# TOXICOLOGY STUDY NO. 87-XE-08JG- 10, SEPTEMBER 2008 – APRIL 2010

PROTOCOL NO. 08JG-80-08-04-02

TOXICOLOGY PORTFOLIO

TWO-GENERATION TEST FOR ENDOCRINE DISRUPTION AND REPRODUCTIVE

TOXICITY IN JAPANESE QUAIL ( COTURNIX JAPONICA ) USING ENDOSULFAN

PREPARED BY MICHAEL J. QUINN JR., HEALTH EFFECTS RESEARCH

PROGRAM

Approved for public release; distribution unlimited.

Specialty: 500C, Toxicity Tests

# Use of trademarked name(s) does not imply endorsement by the U.S. Army

but is intended only to assist in the identification of a specific product. 

# ACKNOWLEDGEMENTS

The authors would like to acknowledge Les T ouart for the opportunity to participate in

this extensive undertaking. We’d also like to thank Patience Browne for her constant

help and support throughout this endeavor. A warm thank you also goes to Pat Beall,

Valerie Adams, William Eck, Emily Lent, Lar ry Williams, Matthew Bazar, Will McCain,

Lee Crouse, Mark Way, Arthur O’Neill, and Christiana Grim for th eir assistance during

the conduct of this study. The authors also appreciate the large amount of endosulfan

that was used to complete this long term study that was donated by Robert Everich of

Makhteshim-Agan of North America Inc. Th is study was done concurrently with two

other similar ones by U.S. Geological Survey , Patuxent Wildlife Research Center and

Wildlife International Ltd.; we’ve support ed each other throughout each study’s conduct

in the development and refinement of this test method, and t he authors are grateful for

having had the opportunity to collaborate with su ch outstanding researchers in this field.

## Toxicology Study No. 87-XE-08JG-10, Protocol No . 08JG-80-08-04-02, September 2008-April 2010



# 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-08JG-10

Protocol No. 08JG-80-08-04-02

Two-Generation Test for Endocrine Disruptio n and Reproductive Toxicity in Japanese

Quail ( Coturnix japonica ) Using Endosulfan

Authors

Michael J. Quinn, Ph.D.

Michelle Cook, M.S.

Terry Hanna

Alicia Shifflet, A.S.

Allison Jackovitz, B.S.

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

Mark S. Johnson, Ph.D.

Study Completion Date

April 2010

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-80-08-04-02

## Toxicology Study No. 87-XE-08JG-10, Protocol No . 08JG-80-08-04-02, September 2008-April 2010



# 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:

No deviations from the aforem entioned regulation affected the quality or integrity of the

study or the interpretation of the results.

________________________ ____ _________________________ Michael J. Quinn Date

Study Director

________________________ _____ _________________________ Mark S. Johnson Date

Program Manager

Health Effects Research

## Toxicology Study No. 87-XE-08JG-10, Protocol No . 08JG-80-08-04-02, September 2008-April 2010



# Prepared by:

________________________ _____ __________________ Michael J. Quinn, Jr. Date

Biologist, Health Effects Research Program

Reviewed by:

________________________ ____ __________________ Craig A. McFarland Date

Toxicologist, Health Effects Research Program

Approved by:

________________________ ____ __________________ Mark S. Johnson Date

Director, Portfolio of Toxicology

# Toxicology Study No. 87-XE-08JG-10, Protocol No. 08JG -80-08-04-02,

September 2008-April 2010

i Table of Contents

Paragraph Page

1. REFERENC ES ................................................................................................ 1

2. PURPOS E ....................................................................................................... 1

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

4. SCOP E ............................................................................................................ 1

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

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

6.1. Experiment al Design ............................................................................... 2

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

6.3. Quality Assuranc e ................................................................................... 4

7. RESULT S ........................................................................................................ 5

7.1. Eggshell Thic kness and Str ength ............................................................ 5

7.2. Egg Pr oduction ........................................................................................ 6

7.3. Sp erm Measur es ..................................................................................... 9

7.4. Growth and Body Wei ght......................................................................... 10

7.5. Feed Cons umption .................................................................................. 16

7.6. Cloacal Gland Area ................................................................................. 21

7.7. Yolk Endosul fan Content ......................................................................... 22

7.8. Onset to Puberty ..................................................................................... 23

7.9. Copulator y Behavior ................................................................................ 24

# Toxicology Study No. 87-XE-08JG-10, Protocol No. 08JG -80-08-04-02,

September 2008-April 2010

ii 7.10. Organ We ights ...................................................................................... 29

7.1. Perivitelline Layer Sperm Penetra tion ..................................................... 32

7.2. Fertility and Hatchability .......................................................................... 33

8. DISCUSSI ON .................................................................................................. 34

9. CONCLUSION ................................................................................................ 36

10. POINT OF CO NTACT .................................................................................... 37

Appendices

A - REFERENC ES ............................................................................................... A-1

B - QUALITY ASSURANCE STATEMEN T .......................................................... B-1

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

D - ANIMAL USE PROTOCOL WI TH MODIFICAT IONS ..................................... D-1

E - DEVIATIONS TO THE PROTOCOL..................................................... E-1

F - DIET PREPARATION REPORT........................................................... F-1

G - CONTRIBUTING SCIENTIST’S STATIS TICAL ANALYSIS REPORT.......... G-1

H - DRAFT PATHOLOGY REPORT......................................................... H-1

I - HORMONE ANALYSIS ME THODS......................................................... I-1

J - STATISTICAL ANALYSIS OF HORMONE DATA..................................... J-1

K - RAW DATA...................................................................................... K-1

LIST OF FIGURES

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 ..................................................... 6

Table 5. F0 Egg Production ................................................................................. 7

Table 6. F1 Egg Production ................................................................................. 7

Table 7. F2 Egg Production ................................................................................. 8

Table 8. F0 Spe rm Measures .............................................................................. 9

Table 9. F1 Spe rm Measures .............................................................................. 9

Table 10. F2 Spe rm Measures ............................................................................ 10

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

# Toxicology Study No. 87-XE-08JG-10, Protocol No. 08JG -80-08-04-02,

September 2008-April 2010

iii Table 12. F1 Young Growth Measures ................................................................ 12

Table 13. F1 Adult Body Weight .......................................................................... 13

Table 14. F2 Young Growth Measures ................................................................ 14

Table 15. F2 Adult Body Weight .......................................................................... 15

Table 16. F0 Female Feed Consumpt ion ............................................................ 17

Table 17. F0 Male F eed Consumptio n ................................................................ 17

Table 18. F0 Paired Feed Consumption .............................................................. 18

Table 19. F1 Female Feed Consumpt ion ............................................................ 18

Table 20. F1 Male F eed Consumptio n ................................................................ 19

Table 21. F1 Paired Feed Consumption .............................................................. 19

Table 22. F2 Female Feed Consumpt ion ............................................................ 20

Table 23. F2 Male F eed Consumptio n ................................................................ 20

Table 24. F0 Cloacal Gland Area ........................................................................ 21

Table 25. F1 Cloacal Gland Area ........................................................................ 21

Table 26. F2 Cloacal Gland Area ........................................................................ 22

Table 27. F0 Onset to Puberty ............................................................................ 23

Table 28. F1 Onset to Puberty ............................................................................ 23

Table 29. F2 Onset to Puberty ............................................................................ 24

Table 30. F0 Number of Mount Attemp ts ............................................................ 24

Table 31. F0 Onse t to Mount ............................................................................... 25

Table 32. F0 Number of Su ccessful Copulat ions ................................................ 25

Table 33. F0 Onset to Su ccessful Copula tion ..................................................... 25

Table 34. F1 Number of Mount Attemp ts ............................................................ 26

Table 35. F1 Onse t to Mount ............................................................................... 26

Table 36. F1 Number of Su ccessful Copulat ions ................................................ 27

Table 37. F1 Onset to Su ccessful Copula tion ..................................................... 27

Table 38. F2 Number of Mount Attemp ts ............................................................ 28

Table 39. F2 Onse t to Mount ............................................................................... 28

Table 40. F2 Number of Su ccessful Copulat ions ................................................ 28

Table 41. F2 Onset to Su ccessful Copula tion ..................................................... 29

Table 42. F0 Female Organ Weight s .................................................................. 29

Table 43. F0 Male Organ Weight s ...................................................................... 30

Table 44. F1 Female Organ Weight s .................................................................. 30

Table 45. F1 Male Organ Weight s ...................................................................... 31

Table 46. F2 Female Organ Weight s .................................................................. 31

Table 47. F2 Male Organ Weight s ...................................................................... 31

Table 48. Male G onadal Weight s ........................................................................ 32

Table 49. Ovarian Fo llicle Counts ....................................................................... 32

Table 50. Perivitelline Lay er Sperm Penet ration ................................................. 33

Table 51. F0 Generation’s Egg Fertility and Hatchability ..................................... 33

Table 52. F1 Generation’s Egg Fertility and Hatchability ..................................... 34

# Toxicology Study No. 87-XE-08JG-10, Protocol No. 08JG -80-08-04-02,

September 2008-April 2010

iv Table 53. F0 Generation Adult Hormone Da ta .................................................... 34

Table 54. F1 Generation Adult Hormone Da ta .................................................... 35

Table 55. F2 Generation Adult Hormone Da ta .................................................... 36

Table 56. Male Japanese Quail Effect s Comparison Across G enerations .......... 37

Table 57. Female Japanese Quail Effects Comparison Across Generations ...... 38

Table 58. Male Copulatory Behavio r Comparison Among G enerations .............. 38

LIST OF FIGURES

Figure 1. Yolk Endo sulfan Conten t ...................................................................... 22

# TOXICOLOGY STUDY NO. 87-XE-08JG-10

PROTOCOL NO. 08JG-80-08-04-02

TWO-GENERATION TEST FOR ENDOCRINE DISRUPTION AND REPRODUCTIVE

TOXICITY IN JAPANESE QUAIL ( COTURNIX JAPONICA ) USING ENDOSULFAN

SEPTEMBER 2008 – APRIL 2010

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). It wa s conducted as part of the on-going effort, under

the U.S. Army Environmental Quality Technol ogy, Ordnance Program, Pollution Prevention Pillar,

under the direction of Research, Devel opment and Engineering Command (RDECOM),

Environmental Acquisition Logistics & Sustainment Program.



# 4 Scope

This report documents the conduct, findings, an d conclusions of using endosulfan to develop and

assess a two-generation test for endocri ne disruption in Japanese Quail.



# 5 Background

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

Screening Program comprised of a battery of Tier 1 screening assays and Tier 2 tests. One of the

Tier 2 tests under development is a two-gener ation avian assay using Japanese quail (Coturnix

japonica). This test is designed to characteri ze dose-response relationships and immediate and

latent adverse consequences associated wi th exposure to putative endocrine modulating

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

endocrine modulating compounds on myriad avian spec ies, and represents terrestrial, oviparous

wildlife species in general. This study will us e endosulfan to fully ev aluate the proposed test

method for its ability to detect adverse effects and characterize dose responses in a two-generation

paradigm, and to develop and optimize a protoc ol for the avian two-generation assay for

characterizing endocrine disruptors. This work will help to determine the value added by examining

endocrine endpoints over two generations in compar ison to tests that include only one generation,

such as the Ecological Effects Guideli nes for Avian Reproduction Tests (EPA, 1996).

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-April 2010

2 6 Materials and Methods

6.1 Experimental Design

The parental (F0) generation was first exposed to endosulfan (CAS No. 115-29-7), a known

estrogenic endocrine disrupting chemical, at four weeks of age and throughout the remainder 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 tr eatments in ovo (embryonic) through maternal

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

incubated and hatched to produce the second generat ion (F2). The F2 generation was exposed to

endosulfan only in ovo through maternal deposition; all feed for the F2 generation was untreated.

Measures of reproductive behavior, circulati ng hormone levels, egg quality, reproduction, and

development were assessed in the P, F1, and F2 generations as described below. Additional

measures of growth, reproductive maturity, imm unocompetence, were assessed in the F1 and F2

generations as described below.

Chemical Treatments:

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 lowest repo rted LC50 from a five-day dietary exposure to

endosulfan in 14-day old Japanese quail was found to be 1250 ppm (FWS, 1975). The treatment

levels for this study were 0 (control), 30, 60, 125, and 250 ppm. The highest dose is one-fifth of the

lowest known LC50 value and assumed not to cause mortality or other severe signs of parental

toxicity that would have precluded the evaluati on of reproductive parameters. The lowest

concentration should produce no effects on adult health or reproductive measures. Endosulfan

was supplied by Makhteshim-Agan of North America Inc., Norwalk, CT. The test diets were

prepared by Wildlife International Ltd., East on, MD, USA. Homogeneity and concentration

verifications for endosulfan were made by USACHP PM Directorate of Laboratory Services. Upon

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

all treatment levels were found to be within acce ptable ranges, each subsequent batch of treated

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

the results of these tests were suggestive of fu rther dose levels being tested, according to GLP

standards.

Eggs:

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

provide sufficient numbers of chicks to establish at least 12 breeding pairs (20 pairs was the goal).

Twenty males and females were used for the study, beginning exposure to the treatments at week

4. Optimally, a goal of at least 6 eggs were set fo r hatch for each parental pair of the F0 generation

to ensure that a minimum of 60 eggs would be hatched for each test level, providing sufficient

numbers of chicks to establish at least 12 breeding pairs (20 is the goal) per F1 test level. If a

treatment level produced insufficient eggs to set the minimum, that level would be dropped from

further analysis in the test. Optimally, a goal of at least 6 eggs was 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.

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

origin. The egg production rate was determined for each hen.

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-April 2010

3 After the 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 the other hatching chicks were considered

unhatched. No assistance was given to chicks du ring hatching; chicks that remained alive but had

no clear ability to hatch on their own were humanely removed from the egg and decapitated

immediately.

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

thickness. Eggshell strength was measured by an Egg Force Reader (Orka Technology Ltd.) that

evaluates the breaking point of an egg’s shell. Eggshell thickness was measured using an Egg

Shell Thickness Gauge (Orka Technology Ltd.) that measures thickness through ultrasound.

General Reproductive Fitness Measures and Observations:

During the treatment period, all birds were observed da ily for overt signs of toxicity or other clinical

signs of illness or trauma; injuries sustained and subsequent treatments were recorded.

Observations included mortality and clinical signs of toxicity such as lethargy, depression, wing

droop, ruffled feathers, etc. If an individual or pair was removed during the study due to nontreatment related injury or death, data from all preceding weeks were reevaluated. Clearly

moribund individuals or those otherwise in severe distress were euthanized by carbon dioxide

asphyxiation.

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

liver, kidneys, gonads, and thyroid glands were re corded immediately following death/euthanasia.

Any morphologic abnormalities were recorded in the study’s notebook or necropsy sheets. Adult

birds that died or were euthanized during the co urse of the treatment periods were necropsied

similarly. Histopathology was performed on the brain, right thyroid gland, liver, bursa of Fabricius,

kidneys, and gonads. Because of the small size of the tissues, the thyroid glands from half of the

adults were fixed in formalin for histopathologic analyses, while thyroid glands from the remaining

half were flash frozen and stored for later glandular thyroxine (T4) content. Immediately following

euthanasia, spermatozoa were released from a we ighed section of epididymis to determine percent

motility and progression and sperm cell concentrat ion per gram epididymis using an IVOS Sperm

Analyzer, Version 12.1 (Hamilton-Thorne Resear ch, Beverly, MA, USA; methods described by

McFarland et al., 2008).

Biochemical/Endocrine Measurements:

All hormone analyses were conducted at the Univer sity of Maryland, Department of Animal and

Avian Sciences. Glandular T3 and T4 content and serum estradiol were measured by

radioimmunoassay (RIA) kits (Siemens) and serum testosterone was measured via an Enzyme

Linked Immuno Sorbent Assay (ELISA) kit (Caymen). Details of their methodologies can be found

in Appendix I.

Behavioral measures:

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 a minimum of 90% of

control males reached reproductive maturity. The re productive behaviors measured included: time

taken for mount attempts to be initiated, number of mount attempts made within the time limit (3

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

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-April 2010

4 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:

Growth was measured every three days in chicks in the F1 and F2 generations 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.



# 6.2 Data Analysis

All observational data were recorded in the labora tory notebook and work sheets where applicable.

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

in the Contributing Scientist Report, Appendix G.



# 6.3 Quality Assurance

The U.S. Army Public Health Command (formerl y USACHPPM) Good Laboratory Practice Policy—

Policy Memorandum 74 states — All experiments and studies conducted by any element of the

AIPH Portfolio of Toxicology will be compliant with the applicable Good Laboratory Practice (GLP)

Standard guideline (USACHPPM 2009). For this study, the test article dictates that the following

regulation applies:

Title 40, Code of Federal Regulations (CFR), Pa rt 792, Toxic Substances Control Act (TSCA),

Good Laboratory Practice Standards: Final Rule

According to this policy and that these results may be used in regulatory decisions involving the

EPA, this study was conducted in compliance with TSCA GLP standards and followed the

appropriate regulatory testing guidelines.. In co mpliance with the GLP requirements, the AIPH

Quality Systems Office audited critical phases of this study. A Quality Assurance Statement is

provided in Appendix B, which provides the dat es of these audits along with the audited phases

and the dates that the results of the audits were reported to Management and the Study Director.

The additional GLP requirement of identifying the lo cation of the archived information is provided in

Appendix C as well as the names of personnel cont ributing to the performance of this study.

In addition, all Investigators and technicians adher ed to The Guide for Care and Use of Laboratory

Animals (U.S. Department of Health, Educatio n, and Welfare Publicati on No. NIH 86-23, 1996).



# Table 1. List of Critical Events

Critical Event Date of Event

Animal Use Protocol 04/18/2008

Animals Received (eggs) 11/14/2008

Study Start 09/03/08

Experimental Start 12/29/08

Experimental Completion 04/01/10

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-April 2010

5 Necropsy Start 07/21/2009

Necropsy Completion 04/01/2010

Study Completion 04/2010

7 Results

 Eggshell Thickness and Strength. Endosulfan did not cause any differences in eggshell

thickness for eggs produced by the F0 and F2 generations (Tables 2-4). Eggshell thickness was

reduced in the two highest dose groups of the F1 generation compared to controls, and eggshells

from the 250 ppm dose group were also significant ly thinner than those from the 30 ppm treatment

level. Average eggshell strength wa s significantly higher in the 250 ppm dose group than controls

for both the F0 and the F1 generations, however endo sulfan had no effect on this trait in the F2

generation.



# Table 2. Eggshell thickness and strengt h from the F0 generation of Japanese

quail (Coturnix japonica) exposed to endosulfan.

F0 Generation

‐ Egg Strength

Thickness (mm) Strength

p

‐ value

= 0.788 p

‐ value

= 0.045*

Dose Average Std. Error 

# Average Std. Error



# 0 0.93 0.07



# 30 0.890 0.060 

# 1.05 0.06



# 60 0.981 0.146 

# 1.05 0.05



# 125 0.846 0.051 

# 1.19 0.05



# 250 0.869 0.066 

# 1.11 0.06



# Table 3. Eggshell thickness and strengt h from the F1 generation of Japanese

quail (Coturnix japonica) exposed to endosulfan.

F1 Generation

‐ Egg Strength

Thickness (mm) Strength

p

‐ value

= 0.000* p

‐ value

= 0.016**

Dose Average Std. Error 

# Average Std. Error



# 0 0.938 0.052 

# 0.96 0.05



# 30 0.845 0.047 

# 1.02 0.05



# 60 0.813 0.052 

# 1.04 0.04



# 125 0.753 0.032 

# 1.09 0.05



# 250 0.647 0.038 

# 1.19 0.04

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-April 2010

6 Table 4. Eggshell thickness and strengt h from the F2 generation of Japanese

quail (Coturnix japonica) exposed to endosulfan.

F2 Generation

‐ Egg Strength

Thickness (mm) Strength

p

‐ value

= 0.924 p

‐ value

= 0.053

Dose Average Std. Error 

# Average Std. Error



# 0 0.476 0.024 

# 0.89 0.06



# 30 0.486 0.024 

# 1.28 0.17



# 60 0.500 0.031 

# 1.03 0.07



# 125 0.471 0.020 

# 1.00 0.05



# 250 0.483 0.021 

# 1.14 0.06

 Egg Production. Egg production was not signi ficantly affected in the F0 and F2 generations

(Tables 5 and 7). In the F1 generation, females from the 250 ppm dose group produced the largest

number of eggs between the first and second weeks and between the second and third weeks

(Table 6).Toxicology Study No. 87-XE-08JG-10, Protocol No. 08JG-80-08-04-02, September 2008-April 20107Table 5. Numbers of eggs produced per week by F0 generation Japanese quail (Coturnix japonica) exposed toendosulfan.Table 6. Numbers of eggs producedper week by F1 generation Japanese quail (Coturnix japonica) exposed toendosulfan.ModelTerm P‐value Dose Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Overall00.45 2.00 3.35 4.65 4.50 3.55 3.15 2.90 3.20 2.85 3.00 2.75 2.40 2.68300.60 2.95 3.80 4.30 4.30 5.20 5.85 6.35 6.65 6.15 6.00 6.25 6.05 4.39600.40 2.40 3.80 4.25 4.55 4.95 4.70 5.60 5.10 5.40 5.55 5.60 5.40 3.861250.05 2.05 4.35 5.45 5.95 5.35 6.20 5.95 5.60 5.75 6.05 6.25 5.85 3.652500.40 2.45 4.15 5.35 5.40 4.75 5.25 5.10 5.75 5.40 4.65 5.20 5.75 3.99Overall0.29 2.35 3.87 4.77 4.90 4.71 4.90 5.00 5.11 4.94 4.90 5.00 4.83Week 0.000*Statisticallysigni ficantinteraction(p‐value<.05)indicatedthateggproductionchangefromweektoweekwassigni ficantlydi fferentbetweendosegroups.GEEEstimatedMarginalMeansF0GenerationEggProductionGEESignificanceTestsInteraction‐Dosebyweek0.000*Dose 0.051ModelTerm P‐value Dose Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19 Week20 Week21 Overall00.95 2.80 4.45 5.45 5.40 5.70 4.90 5.20 4.50 5.30 5.90 5.90 5.30 5.20 4.35 2.75 4.30300.10 1.60 3.50 5.50 6.05 6.30 5.55 5.35 5.00 6.05 5.80 5.60 5.75 5.45 5.05 4.25 3.85601.55 3.50 4.95 5.65 5.40 5.70 5.30 3.95 4.50 5.50 5.55 5.40 5.00 4.30 4.40 3.55 4.451250.25 2.35 4.20 5.60 5.70 6.05 5.55 4.95 4.15 5.00 5.20 5.10 5.95 5.90 4.75 3.70 4.012500.20 3.00 4.95 5.85 5.60 5.45 4.95 5.40 4.35 5.50 5.60 5.25 5.50 5.15 4.45 4.05 4.04Overall0.37 2.56 4.37 5.61 5.62 5.83 5.24 4.94 4.49 5.46 5.60 5.44 5.49 5.17 4.59 3.62Interaction‐Dosebyweek0.000*GEESignificanceTests*Statisticallysigni ficantinteraction(p‐value<.05)indicatedthateggproductionchangefromweektoweekwassigni ficantlydifferentbetweendosegroups.GEEEstimatedMarginalMeansF1GenerationEggProductionDose 0.539Week 0.000Toxicology Study No. 87-XE-08JG-10, Protocol No. 08JG-80-08-04-02, September 2008-April 20108Table 7. Numbers of eggs produced per week by F2 generation Japanese quail (Coturnix japonica) exposed toendosulfan.ModelTerm P‐value Dose Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19 Overall00.35 0.85 3.25 3.95 3.85 4.70 5.95 6.20 5.45 5.70 5.35 5.35 5.85 5.40 3.63300.60 1.60 3.30 3.80 4.35 5.15 5.80 6.35 5.30 6.25 5.70 5.80 5.65 5.70 4.07600.45 0.75 3.15 3.85 4.75 5.10 5.65 6.50 5.15 6.05 5.40 5.85 5.65 5.05 3.731250.05 0.55 2.95 3.40 4.50 5.40 4.95 6.05 4.90 5.15 4.35 5.00 5.90 5.90 2.952500.30 1.00 2.55 2.40 3.45 4.00 3.65 4.50 4.25 4.85 4.05 4.30 4.80 4.80 2.93Overall0.27 0.89 3.03 3.43 4.15 4.84 5.12 5.87 4.99 5.57 4.93 5.22 5.55 5.35Dose 0.087Week 0.000GEESignificanceTestsInteraction‐Dosebyweek0.000**Statisticallysigni ficantinteraction(p‐value<.05)indicatedthateggproductionchangefromweektoweekwassignificantlydifferentbetweendosegroups.GEEEstimatedMarginalMeansF2GenerationEggProduction

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-April 2010

9  Sperm Measures. The number of sperm/g epididymis was the only sperm parameter to be

affected by endosulfan in the F0 generation (Table 8); sperm count in the 125 ppm dose group was

lower than that of controls. Percent progressive sperm was the only parameter affected in the F1

generation; sperm from the 250 ppm group tended to move in a progressive direction more often

than those from the 0, 30, and 60 ppm groups (Table 9). There was also a greater percentage of

progressively moving sperm from the control and 60 ppm dose groups in the F2 generation (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 endosulfan.

F0 Generation

‐ Sperm

% Motile % Progressive Sperm/gram

p

‐ value

= 0.600 p

‐ value

= 0.983 p

‐ value

= 0.033*

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

0 93.5 1.2 17.6 1.7 6917.7 385.4

30 84.3 7.9 17.3 2.2 5278.7 786.3

60 89.9 2.1 16.8 1.3 4786.7 709.7

125 86.8 4.0 18.1 2.6 4212.0 610.7

250 88.4 3.3 16.7 1.7 5132.3 443.6

* Tukey Post

‐ hoc test indicated that dose 125 had a significantly different average sperm per

gram compared to the control group.



# 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 endosulfan.

F1 Generation

– Sperm

% Motile % Progressive Sperm/gram

p

‐ value

= 0.328 p

‐ value

= 0.010* p

‐ value

= 0.338

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

0 37.3 8.2 3.8 1.5 443.2 56.6

30 31.6 5.6 3.1 1.0 342.3 55.0

60 45.3 5.9 5.5 1.3 496.2 46.0

125 44.4 5.9 4.5 0.8 427.7 60.4

250 47.8 3.5 9.8 1.9 465.2 53.0

° ID 177 (dose 250) excluded from analysis: recorded 0's for all three measurements

* Tukey post

‐ hoc test indicated that dose 250 has significantly greater percent progressive

average compared to dose 60, 30, and the control group.

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-April 2010

10 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 endosulfan.

F2 Generation

‐ Sperm

% Motile % Progressive Sperm/gram

p

‐ value

= 0.338* p

‐ value

= 0.014* ° p

‐ value

= 0.303*

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

0 77.8 4.0 16.0 1.6 5585.6 698.2

30 78.4 3.3 12.3 1.5 5167.8 856.9

60 85.4 1.4 15.6 0.8 6879.8 579.1

125 81.4 1.8 12.2 0.8 5663.6 374.8

250 66.2 6.1 11.6 1.7 5284.2 653.5

* P

‐ value calculated from Kruskall

‐ Wallis test

‐ homogeneity of variance assumption violated

for parametric ANOVA test.

° Post hoc evaluation indicates that the control group and dose 60 had significantly greater

average percent progressive sperm compared to dose 30, 125, and 250.



##  Growth and Body Weight. Chick growth was not assessed in the F0 generation because the

birds began exposure to endosulfan at 4 weeks of age. Adult female F0 generation body weights

were not different, however a significant reduction in body weight was observed in males from the

125 and 250 ppm treatment levels (Table 11).

No differences in culmen length were observed for the F1 generation (Table 12), however the

average tarsus length for week old control chicks was larger than that of the 60 ppm dose group.

Statistical differences in body weight for the fi rst five weeks of age were observed for the F1

generation, but these changes were minimal and sp oradically distributed among the does groups.

The dose-by-week interaction was statistically si gnificant for females 6-20 weeks of age in the F1

generation. All dose-by-week interactions were significant, as there was a great amount of

variation between doses from week to week. Average body weights were typically highest in

females in the 250 ppm dose group, with female body weights lowest in the control and 60 ppm

dose groups. The dose-by-week interaction was not statistically significant for the males, however

week and dose individually were. The control gr oup’s average body weight was significantly lower

than the other four dose groups, with the 30 ppm treatment level having a consistently lower

average body weight than observ ed at the 60 ppm level.

No significant differences in body weight, tarsus, or culmen measurements were observed in chicks

from the F2 generation (Table 13). Adult F2 females from the 60 ppm dose group had lower

average body weights than those from the 30 and 125 ppm dose groups. Control male adults from

the F2 generation had lower average body weights compared to all other groups. Additionally,

males at the 125 ppm treatment level had higher average body weights than all other levels.Toxicology Study No. 87-XE-08JG-10, Protocol No. 08JG-80-08-04-02, September 2008-April 201011Table 11. Mean body weightfrom adult female and maleF0 generation Japanese quail (Coturnix japonica)exposed to endosulfan.Factor P‐value Dose Week6 Week7Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Overall0105.1 113.9 124.7 130.0 129.8 132.1 131.5 136.3 137.4 138.2 134.1 141.5 138.0 130.230108.9 120.5 125.5 125.7 129.7 133.3 136.4 138.2 138.2 139.5 139.7 140.4 142.3 132.260103.7 115.2 125.3 127.6 130.5 133.7 131.5 134.6 136.9 137.0 138.3 140.1 142.9 130.6125104.3 116.6 126.7 129.5 131.9 131.5 135.9 135.9 134.0 137.7 140.7 138.6 141.6 131.1250105.1 117.6 125.4 130.3 133.7 132.3 134.3 135.0 141.9 139.4 133.9 140.7 143.0 131.7Overall105.4 116.7 125.5 128.6 131.1 132.5 133.9 136.0 137.7 138.3 137.3 140.3 141.5Factor P‐value Dose Week6 Week7Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Overall098.2 103.6 105.2 107.6 107.8 109.5 108.0 109.2 110.3 111.5 111.9 113.1 115.4 108.53099.5 104.8 106.4 106.7 107.8 110.2 107.9 111.3 112.7 112.0 111.9 113.6 114.8 109.26099.2 103.9 105.6 107.3 109.2 109.9 107.9 108.6 112.0 111.1 113.8 112.4 114.3 108.8125100.0 103.1 103.2 104.0 105.0 106.6 104.4 105.8 102.3 109.5 111.0 110.3 111.1 105.825098.0 1010.9 103.5 105.4 106.8 108.0 106.6 108.3 108.6 108.8 110.4 111.5 113.9 107.0Overall99.0 103.4 104.8 106.2 107.3 108.8 106.9 108.6 109.2 110.6 111.8 112.2 113.9Week 0.000*Interaction‐dosebyweek0.999Dose0.000*Week 0.000**F0GenerationFemaleBodyWeightsF0GenerationMaleBodyWeightsSignificanceTestsEstimatedMarginalMeansEstimatedMarginalMeansSignificanceTestsInteraction‐dosebyweek0.917Dose0.161*Tukeypost‐hoctestsindicatethatweeklyaveragesaresignificantlydi fferentfromeachother.*Tukeypost‐hoctestsindicatethatdose125hadsignificantlowerbodyweightcomparedtothecontrolgroup,dose30,anddose60.Dose250hadasignificantlylowerbodyweightcomparedtodose30anddose60.**Tukeypost‐hoctestsindicatethatweeklyaveragesaresignificantlydi fferentfromeachother.

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-April 2010

12 Table 12. Mean body we ight from young female and male F1 generation

Japanese quail ( Coturnix japonica ) exposed to endosulfan.

Week 1 Week 2 Week 3

Female Male Female Male Female Male

p

‐ value = .13 p

‐ value = .00 N/A* p

‐ value = .00 p

‐ value = .00 p

‐ value = .30 p

‐ value = .58 p

‐ value = .35 p

‐ value = .01

0 13.5 35.0 54.3 78.2 75.5 97.3 91.1 113.9 94.2

30 14.2 36.9 80.0 76.4 75.0 93.1 95.6 110.3 102.9

60 15.8 26.7 51.3 66.9 80.3 100.1 93.1 118.5 98.9

125 18.4 34.8 58.9 79.2 67.9 99.4 95.3 115.5 101.5

250 18.3 35.2 58.0 80.7 76.4 102.1 93.2 113.4 103.6

p

‐ value = .33 p

‐ value = .00 p

‐ value = .63 p

‐ value = .61 p

‐ value = .73 p

‐ value = .17 p

‐ value = .60 p

‐ value = .22 p

‐ value = .65

0 18.1 22.6 20.4 32.7 32.1 33.3 33.1 33.6 33.1

30 19.0 21.3 21.7 32.8 32.1 33.3 32.7 33.4 32.7

60 19.3 19.0 21.4 32.4 32.5 33.3 33.0 33.4 32.8

125 19.9 20.1 21.3 33.3 32.0 33.3 33.0 33.2 33.0

250 19.3 20.5 20.6 32.5 32.1 34.3 33.3 34.1 33.2

p

‐ value = .14 p

‐ value = .09 p

‐ value = .53 p

‐ value = .96 p

‐ value = .52 p

‐ value = .05 p

‐ value = .41 p

‐ value = .48 p

‐ value = .11

0 3.7 6.0 7.4 6.8 6.8 7.6 7.2 7.4 6.9

30 3.9 6.5 7.1 6.9 6.7 7.7 7.1 7.2 7.2

60 3.8 6.3 7.5 6.9 7.0 7.4 7.0 7.4 6.9

125 4.1 6.3 7.4 6.9 6.8 7.2 7.3 7.4 7.4

250 4.1 5.8 7.0 6.8 6.9 7.5 7.2 7.1 7.4

* Dose 30 only had 1 observa tion. Dose 30 va riance was coul d not be calcula ted s o the ANOVA tes t was unable to be executed.



### Dose F1 Generation Tarsus, Culmen, and Body Weight Averages

Tarsus

Culmen Week 6 Week 5 Week 4

Body WeightToxicology Study No. 87-XE-08JG-10, Protocol No. 08JG-80-08-04-02, September 2008-April 201013Table 13. Mean body weightfrom adult female and maleF1 generation Japanese quail (Coturnix japonica)exposed to endosulfan.Factor P‐value Dose Week7 Week8 Week9 Week10Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19 Week20 Week21 Overall0118.4 126.5 128.5 127.5 130.0 131.3 130.1 132.1 135.9 134.7 134.3 134.1 132.8 135.3 127.2 130.630124.2 129.0 136.3 134.9 135.1 136.2 136.6 138.1 136.3 140.9 138.3 140.1 138.8 142.9 143.6 136.760138.2 126.8 131.9 130.7 131.2 131.9 126.8 131.0 131.0 139.6 133.3 125.0 129.2 133.2 135.8 131.7125126.5 132.5 133.7 135.1 134.8 136.0 136.8 131.8 137.0 139.3 138.7 142.0 141.8 136.9 138.9 136.1250130.6 132.6 136.1 137.1 133.6 138.5 139.1 139.3 142.4 142.4 139.3 141.5 143.2 144.6 143.1 138.9Overall127.5 129.4 133.3 133.0 132.9 134.8 133.9 134.5 136.5 139.4 136.8 136.5 137.1 138.6 137.7Factor P‐value Dose Week7 Week8 Week9 Week10Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19 Week20 Week21 Overall0101.9 103.7 105.1 105.1 106.5 105.9 104.6 106.8 111.7 108.3 109.0 112.2 108.3 111.9 108.2 107.330103.8 107.4 108.7 107.9 109.2 107.0 107.0 111.8 111.3 111.1 112.0 112.2 112.6 116.1 114.5 110.160106.2 108.8 110.8 110.4 111.5 111.3 110.0 111.6 113.9 114.5 115.5 109.9 112.1 117.6 116.7 112.0125107.4 109.1 109.9 109.3 110.7 109.7 108.9 114.4 111.2 111.8 112.5 112.2 112.7 114.5 115.9 111.3250104.8 107.7 108.1 107.6 108.4 108.3 110.2 109.5 111.2 111.7 112.1 112.5 113.3 115.5 115.3 110.4Overall104.8 107.3 108.5 108.0 109.2 108.4 108.1 110.6 111.8 111.5 112.2 111.8 111.8 115.1 114.1Week 0.000**F1GenerationMaleBodyWeightsF1GenerationFemaleBodyWeightsWeek 0.000SignificanceTestsInteraction‐dosebyweek0.996SignificanceTestsInteraction‐dosebyweek0.005*Dose0.000EstimatedMarginalMeansEstimatedMarginalMeansDose0.000**Statisticallysignificantinteractionindicatedthatbodyweightchangefromweektoweekwassigni ficantlydifferentbetweenthefivedosegroups.*Tukeypost‐hoctestsindicatedthatthecontrolgroup'sbodyweightwassignificantlylowerthantheotherfourdosegroups.Dose30alsoahdasignificantlylowerbodyweightcomparedtodose60.**Tukeypost‐hoctestsindicatethatweeklyaveragesaresignificantlydi fferentfromeachother.

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-April 2010

14 Table 14. Mean body we ight from young female and male F2 generation

Japanese quail ( Coturnix japonica ) exposed to endosulfan.

Dose Week 1 Week 2

‐ A Week 2

‐ B Week 3

‐ A Week 3

‐ B Week 4

‐ A Week 4

‐ B Week 5

‐ A Week 5

‐ B

p

‐ value = .91 p

‐ value = .94 p

‐ value = .47 p

‐ value = .57 p

‐ value = .95 p

‐ value = .77 p

‐ value = .83 p

‐ value = .65 p

‐ value = .78

0 7.6 16.2 24.8 34.6 46.9 57.0 70.3 78.7 81.8

30 7.2 16.9 25.7 36.2 47.7 60.3 72.3 80.6 84.0

60 7.7 17.2 26.6 37.4 48.2 59.6 72.0 79.2 83.2

125 7.7 17.1 24.6 36.8 47.7 60.3 73.1 82.5 84.6

250 7.6 16.3 23.9 36.0 46.6 58.4 74.0 82.8 86.0

p

‐ value = .80 p

‐ value = .26 p

‐ value = .28 p

‐ value = .31 p

‐ value = .14 p

‐ value = .39 p

‐ value = .16 p

‐ value = .26 p

‐ value = .74

0 15.6 17.8 20.9 23.0 27.2 28.0 30.3 31.0 32.7

30 15.8 19.4 21.4 23.4 26.9 29.7 31.5 31.8 32.8

60 16.1 19.1 21.3 24.1 28.3 29.7 31.7 31.9 32.3

125 15.8 19.8 19.0 23.1 27.1 28.6 31.8 32.8 32.3

250 16.0 18.9 20.1 22.4 26.0 28.5 31.5 32.4 33.1

p

‐ value = .37 p

‐ value = .70 p

‐ value = .43 p

‐ value = .76 p

‐ value = .57 p

‐ value = .56 p

‐ value = .26 p

‐ value = .79 p

‐ value = .82

0 3.8 4.7 5.6 6.3 6.7 7.3 7.4 7.9 7.8

30 3.7 4.7 5.6 6.5 6.6 7.4 7.8 8.0 7.7

60 3.9 4.9 5.7 6.6 5.8 7.7 7.5 8.1 7.9

125 3.7 4.9 5.9 6.3 5.9 7.3 7.4 7.9 7.8

250 4.0 4.8 5.5 6.6 6.6 7.4 7.2 7.9 7.7 Body Weight

Tarsus

Culmen F2 Generation Tarsus, Culmen, and Body Weight AveragesToxicology Study No. 87-XE-08JG-10, Protocol No. 08JG-80-08-04-02, September 2008-April 201015Table 15. Mean body weightfrom adult female and maleF1 generation Japanese quail (Coturnix japonica)exposed to endosulfan.Factor P‐value Dose Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19 Overall0100.9 108.8 124.3 134.5 133.5 132.3 137.8 137.9 140.3 141.6 138.3 138.3 141.1 141.8 132.230106.7 116.0 124.2 130.5 133.9 135.6 137.6 138.5 139.7 143.2 139.8 140.0 140.2 142.1 133.460102.1 108.2 124.5 131.5 131.6 132.5 135.9 138.3 138.5 140.0 136.5 136.5 138.6 137.2 130.9125103.5 112.6 125.2 129.6 136.1 139.3 139.7 140.1 140.4 142.4 139.6 139.6 142.2 145.0 133.9250108.1 115.7 125.8 128.1 131.6 133.4 135.9 137.7 139.3 142.0 140.0 140.1 140.2 140.6 132.7Overall104.2 112.2 124.8 130.8 133.3 134.6 137.5 138.5 139.6 141.8 138.8 138.9 140.4 141.3Factor P‐value Dose Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Overall094.3 98.1 103.4 104.4 104.7 106.1 108.5 108.5 108.7 109.4 109.8 110.1 110.5 105.930100.4 104.2 107.3 109.4 109.5 110.7 112.6 112.7 113.4 114.1 114.1 115.6 114.4 110.66097.6 101.9 106.0 108.0 108.7 110.0 110.8 112.0 111.4 112.9 112.9 112.8 113.2 109.1125102.8 106.9 109.7 111.7 113.4 113.6 115.1 115.6 114.3 115.1 114.3 114.7 114.7 112.4250101.0 103.6 104.1 107.2 108.6 110.2 111.9 113.0 111.2 112.8 113.3 113.5 113.6 109.5Overall99.2 102.9 106.1 108.1 109.0 110.1 111.8 112.3 111.8 112.8 112.9 113.3 113.2SignificanceTestsInteraction‐dosebyweek0.948EstimatedMarginalMeansWeek 0.000**F2GenerationMaleBodyWeightsSignificanceTestsEstimatedMarginalMeans*Tukeypost‐hoctestsindicatedthatdose60hadasigni ficantlylowerbodyweightcomparedtodose30anddose125.**Tukeypost‐hoctestsindicatethatweeklyaveragesaresignificantlydi fferentfromeachother.**Tukeypost‐hoctestsindicatethatweeklyaveragesaresignificantlydi fferentfromeachother.*Tukeypost‐hoctestsindicatethatthecontrolgrouphadasignificantlylowerbodyweightcomparedtotheotherfourdosegroups.Dose125hadasigni ficantlyhigherbodyweightcomparedtothefourotherdosegroups.F2GenerationFemaleBodyWeightsInteraction‐dosebyweek1.000Dose0.000*Week 0.000**Dose0.003*

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-April 2010

16  Feed Consumption. While same-sex paired, the juvenile control males and females from the F0

generation had greater increases in food consumpt ion than all other dose groups (Tables 16 and

17). When males and females were paired as adults, the control, 60, and 250 ppm dose groups

consumed less food than pairs from the 30 and 125 ppm dose groups (Table 18). For the F1

generation, juvenile females from the 250 ppm dose group consumed more food than the other

doses (Table 19); no differences were found with juv enile male feed consumption (Table 20). F1

generation adult male/female pairs from the 125 ppm treatment level had a lower average feed

consumption than those from the control and 250 ppm levels only during the first week of being

paired (Table 21). F2 females from the control dose group had the highest average for feed

consumption at ten weeks of age and greater, and no significant differences were observed in

males (Tables 22 and 23).Toxicology Study No. 87-XE-08JG-10, Protocol No. 08JG-80-08-04-02, September 2008-April 201017Table 16. Mean weekly (from week5 through 10) feed consumption for F0 generation female Japanese quail(Coturnix japonica) exposed to endosulfan.F0GenerationFemaleFoodConsumption(Weeks5‐10)SignificanceTestsEstimatedMarginalMeansFactorP‐valueDoseWeek5Week6Week7Week8Week9Week10OverallInteraction‐dosebyweek0.0030113.1114.9138.7154.6166.7199.8148.030132.0134.8146.1146.0156.8182.5149.7Dose0.00060117.6121.9154.3154.0154.3196.5149.8125123.1141.1146.7187.0191.2187.2162.7Week0.000250125.4145.6147.8175.5175.4186.7159.6Overall122.2131.6146.7163.6168.9190.5Table 17. Mean weekly (from week5 through 10) feed consumption forF0 generation male Japanese quail(Coturnix japonica) exposed to endosulfan.F0GenerationMaleFoodConsumption(Weeks5‐10)SignificanceTestsEstimatedMarginalMeansFactorP‐valueDoseWeek5Week6Week7Week8Week9Week10OverallInteraction‐dosebyweek0.1110107.4121.2131.7123.1121.4136.9123.630109.5117.3122.0107.4106.8118.3113.5Dose0.00060113.5113.2121.3115.9116.1118.8116.5125112.9111.0112.1118.6109.7116.4113.4Week0.000250106.6111.4114.9117.9114.5121.1114.4Overall110.0114.8120.4116.6113.7122.2*ID69‐dose30datapointsremovedforweek9andweek10duetolowfoodconsumptionresults.Toxicology Study No. 87-XE-08JG-10, Protocol No. 08JG-80-08-04-02, September 2008-April 201018Table 18. Mean weekly (from week 11through 18) feed consumption for F0generation female and male Japanesequail (Coturnix japonica) pairs exposed to endosulfan.Factor P‐value Dose Week11 Week12 Week13 Week14 Week15 Week16 Week17Week18 Overall0154.0 290.9 305.4 306.1 282.1 293.6 289.1 113.7 254.430160.4 325.7 324.9 326.5 323.6 302.4 316.2 126.7 275.860159.0 300.9 279.2 330.5 292.1 289.9 295.9 125.1 259.1125163.4 320.2 320.1 323.6 317.9 313.6 315.7 127.7 275.3250161.8 299.5 291.0 305.5 292.0 292.4 318.6 127.1 261.0Overall159.7 307.5 304.1 318.4 301.5 298.4 307.1 124.1F0GenerationFoodConsumption(Week11‐18)EstimatedMarginalMeansSignificanceTestsInteraction‐dosebyweek0.6400.000Week 0.000DoseTable 19. Mean weekly (from week7 through 12) feed consumption for F1 generation female Japanese quail(Coturnix japonica) exposed to endosulfan.Factor P‐value Dose Week7 Week8 Week11 Week12 Overall074.6 126.1 150.1 196.8 131.33078.4 123.4 151.5 190.5 129.960102.9 131.6 147.0 199.8 137.412589.5 124.7 143.4 202.1 133.8250103.3 145.5 163.1 215.1 149.5Overall89.7 130.3 151.0 200.8Interaction‐dosebyweek0.069Dose0.000Week*Id119,12,299,163,and163datapointsremovedforweek7duetolowfoodconsumptionresults.0.000F1GenerationFemaleFoodConsumption(Weeks7‐12)SignificanceTestsEstimatedMarginalMeansToxicology Study No. 87-XE-08JG-10, Protocol No. 08JG-80-08-04-02, September 2008-April 201019Table 20. Mean weekly (from week7 through 12) feed consumption forF1 generation male Japanese quail(Coturnix japonica) exposed to endosulfan.Factor P‐value Dose Week7 Week8 Week11 Week12 Overall079.4 90.1 95.6 124.1 97.63071.9 91.6 93.3 117.9 94.06068.5 97.5 102.7 124.1 99.112570.5 95.5 102.4 126.1 97.125074.7 95.8 100.5 120.3 97.7Overall73.0 94.5 99.1 122.5Week 0.000Dose0.188F1GenerationMaleFoodConsumption(Weeks7‐12)SignificanceTestsEstimatedMarginalMeansInteraction‐dosebyweek0.157Table 21. Mean weekly (from week 13through 21) feed consumption for F1generation female and male Japanesequail (Coturnix japonica) pairs exposed to endosulfan.Factor P‐value Dose Week13 Week14 Week15 Week16 Week17 Week18 Week19 Week20 Week21 Overall0179.3 130.9 346.4 293.4 315.3 309.1 286.7 327.8 257.7 271.930161.7 142.6 338.3 263.2 291.0 298.4 278.4 270.8 248.9 254.860173.4 138.4 329.1 266.5 290.9 286.1 268.1 331.4 241.0 258.3125161.0 130.9 323.3 246.9 297.9 294.5 294.3 279.7 243.6 252.5250172.8 131.8 336.8 281.8 301.5 308.1 304.2 316.7 273.4 269.7Overall169.6 134.9 334.7 270.4 299.3 299.2 286.3 305.3 252.90.001EstimatedMarginalMeansF1GenerationFoodConsumption(Week13‐21)SignificanceTestsInteraction‐dose0.559Dose0.000WeekToxicology Study No. 87-XE-08JG-10, Protocol No. 08JG-80-08-04-02, September 2008-April 201020Table 22. Mean weekly (from week6 through 18) feed consumption for F2 generation female Japanese quail(Coturnix japonica) exposed to endosulfan.Factor P‐value Dose Week6Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Overall091.1 114.7 138.7 98.8 130.7 195.8 219.1 243.9 193.7 231.8 214.3 244.3 243.2 181.830103.4 120.9 125.0 112.4 134.6 183.6 197.7 216.3 202.2 212.5 111.8 217.8 215.5 165.76073.5 108.8 128.3 91.1 136.4 193.9 207.0 236.3 200.7 213.2 107.9 235.1 234.7 166.712594.5 116.0 125.3 91.7 139.3 169.6 178.0 222.6 183.3 198.7 195.2 207.3 223.4 165.025084.5 106.8 123.9 100.8 127.4 187.3 202.5 249.1 193.9 202.0 179.0 222.2 227.3 169.7Overall90.0 113.4 128.2 98.9 133.7 186.2 200.8 233.6 194.7 211.6 161.6 225.3 228.8F2GenerationFemaleFoodConsumption(Week6‐18)EstimatedMarginalMeansWeek 0.000SignificanceTestsInteraction‐dosebyweek0.000Dose0.000*Id305(week9),215(week9),86(week16),73(week16),84(week16)datapointsremovedduetoinaccuratefoodconsumptionresultsTable 23. Mean weekly (from week6 through 18) feed consumption forF2 generation male Japanese quail(Coturnix japonica) exposed to endosulfan.Factor P‐value Dose Week6Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Overall097.8 120.8 122.0 114.6 104.6 121.3 120.9 196.0 119.1 133.7 118.2 116.2 118.1 123.33098.9 115.4 119.6 122.4 107.7 113.6 110.4 200.1 122.8 129.9 112.6 108.5 116.0 121.36089.2 121.7 127.2 123.9 115.2 122.1 130.0 204.1 123.9 127.6 114.0 119.8 124.2 126.412594.2 125.0 129.3 122.0 122.5 120.1 116.1 193.8 119.1 126.0 106.6 113.5 127.9 124.325096.6 116.8 117.3 103.9 132.5 122.1 124.5 198.1 122.3 124.0 109.2 112.2 121.3 123.1Overall95.3 119.9 123.1 117.4 116.5 119.8 120.3 198.4 121.4 128.2 112.1 114.0 121.5F2GenerationMaleFoodConsumption(Week6‐18)EstimatedMarginalMeansDose0.077Week 0.000SignificanceTestsInteraction‐dosebyweek0.003*ID193datapointremovedfromweek7duetoinaccuratefoodconsumptionresult.

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

21  Cloacal Gland Area. Control males from the F0 and F1 generations had larger cloacal glands

during development, however they were only statis tically larger than those from the 60 ppm dose

group in the F0 generation and the 30 ppm dose group in the F1 generation (Tables 23 and 24).

Endosulfan had no effect on cloacal gland size in the F2 generation (Table 25).



# Table 24. Mean cloacal gl and area (mm) from F0 ge neration male Japanese quail

( Coturnix japonica ) exposed to endosulfan.

Factor P

‐ value Dose Week 6 Week 7 Overal l

0 113.4 164.4 138.9

30 75.4 118.6 97.0

60 70.0 114.2 92.1

125 97.7 165.0 131.4

250 84.1 136.7 110.4

Overall 88.1 169.8 F0 Generation Cloaca

Interaction

‐

dose by week

Dose

Week 0.230

0.013*

0.000** Significance Tests

* Tukey post

‐ hoc tests indicate that the control group had a signi ficantly greater CG

area compared to dose 60. 

## Estimated Marginal Means

** Week 7 had significantly greater CG area compared to week 6.



# Table 25. Mean cloacal gl and area (mm) from F1 ge neration male Japanese quail

( Coturnix japonica ) exposed to endosulfan.

Factor P

‐ value Dose Week 6 Week 7 Overal l

0 139.7 189.3 164.5

30 89.2 157.0 123.1

60 150.7 177.2 163.9

125 109.5 173.5 141.5

250 119.6 160.8 140.2

Overall 121.7 171.5 F1 Generation Cloaca

Estimated Marginal Means Significance Tests

Interaction

‐

dose by week 0.588

Dose 0.017*

Week 0.000**

** Week 7 had a significantly greater CG area average compared to week 6. * Tukey post

‐ hoc tests indicated that the control group had a significantly greater CG

area compared to dose 30.

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

22 Table 26. Mean cloacal gl and area (mm) from F2 ge neration male Japanese quail

( Coturnix japonica ) exposed to endosulfan.

Factor P

‐ value Dose Week 6 Week 7 Week 8 Overall

0 104.0 151.5 205.3 153.6

30 100.4 160.8 218.3 159.8

60 76.8 152.8 210.0 146.5

125 97.4 148.6 225.8 157.3

250 74.7 148.9 201.7 141.8

Overall 90.6 152.5 212.2 Dose 0.905 Interaction

‐

dose by week

* All weeks were signi ficantly di fferent from each other. 

### Significance Tests Estimated Marginal Means F2 Generation Cloaca

Week 0.000* 0.785



##  Yolk Content. Figure 1 shows endosulfan concentrations in yolk samples collected from F0

generation eggs. A dose-dependent in crease is apparent for both the α - (endosulfan I) and β

isomers (endosulfan II).



# Figure 1. Yolk concentrati ons of endosulfan I and II fr om eggs collected from the

F0 generation of Japanese quail ( Coturnix japonica ) that were exposed to

endosulfan via feed.

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

23  Onset to Puberty. Onset to puberty was unaltered by endosulfan in the F0 generation (Table

27). Although the p-value for male onset was sli ghtly less than 0.05, there were no significant

differences between treatment levels after account ing for multiple testing. Females in the F1

generation from the 30 ppm dose group had signific antly longer times to onset of reproductive

maturity than females from the control and 60 ppm dose groups. Males from all dose groups

exhibited a similar pattern of onset to puberty (Tab le 28). No differences in onset to puberty were

observed for either gender in the F2 generation (Table 29).



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

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

Table 28. Mean onset to reproductive maturation in male and females Japanese

quail ( Coturnix japonica ) of the F1 generation that were exposed to endosulfan. Dose Average Std. Error 

## Average Std. Error



## 0 57.2 1.68 

## 44.7 0.47



## 30 56.1 1.81 

## 46.8 0.67



## 60 56.6 1.70 

## 45.9 0.64



## 125 56.3 1.05 

## 44.7 0.51



## 250 56.3 1.44 

## 45.8 0.59 

## P

‐ value

= .991 P

‐ value

= .044 Females Males F0 Generation Onset to Puberty (days)



## Dose Average Std. Error 

## Average Std. Error



## 0 49.6 1.44 

## 43.2 2.47



## 30 55.4 1.31 

## 46.0 1.86



## 60 49.9 1.59 

## 41.6 1.92



## 125 54.3 1.27 

## 44.3 1.84



## 250 53.7 1.17 

## 46.5 1.92 

## Females Males

P

‐ value

= .007 * P

‐ value

= .405

* Tukey post

‐ hoc tests indicate that dose 30 has

signi ficantly longer onset to puberty compared to the

control group and dose 60. 

## F1 Generation Onset to Puberty (days)

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

24 Table 29. Mean onset to reproductive maturation in male and females Japanese

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

 Copulatory Behavior. For the F0 generation, males from the control and 125 ppm groups had

significantly greater numbers of mount attempts compared to males from the 30, 60, and 250 ppm

groups in the first of three trials (Table 30). In the second trial, males from the 125 ppm dose group

had a significantly greater number of mount atte mpts compared to the control, 30, and 60 ppm

males. No other differences in copulatory behavior were observed for F0 generation males. Time

to initiate mount (Table 31), number of copulations per trial (Table 32), and time until first copulation

(Table 33) were unaffected by endosulfan in the F0 generation.



# Table 30. 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 endosulfan. Dose Average Std. Error 

## Average Std. Error



## 0 53.2 1.94 

## 48.1 1.32



## 30 54.4 2.18 

## 48.7 1.46



## 60 55.7 1.72 

## 48.1 1.26



## 125 57.3 1.81 

## 47.1 1.23



## 250 55.3 2.66 

## 48.1 1.24 

## F2 Generation Onset to Puberty (days)

Females Males

P

‐ value

= .680 P

‐ value

= .933

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 6.60 4.95 4.40

30 4.60 .53

‐ .91 0.01 4.35 .66

‐ 1.18 0.39 5.00 .81

‐ 1.44 0.38

60 4.20 .48

‐ .84 0.00 4.75 .72

‐ 1.28 0.78 5.35 .89

‐ 1.58 0.17

125 6.55 .78

‐ 1.26 0.95 6.90 1.07

‐ 1.81 0.01 5.21 .92

‐ 1.61 0.25

250 4.40 .51

‐ .87 0.00 5.55 .85

‐ 1.48 0.41 4.75 .81

‐ 1.44 0.61

* 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 F0 Generation

‐ Number of Mounts

Trial 1

Omnibus Likelihood ratio p

‐ value = .000 Trial 2

Omnibus Likelihood ratio p

‐ value = .00 6 Trial 3

Omnibus Likelihood ratio p

‐ value = .676

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

25 Table 31. 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 endosulfan.

Table 32. 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 endosulfan.

Table 33. 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 endosulfan. Dose

0 38.5 12.7 

### 90% 21.9 11.8 

### 90% 21.5 11.8 

### 90%

30 58.1 14.6 

### 80% 27.8 11.9 

### 95% 14.7 8.6 

### 95%

60 77.6 14.8 

### 80% 28.6 10.0 

### 95% 22.5 9.2 

### 95%

125 26.5 9.3 

### 95% 18.7 9.2 

### 95% 15.7 9.0 

### 95%

250 47.9 13.7 

### 85% 16.5 6.3 

### 100% 10.8 4.1 

### 100% F0 Generation

‐ Time to first Mount

Trial 1 Trial 2 Trial 3

Log rank p

‐ value = .069 Log rank p

‐ value = .83 0 Log rank p

‐ value = .688

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

Trial 2

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 0.30 0.42 0.50

30 0.35 .39

‐ 3.47 0.78 0.50 .49

‐ 3.01 0.72 0.65 .57

‐ 2.97 0.53

60 0.10 .07

‐ 1.65 0.18 0.15 .09

‐ 1.34 0.13 0.20 .13

‐ 1.28 0.12

125 0.20 .19

‐ 2.36 0.53 0.58 .55

‐ 3.42 0.49 0.55 .47

‐ 2.59 0.83

250 0.30 .32

‐ 3.10 1.00 0.40 .36

‐ 2.53 0.92 0.55 .47

‐ 2.59 0.83

* 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 Omnibus Likelihood ratio p

‐ value = .226 Omnibus Likelihood ratio p

‐ value = .205 F0 Generation

‐ Number of Copulations

Trial 1

Omnibus Likelihood ratio p

‐ value = .458 Trial 3



### Dose

0 154.7 12.7 

### 20% 121.6 16.5 

### 42% 115.8 16.6 

### 45%

30 144.0 12.5 

### 30% 107.9 16.4 

### 50% 110.5 17.6 

### 45%

60 170.6 7.3 

### 10% 159.8 11.2 

### 15% 151.8 13.0 

### 20%

125 150.3 13.4 

### 20% 120.3 17.4 

### 47% 122.1 17.3 

### 40%

250 139.9 15.7 

### 26% 124.3 17.1 

### 35% 92.9 17.7 

### 55% Copulated

pairs

F0 Generation

‐ Time to first Copulation

Log rank p

‐ value = .624 Log rank p

‐ value = .220 Log rank p

‐ value = .248 Trial 3 Trial 2 Trial 1

Average time

to first

copulation Standard

Error 

### Average time

to first

copulation Standard

Error 

### Copulated

pairs Average time

to first

copulation Standard

Error 

### Copulated

pairs

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

26 No significant differences were observed in either of three trials for number of mount attempts, elapsed

time until first copulatory attempt, and number of succe ssful copulations in male Japanese quail from the

F1 generation (Tables, 34-36). In the last of th ree trials, males from the 125 ppm dose group took

significantly longer to achieve a successful copulation than males from the 30 ppm dose group (Table

37).



# Table 34. 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 endosulfan.

Table 35. 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 endosulfan. 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 2.45 2.80 2.20

30 1.90 .51

‐ 1.19 0.24 2.20 .53

‐ 1.17 0.23 2.21 .66

‐ 1.53 0.98

60 1.95 .52

‐ 1.21 0.29 2.70 .66

‐ 1.40 0.85 2.75 .84

‐ 1.86 0.27

125 2.00 .54

‐ 1.24 0.34 1.95 .46

‐ 1.05 0.08 2.50 .76

‐ 1.70 0.54

250 2.05 .55

‐ 1.27 0.40 2.32 .56

‐ 1.23 0.35 2.65 .79

‐ 1.82 0.38

* 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 Trial 1 Trial 2 Trial 3

Omnibus Likelihood ratio p ‐ value = .740 Omnibus Likelihood ratio p ‐ value = .381 Omnibus Likelihood ratio p ‐ value = .773 F1 Generation

‐ Number of Mounts



### Dose

0 59.4 14.8 

### 85% 44.8 15.8 

### 80% 37.9 15.2 

### 85%

30 43.8 13.2 

### 85% 27.2 11.8 

### 90% 21.6 11.8 

### 90%

60 60.1 13.2 

### 90% 37.8 12.5 

### 90% 10.6 2.9 

### 100%

125 67.9 15.9 

### 75% 37.4 12.0 

### 95% 40.5 13.9 

### 90%

250 44.3 11.9 

### 90% 20.0 9.2 

### 95% 15.8 9.5 

### 94% Mounted

Pairs 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 

### F1 Generation

‐ Time to first Mount

Trial 1 Trial 2 Trial 3

Log rank p

‐ value = .795 Log rank p

‐ value = .790 Log rank p

‐ value = .444

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

27 Table 36. 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 endosulfan.

Table 37. 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 endosulfan.

No differences in number of mount attempts made within the three minute time period for each of the

three trials for the copulatory behavior tests were obs erved for the F2 generation (Table 38). Birds from

the 30 ppm treatment group in the F2 generation init iated their mount attempts earlier than any other

dose group for all three trials, while control males took the longest (Table 39). Males from the 30 ppm

treatment level also had a larger number of successful copulations than controls in the first trial (Table

40), and took the least amount of time to achieve a su ccessful copulation in the first and third trials (Table

41). Trial 1

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 0.35 0.50 0.75

30 0.60 .68

‐ 4.35 0.26 0.55 .47

‐ 2.59 0.83 1.00 .68

‐ 2.62 0.41

60 0.60 .68

‐ 4.35 0.26 0.50 .42

‐ 2.40 1.00 0.75 .49

‐ 2.05 1.00

125 0.35 .35

‐ 2.85 1.00 0.45 .37

‐ 2.22 0.82 0.40 .23

‐ 1.26 0.15

250 0.40 .41

‐ 3.12 0.80 0.71 .61

‐ 3.27 0.42 0.53 .31

‐ 1.61 0.41

* 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 Trial 3

Omnibus Likelihood ratio p

‐ value = .193 Trial 2

Omnibus Likelihood ratio p

‐ value = .873 Omnibus Likelihood ratio p

‐ value = .581 F1 Generation

‐ Number of Copulations



## Dose

0 142.7 13.5 

## 30% 114.4 18.0 

## 40% 91.2 17.8 

## 60%

30 105.6 16.7 

## 55% 113.4 16.0 

## 50% 58.9 14.4 

## 85%

60 110.2 14.7 

## 55% 128.4 15.8 

## 40% 100.5 16.9 

## 60%

125 143.8 14.3 

## 30% 117.6 17.2 

## 40% 130.8 16.1 

## 35%

250 135.3 15.2 

## 35% 91.9 20.2 

## 53% 123.3 18.2 

## 39% 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 F1 Generation

‐ Time to first Copulation

Log rank p

‐ value = .212 Log rank p

‐ value = .755 Log rank p

‐ value = .013 Trial 1 Trial 2 Trial 3

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

28 Table 38. 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 endosulfan.

Table 39. 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 endosulfan.

Table 40. 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 endosulfan. Trial 2

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 1.95 1.90 1.65

30 2.25 .75

‐ 1.77 0.51 2.55 .88

‐ 2.04 0.17 2.21 .85

‐ 2.11 0.21

60 1.95 .64

‐ 1.56 1.00 1.50 .49

‐ 1.27 0.33 2.10 .81

‐ 2.01 0.30

125 2.20 .73

‐ 1.74 0.58 2.05 .69

‐ 1.68 0.74 1.80 .68

‐ 1.75 0.72

250 2.05 .68

‐ 1.63 0.82 2.10 .71

‐ 1.71 0.66 2.15 .83

‐ 2.05 0.25

* 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 F2 Generation

‐ Number of Mounts

Trial 1

Omnibus Likelihood ratio p

‐ value = .945 Trial 3

Omnibus Likelihood ratio p

‐ value = .659 Omnibus Likelihood ratio p

‐ value = .221



### Dose

0 84.9 18.2 

### 60% 62.6 16.4 

### 80% 47.9 15.3 

### 80%

30 17.6 4.5 

### 100% 3.4 0.5 

### 100% 5.1 2.7 

### 100%

60 57.7 16.9 

### 80% 49.4 15.8 

### 80% 22.8 9.3 

### 95%

125 49.8 13.7 

### 85% 26.9 11.1 

### 95% 17.4 8.9 

### 95%

250 50.2 13.7 

### 85% 24.7 10.2 

### 95% 17.1 9.0 

### 95% Mounted

Pairs 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 

### F2 Generation

‐ Time to first Mount

Trial 1 Trial 2 Trial 3

Log rank p

‐ value = .032 Log rank p

‐ value = .000 Log rank p

‐ value = .016

Trial 1 Trial 2

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 0.35 0.60 0.65

30 1.10 1.34

‐ 7.36 0.01 0.75 .59

‐ 2.67 0.57 1.16 .90

‐ 3.54 0.10

60 0.50 .54

‐ 3.75 0.47 0.55 .40

‐ 2.08 0.84 0.65 .46

‐ 2.16 1.00

125 0.50 .54

‐ 3.75 0.47 0.60 .45

‐ 2.23 1.00 0.60 .42

‐ 2.02 0.84

250 0.65 .74

‐ 4.66 0.18 0.65 .49

‐ 2.37 0.84 0.90 .68

‐ 2.83 0.37

* 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 F2 Generation

‐ Number of Copulations

Omnibus Likelihood ratio p

‐ value = .043 Omnibus Likelihood ratio p

‐ value = .950 Trial 3

Omnibus Likelihood ratio p

‐ value = .268

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

29 Table 41. 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 endosulfan.

 Organ Weights. No significant differences we re observed with adult brain,kidney, liver, or

thyroid weights for females from the F0 generati on (Table 42), however kidney wieghts from adult

males of the 250 ppm dose group were significantly smaller than those from the control and 30 ppm

dose groups (Table 43). Additionally, male th yroid glands of the 125 and 250 ppm dose groups

weighed less than those from controls.



# Table 42. Mean brain, kidne y, liver, and thyroid gland weights (g) with standard

error of deviation from adult female Japanese quail ( Coturnix japonica ) of the F0

generation that were exposed to endosulfan (ppm). Dose

0 150.3 13.4 

## 20% 112.8 17.2 

## 50% 104.5 17.6 

## 50%

30 72.8 14.4 

## 80% 83.6 17.7 

## 60% 44.5 13.6 

## 85%

60 114.1 16.8 

## 50% 111.8 17.7 

## 45% 113.1 17.0 

## 45%

125 118.2 16.6 

## 45% 96.7 17.3 

## 55% 96.7 16.0 

## 60%

250 113.3 15.2 

## 50% 105.7 16.1 

## 60% 77.9 15.3 

## 80% 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 F2 Generation

‐ Time to first Copulation

Log rank p

‐ value = .005 Log rank p

‐ value = .863 Log rank p

‐ value = .005 Trial 1 Trial 2 Trial 3

Dose Average Std. Error 

## Average Std. Error 

## Average Std. Error 

## Average Std. Error 

## Average Std. Error



## 0 127.6 7.18 

## 0.73 0.030 

## 1.31 0.110 

## 3.42 0.221 

## 0.014 0.002



## 30 144.8 2.24 

## 0.76 0.018 

## 1.37 0.069 

## 4.21 0.179 

## 0.014 0.002



## 60 136.4 5.40 

## 0.75 0.006 

## 1.23 0.093 

## 3.99 0.272 

## 0.016 0.004



## 125 135.4 2.59 

## 0.78 0.015 

## 1.24 0.105 

## 3.44 0.313 

## 0.017 0.003



## 250 141.9 3.47 

## 0.77 0.010 

## 1.21 0.041 

## 3.78 0.161 

## 0.016 0.002 

## F0 Generation Female Organ Weights

Body Weight Brain Kidneys Liver Thyroid

P

‐ value

= .831 P

‐ value

= .083* P

‐ value

= .34 4 P

‐ value

= .631 P

‐ value

= .082

* P

‐ value calculated from Kruskall

‐ wallis test because homogenei ty of variance assumption violated in parametric ANOVA test.

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

30 Table 43. Mean brain, kidne y, liver, and thyroid gland weights (g) with standard

error of deviation from adult male Japanese quail ( Coturnix japonica ) of the F0

generation that were exposed to endosulfan (ppm).

Females from the 125 ppm treatment level had larger brains than those from controls (Table 44).

Although the p-value for male thyroid weights from the F1 generation was less than 0.05, post-hoc tests

revealed that the differences between dose groups were not significant, due to the adjustment of multiple

testing (Table 45). Cloacal gland size from F1 gener ation males approached significance (p=0.054), with

glands from the 250 ppm group being approximately 13% larger than controls.



# Table 44. Mean brain, kidne y, liver, and thyroid gland weights (g) with standard

error of deviation from adult female Japanese quail ( Coturnix japonica ) of the F1

generation that were exposed to endosulfan (ppm). Dose Average Std. Error 

## Average Std. Error 

## Average Std. Error 

## Average Std. Error 

## Average Std. Error



## 0 112.3 2.35 

## 0.78 0.015 

## 0.79 0.044 

## 1.73 0.070 

## 0.020 0.003



## 30 114.2 3.16 

## 0.77 0.018 

## 0.79 0.036 

## 1.91 0.089 

## 0.013 0.001



## 60†

‐ ‐ 

## ‐ ‐ 

## ‐ ‐ 

## ‐ ‐ 

## ‐ ‐



## 125 108.3 1.57 

## 0.75 0.014 

## 0.61 0.058 

## 1.67 0.047 

## 0.011 0.001



## 250 110.3 1.57 

## 0.79 0.019 

## 0.57 0.057 

## 1.80 0.035 

## 0.012 0.001



## ** Tukey post

‐ hoc results indicate that dose 0 has a significantly greater thyroid weight than dose 125 and dose

250.

† Only 1 observa Ɵ on for dose 60. With only 1 observa Ɵ on, standard devia Ɵ on/error cannot be calculated and

therefore cannot be included in ANOVA test. P

‐ value

= .226 P

‐ value

= .435 P

‐ value

= .012* P

‐ value

= .057 P

‐ value

= .004**

* Tukey post

‐ hoc results indicate that dose 0 and dose 30 have significantly greater kidney weight than dose 250 at the 95%

confidence level. 

## F0 Generation Male Organ Weights

Body Weight Brain Kidneys Liver Thyroid

Dose Average Std. Error 

## Average Std. Error 

## Average Std. Error 

## Average Std. Error 

## Average Std. Error



## 0 137.0 4.24 

## 0.73 0.009 

## 1.39 0.003 

## 4.10 0.206 

## 0.014 0.004



## 30 142.2 2.09 

## 0.76 0.013 

## 1.28 0.072 

## 4.10 0.243 

## 0.011 0.001



## 60 129.7 2.64 

## 0.75 0.006 

## 1.30 0.058 

## 3.69 0.226 

## 0.008 0.001



## 125 144.7 1.32 

## 0.78 0.010 

## 1.32 0.027 

## 4.16 0.241 

## 0.012 0.002



## 250 144.4 3.21 

## 0.75 0.030 

## 1.30 0.026 

## 4.10 0.281 

## 0.016 0.003

† Kruskall

‐ wallis test indicates that dose 125 has a signi fi cantly greater brain weight averages compared to the control group

at 95% confidence level 

## F1 Generation Female Organ Weights

Body Weight Brain Kidneys Liver Thyroid

P

‐ value

= .002° P

‐ value

= .038*† P

‐ value

= .313* P

‐ value

= .641 P

‐ value

= .103*

* P

‐ value calculated from Kruskall

‐ wallis test because homogeneity of variance assumption violated in parametric ANOVA test.

° Tukey post

‐ hoc results indicate that dose 30, 250 and 125 have signi ficantly greater body weight average compared to dose

60 at 95% confidence level.

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

31 Table 45. Mean brain, kidne y, liver, and thyroid gland weights (g) with standard

error of deviation from adult male Japanese quail ( Coturnix japonica ) of the F1

generation that were exposed to endosulfan (ppm).

No differences in brain, kidney, liver, or thyroid gl and weights, or clocal gland size were observed in

Japanese quail from the F2 generation (Tables 46 and 47).



# Table 46. Mean brain, kidne y, liver, and thyroid gland weights (g) with standard

error of deviation from adult female Japanese quail ( Coturnix japonica ) of the F2

generation that were exposed to endosulfan (ppm).

Table 47. Mean brain, kidne y, liver, and thyroid gland weights (g) with standard

error of deviation from adult male Japanese quail ( Coturnix japonica ) of the F2

generation that were exposed to endosulfan (ppm). Dose Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error



### 0 109.0 3.12 

### 0.75 0.020 

### 0.73 0.039 

### 1.76 0.125 

### 0.008 0.002 

### 210.4 18.66



### 30 113.4 1.92 

### 0.72 0.036 

### 0.76 0.030 

### 1.94 0.113 

### 0.017 0.003 

### 179.4 13.50



### 60 114.6 2.88 

### 0.76 0.016 

### 0.77 0.030 

### 1.88 0.074 

### 0.010 0.002 

### 191.7 10.10



### 125 113.1 2.40 

### 0.78 0.023 

### 0.76 0.027 

### 1.86 0.103 

### 0.015 0.003 

### 196.6 14.78



### 250 112.0 2.35 

### 0.76 0.014 

### 0.76 0.019 

### 1.83 0.071 

### 0.008 0.001 

### 239.1 9.92 

### P

‐ value

= .60 8 P

‐ value

= .572 P

‐ value

= .912 P

‐ value

= .767 P

‐ value

= .033 P

‐ value

= .054 F1 Generation Male Organ Weights

Body Weight Brain Kidneys Liver Thyroid Coacal Gland



### Dose Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error



### 0 138.1 2.72 

### 0.77 0.009 

### 1.19 0.035 

### 4.02 0.192 

### 0.014 0.0018



### 30 139.1 2.64 

### 0.75 0.010 

### 1.27 0.049 

### 4.14 0.066 

### 0.014 0.0010



### 60 132.8 1.53 

### 0.75 0.013 

### 1.33 0.035 

### 4.43 0.367 

### 0.011 0.0011



### 125 138.3 2.87 

### 0.77 0.014 

### 1.28 0.062 

### 4.25 0.228 

### 0.012 0.0019



### 250 135.8 1.81 

### 0.77 0.011 

### 1.22 0.053 

### 3.98 0.377 

### 0.015 0.0017 

### F2 Generation Female Organ Weights

Body Weight Brain Kidneys Liver Thyroid

P

‐ value

= .403* P

‐ value

= .248 P

‐ value

= .303 P

‐ value

= .880* P

‐ value

= .597

* P

‐ value calculated from Kruskall

‐ wallis test because homogeneity of variance assumption violated in parametric ANOVA test.



### Dose Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error



### 0 108.1 1.49 

### 0.77 0.011 

### 0.72 0.015 

### 1.66 0.055 

### 0.012 0.0080 

### 221.1 14.23



### 30 111.1 2.41 

### 0.76 0.015 

### 0.77 0.018 

### 1.79 0.053 

### 0.011 0.0083 

### 196.1 13.67



### 60 116.7 1.94 

### 0.78 0.012 

### 0.80 0.030 

### 1.85 0.067 

### 0.012 0.0086 

### 201.3 6.43



### 125 117.4 1.79 

### 0.79 0.017 

### 0.79 0.025 

### 1.92 0.072 

### 0.010 0.0064 

### 222.6 7.22



### 250 110.9 2.39 

### 0.76 0.012 

### 0.77 0.042 

### 1.88 0.109 

### 0.011 0.0074 

### 201.4 16.48

* Tukey post

‐ hoc test indicate that dose 60 and dose 125 have signi ficantly greater body weight compared to dose 0 at a 95% confidence level. 

### P

‐ value

= .009* P

‐ value

= .322 P

‐ value

= .345 P

‐ value

= .157 P

‐ value

= .894 P

‐ value

= .432 F2 Generation Male Organ Weights

Body Weight Brain Kidneys Liver Thyroid Coacal Gland

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

32 Ovarian weights did not differ among treatment leve ls in the F0 and F2 generations, however ovaries

from F1 females from the 60 ppm dose group were smaller than those from all other dose groups (Table

48). Testes weight did not differ among dose groups in any generation. Although the number of

dominant follicles per ovary did not differ among dose groups for any generation, follicle counts were

lower in birds of the 60 ppm treatment level in the F1 generation compared to the other dose groups

(Table 49).



# Table 48. Mean gonad weight s with standard errors of the mean from adult

Japanese quail ( Coturnix japonica ) exposed to endosulfan.

Table 49. Mean ovarian follicle counts wi th standard errors of the mean from

female Japanese quail ( Coturnix japonica ) that were exposed to endosulfan.

Ovarian Follicle Counts

F0 Generation F1 Generation F2 Generation

Dose p = 0.155 p = 0.057 p = 0.273

(ppm) Average Std. Error Average Std. Error Average Std. Error

0 2.56 0.58 4.78 0.28 4.10 0.31

30 4.00 0.14 4.60 0.34 3.90 0.38

60 3.25 0.77 3.20 0.57 3.40 0.53

125 3.46 0.16 4.77 0.15 3.60 0.34

250 3.60 0.27 4.14 0.26 6.55 0.43

 Perivitelline Layer Sperm Penetration. No differences were observed with the number of

hydrolysis points made by spermatozoa around the germinal disc for either the F0 or the F1

generation (Table 50). F0 Generation F1 Generation F2 Generation

Ovary Weight Testes Weight Ovary Weight Testes Weight Ovary Weight Testes Weight

Dose p = 0.407 p = 0.560 p = 0.003* p = 0.750 p = 0.360 p = 0.355

(ppm) Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error Average Std. Error

0 3.27 0.79 3.72 0.29 5.56 0.31 3.34 0.09 2.85 0.64 3.4 0.15

30 4.95 0.45 3.10 0.23 5.02 0.41 2.92 0.35 3.80 0.55 3.32 0.20

60 4.32 0.85 3.14 0.17 3.09 0.63 2.97 0.34 2.85 0.78 3.66 0.11

125 4.49 0.27 3.19 0.22 5.00 0.36 3.08 0.16 3.26 0.57 3.28 0.15

250 5.13 0.31 3.18 0.22 4.91 0.35 1.07 0.34 1.98 0.53 3.13 0.25

*Mean ovarian weights from 60 ppm dpse group weigh less than those from all other treatment levels.

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

33 Table 50. Number of hydrolysis points around the germinal disc made by

spermatozoa from Japanese quail ( Coturnix japonica ) from the F0 and F1

generations of quail that were exposed to endosulfan (ppm).

F1 Generation F2 Generation

# Sperm Holes # Sperm Holes

p

‐ value

= 0.202 p

‐ value

= 0.135

Dose (ppm) Average Std. Error Average Std.

Error

0 26.75 5.53 10.00 2.47

30 14.13 2.18 22.85 5.55

60 20.34 3.61 17.19 3.61

125 18.55 2.57 25.99 6.67

250 17.43 4.27 19.89 2.46

 Fertility and Hatchability. Endosulfan did no appar ent effects on fertility or hatchability in F1 and

F2 chicks (Tables 51 and 52).



# Table 51. Percent fertility, hatchability, deaths occurring at approximately 1 and 2

weeks of development, and F1 chicks that init iated but failed to hatch (pipped) in

eggs produced by the F0 generation ( exposure to endosulfan through feed only)

of Japanese quail ( Coturnix japonica ).

endosulfan (ppm) % fertility % hatchability % died wk 1 % died wk 2 % pipped

0 84.9 79 9.7 8 3.2

30 94.8 86.5 6.1 4.3 3.1

60 91.9 84.6 2.9 11.8 0.7

125 86.6 94.6 0 4 1.3

250 83.3 88.8 4 7.2 0

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

34 Table 52. Percent fertility, hatchability, deaths occurring at approximately 1 and 2

weeks of development, and F2 chicks that init iated but failed to hatch (pipped) in

eggs produced by the F1 gene ration (exposure to endosulfan in ovo and through

feed) of Japanese quail ( Coturnix japonica ).

endosulfan (ppm) % fertility % hatchability % died wk 1 % died wk 2 % pipped

0 99.2 89.8 4.7 2.4 3.1

30 99.2 91.1 3.2 2.4 3.2

60 93.6 94.2 2.9 1.9 1

125 98.2 91.1 7.1 1.8 0

250 96.2 95.3 0 3.9 0.8



#  Hormone Analysis. Endosulfan did not alter adult glandular T3 and T4 in any of the

three generations (Tables 53-55). Adult seru m estradiol and testosterone was likewise

unchanged in the F0 and F1 generations. In the F2 generation serum estradiol was

lower in females from the 250 ppm dose group than the 60 ppm group, and serum

testosterone was lower in males from the 125 and 250 ppm dose groups than control

males.

Table 53. Average glandular thyroxine (T 4) and triiodothyroni ne (T3) and serum

estradiol and testosterone collected fr om F0 generation (exposure through feed

only) adult Japanese quail ( Coturnix japonica ).

Gender Dose Group

(ppm) T3 (ng/ml) 

# T4 (ng/ml) 

# * Estradiol (pg/ml) 

# Testosterone (pg/ml) 

# *

N Mean SE N Mean SE N Mean SE N Mean SE

Female Control 5 1.62 0.20 5 2.54 0.73 5 43.45 6.56 5 522.5 345.9

30 5 1.37 0.09 4 2.32 0.74 5 46.25 3.19 5 868.2 177.7

60 5 1.20 0.21 4 6.98 1.81 5 34.51 2.61 5 339.0 85.1

125 5 1.35 0.22 4 3.74 1.26 5 35.66 7.33 5 453.3 267.9

250 5 1.25 0.10 4 2.86 1.39 5 45.59 7.47 5 467.3 274.3

Male Control 5 1.46 0.11 5 2.49 0.68 5 10.89 4.02 5 1986.3 732.4

30 5 1.50 0.09 4 2.50 0.50 5 14.55 8.85 5 791.8 136.2

60 5 1.60 0.12 5 2.34 1.07 5 10.86 2.89 5 909.6 126.1

125 5 1.58 0.14 5 2.71 0.51 5 9.88 1.37 5 1264.3 363.3

250 5 1.50 0.09 5 3.26 0.92 5 6.90 2.42 5 1157.0 297.8

P

‐ Value Dose = .820 Dose = .568 Dose = .563 Dose = .792

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

35 Gender

= .070 Gender

= .235 Gender

= .000

(Females > Males) Gender

= .000

(Males > Females)

* Analyzed using Log

‐ transformed data due to violations in ANOVA assumptions.



# Table 54. Average glandular thyroxine (T 4) and triiodothyroni ne (T3) and serum

estradiol and testosterone collected from F1 generation (exposure through

embryonic exposure and feed) adult Japanese quail ( Coturnix japonica ).

Gender Dose

Group

(ppm) T3 ( ng/ml) 

# T4 (ng/ml) 

# * Estradiol (pg/ml) 

# Testosterone (pg/ml)



# N Mean SE N Mean SE N Mean SE N Mean SE

Female Control 5 1.39 0.20 4 3.11 0.70 5 30.88 8.40 5 301.8 174.3

30 5 1.99 0.27 4 1.99 0.53 5 54.52 19.76 5 551.6 98.1

60 5 1.89 0.31 5 1.78 0.41 5 57.93 8.33 5 299.2 64.0

125 5 1.24 0.19 4 3.20 1.39 5 44.24 9.25 5 583.4 280.3

250 6 1.40 0.16 3 3.24 2.11 6 46.79 3.65 6 450.8 144.1

Male Control 5 1.60 0.16 5 2.51 0.43 5 10.16 2.24 5 925.8 405.1

30 4 1.65 0.12 3 1.52 0.39 5 9.31 2.22 5 1249.1 437.0

60 5 1.63 0.15 5 2.80 0.58 5 9.23 0.62 5 2114.3 720.8

125 5 1.66 0.16 5 3.21 0.38 5 9.49 1.08 5 3280.4 1241.9

250 4 1.39 0.16 3 2.92 0.92 4 12.88 7.16 4 1693.4 586.0

P

‐ Value Dose = .148 Dose = .459 Dose = .546 Dose = .159

Gender

= .981 Gender

= .607 Gender

= .000

(Females > Males) Gender

= .000

(Males > Females)

* Analyzed using Log

‐ transformed data due to violations in ANOVA assumptions.



# Table 55. Average glandular thyroxine (T 4) and triiodothyroni ne (T3) and serum

estradiol and testosterone collected fr om F2 generation (embryonic exposure

only) adult Japanese quail ( Coturnix japonica ).

Gender Dose

Group

(ppm) T3 (ng/ml) 

# T4 (ng/ml) 

# * Estradiol (pg/ml) 

# Testosterone (pg/ml)



# N Mean SE N Mean SE N Mean SE N Mean SE

Female Control 5 1.84 0.34 2 2.99 1.57 5 30.12 2.75 5 235.3 72.2

30 5 1.42 0.13 4 1.84 0.71 5 39.30 5.30 5 297.5 45.7

60 4 1.18 0.17 3 2.79 1.66 5 49.86 4.67 5 620.5 229.5

125 3 0.89 0.31 3 4.20 2.46 5 36.27 11.38 5 334.6 214.1

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

36 250 5 1.38 0.34 4 3.89 1.45 5 18.83 4.98 5 511.3 279.6

Male Control 5 1.66 0.09 4 2.69 0.09 5 10.17 1.86 5 2191.4 197.0

30 5 1.66 0.32 5 2.96 0.94 5 6.95 1.53 5 1759.1 425.1

60 5 1.80 0.14 5 1.72 0.24 5 5.61 1.96 5 1143.9 300.1

125 4 1.54 0.20 5 1.95 0.82 5 7.65 1.47 5 899.7 213.8

250 5 1.80 0.25 5 3.39 0.92 5 6.42 2.33 5 785.7 252.6

P

‐ Value Dose = .388 Dose = .576

Dose * Gender

=

.022 Dose * Gender

=

.006 Gender

= .033

(Males > Females) Gender

= .712

* Analyzed using Log

‐ transformed data due to violations in ANOVA assumptions.



# 8 Discussion

Although endosulfan is sometimes described as being moderately to highly toxic to birds (Watts,

2009) and is characterized as an endocrine disruptor that causes reproductive and developmental

damage in humans and other mammals (Varayoud et al., 2008), it has often been demonstrated as

a relatively weak endocrine disruptor in avian species. The adult Japanese quail acute oral LD-50

for endosulfan is reported as 46 mg/kg, and the acute dietary LD-50 ranges from 1197-2906 mg/kg

(Prakash et al., 2009; Hill and Camardese, 1986). For in ovo exposures, the chicken (white

leghorn) embryo LD-50 ranged from 5.35 mg endo sulfan/egg for a one-time injection at embryonic

day 3 (Pourmirza, 2000) to 28 mg Endocel (35% endosulfan)/brown leghorn egg injected after 24h

incubation (Mobarak and Al-Asmari, 2011). Moba rak and Al-Asmari (2011) stated 14 mg/egg to be

a teratogenic dose level for brown leghorn chicks while 21 mg/egg was considered to be more of an

embryotoxic dose. Chicks from their lowest dose of 7 mg/egg also had significantly more

deformities compared to control chicks. In addition to skeletal and cartilaginous deformities,

generalized edema, and hematomas were also reported at this lowest dose level. Whereas other

studies have reported neurotoxic e ffects of endosulfan in birds as evidenced by ataxia, tremors,

atypical posturing (Prakash et al., 2009; Hudson et al., 1984), limb paralysis (Mobarak and AlAsmari, 2011), depression and excitation (Qamar et al., 2012), no such effects were observed in

the current study.

As a potential endocrine disrupting chemical, endosulfan has been described as estrogenic and

antiandrogenic, having been shown to cause ch ronic depression of circulating testosterone

(Varayoud et al., 2008; Grunfield and Bonefeld-Jo rgensen, 2004; Chatterjee et al., 2008). The

estrogen-mimicking potential is somewhat limit ed, having been measured to be about six orders of

magnitude lower than that of estradiol as measured by the E-screen test (Soto et al., 1994).

Endosulfan failed to induce a uterotrophic response in ovariectomized rats but induced changes in

progesterone receptor and estrogen receptor alp ha expression (Varayoud et al., 2008). The first

study to assess endosulfan’s potential for reproduc tive toxicity in birds (Japanese quail) found no

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

37 effects on egg laying, eggshell thickness, or hatc hability from birds that were fed feed treated with

Endocel (35% endosulfan) at levels as high as 100 0 ppm for 20 weeks (Prakash et al., 2009). Male

Japanese quail that received 5 mg/kg endosulfan in feed had reduced testes relative weights and

leukocyte counts compared to controls, while birds exposed to 25 and 50 mg/kg Thiodan EC 35

(35% endosulfan) had suppressed onset of puberty (a s measured by appearance of foam in feces)

and a delay in timing of mounting pen mates (Qamar et al., 2012). No alterations in serum

testosterone levels were observed.

Although the information concerning endosulfan toxici ty in an avian species gained from this study

may be useful given the dearth of information avail able for avian species, the main objective of this

study was to assess the value added by use of t he F2 generation compared to a study that would

test no further than the F1 generation. The current guideline for the avian two-generation toxicity

test that has been proposed by the US EPA (Touart 2005) is a large, long-te rm experiment that is

arguably resource expensive. The current study make s use of a large number of animals. In order

to ensure a sufficient sample size for adequate statistical analysis and the production of

subsequent generations for all dose levels, ex tra birds were produced than directly needed for

inclusion in the groups that were measured and assessed for toxicity. The current trend in

toxicology is one of a reduction in animal use and an increase in the reliance on in vitro versus in

vivo studies as applicable. It is acknowledged that t he endpoints in this study require the exposure

of a living system, and non-animal alternatives are not available to replicate the complex processes

of reproduction and development in an intact organi sm. However, the primary objective of this

study is the justification of the additional F2 generation. The current study took approximately

thirteen months to complete all in-life portions, wi th many additional months required for histological

and hormone analyses. Though recent refinements in the methodologies for this avian twogeneration test have reduced the amount of time requi red to conduct this test, the investigation was

temporally as well as financially expensive.

Assessment of the utility of the F2 generation wa s challenging using the mildly estrogenic model

endocrine disrupting chemical endosulfan. Endosulfan induced minimal changes in physiology and

behavior, with many of the effects not partic ularly dose dependent or consistent across

generations. What was fairly consistent was t he lack of effects seen in adult male and female

specific measures in the F2 generation (Tables 53 and 54). While males at the lowest treatment

level did display altered copulatory behaviors co mpared to controls, observed effects (earliest

initiation to mount females, earliest time to su ccessful copulation, and greatest number of

successful copulations) were not considered ad verse (Table 55). Additionally, although some

changes in adult hormone levels were observed, no correlating adverse functional reproductive

effects occurred.



# Table 56. Comparison of adult male Japanese quail ( Coturnix japonica ) measures

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

exposure only) generations. ND = no difference.

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

38 Table 57. Comparison of adult female Japanese quail ( Coturnix japonica )

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

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

Table 58. Comparison of results of F1 and F2 generation male copulatory

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

no difference.

Yamashita et al. (2011) evaluated changes in Japane se quail exposed to low doses of estradiol as

a positive control to determine sensitive endpoint s for use in a one-generation avian endocrine

disrupting toxicity test. Adult quail were fed a diet containing 0.3, 3, and 30 ppm 17 β -estradiol for

six weeks. Decreased body weight, testicular and ovarian atrophy, decreased spermatogenesis,

decreased egg production and fertility rate, and a reduction in eggshell thickness were observed in

birds receiving 30 ppm estradiol compared to controls. Though eggs produced by the F1

generation of the current endosulfan study did have thinner shells than controls at the 125 and 250

ppm dose levels, egg production was not reduced in either generation. In fact, egg production was

increased for a short time in birds from the 250 ppm group. A stimulatory effect of estradiol on egg

production has been documented previously (El-Gh alid, 2009), and a hormetic effect of estradiol

(and xenoestrogens) on egg production is likely. The sperm parameters and fertility endpoints

measured in this study were also unchanged by endosulfan in both generations. Histological Male Specific Measures F1 F2

male onset to maturity ND ND

sperm concentration ND ND

sperm motility ND ND

% progressive sperm 250 ppm more progressive than 0, 30, 60 ppm 0 & 60 ppm more progressive than rest

perivitelline layer penetration ND ND

testes weight ND ND

cloacal gland area 250 ppm largest (p

= 0.054) ND

Female Specific measures F1 F2

female onset to maturity 30 ppm sooner than 0 and 60 ppm ND

egg production 250 ppm highest lay rate btwn. 1st & 3rd week ND

eggshell thickness 125 & 250 ppm less than controls ND

eggshell strength 250 ppm greater than controls ND

ovary weight 60 ppm smallest ND

dominant follicle count ND ND



## Copulatory Behavior Measure F1 F2

time until 1st mount attempt ND 30 ppm earliest initiation in trial 3

time until 1st copulatory success 125 ppm longer than 30 ppm in trial 3 30 ppm earliest success in trials 1 & 3

# attempts / 3 min trial interval ND ND

# successful copulations ND 30 ppm more than controls in trial 1

# Toxicology Study No. 87-XE-08JG-10, Prot ocol No. 08JG-80-08- 04-02, September

2008-March 2010

39 results (Appendix G) suggest that endosulfan caused no effects on male or female reproductive

tissue as well. Yamashita et al. (2011) also reported increased inciden ces of persistent right

oviducts in females caused by low doses of estradi ol. Only one bird in the current study from the

250 ppm dose group exhibited this abnormality, making the interpretation questionable and not

likely associated with exposure to endosulfan.



# 9 Conclusion

There appears to be no clear benefit to produce a generation of Japanese quail beyond the F1

generation for the assessment of low potency en docrine disrupting chemicals exemplified by

endosulfan based on the observed effects that were caused by the exposure levels selected for this

study. In this case, a one-generation or extended on e-generation avian toxicity test may be more

appropriate. It is likely that the results of t he Endocrine Disrupting Screening Program‘s Tier 1

toxicity tests could be used to help determine if a candidate chemical would be more appropriately

screened by an (extended) one- or a full two-generation test.



# 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

Program Manager

Health Effects Research Program

# B-1 APPENDIX A

REFERENCES

Literature Cited

Chatterjee, S., V. Kumar, C.B. Majumder, P. Roy. 2008. Screening of some anti-progestin

endocrine disruptors using a recombinant yeast-based in vitro bioassay. Toxicol. in Vitro , 22:

788-798.

Douthwaite, R.J. 1980. Occurrence of birds in Acacia woodland in northern Botswana related to

endosulfan sprayed for tsetse fly control. Environ. Poll. , 22: 273-279.

Douthwaite, R.J. 1982. Changes in pied kingfisher ( Ceryle rudis ) feeding related to endosulfan

pollution from tsetse fly control operations in the Okavango Delta, Botswana. J. of App. Ecol.

, 19: 133-141.



# Dunachie, J.F. and W.W. Fletcher. 1966. Effe ct 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.

El-Ghalid, O.A.H. 2009. Ex ogenous estradiol: Blood profile and reproductive

performance of female Japanese quails at different stages of production. Asian J Poult.

Sci. , 3: 1-8.

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. Fish and Wildlife Service, Bureau of Sports Fisheries and

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

Garg, U.K., A.K. Pal, G.J. Jha, S.B. Jadhao. 2004a. Haemato-bi ochemical and immunopathophysiological effects of chronic toxici ty with synthetic pyrethroid, organophosphate

and chlorinated pesticides in broiler chicks. Int. Immunopharmacol ., 4: 1709-22.

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

chronic toxicity with synthetic pyrethro id, organophosphate and chlorinated pesticides

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

Grunfeld, H.T., E.C.Bonefeld-Jorgensen. 2004. Effect of in vitro estrogenic pesticides on human

oestrogen receptor alpha and beta mRNA levels. Toxicology Letters , 151: 467-80.



# Hill, E. F. and Camardese, M. B. 1986. Lethal Dietary To xicities of Environmental

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

# Toxicology Study No. 87-XE-09HW-08, Protocol No. 09HW-80-08-03-01,

April–June 2008

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

toxicity of 14 pesticides to mallard ducks of several ages. Toxicol. Appl. Pharmacol ., 22:

556-561.

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

Pesticides to Wildlife, Resource Publicatio n 153. U.S. Department of Interior, Fish and

Wildlife Service, Washington, DC, 6-54.

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

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

Matthews, J., T. Celius, R. Halgren, T. Zacharew ski. 2000. Differential estrogen

receptor binding of estrogenic subs tances: a species comparison. J. Steroid Biochem.

Mol. Biol., 74: 223-234.

Mobarak, Y.M., M.A. Al-Asmari. 2011. Endosulfa n impacts on the developing chick embryos:

morphological, morphometric and skeletal changes. Int. J. Zool. Res. , 7: 107-127.



# Watts, M. 2009. Pesticide Action Netw ork Asia and the Pacific Monograph on

Endosulfan. http://www.panap.net/sites /default/files/monogr aph_endosulfan.pdf.

Pourmirza, A.A. 2000. Toxic effects of malathion and endosulfan on chick embryos. J.

Agric. Sci. Technol. , 2: 161-166.

Prakash, P.J., G. Rajashek har, H. Krishnappa, S.M. Sula iman, K. Venu gopala Rao.

2009. Acute toxic effects of endosulfan 35 EC (Endocel) upon oral gavage and dietary

admixture in Japanese quails. Res. J. Environ. Toxicol. , 3: 124-131.

Qamar, A.B., M.Z. Khan, A. Khan, M.K. Saleemi, I. Javed. 2012. Endosulfan (Thiodan EC 35)

induced toxico-pathological effects in male Japanese quails ( Coturnix japonica ). J. Natn. Sci.

Foundation Sri Lanka , 40: 97-105.

Soto, A.M., K.L. Chung, C. Sonnenschein. 1994. The pesticides endosulfan, toxaphene, and

dieldrin have estrogenic effects on human estrogen-sensitive cells. Environ. Health Perspect.

, 102: 380-383.

US EPA. 2007. Note to reader. Endosulfan Readers Guide. November 16. EPA-HQ-OPP-2002-

0262-0057. http://www.regulations.gov.

Varayoud, J., L. Monje, T. Bernhardt, M. Muñoz-de-Toro, E.H. Luque, J.G. Ramos. 2008.

Endosulfan modulates estrogen-dependent genes like a non-uterotrophic dose of 17betaestradiol. Repro. Toxicol. , 26: 138-145.

Varayoud, J., L. Monje, T. Bernhardt, M. Munoz-de-Toro, E.H. Luque, J.G. Ramos. 2008.

Endosulfan modulates estrogen-dependent genes like a non-uterotrophic dose of 17betaestradiol. Reprod. Toxicol. , 26: 138-145.

# Toxicology Study No. 87-XE-09HW-08, Protocol No. 09HW-80-08-03-01,

April–June 2008

Yamashita, R., A. Oshima, Y. Hasegawa-Baba, M. Wada, K. Shibuya. 2011. Endocrine

disrupting effects of low dose 17 β -estradiol (E 2 

# ) on the Japanese quail ( Coturnix japonica ) were

detected by modified one-generation reproduction study. J. Toxicol. Sci. , 36: 43-54.

# Toxicology Study No. 87-XE-09HW-08, Protocol No. 09HW-80-08-03-01,

April–June 2008

APPENDIX B

QUALITY ASSURANCE STATEMENT

For: DTOX Study No. 87-XE-09HW-08, Protocol No. 09HW-80-08-03-01, titled

“Sensitivity Determination of the Humoral Re sponse to Foreign Red Blood Cells in the

Northern Bobwhite, April-June 2008.” The fo llowing critical phases were inspected/

audited by the USAPHC (Prov)-Quality Systems Office:

PRE IN-LIFE PHASE OF THE STUDY

Critical Phase Inspected/Audited Date Inspected

/Audited Date Reported

to

Management/SD

GLP Protocol Review

IN-LIFE PHASE OF THE STUDY

Critical Phase Inspected/Audited Date

Inspected

/Audited Date Reported to

Management/SD

Test System – Facilities

Test System – Receipt

Test System – Identification

Test Article – Control

Test Article – Dosing

Maintenance and Calibration of Equipment

Compliance with DTOX Protocols

Compliance with DTOX SOPs

Reagents and Solutions

Clinical Testing – Bleeding Technique

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: All findings were made known to the Study Director and the Program Manager at the time

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

reported to Management and the Study Director on the date shown in the table.

_____________________________________ __________________ Michael P. Kefauver Date

GLP Assessor, QSO

_____________________________________ __________________ Gene Sinar Date

Team Leader, QSO

# APPENDIX C

ARCHIVES AND STUDY PERSONNEL

C-1. ARCHIVES

a. All raw data, documentat ion, records, protocol, and a copy of the final report

generated as a result of this study will be ar chived in Bldg. E-2100, USAPHC (Prov), for

a minimum of five (5) years follo wing submission of a final report.

b. Records on animal receipt, diet, and facility environment al parameters will be

archived by the Veterinary Medical Division, Toxicology Directorate, for a minimum of

five (5) years following submissi on 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 ca libration logbooks may be found in Room

1026, Building E-2100, USAPHC (Prov), Aberdeen Proving Ground, Maryland 21010.

d. The present study uses USAPHC (P rov) 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: Dr. Michael Quinn, Michelle Cook, M.S., Alicia Shifflet,

A.S., Allison Jackovitz, B.S., Craig A. McFa rland, D.V.M., Ph.D., Mark S. Johnson,

Ph.D.

b. Quality Systems Office: Mr. Gene Sinar, Mr. Michael Kefauver

# D-1 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 endosulfan

.

PROTOCOL NUMBER:

PRINCIPAL INVESTIGATOR/STUDY DIRECTOR:

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

Biologist

Health Effects Research Program

Toxicology Directorate (410) 436-1064

U.S. Army Center for Health Pr omotion and Preventive Medicine

Aberdeen Proving Ground, MD 21010-5403

CO-INVESTIGATOR(S):

Mark Johnson, Ph.D., DABT

Biologist

Health Effects Research Program

Toxicology Directorate (410) 436-5081

U.S. Army Center for Health Pr omotion and Preventive Medicine

Aberdeen Proving Ground, MD 21010-5403

Emily LaFiandra, Ph.D.

Toxicologist

Health Effects Research Program

Toxicology Directorate (410) 436-7749

U.S. Army Center for Health Pr omotion and Preventive Medicine

Aberdeen Proving Ground, MD 21010-5403

Craig McFarland, Ph.D.

Biologist

Health Effects Research Program

Toxicology Directorate (410) 436-7784

U.S. Army Center for Health Pr omotion and Preventive Medicine

Aberdeen Proving Ground, MD 21010-5403

Amanda Hess-Ruth

Biologist

Toxicity Evaluation Program

Toxicity Directorate (410)436-3973

U.S. Army Center for Health Pr omotion and Preventive Medicine

# Aberdeen Proving Ground, MD 21010-5403

SPONSOR:

Christiana Grim, D.V.M.

Project Officer

Office of Science C oordination and Policy

U.S. Environmental Protection Agency

1200 Pennsylvania Ave., NW (7203)

Washington, DC 20460

Tel: 202-564-8458

I. NON-TECHNICAL SYNOPSIS:

Many military chemicals are known or suspecte d to be endocrine active and have been

found in soil and water on milita ry installations at significant concentrations. Avian species

make up a large percentage of the biodiversity in these areas and ha ve great potential for

exposure to these contaminants. The purpose of this study is to establish a new laboratory

testing procedure that can be used to assess the impact of e ndocrine active chemicals upon avian

health and reproduction. Data coll ected from such tests may be used in the assessment of risks to

birds exposed to military environmental contaminan ts. These data from a two-generation test are

principally intended to evaluate the adverse cons equences of exposure to a “possible” endocrine

disruptor to the reproductive viabil ity of first generation birds, but may also provide information

to confirm a chemical as an endocrine disruptor.

II. BACKGROUND:

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

Endocrine Disruptor Screening Program (EDSP) comprised of a battery of Tier 1 screening

assays and Tier 2 tests. One of the Tier 2 tests under development is a two-generation avian

assay using Japanese quail ( Coturnix japonica ). This test is desi gned to characterize doseresponse relationships and immediate and latent adverse consequences associated with exposure

to putative endocrine modulating compounds. The avian two-generati on assay serves to evaluate

potential population effects of endocrine modul ating compounds on myriad avian species, and

represents terrestrial, oviparous wildlife species in general. This study will use endosulfan to

fully evaluate the proposed test method for its ab ility to detect adverse effects and characterize

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

avian two-generation assay for characterizing endoc rine disruptors. This work will help to

determine the value added by examining e ndocrine endpoints over two generations in

comparison to tests that only include one generati on, such as the Ecologic al Effects Guidelines

for avian reproduction tests (EPA, 1996).

II.2. Literature Search for Duplication :

II.2.1. Literature Source(s) Searched:

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

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

databases checked were:

a. MEDLINE®: National Library of Medicine Databa se (1950 – 2008)

b. RTECS: Registry of Toxic Effect s of Chemical Substances (1960 – 2007)

Science (1996-1999)

c. TOXLINE® (1900 – 2006)

d. PubMed (1980-2008)

e. Adverse Reacti on Database (2007-2008)

f. Toxfile (1965-2008)

g. Inside Conferences (1993-2008)

h. Biological and Ch emical Index (1983-2008)

i. DTIC, STINET: Defense Technical Inform ation Center Private Sc ientific and Technical

Information Network (1980-2008)

j. AGRICOLA: USDA National Ag ricultural Library (1970 – 2008)

k. International Phamacology Abstracts (1970-2007)

l. BIOSIS Toxline ® (1969-2008)

m. Pharm-line ® (1978-2002)

n. Pascal (1973-2008)

o. Global Health (1983-2008)

p. CBRN START: Chemical, Biological, Ra diological and Nuclear. Scientific and

Technical

q. Analysis Research Tool (1980-2008)

r. CAB Abstracts (1972-2008)

s. EMBASE (1974-2008)

t. NTIS: National Technical Information Service (1964-2008)

u. BIOSIS Previews (1926-2008)

II.2.2. Date of Search: 11 February 2008, by the research librarian at Wood Technical

Library.

II.2.3. Period of Search: 1980-2008.

No date limits were applied to any of the databases. The time periods established for each of

the individual databases were th e defaults used, as shown above.

II.2.4. Key Words of Search: Japanese quail, Coturnix, quail, endosulfan, tiovel, thiotox,

thiosulfan tionel, thiosulfan, thionex, thiodan, thimul, thif or, malix, insectophene, hildan,

endosulfan, endocel, devisulpha n, crisulfan, chlorthiepin, beosit, benzoepin, 115-29-7.

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

independent literature search.

1. Acute Exposures. The 5-day dietary LC50 for endosulfan in 14- day old Japanese

quail was found to be 1250 ppm and 2906 ppm (FWS, 1975; Hill and Camardese,

1986). In mallards, the oral LD50s at 36 h, 7 d, 30 d, and 6 months of age were 27.8,

# 6.47, 7.89, and 34.4 mg/kg body weight, respec tively (Hudson et al., 1972). In

pheasants, the LD50 values rage from 80 to >320 mg/kg (Hudson et al., 1984).

2. Chronic Exposures. Broiler chicks expos ed to 2 ppm endosulfan for 8 wk had a

decrease in serum globulin and acetyl cho linesterase activity, total leukocyte and Tlymphocyte counts, and reductions in spleen and bursa of Fabricius weights (Garg et

al., 2004a). Bursal follicle sizes and t hymus lymphocyte numbers also decreased.

Under the same conditions, broiler chicks also exhibited bone effects (Garg et al.,

2004b): the zones of proliferating, maturi ng, degenerating and calcifying cartilage

cells were reduced in width, and a re duction in active osteons was observed.

3. In Ovo Exposures. At 100 and 500 mg/kg, endosulfan in corn oil reduced the

hatchability of white leg horn chickens by 24 and 53%, respectively, compared to

controls (Dunachie and Fletcher, 1969). Previous studies had shown 5 mg/egg to

reduce hatchability in white leghorns by up to 60% (Dunachie and Fletcher, 1966).

Spraying or dipping laye r eggs with endosulfan in alc ohol or acetone at 0.5 to 5%

concentrations resulted in sterility of the gonads after the gon ads were removed at

days 8 and 9 of incubation and cultured on agar medium with 3 drops of a 0.5 to 1.0

g/l endosulfan solution (Lutz and Lutz-Ostertag, 1972).

4. In Vitro Studies. Alpha-endosulfan was observed to bind weakly to the chicken

estrogen receptor (Matthews et al., 2000).

III. OBJECTIVE/HYPOTHESIS:

The objective of this resear ch is to determine the value added by examining endocrine

endpoints in a two-generati on avian reproductive toxi city test paradigm.

IV. MILITARY RELEVANCE:

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

been found in environmental media at many military installations. Currently, few methods exist

to ascertain the magnitude of these effects, al though some have recently been proposed. Most

species of birds are protected by the Migratory Bird Species Act and some are protected by the

Endangered Species Act. As such, the DoD is by executive order obligated to abide by these

laws. Presence of some species at specific in stallations (e.g. Bald Ea gle at Aberdeen Proving

Ground) has had a significant impact on reducin g and curtailing military activity, therefore

affecting readiness of our forces. Proactive co llaboration in the development of methods that

may, in the future, be mandated would serve th e 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 (P) generation at four weeks of age and throughout the rest of the birds’ lives.

Eggs from this generation will be incubate d 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 throughout th e entirety of their lives. Eggs from

this generation will be incubated and ha tched to produce the second generation (F 2 

# ). The

F 2 

# generation will only be exposed to endosulfan in ovo through maternal deposition; all

feed for the F 2 

# generation will be untreated. Measures of reproductive behavior,

circulating hormone levels, egg quality, re production, and development will be assessed

in the P, F 1 

# , and F 2 

# generations as described below. Additional measures of growth,

reproductive maturity, immunocompetence, will be assessed in the F 1 

# and F 2 

# generations

as described below.

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

Chemical Treatments:

The sample size for each treatment will be 16 to ensure that there are 12 breeding pairs in

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

successfully produced eggs. The lowest repor ted LC50 from a five-day dietary exposure

to endosulfan in 14-day old Japanese quail was found to be 1250 ppm (FWS, 1975). The

treatment levels will be 0 (control), 30, 60, 125, and 250 ppm. The highest dose to be

administered is one-fifth of the lowest known LC50 value and should not cause mortality

or other severe signs of parent al toxicity that will preclud e the evaluation of reproductive

parameters. The lowest concentration s hould produce no effects on adult health or

reproductive measures. The test diets will be mixed and assayed at Wildlife International

Ltd., Easton, MD, USA. St ability analyses and hom ogeneity and concentration

verifications will be made every time Wildlife International Ltd. mixes new diet during

the treatment period, according to GLP standards.

# Eggs:

To generate the P generation, a minimum of 60 eggs will be hatched for each treatment

level, to provide sufficient numbers of chicks to establish at leas t 12 breeding pairs (16

pairs is the goal) per P test level. Sixteen 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 P generation to ensure that a minimum of 60 eggs will be

hatched for each test level, providing sufficien t numbers of chicks to establish at least 12

breeding pairs (16 is the goal) per F 1 

# test level. If a treatm ent level produces insufficient

eggs to set the minimum, that level will be dr opped from further analysis in the test and

eggs will not be set to hatch. Fertilized eggs not set to hatch will be frozen prior to being

discarded. Optimally, 6 eggs will be set for each parental pair of th e F1 generation, but a

minimum of 40 eggs will be set and hatched for each treatment level.

During the treatment period, all eggs, with the exception of those that are cracked, broken

or abnormal, or used for eggshell or bioche mical measurements, will be incubated. Only

those eggs from the final week cohorts will be al lowed to hatch. Eggs will be collected at

least once daily, numbered according to pen of origin, and stored in a chilled

environment; broken eggs will be numbered, recorded and then discarded. The total

number of eggs laid per pen will be accounted for at the end of the study.

Fertility and embryo viability will be de termined by removing the eggs out of the

incubator and candling them. Eggs are candled for fertility after 8 days of incubation and

again for viability of embryos approximately 2 days before hatching (day 15 if the quail

population at the laboratory generally hatches on day 17). Except for eggs incubated to

hatch for production of subseque nt generations, the late-sta ge embryos (day 15) will

undergo necropsy and observations. During cand ling, eggs should not be allowed to cool

to room temperature, since this may delay embryonic development. Those chicks that

have not hatched within approximately 24 hour s of the majority of chicks hatching will

be considered unhatched. No assistance will be given to chicks during hatching, however

chicks that remain alive but have no clear ability to hatch will be humanely removed

from the egg and decapitated immediately.

During the treatment periods, one egg per pen will be collected from odd numbered pens

in odd numbered weeks and from even numbe red pens during even numbered weeks.

These eggs will be used to assess eggshe ll strength and eggshell thickness. Eggshell

strength will be measured by an Egg Force Reader (Orka Technology Ltd.) that tests the

breaking point of an egg’s shell. Eggshell thickness will be measured by an Egg Shell

Thickness Gauge (Orka Technology Ltd.) that measures the thickness of egg shells

through ultrasound or by micr ometers or calipers.

General Reproductive Fitness Measures and Observations:

During the treatment period, the parental birds will be observed daily to detect any overt

signs of toxicity or other clinical signs of illness or trauma. Observations will include

mortality and clinical signs of toxicity such as lethar gy, depression, wing droop, ruffled

feathers, etc. Any injuries sustained and subsequent treatment will be recorded. If a pair

is removed during the study due to death, injur y, etc., data from a ll preceding weeks are

# evaluated. If data are used from a pen in wh ich mortality occurred or a bird was injured,

this will be explaine d. Clearly moribund individuals or t hose otherwise in severe distress

will be euthanized, as described below.

Prior to and during the treatment periods, the following information will be recorded in

the study’s notebook or worksheets:

 Eggs laid

 Eggs cracked

 Eggs broken

 Description of all egg abnormalities

 Eggs set

 Eggshell strength

 Eggshell thickness

 Fertile eggs

 Infertile eggs

 Early embryonic deaths

 Late embryonic deaths

 Eggs that hatch

 Eggs that do not hatch

 Egg weight at 8 days an d 24 hours prior to hatch

The following clinical/fitness observations on all hatched offspring will be recorded

during the treatment periods in th e study’s notebook or worksheets:

 Day of hatch

 Hatchling mortality

 Hatchling morphology and/or abnormalities

 Clinical signs of toxicity

 Number of 14-day-old surviving chicks

 Chick body weight at hatching a nd 14 days after hatching

 Sex of F 1 

# /F 2 

# chicks

All adult birds will undergo necropsy and gro ss pathology assessment. The wet weight of

the brain, liver, kidneys, gonads , and thyroid glands will be recorded as soon as possible

after death/euthanasia. Any morphologic abnormalities will be recorded in the study’s

notebook or necropsy sheets. Adul t birds that die, or are euth anized during the course of

the treatment period, will be necropsied sim ilarly. Histopathology will be performed on

the brain, right thyroid gland, liver, bursa of Fabricius, kidneys, and gonads. The left

thyroid gland will be stored for glandular thyr oxine (T4) content. All late-stage embryos

(2 days prior to hatch) that are not allo wed to hatch will be decapitated and undergo

necropsy as described above for adults.

Birds (adults or chicks) which are in severe distress will be euthanized. If one member of

a pair dies or is euthanized during the trea tment period, the other member of the pair will

be euthanized as well.

# Biochemical/Endocrine Measurements:

Measurements of thyroid function will incl ude thyroid gland weight, glandular T4

content, and thyroid histology/histopathol ogy. Measurements will be taken during

embryonic and adult life stages. Eggs that are set, but not in the c ohorts to be hatched,

can be used for embryonic measurements.

Blood from embryos removed from the shell that are used for necropsy and egg yolk

samples shall be collected for steroid hormone (estradiol and test osterone) analysis.

Blood collected from adults at the time of necr opsy shall also have serum steroid analyses

performed.

Vitellogenin is a protein that is generally controlled by estroge n. 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 male s that have been exposed to estrogenic

contaminants, it is expected that vitellogenesi s will be stimulated. Vitellogenin will be

measured from blood of the late embryos used for steroid hormone analysis, thyroid

hormone analysis, and necropsy/pa thology at CHPPM or a contracted lab. It will also be

measured in adult blood at necropsy.

Behavioral measures:

Initiation of normal male sexual behavior is important for succe ssful reproduction. In

reproductively mature males of the F 1 

# and F 2 

# generations, preferably when at least 90%

of control males have reached reproductive ma turity, trials shall be performed to assess

reproductive behavior including time to mount attempts, number of mount attempts, and

number of successful cloacal contacts. Ma les and females will be paired in the F 1 

# generation following completion of the beha vioral measures. Birds from the F 2 

# generation will be euthanized upon completion of this measure.

Growth:

Growth will be measured at least every three days in chicks up to at least week 2 of age.

Growth measures will include body weight, tars us length, and the leng th from the tip of

the beak to the culmen.

Immune Measures:

The measurement of antibody production in re sponse to foreign red blood cells is a

common method for assessing humoral immunity. Quail will be immunized with chukar

partridge ( Alectoris chukar ) erythrocytes (upon availabil ity) because Japanese quail are

minimally sensitive to sheep, human, chicken, turkey, and duck erythrocytes and do not

produce immunoglobulins to human, bovine, an d mouse albumins. The ability to

perform this measure is dependent on the av ailability of chukar pa rtridge red blood cells,

since this material is normally only availa ble right before and during hunting seasons.

Adult quail to be challenged will receive 0.1 ml of a 1.0% suspension of chukar red blood

cells (CRBCs) in saline injected into the j ugular vein. Blood will be collected from the

jugular vein 6 days after the CRBC injecti on, and serum may be frozen until antibody

titers are determined. Total (IgM and IgG) and 2-mercaptoethanol-resistant (IgG)

# antibody activities will be measured by a he magglutination assay at CHPPM or at a

contracted lab.

Cell mediated response will be measured in juveniles by using a 0.1 ml dose of 1 mg/ml

phytohemagglutinin (PHA-P) in phosphate-buffe red saline (PBS). One wing web will

receive an injection with the PHA solution while the other wing web will receive an

injection of PBS alone. The thickness of each wing web will be measured to the nearest

0.01 mm immediately before and 24 hr afte r the injections usi ng pressure-sensitive

micrometers with a low-tension spring that does not crush the skin. A stimulation index

will be calculated as the difference in the ch ange in thickness of the PHA-injected wing

web from the change in thickne ss of the PBS-injected wing web.

This study will be conducted in complia nce with Good Laboratory Practice (GLP)

regulations found in the Toxic Substances Control Act (TSCA): 40 CFR (Code of

Federal Regulations) 792, plus amendments. The investigators and technicians will

adhere to The Guide for Care and Use of Laboratory Animals (U.S. Department of

Health, Education, and Welfare Publication No. NIH 86-23, 1996).

V.2. Data Analysis : All observational data will be r ecorded in the laboratory notebook and

using LabCat ® 

# where applicable. The analysis of variance (ANOVA) assumptions will be

checked, and if they are met, a one-way ANOVA will be applied followed by a multiple

comparisons test to compare treatment groups. If data do not meet the ANOVA assumptions,

data may be transformed appropriately or a nonparametric ANOVA may be applied.

Observational categorical data may be tested with a Fisher’s Exact or Chi S quare 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. Non-animal alternatives would not provide the necessary information.

V.3.2. Animal Model and Sp ecies Justification: The Japanese quail was chosen for this study

because it is an avian species that is both often used in toxicity tests and breeds readily in a

laboratory setting. In addition, these birds only take six weeks to reach sexual maturity, which

greatly reduces the time required to produce multi-g enerations when compared to other similar

species, such as the northern bobwhite which takes an average of six months to reach

reproductive maturity.

V.3.3. Laboratory Animals:

V.3.3.1. Genus and Species: Coturnix japonica

V.3.3.2. Strain/Stock: In-bred; nonspecific

# V.3.3.3. Source/Vendor: University of Maryland, Departme nt 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. P generation: Minimum of 60 eggs set to incubate for 5 treatments = potential

for maximum of 300 chicks; 16 males and 16 females from each treatment

(80 males and 80 females total) will be kept for exposure to endosulfan.

b. F 1 

# generation: Minimum of 60 eggs set to incubate for 5 treatments =

potential for maximum of 300 chicks ; 16 males and 16 females from each

treatment (80 males and 80 females total) will be kept for exposure to

endosulfan.

c. F 2 

# generation: Minimum of 40 eggs set to incubate for 5 treatments =

potential for maximum of 200 chicks; minimum of 12 males and 12 females

from each treatment (60 males and 60 females total) will be kept for behavior

analyses.

V.3.5. Refinement, Reduction, Replacement:

V.3.5.1. Refinement: The exposure levels used in this st udy are based on data from previous

studies that determine toxicity of endosulfan in Japanese qua il and other avian species.

Enrichment will consist of group housing for ha tchlings and juveniles and pair housing for

adults. Singly housed birds will be provided with sisal rope.

V.3.5.2. Reduction: This study will utilize a group of birds to determine reproductive,

developmental, immune, and behavi oral effects in the same indivi duals. This is a large reduction

in the number of birds relative to the number th at would be needed to measure these effects

separately in i ndividual birds.

V.3.5.3. Replacement: No replacement alternatives ar e appropriate for this study.

V.4. Technical Methods :

V.4.1. Pain/Distress Assessment: Pain or distress are not exp ected to be experienced by

any of the birds from the treatments in this study.

V.4.1.1. APHIS Form 7023 Information

# V.4.1.1.1. Number of animals: 440 (maximum potential ~800)

V.4.1.1.1.1. Column C: 440 – ~800 (100%)

V.4.1.1.1.2. Column D: 0

V.4.1.1.1.3. Column E: 0

V.4.1.2. Pain Relief/Prevention : No other substances that could potentially interfere with the

interpretation of these results will be administered in the unlikely event that the animal appears

to be experiencing pain or discomfort. Howe ver, moribund or animals in overt pain will be

humanely euthanized as described in section V .4.6. (Euthanasia). Based on previous experience

with this model, we will consider the following as indications of distress where recovery is

unlikely at continued exposure regimes:

1) Loss of body weight to exceed 20%.

2) Loss of righting response and inability to care and feed without assistance.

3) Indications of respiratory infection (nas al discharge coupled with congested breathing).

A weight of evidence determination will be made on a case-by-case basis after consultation with

the principal investigator a nd the attending veterinarian.

V.4.1.2.1. Anesthesia/Analgesia Tranquilization : n/a

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®: National Library of Medicine Databa se (1950 – 2008)

b. RTECS: Registry of Toxic Effect s of Chemical Substances (1960 – 2007)

Science (1996-1999)

c. TOXLINE® (1900 – 2006)

d. PubMed (1980-2008)

e. Adverse Reacti on Database (2007-2008)

f. Toxfile (1965-2008)

g. Inside Conferences (1993-2008)

h. Biological and Ch emical Index (1983-2008)

i. DTIC, STINET: Defense Technical Inform ation Center Private Sc ientific and Technical

Information Network (1980-2008)

j. AGRICOLA: USDA National Ag ricultural Library (1970 – 2008)

k. International Phamacology Abstracts (1970-2007)

l. BIOSIS Toxline ® (1969-2008)

m. Pharm-line ® (1978-2002)

# n. Pascal (1973-2008)

o. Global Health (1983-2008)

p. CBRN START: Chemical, Biological, Ra diological and Nuclear. Scientific and

Technical

q. Analysis Research Tool (1980-2008)

r. CAB Abstracts (1972-2008)

s. EMBASE (1974-2008)

t. NTIS: National Technical Information Service (1964-2008)

u. BIOSIS Previews (1926-2008)

V.4.1.3.2. Date of Search: 11 February 2008

V.4.1.3.3. Period of Search: The period of search of the database s is the same as that stated in

section II.2.3.

V.4.1.3.4. Key Words of Search: endosulfan, thionex, thioda n, benzoepin, rasayansulfan,

chlorthiepin, devisulfan, Japanese quail, quail, feed aversion, st ress, preven*, control, manage*,

reduce, elimina*, alleviat*, decr eas*, diminish, relief, animal testing reduction, animal testing

refinement, biolog* Model*, theoretical model, biolog* model*, animal testing replacement,

animal testing alternative, alternat*, computer model*, simulate, alternative animal test*, animal

model*. Asterisks indicate that the wo rd roots were used as search terms.

V.4.1.3.5. Results of Search: Few papers were found that discussed avian euthanasia

methods. Several papers were found that evalua ted various anesthesia techniques, including the

use of halothane, ketamine-xylazine, and isof lurane. Nothing was found regarding pain or

distress.

Additionally, an independent lite rature search was conducted us ing a combination of several

academic search engines and by manually tracking down literature cited in captive care

literature. This search yielded several class-sp ecific anesthesia/euthanasia review 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. The housing and husbandry design is appropri ate to the study and in meeting the life

maintenance and enrichment needs of the Japanese quail.

3. The use of asphyxiation through CO 2 

# exposure has been recommended for anesthesia and

euthanasia in birds (AVMA, 2007).

V.4.1.4. Unalleviated Painful/Distre ssful Procedure Justification: Since the goal of this

investigation is to determine the effects from exposure to an environmental contaminant, no

other substances that could potentially interfere with the interpretation of these results will be

administered. However, moribund or animals in overt pain will be humanely euthanized as

described in section V.4.6.

# V.4.2. Prolonged Restraint: Not applicable

V.4.3. Surgery: Not applicable

V.4.3.1. Pre-surgical Provisions: Not applicable

V.4.3.2. Procedure: Not applicable

V.4.3.3. Post-surgical Provisions: Not applicable

V.4.3.4. Location: Not applicable

V.4.3.5. Surgeon: Not applicable

V.4.3.6. Multiple Major Survival Operative Procedures: Not applicable

V.4.3.6.1. Procedures: Not applicable

V.4.3.6.2. Scientific Justification: Not applicable

V.4.4. Animal Manipulations: Quail will be restrained briefly while the injections for the

immune response measures are administered. On e person will present the bird to the injector,

keeping the wings and legs firmly pressed agai nst its body. The injector will then brush the

feathers away from the featherles s tract in the neck re gion and then inject the solution into the

jugular vein. Blood will also be collected from this vein.

V.4.4.1. Injections: Not applicable

V.4.4.2. Biosamples: Blood will be collected according to DTOX SOP 053.

V.4.4.3. Adjuvants: Chukar partridge erythrocytes.

V.4.4.4. Monoclonal Antibody (MAbs) Production: Not applicable.

V.4.4.5. Animal Identification: Individual animals will be identified by cage card and leg

bands according to DTOX SOP 003.

V.4.4.6. Behavioral Studies: Not applicable.

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 reproductiv e behaviors assessed in the F 2 

# generation. Early euthanasia

# may occur when a weight of evidence determin ation (as described in V.4.1.2) indicates that

animals are experiencing significant pain or distress.

V.4.6. Euthanasia: Birds will be euthanized by CO 2 

# asphyxiation as recommended by the

AVMA Guidelines on Euthanasia (2007) and as specified by SOP 066. Although unhatched

birds are not covered by these guidelines, fertilized eggs that are not destined to be set to hatch

will be frozen prior to being discarded, and late stage embryos will be decapitated immediately

following removal from the egg.

V.5. Veterinary Care :

V.5.1. Husbandry Considerations: Treatments will be randomly assigned to birds within

cages and cage units. Cage unit frames are constr ucted of 1” stainless steel tubing. Cages are

constructed of perforated stainless sheet. Each cage unit will have two litter pans per tier that are

easily removed. Each unit will have individual stainless steel feeders and one stainless Edstrom

cup drinker. Watering will be automatic. Cage dimension is 7” wide x 9.5” tall x 11” deep.

Birds will be acclimated to laboratory conditions fo r a minimum of five days prior to the onset of

experiments. Birds will be kept in conditions of constant phot operiod. Birds will be housed in

AAALAC accredited facility at USACHPPM. Each cage will contain a card describing

treatment and animal number. Detailed husba ndry practices are cont ained in DTOX SOP 004.

The photoperiod for chicks and adults will be 16 or 17 hours light and 8 or 7 hours of darkness.

Chicks and juveniles will be group housed, and adults of the P and F 1 

# generations will be pair

housed. Adult males from the F 2 

# generation may be single hous ed for reproductive behavioral

measures, and the F 2 

# females may be single housed for the assessment of onset of reproductive

maturity. Individuals will be paired 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.

Hatchlings will be dried completely before be ing transferred to brooders. Once grouped, chicks

will be checked at least twice daily to ensure that group shunning, hypothermia, and/or starvation

do not occur. Sufficient space for feeding and drinking will be provided, especially during the

first week post-hatch to allow equal access to f ood and water to weaker chicks. Chicks will be

housed together by treatment group and will be iden tifiable individually by le g bands. Sexes will

be separated at 4-5 weeks and placed into in dividual cages to allow for determination of

reproductive maturity. Birds from the F1 gene ration will be pair housed following reproductive

behavior measures.

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 pr events embryo development a nd aids in synchronizing the

development of embryos of eggs laid during that week. All cracked a nd abnormal eggs will be

removed prior to incubation. Eggs will be equilibra ted to room temperature prior to being set in

the incubator. All cracked and abnormal eggs will be recorded. Eggs should be turned in the

incubator at least three times a day. For incubation, temper ature 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 the animal care staff and once on weekend days and holidays. Appropriate methods of animal

care shall be maintained to prevent, control, di agnose, and treat diseases and injuries. If it is

determined that an animal is under distress, comatose, or moribund then the observer will report

to the principal investigator or the attending veterinarian for an examination of its disposition and

medical condition. If the animal is judged (by the PI through consulta tion with the attending

veterinarian) unlikely to recover, as indicated by having met criteria spelled out in V.4.1.2., then

the animal will be humanely euthanized as described.

V.5.2.2. Emergency Veterinary Medical Care: Animals will be observed at least once daily

by the animal care staff on weekend days and holiday s. If an animal is noted to be ill, the

Attending Veterinarian will be contacted. Should the animal require euthanasia as described

previously, the Attending Veterinarian will contact the PI/study director. If the PI/study director

or other co-investigators are not available, the Attending Veterinarian will euthanize the animal

and take as many required samples as possible from the animal according to protocol. 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.

V.5.3. Environmental Enrichment:

V.5.3.1. Enrichment Strategy: Enrichment will consist of group housing for hatchlings and

juveniles and pair housing for adults. Singly hou sed birds will be provided with sisal rope.

V.5.3.2. Enrichment Restriction: NONE.

# VI. STUDY PERSONNEL QUALIFICATIONS AND TRAINING:

All investigators named in this study ha ve demonstrated an understanding of the humane care

and use of research animals. They have take n part in discussions of pertinent laws and

regulations concerning the use of animals in biom edical research in the Department of Defense

as required by Public Laws 89-544, 91-579, a nd 99-198 (The Animal Welfare Act and

Amendments, DOD Directives, and Army Regulations). They are familiar with the concepts for

the reduction or elimination of th e use of animals and have conclude d that there is a need for the

use of animals in this study. They have been familiarized in the proper methods for minimizing

and/or alleviating pain in the anim al species selected for study.

If their training in requir ed procedures has not b een of a formal, documented nature, they will

demonstrate proficiency in these procedures pr ior to performing them on study animals. The

Attending Veterinarian will observe them and determin e proficiency or additional training needs.

Personnel working on this study have been advised on the animal care and use policy at this

institution and are aware of th e established reporting mechanisms for observed deficiencies in

animal care and treatment. The following is a ta ble of participating inve stigators listing training

and qualifications:

Study Personnel Table

Staff Member Procedure(s) Years of Experience Qualifications

Quinn Collection and

handling,

phlebotomy,

euthanasia,

necropsy 10+ yrs animal research.

PI and Co-PI on studies using

the northern bobwhite and

Japanese quail; Quail

necropsy training (June 2005) Ph.D, Animal

Science

Johnson Handling,

euthanasia,

necropsy, dosing 10+ yrs animal research.

Refined the bobwhite model

for its use in ecotoxicology

assessment; Short course on

animal care & use (May

2000, April 2003) Ph.D, Veterinary

Medical Science;

D.A.B.T.,

McFarland Handling,

euthanasia,

necropsy, dosing 10+ years animal and

toxicological investigations:

Northern bobwhite handling,

dosing, prosection training

(Sept 2005); Avian handling

& blood withdrawal (Jan

2007) Ph.D, Biology,

DVM

Amanda HessRuth Handling,

behavior,

necropsy, dosing 5+ years animal and

toxicological investigations,

experience specific to animal

behavior; will receive

specific OTJ training B.S. Animal

Behavior

LaFiandra Handling,

euthanasia, 10 years animal and

toxicological investigations; Ph.D, Natural

Resources and

# necropsy, dosing Avian euthanasia & necropsy

training (Dec 2007); Avian

Handling & Dosing (in-house

training, Dec 2007- Mar

2008) Environmental

Studies

Dr. Michael Quinn has11 years of experience in avian toxicology and has over 20 publications in

the field of wildlife toxicology. He has worked in this field at the Patuxent Wildlife Research

Center USGS, Shippensburg University, and Univ ersity of Maryland, and has worked at CHPPM

for the last three years. Dr. Mark Jo hnson has a background in population ecology and

veterinary medical science. He has worked in the area of environmental risk assessment and has

over 30 publications in the field of wildlife toxi cology and ecology. He has been working in this

field at CHPPM for the last 15 years. Dr. Crai g McFarland has graduate training and experience

in wildlife biology (MS) and environmental t oxicology (PhD). He has 10 years field and

laboratory experience in studies of small mammal toxicology and was a practicing small animal

veterinarian (DVM) for 20 years. Amanda Hess- Ruth has over five years of experience in

animal research specific to toxicology and ha s demonstrated expertise in assessing animal

behavior. Dr. Emily LaFiandra has been a toxicol ogist here at CHPPM for the past year and has

gained extensive on the job experience handling bobwhite.

VII. BIOHAZARD/SAFETY:

Fowl of this type have been reported to carry specific zoonotic diseases that can be

transferred to humans (e.g., psittacosis, histoplamos is), most of them affecting the respiratory

system. The flock from which the eggs will come has been maintained and screened for diseases

for over 15 years. Adherence to standard chem ical and animal handling procedures will be

required during the performance of this study as indicated in CHPPM Regulations 385-1 and

385-5 and DTOX SOP 083. All personnel will use pape r respirators, latex or nitrile gloves, and

lab coats when handling the bird s or endosulfan-treated feed.

VIII. ENCLOSURES: References

CHPPM Standard Operating Procedures

IX. STUDY TIME FRAME:

IX.1. Estimated Experimental Initiation Date : April 2008

IX.2. Estimated Experimental Completion Date : February 2009

# X. ASSURANCES:

As the Study Director/Principal Investig ator on this protocol, I acknowledge my

responsibilities and provide a ssurances 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 desc ribed herein, unless a modificati on is specifically approved by

the IACUC prior to its implementation.

B. Duplication of Effort: I have made every effort to ensu re that this prot ocol 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 resp ect to the statistical analysis, and that the

minimum number of animals needed fo r scientific validity will be used.

D. Biohazard/Safety: I have taken into consider ation and made the proper

coordinations regarding all app licable rules and regulations concerning radiation protection,

biosafety, recombinant issues, and so fort h, in the preparation of this protocol.

E. Training: I verify that the personnel performing the animal

procedures/manipulations/observati ons described in this protocol are technically competent and

have been properly trained to ensure that no unne cessary pain or distress will be caused to the

animals as a result of the procedures/manipulations.

F. Responsibility: I acknowledge the inherent mo ral, ethical and administrative

obligations associated with the performance of this animal use protocol, and I assure that all

individuals associated with this project will demonstrate a co ncern for the health, comfort,

welfare, and well-being of the research animals. Additionally, I pledge to conduct this study in

the spirit of the fourth "R," namely "R esponsibility," which the DOD has embraced for

implementing animal use alternatives where feasible and conducting humane and lawful

research.

G. Scientific Review: This proposed animal use protocol has received appropriate peer

scientific review and is consistent with good scientific research practice.

H. Painful Procedures: I am conducting biomedical experiments, which may

potentially cause more than moment ary or slight pain or distress to animals. This potential pain

and/or distress WILL NOT be relieved with th e use of anesthetics, analgesics, and/or

tranquilizers. I have considered al ternatives to such procedures; however, I have determined that

alternative procedures are not available to accomplish the objectives of this proposed

experiment.

Michael J. Quinn, Jr.

_________________________________ Signature Date (YYYYMMMDD)

## Animal Use Protocol-Two-generation test for endocrine disruption & reproductive t oxicity in the Japanese quail with

endosulfan.



# APPENDIX A

References:

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

hens’ eggs. Nature , 212: 1062.

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

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

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

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

Environmental Protection Ag ency, Washington, D.C.

FWS (U.S. Fish and Wildlife Se rvice). 1975. Lethal Dietary T oxicities of Environmental

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

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

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

and chlorinated pesticides in broiler chicks. Int. Immunopharmacol. , 4: 1709-22.

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

toxicity with synthetic py rethroid, organophosphate and ch lorinated pesticides on bone

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

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

Contaminants to Coturnix, Technical Report Nu mber 2. U.S. Department of Interior, Fish

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

Hudson, R.H., R.K. Tucker, M.A. Haegele. 19 72. Effect of age on sensitivity: acute oral

toxicity of 14 pesticides to mallard ducks of several ages. Toxicol. Appl. Pharmacol. , 22:

556-561.

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

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

Service, Washington, DC, 6-54.

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. Z acharewski. 2000. Differential estrogen receptor

binding of estrogenic substances: a species comparison. J. Steroid Biochem. Mol. Biol.

, 74: 223-234.

## Animal Use Protocol-Two-genera tion test for endocrine disruption & reproductive t oxicity in the Japanese quail with endosulfan.



# APPENDIX B

CHPPM Standard Operating Procedures and Regulations

SOP No. 003.07, Individual Animal Identification.

SOP No. 004.07, Animal Facilit ies and Caretaker Duties.

SOP No. 053.07, Animal Bleeding Technique.

SOP No. 066.07, Animal Euthanasia.

SOP No. 083.07, Health and Safe ty of Laboratory Personnel.

USACHPPM Regulation 385-1, Safety and Occupational Health Program, May 01, 2001.

USACHPPM Regulation 385-5, Occupational Health and Safety of Animal Users, June 1, 2007.

# 22 Protocol Modifications:

# 23

# 24

# 25

# 26

# 27

# 28

# 29

# 30

# 31

# 32

# 33

# 34

# 35 APPENDIX E

DEVIATIONS TO THE PROTOCOL

1. Efforts to include measures of imm une function were withdrawn from the study

design due to a lack of available chukar partridge red blood cells needed to

assess humoral response. The customer de cided that measures specific to the

endocrine and reproductive systems be given pr iority over those of the immune

system.

2. Despite numerous efforts, sample volu mes of blood from day old chicks and late

stage embryos required for hormone and vitellogenin analyses could not be

collected.

3. Although our chemists were able to m easure endosulfan in egg yolk, they were

unable to detect hormones in this matrix.

4. Vitellogenin in adult blood could not be m easured. Efforts to develop methods to

measure vitellogenin via PCR were developed but not able to be included in this

study because since it was not anticipat ed, tissues were not flash frozen upon

collection.

5. The protocol states that eggs shoul d be candled for fertility after 8 days of

incubation and again for viability of embryos approximately 2 days before

hatching. Fertility and viability were det ermined by candling and/or removal of

contents after all chicks were hatched. In this way, determination of embryo age

(week 1 and 2) was easy and prevented disturbance of embryos during

development.

6. The protocol stated that “during the treatment periods, one egg per pen will be

collected from odd numbered pens in odd numbered weeks and from even

numbered pens during even numbered wee ks. These eggs will be used to

assess eggshell strength and eggshell thickness.” We instead measured

eggshell strength and thickness from tw o eggs per hen (when available) that

were produced within the same week.

7. Epididymides were collected and used fo r sperm analysis. As stated in the

protocol, “Histopathology wil l be performed on the brain, ri ght thyroid gland, liver,

bursa of Fabricius, kidneys, and gonads.” Epididymides were not, therefore,

included in the hist opathological analysis.

8. Gender determination could not be co mpleted for all young chicks found dead

while in the brooders, and since there is no evidence of EDCs changing gender

in birds it was agreed that the sex rati os of F1 and F2 chicks would not be

included in this study.

9. Histopathology was perf ormed by Experimental Pat hology Laboratories, Inc.,

Sterling, VA. Their results have been s ent to the EPA under a separate Scope of

Work and will not be included in this report.

# 36 APPENDIX F

DIET PREPARATION REPORT FROM

WILDLIFE INTERNATIONAL, LTD.

# 37 

# Wildlife International, Ltd.

Project Number 167-142

Test Substance

The test substance, endosulfan, was received from Helena Industries, Cordela, GA 31015

on December 12, 2008 and was assigned Wildlife Inte rnational, Ltd. identification number 8838

upon receipt. The test substance was a so lid and was identified in the accompanying

correspondence as: endosulfan 95%. The test mate rial had a reported purity of 95%. The test

substance was held under ambient conditions in locked storage at the Wildlife International, Ltd.

facilities in Easton, Mary land. Concentrations of the test substance in th e diet were “as is” and

not adjusted to 100% active ingredient. Therefore, dietary concentrations are expressed as parts

per million (ppm) in the diet.

Diet Preparation

The basal diet fed to both adults and offspr ing was formulated to Wildlife International,

Ltd. specifications by Cargill Animal Nutriti on, Shippensburg, PA (Appendix I). The basal

ration contained at least 27% pr otein and 2.5% crude fat, and no more than 3.8% crude fiber.

The basal diet contained approximately 1.0% ca lcium, derived from feedstuffs and the 1.0%

limestone used in the formulation of the basal di et by Cargill. While this level of calcium is

sufficient for growth and maintenance rations, additional calcium is required in the ration of

breeding birds for egg shell formation. Theref ore, an additional 5% (w/w) of limestone

(approximately 38.5% Ca) was added to the basal di et for the adults. This raised the calcium

level in the diet for the breeding birds to approximately 3%, slightly above the minimum

recommended for quail. Offspring received di ets prepared without the addition of 5%

supplemental limestone.

Test diets were prepared by mixing endosulfan into a premix that was used for preparation

of the final diet. Control diet and each of th e four treated diets were prepared on December 23,

2008; March 28, 2009, June 22 & 23, 2009 and August 15, 2009. Dietary concentrations were

# 38 not adjusted for purity of the test substance and are presented as parts per million (ppm). Details

of the preparation of test and cont rol diets are show n in Appendix II.

# 39 Appendix I

Diet Formulation

WILDLIFE INTERNATIONAL, LTD. GAME BIRD RATION 1



# INGREDIENTS

Fine Corn Meal Soy Bean Meal, 47.5% Protein

Wheat Midds Agway Special, 60% Protein Alfalfa Meal, 20% Protein Dried Whey

Ground Limestone

Eastman CalPhos GL Ferm (Fermatco) 2



# Salt Iodized

CM T-Premix 561

CM TM Premix 434

CHO CHL 70% Liquimeth 40% Lysine 75%

DL Methionine 055 Vit K 16 g/lb 50#

Selenium

Total PERCENT (%)

42.86

33.39

6.48

9.96

3.00

2.50

0.62

0.11

0.25

0.10

0.05

0.08

0.09

0.25

0.06

0.10

0.05

0.05

100.00

VIT A MIN A ND MINER A L CO NTENT A M O UNT A DDED PER

POUND

Vi tam i n 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 ki u /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)

# 40 Appendix II

Diet Preparation

Premixes for endosulfan were prepared on December 22, 2008; March 27, 2009; June 21,

2009 and August 11, 2009. Nominal preparation was as follows:

Control: 4018.0 g ration + 170 ml acetone + 91 ml corn oil

30 ppm: 3.3825 g Endosulfan + 4014.6 g ration + 170 ml acetone + 91 ml corn oil

60 ppm: 6.7650 g Endosulfan + 4011.2 g ration + 170 ml acetone + 91 ml corn oil

125 ppm: 14.0938 g Endosulfan + 4003.9 g ration + 170 ml acetone + 91 ml corn oil

250 ppm: 28.1875 g Endosulfan + 3989.8 g ration + 170 ml acetone + 91 ml corn oil

For each premix, the appropriate amount of endos ulfan was weighed in a tared plastic weigh

boat on an analytical balance. The test substa nce was transferred to a 400 ml beaker. Acetone

was measured in a graduated syringe and added to the test substance with a portion of the

acetone used to rinse the weigh boat. The rinse was added to the beaker. A magnetic stir-bar

was placed in the beaker and the mixture stirre d for at least two minutes (approximate), on a

magnetic stir plate, until the endosulfan 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.

The required basal ration was weighed into a tared mixing bowl on a top-loading balance.

The test substance mixture was then added to th e 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 20 minutes.

After mixing, 2000.0 g aliquots of the premix were weighed on a top-loading balance. Unless

used immediately for preparation of the final diet s, the premixes were placed in appropriately

labeled plastic bags, reweighed and stored in a freezer.

As needed, the appropriate premix was incorpor ated into the final diet for adult birds as

follows:

0 ppm: 2000 g Premix + 50.250 kg ration + 2750 g limestone

30 ppm: 2000 g Premix + 50.250 kg ration + 2750 g limestone

60 ppm: 2000 g Premix + 50.250 kg ration + 2750 g limestone

125 ppm: 2000 g Premix + 50.250 kg ration + 2750 g limestone

250 ppm: 2000 g Premix + 50.250 kg ration + 2750 g limestone

As needed, the appropriate premix was incorporated into the final diet for chicks as

follows:

0 ppm: 2000 g Premix + 53.000 kg ration

30 ppm: 2000 g Premix + 53.000 kg ration

60 ppm: 2000 g Premix + 53.000 kg ration

125 ppm: 2000 g Premix + 53.000 kg ration

250 ppm: 2000 g Premix + 53.000 kg ration

# 41 The diet was mixed for approximately twenty minutes in a Patterson-Kelly ® 

# Twin Shell

Blender.

APPENDIX G

# 42 CONTRIBUTING SCIENTIST REPORT

STATISTICAL ANALYSIS OF DATA

# 43 

# Statistical Analysis on USAPHC Toxicology Protocol

Number: 08JG-80-08-04-02

Two-Generation Test for Endocrine Disr uption and Reproductive Toxicity in

Japanese Quail ( Corturnix japonica) using Endosulfan

Good Laboratory Practice Compliance Statement

The statistical analysis of the data for t he Two-generation test for endocrine disruption

and reproductive toxicity in Japanese quail (Cortunix japonica) us ing endosulfan was

conducted in compliance with Title 40, Code of Federal Regulations (CFR), Part 792,

Good Laboratory Practice Standards. Statis tical analyses were conducted according to

the procedures outlined in the pr otocol for this study (Protocol Number 08JG-80-08-04-

02).

17 February, 2012

Shane Hall Date

Statistician

# 44 Table of Contents:

ENDOSULFAN ENDOCRINE DISRUPTION

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . 3

Eggshell Thickness and Strength . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . .4

Egg Production . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . 6

Sperm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . .9

Growth and Body Weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . 11

Feed Consumption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . 15

Cloaca Gland (CG) Area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . 19

Yolk Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . .21

Onset to Puberty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . 22

ENDOSULFAN EFFECTS ON REPRODUCTIVE BEHAVIOR

. . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . 24

Number of Mount Attempts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . 25

Time Until First Mount . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . 27

Number of Copulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . 29

Time Until First Copulation . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . 31

ENDOSULFAN EFFECTS ON ORGAN WEIGHTS

. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . 33

# 45 

# ENDOSULFAN ENDOCRINE DISRUPTION

Egg, Sperm, and Grow th Characteristics

This report analyzes the effects that endosul fan had on the endocrine system of male and

female quail through three generations (F0, F1, a nd F2). Data was collected on egg strength and

thickness, egg production, sperm characteristics , body weights, food consumption, coacal gland

(CG) area, P generation yolk conten t, and onset to puberty. All quail s were part of one of five

dose groups: (control) 0 ppm, 30 ppm, 60 ppm, 125 ppm, a nd 250 ppm. For endocrine

disruption measures that contained weekly measurements, the same quails were used from week

to week. SPSS version 16.0 was used to analyze th e behavior data. Sta tistical significance for

this analysis was determ ined using an alpha ( α ) of .05 with a 95% confidence level.

# 46 

# EGGSHELL THICKNESS AND STRENGTH

I. Analysis Methods

An Analysis of Variance (ANOVA) test wa s used to determine if the average eggshell

thickness and/or average eggshell strength for th e dose groups were significantly different. An

F-statistic and corresponding p-valu e were calculated for both th ickness and streng th. If the pvalue was below .05, then it was concluded that at least two dose gr oups had statistically

different averages for that measure. A Tukey pos t-hoc test was then used to find which specific

dose groups were statistically differe nt from one another. A Levene ’s test was used to check to

make sure the dose groups had equa l variances, a major assumption th at must be met in order to

properly run an ANOVA model. If this assumpti on was violated (p-value for Levene’s test <

.05), a non-parametric Kruskal-Wallis te st was run instead of an ANOVA.

II. Summary of Results

The general trend for the egg strength data was that as thickne ss increased, strength

decreased. Larger thickness values tended to be associated with the control group and less

concentrated endosulfan dose groups. The F1 generation had more variation between dose

groups compared to the P and F2 generations, es pecially on thickness. The control group had the

lowest average strength for all three generations , and the highest average thickness for the P and

F1 generations. Strength averages were cons istent across the three generations but thickness

averages slightly decreased from the F0 to F1 generation, and greatly decreased from the F1 to

F2 generation.

III. F0 Generation Results

No statistically significant differences occu rred for thickness averages, averages were

highest for the control group and dose 60. Dose 125 had the lowest average thickness; however

it had the highest strength averag e. The control group’s average strength was directionally lower

than all other dose groups, and was statistically significantly lower than dose 125. Strength

averages generally increased as dose concentration increased.

IV. F1 Generation Results

Statistically significant differences between doses occurred for average thickness and average

strength. The control group’s average thickness was directi onally greater than all other dose

groups, and was significantly greater than dose 125 and dose 250. Dose 30’s thickness was also Dose Average Std. Error 

## Average Std. Error



## 0 0.932 0.074 

## 0.93 0.07



## 30 0.890 0.060 

## 1.05 0.06



## 60 0.981 0.146 

## 1.05 0.05



## 125 0.846 0.051 

## 1.19 0.05



## 250 0.869 0.066 

## 1.11 0.06 

## F0 Generation

‐ Egg Strength

p

‐ value

= .788

* Tukey Pos t

‐ hoc resul ts i ndica te tha t the control group

had signi fi cantly lower s trength compa red to dose group

125. 

## p

‐ value

= .045* Thickness Strength

# 47 significantly greater th an dose group 250. As for strength, dose 250 had a significantly higher

average compared to the control group. Just like the F0 generation, strength averages increased

and thickness averages d ecreased as endosulfan con centration increased.

V. F2 Generation Results

Minimal, non-significant differen ces (p-values >.05) occurred for thickness and strength

averages between dose groups. Negligible fluctu ation occurred in thic kness data across dose

groups, but dose 60 had the highest average thic kness. Dose 30, followed by dose 250 had the

highest average strengths. Unlike the other tw o generations where strength increased and

thickness decreased as dose con centration increased, no true patte rn existed between dose group

concentration and strength or thickness for the F2 generation. Dose Average Std. Error 

## Average Std. Error



## 0 0.938 0.052 

## 0.96 0.05



## 30 0.845 0.047 

## 1.02 0.05



## 60 0.813 0.052 

## 1.04 0.04



## 125 0.753 0.032 

## 1.09 0.05



## 250 0.647 0.038 

## 1.19 0.04 

## p

‐ value

= .000* p

‐ value

= .016** F1 Generation

‐ Egg Strength

Thickness Strength

* Tukey Pos t

‐ hoc res ul ts i ndica te tha t the control group

had signi fica ntly higher thicknes s compa red to dose 250

and dose 125. Dose 30 also grea ter thicknes s compa red to

dose 250.

** Tukey Pos t

‐ hoc res ul ts i ndica te tha t the control group

had signi fica ntly lower s trength compa red to dose 250.



## Dose Average Std. Error 

## Average Std. Error



## 0 0.476 0.024 

## 0.89 0.06



## 30 0.486 0.024 

## 1.28 0.17



## 60 0.500 0.031 

## 1.03 0.07



## 125 0.471 0.020 

## 1.00 0.05



## 250 0.483 0.021 

## 1.14 0.06 

## F2 Generation

‐ Egg Strength

Thickness Strength

p

‐ value

= .924 p

‐ value

= .05348EGG PRODUCTIONI. Analysis MethodsA Generalized Estimating Equation – poisson counts model (GEE)was used to determine if there was a significant differenceamong dose groups in their egg production, accounting for the variability from week to week. P-values were calculated for doseandweek as well as their interaction. Dose,week, and interaction were determined to be significant if the p-value for the GEE was lessthan .05. Estimated marginal means werecalculated for both factors and examined for differences using aTukey post-hoc test,if thep-value for that factor was less than .05.If the interaction was statistically significant, the interaction was investigatedvia marginalmeans. The significance results of the primary factors are considered tobe uninformative and can be ignored.II. Summary of ResultsEgg production increased for the first few weeks of all three generations. After the initial few weeks of each generation, eggproduction from week to week was very similar. Interactionswere significant for all three generations, meaning that the dosegroups’egg production change from week to week werenot statistically equal. Apparent differences in at least one of the dose groupsoccurred in the F0 and F2 generations. The control group had amuch lower egg production compared to the other doses after week 10in the F0 generation, and dose 250 had a much lower egg production compared to the other doses after week 7 in the F2 generation.All generations generally had the same amount of egg production.III. F0 Generation ResultsThe dose by week interaction was statistically significant forthe F0 generation because of the decline in egg production after week 9for the control group (the other dose groups hadeither increases or littlevariation from week to week after week 9). Dose 30and dose125 typically had the highest egg production after the first initial weeks.ModelTerm P‐value Dose Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Overall00.45 2.00 3.35 4.65 4.50 3.55 3.15 2.90 3.20 2.85 3.00 2.75 2.40 2.68300.60 2.95 3.80 4.30 4.30 5.20 5.85 6.35 6.65 6.15 6.00 6.25 6.05 4.39600.40 2.40 3.80 4.25 4.55 4.95 4.70 5.60 5.10 5.40 5.55 5.60 5.40 3.861250.05 2.05 4.35 5.45 5.95 5.35 6.20 5.95 5.60 5.75 6.05 6.25 5.85 3.652500.40 2.45 4.15 5.35 5.40 4.75 5.25 5.10 5.75 5.40 4.65 5.20 5.75 3.99Overall0.29 2.35 3.87 4.77 4.90 4.71 4.90 5.00 5.11 4.94 4.90 5.00 4.83GEEEstimatedMarginalMeansF0GenerationEggProductionGEESignificanceTestsInteraction‐Dosebyweek0.000*Dose 0.051Week 0.000*Statisticallysignificantinteraction(p‐value<.05)indicatedthateggproductionchangefromweektoweekwassigni ficantlydi fferentbetweendosegroups.49IV. F1 Generation ResultsThe dose by week interaction was once again statistically significant.The interaction was significant because of the inconsistentordering of dose groups for egg production. Variation between the doses for each week was much less in the F1 generation comparedto the F0 and F2 generations. However, no dose group consistently had the highestor lowest egg production.V. F2 Generation ResultsAs was in the F0 and F1 generations, the dose by week interaction was statistically significant. The interaction was significantbecause the increase in egg production for dose 250 was not as high as the increase in the other four generations. Dose 250consistently had the lowest egg production after week 8. There was no dose group thatconsistently had the highest egg productionthrough the entire F2 generation. Egg production averages for thefirst few weeks of the F2 generation were lower than they were forthe previous two generations.ModelTerm P‐value Dose Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19 Week20 Week21 Overall00.95 2.80 4.45 5.45 5.40 5.70 4.90 5.20 4.50 5.30 5.90 5.90 5.30 5.20 4.35 2.75 4.30300.10 1.60 3.50 5.50 6.05 6.30 5.55 5.35 5.00 6.05 5.80 5.60 5.75 5.45 5.05 4.25 3.85601.55 3.50 4.95 5.65 5.40 5.70 5.30 3.95 4.50 5.50 5.55 5.40 5.00 4.30 4.40 3.55 4.451250.25 2.35 4.20 5.60 5.70 6.05 5.55 4.95 4.15 5.00 5.20 5.10 5.95 5.90 4.75 3.70 4.012500.20 3.00 4.95 5.85 5.60 5.45 4.95 5.40 4.35 5.50 5.60 5.25 5.50 5.15 4.45 4.05 4.04Overall0.37 2.56 4.37 5.61 5.62 5.83 5.24 4.94 4.49 5.46 5.60 5.44 5.49 5.17 4.59 3.62GEEEstimatedMarginalMeansF1GenerationEggProductionDose 0.539Week 0.000Interaction‐Dosebyweek0.000*GEESignificanceTests*Statisticallysignificantinteraction(p‐value<.05)indicatedthateggproductionchangefromweektoweekwassignificantlydifferentbetweendosegroups.ModelTerm P‐value Dose Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19 Overall00.35 0.85 3.25 3.95 3.85 4.70 5.95 6.20 5.45 5.70 5.35 5.35 5.85 5.40 3.63300.60 1.60 3.30 3.80 4.35 5.15 5.80 6.35 5.30 6.25 5.70 5.80 5.65 5.70 4.07600.45 0.75 3.15 3.85 4.75 5.10 5.65 6.50 5.15 6.05 5.40 5.85 5.65 5.05 3.731250.05 0.55 2.95 3.40 4.50 5.40 4.95 6.05 4.90 5.15 4.35 5.00 5.90 5.90 2.952500.30 1.00 2.55 2.40 3.45 4.00 3.65 4.50 4.25 4.85 4.05 4.30 4.80 4.80 2.93Overall0.27 0.89 3.03 3.43 4.15 4.84 5.12 5.87 4.99 5.57 4.93 5.22 5.55 5.35*Statisticallysigni ficantinteraction(p‐value<.05)indicatedthateggproductionchangefromweektoweekwassigni ficantlydi fferentbetweendosegroups.GEEEstimatedMarginalMeansF2GenerationEggProductionDose 0.087Week 0.000GEESignificanceTestsInteraction‐Dosebyweek0.000*

# 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 for the dose groups

were significantly different. An F-st atistic and corresponding p-value were

calculated for percent motile, percent progressive, and sperm per gram. If the pvalue was below .05, then it was conclude d that at least two dose groups had

statistically different averages for that corresponding measure. A Tukey post-hoc

test was then used to find which specif ic dose groups were statistically different

from one another. A Levene’s test was used to check to make sure the dose

groups had equal variances, a major assumption that must be met in order to

properly run an ANOVA model. If this assumption was violated (p-value for

Levene’s test < .05), a non-parametric Kr uskal-Wallis test was run instead of an

ANOVA.

II. Summary of Results

The F1 generation had severely lowe r averages for all three measures

compared to the F0 and F2 generations. The F0 generation had slightly higher

averages for percent motile and percent progressive compared to the F2

generation, however, in general, sperm per gram averages were higher for the F2

generation. The main exception was th e control group which had about 1,400

units more in the F0 generation compared to the F2 generation. Through all three

generations, differences in the three sp erm measures comparing across all dose

groups was minimal, with very few statistically significant differences.

III. F0 Generation Results

Only sperm per gram had significan t differences among dose groups. The

control group had significantly higher sp erm per gram average than dose 125.

The control group, followed by dose 30, had th e highest sperm per gram averages

for all dose groups. Minimal, non-signifi cant differences occurred between the

five dose groups for percent motile or pe rcent progressive. The control group had

the highest percent motile and the second highest percent progressive out of all

the dose groups.

# 2 IV. F1 Generation Results

Statistically significant differences between dose groups occurred only for

percent progressive in th e F1 generation. Dose 250 had a significantly higher

average percent progressive compared to dose 60, 30, and the control group.

Progressive and motile percents, in general, were higher in the more concentrated

endosulfan dose groups. F1 was the only ge neration where this trend held true.

Sperm per gram averages were highest for dose 60 and lowest for dose 30.

V. F2 Generation Results

In the F2 generation, percent progre ssive was the only factor showing a

significant difference. The control gr oup and dose group 60 had a significantly

greater percent progressive compared to the other three dose groups. No

significant differences occurred between dose groups for either percent motile and

sperm per gram, but dose 60 and dose 125 had the two highest averages. Dose Average Average Average

0 93.5 17.6 6917.7

30 84.3 17.3 5278.7

60 89.9 16.8 4786.7

125 86.8 18.1 4212.0

250 88.4 16.7 5132.3 1.2

7.9

2.1

4.0

3.3 1.7

2.2

1.3

2.6

1.7 385.4

786.3 Std. Erro r Std. Erro r Std. Erro r

709.7

610.7

443.6 F0 Generation

‐ Sperm

% Motile Sperm/gram % Progressive

* Tukey Pos t

‐ hoc tes t indi ca tes tha t dose 125 has a signi ficantly di ffe rent s pe rm per

gram ave rage compa red to the control group. 

## p

‐ value

= .600 p

‐ value

= .983 p

‐ value

= .033*



## Dose Average Average Average

0 37.3 3.8 443.2

30 31.6 3.1 342.3

60 45.3 5.5 496.2

125 44.4 4.5 427.7

250 47.8 9.8 465.2

° ID 177 (dose 250) excl uded from a nalysi s: recorded 0's for all three mea s urements

* Tukey post

‐ hoc tes t i ndi ca ted tha t dose 250 has signi fi ca ntly grea te r pe rcent

progres sive average compa red to dose 60, 30, and the control group. 

## 1.5 Std. Error % Progressive

56.6

55.0

46.0 Std. Error Std. Error

1.0

1.3 8.2

5.6 p

‐ value

= .328 p

‐ value

= .010* p

‐ value

= .338

60.4

53.0 0.8

1.9 5.9

5.9

3.5 Sperm/gram

F1 Generation

‐ Sperm

% Motile

# 3 Dose Average Average Average

0 77.8 16.0 5585.6

30 78.4 12.3 5167.8

60 85.4 15.6 6879.8

125 81.4 12.2 5663.6

250 66.2 11.6 5284.2 Std. Error Std. Error Std. Error

4.0

3.3

1.4

1.8

6.1 698.2

856.9

579.1

374.8

653.5 1.6

1.5

0.8

0.8

1.7 p

‐ value

= .338* p

‐ value

= .014* ° p

‐ value

= .303* % Motile % Progressive Sperm/gram

° Pos t hoc eval ua tion indi ca tes tha t the control group and dose 60 have signi ficantly

grea ter percent progres sive compa red to dose 30, 125, and 250. * P

‐ val ue calcula ted from Kruskall

‐ wallis tes t

‐ homogenei ty of va riance a s s umpti on

viola ted for pa rame tri c ANOVA tes t 

## F2 Generation

‐ Sperm

# 4 

# GROWTH and BODY WEIGHT

I. Analysis Methods

A repeated measured Linear Mixed Mode l was run to determine if there was a

significant difference in chick body wei ghts among the five dose groups. A

repeated measures model was used to test the dose and week factors as well as

their interaction, while accounting for the variation in the da ta from week to

week. Dose, week, and interaction were determined to be significant if the pvalue for the Mixed Model test was less than .05. Estimated marginal means were

calculated for both factors and examined for differences using a Tukey post-hoc

test, if the p-value for that factor wa s less than .05. If the interaction was

statistically significant, the interaction was investigated via marginal means. The

significance results of the primary factors are considered to be uninformative and

can be ignored.

ANOVA tests were used to determin e differences between doses in single

measurement-body weight, tarsus length, and length from the tip of the beak to

the culmen averages. Since the same birds were not measured each week,

ANOVA tests needed to be performed sepa rately for each day of measurements.

If p-values for an ANOVA test were belo w .05, the Tukey post- hoc test was used

to determine which doses had significantly different averages for that particular

measure.

II. Summary of Results

For all three generations, females had greater average body weights compared

to males. Females also tended to gain more weight throughout the duration of the

study compared to males. For both genders , the F1 generation started out with

much higher average body weights compared to the P and F2 generations. At

about week 4, the three gene rations average bodyweights became more similar.

Even with significant differences, body weight differences between dose group,

within each generation, were very minimal.

III. F0 Generation Results (Week 6 through Week 18)

For both genders in the P generation, the interaction was not significant. Week

was a statistically significant factor for both genders, but dose was only

significant for males. In general, as time increased, body weights increased for

both genders. For males, Tukey post-hoc tests revealed that dose 125 had a

significantly lower average body weight compared to the control group, dose 30,

# 5 and dose 60. In addition, dose 250 ha d a significantly lower body weight

compared to dose 30 and dose 60.

IV. F1 Generation Results (Week 2 to Week 7)

Prior to full onset of the study, addi tional information was collected on the

chicks, but it was limited and sporadic. Data was collected for the tarsus length,

the length from the tip of the beak to th e culmen, as well as the full body weight.

Minimal variation in tarsus and culmen averages occurred between the doses

within each measurement da y between week 2 and week 7. The only statistically

significant difference occurred in the ta rsus average on 7/13/2009. The control

group had a significantly greater average compared to dose 60. In general, tarsus

and culmen averages were so similar that no obvious patt erns between dose

groups could be detected. Significantdifferences in average body weight

occurred more frequently than the tarus and culmen averages. However, the

average body weight for the doses within each week were very similar and even

the significant differences were minimal in magnitude. Factor P

‐ value Dose 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 Overall

0 105.1 113.9 124.7 130.0 129.8 132.1 131.5 136.3 137.4 138.2 134.1 141.5 138.0 130.2

30 108.9 120.5 125.5 125.7 129.7 133.3 136.4 138.2 138.2 139.5 139.7 140.4 142.3 132.2

60 103.7 115.2 125.3 127.6 130.5 133.7 131.5 134.6 136.9 137.0 138.3 140.1 142.9 130.6

125 104.3 116.6 126.7 129.5 131.9 131.5 135.9 135.9 134.0 137.7 140.7 138.6 141.6 131.1

250 105.1 117.6 125.4 130.3 133.7 132.3 134.3 135.0 141.9 139.4 133.9 140.7 143.0 131.7

Overall 105.4 116.7 125.5 128.6 131.1 132.5 133.9 136.0 137.7 138.3 137.3 140.3 141.5 F0 Generation Female Body Weights

Estimated Marginal Means Significance Tests

Interaction

‐

dose by week 0.917

Dose 0.161

* Tukey post

‐ hoc tests indicate that weekly averages are signi ficantly different from each other. Week 0.000*

Factor P

‐ value Dose 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 Overall

0 98.2 103.6 105.2 107.6 107.8 109.5 108.0 109.2 110.3 111.5 111.9 113.1 115.4 108.5

30 99.5 104.8 106.4 106.7 107.8 110.2 107.9 111.3 112.7 112.0 111.9 113.6 114.8 109.2

60 99.2 103.9 105.6 107.3 109.2 109.9 107.9 108.6 112.0 111.1 113.8 112.4 114.3 108.8

125 100.0 103.1 103.2 104.0 105.0 106.6 104.4 105.8 102.3 109.5 111.0 110.3 111.1 105.8

250 98.0 1010.9 103.5 105.4 106.8 108.0 106.6 108.3 108.6 108.8 110.4 111.5 113.9 107.0

Overall 99.0 103.4 104.8 106.2 107.3 108.8 106.9 108.6 109.2 110.6 111.8 112.2 113.9

* Tukey post

‐ hoc tests indicate that dose 125 had signi ficant lower body weight compared to the control group, dose 30, and dose 60. Dose 250 had a significantly lower body weight compared to dose 30 and dose

60.

** Tukey post

‐ hoc tests indicate that weekly averages are signi ficantly di fferent from each other. F0 Generation Male Body Weights

Significance Tests Estimated Marginal Means

Interaction

‐

dose by week 0.999

Dose 0.000*

Week 0.000**

# 6 V. F1 Generation Result s (Week 8 through Week 22)

The dose by week interaction was statistically significant for the females in the

F1 generation. Dose 250 generally has the highest body weight average while the

control group and dose 60 had th e lowest body weight averages.

The dose by week interaction was not significant for the males; however, week

and dose were highly significant. As week increased, body weight generally

increased. Tukey post-hoc tests indicat ed that the control group’s average body

weight was significantly lower than the other four dose groups. Additionally,

dose 30 had a significantly lower bod y weight compared to dose 60. Week 2 Week 3 Week 4

Female Male Female Male Female Male

p

‐ value = .13 p

‐ value = .00 N/A* p

‐ value = .00 p

‐ value = .00 p

‐ value = .30 p

‐ value = .58 p

‐ value = .35 p

‐ value = .01

0 13.5 35.0 54.3 78.2 75.5 97.3 91.1 113.9 94.2

30 14.2 36.9 80.0 76.4 75.0 93.1 95.6 110.3 102.9

60 15.8 26.7 51.3 66.9 80.3 100.1 93.1 118.5 98.9

125 18.4 34.8 58.9 79.2 67.9 99.4 95.3 115.5 101.5

250 18.3 35.2 58.0 80.7 76.4 102.1 93.2 113.4 103.6

p

‐ value = .33 p

‐ value = .00 p

‐ value = .63 p

‐ value = .61 p

‐ value = .73 p

‐ value = .17 p

‐ value = .60 p

‐ value = .22 p

‐ value = .65

0 18.1 22.6 20.4 32.7 32.1 33.3 33.1 33.6 33.1

30 19.0 21.3 21.7 32.8 32.1 33.3 32.7 33.4 32.7

60 19.3 19.0 21.4 32.4 32.5 33.3 33.0 33.4 32.8

125 19.9 20.1 21.3 33.3 32.0 33.3 33.0 33.2 33.0

250 19.3 20.5 20.6 32.5 32.1 34.3 33.3 34.1 33.2

p

‐ value = .14 p

‐ value = .09 p

‐ value = .53 p

‐ value = .96 p

‐ value = .52 p

‐ value = .05 p

‐ value = .41 p

‐ value = .48 p

‐ value = .11

0 3.7 6.0 7.4 6.8 6.8 7.6 7.2 7.4 6.9

30 3.9 6.5 7.1 6.9 6.7 7.7 7.1 7.2 7.2

60 3.8 6.3 7.5 6.9 7.0 7.4 7.0 7.4 6.9

125 4.1 6.3 7.4 6.9 6.8 7.2 7.3 7.4 7.4

250 4.1 5.8 7.0 6.8 6.9 7.5 7.2 7.1 7.4

* Dose 30 only had 1 observa tion. Dose 30 va ria nce was could not be calcula ted s o the ANOVA tes t was unable to be executed.

Dose F1 Generation Tarsus, Culmen, and Body Weight Averages

Tarsus

Culmen Week 7 Week 6 Week 5

Body Weight

Factor P

‐ value Dose Week 8 Week 9 Week 10 Week 1 1 Week 12 Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21 Week 22 Overall

0 118.4 126.5 128.5 127.5 130.0 131.3 130.1 132.1 135.9 134.7 134.3 134.1 132.8 135.3 127.2 130.6

30 124.2 129.0 136.3 134.9 135.1 136.2 136.6 138.1 136.3 140.9 138.3 140.1 138.8 142.9 143.6 136.7

60 138.2 126.8 131.9 130.7 131.2 131.9 126.8 131.0 131.0 139.6 133.3 125.0 129.2 133.2 135.8 131.7

125 126.5 132.5 133.7 135.1 134.8 136.0 136.8 131.8 137.0 139.3 138.7 142.0 141.8 136.9 138.9 136.1

250 130.6 132.6 136.1 137.1 133.6 138.5 139.1 139.3 142.4 142.4 139.3 141.5 143.2 144.6 143.1 138.9

Overall 127.5 129.4 133.3 133.0 132.9 134.8 133.9 134.5 136.5 139.4 136.8 136.5 137.1 138.6 137.7

* Statistically signi ficant interaction indicated that body weight change from week to week was signi ficantly di fferent between the five dose groups. Estimated Marginal Means Significance Tests

Interaction

‐

dose by week 0.005*

Dose 0.000

Week 0.000 F1 Generation Female Body Weights

# 7 VI. F2 Generation Results (Week 0 through Week 4)

Prior to full onset of the study, addi tional information was collected on the

chicks, but it was limited and sporadic. Data was collected for the tarsus length,

the length from the tip of the beak to th e culmen, as well as the full body weight.

No significant differences between doses occur for any of the body weight, tarsus,

or culmen data collected in the F2 genera tion from week 0 to week 4. Negligible

differences in averages occurred between doses, but there was no visible pattern

between doses. Factor P

‐ value Dose Week 8 Week 9 Week 10 Week 1 1 Week 12 Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21 Week 22 Overall

0 101.9 103.7 105.1 105.1 106.5 105.9 104.6 106.8 111.7 108.3 109.0 112.2 108.3 111.9 108.2 107.3

30 103.8 107.4 108.7 107.9 109.2 107.0 107.0 111.8 111.3 111.1 112.0 112.2 112.6 116.1 114.5 110.1

60 106.2 108.8 110.8 110.4 111.5 111.3 110.0 111.6 113.9 114.5 115.5 109.9 112.1 117.6 116.7 112.0

125 107.4 109.1 109.9 109.3 110.7 109.7 108.9 114.4 111.2 111.8 112.5 112.2 112.7 114.5 115.9 111.3

250 104.8 107.7 108.1 107.6 108.4 108.3 110.2 109.5 111.2 111.7 112.1 112.5 113.3 115.5 115.3 110.4

Overall 104.8 107.3 108.5 108.0 109.2 108.4 108.1 110.6 111.8 111.5 112.2 111.8 111.8 115.1 114.1

* Tukey post

‐ hoc tests indicated that the control group's body weight was significantly lower than the other four dose groups. Dose 30 also ahd a significantly lower body weight compared to dose 60.

** Tukey post

‐ hoc tests indicate that weekly averages are signi ficantly di fferent from each other. Estimated Marginal Means

Dose 0.000* Significance Tests

Interaction

‐

dose by week 0.996

Week 0.000** F1 Generation Male Body Weights



### Dose Week 1 Week 2

‐ A Week 2

‐ B Week 3

‐ A Week 3

‐ B Week 4

‐ A Week 4

‐ B Week 5

‐ A Week 5

‐ B

p

‐ value = .91 p

‐ value = .94 p

‐ value = .47 p

‐ value = .57 p

‐ value = .95 p

‐ value = .77 p

‐ value = .83 p

‐ value = .65 p

‐ value = .78

0 7.6 16.2 24.8 34.6 46.9 57.0 70.3 78.7 81.8

30 7.2 16.9 25.7 36.2 47.7 60.3 72.3 80.6 84.0

60 7.7 17.2 26.6 37.4 48.2 59.6 72.0 79.2 83.2

125 7.7 17.1 24.6 36.8 47.7 60.3 73.1 82.5 84.6

250 7.6 16.3 23.9 36.0 46.6 58.4 74.0 82.8 86.0

p

‐ value = .80 p

‐ value = .26 p

‐ value = .28 p

‐ value = .31 p

‐ value = .14 p

‐ value = .39 p

‐ value = .16 p

‐ value = .26 p

‐ value = .74

0 15.6 17.8 20.9 23.0 27.2 28.0 30.3 31.0 32.7

30 15.8 19.4 21.4 23.4 26.9 29.7 31.5 31.8 32.8

60 16.1 19.1 21.3 24.1 28.3 29.7 31.7 31.9 32.3

125 15.8 19.8 19.0 23.1 27.1 28.6 31.8 32.8 32.3

250 16.0 18.9 20.1 22.4 26.0 28.5 31.5 32.4 33.1

p

‐ value = .37 p

‐ value = .70 p

‐ value = .43 p

‐ value = .76 p

‐ value = .57 p

‐ value = .56 p

‐ value = .26 p

‐ value = .79 p

‐ value = .82

0 3.8 4.7 5.6 6.3 6.7 7.3 7.4 7.9 7.8

30 3.7 4.7 5.6 6.5 6.6 7.4 7.8 8.0 7.7

60 3.9 4.9 5.7 6.6 5.8 7.7 7.5 8.1 7.9

125 3.7 4.9 5.9 6.3 5.9 7.3 7.4 7.9 7.8

250 4.0 4.8 5.5 6.6 6.6 7.4 7.2 7.9 7.7 Body Weight

Tarsus

Culmen F2 Generation Tarsus, Culmen, and Body Weight Averages

# 8 VII. F2 Generation Result s Week 1 through Week 14

Week and dose were statistically sign ificant or both genders in the F2

generation, and the interaction term wa s not. As week increased, average body

weight for both genders steadily incr eased. In females, dose 60 had a

significantly lower body weight average co mpared to dose 30 and dose 125. For

males, the control group had a signifi cantly lower body weight compared to the

other four dose groups. Furthermore, in males, dose group 125 had a significantly

higher body weight compared to the other four dose groups.

Factor P

‐ value Dose 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 Overall

0 100.9 108.8 124.3 134.5 133.5 132.3 137.8 137.9 140.3 141.6 138.3 138.3 141.1 141.8 132.2

30 106.7 116.0 124.2 130.5 133.9 135.6 137.6 138.5 139.7 143.2 139.8 140.0 140.2 142.1 133.4

60 102.1 108.2 124.5 131.5 131.6 132.5 135.9 138.3 138.5 140.0 136.5 136.5 138.6 137.2 130.9

125 103.5 112.6 125.2 129.6 136.1 139.3 139.7 140.1 140.4 142.4 139.6 139.6 142.2 145.0 133.9

250 108.1 115.7 125.8 128.1 131.6 133.4 135.9 137.7 139.3 142.0 140.0 140.1 140.2 140.6 132.7

Overall 104.2 112.2 124.8 130.8 133.3 134.6 137.5 138.5 139.6 141.8 138.8 138.9 140.4 141.3 F2 Generation Female Body Weights

Dose 0.003*

Week 0.000**

* Tukey post

‐ hoc tests indicated that dose 60 had a significantly lower body weight compared to dose 30 and dose 125.

** Tukey post

‐ hoc tests indicate that weekly averages are signi ficantly different from each other.

Significance Tests

Interaction

‐

dose by week 0.948 Estimated Marginal Means

Factor P

‐ value Dose 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 Overall

0 94.3 98.1 103.4 104.4 104.7 106.1 108.5 108.5 108.7 109.4 109.8 110.1 110.5 105.9

30 100.4 104.2 107.3 109.4 109.5 110.7 112.6 112.7 113.4 114.1 114.1 115.6 114.4 110.6

60 97.6 101.9 106.0 108.0 108.7 110.0 110.8 112.0 111.4 112.9 112.9 112.8 113.2 109.1

125 102.8 106.9 109.7 111.7 113.4 113.6 115.1 115.6 114.3 115.1 114.3 114.7 114.7 112.4

250 101.0 103.6 104.1 107.2 108.6 110.2 111.9 113.0 111.2 112.8 113.3 113.5 113.6 109.5

Overall 99.2 102.9 106.1 108.1 109.0 110.1 111.8 112.3 111.8 112.8 112.9 113.3 113.2

** Tukey post

‐ hoc tests indicate that weekly averages are signi ficantly di fferent from each other. * Tukey post

‐ hoc tests indicate that the control group had a significantly lower body weight compared to the other four dose groups. Dose 125 had a significantly higher body weight compared to the four other dose

groups. Interaction

‐

dose by week 1.000

Dose 0.000*

Week 0.000** F2 Generation Male Body Weights

Significance Tests Estimated Marginal Means

# 9 

# FEED CONSUMPTION

I. Analysis Methods

A repeated measured Linear Mixed Mode l was run to determine if there was a

significant difference in food consump tion between the five dose groups. A

repeated measures model was used to te st the dose and week as well as their

interaction, while accounting for the variati on in the data from week to week.

Dose, week, and interaction were determined to be significant if the p-value for

the Mixed Model test was less than .05. Estimated marginal means were

calculated for both factors and examined for differences using a Tukey post-hoc

test, if the p-value for that factor wa s less than .05. If the interaction was

statistically significant, the interaction wa s investigated via marginal means. The

significance results of the primary factors are considered to be uninformative and

can be ignored. Also, the food consump tion calculations that were incorrectly

recorded as negative were excluded from the analysis since it was impossible for

the birds to eat more feed than was originally in the container.

II. Summary of Results

Males and females typically started out with equal food consumption

averages for the first weeks of data co llection, however female food consumption

increased extensively more throughout th e unpaired weeks than males. No dose

group consistently had higher food consumpti on averages for either the paired or

unpaired weeks. Food consumption averag es, overall, genera lly increased from

one week to the next, however, certain weeks throughout all generations had very

low or very high averages, making one question how valid th e food consumption

measurements really were.

III. F0 Generation Results (Week 5 through Week 10 – unpaired genders)

In the P generation of females, the intera ction was statistically significant. All

doses increased from one week to the next; however, each dose tended to have

higher rate of increase at different week s. Dose 125 and dose 250’s increase in

food consumption during week 8 through 10 were much less than the other dose

groups.

For males, the dose by week interacti on was not significant, but week and dose

were. Food consumption was lowest at w eek one, but generally was steady after

that. The control group had a significantly greater food consumption compared to

all the other dose groups, which had similar food consumption averages.

# 10 IV. F0 Generation Results (Week 11 through Week 18 – paired genders)

From week 11 to week 18, the interac tion was not statistically significant,

however dose and week were. From week 11 to week 12, food consumption

drastically increased. From week 12 to week 17, food consumption was fairly

consistent until week 18, in which food consumption severely dropped. Dose 30

and dose 125’s food consumption averages were significantly greater than the

control group and dose 60 averages.

V. F1 Generation Results (Week 7 through Week 12 – unpaired genders)

For the F1 generation, both genders had non-significant interaction, but

significant differences by week. For females, dose was also statistically

significant. Food consumption generally in creased one week to the next for both Factor P

‐ value Dose Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Overall

0 113.1 114.9 138.7 154.6 166.7 199.8 148.0

30 132.0 134.8 146.1 146.0 156.8 182.5 149.7

60 117.6 121.9 154.3 154.0 154.3 196.5 149.8

125 123.1 141.1 146.7 187.0 191.2 187.2 162.7

250 125.4 145.6 147.8 175.5 175.4 186.7 159.6

Overall 122.2 131.6 146.7 163.6 168.9 190.5 Significance Tests

Interaction

‐

dose by week 0.003

Dose 0.000 F0 Generation Female Food Consumption (Weeks 5

‐ 10)

Estimated Marginal Means

Week 0.000



### Factor P

‐ value Dose Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Overall

0 107.4 121.2 131.7 123.1 121.4 136.9 123.6

30 109.5 117.3 122.0 107.4 106.8 118.3 113.5

60 113.5 113.2 121.3 115.9 116.1 118.8 116.5

125 112.9 111.0 112.1 118.6 109.7 116.4 113.4

250 106.6 111.4 114.9 117.9 114.5 121.1 114.4

Overall 110.0 114.8 120.4 116.6 113.7 122.2 Interaction

‐

dose by week 0.111

* ID 69

‐ dose 30 datapoints removed for week 9 and week 10 due to low food consumption results. 

### Dose 0.000

Week 0.000 F0 Generation Male Food Consumption (Weeks 5

‐ 10)

Estimated Marginal Means Significance Tests

Factor P

‐ value Dose Week 11 Week 12 Week 13 Week 14 Week 15 Week 16 Week 17 W eek 1 8 Overall

0 154.0 290.9 305.4 306.1 282.1 293.6 289.1 113.7 254.4

30 160.4 325.7 324.9 326.5 323.6 302.4 316.2 126.7 275.8

60 159.0 300.9 279.2 330.5 292.1 289.9 295.9 125.1 259.1

125 163.4 320.2 320.1 323.6 317.9 313.6 315.7 127.7 275.3

250 161.8 299.5 291.0 305.5 292.0 292.4 318.6 127.1 261.0

Overall 159.7 307.5 304.1 318.4 301.5 298.4 307.1 124.1 Dose

0.000 F0 Generation Food Consumption (Week 11

‐ 18)

Estimated Marginal Means Significance Tests

Interaction

‐

dose by week 0.640

0.000

Week

# 11 genders. For females, dose group 250 had a significantly larger food consumption

compared to all other doses. Even though significant differe nces did not occur

between dose groups for males, dose 60 had the greatest food consumption

average while dose 30 had the lowest food consumption average.

VI. F1 Generation Results (Week 13 through Week 21 – paired genders)

For the paired birds in week 13 through week 21, week and dose were

significant while their interaction was not. Tukey post-hoc results indicated that

dose 250 and the control group had signi ficantly greater food consumption

averages compared to dose 125. Additionally, the control group had a

significantly greater food consumption av erage compared to dose 30. Week 13

and week 14’s averages were well below th e averages through the rest of the F1

generation. Factor P

‐ value Dose Week 7 Week 8 Week 11 Week 12 Overall

0 74.6 126.1 150.1 196.8 131.3

30 78.4 123.4 151.5 190.5 129.9

60 102.9 131.6 147.0 199.8 137.4

125 89.5 124.7 143.4 202.1 133.8

250 103.3 145.5 163.1 215.1 149.5

Overall 89.7 130.3 151.0 200.8 F1 Generation Female Food Consumption (Weeks 7

‐ 12)

Significance Tests Estimated Marginal Means

0.000 Week

* Id 119, 12, 299, 163, and 163 datapoints removed for week 7 due to low food consumption results.



### Interaction

‐

dose by week 0.069

Dose 0.000



### Factor P

‐ value Dose Week 7 Week 8 Week 9 Week 11 Week 12 Overall

0 79.4 90.1 97.9 95.6 124.1 97.6

30 71.9 91.6 95.1 93.3 117.9 94.0

60 68.5 97.5 102.5 102.7 124.1 99.1

125 70.5 95.5 89.6 102.4 126.1 97.1

250 74.7 95.8 96.0 100.5 120.3 97.7

Overall 73.0 94.5 96.2 99.1 122.5 Interaction

‐

dose by week 0.157

Dose 0.188

Week 0.000 Significance Tests F1 Generation Male Food Consumption (Weeks 7

‐ 12)

Estimated Marginal Means

# 12 VII. F2 Generation Results (Week 6 through Week 18 – unpaired genders)

The interaction was statistically significant for both genders in the F2

generation. For females, the interaction was significant because of the large drop

in food consumption for dose 60 and dose 30 in week 16. The control group for

females did have the highes t overall food consumption av erages, but did not have

the highest average throu ghout all thirteen weeks.

For males, minimal differences in food consumption occurred between dose

groups within each week, and no particular dose group that generally had greater

or lower food consumption averages compared to the other doses. The interaction

was found significant due to the greater fl uctuation in food consumption in the

dose 250 group compared to the other doses.

Overall, both genders had similar food consumption results until week 10

where females started consuming far mo re food. Variation between doses was

much greater for females compared to ma les, and no particular dose constantly

had a higher or lower food consumption compar ed to the others for either gender. Factor P

‐ value Dose Week 13 Week 14 Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21 Overall

0 179.3 130.9 346.4 293.4 315.3 309.1 286.7 327.8 257.7 271.9

30 161.7 142.6 338.3 263.2 291.0 298.4 278.4 270.8 248.9 254.8

60 173.4 138.4 329.1 266.5 290.9 286.1 268.1 331.4 241.0 258.3

125 161.0 130.9 323.3 246.9 297.9 294.5 294.3 279.7 243.6 252.5

250 172.8 131.8 336.8 281.8 301.5 308.1 304.2 316.7 273.4 269.7

Overall 169.6 134.9 334.7 270.4 299.3 299.2 286.3 305.3 252.9 0.000 Week 0.001 Estimated Marginal Means F1 Generation Food Consumption (Week 13

‐ 21)

Significance Tests

Interaction

‐

dose by week 0.559

Dose

Factor P

‐ value Dose 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 Overall

0 91.1 114.7 138.7 98.8 130.7 195.8 219.1 243.9 193.7 231.8 214.3 244.3 243.2 181.8

30 103.4 120.9 125.0 112.4 134.6 183.6 197.7 216.3 202.2 212.5 111.8 217.8 215.5 165.7

60 73.5 108.8 128.3 91.1 136.4 193.9 207.0 236.3 200.7 213.2 107.9 235.1 234.7 166.7

125 94.5 116.0 125.3 91.7 139.3 169.6 178.0 222.6 183.3 198.7 195.2 207.3 223.4 165.0

250 84.5 106.8 123.9 100.8 127.4 187.3 202.5 249.1 193.9 202.0 179.0 222.2 227.3 169.7

Overall 90.0 113.4 128.2 98.9 133.7 186.2 200.8 233.6 194.7 211.6 161.6 225.3 228.8

* Id 305 (week 9), 215 (week 9), 86 (week 16), 73 (week 16), 84 (week 16) datapoints removed due to inaccurate food consumption results Interaction

‐

dose by week 0.000

Dose 0.000 F2 Generation Female Food Consumption (Week 6

‐ 18)

Estimated Marginal Means

Week 0.000 Significance Tests

Factor P

‐ value Dose 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 Overall

0 97.8 120.8 122.0 114.6 104.6 121.3 120.9 196.0 119.1 133.7 118.2 116.2 118.1 123.3

30 98.9 115.4 119.6 122.4 107.7 113.6 110.4 200.1 122.8 129.9 112.6 108.5 116.0 121.3

60 89.2 121.7 127.2 123.9 115.2 122.1 130.0 204.1 123.9 127.6 114.0 119.8 124.2 126.4

125 94.2 125.0 129.3 122.0 122.5 120.1 116.1 193.8 119.1 126.0 106.6 113.5 127.9 124.3

250 96.6 116.8 117.3 103.9 132.5 122.1 124.5 198.1 122.3 124.0 109.2 112.2 121.3 123.1

Overall 95.3 119.9 123.1 117.4 116.5 119.8 120.3 198.4 121.4 128.2 112.1 114.0 121.5

* ID 193 datapoint removed from week 7 due to inaccurate food consumption result. F2 Generation Male Food Consumption (Week 6

‐ 18)

Estimated Marginal Means

Dose 0.077

Week 0.000 Significance Tests

Interaction

‐

dose by week 0.003

# 13 

# 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 five dose groups,

while accounting for the variation throughout weeks. P-values were calculated

for the dose and week as well as their in teraction. Dose, week, and interaction

were determined to be si gnificant if the p-value for the GLM was less than .05.

Estimated marginal means were calculat ed 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 st atistically significan t, the interaction was investigated

via marginal means. The significance results of the primary factors are

considered to be uninformative and can be ignored.

II. Summary of Results

CG area was the largest overall for the F1 generation. Additionally, in all three

generations, CG area, not surprisingly, in creased from one week to the next.

Statistically significant differences occu rred between doses in the P and F1

generations, but there was no distinct pattern between an increased dose

concentration and either an increase or decrease in CG area. In the P and F1

generations, however, the control group did have the largest CG area.

III. F0 Generation Results

The dose by week interaction was not st atistically significant, meaning that

from week 6 to week 7, the five doses increased CG area at the same rate.

However, dose and week were both statisti cally significant. Week 6’s CG area

was significantly larger than week 7’s. The control group has the largest average

CG area for both weeks, and post-hoc analys is revealed that the control group had

a significantly greater CG area compared to dose 60.

# 14 IV. F1 Generation Results

As in the F0 generation, dose and week were statistically significant factors,

while the interaction was not. Week 7’s CG area was significantly larger than

week 6’s CG area. The control group ha s the largest average CG area, and posthoc analysis revealed that the control group had a significantly greater CG area

compared to dose 30. Dose 60 had next larg est CG area, just slightly smaller than

the control group.

V. F2 Generation Results

In the F2 generation, the dose by w eek interaction and dose were not

statistically significant, however week was. The CG area’s increases from week 6

to week 7 and from week 7 to week 8 were both statistically significant. Overall,

dose 30 had the largest and dose 250 ha d the smallest CG area average. Factor P

‐ value Dose Week 6 Week 7 Overall

0 113.4 164.4 138.9

30 75.4 118.6 97.0

60 70.0 114.2 92.1

125 97.7 165.0 131.4

250 84.1 136.7 110.4

Overall 88.1 169.8 Estimated Marginal Means F0 Generation Cloaca

Interaction

‐

dose by week

Dose

Week 0.230

0.013*

0.000** Significance Tests

* Tukey post

‐ hoc tests indicate that the control group had a significantly greater CG

area compared to dose 60.

** Week 7 had significantly greater CG area compared to week 6.



## Factor P

‐ value Dose Week 6 Week 7 Overall

0 139.7 189.3 164.5

30 89.2 157.0 123.1

60 150.7 177.2 163.9

125 109.5 173.5 141.5

250 119.6 160.8 140.2

Overall 121.7 171.5 Interaction

‐

dose by week 0.588

Dose 0.017*

Week 0.000**

** Week 7 had a signi ficantly greater CG area average compared to week 6. * Tukey post

‐ hoc tests indicated that the control group had a signi ficantly greater

CG area compared to dose 30. 

## F1 Generation Cloaca

Estimated Marginal Means Significance Tests

# 15 Factor P

‐ value Dose Week 6 Week 7 Week 8 Overall

0 104.0 151.5 205.3 153.6

30 100.4 160.8 218.3 159.8

60 76.8 152.8 210.0 146.5

125 97.4 148.6 225.8 157.3

250 74.7 148.9 201.7 141.8

Overall 90.6 152.5 212.2

* All weeks were signi ficantly different from each other. 

## Significance Tests Estimated Marginal Means F2 Generation Cloaca

Week 0.000* Interaction

‐

dose by week 0.785

Dose 0.905

# 16 

# YOLK CONTENT

I. Analysis Methods

A non-parametric Kruskal-Wallis test was used to test if there was a significant

difference among doses in total endosulf ans, I endosulfans, and II endosulfans

present in the yolk. A Kruskal-Wallis test is the equivalent of the parametric

ANOVA test, however fewer assumptions ha ve to be met. Non-parametric

models rank the dataset in terms of ma gnitude and test to see if there are

differences in group medians/ranks rather than means. Equal variances of the

groups is not necessary and ample sample size per group is not required. With

unequal and small sample sizes per group, the non-parametric test was more

applicable for the yolk content data than other statistical tests.

II. P Generation Results

Kruskal-wallis average ranks indicated that endosulfan s (Total, I, II)

increased as dose concentration increased. This was true for all three yolk content

measures. The control group and dose 30 were significantly different from dose

125 and dose 250 for all three measures.

Dose 

## Total Endosulfans I II

P

‐ value 0.001 0.002 0.001

0 0.00 0.01 0.00

30 0.12 0.03 0.12

60 0.39 0.07 0.36

125 0.66 0.09 0.64

250 1.50 0.23 1.49 P Generation Yolk Content Medians

# 17 

# ONSET TO PUBERTY

I. Analysis Methods

An Analysis of Variance (ANOVA) te st was used to determine if onset to

puberty averages for dose groups were significantly differe nt. An F-statistic and

corresponding p-value was calculated for each gender within each generation. If

the p-value was below .05, then it was conc luded that at least two dose groups had

statistically different averages for onse t to puberty. A Tukey post-hoc test was

then used to find which specific dose groups were statistically different from one

another. A Levene’s test was used to check to make sure the dose groups had

equal variances, a major assumption that must be met in order to properly run an

ANOVA model. If this assumption was vi olated (p-value for Levene’s test <

.05), a non-parametric Kruskal-Wallis te st was run instead of an ANOVA.

II. Summary of Results

For all dose groups in all three ge nerations, females onset to puberty was

longer than males. Additionally for a ll dose groups in both genders (excluding

dose 30 females and dose 250 males), onset to puberty decreased in length from

the F0 to F1 generation and increased from the F1 to F2 generation. For females,

over all three generations, th ere was no specific dose gr oup that typically had the

shortest or longest onset to puberty aver ages. However, for males, dose 30 and

dose 250 generally had the longest onset to puberty out of all dose groups.

Overall, minimal statistical evidence s howed that there were statistically

significant differences between dose groups fo r onset to puberty in either males or

females.

III. F0 Generation Results

No statistically significant differences occurred between dose groups for either

gender within the F0 generation. Males ha d a p-value slightly below .05, but after

accounting for multiple testing, there were no significant differences between

dose groups. For males, dose 30 had the longest onset to puberty.

# 18 IV. F1 Generation Results

Statistically significant differences occurred between dose groups for the

females in the F1 generation. Dose 30 ha d a significantly longer onset to puberty

compared to the control group and dose 60. For the males, even though there

were no significant differences, dose 30 and dose 250 had the longest averages

and dose 60 had the shortest averages.

V. F2 Generation Results

With p-values well above .05, statisti cally significant differences between dose

groups were not present for either gender, comparing onset to puberty averages.

For females, dose 125 had the longest av erage while the control group had the

shortest averages. For males, dose 30 had the longest and dose 125 had the

shortest average onset to puberty. Dose Average Std. Error 

## Average Std. Error



## 0 57.2 1.68 

## 44.7 0.47



## 30 56.1 1.81 

## 46.8 0.67



## 60 56.6 1.70 

## 45.9 0.64



## 125 56.3 1.05 

## 44.7 0.51



## 250 56.3 1.44 

## 45.8 0.59 

## P

‐ value

= .991 P

‐ value

= .044 Females Males F0 Generation Onset to Puberty (days)



## Dose Average Std. Error 

## Average Std. Error



## 0 49.6 1.44 

## 43.2 2.47



## 30 55.4 1.31 

## 46.0 1.86



## 60 49.9 1.59 

## 41.6 1.92



## 125 54.3 1.27 

## 44.3 1.84



## 250 53.7 1.17 

## 46.5 1.92 

## Females Males

P

‐ value

= .007 * P

‐ value

= .405

* Tukey post

‐ hoc tests indicate that dose 30 has

signi ficantly longer onset to puberty compared to the

control group and dose 60. 

## F1 Generation Onset to Puberty (days)

# 19 Dose Average Std. Error 

### Average Std. Error



### 0 53.2 1.94 

### 48.1 1.32



### 30 54.4 2.18 

### 48.7 1.46



### 60 55.7 1.72 

### 48.1 1.26



### 125 57.3 1.81 

### 47.1 1.23



### 250 55.3 2.66 

### 48.1 1.24 

### F2 Generation Onset to Puberty (days)

Females Males

P

‐ value

= .680 P

‐ value

= .933

# 20 

# ENDOSULFAN EFFECTS ON REPRODUCTIVE

BEHAVIORS

Mounting and Copulation Disruption

This section of the report analyzes th e sexual initiation behavior characteristics

of male Japanese quails through three gene rations (F0, F1, and F2). Specifically,

the number of mount attempts, time to first mount and first copulation, and

number of copulations were an alyzed. Each pair of quail s was part of one of five

dose groups: (control) 0 ppm, 30 ppm, 60 ppm, 125 ppm, and 250 ppm. Data was

collected on the same pairs through three c onsecutive trials in each of the three

generations. SPSS version 16.0 was used to an alyze the behavior data. Statistical

significance for this analysis wa s determined using an alpha ( α ) of .05 with a 95%

confidence level.

# 21 

# NUMBER OF MOUNT ATTEMPTS

I. Analysis Methods

A Generalized Linear Model (GzLM) us ing a poisson loglinear link function

was used to determine if there was a st atistically significant difference in the

number of mounts attempted across the five dose levels. An omnibus likelihood

ratio test in GzLM, which compares the fitted model (intercept plus dose) against

the intercept only model, was used to dete rmine if dose was a significant factor in

predicting the number of m ounts in 180 seconds. If th e p-value for the likelihood

test was less than .05, dose was a signif icant factor in predicting the number of

mount attempts. Parameter estimates (B eta coefficients and Beta confidence

intervals) were then examined to find where the significant differences occurred

between the dose levels if the p-value for the omnibus test was less than .05. In

the GzLM procedure, a reference group was needed for the other dose levels to be

tested against for a significant difference. This was because we fit a categorical

predictor into a linear model. The refe rence group for this analysis was the

‘dose=0’ group which is referred to as the control group thro ughout the report.

An exponentiated β coefficient of 1.00 meant th at the dosed group was 1.00 times

as likely to have a mount compared to the control group, or in other words, the

two groups had an equal chance of having a mount. If the confidence interval

contained 1.00, there was no statistical difference be tween the two groups and

vice versa.

II. Summary of Results

For all dose groups, the numb er of mount attempts was much higher for the F0

generation compared to the latter two generations. Statistically significant

differences between dose groups occurred onl y in the P generation, mainly due to

the high number of mounts attempted fo r dose 125 in the first two trials.

III. F0 Generation Results

Significant differences between dose groups occurred in the first two trials for

the F0 generation. In trial 1, the co ntrol group and dose 125 had significantly

greater number of mount attempts compared to dose 30, dose 60, and dose 250.

In trial 2, dose 125 had a significantly greater number of mount attempts

compared to the control group, dos e 30 and dose 60. Overall, dose 125

consistently had one of the highest es timated number of mount attempts. The

control group was also the only dose group to decline in attempted mounts from

both trial 1 to trial 2 and trial 2 to trial 3.

# 22 IV. F1 Generation Results

Likelihood ratio p-values, which were we ll above .05, indicated that there were

no statistically significant diffe rences between doses within any of the three trials,

comparing the number of mount attempts. From trial to trial, the number of

mount attempts per dose group slightly increased, with the exception of the

control group’s decline from trial 2 to tr ial 3. In trials 1 and 2, the control group

had the highest estimated number of atte mpted mounts, but in trial 3, the control

group’s estimated number of mount attempts was the lowest out of all five dose

groups.

V. F2 Generation Results

Likelihood ratio p-values, which were we ll above .05, indicated that there were

no statistically significant diffe rences between doses within any of the three trials,

comparing the number of mount attempts. Ho wever, in all three trials, the control

group had the lowest number of estima ted mount attmpts, and dose 30 had the

highest number of estimated mount attempts. Overall, variation from trial to trial

was very minimal, with the sharpest increases and decreases, from trial to trial,

occurring in the dose 30 and dose 60 group, respectively. 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 6.60 4.95 4.40

30 4.60 .53

‐ .91 0.01 4.35 .66

‐ 1.18 0.39 5.00 .81

‐ 1.44 0.38

60 4.20 .48

‐ .84 0.00 4.75 .72

‐ 1.28 0.78 5.35 .89

‐ 1.58 0.17

125 6.55 .78

‐ 1.26 0.95 6.90 1.07

‐ 1.81 0.01 5.21 .92

‐ 1.61 0.25

250 4.40 .51

‐ .87 0.00 5.55 .85

‐ 1.48 0.41 4.75 .81

‐ 1.44 0.61

* 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 F0 Generation

‐ Number of Mounts

Trial 1

Omnibus Likelihood ratio p

‐ value = .000 Trial 2

Omnibus Likelihood ratio p

‐ value = .006 Trial 3

Omnibus Likelihood ratio p

‐ value = .676

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 2.45 2.80 2.20

30 1.90 .51

‐ 1.19 0.24 2.20 .53

‐ 1.17 0.23 2.21 .66

‐ 1.53 0.98

60 1.95 .52

‐ 1.21 0.29 2.70 .66

‐ 1.40 0.85 2.75 .84

‐ 1.86 0.27

125 2.00 .54

‐ 1.24 0.34 1.95 .46

‐ 1.05 0.08 2.50 .76

‐ 1.70 0.54

250 2.05 .55

‐ 1.27 0.40 2.32 .56

‐ 1.23 0.35 2.65 .79

‐ 1.82 0.38

* 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 Trial 1 Trial 2 Trial 3

Omnibus Likelihood ratio p ‐ value = .740 Omnibus Likelihood ratio p ‐ value = .381 Omnibus Likelihood ratio p ‐ value = .773 F1 Generation

‐ Number of Mounts

# 23 

# TIME UNTIL FIRST MOUNT ATTEMPT

I. Analysis Methods

Kaplan-Meier survival analysis was used to determine if there was a significant

difference between the five dose levels in terms of the amount of time it took the

birds for their first mount attempt. If th eir time was recorded as 180 seconds, this

meant that the quail did not make a m ount attempt and that observation was

considered censored. A log rank statis tic was computed to test whether the

different dose groups had statis tically different times until the first mount attempt.

If the p-value associated with the log rank statis tic was less than .05, it was

concluded that the times between at l east two of the dose groups were not

statistically equal. To find which dose gr oups did not have equal times, estimated

confidence intervals for the time to attemp ted mount were compared. If two dose

groups had confidence interv als that did not overla p, these two doses were

considered to have statistically differe nt times at the 95% confidence level.

II. Summary of Results Trial 2

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 1.95 1.90 1.65

30 2.25 .75

‐ 1.77 0.51 2.55 .88

‐ 2.04 0.17 2.21 .85

‐ 2.11 0.21

60 1.95 .64

‐ 1.56 1.00 1.50 .49

‐ 1.27 0.33 2.10 .81

‐ 2.01 0.30

125 2.20 .73

‐ 1.74 0.58 2.05 .69

‐ 1.68 0.74 1.80 .68

‐ 1.75 0.72

250 2.05 .68

‐ 1.63 0.82 2.10 .71

‐ 1.71 0.66 2.15 .83

‐ 2.05 0.25

* 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 F2 Generation

‐ Number of Mounts

Trial 1

Omnibus Likelihood ratio p

‐ value = .945 Trial 3

Omnibus Likelihood ratio p

‐ value = .659 Omnibus Likelihood ratio p

‐ value = .221

# 24 In general, through all three genera tions, dose 30 and dose 250 had the

shortest time until first mount attempt. Additionally, almost all dose groups had

the longest time to first attempted mount in trial 1. This was true for all three

generations. In the first two generations , minimal, non-statis tically significant

differences occurred between dose groups within each trial. However, greater

variation between dose groups occurred in the F2 generation. In the F2

generation, the control group and dose 60 ge nerally had the longest time until the

first mount attempt, and dose 30 had, by far, the shortest time until first mount

attempt.

III. F0 Generation Results

Log rank p-values indicated that th ere were no statistically significant

differences between any doses within any of the three trials, comparing the time

to first mount attempt. Time until first mount attempt decreased greatly from trial

1 to trial 2, however, only s lightly decreased from tria l 2 to trial 3. Dose group

125 and dose 250 generally had the shortest estimated time until first mount

attempt in all three trials.

IV. F1 Generation Results

Log rank p-values, which were all well a bove .05, indicated th at there were no

statistically significant differences betw een any doses within any of the three

trials, comparing the time to first mount at tempt. Just like in the P generation, the

estimated time until first attempted mount decreased from one trial to the next.

Mount times were typically lowest for dose 30 and dose 250, and were generally

the highest for the control group and dose 125. Dose

0 38.5 12.7 

### 90% 21.9 11.8 

### 90% 21.5 11.8 

### 90%

30 58.1 14.6 

### 80% 27.8 11.9 

### 95% 14.7 8.6 

### 95%

60 77.6 14.8 

### 80% 28.6 10.0 

### 95% 22.5 9.2 

### 95%

125 26.5 9.3 

### 95% 18.7 9.2 

### 95% 15.7 9.0 

### 95%

250 47.9 13.7 

### 85% 16.5 6.3 

### 100% 10.8 4.1 

### 100% F0 Generation

‐ Time to first Mount

Trial 1 Trial 2 Trial 3

Log rank p

‐ value = .069 Log rank p

‐ value = .83 0 Log rank p

‐ value = .688

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

# 25 V. F2 Generation Results

Log rank p-values indicated that si gnificant differences between doses

occurred in all three trials . In each trial, the control group had a significantly

longer time to mount attempt compared to dose 30. In trial 2, dose 60, dose 125,

and dose 250 also had significantly longer time to first mount attempt compared

to dose 30. Just like the two previous generations, time to first mount attempt

typically decreased from one trial to the next. Dose 30 had a drastically smaller

time to first mount attempt through all th ree generations compared to the other

doses. Conversely, the control group had the longest time until first mount

attempt for all three generations. Dose

0 59.4 14.8 

### 85% 44.8 15.8 

### 80% 37.9 15.2 

### 85%

30 43.8 13.2 

### 85% 27.2 11.8 

### 90% 21.6 11.8 

### 90%

60 60.1 13.2 

### 90% 37.8 12.5 

### 90% 10.6 2.9 

### 100%

125 67.9 15.9 

### 75% 37.4 12.0 

### 95% 40.5 13.9 

### 90%

250 44.3 11.9 

### 90% 20.0 9.2 

### 95% 15.8 9.5 

### 94% Mounted

Pairs 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 

### F1 Generation

‐ Time to first Mount

Trial 1 Trial 2 Trial 3

Log rank p

‐ value = .795 Log rank p

‐ value = .790 Log rank p

‐ value = .444



### Dose

0 84.9 18.2 

### 60% 62.6 16.4 

### 80% 47.9 15.3 

### 80%

30 17.6 4.5 

### 100% 3.4 0.5 

### 100% 5.1 2.7 

### 100%

60 57.7 16.9 

### 80% 49.4 15.8 

### 80% 22.8 9.3 

### 95%

125 49.8 13.7 

### 85% 26.9 11.1 

### 95% 17.4 8.9 

### 95%

250 50.2 13.7 

### 85% 24.7 10.2 

### 95% 17.1 9.0 

### 95% Mounted

Pairs 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 

### F2 Generation

‐ Time to first Mount

Trial 1 Trial 2 Trial 3

Log rank p

‐ value = .032 Log rank p

‐ value = .000 Log rank p

‐ value = .016

# 26 

# NUMBER OF COPULATIONS

I. Analysis Methods

A Generalized Linear Model (GzLM) using a poisson loglinear link function

was used to determine if there was a st atistically significant difference in the

number of copulations (successful cloacal contact) across the five dose levels. An

omnibus likelihood ratio test in GzLM , which compares the fitted model

(intercept plus dose) against the intercep t only model, was used to determine if

dose was a significant factor in predicti ng the number of copulations in 180

seconds. If the p-value for the likelih ood test was less than .05, dose was a

significant factor in predic ting the number of copulations. Parameter estimates

(Beta coefficients and Beta confidence in tervals) and confidence intervals were

then examined to find where the significant differences occurred between the dose

levels if the p-value for the omnibus test was less than .05. In the GzLM

procedure, a reference group was needed for the other dose levels to be tested

against for a significant difference. Th is was because we fit a categorical

predictor into a linear model. The refe rence group for this analysis was the

‘dose=0’ group which is referred to as the control group thro ughout the report.

An exponentiated β coefficient of 1.00 meant th at the dosed group was 1.00 times

as likely to have a copulation compared to the control group, or in other words,

the two groups had an equal chance of having a copulation. If the confidence

interval contained 1.00, there was no statis tical difference between the two groups

and vice versa.

II. Summary of Results

In each generation, particularly the F0 generation, the number of copulations

increased from one trial to the next. Dose 60 had by far the lowest number of

copulations in the F0 generation. For all three generations, dose 30 had one of, if

not the highest estimated number of copul ations of all dose groups. Very little

statistical evidence shows th at there were consistent differences between any of

the dose groups for the number of copulations.

III. F0 Generation Results

Likelihood ratio p-values, which were well above .05, indicated that there were

no statistically significant differences betw een any doses within any of the three

trials, comparing the number of copulati ons. Estimated number of copulations for

all dose groups within e ach trial were all below 1.00, however the number of

copulations did increase from one trial to the next. Dose 60 had the lowest

# 27 number of copulations through all three trials, while the other four dose groups

had almost equal counts.

IV. F1 Generation Results

Likelihood ratio p-values, which were well above .05, indicated that there were

no statistically significant differences betw een any doses within any of the three

trials, comparing the number of copulati ons. Trial 3 had much higher copulation

estimated compared to tria l 1 and trial 2. Patterns between the dose groups were

not apparent throughout the trials furthe r indicating any differences between the

dose groups were very minimal.

V. F2 Generation Results

Statistically significant differences occurred in trial 1 of the F2 generation.

The control group had a si gnificantly lower estimated number of copulations

compared to dose 30. Trends between tria ls or between doses were not apparent,

emphasized by the large p-values in trial 2 and trial 3. Trial 2

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 0.30 0.42 0.50

30 0.35 .39

‐ 3.47 0.78 0.50 .49

‐ 3.01 0.72 0.65 .57

‐ 2.97 0.53

60 0.10 .07

‐ 1.65 0.18 0.15 .09

‐ 1.34 0.13 0.20 .13

‐ 1.28 0.12

125 0.20 .19

‐ 2.36 0.53 0.58 .55

‐ 3.42 0.49 0.55 .47

‐ 2.59 0.83

250 0.30 .32

‐ 3.10 1.00 0.40 .36

‐ 2.53 0.92 0.55 .47

‐ 2.59 0.83

* 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 Omnibus Likelihood ratio p

‐ value = .226 Omnibus Likelihood ratio p

‐ value = .205 F0 Generation

‐ Number of Copulations

Trial 1

Omnibus Likelihood ratio p

‐ value = .458 Trial 3

Trial 1

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 0.35 0.50 0.75

30 0.60 .68

‐ 4.35 0.26 0.55 .47

‐ 2.59 0.83 1.00 .68

‐ 2.62 0.41

60 0.60 .68

‐ 4.35 0.26 0.50 .42

‐ 2.40 1.00 0.75 .49

‐ 2.05 1.00

125 0.35 .35

‐ 2.85 1.00 0.45 .37

‐ 2.22 0.82 0.40 .23

‐ 1.26 0.15

250 0.40 .41

‐ 3.12 0.80 0.71 .61

‐ 3.27 0.42 0.53 .31

‐ 1.61 0.41

* 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 Trial 3

Omnibus Likelihood ratio p

‐ value = .193 Trial 2

Omnibus Likelihood ratio p

‐ value = .873 Omnibus Likelihood ratio p

‐ value = .581 F1 Generation

‐ Number of Copulations

# 28 Trial 1 Trial 2

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 0.35 0.60 0.65

30 1.10 1.34

‐ 7.36 0.01 0.75 .59

‐ 2.67 0.57 1.16 .90

‐ 3.54 0.10

60 0.50 .54

‐ 3.75 0.47 0.55 .40

‐ 2.08 0.84 0.65 .46

‐ 2.16 1.00

125 0.50 .54

‐ 3.75 0.47 0.60 .45

‐ 2.23 1.00 0.60 .42

‐ 2.02 0.84

250 0.65 .74

‐ 4.66 0.18 0.65 .49

‐ 2.37 0.84 0.90 .68

‐ 2.83 0.37

* 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 F2 Generation

‐ Number of Copulations

Omnibus Likelihood ratio p

‐ value = .043 Omnibus Likelihood ratio p

‐ value = .950 Trial 3

Omnibus Likelihood ratio p

‐ value = .268

# 29 

# TIME UNTIL FIRST COPULATION

I. Analysis Methods

Kaplan-Meier survival analysis was used to determine if there was a significant

difference between the five dose levels in terms of the amount of time it took the

birds to achieve their first copulation. If their time was recorded as 180 seconds,

this meant that the quail did not copula te and that observation was considered

censored. A log rank statistic was comput ed to test whether the different dose

groups had statistically different times un til the first copulation. If the p-value

associated with the log rank statistic wa s 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 tim es, estimated confidence intervals for

the time to copulate were compared. If two dose groups had confidence intervals

that did not overlap, these two doses were considered to have statistically

different times at the 95% confidence level.

II. Summary of Results

In all three generations, tr ial 1 had the longest time until first copulation.

The variation between doses in the F2 generation was far greater than the

variation in the P and F1 generation. Mu ch of the F2 variation was due to the

control group’s longer and dose 30’s signifi cantly shorter times until copulation.

Other than the large varia tion between doses in the F2 generation, the dose groups

tended to follow the same decreasing trends from one trial to the next.

III. F0 Generation Results

Log rank p-values, which were well a bove .05, indicated that there were no

statistically significant differences between any doses within each trial, comparing

time to first copulation. Time to first copulation decreased fro m one trial to the

next. Dose 60, however, had the longest estimated copulation time for all three

trials, but was never significantly greater than any of the other dose groups.

# 30 Dose

0 154.7 12.7 

### 20% 121.6 16.5 

### 42% 115.8 16.6 

### 45%

30 144.0 12.5 

### 30% 107.9 16.4 

### 50% 110.5 17.6 

### 45%

60 170.6 7.3 

### 10% 159.8 11.2 

### 15% 151.8 13.0 

### 20%

125 150.3 13.4 

### 20% 120.3 17.4 

### 47% 122.1 17.3 

### 40%

250 139.9 15.7 

### 26% 124.3 17.1 

### 35% 92.9 17.7 

### 55% Copulated

pairs

F0 Generation

‐ Time to first Copulation

Log rank p

‐ value = .624 Log rank p

‐ value = .220 Log rank p

‐ value = .248 Trial 3 Trial 2 Trial 1

Average time

to first

copulation Standard

Error 

### Average time

to first

copulation Standard

Error 

### Copulated

pairs Average time

to first

copulation Standard

Error 

### Copulated

pairs

# 31 IV. F1 Generation Results

For the first two trials, minimal, non- significant differences occurred between

the doses. However, in trial 3, dose 30 ha d a significantly shorter copulation time

estimate compared to dose 125. Dose 30 tended to have one of the shortest times

in all three trials.

V. F2 Generation Results

Significant differences between dose groups for copulation time occurred in

trial 1 and trial 3. For both trials, th e control group had a significantly longer

copulation time than dose 30. The control group had one of the longest, and dose

30 had the shortest copulation times for all three trials. Additionally, the only

doses to shorten their copulation time through each trial were the control group

and dose 250. Dose

0 142.7 13.5 

## 30% 114.4 18.0 

## 40% 91.2 17.8 

## 60%

30 105.6 16.7 

## 55% 113.4 16.0 

## 50% 58.9 14.4 

## 85%

60 110.2 14.7 

## 55% 128.4 15.8 

## 40% 100.5 16.9 

## 60%

125 143.8 14.3 

## 30% 117.6 17.2 

## 40% 130.8 16.1 

## 35%

250 135.3 15.2 

## 35% 91.9 20.2 

## 53% 123.3 18.2 

## 39% 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 F1 Generation

‐ Time to first Copulation

Log rank p

‐ value = .212 Log rank p

‐ value = .755 Log rank p

‐ value = .013 Trial 1 Trial 2 Trial 3

Dose

0 150.3 13.4 

## 20% 112.8 17.2 

## 50% 104.5 17.6 

## 50%

30 72.8 14.4 

## 80% 83.6 17.7 

## 60% 44.5 13.6 

## 85%

60 114.1 16.8 

## 50% 111.8 17.7 

## 45% 113.1 17.0 

## 45%

125 118.2 16.6 

## 45% 96.7 17.3 

## 55% 96.7 16.0 

## 60%

250 113.3 15.2 

## 50% 105.7 16.1 

## 60% 77.9 15.3 

## 80% 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 F2 Generation

‐ Time to first Copulation

Log rank p

‐ value = .005 Log rank p

‐ value = .863 Log rank p

‐ value = .005 Trial 1 Trial 2 Trial 3

# 32 

# ENDOSULFAN EFFECTS ON ORGAN

WEIGHTS

Vital Organ and Total Body Weights

This report analyzes the body and organ weights of Japanese quail that were

collected and weighed after n ecropsy. Weights were take n for quails in one of the

five dose groups ((control) 0 ppm, 30 ppm, 60 ppm, 125 ppm, 250 ppm) in all

three generations (F0, F1, F2). Total body weight was measured as well as brain,

kidney, liver, thyroid and coacal gland (C G) weights. CG weights were only

taken for males in the F1 and F2 generations. SPSS version 16.0 was used to

analyze the body and organ weight data. St atistical significance for this analysis

was determined using an alpha ( α ) of .05 with a 95% confidence level.

# 33 I. Analysis Methods

An Analysis of Variance (ANOVA) test was used to determine if dose group

averages for each measured organ weight were significantly different. An Fstatistic and corresponding p-value were cal culated for each organ. If the p-value

was below .05, then it was concluded that at least two dose groups had

significantly different average weights fo r that organ. To find which dose groups

had significantly different average weight s, 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

non-parametric Kruskal-Wallis test was run instead of an ANOVA.

II. Summary of Results

Statistically significant differences sp oradically occurred throughout all three

generations and in both genders. Tes ting at all three generations and both

genders, thirty-two ANOVA tests were run to see if the organ weight averages

were significantly different between th e five dose groups, and statistically

significant differences (p-value <.05) onl y occurred in six of the ANOVA tests.

Additionally, for both males and females, rankings of dose groups based on their

organ weights were very inconsistent ac ross the three generations. Across all

three generations, kidneys and liver we ights were about twice as heavy for

females as they were for males, and body weights were slightly higher for females

also. Lastly, for each gender, organ weight s from one generation to the next were

similar and had no distinct differences.

III. F0 Generation Results

A. Females

No statistically significant differenc es (ANOVA p-values all above .05) in

body or organ weights occurred between the five dose groups. Inconclusive

trends in organ and body weight aver ages comparing the dosing level of

endosulfan reaffirm that organ and b ody weights for female quails were not

different in the F0 generation. Although no trend in average weights was present

for the degree of endosulfan concentr ation, the control group had the lowest

average for all organ and body weights except kidneys.

# 34 B. Males

Statistically significant di fferences in kidneys (p-value = .012) and thyroid (pvalue = .004) weights occurred between dos e groups. For kidneys, the control

group and dose 30 had significantly greater kidney weights compared to dose 250.

For thyroid, the control group had a signifi cantly greater thyroi d weight compared

to dose 125 and dose 250. Liver weights (p -value =.057) was also very close to

having statistically significant differences between dose groups , with the greatest

difference in dose groups being between dose 30 and dose 125. In general, it was

evident that the control group and dose 30 usually had the two highest averages

for the five organ weights. Addition ally, dose 125 typically had the lowest

average organ weight.

C. Male and Female differences

Kidney and liver weight averages for male s were about half of what they were

for females. Body weight averages for males were also lower than females. Dose Average Std. Error 

## Average Std. Error 

## Average Std. Error 

## Average Std. Error 

## Average Std. Error



## 0 127.6 7.18 

## 0.73 0.030 

## 1.31 0.110 

## 3.42 0.221 

## 0.014 0.002



## 30 144.8 2.24 

## 0.76 0.018 

## 1.37 0.069 

## 4.21 0.179 

## 0.014 0.002



## 60 136.4 5.40 

## 0.75 0.006 

## 1.23 0.093 

## 3.99 0.272 

## 0.016 0.004



## 125 135.4 2.59 

## 0.78 0.015 

## 1.24 0.105 

## 3.44 0.313 

## 0.017 0.003



## 250 141.9 3.47 

## 0.77 0.010 

## 1.21 0.041 

## 3.78 0.161 

## 0.016 0.002 

## F0 Generation Female Organ Weights

Body Weight Brain Kidneys Liver Thyroid

P

‐ value

= .831 P

‐ value

= .083* P

‐ value

= .34 4 P

‐ value

= .631 P

‐ value

= .082

* P

‐ value calculated from Kruskall

‐ wallis test because homogenei ty of variance assumption violated in parametric ANOVA test.



## Dose Average Std. Error 

## Average Std. Error 

## Average Std. Error 

## Average Std. Error 

## Average Std. Error



## 0 112.3 2.35 

## 0.78 0.015 

## 0.79 0.044 

## 1.73 0.070 

## 0.020 0.003



## 30 114.2 3.16 

## 0.77 0.018 

## 0.79 0.036 

## 1.91 0.089 

## 0.013 0.001



## 60†

‐ ‐ 

## ‐ ‐ 

## ‐ ‐ 

## ‐ ‐ 

## ‐ ‐



## 125 108.3 1.57 

## 0.75 0.014 

## 0.61 0.058 

## 1.67 0.047 

## 0.011 0.001



## 250 110.3 1.57 

## 0.79 0.019 

## 0.57 0.057 

## 1.80 0.035 

## 0.012 0.001



## ** Tukey post

‐ hoc results indicate that dose 0 has a significantly greater thyroid weight than dose 125 and dose

250.

† Only 1 observa Ɵ on for dose 60. With only 1 observa Ɵ on, standard devia Ɵ on/error cannot be calculated and

therefore cannot be included in ANOVA test. P

‐ value

= .226 P

‐ value

= .435 P

‐ value

= .012* P

‐ value

= .057 P

‐ value

= .004**

* Tukey post

‐ hoc results indicate that dose 0 and dose 30 have significantly greater kidney weight than dose 250 at the 95%

confidence level. 

## F0 Generation Male Organ Weights

Body Weight Brain Kidneys Liver Thyroid

# 35 More variation in organ weight averages between dose groups occurred in the

males compared to females, indicated by the lower p-values and greater variation

in the averages across dose groups for many of the organ weights, excluding body

weight.

IV. F1 Generation

A. Females

Statistically significant differences in body weight (p-value = .002) and brain

weight (p-value = .038) occurred between dose groups. Dose 30, 125, and 250

had significantly greater body weights comp ared to dose 60. For brain weights,

dose 125 had a significantly greater average weight compared the control group.

Overall, dose 60 had organ weights with the lowest averages. Conversely, dose

125 had averages that were typically the hi ghest for organ weights, especially for

body weight, brain, and liver.

B. Males

Statistically significant di fferences in thyroid (p-val ue = .033) and potentially,

cloacal gland (p-value = .057) averages o ccurred between dose groups. However,

post-hoc tests reveal that differences between dose groups were non-significant,

due to the adjustment of multiple tes ting. Body weight, brain, kidney, and liver

all have dose group averages that are mi nimally different with p-values well

above .05. Overall, the control group’s av erages for organ weights were typically

the lowest average, excluding CG measurements. Dose Average Std. Error 

## Average Std. Error 

## Average Std. Error 

## Average Std. Error 

## Average Std. Error



## 0 137.0 4.24 

## 0.73 0.009 

## 1.39 0.003 

## 4.10 0.206 

## 0.014 0.004



## 30 142.2 2.09 

## 0.76 0.013 

## 1.28 0.072 

## 4.10 0.243 

## 0.011 0.001



## 60 129.7 2.64 

## 0.75 0.006 

## 1.30 0.058 

## 3.69 0.226 

## 0.008 0.001



## 125 144.7 1.32 

## 0.78 0.010 

## 1.32 0.027 

## 4.16 0.241 

## 0.012 0.002



## 250 144.4 3.21 

## 0.75 0.030 

## 1.30 0.026 

## 4.10 0.281 

## 0.016 0.003

† Kruskall

‐ wallis test indicates that dose 125 has a signi fi cantly greater brain weight averages compared to the control group

at 95% confidence level 

## F1 Generation Female Organ Weights

Body Weight Brain Kidneys Liver Thyroid

P

‐ value

= .002° P

‐ value

= .038*† P

‐ value

= .313* P

‐ value

= .641 P

‐ value

= .103*

* P

‐ value calculated from Kruskall

‐ wallis test because homogeneity of variance assumption violated in parametric ANOVA test.

° Tukey post

‐ hoc results indicate that dose 30, 250 and 125 have signi ficantly greater body weight average compared to dose

60 at 95% confidence level.

# 36 C. Male and Female Differences

Kidney and liver weight averages for ma les were about half of what they were

for females. Body weight averages for males were also lower than females. For

males, dose 125’s averages are typically the highest for organ weights.

V. F2 Generation

A. Females

Minimal, non-significant differences in organ weight averages (p-values well

above .05) occurred between dose groups . Additionally, no dose group(s) had

consistently higher or lower averages comp ared to the other dose groups across all

five organ weights.

B. Males

Statistically significant differences in body weight occurred between dose

groups. Post-hoc results indicated th at dose 60 and dose 125 had significantly

greater body weight averages compared to the control group. Besides thyroid,

dose 125 typically had the highest averag e weight for all organs while dose 30

typically had the lowest average weight. Dose Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error



### 0 109.0 3.12 

### 0.75 0.020 

### 0.73 0.039 

### 1.76 0.125 

### 0.008 0.002 

### 210.4 18.66



### 30 113.4 1.92 

### 0.72 0.036 

### 0.76 0.030 

### 1.94 0.113 

### 0.017 0.003 

### 179.4 13.50



### 60 114.6 2.88 

### 0.76 0.016 

### 0.77 0.030 

### 1.88 0.074 

### 0.010 0.002 

### 191.7 10.10



### 125 113.1 2.40 

### 0.78 0.023 

### 0.76 0.027 

### 1.86 0.103 

### 0.015 0.003 

### 196.6 14.78



### 250 112.0 2.35 

### 0.76 0.014 

### 0.76 0.019 

### 1.83 0.071 

### 0.008 0.001 

### 239.1 9.92 

### P

‐ value

= .60 8 P

‐ value

= .572 P

‐ value

= .912 P

‐ value

= .767 P

‐ value

= .033 P

‐ value

= .054 F1 Generation Male Organ Weights

Body Weight Brain Kidneys Liver Thyroid Coacal Gland



### Dose Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error



### 0 138.1 2.72 

### 0.77 0.009 

### 1.19 0.035 

### 4.02 0.192 

### 0.014 0.0018



### 30 139.1 2.64 

### 0.75 0.010 

### 1.27 0.049 

### 4.14 0.066 

### 0.014 0.0010



### 60 132.8 1.53 

### 0.75 0.013 

### 1.33 0.035 

### 4.43 0.367 

### 0.011 0.0011



### 125 138.3 2.87 

### 0.77 0.014 

### 1.28 0.062 

### 4.25 0.228 

### 0.012 0.0019



### 250 135.8 1.81 

### 0.77 0.011 

### 1.22 0.053 

### 3.98 0.377 

### 0.015 0.0017 

### F2 Generation Female Organ Weights

Body Weight Brain Kidneys Liver Thyroid

P

‐ value

= .403* P

‐ value

= .248 P

‐ value

= .303 P

‐ value

= .880* P

‐ value

= .597

* P

‐ value calculated from Kruskall

‐ wallis test because homogeneity of variance assumption violated in parametric ANOVA test.

# 37 C. Male and Female Differences

Just like in the F0 and F1 generations , kidney and liver weights for males were

about half of what they were for fema les, and body weight averages for males

were also lower than females. Dose Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error 

### Average Std. Error



### 0 108.1 1.49 

### 0.77 0.011 

### 0.72 0.015 

### 1.66 0.055 

### 0.012 0.0080 

### 221.1 14.23



### 30 111.1 2.41 

### 0.76 0.015 

### 0.77 0.018 

### 1.79 0.053 

### 0.011 0.0083 

### 196.1 13.67



### 60 116.7 1.94 

### 0.78 0.012 

### 0.80 0.030 

### 1.85 0.067 

### 0.012 0.0086 

### 201.3 6.43



### 125 117.4 1.79 

### 0.79 0.017 

### 0.79 0.025 

### 1.92 0.072 

### 0.010 0.0064 

### 222.6 7.22



### 250 110.9 2.39 

### 0.76 0.012 

### 0.77 0.042 

### 1.88 0.109 

### 0.011 0.0074 

### 201.4 16.48

* Tukey post

‐ hoc test indicate that dose 60 and dose 125 have signi ficantly greater body weight compared to dose 0 at a 95% confidence level. 

### P

‐ value

= .009* P

‐ value

= .322 P

‐ value

= .345 P

‐ value

= .157 P

‐ value

= .894 P

‐ value

= .432 F2 Generation Male Organ Weights

Body Weight Brain Kidneys Liver Thyroid Coacal Gland

# APPENDIX H

EXPERIMENTAL PATHOLOGY LABORATORIES, INC.,

DRAFT PATHOLOGY REPORT

# BATTELLE PACIFIC-NOR THWEST DIVISION

EPA CONTRACT NUMBER EP-W-06-025 (TO17, TD5)

[PROTOCOL # 08JG-80-08-04-02]

EPL PROJECT NUMBER 237-043

PREPARE GLASS SLIDES AND HISTOLOGICALLY

EVALUATE TISSUES FROM HATCHLING/SUBHATCHLING COTURNIX JAPONICA EXPOSED TO

DIFFERENT CONCENTRATIO NS OF ENDOSULFAN

DRAFT PATHOLOGY REPORT

Submitted by:

Experimental Pathology Laboratories, Inc.

P.O. Box 169

Sterling, VA 20167-0169

(703) 471-7060

Submitted to:

Battelle Pacific-Northwest Division

Sequim, WA 98382

March 3, 2010

# 2 TABLE OF CONTENTS



# Page

PATHOLOGY SUMMARY ................................................................................. 1

COMPLIANCE STATEMENT ............................................................................ 7

QUALITY ASSURANCE FINA L CERTIFIC ATION ............................................ 8

APPENDIX A: Figures

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

APPENDIX B: Data Spread sheet ..................................................................... B-1

# 

Experimental Pathology Laboratories, Inc.

DRAFT PATHOLOGY SUMMARY

# 

Experimental Pathology Laboratories, Inc.



# BATTELLE PACIFIC-NOR THWEST DIVISION

EPA CONTRACT NUMBER EP-W-06-025 (TO17, TD5)

[PROTOCOL # 08JG-80-08-04-02]

EPL PROJECT NUMBER 237-043

PREPARE GLASS SLIDES AND HISTOLOGICALLY EVALUATE

TISSUES FROM HATCHLING/SUB-HATCHLING COTURNIX

JAPONICA EXPOSED TO DIFFERENT CONCENTRATIONS OF

ENDOSULFAN

DRAFT PATHOLOGY REPORT

INTRODUCTION

As part of the development and validation of a Tier 2, 2-generation avian toxicity

test to characterize the adverse consequen ces of exposure to put ative endocrine active

substances, a histopathologica l evaluation was performed on tissues from hatchling/ subhatchling Japanese quail ( Coturnix japonica ) that were exposed to various concentrations

of endosulfan via the diet (Table 1).

Table 1. Number of Japanese Quail Hatchlings/Sub-hatchlings Evaluated

Dose

(ppm) No. of Males No. of

Females Total

Number

0.0 8 4 12

30 16 12 28

60 13 16 29

125 10 4 14

250 9 9 18

TOTAL 56 45 101



# METHODS

Necropsies were performed at the U.S. Army Center for Health Promotion &

Preventive Medicine Directorate of Toxicology Health Effects Research.

Tissues that were processed and evaluated included adrenal gla nds, brain within the

skull (for medial preoptic nucleus [POM], pituitary, and pineal gland), Bursa of

Fabricius, cloacal gland, kidney, liver, Me sonephric/Wolffian ducts (ductus deferens),

Müllerian ducts (infundibulum, magnum, isthmus, shell gland, vagina), and testis. At

EPL, tissues that contained bone (the skull, and samples of kidney, adrenal, and genital

tissues that contained spine) were deca lcified in a commercial formic acid/EDTA

solution prior to routine processing for para ffin embedding. All processed and embedded

# - 2 - 

# 

Experimental Pathology Laboratories, Inc.

Battelle Pacific-Northwest Division,

EPA Contract Number EP-W-06-025 (TO17, TD5)

[Protocol # 08JG-80-08-04-02]

3/3/10 DRAFT



# tissues were microtomed to 4-6  m thick sections, and all sections were mounted on glass

slides, stained with hematoxylin and eosin (H&E), and coverslipped according to routine

methods. Sections were evaluated for tr eatment-induced effects via brightfield

microscopy, and the prevalence and severity of findings were assessed as compared to

controls. The severity of lesions was recorded as: Grade 1 = minimal, Grade 2 =

slight/mild, Grade 3 = moderate, and Grade 4 = severe. Results were tabulated in an

Excel spreadsheet (Appendix B).

RESULTS

Based on incidence and severi ty data, there were no clear treatment-related findings

in this study.

There were a few findings that were only observed in endosulfan-treated quail, and

these included: focally extensive hepatocellular necrosis (minimal to slight/mild) in one

female of the 30 ppm dose group (Animal No. E070), two females of the 60 ppm dose

group (Animal Nos. E080 and E082), and one female of the 125 ppm dose group (Animal

No. E040); bile duct hyperplasia (minimal) and peribiliary fibrosis (minimal) in a female

of the 250 ppm dose group (Animal No. E020); and a retained right ovary in a female of

the 250 ppm dose group (Animal No. E061).

In each instance, focally extensive hepa tocellular necrosis (Figs. 1-4) was

characterized by a discrete, r oughly elliptical area of liver cell fragmentation, with

varying degrees of secondary (dystrophic) mi neralization of necrotic cellular debris.

These lesions tended to be located proximate to, but not at, the liver surface, and were

generally situated near the margins of liver lobes.

Bile duct hyperplasia (Figs. 7, with Figs. 5 and 6 included for comparison)

presented as increased bile duct profiles within portal triads, and pe ribiliary fibrosis was

characterized by increased collagen in these same regions.

The retained right ovary (A nimal No. E061), which was smaller than the left, is

illustrated in Fig. 8.

Two other noteworthy or unusual findings that were clearly not treatment-related

included a persistent Müllerian duct (Figs. 9-12) in a cont rol male (Animal No. E065),

and regionally extensive encephalomalacia in a control female (Animal No. E003) (Figs.

14, 15, 17).

It is possible that the single control male (Animal No. E065) that had a persistent

Müllerian duct (Figs. 9-12) may actually have been female, as no gonadal tissue was

recovered for that chick. The Müllerian duc t is an embryonic precursor of the oviduct in

females, whereas the paired Mesonephric/Wol ffian ducts are forerunners of the ductus

deferens. Both types of ducts are typically pr esent in females at this stage of reproductive

# - 3 - 

# 

Experimental Pathology Laboratories, Inc.

Battelle Pacific-Northwest Division,

EPA Contract Number EP-W-06-025 (TO17, TD5)

[Protocol # 08JG-80-08-04-02]

3/3/10 DRAFT



# tract development (the Mesone phric/Wolffian ducts will subsequently atrophy); however,

males are expected to have onl y the Mesonephric/Wolffian ducts.

Regionally extensive encephalomalacia (brain necrosis) in the single control female

(Animal No. E003) was characterized by well-de fined, bilaterally symmetrical areas of

neuropil vacuolation and rarefaction (Figs. 14, 15, 17). This loss of neuropil density was

accompanied by necrosis of neuronal and glial cellular constituents, as evidenced by

marked cytoplasmic shrinkage and dissolu tion accompanied by nuclear pyknosis. The

prominence of small blood vessels in these regions was most likely a consequence of

increased visual contrast as a function of decreased neuropil density. In the examined

sections, areas of encephalomalacia extended throughout the dorsal cerebral cortex and

were located within the granular layer and white matter of the cerebellar folia. Cerebellar

folia were markedly collapsed as a result of the necrosis.

Upon histologic examination of the gonads, two quail that were originally submitted

as females (Animal Nos. E066 and E077) were determined to be males, and one animal

originally submitted as male (Animal No. E079) was determined to be female.

Other findings in this study generally occurred in comparable numbers of treated

and control animals, or were low-frequency (incidental) findings that were observed in

individual animals, and therefore not considered to be treatment-related.



# DISCUSSION AND CONCLUSIONS

In the present study, a cause of the focally -extensive hepatocellu lar necrosis in a

low percentage of endosulfan-treated female quail was not apparent in the histologic

sections. This type of change would not be expected to occur commonly as a

spontaneous finding in Japanese quail embryos or hatchlings; however, the focal (vs.

zonal) distribution of the necrosis would be more consistent with a nontoxic etiology

such as infectious disease. It has been de monstrated that endosulfan administration can

cause histopathologically evident liver lesions in fish and rodents (Altinok and Capkin,

2007; Ballesteros et al., 2007; Glover et al., 2007; Singh et al., 2008). Commonly

reported liver effects include degenerative v acuolar changes in hepatocytes and hepatic

lipidosis (Ballesteros et al., 2007; Glover et al., 2007; Singh et al., 2008). In a prior

report (EP-W-06-025; TO17, TD4), endosulfan exposure was clearly associated with

macro- and microvesicular vacuolar degeneratio n of liver cells in adult female quail.

However, the spatial distribution, extent a nd nature of the lesions in the present study

differed markedly from the changes that were observed in adults (i.e., focal

hepatocellular necrosis vs. zonal vacuolation) , although it is interesting to note that in

both age groups, these unusual types of liv er changes were limited to females.

# - 4 - 

# 

Experimental Pathology Laboratories, Inc.

Battelle Pacific-Northwest Division,

EPA Contract Number EP-W-06-025 (TO17, TD5)

[Protocol # 08JG-80-08-04-02]

3/3/10 DRAFT



# The right ovary was retained in a female (Animal No. E061) of the 250 ppm dose

group. Because this finding only occurred in a single animal, and because vestigial right

ovaries have been observed in untreated Ja panese quail (Fitzgerald, 1969), this result

could not be associated definitively with endos ulfan administration. Similarly, there is

insufficient evidence that endosulfan trea tment was responsible for the bile duct

hyperplasia and peribiliary fibr osis in the single 250 ppm dose group female (Animal No.

E020).

Persistence of the right ovidu ct (Müllerian duct) is a do cumented effect of estrogen

exposure in male Japanese quail (Berg et al., 1999; Ro and Kondo, 1979), although it has

also been known to occur spontaneously in poultry (Wakamatsu et al., 2000). In the

current study, the validity of this finding in one control male is uncertain, because

gonadal tissue was not recovered for this animal, and thus the gender could not be

confirmed.

There was no histopathologically evident cause of the encephalomalacia in the

single control female. A typical cause of this type of lesion in young poultry is vitamin E

deficiency relative to the leve l of oxidized fatty acids in th e feed (Riddell, 1987; Mézes et

al., 1997). The common name for this conditio n is “crazy chick disease”. Although a

nutritional etiology would seem unlikely for a sole animal in a laboratory study in which

the diet did not vary, it is possi ble that this partic ular bird had an idiosyncratic response

related to some anomaly of vitamin E absorption or fatty acid metabolism.

JEFFREY C. WOLF, DVM, Diplomate, ACVP Date

Veterinary Pathologist

JCW/cb

# - 5 - 

# 

Experimental Pathology Laboratories, Inc.

Battelle Pacific-Northwest Division,

EPA Contract Number EP-W-06-025 (TO17, TD5)

[Protocol # 08JG-80-08-04-02]

3/3/10 DRAFT



# REFERENCES

Altinok I, Capkin E (2007) Histopathology of rainbow trout exposed to sublethal

concentrations of methio carb or endosulfan. Toxi col Pathol, 35(3):405-410.

Ballesteros ML, Bianchi GE, Carranza M, Bist oni MA (2007) Endosulfan acute toxicity

and histomorphological alterations in Jenynsia multidentata (Anablepidae,

Cyprinodontiformes). J Environ Sci Health B, 42(4):351-357.

Berg C, Halldin K, Fridolfsson A-K, Brandt I, Brunström B (1999) The avian egg as a

test system for endocrine disrupters: effect s of diethylstilbestrol and ethynylestradiol on

sex organ development. Sci Total Environ, 233(1-3):57-66.

Fitzgerald TC (1969) The Coturnix Quail: Anatomy and Histology. The Iowa State

University Press, Ames, Iowa, p. 260.

Glover CN, Petri D, Tollefsen K-E, Jorum N, Handy RD, Berntssen MHG (2007)

Assessing the sensitivity of Atlantic salmon ( Salmo salar ) to dietary endosulfan exposure

using tissue biochemistry and histol ogy. Aquat Toxicol, 84(3):346-355.

Mézes M, Surai P, Sályi G, Speake BK, Gaál T, Maldjian A (1997) Nutritional metabolic

diseases of poultry and disorders of the biolog ical antioxidant defense system. Acta Vet

Hung, 45(3):349-360.

Riddell C (1987) Avian Histopathology. Ameri can Association of Avian Pathologists,

Inc., Allen Press, Inc., Lawrence, KS, p. 76.

Ro S, Kondo K (1979) The effects of pren atal treatment of exogenous estrogen on the

postnatal development of the sexual organs a nd reproductive ability in the male Japanese

quail. Jap J Zootech Sci, 50(11):821-832.

Singh ND, Sharma AK, Dwivedi P, Patil RD, Kumar M (2008) Experimentally induced

citrinin and endosulfan toxicity in pregnant Wistar rats: histopathological alterations in

liver and kidneys of fetuses. J Appl Toxicol, 28(7):901-907.

Wakamatsu M, Yamagata T, Namikawa T, Mizu tani M (2000) Heredita ry persistent right

oviduct in the chicken PNP/DO line. Poult Sci, 79(8):1075-1081.

# - 6 - 

# 

Experimental Pathology Laboratories, Inc.

3/3/10 DRAFT



# COMPLIANCE STATEMENT

Client

Name 

## Battelle Pacific-Northwest

Division EPL Principal

Investigator 

## Dr. Jeffrey C. Wolf

Client

Study 

## EPA EP-W-06-025 (TO17, TD5)

[Protocol # 08JG-80-08-04-02] EPL

Pathologist 

## Dr. Jeffrey C. Wolf

Species 

## Coturnix japonica

(Japanese quail) EPL Project

Number 

## 237-043

Study

Title 

## Prepare Glass Slides and Histologically Evaluate Tissues From Hatchling/Subhatchling Coturnix japonica Exposed to Different Concentrations of Endosulfan

Test

Article 

## Endosulfan



# The Histopathology portions of the above-referenced study were conducted in

compliance with the Good Laboratory Practice regulations of the Environmental

Protection Agency as stipulated by 40 CFR 160.

EPL Principal Investigator

Date

# - 7 - 

# 

Experimental Pathology Laboratories, Inc.

3/3/10 DRAFT



## QUALITY ASSURANCE FINAL CERTIFICATION

Study Title: Prepare Glass Slides and Histol ogically Evaluate Tissues From Hatchling/Subhatchling Coturnix japonica Exposed to Different Concentrations of Endosulfan

Client Study: EPA EP-W-06-025 (TO 17, TD5) EPL Principal Invest igator: Dr. Jeffrey C. Wolf

[Protocol # 08JG-80-08-04-02]

EPL Project Number: 237-043 EPL Pa thologist: Dr. Jeffrey C. Wolf

The following aspects of this study were inspected by the Quality Assurance Unit of Experimental

Pathology Laboratories, Inc. Dates inspections were performed and findings reported to the EPL Principal

Investigator and Management are indicated below.

Dates

Area Inspected Inspection Reporting

EPL Project Sheets

Project Setup

In-Process

Data Review

Draft Pathology Report

Final Pathology Report

Date of last quarterly facility inspection 11/09

EPL Quality Assurance Unit Date

# APPENDIX A

FIGURES

# A-1 

# FIGURES

Figure 1 (Z15301). Liver from a female quail treated with 60 ppm endosulfan (Animal

No. E080). There is a large, well-circumscrib ed area of necrosis near the liver margin

(arrows). H&E.

# A-2 

# Figure 2 (Z15302). Higher magnification of liver fr om preceding figure. Affected

hepatocytes are fragmented and dissociated. The lack of any appreciable inflammatory

response suggests that this lesion is of short term duration. H&E.

# A-3 

# Figure 3 (Z15303). Liver from a female treated with 125 ppm endosulfan (Animal No.

E040). Another example of fo cal liver necrosis. H&E.

# A-4 

# Figure 4 (Z15304). Higher magnification of liver from preceding figure.

Mineralization (dense dark pur ple deposits; arrows) of necro tic tissue is more obvious in

this animal’s liver compared to the previous example. H&E.

# A-5 

# Figure 5 (Z15299). Liver from a control male (Animal No. E066). At this age, portal

triads often contain multiple bile duct profiles (arrows) that are surrounded by a matrix

of loose fibrous connective tissue. H&E.

# A-6 

# Figure 6 (Z15300). Higher magnification of liver from preceding figure to illustrate

bile duct profile (B) and fibrous connective tissue (F). H&E. 

# B F

# A-7 

# Figure 7 (Z15322). Liver from a female treated with 250 ppm endosulfan (Animal No.

E020). Bile duct profiles (B) are more numerous and tortuous than was generally

observed in controls, and porta l triads are expanded by more abundant fibrosis (F).

These particular changes were only observ ed in a single high dose female. H&E. 

# B

B B B

F F

# A-8 

# Figure 8 (Z15321). Transverse section through the anterior abdomen of a female

treated with 250 ppm endosulfan (Animal No. E061). Evident are spine (S), kidneys

(K), adrenals (A), and ovaries (LO = left ova ry, RO = right ovary). Retained right ovary

can be an indication of exposure to compounds with estrogenic activit y, but in this case

relationship to treatment is questionable, as only a single animal wa s affected, and there

is apparently some degree of spontaneous righ t ovary retention in Japanese quail. H&E. 

# K K A A RO

LO

S

# A-9 

# Figure 9 (Z15317). Transverse section through the mid abdomen of a bird listed as a

control male (Animal No. E065). A precurs or to the oviduct called the Müllerian duct

(arrow) is evident in its typi cal location anterior and lateral to the left metanephric

kidney (K). Although this could be an endoc rine disruptive-type finding, it is also

possible that this animal’s gender was simply misidentified. H&E. 

# K

# A-10 

# Figure 10 (Z15318). Higher magnification of Müllerian duct from preceding figure.

Characteristically, the wall of this duct is thick and densely cellular. H&E.

# A-11 

# Figure 11 (Z15313). Another transverse section at a s lightly different level through the

mid abdomen of Animal No. E065. The Mülleri an duct (closed arrow) is located lateral

to the metanephric (urinary) duct (open arrow). H&E.

# A-12 

# Figure 12 (Z15315). Yet another transverse section at a slightly different level through

the mid abdomen of Animal No. E065. At this level the Müllerian duct is not evident,

but the Mesonephric/Wolffian duc t is present (closed arrow) medial to the metanephric

duct (open arrow). This figur e does not illustrate any abnor mality, but is included for

the purpose of anatomic comparison. The Mesonephric/Wolffian duct, which is the

precursor of the ductus deferens in males, is al so present in females at this age, but will

subsequently atrophy. H&E.

# A-13 

# Figure 13 (Z15305). Transverse section through the br ain of a control female (Animal

No. E018). CC = cerebral cortex, LV = lateral ve ntricle, TV = third ve ntricle, E = eye.

H&E.



# E E LV LV

TV CC CC

# A-14 

# Figure 14 (Z15307). Transverse section through the brain of another control female

(Animal No. E003). Regionally extensive ence phalomalacia (brain necrosis) is evident

as bilaterally symmetrical pale staining areas in the dorsal cerebral cortex.

CC = cerebral cortex, LV = lateral ventricle, TV = third ventricle, E = eye. H&E. 

# CC CC

LV

LV

TV E

E

# A-15 

# Figure 15 (Z15309). Higher magnification of brain fr om previous figure, at border

between less affected (LA) and ence phalomalacic (E) regions. H&E. 

# E

LA

# A-16 

# Figure 16 (Z15306). Sagittal section through the brai n of control female Animal No.

E018 to demonstrate the normal cerebellar folia (CF). H&E. 

# CF

CF

CF

# A-17 

# Figure 17 (Z15310). Sagittal section through the br ain of control female with

encephalomalacia (Animal No. E003). Cerebe llar folia (CF) are markedly collapsed,

most of the granular layer has disappeare d, and the density of the white matter is

decreased. When it is a consequence of vita min E deficiency, the common name for this

disorder is “crazy chick disease”. H&E. 

# CF

CF CF

# APPENDIX B

DATA SPREADSHEET

# B-1 Group Tissue Lesion Severity

Grade Images

Taken

0 ppm ADRENAL No Section . .

0 ppm BURSA Apoptotic Necrosis, Increased 1 .

0 ppm CLOACAL GLAND . . .

0 ppm KIDNEY . . .

0 ppm LIVER . . .

0 ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

0 ppm MULLERIAN DUCTS-M Persistent P .

0 ppm PINEAL GLAND No Section . .

0 ppm PITUITARY . . .

0 ppm PREOPTIC NUCLEUS . . .

0 ppm TESTIS No Section . .

0 ppm ADRENAL No Section . .

0 ppm BURSA . . .

0 ppm CLOACAL GLAND . . .

0 ppm KIDNEY . . .

0 ppm LIVER . . .

0 ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

0 ppm PINEAL GLAND . . .

0 ppm PITUITARY . . .

0 ppm PREOPTIC NUCLEUS . . .

0 ppm TESTIS No Section . .

0 ppm ADRENAL No Section . .

0 ppm BURSA . . .

0 ppm CLOACAL GLAND . . .

0 ppm KIDNEY . . .

0 ppm LIVER . . .

0 ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

0 ppm MESONEPHRIC/WOLFFIAN

DUCTS Missing one of pair . .

0 ppm PINEAL GLAND . . .

0 ppm PITUITARY Cyst(s) P .

0 ppm PREOPTIC NUCLEUS . . .

0 ppm TESTIS . . .

0 ppm TESTIS Missing one of pair . .

0 ppm ADRENAL . . .

0 ppm BURSA . . .

0 ppm CLOACAL GLAND . . .

0 ppm KIDNEY . . .

0 ppm LIVER . . .

0 ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

0 ppm PINEAL GLAND . . .

0 ppm PITUITARY Cyst(s) P .

0 ppm PREOPTIC NUCLEUS . . .

0 ppm TESTIS . . .

# B-1 0 ppm ADRENAL . . .

0 ppm BURSA No Section . .

0 ppm CLOACAL GLAND . . .

0 ppm KIDNEY Tubular Dilation 1 .

0 ppm LIVER . . .

0 ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

0 ppm PINEAL GLAND . . .

0 ppm PITUITARY Cyst(s) P .

0 ppm PREOPTIC NUCLEUS . . .

0 ppm TESTIS . . .

0 ppm ADRENAL . . .

0 ppm BURSA . . .

0 ppm CLOACAL GLAND . . .

0 ppm KIDNEY . . .

0 ppm LIVER . . .

0 ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

0 ppm PINEAL GLAND No Section . .

0 ppm PITUITARY . . .

0 ppm PREOPTIC NUCLEUS . . .

0 ppm TESTIS . . .

0 ppm ADRENAL . . .

0 ppm BURSA . . .

0 ppm CLOACAL GLAND . . .

0 ppm KIDNEY . . .

0 ppm LIVER . . .

0 ppm MESONEPHRIC/WOLFFIAN

DUCTS . . 1

0 ppm MULLERIAN DUCTS-M Persistent P 3

0 ppm PINEAL GLAND No Section . .

0 ppm PITUITARY No Section . .

0 ppm PREOPTIC NUCLEUS . . .

0 ppm TESTIS No Section . .

0 ppm ADRENAL No Section . .

0 ppm BURSA . . .

0 ppm CLOACAL GLAND . . .

0 ppm KIDNEY . . .

0 ppm LIVER . . 2

0 ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

0 ppm PINEAL GLAND No Section . .

0 ppm PITUITARY . . .

0 ppm PREOPTIC NUCLEUS . . .

0 ppm TESTIS Apoptotic Necrosis, Increased 1 .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

# B-1 30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

30

ppm PINEAL GLAND . . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm TESTIS . . .

30

ppm TESTIS Missing one of pair . .

30

ppm ADRENAL . . .

30

ppm ADRENAL Missing one of pair . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS Missing one of pair . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm TESTIS . . .

30

ppm TESTIS Missing one of pair . .

30

ppm ADRENAL . . .

30

ppm BURSA Apoptotic Necrosis, Increased 1 .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY Tubular Dilation 1 .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

30 PINEAL GLAND No Section . .

# B-1 ppm

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm TESTIS . . .

30

ppm TESTIS Missing one of pair . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY Cyst(s) P .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm TESTIS . . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm TESTIS . . .

30

ppm TESTIS Missing one of pair . .

30

ppm ADRENAL . . .

30

ppm BURSA No Section . .

# B-1 30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY Cyst(s) P .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm TESTIS . . .

30

ppm ADRENAL . . .

30

ppm BURSA Apoptotic Necrosis, Increased 1 .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

30

ppm PINEAL GLAND . . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm TESTIS . . .

30

ppm TESTIS Missing one of pair . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY . . .

30 PREOPTIC NUCLEUS . . .

# B-1 ppm

30

ppm TESTIS . . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm TESTIS No Section . .

30

ppm ADRENAL . . .

30

ppm BURSA Apoptotic Necrosis, Increased 1 .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY Cyst(s) P .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm TESTIS . . .

30

ppm ADRENAL . . .

30

ppm BURSA Apoptotic Necrosis, Increased 1 .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

# B-1 30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm TESTIS . . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm TESTIS . . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm TESTIS . . .

30

ppm TESTIS Missing one of pair . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30 CLOACAL GLAND . . .

# B-1 ppm

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

30

ppm PINEAL GLAND . . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm TESTIS . . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

30

ppm PINEAL GLAND . . .

30

ppm PITUITARY Cyst(s) P .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm TESTIS Apoptotic Necrosis, Increased 1 .

30

ppm TESTIS Missing one of pair . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

30

ppm PINEAL GLAND . . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

# B-1 30

ppm TESTIS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm TESTIS . . .

60

ppm TESTIS Missing one of pair . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY Cyst(s) P .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm TESTIS . . .

60

ppm TESTIS Missing one of pair . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60 LIVER . . .

# B-1 ppm

60

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm TESTIS . . .

60

ppm TESTIS Missing one of pair . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

60

ppm PINEAL GLAND . . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm TESTIS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm TESTIS . . .

60

ppm ADRENAL . . .

# B-1 60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

60

ppm PINEAL GLAND . . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm TESTIS . . .

60

ppm ADRENAL . . .

60

ppm BURSA Apoptotic Necrosis, Increased 1 .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

60

ppm PINEAL GLAND . . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm TESTIS . . .

60

ppm TESTIS Missing one of pair . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

60

ppm PINEAL GLAND . . .

60 PITUITARY . . .

# B-1 ppm

60

ppm PREOPTIC NUCLEUS . . .

60

ppm TESTIS . . .

60

ppm TESTIS Missing one of pair . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY No Section . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm TESTIS . . .

60

ppm TESTIS Missing one of pair . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY No Section . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm TESTIS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

# B-1 60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm TESTIS No Section . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm TESTIS . . .

60

ppm TESTIS Missing one of pair . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY Cyst(s) P .

60

ppm PREOPTIC NUCLEUS . . .

60 TESTIS Apoptotic Necrosis, Increased 1 .

# B-1 ppm

60

ppm TESTIS Missing one of pair . .

125

ppm ADRENAL . . .

125

ppm BURSA . . .

125

ppm CLOACAL GLAND . . .

125

ppm KIDNEY . . .

125

ppm LIVER . . .

125

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

125

ppm PINEAL GLAND No Section . .

125

ppm PITUITARY . . .

125

ppm PREOPTIC NUCLEUS . . .

125

ppm TESTIS . . .

125

ppm ADRENAL . . .

125

ppm BURSA . . .

125

ppm CLOACAL GLAND . . .

125

ppm KIDNEY . . .

125

ppm LIVER . . .

125

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

125

ppm PINEAL GLAND . . .

125

ppm PITUITARY . . .

125

ppm PREOPTIC NUCLEUS . . .

125

ppm TESTIS No Section . .

125

ppm ADRENAL . . .

125

ppm BURSA . . .

125

ppm CLOACAL GLAND . . .

125

ppm KIDNEY . . .

125

ppm LIVER . . .

125

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

# B-1 125

ppm PINEAL GLAND . . .

125

ppm PITUITARY . . .

125

ppm PREOPTIC NUCLEUS . . .

125

ppm TESTIS . . .

125

ppm ADRENAL . . .

125

ppm BURSA No Section . .

125

ppm CLOACAL GLAND . . .

125

ppm KIDNEY . . .

125

ppm LIVER . . .

125

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

125

ppm PINEAL GLAND . . .

125

ppm PITUITARY . . .

125

ppm PREOPTIC NUCLEUS . . .

125

ppm TESTIS . . .

125

ppm TESTIS Missing one of pair . .

125

ppm ADRENAL . . .

125

ppm BURSA . . .

125

ppm CLOACAL GLAND . . .

125

ppm KIDNEY . . .

125

ppm LIVER . . .

125

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

125

ppm PINEAL GLAND No Section . .

125

ppm PITUITARY . . .

125

ppm PREOPTIC NUCLEUS . . .

125

ppm TESTIS . . .

125

ppm ADRENAL . . .

125

ppm BURSA . . .

125 CLOACAL GLAND . . .

# B-1 ppm

125

ppm KIDNEY . . .

125

ppm LIVER . . .

125

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

125

ppm PINEAL GLAND . . .

125

ppm PITUITARY . . .

125

ppm PREOPTIC NUCLEUS . . .

125

ppm TESTIS . . .

125

ppm ADRENAL . . .

125

ppm BURSA . . .

125

ppm CLOACAL GLAND . . .

125

ppm KIDNEY . . .

125

ppm LIVER . . .

125

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

125

ppm PINEAL GLAND . . .

125

ppm PITUITARY . . .

125

ppm PREOPTIC NUCLEUS . . .

125

ppm TESTIS . . .

125

ppm TESTIS Missing one of pair . .

125

ppm ADRENAL . . .

125

ppm BURSA . . .

125

ppm CLOACAL GLAND . . .

125

ppm KIDNEY . . .

125

ppm LIVER . . .

125

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

125

ppm PINEAL GLAND No Section . .

125

ppm PITUITARY . . .

125

ppm PREOPTIC NUCLEUS . . .

# B-1 125

ppm TESTIS . . .

125

ppm TESTIS Missing one of pair . .

125

ppm ADRENAL No Section . .

125

ppm BURSA . . .

125

ppm CLOACAL GLAND . . .

125

ppm KIDNEY . . .

125

ppm LIVER . . .

125

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

125

ppm PINEAL GLAND No Section . .

125

ppm PITUITARY . . .

125

ppm PREOPTIC NUCLEUS . . .

125

ppm TESTIS . . .

125

ppm TESTIS Missing one of pair . .

125

ppm ADRENAL . . .

125

ppm BURSA . . .

125

ppm CLOACAL GLAND . . .

125

ppm KIDNEY . . .

125

ppm LIVER . . .

125

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

125

ppm PINEAL GLAND No Section . .

125

ppm PITUITARY . . .

125

ppm PREOPTIC NUCLEUS . . .

125

ppm TESTIS No Section . .

250

ppm ADRENAL . . .

250

ppm BURSA . . .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250 LIVER . . .

# B-1 ppm

250

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

250

ppm PINEAL GLAND No Section . .

250

ppm PITUITARY . . .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm TESTIS . . .

250

ppm TESTIS Missing one of pair . .

250

ppm ADRENAL . . .

250

ppm BURSA . . .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250

ppm LIVER Hepatocellular Vacuolation, Diffuse, Lipid

Type 1 .

250

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

250

ppm PINEAL GLAND No Section . .

250

ppm PITUITARY Cyst(s) P .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm TESTIS . . .

250

ppm ADRENAL . . .

250

ppm BURSA . . .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250

ppm LIVER . . .

250

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

250

ppm PINEAL GLAND . . .

250

ppm PITUITARY . . .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm TESTIS . . .

250

ppm ADRENAL . . .

# B-1 250

ppm BURSA No Section . .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250

ppm LIVER . . .

250

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

250

ppm PINEAL GLAND No Section . .

250

ppm PITUITARY . . .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm TESTIS . . .

250

ppm ADRENAL . . .

250

ppm BURSA . . .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250

ppm LIVER . . .

250

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

250

ppm PINEAL GLAND No Section . .

250

ppm PITUITARY . . .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm TESTIS . . .

250

ppm ADRENAL . . .

250

ppm BURSA . . .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250

ppm LIVER . . .

250

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

250

ppm PINEAL GLAND No Section . .

250

ppm PITUITARY Cyst(s) P .

250 PREOPTIC NUCLEUS . . .

# B-1 ppm

250

ppm TESTIS . . .

250

ppm TESTIS Missing one of pair . .

250

ppm ADRENAL . . .

250

ppm BURSA No Section . .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250

ppm LIVER . . .

250

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

250

ppm PINEAL GLAND . . .

250

ppm PITUITARY . . .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm TESTIS No Section . .

250

ppm ADRENAL . . .

250

ppm BURSA . . .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250

ppm LIVER . . .

250

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

250

ppm PINEAL GLAND No Section . .

250

ppm PITUITARY . . .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm TESTIS . . .

250

ppm TESTIS Missing one of pair . .

250

ppm ADRENAL . . .

250

ppm BURSA . . .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

# B-1 250

ppm LIVER . . .

250

ppm MESONEPHRIC/WOLFFIAN

DUCTS . . .

250

ppm PINEAL GLAND . . .

250

ppm PITUITARY . . .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm TESTIS No Section . .

0 ppm ADRENAL . . .

0 ppm BRAIN Encephalomalacia, Regionally Extensive 3 3

0 ppm BURSA . . .

0 ppm CLOACAL GLAND . . .

0 ppm KIDNEY . . .

0 ppm LIVER . . .

0 ppm MULLERIAN DUCTS . . .

0 ppm OVARY . . .

0 ppm PINEAL GLAND No Section . .

0 ppm PITUITARY . . .

0 ppm PREOPTIC NUCLEUS . . .

0 ppm ADRENAL . . .

0 ppm BURSA . . .

0 ppm CLOACAL GLAND No Section . .

0 ppm KIDNEY . . .

0 ppm LIVER . . .

0 ppm MULLERIAN DUCTS . . .

0 ppm OVARY . . .

0 ppm PINEAL GLAND No Section . .

0 ppm PITUITARY No Section . .

0 ppm PREOPTIC NUCLEUS . . .

0 ppm ADRENAL . . .

0 ppm BURSA Apoptotic Necrosis, Increased 1 .

0 ppm CEREBELLUM . . 1

0 ppm CLOACAL GLAND . . .

0 ppm KIDNEY . . .

0 ppm LIVER . . .

0 ppm MULLERIAN DUCTS No Section . .

0 ppm OVARY . . .

0 ppm PINEAL GLAND No Section . .

0 ppm PITUITARY . . .

0 ppm PREOPTIC NUCLEUS . . 1

0 ppm ADRENAL . . .

0 ppm ADRENAL Missing one of pair . .

0 ppm BURSA . . .

0 ppm CLOACAL GLAND . . .

0 ppm KIDNEY . . .

0 ppm LIVER . . .

0 ppm MULLERIAN DUCTS . . .

# B-1 0 ppm OVARY . . .

0 ppm PINEAL GLAND No Section . .

0 ppm PITUITARY . . .

0 ppm PREOPTIC NUCLEUS . . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MULLERIAN DUCTS . . .

30

ppm OVARY . . .

30

ppm PINEAL GLAND . . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm ADRENAL No Section . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MULLERIAN DUCTS . . .

30

ppm OVARY No Section . .

30

ppm PINEAL GLAND . . .

30

ppm PITUITARY Cyst(s) P .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30 MULLERIAN DUCTS . . .

# B-1 ppm

30

ppm OVARY . . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm ADRENAL . . .

30

ppm BURSA No Section . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MULLERIAN DUCTS . . .

30

ppm OVARY . . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MULLERIAN DUCTS No Section . .

30

ppm OVARY . . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

# B-1 30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MULLERIAN DUCTS . . .

30

ppm OVARY . . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY No Section . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MULLERIAN DUCTS . . .

30

ppm OVARY . . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER Hepatocellular Necrosis, Focally Extensive 1 .

30

ppm MULLERIAN DUCTS . . .

30

ppm OVARY . . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30 ADRENAL . . .

# B-1 ppm

30

ppm BURSA Apoptotic Necrosis, Increased 1 .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MULLERIAN DUCTS . . .

30

ppm OVARY . . .

30

ppm PINEAL GLAND No Section . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MULLERIAN DUCTS . . .

30

ppm OVARY . . .

30

ppm PINEAL GLAND . . .

30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm ADRENAL . . .

30

ppm BURSA Apoptotic Necrosis, Increased 1 .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MULLERIAN DUCTS . . .

30

ppm OVARY . . .

30

ppm PINEAL GLAND No Section . .

# B-1 30

ppm PITUITARY . . .

30

ppm PREOPTIC NUCLEUS . . .

30

ppm ADRENAL . . .

30

ppm BURSA . . .

30

ppm CLOACAL GLAND . . .

30

ppm KIDNEY . . .

30

ppm LIVER . . .

30

ppm MULLERIAN DUCTS No Section . .

30

ppm OVARY . . .

30

ppm PINEAL GLAND . . .

30

ppm PITUITARY Cyst(s) P .

30

ppm PREOPTIC NUCLEUS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MULLERIAN DUCTS . . .

60

ppm OVARY . . .

60

ppm PINEAL GLAND . . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm ADRENAL . . .

60

ppm BURSA Apoptotic Necrosis, Increased 1 .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60 MULLERIAN DUCTS . . .

# B-1 ppm

60

ppm OVARY . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MULLERIAN DUCTS . . .

60

ppm OVARY . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MULLERIAN DUCTS . . .

60

ppm OVARY . . .

60

ppm PINEAL GLAND . . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

# B-1 60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MULLERIAN DUCTS . . .

60

ppm OVARY . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MULLERIAN DUCTS . . .

60

ppm OVARY . . .

60

ppm PINEAL GLAND . . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MULLERIAN DUCTS . . .

60

ppm OVARY . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60 ADRENAL . . .

# B-1 ppm

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MULLERIAN DUCTS . . .

60

ppm OVARY . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MULLERIAN DUCTS No Section . .

60

ppm OVARY . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MULLERIAN DUCTS . . .

60

ppm OVARY . . .

60

ppm PINEAL GLAND No Section . .

# B-1 60

ppm PITUITARY Cyst(s) P .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MULLERIAN DUCTS . . .

60

ppm OVARY . . .

60

ppm PINEAL GLAND . . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MULLERIAN DUCTS No Section . .

60

ppm OVARY . . .

60

ppm PINEAL GLAND . . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60 MULLERIAN DUCTS . . .

# B-1 ppm

60

ppm OVARY . . .

60

ppm PINEAL GLAND . . .

60

ppm PITUITARY No Section . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER Hepatocellular Necrosis, Focally Extensive 2 2

60

ppm MULLERIAN DUCTS . . .

60

ppm OVARY . . .

60

ppm PINEAL GLAND . . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

60

ppm KIDNEY . . .

60

ppm LIVER . . .

60

ppm MULLERIAN DUCTS No Section . .

60

ppm OVARY . . .

60

ppm PINEAL GLAND No Section . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

60

ppm ADRENAL . . .

60

ppm BURSA . . .

60

ppm CLOACAL GLAND . . .

# B-1 60

ppm KIDNEY . . .

60

ppm LIVER Hepatocellular Necrosis, Focally Extensive 1 .

60

ppm MULLERIAN DUCTS . . .

60

ppm OVARY . . .

60

ppm PINEAL GLAND . . .

60

ppm PITUITARY . . .

60

ppm PREOPTIC NUCLEUS . . .

125

ppm ADRENAL . . .

125

ppm BURSA No Section . .

125

ppm CLOACAL GLAND No Section . .

125

ppm KIDNEY . . .

125

ppm LIVER . . .

125

ppm MULLERIAN DUCTS . . .

125

ppm OVARY . . .

125

ppm PINEAL GLAND . . .

125

ppm PITUITARY . . .

125

ppm PREOPTIC NUCLEUS . . .

125

ppm ADRENAL . . .

125

ppm BURSA Apoptotic Necrosis, Increased 1 .

125

ppm CLOACAL GLAND . . .

125

ppm KIDNEY . . .

125

ppm LIVER . . .

125

ppm MULLERIAN DUCTS . . .

125

ppm OVARY . . .

125

ppm PINEAL GLAND . . .

125

ppm PITUITARY . . .

125

ppm PREOPTIC NUCLEUS . . .

125 ADRENAL . . .

# B-1 ppm

125

ppm BURSA Apoptotic Necrosis, Increased 1 .

125

ppm CLOACAL GLAND . . .

125

ppm KIDNEY . . .

125

ppm LIVER Hepatocellular Necrosis, Focally Extensive 2 2

125

ppm MULLERIAN DUCTS . . .

125

ppm OVARY . . .

125

ppm PINEAL GLAND . . .

125

ppm PITUITARY . . .

125

ppm PREOPTIC NUCLEUS . . .

125

ppm ADRENAL . . .

125

ppm BURSA Apoptotic Necrosis, Increased 1 .

125

ppm CLOACAL GLAND . . .

125

ppm KIDNEY . . .

125

ppm LIVER . . .

125

ppm MULLERIAN DUCTS . . .

125

ppm OVARY . . .

125

ppm PINEAL GLAND . . .

125

ppm PITUITARY . . .

125

ppm PREOPTIC NUCLEUS . . .

250

ppm ADRENAL No Section . .

250

ppm BURSA . . .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY Mineralization 1 .

250

ppm LIVER . . .

250

ppm MULLERIAN DUCTS . . .

250

ppm OVARY . . .

250

ppm PINEAL GLAND . . .

# B-1 250

ppm PITUITARY Cyst(s) P .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm ADRENAL No Section . .

250

ppm BURSA Apoptotic Necrosis, Increased 2 .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250

ppm LIVER Bile Duct Hyperplasia 1 1 250

ppm LIVER Fibrosis, Peribiliary 1

250

ppm MULLERIAN DUCTS . . .

250

ppm OVARY . . .

250

ppm PINEAL GLAND No Section . .

250

ppm PITUITARY . . .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm ADRENAL . . .

250

ppm BURSA Apoptotic Necrosis, Increased 1 .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250

ppm LIVER . . .

250

ppm MULLERIAN DUCTS . . .

250

ppm OVARY . . .

250

ppm PINEAL GLAND . . .

250

ppm PITUITARY Cyst(s) P .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm ADRENAL . . .

250

ppm BURSA . . .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250 LIVER . . .

# B-1 ppm

250

ppm MULLERIAN DUCTS . . .

250

ppm OVARY . . .

250

ppm PINEAL GLAND . . .

250

ppm PITUITARY . . .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm ADRENAL . . .

250

ppm BURSA . . .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250

ppm LIVER . . .

250

ppm MULLERIAN DUCTS . . .

250

ppm OVARY . . .

250

ppm PINEAL GLAND . . .

250

ppm PITUITARY . . .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm ADRENAL . . .

250

ppm BURSA . . .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250

ppm LIVER . . .

250

ppm MULLERIAN DUCTS . . .

250

ppm OVARY . . .

250

ppm PINEAL GLAND . . .

250

ppm PITUITARY . . .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm ADRENAL . . .

250

ppm BURSA . . .

# B-1 250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250

ppm LIVER . . .

250

ppm MULLERIAN DUCTS . . .

250

ppm OVARY . . .

250

ppm PINEAL GLAND . . .

250

ppm PITUITARY . . .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm ADRENAL . . .

250

ppm BURSA . . .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250

ppm LIVER . . .

250

ppm MULLERIAN DUCTS . . .

250

ppm OVARY Retained Right Ovary P 1

250

ppm PINEAL GLAND No Section . .

250

ppm PITUITARY . . .

250

ppm PREOPTIC NUCLEUS . . .

250

ppm ADRENAL . . .

250

ppm BURSA . . .

250

ppm CLOACAL GLAND . . .

250

ppm KIDNEY . . .

250

ppm LIVER . . .

250

ppm MULLERIAN DUCTS . . .

250

ppm OVARY . . .

250

ppm PINEAL GLAND . . .

250

ppm PITUITARY No Section . .

250 PREOPTIC NUCLEUS . . .

# B-1 ppm

# APPENDIX I

HORMONE ANALYSES METHODS

# B-1 UNIVERSITY OF MARYLAND

DEPARTMENT OF ANIMAL AND AVIAN SCIENCES

COLLEGE PARK, MD

DRAFT JAPANESE QUAIL HORMONE ANALYSIS METHODS

# B-1 Table of Contents

1.0 Introduction

2.0 Quality Assurance

3.0 Execution of the Technical Directive

4.0 Validation Methods for Avian Samples and Biological Media

5.0 Hormone Assays Selected

5.1 Selected Assays and Vender Information

6.0 Validation of Each Assay

6.1 Estradiol

6.1.1 Extraction

6.1.2 Plasma Samples

6.1.3 Yolk Samples

6.1.4 Fecal Samples

6.2 Testosterone

6.2.1 Extraction

6.2.2 Plasma Samples

6.2.3 Yolk Samples

6.2.4 Fecal Samples

6.3 Thyroid Hormones

6.3.1 Plasma Samples

6.3.2 Thyroid Gland Samples

6.3.2.1 Extraction

7.0 Sample Collection SOPs

7.1 Plasma Samples

7.2 Thyroid Gland Samples

7.3 Egg Yolk Samples

7.4 Fecal Samples

# B-1 1.0 Introduction

The purpose of this Technical Directive 1 (T D1) for Task Order 38 was to validate assay

methods in order to accurately conduct selected hormone measur ements associated with avian

studies of toxicants. Since many of the envi ronmental chemicals currently of concern are

hormonally active or endocrine disrupting chemicals, it is critical to measure pertinent endpoints

to ascertain effects. These endpoints incl ude hormone assays th at reflect impacts on

reproductive, metabolic, and stre ss responsive endocrine systems. Specific hormone assays

validated in TD1 included thyroid hormones (T3 and T4), estradiol and testos terone that occur in

various biological media (e.g. fecal/urate materi al, blood, egg yolk, and/or thyroid gland). TD1

required development of reliable assay methods and subsequent de termination of concentrations

for selected hormones in the various media menti oned above collected from the Tier 2 Japanese

quail two-generation tests.

TD1 required validation of thes e hormone analyses for sensit ivity, accuracy, precision, and

specificity to deliver reliable endocrine data in support of the two-ge neration tests that are

underway at other sites. Sample preparation must support the requi rements of the selected assay

systems, with extraction methods to remove any interfering compounds from the extract prior to

assay. Proper extraction will reduce variability and error due to interference. Sample volumes

or weights must be sufficient to ensure measureable levels of the hormone of interest. As

described below, assays were tested for sensi tivity and to assure serial dilutions of unknown

samples behaved in manner parallel to hormone st andards (i.e. parallelism). Samples used to

determine sensitivity and parallelism were either collected by the testi ng laboratories conducting

the multi-generation tests or from samples collec ted from our Japanese quail, which are of a

similar genetic stock.

2.0 Quality Assurance

The University of Maryland Quality Assuran ce (QA) personnel has been informed about

progress of the validation studies and involved in resolving some issues that arose in working

through the varied assay systems a nd extraction procedures. She ha s been provided with the data

set and conducted QA using the raw data. Based on her assessment, there will be specifications

in the Standard Operating Procedures (SOPs) for the use of the validated assays within

prescribed limits to ensure hi gh quality analyses and accurate da ta. She will prepare a brief audit

report that reviews the data and use of appropr iate methods. Both the summary report and QA

report will be reviewed by Battelle QA.

3.0 Execution of the Technical Directive

A number of commercially available assay kits were tested to select the best-suited assay

systems for the samples generated in the multi-generation avian test. Assays have been validated

for each hormone and in the various matrices. We recommend specific assays on the basis of

assay performance with hormone concentrations as predicted from our test samples. These

recommendations consider both the utility of spec ific analyses for the purpose of screening the

impact of test substances on endocrine activity. In addition, these assays will be critical in

assessing individual endocrine responses and char acterizing adverse consequences of exposure.

Data reports and supporting digita l files including spreadsheets are provided as both hard copy

# B-1 and digital electronic formats. The digital info rmation will be recorded on a DVD and delivered

to the EPA TOPO. The narrative repo rt includes supporting tables and refers to a series of Excel

spreadsheets for each type of assay that contains the complete description of the assay validation,

with reproducibility and detection limits for each hormone in each biological matrix.

A fundamental decision was made regarding the primary assa y methodology using available

commercial assay kits. This decision was whether to use Enzyme Linked Immunoassays

(ELISA) or a radioimmunoa ssay (RIA). Commercial assays we re tested to determine if ELISA

or RIA provides the most advantageous system for analysis of a particular hormone. The assays

tested were those that we have either used or those that have been recently developed for use

with bird samples. An assay system was selected based on the performance capabilities,

including sensitivity, validity, and reliability. Once selected, each assay system was further

tested with avian samples to ascertain the workin g limits for that assay as well as effects of the

matrix within which the hormone resides. These working limits have been incorporated into the

SOPs developed for each assay system to allow for maximal reliability and comparability across

experiments and studies.

4.0 Validation Methods for Avian Samples and Biological Media

Several sources of samples were used to complete the initial testing and subsequent validation of

the assays. For initial testing of the efficacy of assay systems, pools of chicken serum or plasma

were used to establish basic working performan ce of each assay. This allowed for comparison of

the span of the standard curves and establishmen t of the limits of sensitivity as well as collecting

information on the repeatability of the within assay measurements for the points on the standard

curve. Assay of extracted chicken serum or plasma provided information about the accuracy,

precision, and sensitivity of each assay for use wi th an avian sample and if there were any

differences in the measurement of serum or plas ma samples. Once the number of potential assay

systems was reduced, then quail samples were us ed to test the assay systems. These quail

samples were from the University of Maryland Japanese quail col ony; they were archived, frozen

samples collected from adults and hatchlings. These samples provided insight into the working

limits of sensitivity of the assays and allowed us to select assays that would be likely to detect

samples well within the working range of the standard curve for each hormone. In addition,

thyroid, egg yolk and fecal samples from the Univ ersity of Maryland quail colony were used to

provide pooled samples to test the assays in this other matrix.

Two sets of samples were received from the EPA. The first set was intended for assay validation

and included thyroid glands, serum, and plas ma from Bayer Crop Sciences. The blood samples

from this set were entirely unusable due to poor quality. The plasma samples consisted of what

appeared to be packed red bl ood cells, and the serum samples showed unacceptable levels of

hemolysis, which interferes w ith the antibody-antigen interac tions in both ELISAs and RIAs.

The thyroid glands from this set of samples ar e being used to validat e extraction procedures.

A second set of approximately 300 quail samples were received from Smithers Viscient (labeled

TP35) sent by Carol Sloan (RTI). They incl uded F0 adult (175 sample tubes) and embryo

plasma samples (~94 samples), collected using he parin as an anticoagulant. The adult samples

were visually inspected for quality and quant ity. Of the 175 sample tubes, most had ample

# B-1 volume to run all the assays with some surplus and appeared to be of good quality. Seventeen of

the adult samples contained less than the nece ssary volume (see below), and 5 of the samples

were severely hemolyzed. It was been noted whic h samples were inadequate in volume and/or

quality. Embryo samples will have to be pooled, as the volume in most of the samples appears to

be approximately 100μl.

Each matrix was separately validated for use w ith each assay to ensure performance standards.

This meant that samples from blood (plasma), thyroid gland, feces, and yolk were each be tested

and validated according to the appropriate assay sy stem. In addition, samples from different life

stages (adult and hatchling) were tested to ascertain stability of the assay and sufficient detection

sensitivity. Finally, sensitivity was determined for each assay system relative to the predicted

sample volume necessary to achieve the th reshold concentration for detection.

Each assay system was tested for the following criteria: sensitivity, specificity, accuracy, and

precision. The validation criteria are described belo w. These criteria were applied to the extracts

from the different media, including serum/ plasma, thyroid gland, feces, and yolk.

Sensitivity is ability of the assay to distinguish between two sim ilar concentrations. It is

determined by testing the standard deviation of 10 replicate measuremen ts at each of three

selected concentrations. The variability determines the sensitivity of measurements. This

threshold of variability is e xpressed according to pg/ml horm one concentration, which denotes

the sensitivity of the assay. Al so, the test of parallelism provi des additional information about

the volume requirements for each type of sample.

Specificity is defined as the capability to discern a particular horm one as distinct from other

species of hormones that have similar structure. This characteristic is generally determined by

the company that produces the assay kit by comp arison of antibody binding to a range of other

hormones that are likely to be cros s reactive with the antibody. In the case of testosterone, it is

unusual to have an assay with an antibody s howing no cross reactivity with 5dihydrotestosterone (5-DHT). As su ch, most assays for androgenic steroids are termed androgen

assays and this generally denotes a 100% cross reactivity with test osterone and an accompanying

50% cross reactivity with 5-DHT. We did not test the cross reactivity for similar hormones

and relied on the manufacturers’ qualit y assurance and product information.

In addition, a test of parallelism was conducted to ensure that there was no interference due to

compounds present in the extract. Parallelism was conducted usi ng a serum/plasma pool extract

that is serially diluted and assa yed. There were at least 3 series conducted and then averaged to

see where the measured concentrations fall on th e curve. Additionally, the curve produced by

the serial dilution should parall el the standard curve. These determinations also provide an

indication of limits of sensitivity relative to sa mple volume. The slopes were compared to

determine if there is a significant difference in the parallel curves.

Accuracy denotes the closeness of measured concen trations of a hormone with the predicted

concentration as measured by the standard curve. The accuracy of an assay was determined by

replicate assays of three selected standards (n=6/standard concentration). The error of

measurements did not exceed 10% of the standard concentration.

# B-1 Precision is the repeatability of measurements by an assay. There is intra-assay variation and

inter-assay variation. Three point s on the standard curve were selected as targets for pooled

internal controls and analyzed in each assay. The Coefficient of Vari ation around each measured

concentration was less than 10%.

Extraction Methods differed according to the media. Routine extraction methods were tested

for recovery of added hormone to ensure efficien cy of the process. Samples were extracted in a

manner to optimize the reliability of consistent and high percentage recovery. Yolk samples

were tested for the most efficient extraction method and several approaches were tested to

determine if fractional separation was necessa ry. A standardized extraction method was

finalized for each matrix for each assay as desc ribed below and assay validation was performed

using all types of extracts.

5.0 Hormone Assays Selected

5.1 Selected Assays and Vender Information

Assay kits recommended for use in the Avian Tw o-Generation Studies are listed in Table 1.

This table has supporting information below that specifies the criteria th at were used in the

selection of each kit. One of the primary issu es was that of using an Enzyme Linked Immuno

Sorbent Assay (ELISA) or radioimmunoassay (RIA ). We recommend RIAs for most of the

hormones, given sensitivity and sample volume constraints. Additiona lly, the RIA is more

efficient in terms of measuremen t of large numbers of samples. See bulleted points below for

more detailed explanation.

Table 1. Assay kits evaluated for measurement of triiodothyronine (T3), th yroxine (T4), estradiol

(E2), and testosterone (T) in avian serum or plasma samples.

Hormone Assay

Type Company Catalog # Reported

Sensitivity Required Volume

(μl) 1



# T3

Total ELISA Calbiotech T3104T 0.5 ng/ml 100

RIA MP

Biomedicals 06B254215 0.5 ng/ml 200

RIA 2 

# Siemens TKT31 0.2 ng/ml 200

Free RIA Siemens TKF31 0.5 pg/ml 200

T4

Total ELISA Calbiotech T4105T 20 ng/ml 50

RIA MP

Biomedicals 06B254011 20 ng/ml 50

RIA 2 

# Siemens TKT41 10 ng/ml 50

Free RIA Siemens TKF41 1 pg/ml 100

E2 ELISA Caymen 582251 6.6 pg/ml 100

RIA MP

Biomedicals 7238102 10 pg/ml 200

# B-1 RIA 2 

# Siemens TKE21 20 pg/ml 200

T

Total ELISA 2 

# Caymen 582701 3.9 pg/ml 100

RIA MP

Biomedicals 7189102 100 pg/ml 100

Free RIA Siemens TKTF2 0.55 pg/ml 100

1 

# For analysis of each sample in duplicate

2 

# Kits indicated in bold were selected for final an alysis of plasma, thyroid glands, yolk, and feces

from Japanese quail.

 The above kits were all evalua ted for analysis of hormones in avian (chicken) plasma and

serum, both as is and extracted with di ethyl ether and/or methylene chloride.

o All kits worked well and yielded linear standard curves within the reported range

of the assay.

o It was determined that RIAs are pref erred over ELISAs for analysis of large

numbers of samples due to more simp lified methodology with less hands-on time,

ability to assay several hundred samples at one time, and cost. Unfortunately,

neither RIA evaluated for measurement of testosterone in avian samples was

sufficiently sensitive.

 For total T3 and total T4, Siemens RIAs were selected for final use

because of their increased sensitivity. Assays for free T3 and free T4 did

not detect any measurable hormone.

 Although the reported sensitivity of the estradiol RIA from MP

Biomedicals is greater than that of the Siemens RIA, the levels for nonspecific binding measured with that assay were unacceptable. Therefore,

the RIA from Siemens was selected for final use.

 The testosterone RIA from MP Biomed icals was not sufficiently sensitive

for avian samples, and the free testosterone RIA from Siemens did not

detect any measurable hormone. The onl y assay with sufficient sensitivity

for measurement of testosterone in avian samples was the Caymen ELISA,

and it was selected for final use.

o Initial runs with chicken serum and plas ma indicated that hormone measurements

for total T3, total T4, and estradiol were approaching the limit of sensitivities of

the assays, and there was a need to at tempt to improve assay sensitivity.

 For hormone measurement in blood samples, pl asma is preferred ov er serum. While both

worked well in initial evalua tions, the volume of recovery from whole blood is greater in

plasma than in serum. Preparation of plas ma using either heparin or EDTA as the

anticoagulant was compatible with the assa ys, though almost all of the validation was

completed in plasma prepared with heparin as the anticoagulant so this would be the

preferred method for plasma collection.

# B-1  For analysis of all hormones, a minimum volume of 600 μl plasma is required. Of

this, 350 μl must be extracted for measur ement of steroid hormones and 250 μl is

needed for measurement of thyroid hormones in unextracted plasma.

 Thyroid hormones are lost during organic extraction and must be evaluated in

unextracted serum or plasma.

 Initial results for measurement of estradiol and testosterone indicated that serum and

plasma must be organically extracted because of a lack of parallelism in unextracted

samples.

o Diethyl ether proved more suitable for ex traction of steroid hormones from serum

or plasma, due to ease of ex traction and hormone recovery.

o Extraction efficiencies are being conducte d for plasma, yolk, and feces by spiking

samples with tritiated estradiol or tritiated testosterone.

After initial evaluation of both RIAs and ELISAs fr om several companies, the kits indicated in

bold in Table 1 were selected for final use (see a bove for rationale). All selected kits were for

evaluated for specificity, intra- and inter-assay va riation, and parallelism in sample types to be

measured in Japanese quail. The RIAs for T3, T 4, and E2 were also evaluated for the ability to

improve assay sensitivity. Assay characteristi cs are shown in Tabl e 2. Demonstration of

parallelism is shown on the graphs.

 Limits of sensitivity were determined by running a series of dilutions until the % of

maximum binding [(B/B 0 

# )*100] was at least 95% and re peatable in multiple assays.

Actual limits of sensitivity for T3, T4, and E2 were improved approximately 10-fold

(compare Table 2 vs Table 1). When perfor ming these assays, the standard curve is

adjusted accordingly.

 Specificity was determined by measuring a known amount of the i ndicated hormone in

the assay (i.e. T4 in the T3 assay, T3 in the T4 assay, T in the E2 assay, and E2 in the T

assay), and determining cross- reactivity (%) as [(amount me asured/amount added)*100].

 Intra- and inter-assay variati on (coefficient of variation (CV; %) were determined in all

four assays using chic ken plasma because of the large sa mple volume required. In each of

6 independent assay runs, the same sample was analyzed 6 times. The intra-assay CV was

determined in each of the runs, and the average of the 6 runs was determined to be the

intra-assay CV (%). The inter-assay CV (%) wa s determined from the mean value of the

sample in each of the six runs. For total T4 and estradiol, the levels in chicken plasma

approached the limit of sensitivity of the a ssay and contributed to higher variability.

Therefore, additional intra-assay and inter-assa y values for these assays were determined

in the same manner using thyroid extract (t otal T4) and fecal extract (estradiol).

 Parallelism was evaluated in each of the sample types for each of the assays. At least 4

dilutions of a given sample were run on a minimum of 3 independent pools of each

sample type. In addition, 3 independent pools of hatchling plasma were evaluated in each

# B-1 assay at 2 dilutions. This allowed us to furthe r validate the assays in younger birds from a

different colony.

o Total T3: adult plasma (male, fema le, mixed sex), thyroid extracts

o Total T4: adult plasma (male, fema le, mixed sex), thyroid extracts

o E2: ether-extracted plasma (male, female (2), mixed sex), ether-extracted yolk,

ethanol-extracted feces.

o T: ether-extracted plasma (male, fe male, mixed sex), et her-extracted yolk,

ethanol-extracted feces.

Table 2. Assay characteristics of kits selected for final anal ysis of triiodothyronine (T3),

thyroxine (T4), estradiol (E2), and testosterone (T) in Japane se quail plasma, thyroid glands,

yolk, and feces.

Hormon

e Assay Determined

Sensitivity Crossreactivity 1



# (%) Intra-assay CV

(%) Inter-assay CV

(%)

Total T3 Siemens

TKT31 0.03 ng/ml 1.4 (T4) Plasma: 3.7 Plasma: 5.9

Total T4 Siemens

TKT41 1.6 ng/ml 3.3 (T3) Plasma: 9.9

Thyroid 2 

# : 2.9 Plasma: 13.6

Thyroid 2 

# : 15.2

E2 Siemens

TKE21 2.5 pg/ml 0.2 (T) Plasma: 6.9

Feces 2 

# : 3.7 Plasma: 13.7

Feces 2 

# : 10.0

T Caymen

582701 3.9 pg/ml 0.1 (E2) Plasma: 5.7 Plasma: 6.4

1 

# Measured cross-reactivity is with the hormone indicated in parentheses.

2 

# Additional intra- and inter-assay CV values were determined in thyroid extracts for total T4 and

fecal extracts for E2 because plasma measuremen ts were approaching the limitation of sensitivity

for the assays and thought to contribute to in creased variability in these determinations.

6.0 Validation of Each Assay

6.1 Estradiol (see Excel spread sheets for detailed information)

6.1.1 Steroid Extraction from Various Matrices

Samples were tested for assay efficacy with or without organic chemical extraction; however,

steroid hormone assays required ch emical extraction in order to remove interfering lipids and

other compounds that interfere with antibody bind ing. For both the chicken and the quail steroid

hormone assays, samples were chemically extract ed, using ether for the organic solvent. A

protocol for yolk analysis was developed in whic h a series of dilutions was been made with

distilled-deionized water. Becau se the distribution of steroids in the yolk is not homogeneous,

but rather is laid down in layers according to the lay down of the yol k, it was critical to have the

yolk homogenized to have an accurate measure of the steroid content. Adding the water also

helps to evenly distribute the steroid and prepare a mixture of yolk:water that is more easily

extracted with higher steroid hormone recovery from the yolk matrix. Using eggs from the

UMD Japanese quail colony, different amounts of water were tested for making the yolk:water

# B-1 mixture and for extraction efficacy and reconstitution into the assay buffer. The dilutions

included 1:1, 1:2, 1:3 (yolk:water). Results showed that 1:1 pr ovided reasonable extractability,

with the 1:2 dilution providing a better extractio n matrix with less carry over of lipids during

extraction. Extracts were tested in an alcohol clean up procedure to remove excess lipid and

provide an extract that does not interfere with the steroid assays. Fecal samples were extracted

using a 90:10 ethanol:water procedure (see Appendix 1, 2, and 3 for detailed SOPs)

6.1.2 Plasma Samples

6.1.3 Yolk Samples

6.1.4 Fecal Samples

6.2 Testosterone (see Excel spread sheet)

6.2.1 Extraction (see Section 6.1.1)

6.2.2 Plasma Samples

6.2.3 Yolk Samples

6.2.4 Fecal Samples

6.3 Thyroid Hormones (see atta ched spread sheet)

6.3.1 Plasma Samples

6.3.2 Thyroid Gland Samples (see Append ix 4 for extraction and sample

preparation SOP)

# B-1 7.0 Sample Collection SOPs

Recommendations for Preparation of Samples

Plasma collected using either he parin or EDTA both should work well for all the assays (total

T3, total T4, estradiol and testos terone). Since most of the validation was conducted using quail

plasma prepared with heparin, this is the pr eferred anticoagulant for use in the studies. Quail

plasma should be collected into a 2-ml centrifuge t ube that is clearly and legibly labeled with the

bird identification. Sample s should be stored at – 20 o 

# C and shipped on dry ice. A sample

inventory in the form a spreadsheet, clearly indicating bird identification and approximate

plasma volume, must be provided in both an electronic and a hard copy.

The estimated amount of plasma in the embryo samples is 50-150l. Depending on the

hormone concentrations in these samples, we re commend that samples be pooled in order to run

all the assays. The volumes required to run sa mples in duplicate for ea ch assay are T3=200l;

T4=50l; estradiol=200l; testosterone=100l. The T3 and T4 assays use neat plasma, with no

extraction. Assuming the samples cannot be dilute d to run in the assays , the approximate total

amount of plasma required to conduct all assays is 600l.

Thyroid gland samples from adults, hatchling, a nd embryos should be provided frozen in 2-ml

round bottom centrifuge tubes clearly and legibly labeled with the bird identification. The

thyroid glands need to be weighed at collection. If the combined weight of the glands is more

than 10 mg, a piece between 5-10 mg needs to be accurately weighed and placed in the sample

tube. The glands should be stored at -20 C and shippe d on dry ice. A sample inventory in the

form of a spreadsheet, clearly indicating bird identification, total thyroid gl and weight, and

weight of the sample collected into the tube need s to be provided in both an electronic and hard

copy.

Yolk samples should be collected with clean separation from albumin because albumin may

interfere with the assay. The entire yolk shoul d be weighed and homogenized with two volumes

of ultrapure deionized water. A 1-ml aliquot of the homogenized, diluted yolk should be put into

a 1.7-ml centrifuge tube that is cl early and legibly labeled with the bird identification. Samples

should be stored at – 20 o 

# C and shipped on dry ice. A sample inventory in the form of a

spreadsheet, clearly indicating bird identificatio n, total yolk weight, amo unt of water used for

dilution, and volume (if other than 1-ml) needs to be provided in both an electronic and a hard

copy.

Please see below for detailed SOPs.

7.1 Plasma Sample Preparation

1. If collecting during euthanasia, sever the neck and collect trunk blood into a

polypropylene plastic 12x75 mm tube containing anticoagulant [heparin is preferred (10

μl per 1.0 ml blood); EDTA is okay too (10 μl 0.5M per ml of blood)]. No more than 3

ml should be collected per tube. If collec ting by venipuncture, draw blood through needle

into syringe that has been pr e-coated with anticoaculant, remove the needle, and expel

blood into tube. Collect ~2.5X volume of plasma needed.

# B-1 2. Record the volume of total blood collected.

3. Immediately invert the blood sample to allo w the anticoagulant to mix with the blood.

4. Centrifuge the blood for 10 min at 1,500xg and 4 o 

# C. Plasma should be clear or light tan

in color. Reddish color indicates substa ntial red blood cell ly sis and presence of

hemoglobin, which interferes with many assa ys. Opaqueness indicates the presence of

particulates, which s hould be centrifuged out.

5. Transfer the supernatant to a clean 1.7-ml polypropylene microcentrifuge tube. Tubes

should be clearly and legibly labeled with bird identification. Record the volume of

plasma and indicate any discoloratio n and/or opaqueness in each sample.

6. Plasma samples should be stored at -20 o 

# C and shipped on dry ice. A sample inventory in

the form of a spreadsheet, clearly indicating bird identif ication and volume of plasma

collected must be provided with shipme nt, in both an elect ronic and hard copy.

7.2 Thyroid Gland Sample Preparation

1. Weigh the entire thyroid gland and record the total weight.

2. Dissect a small piece of the gland that is between 5-10 mg, weigh the subsample, and

record the exact subsample weight to the tenth of a milligram.

3. Place the thyroid subsample into a 2-ml round bottom microcentrifuge tube (e.g.

Eppendorf catalog # 022363352) on ice. Tubes shoul d be clearly and legibly labeled with

bird identification.

4. Thyroid samples should be stored at -20 o 

# C and shipped on dry ice. A sample inventory in

the form of a spreadsheet, cl early indicating bird identifica tion, total thyroid weight, and

exact weight of the subsample within the tube , must be provided with shipment, in both

electronic and hard copy.

7.3 Egg Yolk Sample Preparation

1. Cleanly separate the yolk from the albumen.

2. Weigh the entire yolk and record the yolk weight.

3. Calculate and record the volume of water so the entire yolk is diluted with 2 volumes of

ultrapure deionized water (i .e. 2 mls water/g yolk).

4. Thoroughly homogenize the entire yolk with the appropriate volume of water determined

in step 3.

# B-1 5. Transfer 1-ml of the diluted, homogen ized yolk into a 1.7-ml polypropylene

microcentrifuge tube on ice. If the volume is other than 1-ml, record the actual volume

transferred. Tubes should be clearly and legibly labeled with bird identification.

6. Yolk samples should be stored at -20 o 

# C and shipped on dry ice. A sample inventory in

the form of a spreadsheet, cl early indicating bird identifica tion, total yolk weight, amount

of water used for dilution, and subsample vol ume (if other than 1-ml ) must be provided

with shipment, in both electronic and hard copy.

7.4 Fecal Sample Preparation

1. Thoroughly mix wet feces to ensure a ra ndomly distributed sample, and place

approximately 0.5 g into a weigh boa t. Record the exact wet weight.

2. Place wet fecal samples into a dessicator for at least 24 hrs. Periodically check the

weight, and remove from desicat or when the sample is no longer losing moisture. Record

the exact dry weight and determine % dry matter.

3. Thoroughly mix the dry fecal sample with a mo rtar and pestle. Weigh approximately 0.2

g dry fecal sample and record the exact weight.

4. Place the weighed dry fecal sample into a 15-ml polypropylene screw cap tube. Tubes

should be clearly and legibly labe led with bird identification.

5. Fecal samples should be stored at -20 o 

# C and shipped on dry ice. A sample inventory in

the form of a spreadsheet, cl early indicating bird identification, total fecal wet weight,

total fecal dry weight, % dry ma tter, and exact weight of th e subsample within the tube,

must be provided shipment, in both electronic and hard copy.

# B-1 Appendix 1: Plasma Extraction for Steroid Hormone Measurement

Reagents

Diethyl ether (Sigma catalog # 346136)

1X EIA Buffer [diluted from 10X EIA buf fer (Caymen Chemical catalog # 400060) using

ultrapure distilled water]

*Note*: Volumes below are for extraction and m easurement of both estradiol and testosterone

using Siemens RIA (catalog # TKE21) and Caym en ELISA (catalog # 582701). If smaller or

larger amounts are to be ex tracted, adjust the volume of ether and reconstitution buffer

accordingly. The important thing is that plasma samples are extracted twi ce with 5X volumes of

ether and reconstituted in 1X volume EIA buffer to provide a consistent volume between the

original and extracted samples. A maximum of 1 mL can be extracted in the tubes below. If

much smaller volumes are being extracted, this can be carried out in 12x75 mm glass culture

tubes (e.g. Fisher catalog # 14-961-26).

Procedure:

1. Label two 18x150 mm glass culture tubes (e.g. VWR catalog # 60825-443) for each

sample.

2. Pipet 400 μl plasma into one of the labeled tubes and cover with foil.

3. Heat plasma in 65 o 

# C water bath for 1 hr to bring st eroids off the glass and partially

denature proteins. Allow samples to cool to room temperature.

4. In the fume hood, add 2 mL diethyl ether to each sample and vortex vigorously for 30

sec.

5. Cover with foil or parafilm and allow samples to stand for ~2 min until fractions separate.

6. Place samples in a -80 o 

# C freezer for ~5 min or snap-fre eze using a dry ice/ethanol bath

until the lower aqueous phase freezes.

7. In the fume hood, decant the ether fraction into a second labeled tube for that sample.

8. Allow the extracted plasma to thaw. In the fume hood, add 2 mL diethyl ether to each

sample and vortex vigorously for 30 sec.

9. Repeat steps 5 through 7, so that samples ar e extracted twice and bot h ether fractions are

combined into the same tube.

# B-1 10. Allow samples to stand overnight in fume hood to evaporate. As they are drying, vortex

periodically to rinse down any steroid that may stick to the side of tube. If necessary, to

speed evaporation, the samples can be dried in the fume hood under a steady stream of

nitrogen.

11. Reconstitute extracted plasma with 400 μl 1X EIA buffer. Vortex for 30 sec and place in

65 o 

# C water bath for 1 hr. Vortex for 15-20 sec every 15 min.

12. Allow samples to stand for ~5 min to drain to the bottom of the tube and transfer to a

clearly and legibly labeled 1.7-mL polypropylene microcentrifuge tube.

13. Store extracted plasma samples at -20 o 

# C.

# B-1 Appendix 2: Yolk Extraction for Steroid Hormone Measurement

Reagents

Diethyl ether (Sigma catalog # 346136)

1X EIA Buffer [diluted from 10X EIA buf fer (Caymen Chemical catalog # 400060) using

ultrapure distilled water]

Procedure:

1. Label two 18x150 mm glass culture tubes (e.g. VWR catalog # 60825-443) for each

sample.

2. Thaw and thoroughly vortex yolk sample, whic h has previously been homogenized with

2 parts ultrapure distilled water.

3. Pipet 300 μl yolk sample into one of the labeled tubes.

4. In the fume hood, add 6 mL diethyl ether to each sample and vortex vigorously for 30

sec.

5. Cover with foil or parafilm and allow samples to stand for 30 min until fractions separate.

Note that the ether phase will remain cloudy, but most of the lipid-rich yolk phase will

settle out.

6. Place samples in a -80 o 

# C freezer for ~5 min or snap-fre eze using a dry ice/ethanol bath

until the lower phase freezes.

7. In the fume hood, decant the ether fraction into a second labeled tube for that sample.

8. Allow the extracted yolk to thaw. In the fu me hood, add 6 mL diethyl ether to each

sample and vortex vigorously for 30 sec.

9. Repeat steps 5 through 7, so that samples ar e extracted twice and bot h ether fractions are

combined into the same tube.

10. Allow samples to stand in fume hood to eva porate (~2 nights). As they are drying, vortex

periodically to rinse down any steroid that may stick to the side of tube. If necessary, to

speed evaporation, the samples can be dried in the fume hood under a steady stream of

nitrogen.

11. Reconstitute extracted yolk with 600 μl 1X EIA buffer. Vortex for 30 sec and place in

65 o 

# C water bath for 1 hr. Vortex for 15-20 sec every 15 min.

# B-1 12. Allow samples to stand for ~5 min to drain to the bottom of the tube and transfer to a

clearly and legibly labeled 1.7-mL polypropylene microcentrifuge tube.

13. Store extracted yolk samples at -20 o 

# C.

# B-1 Appendix 3: Fecal Matter Extraction for Steroid Hormone Measurement

Reagents

Ultrapure distilled water

100% Ethanol

1X EIA Buffer [diluted from 10X EIA buf fer (Caymen Chemical catalog # 400060) using

ultrapure distilled water]

Procedure:

1. Label one 15-mL polypropylene centrifuge tube (VWR catalog # 89004-368) for each

sample.

2. Weigh ~0.2 g dried fecal matter into the labeled 15-mL tube.

3. Add 1 mL of ultrapure water to each sample and vortex.

4. Add 9 mL 100% ethanol to each sample and vor tex vigorously for 30 sec. Be sure the cap

is secured tightly and wrap with parafilm.

5. Secure each tube horizontally to a rocker/shaker and rock vi gorously overnight at room

temperature.

6. Centrifuge samples at 3000 rpm fo r 25 min (room temperature).

7. Transfer supernatant to a clean, labele d 50-mL polypropylene tube (VWR catalog #

89004-364).

8. Place tubes into a 50-60 o 

# C water bath and evaporate samples under a stream of nitrogen.

9. Reconstitute extracted fecal samples with 2 mL 1X EIA buffer. Vortex for 30 sec and

place in 65 o 

# C water bath for 1 hr. Vortex for 15-20 sec every 15 min.

10. Transfer samples to two clearly an d legibly labeled 1.7-mL polypropylene

microcentrifuge tubes.

11. Store at extracted fecal samples at -20 o 

# C.

# B-1 Appendix 4: Thyroid Gland Extraction for Thyroid Hormone Measurement

Reagents

Digestion Mix (100 ml)

1.21 g TRIS

153.65 mg L-Glutathione reduced (Sigma catalog # G4251)

42.55 mg PTU (6-n-propyl-2-t hiouracil; Sigma P-3755)

1ml Triton-X-100 (Fisher catalog # BP151-500)

Bring to final volume of 100 mL with ultrapure distilled water after stirring to dissolve.

Store at 4 o 

# C.

Saturated Ammonium Sulfate (SAS)*

76.7 g ammonium sulfate (Fisher catalog # A702500)

100 mL ultrapure distilled water

Cover the beaker with foil and stir until dissolved while heating to ~65 o 

# C.

Place at 4 o 

# C overnight to allow precipitation to occur.

Filter the solution into a new bottle and store at 4 o 

# C. Further precipitation may occur upon longterm storage.

*SAS can also be purchased fr om Fisher (catalog # PI-45216)

Pronase (Calbiochem catalog # 53702; can be purchased from Fisher)

Procedure:

1. Thyroid gland subsamples weighing ~5 mg should be in a 2-ml roundbottom microfuge

tube (e.g. Eppendorf catalog # 022363352) . Note the exact weight.

2. Calculate the total volume pronase digestion bu ffer needed such that each sample gets

50X volume of a 100 mg/ml solution. For exam ple, a sample weighing 5.2 mg will get

260 μl. To digest 12 samples weighing exactly 5 mg, 3 mL of digestion buffer containing

300 mg of pronase is needed (0.25 mL x 12 = 3 mL x 100 mg/mL = 300 mg). Weigh out

the appropriate amount pronase into the appropriate volume of digestion mix pre-warmed

to 37 o 

# C and vortex to dissolve. Be sure to acc ount for loss during tran sfer and make extra

buffer.

3. Add 50X volumes of 100 mg/ml pronase digest ion buffer made in step 2 pre-warmed to

37 o 

# C to each sample. A piece weighing exactly 5 mg will get 250 μl. Adjust the volume

for the exact weight.

4. Homogenize each sample for 30-60 sec, unt il the tissue is no longer visible.

5. Place samples in a 37 o 

# C water bath for 1-2 hr, and then transfer them to shaking air

incubator set to 37 o 

# C for a total of 24 hr. If a 37 o 

# C shaking water bath is available, it can

be used for the full 24 hr.

# B-1 6. Add one volume of ice-cold SAS solution equa l to the volume of pronase digestion mix.

For example, the 5.2 mg sample will get 260 μl SAS.

7. Place samples on ice for 30 min.

8. Centrifuge samples at 20,000xg and 4 o 

# C for 30 min.

9. Transfer the supernatant to a cl ean 1.7-mL micro centrifuge tube.

10. Store extracted thyroid glands at -20 o 

# C.

# APPENDIX J

CONTRIBUTING SCIENTIST REPORT

STATISTICAL ANALYSIS OF HORMONE DATA

# B-1 

# Statistical Analysis on Hormone Date for USAPHC

Toxicology Protocol Number: 08JG-80-08-04-02

Two-Generation Test for Endocrine Disr uption and Reproductive Toxicity in

Japanese Quail ( Corturnix japonica) using Endosulfan

Good Laboratory Practice Compliance Statement

The statistical analysis of the data for t he Two-generation test for endocrine disruption

and reproductive toxicity in Japanese quail ( Cortunix japonica ) using endosulfan was

conducted in compliance with Title 40, Code of Federal Regulations (CFR), Part 792,

Good Laboratory Practice Standards. Statis tical analyses were conducted according to

the procedures outlined in the pr otocol for this study (Protocol Number 08JG-80-08-04-

02).

29 January, 2013

Shane Hall Date

Statistician

# B-1 I. Analysis Methods

A two-way Analysis Of Varian ce (ANOVA) was used to analyze four hormones measured (T3,

T4, Estradiol, and Testosterone) from two inde pendent three-generation studies that involved

exposing quails to endosulfan. Dosage of eith er endosulfan was altered to determine if the

amount of endosulfan the quails received had an e ffect on their hormone values. Endosulfan was

divided into five dose groups (c ontrol, 30 ppm, 60 ppm, 125 ppm, and 250 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 horm ones as the dependent variable and dose group

and gender as the predictors, determined if at le ast two of the groups with in each predictor were

significantly different from one another (test of main effect). The ANOVA model also

determined if the effect that a change in dose group had on the hormone was significantly

impacted by gender of the quails (test of intera ction). If the interaction was statistically

significant, the main effect results for dose gr oup and gender are potentiall y misleading and must

be interpreted in light of the interaction. Therefore, if an in teraction was significant, p-values for

the component parts are not given. If the inter action was not statistica lly 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 differen ce between males and females and additional posthoc tests were not necessary.

Data was checked to determine if ANOVA assumpti ons were met. A Levene’s test was used to

check equality of variances and ANOVA-model re siduals were examined via a Q-Q plot to

check normality. If assumptions were violated , data was log-transforme d and assumptions were

checked once again to see if an ANOVA was suita ble. For this data, all hormone measures

satisfied ANOVA assumptions using either the un transformed or Log-transformed data, so

additional analysis adjustme nts were not necessary.

Individual data points were occa sionally labeled as “Not Determined – Insufficient Volume”,

“Not Detectable”, or “Not Detectable (Below Assay Limits)”. In the first two cases, the blood

sample for that quail was either not tested or was unsuccessfully tested and was therefore

counted as missing data for the statistical analysis. However, the data points that were labeled as

below the assay limits were tested, but returned values too low for the diagnostic machine to

detect. In this case, limit of detection divided by the square root of tw o was substituted for the

missing value.

SPSS 16.0 was used to execute the statistical an alysis summarized in the report below.

Statistical significance throughout the analysis was defined at α =.05 or 95% confidence level.

II. Results

A. F0 Generation

# B-1 The four hormones were not significantly imp acted by Endosulfan in the F0 generation.

Estradiol and Testosterone were however sign ificantly impacted by gender. Females had a

significantly greater Estradiol average, and ma les had a significantly greater Testosterone

average.

Table 1. Effects of Endosulfan on F0 Generation Quail Hormones

Gender Dose Group

(ppm) T3 (ng/ml) 

# T4 (ng/ml) 

# * Estradiol (pg/ml) 

# Testosterone (pg/ml) 

# *

N Mean SE N Mean SE N Mean SE N Mean SE

Female Control 5 1.62 0.20 5 2.54 0.73 5 43.45 6.56 5 522.5 345.9

30 5 1.37 0.09 4 2.32 0.74 5 46.25 3.19 5 868.2 177.7

60 5 1.20 0.21 4 6.98 1.81 5 34.51 2.61 5 339.0 85.1

125 5 1.35 0.22 4 3.74 1.26 5 35.66 7.33 5 453.3 267.9

250 5 1.25 0.10 4 2.86 1.39 5 45.59 7.47 5 467.3 274.3

Male Control 5 1.46 0.11 5 2.49 0.68 5 10.89 4.02 5 1986.3 732.4

30 5 1.50 0.09 4 2.50 0.50 5 14.55 8.85 5 791.8 136.2

60 5 1.60 0.12 5 2.34 1.07 5 10.86 2.89 5 909.6 126.1

125 5 1.58 0.14 5 2.71 0.51 5 9.88 1.37 5 1264.3 363.3

250 5 1.50 0.09 5 3.26 0.92 5 6.90 2.42 5 1157.0 297.8

P

‐ Value Dose = .820 Dose = .568 Dose = .563 Dose = .792

Gender

= .070 Gender

= .235 Gender

= .000

(Females > Males) Gender

= .000

(Males > Females)

* Analyzed using Log

‐ transformed data due to violations in ANOVA assumptions.

# B-1 B. F1 Generation

The four hormones were not significantly imp acted by Endosulfan in the F1 generation.

Estradiol and Testosterone were however sign ificantly impacted by gender. Females had a

significantly greater Estradiol average, and ma les had a significantly greater Testosterone

average.

Table 2. Effects of Endosulfan on F1 Generation Quail Hormones

Gender Dose

Group

(ppm) T3 ( ng/ml) 

# T4 (ng/ml) 

# * Estradiol (pg/ml) 

# Testosterone (pg/ml)



# N Mean SE N Mean SE N Mean SE N Mean SE

Female Control 5 1.39 0.20 4 3.11 0.70 5 30.88 8.40 5 301.8 174.3

30 5 1.99 0.27 4 1.99 0.53 5 54.52 19.76 5 551.6 98.1

60 5 1.89 0.31 5 1.78 0.41 5 57.93 8.33 5 299.2 64.0

125 5 1.24 0.19 4 3.20 1.39 5 44.24 9.25 5 583.4 280.3

250 6 1.40 0.16 3 3.24 2.11 6 46.79 3.65 6 450.8 144.1

Male Control 5 1.60 0.16 5 2.51 0.43 5 10.16 2.24 5 925.8 405.1

30 4 1.65 0.12 3 1.52 0.39 5 9.31 2.22 5 1249.1 437.0

60 5 1.63 0.15 5 2.80 0.58 5 9.23 0.62 5 2114.3 720.8

125 5 1.66 0.16 5 3.21 0.38 5 9.49 1.08 5 3280.4 1241.9

250 4 1.39 0.16 3 2.92 0.92 4 12.88 7.16 4 1693.4 586.0

P

‐ Value Dose = .148 Dose = .459 Dose = .546 Dose = .159

Gender

= .981 Gender

= .607 Gender

= .000

(Females > Males) Gender

= .000

(Males > Females)

* Analyzed using Log

‐ transformed data due to violations in ANOVA assumptions.

# B-1 C. F2 Generation

In the F2 generation, T3, Estradiol, and Test osterone were all significantly impacted by

Endosulfan dosage and/or gender.

 T3 had similar averages across Endosulfan dose groups, but males had a significantly

greater average compared to females.

 Estradiol was impacted by gender with female s having a greater average within all five

dose groups; however the pattern of Estradiol averages for the dose groups within each

gender were severely different. Males had a fa irly consistent average across all five dose

groups, while females had an average that in creased from the control to dose 60 and

severely decreases from dose 60 to dose 250. For females dose 250 had a significantly

lower mean compared to dose 60, and for ma les there were no significant differences

between groups

 Testosterone was impacted by gender with male s having a greater average within all five

dose groups; however the pattern of testoster one averages for the dose groups within each

gender were severely different. As Endos ulfan dose increased, males had a severely

decreasing average level of testosterone, while females had a generally increasing

average level of testosterone . For males dose 125 and dose 250 had significantly lower

means compared to control, and for fema les no significant differences between groups

were observed.

Table 3. Effects of Endosulfan on F2 Generation Quail Hormones

Gender Dose

Group

(ppm) T3 (ng/ml) 

# T4 (ng/ml) 

# * Estradiol (pg/ml) 

# Testosterone (pg/ml)



# N Mean SE N Mean SE N Mean SE N Mean SE

Female Control 5 1.84 0.34 2 2.99 1.57 5 30.12 2.75 5 235.3 72.2

30 5 1.42 0.13 4 1.84 0.71 5 39.30 5.30 5 297.5 45.7

60 4 1.18 0.17 3 2.79 1.66 5 49.86 4.67 5 620.5 229.5

125 3 0.89 0.31 3 4.20 2.46 5 36.27 11.38 5 334.6 214.1

250 5 1.38 0.34 4 3.89 1.45 5 18.83 4.98 5 511.3 279.6

Male Control 5 1.66 0.09 4 2.69 0.09 5 10.17 1.86 5 2191.4 197.0

30 5 1.66 0.32 5 2.96 0.94 5 6.95 1.53 5 1759.1 425.1

60 5 1.80 0.14 5 1.72 0.24 5 5.61 1.96 5 1143.9 300.1

125 4 1.54 0.20 5 1.95 0.82 5 7.65 1.47 5 899.7 213.8

250 5 1.80 0.25 5 3.39 0.92 5 6.42 2.33 5 785.7 252.6

P

‐ Value Dose = .388 Dose = .576 Dose * Gender

= Dose * Gender

=

# B-1 Gender

= .033

(Males > Females) Gender

= .712 .022 .006

* Analyzed using Log

‐ transformed data due to violations in ANOVA assumptions.



# APPENDIX K

RAW DATA

# B-1 P generation bird weights

DOSE SEX ID 4/20 4/27 5/4 5/11 5/18 5/25 5/30 6/8 6/15 6/22 6/30 7/6 7/13

0 F 144 91 102 109 118 125 126 126 134 135 120 106 125 130

0 F 142 100 112 126 134 130 135 133 137 134 129 127 144 146

0 F 140 120 134 134 134 136 130 139 137 131 147 145 141

0 F 139 97 105 119 119 121 126 119 125 128 132 134 131 134

0 F 141 102 115 116 136 128 131 139 138 149 135 153 150

0 F 143 105 117 128 129 131 134 140 141 143 146 144 146 149

0 F 137 104 139 130 130 128 130 104 128 133 130 138 141 140

0 F 135 121 108 139 138 133 147 145 143 149 142 141 146 103

0 F 133 96 107 126 124 117 128 128 137 133 130 120 134 136

0 F 134 103 119

0 F 136 93 116 132 130 137 135 133 140 145 137 149 145 137

0 F 138 103 119 129 130 125 130 132 140 141 131 119 143 141

0 F 145 105 105 127 134 134 127 129 130 129 140 137 141 144

0 F 146 112 128 119 119 118 118 122 130 134 141 124 142 142

0 F 147 109 102 119 125 120 128 122 131 134 138 129 128 140

0 F 148 98 109 117 135 137 138 131 133 134 143 124 144 145

0 F 149 107 114 140 133 142 137 139 134 150 149 145 114

0 F 150 117 112 133 148 144 145 155 159 154 160 157 153 154

0 F 151 108 107 117 124

0 F 152 110 107 110

30 F 33 106 129 128 118 126 131 128 136 138 138 142 139 140

30 F 34 104 113 113 120 118 119 129 129 121 133 134 127 136

30 F 35 109 115 117 120 128 135 135 126 131 134 138 131 137

30 f 36 101 118 126 107 109 128 127 136 132 140 135 127 143

30 F 37 117 120 122 124 128 127 138 147 142 141 150 142 134

30 F 38 108 130 126 130 132 129 137 136 140 139 141 139 137

30 F 39 107 118 131 126 134 135 138 138 140 137 117 145 146

30 F 40 117 126 125 134 142 141 135 146 149 139 147 143 142

30 F 41 109 132 131 132 141 144 139 151 148 139 140 150 149

30 F 42 131 123 130 129 128 135 140 141 143 142 145 145 146

30 F 43 110 120 136 141 142 136 145 144 143 140 143 150 148

30 F 44 106 114 139 137 141 142 133 147 143 142 142 140 145

30 F 45 109 113 115 114 114 127 132 116 133 134 141 141 141

30 F 46 110 115 117 118 117 124 144 144 143 142 132 145 144

30 F 47 104 116 116 119 127 142 151 145 153 154 155 154 154

30 F 48 109 113 134 128 125 127 132 135 140 138 141 138 142

# B-1 30 F 49 109 136 131 137 138 142 138 142 143 144 145 147 141

30 F 50 100 112 119 119 135 135 133 135 135 136 130 140 140

30 F 51 105 113 120 132 130 130 130 126 102 131 132 133 130

30 F 52 107 134 133 129 139 136 143 144 145 147 144 131 150

60 F 33 104 117 126 126 132 131 134 136 128 130 140 138 145

60 F 34 119 114 127 129 128 135 124 137 132 134 138 139 142

60 F 35 93 98 102 111 111 110 117 118 124 126 126 123 127

60 F 36 101 107 116 115 133 134 133 137 122 134 137 127 139

60 F 37 114 119 128 142 142 141 137 133 144 145 146 147 153

60 F 38 99 103 125 131 132 137 133 131 142 137 112 132 125

60 F 39 98 109 123 121 128 128 120 128 129 126 131 133 133

60 F 40 109 107 110 117 115 127 133 139 142 140 138 142 144

60 F 41 107 116 137 140 145 142 149 144 156 149 157 153 144

60 F 42 102 132 137 132 142 140 134 138 150 145 149 145 151

60 F 43 104 114 130 135 134 142 141 149 147 142 144 140 147

60 F 44 101 115 130 123 121 136 140 140 145 145 145 143 145

60 F 45 102 108 122 118 120 123 129 128 134 136 133 138 136

60 F 46 102 124 126 126 140 142 134 144 147 147 142 147

60 F 47 117 139 135 141 132 143 142 146 149 135 131 154 150

60 F 48 103 116 141 138 140 137 137 137 145 146 147 155

60 F 49 87 99 106 117 120 122 102 103 106

60 F 50 99 120 122 121 127 126 118 131 112 99 121 134 138

60 F 51 98 108

60 F 52 114 138 138 141 137 144 142 147 150 150 147 153 151

125 F 33 105 113 130 129 131 133 133 136 127 137 135 143

125 F 34 112 136 136 136 130 140 142 144 148 145 148 150 150

125 F 35 104 110 132 131 131 135 137 142 136 145 142 147 148

125 F 36 101 123 120 123 125 121 132 135 134 136 136 136 136

125 F 37 100 108 107 105 112 130 120 120 123 133 132 125 137

125 F 38 116 113 131 133 142 137 148 139 142 155 146 159

125 F 39 95 101 124 120 126 119 128 130 116 123 122 122 119

125 F 40 106 129 133 140 136 141 143 148 138 129 146 139 140

125 F 41 110 116 114 131 134 133 141 131 143 139 145 139 142

125 F 42 94 105 108 123 126 129 129 131 135 133 135 137 131

125 F 43 96 102 122 131 136 133 134 136 141 144 140 142 142

125 F 44 104 123 130 127 132 132 140 132 142 142 146 136 143

125 F 45 101 112 122 137 135 138 132 143 142 145 138 142

125 F 46 108 110 127 123 128 130 126 130 131 133 133 132 133

125 F 47 105 121 125 132 132 129 122 133 112 131 135 137 140

125 F 48 109 137 138 139 137 136 138 139 140 148 148 146 146

125 F 49 114 137 146 143 134 114 146 139 139 144 145 147 149

125 F 50 97 111 131 125 133 128 142 137 133 128 137 137 142

125 F 51 105 110 123 141 140 138 143 144 124 149 149 141 151

125 F 52 103 115 135 128 135 136 136 139 141 138 140 139

250 F 33 114 131 128 138 138 131 139 143 143 143 151

250 F 34 107 127 132 143 141 146 143 152 150 153 151

250 F 35 108 128 120 134 139 138 140 133 141 140 143 146 150

250 F 36 112 130 129 134 136 132 133 250 116 143 146

# B-1 250 F 37 98 110 131 121 131 133 132 131 127 133 134

250 F 38 103 111 117 129 129 131 124 131 125 125 140

250 F 39 97 104 117 127 128 129 124 131 133 127 135

250 F 40 107 115 117 123 125 127 124 129 126 130 132

250 F 41 108 117 125 126 132 119 113 132 132 139 140 143 141

250 F 42 106 116 118 124 127 127 134 137 130 135 135

250 F 43 101 108 120 129 131 132 137 134 138 109 120 123 134

250 F 44 103 114 134 139 141 139 135 148 146 146 149

250 F 45 122 120 133 153 158 153 150 152 160 160 155 163 163

250 F 46 104 124 126 131 131 133 136 141 138 143 142 133 145

250 F 47 97 118 128 124 131 124 135 133 114 132 123 163 130

250 F 48 102 123 126 126 127 121 136 127 136 132 138 132 141

250 F 49 103 114 122 130 135 132 139 135 137 142 143 145 145

250 F 50 96 104 107 108 118 129 128 122 121 126 130 131 133

250 F 51 108 120 134 136 142 142 146 150 106 154 158

250 F 52 105 117 143 130 133 127 138 141 138 141 142 145 146

0 M 153 88 100 101 107 104 106 108 110 108 114 114 116 120

0 M 154 90 95 93 98 95 94 92 96 98 100 99 96 101

0 M 155 87 97 103 103 104 105 108 106 107 107 107 108 106

0 M 156 99 104 104 107 107 108 103 107 105 105 106 108 108

0 M 157 104 107 109 110 112 113 110 113 116 114 114 115 115

0 M 158 408 105 102 105 103 106 109 103 104 112 115 117 122

0 M 159 95 104 102 104 107 107 107 109 110 111 111 112 109

0 M 160 100 102 104 109 105 112 107 108 110 111 111 113 133

0 M 161 105 116 117 117 119 118 108 112 113 116 114 116 121

0 M 162 100 103 106 106 108 109 108 107 108 105 113 112 109

0 M 163 95 98 103 103 102 107 106 109 107 107 109 107 107

0 M 164 93 98 103 105 106 108 105 104 108 108 110 109 114

0 M 165 98 102 102 104 107 106 107 108 110 109 111 111 112

0 M 166 94 100 95 101 103 105 105 105 109 111 110 110 114

0 M 167 104 106 109 108 109 109 111 109 108 107 109 110 111

0 M 168 98 102 105 108 108 111 106 104 104 106 107 110 108

0 M 169 101 107 110 112 111 114 113 122 124 126 120 129 134

0 M 170 91 106 110 112 113 114 110 110 111 113 113 115 115

0 M 171 104 106 109 112 112 113 114 117 117 119 117 118 120

0 M 172 109 114 116 120 121 124 123 125 128 129 127 130 129

30 M 53 114 119 124 110 115 121 122 129 131 133 137 140 141

30 M 54 100 109 105 110 112 114 108 110 111 110 112 114 109

30 M 55 104 110 112 112 110 116 114 118 117 118 118 118 119

30 M 56 93 97 100 101 100 104 102 107 109 108 106 106 107

30 M 57 101 108 108 108 107 108 105 107 109 109 111 111 109

30 M 58 96 101 104 105 106 109

30 M 59 100 105 113 103 105 110 107 114 116 113 97 110 121

30 M 60 101 105 108 108 107 108 105 109 112 111 111 111 111

30 M 61 101 105 104 105 106 111 107 111 108 106 109 110 110

30 M 62 99 108 107 110 113 114 108 112 114 115 115 113 115

30 M 63 105 105 101 102 103 104 106 110 107 108 112 115 115

30 M 64 93 101 105 107 107 110 106 108 111 112 111 112 112

# B-1 30 M 65 92 98 101 105 105 108 104 106 104 105 106 107 110

30 M 66 103 108 108 109 110 112 112 115 112 112 113 115 116

30 M 67 97 103 105 108 107 109 103 103 104 106 100 104 107

30 M 68 108 118 122 125 129 129 127 130 132 131 131 130 132

30 M 69 88 90 93 93 95 97 99 100 102 101 104 106 107

30 M 70 94 98 100 99 100 99 96 100 101 102 102 106

30 M 71 99 101 101 102 104 105 104 105 110 111 110 112 113

30 M 72 102 107 107 112 115 116 115 121 120 118 121 122 121

60 M 53 102 107 110 110 112 112 110 115 117 119 117 119 119

60 M 54 102 107 106 110 112 111 110 110 114 115 118 117

60 M 55 92 99 102 103 105 105 101 104 109 109 109 108 111

60 M 56 96 100 103 101 103 107 104 106 108 108 102 104 110

60 M 57 105 111 114 115 116 116 111 113 114 111 114 117 117

60 M 58 97 103 103 106 109 110 107 109 111 113 113 115 117

60 M 59 108 110 116 118 119 118 116 116 119 119 122 123 124

60 M 60 96 98 99 101 102 104 109 109 113 113 114 111 115

60 M 61 96 103 106 110 112 108 108 108 112 112 112 113 114

60 M 62 96 102 104 108 111 110 109 109 112 112 114 111 115

60 M 63 102 112 117 119 121 118 108 100 104 101 104 106 105

60 M 64 99 104 103 101 103 106 104 106 112 110 111 109 112

60 M 65 98 101 103 106 106 109 106 110 105 108 110 110

60 M 66 100 101 105 108 111 113 111 112 114 116 116 112 114

60 M 67 96 102 103 105 107 109 107 113 117 116 119 117 122

60 M 68 105 109 109 110 109 112 111 113 117 112 116 120 119

60 M 69 101 104 104 105 109 107 108 109 109 106 140 112 114

60 M 70 98 97 101 105 109 110 106 102 113 110 112 110 115

60 M 71 107 114 109 109 111 114 112 110 115 113 114 112 117

60 M 72 87 93 95 96 97 99 99 100 99 101 101 102 102

125 M 53 98 104 101 104 105 106 101 106 109 107 109 110 112

125 M 54 103 106 109 110 113 114 112 111 115 117 119 119

125 M 55 108 113 113 114 114 115 113 117 113 118 116 119 120

125 M 56 107 114 119 117 114 115 109 110 112 112 115 115 115

125 M 57 93 98 99 101 97 100 98 98 107 109 109 108 109

125 M 58 106 108 101 108 107 111 112 117 120 120 119 121 122

125 M 59 101 93 97 99 100 99 96 98 100 102 99 101 101

125 M 60 101 101 99 98 99 110 100 105 101 103 103 106 108

125 M 61 96 96 99 99 101 103 100 101 103 104 105 105 104

125 M 62 97 99 106 100 108 109 108 113 116 119 121 122 120

125 M 63 94 99 101 102 100 98 103 104 102 106 108 108 109

125 M 64 98 104 104 107 110 113 106 108 111 110 115 111 109

125 M 65 90 90 89 87 88 89 90 93 93 94 94 93 97

125 M 66 101 99 103 103 103 105 104 105 106 107 107 108 107

125 M 67 105 109 111 112 112 112 111 113 17 115 115 109 118

125 M 68 94 109 97 100 101 103 100 98 98 112 102 104 104

125 M 69 96 101 103 104 105 107 107 106 110 111 111 113 113

125 M 70 103 103 100 99 104 105 100 102 101 103 130 104 104

125 M 71 105 104 105 106 107 106 107 107 107 110 112 114 114

125 M 72 104 111 108 109 111 111 110 109 109 112 113 115 116

# B-1 250 M 53 100 97 100 102 106 109 112 116 109 114 115 119 119

250 M 54 94 99 102 103 107 107 104 108 108 109 108 110 109

250 M 55 92 96 99 99 100 100 98 95 101 104 104 103 107

250 M 56 90 96 97 100 102 104 101 103 104 105 105 107 108

250 M 57 98 101 102 104 107 106 100 105 105 104

250 M 58 108 117 121 123 124 125 122 125 125 126 131

250 M 59 103 106 107 112 112 114 114 110 111 119 122

250 M 60 98 101 102 104 105 108 106 109 109 108 111

250 M 61 95 99 101 100 103 103 106 107 107 111

250 M 62 101 104 104 105 107 106 102 104 105 105 108

250 M 63 106 108 110 110 113 111 110 114 111 111 113 117 118

250 M 64 94 93 100 104 108 110 108 109 108 109 112 112 109

250 M 65 91 102 95 105 100 106 103 106 106 107 107 107 110

250 M 66 105 102 109 114 114 117 107 116 115 117 118 117

250 M 67 102 106 110 112 112 115 110 114 116 114 119 118 119

250 M 68 97 101 101 103 103 105 110 107 106 108 109 111 111

250 M 69 104 111 111 113 113 112 109 111 113 115 114 112 118

250 M 70 90 91 96 97 95 98 101 105 103 104 106 105 109

250 M 71 92 100 100 95 103 100 105 105 104 108 105 109 110

250 M 72 99 107 102 102 102 104 103 107 107 107 109 112 117

P generation cloaca measurements and foam

week 6

‐ 4/20/09 week 7

‐ 4/27/09

DOSE ID cg area foam cg area Foam

0 153 72 150 Y

0 154 36 100

0 155 36 108 Y

0 156 56 130 Y

0 157 154 Y 234 Y

0 158 154 Y 195 Y

0 159 25 117 Y

0 160 144 Y 224 Y

0 161 99 182 Y

0 162 99 154 Y

0 163 182 Y 208 Y

0 164 80 Y 154 Y

0 165 182 Y 195 Y

0 166 81 96 Y

0 167 195 Y 195 Y

0 168 154 Y 195 Y

0 169 154 Y 224 Y

0 170 49 110 Y

0 171 195 Y 208 Y

0 172 120 Y 108 Y

30 53 90 108 Y

30 54 25 24

30 55 25 81

30 56 117 Y 221 Y

# B-1 30 57 25 25

30 58 72 90

30 59 25 25

30 60 100 180 Y

30 61 25 25 Y

30 62 25 64

30 63 72 99 Y

30 64 49 144 Y

30 65 121 Y 168 Y

30 66 120 Y 154 Y

30 67 54 108 Y

30 68 90 130 Y

30 69 81 130 Y

30 70 140 Y 240 Y

30 71 180 Y 224 Y

30 72 72 Y 132 Y

60 53 132 195 Y

60 54 25 80

60 55 72 Y 126 Y

60 56 25 36

60 57 72 180 Y

60 58 60 120 Y

60 59 90 117 Y

60 60 144 Y 182 Y

60 61 72 165 Y

60 62 25 25

60 63 25 25

60 64 25 25

60 65 64 121

60 66 25 25

60 67 120 Y 192 Y

60 68 120 Y 180 Y

60 69 90 Y 135 Y

60 70 132 Y 121 Y

60 71 56 208 Y

60 72 25 25

125 53 168 Y 221 Y

125 54 90 182 Y

125 55 90 Y 182 Y

125 56 80 121 Y

125 57 108 Y 182 Y

125 58 154 Y 210 Y

125 59 99 Y 117 Y

125 60 90 192 Y

125 61 88 154 Y

125 62 25 25

125 63 121 Y 208 Y

125 64 72 165 Y

# B-1 125 65 180 Y 204 Y

125 66 90 Y 154 Y

125 67 25 120

125 68 63 168 Y

125 69 72 154 Y

125 70 120 Y 192 Y

125 71 120 Y 195 Y

125 72 99 Y 154 Y

250 62 120 Y 182 Y

250 55 70 132 Y

250 56 99 168 Y

250 57 180 Y 208 Y

250 58 25 36

250 59 120 Y 224 Y

250 60 180 Y 221 Y

250 61 25 99

250 66 63 121 Y

250 68 25 25

250 69 110 Y 165 Y

250 70 64 180 Y

250 71 92 165 Y

250 72 25 25

250 65 25 25

250 67 154 Y 120 Y

250 54 108 154 Y

250 53 25 120 Y

250 63 81 210 Y

250 64 90 154 Y

P generation egg strength

DOSE ID Thickness Thickness A vg.

thickness Strength

0 135 1.57 1.05 1.31 1.374

0 137 1.36 0.78 1.07 1.302

0 138 0.77 0.92 0.845 0.963

0 140 1.49 0.98 1.235 1.041

0 141 0.44 0.68 0.56 1.194

0 142 1.32 1.16 1.24 0.874

0 143 1.59 1.94 1.765 1.013

0 144 1.02 1.42 1.22 0.855

0 147 1.21 1.43 1.32 1.56

0 148 0.91 1.4 1.155 0.354

0 149 0.89 1.07 0.98 0.982

0 150 1.28 0.104 0.692 1.324

0 135 0.76 0.37 0.565 0.853

0 138 0.96 0.42 0.69 0.506

0 140 0.75 0.59 0.67 0.879

0 142 0.79 0.43 0.61 0.819

0 143 0.47 0.37 0.42 0.454

# B-1 0 144 0.98 0.53 0.755 1.107

0 147 1.43 1.06 1.245 1.122

0 149 0.39 0.57 0.48 0.57

0 150 0.63 0.85 0.74 0.44

0 141 1.16 0.73 0.875

30 33 0.68 1.07 0.875 0.864

30 34 1.23 1.1 1.165 1.248

30 37 1.16 1.19 1.175 0.367

30 38 1.22 1.35 1.285 1.365

30 41 2.03 1.59 1.81 1.94

30 43 1.37 1.15 1.26 1.302

30 49 1.56 1.22 1.39 1.192

30 51 1.17 1.18 1.175 0.325

30 33 0.85 0.91 0.88 1.399

30 44 0.58 0.96 0.77 0.908

30 34 0.38 0.38 0.38 1.36

30 35 0.46 0.77 0.615 1.246

30 36 1.42 1.66 1.54 1.475

30 38 0.47 0.38 0.425 0.778

30 40 0.78 0.95 0.865 1.361

30 41 0.37 0.41 0.39 1.446

30 42 1.16 1.01 1.085 0.433

30 43 0.47 0.5 0.485 0.85

30 44 0.92 0.67 0.795 1.131

30 45 0.84 0.63 0.735 0.691

30 46 0.7 0.48 0.59 1.015

30 47 0.38 0.51 0.445 1.032

30 48 0.42 0.45 0.435 0.709

30 49 0.73 0.81 0.77 1.037

30 50 1.17 0.75 0.96 1.109

30 51 0.36 0.38 0.37 1.005

30 52 0.5 0.32 0.41 1.031

30 35 1.22 0.84 1.41

30 36 0.7 1.39 1.637

30 37 0.65 0.55 0.725

30 39 1.08 0.84 0.537

30 42 0.84 0.7 0.509

30 46 0.89 0.71 0.845

30 47 2.54 0.45 1.245

30 48 1.01 1.02 0.955

30 50 1.11 0.96 1.22

30 52 1.39 0.8 1.227

60 33 2.95 2.87 2.91 1.435

60 35 0.94 1.29 1.115 0.671

60 37 1.36 1.42 1.39 1.295

60 42 1.6 1.5 1.55 1.256

60 43 4.46 3.6 4.03 1.484

60 46 1.48 0.76 1.12 1.198

# B-1 60 52 0.3 0.54 0.42 0.938

60 50 1.51 0.95 1.23 0.687

60 33 0.47 0.43 0.45 1.119

60 36 0.3 0.35 0.325 0.823

60 37 0.77 0.76 0.765 0.813

60 38 0.42 0.32 0.37 1.193

60 39 0.34 0.29 0.315 1.179

60 41 0.51 0.42 0.465 0.974

60 42 1.05 0.99 1.02 1.627

60 43 0.84 0.72 0.78 1.292

60 44 0.36 0.27 0.315 1.62

60 45 0.37 0.38 0.375 0.9

60 47 1.2 0.78 0.99 0.931

60 48 1.24 0.98 1.11 0.957

60 50 0.79 0.46 0.625 1.101

60 35 0.91 0.52 0.803

60 36 1.1 0.91 0.605

60 40 0.67 0.76 0.706

60 41 0.42 0.46 1.104

60 44 1.42 1.01 1.179

60 45 0.88 0.84 0.888

60 48 1.12 0.79 0.729

60 52 1.12 0.61 0.969

125 33 0.51 0.52 0.515 1.016

125 35 1.36 0.67 1.015 1.026

125 36 1.01 0.42 0.715 1.319

125 39 1.29 1.05 1.17 1.216

125 40 0.45 0.37 0.41 1.315

125 41 0.71 1.17 0.94 1.27

125 42 1.07 1.4 1.235 0.383

125 44 0.39 0.95 0.67 1.026

125 46 0.52 0.86 0.69 1.54

125 47 0.63 0.96 0.795 1.309

125 48 0.4 1.45 0.925 1.388

125 49 0.69 0.8 0.745 1.53

125 50 0.94 0.67 0.805 1.239

125 51 1.25 1.08 1.165 1.644

125 52 1.44 1.24 1.34 1.57

125 33 0.8 1.1 0.95 1.148

125 39 0.66 1.05 0.855 1.337

125 40 1.31 1.53 1.42 0.709

125 41 0.86 0.47 0.665 1.604

125 43 1.3 1.67 1.485 1.314

125 45 1.15 1.06 1.105 1.261

125 52 0.42 0.4 0.41 1.3

125 34 0.42 0.33 0.375 1.051

125 35 0.71 0.59 0.65 0.341

125 36 0.91 0.44 0.675 1.258

# B-1 125 37 0.61 1.03 0.82 1.375

125 38 1.32 1.16 1.24 1.455

125 39 0.61 0.55 0.58 1.17

125 42 0.43 0.39 0.41 1.3

125 43 0.87 0.67 0.77 0.823

125 44 1.14 0.81 0.975 1.11

125 45 0.48 0.28 0.38 1.233

125 46 0.66 1.03 0.845 1.41

125 48 0.68 0.39 0.535 1.143

125 49 0.56 0.86 0.71 1.256

125 50 0.88 1.41 1.145 0.914

125 51 0.39 0.37 0.38 1.2

125 34 1.09 1.47 0.824

125 35 0.73 0.43 0.906

125 38 1.68 1.22 1.275

250 33 1.88 0.97 1.425 1.418

250 34 1.77 1.01 1.39 1.566

250 36 0.34 1.05 0.695 1.198

250 37 0.75 0.45 0.6 0.773

250 38 0.93 1.25 1.09 1.009

250 43 0.42 0.95 0.685 1.308

250 44 1.07 0.53 0.8 1.012

250 45 0.78 0.79 0.785 1.6

250 46 1.04 0.67 0.855 1.487

250 47 1.71 1.77 1.74

250 48 1.45 0.87 1.16 1.13

250 49 0.56 0.82 0.69 1.113

250 52 1.11 1.17 1.14 1.356

250 33 1.25 1.81 1.53 0.824

250 35 0.36 1 0.68 0.802

250 39 0.63 0.61 0.62 1.23

250 41 1.07 0.71 0.89 1.739

250 43 1.55 1.27 1.41 0.919

250 44 1.25 1.16 1.205 0.837

250 46 0.77 0.67 0.72 1.475

250 52 0.63 0.69 0.66 1.136

250 35 0.36 0.41 0.385 0.848

250 37 0.38 0.37 0.375 0.514

250 45 1 0.92 0.96 1.49

250 49 0.57 0.46 0.515 0.889

250 50 0.45 0.31 0.38 0.688

250 51 0.63 0.51 0.57 0.471

250 52 0.67 0.45 0.56 1.457

250 38 0.83 0.8 0.571

250 42 0.89 0.56 1.21

P generation yolk content

ug/g yolk

# B-1 DOSE ID total endosulfans I II

0 140A 0 0.0128 0.0017

0 150A 0 0.0113 0.0014

0 141A 0.0007 0.0123 0.0023

30 36A 0.0813 0.0227 0.08

30 49A 0.1528 0.0285 0.1449

30 50A 0.1623 0.0371 0.1505

30 42A 0.0847 0.0177 0.0865

60 52A 0.3268 0.0465 0.316

60 36A 0.3929 0.0695 0.3615

60 47A 0.4758 0.0741 0.448

120 50A 0.6566 0.0856 0.6392

120 42A 0.5114 0.0638 0.5012

120 52A 0.5573 0.0947 0.5143

120 45A 1.2275 0.1587 1.1989

120 48A 0.8998 0.1147 0.8839

250 36A 1.8063 0.2385 1.7664

250 52A 1.4989 0.1734 1.4914

250 42A 1.3745 0.2301 1.2737

250 37A 2.3351 0.3186 2.3012

250 50A 1.2628 0.1304 1.3126

P generation sperm measurements

DOSE ID sperm/gram % motile % progressive

0 164 7735.85 97.8 16.5

0 163 8797.65 94.5 17.5

0 156 8223.95 95.5 18.5

0 157 5467 87 11.5

0 155 7047.45 89.5 12.5

0 170 6363.6 95 29.5

0 169 6361.6 89.5 18

0 171 7945.37 93 11.33

0 154 5994.45 97 21

0 153 5240.5 96.5 19.5

30 56 3236.47 87 20.67

30 66 8328.07 95 24.33

30 71 5467 84.5 13.5

30 67 1744.23 22 2

30 64 5646.4 92 17

30 59 4725.9 94.3 20.7

30 63 4659.6 92.3 21.7

30 65 4325 93.7 16.3

30 68 9375.9 97.5 19.5

60 65 4943.7 93.5 22

60 64 6478.4 92 19.5

60 68 1827.93 93.67 18

60 67 4045.6 84.33 11

60 63 6666.43 89 13

# B-1 60 66 9476.13 134.5 21

60 72 8245.03 96.67 17

60 70 4214.53 75 14

60 71 6421.9 97 20.67

60 69 3977.9 87.67 11.33

125 69 5904.35 95 15

125 67 5259.85 98.5 33

125 65 4191.35 91 16.5

125 70 6747.8 94.5 19.5

125 71 4346.25 86.5 17

125 63 5432.85 96 19

125 66 2502.65 67.5 10.5

125 72 1858.8 85.5 26

125 68 1664.4 67 6.5

250 69 5600.65 97 26.5

250 72 4764.1 89 14.5

250 71 6463.95 95.5 18

250 61 6542.2 96 20

250 60 3549.95 96 18.5

250 65 2763.35 70.5 9

250 62 6411.3 92 18.5

250 56 5426 85.5 13

250 70 4668.8 74 12

# B-1P generation feed consumption4/21week 1consumption4/21 4/28week 2consumption4/28 5/5week 3consumption5/5 5/12week 4consumption5/12 5/19week 5consumption5/19 5/26week 6consumptionDOSE SEX IDinitial final Initial final initial finalinitial final initial final initial final0 F 144 419 315 104 430 312 118 455 332 123 444 335 109 449 320 129 463 292 1710 F 142 415 305 110 427 296 131 425 312 113 447 331 116 439 258 181 454 249 2050 F 140 419 282 137 413 259 154 454 284 170 459 266 193 454 259 195 492 244 2480 F 139 424 324 100 425 320 105 454 341 113 448 338 110 454 342 112 455 303 1520 F 141 382 271 111 422 297 125 450 333 117 446 258 188 443 255 188 465 244 2210 F 143 426 310 116 418 312 106 460 311 149 431 237 194 465 267 198 482 255 2270 F 150 418 320 98 423 315 108 457 327 130 440 315 125 435 247 188 447 249 1980 F 149 406 285 121 422 327 95 462 310 152 440 257 183 450 450 496 269 2270 F 148 402 304 98 419 309 110 456 331 125 436 294 142 450 289 161 446 261 1850 F 147 402 296 106 416 285 131 454 261 193 439 269 170 483 282 201 495 240 2550 F 146 409 278 131 426 312 114 461 355 106 453 370 83 459 386 73 444 353 910 F 145 395 242 153 425 307 118 460 307 153 433 245 188 433 243 190 453 261 1920 F 137 406 303 103 415 315 100 458 322 136 429 253 176 469 278 191 444 254 1900 F 135 388 268 120 416 270 146 436 246 190 443 246 197 441 236 205 506 248 2580 F 133 410 308 102 431 303 128 439 322 117 438 260 178 444 328 116 451 277 1740 F 134 396 259 137 424 321 103 4460 F 136 404 337 67 427 277 150 443 286 157 427 267 160 436 269 167 455 271 1840 F 138 421 309 112 425 327 98 458 321 137 432 272 160 445 273 172 459 240 2190 F 151 416 304 112 418 337 81 445 330 115 441 330 111 446 4460 F 152 426 302 124 424 348 76 45730 F 49 396 288 108 437 307 130 445 279 166 435 268 167 476 269 207 477 259 21830 F 50 393 280 113 449 293 156 460 315 145 439 306 133 442 295 147 439 241 19830 F 51 402 261 141 438 310 128 440 334 106 478 287 191 467 264 203 438 234 20430 F 52 430 299 131 433 273 160 438 253 185 439 260 179 464 276 188 450 257 19330 F 33 402 281 121 441 307 134 441 282 159 449 296 153 442 290 152 487 254 23330 F 34 404 282 122 433 306 127 443 303 140 430 272 158 425 281 144 436 278 15830 F 35 435 299 136 437 315 122 443 344 99 436 353 83 450 375 75 445 327 11830 F 36 403 267 136 437 308 129 457 271 186 448 315 133 450 305 145 448 253 19530 F 37 418 273 145 435 328 107 451 356 95 432 366 66 444 357 87 436 326 11030 F 38 409 247 162 444 282 162 456 249 207 433 251 182 425 233 192 475 258 21730 F 39 414 261 153 430 266 164 453 254 199 432 247 185 461 239 222 639 232 40730 F 40 406 272 134 436 278 158 463 337 126 431 249 182 450 269 181 454 269 18530 F 41 422 291 131 437 295 142 448 258 190 433 254 179 429 236 193 599 272 32730 F 42 404 272 132 433 283 150 454 266 188 445 269 176 427 248 179 467 280 18730 F 43 415 286 129 437 310 127 452 320 132 431 264 167 447 236 211 465 250 21530 F 44 427 309 118 442 311 131 455 295 160 463 286 177 421 242 179 471 255 21630 F 45 401 262 139 437 331 106 455 341 114 441 350 91 437 349 88 444 329 11530 F 46 413 267 146 439 325 114 452 335 117 432 349 83 443 341 102 444 327 11730 F 47 421 316 105 440 305 135 455 355 100 440 358 82 446 347 99 449 322 12730 F 48 411 274 137 439 326 113 447 339 108 435 282 153 415 273 142 474 264 21060 F 33 404 281 123 436 319 117 444 441 303 138 452 278 17460 F 34 391 238 153 439 257 182 443 248 195 430 286 144 437 267 170 491 277 21460 F 35 396 302 94 438 324 114 442 358 84 440 354 86 448 327 121 440 293 14760 F 36 396 290 106 430 320 110 436 312 124 439 321 118 440 329 111 450 287 16360 F 37 407 260 147 433 311 122 443 309 134 438 265 173 474 263 211 493 246 24760 F 38 390 281 109 429 330 99 444 312 132 429 289 140 419 278 141 452 212 240B-160 F 39 401 292 109 437 314 123 440 268 172 433 273 160 428 274 154 453 260 19360 F 40 407 284 123 427 346 81 452 268 184 441 344 97 450 350 100 442 330 11260 F 47 414 266 148 440 303 137 445 273 172 426 254 172 408 261 147 440 234 20660 F 48 445 324 121 434 306 128 445 308 137 428 258 170 417 262 155 474 244 23060 F 49 406 322 84 436 340 96 447 338 109 437 314 123 423 317 106 455 341 11460 F 50 429 307 122 435 321 114 443 292 151 436 288 148 425 259 166 438 266 17260 F 51 420 321 99 435 341 9460 F 52 414 297 117 435 276 159 439 254 185 437 254 183 418 231 187 440 234 20660 F 41 393 269 124 428 302 126 452 269 183 431 267 164 419 236 183 476 271 20560 F 42 423 316 107 434 290 144 441 251 190 448 267 181 418 238 180 483 251 23260 F 43 395 280 115 438 323 115 450 284 166 440 265 175 413 239 174 484 262 22260 F 44 405 294 111 435 322 113 440 283 157 435 266 169 430 279 151 446 248 19860 F 45 411 301 110 436 328 108 447 321 126 422 285 137 428 325 103 478 283 19560 F 46 426 296 130 437 282 155 430 254 176 470 238 232 481 247 234 548 284 264125 F 38 417 266 151 431 323 108 434 265 169 488 269 219 473 235 238 463 244 219125 F 39 431 333 98 431 322 109 430 306 124 434 282 152 439 295 144 423 266 157125 F 37 421 283 138 427 207 220 423 292 131 446 278 168 430 295 135 449 274 175125 F 36 411 284 127 430 241 189 430 251 179 481 262 219 475 247 228 465 271 194125 F 35 391 282 109 430 302 128 434 259 175 442 243 199 432 236 196 476 256 220125 F 34 407 285 122 435 286 149 439 274 165 437 243 194 466 272 194 437 241 196125 F 33 410 286 124 429 304 125 425 307 118 404 257 147 439 279 160 431 264 167125 F 44 407 290 117 437 276 161 431 298 133 474 251 223 516 249 267 419 231 188125 F 43 410 309 101 437 325 112 438 293 145 487 267 220 419 470 266 204125 F 42 411 311 100 430 313 117 444 342 102 442 309 133 430 296 134 448 293 155125 F 41 405 247 158 423 278 145 443 350 93 431 266 165 467 252 215 466 255 211125 F 40 410 296 114 435 281 154 420 252 168 431 248 183 431 236 195 456 278 178125 F 45 426 312 114 435 276 159 436 273 163 435 255 180 426 250 176 421 243 178125 F 46 417 289 128 430 324 106 446 348 98 434 279 155 417 232 185 457 265 192125 F 47 412 304 108 431 309 122 454 305 149 444 265 179 422 247 175 462 274 188125 F 48 425 294 131 441 289 152 437 253 184 480 248 232 420 234 186 460 267 193125 F 49 407 270 137 432 295 137 450 285 165 436 243 193 427 241 186 414 286 128125 F 50 411 301 110 437 294 143 436 286 150 440 244 196 460 251 209 444 244 200125 F 52 410 231 179 434 264 170 437 254 183 433 247 186 429 241 188 459 259 200125 F 51 401 305 96 432 316 116 439 300 139 442 245 197 467 245 222 462 261 201250 F 43 393 272 121 429 312 117 424 264 160 471 439 263 176 419 235 184250 F 44 398 283 115 428 280 148 429 281 148 434 251 183 433 269 164 445 251 194250 F 45 402 273 129 433 316 117 429 333 96 440 292 148 432 294 138 430 265 165250 F 46 410 262 148 436 239 197 432 232 200 439 253 186 433 239 194 466 242 224250 F 47 405 303 102 438 309 129 442 295 147 436 268 168 440 420 252 168250 F 48 401 246 155 430 281 149 430 302 128 442 253 189 438 259 179 451 258 193250 F 49 405 268 137 428 241 187 436 243 193 474 244 230 473 250 223 447 243 204250 F 50 402 291 111 445 330 115 447 356 91 442 355 87 448 360 88 431 323 108250 F 51 401 274 127 424 283 141 421 240 181 472 261 211 437 234 203 495 236 259250 F 52 393 252 141 435 303 132 433 239 194 472 244 228 440 240 200 449 252 197250 F 36 403 278 125 419 248 171 423 254 169 437 243 194 455 249 206 460 241 219250 F 35 408 290 118 421 289 132 427 302 125 448 274 174 414 242 172 450 253 197250 F 34 398 270 128 423 257 166 420 258 162 479 243 236 457 231 226 475 261 214250 F 33 397 278 119 427 267 160 431 278 153 446 267 179 441 261 180 453 267 186250 F 37 399 271 128 430 300 130 424 317 107 434 287 147 442 303 139 427 250 177250 F 38 407 295 112 446 300 146 421 295 126 429 285 144 424 246 178 424 248 176250 F 39 401 256 145 435 245 190 427 235 192 441 267 174 434 246 188 459 256 203250 F 40 404 295 109 431 302 129 431 284 147 446 300 146 430 304 126 427 300 127B-1250 F 41 398 271 127 429 307 122 429 343 86 448 305 143 436 267 169 433 304 129250 F 42 408 298 110 438 304 134 426 275 151 433 246 187 429 245 184 453 244 2090 M 158 419 297 122 431 351 80 462 351 111 437 329 108 446 335 111 455 324 1310 M 157 417 290 127 431 281 150 451 272 179 438 281 157 434 281 153 451 253 1980 M 156 412 307 105 432 305 127 456 263 193 437 286 151 442 319 123 452 277 1750 M 155 424 343 81 424 313 111 449 322 127 438 312 126 443 323 120 459 316 1430 M 154 426 323 103 416 310 106 457 353 104 442 331 111 440 330 110 449 341 1080 M 153 419 335 84 430 304 126 450 336 114 434 312 122 440 329 111 450 332 1180 M 164 427 328 99 423 311 112 463 344 119 436 324 112 445 339 106 454 344 1100 M 163 431 337 94 427 329 98 467 365 102 435 339 96 441 336 105 449 338 1110 M 162 407 279 128 424 293 131 452 308 144 444 311 133 435 300 135 462 317 1450 M 161 420 260 160 435 234 201 455 261 194 441 295 146 442 275 167 450 255 1950 M 160 414 313 101 432 322 110 454 342 112 430 314 116 442 333 109 442 322 1200 M 159 410 301 109 423 294 129 454 330 124 434 305 129 445 304 141 447 306 1410 M 165 409 302 107 418 325 93 456 351 105 432 337 95 453 339 114 445 340 1050 M 166 428 316 112 428 312 116 434 328 106 434 437 328 109 447 326 1210 M 167 399 279 120 428 286 142 456 275 181 430 304 126 450 334 116 452 298 1540 M 168 412 315 97 437 340 97 459 351 108 447 341 106 432 328 104 457 346 1110 M 169 426 320 106 437 303 134 449 329 120 430 325 105 437 325 112 449 317 1320 M 170 399 314 85 437 306 131 446 295 151 438 269 169 429 273 156 444 285 1590 M 171 414 315 99 435 319 116 458 332 126 436 323 113 452 344 108 451 338 1130 M 172 421 312 109 440 326 114 440 326 114 427 309 118 452 335 117 448 301 14730 M 63 395 267 128 437 264 173 418 309 109 432 279 153 429 300 129 437 296 14130 M 64 408 310 98 444 332 112 455 341 114 437 332 105 443 350 93 438 319 11930 M 65 415 314 101 434 326 108 447 326 121 435 320 115 444 346 98 448 325 12330 M 66 408 290 118 441 335 106 453 341 112 444 333 111 458 349 109 446 333 11330 M 67 405 282 123 442 332 110 448 351 97 430 344 86 439 365 74 441 347 9430 M 68 424 312 112 438 303 135 450 308 142 432 300 132 413 299 114 437 305 13230 M 72 409 275 134 440 311 129 444 291 153 438 306 132 456 305 151 455 287 16830 M 71 417 309 108 444 335 109 452 305 147 434 330 104 457 307 150 435 307 12830 M 70 409 303 106 445 343 102 446 329 117 426 351 75 459 366 93 450 344 10630 M 69 401 308 93 442 345 97 446 331 115 402 324 78 449 442 7 358 338 2030 M 53 417 305 112 433 317 116 450 315 135 435 351 84 450 339 111 450 311 13930 M 54 405 307 98 440 337 103 452 362 90 437 330 107 453 352 101 442 333 10930 M 55 422 309 113 442 313 129 446 317 129 433 314 119 448 337 111 434 309 12530 M 56 410 304 106 439 335 104 449 329 120 441 341 100 431 335 96 445 330 11530 M 57 420 311 109 439 310 129 441 328 113 436 329 107 432 307 125 441 325 11630 M 58 420 311 109 444 326 118 447 318 129 438 314 124 436 313 123 446 307 13930 M 59 418 301 117 437 327 110 446 312 134 440 362 78 447 357 90 446 332 11430 M 60 411 309 102 441 321 120 448 322 126 432 320 112 440 324 116 447 328 11930 M 61 413 323 90 451 349 102 452 354 98 433 337 96 450 452 344 10830 M 62 406 293 113 439 306 133 440 302 138 434 305 129 432 294 138 442 304 13860 M 54 418 314 104 439 315 124 441 305 136 431 292 139 443 302 141 441 287 15460 M 53 410 293 117 433 312 121 448 311 137 430 309 121 440 330 110 443 332 11160 M 72 419 331 88 430 336 94 440 339 101 430 324 106 447 338 109 445 323 12260 M 71 411 265 146 430 310 120 432 351 81 431 353 78 429 321 108 448 333 11560 M 70 408 294 114 436 344 92 455 321 134 453 313 140 441 322 119 445 315 13060 M 69 411 303 108 434 333 101 427 321 106 431 310 121 419 315 104 453 325 12860 M 68 415 295 120 434 314 120 432 330 102 434 326 108 441 319 122 439 314 12560 M 67 415 304 111 433 311 122 435 313 122 445 323 122 443 333 110 436 321 11560 M 55 415 291 124 434 314 120 438 322 116 436 329 107 444 275 169 447 320 12760 M 56 414 309 105 437 337 100 449 334 115 429 339 90 450 352 98 445 335 110B-160 M 57 403 284 119 436 319 117 442 312 130 435 324 111 445 346 99 443 330 11360 M 58 398 288 110 429 312 117 452 335 117 432 319 113 435 329 106 449 346 10360 M 59 408 291 117 440 325 115 454 301 153 442 307 135 435 320 115 441 313 12860 M 60 400 296 104 440 321 119 447 310 137 431 290 141 438 294 144 444 309 13560 M 61 410 310 100 436 323 113 444 318 126 438 326 112 449 337 112 440 328 11260 M 62 416 310 106 432 331 101 448 333 115 441 343 98 445 352 93 446 352 9460 M 63 417 289 128 427 289 138 450 330 120 434 337 97 441 362 79 449 369 8060 M 64 432 303 129 432 306 126 437 313 124 432 307 125 435 292 143 441 314 12760 M 65 410 299 111 427 319 108 452 319 133 433 315 118 441 324 117 442 319 12360 M 66 406 297 109 430 334 96 447 326 121 433 297 136 438 315 123 443 319 124125 M 58 411 288 123 436 328 108 445 359 86 438 322 116 435 327 108 441 316 125125 M 57 408 308 100 444 319 125 448 448 432 336 96 436 330 106125 M 56 415 285 130 438 306 132 441 441 444 354 90 454 340 114125 M 55 414 294 120 434 319 115 451 451 444 343 101 421 316 105125 M 54 409 301 108 444 335 109 430 430 432 318 114 429 271 158125 M 53 408 299 109 439 335 104 436 436 443 345 98 438 335 103125 M 59 410 280 130 434 370 64 452 311 141 440 303 137 436 325 111 423 310 113125 M 60 408 294 114 430 320 110 434 317 117 452 331 121 448 331 117 435 316 119125 M 61 433 329 104 447 332 115 440 320 120 445 320 125 439 322 117 425 312 113125 M 62 409 299 110 439 318 121 434 309 125 435 336 99 429 313 116 435 323 112125 M 63 400 300 100 439 318 121 438 307 131 454 328 126 435 336 99 433 324 109125 M 64 420 326 94 438 325 113 430 330 100 437 322 115 431 322 109 430 329 101125 M 69 406 303 103 445 306 139 432 293 139 441 273 168 426 266 160 418 262 156125 M 70 401 271 130 434 358 76 436 345 91 443 339 104 432 324 108 437 321 116125 M 71 399 269 130 424 296 128 436 436 423 324 99 427 320 107125 M 72 400 270 130 435 302 133 432 339 93 440 330 110 443 321 122 427 303 124125 M 68 406 298 108 443 345 98 439 321 118 437 305 132 427 308 119 428 296 132125 M 66 405 299 106 435 337 98 428 331 97 435 341 94 437 338 99 428 329 99125 M 67 395 287 108 435 314 121 422 299 123 442 330 112 438 339 99 431 327 104125 M 65 401 300 101 426 337 89 420 332 88 440 339 101 442 331 111 445 333 112250 M 57 405 310 95 439 337 102 445 322 123 431 323 108 443 331 112 442 323 119250 M 56 399 292 107 438 317 121 422 276 146 438 308 130 450 305 145 449 294 155250 M 55 398 293 105 441 340 101 445 326 119 439 324 115 435 325 110 426 310 116250 M 62 403 290 113 427 313 114 422 300 122 442 303 139 433 309 124 419 292 127250 M 58 403 297 106 436 310 126 420 284 136 432 302 130 447 313 134 438 301 137250 M 59 439 306 133 433 290 143 422 290 132 441 305 136 442 328 114 449 315 134250 M 60 397 298 99 426 325 101 429 316 113 440 315 125 437 312 125 430 303 127250 M 61 395 281 114 428 309 119 429 293 136 439 309 130 442 306 136 424 283 141250 M 66 409 300 109 442 339 103 434 317 117 437 319 118 434 330 104 425 320 105250 M 68 409 294 115 445 335 110 431 301 130 432 306 126 442 322 120 447 326 121250 M 71 426 319 107 438 322 116 434 326 108 439 336 103 450 346 104 455 337 118250 M 72 411 298 113 438 303 135 440 353 87 446 352 94 443 339 104 449 325 124250 M 70 403 307 96 444 355 89 443 340 103 437 341 96 432 349 83 440 336 104250 M 69 409 297 112 438 307 131 441 298 143 440 302 138 439 312 127 437 319 118250 M 65 392 298 94 440 327 113 423 341 82 444 344 100 434 344 90 431 319 112250 M 67 406 305 101 442 337 105 439 332 107 434 435 324 111 419 306 113250 M 54 404 304 100 440 327 113 421 307 114 437 316 121 441 325 116 434 322 112250 M 53 403 296 107 424 343 81 404 339 65 434 328 106 434 333 101 438 325 113250 M 63 400 282 118 428 314 114 421 299 122 441 434 302 132 421 295 126250 M 64 409 321 88 444 353 91 431 338 93 447 340 107 433 336 97 426 327 99P generation feed consumption – pairedB-15/29 6/2 6/5 6/9 6/12 6/16 6/19 6/22 6/26 6/30 7/3 7/7 7/10 7/14 7/17DoseMaleFemaleInitialInitialFinalInitialFinalInitial FinalInitialFinalInitialFinalInitialFinalInitial FinalInitialFinalInitial FinalInitialFinalInitialFinalInitialFinalInitialFinalInitial Final0 136 414 317 416 350 421 315 425 337 426 327 435 354 420 321 432 347 425 332 430 351 429 333 422 344 429 326 431 365 4440 143 405 257 414 347 417 284 411 326 406 311 422 310 420 270 420 312 384 260 414 332 416 252 434 355 402 264 405 334 4300 153 417 335 436 381 425 355 433 376 430 361 431 386 412 335 417 352 402 337 438 390 409 245 438 384 384 318 421 380 4460 154 139 410 272 434 300 416 251 397 269 407 258 423 309 425 278 421 307 405 258 420 267 417 266 403 276 405 247 417 302 4400 155 141 405 236 418 270 422 255 402 259 416 256 427 260 428 241 420 292 403 263 422 281 418 253 417 306 410 260 434 343 4430 156 135 413 231 420 271 420 239 397 244 416 237 415 252 428 241 406 245 421 230 422 260 396 270 431 336 418 270 433 290 4370 157 142 421 418 270 399 246 410 311 425 240 414 270 404 258 416 307 417 242 419 268 423 254 417 288 421 249 421 300 4440 158 144 418 254 409 291 413 266 399 299 424 284 412 302 418 288 416 346 415 337 436 367 425 351 422 356 410 342 422 365 4300 164 145 415 268 427 301 417 241 395 246 427 245 428 307 423 244 425 270 412 254 415 291 405 246 429 260 413 234 420 275 4380 163 146 424 311 433 339 401 278 422 308 423 271 425 314 433 272 432 329 409 274 410 290 408 255 414 286 410 250 428 330 4360 162 140 413 255 422 280 408 236 401 241 418 247 410 278 416 251 404 264 411 243 403 235 415 254 433 261 409 244 417 299 4370 161 148 412 234 419 271 404 252 404 233 414 229 410 229 424 232 427 231 410 242 419 263 397 233 422 292 419 242 433 325 4400 160 149 411 269 424 297 417 264 400 273 410 254 409 294 423 258 409 306 409 255 416 276 409 264 413 284 417 265 423 301 4350 159 150 420 249 421 276 424 258 401 257 421 239 409 253 423 242 431 252 406 251 410 267 413 254 419 272 406 256 425 288 4290 165 137 421 315 431 334 435 284 406 286 413 253 429 307 448 280 413 290 418 260 418 268 405 255 422 290 419 257 438 303 4360 166 412 347 426 377 436 368 420 367 420 361 434 390 441 375 426 385 415 363 428 385 416 368 438 391 391 337 428 385 4270 167 133 415 252 415 305 425 238 407 241 421 243 424 326 430 257 420 315 413 259 410 237 402 248 407 258 403 243 429 323 4480 168 147 413 241 409 268 417 264 403 252 413 242 425 228 429 256 429 268 422 414 249 410 244 423 248 415 227 417 321 4450 169 412 333 418 349 434 344 411 339 418 344 430 369 439 350 420 363 413 356 433 361 411 342 418 350 377 315 435 366 4440 170 421 342 414 365 425 344 417 359 416 277 425 379 436 346 419 366 421 342 423 374 413 328 436 362 389 308 438 390 4460 171 426 358 425 375 432 365 431 377 417 358 429 383 437 371 413 366 419 366 437 388 407 352 436 389 389 335 432 386 4550 172 415 322 424 342 427 335 420 338 426 330 429 352 439 325 428 334 423 315 419 337 427 334 436 345 411 326 432 348 4410 401 236 422 330 420 236 405 241 421 247 416 327 410 247 406 349 425 365 417 338 401 277 415 320 419 287 420 349 42630 36 415 295 427 340 431 300 437 319 437 302 446 339 455 318 434 331 429 311 441 336 410 315 429 354 346 249 436 268 45630 48 426 313 427 337 453 331 426 337 440 325 431 355 452 330 411 326 421 310 416 330 408 299 417 313 417 318 433 243 46030 33 415 317 424 347 446 322 420 311 431 316 419 337 450 323 427 334 422 304 425 340 411 299 430 315 409 252 441 233 45730 52 435 333 434 361 448 327 454 365 434 321 432 345 449 326 422 337 408 316 444 364 425 326 436 347 422 312 437 271 45030 42 418 309 428 357 444 331 436 268 422 342 456 334 436 306 428 312 432 327 403 290 402 323 433 271 426 340 45230 68 423 356 407 346 445 361 446 337 433 360 437 379 460 388 432 375 434 368 449 398 398 341 423 373 404 335 434 381 44630 67 44 425 277 434 316 441 274 418 279 432 249 424 318 454 247 441 312 427 241 431 267 380 243 423 270 413 238 442 301 44930 66 46 431 285 426 286 436 252 420 276 437 256 421 292 446 246 439 273 426 252 436 270 411 252 412 243 413 234 433 264 45430 65 45 434 298 422 295 440 276 417 299 430 250 441 301 444 266 436 317 433 268 436 285 417 259 435 298 419 236 442 311 45030 64 47 429 280 428 300 436 273 417 285 436 260 423 293 447 260 417 292 414 243 419 279 424 261 417 269 420 240 439 318 46330 63 43 435 257 429 274 440 239 412 261 441 263 434 305 440 258 416 301 428 254 434 288 397 239 435 303 432 240 442 334 45730 53 417 333 430 359 441 348 428 362 437 362 447 386 433 353 434 368 420 341 439 371 389 333 413 358 441 367 431 381 46530 54 35 432 304 422 327 438 271 423 309 438 294 442 339 424 251 450 310 415 247 439 321 405 236 402 269 405 235 430 361 45730 55 34 433 279 420 308 431 245 424 291 430 276 427 313 449 247 415 289 424 253 446 324 431 267 428 292 399 261 433 312 44930 56 427 365 432 379 436 365 430 377 445 375 432 391 457 377 435 380 437 373 448 390 424 365 423 375 401 335 435 384 45030 57 37 432 301 425 314 436 272 435 313 428 251 440 324 451 258 428 307 414 232 437 325 426 261 421 282 409 250 436 314 44330 38 423 316 421 338 435 321 443 357 440 319 432 334 457 302 426 318 424 282 440 331 400 275 425 326 429 301 438 333 44430 59 39 433 234 430 245 456 241 404 228 433 231 452 291 448 231 419 231 427 261 426 362 423 259 425 346 417 317 441 389 46030 60 40 431 270 433 293 431 262 425 285 429 258 428 299 439 237 413 273 420 265 436 292 397 260 415 272 430 232 427 301 43630 61 41 428 231 436 247 457 265 416 231 440 250 441 299 447 248 412 261 429 229 443 262 420 239 434 260 410 245 419 261 45230 62 431 342 418 355 444 346 445 365 434 342 428 365 454 357 432 367 428 344 449 373 412 353 411 348 438 338 447 373 45530 72 49 429 244 452 252 445 239 420 231 435 243 417 257 436 245 437 254 434 242 403 232 416 237 431 236 439 231 439 275 45530 71 50 428 257 431 285 433 244 412 241 428 243 437 274 436 232 438 230 432 248 419 251 392 244 435 264 407 233 443 280 44630 70 51 439 277 426 264 453 268 416 279 428 243 423 291 426 244 434 283 421 241 409 266 403 241 420 282 417 258 452 323 45130 69 442 378 430 378 452 375 433 378 424 363 442 393 455 377 438 380 429 366 452 389 441 356 430 375 439 358 458 390 45160 34 425 331 436 353 446 237 422 327 431 338 409 351 462 253 378 296 417 227 419 336 432 259 405 347 434 239 422 343 45960 45 422 314 428 340 439 231 417 271 429 261 436 314 459 292 430 328 429 254 417 298 431 236 427 282 413 255 425 294 458B-160 54 49 440 346 439 313 433 294 435 313 433 298 441 360 462 336 424 334 423 333 453 366 366 311 437 355 433 328 450 379 46160 53 33 434 278 428 295 428 260 412 279 432 268 410 291 441 270 407 282 406 240 428 282 416 255 423 282 420 250 454 334 46960 72 35 414 263 427 327 415 252 415 302 441 267 428 251 418 262 412 284 426 271 433 310 417 260 417 301 416 249 450 332 45860 71 36 425 271 428 320 434 258 404 274 434 247 405 278 440 256 432 283 426 281 431 291 412 259 430 290 426 252 447 310 45460 70 37 419 243 435 318 432 245 429 256 434 266 419 240 442 231 231 235 416 230 427 246 400 237 420 255 424 234 443 258 45760 69 38 433 278 419 328 444 267 428 286 437 248 406 236 461 265 265 278 424 241 409 265 420 249 415 278 415 268 441 348 45860 68 39 431 272 411 296 436 267 416 292 432 261 442 254 462 266 407 267 421 270 414 252 408 240 417 286 426 252 428 278 45760 67 421 352 418 366 442 359 423 365 428 347 418 308 448 360 423 370 424 355 435 382 433 372 456 390 435 355 451 401 45560 55 421 358 416 358 437 346 416 344 431 347 448 390 453 376 433 376 412 327 451 399 399 338 441 389 421 337 439 391 43760 56 40 441 313 441 330 446 427 303 432 277 423 304 447 269 435 304 417 255 418 291 409 254 443 302 420 241 452 328 45760 57 50 432 284 430 300 428 261 409 274 429 272 433 312 452 302 426 299 406 369 453 392 430 364 443 383 444 369 445 397 45260 58 48 433 281 423 293 425 245 408 264 440 235 440 280 437 235 422 267 418 239 407 257 390 250 440 271 415 230 442 301 45060 59 41 431 252 425 284 436 242 428 276 434 244 439 303 446 245 445 294 419 235 420 266 420 255 437 312 439 272 438 321 45960 60 46 437 244 421 250 436 257 423 248 425 230 444 270 437 233 419 237 426 229 420 262 428 261 414 261 422 261 451 307 45960 61 45 438 257 428 313 446 245 415 262 432 391 429 291 444 253 413 287 422 260 422 258 405 233 416 294 427 252 437 297 44360 62 44 428 256 430 309 440 260 412 286 426 372 424 279 440 234 400 250 419 265 416 254 412 241 423 263 407 263 455 301 45560 63 43 426 267 425 314 439 274 418 269 435 372 414 271 446 245 413 250 425 239 403 253 426 251 417 277 429 255 433 326 45760 64 42 430 260 445 327 440 248 414 259 442 385 433 271 455 260 409 275 418 242 429 293 416 258 435 269 426 270 448 314 44760 65 47 423 247 430 284 445 240 411 259 432 393 442 310 450 270 431 311 419 252 413 275 408 259 421 251 416 247 432 289 45460 66 421 341 443 363 452 353 441 373 443 386 423 356 455 303 422 349 425 281 423 361 409 276 430 271 406 252 434 351 45060 58 430 347 447 383 437 361 425 369 441 371 418 366 459 381 439 385 434 375 451 397 429 376 423 374 410 343 437 389 441125 57 34 424 243 435 287 441 241 417 251 430 236 422 268 443 245 432 291 428 245 421 265 417 256 418 252 417 252 429 294 444125 56 36 428 265 430 299 432 261 408 282 423 261 421 298 443 243 438 287 414 243 431 281 408 246 438 295 428 234 450 300 458125 55 35 431 243 414 264 445 232 417 249 438 250 412 287 452 262 410 291 412 244 426 273 422 236 412 253 404 234 459 307 445125 54 37 429 290 417 299 435 267 415 286 435 289 432 305 444 246 415 285 422 261 427 283 426 252 416 276 417 237 435 383 460125 53 423 372 445 400 453 347 432 382 434 374 444 392 460 388 430 386 428 373 438 384 416 366 422 377 432 373 428 376 456125 71 43 432 254 403 275 438 251 420 275 437 247 424 268 443 236 440 267 422 240 423 277 447 255 428 292 427 255 445 297 460125 72 48 418 260 430 282 428 249 406 262 431 250 423 281 461 249 439 289 420 242 423 258 404 237 437 291 423 268 451 299 446125 59 39 434 278 443 294 427 269 422 297 434 311 442 321 441 288 410 309 415 289 442 331 417 285 428 323 431 297 451 369 463125 60 40 417 254 432 294 429 245 414 259 435 265 427 320 451 271 439 285 415 266 428 259 414 247 445 272 413 268 447 316 453125 61 41 435 251 404 262 414 252 397 254 426 236 419 266 440 248 428 285 421 243 425 280 418 261 432 260 405 241 446 276 455125 62 42 415 262 402 288 426 267 407 279 444 283 420 300 448 271 418 288 429 266 423 289 427 275 426 310 432 288 438 340 461125 63 47 421 289 415 300 435 263 426 285 434 294 426 314 453 248 438 302 423 245 429 291 409 266 440 303 420 232 436 307 458125 64 44 414 242 414 252 430 241 419 250 423 241 424 256 439 241 434 241 426 243 420 280 409 244 444 296 419 268 446 324 460125 65 429 365 433 368 449 257 437 364 435 359 449 367 461 356 440 365 433 343 434 352 419 340 433 360 429 250 436 358 456125 66 50 426 263 432 279 438 237 411 254 435 246 422 368 429 243 394 239 432 233 427 239 412 237 433 247 409 237 438 309 459125 67 51 422 252 429 302 439 248 414 262 423 284 428 311 442 243 405 263 417 258 433 308 429 261 430 274 423 248 438 299 459125 68 49 420 250 412 258 427 233 407 232 436 242 415 279 442 247 431 273 425 238 415 248 416 261 425 253 422 232 427 296 460125 69 45 426 267 428 286 432 237 399 232 433 230 437 260 445 251 428 287 430 255 430 280 425 244 402 263 414 250 434 301 457125 70 46 429 280 432 294 433 269 420 289 437 269 417 289 444 247 444 275 422 243 449 298 395 233 416 265 413 254 410 292 461125 38 422 284 413 290 409 251 399 293 436 311 445 350 461 307 420 306 428 273 426 316 436 266 418 274 419 421 455 337 449125 52 414 273 425 342 414 259 402 304 430 308 417 284 450 309 424 305 420 266 432 310 437 276 430 297 428 412 453 360 449125 33 420 314 421 336 442 337 441 360 438 335 428 345 453 331 425 336 432 321 433 317 442 319 448 351 421 418 462 368 442250 53 50 424 291 428 314 428 278 418 303 432 319 444 323 444 272 432 296 428 288 434 305 412 254 429 303 412 248 438 318 450250 54 49 419 247 413 284 438 282 403 288 435 275 410 284 430 254 431 292 415 262 424 275 411 234 413 257 411 251 453 298 446250 55 35 419 261 415 269 404 243 421 266 428 247 414 255 427 247 430 277 420 236 403 248 402 256 425 265 419 232 449 300 451250 56 414 309 433 359 446 335 431 346 436 358 444 368 455 323 426 364 430 360 444 364 419 353 424 335 437 343 441 373 459250 57 33 417 259 419 285 419 250 418 280 427 265 427 295 441 256 437 292 412 250 437 290 414 258 416 275 411 273 431 331 458250 58 37 423 285 420 279 421 245 391 262 427 244 417 276 424 237 437 285 413 238 436 279 401 234 421 255 426 238 448 304 450250 59 38 418 260 408 295 423 255 408 278 433 285 416 321 437 273 426 299 411 272 421 265 408 240 413 267 421 231 451 318 455250 60 39 426 259 418 278 429 244 417 273 429 249 416 279 436 239 439 280 417 243 420 293 413 247 417 277 417 248 447 292 454250 61 40 419 430 315 433 274 410 299 427 289 435 328 453 271 430 297 423 278 413 310 401 270 421 315 427 299 459 332 453B-1250 62 36 417 251 413 270 435 249 406 280 429 242 413 287 436 248 434 320 422 275 415 313 412 242 405 261 407 232 437 274 446250 69 46 417 231 402 250 427 239 405 243 432 244 417 241 425 230 411 240 407 231 410 231 399 235 417 231 436 231 444 374 438250 70 45 436 286 421 316 442 280 424 289 429 270 416 318 443 250 414 280 408 248 440 297 425 354 414 272 431 249 439 302 459250 71 43 424 249 431 300 444 272 411 290 430 259 415 312 407 293 414 295 415 271 426 311 425 289 441 313 432 268 443 334 449250 72 422 358 432 371 447 365 430 368 433 365 440 379 452 362 434 367 407 345 433 356 428 358 421 352 439 338 450 388 463250 63 429 365 440 389 441 249 402 268 424 271 411 299 396 244 434 297 407 243 435 387 439 247 428 391 446 383 435 310 457250 64 52 427 232 414 266 435 247 424 280 430 248 415 273 410 243 439 290 428 229 415 258 428 236 419 242 411 232 445 297 451250 65 51 432 279 426 288 419 376 436 368 420 385 431 432 364 431 387 403 355 433 287 432 385 436 275 415 245 448 387 449250 66 41 425 290 442 302 443 243 411 260 434 263 408 279 423 250 416 276 419 246 432 284 430 254 409 282 410 242 439 316 447250 67 48 414 231 418 265 440 253 436 268 434 248 413 274 409 231 431 268 411 236 434 263 410 241 406 241 416 257 426 285 454250 68 425 334 431 367 443 348 433 361 428 343 423 347 434 350 427 352 418 346 437 368 439 345 435 362 464 372 428 361 460250 34 420 291 405 318 432 295 402 302 427 312 423 445 315 415 314 417 310 432 299 397 299 414 316 420 301 435 330 454250 44 420 260 417 346 443 310 442 344 430 261 419 452 293 409 304 412 301 425 250 421 337 442 301 409 248 441 347 447250 42 423 315 430 343 447 345 439 353 435 327 415 321 443 331 420 321 423 322 419 314 409 328 424 339 412 421 448 370 453250 51 424 289 429 314 442 289 420 300 434 286 429 330 452 252 391 302 425 253 411 265 415 321 451 338 428 265 442 330 445P generation egg productionDOSE ID4/20 4/21 4/22 4/23 4/24 4/25 4/26week64/27 4/28 4/29 4/30 5/1 5/2 5/3week75/45/55/65/75/85/95/105/115/125/135/145/155/165/17week90 144 1 1 1 10 142 1 1 1 1 1 1 1 1 10 140 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 10 1390 141 1 1 1 1 1 1 1 1 1 10 143 1 1 2 1 1 1 1 1 1 1 1 1 1 1 10 150 1 1 1 1 1 1 1 2 1 1 1 10 149 1 1 1 1 1 1 1 1 1 1 1 10 148 1 1 1 1 1 1 1 1 10 147 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 146B-10 145 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 137 2 1 1 1 1 1 1 1 1 1 1 10 135 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 133 1 1 1 1 1 1 1 1 1 10 1340 136 1 1 1 1 1 1 1 1 1 1 1 1 10 138 1 2 2 1 1 1 1 1 1 1 1 1 1 1 10 1510 15230 49 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 50 130 51 1 1 1 1 1 1 130 52 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 33 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 130 34 1 1 2 1 2 1 1 1 1 1 1 1 1 1 1 130 3530 36 1 2 1 1 1 1 1 1 1 1 1 1 130 3730 38 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 39 2 1 1 1 1 1 1 1 1 1 1 1 130 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 41 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 42 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 43 2 1 1 1 1 1 1 1 1 1 1 1 1 130 44 1 1 1 1 1 1 1 1 1 1 1 1 130 4530 4630 4730 48 1 1 1 1 1 1 1 1 1 160 33 1 1 1 1 1 1 1 1 1 1 1 1 160 34 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 35 1 1 160 3660 37 1 1 2 1 1 1 1 1B-160 38 1 1 1 1 1 1 1 1 160 39 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 160 4060 47 1 1 1 1 1 1 1 1 1 1 2 1 2 1 160 48 1 1 1 1 1 1 1 1 1 1 160 4960 50 2 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 160 5160 52 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 260 41 1 1 1 1 1 1 1 1 1 1 160 42 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 160 43 1 1 1 1 1 1 1 1 1 1 1 1 1 160 44 1 2 1 1 1 1 1 1 1 1 1 1 1 1 160 45 1 160 46 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1125 38 1 1 1 1 1 1 1 1 1 1125 39 1 1 1 1 1 1 1 1 1 1 1125 37 1 1125 36 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 35 1 1 1 1 1 1 1 1 1 1 1 1 1125 34 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1125 33 1 1 1 1 1 1 1 1 1125 44 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1125 43 1 1 1 1 1 1 1 1 1 1 1125 42 1 1 1125 41 1 1 1 1 1125 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 45 1 1 1 1 1 1 1 1 1 1125 46 1 1 1 1 1 1 1 1 1 1 1 1 1125 47 1 1 1 1 1 1 1 1 1 1 1 1 1125 48 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 49 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 50 1 1 1 1 1 1 1 1 1 1 1 1125 52 1 1 1 1 1 1 1 1 1 1 1 1B-1125 51 1 1 1 1 1 1 1 1 1250 43 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 44 1 1 1 1 1 1 1 1 1 1 1 1250 45 1 1 1 1 1250 46 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 47 2 1 1 1 1 1 1 1 1 1 1 1 1 1250 48 1 1 1 1 1 1 1 1 1 1 1 1 1250 49 1 1 1 1 1 1 1 1 1 1 1250 50250 51 1 1 1 1 1 1 1 1 1 1 1 1250 52 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1250 36 1 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 35 1 1 2 1 1 1s 1 1 1250 34 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1250 33 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 37 1 1 1 1 1 1 1 1250 38 1 1 1 1 1 1 1250 39 1 1 1 1 1 1250 40250 41 1 1 1 1 1 1 1 1 1250 42 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1DOSE ID5/185/195/205/215/225/235/24week105/25 5/26 5/27 5/28 5/29 5/30 5/31week116/1 6/2 6/3 6/4 6/5 6/6 6/7week126/8 6/9 6/10 6/11 6/12 6/13 6/14week130 144 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 142 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 140 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 2 10 1390 141 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 10 143 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 150 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 149 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 148 2 1 1 1 1 1 1 1 1 1 10 147 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 10 146 1 1 1B-10 145 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 137 1 1 1 1 1 1 1 1 1 1 1 10 135 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20 133 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 10 1340 136 1 1 1 1 1 1 1 1 1 1 10 138 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 2 10 1510 15230 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 1 130 50 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 130 51 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 52 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 33 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 34 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 130 35 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 36 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 37 1 1 1 1 1 1 1 1 1 1 130 38 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 39 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 130 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 130 41 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 130 42 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 130 43 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 44 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 130 45 1 1 1 1 1 1 1 130 46 1 1 1 1 2 1 1 1 1 1 1 1 1 130 47 1 1 1 1 1 1 2 1 1 1 1 1 1 1 130 48 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 1 160 33 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 160 34 1 1 1 1 1 1 1 1 2 1 1 1 1 1 160 35 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 160 36 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 37 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1B-160 38 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 160 39 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 160 40 1 1 1 1 1 1 1 1 1 1 1 160 47 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 48 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 4960 50 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 5160 52 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 160 41 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 160 42 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 160 43 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 44 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 45 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 46 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 38 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 39 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 37 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2125 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 1 1 1125 35 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 34 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 1125 33 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 44 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 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 1125 42 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 1125 41 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 45 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 46 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 47 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 48 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 1125 49 1 2 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1125 50 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1125 52 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 1B-1125 51 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 2 1 1 1 1 1 1 2 1250 43 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 44 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1250 45 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 46 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 47 1 1 1 1 1 1 1 1250 48 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 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 1 1250 50 1 1 1 1 1 1 1 1 1 1 1 1250 51 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 2 1 1 2 2 2 2250 52 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1250 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 1250 35 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 34 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 1250 33 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1250 37 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 38 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 39 1 1 1 1 1 1 1 1 2 1 1 1 1250 40250 41 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1250 42 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1DOSE ID6/15 6/16 6/17 6/18 6/19 6/20 6/21week146/22 6/23 6/24 6/25 6/26 6/27 6/28week15### ### 7/1 7/2 7/3 7/4 7/5week160 144 1 1 1 1 1 1 1 1 10 142 1 1 1 1 1 1 1 1 1 10 140 1 1 1 1 1 1 1 1 1 1 1 10 1390 141 1 1 1 1 1 1 1 2 1 1 1 20 143 1 1 1 1 1 1 1 1 1 1 1 10 150 1 1 1 1 1 1 1 1 1 1 1 1 1 10 149 1 1 1 1 1 1 1 1 1 1 1 10 148 1 1 1 1 1 1 1 1 10 147 1 1 1 1 1 1 1 1 2 1 10 146 1 1 1 1 1 1 1B-10 145 1 1 1 1 1 1 1 1 10 137 1 1 1 1 1 1 1 1 2 2 1 1 1 1 1 1 10 135 1 1 1 1 1 1 1 1 1 1 1 10 133 1 1 1 1 1 1 1 1 1 1 2 1 10 1340 136 1 1 10 138 1 1 1 1 1 1 1 1 1 1 2 10 1510 15230 49 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 50 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 130 51 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 52 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 33 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 34 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 35 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 130 36 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 37 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 38 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 39 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 41 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 42 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 43 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 44 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 130 45 1 1 1 1 1 1 1 2 1 1 1 1 130 46 1 1 1 1 2 1 1 1 1 1 2 1 1 1 1 1 130 47 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 130 48 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 33 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 34 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 160 35 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 2 1 1 1 160 36 1 1 1 1 1 1 2 1 1 1 1 1 1 1 160 37 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1B-160 38 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 39 1 1 1 1 1 1 1 1 1 2 1 1 160 40 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 160 47 1 1 1 1 1 1 1 1 1 160 48 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 4960 50 2 1 1 160 5160 52 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 41 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 160 42 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 160 43 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 44 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 160 45 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 46 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1125 38 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1125 39 1 1 2 1 1125 37 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 36 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 35 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 34 1 1 1 1 1 1 1 1 1 2 1 1 1 2 1125 33 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1125 44 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 43 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1125 42 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1125 41 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1125 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 45 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 46 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1125 47 1 1 1 1 2 1 1 1 1 1 1125 48 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1125 49 1 1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1125 50 2 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1125 52 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1B-1125 51 1 1 1 1 1 1 2 1 1 1 2 1 1 1250 43 1 1 1 1 1 1 1250 44 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 45 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 46 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1250 47250 48 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 49 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 50 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1250 51 2 1 1 1 1 1 1 2 2 2 2 1 1 1 1 1 1 2 1 2 1250 52 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1250 36 1 1 1 1 1 1 1 1 1 1 1 2 1250 35 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1250 34 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 33 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1250 37 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 38 1 1 1 1 1 1 1 1 1 1 1250 39 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 40250 41 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 42 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1DOSE ID 7/6 7/7 7/8 7/9 7/10 7/11 7/12 week

17 7/13 7/14 7/15 7/16 7/17 7/18 7/19 7/20 ### ### ### week

18

0 144 1 1 1 1 1 1 1 1

0 142 1 1 1 1 1 1 1 1 1 1

0 140 1 1 1 1 1 1 2 1 1 1 1

0 139

0 141 1 1 2 1 1 1 1

0 143 1 1 1 1 1 1 1 1 1 1

0 150 1 1 1 2

0 149 1 1 1 1 1 1

0 148 1 2 1

0 147 1 1 1 1

0 146 1 1 1 1 1 2 1 1 1

0 145 1 1 1 1 1 1 1 1

0 137 1 2 1 2 1 1 1 1 1

0 135 1 1 1 1 1

0 133 1 1 1 1 1 1 1 2

0 134

0 136 1 1

0 138 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0 151

0 152

30 49 1 1 1 1 1 1 1 1 1 1 1 1 1 1

30 50 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1

30 51 1 1 1 1 1 1 1 1 1 1 1 1 1 1

30 52 1 1 1 1 1 1 1 1 1 1 1 1 1

30 33 1 1 1 1 1 1 1 2 1 1 1 1 2 1 1 1 1

30 34 1 1 1 1 1 1 1 2 1 1 1 1 1 1

30 35 1 1 1 1 1 2 1 1 1 1 1 1

30 36 1 1 1 2 1 1 1 1 2 1

30 37 1 1 1 1 1 1 1 1 1 1 1 1 1 1

30 38 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

30 39 1 1 1 1 2 1 2 1 1 1 1 1 1 1 1 1

30 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

30 41 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1

30 42 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

30 43 1 1 1 1 1 1 1 1 1 1 1 1

30 44 1 1 1 1 1 1 2 1 1 1 1 1 1

30 45 1 1 1 1 2 1 1 1 1 1 1 1

30 46 1 1 1 1 1 1 1 2 1 1 1 1 1 1

30 47 1 1 1 1 1 1 1 2 1 1 1 1 1 1

30 48 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1

60 33 1 1 1 1 1 1 2 1 1 1 1 1 1

60 34 1 1 1 1 1 1 1 1 1 1 2 1 1 1

60 35 1 1 1 1 1 1 1 1 1 1 1 1 1 1

60 36 1 1 1 1 1 1 1 1 1 1 1 1 1

60 37 1 1 1 1 1 1 2 1 1 1 1 1 1

# B-1 60 38 1 1 1 1 1

60 39 1 1 1 1 1 2 1 1 1 1 1 1 1

60 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

60 47 1 1 1 1 1 1 1 2 1 1 1 1 1

60 48 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2

60 49

60 50 1 1 1 1 2 1 2 1 1 1 1 1 1 1 1 1

60 51

60 52 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1

60 41 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

60 42 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1

60 43 1 1 1 1 1 1 1 1 1 1 1 1

60 44 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1

60 45 1 1 1 1 1 1 1 2 1 1 1 1 1 2 1 1 1

60 46 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1

125 38 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1

125 39 1

125 37 1 1 1 1 1 1 1 1 1 1 1 1

125 36 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

125 35 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1

125 34 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1

125 33 1 1 1 1 1 1 1 2 1 1 1 1 2 1

125 44 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1

125 43 1 1 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1

125 42 1 1 1 1 1 1 2 1 1 1 1

125 41 1 1 1 1 1 1 1 1 1 1 1 1 1 1

125 40 1 1 1 1 1 1 1 1 1 1 1 1 2 1

125 45 1 1 1 1 1 1 1 1 1 1 1

125 46 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 1

125 47 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1

125 48 1 1 1 1 1 1 2 1 1 1 1 1 1 1

125 49 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1

125 50 1 1 1 1 1 1 1 1 1 1 1 1 1 1

125 52 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1

125 51 1 1 1 1 1 1 1 2 1 1 1 1 1 1

250 43 1 1 1 1 1 1 1

250 44 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1

250 45 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

250 46 1 1 1 1 1 2 1 1 1 1 1 1 1

250 47 1 1 1 1 1 1

250 48 1 1 1 1 1 1 1 1 1 1 1 1 1 1

250 49 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1

250 50 1 1 1 1 1 1 1 1 1 2 1 1 1 1

250 51 2 2 1 1 1 1 2 1 1 1 1 1 1 2 1

250 52 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1

250 36 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1

250 35 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1

250 34 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

# B-1 250 33 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

250 37 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

250 38 1 1 2 1 1 1 1

250 39 1 1 1 1 2 1 1 1 1 1

250 40

250 41 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

250 42 1 1 2 1 1 1 1 1 1 1 2 1 1

F1 clocaa measurements and foam

8/3/2009 8/11/2009

DOSE ID cg

area foam DOSE ID cg

area foam DOSE ID first foam ha tch date day of onset

0 218 168 Y 0 221 195 Y 0 205 8/3 6/26 38

0 221 132 Y 0 218 182 Y 0 206 8/3 6/26 38

0 279 144 Y 0 236 72 N 0 213 8/3 6/26 38

0 213 210 Y 0 224 72 N 0 214 8/3 6/26 38

0 214 210 Y 0 216 143 Y 0 216 8/10 6/28 43

0 232 110 Y 0 227 289 Y 0 218 8/3 6/26 38

0 206 143 Y 0 280 132 N 0 220 8/3 6/26 38

0 244 156 Y 0 240 255 Y 0 221 8/3 6/27 37

0 205 156 Y 0 222 72 N 0 222 8/19 6/26 54

0 246 130 Y 0 229 81 N 0 224 9/16 6/26 82

0 224 110 N 0 243 256 Y 0 227 8/3 6/26 38

0 236 72 N 0 220 255 Y 0 229 8/17 6/26 52

0 227 221 Y 0 213 342 Y 0 232 8/3 6/28 36

0 216 90 N 0 278 210 Y 0 236 8/19 6/26 54

0 240 168 Y 0 214 210 Y 0 240 8/3 6/26 38

0 280 90 N 0 232 195 Y 0 243 8/3 6/26 38

0 229 35 N 0 244 240 Y 0 244 8/3 6/25 39

0 222 100 N 0 206 180 Y 0 246 8/3 6/26 38

0 243 154 Y 0 246 132 Y 0 279 8/3 6/29 35

0 220 195 Y 0 205 272 Y 0 280 8/16 6/29 48

30 255 108 Y 30 257 16814 Y 30 204 9/16 6/26 82

30 257 154 Y 30 255 208 Y 30 205 9/16 6/26 82

30 246 144 Y 30 246 323 Y 30 209 9/16 6/26 82

30 237 54 N 30 237 154 Y 30 213 9/16 6/27 81

30 238 100 Y 30 325 100 N 30 215 9/16 6/26 82

30 236 25 N 30 282 196 Y 30 216 8/3 6/26 38

30 223 196 Y 30 223 210 Y 30 219 8/3 6/26 38

30 244 25 N 30 244 121 Y 30 223 8/3 6/26 38

30 215 100 N 30 215 196 Y 30 224 8/3 6/25 39

30 205 25 N 30 205 80 N 30 233 8/3 6/26 38

30 325 25 N 30 224 195 Y 30 234 8/3 6/25 39

30 282 90 Y 30 233 169 Y 30 237 8/10 6/26 45

30 233 108 Y 30 219 240 Y 30 240 9/16 6/27 81

30 224 120 Y 30 248 132 Y 30 242 9/16 6/26 82

30 213 81 N 30 216 240 Y 30 244 8/10 6/27 44

30 219 121 Y 30 217 25 N 30 246 8/3 6/25 39

30 216 143 Y 30 240 132 N 30 255 8/3 6/27 37

# B-1 30 234 132 Y 30 272 54 N 30 257 8/3 6/26 38

30 240 72 N 30 204 100 N 30 282 8/3 6/29 35

30 272 25 N 30 209 40 Y 30 325 9/16 6/26 82

30 204 90 N 60 176 168 Y 60 177 8/7 6/26 42

30 209 25 N 60 167 132 Y 60 109 8/3 6/26 38

60 176 169 Y 60 116 210 Y 60 111 8/3 6/28 36

60 167 130 Y 60 111 234 Y 60 114 8/3 6/29 35

60 111 210 Y 60 164 144 Y 60 116 8/3 6/27 37

60 116 143 Y 60 151 182 Y 60 117 8/3 6/27 37

60 151 144 Y 60 114 156 Y 60 120 8/7 6/26 42

60 164 100 N 60 187 289 Y 60 136 9/1 6/29 64

60 114 100 Y 60 190 64 N 60 151 8/3 6/28 36

60 187 192 Y 60 178 169 Y 60 161 9/16 6/26 82

60 190 120 N 60 177 143 Y 60 164 9/16 6/28 80

60 178 104 Y 60 180 240 Y 60 167 8/3 6/28 36

60 177 100 N 60 161 80 N 60 169 8/3 6/29 35

60 180 168 Y 60 109 225 Y 60 171 8/3 6/25 39

60 109 195 Y 60 173 238 Y 60 173 8/3 6/25 39

60 161 143 N 60 120 256 Y 60 176 8/3 7/4 30

60 120 156 N 60 169 256 Y 60 178 8/3 6/28 36

60 173 240 Y 60 171 168 Y 60 180 8/3 6/26 38

60 171 210 Y 60 117 154 Y 60 187 8/3 6/29 35

60 169 195 Y 60 136 36 N 60 190 8/26 6/26 61

60 117 169 Y 125 110 224 Y 125 109 8/11 6/29 43

60 136 25 N 125 109 81 Y 125 114 8/3 6/28 36

125 109 25 N 125 184 210 Y 125 118 8/3 6/27 37

125 114 168 Y 125 193 208 Y 125 119 8/11 6/25 47

125 193 25 N 125 182 240 Y 125 131 8/3 6/25 39

125 184 144 N 125 155 169 Y 125 134 8/3 6/25 39

125 155 25 N 125 170 165 Y 125 137 8/3 6/26 38

125 182 110 Y 125 106 210 Y 125 151 8/3 6/26 38

125 185 168 Y 125 97 224 Y 125 152 9/16 6/29 79

125 170 108 Y 125 188 48 N 125 155 8/10 6/26 45

125 188 25 N 125 133 169 Y 125 157 9/16 6/26 82

125 189 120 Y 125 151 180 Y 125 161 8/3 6/25 39

125 151 90 Y 125 183 225 Y 125 170 8/3 6/26 38

125 134 165 Y 125 161 169 Y 125 182 8/3 6/29 35

125 161 90 Y 125 131 196 Y 125 183 8/3 6/25 39

125 183 182 Y 125 118 270 Y 125 184 8/9 6/26 44

125 118 195 Y 125 157 56 N 125 185 8/3 6/28 36

125 131 156 Y 125 152 70 N 125 188 9/16 6/28 80

125 152 70 N 125 134 224 Y 125 189 8/3 6/25 39

125 157 63 N 125 119 132 N 125 193 8/10 6/28 43

125 119 80 N 250 158 132 Y 250 103 8/3 6/25 39

125 137 180 Y 250 141 144 N 250 105 8/7 6/27 41

250 135 196 Y 250 138 120 Y 250 106 8/26 6/25 62

250 118 70 N 250 111 210 Y 250 108 8/3 6/26 38

250 120 70 N 250 105 169 Y 250 109 8/3 6/28 36

# B-1 250 140 195 Y 250 112 306 Y 250 111 8/9 6/25 45

250 255 56 N 250 135 156 Y 250 112 8/3 6/26 38

250 109 154 Y 250 118 110 N 250 116 8/3 6/26 38

250 116 247 Y 250 140 210 Y 250 118 9/16 6/26 82

250 131 25 N 250 120 121 N 250 120 9/16 6/26 82

250 106 132 Y 250 155 56 N 250 131 8/19 6/26 54

250 132 143 Y 250 109 210 Y 250 132 8/3 6/27 37

250 158 90 N 250 116 208 Y 250 135 8/3 6/27 37

250 141 54 N 250 131 42 N 250 138 8/7 6/26 42

250 111 80 N 250 132 132 Y 250 140 8/3 6/25 39

250 138 100 N 250 106 100 N 250 141 9/16 6/26 82

250 105 90 N 250 108 256 Y 250 155 9/16 6/26 82

250 112 169 Y 250 174 169 Y 250 158 8/11 6/26 46

250 103 195 Y 250 177 110 N 250 174 8/3 6/30 34

250 177 35 N 250 103 255 Y 250 177 9/16 6/28 80

250 108 210 Y

250 174 80 Y

F1 egg strength

Dose ID Thickness 1 Thickness 2 Thickness avg Strength Yolk

0 202 1.03 0.99 1.01 0.395 Y

0 203 1.28 0.87 1.075 1.223 Y

0 203 1.23 1.27 1.25 1.509 Y

0 207 1.06 0.92 0.99 1.004 Y

0 209 1.1 0.7 0.9 0.543 Y

0 209 0.77 0.8 0.785 0.999 Y

0 211 0.75 0.96 0.855 0.882 Y

0 212 1.06 0.78 0.92 1.145 Y

0 212 0.79 0.67 0.73 0.739 Y

0 215 1.15 1.35 1.25 0.547 Y

0 215 0.92 0.78 0.85 0.87 Y

0 217 0.85 0.92 0.885 0.946 Y

0 219 0.56 0.8 0.68 1.038 Y

0 221 0.72 0.88 0.8 1.283 Y

0 225 0.6 0.81 0.705 1.196 Y

0 225 1.35 1.3 1.325 1.28 Y

0 226 0.72 1.24 0.98 1.09 N

0 226 1.18 0.98 1.08 1.052 Y

# B-1 0 226 0.76 0.84 0.8 0.503 Y

0 228 0.77 0.72 0.745 0.699 Y

0 228 1.02 0.93 0.975 0.988 Y

0 234 1.02 0.81 0.915 1.105 Y

0 234 1.55 1.2 1.375 0.976 Y

0 235 1.12 1.45 1.285 0.893 Y

0 235 1.34 1.6 1.47 1.014 Y

0 237 1 0.87 0.935 0.908 Y

0 237 1.15 1.35 1.25 0.974 Y

0 242 0.34 0.22 0.28 1.027 Y

0 242 0.71 0.82 0.765 1.206 Y

0 245 0.59 0.77 0.68 1.163 Y

0 299 0.41 0.33 0.37 1.063 N

30 202 0.94 0.99 0.965 1.455 Y

30 206 0.85 0.8 0.825 0.472 Y

30 211 0.68 0.55 0.615 0.426 Y

30 211 0.39 0.45 0.42 0.811 Y

30 212 1 0.82 0.91 1.023 Y

30 220 0.73 0.67 0.7 1.117 Y

30 220 1.16 1.12 1.14 0.944 Y

30 221 0.31 0.4 0.355 1.471 Y

30 225 0.85 0.93 0.89 0.438 Y

30 226 1.21 0.78 0.995 0.86 Y

30 227 1.04 1.43 1.235 1.15 Y

30 230 1.16 0.84 1 1.097 Y

30 230 0.59 0.75 0.67 1.122 Y

30 231 0.95 0.9 0.925 1.181 Y

30 232 0.61 0.49 0.55 1.014 Y

30 232 1.07 1.03 1.05 0.808 N

30 232 0.73 0.77 0.75 0.811 Y

30 235 0.91 0.83 0.87 1.192 Y

30 235 1.15 1.03 1.09 1.449 Y

30 242 1.21 0.85 1.03 0.97 Y

30 242 1.23 1.3 1.265 1.239 Y

30 247 1.13 1.03 1.08 1.093 Y

30 247 0.43 0.34 0.385 1.018 Y

30 249 0.41 0.42 0.415 1.301 Y

30 250 1.05 0.84 0.945 0.359 Y

30 250 0.64 0.58 0.61 1.068 Y

30 251 1.27 0.75 1.01 1.431 Y

30 253 0.41 0.55 0.48 1.021 N

30 253 1.22 1.14 1.18 0.98 Y

30 253 1.04 0.92 0.98 1.002 Y

60 102 0.58 0.4 0.49 1.265 Y

60 102 1.34 1.58 1.46 0.918 Y

60 110 0.4 0.32 0.36 1.54 Y

60 115 0.84 0.82 0.83 1.027 Y

60 115 0.59 0.48 0.535 0.813 Y

# B-1 60 119 0.88 0.63 0.755 1.074 Y

60 165 0.79 0.92 0.855 1.002 Y

60 165 0.89 1.2 1.045 1.173 Y

60 166 0.96 1.03 0.995 0.998 Y

60 170 0.93 0.99 0.96 1.239 Y

60 170 0.73 0.63 0.68 1.161 Y

60 172 0.68 0.87 0.775 0.975 Y

60 172 0.92 0.96 0.94 1.21 Y

60 174 1.03 0.94 0.985 0.952 N

60 174 0.5 0.75 0.625 0.477 Y

60 179 0.7 0.62 0.66 0.812 Y

60 179 0.61 0.53 0.57 0.815 Y

60 179 1.38 1.25 1.315 0.996 Y

60 181 0.75 0.64 0.695 0.882 Y

60 181 0.72 0.89 0.805 0.96 Y

60 183 1.2 1.44 1.32 0.945 Y

60 184 0.5 0.44 0.47 1.269 Y

60 184 0.95 0.81 0.88 1.408 Y

60 185 1.07 0.98 1.025 1.111 Y

60 185 1.18 0.69 0.935 1.005 Y

60 189 0.48 0.49 0.485 1.225 Y

60 189 0.89 0.7 0.795 1.12 Y

125 101 Y

125 101 0.38 0.33 0.355 1.383 N

125 101 0.47 0.34 0.405 1.482 N

125 101 1 1.21 1.105 1.767 Y

125 103 0.95 1.02 0.985 1.043 Y

125 106 1.05 0.8 0.925 0.817 N

125 106 0.62 0.97 0.795 0.93 Y

125 107 Y

125 107 0.35 0.34 0.345 1.185 N

125 107 0.79 0.72 0.755 1.243 Y

125 110 0.67 0.51 0.59 0.997 N

125 111 0.39 0.32 0.355 1.159 N

125 111 0.72 0.68 0.7 0.973 Y

125 112 0.87 0.91 0.89 0.591 Y

125 112 0.61 0.53 0.57 1.277 N

125 113 Y

125 113 1.06 0.8 0.93 1.187 N

125 113 1.1 1.22 1.16 0.838 Y

125 114 0.97 0.88 0.925 1.121 N

125 117 0.97 0.99 0.98 1.296 Y

125 117 0.67 0.81 0.74 1.305 Y

125 119 1.09 0.76 0.925 1.292 Y

125 135 Y

125 135 0.9 0.86 0.88 1.364 N

125 135 0.65 0.88 0.765 1.126 Y

125 156 0.72 0.56 0.64 1 N

# B-1 125 156 0.8 0.89 0.845 1.018 Y

125 158 0.69 0.64 0.665 0.408 N

125 158 0.8 0.72 0.76 0.993 Y

125 159 Y

125 159 0.55 0.32 0.435 0.668 N

125 159 0.71 0.96 0.835 0.76 Y

125 160 0.89 0.91 0.9 0.695 N

125 160 0.79 0.97 0.88 0.937 Y

125 163 0.85 0.82 0.835 1.278 N

125 163 0.76 0.81 0.785 0.999 Y

125 165 Y

125 165 0.53 0.5 0.515 1.185 N

125 165 0.7 0.75 0.725 1.371 Y

125 166 0.75 0.65 0.7 0.403 N

125 167 0.6 0.57 0.585 0.814 N

125 167 1.02 1.21 1.115 1.132 Y

125 181 Y

125 181 0.67 0.65 0.66 1.153 N

125 181 0.77 0.88 0.825 1.283 Y

125 191 0.77 0.7 0.735 1.114 Y

125 191 0.78 0.99 0.885 1.414 N

125 191 0.66 0.79 0.725 1.314 Y

250 102 0.89 0.85 0.87 1.524 Y

250 102 0.76 0.68 0.72 1.006 Y

250 107 0.34 0.32 0.33 0.794 N

250 109 1.21 0.85 1.03 1.344 N

250 110 Y

250 110 0.31 0.35 0.33 1.363 N

250 110 0.51 0.42 0.465 0.976 Y

250 114 Y

250 114 0.63 0.54 0.585 0.918 N

250 114 0.85 0.89 0.87 1.153 Y

250 115 0.39 0.46 0.425 1.298 Y

250 115 0.57 0.53 0.55 1.658 Y

250 122 0.8 0.68 0.74 1.275 N

250 131 0.77 0.86 0.815 0.811 N

250 133 0.61 0.46 0.535 1.164 N

250 134 0.39 0.36 0.375 0.8 N

250 136 0.59 0.61 0.6 1.01 Y

250 136 0.77 0.76 0.765 1.191 N

250 151 0.66 0.65 0.655 1.217 Y

250 151 0.41 0.45 0.43 1.396 Y

250 152 1.05 0.86 0.955 1.204 Y

250 152 0.35 0.27 0.31 1.254 Y

250 153 0.74 0.7 0.72 1.257 N

250 153 0.88 0.75 0.815 0.927 Y

250 154 0.7 0.69 0.695 0.673 Y

250 156 Y

# B-1 250 156 0.49 0.46 0.475 1.394 N

250 156 0.66 0.59 0.625 1.456 Y

250 159 Y

250 159 0.58 0.56 0.57 1.282 N

250 159 0.61 0.68 0.645 1.382 Y

250 161 Y

250 161 0.85 0.94 0.895 1.307 N

250 161 0.46 0.44 0.45 1.257 Y

250 171 Y

250 171 0.39 0.28 0.335 1.389 N

250 171 0.9 0.92 0.91 1.485 Y

250 193 1.14 0.86 1 0.943 N

F1 sperm measurements

DOSE ID % motile % progressive sperm/gram

0 214 24 0 690.9

0 214 19 3 597.5

0 214 21 0 728.2

0 218 12 0 295.8

0 218 22 0 254.5

0 218 18 0 385.2

0 220 22 6 427.8

0 220 44 4 641.6

0 220 18 0 451.5

0 221 32 0 228.7

0 221 25 0 465.6

0 221 7 0 220.8

0 222 29 8 606

0 222 5 0 451.5

0 222 15 0 796.1

0 227 77 13 767.9

0 227 29 8 316.6

0 227 42 5 310.4

0 229 11 0 558.5

0 229 6 0 606

0 229 5 0 665.4

0 233 45 7 796.1

0 233 52 3 392.1

0 233 44 6 404

0 240 67 9 401.4

0 240 63 8 550.8

0 240 56 11 532.2

0 279 91 0 143.8

# B-1 0 279 67 0 196.1

0 279 82 0 143.8

0 280 84 22 344

0 280 63 5 275.2

0 280 63 11 240.8

30 205 0 0 13.1

30 205 50 0 52.3

30 205 0 0 39.2

30 215 63 2 453.1

30 215 68 4 601.2

30 215 61 3 313.7

30 217 33 5 274.5

30 217 58 11 248.3

30 217 48 14 274.5

30 219 19 0 104.5

30 219 10 0 963.9

30 219 7 7 718.9

30 223 30 4 252.1

30 223 25 0 186.7

30 223 35 0 158.7

30 237 47 4 508.3

30 237 36 0 428.4

30 237 23 2 472

30 244 3 0 246.9

30 244 12 0 181.5

30 244 8 0 188.8

30 248 38 8 308.9

30 248 44 8 297.1

30 248 27 0 261.4

30 255 19 2 364.6

30 255 30 4 392.1

30 255 13 0 371.5

60 111 49 0 285.9

60 111 59 5 514.6

60 111 64 10 702.5

60 114 50 4 332.7

60 114 57 2 499.1

60 114 40 16 297.1

60 116 73 8 355.8

60 116 70 8 442.9

60 116 56 5 428.4

60 120 71 16 233.4

60 120 56 6 298.8

60 120 56 4 233.4

# B-1 60 136 35 2 562

60 136 33 5 562

60 136 37 0 666.6

60 151 62 15 617.9

60 151 55 7 499.1

60 151 53 7 653.5

60 164 4 0 662.2

60 164 0 0 810.4

60 164 4 0 801.7

60 171 65 16 492.7

60 171 79 8 241.3

60 171 67 11 542.9

60 177 74 7 441.1

60 177 33 0 392.1

60 177 34 0 473.8

60 187 33 2 617.9

60 187 25 0 606

60 187 33 0 617.9

125 109 21 2 575.1

125 109 15 0 357.3

125 109 13 0 470.5

125 119 50 6 348.5

125 119 65 3 337.7

125 119 37 3 326.8

125 131 3 0 413.9

125 131 30 4 294.1

125 131 21 5 206.9

125 134 90 9 632.9

125 134 79 11 454

125 134 68 7 385.2

125 155 69 8 448.1

125 155 62 0 196.1

125 155 55 0 186.7

125 157 67 6 112

125 157 38 8 80.9

125 157 14 0 87.1

125 161 51 1 620.8

125 161 51 5 792.4

125 161 56 9 555.5

125 170 56 4 297.1

125 170 52 9 522.8

125 170 48 4 320.8

125 182 53 2 792.9

125 182 58 7 766.8

125 182 51 6 679.7

125 193 23 5 522.8

125 193 34 0 558.5

125 193 41 2 487.2

# B-1 250 106 55 9 558.5

250 106 61 14 582.2

250 106 33 8 570.3

250 108 61 8 555.5

250 108 58 5 620.8

250 108 52 9 708

250 109 61 6 426.5

250 109 40 6 240.8

250 109 39 3 227

250 116 70 14 597.5

250 116 64 16 746.9

250 116 67 13 802.9

250 131 59 13 246

250 131 66 19 246

250 131 44 6 246

250 132 50 0 294.1

250 132 69 2 400.3

250 132 35 4 465.6

250 135 50 6 426.5

250 135 49 5 433.4

250 135 60 10 467.8

250 158 48 0 297.1

250 158 30 3 392.1

250 158 61 6 368.3

250 174 68 25 748.6

250 174 68 27 522.8

250 174 55 13 368.3

250 177 0 0 0

250 177 0 0 0

250 177 0 0 0F1 growth measurements7/6/2009 7/13/2009 7/20/2009DOSEbabyidBWtarsus1tarsus2avgtarsusculmen1culmen2avgculmenDOSEbabyidBWtarsus1tarsus2avgtarsusculmen1culmen2avgculmenDOSEbabyidBWtarsus1tarsus2avgtarsusculmen1culmen2avgculmen0 372 8 15.6 15.6 15.6 3.3 3.3 3.3 0 371 33 24.2 23.3 23.75 6 5.8 5.9 0 356 50 19.2 19.4 19.3 7 6.8 6.90 371 16 18.9 18.5 18.7 3.9 3.7 3.8 0 377 30 21.8 21.9 21.85 5.5 6 5.75 0 365 56 19.1 19.3 19.2 9.4 9.2 9.30 387 12 17.7 17.7 17.7 3.5 3.7 3.6 0 382 33 22.3 22.5 22.4 5.4 5.5 5.45 0 385 46 17.8 17.7 17.75 6.4 6.6 6.50 362 22 21.3 21.4 21.35 3.7 3.9 3.8 0 387 31 21.9 21.7 21.8 5.4 5.5 5.45 0 232 55 18.9 19 18.95 7.3 7.5 7.40 377 14 19.1 18.6 18.85 4.1 4.2 4.15 0 312 38 21.6 21.6 21.6 6.5 6.9 6.7 0 204 48 22.8 22.7 22.75 6.6 6.9 6.750 384 8 16.4 16.3 16.35 2.9 3.1 3 0 363 38 21.6 22 21.8 6 5.9 5.95 0 231 66 24.8 24.5 24.65 7.3 7.6 7.450 356 13 17.8 17.8 17.8 3.8 3.9 3.85 0 362 43 22.4 22.1 22.25 5.8 6 5.9 0 349 69 21.5 21.5 21.5 8 8.2 8.10 370 15 18.9 19.2 19.05 4.1 4.2 4.15 0 358 27 19.5 19.4 19.45 5.7 6 5.85 0 383 51 21.7 21.6 21.65 7 7.3 7.150 386 10 19.7 19.7 19.7 4.1 4.1 4.1 0 367 33 25.1 24.8 24.95 6.2 6.3 6.25 0 368 54 20.1 19.9 20 7.2 7.5 7.350 357 17 16.1 16.2 16.15 3.2 3.2 3.2 0 355 44 25.6 26 25.8 6.5 6.5 6.5 0 358 48 18.7 18.5 18.6 7.2 7.4 7.330 67 20 21.9 21.9 21.9 4.5 4.4 4.45 30 83 31 21.1 21.3 21.2 7.2 7 7.1 30 96 80 22.6 22.9 22.75 7.2 7.3 7.2530 98 19 20.7 20.7 20.7 4.2 4.3 4.25 30 73 40 23.1 22.9 23 6.4 6.3 6.35 30 82 21.5 21.8 21.65 7.9 8.1 830 425 7 16.7 16.7 16.7 3.7 3.5 3.6 30 418 31 20.9 20.7 20.8 5.7 5.9 5.8 30 414 23.7 24.1 23.9 7.1 7.1 7.130 442 7 15.4 15.3 15.35 3.1 3.1 3.1 30 417 31 21.1 20.7 20.9 5.9 5.7 5.8 30 432 16.8 16.3 16.55 5.6 5.8 5.730 82 17 20.7 21.2 20.95 4.2 4.2 4.2 30 86 40 19.1 19.7 19.4 6.6 6.3 6.45 30 97 23.3 23.4 23.35 7.3 7.4 7.3530 418 13 18 18 18 4.1 4 4.05 30 82 39 23.3 23.6 23.45 6.6 7.3 6.95 30 71 23.1 23.6 23.35 8 8.1 8.0530 414 16 19.9 20 19.95 3.9 3.8 3.85 30 67 40 22.2 21.6 21.9 6.1 6.8 6.45 30 416 20 20.5 20.25 8.7 8.8 8.7530 416 12 18.1 18 18.05 3.5 3.5 3.5 30 409 40 19.3 19 19.15 6.2 6.5 6.35 30 438 19.9 20.1 20 5.9 5.8 5.8530 422 13 18.4 18.3 18.35 3.7 3.6 3.65 30 94 41 21.1 21.2 21.15 7 6.9 6.95 30 87 24.5 24.3 24.4 7.5 7.6 7.5530 71 18 19.8 19.8 19.8 4.5 4.4 4.45 30 23 36 21.9 22.6 22.25 7.1 7.2 7.15 30 424 20.3 20.4 20.35 5.9 5.8 5.8560 283 17 20.6 20.4 20.5 3.6 3.7 3.65 60 510 16 16.6 16.3 16.45 4.8 5.1 4.95 60 262 57 21.9 21.8 21.85 6.7 6.9 6.860 289 16 18.7 19.3 19 4 3.9 3.95 60 296 25 18.4 17.3 17.85 6.7 6.4 6.55 60 268 24 24.4 24.2 6.5 6.7 6.660 305 7 16.3 16.3 16.3 3.6 3.6 3.6 60 309 17 15.9 15.4 15.65 4.9 5.2 5.05 60 486 21.4 21.1 21.25 6.5 6.8 6.6560 249 20 20.3 20.5 20.4 4.5 4.5 4.5 60 282 28 17.8 17.7 17.75 7.4 6.9 7.15 60 266 19.1 19.5 19.3 8.5 8.5 8.560 263 15 18.8 18.9 18.85 3.9 4 3.95 60 223 30 21.3 21.5 21.4 6.2 6.7 6.45 60 298 44 20.5 20.7 20.6 7.1 6.9 760 257 22 21.5 21.5 21.5 4 4.1 4.05 60 269 31 21.2 21.5 21.35 7.1 6.9 7 60 300 21.9 21.8 21.85 7.1 7.4 7.2560 307 12 18.9 18.5 18.7 2.9 3.3 3.1 60 273 27 20.9 20.6 20.75 6.8 6.3 6.55 60 103 19.4 19.5 19.45 8.8 8.6 8.760 295 16 19.3 19.5 19.4 3.6 3.7 3.65 60 303 27 16.9 17.2 17.05 6.1 5.7 5.9 60 283 24.4 24.1 24.25 7.2 7.4 7.360 300 13 17.9 18 17.95 3.4 3.6 3.5 60 276 37 22.4 22.5 22.45 7.1 7 7.05 60 104 54 19.5 19.8 19.65 8.6 8.5 8.55B-160 253 20 20.3 20.4 20.35 3.8 4 3.9 60 279 29 19.1 20 19.55 6 6.1 6.05 60 250 50 21.6 21.8 21.7 7.6 7.5 7.55125 338 13 17.4 17.3 17.35 3.4 3.5 3.45 125 344 25 18.1 17.1 17.6 5.2 5.6 5.4 125 305 58 21.8 21.7 21.75 6.2 6.4 6.3125 341 12 17.3 17.3 17.3 3.5 3.6 3.55 125 329 35 17.8 18.4 18.1 5.2 5.5 5.35 125 104 59 20 19.8 19.9 7.9 8.1 8125 343 7 15.3 15.6 15.45 3.6 3.6 3.6 125 252 50 21.2 23.5 22.35 7.2 6.9 7.05 125 269 57 21.5 22 21.75 7.7 7.4 7.55125 282 23 21.5 21.5 21.5 4.9 4.6 4.75 125 330 29 17.4 17.8 17.6 5.8 6.1 5.95 125 289 60 18.6 18.4 18.5 7 7.2 7.1125 252 30 23.9 23.8 23.85 4.4 4.5 4.45 125 256 35 22.2 20.9 21.55 6.6 6 6.3 125 288 64 22.3 22.2 22.25 7 6.7 6.85125 274 23 22.2 22.2 22.2 4.5 4.5 4.5 125 305 32 22.2 22.6 22.4 7.3 7.3 7.3 125 244 66 21.9 22 21.95 7.7 7.5 7.6125 332 14 19.3 19.3 19.3 3.5 3.4 3.45 125 247 41 21.4 21.5 21.45 6.5 6.5 6.5 125 295 64 21.9 21.8 21.85 7.9 7.8 7.85125 313 20 20.8 20.7 20.75 4.1 4.3 4.2 125 314 31 20 20.2 20.1 6.5 6.1 6.3 125 332 51 22.1 22.5 22.3 7 6.8 6.9125 318 20 20.8 20.7 20.75 4.8 4.7 4.75 125 303 34 20.5 20.6 20.55 6.7 6.2 6.45 125 315 57 20.5 20.7 20.6 7.8 8.1 7.95125 289 22 20.9 21 20.95 4.3 4.5 4.4 125 309 36 19.8 19.6 19.7 6.2 6.1 6.15 125 326 53 22.1 22 22.05 7.3 7.5 7.4250 362 21 21 20.9 20.95 4.7 4.7 4.7 250 367 39 19.2 19.3 19.25 5.9 6.4 6.15 250 336 77 17.1 17.2 17.15 5.9 6.1 6250 358 23 21.3 21.5 21.4 4.2 4.2 4.2 250 355 41 20.5 20.2 20.35 6.6 6 6.3 250 368 48 21.5 21.6 21.55 7.4 7.4 7.4250 384 11 16.4 16.5 16.45 3.9 4.1 4 250 383 35 21.6 21 21.3 6.2 6.5 6.35 250 392 67 18.3 18.6 18.45 6.4 6.4 6.4250 406 11 17 17 17 3.4 3.1 3.25 250 360 43 20.8 21 20.9 5.1 5.5 5.3 250 372 53 23.4 23.5 23.45 7 7.1 7.05250 324 27 21.8 21.8 21.8 4.6 4.7 4.65 250 377 37 21.1 21.5 21.3 5.8 6.1 5.95 250 381 55 17.9 17.9 17.9 7.5 7.7 7.6250 383 19 19.2 19.2 19.2 4.3 4.5 4.4 250 368 39 21.3 20.8 21.05 6.6 6.3 6.45 250 306 56 23 23.2 23.1 7.4 7.2 7.3250 367 21 20 19.8 19.9 4.5 4.8 4.65 250 408 25 19.3 19 19.15 4.7 5.2 4.95 250 358 61 22.9 23.3 23.1 7.1 7.2 7.15250 390 16 17.7 17.9 17.8 3.9 4 3.95 250 336 28 18.9 19 18.95 6.3 6.1 6.2 250 406 54 21 20.9 20.95 6.2 6.4 6.3250 393 12 17.3 17.4 17.35 3.8 3.7 3.75 250 392 26 19.7 18.8 19.25 4.4 4.9 4.65 250 104 61 18 18.1 18.05 7.3 7.2 7.25250 368 22 21.1 21.2 21.15 3.8 3.8 3.8 250 365 39 23 23 23 5.8 6 5.9 250 393 48 22.4 22.7 22.55 7.2 7 7.1F1 growth measurements7/27/098/3/20098/11/2009SEXDOSEIDBWtarsus1tarsus2avgtarsusculmen1culmen2avgculmenSEXDOSEIDBWtarsus1tarsus2avgtarsusculmen1culmen2avgculmenSEXDOSEIDBWtarsus1tarsus2avgtarsusculmen1culmen2avgculmenF 0204 72 33 33 33 6.6 6.9 6.75 F 0207 96 33.1 33.1 33.1 7.2 7.4 7.3 F 0209105 32.8 33 32.9 7.1 7.5 7.3F 0230 83 34.2 34.1 34.15 6.3 6.4 6.35 F 0209 88 32.8 32.7 32.75 7.5 7.9 7.7 F 0211120 35.1 34.9 35 7.3 7.3 7.3F 0248 83 33.2 33.3 33.25 6.5 6.8 6.65 F 0210103 34.5 34.5 34.5 8 7.9 7.95 F 0217115 33.5 33.6 33.55 7.5 7.7 7.6F 0250 86 33 33.2 33.1 6.5 6.4 6.45 F 0211114 34.7 34.8 34.75 7.6 7.7 7.65 F 0219127 33.3 33.6 33.45 7.2 7.4 7.3F 0272 75 31.5 31.8 31.65 6.5 6.8 6.65 F 0215 87 32 32 32 7.3 7.1 7.2 F 0226119 34.9 34.9 34.9 7.1 6.9 7F 0275 79 32 31.7 31.85 7.1 6.9 7 F 0217 88 33.8 33.7 33.75 7.3 7.4 7.35 F 0234118 32.7 32.8 32.75 7.2 7.1 7.15F 0276 77 33.1 33 33.05 7.4 7.3 7.35 F 0219107 33.9 34 33.95 7.6 7.8 7.7 F 0235115 33 33 33 7.8 7.5 7.65F 0292 78 33 33 33 6.8 6.9 6.85 F 0226110 34.9 34.8 34.85 7.1 7.3 7.2 F 0237101 33.5 33.5 33.5 7.9 7.6 7.75B-1F 0297 77 32.4 32.4 32.4 6.3 6.6 6.45 F 0242107 32.5 32.6 32.55 7.6 7.6 7.6 F 0242117 32.4 32.5 32.45 7.9 7.6 7.75F 0299 72 31.1 31 31.05 7.6 7.4 7.5 F 0299 73 31.1 31.1 31.1 8.3 8.4 8.35 F 0245102 34 34.1 34.05 7.2 6.9 7.05F 30271 89 34.1 34.1 34.1 6.9 7 6.95 F 30206 89 31.3 31.4 31.35 8.8 8.7 8.75 F 30202113 33.5 33.3 33.4 6.9 7.1 7F 30273 64 30.6 30.6 30.6 6.4 6.2 6.3 F 30211 88 33.9 33.5 33.7 7.8 7.8 7.8 F 30212103 32.8 32.8 32.8 8.1 8.3 8.2F 30276 75 31.7 32.2 31.95 6.9 6.9 6.9 F 30212 90 32.4 32.5 32.45 7.8 8 7.9 F 30220128 32.9 32.9 32.9 6.9 7 6.95F 30280 81 33.2 33.2 33.2 7.4 7.3 7.35 F 30225 93 32.8 32.8 32.8 7.3 7.5 7.4 F 30226113 33.8 33.6 33.7 7.2 7.2 7.2F 30286 69 32.9 32.9 32.9 7 7 7 F 30226101 33.6 33.7 33.65 7.4 7.3 7.35 F 30230106 33.9 34 33.95 7.2 7.3 7.25F 30288 75 34.1 34.1 34.1 7.1 6.9 7 F 30231 87 33.6 33.8 33.7 8.2 8.1 8.15 F 30232111 33.5 33.5 33.5 7.1 7.4 7.25F 30290 80 32.4 32.5 32.45 7.1 7.1 7.1 F 30235103 34.7 34.7 34.7 7.8 7.7 7.75 F 30242110 33.6 33.5 33.55 7.2 7.2 7.2F 30291 81 33.3 33.3 33.3 6.6 6.5 6.55 F 30242 89 33 33.4 33.2 7.3 7.3 7.3 F 30245109 33.4 33.4 33.4 6.9 6.9 6.9F 30364 74 31.8 31.8 31.8 6.8 6.9 6.85 F 30247 98 34.3 34.4 34.35 7.4 7.5 7.45 F 30251112 33.4 33.4 33.4 6.9 7 6.95F 30369 76 33.1 33 33.05 6.7 6.9 6.8 F 30253 93 32.9 32.7 32.8 7.4 7.4 7.4 F 30253 98 33.2 33.1 33.15 7.3 7.5 7.4F 60105 73 33.8 33.8 33.8 6.6 7 6.8 F 60102101 31.7 31.7 31.7 8.1 8 8.05 F 60102122 31.6 31.6 31.6 8 8 8F 60122 78 33 32.9 32.95 7.1 7.1 7.1 F 60110115 34.3 34.3 34.3 7.4 7.1 7.25 F 60112104 33.4 33.5 33.45 7 7 7F 60124 71 33.7 34 33.85 6.9 6.9 6.9 F 60115109 35.2 35.7 35.45 7.4 7.5 7.45 F 60119127 35.5 35.5 35.5 8.1 8.2 8.15F 60139 62 34.1 34.3 34.2 7.3 7.5 7.4 F 60170 87 32.6 32.7 32.65 7.1 6.9 7 F 60162116 32.8 32.8 32.8 6.7 6.9 6.8F 60142 72 32.2 32 32.1 6.8 7 6.9 F 60174 88 33.1 33.1 33.1 7.9 7.3 7.6 F 60165105 32.2 32.2 32.2 7.8 7.8 7.8F 60143 66 31.3 31.1 31.2 7.4 7.3 7.35 F 60181113 33.1 33 33.05 7.8 8 7.9 F 60166124 33.4 33.3 33.35 7.4 7.4 7.4F 60153 70 32.8 32.9 32.85 6.7 6.9 6.8 F 60183101 33.6 33.6 33.6 6.9 6.8 6.85 F 60175121 33.3 33.6 33.45 7.7 7.4 7.55F 60154 50 29.2 29.4 29.3 5.9 5.8 5.85 F 60184 96 33 32.9 32.95 7.4 7.1 7.25 F 60182124 34.1 34.1 34.1 6.9 6.6 6.75F 60158 72 33.5 33.4 33.45 7.1 7.2 7.15 F 60185 97 33.6 33.5 33.55 7.5 7.2 7.35 F 60185115 33.8 33.8 33.8 7.3 7.4 7.35F 60195 55 30.2 30.2 30.2 7 6.9 6.95 F 60189 94 32.9 32.9 32.9 7.6 7.8 7.7 F 60186127 33.9 33.9 33.9 7.4 7.2 7.3F 125121 77 32.7 32.8 32.75 7.2 6.9 7.05 F 125101106 32.9 32.9 32.9 7.1 7.2 7.15 F 125101133 33.4 33.4 33.4 7.4 7.4 7.4F 125125 77 34.3 34.4 34.35 6.5 6.8 6.65 F 125103112 34.2 34.2 34.2 7.5 7.1 7.3 F 125103129 34 34.4 34.2 7.4 7.4 7.4F 125138 85 34.1 33.9 34 7 7.2 7.1 F 125106115 34.6 34.8 34.7 7.4 7.8 7.6 F 125106122 34.6 34.7 34.65 7.6 7.6 7.6F 125139 82 35.6 35.6 35.6 7 7 7 F 125107 90 32.4 32.4 32.4 7.1 7 7.05 F 125107107 32.3 32.1 32.2 6.6 6.6 6.6F 125143 77 32.1 32.6 32.35 6.3 6.1 6.2 F 125110 85 32.1 32.1 32.1 7.8 7.5 7.65 F 125110107 32.1 32 32.05 7.8 7.8 7.8F 125150 54 28.6 28.6 28.6 5.9 5.7 5.8 F 125160 98 32.5 32.5 32.5 7 7.2 7.1 F 125160115 32.3 32.3 32.3 7.4 7.3 7.35F 125178 81 34 34 34 8 7.6 7.8 F 125163 97 33.7 33.3 33.5 7.3 7.2 7.25 F 125163113 33.3 33.5 33.4 6.8 7.2 7F 125215 90 33.3 33.4 33.35 6.8 6.6 6.7 F 125165 94 33.8 33.8 33.8 6.9 7 6.95 F 125165100 33.7 33.7 33.7 8.2 8.4 8.3F 125220 81 34.3 34.2 34.25 7.2 7.3 7.25 F 125167 98 34.1 34.1 34.1 6.4 6.4 6.4 F 125167121 33.7 33.7 33.7 7.1 7.1 7.1F 125226 88 33.6 33.6 33.6 7.7 7.4 7.55 F 125191 99 33.2 33.2 33.2 7.9 7.5 7.7 F 125191108 33.2 33 33.1 7.7 7.8 7.75F 250122 80 32.6 32.6 32.6 7 7.1 7.05 F 250107 99 34.1 34 34.05 7.1 7.4 7.25 F 250107102 34.2 34.2 34.2 7.2 7.4 7.3F 250125 90 33.1 33 33.05 7.2 7.1 7.15 F 250114106 33.9 33.9 33.9 7.5 7.4 7.45 F 250114111 33.3 33.4 33.35 7.1 7 7.05B-1F 250143 83 33.3 33.3 33.3 6.9 6.9 6.9 F 250115110 34.5 34.7 34.6 7.3 7.5 7.4 F 250115120 34.1 33.8 33.95 6.5 6.8 6.65F 250145 84 32 31.8 31.9 6.2 6.3 6.25 F 250119104 34.6 34.5 34.55 7.6 7.9 7.75 F 250119109 34.4 34.4 34.4 7 7.3 7.15F 250147 89 33.1 33 33.05 6.7 6.5 6.6 F 250152 89 33.6 33.5 33.55 7.1 7.2 7.15 F 250152111 33.8 33.6 33.7 6.7 6.5 6.6F 250166 65 32.6 32.6 32.6 7.5 7.5 7.5 F 250154 97 34.7 34.7 34.7 7.4 7.7 7.55 F 250154105 34.5 34.6 34.55 7.8 7.8 7.8F 250170 68 31.8 31.8 31.8 6.6 6.3 6.45 F 250159103 34.1 34.2 34.15 7.9 7.9 7.9 F 250159122 34.1 34.2 34.15 7.4 7.3 7.35F 250171 86 32.4 32.4 32.4 7.1 7.2 7.15 F 250161109 34.7 34.8 34.75 7.4 7.5 7.45 F 250161127 34.6 34.4 34.5 7.2 7.2 7.2F 250176 91 32.4 32.3 32.35 6.7 6.9 6.8 F 250171 94 32.8 32.7 32.75 6.9 7.1 7 F 250171104 32.5 32.5 32.5 7.5 7.5 7.5F 250179 71 32.3 32.4 32.35 6.4 6.4 6.4 F 250193 98 33.2 33.2 33.2 6.9 7 6.95 F 250193123 32.9 32.9 32.9 6.9 7.1 7M 0201 80 32.4 32.6 32.5 7.4 7.4 7.4 M 0205 88 32.5 32.5 32.5 6.9 6.9 6.9 M 0216 93 33.3 33.4 33.35 7.1 7.1 7.1M 0232 75 30.9 30.7 30.8 6.9 6.5 6.7 M 0206 96 34.1 34.2 34.15 7.2 7.5 7.35 M 0218 97 33.2 33.2 33.2 7.2 7.2 7.2M 0249 85 34.2 34.2 34.2 6.7 6.7 6.7 M 0213 95 33.1 33 33.05 7 7 7 M 0221103 32.9 33 32.95 6.6 7 6.8M 0262 69 31.8 31.9 31.85 5.9 6.2 6.05 M 0214102 34.8 34.8 34.8 7.9 7.7 7.8 M 0222 89 33.5 33.5 33.5 6.9 6.9 6.9M 0265 87 32.5 33.3 32.9 7 7 7 M 0218 94 33.1 33.4 33.25 7.6 7.9 7.75 M 0224 93 33.7 33.6 33.65 6.6 6.7 6.65M 0267 60 29.5 29.4 29.45 5.9 5.9 5.9 M 0221 99 32.8 32.8 32.8 6.9 6.6 6.75 M 0227 98 32.6 32.6 32.6 6.8 6.9 6.85M 0271 74 32 32 32 6.6 6.7 6.65 M 0232 85 31.6 31.7 31.65 7.1 7.1 7.1 M 0229 91 33.3 33.4 33.35 6.8 6.9 6.85M 0279 69 32.2 32.1 32.15 7.3 7.5 7.4 M 0244 87 32.6 32.6 32.6 7.4 7 7.2 M 0236 80 33.7 33.6 33.65 7 6.9 6.95M 0280 80 32.8 32.9 32.85 7 7.3 7.15 M 0246 89 33.8 33.8 33.8 6.9 6.8 6.85 M 0240 95 31.5 31.5 31.5 6.7 6.5 6.6M 0291 76 31.8 32 31.9 7 6.9 6.95 M 0279 76 32.3 32.4 32.35 7.6 7.8 7.7 M 0280103 33.2 33.2 33.2 6.9 6.8 6.85M 30123 75 32 32 32 6.4 6.3 6.35 M 30109 96 33.3 33.3 33.3 7.1 7 7.05 M 30109100 33.6 33.5 33.55 7.4 7.2 7.3M 30129 78 32.2 32.2 32.2 6.5 6.3 6.4 M 30114106 32.8 33.3 33.05 6.4 6.6 6.5 M 30114110 32.9 32.9 32.9 6.3 6.7 6.5M 30141 75 32.7 32.6 32.65 6.5 6.4 6.45 M 30155 88 31.2 31.3 31.25 6.7 6.6 6.65 M 30155 99 31.2 31.1 31.15 7.5 7.4 7.45M 30145 75 32 32.1 32.05 7.1 7.1 7.1 M 30170 91 32.2 32.3 32.25 7.2 7.2 7.2 M 30170101 32.4 32.4 32.4 7.4 7.4 7.4M 30147 80 32.7 32.8 32.75 6.9 6.6 6.75 M 30182 91 32.5 32.6 32.55 6.8 6.4 6.6 M 30182101 32.5 32.3 32.4 6.9 6.6 6.75M 30212 74 32.1 32.2 32.15 6.9 7 6.95 M 30184 96 33 33.1 33.05 7.6 7.4 7.5 M 30184103 33.3 33.3 33.3 7.6 7.6 7.6M 30224 70 31.6 31.6 31.6 7 6.8 6.9 M 30185102 33 33 33 7.8 7.5 7.65 M 30185106 33.1 32.9 33 7.8 7.9 7.85M 30225 63 29.8 29.8 29.8 6.6 6.9 6.75 M 30188 96 32.4 32.3 32.35 6.7 6.5 6.6 M 30188100 32.4 33 32.7 6.6 6.7 6.65M 30228 81 33.1 33.1 33.1 7 6.9 6.95 M 30189 93 33.1 33.1 33.1 7.7 7.6 7.65 M 30189 97 32.9 33 32.95 7.4 7.4 7.4M 30255 79 32.3 32.2 32.25 6.1 6.4 6.25 M 30193 97 33.1 33.2 33.15 6.9 7.3 7.1 M 30193112 33.2 33 33.1 7.2 7.5 7.35M 60104 83 32.5 32.4 32.45 7.1 7.4 7.25 M 60106106 33 32.9 32.95 7.3 7.5 7.4 M 60105103 32.6 32.9 32.75 8.1 8.1 8.1M 60121 79 32.8 32.7 32.75 6.6 6.7 6.65 M 60109 92 33.4 33.6 33.5 6.8 7 6.9 M 60111104 33.3 33.3 33.3 7.1 7 7.05M 60127 75 33.3 33.3 33.3 7.2 6.9 7.05 M 60116 92 33.6 33.7 33.65 7.4 7 7.2 M 60112 95 32.7 32.7 32.7 6.7 6.6 6.65M 60149 88 31.7 31.8 31.75 6.7 6.8 6.75 M 60118 82 33 33 33 7.4 703 355.2 M 60118100 33 33.6 33.3 8.1 8 8.05M 60163 81 31.8 31.9 31.85 7.2 7 7.1 M 60120 93 32 32.3 32.15 6.7 6.6 6.65 M 60120 98 32.4 32.3 32.35 7.2 7.4 7.3M 60167 66 32.1 31.9 32 7 6.9 6.95 M 60131 93 32.6 32.7 32.65 6.9 6.9 6.9 M 60135 93 32.4 32.5 32.45 7.2 6.9 7.05B-1M 60168 84 33.4 33.3 33.35 7.3 7.3 7.3 M 60132 93 32.4 32.4 32.4 7 7 7 M 60138 95 32.4 32.3 32.35 3.4 6.4 4.9M 60169 83 33.3 33.2 33.25 7.1 7.2 7.15 M 60135 92 32.6 32.5 32.55 7.1 6.9 7 M 60140100 34.1 34 34.05 7.4 7.5 7.45M 60177 82 33 33.1 33.05 7.1 6.9 7 M 60140 93 34 33.9 33.95 7 7 7 M 60141102 32.4 32.4 32.4 4.1 7.1 5.6M 60195 82 31.7 31.6 31.65 6.6 6.7 6.65 M 60155 95 32.4 33 32.7 7 7.2 7.1 M 60158 99 32.7 32.6 32.65 7.2 7.1 7.15M 125121 71 31.7 31.7 31.7 7.5 7.5 7.5 M 125111103 33.7 33.6 33.65 7.6 7.7 7.65 M 125111108 33.9 33.7 33.8 7.3 7.5 7.4M 125128 67 32.8 32.9 32.85 7.4 7.3 7.35 M 125114 82 31 30.9 30.95 7.2 7.4 7.3 M 125114 89 30.4 30 30.2 7 7.4 7.2M 125132 74 33 32.8 32.9 6.8 7 6.9 M 125116 80 32 32.1 32.05 6.6 6.9 6.75 M 125116 95 32.1 32.2 32.15 6.9 7 6.95M 125134 54 31.3 31.4 31.35 6.5 6.5 6.5 M 125151100 33.3 33.4 33.35 8.1 7.8 7.95 M 125151107 33.3 33.3 33.3 7.5 7.7 7.6M 125136 79 33 33.1 33.05 6.7 6.6 6.65 M 125164 86 32 32.4 32.2 7.6 7.5 7.55 M 125164106 32.3 32.3 32.3 8.2 7.8 8M 125149 69 31.1 31.2 31.15 7 6.8 6.9 M 125167 98 33.3 33.3 33.3 7 7.1 7.05 M 125167 98 33.5 33.7 33.6 7.1 6.9 7M 125193 72 31.4 31.5 31.45 6.5 6.5 6.5 M 125176112 34.3 34.1 34.2 6.9 6.9 6.9 M 125176111 34.7 34.5 34.6 7.1 7.1 7.1M 125196 61 31.6 31.6 31.6 6.6 6.4 6.5 M 125178 98 33.2 33.2 33.2 6.9 7.1 7 M 125178100 32.9 33 32.95 7.7 7.7 7.7M 125213 65 31.4 31.4 31.4 6.8 7.1 6.95 M 125187 99 33.6 33.5 33.55 7.4 7.3 7.35 M 125187100 33.6 33.6 33.6 7.4 7.4 7.4M 125214 67 32.7 32.7 32.7 6.6 6.5 6.55 M 125190 95 33.4 33.4 33.4 7.4 7.4 7.4 M 125190101 33.6 33.4 33.5 7.3 7 7.15M 250267 79 34.2 34.3 34.25 7.2 6.8 7 M 250205 93 34.1 34 34.05 7.3 7.1 7.2 M 250205103 34.1 34.1 34.1 7.5 7.5 7.5M 250275 85 31.5 31.5 31.5 7.3 7.5 7.4 M 250215 89 32.9 32.6 32.75 7.1 7 7.05 M 250215108 33 33 33 8 8.1 8.05M 250279 69 32 32.1 32.05 6.5 6.6 6.55 M 250223 93 33.8 33.9 33.85 7.3 7.4 7.35 M 250223 97 33.4 33.3 33.35 7.4 7.1 7.25M 250285 73 32.9 33.3 33.1 7 6.7 6.85 M 250237 96 33.6 33.6 33.6 6.8 6.7 6.75 M 250237105 33.7 33.7 33.7 7.7 7.6 7.65M 250294 80 32.1 32.4 32.25 7.6 7.3 7.45 M 250244 90 32.9 33 32.95 6.7 7 6.85 M 250244 98 32.9 32.9 32.9 7.4 7.3 7.35M 250296 81 31.1 31.2 31.15 6.3 6.6 6.45 M 250246 96 34.2 34.2 34.2 8 7.8 7.9 M 250246104 34.1 34.4 34.25 7.4 7.2 7.3M 250297 74 32.3 32.5 32.4 6.7 6.5 6.6 M 250255 87 31.8 31.6 31.7 7.6 7.3 7.45 M 250255 94 32 32.2 32.1 7.3 7.5 7.4M 250322 76 30.8 30.8 30.8 7.2 7.3 7.25 M 250257 96 33.7 33.7 33.7 7.3 7.2 7.25 M 250257100 33.6 33.5 33.55 7 6.9 6.95M 250326 68 30.3 30.2 30.25 6.3 6 6.15 M 250282 92 32.6 32.5 32.55 7.1 7.3 7.2 M 250282 96 32.5 32.4 32.45 7.3 7.2 7.25M 250327 79 32.8 32.8 32.8 6.8 6.7 6.75 M 250325 94 33.2 32.8 33 7.5 7.3 7.4 M 250325102 32.7 32.7 32.7 7.5 7.3 7.4F1 bird weights8/19 8/24 9/1 9/9 9/14 9/21 9/29 10/6 10/13 10/20 10/27 11/2 11/10 11/17 11/248/19 8/24 9/1 9/9 9/14 9/21 9/29 10/6 10/13 10/20 10/27 11/2 11/10 11/17 11/24SEXDOSEIDBWBWBWBWBWBWBWBWBWBWBWBWBWBWBWSEXDOSEIDBWBWBWBWBWBWBWBWBWBWBWBWBWBWBWM 0 221 110 113 114 113 116 117 114 116 118 117 117 116 116 122 114F 0 217 128 129 122 129 126 134 125 135 138 134 135 139 127 135 119M 0 218 105 107 106 105 108 107 107 109 110 110 110 111 109 111 107F 0 211 137 138 137 144 142 123 135 145 144 144 146 146 143 147 125M 0 236 104 108 108 107 109 108 107 111 112 111 113 115 113 116 111F 0 234 114 130 124 131 127 133 134 134 125 133 127 134 127 128M 0 224 110 115 116 116 115 116 111 113 116 115 118 117 114 120 115F 0 237 122 126 138 127 136 137 131 132 144 124M 0 216 100 102 104 106 105 104 104 109 110 109 109 110 104 104 101F 0 235 113 114 120 120 118 130 126 128 134 133 128 124 132 121 117M 0 227 101 101 103 102 106 101 105 109 111 109 110 111 111 113 110F 0 245 112 128 127 136 135 131 133 142 146 142 148 132B-1M 0 280 104 103 106 106 108 104 107 113 109 114 115 113 115 118 117F 0 219 101 121 127 133 132 126 137 136 137 137 136 140 125 140 130M 0 240 97 98 98 98 103 103 101 100 102 103 102 102 103 102 104F 0 242 115 132 128 130 132 132 127 119 134 135 135 139 140 148 151M 0 222 95 97 99 97 98 101 99 101 102 101 101 104 103 104 103F 0 226 123 132 133 126 136 138 138 142 143 143 145 145 142 145 139M 0 229 99 101 102 103 104 103 101 101 104 104 105 105 105 107 108F 0 209 130 123 125 130 126 135 134 137 138 136 138 138 141 138 140M 0 243 98 101 106 107 108 108 98 102 103 103 105 107 106 108 110F 0 215 103 119 123 127 124 125 127 130 125 126 124 123 119 128 126M 0 220 109 111 112 111 113 117 115 116 117 116 119 120 122 123 122F 0 299 86 92 106 114 116 119 119 122 122 124 123 117 126 118 117M 0 213 103 104 107 104 104 104F 0 203 123 136 134 129 136 139 138 141 141 132 145 142 142 140 138M 0 279 90 89 93 94 96 93 93 94 96 94 97 97 91 94F 0 228 121 130 130 101 129 129 129 130 131 132 120 132 128 137 109M 0 214 115 116 117 117 117 118 112 116 119 120 120 121 116 121 113F 0 225 128 137 135 139 134 134 137 129 146 144 142 142 139 150 129M 0 232 97 99 99 99 100 100 104 105 108 107 104 103 104 108 101F 0 241 133 136 129 129 124 127 134 147 128 141 143 143 132 143 130M 0 244 97 98 101 104 104 103 103 103 105 106 108 143 104 107 100F 0 212 124 128 129 118 131 131 131 123 134 132 136 128 135 131 127M 0 206 105 110 110 111 113 112 107 108 111 114 112 113 112 115 110F 0 202 124 129 133 130 134 136 123 132 135 130 133 123 122 122 107M 0 246 102 103 102 104 104 101 102 104 107 106 105 108 109F 0 207 115 120 133 129 132 136 118 106 136 134 108 131 122 134M 0 205 97 98 99 98 99 98 97 100 162 99 101 102 104 106F 0 210 115 129 136M 30 257 104 105 104 104 104 105 106 110 110 105 109 109 109 109 110F 30 253 108 109 114 125 128 128 132 134 138 136 135 139 141 133 129M 30 255 99 102 103 103 105 106 104 107 107 108 108 110 112 133 114F 30 226 144 143 148 139 144 140 141 147 143 143 132 148 149 146 148M 30 246 109 110 109 110 114 112 110 115 117 113 115 116 116 118 117F 30 212 107 124 138 136 126 136 138 134 120 140 127 118 125 150 142M 30 237 113 117 118 116 119 116 116 118 117 118 120 120 121 125 125F 30 242 116 126 144 137 142 138 130 144 125 146 145 146 129 145 149M 30 325 112 115 115 116 116 115 115 120 119 117 119 121 123 122 123F 30 202 128 140 136 139 138 130 138 141 139 139 138 143 138 145 145M 30 282 104 106 109 106 108 108 105 110 109 110 110 113 109 111 112F 30 230 136 137 137 138 133 141 139 146 143 146 146 119 120 151 148M 30 223 108 109 108 111 111 115 106 110 112 110 111 111 111 114 112F 30 232 115 120 145 138 145 140 131 145 146 146 147 149 146 105 151M 30 244 97 100 102 100 102 100 105 105 106 107 107 106 106 109 109F 30 220 126 123 138 134 130 134 128 135 134 135 135 136 133 141 138M 30 215 97 115 117 117 116 112 118 119 119 121 121 118 122 125 121F 30 245 125 125 127 124 127 127 130 129 133 132 131 133 133 134 134M 30 205 105 112 113 111 112 113 113 115 115 115 114 116 113 117 118F 30 251 137 126 139 136 139 138 146 138 139 149 140 153 151 146M 30 224 102 107 108 108 110 103 109 110 111 109 112 111 112 113 111F 30 227 126 121 133 126 134 130 137 135 129 138 136 132 138 141 135M 30 233 97 104 105 103 105 108 105 108 109 109 110 109 110 110 109F 30 250 130 143 142 144 142 146 139 144 139 149 146 149 153 152 143M 30 219 102 103 104 104 106 108 108 106 106 104 103 105 104 106 107F 30 247 127 131 137 139 139 139 145 143 145 142 145 145 146 147 140M 30 248 101 102 105 106 108 104 107 110 111 111 111 112 114 114 116F 30 211 127 140 136 136 135 138 137 133 139 145 141 149 144 144 151M 30 216 113 115 117 115 115 115 111 115 118 116 117 120 122 122 123F 30 231 115 131 142 144 145 137 139 147 136 150 141 149 148 150 151M 30 217 101 106 109 112 107 108 101 107 109 108 110 109 111 115F 30 235 135 133 143 137 135 142 144 133 147 147 149 148 137 156 149M 30 240 98 99 102 99 102 104 102 108 107 105 108 108 108 108 107F 30 225 115 120 126 119 127 125 127 131 130 129 132 134 127 138 137M 30 272 104 105 105 103 103 101 100 104 103 108 107 100 96 107F 30 206 110 124 127 128 123 140 136 130 123 134 139 142 129 139 140M 30 204 109 113 113 106 110 87 93 113 115 120 121 122 123 123 123F 30 249 129 130 140 140 138 134 137 128 134 136 117 130 144 148 145M 30 209 100 103 108 108 110 100 106 105 106 107 107 107 109 110 110F 30 221 127 133 134 138 132 141 137 145 143 136 144 140 145 151 150B-1M 60 176 113 117 119 119 120 114 99 120 123 130 129 128 131 130F 60 162 96 112 132 133 131 129 111 134 113 134 134 137 136 132 136M 60 167 102 103 103 103 107 102 108 109 108 109 112 112 118 116F 60 166 128 133 134 134 135 132 136 134 137 144 139 140 133 145 144M 60 116 101 102 103 100 101 101 100 103 105 104 106 106 105 110 107F 60 182 135 132 139 131 138 103 108 125 128 127 114 101 108 114 118M 60 111 117 120 121 121 123 122 116 120 122 123 114 123 124 127 123F 60 112 118 120 128 131 126 133 128 129 123 140 126 132 136 134 134M 60 164 104 105 108 108 110 109 107 108 112 114 124 116 116 120 118F 60 119 139 140 145 142 137 147 133 106 142 129 146 151 142 130M 60 151 116 121 127 130 133 131 128 129 127 128 124 126 127 130F 60 186 135 134 140 135 146 143 147 136 137 149 146 142 150 151M 60 114 97 98 100 100 101 101 99 100 102 102 103 104 102 106 104F 60 165 113 115 113 115 102 119 117 116 125 125 124 125 112 119 123M 60 187 103 104 104 105 105 106 108 110 112 112 112 114 115 118 117F 60 175 134 131 136 135 133 131 121 146 121 144 126 148 142 133 152M 60 190 107 108 107 106 107 110 108 112 114 110 114 105 109 118 118F 60 102 102 125 132 122 134 129 129 136 135 193 137 135 140 144 134M 60 178 105 107 110 111 110 109 112 115 113 114 107 115 114F 60 185 185 132 133 134 134 137 132 134 131 132 121 108 109 129 137M 60 177 103 105 107 107 106 104 106 110 109 112 113 95 102 114 111F 60 184 184 124 137 136 136 133 125 137 136 136 137 137 133 137 144M 60 180 96 99 100 100 99 97 98 99 99 101 101 94 97 103 105F 60 181 181 128 135 120 137 136 124 131 140 141 137 101 110 139 126M 60 161 110 118 124 125 129 126 126 131 135 135 136 132 130 137F 60 183 183 123 131 139 136 142 141 141 146 146 143 109 125 131 150M 60 109 101 101 104 104 103 104 107 109 109 110 111 96 105 113 114F 60 174 174 110 111 121 112 121 122 126 125 127 121 101 125 115 113M 60 173 110 113 116 114 117 117 115 118 114 113 116 104 113 116 119F 60 189 109 121 119 123 123 122 122 127 118 126 130 96 131 123 122M 60 120 103 105 105 104 105 107 106 109 108 109 109 99 103 110 114F 60 170 130 137 136 129 136 130 121 124 131 137 124 139 133 147 143M 60 169 104 107 110 112 114 112 112 115 118 119 118 112 105 119F 60 110 129 129 137 138 136 137 132 112 133 143 143 113 122 139 139M 60 171 111 112 115 111 112 110 108 110 115 118 120 107 111 118 120F 60 115 136 143 144 131 142 143 140 146 142 144 142 135 139 143 139M 60 117 113 115 116 112 116 116 116 115 117 117 116 107 112 122 121F 60 179 130 114 137 134 118 136 116 142 129 136 135 138 134 144 142M 60 136 107 116 117 116 115 116 114 114 115 118 119 108 119 120 120F 60 172 122 132 119 130 131 134 131 137 128 139 141 111 123 132 133M 125 114 119 120 121 122 123 122 124 126 127 128 127 127 126 127 126F 125 101 131 126 135 131 137 134 134 138 138 139 158 137 128 114 131M 125 109 112 112 115 115 119 121 113 113 116 118 119 116 118 122 118F 125 103 139 143 142 144 138 130 144 148 140 148 106 149 153 135 156M 125 184 113 112 114 115 117 115 112 119 120 118 121 122 120 125 124F 125 110 131 132 127 128 132 138 128 120 132 139 138 140 142 161 134M 125 193 124 128 131 130 134 133 129 130 125 128 127 131 133 129 132F 125 165 120 135 138 136 136 138 133 94M 125 182 103 104 105 105 105 106 107 107 107 105 108 107 109 111 110F 125 107 127 141 132 136 141 138 145 133 148 145 144 135 138 128 146M 125 155 101 105 104 103 104 104 103 138 105 105 104 104 104 109 113F 125 167 119 130 135 132 127 133 129 134 136 139 138 143 132 135 135M 125 170 103 103 101 101 101 88 102 145 105 104 104 100 107 109 108F 125 191 127 136 135 134 136 135 138 127 122 134 141 140 143M 125 185 112 114 114 113 116 114 112 110 111 116 117 114 113 120 119F 125 106 121 121 130 131 141 128 142 136 146 141 142 146 150 152 151M 125 189 102 104 106 103 104 104 104 105 107 109 109 108 108 111 108F 125 160 130 132 135 137 134 138 140 136 141 135 144 142 144 137 142M 125 188 104 108 111 110 112 110 109 110 110 112 114 113 113 115 115F 125 163 132 131 133 135 136 132 130 137 138 137 139 139 138 131 133M 125 133 98 101 101 100 102 101 99 100 99 103 106 105 106 110 110F 125 112 129 139 139 139 142 133 145 102 135 146 147 146 142 149 142M 125 151 98 103 103 102 103 104 102 103 104 105 108 105 106 108F 125 166 122 123 131 133 132 124 144 110 120 137 137 152 142 138 133M 125 183 102 107 107 108 107 106 106 111 110 109 109 111 108 113 111F 125 117 139 137 139 138 130 142 140 144 146 148 141 148 146 145 140M 125 161 100 99 99 97 100 101 100 101 103 102 102 102 103 90 103F 125 156 120 134 129 136 122 136 130 135 140 140 141 137 141 140 139B-1M 125 131 112 109 110 107 110 112 109 113 115 115 113 113 115 116 115F 125 158 120 123 130 136 133 138 109 140 140 138 139 134 144 146 140M 125 118 107 106 111 114 114 109 102 105 109 110 110 112 110 113 115F 125 159 119 129 128 129 136 140 132 135 122 126 114 140 138 135 144M 125 157 109 111 114 112 113 111 111 112 113 112 112 113 112 112 120F 125 135 126 127 131 133 120 133 136 125 122 119 127 138 136 125 115M 125 152 116 118 113 113 111 114 112 117 116 116 117 115 117 118 119F 125 181 124 136 136 137 135 143 137 145 145 141 146 145 148 102M 125 134 108 110 110 109 111 112 113 116 113 112 113 115 116 118 119F 125 111 125 138 143 139 149 146 155 152 146 146 145 137 144 150 133M 125 119 105 107 107 106 107 106 108 107 108 109 110 110 110 113 117F 125 113 128 136 125 138 139 140 145 145 146 149 148 150 147 141 143M 250 158 105 107 110 112 112 109 142 112 112 112 117 116 117 118 118F 250 161 136 136 141 140 137 147 142 141 147 148 151 102 147 153 152M 250 141 106 107 110 109 110 111 110 112 114 113 113 114 116 118 117F 250 152 125 132 133 131 112 139 136 138 137 132 138 133 135 133M 250 138 105 107 110 108 105 103 105 110 113 113 116 115 115 118 117F 250 107 133 122 130 140 137 138 137 141 138 138 139 141 138 129 138M 250 111 105 109 104 103 105 107 109 112 110 116 115 118 117 119 121F 250 115 137 143 136 142 145 139 150 150 151 150 149 145 152 159 148M 250 105 108 110 107 107 108 109 108 110 114 115 113 112 116 115 114F 250 114 123 136 137 135 125 136 139 149 145 139 148 145 146 146 141M 250 112 102 103 102 103 105 104 106 108 110 109 110 111 111 111 114F 250 159 136 138 135 138 139 139 128 140 139 139 140 141 142 144 145M 250 135 100 103 100 101 103 101 103 102 103 103 101 103 103 106 105F 250 193 125 128 131 131 130 132 131 134 140 139 138 140 140 140 134M 250 118 110 111 113 115 116 116 111 114 114 114 117 116 115 120 120F 250 171 120 131 137 139 140 137 140 122 142 144 129 146 142 139 135M 250 140 106 106 107 109 105 107 107 112 115 116 119 115 115 116 119F 250 154 117 121 127 133 124 128 139 131 139 135 128 140 142 144 129M 250 120 97 99 101 100 103 103 102 104 104 104 107 108 107 111 109F 250 119 132 130 140 140 132 143M 250 155 114 118 118 114 118 116 114 105 117 118 118 118 119 119 119F 250 156 149 147 143 149 144 150 149 154 153 151 153 153 154 159 153M 250 109 94 107 99 100 102 102 105 105 107 106 105 106 107 110 109F 250 134 119 134 132 137 122 138 134 131 143 142 143 146 124 141 140M 250 116 105 106 112 112 115 114 114 115 118 117 118 118 119 121 121F 250 110 143 144 148 139 149 149 153M 250 131 109 110 106 105 105 106 105 107 108 109 109 109 111 110 111F 250 102 140 142 142 150 146 150 144 150 148 156 145 154 156 160 158M 250 132 99 104 115 105 104 105 105 104 105 106 105 103 106 109 107F 250 153 126 128 128 124 130 129 126 135 134 139 127 134 138 134 132M 250 106 110 114 114 118 118 117 120 120 121 120 119 121 122 129 129F 250 133 125 122 130 133 124 128 136 137 133 137 132 137 136 142 159M 250 108 111 113 117 115 117 116 115 117 115 118 117 120 118 122 121F 250 151 139 135 141 141 140 134 142 128 143 145 129 146 149 144 146M 250 174 101 106 103 103 102 104 106 103 105 106 106 107 109 112 110F 250 113 130 120 125 128 123 133 131 134 137 137 134 146 138 133 134M 250 177 107 109 111 108 110 108 110 111 111 111 110 112 113 115F 250 122 113 123 140 135 128 139 136 145 144 146 145 147 148 143 151M 250 103 102 104 103 104 105 108 106 107 108 107 107 108 109 110 110F 250 136 143 139 145 136 145 141 149 148 150 147 139 151 150 149 148F1 feed consumption

8/18 8/25 8/31 9/4 9/8 9/14 9/18 9/21

DOSE SEX ID Initial Final Final Initial Final Initial Fina l Initial Final Initial Final Initial Final Initial Final Initial

0 F 217 402 306 434 229 434 348 467 343 438 305 443 335 437 366

0 F 211 387 292 434 296 449 344 451 329 440 292 444 365 433 381

0 F 234 389 293 435 301 456 346 449 334 451 281 442 337 458 376

0 F 237 375 309 428 268 445 344 452 332 439 267 442 309 451 381

0 F 235 395 341 445 332 450 343 454 347 445 299 448 337 431 353

0 F 245 300 347 425 321 452 367 457 357 448 304 450 331 425 355

0 F 219 399 348 433 358 450 362 460 354 453 311 453 361 436 367

0 F 242 400 331 419 300 446 354 465 368 458 298 437 310 434 341

0 F 226 399 299 425 274 443 357 457 344 448 293 448 329 433 355

0 F 209 369 308 438 298 443 351 445 329 440 250 467 341 434 351

0 F 215 367 299 435 361 444 359 456 346 450 296 469 333 447 347

0 F 299 381 367 424 341 433 379 451 365 448 326 435 344 437 368

0 F 203 400 303 431 285 451 332 464 328 455 281 448 300 437 341

0 F 228 391 295 434 295 437 345 444 362 460 349 431 298 421 352

0 F 225 408 343 439 294 438 336 449 332 442 265 444 298 429 336

0 F 241 399 302 447 299 451 356 464 358 450 343 432 346 446 370

0 F 212 395 348 445 317 451 360 457 358 455 317 446 336 441 373

0 F 202 399 328 430 309 435 348 453 339 447 293 458 321 444 356

0 F 207 403 350 454 360 441 362 458 353 447 293 453 327 438 336

0 F 210 390 329 447 343 433 388 455

0 M 221 408 351 445 359 438 350 453 347 436 356 438 331

0 M 218 386 334 423 329 448 348 454 327 445 343 442 314

0 M 236 435 351 430 329 440 341 447 335 447 365 440 332

0 M 224 421 331 421 325 453 352 453 337 447 340 439 297

0 M 216 410 342 426 326 445 339 450 322 441 338 436 307

0 M 227 398 332 425 353 452 356 456 319 442 338 454 326

0 M 280 433 344 435 357 452 357 458 331 437 336 434 313

0 M 240 431 346 437 365 424 348 456 358 442 351 455 344

0 M 222 434 337 430 336 444 341 449 324 442 352 443 321

0 M 229 421 354 439 349 443 346 455 337 440 354 452 337

0 M 243 435 344 430 353 437 348 450 336 442 372 431 343

0 M 220 425 329 432 311 435 314 440 294 438 320 443 283

0 M 213 425 351 446 358 437 343 457 335 440 342 437 311

0 M 279 400 343 451 373 432 345 447 331 443 354 442 309

0 M 214 405 330 435 309 429 305 459 300 443 315 448 290

0 M 232 420 314 438 360 443 353 440 327 448 354 447 312

0 M 244 420 342 439 365 438 343 444 320 447 353 439 331

0 M 206 417 318 440 336 409 302 446 306 427 318 434 321

0 M 246 438 335 444 357 438 338 448 322 441 338 448 320

0 M 205 410 357 441 356 447 357 443 327 444 361 450 328

30 F 253 429 341 446 358 427 388 441 370 424 291 442 342 444 361

30 F 226 411 313 434 254 453 337 447 337 429 275 428 322 439 352

30 F 212 435 376 451 344 438 355 449 345 442 285 439 338 447 379

30 F 242 419 340 460 354 437 361 437 344 436 290 450 334 458 369

30 F 202 423 323 452 289 442 336 437 304 440 256 442 319 440 338

# B-1 30 F 230 395 324 446 313 446 330 453 331 439 278 447 325 440 350

30 F 232 412 341 443 348 455 350 450 349 454 300 443 317 451 373

30 F 220 401 331 450 319 432 345 440 342 441 305 462 353 450 376

30 F 245 418 330 460 343 438 366 456 360 455 302 446 348 461 392

30 F 251 400 325 443 314 440 350 439 344 452 289 439 337 441 360

30 F 250 396 311 443 307 434 320 439 292 446 265 439 308 438 342

30 F 247 410 327 447 315 452 334 447 326 444 291 454 439 447 357

30 F 211 398 323 427 317 436 349 444 350 447 304 420 321 431 360

30 F 231 411 325 449 321 432 352 448 344 450 298 442 310 444 356

30 F 235 405 361 438 327 434 335 448 340 445 299 450 328 446 359

30 F 225 419 329 444 320 453 356 447 349 448 300 439 318 450 368

30 F 206 403 348 441 321 442 353 446 344 449 322 441 340 439 374

30 F 249 410 333 445 318 433 351 448 336 447 301 437 324 455 374

30 F 221 407 312 449 342 442 359 450 346 457 315 461 340 446 366

30 M 257 417 353 421 328 431 338 435 279 444 340 422 286

30 M 255 415 361 437 359 434 351 445 344 450 370 441 314

30 M 246 413 332 440 327 424 326 434 307 450 337 445 301

30 M 237 402 353 449 358 431 339 440 331 445 364 438 321

30 M 325 427 357 444 353 443 347 448 332 462 363 439 310

30 M 282 425 348 436 336 439 331 441 323 449 353 412 300

30 M 223 415 333 440 340 433 332 454 317 448 342 439 312

30 M 244 413 358 444 365 440 355 437 330 446 366 449 345

30 M 215 426 349 441 355 438 351 438 331 451 370 434 332

30 M 205 423 355 435 338 439 344 446 307 447 357 429 292

30 M 224 425 338 433 349 434 350 445 335 440 351 416 323

30 M 233 431 348 423 334 436 329 438 310 446 340 442 305

30 M 219 409 356 442 354 439 348 439 322 435 338 442 306

30 M 248 421 341 437 346 437 342 438 300 456 350 427 328

30 M 216 432 352 431 344 438 348 445 327 453 367 433 334

30 M 217 421 342 438 357 438 341 440 315 459 376 458 344

30 M 240 424 357 422 346 421 338 444 347 443 362 437 323

30 M 272 415 368 446 358 452 359 445 342 443 371 437 339

30 M 204 433 361 445 341 451 356 450 347 456 359 460 355

30 M 209 431 318 439 323 424 295 457 305 453 334 444 316

60 F 162 424 395 443 354 443 352 445 345 440 299 448 336 431 364

60 F 166 417 326 428 303 446 367 439 346 451 316 448 348 443 385

60 F 182 426 321 444 307 440 352 449 345 457 318 451 360 454 429

60 F 112 417 332 436 336 445 363 445 360 454 327 442 342 449 385

60 F 119 407 270 445 255 457 323 453 319 452 268 451 317 435 340

60 F 186 426 320 461 312 451 348 439 351 453 322 453 346 457 381

60 F 165 412 312 441 307 456 347 444 321 458 322 453 327 445 353

60 F 175 424 322 449 309 452 352 448 341 452 305 447 324 440 362

60 F 102 418 311 434 293 436 339 435 317 451 283 429 278 442 355

60 F 185 418 321 443 314 452 334 440 322 442 276 445 333 437 347

60 F 184 425 308 448 309 434 346 453 330 439 277 442 305 438 341

60 F 181 418 329 437 332 440 359 455 359 461 331 438 302 426 344

60 F 183 416 353 452 346 443 355 454 358 447 302 440 299 434 354

60 F 174 416 317 449 320 433 346 453 334 452 319 444 344 444 362

# B-1 60 F 189 430 322 450 326 432 356 437 342 449 296 462 338 436 352

60 F 170 411 323 443 315 442 358 437 329 445 286 446 318 435 351

60 F 110 429 317 438 297 433 350 437 335 441 287 443 336 445 365

60 F 115 428 311 452 306 451 351 450 363 453 301 441 310 448 361

60 F 179 423 303 448 320 444 356 450 335 446 333 438 327 445 367

60 F 172 422 309 449 297 442 359 447 342 445 281 437 277 424 346

60 M 176 424 376 424 334 439 340 441 305 438 324 429 295

60 M 167 423 361 449 352 448 342 452 305 453 336 448 317

60 M 116 405 348 430 349 456 378 454 355 451 383 447 347

60 M 171 409 318 461 334 443 331 436 312 458 356 440 293

60 M 164 421 353 444 340 454 317 444 299 464 335 438 285

60 M 151 412 328 436 317 429 308 436 291 456 346 429 315

60 M 114 420 351 429 345 442 357 446 345 454 381 423 328

60 M 187 401 342 435 336 438 337 443 355 456 355 438 319

60 M 190 413 343 432 350 433 355 445 322 458 353 438 310

60 M 178 416 358 435 355 423 355 438 330 442 345 435 316

60 M 177 431 352 442 348 460 368 452 333 444 358 454 338

60 M 180 411 355 443 364 454 368 452 340 450 362 443 337

60 M 161 413 336 440 350 457 327 444 294 453 315 433 281

60 M 109 406 353 431 334 451 338 435 313 444 342 436 295

60 M 173 413 358 434 337 444 344 444 320 440 337 428 302

60 M 120 423 337 433 325 439 326 440 284 451 311 432 334

60 M 169 419 355 437 329 433 341 445 336 453 366 433 322

60 M 171 412 318 461 334 443 319 457 312 458 356 440 293

60 M 117 423 349 439 349 450 352 456 352 451 355 424 279

60 M 136 416 350 439 342 441 324 447 319 449 354 430 330

125 F 101 416 302 428 289 423 338 448 346 442 294 454 323 445 334

125 F 103 421 344 427 290 429 355 437 345 445 312 462 346 453 364

125 F 110 409 315 436 307 423 353 438 339 444 308 451 346 448 372

125 F 165 403 328 449 331 448 350 447 343 451 323 436 318 459 381

125 F 107 418 338 450 303 432 327 441 314 448 271 449 316 448 349

125 F 167 400 306 439 304 446 336 445 332 439 289 436 319 456 368

125 F 191 440 331 456 324 436 339 438 332 452 298 444 332 457 365

125 F 106 431 355 446 369 441 391 448 371 444 320 431 318 450 370

125 F 160 439 307 448 340 440 327 442 330 447 284 448 324 459 373

125 F 163 429 217 444 295 423 343 441 325 450 296 420 303 454 360

125 F 112 407 333 447 302 422 346 445 237 429 277 447 330 452 366

125 F 166 415 374 448 338 441 339 457 340 450 310 435 316 446 369

125 F 117 428 319 450 318 439 342 441 335 440 296 441 330 452 371

125 F 156 432 345 452 330 443 350 435 329 442 323 445 327 461 370

125 F 158 428 345 445 358 439 371 448 358 450 318 435 339 457 393

125 F 159 427 337 448 351 448 387 436 359 444 317 430 316 448 372

125 F 135 415 365 445 326 437 353 437 337 447 326 435 337 453 379

125 F 181 431 311 452 309 438 341 446 335 453 313 423 302 441 366

125 F 111 433 321 448 313 452 348 449 327 443 277 443 303 448 354

125 F 113 425 342 456 323 429 355 448 341 449 289 443 323 460 370

125 M 114 418 342 454 345 441 341 440 327 452 331 438 292

125 M 109 410 331 458 367 445 354 420 278 446 347 437 306

# B-1 125 M 184 410 348 438 349 431 346 402 313 442 354 427 321

125 M 193 401 309 428 311 441 340 413 291 446 330 437 284

125 M 182 419 357 437 344 438 347 410 293 459 349 446 322

125 M 155 405 353 441 354 431 349 421 321 451 367 435 330

125 M 170 424 368 451 368 425 367 432 326 455 361 453 352

125 M 185 411 324 443 330 410 317 410 294 446 322 428 313

125 M 189 432 362 444 352 437 352 409 311 453 377 445 341

125 M 188 434 371 450 350 438 344 414 308 449 357 431 321

125 M 133 427 364 439 354 443 366 413 311 461 361 438 331

125 M 151 415 344 440 329 429 335 411 297 438 326 435 307

125 M 183 412 347 436 322 448 383 440 307 442 327 427 282

125 M 161 407 344 439 349 435 351 434 316 437 324 434 283

125 M 131 403 327 442 358 431 338 435 311 441 325 424 290

125 M 118 413 324 436 356 432 345 444 322 436 336 435 326

125 M 157 410 347 444 332 443 321 413 308 452 339 437 285

125 M 152 405 330 438 329 439 345 424 316 446 350 434 280

125 M 134 423 329 424 313 432 320 424 284 437 319 432 290

125 M 119 406 338 433 356 429 342 427 317 433 350 447 354

250 F 161 413 321 433 299 434 351 439 348 453 322 457 345 438 369

250 F 152 418 319 428 295 450 327 445 329 448 315 450 342 448 369

250 F 107 406 329 444 298 426 341 442 325 448 278 441 309 454 339

250 F 115 408 338 448 306 423 344 437 337 446 288 445 337 453 378

250 F 114 431 335 448 330 440 355 454 347 446 326 449 334 465 392

250 F 159 401 278 453 298 432 332 440 322 449 275 447 329 457 362

250 F 156 418 281 450 243 459 305 430 276 450 238 445 253 443 324

250 F 134 427 334 454 321 441 339 448 330 446 297 453 328 452 359

250 F 110 425 299 438 247 446 316 443 304 450 258 429 344 447 346

250 F 102 418 310 428 291 435 339 439 298 446 262 428 282 450 337

250 F 153 405 310 445 288 437 329 439 301 455 273 432 289 446 340

250 F 133 413 317 451 332 435 343 441 327 451 285 431 315 461 363

250 F 151 422 300 440 271 443 307 432 300 452 249 430 283 438 331

250 F 113 413 302 432 295 425 353 437 334 445 288 434 322 452 383

250 F 122 423 335 437 333 440 347 444 330 444 306 429 318 461 376

250 F 136 431 312 439 281 446 322 438 306 453 286 446 295 463 348

250 F 193 414 291 448 318 447 278 443 318 458 371

250 F 171 428 330 437 353 442 297 444 330 460 376

250 F 154 433 336 441 365 450 329 444 361 466 398

250 F 119 326 329 438 304 432 339 448 330 453 316 443 327 450 358

250 M 158 422 352 450 361 436 346 441 316 447 359 459 335

250 M 141 416 344 442 358 438 351 439 336 452 363 415 305

250 M 138 426 345 430 334 446 351 436 324 452 360 440 351

250 M 111 422 362 443 364 439 361 449 341 450 358 438 323

250 M 105 420 339 438 331 430 331 431 305 451 339 441 303

250 M 112 422 334 450 336 451 351 449 319 444 341 450 312

250 M 135 424 356 453 366 451 368 458 346 454 363 433 325

250 M 118 411 330 424 308 431 313 437 298 445 335 428 299

250 M 140 430 345 449 352 453 344 451 293 453 320 437 321

250 M 120 416 365 444 368 440 341 429 312 461 350 432 333

# B-1 250 M 155 420 304 442 335 422 330 445 354 458 364 450 336

250 M 109 411 345 442 365 434 352 442 334 447 354 426 304

250 M 116 427 348 426 340 440 346 444 325 452 367 431 311

250 M 131 424 331 427 345 439 351 446 341 461 373 440 331

250 M 132 413 348 449 361 444 357 454 334 461 369 434 324

250 M 106 405 352 437 343 437 342 435 306 453 342 436 314

250 M 108 408 326 438 292 448 318 437 286 449 318 429 284

250 M 174 409 352 435 316 438 330 420 304 442 335 423 290

250 M 177 421 331 432 328 449 347 435 332 445 344 441 301

250 M 103 412 357 436 348 449 365 448 336 450 363 447 322F1 feed consumption9/28 10/2 10/4 10/910/1210/1610/1910/2310/2610/30 11/2 11/4 11/911/1311/1611/2011/2311/27DOSE ID FinalInitialFinalInitialFinalInitialFinalInitialFinalInitialFinalInitialFinalInitialFinalInitialFinalInitialFinalInitialFinalInitialFinalInitialFinalInitialFinalInitialFinalInitialFinalInitialFinalInitialFinal0221/217 451 271 452 323 428 254 420 261 409 282 450 321 430 272 446 310 445 255 455 329 436 300 419 297 431 246 438 270 445 312 428310 428 243 4230218/211 451 294 449 328 427 253 442 267 424 292 433 274 435 268 438 282 432 261 461 323 441 309 440 277 438 281 446 278 435 307 427322 432 275 4270 236 435 368 498 402 433 374 429 356 406 355 453 397 429 359 441 384 416 356 465 409 443 394 442 376 452 392 448 401 448 395 454 406 444 380 4300224/237 463 235 454 323 440 247 439 257 400 251 449 303 443 257 429 288 429 259 463 322 435 283 451 317 435 242 438 235 433 278 416258 419 236 4170 216 434 334 457 378 429 350 441 346 434 364 444 375 438 362 444 367 413 343 462 386 442 375 459 367 451 366 449 372 451 381 448 387 434 318 4370227/245 450 279 461 331 437 252 450 260 412 272 440 314 437 248 458 307 429 255 453 319 439 357 447 369 451 379 434 365 432 388 450402 479 364 4110280/215 427 259 459 321 422 244 454 291 411 270 449 303 448 270 434 284 440 264 449 330 435 295 447 261 437 259 430 263 440 299 436280 407 260 4170240/242 463 280 450 342 439 287 451 258 431 293 456 310 451 284 436 303 443 273 453 314 427 269 439 284 445 283 428 280 435 274 431277 415 230 4150222/226 452 279 459 319 422 247 453 274 413 238 423 262 451 279 425 266 441 244 450 317 444 296 451 281 456 391 433 302 434 291 434262 416 231 4210229/209 465 267 466 313 426 362 427 257 422 256 446 267 446 242 434 279 447 235 462 313 430 262 438 280 448 250 421 254 434 265 429237 424 242 4160 243 439 388 464 418 418 246 446 352 404 358 450 393 436 365 424 381 447 386 458 410 433 387 448 390 437 378 437 374 421 404 447 405 456 400 4120220/234 457 262 456 292 433 260 436 281 436 279 439 306 444 278 432 287 437 255 460 339 438 295 453 268 445 254 428 250 428 309 437265 403 229 4350279/228 440 257 461 304 457 264 456 257 410 255 439 276 447 281 430 284 441 254 454 321 443 296 440 305 442 242 424 244 427 302 435362 435 290 4120214/225 459 273 462 321 450 264 456 267 421 246 435 274 440 250 430 253 447 248 442 290 447 290 448 308 446 260 434 262 431 303 441308 428 251 4270232/241 441 285 452 316 436 235 451 284 427 291 452 308 456 287 453 307 446 285 444 319 430 270 432 292 447 273 435 274 437 319 435327 424 271 4200244/212 457 288 461 353 453 289 453 293 428 308 450 331 450 295 460 338 440 271 453 346 449 320 447 313 441 279 436 280 430 335 439339 436 252 4360206/202 460 297 465 371 448 287 441 257 425 283 439 294 438 255 437 376 427 260 439 311 442 292 430 288 437 249 427 244 427 299 438323 424 234 4320246/207 257 285 467 353 453 269 444 270 438 289 441 299 443 264 451 328 448 300 445 327 444 307 435 315 445 259 430 261 445 314 434290 426 287 4180 205 452 365 464 389 402 342 437 323 428 371 444 384 428 359 441 372 436 358 460 385 454 379 440 358 439 356 447 357 432 391 447 387 444 372 4300 235 422 318 462 373 428 328 438 340 388 319 442 367 437 335 441 347 445 322 465 370 434 348 447 336 444 307 450 345 448 385 452 408 439 348 4230 299 431 352 460 388 388 319 441 345 386 313 446 366 437 338 466 378 426 347 436 376 436 365 454 359 449 344 448 348 450 360 434 391 417 321 4220 219 436 325 455 383 441 344 439 306 386 305 436 351 430 330 439 349 401 396 456 365 440 348 438 341 460 348 454 306 432 337 433 346 435 300 4020 203 454 323 458 347 421 276 446 323 389 309 456 347 429 305 444 336 416 283 461 354 446 336 446 313 440 273 433 252 428 346 422 306 40330 257 437 337 449 355 429 340 448 342 395 329 439 363 428 361 434 334 440 369 443 366 552 348 435 349 414 340 430 361 449364 448 357 42730255/212 449 284 454 336 430 260 434 319 382 279 438 331 454 337 426 368 435 311 444 359 418 321 422 280 433 292 415 297 430 325 448326 434 257 43630 246 447 354 462 368 442 347 465 346 393 339 436 371 452 355 433 362 429 333 431 361 412 357 431 350 433 358 424 349 433 363450 373 428 338 43830237/242 462 288 451 312 429 262 442 294 402 292 436 308 445 297 434 318 438 266 440 318 426 303 441 281 439 389 416 392 431 322 447300 425 264 43830 305 457 360 460 395 423 351 452 363 403 343 439 386 414 345 445 387 433 346 440 383 419 365 428 360 430 369 432 371 440 388451 394 436 359 42630 282 439 358 459 390 429 351 434 342 404 345 431 370 408 327 453 406 450 356 441 394 421 357 426 357 431 365 429 362 435 389450 393 442 371 45030223/232 463 296 457 312 435 250 448 257 411 269 435 294 444 253 420 286 441 254 444 308 426 297 438 267 434 374 452 370 433 296 440294 437 272 41330244/220 450 288 441 316 439 276 443 281 401 289 442 386 425 301 436 304 439 270 443 312 429 300 421 278 441 291 417 292 414 300 437308 438 279 43230215/245 452 286 452 310 449 269 443 280 423 288 447 315 441 276 442 310 430 271 428 299 423 291 451 301 435 279 419 280 420 340 449313 439 259 441B-130205/251 453 308 457 302 429 254 437 274 415 275 436 265 449 260 432 319 437 239 434 287 428 287 449 273 442 269 421 272 421 322 446282 415 321 43130 224 343 459 371 426 337 437 386 425 260 427 377 450 356 440 392 426 383 432 339 436 367 427 367 425 398 453 397 438 372 41930224/227 460 398 430 363 398 356 44030233/250 258 279 458 279 444 245 433 253 405 252 438 293 449 266 432 264 441 264 431 257 424 243 445 272 430 275 414 270 439 288 448286 427 229 43530219/247 454 278 448 306 423 263 438 255 408 280 441 313 443 276 451 303 443 259 414 289 430 291 440 267 431 262 426 261 419 331 446316 443 231 42630248/211 458 305 454 315 433 264 443 280 428 288 441 311 448 290 440 307 424 253 438 306 430 295 441 300 440 264 422 271 436 306 434308 422 232 44630 216 437 369 460 401 428 366 429 355 440 280 435 379 441 367 422 362 441 356 429 373 426 369 421 345 428 389 454 322429 355 43130217/235 454 315 456 327 436 274 452 299 421 279 431 284 448 299 431 298 433 259 439 311 412 277 451 317 450 355 439 350 412 321 440392 442 259 40630 240 458 378 458 365 440 368 452 362 432 345 426 379 417 348 435 374 439 351 440 386 429 377 447 363 433 351 427 360 422 387451 390 441 360 42730272/230 455 302 451 294 435 241 442 281 410 291 433 270 439 246 427 280 439 275 467 361 444 348 460 396 458 280 435 279 428 309 447279 449 242 43130 204 265 368 466 385 459 385 465 373 458 341 433 356 438 337 428 360 445 335 439 375 431 374 439 353 420 335 421 330 433 375451 380 439 355 42230209/221 454 275 462 321 439 254 452 283 433 284 430 293 437 274 421 290 438 286 441 306 430 296 458 315 447 243 435 235 440 308 437283 433 245 42430 253 456 353 454 367 430 325 455 339 413 331 437 341 443 331 415 324 329 303 432 345 436 339 415 293 449 320 460 322 427 354452 366 441 306 42830 226 429 261 446 351 439 303 448 322 427 308 438 329 433 302 435 265 434 294 445 330 417 304 411 247 428 254 450 250 430 340448 340 444 300 45030 202 450 314 457 350 432 275 445 329 403 267 440 334 438 246 418 328 441 243 452 351 424 323 423 259 440 298 454 300 433 313445 260 432 238 44530 231 454 438 454 338 436 319 463 340 409 316 442 344 424 304 421 321 418 278 437 351 419 334 419 302 441 294 456 296 441 339451 336 430 265 43530 225 453 340 460 345 432 325 421 314 408 327 436 351 430 322 418 325 428 316 434 348 425 343 432 324 424 316 448 317 440 338456 358 435 318 44030 206 453 357 452 376 435 335 451 345 395 269 434 366 434 342 416 271 446 336 441 345 421 332 427 326 429 337 440 335 438 343453 360 434 330 43230 249 455 354 446 383 444 357 449 353 405 351 447 367 444 362 430 385 429 390 450 404 436 364 437 335 454 327 442 330 429 350447 363 453 337 43330 227 457 281 442 296 427 341 454 281 428 343 426 321 437 354 414 297 412 267 426 278 423 375 435 345 448 352 454304 43760176/162 436 274 448 340 428 376 444 348 411 335 424 351 446 340 447 353 437 313 453 368 445 372 453 331 453 318 436 315 421 347 438341 418 347 41860 167 464 376 440 375 452 355 452 357 420 357 440 374 447 345 433 368 441 338 448 378 432 362 451 349 426 322 433 330 429 360438 374 426 352 41560116/182 437 322 464 345 436 292 461 326 411 302 433 337 442 303 438 361 429 304 443 362 418 339 455 326 437 311 428 310 428 337 445337 426 290 42660111/112 447 189 450 318 429 258 451 302 401 260 435 313 432 249 425 298 424 263 442 314 433 297 449 274 422 271 435 275 420 298 440292 429 262 43860164/170 460 285 462 320 445 246 447 267 419 269 433 280 440 257 432 303 428 271 444 294 421 265 445 284 422 255 427 256 422 294 439269 447 250 43360151/186 453 300 452 301 443 266 436 272 410 266 442 301 438 274 428 302 439 262 459 335 430 301 458 287 452 269 431 263 439 296 438285 445 257 43260114/465 456 293 447 310 439 273 448 277 428 315 437 323 450 275 436 308 438 273 450 325 437 320 459 305 455 296 435 288 429 308 451325 438 278 43760187/175 450 285 446 296 441 253 442 306 423 286 442 293 439 277 438 322 410 258 452 310 432 304 455 298 447 276 436 272 435 277 453307 450 304 45460 173 437 360 460 392 448 382 450 382 401 361 439 385 443 350 434 382 435 339 451 399 436 386 441 365 436 359 433 362 440 371455 399 442 360 42760120/185 447 249 459 289 443 243 449 250 415 282 439 298 435 284 446 314 419 272 440 316 429 306 441 311 449 237 424 242 429 308 451284 444 240 45460 169 441 365 462 386 433 357 427 342 420 361 444 374 432 343 416 343 440 328 436 357 431 363 436 354 421 341 431 345 427 373456 376 444 350 41060171/181 436 253 461 319 451 268 447 263 418 264 439 273 434 265 425 270 433 241 446 323 423 329 439 287 423 249 433 253 417 317 452334 445 266 42560117/183 455 274 456 319 451 255 430 250 424 274 432 281 443 258 421 289 433 245 439 308 424 333 445 292 426 237 424 237 430 320 448328 428 261 42660136/174 446 280 465 322 452 276 425 269 415 278 435 298 440 288 429 304 432 260 448 319 426 306 449 280 429 265 425 260 434 327 443337 430 285 436B-160 190 441 354 459 373 436 333 430 330 424 325 437 357 432 337 430 345 426 302 437 337 440 336 451 358 418 339 427 302 429 374445 392 443 353 42260 178 442 353 460 389 446 367 440 366 424 361 447 388 432 354 447 379 442 367 442 392 431 376 441 366 427 353 434 352 430 399455 409 440 376 42860177/110 459 317 467 357 455 314 4433007 438 285 438 299 427 263 434 299 423 244 410 300 423 325 450 289 427 259 428 245 441 339 443 331 434 274 42960180/115 443 269 445 305 434 251 431 260 412 292 436 296 440 258 434 281 435 257 430 298 421 289 442 269 430 236 421 240 442 326 425321 434 277 43460 161 442 330 467 373 447 347 436 328 441 356 434 359 435 346 432 355 445 344 443 367 426 359 451 361 420 327 435 371 430 366441 361 422 331 44260109/172 449 257 451 295 425 236 428 242 421 283 438 284 446 258 417 276 437 257 422 298 425 312 450 293 434 269 427 272 425 340 440319 443 279 43360 166 449 357 452 389 437 349 432 344 421 353 445 367 435 328 452 377 437 338 437 366 421 359 453 359 432 333 441 330 420 360438 363 430 338 42460 119 442 336 444 341 435 283 438 322 425 323 440 356 434 324 455 360 435 311 449 353 425 333 456 320 447 311 435 315 43260 102 449 328 452 353 419 304 453 326 430 341 436 343 432 317 444 347 430 283 434 347 421 331 454 320 444 299 435 300 419 319434 335 414 307 42860 184 449 342 462 354 433 319 448 309 429 334 433 344 426 315 438 349 443 312 442 336 420 317 443 308 439 289 434 288 425 311446 295 417 295 43460 189 465 350 462 370 441 332 447 324 453 347 442 353 430 324 451 360 443 311 446 350 424 332 458 338 440 295 423 290 402 340443 340 422 313 42860 179 456 360 464 376 445 355 431 346 422 343 437 344 425 301 423 338 434 303 446 351 436 337 447 325 454 311 436 302 420 317436 314 414 289 430125 183 448 343 459 382 415 344 438 341 402 360 445 360 425 339 428 347 437 327 449 353 422 341 437 333 434 343 412 291 436 348443 368 444 350 428125161/103 451 259 450 315 437 246 422 266 415 291 426 293 441 267 422 291 428 261 437 288 435 287 439 264 424 240 417 340 420 336 447292 440 231 450125131/110 437 262 456 317 440 260 430 262 422 277 434 270 434 244 418 268 427 236 423 276 432 278 443 265 443 253 422 249 433 275 441281 444 240 447125118/165 447 319 450 354 428 288 427 350 430 360 450 370 446 342 444 376 442 346 437 375 439 368 437 355 419 343 410 249 428 373 436378 437 370 422125157/107 459 286 468 334 442 259 447 267 423 253 439 276 437 235 436 294 424 240 450 287 434 279 444 251 422 328 433 341 427 344 454323 430 231 457125 152 465 348 458 358 442 340 427 325 417 331 433 346 439 328 418 335 432 319 448 362 433 353 439 345 433 329 430 325 431 351432 344 427 327 435125134/191 450 314 446 376 438 259 419 277 407 276 452 325 439 280 422 270 431 261 445 312 432 297 448 292 453 248 408 331 435 316 442306 436 265 439125119/106 458 290 450 331 442 266 423 273 413 293 446 315 438 271 420 273 426 262 440 300 442 303 450 266 440 272 416 245 436 301 448305 444 265 452125 114 451 342 458 388 430 352 450 363 443 371 444 371 444 330 427 364 426 340 437 386 437 373 439 357 426 330 426 268 433 360439 373 445 358 426125109/156 464 307 461 314 426 272 448 275 408 261 449 316 435 271 420 286 430 271 443 310 429 288 442 275 429 268 429 333 427 303 438313 434 270 443125 184 460 372 465 336 433 347 428 353 414 358 455 388 419 325 419 360 424 334 445 380 432 369 439 353 449 346 424 271 426 375443 375 444 354 405125193/159 457 288 454 323 434 239 435 293 423 340 457 341 459 330 437 337 441 257 449 308 433 273 432 274 422 240 414 350 422 304 443294 425 260 419125182/135 462 295 457 328 436 254 419 278 421 321 444 331 443 305 436 305 441 254 440 305 417 254 425 271 421 235 422 242 439 309 444313 434 243 422125155/181 462 305 456 302 422 269 440 276 413 303 437 310 439 256 434 310 432 269 443 312 440 311 431 284 436 279 420 236 427 371 451384 444 368 445125170/111 453 292 446 313 441 287 425 278 424 286 456 308 449 272 425 293 418 252 451 306 433 297 433 282 423 263 413 277 435 283 445298 420 263 445125185/113 457 280 446 292 442 267 434 259 422 269 449 274 445 236 445 287 425 243 441 300 430 259 429 258 427 255 412 260 421 295 445262 443 242 452125189/160 455 296 447 309 449 287 422 268 408 279 451 320 444 254 413 292 435 256 442 313 426 265 417 274 422 280 424 261 435 311 441313 432 275 434125188/158 446 285 461 317 448 246 423 243 417 269 445 299 451 254 423 252 432 253 446 306 444 308 433 278 434 276 421 275 434 300 448298 426 251 431125133/112 462 336 461 338 444 276 427 290 419 391 453 312 443 265 413 251 431 251 446 324 448 308 442 236 434 262 415 242 437 306 447303 447 255 449125151/166 459 289 449 300 431 277 420 282 423 363 450 383 430 336 427 338 427 343 446 380 433 368 444 360 433 417 436 429 436 348 447373 428 350 445125 117 448 337 450 352 439 319 444 330 435 312 432 340 432 315 426 328 417 318 442 347 420 334 434 310 431 310 436 330 441 332430 342 440 331 428125 167 455 353 455 360 439 328 418 289 413 317 424 280 440 313 446 336 419 297 444 353 437 346 438 312 421 329 424 316 434 341437 274 428 297 441B-1125 163 431 320 445 347 444 326 435 318 407 333 442 346 432 318 443 342 441 304 444 351 425 332 448 318 437 315 426 328 435 350435 347 440 295 441125 101 460 325 446 345 438 318 437 304 413 323 445 335 427 308 418 284 433 332 442 352 428 340 438 321 444 285 427 322 423 372438 349 429 314 447250135/156 444 237 452 285 435 230 427 261 395 242 437 264 441 242 446 262 429 262 447 261 435 246 441 270 440 256 414 260 413 278 438340 439 231 440250 118 458 357 453 368 446 349 444 354 386 331 438 364 442 332 446 374 438 342 447 373 440 372 436 356 437 345 425 242 420 361449 364 435 342 453250140/110 459 334 452 338 429 334 420 337 402 326 435 344 432 334 433 348 433 344 449 380 440 374 441 354 423 333 429 342 422 347 443355 429 333 450250 120 453 373 442 379 442 361 427 345 405 340 428 365 430 336 439 352 431 339 436 368 428 362 438 345 437 340 410 341 414 380458 380 440 349 435250 155 460 391 443 393 445 375 447 342 399 338 425 374 435 357 423 381 425 359 448 302 441 396 445 378 454 388 431 392 424 401458 402 445 382 435250109/133 456 291 450 388 433 230 434 239 392 267 436 305 443 275 435 298 440 265 440 309 439 296 447 287 430 264 401 270 429 315 447292 420 242 439250116/151 461 277 448 319 440 249 426 260 393 263 445 296 445 289 439 294 423 280 433 255 438 291 448 251 469 234 415 241 426 255 457301 440 240 444250131/113 457 292 454 317 436 261 429 269 393 261 435 291 432 253 424 297 425 276 425 287 427 292 447 257 411 286 429 269 416 329 456317 427 255 438250132/122 462 279 450 297 441 255 437 273 394 267 440 283 443 261 445 280 439 259 446 293 450 285 440 266 425 256 417 265 427 282 450284 421 233 442250106/136 453 251 446 294 431 230 425 256 415 282 445 299 436 296 434 305 438 273 448 303 442 303 449 283 431 253 421 252 431 274 444273 426 237 431250108/102 452 255 452 279 432 234 429 262 400 246 443 242 434 243 435 290 435 245 443 263 437 258 413 268 434 251 410 254 427 295 442261 433 230 418250174/171 448 298 442 289 438 266 420 267 394 261 435 290 443 276 434 302 438 258 444 293 446 302 426 294 431 252 407 250 435 288 428299 440 258 446250177/154 452 280 448 320 433 253 426 263 399 228 447 313 444 283 435 314 425 269 443 294 437 290 420 291 425 256 422 251 441 313 455326 433 356 419250 103 459 358 447 393 441 373 413 336 400 350 435 371 452 382 434 375 440 366 449 399 438 389 442 371 410 343 419 340 428 372446 380 427 356 419250158/161 458 307 460 399 439 278 442 279 407 284 435 296 440 275 423 280 431 261 426 292 439 287 421 273 428 273 413 268 434 307 444285 442 256 446250 141 460 376 455 390 434 360 428 361 431 367 437 382 441 369 440 385 430 368 444 389 448 388 438 358 442 353 431 349 427 384435 382 442 374 430250 138 456 390 458 398 434 364 410 339 406 351 438 384 428 357 442 390 438 367 448 397 447 396 431 337 430 357 433 352 425 393448 395 451 278 429250111/115 450 298 445 305 433 257 425 287 407 306 444 303 446 288 430 305 436 264 453 332 425 305 445 263 445 271 420 270 424 309 440305 424 260 425250105/115 450 257 445 300 434 234 416 267 427 259 448 262 446 260 427 295 427 253 426 266 423 251 435 259 415 264 418 265 433 304 429243 448 232 424250 112 459 380 461 401 443 370 433 335 438 381 446 389 436 371 424 374 440 342 441 386 430 377 438 368 449 353 437 348 431 390435 384 436 327 428250 107 450 331 452 338 429 299 453 328 381 264 432 323 438 270 422 327 429 248 436 329 421 287 429 278 434 335 435 432 432 357453 366 440 332 439250 152 447 328 457 351 419 311 438 323 388 335 434 355 432 335 443 337 427 306 450 347 422 344 444 329 442 317 429 309 417 344436 326 437 332 445250 159 437 328 451 350 434 297 430 319 399 297 426 328 426 305 431 335 425 276 442 345 434 336 437 307 443 284 414 282 415 354444 357 441 307 442250 134 465 352 456 366 443 333 427 312 387 275 425 330 432 307 441 319 421 304 447 355 433 336 444 333 442 287 432 285 421 311437 234 439 305 448250 153 452 301 444 337 433 286 439 302 401 308 401 285 429 300 444 335 429 301 449 347 432 323 454 300 440 274 428 307 421 329447 301 442 304 439250 193 449 315 456 327 437 297 441 317 410 330 419 319 434 314 442 337 431 269 437 348 436 332 433 273 441 308 422 275 412 329448 345 423 296 449F1eggproductionDOSEID8/78/88/98/108/118/128/13week68/148/158/168/178/188/198/20week78/218/228/238/248/258/268/27week88/288/298/308/319/19/29/3week99/49/59/69/79/89/99/10week10021711111111111111111111111111021111112111111111111111111023411111111111111111111111111B-10237111111111111111111023511111111111111111111111102451111111111111021911111111111111111111102421111111111111111111102261111111111111111111111111110209111111111111111111111110215111111111111111029911110203111111111111211111111111110228111111111111111111111110225111111111111111111111024111111111111111111021211111111111111111110202111111111111111110207111111111111110210230253111302261111111111111111111111302121111111111113024211111113020211111111111111111111302301111111111111111111111302321111113022011111111111111111111111302451111111111111111111111111302511111111111111111111111130227111111111211111113025011111111111111111302471111111111211111111111130211111111111111111130231111111111111302351111111111111111111302251111111111111111111111111B-1302061111111130249111111111111111113022111111111111111116016211111111111111116016611111111111111111111111111160182111111111111111111111111601121111111160119111111111111111111111211111116018611111111111111111111111116016511111111111111111111111111160175111111211111111111111111601021111111111111111111111111116018511111111111111111111116018411111111111111116018111111111111111111116018311111111601741111111111111111111111111601891111111111111111111111111601701111111111111116011011111111111111111111121111116011511111111111116017911111111111111111111111160172111111111111111112510121111111111111111111111111251031111111111111111111251101111111111111111111111112516511111111111111111251071111111111111111112516711111111111111111112111111125191111111111111111111112510611111251601111111111111111111112516311111111111111111111111111251121111111111111111111111111111B-1125166111111111111111111251171111111111111112111111112515611111111111111111251581111111111125159111125135111111111111111111125181111111111112511121111111111111111111125113111111111111211111125016111111111111111111111111112501521111111111111111111111112501071111111111112501151111111111111111111111125011411111111111111250159111111111111111111111111125019311111111111111111111125017111111111111250154111111111112501191111111111111111 1250156111111111111111111111125013411111111111111125011011111111111111111111111111250102111111111111111112501531111111111111111112111111125013311111111111111111111125015111111111111111111111111112501131111111111111111111250122111111111250136111111121111111111111111111F1eggproductionDOSEID9/119/129/139/149/159/169/17week119/189/199/209/219/229/239/24week129/259/269/279/289/299/3010/1week1310/210/310/410/510/610/710/8week1410/9####################10/15week150217111111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 102111111111 1 1 2 1 1 1 1 1 1 1 1B-1023411111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 102371111111121 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1023511111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10245111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 10219111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 102421111111111111111111 1 1 1 1 2 1 102261111111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 10209111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10215111111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 102991111111 1 1 1 1 1 1 1 1 1 1 1 1 1 10203111111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1022811121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 102251111111121 1 1 1 2 1 1 1 1 1 1 10241111 1 1 1 1 1 1 1 1 1 1021211111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10202111111111 1 1 1 1 1 1 1 1 10207111111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1021030253111111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1302261111111111111111111 1 1 1 1 1 1 1 1 1 1 1 1 13021211111 1 1 1 1 1 1 2 2 1 1 1 1 1 1 1 1 1302421111111111111 1 1 1 1 1 1 1 1 1 1 1 1302021111111111111111111111111111111111302301111111211 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 13023211111111111111111 1 1 1 1 1 1 1 1 1 1 1 13022011111112111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 13024511111112111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 130251111111111111 1 1 1 1 1 1 1 1 1 130227111111111112 111 1 1 1 1 1 1 1 1 1 1 1 1 1302501111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1B-13024711111111111111111111 1 1 1 1 1 1 1 1 1 1 1 1 1302111111111211 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1302312111111111111 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 13023521111211 1 1 1 1 1 1 1 1 1 1 1 1302251111111211 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130206111111 1 1 1 1 1 1 1 1 1 1 13024911111111111 1 1 1 1 1 1 1 1 1 1 1 1302211111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1601621111111111111 1 1 1 1 1 1 16016611111111111111211111 1 1 1 1 1 1 1 1 1 1 1 1601821111111 1 160112111111 1 1 1 1 1 1 1 1 1 1 160119111111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160186211111 1 1 1 1 1 1 1 1 1 1 1 1 160165111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160175111111111 1 1 2 1 1 1 160102111111111111111 1 1 1 1 1 1 1 1 1 1 1 1 160185111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160184111111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160181111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1601831111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160174111121 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1601891111111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1601701111111111111111 1 1 1 1601101111111121 1 2 1 1 1 1 1 1 1 1 1 160115111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160179111121 1 2 1 1 1 1 1 1 16017211111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11251011112111111121111111 1 1 1 1 1 1 1 1 1 1 1 1 11251031111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1B-112511011111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125165111111121 1 1 1 1 1 1 1 1 1125107111111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 112516711111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11251911111111121 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 11251061111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 112516011111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11251631111111111111111111 1 1 1 1 1 1 1 1 1 1 1 1 11251121111111111 1 1 1 1 1 1 1 1 1 112516611111111112511711111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 112515611121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125158111111121 1 1 1 1 1 1 1 1 1 1 112515911111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11251351111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 112518111111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11251111111111111111111111 1 1 1 1 1 1 1 11251131111111121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 125016111111112111 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 125015211111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250107111111111 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1250115111111111111111112111112111111112501141111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12501591111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12501931111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250171111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1250154111 1 1 1 1 1 1 1 1 1 1 1 1 12501191112 1 1 1250156111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250134111 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1B-125011011111111111111111125010211111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250153111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12501331111111111111111111111111111111250151111111111111121111111 1 1 1 1 12501131111111111 111111 1 1 1 1 1 1 1 1 1 1 1250122111111 1 1111 1 1 1 1 1 1 1 1 1 1 1 12501361111111111111111111111111111 1 1F1eggproductionDOSEID############10/1910/2010/2110/22week 1610/2310/2410/2510/2610/2710/2810/29week 1710/3010/3111/111/211/311/411/5week 1811/611/711/811/9############week 19########################11/19week 2002171 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 102111 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 1 102341 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 102371 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 1 102351 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 1 1 102451 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 102191 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 102421 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 1 1 1 102261 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 102091 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 102151 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 102991 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 102031 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 1 1 1 102281 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 1 102251 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 102411 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 102121 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 102021 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 102071 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1B-10210302531 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 1 1 1 1 1302261 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 1 1 1 1 2 1302121 1 1 1 1 1 1 1302421 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 13020211111111111111111111111111111 1 1 1 1302301 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1302321 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 1 1 1 1302201 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 1 1 1 1 1302451 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 1 1 1 1 1 1302511 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1302271 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1302501 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 1 1 1302471 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 1 1 1 1302111 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 1 1 1302311 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 1302351 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1302251 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 1 1 1 1302061 1 1 1 1 1 1 1 1 1 1302491 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1302211 1 1 1 1 1 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 1601621 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1601661 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 1 2 1601821 1 1 1 2 1601121 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1601191 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 1601861 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1601651 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 1 1 1601751 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1601021 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 1 1B-1601851 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1601841 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 1 1601811 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1601831 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1601741 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1601891 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1601701 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 1601101 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 1601151 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 1601791 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1601721 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 11251011 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 11251031 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 11251101 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 1 1 1 11251651251071 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 11251671 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 11251911 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 11251061 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11251601 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 1 11251631 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 1 1 11251121 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 1 1 1 11251661 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 11251171 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 11251562 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 11251581 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11251591 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11251351 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 11251812 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 1 1 1 1 1 11251111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1B-11251131 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 12501612 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 1 12501522 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 12501071 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 125011511111111111111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12501141 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 1 1 12501591 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 1 1 12501931 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 12501711 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 12501541 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12501192501561 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 1 1 1 1 12501341 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 12501102501021 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12501531 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 12501331 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 12501511 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12501131 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 12501221 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 12501361 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 1 1F1 egg production

DOSE ID #### #### #### 11/23 11/24 11/25 11/26 week

21 11/27 11/28 11/29 11/30 12/1 12/2 12/3 week

22

0 217 1 1 1 2 1

0 211 1 1 1 1 2 1 1

0 234 1 1 1

0 237 1 1 1 1 1 1 1

0 235 1 1 1 1

0 245

0 219 1 1 1 1 2

0 242 1 1 1 1 1 1 1 1 1

0 226 1 1 1 1 1 1 1 1 1 1 2

0 209 1 1 1 1 2 1 1

0 215 1 1 1 1 1 1 1 2 1

0 299 1 1 1 1 1 1

0 203 1 1 1 1 1 1

0 228 1 1 2 1

0 225 1 1 2 1 1

0 241 1 1 1

0 212 1 1 1 1 1 1

0 202 1 1

0 207 1 1 1 1 1 1 1

0 210

30 253 1 1 1 1 1 1 1 1

30 226 1 1 1 1 1 1 1 1

30 212 1 1 1 1 1 1 1

30 242 1 1 1 1 1 1 1 1 1

30 202 1 1 1 1 1 1 1 1 1 1 1 1

30 230 1 1 1 1 1 1 1 1 1 1

30 232 1 1 1 1 1 1 2 1 1

30 220 1 1 1 1 1 1 1 1 1 1

30 245 1 1 2 1 1 1 1 1 1

30 251 1 1 1 1 1 1 1

30 227 1 1 1 1 1 1 1 1

30 250 1 1 1 1 1 1 1 1 1 1 1 1 1

30 247 1 1 1 1 1 1 1 1 1

30 211 1 1 1 1 1 1 1 1 1

30 231 1 1 1 1 1 1 1 1 1 1

30 235 1 1 1 1 1 1 1 1 1

30 225 1 1 1 1 1 1 2 1 1 1

30 206 1 1 1

30 249 1 1 1 1 1 1 1 1 1 1

30 221 1 1 1 1 1 1 1 1 1 1 1 1

60 162 1 1 1 1 1

60 166 1 1 1 1 1 1 1 1 1 1 1

60 182

60 112 1 2 1 1 1 1

60 119

60 186 1 1 1 1 1 1 1 1 1 1 1 1

# B-1 60 165 1 1 1 1 1 1 1 1 1 1 1 1

60 175 1 1 1 1 1 1 1 1

60 102 1 1 1 2 1 1 1 1 1 1

60 185 1 2 1 1 1

60 184 1 1 1 1 1 1 1 1 1 1 1 1

60 181 1

60 183 1 1

60 174 1 2 1

60 189 1 1 2 1 1 1 1 1 1

60 170 1 1 1 1 1 1 1 1 1 1 1

60 110 1 1 1 1 1 1 1

60 115 1 1 1

60 179 1 1 1 1 1 1 1

60 172 2 1 1 1 1

125 101 1 1 2 1 1

125 103 1 1 1 1 1 1 2 1

125 110 1 1 1 1 1 1 1 1 2 1 1

125 165 1

125 107 1 1 2 1

125 167 1 1 1 1 1 1 1 1 1 1 1 1

125 191 1 1 1 1 1 2 1

125 106 1 1 1 1 1 1 1 1 1 1 1 1

125 160 1 1 1 1 1 1 1 1 1 1 1

125 163 1 1 1 1 1 1 1 2 1 1 1 1

125 112 1 1 1 1 1 1 2 1 1 1

125 166 1 1 1 2 1 1 1 1 1 1

125 117 1 1 1 2 1 1 1 2 1 1

125 156 1 1 1 1 1 1 1 1 1 1 1

125 158 1 1 1 1 1 2 1

125 159 1 1 1 1 1 1 1 1 1 1 1

125 135

125 181

125 111 1 1 1 1 1 1 1

125 113 1 1 1 1 1 1 1 1 1 1 1 1

250 161 1 1 1 1 1 1 1 1 1 1

250 152 1 1 1 1 1 2 1 2 1 1

250 107 1 1 1 1

250 115 1 1 1 1 1 2 1

250 114 1 1 1 1 1 1 1 1 1 1 1

250 159 1 1 1 1 1 1 1 1

250 193 1 1 1 1 1 1 1 1 1

250 171 1 1 1 1 1 1 1 1 2 1 1 1

250 154 1 1 1

250 119

250 156 1 1 1 1 1 1 1

250 134 1 1 1 1 1 1 1 1 2 1

250 110

250 102 1 1 1 1 1 1 1 1 1 1 1 1

# B-1 250 153 1 1 1 1 1 1 1

250 133 1 1 1 1 1 1 1 1 1 1

250 151 1 1 1 1 1 1

250 113 1 1 1 1 1 1 1 1

250 122 1 1 1 1 1 1 1 1 1 1 1 1

250 136 1 1 1 1 1 1 1 1 1 1 1 1

F2 cloaca measurements and foam

12/23 12/30 1/6

DOSE ID CG area foam CG area foam CG area foam DOSE ID first day of foam

0 193 25 N 25 Y 25 Y 0 193 1/19

0 195 238 Y 210 Y 210 Y 0 195 12/23

0 196 252 Y 255 Y 289 Y 0 196 12/23

0 198 25 N 143 Y 169 Y 0 198 12/28

0 184 270 Y 272 Y 240 Y 0 184 12/23

0 192 120 Y 182 Y 210 Y 0 192 12/23

0 177 25 N 25 N 169 N 0 177

0 180 25 N 25 N 182 Y 0 180 1/5

0 304 25 N 100 N 225 Y 0 304 12/31

0 308 156 Y 240 Y 272 Y 0 308 12/23

0 322 156 Y 196 Y 210 Y 0 322 12/23

0 321 25 N 25 N 25 Y 0 321 1/6

0 30 182 Y 450 Y 323 Y 0 301 12/23

0 182 25 N 25 N 225 Y 0 182 1/3

0 185 156 Y 156 Y 238 Y 0 185 12/23

0 173 144 Y 156 Y 224 Y 0 173 12/23

0 187 25 N 25 N 195 Y 0 187 1/3

0 303 156 Y 182 Y 225 Y 0 303 12/23

0 310 25 N 169 Y 225 Y 0 310 12/27

0 329 25 N 169 Y 224 Y 0 329 12/29

30 94 25 N 25 N 210 Y 30 94 1/6

30 93 25 N 210 Y 255 Y 30 93 12/28

30 90 25 N 25 N 210 Y 30 90 1/6

30 80 156 Y 255 Y 272 Y 30 80 12/23

30 78 196 Y 238 Y 255 Y 30 78 12/23

30 76 25 N 182 Y 195 Y 30 76 12/28

30 407 132 N 225 Y 255 Y 30 407 12/24

30 402 256 Y 255 Y 255 Y 30 402 12/23

30 417 25 N 25 N 196 Y 30 417 1/5

30 406 169 Y 224 Y 255 Y 30 406 12/23

30 92 25 N 240 N 304 N 30 92 12/28

30 88 25 N 25 Y 25 Y 30 88

30 95 25 N 25 N 25 N 30 95

30 91 210 Y 270 Y 288 Y 30 91 12/23

30 77 156 Y 182 Y 240 Y 30 77 12/23

30 83 182 Y 210 Y 288 Y 30 83 12/23

30 404 25 N 169 Y 270 Y 30 404

30 100 132 Y 196 Y 240 Y 30 100 12/23

# B-1 30 99 169 Y 210 Y 240 Y 30 99 12/23

30 97 25 N 25 N 88 Y 30 97 1/4

60 90 182 Y 195 Y 210 Y 60 90 12/23

60 88 25 N 210 Y 210 Y 60 88

60 205 25 Y 195 Y 255 Y 60 205 12/23

60 208 169 Y 255 Y 285 Y 60 208 12/23

60 81 144 Y 195 Y 210 Y 60 81 12/23

60 84 168 Y 210 Y 238 Y 60 84 12/23

60 80 182 Y 210 Y 240 Y 60 80 12/23

60 86 144 Y 195 Y 182 Y 60 86 12/23

60 96 25 N 25 Y 195 Y 60 96 1/3

60 79 25 N 182 Y 196 Y 60 79 12/28

60 203 25 N 182 Y 210 Y 60 203 12/28

60 95 25 N 25 N 210 Y 60 95 1/5

60 99 25 N 25 Y 182 Y 60 99 1/3

60 98 25 N 25 N 182 Y 60 98 1/4

60 201 25 N 25 N 30 N 60 201

60 204 25 N 196 Y 240 Y 60 204 12/28

60 209 156 Y 196 Y 225 Y 60 209 12/23

60 210 25 N 132 N 156 N 60 210 12/28

60 219 25 N 182 Y 272 Y 60 219 12/27

60 218 90 N 196 Y 272 Y 60 218 12/24

125 75 169 Y 210 Y 240 Y 125 75 12/23

125 77 168 Y 195 Y 240 Y 125 77 12/23

125 95 144 Y 210 Y 255 Y 125 95 12/23

125 233 25 N 25 N 182 Y 125 233 1/5

125 100 144 Y 225 Y 288 Y 125 100 12/23

125 81 25 N 169 Y 225 Y 125 81 12/27

125 98 121 Y 169 Y 210 Y 125 98 12/23

125 97 25 N 182 Y 225 Y 125 97 12/28

125 208 25 N 169 Y 224 Y 125 208 12/27

125 76 156 Y 210 Y 238 Y 125 76 12/23

125 235 25 N 210 Y 288 Y 125 235 12/27

125 93 182 Y 169 Y 210 Y 125 93 12/23

125 82 156 Y 196 Y 272 Y 125 82 12/23

125 94 182 Y 169 Y 240 Y 125 94 12/23

125 90 156 Y 182 Y 224 Y 125 90 12/23

125 99 25 N 25 N 210 Y 125 99 1/3

125 78 25 N 25 N 240 Y 125 78 1/5

125 85 144 Y 182 Y 270 Y 125 85 12/23

125 204 25 N 25 N 210 Y 125 204 1/4

125 79 25 N 25 N 25 N 125 79

250 84 182 Y 195 Y 270 Y 250 84 12/23

250 83 156 Y 196 Y 240 Y 250 83 12/23

250 87 25 N 182 Y 224 Y 250 87 12/28

250 91 100 Y 196 Y 210 Y 250 91 12/23

250 77 25 N 210 Y 270 Y 250 77 12/28

250 76 25 N 182 Y 225 Y 250 76 12/28

# B-1 250 98 156 Y 182 Y 210 Y 250 98 12/23

250 100 25 N 25 N 25 N 250 100

250 95 25 N 169 Y 195 Y 250 95 12/27

250 99 25 N 25 N 25 N 250 99

250 231 25 N 25 N 25 N 250 231

250 189 144 Y 210 Y 238 Y 250 189 12/23

250 230 25 N 25 Y 210 Y 250 230 1/4

250 90 25 N 182 Y 272 Y 250 90 12/28

250 75 25 N 210 Y 255 Y 250 75 12/28

250 81 143 Y 182 Y 240 Y 250 81 12/23

250 85 25 N 25 Y 210 Y 250 85 1/3

250 80 144 Y 132 Y 225 Y 250 80 12/23

250 181 169 Y 255 Y 255 Y 250 181 12/23

250 190 25 N 169 Y 210 Y 250 190 12/28

F2 egg strength

DATE DOSE ID Thickness 1 Thickness 2 Strength Yolk DATE Thickness 1 Thickness 2 Strength DATE Thickness 1 Thickness 2 Strength

0 183

0 199

1/26 0 200 0.43 0.6 1.414 1/28 0.3 0.37 1.364

1/27 0 194 0.36 0.41 1.193

1/25 0 179 0.5 0.34 0.729 1/27 1.04 0.86 0.495 1/28 0.58 0.42 0.93

1/25 0 191 0.4 0.35 0.761 1/27 0.46 0.3 0.691 1/28 0.33 0.35 0.345

1/28 0 188 0.37 0.34 0.463 1/26 0.37 0.4 1.232

1/25 0 189 0.39 0.43 0.939

1/27 0 313 0.35 0.41 0.598

1/25 0 174 0.54 0.49 0.696 N 1/26 0.45 0.37 0.382 1/27 0.48 0.5 1.071

1/25 0 314 0.45 0.49 1.124 1/26 0.43 0.38 1.02

1/25 0 309 0.31 0.36 0.785 1/26 0.68 0.7 0.757

1/25 0 186 0.55 0.38 0.931 1/26 0.42 0.33 1.371

1/25 0 197 0.49 0.62 1.155 1/26 0.54 0.54 1.439

1/25 0 178 0.62 0.62 1.017 1/26 0.76 0.84 0.409

0 181

1/26 0 175 0.78 0.44 0.851 1/27 0.44 0.45 0.699 1/28 0.59 0.61 0.764

0 190

1/25 0 323 0.43 0.37 0.953 1/26 0.52 0.48 1.551

1/25 0 305 0.39 0.47 0.7

1/25 30 81 0.37 0.38 1.055 1/26 0.57 0.54 1.004

1/25 30 96 0.51 0.36 1.172 1/27 0.5 0.42 1.981 1/28 0.38 0.34 1.503

1/26 30 89 0.35 0.38 1.534 1/27 0.65 0.47 1.003

1/25 30 85 0.41 0.43 0.911 1/26 0.51 0.38 0.778

1/27 30 403 0.66 0.78 0.738

1/25 30 405 1.06 0.71 1.249 1/26 0.57 0.64 1.252

1/25 30 408 0.62 0.45 1.215 1/26 0.53 0.41 1.157

1/26 30 427 0.38 0.41 1.118 1/27 0.37 0.44 1.185 1/28 0.42 0.39 0.393

1/26 30 409 0.41 0.47 1.002 1/28 0.35 0.32 0.72

30 432

30 84 1/26 0.72 0.53 1.105

# B-1 1/25 30 86 0.35 0.3 1.257 1/28 0.35 0.28 1.024

1/25 30 82 0.49 0.45 1.02 1/26 0.42 0.68 0.996

1/25 30 87 0.43 0.45 1.027 1/28 0.39 0.33 1.106

1/25 30 401 0.44 0.56 0.943 N 1/26 0.3 0.25 1.024 1/27 0.89 0.9 0.977

1/25 30 74 0.6 0.48 1.528 1/26 0.72 0.52

1/25 30 73 0.65 0.41 0.792 1/26 0.34 0.45

1/27 30 86 0.52 0.45 1.065

1/25 30 75 0.41 0.42 1.096 1/26 0.43 0.4 1.074

30 79

1/25 60 75 0.91 0.84 0.987 1/26 0.57 0.75 0.764

1/25 60 91 0.45 0.46 1.344 1/26 0.33 0.35 1.11

1/25 60 89 0.43 0.47 1.707 1/27 0.4 0.35 0.34 1/28 0.32 0.34 1

1/25 60 73 0.41 0.38 1.47 N 1/28 0.45 0.39 1.447

1/28 60 85 0.39 0.34 1.149

1/25 60 100 0.29 0.28 1.214 1/26 0.39 0.41 1.299

1/25 60 94 0.42 0.41 0.834 1/26 0.35 0.42 1.175

1/25 60 76 0.3 0.41 0.35 1/26 0.87 0.81 1.452

60 78

1/25 60 93 0.6 0.45 1.358 1/26 0.81 1.05 1.091

1/25 60 82 0.56 0.35 0.974 1/26 0.83 0.97 0.911

1/25 60 74 0.65 0.63 1.781 1/26 0.41 0.42 1.228

1/25 60 92 0.53 0.56 0.292 1/26 0.87 0.57 0.871

60 87

1/25 60 202 0.44 0.56 0.887 1/27 0.42 0.36 1.157 1/28 0.38 0.31 0.822

1/25 60 77 0.71 0.89 1.3 1/26 0.42 0.38 1.231

1/25 60 222 0.32 0.36 1.562 1/26 0.61 0.85 1.493

1/25 60 97 0.74 0.5 1.158

1/26 60 215 0.6 0.48 0.333 1/27 0.3 0.32 0.777 1/28 0.32 0.35 0.425

1/25 60 216 0.37 0.26 0.347

1/25 125 74 0.52 0.41 0.935 1/27 0.47 0.36 0.898 1/28 0.39 0.35 1.12

1/25 125 80 0.42 0.34 0.979 1/26 0.62 0.65 1.369

1/26 125 83 0.67 0.6 0.902 1/27 0.62 0.6 1.041

1/25 125 73 0.36 0.32 0.45 1/28 0.38 0.33 0.967

1/25 125 206 0.61 0.4 0.924 1/27 0.29 0.32 0.808

1/25 125 205 0.37 0.33 0.921 1/26 0.64 0.73 0.789

1/25 125 234 0.32 0.33 1.152 1/26 0.46 0.53 1.353

1/25 125 201 0.42 0.37 0.495 1/27 0.45 0.47 0.557 1/28 0.28 0.32 1.392

1/27 125 239 0.52 0.46 0.559

1/25 125 237 0.45 0.59 0.821 1/26 0.36 0.41 1.557

1/26 125 88 0.72 0.78 0.476 1/28 0.27 0.37 0.464 1/28 0.4 0.42 0.75

125 87 1/26 0.39 0.42 1.602

1/25 125 89 0.4 0.39 1.181 1/26 0.47 0.67 1.315

1/25 125 84 0.53 0.43 1.287 1/26 0.6 0.48 1.227

1/25 125 207 0.64 0.48 1.31 1/26 0.69 0.57 1.296

1/25 125 202 0.4 0.45 1.325 1/26 0.61 0.67 1.107

1/25 125 259 0.52 0.39 1.004 1/26 0.3 0.32 0.829

1/25 125 209 0.88 0.65 1.042 1/26 0.52 0.36 1.318

1/25 125 258 0.48 0.35 1.153 1/26 0.62 0.61 0.819

# B-1 1/25 125 260 0.34 0.39 1.013 1/26 0.37 0.42 1.101

1/25 250 89 0.49 0.33 1.182 1/26 0.65 0.62 1.322

1/25 250 82 0.77 0.8 0.909 1/26 0.35 0.46 1.414

1/25 250 79 0.37 0.38 1.233 1/26 0.41 0.43 1.555

250 92

1/25 250 94 0.41 0.45 1.33 1/26 0.61 0.53 1.582

1/25 250 228 0.46 0.53 1.258 1/26 0.47 0.37 0.835

250 188

1/25 250 184 0.4 0.59 1.342 1/26 0.43 0.39 1.081

1/25 250 97 0.62 0.72 0.332 1/26 0.53 0.64 1.181

1/25 250 74 0.48 0.52 1.623 1/26 0.32 0.39 1.316

1/25 250 78 0.63 0.72 1.046 1/26 0.47 0.42 1.229

1/25 250 88 0.48 0.44 0.996 1/26 0.38 0.4 0.89

1/25 250 96 0.59 0.42 0.357 1/26 0.34 0.37 1.397

1/25 250 93 0.29 0.38 1.119 1/26 0.48 0.43 1.305

1/26 250 225 0.41 0.55 0.86 1/27 0.56 0.58 0.683

1/25 250 221 0.46 0.49 1.358 1/26 0.38 0.46 1.294

F2 sperm measurements

ID Dose Volume Weight # sperm/gram % Motile % Progressive

182 0 0.5 0.019 178.9 42 4

182 0 0.5 0.019 151.3 27 1

303 0 0.5 0.019 31 61 6

303 0 0.5 0.019 36.1 48 10

303 0 0.5 0.019 27.5 44 0

173 0 0.5 0.02 313.7 42 8

173 0 0.5 0.02 398.6 66 11

173 0 0.5 0.02 346.4 47 11

329 0 0.5 0.015 8200.5 98 37

329 0 0.5 0.015 7125.3 97 35

329 0 0.5 0.015 4501.2 94 26

198 0 0.5 0.016 5125.3 90 15

198 0 0.5 0.016 7209.6 92 21

198 0 0.5 0.016 8593.4 93 19

195 0 0.5 0.011 9244.2 94 23

195 0 0.5 0.011 6063.4 94 20

195 0 0.5 0.011 5144 96 19

192 0 0.5 0.011 12151.7 97 20

192 0 0.5 0.011 8573.3 94 21

192 0 0.5 0.011 7802.9 95 14

308 0 0.5 0.008 9328.1 92 20

308 0 0.5 0.008 9157.2 88 18

308 0 0.5 0.008 8371.3 84 17

321 0 0.5 0.006 6195.9 85 13

321 0 0.5 0.006 6514.8 82 15

321 0 0.5 0.006 4647 55 7

# B-1 180 0 0.5 0.008 8200.5 83 21

180 0 0.5 0.008 9430.6 91 20

180 0 0.5 0.008 8918 86 14

93 30 0.5 0.018 319.5 30 0

93 30 0.5 0.018 283.2 38 5

76 30 0.5 0.02 35.9 73 0

76 30 0.5 0.02 29.4 72 6

76 30 0.5 0.02 39.2 88 4

80 30 0.5 0.019 39.6 74 9

80 30 0.5 0.019 48.2 89 21

80 30 0.5 0.019 34.4 60 5

402 30 0.5 0.008 3006.9 67 8

402 30 0.5 0.08 4100.3 84 11

402 30 0.5 0.008 3656.1 82 7

88 30 0.5 0.008 7927.2 95 14

88 30 0.5 0.008 7141.3 94 12

88 30 0.5 0.008 9191.4 96 9

91 30 0.5 0.008 10489.8 89 12

91 30 0.5 0.008 7483 85 13

91 30 0.5 0.008 9054.7 91 12

406 30 0.5 0.009 1974.2 58 5

406 30 0.5 0.009 4221.7 59 9

406 30 0.5 0.009 5710 74 13

97 30 0.5 0.009 1549 57 10

97 30 0.5 0.009 1640.1 74 20

97 30 0.5 0.009 2460.2 81 10

83 30 0.5 0.008 9840.6 93 29

83 30 0.5 0.008 12027.4 95 32

83 30 0.5 0.008 9943.1 92 27

100 30 0.5 0.008 10353.1 95 19

100 30 0.5 0.008 13940.9 94 18

100 30 0.5 0.008 13325.8 95 18

79 60 0.5 0.014 420.1 64 9

79 60 0.5 0.014 373.4 60 20

79 60 0.5 0.014 560.2 75 10

79 60 0.5 0.014 2050.1 86 25

79 60 0.5 0.014 8630.1 89 20

84 60 0.5 0.014 9489.2 91 15

84 60 0.5 0.014 6658 81 10

84 60 0.5 0.014 6130.9 86 13

86 60 0.4 0.013 11459.7 92 21

86 60 0.5 0.013 10429.4 93 17

86 60 0.5 0.013 10744.8 91 20

95 60 0.5 0.006 9020.6 90 12

95 60 0.5 0.006 9248.3 93 23

95 60 0.5 0.006 5831.5 77 16

98 60 0.5 0.008 7483 85 19

98 60 0.5 0.008 6047.9 91 18

# B-1 98 60 0.5 0.008 5740.4 93 15

204 60 0.5 0.009 7167.8 90 16

204 60 0.5 0.009 5254.4 82 10

204 60 0.5 0.009 6712.3 85 14

88 60 0.5 0.007 12457 88 19

88 60 0.5 0.007 13042.7 88 20

88 60 0.5 0.007 9567.3 90 13

218 60 0.5 0.008 4271.1 82 12

218 60 0.5 0.008 3861.1 90 17

218 60 0.5 0.008 3826.9 76 9

208 60 0.5 0.007 7458.6 93 19

208 60 0.5 0.007 8512.9 93 22

208 60 0.5 0.007 6091.8 90 10

210 60 0.5 0.007 8708.2 84 10

210 60 0.5 0.007 5701.3 81 10

210 60 0.5 0.007 7107.1 85 14

81 125 0.5 0.02 3362.2 91 18

81 125 0.5 0.02 3854.2 90 15

81 125 0.5 0.02 4018.2 90 22

77 125 0.5 0.015 4391.8 87 14

77 125 0.5 0.015 4045.6 79 11

77 125 0.5 0.015 5703.9 79 9

233 125 0.5 0.019 4675.7 92 15

233 125 0.5 0.019 4013.9 90 10

233 125 0.5 0.019 3913.2 86 15

76 125 0.5 0.005 5303 79 10

76 125 0.5 0.005 5849.7 87 9

76 125 0.5 0.005 5084.3 80 9

97 125 0.5 0.008 6492.1 89 17

97 125 0.5 0.008 6389.6 84 7

97 125 0.5 0.008 7312.1 80 14

93 125 0.5 0.008 8610.5 92 14

93 125 0.5 0.008 6526.2 86 11

93 125 0.5 0.008 6082 89 12

94 125 0.5 0.005 4100.3 80 15

94 125 0.5 0.005 3006.9 67 13

94 125 0.5 0.005 4647 66 7

79 125 0.5 0.007 3241.2 55 5

79 125 0.5 0.007 6794.7 86 9

79 125 0.5 0.007 4295.5 62 5

85 125 0.5 0.007 5467 62 11

85 125 0.5 0.007 5232.7 81 14

85 125 0.5 0.007 8786.3 80 8

99 125 0.5 0.006 11754.1 90 19

99 125 0.5 0.006 9795 89 19

99 125 0.4 0.006 7157.7 75 8

81 250 0.5 0.013 9146.7 93 20

81 250 0.5 0.013 10744.8 95 18

# B-1 81 250 0.5 0.013 10892 94 16

90 250 0.5 0.009 880.8 17 0

90 250 0.5 0.009 850.4 54 4

90 250 0.5 0.009 789.7 58 0

80 250 0.5 0.013 10303.2 90 17

80 250 0.5 0.013 10807.8 93 14

80 250 0.5 0.013 10050.9 93 13

91 250 0.5 0.01 6587.7 89 17

91 250 0.5 0.01 7517.1 89 18

91 250 0.5 0.01 6806.4 94 12

190 250 0.5 0.011 2982 61 7

190 250 0.5 0.011 4249.4 85 12

190 250 0.5 0.011 4547.6 79 15

76 250 0.5 0.011 7305.9 93 31

76 250 0.5 0.011 8051.4 96 33

76 250 0.5 0.011 8076.3 90 26

83 250 0.5 0.008 4066.1 73 12

83 250 0.5 0.008 5262 87 13

83 250 0.5 0.008 4339.4 84 7

100 250 0.5 0.007 7653.8 81 18

100 250 0.5 0.007 6248 68 13

100 250 0.5 0.007 4490.8 59 11

189 250 0.5 0.01 1503.4 11 0

189 250 0.5 0.01 1312.1 8 0

189 250 0.5 0.01 1530.8 5 0

99 250 0.5 0.005 273.4 0 0

99 250 0.5 0.005 710.7 46 0

99 250 0.5 0.005 546.7 0 0

F2 growth measurements

11/2

DOSE baby

id BW tarsus

1 tarsus

2 avg

tarsus culmen

1 culmen

2 avg

culmen

0 394 8 14.9 14.4 14.65 4.9 5.3 5.1

0 392 9 17.8 17.4 17.6 4 4.1 4.05

0 401 7 15.9 16.3 16.1 4.1 4.5 4.3

0 391 8 16.4 16.5 16.45 4.2 4.5 4.35

0 396 19 16.1 16.2 16.15 4.2 4.6 4.4

0 388 9 14.6 14.3 14.45 4.2 4.4 4.3

0 403 6 13.5 13.8 13.65 3 3 3

0 395 7 17.2 17.5 17.35 3.4 3.1 3.25

0 398 8 15.4 15.5 15.45 3.7 3.9 3.8

0 404 8 16.1 16.6 16.35 2.7 3 2.85

0 426 7 13.4 13.9 13.65 4.5 4.1 4.3

0 432 6 13.2 13.4 13.3 3.2 3.6 3.4

0 424 6 16.2 16.2 16.2 3 3.2 3.1

0 431 6 15.8 16.1 15.95 3.5 3.6 3.55

0 440 6 16 16.1 16.05 3.5 3.6 3.55

# B-1 0 421 7 16 16.3 16.15 4 4.1 4.05

0 439 6 16.1 16.5 16.3 3.8 4 3.9

0 437 6 15.7 15.6 15.65 3.8 3.9 3.85

0 436 6 15.2 15.3 15.25 3.5 3.8 3.65

0 420 7 15.5 15.7 15.6 3 3.2 3.1

30 446 7 13.5 12.6 13.05 3.1 2.8 2.95

30 450 7 12.9 13 12.95 4.2 3.8 4

30 456 8 14.5 15 14.75 3.9 3.9 3.9

30 454 8 16.3 15.6 15.95 4.1 3.9 4

30 455 9 17 17.3 17.15 3.7 3.7 3.7

30 451 6 15.6 15.9 15.75 3.8 4 3.9

30 457 7 12.3 12.5 12.4 3.2 3.7 3.45

30 460 9 18 18.3 18.15 3.1 3.4 3.25

30 445 7 15.5 15.9 15.7 3.9 4.2 4.05

30 453 9 18.2 18.4 18.3 3.3 3.6 3.45

30 393 7 16.6 17 16.8 3.3 3.5 3.4

30 487 8 17.1 17.4 17.25 3.8 4.1 3.95

30 486 6 16.2 15.9 16.05 4 4.2 4.1

30 492 6 14.7 14.9 14.8 3.8 4.1 3.95

30 484 6 16.4 16.6 16.5 3.8 3.9 3.85

30 489 7 17.1 17.2 17.15 3.6 3.7 3.65

30 493 7 14 14.2 14.1 3.5 3.8 3.65

30 491 7 17.4 17.1 17.25 3.8 4.1 3.95

30 495 7 16.8 17.1 16.95 3.8 4 3.9

30 490 6 14.8 15 14.9 3.4 3.6 3.5

60 373 8 15.3 15.6 15.45 4 3.6 3.8

60 375 9 17 17.6 17.3 4.6 4.3 4.45

60 363 10 16.5 16.3 16.4 3.8 3.5 3.65

60 359 9 16.7 16.9 16.8 4.3 4.4 4.35

60 365 10 15.9 15.7 15.8 4.2 4.1 4.15

60 370 10 17.4 17.6 17.5 3.7 4.1 3.9

60 372 9 13 13.1 13.05 4.2 4.2 4.2

60 376 6 15.8 16 15.9 3 3.4 3.2

60 377 6 15.7 15.9 15.8 3.7 3.8 3.75

60 364 7 17.4 17.5 17.45 3.7 3.8 3.75

60 399 7 16 16.4 16.2 3.6 3.9 3.75

60 321 6 16.4 16.6 16.5 4.2 4.4 4.3

60 400 8 16.5 15.7 16.1 3.8 3.9 3.85

60 397 8 16.1 16.3 16.2 3.4 3.6 3.5

60 396 6 16.7 16.8 16.75 3.3 3.4 3.35

60 395 7 15.5 15.7 15.6 4.1 4.3 4.2

60 394 6 15.7 15.8 15.75 3.6 3.6 3.6

60 398 7 14.9 15.2 15.05 3.7 3.9 3.8

125 387 10 17.6 17 17.3 4.9 4.4 4.65

125 382 7 15.1 15.7 15.4 3.2 3.7 3.45

125 391 9 13.2 13.9 13.55 3.3 3.7 3.5

125 392 9 15.7 15.2 15.45 3.5 3.7 3.6

125 386 9 16.6 17 16.8 3.8 4 3.9

# B-1 125 389 7 16.4 16.5 16.45 3.4 3.8 3.6

125 396 8 14.9 15.2 15.05 3.2 3.8 3.5

125 398 7 15.7 16.1 15.9 3.8 3.8 3.8

125 395 8 15.3 15.6 15.45 4.2 4 4.1

125 388 10 15.4 15.1 15.25 4.1 3.8 3.95

125 356 7 18.3 18 18.15 3.6 3.8 3.7

125 354 7 15.8 16 15.9 3.5 3.7 3.6

125 353 8 14.7 15 14.85 3.6 3.7 3.65

125 356 8 16.4 16.5 16.45 4.1 4.5 4.3

125 357 6 15.8 16 15.9 3.7 3.4 3.55

125 359 6 15.7 15.9 15.8 3.1 3.3 3.2

125 361 6 15.3 15.4 15.35 3.9 3.4 3.65

125 362 7 16 16.2 16.1 3.4 3.6 3.5

125 363 7 14.8 15 14.9 3.8 3.4 3.6

125 352 7 16 15.8 15.9 4 4.2 4.1

250 415 11 16.3 16.6 16.45 4.7 4.9 4.8

250 420 10 16.8 17.2 17 4.5 4.8 4.65

250 422 7 16.7 16.9 16.8 3.8 4.1 3.95

250 412 11 17 16.8 16.9 4 4.3 4.15

250 427 7 13.3 13.6 13.45 3.6 3.8 3.7

250 419 8 15.2 15.4 15.3 3.8 4.1 3.95

250 425 6 13.4 13.7 13.55 3.5 3.6 3.55

250 414 10 15.5 15.8 15.65 3.2 3.6 3.4

250 418 8 18 18.4 18.2 4.1 4.2 4.15

250 416 7 17.5 17.7 17.6 4 4.2 4.1

250 445 8 15.8 16.1 15.95 4.1 4.3 4.2

250 453 7 14.5 14.7 14.6 3.6 3.8 3.7

250 455 6 15.8 16.2 16 3.8 3.9 3.85

250 448 7 16.3 16.4 16.35 3.6 3.9 3.75

250 450 7 14 14.1 14.05 4.1 4.4 4.25

250 444 6 17.1 17.3 17.2 4.1 4.5 4.3

250 460 6 16.3 16.4 16.35 3.6 3.8 3.7

250 458 7 16.4 16.5 16.45 4 4.1 4.05

250 452 6 15.6 15.9 15.75 3.6 3.7 3.65

250 469 6 16 16.4 16.2 3.2 3.4 3.3F2 growth measurements11/6 11/19DOSEbabyidBWtarsus1tarsus2avgtarsusculmen1culmen2avgculmenDOSEbabyidBWtarsus1tarsus2avgtarsusculmen1culmen2avgculmen0 388 19 6.2 6.4 6.3 19.9 20 19.95 0 397 30 5.7 5.9 5.8 30.9 21 25.950 395 19 3.3 3.5 3.4 190.6 19.8 105.2 0 394 22 5.1 5 5.05 25.5 25.2 25.350 389 16 5 5.3 5.15 18.8 19.2 19 0 401 23 5.8 5.9 5.85 22.7 23 22.850 396 18 5.1 5.2 5.15 19.3 19.4 19.35 0 396 25 6.3 6.6 6.45 19.2 19.4 19.30 402 13 4.5 4.7 4.6 18.1 18.5 18.3 0 403 25 5.1 4.8 4.95 19.4 19.5 19.450 404 19 4.8 4.9 4.85 17.7 17.9 17.8 0 403 22 5.2 5.6 5.4 16.8 17 16.90 394 17 5.6 5.6 5.6 19.9 20.1 20 0 405 22 6 6.1 6.05 18.4 18.6 18.50 427 8 3.1 3.4 3.25 12.3 12.4 12.35 0 404 27 5.6 5.7 5.65 20.8 20.9 20.850 401 15 4.3 4.1 4.2 18.4 18.6 18.5 0 393 26 5.4 5.5 5.45 19.9 20.3 20.10 392 18 4 4.1 4.05 17.8 18 17.9 0 392 26 4.9 5.2 5.05 19.4 19.7 19.5530 496 15 4.7 4.5 4.6 21.8 21.6 21.7 30 455 82 5.4 5.7 5.55 25.5 25.3 25.430 453 19 4.4 4.5 4.45 21.2 20.9 21.05 30 444 29 5.7 5.8 5.75 24.7 25 24.8530 456 12 4.3 4.3 4.3 17.4 17.6 17.5 30 447 27 5.8 6.1 5.95 25.1 25.3 25.230 457 14 5.1 5.2 5.15 18.4 18.7 18.55 30 453 26 5.2 5.3 5.25 23.8 23.9 23.8530 444 20 4.8 5 4.9 20.1 20.2 20.15 30 449 27 5.8 5.8 5.8 22 22.2 22.130 448 21 5.2 5.3 5.25 21.1 21.2 21.15 30 454 25 6.1 6.2 6.15 17.9 18.2 18.0530 455 20 4.4 4.7 4.55 20.5 20.7 20.6 30 458 28 5.3 5.4 5.35 22.1 22.2 22.1530 451 14 4.4 4.3 4.35 16.4 16.8 16.6 30 446 23 4.7 4.8 4.75 17.1 17.3 17.230 445 15 4.7 4.9 4.8 18.1 18.3 18.2 30 457 23 5.5 5.6 5.55 17.8 17.9 17.8530 458 19 4.1 4.2 4.15 18.4 18.5 18.45 30 451 21 5.7 6 5.85 16.8 17 16.960 369 19 5.2 5.4 5.3 20 20.2 20.1 60 370 25 6.1 6.3 6.2 22.2 22.3 22.2560 370 17 4.8 4.9 4.85 19.6 19.9 19.75 60 365 29 6.1 6.5 6.3 26.3 26.7 26.560 376 18 4.6 4.7 4.65 19 19.5 19.25 60 366 28 4.2 4.5 4.35 22.9 23.4 23.1560 378 14 4.7 4.5 4.6 18 18.5 18.25 60 367 23 5.6 5.6 5.6 20.8 20.9 20.8560 367 17 5.3 5.5 5.4 18.5 18.4 18.45 60 362 29 5.2 5.5 5.35 23.5 23.7 23.660 381 15 4.6 4.6 4.6 20 20.1 20.05 60 375 29 5.3 5.6 5.45 20.5 20.7 20.660 366 19 5 5.3 5.15 19.6 19.7 19.65 60 377 24 5.1 5.3 5.2 17.6 17.9 17.7560 362 19 5.6 5.7 5.65 20 19.7 19.85 60 361 24 5.8 5.9 5.85 20.2 20 20.160 373 16 4.1 4.4 4.25 20 20 20 60 379 30 6 6.2 6.1 19.1 18.8 18.9560 369 18 5 4.8 4.9 15.7 16 15.85 60 380 25 6.7 6.8 6.75 19.4 19.6 19.5125 381 20 4.8 5 4.9 21.8 23 22.4 125 395 23 6.2 6.4 6.3 23.2 23.4 23.3B-1125 380 19 5.1 5.2 5.15 22.2 22.5 22.35 125 384 27 6.6 6.4 6.5 21.1 21.3 21.2125 396 19 4.7 4.7 4.7 21.4 21.7 21.55 125 396 27 5.8 5.9 5.85 19.6 20 19.8125 398 17 4.4 4.6 4.5 19.7 19.9 19.8 125 366 23 6.4 6.8 6.6 18.2 18.4 18.3125 382 19 3.8 4 3.9 17.5 17.7 17.6 125 383 26 5.1 5.2 5.15 18.9 19.2 19.05125 375 16 6.1 6.3 6.2 21.1 21.2 21.15 125 381 26 6.4 6.6 6.5 18.1 18.4 18.25125 394 13 4.6 4.8 4.7 18.6 18.7 18.65 125 399 23 5.5 5.4 5.45 17.8 17.7 17.75125 399 16 4.8 5 4.9 19.7 19.9 19.8 125 390 24 5.3 5.4 5.35 18 18.1 18.05125 345 15 5.2 5.1 5.15 17.2 17.3 17.25 125 397 24 5.3 5.6 5.45 16.9 17.2 17.05125 400 17 4.7 4.8 4.75 17.7 17.9 17.8 125 345 23 6.1 6.2 6.15 17.6 17.7 17.65250 414 4.7 4.8 4.8 20.1 20.2 20.15 250 418 24 5.4 5.5 5.45 22.8 23.1 22.95250 420 21 4.7 5 4.85 21.1 21.5 21.3 250 422 26 5.2 5.3 5.25 22.2 22.5 22.35250 426 17 5.2 5 5.1 19 19.3 19.15 250 415 30 5.7 5.4 5.55 22 22.4 22.2250 413 21 4.7 4.6 4.65 22.2 22.4 22.3 250 412 30 4.4 4.9 4.65 22.4 21.9 22.15250 417 13 4.8 5 4.9 18.5 18.7 18.6 250 427 23 5.2 5.3 5.25 18.2 18.4 18.3250 418 15 4.8 5 4.9 16.1 16.4 16.25 250 425 19 5.3 5.6 5.45 17.5 17.8 17.65250 427 15 4.7 4.9 4.8 15.9 16 15.95 250 426 24 6.3 6.2 6.25 18.3 18.4 18.35250 412 22 4.8 5 4.9 22 22.2 22.1 250 413 31 6.4 6.5 6.45 19.8 20.1 19.95250 422 17 4.4 4.7 4.55 16.5 16.9 16.7 250 428 11 4.8 5.1 4.95 16.8 17 16.9250 429 17 4.8 5 4.9 16.6 16.7 16.65 250 413 21 5.6 5.9 5.75 20 20.3 20.15F2 growth measurements11/23 11/27 11/30DOSEbabyidIDBWtarsus1tarsus2avgtarsusculmen1culmen2avgculmenDOSEIDBWtarsus1tarsus2avgtarsusculmen1culmen2avgculmenDOSEIDBWtarsus1tarsus2avgtarsusculmen1culmen2avgculmen0 393 191 38.3 6.8 6.7 6.75 22.5 22.9 22.7 0 192 52 7.1 7.3 7.2 29.2 30.3 29.75 0 183 56 7.8 8 7.9 26.6 27 26.80 392 192 38 4.8 5.1 4.95 24.5 24.7 24.6 0 195 48 7 7.3 7.15 27.5 27.6 27.55 0 200 57 7.9 8 7.95 28.8 28.6 28.70 402 193 30.3 6.9 7.2 7.05 23 22.7 22.85 0 191 49 6.5 6.9 6.7 27.4 27.5 27.45 0 181 65 7.2 7.5 7.35 29.6 29.8 29.70 394 194 35.8 6.1 6 6.05 23.3 23.7 23.5 0 185 49 7.3 7.4 7.35 28.9 29 28.95 0 197 58 6.9 7.1 7 29.4 29.8 29.60 356 195 36.2 6.6 6.8 6.7 21.7 21.8 21.75 0 193 42 6.1 6.5 6.3 27.8 27.7 27.75 0 196 52 6.8 6.9 6.85 26 26.2 26.10 397 196 29.3 7.6 7.8 7.7 21.2 21.3 21.25 0 196 40 6.1 6.5 6.3 24.6 24.9 24.75 0 198 57 8 8.1 8.05 30 30.2 30.10 1 197 33.6 6.1 6.4 6.25 25.5 24.9 25.2 0 194 49 7.1 7.3 7.2 27.1 27.3 27.2 0 182 47 6.2 6.3 6.25 24 24.4 24.20 389 198 34.3 5.4 5.3 5.35 22.4 22.7 22.55 0 184 48 5.9 6 5.95 27.5 27.6 27.55 0 199 62 8 8.1 8.05 31.2 31.2 31.20 388 199 36.8 6.2 6.4 6.3 22 22.1 22.05 0 198 46 6.6 6.3 6.45 27.2 27.5 27.35 0 184 62 7.1 7.3 7.2 30 29.9 29.950 351 200 33.5 5.7 5.7 5.7 23.5 23.9 23.7 0 103 42 6.2 6.5 6.35 22.1 22.5 22.3 0 193 54 6.3 6.4 6.35 23.3 23.4 23.3530 455 81 40.2 7.1 7.3 7.2 24.9 25 24.95 0 186 51 6.6 6.8 6.7 28.1 28.5 28.3 30 68 55 6.1 6.2 6.15 29.3 29.5 29.4B-130 449 82 38.2 6.1 6 6.05 25.5 25.9 25.7 30 18 51 5.8 6 5.9 29.9 30 29.95 30 95 52 7.9 8 7.95 28.9 29 28.9530 444 83 40.5 6.6 6.7 6.65 25.2 25 25.1 30 93 46 6.2 6.5 6.35 26 26.1 26.05 30 91 66 7.3 7.4 7.35 29.5 29.6 29.5530 452 84 33.4 7.1 7.5 7.3 19.9 19.7 19.8 30 92 52 7.3 7.4 7.35 24.6 24.8 24.7 30 93 60 7.1 7.3 7.2 28.1 28.2 28.1530 456 85 31.7 6.3 6.5 6.4 20 20.1 20.05 30 91 52 7.3 7.3 7.3 28.2 28.4 28.3 30 92 64 8 8.4 8.2 30.1 29.9 3030 446 86 34 5.9 6.2 6.05 21.1 21.3 21.2 30 83 52 7 7.2 7.1 25.6 25.9 25.75 30 87 71 7.9 8.1 8 33.9 34 33.9530 448 87 42.3 7.5 7.6 7.55 24.7 25 24.85 30 96 49 6.2 6.2 6.2 28.8 28.9 28.85 30 84 58 7.5 7.5 7.5 29.7 30 29.8530 451 89 31.6 5.9 5.8 5.85 22.8 23 22.9 30 94 41 6.2 6 6.1 26.2 26.4 26.3 30 98 58 6.7 6.5 6.6 29.8 29.5 29.6530 459 90 35.7 5.8 6 5.9 24.4 24.7 24.55 30 95 40 6.3 6.8 6.55 26.1 26.5 26.3 30 90 62 7.9 8.1 8 29.7 29.8 29.7530 454 99 34.5 6.4 6.6 6.5 24.4 24.5 24.45 30 86 46 6.2 6.3 6.25 25.8 26.2 26 30 85 57 7 7.3 7.15 27.7 27.5 27.660 365 73 38.7 6.8 7.1 6.95 23.5 23.2 23.35 30 82 50 7 7.4 7.2 27.8 27.5 27.65 60 80 65 7.7 8 7.85 29.6 29.6 29.660 370 81 34.2 7.1 7.3 7.2 24.5 24.6 24.55 30 88 46 6.2 6.3 6.25 26.1 26.5 26.3 60 96 44 7.1 7.5 7.3 26.5 26.3 26.460 369 82 39.8 7 7.1 7.05 24.5 24.6 24.55 60 98 48 7.3 7.2 7.25 31.1 31.3 31.2 60 91 68 7.5 7.6 7.55 27.3 27.4 27.3560 380 84 36.6 7.2 7.3 7.25 22.5 22.5 22.5 60 89 51 6.6 6 6.3 29.3 29.5 29.4 60 96 59 8.3 8.1 8.2 27.1 27.2 27.1560 376 85 33.3 5.7 5.9 5.8 24.6 24.6 24.6 60 82 51 6.8 6.5 6.65 28.8 29 28.9 60 78 57 6.3 6.6 6.45 29.9 30.3 30.160 372 86 37.5 6 6.3 6.15 21.3 21.6 21.45 60 88 54 6.5 6.9 6.7 29.6 30 29.8 60 79 60 7.4 7.7 7.55 31.4 31.5 31.4560 374 87 37.3 5.2 5.3 5.25 24.1 24.3 24.2 60 87 48 7.1 7.3 7.2 25.7 26.1 25.9 60 76 64 7.8 8 7.9 31.5 31.9 31.760 320 88 41.6 7.1 7.4 7.25 26.2 26.5 26.35 60 86 50 7 7.4 7.2 25.7 25.8 25.75 60 77 50 7.7 7.8 7.75 29.8 29.7 29.7560 366 89 39.7 6.4 6.3 6.35 25.7 25.9 25.8 60 73 50 6.6 6.8 6.7 30 29.8 29.9 60 75 69 8.8 8.9 8.85 31.5 31.6 31.5560 196 90 35.7 6.3 6.3 6.3 23.8 23.9 23.85 60 90 46 6.8 7.1 6.95 29.5 29.4 29.45 60 95 60 7.8 7.9 7.85 31.9 31.7 31.8125 392 73 37.2 6.3 6.4 6.35 21.9 22.1 22 60 74 40 6.3 6 6.15 25.4 25.8 25.6 125 94 62 7.2 7.3 7.25 29.4 29.7 29.55125 396 74 40 6.5 6.9 6.7 23.3 23.6 23.45 60 81 44 6.7 7 6.85 27.5 26.9 27.2 125 81 58 7.9 7.9 7.9 29.3 29.6 29.45125 382 75 40.9 6.8 7.1 6.95 22.2 22.4 22.3 125 90 46 7.4 7 7.2 27.7 28 27.85 125 96 52 7.5 7.8 7.65 27.8 28.2 28125 398 76 36.4 6.3 6.5 6.4 24.2 24.3 24.25 125 91 49 6.9 6.9 6.9 28.8 28.5 28.65 125 82 66 7.3 7.5 7.4 31.3 31.2 31.25125 345 77 33.6 6.1 6.5 6.3 21.7 21.9 21.8 125 75 51 6 6.3 6.15 28.4 28.2 28.3 125 99 62 7.5 7.6 7.55 29.2 29.3 29.25125 393 78 35 6.1 6.3 6.2 23.5 23.5 23.5 125 74 52 7.1 7.1 7.1 25.8 26.1 25.95 125 95 63 6.9 7 6.95 28.4 28.5 28.45125 391 79 36.2 7.8 4.3 6.05 23.6 23.8 23.7 125 76 47 6.1 6.4 6.25 27.4 27.5 27.45 125 100 59 7 7.3 7.15 25 25.4 25.2125 375 80 35.2 7 7.2 7.1 22.2 22.6 22.4 125 77 45 7.6 7.4 7.5 27.6 27.6 27.6 125 98 57 7.3 7.3 7.3 30 29.7 29.85125 387 90 36 6.4 6.6 6.5 25.5 25.8 25.65 125 83 50 6.8 6.9 6.85 26.6 26.8 26.7 125 97 65 6.8 6.9 6.85 28.2 28 28.1125 383 91 37.7 2 6.3 4.15 22.2 22.5 22.35 125 80 46 6 6.1 6.05 24.2 24.5 24.35 125 90 59 7 7.1 7.05 26.6 26.9 26.75250 415 81 42.2 6.1 6.3 6.2 22.2 22.5 22.35 125 92 41 7.1 7.2 7.15 26.3 25.9 26.1 250 87 65 7.9 8 7.95 25.8 25.7 25.75250 419 82 40.2 7.1 7 7.05 23.2 23.3 23.25 125 93 50 8.1 8.3 8.2 28.2 28.4 28.3 250 88 74 7.8 8 7.9 32.8 32.9 32.85250 427 83 33.7 7.1 7.3 7.2 24.1 24.3 24.2 250 30 35 7.1 7.4 7.25 22.1 22 22.05 250 89 61 7.9 8 7.95 30 30.1 30.05250 414 84 36.8 6.9 7 6.95 21.2 21.5 21.35 250 78 45 6.9 6.7 6.8 28.2 28.6 28.4 250 90 48 5.6 5.6 5.6 23 23.4 23.2250 125 85 30.1 6.3 6.7 6.5 21.2 21.4 21.3 250 89 49 7.2 7.3 7.25 24.2 24.4 24.3 250 74 49 7.1 7.3 7.2 26.1 26.6 26.35B-1250 413 85 46 6.7 6.9 6.8 25.5 25.2 25.35 250 73 35 5.2 5.4 5.3 23.2 23.6 23.4 250 76 60 6.9 7.1 7 30 30.2 30.1250 429 87 37.5 6 6.2 6.1 25.5 25.2 25.35 250 84 48 7 7.1 7.05 26.3 26.5 26.4 250 75 66 7.8 8 7.9 32.4 32.5 32.45250 422 89 36.8 6.8 6.9 6.85 21.9 21.8 21.85 250 83 44 6.5 6.7 6.6 24.5 25.3 24.9 250 73 45 6.7 6.8 6.75 24.9 25.1 25250 428 90 23.8 5.4 5.9 5.65 17 17.6 17.3 250 81 54 6.4 6.7 6.55 27.7 28 27.85 250 78 59 7.9 8 7.95 30.3 30.5 30.4250 426 91 32.8 6.4 6 6.2 21.9 21.4 21.65 250 85 41 5.9 6.2 6.05 25 25.1 25.05 250 91 57 7.8 7.9 7.85 28.7 28.8 28.75250 87 49 6.4 6 6.2 26.6 26.3 26.45250 82 53 7 7.1 7.05 27.1 26.8 26.95250 88 60 6.5 6.5 6.5 30.6 30.8 30.7F2 growth measurements12/4 12/7 12/10DOSEIDBWtarsus1tarsus2avgtarsusculmen1culmen2avgculmenSEXDOSEIDBWtarsus1tarsus2avgtarsusculmen1culmen2avgculmenSEXDOSEIDBWtarsus1tarsus2avgtarsusculmen1culmen2avgculmen0 193 73 9 9.2 9.1 30.3 30.5 30.4 F 0 181 90 8.7 9 8.85 33.7 33.8 33.75 F 0 200 82 8.4 8.4 8.4 32.5 32.6 32.550 198 69 6.6 6.7 6.65 32.8 33 32.9 F 0 183 77 8.1 8.5 8.3 31.3 30.9 31.1 F 0 181 91 7.9 8 7.95 34.8 35 34.90 181 80 8.2 8.3 8.25 33.1 33 33.05 F 0 197 80 7.4 7.7 7.55 28.4 28.6 28.5 F 0 199 83 7.5 8 7.75 33 33.1 33.050 183 69 6.8 6.9 6.85 30.6 30.8 30.7 F 0 199 79 6.7 6.9 6.8 32 32.4 32.2 F 0 183 81 8.1 8.3 8.2 32.6 32.7 32.650 184 76 7.4 7.4 7.4 30.1 30.5 30.3 F 0 200 75 8.4 8.7 8.55 29.4 29.8 29.6 F 0 197 84 6 6.3 6.15 32.9 33 32.950 196 61 6.8 6.8 6.8 29.6 29.8 29.7 F 30 68 80 8.4 8.7 8.55 33.6 32.9 33.25 F 30 86 83 8 8 8 33.3 33.2 33.250 200 68 7.1 7.2 7.15 30.7 21 25.85 F 30 84 84 8.2 8.6 8.4 32.8 32.9 32.85 F 30 85 87 6.9 7.1 7 32.9 32.8 32.850 197 71 7 7.2 7.1 29.9 29.7 29.8 F 30 85 83 8 8.2 8.1 31.7 32 31.85 F 30 68 81 8.5 8.7 8.6 33.1 32.9 330 182 60 8.1 8.3 8.2 28.9 29 28.95 F 30 86 80 7.5 7.7 7.6 30.6 30.4 30.5 F 30 87 101 7.6 7.7 7.65 35.4 35.7 35.550 192 76 6.8 7 6.9 31.6 31.8 31.7 F 30 87 97 7.7 7.8 7.75 33.6 34 33.8 F 60 91 87 8 8.2 8.1 33.5 33.8 33.6530 86 72 7.1 7.3 7.2 32.3 32.4 32.35 F 60 75 89 9 9.4 9.2 34.6 34.9 34.75 F 60 92 84 7.9 8.1 8 34.7 34.9 34.830 89 68 7.7 7.5 7.6 31.2 31.1 31.15 F 60 77 73 8.2 8.4 8.3 33.8 33.9 33.85 F 60 75 94 8.3 8 8.15 31.6 31.8 31.730 91 79 8.4 8.5 8.45 32.7 33 32.85 F 60 78 82 8.1 8.4 8.25 33.7 33.9 33.8 F 60 76 88 8 8.1 8.05 30.5 30.7 30.630 84 73 8 8.4 8.2 31.6 31.5 31.55 F 60 92 83 7.6 7.8 7.7 27.3 27.5 27.4 F 60 93 76 7.1 7.2 7.15 28.8 28.9 28.8530 95 63 7.1 7.4 7.25 30.6 31 30.8 F 60 93 74 7.7 7.9 7.8 29 29.4 29.2 F 60 94 82 8.6 8.7 8.65 33.1 33.4 33.2530 94 63 7.8 8 7.9 30.5 30.7 30.6 F 60 94 79 8.5 8.8 8.65 33.1 33.2 33.15 F 125 74 86 7.7 7.5 7.6 30.5 30.7 30.630 93 71 8.1 8.3 8.2 30 30.8 30.4 F 125 74 85 7.4 7.5 7.45 34.9 34.5 34.7 F 250 78 91 7.4 7.5 7.45 34.3 34.4 34.3530 87 86 7.2 7.5 7.35 32.5 32.4 32.45 F 250 78 82 7.8 7.9 7.85 32.2 32.6 32.4 F 250 74 74 7.6 7.8 7.7 33.6 33.9 33.7530 85 71 7.2 7.4 7.3 28.7 28.9 28.8 F 250 88 95 7.3 7.5 7.4 33.3 33.3 33.3 F 250 88 97 8.5 8.9 8.7 34.1 34.2 34.1530 90 77 8.1 8.3 8.2 33.8 33.9 33.85 F 250 89 83 7.4 7.5 7.45 32.7 33 32.85 M 0 196 73 7.4 7.6 7.5 29.8 30 29.960 78 69 7.1 7.2 7.15 32 32.3 32.15 M 0 182 69 7.7 7.5 7.6 32.5 32.7 32.6 M 0 184 87 8.5 8.6 8.55 31.3 31.6 31.4560 77 62 7.2 7.2 7.2 32.6 32.3 32.45 M 0 196 71 8.2 8.3 8.25 27.3 27.4 27.35 M 0 192 86 7.7 7.8 7.75 33.5 33.6 33.55B-160 76 77 7.5 7.8 7.65 31.8 32.2 32 M 0 198 77 7.9 8.2 8.05 30 30.1 30.05 M 0 182 72 7.5 7.6 7.55 33.6 33.3 33.4560 92 75 7.1 7.1 7.1 32.2 31.5 31.85 M 30 91 86 8.4 8.5 8.45 31.7 32 31.85 M 0 198 79 7.6 7.8 7.7 32.3 32.1 32.260 96 57 7.5 7.8 7.65 29.7 30 29.85 M 30 92 70 7.9 8.1 8 29.8 29.7 29.75 M 30 94 74 7.3 7.5 7.4 30.8 31 30.960 94 70 8.6 8.6 8.6 31.8 32.2 32 M 30 93 82 7.4 7.5 7.45 32.2 32.3 32.25 M 30 90 89 7.8 8 7.9 33.3 33.4 33.3560 75 83 7.8 7.9 7.85 32.7 32.9 32.8 M 30 95 71 7.1 7.2 7.15 29.2 29.2 29.2 M 30 92 78 7.5 7.4 7.45 29.9 30 29.9560 79 73 7.4 7.4 7.4 30.9 31.1 31 M 60 79 80 8.1 8.5 8.3 32.8 32.9 32.85 M 30 95 74 7.3 7.4 7.35 31.3 31.4 31.3560 95 74 7 7.2 7.1 30.7 30.8 30.75 M 60 90 85 6.9 7 6.95 31.2 31.2 31.2 M 30 93 84 7.1 7.2 7.15 34.2 34.2 34.260 91 80 7.1 7.2 7.15 32.5 32.6 32.55 M 60 95 82 8.6 8.9 8.75 30.7 30.6 30.65 M 30 91 89 8.1 7.9 8 33.1 33.4 33.25125 95 76 6.9 6.7 6.8 31.2 31.2 31.2 M 60 96 65 7.3 7.5 7.4 32.2 31.9 32.05 M 60 96 69 8.2 8.4 8.3 30.8 31 30.9125 96 63 7.1 7.2 7.15 31.4 31.6 31.5 M 125 81 81 7.8 8 7.9 32.1 32.2 32.15 M 60 80 87 6.8 6.9 6.85 33.2 33.4 33.3125 99 73 7.8 7.9 7.85 32.3 32 32.15 M 125 82 90 7.8 7.8 7.8 33.6 33.7 33.65 M 60 95 83 7.4 7.9 7.65 33 32.9 32.95125 81 72 7.5 7.6 7.55 31.7 32 31.85 M 125 94 84 8.2 8.5 8.35 28.9 29.2 29.05 M 60 79 82 8.2 8.3 8.25 32.4 32.6 32.5125 98 68 7.2 7.4 7.3 31.1 31.5 31.3 M 125 95 84 7.3 7.4 7.35 33.7 33.9 33.8 M 125 81 86 8.3 8.5 8.4 32.4 32.6 32.5125 97 77 7.7 7.8 7.75 32.5 32.3 32.4 M 125 96 72 8.4 8.4 8.4 31.5 31.8 31.65 M 125 96 75 8 8.3 8.15 32 32.3 32.15125 82 80 7.1 7.5 7.3 32.6 32.9 32.75 M 125 97 88 8.1 8.1 8.1 34.4 34.3 34.35 M 125 97 88 7.9 8 7.95 34.2 34.5 34.35125 100 72 7.1 7.3 7.2 32.7 32.9 32.8 M 125 98 80 8.4 8.7 8.55 34.6 34.7 34.65 M 125 100 85 7.1 7.2 7.15 34.3 34.5 34.4125 94 74 7.1 7.2 7.15 29 29.2 29.1 M 125 99 80 7.2 7.5 7.35 32.6 32.7 32.65 M 125 82 94 7.4 7.6 7.5 32.2 32.3 32.25125 74 76 7.8 7.8 7.8 32.8 33 32.9 M 125 100 81 7.5 7.5 7.5 30.8 30.9 30.85 M 125 98 82 7.6 7.6 7.6 33.9 33.8 33.85250 16 71 6.8 6.8 6.8 30.3 30.6 30.45 M 250 73 64 7.5 7.7 7.6 33.5 33.6 33.55 M 125 99 81 7.1 7.2 7.15 32.7 32.4 32.55250 89 77 6.6 6.8 6.7 32.8 32.8 32.8 M 250 75 91 8.4 8.7 8.55 33.2 33.5 33.35 M 125 95 84 8.2 8.3 8.25 33.1 33.3 33.2250 73 57 6.9 7 6.95 29.3 29.5 29.4 M 250 76 78 6.8 7.2 7 33 33.1 33.05 M 125 94 85 8.2 8.5 8.35 27.6 27.4 27.5250 74 63 7.5 7.6 7.55 29.5 29.9 29.7 M 250 77 96 7.7 8 7.85 30.1 30.4 30.25 M 250 75 92 7.9 8 7.95 33.2 33.2 33.2250 88 87 8.3 8.5 8.4 33.3 33.4 33.35 M 250 81 88 8.2 8.5 8.35 31.5 32 31.75 M 250 91 81 7.8 7.9 7.85 30.9 30.9 30.9250 87 76 7.3 7.4 7.35 33.2 32.9 33.05 M 250 90 73 8 8.2 8.1 32.6 32.7 32.65 M 250 81 90 7.7 7.7 7.7 34.4 34.8 34.6250 77 86 8 8.3 8.15 31.7 31.6 31.65 250 16 78 8.5 8.6 8.55 31.2 31 31.1 M 250 77 99 7.5 7.5 7.5 32.8 32.9 32.85250 76 79 6.7 7 6.85 32.1 32.2 32.15 30 94 73 8 8.4 8.2 32.4 32.5 32.45 M 250 87 89 7 7.1 7.05 33 33.2 33.1250 90 62 6.6 6.9 6.75 30.6 30.7 30.65 0 192 84 7.4 7.5 7.45 33.7 33.8 33.75 M 250 73 67 7.3 7.4 7.35 31.4 31.5 31.45250 75 79 7 7.4 7.2 30.1 30.5 30.3 0 184 85 7 7.2 7.1 30.9 31 30.95 M 250 76 80 7.7 7.8 7.75 33.2 33 33.1250 81 79 6.4 6.5 6.45 32 32.3 32.15250 78 72 7.2 7.5 7.35 32.3 32.4 32.35F2 bird weights12/22 12/28 1/5 1/12 1/19 1/26 2/2 2/9 2/16 2/22 3/2 3/9 3/16 3/22 12/22 12/28 1/5 1/12 1/19 1/26 2/2 2/9 2/16 2/22 3/2 3/9 3/16B-1SEXDOSEIDBWBWBWBWBWBW BW BW BW BW BW BW BW BWSEXDOSEIDBWBWBWBWBWBW BW BW BW BW BW BW BWF 0 183 92 103 113 134 127 120 133 132 139 142 137 136 138 140 M 0 1939186 97 101 103 105 106 107 109 108 108 109 108F 0 199 97 91 121 131 125 113 127 139 139 141 130 131 143 141 M 0 195 89 96 100 102 101 105 105 104 103 106 107 107 107F 0 200 97 107 123 130 129 118 127 132 128 133 120 122 129 135 M 0 196 94 99 103 104 104 105 101 105 109 104 105 104 106F 0 194 96 110 127 136 131 135 135 129 140 110 134 134 139 137 M 0 198 94 89 93 98 91 99 102 99 99 103 103 104 104F 0 179 115 122 109 131 130 136 134 142 140 147 145 144 144 136 M 0 184 100 104 106 108 111 111 111 113 112 113 115 116 116F 0 191 92 92 100 121 126 123 127 126 126 135 133 137 131 132 M 0 192 95 100 99 105 103 103 105 108 109 110 110 111 110F 0 188 96 113 116 124 133 129 131 133 133 137 134 133 135 137 M 0 177 91 102 110 115 116 119 120 120 121 123 120 119 123F 0 189 123 138 139 136 128 137 147 145 147 147 144 143 140 144 M 0 180 92 104 113 114 109 115 116 117 118 117 115 116 117F 0 313 102 109 115 121 130 148 145 133 144 139 138 137 133 133 M 0 304 95 99 102 105 108 106 109 111 111 114 114 114 115F 0 174 106 115 122 137 133 136 142 140 141 146 142 143 140 137 M 0 308 95 98 103 107 107 107 112 112 111 112 111 110 112F 0 314 96 106 127 138 140 145 147 142 143 141 132 132 147 148 M 0 322 95 99 104 95 98 102 106 105 106 106 107 108 110F 0 309 95 107 121 125 127 130 130 126 126 129 128 129 131 135 M 0 321 91 102 112 102 108 110 113 114 113 114 115 116 115F 0 186 101 94 133 145 144 144 146 147 147 153 146 144 152 156 M 0 301 100 102 107 109 109 109 114 114 114 114 115 114 114F 0 197 92 105 133 134 141 134 135 137 139 142 135 134 137 133 M 0 182 91 97 103 103 104 106 110 106 105 106 106 107 107F 0 178 104 101 140 141 143 141 134 142 147 144 135 135 147 138 M 0 185 104 102 107 109 108 108 108 108 109 112 112 113 112F 0 181 101 105 127 144 141 130 143 144 144 150 145 146 139 149 M 0 173 93 91 97 97 98 98 100 99 98 99 101 101 100F 0 175 107 111 134 144 137 146 146 145 149 143 144 145 139 140 M 0 187 91 88 94 97 96 95 104 99 99 98 100 101 101F 0 190 81 94 112 128 141 118 142 138 146 145 147 146 152 151 M 0 303 93 98 102 100 102 106 105 106 104 106 108 107 106F 0 323 106 129 144 142 138 142 138 144 149 152 152 152 153 153 M 0 310 90 96 100 103 104 106 107 109 110 109 110 109 110F 0 305 119 123 129 147 125 121 147 141 139 155 144 143 153 160 M 0 329 102 109 115 113 114 107 115 113 114 114 113 115 116F 30 81 126 131 129 135 135 132 130 140 140 141 141 140 138 133 M 30 94 96 97 103 108 105 107 110 111 108 108 109 108 109F 30 96 102 115 127 114 132 131 128 132 128 135 1282 129 134 134 M 30 93 103 104 106 108 106 109 108 106 107 106 107 107 109F 30 89 101 105 122 138 142 140 138 140 141 146 134 133 145 140 M 30 90 110 119 118 118 118 121 123 122 121 124 125 125 124F 30 85 110 114 132 132 138 139 138 127 139 143 138 139 139 143 M 30 80 110 114 118 122 123 129 130 133 134 129 130 131 130F 30 403 91 106 112 119 131 136 134 140 139 141 144 143 138 143 M 30 78 97 100 105 108 110 112 113 115 114 114 115 141 115F 30 405 97 119 121 131 128 127 134 137 143 144 140 140 135 142 M 30 76 96 95 101 103 105 109 109 107 106 106 104 104 108F 30 408 118 129 133 137 138 138 137 139 141 143 135 137 141 138 M 30 407 100 102 103 106 108 110 110 112 112 113 113 112 111F 30 427 108 133 134 140 143 142 143 146 141 144 140 141 141 144 M 30 402 100 102 105 107 106 106 112 110 115 115 114 113 114F 30 409 101 106 111 119 130 152 144 145 140 149 145 145 148 151 M 30 417 101 105 106 109 97 100 106 96 107 110 114 114 112F 30 432 106 119 127 139 146 133 149 151 150 154 152 151 152 150 M 30 406 93 98 97 102 103 105 107 108 107 112 113 114 110F 30 84 105 122 122 132 123 135 132 132 136 137 127 128 131 136 M 30 92 100 110 115 118 123 120 123 126 126 126 126 126 125F 30 86 101 107 111 122 128 127 128 125 132 132 131 132 129 133 M 30 88 103 104 105 106 108 105 109 107 107 109 112 113 111F 30 82 107 116 124 134 131 132 140 139 133 141 140 141 143 140 M 30 95 93 95 100 102 118 105 105 105 106 106 108 109 108B-1F 30 87 117 127 149 145 151 140 150 156 159 163 167 166 162 170 M 30 91 105 107 112 111 107 108 111 114 115 115 116 115 112F 30 401 104 111 122 126 128 148 148 150 149 154 150 149 140 139 M 30 77 102 105 115 116 122 119 121 123 122 122 124 124 124F 30 74 106 117 125 123 126 138 140 141 143 145 144 145 142 146 M 30 83 104 104 106 106 105 105 106 106 108 109 108 110 109F 30 73 107 112 118 125 135 137 138 138 141 133 143 143 142 143 M 30 404 106 120 120 123 108 126 126 128 128 131 132 131 131F 30 86 88 100 118 130 126 133 131 128 135 135 135 135 125 136 M 30 100 89 97 102 103 103 107 110 107 107 108 107 107 108F 30 75 119 124 132 138 138 136 141 139 139 146 144 143 144 147 M 30 99 98 102 104 107 103 108 109 111 111 112 110 111 113F 30 79 120 106 114 130 128 115 128 125 125 137 118 119 135 133 M 30 97 102 104 105 105 111 103 104 107 106 106 95 96 104F 60 75 114 128 149 149 150 151 147 159 151 150 157 155 159 159 M 60 90 98 100 103 106 104 108 107 108 109 108 106 105 111F 60 91 101 110 128 136 129 143 136 140 140 144 144 143 139 145 M 60 88 100 102 108 109 110 109 112 114 113 115 113 114 115F 60 89 113 120 126 135 140 149 144 150 143 147 144 145 142 147 M 60 205 85 88 90 94 95 97 98 100 100 99 100 101 99F 60 73 102 106 121 134 141 134 140 146 139 137 144 144 139 142 M 60 208 103 104 107 110 110 115 119 118 117 118 117 116 114F 60 85 101 108 126 144 127 135 148 140 147 148 135 137 143 129 M 60 81 94 99 100 99 101 101 103 103 101 101 105 104 102F 60 100 121 129 137 138 135 145 142 139 148 148 150 146 143 133 M 60 84 94 99 101 105 105 107 108 112 111 115 112 111 110F 60 94 97 120 124 123 129 127 132 124 123 135 116 117 131 118 M 60 80 105 107 111 114 113 109 108 111 110 114 113 111 114F 60 76 105 112 125 132 142 139 134 139 138 135 140 140 140 138 M 60 86 104 105 109 113 114 116 117 118 119 119 120 121 120F 60 78 98 104 124 134 109 108 138 139 143 142 134 133 133 142 M 60 96 93 97 104 103 104 106 105 106 105 108 107 106 106F 60 93 93 110 122 124 123 123 118 125 120 128 125 125 128 112 M 60 79 104 108 117 112 106 106 112 111 114 115 120 120 122F 60 82 95 103 116 122 127 126 122 131 127 123 120 121 130 128 M 60 203 98 103 109 109 113 108 118 119 118 120 122 121 125F 60 74 96 104 119 129 126 125 129 129 130 133 122 122 126 134 M 60 95 102 107 113 115 114 117 115 116 115 117 117 118 113F 60 92 110 117 127 132 125 135 137 135 143 139 140 140 142 131 M 60 99 112 112 112 112 115 115 121 120 119 123 123 122 126F 60 87 94 97 124 133 135 113 136 140 140 131 124 125 141 145 M 60 98 94 103 109 111 112 120 112 114 115 117 117 117 116F 60 202 89 93 115 136 122 138 144 139 140 145 136 135 145 146 M 60 201 85 92 99 101 102 109 101 100 101 102 104 104 102F 60 77 95 83 96 118 135 135 137 137 137 140 128 129 136 136 M 60 204 86 95 100 103 105 106 104 105 106 104 104 104 109F 60 222 97 96 112 127 137 144 143 143 142 152 150 151 150 151 M 60 209 104 104 101 107 110 113 111 116 113 114 111 112 109F 60 97 123 118 135 115 133 122 137 136 136 142 136 137 123 133 M 60 210 95 103 111 115 116 115 119 118 118 120 118 117 120F 60 215 98 104 133 131 131 135 136 136 139 134 140 140 137 140 M 60 219 95 106 104 107 108 107 108 110 109 110 109 109 109F 60 216 99 102 130 137 136 122 137 139 143 146 144 144 144 135 M 60 218 101 103 111 114 117 116 117 120 115 118 120 122 122F 125 88 93 109 119 133 137 140 146 150 149 145 141 139 142 152 M 125 75 101 103 107 106 106 112 112 113 113 112 113 115 111F 125 87 100 102 121 123 132 125 136 136 134 137 139 141 128 130 M 125 77 100 103 104 105 106 107 109 110 102 109 108 109 108F 125 89 99 115 128 133 124 133 135 137 123 123 131 130 136 138 M 125 95 107 107 110 116 114 115 121 122 122 124 121 122 123F 125 84 92 103 119 108 134 134 129 111 135 139 136 135 138 140 M 125 233 88 101 109 110 110 109 111 112 110 114 113 112 112F 125 207 105 119 136 122 145 148 149 152 153 153 150 150 149 156 M 125 100 101 102 102 107 110 110 109 111 113 112 111 110 111F 125 202 105 118 120 135 141 145 146 149 152 150 150 151 150 151 M 125 81 104 110 114 118 119 124 124 126 124 124 119 119 122F 125 259 104 113 123 134 133 137 135 131 132 142 126 127 140 140 M 125 98 105 106 107 111 113 113 114 115 108 112 113 112 116B-1F 125 209 104 123 125 131 141 143 144 145 144 146 139 138 147 147 M 125 97 99 109 115 116 117 120 122 124 119 119 120 122 120F 125 258 103 106 116 124 140 142 133 144 144 132 129 129 145 149 M 125 208 96 99 102 106 118 107 110 110 112 110 111 111 112F 125 260 98 115 147 152 155 154 147 157 153 148 155 157 155 155 M 125 76 107 113 114 111 114 115 117 115 115 117 117 116 115F 125 74 103 106 109 117 134 137 136 134 126 141 135 134 137 143 M 125 235 103 112 114 114 114 115 115 117 115 118 115 116 116F 125 80 100 94 119 130 134 125 121 126 120 137 128 129 122 141 M 125 93 106 105 107 110 111 109 114 114 115 113 111 111 110F 125 83 115 118 132 135 145 159 154 149 146 155 151 152 157 157 M 125 82 112 110 109 108 114 113 116 116 114 112 110 111 110F 125 73 111 120 135 127 123 140 139 131 136 147 129 128 145 147 M 125 94 108 109 114 116 118 116 119 118 119 119 116 115 118F 125 206 105 116 134 128 130 132 139 141 139 143 140 141 135 142 M 125 90 100 102 107 106 109 111 112 113 110 111 112 113 111F 125 205 118 130 136 142 159 160 165 162 162 162 164 162 161 161 M 125 99 102 109 112 115 116 119 120 121 121 122 123 125 123F 125 234 106 113 121 136 134 129 137 142 141 145 143 141 141 136 M 125 78 101 109 113 117 116 115 116 115 114 112 113 114 114F 125 201 111 117 132 126 124 126 133 135 136 116 131 132 135 134 M 125 85 103 108 111 112 112 115 115 117 115 117 119 120 119F 125 239 102 111 115 122 127 140 133 141 144 147 145 146 140 146 M 125 204 109 113 110 112 113 112 113 113 115 115 113 113 113F 125 237 95 104 117 134 130 137 136 128 138 139 130 130 140 135 M 125 79 104 108 112 117 117 114 113 109 110 110 108 107 109F 250 89 118 136 140 140 145 138 148 142 150 148 150 147 146 149 M 250 84 97 104 109 113 115 114 116 116 110 113 116 117 118F 250 82 119 112 121 124 129 130 135 139 136 141 138 137 140 140 M 250 83 106 101 103 103 108 109 113 115 111 114 112 111 111F 250 79 107 113 139 130 126 137 134 145 141 148 144 143 148 139 M 250 87 104 113 117 118 121 121 122 123 117 121 122 122 125F 250 92 102 104 116 113 120 119 132 139 140 132 109 109 114 120 M 250 91 101 106 106 105 108 110 114 114 113 117 115 115 112F 250 94 102 107 116 125 138 135 141 140 134 140 141 142 142 136 M 250 77 121 125 101 130 130 133 135 136 138 137 132 133 139F 250 228 101 107 129 132 134 132 136 122 136 137 138 137 139 128 M 250 76 94 94 94 97 98 99 100 101 96 100 95 97 99F 250 188 95 98 102 105 105 111 121 117 128 134 138 140 137 139 M 250 98 100 98 98 93 98 102 103 102 96 96 103 102 100F 250 184 105 126 128 123 132 129 125 128 133 139 134 135 138 133 M 250 100 99 103 104 111 110 120 119 122 121 121 121 120 121F 250 97 112 114 134 139 134 138 113 140 142 141 146 145 144 145 M 250 95 113 98 104 106 110 109 110 114 113 113 115 115 117F 250 74 99 116 141 132 132 146 141 152 140 154 148 149 148 154 M 250 99 100 103 102 103 103 105 108 107 106 107 111 112 108F 250 78 118 123 136 140 139 145 140 147 145 139 140 142 149 148 M 250 231 94 98 105 104 105 106 108 106 106 108 110 110 111F 250 88 122 133 141 149 149 142 147 141 150 147 148 147 139 151 M 250 189 95 99 103 108 109 110 112 114 114 117 117 116 115F 250 96 118 125 132 144 133 147 147 142 151 151 152 151 152 151 M 250 230 95 103 106 106 108 107 107 110 110 111 111 110 111F 250 93 117 124 135 134 140 140 145 146 142 153 142 144 148 146 M 250 90 99 101 107 109 108 111 112 115 115 115 114 114 115F 250 225 110 116 121 125 137 143 148 145 143 150 145 146 140 144 M 250 75 103 107 103 105 107 107 111 112 111 112 112 113 115F 250 221 109 118 128 140 147 143 149 150 145 156 153 153 150 157 M 250 81 106 109 111 112 112 111 114 112 109 112 112 112 110F 250 223 102 107 109 113 118 120 124 128 131 136 136 135 138 138 M 250 85 106 112 110 117 119 120 123 125 122 124 125 124 127F 250 183 105 107 107 104 104 105 107 108 110 111 110 111 111 112 M 250 80 92 93 91 93 93 94 95 96 96 96 97 99 97F 250 182 100 114 123 128 129 129 129 130 132 132 132 133 133 133 M 250 181 99 104 107 111 110 114 112 116 115 116 120 121 117F 250 187 100 114 117 121 140 138 155 152 156 151 155 155 147 148 M 250 190 96 100 101 100 100 101 104 104 104 105 106 107 103B-1F2 feed consumption12/21 12/28 1/4 1/11 1/18 1/25 1/29 2/1 2/5 2/8 2/12 2/15 2/19DOSE SEX ID Initial Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final0 M 193 389 324 326 322 433 306 425 320 426 314 428 313 426 305 431 362 429 312 4580 M 195 386 319 431 324 432 317 426 325 427 329 419 312 426 326 444 378 427 317 4400 M 196 386 308 429 319 437 330 433 322 422 327 421 323 430 325 442 365 412 318 4410 M 198 388 325 435 325 431 305 421 320 429 302 412 322 445 355 439 306 432 359 428 313 4570 M 194 428 324 434 311 411 334 414 313 433 317 402 283 430 305 442 363 437 266 4550 M 192 438 322 432 331 433 310 419 334 418 331 432 322 421 319 423 354 429 327 4600 M 177 425 321 432 310 430 303 418 308 412 313 413 286 428 324 441 355 424 310 4470 M 180 428 320 435 291 435 293 424 297 428 306 418 279 431 303 439 338 418 347 4630 M 304 427 309 435 318 427 310 421 317 416 338 421 313 426 282 436 363 424 290 4650 M 308 421 315 427 324 434 311 419 326 426 319 424 319 423 322 437 366 416 307 4500 M 322 410 316 431 322 430 339 406 328 404 310 425 321 425 297 425 369 424 314 4590 M 321 404 311 426 285 432 318 421 290 411 290 428 298 438 312 430 350 445 325 4500 M 301 436 312 424 308 440 317 438 307 425 314 407 295 438 310 433 352 443 315 4520 M 182 422 282 437 305 445 325 439 308 420 318 407 301 439 376 426 298 424 348 452 311 4580 M 185 413 317 434 306 441 305 439 307 404 313 433 311 439 320 438 364 409 287 4490 M 173 431 317 435 282 436 272 436 284 425 303 423 285 435 284 434 352 419 286 4640 M 187 411 330 427 308 434 291 434 309 433 321 435 311 435 301 441 361 458 350 4650 M 303 412 317 424 316 443 331 441 311 418 330 421 320 427 323 440 368 433 311 4490 M 310 434 325 434 309 440 315 426 312 436 326 433 310 430 318 424 352 435 329 4610 M 329 400 320 431 303 440 312 436 306 430 331 426 331 432 307 441 340 420 265 4540 F 183 406 316 431 309 433 301 427 346 421 298 418 275 439 326 455 369 415 315 452 305 405 332 4420 F 199 424 318 433 300 428 276 406 335 419 300 434 278 436 341 435 353 428 353 431 281 439 335 4270 F 200 409 335 425 275 432 251 425 276 427 268 404 307 431 331 438 316 449 356 421 326 435 307 428 346 4410 F 194 405 294 426 336 424 268 433 332 442 231 412 242 439 343 449 333 422 336 421 307 448 243 419 336 4380 F 179 408 308 434 295 426 309 429 299 326 264 421 310 437 337 429 310 445 328 433 323 424 311 425 332 4340 F 191 409 332 434 325 426 315 432 330 426 265 411 315 429 352 447 335 440 343 432 343 419 314 440 328 4330 F 188 395 319 436 381 425 318 428 387 427 305 423 272 413 326 431 279 450 337 418 301 440 271 432 305 4420 F 189 493 330 423 344 432 325 433 342 430 344 430 298 421 335 437 355 430 332 430 294 422 333 4270 F 313 427 301 425 339 432 286 432 335 431 273 405 302 418 329 429 313 443 342 428 320 419 303 412 312 4500 F 174 413 295 430 304 431 301 430 350 442 337 450 296 429 311 432 285 437 319 417 285 443 296 419 296 4420 F 314 433 332 428 286 425 296 433 291 423 310 419 298 424 334 439 316 451 318 419 323 417 297 406 297 441B-10 F 309 348 316 436 316 421 295 424 320 434 313 424 278 436 316 441 341 427 320 426 293 410 310 4510 F 186 410 363 435 257 423 241 429 351 446 293 408 311 430 252 437 356 420 298 442 303 417 334 4260 F 197 432 331 442 280 426 271 433 338 430 332 424 295 421 330 438 312 437 339 425 306 438 290 424 304 4430 F 178 421 312 431 404 419 281 426 356 441 339 415 285 438 337 438 322 429 342 431 305 442 284 430 329 4320 F 181 440 315 433 331 399 269 437 327 445 292 429 285 433 327 445 330 435 327 430 331 431 281 438 331 4350 F 175 428 319 434 287 413 268 432 281 428 263 402 308 433 343 434 313 437 338 419 336 439 302 417 335 4370 F 190 408 346 430 307 421 293 437 331 432 302 423 280 434 337 431 296 436 357 432 321 410 281 410 342 4390 F 323 405 320 432 344 422 282 418 357 428 302 413 323 432 369 440 337 445 350 429 305 422 323 429 336 4290 F 305 398 332 427 304 423 268 414 399 434 294 429 277 437 317 436 365 419 301 415 305 426 331 44230 M 94 415 323 422 301 427 313 433 295 431 321 422 316 424 315 432 373 431 332 45030 M 93 423 320 433 316 432 325 438 312 436 304 427 301 415 327 443 354 442 321 45230 M 90 401 325 422 305 423 318 438 300 435 300 422 307 436 326 435 352 436 313 45630 M 80 411 315 431 315 421 295 420 302 422 302 418 289 423 291 436 346 436 272 45430 M 78 404 314 432 316 435 331 423 303 423 320 411 303 434 333 435 359 437 312 45030 M 76 403 305 428 316 420 299 429 303 413 301 421 313 427 313 437 367 429 308 45430 M 407 402 317 432 325 429 297 427 319 405 278 407 271 429 331 431 344 452 329 45630 M 402 393 308 434 314 429 301 420 310 406 321 415 292 435 310 432 353 437 275 45030 M 417 382 319 425 327 443 325 420 322 417 343 404 316 425 337 437 366 429 328 45230 M 406 395 315 437 314 428 305 430 309 422 304 429 309 432 308 438 359 429 295 45630 M 92 446 311 421 302 432 276 437 295 423 299 420 299 426 305 431 357 415 294 44830 M 88 422 289 430 326 433 346 433 319 425 332 435 305 437 336 445 361 416 329 45530 M 95 433 316 428 306 442 311 433 302 423 320 419 315 439 311 434 344 411 274 45530 M 91 429 317 428 322 429 315 433 317 417 335 422 320 426 330 435 372 440 330 44830 M 77 430 330 430 292 439 301 424 290 429 300 423 317 433 314 437 356 438 307 45730 M 83 425 316 432 321 430 310 420 317 433 344 432 299 423 332 438 357 434 313 45530 M 404 421 321 421 306 437 296 427 299 450 307 418 302 425 292 430 345 413 271 44930 M 100 431 325 432 315 434 325 436 298 413 313 408 305 433 325 431 366 431 314 45430 M 99 441 324 431 312 437 317 425 310 410 326 421 324 435 328 435 374 433 320 45330 M 97 412 332 433 324 427 330 442 318 428 337 418 317 428 314 439 361 437 333 45230 F 81 438 305 432 294 426 242 420 327 413 307 421 297 420 309 432 312 436 320 426 305 436 296 437 304 44630 F 96 424 335 434 345 428 310 436 338 420 325 434 304 426 326 445 367 408 312 437 292 434 338 44730 F 89 424 309 432 338 406 306 429 330 430 283 421 293 420 320 433 327 441 339 428 323 428 294 438 319 42930 F 85 415 315 423 314 418 297 423 362 442 310 423 299 432 323 442 331 417 320 433 335 435 298 428 324 45130 F 403 424 316 433 266 431 312 423 266 424 285 409 305 421 313 436 326 433 299 444 303 431 308 445B-130 F 405 418 327 432 313 427 297 416 310 437 284 414 296 418 323 430 323 427 338 421 329 444 298 434 337 44730 F 408 420 317 428 329 426 308 434 322 426 313 424 301 423 324 429 314 420 317 431 305 431 305 430 304 44230 F 427 415 330 428 303 426 304 420 301 448 290 429 294 428 334 424 365 427 339 441 308 411 351 43430 F 409 411 328 430 301 421 301 425 298 435 281 410 304 420 343 439 347 433 357 422 319 449 312 426 322 44730 F 432 423 317 428 293 420 318 432 291 437 322 431 318 430 351 445 345 443 352 435 342 447 315 443 345 45030 F 84 428 318 436 276 428 293 422 275 436 300 423 301 431 345 435 350 325 343 429 314 438 344 43530 F 86 410 334 425 315 424 292 427 310 429 290 422 292 416 328 438 356 333 349 413 310 435 320 44630 F 82 419 299 424 312 417 321 423 311 425 259 420 314 423 356 443 342 444 366 329 328 421 313 437 350 45130 F 87 491 308 425 251 403 272 415 259 428 284 426 287 430 330 448 320 434 319 429 309 441 288 427 314 44330 F 401 411 331 434 297 419 283 416 287 437 293 423 309 414 328 443 327 421 337 332 291 450 310 430 336 44630 F 74 420 302 432 308 431 288 416 301 438 273 422 321 425 346 449 333 427 327 422 304 443 322 435 310 45630 F 73 411 296 422 314 421 315 417 313 427 319 416 310 421 344 432 339 431 319 424 305 433 312 429 322 45030 F 98 413 331 431 313 414 299 427 310 422 270 424 310 424 353 446 346 429 340 429 293 444 310 412 320 43030 F 75 407 333 429 330 431 295 419 339 418 321 430 313 428 336 438 316 442 322 425 319 428 312 427 337 44830 F 79 413 317 435 363 419 283 418 361 433 305 416 319 423 344 442 345 433 365 409 338 429 320 442 305 44560 M 90 435 340 425 274 424 265 410 276 422 303 422 307 431 325 431 339 428 290 44460 M 88 423 325 434 331 425 305 427 326 434 308 415 305 430 309 427 365 416 292 44160 M 205 419 332 423 309 434 300 426 301 440 319 426 302 425 318 436 347 446 298 44960 M 208 417 327 432 319 422 301 426 312 432 300 412 303 417 309 427 341 433 322 44060 M 81 408 341 435 323 432 326 468 318 427 304 421 287 424 282 435 356 415 298 45060 M 84 422 340 424 304 435 300 433 297 422 320 430 291 434 329 441 347 434 310 44760 M 80 424 332 423 308 431 305 421 308 405 317 410 287 415 306 432 363 415 291 44560 M 86 413 340 438 313 429 298 423 310 423 314 426 285 431 281 428 327 428 290 44060 M 96 421 319 435 310 448 310 429 305 432 320 428 312 424 302 438 350 415 300 44860 M 79 423 322 439 302 437 316 418 198 419 311 427 301 428 319 431 356 433 307 44160 M 203 414 340 426 301 438 307 423 302 432 301 420 268 435 308 431 342 414 274 44760 M 95 408 335 429 307 454 310 426 301 421 336 426 320 425 326 434 366 439 337 44260 M 99 443 325 431 319 430 316 423 311 427 302 419 290 420 308 431 358 414 284 45260 M 98 413 320 434 320 435 324 419 315 426 341 423 308 439 319 423 353 432 320 44260 M 201 407 321 425 299 434 324 417 286 425 322 418 317 423 318 436 367 428 299 45160 M 204 412 336 432 310 423 306 423 299 423 323 423 320 445 309 442 368 420 311 44660 M 209 417 342 425 315 440 312 426 309 440 301 418 298 438 282 429 355 417 285 44860 M 210 423 331 433 296 419 286 419 297 442 304 425 298 424 304 436 352 446 338 44160 M 219 430 324 430 317 427 317 430 315 429 333 426 330 440 328 432 368 412 273 453B-160 M 218 422 318 425 287 433 279 424 347 430 269 430 275 435 257 436 281 425 322 418 306 44160 F 75 420 303 426 286 417 275 424 341 432 299 413 267 432 313 425 328 435 322 412 263 443 268 434 309 44860 F 91 415 337 428 286 420 308 420 290 434 283 410 296 418 341 434 318 434 344 406 308 446 295 431 320 44360 F 89 422 341 432 301 428 306 416 287 432 311 425 306 437 305 436 344 427 334 415 272 437 327 44260 F 73 410 360 426 304 412 255 413 353 430 328 430 267 424 324 438 336 429 324 423 306 442 365 441 296 44360 F 85 401 349 433 284 426 293 425 279 440 307 419 277 430 336 429 360 425 339 414 255 436 344 44060 F 100 387 299 424 300 424 299 422 294 436 267 429 251 431 327 437 317 430 334 423 313 423 250 408 317 45260 F 94 399 340 434 358 422 327 430 349 439 258 427 305 424 342 432 321 420 322 404 308 420 304 437 314 45260 F 76 388 341 432 340 420 282 410 334 435 287 417 290 429 279 427 267 438 328 411 309 424 292 443 308 43560 F 78 381 336 430 352 419 248 403 349 429 352 432 292 450 340 437 337 408 305 437 310 433 330 44060 F 93 438 338 427 366 434 302 427 361 434 287 434 328 428 352 444 345 424 354 431 335 428 326 432 345 44760 F 82 426 341 432 304 422 308 421 361 435 267 429 297 443 364 445 348 440 339 413 346 418 299 435 346 45160 F 74 428 340 433 305 437 299 425 299 428 288 431 313 436 357 440 325 421 353 405 308 445 310 428 332 43160 F 92 417 302 431 359 422 281 413 349 425 285 434 277 442 349 443 321 432 324 422 328 407 275 422 310 43960 F 87 401 339 436 305 417 292 424 300 423 314 431 295 440 332 429 326 429 325 449 283 428 319 44660 F 202 431 342 434 295 404 302 423 352 424 279 419 298 431 348 437 331 426 336 425 316 448 296 423 328 45960 F 77 407 359 436 339 418 281 410 364 435 349 419 282 443 344 421 358 423 329 430 295 416 397 44260 F 222 410 360 437 317 415 299 421 311 425 338 430 339 436 309 433 323 405 319 418 292 422 346 44660 F 97 399 298 433 359 426 313 422 342 438 267 412 299 425 330 430 317 423 341 410 313 403 296 425 319 44460 F 215 394 340 427 321 419 281 427 440 258 429 311 427 344 447 335 435 343 429 313 450 310 437 312 44160 F 216 400 340 432 367 415 300 413 317 421 283 418 311 429 338 447 324 440 323 410 308 444 302 471 306 446125 M 75 415 322 436 304 429 312 423 300 419 306 424 319 428 324 442 371 421 304 456125 M 77 394 309 425 308 438 296 428 302 421 291 418 295 423 286 427 329 418 277 449125 M 95 393 320 438 315 424 291 425 313 425 318 418 309 428 340 412 340 426 313 451125 M 233 393 301 424 306 428 308 422 299 427 328 427 313 427 333 429 358 428 314 447125 M 100 392 321 436 319 437 303 424 313 412 298 416 306 419 302 435 356 436 321 456125 M 81 399 332 435 308 437 315 431 301 414 289 415 282 445 334 430 351 438 322 452125 M 98 405 316 439 267 438 278 412 270 425 295 411 282 439 319 427 342 412 311 436125 M 97 402 317 435 307 436 297 432 302 428 303 418 291 436 310 437 361 423 317 438125 M 208 386 312 438 329 429 300 418 321 426 324 425 319 427 309 435 371 431 323 445125 M 76 395 297 434 286 451 310 425 283 421 302 417 321 448 355 444 296 438 327 444 293 444125 M 235 422 285 430 273 444 299 426 243 430 299 418 294 432 296 443 358 430 242 450125 M 93 431 320 428 319 428 304 421 315 422 279 421 299 426 310 426 343 411 305 446125 M 82 427 337 432 326 430 335 430 325 437 307 425 298 427 309 411 358 418 276 446B-1125 M 94 429 317 436 299 437 322 427 297 435 317 427 311 426 321 430 360 437 320 440125 M 90 424 321 425 310 441 310 415 305 430 315 428 308 440 332 434 356 412 315 449125 M 99 419 332 432 303 426 306 423 304 440 295 421 300 443 305 431 335 414 319 442125 M 78 432 327 435 317 437 321 430 308 438 311 412 315 430 331 428 369 416 313 446125 M 85 444 318 437 304 424 301 430 298 436 289 418 290 428 338 438 336 429 317 443125 M 204 415 325 430 338 442 320 430 331 437 307 414 300 419 289 334 349 430 320 450125 M 79 431 336 434 322 430 273 413 315 431 331 424 337 438 320 437 361 425 295 445125 F 88 425 312 435 306 436 296 413 304 433 316 426 282 428 323 424 342 410 303 446 295 420 357 439125 F 87 423 325 426 296 425 311 421 366 437 303 431 273 421 318 426 343 417 320 432 293 430 319 436125 F 89 417 327 437 312 420 326 425 299 446 299 423 320 433 352 437 346 428 356 424 332 403 321 415 317 451125 F 84 422 327 424 307 413 281 414 302 445 307 436 289 428 331 436 408 430 341 440 355 444 330 438125 F 207 419 338 435 277 422 284 414 365 441 312 426 285 430 319 442 283 441 333 438 307 414 283 429 353 440125 F 202 421 312 436 330 417 262 420 351 436 307 426 313 434 362 335 335 440 341 417 304 447 310 421 341 440125 F 259 412 316 439 310 407 306 413 301 434 288 415 314 419 352 442 355 442 361 429 330 454 312 431 304 452125 F 209 414 317 432 355 405 266 420 349 443 281 422 305 418 320 438 339 430 350 426 329 414 300 430 334 449125 F 258 423 322 435 295 413 309 415 287 440 291 422 314 425 347 432 364 440 347 445 298 440 332 455125 F 260 429 315 438 397 421 279 403 350 432 331 427 303 431 328 435 319 448 343 406 309 429 301 427 322 433125 F 74 408 336 434 344 430 284 430 338 430 269 426 292 436 340 444 362 419 345 400 289 431 330 439125 F 80 415 338 427 299 411 280 410 348 432 264 427 279 432 319 437 341 412 330 423 291 421 321 436125 F 83 402 316 432 286 412 289 416 288 434 253 430 271 430 329 447 352 422 320 405 295 429 355 451125 F 73 409 310 430 298 429 312 424 295 432 271 418 285 433 342 445 325 423 363 432 320 405 280 444 317 452125 F 206 415 332 434 300 431 281 414 357 432 336 431 295 434 338 437 340 440 330 421 296 415 320 440125 F 205 414 324 434 297 427 313 426 287 432 299 433 316 424 341 440 337 420 332 425 310 446 314 421 332 442125 F 234 412 320 433 300 418 316 428 301 426 289 421 310 443 345 420 407 419 347 415 311 437 350 431125 F 201 409 318 428 320 422 310 432 320 422 293 420 317 423 355 442 349 433 345 428 331 450 313 425 342 450125 F 239 414 334 433 368 424 310 419 361 423 308 418 287 429 330 429 357 439 325 407 315 422 301 433125 F 327 434 309 430 335 426 288 405 359 426 273 433 320 418 342 449 336 438 343 405 308 447 310 416 331 438250 M 84 390 321 435 319 430 312 414 309 432 297 421 307 425 315 428 342 432 331 448250 M 83 385 310 438 314 436 316 426 350 434 290 422 302 423 288 436 357 426 304 441250 M 87 386 322 431 289 424 286 425 287 425 290 428 290 435 301 441 344 433 285 452250 M 91 386 305 425 304 430 355 430 306 432 289 438 303 445 300 434 358 428 289 451250 M 77 403 318 434 278 428 285 426 282 429 286 421 286 441 284 431 332 420 270 431250 M 76 437 328 435 322 445 317 442 320 440 300 423 308 428 316 434 359 427 321 441250 M 98 411 317 436 340 441 370 442 338 442 314 421 316 437 343 429 388 416 335 448B-1250 M 100 420 315 439 341 446 336 443 338 445 295 420 306 417 296 423 361 418 300 450250 M 95 428 300 438 300 432 290 425 302 432 300 412 301 434 313 430 364 423 299 447250 M 99 426 297 438 320 445 345 436 318 442 303 424 303 422 314 430 349 421 295 440250 M 231 427 285 432 311 433 321 421 313 432 300 422 313 432 314 430 360 412 330 441250 M 189 424 324 423 327 432 306 415 325 423 316 421 304 439 312 437 353 435 306 447250 M 230 439 322 427 325 441 328 408 324 441 320 418 310 434 297 443 366 419 287 442250 M 90 436 333 429 307 439 317 413 306 437 315 417 320 445 342 433 360 435 287 450250 M 75 429 328 428 319 432 335 409 324 429 307 426 304 421 315 432 357 424 271 441250 M 81 391 319 432 305 428 307 410 299 438 293 417 294 417 285 435 347 432 323 443250 M 85 413 328 433 324 427 258 402 346 427 294 418 259 426 319 436 295 428 311 426 334 447250 M 80 407 335 424 335 436 331 414 334 442 310 420 318 424 306 422 364 425 301 442250 M 181 435 324 435 311 442 310 415 309 436 303 413 329 429 287 428 374 426 298 445250 M 190 423 333 436 321 427 323 411 320 427 313 422 316 442 314 423 364 426 293 441250 F 89 411 317 435 310 415 263 413 340 432 289 416 232 431 310 440 272 434 320 416 256 418 229 420 295 443250 F 82 410 317 435 324 420 275 414 353 429 327 422 322 428 336 440 319 428 340 422 319 443 320 435 333 444250 F 79 428 334 436 317 426 288 433 310 431 283 481 310 424 342 437 335 423 342 425 332 412 308 441 339 439250 F 92 430 318 425 290 421 329 435 285 425 328 423 322 440 262 437 369 437 359 446 310 433 359 447250 F 94 421 332 424 290 403 264 424 283 455 318 420 309 426 335 439 324 426 335 421 300 442 309 432 309 449250 F 228 405 335 432 321 422 301 421 316 427 285 416 318 421 347 440 336 445 364 415 337 428 315 433 340 439250 F 188 396 298 428 384 433 315 428 379 421 311 418 321 443 354 442 384 444 360 449 310 429 347 452250 F 184 407 320 430 355 419 296 424 349 433 297 411 311 427 347 437 358 427 349 429 254 427 308 423 341 445250 F 97 405 331 431 355 426 299 431 348 425 284 425 300 423 344 440 340 435 340 421 318 451 300 430 325 449250 F 74 432 330 432 328 427 319 435 321 422 264 423 308 426 323 436 324 422 322 431 308 450 304 425 309 438250 F 78 427 305 429 285 422 301 422 321 423 307 415 261 435 298 426 303 444 321 430 309 444 262 429 310 444250 F 88 402 335 432 313 429 265 419 316 413 297 420 269 428 285 433 276 445 341 420 318 418 265 435 296 442250 F 96 425 340 432 381 433 277 421 349 421 297 418 289 437 321 425 272 423 348 427 287 445 284 440 330 440250 F 93 420 359 428 315 418 289 426 342 425 276 420 283 430 327 430 301 441 383 440 301 453 281 432 336 448250 F 225 433 339 430 308 412 304 415 301 432 274 409 319 426 367 447 336 443 365 417 302 444 300 433 394 449250 F 221 431 341 424 295 428 293 424 291 423 332 420 289 429 329 441 336 420 331 436 398 423 332 440250 F 223 388 342 431 317 426 306 420 309 421 299 423 277 431 358 429 365 433 349 437 282 427 337 446250 F 183 410 343 434 317 416 320 425 310 422 301 424 296 425 352 424 340 422 364 446 283 422 327 453250 F 182 431 341 435 338 404 303 430 339 429 285 409 340 435 319 437 341 424 340 424 263 428 348 448250 F 187 393 339 425 329 411 327 439 322 433 341 418 290 431 337 433 322 420 318 411 264 435 342 437B-1F2 feed consumption2/22 2/26 3/1 3/5 3/8 3/12 3/15 3/19 3/22 3/26DOSE SEX ID Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final0 M 193 326 451 324 438 322 437 325 4440 M 195 317 445 319 438 340 430 338 4460 M 196 336 440 330 440 325 433 330 4550 M 198 306 438 316 435 303 463 307 4590 M 194 316 438 315 440 310 453 312 4380 M 192 325 436 321 439 353 446 349 4330 M 177 316 444 315 435 326 432 331 4370 M 180 302 449 305 440 313 440 317 4420 M 304 317 443 314 448 348 442 342 4320 M 308 341 436 342 450 337 453 335 4440 M 322 313 435 320 437 340 430 336 4480 M 321 304 447 303 440 310 455 314 4540 M 301 328 419 330 445 311 436 309 4500 M 182 314 447 314 436 302 453 305 4320 M 185 321 439 323 434 323 427 318 4510 M 173 308 450 310 440 309 445 307 4430 M 187 350 426 349 445 317 454 314 4420 M 303 321 441 323 442 350 444 351 4290 M 310 347 428 345 444 336 430 338 4320 M 329 315 441 312 435 303 442 305 4380 F 183 351 455 339 430 331 403 343 425 328 440 331 454 312 442 327 452 325 451 333 4340 F 199 344 456 346 430 342 422 342 444 340 444 340 440 310 440 345 450 337 442 341 4280 F 200 364 457 342 429 340 414 340 433 339 451 342 439 320 444 340 446 339 440 327 4400 F 194 373 460 360 429 344 419 352 442 342 453 333 446 322 453 331 448 340 439 341 4250 F 179 332 460 316 430 326 393 318 447 329 448 334 437 321 451 332 443 325 452 329 4300 F 191 340 453 331 435 332 400 330 444 332 459 339 435 323 453 342 438 330 454 308 4290 F 188 322 448 297 448 326 438 303 441 321 447 279 461 311 442 282 434 317 440 289 4580 F 189 351 451 360 423 340 417 356 446 332 446 351 464 307 452 352 452 329 460 337 4330 F 313 335 435 300 444 309 450 305 422 312 440 327 436 314 460 324 445 309 452 339 4310 F 174 315 443 294 441 302 441 294 429 305 438 297 430 332 440 299 446 301 455 310 4470 F 314 341 442 327 408 317 399 329 435 316 452 345 438 329 442 350 439 317 443 317 447B-10 F 309 354 447 340 427 332 427 333 444 339 460 355 470 321 448 354 442 342 441 321 4670 F 186 325 441 296 441 312 438 292 437 308 455 279 460 339 455 283 449 305 449 289 4570 F 197 375 460 354 432 329 424 351 441 321 641 322 467 310 457 323 450 317 451 330 4390 F 178 339 443 319 427 318 409 322 430 322 453 317 427 339 438 320 451 321 438 349 4570 F 181 339 443 310 439 324 441 313 448 319 440 329 431 342 440 327 448 323 444 319 4310 F 175 347 441 329 435 325 414 320 450 320 438 327 439 306 441 330 432 322 445 313 4490 F 190 335 437 299 433 317 438 304 425 322 439 320 432 316 452 318 441 319 446 304 4320 F 323 330 442 301 427 304 416 300 437 309 444 311 453 321 455 312 443 307 438 305 4270 F 305 339 447 323 416 313 413 320 425 310 452 350 428 312 449 348 432 308 445 328 44330 M 94 323 430 314 433 325 448 324 44230 M 93 311 432 321 430 326 464 327 45530 M 90 321 437 345 440 333 431 329 43230 M 80 342 440 350 443 314 463 317 44130 M 78 317 443 331 435 350 412 348 44030 M 76 331 430 311 445 299 455 303 44230 M 407 299 433 323 440 307 435 302 44830 M 402 308 441 320 432 325 432 321 45230 M 417 327 435 330 434 328 444 322 43830 M 406 312 447 317 432 340 430 338 43730 M 92 318 436 313 440 319 458 322 43030 M 88 325 427 325 432 337 464 339 42930 M 95 300 449 310 445 325 454 332 43230 M 91 336 422 300 420 337 448 341 43730 M 77 315 436 328 436 312 426 319 44230 M 83 333 427 319 434 324 432 329 44030 M 404 308 429 340 430 309 448 301 43930 M 100 326 435 325 428 343 446 342 44830 M 99 358 445 315 434 341 440 335 45330 M 97 349 434 320 432 331 415 335 45330 F 81 315 444 320 430 311 451 323 342 310 457 302 454 332 448 329 402 313 441 314 42030 F 96 340 439 365 431 350 452 335 340 348 458 361 451 330 440 299 455 341 439 353 44230 F 89 332 457 336 428 333 407 350 345 330 440 341 435 315 452 337 429 332 452 323 44530 F 85 348 445 334 429 334 433 342 343 333 452 336 445 319 441 332 443 331 461 324 43430 F 403 333 449 303 440 316 433 356 439 317 441 315 453 340 457 321 438 322 459 310 436B-130 F 405 363 443 337 425 338 445 342 352 342 463 344 438 322 442 339 448 339 456 327 45130 F 408 321 451 309 424 309 435 336 344 315 454 313 410 330 455 319 451 319 445 305 43730 F 427 352 449 334 426 342 443 306 3400 338 451 346 447 336 442 338 449 341 454 323 44630 F 409 350 440 322 427 330 430 309 338 334 441 328 427 332 463 329 443 331 443 315 45830 F 432 348 456 337 456 351 456 332 316 350 408 354 458 348 452 342 449 352 450 324 42730 F 84 358 456 359 420 349 321 328 328 351 442 354 443 350 461 355 455 357 453 351 43730 F 86 367 443 339 420 348 319 321 330 352 465 342 446 351 455 340 427 351 463 335 41930 F 82 362 438 337 445 363 417 335 334 347 450 344 448 349 458 341 450 344 452 340 43530 F 87 335 441 304 449 326 426 342 342 362 452 310 420 361 443 316 444 359 451 299 43530 F 401 353 443 308 451 337 436 322 330 321 461 337 448 318 459 332 443 323 434 346 43930 F 74 358 449 334 434 341 416 336 325 342 443 331 423 336 449 336 448 345 442 334 45430 F 73 358 437 329 440 352 404 334 303 349 450 351 423 341 444 355 446 352 456 325 41830 F 98 342 447 317 427 312 446 302 335 317 443 315 461 329 442 317 425 321 443 306 44930 F 75 366 454 337 426 339 447 337 312 340 457 358 451 338 356 352 427 343 442 336 45230 F 79 357 450 346 421 342 424 310 329 342 456 341 431 340 450 338 451 339 450 347 44060 M 90 313 448 325 432 309 439 311 45760 M 88 343 445 323 433 342 456 344 45560 M 205 319 441 340 445 321 454 317 43860 M 208 322 438 353 442 277 447 282 44060 M 81 333 443 334 432 322 427 329 40260 M 84 294 440 295 430 333 429 331 43860 M 80 323 444 320 436 318 458 315 45560 M 86 283 435 318 434 283 446 291 45660 M 96 308 453 326 431 302 438 299 45560 M 79 314 448 314 431 323 431 319 45460 M 203 295 432 307 438 278 434 277 44860 M 95 326 441 283 440 354 463 354 43060 M 99 313 442 335 442 322 461 326 42960 M 98 318 458 353 436 320 424 321 45760 M 201 320 448 342 442 324 456 319 46160 M 204 335 429 343 440 333 441 330 43960 M 209 342 423 342 438 335 429 329 44160 M 210 306 426 302 435 309 460 311 44760 M 219 353 423 348 432 339 404 342 455B-160 M 218 296 427 301 448 297 433 299 45960 F 75 334 447 318 414 305 455 425 428 302 450 305 448 322 462 300 448 300 456 305 45160 F 91 347 435 330 412 322 423 427 434 321 452 329 457 320 443 328 455 317 455 328 43460 F 89 342 452 325 414 323 434 432 440 325 448 316 461 323 442 319 462 320 450 319 45160 F 73 323 444 296 427 302 438 430 445 306 440 298 442 310 443 298 449 309 459 297 44160 F 85 356 453 346 443 356 450 327 445 352 447 346 429 345 444 342 451 355 443 344 43660 F 100 343 439 318 418 321 384 435 442 317 446 322 441 320 445 318 444 322 461 344 42960 F 94 339 451 317 420 311 415 438 442 312 445 317 435 315 443 318 460 309 459 319 44560 F 76 325 435 300 436 331 419 440 446 327 466 312 463 325 436 332 457 322 443 314 43360 F 78 347 437 323 415 319 407 437 436 322 448 338 443 320 435 347 442 321 440 332 43460 F 93 371 439 339 415 339 402 437 439 333 456 353 428 328 439 352 438 339 445 348 43360 F 82 385 448 353 419 355 412 433 444 342 447 343 440 336 438 337 443 338 443 385 43260 F 74 350 447 329 403 322 398 421 441 324 452 337 435 326 440 339 444 342 441 332 40760 F 92 364 440 332 416 322 426 424 448 322 453 344 442 325 443 360 441 324 441 335 42460 F 87 374 443 336 418 322 419 432 450 324 462 369 410 319 446 332 436 321 443 334 43760 F 202 353 442 327 424 321 438 430 451 319 446 335 464 321 455 351 435 323 445 325 45560 F 77 363 452 366 427 342 408 446 450 341 446 354 418 337 457 352 459 324 438 333 41760 F 222 340 435 308 455 318 421 437 440 322 452 312 470 324 456 306 437 351 448 310 41760 F 97 334 425 308 436 353 437 429 432 355 451 330 435 348 459 329 442 342 455 344 41060 F 215 360 452 340 428 337 402 434 437 333 448 334 434 329 448 331 445 337 456 331 45760 F 216 338 449 327 434 317 410 433 435 320 445 325 441 323 443 329 442 317 450 333 423125 M 75 321 437 320 430 327 440 331 447125 M 77 281 423 284 433 313 439 319 442125 M 95 328 408 325 447 317 445 313 448125 M 233 323 448 327 444 326 465 320 441125 M 100 335 425 330 425 323 474 326 452125 M 81 333 419 332 422 319 457 323 449125 M 98 315 444 317 425 290 448 320 451125 M 97 300 447 299 435 337 464 332 439125 M 208 328 430 325 430 303 455 310 443125 M 76 296 433 291 432 308 466 316 441125 M 235 324 416 319 442 321 436 318 444125 M 93 324 422 324 435 315 420 316 447125 M 82 321 420 319 440 305 431 309 442B-1125 M 94 333 432 332 422 308 434 307 450125 M 90 321 410 327 415 292 456 317 442125 M 99 317 414 320 412 320 429 315 443125 M 78 348 423 341 431 343 445 342 436125 M 85 308 414 310 422 301 444 309 439125 M 204 318 438 315 421 312 444 308 440125 M 79 338 420 334 417 331 441 325 435125 F 88 336 430 303 443 336 416 308 440 358 438 339 456 355 434 335 453 332 468 310 449125 F 87 347 431 326 414 324 448 320 441 334 443 348 433 339 440 339 448 329 450 345 442125 F 89 376 435 352 427 347 411 349 442 342 455 335 452 337 439 342 443 341 457 316 416125 F 84 348 440 316 446 341 399 318 432 328 456 337 431 331 445 346 438 332 458 323 414125 F 207 338 440 309 421 312 413 312 430 342 449 309 459 345 449 312 439 339 451 317 462125 F 202 349 439 315 440 324 439 316 436 337 462 329 456 337 459 317 436 338 452 320 460125 F 259 376 438 350 401 340 414 345 447 315 443 356 451 319 456 349 440 317 448 343 440125 F 209 331 441 316 445 342 413 317 422 321 440 316 415 320 451 315 452 322 441 327 429125 F 258 367 452 361 429 362 426 357 421 345 443 360 445 340 464 358 451 342 442 351 428125 F 260 340 449 319 442 334 432 320 433 341 445 363 427 354 462 350 446 341 441 312 456125 F 74 388 445 363 434 359 422 357 428 352 443 369 420 351 442 352 437 351 452 336 439125 F 80 346 438 328 414 328 417 333 439 325 436 345 425 332 454 350 447 323 443 317 436125 F 83 340 437 308 407 309 413 310 442 310 445 349 423 312 445 332 452 312 446 325 436125 F 73 348 433 316 396 314 414 317 438 311 438 336 442 310 455 348 448 310 450 325 436125 F 206 374 441 331 423 335 418 331 445 336 440 335 424 329 456 341 455 334 457 329 430125 F 205 357 433 317 421 323 421 322 425 327 461 326 423 334 447 330 447 332 456 337 430125 F 234 369 437 346 423 343 423 338 423 339 448 345 466 337 444 342 442 341 457 359 430125 F 201 388 436 377 433 357 426 376 446 362 4555 353 441 361 443 349 440 359 458 441125 F 239 348 432 320 430 342 410 329 447 341 456 327 452 359 448 336 443 337 452 342 438125 F 327 353 431 320 415 323 497 317 432 320 450 340 434 357 452 332 451 318 441 318 439250 M 84 302 456 303 439 317 460 321 455250 M 83 312 419 315 442 346 453 342 451250 M 87 316 448 320 448 307 442 311 449250 M 91 308 438 294 438 341 439 338 451250 M 77 293 426 301 450 311 439 315 446250 M 76 337 415 330 448 328 441 314 447250 M 98 349 438 352 435 348 436 334 455B-1250 M 100 325 458 326 432 356 444 342 453250 M 95 328 423 329 438 329 458 351 437250 M 99 314 415 313 440 324 434 337 452250 M 231 328 418 329 442 320 429 315 445250 M 189 326 421 324 437 332 443 319 443250 M 230 337 422 340 428 329 467 322 449250 M 90 342 428 341 423 332 433 337 451250 M 75 324 423 320 423 315 443 316 450250 M 81 300 435 301 436 300 443 299 442250 M 85 292 421 303 420 257 448 267 446250 M 80 333 438 340 428 321 454 322 444250 M 181 316 428 319 437 337 463 333 450250 M 190 327 438 325 452 343 431 339 445250 F 89 302 445 292 431 301 414 288 436 305 456 302 455 312 456 305 439 307 455 302 448250 F 82 349 424 313 432 319 429 310 425 320 442 330 463 313 454 329 433 319 451 339 417250 F 79 352 437 334 429 340 405 337 440 337 434 343 427 335 450 336 438 335 450 348 396250 F 92 383 451 396 456 415 433 387 425 410 463 381 456 392 461 377 442 411 443 354 427250 F 94 337 423 300 412 302 395 301 430 305 451 306 448 316 449 309 450 309 442 321 423250 F 228 352 423 308 415 318 393 302 431 315 451 368 429 317 455 352 438 311 452 336 403250 F 188 371 450 345 437 358 447 335 429 352 453 335 451 349 454 335 442 349 461 328 421250 F 184 363 434 329 434 337 436 332 427 340 450 345 438 344 461 339 440 342 447 329 439250 F 97 349 442 328 423 325 406 329 425 327 443 338 445 329 447 333 449 329 459 322 453250 F 74 337 438 313 440 330 420 317 436 332 454 331 440 333 445 332 455 330 445 327 429250 F 78 336 445 317 426 323 408 320 433 327 445 316 464 325 453 308 452 331 443 285 442250 F 88 341 436 320 426 322 426 322 424 323 456 318 447 321 452 321 439 327 444 306 462250 F 96 321 428 290 415 304 427 301 422 306 459 275 431 310 449 279 437 325 454 302 433250 F 93 330 420 325 422 319 404 327 423 322 447 320 422 324 446 322 433 310 452 322 431250 F 225 341 446 327 417 322 399 334 426 322 462 318 440 325 451 311 432 320 451 340 426250 F 221 368 441 338 431 353 410 376 436 349 450 358 439 352 455 357 439 350 450 360 431250 F 223 372 444 375 421 366 405 365 428 360 452 362 438 362 457 359 451 360 463 380 422250 F 183 384 447 370 459 395 416 369 436 400 443 384 435 370 449 356 452 404 439 359 428250 F 182 355 442 342 433 337 420 335 440 341 444 331 439 340 462 335 442 342 443 333 425250 F 187 328 433 316 415 319 403 317 430 322 451 317 401 324 441 321 443 320 455 326 438B-1F2 egg productionDOSE ID12/21 12/22 12/23 12/24 12/25 12/26 12/27week612/28 12/29 12/30 12/31 1/1 1/2 1/3week71/4 1/5 1/6 1/7 1/8 1/9 1/10week81/11 1/12 1/13 1/14 1/15 1/16 1/17week90 183 1 1 1 1 1 1 1 10 199 1 1 1 1 1 10 200 1 1 1 1 1 1 1 2 1 1 1 1 1 10 194 1 1 1 1 1 1 1 1 1 1 1 1 10 179 1 1 1 1 1 1 1 1 1 1 1 1 10 191 1 1 1 10 188 1 1 1 10 189 1 1 1 1 1 1 1 1 1 1 1 10 3130 174 1 1 1 1 1 1 1 1 1 1 1 1 10 314 1 1 1 1 1 1 1 10 309 1 1 1 1 1 1 1 1 1 10 186 1 1 1 1 2 1 1 1 10 197 1 1 1 1 1 1 1 1 10 178 1 1 1 1 2 10 181 1 10 175 1 1 1 1 10 190 10 323 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 10 305 2 1 1 1 1 1 1 1 1 130 81 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 96 1 1 1 1 1 1 1 1 1 1 1 1 1 130 89 1 1 1 1 1 1 130 85 1 1 1 1 1 1 1 1 1 1 130 403 130 405 1 1 1 1 1 1 1 1 130 408 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 427 1 1 1 1 1 1 1 1 1 1 130 40930 432 1 130 84 1 1 1 1 1 1 1 1 1 1 1 1 1 130 86 1 1 1 1 1 1 1 1B-130 82 1 1 1 1 130 87 1 1 1 1 1 1 1 1 1 1 1 1 130 40130 74 1 1 1 1 130 73 1 1 1 130 98 1 1 1 1 1 130 75 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 79 1 1 1 1 1 1 1 1 1 1 1 1 1 160 75 1 1 1 1 1 1 1 1 1 160 91 1 1 1 1 1 1 1 1 1 160 8960 73 1 1 1 1 160 85 1 1 1 1 1 160 100 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 94 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 7660 78 1 1 1 1 1 1 1 1 160 93 1 1 1 1 1 1 1 1 1 1 1 1 1 160 82 1 1 1 1 1 1 1 1 1 1 160 74 1 1 1 1 1 1 1 160 92 1 1 1 1 160 87 1 1 1 1 1 1 160 202 1 1 1 160 77 160 222 160 97 2 2 1 1 2 1 1 1 1 1 1 1 1 1 1 160 215 1 1 1 1 1 1 1 1 1 160 216 1 1 1 1 1 1 1 1 1125 88 1125 87 1 1 2 1 1 1 1 1 1125 89 1 1 1 1 1 1 1 1125 84 1 1 1 2 1125 207 1 1 1 1 1 1 1 1 1 2125 202 2 1 1 1 1 1 1 1 1B-1125 259 1 1 1 1 1125 209 1 1 1 1 1 1 1 1 1 1 1 1125 258 1125 260 1 1 1 1 1 1 1 1 1 1 1 1125 74125 80 1 1 1 1 1 1 1125 83 1 1 1 1125 73 1 1 1 1 1 1 1 1 1 1 1125 206 1 1 1 1 1 1 1 1 1 1 1 1 1125 205125 234 1 1 1125 201 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1125 239125 237 1 1 1 1 1 1 1 1250 89 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 82 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 79 1 1 1 1 1 1250 92250 94250 228 1 1 1 1 1 1 1 1 1250 188250 184 1 1 1 1 1 1 1 1 1 1 1 1 1250 97 1 1 1 1 1250 74 1 1 1 1 1 1 1 1 1 1 1250 78 1 1 1 1 1 1 1 1 1 1 1250 88 1 1 1 1 1 1 1 1 1 1250 96 1 1 1 1 1250 93 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1250 225250 221 1 1 1 1250 223250 183250 182250 187B-1F2 egg productionDOSE ID1/18 1/19 1/20 1/21 1/22 1/23 1/24week 101/25 1/26 1/27 1/28 1/29 1/30 1/31week 112/1 2/2 2/3 2/4 2/5 2/6 2/7week 122/8 2/9 2/10 2/11 2/12 2/13 2/14week 130 183 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 199 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 200 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 194 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 10 179 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 191 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 188 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 189 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 313 1 1 1 1 1 1 1 1 1 1 1 1 10 174 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 10 314 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 309 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 186 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 10 197 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 10 178 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 181 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 175 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 190 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20 323 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 305 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 81 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 1 130 96 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 89 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 85 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 403 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 405 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 408 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 427 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 409 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 432 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 84 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 86 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1B-130 82 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 87 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 401 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 74 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 73 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 98 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 75 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 79 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 75 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 91 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 160 89 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 73 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 85 1 1 1 1 1 1 1 1 1 1 1 1 1 160 100 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 160 94 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 76 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 78 1 1 1 1 1 1 1 1 1 1 1 1 160 93 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 82 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 74 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 160 92 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 87 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 160 202 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 77 1 1 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 222 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 97 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 215 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 216 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 88 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 87 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 89 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 84 1 1 1 1 1 1 1 1 1 1 1 1 2 1125 207 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 1125 202 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1B-1125 259 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 209 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 258 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 260 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 74 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 80 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 83 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 73 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 206 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 205 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 234 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 201 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 239 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 237 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 89 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 1250 82 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 1250 79 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 92 1 1 1 1 1250 94 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 1250 228 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 188 1 1 1 1250 184 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 97 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 74 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 78 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 88 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 96 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 93 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1250 225 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 221 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 223250 183250 182250 187 1 1 1 1 1 1B-1F2 egg productionDOSE ID2/15 2/16 2/17 2/18 2/19 2/20 2/21week 142/22 2/23 2/24 2/25 2/26 2/27 2/28week 15 3/1 3/2 3/3 3/4 3/5 3/6 3/7 week 163/8 3/9 3/10 3/11 3/12 3/13 3/14week 170 183 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 10 199 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 200 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 194 1 1 1 1 1 1 1 1 2 2 1 1 1 20 179 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 191 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 10 188 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 189 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 313 1 1 1 1 1 1 1 1 1 1 1 1 10 174 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 10 314 1 1 1 1 1 1 1 1 1 1 10 309 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 186 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 10 197 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 20 178 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 181 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 175 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 190 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 10 323 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 305 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 230 81 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 96 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 89 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 85 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 403 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 405 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 408 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 427 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 409 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 432 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 84 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 86 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1B-130 82 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 87 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 401 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 74 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 1 130 73 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 130 98 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 75 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 79 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 75 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 260 91 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 89 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 73 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 85 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 100 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 94 1 1 1 1 2 1 160 76 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 78 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 93 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 82 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 74 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 92 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 87 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 260 202 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 160 77 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 222 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 97 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 215 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 160 216 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 1125 88 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 87 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 1125 89 1 1 1 1 1 1125 84 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 207 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 2125 202 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2B-1125 259 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 209 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2125 258 1 1 1 1 1 1 1 1 1 1 1 1125 260 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 74 1 1 1 1 1 1 1 1 1125 80 1 1 1 1 1 1 1 1 1 1 1 1125 83 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2125 73 1 1 1 1 1 1 1 1 1 1125 206 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 205 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 1125 234 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 201 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 239 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 237 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2250 89 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 82 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 79 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 92 1 1 1 1250 94 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 228 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 188 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 184 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 97 1 1 1 1 1 1 1 1 1 1 1 1 1250 74 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 78 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 88 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 96 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 1250 93 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2250 225 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1250 221 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 1250 223250 183250 182250 187 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1B-1F2 egg productionDOSE ID3/15 3/16 3/17 3/18 3/19 3/20 3/21week183/22 3/22 3/23 3/24 3/25 3/26 3/27 3/28 3/29 3/30 3/31 4/1week190 183 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 199 1 1 1 1 1 1 1 1 1 1 10 200 1 1 1 1 1 1 1 1 1 1 1 1 1 10 194 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 179 1 1 1 1 1 1 1 1 1 1 10 191 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 188 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 189 1 1 1 1 1 1 1 1 10 313 1 10 174 1 1 1 1 1 1 1 1 1 1 1 10 314 1 1 1 1 1 1 1 1 1 1 1 1 10 309 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 10 186 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 197 1 1 2 1 1 1 1 1 1 10 178 1 1 1 1 1 1 1 10 181 1 1 1 1 1 1 1 1 1 1 1 10 175 1 1 1 1 1 1 1 1 10 190 1 1 1 1 1 1 1 1 1 1 1 1 10 323 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 305 1 1 1 1 1 1 1 1 1 1 130 81 1 1 1 1 1 2 1 130 96 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 89 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 85 1 1 1 1 1 1 1 1 1 1 1 1 130 403 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 405 1 1 1 1 1 1 1 1 1 1 130 408 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 427 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 409 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 432 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 84 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 86 1 1 1 1 1 1 1 1 1 1 1 1 1B-130 82 1 1 1 1 1 1 1 1 1 1 130 87 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 401 1 1 1 130 74 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 73 1 1 1 1 1 1 1 1 1 130 98 1 1 1 1 1 2 1 1 1 1 1 1 1 1 130 75 1 1 1 1 1 1 1 1 1 1 1 1 1 1 130 79 1 1 1 1 1 1 1 1 1 1 160 75 1 1 1 1 1 1 1 1 1 1 1 160 91 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 89 1 2 1 1 1 1 1 1 1 1 1 1 1 1 160 73 1 1 1 1 1 1 1 1 1 160 85 1 1 1 1 1 1 1 1 1 160 100 1 1 1 1 1 1 1 1 1 160 94 1 1 160 76 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 78 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 93 1 2 1 1 1 1 1 1 1 1 1 1 1 1 160 82 1 1 1 1 1 1 1 1 160 74 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 92 1 1 1 1 1 1 1 1 1 1 1 160 87 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 202 1 1 1 1 1 2 1 1 1 1 1 160 77 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 222 1 1 1 1 1 1 1 1 1 1 1 1 160 97 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 160 215 1 1 1 1 1 1 1 1 1 1 1 1 1 160 216 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 88 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 87 1 1 1 1 1 1 1 1 1125 89 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 84 1 1 1 1 1 1 1 1 1 1 1125 207 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 202 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1B-1125 259 1 1 1 1 1 1 1 1 1 1 1 1 1125 209 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 258 1 1 1 1 1 1 1 1 1 1 1 1 1125 260 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 74 1 1 1 1 1 1 1 1 1 1 1 1 1 1125 80 1 1 1 1 1 1 1 1 1 1 1125 83 1 1 1 1 1 1 2 1 1 1 1 1 1125 73 1 1 1 1 1 1 1 1 1 1125 206 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2125 205 1 1 1 1 1 1 1 1 1 1 1 1 1125 234 1 1 1 1 1 1 1 1 1 1 1 1 1125 201 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2125 239 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1125 237 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1250 89 1 1 1 1 1 2 1 1 1 1 1 1 1250 82 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 79 1 1 1 1 1 1 1 1 1250 92250 94 1 1 1 1 1 1 1 1 1 1 1 1250 228 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 188 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 184 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 97 1 1 1 1 1 1 1 1 1 1 1 1 1250 74 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 78 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 88 1 1 1 1 1 1 1 1 1 1 1 1250 96 1 1 1 1 1 1 1 1 1 1250 93 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 225 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 221 1 1 1 1 1 1 1 1 1 1 1 1 1 1250 223250 183250 182250 187 1 1 1 1 1 1 1 1 1 1 1 1B-1