

200434507

200434508

200434509 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434510

200434511 ■■■■■■■■ ↓ ■■■■■■■■■ 1 / 18

200434512

200434601

200434602 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434603 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200434604 ■■■■■ ↑ ■ ■ ↑ ■■■■■■■■■ 2 / 18

200434605 ■■■■■■■■■■■■■ ↑ ■■■■ 1 / 18

200434606 ■■■■■■■■ ↓ ■■■■ ↑ ■■■■ 2 / 18

200434607 ■■■■■■■■■■■■■■■■■■ 0 / 18

200434608 ■■■■■■■■■■■■■■■■■■ 0 / 18

200435801 ■■■■■■■■■■■■■■■■■■ 0 / 18

200435802

200435803 ■■■■■■■■■■■■■ ↑ ■■■■ 1 / 18

200436001 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436002 ■■■■■■■■■■■■■ ↑ ■■■■ 1 / 18

200436003 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436004 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436005 ■■■■■ ↑ ■■■■■■■■■■■■ 1 / 18

200436006 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436007 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436008 ■■■ ↑ ↑ ■■■■■■■■ ↑ ■■■■ 3 / 18

200436009

200436010

200436011

200436012

200436013 ■■■■■■■■■■■■■ ↑ ■■■■ 1 / 18

200436014

200436401 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436402

200436403 ■■■■■ ↑ ■■■■■■■■■■ ↑ ■ 2 / 18

200436404 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436405

200436601 ■■■■ ↑ ■■■■■■■■■■■■■ 1 / 18

200436602 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436603 ■■■■■■■■■■■■■■■■■■ 0 / 18

Chart F-6 (Page 6): Summary of Hematology Data for Viagen Clone Progeny and Comparators (January 2005) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

Summary

200436604 ■■■■ ↑ ↑ ■■■■■■■■■■■■ 2 / 18

200436605

200436606 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436607 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

200436608 ■■■■■ ↑ ■■■■■■■■ ↑ ■ ↑ ■ 3 / 18

200436609

200436610 ■■■■■ ↑ ■■■■■■■■■■ ↑ ■ 2 / 18

200436611

200436612 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

200436901 ■■■■ ↑ ■■■■■■■■ ↑ ■■■■ 2 / 18

200436902 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436903 ■■■■■■■■■■■■■■■■■■ 0 / 18

200436904

200436905 ■■■■■■■■■■■■■ ↑ ■■■■ 1 / 18

200436906

200436907

200436908

200436909

200436910

200436911

200436912 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200436913 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437401 ■■■■■■■■ ↑ ■■■■■■■■■ 1 / 18

200437402 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437403

200437404

200437405

200437406 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437407

200437408 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437409

200437410

200437411

200437501 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437502 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437503

200437504

200437505

200437506

200437507

Chart F-6 (Page 7): Summary of Hematology Data for Viagen Clone Progeny and Comparators (January 2005) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

Retic absBasophilsEosinophilsHemoglobinHematocritLUCLymphocytesMCHMCHCMCVMonocytesMPVPlateletsRBCRDWReticSeg NeutWBC

Summary

200437508

200437509 ■■■ ↑ ↑ ■■■■■■■■ ↑ ■ ■ ↑ ■ 4 / 18

200437510 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

200437801

200437802

200437803 ■■■■■■■■■■■■■■■■■■ 0 / 18

200437804 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

200438101 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438102 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438103

200438104 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438105 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438106 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438107

200438601

200438602 ■■■■■■■■■■■■■ ↑ ■■■■ 1 / 18

200438603 ■■■■■■■■■■■■■■ ↓ ■■■ 1 / 18

200438604 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438605 ■■■■■■■■ ↑ ■■■■■■■■■ 1 / 18

200438606 ■ ■ ↑ ■■■■■■■■■■■■■■■ 1 / 18

200438607 ■■■■■ ↑ ■■■■■■■■■■ ↑ ■ 2 / 18

200438608 ■■■■■ ↑ ■■■■■■■■■■ ↑ ↑ 3 / 18

200438609 ■■■■■■■■■■■■■■■■■■ 0 / 18

200438610 ■■■■■■■■■■■■■■■■ ↑ ■ 1 / 18

200438611 ■■■■■■■■■■■■ ↑ ■■■ ↑ ↑ 3 / 18

200439501 ■■■■■ ↑ ■■■■■■■ ↑ ↑ ■■■ 3 / 18

200439502 ■■■■■■■■■■■■■■■■■■ 0 / 18

200439503 ■■■■■■■■■■■■■■■■■■ 0 / 18

200439504 ■■■■■■■■■■■■■■■■■■ 0 / 18

200439505 ■■■■■■■■■■■■■■ ↑ ■■■ 1 / 18

5 0 6 4 11 19 0 0 11 2 1 0 4 19 11 3 21 5

//////// / / / / / / / / / /

138 138 138 138 138 138 138 138 138 138 138 138 138 138 138 138 138 138

Summary

Chart F-6 (Page 8): Summary of Hematology Data for Viagen Clone Progeny and Comparators (January 2005) ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

TreatmentBloodpHProteinBloodpHProtein

Summary

200430501 Control 0 8 0.3 ■■■ 0 /3

200430502 Control 0 8 1 ■■■ 0 /3

200430503 Control 0 7 0.1 ■ ↓ ■ 1 /3

200430504 Control 0 7.5 0.1 ■■■ 0 /3

200430506 Control 0 8 1 ■■■ 0 /3

200430507 Control 0 7 1 ■ ↓ ■ 1 /3

200430508 Control 0 6 0.3 ■ ↓ ■ 1 /3

200430509 Control 80 8 1 ↑ ■ ■ 1 /3

200430510 Control 0 8 3 ■■■ 0 /3

200431801 Control 0 8 3 ■■■ 0 /3

200431802 Control 0 8 0.1 ■■■ 0 /3

200431803 Control 0 8 0.3 ■■■ 0 /3

200431804 Control 0 7 3 ■ ↓ ■ 1 /3

200431805 Control 0 8 0.1 ■■■ 0 /3

200432601 Control 0 6.5 1 ■ ↓ ■ 1 /3

200432602 Control 0 8 0.3 ■■■ 0 /3

200432604 Control 10 6.5 0.3 ↑ ↓ ■ 2 /3

200432607 Control 0 8 3 ■■■ 0 /3

200432609 Control 0 8 0.1 ■■■ 0 /3

200432610 Control 10 8 0.1 ↑ ■ ■ 1 /3

200432901 Control 0 8 0.3 ■■■ 0 /3

200432904 Control 0 8 3 ■■■ 0 /3

200432906 Control 200 7 3 ↑ ↓ ■ 2 /3

200432907 Control 0 8 1 ■■■ 0 /3

200432909 Control 0 8 3 ■■■ 0 /3

200432911 Control 0 8 0.1 ■■■ 0 /3

200432913 Control 25 6 1 ↑ ↓ ■ 2 /3

200433301 Control 0 8 0.1 ■■■ 0 /3

200433302 Control 0 7.5 0.1 ■■■ 0 /3

200433303 Control 0 8 0.3 ■■■ 0 /3

200433306 Control 0 8 0.1 ■■■ 0 /3

200433307 Control 0 7.5 0.1 ■■■ 0 /3

200433401 Control 0 8 0.3 ■■■ 0 /3

200433402 Control 0 7.5 1 ■■■ 0 /3

200433403 Control 0 8 0.3 ■■■ 0 /3

200433404 Control 0 8 0.3 ■■■ 0 /3

200433405 Control 0 8 1 ■■■ 0 /3

200433406 Control 0 7.5 1 ■■■ 0 /3

200433408 Control 10 8 0.1 ↑ ■ ■ 1 /3

200433409 Control 0 7.5 0.3 ■■■ 0 /3

Chart F-7 (Page 1): Summary of Urinalysis Data for Viagen Clone Progeny and Comparators ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

TreatmentBloodpHProteinBloodpHProtein

Summary

200433501 Control 25 7.5 0.1 ↑ ■ ■ 1 /3

200433502 Control 0 8 0.3 ■■■ 0 /3

200433503 Control 0 7.5 0.3 ■■■ 0 /3

200433504 Control 0 7.5 1 ■■■ 0 /3

200433505 Control 0 8 0.3 ■■■ 0 /3

200433506 Control 0 7.5 0.3 ■■■ 0 /3

200433507 Control 200 7.5 1 ↑ ■ ■ 1 /3

200433510 Control 0 7 0.1 ■ ↓ ■ 1 /3

200433511 Control 0 8 0.1 ■■■ 0 /3

200433512 Control 10 8 0.3 ↑ ■ ■ 1 /3

200434001 Control 0 8 0.1 ■■■ 0 /3

200434003 Control 0 8 3 ■■■ 0 /3

200434004 Control 200 8 1 ↑ ■ ■ 1 /3

200434005 Control 0 8 0 ■■■ 0 /3

200434006 Control 200 6.5 1 ↑ ↓ ■ 2 /3

200434007 Control 0 8 1 ■■■ 0 /3

200434008 Control 0 7.5 3 ■■■ 0 /3

200434009 Control 25 8 0.1 ↑ ■ ■ 1 /3

200434201 Control 0 8.5 3 ■ ↑ ■ 1 /3

200434202 Control 80 8 0.1 ↑ ■ ■ 1 /3

200434203 Control 0 8 0.1 ■■■ 0 /3

200434206 Control 10 7.5 3 ↑ ■ ■ 1 /3

200434301 Control 0 7.5 0.1 ■■■ 0 /3

200434302 Control 0 7.5 0.3 ■■■ 0 /3

200434303 Control 0 8 1 ■■■ 0 /3

200434304 Control 0 6.5 0.3 ■ ↓ ■ 1 /3

200434306 Control 0 7.5 0.3 ■■■ 0 /3

200434310 Control 10 8 3 ↑ ■ ■ 1 /3

200434802 Control 0 8 1 ■■■ 0 /3

200434803 Control 0 7.5 0.3 ■■■ 0 /3

200434804 Control 0 8 1 ■■■ 0 /3

200434805 Control 0 7.5 1 ■■■ 0 /3

200434806 Control 0 8 0.1 ■■■ 0 /3

200434807 Control 0 8 0.1 ■■■ 0 /3

200434808 Control 0 8 1 ■■■ 0 /3

200434809 Control 0 8 1 ■■■ 0 /3

200434901 Control 10 8 0.3 ↑ ■ ■ 1 /3

200434902 Control 0 8 3 ■■■ 0 /3

200434903 Control 0 8 0.3 ■■■ 0 /3

200434904 Control 0 8 3 ■■■ 0 /3

Chart F-7 (Page 2): Summary of Urinalysis Data for Viagen Clone Progeny and Comparators ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

TreatmentBloodpHProteinBloodpHProtein

Summary

200434905 Control 25 7 3 ↑ ↓ ■ 2 /3

200435301 Control 0 8 0.3 ■■■ 0 /3

200435302 Control 0 8 0.1 ■■■ 0 /3

200435303 Control 0 8 0.1 ■■■ 0 /3

200435304 Control 0 8 3 ■■■ 0 /3

200435305 Control 80 6 0.3 ↑ ↓ ■ 2 /3

200435306 Control 0 8 1 ■■■ 0 /3

200435307 Control 10 8 0.3 ↑ ■ ■ 1 /3

200435308 Control 10 7.5 0.1 ↑ ■ ■ 1 /3

200435309 Control 0 7 0.3 ■ ↓ ■ 1 /3

200435310 Control 10 7.5 0.3 ↑ ■ ■ 1 /3

200435311 Control 200 7.5 0.3 ↑ ■ ■ 1 /3

200435401 Control 0 8 1 ■■■ 0 /3

200435402 Control 0 8 1 ■■■ 0 /3

200435403 Control 0 8 1 ■■■ 0 /3

200435404 Control 0 8 0.1 ■■■ 0 /3

200435405 Control 10 6.5 1 ↑ ↓ ■ 2 /3

200435407 Control 0 8 0.3 ■■■ 0 /3

200435409 Control 200 8 1 ↑ ■ ■ 1 /3

200435410 Control 0 6 0.1 ■ ↓ ■ 1 /3

200435411 Control 10 7 0.3 ↑ ↓ ■ 2 /3

200435412 Control 10 8 3 ↑ ■ ■ 1 /3

200435413 Control 10 8 3 ↑ ■ ■ 1 /3

200436301 Control 0 7 0.1 ■ ↓ ■ 1 /3

200436303 Control 0 8 0.3 ■■■ 0 /3

200436305 Control 0 7.5 0.1 ■■■ 0 /3

200436309 Control 0 8 3 ■■■ 0 /3

200436310 Control 0 6 1 ■ ↓ ■ 1 /3

200436312 Control 0 7.5 3 ■■■ 0 /3

200437301 Control 0 7.5 0.3 ■■■ 0 /3

200437302 Control 80 6.5 1 ↑ ↓ ■ 2 /3

200437303 Control 0 8 0.1 ■■■ 0 /3

200437305 Control 0 8 0.1 ■■■ 0 /3

200437307 Control 0 8 0.3 ■■■ 0 /3

200437309 Control 0 8 0.3 ■■■ 0 /3

200438401 Control 0 8 1 ■■■ 0 /3

200438402 Control 10 7.5 1 ↑ ■ ■ 1 /3

200438403 Control 0 8 0.1 ■■■ 0 /3

200438404 Control 10 8 0.3 ↑ ■ ■ 1 /3

200438406 Control 10 8 1 ↑ ■ ■ 1 /3

Chart F-7 (Page 3): Summary of Urinalysis Data for Viagen Clone Progeny and Comparators ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

TreatmentBloodpHProteinBloodpHProtein

Summary

200438408 Control 10 8 3 ↑ ■ ■ 1 /3

200438501 Control 0 7.5 0.3 ■■■ 0 /3

200438503 Control 0 7 3 ■ ↓ ■ 1 /3

200438505 Control 0 8 3 ■■■ 0 /3

200438511 Control 0 8 1 ■■■ 0 /3

200438513 Control 0 8 3 ■■■ 0 /3

200438701 Control 0 7.5 0.3 ■■■ 0 /3

200438703 Control 0 7 0.1 ■ ↓ ■ 1 /3

200438705 Control 0 8 3 ■■■ 0 /3

200438706 Control 0 8 0.3 ■■■ 0 /3

200438707 Control 0 8 0.1 ■■■ 0 /3

200438709 Control 0 7.5 0.1 ■■■ 0 /3

200439202 Control 0 8 0.3 ■■■ 0 /3

200439204 Control 0 7.5 3 ■■■ 0 /3

200439205 Control 0 8 0.3 ■■■ 0 /3

200439206 Control 10 7.5 3 ↑ ■ ■ 1 /3

200439208 Control 200 7.5 1 ↑ ■ ■ 1 /3

200439210 Control 0 8 0.3 ■■■ 0 /3

200439401 Control 0 6 1 ■ ↓ ■ 1 /3

200439404 Control 0 6.5 3 ■ ↓ ■ 1 /3

200439406 Control 0 8 3 ■■■ 0 /3

200439901 Control 10 8 0.1 ↑ ■ ■ 1 /3

200439902 Control 0 7 0.1 ■ ↓ ■ 1 /3

200439903 Control 200 7.5 0.1 ↑ ■ ■ 1 /3

200439904 Control 0 7.5 1 ■■■ 0 /3

200440601 Control 0 6.5 1 ■ ↓ ■ 1 /3

200440602 Control 10 6 1 ↑ ↓ ■ 2 /3

200440603 Control 0 7 1 ■ ↓ ■ 1 /3

200440605 Control 0 8 0.3 ■■■ 0 /3

200440901 Control 0 8 0.3 ■■■ 0 /3

200440903 Control 80 8 3 ↑ ■ ■ 1 /3

200440906 Control 0 7.5 0.3 ■■■ 0 /3

200440907 Control 80 6.5 3 ↑ ↓ ■ 2 /3

200440909 Control 0 6 1 ■ ↓ ■ 1 /3

200440911 Control 10 6 1 ↑ ↓ ■ 2 /3

200441201 Control 0 6.5 1 ■ ↓ ■ 1 /3

200441202 Control 0 7.5 1 ■■■ 0 /3

200441203 Control 0 6 0.3 ■ ↓ ■ 1 /3

200441205 Control 10 6 0.3 ↑ ↓ ■ 2 /3

200441207 Control 0 6 0.3 ■ ↓ ■ 1 /3

Chart F-7 (Page 4): Summary of Urinalysis Data for Viagen Clone Progeny and Comparators ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

TreatmentBloodpHProteinBloodpHProtein

Summary

200441209 Control 0 6 0.3 ■ ↓ ■ 1 /3

200441211 Control 0 7.5 0.3 ■■■ 0 /3

200430601

200430602 Clone 0 8 1 ■■■ 0 /3

200430603 Clone 0 8.5 1 ■ ↑ ■ 1 /3

200430604 Clone 10 8 0.1 ↑ ■ ■ 1 /3

200430605 Clone 0 7.5 0.1 ■■■ 0 /3

200430606 Clone 10 7.5 0.3 ↑ ■ ■ 1 /3

200430607 Clone 200 8 0.1 ↑ ■ ■ 1 /3

200430608 Clone 10 8 0.3 ↑ ■ ■ 1 /3

200430609 Clone 0 8 0.1 ■■■ 0 /3

200430610 Clone 80 7.5 0.3 ↑ ■ ■ 1 /3

200430611 Clone 10 6 0.1 ↑ ↓ ■ 2 /3

200430701 Clone 0 7 1 ■ ↓ ■ 1 /3

200430702 Clone 0 8 0.3 ■■■ 0 /3

200430703 Clone 0 8 1 ■■■ 0 /3

200430704 Clone 0 8 3 ■■■ 0 /3

200430705 Clone 0 8 0.1 ■■■ 0 /3

200430706 Clone 10 7 0.1 ↑ ↓ ■ 2 /3

200430707 Clone 0 8 0.1 ■■■ 0 /3

200430708 Clone 0 8 0.1 ■■■ 0 /3

200430709 Clone 0 8 0.1 ■■■ 0 /3

200430710 Clone 10 7.5 0.1 ↑ ■ ■ 1 /3

200430711 Clone 0 8 0.3 ■■■ 0 /3

200430801 Clone 0 7 3 ■ ↓ ■ 1 /3

200430802 Clone 0 8 1 ■■■ 0 /3

200430803 Clone 10 7.5 0.1 ↑ ■ ■ 1 /3

200430804 Clone 10 7.5 0 ↑ ■ ■ 1 /3

200430805 Clone 0 7.5 0.1 ■■■ 0 /3

200430901

200430902 Clone 0 6.5 0 ■ ↓ ■ 1 /3

200430903 Clone 0 7 0.3 ■ ↓ ■ 1 /3

200430904

200430905 Clone 0 7.5 0.1 ■■■ 0 /3

200430906 Clone 0 7 0.3 ■ ↓ ■ 1 /3

200431001

200431002 Clone 0 7.5 0.3 ■■■ 0 /3

200431003 Clone 0 8 0.1 ■■■ 0 /3

200431004 Clone 0 8 0.3 ■■■ 0 /3

200431005 Clone 0 8 0.1 ■■■ 0 /3

Chart F-7 (Page 5): Summary of Urinalysis Data for Viagen Clone Progeny and Comparators ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

TreatmentBloodpHProteinBloodpHProtein

Summary

200431006 Clone 0 6.5 0.1 ■ ↓ ■ 1 /3

200431007 Clone 80 8 0 ↑ ■ ■ 1 /3

200431008 Clone 0 7.5 0.1 ■■■ 0 /3

200431009 Clone 10 7.5 0.1 ↑ ■ ■ 1 /3

200431010 Clone 0 8 1 ■■■ 0 /3

200431011 Clone 0 8 3 ■■■ 0 /3

200431012 Clone 25 7.5 0.3 ↑ ■ ■ 1 /3

200431013

200431401 Clone 0 8 0.1 ■■■ 0 /3

200431402 Clone 0 8 0.3 ■■■ 0 /3

200431403 Clone 0 8 1 ■■■ 0 /3

200431404 Clone 0 7.5 0.1 ■■■ 0 /3

200431405 Clone 0 7.5 0.1 ■■■ 0 /3

200431406 Clone 0 8 0.1 ■■■ 0 /3

200431407 Clone 0 8 0.1 ■■■ 0 /3

200431408 Clone 0 6.5 0.1 ■ ↓ ■ 1 /3

200431409 Clone 0 8 0.1 ■■■ 0 /3

200431410 Clone 10 7.5 0.1 ↑ ■ ■ 1 /3

200431411 Clone 0 7.5 0.1 ■■■ 0 /3

200431501 Clone 0 7.5 0.1 ■■■ 0 /3

200431502 Clone 0 8 0 ■■■ 0 /3

200431503 Clone 0 7 0.1 ■ ↓ ■ 1 /3

200431504 Clone 0 8 0.1 ■■■ 0 /3

200431505 Clone 0 6.5 0.1 ■ ↓ ■ 1 /3

200431506 Clone 0 8 0.3 ■■■ 0 /3

200431601 Clone 0 8 1 ■■■ 0 /3

200431602 Clone 0 8 0.3 ■■■ 0 /3

200431603 Clone 0 8 1 ■■■ 0 /3

200431604 Clone 0 8 0.3 ■■■ 0 /3

200431605 Clone 0 8 0.3 ■■■ 0 /3

200431606 Clone 0 8 0.1 ■■■ 0 /3

200431607 Clone 0 8 0.3 ■■■ 0 /3

200431608 Clone 0 7.5 0 ■■■ 0 /3

200431609

200431610 Clone 10 8 0.3 ↑ ■ ■ 1 /3

200431701 Clone 0 8 0.3 ■■■ 0 /3

200431702 Clone 0 8 0.1 ■■■ 0 /3

200431703 Clone 80 8 0.3 ↑ ■ ■ 1 /3

200431704 Clone 80 8 0.1 ↑ ■ ■ 1 /3

200431705

Chart F-7 (Page 6): Summary of Urinalysis Data for Viagen Clone Progeny and Comparators ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

TreatmentBloodpHProteinBloodpHProtein

Summary

200431706 Clone 0 8 3 ■■■ 0 /3

200431707

200431708 Clone 200 8 3 ↑ ■ ■ 1 /3

200432001 Clone 0 8 1 ■■■ 0 /3

200432002 Clone 0 8 3 ■■■ 0 /3

200432003 Clone 0 7 0.3 ■ ↓ ■ 1 /3

200432004 Clone 0 8 0.3 ■■■ 0 /3

200432005 Clone 80 8 0.1 ↑ ■ ■ 1 /3

200432006 Clone 10 8 0.1 ↑ ■ ■ 1 /3

200432007 Clone 0 8 1 ■■■ 0 /3

200432008 Clone 0 7.5 0.3 ■■■ 0 /3

200432101 Clone 0 6 0 ■ ↓ ■ 1 /3

200432102 Clone 0 8 1 ■■■ 0 /3

200432103 Clone 0 7.5 0.3 ■■■ 0 /3

200432104 Clone 10 8 0.3 ↑ ■ ■ 1 /3

200432105 Clone 0 8 0.3 ■■■ 0 /3

200432106

200432107

200432108 Clone 0 8 3 ■■■ 0 /3

200432109

200432301

200432302 Clone 10 8 0.1 ↑ ■ ■ 1 /3

200432303

200432304 Clone 0 6.5 0.1 ■ ↓ ■ 1 /3

200432305

200432306 Clone 0 7.5 0.3 ■■■ 0 /3

200432307

200432308 Clone 200 8 1 ↑ ■ ■ 1 /3

200432309

200432310 Clone 0 8 1 ■■■ 0 /3

200432311

200432312 Clone 200 6 3 ↑ ↓ ■ 2 /3

200432401 Clone 10 8 3 ↑ ■ ■ 1 /3

200432402 Clone 0 8 0.3 ■■■ 0 /3

200432403 Clone 0 8 1 ■■■ 0 /3

200432404 Clone 0 7.5 0.3 ■■■ 0 /3

200432405 Clone 0 7.5 0.1 ■■■ 0 /3

200432406 Clone 0 8 0.3 ■■■ 0 /3

200432407 Clone 0 8 1 ■■■ 0 /3

200432408

Chart F-7 (Page 7): Summary of Urinalysis Data for Viagen Clone Progeny and Comparators ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

TreatmentBloodpHProteinBloodpHProtein

Summary

200432501 Clone 0 7.5 0.1 ■■■ 0 /3

200432502

200432503 Clone 0 7.5 0.1 ■■■ 0 /3

200432504 Clone 0 8 0.3 ■■■ 0 /3

200432505

200432506 Clone 0 8 0.3 ■■■ 0 /3

200432507 Clone 0 7.5 0.3 ■■■ 0 /3

200432508

200432701

200432702 Clone 0 8 0.3 ■■■ 0 /3

200432703

200432704 Clone 10 8 0.3 ↑ ■ ■ 1 /3

200432705 Clone 10 8 0.3 ↑ ■ ■ 1 /3

200432706 Clone 0 8 0.1 ■■■ 0 /3

200433001 Clone 0 8 0.3 ■■■ 0 /3

200433002 Clone 0 8 0.3 ■■■ 0 /3

200433003 Clone 0 7.5 0.3 ■■■ 0 /3

200433004

200433005

200433006

200433007 Clone 0 8 0.3 ■■■ 0 /3

200433008

200433009

200433010 Clone 10 8 0.1 ↑ ■ ■ 1 /3

200433011 Clone 10 6 0.3 ↑ ↓ ■ 2 /3

200433201 Clone 0 8 0.1 ■■■ 0 /3

200433202 Clone 0 8 0.1 ■■■ 0 /3

200433203 Clone 0 6 0.1 ■ ↓ ■ 1 /3

200433204 Clone 0 8 1 ■■■ 0 /3

200433205 Clone 10 8 1 ↑ ■ ■ 1 /3

200433206 Clone 0 7.5 0.1 ■■■ 0 /3

200433207 Clone 10 8 0.1 ↑ ■ ■ 1 /3

200433208 Clone 10 8 1 ↑ ■ ■ 1 /3

200433601 Clone 0 8 0.1 ■■■ 0 /3

200433602 Clone 0 8 1 ■■■ 0 /3

200433603 Clone 0 8 0.1 ■■■ 0 /3

200433604 Clone 0 8 0.1 ■■■ 0 /3

200433605 Clone 0 8 0.1 ■■■ 0 /3

200433701

200433702

Chart F-7 (Page 8): Summary of Urinalysis Data for Viagen Clone Progeny and Comparators ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

TreatmentBloodpHProteinBloodpHProtein

Summary

200433703

200433704

200433705

200433706

200433707

200433708 Clone 0 8.5 0.3 ■ ↑ ■ 1 /3

200433709 Clone 0 6 0.1 ■ ↓ ■ 1 /3

200433710 Clone 10 7.5 0.1 ↑ ■ ■ 1 /3

200433711 Clone 80 8 0.3 ↑ ■ ■ 1 /3

200433712 Clone 0 8 0.1 ■■■ 0 /3

200433801 Clone 0 7 0.3 ■ ↓ ■ 1 /3

200433802 Clone 10 7 3 ↑ ↓ ■ 2 /3

200433803 Clone 0 8 3 ■■■ 0 /3

200433804 Clone 0 8.5 0.3 ■ ↑ ■ 1 /3

200433805

200433806 Clone 10 6.5 0.3 ↑ ↓ ■ 2 /3

200433807 Clone 0 7.5 0.1 ■■■ 0 /3

200433808 Clone 10 8 0.1 ↑ ■ ■ 1 /3

200433809 Clone 0 8 1 ■■■ 0 /3

200434101 Clone 0 7.5 0.1 ■■■ 0 /3

200434102 Clone 0 8 0.1 ■■■ 0 /3

200434103

200434104 Clone 0 8 0.3 ■■■ 0 /3

200434105 Clone 0 8 0.1 ■■■ 0 /3

200434106

200434107 Clone 0 8 0.1 ■■■ 0 /3

200434108

200434109 Clone 0 8 0.3 ■■■ 0 /3

200434110

200434111 Clone 0 7.5 0.3 ■■■ 0 /3

200434112

200434113 Clone 0 8 20 ■ ■ ↑ 1 /3

200434114

200434115

200434401 Clone 0 8 1 ■■■ 0 /3

200434402 Clone 0 8 1 ■■■ 0 /3

200434403 Clone 0 8 0.3 ■■■ 0 /3

200434501 Clone 0 8 1 ■■■ 0 /3

200434502 Clone 0 8 0.1 ■■■ 0 /3

200434503 Clone 0 6 0.1 ■ ↓ ■ 1 /3

Chart F-7 (Page 9): Summary of Urinalysis Data for Viagen Clone Progeny and Comparators ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

TreatmentBloodpHProteinBloodpHProtein

Summary

200434504 Clone 0 8 0.3 ■■■ 0 /3

200434505

200434506

200434507 Clone 80 6 0.1 ↑ ↓ ■ 2 /3

200434508

200434509 Clone 25 7 0.3 ↑ ↓ ■ 2 /3

200434510

200434511 Clone 0 7 0.1 ■ ↓ ■ 1 /3

200434512 Clone 0 8 1 ■■■ 0 /3

200434601 Clone 0 8 0.3 ■■■ 0 /3

200434602 Clone 0 8 1 ■■■ 0 /3

200434603 Clone 0 8 3 ■■■ 0 /3

200434604 Clone 0 6.5 0.3 ■ ↓ ■ 1 /3

200434605 Clone 0 8 1 ■■■ 0 /3

200434606 Clone 0 8 0.3 ■■■ 0 /3

200434607 Clone 0 7 0.3 ■ ↓ ■ 1 /3

200434608 Clone 10 8 1 ↑ ■ ■ 1 /3

200435801 Clone 0 7.5 0.1 ■■■ 0 /3

200435802 Clone 10 8.5 0.1 ↑ ↑ ■ 2 /3

200435803 Clone 200 8 1 ↑ ■ ■ 1 /3

200436001 Clone 0 8 0.3 ■■■ 0 /3

200436002 Clone 0 7.5 0.3 ■■■ 0 /3

200436003 Clone 0 8 3 ■■■ 0 /3

200436004 Clone 0 7.5 0.1 ■■■ 0 /3

200436005 Clone 80 8 0.3 ↑ ■ ■ 1 /3

200436006 Clone 25 8 3 ↑ ■ ■ 1 /3

200436007 Clone 0 8 0.3 ■■■ 0 /3

200436008 Clone 0 8 0.3 ■■■ 0 /3

200436009 Clone 0 7.5 0.1 ■■■ 0 /3

200436010 Clone 0 8 0.1 ■■■ 0 /3

200436011 Clone 0 8 0.1 ■■■ 0 /3

200436012 Clone 0 7.5 0.1 ■■■ 0 /3

200436013 Clone 0 6.5 0.3 ■ ↓ ■ 1 /3

200436014

200436401 Clone 0 8 3 ■■■ 0 /3

200436402 Clone 0 7.5 0.3 ■■■ 0 /3

200436403 Clone 0 8 1 ■■■ 0 /3

200436404 Clone 0 8 0.1 ■■■ 0 /3

200436405 Clone 0 8 0.3 ■■■ 0 /3

200436601 Clone 0 8 0.3 ■■■ 0 /3

Chart F-7 (Page 10): Summary of Urinalysis Data for Viagen Clone Progeny and Comparators ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

TreatmentBloodpHProteinBloodpHProtein

Summary

200436602 Clone 0 8 3 ■■■ 0 /3

200436603 Clone 0 7.5 0.1 ■■■ 0 /3

200436604 Clone 0 7.5 3 ■■■ 0 /3

200436605

200436606 Clone 0 7.5 0.3 ■■■ 0 /3

200436607 Clone 200 7.5 1 ↑ ■ ■ 1 /3

200436608 Clone 0 6.5 0.1 ■ ↓ ■ 1 /3

200436609

200436610 Clone 0 7 0.1 ■ ↓ ■ 1 /3

200436611

200436612 Clone 0 8 1 ■■■ 0 /3

200436901 Clone 0 8 0.1 ■■■ 0 /3

200436902 Clone 0 7.5 0.1 ■■■ 0 /3

200436903 Clone 0 7.5 3 ■■■ 0 /3

200436904

200436905 Clone 0 8 0.3 ■■■ 0 /3

200436906

200436907

200436908

200436909 Clone 0 8 0.1 ■■■ 0 /3

200436910

200436911 Clone 0 8 0.1 ■■■ 0 /3

200436912 Clone 0 6 0.1 ■ ↓ ■ 1 /3

200436913 Clone 10 8 1 ↑ ■ ■ 1 /3

200437401 Clone 0 7.5 0.1 ■■■ 0 /3

200437402 Clone 0 8 1 ■■■ 0 /3

200437403

200437404 Clone 0 8 1 ■■■ 0 /3

200437405

200437406 Clone 0 8 0.3 ■■■ 0 /3

200437407

200437408 Clone 0 7 1 ■ ↓ ■ 1 /3

200437409

200437410

200437411

200437501 Clone 80 8 1 ↑ ■ ■ 1 /3

200437502 Clone 0 8 1 ■■■ 0 /3

200437503

200437504 Clone 0 8 1 ■■■ 0 /3

200437505

Chart F-7 (Page 11): Summary of Urinalysis Data for Viagen Clone Progeny and Comparators ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal_ID

TreatmentBloodpHProteinBloodpHProtein

Summary

200437506 Clone 10 7.5 0.1 ↑ ■ ■ 1 /3

200437507

200437508

200437509 Clone 200 8 1 ↑ ■ ■ 1 /3

200437510 Clone 25 7.5 0.3 ↑ ■ ■ 1 /3

200437801 Clone 0 8 3 ■■■ 0 /3

200437802 Clone 0 6.5 0.1 ■ ↓ ■ 1 /3

200437803 Clone 0 7 0.3 ■ ↓ ■ 1 /3

200437804 Clone 10 8 0.3 ↑ ■ ■ 1 /3

200438101 Clone 0 8 0.3 ■■■ 0 /3

200438102 Clone 0 8 0.3 ■■■ 0 /3

200438103

200438104 Clone 0 8 1 ■■■ 0 /3

200438105 Clone 0 8 1 ■■■ 0 /3

200438106 Clone 0 8 0.1 ■■■ 0 /3

200438107 Clone 0 7.5 0.1 ■■■ 0 /3

200438601

200438602 Clone 0 7.5 0.3 ■■■ 0 /3

200438603 Clone 0 7 0.1 ■ ↓ ■ 1 /3

200438604 Clone 0 8 1 ■■■ 0 /3

200438605 Clone 0 8 1 ■■■ 0 /3

200438606 Clone 0 7.5 0.1 ■■■ 0 /3

200438607 Clone 0 8 0.1 ■■■ 0 /3

200438608 Clone 0 7.5 1 ■■■ 0 /3

200438609 Clone 0 6.5 0.1 ■ ↓ ■ 1 /3

200438610 Clone 0 6.5 0.1 ■ ↓ ■ 1 /3

200438611 Clone 0 8 0.3 ■■■ 0 /3

200439501 Clone 0 7.5 0.3 ■■■ 0 /3

200439502 Clone 10 6 1 ↑ ↓ ■ 2 /3

200439503 Clone 0 8 0.1 ■■■ 0 /3

200439504 Clone 0 7.5 0.3 ■■■ 0 /3

200439505 Clone 0 7.5 0.3 ■■■ 0 /3

Chart F-7 (Page 12): Summary of Urinalysis Data for Viagen Clone Progeny and Comparators ■ In Comparison Range

↑/↓ Animal is above/below reference range

Blank rows indicate values that were missing
Animal Cloning: A Risk Assessment DRAFT 

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Animal Cloning: A Risk Assessment DRAFT

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Animal Cloning: A Risk Assessment DRAFT

Appendix G 

Investigation on the Attributes of Cloned 

Bovine Products 
Animal Cloning: A Risk Assessment DRAFT 

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Animal Cloning: A Risk Assessment DRAFT

Appendix G 

Investigation on the Attributes of Cloned 

Bovine Products 

The Japanese Research Institute for Animal Science in Biochemistry and Toxicology provided 

an unpublished bound report “Investigation on the Attributes of Cloned Bovine Products” by the 

Japan Livestock Technology Association (Japan, 2002). The 489 page report, provided in the 

original Japanese, and was accompanied by an eight page August 2002 English-language 

summary. 

This appendix contains a translation of the first three pages of the bound report and the eight 

page English summary. These are followed by tables from the original bound report. The tables 

present the results of a feeding study in which rats were fed diets containing freeze dried milk or 

freeze dried beef from ordinary cattle and clone cattle at concentrations of 0, 2.5, 5, or 10% of 

the diet for 28days.General signs, body weight, food consumption, urinalysis, sensory and reflex 

function, spontaneous movement frequency, general function, reproductive cycle, hematology at 

autopsy, blood chemistry, organ weights, pathology and histopathology were compared between 

groups. English-language tables were provided in the original Japanese-language report with the 

results.These tables are included in this appendix. 
Animal Cloning: A Risk Assessment DRAFT 

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Appendix G: The Comprehensive Veterinary Examination 2 

Animal Cloning: A Risk Assessment DRAFT

English language translation of the first three 

pages of the report “Investigation on the 

Attributes of Cloned Bovine Products” 
Animal Cloning: A Risk Assessment DRAFT 

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PROJECT REPORT OF AN INVESTIGATION ON THE PROPERTIES 

OF PRODUCTS FROM CLONED CATTLE 

(An Urgent Study Project for the Utilization of Cloned Cattle) 

(1999 – 2001)

September 2002 

Japan Livestock Technology Association

A project aided by the Agriculture and 

Livestock Industries Corporation 
 2

Results of an investigation on the properties of products from cloned cattle 

 Rapid advances have been made in the application of cloning technology in 

cattle multiplication. Products from embryonic clones of cattle are already on the 

market as safe foods. The Ministry of Health, Labor and Welfare (MHLW) is currently 

gathering data on the safety of products from somatic cell cloned cattle and has released 

an interim report which states that so far there is no reason to anticipate safety-related 

problems. 

 Cloning technology is expected to advance further and come into wide use as a 

technology that can provide inexpensive meat, milk, etc to consumers. However, for this 

to happen, it is essential for the meat and milk of cloned cattle to become widely 

accepted by consumers as safe, high-quality commodities. 

 This Association conducted various investigations on cloned cattle, with grants 

from the Agriculture and Livestock Industries Corporation (ALIC), during 1999 to 2001. 

An investigation on the properties of products from cloned cattle, which was a part of 

these investigations, was commissioned to the Research Institute for Animal Science in 

Biochemistry and Toxicology (RIAS). 

 The objective of this investigation was to collect data that would confirm the 

safety of products from embryonic clones of cattle, which are already in use as food 

items, and also data that would be useful in evaluating the safety of products from 

somatic cell clones. For this purpose, the properties of the blood, and the composition of 

nutritional components such as proteins, lipids, amino acids and fatty acids of raw milk 

and meat were analyzed and compared among ordinary cattle, embryonic clones and 

somatic cell clones of Holstein and Black Japanese breeds. Digestibility studies with 

artificial digestive fluid and with rats, allergenicity and mutagenicity (micronucleus) 

tests with mice, and a 14-week feeding study with rats were also conducted and the 

digestibility, allergenicity, and mutagenicity of the products, and their effects on the 

growth, functions and morphology of the test animals were compared. The results of 

none of the analyses or tests showed any significant differences between products from 

ordinary cattle and the two types of cloned cattle. Also, no harmful effect attributable to 

the raw milk or meat of the two types of clones was observed. 

September 10, 2002 

Cloned Cattle Investigation Committee 

Japan Livestock Technology Association 
 3

– Contents – 

Results of an investigation on the properties of products from cloned cattle 

Urgent study project on the utilization of cloned cattle – List of the “Cloned Cattle 

Investigation Committee” members 

1. Summary results of the investigation on the properties of products from cloned 

cattle 

2. Tests on the properties of blood of cattle covered by the investigation on the 

properties of products from cloned cattle 

3. Analysis of nutritional components of raw milk and meat of cloned cattle 

4. Allergenicity tests of raw milk and meat of cloned cattle by the mouse abdominal 

wall method 

5. Studies on digestibility of raw milk and meat of cloned cattle 

6. Mouse micronucleus test on raw milk and meat of cloned cattle 

7-1 14-week feeding study on rats, with raw milk of cloned cattle 

7-2 Preliminary study for the “14-week feeding study on rats, with raw milk of cloned 

cattle”– Raw milk dry powder mixing concentration-setting study 

8-1 14-week feeding study on rats, with beef from cloned cattle 

8-2 Preliminary study for the “14-week feeding study on rats, with beef from cloned 

cattle” – Dry meat powder mixing concentration-setting study 
Animal Cloning: A Risk Assessment DRAFT 

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Appendix G: The Comprehensive Veterinary Examination 

Animal Cloning: A Risk Assessment DRAFT

English language summary of the report 

“Investigation on the Attributes of Cloned 

Bovine Products” 
Animal Cloning: A Risk Assessment DRAFT 

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1 

August 13, 2002 

Livestock Technology Division, 

Livestock Industry Department, 

Agricultural Production Bureau, 

Ministry of Agriculture, 

Forestry and Fisheries 

RE: Outline of Investigative Results on the Attributes of Cloned Bovine Products 

An interim report of the Ministry of Health and Welfare (MHW) released in June 2000 

concerning the safety of foods made from cloned cattle states, “There is no scientific basis for 

fearing the safety of foods”, and it recommended that foods derived from BNT cloned cattle 

be sold with labeling to that effect (optional labeling). The interim report also stated that, “It 

would be desirable to obtain data on a greater number of cloned cattle that would support 

safety.” Because somatic cloning technology is a newer technology, the Ministry of 

Agriculture, Forestry and Fisheries (MAFF) is requesting self-restraint on the shipment of 

SCNT cloned cattle. The Ministry of Health, Labour and Welfare (MHLW: a new Ministry 

changed from MHW) are currently conducting an investigative study on safety. 

In response, the Research Institute for Animal Science in Biochemistry and Toxicology has 

been conducting a study on the attributes of cloned bovine products (emergency study project 

on cloned bovine usage). The results of this study have now been gathered so a summary is 

attached separately. 

It is intended that the results of this study will be submitted as reference material for the 

“investigative research on the safety of animal foods that use cloning technology” currently 

being conducted at the MHLW. 

For further information, please contact: 

Yoshitake 

Livestock Breeding Technology Center 

Livestock Technology Division, 

Livestock Industry Department, 

 Agricultural Production Bureau, 

Ministry of Agriculture, Forestry and Fisheries 

TEL: 03-3502-8111 (ext. 3911) 

Direct TEL: 03-3591-3656 

FAX: 03-3593-7233

Press Release 
2 

August 13, 2002 

Research Institute for Animal 

Science in Biochemistry and Toxicology 

Outline of Investigation on the Attributes of Cloned Bovine Products 

Commissioned by the Livestock Technology Association from FY1999, we conducted an 

investigation on the attributes of cloned bovine products. The following are the results of this 

investigation. 

1. Objectives 

To conduct an investigation on the blood attributes of cloned cattle (BNT cloned cattle or 

SCNT cloned cattle), and analyze the components of cloned bovine products (milk and beef), 

as well as to conduct a study on animal feeding of feed additives from cloned cattle, and 

obtain data comparing cloned bovine products and existing foods (products from ordinary 

cattle produced by artificial insemination, etc). 

2. Outline of investigation 

(1) Blood test 

(Material and method) 

Blood was sampled from ordinary cattle and cloned cattle at 3, 6, and 9 months of 

pregnancy and 3 and 6 weeks after birth in the case of dairy cattle (Holstein), and 

3 to 4 times during a period from 21 to 28 weeks after birth in the case of beef 

cattle (kuroge-wagyu). The sampled blood was subject to hematological testing 

(12 items including red blood cell count, white blood cell count, and hemoglobin) 

and biochemical examination of blood (25 items including total protein, and total 

cholesterol) and compared. 

(Results) 

None of the animals showed abnormalities in performance status. There were 

also no biologically significant differences* in any of the test values between 

ordinary cattle and cloned cattle, for both the dairy and beef types. 

* A biologically significant difference means a difference that could possibly 

have an effect on factors such as health and survival evident between the 

study groups. There are no problems if a biologically significant difference 

and statistically significant difference are in accord, but even if there is a 

statistically significant difference between the study groups in general, and 

that the difference is within the range of normal values it is unlikely that 

health would be affected, so one could not say that a biologically significant 

difference exists. In the investigative report, biologically significant 

difference was studied in addition to statistically significant difference. The 

same applies hereafter. 

(2) Analytical study of milk and meat components 
3 

(Material and method) 

The general components (water content, protein, lipids, and sugars), amino acids 

(18 types), and fatty acid (21 types) content (content per 100 g) in milk and slices 

of meat (9 sites) sampled from ordinary cattle and cloned cattle were measured 

and compared. 

(Results) 

Although there were slight variations seen among individual cattle, no 

biologically significant differences were evident in the general components, 

amino acids, and fatty acid content between ordinary cattle and cloned cattle for 

both milk and different sites of meat (Table 1). 

(3) Milk and meat digestion study 

(Material and method) 

A study of digestion of pieces of meat sampled from ordinary cattle and cloned 

cattle by artificial gastric juice and intestinal juice, and a study of digestion of 

milk and meat that had been frozen, dried, and powdered (freeze-dried food) and 

added to feed, using rats were conducted, and the digestion rates that were 

regarded as parameters of protein were compared. 

(Results) 

There were no biologically significant differences in the rates of digestion of the 

feed additives due to artificial gastric juice and intestinal juice using rats between 

ordinary cattle and cloned cattle (Tables 2 and 3). 

(4) Allergen testing of milk and meat by mouse abdominal wall method 

(Material and method) 

We sensitized mice with an intraperitoneal injection of extract from freeze-dried 

milk and meat slices sampled from ordinary cattle and cloned cattle. Fourteen 

days later we retracted the abdomen and induced an allergic reaction by reinjection in the abdominal wall. Allergen activity was compared based on the 

extent of vascular permeability (diameter of dye leakage) seen due to the 

sensitization treatment. 

(Results) 

For both milk and meat slices there were no statistically significant differences in 

allergen activity between ordinary cattle and cloned cattle (Table 4). 

(5) Feeding test by the supply of a combination feed of milk and meat using rats 

(Material and method) 

Freeze-dried milk and meat of ordinary cattle and cloned cattle were each 

combined with basic feed at concentrations of 2.5%, 5%, and 10% in the case of 

freeze-dried milk, and 1%, 2.5%, and 5% in the case of freeze-dried meat, and fed 

to rats (20 per group (10 males and 10 females)) for 14 weeks. 
4 

The general sign, body weight, food consumption, urinalysis (8 items), sensory 

and reflex function, spontaneous movement frequency, general function, 

reproductive cycle, hematology at autopsy (11 items), blood chemistry (23 items), 

autopsy and organ weights (brain, pituitary gland, cerebral gland, thyroid gland, 

heart, lungs, liver, pancreas, adrenal bodies, and reproductive organs) of rats given 

the feed were compared among a basic feed group, ordinary cattle group, and 

cloned cattle group. 

(Results) 

There were no biologically significant differences in each of the items observed 

and tested over time in rats at any concentration of feed additive for milk and 

meat between ordinary cattle and cloned cattle (Table 5). 

(6) Mutagenicity by milk and meat supply using mice (micronucleus test) 

(Material and method) 

Feed produced in the feed test by the supply of a combination feed of milk and 

meat in (5) was given to mice for 14 days whereupon the incidence of bone 

marrow micronucleus-possessing erythrocytes appearing was tested 

(micronucleus) and mutagenicity (clastogenicity) was studied. 

(Results) 

Clastogenicity was negative and mutagenicity was not evident for milk and meat 

feed additives from ordinary cattle and cloned cattle (Table 6). 

3. Summary 

The above results revealed no biologically significant differences in component analysis 

testing and feed additive animal testing between products of BNT cloned cattle and SCNT 

cloned cattle (milk and meat), and the products of ordinary cattle. 
5 

Table 1. General components 

(1) Milk 

Classification Cattle No. Protein 

(g/100 g) 

Fats 

(g/100 g) 

Sugars 

(g/100 g) 

Ash 

content 

(g/100 g) 

Water 

content 

(g/100 g) 

Calcium 

(mg/100 g) 

Cholesterol 

(mg/100 g) 

Min. value 

Max value 

3.0 

3.4 

2.2 

3.3 

4.6 

4.6 

0.7 

0.7 

88.1 

89.7 

100 

110 

8 

Ordinary cattle 10

Mean value 3.3 2.7 4.6 0.7 88.9 105 9

BNT cloned 

cattle 

No.1 

No.2 

2.9 

2.9 

2.3 

3.6 

3.0 

3.5 

0.8 

0.7 

91.1 

89.3 

95 

105 

9 

9

SCNT cloned 

cattle 

No 1 

No.2 

No.3 

3.1 

3.3 

3.3 

4.3 

2.6 

3.1 

4.6 

4.4 

4.5 

0.7 

0.7 

0.7 

87.4 

89.1 

88.5 

120 

115 

115 

9 

11 

10

Note: The analytical values for each animal are the mean of the analytical values for milk sampled at two points – 3 weeks 

and 6 weeks after delivery. 

(2) Meat 

Classification Cattle No. Protein 

(g/100 g) 

Fats 

(g/100 g) 

Sugars 

(g/100 g) 

Ash content 

(g/100 g) 

Water 

content 

(g/100 g) 

Cholesterol 

(mg/100 g) 

Min. value 

Max value 

17.8 

19.6 

13.8 

22.9 

0.4 

0.8 

0.9 

1.0 

58.0 

64.8 

50 

68 Ordinary 

cattle 

Mean value 18.4 19.3 0.6 0.9 60.8 59

BNT cloned cattle 17.4 21.2 0.4 0.9 60.2 56

SCNT cloned cattle 16.8 23.8 0.5 0.9 57.9 68

Note: The analytical value for each animal is the mean value of the analytical values of 9 sites: shoulder, chuck loin, rib loin, 

loin end, brisket, round, silver side, rump, and tender loin. 
6 

Table 2. Rates of meat digestion by artificial digestive juices 

Rate of digestion after the start of incubation Digestive 

juice Sample 

Course Start 0.75 hr 1.5 hr 3 hr 6 hr 12 hr

Ordinary beef 0 68 79 - 95 90 Artificial 

gastric juice 

Somatic cloned beef 0 59 78 - 91 90

Artificial Ordinary beef 0 - 20 40 66 67

intestinal 

juice Somatic cloned beef 0 - 28 38 67 63

Note: The digestion rate shows the protein rate of digestion. 

Table 3. Digestion rates of milk and meat in rats 

Sample Test group Number of animals Digestion rate (mean±

standard deviation) 

Ordinary cattle 5 83.0±2.6 

Milk BNT cloned cattle 5 82.7±2.0 

SCNT cloned cattle 5 81.3±3.4 

Ordinary cattle 5 83.8±6.6 

Meat BNT cloned cattle 5 82.3±4.7 

SCNT cloned cattle 5 84.9±3.6 

Note: Milk and meat were each freeze-dried and combined in feed. The digestion rate shows the protein digestion rate. 

Table 4. Allergen study of milk and meat by mouse abdominal wall method 

Sample Test group Number of 

animals 

Diameter of dye leakage (mm) 

(mean±SD) 

Ordinary cattle Control group 

Test group 

7 

10 

7.0±3.7 

18.0±2.9 

BNT cloned cattle Control group 

Test group 

7 

10 

4.7±3.2 

18.0±3.9 

Milk 

SCNT cloned cattle Control group 

Test group 

7 

10 

4.9±4.6 

17.9±4.2 

Ordinary cattle Control group 

Test group 

7 

10 

5.3±5.0 

13.0±5.9 

BNT cloned cattle Control group 

Test group 

7 

10 

7.0±4.9 

12.5±3.5 

Meat 

SCNT cloned cattle Control group 

Test group 

7 

10 

5.7±4.2 

13.1±5.0 

Note: Milk and meat were each freeze-dried and the extracts were used as samples. The test groups underwent sensitization 

treatment and elicitation, while the control groups underwent elicitation only. 
7 

Table 5. Feed study of milk and meat by formula feed supply using rats 

(1) Changes in rat body weight by milk formula feed supply (mean±standard deviation) 

(g)

Feeding period (weeks) 

Study group 

Number 

of 

animals 1 2 3 4 5 6 8 10 12 14 

Amount of 

body 

weight 

increase in 

1-14 weeks

Basal diet 10 146±6 189±20 255±34 299±45 344±59 373±58 428±56 448±58 516±47 547±77 401±76 

Ordinary cattle 

High concentration (10%) 10 146±6 194±11 260±13 304±17 350±19 378±22 426±36 475±43 515±43 544±48 398±48 

BNT cloned cattle 

High concentration (10%) 10 146±6 175±19 245±15 295±14 333±18 367±16 425±22 462±34 503±41 530±44 384±41 

Male

SCNT cloned cattle 

High concentration (10%) 10 146±7 196±10 261±17 310±23 353±27 379±30 432±41 473±41 519±43 545±48 399±43 

Basal diet 10 117±5 150±6 184±12 208±9 229±10 242±10 267±16 283±15 304±18 310±20 193±18 

Ordinary cattle 

High concentration (10%) 10 118±5 152±7 181±7 209±12 234±16 247±15 273±20 298±21 316±28 329±40 211±42 

BNT cloned cattle 

High concentration (10%) 10 119±7 157±11 186±12 208±22 223±27 244±26 272±19 292±21 313±26 326±30 207±27 

Female 

SCNT cloned cattle 

High concentration (10%) 10 118±7 151±12 181±12 209±13 229±16 247±19 274±18 293±19 317±21 330±33 213±33 

Note: Aside from studying a 10% milk powder concentration, 10 cattle each were also fed a low concentration (2.5%) and a 

medium concentration (5%), but no significant differences were noted. 

(2) Changes in rat body weight by meat formula feed supply (mean±standard deviation) 

(g) 

Feeding period (weeks) 

Study group 

Number 

of 

animals 1 2 3 4 5 6 8 10 12 14 

Amount of 

body 

weight 

increase in 

1-14 weeks

Basal diet 10 143±5 216±12 279±14 336±16 382±18 426±22 489±31 534±37 560±42 590±50 447±51 

Ordinary cattle 

High concentration (5%) 10 143±5 221±9 284±17 337±24 386±30 432±36 492±44 541±55 575±62 604±65 462±65 

BNT cloned cattle 

High concentration (5%) 10 143±5 219±10 286±14 343±18 392±21 431±26 488±34 535±39 564±43 591±49 448±51 

Male

SCNT cloned cattle 

High concentration (5%) 10 143±6 215±9 278±14 336±20 392±29 435±35 499±48 551±60 581±70 613±80 469±76 

Basal diet 10 120±4 167±14 198±15 228±18 253±26 272±26 290±26 313±33 330±38 338±35 218±38 

Ordinary cattle 

High concentration (5%) 10 120±4 169±11 200±12 230±15 254±19 274±18 297±23 316±28 331±31 341±30 221±30 

BNT cloned cattle 

High concentration (5%) 10 120±5 171±8 201±10 236±15 260±20 280±23 311±29 330±27 347±35 361±40 241±39 

Female 

SCNT cloned cattle 

High concentration (5%) 10 120±4 167±8 195±9 227±11 250±12 268±12 292±16 310±19 329±20 336±20 216±17 

Note: Aside from studying a 5% meat powder concentration, 10 cattle each were also fed a low concentration (1.0%) and a 

medium concentration (2.5%), but no significant differences were noted. 
8 

Table 6. Mutagenicity by the supply (14 days) of milk and meat using mice 

(micronucleus test) 

(1) Milk 

Test group 

Number 

of 

animals 

Incidence (%) of micronucleus 

appearance 

(Min – max) 

Polychromatic erythrocyte rate (%) 

(Min – max) Assessment 

Negative control 

group (basal diet) 6 0.27±0.10 (0.1 – 0.4) 49.2±6.6 (42.2 – 57.1) 

Ordinary cattle 

2.5% group 6 0.22±0.17 (0.0 – 0.4) 49.4±3.8 (43.1 – 53.1) Negative

5% group 6 0.20±0.14 (0.0 – 0.4) 45.7±5.0 (36.8 – 50.3) Negative

10% group 6 0.12±0.10 (0.0 – 0.2) 44.5±7.5 (35.4 – 56.9) Negative

BNT cloned cattle 

2.5% group 6 0.30±0.14 (0.1 – 0.5) 44.0±6.7 (36.5 – 55.2) Negative

5% group 6 0.25±0.12 (0.1 – 0.4) 47.4±8.1 (36.5 – 56.3) Negative

10% group 6 0.17±0.08 (0.1 – 0.3) 44.7±8.4 (32.3 – 56.8) Negative

SCNT cloned cattle 

2.5% group 6 0.22±0.13 (0.0 – 0.3) 49.7±7.4 (35.8 – 56.3) Negative

5% group 6 0.28±0.15 (0.1 – 0.5) 49.5±7.8 (41.2 – 60.9) Negative

10% group 6 0.25±0.05 (0.2 – 0.3) 44.0±6.2 (34.2 – 52.8) Negative

Positive control 

group 

(Mitomycin C) 

6 6.02±1.03** (4.6 – 7.6) 34.6±5.5 (26.3 – 40.3) Positive

(2) Meat 

Test group 

Number 

of 

animals 

Incidence (%) of micronucleus 

appearance 

(Min – max) 

Polychromatic erythrocyte rate (%) 

(Min – max) Assessment 

Negative control 

group (basal diet) 6 0.20±0.18 (0.0 – 0.5) 47.7±9.7 (30.2 – 59.7) 

Ordinary cattle 

1% group 6 0.17±0.12 (0.1 – 0.4) 50.0±9.1 (37.9 – 61.3) Negative

2.5% group 6 0.13±0.08 (0.0 – 0.2) 47.3±13.1 (22.3 – 60.2) Negative

 

5% group 6 0.12±0.15 (0.0 – 0.3) 46.8±10.5 (37.2 – 63.5) Negative

BNT cloned cattle 

1% group 6 0.20±0.06 (0.1 – 0.3) 51.0±7.3 (41.3 – 59.3) Negative

2.5% group 6 0.23±0.14 (0.0 – 0.4) 47.1±4.3 (40.6 – 51.1) Negative

5% group 6 0.12±0.08 (0.1 – 0.2) 49.6±9.6 (37.9 – 61.9) Negative

SCNT cloned cattle 

1% group 6 0.18±0.10 (0.1 – 0.3) 48.3±8.4 (35.6 – 55.1) Negative

2.5% group 6 0.22±0.10 (0.1 – 0.4) 51.7±7.3 (44.3 – 63.9) Negative

5% group 6 0.22±0.08 (0.1 – 0.3) 48.4±8.1 (38.4 – 58.1) Negative

Positive control 

group 

(Mitomycin C) 

6 6.95±1.56** (4.1 – 8.4) 25.7±6.5 (19.4 – 36.4) Positive

Note: Milk and meat were freeze-dried and powdered and combined in feed. The positive control group was administered a 

single dose of 2 mg/kg of mitomycin C intraperitoneally. Values were shown as mean±standard deviation. 

** denotes a significant difference at p<0.01 against the positive control group. 
Appendix G: The Comprehensive Veterinary Examination 

Animal Cloning: A Risk Assessment DRAFT

Results of a 28-Day Feeding Study in Rats 

Fed Diets Containing Freeze Dried Milk or 

Beef from Ordinary Cattle and Clone Cattle
Animal Cloning: A Risk Assessment DRAFT 

This page intentionally left blank.
Table 1 Analyzed nutrient composition of the milk powder 

Macronutr ient (X) 

Crude protein 23.3 

Crude fat 23.4 

Carbohydrate 45.0 

Crude fiber 0.0 

Ash 5.4 

Water 2.9 

Vitamin (mgA00g) 

A 113IU/lOOg 

B 1 0.27 

Bz 1.11 

B s 0.17 

B 12 0.0017 

D3 ND 

E 0.4 

KI ND 

Kz 0.0070 

Niacin 0.86 

Pantotheinic 

acid 2.90 

Folic acid 0.05 

Biotin 0.00919 

Chol ine 0.10 (XI 

Mineral (mgA00g) 
Table 2-1 Comosition of the milk powder-contained diets 

(glkg diets) 

I Innredient I Basal diet ( 5% diet 110% diet 120% diet 1 

Milk powder 

Cornstarch 

Casein (93.5% protein) 

Alfa-cornstarch 

Sucrose 

Soybean oil 

Cellulose 

Mineral Mix * 

Vitmin Mix ' 

L-Cyst ine 

Choline Bitartrate (41.11 

chol ine) 

t-Butylbydroqinone 

Calollc value (kcal) 395 396 397 398 

Crude protein (X) 18.7 18.7 18.7 18.7 

Crude fat (X) 7.0 7.0 7.0 7.0 

Carbohydrate (X) 64.4 64.6 64.8 65. 1 

Crude fiber 0 5.0 5.0 5.0 5.0 

* : Ingredients of the mineral mix and vitamin mix are sbwn in table 2-2. -. 

Table 2-2 lnaredienls of tbe mineral mix wd vituin mix for the milk pmder-contained diet 

I Vituin - Mix 

% diet 

- 

0.687 

0.560 

0.510 

0.690 

2 400 

0. W) 

15. WO 

a 015 

3.m 

l.m 

0.200 

a 020 

875.048 

7 

I 

- 

, 

I 

- i : Caapotitian is differ frm that 

The mineral mix and vituin mix lo! 

at each additional level. 

1111 dlet % diet 1% diet 1% diet 1 lmredients I Basal dlet 

Calcium Clrbonate 

Potassim Phosphate 

(mnohas!c) 

Poturlum Cltrate Ha0 

ca:UI.M 

P:P78 

K:2873 

K:M16 

Ha : 38.34 

S :la3 

K:UBI 

Ug:W.32 

Pe : 16.50 

Zn : 52 14 

b:47.70 

Cu : 51.47 

1 :58.30 

Se : 41.79 

Yo:51.34 

Si: 9.88 

Cr : 10.42 

Li : 16.38 

B :17.50 

F:45U 

Ri:45.00 

V :4LB - 

I Sodium Chloride 

Potasslum Sulfale Vituine 1,. (0.W 2.m 

18 565 

5.881 Viluine 0, 0. W 

1.32 (rn. WOIU/8) 

Pa 

0.285 Vituine B Acetate l5WO 

0.003 (5001U/d 

0.- Vltul~e KI 0.075 

0.00785 ~IOCI~ 3000 

hnesiw Oxide 

Perric Citrate 

Zinc Cc.rbonate 

Ylaganous Carbonate 

Cipric Carbonate 

Polassiw iodate 

Sodium Selenate 

hniw Paramlybdalc 

4HaO 

Sodica Yetasllicate 9Rd 

Chromium Potassium 

S~lfate lW10 

Lithium Chloride 

Boric Acid 

Sodiu Plouride 

Nickel Carbooate 

Amnium Vwadate 

1.45 POUC ~cid a zw 

o. m 

~i~ti~ a 020 

0.0174 

0.0815 

0.0835 

0.0318 

0. am 

Sncrose 1 inel? 

713.18m powdered 74.655 I 

the AIN standared diet. 

tb CD.PO~I~IOD N deailed to reflect the mineral (casen1ia1 miterals) ud vituin contents of tbe milk pder 

Suerow finely p~dered 
Table 3 Body weights of male rats fed the milk powder-conlained diet in s 28day dose 

rangtf inding test 

0 

Conc. in Number of Day Gain 

diet animals 1 7 I4 2 1 28 0-28 

OX 6 125 I87 239 290 331 205 

f3*6*7*10 f13 12 

5% 6 125 183 241 297 340 215 

f 3f 6 11 f16 f22 f21 

I OX 6 I25 183 239 290 332 208 

f 3 f 7 f 12 f 18 f 20 f 17 

20% 6 125 183 238 281 320 196 

f 4 f 9 *I2 f21 f26 27 

Each value is expressed as mean* S.D. r 

Table 4 Body weights of fena!e rats fed the mllk powder-contained diet in a 28day dose 

range-finding test 

- - - - - - - - - - 

Cone. in Number of Day Gain 

diet animals 1 1 I4 2 1 28 0-28 

20% 6 117 153 181 207 229 112 

f 8 f 10 f I1 f 22 f 25 f 18 

Eacb value is expressed as reanf S.D. 
Table 5 Food consumption of male rals fed the milk powder-conlained 

diet in a 28day dose range-finding test 

(9) 

Conc. in Number of Week 

diet animals 1 2 3 4 

Each value is expressed 'as meanf S.D. 

Table 6 Food consumption of female rats fed the ilk powder-contained 

dlet in a 28day dose rangelinding test 

k) 

Conc. in Number of Week 

diet anlmals I 2 3 4 

I I I4 15 I5 

20% 6 i 2 i 2 i 2 i 1 

Each value is expressed as meanf S.D. 
Table 7 Urinary findings of male rats fed the milk powderxontained diet 

in a 28day dose range-finding test 

Conc. in No. of Color Cloudy pH Protein Glucose 

diet animals PY - + 6.0 6.0 6.6 7.0 7.6 8.0 8.6 - + ++ +++ - * + ++ +++ 

Conc. in No. of Ketone body Occult blood Urobilinogen Bilirubin 

diet animals - * + ++ +++ - + ++ +++ 0.11 2 4 8 - + ++ +++ 

" 

Color : PY(pale yellow) 

Cloudy : - begligible), +(cloudy) 

Protein : -(negligible), *(15-SOmgldL), +(3OmgldL), ++(100mgldL), +++(300mgldL) 

Glucose : ,- begligible), *(O.lgldL), + (O.PbgldL), ++(0.6gldL), +++(lgldL) 

Ketone body ': -(negligible), *(6mgldLj, + (lSmgldL), ++(40mgldL), +++(80mgldL) 

Occult blood : -(negligible). *(trace), +(slight). ++(moderate). +++(marked) 

Urobilinogen : Ehrlich unit/dL 

Bilirubin : - (negligible), +(slight), ++(moderate), +++(marked) 

Table 8 Urinary findings of female rats fed the milk powder-contained diet 

in a 28day dose range.finding test 

Conc. in No. of Color Cloudy pH Protein Glucose 

diet animal8 PY - + 6.0 6.0 6.6 7.0 7.6 8.0 8.6 - t + ++ +++ - t + ++ +++ 

Conc. in No. of Ketone body Occult blood Urobilinogen Bilirubin 

diet animals - * + ++ +++ - + + ++ +++ 0.1 1 2 4 8 - + ++ +++ 

Color : PY(pa1e yellow) 

Cloudy : -(negligible), +(cloudy) 

Protein : -(negligible), *(I65 BOmgldL), + (3OmgldL), ++(100mgldL), +++(300mgldL) 

Glucose : -(negligible). *(O.lgldL). + (0.26gldL), ++(O.bgldL), +++(lgldL) 

Ketone body : -(negligible), *(6mgldL), +(16mgldL), ++(4OmgldL), +++(80mgldL) 

Occult blood : -(negligible), *(trace). +(slight), ++(moderate), +++(marked) 

Urobilinogen : Ehrlich unit/dL 

Bilirubin : - (negligible), +(alight), ++(moderate), +++(marked) 
Table 9 Hematological findings of male rate fed the milk powdercontained diet 

in a 28by dom rmge.finding test 

Each value ia exuressed as meaniS.D. 

Table 10 Hematological fin(lmpp of female rate fed he milk powdwcontained diet 

in the =day repeat dose toxicity test 

Differential leukocyta wuuts 06) 

Cone. in No. of Neutro. Plat. 

diet animals Barn. Eom. Stab. Seg. Lymph Mono. Other (10'lpL) 

ac va ue 18 emrease aa me : &~&cantJ~ diffeAt hom%%iat 6% level of probability 
Table 11 Blood biochemical 6ndings of male rats fed the milk powdercontained diet 

in a 28day doae range-6nding test 

Each value a ex~resaad as mean;tS.D. : ~ignificantlyhifferent from control at 6% level of probability ** : Sdcantly dSerent from control at 1% level of probability 

Table 12 Blood biochemical findings of female rats fed the miJk powdercontained diet 

in s 28day dose rangwfinding test 

Each value la expressed as mennS3.D. 
0 6 312 1.89 8.72 2.37 0.63 1.09 1.21 0.62 21.0 10.0 

Absolute f12 fO.10 f0.69 20.20 fO.07 20.06 fO.10 20.10 1. 21.5 

Each due u expressed M mean f S.D. 

a) : Rekttve cfm wekht per 1% b& wei& 

Tatle 13-2 Absolute and rskh organ wd&b d mde nu fed dry beef-connined dist in a 28day dose ranasfindinn study 

0 6 312 53.7 2.70 0.49 1.23 0.77 

Absolute f12 25.3 *a24 *am fo.18 20.06 

Each due Ls emessed as mean f S.D. 

a) : Relative cqan wekht rn 1lOO tcdy weinht 
TaUe 14 Absduw and rehtiw arm weights d female rats fed dk powdercontained diet in a Bday dose nnm-kdinn st& 

CWC. in Naof B.W. Brain Liver KidnW Sdeen Heart Lwl( Thymus Ihyr. Pitui Adred hv Um 

dietOAni 0 0 (R) (R) (R) (R) (R) (R) (d d (md (4 (R) 

0 6 211 1.85 569 1.63 0.42 0.75 0.98 0.52 21.0 12.9 54.9 78.3 0.47 

Absolute f10 f 0.10 f 0.25 '0.14 f0.M f 0.06 f 0.03 f 0.10 f 2.4 f 1.2 f6.0 f10.7 f 0.13 

Each due k exmessed as mean f S.D. 

a) : Rekh axan weight per 100~ bcdy wekht 
L11 CSI 281 LOZ 622 usan 

211 8P1 Ell 261 91Z tZS 

E2S tll OS1 811 EOZ OZZ 

ZZS 611 191 681 1lZ 822 

1ZS EEI 191 112 LPZ --- 

RLZ 

011 ZS1 E81 soz SZZ 

S11 Otl 091 S81 SOZ 

s oa 

XOZ i 61s 

81s tll IS1 SLI 661 612 

11s 121 991 P81 LO2 ZEZ 

915 611 t91 061 OZZ 8PZ 

SIS 911 ESl 581 EIZ ZEZ 

+IS Zll tP1 691 181 S1Z 

1 XO ElS 011 6E1 191 881 Z1Z 

Rean 911 ESl 8LI EOZ 622 

911 1S1 181 802 SZZ 

311 Ltl .- ZLI . . . IOZ --- 

kCZ 

01s 811 IS1 PLl 061 802 

60s I11 1P1 191 081 002 

211 PPI 691 861 IZZ 

SZ1 t81 602 OPZ 182 

80s 

XS LOS 

911 OSI L11 ZOZ PZZ usan 

90s ZZ1 PSI 811 802 8ZZ 

SOS 011 Etl ELI S61 112 

tOS 021 CSl 8L1 L61 LIZ 

EOS Zll ttl 891 P61 91Z 

ZOS 911 ESI 981 OIZ EPZ 

XO 10s tlI HI 6L1 SOZ 1EZ 

tZO 1El 181 St2 1SZ 182 

EZO tZ1 081 9EZ 682 LZE 

zzo 811 991 812 t9Z 662 

120 121 261 SSZ 60E tSE 

020 EZ1 L81 8EZ 882 6ZE 

XOZ 610 $21 981 LEZ L8Z 1st 

SZI E81 6EZ 062 ZEC oean 

810 621 681 EtZ t6Z 6EE 

110 LZ1 Z61 ESZ 60E 1% 

910 021 1L1 1ZZ 092 662 

$10 CZ1 181 SEZ 282 tZE 

t10 921 281 OSZ LOE ESE 

I XO EIO tZ1 281 EEZ 982 8ZE 

man SZ1 E81 It2 L6Z OtE 

ZIO LZI E81 ZEZ 062 OEE 

110 621 811 6EZ 962 LEE 

010 SZ1 981 8+Z tOE OSE 

600 tZI 081 LEZ 682 EEE 

800 611 SLl OEZ LLZ Z1E 

XS LOO $21 E61 8SZ PZE 9LE 

$21 L81 6EZ 062 ICE osan 

900 921 061 1EZ S8Z ItE 

SO0 tZI 8L1 ZEZ t8Z EZE 

to0 OEl E61 6tZ 90E SPE 

COO LZI t81 SEZ 6LZ I1E 

ZOO OZI E81 EtZ 062 9ZE 

XO 100 SZ1 161 EPZ S62 LEE 

paololuo~-iaprod 1a!p ol o hspgz asop Bo!po(j-aausi lsal 

x!PoJddV I lenp!r!pol hpoq slppr lo alw slsi pal aqi ql!w 
Appendix 3 Individual food consumption 01 male rats fed 

the milk powder-containd diet In a 28day dose 

range-f inding test 

(a/dav/rat) 

Conc. in Cage Week 

dlet oomber I 2 3 4 

Mean 16 19 20 20 

Mean 16 19 20 20 

Mean 15 19 20 20 

Mean' I5 18 17 19 

Appendix 4 Individnal food consumption of female rats fed 

the mi Ik powder-containd diet in a 28day dose 

range-findlng test 

(g/day/rat) 

Conc. in Cage Week 

diet number I 2 3 4 

OX 13 12 I4 16 17 

I4 I I I4 I5 16 

I5 I I 14 14 14 

Mean I I I4 16 16 

5% 16 13 15 19 19 

I7 I I 13 13 13 

18 I I 15 14 16 

Mean 12 I4 15 16 

I 0% 19 10 12 13 13 

20 13 15 I5 16 

2 1 I I 13 14 15 

Mean I I 13 14 15 

20% 22 10 13 14 14 

23 13 16 17 16 

24 I I 12 13 14 

9 Mean I I 14 15 15 
Appendix 6 lndividual urinary findings of male rats fed the milk powder.contained diet 

in a 28day done range.fiding test 

Conc, in Animal Color Cloudy pH Protein Glucose Ketone Occult Urobilinogen Bilirubin 

diet number body blood 

- + - - - 0.1 - 0% 001 PY 6.0 

- + - - - 0.1 - 002 PY 6.0 

023 PY - 6.0 - - 0.1 - 

024 PY - 6.5 * - - - r. 0.1 - 

Color : PYbrle vellow) 

Cloudy : -?negigible) 

Protein : -(negligible), t(l6-30nigldL), +(aOmg/dL) 

Glucose : -(negligible) 

Ketone body : -(negligible), i(5mgldL) 

Occult blood : -(negligible) 

Urobilinogen : Ehrlich uniUdL 

Bilirubin : -(negligible) 

Appendix 6 Individual urinary fidings of female rats fed the milk powder.contained diet 

0 

in a 28day dose range.finding test 

diet number body blood 

0% 601 PY - 7.0 - - - - 0.1 - 

602 PY - 6.6 - - - 0.1 - 

523 PY - 6.0 - - - 0.1 - 

524 PY - 6.0 - - - 0.1 - 

Color : PY(pa1e yellow) 

Cloudy : -inegligible) 

Protein : -(negligible), *(16-3OmgldL) 

Glucose : -(negligible) 

Ketone body : -(negligible), *(SmgldL) 

Occult blood : -(negligible). *(trace) 

Urobilinogen : Ehrlich uniUdL 

Bilirubin : -(negligible) 
Appendix 7 Individual hematological findings d male rat8 fed the milk powderwntained diet 

in a 28day dona range.Bnding test 

Conc. m Anunal Hb Ht MCV WBC Plat. PT 

diet number (lo'/& (gldW % (fW (ps) (%) 101 10 (see) 

0% M)1 770 14.8 42.2 66 36.1 67 110 13.2 20.1 

@I2 764 16.6 43.7 67 20.3 36.6 61 95 12.9 22.4 

M2 7% 14 7 41.2 66 20.0 35.7 49 94 13.4 20.6 

Mean 734. 14.6 41.6 67 19.9 36.0 60 121 12.7 19.2 -- .-f M. - * --- -*Yr -. -- -yfK * ---- y4,-2 -.- - -. *do..3-.--- .-5g --.. .Yg.-6. -.- -.3b:y.-- .- --6y- - -. - --f lE- .- -g--- - 2u.x-----. 

014 781 15.6 42.9 56 19.8 36.1 52 118 12:7 19.1 

016 743 16.4 43.8 69 20.7 35.2 60 120 13.5 18.7 

016 822 14.2 40.6 49 17.3 35.0 28 150 12.9 18.6 

017 753 14.8 42.6 67 19.7 34.7 46 118 13.0 16.8 

018 747 14.7 43.2 68 19.7 34.0 39 1,!0 12.8 19.5 

Mean 760 14.8 42.2 66 19.6 36.0 46 122 13.0 - 18.8 --.-2 ------.v $.-----T49------y4~y--....-di~r..---.EK--.--- c----..3gK------.6T------- f2 s.---.. rg------ itr.7-----. 

020 119 14.8 42.3 69 20.6 35.0 45 116 13.0 21.2 

021 718 14.6 42.0 58 20.3 34.8 30 129 13.1 18.4 

022 780 14.8 42.0 64 19.0 35.2 40 132 12.7 18.3 

023 734 14.4 41.6 67 19.6 34.6 67 142 13.2 20.3 

024 411 7.2 23.6 57 17.6 30.6 61 87 13.1 16.8 

Mean 685 13.4 38.8 57 19.4 34.3 48 122 13.0 19.3 
Appendix 8. 1 Individual hematologid findings of female rats fed the milk powdercontained diet 

in a 28day dose range-fink teat 

unc. m Anma1 Hb Ht MCV MCH MCHC FT m WBC 

diet number (10'bW (%dU (%) (fu 9, ("A) (4 (set) (1O'hL) 

0% 601 1. 1. 1. 13. 64 

602 1 1 !"82"0.",::! 13.: i:: 34 

Mean ......5'k' . '-""W ....... 

608 

609 

610 

611 

612 

....m * A ........Kf9.........7g6........lE.$......... Mean 744 14.7 41.5 3.3 66 * 19.8 o.r .......3K.3......... 36.6 13.2 .3..x .........i r. 18.6 26 

620 687 14:4 38.7 66 21:0 37.2 12.9 18:7 37 

521 726 14.6 40.6 66 20.0 36.8 14.1 17.4 26 

Mean 742 14.8 41.1 66 20.0 36.1 13.0 17.5 33 

Appendix 8 . 2 Individual hematological findings of female rats fed the milk powderxontained diet 

in the ?.&day repeat doos toxicity tea 

rn renh 

Conc. in Animal Neutro. Plat 

diet number Baso. Em. Stab. Seg. Lymph. Mono. Other (1041pL) 

0% 601 0 1 0 14 82 3 0 102 

...... .-.... Mean .b.oy ....--.. .K 0 .......... -@. 1 .......... 0 ......... 12 ......... 86 -....... 2 0 119 ........-... -...-...-...-. 90----- 

608 0 1 1 16 81 1 0 139 

604 0 3 0 18 78 1 0 120 
Appendix 9 - 1 Individual blood biochemical fmdings of male rats ted the milk ~owderwntained diet 

in a 28day dom range-finding test 

Mean 269 77 31 663 0.42 66 194 6.94 49.9 22.1 ....... i:i ...... igi ....... 3:? .... .@O '.".l.oK." ...Bi. ....... 3ti." ... ..8a" ...... Y3'...3'.3' 5B .....K @ 4444444444 $9'-.9'9'.. 24 6'6'-... b:m .. " .6.1..4 ..e-l. E-. -.-.-. 4.2- 1.w014 331 88 29 834 0:26 96 214 6.90 62:2 2216 3.8 4.7 13.2 3.6 1.09 

016 309 80 32 953 0.64 86 247 6.83 60.8 21.9 4.8 6.9 13.0 3.6 1.08 

016 291 87 34 803 0.72 61 189 6.10 48.4 21.7 6.4 6.2 14.6 3.8 0.94 

017 366 73 26 610 0.83 38 213 6.90 49.6 18.0 6.3 6.9 16.0 3.3 0.98 

....mK...... Mean .$.. .... ;r? 301 ... .....18....... 80 .$ ....... ;;; .... $$ ........ :; ...... .g .-.... g;.. g; ..... ;.:; ...... ;g. ......$ .... !?:1 ....... ;; ...-- ig.. 13.0 . 020 323 71 28 747 0:39 42 188 6.98 60:1 19.4 6.6 6.1 14.8 4.1 1:00 

021 177 66 25 686 0.45 36 163 6.14 64.0 21.1 6.4 3.8 12.7 3.0 1.17 

022 321 72 32 463 0.67 32 220 6.26 62.6 23.1 4.6 3.7 11.6 4.4 1.11 

023 211 59 22 613 0.33 40 176 6.29 46.3 17.6 6.3 7.0 17.7 6.2 0.86 

024 616 90 26 283 1.32 40 264 6.12 62.0 21.0 6.7 4.4 14.1 2.8 1.08 

Mean 337 73 27 643 0.61 40 207 6.14 60.8 20.7 6.9 4.8 14.0 3.9 1.04 

Appendix 9 - 2 Individual blood biochemical finding8 of male rats fed the mik powder-wntained diet 

in a 28day do= ranga-hding teat ' 

......K~.......m7.........6'6......y25........ Mean 68 71 ? ....... Gl .... Ti:? ..-.-. ;:g ..... ;:;; ...... ;:g --..- ;;:; ....... .....- ;.-.... ;$; ...... ..... 1Oil 122 12.0 ; 008 46 71 81 119 13.3 0:76 0:64 0133 10:2 7:0 144 4.37 106 

009 66 86 106 122 11.4 0.93 0.64 0.27 10.0 7.2 143 4.71 106 

010 63 72 83 132 12.7 0.93 0.48 0.26 10.2 8.3 142 4.84 106 

* %. ......dy3'........62........ Mean 66 73 ...... 89 127 12.8 0.83 0.60 0.30 10.2 7.7 144 4.66 106 .$. 9T-... .&.-. ...iPPB......SSB6 --.... bb K-....b' 99'.... ib'b'l ......... 7.738...i 5d ......;r..6 TTTTTTTTTT 

11b'--- 

014 62 98 90 137 11.8 0.78 061 0:24 1012 619 144 4:48 107 

016 39 76 84 126 11.6 1.06 0.66 0.30 10.1 7.3 143 4.69 107 

016 36 31 66 168 12.3 0.79 0.60 0.21 9.7 7.6 144 6.01 110 

017 32 43 63 162 12.8 0.98 0.66 0.30 10.2 8.2 143 4.87 106 

'--'m*.-".' ..6iS'........ Mean 43 68 78 184 12.0 0.86 0.66 0.27 10.1 7.6 144 4.70 108 47 ........ ?. ......... $.. .............. i2.,7 .-...- ti gr... ..b',gr... ..b.2r .....ib.. ....... ......fx6......6B...... .lw .... 131 

020 30 40 60 130 10.1 0:80 0.47 0:24 10:l 7:0 143 6:19 108 

021 61 66 100 130 10.8 0.79 0.47 0.29 10.4 7.9 146 4.72 106 

022 69 104 110 126 12.6 0.74 0.63 0.26 10.3 7.6 146 4.76 108 

023 48 96 93 135 8.6 0.80 0.60 0.24 10.7 6.9 146 4.66 108 

Mean 66 78 94 128 10.7 0.91 0.62 0.26 10.3 7.6 146 6.06 108 
&pendiX 10 - 1 Individual blood biochemical findings of female rats fed the milk powdercontained diet ~n a %day dose range.finding tea 

nc. a1.G a8.G or in ima Y' kd B $% 2% ai $$ $v"Z 8 &% &" (e (o (2 (z NG 

- 0% 501 343 480 0.31 390 166 6.66 56.4 19.8 4.8 2.2 12.6 4.2 1.29 

502 300 66 21 330 0.68 199 156 6.44 67.6 18.9 6.6 2.2 11.6 4.3 1.86 

Mean 320 76 25 441 0.39 316 171 6.39 66.2 19.6 6.4 2.1 12.6 4.2 1.28 .................$ Di ........2g3........6.*........zg...... i.2 ......63*....... .z...... x6 ......c.57.....$ ..2-...-- ,...$ ........ 4,.i ........ $;* .... .i .2.4..... ..4.. * . .....] ..r 4... 

5% 

508 567 85 31 432 0:27 443 209 6.41 57:3 17.4 6.9 2.6 12.3 4:6 1:34 

509 236 79 22 396 0.63 4W 117 6.70 51.7 20.4 7.3 3.9 12.3 4.4 1.07 

Mean 453 82 25 404 0.38 412 198 6.38 56.1 17.7 6.8 3.0 ..-l2:9 4.6 L29 ... .,* ii... .... .... ....4u2 ..... ...is. ....... z4 .b.6i68 .....d .6 ........z5r......8 w' ... 1.3' .....6;8'8'.g...... 8'8'.1 ........ 44.6'.... ...* ;B-- 3.T ......2...,......l..Ax.. 

514 252 71 21 467 0.35 161 154 6.02 55.2 19.9 4.8 2.8 12.6 4.8 . 1.23 

515 381 81 24 468 0.31 420 198 6.18 66.1 20.9 3.7 2.8 13.2 3.3 1.28 

516 392 87 26 392 0.41 217 201 6.75 57.6 19.5 3.7 2.1 13.4 3.7 1.36 

517 320 89 22 539 0.51 368 159 6.64 59.0 18.8 6.5 1.9 12.7 2.1 1.44 

618 334 81 24 471 0.61 646 195 6.84 56.4 20.1 4.4 2.1 12.7 4.3 1.29 

Mean 347 .... H,* -....- .6 r9 -....-. 3,-o --... 

620 334 

521 412 

Mean 430 85 27 388 0.32 378 195 6.48 56.6 18.6 5.5 2.6 12.6 4.1 1.31 

Appendix 10 - 2 Individual blood biochemical tindings of female rate ted the milk powder-contained diet 

in a 28day dose range.finding test 

Mean 64 30 111 ..""l;ii...... g6i ......... * .8 ........ 3'33..3.3.3 ... 

144 

508 74 56 130 

L- Mean 65 25 99 120 16.1 0.97 0.66 0.27 9.8 6.1 146 4.49 107 
Appsndix 11 Ahrdute -weights of individual rmle rats fed milk powdei-mnhined diet in a 28-day dore range-hdinp study 

Mean 312 1.89 8.72 2.37 0.63 1.09 1.21 0.62 21.0 10.0 53.7 2.70 0.49 1.23 0.77 

Mean 320 1.93 8.98 2.49 0.62 1.15 1.25 0.67 21.4 10.6 53.1 2.68 0.50 1.22 0.75 

10 013 312 1.93 8.02 2.35 0.56 1.04 1.19 0.55 23.9 10.2 44.6 2.77 0.55 1.27 0.711 

. . 

20 019 308 1.94 8.28 2.28 0.62 1.13 1.30 0.72 25.5 10.0 65.2 rS.12 0.49 1.14 0.68 

020 309 1.88 8.50 2.38 0.69 1.08 1.21 0.71 22.6 10.7 54.3 2.65 0.37 1.89 0.75 

02 1 334 1.91 9.66 211 0.57 1.11 1.22 0.72 20.6 11.1 56.3 2.97 0.51 1.31 0.68 

022 278 1.91 8.33 217 0.56 1.02 1.05 0.51 19.8 10.3 41.2 2.64 0.60 1.43 0.76 

023 307 1.88 1l.M 2.26 0.68 1.00 1.19 0.70 25.3 10.2 58.1 267 0.60 1.23 0.74 

07.4 265 1.93 7.48 2.31 0.71 0.99 1.14 0.61 166 9.7 46.8 2.63 0.37 1.16 0.64 

Mean 300 1.91 8.88 235 0. 1.06 1.19 0.66 21.7 10.3 53.7 2.61 0.49 1.36 0.71 

Appendix 12 Rehtive weights of indivimvl de rats fsd milk powdermnWl rd diet in a 28-day daM ranee-hding ah 
~~~dh 13 Absolute orpan weights of individual female rats fed milk powdercontained dlet in a 28-day dose ranee-finding study 

cone. in Animal B.W. Brain Liver Kidney Spleen Hesrt Lung Thymus Thyr. Pitui ~drene.1 ovary Uterus 

diet(%)numbers (g) (&) (8) (g) (8) (8) (8) (id (mid hid hg) @ 

- 

0 501 218 1.92 5.91 1.61 0.46 0.76 0.96 0.46 25.7 14.4 50.2 76.3 0.53 

502 226 1.88 6.23 1.88 0.42 0.85 1.03 0.67 19.1 12.9 59.9 89.1 0.44 

503 201 2.01 6.05 1.64 0.45 0.75 0.97 0.49 19.4 13.4 60.1 91.8 0.47 

504 209 1.78 5.76 1.64 0.35 0.70 0.98 0.42 20.1 13.4 51.9 63.8 0.38 

505 199 1.74 5.50 1.52 0.44 0.69 0.95 0.61 21.5 12.2 60.4 78.0 0.31 

506 212 1.77 5.86 1.47 0.40 0.73 0.99 0.46 20.4 10.8 46.6 70.6 0.69 

Mean 211 1.85 5.89 1.63 0.42 0.75 0.98 0.52 21.0 12.9 54.9 78.3 0.47 

- 

5 507 265 1.82 8.51 1.88 0.58 0.95 1.09 0.55 19.2 17.1 71.7 77.9 0.63 

508 211 1.87 5.96 1.47 0.49 0.79 1.02 0.57 17.8 12.2 €4.3 80.9 0.46 

509 186 1.79 5.39 1.47 0.37 0.67 1.02 0.33 17.3 .13.8 63.3 70.6 0.66 

510 196 1.87 5.53 1.57 0.43 0.67 1.04 0.44 23.2 13.9 44.0 80.1 0.40 

511 227 1.83 6.05 1.42 0.37 0.75 0.90 0.58 26.7 11.0 49.3 60.5 0.24 

512 215 1.78 5.63 1.59 0.43 0.81 0.92 0.48 17.8 11.7 67.2 75.5 0.42 

Mean 217 1.83 6.18 1.57 0.45 0.77 1.00 0.49 20.3 13.3 60.0 74.3 0.47 

10 513 193 1.70 5.26 1.41 0.44 0.70 0.91 0.51 22.4 9.9 52.2 60.8 0.53 

514 201 1.79 5.49 1.56 0.39 0.67 0.93 0.39 17.8 11.1 47.9 106.9 0.37 

515 218 1.93 5.86 1.80 0.46 0.79 0.97 0.47 22.5 11.2 50.7 76.2 0.43 

516 232 1.77 6.54 1.91 0.39 0.88 1.01 0.50 20.5 12.4 55.7 97.1 0.65 

517 217 1.92 6.69 1.52 0.48 0.75 0.95 0.49 19.6 14.1 55.1 96.6 0.45 

518 204 1.79 5.44 1.64 0.37 0.79 0.94 0.42 19.1 12.2 52.7 79.1 0.47 

Mean 211 1.82 5.88 1.64 0.42 0.76 0.95 0.46 20.3 11.8 52.4 86.1 0.48 

20 519 198 1.76 5.64 1.64 0.44 0.69 0.99 0.63 20.9 1O.V' 65.6 64.2 0.56 

520 214 1.83 6.22 1.81 0.37 0.76 1.00 0.58 20.6 13.9 53.3 101.5 0.40 

521 263 1.89 7.29 1.93 0.43 0.91 1.18 0.55 23.4 14.6 61.7 84.4 0.40 

522 211 1.74 5.69 1. 0.37 0.73 1.00 0.52 24.8 11.9 58.9 74.9 0.46 

523 211 1.81 5.78 1.85 0.42 0.77 1.W 0.38 17.1 12.6 55.0 74.2 0.48 

524 201 1.80 5.92 1.62 0.48 0.73 0.89 0.48 20.9 13.2 59.7 76.6 0.45 

Appendix 14 Relative organ weights ofindividual female raLI fed mlk powdercontained diet in a 28-day dose range-findi study 

506 212 0.83 2.76 0.69 0.19 0.34 0.47 0.22 9.6 5.1 22.0 33.3 0.33 

Mean 211 0.88 2.79 0.77 0.20 0.35 0.47 0.25 10.0 6.1 26.1 37.2 0.22 

512 215 0.83 2.62 0.74 0.20 0.38 0.43 0.22 8.3 5.4 31.3 35.1 0.20 

Mean 217 0.86 2.84 0.73 0.21 0.36 0.47 0.23 9.5 6.2 27.8 34.7 0.22 
Table 1 Analyzed nutrient composition of the dried beef 

Macronu t r ien t (%) 

:rude protein 

:rude fat 

:arbohydrate 

:rude fiber 

\sh 

Rater 

V i tam i n (mg/lOOg) 

A 

B .I 

Bz 

Ba 

B 12 

Ds 

E 

K I 

K z 

Niacin 

Pantotheinic 

acid 

Folic acid 

Biotin 

Cho 1 ine 

Mineral (mg/lOOg) 
I Ingredient Basal diet 

I 

Dried beef 

Cornstarch 

Casein (93.5% protein) 

Alfa-cornstarch 

Sucrose 

Soybean oi 1 

Cellulose 

Mineral Mix ' 

Vitamin Mix ' 

1-Cyst ine 1 Choline Bitartrate (41.1% 1 1-Bu%&% inone 

Calolic value (kcal) 

Crude protein (X) 

Crude fat a0 

Carbohydrate (X) 

Crude fiber 0 

Wkg diets) 

5% diet 10% diet Basal diet 20% diet 

11 

* : Ingredients of the mlneral mix and vitamin mix are shorn in table 2-2. 

Table 2-2 Intred~ents of the mineral mix md vituln mix for the dried basf-contained diet 

Mid 

Mineral Mix I 

Ingredients 

ESEUrIAL YlWW 

hlciw Carbonats 

(mnobasic) K:2873 

Sodium Chloride 

Potassilo Sulfate 

Yapnesium Oxide 

Ferric Citrate 

Zinc Carbonate 

YInganous Carbonate 

Cupric Carbonate 

Potassium Iodate 

Sodium Selenate 

haniw Parmi ybdaj 

4HaO 

Sodlum Metaaillcate 9H.0 

Chromium Potasalum 

Sulfate 12hO 

Llthiw Chloride 

Boric kid 

Sodium Flouride 

Nickel Carbonate 

haaium Vanadate 

Sucrose finely mdered I I 223. 646 

5% 10% &ad 20% Ingredients 

diet diet diet U diet 

Vituine A Palmitate 

600. mlU/g) 

356357 355786 357.00 3x.m 

192359 lBB843 196.00 181.688 Thluine ACl (8,) 

51.612 45 832 1228444 Riboflavin (8.) 

Rrldoxlne UCl (8,) 

Vitulne 8,~ (O.lX) 

Vitulne 0, 

(400.0001U/g) 

I Vltulne B Acetate 

(5M)IU/g) Vltulne K, 

I I I I Calclu Pantothenate 

1.45 1.45 1.45 1.45 Polic Acid 

o.m 0.275 am am 

Biotin 

0.0174 0.0174 0.0174 0.0174 

0.~15 0.0815 a onis o.0~15 

0.0835 0.0635 0.0835 0.0895 

0.0318 0.0318 O.Ml8 a0318 

0.m 0.0066 am am 

Sucrose finely !%?.92688 '381.511 160.562 436.999 powdered 

Vituin Mix 

4 : CamPotition is differ from that of tho AIN 11 

The mineral mix and vituin mix for each compotil 

at each additional levsl. 

~sal d- 

!t I/P 

0.800 

tandared diet. 

tion ns designed lo reflect the mineral (esrenti~l miterals) and vituin eontents of the drled beef 

5% 

diet 

0.800 

10% 

diet 

0.800 

20% 

diet 

0.800 
4 

! 

Table 3 Body velghts of male rats fed the dry beef-contained diet in a 28day dose 

range-finding lest 

(8) 

Conc. in Number of Day Gain 

d~el animals 1 7 14 2 1 28 0-28 

Each value is expressed as mean* S.D. 

a):Control I b):Control U P- 

*:Significanlly differenl from conlrol II at 5% level of probabilily 

Table 4 Body veighls of female rats fed the dry beef-contained diet in a 28day dose 

range-f inding test 

(9) 

Conc. in Number 'of Day Gain 

diet animals I 1 I4 21 28 0-28 

Each value is expressed as meanfS. D. 

a):Control I b):Control U 
Table 5 Pood consumplion of dale rats fed the dry beef-contained 

diet in a 28day dose range-finding lest 

(ddayhal) 

Conc. in Number of Week 

diet animals I 2 3 4 

Each value is expressed as meanfS. D. 

a):Conlrol I b):Conlrol 11 

Table 6 Pood consumption or female rals fed the dry beef-contained 

diel in a 28day dose range-finding test 

(g/day/ral) 

Conc. in Number of Week 

diet animals I 2 3 4 

Each value is expressed as meanf S. D. 

a):Control I b):Control ll 

Table 

Conc. 

diet 

0' 

6' 

lo! 

. . . . . . . . . 

O! 

203 

Canc. 

diet 

O! 

6! 

109 

. -. . . . . . . 

09 

20% 

7!&c 

Cloud: 

Protei. 

Glucol 

Ksbm 

Occult 

Urobil 

Bilirut 

a) : Co 

Tab11 

CDnc 

- diet 

( 

I 

10 

. . . . . . . .

( 

20 

Cone 

- diet 

( 

t 

10 

. . --. . ,

( 

20 

rn 

Clou, 

Protc 

Glue, 

Ketn 

Oceu 

Urob 

Bilir 

a) : C 

Table 9 Hematolo 'cal Kndin a of male rata fed the dry beefantamed diet 

in the 28-fay repeat ioae toxicity test 

Cone, in No. of RBC Hb Ht MCV MCH MCHC WBC 

diet animale (10'hL) (gldL) % W bg) ( (set) (sed (IO'CL) 

PA" 6 737 14.0 40.7 66 19 0 34.4 12.8 19.4 60 * 24 * 0.7 1.9 4 + 1.2 0.7 * 0.3 17 7 

6% 6 773 14.6 42.6 66 18.8 34.2 13.3 18.8 62 

20 0.6 1.1 2 0.7 0.6 0.4 * 1.1 22 

1VA 6 73 1 14.2 40.9 66 19.4 34.6 13.6 19.1 46 

66 * 1.3 t 3.4 2 0.8 1.2 0.8 1.4 6 

Differential leukocyte counts (%) 

Cone. in No. of Neutro. Plat. 

diet animala Baso. Eoein. Stab. Seg. Lymph. Mono. Other (10'1~/11U 

Each value is e rewd as meanS.D. 

a) : Contml I. 8 : Control II 

6 : Significantly different from wnhlII at 6% level ofprobability 

Table 10 Hematological findings of female rats fed the dry beef-wntained diet 

in a 28day dose rangefidiag teat 

Conc. in No. of RBC Hb Ht MCV MCH MCHC WBC Plat FT AFlT 

diet animala (lo'/$) @dL) (%) (a bd (%) (1O'bW (10'lpL) (e3 (ad 

0%" 6 762 16.0 42.0 66 19.7 36.8 37 104 12.8 18.1 

28 * 0.3 * 1.2 2 S 0.6 * 0.6 6 * 7 0.3 1.4 

6% 6 767 14.8 42.2 66 19.4 36.2 46 120 12.9 17.7 

21 0.4 0.6 2 0.7 0.7 30 13 * 0.3 1.4 

10% 6 787 16.3 43.1 66 19.6 35.6 29 126 ** 13.0 18.0 * 38 0.4 t 0.8 i 2 0.8 0.7 6 10 0.4 0.6 

..................................................................................................................................................... 

0%" 6 748 16.3 43.0 68 20.4 36.6 34 114 13.2 18.1 

23 0.4 0.9 1 0.6 0.7 9 16 0.6 i 1.6 

20% 6 766 14.2 40.W 63## 18.- 34.9 33 124 12.9 18.3 

28 * 1.2 2.2 2 1.2 1.6 10 17 0.3 0.6 

Each value is e remd as mea&.D. 

a) : ~ontrol I. 3 : control 11 ' : SignScantly diffirent from wntml I at 6% level of probability antl different from mn-1 I at 1% level of probability *=:Si* y,.tr ,& # . S~phan y I eren m mntmlII at 6% level of probability ## : signiticantly different fmmmntmlI1 at 1% level ofprobability 

Tabla 11 

-isiz 

diet 

0.A" 
Table 11 Blood biochemical tindinga of male rats fed the dry beef.contained diet 

in a %day do@ rangwinding teat 

-bnc. & in 0.0 L H 

diet animals QUU QUIL) aUm ~un) sun) sun) @dL) (%) ()o (%) (%) ('4 (fC) 

0%'' 6 323 82 30 666 0.66 60 223 5.84 60.7 22.8 5.6 2.9 14.3 3.9 1.03 

* 119 * 8 i 3 i 16410.26 i 10 + 34 +0.22 i 2.1 + 1.6 + 0.9 * 0.5 l 1.0 0.9 i0.09 

5% 6 238 15 28 136 0.41 49 220 5.88 62.6 21.9 6.0 2.8 13.5 3.2 1.11 

* 84 * 8 i 2 i 135 i0.12 i 6 i 81 +0.32 i 1.6 + 0.9 l 1.0 l 0.6 l 1.0 0.1 i0.01 

10% 6 288 5.83 52.2 23.2 6.2 2.1 13.4 3.3 1.12 * i 1; i 3z i :g t!:r* 4: + 42 +0.19 + 6.0 f 2.9 + 1.3 f 0.7 i 3.8 t 1.4 i0.26 

Ea value is ex ressed as meaniS.D. 

a) %ntm~ I, bP : ~ontrol II ' : Sign/tieantly different fmm mntrol I at 5% level of probability " : S@~ficantl ditYennt fmm control I at 1% lev$ of probablllty # : ~gnitiindifferent fmm control11 at 5% level of probability 

Table 12 Blood biochemical findings of $male rata fed the dry beef-mntahed diet 

in a 28day do- rangehding test 

Conc.inNo.of LDH GOT GFT ALP yGTP QE CK T.P. Alb at-G ar-G or-G 8-G yG AIC 

diet animale QUn) aU/L) OUR3 OUn) QUlW QUIL) QUn) (gldu (%) (%) ()o (%) (%) (%) 

0 6 318 &1 26 416 0.49 251 221 6.33 61.4 16.6 6.9 2.1 12.6 3.1 1.36 * 106 i 5 i 6 i 55 i0.19 86* 13 i0.32 + 1.3 i 1.9 * 0.8 l 0.3 l 0.7 0.4 10.07 

5% 6 414 18 26 416 0.44 319 206 6.34 66.6 19.1 5.9 2.5 12.3 3.1 1.30 

115 + 11 i 2 i I0 i0.25 + 104 + 46 10.44 i 2.2 i 1.7 l 0.4 * 0.6 l 0.9 i 0.3 i0.12 

10% 6 661 El 24 496 0.64 349 219 6.19 68.0 11.8 4.1' 2.8 13.1 4.2'" 1.39 

134 * 11 i 2 + 153 +0.32 i 19 i 34 +0.26 i 2.5 f 2.3 * 1.3 l 1.0 * 0.9 + 0.6 iO.14 

0%" 6 609 81 26 413 0.44 253 196 6.40 69.1 16.6 4.0 2.1 12.3 3.3 1.49 i 200 + 6 i 2 i 42 i0.21 i 12i 32 i0.39 i 2.0 t 0.6 l 0.7 * 0.9 + 1.0 1.3 i0.12 

20K 6 496 18 26 462 0.2s 311X 228 6.37 56.8 18.8 6.W 2.3 12.8 8.8 1.32 * 229 + 4 i 2 i 66 iO.06 f 61+ 88 i0.42 i 2.9 i 1.3 * 1.0 0.7 l 0.8 l 1.6 i0.16 

Cow. in No. of T-Cho. T.G. PL Glu. BUN UA Cma. T-Bil. Ca P Na K C1 

diet animala (mgldL) hgldW bg/dW hgldW hgldu GngldL) (mgldW (mg/dL) bg/dW GngldW (mEqm hEqU (mEqU 

0%'' 6 64 20 99 111 16.6 0.89 0.64 0.25 9.7 6.1 143 4.11 106 l 12 t 6 l 16 i 12 i 2.1 + 0.22 i 0.09 i 0.03 i 0.4 i 0.9 1 0.32 * 2 

6% 6 61 20 93 122 141 1.20 0.63 0.21 9.8 6.8 144 4.14 101 l 9 + 12 * 18 i 11 i 3.6 i 0.29 + 0.10 + 0.03 i 0.2 i 1.0 f 1 i 0.31 * 1 

10% 6 62 13 96 101 15.1 1.13 0.68 0.21 9.6 6.6 146 4.28 110' i 12 t 6 * 16 I i 3.0 i 0.11 t 0.01 * 0.03 i 0.2 i 0.6 t 1 t 0.22 * 2 
Tlble 13-1 Absolute md relativa arm we&hta dda nta led dry dryFc~tsined diet In a 28-d.y dms mRflndhn study 

Canc. in No.of B.W. Brain Ln Khw Spbm Ha? Luna nUrmus . Phi 

dletDL) Ani a (A (a) (a) W (d) W (R) (mil (md 

Ench valw L sxsnuscd M mean f S.D. 

$:Contrd I; b):Conuol n 

c) : Rehrive orm wet& DW lOOn bodv wewt 

Tabb 13-2 AbsoluU and nklive arm weMta dnvb rats led drr babwtained dlst ln 8 28-d¶Y doW mne-&ldlnn mdy 

CMr ln No.of B.W. Adrenal TsrtiP RorL Semiv Epidi 

d*tOl) And 0 (mr) W W WW 

20 6 257 17.1 1.02 0.12 W 0.42 0.25 

+.29 f 2.0 kO.11 f 0.02 20.03 f 0.03 

Each vllw L axpeosed as mean f SD. 

I): Control I ; b) : Contral n 

C) : Rektive orm weinht oer lOOa body weipht 
- -- 

a& value is expresed as mean f S.D. I : Control I ; b) : Control U 

I : Relative arm webht per la body weight 

Simficantly different &om control U at 5% lwel of rrobnbdity 
Appendix 2 lndivldnal body weights of female rals fed the dry 

beef-contained dlet in a 28day dose r~ngrfinding tesl 

Conc. In Aninal Day 

dlel number 0 7 14 2t 28 

Mean 116 146 174 198 218 

Mean 115 149 171 201 223 

Mean 116 145 174 197 217 

Appendix 1 Individnal body weights of male rats fed the dry 

beef-contained diet in a 28day dose range-finding test 

(8) 

Conc. in Animal Day 

diet number 0 7 14 21 28 

Mean 121 176 236 291 335 

Mern 

Mean 121 172 226 280 308 

7 Mean 121 I80 240 286 319 

I-.- .I- __----.- 

Mean 121 164 208 246 ,271 

Appendix 4 Individual food consumplion of female rals fed Ihe dry 

beef-conlrlned die1 in a 28day dose range-finding lesl 

Appendlx 3 Individual food consunptioo of male rals fed Ibe dry 

beef-conlained die1 in a Uday dose range-findl?~ test 
Appendix 4 Individual food consumption of female rats fed the dry 

beef-contained diet in a 28day dose range-finding test 

(dda~/ral) 

Conc. in Cage week 

diet number 1 2 3 4 

OX &I 16 13 13 15 15 

17 13 14 I6 I6 

18 14 I6 14 13 

Mean , 13 14 15 15 

-- -.-.-.-.--..---...-...--- 

5% 19 12 10 16 14 

20 12 15 15 16 

2 1 16 I4 15 14 

Mean 13 13 15 15 

-- 

OX b' 25 14 14 15 16 

Z6 14 14 15 13 

27 13 15 15 16 

Mean 14 14 15 15 

-------- -- ---- -- -----..- 

20% 28 14 15 14 15 

29 12 14 13 13 

30 13 13 12 13 

Mean 13 14 13 14 

Appendix 3 Individual food consumption of male rats fed the dry 

beef-contained die1 in a 28day dose range-finding test 

(dday/rat) 

Conc. in Cage reek 

diet number I 2 3 4 

Mean 18 2 1 22 22 

Mean 18 19 2 1 21 

.-- -.-.- - --- --.. 

Mean 17 19 20 17 

Mean 16 18 17 15 
Appendix 6 - 1 Individual urinary findings of male rats fed the dry beef-contained diet 

in a 28day dose range.finding test 

Conc. in Animal Color Cloudy pH Protein Glucose Ketone Occult Urobilinogen Bilirubin 

diet number body blood 

0% &) 001 PY - 6.6 - 0.1 - - 

018 PY - 6.0 - - - + 0.1 - 

Color : PY(pa1e yellow) 

Cloudy : -(negligible). r' 

Protein : -(negligible), *(16-30mgldL). + (30mgldL) 

Glucose :.- (negligible) 

Ketone body : -(negligible), i(6mgldL) 

Occult blood : -(negligible), *(trace), +(slight) 

Urobilinogen : Ehrlich unit/dL 

Bilirubin : -(negligible) 

a) : Control I 

Appendix 6 - 2 Individual urinary findings of male rata fed the dry beef.contained diet 

in a 28day dose range3inding test 

Conc. in Animal Color Cloudy pH Protein Glucose Ketone Occult Urobilinogen Bilirubin 

diet number body blood 

0%" 019 PY - 6.0 - - - 0.1 - 

020 PY - 6.0 f - - - 0.1 - 

021 PY - 6.0 + - - - 0.1 - 

022 P Y - 6.0 - - - 0.1 - 

023 PY - 6.0 - - - - 0.1 - 

024 PY - t - - - 0.1 - ..................................................................... .!:! ...................................................................................................... 20% 026 PY - 6.6 - - - i 0.1 - 

026 PY - 6.0 i - - - 0.1 - 

027 PY - 6.0 - - - 0.1 - 

028 PY - 6.0 - - - - 0.1 - 

029 PY - 6.0 - - - - 0.1 - 

030 PY - 6.0 + - - - 0.1 - 

b) : Control 11 

Appendix 

Conc, in 

diet 

0% *' 

.............. 6% 

............ 10% 

Color : F 

Cloudy : 

Protein 

Glucose 

Ketone l 

Occult b 

Urobilin 

Bilirubi~ 

a) : Con! 

Appenc 

- 

Conc. 

- diet 

0% 

........... 20% 

iim% 
Appendix 6 - 1 Individual urinary findings of female rats fed the dry beef-contained diet 

in a 28day dose range-finding teat 

Conc. in Animal Color cloudy y~ probin Glucose Ketone Occult Urobilinogen Bilirubin 

diet number body blood 

OX .' 601 PY - 6.0 t - - - 0.1 - 

602 PY - 6.0 * - - - 0.1 - 

603 PY - 6.0 - - - - 0.1 - 

604 PY - 6,6 t - - - 0.1 - 

606 P Y - 6.0 - - - - 0.1 - 

606 PY - * - - - 0.1 - ........ 6:O ...................................................................................................... 6% 607 PY - 7.0 - - - - 0.1 - 

608 P Y - . 6.0 - - - - 0.1 - 

509 PY - 6.6 - - - - 0.1 - 

610 PY - 6.6 - - - + 0.1 - 

611 P Y - 6.0 t - - t 0.1 - 

612 PY - 6.0 - - - I 0.1 - 

P Y - 1.5 + - - - 0.1 - 

614 PY - 6.0 - - - - 0.1 - 

516 PY - 6.0 t - - - 0.1 - 

616 PY - 7.0 - - - - 0.1 - 

617 PY - 6.0 * - - - 0.1 - 

618 P Y - 7.0 - - - - 0.1 - 

Color : PY(pa1e yellow) 

Cloudy : -(negligible) 

Protein : -(negligible), *(16--3OmgldL). + (30mgldL) 

Glucose : -(negligible) 

Ketone body : -(negligible) 

Occult blood : - begligible), *(trace), +(slight) 

Urobilinogen : Ehrlich uniUdL 

Bilirubin : - (negligible) 

a) : Control I 

Appendix 6 .2 Individual urinary findings offemale rats fed the dry beeIcontained diet 

in a 28day dose range-finding test 

Conc, in Animal Color Cloudy pH Protein Glucoee Ketane Occult Urobilinogen Bilirubin 

diet number body blood 

OX "' 6 19 PY - 7.6 + - - - 0.1 - 620 PY - 6.0 - - - - 0.1 - 

621 PY - 6.0 * - - - 0.1 - 

522 PY - 6.0 - - - - 0.1 - 

523 PY - 6.6 t - - t 0.1 - 

624 P Y - 6.0 t - - - 0 1 ................................................................................................................................................................................. - 

20% 626 PY - 6.6 - - - - 0.1 - 

626 PY - 6.0 - - - - 0.1 - 

627 PY - 6.0 - - -. - 0.1 - 

628 PY - 7.6 * - - - 0.1 - 

629 PY - 6.0 - - - - 0.1 - 

630 PY - 6.0 * - - - 0.1 - 

b) : Control I1 
Appendix I - 1 Individual hematological fipdinga of male rat$ fed the dry beefwntained diet m a 28day dose mngefindmg test 

Conc. in Animal RBC Hb Ht MCV MCH MCHC Am WBC 

diet number (lO'/lrL) (gIdL) (%) W (pg) (%) (sed he) (10'lpL) 

0%" 001 711 13.1 39.3 66 19.3 34.9 13.2 21.7 69 

002 Ill 13.2 38.6 60 17.0 34.2 12.9 11.9 66 

008 749 15.0 42.3 66 20.0 36.6 12.9 20.2 68 

004 739 13.3 39.2 63 18.0 33.9 12.3 18.6 61 

006 121 14.3 42.4 59 19.8 33.1 13.0 17.4 66 

006 722 14.4 42.6 69 19.9 33.9 12.6 20.7 63 

."".dSi"'.." Mean 131 14.2 40.9 66 19.4 34.6 13.6 19.1 46 ............................. """ .. 019 194 13.1 39.1 49 16.6 33.6 12.8 16.6 49 

020 168 14.3 41.4 65 18.9 34.6 12.4 16.1 69 

021 696 14.6 42.6 61 21.0 34.4 1.6 18.6 61 

022 820 15.8 46.6 51 19.3 34.0 15.2 23.4 32 ' 023 107 14.1 43.2 61 20.8 34.0 13.3 22.1 " 31 

024 762 14 44.2 68 19.4 33.6 13.4 20.1 67. 

".= ........U21i......... Mean 

026 

021 

028 

029 

030 

Mean 822 14.3 41.4 61 11.3 34.4 13.6 18.2 38 

a) : Conk01 I, b) : Control 11 

Appendix 1.2 Individual hematological findinp of male rau fed the dry beekontained diet 

in the 28day repeat dose toxicity bat 

Pirentid leukocytn countl, (%) 

Cone. in Animal Neutro. Plat 

diet number Barn. Eosin. Stab. Set Lymph. Mono. Other (10'bW 

On.' 001 0 1 0 14 83 2 0 130 

002 1 0 0 10 87 2 0 138 

003 0 0 0 16 83 1 0 17.8 

004 0 2 0 22 14 2 0 146 

006 0 1 0 16 80 3 0 119 

006 0 0 0 8 92 0 0 130 

Mean 0 1 0 14 83 2 0 132 ..".K~.-""- WI ..........tl...........0'...........U...........w........78......... ............. '0' ........1g7..... 

008 0 0 0 10 81 3 0 126 

009 0 1 0 10 88 1 0 96 

010 0 0 0 12 87 1 0 131 

011 0 1 0 19 80 0 0 106 

012 0 1 0 13 86 0 0 118 

Mean 0 1 0 14 84 1 0 @ 119 ..'~w..""..ul 3....... ... ...........y...........u...........l~........ ........................ ........ 125 ..... 

014 0 0 0 8 92 0 0 122 

016 0 1 0 I 90 2 0 127 

016 0 0 0 12 81 1 0 131 

017 0 0 0 19 I1 4 0 239 

018 0 4 0 12 83 1 0 113 

Mean 0 1 0 143 : .....& " ............................................... !? ........... !.? ......... 66 ......... ? ................................... 019 0 0 0 12 88 0 0 126 

020 0 0 0 11 81 2 0 110 

021 0 1 0 22 16 1 0 91 

022 0 1 0 17 80 2 0 142 

023 0 0 1 18 81 0 0 142 

024 0 0 0 12 87 1 0 124 

Mean 

026 

027 

028 

029 

030 

Mean 0 1 0 12 86 1 0 134 

a) : Conhul I, b) : Control I1 

Appendi 

XEZi 

diet 

OW" 
Appendix 8 Individual hematological findings offemale tars fed the dry beepcontained diet UI a 28day dose rangahding test 

Conc. in Animal RBC Hb t M M MCHC WBC Plat AF''I"T 

diet number (10' W d~) ) (pCH (%) (1OzbW (10'bL) (md (see) 

OH" 601 7!5 y4.7 40.7 53 19% 36.1 29 111 12.9 17.1 

502 717 14.7 40.7 67 20.6 36.1 31 103 13.1 20.6 

603 780 16.0 42.4 64 19.2 35.4 42 100 12.6 16.6 

504 751 14.9 42.6 67 19.8 36.0 42 107 12.3 18.3 

505 768 14 4.1 66 20.3 36.6 36 94 12.7 17.7 

606 800 1 43.7 55 19.4 36.6 43 111 13.2 18.4 

.... *&&. Mean 767 14.8 42.2 66 19.4 35.2 46 120 12.9 17.7 ......... l3 ........ 27 ...... ;f ......a.o........62..... ..,. .......3E.l.......2 8. .......12K........12.7........ 7,.* ....-. 

614 821 16.6 44.0 64 19.0 35.6 23 123 12.4 17.3 

516 761 15.4 42.2 66 20.6 36.6 22 116 13.1 18.1 

516 772 15.0 43.3 66 19.4 34.6 33 131 13.4 18.8 

517 811 16.9 44.1 64 19.6 36.1 38 143 12.7 18.4 

618 739 14.9 42.2 67 20.2 36.3 28 118 13.4 18.0 

Mean .......... .".""'U"'.'."' 0% 619 

620 

621 

622 

623 

624 

Mean 766 14.2 40.8 53 18.6 34.9 33 124 12.9 18.3 

a) : Control I, b) : Control I1 

Appendix 9 . 1 Individual blood bioehemical findings ofmale rata fed the dry beef.mntained diet 

in a 28day dose rangs-finding tast 

Cone. in Aqimal LDH GOT GFT ALP y.GTP BE CK T.P. ALb a,-G aa-G as-G B-G y.G AIG 

diet number aum aun) aum arm) aum aum aun) @/do (n) H o() (H) (H) (n) 

OH" 001 337 88 34 843 0.99 59 229 6.58 49.7 21.1 5.6 3.4 16.7 4.6 0.99 

002 344 87 33 630 0.63 45 262 6.06 49.4 22.6 6.6 3.5 13.8 4.1 0.98 

003 267 74 29 472 0.38 46 242 6.06 61.8 21.2 6.4 2.6 14.7 4.3 1.07 

001 637 82 29 8M.0.68 66 239 6.61 48.9 26.3 4.1 3.1 14.6 4.1 0.96 

CC5 267 90 30 764 0.63 46 218 6.76 54.6 23.4 6.1 2.3 12.6 2.1 1.20 

006 188 69 26 480 0.23 42 167 6.98 49.6 29.1 6.4 2.6 14.3 4.0 0.98 

Mean 323 82 ........ 30 666 0.66 60 .". 'liJ('..."..60r' ...... m-m-m-.m-.m- .'lg 3r... '.'737." ..cl........ o.... 

008 227 84 30 871 0140 48 

009 129 77 27 630 0.32 64 010 242 66 26 786 0.46 46 011 183 65 28 866 0.43 42 

012 270 77 29 6n 0.66 66 

Mean 339 96 32 1086 0.65 41 228 6.66 51.5 21.0 5.6 3.7 13.9 4.3 1.07 

a) : Control I, b) : Control I1 
Appendix 9 .2 !ndividual blood biochemical finding8 of male rats fed the dry beef-contained diet ma 28day does range-fioding test 

Mean "".L;Y'." ....o ........ 

008 

W9 

010 

011 

012 

.09i;..' ...o Mean '3' ........ 41 60 86 ....... f3 136 B...... 10.6 ......1'D9..... 1.07 b:li 0.63 22222.2 0.26 b'b'ZN ......gB.......77. 9.9 8.3 ....... 144 ir6' 4.53 4' ..... 106 iw.. 

014 48 69 86 118 13.6 1:26 0.69 0.29 9:6 1.9 146 3.98 108 

016 40 28 76 122 12.6 1.42 0.49 0.21 9.4 1.8 143 4.64 106 

016 46 61 16 138 10.9 1.13 0.43 0.24 9.8 8.8 144 4.86 106 

017 43 29 66 114 10.0 1.46 0.63 0.31 9.6 8.1 146 5.86 105 

018 39 26 61 126 13.3 1.18 0.60 0.26 9.5 8.4 146 4.16 107 

Mean 39 31 15 129 12.8 1.31 0.62 0.28 9.4 1.3 144 4.26 106 

a) : Control I, b) : Contml I1 

Appendix 10 - 1 !ndividual blood biocbamical findings of female rata led the dry beef-sntained diet ma 28day does rangefinding test 

OK" 601 466 86 26 410 0.28 216 264 6.23 58.2 19.6 4.1 2.1 11.7 3.2 1.39 

602 319 19 22 340 0.46 187 148 6.96 58.4 15.9 6.5 2.5 13.1 3.6 1.40 

609 631 86 34 361 0.38 413 236 6.66 59.1 16.6 6.0 2.9 12.1 3.4 1.44 

604 241 B8 29 410 0.61 260 126 6.40 66.3 20.8 6.7 3.0 11.9 2.6 1.29 

606 334 16 22 498 0.48 276 318 6.73 56.3 18.2 6.8 2.4 13.1 3.2 1.29 

MI6 368 88 26 472 0.85 189 237 6.02 66.2 20.0 5.4 2.4 13.4 2.6 1.28 

..'..K~"... Mean 318 84 26 416 0.49 261 221 6.33 67.4 18.6 5.9 2.1 12.6 3.1 1.36 " .6w...... 67.. ......,I........ ...... ?'4 8;1..... 6' 6'J ....... 2K 2'...... g6 .....6'6' * ...... ,'d...... iB:6' ...... 6'6:2 ......... .K ..... yr 4...4...ggK ;3x.. 

508 598 98 25 608 0167 202 196 6.80 61.4 20.0 6.0 1.4 12:0 3.2 1.36 

509 341 13 25 366 0.30 440 170 6.74 58.5 18.6 6.1 2.8 10.6 3.9 1.41 

610 364 17 26 326 0.13 225 216 6.06 58.1 16.4 6.4 2.4 12.8 3.9 1.39 

611 573 12 22 391 0.33 392 192 6.10 62.1 21.2 6.9 3.1 13.1 4.0 1.11 

512 411 68 22 413 0.07 404 166 6.11 66.0 20.2 6.3 2.8 13.0 3.1 1.22 

---To U'....... Mean as ...... 474 ........ 78 25 ... ;;i ...... ;.: ..... g:; ..... g:; ....... ;; ........ g, ...... ....... ..-.. +;;. 

514 632 99 26 606 0.31 315 213 6.08 61:3 18.1 4.5 3.9 12.4 3.8 1.34 

616 645 73 27 385 0.46 193 239 6.08 69.0 19.4 2.9 2.2 12.9 3.6 1.44 

516 617 80 21 369 0.30 318 214 6.45 61.1 11.8 2.5 1.2 13.0 3.8 1.61 

611 321 99 26 164 0.69 311 199 6.10 64.4 18.8 6.7 3.1 13.3 4.1 1.19 

618 685 88 23 444 1.11 366 213 6.62 66.6 19.3 3.9 2.1 12.3 6.2 1.30 

Mean 609 81 26 2.1 12 3 3.3 1.49 ...zox.......azs. .....71K.---..., a ........'i3....... .... :j; ...... .$: ...... ii! ...... ..... ..... .$$ ....... $$ ......... .o ..... i2. ......s.r....m .. 

626 649 74 26 504 0.11 336 233 6.63 59.1 18.6 6.0 1.4 13.5 1.9 

621 114 81 28 466 0.34 382 110 5.70 58.2 19.9 6.0 2.1 11.3 2.5 

528 642 86 26 637 0.21 483 332 6.63 58.7 11.9 6.7 2.0 12.3 2.4 

629 244 76 28 434 0.24 294 146 6.12 61.8 20.2 5.9 3.1 13.3 5.l 

530 546 18 24 452 0.28 329 314 6.30 66.1 19.2 6.9 2.0 12.9 3.9 1.23 

Mean 496 78 26 462 0.23 371 228 6.31 66.8 18.8 6.8 2.3 12.8 3.6 

a) : Control I, b) : Conk01 I1 
Appendix 10.2 Individual blood biochemi-$ findings offemale rats fed the dry beef.contsined diet 

in a 28day dow rangwind~ng test 

"..'gJ(" . .....6 Mean M'.....M'M'. 54 11 ........ 20 ...Q.."..... 99 117 166 ......i-.4g' 0.89 0.64 ." ..E,7 * ......bbZ&.. 0.26 --. 9.7 6.7 143 4.11 ...>FA. 1w 

608 38 11 68 121 20.3 1114 0.79 0.22 9.6 6.6 146 3.58 107 

609 60 42 110 135 117 0.97 0.61 0.27 10.0 6.2 146 4.46 106 

610 62 28 110 115 12.4 0.90 0.66 0.27 10.1 8.2 144 3.90 106 

511 50 14 96 128 12.8 1.62 0.52 0.31 9.8 1.4 144 . 4.46 107 

612 48 13 94 129 13.0 1.10 0.68 0.27 9.9 7.1 144 4.02 108 

Mean 45 20 90 127 17.8 1.18 0.68 0.27 9.8 6.7 146 4.46 108 

a) : Canhol I, b) : Contml 11 

AppndU 11 AbuJuu orlan webhu of individd nu fed diy bwf-conlsind dht A 128-dry he ma-flndLU study 

Conr in Animal B.W. Bnh Uvu Kidney Sdmn Hsvt Lunl thymu Th. Pltui. Adnrul Tutia hL %i.v WW. 

dh~8)nnumb.n @ @ O O O O O 0 Gnd hd hd @ @ 0 @ 

001 312 1.90 8.66 2.43 0.61 1.16 1.41 0.66 26.7 10.1 48.8 2.62 0.40 1.51 0.73 

002 308 1.76 9.45 2.15 0.61 1.W 1.24 0.61 30.5 9.5 47.1 2.46 0.51 1.51 0.72 

003 315 1.89 9.00 228 0.59 1.kl 1.22 0.57 28.4 8.9 58.1 2.56 0.53 1.37 0.72 

OM 313 1.73 8.59 2.24 0.57 1.10 1.36 0.58 N.9 la6 52.6 2.60 0.35 1.30 0.74 

005 331 1.89 9.43 2.58 0.69 1.09 1.35 0.53 19.9 10.7 60.1 2.85 0.49 1.76 0.63 

OM 307 1.93 8.48 2.32 0.51 1.11 1.32 0.45 26.7 12.3 54.1 2.83 0.48 1.31 0.76 

Mw 314 1.85 8.94 2.33 0.60 1.11 1.32 0.57 25.5 10.4 53.5 2.S 0.46 1.46 0.72 

5 007 287 1.94 7.88 249 0.48 1.02 1.22 0.48 18.7 8.8 51.3 2.68 0.40 1.26 0.67 

WB 296 1.91 7.60 2.49 0.48 0.97 1.29 0.59 16.1 9.5 49.5 2.63 0.37 1.32 0.65 

M)s 362 2.03 9.72 2.59 0.73 1.29 1.24 0.61 18.1 12.3 53.1 2.45 0.47 1.40 0.71 

010 329 1.97 9.45 2.62 0.57 1.18 1.26 0.85 19.1 10.1 45.6 2.75 0.54 1.61 0.67 

011 341 1.85 10.76 2.73 0.62 1.05 1.31 0.86 22.9 10.8 63.6 2.83 0.37 1.30 0.67 

012 295 1.82 8.41 2.45 0.49 1.09 1.25 0.61 21.0 8.7 42.8 2.49 0.43 1.10 0.71 

""" --- - - 

Mean 257 1.90 6.32 2.07 0.54 0% 1.13 0.47 21.4 9.6 43.7 2.58 0.30 1.09 0.63 

a) : Control I ; b) : Control II 

- 10- - 
~p - ~p -- 

Cons. in Animal B.W. Bnln Liver Kidney Wean Hwt Lunl thmut Thyr. Pitul. Ahrd Test~ Prtrrl LmLv Epidid. 

did%) mmkn 0 % (Q (Q (Q (Q 6iu%) 6iu%) (mB (%) 6) (I) (s) 

001 312 0.61 2.78 0.78 0.20 0.37 0.45 0.21 8.6 3.2 15.6 0 0.13 0.48 0.23 

002 308 0.57 3.07 0.70 0.20 0.35 0.40 0.20 9.9 3.1 15.3 0.80 0.17 0.49 0.23 

003 315 0.60 2.86 0.72 0.19 0.25 039 0.18 9.0 2.8 18.4 0.81 0.17 0.43 Om 

O* 019 3W 0.64 2.63 

020 313 0.62 2.90 

021 348 0.58 3.15 

022 . 238 0.75 268 

023 285 0.70 2.71 

024 310 0.63 2.91 

MEM 294 0.65 2.83 

20 025 252 0.79 2.20 

026 237 0.78 3.03 

027 274 0.65 2.45 

028 308 0.65 2.32 

029 233 0.82 2.45 

030 236 0.79 2.37 

M4.n 257 0.75 2.47 

3 : Contrd 1 ; b) : Control U 

Amndix 13 , Absolute om nUhU dindMdrul hda nu hd dn baef-canalnod dlet in a 28-dw dow mn~hndiig study 

MOM 203 1.81 5.39 1.63 0.43 0.75 0.99 0.49 20.1 12.9 53.8 69.0 0.45 

3 : Convd I ; b) : Control u 

a) : Cmt 
Conrin AnLML B.W. Brain Liver Kdney Spleen Heart LW m~ f"sj ("2) 

died%)nnumberr 6) (I) (%I (%I (W (%I 6) 6) (%) 

Mean 204 0.88 2.98 0.86 0.21 0.36 0.48 0.24 10.1 6.4 27.4 33.1 0.23 

5 507 168 0.91 2.72 0.75 0.21 0.34 0.53 0.24 9.9 5.8 21.6 41.0 0.19 

508 721 0.82 283 0.68 0.21 0.40 0.48 0.16 7.9 6.6 27.9 43.6 0.22 

509 201 0.88 3.27 0.79 0.26 0.39 0.56 0.23 12.1 6.4 28.6 36.5 0.32 

510 197 0.90 2.71 0.84 0.21 0.36 0.48 0.27 10.5 5.7 24.0 28.2 0.14 

511 228 0.82 3.29 0.80 0.24 0.38 0.46 0.21 7.5 6.4 27.6 36.0 0.22 

512 213 0.88 2.93 0.77 0.17 0.35 0.46 0.20 11.8 7.3 21.4 27.9 0.29 

Mean 208 0.87 2.96 0.80 0.22 0.31 0.50 0.72 10.0 6.4 25.2 35.5 0.23 

10 513 181 0.98 2.55 0.81 0.25 0.38 0.51 0.22 10.8 6.5 27.7 33.8 0.19 

514 179 1.07 280 0.85 0.18 038 0.51 0.23 12.0 6.8 30.2 43.4 0.25 

515 201 0.94 2.80 0.82 0.24 0.37 0.51 0.23 10.8 7.4 28.8 50.0 0.28 

516 204 0.86 2.56 0.79 0.21 b.35 0.48 0.24 10.3 6.6 23.6 34.5 0.16 

517 204 0.67 2.81 0.80 0.21 0.33 0.50 0.20 8.8 6.0 32.2 37.9 0.25 

518 219 0.84 2.82 0.92 0.22 0.33 0.47 0.25 10.0 6.6 30.0 33.2 0.26 

Mean 198 0.93 2.72 0.83 0.22 0.36 0.50 0.23 10.5 6.7 28.8 38.8 0.23 

524 224 

Mean 215 

20 525 218 

526 209 

527 191 

528 201 

529 172 

530 226 

Mean 203 

a) : Contml I ; b) : Control U 
Animal Cloning: A Risk Assessment DRAFT 

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Animal Cloning: A Risk Assessment DRAFT

Appendix H: 

The Comprehensive Veterinary Examination 
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Animal Cloning: A Risk Assessment DRAFT

Appendix H: The Comprehensive 

Veterinary Examination 

CVM recognizes that readers of this Risk Assessment will have diverse training and 

expertise, and not all will be familiar with the practice of veterinary medicine. The 

purpose of this Appendix is to (1) acquaint readers with a process used by veterinarians 

(and specifically veterinarians working with livestock) as part of the evaluation of the 

health of an animal, and (2) how we interpreted veterinary examination reports on animal 

clones as part of the evaluation of the overall risk assessment process. It is not intended to 

be a guidance for veterinarians working with animal clones or any other livestock. 

A. Introduction to the Comprehensive Veterinary Examination 

The standard veterinary procedure that forms the basis of the CVE is referred to as the 

“Problem Oriented Medical Approach (POM).” Traditionally, this approach involves a 

Chief Complaint followed by a Physical Exam, which then generates a Problem List. The 

veterinarian then develops a list of Differential Diagnoses (probable causes), from which 

a Diagnostic Plan is formulated, including the conduct and interpretation of laboratory 

work. This leads to a revised Problem List based on laboratory data that implicate 

particular diseases or conditions. The new information results in a revised Differential 

Diagnosis and revised Diagnostic Plan (if additional work is still needed). All of the data 

finally results in a Therapeutic Plan. The owner of the animal is then informed by a 

Client Education Plan (Weed 1970). 

Comprehensive veterinary examinations (CVEs) are systematic approaches for 

examining domestic livestock animals, and making informed judgments as to their health. 

The CVE contains both objective and subjective information, and requires experienced 

veterinarians familiar with the animal breeds and species to be evaluated. For each breed 

and species, the veterinarian is able to judge whether any observation is “normal” within 

the range of biologic variability. The POM is the standard medical examination and 

evaluation technique. This approach compares the non-healthy patient to the clinically 

normal animal. Each abnormal observation is recorded in a “problem list.” The 

veterinarian ultimately refines and compiles the problem list to develop a list of 

differential diagnoses that would account for the problems found on the exam. The notion 

of “normal” is difficult to define and articulate, and, of necessity, requires subjective 

assessment and expert judgment. In contrast, measurements such as temperature, pulse, 

respiratory rate, weight, height, etc., are objective and, in principle, easier to define as 

“within normal range.” Experienced examiners will agree that published “normal ranges” 
Appendix H: The Comprehensive Veterinary Examination H-4 

Animal Cloning: A Risk Assessment DRAFT

of these values can be slightly exceeded without judging the animal to be unhealthy. The 

CVE, by an experienced practitioner, employs both objective and subjective information 

to maximize the information gathered in examinations. 

B. The Importance of Species-Specific Standards 

As the purpose of this Appendix is to provide an overview of the CVE, it will not 

explicitly address every difference among the four species under consideration in this 

Risk Assessment. This Appendix will discuss the general approaches that are employed 

in performing such examinations. Species differ in, among other things, size, shape, 

demeanor, physiology, nutritional needs, and husbandry. Because of this, cows are 

examined differently from swine, goats, or sheep. For example, an experienced 

veterinarian or animal handler would not approach a dairy cow by moving toward her 

head first. Dairy cows are accustomed to being approached from the rear because that is 

how they are approached when they are milked. Other species-specific differences may 

be physiologically determined (e.g., birth weight ranges, reproductive behavior, gestation 

length) or as the result of species- or breed-specific husbandry (e.g., age of weaning, 

handling, and feeding practices). Thus, comparing the birth weight of Holstein and 

Longhorn calves would be inappropriate, as is comparing piglet weaning weights in 

facilities that wean at different ages. 

C. How a Veterinarian Performs a Comprehensive Veterinary Examination 

The physical examination portion of a CVE for any age of animal usually begins with an 

observation from a distance, so as not to influence the animal’s response. An animal 

approached by a human who is not normally involved in the animal’s care can affect its 

demeanor and respiratory rate. First, the consulting veterinarian observes the animal’s 

behavior and general appearance from a distance far enough not to provoke anxiety. The 

veterinarian notes the animal’s posture, resting respiratory rate, vocalizations, eating/cud 

chewing, excretory functions (defecation/feces and urination), gait, body condition, 

conformation, and general skin and coat appearance. 

The subsequent close examination occurs in a systematic fashion. A CVE is generally 

carried out in anatomical order, usually from head to tail (except for dairy cows). If any 

abnormalities are discovered during the course of the examination, they are listed by 

functional systems, which include 

• Special senses (eyes, ears, nose, mouth) 

• Skin and Coat 
Appendix H: The Comprehensive Veterinary Examination H-5 

Animal Cloning: A Risk Assessment DRAFT

• Musculo/Skeletal (including a body condition score) 

• Respiratory 

• Cardio/Vascular 

• Neurological 

• Gastro/Intestinal 

• Genito/Urinary 

• Peripheral Lymph Nodes. 

Often, the veterinarian begins with inserting a thermometer in the rectum to measure 

body temperature, and, if appropriate for the species, takes a peripheral pulse in the tail 

(sheep with docked tails are the exception). The veterinarian then begins the examination 

at the head. The eyes, ears, mouth, and nose are examined. Hydration, anemia status, and 

other physiological measures can be assessed by examining mucous membrane color, 

feeling the gums for moistness, and pinching the skin to look for tenting, a sign of 

dehydration. The veterinarian then looks at the eyes to determine the degree to which 

they may be sunken from possible dehydration, whether there is any yellow coloration 

(jaundice from liver dysfunction), abnormal odors or discharges, whether the pupils are 

of similar size and symmetry (neurological function), and may perform a retinal fundic 

exam. While working on the head, the veterinarian can look at, smell, and touch the 

mouth, teeth, tongue, gums, lips, ear position, eyelid position, nostril flaring, feel for 

muscle atrophy, even breath from the nostrils, tongue tone, and look for drooling 

(ptyalism). The veterinarian then proceeds to the neck, where s/he looks, listens 

(auscults) and feels (palpates) for lumps, enlarged lymph nodes, salivary gland 

abnormalities, larynx, changes in the size or symmetry of the thyroid gland or trachea, the 

strength and regularity of the jugular pulse, and abnormal tracheal and pharyngeal 

sounds. 

The next area examined is the thorax. The veterinarian observes respiratory excursions 

more carefully than from the initial distance overview, and notes abnormalities such as 

abdominal breathing or forced expirations. The veterinarian auscults (listens with a 

stethoscope) the heart and lungs and listens for signs of thoracic disease. Some 

veterinarians choose to look at the front legs and feet at this time and note any skin, 

musculoskeletal, hoof, or other lesions. Another approach is to look at all four legs and 

gait separately as its own system. 

The abdomen of the animal is examined by auscultation and palpation. In ruminants, for 

example, the veterinarian watches the left side of the animal’s abdomen for rumen 

movements; these are related to cud chewing. S/he then listens to the left side of the 

abdomen to hear the rumen moving, and palpates the prefemoral and prescapular lymph 
Appendix H: The Comprehensive Veterinary Examination H-6 

Animal Cloning: A Risk Assessment DRAFT

nodes. Peritonitis (in cows) is investigated by pinching the withers to see if the cow 

buckles down or lordoses (arches downward) her spine. If lordosis is not observed, the 

animal may have peritonitis caused by hardware disease (traumatic reticulo-pericarditis, 

usually caused by accidental ingestion of nails or wire). In addition, while on the left side, 

the veterinarian pushes in on the rumen to determine fill and consistency, uses a 

stethoscope to listen for “normal” borborygmi (intestinal sounds), and thumps on the 

cow’s side to determine whether a displaced abomasum is present by listening for the 

characteristic pinging sound of the displacement. While examining the abdomen, s/he 

also has access to the umbilicus, the udder (females), prepuce (males), tail, perineum, 

escutcheon, and back legs. The right side of the abdomen is examined similar to the left. 

Pings on the right have different meanings than those on the left in all species. Lymph 

nodes are palpated on the right as well. The abdomen may be balloted (punched to feel 

for a wave of fluid). In smaller ruminants and pigs, the veterinarian palpates the abdomen 

by placing her/his hands behind the rib cage and pushing in and up (toward the spine) to 

feel for any abnormal viscera and for a gravid uterus. 

In mature female animals, the veterinarian checks the mammary glands for symmetry, 

temperature fluctuations, swelling, or pain. The veterinarian then examines the teats at 

their distal end for streak canal abnormalities. Next s/he examines the milk for 

abnormalities such as watery or clumpy consistency, clotting, an off-color or odor, or the 

presence of blood. The mammary lymph nodes are examined. 

External genitalia are also examined as part of the CVE (additional examinations are 

required for Breeding Soundness Examinations), including the vulva, prepuce, scrotum 

and testicles. Internal female and male genitalia are examined during rectal palpation. 

If the limbs had not been examined during the thoracic and abdominal exam, they would 

be examined next. Skin, hoof, conformation, swollen or hot joints, sore ligaments, and 

lameness (gait abnormalities) are evaluated. 

As ancillary tests, the veterinarian may elect to perform rectal palpation for 

gastrointestinal, genitourinary, cardiovascular, renal, lymph nodes, and musculoskeletal 

abnormalities. 

1. Specific Considerations for Neonatal CVEs 

Practitioners generally recommend examining calves, piglets, kids, and lambs within a 

few hours of birth for general demeanor including alertness, ability to stand, ability to 

suckle, respiratory excursions, mobility, etc. The neonate is then examined for cleft 
Appendix H: The Comprehensive Veterinary Examination H-7 

Animal Cloning: A Risk Assessment DRAFT

palate, naval abnormalities, and joint and limb abnormalities, and their chests are 

ausculted for cardiac abnormalities. Body temperature is also measured. Non-patent anus, 

or atresia ani may be discovered. Pigs, specifically, are examined for umbilical or 

inguinal hernias. 

The newborn animals are weighed, and those measurements entered into a birth weight 

database used to calculate Expected Progeny Differences (EPD), especially in beef cattle. 

(Cattle producers use these real data to improve their herds.) Birth weights also determine 

“calving ease” for the dams. Passive transfer of immunity from colostrum is measured by 

taking blood samples and measuring IgG and total protein at approximately 24 hours post 

partum after the calf has had an opportunity to consume colostrum. 

2. Specific Considerations for Juvenile CVEs 

In addition to the general physical exam information on weaning weight, age at weaning, 

and weight gain is often collected. The producer often keeps health records, which may 

include sick days, diagnoses, therapeutic records, laboratory data, etc. In particular, 

animals may be monitored for infections at the navel, in the joints, lungs (pneumonia), 

and gastrointestinal system (diarrhea). 

3. Specific Considerations for Mature Animal CVEs 

Most cattle, swine, sheep, and goats do not live out their natural lifespans in commercial 

production facilities. Thus, for utility in assessing health of the animal prior to use, 

producers may perform formal CVEs as close to the animal’s final use (e.g., slaughter) as 

possible, although health records may be maintained by producers for as long as the 

animal is alive. Health records include illnesses, therapies, growth, weight gain, and 

might also include feed efficiency (for meat animals), milk production, reproductive 

records, and dry matter intake in dairy cows, among others. 

4. Specific Consideration for Reproductive Stage Examinations 

a. Males 

Breeding Soundness Examinations (BSEs) have been developed and standardized by 

many professional societies and graduate veterinary schools. Among others, the Society 

of Theriogenology has a standardized protocol for bulls (www.therio.org). Its utility in 

comparison with others has been reviewed by Higdon et al. (2000). Another BSE 

protocol for beef bulls has been published by Gosey from the University of Nebraska 

(www.ianr.unl.edu/pubs/beef/g666.htm). Shipley (1999) and Levis at the Ohio Pork 
Appendix H: The Comprehensive Veterinary Examination H-8 

Animal Cloning: A Risk Assessment DRAFT

Industry Center of the Ohio State University Extension 

(http://porkinfo.osu.edu/levis.html#Top) have published guides for evaluating 

reproductive performance in boars (intact male pigs). Breeding soundness exams have 

been developed for the small ruminants (e.g., rams and bucks) and can be found at web 

sites of the Utah State University Extension and the University of Minnesota, among 

others. 

Briefly, a BSE includes evaluation of reproductive behavior, anatomical status, and 

evaluation of semen quality. Behavioral observations include libido, recognition of 

receptive females, and appropriate mounting behavior. Anatomic evaluations include 

body condition, with particular attention to the eyes, feet, and legs. Physical examinations 

are generally performed externally and rectally. The testes, scrotum, and epididymis are 

palpated, and scrotal circumference measured. The penis and prepuce are examined for 

appropriate anatomic development (e.g., penile deviations) as well as inflammation, 

abscesses, or adhesions. The prostate and seminal vesicles are evaluated by rectal 

palpation. Semen quality is evaluated by looking at volume, color, motility, and 

morphology. Evaluations are made in the context of age, breed, and species-specifics. 

Once breeding has occurred, measures of fertility generally are recorded. 

b. Females 

Evaluations of female reproductive function have not been formalized in the same 

manner as those for males, largely due to the role of the stud in modern agricultural 

practice. Nonetheless, there are standard reproductive function examinations that carry 

the same importance in female animals as do the male examinations. The female-specific 

examinations include behavioral and anatomic components. Evaluation of germ cell 

production is generally not measured except by fertility and fecundity. Behavioral 

observations include age at onset of estrous behavior (puberty), behavior at parturition, 

offspring acceptance, and mothering behavior. Anatomical evaluations include body 

condition, examination of external genitalia, and evaluation of the internal reproductive 

tract by rectal palpation, vaginal speculum, and ultrasound (if clinically indicated). 

Fertility is monitored by recording the number of coital or insemination attempts needed 

for conception, (although, in the event of conception failure, the male contribution is also 

considered). The producer often keeps records of abortion incidence, birthing interval, 

retained placenta (especially for cows), mastitis (inflamed mammary gland or udder), and 

metritis (infected uterus). The colostrum management program is often monitored and 

evaluated using a colostrometer for colostrum quality and blood levels of IgG in the calf 

for transfer of passive immunity. 
Appendix H: The Comprehensive Veterinary Examination H-9 

Animal Cloning: A Risk Assessment DRAFT

D. Interpreting the Comprehensive Veterinary Examination for Animal Clones 

The CVE was devised for sick animals, and works well when the patient has a chief 

complaint requiring diagnosis and therapy. Laboratory work is designed to gather 

evidence to support the clinical diagnosis. For animal clones, however, the CVE is 

attempting to prove the negative (i.e., this animal is not materially different from a 

sexually reproduced animal), and therefore attempting to “prove” that the animal clone is 

healthy. Examination and diagnostic testing (such as blood work) as a screen for healthy 

animals (or animal clones) to determine if the animals have a general, non-specific occult 

abnormality is not straightforward. In the absence of the “complaint, diagnosis, and 

treatment” paradigm, if one value is out of range in an otherwise apparently healthy 

animal with an otherwise “normal” clinical chemistry screen, it is difficult to determine 

what level of concern to place on the anomalous value. 

Reference ranges are based on a population of “normal” animals for a particular 

laboratory and species. In general, the reference ranges result from the collection of all of 

the measurements that have been taken by that laboratory, and calculation of the mean. 

The reference range is then set at the mean plus or minus two standard deviations, or a 

p<0.05 or 95 percent confidence interval. In other words, of any 20 test runs, one will 

have a value that is out of the range based on the statistical cut-offs as previously defined. 

There may also be biological explanations for “out of range” values. For example, 

alkaline phosphatase is an enzyme found in every tissue, and is released when the cells of 

that particular organ or tissue are damaged. Elevated levels can be associated with 

diseases of the bones such as rickets, osteomalacia, hyperparathyroidism, and bone 

tumors, or as part of such normal processes as healing fractures, pregnancy, or growth. 

To place this discussion in the context of the whole animal, we refer to the adage that 

“one treats the patient, not the blood work.” CVM’s determination that there is a 

biologically relevant difference between an animal clone and a sexually reproduced 

animal was based on a combination of statistical validity, weight of evidence, biological 

plausibility, and clinical impression; that is, a reliance on the body of evidence and expert 

veterinary judgment. 

E. Parameters Evaluated in the Comprehensive Veterinary Exam for a Risk 

Assessment 

The following series of tables presents the parameters used in evaluating the health status 

of animal clones when incorporated into a CVE. 
Appendix H: The Comprehensive Veterinary Examination H-10 

Animal Cloning: A Risk Assessment DRAFT

Table H-1: Clinical Chemistry Parameters Associated with Organs and Organ Systems 

(adapted from Hayes Principles and Methods of Toxicology 1994)

Heart

Creatine kinase (creatine phosphokinase) and isoenzymes 

Lactate dehydrogenase and isoenzymes 

Liver

Alanine aminotransferase 

Albumin 

Alkaline phosphatase 

Ammonia and Blood Urea Nitrogen 

Aspartate aminotransferase 

Bile acids 

Bilirubin 

Gamma glutamyl transferase 

Lactate dehydrogenase and isoenzymes 

Sorbitol dehydrogenase 

Total protein 

Kidney

Albumin 

Blood Urea Nitrogen 

Chloride 

Creatine (urine and serum) 

Glucose (urine and serum) 

Potassium 

Protein 

Sodium 

Pancreas

Amylase 

Calcium 

Glucose 

Lipase 

Bone

Alkaline phosphatase and isoenzymes 

Calcium 

Phosphorus 

Uric Acid 
Appendix H: The Comprehensive Veterinary Examination H-11 

Animal Cloning: A Risk Assessment DRAFT

Table H-2: Animal Health Measures for Evaluating Livestock, Including Animal Clones - General Health 

Status

Measurement Comments

Condition at Birth and Perinatal (+48 hours) Period

Caesarian/Vaginal Delivery Birthing ease 

Examination of Placenta Cotyledon numbers 

Immediate Post-parturition assistance Fetal-neonatal transition 

Birth weight LOS 

Comprehensive Neonatal Examination 

Generalized

- Demeanor 

- Posture/Gait 

- Respiratory rate 

- Vocalization 

- Nursing behavior 

- Urination/Defecation 

- Body condition 

- Body conformation 

- Skin/coat 

System Specific

- Special Senses (ear, eyes, nose, throat) 

- Integumentary 

- Musculoskeletal 

- Cardiovascular 

- Respiratory 

- Oropharyngeal 

- Gastrointestinal 

- Genitourinary 

- Neurological 

- Peripheral lymph nodes 

Take notice if LOS is present, 

especially in cattle and sheep. 

Umbilicus 

Contracted tendons 

Bloat, diarrhea 

IgG at 24-48 hours after birth 
Appendix H: The Comprehensive Veterinary Examination H-12 

Animal Cloning: A Risk Assessment DRAFT

Table H-3: Animal Health Measures for Evaluating Livestock, Including Animal Clones - Condition During 

Juvenile Post-Weaning Period (All Species)

Measurement Comments

Comprehensive Juvenile Animal Examination

Weaning weight 

Generalized

- Demeanor 

- Posture/Gait 

- Respiratory rate 

- Vocalization 

- Appetite/Feed consumption 

- Urination/Defecation 

- Body condition 

- Body conformation 

- Skin/coat 

- Body weight/gain 

System Specific

- Special Senses (ear, eyes, nose, throat) 

- Integumentary 

- Musculoskeletal 

- Cardiovascular 

- Respiratory 

- Oropharyngeal 

- Gastrointestinal 

- Genitourinary 

- Neurological 

- Peripheral lymph nodes 

Normal growth rate 

Contracted tendons 

Health/therapeutic records, with special attention to 

- Diarrhea 

- Pneumonia 

Diabetes previously noted in animal clones 

Nervousness previously noted in animal clones 
Appendix H: The Comprehensive Veterinary Examination H-13 

Animal Cloning: A Risk Assessment DRAFT

Table H-4: Animal Health Measures for Evaluating Livestock, Including Animal Clones - Reproductive (for 

males and non-dairy females, during first breeding cycle; early in lactation for dairy animals) 

Measurement Comments

Comprehensive Breeding Soundness Examination (Males)

-Comprehensive physical examination with special attention 

to 

-eyes, 

-feet/legs 

-prepuce 

-penis 

-palpation of testes and epididymides 

-Scrotal circumference 

-Rectal Examination, including 

-hernia, 

-spinal lesions, 

-internal abscesses, 

-accessory sex glands 

-Semen analysis (motility and morphology) 

-Libido 

per established guidance on a speciesspecific basis 

For seasonal breeders, exam should 

be performed during breeding season. 

Comprehensive Reproductive Exam (Females)

- Age of puberty onset (behavioral and/or physiological 

measures) 

-Comprehensive physical examination 

- Physical exam of genitalia 

-External 

- Internal 

-Rectal palpation, vaginal speculum, 

-Ultrasound if indicated by clinical findings 

-Record review for 

 -calving assistance 

- abortion incidence 

- calving interval 

- retained placenta 

- metritis 

- mastitis 

- maternal behavioral traits 

- colostrum quality using colostrometer 

Concomitant IgG measure in calf for 

measure of colostrum quality in beef 

calves, kids, and lambs. First parity 

dairy animals may not produce 

adequate quality colostrum. 

Comparisons should thus be made for 

appropriate parity level. 
Appendix H: The Comprehensive Veterinary Examination H-14 

Animal Cloning: A Risk Assessment DRAFT

Table H-5: Animal Health Measures for Evaluating Livestock, Including Animal Clones - Maturity, Aging, 

Lifespan (immediately before animal use (e.g., slaughter)) 

Measurement Comments

Comprehensive Mature Animal Examination

Generalized

- Demeanor 

- Posture/Gait 

- Respiratory rate 

- Vocalization 

- Appetite/Feed consumption 

- Urination/Defecation 

- Body condition 

- Body conformation 

- Skin/coat 

System Specific

- Special Senses (ear, eyes, nose, throat) 

- Integumentary 

- Musculoskeletal 

- Cardiovascular 

- Respiratory 

- Oropharyngeal 

- Gastrointestinal 

- Genitourinary 

- Neurological 

- Peripheral lymph nodes 

Health records kept on all breeding 

animals, and on all primary animal 

clones. They include all veterinary 

diagnoses, therapies, and vital 

statistics such as birth weight, 

weaning weight, physical exam 

findings, etc. 

Growth Performance/ 

Weight gain 

Signs and Symptoms Observed in Animal Clones 

-Arthritis 

-Diabetes 

-Nervousness 

-Seizures 

-Neoplasms 

-Other unspecified signs of early aging 
Appendix H: The Comprehensive Veterinary Examination H-15 

Animal Cloning: A Risk Assessment DRAFT

Table H-6: Animal Health Measures for Evaluating Livestock, Including Animal Clones - Clinical 

Measurements

Measurement Comments

Biochemistry 

Albumin 

Alkaline Phosphatase 

Amylase 

ALT (SGPT) 

AST (SGOT) 

Bile acids 

BUN 

CIAP (calf intestinal alkaline phosphatase) for bovine species 

Creatine phosphokinase 

Gamma glutamyl transferase 

Lipase 

Sorbitol Dehydrogenase 

Cholesterol 

Creatinine 

Glucose 

Serum Protein 

Calcium 

Chloride 

Potassium 

Sodium 

Hemo/Leukograms (CBC) 

 Red Blood Cell count 

WBC count including Differential 

Platelet count 

Urinalysis

Specific gravity 

Glucose 

Ketones 

Bilirubin 

pH 

Cells 

Protein 

Bacteria 

Blood (including leukocytes) 

Nitrate 
Appendix H: The Comprehensive Veterinary Examination H-16 

Animal Cloning: A Risk Assessment DRAFT

The following table describes the nature of the laboratory tests that have been performed 

during a CVE, and what they measure. Some common abbreviations, expanded 

explanations of the functional descriptions, and how the tests are used in differential 

diagnosis were added during the review of the submitted data as part of the risk 

assessment. 

Table H-7: Standard Large Animal Panel (Blood Biochemistry) Often Performed During a CVE

Test Origin Functional Description

Sodium (Na+) Diet

Principle cation of extracellular fluid (ECF or plasma). Maintains 

osmotic pressure of ECF. Cannot evaluate the electrolytes (Na+

, 

K+, Cl-, HCO3) by themselves. They are interdependent. Their 

regulation depends on hydration status, disease, aldosterone, 

renin-angiotensin, acid/base status, etc. Low Na+ may indicate - 

diarrhea, vomiting, congestive heart failure, renal disease, 

ruptured bladder. Elevations may indicate - dehydration, 

vomiting and diarrhea, inadequate intake, renal failure, increased 

salt intake, artifact from improper sample handling.

Potassium (K+) Diet

Principle cation of intracellular fluid (ICF). Maintains osmotic 

pressure within the cell (osmotic balance). Elevations effect 

cardiac function. Plasma levels may be altered by diarrhea, renal 

failure, metabolic or respiratory acidosis (causes elevated K+

, 

a.k.a. hyperkalemia), anorexia, hypoadrenocorticism (Addison’s 

disease), ruptured bladder, artifact from hemolysis of sample.

Chloride (Cl-) Diet

Major plasma (ECF) anion. Used to calculate the anion gap. Also 

affected by acid/base status. Reductions seen with chronic 

vomiting. 

Bicarbonate

(HCO3

-

) Metabolism

Indicative of the CO2 concentration in blood, buffers blood from 

radical pH changes. Used to diagnose metabolic or respiratory 

acidosis or alkalosis. 

Anion Gap Calculated

Calculated value to represent the unmeasured anions in the 

blood. High anion gap may indicate ketoacidosis, lactic acidosis, 

renal failure (uremic acidosis) and ethylene glycol toxicosis 

(organic acidosis). Low anion gap is RARE. It may indicate low 

plasma albumin, or high calcium

Blood Urea Nitrogen 

(BUN) Liver

Urea is produced by liver and excreted by kidneys. High values 

may be pre-renal (dehydration), renal (disease of kidney), or post 

renal (disease of ureter, bladder or urethra). Low values may 

indicate chronic liver failure (lack of production) or dietary 

protein deficiency.

Creatinine Muscle

Product of creatine metabolism by muscle tissue and excreted by 

kidneys. Used as an important measurement of kidney function. 

Resting level directly related to muscle mass. Higher in noncastrated males than females.

Calcium (Ca++) Diet, bone

Regulated by parathyroid hormone and calcitonin (from the 

thyroid). Vitamin D synthesized in liver important in dietary 

absorption. Acid base status and albumin level can affect blood 

levels. Co-factor for many enzymes. Key role in bone 
Appendix H: The Comprehensive Veterinary Examination H-17 

Animal Cloning: A Risk Assessment DRAFT

development, blood coagulation, cell growth, neuro-muscular 

transmission. Intestinal absorption may be affected by diarrhea 

or vitamin D deficiency.

Phosphorus (P) Diet, bone 

Similar to Ca++ Indicative of parathyroid and thyroid gland 

function, renal function. Important as a buffer for the blood. 

Blood level primarily regulated by the kidneys. Abnormalities 

related to dietary deficiency, renal excretion and hormonal 

imbalances that would also affect Ca++. 

Magnesium (Mg++) Diet

Required for normal muscle and nervous tissue function. Cofactor for many enzymes, especially kinases and phosphatases. 

Influences the regulation of serum calcium. Mostly clinical 

relevance in a deficiency known as “Grass Tetany” usually from 

acutely feeding the cattle lush rye pasture which is notorious for 

Mg++ deficiency.

Total Protein (TP) 

Liver, 

Immune 

System 

Consists mostly of proteins produced by liver (albumin, carrier 

proteins) and immunoglobulins. Used as indicator of total plasma 

volume. 

Albumin (alb) Liver 

Regulates plasma osmolarity. Binds certain molecules (some 

drugs, Ca++). Deficiency can be increased loss or decreased 

production. Loss may come from malfunctioning kidneys, 

intestine, or leakage into a body cavity. Decreased production 

may come from chronic liver failure. 

Globulin (glob) 

alpha-, betaliver; 

gamma-B 

lymphocytes 

Sum of globulins produced by the liver (transport proteins, like 

haptoglobin) and immunoglobulins (passive acquired from 

colostrum or endogenous production from B lymphocytes that 

have matured into plasma cells). 

Albumin/Globulin 

Ratio (A/G) Calculated 

Used to determine if there is an overproduction of gamma 

globulin which may occur in autoimmune disease. Low values 

may be due to insufficient albumin (see albumin for discussion) 

Glucose 

Digestion, 

liver 

glycogen, 

synthesis 

Indicative of the energy state of the animal. Can be reduced 

(hypoglycemia) in anorexia or due to artifact (sample handling), 

increased in diabetes mellitus (rare in cattle) or stress (common 

in cattle) 

Alanine 

Aminotransferase 

(AST) 

Liver, 

muscle 

Present in liver and released if liver is damaged. May also 

indicate skeletal and cardiac muscle damage. 

Sorbitol 

Dehydrogenase (SDH) Liver Present in cytosol of liver cells. High serum levels may indicate liver damage. 

Alkaline Phosphatase 

(Alk Phos) 

Organ 

Membranes 

Present in hepatocytes, biliary epithelium, osteoblasts, placenta, 

intestine, and kidney. May be high in young animals due to bone 

growth. Also an indicator of cholestasis (impaired bile flow). 

Gamma 

glutamyltransferase 

(GGT) 

Organ 

Membranes 

Present in hepatocytes, biliary epithelium, and kidney. Used to 

detect cholestasis. Has no involvement with the skeletal system 

to differentiate it from alkaline phosphatase. It is a better 

indicator of biliary stasis in large animals. 

Total Bilirubin (T bili) Hemoglobin 

Degradation 

Increase in bilirubin can result in icterus (jaundice). Used as a 

measure of liver maturity and function. 

Indirect Bilirubin 

Hemoglobin 

Degradation 

(NonConjugated) 

Bilirubin not conjugated to various carbohydrates for transport 

into bile in to the liver (elevations are from pre-hepatic 

sources). Value may be elevated by hemolysis or internal 

hemorrhage. Used to assess hepatobiliary function. 
Appendix H: The Comprehensive Veterinary Examination H-18 

Animal Cloning: A Risk Assessment DRAFT

Direct Bilirubin 

Hemoglobin 

Degradation 

(Conjugated) 

Bilirubin conjugated to various carbohydrates for transport to 

allow for inclusion into micelles in bile for transport from the 

liver (elevated in post-hepatic obstruction). Elevated with biliary 

outflow obstruction. Used to assess hepatobiliary function. 

Amylase Pancreas 

Used by GI system to aid in digestion of starch and sugars. 

Elevations indicate pancreatic inflammation. Not important in 

ruminant species. 

Cholesterol

Liver 

Synthesis, 

diet 

Precursor for synthesis of steroid hormones, bile acids, and 

vitamin D. Constituent of cell membranes and bile micelles. 

Variations may be secondary to endocrine, hepatic, or renal 

disease. 

Creatinine Phosphokinase 

(CK or CPK) 

Muscle 

(skeletal and 

cardiac), 

Brain 

Intracellular enzyme in skeletal and cardiac muscle. Used to 

detect damage to muscle. 

Iron (Fe++) Diet Ferric Iron associated with transferrin. Iron deficiency is often 

suspected as the cause of anemia. 

TIBC (Total Iron Binding 

Capacity) 

Transferrin 

production 

by Liver 

Used as measurement of the total amount of transferrin. 

Random Bile Acids 

(hBA) Liver 

Produced by liver and secreted into bile. Elevations indicate 

reduced liver function and not necessarily inflammation or 

biliary stasis. 

Lipemia - Index Lipids in 

plasma 

Measure of the level of lipids in the circulation. Can be caused 

by diets high in fats and influenced by postprandial (after eating) 

sampling. 

Hemolysis - Index Lysis of red 

blood cells 

Caused by lysis of red blood cells which results in release of 

hemoglobin into the plasma. Also used as an indicator of sample 

quality. 

Icterus - Index Bilirubin in 

plasma 

Index measured by the color of plasma to indicate the amount of 

bilirubin. It is qualitatively measured by comparing to standard 

colors. It varies with labs, species, liver disease, dietary intake of 

carotene in cattle, among other things. 

Insulin Like Growth 

Factor I (IGF-I) Liver 

Synthesized by the liver in response to growth hormone. Used as 

an indicator of the amount of growth hormone being 

produced. Elevations can be related to increased nutritional 

status, low values to negative energy balance. Higher in growing 

animals. 

Estradiol (E2) Ovary 

Synthesis is controlled by gonadotropins. Synthesis rates related 

to ovarian function in females. Used to monitor follicular and 

luteal activity. 

Cortisol Adrenal 

Cortex 

Synthesis is regulated by the hypothalamus and pituitary. 

Involved in normal metabolism. Elevated levels are associated 

with stress. 

Triiodothyronine (T3) Thyroid 

Gland 

Synthesis is regulated by the hypothalamus and pituitary. 

Involved in normal metabolism. 

Complete Blood Count 

(Hemogram) Description 

Hematocrit (Hct) Refers to the percent of blood that is occupied by red blood cells. 

Low values are good indicators of anemia.

Hemoglobin (Hb) Protein used by red blood cells to distribute oxygen to other tissues 
Appendix H: The Comprehensive Veterinary Examination H-19 

Animal Cloning: A Risk Assessment DRAFT

and cells in the body. Low values are good indicators of anemia. 

Red Blood Cells (RBC) 

The absolute concentration of red blood cells in the blood. A low 

red blood cell count is defined as anemia. High count is 

polycythemia. 

Mean Corpuscular Volume (MCV) 

The actual volume of the red blood cells. Larger red blood cells 

may indicate anemia due to B12 or folic acid deficiency, also may 

caused by increase in reticulocytes; smaller red blood cells may 

indicate anemia due to iron deficiency. 

Mean Corpuscular Hemoglobin (MCH) 

This test measures the amount of hemoglobin in red blood cells. 

Both hemoglobin and hematocrit are used to calculate this number. 

Low levels indicate anemia. Of limited diagnostic value. 

Mean Corpuscular 

Hemoglobin Concentration (MCHC) 

This test measures the amount of hemoglobin in red blood cells. 

Both hemoglobin and hematocrit are used to calculate this number. 

Low levels indicate anemia. 

Red Cell 

Distribution Width(RDW) 

RDW evaluates the range of sizes of RBCs in a blood sample. If 

anemia is suspected, based on other blood counts, RDW test results 

are often used together with MCV results to determine the cause of 

anemia. 

White Blood Cells (WBC) 

Leukocytes (WBCs) are produced by the immune system (in bone 

marrow) to help defend against infection. A high WBC count likely 

indicates an infection, whereas a low number might be an acute 

response where readily available cells are summoned to the site of 

infection or due to immunosuppression. 

Segmented Neutrophils (segs) 

Phagocytic cells present to guard against infection, particularly 

bacterial. Segmented neutrophils are mature neutrophils, and are 

the predominant white cell in non-ruminant mammals. 

Banded Neutrophils (bands) Immature neutrophils. Elevated levels occur in response to a recent 

infection. 

Lymphocytes (lymphs) 

T-cells, B-cells, and natural killer (NK) cells. Viral infections can 

either increase or decrease the total percentage of lymphocytes. It 

is the predominant white cell in ruminants. 

Monocytes (monos) 

Monocytes are a type of phagocyte that mature into 

macrophages. A low number can indicate a higher risk of bacterial 

infection. 

Eosinophils (eos) 

Active in killing parasites, can inhibit mast cells or release 

mediators of inflammation. A high number of eosinophils can 

indicate allergies or parasitic infections. 

Basophils (basos) Function unclear. A type of phagocyte that produces the antiinflammatory protein histamine. 

Platelets (Thrombocytes) 

Tiny cells produced by the bone marrow to help blood clot 

formation. High number might indicate a blood disease. A 

decreased platelet count is called thrombocytopenia. Used to 

measure immune system function. 

Mean Platelet

Volume (MPV) 

Measures the average volume of platelets. May be artifactually 

high due to clumping of platelets in blood sample. 

Total Protein-refractometer (TP-ref) The total amount of protein in the plasma measured by a 

refractometer. 

RBC Morphology 

General morphology (shape) of red blood cells. Poikilocytes - 

RBCs of irregular shape. Schizocytes - poikilocytes from 

fragmentation due to flowing through damaged small vessels. 
Appendix H: The Comprehensive Veterinary Examination H-20 

Animal Cloning: A Risk Assessment DRAFT

Parasites The blood sample is examined for the presence of parasites. 

Complete Blood Count 

(Hemogram) Description 

WBC Exam Morphological appearance of the white blood cells.

Plasma Appearance General appearance of the plasma.