Approved for public release; distribution unlimited. 

Specialty: 500C, Toxicity Tests 

Toxicology Study No. 87-XE-0DUZ-11, June 2010 – September 2011 

Protocol No. 08JG-80-10-03-01 

Toxicology Portfolio 

Two-Generation Test for Endocrine Disruption and Reproductive 

Toxicity in Japanese Quail (Coturnix japonica) Using Trenbolone 

Prepared by Michael Quinn, Health Effects Research Program 
ACKNOWLEDGEMENTS 

The authors would like to acknowledge Pat Beall, Valerie Adams, William Eck, Emily 

Lent, Larry Williams, Matthew Bazar, Will McCain, Lee Crouse, Mark Way, Arthur 

O’Neill, and Christiana Grim for their support during the conduct of this study. We 

would also like to thank Shane Hall for performing the statistics for this study. This 

study was done concurrently with two other similar ones by U.S. Geological Survey, 

Patuxent Wildlife Research Center and Wildlife International Ltd.; we’ve supported each 

other throughout each study’s conduct in the development and refinement of this test 

method, and the authors are grateful for having had the opportunity to collaborate with 

such outstanding researchers in this field. 
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

Sponsor

U.S. Environmental Protection Agency 

Office of Science Coordination and Policy 

1200 Pennsylvania Avenue, NW (7203), Washington, DC 20460 

Study Title

Toxicology Study No. 87-XE-0DUZ-11 

Protocol No. 08JG-80-10-03-01 

Two-generation test for endocrine disruption and reproductive toxicity in Japanese quail 

(Coturnix japonica) using trenbolone 

Study Completion Date

September 2011 

Performing Laboratory

U.S. Army Public Health Command 

Portfolio of Toxicology 

Health Effects Research Program (MCHB-IP-THE) 

5158 Blackhawk Road 

Aberdeen Proving Ground, Maryland 21010-5403 

Laboratory Project ID

08JG-80-10-03-01 

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT 

The study described in this report was conducted in compliance with Title 40, Code of 

Federal Regulations (CFR), Part 792, Good Laboratory Practice Standards, except for 

the following: 
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

No deviations from the aforementioned regulation affected the quality or integrity of the 

study or the interpretation of the results. 

______________________________ ______________________________ 

Michael J. Quinn Date 

Study Director 

______________________________ ______________________________ 

Mark S. Johnson Date 

Program Manager 

Health Effects Research 

Prepared by: 

_____________________________ __________________ 
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

Michael J. Quinn, Jr., Ph.D. Date 

Biologist, Health Effects Research Program 

Reviewed by: 

____________________________ __________________ 

Craig A. McFarland, Ph.D., DVM Date 

Approved by: 

____________________________ __________________ 

Mark S. Johnson, Ph.D. Date 
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

i

Table of Contents 

Paragraph Page 

1. REFERENCES ............................................................................................. 1 

2. PURPOSE .................................................................................................... 1 

3. AUTHORITY ................................................................................................ 1 

4. SCOPE ......................................................................................................... 1 

5. BACKGROUND ........................................................................................... 1 

6. MATERIALS AND METHODS ..................................................................... 2 

 6.1 Experimental Design ............................................................................. 2 

 6.2 Data Analysis ........................................................................................ 4 

 6.3 Quality Assurance ................................................................................. 4 

7. RESULTS ..................................................................................................... 4 

 7.1. Eggshell Thickness and Strength ............................................................ 4 

 7.2. Egg Production ........................................................................................ 5 

 7.3. Sperm Measures ..................................................................................... 6 

 7.4. Growth and Body Weight......................................................................... 8 

 

 7.5. Feed Consumption .................................................................................. 9 

 7.6. Cloacal Gland Area ................................................................................. 12 

 7.7. Onset to Puberty ..................................................................................... 13 

 

 7.8. Copulatory Behavior ................................................................................ 14 

 7.9. Organ Weights ........................................................................................ 19 

 7.10. Hormone Analyses ................................................................................ 20 

 
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

8. DISCUSSION ............................................................................................... 21 

9. CONCLUSION ............................................................................................. 23 

10. POINT OF CONTACT .................................................................................. 23 

Appendices 

A REFERENCES ............................................................................................. A-1 

B QUALITY ASSURANCE STATEMENT ........................................................ B-1 

C ARCHIVES AND STUDY PERSONNEL ...................................................... C-1 

D ANIMAL USE PROTOCOL WITH MODIFICATIONS ................................... D-1 

List of Tables 

Table 1. List of Critical Events ............................................................................. 4 

Table 2. F0 Eggshell Thickness and Strength ..................................................... 5 

Table 3. F1 Eggshell Thickness and Strength ..................................................... 5 

Table 4. F2 Eggshell Thickness and Strength ..................................................... 5 

Table 5. F0 Egg Production ................................................................................. 6 

Table 6. F1 Egg Production ................................................................................. 6 

Table 7. F2 Egg Production ................................................................................. 6 

Table 8. F0 Sperm Measures .............................................................................. 7 

Table 9. F1 Sperm Measures .............................................................................. 7 

Table 10. F2 Sperm Measures ............................................................................ 8 

Table 11. F0 Adult Body Weight .......................................................................... 8 

Table 12. F1 Adult Body Weight .......................................................................... 8 

Table 13. F2 Adult Body Weight .......................................................................... 9 

Table 14. F0 Female Feed Consumption ............................................................ 10 

Table 15. F0 Male Feed Consumption ................................................................ 10 

Table 16. F0 Paired Feed Consumption .............................................................. 10 

Table 17. F1 Female Feed Consumption ............................................................ 10 

Table 18. F1 Male Feed Consumption ................................................................ 11 

Table 19. F1 Paired Feed Consumption .............................................................. 11 

Table 20. F2 Female Feed Consumption ............................................................ 11 

Table 21. F2 Male Feed Consumption ................................................................ 12 

Table 22. F0 Cloacal Gland Area ........................................................................ 12 

Table 23. F1 Cloacal Gland Area ........................................................................ 12 

Table 24. F2 Cloacal Gland Area ........................................................................ 13 

Table 25. F0 Onset to Puberty ............................................................................ 13 

Table 26. F1 Onset to Puberty ............................................................................ 14 

Table 27. F2 Onset to Puberty ............................................................................ 14 
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

Table 28. F0 Number of Mount Attempts ............................................................ 15 

Table 29. F0 Onset to Mount ............................................................................... 15 

Table 30. F0 Number of Successful Copulations ................................................ 15 

Table 31. F0 Onset to Successful Copulation ..................................................... 16 

Table 32. F1 Number of Mount Attempts ............................................................ 16 

Table 33. F1 Onset to Mount ............................................................................... 17 

Table 34. F1 Number of Successful Copulations ................................................ 17 

Table 35. F1 Onset to Successful Copulation ..................................................... 17 

Table 36. F2 Number of Mount Attempts ............................................................ 18 

Table 37. F2 Onset to Mount ............................................................................... 18 

Table 38. F2 Number of Successful Copulations ................................................ 18 

Table 39. F2 Onset to Successful Copulation ..................................................... 18 

Table 40. F0 Female Organ Weights .................................................................. 19 

Table 41. F0 Male Organ Weights ...................................................................... 19 

Table 42. F1 Female Organ Weights .................................................................. 19 

Table 43. F1 Male Organ Weights ...................................................................... 20 

Table 44. F2 Female Organ Weights .................................................................. 20 

Table 45. F2 Male Organ Weights ...................................................................... 20 

Table 46. F2 Generation Adult Hormone Data .................................................... 20 

Table 47. Comparison Across Generations of Male Japanese Quail Effects ...... 20 

Table 48. Comparison Across Generations of Female Japanese Quail Effects .. 20 

Table 49. Comparison Across Generations of Male Copulatory Behavior ........... 20 
Toxicology Study No. 87-XE-0DUZ-11 

Protocol No. 08JG-80-10-03-01 

Two-Generation Test for Endocrine Disruption and Reproductive Toxicity in 

Japanese Quail (Coturnix japonica) Using Trenbolone 

June 2010 – September 2011

1 References 

See Appendix A for a listing of references. 

2 Purpose 

The objective of this research is to determine the value added by examining endocrine endpoints in 

a two-generation avian reproductive toxicity test paradigm. 

3 Authority 

This research and assessment addresses part of the environmental safety and occupational health 

(ESOH) objectives outlined in Army Regulation (AR) 200-1, AR 40-5 and AR 70-1, Department of 

Defense Instruction (DODI) 4715.4, and the Army Environmental Research and Technology 

Assessment (AERTA) requirement A (3.3.c) (references 1-5). It was conducted as part of the ongoing effort, under the U.S. Army Environmental Quality Technology, Ordnance Program, Pollution 

Prevention Pillar, under the direction of Research, Development and Engineering Command 

(RDECOM), Environmental Acquisition Logistics & Sustainment Program. 

4 Scope 

This report documents the conduct, findings, and conclusions of using trenbolone to develop and 

assess a two-generation test for endocrine disruption in Japanese quail. 

5 Background 

The U.S. Environmental Protection Agency (EPA) is implementing an Endocrine Disruptor 

Screening Program (EDSP) comprised of a battery of Tier 1 screening assays and Tier 2 tests. 

One of the Tier 2 tests under development is a two-generation avian assay using Japanese quail 

(Coturnix japonica). This test is designed to characterize dose-response relationships and 

immediate and latent adverse consequences associated with exposure to putative endocrine 

modulating compounds. The avian two-generation assay serves to evaluate potential population 

effects of endocrine modulating compounds on myriad avian species, and represents terrestrial, 

oviparous wildlife species in general. This study will use trenbolone, a synthetic androgen, to 

further evaluate the proposed test method for its ability to detect adverse effects and characterize 

dose responses in a two-generation paradigm, and to develop and optimize a protocol for the avian 

two-generation assay for characterizing endocrine disruptors. In combination with information 

gained from a prior study using endosulfan as an estrogen-active endocrine disruptor and a future 

study using a thyroid-active chemical, this work will help to determine the value added by 

examining endocrine endpoints over two generations in comparison to tests that only include one 

generation, such as the Ecological Effects Guidelines for avian reproduction tests (EPA, 1996).
Toxicology Study No. 87-XE-0FV5-12, Protocol No. 0FV5-96-12-05-01, June-August 2012 

2 

6 Materials and Methods 

6.1 Experimental Design 

The parental (F0) generation was first exposed to trenbolone, a known androgenic endocrine 

disrupting chemical (CAS No. 10161-33-8), at four weeks of age and throughout the rest of the 

birds’ lives. Eggs from this generation were incubated and hatched to produce the first generation 

(F1). The F1 generation was exposed to the trenbolone in ovo (embryonic) through maternal 

deposition and through feed throughout the entirety of their lives. Eggs from this generation were 

incubated and hatched to produce the second generation (F2). The F2 generation was only 

exposed to trenbolone in ovo through maternal deposition; all feed for the F2 generation was 

untreated. Measures of reproductive behavior, circulating hormone levels, egg quality, 

reproduction, and development were assessed in the F0, F1, and F2 generations as described 

below. Additional measures of growth and reproductive development were assessed in the F1 and 

F2 generations as described below. 

The sample size for each treatment was 20 males and 20 females to ensure that there were at 

least 12 breeding pairs in the parental generation control group at the end of the treatment period to 

have successfully produced eggs. The treatment levels for this study were 0 (control), 0.5, 5, 10, 

and 20 ppm. Dose levels were determined by the U.S. EPA following completion of a range finding 

study done by the U.S. Geological Service, Patuxent Wildlife Research Center, Laurel, MD. The 

test diets were mixed and assayed at Wildlife International Ltd., Easton, MD, USA. Homogeneity 

and concentration verifications were made by USACHPPM Directorate of Laboratory Services. 

Upon receipt of the first batch of treated feed, all doses were tested for concentration verification. 

Once all treatment levels were found to be within acceptable ranges, each subsequent batch of 

treated feed only required two randomly selected doses to be tested for concentration verification 

unless the results of these tests were suggested the need for further testing, according to GLP 

standards. 

To generate the F0 generation, a minimum of 60 eggs were hatched for each treatment level., At 

least 12 breeding pairs per F0 test level were needed for the study to proceed with 20 pairs per 

level as the goal.. Twenty males and females were used for the study, beginning exposure to the 

treatments at week 4. Optimally, 6 eggs were set for hatch for each parental pair of the F0 

generation to ensure that a minimum of 60 eggs would be hatched for each test level. Once again, 

sufficient numbers of chicks were needed to establish at least 12 breeding pairs (20 is the goal) per 

F1 test level. , If a treatment level produced insufficient eggs to set the minimum, that level was to 

be dropped from further analysis in the test. Optimally, 6 eggs were set for each parental pair of 

the F1 generation to ensure that a minimum of 40 eggs would be set and hatched for each 

treatment level of the F2 generation.. 

During the treatment period, all eggs were collected at least once daily and recorded by hen of 

origin. The total numbers of eggs laid per hen were calculated and recorded at the end of the 

study. 

After hatch of each generation, fertility and embryo viability were determined by candling and/or 

dissecting the unhatched eggs. It was decided that candling would not be performed during 

incubation of each generation to prevent delay of embryonic development. Chicks that did not 

hatch within approximately 24 hours of the majority of chicks hatching were considered unhatched. 

No assistance was given to chicks during hatching; chicks that remained alive but had no clear 

ability to hatch on their own were humanely removed from the egg and decapitated immediately. 
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

3

Prior to incubation of subsequent generations, eggs were collected to assess eggshell strength and 

eggshell thickness. Eggshell strength was measured by an Egg Force Reader (Orka Technology 

Ltd.) that tests the breaking point of an egg’s shell. Eggshell strength was measured in two eggs 

per hen and the average value per hen was calculated. Eggshell thickness was measured by 

digital calipers to the nearest 0.00 mm. Two dry sections of shell taken from opposite sides of each 

egg were measured for thickness and the average value per egg was calculated. 

During the treatment period, all birds were observed daily to detect any overt signs of toxicity or 

other clinical signs of illness or trauma. Observations included mortality and clinical signs of toxicity 

such as lethargy, depression, wing droop, ruffled feathers, etc. Any injuries sustained and 

subsequent treatments were recorded. If an individual or pair was removed during the study due to 

death, injury, etc., data from all preceding weeks are evaluated. Clearly moribund individuals or 

those otherwise in severe distress were euthanized by CO2 asphyxiation. 

All adult birds underwent necropsy and gross pathology assessment. The wet weight of the brain, 

liver, kidneys, gonads, and thyroid glands were recorded as soon as possible after 

death/euthanasia. Any morphologic abnormalities were recorded in the study’s notebook or 

necropsy sheets. Adult birds that died or were euthanized during the course of the treatment 

periods were necropsied similarly. Histopathology was performed on the brain, right thyroid gland, 

liver, bursa of Fabricius, kidneys, and gonads. Upon the histopathologist’s suggestion due to the 

small size of the tissues, the thyroid glands from half of the adults were fixed in formalin for 

histopathologic analyses, and thyroid glands from the remaining half were flash frozen and stored 

for later glandular thyroxine (T4) content. Immediately following euthanasia, spermatozoa were 

released from a weighed section of epididymis by mincing the tissues with scissors into 200 μl 

buffered Roswell Park Memorial Institute-1640 medium (Sigma Chemical, St. Louis, MO, USA) as 

described by McFarland et al., (2008). Two μl of the suspension was loaded onto 20-μm 4-

chamber slides (Leja Products, Nieuw-Vennep, the Netherlands) to determine cell concentration 

per gram epididymis, percent motility, and percent progressively motile using a IVOS Sperm 

Analyzer, Version 12.1 (Hamilton-Thorne Research, Beverly, MA, USA). 

Measurements of thyroid function include thyroid gland weight, adult glandular T4 content, and 

thyroid histology/histopathology. Blood samples collected from adults at the time of necropsy were 

analyzed for circulating estradiol and testosterone. All hormone analyses were conducted at the 

University of Maryland, Department of Animal and Avian Sciences. Glandular T3 and T4 content 

and serum estradiol were measured by radioimmunoassay (RIA) kits (Siemens) and serum 

testosterone was measured via an Enzyme Linked Immuno Sorbent Assay (ELISA) kit (Caymen). 

Details of their methodologies can be found in Appendix H. 

Initiation of normal male sexual behavior is important for successful reproduction. In reproductively 

mature males of the F1 and F2 generations, trials were preformed when at least 90% of control 

males reached reproductive maturity. The reproductive behaviors that were measured included: 

time taken for mount attempts to be initiated, number of mount attempts made within the time limit 

(3 minutes), time taken to achieve a successful copulation, and number of successful cloacal 

contacts. Males and females of the F1 generation were paired following completion of the 

behavioral measures. Birds from the F2 generation were euthanized upon completion of this 

measure. 

Growth was measured every three days in chicks from day of hatch to up to at least week 2 of age. 

Growth measures included body weight, tarsus length, and the length from the tip of the beak to the 

culmen. 
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

4

This study was conducted in compliance with Good Laboratory Practice (GLP) regulations found in 

the Toxic Substances Control Act (TSCA): 40 CFR (Code of Federal Regulations) 792, plus 

amendments. All investigators and technicians adhered to The Guide for Care and Use of 

Laboratory Animals (U.S. Department of Health, Education, and Welfare Publication No. NIH 86-23, 

1996). 

6.2 Data Analysis 

All observational data was recorded in the laboratory notebook and work sheets where applicable. 

Data was analyzed by PHC statistician Shane Hall. Specifics regarding this analysis can be found 

in the Contributing Scientist Report, Appendix G. 

 

6.3 Quality Assurance 

Critical phases of this study were audited by the USAPHC) Quality Systems Office (Appendix B). 

The Good Laboratory Practices (GLP) regulation followed is according to the Toxic Substances 

Control Act. 

Table 1. List of Critical Events 

Date of Event 

Animal Use Protocol 

Animals Received 

Study Start 

Experimental Start 

Experimental Completion 

Necropsy Start 

Necropsy Completion 

Study Completion 

7 Results 

 Eggshell Thickness and Strength. Trenbolone did not cause any changes in eggshell thickness 

or strength in eggs produced by either generation (Tables 2-4). 

Table 2. Eggshell thickness and strength (force) from the F0 generation of 

Japanese quail (Coturnix japonica) exposed to trenbolone. 
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

5

Table 3. Eggshell thickness and strength (force) from the F1 generation of 

Japanese quail (Coturnix japonica) exposed to trenbolone. 

Table 4. Eggshell thickness and strength (force) from the F2 generation of 

Japanese quail (Coturnix japonica) exposed to trenbolone. 

 Egg Production. F0 generation hens from the 10 and 20 ppm dose levels produced fewer eggs 

than those from the other dose groups from week 7 through 10 (Table 5). Similarly, F1 generation 

hens from the 20 ppm group produced fewer eggs than hens from all other treatment levels from 

ppm Average Std. Error Average Std. Error

0 0.381 0.010 1.24 0.05

0.5 0.385 0.012 1.12 0.09

5 0.382 0.011 1.30 0.06

10 0.379 0.013 1.23 0.08

20 0.366 0.008 1.07 0.13

F0Generation ‐ Egg Strength

p‐value = .795 p‐value = .558*

* P‐value calcula ted from Kruskall‐wallis tes t ‐ 

homogenei ty of va ria nce a s s umption viola ted for

pa rame tric ANOVA tes t

Thickness Force

ppm Average Std. Error Average Std. Error

0 0.320 0.006 1.31 0.09

0.5 0.327 0.007 1.41 0.06

5 0.315 0.006 1.38 0.07

10 0.329 0.008 1.37 0.08

20* 0.345 0.006 0.53 0.27

Thickness Force

* ppm level 20 was excluded from ANOVA test due to

small n (thickness: n=4, force: n=3)

p‐value = .409 p‐value = .787

F1 Generation ‐ Egg Strength

ppm Average Std. Error Average Std. Error

0 0.199 0.005 1.15 0.07

0.5 0.199 0.004 1.01 0.06

5 0.203 0.005 1.09 0.04

10 0.206 0.005 1.17 0.06

F2 Generation ‐ Egg Strength

Thickness Force

p‐value = .590 p‐value = .225
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

6

week 7 through 10 (Table 6). Egg production was so low in this dose group that the 20 ppm dose 

level was dropped from the F2 generation. No differences in egg production were observed in the 

F2 generation (Table 7). 

Table 5. Numbers of eggs produced per week by F0 generation Japanese quail 

(Coturnix japonica) exposed to trenbolone. 

Table 6. Numbers of eggs produced per week by F1 generation Japanese quail 

(Coturnix japonica) exposed to trenbolone. 

Table 7. Numbers of eggs produced per week by F2 generation Japanese quail 

(Coturnix japonica) exposed to trenbolone. 

 Sperm Measures. Sperm from F0 generation males of the 20 ppm dose group displayed higher 

motility than those from the other dose groups (Table 8). No other sperm characteristics were 

altered by trenbolone in this generation. Percent motility and sperm concentration (number of 

sperm per gram vas deferens) in the F1 generation remained consistent among treatment levels, 

but progressive motion of sperm from the 0.5 ppm dose group was lower than that from the 20 ppm 

group (Table 9). Percent motility and progressive movement were unaltered in the F2 generation; 

however control males had a higher sperm count than males from the 5 ppm level. (Table 10). 

Table 8. Percent motility, percent of motile sperm that moved progressively, and 

number of sperm per gram tissue epididymis from the F0 generation of Japanese 

quail (Coturnix japonica) exposed to trenbolone. 

Factor P‐value ppm Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21 Overall

0 0.65 2.35 3.35 5.25 4.40 3.85 5.30 5.65 6.10 5.55 5.70 5.80 5.45 6.00 5.45 4.26

0.5 0.5 1.3 2.65 4.05 4.15 3.9 4.4 5.15 4.8 4.9 5.05 4.85 5.25 5.15 4.4 3.54

5 1.20 2.80 4.30 4.75 5.30 4.45 5.20 5.55 5.65 5.20 5.05 4.10 5.05 4.70 5.10 4.32

10 0.25 1.65 2.55 3.40 3.45 3.20 3.60 3.80 3.80 4.35 3.40 3.75 3.95 4.00 3.85 2.86

20 0.30 1.15 1.65 2.40 2.45 2.50 2.80 2.60 3.10 3.05 2.25 2.40 2.75 2.95 2.55 2.09

Overall 0.49 1.75 2.76 3.83 3.82 3.51 4.14 4.37 4.55 4.52 4.07 4.01 4.36 4.43 4.13

** Week 6 was not included in the analysis ‐ only two quails produced any eggs in week 6.

F0Generation Egg Production

GEE Significance Tests GEE Estimated Marginal Means

Interaction ‐            

ppm by week 0.000

ppm 0.000

Week 0.000

Factor P‐value ppm Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21 Week 22 Week 23 Week 24 Week 25 Overall

0 0.45 1.25 2.85 3.65 2.25 5.10 4.00 3.45 4.40 5.30 5.40 5.25 5.55 4.70 5.10 5.25 5.30 6.15 6.30 3.90

0.5 0.40 2.30 2.90 3.00 4.20 3.65 4.25 4.10 4.85 4.80 5.45 5.30 5.45 5.40 5.25 5.20 5.50 5.90 5.40 3.94

5 0.14 0.76 2.10 2.38 2.90 3.19 3.33 3.38 4.14 3.90 2.44 4.14 4.10 3.19 4.57 4.43 4.57 4.48 4.71 2.84

10 0.25 1.11 1.32 1.93 2.57 1.93 2.43 2.64 3.04 2.61 2.54 3.25 2.79 2.79 3.39 3.11 3.36 3.21 3.11 2.23

20 0.00 0.00 0.17 0.22 0.28 0.67 0.56 0.11 0.50 0.72 0.83 0.83 1.00 0.94 0.89 0.89 1.06 0.67 1.06 0.03

Overall 0.00 0.00 1.31 1.62 2.06 2.38 2.38 1.70 2.66 2.85 3.05 3.15 3.22 2.92 3.26 3.20 3.43 3.22 3.50

** Week 6 was not included in the analysis ‐ only one quail produced any eggs in week 6.

F1Generation Egg Production

GEE Estimated Marginal Means

ppm 0.000

Week 0.000

Interaction ‐            

ppm by week 0.000

GEE Significance Tests

Factor P‐value ppm Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Overall

0 0.10 2.00 3.25 4.30 5.30 5.45 3.85 2.27

0.5 0.50 1.90 3.70 4.10 4.25 4.55 4.10 2.73

5 0.45 2.25 4.05 5.15 5.00 5.05 5.75 3.15

10 0.10 1.50 2.85 4.45 4.80 5.30 5.35 2.21

Overall 0.22 1.89 3.43 4.47 4.82 5.08 4.69

** Week 5 was not included in the analysis ‐ only one quail produced any eggs in week 6.

ppm 0.268

Week 0.000

GEE Significance Tests

Interaction ‐            

ppm by week

0.080

GEE Estimated Marginal Means

F2 Generation Egg Production
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

7

Table 9. Percent motility, percent of motile sperm that moved progressively, and 

number of sperm per gram tissue epididymis from the F1 generation of Japanese 

quail (Coturnix japonica) exposed to trenbolone. 

Table 10. Percent motility, percent of motile sperm that moved progressively, and 

number of sperm per gram tissue epididymis from the F2 generation of Japanese 

quail (Coturnix japonica) exposed to trenbolone. 

ppm Average Std. Error Average Std. Error Average Std. Error

0 54.3 5.9 11.0 2.8 1259.8 248.4

0.5 66.1 5.3 18.6 5.6 1738.8 301.0

5 61.7 6.5 14.9 5.4 1580.2 489.7

10 49.5 6.1 10.9 3.6 1198.1 230.4

20 73.8 5.1 18.8 3.4 2100.1 429.8

F0 Generation ‐ Sperm

% Motile Sperm/gram % Progressive

p‐value = .042* p‐value = .520 p‐value = .377

* ppm level 10 and level 20 a re signi fica ntly di fferent from each othe r

determined by Tukey Pos t‐hoc tes t using a 95% confidence level.

§ ID 488 (ppm = .5) excluded from analysi s because da ta poi nts for each of the

three mea s urements were extreme outliers

ppm Average Std. Error Average Std. Error Average Std. Error

0 48.2 6.9 26.2 4.1 3852.1 685.9

0.5 30.0 8.8 16.6 4.8 3063.2 1021.7

5 48.9 8.2 29.2 5.2 4516.0 898.4

10 60.1 5.8 35.6 4.5 3672.5 918.9

20 60.3 3.2 35.8 2.2 5555.3 1044.0

F1 Generation ‐ Sperm

% Motile % Progressive Sperm/gram

p‐value = .066 p‐value = .030* p‐value = .390

* Tukey Pos t‐hoc res ul ts indi ca te tha t level .5 and level 20 have signi fi ca ntly

di fferent % progres sive averages.

§ ID 46 (ppm = .5) excl uded from a nalysi s beca use da ta points for each of the

three mea s urements were extreme outliers
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

8

 Growth and Body Weight. F0 generation females from the control group tended to have a higher 

mean body weight from week 5 through week 21 than all other dose groups, and females from the 

20 ppm group had a significantly lower average body weight than all other dose groups (Table 11). 

Control males from this generation weighed more than males from the 0.5 and 10 ppm dose 

groups. Similarly, F1 generation females and males from the 20 ppm treatment level had a lower 

mean body weight than all other levels (Table 12). In the F2 generation, females receiving 10 ppm 

trenbolone in feed weighed more than birds from the control and 5 ppm dose groups (Table 13). 

Males from the 0.5 ppm dose group had lower average body weights than all others from this 

generation. 

Table 11. Mean body weight from adult female and male F0 generation Japanese 

quail (Coturnix japonica) exposed to trenbolone. 

Table 12. Mean body weight from female and male F1 generation Japanese quail 

(Coturnix japonica) exposed to trenbolone. 

ppm Average Std. Error Average Std. Error Average Std. Error

0 29.5 4.9 8.1 1.7 266.1 29.4

0.5 38.3 4.5 12.4 1.6 217.3 15.8

5 30.9 5.6 9.4 2.5 166.5 22.4

10 44.4 3.8 12.8 1.8 179.2 15.3

% Motile % Progressive Sperm/gram

F2Generation ‐ Sperm

p‐value = .115 p‐value = .258 p‐value = .039*°

* Non‐pa rame tric ranks tes t i ndica tes tha t control group i s signi ficantly di fferent

from ppm level 5.

°P‐value calcula ted from Kruskall‐wallis tes t ‐ homogenei ty of va riance

a s s umption viola ted for pa rame tric ANOVA tes t

Model Term P‐value ppm Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21 Overall

0 98.5 106.1 118.0 124.3 128.4 134.6 135.4 135.7 139.3 140.0 142.2 141.6 141.1 142.5 142.4 142.6 143.2 132.7

0.5 92.5 100.9 110.9 115.5 124.5 129.7 130.8 135.1 135.8 137.0 138.6 138.7 138.7 141.9 141.9 140.2 138.3 128.9

5 93.0 100.9 115.0 120.2 128.3 132.8 134.9 136.2 139.2 139.2 139.9 138.2 135.7 138.2 138.6 138.2 143.4 130.1

10 94.0 101.8 111.4 120.2 125.0 130.3 132.3 133.8 132.9 134.8 138.1 137.2 139.5 139.7 139.6 139.2 139.6 128.8

20 89.2 98.1 107.5 114.5 120.3 122.6 126.6 126.1 130.1 130.6 133.1 130.6 129.2 134.2 133.9 133.8 132.3 123.1

Overall 93.4 101.6 112.5 118.9 125.3 129.9 132.0 133.4 135.5 136.3 138.4 137.2 136.8 139.3 139.3 138.8 139.4

ppm 0.000

F0 Generation Female Body Weights

Significance Tests Estimated Marginal Means

Interaction ‐          

ppm by week 1.000

Week 0.000

Model Term P‐value ppm Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Overall

0 90.6 98.8 104.5 106.5 107.8 110.0 111.1 111.6 112.9 113.1 113.6 114.0 115.0 115.3 115.0 115.0 109.7

0.5 88.5 98.2 101.9 102.7 104.2 106.0 107.4 108.4 110.9 111.7 111.9 112.1 112.8 113.4 115.3 114.1 107.5

5 90.9 98.1 103.0 104.7 106.0 107.9 109.2 107.2 111.1 112.2 112.2 112.7 112.6 113.1 113.2 113.8 108.0

10 86.7 95.4 101.5 104.3 105.8 106.8 107.9 108.4 110.2 111.1 111.7 112.3 113.1 114.4 114.8 114.8 107.4

20 89.2 96.7 102.8 104.4 106.6 107.6 110.1 109.4 111.7 113.5 111.5 112.5 113.2 113.7 112.9 114.1 108.1

Overall 89.2 97.4 102.7 104.5 106.1 107.6 109.1 109.0 111.3 112.3 112.2 112.7 113.3 114.0 114.2 114.3

Week 0.000

F0Generation Male Body Weights

Significance Tests Estimated Marginal Means

Interaction ‐          

ppm by week 1.000

ppm 0.002

Model Term P‐value ppm Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21 Week 22 Week 23 Overall

0 94.1 103.6 111.8 114.3 125.5 125.5 132.0 131.1 133.4 133.5 137.5 141.3 140.0 138.6 143.1 142.8 141.1 139.1 141.3 130.0

0.5 92.2 103.6 113.3 118.9 125.8 126.5 131.6 133.3 133.6 133.6 139.3 135.9 139.2 139.2 143.5 141.9 142.3 143.6 144.9 130.6

5 92.5 101.4 107.3 113.2 120.6 125.1 126.7 134.0 134.3 134.9 135.7 134.7 136.3 134.4 140.1 140.6 141.6 138.2 142.0 128.1

10 91.9 101.9 113.6 115.5 122.7 123.5 129.0 127.2 134.6 137.7 136.3 135.5 137.4 134.6 141.7 145.7 142.5 142.9 142.2 129.3

20 82.8 95.9 101.8 104.3 110.3 113.0 116.1 120.8 120.1 122.4 123.3 124.9 126.4 130.2 126.4 131.0 132.3 134.1 134.3 118.5

Overall 90.7 101.3 109.5 113.2 121.0 122.7 127.1 129.4 131.2 132.4 134.4 134.5 135.9 135.4 138.9 140.4 140.0 139.6 140.9

0.000

Significance Tests Estimated Marginal Means

F1 Generation Female Body Weights

Interaction ‐          

ppm by week 0.988

ppm 0.000

Week
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

9

Table 13. Mean body weight from female and male F2 generation Japanese quail 

(Coturnix japonica) exposed to trenbolone. 

 Feed Consumption. Between weeks 5 and 10, females from the 5 ppm dose group in the F0 

generation had a higher average food consumption than birds from the control, 0.5, and 20 ppm 

groups. Throughout weeks 5-10, control birds ate less than all other birds except for those in the 

0.5 ppm group (Table 14). Control males in the same age range consumed less feed than those 

from all other dose groups except for those from the 10 ppm group (Table 15). Feed consumption 

in the 5 ppm males exceeded that of the 10 ppm males. When the birds were paired during weeks 

13-21, birds from the 10 ppm dose group consumed more feed than all other groups, and pairs 

form the 20 ppm group consumed less than all others (Table 16). 

Female F1 generation quail from the 0.5 ppm group consumed more feed than all other groups 

whereas control females ate the least during weeks 6 through 11 (Table 17). Control males had the 

lowest overall average feed consumption during this time, while males from the 10 ppm level had 

the highest (Table 18). F1 generation pairs from the 20 ppm dose group typically had the lowest 

average feed consumption during weeks 14-23 (Table 19). 

Feed consumption did not differ in F2 females (Table 20), however F2 males from the 5 and 10 

ppm dose groups had the highest feed consumption averages during weeks 6 through 11 (Table 

21). 

Table 14. Mean weekly (from week 5 through 10) feed consumption for F0 

generation female Japanese quail (Coturnix japonica) exposed to trenbolone. 

Model Term P‐value ppm Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21 Week 22 Week 23 Overall

0 92.8 101.8 106.7 106.8 109.1 110.1 111.9 111.4 112.8 112.7 114.6 115.5 115.7 116.1 117.3 117.6 117.9 117.6 117.4 111.9

0.5 90.4 97.3 105.1 107.3 109.2 111.5 113.0 112.1 110.2 111.8 115.1 118.0 116.6 115.2 117.2 116.9 115.9 115.9 117.5 111.4

5 91.6 100.6 105.2 106.7 108.9 110.7 110.9 111.9 112.0 112.0 113.6 115.5 115.0 115.7 117.8 116.9 117.3 116.1 117.3 111.3

10 85.0 96.5 104.1 105.1 106.4 108.4 110.0 110.6 110.4 110.6 114.1 113.6 117.9 113.9 117.1 116.9 117.7 115.7 116.0 110.0

20 77.7 89.8 98.4 100.6 104.9 106.9 106.8 108.5 108.5 108.8 112.5 112.2 112.6 115.1 118.2 117.9 117.5 117.7 116.0 107.9

Overall 87.5 97.2 103.9 105.3 107.7 109.5 110.5 110.9 110.8 111.2 114.0 115.0 115.5 115.2 117.5 117.2 117.2 116.6 116.8

ppm 0.000

Week 0.000

F1 Generation Male Body Weights

Significance Tests Estimated Marginal Means

Interaction ‐          

ppm by week 0.177

Model Term P‐value ppm Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Overall

0 83.2 97.4 101.7 112.4 126.1 128.4 129.4 132.4 136.9 131.9 118.0

0.5 86.9 98.3 103.7 112.8 125.8 128.8 129.3 130.6 133.2 131.6 118.1

5 88.1 98.9 103.1 115.3 128.3 132.0 134.0 130.2 132.4 137.2 119.9

10 86.0 102.3 106.7 115.4 126.5 128.5 136.9 134.7 137.5 140.2 121.4

Overall 86.1 99.2 103.7 113.9 126.7 129.4 132.4 132.0 135.0 135.2

Estimated Marginal Means

Week 0.000

Significance Tests

Interaction ‐          

ppm by week 0.727

ppm 0.001

F2Generation Female Body Weights

Model Term P‐value ppm Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Overall

0 83.9 93.0 96.6 104.1 108.2 107.1 107.9 108.7 109.7 102.1

0.5 77.5 91.4 95.7 103.3 105.9 104.0 104.9 106.7 107.7 99.7

5 81.5 95.8 99.1 104.6 107.2 107.0 109.4 110.9 112.4 103.1

10 82.5 94.8 99.0 104.9 107.8 107.7 109.8 109.5 112.3 103.1

Overall 81.4 93.7 97.6 104.2 107.3 106.4 108.0 108.9 110.5

Estimated Marginal Means

ppm 0.000

Week 0.000

Significance Tests

Interaction ‐          

ppm by week 0.988

F2 Generation Male Body Weights
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

10

Table 15. Mean weekly (from week 5 through 10) feed consumption for F0 

generation male Japanese quail (Coturnix japonica) exposed to trenbolone. 

Table 16. Mean weekly (from week 13 through 21) feed consumption for F0 

generation female and male Japanese quail (Coturnix japonica) pairs exposed to 

trenbolone. 

Table 17. Mean weekly (from week 6 through 11) feed consumption for F1 

generation female Japanese quail (Coturnix japonica) exposed to trenbolone. 

Factor P‐value ppm Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Overall

0 119.2 124.7 130.1 137.3 156.9 175.6 140.6

0.5 116.4 114.1 128.7 114.7 150.5 169.4 132.3

5 109.1 120.1 121.6 132.9 173.3 175.9 138.8

10 137.1 128.5 139.4 132.9 167.1 169.1 145.7

20 113.1 125.6 129.6 132.3 165.9 150.7 136.2

Overall 119.0 122.6 129.8 130.0 162.7 168.1

Significance Tests Estimated Marginal Means

Week 0.000

Interaction ‐ 

ppm by week

0.005

ppm 0.000

F0Generation Female Food Consumption

Factor P‐value ppm Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Overall

0 110.7 111.2 111.8 126.0 110.2 114.4 114.0

0.5 110.6 109.9 105.3 132.5 121.8 122.6 117.1

5 122.3 134.2 128.7 143.1 135.3 135.2 133.1

10 117.1 123.1 135.8 128.1 130.8 131.4 127.7

20 111.4 118.6 126.6 124.8 131.9 126.2 123.2

Overall 114.4 115.9 121.6 130.9 126.0 125.9

Week 0.000

F0 Generation Male Food Consumption

Significance Tests Estimated Marginal Means

Interaction ‐ 

dose by week

0.060

ppm Level 0.000

Factor P‐value ppm Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21 Overall

0 300.1 302.9 316.2 282.9 308.2 305.6 322.7 326.9 320.8 309.6

0.5 290.1 282.0 282.6 290.9 295.3 286.2 316.5 331.5 312.8 298.7

5 287.7 284.8 294.5 276.7 292.5 320.1 357.4 337.1 325.8 308.5

10 282.8 293.1 303.8 311.2 336.3 326.6 387.4 357.5 344.5 327.0

20 252.2 264.2 255.3 260.5 277.9 286.3 344.4 296.1 303.6 282.3

Overall 282.6 285.4 290.5 284.4 302.1 305.0 345.7 329.8 321.5

Significance Tests

Week

Estimated Marginal Means

F0Generation Paired‐Gender Food Consumption

0.000

Interaction ‐ 

ppm by week

0.155

ppm 0.000

Factor P‐value ppm Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Overall

0 127.5 136.4 123.3 103.9 144.7 139.1 129.1

0.5 125.0 141.0 160.3 161.3 164.8 178.8 155.2

5 118.2 115.3 128.6 149.0 169.3 154.0 139.1

10 130.6 141.4 128.1 148.4 148.7 162.5 143.3

20 118.4 122.1 112.7 168.5 223.5 132.9 146.4

Overall 124.0 131.2 130.6 146.2 170.2 153.5

Week 0.000

Significance Tests Estimated Marginal Means

F1 Generation Female Food Consumption

Interaction ‐ 

ppm by week

0.003

ppm 0.020
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

11

Table 18. Mean weekly (from week 6 through 11) feed consumption for F1 

generation male Japanese quail (Coturnix japonica) exposed to trenbolone. 

Table 19. Mean weekly (from week 14 through 23) feed consumption for F1 

generation female and male Japanese quail (Coturnix japonica) pairs exposed to 

trenbolone. 

Table 20. Mean weekly (from week 6 through 11) feed consumption for F2 

generation female Japanese quail (Coturnix japonica) exposed to trenbolone. 

Table 21. Mean weekly (from week 6 through 11) feed consumption for F2 

generation male Japanese quail (Coturnix japonica) exposed to trenbolone. 

Factor P‐value ppm Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Overall

0 116.2 112.5 114.3 108.5 126.2 108.3 114.3

0.5 114.4 117.6 128.1 141.1 145.7 120.0 127.8

5 114.5 118.3 120.0 135.6 159.4 122.2 128.3

10 131.8 124.5 129.9 159.4 148.9 148.2 140.5

20 116.7 123.6 114.4 122.6 135.8 115.8 121.5

Overall 118.7 119.3 121.3 133.4 143.2 122.9

F1 Generation Male Food Consumption

Interaction ‐ 

ppm by week

0.047

ppm 0.000

Week 0.000

Significance Tests Estimated Marginal Means

Factor P‐value ppm Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21 Week 22 Week 23 Overall

0 289.2 244.8 272.6 305.8 192.2 301.0 305.1 262.5 348.9 305.2 282.7

0.5 318.1 242.6 263.2 178.1 210.9 307.8 303.9 256.0 350.2 311.6 274.2

5 286.6 221.3 246.9 260.8 183.2 257.4 298.8 250.3 299.8 323.1 262.8

10 268.0 224.4 243.1 252.8 238.5 281.8 304.1 250.3 329.3 292.8 268.5

20 258.5 175.7 202.2 244.1 202.6 236.5 217.5 229.0 303.3 255.3 232.5

Overall 284.1 221.8 245.6 248.3 205.5 276.9 285.9 249.6 326.3 297.6

Significance Tests

Interaction ‐ 

ppm by week 0.000

ppm 0.000

Week 0.000

F1 Generation Paired‐Gender Food Consumption

Estimated Marginal Means

Factor P‐value ppm Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Overall

0 112.4 105.3 137.4 155.6 136.9 150.5 133.0

0.5 104.4 116.2 133.8 149.6 132.6 147.5 130.7

5 105.3 108.1 148.1 159.3 141.2 156.9 136.5

10 108.0 101.6 139.3 154.4 146.5 181.6 138.7

Overall 107.5 107.8 139.6 155.0 139.3 159.1

F2 Generation Female Food Consumption

Estimated Marginal Means

Week 0.000

Significance Tests

Interaction ‐ 

ppm by week

0.004

ppm 0.054
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

12

 Cloacal Gland Area. Cloacal gland size was only affected by trenbolone in the F1 generation 

(Tables 22-24). Cloacal foam glands in F1 males from the 0.5 ppm treatment level were smaller 

than those from the 5 and 20 ppm groups. 

Table 22. Mean cloacal gland area (mm) from F0 generation male Japanese quail 

(Coturnix japonica) exposed to trenbolone. 

Table 23. Mean cloacal gland area (mm) from F1 generation male Japanese quail 

(Coturnix japonica) exposed to trenbolone. 

Table 24. Mean cloacal gland area (mm) from F2 generation male Japanese quail 

(Coturnix japonica) exposed to trenbolone. 

Factor P‐value ppm Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Overall

0 92.0 105.4 105.2 111.5 108.6 117.5 106.7

0.5 92.7 97.8 107.4 116.4 109.5 117.0 106.8

5 99.4 103.4 114.8 116.4 113.4 118.2 110.9

10 98.7 99.7 113.8 112.3 116.0 123.7 110.7

Overall 95.7 101.5 110.3 114.1 111.9 119.1

ppm 0.040

Week 0.000

Estimated Marginal Means

Interaction ‐ 

ppm by week

0.669

Significance Tests

F2 Generation Male Food Consumption

Factor P‐value ppm Week 6 Week 7 Week 8 Week 9 Overall

0 136.5 157.5 230.4 300.2 206.1

0.5 146.7 178.6 243.2 292.5 215.3

5 153.0 175.7 248.2 315.1 222.9

10 151.2 228.3 258.9 300.9 234.8

20 124.2 212.4 255.3 302.3 223.6

Overall 142.3 190.5 247.2 302.2

*  95% confi dence limi ts reveal each week i s signi ficantly di fferent from the remaining three weeks.

Interaction ‐          

ppm by Week

ppm

Week

0.000°

0.197

0.000*

F0Generation Cloaca

GLM Significance Tests

° ppm level 10 and level 20 increa se cg a rea signi ficantly from week 6 to week 7, while the two low ppm level s and

control group signi ficantly increa se cg a rea from week 7 to week 8.

GLM Estimated Marginal Means

Factor P‐value ppm Week 5 Week 6 Week 7 Week 8 Overall

0 71.6 97.5 168.6 218.1 138.9

0.5 60.7 86.3 101.9 149.7 99.6

5 85.3 114.7 160.9 208.8 142.4

10 86.9 127.2 147.2 186.9 137.1

20 110.0 129.3 141.7 234.6 153.9

Overall 82.9 110.9 144 199.6

* Using 95% confidence limi ts, each week i s s igni ficantly di fferent from the remaining three weeks.

GLM Significance Tests GLM Estimated Marginal Means

° Res ul ts from Tukey Pos t‐hoc tes t indica te tha t ppm level .5 i s signi ficantly di fferent from ppm level 5 and ppm level

20.

F1Generation Cloaca

Week 0.000*

Interaction ‐          

ppm Level by Week

0.289

ppm 0.006°
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

13

 Onset to Puberty. Male onset to puberty was unaffected by exposure to trenbolone in all three 

generations (Tables 25-27). Reproductive maturation in F0 generation females was delayed in the 

0.5 and 20 ppm dose groups compared to the 5 ppm dose level. Female onset to puberty was also 

delayed in F1 generation females from the highest dose group relative to others. Female 

reproductive maturation was unaffected by trenbolone exposure in the F2 generation. 

Table 25. Mean onset to reproductive maturation in male and females Japanese 

quail (Coturnix japonica) of the F0 generation that were exposed to trenbolone. 

Table 26. Mean onset to reproductive maturation in male and females Japanese 

quail (Coturnix japonica) of the F1 generation that were exposed to trenbolone.

Factor P‐value ppm Week 4 Week 5 Week 6 Week 7 Overall

0 70.4 106.7 180.4 219.8 144.3

0.5 58.9 107.5 166.2 232.1 141.2

5 73.0 121.1 198.4 267.4 164.9

10 75.3 117.6 186.8 266.1 161.5

Overall 69.4 113.2 182.9 246.3

* Using 95% confidence limi ts, ea ch week i s signi fica ntly di ffe rent from the remai ning three weeks.

Interaction ‐          

ppm by Week

0.369

ppm 0.277

Week 0.000*

F2 Generation Cloaca

GLM Significance Tests GLM Estimated Marginal Means

ppm Average Std. Error Average Std. Error

0 61.4 2.14 45.6 0.70

0.5 71.6 4.96 46.1 1.00

5 59.4 1.71 45.5 0.77

10 72.8 6.14 46.2 0.86

20 73.4 5.60 47.8 0.68

0P‐value calculated from Kruskal‐Wallis test ‐ 

homogeneity of variance assumption violated.

*Post‐hoc test indicate that ppm level 5 has significantly

shorter onset to puberty compared to level 20 and .5.

F0Generation Onset to Puberty (days)

Females Males

P‐value = .036*0 P‐value = .256
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

14

Table 27. Mean onset to reproductive maturation in male and females Japanese 

quail (Coturnix japonica) of the F2 generation that were exposed to trenbolone. 

 Copulatory Behavior. For the F0 generation, males from the control group displayed a higher 

number of mount attempts than males from the 5 and 20 ppm groups on the second and third days 

of the behavioral trial (Table 28). Control males also had a higher number of attempts than the 10 

ppm birds on day two. Time to initiate a mount attempt was delayed in the 20 ppm dose group on 

the second and third days of the test compared to controls (Table 29). No differences in number of 

successful copulations among dose groups were observed in the F0 generation (Table 30). Time 

until first successful copulation was reduced by trenbolone exposure in the 0.5, 10, and 20 ppm 

dose groups for all three days (Table 31). 

Table 28. Mean number of mount attempts initiated within a 3 minute time period 

per trial by male Japanese quail (Coturnix japonica) from the F0 generation that 

were exposed to trenbolone. 

ppm Average Std. Error Average Std. Error

0 67.7 4.02 49.1 6.86

0.5 75.6 6.71 58.3 6.65

5 75.5 3.39 39.8 1.50

10 72.0 5.81 43.9 8.80

20 113.9 12.76 42.9 7.17

*Tukey post‐hoc test indicate that ppm 20 has

signi ficantly longer onset to puberty compared to all

other ppm levels.

F1Generation Onset to Puberty (days)

Females Males

P‐value = .000* P‐value = .283

ppm Average Std. Error Average Std. Error

0 64.9 2.10 51.1 2.52

0.5 62.0 2.14 51.0 2.86

5 60.8 1.19 48.3 1.11

10 65.6 1.81 48.5 2.08

F2Generation Onset to Puberty (days)

Females Males

P‐value = .202 P‐value = .700
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

15

Table 29. Mean amount of time (s) from start of test until first mount attempt per 

trial by male Japanese quail (Coturnix japonica) from the F0 generation exposed 

to trenbolone. 

Table 30. Mean number of successful copulations within a 3 minute period per 

trial completed by Japanese quail (Coturnix japonica) from the F0 generation that 

were exposed to trenbolone. 

Table 31. Mean amount of time (s) from start of test until first successful 

copulation per trial by male Japanese quail (Coturnix japonica) from the F0 

generation exposed to trenbolone. 

Dose

Average number

of Mounts

95% β Confidence

Interval* P‐value Average number

of Mounts

95% β Confidence

Interval* P‐value Average number

of Mounts

95% β Confidence

Interval* P‐value

0 3.50 3.17 3.08

0.5 2.75 .50‐1.24 0.30 1.92 .361‐1.016 0.06 2.08 .41‐1.12 0.13

5 2.08 .36‐.98 0.04 1.58 .288‐.867 0.01 1.67 .31‐.93 0.03

10 2.08 .36‐.98 0.04 1.50 .270‐.830 0.01 2.08 .41‐1.12 0.13

20 1.83 .32‐.88 0.01 1.08 .182‐.642 0.00 0.75 .12‐.50 0.00

F0 Generation ‐ Number of Mounts

Day 1

Omnibus Likelihood ratio p‐value = .071

Day 2

Omnibus Likelihood ratio p‐value = .004

Day 3

Omnibus Likelihood ratio p‐value = .001

* Confidence Intervals reflect comparison to control group (ppm = 0).  Ppm levels with a confidence interval not including 1.00 is considered to be significantly

different from the control group.

Dose

Average time

to first mount

Standard

Error

Mounted

pairs

Average time

to first mount

Standard

Error

Mounted

pairs

Average time

to first mount

Standard

Error

Mounted

pairs

0 11.6 4.69 100% 2.8 0.56 100% 2.4 0.50 100%

0.5 14.6 4.73 100% 15.9 13.38 100% 23.0 14.45 92%

5 33.1 14.98 100% 17.4 14.18 92% 18.3 14.15 92%

10 47.3 18.18 83% 28.4 14.07 100% 43.5 18.39 83%

20 71.4 22.72 67% 77.6 25.00 58% 77.7 25.03 58%

F0 Generation ‐ Time to first Mount

Day 1 Day 2 Day 3

Log rank p‐value = .066 Log rank p‐value = .013 Log rank p‐value = .031

Dose

Average number

of Copulations

95% β Confidence

Interval* P‐value Average number

of Copulations

95% β Confidence

Interval* P‐value Average number

of Copulations

95% β Confidence

Interval* P‐value

0 1.25 1.58 1.42

0.5 0.58 .19‐1.15 0.10 0.67 .18‐.96 0.04 0.58 .17‐.99 0.05

5 0.75 .26‐1.37 0.23 1.17 .37‐1.47 0.39 0.92 .30‐1.38 0.26

10 0.50 .16‐1.03 0.06 0.33 .07‐.62 0.01 0.83 .27‐1.29 0.18

20 0.33 .09‐.80 0.02 0.67 .18‐.96 0.04 0.42 .11‐.80 0.02

F0 Generation ‐ Number of Copulations

Day 1 Day 2 Day 3

Omnibus Likelihood ratio p‐value = .088 Omnibus Likelihood ratio p‐value = .011 Omnibus Likelihood ratio p‐value = .084

* Confidence Intervals reflect comparison to control group (ppm = 0).  Ppm levels with a confidence interval not including 1.00 is considered to be significantly

different from the control group.
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

16

Control males from the F1 generation had a higher average number of mount attempts than males from 

the 5 and 20 ppm groups for all three days of the behavioral test (Table 32). Males from the 5 and 20 

ppm dose groups had delayed onset to mounting behavior compared to controls over all three days, and 

onset was also delayed in the 0.5 ppm dose group relative to controls on the first and second days (Table 

33). F1 generation control males had the greatest number of successful copulations and males from the 

20 ppm dose group had the lowest number of successes in all three trial days (Table 34). All males from 

the 20 ppm group failed to successfully copulate on the first day of the test. On the second and third 

days, 20 ppm dose group males had statistically fewer successes than control males. Onset to first 

successful copulation was only changed in the F1 generation on the third day of behavioral testing (Table 

35). Males from the highest treatment level took longer to achieve a successful copulation than control 

males. 

Table 32. Mean number of mount attempts initiated within a 3 minute time period 

per trial by male Japanese quail (Coturnix japonica) from the F1 generation that 

were exposed to trenbolone. 

Table 33. Mean amount of time (s) from start of test until first mount attempt per 

trial by male Japanese quail (Coturnix japonica) from the F1 generation exposed 

to trenbolone. 

Dose

Average time to

first copulation

Standard

Error

Copulated

pairs

Average time to

first copulation

Standard

Error

Copulated

pairs

Average time to

first copulation

Standard

Error

Copulated

pairs

0 39.5 13.44 92% 38.8 18.36 83% 41.2 18.16 83%

0.5 111.6 20.91 50% 93.9 24.87 50% 109.8 24.02 42%

5 94.2 20.72 67% 40.7 18.24 83% 53.6 21.18 75%

10 108.8 21.15 50% 123.3 23.15 33% 99.6 23.38 50%

20 127.7 21.51 33% 110.7 23.76 42% 127.7 21.72 33%

F0Generation ‐ Time to first Copulation

Day 1 Day 2 Day 3

Log rank p‐value = .009 Log rank p‐value = .031 Log rank p‐value = .037

Dose

Average number

of Mounts

95% β Confidence

Interval* P‐value Average number

of Mounts

95% β Confidence

Interval* P‐value Average number

of Mounts

95% β Confidence

Interval* P‐value

0 3.25 2.85 3.60

0.5 2.00 .42‐.91 0.02 1.75 .40‐.94 0.02 2.80 .55‐1.10 0.16

5 1.60 .32‐.75 0.00 1.45 .33‐.80 0.00 2.20 .42‐.89 0.01

10 1.69 .32‐.84 0.02 2.62 .60‐1.40 0.69 2.61 .48‐1.09 0.13

20 1.00 .17‐.56 0.00 1.69 .36‐.97 0.04 1.38 .23‐.65 0.00

Day 2 Day 3

Omnibus Likelihood ratio p‐value = .000 Omnibus Likelihood ratio p‐value = .011 Omnibus Likelihood ratio p‐value = .002

* Confidence Intervals reflect comparison to control group (ppm = 0).  Ppm levels with a confidence interval not including 1.00 is considered to be significantly

different from the control group.

F1 Generation ‐ Number of Mounts

Day 1
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

17

Table 34. Mean number of successful copulations within a 3 minute period per 

trial completed by Japanese quail (Coturnix japonica) from the F1 generation that 

were exposed to trenbolone. 

Table 35. Mean amount of time (s) from start of test until first successful 

copulation per trial by male Japanese quail (Coturnix japonica) from the F1 

generation exposed to trenbolone. 

Male copulatory behavior in the F2 generation remained unchanged by in ovo exposure to trenbolone in 

the F2 generation (Tables 36-39). 

Table 36. Mean number of mount attempts initiated within a 3 minute time period 

per trial by male Japanese quail (Coturnix japonica) from the F2 generation that 

were exposed to trenbolone. 

Dose

Average time

to first mount

Standard

Error

Mounted

pairs

Average time

to first mount

Standard

Error

Mounted

pairs

Average time

to first mount

Standard

Error

Mounted

pairs

0 21.4 4.82 100% 6.6 2.34 100% 7.2 1.78 100%

0.5 68.8 14.80 80% 49.9 15.89 80% 29.8 11.63 90%

5 70.8 16.32 70% 50.9 16.75 75% 45.6 15.77 80%

10 73.4 20.11 69% 44.2 19.07 85% 26.2 13.40 92%

20 116.2 21.56 46% 97.7 22.83 54% 113.2 23.43 39%

Day 1 Day 2 Day 3

Log rank p‐value = .001 Log rank p‐value = .002 Log rank p‐value = .002

F1 Generation ‐ Time to first Mount

Dose

Average number

of Copulations

95% β Confidence

Interval* P‐value Average number

of Copulations

95% β Confidence

Interval* P‐value Average number

of Copulations

95% β Confidence

Interval* P‐value

0 0.90 1.05 1.10

0.5 0.45 .23‐1.11 0.09 0.45 .20‐.94 0.03 0.60 .27‐1.10 0.09

5 0.35 .16‐.93 0.03 0.80 .40‐1.46 0.41 1.00 .50‐1.67 0.76

10 0.38 .16‐1.15 0.09 0.62 .26‐1.32 0.20 0.62 .25‐1.26 0.16

20 0.23 .07‐.74 0.01 0.08 .01‐.52 0.01

* Confidence Intervals reflect comparison to control group (ppm = 0).  Ppm levels with a confidence interval not including 1.00 is considered to be significantly

different from the control group.

F1 Generation ‐ Number of Copulations

Day 1 Day 2 Day 3

Omnibus Likelihood ratio p‐value = .090 Omnibus Likelihood ratio p‐value = .028 Omnibus Likelihood ratio p‐value = .001

All pairs failed to Copulate

Dose

Average time to

first copulation

Standard

Error

Copulated

pairs

Average time to

first copulation

Standard

Error

Copulated

pairs

Average time to

first copulation

Standard

Error

Copulated

pairs

0 86.8 16.57 65% 74.5 17.44 65% 65.6 16.98 70%

0.5 119.0 16.87 40% 113.7 18.24 40% 97.4 18.51 50%

5 131.5 14.98 35% 92.5 17.87 55% 84.5 18.51 60%

10 120.5 21.03 39% 117.0 22.14 39% 114.8 22.91 39%

20 162.2 All pairs failed to Copulate 12.52 23% 166.9 12.56 7%

F1Generation ‐ Time to first Copulation

Day 1 Day 2 Day 3

Log rank p‐value = .170 Log rank p‐value = .141 Log rank p‐value = .016
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

18

Table 37. Mean amount of time (s) from start of test until first mount attempt per 

trial by male Japanese quail (Coturnix japonica) from the F2 generation exposed 

to trenbolone. 

Table 38. Mean number of successful copulations within a 3 minute period per 

trial completed by Japanese quail (Coturnix japonica) from the F2 generation that 

were exposed to trenbolone. 

Table 39. Mean amount of time (s) from start of test until first successful 

copulation per trial by male Japanese quail (Coturnix japonica) from the F2 

generation exposed to trenbolone. 

Dose

Average number

of Mounts

95% β Confidence

Interval* P‐value Average number

of Mounts

95% β Confidence

Interval* P‐value Average number

of Mounts

95% β Confidence

Interval* P‐value

0 2.85 3.25 3.05

0.5 3.55 .88‐1.76 0.22 3.00 .65‐1.31 0.66 2.70 .61‐1.28 0.51

5 2.45 .59‐1.26 0.44 2.45 .52‐1.09 0.14 2.50 .56‐1.19 0.30

10 2.94 .72‐1.49 0.95 3.10 .67‐1.35 0.79 2.95 .68‐1.38 0.86

* Confidence Intervals reflect comparison to control group (ppm = 0).  Ppm levels with a confidence interval not including 1.00 is considered to be significantly

different from the control group.

F2 Generation ‐ Number of Mounts

Day 1 Day 2 Day 3

Omnibus Likelihood ratio p‐value = .245 Omnibus Likelihood ratio p‐value = .465 Omnibus Likelihood ratio p‐value = .722

Dose

Average time

to first mount

Standard

Error

Mounted

pairs

Average time

to first mount

Standard

Error

Mounted

pairs

Average time

to first mount

Standard

Error

Mounted

pairs

0 40.4 12.05 90% 20.3 11.18 95% 16.7 8.80 95%

0.5 42.4 13.11 90% 15.0 8.53 95% 13.8 8.60 95%

5 29.1 7.30 100% 16.9 9.11 95% 7.1 3.09 100%

10 28.7 11.51 90% 12.3 8.63 95% 2.9 0.71 100%

F2 Generation ‐ Time to first Mount

Day 1 Day 2 Day 3

Log rank p‐value = .858 Log rank p‐value = .558 Log rank p‐value = .149

Dose

Average number

of Copulations

95% β Confidence

Interval* P‐value Average number

of Copulations

95% β Confidence

Interval* P‐value Average number

of Copulations

95% β Confidence

Interval* P‐value

0 0.95 0.80 1.10

0.5 0.35 .16‐.876 0.02 0.65 .39‐1.69 0.58 1.05 .53‐1.74 0.88

5 0.90 .50‐1.81 0.87 1.05 .69‐2.52 0.41 1.00 .50‐1.67 0.76

10 0.85 .47‐1.72 0.74 1.15 .76‐2.72 0.27 0.86 .73‐2.23 0.40

* Confidence Intervals reflect comparison to control group (ppm = 0).  Ppm levels with a confidence interval not including 1.00 is considered to be significantly

different from the control group.

F2 Generation ‐ Number of Copulations

Day 1 Day 2 Day 3

Omnibus Likelihood ratio p‐value = .068 Omnibus Likelihood ratio p‐value = .320 Omnibus Likelihood ratio p‐value = .651

Dose

Average time to

first copulation

Standard

Error

Copulated

pairs

Average time to

first copulation

Standard

Error

Copulated

pairs

Average time to

first copulation

Standard

Error

Copulated

pairs

0 93.6 17.28 60% 78.6 17.15 65% 80.0 17.45 65%

0.5 137.5 14.46 35% 95.9 18.32 55% 62.8 17.17 75%

5 93.9 16.65 65% 53.8 16.39 75% 69.9 18.12 65%

10 83.5 16.41 65% 58.8 15.95 75% 44.9 15.02 85%

F2Generation ‐ Time to first Copulation

Log rank p‐value = .160 Log rank p‐value = .308 Log rank p‐value = .372

Day 1 Day 2 Day 3
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

19

 Organ Weights. No differences in body weight on day of necropsy and organ weights were 

observed in males and females from the F0 generation (Tables 40-41). In the F1 generation, body 

weight was only alteres in females (Tables 42-43). Females from the 0.5 and 20 ppm dose levels 

weighed less than those from the 10 ppm group. Female kidney weight was also significantly 

reduced compared to the control and 0.5 ppm dose groups. In F1 generation males, only bursa 

weight was affected by trenbolone; bursas of Fabricius from 20 ppm treatment level males were 

smaller than controls. In the F2 generation, spleen weight was the only organ wieght altered by 

trenbolone expsoure (Tables 44-45). Female and male spleens were larger in the 10 ppm group 

compared to all other treatment levels. 

Table 40. Mean body weight at day of necropsy and mean organ weights (g) with 

standard error of deviation from adult female Japanese quail (Coturnix japonica) 

of the F0 generation that were exposed to trenbolone (ppm). 

Table 41. Mean body weight at day of necropsy and mean organ weights (g) with 

standard error of deviation from adult male Japanese quail (Coturnix japonica) of 

the F0 generation that were exposed to trenbolone (ppm). 

Table 42. Mean body weight at day of necropsy and mean organ weights (g) with 

standard error of deviation from adult female Japanese quail (Coturnix japonica) 

of the F1 generation that were exposed to trenbolone (ppm). 

Dose Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error

0 141.5 3.23 0.764 0.012 1.27 0.041 1.40 0.062 4.24 0.205 4.60 0.469 0.028 0.0039 6.29 0.365 0.0079 0.0011 0.024 0.0045

0.5 136.2 4.20 0.754 0.017 1.19 0.047 1.27 0.066 3.51 0.282 3.96 0.899 0.026 0.0022 4.90 0.910 0.0085 0.0009 0.022 0.0042

5 139.9 2.94 0.746 0.014 1.14 0.040 1.37 0.048 4.16 0.188 4.27 0.467 0.034 0.0039 6.47 0.212 0.0053 0.0011 0.016 0.0029

10 138.8 3.58 0.736 0.014 1.20 0.050 1.29 0.065 3.64 0.301 3.07 0.376 0.030 0.0039 6.12 0.338 0.0057 0.0012 0.017 0.0047

20 134.9 3.03 0.748 0.011 1.22 0.048 1.31 0.045 3.65 0.165 3.30 0.361 0.031 0.0017 5.76 0.306 0.0069 0.0009 0.019 0.0043

* P‐value calculated from Kruskal‐wallis test because homogeneity of variance assumption violated in parametric ANOVA test.

Thyroid

P‐value = .648* P‐value = .144

Bursa

P‐value = .615

Ovaries

P‐value = .139*

Spleen

P‐value = .492

Oviduct

F0 Generation Female Organ Weights

P‐value = .673 P‐value = .705 P‐value = .346 P‐value = .457 P‐value = .102

Body Weight Brain Heart Kidneys Liver

Dose Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error

0 116.2 1.97 0.759 0.012 1.20 0.042 0.810 0.0228 0.204 0.014 2.05 0.076 0.0197 0.0021 3.46 0.232 0.0095 0.0018 0.038 0.0072 259.1 8.64

0.5 113.7 1.34 0.763 0.012 1.22 0.038 0.825 0.0297 0.214 0.012 1.99 0.104 0.0204 0.0019 3.38 0.173 0.0075 0.0018 0.035 0.0053 255.8 7.19

5 114.4 2.35 0.774 0.019 1.20 0.042 0.804 0.0261 0.185 0.018 2.07 0.123 0.0206 0.0016 3.52 0.228 0.0082 0.0019 0.033 0.0042 255.6 11.96

10 113.8 1.78 0.746 0.012 1.26 0.024 0.803 0.0288 0.176 0.013 1.96 0.091 0.0210 0.0013 3.25 0.128 0.0079 0.0021 0.031 0.0030 260.2 12.92

20 114.1 1.26 0.753 0.015 1.13 0.047 0.806 0.0152 0.207 0.011 2.13 0.108 0.0212 0.0024 3.23 0.174 0.0052 0.0010 0.034 0.0029 276.4 10.09

* P‐value calculated from Kruskal‐wallis test because homogeneity of variance assumption violated in parametric ANOVA test.

CG area

P‐value = .605

F0 Generation Male Organ Weights

P‐value = .855 P‐value = .698 P‐value = .270 P‐value = .971 P‐value = .253 P‐value = .773 P‐value = .983 P‐value = .779 P‐value = .536 P‐value = .961*

Body Weight Brain Heart Kidneys Epididymides Liver Spleen Testes Thyroid Bursa

Dose Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error

0 138.1 2.47 0.777 0.011 1.15 0.041 1.40 0.061 4.13 0.230 4.66 0.417 0.036 0.0065 5.72 0.398 0.0094 0.0014 0.022 0.0028

0.5 143.1 2.40 0.798 0.025 1.28 0.036 1.39 0.037 4.11 0.217 4.50 0.279 0.035 0.0029 5.89 0.175 0.0098 0.0017 0.021 0.0036

5 138.5 3.32 0.798 0.014 1.20 0.050 1.28 0.047 4.05 0.331 4.04 0.578 0.035 0.0055 5.01 0.566 0.0065 0.0015 0.012 0.0017

10 143.6 2.69 0.810 0.019 1.22 0.067 1.25 0.053 3.73 0.177 4.45 0.458 0.038 0.0026 6.02 0.403 0.0083 0.0018 0.021 0.0052

20 129.3 3.69 0.800 0.016 1.18 0.046 1.05 0.101 2.82 0.352 3.72 0.759 0.031 0.0083 4.46 1.010 0.0092 0.0030 0.014 0.0069

* P‐value calculated from Kruskal‐wallis test because homogeneity of variance assumption violated in parametric ANOVA test.

° Tukey post‐hoc test indicated that dose 10 had a signi ficantly greater body weight average compared to dose 20.

† Tukey post‐hoc test indicated that dose .50 and the control group had significantly greater kidney weight averages compared to dose 20.

P‐value = .048° P‐value = .760 P‐value = .446 P‐value = .005† P‐value = .053 P‐value = .785 P‐value = .822* P‐value = .217 P‐value = .624 P‐value = .203*

F1 Generation Female Organ Weights

Body Weight Brain Heart Kidneys Liver Ovaries Spleen Oviduct Thyroid Bursa
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

20

Table 43. Mean body weight at day of necropsy and mean organ weights (g) with 

standard error of deviation from adult male Japanese quail (Coturnix japonica) of 

the F1 generation that were exposed to trenbolone (ppm). 

Table 44. Mean body weight at day of necropsy and mean organ weights (g) with 

standard error of deviation from adult female Japanese quail (Coturnix japonica) 

of the F2 generation that were exposed to trenbolone (ppm). 

Table 45. Mean body weight at day of necropsy and mean organ weights (g) with 

standard error of deviation from adult male Japanese quail (Coturnix japonica) of 

the F2 generation that were exposed to trenbolone (ppm). 

 Hormone Analysis. Trenbolone did not alter adult serum estradiol concentrations (Table 

46). Testosterone was affected in males only; serum testosterone was elevated in 10 ppm 

dose level males relative to controls. 

Table 46. Average serum estradiol and testosterone collected from F2 generation 

adult Japanese quail (Coturnix japonica). 

Table 4. Effects of Trenbolone on F2 Generation Quail Hormones

Gender Dose Group

(ppm)

Estradiol (pg/ml) Testosterone (pg/ml)

N Mean SE N Mean SE

Female

Control 4 32.91 4.01 4 683.6 252.3

0.5 5 28.66 11.34 5 475.6 293.8

5 3 32.23 5.97 3 527.0 97.1

10 3 36.49 6.71 3 987.7 463.8

Male Control 3 1.58 0.00 3 769.4 233.2

0.5 4 9.83 1.30 4 2405.8 148.5

Dose Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error

0 119.5 1.69 0.787 0.012 1.13 0.045 0.792 0.0286 0.194 0.019 1.77 0.041 0.0212 0.0015 3.65 0.174 0.0129 0.0023 0.034 0.0039 234.9 10.84

0.5 119.0 1.81 0.745 0.018 1.15 0.057 0.813 0.0260 0.164 0.015 1.95 0.055 0.0191 0.0029 3.31 0.275 0.0107 0.0016 0.025 0.0030 221.5 8.45

5 119.9 2.39 0.762 0.017 1.17 0.040 0.825 0.0342 0.190 0.019 1.96 0.094 0.0202 0.0026 3.55 0.166 0.0112 0.0011 0.025 0.0039 226.0 8.23

10 119.2 1.97 0.803 0.020 1.18 0.058 0.881 0.0348 0.151 0.011 1.94 0.097 0.0224 0.0040 3.63 0.211 0.0077 0.0018 0.030 0.0033 238.8 8.87

20 117.7 2.24 0.781 0.012 1.09 0.047 0.759 0.0238 0.154 0.014 1.82 0.081 0.0221 0.0034 2.97 0.134 0.0068 0.0011 0.018 0.0024 202.6 11.15

° Tukey post‐hoc test indicated that the control group had a significantly greater bursa weight average compared to dose 20.

F1 Generation Male Organ Weights

Body Weight Brain Heart Kidneys Epididymides Liver Spleen Testes Thyroid Bursa CG area

P‐value = .958 P‐value = .124 P‐value = .769 P‐value = .130 P‐value = .251 P‐value = .252 P‐value = .920 P‐value = .119 P‐value = .090 P‐value = .028° P‐value = .110

Dose Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error

0 134.5 3.13 0.768 0.012 1.18 0.057 1.26 0.043 3.80 0.223 4.02 0.452 0.036 0.0034 6.11 0.678 0.012 0.0020 0.041 0.0063

0.5 131.4 2.71 0.759 0.015 1.21 0.068 1.27 0.046 4.20 0.300 3.51 0.583 0.039 0.0042 6.60 0.533 0.010 0.0014 0.044 0.0073

5 134.7 1.88 0.744 0.013 1.25 0.067 1.28 0.029 4.30 0.158 3.23 0.474 0.039 0.0029 5.62 0.520 0.009 0.0013 0.031 0.0059

10 137.8 2.20 0.775 0.014 1.29 0.056 1.37 0.047 4.44 0.173 3.45 0.484 0.053 0.0057 5.91 0.498 0.011 0.0024 0.048 0.0042

° Tukey post‐hoc test indicated that dose 10 had a signi ficantly greater spleen weight average compared to the control group.

F2 Generation Female Organ Weights

Body Weight Brain Heart Kidneys Liver Ovaries Spleen Oviduct Thyroid Bursa

P‐value = .374 P‐value = .390 P‐value = .646 P‐value = .231 P‐value = .189 P‐value = .720 P‐value = .033° P‐value = .674 P‐value = .759 P‐value = .212

Dose Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error

0 111.0 1.29 0.722 0.022 1.08 0.038 0.84 0.028 0.205 0.011 1.97 0.042 0.0224 0.0020 3.50 0.119 0.0093 0.0015 0.053 0.0067 244.1 11.98

0.5 108.6 1.62 0.733 0.029 1.14 0.045 0.77 0.034 0.231 0.017 1.85 0.040 0.0226 0.0013 3.66 0.184 0.0077 0.0011 0.054 0.0050 252.0 10.98

5 113.3 1.69 0.737 0.031 1.19 0.043 0.78 0.030 0.229 0.016 1.94 0.049 0.0228 0.0020 3.49 0.137 0.0108 0.0015 0.047 0.0071 248.7 12.91

10 112.9 0.89 0.751 0.023 1.18 0.040 0.83 0.024 0.214 0.012 2.05 0.090 0.0311 0.0025 3.58 0.118 0.0081 0.0015 0.051 0.0062 270.0 9.47

P‐value = .402

F2 Generation Male Organ Weights

° Tukey post‐hoc test indicated that dose 10 had a significantly greater spleen weight average compared to the other three dose groups

P‐value = .085 P‐value = .893 P‐value = .267 P‐value = .316 P‐value = .508 P‐value = .135 P‐value = .007° P‐value = .800 P‐value = .425 P‐value = .879

Body Weight Brain Heart Kidneys Epididymides Liver Spleen Testes Thyroid Bursa CG area
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

21

5 3 5.53 2.01 3 2090.4 368.8

10 3 7.85 0.90 3 3962.8 860.4

P‐Value

Dose = .922

Gender = .000                       Dose * Gender = .011

(Females > Males)

8 Discussion 

Trenbolone is a synthetic androgen that is used to increase muscle mass in cattle and to maintain 

muscle mass of cattle during shipment to slaughter. The active metabolite, trenbolone 17β (TBOH17β), then enters the environment though excreta where it can accumulate in soils and in higher 

trophic levels of food webs. TBOH-17β has been shown to have an affinity for the recombinant 

human androgen receptor similar to that of dihydrotestosterone (Bauer et al., 2000). Although few 

studies have been done to test trenbolone’s effects on wildlife, it has been shown to be a relatively 

potent androgenic endocrine disrupting chemical that can cause developmental and functional 

changes in birds (reviewed in Quinn et al., 2007). 

In a feed exposure study similar to the current study, Henry et al. (2012) observed no differences in 

body and testes weight, testes histology, plasma testosterone concentrations, or cloacal gland size 

and weight at the maximum dose of 20 ppm trenbolone acetate. Age of onset of male reproductive 

maturation was lowest in males from the 20 ppm treatment level. The same dose reduced overall 

egg production and plasma testosterone in females. 

F0 and F1 generation birds from the 20 ppm dose group had consistently lower body weights 

throughout the course of the study. Birds from the F2 generation’s high dose (10 ppm) tended to 

have greater body weights than those from the other groups. The decreased weight in the F0 and 

F1 generations might have been due to overall toxic effects, while the 10 ppm dose might have 

acted similar to doses used in cattle as growth promoters. Changes in feed consumption were 

inconsistent and difficult to interpret, especially once the birds were paired. Significant spillage of 

feed was observed in all birds from all dose groups and generations, compromising the accuracy of 

this measurement. Feed consumption may be used to provide a very rough estimate of exposure; 

however this measure is not used in the current assessment of the usefulness of the F2 generation. 

What makes this avian two-generation toxicity test different from others done at USAPHC is the 

elimination of the highest dose group in the F2 generation due to poor egg production by the F1 

generation 20 ppm dose group females. Removal of the 20 ppm dose group from the F2 

generation is assuredly one of the main causes of the discrepancy between the number of 

observed trenbolone induced changes in the F1 and F2 generations (summarized in Tables 56-58). 

Additionally, previous avian toxicology studies have shown that some endocrine active systems that 

suffer effects from embryonic exposure to endocrine disrupting chemicals may be resilient enough 

to overcome these effects in later development provided that exposure does not continue post 

hatch (Quinn et al., 2006; Quinn et al., 2007a). Specifically, the neuroendocrine system has been 

suggested to be one of the most sensitive areas to be affected by endocrine disrupting chemicals 

during development. Japanese quail that were exposed to trenbolone acetate or 

dichlorodiphenyldichloroethylene (p,p’-DDE), an anti-androgenic endocrine disrupting chemical, 

only in ovo were shown to have normal gonadal structure and function as adults; however male 

reproductive behaviors remained inhibited (Quinn et al., 2006 and 2007b), suggesting that the 
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

22

neuroendocrine system may be less resilient to embryonic contaminant exposure. No copulatory 

behavior effects were observed in F2 generation males. Of course, the lack of a 20 ppm dose 

group is the most likely reason for the absence of F2 generation behavioral effects. 

Table 47. Comparison of adult male Japanese quail (Coturnix japonica) measures 

between the F1 (exposed to trenbolone in ovo and through feed) and F2 (in ovo

exposure only) generations. ND = no difference. 

Male Specific Measures F1 F2

male onset to maturity ND ND

sperm concentration ND 5 ppm lower than 20 ppm

sperm motility ND ND

% progressive sperm 20 ppm higher than 5 ppm ND

testes weight ND ND

clocal gland area 5 & 20 ppm lower than 0.5 ppm ND

Table 48. Comparison of adult female Japanese quail (Coturnix japonica) 

measures between the F1 (exposed to trenbolone in ovo and through feed) and 

F2 (in ovo exposure only) generations. ND = no difference. 

Female Specific Measures F1 F2

female onset to maturity 20 ppm delayed ND

egg production 20 ppm reduced from week 7‐10 ND

eggshell thickness ND ND

eggshell strength ND ND

ovary weight ND ND

dominant follicle count ND ND

Table 49. Comparison of results of F1 and F2 generation male copulatory 

behavior tests in Japanese quail (Coturnix japonica) exposed to trenbolone. ND = 

no difference. 

Copulatory Behavior Measure F1 F2

time until 1st mount attempt 5 & 20 ppm delayed ND

time until 1st copulatory success 20 ppm lower than controls ND

# attempts / 3 min trial interval 5 & 20 ppm lower than controls ND

# successful copulations 20 ppm lowest ND

The absence of effects from a potent androgen active chemical in the F2 generation suggests that 

a full two-generation test may not be appropriate for every candidate endocrine disrupting chemical. 
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

23

Janer et al., (2007) analyzed the added value of F2 generations in rat toxicity tests by evaluating 

176 multigenerational studies. Although F1 rats often displayed increased sensitivity to several 

substances over the F0 generation, only 3 of the 176 studies observed reproductive effects in the 

F2 generation that were not present in the F1. It should be noted that in these rare examples, the 

F2 effects were not severe enough to cause changes in studies’ critical effects or overall No 

Observed Adverse Effect Levels (NOAELs). Since results from the F2 generations did not have 

any effects on subsequent risk assessments or classifications that were determined from F1 

generation effects, a robust one-generation rat toxicity study seemed more favorable, with the 

significant reduction in animal numbers and resources such as time and money, than currently 

established two-generation tests. Care in the decision to use a one- or extended one-generation 

test over a two-generation test based on retrospective analysis of existing data should be taken as 

these results may be biased by what chemical specific data is currently available. Continued 

assessment of avian specific multi-generation studies is needed to be able to adequately assess 

the added value of F2 generations in avian test paradigms. 

9 Conclusion 

The information gained from the F2 generation does not appear to improve understanding of the 

toxic effects of trenbolone in Japanese quail at the dose levels used in this study. In this case, a 

one-generation or extended one-generation avian toxicity test would likely have been sufficient for 

multi-generational assessment of trenbolone’s endocrine active potential. Unlike a prior avian twogeneration toxicity test done at USAPHC with the relatively weak endocrine disrupting chemical 

endosulfan, trenbolone has been demonstrated to be a potent androgen active chemical that can 

alter reproductive development and function in this species It is likely that the results of the 

Endocrine Disrupting Screening Program‘s Tier 1 toxicity tests and the results from the initial F0 

and F1 generations of a two-generation test could be useful in determining if an additional F2 

generation is warranted. 

10 Point of Contact 

If you have questions concerning this study, contact Dr. Michael J. Quinn, Health Effects Research 

Program at 410-436-1064. 

 MICHAEL J. QUINN 

 Biologist 

 Health Effects Research Program 

APPROVED: 

MARK S. JOHNSON 
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

24

Program Manager 

Health Effects Research Program 
Toxicology Study No. 87-XE-0FV5-12, Protocol No. 0FV5-96-12-05-01, June-August 2012 

25 

APPENDIX A 

REFERENCES 

Bauer, E., A. Daxenberger, T. Petri, H. Sauerwein, H. Meyer. 2000. Characterisation of 

the affinity of different anabolics and synthetic hormones to the human androgen 

receptor, human sex hormone binding globulin and to the bovine progestin receptor. 

APPMIS 108, 838-846. 

Dunachie, J.F. and W.W. Fletcher. 1966. Effect of some insecticides on the hatching 

 rate of hens’ eggs. Nature, 212: 1062. 

Dunachie and W.W. Fletcher. 1969. An investigation of the toxicity of insecticides to 

 birds’ eggs using the egg injection technique. Annu. Appl. Biol., 64: 409-423. 

EPA (U.S. Environmental Protection Agency). 1996. Avian reproduction test. OPPTS 

 850.2300 “Public Draft” Ecological Effects Test Guidelines. EPA 712-C-96-141. U.S. 

 Environmental Protection Agency, Washington, D.C. 

FWS (U.S. Fish and Wildlife Service). 1975. Lethal Dietary Toxicities of Environmental 

Pollutants to Birds. U.S. Fish and Wildlife Service, Bureau of Sports Fisheries and 

Wildlife. Special Scientific Report - Wildlife No. 191. Washington, DC, U.S. 

Garg, U.K., A.K. Pal, G.J. Jha, S.B. Jadhao. 2004a. Haemato-biochemical and immunopathophysiological effects of chronic toxicity with synthetic pyrethroid, 

organophosphate and chlorinated pesticides in broiler chicks. Int. 

Immunopharmacol., 4: 1709-22. 

Garg, U.K., A.K. Pal, G.J. Jha, S.B. Jadhao. 2004b. Pathophysiological effects of 

chronic toxicity with synthetic pyrethroid, organophosphate and chlorinated 

pesticides on bone health of broiler chicks. Toxicol. Pathol., 32: 364-9. 

Henry, P.F.P., V.G. Akuffo, Y. Chen, N.K. Karouna-Renier, D.T. Sprague, M.R. Bakst. 

 2012. Avian Biol. Res., 5: 61-. 

Hill, E. F. and Camardese, M. B. 1986. Lethal Dietary Toxicities of Environmental 

 Contaminants to Coturnix, Technical Report Number 2. U.S. Department of Interior, 

 Fish and Wildlife Service, Washington, DC, 6-55. 

Hudson, R.H., R.K. Tucker, M.A. Haegele. 1972. Effect of age on sensitivity: acute oral 
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

26

toxicity of 14 pesticides to mallard ducks of several ages. Toxicol. Appl. 

Pharmacolg., 22: 556-561. 

Hudson, R.H., R.K.Tucker, M.A. Haegele. 1984. Handbook of Acute Toxicity of 

Pesticides to Wildlife, Resource Publication 153. U.S. Department of Interior, Fish 

and Wildlife Service, Washington, DC, 6-54. 

Janer, G., B.C. Hakkert, W. Slob, T. Vermeire, A.H. Piersma. 2007. A retrospective 

analysis of the two-generation study: What is the added value of the second 

generation? Reproductive Toxicol., 24: 97-102. 

Lutz, H. and Y. Lutz-Ostertag. 1972. The action of different pesticides on the 

 development of bird embryos. Adv. Exp. Med. Biol., 27: 127-150. 

Matthews, J., T. Celius, R. Halgren, T. Zacharewski. 2000. Differential estrogen 

receptor binding of estrogenic substances: a species comparison. J. Steroid 

Biochem. Mol. Biol., 74: 223-234. 

Quinn, M.J., Jr., E.T. Lavoie, M.A. Ottinger. 2006. Reproductive toxicity of trenbolone 

 acetate in embryonically exposed Japanese quail. Chemosphere, 66: 1191-1196. 

Quinn, M.J., Jr., M. McKernan, E.T. Lavoie, M.A. Ottinger. 2007a. Immunotoxicity of 

 trenbolone acetate in Japanese quail. J. Toxicol. Environ. Health, 70: 1-6. 

Quinn, M.J. Jr., C.L. Summitt, M.A. Ottinger. 2007b. Consequences of in ovo exposure 

to p,p’-DDE on reproductive development and function in the Japanese quail. 

Hormones Behav., 53: 249-253. 
Toxicology Study No. 87-XE-0FV5-12, Protocol No. 0FV5-96-12-05-01, June-August 2012 

27 

APPENDIX B 

QUALITY ASSURANCE STATEMENT 

For: Toxicology Study No. 87-XE-08JG-10, Protocol No. 0FV5-96-12-05-01, entitled: 

“Assessment of 3-nitro-1,2,4-triazol-5-one (NTO) as a potential endocrine disrupting chemical in 

rats using the Hershberger and uterotrophic bioassays”, the following critical phases were 

audited by the Quality Systems Office: 

PRE IN-LIFE PHASE OF THE STUDY

Critical Phase Inspected/Audited Date Inspected 

/Audited

Date Reported to 

Management/SD

Study Protocol GLP Review 

IN-LIFE PHASE OF THE STUDY

Critical Phase Inspected/Audited Date Inspected 

/Audited

Date Reported to 

Management/SD

Test System - Facilities, Identification, 

Husbandry & Food and Water Supply

 

Compliance with the OPPTS 890.1600 Test 

Guideline

 

Euthanasia, Necropsy and Tissue Collection 

Procedures

 

Maintenance and Calibration of Equipment 

Test Article Control, Handling and 

Administration

 

Study Personnel Training Records 

Study Final Animal In-Life Endpoint Criteria 

Euthanasia, Necropsy and Tissue Collection 

Procedures

 

POST IN-LIFE PHASE OF THE STUDY

Critical Phase Inspected/Audited Date Inspected 

/Audited

Date Reported to 

Management/SD

Final Study Report Review 

Study Raw Data Review 

Note 1 All findings were made known to the Study Director and the Program Manager at the time of the 

audit/inspection. If there were no findings during the inspection, the inspection was reported to 

Management and the Study Director on the date shown in the table. 
Toxicology Study No. 87-XE-0DUZ-11, Protocol No. 08JG-80-10-03-01, June 2010-September 2011 

28

Note 2 In addition to the study specific critical phase inspections listed here, general facility and process 

based inspection not specifically related to this study are done monthly or annually in accordance with QA 

Standard Procedure. 

Note 3 This report has been audited by the Quality Assurance Unit (QSO), and is considered to be an 

accurate account of the data generated and of the procedures followed 

___________________________________ ____________________ 

Michael P. Kefauver Date 

GLP Assessor, QSO 
Toxicology Study No. 87-XE-0FV5-12, Protocol No. 0FV5-96-12-05-01, June-August 2012 

29 

APPENDIX C 

ARCHIVES AND STUDY PERSONNEL 

C-1. ARCHIVES. 

 a. All raw data, documentation, records, protocol, and a copy of the final report generated as a 

result of this study will be archived in Bldg. E-2100, USAPHC (Prov), for a minimum of five (5) years 

following submission of a final report. 

 b. Records on animal receipt, diet, and facility environmental parameters will be archived by the 

Veterinary Medical Division, Toxicology Directorate, for a minimum of five (5) years following submission 

of the final report to the Sponsor. If the report is used to support a regulatory action, it shall, along with all 

supporting data, be retained indefinitely. 

 c. Archived SOPs and maintenance and calibration logbooks may be found in Room 1026, 

Building E-2100, USAPHC (Prov), Aberdeen Proving Ground, Maryland 21010. 

 d. The present study uses USAPHC (Prov) Toxicology Study No. 87-XE-08JG-10, Protocol 

08JG-80-08-04-02 for all filings. 

C-2. PERSONNEL. The following USAPHC (Prov) personnel participated in the studies reported 

herein. 

 a. Directorate of Toxicology: Michael J. Quinn Jr., Michelle Cook, Alicia Shifflet, Allison 

Jackovitz, Terry Hanna, Craig A. McFarland, Mark S. Johnson 

 b. Quality Systems Office: Gene Sinar, Michael Kefauver 
Toxicology Study No. 87-XE-0FV5-12, Protocol No. 0FV5-96-12-05-01, June-August 2012 

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APPENDIX D 

ANIMAL USE PROTOCOL WITH MODIFICATIONS 

ANIMAL USE PROTOCOL 

TOXICOLOGY DIRECTORATE 

U.S. ARMY CENTER FOR HEALTH PROMOTION 

AND PREVENTIVE MEDICINE 

ABERDEEN PROVING GROUND, MD 21010-5403

PROTOCOL TITLE: Two-generation test for endocrine disruption and reproductive toxicity in Japanese 

quail (Corturnix japonica) using trenbolone. 

 

PROTOCOL NUMBER: 

PRINCIPAL INVESTIGATOR/STUDY DIRECTOR: 

Michael J. Quinn, Jr., Ph.D. 

Biologist 

Health Effects Research Program 

Toxicology Directorate 410-436-1064 

CO-INVESTIGATOR(S): 

Michelle Cook, M.S. (PRIMARY)

Biologist 

Health Effects Research Program 

410-436-8772 

Craig McFarland, Ph.D., D.V.M. 

Biologist 

Health Effects Research Program 

410-436-7784 

Terry Hanna 

Biologist 

Toxicity Evaluation Program 

410-436-5072 

Alicia Bonney 

Biologist 

Health Effects Research Program 

410-436-1305 

SPONSOR: 

Les Touart, Ph.D. 

Project Officer 

Office of Science Coordination and Policy 

U.S. Environmental Protection Agency 

1200 Pennsylvania Ave., NW (7203) 

Washington, DC 20460 

 Tel: 202-564-8468 
Animal Use Protocol-Two-generation test for endocrine disruption & reproductive toxicity in the Japanese quail with 

trenbolone. 

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I. NON-TECHNICAL SYNOPSIS: 

 The purpose of this study is to establish a new laboratory testing procedure that can be 

used to assess the impact of endocrine active chemicals upon avian health and reproduction. Birds will 

be exposed to trenbolone in feed. Blood will be drawn prior to euthanasia for blood cellularity and 

chemistry analyses. Tissues will be collected for histopathological evaluation. Data from a twogeneration test that this study attempts to develop are principally intended to evaluate the adverse 

consequences of exposure to a “possible” endocrine disruptor to the reproductive viability of first 

generation birds, but may also provide information to confirm a chemical as an endocrine disruptor. 

II. BACKGROUND: 

II.1. Background: The U.S. Environmental Protection Agency (EPA) is implementing an Endocrine 

Disruptor Screening Program (EDSP) comprised of a battery of Tier 1 screening assays and Tier 2 tests. 

One of the Tier 2 tests under development is a two-generation avian assay using Japanese quail 

(Coturnix japonica). This test is designed to characterize dose-response relationships and immediate and 

latent adverse consequences associated with exposure to putative endocrine modulating compounds. 

The avian two-generation assay serves to evaluate potential population effects of endocrine modulating 

compounds on myriad avian species, and represents terrestrial, oviparous wildlife species in general. 

This study will use trenbolone, a synthetic androgen, to further evaluate the proposed test method for its 

ability to detect adverse effects and characterize dose responses in a two-generation paradigm, and to 

develop and optimize a protocol for the avian two-generation assay for characterizing endocrine 

disruptors. In combination with information gained from a prior study using endosulfan as an estrogenactive endocrine disruptor and a potential future study using a thyroid-active chemical, this work will help 

to determine the value added by examining endocrine endpoints over two generations in comparison to 

tests that only include one generation, such as the Ecological Effects Guidelines for avian reproduction 

tests (EPA, 1996). 

II.2. Literature Search for Duplication: 

II.2.1. Literature Source(s) Searched: 

 A number of databases were consulted in an effort to obtain information on the toxicity of 

trenbolone in the Japanese quail as well as a determination for duplication of effort. The databases 

checked were: 

 

a. Medline 

b. ToxFile 

c. FEDRIP 

d. Biosis 

e. EMBASE 

f. CA SEARCH 

g. DTIC (Defense Technical Information Center) 

h. BRD (Biomedical Research Database) 

II.2.2. Date of Search: 29 December 2009

II.2.3. Period of Search: 1926-2009. 

II.2.4. Key Words of Search: trenbolone, parabolan, 10161-33-8, oral exposure, aerosol, inhalation, 

toxic, Japanese quail, quail, coturnix, bird. 

II.2.5. Results of Search: Few relevant studies were located using the above described search and an 

independent literature search. 
Animal Use Protocol-Two-generation test for endocrine disruption & reproductive toxicity in the Japanese quail with 

trenbolone. 

32 of 18 

1. Avian Specific. Two main types of papers specific to birds were located: general endocrine 

disruption in avian species review papers, and reports specific to trenbolone acetate exposure 

in Japanese quail. The PI is intimately aware of these since he is an author of most of them. 

The review papers have been helpful in the general design of the current study. The 

trenbolone-specific papers, however, are limited in their usefulness. The studies that these 

papers describe had all used yolk injections of trenbolone acetate; no information yet enables 

the extrapolation of how much oral maternal exposure would result in a specific amount being 

deposited into the yolk. The current proposed study will help to address these needs which 

will make yolk concentration data from field studies more meaningful. Quinn et al. (2006) 

have demonstrated in ovo exposure to trenbolone acetate to negatively impact reproductive 

maturation and behavior, thus giving us confidence of using this chemical as a model 

androgen-active endocrine disruptor in the assessment of the avian two-generation test. 

2. Non-avian Studies. Only one study was found that assessed fetal exposure to trenbolone from 

maternal exposure using rabbits (Lange et al., 2002), however this study only sought to 

determine if the chemical crossed the placental barrier. 

3. Other. Most of the remainder of the papers found described various analytical methods for the 

detection of trenbolone acetate metabolites in urine and tissue of various species for the 

purposes of environmental health assessment and antidoping screening in human and equine 

athletes, and are not pertinent to the current proposed study. 

III. OBJECTIVE/HYPOTHESIS: 

 The objective of this research is to determine the value added by examining endocrine endpoints 

in a two-generation avian reproductive toxicity test paradigm. 

IV. MILITARY RELEVANCE: 

Many chemicals used by the military that are known or suspected to be endocrine active have been 

found in environmental media at many military installations. Currently, few methods exist to ascertain the 

magnitude of these effects, although some have recently been proposed. Most species of birds are 

protected by the Migratory Bird Species Act and some are protected by the Endangered Species Act. As 

such, the DoD is, by executive order, obligated to abide by these laws. The presence of some species at 

specific installations (e.g. Bald Eagle at Aberdeen Proving Ground) has had a significant impact on 

reducing and curtailing military activity, therefore affecting readiness of our forces. Proactive 

collaboration in the development of methods that may, in the future, be mandated would serve the 

military, the Environmental Protection Agency, and the federal government at large. 

V. MATERIALS AND METHODS: 

V.1. Experimental Design and General Procedures: 

V.1.1. Experimental Design: 

 General Design: 

The general design of this study is illustrated in Figure 1. We will begin to expose the 

parental (F0) generation to the trenbolone via treated feed at four weeks of age and throughout 

the rest of the birds’ lives. Eggs from this generation will be incubated and hatched to produce 

the first generation (F1). The F1 generation will be exposed to the treatments in ovo (embryonic) 

through maternal deposition and through feed throughout the entirety of their lives. Eggs from 

this generation will be incubated and hatched to produce the second generation (F2). The F2

generation will only be exposed to trenbolone in ovo through maternal deposition; all feed for the 

F2 generation will be untreated. Measures of reproductive behavior, circulating hormone levels, 

egg quality, reproduction, and development will be assessed in the F0, F1, and F2 generations as 

described below. Additional measures of growth and reproductive maturity will be assessed in 

the F1 and F2 generations as described below. 
Animal Use Protocol-Two-generation test for endocrine disruption & reproductive toxicity in the Japanese quail with 

trenbolone. 

33 of 18 

Figure 1. Timeline of major events in avian two-generation toxicity test. 

Chemical Treatments: 

The sample size for each treatment will be 20 to ensure that there are 12 breeding pairs in the 

parental generation control group at the end of the treatment period that have successfully 

produced eggs. The treatment levels will be determined by the EPA following completion of a 

range-finding study currently being done by the U.S. Geological Service at Patuxent Wildlife 

Research Center, Laurel, MD. The test diets will be mixed and assayed at Wildlife International 

Ltd., Easton, MD, USA. Stability analyses and homogeneity and concentration verifications will 

be made for new diets made during the treatment period, according to GLP standards (DLS SOP 

CAD 98.1). 

Eggs: 

To generate the F0 generation, a minimum of 60 eggs will be hatched for each treatment level, to 

provide sufficient numbers of chicks to establish at least 12 per F0 test level. Twenty males and 

females will be used for the study, beginning exposure to the treatments at week 4. Optimally, 6 

eggs will be set for hatch for each parental pair of the F0 generation to ensure that a minimum of 

60 eggs will be hatched for each test level, providing sufficient numbers of chicks to establish at 

least 12 breeding pairs per F1 test level. If a treatment level produces insufficient eggs to set the 

minimum, that level will be used to its fullest extent. Fertilized eggs not set to hatch will be frozen 

prior to being discarded. Optimally, 6 eggs will be set for each parental pair of the F1 generation, 

but a minimum of 40 eggs will be set and hatched for each treatment level. 

Fertility and embryo viability will be determined by either removing the eggs out of the incubator 

and candling them or assessing them after the incubation has ended. Except for eggs incubated 

to hatch for production of subsequent generations, the late-stage embryos (day 15-16) will 

undergo necropsy for collection of thyroid glands as described below. Those chicks that have not 

hatched within approximately 24 hours of the majority of chicks hatching will be considered 

unhatched. No assistance will be given to chicks during hatching, however chicks that remain 

alive but have no clear ability to hatch (i.e. shell membranes dry) will be humanely removed from 

the egg and decapitated immediately. 

During the treatment periods, eggs will be used to assess eggshell strength and eggshell 

thickness. Eggshell strength will be measured by an Egg Force Reader (Orka Technology Ltd.) 
Animal Use Protocol-Two-generation test for endocrine disruption & reproductive toxicity in the Japanese quail with 

trenbolone. 

34 of 18 

that tests the breaking point of an egg’s shell. Eggshell thickness will be measured by an Egg 

Shell Thickness Gauge (Orka Technology Ltd.) that measures the thickness of egg shells through 

ultrasound or by micrometers or calipers. 

General Reproductive Fitness Measures and Observations: 

During the treatment period, the parental birds will be observed daily to detect any overt signs of 

toxicity or other clinical signs of illness or trauma. Observations will include mortality and clinical 

signs of toxicity such as lethargy, depression, wing droop, ruffled feathers, etc. Any injuries 

sustained and subsequent treatment will be recorded. If a pair is removed during the study due 

to death, injury, etc., data from all preceding weeks are evaluated. If data are used from a pen in 

which mortality occurred or a bird was injured, this will be explained. Clearly moribund individuals 

or those otherwise in severe distress will be euthanized, as described below. 

All adult birds will undergo necropsy and gross pathology assessment. The wet weight of the 

brain, liver, heart, spleen, kidneys, gonads, oviduct, and thyroid glands will be recorded as soon 

as possible after death/euthanasia. The size of the male cloacal gland will also be measured. 

Any morphologic abnormalities will be recorded in the study’s notebook or necropsy sheets. 

Adult birds that die, or are euthanized during the course of the treatment period, will be 

necropsied similarly. Histopathology will be performed on the brain, thyroid glands, liver, bursa of 

Fabricius, heart, spleen, kidneys, and gonads. Upon necropsy, half of the study animals’ thyroid 

glands will be stored for glandular thyroxine (T4) content, and the other half with be 

histopathologically assessed. 

Biochemical/Endocrine Measurements: 

Measurements of thyroid function will include thyroid gland weight, glandular T4 content (upon 

assay availability), and thyroid histology/histopathology. Measurements will be taken during 

embryonic and adult life stages. Eggs that are set, but not in the cohorts to be hatched, can be 

used for embryonic measurements. 

Blood collected from adults at the time of necropsy shall also have serum steroid analyses 

performed. 

Vitellogenin is a protein that is generally controlled by estrogen. It is present in females of 

oviparous species during egg development. It is not expressed in normal males due to lack of 

estrogen signal, however in males that have been exposed to estrogenic contaminants, it is 

expected that vitellogenesis will be stimulated. Vitellogenin will be measured in adult blood at 

necropsy. 

Behavioral measures: 

Initiation of normal male sexual behavior is important for successful reproduction. In 

reproductively mature males of the F1 and F2 generations, preferably when at least 90% of control 

males have reached reproductive maturity, trials shall be performed to assess reproductive 

behavior including time to mount attempts, number of mount attempts, and number of successful 

cloacal contacts. Males and females will be paired in the F1 generation following completion of 

the behavioral measures. Birds from the F2 generation will be euthanized upon completion of this 

measure. 

This study will be conducted in compliance with Good Laboratory Practice (GLP) regulations 

found in the Toxic Substances Control Act (TSCA): 40 CFR (Code of Federal Regulations) 792, 

plus amendments. The investigators and technicians will adhere to The Guide for Care and Use 

of Laboratory Animals (U.S. Department of Health, Education, and Welfare Publication No. NIH 

86-23, 1996). 

 

V.2. Data Analysis: All observational data will be recorded in the laboratory notebook. The analysis of 

variance (ANOVA) assumptions will be checked, and if they are met, a one-way ANOVA will be applied 

followed by a multiple comparisons test to compare treatment groups. If data do not meet the ANOVA 
Animal Use Protocol-Two-generation test for endocrine disruption & reproductive toxicity in the Japanese quail with 

trenbolone. 

35 of 18 

assumptions, data may be transformed appropriately or a nonparametric ANOVA may be applied. 

Observational categorical data may be tested with a Fisher’s Exact or Chi Square test. Statistical 

significance is defined as p < 0.05. 

V.3. Laboratory Animals Required and Justification: 

V.3.1. Non-animal Alternatives Considered: The objective addressed by this study is to determine 

the value added by examining endocrine endpoints in an avian reproductive toxicity test paradigm. Nonanimal alternatives would not provide the necessary information.

V.3.2. Animal Model and Species Justification: The Japanese quail was chosen for this study 

because it is an avian species that is both often used in toxicity tests and breeds readily in a laboratory 

setting. In addition, these birds only take six weeks to reach sexual maturity, which greatly reduces the 

time required to produce multi-generations when compared to other similar species, such as the northern 

bobwhite which takes an average of six months to reach reproductive maturity. 

 

V.3.3. Laboratory Animals: 

V.3.3.1. Genus and Species: Coturnix japonica

V.3.3.2. Strain/Stock: In-bred; nonspecific 

V.3.3.3. Source/Vendor: University of Maryland, Department of Animal and Avian Sciences, 

College Park, MD. 

V.3.3.4. Age: Eggs - adult

V.3.3.5. Weight: Adult weight = ~150g 

V.3.3.6. Sex: Males and females

V.3.3.7. Special Considerations: None

V.3.4. Number of Animals Required (By Species): 

1) Japanese quail: 

a. F0 generation: Minimum of 60 eggs set to incubate for 5 treatments = potential for 

maximum of 300 chicks; 20 males and 20 females from each treatment (100 males 

and 100 females total) will be kept for exposure to trenbolone. 

b. F1 generation: Minimum of 60 eggs set to incubate for 5 treatments = potential for 

maximum of 300 chicks; 20 males and 20 females from each treatment (100 males 

and 100 females total) will be kept for exposure to trenbolone. 

c. F2 generation: Minimum of 40 eggs set to incubate for 5 treatments = potential for 

maximum of 200 chicks; 20 males and 20 females from each treatment (100 males 

and 100 females total) will be kept for behavioral analyses. 

d. Sentinels: 20 birds will be used as sentinels per each generation (n = 60). 

e. Potential Replacements: 12 birds will be used as potential replacements for birds 

unable to adapt to the new water system upon transfer from the chick brooders to 

adult caging (n = 36). 

V.3.5. Refinement, Reduction, Replacement: 

V.3.5.1. Refinement: No additional refinements will be employed other than the environmental 

enrichment strategy. 
Animal Use Protocol-Two-generation test for endocrine disruption & reproductive toxicity in the Japanese quail with 

trenbolone. 

36 of 18 

V.3.5.2. Reduction: This study will utilize a group of birds to determine reproductive, 

developmental, and behavioral effects in the same individuals. This is a large reduction in the number of 

birds relative to the number that would be needed to measure these effects separately in individual birds. 

V.3.5.3. Replacement: None. 

V.4. Technical Methods: 

V.4.1. Pain/Distress Assessment: 

V.4.1.1. APHIS Form 7023 Information 

V.4.1.1.1. Number of animals: 696 (maximum potential ~896)

V.4.1.1.1.1. Column C: 696 – ~896 (100%) 

V.4.1.1.1.2. Column D: 0 

V.4.1.1.1.3. Column E: 0 

V.4.1.2. Pain Relief/Prevention: No other substances that could potentially interfere with the 

interpretation of these results will be administered in the unlikely event that the animal appears to be 

experiencing pain or discomfort. However, moribund or animals in overt pain will be humanely 

euthanized as described in section V.4.6. (Euthanasia). Based on previous experience with this model, 

we will consider the following as indications of distress where recovery is unlikely at continued exposure 

regimes: 

1) Loss of body weight to exceed 20%. 

2) Loss of righting response and inability to care and feed without assistance. 

A weight of evidence determination will be made on a case-by-case basis after consultation with the 

principal investigator and the attending veterinarian. 

V.4.1.2.1. Anesthesia/Analgesia Tranquilization: None 

V.4.1.2.2. Pre- and Post-procedural Provisions: None

V.4.1.2.3. Paralytics: None

V.4.1.3. Literature Search for Alternatives to Painful or Distressful Procedures: 

V.4.1.3.1. Sources Searched:

 

a. Medline 

b. ToxFile 

c. AGRICOLA 

d. Biosis 

e. EMBASE 

f. CA SEARCH 

V.4.1.3.2. Date of Search: 29 December 2009 

V.4.1.3.3. Period of Search: 1926-2009 

V.4.1.3.4. Key Words of Search: trenbolone, parabolan, 10161-33-8, oral exposure, aerosol, 

inhalation, toxic, alternative, welfare, method, model, in vitro, pain, distress, simulate, video, computer, 

replacement, refinement, reduction. 
Animal Use Protocol-Two-generation test for endocrine disruption & reproductive toxicity in the Japanese quail with 

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V.4.1.3.5. Results of Search: Few papers were found that discussed avian euthanasia methods. 

Several papers were found that evaluated various anesthesia techniques, including the use of halothane, 

ketamine-xylazine, and isoflurane. Nothing was found regarding pain or distress. The only in vitro

studies that were found that used trenbolone were genotoxicity, carcinogenicity, and mutagenicity tests, 

which have no relevance to the current proposed study and offer no alternative to the in vivo methods 

presented here. 

Additionally, an independent literature search was conducted using a combination of several academic 

search engines and by manually tracking down literature cited in captive care literature. This search 

yielded several class-specific anesthesia/euthanasia review papers. The following synopses summarize 

our most relevant findings: 

1. There are no alternatives to using live birds for addressing the purpose of this study. 

2. No substances can be used to alleviate pain since the interaction of such substances with the study 

compound are unknown, which may compromise the results. 

V.4.1.4. Unalleviated Painful/Distressful Procedure Justification: The nature of this study 

precludes the use of totally painless procedures, and attempts to alleviate pain or distress by the 

administration of anesthetics, analgesics, or other drug interventions may alter the manifestation of the 

toxic responses and/or interfere with the mechanism of toxicity. The observation of the onset, duration, 

and/or reversibility of toxic signs is critical to mechanistic interpretation and understanding toxicologic 

processes. “Toxic signs” are defined in TOX SOP 063. Moribund animals will be humanely euthanized as 

described in Section V.4.5. (Study Endpoint) in accordance with TOX SOP 066. 

 Moribund animals will be euthanized to alleviate distress. The minimal number of animals 

needed for statistical significance will be used. The final number of birds in each pain category will be 

reported to the IACUC annually and at the completion of the protocol. 

V.4.2. Prolonged Restraint: None

V.4.3. Surgery: None 

V.4.3.1. Pre-surgical Provisions: None

V.4.3.2. Procedure: None

V.4.3.3. Post-surgical Provisions: None

V.4.3.4. Location: None

V.4.3.5. Surgeon: None

V.4.3.6. Multiple Major Survival Operative Procedures: None

V.4.3.6.1. Procedures: Not applicable

V.4.3.6.2. Scientific Justification: Not applicable

V.4.4. Animal Manipulations: One person will present the bird to the injector, keeping the wings and 

legs firmly pressed against its body. The injector will then brush the feathers away from the featherless 

tract in the neck region and then inject the solution into the jugular vein. Blood will also be collected from 

this vein. 

V.4.4.1. Injections: Not applicable 
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V.4.4.2. Biosamples: Blood will be collected IAW DTOX SOP 053 as described in section V.1.1. Experimental 

Design. Blood will be collected either from the jugular vein or following euthanasia via cardiac stick with a 25-

gauge needle and 1-3 ml syringe. 

V.4.4.3. Adjuvants: None

V.4.4.4. Monoclonal Antibody (MAbs) Production: Not applicable

V.4.4.5. Animal Identification: Individual animals will be identified by cage cards and leg bands 

(IAW DTOX SOP 003).

V.4.4.6. Behavioral Studies: Male copulatory behavior will be assessed as described in section 

V.1.1. Experimental Design, Behavioral measures.

V.4.4.7. Other Procedures: None. 

V.4.4.8. Tissue Sharing: Tissues may be shared with U.S. Army Corps of Engineers, 

Engineering Research and Development Center for toxicogenomics research.

V.4.5. Study Endpoint: The study will be terminated within one week via euthanasia following 

completion of reproductive behaviors assessed in the F2 generation. Moribund or animals in overt pain 

will be humanely euthanized as described in section V.4.6. (Euthanasia). Based on previous experience 

with these models we will consider the following as indications of distress where recovery is unlikely at 

continued exposure regimes: 

1) Loss of body weight (from day 0) to exceed 20%. 

2) Loss of righting response and inability to feed or drink without assistance. 

A weight of evidence determination will be made on a case-by-case basis after consultation with the 

principal investigator and the attending veterinarian. 

V.4.6. Euthanasia: Birds will be euthanized by CO2 asphyxiation as recommended by the AVMA 

Guidelines on Euthanasia (2007). Although unhatched birds are not covered by these guidelines, 

fertilized eggs that are not destined to be set to hatch will be frozen prior to being discarded, and late 

stage embryos will be decapitated immediately following removal from the egg. 

 Decapitation will occur with shears that will be kept sharp and will only be performed by the study 

director or the Attending Vet until other study personnel are trained. Study staff and veterinary medicine 

personnel who are trained may perform the euthanasia. If the SD/PI or other study staff personnel are 

not available, the veterinary staff will euthanize the animal and take as many required samples as 

possible from the animal, according to the protocol and upon training. The animal carcass will be held at 

the facility under refrigeration for a minimum of three days in order for investigators to take any additional 

required samples. Euthanasia will be performed IAW TOX SOP 066. 

V.5. Veterinary Care: 

V.5.1. Husbandry Considerations: Birds will be held according to DTOX SOPs 028 and 150 where 

applicable. Treatments will be randomly assigned to birds within cages and cage units. Cage unit frames 

are constructed of 1” stainless steel tubing. Cages are constructed of perforated stainless sheet. Each 

cage unit will have two litter pans per tier that are easily removed. Each unit will have individual stainless 

steel feeders and one stainless Edstrom cup drinker. Watering will be automatic. Cage dimension is 7” 

wide x 9.5” tall x 11” deep. Birds will be acclimated to laboratory conditions for a minimum of five days 

prior to the onset of experiments. Birds will be kept in conditions of constant photoperiod. Birds will be 

housed in AAALAC accredited facility at USACHPPM. Each cage will contain a card describing treatment 

and animal number. 

The photoperiod for chicks and adults will be 16 or 17 hours light and 8 or 7 hours of darkness. Chicks 

and juveniles will be group housed. Birds will be pair housed upon transition to adult caging and 

separated to single cages until copulatory behaviors have been completed. Individuals will be paired 
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according to similar weights to minimize the potential for aggression. Pairs will be monitored daily for pen 

mate aggression, and incompatible pairs will be separated and repaired as appropriate. If individuals are 

too aggressive to be paired, they may be singly housed and mating will occur via daily pairings that may 

last approximately 30 minutes; this is a time that has shown to be effective in allowing mating to occur 

before effects of aggression can be made.

Hatchlings will be dried completely before being transferred to brooders. Once grouped, chicks will be 

checked at least twice daily to ensure that group shunning, hypothermia, and/or starvation do not occur. 

Sufficient space for feeding and drinking will be provided, especially during the first week post-hatch to 

allow equal access to food and water to weaker chicks. Chicks will be housed together by treatment 

group when receiving treated feed and will be identifiable individually by leg bands. Juveniles will be 

moved to adult cages as pairs to facilitate learning how to use the cage rack’s water system during the 

fifth week of age. Twelve extra birds may be brought over to the adult caging at this time (six male, six 

female) to replace any birds that may not have learned how to use the stainless steel water valves. After 

one week, pairs will be separated until after the copulatory tests are completed so onset of reproductive 

maturity and reproductive behavior naivety can be maintained. 

Housing conditions: 

Age (weeks) Temp. (o

C) RH (%) 

1 35-38 40-80 

2 30-35 40-80 

3-4 23-27 40-80 

>4 16-27 40-80 

Feed and water will be provided ad libitum. Chicks will receive a growth diet and adults will be given a 

laying diet that will accommodate for expenditure of calcium through egg laying. 

Eggs will be stored in a cold storage facility for a maximum of one week prior to setting in an incubator. 

Cool storage prevents embryo development and aids in synchronizing the development of embryos of 

eggs laid during that week. All cracked and abnormal eggs will be removed prior to incubation. Eggs will 

be equilibrated to room temperature prior to being set in the incubator. All cracked and abnormal eggs 

will be recorded. Eggs should be turned in the incubator at least three times a day. For incubation, 

temperature should be between 37.5-37.8o

C and relative humidity should be between 50-70%. Eggs 

should be hatched at 37-37.5o

C and relative humidity should be between 70-75%. 

V.5.1.1. Study Room: Housing- as assigned, E2100/ E-2101. 

 Dosing- rm. E2100 / E2101 

 Necropsy- rm. 3201, E2100 

V.5.1.2. Special Husbandry Provisions: Not applicable 

V.5.1.3. Exceptions: None

V.5.2. Veterinary Medical Care: 

 

V.5.2.1. Routine Veterinary Medical Care: All animals will be observed at least twice daily by 

assigned veterinary personnel and once on weekend days and holidays for husbandry conditions, 

humane care, and general health. After exposure begins, animals will be observed at least once daily by 

the PI or study staff. Frequency of observations may be increased as needed following agreement 

between the AV and SD/PI. 

V.5.2.2. Emergency Veterinary Medical Care: All emergency animal health care will be 

provided by the veterinary staff in consultation with the PI whenever possible or by the study staff as 

appropriate. 

V.5.3. Environmental Enrichment 
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V.5.3.1. Enrichment Strategy: Enrichment will consist of group housing for hatchlings and 

juveniles and pair housing for adults. Additional enrichment may be used as available following 

agreement between the AV and SD/PI.

V.5.3.2. Enrichment Restriction: Feed enrichment will be restricted since feed consumption will 

be monitored.

VI. STUDY PERSONNEL QUALIFICATIONS AND TRAINING: 

 

 If training in required procedures has not been of a formal, documented nature, proficiency in these 

procedures will be demonstrated prior to performing them on study animals. The Attending Veterinarian 

will observe them and determine proficiency or additional training needs. 

 

Study Personnel Table 

Staff 

Member 

Procedure(s) Training Qualifications 

Michael 

Quinn 

Collection 

and handling, 

feeding, 

weighing, 

phlebotomy, 

euthanasia, 

necropsy 

Necropsy- Quail (6/2005); Avian 

(Northern Bobwhite) handling, 

dosing, & prosection (9/2005); 

Avian Handling and Blood 

Withdrawal Techniques (Cardiac, 

Jugular) in the Bobwhite Quail 

(1/2007); Avian Handling and 

Dosing in the Bobwhite Quail 

(3/2008); Avian euthanasia (CO2) 

& necropsy (Japanese quail) 

(9/2008); Avian handling & health 

surveillance (Japanese quail): 

juvenile thru adult, normal phys. 

signs, feces, behavior (1/2009); 

Avian handling, health 

surveillance, normal physiological 

signs (feces, behavior) (Japanese 

quail) (5/2009); Avian jugular 

bleeding training (Japanese quail) 

(6/2009); Japanese quail CO2 

euthanasia & necropsy training 

(10/2009) 

12+ yrs animal research; Served as PI & Co-PI 

on previous studies using various avian species. 

Ph.D, Animal Science 

Michelle 

Cook 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

Japanese quail - incubation, 

hatching, and care from day 1 

through adult (including operation 

of equipment) (8/2008); Avian inhouse training at DTOX: 

Japanese quail euthanasia (CO2) 

& necropsy (9/2008); Japanese 

quail handling & health 

surveillance juvenile through 

adult, normal phys. signs, feces, 

behavior (1/2009); normal 

physiological signs (feces, 

6+ years animal research. Extensive in-house 

avian training. M.S. Biology. 
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behavior) (5/2009); Avian jugular 

bleeding training (Japanese quail) 

(6/2009) 

Craig 

McFarlan

d 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

Avian (Northern Bobwhite) 

handling, dosing, & prosection 

(9/2005); Avian Handling and 

Blood Withdrawal Techniques 

(Cardiac, Jugular) in the Bobwhite 

Quail (1/2007); Avian jugular 

bleeding training (Japanese quail) 

(6/2009) 

10+ years animalresearch. 

Ph.D, Biology, DVM 

Terry 

Hanna 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

Northern Bobwhite- care & 

handling (dosing & cage 

requirements) (11/2005); Avian 

Handling and Blood Withdrawal 

Techniques (Cardiac, Jugular) in 

the Bobwhite Quail (1/2007); 

Japanese quail - incubation, 

hatching, and care from day 1 

through adult (including operation 

of equipment) (8/2008); Avian 

euthanasia (CO2) techniques (in 

Japanese quail) 6/2009); 

Japanese quail - CO2 euthanasia 

(10/2009); Japanese quail – 

Necropsy (10/2009) 

ALAT (1992), 

Alicia 

Bonney 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

Avian handling & health 

surveillance (Japanese quail): 

juvenile thru adult; normal phys. 

signs, feces, behavior (1/2009); 

Japanese quail - avian bleeding 

training, jugular (6/2009); 

Japanese quail - observation of 

mating, body and food weight 

measurements (6/2009); 

Japanese quail - CO2 euthanasia 

(10/2009); Japanese quail – 

Necropsy (10/2009) 

Lee 

Crouse 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

Avian Handling and Blood 

Withdrawal Techniques (Cardiac, 

Jugular) in the Bobwhite Quail 

(1/2007); 

Arthur 

O’Neill 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

Avian Handling and Blood 

Withdrawal Techniques in the 

Bobwhite Quail (3/2008) 

Will 

McCain 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

 
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Emily 

LaFiandr

a 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

Northern bobwhite euthanasia & 

necropsy (12/2007); Avian 

Handling and Dosing in the 

Bobwhite Quail (3/2008); Avian 

handling & health surveillance 

(Japanese quail): juvenile thru 

adult; normal phys. signs, feces, 

behavior (1/2009); Japanese 

quail - CO2 euthanasia (10/2009); 

Japanese quail – Necropsy 

(10/2009); 

10+ years animal research. 

Ph.D., Toxicology 

Matthew 

Bazar 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

 

Valerie 

Adams 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

Avian handling & health 

surveillance (Japanese quail): 

juvenile thru adult; normal phys. 

signs, feces, behavior (1/2009) 

William 

Eck 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

 

Larry 

Williams 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

Avian handling & health 

surveillance (Japanese quail): 

juvenile thru adult; normal phys. 

signs, feces, behavior (1/2009); 

Japanese quail - CO2 euthanasia 

(10/2009); 

Mark 

Way 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

Avian Handling and Dosing in the 

Bobwhite Quail (3/2008); Avian 

handling & health surveillance 

(Japanese quail): juvenile thru 

adult; normal phys. signs, feces, 

behavior (1/2009); Japanese 

quail - avian bleeding training, 

jugular (6/2009); Japanese quail 

- CO2 euthanasia (10/2009); 

Japanese quail – Necropsy 

(10/2009) 

15+ yrs animal research. 

LAT (1990), 

B.S. Biology 

Patricia 

Beall 

 Necropsy- Quail (6/2005); 

John 

Houpt 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

Japanese quail - CO2 euthanasia 

(10/2009); Japanese quail – 

Necropsy (10/2009); 

Rebecca Handling, Northern Bobwhite- care & ALAT (1995); Registered Veterinary Technician 
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Kilby feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

handling (dosing & cage 

requirements) (11/2005); Avian 

Handling and Blood Withdrawal 

Techniques (Cardiac, Jugular) in 

the Bobwhite Quail (1/2007); 

Japanese quail - incubation, 

hatching, and care from day 1 

through adult (including operation 

of equipment) (8/2008); Avian 

handling & health surveillance 

(Japanese quail): juvenile thru 

adult; normal phys. signs, feces, 

behavior (1/2009); Avian 

euthanasia (CO2) techniques (in 

Japanese quail) (6/2009) 

Robert 

Sunderla

nd 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

Avian Handling and Blood 

Withdrawal Techniques (Cardiac, 

Jugular) in the Bobwhite Quail 

(1/2007); Japanese quail - 

incubation, hatching, and care 

from day 1 through adult 

(including operation of 

equipment) (8/2008); Avian 

handling & health surveillance 

(Japanese quail): juvenile thru 

adult; normal phys. signs, feces, 

behavior (1/2009); Avian 

euthanasia (CO2) techniques (in 

Japanese quail) 6/2009); 

Japanese quail - CO2 euthanasia 

(10/2009) 

LAT (1991), 

Jason 

Williams 

Handling, 

feeding, 

weighing, 

phlebotomy, 

necropsy, 

euthanasia 

Japanese quail - CO2 euthanasia 

(10/2009) 

 

VII. BIOHAZARD/SAFETY:

Birds of this type have been reported to carry specific zoonotic diseases that can be transferred to 

humans (e.g., psittacosis, histoplasmosis), most of them affecting the respiratory system. The eggs will 

be procured from a reputable supplier who will screen the health of the bird colony before the eggs are 

delivered. Following hatching, brooder housing, and transition to adult caging, all birds will be 

quarantined for a minimum of five days and monitored to ensure they appear free from disease and other 

health related effects. However, since the possibility exists that some animals may be infected, personnel 

will wear dust masks to reduce the possibility of infection and inhalation of dander. 

Adherence to standard chemical and animal handling procedures will be required during the 

performance of this study. In addition, all personnel participating in this study will act according to 

CHPPM Regulations 385-1 and 385-5 and use proper PPE according to DTOX SOP 083. 

VIII. ENCLOSURES: Appendix A: References 

IX. STUDY TIME FRAME: 

IX.1. Estimated Experimental Initiation Date: April 2010 
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IX.2. Estimated Experimental Completion Date: April 2011 
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X. ASSURANCES: 

As the Study Director/Principal Investigator on this protocol, I acknowledge my responsibilities 

and provide assurances for the following: 

A. Animal Use: The animals authorized for use in this protocol will be used only in the 

activities and in the manner described herein, unless a modification is specifically approved by the IACUC 

prior to its implementation.

B. Duplication of Effort: I have made every effort to ensure that this protocol is not an 

unnecessary duplication of previous experiments. 

C. Statistical Assurance: : I assure that I have consulted with a qualified individual who 

evaluated the experimental design with respect to the statistical analysis, and that the minimum number 

of animals needed for scientific validity will be used. 

D. Biohazard/Safety: I have taken into consideration and made the proper coordinations 

regarding all applicable rules and regulations concerning radiation protection, biosafety, recombinant 

issues, and so forth, in the preparation of this protocol. 

E. Training: I verify that the personnel performing the animal 

procedures/manipulations/observations described in this protocol are technically competent and have 

been properly trained to ensure that no unnecessary pain or distress will be caused to the animals as a 

result of the procedures/manipulations. 

F. Responsibility: I acknowledge the inherent moral, ethical and administrative obligations 

associated with the performance of this animal use protocol, and I assure that all individuals associated 

with this project will demonstrate a concern for the health, comfort, welfare, and well-being of the 

research animals. Additionally, I pledge to conduct this study in the spirit of the fourth "R," namely 

"Responsibility," which the DOD has embraced for implementing animal use alternatives where feasible 

and conducting humane and lawful research. 

G. Scientific Review: This proposed animal use protocol has received appropriate peer 

scientific review and is consistent with good scientific research practice. 

H. Painful Procedures: I am conducting biomedical experiments, which may potentially 

cause more than momentary or slight pain or distress to animals. This potential pain and/or distress WILL 

NOT be relieved with the use of anesthetics, analgesics, and/or tranquilizers. I have considered 

alternatives to such procedures; however, I have determined that alternative procedures are not available 

to accomplish the objectives of this proposed experiment. 

 

Michael J. Quinn, Jr. 

 _________________________________ 

Signature Date 

(YYYYMMMDD) 
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IX.2. As the Primary Co-Investigator on this protocol, I acknowledge my responsibilities and provide 

assurances for the following: 

 A. Animal Use: The animals authorized for use in this protocol will be used only in the activities 

and in the manner described herein, unless a modification is specifically approved by the IACUC prior to 

its implementation. 

 B. Duplication of Effort: I have made every effort to ensure that this protocol is not an 

unnecessary duplication of previous experiments. 

 C. Statistical Assurance: I assure that I have consulted with a qualified individual who evaluated 

the experimental design with respect to the statistical analysis, and that the minimum number of animals 

needed for scientific validity will be used. 

 D. Biohazard/Safety: I have taken into consideration and made the proper coordinations 

regarding all applicable rules and regulations concerning radiation protection, biosafety, recombinant 

issues, and so forth, in the preparation of this protocol. 

 E. Training: I verify that the personnel performing the animal 

procedures/manipulations/observations described in this protocol are technically competent and have 

been properly trained to ensure that no unnecessary pain or distress will be caused to the animals as a 

result of the procedures/manipulations. 

 F. Responsibility: I acknowledge the inherent moral, ethical and administrative obligations 

associated with the performance of this animal use protocol, and I assure that all individuals associated 

with this project will demonstrate a concern for the health, comfort, welfare, and well-being of the 

research animals. Additionally, I pledge to conduct this study in the spirit of the fourth "R," namely 

"Responsibility," which the DOD has embraced for implementing animal use alternatives where feasible 

and conducting humane and lawful research. 

 G. Scientific Review: This proposed animal use protocol has received appropriate peer scientific 

review and is consistent with good scientific research practice. 

 H. Painful Procedures: I am conducting biomedical experiments, which may potentially cause 

more than momentary or slight pain or distress to animals. This potential pain and/or distress WILL NOT 

be relieved with the use of anesthetics, analgesics, and/or tranquilizers. I have considered alternatives to 

such procedures; however, I have determined that alternative procedures are not available to accomplish 

the objectives of this proposed experiment. 

 

 Michelle Cook_____________________ 

First name, MI, Last name of Primary Co-investigator 

 

______________________________________________________________________________ 

Signature Date (YYYYMMDD) 
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IX.3. As a Co-Investigator on this protocol, I provide the following assurances: 

A. Animal Use: The animals authorized for use in this protocol will be used only in the activities 

and in the manner described herein, unless a modification is specifically approved by the IACUC prior to 

its implementation. 

B. Authority: I understand that, as a Co-Investigator, I am authorized, responsible for, and 

willing to perform all procedures and manipulations as assigned to me by the SD/PI. 

C. Training: I verify that I am technically competent and have been or will be properly trained to 

ensure that no unnecessary pain or distress will be caused to the animals as a result of the assigned 

procedures/manipulations performed by me. 

D. Responsibility: I acknowledge the inherent moral and administrative obligations associated 

with the performance of this animal use protocol, and I assure that I will demonstrate a concern for the 

health, comfort, welfare, and well-being of the research animals. Additionally, I pledge to participate in 

this study in the spirit of the fourth “R”, namely “Responsibility,” which the DOD has embraced for 

implementing animal use alternatives where feasible, and conducting humane and lawful research. 

E. Painful Procedures: I am participating in biomedical experiments, which may potentially 

cause more than momentary or slight pain or distress to animals. I will follow the direction of the SD/PI 

relative to potential pain and/or distress and relief by the use of anesthetics, analgesics and/or 

tranquilizers if the protocol specifies for their use. 

______________________________________________________________________________ 

(PRINT) 

 (Signature) (Date) 

First name, MI, Last name of Co-Investigator 

______________________________________________________________________________ 

(PRINT) 

 (Signature) (Date) 

First name, MI, Last name of Co-Investigator 

______________________________________________________________________________ 

(PRINT) 

 (Signature) (Date) 

First name, MI, Last name of Co-Investigator 

______________________________________________________________________________ 

(PRINT) 

 (Signature) (Date) 

First name, MI, Last name of Co-Investigator 
48 of 18 

APPENDIX A 

References:

Directorate of Laboratory Services SOP CAD 98.1. 2003. Chromatographic/Spectrophotometric Analysis 

of Toxicology Directorate Samples. 

Directorate of Toxicology, Standing Operating Procedure (DTOX SOP) AP003-P-001. Individual 

 Animal Identification. USACHPPM, Aberdeen Proving Ground, MD. 

Directorate of Toxicology, Standing Operating Procedure (DTOX SOP) AP028-P-001. Animal Quality 

Control Procedures. USACHPPM, Aberdeen Proving Ground, MD. 

Directorate of Toxicology, Standing Operating Procedure (DTOX SOP) AP053-P-001. Animal Bleeding 

Technique. USACHPPM, Aberdeen Proving Ground, MD. 

Directorate of Toxicology, Standing Operating Procedure (DTOX SOP) AP063-P-001. Test System 

Observations. USACHPPM, Aberdeen Proving Ground, MD. 

Directorate of Toxicology, Standing Operating Procedure (DTOX SOP) AP066-P-001. Animal Euthanasia. 

USACHPPM, Aberdeen Proving Ground, MD. 

Directorate of Toxicology, Standing Operating Procedure (DTOX SOP) GL083-P-001. Health and Safety 

of Laboratory Personnel. USACHPPM, Aberdeen Proving Ground, MD. 

Directorate of Toxicology, Standing Operating Procedure (DTOX SOP) AP150-P-001. Avian 

 Husbandry and Health Surveillance. USACHPPM, Aberdeen Proving Ground, 

Feder, P.I.,D.W. Hobson, C.T. Olson, R.L. Joiner, M.C. Matthews. 1991. Stagewise, adaptive 

dose allocation for quantal response dose-response studies. Neurosci Biobehav Rev, 15: 109-14. 

EPA (U.S. Environmental Protection Agency). 1996. Avian reproduction test. OPPTS 850.2300 “Public 

Draft” Ecological Effects Test Guidelines. EPA 712-C-96-141. U.S. Environmental Protection Agency, 

Washington, D.C. 

Lange, I.G., A. Daxenberger, H.H.D. Meyer, E. Rajpert-De Meyts, N.E. Skakkebaek, D.N.R. 

Veeramachaneni. 2002. Quantitative assessment of foetal exposure to trenbolone acetate, zeranol 

and melengestrol acetate, following maternal dosing in rabbits. Xenobiotica, 32: 641-651. 

Quinn, M.J., Jr., E.T. Lavoie, M.A. Ottinger. 2006. Reproductive toxicity of trenbolone acetate in 

embryonically exposed Japanese quail. Chemosphere, 66: 1191-1196. 
49 of 18 

APPENDIX B 

Modifications:

1. Modification: The addition of 5-7 sentinel birds per generation, a range which has been used in 

previous avian toxicity tests and is approved by the Attending Veterinarian. 

Justification/Reason: Although the current Avian Toxicity Test SOP does not mention use of sentinel 

individuals, previous veterinarians have made their inclusion in toxicity tests mandatory as an 

additional means of health surveillance. The use of sentinel quail in this study will help the 

investigators and caretakers be better able to monitor the overall condition of this study’s three 

generations. 

2. Modification: The addition of Matthew Bazar as a co-investigator. 

 Justification/Reason: Matthew Bazar WILL NOT handle live animals from this study. He will perform routine 

histological measures and also develop novel techniques to measure new endpoints that will enable us to better 

assess the potential for endocrine disruption. The development of these new methods will be a great 

undertaking; his clarification as co-PI is better reflective of his efforts. 

3. Modification: The number of breeding pairs per dose group will increase from 16 to 20 to better enable the 

threshold of 12 breeding pairs per treatment to be maintained throughout the study. A maximum of 20 birds will 

be used as sentinels, rather than the previous seven. 

Justification/Reason: A larger initial number of birds used will help prevent the study from having to be 

prematurely ceased by better ensuring the study’s threshold of 12 breeding pairs per treatment group throughout 

the study. An increase in the number of sentinels will also help to ensure that some remain pending another 

potential unexpected decline in numbers. 

4. Modification: To better enable birds to learn how to use the adult caging water system, birds should be housed in 

pairs upon transition from a group hosed situation to being singly housed for at least three days and provided 

treated feed at least one week after the move from the brooder system. Water jars will be provided upon first 

signs of dehydration. 

 Justification/Reason: Birds will be better able to learn how to use the water system in the adult caging by 

watching each other in a pair housed situation and the supplemental water source will give the birds more time to 

learn how to use the water system. A delay in separation and administration of treated feed will help to delay the 

impact of numerous stress events from happening at one time. 

5. Modification: The allowance of enrichment strategies in addition to or replacements of sisal rope as approved by 

the Attending Veterinarian and the Study Director. 

Justification/Reason: To allow for the use of better enrichment strategies that may enhance the mental health of 

the birds. 

6. Additional Study Personnel will be added to this protocol: Rebecca Kilby, Robert Sunderland, Shenell Tolson, 

Mark Way, Larry Williams, Michelle Cook, Valerie Adams, and Terry Hanna. 

 Justification/Reason: Additional trained personnel participating in this study will enhance our ability to recognize 

 health problems that will enable us to provide solutions sooner. 

7. Study Personnel to be able to perform CO2 euthanasia on Japanese quail now include Alicia Bonney, Mark Way, 

Terry Hanna, Emily LaFiandra, and John Houpt. 

 Justification/Reason: During holidays and conferences, the number of available Study Staff listed in the current 
50 of 18 

protocol to be able to perform euthanasia on Japanese quail may be limited; this additional number of trained 

individuals will alleviate this problem. Training forms are available. 
51 of 18 

APPENDIX E 

DEVIATIONS TO THE PROTOCOL 
52 of 18 

Wildlife International, Ltd. 

APPENDIX F 

DIET PREPARATION FROM WILDLIFE INTERNATIONAL, LTD. 

Project Number 167-143

Test Substance 

 The test substance, trenbolone C-III, was received from Battelle Marine Sciences Laboratory, 

Sequim, WA 98362 on February 16, 2010 and was assigned Wildlife International, Ltd. identification 

number 9399 upon receipt. The test substance was a solid and was identified as: trenbolone C-III, Lot # 

099K0968, CAS # 10161-33-8. The test material had a reported purity of  95%. The test substance was 

held under refrigerated conditions in a locked safe at the Wildlife International, Ltd. facilities in Easton, 

Maryland. Concentrations of the test substance in the diet were adjusted to 100% active ingredient 

based upon a reported purity of 95%. Therefore, dietary concentrations are expressed as parts per 

million active ingredient (ppm a.i.) in the diet. 

Diet Preparation 

 The basal diet fed to both adults and offspring was formulated to Wildlife International, Ltd. 

specifications by Cargill Animal Nutrition, Shippensburg, PA (Appendix I). The basal ration contained at 

least 27% protein and 2.5% crude fat, and no more than 3.8% crude fiber. The basal diet contained 

approximately 1.0% calcium, derived from feedstuffs and the 1.0% limestone used in the formulation of 

the basal diet by Cargill. While this level of calcium is sufficient for growth and maintenance rations, 

additional calcium is required in the ration of breeding birds for egg shell formation. Therefore, an 

additional 5% (w/w) of limestone (approximately 38.5% Ca) was added to the basal diet for the adults. 

This raised the calcium level in the diet for the breeding birds to approximately 3%, slightly above the 

minimum recommended for quail. Offspring received diets prepared without the addition of 5% 

supplemental limestone. 

 Test diets were prepared by mixing trenbolone into a premix that was used for preparation of the 

final diet. Details of the preparation of test and control diets are shown in Appendix II. 
53 

Appendix I 

Diet Formulation 

WILDLIFE INTERNATIONAL, LTD. GAME BIRD RATION1 

INGREDIENTS

Fine Corn Meal 

Soy Bean Meal, 47.5% Protein 

Wheat Midds 

Agway Special, 60% Protein 

Alfalfa Meal, 20% Protein 

Dried Whey 

Ground Limestone 

Eastman CalPhos 

GL Ferm (Fermatco)2

Salt Iodized 

CM T-Premix 561 

CM TM Premix 434 

CHO CHL 70% 

Liquimeth 40% 

Lysine 75% 

DL Methionine 055 

Vit K 16 g/lb 50# 

Selenium 

Total 

PERCENT (%)

42.86 

33.39 

6.48 

9.96 

3.00 

2.50 

0.62 

0.11 

0.25 

0.10 

0.05 

0.08 

0.09 

0.25 

0.06 

0.10 

0.05 

0.05 

100.00 

VITAMIN AND MINERAL CONTENT AMOUNT ADDED PER POUND

Vitamin D3

Vitamin A 

Riboflavin 

Niacin 

Pantothenic Acid 

Vitamin B12

Folic Acid 

Biotin 

Pyridoxine 

Thiamine 

Vitamin E 

Vitamin K (Menadione Dimethylpyrimidinol Bisulfite) 

Manganese 

Zinc 

Copper 

Iodine 

Iron 

Selenium 

Beta-Carotene 

Calcium 

Chloride 

Choline 

Cobalt 

Magnesium 

Phos 30 

Phos 50 

Potassium 

Sodium 

Sulfur 

Phosphorus 

Ca:PHOS 

0.600 kiu/lb 

 2.000 kiu/lb 

 2.1992 mg/lb 

 21.6932 mg/lb 

 5.2241 mg/lb 

 6.0000 ug/lb 

 0.5169 mg/lb 

 65.9360 ug/lb 

 1.4639 mg/lb 

 0.9200 mg/lb 

 5.0000 iu/lb 

 3.2400 mg/lb 

 115.0203 mg/kg 

 194.6601 mg/kg 

 24.3007 mg/kg 

 2.4410 mg/kg 

 150.4117 mg/kg 

 0.4749 mg/kg 

 1.1413 mg/lb 

 0.9750 % 

 0.2164 % 

 1882.4116 mg/kg 

 0.1139 mg/kg 

 0.2503 % 

 0.3908 % 

 0.4719 % 

 1.1148 % 

 0.1335 % 

 0.3478 % 

 0.6800 % 

1.4338 ratio 1

 The guaranteed analysis is a minimum of 27% protein, a minimum of 2.5% crude fat and a maximum of 

3.8% crude fiber. 2

 Fermentation By-Products (Source of Unidentified Growth Factors) 
54 

Appendix II 

Diet Preparation 

 As needed, premixes for trenbolone diets were prepared on August 5, 2010; November 30, 2010; 

February 28, 2010 and for Control (0 ppm) only on July 27, 2011. Nominal preparation was as follows: 

 Control: 7938.0 g ration + 250 ml acetone + 180 ml corn oil 

 0.5 ppm: 0.0586 g Trenbolone + 7937.9 g ration + 250 ml acetone + 180 ml corn oil 

 5 ppm: 0.5862 g Trenbolone + 7937.4 g ration + 250 ml acetone + 180 ml corn oil 

 10 ppm: 1.1724 g Trenbolone + 7936.8 g ration + 250 ml acetone + 180 ml corn oil 

 20 ppm: 2.3447 g Trenbolone + 7935.7 g ration + 250 ml acetone + 180 ml corn oil 

For each premix, the appropriate amount of trenbolone was weighed in a tared glass Petri dish on an 

analytical balance. The test substance was transferred to a beaker. Acetone was measured in a 

graduated syringe and added to the test substance with a portion of the acetone used to rinse the 

Petri dish. The rinse was added to the beaker. A magnetic stir-bar was placed in the beaker and the 

mixture stirred for at least two minutes (approximate) on a magnetic stir plate, until the trenbolone 

dissolved. Corn oil was then measured in a graduated cylinder and added to the test substance 

solution. Mixing then was continued for at least one additional minute. 

Approximately half of the required basal ration was weighed into a tared mixing bowl on a toploading balance. The test substance mixture was then added to the basal ration in the mixing bowl. The 

beaker was rinsed with additional acetone measured in a graduated syringe. The rinse was added to the 

mixing bowl and the contents of the bowl mixed on a stand mixer for approximately 15 minutes. The 

remainder of the basal ration needed for the premix was weighed on a top-loading balance and added to 

the mixing bowl. Mixing was the continued for approximately ten additional minutes. After mixing, 

aliquots of the premix were weighed on a top-loading balance. Unless used immediately for preparation 

of the final diets, the premixes were placed in appropriately labeled plastic bags, re-weighed and stored 

frozen. 

 

 Final diets for adult birds were prepared on August 6, 2010; December 1, 2010; March 4, 2011 and 

Control (0 ppm) only on August 2, 2011. As needed, the appropriate premix was incorporated into the 

final diet for adult birds as follows: 

 0 ppm: 4000 g Premix + 48.250 kg ration + 2750 g limestone 

 0.5 ppm: 4000 g Premix + 48.250 kg ration + 2750 g limestone 

 5 ppm: 4000 g Premix + 48.250 kg ration + 2750 g limestone 

 10 ppm: 4000 g Premix + 48.250 kg ration + 2750 g limestone 
55 

 20 ppm: 4000 g Premix + 48.250 kg ration + 2750 g limestone 

 

 Final diets for chicks (no limestone) were prepared on December 2, 2010. As needed, the 

appropriate premix was incorporated into the final diet for chicks as follows: 

 0 ppm: 4000 g Premix + 51.000 kg ration 

 0.5 ppm: 4000 g Premix + 51.000 kg ration 

 5 ppm: 4000 g Premix + 51.000 kg ration 

 10 ppm: 4000 g Premix + 51.000 kg ration 

 20 ppm: 4000 g Premix + 51.000 kg ration 

 All final diets were mixed for approximately twenty minutes in a Patterson-Kelly®

 Twin Shell 

Blender. 
56 

APPENDIX G 

CONTRIBUTING SCIENTIST REPORT 

STATISTICAL ANALYSIS OF DATA 
57 

Statistical Analysis on USAPHC Toxicology Protocol Number: 08JG-80-08-04-02 

Two-Generation Test for Endocrine Disruption and Reproductive Toxicity in 

Japanese Quail (Corturnix japonica) using Trenbolone 

Good Laboratory Practice Compliance Statement 

The statistical analysis of the data for the Two-generation test for endocrine disruption and reproductive 

toxicity in Japanese quail (Cortunix japonica) using Trenbolone was conducted in compliance with Title 

40, Code of Federal Regulations (CFR), Part 792, Good Laboratory Practice Standards. Statistical 

analyses were conducted according to the procedures outlined in the protocol for this study (Protocol 

Number 08JG-80-08-04-02). 

                   9 May 2012

        

Shane Hall Date 

Statistician 
58 

TRENBOLONE ENDOCRINE DISRUPTION

Egg, Sperm, and Growth Characteristics 

 

 This report analyzes the effects that trenbolone had on the endocrine system of male and female 

quails through three generations (F0, F1, and F2). Data was collected on egg strength and thickness, 

egg production, sperm characteristics, body weights, food consumption, coacal gland (CG) area, and 

days until puberty. All quails were part of one of five dose groups ((control) 0 ppm, 0.5 ppm, 5 ppm, 10 

ppm, and 20 ppm). Ppm level 20 was not included in the F2 generation study. For the endocrine 

disruption metrics that contained weekly measurements, the same quails were tested from week to week. 

SPSS version 16.0 was used to analyze the endocrine disruption data. Statistically significant results 

were determined using α=.05, resulting in a 95% confidence level. 

 
59 

EGGSHELL THICKNESS and STRENGTH 

I. Analysis Methods 

 An Analysis of Variance (ANOVA) test was used to determine if the average eggshell thickness 

and/or average eggshell force among the ppm levels was significantly different. An F-statistic and 

corresponding p-value were calculated for both thickness and strength. If the p-value was below .05, 

then it was concluded that at least two ppm levels had statistically different averages for that measure. A 

Tukey post-hoc test was then used to find which specific ppm levels were statistically different from one 

another. A Levene’s test was used to check to make sure the ppm levels had equal variances, a major 

assumption that must be met in order to properly run an ANOVA model. If this assumption was violated 

(p-value for Levene’s test < .05), a non-parametric Kruskal-Wallis test was run instead of an ANOVA. 

II. Summary of Results 

Thickness averages for each ppm level decreased from the F0 to F1 generation and from the F1 

to F2 generation. Force measurements for each ppm level slightly increased from the F0 to F1 

generation, but decreased from the F1 to F2 generation. Minimal, non-significant differences, especially 

in thickness, occurred among all ppm levels within each of the three generations. 

III. F0 Generation Results 

Non-significant, minimal differences occurred between ppm levels for average thickness and 

average force. Average thickness generally decreased as trenbolone concentration increased. However, 

there was no evidence that showed that force averages were dependent on the degree of concentration 

of trenbolone. Interestingly, a Kruskal-Wallis test was needed for force because the variation of force 

measurements in ppm level 20 was almost two times greater than the other four ppm groups. 

IV. F1 Generation Results 

Non-significant, negligible differences occurred among ppm levels between thickness and force in 

the F1 generation. Additionally, trends in thickness and force averages based on the severity of 

concentration of trenbolone were not present. 

ppm Average Std. Error Average Std. Error

0 0.381 0.010 1.24 0.05

0.5 0.385 0.012 1.12 0.09

5 0.382 0.011 1.30 0.06

10 0.379 0.013 1.23 0.08

20 0.366 0.008 1.07 0.13

F0Generation ‐ Egg Strength

p‐value = .795 p‐value = .558*

* P‐value calcula ted from Krus kall‐wallis tes t ‐ 

homogenei ty of va riance a s s umption viola ted for

pa rame tric ANOVA tes t

Thickness Force
60 

V. F2 Generation Results 

Just like in the F0 and F1 generations, non-significant, minimal differences occurred between 

ppm levels for thickness and force in the F2 generation. Additionally, trends in thickness and force 

averages based on the concentration of trenbolone were not present. 

ppm Average Std. Error Average Std. Error

0 0.320 0.006 1.31 0.09

0.5 0.327 0.007 1.41 0.06

5 0.315 0.006 1.38 0.07

10 0.329 0.008 1.37 0.08

20* 0.345 0.006 0.53 0.27

Thickness Force

* ppm level 20 was excluded from ANOVA test due to

small n (thickness: n=4, force: n=3)

p‐value = .409 p‐value = .787

F1 Generation ‐ Egg Strength

ppm Average Std. Error Average Std. Error

0 0.199 0.005 1.15 0.07

0.5 0.199 0.004 1.01 0.06

5 0.203 0.005 1.09 0.04

10 0.206 0.005 1.17 0.06

F2 Generation ‐ Egg Strength

Thickness Force

p‐value = .590 p‐value = .225
EGG PRODUCTION 

I. Analysis Methods 

A Generalized Estimating Equation – poisson counts model (GEE) was used to determine if there was a significant 

levels in their egg production, accounting for the variability from week to week. P-values were calculated for the ppm level 

well as their interaction. The dose, week, and interaction were determined to be significant if the p-value for that term from 

.05. Estimated marginal means were calculated for both factors and examined for differences using a Tukey post-hoc test, factor was less than .05. If the interaction was statistically significant, the interaction was investigated via marginal means, 

results of the primary factors are considered to be uninformative and can be ignored. II. Summary of Results  In the F0 and F1 generations, there was a high correlation between egg production and the dosing severity of trenb

that the lower the dosing concentration of trenbolone, the higher the egg production. This trend didn’t seem to follow throu

as all four dosing groups had the relatively same egg production, however only seven weeks of data was collected in the FComparing across generations, the F1 generation had lower egg production averages across the weeks, especially for ppm 

III. F0 Generation Results  The ppm by week interaction was statistically significant (p-value = .000) indicating there was a significant differenctrends among the five dose groups through the fifteen weeks. From week seven to week ten, the rate of egg production indose 10 and especially dose 20 compared to the other ppm levels. After week ten, egg production was consistent for each 

dose 20 and dose 10 had the lowest egg production. Dose 5, .5 and the control group had egg production that was similar weeks. IV. F1 Generation Results 

Just like the F0 generation, the interaction was statistically significant (p-value = .000). The increase in egg produc

much less than the other four dose groups in weeks seven through ten. Excluding dose 20, all dose groups had a slight in

production after week ten. Dose 20’s egg production stayed fairly consistent through all nineteen weeks. Overall, egg prod

the control group and dose .5. Dose 5 and dose 10 had the similar egg production counts through the nineteen weeks, but 

Factor P‐value ppm Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 We

0 0.65 2.35 3.35 5.25 4.40 3.85 5.30 5.65 6.10 5.55 5.70 5.80 5.45 6

0.5 0.5 1.3 2.65 4.05 4.15 3.9 4.4 5.15 4.8 4.9 5.05 4.85 5.25 5

5 1.20 2.80 4.30 4.75 5.30 4.45 5.20 5.55 5.65 5.20 5.05 4.10 5.05 4

10 0.25 1.65 2.55 3.40 3.45 3.20 3.60 3.80 3.80 4.35 3.40 3.75 3.95 420 0.30 1.15 1.65 2.40 2.45 2.50 2.80 2.60 3.10 3.05 2.25 2.40 2.75 2

Overall 0.49 1.75 2.76 3.83 3.82 3.51 4.14 4.37 4.55 4.52 4.07 4.01 4.36 4

** Week 6 was not included in the analysis ‐ only two quails produced any eggs in week 6.

F0 Generation Egg Production

GEE Significance Tests GEE Estimated Marginal Means

Interaction ‐            

ppm by week

0.000

ppm 0.000

Week 0.000
compared to the control and .5 dose groups. Dose 20 had a much lower egg production compared to the other four dose gentire nineteen weeks. V. F2 Generation Results 

Significant differences in egg production among dose groups did not occur in the F2 generation. Additionally, all do

had the same fluctuation in egg production through the seven weeks. There was a significant difference among weeks. Fr

four, there was a large increase in egg production. After week four, egg production stayed fairly consistent with the previou

Factor P‐value ppm Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21 Week 22 W

0 0.45 1.25 2.85 3.65 2.25 5.10 4.00 3.45 4.40 5.30 5.40 5.25 5.55 4.70 5.10 5.25 

0.5 0.40 2.30 2.90 3.00 4.20 3.65 4.25 4.10 4.85 4.80 5.45 5.30 5.45 5.40 5.25 5.20 

5 0.14 0.76 2.10 2.38 2.90 3.19 3.33 3.38 4.14 3.90 2.44 4.14 4.10 3.19 4.57 4.43 

10 0.25 1.11 1.32 1.93 2.57 1.93 2.43 2.64 3.04 2.61 2.54 3.25 2.79 2.79 3.39 3.11 20 0.00 0.00 0.17 0.22 0.28 0.67 0.56 0.11 0.50 0.72 0.83 0.83 1.00 0.94 0.89 0.89 

Overall 0.00 0.00 1.31 1.62 2.06 2.38 2.38 1.70 2.66 2.85 3.05 3.15 3.22 2.92 3.26 3.20 

** Week 6 was not included in the analysis ‐ only one quail produced any eggs in week 6.

F1Generation Egg Production

GEE Estimated Marginal Means

ppm 0.000

Week 0.000

Interaction ‐            

ppm by week

0.000

GEE Significance Tests

Factor P‐value ppm Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Overall

0 0.10 2.00 3.25 4.30 5.30 5.45 3.85 2.27

0.5 0.50 1.90 3.70 4.10 4.25 4.55 4.10 2.73

5 0.45 2.25 4.05 5.15 5.00 5.05 5.75 3.15

10 0.10 1.50 2.85 4.45 4.80 5.30 5.35 2.21

Overall 0.22 1.89 3.43 4.47 4.82 5.08 4.69

** Week 5 was not included in the analysis ‐ only one quail produced any eggs in week 6.

ppm 0.268

Week 0.000

GEE Significance Tests

Interaction ‐            

ppm by week

0.080

GEE Estimated Marginal Means

F2 Generation Egg Production
Toxicology Study No. 87-XE-0FV5-12, Protocol No. 0FV5-96-12-05-01, June-August 2012 

9 

SPERM 

I. Analysis Methods 

 An Analysis of Variance (ANOVA) test was used to determine if the averages for percent motile, 

percent progressive, and sperm per gram between the ppm levels were significantly different. An Fstatistic and corresponding p-value were calculated for percent motile, percent progressive, and sperm 

per gram. If the p-value was below .05, then it was concluded that at least two ppm levels had 

statistically different averages for that corresponding measure. A Tukey post-hoc test was then used to 

find which specific ppm levels were statistically different from one another. A Levene’s test was used to 

check to make sure the ppm levels had equal variances, a major assumption that must be met in order to 

properly run an ANOVA model. If this assumption was violated (p-value for Levene’s test < .05), a nonparametric Kruskal-Wallis test was run instead of an ANOVA. 

II. Summary of Results 

Sperm per gram averages increased from the F0 to F1 generation, but then declined drastically in 

the F2 generation. Overall averages were approximately 1,600, 4,100, and 200 for the F0, F1, and F2 

generations, respectively. Percent progressive averages also increased from the F0 to F1 generation, 

and then declined in the F2 generation. However, this decline was not as extreme in magnitude as the 

averages for sperm per gram. Lastly, percent motile averages generally decreased from one parental 

generation to the next. 

 Even though statistical differences occurred within each generation, very little evidence suggests 

that, within each generation, there were substantial differences between ppm levels that didn’t happen 

just because multiple variables were being measured. 

III. F0 Generation Results 

For percent motile, percent progressive, and sperm per gram, comparing ppm level’s averages 

were very consistent. Averages (in all three measures) for ppm levels, ranked from highest to lowest 

were: level 20, level .5, level 5, control group, and finally level 10. However, statistically significant 

differences only occurred for percent motile, in which ppm level 20 had a significantly higher percent 

compared to ppm level 10. 

IV. F1 Generation Results 

Ppm level 20 consistently had the highest averages for all three metrics. Ppm level .5, having 

the lowest averages for all three metrics, did not follow its trend in the F0 generation. However, 

statistically significant differences only occurred for percent progressive in which ppm level .5 had a 

significantly lower percent compared to ppm level 20. 

ppm Average Std. Error Average Std. Error Average Std. Error

0 54.3 5.9 11.0 2.8 1259.8 248.4

0.5 66.1 5.3 18.6 5.6 1738.8 301.0

5 61.7 6.5 14.9 5.4 1580.2 489.7

10 49.5 6.1 10.9 3.6 1198.1 230.4

20 73.8 5.1 18.8 3.4 2100.1 429.8

F0 Generation ‐ Sperm

% Motile Sperm/gram % Progressive

p‐value = .042* p‐value = .520 p‐value = .377

* ppm level 10 and level 20 a re signi fica ntly di fferent from each othe r

determined by Tukey Pos t‐hoc tes t using a 95% confidence level.

§ ID 488 (ppm = .5) excluded from analysi s because da ta poi nts for each of the

three mea s urements were extreme outliers
64 

V. F2 Generation Results 

Percent motile and percent progressive averages were lowest for the control group and ppm level 

5. Sperm per gram averages were lowest for ppm level 5 but were highest for the control group. Sperm 

per gram was the only measurement that had statistically significant differences between ppm levels (pvalue =.030). Sperm per gram averages for the control group were significantly greater than ppm level 5. 

 

ppm Average Std. Error Average Std. Error Average Std. Error

0 48.2 6.9 26.2 4.1 3852.1 685.9

0.5 30.0 8.8 16.6 4.8 3063.2 1021.7

5 48.9 8.2 29.2 5.2 4516.0 898.4

10 60.1 5.8 35.6 4.5 3672.5 918.9

20 60.3 3.2 35.8 2.2 5555.3 1044.0

F1Generation ‐ Sperm

% Motile % Progressive Sperm/gram

p‐value = .066 p‐value = .030* p‐value = .390

* Tukey Pos t‐hoc res ul ts indica te tha t level .5 and level 20 have signi fi ca ntly

di ffe rent % progres sive averages.

§ ID 46 (ppm = .5) excl uded from a nalysi s beca use da ta points for each of the

three mea s urements were extreme outliers

ppm Average Std. Error Average Std. Error Average Std. Error

0 29.5 4.9 8.1 1.7 266.1 29.4

0.5 38.3 4.5 12.4 1.6 217.3 15.8

5 30.9 5.6 9.4 2.5 166.5 22.4

10 44.4 3.8 12.8 1.8 179.2 15.3

% Motile % Progressive Sperm/gram

F2Generation ‐ Sperm

p‐value = .115 p‐value = .258 p‐value = .039*°

* Non‐pa rame tric ranks tes t i ndica tes tha t control group i s signi fica ntly di fferent

from ppm level 5.

°P‐value calcula ted from Kruskall‐wallis tes t ‐ homogenei ty of va riance

a s s umpti on vi ola ted for pa rame tric ANOVA tes t
CHICK GROWTH 

I. Analysis Methods 

A repeated measured Linear Mixed Model was run to determine if there was a significant difference in body weight

levels. A repeated measures model was used to test the dose and week factors as well as their interaction, while accountithe data from week to week. The dose, week, and interaction were determined to be significant if the p-value for the Mixed than .05. Estimated marginal means were calculated for both factors and examined for differences using a Tukey post-hoc that factor was less than .05. If the interaction was statistically significant, the interaction was investigated via marginal me

significance results of the primary factors are considered to be uninformative and can be ignored. 

II. Summary of Results 

Overall, females had greater body weights than males through all three generations. At the start of each generatio

were slightly greater than males, but by the end of each generation, bodyweights for females were well above the bodywei

 For females, comparing the same week at each generation, overall bodyweights were similar across all three genedifference though, was that at the start of the F2 generation, bodyweights were lower than they were at the start of the F0 a

Comparing ppm levels, ppm level 20 had a much lower bodyweight in the first two generations compared to the other four  Male bodyweights, comparing the same week at each generation, were higher for the F1 generation compared to t

generation bodyweights were more comparable to the F1 rather than F0 generation, albeit only ten weeks of bodyweights wF2 generation. Just like in the females, the F2 generation bodyweights at the start of the generation were lower than they w

F0 and F1 generations. Ppm level 20 had a lower bodyweight compared to the other levels in the F1 generation, however as drastic as it was for the females. III. F0 Generation Results 

For both genders, week and ppm level were statistically significant, but their interaction was not. For both genders

from one week to the next, especially in the first seven to ten weeks. For females, the control group had a significantly gre

compared to the other four ppm levels. Additionally, ppm level 20 had a body weight that was significantly less than the oththe males, body weights were lower than females and the variation between the ppm levels was much less compared to thgroup had the highest overall average body weight and was statistically greater than ppm level .5 and level 10. 

Model Term P‐value ppm Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 

0 98.5 106.1 118.0 124.3 128.4 134.6 135.4 135.7 139.3 140.0 142.2 141.6 141.1 142.5 142.4 

0.5 92.5 100.9 110.9 115.5 124.5 129.7 130.8 135.1 135.8 137.0 138.6 138.7 138.7 141.9 141.9 

5 93.0 100.9 115.0 120.2 128.3 132.8 134.9 136.2 139.2 139.2 139.9 138.2 135.7 138.2 138.6 

10 94.0 101.8 111.4 120.2 125.0 130.3 132.3 133.8 132.9 134.8 138.1 137.2 139.5 139.7 139.6 20 89.2 98.1 107.5 114.5 120.3 122.6 126.6 126.1 130.1 130.6 133.1 130.6 129.2 134.2 133.9 

Overall 93.4 101.6 112.5 118.9 125.3 129.9 132.0 133.4 135.5 136.3 138.4 137.2 136.8 139.3 139.3 

ppm 0.000

F0 Generation Female Body Weights

Significance Tests Estimated Marginal Means

Interaction ‐          

ppm by week

1.000

Week 0.000
IV. F1 Generation Results 

For both genders, week and ppm level were statistically significant, but the interaction was not. Body weights gene

one week to the next; however female body weights increased at a higher rate compared to males in the latter weeks. Ove

weights were higher than the male body weights. Comparing the ppm levels within females, the only statistically significanppm level 20 had a lower body weight average compared to the other four ppm levels. For males, ppm level 20 once again 

lower body weight average compared to the other four ppm levels. Bodyweights collected on 12/10, 12/16, 12/22, 12/29/ afrom the statistical analysis due to very sparse data collection. 

V. F2 Generation Results 

Just like in the F0 and F1 generations, week and ppm level were statistically significant, but the interaction was not 

females. For both genders, body weights increased far less severely after week 5, however there was still a slight increaseweight for the ppm levels were minimal for males and females, however statistically significant differences did exist. In fema significantly higher body weight compared to the control group and ppm level .5. For males, ppm level .5 had a significancompared to the other three ppm levels. Bodyweights collected on 6/9, 6/18 and 6/26 were excluded from the statistical ansparse data collection. 

Model Term P‐value ppm Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 W

0 90.6 98.8 104.5 106.5 107.8 110.0 111.1 111.6 112.9 113.1 113.6 114.0 115.0 115.3 

0.5 88.5 98.2 101.9 102.7 104.2 106.0 107.4 108.4 110.9 111.7 111.9 112.1 112.8 113.4 

5 90.9 98.1 103.0 104.7 106.0 107.9 109.2 107.2 111.1 112.2 112.2 112.7 112.6 113.1 

10 86.7 95.4 101.5 104.3 105.8 106.8 107.9 108.4 110.2 111.1 111.7 112.3 113.1 114.4 20 89.2 96.7 102.8 104.4 106.6 107.6 110.1 109.4 111.7 113.5 111.5 112.5 113.2 113.7 

Overall 89.2 97.4 102.7 104.5 106.1 107.6 109.1 109.0 111.3 112.3 112.2 112.7 113.3 114.0 

Week 0.000

F0 Generation Male Body Weights

Significance Tests Estimated Marginal Means

Interaction ‐          

ppm by week

1.000

ppm 0.002

Model Term P‐value ppm Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 W

0 94.1 103.6 111.8 114.3 125.5 125.5 132.0 131.1 133.4 133.5 137.5 141.3 140.0 138.6 143.1 142.8 1

0.5 92.2 103.6 113.3 118.9 125.8 126.5 131.6 133.3 133.6 133.6 139.3 135.9 139.2 139.2 143.5 141.9 1

5 92.5 101.4 107.3 113.2 120.6 125.1 126.7 134.0 134.3 134.9 135.7 134.7 136.3 134.4 140.1 140.6 1

10 91.9 101.9 113.6 115.5 122.7 123.5 129.0 127.2 134.6 137.7 136.3 135.5 137.4 134.6 141.7 145.7 120 82.8 95.9 101.8 104.3 110.3 113.0 116.1 120.8 120.1 122.4 123.3 124.9 126.4 130.2 126.4 131.0 1

Overall 90.7 101.3 109.5 113.2 121.0 122.7 127.1 129.4 131.2 132.4 134.4 134.5 135.9 135.4 138.9 140.4 1

0.000

Significance Tests Estimated Marginal Means

F1 Generation Female Body Weights

Interaction ‐          

ppm by week

0.988

ppm 0.000

Week

Model Term P‐value ppm Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 W

0 92.8 101.8 106.7 106.8 109.1 110.1 111.9 111.4 112.8 112.7 114.6 115.5 115.7 116.1 117.3 117.6 1

0.5 90.4 97.3 105.1 107.3 109.2 111.5 113.0 112.1 110.2 111.8 115.1 118.0 116.6 115.2 117.2 116.9 1

5 91.6 100.6 105.2 106.7 108.9 110.7 110.9 111.9 112.0 112.0 113.6 115.5 115.0 115.7 117.8 116.9 1

10 85.0 96.5 104.1 105.1 106.4 108.4 110.0 110.6 110.4 110.6 114.1 113.6 117.9 113.9 117.1 116.9 120 77.7 89.8 98.4 100.6 104.9 106.9 106.8 108.5 108.5 108.8 112.5 112.2 112.6 115.1 118.2 117.9 1

Overall 87.5 97.2 103.9 105.3 107.7 109.5 110.5 110.9 110.8 111.2 114.0 115.0 115.5 115.2 117.5 117.2 1

ppm 0.000

Week 0.000

F1Generation Male Body Weights

Significance Tests Estimated Marginal Means

Interaction ‐          

ppm by week

0.177
Model Term P‐value ppm Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 

0 83.2 97.4 101.7 112.4 126.1 128.4 129.4 132.4 136.9 131.9 

0.5 86.9 98.3 103.7 112.8 125.8 128.8 129.3 130.6 133.2 131.6 

5 88.1 98.9 103.1 115.3 128.3 132.0 134.0 130.2 132.4 137.2 

10 86.0 102.3 106.7 115.4 126.5 128.5 136.9 134.7 137.5 140.2 

Overall 86.1 99.2 103.7 113.9 126.7 129.4 132.4 132.0 135.0 135.2

Estimated Marginal Means

Week 0.000

Significance Tests

Interaction ‐          

ppm by week

0.727

ppm 0.001

F2 Generation Female Body Weights

Model Term P‐value ppm Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 

0 83.9 93.0 96.6 104.1 108.2 107.1 107.9 108.7 109.7 

0.5 77.5 91.4 95.7 103.3 105.9 104.0 104.9 106.7 107.7 

5 81.5 95.8 99.1 104.6 107.2 107.0 109.4 110.9 112.4 

10 82.5 94.8 99.0 104.9 107.8 107.7 109.8 109.5 112.3 

Overall 81.4 93.7 97.6 104.2 107.3 106.4 108.0 108.9 110.5

Estimated Marginal Means

ppm 0.000

Week 0.000

Significance Tests

Interaction ‐          

ppm by week

0.988

F2 Generation Male Body Weights
68 

FEED CONSUMPTION 

I. Analysis Methods 

A repeated measured Linear Mixed Model was run to determine if there was a significant 

difference in food consumption between the five dose groups. A repeated measures model was used to 

test the dose and week factors as well as their interaction, while accounting for the variation in the data 

from week to week. The dose, week, and interaction were determined to be significant if the p-value for 

the Mixed Model test was less than .05. Estimated marginal means were calculated for both factors and 

examined for differences using a Tukey post-hoc test, if the p-value for that factor was less than .05. If 

the interaction was statistically significant, the interaction was investigated via marginal means. The 

significance results of the primary factors are considered to be uninformative and can be ignored. Also, 

the food consumption calculations that were incorrectly recorded as negative were excluded from the 

analysis since it was impossible for the birds to eat more feed than was originally in the container. 

II. Summary of Results 

Differences in food consumption occurred among ppm levels in all three generations and in both 

genders. However, food consumption averages and these differences did not seem to be completely 

dependent upon ppm level, as there was never a uniform trend of increase or decrease in food 

consumption as trenbolone concentration increased. 

 

III. F0 Generation Results (Week 5 through Week 10 – unpaired genders) 

The ppm by week interaction was significant for females, but not for males. Statistically 

significant differences in weeks were present for both genders. For females, ppm levels .5 and 10’s 

results were not as uniform as the other ppm levels. Ppm level .10 started out higher than the other ppm 

levels, and ppm level .5 had a lower food consumption average compared to the other groups at weeks 

eight and nine. Overall, food consumption was consistent from week to week until week nine and ten 

where it greatly increased. 

 Overall significant differences occurred among ppm levels for the males in the F0 generation. 

Ppm level 5 had a significantly greater food consumption compared to all other levels except ppm level 

10. The control group had a significantly lower food consumption compared to all ppm levels except ppm 

level .5. 

IV. P Generation Results Week 13-21 

Significant differences in food consumption occurred among the ppm levels and throughout the 

weeks of the study, and their interaction was not statistically significant. Ppm level 10 had a significantly 

Factor P‐value ppm Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Overall

0 119.2 124.7 130.1 137.3 156.9 175.6 140.6

0.5 116.4 114.1 128.7 114.7 150.5 169.4 132.3

5 109.1 120.1 121.6 132.9 173.3 175.9 138.8

10 137.1 128.5 139.4 132.9 167.1 169.1 145.7

20 113.1 125.6 129.6 132.3 165.9 150.7 136.2

Overall 119.0 122.6 129.8 130.0 162.7 168.1

Significance Tests Estimated Marginal Means

Week 0.000

Interaction ‐ 

ppm by week

0.005

ppm 0.000

F0Generation Female Food Consumption

Factor P‐value ppm Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Overall

0 110.7 111.2 111.8 126.0 110.2 114.4 114.0

0.5 110.6 109.9 105.3 132.5 121.8 122.6 117.1

5 122.3 134.2 128.7 143.1 135.3 135.2 133.1

10 117.1 123.1 135.8 128.1 130.8 131.4 127.7

20 111.4 118.6 126.6 124.8 131.9 126.2 123.2

Overall 114.4 115.9 121.6 130.9 126.0 125.9

Week 0.000

F0 Generation Male Food Consumption

Significance Tests Estimated Marginal Means

Interaction ‐ 

dose by week

0.060

ppm Level 0.000
69 

greater food consumption compared to all other levels, while ppm level 20 had a significantly lower food 

consumption compared to all other levels. Additionally, food consumption did not have any trend in 

magnitude based on the degree of trenbolone concentration. These results indicate that there were 

major differences between dose groups, but these differences are not solely based on the concentration 

of trenbolone. Lastly, food consumption generally increased from week 13 through week 21, but peaked 

at week 19. 

 

V. F1 Generation Results 

Ppm by week interactions were significant for both females and males (p-values = .003 and .047). 

For females and males, the interaction was significant because there were increasing differences in food 

consumption among ppm levels as week increased. Overall in females, ppm level .5 had the highest food 

consumption and the control group had the lowest food consumption. In males, ppm level 10 had the 

highest food consumption while the control group had the lowest food consumption. 

VI. F1 Generation Results Week 14-23 

With a p-value of .000, the ppm level by week interaction was statistically significant, fluctuation of 

ppm levels at each week likely caused the significant p-value. Even though ppm level 20 typically had the 

lowest food consumption at each week, there was fluctuation between the other levels as to which had 

the highest average, which likely caused the significant interaction. With a slight trend upward in food 

consumption from week fourteen to week twenty-three, food consumption peaked at week twenty-two and 

was lowest at week fifteen. 

Factor P‐value ppm Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21 Overall

0 300.1 302.9 316.2 282.9 308.2 305.6 322.7 326.9 320.8 309.6

0.5 290.1 282.0 282.6 290.9 295.3 286.2 316.5 331.5 312.8 298.7

5 287.7 284.8 294.5 276.7 292.5 320.1 357.4 337.1 325.8 308.5

10 282.8 293.1 303.8 311.2 336.3 326.6 387.4 357.5 344.5 327.0

20 252.2 264.2 255.3 260.5 277.9 286.3 344.4 296.1 303.6 282.3

Overall 282.6 285.4 290.5 284.4 302.1 305.0 345.7 329.8 321.5

Significance Tests

Week

Estimated Marginal Means

F0Generation Paired‐Gender Food Consumption

0.000

Interaction ‐ 

ppm by week

0.155

ppm 0.000

Factor P‐value ppm Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Overall

0 127.5 136.4 123.3 103.9 144.7 139.1 129.1

0.5 125.0 141.0 160.3 161.3 164.8 178.8 155.2

5 118.2 115.3 128.6 149.0 169.3 154.0 139.1

10 130.6 141.4 128.1 148.4 148.7 162.5 143.3

20 118.4 122.1 112.7 168.5 223.5 132.9 146.4

Overall 124.0 131.2 130.6 146.2 170.2 153.5

Week 0.000

Significance Tests Estimated Marginal Means

F1 Generation Female Food Consumption

Interaction ‐ 

ppm by week

0.003

ppm 0.020

Factor P‐value ppm Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Overall

0 116.2 112.5 114.3 108.5 126.2 108.3 114.3

0.5 114.4 117.6 128.1 141.1 145.7 120.0 127.8

5 114.5 118.3 120.0 135.6 159.4 122.2 128.3

10 131.8 124.5 129.9 159.4 148.9 148.2 140.5

20 116.7 123.6 114.4 122.6 135.8 115.8 121.5

Overall 118.7 119.3 121.3 133.4 143.2 122.9

F1 Generation Male Food Consumption

Interaction ‐ 

ppm by week

0.047

ppm 0.000

Week 0.000

Significance Tests Estimated Marginal Means
70 

VII. F2 Generation Results 

For females, overall and weekly averages were very similar for the four ppm levels with the 

exception of the final week of data collection where ppm level 10 has a spike in food consumption. This 

likely caused the significant interaction. Food consumption weekly averages generally increased from 

week to week, with the first two weeks having much lower consumption averages compared to the others. 

Overall, ppm level 5 and 10 had higher food consumption averages compared to the control and .5 levels. 

 For males, just like in females, ppm level 5 and 10 had the highest food consumption averages. 

Even with a p-value less than .05 (p-value =.04), after adjusting for multiple testing, there were no 

significant differences between ppm levels. From week to week, food consumption generally increased, 

but the four ppm levels had very similar results. 

 

Factor P‐value ppm Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21 Week 22 Week 23 Overall

0 289.2 244.8 272.6 305.8 192.2 301.0 305.1 262.5 348.9 305.2 282.7

0.5 318.1 242.6 263.2 178.1 210.9 307.8 303.9 256.0 350.2 311.6 274.2

5 286.6 221.3 246.9 260.8 183.2 257.4 298.8 250.3 299.8 323.1 262.8

10 268.0 224.4 243.1 252.8 238.5 281.8 304.1 250.3 329.3 292.8 268.5

20 258.5 175.7 202.2 244.1 202.6 236.5 217.5 229.0 303.3 255.3 232.5

Overall 284.1 221.8 245.6 248.3 205.5 276.9 285.9 249.6 326.3 297.6

Significance Tests

Interaction ‐ 

ppm by week 0.000

ppm 0.000

Week 0.000

F1 Generation Paired‐Gender Food Consumption

Estimated Marginal Means

Factor P‐value ppm Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Overall

0 112.4 105.3 137.4 155.6 136.9 150.5 133.0

0.5 104.4 116.2 133.8 149.6 132.6 147.5 130.7

5 105.3 108.1 148.1 159.3 141.2 156.9 136.5

10 108.0 101.6 139.3 154.4 146.5 181.6 138.7

Overall 107.5 107.8 139.6 155.0 139.3 159.1

F2Generation Female Food Consumption

Estimated Marginal Means

Week 0.000

Significance Tests

Interaction ‐ 

ppm by week

0.004

ppm 0.054

Factor P‐value ppm Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Overall

0 92.0 105.4 105.2 111.5 108.6 117.5 106.7

0.5 92.7 97.8 107.4 116.4 109.5 117.0 106.8

5 99.4 103.4 114.8 116.4 113.4 118.2 110.9

10 98.7 99.7 113.8 112.3 116.0 123.7 110.7

Overall 95.7 101.5 110.3 114.1 111.9 119.1

ppm 0.040

Week 0.000

Estimated Marginal Means

Interaction ‐ 

ppm by week

0.669

Significance Tests

F2 Generation Male Food Consumption
71 

CLOACA GLAND (CG) AREA 

I. Analysis Methods 

 A General Linear Model – Repeated Measures (GLMREP) test was used to determine if the CG 

area was statistically difference between the ppm levels, while accounting for the variation throughout 

weeks. P-values were calculated for ppm level and week factors as well as their interaction. The ppm 

level, week, and interaction were determined to be significant if the p-value for the GLM was less than 

.05. Estimated marginal means were calculated for both factors and examined for differences using a 

Tukey post-hoc test, if the p-value for that factor was less than .05. If the interaction was statistically 

significant, the interaction was investigated via marginal means, and the significance results of the 

primary factors are considered to be uninformative and can be ignored. 

II. Summary of Results 

Not surprisingly, CG area increased from week one to week four in all three generations. 

Comparing the three generations, the F0 generation had the highest CG area averages at each particular 

week. F1 and F2 generations from week to week were generally similar, however F2’s CG area in the 

later weeks was increasingly greater than the F1 generation. Only ppm level .5 had clear differences in 

CG area compared to the other ppm levels, and that was mainly in the F1 and F2 generations. 

III. F0 Generation Results 

In the P generation, the ppm by week interaction was statistically significant indicating that as 

time (week) increased, the rate of increase for each ppm level was not equal. The interaction was 

significant because the two most concentrated ppm levels increased CG area at a much higher rate from 

week one to week two compared to the other three levels. Then from week seven to week nine, the three 

low concentration levels increased CG area at a much higher rate compared to ppm level 10 and level 20. 

Overall, all five ppm levels started (week six) and finished (week nine) with approximately the same CG 

area averages. 

IV. F1 Generation Results 

P-values indicated that ppm level and week were statistically significant, however their interaction 

was not. We can concluded that there were overall differences within the ppm levels, however the rate of 

increase from week to week was equal for all ppm levels. A Tukey post-hoc test revealed that ppm level 

.5 had a significantly lower CG area compared to ppm level 5 and ppm level 20. 

Factor P‐value ppm Week 6 Week 7 Week 8 Week 9 Overall

0 136.5 157.5 230.4 300.2 206.1

0.5 146.7 178.6 243.2 292.5 215.3

5 153.0 175.7 248.2 315.1 222.9

10 151.2 228.3 258.9 300.9 234.8

20 124.2 212.4 255.3 302.3 223.6

Overall 142.3 190.5 247.2 302.2

*  95% confidence limi ts reveal each week i s signi fica ntly di fferent from the remaini ng three weeks.

Interaction ‐          

ppm by Week

ppm

Week

0.000°

0.197

0.000*

F0 Generation Cloaca

GLM Significance Tests

° ppm level 10 and level 20 increa se cg a rea signi ficantly from week 6 to week 7, while the two low ppm level s and

control group signi ficantly increa se cg a rea from week 7 to week 8.

GLM Estimated Marginal Means
72 

V. F2 Generation Results 

With p-values well above .05 for ppm level and the interaction, the only statistically significant 

variation in the F2 generation data was a change from week to week. Week was statistically significant 

because from one week to the next, CG area greatly increased. Even though statistically significance 

could not be claimed, ppm level 20 and level 10 had much higher overall CG areas compared to the 

control and .5 ppm level. 

 

Factor P‐value ppm Week 5 Week 6 Week 7 Week 8 Overall

0 71.6 97.5 168.6 218.1 138.9

0.5 60.7 86.3 101.9 149.7 99.6

5 85.3 114.7 160.9 208.8 142.4

10 86.9 127.2 147.2 186.9 137.1

20 110.0 129.3 141.7 234.6 153.9

Overall 82.9 110.9 144 199.6

* Using 95% confi dence limi ts, each week i s signi fica ntly di fferent from the remai ning three weeks.

GLM Significance Tests GLM Estimated Marginal Means

° Res ul ts from Tukey Pos t‐hoc tes t i ndica te tha t ppm level .5 i s signi fica ntly di fferent from ppm level 5 and ppm level

20.

F1 Generation Cloaca

Week 0.000*

Interaction ‐          

ppm Level by Week

0.289

ppm 0.006°

Factor P‐value ppm Week 4 Week 5 Week 6 Week 7 Overall

0 70.4 106.7 180.4 219.8 144.3

0.5 58.9 107.5 166.2 232.1 141.2

5 73.0 121.1 198.4 267.4 164.9

10 75.3 117.6 186.8 266.1 161.5

Overall 69.4 113.2 182.9 246.3

* Using 95% confidence limi ts, each week i s signi ficantly di fferent from the remaining three weeks.

Interaction ‐          

ppm by Week

0.369

ppm 0.277

Week 0.000*

F2Generation Cloaca

GLM Significance Tests GLM Estimated Marginal Means
73 

ONSET TO PUBERTY 

I. Analysis Methods 

 An Analysis of Variance (ANOVA) test was used to determine if onset to puberty averages for 

dose groups were significantly different. An F-statistic and corresponding p-value was calculated for each 

gender within each generation. If the p-value was below .05, then it was concluded that at least two dose 

groups had statistically different averages for onset to puberty. A Tukey post-hoc test was then used to 

find which specific dose groups were statistically different from one another. A Levene’s test was used to 

check to make sure the dose groups had equal variances, a major assumption that must be met in order 

to properly run an ANOVA model. If this assumption was violated (p-value for Levene’s test < .05), a nonparametric Kruskal-Wallis test was run instead of an ANOVA.

II. Summary of Results 

At each generation, variation among ppm levels was much greater for females compared to 

males. Additionally, females had longer averages compared to males for each generation. Besides the 

gender differences, there weren’t any apparent differences between ppm levels or between generations 

that were consistent. 

III. F0 Generation Results 

 Statistically significant differences occurred among female ppm levels, but not male ppm levels. 

For females, ppm level 5 had a significantly shorter onset to puberty compared to ppm level 20 and level 

.5. Ppm levels 5 and the control group had very similar onset to puberty averages, while ppm levels .5, 

10, and 20 have very similar averages. 

 For males, ppm level 20 had the longest onset to puberty average, just like the females. 

However, differences between onset to puberty averages were very minimal for the five ppm level groups. 

IV. F1 Generation Results 

 Just like in the F0 generation, statistically significant differences occurred among female ppm 

levels, but not male ppm levels. For females, ppm level 20 had a significantly longer onset to puberty 

compared to all other ppm levels. All four other ppm levels had similar onset to puberty averages. 

 In males, ppm level .5, followed by the control group had the longest onset to puberty, but was 

not different enough to be significantly different. 

ppm Average Std. Error Average Std. Error

0 61.4 2.14 45.6 0.70

0.5 71.6 4.96 46.1 1.00

5 59.4 1.71 45.5 0.77

10 72.8 6.14 46.2 0.86

20 73.4 5.60 47.8 0.68

0P‐value calculated from Kruskal‐Wallis test ‐ 

homogeneity of variance assumption violated.

*Post‐hoc test indicate that ppm level 5 has significantly

shorter onset to puberty compared to level 20 and .5.

F0 Generation Onset to Puberty (days)

Females Males

P‐value = .036*0 P‐value = .256
74 

V. F2 Generation Results 

 No statistically significant differences in onset to puberty averages occurred among ppm levels for 

males or females in the F2 generation. Both genders had very similar onset to puberty averages among 

the dose groups with females ranging from 60.8 to 65.6 days and males ranging from 48.3 to 51.1 days. 

 

ppm Average Std. Error Average Std. Error

0 67.7 4.02 49.1 6.86

0.5 75.6 6.71 58.3 6.65

5 75.5 3.39 39.8 1.50

10 72.0 5.81 43.9 8.80

20 113.9 12.76 42.9 7.17

*Tukey post‐hoc test indicate that ppm 20 has

signi ficantly longer onset to puberty compared to all

other ppm levels.

F1Generation Onset to Puberty (days)

Females Males

P‐value = .000* P‐value = .283

ppm Average Std. Error Average Std. Error

0 64.9 2.10 51.1 2.52

0.5 62.0 2.14 51.0 2.86

5 60.8 1.19 48.3 1.11

10 65.6 1.81 48.5 2.08

F2Generation Onset to Puberty (days)

Females Males

P‐value = .202 P‐value = .700
75 

TRENBOLONE EFFECTS ON REPRODUCTIVE 

BEHAVIORS

Mounting and Copulation Disruption 

 

 This section of the report analyzes the sexual initiation behavior characteristics of male Japanese 

quails through three generations (F0, F1, and F2). Specifically, the number of mount attempts, time to 

first mount and first copulation, and number of copulations were analyzed. Each pair of quails was part of 

one of five dose groups: (control) 0 ppm, .5 ppm, 5 ppm, 10 ppm, and 20 ppm. Data was collected on the 

same pairs through three consecutive trials in each of the three generations. SPSS version 16.0 was 

used to analyze the behavior data. Statistical significance for this analysis was determined using an 

alpha (α) of .05 with a 95% confidence level. 
76 

NUMBER OF MOUNT ATTEMPTS 

I. Analysis Methods 

A Generalized Linear Model (GzLM) using a poisson loglinear link function was used to determine 

if there was a statistically significant difference in the number of mounts attempted among the five dose 

levels. An omnibus likelihood ratio test in GzLM, which compares the fitted model (intercept plus dose) 

against the intercept only model, was used to determine if dose was a significant factor in predicting the 

number of mounts in 180 seconds. If the p-value for the likelihood test was less than .05, dose was a 

significant factor in predicting the number of mount attempts. Parameter estimates (Beta coefficients and 

Beta confidence intervals) were then examined to find where the significant differences occurred among 

the dose levels if the p-value for the omnibus test was less than .05. In the GzLM procedure, a reference 

group was needed for the other dose levels to be tested against for a significant difference. This was 

because we fit a categorical predictor into a linear model. The reference group for this analysis was the 

‘dose=0’ group which is referred to as the control group throughout the report. An exponentiated β

coefficient of 1.00 meant that the dosed group was 1.00 times as likely to have a mount compared to the 

control group, or in other words, the two groups had an equal chance of having a mount. If the 

confidence interval contained 1.00, there was no statistical difference between the two groups and vice 

versa. 

II. Summary of Results 

For each dose level, the number of mount attempts stayed comparatively consistent from day 

one of the F0 generation to day three of the F1 generation. However, in the F2 generation, all dose 

groups (besides the control) greatly increased their number of mount attempts compared to the other two 

generations. The control group’s number of mounts slightly decreased, compared to the F0 and F1 

generation. Comparing the five dose groups, in all three days of the F0 and F1 generations, the control 

group consistently had the greatest number of mounts while ppm level 20 had the lowest number of 

mounts. F2 generation results, as suggested above, were not as consistent as the F0 and F1 

generations. 

III. F0 Generation Results 

For all three days, the number of estimated mounts increased as dose concentration decreased, 

with the control group consistently having the highest number of average mounts. Significant differences 

among dose levels, however only occurred in the last two days of the F0 generation. In days two and 

three, the control group had a significantly greater number of mount attempts compared to dose 5 and 

dose 20. Additionally, on day two, the control group also had a significantly greater number of mount 

attempts compared to dose 10. 

IV. F1 Generation Results 

As was the case with the F0 generation, the number of estimated mounts generally increased as 

dose concentration decreased. In all three days of the F1 generation, the control group had significantly 

greater number of estimated mounts compared to at least two of the dosed groups. Dose 5 and dose 20 

had significantly less mounts compared to the control for all three trials. 

Dose

Average number

of Mounts

95% β Confidence

Interval* P‐value Average number

of Mounts

95% β Confidence

Interval* P‐value Average number

of Mounts

95% β Confidence

Interval* P‐value

0 3.50 3.17 3.08

0.5 2.75 .50‐1.24 0.30 1.92 .361‐1.016 0.06 2.08 .41‐1.12 0.13

5 2.08 .36‐.98 0.04 1.58 .288‐.867 0.01 1.67 .31‐.93 0.03

10 2.08 .36‐.98 0.04 1.50 .270‐.830 0.01 2.08 .41‐1.12 0.13

20 1.83 .32‐.88 0.01 1.08 .182‐.642 0.00 0.75 .12‐.50 0.00

F0 Generation ‐ Number of Mounts

Day 1

Omnibus Likelihood ratio p‐value = .071

Day 2

Omnibus Likelihood ratio p‐value = .004

Day 3

Omnibus Likelihood ratio p‐value = .001

* Confidence Intervals reflect comparison to control group (ppm = 0).  Ppm levels with a confidence interval not including 1.00 is considered to be significantly

different from the control group.
77 

V. F2 Generation Results 

In all three days of the F2 generation, no statistically significant differences in number of mount 

attempts occurred among the dose groups. Additionally, even though the control group typically had one 

of the highest estimated mount totals, there was no apparent increase in the number of mount attempts 

as trenbolone concentration decreased, like there was in the two previous generations. 

 

Dose

Average number

of Mounts

95% β Confidence

Interval* P‐value Average number

of Mounts

95% β Confidence

Interval* P‐value Average number

of Mounts

95% β Confidence

Interval* P‐value

0 3.25 2.85 3.60

0.5 2.00 .42‐.91 0.02 1.75 .40‐.94 0.02 2.80 .55‐1.10 0.16

5 1.60 .32‐.75 0.00 1.45 .33‐.80 0.00 2.20 .42‐.89 0.01

10 1.69 .32‐.84 0.02 2.62 .60‐1.40 0.69 2.61 .48‐1.09 0.13

20 1.00 .17‐.56 0.00 1.69 .36‐.97 0.04 1.38 .23‐.65 0.00

Day 2 Day 3

Omnibus Likelihood ratio p‐value = .000 Omnibus Likelihood ratio p‐value = .011 Omnibus Likelihood ratio p‐value = .002

* Confidence Intervals reflect comparison to control group (ppm = 0).  Ppm levels with a confidence interval not including 1.00 is considered to be significantly

different from the control group.

F1 Generation ‐ Number of Mounts

Day 1

Dose

Average number

of Mounts

95% β Confidence

Interval* P‐value Average number

of Mounts

95% β Confidence

Interval* P‐value Average number

of Mounts

95% β Confidence

Interval* P‐value

0 2.85 3.25 3.05

0.5 3.55 .88‐1.76 0.22 3.00 .65‐1.31 0.66 2.70 .61‐1.28 0.51

5 2.45 .59‐1.26 0.44 2.45 .52‐1.09 0.14 2.50 .56‐1.19 0.30

10 2.94 .72‐1.49 0.95 3.10 .67‐1.35 0.79 2.95 .68‐1.38 0.86

* Confidence Intervals reflect comparison to control group (ppm = 0).  Ppm levels with a confidence interval not including 1.00 is considered to be significantly

different from the control group.

F2 Generation ‐ Number of Mounts

Day 1 Day 2 Day 3

Omnibus Likelihood ratio p‐value = .245 Omnibus Likelihood ratio p‐value = .465 Omnibus Likelihood ratio p‐value = .722
78 

TIME UNTIL FIRST MOUNT ATTEMPT 

I. Analysis Methods 

Kaplan-Meier survival analysis was used to determine if there was a significant difference among 

the five dose levels in terms of the amount of time it took the birds for their first mount attempt. If their 

time was recorded as 180 seconds, this meant that the quail did not make a mount attempt and that 

observation was considered censored. A log rank statistic was computed to test whether the different 

dose groups had statistically different times until the first mount attempt. If the p-value associated with 

the log rank statistic was less than .05, it was concluded that the times between at least two of the dose 

groups were not statistically equal. To find which dose groups did not have equal times, confidence 

intervals for the time to attempted mount were compared. If two dose groups had confidence intervals 

that did not overlap, these two doses were considered to have statistically different times. 

II. Summary of Results 

Mirroring the major differences in the number of mount attempts, in all three days of the F0 and 

F1 generations, the control group consistently had the shortest time until first mount attempt while dose 

20 had the longest time until first mount attempt. The other three dose groups’ times were very similar 

compared to each other throughout all three generations of the study. In the F1 and F2 generations, day 

one typically had the longest time until first mount and day two and day three’s times gradually decreased 

from day one. Lastly, in the F2 generation, the control group’s times were very similar to the other dose 

groups, which was not the case in the F0 and F1 generation. 

III. F0 Generation Results 

For all three days, the lower the concentration of trenbolone, the shorter the time until the first 

mount. However, statistically significant differences among dose groups only occurred for day two and 

day three. In both days, the control group had a significantly shorter time to mount compared to dose 20. 

IV. F1 Generation Results 

The control group had a much shorter time until first mount, and dose 20 had consistently longer 

times until first mount, which was the same pattern present in the F0 generation. In fact, the control 

group had a significantly shorter time to mount compared to dose 20 and dose 5 for all three days. The 

control group also had a significantly shorter time to mount compared to dose .5 for days one and two. 

Dose

Average time

to first mount

Standard

Error

Mounted

pairs

Average time

to first mount

Standard

Error

Mounted

pairs

Average time

to first mount

Standard

Error

Mounted

pairs

0 11.6 4.69 100% 2.8 0.56 100% 2.4 0.50 100%

0.5 14.6 4.73 100% 15.9 13.38 100% 23.0 14.45 92%

5 33.1 14.98 100% 17.4 14.18 92% 18.3 14.15 92%

10 47.3 18.18 83% 28.4 14.07 100% 43.5 18.39 83%

20 71.4 22.72 67% 77.6 25.00 58% 77.7 25.03 58%

F0 Generation ‐ Time to first Mount

Day 1 Day 2 Day 3

Log rank p‐value = .066 Log rank p‐value = .013 Log rank p‐value = .031

Dose

Average time

to first mount

Standard

Error

Mounted

pairs

Average time

to first mount

Standard

Error

Mounted

pairs

Average time

to first mount

Standard

Error

Mounted

pairs

0 21.4 4.82 100% 6.6 2.34 100% 7.2 1.78 100%

0.5 68.8 14.80 80% 49.9 15.89 80% 29.8 11.63 90%

5 70.8 16.32 70% 50.9 16.75 75% 45.6 15.77 80%

10 73.4 20.11 69% 44.2 19.07 85% 26.2 13.40 92%

20 116.2 21.56 46% 97.7 22.83 54% 113.2 23.43 39%

Day 1 Day 2 Day 3

Log rank p‐value = .001 Log rank p‐value = .002 Log rank p‐value = .002

F1 Generation ‐ Time to first Mount
79 

V. F2 Generation Results 

The times to first mount in the F2 generation data did not follow the same trend as the F0 and F1 

generations. In this generation, the higher the concentration of trenbolone, the shorter the time to first 

mount, a reversal of the other two generations, especially the F0 generation. Even though these 

differences were not statistically significant, the control group and dose .5 typically had very similar 

results, while the two higher-concentrated dose groups generally had more similar results (usually a 

shorter time to first mount). 

 

Dose

Average time

to first mount

Standard

Error

Mounted

pairs

Average time

to first mount

Standard

Error

Mounted

pairs

Average time

to first mount

Standard

Error

Mounted

pairs

0 40.4 12.05 90% 20.3 11.18 95% 16.7 8.80 95%

0.5 42.4 13.11 90% 15.0 8.53 95% 13.8 8.60 95%

5 29.1 7.30 100% 16.9 9.11 95% 7.1 3.09 100%

10 28.7 11.51 90% 12.3 8.63 95% 2.9 0.71 100%

F2 Generation ‐ Time to first Mount

Day 1 Day 2 Day 3

Log rank p‐value = .858 Log rank p‐value = .558 Log rank p‐value = .149
80 

NUMBER OF COPULATIONS 

I. Analysis Methods 

 A Generalized Linear Model (GzLM) using a poisson loglinear link function was used to determine 

if there was a statistically significant difference in the number of copulations (successful cloacal contact) 

across the five dose levels. An omnibus likelihood ratio test in GzLM, which compares the fitted model 

(intercept plus dose) against the intercept only model, was used to determine if dose was a significant 

factor in predicting the number of copulations in 180 seconds. If the p-value for the likelihood test was 

less than .05, dose was a significant factor in predicting the number of copulations. Parameter estimates 

(Beta coefficients and Beta confidence intervals) and confidence intervals were then examined to find 

where the significant differences occurred among the dose levels if the p-value for the omnibus test was 

less than .05. In the GzLM procedure, a reference group was needed for the other dose levels to be 

tested against for a significant difference. This was because we fit a categorical predictor into a linear 

model. The reference group for this analysis was the ‘dose=0’ group which is referred to as the control 

group throughout the report. An exponentiated β coefficient of 1.00 meant that the dosed group was 1.00 

times as likely to have a copulation compared to the control group, or in other words, the two groups had 

an equal chance of having a copulation. If the confidence interval contained 1.00, there was no statistical 

difference between the two groups and vice versa. 

II. Summary of Results

With the exception of day one of F1 generation, the control group, followed by dose 5 had the 

greatest number of copulations for any of the ppm levels. Comparing results across the three 

generations, the control group was the only group that drastically decreased in the number of copulations 

from the F0 generation to the F1 and F2 generations. Also, dose .5 and dose 10 had a much higher 

number of copulations in the F2 generation, compared to the F0 and F1 generations. 

III. F0 Generation Results 

The control group had the highest number of copulations for all three days of the F0 generation. 

However, only in day two, did the control group have a statistically significantly greater number of 

copulations than any of the other dose groups (dose .5, 10, and 20). Overall, the number of copulations 

was highest for day two in the F0 generation. 

 

IV. F1 Generation Results 

The control group had the greatest number of copulations for all three days. Dose 20, on the 

other hand, consistently had the lowest number of copulations. In fact, on day one, all dose 20 pairs 

failed to copulate even once. The only statistically significant difference between any of the dose groups 

was that on day two and day three, the control group had a greater number of copulations compared to 

the dose 20. 

Dose

Average number

of Copulations

95% β Confidence

Interval* P‐value Average number

of Copulations

95% β Confidence

Interval* P‐value Average number

of Copulations

95% β Confidence

Interval* P‐value

0 1.25 1.58 1.42

0.5 0.58 .19‐1.15 0.10 0.67 .18‐.96 0.04 0.58 .17‐.99 0.05

5 0.75 .26‐1.37 0.23 1.17 .37‐1.47 0.39 0.92 .30‐1.38 0.26

10 0.50 .16‐1.03 0.06 0.33 .07‐.62 0.01 0.83 .27‐1.29 0.18

20 0.33 .09‐.80 0.02 0.67 .18‐.96 0.04 0.42 .11‐.80 0.02

F0 Generation ‐ Number of Copulations

Day 1 Day 2 Day 3

Omnibus Likelihood ratio p‐value = .088 Omnibus Likelihood ratio p‐value = .011 Omnibus Likelihood ratio p‐value = .084

* Confidence Intervals reflect comparison to control group (ppm = 0).  Ppm levels with a confidence interval not including 1.00 is considered to be significantly

different from the control group.
81 

V. F2 Generation Results 

No statistically significant differences occurred among dose groups within any of the three days in 

the F2 generation. Additionally, no common trends occur among the dose groups within each day or 

among the three days. 

 

Dose

Average number

of Copulations

95% β Confidence

Interval* P‐value Average number

of Copulations

95% β Confidence

Interval* P‐value Average number

of Copulations

95% β Confidence

Interval* P‐value

0 0.90 1.05 1.10

0.5 0.45 .23‐1.11 0.09 0.45 .20‐.94 0.03 0.60 .27‐1.10 0.09

5 0.35 .16‐.93 0.03 0.80 .40‐1.46 0.41 1.00 .50‐1.67 0.76

10 0.38 .16‐1.15 0.09 0.62 .26‐1.32 0.20 0.62 .25‐1.26 0.16

20 0.23 .07‐.74 0.01 0.08 .01‐.52 0.01

* Confidence Intervals reflect comparison to control group (ppm = 0).  Ppm levels with a confidence interval not including 1.00 is considered to be significantly

different from the control group.

F1 Generation ‐ Number of Copulations

Day 1 Day 2 Day 3

Omnibus Likelihood ratio p‐value = .090 Omnibus Likelihood ratio p‐value = .028 Omnibus Likelihood ratio p‐value = .001

All pairs failed to Copulate

Dose

Average number

of Copulations

95% β Confidence

Interval* P‐value Average number

of Copulations

95% β Confidence

Interval* P‐value Average number

of Copulations

95% β Confidence

Interval* P‐value

0 0.95 0.80 1.10

0.5 0.35 .16‐.876 0.02 0.65 .39‐1.69 0.58 1.05 .53‐1.74 0.88

5 0.90 .50‐1.81 0.87 1.05 .69‐2.52 0.41 1.00 .50‐1.67 0.76

10 0.85 .47‐1.72 0.74 1.15 .76‐2.72 0.27 0.86 .73‐2.23 0.40

* Confidence Intervals reflect comparison to control group (ppm = 0).  Ppm levels with a confidence interval not including 1.00 is considered to be significantly

different from the control group.

F2 Generation ‐ Number of Copulations

Day 1 Day 2 Day 3

Omnibus Likelihood ratio p‐value = .068 Omnibus Likelihood ratio p‐value = .320 Omnibus Likelihood ratio p‐value = .651
82 

TIME UNTIL FIRST COPULATION 

I. Analysis Methods 

Kaplan-Meier survival analysis was used to determine if there was a significant difference among 

the five dose levels in terms of the amount of time it took the birds to achieve their first copulation. If their 

time was recorded as 180 seconds, this meant that the quail did not copulate and that observation was 

considered censored. A log rank statistic was computed to test whether the different dose groups had 

statistically different times until the first copulation. If the p-value associated with the log rank statistic was 

less than .05, it was concluded that the times between at least two of the dose groups were not 

statistically equal. To find which dose groups did not have equal times, estimated confidence intervals for 

the time to copulate were compared. If two dose groups had confidence intervals that did not overlap, 

these two doses were considered to have statistically different times at the 95% confidence level. 

II. Summary of Results 

The time until first copulation stayed fairly consistent through all three generations for all the dose 

groups with the exception of the control group and ppm level 20. The control group greatly increased its 

time until first copulation in the F1 and F2 generations compared to the F0 generation. The control group 

also had the shortest time until first copulation for every day in the first two generations, but then had one 

of the longest times for the final generation. Dose 20 also greatly increased its time to first copulation in 

the F1 generation compared to the F0 generation. Additionally, through the F0 and F1 generations, dose 

20 characteristically had the longest time until first copulation out of all the dose groups. 

III. F0 Generation Results 

Statistically significant differences among dose groups occurred for all three days in the F0 

generation. Consistently, the control group had the shortest time and had significantly shorter times than: 

dose 10 and dose 20 on day one, dose 10 on day two, and dose 20 on day three. Dose 20, 10, and .5 

had the three longest times to copulation for all three days. 

IV. F1 Generation Results 

For all three days, the control group had the shortest time until first copulation and dose 20 had 

the longest time to first copulation. Furthermore, in day one, all pairs of dose 20 quails failed to copulate. 

Statistically significant differences only occurred in day three, in which the control group had a 

significantly shorter time to copulation compared to dose 20. As for the three groups with mid-level 

concentrations of trenbolone, there were no apparent trends or differences that were consistent from day 

to day. 

Dose

Average time to

first copulation

Standard

Error

Copulated

pairs

Average time to

first copulation

Standard

Error

Copulated

pairs

Average time to

first copulation

Standard

Error

Copulated

pairs

0 39.5 13.44 92% 38.8 18.36 83% 41.2 18.16 83%

0.5 111.6 20.91 50% 93.9 24.87 50% 109.8 24.02 42%

5 94.2 20.72 67% 40.7 18.24 83% 53.6 21.18 75%

10 108.8 21.15 50% 123.3 23.15 33% 99.6 23.38 50%

20 127.7 21.51 33% 110.7 23.76 42% 127.7 21.72 33%

F0Generation ‐ Time to first Copulation

Day 1 Day 2 Day 3

Log rank p‐value = .009 Log rank p‐value = .031 Log rank p‐value = .037
83 

V. F2 Generation Results 

No statistically significant differences occurred among dose groups within any of the three days of 

the F2 generation. Additionally, other than dose 10 typically having one of the shortest times to first 

copulation, no apparent trends were present comparing the four dose levels or comparing from day to 

day. 

Dose

Average time to

first copulation

Standard

Error

Copulated

pairs

Average time to

first copulation

Standard

Error

Copulated

pairs

Average time to

first copulation

Standard

Error

Copulated

pairs

0 86.8 16.57 65% 74.5 17.44 65% 65.6 16.98 70%

0.5 119.0 16.87 40% 113.7 18.24 40% 97.4 18.51 50%

5 131.5 14.98 35% 92.5 17.87 55% 84.5 18.51 60%

10 120.5 21.03 39% 117.0 22.14 39% 114.8 22.91 39%

20 162.2 All pairs failed to Copulate 12.52 23% 166.9 12.56 7%

F1Generation ‐ Time to first Copulation

Day 1 Day 2 Day 3

Log rank p‐value = .170 Log rank p‐value = .141 Log rank p‐value = .016

Dose

Average time to

first copulation

Standard

Error

Copulated

pairs

Average time to

first copulation

Standard

Error

Copulated

pairs

Average time to

first copulation

Standard

Error

Copulated

pairs

0 93.6 17.28 60% 78.6 17.15 65% 80.0 17.45 65%

0.5 137.5 14.46 35% 95.9 18.32 55% 62.8 17.17 75%

5 93.9 16.65 65% 53.8 16.39 75% 69.9 18.12 65%

10 83.5 16.41 65% 58.8 15.95 75% 44.9 15.02 85%

F2Generation ‐ Time to first Copulation

Log rank p‐value = .160 Log rank p‐value = .308 Log rank p‐value = .372

Day 1 Day 2 Day 3
84 

TRENBOLONE EFFECTS ON ORGAN WEIGHTS

Vital Organ and Total Body Weights 

 

 This section of the report analyzes the body and organ weights of Japanese quail that were collected and 

weighed after necropsy. Weights were taken for quails in one of the five dose groups ((control) 0 ppm, .50 ppm, 5 ppm, 

10 ppm, 20 ppm) in all three generations (F0, F1, and F2). Total body weight was measured as well as brain, heart, 

kidneys, liver, ovaries, spleen, oviduct, tyroid, bursa, epididymides, testes and coacal gland (CG) weights. Ovaries, 

oviduct, epididymides and testes are gender-specific organs therefore only having weights for one of the genders. Quails 

in dose 20 were not present in the F2 generation, therefore only leaving statistical analysis to determine differences in the 

four lower concentrated dose groups of trenbolone. SPSS version 16.0 was used to analyze the body and organ weight 

data. Statistical significance for this analysis was determined using an alpha (α) of .05 with a 95% confidence level. 

 
85 

I. Analysis Methods 

 An Analysis of Variance (ANOVA) test was used to determine if dose group averages for each measured organ 

weight were significantly different. An F-statistic and corresponding p-value were calculated for each organ. If the p-value 

was below .05, then it was concluded that at least two dose groups had significantly different average weights for that 

organ. To find which dose groups had significantly different average weights, a Tukey post-hoc test was used. A 

Levene’s test was used to check to make sure the dose groups had equal variances, a major assumption that must be 

met in order to validly use ANOVA test results. If this assumption was violated (p-value for Levene’s test <.05), a nonparametric Kruskal-Wallis test was run instead of an ANOVA. 

II. Summary of Results 

Statistically significant differences sparingly occurred among dose groups throughout the F1 and F2 generations, 

and no statistically significant differences occurred in any of the organ weights for either gender within the F0 generation. 

With very few statistically significant differences in organ weights, plus p-values for a majority of the other organ weights 

well above .05, evidence suggests that overall, in all three generations for all organ weights, there was no substantial 

difference among the five dose groups in terms of their vital organ or overall body weights. One exception to this was that 

for both genders in the F2 generation, dose 10 (which was the highest-concentrated dose level for the F2 generation) had 

significantly greater spleen weights than at least one of the other dose groups. 

Some notable differences in organ weights comparing across the three generations occurred for both genders. In 

females: brain weights were highest in the F1 generation, bursa weights were highest in the F2 generation, thyroid 

weights were highest in the F2 generation, and spleen weights were highest in the F2 generation. In males: bodyweights 

were highest in the F1 generation and lowest in the F2 generation, heart weights were highest in the F0 generation, 

spleen weights were highest in the F2 generation, bursa weights were highest in the F2 generation, and CG area weights 

were highest in the F0 generation. 

III. F0 Generation Results 

 A. Females 

No statistically significant differences (ANOVA p-values all above .05) in body or organ weights occurred among 

the five dose groups. The control group (excluding spleen, oviduct, and thyroid) had the highest average for all organ 

weights. However, there were inconclusive trends in organ and bodyweight averages, other than the notably high 

average in the control group, comparing the dosing levels of trenbolone. This overall evidence showed that organ and 

bodyweights for female quails were not different among the five dose groups in the F0 generation. 

B. Males 

Again, no statistically significant differences (ANOVA p-values all above .05) in body or organ weights occurred 

among the five dose groups. The control group typically had one of the highest organ weight averages, but differences 

between the control and other dose groups were not as definitive as the females. For the males, one of the two highestconcentrated dose groups always had the lowest average organ weight, excluding overall body weight and CG area. 

Overall however, there were inconclusive trends in organ weight averages across all organ weights comparing the dosing 

levels of trenbolone. 

Dose Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error

0 141.5 3.23 0.764 0.012 1.27 0.041 1.40 0.062 4.24 0.205 4.60 0.469 0.028 0.0039 6.29 0.365 0.0079 0.0011 0.024 0.0045

0.5 136.2 4.20 0.754 0.017 1.19 0.047 1.27 0.066 3.51 0.282 3.96 0.899 0.026 0.0022 4.90 0.910 0.0085 0.0009 0.022 0.0042

5 139.9 2.94 0.746 0.014 1.14 0.040 1.37 0.048 4.16 0.188 4.27 0.467 0.034 0.0039 6.47 0.212 0.0053 0.0011 0.016 0.0029

10 138.8 3.58 0.736 0.014 1.20 0.050 1.29 0.065 3.64 0.301 3.07 0.376 0.030 0.0039 6.12 0.338 0.0057 0.0012 0.017 0.0047

20 134.9 3.03 0.748 0.011 1.22 0.048 1.31 0.045 3.65 0.165 3.30 0.361 0.031 0.0017 5.76 0.306 0.0069 0.0009 0.019 0.0043

P‐value calculated from Kruskal‐wallis test because homogeneity of variance assumption violated in parametric ANOVA test.

Thyroid

P‐value = .648* P‐value = .144

Bursa

P‐value = .615

Ovaries

P‐value = .139*

Spleen

P‐value = .492

Oviduct

F0 Generation Female Organ Weights

P‐value = .673 P‐value = .705 P‐value = .346 P‐value = .457 P‐value = .102

Body Weight Brain Heart Kidneys Liver

Dose Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error

0 116.2 1.97 0.759 0.012 1.20 0.042 0.810 0.0228 0.204 0.014 2.05 0.076 0.0197 0.0021 3.46 0.232 0.0095 0.0018 0.038 0.0072 259.1 8.64

0.5 113.7 1.34 0.763 0.012 1.22 0.038 0.825 0.0297 0.214 0.012 1.99 0.104 0.0204 0.0019 3.38 0.173 0.0075 0.0018 0.035 0.0053 255.8 7.19

5 114.4 2.35 0.774 0.019 1.20 0.042 0.804 0.0261 0.185 0.018 2.07 0.123 0.0206 0.0016 3.52 0.228 0.0082 0.0019 0.033 0.0042 255.6 11.96

10 113.8 1.78 0.746 0.012 1.26 0.024 0.803 0.0288 0.176 0.013 1.96 0.091 0.0210 0.0013 3.25 0.128 0.0079 0.0021 0.031 0.0030 260.2 12.92

20 114.1 1.26 0.753 0.015 1.13 0.047 0.806 0.0152 0.207 0.011 2.13 0.108 0.0212 0.0024 3.23 0.174 0.0052 0.0010 0.034 0.0029 276.4 10.09

* P‐value calculated from Kruskal‐wallis test because homogeneity of variance assumption violated in parametric ANOVA test.

CG area

P‐value = .605

F0 Generation Male Organ Weights

P‐value = .855 P‐value = .698 P‐value = .270 P‐value = .971 P‐value = .253 P‐value = .773 P‐value = .983 P‐value = .779 P‐value = .536 P‐value = .961*

Body Weight Brain Heart Kidneys Epididymides Liver Spleen Testes Thyroid Bursa
86 

C. Male and Female differences 

Body, kidney, liver, and spleen weights were much greater for females compared to males. However, bursa 

weights were much greater for males compared to females. Only in the females was it very clear that the control group 

consistently had greater organ weight averages compared to the other dose groups. Lastly, in the males, one of the two 

highest concentrated dose groups typically had the lowest average organ weight; this was not the case for the females. 

IV. F1 Generation Results 

 A. Females 

Significant differences among dose groups occurred for overall bodyweight and kidney weight. Dose 10 had a 

significantly greater body weight compared to dose 20, and dose .5 and the control group had significantly greater kidney 

weights compared to dose 20. Statistically significant differences among doses were not present for any of the other 

organ weights. Other than the two organs with significant differences among dose groups, inconclusive overall trends 

were present in organ weight averages comparing dosing levels of trenbolone. 

B. Males 

The control group had a statistically greater bursa average compared to dose 20. Statistically significant 

differences in doses were not present for any of the other organ weights. Dose 20 typically had the lowest average organ 

weight, however overall trends in organ weights were not present comparing dosing levels of trenbolone. 

C. Male and Female differences 

As in the F0 generation, bodyweight, kidney, liver, and spleen averages were much higher for the females 

compared to males, and bursa averages were higher for males. 

V. F2 Generation Results 

 A. Females 

Dose 10 had a significantly greater spleen weight average compared to the control group for the males in the F2 

generation. Statistically significant differences in doses were not present for any of the other organ weights. In fact, all 

other p-values were well above the cutoff of .05. Typically, dose 10 had the highest average organ weight, however there 

was inconclusive evidence of any trends among trenbolone dosing levels. 

Dose Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error

0 138.1 2.47 0.777 0.011 1.15 0.041 1.40 0.061 4.13 0.230 4.66 0.417 0.036 0.0065 5.72 0.398 0.0094 0.0014 0.022 0.0028

0.5 143.1 2.40 0.798 0.025 1.28 0.036 1.39 0.037 4.11 0.217 4.50 0.279 0.035 0.0029 5.89 0.175 0.0098 0.0017 0.021 0.0036

5 138.5 3.32 0.798 0.014 1.20 0.050 1.28 0.047 4.05 0.331 4.04 0.578 0.035 0.0055 5.01 0.566 0.0065 0.0015 0.012 0.0017

10 143.6 2.69 0.810 0.019 1.22 0.067 1.25 0.053 3.73 0.177 4.45 0.458 0.038 0.0026 6.02 0.403 0.0083 0.0018 0.021 0.0052

20 129.3 3.69 0.800 0.016 1.18 0.046 1.05 0.101 2.82 0.352 3.72 0.759 0.031 0.0083 4.46 1.010 0.0092 0.0030 0.014 0.0069

* P‐value calculated from Kruskal‐wallis test because homogeneity of variance assumption violated in parametric ANOVA test.

° Tukey post‐hoc test indicated that dose 10 had a significantly greater body weight average compared to dose 20.

† Tukey post‐hoc test indicated that dose .50 and the control group had significantly greater kidney weight averages compared to dose 20.

P‐value = .048° P‐value = .760 P‐value = .446 P‐value = .005† P‐value = .053 P‐value = .785 P‐value = .822* P‐value = .217 P‐value = .624 P‐value = .203*

F1Generation Female Organ Weights

Body Weight Brain Heart Kidneys Liver Ovaries Spleen Oviduct Thyroid Bursa

Dose Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error

0 119.5 1.69 0.787 0.012 1.13 0.045 0.792 0.0286 0.194 0.019 1.77 0.041 0.0212 0.0015 3.65 0.174 0.0129 0.0023 0.034 0.0039 234.9 10.84

0.5 119.0 1.81 0.745 0.018 1.15 0.057 0.813 0.0260 0.164 0.015 1.95 0.055 0.0191 0.0029 3.31 0.275 0.0107 0.0016 0.025 0.0030 221.5 8.45

5 119.9 2.39 0.762 0.017 1.17 0.040 0.825 0.0342 0.190 0.019 1.96 0.094 0.0202 0.0026 3.55 0.166 0.0112 0.0011 0.025 0.0039 226.0 8.23

10 119.2 1.97 0.803 0.020 1.18 0.058 0.881 0.0348 0.151 0.011 1.94 0.097 0.0224 0.0040 3.63 0.211 0.0077 0.0018 0.030 0.0033 238.8 8.87

20 117.7 2.24 0.781 0.012 1.09 0.047 0.759 0.0238 0.154 0.014 1.82 0.081 0.0221 0.0034 2.97 0.134 0.0068 0.0011 0.018 0.0024 202.6 11.15

° Tukey post‐hoc test indicated that the control group had a signi ficantly greater bursa weight average compared to dose 20.

F1 Generation Male Organ Weights

Body Weight Brain Heart Kidneys Epididymides Liver Spleen Testes Thyroid Bursa CG area

P‐value = .958 P‐value = .124 P‐value = .769 P‐value = .130 P‐value = .251 P‐value = .252 P‐value = .920 P‐value = .119 P‐value = .090 P‐value = .028° P‐value = .110

Dose Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error

0 134.5 3.13 0.768 0.012 1.18 0.057 1.26 0.043 3.80 0.223 4.02 0.452 0.036 0.0034 6.11 0.678 0.012 0.0020 0.041 0.0063

0.5 131.4 2.71 0.759 0.015 1.21 0.068 1.27 0.046 4.20 0.300 3.51 0.583 0.039 0.0042 6.60 0.533 0.010 0.0014 0.044 0.0073

5 134.7 1.88 0.744 0.013 1.25 0.067 1.28 0.029 4.30 0.158 3.23 0.474 0.039 0.0029 5.62 0.520 0.009 0.0013 0.031 0.0059

10 137.8 2.20 0.775 0.014 1.29 0.056 1.37 0.047 4.44 0.173 3.45 0.484 0.053 0.0057 5.91 0.498 0.011 0.0024 0.048 0.0042

° Tukey post‐hoc test indicated that dose 10 had a significantly greater spleen weight average compared to the control group.

F2Generation Female Organ Weights

Body Weight Brain Heart Kidneys Liver Ovaries Spleen Oviduct Thyroid Bursa

P‐value = .374 P‐value = .390 P‐value = .646 P‐value = .231 P‐value = .189 P‐value = .720 P‐value = .033° P‐value = .674 P‐value = .759 P‐value = .212
87 

B. Males 

Dose 10 had a significantly greater spleen weight compared to the other three dose groups. Statistically 

significant differences in doses were not present for any of the other organ weights. Inconclusive trends in organ and 

body weight averages among dosing levels reaffirmed that organ and body weights for male quails were not different in 

the F2 generation. 

C. Male and Female differences 

Just like in the F0 and F1 generations, bodyweight, kidneys, liver, and spleen averages were much higher for 

females compared to males, while males had higher bursa averages compared to females. 

Dose Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error

0 111.0 1.29 0.722 0.022 1.08 0.038 0.84 0.028 0.205 0.011 1.97 0.042 0.0224 0.0020 3.50 0.119 0.0093 0.0015 0.053 0.0067 244.1 11.98

0.5 108.6 1.62 0.733 0.029 1.14 0.045 0.77 0.034 0.231 0.017 1.85 0.040 0.0226 0.0013 3.66 0.184 0.0077 0.0011 0.054 0.0050 252.0 10.98

5 113.3 1.69 0.737 0.031 1.19 0.043 0.78 0.030 0.229 0.016 1.94 0.049 0.0228 0.0020 3.49 0.137 0.0108 0.0015 0.047 0.0071 248.7 12.91

10 112.9 0.89 0.751 0.023 1.18 0.040 0.83 0.024 0.214 0.012 2.05 0.090 0.0311 0.0025 3.58 0.118 0.0081 0.0015 0.051 0.0062 270.0 9.47

P‐value = .402

F2Generation Male Organ Weights

° Tukey post‐hoc test indicated that dose 10 had a significantly greater spleen weight average compared to the other three dose groups

P‐value = .085 P‐value = .893 P‐value = .267 P‐value = .316 P‐value = .508 P‐value = .135 P‐value = .007° P‐value = .800 P‐value = .425 P‐value = .879

Body Weight Brain Heart Kidneys Epididymides Liver Spleen Testes Thyroid Bursa CG area
88 

APPENDIX J 

CONTRIBUTING SCIENTIST REPORT 

STATISTICAL ANALYSIS OF HORMONE DATA 
89 

Statistical Analysis on Hormone Data for USAPHC 

Toxicology Protocol Number: 08JG-80-10-03-01 

Two-Generation Test for Endocrine Disruption and Reproductive Toxicity in 

Japanese Quail (Corturnix japonica) using Trenbolone 

Good Laboratory Practice Compliance Statement 

The statistical analysis of the data for the Two-generation test for endocrine disruption and reproductive toxicity in 

Japanese quail (Cortunix japonica) using trenbolone was conducted in compliance with Title 40, Code of Federal 

Regulations (CFR), Part 792, Good Laboratory Practice Standards. Statistical analyses were conducted according to the 

procedures outlined in the protocol for this study (Protocol Number 08JG-80-08-04-02). 

 

 

 

 29 January, 2013 

Shane Hall 

 Date 

Statistician 
90 

I. Analysis Methods 

A two-way Analysis Of Variance (ANOVA) was used to analyze Estradiol, and Testosterone from a two-generation study 

that involved exposing quails to trenbolone. Trenbolone was divided into five dose groups (control, 0.5 ppm, 5 ppm, 10 

ppm, and 20 ppm). In addition to dosage, both male and female quails were used to determine if gender also had an 

effect on their hormone values. 

The ANOVA model, using each of the four hormones as the dependent variable and dose group and gender as the 

predictors, determined if at least two of the groups within each predictor were significantly different from one another (test 

of main effect). The ANOVA model also determined if the effect that a change in dose group had on the hormone was 

significantly impacted by gender of the quails (test of interaction). If the interaction was statistically significant, the main 

effect results for dose group and gender are potentially misleading and must be interpreted in light of the interaction. 

Therefore, if an interaction was significant, p-values for the component parts are not given. If the interaction was not 

statistically significant, and dose group was, a Tukey post-hoc test was used to determine which groups were significantly 

different from one another. Because gender only consisted of males and females, the ANOVA p-value was sufficient to 

determine a difference between males and females and additional post-hoc tests were not necessary. 

Data was checked to determine if ANOVA assumptions were met. A Levene’s test was used to check equality of 

variances and ANOVA-model residuals were examined via a Q-Q plot to check normality. If assumptions were violated, 

data was log-transformed and assumptions were checked once again to see if an ANOVA was suitable. For this data, all 

hormone measures satisfied ANOVA assumptions using either the untransformed or Log-transformed data, so additional 

analysis adjustments were not necessary. 

Individual data points were occasionally labeled as “Not Determined – Insufficient Volume”, “Not Detectable”, or “Not 

Detectable (Below Assay Limits)”. In the first two cases, the blood sample for that quail was either not tested or was 

unsuccessfully tested and was therefore counted as missing data for the statistical analysis. However, the data points 

that were labeled as below the assay limits were tested, but returned values too low for the diagnostic machine to detect. 

In this case, limit of detection divided by the square root of two was substituted for the missing value. 

SPSS 16.0 was used to execute the statistical analysis summarized in the report below. Statistical significance 

throughout the analysis was defined at α=.05 or 95% confidence level. 

II. Results 

Estradiol and Testosterone were both significantly impacted by Trenbolone dosage and/or gender. 

 Estradiol had similar averages across dose groups, but females had a significantly greater average compared to 

males. For females, dose 250 had a significantly lower mean compared to dose 60, and no significant differences 

between groups were observed for males. 

 Testosterone was impacted by gender with males having a greater average within all five dose groups; however 

the pattern of testosterone averages for the dose groups within each gender were severely different. Females 

had a fairly consistent testosterone average across all four dose groups, while males have a definitive increasing 

tend from the control to dose 10. No significant differences between female groups were observed; however for 

males, dose 125 and dose 250 had significantly lower means compared to the control dose. 

Table 1. Effects of Trenbolone on F2 Generation Quail Hormones

Gender Dose Group

(ppm)

Estradiol (pg/ml) Testosterone (pg/ml)

N Mean SE N Mean SE

Female

Control 4 32.91 4.01 4 683.6 252.3

0.5 5 28.66 11.34 5 475.6 293.8

5 3 32.23 5.97 3 527.0 97.1

10 3 36.49 6.71 3 987.7 463.8

Male Control 3 1.58 0.00 3 769.4 233.2

0.5 4 9.83 1.30 4 2405.8 148.5
91 

5 3 5.53 2.01 3 2090.4 368.8

10 3 7.85 0.90 3 3962.8 860.4

P‐Value

Dose = .922

Gender = .000                       Dose * Gender = .011

(Females > Males)

 
92 

APPENDIX K 

RAW DATA 
93 

ppm ID 8/19 8/20 8/21 8/22 

week 

6 8/23 8/24 8/25 8/26 8/27 8/28 8/29 

week 

7 8/30 8/31 9/1 9/2 9/3 9/4 9/5 

week 

8 9/6 9/7 9/8 9/9 9/10 9/11 9/12 

week 

9 9/13 9/14 9/

0 391 1 

0 392 

0 393 1 1 1 1 

0 394 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 395 1 1 1 1 1 1 1 1 1 

0 396 1 1 1 1 1 1 1 

0 397 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 398 1 1 1 1 

0 399 1 1 1 1 2 1 

0 400 1 2 1 1 1 1 1 1 1 1 1 1 1 1 

0 201 

0 202 

0 203 1 1 1 1 1 1 1 1 1 

0 204 1 1 1 1 1 2 1 1 1 

0 205 1 1 1 1 1 1 1 1 

0 206 1 1 1 1 1 1 1 1 1 

0 207 1 1 1 1 1 1 1 1 1 1 2 1 

0 208 1 1 

0 209 1 1 1 1 1 1 1 

0 210 1 1 1 1 1 1 1 1 1 1 

0.5 11 

0.5 498 

0.5 13 1 1 1 1 

0.5 14 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 

0.5 15 1 1 1 1 1 1 1 

0.5 16 

0.5 17 1 1 1 1 1 

0.5 18 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 19 

0.5 20 1 1 1 1 1 1 1 1 1 1 1 

0.5 1 

0.5 2 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 3 

0.5 4 1 

0.5 5 1 1 1 1 1 1 1 

0.5 6 1 1 

0.5 7 1 1 1 

0.5 8 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 9 1 1 1 

0.5 10 1 1 1 1 1 
94 

5 280 1 1 1 1 1 1 

5 281 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 283 1 1 1 1 1 1 1 

5 284 1 1 1 1 

5 285 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 286 1 1 1 1 1 1 1 1 1 

5 287 

5 288 1 2 

5 289 1 1 1 1 1 1 1 1 1 

5 290 2 1 1 1 1 1 1 1 1 1 2 1 1 1 1 

5 291 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 292 

5 293 1 1 1 1 1 1 1 1 1 1 1 

5 294 1 1 1 1 1 1 1 1 

5 295 1 1 1 1 1 1 1 1 1 1 

5 296 1 1 1 1 1 1 1 1 1 1 1 

5 297 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 298 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 299 1 1 1 1 1 1 1 1 1 1 

5 300 1 1 1 1 1 1 

10 33 

10 34 

10 35 1 

10 36 1 1 1 1 1 1 1 

10 37 1 1 1 1 1 1 1 1 1 

10 94 1 1 1 1 1 1 

10 39 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 40 

10 27 1 

10 28 1 

10 29 

10 30 1 1 1 1 1 1 1 1 

10 31 1 1 1 

10 32 

10 21 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 22 1 1 1 1 1 1 1 1 1 

10 23 1 1 1 1 1 1 1 1 1 1 1 

10 24 1 1 1 1 1 1 1 1 1 1 

10 25 1 1 

10 26 1 1 1 1 1 1 1 1 1 1 1 

20 301 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 290 1 1 1 1 
95 

20 303 

20 380 1 1 1 

20 305 1 1 

20 306 1 1 1 1 

20 307 1 1 1 1 1 1 

20 308 1 1 1 

20 309 

20 310 

20 291 1 1 1 1 1 1 1 1 1 

20 292 1 1 1 1 1 1 

20 293 1 1 1 1 1 1 1 1 1 1 

20 294 1 

20 295 1 

20 296 

20 297 1 1 1 1 1 1 1 1 1 

20 379 1 1 1 1 1 

20 299 

20 300 

ppm ID 9/20 9/21 9/22 9/23 9/24 9/25 9/26 

week 

11 9/27 9/28 9/29 9/30 10/1 10/2 10/3 week 12 10/4 10/5 10/6 10/7 10/8 10/9 10/10 

week 

13 10/11 10/

0 391 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 392 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 393 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 394 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 395 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 396 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 397 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 398 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 399 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 400 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 201 1 1 1 1 1 1 1 1 

0 202 1 1 

0 203 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 204 1 1 1 1 1 1 1 1 1 1 1 1 

0 205 1 1 1 1 1 1 1 1 1 1 1 1 

0 206 1 1 1 1 1 1 1 

0 207 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 208 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 209 1 1 1 1 1 1 1 1 1 1 1 1 

0 210 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 11 1 1 1 1 1 1 1 

0.5 498 1 1 1 1 1 1 

0.5 13 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 
96 

0.5 14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 15 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 16 

0.5 17 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 18 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 19 1 1 

0.5 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 3 

0.5 4 1 1 1 1 1 1 1 1 1 1 1 2 1 2 1 1 1 

0.5 5 1 1 1 1 1 1 1 1 1 

0.5 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 9 1 1 1 1 1 1 1 1 1 

0.5 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 280 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 281 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 283 1 1 1 1 1 1 1 1 

5 284 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 285 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 286 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 287 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 288 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 289 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 290 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 291 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 292 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 293 1 1 1 1 1 1 1 1 1 1 1 

5 294 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 295 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 296 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 297 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 298 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 299 1 1 1 1 1 1 1 1 1 1 1 

5 300 1 1 1 1 1 1 1 1 1 1 

10 33 

10 34 

10 35 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 36 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 37 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 
97 

10 94 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 39 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 27 1 1 1 

10 28 1 1 1 1 1 1 1 1 1 1 1 

10 29 

10 30 1 1 1 1 1 1 1 1 1 

10 31 1 1 1 1 1 1 1 1 1 1 

10 32 1 1 1 1 1 1 1 1 

10 21 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 22 1 1 1 1 1 1 1 1 1 1 1 1 

10 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 24 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 26 1 1 1 1 1 1 1 1 1 1 1 1 

20 301 1 1 1 1 1 1 1 1 1 

20 290 1 1 1 1 1 1 1 1 

20 303 1 1 1 1 1 1 1 1 1 1 1 1 

20 380 1 1 1 1 1 1 1 1 1 1 

20 305 1 1 1 1 1 1 1 1 1 1 1 

20 306 1 1 1 1 1 1 1 1 1 1 1 

20 307 1 1 1 1 1 1 1 1 1 1 

20 308 1 1 1 1 1 1 1 1 1 1 1 1 

20 309 

20 310 1 1 1 1 

20 291 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 292 1 1 1 1 1 1 1 1 

20 293 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 294 1 

20 295 1 1 1 1 1 1 1 1 1 

20 296 

20 297 1 1 1 1 1 1 1 1 1 1 

20 379 1 1 1 2 1 1 1 1 1 1 1 1 1 1 

20 299 

20 300 1 1 1 1 1 1 1 1 1 

p

p

m 

I

D 

10

/1

8 

10

/1

9 

10

/2

0 

10

/2

1 

10

/2

2 

10

/2

3 

10

/2

4 

we

ek 

15 

10

/2

5 

10

/2

6 

10

/2

7 

10

/2

8 

10

/2

9 

10

/3

0 

10

/3

1 

we

ek 

16 

1

1/

1 

1

1/

2 

1

1/

3 

1

1/

4 

1

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5 

1

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6 

1

1/

7 

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ek 

17 

1

1/

8 

1

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9 

11

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0 

11

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11

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11

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3 

11

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4 

we

ek 

18 

0 

3

9

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

3

9

2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

3

9

3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

3

9

4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

3

9

5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 
98 

0 

3

9

6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

3

9

7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

3

9

8 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 

0 

3

9

9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

4

0

0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

2

0

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

2

0

2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

2

0

3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

2

0

4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

2

0

5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

2

0

6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

2

0

7 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 

0 

2

0

8 1 1 1 1 1 1 1 

0 

2

0

9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

2

1

0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.

5 

1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.

5 

4

9

8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.

5 

1

3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.

5 

1

4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.

5 

1

5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.

5 

1

6 

0.

5 

1

7 1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 

0.

5 

1

8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.

5 

1

9 1 1 1 1 1 1 

0.

5 

2

0 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 1 1 1 1 1 1 

0.

5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.

5 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.

5 3 1 1 1 1 1 1 1 1 1 1 2 1 1 

0.

5 4 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 

0.

5 5 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.

5 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.

5 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.

5 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.

5 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.

5 

1

0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

8

0 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 

5 

2

8

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

8

3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

8

4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 

5 

2

8

5 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

8

6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

8

7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

8

8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 
99 

5 

2

8

9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

9

0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

9

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

9

2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

9

3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

9

4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

9

5 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

9

6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

9

7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

9

8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

2

9

9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

3

0

0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 

3

3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 

3

4 

10 

3

5 1 1 1 1 1 1 1 1 

10 

3

6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 

3

7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 

9

4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 

3

9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 

4

0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 

2

7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 

2

8 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 

10 

2

9 

10 

3

0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 

3

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 

3

2 1 1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 

10 

2

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 

2

2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 

2

3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 

2

4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 

2

5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 

2

6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

3

0

1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

2

9

0 1 1 1 1 1 1 1 1 1 

20 

3

0

3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

3

8

0 1 1 1 1 1 1 1 1 1 1 1 

20 

3

0

5 1 1 1 1 1 1 1 1 1 

20 

3

0

6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

3

0

7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

3

0

8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

3

0

9 1 1 1 1 1 1 

20 

3

1

0 1 
100 

20 

2

9

1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 

20 

2

9

2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

2

9

3 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

2

9

4 1 1 1 

20 

2

9

5 1 1 1 1 1 2 1 1 1 1 1 1 1 

20 

2

9

6 

20 

2

9

7 1 1 1 1 1 1 1 1 

20 

3

7

9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

2

9

9 1 1 

20 

3

0

0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

pp

m ID 

11/1

5 

11/1

6 

11/1

7 

11/1

8 

11/1

9 

11/2

0 

11/2

1 

wee

k 19 

11/2

2 

11/2

3 

11/2

4 

11/2

5 

11/2

6 

11/2

7 

11/2

8 

wee

k 20 

11/2

9 

11/3

0 

12/

1 

12/

2 

12/

3 

12/

4 

12/

5 

wee

k 21 

12/

6 

12/

7 

12/

8 

12/

9 

0 

39

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

39

2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

39

3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

39

4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

39

5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

39

6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

39

7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

39

8 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

39

9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

40

0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 

0 

20

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

20

2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

20

3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

20

4 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

20

5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

20

6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

20

7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

20

8 1 1 1 

0 

20

9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

21

0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 

0.5 11 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 

0.5 

49

8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 13 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 14 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 

0.5 15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 16 

0.5 17 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 18 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 
101 

0.5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 3 1 1 1 1 

0.5 4 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 

0.5 5 1 1 1 

0.5 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 8 2 1 1 1 2 1 1 2 1 1 1 1 1 1 1 1 1 1 

0.5 9 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0.5 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

28

0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

28

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

28

3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

28

4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

28

5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

28

6 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 

5 

28

7 1 1 

5 

28

8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

28

9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

29

0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

29

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

29

2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

29

3 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

29

4 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 

5 

29

5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 

5 

29

6 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 

5 

29

7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

29

8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

29

9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

5 

30

0 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 33 1 2 1 1 1 1 1 1 1 1 1 1 

10 34 

10 35 1 1 1 1 1 1 1 1 1 1 

10 36 1 1 1 1 1 1 1 1 1 1 1 

10 37 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 

10 94 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 

10 39 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 28 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 29 

10 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 31 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 
102 

10 32 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 21 1 1 1 1 1 1 1 1 1 

10 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 24 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

10 25 1 1 1 1 1 1 1 1 1 1 1 

10 26 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

30

1 1 1 1 1 1 1 1 1 

20 

29

0 1 1 1 1 1 1 1 1 

20 

30

3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

38

0 1 1 1 1 1 1 1 1 1 1 1 

20 

30

5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

30

6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

30

7 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

30

8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

30

9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

31

0 

20 

29

1 1 1 1 1 1 1 1 1 1 

20 

29

2 1 1 1 1 1 1 1 1 1 1 1 1 

20 

29

3 1 1 1 1 1 1 1 1 1 1 1 1 1 

20 

29

4 1 1 

20 

29

5 1 1 1 1 1 1 1 1 1 1 1 1 

20 

29

6 

20 

29

7 1 1 1 1 1 

20 

37

9 1 

20 

29

9 1 1 1 1 

20 

30

0 1 1 1 1 1 1 1 1 1 1 1 

FO unpaired feed consumption 

 10/4/10 10/7/10 10/11/10 10/14/10 10/18/10 10/22/10 10/25/10 10/29/10 11/1/10 11/4

DOSE PAIR PRE POST PRE 

week 

13 POST PRE POST PRE 

week 

14 POST PRE POST PRE 

week 

15 POST PRE POST PRE 

week 

16 POST PRE

0 391 446 369 446 332 191 442 372 445 335 180 441 345 444 359 181 437 355 450 313 219 443 355

0 392 449 343 444 300 250 443 341 438 310 230 447 362 447 363 169 436 342 452 298 248 432 362

0 393 438 301 442 312 267 436 315 442 301 262 435 339 445 325 216 447 338 452 303 258 444 341

0 397 436 348 438 291 235 446 342 446 314 236 436 328 451 214 345 442 329 454 287 280 443 350

0 398 452 367 441 315 211 441 360 444 291 234 442 352 450 326 214 445 350 451 311 235 437 362

0 205 440 355 445 306 224 436 352 444 312 216 441 350 443 329 205 451 352 452 280 271 443 340

0 371 440 389 451 364 138 445 390 440 365 130 440 387 447 370 130 448 386 459 359 162 433 379

0 372 442 386 446 363 139 453 380 439 365 147 440 386 442 358 138 449 387 455 356 161 428 371

0 373 446 388 449 361 146 449 382 429 364 132 441 390 442 367 126 449 302 452 355 244 429 384

0 378 436 374 447 348 161 439 379 441 353 148 449 374 450 351 174 442 390 446 344 154 435 362

0 385 447 373 441 279 236 451 371 440 269 251 432 365 437 272 232 445 269 448 332 292 442 366
103 

0.5 8 

0.5 3 440 378 445 334 173 440 387 425 352 126 435 371 443 350 157 433 369 449 327 186 437 370

0.5 5 440 388 442 358 136 440 379 423 339 145 433 379 448 329 173 441 380 446 322 185 436 352

0.5 17 439 337 441 311 232 440 345 428 320 203 439 353 452 317 221 440 373 448 301 214 440 355

0.5 20 433 346 439 325 201 432 351 429 317 193 437 346 453 338 206 436 358 449 316 211 439 343

0.5 499 444 365 447 348 178 448 380 436 352 152 448 380 446 326 188 445 374 445 353 163 431 378

0.5 493 430 374 446 344 158 447 371 432 346 162 442 388 445 351 148 453 380 448 337 184 443 359

0.5 485 437 360 446 342 181 437 364 431 330 174 441 352 433 341 181 452 359 445 336 202 437 354

0.5 8/496 441 302 435 250 324 453 316 437 246 328 453 311 442 313 271 449 325 450 237 337 437 309

5 280 443 359 432 350 166 420 351 439 330 178 453 340 435 344 204 438 340 435 322 211 417 349

5 284 423 286 429 323 243 417 299 442 346 214 452 282 429 329 270 439 341 437 317 218 421 280

5 297 429 355 436 308 202 428 367 445 313 193 445 356 422 318 193 442 328 439 307 246 428 351

5 299 431 352 445 317 207 423 350 440 325 188 447 309 423 303 258 440 332 434 326 216 420 348

5 294 440 336 444 270 278 416 349 441 291 217 450 347 430 282 251 441 336 435 281 259 419 344

5 259 427 375 429 328 153 425 369 445 325 176 441 361 435 330 185 432 349 429 321 191 422 352

5 262 427 354 440 328 185 425 349 447 337 186 445 355 442 321 211 437 342 439 320 214 424 344

5 269 416 352 440 346 158 430 355 447 344 178 449 342 431 351 187 434 340 432 258 268 432 343

5 278 446 380 431 330 167 439 369 446 335 181 440 352 442 319 211 438 350 437 319 206 441 350

10 39 433 354 440 324 195 435 347 436 327 197 441 352 436 320 205 438 345 439 321 211 436 352

10 21 429 353 437 324 189 440 358 438 330 190 432 351 441 337 185 439 339 443 320 223 444 347

10 24 433 360 428 328 173 429 358 444 324 191 439 340 452 322 229 431 348 440 331 192 439 341

10 26 444 359 425 326 184 427 351 444 331 189 436 337 449 331 217 441 349 437 329 200 442 338

10 28 435 356 419 342 156 421 359 446 339 169 429 361 449 350 167 429 360 441 321 189 443 352

10 2 439 384 437 362 130 438 381 438 369 126 436 373 436 253 246 435 365 442 357 155 450 377

10 7 441 386 426 379 102 429 381 451 370 129 435 374 441 368 134 441 370 437 374 134 449 379

10 12 428 358 438 355 153 433 362 449 345 175 444 384 436 350 146 428 375 439 348 144 448 357

10 13 430 351 428 367 140 434 367 452 365 154 441 374 442 358 151 431 370 449 357 153 450 352

10 16 442 392 440 362 128 445 393 441 352 141 440 388 442 352 142 436 372 456 352 168 443 370

10 18 440 388 429 364 117 436 391 445 352 138 441 382 449 357 151 436 380 457 353 160 442 371

10 20 426 362 430 370 124 430 358 433 358 147 438 341 445 358 184 441 347 459 363 190 445 339

20 379 418 337 420 337 164 435 340 422 333 184 444 333 429 367 173 432 342 445 340 195 439 372

20 380 423 338 423 330 178 442 337 435 347 193 447 350 423 350 170 429 347 440 352 170 440 375

20 307 433 358 428 333 170 445 356 423 342 170 444 342 425 340 187 434 342 442 339 195 439 309

20 300 436 364 432 353 151 445 338 434 337 204 440 341 433 339 193 435 350 441 337 189 442 280

20 287 431 369 440 371 131 432 382 442 365 127 433 379 432 361 125 437 363 442 365 151 443 363

20 278 432 366 435 369 132 445 358 442 289 240 435 347 442 272 258 446 344 441 352 191 437 342

20 281 432 371 430 375 116 446 376 436 360 146 446 362 447 372 159 438 359 445 364 160 439 369

20 284 429 376 436 365 124 429 380 435 355 129 432 375 450 361 146 442 386 450 358 148 441 375

0 204 

0 377 443 385 432 353 137 432 391 439 359 121 450 302 454 352 250 439 374 447 350 162 439 387

0.5 490 438 382 450 366 140 429 371 436 362 132 447 369 450 358 170 441 381 449 350 159 427 374
104 

 11/12/10 11/15/10 11/19/10 11/22/10 11/25/10 11/29/10 12/2/10 12/6/10 

DOSE PAIR POST PRE POST PRE 

week 

18 POST PRE POST PRE 

week 

19 POST PRE POST PRE 

week 

20 POST PRE POST PRE 

week 

21 

0 391 453 347 455 373 188 436 339 446 341 202 448 327 451 337 235 443 309 435 331 238 

0 392 454 335 455 311 263 446 322 450 331 243 448 318 441 329 242 442 266 433 328 281 

0 393 436 273 445 283 325 436 289 455 271 331 452 307 448 265 328 443 282 435 272 324 

0 397 448 303 443 361 227 457 300 456 316 297 449 309 446 311 275 443 289 441 317 278 

0 398 443 312 439 332 238 439 303 445 328 253 450 321 432 317 244 443 321 439 313 248 

0 205 449 318 434 341 224 418 322 443 335 204 452 325 446 333 240 447 339 440 330 218 

0 371 448 393 447 405 97 434 387 442 374 115 452 365 440 373 154 441 339 417 360 159 

0 372 438 370 445 388 125 434 365 450 367 152 448 363 435 364 156 439 346 429 351 171 

0 373 454 396 452 409 101 433 389 443 382 105 449 264 455 378 262 448 344 417 373 148 

0 378 464 374 464 396 158 448 362 442 370 158 445 347 443 365 176 447 329 434 370 182 

0 385 455 381 454 400 128 443 286 452 376 233 452 350 453 371 184 438 361 433 375 135 

0.5 8 230 457 367 425 236 449 241 397 454 360 436 245 285 447 437 456 259 207 

0.5 3 473 391 467 394 155 435 380 450 379 126 446 379 436 369 134 432 382 452 360 142 

0.5 5 465 354 447 366 192 424 362 447 351 158 437 354 454 354 183 452 394 463 358 163 

0.5 17 453 318 437 332 240 451 322 444 310 263 449 341 454 313 249 450 331 458 311 266 

0.5 20 455 360 451 391 155 426 351 445 351 169 446 318 434 348 214 446 332 425 342 197 

0.5 499 454 352 451 378 175 429 349 442 342 180 448 337 461 331 241 451 336 429 333 211 

0.5 493 453 373 442 384 138 446 370 442 370 148 446 356 446 367 169 451 367 440 369 155 

0.5 485 444 371 445 340 178 442 368 454 369 159 447 361 474 352 208 444 386 425 348 135 

0.5 8/496 455 315 439 375 204 437 304 447 321 259 453 359 453 326 221 449 343 443 324 225 

5 280 448 345 449 374 178 434 279 445 333 267 450 338 454 327 239 439 317 429 329 222 

5 284 310 256 446 343 157 433 261 448 249 371 452 291 447 254 354 440 352 437 241 284 

5 297 434 309 435 342 218 461 303 446 287 317 449 319 445 281 294 442 328 433 272 275 

5 299 330 291 446 352 133 441 299 445 282 305 450 326 449 273 300 442 328 455 265 304 

5 294 315 241 446 344 176 440 252 445 263 370 449 315 455 265 324 440 323 450 260 307 

5 259 462 370 445 380 157 439 363 440 369 147 452 368 430 359 155 441 304 439 349 227 

5 262 449 384 452 407 110 440 371 444 381 132 453 366 445 257 275 447 299 434 253 329 

5 269 460 371 446 377 158 430 365 451 358 158 450 350 449 350 199 442 252 425 343 272 

5 278 448 373 455 400 130 445 362 442 365 160 449 370 447 361 165 445 274 438 348 261 

10 39 457 343 452 364 202 438 338 443 332 211 445 313 449 338 243 448 344 433 330 207 

10 21 436 326 446 369 187 413 320 442 339 196 449 300 434 331 252 446 354 457 333 216 

10 24 453 261 455 333 314 438 279 443 279 323 447 281 432 281 317 445 284 446 279 328 

10 26 445 287 448 361 245 425 288 441 303 275 445 308 443 303 277 445 326 439 310 248 

10 28 445 343 442 369 175 444 336 443 331 220 450 349 444 324 221 451 361 447 318 219 

10 2 453 385 445 401 112 445 370 443 378 140 444 358 455 376 165 447 378 424 370 123 

10 7 446 374 443 359 156 454 365 457 363 183 449 324 430 354 201 444 373 448 351 168 

10 12 447 374 444 353 164 448 356 442 361 173 446 321 435 369 191 449 368 433 362 152 

10 13 446 339 446 357 196 436 362 441 358 157 445 297 432 361 219 451 330 455 369 207 

10 16 446 381 446 357 154 432 368 443 362 145 452 367 431 360 156 442 348 422 368 148 

10 18 457 391 461 410 117 435 383 449 379 122 446 343 436 375 164 452 361 433 361 163 
105 

10 20 446 375 451 400 122 468 372 442 364 174 449 365 433 368 149 450 364 420 361 145 

20 379 460 341 452 389 182 431 

20 380 458 366 449 381 160 446 368 451 350 179 448 358 435 340 185 445 334 448 332 227 

20 307 455 291 443 364 243 429 378 448 318 181 448 287 436 322 275 443 309 461 335 260 

20 300 457 348 450 377 182 437 343 446 316 224 446 338 434 319 223 443 326 450 338 229 

20 287 458 384 449 401 122 457 374 449 371 161 447 371 445 370 151 446 377 422 360 131 

20 278 449 384 452 406 111 459 378 452 378 155 443 366 447 368 156 441 366 453 342 186 

20 281 448 358 446 377 159 447 303 446 351 239 449 370 453 346 186 447 354 442 349 186 

20 284 443 373 446 391 125 432 362 444 370 144 451 343 438 369 177 450 366 413 358 139 

0 204 453 318 445 263 317 440 332 455 312 251 449 317 459 300 291 445 328 431 319 229 

0 377 463 293 464 411 223 430 387 449 383 109 455 355 451 376 175 447 329 422 371 169 

0.5 490 457 282 463 403 235 446 273 438 287 324 462 360 452 281 273 453 371 433 277 238 

F0 paired feed consumption 

 10/4/2010 10/7/2010 10/11/2010 10/14/2010 10/18/2012 10/22/2012 10/25/2010 10/29/2010 11/1/2010 

DOSE PAIR PRE POST PRE 

week 

13 POST PRE POST PRE 

week 

14 POST PRE POST PRE 

week 

15 POST PRE POST PRE 

week 

16 

0 394/374 437 281 442 286 312 451 274 427 291 313 445 269 445 295 326 445 309 444 278 302 

0 375/395 433 313 443 290 273 451 311 432 284 288 444 307 449 291 295 447 279 447 288 327 

0 376/396 441 299 436 278 300 442 305 433 280 290 452 301 452 273 330 447 307 446 273 313 

0 379/399 436 277 443 269 333 442 282 438 262 336 451 283 452 257 363 441 293 442 252 338 

0 380/400 449 323 440 274 292 445 323 438 269 291 451 333 450 263 305 442 338 441 271 274 

0 381/209 437 303 442 285 291 456 310 445 278 313 453 295 444 280 322 439 385 445 277 222 

0 382/202 448 344 447 348 203 448 352 442 332 206 450 349 439 351 189 443 293 450 346 254 

0 383/203 447 287 448 285 323 449 276 443 280 336 442 280 447 288 321 439 340 439 279 259 

0 384/204 438 271 446 287 326 450 276 441 281 334 439 279 445 283 322 440 359 444 278 247 

0 386/206 439 315 441 270 295 450 302 424 268 304 441 300 438 267 312 439 370 440 267 242 

0 387/207 442 287 446 267 334 445 285 431 271 320 457 278 440 261 358 442 309 448 270 311 

0 388/208 443 307 449 272 313 444 311 435 273 295 449 307 440 274 308 445 308 440 286 291 

0 389/210 435 291 449 282 311 441 287 433 286 301 448 281 448 275 340 444 377 447 281 233 

0 390/201 445 299 436 286 296 441 282 434 279 314 441 272 447 280 336 441 271 446 269 347 

0.5 1/488 424 298 446 298 274 435 303 435 301 266 435 300 455 302 288 436 302 452 290 296 

0.5 2/489 427 271 445 258 343 436 279 433 263 327 439 286 451 269 335 438 289 451 287 313 

0.5 4/492 435 308 449 306 270 442 299 440 302 281 443 307 442 301 277 449 317 450 305 277 

0.5 6/494 423 291 442 270 304 445 285 433 274 319 442 311 448 263 316 447 301 440 266 320 

0.5 7/495 430 304 440 313 253 448 310 430 317 251 452 307 448 350 243 449 317 444 310 266 

0.5 9/497 443 329 441 304 251 452 339 441 301 253 453 320 447 312 268 442 321 451 301 271 

0.5 13/478 442 301 436 295 282 455 311 439 295 288 442 209 443 301 375 443 316 455 289 293 

0.5 14/479 444 304 432 263 309 447 324 442 270 295 442 317 424 273 276 441 329 445 252 305 

0.5 15/480 445 306 447 301 285 449 318 441 308 264 443 321 449 306 265 444 312 445 303 274 

0.5 16/481 441 292 439 268 320 442 302 436 264 312 435 267 445 269 344 445 333 449 268 293 

0.5 10/482 442 282 443 271 332 443 274 435 263 341 437 273 443 264 343 441 316 444 261 308 

0.5 11/483 443 324 442 292 269 443 318 428 302 251 441 312 445 288 286 449 324 442 252 315 
106 

0.5 484/498 435 321 435 275 274 440 329 430 279 262 440 337 443 275 271 450 335 442 272 285 

0.5 486/18 443 289 447 301 300 440 301 432 297 274 439 307 434 498 68 449 316 442 304 271 

0.5 487/19 445 311 448 297 285 439 320 428 301 246 451 302 435 300 284 447 320 439 289 277 

5 260/281 433 306 424 298 253 421 317 446 303 247 447 311 433 301 268 436 317 432 288 263 

5 261/283 439 316 430 305 248 429 309 449 304 265 447 303 437 311 270 430 325 435 309 231 

5 263/285 439 294 441 289 297 427 287 450 291 299 442 284 444 307 295 431 316 442 279 278 

5 264/286 446 313 443 301 275 429 323 436 306 236 439 319 445 286 279 435 345 436 292 234 

5 279/287 445 313 445 291 286 435 329 435 282 259 446 320 448 288 286 432 328 444 286 262 

5 266/288 426 263 442 285 320 432 284 441 290 299 449 288 441 275 327 421 370 441 273 219 

5 267/295 438 307 438 308 261 436 316 440 300 260 450 311 435 303 271 424 313 435 301 245 

5 268/296 437 280 439 288 308 432 284 442 292 298 456 270 430 279 337 429 281 432 272 308 

5 270/298 438 290 432 296 284 431 285 439 291 294 451 281 432 303 299 435 276 436 292 303 

5 272/300 444 321 426 309 240 432 329 442 299 246 442 335 437 320 224 432 322 436 301 245 

5 273/289 442 300 427 270 299 429 308 436 278 279 442 319 442 267 298 432 308 437 265 296 

5 274/290 440 292 439 265 322 432 293 441 273 307 439 306 444 279 298 444 315 431 264 296 

5 275/291 437 312 432 282 275 433 299 435 274 295 440 314 435 263 298 440 317 432 273 282 

5 276/292 427 279 446 275 319 444 261 435 271 347 445 272 440 280 333 438 263 439 270 344 

5 277/293 444 275 429 270 328 440 271 448 276 341 446 267 437 282 334 436 281 442 253 344 

10 1/33 440 334 438 282 262 439 330 442 271 280 433 225 442 283 367 432 229 440 276 367 

10 3/35 446 285 440 265 336 441 290 436 273 314 435 284 437 264 324 428 278 442 263 329 

10 4/36 437 297 433 295 278 431 289 441 279 304 434 292 443 285 300 431 294 445 292 290 

10 91/37 433 296 422 323 236 425 292 444 309 268 435 291 445 329 260 412 287 440 310 255 

10 92/94 441 291 433 304 279 434 297 445 292 290 434 256 446 319 305 437 266 442 300 313 

10 8/40 432 250 428 269 341 427 240 446 270 363 433 245 440 251 377 439 242 439 261 375 

10 93/30 423 316 444 303 248 432 310 442 291 273 439 299 440 301 279 445 301 444 297 291 

10 10/31 426 296 442 321 251 442 301 443 324 260 445 312 438 327 244 444 302 442 317 267 

10 11/32 424 311 440 303 250 439 312 445 303 269 442 301 439 300 280 442 302 443 308 275 

10 14/22 435 274 435 263 333 435 274 449 270 340 438 256 441 269 354 427 251 452 251 377 

10 15/23 439 253 443 281 348 443 261 441 279 344 436 263 442 288 327 430 251 455 270 364 

10 17/25 444 322 446 301 267 442 326 446 309 253 447 327 450 312 258 430 319 455 309 257 

10 19/27 437 302 432 320 247 433 305 436 312 252 445 300 448 319 274 444 303 457 312 286 

20 269/210 417 320 418 302 213 444 316 429 305 252 439 310 436 289 276 445 298 445 300 292 

20 270/291 415 264 417 286 282 436 279 433 287 303 442 278 434 307 291 442 282 441 288 313 

20 271/292 430 300 425 289 266 432 301 435 289 277 444 292 441 281 312 445 297 440 285 303 

20 272/293 428 302 429 306 249 429 300 440 292 277 439 297 433 307 268 445 291 450 300 304 

20 289/294 445 323 429 309 242 427 323 437 317 224 443 313 430 319 241 445 305 448 301 287 

20 274/295 447 324 433 319 237 441 325 436 311 241 442 326 432 309 239 441 310 447 311 267 

20 285/296 423 340 435 298 220 443 337 437 302 241 443 334 435 303 241 438 329 445 303 251 

20 286/297 434 306 436 324 240 438 311 437 318 246 439 317 441 315 248 435 319 441 324 233 

20 288/299 445 303 442 287 297 448 303 448 278 315 432 309 431 288 266 430 313 433 380 170 

20 275/301 429 310 445 301 263 427 319 445 303 250 438 327 440 305 246 429 323 436 299 243 

20 276/290 432 329 448 292 259 438 334 448 293 259 428 330 445 296 247 434 338 431 291 236 

20 277/303 438 291 432 287 292 439 300 444 287 296 430 305 441 283 283 432 314 435 290 263 
107 

20 279/305 431 327 428 285 247 439 331 432 282 258 444 334 440 281 269 445 340 442 311 236 

20 280/306 430 316 432 310 236 444 310 430 283 281 439 316 436 303 256 427 311 442 295 263 

20 282/308 433 332 431 270 262 446 335 430 274 267 442 347 445 374 166 443 352 443 263 271 

20 283/309 436 335 430 301 230 432 342 431 281 240 442 348 448 306 236 440 351 447 300 236 

PAIRED 

 

11/12/201

0 

11/15/201

0 

11/19/201

0 

11/22/201

0 

11/25/201

0 

11/29/201

0 

12/2/201

0 

12/6/201

0 

DOSE PAIR 

POS

T PRE POST PRE 

week 

18 

POS

T PRE 

POS

T PRE 

week 

19 

POS

T PRE 

POS

T PRE 

week 

20 

POS

T PRE 

POS

T PRE 

week 

21 

0 

394/37

4 447 261 451 297 340 451 267 440 253 371 452 274 455 252 381 447 272 436 254 357 

0 

375/39

5 455 297 449 331 276 419 305 451 282 283 450 285 440 281 324 447 275 419 275 316 

0 

376/39

6 450 278 452 329 295 438 280 445 373 230 455 300 442 374 223 449 285 437 274 327 

0 

379/39

9 454 262 450 296 346 455 269 446 255 377 443 284 425 257 327 445 271 431 252 353 

0 

380/40

0 445 241 461 317 348 442 251 446 259 378 445 254 432 250 373 446 255 407 248 350 

0 

381/20

9 460 304 449 339 266 436 396 459 302 197 439 273 432 310 288 442 306 428 309 255 

0 

382/20

2 455 261 454 323 325 455 275 460 257 383 435 266 432 249 352 439 310 428 248 309 

0 

383/20

3 441 266 446 296 325 435 252 455 258 380 446 271 439 259 355 443 288 434 252 337 

0 

384/20

4 458 372 461 390 157 436 388 454 370 132 449 356 459 373 179 445 365 437 373 144 

0 

386/20

6 448 273 460 337 298 442 259 450 276 357 461 269 434 264 362 446 267 423 270 332 

0 

387/20

7 457 278 454 324 309 445 284 452 278 335 454 272 436 274 344 445 276 417 264 322 

0 

388/20

8 451 266 454 338 301 435 261 454 263 365 452 272 442 262 360 447 292 435 257 333 

0 

389/21

0 456 261 454 315 334 430 261 452 259 362 446 275 436 265 342 446 289 446 263 340 

0 

390/20

1 460 269 452 284 359 450 268 447 261 368 449 259 433 257 366 447 230 450 251 416 

0.5 1/488 452 307 441 334 252 423 300 444 303 264 446 288 454 304 308 446 299 437 307 277 

0.5 2/489 449 255 459 316 337 431 259 440 260 352 450 255 444 252 387 446 250 441 257 380 

0.5 4/492 448 277 445 337 279 464 269 443 262 376 441 302 438 260 317 447 296 428 265 314 

0.5 6/494 435 282 443 321 275 439 286 443 278 318 450 284 431 276 321 453 273 406 279 307 

0.5 7/495 450 301 444 332 261 433 305 450 308 270 449 269 437 315 302 450 297 445 318 280 

0.5 9/497 446 275 453 328 296 425 268 450 264 343 449 280 433 251 351 451 270 433 253 361 

0.5 13/478 448 379 447 310 206 420 370 454 379 125 454 282 447 279 340 447 273 427 371 230 

0.5 14/479 453 297 454 309 301 437 283 442 268 328 445 277 441 266 343 442 292 437 262 325 

0.5 15/480 448 365 442 330 195 451 261 448 257 381 455 298 440 247 350 443 301 431 241 332 

0.5 16/481 443 281 451 334 279 454 267 450 277 360 454 307 444 263 328 440 314 427 262 291 

0.5 10/482 446 290 446 264 338 421 281 449 287 302 454 273 421 273 329 442 237 422 271 356 

0.5 11/483 444 288 450 323 283 433 380 457 288 222 452 282 437 277 330 445 283 417 276 303 

0.5 

484/49

8 460 290 446 321 295 449 287 455 276 341 443 295 445 278 315 444 284 421 270 311 

0.5 486/18 445 155 449 308 431 454 244 454 261 403 448 269 428 275 332 442 288 411 273 292 

0.5 487/19 439 288 442 328 265 452 271 451 270 362 449 292 430 267 320 445 292 445 265 333 

5 

260/28

1 462 250 448 339 321 453 268 439 248 376 456 292 449 254 359 445 280 441 259 347 

5 

261/28

3 454 263 458 310 339 458 259 441 260 380 458 266 415 258 349 447 312 451 244 342 

5 

263/28

5 451 270 442 319 304 449 263 449 262 373 446 263 431 267 347 444 276 442 255 355 

5 

264/28

6 443 266 449 304 322 448 254 446 254 386 443 289 445 252 347 446 290 445 259 342 

5 

279/28

7 448 304 433 346 231 444 310 445 311 268 459 324 453 318 270 442 311 449 303 277 
108 

5 

266/28

8 449 240 472 317 364 445 256 440 233 396 455 279 437 230 383 442 282 422 250 332 

5 

267/29

5 433 272 466 334 293 441 260 447 271 357 444 287 425 264 318 441 271 420 259 331 

5 

268/29

6 463 260 448 281 370 439 271 445 250 363 453 279 437 258 353 439 270 422 258 333 

5 

270/29

8 457 304 441 286 308 434 309 445 324 246 449 293 445 322 279 442 359 438 304 217 

5 

272/30

0 453 295 452 341 269 437 290 446 291 302 442 294 427 288 287 440 277 429 284 308 

5 

273/28

9 455 272 449 320 312 454 280 445 276 343 447 281 435 289 312 442 265 426 277 326 

5 

274/29

0 447 250 450 291 356 468 254 449 243 420 448 272 435 237 374 442 323 419 231 307 

5 

275/29

1 454 269 449 320 314 443 263 444 253 371 441 277 434 241 357 441 268 428 245 356 

5 

276/29

2 460 274 454 313 327 466 241 442 259 408 444 292 447 240 359 438 266 424 230 366 

5 

277/29

3 453 247 455 290 371 443 263 444 252 372 446 263 433 253 363 440 279 429 242 348 

10 1/33 448 283 444 313 296 451 278 446 278 341 445 306 459 272 326 445 312 433 267 299 

10 3/35 445 260 441 304 322 436 251 442 252 375 448 280 441 251 358 446 296 438 256 332 

10 4/36 447 266 445 318 308 459 254 449 266 388 452 271 454 261 374 449 289 451 268 343 

10 91/37 449 261 439 307 320 452 259 450 263 380 499 268 442 258 415 445 291 433 254 333 

10 92/94 452 286 444 321 289 458 277 458 261 378 448 265 430 258 355 445 288 434 245 346 

10 8/40 452 247 440 267 378 456 253 455 239 419 449 242 418 244 381 446 264 445 243 384 

10 93/30 452 244 444 274 378 454 244 460 252 418 452 273 419 243 355 451 278 411 234 350 

10 10/31 447 254 445 306 332 448 248 441 245 396 448 289 421 230 350 452 275 416 243 350 

10 11/32 453 237 445 280 381 445 242 443 244 402 444 281 434 242 355 447 294 432 244 341 

10 14/22 440 246 447 317 324 454 240 441 240 415 460 255 421 235 391 446 261 416 231 370 

10 15/23 447 240 446 313 340 454 241 442 256 399 455 296 433 260 332 444 246 404 251 351 

10 17/25 444 288 445 345 256 469 279 455 273 372 448 303 433 267 311 448 310 432 257 313 

10 19/27 450 282 442 288 322 451 280 442 260 353 451 283 431 254 345 449 279 439 242 367 

20 

269/21

0 451 298 444 339 258 463 287 442 286 332 447 295 458 280 330 441 305 450 240 346 

20 

270/29

1 452 265 450 319 318 460 252 447 267 388 445 281 438 261 341 446 317 451 259 321 

20 

271/29

2 455 256 449 301 347 460 259 446 241 406 447 278 433 326 276 442 343 452 241 310 

20 

272/29

3 453 287 453 332 287 474 280 446 263 377 448 300 437 259 326 442 291 438 243 346 

20 

289/29

4 446 319 444 348 223 469 312 442 300 299 444 313 454 314 271 443 325 436 302 252 

20 

274/29

5 448 254 448 322 320 469 248 446 247 420 450 292 442 242 358 446 286 444 237 367 

20 

285/29

6 452 311 451 342 250 465 301 448 313 299 448 327 447 317 251 449 312 441 314 264 

20 

286/29

7 447 284 441 321 283 452 281 452 298 325 452 301 454 294 311 443 302 439 284 296 

20 

288/29

9 451 251 449 329 320 462 242 442 241 421 447 302 439 236 348 449 306 444 248 339 

20 

275/30

1 440 294 440 323 263 468 290 443 297 324 444 310 429 299 264 443 363 451 283 248 

20 

276/29

0 447 311 445 352 229 463 333 442 317 255 442 322 448 313 255 441 324 451 304 264 

20 

277/30

3 449 269 444 324 300 446 267 451 262 368 439 283 428 265 319 441 285 431 265 322 

20 

279/30

5 453 271 457 308 331 463 244 449 261 407 444 295 428 380 197 443 267 443 363 256 

20 

280/30

6 458 263 449 330 314 465 354 442 273 280 442 294 428 262 314 450 282 435 257 346 

20 

282/30

8 445 286 448 337 270 456 275 448 279 350 452 302 440 271 319 449 302 437 260 324 

20 

283/30

9 440 303 439 308 268 453 321 444 316 260 452 301 431 325 257 451 294 428 329 256 
109 

F0 bird weights 

ppm Sex ID 8/10/10 8/17/10 8/24/10 8/31/10 9/7/10 9/14/10 9/21/10 10/5/10 10/12/10 10/19/10 10/26/10 11/2/10 11/9/10 11/16/10 11/2

0 F 391 106 118 127 128 135 130 141 134 144 141 148 149 138 147 147

0 F 392 92 100 109 112 119 131 139 134 142 150 141 141 141 139 140

0 F 393 108 100 117 122 135 144 143 147 149 159 148 153 151 153 152

0 F 394 100 110 125 132 133 137 136 140 140 150 136 138 138 140 140

0 F 395 98 103 113 122 138 124 138 134 136 138 138 136 134 136 135

0 F 396 101 111 121 125 144 144 144 141 146 144 147 148 148 145 144

0 F 397 98 118 125 122 126 128 127 126 135 132 135 136 135 136 136

0 F 398 89 100 108 116 112 137 129 136 136 135 137 141 143 141 140

0 F 399 96 109 126 97 123 134 135 121 137 136 138 137 135 138 139

0 F 400 90 91 104 127 120 128 119 126 130 130 123 1334 130 136 136

0 F 201 109 116 123 131 122 137 133 137 145 141 142 134 135 139 139

0 F 202 99 102 103 114 112 128 132 136 132 135 144 142 144 142 142

0 F 203 92 104 116 123 132 138 139 144 148 150 148 145 147 149 147

0 F 204 88 104 119 132 129 130 119 131 106 105 135 129 135 141 141

0 F 205 104 107 118 125 135 131 135 127 139 139 143 136 130 138 138

0 F 206 89 95 102 118 128 127 126 126 132 133 134 128 135 135 135

0 F 207 93 98 117 132 130 137 138 139 139 135 147 150 149 142 142

0 F 208 112 119 129 131 136 139 147 153 158 155 158 157 156 139 140

0 F 209 99 107 123 135 121 140 141 136 150 150 144 143 140 152 153

0 F 210 107 110 135 142 137 148 146 145 142 142 158 154 158 162 162

0 M 371 93 105 109 111 113 114 112 113 116 115 121 125 122 121 120

0 M 372 90 97 111 109 108 111 114 117 118 119 117 120 120 122 121

0 M 373 90 94 99 102 99 102 101 101 102 103 105 106 106 104 104

0 M 374 91 98 107 106 107 112 112 113 113 115 117 118 118 117 117

0 M 375 94 109 102 104 109 116 122 116 126 125 127 126 127 125 124

0 M 376 76 93 95 94 95 96 97 97 101 100 99 98 101 100 101

0 M 377 89 96 103 106 105 107 109 105 110 109 110 109 114 113 112

0 M 378 101 93 106 113 114 118 117 122 125 125 114 112 120 122 120

0 M 379 103 118 127 130 131 135 132 130 135 132 129 133 133 134 133

0 M 380 97 95 103 102 100 103 102 109 107 110 111 113 113 112 112

0 M 381 101 105 113 116 116 120 120 119 123 120 123 123 122 122 121

0 M 382 91 103 105 105 111 110 115 113 100 101 107 110 110 115 115

0 M 383 73 88 101 100 99 102 104 103 103 104 105 102 108 109 108

0 M 384 85 94 93 98 101 103 104 103 106 106 109 111 112 112 113

0 M 385 92 99 99 104 106 105 107 112 112 115 112 110 109 110 112
110 

0 M 386 82 95 102 102 106 108 110 112 113 114 113 110 112 113 113

0 M 387 84 93 100 104 106 105 109 106 104 107 106 106 108 106 105

0 M 388 93 102 105 111 114 116 119 120 121 120 123 123 122 123 122

0 M 389 92 97 99 104 106 106 107 107 110 111 110 111 111 111 111

0 M 390 94 101 110 109 110 110 109 114 113 111 114 113 111 115 115

0.5 F 499 80 100 407 108 110 122 129 127 131 133 131 134 135 133 130

0.5 F 11 96 100 90 101 105 111 117 130 121 120 135 135 129 138 138

0.5 F 498 73 85 95 98 103 102 115 132 129 128 125 113 129 130 130

0.5 F 13 97 96 112 121 129 139 139 144 140 139 147 148 151 151 150

0.5 F 14 105 108 141 122 135 138 141 147 140 140 145 142 154 158 159

0.5 F 15 81 91 98 109 123 126 124 130 136 138 126 131 136 136 137

0.5 F 16 83 88 97 97 106 116 115 113 120 122 124 124 126 126 128

0.5 F 17 102 110 121 123 141 145 133 144 152 152 144 153 152 156 156

0.5 F 18 103 112 141 133 134 146 145 157 157 155 157 153 158 159 158

0.5 F 19 87 93 99 84 100 104 107 120 143 127 122 138 113 137 137

0.5 F 20 94 101 113 129 130 136 129 143 143 143 146 146 149 146 148

0.5 F 1 92 90 100 105 111 117 130 130 128 128 129 129 118 130 131

0.5 F 2 84 92 105 117 124 126 118 126 128 126 130 133 133 131 130

0.5 F 3 84 92 98 99 105 114 116 116 125 129 132 121 117 124 124

0.5 F 4 98 104 114 122 134 130 142 137 141 137 141 127 148 146 146

0.5 F 5 95 107 115 125 149 149 140 124 127 150 142 152 152 133 132

0.5 F 6 99 112 115 126 130 138 147 146 153 130 151 154 154 154 154

0.5 F 7 93 107 109 109 130 131 131 139 129 143 141 143 135 147 147

0.5 F 8 91 104 110 127 136 131 140 135 110 142 139 140 125 140 141

0.5 F 9 103 112 122 127 136 145 138 141 143 141 150 144 136 143 143

0.5 F 10 102 115 127 143 144 158 151 157 156 154 154 152 162 161 160

0.5 M 488 79 95 99 101 99 95 98 104 106 103 105 106 107 106 106

0.5 M 489 88 94 100 99 102 102 104 109 113 115 115 116 116 117 117

0.5 M 490 90 99 104 100 102 105 104 103 106 107 108 107 110 112 112

0.5 M 492 89 94 96 96 93 99 99 102 104 108 101 103 104 106 107

0.5 M 493 88 99 103 106 112 112 116 116 121 122 121 120 120 120 119

0.5 M 494 83 96 100 101 106 106 109 108 109 111 111 114 112 111 112

0.5 M 495 82 92 102 102 99 102 101 103 107 108 107 108 109 105 104

0.5 M 496/491 93 106 105 105 111 112 111 117 120 122 120 120 114 119 154

0.5 M 497 90 98 99 97 100 104 106 100 103 105 106 108 107 106 106

0.5 M 478 91 105 106 106 107 116 115 114 118 115 119 118 117 120 120

0.5 M 479 92 100 103 106 104 109 110 109 112 113 116 115 116 117 117

0.5 M 480 95 99 105 106 103 107 108 110 110 112 111 110 112 113 112

0.5 M 481 95 103 108 109 111 111 113 111 113 112 115 114 116 117 116

0.5 M 482 90 102 110 115 117 122 122 119 121 117 122 121 124 125 125

0.5 M 483 84 94 98 100 102 105 104 111 112 110 116 114 117 116 116

0.5 M 484 92 100 101 99 103 104 107 104 107 108 105 106 109 113 115

0.5 M 485 84 94 103 103 103 104 107 107 109 111 112 111 111 112 112
111 

0.5 M 486 88 98 99 104 98 100 102 104 105 108 105 105 105 104 105

0.5 M 487 89 97 96 96 108 99 104 109 112 116 112 114 117 116 115

5 F 280 98 105 110 117 139 140 133 139 145 150 149 138 145 142 141

5 F 281 102 119 143 137 143 147 149 147 154 156 152 155 134 147 147

5 F 283 80 88 104 105 126 107 127 129 131 130 131 123 116 135 135

5 F 284 89 90 103 111 119 135 138 137 137 135 141 135 140 141 141

5 F 285 93 101 126 121 137 130 140 144 143 140 142 134 138 148 148

5 F 286 91 97 108 111 108 121 115 126 123 121 126 121 126 122 121

5 F 287 100 100 117 125 135 149 143 140 140 138 149 151 138 131 135

5 F 288 82 88 102 105 121 133 133 127 133 130 136 126 128 126 127

5 F 289 95 92 98 107 129 129 131 132 133 136 135 137 137 136 136

5 F 290 97 111 132 130 118 131 133 139 140 143 140 137 135 133 133

5 F 291 87 90 114 117 109 111 113 125 127 123 128 119 128 129 130

5 F 292 94 104 116 109 117 133 147 134 140 143 141 129 128 134 134

5 F 293 102 110 124 136 137 146 144 150 155 151 154 155 146 154 154

5 F 294 89 95 109 109 110 117 124 129 130 134 122 132 130 125 125

5 F 295 96 112 113 128 144 144 138 150 149 144 152 153 155 153 153

5 F 296 100 103 107 130 133 138 140 143 143 140 148 151 149 147 146

5 F 297 92 109 126 141 142 142 145 137 145 149 146 148 148 149 149

5 F 298 96 112 128 118 125 126 127 129 131 135 131 128 128 124 126

5 F 299 96 97 114 137 143 148 145 138 152 150 151 158 144 154 154

5 F 300 81 95 105 110 131 128 133 128 132 136 124 133 120 133 136

5 M 267 92 93 106 110 111 109 111 107 108 110 108 110 111 110 111

5 M 268 102 105 107 110 112 115 116 114 117 120 117 117 119 120 120

5 M 269 93 97 97 98 97 99 99 110 107 105 105 110 108 107 106

5 M 270 82 86 89 91 92 92 95 97 97 99 96 97 96 97 97 

5 M 271 76 103 114 113 114 114 118 115 116 113 120 119 119 120 120

5 M 272 88 90 92 95 93 100 100 104 103 102 106 107 105 105 105

5 M 273 97 107 114 108 115 119 121 108 127 115 126 127 129 129 128

5 M 274 91 101 107 106 112 112 114 121 111 129 112 112 113 113 113

5 M 275 80 101 105 105 103 104 104 122 103 127 104 103 106 107 107

5 M 276 109 99 106 110 113 126 136 112 140 110 141 140 136 139 139

5 M 277 99 100 103 104 101 101 103 103 105 100 104 105 105 106 106

5 M 278 90 94 96 97 95 96 96 96 97 143 101 102 101 103 101

5 M 259 89 92 104 106 110 113 116 104 115 103 120 122 121 120 120

5 M 260 93 103 115 116 113 123 125 96 126 99 129 130 131 128 127

5 M 261 88 97 104 106 107 109 107 102 110 112 113 113 109 115 115

5 M 262 95 105 101 102 104 106 106 108 113 119 115 114 115 115 116

5 M 263 97 100 106 107 109 109 109 112 114 116 114 111 112 112 113

5 M 264 98 108 110 106 112 115 115 115 115 119 114 115 117 116 117

5 M 279 77 89 85 97 98 98 100 98 100 103 100 100 100 104 104

5 M 266 81 91 99 107 108 97 93 99 97 100 99 99 99 96 98 

10 F 33 97 110 116 116 124 126 127 119 133 136 139 133 143 138 137
112 

10 F 34 99 108 116 120 121 129 138 

10 F 35 90 100 110 116 118 133 127 126 128 130 126 122 128 136 136

10 F 36 98 98 111 125 129 137 140 138 135 134 144 145 149 151 152

10 F 37 103 112 117 121 126 123 126 141 123 122 144 138 144 148 145

10 F 94 85 89 102 109 126 121 119 125 118 119 118 125 120 128 128

10 F 39 97 111 120 135 138 139 142 141 143 145 145 143 144 144 144

10 F 40 81 94 102 109 115 124 128 137 129 130 140 131 140 141 140

10 F 27 87 95 99 109 115 124 118 125 125 126 130 133 139 133 133

10 F 28 92 101 108 116 121 129 131 132 134 145 140 136 136 136 136

10 F 29 78 87 100 102 112 114 122 

10 F 30 102 112 123 142 138 145 151 147 147 147 154 154 158 155 157

10 F 31 95 102 112 116 136 137 138 130 136 138 138 139 130 136 137

10 F 32 100 103 112 117 121 125 139 144 145 149 151 150 153 156 156

10 F 21 104 117 129 137 130 140 141 145 138 140 141 147 150 149 149

10 F 22 89 96 102 99 106 116 119 117 121 122 117 121 122 116 116

10 F 23 94 102 109 132 134 135 133 131 128 131 135 134 139 143 142

10 F 24 102 108 118 126 123 137 136 132 141 140 143 138 145 137 137

10 F 25 101 96 115 131 136 142 144 147 142 143 145 154 143 141 141

10 F 26 85 94 107 126 130 130 126 132 127 130 135 126 128 127 127

10 M 1 90 93 102 107 108 109 110 109 112 118 112 114 111 115 116

10 M 2 92 87 94 101 107 111 112 112 116 115 119 118 118 118 118

10 M 3 84 97 104 109 110 111 111 114 111 113 115 115 117 116 116

10 M 4 83 91 101 104 96 98 101 103 106 105 111 111 110 110 111

10 M 91 97 103 110 111 114 117 115 119 121 120 123 123 122 125 125

10 M 92 77 88 89 92 94 96 96 97 99 100 99 99 99 98 99 

10 M 7 76 85 90 89 98 95 97 98 102 105 104 101 102 107 107

10 M 8 84 95 100 106 103 101 106 108 112 110 109 114 114 120 121

10 M 93 98 106 115 114 116 118 119 117 120 126 120 122 120 121 121

10 M 10 96 99 110 107 119 108 110 110 112 111 116 118 121 123 122

10 M 11 93 97 99 101 100 103 101 104 108 106 107 110 112 114 114

10 M 12 81 88 100 103 103 101 101 102 107 106 108 105 108 107 107

10 M 13 97 112 115 118 118 121 121 126 128 122 127 126 128 131 132

10 M 14 89 100 101 102 106 108 111 108 107 111 106 110 111 111 113

10 M 15 91 99 97 107 110 116 116 115 115 111 117 119 121 121 121

10 M 16 84 91 96 98 98 100 101 99 100 103 105 103 105 107 107

10 M 17 92 97 104 105 105 107 108 105 104 105 108 107 106 109 109

10 M 18 92 92 96 98 100 100 102 101 104 107 103 105 106 106 106

10 M 19 63 89 96 99 98 99 100 101 102 107 103 104 106 105 106

10 M 20 8 99 11 114 113 117 119 119 117 120 121 122 124 124 124

20 F 301 98 112 127 119 115 118 124 126 127 128 140 130 133 142 142

20 F 290 86 95 103 105 118 105 111 110 114 116 140 116 107 114 114

20 F 303 96 108 119 133 114 130 134 132 148 145 154 143 154 154 155

20 F 380 83 97 101 104 130 126 131 142 146 144 151 146 153 153 150
113 

20 F 305 87 84 100 100 117 119 124 112 127 130 131 131 119 128 128

20 F 306 86 98 112 114 136 131 132 142 139 140 141 143 142 143 143

20 F 307 89 97 107 121 131 126 139 131 129 132 127 128 138 132 133

20 F 308 90 99 110 111 120 125 137 139 131 133 147 137 134 149 146

20 F 309 88 90 98 91 108 115 123 113 113 114 124 131 127 120 120

20 F 310 83 91 102 110 107 119 116 127 127 128 109 103 108 112 114

20 F 291 104 119 119 139 142 137 144 133 143 140 139 146 130 141 140

20 F 292 91 103 112 123 124 125 126 133 125 130 136 136 139 138 138

20 F 293 100 106 114 132 132 133 140 136 170 165 142 144 127 144 144

20 F 294 71 83 90 95 95 98 97 99 102 103 104 109 104 109 109

20 F 295 93 99 108 117 118 142 135 134 115 115 140 136 145 146 146

20 F 296 92 98 107 106 110 111 111 103 131 131 109 110 110 116 116

20 F 297 89 95 108 116 120 123 131 137 126 127 137 124 125 128 126

20 F 379 77 90 98 117 125 117 121 119 129 130 130 131 120 

20 F 299 95 102 108 123 125 129 130 127 131 130 135 138 136 145 145

20 F 300 86 96 106 113 118 123 126 127 129 131 126 130 132 135 136

20 M 269 89 96 100 98 103 103 104 106 107 110 108 108 109 110 111

20 M 270 101 106 116 123 123 129 132 130 130 131 131 130 127 130 129

20 M 271 90 99 100 102 105 106 109 107 111 114 111 112 113 112 111

20 M 272 90 97 101 104 103 106 106 103 109 111 109 108 107 110 111

20 M 289 96 99 112 113 111 113 114 113 112 113 90 105 107 112 112

20 M 274 94 101 105 109 112 112 116 111 113 113 114 115 116 118 118

20 M 285 95 102 105 105 108 109 112 108 112 113 113 114 114 113 118

20 M 286 87 96 107 104 109 112 116 111 119 123 118 119 119 119 105

20 M 287 81 91 96 99 104 106 107 106 107 111 109 109 107 110 107

20 M 288 85 102 100 110 109 113 115 115 119 120 121 119 118 118 112

20 M 279 94 94 103 100 111 109 113 106 115 118 109 118 113 114 114

20 M 280 78 83 93 95 99 93 96 97 105 107 100 107 106 104 107

20 M 281 87 95 102 102 108 102 103 109 105 106 113 105 113 112 111

20 M 282 96 102 107 104 100 108 112 111 108 111 118 111 117 116 116

20 M 283 80 87 99 103 107 108 109 110 113 112 115 113 117 115 115

20 M 284 89 99 99 105 97 102 109 117 97 98 110 105 117 118 118

20 M 275 92 101 107 110 104 114 116 116 108 110 118 115 118 119 118

20 M 276 84 92 96 98 110 98 102 105 115 115 108 104 106 105 105

20 M 277 89 100 108 109 105 105 106 103 113 116 106 118 107 107 107

20 M 278 87 91 100 95 103 103 105 104 116 117 109 114 112 112 112

ppm ID Motile 1 Motile 2 Motile 3 Motile Average Progressive 1 Progressive 2 Progresive 3 Progressive Average Sperm/gram 1 Sperm/gram 2 Sperm

0 371 92 59 69 73.33333333 66 11 14 30.33333333 4592.3 2175.3 2428.5

0 372 53 62 57.5 8 6 7 863.2 1070.4 

0 373 66 31 48.5 0 0 0 283 225.1 
114 

0 378 46 46 33 41.66666667 6 6 5 5.666666667 1519.8 1629.2 1394.1

0 379 65 57 61 13 15 14 743.5 727.1 

0 380 54 59 56.5 11 10 10.5 822.9 1041.6 

0 384 39 26 32.5 7 7 7 1416 1213.7 

0 385 19 23 21 3 4 3.5 688.8 863.8 

0 386 90 67 78.5 28 12 20 2375.1 1530.8 

0 387 75 71 73 3 21 12 924.4 1247.5 

0.5 478 52 64 55 57 10 23 18 17 1251.9 1667.4 1344.9

0.5 482 77 74 75.5 9 10 9.5 1469.3 1489.8 

0.5 483 60 63 61.5 12 16 14 1339.4 1711.2 

0.5 484 59 61 60 22 25 23.5 3318.2 3403.8 

0.5 485 34 38 36 7 7 7 1281.7 1281.7 

0.5 488 9 3 6 0 0 0 180.4 196.8 

0.5 489 92 90 80 87.33333333 91 35 39 55 4175.7 2218 2613.7

0.5 490 73 79 76 30 38 34 1910.6 2066 

0.5 496 82 85 83.5 2 3 2.5 765.4 585.3 

0.5 497 73 43 58 9 1 5 1098.6 728.9 

5 259 89 84 64 79 42 31 25 32.66666667 2284 1785.9 1160.2

5 260 72 72 72 0 1 0.5 256.7 275.7 

5 261 84 82 83 38 48 43 1491 1580.5 

5 266 56 64 53 57.66666667 3 9 6 6 623.2 623.2 672.4 

5 267 63 55 59 7 2 4.5 574 541.2 

5 268 77 84 80.5 37 47 42 5418.4 5977.3 

5 272 75 33 54 13 2 7.5 2159.5 1547.2 

5 273 83 68 75.5 9 7 8 1935.3 1361.3 

5 274 24 16 20 3 0 1.5 855.7 808.2 

5 275 30 43 36.5 4 2 3 1022.3 1033.3 

10 1 25 22 23.5 2 2 2 1007.1 943.8 

10 10 25 54 46 41.66666667 2 8 13 7.666666667 584.7 884.2 741.6 

10 14 26 18 22 1 3 2 1191.2 909.2 

10 15 59 70 64.5 22 43 32.5 1946.3 2618.7 

10 16 62 33 47.5 6 1 3.5 441.9 291.6 

10 17 52 45 48.5 37 23 30 2611.3 2238.3 

10 2 78 54 50 60.66666667 9 10 17 12 1762.3 1054.8 1633.7

10 3 68 72 70 9 18 13.5 1498.6 1672.3 

10 8 87 72 79.5 1 2 1.5 492 404.6 

10 93 33 41 37 2 7 4.5 688.2 566 

20 269 72 26 30 42.66666667 4 0 1 1.666666667 1077 661.5 574 

20 270 88 76 82 39 22 30.5 1377.7 1055.1 

20 271 80 84 82 3 7 5 1292.8 1138.4 

20 278 47 67 84 66 6 17 20 14.33333333 869.3 1454.2 1760.4

20 279 92 89 63 81.33333333 36 35 16 29 4056.5 3608.2 2493 

20 280 97 93 97 95.66666667 124 60 101 95 6316 3421.7 5055.4
115 

20 281 67 79 86 77.33333333 20 36 40 32 2284 3669 3450.3

20 286 91 74 82.5 33 14 23.5 1665.8 1279.9 

20 287 44 57 50.5 11 24 17.5 2379.8 2797.8 

20 288 79 76 77.5 12 8 10 1041.3 807 
F0 male onset to puberty 7/7/10 “birthday” 100% of controls became foamy on 8/30/10 

ppm ID puberty day of onset 0 371 8/19/10 43 0 372 8/23/10 47 0 373 8/24/10 48 0 374 8/22/10 46 0 375 8/30/11 54 0 376 8/20/10 44 0 377 8/20/10 44 0 378 8/27/10 51 0 379 8/19/10 43 0 380 8/23/10 47 0 381 8/20/10 44 0 382 8/18/10 42 0 383 8/25/10 49 0 384 8/20/10 44 0 385 8/18/10 42 0 386 8/22/10 46 0 387 8/21/10 45 0 388 8/20/10 44 0 389 8/18/10 42 0 390 8/22/10 46 0.5 488 8/22/10 46 0.5 489 8/26/10 50 0.5 490 8/28/10 52 0.5 492 8/19/10 43 0.5 493 8/20/10 44 0.5 494 8/21/10 45 0.5 495 8/22/10 46 0.5 496 8/18/10 42 0.5 497 8/18/10 42 
0.5 478 8/19/10 43 

0.5 479 8/21/10 45 0.5 480 8/21/10 45 0.5 481 8/20/10 44 0.5 482 8/23/10 47 0.5 483 8/22/10 46 0.5 484 8/18/10 42 0.5 485 8/28/10 52 0.5 486 9/4/10 59 0.5 487 8/19/10 43 5 259 8/20/10 44 5 260 8/23/10 47 5 261 8/25/10 49 5 262 8/18/10 42 5 263 8/20/10 44 5 264 8/27/10 51 5 279 8/24/10 48 5 266 8/30/10 54 5 267 8/22/10 46 5 268 8/18/10 42 5 269 8/18/10 42 5 270 8/20/10 44 5 271 8/21/10 45 5 272 8/19/10 43 5 273 8/20/10 44 5 274 8/18/10 42 5 275 8/23/10 47 5 276 8/26/10 50 5 277 8/19/10 43 5 278 8/19/10 43 10 1 8/18/10 42 
10 2 8/20/10 44 

10 3 8/18/10 42 10 4 8/19/10 43 10 91 8/19/10 43 10 92 8/25/10 49 10 7 8/28/10 52 10 8 8/31/10 55 10 93 8/18/10 42 10 10 8/20/10 44 10 11 8/20/10 44 10 12 8/23/10 47 10 13 8/23/10 47 10 14 8/19/10 43 10 15 8/28/10 52 10 16 8/21/10 45 10 17 8/22/10 46 10 18 8/20/10 44 10 19 8/25/10 49 10 20 8/26/10 50 20 269 8/20/10 44 20 270 8/27/10 51 20 271 8/22/10 46 20 272 8/20/10 44 20 289 8/21/10 45 20 274 8/23/10 47 20 285 8/19/10 43 20 286 8/22/10 46 20 287 8/27/10 51 20 288 8/23/10 47 20 275 8/23/10 47 20 276 8/31/10 55 
20 277 8/21/10 45 

20 278 8/26/10 50 20 279 8/24/10 48 20 280 8/25/10 49 20 281 8/24/10 48 20 282 8/25/10 49 20 283 8/25/10 49 20 284 8/28/10 52 F0 cloaca measurements (mm2) ppm ID 8/18/10 8/25/10 9/1/10 9/8/10 0 371 180 165 324 361 0 372 88 100 195 304 0 373 80 132 224 255 0 374 80 99 196 256 0 375 162 100 169 225 0 376 187 143 182 255 0 377 143 180 196 288 0 378 72 140 208 240 0 379 165 185 289 360 0 380 90 182 224 288 0 381 165 266 306 342 0 382 196 204 154 324 0 383 49 99 182 238 0 384 120 132 240 285 0 385 210 192 272 289 0 386 100 168 255 323 0 387 108 143 221 306 0 388 196 154 272 380 0 389 238 221 288 324 0 390 100 144 210 361 
0.5 488 120 196 126 210 

0.5 489 81 144 180 182 0.5 490 90 140 240 288 0.5 499 49 56 96 0.5 492 180 224 288 288 0.5 493 154 196 256 270 0.5 494 117 182 224 255 0.5 495 80 182 224 270 0.5 496 270 240 256 360 0.5 497 225 224 270 323 0.5 478 216 182 272 361 0.5 479 132 168 288 289 0.5 480 130 195 306 324 0.5 481 182 154 240 361 0.5 482 99 168 256 255 0.5 483 180 182 288 342 0.5 484 224 210 255 342 0.5 485 64 100 195 256 0.5 486 63 99 169 342 0.5 487 180 208 288 240 5 259 100 210 288 323 5 260 120 168 272 270 5 261 99 132 225 272 5 262 208 240 304 272 5 263 195 238 306 340 5 264 221 168 255 400 5 279 132 110 210 285 5 266 77 99 168 255 5 267 90 117 196 342 5 268 238 221 323 400 5 269 195 187 255 289 
5 270 156 165 225 306 

5 271 130 195 288 418 5 272 154 192 255 306 5 273 195 304 272 342 5 274 156 180 154 255 5 275 70 132 256 272 5 276 90 110 168 324 5 277 210 180 255 324 5 278 224 165 288 306 10 1 192 272 272 361 10 2 143 270 288 380 10 3 180 255 306 288 10 4 143 238 240 272 10 91 288 306 342 360 10 92 63 100 195 256 10 7 77 156 180 240 10 8 80 169 225 195 10 93 224 289 289 323 10 10 192 288 289 342 10 11 195 288 306 342 10 12 121 210 210 266 10 13 182 240 270 256 10 14 210 306 255 304 10 15 90 132 210 240 10 16 132 192 238 323 10 17 132 224 270 323 10 18 168 240 252 323 10 19 121 195 270 324 10 20 90 196 270 300 20 269 195 210 304 285 20 270 110 224 304 270 
20 271 192 210 255 289 

20 272 165 285 255 272 20 289 108 210 240 285 20 274 195 238 255 288 20 285 120 224 255 288 20 286 120 225 306 360 20 287 80 210 238 285 20 288 80 132 289 255 20 275 99 182 255 323 20 276 108 252 238 289 20 277 108 195 255 360 20 278 70 210 238 288 20 279 144 240 225 323 20 280 120 225 210 270 20 281 132 240 255 306 20 282 143 182 306 361 20 283 96 210 255 361 20 284 99 144 168 288 F0 egg strength ppm ID Thickness Thickness Force 0 201 0.32 0.34 0.857 0 201 0.39 0.35 1.424 0 203 0.5 0.42 1.164 0 203 0.29 0.3 1.164 0 204 0.39 0.55 1.653 0 204 0.35 0.32 0.929 0 205 0.33 0.45 0.848 0 205 0.38 0.45 1.349 0 206 0.36 0.32 1.912 0 206 0.32 0.42 1.497 
0 207 0.29 0.33 1.418 

0 207 0.34 0.32 1.635 0 209 0.38 0.4 1.165 0 209 0.38 0.32 1.567 0 210 0.4 0.43 0 210 0.3 0.37 1.503 0 210 0.46 0.32 1.522 0 391 0.39 0.33 1.102 0 391 0.32 0.36 1.328 0 392 0.47 0.35 1.194 0 392 0.31 0.29 1.136 0 393 0.3 0.32 1.296 0 393 0.42 0.38 0.964 0 394 0.42 0.47 1.006 0 394 0.52 0.6 1.37 0 395 0.31 0.26 1.234 0 395 0.42 0.46 0.917 0 396 0.43 0.47 1.235 0 396 0.41 0.42 1.043 0 398 0.42 0.37 1.514 0 398 0.37 0.33 1.477 0 399 0.45 0.37 0.638 0 399 0.39 0.4 1.342 0 400 0.41 0.4 1.151 0 400 0.32 0.37 0.645 0.5 1 0.36 0.39 1.534 0.5 1 0.34 0.43 1.455 0.5 4 0.54 0.45 0.673 0.5 4 0.48 0.34 1.11 0.5 6 0.42 0.5 1.079 0.5 6 0.46 0.38 0.372 
0.5 7 0.43 0.46 1.067 

0.5 7 0.31 0.27 1.127 0.5 8 0.44 0.43 1.817 0.5 8 0.32 0.39 1.56 0.5 9 0.35 0.28 1.208 0.5 9 0.43 0.45 1.375 0.5 10 0.45 0.42 1.289 0.5 10 0.28 0.26 0.5 10 0.46 0.44 1.536 0.5 11 0.32 0.42 1.367 0.5 11 0.37 0.32 1.871 0.5 13 0.37 0.34 1.523 0.5 13 0.33 0.3 1.68 0.5 14 0.39 0.44 1.053 0.5 14 0.36 0.35 0.486 0.5 15 0.31 0.35 0.5 15 0.29 0.28 0.93 0.5 17 0.38 0.42 1.229 0.5 17 0.4 0.48 0.649 0.5 18 0.27 0.37 0.614 0.5 18 0.29 0.33 1.139 0.5 19 0.39 0.3 0.626 0.5 20 0.33 0.29 1.319 0.5 20 0.32 0.4 1.436 0.5 498 0.47 0.34 0.896 0.5 498 0.43 0.38 0.558 0.5 499 0.58 0.56 0.813 0.5 499 0.42 0.39 1.043 5 280 0.29 0.34 1.336 5 280 0.3 0.29 1.506 5 283 0.33 0.35 1.285 
5 283 0.36 0.33 1.365 

5 284 0.4 0.33 1.374 5 284 0.47 0.57 0.469 5 285 0.33 0.35 1.429 5 285 0.41 0.38 1.624 5 286 0.35 0.43 1.304 5 286 0.41 0.35 1.586 5 286 0.41 0.39 1.719 5 286 0.42 0.47 1.673 5 288 0.4 0.34 1.332 5 288 0.39 0.42 1.527 5 289 0.47 0.46 0.945 5 289 0.39 0.33 1.209 5 290 0.33 0.28 1.624 5 290 0.43 0.41 1.021 5 291 0.39 0.43 5 291 0.26 0.28 1.724 5 291 0.35 0.27 1.525 5 292 0.27 0.29 0.967 5 292 0.42 0.44 1.349 5 293 0.36 0.41 1.738 5 293 0.47 0.38 1.369 5 294 0.49 0.4 0.655 5 294 0.35 0.55 1.354 5 295 0.88 0.92 1.633 5 295 0.37 0.32 1.613 5 296 0.33 0.39 5 296 0.34 0.43 1.353 5 296 0.41 0.36 1.159 5 297 0.38 0.34 1.382 5 297 0.35 0.38 0.967 
5 298 0.53 0.62 1.03 

5 298 0.39 0.34 0.989 5 299 0.31 0.29 1.415 5 299 0.41 0.47 0.665 10 22 0.37 0.33 0.564 10 22 0.34 0.43 0.926 10 23 0.38 0.56 1.208 10 23 0.43 0.45 1.163 10 24 0.34 0.29 1.547 10 24 0.33 0.44 1.698 10 25 0.59 0.52 1.519 10 25 0.42 0.4 1.608 10 26 0.37 0.39 0.613 10 27 0.41 0.35 10 27 0.38 0.35 1.3 10 28 0.44 0.47 10 27 0.4 0.33 1.125 10 28 0.42 0.39 1.334 10 30 0.32 0.33 1.271 10 30 0.31 0.29 1.582 10 31 0.36 0.29 1.068 10 31 0.27 0.3 1.249 10 32 0.28 0.36 1.778 10 32 0.59 0.54 1.682 10 33 0.31 0.35 1.178 10 33 0.33 0.29 1.268 10 34 0.52 0.37 1.37 10 36 0.38 0.39 0.257 10 36 0.31 0.39 1.506 10 37 0.4 0.37 1.444 10 37 0.29 0.34 0.932 
10 39 0.39 0.35 0.498 

10 39 0.41 0.28 1.053 10 40 0.32 0.37 1.511 10 94 0.42 0.37 1.782 10 94 0.29 0.33 1.048 20 290 0.41 0.32 0.394 20 292 0.38 0.42 20 292 0.32 0.38 1.609 20 292 0.35 0.29 1.321 20 293 0.3 0.42 1.505 20 293 0.31 0.28 1.242 20 294 0.39 0.41 0.603 20 295 0.37 0.35 1.467 20 295 0.34 0.36 1.643 20 300 0.4 0.42 0.908 20 300 0.41 0.51 1.556 20 300 0.33 0.36 1.71 20 301 0.29 0.3 20 303 0.3 0.31 1.116 20 303 0.48 0.51 1.647 20 305 0.31 0.33 20 305 0.4 0.37 0.351 20 306 0.33 0.31 0.413 20 306 0.43 0.37 1.183 20 307 0.36 0.37 1.39 20 307 0.37 0.38 1.415 20 308 0.28 0.31 1.44 20 308 0.4 0.31 1.501 20 309 0.31 0.39 1.144 20 309 0.4 0.49 1.364 20 380 0.43 0.38 0.545 
20 380 0.3 0.39 0.395 

F1 egg production ppm ID 

1/2

4 

1/2

5 

1/2

6 week 6 

1/2

7 

1/2

8 

1/2

9 

1/3

0 

1/3

1 

2/

1 

2/

2 

week 7 

2/

3 

2/

4 2/5 

2/

6 

2/

7 

2/

8 

2/

9 

week 8 

2/1

0 

2/1

1 

2/1

2 

2/1

3 

2/1

4 2/15 

2/1

6 

week 9 

2/

0 

211 2 1 1 1 1 1 1 1 1 1 

0 

224 1 

0 

228 1 1 1 

0 

212 

0 

218 1 2 1 1 1 1 1 1 1 1 

0 

230 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

0 

216 

0 

270 

0 

226 1 1 1 1 1 1 1 

0 

231 1 1 1 1 1 1 

0 

276 

0 

299 

0 

294 

0 

292 1 1 1 1 1 1 1 1 1 1 1 

0 

237 1 1 1 1 1 

0 

227 1 1 1 1 1 1 

0 

283 1 1 1 1 1 1 1 1 1 

0 

217 1 1 1 1 1 1 

0 

235 1 1 1 1 1 1 1 1 1 1 

0 

215 

0.5 

110 1 1 1 1 

0.5 26 0.5 90 0.5 36 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 

107 
0.5 

108 

0.5 73 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 

105 1 1 1 1 1 1 1 1 1 1 

0.5 35 0.5 37 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 0.5 40 1 1 1 2 1 1 1 1 1 1 1 1 1 0.5 50 2 1 1 1 1 1 1 1 1 1 0.5 38 0.5 24 0.5 27 1 1 1 1 1 1 0.5 22 1 1 1 1 1 1 1 0.5 30 1 1 0.5 25 1 1 0.5 34 1 1 1 5 68 1 1 1 1 1 1 2 5 70 5 15 1 1 5 76 5 78 5 64 1 1 1 1 1 2 1 1 1 1 1 1 5 77 5 61 1 1 1 1 1 1 1 5 49 1 1 1 1 1 2 1 1 1 5 45 5 47 1 1 1 1 5 43 1 1 1 1 1 5 23 1 1 1 1 1 1 2 1 1 1 1 5 27 1 1 1 1 1 1 1 1 1 5 39 5 54 
5 53 5 38 5 7 5 1 5 66 1 10 

352 1 1 1 1 1 1 1 1 1 1 

10 

359 1 1 1 1 1 1 1 1 

10 95 1 1 1 1 1 1 1 1 10 

398 1 1 1 1 2 1 

10 

390 1 

10 

376 1 1 1 1 1 1 1 1 1 

10 

371 

10 

368 

10 

370 

10 

373 

10 

349 

10 

369 

10 

361 

10 

380 

10 

367 1 1 1 1 1 

euthanized 

10 

351 1 1 1 1 2 2 

10 

350 1 1 1 1 1 

10 

374 1 1 

10 96 10 

357 1 1 1 1 1 1 1 1 

10 

372 1 

10 

358 1 

10 

385 1 1 1 2 

10 

395 1 1 1 1 1 

10 

379 

10 

360 
10 

389 

10 

400 

20 

316 

20 

321 

20 

318 

20 

312 

20 

311 

20 

339 

20 

358 

20 

352 

20 

317 

20 

359 

20 

351 

20 

343 

20 

328 

20 

342 

eggbd 

20 

320 1 1 1 

20 

333 1 

20 

313 

20 

324 

ppm ID 2/24 2/25 2/26 2/27 2/28 3/1 3/2 

week 11 3/3 3/4 3/5 3/6 3/7 3/8 3/9 week 12 3/10 3/11 3/12 3/13 3/14 3/15 3/16 week 13 3/17 3/18 3/19 3/20 

0 211 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 0 224 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 228 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 212 0 218 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 230 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 216 0 270 
0 226 1 1 1 1 1 1 1 1 1 1 1 1 1 0 231 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 276 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 299 1 1 1 1 1 1 1 1 1 0 294 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 292 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 237 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 227 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 283 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 217 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 235 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 215 1 1 1 1 1 1 2 1 1 1 0.5 110 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 26 0.5 90 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 36 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 107 1 1 1 1 1 1 1 1 1 0.5 108 1 1 1 1 0.5 73 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 105 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 35 0.5 37 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 50 1 1 1 1 1 1 1 1 1 0.5 38 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 24 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 27 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 30 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 
0.5 34 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 68 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 70 1 1 1 5 15 1 1 1 1 1 5 76 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 78 1 1 1 1 1 1 5 64 1 1 1 1 1 1 1 1 1 1 1 1 1 5 77 1 1 1 1 1 1 1 1 5 61 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 49 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 45 1 1 1 1 1 1 5 47 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 43 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 23 1 1 1 1 1 1 1 1 1 1 1 1 5 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 39 1 1 1 1 1 1 1 1 1 1 5 54 1 1 1 1 1 1 1 1 1 5 53 5 38 5 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 1 1 1 5 66 1 1 1 1 1 1 1 1 1 2 10 352 1 1 1 1 1 10 359 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 95 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 398 1 1 1 1 1 1 1 1 1 10 390 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 376 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 2 10 371 10 368 1 1 1 1 1 1 1 1 1 1 1 10 370 1 1 1 1 1 1 1 1 
10 373 10 349 10 369 10 361 10 380 1 1 1 1 1 1 10 367 10 351 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 350 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 374 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 96 10 357 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 372 10 358 1 1 1 10 385 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 395 1 1 1 1 1 1 1 1 1 1 1 10 379 1 1 10 360 2 1 1 1 1 1 1 1 1 1 10 389 1 1 1 1 1 1 1 1 1 1 1 1 10 400 1 1 1 1 1 20 316 eggbd 20 321 20 318 20 312 1 1 1 20 311 eggbd 20 339 1 1 1 1 1 1 1 20 358 1 1 20 352 20 317 1 20 359 1 2 1 20 351 eggbd 20 343 
20 328 20 342 20 320 1 1 1 1 20 333 1 1 1 20 313 1 1 1 1 1 1 20 324 ppm ID 3/24 3/25 3/26 3/27 3/28 3/29 3/30 week 15 3/31 4/1 4/2 4/3 4/4 4/5 4/6 week 16 4/7 4/8 4/9 4/10 4/11 4/12 4/13 week 17 4/14 4/15 4/16 4/17 4/18 0 211 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 224 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 228 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 212 0 218 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 230 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 216 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 270 1 1 0 226 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 231 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 276 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 299 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 294 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 292 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 237 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 227 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 283 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 217 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 235 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 215 1 1 1 0.5 110 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 26 1 1 1 1 1 1 1 1 1 1 1 1 
0.5 90 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 36 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 107 1 1 1 1 1 1 1 1 1 1 1 0.5 108 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 73 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 105 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 0.5 35 0.5 37 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 50 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 38 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 24 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 0.5 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 34 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 68 1 1 1 1 1 1 1 1 5 70 1 5 15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 76 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 78 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 64 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 77 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 61 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 49 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 45 1 1 1 1 1 1 1 1 1 5 47 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 43 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 
5 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 39 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 54 1 1 1 1 1 1 1 1 1 1 5 53 1 1 1 1 1 1 1 1 1 1 1 5 38 5 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 1 5 66 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 352 1 1 1 1 1 1 1 1 1 1 1 1 1 10 359 1 1 1 1 1 10 95 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 398 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 390 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 10 376 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 371 10 368 1 1 1 1 1 2 1 1 1 1 1 1 10 370 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 373 10 349 10 369 10 361 10 380 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 367 10 351 1 1 1 1 1 1 1 1 1 1 1 2 1 1 10 350 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 374 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 96 10 357 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 10 372 10 358 10 385 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 
10 395 2 1 1 1 1 1 1 1 1 1 1 1 1 1 10 379 1 1 1 1 1 1 10 360 1 1 1 1 1 1 1 1 1 1 1 1 1 10 389 1 1 1 1 1 1 1 1 1 1 1 1 1 2 10 400 1 1 1 1 1 1 1 1 1 1 1 1 1 20 316 20 321 20 318 20 312 1 1 1 1 1 1 1 1 1 1 20 311 20 339 2 2 1 1 1 1 1 1 1 20 358 20 352 20 317 1 1 20 359 1 1 20 351 1 20 343 20 328 20 342 20 320 1 1 1 1 20 333 1 1 1 1 1 20 313 1 1 1 1 1 1 1 1 1 1 1 20 324 ppm ID 4/21 4/22 4/23 4/24 4/25 4/26 4/27 week 19 4/28 4/29 4/30 5/1 5/2 5/3 5/4 week 20 5/5 5/6 5/7 5/8 5/9 5/10 5/11 week 21 5/12 5/13 5/14 5/15 5/16 0 211 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 224 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 228 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 212 1 1 1 1 1 1 1 1 0 218 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 
0 230 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 216 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 270 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 226 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 231 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 276 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 299 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 294 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 292 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 237 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 227 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 0 283 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 217 1 1 1 1 1 1 1 1 1 1 1 1 0 235 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 215 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 110 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 26 1 1 1 1 1 1 1 1 1 1 1 1 0.5 90 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 36 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 107 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 108 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 73 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 105 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 35 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 37 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 0.5 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 50 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 38 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 24 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 
0.5 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 34 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 68 1 1 1 5 70 1 1 1 1 1 1 1 1 1 1 1 1 1 5 15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 76 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 5 78 1 1 1 1 1 1 1 1 1 1 1 5 64 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 77 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 61 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 5 49 1 1 1 1 1 5 45 1 1 1 1 1 1 1 1 5 47 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 43 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 39 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 54 1 1 1 1 1 1 1 1 1 1 5 53 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 38 5 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 1 5 66 1 1 1 1 1 1 1 1 1 1 1 1 1 10 352 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 359 1 1 1 1 1 1 1 1 10 95 1 1 1 1 1 1 1 1 1 1 1 1 10 398 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 390 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 376 1 1 1 1 1 1 1 1 1 1 1 1 1 
10 371 10 368 1 1 1 1 1 1 1 2 1 1 1 10 370 1 1 1 1 1 1 1 1 1 1 10 373 10 349 10 369 10 361 1 1 1 2 1 1 1 1 10 380 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 367 10 351 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 350 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 10 374 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 96 10 357 2 1 1 1 1 2 1 1 1 1 2 1 1 1 1 1 1 1 1 10 372 10 358 1 10 385 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 2 1 1 10 395 1 1 1 1 1 2 1 1 1 1 2 1 2 2 1 10 379 1 1 1 1 2 1 1 10 360 1 1 1 1 1 1 1 1 1 1 1 1 1 10 389 1 1 1 1 1 1 1 1 1 1 1 1 10 400 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20 316 20 321 20 318 20 312 1 1 1 1 1 1 1 1 1 1 1 1 1 20 311 20 339 1 1 1 1 1 1 1 1 1 20 358 1 1 1 1 1 1 1 1 1 1 1 20 352 20 317 1 1 1 1 1 1 1 1 1 
20 359 1 20 351 1 1 1 1 1 20 343 20 328 20 342 20 320 1 20 333 1 1 1 1 1 1 1 20 313 1 1 1 1 1 20 324 ppm ID 5/19 5/20 5/21 5/22 5/23 5/24 5/25 

week 23 5/26 5/27 5/28 5/29 5/30 5/31 6/1 week 24 6/2 6/3 6/4 6/5 6/6 6/7 6/8 week 25 6/9 6/10 6/11 6/12 6/13 

0 211 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 224 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 228 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 212 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 218 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 230 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 216 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 270 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 226 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 231 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 0 276 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 2 1 0 299 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 294 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 292 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 237 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 0 227 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 283 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 217 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 0 235 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 
0 215 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 110 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 0.5 26 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 90 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 36 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 0.5 107 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 108 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 73 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 0.5 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 105 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 35 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 37 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 40 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 0.5 50 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 38 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 24 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 0.5 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 0.5 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 34 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 68 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 70 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 76 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 78 1 1 1 1 1 1 1 5 64 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 77 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 61 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 49 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 45 1 1 1 1 1 1 1 1 1 1 1 1 
5 47 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 43 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 39 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 54 1 1 1 1 1 1 1 1 1 1 1 5 53 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 38 5 7 1 1 1 1 1 1 1 1 5 1 1 1 5 66 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 10 352 1 1 1 1 1 1 1 1 1 1 1 1 10 359 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 95 1 1 1 1 1 1 1 1 1 10 398 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 390 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 10 376 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 371 10 368 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 370 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 373 1 1 10 349 10 369 10 361 1 1 1 1 1 1 1 1 10 380 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 367 10 351 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 350 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 10 374 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 96 10 357 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 
10 372 10 358 10 385 1 1 2 1 1 1 1 2 1 1 2 1 1 1 1 1 1 1 10 395 1 1 1 1 1 1 1 1 1 1 1 2 1 10 379 1 1 1 1 10 360 1 1 1 1 1 1 2 10 389 1 1 2 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 10 400 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 20 316 20 321 20 318 20 312 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20 311 20 339 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20 358 1 1 1 1 1 1 1 1 1 1 1 20 352 20 317 20 359 20 351 1 1 1 20 343 20 328 20 342 20 320 20 333 1 1 1 1 20 313 1 1 1 1 1 1 1 20 324 

F1 paired feed consumptionDOSE ID ID 3/21 3/24 3/24 3/28 

week 14 3/28 4/1 4/1 4/4 week 15 4/4 4/6 4/6 4/11 week 16 4/11 4/14 4/14 4/18 week 17 4/18 4/22 4/22 4/2

0 213 224 442 297 409 252 302 413 300 420 292 241 420 297 414 247 290 422 270 415 245 322 424 304 413 3
0 219 202 438 294 396 251 289 417 299 419 263 274 421 301 419 250 289 415 281 413 274 273 419 303 420 320 216 214 445 283 162 406 309 415 257 255 414 315 412 251 260 420 251 413 273 309 418 319 417 30 270 236 445 303 407 275 274 402 331 415 257 229 419 325 409 293 210 413 269 418 283 279 414 312 401 340 226 264 442 335 405 239 273 404 329 413 294 194 412 331 418 255 244 419 278 428 241 328 417 315 400 30 259 294 422 303 397 244 272 397 301 408 259 245 411 307 413 246 271 420 279 415 246 310 423 296 425 340 234 292 437 284 406 252 307 405 291 406 290 230 416 300 413 261 268 424 286 414 258 294 412 299 411 30 255 237 436 277 417 269 307 401 279 414 276 260 417 289 421 258 291 418 275 411 244 310 408 300 409 320 247 227 443 292 411 256 306 401 281 411 279 252 413 280 412 255 290 412 273 412 233 318 419 317 410 30 242 283 449 275 422 249 347 400 283 412 258 271 413 279 425 250 309 424 264 417 237 340 422 308 421 320 261 217 447 280 419 252 334 406 276 419 290 259 416 282 409 255 288 424 275 418 254 313 424 310 418 320 280 235 443 295 413 248 313 406 303 403 277 229 409 306 411 253 261 413 283 429 274 285 413 335 411 320 246 215 442 304 413 277 274 411 291 420 297 243 422 289 412 272 273 421 291 421 256 295 419 331 413 32

0.5 21 26 449 323 408 259 275 407 319 399 292 195 411 262 149 410 300 425 282 253 421 301 417 20.5 44 36 444 236 208 405 315 416 263 243 418 320 420 248 270 417 406 411 234 188 415 302 415 20.5 48 107 448 310 410 231 317 411 282 412 279 262 411 292 412 252 279 414 315 409 260 248 414 305 430 20.5 31 108 448 271 399 245 331 407 291 411 292 235 416 287 416 287 258 420 411 422 251 180 412 299 422 320.5 39 29 442 310 399 236 295 415 304 414 273 252 423 311 423 240 295 417 415 403 231 174 417 315 418 20.5 46 105 440 283 403 231 329 407 288 411 258 272 400 295 401 236 270 410 407 407 231 179 420 311 415 30.5 45 35 441 281 408 251 317 413 284 405 307 227 409 266 400 288 255 421 412 409 262 156 415 297 425 20.5 104 37 438 323 409 230 294 406 317 407 277 219 418 304 422 368 168 414 398 409 264 161 416 312 407 340.5 116 40 441 293 393 230 311 406 297 416 258 267 409 292 417 235 299 413 419 436 231 199 413 301 419 30.5 88 50 439 28 399 249 561 413 289 414 280 258 413 289 415 239 300 413 415 412 266 144 423 300 408 30.5 47 38 452 311 407 236 312 420 304 407 298 225 420 294 418 267 277 411 408 413 316 100 422 328 411 20.5 106 24 447 331 412 247 281 407 287 393 270 243 423 280 415 253 305 416 416 418 266 152 419 315 425 30.5 41 27 448 279 397 241 325 403 286 402 278 241 424 282 416 254 304 426 404 414 257 179 418 300 413 340.5 81 22 446 273 405 251 327 415 274 399 263 277 416 279 412 234 315 419 404 419 269 165 418 329 415 30.5 49 30 433 285 403 231 320 418 278 420 288 272 412 291 409 249 281 416 412 423 252 175 413 333 420 30.5 42 25 434 296 412 238 312 416 301 411 287 239 409 307 411 261 252 429 392 425 251 211 412 306 415 30.5 33 34 445 300 405 257 293 412 309 409 315 197 412 309 401 306 198 430 410 420 277 163 412 313 398 32

5 52 68 440 300 418 256 302 408 304 410 335 179 425 301 427 330 221 416 329 419 322 184 411 313 413 35 35 70 448 298 422 308 264 415 310 420 309 216 416 305 409 313 207 424 303 407 257 271 410 307 400 3
5 37 15 450 291 410 257 312 421 297 411 285 250 411 303 412 254 266 413 298 412 236 291 419 305 414 35 34 76 441 301 404 283 261 409 309 407 315 192 411 296 420 240 295 410 312 412 274 236 398 292 412 345 41 78 440 313 405 260 272 419 310 409 302 216 412 302 423 250 283 406 306 411 252 259 412 325 399 325 36 64 443 313 403 251 282 413 306 405 295 217 419 301 407 262 263 417 284 416 277 272 416 330 416 325 46 77 439 296 414 269 288 414 303 404 276 239 409 298 412 247 276 400 280 415 254 281 415 296 421 35 33 61 437 319 413 270 261 413 319 401 283 212 415 312 409 266 246 410 274 423 268 291 417 320 425 35 58 49 448 310 396 243 291 409 320 418 272 235 413 315 411 269 240 409 266 412 251 304 422 315 415 35 44 45 450 305 410 277 278 419 302 415 289 243 418 300 416 306 228 421 290 418 264 285 405 317 413 35 11 39 451 281 413 236 347 416 294 411 292 241 419 299 414 251 283 419 305 421 250 285 421 305 416 325 16 54 454 312 423 268 297 424 307 419 286 250 422 296 413 291 248 428 315 415 259 269 409 304 442 35 29 53 450 350 400 258 242 418 345 417 303 187 428 335 412 276 229 414 317 417 265 249 423 321 421 35 4 38 447 323 410 263 271 433 325 401 330 179 417 331 419 303 202 414 322 411 298 205 430 333 419 35 28 7 446 306 410 249 301 406 312 403 269 228 407 310 417 266 248 412 310 416 251 267 425 316 410 325 1 66 452 309 420 247 316 416 302 405 262 257 420 308 421 317 216 422 308 415 305 224 419 318 413 34

10 382 352 440 327 393 231 275 406 297 404 298 215 418 301 427 257 287 407 300 410 257 260 408 331 424 210 98 95 448 297 412 254 309 411 305 411 285 232 410 305 409 291 223 421 292 427 245 311 408 319 418 210 386 390 437 313 411 261 274 418 322 412 298 210 416 317 420 285 234 414 284 408 274 264 401 325 415 310 394 374 437 303 416 275 275 419 310 408 284 233 414 318 421 250 267 412 286 413 237 302 402 322 416 210 362 96 435 292 143 411 311 422 306 216 415 304 419 282 248 406 311 415 270 240 416 307 415 310 392 357 440 312 414 292 250 411 319 416 287 221 414 299 417 250 282 413 287 416 329 213 418 303 401 210 381 461 448 341 412 284 235 421 341 409 298 191 428 332 415 318 193 406 391 417 303 129 407 325 409 2210 97 380 455 307 408 235 321 414 308 411 268 249 410 296 409 260 263 420 273 400 257 290 422 340 417 2210 358 376 452 294 405 232 331 411 286 412 271 266 416 296 399 306 213 411 269 419 297 264 413 333 415 210 356 370 452 330 406 261 267 414 305 406 304 211 407 317 408 277 221 405 298 415 267 255 403 341 409 2220 350 318 449 328 412 291 242 423 334 403 318 174 428 332 400 303 193 422 310 416 300 228 413 323 419 3420 348 312 447 298 411 249 311 406 301 399 306 198 411 322 417 265 241 412 312 415 253 262 429 304 413 220 331 339 453 321 407 271 268 412 316 393 311 178 422 316 425 290 241 413 305 416 264 260 433 338 418 320 314 458 452 307 414 271 288 414 311 407 302 208 414 310 413 332 185 418 310 415 20 326 352 444 323 402 285 238 408 324 84 415 305 422 341 191 430 316 412 20 335 327 439 319 408 309 219 416 313 410 313 200 419 339 407 304 183 433 320 412 304 221 420 328 421 320 361 313 435 341 414 257 251 412 333 405 294 190 418 337 411 285 207 419 322 419 265 251 419 318 412 3
20 325 351 450 334 419 269 266 418 342 404 318 162 416 325 409 302 198 414 321 421 264 250 414 326 425 320 334 320 450 325 402 282 245 424 321 401 300 204 411 326 418 338 165 420 329 417 283 225 395 325 409 320 319 333 447 354 421 257 257 430 350 400 321 159 416 340 411 269 218 409 318 418 253 256 423 311 412 3

F1 paired feed consumption DOSE ID ID 5/2 5/5 5/5 5/9 week 20 5/9 5/13 5/13 5/16 week 21 5/16 5/20 5/20 5/24 week 22 5/24 5/27 5/27 5/31 

week 23 

0 213 224 433 282 425 275 301 412 290 420 288 254 413 253 470 266 364 466 285 426 256 351 0 219 202 437 283 437 271 320 416 278 422 289 271 420 278 450 269 323 451 317 431 252 313 0 216 214 422 299 429 314 238 414 303 412 111 430 340 453 304 239 450 290 160 0 270 236 420 291 434 268 295 420 298 423 294 251 425 280 456 279 322 451 309 431 281 292 0 226 264 429 271 438 240 356 418 264 411 279 286 415 251 461 252 373 467 321 441 276 311 0 259 294 425 268 436 295 298 432 267 409 289 285 422 262 469 302 327 483 323 424 271 313 0 234 292 434 274 436 301 295 422 279 415 330 228 417 235 478 269 391 457 291 413 242 337 0 255 237 421 267 422 244 332 420 261 408 290 277 409 254 473 253 375 456 320 418 254 300 0 247 227 424 233 425 238 378 411 225 407 278 315 417 241 458 247 387 463 283 416 298 298 0 242 283 424 273 430 270 311 426 260 416 280 302 409 249 461 243 378 461 298 426 256 333 0 261 217 420 287 427 312 248 424 282 415 302 255 411 254 456 289 324 467 322 423 272 296 0 280 235 418 288 428 271 287 423 272 419 266 304 410 256 455 262 347 462 298 425 259 330 0 246 215 415 288 425 245 307 427 277 417 293 274 419 237 466 262 386 460 295 423 255 333 

0.5 21 26 431 279 419 296 275 420 265 413 349 219 433 326 452 306 253 456 312 144 0.5 44 36 428 327 424 302 223 411 319 415 317 190 429 261 479 254 393 455 252 422 329 296 0.5 48 107 430 298 424 232 324 427 279 418 231 335 413 249 461 239 386 458 263 422 229 388 0.5 31 108 439 303 429 248 317 428 295 427 289 271 419 260 473 309 323 466 323 430 263 310 0.5 39 29 437 307 422 282 270 410 300 415 285 240 424 267 481 239 399 472 271 423 288 336 0.5 46 105 431 291 431 243 328 430 288 415 280 277 413 272 462 244 359 470 290 417 231 366 0.5 45 35 425 268 435 247 345 425 273 416 280 288 422 277 465 270 340 451 289 434 252 344 0.5 104 37 434 305 428 305 252 432 296 415 299 252 416 259 470 287 340 465 307 415 260 313 0.5 116 40 432 296 422 240 318 415 284 413 274 270 426 230 471 231 436 457 259 413 230 381 0.5 88 50 426 292 421 239 316 428 283 415 281 279 415 247 463 255 376 469 300 418 248 339 0.5 47 38 426 292 430 244 320 423 286 417 293 261 417 261 467 290 333 470 325 414 269 290 
0.5 106 24 430 267 438 291 310 415 266 411 293 267 417 271 459 276 329 473 312 426 266 321 0.5 41 27 431 303 435 289 274 421 306 420 327 208 423 291 463 333 262 471 305 166 0.5 81 22 425 266 437 246 350 407 256 424 274 301 427 256 472 260 383 449 289 427 266 321 0.5 49 30 434 283 433 289 295 409 289 418 321 217 424 274 464 261 353 468 309 413 243 329 0.5 42 25 433 315 437 243 312 411 308 418 251 270 421 264 469 244 382 478 292 421 230 377 0.5 33 34 436 301 438 236 337 419 312 419 319 207 405 261 470 307 307 459 337 420 266 276 

5 52 68 427 314 432 256 289 425 327 426 303 221 420 314 468 270 304 447 274 416 288 301 5 35 70 422 306 429 289 256 428 309 422 295 246 424 288 469 293 312 475 308 423 285 305 5 37 15 422 274 422 241 329 413 280 423 302 254 415 306 474 307 276 446 282 420 239 345 5 34 76 425 273 429 241 340 413 281 429 283 278 426 309 468 249 336 470 268 419 234 387 5 41 78 430 297 426 254 305 417 285 434 291 275 413 290 454 277 300 458 300 411 260 309 5 36 64 427 289 431 269 300 420 273 421 303 265 409 303 470 266 310 471 306 429 264 330 5 46 77 431 266 425 286 304 404 263 420 284 277 411 278 477 280 330 474 279 417 261 351 5 33 61 433 286 420 251 316 409 274 420 308 247 408 302 472 270 308 475 315 421 292 289 5 58 49 424 310 425 305 234 403 308 408 296 207 416 322 454 302 246 482 339 419 269 293 5 44 45 435 289 422 238 330 419 278 406 302 245 419 273 473 297 322 458 327 427 254 304 5 11 39 419 283 422 249 309 402 280 401 278 245 413 274 444 243 340 473 262 413 236 388 5 16 54 421 283 421 253 306 422 272 417 291 276 413 292 459 301 279 455 315 421 257 304 5 29 53 418 301 429 279 267 413 293 409 304 225 415 299 452 279 289 479 338 432 174 399 5 4 38 415 320 422 244 273 415 322 412 330 175 421 331 455 317 228 478 357 425 306 240 5 28 7 420 280 426 251 315 418 274 420 293 271 418 290 437 249 316 467 339 430 279 279 5 1 66 415 268 425 264 308 417 255 414 278 298 423 308 436 251 300 474 295 430 263 346 

10 382 352 420 286 421 255 300 420 280 416 280 276 421 306 464 289 290 462 309 429 257 325 10 98 95 417 293 429 277 276 417 281 412 290 258 420 250 468 300 338 468 330 425 266 297 10 386 390 420 284 430 255 311 420 286 413 317 230 422 253 459 284 344 457 322 424 262 297 10 394 374 421 286 434 240 329 418 279 433 313 259 430 255 476 251 400 472 298 422 237 359 10 362 96 429 308 437 332 226 410 300 414 338 186 421 332 470 356 203 454 330 124 10 392 357 427 245 438 231 389 413 252 423 307 277 426 271 467 250 372 471 290 429 260 350 10 381 461 424 298 435 258 303 408 285 415 327 211 424 297 443 285 285 478 340 424 283 279 10 97 380 423 262 436 267 330 410 254 416 286 286 402 252 458 268 340 467 338 426 265 290 10 358 376 418 274 419 264 299 423 268 428 290 293 416 233 455 268 370 482 316 422 283 305 
10 356 370 420 311 417 248 278 419 313 417 296 227 421 252 470 288 351 466 319 413 258 302 20 350 318 422 327 95 425 361 64 422 355 465 376 156 464 343 121 20 348 312 425 315 434 253 291 413 320 417 292 218 420 250 467 279 358 467 296 422 260 333 20 331 339 428 325 439 284 258 424 331 423 316 200 422 274 464 253 359 464 319 418 275 288 20 314 458 424 320 104 428 428 0 0 20 326 352 451 344 107 431 431 0 0 20 335 327 433 310 431 299 255 414 314 422 321 201 425 300 465 315 275 472 344 428 312 244 20 361 313 427 305 438 255 305 406 304 421 302 221 427 273 480 287 347 463 315 422 348 222 20 325 351 430 284 432 279 299 416 378 413 319 132 430 299 460 288 303 470 349 427 296 252 20 334 320 436 307 433 268 294 412 305 419 325 201 425 271 469 326 297 460 333 445 312 260 20 319 333 335 327 430 271 167 413 319 407 307 194 421 256 434 268 331 468 304 425 267 322 

F1 unpaired feed consumption 

ID 3/21 3/28 

week 14 3/28 4/1 4/1 4/4 week 15 4/4 4/6 4/6 4/11 week 16 4/11 4/14 4/14 4/18 week 17 4/18 4/22 4/22 4/24 week 18 4

 426 279 147 406 285 121 407 288 119 425 296 129 413 328 85 4

0 230 427 233 194 420 254 166 412 282 130 420 269 151 421 338 83 40 276 444 256 188 420 273 147 412 257 155 423 256 167 402 340 62 40 218 449 271 178 426 280 146 419 276 143 431 264 167 419 325 94 40 211 440 233 207 414 271 143 419 357 62 429 253 176 420 324 96 40 231 435 240 195 419 285 134 413 230 183 426 259 167 414 330 84 40 228 427 246 181 419 280 139 418 264 154 431 286 145 419 319 100 40 225 439 295 144 418 312 106 419 318 101 405 292 113 420 331 89 40 221 441 320 121 413 319 94 418 312 106 412 306 106 427 353 74 40 232 438 331 107 403 305 98 413 313 100 413 302 111 412 344 68 40 238 436 325 111 418 333 85 400 300 100 411 307 104 417 332 85 40 233 420 295 125 406 301 105 425 313 112 410 326 84 415 359 56 40 223 442 315 127 407 319 88 428 331 97 410 330 80 410 365 45 40 220 442 314 128 418 300 118 418 283 135 416 295 121 408 353 55 4

0.5 110 449 263 186 416 318 98 411 278 133 416 280 136 417 275 142 40.5 90 452 283 169 415 302 113 410 285 125 413 269 144 418 304 114 4
0.5 73 450 277 173 413 300 113 422 255 167 416 313 103 422 293 129 40.5 43 447 283 164 421 330 91 423 315 108 412 316 96 424 361 63 40.5 23 445 280 165 409 302 107 417 322 423 312 206 414 297 117 420 340 80 40.5 32 440 274 166 406 304 102 416 311 105 411 309 102 410 336 74 4

5 27 440 280 160 417 278 139 394 298 96 413 284 129 412 323 89 45 43 436 280 156 411 297 114 420 262 158 420 292 128 413 342 71 45 23 425 261 164 412 279 133 417 295 122 411 277 134 407 325 82 45 47 437 257 180 411 291 120 415 267 148 409 268 141 409 321 88 45 18 451 291 160 416 308 108 403 345 58 425 308 117 407 349 58 45 26 453 270 183 423 300 123 413 309 104 425 291 134 427 350 77 45 19 439 334 105 424 323 101 415 355 60 413 317 96 411 365 46 45 20 448 342 106 415 335 80 417 363 54 415 308 107 412 365 47 4

10 351 435 231 204 411 290 121 411 304 107 425 25 400 415 290 125 410 373 436 216 220 414 379 35 427 368 59 434 341 93 413 355 58 410 368 440 243 197 406 328 78 407 327 80 417 323 94 414 327 87 410 398 435 277 158 407 276 131 409 325 84 416 289 127 418 330 88 410 359 442 329 113 426 327 99 425 323 102 425 292 133 425 364 61 410 350 442 245 197 408 259 149 411 365 46 408 257 151 405 292 113 410 393 435 296 139 413 292 121 417 356 61 423 337 86 418 351 67 410 360 445 231 214 405 327 78 404 310 94 419 233 186 408 279 129 410 348 442 270 172 407 328 79 409 340 69 392 338 54 404 349 55 410 388 439 321 118 404 316 88 415 371 44 426 312 114 413 367 46 410 385 441 269 172 419 280 139 411 308 103 410 288 122 422 318 104 410 395 437 302 135 422 294 128 421 312 109 390 297 93 418 325 93 410 389 436 322 114 417 298 119 424 350 74 426 322 104 415 314 101 410 400 442 324 118 421 315 106 422 302 120 418 280 138 421 244 177 410 379 443 291 152 411 314 97 418 327 91 413 293 120 409 283 126 410 353 458 317 141 409 301 108 414 370 44 437 346 91 417 313 104 420 344 444 329 115 415 318 97 412 307 105 425 315 110 428 352 76 420 328 436 397 39 424 317 107 434 348 86 423 348 75 417 353 64 420 317 438 337 101 425 333 92 418 307 111 423 347 76 425 353 72 4
20 359 441 348 93 421 421 305 -305 430 307 123 417 319 98 420 345 450 321 129 411 411 345 -345 412 323 89 409 362 47 420 338 437 320 117 414 340 74 424 353 71 433 330 103 478 344 134 420 322 427 298 129 413 304 109 414 342 72 423 324 99 415 351 64 420 337 451 305 146 410 327 83 411 353 58 421 352 69 414 351 63 420 324 443 384 59 428 353 75 416 347 69 415 311 104 416 344 72 420 356 440 310 130 415 321 94 415 348 67 421 340 81 419 356 63 420 330 445 323 122 426 318 108 418 331 87 413 335 78 425 358 67 4

DOSE ID ID 5/2 5/5 5/5 5/9 week 20 5/9 5/13 5/13 5/16 week 21 5/16 5/20 5/20 5/24 week 22 5/24 5/27 5/27 5/31 

week 23 

0 299 425 312 113 407 293 114 422 312 447 331 226 445 303 142 0 230 422 372 50 435 337 98 420 325 460 344 211 448 303 145 0 276 426 297 129 412 312 100 422 290 433 378 187 462 291 171 0 218 431 317 114 412 327 85 415 331 446 320 210 456 299 157 0 211 426 279 147 416 316 100 416 302 449 348 215 454 269 185 0 231 428 293 135 413 317 96 424 333 459 334 216 455 318 137 0 228 430 321 109 420 326 94 414 329 455 361 179 452 318 134 0 225 425 319 106 407 356 51 429 367 461 383 140 464 341 123 0 221 424 347 77 422 356 66 430 375 454 393 116 451 343 108 0 232 430 328 102 436 350 86 416 347 457 361 165 454 291 163 0 238 430 330 100 414 366 48 435 382 454 385 122 454 345 109 0 233 431 316 115 415 343 72 408 354 454 390 118 462 351 111 0 223 430 333 97 408 337 71 417 356 451 384 128 462 344 118 0 220 433 323 110 424 338 86 427 361 447 370 143 463 319 144 

0.5 110 420 277 143 407 278 129 424 327 425 288 234 453 275 178 0.5 90 422 281 141 412 315 97 413 315 456 343 211 462 302 160 0.5 73 428 272 156 436 288 148 424 285 448 281 306 459 254 205 0.5 43 429 335 94 423 356 67 428 320 454 390 172 454 329 125 0.5 23 434 317 117 405 334 71 419 301 455 338 235 460 302 158 0.5 32 433 335 98 421 358 63 428 338 446 364 172 453 333 120 
5 27 420 292 128 423 298 125 419 333 473 370 189 465 330 135 5 43 418 274 144 415 319 96 421 343 455 332 201 461 296 165 5 23 415 292 123 418 344 74 427 353 466 345 195 471 290 181 5 47 415 289 126 421 313 108 418 328 439 312 217 443 262 181 5 18 434 339 95 423 346 77 427 350 455 363 169 461 325 136 5 26 431 336 95 418 323 95 423 331 466 367 191 465 285 180 5 19 426 350 76 415 357 58 430 366 459 395 128 454 341 113 5 20 427 347 80 413 343 70 419 362 468 400 125 473 354 119 

10 351 424 257 167 415 304 111 409 279 452 332 250 450 298 152 10 373 424 330 94 419 349 70 424 332 92 462 374 88 10 368 423 280 143 412 315 97 416 409 548 321 234 447 243 204 10 398 415 270 145 423 335 88 398 327 457 347 181 456 322 134 10 359 411 346 65 422 286 136 414 306 455 338 225 453 313 140 10 350 427 242 185 423 297 126 421 287 455 331 258 453 320 133 10 393 415 316 99 415 340 75 428 351 464 366 175 480 340 140 10 360 411 294 117 415 330 85 413 319 468 368 194 463 326 137 10 348 412 318 94 421 331 90 413 331 471 370 183 446 332 114 10 388 422 325 97 417 345 72 421 338 473 386 170 459 347 112 10 385 420 285 135 412 320 92 418 299 454 310 263 455 287 168 10 395 425 290 135 413 333 80 418 309 462 353 218 433 305 128 10 389 425 305 120 412 336 76 438 352 467 350 203 467 336 131 10 400 422 323 99 405 303 102 416 302 448 333 229 467 269 198 10 379 420 317 103 408 322 86 421 314 449 333 223 460 319 141 10 353 428 252 176 417 357 60 436 361 457 371 161 457 351 106 20 344 417 335 82 417 350 67 415 353 450 364 148 465 361 104 20 328 410 326 84 417 344 73 422 351 437 356 152 460 374 86 20 317 413 413 0 0 0 20 359 418 291 127 413 312 101 423 333 435 331 194 452 310 142 20 345 412 329 83 414 345 69 426 372 460 390 124 463 360 103 20 338 431 342 89 405 356 49 425 358 474 409 132 460 460 20 322 437 334 103 415 357 58 435 359 460 376 160 458 458 
20 337 432 339 93 421 358 63 427 354 447 361 159 452 355 97 20 324 438 341 97 412 369 43 434 370 458 365 157 474 387 87 20 356 433 324 109 432 354 78 419 338 473 352 202 463 340 123 20 330 430 335 95 416 355 61 424 369 468 402 121 467 378 89 

F1 bird weights ppm Sex 

Baby ID 12/10/10 12/16/10 ID 12/22/10 12/29/10 1/4/11 1/13/11 1/19/11 1/25/11 2/1/11 2/8/11 2/15/11 2/24/11 3/1/11 3/9/11 3/22/11 3/29/11 4/5/11 4/12/11 4/20/11 

0 F 4 18 211 90.2 104 106 112 126 127 124 140 127 140 141 140 149 147 0 F 3 19 212 71.9 102 103 100 112 116 114 120 122 113 113 120 125 125 0 F 36 215 91 103 107 112 117 118 125 139 122 125 132 147 136 0 F 11 216 75.9 95.1 113 125 125 133 141 134 128 130 136 135 153 152 153 0 F 23 217 92 101 108 111 128 136 140 138 139 140 139 141 140 0 F 3 19 218 81.2 94 97 103 123 120 115 123 119 124 126 134 122 135 0 F 30 222 63.8 85.1 87 99 0 F 66 270 105 92 110 114 118 122 111 111 125 136 129 0 F 15 20 224 91.2 103 110 120 129 145 130 147 140 140 139 147 147 142 0 F 13 18 226 71 92 103 109 131 103 111 144 144 139 142 143 145 150 146 0 F 40 12 227 102 116 113 114 126 128 134 140 142 141 126 146 144 0 F 7 228 89.7 100 105 119 111 123 122 123 110 131 130 147 134 129 0 F 9 18 230 70.7 85.8 94 95 119 124 134 126 141 141 142 140 145 134 148 0 F 37 12 231 70.8 92.1 108 115 104 117 131 134 138 136 135 136 138 149 147 0 F 45 235 91 97 116 102 134 121 132 134 141 140 131 140 140 0 F 22 237 72.7 86.7 94 103 105 92 110 114 118 122 111 111 125 139 133 0 F 79 276 91 106 120 121 126 124 134 143 140 141 143 143 145 0 F 82 283 93 102 103 115 133 139 138 133 146 144 149 147 147 0 F 74 292 71 97 111 125 140 135 135 132 135 135 137 141 136 0 F 89 294 54.8 66 83 102 108 117 118 129 136 139 139 136 143 137 0 F 20 19 299 83 99 112 116 121 120 132 131 138 139 138 141 140 0 M 33 213 81.7 92 102 104 102 103 102 103 104 107 107 108 109 104 0 M 17 21 214 78.1 87 90 97 90 98 93 102 103 103 104 108 109 110 
0 M 35 219 83.6 99 111 116 120 121 124 126 122 127 126 133 133 132 0 M 2 21 220 81.8 89 99 104 104 106 110 113 113 113 112 112 113 112 0 M 26 221 86.6 101 109 115 111 112 114 114 117 116 114 117 119 115 0 M 27 223 98 107 111 113 115 117 118 118 115 116 114 113 115 0 M 1 19 225 82.3 92 104 107 108 108 111 105 102 102 103 104 109 107 0 M 16 15 232 90.1 105 101 111 107 113 108 116 120 122 124 121 122 120 0 M 5 233 68.4 85.1 90 102 102 101 101 103 105 106 109 108 111 112 112 0 M 6 234 97 112 111 104 103 105 107 104 111 113 115 114 113 0 M 19 20 236 64.1 76.4 91 99 103 101 106 109 112 111 111 111 113 111 111 0 M 18 238 62.8 81 93 102 101 101 105 108 111 111 109 107 110 115 112 0 M 59 242 90 106 118 120 117 117 118 116 119 119 123 125 128 0 M 29 20 246 89 97 105 108 110 114 116 108 114 113 114 113 116 0 M 46 40.5 247 95 106 118 124 124 123 122 124 126 123 127 129 125 0 M 57 30.7 255 99 108 103 114 118 112 109 121 120 119 120 121 121 0 M 51 37.3 259 84 95 102 100 105 108 110 108 107 108 109 111 115 0 M 47 261 91 100 103 106 107 109 112 111 113 111 115 113 119 0 M 34 264 90 95 102 102 105 106 107 106 108 109 109 111 115 0 M 75 280 84 90 101 99 105 108 112 102 104 106 108 108 111 0.5 F 1 22 45.2 92.9 108 115 127 118 137 124 129 141 147 147 145 137 140 0.5 F 6 24 109 116 122 125 130 129 132 146 147 144 153 118 150 0.5 F 35 38.4 25 74.3 73.1 78 90 100 99 100 123 132 128 129 129 127 129 132 0.5 F 29 26 66.2 85.7 99 108 117 119 121 122 121 121 119 120 138 142 140 0.5 F 24 32.7 27 68.4 80.4 99 108 120 124 146 130 134 148 147 147 143 128 146 0.5 F 2 28 68.4 80 0.5 F 63 73 114 120 139 140 146 144 140 146 147 157 155 157 0.5 F 38 40.1 29 90 111 115 110 126 123 134 133 140 145 141 147 139 0.5 F 4 30 52.5 107 95 106 107 111 119 127 119 123 126 116 134 145 0.5 F 19 34 88 99 104 101 107 111 114 114 111 110 113 103 117 0.5 F 16 31.7 35 47.1 93 96 122 122 132 131 138 135 136 134 142 122 117 0.5 F 37 32.9 36 71.2 81.7 99 100 105 119 122 122 128 128 112 111 137 143 143 0.5 F 14 37 78.2 93 88 94 97 102 115 123 123 129 130 130 137 138 
0.5 F 9 38 65.8 69.7 74.2 65 111 127 144 144 146 145 150 152 150 155 127 125 0.5 F 40 40 72.1 86.5 103 109 119 135 147 146 146 153 141 140 148 156 155 0.5 F 60 50 78.8 96 114 120 139 140 146 144 140 146 147 157 156 149 0.5 F 53 90 88 97 106 113 114 119 137 131 131 133 138 140 141 0.5 F 100 105 85 95 105 116 136 138 136 136 130 127 140 149 143 0.5 F 96 107 74 90 97 104 107 114 118 125 113 114 123 135 123 0.5 F 106 108 86 103 114 119 111 102 116 117 128 125 134 121 136 0.5 F 71 110 103 112 125 128 142 124 134 138 145 145 148 139 148 0.5 M 8 37 21 79.8 97 103 108 110 110 113 115 114 116 115 114 116 115 0.5 M 5 23 60.3 78.3 91 102 108 112 109 111 115 113 114 115 120 121 121 0.5 M 3 31 77.8 93 99 104 103 102 105 107 106 112 114 112 112 113 0.5 M 11 32 64.6 82.9 95 106 110 113 112 117 110 109 106 106 111 109 112 0.5 M 13 41.8 33 62.3 61.9 77 88 101 107 109 110 112 118 108 118 108 110 109 111 0.5 M 12 39 85.2 98 106 112 114 118 120 122 124 120 118 121 123 120 0.5 M 15 38.5 41 68.2 63.9 80.7 98 106 111 115 119 123 1251 123 112 125 128 155 128 0.5 M 20 37.2 42 58.2 71.2 85 97 105 110 113 115 117 117 102 117 118 117 117 0.5 M 26 43 62.4 85 88 97 97 98 100 100 100 104 105 102 109 105 0.5 M 33 35.8 44 57.3 82.6 96 104 111 110 115 115 118 118 114 115 122 121 122 0.5 M 22 45 62.5 76.3 88 90 98 99 102 102 104 104 103 104 105 108 107 0.5 M 17 46 92 102 110 114 111 113 114 116 108 108 121 127 121 0.5 M 27 47 44 77.9 91 97 98 100 103 103 110 105 110 103 107 108 108 0.5 M 28 35.6 48 94 95 104 103 105 113 116 118 120 117 124 124 141 0.5 M 21 35.6 49 65.6 83 96 92 94 96 99 100 102 105 105 106 105 0.5 M 39 81 83 96 98 102 104 108 96 97 99 102 103 104 0.5 M 62 88 78 95 101 107 115 116 118 112 119 112 113 113 117 0.5 M 68 104 81 98 110 115 118 121 120 124 107 119 126 123 121 0.5 M 87 106 88 97 106 110 113 114 113 117 102 114 118 132 119 0.5 M 88 116 90 94 109 115 115 117 115 117 117 117 122 123 125 5 F 36 37.3 23 82.1 99 110 104 116 117 127 129 137 137 138 140 125 136 5 F 40 27 80.1 98 107 114 111 120 127 115 121 137 134 135 145 160 159 5 F 53 38 87 102 119 124 134 133 135 139 135 136 135 113 116 
5 F 61 39 91 102 120 128 133 141 139 143 155 153 162 154 141 5 F 79 43 93 104 105 103 105 109 112 122 113 116 115 148 133 5 F 55 45 87 91 105 113 119 120 124 133 136 137 138 109 136 5 F 26 47 92 101 113 123 138 140 138 145 150 149 130 140 133 5 F 76 49 97 104 100 106 102 109 118 120 124 123 130 154 150 5 F 46 53 99 105 102 104 109 125 126 132 111 115 130 144 137 5 F 29 54 97 110 106 123 140 146 145 150 152 150 152 135 138 5 F 99 55 83 99 5 F 18 7 70.1 104 116 117 127 129 137 137 138 140 140 134 5 F 49 61 94 101 113 119 127 130 129 142 143 141 137 142 141 5 F 39 64 95 104 107 110 129 134 136 140 132 131 138 110 131 5 F 21 16 66 88 95 119 117 124 127 124 134 130 133 135 122 137 5 F 44 13 68 95 100 99 105 113 127 126 112 138 138 131 117 120 5 F 63 70 101 108 95 106 128 131 123 131 133 136 117 130 136 5 F 82 75 86 94 5 F 6 15 81.7 111 120 127 115 121 137 134 135 145 142 145 5 F 89 76 86 90 97 95 97 105 115 131 130 130 120 136 125 5 F 95 77 93 106 121 120 124 131 130 141 140 140 149 149 150 5 F 87 78 79 87 94 96 101 109 113 117 121 123 124 124 128 5 M 38 42.5 1 79.6 80 101 105 100 108 111 109 117 112 111 116 114 114 5 M 30 14 35.4 4 59 96 105 112 114 116 117 115 117 119 120 120 118 121 5 M 12 38.7 11 57.5 70.2 95 105 109 110 114 120 118 122 115 113 118 125 126 5 M 15 16 67.3 82.8 98 97 102 100 104 101 104 107 108 109 110 110 112 5 M 41 18 80 87 99 107 106 106 108 109 108 110 108 113 112 115 5 M 22 17 19 69.6 69 81 93 95 96 96 99 101 101 102 103 101 103 99 5 M 8 20 80.8 88 98 102 103 103 103 102 107 107 104 101 110 100 5 M 14 36.8 26 55.7 82.2 96 102 107 104 106 109 108 112 114 114 117 121 115 5 M 16 38.6 28 58.8 82 89 95 98 99 100 101 100 97 99 99 98 101 5 M 20 29 69.7 89 94 91 95 101 95 98 106 106 103 107 109 108 5 M 51 33 92 103 113 115 102 108 107 110 110 114 114 114 114 5 M 31 34 102 109 107 107 109 111 118 115 119 122 123 122 127 
5 M 58 35 97 104 112 109 113 124 129 126 123 124 127 127 126 5 M 56 36 89 96 104 109 109 108 99 102 114 115 110 128 112 5 M 59 37 98 108 117 117 120 119 120 121 115 114 121 119 123 5 M 65 41 103 105 115 119 121 125 125 125 127 125 131 124 130 5 M 57 44 100 108 105 111 119 120 125 119 123 120 123 132 121 5 M 25 46 99 115 111 116 132 135 129 123 116 118 116 117 126 5 M 50 52 80 87 101 103 100 102 100 100 100 100 99 99 100 5 M 37 58 80 93 94 101 100 98 100 100 103 103 106 108 109 10 F 96 74 80 90 92 97 100 101 99 96 99 101 106 100 10 F 42 349 68.6 64.5 66 83 93 107 112 126 10 F 6 350 65.8 97 106 111 118 124 126 128 131 125 127 141 139 144 10 F 9 351 62.1 80.1 93 104 115 110 140 128 127 118 134 135 137 134 138 10 F 7 22 352 99.7 118 126 142 140 141 122 147 149 150 149 162 148 163 10 F 3 357 54.9 92.3 104 118 126 136 143 145 142 144 145 146 149 153 150 10 F 28 359 84.1 93 106 115 134 123 119 129 128 139 140 127 135 130 10 F 44 360 45.4 76.9 91 95 102 107 112 131 132 116 140 141 141 137 142 10 F 16 15 37.7 361 80 96 100 92 102 94 97 99 105 107 110 103 111 10 F 25 367 73.2 102 111 131 133 97 99 105 143 10 F 13 368 57.1 87.4 78 85 105 109 117 129 148 141 141 143 131 131 134 10 F 26 35.6 369 68.2 73.4 85.7 91 101 109 114 118 124 148 141 141 10 F 5 370 84.8 99 105 118 118 120 128 140 142 133 136 145 140 147 10 F 35 371 67 84 97 91 100 98 107 109 133 136 10 F 12 21 373 81.1 94 109 118 111 116 118 120 112 134 135 100 113 102 10 F 47 374 66.9 71.7 81 90 103 109 124 119 123 125 137 137 119 136 122 10 F 52 35.5 376 93 97 116 118 138 132 139 142 145 146 153 145 153 10 F 30 15 33 379 89 97 101 104 113 111 117 124 121 124 126 130 131 10 F 43 385 101 108 122 130 142 141 143 139 149 149 146 150 148 10 F 56 389 99 108 121 115 121 123 129 119 137 139 144 145 146 10 F 78 390 80 102 106 115 107 123 138 135 136 138 144 114 140 10 F 51 395 100 101 117 123 136 147 145 134 144 145 140 150 144 10 F 48 398 103 114 124 126 139 133 140 148 151 152 150 139 150 
10 F 56 400 97 108 118 112 118 125 129 123 137 150 144 154 10 F 12 21 372 98 109 123 105 10 F 49 new 380 106 107 110 126 138 138 139 143 141 10 F 49 old 380 84 97 10 F 95 117 130 144 142 149 137 150 149 149 136 150 10 M 97 76 89 102 99 108 114 118 119 117 115 122 121 170 10 M 98 82 92 102 100 100 103 103 103 106 105 108 109 108 10 M 54 348 85 95 98 101 103 102 105 104 106 107 111 112 114 10 M 23 353 77.9 86 102 97 97 100 101 105 104 103 104 109 110 108 10 M 36 356 63.3 75 89 96 103 103 108 110 109 113 111 112 111 111 112 10 M 39 358 78 75.1 92 102 109 108 112 114 117 117 110 107 115 113 110 10 M 18 362 54.8 76.5 86 99 103 108 100 104 105 106 112 110 115 116 117 10 M 64 381 80 91 99 100 104 104 107 105 107 105 107 110 123 10 M 45 382 96 107 115 114 114 113 114 115 110 111 115 117 116 10 M 57 386 71 85 93 97 94 98 98 96 96 98 100 99 101 10 M 46 388 89 101 109 112 113 117 116 119 119 121 122 123 122 10 M 69 392 81 93 105 104 108 110 111 114 114 115 117 120 110 10 M 77 393 82 95 110 116 115 113 116 118 122 123 127 125 10 M 11 394 95 104 113 112 111 114 116 116 113 115 118 116 115 20 F 11 19 311 87 96 105 106 114 105 20 F 9 19 312 83.2 92 106 114 115 115 118 125 115 116 136 143 140 20 F 8 313 66.9 68.9 80 91 94 92 98 95 110 119 129 131 123 125 125 20 F 21 315 57.3 71.3 63 129 20 F 20 13 316 70.6 85 87 99 102 105 110 130 111 20 F 4 317 85 94 97 100 104 106 105 111 111 115 118 119 125 20 F 5 318 68.9 86.5 102 115 121 126 128 122 131 125 129 128 122 128 125 20 F 3 320 72.3 85 97 102 100 110 124 120 104 112 116 128 96 131 20 F 47 321 82 97 103 109 117 123 127 136 20 F 46 327 65.1 76 98 101 108 116 118 126 130 115 114 119 122 123 20 F 26 35.7 333 56.1 82 94 95 103 117 108 110 120 129 130 133 138 135 20 F 1 20 339 70.2 81 90 90 85 93 105 107 117 116 119 111 115 113 
20 F 18 342 79.5 90 100 104 116 128 106 20 F 7 19 343 80.4 97 104 111 113 119 125 20 F 30 33.2 351 49.5 75 91 98 95 100 111 102 105 125 127 134 139 135 20 F 49 352 62 75 85 94 100 89 92 104 111 114 20 F 39 358 72 89 98 103 114 125 118 133 115 116 109 124 112 20 F 37 359 82 96 105 100 108 125 128 135 132 134 20 M 13 16 314 68 78 90 98 88 94 101 103 107 108 105 111 113 113 20 M 25 319 69.5 81 90 96 102 108 108 107 111 118 118 116 114 111 20 M 27 34.5 322 71.7 68.8 86 91 105 105 111 116 111 114 109 110 119 119 121 20 M 19 324 68.1 84 85 94 94 100 99 102 118 110 110 106 112 20 M 12 35 325 75.2 70 96 107 110 110 112 113 118 116 114 120 119 120 20 M 2 20 326 69.6 81 84 90 88 93 93 100 96 100 100 105 105 106 20 M 41 33.2 328 68 80 89 93 99 102 105 107 111 111 118 102 114 20 M 31 330 75.4 79 91 98 106 112 115 113 104 106 110 115 113 20 M 10 331 55.4 71.2 74 88 89 93 94 97 95 97 98 99 102 101 103 20 M 16 14 334 65.8 79 87 96 100 102 104 101 102 103 106 106 110 106 20 M 17 335 74.8 87 95 108 108 110 107 111 113 109 111 122 120 122 20 M 15 37.4 337 47.4 71 72.9 86 98 111 118 122 119 114 117 124 124 128 126 125 20 M 40 36.6 338 73.2 70 82 89 94 97 100 103 102 106 105 107 107 107 20 M 42 344 65.2 68 85 105 101 102 107 106 106 109 108 110 20 M 48 345 52.7 71.3 67 79 89 92 98 101 98 100 102 103 104 109 106 20 M 6 348 78.7 84 104 111 111 113 118 117 115 113 113 113 112 110 20 M 45 40.2 350 77 89 97 101 109 115 111 105 96 98 107 107 106 20 M 32 32.7 356 57.2 73 89 99 106 113 119 123 119 122 120 120 126 125 126 20 M 33 361 69 93 99 94 98 100 104 106 107 105 109 108 107 F1 sperm measurements ppm ID Motile 1 Motile 2 Motile Average Progressive 1 Progressive 2 Progressive Average Sperm/gram 1 Sperm/gram 2 Sperm/gram Average 0 213 39 30 34.5 24 22 23 1432.5 2230.6 1831.55 0 221 61 39 50 37 16 26.5 2589.6 2366.4 2478 
0 225 27 20 23.5 9 13 11 2025.9 1381.3 1703.6 

0 233 58 62 60 3 42 22.5 4011 3990.5 4000.75 0 234 70 65 67.5 38 50 44 2578.5 1964.6 2271.55 0 236 3 1 2 2 1 1.5 1317.1 2143.2 1730.15 0 242 67 67 67 29 38 33.5 6671.3 5566.2 6118.75 0 247 50 56 53 23 37 30 5770.9 6016.5 5893.7 0 255 65 74 69.5 39 43 41 6221.1 9086.1 7653.6 0 264 54 55 54.5 27 31 29 4133.8 5545.8 4839.8 0.5 21 48 45 46.5 27 27 27 2718.8 4595.7 3657.25 0.5 23 12 8 10 12 8 10 244.1 188.2 216.15 0.5 39 48 34 41 23 17 20 4338.4 2169.2 3253.8 0.5 41 43 48 45.5 24 30 27 2762.7 2670.6 2716.65 0.5 43 4 6 5 0 0 0 581.8 400 490.9 0.5 45 71 69 70 40 36 38 10514.8 10492.5 10503.65 0.5 46 0 0 0 0 0 0 51.4 51.4 51.4 0.5 47 0 0 0 0 0 0 2553.9 1964.6 2259.25 0.5 81 45 59 52 21 34 27.5 557.9 6174.3 3366.1 0.5 106 0 0 0 0 0 0 920.9 1289.2 1105.05 5 394 33 13 23 17 8 12.5 1824.2 1333.1 1578.65 5 11 68 69 68.5 38 35 36.5 7029.4 4512.3 5770.85 5 16 75 74 74.5 46 51 48.5 8871.2 7489.9 8180.55 5 18 42 38 40 27 21 24 4027.3 5328.9 4678.1 5 29 53 86 69.5 36 66 51 3585.3 2455.7 3020.5 5 33 65 74 69.5 35 39 37 8865.1 8717.7 8791.4 5 35 15 26 20.5 15 9 12 425.7 753.1 589.4 5 37 61 58 59.5 38 34 36 3683.5 3862.1 3772.8 5 44 64 62 63 32 34 33 8840.5 5893.7 7367.1 5 58 1 1 1 1 1 1 2373.8 2428.4 2401.1 10 98 55 29 42 38 14 26 1289.2 1074.4 1181.8 10 353 74 92 83 50 65 57.5 2087.3 3131 2609.15 10 362 46 50 48 30 33 31.5 2739 2247.9 2493.45 
10 382 64 59 61.5 31 35 33 3781.8 1817.2 2799.5 

10 392 63 66 64.5 33 32 32.5 5975.5 6466.7 6221.1 10 393 67 56 61.5 36 30 33 6492.2 6968 6730.1 20 319 56 35 45.5 33 17 25 1578.7 806.9 1192.8 20 326 52 54 53 37 32 34.5 4850 4236.1 4543.05 20 335 68 65 66.5 44 38 41 5689 9004.2 7346.6 20 338 75 75 75 47 42 44.5 8656.3 13751.9 11204.1 20 344 64 57 60.5 41 34 37.5 4474.8 3492.5 3983.65 20 345 55 63 59 30 35 32.5 2578.5 5371.8 3975.15 20 356 61 69 65 39 39 39 4706.8 6712.2 5709.5 20 361 53 62 57.5 30 34 32 6671.3 6303 6487.15 
ppm ID first day of foam 

date of hatch date of onset 

0 213 1/12/2011 12/9/2010 34 0 214 1/12/2011 12/8/2010 35 0 219 2/2/2011 12/9/2010 0 220 1/19/2011 12/6/2010 44 0 221 1/12/2011 12/8/2010 35 0 223 1/15/2011 12/8/2010 38 0 225 1/12/2011 12/6/2010 37 0 232 1/12/2011 12/8/2010 35 0 233 1/12/2011 12/6/2010 37 0 234 1/21/2011 12/6/2010 46 0 236 1/26/2011 12/8/2010 49 0 238 1/12/2011 12/8/2010 35 0 242 1/19/2011 12/11/2010 39 0 246 2/14/2011 12/9/2010 67 0 247 2/1/2011 12/11/2010 52 0 255 1/26/2011 12/11/2010 46 0 259 1/19/2011 12/11/2010 39 0 261 1/24/2011 12/11/2010 44 0 264 1/26/2011 12/9/2010 48 0 280 1/29/2011 12/13/2010 47 0.5 21 1/12/2011 12/6/2010 37 0.5 23 2/2/2011 12/6/2010 58 0.5 31 1/12/2011 12/6/2010 37 0.5 32 1/17/2011 12/7/2010 41 0.5 33 1/28/2011 12/7/2010 52 0.5 39 1/17/2011 12/7/2010 41 0.5 41 2/11/2011 12/7/2010 66 0.5 42 2/10/2011 12/8/2010 64 0.5 43 1/12/2011 12/8/2010 35 0.5 44 2/11/2011 12/9/2010 64 
0.5 45 1/28/2011 12/8/2010 51 

0.5 46 12/7/2011 0.5 47 1/17/2011 12/8/2010 40 0.5 48 1/28/2011 12/8/2010 51 0.5 49 1/26/2011 12/8/2010 49 0.5 81 2/18/2011 12/9/2010 71 0.5 88 2/13/2011 12/11/2010 64 0.5 104 2/15/2011 12/12/2010 65 0.5 106 1/26/2011 12/13/2010 44 0.5 116 2/11/2011 12/13/2010 60 5 1 1/14/2011 12/9/2010 36 5 4 1/12/2011 12/7/2010 36 5 11 1/30/2011 12/7/2010 54 5 16 1/12/2011 12/7/2010 36 5 18 1/17/2011 12/9/2010 39 5 19 1/12/2011 12/8/2010 35 5 20 1/12/2011 12/7/2010 36 5 26 1/12/2011 12/7/2010 36 5 28 1/19/2011 12/7/2010 43 5 29 1/12/2011 12/7/2010 36 5 33 1/30/2011 12/11/2010 50 5 34 1/16/2011 12/8/2010 39 5 35 2/2/2011 12/11/2010 53 5 36 1/14/2011 12/11/2010 34 5 37 1/16/2011 12/11/2010 36 5 41 1/14/2011 12/12/2010 33 5 44 1/14/2011 12/11/2010 34 5 46 1/17/2011 12/8/2010 40 5 52 1/31/2011 12/11/2010 51 5 58 1/17/2011 12/9/2010 39 10 97 1/16/2011 12/8/2010 39 
10 98 1/12/2011 12/11/2010 32 

10 348 1/12/2011 12/12/2010 31 10 353 1/16/2011 12/8/2010 39 10 356 1/12/2011 12/9/2010 34 10 358 1/12/2011 12/10/2010 33 10 360 female! 10 362 1/12/2011 12/7/2010 36 10 371 male? 10 379 female! 10 381 1/16/2011 12/13/2010 34 10 382 1/12/2011 12/11/2010 32 10 385 female! 10 386 1/19/2011 12/12/2010 38 10 388 1/12/2011 12/11/2010 32 10 389 1/26/2011 12/12/2010 45 10 392 1/14/2011 12/15/2010 30 10 393 1/16/2011 12/15/2010 32 10 394 1/16/2011 12/7/2010 40 10 395 female! 10 400 12/7/2010 20 313 female! 20 314 1/12/2011 12/9/2010 34 20 317 12/7/2010 20 319 1/12/2011 12/11/2010 32 20 322 1/12/2011 12/11/2010 32 20 324 1/26/2011 12/10/2010 47 20 325 1/12/2011 12/10/2010 33 20 326 1/12/2011 12/6/2010 37 20 330 1/12/2011 12/12/2010 31 20 331 1/12/2011 12/8/2010 35 20 333 1/23/2011 12/11/2010 43 
20 334 1/16/2011 12/7/2010 40 

20 335 1/12/2011 12/7/2010 36 20 337 1/16/2011 12/9/2010 38 20 338 1/12/2011 12/14/2010 29 20 344 2/15/2011 12/15/2010 62 20 345 1/12/2011 12/15/2010 28 20 348 1/12/2011 12/7/2010 36 20 350 1/12/2011 12/15/2010 28 20 356 1/12/2011 12/12/2010 31 20 361 1/12/2011 12/13/2010 30 F1 cloaca measuremets (mm2) ppm ID 1/12/2011 1/20/2011 1/26/2011 2/2/2011 0 225 156 130 252 225 0 213 132 168 224 342 0 223 80 81 225 289 0 220 72 99 25 224 0 221 144 143 80 210 0 219 25 48 285 168 0 232 110 80 225 90 0 238 143 156 132 240 0 214 153 120 121 210 0 236 25 64 156 196 0 233 100 132 195 256 0 264 25 49 182 224 0 234 72 110 182 256 0 259 25 110 240 256 0 255 25 72 224 224 0 247 35 72 90 210 0 242 25 90 169 272 
0 261 35 72 195 182 

0 280 25 72 144 224 0 246 25 81 25 64 0.5 43 100 110 132 210 0.5 21 156 154 176 255 0.5 23 72 64 49 120 0.5 44 25 56 49 49 0.5 48 42 49 81 196 0.5 31 120 120 240 256 0.5 32 126 100 208 108 0.5 39 99 143 210 272 0.5 46 54 90 50 49 0.5 45 64 72 49 168 0.5 104 25 72 25 81 0.5 116 25 64 49 110 0.5 88 25 91 49 64 0.5 47 49 110 156 210 0.5 106 25 49 80 169 0.5 41 25 56 49 72 0.5 81 63 49 90 0.5 49 63 81 132 0.5 42 25 72 49 81 0.5 33 35 81 132 225 0.5 67 25 0.5 79 63 5 52 35 73 64 90 5 35 56 90 80 108 5 37 56 100 110 180 5 34 80 110 225 240 5 41 64 110 252 272 5 36 49 100 225 240 
5 46 64 121 132 156 

5 33 64 81 64 168 5 58 25 91 90 210 5 44 88 100 110 182 5 18 110 132 272 289 5 26 100 182 255 288 5 19 81 100 182 225 5 20 143 196 225 272 5 11 81 100 81 144 5 16 169 132 225 306 5 29 156 156 144 225 5 4 132 100 225 255 5 28 81 110 100 182 5 1 72 110 156 144 10 382 90 110 210 169 10 393 64 91 156 182 10 360 81 100 100 99 10 98 81 132 120 210 10 348 81 100 144 196 10 386 72 132 110 144 10 388 90 121 180 210 10 394 88 156 156 255 10 362 110 144 165 224 10 392 90 168 240 288 10 389 72 121 144 168 10 400 72 121 441 110 10 379 90 132 120 143 10 381 81 144 156 210 10 97 63 110 120 169 10 358 110 132 120 156 10 353 120 143 144 288 
10 356 110 132 154 144 

20 345 144 132 121 169 20 350 90 121 144 182 20 348 110 154 196 272 20 331 110 144 121 168 20 314 110 156 144 195 20 326 156 144 144 168 20 335 130 121 182 210 20 338 132 132 132 210 20 361 110 110 144 156 20 313 90 110 100 132 20 322 110 121 156 182 20 325 99 156 182 240 20 337 100 156 182 210 20 334 110 132 130 225 20 324 81 100 110 143 20 356 144 144 168 225 20 317 64 110 81 132 20 319 100 121 144 120 20 333 90 100 121 156 20 330 120 121 132 1196 10 385 64 121 100 182 10 395 81 90 80 144 F1 egg strength ppm ID Thickness Thickness Force 0 211 0.3 0.29 0.539 0 211 0.32 0.31 1.055 0 217 0.33 0.29 0.978 
0 217 0.33 0.37 1.182 

0 218 0.32 0.31 1.718 0 218 0.31 0.29 1.691 0 227 0.43 0.33 1.325 0 227 0.29 0.29 1.481 0 228 0.3 0.29 1.36 0 228 0.29 0.28 1.273 0 230 0.33 0.29 1.244 0 230 0.29 0.29 0 231 0.42 0.43 1.248 0 231 0.3 0.28 0.596 0 235 0.27 0.36 1.287 0 235 0.29 0.28 1.243 0 237 0.4 0.27 1.67 0 237 0.3 0.29 1.543 0 276 0.3 0.3 1.557 0 276 0.32 0.3 1.565 0 283 0.45 0.31 1.877 0 283 0.32 0.3 2.032 0 292 0.29 0.36 0.914 0 292 0.27 0.27 1.472 0 294 0.35 0.32 1.423 0 294 0.39 0.29 1.331 0 299 0.31 0.29 1.583 0 299 0.35 0.33 0.254 0.5 22 0.29 0.3 1.335 0.5 22 0.29 0.28 1.328 0.5 24 0.29 0.31 1.613 0.5 24 0.29 0.29 1.629 0.5 25 0.37 0.27 1.217 0.5 25 0.43 0.38 1.523 
0.5 27 0.39 0.38 1.147 

0.5 27 0.36 0.29 1.203 0.5 29 0.32 0.29 1.404 0.5 29 0.44 0.31 0.983 0.5 30 0.29 0.32 1.196 0.5 30 0.29 0.29 1.107 0.5 34 0.34 0.29 1.207 0.5 34 0.29 0.31 1.259 0.5 36 0.28 0.29 1.665 0.5 36 0.4 0.27 1.279 0.5 37 0.36 0.37 1.367 0.5 37 0.33 0.31 0.891 0.5 38 0.36 0.36 1.647 0.5 38 0.38 0.39 1.616 0.5 40 0.29 0.29 1.846 0.5 40 0.38 0.37 1.687 0.5 73 0.33 0.3 1.582 0.5 73 0.29 0.3 1.771 0.5 90 0.34 0.29 1.647 0.5 90 0.3 0.29 1.047 0.5 105 0.31 0.33 1.91 0.5 105 0.33 0.3 1.104 0.5 108 0.4 0.3 1.164 0.5 108 0.43 0.36 1.296 0.5 110 0.32 0.32 1.897 0.5 110 0.3 0.29 1.588 5 7 0.27 0.29 1.817 5 7 0.28 0.3 1.613 5 27 0.3 0.31 1.958 5 27 0.28 0.29 1.282 5 39 0.28 0.31 1.786 
5 39 0.32 0.32 1.859 

5 43 0.29 0.3 1.136 5 43 0.31 0.3 1.74 5 49 0.3 0.31 1.146 5 49 0.3 0.31 1.29 5 54 0.43 0.28 1.118 5 54 0.26 0.28 1.677 5 61 0.27 0.29 1.428 5 61 0.34 0.28 1.522 5 64 0.28 0.29 1.21 5 64 0.28 0.31 1.233 5 66 0.38 0.41 1.236 5 66 0.29 0.29 1.79 5 68 0.31 0.29 1.217 5 68 0.3 0.42 0.579 5 70 0.32 0.38 1.196 5 70 0.28 0.42 1.357 5 76 0.29 0.33 1.301 5 76 0.33 0.29 1.184 5 68 0.35 0.37 1.03 5 68 0.37 0.31 1.26 10 350 0.36 0.41 1.203 10 350 0.27 0.29 1.226 10 351 0.28 0.3 1.174 10 351 0.3 0.31 1.274 10 357 0.29 0.3 1.178 10 357 0.29 0.3 1.267 10 359 0.3 0.28 1.205 10 359 0.27 0.28 1.356 10 360 0.38 0.35 1.523 10 360 0.28 0.34 1.102 
10 374 0.41 0.37 1.474 

10 374 0.31 0.41 1.254 10 376 0.34 0.32 1.494 10 376 0.38 0.35 1.335 10 379 0.39 0.33 10 380 0.42 0.28 1.292 10 380 0.28 0.31 1.227 10 385 0.35 0.4 1.149 10 385 0.29 0.32 1.253 10 389 0.28 0.29 0.567 10 389 0.4 0.42 1.299 10 390 0.4 0.41 1.923 10 390 0.33 0.33 1.634 10 395 0.32 0.29 1.297 10 395 0.29 0.29 0.657 10 395 0.39 0.37 1.166 10 395 0.3 0.41 2.04 10 398 0.28 0.29 2.064 10 398 0.28 0.3 1.1 10 400 0.29 0.29 2.156 10 400 0.28 0.32 2.167 20 313 0.4 0.41 1.139 20 313 0.33 0.29 1.018 20 313 0.3 0.28 1.032 20 324 0.34 0.36 20 333 0.31 0.34 0.263 20 339 0.38 0.31 20 339 0.36 0.39 20 359 0.35 0.35 0.264 
F2 egg production ppm ID 7/23 7/24 

week 5 7/25 7/26 7/27 7/28 7/29 7/30 7/31 

week 6 8/1 8/2 8/3 8/4 8/5 8/6 8/7 

week 7 8/8 8/9 8/10 8/11 8/12 8/13 8/14 

week 8 8/15 8/16 

0 254 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 257 1 1 1 1 1 1 0 0 258 1 1 1 1 1 1 1 1 1 1 1 1 0 259 0 281 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 283 0 284 1 1 1 1 1 1 0 286 0 413 1 1 1 1 1 1 1 1 1 0 415 1 1 1 0 417 0 420 0 429 1 1 1 1 1 1 1 1 1 1 0 437 1 1 1 1 1 1 1 1 1 0 440 1 1 1 1 1 1 1 1 1 1 0 445 1 1 1 1 0 453 1 1 1 1 1 1 1 1 1 0 454 1 1 1 1 1 0 457 1 1 1 1 1 1 0 458 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 118 1 1 1 1 1 1 1 1 1 1 0.5 119 1 1 1 1 0.5 120 0.5 125 0.5 126 1 1 1 1 1 1 1 1 1 1 1 1 0.5 127 1 0.5 129 1 1 1 1 1 1 1 1 1 1 1 0.5 130 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 131 1 1 1 1 1 1 
0.5 134 1 1 1 1 1 1 1 1 1 1 0.5 135 0.5 136 1 1 1 1 1 1 1 1 1 1 0.5 137 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 138 1 1 1 1 1 1 1 0.5 141 0.5 143 1 1 1 1 1 1 0.5 144 1 1 1 1 1 1 1 1 1 1 1 1 0.5 147 1 1 1 1 1 1 1 1 1 1 0.5 148 1 1 1 1 0.5 153 1 1 1 1 1 1 1 1 1 1 5 91 1 1 5 93 1 1 1 1 1 1 5 96 1 1 1 1 5 97 1 1 1 1 1 1 1 5 100 1 1 1 1 1 5 104 1 1 1 1 1 1 1 1 1 1 1 1 5 105 1 1 1 1 1 1 1 1 1 1 5 106 1 1 1 1 1 1 1 1 1 1 1 1 1 5 107 1 1 1 5 111 1 1 1 1 1 1 1 1 5 113 1 1 1 1 5 114 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 115 1 1 1 1 1 5 120 1 1 1 1 1 1 5 123 1 1 1 1 1 1 1 1 1 5 130 1 1 1 1 1 1 1 1 1 1 1 5 140 1 1 1 1 1 1 1 1 1 1 1 5 148 1 1 1 1 1 1 1 1 1 1 1 1 1 5 151 1 1 1 1 1 5 155 1 1 1 1 1 1 1 1 
10 41 10 44 1 1 1 10 48 1 1 1 2 10 50 1 1 1 1 1 1 1 10 52 1 10 57 10 60 1 1 1 10 63 1 1 1 1 1 1 1 1 2 10 69 10 70 10 71 1 1 1 1 1 1 1 1 1 1 10 77 1 2 1 1 10 79 1 1 1 1 1 1 1 10 80 1 1 1 1 1 1 1 1 10 82 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 83 10 84 1 1 1 1 10 85 1 1 1 1 1 1 1 1 1 1 10 87 1 1 1 1 1 1 1 1 1 1 10 88 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ppm ID 8/22 8/23 8/24 8/25 8/26 8/27 8/28 

week 10 8/29 8/30 8/31 9/1 9/2 9/3 9/4 

week 11 9/5 9/6 9/7 9/8 9/9 9/10 9/11 

week 12 

0 254 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 257 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 258 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 259 1 1 1 1 1 1 1 1 1 0 281 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 283 1 1 1 1 0 284 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 286 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 
0 413 1 1 1 1 1 1 1 1 1 1 1 1 0 415 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 0 417 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 420 1 1 1 1 0 429 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 437 1 1 1 1 1 1 1 1 1 1 1 1 0 440 1 1 1 1 1 1 1 1 1 0 445 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 453 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 454 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 457 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 458 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 118 1 1 1 1 1 1 1 0 1 1 1 0.5 119 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 0.5 120 1 1 1 1 1 1 1 0.5 125 0.5 126 1 2 2 1 2 1 1 1 2 1 2 2 0.5 127 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 129 1 1 1 1 1 1 1 1 1 1 1 1 0.5 130 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 131 1 1 1 1 1 1 1 1 0.5 134 1 1 1 1 1 1 1 1 1 1 0.5 135 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 136 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 137 0.5 138 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 141 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 143 1 1 1 1 1 1 1 1 0.5 144 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 147 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.5 148 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 
0.5 153 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 91 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 93 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 96 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 97 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 100 1 1 1 1 1 1 1 1 1 1 1 1 5 104 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 105 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 106 1 1 1 1 1 1 1 1 1 5 107 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 113 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 114 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 115 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 120 1 1 1 1 1 1 1 1 1 1 1 1 5 123 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 130 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 140 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 5 148 1 1 1 1 1 5 151 1 1 1 1 1 1 1 1 1 1 1 1 5 155 1 1 1 1 1 1 1 1 10 41 1 1 1 1 1 1 1 1 1 1 1 10 44 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 48 1 1 1 1 1 2 1 1 1 1 1 1 1 1 10 50 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 52 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 57 10 60 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 63 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 69 1 1 1 1 1 1 1 1 1 1 10 70 1 1 1 1 1 1 1 1 1 1 1 1 
10 71 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 77 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 79 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 80 1 1 1 1 1 1 1 1 1 1 1 10 82 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 83 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 84 1 1 1 1 1 1 1 1 1 1 1 1 1 10 85 1 1 1 1 1 1 1 1 1 1 1 1 10 87 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 88 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

F2 feed consumption DOSE SEX ID 7/25 8/1 week 6 8/1 8/8 week 7 8/8 8/15 week 8 8/15 8/22 week 9 8/22 8/29 week 10 8

0 F 284 389 281 108 419 286 133 409 256 153 420 238 182 419 287 132 40 F 286 408 289 119 435 311 124 422 324 98 420 305 115 415 304 111 40 F 281 396 268 128 422 261 161 407 254 153 444 271 173 418 269 149 40 F 283 406 293 113 434 334 100 424 316 108 428 330 98 416 299 117 40 F 415 404 303 101 420 296 124 413 271 142 428 239 189 420 321 99 40 F 254 393 288 105 413 282 131 415 229 186 425 255 170 433 320 113 40 F 417 407 280 127 429 349 80 419 316 103 415 280 135 416 273 143 40 F 420 405 276 129 416 352 64 422 307 115 423 326 97 428 328 100 40 F 257 407 320 87 427 305 122 420 266 154 430 245 185 406 295 111 40 F 259 400 273 0 419 350 69 401 305 96 438 343 95 399 290 109 40 F 258 404 278 126 422 305 117 421 287 134 423 282 141 415 268 147 40 F 429 403 321 82 427 317 110 411 243 168 436 244 192 426 290 136 40 F 413 405 294 111 415 293 122 408 274 134 432 259 173 432 277 155 4
0 F 437 399 294 105 420 300 120 407 246 161 423 242 181 426 250 176 40 F 457 403 267 136 411 323 88 414 266 148 419 232 187 440 241 199 40 F 454 400 291 109 421 361 60 426 333 93 433 281 152 429 274 155 40 F 453 406 290 116 421 318 103 416 245 171 428 247 181 432 254 178 40 F 458 410 313 97 423 316 107 418 265 153 441 281 160 442 270 172 40 F 440 416 305 111 415 325 90 411 264 147 427 270 157 413 324 89 40 F 445 398 288 110 415 334 81 415 285 130 428 279 149 420 274 146 40 M 282 411 322 89 415 316 99 412 320 92 412 310 102 419 314 105 40 M 260 417 325 92 418 314 104 413 317 96 416 309 107 426 327 99 40 M 251 405 300 105 424 328 96 427 322 105 410 307 103 431 327 104 40 M 255 410 278 132 431 329 102 425 337 88 427 335 92 429 335 94 40 M 412 403 300 103 423 319 104 410 295 115 420 314 106 416 321 95 40 M 419 405 353 52 430 339 91 431 336 95 404 307 97 435 329 106 40 M 285 409 318 91 419 289 130 411 299 112 419 298 121 406 310 96 40 M 288 406 315 91 421 315 106 419 314 105 430 324 106 437 335 102 40 M 252 407 347 60 431 329 102 438 332 106 435 330 105 432 320 112 40 M 422 404 311 93 429 331 98 417 306 111 427 309 118 440 333 107 40 M 416 414 348 66 433 304 129 424 325 99 420 304 116 431 317 114 40 M 418 403 324 79 419 351 68 414 308 106 430 307 123 426 334 92 40 M 253 411 297 114 428 335 93 427 315 112 405 277 128 425 313 112 40 M 256 394 270 124 418 289 129 423 292 131 413 291 122 427 308 119 40 M 287 410 316 94 420 311 109 422 319 103 430 304 126 445 327 118 40 M 426 395 305 90 413 310 103 417 297 120 426 307 119 414 334 80 40 M 290 409 317 92 423 330 93 421 333 88 419 332 87 433 338 95 40 M 425 400 312 88 410 294 116 428 331 97 429 286 143 434 309 125 40 M 424 420 331 89 427 308 119 431 330 101 428 320 108 442 280 162 40 M 289 400 305 95 435 319 116 430 308 122 411 310 101 441 306 135 4

0.5 F 130 399 281 118 418 289 129 412 266 146 422 241 181 411 281 130 40.5 F 136 385 309 76 423 290 133 417 268 149 434 255 179 416 298 118 40.5 F 131 400 300 100 418 292 126 408 277 131 415 287 128 423 287 136 40.5 F 134 398 254 144 422 312 110 410 298 112 436 265 171 426 305 121 4
0.5 F 119 402 323 79 430 286 144 413 260 153 426 270 156 437 282 155 40.5 F 126 399 299 100 428 281 147 407 258 149 429 249 180 402 289 113 40.5 F 120 401 325 76 427 322 105 420 354 66 429 332 97 424 296 128 40.5 F 125 408 289 119 416 307 109 411 284 127 432 379 53 421 333 88 40.5 F 118 407 327 80 420 307 113 417 253 164 423 251 172 411 306 105 40.5 F 141 395 299 96 427 324 103 396 288 108 424 284 140 433 269 164 40.5 F 138 414 356 58 411 339 72 424 296 128 425 276 149 417 276 141 40.5 F 144 414 310 104 420 304 116 417 273 144 424 247 177 413 296 117 40.5 F 135 401 301 100 424 316 108 417 318 99 418 296 122 425 284 141 40.5 F 143 401 281 120 412 305 107 423 263 160 434 271 163 430 291 139 40.5 F 127 408 296 112 423 323 100 423 311 112 429 284 145 426 278 148 40.5 F 147 403 262 141 418 311 107 405 271 134 420 249 171 417 276 141 40.5 F 137 398 276 122 427 313 114 407 278 129 417 0.5 F 148 397 307 90 411 297 114 408 272 136 420 273 147 422 267 155 40.5 F 129 401 272 129 421 285 136 406 245 161 413 296 117 429 293 136 40.5 F 153 395 271 124 418 288 130 421 253 168 435 241 194 419 275 144 40.5 M 155 410 328 82 415 335 80 424 332 92 406 302 104 421 326 95 40.5 M 161 407 312 95 427 323 104 412 323 89 426 310 116 432 326 106 40.5 M 133 412 326 86 421 317 104 420 283 137 425 289 136 421 306 115 40.5 M 123 412 359 53 432 353 79 402 293 109 420 326 94 432 341 91 40.5 M 142 418 334 84 418 310 108 425 313 112 425 328 97 416 312 104 40.5 M 146 405 328 77 417 320 97 416 305 111 436 299 137 420 294 126 40.5 M 132 408 328 80 424 322 102 429 312 117 418 299 119 423 310 113 40.5 M 121 407 344 63 424 330 94 295 320 -25 420 302 118 425 317 108 40.5 M 122 424 349 75 418 332 86 424 318 106 430 316 114 424 331 93 40.5 M 128 416 301 115 419 295 124 412 290 122 425 276 149 419 295 124 40.5 M 145 402 283 119 412 334 78 415 318 97 430 313 117 421 327 94 40.5 M 156 405 310 95 415 337 78 415 321 94 421 320 101 426 329 97 40.5 M 151 406 311 95 424 311 113 421 304 117 421 295 126 420 294 126 40.5 M 157 408 302 106 427 338 89 426 332 94 423 314 109 432 315 117 40.5 M 152 405 306 99 421 307 114 415 316 99 401 287 114 419 305 114 4
0.5 M 140 383 307 76 421 312 109 410 291 119 421 297 124 433 306 127 40.5 M 160 412 305 107 420 323 97 416 301 115 419 312 107 416 319 97 40.5 M 190 413 298 115 437 303 134 420 303 117 425 285 140 418 306 112 40.5 M 186 410 294 116 438 347 91 428 339 89 422 333 89 425 314 111 40.5 M 188 414 299 115 427 352 75 432 327 105 422 305 117 445 326 119 4

5 F 100 406 325 81 426 334 92 423 283 140 423 258 165 429 315 114 45 F 120 405 316 89 403 322 81 421 294 127 431 276 155 433 268 165 45 F 104 398 310 88 427 314 113 419 266 153 419 257 162 420 259 161 45 F 114 406 279 127 427 283 144 417 245 172 423 262 161 419 270 149 45 F 123 401 293 108 417 302 115 408 266 142 418 255 163 410 289 121 45 F 113 406 306 100 438 322 116 414 280 134 418 243 175 424 270 154 45 F 130 409 311 98 426 312 114 422 280 142 420 249 171 432 289 143 45 F 140 417 300 117 417 303 114 426 263 163 427 244 183 419 276 143 45 F 105 408 317 91 428 309 119 424 269 155 424 230 194 426 244 182 45 F 106 414 320 94 421 275 146 414 242 172 418 281 137 431 299 132 45 F 107 403 295 108 412 332 80 416 294 122 425 250 175 406 284 122 45 F 111 407 315 92 414 325 89 424 232 192 417 233 184 418 255 163 45 F 93 409 286 123 425 337 88 412 278 134 424 259 165 415 276 139 45 F 97 408 318 90 408 317 91 412 276 136 426 271 155 419 288 131 45 F 91 410 418 -8 432 342 90 430 337 93 426 267 159 409 246 163 45 F 96 405 316 89 432 341 91 423 312 111 417 259 158 408 276 132 45 F 115 402 310 92 417 309 108 419 231 188 412 251 161 425 264 161 45 F 148 414 321 93 423 293 130 422 263 159 434 335 99 429 343 86 45 F 151 407 290 117 424 319 105 420 281 139 426 291 135 419 301 118 45 F 155 497 293 204 416 280 136 420 232 188 410 281 129 434 289 145 45 M 133 403 304 99 427 317 110 415 281 134 427 302 125 426 319 107 45 M 131 399 305 94 428 317 111 427 305 122 417 284 133 434 320 114 45 M 99 398 295 103 417 331 86 431 334 97 407 310 97 433 309 124 45 M 122 393 282 111 418 305 113 420 302 118 429 310 119 437 310 127 45 M 102 411 307 104 433 326 107 418 316 102 413 308 105 424 318 106 45 M 103 394 293 101 428 333 95 419 308 111 418 302 116 432 305 127 4
5 M 118 416 304 112 426 332 94 442 329 113 415 306 109 430 309 121 45 M 134 396 302 94 431 307 124 426 292 134 409 277 132 421 299 122 45 M 108 405 317 88 418 305 113 425 304 121 416 300 116 416 324 92 45 M 110 406 303 103 418 315 103 422 313 109 397 292 105 429 318 111 45 M 109 407 296 111 420 302 118 429 301 128 414 274 140 421 297 124 45 M 112 409 312 97 422 328 94 401 286 115 415 297 118 449 331 118 45 M 119 412 303 109 426 309 117 425 314 111 421 309 112 424 332 92 45 M 95 397 332 65 422 330 92 429 320 109 431 321 110 441 340 101 45 M 94 404 313 91 410 331 79 424 312 112 411 301 110 420 312 108 45 M 117 404 298 106 426 339 87 434 313 121 412 290 122 433 315 118 45 M 101 409 296 113 425 301 124 434 312 122 402 273 129 407 307 100 45 M 98 408 320 88 413 299 114 428 313 115 395 273 122 434 301 133 45 M 121 398 288 110 424 316 108 427 317 110 410 300 110 426 318 108 45 M 116 409 321 88 421 343 78 427 336 91 397 299 98 437 322 115 4

10 F 44 406 315 91 422 346 76 420 313 107 412 264 148 431 299 132 410 F 50 409 328 81 403 296 107 421 283 138 406 242 164 419 245 174 410 F 41 406 307 99 413 316 97 405 292 113 415 264 151 433 269 164 410 F 44 410 322 88 414 346 68 414 299 115 428 244 184 415 277 138 410 F 52 498 315 183 420 335 85 417 315 102 423 279 144 416 297 119 410 F 57 410 302 108 419 310 109 418 265 153 423 261 162 426 268 158 410 F 85 403 289 114 421 288 133 419 271 148 423 275 148 430 291 139 410 F 88 405 299 106 406 306 100 414 253 161 413 250 163 421 293 128 410 F 82 395 258 137 417 315 102 425 244 181 422 243 179 431 244 187 410 F 83 405 295 110 413 333 80 426 300 126 419 284 135 424 291 133 410 F 69 405 302 103 430 336 94 432 277 155 426 305 121 421 293 128 410 F 84 402 324 78 437 335 102 422 290 132 419 283 136 430 291 139 410 F 60 409 332 77 413 333 80 426 301 125 433 312 121 407 255 152 410 F 63 403 331 72 430 297 133 424 286 138 426 250 176 417 258 159 410 F 80 489 274 215 424 307 117 415 256 159 425 243 182 415 258 157 410 F 87 406 325 81 428 312 116 421 252 169 427 248 179 426 289 137 410 F 71 402 283 119 415 264 151 412 254 158 415 244 171 411 283 128 4
10 F 70 406 314 92 418 337 81 420 294 126 429 276 153 421 273 148 410 F 77 399 304 95 428 332 96 444 320 124 393 253 140 432 264 168 410 F 79 412 301 111 416 311 105 416 260 156 434 283 151 433 291 142 410 M 47 411 327 84 411 321 90 418 301 117 415 303 112 406 270 136 410 M 42 407 304 103 420 340 80 426 341 85 399 304 95 445 269 176 410 M 72 413 339 74 424 332 92 421 306 115 417 298 119 426 301 125 410 M 89 413 307 106 417 300 117 423 311 112 408 295 113 432 315 117 410 M 67 407 321 86 419 320 99 426 305 121 418 289 129 410 310 100 410 M 66 416 328 88 415 330 85 430 332 98 407 283 124 437 322 115 410 M 58 412 296 116 420 304 116 424 301 123 421 300 121 445 327 118 410 M 51 405 302 103 422 317 105 435 294 141 423 296 127 429 315 114 410 M 54 413 310 103 410 295 115 428 305 123 418 313 105 421 322 99 410 M 65 405 305 100 424 306 118 423 305 118 411 299 112 420 330 90 410 M 49 378 306 72 418 303 115 426 312 114 408 304 104 417 319 98 410 M 46 406 295 111 414 310 104 421 328 93 413 336 77 430 313 117 410 M 55 408 313 95 422 339 83 420 283 137 413 269 144 430 306 124 410 M 45 392 292 100 417 326 91 414 320 94 405 307 98 432 331 101 410 M 53 412 311 101 419 320 99 427 316 111 397 288 109 428 323 105 410 M 59 411 288 123 416 320 96 419 293 126 416 269 147 427 311 116 410 M 61 411 344 67 419 342 77 425 338 87 389 289 100 437 323 114 410 M 56 414 300 114 416 324 92 419 292 127 384 301 83 448 320 128 410 M 62 411 299 112 418 302 116 450 330 120 419 312 107 431 325 106 410 M 61 406 290 116 414 311 103 433 319 114 419 300 119 446 325 121 4
F2 bird weights 

ppm Sex 

Baby ID 6/9/11 ID 6/18/11 6/26/11 7/5/11 7/12/11 7/18/11 7/26/11 8/2/11 8/9/11 8/16/11 8/22/11 8/29/11 9/7/11 

0 F 11 254 40 86 95 104 114 125 131 120 128 130 130 0 F 10 257 36 83 99 105 113 120 129 126 139 141 142 0 F 12 258 38 97 102 110 124 133 117 133 131 133 0 F 7 259 38 98 103 111 114 111 114 117 131 121 0 F 24 281 35 88 101 105 126 142 146 131 147 150 140 0 F 21 283 25.9 76 90 91 101 110 107 112 115 134 119 0 F 26 284 41 94 98 106 115 138 137 144 144 147 150 0 F 17 286 35 88 103 103 114 124 121 146 140 139 139 0 F 29 413 74 98 100 114 137 136 123 140 145 144 0 F 41 415 83 104 109 117 121 130 148 145 156 154 0 F 35 417 81 97 88 110 122 119 135 138 138 123 0 F 47 420 79 94 99 95 115 113 120 121 121 129 0 F 57 6.1 429 96 105 120 138 134 125 135 139 130 0 F 60 5.9 437 94 103 115 131 139 136 141 143 122 0 F 67 440 38 104 115 138 119 139 124 123 119 0 F 89 445 47 111 117 128 142 148 143 145 141 0 F 70 453 37 93 107 118 126 124 124 125 126 0 F 105 454 43 102 114 126 128 131 133 140 125 0 F 88 457 40 105 114 127 139 130 121 130 131 0 F 97 458 38 96 105 124 127 118 120 130 120 0 M 14 251 33.2 78 91 95 107 105 101 100 104 106 0 M 9 252 37.4 82 95 99 107 106 106 106 107 110 0 M 15 253 33.3 84 95 102 108 114 111 112 110 109 0 M 13 255 40.2 92 105 118 123 126 121 118 117 116 0 M 8 256 36 87 100 98 106 114 116 119 121 118 0 M 25 260 40 90 98 104 111 109 108 110 113 0 M 19 282 37.9 84 91 95 103 105 106 104 107 108 0 M 20 285 40 87 96 98 104 112 109 113 114 110 0 M 27 287 40 95 101 105 107 108 111 109 112 
0 M 22 288 30 81 95 99 107 115 114 114 115 114 

0 M 16 289 34 93 96 102 108 106 106 108 112 0 M 23 290 39 93 95 106 109 108 107 103 104 0 M 43 412 74 102 105 108 107 109 110 111 0 M 42 416 94 85 107 111 108 110 114 112 0 M 37 418 91 93 102 106 105 106 106 106 0 M 46 419 85 89 92 92 91 91 93 94 0 M 48 422 86 82 91 96 98 100 104 107 0 M 28 424 85 88 92 100 99 100 97 109 0 M 44 425 96 102 113 119 117 119 120 121 0 M 45 426 88 96 98 99 101 105 105 101 

0.5 F 160 118 41 94 106 118 120 126 138 139 134 134 118 0.5 F 171 119 41 87 102 113 123 128 148 142 148 151 149 0.5 F 165 120 38 86 102 111 117 119 117 113 114 131 134 0.5 F 168 125 31 78 97 109 108 114 117 134 93 106 109 0.5 F 167 126 37.5 86 97 97 107 124 119 128 122 120 132 0.5 F 159 127 41.8 88 97 107 111 122 121 128 134 124 133 0.5 F 16 129 41.9 97 104 113 132 131 139 119 129 128 0.5 F 173 130 42.5 97 109 119 138 141 139 145 139 147 138 0.5 F 182 131 35.5 84 90 90 101 117 134 132 118 134 125 0.5 F 183 134 40 86 98 105 123 131 128 109 129 129 126 0.5 F 181 135 40.1 107 111 119 126 126 134 147 148 145 0.5 F 179 136 33.1 83 97 102 101 122 138 137 142 137 141 0.5 F 174 137 42 104 108 131 142 121 90 0.5 F 186 138 84 88 86 116 131 133 130 136 131 0.5 F 194 141 99 99 111 116 113 121 142 136 130 0.5 F 196 143 96 95 107 125 126 128 126 131 118 0.5 F 192 144 91 94 105 128 128 132 133 131 126 0.5 F 190 147 102 102 116 140 126 137 136 143 137 0.5 F 193 148 89 92 98 106 129 125 130 132 0.5 F 205 7.5 153 101 109 120 141 146 135 151 134 149 
0.5 M 163 121 39.2 83 96 107 108 105 103 103 106 108 

0.5 M 172 122 38.5 84 95 103 104 106 106 108 109 109 0.5 M 166 123 29.2 70 82 90 94 90 86 93 94 96 0.5 M 162 128 41.8 103 109 114 117 117 121 122 122 0.5 M 178 132 33 79 91 96 102 105 106 108 108 113 0.5 M 127 133 35 79 88 89 100 102 102 105 110 110 0.5 M 185 140 79 97 97 109 114 114 115 118 117 0.5 M 195 142 75 90 92 101 103 103 103 102 105 0.5 M 197 145 71 84 92 95 95 96 99 95 98 0.5 M 188 146 91 91 99 102 101 104 106 106 0.5 M 206 6.2 151 101 108 110 114 114 110 116 118 0.5 M 204 6.7 152 70 85 88 102 104 103 104 106 106 0.5 M 203 6.2 155 87 93 96 97 94 96 97 98 0.5 M 202 6.6 156 85 97 107 119 118 113 113 112 112 0.5 M 200 6.7 157 85 91 106 108 101 100 101 103 0.5 M 122 160 88 93 96 95 95 98 99 0.5 M 219 7.2 161 90 85 99 104 101 101 104 103 0.5 M 249 186 43 98 102 112 109 106 106 108 0.5 M 248 188 35 96 109 118 106 103 106 107 0.5 M 150 190 36 93 103 108 110 111 117 116 

5 F 21 91 35.8 85 94 100 112 122 120 127 135 122 131 5 F 17 93 39 91 106 106 124 135 137 144 142 143 142 5 F 22 96 33.8 87 102 107 124 134 134 139 140 139 143 5 F 19 97 35 79 91 96 109 116 118 124 117 125 116 5 F 18 100 33 92 102 108 117 132 127 140 139 132 135 5 F 20 104 41 104 104 122 131 128 133 137 140 138 5 F 37 105 40.5 91 102 109 117 136 124 124 137 133 140 5 F 32 106 34.8 83 93 103 114 132 127 136 108 124 134 5 F 38 107 39.8 92 106 111 128 138 135 148 144 142 142 5 F 36 111 41 93 105 108 122 138 139 138 139 141 145 5 F 44 113 41 103 99 110 119 134 145 143 153 155 
5 F 43 114 42 108 110 136 131 143 136 135 138 135 

5 F 54 115 44 118 121 147 153 140 153 155 5 F 50 120 91 94 104 110 129 118 130 126 108 5 F 62 123 92 92 101 123 121 127 120 125 129 5 F 86 6 130 95 103 118 143 142 141 132 142 145 5 F 78 140 89 90 102 129 123 131 131 134 125 5 F 94 7.1 148 117 124 138 144 142 113 120 150 5 F 106 151 45 96 98 107 125 128 116 111 125 5 F 27 155 43 106 130 142 130 128 130 150 5 F 29 158 42 105 109 109 108 5 M 16 94 30.4 76 90 93 101 107 106 106 109 110 5 M 31 95 40.8 93 106 114 110 109 105 116 117 118 5 M 26 98 39 87 89 104 100 109 109 95 95 97 5 M 30 99 33.6 76 90 91 96 97 108 102 103 103 5 M 23 101 31 77 88 89 94 101 99 106 108 109 5 M 42 102 37 96 95 104 107 95 103 107 114 5 M 34 103 40 96 97 101 98 112 124 124 121 5 M 40 108 39 93 110 115 119 119 122 105 105 106 5 M 39 109 40 83 96 102 107 105 102 110 114 123 5 M 41 110 35 77 90 95 101 109 109 109 110 108 5 M 47 112 40 90 97 103 109 109 106 114 113 116 5 M 48 116 39 99 104 116 115 114 104 106 107 5 M 53 117 70 85 85 96 100 102 117 120 124 5 M 52 118 102 104 114 119 118 105 104 104 5 M 58 119 75 89 89 98 102 102 118 119 120 5 M 68 121 101 104 110 116 118 111 112 114 5 M 59 122 97 102 105 109 100 100 102 105 5 M 67 6.7 127 105 122 125 127 5 M 66 6.3 131 94 98 101 99 99 112 114 109 

10 F 40 41 40 91 103 109 117 124 124 137 147 131 151 10 F 36 44 38 90 105 110 119 123 127 137 135 138 138 
10 F 52 48 35 98 104 114 116 125 131 121 132 136 

10 F 47 50 42 94 111 114 126 135 139 140 137 133 143 10 F 45 52 41 99 103 111 119 126 132 131 139 140 10 F 53 57 100 99 112 128 127 131 137 140 135 10 F 62 7.1 60 82 98 106 117 119 121 135 115 135 139 10 F 59 6.4 63 86 104 105 122 136 125 134 137 136 136 10 F 71 7.5 69 84 111 109 115 112 150 125 147 142 10 F 65 5.9 70 77 103 102 106 112 137 136 132 138 10 F 69 6.7 71 92 108 141 133 141 137 134 139 10 F 66 6.3 77 108 108 119 127 125 120 133 139 10 F 185 79 47 105 114 125 139 139 142 135 141 10 F 189 80 46 108 119 132 145 141 141 138 130 10 F 184 82 44 103 114 137 129 144 140 146 143 10 F 183 83 50 117 131 138 135 147 159 157 159 10 F 187 84 38 97 102 101 113 127 108 121 132 10 F 188 85 44 110 115 140 138 129 146 138 134 10 F 96 87 51 84 121 127 135 143 140 142 148 146 10 F 93 88 45 108 120 141 130 140 138 137 142 10 M 95 42 37.9 83 97 96 106 113 109 110 106 109 10 M 37 45 34.6 88 92 96 99 99 99 102 106 10 M 41 46 36 88 97 97 102 109 111 110 105 109 10 M 43 47 40 92 104 107 108 107 107 108 107 114 10 M 44 49 37 85 99 106 110 114 114 115 117 118 10 M 51 51 40 98 97 104 107 108 110 113 113 10 M 42 53 39.5 95 98 102 104 104 103 103 108 10 M 46 54 36 95 95 102 109 111 116 114 117 10 M 49 55 80 94 95 101 105 103 108 108 111 10 M 58 56 74 91 92 99 103 103 105 106 110 10 M 57 58 75 93 94 104 108 110 114 115 114 10 M 55 59 89 85 89 103 108 106 111 115 112 10 M 54 6 61 80 96 109 116 110 110 109 108 111 
10 M 80 6.8 62 77 91 93 99 105 107 107 107 109 

10 M 64 6 64 85 97 95 103 109 108 109 108 112 10 M 60 65 96 97 106 110 112 115 115 118 10 M 80 6 66 103 102 102 102 110 110 112 10 M 72 67 99 102 109 107 110 110 113 10 M 79 72 50 111 117 114 111 113 109 115 10 M 75 89 44 114 115 110 112 113 111 114 

F2 sperm measurements ppm ID Motile 1 Motile 2 Motile Average Progressive 1 Progressive 2 Progressive Average Sperm/gram 1 Sperm/gram 2 Sperm/gram Avera0 251 23 34 28.5 4 9 6.5 146.4 258.8 202.6 0 252 14 30 22 4 6 5 222.2 305 263.6 0 260 10 12 11 3 3 3 74.7 146.9 110.8 0 282 9 8 8.5 2 1 1.5 316.4 379.7 348.05 0 287 15 47 31 3 11 7 212 386.3 299.15 0 288 50 65 57.5 16 17 16.5 298 347.7 322.85 0 290 28 34 31 10 19 14.5 303.2 386.9 345.05 0 416 48 53 50.5 11 16 13.5 334.6 470.5 402.55 0 422 28 42 35 3 18 10.5 139.4 217.8 178.6 0 426 22 18 20 3 2 2.5 161.2 213.5 187.35 0.5 121 26 28 27 6 4 5 273.5 273.5 273.5 0.5 122 46 23 34.5 21 11 16 305 204.8 254.9 0.5 128 46 39 42.5 17 17 17 153.1 153.1 153.1 
0.5 133 28 25 26.5 13 21 17 152.1 114.1 133.1 

0.5 140 36 36 36 16 7 11.5 181 237.3 209.15 0.5 145 51 62 56.5 15 19 17 239.6 274.5 257.05 0.5 152 47 44 45.5 6 13 9.5 139.4 196.1 167.75 0.5 155 15 9 12 5 1 3 190.7 264.5 227.6 0.5 160 63 55 59 14 13 13.5 256.2 209.1 232.65 0.5 161 55 32 43.5 17 12 14.5 279.7 248.3 264 5 94 39 42 40.5 9 12 10.5 115 90.6 102.8 5 99 37 42 39.5 13 14 13.5 267.9 235.3 251.6 5 101 17 4 10.5 0 0 0 137.8 123.6 130.7 5 108 31 28 29.5 11 3 7 140.8 128.7 134.75 5 109 55 73 64 26 30 28 168.9 309.7 239.3 5 110 32 39 35.5 4 11 7.5 244 180.1 212.05 5 117 37 38 37.5 6 14 10 283.2 252.7 267.95 5 131 19 17 18 4 6 5 90.6 62.7 76.65 5 133 0 0 0 0 0 0 134.4 181.7 158.05 5 134 31 36 33.5 13 12 12.5 59.8 123.2 91.5 10 42 47 27 37 7 7 7 112 112 112 10 47 47 29 38 9 8 8.5 144.5 147.6 146.05 10 49 29 57 43 7 23 15 213.9 166.4 190.15 10 51 48 37 42.5 11 4 7.5 217.8 148.1 182.95 10 53 46 59 52.5 16 18 17 277.5 205.1 241.3 10 54 49 30 39.5 17 5 11 140.8 172.9 156.85 10 59 38 36 37 17 14 15.5 125.5 115 120.25 10 61 74 64 69 25 22 23.5 230.9 196.1 213.5 10 65 30 26 28 8 5 6.5 257.9 257.9 257.9 10 72 61 54 57.5 18 14 16 162.1 180.4 171.25 
Toxicology Study No. 87-XE-0FV5-12, Protocol No. 0FV5-96-12-05-01, June-August 2012 

9 

F2 cloaca measurements 

Ppm ID 7/12/2011 7/21/2011 7/27/2011 8/5/2011 

0 285 81 196 240 196 

0 282 25 90 225 240 

0 260 25 49 156 210 

0 288 56 49 143 289 

0 252 63 110 225 210 

0 251 25 121 256 240 

0 422 72 30 80 64 

0 255 56 168 255 289 

0 419 121 156 196 255 

0 418 49 56 90 196 

0 424 25 36 64 64 

0 253 64 169 196 240 

0 289 90 195 182 252 

0 256 99 64 182 255 

0 425 90 56 143 224 

0 287 90 88 240 288 

0 426 81 132 210 224 

0 290 143 120 169 255 

0 416 42 25 49 80 

0 412 110 224 306 324 

0.5 133 42 72 169 324 

0.5 132 64 144 169 289 

0.5 121 99 195 210 255 

0.5 123 56 132 195 240 

0.5 142 72 64 80 100 

0.5 122 100 169 240 224 

0.5 128 72 144 180 306 

0.5 146 64 100 182 240 

0.5 155 72 132 225 225 

0.5 152 25 72 180 240 

0.5 151 49 182 240 288 

0.5 145 25 36 81 224 

0.5 156 25 49 144 240 

0.5 157 25 121 225 272 

0.5 140 81 72 90 182 

0.5 161 72 36 49 64 
193 

5 108 100 270 240 240 

5 109 72 240 306 342 

5 110 25 25 81 342 

5 112 54 100 182 238 

5 94 54 169 256 288 

5 119 90 110 210 256 

5 95 99 240 270 360 

5 117 72 132 240 256 

5 101 56 132 221 270 

5 99 40 90 240 289 

5 122 90 100 169 210 

5 98 64 99 156 255 

5 121 110 72 234 306 

5 102 121 156 224 289 

5 116 81 90 121 240 

5 103 81 81 132 225 

5 118 80 30 100 169 

5 133 35 110 195 270 

5 131 72 56 195 196 

5 134 64 120 195 306 

10 49 72 144 168 304 

10 47 72 143 224 324 

10 46 64 81 165 289 

10 42 42 72 81 272 

10 58 64 132 182 270 

10 55 72 100 208 240 

10 45 121 168 306 306 

10 51 121 108 182 238 

10 53 120 169 240 289 

10 54 90 90 195 272 

10 59 25 72 100 144 

10 65 90 132 224 380 

10 61 90 195 240 324 

10 56 81 56 25 30 

10 64 25 99 225 270 

10 62 56 121 224 306 
194 

F2 onset to pubery 

Trenbolone (ppm) ID first day of foam day of onset 

0 251 7/26/2011 50 

0 252 7/27/2011 51 

0 253 7/18/2011 42 

0 255 7/22/2011 46 

0 256 7/29/2011 53 

0 260 7/27/2011 50 

0 282 7/25/2011 48 

0 285 7/19/2011 42 

0 287 7/25/2011 48 

0 288 7/28/2011 51 

0 289 7/20/2011 43 

0 290 7/20/2011 43 

0 412 7/18/2011 40 

0 416 8/8/2011 61 

0 418 8/3/2011 56 

0 419 7/20/2011 42 

0 422 8/16/2011 76 

0 424 

0 425 7/28/2011 50 

0 426 7/23/2011 45 

0.5 121 7/19/2011 43 

0.5 122 7/21/2011 45 

0.5 123 7/21/2011 45 

0.5 128 7/22/2011 46 

0.5 132 7/21/2011 44 

0.5 133 

0.5 140 7/30/2011 53 

0.5 142 8/9/2011 46 

0.5 145 7/29/2011 51 

0.5 146 7/25/2011 47 

0.5 151 

0.5 152 7/26/2011 47 

0.5 155 7/19/2011 40 

0.5 156 7/29/2011 50 
195 

0.5 157 7/19/2011 40 

0.5 160 7/19/2011 39 

0.5 161 8/8/2011 59 

0.5 186 8/16/2011 64 

0.5 188 7/31/2011 48 

0.5 190 7/28/2011 45 

5 94 7/22/2011 46 

5 95 7/20/2011 44 

5 98 7/27/2011 51 

5 99 7/26/2011 50 

5 101 7/21/2011 45 

5 102 7/20/2011 44 

5 103 7/29/2011 53 

5 108 7/17/2011 40 

5 109 7/17/2011 45 

5 110 8/1/2011 54 

5 112 7/25/2011 48 

5 116 7/30/2011 53 

5 117 7/22/2011 45 

5 118 8/9/2011 63 

5 119 7/25/2011 47 

5 121 7/27/2011 49 

5 122 7/26/2011 48 

5 131 7/27/2011 48 

5 133 7/23/2011 44 

5 134 7/27/2011 48 

10 42 8/2/2011 57 

10 45 7/19/2011 43 

10 46 7/28/2011 51 

10 47 7/21/2011 44 

10 49 7/23/2011 46 

10 51 7/26/2011 49 

10 53 7/19/2011 42 

10 54 7/28/2011 51 

10 55 7/22/2011 43 

10 56 8/1/2011 53 

10 58 7/25/2011 48 

10 59 
196 

10 61 7/19/2011 43 

10 62 7/21/2011 45 

10 64 7/24/2011 48 

10 65 7/25/2011 44 

10 66 7/29/2011 49 

10 67 7/20/2011 44 

10 72 7/22/2011 42 

10 89 7/26/2011 43 

Trenbolone (ppm) ID Thickness Thickness Force 

0 254 0.203 0.198 1.034 

0 254 0.194 0.195 1.052 

0 257 0.196 0.217 1.412 

0 257 0.219 0.22 1.308 

0 258 0.176 0.197 1.243 

0 258 0.205 0.198 1.452 

0 259 0.169 0.168 0.694 

0 259 0.171 0.168 0.282 

0 281 0.205 0.218 1.015 

0 281 0.217 0.213 2.148 

0 283 0.179 0.182 0.65 

0 283 0.194 0.194 0.41 

0 284 0.229 0.244 1.908 

0 284 0.237 0.254 1.694 

0 286 0.205 0.224 1.266 

0 286 0.182 0.198 1.266 

0 413 0.214 0.219 1.075 

0 413 0.235 0.227 1.444 

0 415 0.212 0.211 1.512 

0 415 0.224 0.217 1.368 

0 417 0.203 0.203 0.916 

0 417 0.199 0.198 1.068 

0 420 0.174 0.181 0.917 

0 420 0.174 0.168 1.274 

0 429 0.212 0.225 1.047 

0 429 0.215 0.198 1.008 

0 437 0.206 0.202 0.939 
197 

0 437 0.213 0.214 0.949 

0 440 0.182 0.202 0.754 

0 440 0.184 0.187 0.789 

0 445 0.191 0.191 1.022 

0 445 0.199 0.198 1.145 

0 453 0.171 0.18 1.476 

0 453 0.165 0.18 1.005 

0 454 0.197 0.201 1.38 

0 454 0.26 0.198 1.451 

0 457 0.151 0.149 1.092 

0 457 0.174 0.169 1.21 

0 458 0.221 0.232 1.525 

0 458 0.229 0.213 0.93 

0.5 118 0.143 0.144 0.337 

0.5 118 0.189 0.189 0.98 

0.5 119 0.202 0.206 1.114 

0.5 119 0.198 0.196 1.416 

0.5 120 0.182 0.187 0.941 

0.5 120 0.174 0.18 0.822 

0.5 126 0.181 0.197 1.12 

0.5 126 0.19 0.19 0.73 

0.5 127 0.231 0.219 1.199 

0.5 127 0.241 0.217 1.423 

0.5 129 0.185 0.19 0.932 

0.5 129 0.187 0.23 1.037 

0.5 130 0.197 0.206 1.586 

0.5 130 0.195 0.195 1.618 

0.5 131 0.174 0.171 0.953 

0.5 131 0.182 0.173 1.004 

0.5 134 0.191 0.178 0.877 

0.5 134 0.172 0.181 1.057 

0.5 135 0.202 0.186 0.949 

0.5 135 0.194 0.186 0.838 

0.5 136 0.217 0.255 1.446 

0.5 136 0.222 0.229 1.519 

0.5 138 0.256 0.232 0.874 

0.5 138 0.225 0.236 0.923 

0.5 141 0.193 0.198 0.62 
198 

0.5 141 0.223 0.209 0.766 

0.5 143 0.205 0.187 0.706 

0.5 143 0.214 0.213 0.872 

0.5 144 0.202 0.197 0.777 

0.5 144 0.191 0.195 0.918 

0.5 147 0.198 0.192 0.958 

0.5 147 0.21 0.205 1.301 

0.5 148 0.205 0.212 1.173 

0.5 148 0.192 0.209 0.967 

0.5 153 0.236 0.208 1.106 

0.5 153 0.175 0.158 0.379 

5 91 0.177 0.191 0.835 

5 91 0.175 0.158 0.889 

5 93 0.196 0.216 0.894 

5 93 0.212 0.201 1.073 

5 96 0.185 0.203 0.91 

5 96 0.185 0.191 1.088 

5 97 0.186 0.181 1.327 

5 97 0.181 0.19 1.335 

5 100 0.175 0.169 1.184 

5 100 0.202 0.194 0.779 

5 104 0.23 0.244 0.892 

5 104 0.236 0.236 1.077 

5 105 0.212 0.199 0.879 

5 105 0.16 0.166 0.799 

5 106 0.182 0.181 0.844 

5 106 0.193 0.198 0.723 

5 107 0.185 0.187 0.845 

5 107 0.183 0.178 1.167 

5 111 0.19 0.192 1.211 

5 111 0.191 0.187 1.262 

5 113 0.214 0.217 1.114 

5 113 0.223 0.218 1.234 

5 114 0.23 0.228 1.419 

5 114 0.206 0.219 1.837 

5 115 0.202 0.198 1.065 

5 115 0.226 0.209 1.315 

5 120 0.229 0.231 1.013 
199 

5 120 0.239 0.236 1.154 

5 123 0.225 0.22 1.283 

5 123 0.249 0.211 1.111 

5 130 0.209 0.201 0.965 

5 130 0.199 0.205 1.207 

5 140 0.254 0.244 1.104 

5 140 0.221 0.231 0.892 

5 148 0.18 0.196 1.267 

5 148 0.197 0.215 0.981 

5 151 0.195 0.186 1.125 

5 151 0.191 0.195 1.097 

5 155 0.171 0.171 1.07 

5 155 0.203 0.215 1.159 

10 41 0.223 0.222 0.506 

10 41 0.22 0.235 0.641 

10 44 0.228 0.219 1.741 

10 44 0.24 0.234 1.262 

10 48 0.203 0.204 1.162 

10 48 0.208 0.188 1.103 

10 50 0.183 0.197 1.163 

10 50 0.194 0.193 1.23 

10 52 0.22 0.214 0.995 

10 52 0.223 0.217 1.126 

10 60 0.197 0.214 1.173 

10 60 0.201 0.209 1.172 

10 63 0.214 0.199 1.285 

10 63 0.208 0.205 1.404 

10 69 0.168 0.194 0.82 

10 69 0.175 0.177 0.63 

10 70 0.187 0.203 1.424 

10 70 0.212 0.202 1.501 

10 71 0.235 0.217 1.282 

10 71 0.223 0.234 1.062 

10 77 0.222 0.23 1.525 

10 77 0.199 0.208 1.277 

10 79 0.268 0.226 1.152 

10 79 0.261 0.231 1.449 

10 80 0.203 0.204 0.921 
200 

10 80 0.163 0.164 0.918 

10 82 0.207 0.246 1.174 

10 82 0.209 0.22 1.344 

10 83 0.196 0.203 1.262 

10 83 0.189 0.205 1.279 

10 0.187 0.189 1.408 

10 84 0.181 0.184 1.044 

10 85 0.226 0.224 1.147 

10 85 0.204 0.201 1.021 

10 87 0.19 0.183 1.414 

10 87 0.179 0.187 1.525 

10 88 0.194 0.189 0.978 

10 88 0.198 0.188 0.817