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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 Endocri ne 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 B eall, 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 perfo rming the statistics for this study. This study was done concurrently with two other si milar ones by U.S. Geological Survey,

Patuxent Wildlife Research Center and Wild life 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 grat eful 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 conduc ted in compliance with Title 40, Code of Federal Regulations (CFR), Part 792, Good Laboratory Practi ce 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 aforem entioned regulation affected the quality or int egrity 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. REFERENC ES ............................................................................................. 1

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

3. AUTHOR ITY ................................................................................................ 1

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

5. BACKGRO UND ........................................................................................... 1

6. MATERIALS A ND METHOD S ..................................................................... 2 6.1 Experimental Design ............................................................................. 2

6.2 Data Analysis ........................................................................................ 4 6.3 Quality Assuranc e ................................................................................. 4

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

7.1. Eggshell Thic kness and Str ength ............................................................ 4

7.2. Egg Pr oduction ........................................................................................ 5

7.3. Sp erm Measur es ..................................................................................... 6

7.4. Growth and Body Wei ght......................................................................... 8

7.5. Feed C onsumpti on .................................................................................. 9

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

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

7.8. Copulator y Behavior ................................................................................ 14

7.9. Organ We ights ........................................................................................ 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. DISCUSSI ON ............................................................................................... 21

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

10. POINT OF CO NTACT .................................................................................. 23

Appendices

A REFERENCES ............................................................................................. A-1
B QUALITY ASSURANCE ST ATEMENT ........................................................ B-1

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

D ANIMAL USE PROTOCOL WITH MODIFICATION S ................................... D-1

List of Tables

Table 1. List of Critical Ev ents ............................................................................. 4 Table 2. F0 Eggshell Th ickness and St rength ..................................................... 5 Table 3. F1 Eggshell Th ickness and St rength ..................................................... 5 Table 4. F2 Eggshell Th ickness and St rength ..................................................... 5 Table 5. F0 Egg Production ................................................................................. 6 Table 6. F1 Egg Production ................................................................................. 6 Table 7. F2 Egg Production ................................................................................. 6 Table 8. F0 Spe rm Measures .............................................................................. 7 Table 9. F1 Spe rm Measures .............................................................................. 7 Table 10. F2 Spe rm Measures ............................................................................ 8

Table 11. F0 Adult Body Wei ght .......................................................................... 8

Table 12. F1 Adult Body Wei ght .......................................................................... 8

Table 13. F2 Adult Body Wei ght .......................................................................... 9

Table 14. F0 Female Feed Consumpt ion ............................................................ 10

Table 15. F0 Male F eed Consumptio n ................................................................ 10 Table 16. F0 Paired Feed Consumption .............................................................. 10

Table 17. F1 Female Feed Consumpt ion ............................................................ 10

Table 18. F1 Male F eed Consumptio n ................................................................ 11 Table 19. F1 Paired Feed Consumption .............................................................. 11

Table 20. F2 Female Feed Consumpt ion ............................................................ 11

Table 21. F2 Male F eed Consumptio n ................................................................ 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 Attemp ts ............................................................ 15

Table 29. F0 Onse t to Mount ............................................................................... 15

Table 30. F0 Number of Su ccessful Copulat ions ................................................ 15

Table 31. F0 Onset to Su ccessful Copula tion ..................................................... 16

Table 32. F1 Number of Mount Attemp ts ............................................................ 16

Table 33. F1 Onse t to Mount ............................................................................... 17

Table 34. F1 Number of Su ccessful Copulat ions ................................................ 17

Table 35. F1 Onset to Su ccessful Copula tion ..................................................... 17

Table 36. F2 Number of Mount Attemp ts ............................................................ 18

Table 37. F2 Onse t to Mount ............................................................................... 18

Table 38. F2 Number of Su ccessful Copulat ions ................................................ 18

Table 39. F2 Onset to Su ccessful Copula tion ..................................................... 18

Table 40. F0 Female Organ Weight s .................................................................. 19

Table 41. F0 Male Organ Weight s ...................................................................... 19

Table 42. F1 Female Organ Weight s .................................................................. 19

Table 43. F1 Male Organ Weight s ...................................................................... 20

Table 44. F2 Female Organ Weight s .................................................................. 20

Table 45. F2 Male Organ Weight s ...................................................................... 20

Table 46. F2 Generation Adult Hormone Da ta .................................................... 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 Generat ions of Male Copulatory Behavior ........... 20

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

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

Two-Generation Test for Endocrine Di sruption 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 (A R) 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) (refer ences 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 Re search, Development and Engineering Command (RDECOM), Environmental Acquisition Logistics & Sustainment Program.



# 4 Scope

This report documents the conduct, findings, an d conclusions of using trenbolone to develop and assess a two-generation test for endoc rine 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 as sociated with exposure to putative endocrine modulating compounds. The avian two-generation assay serves to evaluate potential population effects of endocrine modulating compounds on myri ad avian species, and represents terrestrial, oviparous wildlife species in general. This st udy will use trenbolone, a synthetic androgen, to further evaluate the propo sed test method for its ability to det ect 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 Gui delines for avian reprodu ction 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 behav ior, 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 male s and 20 females to ensure that there were at least 12 breeding pairs in the parental generation co ntrol 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, Patu xent Wildlife Research Center, Laurel, MD. The test diets were mixed and assayed at Wildlife Inte rnational Ltd., Easton, MD, USA. Homogeneity and concentration verifications were made by USAC HPPM Directorate of Laboratory Services. Upon receipt of the first batch of treated feed, a ll doses were tested for concentration verification.

Once all treatment levels were found to be wi thin acceptable ranges, each subsequent batch of treated feed only required two randomly selected dose s 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 woul d be hatched for each test level. Once again,

sufficient numbers of chicks were needed to establis h at least 12 breeding pairs (20 is the goal) per F1 test level. , If a treatment level produced insu fficient eggs to set the minimum, that level was to be dropped from further analysis in the test. Opti mally, 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 embr yo 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 devel opment. 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 hat ching; 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 meas ured by an Egg Force Reader (Orka Technology Ltd.) that tests the breaking poi nt 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 se ctions 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. Observatio ns included mortality and clinical signs of toxicity such as lethargy, depression, wing droop, ruffl ed feathers, etc. Any injuries sustained and subsequent treatments were recorded. If an indivi dual 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. I mmediately following euthanasia, spermatozoa were released from a weighed section of epididymis by mincing the tissues with scissors into 200 μ l

buffered Roswell Park Memorial Institute-1640 me dium (Sigma Chemical, St. Louis, MO, USA) as described by McFarland et al., (2008). Two μ l of the suspension was loaded onto 20- μ m 4- chamber slides (Leja Products, Nieuw-Vennep, t he Netherlands) to determine cell concentration per gram epididymis, percent mo tility, and percent progressively motile using a IVOS Sperm Analyzer, Version 12.1 (Hamilton-Thor ne Research, Beverly, MA, USA).

Measurements of thyroid function include thyroid gland weight, adult glandular T4 content, and thyroid histology/histopathology. Blood samples colle cted 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 Link ed Immuno Sorbent Assay (ELISA) kit (Caymen). Details of their methodologies can be found in Appendix H.

Initiation of normal male sexual behavior is importan t for successful reproducti on. 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, numbe r of mount attempts made within the time limit (3 minutes), time taken to achieve a successf ul copulation, and number of successful cloacal contacts. Males and females of the F1 gener ation 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 G ood Laboratory Practice (GLP) regulations found in the Toxic Substances Control Act (TSCA): 40 CFR (Code of Federal Regulations) 792, plus amendments. All investigators and technician s adhered to The Guide for Care and Use of Laboratory Animals (U.S. Department of Health, E ducation, and Welfare Publication No. NIH 86-23, 1996).



# 6.2 Data Analysis

All observational data was recorded in the laborat ory 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 we re audited by the USAPHC) Qualit y 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 th an 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 

## F0 Generation ‐ 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 = .225 p

‐ value = .590

## 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 (T able 8). No other sperm characteristics were altered by trenbolone in this generation. Pe rcent 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 epididym is 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. F0 Generation Egg Production

GEE Estimated Marginal Means GEE Significance Tests

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. F1 Generation 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 epididym is 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 epididym is 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 = .52 0 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 = .39 0 p

‐ value = .030* p

‐ value = .066

* 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 th an 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 t he control and 5 ppm dose groups (Table 13). Males from the 0.5 ppm dose group had lower av erage body weights than all others from this generation.



# Table 11. Mean body weight from adult female and ma le F0 generation Japanese quail ( Coturnix japonica ) exposed to trenbolone.

Table 12. Mean body wei ght 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 F2 Generation ‐ 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 F0 Generation Male Body Weights

Estimated Marginal Means Significance Tests

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 Estimated Marginal Means Significance Tests 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 wei ght 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 le ss 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 ma les. 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 consumpt ion 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

Estimated Marginal Means Significance Tests

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 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 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 F0 Generation 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 F0 Generation 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 gl and area (mm) from F0 ge neration male Japanese quail ( Coturnix japonica ) exposed to trenbolone.

Table 23. Mean cloacal gl and area (mm) from F1 ge neration male Japanese quail ( Coturnix japonica ) exposed to trenbolone.

Table 24. Mean cloacal gl and area (mm) from F2 ge neration 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* 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

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 Estimated Marginal Means GLM Significance Tests

° 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. 

### F1 Generation 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 u naffected 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 Estimated Marginal Means GLM Significance Tests



## 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

0 P

‐ 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

## 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, ma les 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 co ntrols (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 wi thin 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. 

## F1 Generation 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 

## F2 Generation 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 ti me (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 c opulations 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 t est 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 beh avioral test (Table 32). Males from the 5 and 20 ppm dose groups had delayed onset to mounting behavi or compared to controls over all three days, and onset was also delayed in the 0.5 ppm dose group rela tive to controls on the first and second days (Table 33). F1 generation control males had the greatest nu mber 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 fewe r successes than control males. Onset to first successful copulation was only changed in the F1 gener ation on the third day of behavioral testing (Table 35). Males from the highest treatment level took lo nger to achieve a successf ul copulation than control

males.



# Table 32. Mean number of mount attempts initiated wi thin 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 ti me (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% F0 Generation ‐ Time to first Copulation

Day 1 Day 2 Day 3

Log rank p

‐ value = .00 9 Log rank p

‐ value = .031 Log rank p

‐ value = .03 7

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 = .002 Omnibus Likelihood ratio p

‐ value = .011 Omnibus Likelihood ratio p

‐ value = .000

* 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 c opulations 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 t est 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 rema ined unchanged by in ovo exposure to trenbolone in the F2 generation (Tables 36-39).



# Table 36. Mean number of mount attempts initiated wi thin 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 = .00 2 Log rank p

‐ value = .00 2 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 12.52 

### 23% 166.9 12.56 

### 7% All pairs failed to Copulate F1 Generation ‐ Time to first Copulation

Day 1 Day 2 Day 3

Log rank p

‐ value = .170 Log rank p

‐ value = .141 Log rank p

‐ value = .01 6

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

18 

# Table 37. Mean amount of ti me (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 c opulations 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 t est 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 = .465 Omnibus Likelihood ratio p

‐ value = .722 Omnibus Likelihood ratio p

‐ value = .245



### 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 = .32 0 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% F2 Generation ‐ 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. Fe male kidney weight was also significantly reduced compared to the control and 0.5 ppm dos e groups. In F1 generation males, only bursa weight was affected by trenbolone; bursas of F abricius from 20 ppm treatment level males were smaller than controls. In the F2 generation, sp leen 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 m ean organ weights (g) with

standard error of deviation fr om 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 m ean organ weights (g) with

standard error of deviation fr om 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 m ean organ weights (g) with

standard error of deviation fr om 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 = .102 P

‐ value = .673 P

‐ value = .705 P

‐ value = .346 P

‐ value = .45 7 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 = .773 P

‐ value = .983 P

‐ value = .779 P

‐ value = .536 P

‐ value = .961* P

‐ value = .855 P

‐ value = .698 P

‐ value = .270 P

‐ value = .971 P

‐ value = .253 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 signi fi cantly greater kidney weight averages compared to dose 20. P

‐ value = .785 P

‐ value = .822* P

‐ value = .217 P

‐ value = .624 P

‐ value = .203* P

‐ value = .048° P

‐ value = .760 P

‐ value = .446 P

‐ value = .005† P

‐ value = .053 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 m ean organ weights (g) with

standard error of deviation fr om 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 m ean organ weights (g) with

standard error of deviation fr om 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 m ean organ weights (g) with

standard error of deviation fr om 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 te stosterone 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 = .110 P

‐ value = .252 P

‐ value = .920 P

‐ value = .119 P

‐ value = .090 P

‐ value = .028 ° P

‐ value = .958 P

‐ value = .124 P

‐ value = .769 P

‐ value = .130 P

‐ value = .251

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 = .720 P

‐ value = .033 ° P

‐ value = .674 P

‐ value = .759 P

‐ value = .212 P

‐ value = .374 P

‐ value = .390 P

‐ value = .646 P

‐ value = .231 P

‐ value = .189

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 = .135 P

‐ value = .007 ° P

‐ value = .800 P

‐ value = .425 P

‐ value = .879 P

‐ value = .085 P

‐ value = .893 P

‐ value = .267 P

‐ value = .316 P

‐ value = .508 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

Dose * Gender = .011 Gender = .000
(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 sl aughter. The active metabolite, trenbolone 17 β (TBOH17 β ), then enters the environment t hough excreta where it can accumulate in soils and in higher trophic levels of food webs. TBOH-17 β has been shown to have an affinity for the recombinant human androgen receptor similar to that of dihydr otestosterone (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 t hat can cause developmental and functional changes in birds (reviewed in Quinn et al., 2007).

In a feed exposure study similar to the current st udy, Henry et al. (2012) observed no differences in body and testes weight, testes histology, plasma te stosterone 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 do se 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 thos e 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 on ce the birds were paired. Significant spillage of feed was observed in all birds from all dose grou ps 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 as sessment 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. Remova l of the 20 ppm dose group from the F2 generation is assuredly one of the main caus es 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 endocrin e active systems that suffer effects from embryonic exposure to endocr ine 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 em bryonic contaminant exposure. No copulatory behavior effects were observed in F2 generation ma les. Of course, the lack of a 20 ppm dose group is the most likely reason for the ab sence 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 t he 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). Sinc e 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 toxi city study seemed more favorable, with the significant reduction in animal numbers and reso urces 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 retrospe ctive analysis of existing data should be taken as these results may be biased by what chemical sp ecific 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-gener ation avian toxicity test would likely have been sufficient for multi-generational assessment of trenbolone’s endocri ne 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 toxi city 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, He alth 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. Pe tri, H. Sauerwein, H. Meyer. 2000. Characterisation of the affinity of different anabolics and synthetic horm ones to the human androgen receptor, human sex hormone binding globulin and to t he 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 investigat ion 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 Te st Guidelines. EPA 7 12-C-96-141. U.S. Environmental Protection Agency, Washington, D.C.

FWS (U.S. Fish and Wildlife Serv ice). 1975. Lethal Dietary Toxi cities of Environmental

Pollutants to Birds. U.S. Fi sh and Wildlife Service, Bu reau 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. J adhao. 2004a. Haemato-bioc hemical and immunopathophysiological effects of chronic toxicity with synthetic pyrethroid, organophosphate and chlorinated pes ticides in broiler chicks. Int. Immunopharmacol. , 4: 1709-22.

Garg, U.K., A.K. Pal, G.J. Jha, S.B. J adhao. 2004b. Pathophysiological effects of chronic toxicity with synthetic py rethroid, 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 Toxi cities 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 Publicat ion 153. U.S. Department of Interior, Fish and Wildlife Service, Washington, DC, 6-54.

Janer, G., B.C. Hakkert, W. Slob, T. Vermeir e, A.H. Piersma. 2007. A retrospective analysis of the two-generation study: W hat 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 subs tances: a species comparison. J. Steroid

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

Quinn, M.J., Jr., E.T. Lavoie , M.A. Ottinger. 2006. Reproducti ve toxicity of trenbolone acetate in embryonically exposed Japanese quail. Chemosphere , 66: 1191-1196.

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

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

Quinn, M.J. Jr., C.L. Summi tt, M.A. Ottinger. 2007b. Consequenc es 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

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# 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 Direct or and the Program Manager at the time of the audit/inspection. If there were no findings during t he 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 pha se 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 gener ated 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-210 0, 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 disrupt ion & 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 chemic als upon avian health and reproduction. Birds will be exposed to trenbolone in feed. Blood will be dr awn prior to euthanasia for blood cellularity and chemistry analyses. Tissues will be collected fo r histopathological evaluation. Data from a twogeneration test that this study attempts to devel op are principally intended to evaluate the adverse consequences of exposure to a “pos sible” endocrine disruptor to t he 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 batte ry 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 ex posure 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 te rrestrial, 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 a nd characterize dose responses in a two-generation paradigm, and to develop and optimize a protocol for the avian tw o-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 endocri ne endpoints over two generations in comparison to tests that only include one generation, such as the Ec ological 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 effo rt 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, or al exposure, aerosol, inhalation, toxic, Japanese quail, quail, coturnix, bird.

II.2.5. Results of Search: Few relevant studies were locat ed using the above described search and an independent literature search.

## Animal Use Protocol-Two-generation test for endocrine disrupt ion & reproductive toxicity in the Japanese quail with

trenbolone.

32 of 18 

## 1. Avian Specific. Two main types of papers sp ecific 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 expo sure would result in a specific amount being deposited into the yolk. The current propos ed 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 asse ssment 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 cros sed the placental barrier. 3. Other. Most of the remainder of the papers fo und 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 determi ne 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 k nown or suspected to be endocrine active have been found in environmental media at many military installati ons. Currently, few methods exist to ascertain the magnitude of these effects, although some have rece ntly 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 Aber deen 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 (F 0 

## ) 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 (F 1 

## ). The F 1 

## generation will be exposed to the treatments in ovo (embryonic) through maternal deposition and through feed thro ughout the entirety of their lives. Eggs from this generation will be incubated and hatched to produce the second generation (F 2 

## ). The F 2 

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

F 2 

## generation will be untreated. Measures of repr oductive behavior, circulating hormone levels, egg quality, reproduction, and development will be assessed in the F 0 

## , F 1 

## , and F 2 

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

the F 1 

## and F 2 

## generations as described below.

## Animal Use Protocol-Two-generation test for endocrine disrupt ion & 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 t hat 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 pe riod, according to GLP standards (DLS SOP CAD 98.1).

Eggs: To generate the F 0 

## 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 F 0 

## 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 F 0 

## generation to ensure that a minimum of 60 eggs will be hatched for each test level, providing sufficient num bers of chicks to establish at least 12 breeding pairs per F 1 

## test level. If a treatment level produces insufficient eggs to set the minimum, that level will be used to its fullest ext ent. 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 ei ther removing the eggs out of the incubator and candling them or assessing them after the in cubation has ended. Except for eggs incubated to hatch for production of s ubsequent generations, the late-s tage 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 majo rity 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. she ll 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 disrupt ion & reproductive toxicity in the Japanese quail with

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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.) t hat 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 w ill be observed daily to det ect 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 recor ded. If a pair is removed during the study due to death, injury, etc., data from all preceding week s are evaluated. If data are used from a pen in which mortality occurred or a bird was injured, th is 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 pat hology assessment. The wet weight of the brain, liver, heart, spleen, kidneys, gonads, ov iduct, 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. Histopatholog y 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 th yroid gland weight, glandular T4 content (upon assay availability), and thyroid histology/histopa thology. Measurements will be taken during embryonic and adult life stages. Eggs that are se t, 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 controlle d 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 F 1 

## and F 2 

## 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 F 1 

## generation following completion of

the behavioral measures. Birds from the F 2 

## 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 techni cians 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, a nd if they are met, a one-way ANOVA will be applied followed by a multiple comparisons test to compar e treatment groups. If data do not meet the ANOVA

## Animal Use Protocol-Two-generation test for endocrine disrupt ion & reproductive toxicity in the Japanese quail with

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## assumptions, data may be transformed appropriately or a nonparametric ANOVA may be applied. Observational categorical data may be tested with a Fi sher’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 pr ovide 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 us ed 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 compar ed 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. F 0 

## 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. F 1 

## 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. F 2 

## 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.

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## 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 m easure 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 coul d 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, mori bund 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 distre ss where recovery is unlik ely 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/A nalgesia 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, welf are, method, model, in vitro, pain, distress, simulate, video, computer, replacement, refinement, reduction.

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## V.4.1.3.5. Results of Search: Few papers were found that discus sed avian euthanasia methods. Several papers were found that evaluated various anest hesia 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 ge notoxicity, carcinogenicity, and mutagenicity tests, which have no relevance to the current prop osed study and offer no alternative to the in vivo methods presented here.

Additionally, an independent literature search was co nducted 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 re view papers. The following synopses summarize our most relevant findings:

1. There are no alternatives to using live bi rds for addressing the purpose of this study.

2. No substances can be used to alleviate pain sinc e 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 dr ug 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 me chanistic 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 Endpoi nt) 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 fi nal 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. Scientif ic 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 aw ay from the featherless tract in the neck region and then inject the solution in to 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 de scribed in section V.1.1. Experimental Design. Blood will be collected either from the jugular ve in 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 F 2 

## 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 in dications 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 CO 2 

## asphyxiation as recommended by the AVMA Guidelines on Euthanasia (2007). Although unhatch ed birds are not covered by these guidelines, fertilized eggs that are not destined to be set to hatch will be frozen pr ior 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 ot her 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 t he 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 ti er that are easily removed. Each unit will have individual stainless

steel feeders and one stainless Edst rom 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 facilit y at USACHPPM. Each cage will co ntain a card describing treatment

and animal number.

The photoperiod for chicks and adults will be 16 or 17 hour s 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 house d 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 tran sferred to brooders. On ce grouped, chicks will be checked at least twice daily to ensure that group sh unning, 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 ch icks. Chicks will be housed together by treatment group when receiving treated feed and will be identifi able individually by leg bands. Juveniles will be moved to adult cages as pairs to facilitate learning how to use the ca ge 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 copul atory 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 pr ior to being set in the incubator. All cracked and abnormal eggs will be recorded. Eggs should be tu rned in the incubator at least thr ee times a day. For incubation, temperature should be between 37.5-37.8 o 

## C and relative humidity should be between 50-70%. Eggs

should be hatched at 37-37.5 o 

## 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 week end 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 increase d 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 gr oup 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 (CO 2 

## ) & 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 operationof equipment) (8/2008); Avian inhouse training at DTOX: Japanese quail euthanasia (CO 2 

## ) & 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 specif ic zoonotic diseases that can be transferred to humans (e.g., psittacosis, histoplasmo sis), 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 specific ally 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 t he 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 concer ning 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 th is protocol are technically competent and have been properly trained to ensure that no unnecessary pa in 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 pr otocol, and I assure that all individuals associated with this project will demonstrate a concern for t he health, comfort, welfar e, 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 implem enting 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 wi th 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, an algesics, and/or tranquilizers. I have considered alternatives to such procedures; however, I have deter mined 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 protoc ol, 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 modifica tion 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 statisti cal 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 concer ning 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 th is protocol are technically competent and have been properly trained to ensure that no unnecessary pa in 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 pr otocol, and I assure that all individuals associated with this project will demonstrate a concern for t he health, comfort, welfar e, 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 implem enting 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 det ermined 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 protoc ol, I provide the following assurances:

A. Animal Use: The animals authorized for use in this pr otocol will be used only in the activities and in the manner described herein, unless a modifica tion is specifically approved by the IACUC prior to its implementation.

B. Authority: I understand that, as a Co-Investigato r, I am authorized, responsible for, and willing to perform all procedur es and manipulations as assi gned 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 ca used 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 resear ch animals. Additionally, I pledge to participate in this study in the spirit of the fourth “R”, nam ely “Responsibility,” which the DOD has embraced for implementing animal use alternatives where feas ible, 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 reli ef by the use of anest hetics, 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

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## APPENDIX A

References: Directorate of Laboratory Services SOP CAD 98.1. 20 03. Chromatographic/Spectrophotometric Analysis of Toxicology Directorate Samples.

Directorate of Toxicology, Standing Operating Pr ocedure (DTOX SOP) AP003-P-001. Individual Animal Identification. USACHPPM, Aberdeen Proving Ground, MD.

Directorate of Toxicology, Standing Operating Proc edure (DTOX SOP) AP028-P-001. Animal Quality Control Procedures. USACHPPM, Aberdeen Proving Ground, MD.

Directorate of Toxicology, Standing Operating Pr ocedure (DTOX SOP) AP053-P-001. Animal Bleeding Technique. USACHPPM, Aberdeen Proving Ground, MD.

Directorate of Toxicology, Standing Operating Pr ocedure (DTOX SOP) AP063-P-001. Test System Observations. USACHPPM, Aberdeen Proving Ground, MD.

Directorate of Toxicology, Standing Operating Procedu re (DTOX SOP) AP066-P-001. Animal Euthanasia. USACHPPM, Aberdeen Proving Ground, MD.

Directorate of Toxicology, Standing Operating Proc edure (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. Jo iner, 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 reproduc tion test. OPPTS 850.2300 “Public Draft” Ecological Effects Test Guidelines. EPA 712-C- 96-141. U.S. Environmen tal 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. 200 6. 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 Toxi city Test SOP does not mention use of sentinel individuals, previous veterinarians have made thei r inclusion in toxicity tests mandatory as an

additional means of health surveillance. The us e of sentinel quail in this study will help the investigators and caretakers be better able to moni tor 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 liv e 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 en docrine 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 main tained 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 bi rds used will help prevent the study from having to be prematurely ceased by better ensuring the study’s thres hold 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 be ing 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 si tuation and the supplemental water sour ce will give the birds more time to learn how to use the water system. A delay in separati on 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 enr ichment strategies that may enhance the mental health of

the birds.

6. Additional Study Personnel will be added to this pr otocol: Rebecca Kilby, Robert Sunderland, Shenell Tolson, Mark Way, Larry Williams, Michelle Cook, Valerie Adams, and Terry Hanna.

Justification/Reason: Additional trained personnel partic ipating 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, t he number of available Study St aff listed in the current50 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 PROTOCOL52 of 18 

# W ildlife International, Ltd. APPENDIX F

DIET PREPARATION FROM WI LDLIFE INTERNATIONAL, LTD.

Project Number 167-143

Test Substance
The test substance, trenbolone C-III, was received from Ba ttelle Marine Sciences Laboratory,

Sequim, WA 98362 on February 16, 2010 and was assigne d 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 offspri ng was formulated to Wildlife International, Ltd.

specifications by Cargill Animal Nutrition, Shippensb urg, PA (Appendix I). The basal ration contained at

least 27% protein and 2.5% crude fat, and no more t han 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 calc ium is sufficient for growth and maintenance rations,

additional calcium is required in the ration of br eeding 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 approximat ely 3%, slightly above the

minimum recommended for quail. Offspring rece ived diets prepared wit hout the addition of 5%

supplemental limestone.

Test diets were prepared by mixing trenbolone in to a premix that was used for preparation of the

final diet. Details of the preparation of te st and control diets are shown in Appendix II.53 

## Appendix I

Diet Formulation WILDLIFE INTERNATIONAL, LTD. GAME BIRD RATION 1



## IN G REDIENT S

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 PER C ENT (%)

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 A MOUNT ADDED PER POUND

Vitamin D 3 

## Vitamin A Riboflavin Niacin Pantothenic Acid Vitamin B 12 

## 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 pr epared 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 tran sferred to a beaker. Acetone was measured in a

graduated syringe and added to the test substance wi th 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 appr oximately 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 Augu st 6, 2010; December 1, 2010; March 4, 2011 and

Control (0 ppm) only on August 2, 2011. As needed , the appropriate premix wa s 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 limestone55 

## 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 DATA57 

# Statistical Analysis on USAPHC Toxicol ogy Protocol Number: 08JG-80-08-04-02

Two-Generation Test for Endocrine Di sruption and Reproductive Toxicity in Japanese Quail (Corturnix japonica) using Trenbolone

Good Laboratory Practice Compliance Statement The statistical analysis of the data for the Two-gen eration 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

Statistician58 

# TRENBOLONE ENDOCRINE DISRUPTION

Egg, Sperm, and Growth Characteristics

This report analyzes the effects that trenbol one 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 characteristic s, body weights, food consumption, coacal gland (CG) area, and days until puberty. All quails were part of one of fi ve 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 week ly measurements, the same quails were tested from week to week. SPSS version 16.0 was used to analyze the endocrine disr uption data. Statistically significant results were determined using α =.05, resulting in a 95% confidence level.59 

# EGGSHELL THICKNES S and STRENGTH

I. Analysis Methods An Analysis of Variance (ANOVA) test was us ed 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 thic kness and strength. If the p-value was below .05, then it was concluded that at least two ppm levels had statistically different aver ages 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 r un 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 aver ages 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 thickne ss 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 

## F0 Generation ‐ 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 Force60 

## 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 gene ration. 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 = .225 p

‐ value = .59061EGG PRODUCTIONI. Analysis MethodsA Generalized Estimating Equation – poisson counts model (GEE) was used to determine if there was a significant difference among ppmlevels in their egg production, accounting for the variability from week to week. P-values were calculated for the ppm level and week factors aswell as their interaction. The dose, week, and interaction weredetermined to be significant if the p-value for that term fromthe GEE 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 thatfactor was less than .05. If the interaction was statisticallysignificant, the interaction was investigated via marginal means, and the significanceresults of the primary factors are considered to be uninformative and can be ignored.II. Summary of ResultsIn the F0 and F1 generations, there was a high correlation between egg production and the dosing severity of trenbolone. It was apparentthat the lower the dosing concentration oftrenbolone, the higher the egg production. This trend didn’t seem to follow throughto the F2 generationas all four dosing groups had the relatively same egg production, however only seven weeks of data was collected in the F2 generation.Comparing across generations, the F1 generation had lower eggproduction averages across the weeks, especially for ppm level 20, 10, and 5.III. F0 Generation ResultsThe ppm by week interaction was statistically significant (p-value = .000) indicating there was a significant difference in egg productiontrends among the five dose groups through the fifteen weeks. From week seven to week ten, the rate of egg production increasewas far less fordose 10 and especially dose 20 compared to the other ppm levels.After week ten, egg production was consistent for each of thedoses. Overall,dose 20 and dose 10 had the lowest egg production. Dose 5, .5and the control group had egg production that was similar in magnitude for allweeks.IV. F1 Generation ResultsJust like the F0 generation, the interactionwas statistically significant(p-value = .000). The increase in egg production for dose 20 wasmuch less than the other four dose groups in weeks seven through ten. Excluding dose 20, all dose groups had a slight increasein eggproduction after week ten. Dose 20’s egg production stayed fairly consistent through all nineteen weeks. Overall, egg production was highest forthe control group and dose .5. Dose 5 and dose 10 had thesimilar egg production counts through the nineteen weeks, but had lower averagesFactor P‐value ppmWeek7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19 Week20 Week21 Overall00.65 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 4.260.50.5 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 3.5451.20 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 4.32100.25 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 2.86200.30 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 2.09Overall0.49 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**Week6wasnotincludedintheanalysis‐onlytwoquailsproducedanyeggsinweek6.F0GenerationEggProductionGEEEstimatedMarginalMeansGEESignificanceTestsInteraction‐ppmbyweek0.000ppm0.000Week 0.00062compared to the control and .5 dose groups. Dose 20 had a muchlower egg production compared to the other four dose groups throughout theentire nineteen weeks.V. F2 Generation ResultsSignificant differences in egg production among dose groups did not occur in the F2 generation. Additionally, all dose groupsgenerallyhad the same fluctuation in egg production through the seven weeks.There was a significant difference among weeks. From weeks one throughfour, there was a large increase in egg production. After weekfour, egg production stayed fairlyconsistent with the previousweek.Factor P‐value ppm Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19 Week20 Week21 Week22 Week23 Week24 Week25 Overall00.45 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 3.900.50.40 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 3.9450.14 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 2.84100.25 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 2.23200.00 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 0.03Overall0.00 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**Week6wasnotincludedintheanalysis‐onlyonequailproducedanyeggsinweek6.F1GenerationEggProductionGEEEstimatedMarginalMeansppm0.000Week 0.000Interaction‐ppmbyweek0.000GEESignificanceTestsFactor P‐value ppm Week6 Week7 Week8 Week9 Week10 Week11 Week12 Overall00.10 2.00 3.25 4.30 5.30 5.45 3.85 2.270.50.50 1.90 3.70 4.10 4.25 4.55 4.10 2.7350.45 2.25 4.05 5.15 5.00 5.05 5.75 3.15100.10 1.50 2.85 4.45 4.80 5.30 5.35 2.21Overall0.22 1.89 3.43 4.47 4.82 5.08 4.69**Week5wasnotincludedintheanalysis‐onlyonequailproducedanyeggsinweek6.ppm 0.268Week 0.000GEESignificanceTestsInteraction‐ppmbyweek0.080GEEEstimatedMarginalMeansF2GenerationEggProduction

## 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 correspondi ng measure. A Tukey post-hoc test was then used to find which specific ppm levels were statistically di fferent 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 toproperly 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 approx imately 1,600, 4,100, and 200 for the F0, F1, and F2 generations, respectively. Percent progressive avera ges 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 mot ile 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 sper m per gram, comparing ppm level’s averages were very consistent. Averages (in all three measur es) for ppm levels, ranked from highest to lowest were: level 20, level .5, level 5, control group, and fi nally level 10. However, statistically significant differences only occurred for percent motile, in wh ich 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 fo r 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 = .52 0 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 outliers64 

## 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 le vel 5 but were highest for the control group. Sperm per gram was the only measurement that had statistica lly significant differences between ppm levels (pvalue =.030). Sperm per gram averages for the contro l 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 

## F1 Generation ‐ Sperm

% Motile % Progressive Sperm/gram

p

‐ value = .39 0 p

‐ value = .030* p

‐ value = .06 6

* 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 F2 Generation ‐ 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 t65CHICK GROWTHI. Analysis MethodsA repeated measured Linear Mixed Model was run to determine ifthere was a significant difference in body weights among the ppmlevels. A repeated measures model was used to test the dose and week factors as well as their interaction, while accounting for the variation inthe data from week to week. The dose, week, and interaction weredetermined to be significant if the p-value for the Mixed Model test was lessthan .05. Estimated marginal means were calculated for bothfactors and examined for differences using a Tukey post-hoc test,if the p-value forthat factor was less than .05. If the interaction was statistically significant, the interaction was investigated via marginalmeans, and thesignificance results of the primary factors areconsidered to be uninformative and can be ignored.II. Summary of ResultsOverall, females had greater body weights than males through allthree generations. At the start of each generation, female bodyweightswere slightly greater than males, but by the end of each generation, bodyweights for females were well above the bodyweights ofthe males.For females, comparing the same week at each generation, overall bodyweights were similar across all three generations. One noticeabledifference though, was that at the start of the F2 generation, bodyweights were lower than they were at the start of the F0 andF1 generations.Comparing ppm levels, ppm level 20 had a much lower bodyweight inthe first two generations compared to the other four levels.Male bodyweights, comparing the same week at each generation,were higher for the F1 generation compared to the F0 generation. F2generation bodyweights were more comparable to the F1 ratherthan F0 generation, albeit only ten weeks of bodyweights were collected for theF2 generation. Just like in the females, the F2 generation bodyweights at the start of the generation were lower than they were at the start of theF0 and F1 generations. Ppm level 20 had a lower bodyweight comparedto the other levels in the F1 generation, however this difference was notas drastic as it was for the females.III. F0 Generation ResultsFor both genders, week and ppm level were statistically significant, but their interaction was not. For both genders, body weight increasedfrom one week to the next, especially in the first seven to tenweeks. For females, the control group had a significantly greater body weightcompared to the other four ppm levels. Additionally, ppm level20 had a body weight that was significantly less than the otherfour ppm levels. Inthe males, body weights were lower than females and the variation between the ppm levels was much less compared to the females.The controlgroup had the highest overall average body weight and was statistically greater than ppm level .5 and level 10.ModelTerm P‐value ppm Week5 Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19 Week20 Week21 Overall098.5 106.1 118.0 124.3 128.4 134.6 135.4 135.7 139.3 140.0 142.2 141.6 141.1 142.5 142.4 142.6 143.2 132.70.592.5 100.9 110.9 115.5 124.5 129.7 130.8 135.1 135.8 137.0 138.6 138.7 138.7 141.9 141.9 140.2 138.3 128.9593.0 100.9 115.0 120.2 128.3 132.8 134.9 136.2 139.2 139.2 139.9 138.2 135.7 138.2 138.6 138.2 143.4 130.11094.0 101.8 111.4 120.2 125.0 130.3 132.3 133.8 132.9 134.8 138.1 137.2 139.5 139.7 139.6 139.2 139.6 128.82089.2 98.1 107.5 114.5 120.3 122.6 126.6 126.1 130.1 130.6 133.1 130.6 129.2 134.2 133.9 133.8 132.3 123.1Overall93.4 101.6 112.5 118.9 125.3 129.9 132.0 133.4 135.5 136.3 138.4 137.2 136.8 139.3 139.3 138.8 139.4ppm 0.000F0GenerationFemaleBodyWeightsSignificanceTestsEstimatedMarginalMeansInteraction‐ppmbyweek1.000Week 0.00066IV. F1 Generation ResultsFor both genders, week and ppm level were statistically significant, but the interaction was not.Body weights generally increased fromone week to the next; however female body weights increased at a higher rate compared tomales in the latter weeks. Overall, female bodyweights were higher than the male body weights. Comparing the ppm levels within females, the only statistically significant difference was thatppm level 20 had a lower body weight average compared to the other four ppm levels. For males, ppm level 20 once again had a significantlylower body weight average compared to the other four ppm levels. Bodyweights collected on12/10, 12/16, 12/22, 12/29/ and 1/4were excludedfrom the statistical analysis due to very sparse data collection.V. F2 Generation ResultsJust like in the F0 and F1 generations, week and ppm level werestatistically significant, butthe interaction was not for both, males andfemales. For both genders, body weights increased far less severely after week 5, however there was still a slight increase.Differences in bodyweight for the ppm levels were minimal for males and females,however statistically significant differences did exist. In females, ppm level 10 hada significantly higher body weight compared to the control groupand ppm level .5. For males, ppm level .5 had a significantlylower body weightcompared to the other three ppm levels. Bodyweights collectedon 6/9, 6/18 and 6/26 were excluded from the statistical analysis due to verysparse data collection.ModelTerm P‐value ppm Week5 Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19 Week20 Overall090.6 98.8 104.5 106.5 107.8 110.0 111.1 111.6 112.9 113.1 113.6 114.0 115.0 115.3 115.0 115.0 109.70.588.5 98.2 101.9 102.7 104.2 106.0 107.4 108.4 110.9 111.7 111.9 112.1 112.8 113.4 115.3 114.1 107.5590.9 98.1 103.0 104.7 106.0 107.9 109.2 107.2 111.1 112.2 112.2 112.7 112.6 113.1 113.2 113.8 108.01086.7 95.4 101.5 104.3 105.8 106.8 107.9 108.4 110.2 111.1 111.7 112.3 113.1 114.4 114.8 114.8 107.42089.2 96.7 102.8 104.4 106.6 107.6 110.1 109.4 111.7 113.5 111.5 112.5 113.2 113.7 112.9 114.1 108.1Overall89.2 97.4 102.7 104.5 106.1 107.6 109.1 109.0 111.3 112.3 112.2 112.7 113.3 114.0 114.2 114.3Week 0.000F0GenerationMaleBodyWeightsEstimatedMarginalMeansSignificanceTestsInteraction‐ppmbyweek1.000ppm 0.002ModelTerm P‐value ppm Week5 Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19 Week20 Week21 Week22 Week23 Overall094.1 103.6 111.8 114.3 125.5 125.5 132.0 131.1 133.4 133.5 137.5 141.3 140.0 138.6 143.1 142.8 141.1 139.1 141.3 130.00.592.2 103.6 113.3 118.9 125.8 126.5 131.6 133.3 133.6 133.6 139.3 135.9 139.2 139.2 143.5 141.9 142.3 143.6 144.9 130.6592.5 101.4 107.3 113.2 120.6 125.1 126.7 134.0 134.3 134.9 135.7 134.7 136.3 134.4 140.1 140.6 141.6 138.2 142.0 128.11091.9 101.9 113.6 115.5 122.7 123.5 129.0 127.2 134.6 137.7 136.3 135.5 137.4 134.6 141.7 145.7 142.5 142.9 142.2 129.32082.8 95.9 101.8 104.3 110.3 113.0 116.1 120.8 120.1 122.4 123.3 124.9 126.4 130.2 126.4 131.0 132.3 134.1 134.3 118.5Overall90.7 101.3 109.5 113.2 121.0 122.7 127.1 129.4 131.2 132.4 134.4 134.5 135.9 135.4 138.9 140.4 140.0 139.6 140.90.000EstimatedMarginalMeansSignificanceTestsF1GenerationFemaleBodyWeightsInteraction‐ppmbyweek0.988ppm0.000WeekModelTerm P‐value ppm Week5 Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19 Week20 Week21 Week22 Week23 Overall092.8 101.8 106.7 106.8 109.1 110.1 111.9 111.4 112.8 112.7 114.6 115.5 115.7 116.1 117.3 117.6 117.9 117.6 117.4 111.90.590.4 97.3 105.1 107.3 109.2 111.5 113.0 112.1 110.2 111.8 115.1 118.0 116.6 115.2 117.2 116.9 115.9 115.9 117.5 111.4591.6 100.6 105.2 106.7 108.9 110.7 110.9 111.9 112.0 112.0 113.6 115.5 115.0 115.7 117.8 116.9 117.3 116.1 117.3 111.31085.0 96.5 104.1 105.1 106.4 108.4 110.0 110.6 110.4 110.6 114.1 113.6 117.9 113.9 117.1 116.9 117.7 115.7 116.0 110.02077.7 89.8 98.4 100.6 104.9 106.9 106.8 108.5 108.5 108.8 112.5 112.2 112.6 115.1 118.2 117.9 117.5 117.7 116.0 107.9Overall87.5 97.2 103.9 105.3 107.7 109.5 110.5 110.9 110.8 111.2 114.0 115.0 115.5 115.2 117.5 117.2 117.2 116.6 116.8ppm 0.000Week 0.000F1GenerationMaleBodyWeightsEstimatedMarginalMeansSignificanceTestsInteraction‐ppmbyweek0.17767ModelTerm P‐value ppm Week3 Week4 Week5 Week6 Week7 Week8 Week9 Week10 Week11 Week12 Overall083.2 97.4 101.7 112.4 126.1 128.4 129.4 132.4 136.9 131.9 118.00.586.9 98.3 103.7 112.8 125.8 128.8 129.3 130.6 133.2 131.6 118.1588.1 98.9 103.1 115.3 128.3 132.0 134.0 130.2 132.4 137.2 119.91086.0 102.3 106.7 115.4 126.5 128.5 136.9 134.7 137.5 140.2 121.4Overall86.1 99.2 103.7 113.9 126.7 129.4 132.4 132.0 135.0 135.2EstimatedMarginalMeansWeek 0.000SignificanceTestsInteraction‐ppmbyweek0.727ppm 0.001F2GenerationFemaleBodyWeightsModelTerm P‐value ppm Week3 Week4 Week5 Week6 Week7 Week8 Week9 Week10 Week11 Overall083.9 93.0 96.6 104.1 108.2 107.1 107.9 108.7 109.7 102.10.577.5 91.4 95.7 103.3 105.9 104.0 104.9 106.7 107.7 99.7581.5 95.8 99.1 104.6 107.2 107.0 109.4 110.9 112.4 103.11082.5 94.8 99.0 104.9 107.8 107.7 109.8 109.5 112.3 103.1Overall81.4 93.7 97.6 104.2 107.3 106.4 108.0 108.9 110.5EstimatedMarginalMeansppm 0.000Week 0.000SignificanceTestsInteraction‐ppmbyweek0.988F2GenerationMaleBodyWeights68 

# 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 intera ction, 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. Estimat ed 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 si gnificant, the interaction was investigated via marginal means. The significance results of the primary factors are cons idered to be uninformative and can be ignored. Also, the food consumption calculations that were incorr ectly 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 consumpti on 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 fo r 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 consumpti on average compared to the other groups at weeks eight and nine. Overall, food consumption was consis tent from week to week until week nine and ten where it greatly increased.

Overall significant differences occurred among pp m levels for the males in the F0 generation. Ppm level 5 had a significantly greater food consumpti on 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 statis tically 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 F0 Generation 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 0.00069 

## greater food consumption compared to all other leve ls, while ppm level 20 had a significantly lower food consumption compared to all other levels. Addi tionally, food consumption did not have any trend in magnitude based on the degree of trenbolone concentra tion. These results indicate that there were major differences between dose groups, but these diff erences are not solely based on the concentration of trenbolone. Lastly, food consumption generally in creased 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. Ov erall in females, ppm level .5 had the highest food consumption and the control group had the lowest f ood 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 inte raction was statistically significant, fluctuation of ppm levels at each week likely caused the signific ant p-value. Even though ppm level 20 typically had the lowest food consumption at each week , there was fluctuation between t he other levels as to which had the highest average, which likely caused the significant in teraction. 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 F0 Generation 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 Means70 

## VII. F2 Generation Results For females, overall and weekly averages were very si milar 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 co nsumption 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), afte r adjusting for multiple testing, there were no significant differences between ppm levels. From we ek 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 F2 Generation 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 Consumption71 

# CLOACA GLAND (CG) AREA

I. Analysis Methods A General Linear Model – Repeated Measures (GLM REP 

## ) test was used to determine if the CG area was statistically difference between the ppm le vels, while accounting for the variation throughout weeks. P-values were calculated for ppm level and w eek 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 fo r both factors and examined for differences using a Tukey post-hoc test, if the p-value for that factor wa s less than .05. If the interaction was statistically significant, the interaction was investigated via marg inal means, and the sign ificance 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 leve ls increased CG area at a much higher rate from week one to week two compared to the other three leve ls. 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 st atistically significant, however their interaction

was not. We can concluded that there were overall di fferences 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 Means72 

## 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 in creased. Even though stat istically 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 Estimated Marginal Means GLM Significance Tests

° 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* F2 Generation Cloaca

GLM Estimated Marginal Means GLM Significance Tests73 

# ONSET TO PUBERTY

I. Analysis Methods An Analysis of Variance (ANOVA) test was us ed to determine if onset to puberty averages for dose groups were significantly different. An F-stat istic and corresponding p-value was calculated for each gender within each generation. If the p-value was belo w .05, then it was conclude d 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 diffe rent 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 orderto 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 co mpared 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 amon g 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 simila r 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 si gnificant 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 c ontrol 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

0 P

‐ 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 = .25674 

## 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. 

## F1 Generation 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 

## F2 Generation Onset to Puberty (days)

Females Males

P

‐ value = .202 P

‐ value = .70075 

# 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). S pecifically, 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 si gnificance 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 poiss on 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, wh ich 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 fo r 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 fi nd 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 test ed 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 cont rol 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 differe nce between the two groups and vice

versa.

II. Summary of Results For each dose level, the number of mount atte mpts stayed comparatively consistent from day one of the F0 generation to day three of the F1 gener ation. 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 moun ts while ppm level 20 had the lowest number of mounts. F2 generation results, as suggested a bove, were not as consistent as the F0 and F1 generations.

III. F0 Generation Results For all three days, the number of estimated m ounts 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 signifi cantly greater number of mount attempts compared to dose 10.

IV. F1 Generation Results As was the case with the F0 generation, the num ber 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 statisti cally significant differences in number of mount attempts occurred among the dose groups. Addi tionally, 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 t here 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 = .002 Omnibus Likelihood ratio p

‐ value = .011 Omnibus Likelihood ratio p

‐ value = .000

* 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 = .465 Omnibus Likelihood ratio p

‐ value = .722 Omnibus Likelihood ratio p

‐ value = .24578 

# TIME UNTIL FIRST MOUNT ATTEMPT

I. Analysis Methods Kaplan-Meier survival analysis was used to deter mine 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 stat istic 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 conc luded 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 attempt ed 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 contro l 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 differenc es among dose groups only occurred for day two and day three. In both days, the control group had a signific antly 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 pres ent 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 = .00 2 Log rank p

‐ value = .00 2 F1 Generation ‐ Time to first Mount79 

## 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 concen tration 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 grou ps 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 = .14980 

# NUMBER OF COPULATIONS

I. Analysis Methods A Generalized Linear Model (GzLM) using a poiss on loglinear link function was used to determine if there was a statistically significant difference in t he 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 m odel, was used to determine if dose was a significant factor in predicting the number of copulations in 1 80 seconds. If the p-value for the likelihood test was less than .05, dose was a significant factor in predi cting 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 dos e levels if the p-value for the omnibus test was less than .05. In the GzLM procedure, a referenc e 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 t he ‘dose=0’ group which is referred to as the control group throughout the report. An exponentiated β coefficient of 1.00 meant t hat the dosed group was 1.00 times as likely to have a copulation compared to the cont rol group, or in other words, the two groups had an equal chance of having a copulation. If the conf idence 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 copulat ions 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 copul ations for all three days. Dose 20, on the other hand, consistently had the lo west number of copulations. In fact, on day one, all dose 20 pairs failed to copulate even once. The only statistically si gnificant difference between any of the dose groups was that on day two and day three, the control gr oup 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 = .01 1 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 am ong 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 = .32 0 Omnibus Likelihood ratio p

‐ value = .65182 

# TIME UNTIL FIRS T COPULATION

I. Analysis Methods Kaplan-Meier survival analysis was used to deter mine 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 t hat the quail did not copulate and that observation was considered censored. A log rank statistic was co mputed 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 gr oups had confidence interval s that did not overlap,

these two doses were considered to have statistica lly different times at the 95% confidence level.

II. Summary of Results The time until first copulation stayed fairly consist ent 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 al so 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 fi rst 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 s hortest 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 da y 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 tr ends or differences that we re 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% F0 Generation ‐ Time to first Copulation

Day 1 Day 2 Day 3

Log rank p

‐ value = .00 9 Log rank p

‐ value = .031 Log rank p

‐ value = .03 783 

## V. F2 Generation Results No statistically significant differences occurred am ong dose groups within any of the three days of the F2 generation. Additionally, other than dose 10 ty pically 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 12.52 

### 23% 166.9 12.56 

### 7% All pairs failed to Copulate F1 Generation ‐ Time to first Copulation

Day 1 Day 2 Day 3

Log rank p

‐ value = .170 Log rank p

‐ value = .141 Log rank p

‐ value = .01 6

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% F2 Generation ‐ Time to first Copulation

Log rank p

‐ value = .160 Log rank p

‐ value = .308 Log rank p

‐ value = .372 Day 1 Day 2 Day 384 

# TRENBOLONE EFFECTS ON ORGAN WEIGHTS

Vital Organ and Total Body Weights

This section of the report analyzes the body and org an 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 th erefore only having weights for one of the genders. Quails in dose 20 were not present in the F2 generation, therefore onl y 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 de termine if dose group averages for each measured organ weight were significantly different. An F-statistic and corres ponding p-value were calculated for each organ. If the p-value
was below .05, then it was concluded that at least two dos e groups had significantly diffe rent average weights for that organ. To find which dose groups had significantly differ ent 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 violat ed (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 t he organ weights for either gender within the F0 generation. With very few statistically significant differences in organ we ights, 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 or gan or overall body weights. On e exception to this was that for both genders in the F2 generation, dose 10 (which was t he 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 we ights 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-va lues 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 definitiv e 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 we ight 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 = .102 P

‐ value = .673 P

‐ value = .705 P

‐ value = .346 P

‐ value = .457 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 = .773 P

‐ value = .983 P

‐ value = .779 P

‐ value = .536 P

‐ value = .961* P

‐ value = .855 P

‐ value = .698 P

‐ value = .270 P

‐ value = .971 P

‐ value = .253 Body Weight Brain Heart Kidneys Epididymides Liver Spleen Testes Thyroid Bursa86 

## 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 ve ry 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 fo r overall bodyweight and kidney weight. Dose 10 had a significantly greater body weight compared to dose 20, and dos e .5 and the control group had significantly greater kidney weights compared to dose 20. Statistically significant di fferences 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 do se 20. Statistically significant differences in doses were not present for any of the other orga n 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. Typica lly, 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 signi fi cantly greater kidney weight averages compared to dose 20. P

‐ value = .785 P

‐ value = .822* P

‐ value = .217 P

‐ value = .624 P

‐ value = .203* P

‐ value = .048° P

‐ value = .760 P

‐ value = .446 P

‐ value = .005† P

‐ value = .053 F1 Generation 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 = .110 P

‐ value = .252 P

‐ value = .92 0 P

‐ value = .119 P

‐ value = .09 0 P

‐ value = .028 ° P

‐ value = .958 P

‐ value = .124 P

‐ value = .769 P

‐ value = .130 P

‐ value = .251

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. F2 Generation Female Organ Weights

Body Weight Brain Heart Kidneys Liver Ovaries Spleen Oviduct Thyroid Bursa

P

‐ value = .720 P

‐ value = .033 ° P

‐ value = .674 P

‐ value = .759 P

‐ value = .212 P

‐ value = .374 P

‐ value = .390 P

‐ value = .646 P

‐ value = .231 P

‐ value = .18987 

## B. Males Dose 10 had a significantly greater spleen weight com pared 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 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 = .135 P

‐ value = .007 ° P

‐ value = .800 P

‐ value = .425 P

‐ value = .879 P

‐ value = .085 P

‐ value = .893 P

‐ value = .267 P

‐ value = .316 P

‐ value = .508 Body Weight Brain Heart Kidneys Epididymides Liver Spleen Testes Thyroid Bursa CG area88 

# APPENDIX J

CONTRIBUTING SCIENTIST REPORT

STATISTICAL ANALYSIS OF HORMONE DATA89 

# Statistical Analysis on Hormone Data for USAPHC Toxicology Protocol Number: 08JG-80-10-03-01

Two-Generation Test for Endocrine Disr uption and Reproductive Toxicity in Japanese Quail ( Corturnix japonica) using Trenbolone

Good Laboratory Practice Compliance Statement The statistical analysis of the data fo r 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 Standa rds. 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

Statistician90 

## 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 wa s 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 ea ch predictor were significantly different from one another (test of main effect). The ANOVA model also determined if t he effect that a change in dose group had on the hormone was significantly impacted by gender of the quails (test of interact ion). If the interaction was statistically significant, the ma in 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 we re 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 Dete rmined – 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 c ounted as missing data for the statistica l analysis. However, the data points that were labeled as below the assay limits were tested, but re turned 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 summa rized 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, bu t 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 differ ences 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 2405.8 148.591 

## 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

Dose * Gender = .011 Gender = .000
(Females > Males)92 

# APPENDIX K

RAW DATA93 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 1 194 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 1 195 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 1 196 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 1 197 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

1/

5 1

1/

6 1

1/

7 we ek

17 1

1/

8 1

1/

9 11

/1

0 11

/1

1 11

/1

2 11

/1

3 11

/1

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 1 198 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 1 199 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 1100 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 2101 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 1102 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 PR E

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 366103 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 374104 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 163105 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 315106 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 263107 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 277108 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 256109 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 1 1/ 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 112110 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 112111 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 137112 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 150113 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 Progr essive Average Sperm/gram 1 Sper m/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 0 283 225.1114 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 5055.4115 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 807116F0 male onset to puberty 7/7/10 “birthday”100% of controls became foamy on 8/30/10ppm ID puberty day of onset0 371 8/19/10 430 372 8/23/10 470 373 8/24/10 480 374 8/22/10 460 375 8/30/11 540 376 8/20/10 440 377 8/20/10 440 378 8/27/10 510 379 8/19/10 430 380 8/23/10 470 381 8/20/10 440 382 8/18/10 420 383 8/25/10 490 384 8/20/10 440 385 8/18/10 420 386 8/22/10 460 387 8/21/10 450 388 8/20/10 440 389 8/18/10 420 390 8/22/10 460.5 488 8/22/10 460.5 489 8/26/10 500.5 490 8/28/10 520.5 492 8/19/10 430.5 493 8/20/10 440.5 494 8/21/10 450.5 495 8/22/10 460.5 496 8/18/10 420.5 497 8/18/10 421170.5 478 8/19/10 430.5 479 8/21/10 450.5 480 8/21/10 450.5 481 8/20/10 440.5 482 8/23/10 470.5 483 8/22/10 460.5 484 8/18/10 420.5 485 8/28/10 520.5 486 9/4/10 590.5 487 8/19/10 435 259 8/20/10 445 260 8/23/10 475 261 8/25/10 495 262 8/18/10 425 263 8/20/10 445 264 8/27/10 515 279 8/24/10 485 266 8/30/10 545 267 8/22/10 465 268 8/18/10 425 269 8/18/10 425 270 8/20/10 445 271 8/21/10 455 272 8/19/10 435 273 8/20/10 445 274 8/18/10 425 275 8/23/10 475 276 8/26/10 505 277 8/19/10 435 278 8/19/10 4310 1 8/18/10 4211810 2 8/20/10 4410 3 8/18/10 4210 4 8/19/10 4310 91 8/19/10 4310 92 8/25/10 4910 7 8/28/10 5210 8 8/31/10 5510 93 8/18/10 4210 10 8/20/10 4410 11 8/20/10 4410 12 8/23/10 4710 13 8/23/10 4710 14 8/19/10 4310 15 8/28/10 5210 16 8/21/10 4510 17 8/22/10 4610 18 8/20/10 4410 19 8/25/10 4910 20 8/26/10 5020 269 8/20/10 4420 270 8/27/10 5120 271 8/22/10 4620 272 8/20/10 4420 289 8/21/10 4520 274 8/23/10 4720 285 8/19/10 4320 286 8/22/10 4620 287 8/27/10 5120 288 8/23/10 4720 275 8/23/10 4720 276 8/31/10 5511920 277 8/21/10 4520 278 8/26/10 5020 279 8/24/10 4820 280 8/25/10 4920 281 8/24/10 4820 282 8/25/10 4920 283 8/25/10 4920 284 8/28/10 52F0 cloaca measurements (mm2)ppm ID 8/18/10 8/25/10 9/1/10 9/8/100 371 180 165 324 3610 372 88 100 195 3040 373 80 132 224 2550 374 80 99 196 2560 375 162 100 169 2250 376 187 143 182 2550 377 143 180 196 2880 378 72 140 208 2400 379 165 185 289 3600 380 90 182 224 2880 381 165 266 306 3420 382 196 204 154 3240 383 49 99 182 2380 384 120 132 240 2850 385 210 192 272 2890 386 100 168 255 3230 387 108 143 221 3060 388 196 154 272 3800 389 238 221 288 3240 390 100 144 210 3611200.5 488 120 196 126 2100.5 489 81 144 180 1820.5 490 90 140 240 2880.5 499 49 56 960.5 492 180 224 288 2880.5 493 154 196 256 2700.5 494 117 182 224 2550.5 495 80 182 224 2700.5 496 270 240 256 3600.5 497 225 224 270 3230.5 478 216 182 272 3610.5 479 132 168 288 2890.5 480 130 195 306 3240.5 481 182 154 240 3610.5 482 99 168 256 2550.5 483 180 182 288 3420.5 484 224 210 255 3420.5 485 64 100 195 2560.5 486 63 99 169 3420.5 487 180 208 288 2405 259 100 210 288 3235 260 120 168 272 2705 261 99 132 225 2725 262 208 240 304 2725 263 195 238 306 3405 264 221 168 255 4005 279 132 110 210 2855 266 77 99 168 2555 267 90 117 196 3425 268 238 221 323 4005 269 195 187 255 2891215 270 156 165 225 3065 271 130 195 288 4185 272 154 192 255 3065 273 195 304 272 3425 274 156 180 154 2555 275 70 132 256 2725 276 90 110 168 3245 277 210 180 255 3245 278 224 165 288 30610 1 192 272 272 36110 2 143 270 288 38010 3 180 255 306 28810 4 143 238 240 27210 91 288 306 342 36010 92 63 100 195 25610 7 77 156 180 24010 8 80 169 225 19510 93 224 289 289 32310 10 192 288 289 34210 11 195 288 306 34210 12 121 210 210 26610 13 182 240 270 25610 14 210 306 255 30410 15 90 132 210 24010 16 132 192 238 32310 17 132 224 270 32310 18 168 240 252 32310 19 121 195 270 32410 20 90 196 270 30020 269 195 210 304 28520 270 110 224 304 27012220 271 192 210 255 28920 272 165 285 255 27220 289 108 210 240 28520 274 195 238 255 28820 285 120 224 255 28820 286 120 225 306 36020 287 80 210 238 28520 288 80 132 289 25520 275 99 182 255 32320 276 108 252 238 28920 277 108 195 255 36020 278 70 210 238 28820 279 144 240 225 32320 280 120 225 210 27020 281 132 240 255 30620 282 143 182 306 36120 283 96 210 255 36120 284 99 144 168 288F0 egg strengthppm ID Thickness Thickness Force0 201 0.32 0.34 0.8570 201 0.39 0.35 1.4240 203 0.5 0.42 1.1640 203 0.29 0.3 1.1640 204 0.39 0.55 1.6530 204 0.35 0.32 0.9290 205 0.33 0.45 0.8480 205 0.38 0.45 1.3490 206 0.36 0.32 1.9120 206 0.32 0.42 1.4971230 207 0.29 0.33 1.4180 207 0.34 0.32 1.6350 209 0.38 0.4 1.1650 209 0.38 0.32 1.5670 210 0.4 0.430 210 0.3 0.37 1.5030 210 0.46 0.32 1.5220 391 0.39 0.33 1.1020 391 0.32 0.36 1.3280 392 0.47 0.35 1.1940 392 0.31 0.29 1.1360 393 0.3 0.32 1.2960 393 0.42 0.38 0.9640 394 0.42 0.47 1.0060 394 0.52 0.6 1.370 395 0.31 0.26 1.2340 395 0.42 0.46 0.9170 396 0.43 0.47 1.2350 396 0.41 0.42 1.0430 398 0.42 0.37 1.5140 398 0.37 0.33 1.4770 399 0.45 0.37 0.6380 399 0.39 0.4 1.3420 400 0.41 0.4 1.1510 400 0.32 0.37 0.6450.5 1 0.36 0.39 1.5340.5 1 0.34 0.43 1.4550.5 4 0.54 0.45 0.6730.5 4 0.48 0.34 1.110.5 6 0.42 0.5 1.0790.5 6 0.46 0.38 0.3721240.5 7 0.43 0.46 1.0670.5 7 0.31 0.27 1.1270.5 8 0.44 0.43 1.8170.5 8 0.32 0.39 1.560.5 9 0.35 0.28 1.2080.5 9 0.43 0.45 1.3750.5 10 0.45 0.42 1.2890.5 10 0.28 0.260.5 10 0.46 0.44 1.5360.5 11 0.32 0.42 1.3670.5 11 0.37 0.32 1.8710.5 13 0.37 0.34 1.5230.5 13 0.33 0.3 1.680.5 14 0.39 0.44 1.0530.5 14 0.36 0.35 0.4860.5 15 0.31 0.350.5 15 0.29 0.28 0.930.5 17 0.38 0.42 1.2290.5 17 0.4 0.48 0.6490.5 18 0.27 0.37 0.6140.5 18 0.29 0.33 1.1390.5 19 0.39 0.3 0.6260.5 20 0.33 0.29 1.3190.5 20 0.32 0.4 1.4360.5 498 0.47 0.34 0.8960.5 498 0.43 0.38 0.5580.5 499 0.58 0.56 0.8130.5 499 0.42 0.39 1.0435 280 0.29 0.34 1.3365 280 0.3 0.29 1.5065 283 0.33 0.35 1.2851255 283 0.36 0.33 1.3655 284 0.4 0.33 1.3745 284 0.47 0.57 0.4695 285 0.33 0.35 1.4295 285 0.41 0.38 1.6245 286 0.35 0.43 1.3045 286 0.41 0.35 1.5865 286 0.41 0.39 1.7195 286 0.42 0.47 1.6735 288 0.4 0.34 1.3325 288 0.39 0.42 1.5275 289 0.47 0.46 0.9455 289 0.39 0.33 1.2095 290 0.33 0.28 1.6245 290 0.43 0.41 1.0215 291 0.39 0.435 291 0.26 0.28 1.7245 291 0.35 0.27 1.5255 292 0.27 0.29 0.9675 292 0.42 0.44 1.3495 293 0.36 0.41 1.7385 293 0.47 0.38 1.3695 294 0.49 0.4 0.6555 294 0.35 0.55 1.3545 295 0.88 0.92 1.6335 295 0.37 0.32 1.6135 296 0.33 0.395 296 0.34 0.43 1.3535 296 0.41 0.36 1.1595 297 0.38 0.34 1.3825 297 0.35 0.38 0.9671265 298 0.53 0.62 1.035 298 0.39 0.34 0.9895 299 0.31 0.29 1.4155 299 0.41 0.47 0.66510 22 0.37 0.33 0.56410 22 0.34 0.43 0.92610 23 0.38 0.56 1.20810 23 0.43 0.45 1.16310 24 0.34 0.29 1.54710 24 0.33 0.44 1.69810 25 0.59 0.52 1.51910 25 0.42 0.4 1.60810 26 0.37 0.39 0.61310 27 0.41 0.3510 27 0.38 0.35 1.310 28 0.44 0.4710 27 0.4 0.33 1.12510 28 0.42 0.39 1.33410 30 0.32 0.33 1.27110 30 0.31 0.29 1.58210 31 0.36 0.29 1.06810 31 0.27 0.3 1.24910 32 0.28 0.36 1.77810 32 0.59 0.54 1.68210 33 0.31 0.35 1.17810 33 0.33 0.29 1.26810 34 0.52 0.37 1.3710 36 0.38 0.39 0.25710 36 0.31 0.39 1.50610 37 0.4 0.37 1.44410 37 0.29 0.34 0.93212710 39 0.39 0.35 0.49810 39 0.41 0.28 1.05310 40 0.32 0.37 1.51110 94 0.42 0.37 1.78210 94 0.29 0.33 1.04820 290 0.41 0.32 0.39420 292 0.38 0.4220 292 0.32 0.38 1.60920 292 0.35 0.29 1.32120 293 0.3 0.42 1.50520 293 0.31 0.28 1.24220 294 0.39 0.41 0.60320 295 0.37 0.35 1.46720 295 0.34 0.36 1.64320 300 0.4 0.42 0.90820 300 0.41 0.51 1.55620 300 0.33 0.36 1.7120 301 0.29 0.320 303 0.3 0.31 1.11620 303 0.48 0.51 1.64720 305 0.31 0.3320 305 0.4 0.37 0.35120 306 0.33 0.31 0.41320 306 0.43 0.37 1.18320 307 0.36 0.37 1.3920 307 0.37 0.38 1.41520 308 0.28 0.31 1.4420 308 0.4 0.31 1.50120 309 0.31 0.39 1.14420 309 0.4 0.49 1.36420 380 0.43 0.38 0.54512820 380 0.3 0.39 0.395F1 egg productionppm ID1/241/251/26week 61/271/281/291/301/312/12/2week72/32/4 2/52/62/72/82/9week82/102/112/122/132/14 2/152/16week92/172/182/19 2/202/212/22 2/23week100211 2 1 1 1 1 1 1 1 1 1 1 1 1 1 10224 1 1 1 1 1 10228 1 1 1 1 1 1 1 1 102120218 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 10230 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1021602700226 1 1 1 1 1 1 1 1 1 1 10231 1 1 1 1 1 1 1 1027602990294 10292 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10237 1 1 1 1 1 1 1 10227 1 1 1 1 1 1 1 1 1 1 1 10283 1 1 1 1 1 1 1 1 1 1 1 1 1 10217 1 1 1 1 1 1 1 1 1 1 10235 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 102150.5110 1 1 1 10.5 260.5 900.5 36 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.51071290.51080.5 73 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5105 1 1 1 1 1 1 1 1 1 1 1 1 10.5 350.5 37 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 40 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 10.5 50 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 38 1 1 10.5 24 1 10.5 27 1 1 1 1 1 1 1 10.5 22 1 1 1 1 1 1 1 1 1 1 1 1 10.5 30 1 1 10.5 25 1 1 1 1 1 1 1 10.5 34 1 1 1 1 1 15 68 1 1 1 1 1 1 2 1 1 15 705 15 1 15 765 785 64 1 1 1 1 1 2 1 1 1 1 1 1 1 1 15 775 61 1 1 1 1 1 1 1 1 15 49 1 1 1 1 1 2 1 1 1 1 1 1 1 1 15 455 47 1 1 1 1 1 1 1 1 15 43 1 1 1 1 1 1 1 1 1 15 23 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 15 27 1 1 1 1 1 1 1 1 1 1 1 1 1 15 395 54 11305 535 385 7 1 15 15 66 1 1 1 1 110352 1 1 1 1 1 1 1 1 1 110359 1 1 1 1 1 1 1 1 1 110 95 1 1 1 1 1 1 1 1 1 s10398 1 1 1 1 2 1 1 110390 1 1 1 110376 1 1 1 1 1 1 1 1 1 1 1 1 1 11037110368 110370 1 1 1103731034910369eggbd103611038010367 1 1 1 1 1euthanized10351 1 1 1 1 2 210350 1 1 1 1 1 2 1 1 110374 1 1 1 1 1 1 1 110 9610357 1 1 1 1 1 1 1 1 2 1 1 1 110372 110358 1 110385 1 1 1 2 1 1 1 1 1 110395 1 1 1 1 1 1 110379 110360 113110389 1 110400 1203162032120318203122031120339203582035220317203592035120343eggbd2032820342eggbd20320 1 1 1 1 1 120333 12031320324ppm ID2/24 2/25 2/26 2/27 2/28 3/1 3/2week113/3 3/4 3/5 3/6 3/7 3/8 3/9week 123/10 3/11 3/12 3/13 3/14 3/15 3/16week 133/17 3/18 3/19 3/20 3/21 3/22 3/23week 140 211 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 10 224 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 228 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 2120 218 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 230 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 2160 2701320 226 1 1 1 1 1 1 1 1 1 1 1 1 10 231 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 276 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 299 1 1 1 1 1 1 1 1 1 10 294 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 292 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 1 1 10 237 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 227 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 283 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 217 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 235 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 215 1 1 1 1 1 1 2 1 1 10.5 110 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 260.5 90 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 36 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 107 1 1 1 1 1 1 1 1 1 10.5 108 1 1 1 10.5 73 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 105 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 350.5 37 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 50 1 1 1 1 1 1 1 1 10.5 38 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 24 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 27 1 1 1 1 1 1 1 1 1 1 1 1 10.5 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11330.5 34 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 68 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 70 1 1 1 15 15 1 1 1 1 15 76 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 78 1 1 1 1 1 1 1 15 64 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 77 1 1 1 1 1 1 1 15 61 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 49 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 45 1 1 1 1 1 1 15 47 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 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 15 23 1 1 1 1 1 1 1 1 1 1 1 1 1 15 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 39 1 1 1 1 1 1 1 1 1 1 1 15 54 1 1 1 1 1 1 1 1 1 1 15 535 385 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 1 1 15 66 1 1 1 1 1 1 1 1 1 2 110 352 1 1 1 1 110 359 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 95 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 110 398 1 1 1 1 1 1 1 1 1 1 110 390 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 376 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 2 110 37110 368 1 1 1 1 1 1 1 1 1 1 1 1 110 370 1 1 1 1 1 1 1 113410 37310 34910 36910 36110 380 1 1 1 1 1 1 1 110 36710 351 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 350 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 374 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 9610 357 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 37210 358 1 1 110 385 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 395 1 1 1 1 1 1 1 1 1 1 1 210 379 1 1 110 360 2 1 1 1 1 1 1 1 1 1 110 389 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 400 1 1 1 1 1 1 1 120 316 eggbd20 32120 31820 312 1 1 120 311 eggbd20 339 1 1 1 1 1 1 120 358 1 120 35220 317 120 359 1 2 120 351 eggbd20 34313520 32820 34220 320 1 1 1 120 333 1 1 120 313 1 1 1 1 1 1 120 324ppm ID3/24 3/25 3/26 3/27 3/28 3/29 3/30week 153/31 4/1 4/2 4/3 4/4 4/5 4/6week 164/7 4/8 4/9 4/10 4/11 4/12 4/13week 174/14 4/15 4/16 4/17 4/18 4/19 4/20week 180 211 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 224 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 228 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 2120 218 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 230 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 216 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 270 1 1 10 226 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 231 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 1 10 276 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 10 299 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 294 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 292 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 237 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 227 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 283 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 217 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 235 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 215 1 1 1 1 10.5 110 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 26 1 1 1 1 1 1 1 1 1 1 1 1 11360.5 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 1 10.5 36 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 107 1 1 1 1 1 1 1 1 1 1 1 1 10.5 108 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 73 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 105 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 10.5 35 10.5 37 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 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 1 10.5 50 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 38 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 24 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 10.5 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 1 10.5 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 10.5 34 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 68 1 1 1 1 1 1 1 15 70 1 15 15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 76 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 78 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 64 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 77 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 61 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 49 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 45 1 1 1 1 1 1 1 1 1 15 47 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 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 15 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11375 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 39 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 54 1 1 1 1 1 1 1 1 1 1 15 53 1 1 1 1 1 1 1 1 1 1 1 1 15385 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 15 66 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 352 1 1 1 1 1 1 1 1 1 1 1 1 110 359 1 1 1 1 1 110 95 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 398 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 390 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 110 376 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11037110 368 1 1 1 1 1 2 1 1 1 1 1 1 110 370 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 373103491036910 36110 380 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 36710 351 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 110 350 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 110 374 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1109610 357 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 110 37210 35810 385 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 213810 395 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 379 1 1 1 1 1 110 360 1 1 1 1 1 1 1 1 1 1 1 1 1 110 389 1 1 1 1 1 1 1 1 1 1 1 1 1 2 110 400 1 1 1 1 1 1 1 1 1 1 1 1 1 120316203212031820 312 1 1 1 1 1 1 1 1 1 1 1 12031120 339 2 2 1 1 1 1 1 1 1 1 120 358 12035220 317 1 120 359 1 120 351 120343203282034220 320 1 1 1 120 333 1 1 1 1 1 120 313 1 1 1 1 1 1 1 1 1 1 120324ppm ID4/21 4/22 4/23 4/24 4/25 4/26 4/27week 194/28 4/29 4/30 5/1 5/2 5/3 5/4week 205/5 5/6 5/7 5/8 5/9 5/10 5/11week 215/12 5/13 5/14 5/15 5/16 5/17 5/18week 220 211 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 224 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 228 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 212 1 1 1 1 1 1 1 10 218 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11390 230 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 216 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 270 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 226 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 231 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 276 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 1 10 299 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 294 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 292 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 237 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 10 227 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 10 283 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 217 1 1 1 1 1 1 1 1 1 1 1 1 1 10 235 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 1 10 215 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 110 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 26 1 1 1 1 1 1 1 1 1 1 1 10.5 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 1 10.5 36 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 10.5 107 1 1 1 1 1 1 1 1 1 1 1 1 10.5 108 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 73 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 105 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 35 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 37 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 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 1 10.5 50 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 38 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 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 1 10.5 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 1 11400.5 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20.5 34 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 68 1 1 1 1 15 70 1 1 1 1 1 1 1 1 1 1 1 1 1 15 15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 76 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 78 1 1 1 1 1 1 1 1 1 1 1 15 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 1 15 77 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 61 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 15 49 1 1 1 1 1 15 45 1 1 1 1 1 1 1 1 15 47 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 43 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 39 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 54 1 1 1 1 1 1 1 1 1 1 15 53 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15385 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 15 66 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 352 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 359 1 1 1 1 1 1 1 1 1 110 95 1 1 1 1 1 1 1 1 1 1 1 1 1 110 398 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 390 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 376 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11411037110 368 1 1 1 1 1 1 1 2 1 1 1 1 110 370 1 1 1 1 1 1 1 1 1 1 1 110 373103491036910 361 1 1 1 2 1 1 1 110 380 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 36710 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 1 110 350 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 110 374 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1109610 357 2 1 1 1 1 2 1 1 1 1 2 1 1 1 1 1 1 1 110 37210 358 110 385 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 2 1 1 110 395 1 1 1 1 1 2 1 1 1 1 2 1 2 2 1 1 110 379 1 1 1 1 2 1 110 360 1 1 1 1 1 1 1 1 1 1 1 1 1 110 389 1 1 1 1 1 1 1 1 1 1 1 1 110 400 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 120316203212031820 312 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12031120 339 1 1 1 1 1 1 1 1 120 358 1 1 1 1 1 1 1 1 1 1 1 12035220 317 1 1 1 1 1 1 1 1 114220 359 120 351 1 1 1 1 1 120343203282034220 320 120 333 1 1 1 1 1 1 1 1 120 313 1 1 1 1 120324ppm ID5/19 5/20 5/21 5/22 5/23 5/24 5/25week235/26 5/27 5/28 5/29 5/30 5/31 6/1week 246/2 6/3 6/4 6/5 6/6 6/7 6/8week 256/9 6/10 6/11 6/12 6/130 211 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 224 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 228 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 212 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 218 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 230 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 216 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 270 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 226 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 231 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 10 276 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 2 10 299 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 294 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 292 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 237 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 10 227 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 283 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 217 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 10 235 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 11430 215 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 110 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 10.5 26 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 90 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 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 10.5 107 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 108 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 73 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 10.5 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 105 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 35 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 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 10.5 40 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20.5 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 10.5 38 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 24 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 10.5 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 10.5 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 34 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 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 15 70 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 76 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 78 1 1 1 1 1 1 15 64 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 77 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 61 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 49 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 45 1 1 1 1 1 1 1 1 1 1 1 11445 47 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 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 15 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 39 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 54 1 1 1 1 1 1 1 1 1 1 15 53 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15385 7 1 1 1 1 1 1 1 15 1 1 15 66 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 110 352 1 1 1 1 1 1 1 1 1 1 1 110 359 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 95 1 1 1 1 1 1 1 1 110 398 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 390 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 2 1 110 376 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11037110 368 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 370 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 373 1 1103491036910 361 1 1 1 1 1 1 1 110 380 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 36710 351 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 350 1 1 1 1 1 1 1 1 1 1 1 1 1 2 210 374 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1109610 357 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 114510 37210 35810 385 1 1 2 1 1 1 1 2 1 1 2 1 1 1 1 1 1 110 395 1 1 1 1 1 1 1 1 1 1 1 2 110 379 1 1 1 110 360 1 1 1 1 1 1 210 389 1 1 2 1 1 1 2 1 1 1 1 1 1 1 1 1 1 110 400 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 220316203212031820 312 1 1 1 1 1 1 1 1 1 1 1 1 1 12031120 339 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 120 358 1 1 1 1 1 1 1 1 1 1 12035220 31720 35920 351 1 1 120343203282034220 32020 333 1 1 1 120 313 1 1 1 1 1 1 120324F1 paired feed consumptionDOSE ID ID3/21 3/24 3/24 3/28week143/28 4/1 4/1 4/4week 154/4 4/6 4/6 4/11week 164/11 4/14 4/14 4/18week 174/18 4/22 4/22 4/24week 184/24 4/29 4/29 5/2week 190 213 224 442 297 409 252 302 413 300 420 292 241 420 297 414 247 290 422 270 415 245 322 424 304 413 315 218 435 314 422 241 3021460 219 202 438 294 396 251 289 417 299 419 263 274 421 301 419 250 289 415 281 413 274 273 419 303 420 329 207 443 307 420 235 3210 216 214 445 283 162 406 309 415 257 255 414 315 412 251 260 420 251 413 273 309 418 319 417 332 184 444 315 425 328 2260 270 236 445 303 407 275 274 402 331 415 257 229 419 325 409 293 210 413 269 418 283 279 414 312 401 340 163 446 318 418 317 2290 226 264 442 335 405 239 273 404 329 413 294 194 412 331 418 255 244 419 278 428 241 328 417 315 400 313 189 440 313 418 281 2640 259 294 422 303 397 244 272 397 301 408 259 245 411 307 413 246 271 420 279 415 246 310 423 296 425 340 212 453 300 421 225 3490 234 292 437 284 406 252 307 405 291 406 290 230 416 300 413 261 268 424 286 414 258 294 412 299 411 334 190 448 297 405 231 3250 255 237 436 277 417 269 307 401 279 414 276 260 417 289 421 258 291 418 275 411 244 310 408 300 409 327 190 441 299 413 220 3350 247 227 443 292 411 256 306 401 281 411 279 252 413 280 412 255 290 412 273 412 233 318 419 317 410 318 194 440 320 415 215 3200 242 283 449 275 422 249 347 400 283 412 258 271 413 279 425 250 309 424 264 417 237 340 422 308 421 327 208 450 310 422 239 3230 261 217 447 280 419 252 334 406 276 419 290 259 416 282 409 255 288 424 275 418 254 313 424 310 418 326 206 455 305 426 241 3350 280 235 443 295 413 248 313 406 303 403 277 229 409 306 411 253 261 413 283 429 274 285 413 335 411 326 163 450 330 423 235 3080 246 215 442 304 413 277 274 411 291 420 297 243 422 289 412 272 273 421 291 421 256 295 419 331 413 327 174 448 328 424 268 2760.5 21 26 449 323 408 259 275 407 319 399 292 195 411 262 149 410 300 425 282 253 421 301 417 295 242 466 307 430 280 3090.5 44 36 444 236 208 405 315 416 263 243 418 320 420 248 270 417 406 411 234 188 415 302 415 292 236 450 306 425 250 3190.5 48 107 448 310 410 231 317 411 282 412 279 262 411 292 412 252 279 414 315 409 260 248 414 305 430 287 252 447 300 423 2453250.5 31 108 448 271 399 245 331 407 291 411 292 235 416 287 416 287 258 420 411 422 251 180 412 299 422 320 215 458 301 422 2872920.5 39 29 442 310 399 236 295 415 304 414 273 252 423 311 423 240 295 417 415 403 231 174 417 315 418 289 231 468 320 423 264 3070.5 46 105 440 283 403 231 329 407 288 411 258 272 400 295 401 236 270 410 407 407 231 179 420 311 415 300 224 460 316 422 2553110.5 45 35 441 281 408 251 317 413 284 405 307 227 409 266 400 288 255 421 412 409 262 156 415 297 425 276 267 455 300 428 260 3230.5 104 37 438 323 409 230 294 406 317 407 277 219 418 304 422 368 168 414 398 409 264 161 416 312 407 346 165 458 318 432 2573150.5 116 40 441 293 393 230 311 406 297 416 258 267 409 292 417 235 299 413 419 436 231 199 413 301 419 337 194 449 298 429 2513290.5 88 50 439 28 399 249 561 413 289 414 280 258 413 289 415 239 300 413 415 412 266 144 423 300 408 337 194 463 297 427 273 3200.5 47 38 452 311 407 236 312 420 304 407 298 225 420 294 418 267 277 411 408 413 316 100 422 328 411 298 207 453 324 430 279 2800.5 106 24 447 331 412 247 281 407 287 393 270 243 423 280 415 253 305 416 416 418 266 152 419 315 425 317 212 445 313 432 2613030.5 41 27 448 279 397 241 325 403 286 402 278 241 424 282 416 254 304 426 404 414 257 179 418 300 413 349 182 454 310 440 265 3190.5 81 22 446 273 405 251 327 415 274 399 263 277 416 279 412 234 315 419 404 419 269 165 418 329 415 308 196 447 330 431 272 2760.5 49 30 433 285 403 231 320 418 278 420 288 272 412 291 409 249 281 416 412 423 252 175 413 333 420 312 188 451 325 434 279 2810.5 42 25 434 296 412 238 312 416 301 411 287 239 409 307 411 261 252 429 392 425 251 211 412 306 415 311 210 448 311 431 254 3140.5 33 34 445 300 405 257 293 412 309 409 315 197 412 309 401 306 198 430 410 420 277 163 412 313 398 326 171 455 307 425 263 3105 52 68 440 300 418 256 302 408 304 410 335 179 425 301 427 330 221 416 329 419 322 184 411 313 413 337 174 449 312 416 322 2315 35 70 448 298 422 308 264 415 310 420 309 216 416 305 409 313 207 424 303 407 257 271 410 307 400 337 166 463 310 415 280 2881475 37 15 450 291 410 257 312 421 297 411 285 250 411 303 412 254 266 413 298 412 236 291 419 305 414 332 196 452 309 420 287 2765 34 76 441 301 404 283 261 409 309 407 315 192 411 296 420 240 295 410 312 412 274 236 398 292 412 340 178 449 301 419 277 2905 41 78 440 313 405 260 272 419 310 409 302 216 412 302 423 250 283 406 306 411 252 259 412 325 399 327 159 462 313 410 289 2705 36 64 443 313 403 251 282 413 306 405 295 217 419 301 407 262 263 417 284 416 277 272 416 330 416 328 174 466 324 424 281 2855 46 77 439 296 414 269 288 414 303 404 276 239 409 298 412 247 276 400 280 415 254 281 415 296 421 307 233 441 395 412 266 1925 33 61 437 319 413 270 261 413 319 401 283 212 415 312 409 266 246 410 274 423 268 291 417 320 425 337 185 453 326 416 293 2505 58 49 448 310 396 243 291 409 320 418 272 235 413 315 411 269 240 409 266 412 251 304 422 315 415 336 186 447 318 413 317 2255 44 45 450 305 410 277 278 419 302 415 289 243 418 300 416 306 228 421 290 418 264 285 405 317 413 338 163 452 311 415 293 2635 11 39 451 281 413 236 347 416 294 411 292 241 419 299 414 251 283 419 305 421 250 285 421 305 416 322 210 460 308 414 302 2645 16 54 454 312 423 268 297 424 307 419 286 250 422 296 413 291 248 428 315 415 259 269 409 304 442 339 208 462 309 400 319 2345 29 53 450 350 400 258 242 418 345 417 303 187 428 335 412 276 229 414 317 417 265 249 423 321 421 336 187 457 317 409 296 2535 4 38 447 323 410 263 271 433 325 401 330 179 417 331 419 303 202 414 322 411 298 205 430 333 419 362 154 460 326 412 278 2685 28 7 446 306 410 249 301 406 312 403 269 228 407 310 417 266 248 412 310 416 251 267 425 316 410 328 191 460 320 413 285 2685 1 66 452 309 420 247 316 416 302 405 262 257 420 308 421 317 216 422 308 415 305 224 419 318 413 347 167 459 327 410 281 26110 382 352 440 327 393 231 275 406 297 404 298 215 418 301 427 257 287 407 300 410 257 260 408 331 424 257 244 440 328 421 28025310 98 95 448 297 412 254 309 411 305 411 285 232 410 305 409 291 223 421 292 427 245 311 408 319 418 279 228 449 321 425 282 27110 386 390 437 313 411 261 274 418 322 412 298 210 416 317 420 285 234 414 284 408 274 264 401 325 415 300 191 453 320 424 28127610 394 374 437 303 416 275 275 419 310 408 284 233 414 318 421 250 267 412 286 413 237 302 402 322 416 273 223 454 319 419 26229210 362 96 435 292 143 411 311 422 306 216 415 304 419 282 248 406 311 415 270 240 416 307 415 314 210 474 310 422 299 28710 392 357 440 312 414 292 250 411 319 416 287 221 414 299 417 250 282 413 287 416 329 213 418 303 401 268 248 455 308 421 25031810 381 461 448 341 412 284 235 421 341 409 298 191 428 332 415 318 193 406 391 417 303 129 407 325 409 229 262 471 321 424 24832610 97 380 455 307 408 235 321 414 308 411 268 249 410 296 409 260 263 420 273 400 257 290 422 340 417 228 271 478 330 427 300 27510 358 376 452 294 405 232 331 411 286 412 271 266 416 296 399 306 213 411 269 419 297 264 413 333 415 230 265 470 337 423 27727910 356 370 452 330 406 261 267 414 305 406 304 211 407 317 408 277 221 405 298 415 267 255 403 341 409 228 243 476 333 422 32424120 350 318 449 328 412 291 242 423 334 403 318 174 428 332 400 303 193 422 310 416 300 228 413 323 419 349 160 455 327 424 34720520 348 312 447 298 411 249 311 406 301 399 306 198 411 322 417 265 241 412 312 415 253 262 429 304 413 292 246 459 318 413 28826620 331 339 453 321 407 271 268 412 316 393 311 178 422 316 425 290 241 413 305 416 264 260 433 338 418 305 208 463 322 410 30324820 314 458 452 307 414 271 288 414 311 407 302 208 414 310 413 332 185 418 310 415 463 331 13220 326 352 444 323 402 285 238 408 324 84 415 305 422 341 191 430 316 412 458 328 13020 335 327 439 319 408 309 219 416 313 410 313 200 419 339 407 304 183 433 320 412 304 221 420 328 421 304 209 465 320 425 30526520 361 313 435 341 414 257 251 412 333 405 294 190 418 337 411 285 207 419 322 419 265 251 419 318 412 308 205 446 324 428 25529514820 325 351 450 334 419 269 266 418 342 404 318 162 416 325 409 302 198 414 321 421 264 250 414 326 425 304 209 456 337 427 29325320 334 320 450 325 402 282 245 424 321 401 300 204 411 326 418 338 165 420 329 417 283 225 395 325 409 317 162 451 324 434 28727420 319 333 447 354 421 257 257 430 350 400 321 159 416 340 411 269 218 409 318 418 253 256 423 311 412 302 222 436 318 438 259297F1 paired feed consumptionDOSE ID ID5/2 5/5 5/5 5/9week 205/9 5/13 5/13 5/16week 215/16 5/20 5/20 5/24week 225/24 5/27 5/27 5/31week230 213 224 433 282 425 275 301 412 290 420 288 254 413 253 470 266 364 466 285 426 256 3510 219 202 437 283 437 271 320 416 278 422 289 271 420 278 450 269 323 451 317 431 252 3130 216 214 422 299 429 314 238 414 303 412 111 430 340 453 304 239 450 290 1600 270 236 420 291 434 268 295 420 298 423 294 251 425 280 456 279 322 451 309 431 281 2920 226 264 429 271 438 240 356 418 264 411 279 286 415 251 461 252 373 467 321 441 276 3110 259 294 425 268 436 295 298 432 267 409 289 285 422 262 469 302 327 483 323 424 271 3130 234 292 434 274 436 301 295 422 279 415 330 228 417 235 478 269 391 457 291 413 242 3370 255 237 421 267 422 244 332 420 261 408 290 277 409 254 473 253 375 456 320 418 254 3000 247 227 424 233 425 238 378 411 225 407 278 315 417 241 458 247 387 463 283 416 298 2980 242 283 424 273 430 270 311 426 260 416 280 302 409 249 461 243 378 461 298 426 256 3330 261 217 420 287 427 312 248 424 282 415 302 255 411 254 456 289 324 467 322 423 272 2960 280 235 418 288 428 271 287 423 272 419 266 304 410 256 455 262 347 462 298 425 259 3300 246 215 415 288 425 245 307 427 277 417 293 274 419 237 466 262 386 460 295 423 255 3330.5 21 26 431 279 419 296 275 420 265 413 349 219 433 326 452 306 253 456 312 1440.5 44 36 428 327 424 302 223 411 319 415 317 190 429 261 479 254 393 455 252 422 329 2960.5 48 107 430 298 424 232 324 427 279 418 231 335 413 249 461 239 386 458 263 422 229 3880.5 31 108 439 303 429 248 317 428 295 427 289 271 419 260 473 309 323 466 323 430 263 3100.5 39 29 437 307 422 282 270 410 300 415 285 240 424 267 481 239 399 472 271 423 288 3360.5 46 105 431 291 431 243 328 430 288 415 280 277 413 272 462 244 359 470 290 417 231 3660.5 45 35 425 268 435 247 345 425 273 416 280 288 422 277 465 270 340 451 289 434 252 3440.5 104 37 434 305 428 305 252 432 296 415 299 252 416 259 470 287 340 465 307 415 260 3130.5 116 40 432 296 422 240 318 415 284 413 274 270 426 230 471 231 436 457 259 413 230 3810.5 88 50 426 292 421 239 316 428 283 415 281 279 415 247 463 255 376 469 300 418 248 3390.5 47 38 426 292 430 244 320 423 286 417 293 261 417 261 467 290 333 470 325 414 269 2901490.5 106 24 430 267 438 291 310 415 266 411 293 267 417 271 459 276 329 473 312 426 266 3210.5 41 27 431 303 435 289 274 421 306 420 327 208 423 291 463 333 262 471 305 1660.5 81 22 425 266 437 246 350 407 256 424 274 301 427 256 472 260 383 449 289 427 266 3210.5 49 30 434 283 433 289 295 409 289 418 321 217 424 274 464 261 353 468 309 413 243 3290.5 42 25 433 315 437 243 312 411 308 418 251 270 421 264 469 244 382 478 292 421 230 3770.5 33 34 436 301 438 236 337 419 312 419 319 207 405 261 470 307 307 459 337 420 266 2765 52 68 427 314 432 256 289 425 327 426 303 221 420 314 468 270 304 447 274 416 288 3015 35 70 422 306 429 289 256 428 309 422 295 246 424 288 469 293 312 475 308 423 285 3055 37 15 422 274 422 241 329 413 280 423 302 254 415 306 474 307 276 446 282 420 239 3455 34 76 425 273 429 241 340 413 281 429 283 278 426 309 468 249 336 470 268 419 234 3875 41 78 430 297 426 254 305 417 285 434 291 275 413 290 454 277 300 458 300 411 260 3095 36 64 427 289 431 269 300 420 273 421 303 265 409 303 470 266 310 471 306 429 264 3305 46 77 431 266 425 286 304 404 263 420 284 277 411 278 477 280 330 474 279 417 261 3515 33 61 433 286 420 251 316 409 274 420 308 247 408 302 472 270 308 475 315 421 292 2895 58 49 424 310 425 305 234 403 308 408 296 207 416 322 454 302 246 482 339 419 269 2935 44 45 435 289 422 238 330 419 278 406 302 245 419 273 473 297 322 458 327 427 254 3045 11 39 419 283 422 249 309 402 280 401 278 245 413 274 444 243 340 473 262 413 236 3885 16 54 421 283 421 253 306 422 272 417 291 276 413 292 459 301 279 455 315 421 257 3045 29 53 418 301 429 279 267 413 293 409 304 225 415 299 452 279 289 479 338 432 174 3995 4 38 415 320 422 244 273 415 322 412 330 175 421 331 455 317 228 478 357 425 306 2405 28 7 420 280 426 251 315 418 274 420 293 271 418 290 437 249 316 467 339 430 279 2795 1 66 415 268 425 264 308 417 255 414 278 298 423 308 436 251 300 474 295 430 263 34610 382 352 420 286 421 255 300 420 280 416 280 276 421 306 464 289 290 462 309 429 257 32510 98 95 417 293 429 277 276 417 281 412 290 258 420 250 468 300 338 468 330 425 266 29710 386 390 420 284 430 255 311 420 286 413 317 230 422 253 459 284 344 457 322 424 262 29710 394 374 421 286 434 240 329 418 279 433 313 259 430 255 476 251 400 472 298 422 237 35910 362 96 429 308 437 332 226 410 300 414 338 186 421 332 470 356 203 454 330 12410 392 357 427 245 438 231 389 413 252 423 307 277 426 271 467 250 372 471 290 429 260 35010 381 461 424 298 435 258 303 408 285 415 327 211 424 297 443 285 285 478 340 424 283 27910 97 380 423 262 436 267 330 410 254 416 286 286 402 252 458 268 340 467 338 426 265 29010 358 376 418 274 419 264 299 423 268 428 290 293 416 233 455 268 370 482 316 422 283 30515010 356 370 420 311 417 248 278 419 313 417 296 227 421 252 470 288 351 466 319 413 258 30220 350 318 422 327 95 425 361 64 422 355 465 376 156 464 343 12120 348 312 425 315 434 253 291 413 320 417 292 218 420 250 467 279 358 467 296 422 260 33320 331 339 428 325 439 284 258 424 331 423 316 200 422 274 464 253 359 464 319 418 275 28820 314 458 424 320 104 428 428 0 020 326 352 451 344 107 431 431 0 020 335 327 433 310 431 299 255 414 314 422 321 201 425 300 465 315 275 472 344 428 312 24420 361 313 427 305 438 255 305 406 304 421 302 221 427 273 480 287 347 463 315 422 348 22220 325 351 430 284 432 279 299 416 378 413 319 132 430 299 460 288 303 470 349 427 296 25220 334 320 436 307 433 268 294 412 305 419 325 201 425 271 469 326 297 460 333 445 312 26020 319 333 335 327 430 271 167 413 319 407 307 194 421 256 434 268 331 468 304 425 267 322F1 unpaired feed consumptionID3/21 3/28week143/28 4/1 4/1 4/4week 154/4 4/6 4/6 4/11week 164/11 4/14 4/14 4/18week 174/18 4/22 4/22 4/24week 184/24 4/29 4/29 5/2week 19426 279 147 406 285 121 407 288 119 425 296 129 413 328 85 444 268 1760 230 427 233 194 420 254 166 412 282 130 420 269 151 421 338 83 453 274 1790 276 444 256 188 420 273 147 412 257 155 423 256 167 402 340 62 456 264 1920 218 449 271 178 426 280 146 419 276 143 431 264 167 419 325 94 447 290 1570 211 440 233 207 414 271 143 419 357 62 429 253 176 420 324 96 444 270 1740 231 435 240 195 419 285 134 413 230 183 426 259 167 414 330 84 452 304 1480 228 427 246 181 419 280 139 418 264 154 431 286 145 419 319 100 435 276 1590 225 439 295 144 418 312 106 419 318 101 405 292 113 420 331 89 437 270 1670 221 441 320 121 413 319 94 418 312 106 412 306 106 427 353 74 457 273 1840 232 438 331 107 403 305 98 413 313 100 413 302 111 412 344 68 441 324 1170 238 436 325 111 418 333 85 400 300 100 411 307 104 417 332 85 441 329 1120 233 420 295 125 406 301 105 425 313 112 410 326 84 415 359 56 452 353 990 223 442 315 127 407 319 88 428 331 97 410 330 80 410 365 45 453 337 1160 220 442 314 128 418 300 118 418 283 135 416 295 121 408 353 55 443 331 1120.5 110 449 263 186 416 318 98 411 278 133 416 280 136 417 275 142 463 318 1450.5 90 452 283 169 415 302 113 410 285 125 413 269 144 418 304 114 450 281 1691510.5 73 450 277 173 413 300 113 422 255 167 416 313 103 422 293 129 461 274 1870.5 43 447 283 164 421 330 91 423 315 108 412 316 96 424 361 63 474 335 1390.5 23 445 280 165 409 302 107 417 322 423 312 206 414 297 117 420 340 80 458 339 1190.5 32 440 274 166 406 304 102 416 311 105 411 309 102 410 336 74 448 337 1115 27 440 280 160 417 278 139 394 298 96 413 284 129 412 323 89 452 332 1205 43 436 280 156 411 297 114 420 262 158 420 292 128 413 342 71 460 334 1265 23 425 261 164 412 279 133 417 295 122 411 277 134 407 325 82 447 324 1235 47 437 257 180 411 291 120 415 267 148 409 268 141 409 321 88 466 318 1485 18 451 291 160 416 308 108 403 345 58 425 308 117 407 349 58 461 340 1215 26 453 270 183 423 300 123 413 309 104 425 291 134 427 350 77 463 333 1305 19 439 334 105 424 323 101 415 355 60 413 317 96 411 365 46 450 349 1015 20 448 342 106 415 335 80 417 363 54 415 308 107 412 365 47 454 350 10410 351 435 231 204 411 290 121 411 304 107 425 25 400 415 290 125 474 312 16210 373 436 216 220 414 379 35 427 368 59 434 341 93 413 355 58 457 368 8910 368 440 243 197 406 328 78 407 327 80 417 323 94 414 327 87 447 341 10610 398 435 277 158 407 276 131 409 325 84 416 289 127 418 330 88 457 294 16310 359 442 329 113 426 327 99 425 323 102 425 292 133 425 364 61 468 348 12010 350 442 245 197 408 259 149 411 365 46 408 257 151 405 292 113 457 266 19110 393 435 296 139 413 292 121 417 356 61 423 337 86 418 351 67 453 341 11210 360 445 231 214 405 327 78 404 310 94 419 233 186 408 279 129 463 310 15310 348 442 270 172 407 328 79 409 340 69 392 338 54 404 349 55 474 334 14010 388 439 321 118 404 316 88 415 371 44 426 312 114 413 367 46 470 332 13810 385 441 269 172 419 280 139 411 308 103 410 288 122 422 318 104 455 308 14710 395 437 302 135 422 294 128 421 312 109 390 297 93 418 325 93 454 302 15210 389 436 322 114 417 298 119 424 350 74 426 322 104 415 314 101 466 305 16110 400 442 324 118 421 315 106 422 302 120 418 280 138 421 244 177 453 312 14110 379 443 291 152 411 314 97 418 327 91 413 293 120 409 283 126 458 329 12910 353 458 317 141 409 301 108 414 370 44 437 346 91 417 313 104 475 333 14220 344 444 329 115 415 318 97 412 307 105 425 315 110 428 352 76 470 348 12220 328 436 397 39 424 317 107 434 348 86 423 348 75 417 353 64 458 367 9120 317 438 337 101 425 333 92 418 307 111 423 347 76 425 353 72 465 352 11315220 359 441 348 93 421 421 305 -305 430 307 123 417 319 98 446 294 15220 345 450 321 129 411 411 345 -345 412 323 89 409 362 47 468 361 10720 338 437 320 117 414 340 74 424 353 71 433 330 103 478 344 134 465 329 13620 322 427 298 129 413 304 109 414 342 72 423 324 99 415 351 64 465 332 13320 337 451 305 146 410 327 83 411 353 58 421 352 69 414 351 63 452 343 10920 324 443 384 59 428 353 75 416 347 69 415 311 104 416 344 72 445 340 10520 356 440 310 130 415 321 94 415 348 67 421 340 81 419 356 63 455 336 11920 330 445 323 122 426 318 108 418 331 87 413 335 78 425 358 67 462 334 128DOSE ID ID5/2 5/5 5/5 5/9week 205/9 5/13 5/13 5/16week 215/16 5/20 5/20 5/24week 225/24 5/27 5/27 5/31week230 299 425 312 113 407 293 114 422 312 447 331 226 445 303 1420 230 422 372 50 435 337 98 420 325 460 344 211 448 303 1450 276 426 297 129 412 312 100 422 290 433 378 187 462 291 1710 218 431 317 114 412 327 85 415 331 446 320 210 456 299 1570 211 426 279 147 416 316 100 416 302 449 348 215 454 269 1850 231 428 293 135 413 317 96 424 333 459 334 216 455 318 1370 228 430 321 109 420 326 94 414 329 455 361 179 452 318 1340 225 425 319 106 407 356 51 429 367 461 383 140 464 341 1230 221 424 347 77 422 356 66 430 375 454 393 116 451 343 1080 232 430 328 102 436 350 86 416 347 457 361 165 454 291 1630 238 430 330 100 414 366 48 435 382 454 385 122 454 345 1090 233 431 316 115 415 343 72 408 354 454 390 118 462 351 1110 223 430 333 97 408 337 71 417 356 451 384 128 462 344 1180 220 433 323 110 424 338 86 427 361 447 370 143 463 319 1440.5 110 420 277 143 407 278 129 424 327 425 288 234 453 275 1780.5 90 422 281 141 412 315 97 413 315 456 343 211 462 302 1600.5 73 428 272 156 436 288 148 424 285 448 281 306 459 254 2050.5 43 429 335 94 423 356 67 428 320 454 390 172 454 329 1250.5 23 434 317 117 405 334 71 419 301 455 338 235 460 302 1580.5 32 433 335 98 421 358 63 428 338 446 364 172 453 333 1201535 27 420 292 128 423 298 125 419 333 473 370 189 465 330 1355 43 418 274 144 415 319 96 421 343 455 332 201 461 296 1655 23 415 292 123 418 344 74 427 353 466 345 195 471 290 1815 47 415 289 126 421 313 108 418 328 439 312 217 443 262 1815 18 434 339 95 423 346 77 427 350 455 363 169 461 325 1365 26 431 336 95 418 323 95 423 331 466 367 191 465 285 1805 19 426 350 76 415 357 58 430 366 459 395 128 454 341 1135 20 427 347 80 413 343 70 419 362 468 400 125 473 354 11910 351 424 257 167 415 304 111 409 279 452 332 250 450 298 15210 373 424 330 94 419 349 70 424 332 92 462 374 8810 368 423 280 143 412 315 97 416 409 548 321 234 447 243 20410 398 415 270 145 423 335 88 398 327 457 347 181 456 322 13410 359 411 346 65 422 286 136 414 306 455 338 225 453 313 14010 350 427 242 185 423 297 126 421 287 455 331 258 453 320 13310 393 415 316 99 415 340 75 428 351 464 366 175 480 340 14010 360 411 294 117 415 330 85 413 319 468 368 194 463 326 13710 348 412 318 94 421 331 90 413 331 471 370 183 446 332 11410 388 422 325 97 417 345 72 421 338 473 386 170 459 347 11210 385 420 285 135 412 320 92 418 299 454 310 263 455 287 16810 395 425 290 135 413 333 80 418 309 462 353 218 433 305 12810 389 425 305 120 412 336 76 438 352 467 350 203 467 336 13110 400 422 323 99 405 303 102 416 302 448 333 229 467 269 19810 379 420 317 103 408 322 86 421 314 449 333 223 460 319 14110 353 428 252 176 417 357 60 436 361 457 371 161 457 351 10620 344 417 335 82 417 350 67 415 353 450 364 148 465 361 10420 328 410 326 84 417 344 73 422 351 437 356 152 460 374 8620 317 413 413 0 0 020 359 418 291 127 413 312 101 423 333 435 331 194 452 310 14220 345 412 329 83 414 345 69 426 372 460 390 124 463 360 10320 338 431 342 89 405 356 49 425 358 474 409 132 460 46020 322 437 334 103 415 357 58 435 359 460 376 160 458 45815420 337 432 339 93 421 358 63 427 354 447 361 159 452 355 9720 324 438 341 97 412 369 43 434 370 458 365 157 474 387 8720 356 433 324 109 432 354 78 419 338 473 352 202 463 340 12320 330 430 335 95 416 355 61 424 369 468 402 121 467 378 89F1 bird weightsppm SexBabyID12/10/10 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 4/25/11 5/3/11 5/10/11 5/17/11 5/23/11 6/1/110 F 4 18 211 90.2 104 106 112 126 127 124 140 127 140 141 140 149 147 140 145 141 145 130 1420 F 3 19 212 71.9 102 103 100 112 116 114 120 122 113 113 120 125 125 138 147 142 104 133 1470 F 36 215 91 103 107 112 117 118 125 139 122 125 132 147 136 126 147 142 144 133 1450 F 11 216 75.9 95.1 113 125 125 133 141 134 128 130 136 135 153 152 153 147 153 158 153 146 1500 F 23 217 92 101 108 111 128 136 140 138 139 140 139 141 140 140 132 132 141 140 1400 F 3 19 218 81.2 94 97 103 123 120 115 123 119 124 126 134 122 135 124 123 131 129 119 1250 F 30 222 63.8 85.1 87 990 F 66 270 105 92 110 114 118 122 111 111 125 136 129 135 138 140 139 135 1350 F 15 20 224 91.2 103 110 120 129 145 130 147 140 140 139 147 147 142 147 149 148 148 146 1490 F 13 18 226 71 92 103 109 131 103 111 144 144 139 142 143 145 150 146 152 153 158 157 156 1540 F 40 12 227 102 116 113 114 126 128 134 140 142 141 126 146 144 142 148 147 145 140 1180 F 7 228 89.7 100 105 119 111 123 122 123 110 131 130 147 134 129 148 140 148 149 152 1410 F 9 18 230 70.7 85.8 94 95 119 124 134 126 141 141 142 140 145 134 148 135 141 111 130 121 1300 F 37 12 231 70.8 92.1 108 115 104 117 131 134 138 136 135 136 138 149 147 149 153 157 156 136 1500 F 45 235 91 97 116 102 134 121 132 134 141 140 131 140 140 137 141 140 139 138 1370 F 22 237 72.7 86.7 94 103 105 92 110 114 118 122 111 111 125 139 133 125 145 142 141 140 1440 F 79 276 91 106 120 121 126 124 134 143 140 141 143 143 145 142 132 141 139 140 1410 F 82 283 93 102 103 115 133 139 138 133 146 144 149 147 147 137 149 143 148 152 1480 F 74 292 71 97 111 125 140 135 135 132 135 135 137 141 136 133 143 141 130 140 1420 F 89 294 54.8 66 83 102 108 117 118 129 136 139 139 136 143 137 132 141 146 145 140 1430 F 20 19 299 83 99 112 116 121 120 132 131 138 139 138 141 140 142 141 148 140 144 1450 M 33 213 81.7 92 102 104 102 103 102 103 104 107 107 108 109 104 107 111 110 111 109 1110 M 17 21 214 78.1 87 90 97 90 98 93 102 103 103 104 108 109 110 110 110 106 106 111 1141550 M 35 219 83.6 99 111 116 120 121 124 126 122 127 126 133 133 132 128 129 130 133 121 1240 M 2 21 220 81.8 89 99 104 104 106 110 113 113 113 112 112 113 112 117 121 121 121 122 1240 M 26 221 86.6 101 109 115 111 112 114 114 117 116 114 117 119 115 120 121 120 118 117 1160 M 27 223 98 107 111 113 115 117 118 118 115 116 114 113 115 118 120 122 123 122 1230 M 1 19 225 82.3 92 104 107 108 108 111 105 102 102 103 104 109 107 109 107 107 108 107 1100 M 16 15 232 90.1 105 101 111 107 113 108 116 120 122 124 121 122 120 125 127 126 126 125 1280 M 5 233 68.4 85.1 90 102 102 101 101 103 105 106 109 108 111 112 112 111 112 113 112 133 1120 M 6 234 97 112 111 104 103 105 107 104 111 113 115 114 113 111 118 116 116 115 1120 M 19 20 236 64.1 76.4 91 99 103 101 106 109 112 111 111 111 113 111 111 110 113 111 111 110 1140 M 18 238 62.8 81 93 102 101 101 105 108 111 111 109 107 110 115 112 119 121 121 120 120 1200 M 59 242 90 106 118 120 117 117 118 116 119 119 123 125 128 126 128 128 128 128 1290 M 29 20 246 89 97 105 108 110 114 116 108 114 113 114 113 116 116 113 116 117 116 1190 M 46 40.5 247 95 106 118 124 124 123 122 124 126 123 127 129 125 130 129 133 131 130 1290 M 57 30.7 255 99 108 103 114 118 112 109 121 120 119 120 121 121 119 122 120 123 121 1230 M 51 37.3 259 84 95 102 100 105 108 110 108 107 108 109 111 115 110 111 113 115 113 1130 M 47 261 91 100 103 106 107 109 112 111 113 111 115 113 119 116 114 116 116 110 1040 M 34 264 90 95 102 102 105 106 107 106 108 109 109 111 115 111 111 113 114 112 1110 M 75 280 84 90 101 99 105 108 112 102 104 106 108 108 111 108 107 109 108 109 1110.5 F 1 22 45.2 92.9 108 115 127 118 137 124 129 141 147 147 145 137 140 142 151 152 153 144 1490.5 F 6 24 109 116 122 125 130 129 132 146 147 144 153 118 150 152 155 155 156 154 1550.5 F 35 38.4 25 74.3 73.1 78 90 100 99 100 123 132 128 129 129 127 129 132 116 132 130 123 129 1320.5 F 29 26 66.2 85.7 99 108 117 119 121 122 121 121 119 120 138 142 140 143 145 120 115 143 1460.5 F 24 32.7 27 68.4 80.4 99 108 120 124 146 130 134 148 147 147 143 128 146 147 157 158 155 155 1570.5 F 2 28 68.4 800.5 F 63 73 114 120 139 140 146 144 140 146 147 157 155 157 148 153 161 156 159 1580.5 F 38 40.1 29 90 111 115 110 126 123 134 133 140 145 141 147 139 147 135 147 146 145 1400.5 F 4 30 52.5 107 95 106 107 111 119 127 119 123 126 116 134 145 139 142 143 148 139 1450.5 F 19 34 88 99 104 101 107 111 114 114 111 110 113 103 117 114 129 131 131 124 1280.5 F 16 31.7 35 47.1 93 96 122 122 132 131 138 135 136 134 142 122 117 141 143 143 132 141 1420.5 F 37 32.9 36 71.2 81.7 99 100 105 119 122 122 128 128 112 111 137 143 143 140 146 123 145 141 1420.5 F 14 37 78.2 93 88 94 97 102 115 123 123 129 130 130 137 138 136 136 138 138 136 1371560.5 F 9 38 65.8 69.7 74.2 65 111 127 144 144 146 145 150 152 150 155 127 125 114 131 125 129 127 1290.5 F 40 40 72.1 86.5 103 109 119 135 147 146 146 153 141 140 148 156 155 157 161 153 159 165 1600.5 F 60 50 78.8 96 114 120 139 140 146 144 140 146 147 157 156 149 153 155 131 159 157 1570.5 F 53 90 88 97 106 113 114 119 137 131 131 133 138 140 141 137 136 147 142 146 1420.5 F 100 105 85 95 105 116 136 138 136 136 130 127 140 149 143 146 143 152 147 143 1530.5 F 96 107 74 90 97 104 107 114 118 125 113 114 123 135 123 135 134 137 116 142 1370.5 F 106 108 86 103 114 119 111 102 116 117 128 125 134 121 136 122 136 137 138 132 1380.5 F 71 110 103 112 125 128 142 124 134 138 145 145 148 139 148 154 149 155 157 150 1510.5 M 8 37 21 79.8 97 103 108 110 110 113 115 114 116 115 114 116 115 115 116 117 120 122 1230.5 M 5 23 60.3 78.3 91 102 108 112 109 111 115 113 114 115 120 121 121 120 122 119 120 119 1220.5 M 3 31 77.8 93 99 104 103 102 105 107 106 112 114 112 112 113 114 115 117 115 112 1150.5 M 11 32 64.6 82.9 95 106 110 113 112 117 110 109 106 106 111 109 112 108 109 109 110 110 1120.5 M 13 41.8 33 62.3 61.9 77 88 101 107 109 110 112 118 108 118 108 110 109 111 111 115 114 115 113 1140.5 M 12 39 85.2 98 106 112 114 118 120 122 124 120 118 121 123 120 123 124 126 126 130 1290.5 M 15 38.5 41 68.2 63.9 80.7 98 106 111 115 119 123 1251 123 112 125 128 155 128 129 129 129 115 118 1310.5 M 20 37.2 42 58.2 71.2 85 97 105 110 113 115 117 117 102 117 118 117 117 118 118 119 119 116 1200.5 M 26 43 62.4 85 88 97 97 98 100 100 100 104 105 102 109 105 111 113 110 108 106 1080.5 M 33 35.8 44 57.3 82.6 96 104 111 110 115 115 118 118 114 115 122 121 122 121 123 122 120 121 1220.5 M 22 45 62.5 76.3 88 90 98 99 102 102 104 104 103 104 105 108 107 107 107 108 109 110 1090.5 M 17 46 92 102 110 114 111 113 114 116 108 108 121 127 121 107 129 124 122 124 1240.5 M 27 47 44 77.9 91 97 98 100 103 103 110 105 110 103 107 108 108 106 108 110 110 108 1100.5 M 28 35.6 48 94 95 104 103 105 113 116 118 120 117 124 124 141 125 125 127 121 125 1250.5 M 21 35.6 49 65.6 83 96 92 94 96 99 100 102 105 105 106 105 106 109 103 103 101 1030.5 M 39 81 83 96 98 102 104 108 96 97 99 102 103 104 103 105 105 105 104 1050.5 M 62 88 78 95 101 107 115 116 118 112 119 112 113 113 117 113 109 111 111 112 1120.5 M 68 104 81 98 110 115 118 121 120 124 107 119 126 123 121 120 121 119 120 118 1180.5 M 87 106 88 97 106 110 113 114 113 117 102 114 118 132 119 121 123 123 123 121 1220.5 M 88 116 90 94 109 115 115 117 115 117 117 117 122 123 125 126 123 125 125 127 1255 F 36 37.3 23 82.1 99 110 104 116 117 127 129 137 137 138 140 125 136 132 148 145 134 149 1465 F 40 27 80.1 98 107 114 111 120 127 115 121 137 134 135 145 160 159 157 162 156 160 155 1435 F 53 38 87 102 119 124 134 133 135 139 135 136 135 113 116 116 116 119 118 119 1191575 F 61 39 91 102 120 128 133 141 139 143 155 153 162 154 141 149 151 150 153 154 1425 F 79 43 93 104 105 103 105 109 112 122 113 116 115 148 133 149 151 156 150 149 1575 F 55 45 87 91 105 113 119 120 124 133 136 137 138 109 136 115 126 130 130 126 1265 F 26 47 92 101 113 123 138 140 138 145 150 149 130 140 133 133 138 141 140 140 1425 F 76 49 97 104 100 106 102 109 118 120 124 123 130 154 150 129 127 125 154 134 1405 F 46 53 99 105 102 104 109 125 126 132 111 115 130 144 137 146 147 147 144 145 1485 F 29 54 97 110 106 123 140 146 145 150 152 150 152 135 138 147 138 144 136 136 1425 F 99 55 83 995 F 18 7 70.1 104 116 117 127 129 137 137 138 140 140 134 133 138 142 136 138 1265 F 49 61 94 101 113 119 127 130 129 142 143 141 137 142 141 143 135 132 151 136 1525 F 39 64 95 104 107 110 129 134 136 140 132 131 138 110 131 132 138 139 132 138 1355 F 21 16 66 88 95 119 117 124 127 124 134 130 133 135 122 137 125 143 142 144 133 1425 F 44 13 68 95 100 99 105 113 127 126 112 138 138 131 117 120 123 120 137 139 137 1395 F 63 70 101 108 95 106 128 131 123 131 133 136 117 130 136 132 150 140 142 123 1545 F 82 75 86 945 F 6 15 81.7 111 120 127 115 121 137 134 135 145 142 145 143 155 152 154 151 1505 F 89 76 86 90 97 95 97 105 115 131 130 130 120 136 125 124 141 141 139 137 1445 F 95 77 93 106 121 120 124 131 130 141 140 140 149 149 150 136 152 143 150 141 1535 F 87 78 79 87 94 96 101 109 113 117 121 123 124 124 128 124 125 130 125 122 1405 M 38 42.5 1 79.6 80 101 105 100 108 111 109 117 112 111 116 114 114 113 118 120 117 117 1185 M 30 14 35.4 4 59 96 105 112 114 116 117 115 117 119 120 120 118 121 121 123 123 122 120 1215 M 12 38.7 11 57.5 70.2 95 105 109 110 114 120 118 122 115 113 118 125 126 126 128 128 128 129 1285 M 15 16 67.3 82.8 98 97 102 100 104 101 104 107 108 109 110 110 112 112 114 114 112 114 1125 M 41 18 80 87 99 107 106 106 108 109 108 110 108 113 112 115 113 113 113 115 115 1155 M 22 17 19 69.6 69 81 93 95 96 96 99 101 101 102 103 101 103 99 101 102 103 104 103 1045 M 8 20 80.8 88 98 102 103 103 103 102 107 107 104 101 110 100 110 114 116 113 111 1135 M 14 36.8 26 55.7 82.2 96 102 107 104 106 109 108 112 114 114 117 121 115 122 120 122 122 120 1215 M 16 38.6 28 58.8 82 89 95 98 99 100 101 100 97 99 99 98 101 97 101 100 103 102 1015 M 20 29 69.7 89 94 91 95 101 95 98 106 106 103 107 109 108 109 110 110 109 111 1125 M 51 33 92 103 113 115 102 108 107 110 110 114 114 114 114 115 117 116 117 117 1185 M 31 34 102 109 107 107 109 111 118 115 119 122 123 122 127 120 124 126 127 130 1311585 M 58 35 97 104 112 109 113 124 129 126 123 124 127 127 126 133 135 116 123 118 1325 M 56 36 89 96 104 109 109 108 99 102 114 115 110 128 112 115 116 113 115 117 1175 M 59 37 98 108 117 117 120 119 120 121 115 114 121 119 123 121 123 121 121 109 1165 M 65 41 103 105 115 119 121 125 125 125 127 125 131 124 130 134 138 140 139 140 1255 M 57 44 100 108 105 111 119 120 125 119 123 120 123 132 121 126 131 131 132 127 1305 M 25 46 99 115 111 116 132 135 129 123 116 118 116 117 126 117 119 119 120 122 1225 M 50 52 80 87 101 103 100 102 100 100 100 100 99 99 100 102 103 103 104 105 1035 M 37 58 80 93 94 101 100 98 100 100 103 103 106 108 109 106 106 104 103 94 10610 F 96 74 80 90 92 97 100 101 99 96 99 101 106 100 116 12010 F 42 349 68.6 64.5 66 83 93 107 112 12610 F 6 350 65.8 97 106 111 118 124 126 128 131 125 127 141 139 144 141 141 146 145 135 15010 F 9 351 62.1 80.1 93 104 115 110 140 128 127 118 134 135 137 134 138 138 144 145 144 149 13810 F 7 22 352 99.7 118 126 142 140 141 122 147 149 150 149 162 148 163 167 158 168 163 146 16310 F 3 357 54.9 92.3 104 118 126 136 143 145 142 144 145 146 149 153 150 152 151 151 140 152 15510 F 28 359 84.1 93 106 115 134 123 119 129 128 139 140 127 135 130 122 138 132 149 147 14610 F 44 360 45.4 76.9 91 95 102 107 112 131 132 116 140 141 141 137 142 142 145 143 133 128 13410 F 16 15 37.7 361 80 96 100 92 102 94 97 99 105 107 110 103 111 106 117 116 109 122 12210 F 25 367 73.2 102 111 131 133 97 99 105 14310 F 13 368 57.1 87.4 78 85 105 109 117 129 148 141 141 143 131 131 134 145 132 150 151 149 13810 F 26 35.6 369 68.2 73.4 85.7 91 101 109 114 118 124 148 141 14110 F 5 370 84.8 99 105 118 118 120 128 140 142 133 136 145 140 147 123 140 143 151 15410 F 35 371 67 84 97 91 100 98 107 109 133 13610 F 12 21 373 81.1 94 109 118 111 116 118 120 112 134 135 100 113 102 109 122 131 129 141 12110 F 47 374 66.9 71.7 81 90 103 109 124 119 123 125 137 137 119 136 122 135 134 136 116 123 13110 F 52 35.5 376 93 97 116 118 138 132 139 142 145 146 153 145 153 134 156 155 157 148 12910 F 30 15 33 379 89 97 101 104 113 111 117 124 121 124 126 130 131 129 134 141 134 135 13310 F 43 385 101 108 122 130 142 141 143 139 149 149 146 150 148 148 146 147 142 143 14810 F 56 389 99 108 121 115 121 123 129 119 137 139 144 145 146 121 150 150 140 139 14110 F 78 390 80 102 106 115 107 123 138 135 136 138 144 114 140 140 146 148 149 150 15110 F 51 395 100 101 117 123 136 147 145 134 144 145 140 150 144 142 148 151 155 145 13210 F 48 398 103 114 124 126 139 133 140 148 151 152 150 139 150 158 148 161 150 161 15915910 F 56 400 97 108 118 112 118 125 129 123 137 150 144 154 141 150 159 154 15010 F 12 21 372 98 109 123 10510 F 49 new 380 106 107 110 126 138 138 139 143 141 111 144 145 147 143 14610 F 49 old 380 84 9710 F 95 117 130 144 142 149 137 150 149 149 136 150 141 154 141 154 140 14510 M 97 76 89 102 99 108 114 118 119 117 115 122 121 170 109 123 126 128 127 12410 M 98 82 92 102 100 100 103 103 103 106 105 108 109 108 111 112 110 112 109 10910 M 54 348 85 95 98 101 103 102 105 104 106 107 111 112 114 113 114 113 112 114 11310 M 23 353 77.9 86 102 97 97 100 101 105 104 103 104 109 110 108 109 114 113 112 111 11210 M 36 356 63.3 75 89 96 103 103 108 110 109 113 111 112 111 111 112 104 111 111 112 110 11310 M 39 358 78 75.1 92 102 109 108 112 114 117 117 110 107 115 113 110 107 122 126 125 120 12210 M 18 362 54.8 76.5 86 99 103 108 100 104 105 106 112 110 115 116 117 117 115 118 118 118 11810 M 64 381 80 91 99 100 104 104 107 105 107 105 107 110 123 112 112 112 112 110 11110 M 45 382 96 107 115 114 114 113 114 115 110 111 115 117 116 120 122 121 122 120 12910 M 57 386 71 85 93 97 94 98 98 96 96 98 100 99 101 102 103 100 102 102 10110 M 46 388 89 101 109 112 113 117 116 119 119 121 122 123 122 125 125 123 124 124 12510 M 69 392 81 93 105 104 108 110 111 114 114 115 117 120 110 120 121 120 121 115 11810 M 77 393 82 95 110 116 115 113 116 118 122 123 127 125 126 127 127 129 127 11110 M 11 394 95 104 113 112 111 114 116 116 113 115 118 116 115 120 118 117 119 113 11820 F 11 19 311 87 96 105 106 114 10520 F 9 19 312 83.2 92 106 114 115 115 118 125 115 116 136 143 140 143 141 142 148 139 14520 F 8 313 66.9 68.9 80 91 94 92 98 95 110 119 129 131 123 125 125 122 113 135 129 131 13220 F 21 315 57.3 71.3 63 12920 F 20 13 316 70.6 85 87 99 102 105 110 130 11120 F 4 317 85 94 97 100 104 106 105 111 111 115 118 119 125 133 13320 F 5 318 68.9 86.5 102 115 121 126 128 122 131 125 129 128 122 128 12520 F 3 320 72.3 85 97 102 100 110 124 120 104 112 116 128 96 131 124 103 109 118 123 12620 F 47 321 82 97 103 109 117 123 127 13620 F 46 327 65.1 76 98 101 108 116 118 126 130 115 114 119 122 123 126 129 135 132 12920 F 26 35.7 333 56.1 82 94 95 103 117 108 110 120 129 130 133 138 135 141 137 137 147 137 13920 F 1 20 339 70.2 81 90 90 85 93 105 107 117 116 119 111 115 113 109 115 116 12616020 F 18 342 79.5 90 100 104 116 128 10620 F 7 19 343 80.4 97 104 111 113 119 12520 F 30 33.2 351 49.5 75 91 98 95 100 111 102 105 125 127 134 139 135 145 132 135 137 137 13220 F 49 352 62 75 85 94 100 89 92 104 111 11420 F 39 358 72 89 98 103 114 125 118 133 115 116 109 124 112 129 135 140 132 143 14020 F 37 359 82 96 105 100 108 125 128 135 132 134 11520 M 13 16 314 68 78 90 98 88 94 101 103 107 108 105 111 113 113 115 112 11420 M 25 319 69.5 81 90 96 102 108 108 107 111 118 118 116 114 111 118 120 118 122 12220 M 27 34.5 322 71.7 68.8 86 91 105 105 111 116 111 114 109 110 119 119 121 122 125 122 124 121 12020 M 19 324 68.1 84 85 94 94 100 99 102 118 110 110 106 112 121 127 125 117 132 10420 M 12 35 325 75.2 70 96 107 110 110 112 113 118 116 114 120 119 120 124 125 126 127 128 12420 M 2 20 326 69.6 81 84 90 88 93 93 100 96 100 100 105 105 106 105 105 105 105 104 10520 M 41 33.2 328 68 80 89 93 99 102 105 107 111 111 118 102 114 119 121 129 129 12920 M 31 330 75.4 79 91 98 106 112 115 113 104 106 110 115 113 122 124 127 127 126 12820 M 10 331 55.4 71.2 74 88 89 93 94 97 95 97 98 99 102 101 103 101 102 102 102 102 10220 M 16 14 334 65.8 79 87 96 100 102 104 101 102 103 106 106 110 106 107 113 113 111 110 11420 M 17 335 74.8 87 95 108 108 110 107 111 113 109 111 122 120 122 116 121 120 121 119 12120 M 15 37.4 337 47.4 71 72.9 86 98 111 118 122 119 114 117 124 124 128 126 125 125 129 132 133 134 13320 M 40 36.6 338 73.2 70 82 89 94 97 100 103 102 106 105 107 107 107 110 112 112 108 104 10720 M 42 344 65.2 68 85 105 101 102 107 106 106 109 108 110 109 113 112 113 114 11420 M 48 345 52.7 71.3 67 79 89 92 98 101 98 100 102 103 104 109 106 109 109 109 110 110 11120 M 6 348 78.7 84 104 111 111 113 118 117 115 113 113 113 112 110 119 118 119 121 114 11920 M 45 40.2 350 77 89 97 101 109 115 111 105 96 98 107 107 106 108 112 116 109 109 10920 M 32 32.7 356 57.2 73 89 99 106 113 119 123 119 122 120 120 126 125 126 127 125 126 128 129 12720 M 33 361 69 93 99 94 98 100 104 106 107 105 109 108 107 109 135 112 112 111 112F1 sperm measurementsppm ID Motile 1 Motile 2 Motile Average Progressive 1 Progressive2 Progressive Average Sperm/gram 1 Sperm/gram 2 Sperm/gram Average0 213 39 30 34.5 24 22 23 1432.5 2230.6 1831.550 221 61 39 50 37 16 26.5 2589.6 2366.4 24781610 225 27 20 23.5 9 13 11 2025.9 1381.3 1703.60 233 58 62 60 3 42 22.5 4011 3990.5 4000.750 234 70 65 67.5 38 50 44 2578.5 1964.6 2271.550 236 3 1 2 2 1 1.5 1317.1 2143.2 1730.150 242 67 67 67 29 38 33.5 6671.3 5566.2 6118.750 247 50 56 53 23 37 30 5770.9 6016.5 5893.70 255 65 74 69.5 39 43 41 6221.1 9086.1 7653.60 264 54 55 54.5 27 31 29 4133.8 5545.8 4839.80.5 21 48 45 46.5 27 27 27 2718.8 4595.7 3657.250.5 23 12 8 10 12 8 10 244.1 188.2 216.150.5 39 48 34 41 23 17 20 4338.4 2169.2 3253.80.5 41 43 48 45.5 24 30 27 2762.7 2670.6 2716.650.5 43 4 6 5 0 0 0 581.8 400 490.90.5 45 71 69 70 40 36 38 10514.8 10492.5 10503.650.5 46 0 0 0 0 0 0 51.4 51.4 51.40.5 47 0 0 0 0 0 0 2553.9 1964.6 2259.250.5 81 45 59 52 21 34 27.5 557.9 6174.3 3366.10.5 106 0 0 0 0 0 0 920.9 1289.2 1105.055 394 33 13 23 17 8 12.5 1824.2 1333.1 1578.655 11 68 69 68.5 38 35 36.5 7029.4 4512.3 5770.855 16 75 74 74.5 46 51 48.5 8871.2 7489.9 8180.555 18 42 38 40 27 21 24 4027.3 5328.9 4678.15 29 53 86 69.5 36 66 51 3585.3 2455.7 3020.55 33 65 74 69.5 35 39 37 8865.1 8717.7 8791.45 35 15 26 20.5 15 9 12 425.7 753.1 589.45 37 61 58 59.5 38 34 36 3683.5 3862.1 3772.85 44 64 62 63 32 34 33 8840.5 5893.7 7367.15 58 1 1 1 1 1 1 2373.8 2428.4 2401.110 98 55 29 42 38 14 26 1289.2 1074.4 1181.810 353 74 92 83 50 65 57.5 2087.3 3131 2609.1510 362 46 50 48 30 33 31.5 2739 2247.9 2493.4516210 382 64 59 61.5 31 35 33 3781.8 1817.2 2799.510 392 63 66 64.5 33 32 32.5 5975.5 6466.7 6221.110 393 67 56 61.5 36 30 33 6492.2 6968 6730.120 319 56 35 45.5 33 17 25 1578.7 806.9 1192.820 326 52 54 53 37 32 34.5 4850 4236.1 4543.0520 335 68 65 66.5 44 38 41 5689 9004.2 7346.620 338 75 75 75 47 42 44.5 8656.3 13751.9 11204.120 344 64 57 60.5 41 34 37.5 4474.8 3492.5 3983.6520 345 55 63 59 30 35 32.5 2578.5 5371.8 3975.1520 356 61 69 65 39 39 39 4706.8 6712.2 5709.520 361 53 62 57.5 30 34 32 6671.3 6303 6487.15163ppm ID first day of foamdate ofhatchdate of onset0 213 1/12/2011 12/9/2010 340 214 1/12/2011 12/8/2010 350 219 2/2/2011 12/9/20100 220 1/19/2011 12/6/2010 440 221 1/12/2011 12/8/2010 350 223 1/15/2011 12/8/2010 380 225 1/12/2011 12/6/2010 370 232 1/12/2011 12/8/2010 350 233 1/12/2011 12/6/2010 370 234 1/21/2011 12/6/2010 460 236 1/26/2011 12/8/2010 490 238 1/12/2011 12/8/2010 350 242 1/19/2011 12/11/2010 390 246 2/14/2011 12/9/2010 670 247 2/1/2011 12/11/2010 520 255 1/26/2011 12/11/2010 460 259 1/19/2011 12/11/2010 390 261 1/24/2011 12/11/2010 440 264 1/26/2011 12/9/2010 480 280 1/29/2011 12/13/2010 470.5 21 1/12/2011 12/6/2010 370.5 23 2/2/2011 12/6/2010 580.5 31 1/12/2011 12/6/2010 370.5 32 1/17/2011 12/7/2010 410.5 33 1/28/2011 12/7/2010 520.5 39 1/17/2011 12/7/2010 410.5 41 2/11/2011 12/7/2010 660.5 42 2/10/2011 12/8/2010 640.5 43 1/12/2011 12/8/2010 350.5 44 2/11/2011 12/9/2010 641640.5 45 1/28/2011 12/8/2010 510.5 46 12/7/20110.5 47 1/17/2011 12/8/2010 400.5 48 1/28/2011 12/8/2010 510.5 49 1/26/2011 12/8/2010 490.5 81 2/18/2011 12/9/2010 710.5 88 2/13/2011 12/11/2010 640.5 104 2/15/2011 12/12/2010 650.5 106 1/26/2011 12/13/2010 440.5 116 2/11/2011 12/13/2010 605 1 1/14/2011 12/9/2010 365 4 1/12/2011 12/7/2010 365 11 1/30/2011 12/7/2010 545 16 1/12/2011 12/7/2010 365 18 1/17/2011 12/9/2010 395 19 1/12/2011 12/8/2010 355 20 1/12/2011 12/7/2010 365 26 1/12/2011 12/7/2010 365 28 1/19/2011 12/7/2010 435 29 1/12/2011 12/7/2010 365 33 1/30/2011 12/11/2010 505 34 1/16/2011 12/8/2010 395 35 2/2/2011 12/11/2010 535 36 1/14/2011 12/11/2010 345 37 1/16/2011 12/11/2010 365 41 1/14/2011 12/12/2010 335 44 1/14/2011 12/11/2010 345 46 1/17/2011 12/8/2010 405 52 1/31/2011 12/11/2010 515 58 1/17/2011 12/9/2010 3910 97 1/16/2011 12/8/2010 3916510 98 1/12/2011 12/11/2010 3210 348 1/12/2011 12/12/2010 3110 353 1/16/2011 12/8/2010 3910 356 1/12/2011 12/9/2010 3410 358 1/12/2011 12/10/2010 3310 360 female!10 362 1/12/2011 12/7/2010 3610 371 male?10 379 female!10 381 1/16/2011 12/13/2010 3410 382 1/12/2011 12/11/2010 3210 385 female!10 386 1/19/2011 12/12/2010 3810 388 1/12/2011 12/11/2010 3210 389 1/26/2011 12/12/2010 4510 392 1/14/2011 12/15/2010 3010 393 1/16/2011 12/15/2010 3210 394 1/16/2011 12/7/2010 4010 395 female!10 400 12/7/201020 313 female!20 314 1/12/2011 12/9/2010 3420 317 12/7/201020 319 1/12/2011 12/11/2010 3220 322 1/12/2011 12/11/2010 3220 324 1/26/2011 12/10/2010 4720 325 1/12/2011 12/10/2010 3320 326 1/12/2011 12/6/2010 3720 330 1/12/2011 12/12/2010 3120 331 1/12/2011 12/8/2010 3520 333 1/23/2011 12/11/2010 4316620 334 1/16/2011 12/7/2010 4020 335 1/12/2011 12/7/2010 3620 337 1/16/2011 12/9/2010 3820 338 1/12/2011 12/14/2010 2920 344 2/15/2011 12/15/2010 6220 345 1/12/2011 12/15/2010 2820 348 1/12/2011 12/7/2010 3620 350 1/12/2011 12/15/2010 2820 356 1/12/2011 12/12/2010 3120 361 1/12/2011 12/13/2010 30F1 cloaca measuremets (mm2)ppm ID 1/12/2011 1/20/2011 1/26/2011 2/2/20110 225 156 130 252 2250 213 132 168 224 3420 223 80 81 225 2890 220 72 99 25 2240 221 144 143 80 2100 219 25 48 285 1680 232 110 80 225 900 238 143 156 132 2400 214 153 120 121 2100 236 25 64 156 1960 233 100 132 195 2560 264 25 49 182 2240 234 72 110 182 2560 259 25 110 240 2560 255 25 72 224 2240 247 35 72 90 2100 242 25 90 169 2721670 261 35 72 195 1820 280 25 72 144 2240 246 25 81 25 640.5 43 100 110 132 2100.5 21 156 154 176 2550.5 23 72 64 49 1200.5 44 25 56 49 490.5 48 42 49 81 1960.5 31 120 120 240 2560.5 32 126 100 208 1080.5 39 99 143 210 2720.5 46 54 90 50 490.5 45 64 72 49 1680.5 104 25 72 25 810.5 116 25 64 49 1100.5 88 25 91 49 640.5 47 49 110 156 2100.5 106 25 49 80 1690.5 41 25 56 49 720.5 81 63 49 900.5 49 63 81 1320.5 42 25 72 49 810.5 33 35 81 132 2250.5 67 250.5 79 635 52 35 73 64 905 35 56 90 80 1085 37 56 100 110 1805 34 80 110 225 2405 41 64 110 252 2725 36 49 100 225 2401685 46 64 121 132 1565 33 64 81 64 1685 58 25 91 90 2105 44 88 100 110 1825 18 110 132 272 2895 26 100 182 255 2885 19 81 100 182 2255 20 143 196 225 2725 11 81 100 81 1445 16 169 132 225 3065 29 156 156 144 2255 4 132 100 225 2555 28 81 110 100 1825 1 72 110 156 14410 382 90 110 210 16910 393 64 91 156 18210 360 81 100 100 9910 98 81 132 120 21010 348 81 100 144 19610 386 72 132 110 14410 388 90 121 180 21010 394 88 156 156 25510 362 110 144 165 22410 392 90 168 240 28810 389 72 121 144 16810 400 72 121 441 11010 379 90 132 120 14310 381 81 144 156 21010 97 63 110 120 16910 358 110 132 120 15610 353 120 143 144 28816910 356 110 132 154 14420 345 144 132 121 16920 350 90 121 144 18220 348 110 154 196 27220 331 110 144 121 16820 314 110 156 144 19520 326 156 144 144 16820 335 130 121 182 21020 338 132 132 132 21020 361 110 110 144 15620 313 90 110 100 13220 322 110 121 156 18220 325 99 156 182 24020 337 100 156 182 21020 334 110 132 130 22520 324 81 100 110 14320 356 144 144 168 22520 317 64 110 81 13220 319 100 121 144 12020 333 90 100 121 15620 330 120 121 132 119610 385 64 121 100 18210 395 81 90 80 144F1 egg strengthppm ID Thickness Thickness Force0 211 0.3 0.29 0.5390 211 0.32 0.31 1.0550 217 0.33 0.29 0.9781700 217 0.33 0.37 1.1820 218 0.32 0.31 1.7180 218 0.31 0.29 1.6910 227 0.43 0.33 1.3250 227 0.29 0.29 1.4810 228 0.3 0.29 1.360 228 0.29 0.28 1.2730 230 0.33 0.29 1.2440 230 0.29 0.290 231 0.42 0.43 1.2480 231 0.3 0.28 0.5960 235 0.27 0.36 1.2870 235 0.29 0.28 1.2430 237 0.4 0.27 1.670 237 0.3 0.29 1.5430 276 0.3 0.3 1.5570 276 0.32 0.3 1.5650 283 0.45 0.31 1.8770 283 0.32 0.3 2.0320 292 0.29 0.36 0.9140 292 0.27 0.27 1.4720 294 0.35 0.32 1.4230 294 0.39 0.29 1.3310 299 0.31 0.29 1.5830 299 0.35 0.33 0.2540.5 22 0.29 0.3 1.3350.5 22 0.29 0.28 1.3280.5 24 0.29 0.31 1.6130.5 24 0.29 0.29 1.6290.5 25 0.37 0.27 1.2170.5 25 0.43 0.38 1.5231710.5 27 0.39 0.38 1.1470.5 27 0.36 0.29 1.2030.5 29 0.32 0.29 1.4040.5 29 0.44 0.31 0.9830.5 30 0.29 0.32 1.1960.5 30 0.29 0.29 1.1070.5 34 0.34 0.29 1.2070.5 34 0.29 0.31 1.2590.5 36 0.28 0.29 1.6650.5 36 0.4 0.27 1.2790.5 37 0.36 0.37 1.3670.5 37 0.33 0.31 0.8910.5 38 0.36 0.36 1.6470.5 38 0.38 0.39 1.6160.5 40 0.29 0.29 1.8460.5 40 0.38 0.37 1.6870.5 73 0.33 0.3 1.5820.5 73 0.29 0.3 1.7710.5 90 0.34 0.29 1.6470.5 90 0.3 0.29 1.0470.5 105 0.31 0.33 1.910.5 105 0.33 0.3 1.1040.5 108 0.4 0.3 1.1640.5 108 0.43 0.36 1.2960.5 110 0.32 0.32 1.8970.5 110 0.3 0.29 1.5885 7 0.27 0.29 1.8175 7 0.28 0.3 1.6135 27 0.3 0.31 1.9585 27 0.28 0.29 1.2825 39 0.28 0.31 1.7861725 39 0.32 0.32 1.8595 43 0.29 0.3 1.1365 43 0.31 0.3 1.745 49 0.3 0.31 1.1465 49 0.3 0.31 1.295 54 0.43 0.28 1.1185 54 0.26 0.28 1.6775 61 0.27 0.29 1.4285 61 0.34 0.28 1.5225 64 0.28 0.29 1.215 64 0.28 0.31 1.2335 66 0.38 0.41 1.2365 66 0.29 0.29 1.795 68 0.31 0.29 1.2175 68 0.3 0.42 0.5795 70 0.32 0.38 1.1965 70 0.28 0.42 1.3575 76 0.29 0.33 1.3015 76 0.33 0.29 1.1845 68 0.35 0.37 1.035 68 0.37 0.31 1.2610 350 0.36 0.41 1.20310 350 0.27 0.29 1.22610 351 0.28 0.3 1.17410 351 0.3 0.31 1.27410 357 0.29 0.3 1.17810 357 0.29 0.3 1.26710 359 0.3 0.28 1.20510 359 0.27 0.28 1.35610 360 0.38 0.35 1.52310 360 0.28 0.34 1.10217310 374 0.41 0.37 1.47410 374 0.31 0.41 1.25410 376 0.34 0.32 1.49410 376 0.38 0.35 1.33510 379 0.39 0.3310 380 0.42 0.28 1.29210 380 0.28 0.31 1.22710 385 0.35 0.4 1.14910 385 0.29 0.32 1.25310 389 0.28 0.29 0.56710 389 0.4 0.42 1.29910 390 0.4 0.41 1.92310 390 0.33 0.33 1.63410 395 0.32 0.29 1.29710 395 0.29 0.29 0.65710 395 0.39 0.37 1.16610 395 0.3 0.41 2.0410 398 0.28 0.29 2.06410 398 0.28 0.3 1.110 400 0.29 0.29 2.15610 400 0.28 0.32 2.16720 313 0.4 0.41 1.13920 313 0.33 0.29 1.01820 313 0.3 0.28 1.03220 324 0.34 0.3620 333 0.31 0.34 0.26320 339 0.38 0.3120 339 0.36 0.3920 359 0.35 0.35 0.264174F2 egg productionppm ID7/23 7/24week57/25 7/26 7/27 7/28 7/29 7/30 7/31week68/1 8/2 8/3 8/4 8/5 8/6 8/7week78/8 8/9 8/10 8/11 8/12 8/13 8/14week88/15 8/16 8/17 8/18 8/19 8/20 8/21week90 254 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 257 1 1 1 1 1 1 0 1 1 1 10 258 1 1 1 1 1 1 1 1 1 1 1 1 10 2590 281 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 2830 284 1 1 1 1 1 1 1 1 10 286 1 1 1 10 413 1 1 1 1 1 1 1 1 1 1 1 1 10 415 1 1 1 1 1 1 1 10 417 10 420 10 429 1 1 1 1 1 1 1 1 1 1 1 1 10 437 1 1 1 1 1 1 1 1 1 1 1 1 1 10 440 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 445 1 1 1 1 1 1 1 1 10 453 1 1 1 1 1 1 1 1 1 1 1 1 1 10 454 1 1 1 1 1 1 1 1 10 457 1 1 1 1 1 1 1 1 1 1 10 458 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 118 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 119 1 1 1 1 1 1 10.5 1200.51250.5 126 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 127 1 1 1 10.5 129 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 130 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 10.5 131 1 1 1 1 1 1 1 1 1 11750.5 134 1 1 1 1 1 1 1 1 1 10.5 135 10.5 136 1 1 1 1 1 1 1 1 1 1 1 10.5 137 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 138 1 1 1 1 1 1 1 1 1 1 10.5 141 1 10.5 143 1 1 1 1 1 1 1 1 1 10.5 144 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 147 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 148 1 1 1 1 1 1 1 1 10.5 153 1 1 1 1 1 1 1 1 1 1 1 15 91 1 1 1 1 1 15 93 1 1 1 1 1 1 1 1 1 15 96 1 1 1 1 1 1 1 15 97 1 1 1 1 1 1 1 1 1 1 15 100 1 1 1 1 1 1 1 15 104 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 105 1 1 1 1 1 1 1 1 1 1 1 15 106 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 107 1 1 1 1 1 1 15 111 1 1 1 1 1 1 1 1 1 1 1 1 15 113 1 1 1 1 1 1 1 1 15 114 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 115 1 1 1 1 1 1 1 1 1 15 120 1 1 1 1 1 1 1 15 123 1 1 1 1 1 1 1 1 1 1 1 1 1 15 130 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 140 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 148 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 151 1 1 1 1 1 1 1 1 15 155 1 1 1 1 1 1 1 1 117610 4110 44 1 1 1 1 1 1 110 48 1 1 1 2 1 1 1 110 50 1 1 1 1 1 1 1 1 1 1 110 52 1 1 1 1105710 60 1 1 1 1 1 1 1 110 63 1 1 1 1 1 1 1 1 2 1 1 1 110 69 1 1 1 110 70 1 1 1 110 71 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 77 1 2 1 1 1 1 110 79 1 1 1 1 1 1 1 110 80 1 1 1 1 1 1 1 1 1 1 1 1 110 82 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 83 110 84 1 1 1 1 1 1 1 110 85 1 1 1 1 1 1 1 1 1 110 87 1 1 1 1 1 1 1 1 1 1 1 1 1 110 88 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1ppm ID8/22 8/23 8/24 8/25 8/26 8/27 8/28week108/29 8/30 8/31 9/1 9/2 9/3 9/4week119/5 9/6 9/7 9/8 9/9 9/10 9/11week120 254 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 257 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 258 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 259 1 1 1 1 1 1 1 1 10 281 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 283 1 1 1 10 284 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 286 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11770 413 1 1 1 1 1 1 1 1 1 1 1 10 415 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 10 417 1 1 1 1 1 1 1 1 1 1 1 1 1 10 420 1 1 1 10 429 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 437 1 1 1 1 1 1 1 1 1 1 1 10 440 1 1 1 1 1 1 1 1 10 445 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 453 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 454 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 457 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 458 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 118 1 1 1 1 1 1 1 0 1 1 10.5 119 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 10.5 120 1 1 1 1 1 1 10.51250.5 126 1 2 2 1 2 1 1 1 2 1 2 20.5 127 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 129 1 1 1 1 1 1 1 1 1 1 1 10.5 130 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 131 1 1 1 1 1 1 1 10.5 134 1 1 1 1 1 1 1 1 1 10.5 135 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 136 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 1370.5 138 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 141 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 143 1 1 1 1 1 1 1 10.5 144 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 147 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10.5 148 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11780.5 153 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 91 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 93 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 96 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 97 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 1 1 1 1 1 1 1 1 1 1 1 15 104 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 105 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 106 1 1 1 1 1 1 1 1 15 107 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 113 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 114 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 115 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 120 1 1 1 1 1 1 1 1 1 1 1 15 123 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 130 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 140 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 15 148 1 1 1 1 15 151 1 1 1 1 1 1 1 1 1 1 1 15 155 1 1 1 1 1 1 1 110 41 1 1 1 1 1 1 1 1 1 1 110 44 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 48 1 1 1 1 1 2 1 1 1 1 1 1 1 110 50 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 52 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1105710 60 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 63 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 69 1 1 1 1 1 1 1 1 1 110 70 1 1 1 1 1 1 1 1 1 1 1 117910 71 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 77 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 79 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 80 1 1 1 1 1 1 1 1 1 1 110 82 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 83 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 84 1 1 1 1 1 1 1 1 1 1 1 1 110 85 1 1 1 1 1 1 1 1 1 1 1 110 87 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 110 88 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1F2 feed consumptionDOSE SEX ID7/25 8/1week 68/1 8/8week 78/8 8/15week 88/15 8/22week 98/22 8/29week 108/29 9/5week 110 F 284 389 281 108 419 286 133 409 256 153 420 238 182 419 287 132 448 252 1960 F 286 408 289 119 435 311 124 422 324 98 420 305 115 415 304 111 444 293 1510 F 281 396 268 128 422 261 161 407 254 153 444 271 173 418 269 149 442 277 1650 F 283 406 293 113 434 334 100 424 316 108 428 330 98 416 299 117 451 332 1190 F 415 404 303 101 420 296 124 413 271 142 428 239 189 420 321 99 439 284 1550 F 254 393 288 105 413 282 131 415 229 186 425 255 170 433 320 113 446 266 1800 F 417 407 280 127 429 349 80 419 316 103 415 280 135 416 273 143 446 304 1420 F 420 405 276 129 416 352 64 422 307 115 423 326 97 428 328 100 425 307 1180 F 257 407 320 87 427 305 122 420 266 154 430 245 185 406 295 111 434 270 1640 F 259 400 273 0 419 350 69 401 305 96 438 343 95 399 290 109 442 323 1190 F 258 404 278 126 422 305 117 421 287 134 423 282 141 415 268 147 418 269 1490 F 429 403 321 82 427 317 110 411 243 168 436 244 192 426 290 136 456 273 1830 F 413 405 294 111 415 293 122 408 274 134 432 259 173 432 277 155 438 270 1681800 F 437 399 294 105 420 300 120 407 246 161 423 242 181 426 250 176 443 313 1300 F 457 403 267 136 411 323 88 414 266 148 419 232 187 440 241 199 420 259 1610 F 454 400 291 109 421 361 60 426 333 93 433 281 152 429 274 155 438 303 1350 F 453 406 290 116 421 318 103 416 245 171 428 247 181 432 254 178 440 280 1600 F 458 410 313 97 423 316 107 418 265 153 441 281 160 442 270 172 441 284 1570 F 440 416 305 111 415 325 90 411 264 147 427 270 157 413 324 89 445 330 1150 F 445 398 288 110 415 334 81 415 285 130 428 279 149 420 274 146 435 292 1430 M 282 411 322 89 415 316 99 412 320 92 412 310 102 419 314 105 447 332 1150 M 260 417 325 92 418 314 104 413 317 96 416 309 107 426 327 99 445 339 1060 M 251 405 300 105 424 328 96 427 322 105 410 307 103 431 327 104 422 293 1290 M 255 410 278 132 431 329 102 425 337 88 427 335 92 429 335 94 440 334 1060 M 412 403 300 103 423 319 104 410 295 115 420 314 106 416 321 95 448 322 1260 M 419 405 353 52 430 339 91 431 336 95 404 307 97 435 329 106 447 322 1250 M 285 409 318 91 419 289 130 411 299 112 419 298 121 406 310 96 431 297 1340 M 288 406 315 91 421 315 106 419 314 105 430 324 106 437 335 102 446 343 1030 M 252 407 347 60 431 329 102 438 332 106 435 330 105 432 320 112 438 325 1130 M 422 404 311 93 429 331 98 417 306 111 427 309 118 440 333 107 423 315 1080 M 416 414 348 66 433 304 129 424 325 99 420 304 116 431 317 114 439 319 1200 M 418 403 324 79 419 351 68 414 308 106 430 307 123 426 334 92 426 301 1250 M 253 411 297 114 428 335 93 427 315 112 405 277 128 425 313 112 429 289 1400 M 256 394 270 124 418 289 129 423 292 131 413 291 122 427 308 119 426 302 1240 M 287 410 316 94 420 311 109 422 319 103 430 304 126 445 327 118 415 298 1170 M 426 395 305 90 413 310 103 417 297 120 426 307 119 414 334 80 428 332 960 M 290 409 317 92 423 330 93 421 333 88 419 332 87 433 338 95 444 339 1050 M 425 400 312 88 410 294 116 428 331 97 429 286 143 434 309 125 453 318 1350 M 424 420 331 89 427 308 119 431 330 101 428 320 108 442 280 162 430 316 1140 M 289 400 305 95 435 319 116 430 308 122 411 310 101 441 306 135 444 335 1090.5 F 130 399 281 118 418 289 129 412 266 146 422 241 181 411 281 130 434 278 1560.5 F 136 385 309 76 423 290 133 417 268 149 434 255 179 416 298 118 453 281 1720.5 F 131 400 300 100 418 292 126 408 277 131 415 287 128 423 287 136 438 311 1270.5 F 134 398 254 144 422 312 110 410 298 112 436 265 171 426 305 121 453 306 1471810.5 F 119 402 323 79 430 286 144 413 260 153 426 270 156 437 282 155 440 287 1530.5 F 126 399 299 100 428 281 147 407 258 149 429 249 180 402 289 113 436 267 1690.5 F 120 401 325 76 427 322 105 420 354 66 429 332 97 424 296 128 450 294 1560.5 F 125 408 289 119 416 307 109 411 284 127 432 379 53 421 333 88 445 357 880.5 F 118 407 327 80 420 307 113 417 253 164 423 251 172 411 306 105 434 333 1010.5 F 141 395 299 96 427 324 103 396 288 108 424 284 140 433 269 164 442 290 1520.5 F 138 414 356 58 411 339 72 424 296 128 425 276 149 417 276 141 426 257 1690.5 F 144 414 310 104 420 304 116 417 273 144 424 247 177 413 296 117 447 264 1830.5 F 135 401 301 100 424 316 108 417 318 99 418 296 122 425 284 141 439 269 1700.5 F 143 401 281 120 412 305 107 423 263 160 434 271 163 430 291 139 445 310 1350.5 F 127 408 296 112 423 323 100 423 311 112 429 284 145 426 278 148 437 307 1300.5 F 147 403 262 141 418 311 107 405 271 134 420 249 171 417 276 141 439 282 1570.5 F 137 398 276 122 427 313 114 407 278 129 4170.5 F 148 397 307 90 411 297 114 408 272 136 420 273 147 422 267 155 435 297 1380.5 F 129 401 272 129 421 285 136 406 245 161 413 296 117 429 293 136 443 290 1530.5 F 153 395 271 124 418 288 130 421 253 168 435 241 194 419 275 144 445 298 1470.5 M 155 410 328 82 415 335 80 424 332 92 406 302 104 421 326 95 429 319 1100.5 M 161 407 312 95 427 323 104 412 323 89 426 310 116 432 326 106 431 310 1210.5 M 133 412 326 86 421 317 104 420 283 137 425 289 136 421 306 115 445 293 1520.5 M 123 412 359 53 432 353 79 402 293 109 420 326 94 432 341 91 430 326 1040.5 M 142 418 334 84 418 310 108 425 313 112 425 328 97 416 312 104 440 343 970.5 M 146 405 328 77 417 320 97 416 305 111 436 299 137 420 294 126 445 315 1300.5 M 132 408 328 80 424 322 102 429 312 117 418 299 119 423 310 113 437 300 1370.5 M 121 407 344 63 424 330 94 295 320 -25 420 302 118 425 317 108 444 311 1330.5 M 122 424 349 75 418 332 86 424 318 106 430 316 114 424 331 93 421 319 1020.5 M 128 416 301 115 419 295 124 412 290 122 425 276 149 419 295 124 439 296 1430.5 M 145 402 283 119 412 334 78 415 318 97 430 313 117 421 327 94 418 313 1050.5 M 156 405 310 95 415 337 78 415 321 94 421 320 101 426 329 97 445 344 1010.5 M 151 406 311 95 424 311 113 421 304 117 421 295 126 420 294 126 437 312 1250.5 M 157 408 302 106 427 338 89 426 332 94 423 314 109 432 315 117 449 336 1130.5 M 152 405 306 99 421 307 114 415 316 99 401 287 114 419 305 114 436 324 1121820.5 M 140 383 307 76 421 312 109 410 291 119 421 297 124 433 306 127 436 313 1230.5 M 160 412 305 107 420 323 97 416 301 115 419 312 107 416 319 97 419 312 1070.5 M 190 413 298 115 437 303 134 420 303 117 425 285 140 418 306 112 433 323 1100.5 M 186 410 294 116 438 347 91 428 339 89 422 333 89 425 314 111 436 328 1080.5 M 188 414 299 115 427 352 75 432 327 105 422 305 117 445 326 119 418 311 1075 F 100 406 325 81 426 334 92 423 283 140 423 258 165 429 315 114 437 298 1395 F 120 405 316 89 403 322 81 421 294 127 431 276 155 433 268 165 433 331 1025 F 104 398 310 88 427 314 113 419 266 153 419 257 162 420 259 161 426 304 1225 F 114 406 279 127 427 283 144 417 245 172 423 262 161 419 270 149 426 270 1565 F 123 401 293 108 417 302 115 408 266 142 418 255 163 410 289 121 437 285 1525 F 113 406 306 100 438 322 116 414 280 134 418 243 175 424 270 154 430 269 1615 F 130 409 311 98 426 312 114 422 280 142 420 249 171 432 289 143 437 284 1535 F 140 417 300 117 417 303 114 426 263 163 427 244 183 419 276 143 429 299 1305 F 105 408 317 91 428 309 119 424 269 155 424 230 194 426 244 182 431 252 1795 F 106 414 320 94 421 275 146 414 242 172 418 281 137 431 299 132 445 273 1725 F 107 403 295 108 412 332 80 416 294 122 425 250 175 406 284 122 423 274 1495 F 111 407 315 92 414 325 89 424 232 192 417 233 184 418 255 163 433 266 1675 F 93 409 286 123 425 337 88 412 278 134 424 259 165 415 276 139 422 273 1495 F 97 408 318 90 408 317 91 412 276 136 426 271 155 419 288 131 454 284 1705 F 91 410 418 -8 432 342 90 430 337 93 426 267 159 409 246 163 435 272 1635 F 96 405 316 89 432 341 91 423 312 111 417 259 158 408 276 132 442 269 1735 F 115 402 310 92 417 309 108 419 231 188 412 251 161 425 264 161 445 258 1875 F 148 414 321 93 423 293 130 422 263 159 434 335 99 429 343 86 440 275 1655 F 151 407 290 117 424 319 105 420 281 139 426 291 135 419 301 118 427 275 1525 F 155 497 293 204 416 280 136 420 232 188 410 281 129 434 289 145 446 249 1975 M 133 403 304 99 427 317 110 415 281 134 427 302 125 426 319 107 445 329 1165 M 131 399 305 94 428 317 111 427 305 122 417 284 133 434 320 114 451 314 1375 M 99 398 295 103 417 331 86 431 334 97 407 310 97 433 309 124 432 327 1055 M 122 393 282 111 418 305 113 420 302 118 429 310 119 437 310 127 422 293 1295 M 102 411 307 104 433 326 107 418 316 102 413 308 105 424 318 106 439 329 1105 M 103 394 293 101 428 333 95 419 308 111 418 302 116 432 305 127 432 331 1011835 M 118 416 304 112 426 332 94 442 329 113 415 306 109 430 309 121 443 320 1235 M 134 396 302 94 431 307 124 426 292 134 409 277 132 421 299 122 443 319 1245 M 108 405 317 88 418 305 113 425 304 121 416 300 116 416 324 92 413 307 1065 M 110 406 303 103 418 315 103 422 313 109 397 292 105 429 318 111 425 317 1085 M 109 407 296 111 420 302 118 429 301 128 414 274 140 421 297 124 428 288 1405 M 112 409 312 97 422 328 94 401 286 115 415 297 118 449 331 118 421 286 1355 M 119 412 303 109 426 309 117 425 314 111 421 309 112 424 332 92 422 324 985 M 95 397 332 65 422 330 92 429 320 109 431 321 110 441 340 101 430 334 965 M 94 404 313 91 410 331 79 424 312 112 411 301 110 420 312 108 471 323 1485 M 117 404 298 106 426 339 87 434 313 121 412 290 122 433 315 118 427 313 1145 M 101 409 296 113 425 301 124 434 312 122 402 273 129 407 307 100 425 310 1155 M 98 408 320 88 413 299 114 428 313 115 395 273 122 434 301 133 422 308 1145 M 121 398 288 110 424 316 108 427 317 110 410 300 110 426 318 108 439 310 1295 M 116 409 321 88 421 343 78 427 336 91 397 299 98 437 322 115 428 312 11610 F 44 406 315 91 422 346 76 420 313 107 412 264 148 431 299 132 447 262 18510 F 50 409 328 81 403 296 107 421 283 138 406 242 164 419 245 174 439 244 19510 F 41 406 307 99 413 316 97 405 292 113 415 264 151 433 269 164 430 233 19710 F 44 410 322 88 414 346 68 414 299 115 428 244 184 415 277 138 439 246 19310 F 52 498 315 183 420 335 85 417 315 102 423 279 144 416 297 119 450 255 19510 F 57 410 302 108 419 310 109 418 265 153 423 261 162 426 268 158 442 286 15610 F 85 403 289 114 421 288 133 419 271 148 423 275 148 430 291 139 427 284 14310 F 88 405 299 106 406 306 100 414 253 161 413 250 163 421 293 128 451 253 19810 F 82 395 258 137 417 315 102 425 244 181 422 243 179 431 244 187 441 241 20010 F 83 405 295 110 413 333 80 426 300 126 419 284 135 424 291 133 427 248 17910 F 69 405 302 103 430 336 94 432 277 155 426 305 121 421 293 128 451 264 18710 F 84 402 324 78 437 335 102 422 290 132 419 283 136 430 291 139 448 271 17710 F 60 409 332 77 413 333 80 426 301 125 433 312 121 407 255 152 418 237 18110 F 63 403 331 72 430 297 133 424 286 138 426 250 176 417 258 159 441 241 20010 F 80 489 274 215 424 307 117 415 256 159 425 243 182 415 258 157 426 275 15110 F 87 406 325 81 428 312 116 421 252 169 427 248 179 426 289 137 443 238 20510 F 71 402 283 119 415 264 151 412 254 158 415 244 171 411 283 128 434 243 19118410 F 70 406 314 92 418 337 81 420 294 126 429 276 153 421 273 148 447 284 16310 F 77 399 304 95 428 332 96 444 320 124 393 253 140 432 264 168 425 244 18110 F 79 412 301 111 416 311 105 416 260 156 434 283 151 433 291 142 438 283 15510 M 47 411 327 84 411 321 90 418 301 117 415 303 112 406 270 136 440 318 12210 M 42 407 304 103 420 340 80 426 341 85 399 304 95 445 269 176 436 300 13610 M 72 413 339 74 424 332 92 421 306 115 417 298 119 426 301 125 432 281 15110 M 89 413 307 106 417 300 117 423 311 112 408 295 113 432 315 117 438 305 13310 M 67 407 321 86 419 320 99 426 305 121 418 289 129 410 310 100 424 294 13010 M 66 416 328 88 415 330 85 430 332 98 407 283 124 437 322 115 439 400 3910 M 58 412 296 116 420 304 116 424 301 123 421 300 121 445 327 118 432 304 12810 M 51 405 302 103 422 317 105 435 294 141 423 296 127 429 315 114 431 279 15210 M 54 413 310 103 410 295 115 428 305 123 418 313 105 421 322 99 435 303 13210 M 65 405 305 100 424 306 118 423 305 118 411 299 112 420 330 90 440 305 13510 M 49 378 306 72 418 303 115 426 312 114 408 304 104 417 319 98 436 303 13310 M 46 406 295 111 414 310 104 421 328 93 413 336 77 430 313 117 430 329 10110 M 55 408 313 95 422 339 83 420 283 137 413 269 144 430 306 124 420 290 13010 M 45 392 292 100 417 326 91 414 320 94 405 307 98 432 331 101 427 314 11310 M 53 412 311 101 419 320 99 427 316 111 397 288 109 428 323 105 424 307 11710 M 59 411 288 123 416 320 96 419 293 126 416 269 147 427 311 116 425 273 15210 M 61 411 344 67 419 342 77 425 338 87 389 289 100 437 323 114 419 315 10410 M 56 414 300 114 416 324 92 419 292 127 384 301 83 448 320 128 425 283 14210 M 62 411 299 112 418 302 116 450 330 120 419 312 107 431 325 106 419 304 11510 M 61 406 290 116 414 311 103 433 319 114 419 300 119 446 325 121 426 318 108185F2 bird weightsppm SexBabyID6/9/11 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 9/7/110 F 11 254 40 86 95 104 114 125 131 120 128 130 1300 F 10 257 36 83 99 105 113 120 129 126 139 141 1420 F 12 258 38 97 102 110 124 133 117 133 131 1330 F 7 259 38 98 103 111 114 111 114 117 131 1210 F 24 281 35 88 101 105 126 142 146 131 147 150 1400 F 21 283 25.9 76 90 91 101 110 107 112 115 134 1190 F 26 284 41 94 98 106 115 138 137 144 144 147 1500 F 17 286 35 88 103 103 114 124 121 146 140 139 1390 F 29 413 74 98 100 114 137 136 123 140 145 1440 F 41 415 83 104 109 117 121 130 148 145 156 1540 F 35 417 81 97 88 110 122 119 135 138 138 1230 F 47 420 79 94 99 95 115 113 120 121 121 1290 F 57 6.1 429 96 105 120 138 134 125 135 139 1300 F 60 5.9 437 94 103 115 131 139 136 141 143 1220 F 67 440 38 104 115 138 119 139 124 123 1190 F 89 445 47 111 117 128 142 148 143 145 1410 F 70 453 37 93 107 118 126 124 124 125 1260 F 105 454 43 102 114 126 128 131 133 140 1250 F 88 457 40 105 114 127 139 130 121 130 1310 F 97 458 38 96 105 124 127 118 120 130 1200 M 14 251 33.2 78 91 95 107 105 101 100 104 1060 M 9 252 37.4 82 95 99 107 106 106 106 107 1100 M 15 253 33.3 84 95 102 108 114 111 112 110 1090 M 13 255 40.2 92 105 118 123 126 121 118 117 1160 M 8 256 36 87 100 98 106 114 116 119 121 1180 M 25 260 40 90 98 104 111 109 108 110 1130 M 19 282 37.9 84 91 95 103 105 106 104 107 1080 M 20 285 40 87 96 98 104 112 109 113 114 1100 M 27 287 40 95 101 105 107 108 111 109 1121860 M 22 288 30 81 95 99 107 115 114 114 115 1140 M 16 289 34 93 96 102 108 106 106 108 1120 M 23 290 39 93 95 106 109 108 107 103 1040 M 43 412 74 102 105 108 107 109 110 1110 M 42 416 94 85 107 111 108 110 114 1120 M 37 418 91 93 102 106 105 106 106 1060 M 46 419 85 89 92 92 91 91 93 940 M 48 422 86 82 91 96 98 100 104 1070 M 28 424 85 88 92 100 99 100 97 1090 M 44 425 96 102 113 119 117 119 120 1210 M 45 426 88 96 98 99 101 105 105 1010.5 F 160 118 41 94 106 118 120 126 138 139 134 134 1180.5 F 171 119 41 87 102 113 123 128 148 142 148 151 1490.5 F 165 120 38 86 102 111 117 119 117 113 114 131 1340.5 F 168 125 31 78 97 109 108 114 117 134 93 106 1090.5 F 167 126 37.5 86 97 97 107 124 119 128 122 120 1320.5 F 159 127 41.8 88 97 107 111 122 121 128 134 124 1330.5 F 16 129 41.9 97 104 113 132 131 139 119 129 1280.5 F 173 130 42.5 97 109 119 138 141 139 145 139 147 1380.5 F 182 131 35.5 84 90 90 101 117 134 132 118 134 1250.5 F 183 134 40 86 98 105 123 131 128 109 129 129 1260.5 F 181 135 40.1 107 111 119 126 126 134 147 148 1450.5 F 179 136 33.1 83 97 102 101 122 138 137 142 137 1410.5 F 174 137 42 104 108 131 142 121 900.5 F 186 138 84 88 86 116 131 133 130 136 1310.5 F 194 141 99 99 111 116 113 121 142 136 1300.5 F 196 143 96 95 107 125 126 128 126 131 1180.5 F 192 144 91 94 105 128 128 132 133 131 1260.5 F 190 147 102 102 116 140 126 137 136 143 1370.5 F 193 148 89 92 98 106 129 125 130 1320.5 F 205 7.5 153 101 109 120 141 146 135 151 134 1491870.5 M 163 121 39.2 83 96 107 108 105 103 103 106 1080.5 M 172 122 38.5 84 95 103 104 106 106 108 109 1090.5 M 166 123 29.2 70 82 90 94 90 86 93 94 960.5 M 162 128 41.8 103 109 114 117 117 121 122 1220.5 M 178 132 33 79 91 96 102 105 106 108 108 1130.5 M 127 133 35 79 88 89 100 102 102 105 110 1100.5 M 185 140 79 97 97 109 114 114 115 118 1170.5 M 195 142 75 90 92 101 103 103 103 102 1050.5 M 197 145 71 84 92 95 95 96 99 95 980.5 M 188 146 91 91 99 102 101 104 106 1060.5 M 206 6.2 151 101 108 110 114 114 110 116 1180.5 M 204 6.7 152 70 85 88 102 104 103 104 106 1060.5 M 203 6.2 155 87 93 96 97 94 96 97 980.5 M 202 6.6 156 85 97 107 119 118 113 113 112 1120.5 M 200 6.7 157 85 91 106 108 101 100 101 1030.5 M 122 160 88 93 96 95 95 98 990.5 M 219 7.2 161 90 85 99 104 101 101 104 1030.5 M 249 186 43 98 102 112 109 106 106 1080.5 M 248 188 35 96 109 118 106 103 106 1070.5 M 150 190 36 93 103 108 110 111 117 1165 F 21 91 35.8 85 94 100 112 122 120 127 135 122 1315 F 17 93 39 91 106 106 124 135 137 144 142 143 1425 F 22 96 33.8 87 102 107 124 134 134 139 140 139 1435 F 19 97 35 79 91 96 109 116 118 124 117 125 1165 F 18 100 33 92 102 108 117 132 127 140 139 132 1355 F 20 104 41 104 104 122 131 128 133 137 140 1385 F 37 105 40.5 91 102 109 117 136 124 124 137 133 1405 F 32 106 34.8 83 93 103 114 132 127 136 108 124 1345 F 38 107 39.8 92 106 111 128 138 135 148 144 142 1425 F 36 111 41 93 105 108 122 138 139 138 139 141 1455 F 44 113 41 103 99 110 119 134 145 143 153 1551885 F 43 114 42 108 110 136 131 143 136 135 138 1355 F 54 115 44 118 121 147 153 140 153 1555 F 50 120 91 94 104 110 129 118 130 126 1085 F 62 123 92 92 101 123 121 127 120 125 1295 F 86 6 130 95 103 118 143 142 141 132 142 1455 F 78 140 89 90 102 129 123 131 131 134 1255 F 94 7.1 148 117 124 138 144 142 113 120 1505 F 106 151 45 96 98 107 125 128 116 111 1255 F 27 155 43 106 130 142 130 128 130 1505 F 29 158 42 105 109 109 1085 M 16 94 30.4 76 90 93 101 107 106 106 109 1105 M 31 95 40.8 93 106 114 110 109 105 116 117 1185 M 26 98 39 87 89 104 100 109 109 95 95 975 M 30 99 33.6 76 90 91 96 97 108 102 103 1035 M 23 101 31 77 88 89 94 101 99 106 108 1095 M 42 102 37 96 95 104 107 95 103 107 1145 M 34 103 40 96 97 101 98 112 124 124 1215 M 40 108 39 93 110 115 119 119 122 105 105 1065 M 39 109 40 83 96 102 107 105 102 110 114 1235 M 41 110 35 77 90 95 101 109 109 109 110 1085 M 47 112 40 90 97 103 109 109 106 114 113 1165 M 48 116 39 99 104 116 115 114 104 106 1075 M 53 117 70 85 85 96 100 102 117 120 1245 M 52 118 102 104 114 119 118 105 104 1045 M 58 119 75 89 89 98 102 102 118 119 1205 M 68 121 101 104 110 116 118 111 112 1145 M 59 122 97 102 105 109 100 100 102 1055 M 67 6.7 127 105 122 125 1275 M 66 6.3 131 94 98 101 99 99 112 114 10910 F 40 41 40 91 103 109 117 124 124 137 147 131 15110 F 36 44 38 90 105 110 119 123 127 137 135 138 13818910 F 52 48 35 98 104 114 116 125 131 121 132 13610 F 47 50 42 94 111 114 126 135 139 140 137 133 14310 F 45 52 41 99 103 111 119 126 132 131 139 14010 F 53 57 100 99 112 128 127 131 137 140 13510 F 62 7.1 60 82 98 106 117 119 121 135 115 135 13910 F 59 6.4 63 86 104 105 122 136 125 134 137 136 13610 F 71 7.5 69 84 111 109 115 112 150 125 147 14210 F 65 5.9 70 77 103 102 106 112 137 136 132 13810 F 69 6.7 71 92 108 141 133 141 137 134 13910 F 66 6.3 77 108 108 119 127 125 120 133 13910 F 185 79 47 105 114 125 139 139 142 135 14110 F 189 80 46 108 119 132 145 141 141 138 13010 F 184 82 44 103 114 137 129 144 140 146 14310 F 183 83 50 117 131 138 135 147 159 157 15910 F 187 84 38 97 102 101 113 127 108 121 13210 F 188 85 44 110 115 140 138 129 146 138 13410 F 96 87 51 84 121 127 135 143 140 142 148 14610 F 93 88 45 108 120 141 130 140 138 137 14210 M 95 42 37.9 83 97 96 106 113 109 110 106 10910 M 37 45 34.6 88 92 96 99 99 99 102 10610 M 41 46 36 88 97 97 102 109 111 110 105 10910 M 43 47 40 92 104 107 108 107 107 108 107 11410 M 44 49 37 85 99 106 110 114 114 115 117 11810 M 51 51 40 98 97 104 107 108 110 113 11310 M 42 53 39.5 95 98 102 104 104 103 103 10810 M 46 54 36 95 95 102 109 111 116 114 11710 M 49 55 80 94 95 101 105 103 108 108 11110 M 58 56 74 91 92 99 103 103 105 106 11010 M 57 58 75 93 94 104 108 110 114 115 11410 M 55 59 89 85 89 103 108 106 111 115 11210 M 54 6 61 80 96 109 116 110 110 109 108 11119010 M 80 6.8 62 77 91 93 99 105 107 107 107 10910 M 64 6 64 85 97 95 103 109 108 109 108 11210 M 60 65 96 97 106 110 112 115 115 11810 M 80 6 66 103 102 102 102 110 110 11210 M 72 67 99 102 109 107 110 110 11310 M 79 72 50 111 117 114 111 113 109 11510 M 75 89 44 114 115 110 112 113 111 114F2 sperm measurementsppm ID Motile 1 Motile 2 Motile Average Progressive 1 Progressive2 Progressive Average Sperm/gram 1 Sperm/gram 2 Sperm/gram Average0 251 23 34 28.5 4 9 6.5 146.4 258.8 202.60 252 14 30 22 4 6 5 222.2 305 263.60 260 10 12 11 3 3 3 74.7 146.9 110.80 282 9 8 8.5 2 1 1.5 316.4 379.7 348.050 287 15 47 31 3 11 7 212 386.3 299.150 288 50 65 57.5 16 17 16.5 298 347.7 322.850 290 28 34 31 10 19 14.5 303.2 386.9 345.050 416 48 53 50.5 11 16 13.5 334.6 470.5 402.550 422 28 42 35 3 18 10.5 139.4 217.8 178.60 426 22 18 20 3 2 2.5 161.2 213.5 187.350.5 121 26 28 27 6 4 5 273.5 273.5 273.50.5 122 46 23 34.5 21 11 16 305 204.8 254.90.5 128 46 39 42.5 17 17 17 153.1 153.1 153.11910.5 133 28 25 26.5 13 21 17 152.1 114.1 133.10.5 140 36 36 36 16 7 11.5 181 237.3 209.150.5 145 51 62 56.5 15 19 17 239.6 274.5 257.050.5 152 47 44 45.5 6 13 9.5 139.4 196.1 167.750.5 155 15 9 12 5 1 3 190.7 264.5 227.60.5 160 63 55 59 14 13 13.5 256.2 209.1 232.650.5 161 55 32 43.5 17 12 14.5 279.7 248.3 2645 94 39 42 40.5 9 12 10.5 115 90.6 102.85 99 37 42 39.5 13 14 13.5 267.9 235.3 251.65 101 17 4 10.5 0 0 0 137.8 123.6 130.75 108 31 28 29.5 11 3 7 140.8 128.7 134.755 109 55 73 64 26 30 28 168.9 309.7 239.35 110 32 39 35.5 4 11 7.5 244 180.1 212.055 117 37 38 37.5 6 14 10 283.2 252.7 267.955 131 19 17 18 4 6 5 90.6 62.7 76.655 133 0 0 0 0 0 0 134.4 181.7 158.055 134 31 36 33.5 13 12 12.5 59.8 123.2 91.510 42 47 27 37 7 7 7 112 112 11210 47 47 29 38 9 8 8.5 144.5 147.6 146.0510 49 29 57 43 7 23 15 213.9 166.4 190.1510 51 48 37 42.5 11 4 7.5 217.8 148.1 182.9510 53 46 59 52.5 16 18 17 277.5 205.1 241.310 54 49 30 39.5 17 5 11 140.8 172.9 156.8510 59 38 36 37 17 14 15.5 125.5 115 120.2510 61 74 64 69 25 22 23.5 230.9 196.1 213.510 65 30 26 28 8 5 6.5 257.9 257.9 257.910 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 64193 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 306194 F2 onset to pubery

Trenbolone (ppm) ID first da y 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 50195 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 59196 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 0.939197 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 0.62198 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 1.013199 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 0.921200 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