
     Chemical Safety
     and Pollution Prevention
     (7101)
EPA No. 740-C-15-003
July 2015


Endocrine Disruptor Screening Program Test Guidelines



OCSPP 890.2100:

Avian Two-generation Toxicity Test in the Japanese Quail










                                       




NOTICE

This guideline is one of a series of test guidelines established by the United States Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention (OCSPP) for use in testing pesticides and chemical substances to develop data for submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and Cosmetic Act (FFDCA) (21 U.S.C. 346a).  Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic Substances (OPPTS).  To distinguish these guidelines from guidelines issued by other organizations, the numbering convention adopted in 1994 specifically included OPPTS as part of the guideline's number.  Any test guidelines developed after April 22, 2010 will use the new acronym (OCSPP) in their title.

The OCSPP test guidelines serve as a compendium of accepted scientific methodologies and protocols that are intended to provide data to inform regulatory decisions under TSCA, FIFRA and/or FFDCA.  This document provides guidance for conducting the test, and is also used by EPA, the public and the companies that are subject to data submission requirements under TSCA, FIFRA and/or the FFDCA.  As a guidance document, these guidelines are not binding on either EPA or any outside parties, and the EPA may depart from the guidelines where circumstances warrant and without prior notice.  At places in this guidance, the Agency uses the word "should."  In this guidance, the use of "should" with regard to an action means that the action is recommended rather than mandatory.  The procedures contained in this guideline are strongly recommended for generating the data that are the subject of the guideline, but EPA recognizes that departures may be appropriate in specific situations. You may propose alternatives to the recommendations described in these guidelines, and the Agency will assess them for appropriateness on a case-by-case basis.  

For additional information about these test guidelines and to access these guidelines electronically, please go to http://www.epa.gov/ocspp and select "Test Methods & Guidelines" on the left side navigation menu.  You may also access the guidelines in http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.



OPPTS 890.2100:	Avian Two-generation Toxicity Test in the Japanese Quail.

      (a)  Scope.

	(1)  Applicability.  This guideline is intended to meet testing requirements of the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).

	(2)  Background.  The Endocrine Disruptor Screening Program (EDSP) reflects a two-tiered approach to implement the statutory testing requirements of FFDCA section 408(p) (21 U.S.C. 346a).  In general, EPA intends to use the data collected under the EDSP, along with other information, to determine if a pesticide chemical, or other substances, may pose a risk to human health or the environment due to disruption of the endocrine system. This test guideline is part of the EDSP Tier 2 tests included in the OCSPP 890 series.

The Japanese Quail Two-Generation Toxicity Test is used to characterize the nature and dose-response relationship of chemical with endocrine bioactivity on birds. This test guideline is intended to be used for a definitive test in characterizing the potential adverse apical effects posed to avian fauna from a putative vertebrate hormone active substance. Chemicals found to have potential bioactivity in the estrogen, androgen, or thyroid hormone systems through EDSP Tier 1 screening or other scientifically relevant information may require Tier 2 testing.  However, potential endocrine bioactivity does not necessarily mean it will cause adverse effects in humans or ecological systems.  The decision to require the Japanese Quail Two-Generation Test is based on EPA's weight-of-evidence determination which, if any, of the Tier 2 tests are necessary based on the Tier 1 screening data, and other scientifically relevant information.  

	(3)  Source.  The source material used in developing this harmonized OCSPP 890 series test guideline includes the OCSPP 850.2300, Avian Reproduction Test (Ref. 1), Organization for Economic Cooperation and Development (OECD) test guideline, OECD 206, Avian Reproduction Test (Ref. 2), OECD detailed review paper for avian two-generation toxicity test (Ref. 3), OECD draft test guideline for avian two-generation toxicity test (Ref. 4), and the report from the 1996 SETAC/OECD Workshop on avian toxicity (Ref. 5).

      (b)  Purpose.  The purpose of this guideline is to describe a laboratory testing procedure that can be used to assess the impact of chemicals upon Japanese quail (Coturnix japonica) and includes test chemical exposure at four life stages: in ovo, juvenile, subadults, and adults.  This test is designed to evaluate health and reproductive viability of the first filial (F1) generation following parental exposure (F0), following dietary exposure of the test chemical, but may be adapted to exposure through drinking water if this is the anticipated route of exposure of a chemical substance.  The number of second generation (F2) 14-day old survivors per F1 generation hen is the primary biological endpoint of this test, although the test includes an option of extending the study through reproductive maturity of the second filial (F2) generation.  Additional biochemical, histological, and morphological endpoints are included to assess and confirm potential endocrine disruption.  The decision to extend the test to the second generation should balance the value added of reproductive viability data for a second generation with the outcomes from the first generation, existing knowledge for the chemical being evaluated (e.g., data from avian one-generation reproduction toxicity tests (Ref. 1, Ref. 2), and animal welfare.

      (c)  Definitions.  The definitions in this test guideline are analogous to those used in the test guidelines OECD 206 (Ref. 2), and OCSPP 850.2300 (Ref. 1) with specific application to the development of Japanese quail.

14-day-old survivors are birds that survive for two weeks following hatch.

Cracked eggs are eggs determined to have cracked shells when inspected with a candling lamp. Fine cracks cannot be detected without using a candling lamp and if undetected will bias data by adversely affecting measures of embryo development.

Eggs set refers to all eggs placed under incubation, i.e. total eggs produced minus cracked eggs and those selected for analysis of eggshell thickness. The number of eggs set, itself, is an artificial number, but it is essential for the statistical analysis of other development parameters.

Eggshell thickness refers to the thickness of the shell and the membrane of an egg at several points around the girth after the egg has been opened, washed out, and the shell and membrane dried for at least 48 hours at room temperature. Values are expressed as the average thickness of these several measured points in millimeters (mm).

Fertile eggs refers to eggs in which fertilization has occurred and embryonic development has begun. This is determined by candling the eggs 8 days after incubation has begun. It is difficult to distinguish between the absence of fertilization and early embryonic death. The distinction can be made by breaking open eggs that appear infertile and examining further. This distinction is especially important when a test substance induces early embryo mortality.

Hatchlings, normal refers to embryos that mature, pip the shell, and liberate themselves from the eggs on day 17-22 of incubation.

Live  15-day embryos refers to viable embryos that are developing normally after 15 days of incubation. This is determined by candling the eggs.

      (d)  General Experimental Design.
      
      Principle of the Test.  The parental (F0) generation is exposed to test chemical beginning at 4 weeks post-hatch.  The first filial (F1) generation is exposed to test chemical in ovo (through parental exposures) and from hatch through termination.  The second filial (F2) generation is fed an untreated diet from hatch through termination. Effects of test chemical exposure on growth and development, reproduction, histology, and biochemical endpoints are determined by statistically comparing treated birds to control birds (Appendix 1).  The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) are determined for each endpoint that is statistically tested (Appendix 1).  

During the treatment period, adult birds in the F0 and F1 generations are observed daily for indications of overt toxicity or other clinical signs.  Reproductive parameters are monitored in F0 and F1 breeding pairs.  Offspring growth and development is monitored in the F1 and F2 generations.  Additionally, gross morphology, histopathology, developmental landmarks, and hormone levels are also measured. 
      
      (e)  Description of the Method.

      (1)  Test animals.  The test species, Japanese quail, is a domesticated species, easy to raise in captivity, have a short generation time, and their precocial development allows for the production of large numbers of eggs that can be artificially incubated to produce the next generation.  Japanese quail generally start laying eggs at approximately six weeks of age. Once egg-laying has begun it will take about two to three weeks for birds to reach full egg production (eight to nine weeks of age), at which point eggs are generally fertile.
      
Numerous Japanese quail strains have been established for egg or meat production.  Strains selected for egg production, with an average adult female weight ranging of 120 to 160 g and an average adult male weight range of 100 to 140 g, are recommended.  Birds reared from eggs hatched at a laboratory facility are preferred, to ensure all animals are the same age at the time the study is initiated.  Birds used in the test should appear healthy and be free of abnormalities or injuries. Birds should not receive any medications beginning one week prior to the start of the administration of treated diet until the test is terminated. It is recommended that F0 birds used in the test are from the same hatch. 
      
      (2)  Husbandry.  Suitable indoor facilities for rearing Japanese quail that allow for adequate space and control of temperature, humidity, ventilation, and light are need throughout the test. Appropriate temperature and humidity ranges and minimum area for housing birds is specified below (Table 1). Recommended ventilation is about 8 to 15 air changes per hour. The photoperiod should be 16 or 17 hours light and 7 or 8 hours of darkness, with a 15 to 30 minute transition period at dawn and dusk is recommended. Birds should be exposed to light intensity of at least 10 lux, measured at the level of the feeder. Breeding pairs should be observed to ensure that light intensity is not too high and does not promote aggressive behavior between birds.  Sound and vibrations or other disturbances in the rooms should be kept to a minimum to minimize stress.
      
Incubators and hatchers, with automatic temperature and humidity controls and an egg-turning device, are recommended. In addition, suitable equipment is required to maintain stored eggs within the temperature and humidity ranges specified (Table 1).  A strength tester is needed to measure eggshell strength.
 
 Table 1.  Housing Conditions
 Age
 (week)
 Temperature
 ([o]C)
 Relative humidity
 (%)
 Minimum floor area
 (cm[2]/bird)
 1
 35-38
 40-80
 50
 2
 30-35
 40-80
 75
 3-4
 23-27
 40-80
 100
 >4
 16-27
 40-80
 625
 Note:	The acceptability of housing conditions of adults will be evaluated on the basis of results of reproductive performance.

Wire pens with slanting floors and egg-catchers or other measures to prevent breakage of eggs are recommended for adults.  Pens for both adults and chicks should preferably be of stainless or galvanized steel or other inert materials.  If the floor of the pens is wire mesh, the wire mesh should be of a size sufficient to prevent foot injury (trauma or pododermatitis) but large enough to allow excreta to drop through.  Measures should be taken to minimize spillage of diet (e.g., covering the food troughs with a wire grid).

Adult birds should be housed in pairs (one male/one female), and pens should be distributed to prevent cross-contamination of treated feed and avoid positional effects (e.g., all birds of one treatment group are closet to a door).  Measures should be taken to minimize injuries, stress and mortality due to aggression between pen mates.  If separation becomes necessary, pairs should be reintroduced daily to maintain fertility.

Chicks should be reared in thermostatically controlled rearing pens or cages free of drafts and radiators (e.g., ceramics) are recommended to maintain adequate temperatures (Table 1). Sufficient space should be provided, especially during the first week after hatching, to allow access to feed and water for weaker birds. Chicks should be identified individually or by pen of origin, and may be housed together, in groups of approximately equal number, preferably by treatment group. The optimal number of chicks per pen depends on pen size, but stress should be minimized.  The F1 birds should be separated into pairs (one male/one female) at 4 weeks post-hatch, or as soon as sex can be discriminated.
 
      (3)  Feeding.  Diet and drinking water are provided ad libitum.  The diet should be described in the study report, including caloric content, and should meet the specific nutrient requirements of Japanese quail (Table 2).  If the same diet is used for chicks and adults, extra calcium should be added to the adult diet to accommodate egg production.  During the treatment period, birds are fed basal diet mixed with the test substance at specified concentrations.  Sufficient space for drinking and feeding should be provided during the first week after hatching so weak birds have access.

Table 2.  Recommended Nutritional Values
Nutrient
Adults (> 4weeks)

Recommended range (%)
Chicks

Recommended range (%)
Crude protein
27 to 29
27 to 30
Crude fibre
3.5 to 5.0
3.0 to 6.0
Crude fat
2.5 to 7.0
5.5 to 7.5
Calcium
2.6 to 3.6
0.75 to 1.2
Phosphorous
0.9 to 1.1
0.6 to 1.0

      (f)  Procedure.

      (1) Test Animals.  

      (i) F0 Generation.  At approximately two weeks of age, individual F0 birds are genetically-sexed (preferred method).  At four weeks post-hatch, individual F0 birds are weighed, randomly paired and allocated to pens based on genetic sex. Birds are paired, preferably by known lineage, such that males from one line are paired with females from another line, thus, avoiding mating between closely related birds.  There should be no significant difference in mean body weight between test groups at the start of the treatment period.  At least 16 pairs of adult birds should be allocated to the control group to ensure that there are at least 12 breeding pairs that have successfully produced eggs remain at the end of the test period.  Breeding pairs should be observed for signs of aggression, and if necessary separated (e.g, by a translucent separation in the pen) except for periodic conjugal visits.  Breeding pairs that do not lay eggs by the 2[nd] week post-treatment (8 weeks post-hatch) should be excluded from further study.  Reproductive data are collected until the end of the 7th week post-treatment, at which point birds are humanely killed for necropsy, histopathology, and measurement of specific endocrine and physiological parameters (Appendices 1 and 3).

      (ii) F1 Generation.  Brood from the F0 6[th] and 7[th] weeks of egg production (i.e., weeks 12 and 13 post-hatch) are used to establish the F1 generation.  Optimally, six eggs are set for each parental pair, but a minimum of 40 eggs should be set for each test level.  If a treatment level produces fewer than 40 eggs, that level is dropped from further analysis in the test. During week 4 post-hatch, birds from F1 control and treatment groups are weighed, genetically sexed, and randomly allocated to pens by pairs, with odd numbered pen males being paired with even numbered females and even numbered pen males being paired with odd numbered females.  F1 breeding pairs are maintained on treated diet (with the exception of the control pairs) from hatch until test termination (approximately 6 weeks post-fertility).  All other experimental conditions are maintained in the same manner as the F0 generation (Appendices 1 and 3). 

      (iii) F2 Generation.  Brood from the F1 6[th] and 7[th] weeks of egg production (i.e. weeks 12 and 13 post-hatch) are used to establish the F2 generation. Similarly to the F1 generation, an optimum of six eggs are set for each breeding pair with a minimum of 40 eggs for each test level.  F2 offspring will be maintained on untreated diet from hatch until test termination, at either 14 days post-hatch or until F2 offspring reach sexual maturity at approximately 6 weeks post-hatch (if the option to extend the test is exercised).  

      (2)  Egg Collection, Incubation, and Hatching.  To determine the effect of the test chemical on reproductive fitness, eggs collected for incubation and hatching should be collected at a time associated with peak fertility and after test chemicals concentrations are expected to reach steady state within the egg.  For this reason, eggs are collected several weeks after the onset of egg production and after adults have been exposed to the test substance (Appendices 1-3).  
      
Beginning the 5[th] week of egg production, eggs are set and incubated to determine embryo viability.  Eggs and candled at embryonic day (ED) 8 and 15, to determine early and late embryonic viability, respectively.  Eggs collected during the 5[th] week of egg production will be used to determine eggshell thickness and for embryo tissue collection after the ED15 viability measure is determined.  Eggs collected during the 6[th] and 7[th] week of egg production will be allowed to hatch (see Section F.1) or used to determine eggshell thickness after the ED15 viability measure is determined.  Eggs are stored in a cold storage facility prior to incubation.  

Incubation is best performed when eggs are set in an incubator with temperature and humidity control and an automatic turning device (Table 3).  From the second day of incubation onwards, eggs should be turned at least three times per day.  If turned by hand, eggs should be turned an odd number of times a day.  Eggs selected for hatching are moved from an incubator to a hatching chamber.

Table 3.  Recommended conditions for egg storage, incubation and hatching†.
 
 Temperature ([o]C)
 Relative humidity (%)
 Turning
 Storage
 13-16
 55 - 80 
 Yes
 Incubation
 37.5
 50 - 70
 Yes
 Hatching
 37.5
 70 - 75
 No
†Temperature and relative humidities given are for forced draft incubators and hatchers.  In still-air, gravity-vented incubators and hatchers, temperatures should be 1.5 to 2 [o]C higher and relative humidity should be increased by about 10 percent.  At higher elevations, higher relative humidity is necessary.
      (3)  Dietary Concentrations.  The concentration of the test substance in the diet is expressed as weight of the test substance per unit weight of feed (mg/kg of feed) if the test substance is introduced through a dietary exposure.

Dietary concentrations of the test substance should be chosen on the basis of toxicological data from a range-finding study or other avian toxicity tests (e.g., Ref. 1; Ref. 2).  Additional information from toxicity tests with rodents or other mammals may be helpful in selecting the dietary concentrations.  

A range-finding test is recommended to determine the concentrations of the test substance to be used in the definitive test. A minimum six week dietary exposure period should provide information useful in determining appropriate test concentrations and evaluating any potential avoidance of treated feed.  Results of the range-finding study and/or existing avian toxicity data can be used to determine the highest test substance concentration chosen and is expected to affect development and reproduction, though not cause mortality or other overt toxicity that precludes evaluation of reproductive parameters.  If no significant effects are were observed in the range-finding study or reported in the literature, then the maximum recommended test concentration for avian studies is 5000 ppm.   Ideally, selected test concentrations should allow determination of the lowest observed effect level (LOEC) and no observed effect level (NOEC) for the test substance.  Intermediate concentrations should be geometrically spaced between the highest and lowest doses, e.g., 1/6 and 1/36 of the highest dose.  A minimum of four concentrations and a control are required for the definitive test.

      (3)  Preparation and monitoring of the test diet.  To prepare the test diets, the appropriate amounts of test substance are mixed into the diet.  The mixing method should be developed so as to obtain a homogeneous distribution of test substance in the diet.  
      
If possible, the test substance should be added to the diet without the use of a vehicle or diluent.  If necessary, a vehicle of negligible toxicity (e.g., food grade corn oil) may be used to ensure a uniform distribution.  Acetone may be used to dissolve a test substance, provided it is allowed to evaporate before feeding the diet to the birds.  The amount of vehicle used should be as low as possible and should not exceed 2% by weight of the diet.  When used, a constant amount of vehicle should be added to each test group and the control group diet, in order to keep the caloric value of the diet equal between dosage groups.
      
The frequency of diet preparation should be chosen so that degradation or volatilization of the test substance in the diet does not allow the actual concentration to fall below 80% relative to initial concentration.  The frequency of diet renewal should not be more than once a day and not less than once a week.  Residual feed should be visibly inspected for mold and if acceptable, weighed and mixed with fresh feed supplied to the birds.  It may be desirable to keep all feed frozen until use. Homogeneity of the test substance in the diet may be evaluated prior to the test or samples for homogeneity measurements may be taken at the first mix prepared for the study.

A validated analytical method should be used to determine test substance concentrations prior to the beginning of the test (e.g., Ref 2).  Stability of the chemical in feed should be analyzed under typical test conditions prior to the start of the test or in parallel with the range-finding study and verified during the definitive reproduction test.  Stability data from previous avian reproduction toxicity test (e.g., Ref. 2) or mammalian feeding studies may be used as guidance, if available.  If chemical stability data are unavailable then stability should be determined using guidance in this test guideline.  Samples should be analyzed biweekly.

To verify test concentrations, samples of diets fed to the birds should be taken each time a new batch of treated diet is prepared to measure the actual (as opposed to nominal) concentration of test substance.
      
Both F0 and F1 generations are maintained on treated diet until test termination.  All F2 chicks are maintained on untreated diet.
      
      (4)  Measurements and Observations. The sampling design, endpoints, and sampling schedule for each generation are summarized in materials accompanying this guideline (Appendices 1-3).

      (i)  Adults.  Prior to start of the treatment period, egg production of all bird pairs from the F0 generation (including potential replacements) should be recorded.  Pairs that do not lay eggs by the 2[nd] week post-treatment (8 weeks post-hatch) should be excluded from further study.
            
The following data should be collected on adult during the study:
   * Clinical signs of toxicity and general health (e.g. mortality, lethargy, depression, wing droop, ruffled feathers, lacrimation, feces, etc.) should be evaluated at least once daily, during the acclimation and treatment periods.  Any injuries sustained and subsequent treatment should also be recorded.  Moribund individuals or those otherwise in severe distress should be immediately and humanely euthanized.
   * Food consumption (per pair) should be recorded at least weekly as often as food is replaced in the feeders, noting any apparent food spillage.
   * Body weights should be determined at pairing and at the end of the treatment period. There should be no significant difference in mean body weight between test groups at the start of the treatment period.  If there is a significant difference between test groups, birds should be again randomized to avoid differences in body weights between control and treatment groups prior to the beginning of treatment. 
   * Observations of secondary sex characteristics and timing of sexual maturity (egg or foam production) or absence thereof will be recorded for the F1 generation and F2 generation if the study is extended.  The timing of sexual maturity will be considered when egg or foam production occurs in 90% of the control group.  If time to sexual maturation is monitored in the F2 generation, F2 observations will extend to the period required for sexual maturation of the control group.  This should suffice to adequately assess potential pubertal delays among treated birds. 
         
The following information is recorded per pair and per treatment/control:
   * date of production of first egg
   * date of production of first foam
   * plumage (recorded as "male" (rusty), "female" (mottled), or "ambiguous". 
      
	(ii)  Eggs.  Once egg laying begins, eggs should be collected daily and marked according to pen.  Eggs lots from weeks 5 through 7 of egg production should be set for the F0 generation and weeks 1 through 7 of egg production should be set for the F1 generation (Appendix 2).  Eggs should be stored and set weekly or every other week for incubation (see Table 3 for conditions).  Prior to incubation, all eggs should be candled to detect cracks.  Cracked eggs should not be incubated but are counted.  Eggs set for incubation should be candled at embryonic day (ED) 8 to determine fertility.  In addition, eggs set for hatch or sample collection should be candled again at ED 15 to determine embryo viability (Appendices 2 and 3).  The total number of eggs laid per pen should be accounted for at the end of the study.

Eggs collected from F0 and F1 breeding pairs during weeks 11, 12, and 13 post-hatch should be transferred from incubation conditions to hatching conditions on day 16.  Hatching should be complete by day 17 or 18.  On ED 15, two eggs per pen will be opened and embryo histopathology will be determined from incubated eggs collected during week 11 post-hatch.  

Eggs collected from F0 and F1 breeding pairs during weeks 12 and 13 post-hatch will be incubated to establish the F1 and F2 generations.  Six eggs lacking cracks, breaks, or abnormalities will be randomly selected for incubation.  If during weeks 12 and 13 post-hatch, F0 and F1 breeding pairs in a treatment level do not collectively produce at least 40 eggs, that level is dropped from further analysis.

After hatching, chicks should be dry before they are removed from the hatcher.  Chicks that have not hatched within approximately 24 hours of the majority of chicks hatching should be considered unhatched.  No assistance should be given to chicks during hatching. 

From the onset of egg production, one egg per pen is collected from odd number pens in odd numbered weeks and from even numbered pens in even numbered weeks to measure eggshell strength and thickness from all F0 and F1 breeding pairs. Eggshell strength is measured in Newtons using a strength tester. The egg is placed on its side on the test stand so the compression head will contact the egg at the equator.  Eggshell thickness is measured in eggs cut open at the equator, washed, and dried with the membrane intact for at least 48 hours at room temperature.  Eggshell thickness is measured three points using a calibrated micrometer.  Eggshells are measured to 0.01 mm and the mean value is calculated per egg.  Egg yolks from eggs broken during shell measurements can be used for biochemical analyses.

During the treatment periods, the following egg information is recorded for control and treated birds:
      *       Total egg production per hen
      *       Number of eggs cracked 
      *       Number of eggs broken
      *       Number of abnormal eggs (plus description of abnormalities)
      *       Number of eggs set
      *       Egg fertility (ED 8) 
      *       Embryo viability (viability and embryonic deaths; ED 15) 
      *       Eggshell thickness (nm)
      *       Eggshell strength (Newtons)
      *       Number of eggs that hatch 
      *       Number of 14-day old survivors per hen 

If a pair is removed during the study (e.g. death, injury, etc.), data from all preceding weeks are evaluated.  If data from a pen in which mortality occurred or a bird was injured are used in study results, this should be justified.

Typical values for reproductive parameters in Japanese quail are shown in Table 4.  Minor differences in control values may be due to environmental differences inherent to each laboratory, however, considerable deviations from values reported below may be due to husbandry conditions. 

Table 4.  Typical values for reproductive parameters in Japanese quail. 
Parameter
Units
Normal JQ values†
Egg laid/hen
#/day
0.61 to 0.89
Eggs cracked/broken* 
% of eggs laid
0 to 10%
Eggs set
#/pair
[1]
Eggshell strength
Per egg (Newtons)
[1]
Eggshell thickness
Mean/egg (0.01 mm)
0.19 
Fertile eggs (ED8)
%eggs set
80-100%
Viable embryos (ED15)
% of eggs set
80 to 96%
Hatchability
% hatching of viable embryos
65 to 100%
Hatchling survival to day 14
% of hatched eggs that survive to 14 days
85 to 97%
†Values obtained from OCSPP 850.2300 (Ref 1) and OECD Test No. 206 (Ref. 2).
* Describe any egg abnormalities.
1 No specific ranges provided.
      
      (iii) Chicks.  The following observations on F1 and F2 chicks are recorded during the treatment periods
   * Day of hatch
   * Abnormal hatchling morphology/clinical signs of toxicity or disease
   * Hatching mortality
   * Chick body weight at hatching
   * Number of 14-day-old surviving chicks
   * Chick body weight at 14 days after hatching
   * Phenotypic sex (F1, F2*)
   * Genetic sex (F1, F2*)
   * Time of sexual maturity (F1, F2*)
         *If study is extended to include sexual maturity of F2 generation.  
       
	(5)  Performance Criteria.  Sample concentration should be at least 80% of the initial concentration.  Higher rates of loss should be investigated. When possible, all mortalities in the control group should be explained. At least 12 breeding F0 and (F1) pairs that have produced eggs should be available in the control group at the end of the test period.  Study quality is based on the performance of the control group summarized in Table 5.

Table 5. Performance Criteria.

Parameter
Acceptable level
Sample stability in diet 
Losses of 20% or less relative to initial chemical concentration
Morbidity/Mortality during test duration
<10% in the F0 and F1 control groups
Parental mortality during acclimation
<3%
Eggs laid/pair
>4/wk 
Viable embryos
>80% of eggs set in the F0 and F1 control groups
Normal hatchlings
>85% of viable embryos in the F0 and F1 control groups
Cracked eggs
< 10% in the F0 and F1 control groups
Successful egg production
> 12 breeding pairs in the F0 and F1 control groups 

	(6)  Necropsy. Birds should be humanely euthanized during culls and at the study termination.  In addition, birds (adults or chicks) which are in severe distress will be killed in extremis. If one member of a pair dies or is killed in extremis during the treatment period, the other member of the pair is killed as well.

      (i)  Adults.  All surviving F0 and F1 birds should be humanely terminated at the end of the treatment period.  All adult birds undergo necropsy and gross examination (see guidance in Appendix 5). Adult birds that die, or are killed during the course of the treatment period, will be subjected to the same procedures.  Additional biochemical and histopathology endpoints will be measured in eight (8) males and eight (8) females randomly selected from each treatment and control group.  If the study is extended through sexual maturation of the F2 generation, F2 birds will be treated as adult at necropsy.  In all other cases, samples collected from 14 day old F2 birds are described in section 6(ii) below.  
      
The following samples are collected from adult birds at necropsy:
   * Body weight
   * Morphologic abnormalities. 
   * Trunk blood will be collected for hormone assay (Appendix 4).  If adult blood volumes are limited, the left thyroid gland can be excised and used to determine glandular thyroid hormone levels (see Section 7 below).
         *       Kidneys, liver, adrenal glands, reproductive organs and associated structures (cloacal gland and bursa), and right thyroid gland will be excised and preserved in Davidson's fixative (or a suitable alternative) for histopathological examination.  A detailed description of excision, fixation, and tissue preparation is provided in Appendix 5 of this document. 
             
	(ii)  Chicks (14 days).  At 14 days post-hatch, eight (8) male and eight (8) female F2 chicks will be randomly selected and humanely terminated.  

The following samples will be collected from F2 chicks at necropsy:
   *    Body weight
   *    Morphologic abnormalities. 
   * Trunk blood will be collected for hormone assay (Appendix 4).  If chick blood volumes are limited, the left thyroid gland can be excised and used to determine glandular thyroid hormone levels (see section 7 below).
         *       Kidneys, liver, adrenal glands, and thyroid glands will be excised; reproductive organs and associated structures (cloacal gland and bursa) will be fixed in situ.  Tissues will be preserved with Davidson's fixative (or a suitable alternative) for histopathological examination.  A detailed description of excision, fixation, and tissue preparation is provided in Appendix 5 of this document. 
            
      (iii)  Embryos.  After assessing late embryo viability at ED 15, two embryos for each mating pair are randomly selected and tissues are preserved for histopathology as described above for 14 day old chicks.  The embryo is removed from the shell and weighed.  Thyroid hormone is measured from excised thyroid gland and egg yolks are extracted for steroid hormone analysis.  Eggs collected from birds during 11 week post-hatch (treatment week 5) and incubated to ED 15 can be used in lieu of measuring embryo glandular T4 measurements, as embryo tissues are fixed in situ.
      
The following samples will be collected from embryos at necropsy:
   *    Body weight
   *    Morphologic abnormalities. 
         *       Kidneys, liver, adrenal glands, and thyroid glands will be excised; reproductive organs and associated structures (cloacal gland and bursa) will be fixed in situ.  Tissues will be preserved with Davidson's fixative (or a suitable alternative) for histopathological examination.  A detailed description of excision, fixation, and tissue preparation is provided in Appendix 5 of this document. 
         *       Steroid hormones are measured from egg yolks extracts.
         *       Glandular thyroid hormone levels are measured from excised thyroid glands.
             
	(iv) Examination of tissues.  F1 birds, exposed to test chemical in ovo and throughout their life cycle, are expected to have the greatest treatment-related effects.  For this reason, tissues in only the control and highest dose tested in F1 birds will be examined to assure that endocrine effects are neither masked nor imitated by systemic toxicity.  Tissues examined from F1 embryos, 14 day old hatchlings, and adult will include liver, kidney, adrenal, reproductive tissues (gonads, accessory sex tissues, reproductive glands and ducts) and thyroid gland (Table 6). 
 
Table 6:  Tissues examined for general toxicity from adult F1 birds.
Treatment
Number
Tissues
Purpose
Control
8M / 8F
Liver, kidney, adrenal, thyroid, reproductive tissues
Examine tissues for potential systemic (non-endocrine related) toxicity
Highest treatment concentration
8M / 8F



If no signs of overt general toxicity are observed among F1 birds in the high treatment group, histopathological samples from F0, F1, and F2 birds will be limited to reproductive tissues and thyroid glands (Tables 7 and 8).  If signs of overt toxicity are observed in the high treatment group, the potential of overt toxicity mimicking or masking endocrine related effects cannot be ruled out.  Tissues in Table 6 should be examined in the next highest until indications of overt toxicity are not observed.

Table 7.  Tissues examined for endocrine-specific effects from all adult F0 and F1, and 14 day-old chicks or adult F2 birds.
Generation
Treatment
Sample size
Tissues
Purpose
F0
Control
8M / 8F
Reproductive tissues:
Ovary, oviduct, shell gland, cloacal glands, bursa (F)
Testis, cloacal glands, bursa epididymis/vas  (when present; M)

Thyroid gland
To examine treatment-related effects on the endocrine system of Japanese quail exposed to a putative endocrine disrupting compound.

Low
8M / 8F



low mid
8M / 8F



high mid
8M / 8F



high 
8M / 8F


F1
Control*
8M / 8F



Low
8M / 8F



low mid
8M / 8F



high mid
8M / 8F



high* 
8M / 8F


F2†
Control
8M / 8F



Low
8M / 8F



low mid
8M / 8F



high mid
8M / 8F



high 
8M / 8F


 * Previously examined in initial determination of potential overt toxicity (see Table 6).
 †F2 birds may be 14 day old chicks or sexually mature adult birds.
 
Additionally, if no indications of overt toxicity are observed among F1 birds in the high treatment group, histopathological samples will be examined in F1 and F2 embryos (Table 8).  
 
Table 8. Samples examined for endocrine and developmental effects on embryonic day (ED) 15 in ovo quail in the F1 and F2 generations.
Generation
Treatment
Sample size
Tissues
Purpose
F1
Control
5
Liver, kidney, adrenal, thyroid, reproductive structures, gonad
Examine potential developmental effects of exposure to putative endocrine disrupting compound in ovo.

Low
5



low mid
5



high mid
5



high 
5


F2
Control
5



Low
5



low mid
5



high mid
5



high 
5



      (7)  Hormone Measurements.  Hormones will be measured from samples collected at necropsy (see section 6).  Depending on the age of bird and volume of blood collected, hormones may be measured in blood, egg yolk, or thyroid glands.  Plasma treated with heparin or EDTA is preferred over serum for hormone measurements due to greater hormone recovery. A minimum volume of 600 ul is required for thyroid (250 ul of unextracted plasma) and steroid hormone analyses (350 ul of diethyl ether extracted plasma; see guidance in Appendix 4 of this document). 
      
        (i)  Thyroid hormone measurements.  Measurements of thyroid function include thyroid gland weight, plasma thyroxine (T4), glandular T4 content, and thyroid histology/histopathology.  Samples will be collected from embryonic and adult life stages.  Glandular T4 levels will be measured in from ED 15 two embryos from each breeding pair and can be determined from the left, unfixed thyroid gland of adults and chicks if blood volumes are limited.  Thyroxine content in eggs collected from 11 weeks post hatch (treatment week 5) and incubated to ED 15, can be used in lieu of embryo glandular T4 measurements.  A detailed description of blood, thyroid gland, and yolk preparation for thyroid hormone analysis and immunoassay results are provided in Appendix 4 of this document.  If alternative sample preparation or analytical methods are used, validation should be adequately demonstrated.
 
       (ii)  Steroid hormone measurements.  Steroid hormones may be determined from blood or egg yolk samples.   Steroid content is measured in blood collected from adults and 14 day-old chicks and eggs collected from 11 weeks post hatch (treatment week 5) and incubated to ED 15.  A detailed description of blood, thyroid gland, and yolk preparation for steroid hormone analysis and immunoassay results are provided in Appendix 4 of this document.  If alternative sample preparation or analytical methods are used, validation should be adequately demonstrated.
 
       (8)  Data Collection and Reporting
 
      (i)  Treatment of Results.  Data are collected in a spreadsheet form and analyzed with a statistical software package.  Statistical analyses of the data should preferably follow procedures described in Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application (Ref. 6).  All adult health data should be recorded per individual bird or breeding cage (e.g. body weight, number of eggs per hen, food consumption per pair).  The F0 parental pair (or pen) is the primary statistical unit and the F1 parental pair (or pen) is the secondary unit; all reproductive data should be related by F0 lineage.  

      (ii)  Statistical Treatment.  Treatment related effects will be determined by statistically comparing treated birds to controls.  Statistical comparisons of endpoints between generations, beyond noting the NOEC/LOEC for a particular endpoint for each generation, are not desired as the experimental design and timing of sample collections vary by generation.  A description of test variables by type for statistical analysis is provided in Appendix 1 of this document.  The raw data values should be reported by pen to provide sufficient detail for an independent statistical analysis.

Prior to using parametric statistical analysis, variables should be checked for normality.  If the assumptions of parametric statistics are violated, data should be transformed and retested.  Transformations should follow commonly accepted, scientific approaches.  For example, proportions should be arcsine transformed, and skewed data should be retested after applying log or negative log transformation.  If data are still not normally distributed, EPA recommends examining data for possible alternate (e.g., binomial) distributions.  Monotonicity and homogeneity of variance can be tested within the statistical model.  EPA recommends an analysis of other statistical approaches be exhausted prior to considering an analysis of ranked data.

The following equations are used to calculate: adult body weight gain, average hatchling weight, average 14-day hatchling survivor weight, average eggshell thickness, total food consumption per adult bird per pen, proportion of uncracked eggs, and proportion of normal eggs.

		(A)  Response variable calculation.  For all equations in this guideline, the index j = 1 to the total number of pens per treatment group (typically 16 pairs).
   
		(B)  Adult body weight gain.  The change in adult body weight (males and females are tracked separately) between test initiation and test termination,, for pen j is the measure used in this test guideline to evaluate the inhibitory effects of the test substance on adult growth.  The change in adult body weight (male or female), assuming one bird of each sex per pen, is calculated using Equation 1.  

--------------------------------------------------------------------------------
                                    Equation 1
--------------------------------------------------------------------------------
where:
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------
bwjt = male (or female) body weight in pen j at time t, where t1 is test initiation; and t2 is test termination.

		(C)  Average hatchling weight.  The response measure for hatchling weight is the average hatchling weight per pen which is calculated using Equation 2.

--------------------------------------------------------------------------------
			Equation 2
--------------------------------------------------------------------------------
where:
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------
k = index number of a hatchling in pen j from 1 to NHj;
--------------------------------------------------------------------------------
NHj = total number of hatchlings in pen j; and
--------------------------------------------------------------------------------
HATWTkj = body weight of hatchling k in pen j.
   
      (D)  Average 14-d hatchling survivor weight.  The response measure for 14-d hatchling survivor weight is the average survivor weight per pen which is calculated using Equation 3.

--------------------------------------------------------------------------------
                     Equation 3
--------------------------------------------------------------------------------
where:
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------
k = index number of a 14-d old hatchling in pen j from 1 to HSj;
--------------------------------------------------------------------------------
HSj = total number of 14-d old surviving hatchlings in pen j; and
--------------------------------------------------------------------------------
SURVWTkj = body weight of 14-d old surviving hatchling k in pen j.
	
	(E)  Average egg shell thickness.  The response variable for egg shell thickness is the average thickness per pen which is calculated using Equation 4.

--------------------------------------------------------------------------------
                                 Equation 4
--------------------------------------------------------------------------------
where:
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------
k = index number of egg shell thickness measurement in pen j from 1 to mj;
--------------------------------------------------------------------------------
mj = total number of eggs with shell thickness measured  in pen j; and
--------------------------------------------------------------------------------
THICKkj = average shell thickness of egg k in pen j., measured as described in Section (f) 
--------------------------------------------------------------------------------
4(ii) of this guideline.
--------------------------------------------------------------------------------


	(F)  Total food consumption per adult per pen.  Total food consumption per adult bird between test initiation and test termination, TFOODj, for pen j is the measure used in this test guideline to evaluate aversion or inhibitory effects of the test substance on consumption of food by adults.  The total food consumption per adult bird per pen is calculated using Equation 5.  Additionally, the weekly (or biweekly after the onset of laying) food consumption rate per adult (e.g., t1, t2, etc.) during the course of the test is calculated and plotted to assess effects on the pattern of food consumption.

--------------------------------------------------------------------------------
                                Equation 5
--------------------------------------------------------------------------------
where:
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------
t = index of weekly and biweekly measurements of food consumption, with t=1 being week 1 of the study and t=term being the test or exposure termination week;
--------------------------------------------------------------------------------
FOODjt = total food consumption in pen j at time t;
--------------------------------------------------------------------------------
mjt = number of adult birds in pen j at time t.
--------------------------------------------------------------------------------


	(G)  Proportion of uncracked eggs.  The proportion of uncracked eggs is calculated using Equation 6.

--------------------------------------------------------------------------------
                                Equation 6
--------------------------------------------------------------------------------
where:
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------
ELj = total number of eggs laid in pen j; and
--------------------------------------------------------------------------------
ECj = number of eggs cracked in pen j.
	
	(H)  Proportion of normal eggs.  The proportion of normal eggs is calculated using Equation 7.
            
--------------------------------------------------------------------------------
                      Equation 7
--------------------------------------------------------------------------------
where:
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------
ELj = total number of eggs laid in pen j;
--------------------------------------------------------------------------------
ECj = number of eggs cracked in pen j; and
--------------------------------------------------------------------------------
EAj = number of irregular or abnormal eggs in pen j.
--------------------------------------------------------------------------------


      (iii)  Descriptive Statistics.

      (A)  Environmental conditions.  Calculate descriptive statistics (mean, standard deviation, minimum, maximum) for the following parameters: 
   * temperature,  relative humidity, and light intensity during the three exposure phases(initial, photostimulation, and egg production) for adults;
   * temperature,  relative humidity, and light intensity for chicks in brooder pens;
   * temperature and relative humidity during egg storage and incubation.
      
      (B)  Test substance stability in diet.  Calculate descriptive statistics (mean, standard deviation, coefficient of variation, minimum, maximum) by treatment level of the test substance concentration in the diet.
      
      (C)  Reproductive response variables.  For each treatment group including the control, calculate and plot summary statistics (mean, median, minimum, maximum, first quartile, and third quartile) for each reproductive response variable that is statistically analyzed (Appendix 1).  Additionally, calculate the standard deviation, coefficient of variation, standard error of mean, and 95% confidence interval of mean for each treatment group including the controls.
      
      (D)  Inhibitory effects (Percent inhibition).  Except for the two response variables, number of cracked eggs and number of irregular and abnormal eggs, all other response variables are expected to exhibit increasing inhibition or reduction in the measured response with increasing test substance concentration in the diet.  For all response variables in Appendix 1 the percent inhibition (%I) as compared to the control at each test substance concentration is calculated using Equation 8.
            
--------------------------------------------------------------------------------
	                                      Equation 8
--------------------------------------------------------------------------------
where:
--------------------------------------------------------------------------------

--------------------------------------------------------------------------------
C = the control mean treatment response value (e.g., number of eggs laid); and X = the test substance treatment mean response value (e.g., number of eggs laid).  Stimulation or a greater response in the test substance treatment than the control is reported as negative %I. 
--------------------------------------------------------------------------------

      
      (E)  Stimulatory effects.  For the response variables number of cracked eggs and number of irregular and abnormal eggs, the interest is in the increase or stimulation of these events with increasing test substance concentrations rather than in their reduction or inhibition.  The percent stimulation or increase is also calculated using Equation 8 except stimulation is reported as negative values of %I.  Negative %I values indicate an increased or stimulatory effect over the control response.  If working with negative numbers is confusing, the analyst may find multiplying the %I value by -1 reduces confusion when discussing the increase in cracked eggs and irregular or abnormal eggs with increased test substance concentration.
      
	(F)  No observed effect concentration (NOEC) and lowest observed effect concentration (LOEC). A NOEC and LOEC are determined for each response variable in Appendix 1 using appropriate statistical methods.  All methods used for statistical analysis should be described completely.  Experimental units (replicates) are the individual pens within each treatment level.  The overall study NOEC and LOEC are the lowest values (most sensitive) of all response variables considered.

      (iv)  Statistical Analyses By Variable Class
      
      (A)  Continuous.  Continuous data should be analyzed using a simple ANOVA by generation with treatment as the effect (Appendix 1). For most continuous variables, a normal distribution can be assumed and tested within the model (normality preferably using the Shapiro-Wilk or Anderson-Darling test and variance homogeneity tested using the Levene test; both tests are performed on the residuals from an ANOVA).  The exception may be endpoints, where the range values within a treatment group is greater than one order of magnitude (e.g., blood, yolk, and glandular endocrine data).  In this case, a log transformation can be applied to normalize data prior to the one factor ANOVA.
      
Where non-normality or variance heterogeneity is found, a normalizing, variance stabilizing transformation should be sought.  If the data (perhaps after a transformation) are normally distributed with homogeneous variance, a significant treatment effect is determined from Dunnett's test.  If the data (perhaps after a transformation) are normally distributed with heterogeneous variance, a significant treatment effect is determined from the Tamhane-Dunnett or T3 test or from the Mann-Whitney-Wilcoxon U test.  Where no normalizing transformation can be found, a significant treatment effect is determined from the Mann-Whitney-Wilcoxon U test using a Bonferroni-Holm adjustment to the p-values.  The Dunnett test is applied independently of any ANOVA F-test and the Mann-Whitney test is applied independently of any overall Kruskall-Wallis test.  In addition, regardless of the results of the previous tests, a step-down Jonckheere-Terpstra trend (JT) test should also be applied to the data as long as a monotonic response was observed.

	(B) Proportional.  A binomial model is recommend for anlysis of proportional variables (Appendix 1).   For monotonic data, the Rao Scott Cochran-Armitage test is preferred.  When the denominators of the proportions representing responses of replicate pairs of birds are very large, generally approaching 50 for example, it was recommended that analysis of the response be performed on the means of the arcsine-square root transformed replicate proportions using Dunnett's test. Where the data differ significantly from monotonicity, the Rao-Scott adjustment can be applied to the Fisher-Exact test. A Dunnett test on arcsine-square root transformed proportions is also acceptable, but should be weighted by replicate size if there are marked variations in replicate sizes.

	(C) Time-to-event.  Time to event endpoints are often right censored (i.e., the event does not occur within the observation period; Appendix 1).  The recommended statistical analysis for these types of endpoints is the Cox Proportional Hazard mixed effects model, so long as the event occurs over a range of at least six days.

	(D) Ordered categorical.  Histopathology severity scores can be analyzed using a relatively new approach, Rao Scott Cochran-Armitage by slices (RSCABS; Ref. 7).  Both SAS and R-based software versions are available free of charge from the publication's authors.  This approach accounts for both severity and frequency of specific pathologies.  Other valid statistical approach can be used, if appropriately explained and justified.

Phenotypic sex based on secondary sex characteristics (i.e., plumage) may be recorded as "male", "ambiguous", or "female". Analyses of severity of phenotypic deviation from genetic sex should be assigned on an ordinal scale of 0 to 3 indicating divergence of each birds secondary sex characteristics expectation based on its known genotypic sex.  For a male, for example, the scale would be 0=normal male phenotype, 1=mostly male phenotype, 2=indeterminate phenotype, 3=female phenotype.  Thus, statistical analysis of each genetic sex will be performed independently.

      (v) Test Report.  In addition to data indicated in the sections above and described in Appendices 1  -  4, the final report should include the following information:

Test substance:
   * Identification, including chemical name
   * Batch or lot number
   * Degree of purity
   * Chemical stability under the conditions of the test
   * Volatility

Test species:
   * Name of species tested (scientific name)
   * Strain or origin
   * Source information
   * Age of birds at start of test (in weeks) 
Description of Test Conditions:
   *      Description of housing conditions (type, size and material of pens for adults and for chicks, additional floor covering, adjustments made to pen floors to facilitate egg collection and prevent breakage, temperature, humidity, ventilation, illumination intensity and photoperiod, water supply); any changes to these during the test; measures taken to minimize food spillage;
   *      Description of the untreated diet, manufacturer, composition, caloric content of diet and carrier, results of periodically performed contaminant and nutrient analysis of diet and drinking water contaminant analysis;
   *      Test groups (number of concentrations used, nominal concentrations); details of type of carrier used and concentration as percentage of diet; test substance concentrations should be reported as mg/kg diet and as mg/kg body weight;
   *      Specific analytical method used to determine the concentrations of test substance in the diet as well as actual values of homogeneity, stability and accuracy of preparation in diet under test conditions, as recorded in the study
   *      Specific analytical method used to conduct hormone analyses (and other biochemical analyses)
   *      Description of the type and frequency of the procedure used to prepare the test diets; description of the manner of administrating the test diets; storage conditions
   *      Description of acclimation, stabilization and pre-treatment procedures, and method of assigning pairs to test groups (F0 and F1);  arrangement of pens; number of pairs per dose group; measures taken, if any, to reduce pen-mate aggression
   *      Frequency, duration and methods of observation
   *      Information on the period and conditions of storage of eggs and on the incubation method
   *      Method of marking all birds and eggs.
     
Results:
   *      Adherence to performance criteria
   *      Description of genotyping methods
   *      Description of all biochemical analysis methods (i.e., thyroid and steroid hormone analyses), data for adult, chick, and embryos (yolk) should be tabulated (separately for F0 and F1)
   *      Description of incidence of mortality, indicating the number of dead animals and the time of death during the test
   *      Description of all clinical signs of toxicity and other abnormal behavior, including time of  onset, duration and severity, and number of affected birds per test group and in the control group; any injuries sustained and subsequent treatment
   *      Description of individual weights of the birds that died or were killed in extremis during the test along with macroscopic pathological findings and adult fixed organ weights and results of histopathological investigations
   *      Description of weekly food consumption per test group during treatment periods and extrapolated mean food consumption per pair; and expressed as mg/kg body weight per bird per day; an indication of any apparent food spillage
   *      Results of range-finding test (when performed)
   *      Description of reproductive effects during treatment periods (tabulated separately for F0 and F1) as described above under statistical treatment (Section 8ii). 
   *      NOEC and LOEC per parameter that was evaluated statistically 
   *      Description of statistical methods used in the data analysis.
     
 

       (h)  References.
   
   1) US EPA. 2012. Ecological Effects Test Guidelines. OCSPP 850.2300: Avian Reproduction Test.  US EPA, Washington, DC.
   2) OECD Guidelines for the Testing of Chemicals.  1993.   Section 2 - Effect on Biotic Systems: Test Guideline 206: Avian Reproduction Test (adopted April 1984).  Paris, OECD.  
   3) US EPA. 2005. Final Detailed Review Paper for Avian Two-Generation Toxicity Test. Prepared for: US EPA, Washington, DC.
   4) OECD Guidelines for Testing of Chemicals.  Proposal for a new test guideline: Avian Two-generation Toxicity Test in the Japanese Quail, Draft November 2005.
   5) Report of the SETAC/OECD Workshop on Avian Toxicity testing.  (1996). Environment Health and Safety Publication - Series on Testing and Assessment: No. 5.  OECD/GD (96)166, Paris.
   6) OECD. 2006. Guidance Document on Current Approaches in the Statistical Analysis of Ecotoxicity Data, OECD Series on Testing and Assessment, No. 54. ENV/JM/MONO(2006)18. OECD Publishing, Paris.
      
   7) Green J.W., Springer T.A., Saulnier A.N., Swintek J. (2014). Statistical analysis of histopathology endpoints.  Environ Toxicol Chem. 33(5):1108-1116.
      


      (i)  List of JQTT Appendices.																				Page
(1)  Description of Test Variables by Type for Statistical Analysis ...........................	25	
(2)  Sampling Design for the Avian Two-Generation Toxicity Test in Japanese Quail...... 27
(3)  Weekly Schedule of Actions by Generation for the Avian Two-Generation Toxicity
 	Test in Japanese Quail .................................................................................	28
(4)  Recommendations for Preparation of Samples for Biochemical Analyses...............	29
(5)  Histopathology Guidance for the Avian Two Generation Toxicity Test 
	in Japanese Quail .................................................................................	32


APPENDIX 1:  Description of Test Variables by Type for Statistical Analysis. 





[1]Stats: Statistical differences between controls and treatment groups should be performed (Y), are not necessary (N), or are optional (op) for variables identified.  For details on suggested statistical analyses for variable types, please see text.


APPENDIX 2:  Sampling Design for the Avian 2-Generation Toxicity Test in Japanese Quail.  

                                       
                                  Generation
                                    Timing
                                      F0
                                      F1
                                      F2
ED 8

Fertility

ED 15

Embryo viability
Embryo weight
Glandular thyroid hormone
Yolk steroid hormones
Histopathology
Embryo viability
Embryo weight
Glandular thyroid hormone
Yolk steroid hormones
Histopathology
HATCH

Begin treated diet
Untreated diet
Week 1

Hatchling success
Hatchling weight
Hatchling success
Hatchling weight
Week 2

Body weight

Body weight
Termination
Trunk blood
Glandular thyroid hormone
Histopathology
Weeks 1-4

Observations of secondary sex characteristics

Week 4
Pairs established
2 week acclimation period*
Pairs established 
Pairs established (if extended)
Weeks 4-6
Sexual maturity
   * Egg production
   * Foam production
Sexual maturity
   * Egg production
   * Foam production
Sexual maturity (if extended)
   * Egg production
   * Foam production 
Body weight
Termination
Trunk blood
Glandular thyroid hormone
Histopathology
Week 6
Begin treated diet*


All wks post-treatment
Daily clinical observations
Daily egg collections
Weekly egg collection (shell thickness/strength; biweekly from each pen)
Weekly food consumption
Daily clinical observations
Daily egg collections
Biweekly egg collection (shell thickness/strength)
Weekly food consumption
Set eggs from wks 1-4 to ED8
Daily clinical observations
Weekly food consumption
5  wk post-treatment
Egg collection
Set to ED 15 to determine fertility/viability
Egg collection
Set to ED 15 to determine fertility/viability

6-7 wk post-treatment
Egg collection
Set for F1
Termination 
Body weight
Trunk blood
Glandular thyroid hormone
Histopathology
Egg collection
Set for F1
Termination 
Body weight
Trunk blood
Glandular thyroid hormone
Histopathology
      
ED = embryonic day.
*F0 birds are given two weeks to acclimate after sexual maturity is reached, as indicated by the initiation of foam production, egg laying, and mating behavior among pairs.  The earliest treatment would occur when parental birds are 6 weeks old.

APPENDIX 3:  Weekly Schedule of Actions by Generation for the Avian Two-Generation Toxicity Test in Japanese Quail.  See section 4ii in the test guideline for details.
 

Week
 
F0
 
 
F1
 
 
F2

Age
egg lot
Action
Age
egg lot
Action
Age
            Action
   
 
 
set eggs





2
 
 
 





3
0
 
hatch





4
1
 
 





5
2
 
genetic sex





6
3
 
 





7
4
 
pair





8
5
 
 





9
6
 
treated diet/eggs?





10
7
1
not set





11
8
2
not set





12
9
3
not set





13
10
4
not set





14
11
5
set eggs (to ED15)





15
12
6
set eggs (F1)





16
13
7
set eggs (F1)
 
 
hatch ED15 (wk 5)


17
14
 
terminate F0 adults
0
 
hatch (wk 6)/treated diet


18



1
 
 


19



2
 
genetic sex


20



3
 
 


21



4
 
pair


22



5
 
 


23



6
 
eggs?


24



7
1
set eggs (to ED8)


25



8
2
set eggs (to ED8)


26



9
3
set eggs (to ED8)


27



10
4
set eggs (to ED8)


28



11
5
set eggs (10 ED15)


29



12
6
set eggs (F2)


30



13
7
set eggs (F2)
 
hatch ED15 (wk 5)
31



14
8
terminate F1 adults
0
hatch (wk 6)/untreated diet
32






1
 
33






2
genetic sex; terminate F2
34






3
 
35






4
 
36






5
 
37






6
Reproductive maturity, if extended
38






7
terminate F2 if extended
APPENDIX 4:  Recommendations for Preparation of Samples for Biochemical Analyses.

     (a)  Blood Collection for hormone analyses.
      
   (1) The tube for blood collection may be a hard plastic centrifuge tube (no plasticizers) or borosilicate glass, depending on the size needed for volume and centrifuge. 
   (2) The volume of whole blood collected should be ~2.5X the volume of serum or plasma needed. 
   (3) For serum, the collection tubes contain no additives. 
   (4) For plasma, an anticoagulant (0.5 M EDTA, 10 ul per ml blood) is added to each tube prior to collection and the syringe (if applicable) is rinsed with the EDTA solution by drawing it up through the needle into the syringe and expelling it, leaving a small amount in the needle. 
   (5) Samples may be collected at the time of euthanasia; if the neck is severed, then trunk blood can be collected into the tube.  
   (6) If the sample is taken by venipuncture from older animals, the needle size is 16-20 gauge; smaller gauge results in hemolysis if the blood sample is squeezed back through the barrel of the needle (better to remove the needle and then expel the blood sample into the tube). 
   (7) For serum, allow samples to stand for 30-45 min at room temperature to clot and then centrifuge at 1,500xg for 10 min at 4[o]C. 
   (8) For plasma, invert the tubes immediately after collection to allow the anticoagulant to mix with the blood. 
   (9) If a number of samples are to be collected prior to centrifuging in order to fill the centrifuge rotors, the tubes should be kept chilled by putting them into a rack on ice. 
   (10) Samples should be centrifuged at 1,500xg for 10 min at 4[o]C. Serum and plasma should be clear and light tan in color. 
   (11) A Pasteur pipet with a bulb may then be used to siphon the serum or plasma sample and transfer it to a labeled polypropylene microcentrifuge tube for storage.  
   (12) The sample should be kept frozen at -20[o]C or below. Labeling, using indelible pen should include code for the sample, date, and other identifiers specific for the sample. 
   (13) A sample inventory in the form a spreadsheet, clearly indicating bird identification and approximate plasma volume should be kept.
   (14) If shipped for analysis use dry ice and include a copy of the sample inventory. 
   (15) If the samples cannot be diluted to run in the assays, the approximate total amount of plasma required to conduct all assays is 600ul.  Ideally samples are not pooled.  If plasma volume is limited, thyroid hormone levels can be determined from left thyroid glands. 
      
   (b)  Left thyroid gland samples.
  
   (1)    The thyroid glands are weighed at collection
   (2)    The right thyroid gland is used for histopathology and the left thyroid gland is used for biochemical analyses. 
   (3)    If the weight of the left thyroid gland is more than 10 mg, a piece between 5-10 mg needs to be accurately weighed and placed in the sample tube.  Thyroid gland samples from adults, hatchling, and embryos are placed into 2-ml round bottom centrifuge tubes clearly and legibly labeled in indelible ink and stored frozen at -20[o]C or below.  The label should include code for the sample, date, and other identifiers specific for the sample.  A sample inventory in the form a spreadsheet, clearly indicating bird identification, total thyroid gland weight, and weight of the sample collected into the tube is retained.
   (4)    If shipped for analysis use dry ice and include a copy of the sample inventory. 
   
   (c)  Yolk samples.

    (1)    Yolk samples should be collected with clean separation from albumin because albumin may interfere with the assay. 
    (2)    The entire yolk should be weighed and homogenized with an equal volume of ultrapure deionized water. 
    (3)    A 1-ml aliquot of the homogenized, diluted yolk should be put into a 1.7-ml centrifuge tube that is clearly and legibly labeled with the bird identification. 
    (4)    A sample inventory in the form of a spreadsheet, clearly indicating bird identification, total yolk weight, amount of water used for dilution, and volume (if other than 1-ml) should be retained.
    (5)    Samples should be stored at  -  20[o]C.
    (6)    If shipped for analysis, use dry ice and include a copy of the sample inventory. 

   (d)  Biochemical assays.  Radioimmunoassays (RIAs) are recommended, given sensitivity and sample volume constraints.  The following kits were validated for biochemical analysis of Japanese quail samples are recommend for analyses.  Other assays may be used but must be validated (e.g., demonstrate parallelism and specificity) for Japanese quail samples prior to use to be considered as alternatives.

Table 1. Example assay kits evaluated for measurement of triiodothyronine (T3), thyroxine (T4), estradiol (E2), and testosterone (T) in avian serum or plasma samples. 
Hormone
Assay Type
Company
Catalog #
                             Reported Sensitivity
                           Required Volume (ul)[1]
T3



                                       
                                       
Free
RIA
Siemens
TKT31
                                   0.2 ng/ml
                                      200
T4



                                       
                                       
Free
RIA
Siemens
TKT41
                                   10 ng/ml
                                      50
E2



                                       
                                       
Free
RIA
Siemens
TKE21
                                   20 pg/ml
                                      200
T



                                       
                                       
Total
ELISA
Caymen
582701
                                   3.9 pg/ml
                                      100
 - 



                                       
                                       
[1]For analysis of each sample in duplicate

Hormones should be analyzed in plasma because the volume of recovery from whole blood is greater than that of serum.  Collect plasma using either heparin as the anticoagulant.  
   * For analysis of all hormones, a minimum volume of 600 ul plasma is required. Of this, 350 ul is extracted for measurement of steroid hormones and 250 ul is needed for measurement of thyroid hormones in unextracted plasma. 
   * Thyroid hormones are lost during organic extraction and should be evaluated in unextracted plasma.
   * Initial results for measurement of estradiol and testosterone indicated plasma should be organically extracted because of a lack of parallelism in unextracted samples.
         o Diethyl ether proved more suitable for extraction of steroid hormones from plasma, due to ease of extraction and hormone recovery.
         o Extraction efficiencies should being conducted for plasma and yolk by spiking samples with tritiated estradiol or tritiated testosterone.

Assay characteristics are shown in Table 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/B0)*100] was at least 95% and repeatable in multiple assays. Actual limits of sensitivity for T3, T4, and E2 were improved approximately 10-fold (compare Table 2 vs Table 1). When performing these assays, the standard curve is adjusted accordingly.
   * Specificity was determined by measuring a known amount of the indicated 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 measured/amount added)*100]. 
   * Intra- and inter-assay variation (coefficient of variation (CV; %) were determined in all four assays using chicken plasma because of the large sample 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 (%) was 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 assay and contributed to higher variability. Therefore, additional intra-assay and inter-assay values for these assays were determined in the same manner using thyroid extract (total 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 assay at 2 dilutions. This allowed us to further validate the assays in younger birds from a different colony.  
         o Total T3: adult plasma (male, female, mixed sex), thyroid extracts
         o Total T4: adult plasma (male, female, 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, female, mixed sex), ether-extracted yolk, ethanol-extracted feces.
            
Table 2. Assay characteristics of kits selected for final analysis of triiodothyronine (T3), thyroxine (T4), estradiol (E2), and testosterone (T) in Japanese quail plasma, thyroid glands, yolk, and feces.    
Hormone
Assay 
                            Determined Sensitivity
                           Cross-reactivity[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. 
2Additional intra- and inter-assay CV values were determined in thyroid extracts for total T4 and fecal extracts for E2 because plasma measurements were approaching the limitation of sensitivity for the assays and thought to contribute to increased variability in these determinations.

APPENDIX 5:  Histopathology Guidance for the Avian Two-Generation Toxicity Test in Japanese Quail.

      (a) Introduction.  This guidance is provided to help ensure that histological procedures and pathological evaluations are performed accurately and consistently. The guidance provided is based on procedures developed by the U.S. Environmental Protection Agency in consultation with other scientific experts.  Specific products and/or equipment listed can be substituted with comparable materials.  Laboratories may depart from some aspects of this guidance due to variability in some standard laboratory practices (e.g., how much information is included on slide labels, etc.).
      
The intent of this photographic atlas is to provide an optimized histopathologic protocol for evaluating potential effects of endocrine disruptive compounds (EDCs) in a variety of tissue types collected from Japanese quail (Coturnix japonica), as part of the Avian Two-generation Toxicity Test in Japanese Quail. To achieve this goal, images were obtained from gross and microscopic specimens from male and female quail of different age groups, and standardized methodology was developed and described for necropsy, gross trimming, decalcification, tissue processing, microtomy, and other histologic procedures. At minimum, professionals who use this document are presumed to have achieved a basic level of proficiency in the areas of post-mortem dissection, histology, histopathologic slide examination and diagnostic terminology, because comprehensive fundamental training in those subjects it is beyond the scope of this atlas. 

There are two primary reasons why standardization of post-mortem procedures are imperative for EDC testing of quail. First, with the notable exception of hermaphroditism, morphologic alterations induced by EDCs often tend to be incremental and comparative rather than categorical. For example, in addition to obvious changes such as feminization of the left testis (Berg et al., 1999; Perrin et al., 1995; Ro and Kondo, 1977; Scheib and Reyss-Brion, 1979) and persistence of Müllerian ducts (Berg et al., 1999; Ro and Kondo, 1977; Scheib and Reyss-Brion, 1979), effects of estrogenic substance administration in male Japanese quail have included incremental findings such as attenuation of the ductus deferens (Masuda and Koyanagi, 2001; Ro and Kondo, 1979), reduced cloacal gland development (Halldin et al., 2003; Ro and Kondo, 1979; Yoshimura and Kawai, 2002), and decreased numbers of large perikaryons in the medial preoptic nucleus of the hypothalamus (Masuda and Koyanagi, 1998). Other types of EDC experiments that may produce histomorphologic effects that are only evident after careful comparison of compound-treated and negative control birds include trials that employ weakly-active substances, low doses, short durations, and/or adult (versus juvenile) exposures.

The second major impetus for protocol standardization involves the anticipation that identical or similar quail EDC investigations will be performed concurrently by multiple facilities.  Consequently, the use of uniform post-mortem procedures can help to minimize inter-laboratory variability in terms of histologic slide quality, tissue accountability, and lesion representativeness, which in turn may improve the consistency of inter-study histopathologic results. The generation of consistent results would tend to promote confidence in the validity of histopathology as an endpoint, and in the reliability of data intended for use in regulatory decision-making.

The primary tissues of interest covered in this document include representatives of the endocrine system, reproductive system, and immune system, and other anatomic structures that are known to exhibit hormonally-dependent sexual dimorphism. The post-mortem procedures developed to collect these tissues were intended to: 1) maximize tissue recovery, accountability, and collection efficiency; 2) minimize artifactual changes caused by tissue handling trauma and autolysis; and 3) optimize and standardize organ-specific planes of specimen sectioning to permit appropriate diagnostic comparisons and enhance inter-animal consistency. Toward these ends, and given the diminutive size and fragile nature of many quail tissues, this protocol recommends that certain individual organs not be collected during the initial necropsy procedure; instead, such specimens are acquired post-fixation during the gross trimming phase (e.g., heart, adult gonads), or are captured via microtomy of larger anatomic regions (e.g., thyroid glands, chick gonads).

Unless otherwise noted, all of the photomicrographic figures included in this atlas were obtained from histologic sections that were stained with hematoxylin and eosin (H&E).

(b)  Post-Mortem Procedures  -  Adults
	(1)  Specimen Sampling and Processing
 (I)  Necropsy and Tissue Collection.  Tissues from each animal are placed in an individually labeled container that is partially filled (>10-fold fixative volume to tissue volume) with modified Davidson's solution (the primary fixative, see Section (e)).  The necropsy procedures are:
   * A T-shaped incision is created in the ventral abdominal skin and body wall, and the newly created triangular flaps of abdominal skin are excised.  Two lateral incisions are made through the skin, breast muscles and ribs in the caudal to cranial direction, terminating at the thoracic inlet, and the resulting triangular segment of ribs and sternum is removed to expose the thoracic cavity (Fig. 1).
   * The carcass is transected distal to the tracheal bifurcation and proximal to the adrenal glands (Fig. 2), and the anterior portion of the carcass, which contains the thyroids, is placed in the primary fixative. 
   * The spleen is identified and excised from the viscera (Fig. 3), and inserted into a filter paper envelope which is placed in the primary fixative.
   * The liver is excised from the other viscera and placed in the primary fixative.  Several partial-thickness longitudinal incisions may be made in the surface of the liver to enhance fixation (Fig. 4). 
   * The distal gastrointestinal tract is severed at the rectum, and the viscera are reflected proximally, carefully severing any mesenteric attachments as the gut is retracted (a short stump of rectum is intentionally left attached to the carcass to provide a landmark for identification of the cloacal bursa at gross trimming).  The esophagus is severed anterior to the crop, and the viscera are removed en masse (Fig. 5). 
   * The duodenal loop, which contains the pancreas, is excised and placed in the primary fixative (Figs. 6-7).  The remaining viscera are placed in the primary fixative for possible subsequent evaluation.
   * The reproductive tracts are excised in toto.  For males this includes the left and right testes, epididymides, and deferent ducts (Figs. 8-10).  An attempt is made to collect these structures intact and interconnected.  It is important to note any abnormalities regarding the relative size, shape, texture, color, or orientation of the left and right reproductive tracts.  THE LEFT AND RIGHT REPRODUCTIVE TISSUES SHOULD BE LABELED INDIVIDUALLY.  
      Although the left testis is often longer and more kidney-shaped than the right, this is not always the case; consequently, for accurate identification at gross trimming, the left reproductive tract is loosely wrapped in wet gauze.  If the testis is greater than 1 cm thick in its narrowest dimension, a small (~1cm) stab incision is made through the tunica albuginea to promote internal fixation.  The male reproductive tract is placed in the primary fixative.
   * For females, the reproductive tract includes the left ovary (and right ovary remnant if present), oviduct (infundibulum, magnum, isthmus), uterus (shell gland), and vagina (Fig. 11).  To enhance internal fixation, the oviduct and uterus are incised longitudinally, leaving intact a short (e.g., 3-4 mm) napkin-ring-like transverse segment at each of the five aforementioned anatomic sites (Fig. 12).  The ovary is trimmed so that the thickness of its smallest dimension is no greater than 1 cm.  If there is a right ovary remnant, or other anomalous structure in the general vicinity, it should be collected and identified separately (e.g., placed in a labeled tissue cassette).  The female reproductive tract is placed in the primary fixative.
   * The leg and lateral body wall are excised from each side of the posterior carcass segment, with care taken not to disrupt the cloacal region (Figs. 13 and 89).  The posterior carcass segment, which contains the adrenal glands, urinary tract, bursa and cloacal gland, is placed in the primary fixative (Fig. 14).  The legs and lateral body wall are discarded.
      
Following approximately 24 hours, tissues (Fig. 15) are rinsed in 70% ethanol and transferred to 10% neutral buffered formalin (NBF) or ethanol for storage.  Tissues may be shipped in NBF, ethanol, or water (e.g., packed in wet gauze), in sufficient amount to ensure that they are not allowed to dry out during transit.  Tissues shipped in water are transferred to NBF upon arrival at the receiving facility.
	(ii)  Gross Trimming and Embedding.  Prior to gross trimming and embedding, adult bird tissues that contain bone, such as the anterior and posterior carcass segments, are decalcified in a formic acid / EDTA solution (e.g., Formical-2000(TM), Decal Chemical Corporation, Tallman, NY) for 20-24 hours.  Between the gross trimming and embedding steps, tissues are processed routinely for paraffin embedding.  An example automatic processor schedule is presented in Section (f).  The gross trimming and embedding procedures are:
   * A transverse cut is made across the anterior portion of the carcass, cranial to the thyroid glands (Figs. 16 and 87-88), and the slab that contains the thyroid glands (~ 3-4 mm thick) is embedded so that sectioning can begin at the caudal surface.
   * A longitudinal slab (~ 2-3 mm thick) is cut from the approximate center of each of the right and left major liver lobes (Figs. 17-18), and the two slabs are embedded so that their cut surfaces are sectioned.
   * For females (Figs. 19-22), the largest yellow follicles (> 3-4 mm) are excised from the ovary and returned to the fixative, and the remaining hilar region, which contains smaller yellow and white follicles, is embedded in toto. If the trimmed ovary is still too large to fit into a single cassette, it may be trimmed further and split into two or more cassettes as needed. An intact 2-3 mm napkin-ring-like transverse segment is excised from each of the following sites: infundibulum, magnum, isthmus, uterus (caudal pouch portion), and vagina, and each segment is embedded so that one of the cut surfaces is sectioned.
   * For males, a longitudinal cut is made through each testis (Fig. 23).  This cut is made slightly to one side of the central axis of the testis, so that the attachment sites of the epididymis and ductus deferens are immediately adjacent to the cut surface of the larger portion (the smaller portion is returned to the fixative).  To better fit the tissue cassette, a small amount of tissue is shaved off the side opposite the initial cut surface, and the testis is embedded so that sectioning can begin at the initial cut surface, and would include the epididymis, and ductus deferens.  Similar to the ovary, if the testis sections are too large to fit into standard cassettes they may be trimmed further, but the connection between the epididymis and the testis should be maintained if possible.
   * The pancreas is excised from the duodenal loop (Fig. 24) and embedded in toto.  The spleen is not trimmed and is embedded in toto.
   * The cloacal region is excised from the posterior portion of carcass.  The conical cloacal bursa (bursa of Fabricius), which is situated just proximal to the rectal stump, may be exposed by gentle dissection and everted by manual pressure applied to the external surface of the cloacal region (Fig. 25).  One approach is to sever the bursa at its base, and embed it for transverse sectioning, beginning at the apex of the cone.  However, it is often easier to leave the bursa in situ, and have it appear in the cloacal gland section.
   * Two parallel parasagittal cuts are made through the cloacal region and the intervening midline sagittal slab (~3-4 mm), which contains the cloacal glands (Figs. 26-27), is embedded so that either lateral side is sectioned.
   * Any remaining lateral skin flaps are trimmed off the posterior portion of carcass.  Two parallel transverse cuts are made across the posterior portion of carcass, so that substantial portions of adrenal glands and cranial (proximal) kidney are included in the intervening slab (Fig. 28).  Two more sets of parallel transverse cuts are made at midway levels of the middle and caudal (distal) kidney lobes, respectively (Fig. 29).  Each slab (~ 2-3 mm) is embedded so that sectioning can begin at the caudal surface.  
   * The final appearance of the tissues post-trimming is illustrated in Fig. 30.
      
	(iii)  Microtomy.  Section thickness for all tissues is ~ 4-6 microns.  The term "full face" is used here to indicate a section in which the entire circumference of the anatomic structure of interest is represented.  The microtomy procedures are:
   * The two liver slabs, which are in the same paraffin block, are microtomed full face (Figs. 31-34).
   * The transverse posterior carcass slab that contains the adrenal glands and cranial kidney are microtomed until the adrenal glands are full face (Figs. 35-38).  Additional sections are acquired if these structures could not be represented in the same section.  The posterior carcass slabs that contains the middle kidney and caudal kidney are each microtomed so that the renal tissue is full face (Figs. 39-40).
   * The pancreas and spleen are microtomed full face (Figs. 41-44).
   * If the cloacal bursa is to be excised, then three sections are obtained.  The first section is obtained at the grossly visible dark region of the bursa, and the second and third sections are taken at approximately 100 micron intervals.  Alternatively, if the bursa is to be left in situ, it will appear in the cloacal gland section (Fig. 114).  Figs. 45-48 represent the four grades of bursa involution.
   * The anterior carcass slab that contains the thyroid glands is microtomed until the left and right thyroids are full face (Figs. 49-50).  It is usually possible to obtain both thyroid glands in a single section, but not always.
   * The midline sagittal tissue slab that contains the cloacal glands is microtomed until the glands are full face (Figs. 51-56).
   * The testis is microtomed full face, to include portions of the epididymis and ductus deferens in the same section (Figs. 57-63).  Additional sections are acquired if, due to tissue positioning in the block, the latter two structures could not be represented in the initial section.
   * The ovary is microtomed full face (Figs. 64-74).  The oval profiles of infundibulum, magnum, isthmus, uterus, and vagina are microtomed full face (Figs. 75-86). 

      
       (2) Gross Figures.

                                     Fig 1
                                     Fig 1
            
            Figure 1. To expose the abdominal and thoracic cavities, incisions made through the abdominal wall and around the keel.
            
            
                                       V
V
L
L
H
H
A
A
                                     Fig 2
                                     Fig 2
            
            Figure 2.  The abdominal and thoracic cavities are exposed, revealing the liver (L), and abdominal viscera (v).  The anterior carcass (A), which contains the thyroid glands, is divided (dashed line) from the posterior carcass.  For a more detailed view of this procedure see Figs. 86-87, which illustrate collection of the thyroid glands in the chick.
                                       
                                       
                                       
                                       S
S
G
G
P
P
T
T
                                     Fig 3
                                     Fig 3
            
            Figure 3.  The small, pink, triangular to ovoid, spleen (S) is identified proximate to the proventriculus (P), gizzard (G), and testis (T).  The fresh spleen is collected at this point because it can be difficult to locate after the viscera have been removed or following fixation.
            
                                     Fig 4
                                     Fig 4
            
            Figure 4.  Following removal of the bilobed liver (shown here in situ), several partial-thickness longitudinal incisions (dotted lines) are made to facilitate fixation.  
                                       
                                       
                                       
                                     Fig 5
                                     Fig 5
            
            Figure 5.  In this image the posterior end of the carcass is facing away from the viewer, the anterior end toward the viewer.  The gloved hand holds the rectum, as the viscera are retracted anteriorly and severed at the esophagus (dotted line).
            
            
                                     Fig 6
                                     Fig 6
             
            Figure 6.  The duodenal loop, which contains the pancreas, is identified.
                                       
                                       
                                       
                                       D
D
P
P
                                     Fig 7
                                     Fig 7
            
            Figure 7.  The excised duodenal loop (D) and pancreas (P).
            
            
                                     Fig 8
                                     Fig 8
            
            Figure 8.  As much as possible, the male reproductive tract, which includes the testis, epididymis, and ductus deferens is removed intact and interconnected.
                                       
                                       
                                       
                                       L
L
R
R
                                     Fig 9
                                     Fig 9
            
            Figure 9.  The excised right (R) and left (L) male reproductive tracts.  For identification purposes, the left reproductive tract can be loosely wrapped in wet gauze prior to placement in a container of fixative solution.
            
                                       D
D
E
E
T
T
                                    Fig 10
                                    Fig 10
            
            Figure 10.  Higher magnification of the male reproductive tract to illustrate the testis (T), epididymis (E), and ductus deferens (D).
            
                                    Fig 11
                                    Fig 11
V
V
U
U
M
M
In
In
O
O
Is
Is
            
            Figure 11.  Components of the female reproductive tract include the ovary (O), infundibulum (In), magnum (M), isthmus (I), uterus (U), and vagina (V).
            
            
            
                                    Fig 12
                                    Fig 12

            
            Figure 12.  To promote fixation, large areas of the oviduct and uterus are incised, but certain short segments destined for histopathologic evaluation are kept intact (arrows).
            
                                    Fig 13
                                    Fig 13
            
            Figure 13.  The legs are removed from the posterior carcass and cloacal region.
            
            
            
                                    Fig 14
                                    Fig 14
K
K
K
K
            
            Figure 14.  The posterior carcass segment and cloacal region following removal of the legs.  Scissors indicate the position of the cloacal bursa.  Also evident are the left and right kidneys (K).
            
                                    Fig 15
                                    Fig 15
j
j
d
d
i
i
h
h
g
g
f
f
e
e
a
a
c
c
b
b
      
      Figure 15.  Fixed tissue samples post-necropsy.  In this instance, the cloacal region was separated from the posterior carcass (the orientation of the cloacal region is more easily appreciated if kept connected to the posterior carcass).  KEY: a = skull; b = posterior carcass segment; c = liver (this particular liver was not sliced to facilitate fixation); d = male reproductive tract; e = anterior carcass segment; f = cloacal region; g = duodenal loop; h = ovary; i = heart; j = remainder of female reproductive tract.  The spleen, submitted in a lens paper envelope, is not pictured here. NOTE: THE HEART, AND BRAIN COMPONENTS INCLUDING THE PITUITARY GLAND, MEDIAL PREOPTIC NUCLEUS, AND PINEAL GLAND ARE NO LONGER REQUIRED TISSUES FOR THIS ASSAY.
                                       
                                    Fig 16
                                    Fig 16

      
      Figure 16.  The paired thyroid glands are located lateral to the esophagus, at a level that is just anterior to the tracheal bifurcation.  Two transverse cuts are made through the anterior carcass segment, above and below the thyroid glands, and the intervening slab is submitted for histology.
      
      
      
      
                                       Fig 17
                                    Fig 17
                                    Fig 18
                                    Fig 18

      Figures 17 and 18.  Two liver samples are obtained, one from each of the right and left major lobes along its long axis.  To obtain a sample, two transverse cuts are made from the diaphragmatic to the visceral surface, and the intervening slab is submitted for histology.
      
      
      
                                      in
in
m
m
is
is
u
u
v
v
o
o
                                    Fig 19
                                    Fig 19
      
      Figure 19.  The female reproductive tract.  Tubular portions have been opened adjacent to sampling sites to facilitate fixation.  KEY: o = ovary; in = infundibulum; m = magnum; is = isthmus; v = vagina.  Typically, only the left reproductive tract is present.  IF RESIDUAL PORTIONS OF THE RIGHT SIDE REPRODUCTIVE TRACT ARE FOUND, THEY SHOULD BE CLEARLY IDENTIFIED AND SUBMITTED FOR HISTOLOGY.
                                    Fig 23
                                    Fig 23
                                    Fig 22
                                    Fig 22
                                    Fig 21
                                    Fig 21
                                    Fig 20
                                    Fig 20
      
      Figures 20 through 23.  Trimming the female and male reproductive tract.  Fig. 20: A slab is trimmed through the central region of the ovary to include the hilus.  Larger yellow follicles often become detached during trimming and are therefore not submitted. If the trimmed ovary is still too large to fit into a single cassette, or if adequate fixation may be impeded by particularly large specimens (i.e., > 1 cm thick), the ovary may be trimmed further and split into two or more cassettes as needed.  Fig. 21: A transverse ring of tissue is trimmed from the uterus (shell gland).  Similarly, transverse rings are trimmed from each of the infundibulum, magnum, and isthmus regions of the oviduct.  Fig. 22: A transverse ring of tissue is trimmed from the vagina, the mucosal surface of which often becomes everted (protrudes externally) during fixation.  Fig. 23: A central slab is trimmed from each testis along its long axis.  The slab should include the epididymis and ductus deferens (submit these latter tissues for histology should they become detached).  Similar to the ovary, if the testis sections are too large to fit into standard cassettes they may be trimmed further, but the connection between the epididymis and the testis should be maintained if possible. THE RIGHT AND LEFT REPRODUCTIVE TRACTS SHOULD ALWAYS BE KEPT SEPARATE AND CLEARLY IDENTIFIED.


                                    Fig 24
                                    Fig 24
                                       
      
      Figure 24.  The pancreas is trimmed from within the duodenal loop.  It is embedded whole.
      
                                       
                                    Fig 25
                                    Fig 25
R
R

      
      Figure 25.  The cloacal bursa (bursa of Fabricius, arrow) is a pigmented conical structure that is located dorsal to the rectum (R).  Although the bursa can be excised at its base and embedded for transverse sectioning, it is usually easier to leave this structure in situ and thus include it in the clocal gland section (see Figs. 26-27).
      
                                       
                                    Fig 26
                                    Fig 26
                                    Fig 27
                                    Fig 27
         
      
      Figures 26 and 27.  Fig. 26 : Trimming of a midline sagittal slab from the cloacal region in order to obtain histologic sections of the cloacal glands and cloacal bursa.  Fig. 27: The slab specimen to be embedded.  The site of the cloacal glands is indicated by the arrows. 




                                    Fig 29
                                    Fig 29
                                    Fig 28
                                    Fig 28
MK
MK
CaK
CaK
CrK
CrK
    
      
      Figures 28 and 29.  Fig.  28: Trimming of the cranial kidney (CrK) slab.  This slab should also contain the paired adrenal glands.  Fig. 29: Trimming of the middle kidney (MK) and caudal kidney (CaK) slabs.
      

                                       
                                       o
o
n
n
m
m
l
l
k
k
j
j
i
i
h
h
g
g
f
f
e
e
d
d
c
c
a
a
b
b
                                    Fig 30
                                    Fig 30
      Figure 30.  Trimmed tissue samples pre-embedding.  KEY: a = cross section of brain/skull for medial preoptic nucleus and pituitary gland; b = sagittal section of brain/skull for pineal gland; c = liver; d = cranial kidney and adrenal glands; e = middle kidney and caudal kidney; f = heart; g = spleen and pancreas; h = bursa of Fabricius; i = anterior carcass for paired thyroid glands; j = cloacal region for cloacal glands; k =left testis, epididymis, and ductus deferens; l = right testis and epididymis (ductus deferens inadvertently not included); m = ovary; n = infundibulum and uterus; o = magnum, isthmus, and vagina.  NOTE: THE HEART, AND BRAIN COMPONENTS INCLUDING THE PITUITARY GLAND, MEDIAL PREOPTIC NUCLEUS, AND PINEAL GLAND ARE NO LONGER REQUIRED TISSUES FOR THIS ASSAY.  

      (3) Histologic Figures.

                                    Fig 34
                                    Fig 34
                                    Fig 32
                                    Fig 32
                                    Fig 31
                                    Fig 31
                                    Fig 33
                                    Fig 33
                                      BD
                                      BD
                                      PV
                                      PV
                                      HA
                                      HA
                                      CV
                                      CV
                                      PV
                                      PV
                                      HA
                                      HA
                                      BD
                                      BD
Figures 31-34.  Medium and high magnification views of liver from 6-week-old quail (male series on left, female on right).  Hepatocytes have variable degrees of lipid-type vacuolation (small, clear holes in the cytoplasm).  Compared to many mammalian livers, the portal areas (which consist of a portal vein [PV], a hepatic artery [HA], and bile ducts [BD]) have comparatively less fibrous connective tissue.  The livers of male and female quail appear essentially identical at this age.  CV = central vein.  Figs. 31 and 32, 
bar = 100 um; Figs. 33 and 34, bar = 25 um.
                                    Fig 35
                                    Fig 35
                                    ventrum
                                    ventrum
                                      AG
                                      AG
                                      AG
                                      AG
                                      CK
                                      CK
                                      CK
                                      CK
                                    dorsum
                                    dorsum
      Figure 35.  Cranial kidneys (CK) and adrenal glands (AG) from a 12-week-old male.  The reproductive tract was removed at gross trimming.  Bar = 800 um.
      
      
      
                                    Fig 36
                                    Fig 36
      Figure 36.  Medium magnification of cranial kidney (top) and adrenal gland (bottom) from a 12-week-old male.  Bar = 250 um.
                                    Fig 37
                                    Fig 37
                                       C
                                       C
                                       M
                                       M
                                       C
                                       C
                                       C
                                       C
                                       M
                                       M
                                       M
                                       M
      Figure 37.  High magnification of adrenal gland from a 12-week-old male.  Unlike mammalian adrenal glands, which have distinct outer cortical (steroidogenic) and inner medullary (adrenergic, chromaffin) regions, the avian cortical (C) tissue is intermingled with the medullary (M) tissue.  The relative proportions of cortical versus medullary tissue can be quite variable in different sections from the same bird or among sections from different birds.  Bar = 25 um.
      
      
      
                                       G
                                       G
                                       P
                                       P
                                       G
                                       G
                                       P
                                       P
                                       D
                                       D
                                       P
                                       P
                                    Fig 38
                                    Fig 38
                                       G
                                       G
      Figure 38.  High magnification of cranial kidney from a 12-week-old male.  Structures in this image of the cortical region include glomeruli (G), proximal convoluted tubules (P), and the far smaller distal convoluted tubules (D).  Bar = 25 um.
                                       S
                                       S
                                       N
                                       N
                                       S
                                       S
                                       K
                                       K
                                       K
                                       K
                                    dorsum
                                    dorsum
                                    Fig 39
                                    Fig 39
                                    ventrum
                                    ventrum
      Figure 39.  Transverse section through mid-kidney region from a 12-week old male.  
S = spinal cord, N = notochord, K = kidney. Bar = 800 um.
      
      
      
                                    ventrum
                                    ventrum
                                    Fig 40
                                    Fig 40
                                       S
                                       S
                                       K
                                       K
                                       K
                                       K
                                    dorsum
                                    dorsum
      Figure 40.  Transverse section through caudal kidney region from a 12-week-old male.  S = spinal cord, K = kidney. Bar = 800 um.


                                    Fig 41
                                    Fig 41
                                     S __
                                     S __
      Figure 41.  Pancreas from a 12-week-old male.  Coturnix spp. have two distinct types of islets of Langerhans, alpha and beta, and this image illustrates the very large, slightly basophilic alpha islets (arrows) that are often located in the splenic limb.  S = spleen.  
      Bar = 250 um. 
      
      
      
                                       B

                                       B

                                    Fig 42
                                    Fig 42
                                       E

                                       E

                                       A

                                       A

                                       E

                                       E

      Figure 42.  Pancreas from a 12-week-old male.  High magnification to illustrate alpha islet (A), beta islets (B), and exocrine (acinar) regions (E).  Bar = 50 um.
                                    Fig 43
                                    Fig 43
      Figure 43.  Spleen from a 12-week-old male.  In this particular case, lymphoid follicles (arrows) are few and sparsely populated.  Bar = 500 um.
      
      
      
                                       R
                                       R
                                    Fig 44
                                    Fig 44
                                       W
                                       W
      Figure 44.  Spleen from a 12-week-old male.  Indicated are the lymphoid tissues of the white pulp (W), the blood sinuses of the red pulp (R), and the pink, perivascular splenic ellipsoids (arrows).  Bar = 25 um.

                                    Fig 46
                                    Fig 46
                                    Fig 45
                                    Fig 45
                                    Fig 48
                                    Fig 48
                                    Fig 47
                                    Fig 47
Figures 45-48.  Bursa of Fabricius from four 12-week-old quail. Figs. 45 through 48 represent progressive grades of involution, from Grade 1 to Grade 4, respectively.  The hallmark of involution is lymphoid tissue depletion, and as this occurs the intervening connective tissue (fibrous tissue, adipose tissue) becomes relatively more prominent.  Bar = 250 um.


                                    ventrum
                                    ventrum
                                       T
                                       T
                                    dorsum
                                    dorsum
                                    Fig 49
                                    Fig 49
                                       E
                                       E
      Figure 49.  Transverse section through the anterior carcass segment from a 12-week-old male to evaluate the paired thyroid glands (arrows).  As evident here, the thyroid glands are more closely associated with the esophagus (E) than the trachea (T).  It may not always be possible to capture both the left and right thyroid glands in a single section.  
Bar = 800 um.
      
      
      
                                    Fig 50
                                    Fig 50
      Figure 50.  High magnification of thyroid gland from a 12-week-old male.  Variably-sized spherical or angular follicles are lined by a single layer of low cuboidal epithelium and are filled with homogeneous pink colloid.  Avian thyroid glands do not contain parafollicular "C" cells, but cells with similar function exist in the ultimobranchial bodies (not shown). 
Bar = 25 um.
                                    Fig 51
                                    Fig 51
                                      CG
                                      CG
                                       C
                                       C
                                       U
                                       U
                                       P
                                       P
                                       V
                                       V
                                      UG
                                      UG
Figure 51.  Cloacal region from a 6-week-old male to demonstrate cloacal glands (CG) which are located within the dorsal wall of the proctodeum (P).  UG = uropygial (preen) gland, V = vertebrae, C = coprodeum, U = urodeum.  Although not present in this particular section because it was excised, the cloacal bursa can also be found in this region (see Figs. 55 and 114).  Bar = 1000 um.


                                    Fig 55
                                    Fig 55
                                    Fig 53
                                    Fig 53
                                    Fig 52
                                    Fig 52
                                    Fig 54
                                    Fig 54
                                       B
                                       B
Figures 52-55.  The deeply basophilic cloacal glands from two 12-week-old males (top) and two 12-week-old females (bottom).  Figs. 52 through 55 represent progressive grades of cloacal gland development, from Grade 4 to Grade 1, respectively.  As evident here, both males and females possess these foam producing glands, but in adults the glands of males are typically more developed than those of females.  Cloacal gland development is androgen-dependent, and estrogens are inhibitory.  In Fig. 55, the bursa of Fabricius (B) is present.  Bar = 800 um.

                                    Fig 56
                                    Fig 56
L
L
GC
GC
Figure 56.  Higher magnification of cloacal glands (CG).  The cytoplasm of the tall columnar glandular cells (GC) is filled with basophilic mucinous material.  Because of the associated presence of lymphoid tissue (L), some texts refer to this anatomic site as the "lymphoglandular ridge".  Bar = 25 um.
                                       T
                                       T
                                       E
                                       E
                                       D
                                       D
                                    Fig 57
                                    Fig 57
      Figure 57.  Testis (T), epididymis (E), and ductus deferens (D) from an adult male.  If tissues are collected, embedded, and microtomed carefully, it is not extraordinarily difficult to capture all three of these structures in a single histologic section.  Bar = 800 um.
      
      
                                    Fig 61
                                    Fig 61
                                    Fig 60
                                    Fig 60
                                    Fig 59
                                    Fig 59
                                    Fig 58
                                    Fig 58
Figures 58-61.  Higher magnification of various areas of a single testis, this time from a 6-week-old male.  Unlike the testes of rats, for example, in which each cross-sectional profile of seminiferous tubule represents a different phase of development, a given quail tubule contains multiple (e.g., 7 or more) of 10 previously
defined developmental phases.  Thus, semi-quantitative staging of individual tubules, much less the entire testis, is not considered practicable.  Bar = 25 um. 
 


                                    Fig 62
                                    Fig 62
sg
sg
sc
sc
sc
sc
sc
sc
es
es
sc
sc
sg
sg
sc
sc
ls
ls
sz
sz
es
es
sg
sg
Figure 62.  Still higher magnification of testis from a 6-week-old male.  Various germ cell stages can be appreciated in this image, including spermatogonia (sg), primary and secondary spermatocytes (sc), early [round] spermatids (es), late [elongated] spermatids (ls), and spermatozoa (sz).  Note that in quail the nuclei of spermatogonia are actually smaller than the nuclei of spermatocytes.  Sertoli cells (arrows) can be difficult to distinguish from germ cells; Sertoli cells also have a distinct nucleolus, but they have less nuclear chromatin material and their cytoplasm is less obvious.  Additionally, the Sertoli cell nucleus often has a slightly angular profile as compared to the more rounded nuclei of germ cells.  Quail Sertoli cells are not always located immediately adjacent to the tubular basement membrane.  Leydig (interstitial) cells (arrowhead) are typically sparse in the coturnix testis.  Bar = 25 um.



PED
PED
                                    Fig 63
                                    Fig 63
C
C
PED
PED
DED
DED
PED
PED
PED
PED
PED
PED
DED
DED
DED
DED
CD
CD
C
C
RT
RT
RT
RT
RT
RT
ST
ST
TR
TR
Figure 63.  Epididymis from an adult male.  The epididymis runs from the cranial to the caudal pole of the testis, along the dorsomedial surface, and it receives spermatozoa from the testis along much of its length.  The sequence of sperm travel outward from the testis parenchyma is as follows: seminiferous tubule (ST)  tubulus rectus (TR)  rete testis (RT)  proximal efferent ductule (PED)  distal efferent ductule (DED)  collecting ductules (CD)  ductus epididymis  ductus deferens.  The abundant eosinophilic cytoplasm of the large, folded PED makes them readily distinguishable from the smaller, but more plentiful, profiles of the darker DED.  It is normal to observe large amounts of exfoliated cellular debris in the lumen of the RT and PED; however, cellular debris is typically only sparse downstream in the DED, CD, epididymal duct and ductus deferens.  Bar = 100 um.



                                    Fig 68
                                    Fig 68
                                    Fig 64
                                    Fig 64
                                    Fig 65
                                    Fig 65
                                    Fig 69
                                    Fig 69
                                    Fig 66
                                    Fig 66
                                    Fig 67
                                    Fig 67
G
G
TE
TE
TI
TI
V
V
Y
Y
N
N
Figures 64-69.  Sections of ovary from several 6-week-old females, with follicles in various phases of development.  Nuclei (germinal vesicles, N) are evident in some follicles due to the plane of sectioning.  Other follicular structures that are evident in the late stage follicle in Fig. 69 include the yolk cytoplasm (Y), vitellogenic membrane (V), granulosa cell layer (G), theca interna (TI) and theca externa (TE).  Note persistence of the nucleus (N) in the smaller early stage follicles.  Bar = 800 um (Figs. 64 to 66), 250 um (Fig. 67), 100 um (Fig. 68), and 50 um (Fig. 69).

late
POF
late
POF
early
POF
early
POF
                                    Fig 70
                                    Fig 70
Figure 70.  Post-ovulatory follicles (POF) in the ovary of an adult female.  Of the two POFs in this image (arrows), the incomplete one on the left is a remnant of the more recent ovulation.  In birds, the term post-ovulatory follicle is preferred over the corpus luteum, the latter of which refers to a post-ovulatory structure in the mammalian ovary.  There may be some functional similarities between the POF and the corpus luteum in terms of steroid hormone production.  Bar = 500 um. 


                                    Fig 72
                                    Fig 72
                                    Fig 71
                                    Fig 71
  Figures 71 and 72.  Higher magnification of early and late post-ovulatory follicles, respectively.  The major difference between these two structures is the extensive proliferation of granulosa cells in the late POF (for a more detailed view, see the next two figures).  Bar = 100 um.


TE
TE
TI
TI
GC
GC
TE
TE
GC
GC
TI
TI
                                    Fig 74
                                    Fig 74
                                    Fig 73
                                    Fig 73
 Figures 73 and 74.  Still higher magnification early and late post-ovulatory follicles, respectively, to better appreciate the different cell layers: GC = granulosa cells, TI = theca interna, TE = theca externa.  
Bar = 25 um.
S
S
M
M
S
S
                                    Fig 80
                                    Fig 80
                                    Fig 79
                                    Fig 79
                                    Fig 78
                                    Fig 78
                                    Fig 77
                                    Fig 77
                                    Fig 76
                                    Fig 76
                                    Fig 75
                                    Fig 75
Figures 75-80.  Several regions of proximal oviduct from adult quail.  Figs. 75 and 76 are low and high magnification views, respectively, of transverse sections of infundibulum, which has a highly folded, densely basophilic mucosal epithelium.  Figs. 77 and 78 represent a transverse section of the larger diameter magnum, in which the glands have decreased pink secretory product (S) due to the recent passage of an egg.  Figs. 79 and 80 are from a section of magnum in which the glands are filled with secretory material (S) prior to egg passage.  The mucus cell lining (M) of the magnum is also more prominent prior to egg passage.  Bar = 250 um (Fig. 75), 25 um (Figs 76, 78 and 80), and 500 um (Figs. 77 and 79).

                                    Fig 82
                                    Fig 82
                                    Fig 81
                                    Fig 81
                                    Fig 86
                                    Fig 86
                                    Fig 85
                                    Fig 85
                                    Fig 84
                                    Fig 84
                                    Fig 83
                                    Fig 83
Figures 81-86.  Regions of distal oviduct.  Figs. 81 and 82 are low and high magnification views of transverse sections of isthmus.  The amount of secretory product is highly variable in this segment.  The isthmus mucosal lining is more folded than that of the magnum, but the major difference between these two segments is that the mucosal lining of the isthmus lacks mucous cells.  The shell gland (uterus) (Figs. 83 and 84) has more tertiary folding than the previous segments, and the complete absence of pink secretory material in the mucosal glands distinguishes this shell gland from the isthmus.  Mucosal glands are completely absent in the vagina (Figs. 85 and 86).  Bar = 500 um (Figs. 81 and 83), 25 um (Figs 82, 84 and 86), and 250 um (Fig. 85).

(c)  Pathologic Evaluation.

      (1)  General Approach to Pathologic Evaluations.  Studies are to be read by individuals experienced in reading toxicologic pathology studies, and who are familiar with normal avian thyroid and gonad histology, physiology, and general responses of these organs to toxicologic insult.  Pathologists may be board certified (e.g. American College of Veterinary Pathologists, The European Centre of Toxicologic Pathology, or other certifying organizations); however, certification is not a requirement as long as the pathologist has obtained sufficient experience with, and knowledge of, avian histology and toxicologic pathology.  Technicians should not be used to conduct readings due to the subtle nature of some changes and the need for subjective judgments based on past experience.

It is recognized that there is a limited pool of pathologists with the necessary training and experience that are available to read the histopathology endpoint for the JQTT assay.  If an individual has toxicological pathology experience and is familiar with thyroid, liver, and gonadal histology in avian species, he/she may be trained to read the JQTT assay.  If pathologists with little experience are used to conduct the histopathological analysis, informal peer review may be necessary.

Pathologists are to read these studies unblinded (i.e., without knowledge of the treatment group status of individual frogs).  This is because endocrinological effects on histomorphology tend to be incremental, and subtle differences between exposed and unexposed animals may not be recognizable unless tissue sections from high dose animals can be knowingly compared to those from controls.  Thus the aim of the initial evaluation is to ensure that diagnoses are not missed (i.e., to avoid false-negative results).  On the other hand, it is expected that all potential treatment-related findings will be re-evaluated by the pathologist in a blinded manner, in order to prevent the reporting of false-positive results.  As a rule, treatment groups should be evaluated in the following order: Control, High-dose, Mid-dose, and Low-dose.

Pathologists should specifically evaluate the target tissues identified in the guidelines; however, changes observed in other tissue types may also be recorded.  This especially pertains to findings suspected to be treatment-related, or findings that might otherwise impact the study results (e.g., systemic inflammation or neoplasia). 

It is suggested that the pathologist be provided with all available information related to the study prior to conducting their readings.  Information regarding gross morphologic abnormalities, mortality rates, and general test population performance and health are useful for pathologists to provide comprehensive reports and to aid in the interpretation of findings.  For a more comprehensive discussion of standard reading approaches for toxicologic pathology studies, please refer to the Society of Toxicologic Pathology Best Practices for reading toxicologic histopathology studies (Crissman et al., 2004).

      (2)  Severity Grading.  In toxicologic pathology, it is recognized that compounds may exert subtle effects on tissues that are not adequately represented by simple binary (positive or negative) responses.  Severity grading involves a semi-quantitative estimation of the degree to which a particular histomorphologic change is present in a tissue section (Shackelford et al., 2002).  The purpose of severity grading is to provide an efficient, semi-objective mechanism for comparing changes (including potential compound-related effects) among animals, treatment groups, and studies.

Severity grading should usually use the following system: 
   * NR (not remarkable)
   * Grade 1 (minimal)
   * Grade 2 (mild)
   * Grade 3 (moderate)
   * Grade 4 (severe)
   
Findings that are not present are not graded (i.e., there is no Grade 0); however, if there are no findings for an entire tissue section, that section is scored as NR (not remarkable).  

A grading system needs to be flexible enough to encompass a variety of different tissue changes.  In theory, there are three broad categories of changes based on the intuitive manner in which people tend to quantify observations in tissue sections:

                     *       Discrete: these are changes that could be readily counted.  Examples include atretic follicles, oocytes in the testis, and clusters of apoptotic cells.
   
                     *       Spatial: these are changes that could be quantified by area measurements.  Includes lesions that are typically classified as focal, multifocal, coalescing, or diffuse.  Specific examples include granulomatous inflammation and tissue necrosis.
   
                     *       Global: these are generalized changes that would usually require more sophisticated measurement techniques for quantification.  Examples include increased hepatocyte basophilia, thyroid follicular cell hypertrophy, or quantitative alterations in cell populations.

Listed below are general guidelines for the use of a severity grading system, with examples of how the system could be applied to each of the above categories.  Please understand that the terms Discrete, Spatial, and Global are used for illustrative purposes only; it is not intended that these terms be incorporated into any diagnosis or grade.  It should be stressed that the examples below should be modified as needed for each particular type of change (diagnosis).  
      
Grade 1:
         *       Discrete change example: 0 to 2 occurrences per microscopic field, or 1 to 2 occurrences per tissue section.
   
         *       Spatial change example: the change occupies a miniscule area of either a specific tissue type or the entire tissue section.
   
         *       Global change example: the least perceptible alteration relative to control animals or prior experience.
   
Grade 2:
         *       Discrete change example: 3 to 5 occurrences per microscopic field or tissue section.
   
         *       Spatial change example: the change occupies a larger area than Grade 1, but still less than or equal to 25% of either a specific tissue type or the tissue section.
   
         *       Global change example: the alteration is easily appreciated, but still not dramatic.
   
Grade 3:
         *       Discrete change example: 6 to 8 occurrences per microscopic field or tissue section.
   
         *       Spatial change example: the change occupies more than 25% but less than or equal to 50% of either a specific tissue type or the entire tissue section.
   
         *       Global change example: the alteration is dramatic, but a more pronounced alteration can be envisioned.
   
Grade 4:
         *       Discrete change example: 9 or more occurrences per microscopic field or tissue section.
   
         *       Spatial change example: the change occupies more than 50% of either a specific tissue type or the entire tissue section.
   
         *       Global change example: essentially, the most pronounced imaginable alteration.
      
At least some of the histomorphologic changes that have been associated with EDCs in wildlife are considered to be exacerbations of "normal", physiologic findings.  Whenever possible, the severity of a given change should be scored relative to the severity of the same change in concurrent control animals.  For each important (i.e., treatment-associated) finding, the severity scoring criteria should be stated in the Materials and Methods section of the pathology narrative report.  By convention, it is recommended that severity grading should not be influenced by the estimated physiologic importance of the change.  For example, the presence of two oocytes in the testis should not be graded as "severe", even if the pathologist considers this finding to be highly significant in terms of endocrine modulation.  The reason is that estimating the physiologic importance adds a further layer of subjectivity to the findings that complicates interlaboratory results comparisons.

      (3)  Data Recording.  The pathologist records the results on a spreadsheet template provided by EPA.  For each frog, the pathologist records the presence of a diagnosis by indicating the severity grade.  In rare instances (e.g., tumor diagnoses), severity grading may not be applicable.  If there are no findings for a particular frog, this should be recorded specifically.  It is also important to record a notation if the target tissue is missing or if the amount of tissue present is insufficient to make a diagnosis.  Adding modifiers to a diagnosis may help to further describe or categorize a finding in terms of chronicity, spatial distribution, color, etc.  In many instances, modifiers are superfluous or redundant (e.g., fibrosis is always chronic); therefore, the use of modifiers should be kept to a minimum.  An occasionally important modifier for evaluating paired organs is unilateral; unless specified in this manner, all diagnoses for paired organs are assumed to be bilateral.  Other modifiers can be created sparingly as needed by the pathologist.

      (4)  Statistical Analysis.  Histopathology data are analyzed using a recently described method, the Rao-Scott Cochran-Armitage by Slices, or RSCABS (Green et al., 2014).  RSCABS is implemented through the use of an SAS- or R-based software package called StatCHARRMS.  Advantages of using RSCABS as a statistical method for analyzing histopathology data include the ability to account for: 1) experimental designs with multiple replicates, 2) lesion severity scores of individual animals in addition to group-wise lesion prevalence, and 3) dose-response relationships.  Additionally, the RSCABS test is easy to perform and interpret.

      (5)  Data Interpretation.  Once the microscopic examinations have been completed and statistical analyses have been performed on the resulting data, the pathologist interprets the histopathologic findings.  The initial task is to determine which, if any, of the recorded findings are related to administration of the test article, and which are not.  The goal is to classify each type of recorded finding (i.e., diagnosis) into one of three categories: 1) Treatment-related, 2) Potentially treatment-related, and 3) Non-treatment-related.  Criteria for these determinations are listed below.  
      
      (i)  Determining Relationship to Treatment.  A weight-of-evidence (WOE) approach is used to determine if a particular finding should be considered treatment-related.  Such evidence may include any or all of the following as available:

         *       Differences between groups of control and treated animals in terms of lesion prevalence and severity, utilizing statistical analytical results to test for significance as warranted.
   
         *       Ancillary data from the current study, involving information such as behavioral observations, liver and body weights, genotypic sex, time-to-metamorphosis, and age at sacrifice.
   
         *       Results from other submitted or pending agency studies.
   
         *       The at-large scientific literature, giving greater weight to studies in which the quality of the research can be established and is considered superior.
   
         *       Overall biological, physiological, and toxicological plausibility.

Findings that are considered potentially treatment-related may be those that have borderline statistical significance, or those in which the relationship to treatment is considered equivocal for other reasons (e.g., lack of corroborating evidence from other sacrifices or other studies, biological or toxicological implausibility, or commonality of the diagnosis as a background finding).

There are several points to be made regarding the determination of treatment-relatedness. First, it is possible for a finding to be treatment-related but not be caused by the test article.  This can include situations in which group-wise differences may be associated with an uncontrolled (and possibly unrecognized) variable involving conduct of the in-life assay, specimen preparation, or some other non-systemic bias.  Second, not all statistically significant differences are real, as a p-value significance level of 0.05 allows for the probability that in 5% of cases the result occurred by chance.  Third, a finding may be statistically significant and not necessarily biologically or toxicologically important.  Fourth, in some instances, treatment-related findings may not be statistically significant.  For example, this can occur when treatment induces a low frequency of a lesion type that rarely occurs spontaneously.

      (ii)  Determining Relationship to Endocrine Disruption.  A similar weight-of-evidence (WOE) approach can be used to determine if a particular finding is likely to be endocrine-related; however, in this case the WOE will more heavily depend on ancillary data, results of other assays, and the published literature, including mechanistic studies where available.

      (6)  Report Format.  The pathologist is responsible for deliverables that include: 1) Pathology Narrative Report, 2) Spreadsheet with recorded data, and 3) TIFF image files of figures.

      (i)  Pathology Narrative.  Each histopathology narrative report should contain at least the first five of the following sections: Introduction, Materials and Methods, Results, Discussion, Summary/Conclusions, References, Tables, and Figures.  The Introduction section briefly outlines the experimental design.  The Materials and Methods section briefly describes procedures used during the slide preparation and examination phases of the study.  If specific severity grading criteria were created for a particular finding, they should also be listed in this section.  The Results section should report findings that are: 1) treatment-related; 2) potentially treatment-related; 3) non-treatment-related findings that are novel or unusual.  Detailed histomorphologic descriptions need only be included for findings that differ substantially from diagnoses presented the Histopathology Atlas.  It is intended that the Results section should be as objective as possible (i.e., opinions and theories should be reserved for the Discussion section).  The Discussion section, which contains subjective information, should address relevant findings that were reported in the Results section.  Opinions and theories can be included in this section, preferably backed by references from peer-reviewed sources, but unsupported speculation should be avoided.  The Summary/Conclusions section should encapsulate the most important information from the Results and Discussion sections.  The References section should include only material that is cited specifically in the narrative report.  A separate Tables section may not be necessary if tables are embedded in the Results section.  The Figures section should include photomicrographic examples of treatment-related findings, plus unusual or noteworthy lesions.  The Figures section should include normal tissues for comparison, and digital images should be taken at magnifications that clearly illustrate the salient features of the findings.  Figures embedded in the narrative should be in a universally readable compressed file format such as JPEG.

      (ii)  Spreadsheet.  In addition to the recorded histopathology findings, the completed spreadsheet should indicate the animals from which figure images were photographed, and the number of images obtained per photographed fish.

      (iii)  Figures.  A complete set of unembedded and unannotated photomicrographic figures should be submitted electronically on portable media as uncompressed TIFF files.

      (7)  Pathology Peer Review.  Following the initial slide evaluation and creation of a draft report by the study pathologist (SP), it is encouraged that at least a subset of the original histologic sections be assessed by a second reviewing pathologist (RP).  Known as pathology peer review, the purpose of this exercise is to increase confidence in the histopathology data by ensuring diagnostic accuracy and consistency.  Commonly, this procedure involves the targeted examination of one or more tissue tissue types in which treatment-related findings were initially detected (this helps to guard against false positive results), plus all tissues from a randomly selected percentage (e.g., 10-20%) of animals from the control and high-dose groups (this helps guard against false negatives).  The RP is tasked with determining the accuracy and consistency of diagnostic criteria, diagnostic terminology, severity grading, and the interpretation of findings.  The peer review can be performed in-house or (preferably) by an external pathologist, and frequently the reviewing pathologist has at least equal or greater expertise than the SP.  Following the peer review, the SP and RP typically meet to resolve diagnostic differences.  In unusual cases in which such differences cannot be resolved, a panel of experts (Pathology Working Group) may be convened to determine the final diagnoses.  In addition to enhancing confidence in the histopathology results, benefits of peer review may include decreased inter-laboratory variability, and cross-training of pathologists (i.e., the initial study pathologist may not always need to be an avian expert).  Recommended procedures for conducting pathology peer reviews have been described elsewhere (Morton et al., 2010; The Society of Toxicologic Pathologists, 1991; 1997).

(8)  Interpretation of Histopathologic Findings.
(I)  THYROID GLANDS.  The paired thyroid glands should be evaluated for potential changes in: 1) overall thyroid size; 2) the overall size and shape of follicles; 3) the overall size and relative number of thyroid follicular epithelial cells; and 4) the relative quantity and quality of colloid.  Pericapsular, subcapsular, and/or parenchymal lymphocytic infiltrates may be present to varying degrees, and in some cases, these infiltrates may contain epithelioid cells and keratin-like material, which suggests that these infiltrates represent congenitally-displaced thymic tissue.  Portions of thymus and/or parathyroid gland may also be found adjacent to one or both thyroid glands.  Because they are considered constituent, it is not necessary to record the presence of these lymphoid tissues as a finding, unless the pathologist happens to notice obvious group-wise differences between treated and control birds in the prevalence or amount of lymphoid tissue.  It is preferable to assess changes of thyroid gland follicular size/height and thyroid gland follicular cell number independently.  Preferred terms for increases in these parameters are "thyroid gland follicular epithelium, increased cell size" and "thyroid gland, follicular epithelium, increased cell number", respectively.  When in doubt, it is often better to use descriptive terminology as opposed to diagnoses that may have presumptive mechanistic connotations.  For example, if, in a particular study, the thyroid glands appear to be smaller on average than those of most untreated controls, the preferred diagnostic term would be "thyroid gland, decreased size" rather than "thyroid gland atrophy" or "thyroid gland hypoplasia".   

      (ii)  Liver.  All livers should be assessed for the overall amount of hepatocellular cytoplasmic vacuolation, the morphologic appearance of which is usually consistent with lipid droplets (clear round vacuoles, with nuclear displacement if vacuoles are very large) but may be more consistent with glycogen in some cases (slightly angular vacuoles with a flocculent appearance).  Cytoplasmic vacuolation may occur diffusely and/or focally throughout the liver, and the distribution and pattern (e.g., centrilobular, periportal, random, etc.) of vacuolation may be important in some cases.  It should be noted that degrees of vacuolation that are considered "normal" in some studies may be abnormal in other studies if it can be demonstrated that there are relative differences in the degree, type or distribution of vacuolation in treated quail as compared to controls.  Common findings in the liver that may be either incidental or treatment-related include focal areas of hepatocellular necrosis, focal inflammatory cell infiltrates with or without necrosis (`focal granuloma"), and peribiliary inflammation.

      (iii)  Adrenal Glands.  Other than the presence of occasional inflammatory cell foci, adrenal gland lesions are unlikely to be common in most endocrine disruptor studies.  However, attention should be paid to the relative proportion of cortical to medullary tissue, although this can be difficult to assess, as it tends to vary from animal to animal, and also among different levels of tissue sectioning. 

      (iv)  Kidneys.  Findings that are common in the cranial, middle, or caudal kidneys include tubular dilation, tubular casts, and small foci of mononuclear cell infiltrates.  The presence of mesonephric kidney remnants in adults should be documented.

      (v)  Pancreas.  The exocrine pancreas (acinar cells) should be examined for zymogen granule depletion, and potential proliferative effects (e.g., acinar cell hypertrophy, foci of acinar cell alteration).  The relative abundance of endocrine tissue should be assessed for the two different types of islets of Langerhans, alpha and beta, which are normally present in the quail pancreas.

      (vi)  Spleen.  Endocrine-related lesions probably occur infrequently in the spleen; however, it is conceivable that some administered compounds may additionally have immunotoxic effects.  Consequently, it is important to evaluate the relative abundance of lymphoid tissue, look for evidence of lymphoid necrosis, and pay attention to the prominence of the periarteriolar sheaths.

      (vii)  Cloacal Bursa (Bursa of Fabricius).  Like the spleen, the cloacal bursa should be examined for potential immunotoxic effects.  Additionally, the degree of bursal involution should be scored for adult birds.

      (viii)  Cloacal Glands.  Cloacal gland development is known to be androgen dependent, and it has been demonstrated that administration of estrogenic substances can inhibit such development.  Therefore, the degree of cloacal gland development should be scored for all adult birds.  Estrogen-treated adult males might be expected to have decreased cloacal gland development, whereas the opposite effect may occur in females exposed to an androgenic compound.

      (ix)  Testis.  Feminization of the testes is a signature effect of estrogenic substance administration in male quail.  Specific morphologic effects include flattening of the testes, predominance of the cortex over the medulla, regression of spermatogenic tissue, the presence of oogenic tissue in the testes, a decrease in the size of the right testis, and possibly an increase in the size of the left testis.  Changes that are present in a given study are likely to depend on: 1) the precise mode(s) of action of the test article; 2) the relative potency of the test article; 3) the timing and duration of test article administration; and 4) the age at which the birds are sacrificed.  Although staging of the testis is not likely to be a practical or useful exercise in quail, the seminiferous tubules of adult males should be examined to determine if one or more spermatogenic cell types (e.g., spermatogonia, spermatocytes, spermatids, spermatozoa) are consistently absent in a particular testis section.

      (x)  Epididymis and Ductus Deferens.  Historically, endocrine-related findings have been reported less frequently in the epididymis and ductus deferens as compared to the testis.  However, documented effects of estrogenic substances generally manifest as attenuated development or degeneration of these structures.  It should be recognized that that subtle degenerative or necrotic changes in the testis may be most reliably visualized downstream as increased intraluminal cells and cellular debris in these ducts.  But because a certain amount of cellular debris is almost always present in the rete testis and proximal efferent ductule of the epididymis, care should be taken not to interpret this as a pathologic finding.  

      (xi)  Ovary.  To date there have been surprisingly few reported effects of endocrine-active compounds on the quail ovary.  This is likely due to the fact that most of the studied compounds have had estrogenic activity.  Reported effects of estrogenic substance administration include intraperitoneal ovulations and the production of eggs that lack yolks.

      (xii)  Oviduct, Shell Gland (Uterus), and Vagina.  Persistence of the right oviduct (Müllerian duct) is a signature effect of estrogenic substance administration in female quail.  This may be accompanied by a concomitant decrease in left oviduct development, and oviduct developmental abnormalities such as cyst formation.  Decreased density of the tubular glands is a reported estrogenic effect in the shell gland.

      
             (9)  Atlas of Histopathologic Findings

                                       

Figure 87.  A and B:  Normal adrenal gland.  C and D:  Adrenal gland with increased cortical vacuolation.  Vacuolation is due to the presence of fine lipid droplets within the cytoplasm.  Typically, there is substantial inter-animal variability in the amount of vacuolation among the adrenal glands of control birds, and a relationship to treatment has not yet been well established.  Bar = 100 um (A and C), 25 um (B and D).  

                                       
                                       

Figure 88.  Normal thyroid (A) and decreased follicle size, grade 1 (B) in adult quail.  Normal thyroid (C) and increased follicle size, grade 3 (D) in adult quail.  Increased follicle size is accompanied by attenuation of the typically cuboidal follicular epithelium.  Follicle sizes in compound treated birds are assessed relative to concurrent controls.  Bar = 50 um (A and B), 25 um (C and D).  

                                       
                                       
Figure 89.  Grading thyroid gland, follicular epithelium, increased follicular cell size and increased follicular cell number in quail embryos.  In this case, affected birds were exposed to sodium perchlorate, which inhibits iodine uptake.  Increased follicular cell size is most often characterized by increased cell height.  Moderate to severe increases in follicular cell number may be accompanied by infolding of the epithelium.  These changes are consistent with elevated thyroid stimulating hormone activity, which also tends to cause follicle sizes to decrease.  It is preferable to grade changes in cell size and number independently.  Bar = 25 um.

                                       

Figure 90.  Grading thyroid gland, arteriolar hypertrophy in adult quail.  Perchlorate exposure was associated with an unusual dose-responsive expansion of the tunica media, and tunica intima to a lesser extent, of small arterioles.  The increased number of arteriolar profiles (arrows) as compared to controls may be due to the increased prominence and tortuosity displayed by these vessels.  Bar = 25 um.
                                       

Figure 91.  Mononuclear cell infiltrates (thymic lymphoid tissue) in the thyroid gland versus thyroiditis.  A and B: It is common for quail thyroid glands to contain discrete, well-circumscribed aggregates of small lymphocytes (arrow), which are usually located in the peri- or intra-capsular region.  Such lymphoid tissues may additionally contain epithelial cells and/or cystic structures, and the composition and positioning of such tissue suggests that they probably represent foci of misplaced thymic tissue.  The presence or absence of thymic tissue in a given section is likely dependent on individual animal variability and chance encounter during microtomy.  Because they are considered constituent, it is not necessary to record the presence of these infiltrates as a finding, unless the pathologist happens to notice obvious group-wise differences between treated and control birds in the prevalence or amount of lymphoid tissue.  Bar = 250 um (A), 25 um (B).  C and D: In this particular case, the additional presence of numerous granulocytes (arrows) is consistent with an inflammatory process, the cause of which was undetermined.  Bar = 100 um (C), 25 um (D).

                                       

Figure 92.  The pathologist should recognize that the thyroid glands of control quail may vary in appearance from animal to animal, and that variation can occur even among different areas within a single gland.  Furthermore, artifacts may be caused by histologic processing, and the normal three-dimensional follicular architecture may mimic lesions when viewed in two-dimensional histologic sections.  Various artifacts in this particular section of normal thyroid gland include colloid that appears hypereosinophilic (caused by curling of colloid in the section), colloid "dropout" (loss of tissue during microtomy), and the apparent "piling up" (arrows) of follicular epithelium (follicles sectioned tangentially).  Bar = 50 um.

                                       

Figure 93.  Accessory parathyroid tissue.  A: Irregular accumulation of glandular tissue is evident near the right thyroid gland (t).  This tissue does not entirely resemble the normal quail parathyroid gland, which is typically a discrete, encapsulated structure (see Fig. 97).  Bar = 250 um.  B: Higher magnification of parathyroid tissue.  This was observed as an incidental finding in a single bird.  Bar = 50 um.

                                       

Figure 94.  Hepatocellular vacuolation.  A: Liver with no vacuolation.  B: Liver with mild vacuolation from a comparably-aged female control quail from the same study as A.  The clear, round appearance of these vacuoles suggests that they likely contained lipid in vivo.  These images are included to demonstrate the variability in hepatocyte vacuolation that can occur without treatment.  Despite this variability, livers should be assessed for group-wise differences in the degree of hepatocellular vacuolation.  C and D: Micro and macrovesicular vacuolation in the liver of an adult female quail treated with 30 ppm endosulfan.  The distribution of the vacuolization is distinctly perivascular.  As compared to the macrovesicular type, the presence of microvesicular vacuolization is thought to indicate a more rapid course of lipid accumulation.  This lesion, which was thought to be treatment-related, was also found to a relatively minor degree in untreated quail.  Bar = 25 um (A, B, and D), 250 um (C).



                                       

Figure 95.  Grading of hepatocellular vacuolation.  Grade 1 encompasses zero to occasional small cytoplasmic vacuoles, whereas the vast majority of hepatocytes have large cytoplasmic vacuoles in a Grade 4 liver.  Bar = 25 um (Grades 1 and 2), 250 um (Grades 3 and 4).
 
                                       

Figure 96.  A:  Hepatic telangiectasis in the liver of an adult female quail treated with 250 ppm endosulfan.  This lesion is characterized by randomly distributed, patchy areas of sinusoidal capillary dilation.  Bar = 500 um.  B: Higher magnification of telangiectasis lesions from A.  Although there is a superficial resemblance to tumors of endothelial cell origin, the lack of cellular atypia is consistent with a non-neoplastic diagnosis.  A similar lesion may also be produced as an artifact if the liver is squeezed between two fingers inadvertently during post-mortem collection.  Bar = 25 um.

                                       

Figure 97.  Hepatic telangiectasis, grading. Grade 1 is typically one small focus of telangiectasis, as opposed to Grade 4, in which the vast majority of the liver section is affected.  Bar = 100 um (Grades 1 through 4).





Figure 98.  Necrosis and inflammation in the livers of adult quail.  These represent a spectrum of changes that are likely related, although the cause is unknown.  All may be observed at a low degree of severity in untreated control birds; however, they may also be exacerbated by toxic or infectious causes.  A: Individual hepatocyte necrosis (arrow).  This is a subtle lesion that can easily be missed.  B: Focal liver necrosis (arrow).  This particular example is an acute lesion; with time, it will induce at least a minor inflammatory response.  C and D: Focal granuloma.  Focal granuloma (not to be confused with granulomatous inflammation) is a term used to indicate small nodular lesions that consist primarily of mononuclear cell infiltrates (most of which are lymphocytes).  The lesions may also contain small numbers of granulocytes and necrotic hepatocytes.  Focal granulomas are often observed as incidental findings in the livers of quail and almost every other vertebrate species that have been evaluated in toxicological studies, and they may represent sequelae to individual hepatocyte necrosis or focal liver necrosis.  All figures, bar = 25 um.
                                       

Figure 99.  A: Focal necrosis with secondary mineralization in the liver of a hatchling female that had been treated with 125 ppm endosulfan.  Bar = 100 um.  B: Higher magnification of the liver from the preceding figure.  Mineralization is evident as dense dark purple deposits.  Bar = 25 um.



Figure 100.  Liver necrosis, grading.  Grade 1 is typically represented by one to a few individual necrotic hepatocytes.  In Grade 4 (no representative image yet), the vast majority of the liver section would be necrotic. Bar = 25 um (Grade 1), 100 um (Grade 2).




Figure 101.  Bile pigment accumulation in the liver.  Most of the pigment appears to be within the distended cytoplasm of hepatocytes.  Special stains may be required to confirm that the pigment is actually bile, versus cell breakdown products such as lipofuscin, ceroid, or hemosiderin.  Bar = 25 um.


Figure 102.  A: Eosinophilic focus of cellular alteration (arrows) in the liver of an adult female quail.  This lesion is characterized by distinct, localized change in the size (usually enlarged with eosinophilic foci) and coloration of hepatocytes.  Although not considered tumors per se, these lesions may be precursors of primary hepatocellular neoplasms.  Bar = 50 um.  B: Higher magnification of focus from preceding figure.  The lack of a sharp margin, peripheral tissue compression, and cellular atypia are consistent with a diagnosis of altered focus rather than hepatocellular adenoma.  Bar = 25 um.



Figure 103.  Metanephric kidney from a 2-week-old control male.  The presence of immature (embryonic) nephrons in the subcapsular region (arrows) is not unusual at this age and should not be confused with neoplastic transformation.  Bar = 50 um.



Figure 104.  Mature versus immature testes in adult male quail.  As compared to the mature testis (A and B), the germinal epithelium of immature testes (C and D) contains fewer numbers of the more mature germ cell types (especially spermatids and spermatozoa), and there may be a slight increase in exfoliated cells and intraluminal cellular debris.  The animal represented by C and D was from a group of males in which the testes were otherwise unaffected, and the reason for the relative immaturity of the testis in this particular bird was undetermined.  Differentiating immature testes from testicular degeneration with decreased sperm formation can be challenging.  In the latter case, the lesions are more likely to be non-uniform throughout the two testes, the testis may have a more "moth-eaten" appearance, and there will usually be more degenerating cells (i.e., apoptotic cells and germ cell syncytia).  Bar = 100 um (A and C), Bar = 25 um (B and D).





Figure 105.  Testis immaturity, grading.  Grade 1 immaturity is characterized by the presence of few elongating spermatids and spermatozoa relative to mature testes.  In Grade 2, no elongating spermatids or spermatozoa are evident.  In Grade 3 (image not yet available), the testes would consist predominantly of dividing spermatogonia and few spermatocytes, whereas the germ cell component of Grade 4 testes consists almost entirely of resting spermatogonia.  Bar = 100 um (Grades 1 and 2), 25 um (Grade 4).






Figure 106.  Testicular degeneration, grading.  Features of degeneration vary and may include: detachment of spermatozoa and spermatids; loss of specific spermatogenic cell types (the most mature cells are frequently affected first), presence of apoptotic cells, spermatid giant cells, and/or cellular debris in tubular lumina; and loss and/or displacement of Sertoli cells.  Severe degeneration is often characterized by the presence of atrophic tubules that are lined solely by Sertoli cells.  In the examples above, Grade 2 may appear more severely affected than Grade 3, but the degenerative changes were more localized in the Grade 2 testis.  Bar = 250 um (Grades 1-3).




Figure 107.  A and B: Inflammation of the testis (orchitis).  In this case, granulocytic and granulomatous inflammation (gi) occurred in response to a bacterial infection of the testis.  The inflammatory lesion is surrounded by an eosinophilic ring of Splendore-Hoeppli material (sh); consisting of antigen-antibody complexes, fibrin, and tissue debris) and necrotic testicular tissue (nt).  In the higher magnification view, the arrows indicate multinucleated giant cell macrophages.  Bar = 250 um (A), 50 um (B).



Figure 108.  Vacuolation of the epididymal duct epithelium.  Dose-responsive vacuolation of the proximal efferent duct epithelium occurred in adult quail exposed to vinclozolin.  A and B: Normal epididymis, p = proximal efferent duct.  C and D: Severe vacuolation of the proximal efferent duct (p) epithelium.  Bar = 250 um (A and C), 100 um (B and D).



Figure 109.  Grading epididymal duct vacuolation.  Bar = 25 um.

Figure 110.  Dilation of the proximal efferent ducts.  This occurred as a dose-responsive finding in adult quail exposed to vinclozolin.  A and B: Normal epididymis, p = proximal efferent duct. Note the convoluted folding of the ductular epithelium (arrow).  C and D: Severe dilation of the proximal efferent duct (p).  Here the ductular epithelium is flattened (arrow).  Bar = 250 um (A and C), 100 um (B and D).


Figure 111.  Grading epididymal duct dilation.  Areas of ductular epithelial flattening are evident in the Grade 1 example (arrows).  Bar = 250 um.






Figure 112.  Increased interstitium in the epididymis.  Vinclozolin administration was associated with a relative increase in the amount of interstitium relative to ducts in the epididymides of hatchling quail.  A and B: Left and right epididymides (arrows) and higher magnification view of epididymis in control male.  C and D: A and B: Left and right epididymides (arrows) and higher magnification view of epididymis in male exposed to vinclozolin. i = interstitium.  Bar = 250 um (A and C), 50 um (B and D).


    

Figure 113.  Epididymal lesions in adult males.  A and B:  Mucosal epithelial cysts of the epididymis in an adult male quail.  Each of the multifocal cysts (arrows) appears to contain homogeneous, pink, proteinaceous fluid.  This lesion, which occurred at the junction between the epididymis and ductus deferens, was observed in a single male of a toxicity study, and the cause was undetermined.  C and D:  Mineralization in the epididymis of an adult male quail.  This was an incidental finding in a toxicity study. In this case, the mineralized material appears to be confined to the tubule lumen.  Bar = 100 um (A and C), Bar = 25 um 
(B and D).




Figure 114.  A: Focal vacuolar hypertrophy and hyperplasia of the epididymal epithelium in an adult male quail.  A mass-like proliferation of enlarged ductular epithelial cells protrudes into the ductular lumen (arrows).  Bar = 100 um.  B and C: Higher magnifications of the lesion.  The arrows indicate sites at which the ductular epithelial cells are enlarged in situ, confirming the epithelium as the origin of this lesion. This finding occurred in only a single male exposed to hexabromocyclododecane (HBCD); consequently, relationship to treatment was not established.  Bar = 25 um.



Figure 115.  A: Involution (invasion) atresia in the ovary of an adult female quail (arrows).  B: Higher magnification of atretic follicle from A.  This type of atresia, which affects predominantly small follicles, is characterized by invasion of the oocyte by cells of the granulosa and/or theca (t) layers, accompanied by the in situ absorption of yolk (y).  The collapsed follicles are typically enveloped by histiocytic macrophages (h).  It is not necessary to record the presence of these histiocytic cells as a diagnostic finding, because they are a normal physiological response to invasion atresia.  Bar = 100 um (A), 25 um (B).



Figure 116.  Grading involution atresia.  Arrows indicate affected follicles.  Bar = 250 um (Grades 1 and 2).




Figure 117.  Burst atresia in an adult female quail.  This subtype of oocyte atresia, which tends to affect larger follicles, is characterized by the presence of free yolk within the ovarian interstitium and abdominal cavity, and one or more collapsed follicles that contain oocyte remnants.  Burst atresia has been attributed to disorders that interfere with laying, such as infectious disease or stress.  It may be necessary to differentiate burst atresia from incidental damage to follicles caused by post-mortem handling.  A and B (the boxed area in A corresponds to B): The ovarian interstitium and crevices along the ovarian surface contain granular yolk material (y).  C and D (D is a higher magnification of C):  Burst follicle (bf) that contains inspissated yolk (iy).  Bar = 500 um (A), 100 um (B), 250 um (C), and 50 um (D).



Figure 118.  A: Retained right ovary (RO) in a hatchling female.  Although this is a type of change that could possibly be related to estrogenic substance administration, it is important to remember that a low level of spontaneous right ovary retention is thought to occur in Japanese quail.  LO = left ovary, SV = spinal vertebra, Kcr = cranial kidney, AG = adrenal gland.  Bar = 250 um.  B: Retained right oviduct, magnum region, in an adult female.  The luminal configuration and mucosal fold architecture are malformed. Bar = 500 um.  C: Magnum region of normal (left) oviduct included for comparison.  Bar = 500 um.  D: Cystic degeneration of the oviduct is evident in the magnum region of this persistent right oviduct from Fig. 201.  Note gaps (arrows) in the epithelium that lines the cystic spaces.  Cystic degeneration is also observed occasionally in the magnum and isthmus regions of normal (left) oviducts.  Bar = 50 um.  



Figure 119.  Retained right oviduct in a quail embryo.  ro = right oviduct, lo = left oviduct, rw = right Wolffian duct, lw = left Wolffian duct, ru = right ureter, lu = left ureter, b = bursa of Fabricius, c = colon.  Bar = 250 um (A), 50 um (B).  


   

Figure 120.  A: Ovarian abscess (granulocytic oophoritis) in an adult female quail.  Bar = 800 um.  B:  Higher magnification of ovarian abscess from preceding figure.  The intralesional presence of large bacterial colonies (arrows), and numerous smaller colonies, suggests that they were the cause of the granulocytic inflammatory response in this case.  Bar = 25 um.



Figure 121.  Examples of decreased development (relative to controls).  A and B: Normal magnum region from the oviduct of an adult female quail.  G = mucosal glands.  C and D: Comparatively less developed magnum.  The mucosal glands contain little if any secretory material.  E and F: Normal shell gland region from the oviduct of an adult female quail.  G and H: Comparatively less developed shell gland.  Bar = 500 um (A, C, E, and G), 50 um (B, D, F, and H).





Figure 122.  Grading oviduct immaturity/inactivity.  The shell gland (low magnification and high magnification inset) is used for grading comparison in this case.  Bar = 500 um.



Figure 123.  A: Ductular cyst.  This cyst (c) within the mucosa of the magnum appears to be a dilation of the excretory duct, adjacent to an invagination of the surface epithelium (s).  Bar = 50 um.  B: Higher magnification of preceding figure.  Note that the cells lining the cyst (black arrow) resemble the epithelial cells of the surface epithelium (white arrow) rather than those of the glands. Bar = 25 um.  

     


Figure 124.  A: Oviduct cyst.  These fluid-filled cysts, which are often grossly visible, are attached to the external surface of the oviduct.  They are relatively simple structures, consisting of a non-uniform wall comprised chiefly of smooth muscle, and a relatively well-developed mucosal epithelium.  Such cysts may occur in association with a retained right oviduct, or on the left oviduct, and although cyst development may occur spontaneously, it is also a documented effect of EDC administration.  Bar = 100 um.  B: Higher magnification of preceding figure.  In this cyst the mucosal epithelium, which consists of goblet cells interspersed with ciliated cells, is essentially identical to that of the magnum region of the normal oviduct.  Bar = 25 um.  





Figure 125.  Shell gland findings.  A and B: Normal shell gland (uterus) from a laying adult female.  C and D: Salpingitis and mucosal gland atrophy.  In this individual, the tubular glands are small and sparse, with diminished cytoplasm.  There are granulocytic and mononuclear cell infiltrates (arrows) in the propria mucosa.  Minimal to mild glandular atrophy was observed as a treatment-related response to trenbolone exposure.  This change may resemble physiological involution of the shell gland that occurs at the end of the laying period.  E and F: Mucosal epithelial hyperplasia.  The normal simple columnar epithelium has been replaced by a thickened pseudostratified hyperplastic epithelium (he).  This effect was also observed in trenbolone-exposed females, as a change that was independent from uterine gland atrophy.  Bar = 100 um (A, C, and E), 25 um (B, D, and F).  




Figure 126.  A: Shell gland inflammation (salpingitis).  Granulocytes and mineralized material are evident in the shell gland lumen.  Bar = 100 um.  B: Higher magnification of A.  By itself, the presence of mineralized concretions (arrows) in the shell gland is not abnormal, as it represents residual shell material; however, in the normal shell gland this material should not be accompanied by the presence of granulocytic inflammatory cells.  Bar = 25 um.  



Figure 127.  Vaginal inflammation, with hyperplasia and squamous metaplasia of the mucosal epithelium in an adult female.  A and B: Normal vagina.  C and D: The mucosal epithelium is thickened, and there are inflammatory cell infiltrates (i) in the lamina propria.  The arrow indicates the site of squamous metaplasia.  
h = hyperplasia, sm = squamous metaplasia.  Bar = 100 um (A and C), 25 um (B and D).  



Figure 128.  Bursal involution versus follicular degeneration.  A and B:  Normal bursa, Grade 1 involution score.  At high magnification (B), a fine membrane (arrow) divides the cortex (c) from the medulla (m).  C and D: Normal bursa, Grade 2 involution score.  The delineation between the cortex and medulla is still apparent (arrow).  E and F: Follicular degeneration of the bursa (minimal).  Changes include increased apoptotic necrosis and comparative sparseness of medullary lymphocytes (arrow), thinning of the cortex, and decreased distinction between the cortex and medulla.  Bar = 250 um (A, C, and E), 50 um (B, D, and F).  

(e)  Fixative Formulations

                           Modified Davidson's Fixative
                            Formaldehyde (37-40%)	220 ml
                             Glacial acetic acid		115 ml
                              95% Ethyl alcohol		330 ml
                              Distilled water			335 ml
                                          
                   Davidson's Fixative (included for comparison)
                            Formaldehyde (37-40%)	200 ml
                                  Glycerol			100 ml
                             Glacial acetic acid		100 ml
                              Absolute alcohol		300 ml
                              Distilled water			300 ml
                                          
(f)  Automatic Processor Schedule
     The following is an example automated processor schedule that can be used to create paraffin-embedded sections of most types of quail tissues.

                                  Station No.
                                    Reagent
                                   Pressure/
                                    Vacuum
                                     Cycle
                                   Heat (C)
                                Time (minutes)
                                       1
                               As appropriate[c]
                                      On
                                    Ambient
                                      40
                                       2
                                  70% alcohol
                                      On
                                    Ambient
                                      40
                                       3
                                  80% alcohol
                                      On
                                    Ambient
                                      40
                                       4
                                  95% alcohol
                                      On
                                    Ambient
                                      40
                                       5
                                  95% alcohol
                                      On
                                    Ambient
                                      40
                                       6
                                 100% alcohol
                                      On
                                    Ambient
                                      40
                                       7
                                 100% alcohol
                                      On
                                    Ambient
                                      40
                                       8
                                 100% alcohol
                                      On
                                    Ambient
                                      40
                                       9
                                 Clear Rite 3
                                      On
                                    Ambient
                                      60
                                      10
                                 Clear Rite 3
                                      On
                                    Ambient
                                      60
                                      11
                                   Paraffin
                                      On
                                      60
                                    45(60*)
                                      12
                                   Paraffin
                                      On
                                      60
                                    45(60*)
                                      13
                                   Paraffin
                                      On
                                      60
                                    45(60*)
                                      14
                                   Paraffin
                                      On
                                      60
                                      45
                                      15
                                    Xylene
                                     Auto
                                     Auto
                                     Auto
                                      16
                                 100% alcohol
                                     Auto
                                     Auto
                                     Auto
                                      17
                           Tap water/cleaning water
                                     Auto
                                     Auto
                                     Auto
                                      18
                                Charcoal/Waste
                                     Auto
                                     Auto
                                     Auto
                                     19[e]
                              Fume Control Water
                                     Auto
                                     Auto
                                     Auto

(g) Post-Mortem Procedures  -  Chicks
      Although most of the post-mortem procedures used for chicks are similar to those employed for adults, there are two important differences: 1) Because of the relative immaturity and diminutive size of the chick reproductive tract, the relevant reproductive structures (gonads and Wolffian or Müllerian duct systems) are sampled in transverse sections with the kidneys, as opposed to excising those organs separately; and 2) For similar reasons, in chicks, the bursa of Fabricius (cloacal bursa) is sampled in a single sagittal midline section with the cloacal glands, instead of collecting it separately as for adults.

      (1)  Specimen Sampling and Processing

	(i)  Necropsy and Tissue Collection.  Tissues from each animal are placed in an individually labeled container that is partially filled (>10-fold fixative volume to tissue volume) with modified Davidson's solution (the primary fixative, see Section (e)).  The necropsy procedures are as follows:

   * A T-shaped incision is created in the ventral abdominal skin and body wall, and the newly created triangular flaps of abdominal skin are excised.  Two lateral incisions are made through the skin, breast muscles and ribs in the caudal to cranial direction, terminating at the thoracic inlet, and the resulting triangular segment of ribs and sternum is removed to expose the thoracic cavity.
   * The carcass is transected distal to the tracheal bifurcation and proximal to the adrenal glands, and the anterior portion of the carcass, which contains the thyroid glands, is placed in the primary fixative (Figs. 129-130).
   * The spleen is identified and excised from the viscera, and inserted into a filter paper envelope or plastic tissue cassette which is placed in the primary fixative.  
   * The liver is excised from the other viscera and placed in the primary fixative.  Several partial-thickness longitudinal incisions may be made in the surface of the liver to enhance fixation. 
   * The distal gastrointestinal tract is severed at the rectum, and the viscera are reflected proximally, carefully severing any mesenteric attachments as the gut is retracted (a short stump of rectum is intentionally left attached to the carcass to provide a landmark for identification of the cloacal bursa at gross trimming).  The esophagus is severed, and the viscera are removed en masse.
   * The duodenal loop, which contains the pancreas, is excised and placed in the primary fixative.  The remaining viscera are placed in the primary fixative for possible subsequent evaluation.
   * The legs and lateral body wall are excised from each side of the posterior carcass segment, with care taken not to disrupt the cloacal region (Fig. 132).  The posterior carcass segment, which contains the adrenal glands, urogenital tracts, bursa and cloacal gland, is placed in the primary fixative.  The reproductive organs are not excised from the carcass.  
Following approximately 24 hours, tissues are rinsed in 70% ethanol and transferred to 10% neutral buffered formalin (NBF) or ethanol for storage.  Tissues may be shipped in NBF, ethanol, or water (e.g., packed in wet gauze), in sufficient amount to ensure that they are not allowed to dry out during transit.  Tissues shipped in water are transferred to NBF upon arrival at the receiving facility.
      (ii)  Gross Trimming and Embedding.  Prior to gross trimming and embedding, adult bird tissues that contain bone, which include the skull, and anterior and posterior carcass segments, are decalcified in a formic acid / EDTA solution (Formical-2000[(TM)], Decal Chemical Corporation, Tallman, NY) for 20-24 hours.  Between the gross trimming and embedding steps, tissues are processed routinely in an automatic processor according to a standardized schedule (see Section (f)). The gross trimming and embedding procedures are:

     *       A transverse cut is made across the anterior portion of the carcass, cranial to the thyroid glands, and the slab that contains the thyroid glands (~ 3-4 mm thick) is embedded so that sectioning can begin at the caudal surface (Figs. 130-131).
     *       A longitudinal slab (~ 2-3 mm thick) is cut from the approximate center of each of the right and left major liver lobes, and the two slabs are embedded so that their cut surfaces are sectioned.
     *       The pancreas is excised from the duodenal loop and embedded in toto.  The spleen is not trimmed and is embedded in toto.
     *       The cloacal region is excised from the posterior portion of carcass.  The conical cloacal bursa (bursa of Fabricius), which is situated just proximal to the rectal stump, is exposed by gentle dissection, everted by manual pressure applied to the external surface of the cloacal region, severed at its base, and embedded for transverse sectioning, beginning at the apex of the cone.  
     *       Two parallel parasagittal cuts are made through the cloacal region and the intervening midline sagittal slab (~3-4 mm), which contains the cloacal glands, are embedded so that either lateral side is sectioned.
     *       Trimming of the posterior carcass segment is illustrated in Figs. 131-136.  Any remaining lateral skin flaps are trimmed off the posterior portion of carcass.  Two parallel transverse cuts are made at a proximal level of the posterior carcass, so that substantial portions of adrenal glands, cranial (proximal) kidney, and male or female gonads are included in the intervening slab.  NOTE: THE LEFT VS. RIGHT ORIENTATION OF THIS TISSUE SHOULD BE IDENTIFIED AND MAINTAINED, so that the identities of the left and right gonads are evident during the histopathologic evaluation.  Two more sets of parallel transverse cuts are made at the halfway points of the middle and caudal (distal) kidney lobes, to create the middle and distal slabs, respectively.  Each slab (~ 2-3 mm) is embedded so that sectioning can begin at the caudal surface.  
      (ii)  Microtomy.  Section thickness for all tissues is ~ 4-6 microns.  The term "full face" is used here to indicate a section in which the entire circumference of the anatomic structure of interest is represented.  The microtomy procedures are:

     *       The anterior carcass slab that contains the thyroid glands is microtomed until the left and right thyroids are full face (Figs137-1139).  It is usually possible to obtain both thyroid glands in a single section, but not always.
     *       The two liver slabs, which are in the same paraffin block, are microtomed full face (Fig. 140).
     *        The pancreas and spleen are microtomed full face.
     *       In the posterior carcass, the transverse proximal slab that contains the adrenal glands, cranial kidney and male or female gonads are microtomed until the adrenal glands are full face (Figs. 141-155).  Additional sections are acquired if these structures could not all be represented in the same section.  The middle and distal slabs that contains the middle kidney and caudal kidney (and also the remainder of the reproductive tract), respectively, are each microtomed so that the renal tissue is full face.
     *       A midline sagittal section is obtained of the cloacal bursa (bursa of Fabricius) and the cloacal glands (Figs. 156-160).  Additional sections may have to be acquired if both structures are not adequately represented in the initial section. (2)  Gross Figures


                                    Fig 130
                                    Fig 130
                                    Fig 129
                                    Fig 129

Figures 129-130.  Collection of thyroid glands.  The white line in Fig. 129 illustrates the initial transverse cut though the thorax, which is made posterior to the tracheal bifurcation and just cranial to the heart.  The arrows indicate the slightly translucent, ovoid, left and right thyroid glands.  The white line in Fig. 130 depicts the second transverse cut which is made proximal to the thyroid glands (arrows).  The entire transverse slab of tissue is embedded so that the caudal surface of the thyroids is microtomed first.  This image was used for illustrative purposes; in reality these cuts would be made on fixed, not fresh, specimens.
                                    Fig 131
                                    Fig 131

                                       
                                       
Figure 131.  The legs and lateral flaps of skin are trimmed off the central portion of the carcass, as indicated by the dashed lines.

                                       
                                    Fig 132
                                    Fig 132
T
T
A
A
K
K
K
K
K
K
K
K
T
T
A
A
T
T
K
K
K
K
T
T
                                    Fig 133
                                    Fig 133
                                             
                                       
                                       
                                       
                                       
                                       A
A
A
A
O
O
K
K
K
K
O
O
K
K
K
K
K
K
K
K
                                    Fig 135
                                    Fig 135
                                    Fig 134
                                    Fig 134
                                           
Figures 132-135.  Relative position and appearance of the adrenal glands (A), testes (T), ovary (O), and kidneys (K) in male and female quail chicks.  Figs. 132 and 133 are low and high magnification views of these tissues in a male chick, whereas Figs. 134 and 135 are low and high magnifications of female chick tissues.  Note that the testes and ovary are slightly more translucent than the more proximal adrenal glands, and that the ovary is a much less distinct structure when compared to the right and left testes.

                            Fig 136 13713613137713
                            Fig 136 13713613137713
                                        
Figure 136.  Illustration of gross trimming to obtain three segments that contain the adrenal glands (A), testes (T) or left ovary (not depicted) and mid-kidney (Km) and caudal kidney (Kc) samples.  Knife cuts are indicated by white lines, and are numbered in the approximate order in which they would occur.  Note that the cranial kidneys, which are hidden behind the adrenal glands and testes, would be sampled automatically in the same slab as those other two types of structures.  [The yellow coloration of this particular specimen is a result of fixation in Bouin's solution rather than modified Davidson's.]

      (3)  Histologic Figures

                                       T
T
E
E
SV
SV
                                    Fig 137
                                    Fig 137

Figure 137.  Transverse section through the anterior carcass at the level of the thyroid glands (arrows) in a 
3-day-old quail.  SV = spinal vertebra, E = esophagus, T = trachea.  Bar = 500 um.



                                      PG
PG
TH
TH
TH
TH
TG
TG
                                    Fig 138
                                    Fig 138
                                    Fig 139
                                    Fig 139
                                               
Figure 138.  Higher magnification of the preceding figure to illustrate the thyroid gland (TG) and thymus (TH).  Bar = 100 um.  Figure 139.  Thyroid gland (TG) and parathyroid gland (PG) from a 2-week-old chick.  Thymus and parathyroid gland may or may not be present in the thyroid sections.  Bar = 50 um. 
                                        



                                    Fig 140
                                    Fig 140
                                      CV
                                      CV
                                       P
                                       P
Figure 140.  Section of liver from a hatchling quail.  P = portal region, CV = central vein.  It can be difficult to distinguish individual portal elements at this stage of development.  Most of the hepatocellular cytoplasmic vacuolation in this liver is of the glycogen type.  Bar = 25 um.  

                                       
LT
LT
MK
MK
AG
AG
AG
AG
Kc
Kc
Kc
Kc
SV
SV
SC
SC
EM
EM
EM
EM
                                    Fig 141
                                    Fig 141

Figure 141.  Transverse section through the proximal segment of the posterior carcass.  Structures evident in this image include the epaxial musculature (EM), spinal cord (SC), spinal vertebra (SV), cranial kidney lobes (Kc), adrenal glands (AG), mesonephric kidney remnant (MK), and left testis (LT).  Bar = 500 um.  

T
T
MK
MK
                                    Fig 142
                                    Fig 142
AG
AG
Figure 142.  Higher magnification of preceding figure to demonstrate the relationship between the testis (T), adrenal gland (AG), and mesonephric kidney remnant (MK) in this hatchling male.  As males mature, the mesonephric kidney remnant will eventually be modified and become the epididymis.  Bar = 100 um.  


                                    Fig 143
                                    Fig 143

Figure 143. High magnification of testis from a hatchling male.  At this stage of development, tubule formation is primitive, and the spermatogenic cell precursors appear highly pleomorphic.  Bar = 25 um.  

                                    Fig 144

                                    Fig 144

M
M
C
C

Figure 144.  High magnification of adrenal gland from a hatchling male.  Differentiating cortical tissue (C) from the very slightly paler medullary tissue (M) is difficult at this early stage of development.  Bar = 25 um.
                                       

                                    Fig 145
                                    Fig 145
T
T
T
T
RG
RG
RG
RG

Figure 145.  High magnification of mesonephric kidney from a hatchling male.  Renal tubules (T) and rudimentary glomerulus-like structures (RG) are evident.  Bar = 25 um.  


                                      Kcr
Kcr
SV
SV
T
T
MK
MK
SC
SC
T
T
MK
MK
Kcr
Kcr
                                    Fig 146
                                    Fig 146
                                       
Figure 146.  Transverse section through the proximal segment of the posterior carcass, at a more caudal level than in Fig. 141.  Structures evident in this image include the spinal cord (SC), spinal vertebra (SV), cranial kidney lobes (Kcr), mesonephric kidney remnants (MK), and left and right testes (T).  Bar = 250 um.  
                                       
                                       
                                       
                                      Km
Km
Km
Km
SC
SC
SC
SC
NC
NC
                                    Fig 147
                                    Fig 147
SV
SV
                                       
Figure 147.  Transverse section through the middle segment of the posterior carcass of a hatchling male.  Structures evident in this image include the notochord (NC), spinal cord (SC), spinal vertebra (SV), and middle kidney lobes (Km).  The testes and adrenal glands are not present at this sectioning level.  The box indicates the position of the next figure.  Bar = 500 um.
                                       
                                       W
W
U
U
                                    Fig 148
                                    Fig 148
                                       
Figure 148.  Higher magnification of the boxed area in the preceding figure to illustrate the relationship between the ureter (metanephric duct) (U) and the Wolffian (mesonephric) duct (W).  In males, the bilateral Wolffian ducts will eventually become the left and right ductus deferens (in females, these ducts attenuate and become non-functional).  Bar = 25 um.
                                       
                                       
                                       
                                       W
W
U
U
Kca
Kca
SC
SC
SV
SV
Kca
Kca
W
W
U
U
                                    Fig 149
                                    Fig 149
Kca
Kca
Kca
Kca
                                       
Figure 149.  Transverse section through the distal segment of the posterior carcass of a hatchling male.  Structures evident in this image include the spinal cord (SC), spinal vertebra (SV), caudal kidney lobes (Kca), ureters (U), and Wolffian ducts (W).  Bar = 250 um.
                                      MK
MK
SV
SV
SC
SC
SV
SV
Kcr
Kcr
MK
MK
AG
AG
O
O
AG
AG
                                    Fig 150
                                    Fig 150
Kcr
Kcr

Figure 150.  Transverse section through the proximal segment of the posterior carcass of a hatchling female.  Structures evident in this image include the spinal cord (SC), spinal vertebra (SV), cranial kidney lobes (Kca), adrenal glands (A), mesonephric kidney remnants (MK), and ovary (O).  Note that the ovary is a flattened structure compared to the testis (Fig. 146), and that normally only the left ovary is present.  Bar = 500 um.
                                       
                                       
                                       
                                      Om
Om
Oc
Oc
AG
AG
                                    Fig 151
                                    Fig 151
  
Figure 151.  Higher magnification of the adrenal gland (AG) and ovary from the preceding figure.  In this image, the ovarian cortex (Oc) is paler than the medulla (Om), and the two layers are divided by an irregular border.  Bar = 100 um.
                                       
                                       
                                    Fig 152
                                    Fig 152
Om
Om
Oc
Oc
OC
OC
Om
Om

Figure 152.  Higher magnification of ovary from the preceding figure to further demonstrate the relative appearance of the cortex (Oc) and medulla (Om).  The cortex is the precursor of the ovarian germinal epithelium.  Like the testis at this developmental stage, the ovary is primitive and does not resemble the adult organ.  Bar = 25 um.



                                    Fig 153
                                    Fig 153
Km
Km
SV
SV
Km
Km
SC
SC
SC
SC
NC
NC

Figure 153.  Transverse section through the middle segment of the posterior carcass of a hatchling female.  Structures evident in this image include the notochord (NC), spinal cord (SC), spinal vertebra (SV), and middle kidney lobes (Km).  The testes and adrenal glands are not present at this sectioning level.  The box indicates the position of the next figure.  Bar = 500 um.

                                    Fig 154
                                    Fig 154
M
M
W
W
U
U
                                       
Figure 154.  Higher magnification of the boxed area in the preceding figure to illustrate the relationship between the ureter (metanephric duct) (U), the Wolffian (mesonephric) duct (W), and the Müllerian (paramesonephric) duct (M).  The Müllerian duct, which is generally larger than the Wolffian duct and has a densely-cellular adventitial (outer) layer, eventually becomes the oviduct.  Like the ovary, only the left Müllerian duct is normally present.  In hatchling males, the Müllerian duct degenerates and is not normally evident at this stage (conversely, Wolffian ducts can be found in both males and females).  Bar = 50 um.

                                    Fig 155
                                    Fig 155
M
M
SV
SV
Kca
Kca
SC
SC
Kca
Kca
                                       
Figure 155.  Transverse section through the distal segment of the posterior carcass of a hatchling female.  Structures evident in this image include the spinal cord (SC), spinal vertebra (SV), middle kidney lobes (Km), and Müllerian duct (M).  Bar = 500 um.

U
U
B
B
CG
CG
P
P
C
C
                                    Fig 156
                                    Fig 156

Figure 156.  Midline sagittal section through the cloacal region of a hatchling quail.  Cranial is to the left, and caudal is to the right.  Structures evident in this image include the bursa of Fabricius (B), the cloacal glands (CG), and the coprodeum (C), proctodeum (P) and urodeum (U).  The bursa of Fabricius and cloacal glands are relatively underdeveloped at this stage, and there is little difference between the cloacal glands of males and females.  Bar = 500 um.


                                    Fig 157
                                    Fig 157
                                       
Figure 157.  Higher magnification of the bursa of Fabricius from the preceding figure.  Because of the diminutive size of the bursa in juvenile quail, it is more practical to section the bursa longitudinally rather than transversely.  Bar = 100 um.



                                    Fig 158
                                    Fig 158
                                       
Figure 158.  Higher magnification of the bursa of Fabricius from the preceding figure.  The spherical bursal follicles appear to be populated predominantly by epithelial cells rather than lymphocytes.  It is not uncommon to see cells undergoing mitotic division and a few necrotic/apoptotic cells in the centers of follicles.  Bar = 25 um.
                                       
                                    Fig 159
                                    Fig 159
CG
CG
                                       
Figure 159.  Higher magnification of the cloacal glands (CG) from Fig. 156.  The glands are relatively underdeveloped in both male and female hatchlings.  Bar = 100 um.
                                       
                                       
                                       
                                    Fig 160
                                    Fig 160
                                       
Figure 160.  Higher magnification of the rudimentary cloacal glands (CG) from the preceding figure.  Bar = 25 um.

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