# Presentation: 409444

## Summary Data from Animal Clones

- Eric S. Dubbin, D.V.M.
- Ruminant Drugs Team
- Center for Veterinary Medicine
- Food and Drug Administration

## Critical Biological Systems Approach

**Critical Biological Systems Approach**

**Perinatal**
**Development **
**and Function**

**Reproductive Development**
**And Function**

**Post-Pubertal**
**Maturation**

**Cell Fusion Through **
**Fetal Development**

**Offspring**** **

**Juvenile Development**
**And Function**

**Notes:**
This slide is to remind all of us that we are evaluating the data along developmental nodes corresponding to critical stages in life.

## Presentation Goals

- Overview of literature findings
- Species-by-species evaluations
- Key outcomes in CBSA context
- Emphasis on Development Node data relevant to
- Animal health risk
- Food consumption risk
- More detailed presentation of Cyagra data
- Largest single data set
- Most detailed

## CBSA: Summary of 1st Developmental Node

- Period of highest risk for developing clone
- Failure to divide or implant
- Reprogramming defects
- Placentation difficulties
- Common across all species evaluated
- Percentage of successful outcomes very low
- Data of limited utility for further assessment of risks

**Cell Fusion Through **
**Fetal Development**

**Notes:**
The data are of limited utility except that they set the stage for the next node.

## Bovine Clones

**Critical Biological Systems Approach**

## CBSA: Bovine Perinatal Clones

- Key studies from a few laboratories
- Many address/intermingle transgenic clones
- Number of “just” clones relatively small (~50)
- Most information on overall health
- Few physiological/biochemical studies
- Large Offspring Syndrome most common

**Notes:**
Large offspring syndrome is a collection of findings where the calf is larger than expected for the breed, the placenta is edematous, parturition is delayed, and the calf is unthrifty.  These calves may need feeding and respiratory support.  Many survive.

## CBSA: Bovine Perinatal Period Summary 

**Newborn clones fragile during first days**
**Higher incidence of death early in technology development **
**No qualitative differences relative to other ARTs**
**Frequencies often increased**
**Most common anomalies LOS related**
**BW at birth often higher than comparators**
**Cardiovascular malformations**
**Respiratory difficulties**
**Flexor tendon contracture**

**Notes:**
Dr. Adams talk will address LOS in more depth

## Critical Biological Systems Approach

**Perinatal**
**Development **
**and Function**

**Cell Fusion Through **
**Fetal Development**

**Juvenile Development**
**And Function**

**Notes:**
The next node encompasses juvenile development in and around the time of weaning depending on the species being discussed.

## CBSA:  Bovine Juvenile Clones

- Period of resolution of initial instabilities
- ~100 clones in all the reports
- 3 reported deaths in otherwise healthy-appearing clones
- 1 study has **** in GH and IGF-I levels in clones than controls (Govoni et al. 2002)
- Growth and general health usually reported as “normal”
- Behavior normal; resembles donor animal (Savage 2003)

## CBSA:  Bovine Juvenile Clones

**CBSA:  Bovine Juvenile Clones**

- One clone w lymphoid hypoplasia; dies at day 51 (Renard et al 1999)
- Japanese Black Beef clones of the 12 clones surviving perinatal period, all were “healthy and normal” up to 350 days (Kato 1998, 2000)
- 4 clones from ear cells of a 17 yr old Japanese Black Beef bull alive and healthy at 10-12 months. Vet exams, growth curves, and 30 blood parameters reported as “normal”.  No data provided. (Kubota et al. 2000)

## CBSA:  Bovine Juvenile Clones

**CBSA:  Bovine Juvenile Clones**

- Physiological measurements on 21 healthy-appearing clones (Chavatte-Palmer et al. 2002)
****** **body temp w/o infection resolved by 50 days
- Some blood parameters initially unstable; resolve in a few weeks
- Basic clinical chemistry normal
- Hormone levels: IGF-I, IGF binding protein, leptin, insulin (post-prandial glucose) all normal
- Initial thyroxine, leptin, IGF-II differences resolved within 7- 15 days

## CBSA: Juvenile Developmental Node Summary

- Initial physiological instabilities resolve during this time period
- Lab results –> appropriate responses to growth and development
- Physical anomalies clearly detected
- Normal and healthy animals behave and appear functionally indistinguishable from Comparators

## Critical Biological Systems Approach

**Perinatal**
**Development **
**and Function**

**Reproductive Development**
**And Function**

**Cell Fusion Through **
**Fetal Development**

**Juvenile Development**
**And Function**

**Notes:**
The reproductive system is a complex set of interactions.  We considered that if a clone could successfully breed and reproduce, that would demonstrate appropriate control of this process.  Success at this node indicates that the clones are genetically well integrated because the subtle and complex process is functional. 

## CBSA: Reproductive Development and Function

- Data quite limited
- Often cursory mention of “normal activity”
- Explicit evaluation of bovine clone reproductive function (Enright et al. 2002; Govoni, Savage)
- Puberty somewhat later (314 d v. 272 d)
- Higher BW at first estrus (336 kg v. 302 kg)
- No difference in cycle length, follicular development, hormonal profiles (daily LH, FSH, E2, & P4 similar)
- 3/4 clones and 4/4 controls preg post AI (# of inseminations not reported)

**Notes:**
The this report is one study and includes 4 cows (Enright)

Another report indicated clone Holstein heifers were cyclic by 10-11 months (normal 9-12mo), Pace 2002

Govoni says that low IGF-I may delay puberty

## CBSA: Reproductive Development and Function

- 5 live clones healthy, normal, and normally cyclic at 1yr of age** ****(Heyman et al.** 2002 )
- “Gene” the bull, healthy, fertile, sired calves (AI/IVF)** **Forsberg et al. (2002)
- One clone bred via AI, conceived, delivered normal calf** **Kato et al. (2000)
- 2 Holstein clones Aoki et al. (2003) abstract
- First post partum ovulations delayed
- Follicular waves (2/cycle) normal
- Calved normally

**Notes:**
Other reports give a passing mention on reproduction

## CBSA: Reproductive Development and Function Summary

- Clones appear to develop normally
- Puberty reached ~ normally, but may be slightly delayed
- Reproductive function appears normal in males and females

## Critical Biological Systems Approach

**And Function**

**Critical Biological Systems Approach**

**Perinatal**
**Development **
**and Function**

**Reproductive Development**
**And Function**

**Cell Fusion Through **
**Fetal Development**

**Post Pubertal **
**Maturation**

**Notes:**
The concept of life span is different for food animals.  
There are 2 classes of food animals, brood animals for reproduction and market animals for consumption
Market animals are raised until they reach market weight. They are then slaughtered for food.  That’s about 18-24 mo for bovine and 6-8 mo for swine.
Brood animals are the breeder stock.  Those animals remain alive to produce offspring until they are culled for some reason.  Reasons include: infertility, lameness, disease, poor production.  Cattle, pigs, sheep and goats can live for up to 15-20 years but are usually culled before that.

## CBSA:  Post Pubertal Maturation

- Few bovine clones have reached advanced age
- No reports of sudden abnormalities arising
**Hill et al.** (2000)
-  “Second Chance”, overcame neonatal diabetes, now 3 years of age w/ normal BW, growth, behavior, and semen production

## Data Provided by from Cyagra, Inc.

- Late Spring 2003, influx of new data from Cyagra
- Physical exams
- Medical records
- Systematically collected laboratory data

**Notes:**
In the Spring of 2000, we encouraged producers to publish data on the health of clones..
We presented our draft risk assessment methodology in the  Fall of 2002.

In the Spring 2003, the Cyagra Co. sent in this set of data.  
The set includes Physical exams, Medical Records, Systematic Laboratory Data
Clinical Data are evaluated using the method of clinical medicine 

## Clinical Medicine

- History – chief complaint
- Physical Exam
- Systematic approach
- Done the same way each time
- Systems based
- Clinical Pathology
- Serum Chemistry
- Electrolytes
- Blood Cells
- Red Blood Cells
- White Blood Cells
- Platelets
- Urinalysis
- Summary – Lab Data must be in Context

**Notes:**
Problem Based Medical Approach – clinical medicine uses this approach which contingent on the animal having a problem.  In a survey type of study like this, we are looking at lab values on animals that are well.  They have no problem.  This is one of the dilemmas of running lab work.
	Chief Complaint
	Physical Exam
	Problem List
	Differential Diagnosis
	Plan – Dx, Tx, CE
	Diagnostic Test
		Lab work
	Revised Problem List
	Revised Differential Diagnosis
	Revised Plan
Systems based: Demeanor, temperature, Skin – color, character, elasticity; Ear eyes nose throat – mucous membranes, hydration; Musculo-skeletal – lameness; G.I. – vomiting, diarrhea, bloating; Cardio-Pulmonary-Vascular – auscult, edema, peripheral pulses; Genito-urinary; Neurological; Peripheral LN

## Clinical Pathology

- Complements the physical exam
- Assist diagnosis for a disease process
- Reference Ranges have limitations
- Based on a population of healthy animals
- Mean +/- 2 SD
- 5% of normal values may be out of range
- Lab’s reference population
- Artifact
- Clinical Judgment Still Necessary

**Notes:**
Artifact – Unexplainable value caused by for example
Lab error
Poor sample handling
Unforeseen circumstances
Lipemia
Hemolysis
Stress
Drug interactions
Diff labs, reagents, techniques yield different results

## Cyagra Data Set

| Distribution of Cattle Clones and Comparator Populations |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | Number of Animals |  |  |  |
|  | Neo-
nates | 1 to 6 months | 6 to 18 months | Totals |
| Clones | 10 | 46
(7 neonates) | 18 | 74
(67) |
| Comparators | 17 | 47 | 21 
(1 rep) | 86
(85) |

**Notes:**
The 74 total clone values includes the 7 neonates/1-6 month subcohort
On the second line, we have approximate, age, breed, and environmentally matched comparators.

## Cyagra Health Data

- Mortality
- Physical Exam
- Clinical & Surgery– Umbilical problems
- Cull

**Notes:**
Before we looked at the clin path, we looked at the overall health of the animals.  We looked at mortality, PE, clinical, surgery

## Cyagra Health Data

- Mortality
- 134 clone calves
- 28 stillborn, died, or euthanized by 48 hrs
- 11 calves died from 2d to 1 yr
- 8 from d2- 29
- 3 from d30-180
- 0 after 180

**Notes:**
The numbers do not add up, so don’t try.  The 7 of the 67 calves have values at neonatal and juvenile nodes.  These calves with data were submitted first.  We were able to examine their health and lab data.  The remaining 60 calves were submitted later as the info became available and none had blood data.

## Cyagra Health Data

- Mortality (of 134 clones)
- 28 stillborn, died, or euthanized by 48 hrs
- Stillborn – 11
- Euthanized – 12
- Abnormal development 7
- Contracture 5
- Diarrhea – 2 – rotavirus
- Unknown – 3
- Appeared normal
- thick placenta & pericarditis
- Moist umbilicus, depressed

**Notes:**
1 euthanized for failure to thrive 

## Cyagra Health Data

- Mortality (of 134 clones)
- 11 clones died after 2d
- 3 septic umbilical problems
- 3 G.I. bloat, adhesions, abomasal rupture
- 3 neonatal circulatory problems
- 1 contracture, non responsive
- 1 accidental trauma

## Cyagra Health Data of Surviving Clones

- Physical Exams
- 11 clones - musculo skeletal abnormalities
- 9 clones with contracted tendons – survived
- 1 thick withers, enl. carpus, leg dev. laterally – culled
- Dwarf with frequent bloat, eventually culled
- 1 clone - early mammary development
- 2 clones - harsh lung sounds
- 3 bull clones - a retained testicle (cryptorchid) all were from the same donor bull
- 1 bull clone - cardiac arrhythmia

## Cyagra Health Data

- Umbilical
- 41 umbilical difficulties
- Bleeding, infection, adhesions,
- 2 had subsequent pyelonephritis
- Surgeries
- 29 clones had umbilical surgery
- Miscellaneous health problems
- 3 clones had rotavirus
- 2 clones died of that disease
- 2 G.I.- bloat, adhesions, ruptured abomasum
- 1 Failure to transition to fetal circulation
- 1 hypoxia, rapid deterioration
- Cull
- 2 -1 dwarf and 1 with thick withers, enlarged carpus, deviated leg

**Notes:**
Umbilical extirpation is the removal of the entire umbilicus.  This includes the external umbilical stump and the internal vessels: the umbilical vein, both umbilical arteries, and the urachal remnant (that leads to the bladder).  Sometime the urachal remnant will not have closed.  This is defined as a patent urachus.  A patent urachus leaks urine

## Clinical Chemistry, CBC, and Urinalysis

- Organization and interpretation of data
- Decide on most appropriate comparison

## Summary of Laboratory Value Comparisons

| Table Cyagra 3a:  Fraction of Blood Values Within Comparison Range |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Animals | Analysis | Clones: Reference Range |  | Clones: Comparator Population |  | Comparator Population: Reference  Range |  |
|  |  | Chart  | FCWR | Chart  | FCWR | Chart   | FCWR |
| (6-18 months) | Clinical Chemistry | 300 | 0.75 | 301 | 0.99
586/592 | 302 | 0.73 |
|  | Hematology | 310 | 0.73 | 311 | 0.99
291/294 | 312 | 0.71 |
| (1-6 months) | Clinical Chemistry | 200 | 0.62 | 201 | 0.96
1,434/
1,492 | 202 | 0.70 |
|  | Hematology | 210 | 0.61 | 211 | 0.96
711/742 | 212 | 0.61 |
| Neonates
(<48 hours) | Clinical Chemistry | 100 | 0.31 | 101 | 0.92
293/319 | 102 | 0.36 |
|  | Hematology | 110 | 0.61 | 111 | 0.90
107/119 | 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. |  |  |  |  |  |  |  |

**Notes:**
This slide illustrates which comparison gave the closest alignment of values.  

We took the data from three different populations.  The clones, the age matched comparators, and lab reference range.  We then compared individual analyte values from one population with the range exhibited in the other populations.  We then counted the number of analytes from the target population that were within the range of value exhibited in the standard.  Look at the top row, the chart demonstrates that when the clinical chemistry of the 6-18mo group clones is compared to the lab reference range, 75% of the clones have analyte values within the Lab reference range.  Continuing across the top, 99% of the clones have analyte values within the range demonstrated by the age-matched comparators.  Finally on the top left, 73% the age-matched comparators have analyte values with the range of the reference lab.

The chart shows the comparisons for three nodes.  The central column gives the actual numbers on which the percentages are based.

At birth
CBC - 90% (107 of 119 values similar) values, 
Clin chem - 92% (293 of 319 values similar). 
1 to 6 month age cohorts - even more similar 
CBC – 96% (711 of 742 values similar ) 
Clin Chem – 96% (1,434 of 1,492 values similar) 
6 to 18 month cohorts - are almost superimposable 
CBC – 99% (291 of 294 values similar)
Clin Chem – 99% (586 of 592 values similar) 
significantly less than would be expected by chance alone

Conclusion:
Using age matched calves is more appropriate for a reference range than the laboratory’s own reference range.
Cornell’s Lab reference range is derived from 50 adult cattle (dairy or beef not mentioned), thus the cattle used for the lab’s reference range are not the most appropriate comparisons.

We also notice from this table, that as the calves get older, their lab values stablize.
There is less variability between the clones and that age matched comparators with the passage of each node.
Out of range values that occur early in life appear to self correct with each developmental node

## Clinical Chemistry, CBC, and Urinalysis

- Creation of comparative chart approach
-  Good visual comparison
-  Concise, but informative

## Chart 311: Hemogram Clones v Comparator [6-18 mo]

**Notes:**
This slide is for demonstration purposes.  It demonstrates how we organized the individual analytes to give a quick view of overall sameness/difference and totals.
Across the top, animal means CVM’s animal identifier
The first column lists the analytes measured.
Black Squares for clone values within comparator range
Grey squares for out of range but not that much (less than 10%, considered to be within acceptable clinical variation)
Up or down arrow for out of range
Asterisk means no data
X means presumed artifact
We then added the arrows for a total of out of range values.
Across totals (rows) – are the number of times an analyte was out of range
Down totals (columns)- are the number of out of range analytes for that calf identified at the top of the column
The column on the far right lists how many analytes are out of the comparator range
The row on the bottom is how many analytes for that animal are out of the comparator range 
47 arrows out of 119 boxes (39%) indicates low similarity b/t the two populations
Note we dropped Urinalysis, results were sparse and mostly qualitative, used only for clarification as needed
Conclusions made in the context of the entire animal

The purpose of this slide is to note that on the whole chart, there are only 3 arrows (out of 306).  The rest are boxes.  These few out of range points represent close similarity between the two populations compared.

Evaluation Method
If only one analyte was out of range, we did not address it.  It could be due to chance, it is a soft finding.

If two or more analytes were out of range, these records were further scrutinized to look for causes and trends.

## Clinical Chemistry and CBC

- What questions do we want these data to answer?
- Similarity/Differences
- Response to internal and external stimuli
- Predictability

**Notes:**
We wanted to know if the data could demonstrate:

Similarity/Differences – are the two populations are similar physiologically. Are there subtle biochemical changes that are not grossly visible. 
1. A finding of similar laboratory values would provide confidence that there were no material differences in metabolic, immunologic, and hematopoeitic (blood producing) functions between clones and conventional animals
Internal and External stimuli – Do the clones respond to the internal (growth and maturation) and normal external (stressors, disease) stimuli appropriately?  For example,  Did calves with an infection develop a high white count?  
Predictability 
Do the blood data indicate some predictor of long-term viability of that animal or that cohort.  Were any consistent findings that would allow us to predict the fate of the animal.  For example, were there a high number of anemic animals that were unthrifty and poor doers?

## Trend 1- Age Appropriate Responses in Growth Analytes

- Growing animals, both clones and comparators, have elevations in growth specific analytes relative to adults
- Alkaline phosphatase
- Ca++
- P

**Notes:**
When clones and age matched comparators are compared throughout the three nodes to the adult population reference range

This is an age appropriate elevation indicating proper metabolic growth pathways involving vitamin D synth, and functioning kidney, bone, parathyroid, thyroid gland, and appropriate intestinal absorption.

We conclude that the clones growth metabolism is similar to that of the controls.  Remember that other growth factors were studied in the literature and found GH, IGF-I, T3 to be similar as well.

  we looked at the clones, and as would be expected of normal animals in this age group we saw changes in physisol parameters consistent with animals of that age  

## Trend 2 – Age Appropriate Responses in 		Immunoglobulin 

- Endogenous Immunoglobulin Production
- Total protein
- Globulin
- Young calves, both clones and comparators, have low globulin levels
- Older calves have higher globulin levels

**Notes:**
Globulin consists mostly of  Immunoglobulin (Ig) derived from maternal antibody in colostrum in neonates.  As the calves age endogenous production increases.  

At about 6 weeks colostral Ab dissipates.  As calves age, the calves own B lymphocytes began to produce their own Ig (Ab) as evidenced by increase globulin levels.  

We concluded that the clones own antibody production matures similar to the comparators

In the clones, and as would be expected of normal animals in this age group,  we saw changes in physiological parameters consistent with animals of that age  

## Cyagra Neonates: Clinical Chemistry

**Notes:**
10 live clones, 17 comparators
92% agreement b/t clones and controls
Chemistry - Across
Liver – low AST, Cholesterol, bile acids
Colostrum – low GGT 
Down
#43 many analytes out of range, died from rotavirus
#75 – low AST, chol, bile acids; no follow up
#80 – high Na, low AST, high % saturation, rotavirus diarrhea, died

## Cyagra Neonates: Hematology

**Notes:**
90% agreement b/t clones and controls

Hematology - Across
15/17 analytes were within range
3 calves had low lymphocytes 
Calf #43 died of rotavirus diarrhea
2 platelets out of range, one high, one low
High - died from diarrhea, Low - survived to weaning
Down
#43 many analytes out of range, died from rotavirus
#71, 72, 73, 78, 79, 119, 132 – discussed in the neonate to 1-6mo section
	#78 – low WBC, low lymph, low platelets, survived
	#79 – low AST, low bile acids, culled posture & gait
	#132 – low AST, chol; survived
	#79 – poor conformation, culled eventually

## Neonates: Summary

- 10 clones, 2 died
**Similarity:**  92% clin chem; 90% hematology
- Liver enzymes different, normalize by next node
**Response**
- Growth factors and immune factors show expected distribution
**Predictability**
- Dead clone with many analytes out of range
- Dead clone with few analytes out of range
- Laboratory values must be interpreted in the context of the animal.

## Cyagra 1-6 Month Group: Clin Chem

**Notes:**
46 clones and 47 comparators
96% agreement

Chemistry across
Ca, P, Alk Phos – growth related.
7/42 creatinine: 6 lower, 1 higher – clinical relevance of low values?
6/42 elevated glucose, transient (urinalysis)
TP 2 low, in the hematology there is 1 high and 1 low.  They are not the same calves.  This illustrates some variability with the tests themselves
SDH 3 low, GGT 3 low, not the same 3 calves, clinical relevance of low values?
Chemistry – Down
	#71, 72, & 73 had many analytes out of range, no health problems
	#79 had 5 analytes out of range, culled for poor posture and gait.

## Cyagra 1-6 Month Group: Hematology

**Notes:**
96%

Hematology across
No anemia or leukopenia
MCHC 4/44 elevated, RDW 3/44 elevated – indicators of red cell maturation.  Used to determine causes of anemia.  Their value
	in health animal with out anemia is unknown
Basophils 4/44 elevated – no known significance
MPV 9/41 elevated can be artifact from clumping, blood smears indicated adequate platelet numbers and morphology
TP ref 1 high and 1 low

Individual animals- Down
#58 had 7 analytes out of range, elevated TP may be related to Ag stim from umbil abscess
#100 had elevated WBC, umbilical abscess, appropriate response
#102 had dec platelets, inc MPV, inc MCHC, had umbilical surgery

## Cyagra 1-6 Month Group: Summary

**46 clones with blood work**, one culled
- 3 died in this age group (no blood work)
**Similarity to comparators:**
- 96% clin chem; 96% hematology
- Elevated growth analytes, glucose relative to Comparators
- 4 animals, same donor, different phenotypes
- 3 cryptorchid, same bull donor
**Response**
- Appropriate immune response & WBC elevation in response to abscess
**Predictability**
- Clone had only one analyte out of range, health problems
- 3 clones with many analytes out of range, no health problems
- Laboratory values must be interpreted in the context of the animal.

**Notes:**
Growth analytes – Ca, P, and Alk phos, may be related to superior genetics or care received.  
	Glucose may be involved here but we saw that it was transient (based on urinalysis), may be stress related
#87, 88, 89 Jersey heifers, same cell line
A fourth heifer in this line died from LOS, that at about 5 months - 
#87 (141 days) weighed 282 lbs
#88 (140 days) weighed 197 lbs
#89 (131 days) weighed 215 lbs
All dehorned and had umbilical surgery, it is interesting to note that 4 clones from the same cell line, 1 died and the 3 other exhibit different phenotypes.
# 128, 130, 131 
All three were cryptorchid, same cell line

## 7 Clone Subcohort Analysis 

- Neonatal Chemistry
- Colostrum
- Globulin
- 0/7 neonates low
- 3/6 1-6mo low, reflects maternal Ig waning
- Lower GGT
- 4/7 neonates, pre-colostrum, normalized by 1-6mo
- Liver
- Lower AST
- 6/7 neonates, normalized by 1-6mo
- Lower Cholesterol & Bile Acids
- 5/7 neonates, normalized by 1-6mo
- Lower GGT
- 4/7 neonates, normalized by 1-6mo

**Notes:**
There are 7 calves for which we have data at the neonatal node and the juvenile node.
On the neonatal chart, none of the calves had an out of range globulin, but this is because of a low globulin level in the comparator group CHECK THIS OUT PLEASE, its true, it was 1.3 g/dl.  The three low globulins in the 1-6 mo period are because those three calves were at the lower end of the age group. 
For GGT, 4 calves were lower than the comparators, this is a soft finding for colostrum deprivation and because this analyte has liver implications, I will discuss it further there.  Note that no clones are low in the next node.
4 liver related analytes are low for neonates.  They all normalize by the next node.  The cause of elevated liver values is usually liver damage.  low liver values are uncommon. The cause is unknown.   I have speculated that the cause may be from the clone calve’s transition from fetal to neonatal circulation.  In fetal circulation the liver is largely by-passed by the ductus venosus.  Upon birth this vessel closes and the liver becomes perfused with blood.  Neonatal clones have a higher incidence of umbilical problems and these enzyme differences may be evidence of that.  We do not have specific data describing the umbilicus of these 7 calves.

##  7 Clone Subcohort Analysis (cont.)

- Neonatal Hematology
- One clone with decreased lymphocytes and platelets
- Normalized by 1-6mo
- #79
- Neonate
- Decrease AST, decreased bile acids
- Had no hematology data
- Juvenile (1-6 mo)
- Increased creatinine, dec glob, inc alk phos, inc cholesterol
- Hematology - dec TP-ref, RDW, and inc MPV.
- Clone culled for  poor conformation

**Notes:**
Calf #79, was the cull with thick withers and enlarged carpus.  Note that it had a decreased total protein as measured by refractometer (on the hematology panel).  The total protein from the chemistry panel was within range.  The same analyte from the same blood sample determined by to different methods yielded the same result for must calves.  But for this one calf, there was a difference.  Is this difference real?  Again, must be taken in context of the animal and the other blood work.

## 7 Clone Subcohort Analysis (cont.)

- 1-6 Month Clones
- 4 elevated glucose, no glycosuria
- 4 elevated alk phos, younger animals
- 3 elevated P, younger animals
- 3 increased A/G ratio (assoc with dec glob)
- 1 elevated creatinine, only marginal, no other renal signs
- 2 decreased anion gap, unknown cause
- These out of range values do not indicate any consistent trends other than expected physiological changes

**Notes:**
We looked at the 1-6mo chart to see if differences showed up.  Remember, elevations are relative to the comparator group
The elevated glucose was transient
The elevated alk phos  and elevated Pin 1-6 group is b/c these 4 animals were among the youngest of the 1-6mo group (about 6 weeks of age), which is age appropriate
The elevated A/G also occurred in the 3 animals among the youngest of the 1-6mo group.  Again an appropriate response for a 6 week old calf with maternal Ab waning and prior to endogenous Ab prod’n

## 7 Clone Subcohort Analysis (cont.)

- Summary
- Out of range neonatal values improved by 1-6mo
- Out of range 1-6mo values, when taken in context, were not of clinical concern
- 1 clone later culled for poor conformation

## Cyagra 6-18 Month Group: Chemistry

**Notes:**
18 clones and 21 comparators 
99% agreement, this is a greater agreement than we would expect by chance alone.
IGF-I – 4 bulls with slight elevations IGF-I increase may be due to superior genetics.  924, 916, and 938 ng/ml compared to 33-875 ng/ml ref range.  This ref range shows that IGF-I can fluctuate dramatically.  <10% increase is not that much
Estrogen – 5 bulls slightly outside comparator range but not Cornell reference lab range,  E2 – range in this study = 4.28-24.33 pg/ml.  Comparator range 4.1-11.41 pg/ml.  Cornell lab reference range 10-40 pg/ml
Hormones are released in a pulse.
Creatinine – since we discussed grey boxes for E2 and IGF, notice that creat has 7 grey boxes.  These values were very close to the high comparator range (2.1 v. 2.0)

Animals down
#103 – clinically normal animal with 3 analytes out of range, 

## Cyagra 6-18 Month Group: Hematology

**Notes:**
99% agreement

Analytes Across
MCV - 2 low 
Low MCV and are discussed before and in this context, not indicative of a problem

Animals Down
#108 – few analyte abnormalities, but was a dwarf with frequent bloat.  Only 3 minor analytes out of range (TIBC low, RBC high – mild with no influence on Hct, MCV low)

## Summary: Cyagra 6-18 Month Group 

- 18 clones with blood work
- 0 died in this age group
**Similarity:**
- 99% clin chem;
- 99% hematology
- IGF-I and GH, only slightly elevated to comparator, but within published and physiologic ranges
**Response ******** **no disease to monitor

**Notes:**
Again, IGF-I and GH, endocrine metabolism is complex.  Slight elevations may be seen due to better genetics, better care.

Not predictive

## Swine

- Archer 2003 –
- determine variation within a population of clones and compare to pigs of
- genetically similar age,
- breed (Duroc), and
- sex matched naturally bred females
- Half siblings

## Swine

**Archer et al**. 2003
- Behavioral Traits
- Food preferences
- Temperament
- Time budgets
- Behavior was no more homogeneous than siblings’
- Behave as conventional animals; no behavioral abnormalities observed

**Notes:**
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 and being placed on their backs and being lifted off the ground)
time budgets (the amount of time spent engaged in a particular activity in their pens). 

##  Swine

- Physical traits
- Body Weights
- No differences, normal range for 27 week old pigs
- Phenotypic Variation
- Teat number –1 with a 6,7 pattern all others had 6,6.
- Skin
- Hair growth pattern – 1 with a unique growth pattern
- 1 had hyperkeratosis.  The paper does not say whether this was the same pig

## Swine

-  Archer et al. 2003, used with permission

**Notes:**
Phenotypic variation, coarse hair coat

## Swine

- Phenotypic Variation
- The largest and smallest clones by size are shown side by side.
- The smaller clone never achieved the weight/size of the other clones.
- These two clones are from the same nuclear donor and farrowed to the same recipient dam.

## Swine

- Archer et al. 2003, used with permission

**Notes:**
Phenotypic variation, difference in body weight and size with nuclear cell line, same dam
We saw this with Jersey cows

##   Swine

- Archer 2003

**Notes:**
Notice the growth factors (Alk phos, P, T3), with elevations early and less so later.  The clones respond as we expect normal animals in that stage of growth.

Next notice that the globulins also increase over time.  

## Swine

- Clones and comparators indistinguishable
- Lab values and behavior studies
- Growth analytes
- Alk Phos, P
- T3, glucose
- Immune status
- Globulin, Serum Protein, A/G ratio

**Notes:**
Ref range from Merck Manual.  Likely adult population.  Notice for example that all the alk phos from 15 weeks of age are higher than the range.

Growth factors are higher for the 15 week old pigs than for the 27 week old pigs.  This age appropriate change is similar for the clones and controls.  This is inc at 15 and dec at 27 is not seen in Ca.

Globulins and the related analytes (TP and A/G of which Globulins are components) show an age related increase from the 15 week period to the 27 week period.  This change was discussed in the cattle section.  But briefly, younger animals must make their own immunoglobulin as they get older and the Ig they consumed in the colostrum wanes.  Ig comes from B lymphocytes which are stimulated to produce Ig with age and antigenic exposure.  These data show the young clones and controls have a similar profile.  Both are low compared to the Merck ref range at 15 and approach the ref range similarly with age. 

## Goats

- 27 transgenic and 70 non-transgenic (97 total)
- 13 recips (avg 7 embryos/dam)
- 5 (38%) confirmed pregnant d35
- 4 recips delivered 5 male kids
- (7.1% of embryos for that cell line)
- 1 recips delivered a female kid
- (7.7% of embryos for that cell line)
- (Keefer et al. 2001)

## Goats

- Birth weights (2.35 avg) and cotyledon numbers (avg 60) not significantly different from naturally bred goats at the facility
- 3 deaths due to bacterial lung infection
- 1 kid died w/in 24 h
- 1 kid died at 1mo
- 1 kid died at 3mo

## Goats

- 91 embryos into 8 recips.
- 4 pregnant at day 30 delivered 7 female kids, 3 sets of twins
- 1 died at birth
- 54 male embryos in 6 recips.
- 1 confirmed pregnant delivered 2 male kids in another study, Keefer 2002
- 1 died at birth
***All maintained pregnancy to term***

## Goats

- Average birth weights for clones were similar to kids derived from natural mating (NM)
- 1.7 +/-0.13 kg and 1.3+/-0.06 kg respectively
- The number of cotyledons per placenta
- 40+/-6.8 clones and 60+/-9.3 for NM
- Keefer 2002-

## Goats

- Half derived clones poor suckling reflex
- Fed by oro-gastric tube for  1 day
- Good suckling behavior began on day 2
- Clones appeared healthy
- Routine blood profiles monitored until 1 yr of age
- “...within normal ranges for growing Nigerian Dwarf kids”
- Keefer 2002-

**Notes:**

Hematology and chem panel info not provided.

## Summary		

- Clones that survive the critical few days after birth are mostly normal and healthy.

## In Summary

- Anomalies not qualitatively different from other ARTs; increase in frequency
- Perinatal node
- mortality, placentation, LOS/BW, body temp, respiratory, circulatory, defects, musculoskeletal, umbilical, lab values.
- Not common across species
- Placentation, LOS not issues in swine and goats
- Juvenile node
- less problems:
- hormone levels-similar, behavior-same, mortality low
**Reproductive and Maturation** nodes
- No apparent problems
- Data sparse,
- Growth parameters and fertility very similar