United States
Environmental Protection
^yr L—1 M * Agency
Chemical Safety EPA No. 740-C-15-003
and Pollution Prevention July 2015
(7101)
Endocrine Disruptor
Screening Program
Test Guidelines
OCSPP 890.2100:
Avian Two-generation
Toxicity Test in the
Japanese Quail
-------
Final July 2015
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, etseq.), the Federal Insecticide, Fungicide and
Rodenticide Act (FIFRA) (7 U.S.C. 136, el 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.
Page 2 of 155
-------
Final July 2015
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 (Fl) 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 Fl
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
Page 3 of 155
-------
Final July 2015
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 (Fl) 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 Fl generations are observed daily for
indications of overt toxicity or other clinical signs. Reproductive parameters are monitored in F0
Page 4 of 155
-------
Final July 2015
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
(°C)
Relative humidity
(%)
Minimum floor area
(cm2/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
\Tote: The acceptability of housing conditions of adults will be evaluated on the basis of results
of reproductive performance.
Page 5 of 155
-------
Final July 2015
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. Recommendec
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) FO Generation. At approximately two weeks of age, individual FO birds are
genetically-sexed (preferred method). At four weeks post-hatch, individual FO birds are
Page 6 of 155
-------
Final July 2015
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 2nd 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 FO 6th and 7th 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 FO generation (Appendices 1 and 3).
(iii) F2 Generation. Brood from the F1 6th and 7th 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 5th 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 5th week of egg production will be
used to determine eggshell thickness and for embryo tissue collection after the ED 15 viability
measure is determined. Eggs collected during the 6th and 7th week of egg production will be
allowed to hatch (see Section F.l) or used to determine eggshell thickness after the ED 15
viability measure is determined. Eggs are stored in a cold storage facility prior to incubation.
Page 7 of 155
-------
Final July 2015
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
latchingf.
Temperature (°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 °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
Page 8 of 155
-------
Final July 2015
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 2nd 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
Page 9 of 155
-------
Final July 2015
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
Page 10 of 155
-------
Final July 2015
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 f
Egg laid/hen
#/day
0.61 to 0.89
Eggs cracked/broken*
% of eggs laid
0 to 10%
Eggs set
#/pair
i
Eggshell strength
Per egg (Newtons)
i
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%
ues obtained from OCSPP 850.2300 (Ref 1) and OECD Test No. 206 (Ref. 2).
* Describe any egg abnormalities.
1 No specific ranges provided.
Page 11 of 155
-------
Final July 2015
(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 (Fl, F2*)
• Genetic sex (Fl, F2*)
• Time of sexual maturity (Fl, 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 (Fl) 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 Fl control groups
Parental mortality during acclimation
<3%
Eggs laid/pair
>4/wk
Viable embryos
>80% of eggs set in the F0 and Fl control
groups
Normal hatchlings
>85% of viable embryos in the F0 and Fl
control groups
Cracked eggs
< 10% in the F0 and Fl control groups
Successful egg production
>12 breeding pairs in the F0 and Fl 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.
Page 12 of 155
-------
Final July 2015
(i) Adults. All surviving FO 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:
Page 13 of 155
-------
Final July 2015
• 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
Tom adult Fl birds.
Treatment
Number
Tissues
Purpose
Control
8M/8F
Examine tissues for
potential systemic (non-
endocrine related)
toxicity
Highest treatment
concentration
8M/8F
Liver, kidney, adrenal,
thyroid, reproductive
tissues
If no signs of overt general toxicity are observed among F1 birds in the high treatment group,
histopathological samples from FO, Fl, 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 FO and Fl, and 14 day-
old chicks or adult F2 birds.
Generation
Treatment
Sample size
Tissues
Purpose
FO
Control
8M/8F
Reproductive tissues:
Ovary, oviduct, shell
gland, cloacal glands,
bursa (F)
Testis, cloacal glands,
bursa epididymis/vas
(when present; M)
Low
8M/8F
low mid
8M/8F
To examine treatment-
high mid
8M/8F
related effects on the
high
8M/8F
endocrine system of
Fl
Control*
8M/8F
Japanese quail exposed
Low
8M/8F
to a putative endocrine
low mid
8M/8F
disrupting compound.
high mid
8M/8F
Thyroid gland
high*
8M/8F
Page 14 of 155
-------
Final July 2015
Generation
Treatment
Sample size
Tissues
Purpose
F2f
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).
fF2 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 t
ie F1 and F2 generations.
Generation
Treatment
Sample size
Tissues
Purpose
F1
Control
5
Low
5
low mid
5
Examine potential
developmental effects
of exposure to putative
endocrine disrupting
compound in ovo.
high mid
5
Liver, kidney,
high
5
adrenal, thyroid,
F2
Control
5
reproductive
Low
5
structures, gonad
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 |il is required for thyroid (250 pi of unextracted plasma) and steroid hormone
analyses (350 pi 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
Page 15 of 155
-------
Final July 2015
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 FO 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 FO 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, M>w., 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.
Page 16 of 155
-------
Final July 2015
Abw, = bw 2 - bw , Equation 1
j jtl J'tl
where:
bwjt = male (or female) body weight in pen j at time t, where tl 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.
NHj
£ HATWTk
HATWTj = k=l 'nH Equation 2
where:
k = index number of a hatchling in pen j from 1 to NHf,
NHj = total number of hatchlings in pen /: and
HATWT/g = 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.
HS,
Y SURVWTk
SURVWTj = k~1 Equation 3
where:
k = index number of a 14-d old hatchling in pen j from 1 to HSf.
HSj = total number of 14-d old surviving hatchlings in pen /: 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.
mj
YTHICK
THICK j = k 1 /_ Equation 4
where:
k = index number of egg shell thickness measurement in pen j from 1 to m/.
ntj = total number of eggs with shell thickness measured in pen /: and
THICKiq = average shell thickness of egg k in pen j., measured as described in Section (f)
4(ii) of this guideline.
Page 17 of 155
-------
Final July 2015
(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., tl, /2, etc.) during the course of the test is calculated and plotted to assess effects on the
pattern of food consumption.
TFOODj = £
where:
termf /.()() J) '
/£_
m „
v j' J
Equation 5
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 I :
m]t = 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.
UEj = ' ^ Equation 6
j j
where:
ELj = total number of eggs laid in pen /: and
ECj = number of eggs cracked in pen j.
(H) Proportion of normal eggs. The proportion of normal eggs is calculated using
Equation 7.
NEj = ^ELj ~ EC} ~ EAj)eL Equation 7
where:
ELj = total number of eggs laid in pen /:
ECj = number of eggs cracked in pen /: 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
Page 18 of 155
-------
Final July 2015
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.
%I = (C ^)(1QQ) 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.
Page 19 of 155
-------
Final July 2015
(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
Page 20 of 155
-------
Final July 2015
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, l=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 (FO and Fl); 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
Page 21 of 155
-------
Final July 2015
• 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 FO
and Fl)
• 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 FO
and Fl) 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.
Page 22 of 155
-------
Final July 2015
(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. Preparedfor: 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.
Page 23 of 155
-------
Final July 2015
(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
Page 24 of 155
-------
Final July 2015
APPENDIX 1: Description of Test Variables by Type for Statistical Analysis.
Stats1
Gen
Endpoint
Variable Type
Growth and Development
op
FO, Fl, F2
Mortality (adult)
Proportion
op
FO, Fl, F2
Clinical observations (e.g. lethargy, depression, wing droop, ruffled
feathers, lacrimation)
Proportion/descriptive
Y
Fl, F2
Day of hatch
Time to event, right censored
Y
Fl, F2
Hatching mortality
Proportion
N
Fl, F2
Hatchling morphology and/or abnormalities
Proportion/descriptive
N
FO, Fl, F2
1 nitial/14d body weight
Continuous
Y
FO, Fl, F2
Terminal body weight
Continuous
N
FO, Fl, F2
Genetic sex [Note that may wish to analyze for concordance of
genetic and phenotypic sex]
Dichotomous
N
FO, Fl, F2
Pheotypic sex/Plumage
Ordered categorical
Y
Fl, F2op
Time to female reproductive maturation (first egg production)* *The
timing of sexual maturity will be considered egg or foam production
in 90% of the control and appropriate behavioral responses among
paired birds.
Time to event, right censored
Y
Fl, F2op
Time to male reproductive maturation (first foam production)* *The
timing of sexual maturity will be considered egg or foam production
in 90% of the control and appropriate behavioral responses among
paired birds.
Time to event, right censored
Y
Fl, F2
cloacal length
Continuous
Reproduction
N
FO, Fl
Number of eggs laid per pen (ELj)
Count
Y
FO, Fl
Number of eggs laid per pen/day [Elj/ftreproductive days)
Count
N
FO, Fl
Number of irregular or abnormal eggs per pen [EAy-)
Count
N
FO, Fl
Number of cracked eggs per pen (ECj)
Count
N
FO, Fl
Number of eggs set per pen. [ESj)
Count
N
Fl, F2
Number of viable ED8 embryos per pen (VEj)
Count
N
Fl, F2
Number of live EDI5 embryos per pen [LEj)
Count
N
Fl, F2
Number of normal hatchlings per pen (NHj)
Count
N
Fl, F2
Number of 14 day-old survivors per pen (HSj)
Count
Y
FO, Fl
Proportion of uncracked eggs per pen (ELj - ECj)/[ELj)
Proportion
Y
Fl, F2
Proportion viable ED8 embryos of eggs set per pen [VEj/ES j)
Proportion
Y
Fl, F2
Proportion of EDI5 live embryos of viable embryos per pen (LE5fVE5)
Proportion
Y
Fl, F2
Proportion of normal hatchlings of eggs set per pen {NHj/ESj)
Proportion
Y
Fl, F2
Proportion of normal hatchlings of live ED15 embryos per pen
(NHj/LEj)
Proportion
Y
Fl, F2
Proportion of 14 day-old survivors of eggs set per pen (HSj/ESj)
Proportion
Y
Fl, F2
Proportion of 14 day-old survivors of normal hatchlings per pen
[HSj/NH j)
Proportion
Y
FO, Fl
Average eggshell thickness per pen [THICK j)
Continuous
Y
Fl, F2
Average hatchling body weight per pen [HATWTj)
Continuous
Y
Fl, F2
Average 14 day-old survivor body weight per pen [SURVWTj)
Continuous
Y
FO, Fl, F2op
Adult male body weight gain per pen ( Ahw/)
Continuous
Y
FO, Fl, F2op
Adult female body weight gain per pen ( A bwj}
Continuous
Y
FO, Fl, F2op
Total food consumption per adult bird per pen [FOODj)
Continuous
N
Fl, F2
Sex ratio of chicks (# female chicks /total chicks)
Proportion
Page 25 of 155
-------
Final July 2015
Stats1
Gen
Endpoint
Variable Type
Histology
Y
FO, Fl, F2
Gross anomalies of the reproductive tract
Proportion/descripti ve
op
Fl(818); possible
additional
Histology-overt toxicity
Ordered
categorical/proportional
op
F0(8|8), Fl(8|8),
F2(8|8)
Histology - reproductive tissues
Ordered
categorical/proportional
op
F0(2/pr),
Fl(2/pr),
Embryo Histology - reproductive tissues
Ordered
categorical/proportional
Biochemical
Y
0(x2/pr),
l(x2/pr),
Estradiol and testosterone (egg yolk); thyroid hormone (ED15 thyroid
gland)
Continuous
Y
F0(8|8), Fl(8|8),
F2(8|8)
Adult serum hormones (estradiol, testosterone, thyroid hormone);
alternatively thyroid hormone can be measure from thyroid gland
Continuous
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.
Page 26 of 155
-------
Final July 2015
APPENDIX 2: Sampling Design for the Avian 2-Generation Toxicity Test in Japanese
Quail.
Generation
Timing
FO
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 irealed diel
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.
Page 27 of 155
-------
Final July 2015
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
8
4
5
pair
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 ED 15 (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 ED 15 (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
Reproductive maturity, if
6 extended
38
7 terminate F2 if extended
Page 28 of 155
-------
Final July 2015
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 |il 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 l,500xg for 10 min at 4°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 l,500xg for 10 min at 4°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°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 600|il. 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°C or below. The label should include
code for the sample, date, and other identifiers specific for the sample. A sample inventory
Page 29 of 155
-------
Final July 2015
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°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
'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 |il plasma is required. Of this, 350
|il is extracted for measurement of steroid hormones and 250 |il 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.
Page 30 of 155
-------
Final July 2015
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/Bo)* 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-
reactivity1 (%)
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
Thyroid2: 2.9
Plasma: 13.6
Thyroid2: 15.2
E2
Siemens TKE21
2.5 pg/ml
0.2 (T)
Plasma: 6.9
Feces2: 3.7
Plasma: 13.7
Feces2: 10.0
T
Caymen 582701
3.9 pg/ml
0.1 (E2)
Plasma: 5.7
Plasma: 6.4
Measured cross-reactivity is with the hormone indicated in parentheses.
Page 31 of 155
-------
Final July 2015
Additional 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.
Page 32 of 155
-------
Final July 2015
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 Miillerian 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
Page 32 of 155
-------
Final July 2015
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
Page 33 of 155
-------
Final July 2015
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 (~lcm) 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™, 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.
Page 34 of 155
-------
Final July 2015
• 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
cloacaI 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
Page 35 of 155
-------
Final July 2015
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).
Page 36 of 155
-------
Final July 2015
(2) Gross Figures.
Figure 1. To expose the abdominal and thoracic cavities, incisions made
through the abdominal wall and around the keel.
Figure 2. The abdominal and thoracic cavities are exposed, revealing the liver
il i. 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.
Page 37 of 155
-------
Final July 2015
Figure 3. The small, pink, triangular to ovoid, spleen (S) is identified
proximate to the proventriculus (P), gizzard (Cr), 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.
Figure 4. Following removal of the bilobed liver (shown here in situ), several
partial-thickness longitudinal incisions (dotted lines) are made to facilitate
fixation.
Page 38 of 155
-------
Final July 2015
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).
Figure 6. The duodenal loop, which contains the pancreas, is identified.
Page 39 of 155
-------
Final July 2015
Fig 7
Figure 7. The excised duodenal loop (D) and pancreas (P).
Figure 8. As much as possible, the male reproductive tract, which includes
the testis, epididymis, and ductus deferens is removed intact and
interconnected.
Page 40 of 155
-------
Final July 2015
Fig 9
L
rV
7 \
> i
1
I
(
$
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.
Page 41 of 155
-------
Final July 2015
Fig 10
¦
D
1
J
Figure 10. Higher magnification of the male reproductive tract to illustrate
the testis (T), epididymis (E), and ductus deferens (D).
Page 42 of 155
-------
Final July 2015
Figure 11. Components of ths female reproductive tract include the ovary
(O.i. infundibulum (In), magnum (M), isthmus (I), uterus (U), and vagina (V).
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).
Page 43 of 155
-------
Final July
2015
Figure 13. The legs are removed from the posterior carcass and cloacal
region.
Figure 14. The posterior carcass segment and cloacal region following
removal of the legs. Scissors indicate the position of the chiacal bursa Also
evident are the left and right kidneys (K).
Page 44 of 155
-------
Final July 2015
a b c
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 = cluneal region, g = duodenal hop, 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.
Page 45 of 155
-------
Final July 2015
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.
Page 46 of 155
-------
Final July 2015
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.
HH
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, Till V SHOULD
BE CLEARLY IDENTIFIED AND SUBMITTED FOR HISTOLOGY.
Fig 18
Page 47 of 155
-------
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 inftindibukim, 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 I 111 REPRODUCTIVE TRACTS SHOULD ALWAYS BE KEPT
SEPARATE AND CLEARLY IDENTIFIED.
Figure 24. The pancreas is trimmed from within the duodenal loop. It is embedded whole.
Fig 21
Page 48 of 155
-------
Final July 2015
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).
Page 49 of 155
-------
Final July 2015
Figures 26 and 21. Fig. 26 : Trimming of a midline sagittal slab from the cloacal region in order
to obtain histologic sections of the chiacal glands and cloacal bursa. Fig. 27: The slab specimen to
be embedded. The site of the cloacal glands is indicated by the arrows.
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.
Fig 27
MK CaK
Page 50 of 155
-------
Final July 2015
V43Ptfl «~
I It£?«j
b%.0*-o*»
V43946 I
170 _ _
Fig 30
V439
-------
Final July 2015
(3) Histologic Figures.
ilJ nff M wu W W
:
Figures 31-34. Medium and high magnification views of liver from 6-week-old quail (male series on left, female 011
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. Tags. 31 and 32,
bar =100 Jim; Tigs. 33 and 34, bar = 25 Jiffi,
Page 52 of 155
-------
Final July 2015
ventrum
Figure 35. Cranial kidneys i'CK ) and adrenal glands ( AG ) from a 12-week-old male. The
reproductive tract was removed at gross trimming. Bar = 800 jatL
Figure 36. Medium magnification of cranial kidney (top) and adrenal gland (bottom) from a
week-old male. Bar = 250 um.
Page 53 of 155
-------
Final July 2015
Figure 37. High magnification of adrenal gland from a 12-week-oldmale. 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 medullar)' (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 uni.
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.
Page 54 of 155
-------
Final July 2015
ventrum
Figure 39. Transverse section through mid-kidney region from a 12-week old male.
S = spinal cord, N = notochord, K = kidney. Bar = 800 urn
ventrum
Figure 40. Transverse section through caudal kidney region from a 12-week-old male. S = spinal
cord, K = kidney. Bar = 800 fim.
Page 55 of 155
-------
Final July 2015
Figure 41. Pancreas from a 12-week-old male. Cohmrix 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 |im.
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 urn.
Page 56 of 155
-------
Final July 2015
Figure 43. Spleen from a 12-week-old male. In this particular case, lymphoid follicles (arrows)
are few and sparsely populated. Bar = 500 p.jil
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 jun.
Page 57 of 155
-------
Final July 2015
Fig 48
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.
Page 58 of 155
-------
Final July 2015
ventrum
Fig 49
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 (.1111.
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 "G" cells, but
cells with similar function exist in the ultimobranchial bodies (not shown).
Bar = 25 urn.
Page 59 of 155
-------
Final July 2015
Figure 51. Cloacal region from a 6-week-old male to demonstrate clinical 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 clinical bursa can also be found in this region
(see Figs. 55 and 114). Bar= 1000 [xm.
Page 60 of 155
-------
Final July 2015
Figures 52-55. The deeply basophilic cloacalglands 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 iim.
Page 61 of 155
-------
Final July 2015
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.
Page 62 of 155
-------
Final July 2015
Figure 57. Testis (T), epididymis 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.
Page 63 of 155
-------
Final July 2015
mm
Vo
#5
V
-.1. .r.": * k
>•»?» *afe-4'e.js'5s2E®t - : 'it-;. ¦ * ... Q
St
Eft®
UfA^e ' ; w (3Fj
1 £¦ 4
Ktapfe
-
I# - $4#J5#fl
V A -Jit ±J<&&7Q
•r *ikl i fm\x& a yfW® y ips
1*8 as* ^ H © J s- ,#E
I <£>i ¦ a5*# JS - 5 ££
fliSS^M. O > J?//® .?b- '*">''' %
iltfU -» v-: ; i> * V f*
.^-aSr* i ¦# • •- *%. , ! •*
• «5*V 'V
J,".'-
;
IR961 \stj ,
f) -_<_c ,5- ttT^I
¦¦*^jS? ¦:" •'® 14l- -
4, •• . ,«..»r'£ a, *"
VMr ~> v\ -. h': ¦•/• .
V;<^ ft*.'* JSmV V- - ^ lJ
*••» *£.¦•». •»* "y«@pw ¦*.« -•«** *j?&& Msop
:£'*: ¦* >' v#V5mTi. - •«, .*»*>
4 ¦• *•• •'*" ** < % .:. j ;*>.
P^ke'^8
>. V-' -
iHPPPffjfr^ s&iuSf
^ x1 »JSD1m
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 urn.
Page 64 of 155
-------
Final July 2015
Fig 62
es
sz
sc
fr-
^
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 (Is), 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 inn.
Page 65 of 155
-------
Final July 2015
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) 4 proximal efferent ductule (FED) distal efferent ductule (DED) collecting ductules (CD) 4 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.
Page 66 of 155
-------
Final July 2015
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 ('II) and theca externa (TE). Note persistence of the nucleus (N) in the smaller
early stage follicles. Bar = 800 pin (Figs. 64 to 66), 250 (jm (Fig. 67), 100 |im (Fig. 68), and 50 (jm (Fig. 69).
Page 67 of 155
-------
Final July 2015
.9
Figure 70. Post-ovulatory follicles (PQF) 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 luteiun, the latter of which refers to a post-ovulatory structure in the mammalian ovary.
There may be some functional simi larities between the POF and the corpus luteum in terms of steroid hormone
production. Bar = 500 urn.
Page 68 of 155
-------
Final July 2015
Figures 71 and 72. Higher magnification of early and late post-ovukitory follicles, respectively. The major difference
between these two structures is the extensive proliferation of granulosa cells in the late PQF (for a more detailed view,
see the next two figures). Bar = 100 urn.
Figures 73 and 74. Still higher magnification early and late post-omlatory follicles, respectively, to better appreciate
the different cell layers: GC = granulosa cells, TI = theca interna, IT' = theca externa.
Bar = 25 mn.
Page 69 of 155
-------
Final July 2015
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 nifundibuhun, 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. Fhe mucus cell lining (M) of
the magnum is also more prominent prior to egg passage. Bar = 250 (jm (Fig. 75), 25 um (Figs 76, 78 and 80), and 500
um (Figs. 77 and 79).
Page 70 of 155
-------
Final July 2015
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 iim (Figs. 81 and 83),
25 (tm (Figs 82, 84 and 86), and 250 (.mi (Fig. 85).
Page 71 of 155
-------
Final July 2015
(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-relatedfindings 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
Page 72 of 155
-------
Final July 2015
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.
Page 73 of 155
-------
Final July 2015
• 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
Page 74 of 155
-------
Final July 2015
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.
Page 75 of 155
-------
Final July 2015
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
Page 76 of 155
-------
Final July 2015
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
Page 77 of 155
-------
Final July 2015
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
Page 78 of 155
-------
Final July 2015
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 (Miillerian duct) is a signature effect of estrogenic substance administration in
female quail. This may be accompanied by a concomitant decrease in left oviduct
Page 79 of 155
-------
Final July 2015
development, and oviduct developmental abnormalities such as cyst formation.
Decreased density of the tubular glands is a reported estrogenic effect in the shell gland.
Page 80 of 155
-------
Final July 2015
(9) Atlas of Histopathologic Findings
Figure 87. A andB: Normal adrenal gland. C and D: Adrenal eland with increased cortical vacuolation.
Vacuo lation is due to the presence of fine lipid droplets within the cytoplasm. Typically, there is substantial inter-
animal vari ability 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).
Page 81 of 155
-------
Final July 2015
Figure 88. Normal thyroid (A) and decreasedfollicle 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 jxm (A and B), 25 (iitt (C and D).
Page 82 of 155
-------
Final July 2015
Grade 2
Grade 3
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 perclilorate,
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 (im.
Page 83 of 155
-------
Final July 2015
Figure 90. Grading thyroid gland, arteriolar hypertrophy ill adult quail. Perchlorate exposure was associated with an
unusual dose-responsive expansion of the tunica media, and tunica intirna 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 urn.
Page 84 of 155
-------
Final July 2015
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 uni (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 jun
(C), 25 (.tm (D).
Page 85 of 155
-------
Final July 2015
v ir
r/
•7
Mf
V
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 jim»
Page 86 of 155
-------
Final July 2015
. ¦
|Li I, J. I,*
¥.. A-?*. ;. 4 >¦> ^
-
¦ • .¦-¦'•¦
. • • I. ;:¦•¦ -..
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 urn.
Page 87 of 155
-------
Final July 2015
Figure 94. Hepatocellular vacuolation. A: Liver with no vacuolation. R Liver with mild vacuolation from a
comparably-aged female control quail from the same study as A. Lhe 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 macro vesicular vacuolation in the liver of
an adult female quail treated with 30 ppm endosulfan. Lhe distribution of the vacuolization is distinctly perivascular.
As compared to the macro vesicular' type, the presence of microvesicular vacuolization is thought to indicate a more
rapid course of lipid accumulation. Lhis lesion, which was thought to be treatment-related, was also found to a
relatively minor degree in untreated quail. Bar = 25 jxtn (A, B, and D), 250 pin (C).
Page 88 of 155
-------
Final July 2015
Grade 1
Grade 2
Grade 3
Grade 4
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 jini
(Grades 1 and 2), 250 jim (Grades 3 and 4).
Page 89 of 155
-------
Final July 2015
,
-------
Final July 2015
Vv .'• $ r •.f|k'& '¦ .'
iterate
$$&£¦
Grade 1
Wm
Grade 3
Grade 4
& &»s3sSl8rf£
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 urn (Grades 1 through 4),
Page 91 of 155
-------
Final July 2015
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 winch 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 uni.
Page 92 of 155
-------
Final July 2015
>-¦
¦MftflN
Figure 99. A: Focal necrosis with secondary mineralization in the liver of a hatchling female that had been treated
with 125 ppm endosult'mi. Bar =100 um. B; Higher magnification of the liver from the preceding figure.
Mineralization is evident as dense dark purple deposits. Bar = 25 pm.
Page 93 of 155
-------
Final July 2015
Grade 1
Grade 3 Grade 4
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 uin (Grade
1), 100 pit (Grade 2).
Page 94 of 155
-------
Final July 2015
•* •;'
Fisiure 101. Bile pigment accumulation in the liver. Most of the pigment appears to be w ithin 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.
Page 95 of 155
-------
Final July 2015
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
hepatoeytes. Although not considered tumors per se, these lesions may be precursors of primary hepatocellular
neoplasms. Bar = 50 uin. 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.
Page 96 of 155
-------
Final July 2015
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 urn.
Page 97 of 155
-------
Final July 2015
jfoai
¦ •
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
Jim (A and C), Bar = 25 um (B and D).
Page 98 of 155
-------
Final July 2015
Grade 2
Grade 3
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 jun (Grades 1 and 2), 25 fun (Grade 4).
Page 99 of 155
-------
Final July 2015
Grade 1
Grade 4
Grade 3 |r
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 luminal 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 uni (Grades 1-3).
Page 100 of 155
-------
Final July 2015
Figure 107. A andB: 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 (sli); 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 urn (A), 50 jun (B).
Page 101 of 155
-------
Final July 2015
Figure 108. I'denotation of the epidldymal duct epithelium. Dose-responsive vacuo lation of the proximal efferent
duct epithelium occurred in adult quail exposed to vinclozolin. A andB: Normal epididymis, p = proximal efferent
duct. C and D: Severe vacuolation of the proximal efferent duct (p) epithelium. Bar = 250 una (A and G), 100 tun IB
and D).
Grade 1
Figure 109. Grading epididyntal duct vacuolation. Bar = 25 (mi.
Page 102 of 155
-------
Final July 2015
Figure 110. Dilution 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 jiin (A and C), 100 (im(B and I)).
Grade 2
Figure 111. Grading epididymal duct dilation. Areas of ductular epithelial flattening are evident in the Grade 1
example (arrows). Bar = 250 fun.
Page 103 of 155
-------
Final July 2015
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 andB: 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 |.im (A and C), 50 |im (B and D).
Page 104 of 155
-------
Final July 2015
Figure 113. Epididymal lesions in adult males. A andB: Mucosal epithelial cysts of the epididymis in an adult male
quail. Each of the multifocal cy sts [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 urn (A and C),Bar = 25 urn
(B and D).
Page 105 of 155
-------
Final July 2015
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 jim.
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
Jim..
Page 106 of 155
-------
Final July 2015
MS * i
5i »*'
K H
*5L%*
^ « *3* vSr*- ?-
*\* c
X » %*.
V , **
¦ Sfflti*'1* 'ftSl
HBmtair' t>
^>4*#£ Jm*
+ y*k ^ ¦ %,'¦ -v
r
j^'ji
Figure 115. A: Involution (invasion) atresia in the ovary of an adult female quail (arrows), jjfc 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 nm (A), 25 pm (B).
Page 107 of 155
-------
Final July 2015
Grade 1
Grade 2
Grade 3 Grade 4
Figure 116. Grading involution atresia. Arrows indicate affected follicles. Bar = 250 pm (Grades 1 and 2).
Page 108 of 155
-------
Final July 2015
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 \xm (A), 100 p,m (B), 250 (.mi (C), and 50 jjia (D).
Page 109 of 155
-------
Final July 2015
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, AO
= adrenal gland. Bar = 250 tun. B: Retained right oviduct, magnum region, in an adult female. The luminal
configuration and mucosal fold architecture are malformed. Bar = 500 pm. 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 pm.
Page 110 of 155
-------
Final July 2015
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 (j,m (A), 50 um
(B).
Page 111 of 155
-------
Final July 2015
Fisiure 120. A: Ovarian abscess (granulocytic oophoritis) in an adult female quail. Bar = 800 itm. R 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 uin.
Page 112 of 155
-------
Final July 2015
~
-
Figure 121. Examples of decreased development (relative to controls). A andB: 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 una (A, C,. E, and G), 50 jun (B, D, F, and H).
Page 113 of 155
-------
Final July 2015
Grade 1
Grade 2
Grade 3
Grade 4
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 inn.
Page 114 of 155
-------
Final July 2015
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 Jim. & 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.
Page 115 of 155
-------
Final July 2015
Figure 124. A: Oviduct cyst. These fluid-filled cy sts, 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 (.im. R 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 uin.
Page 116 of 155
-------
Final July 2015
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 jim (A, C. and E),25 ,um (B, D, and F).
Page 117 of 155
-------
Final July 2015
Figure 126. A: Shell gland inflammation (salpingitis). Granulocytes and mineralized material are evident in the shell
gland lumen. Bar = 100 una. 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 pm.
Page 118 of 155
-------
Final July 2015
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 Jim (A and C), 25 tun IB and D).
Page 119 of 155
-------
Final July 2015
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 medullar)' lymphocytes (arrow), thinning of the cortex, and decreased distinction between the cortex and
medulla. Bar = 250 |mi (A, C, and E), 50 |mi (B, D, and F).
Page 120 of 155
-------
Final July 2015
(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 appropriate0
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%o alcohol
On
Ambient
40
8
100%o 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%o alcohol
Auto
Auto
Auto
17
Tap water/cleaning water
Auto
Auto
Auto
18
Charcoal/Waste
Auto
Auto
Auto
19e
Fume Control Water
Auto
Auto
Auto
Page 121 of 155
-------
Final July 2015
(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 Miillerian 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,
Page 122 of 155
-------
Final July 2015
bursa and cloacalgland, 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™,
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
Page 123 of 155
-------
Final July 2015
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 (Figsl37-1139). 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.
Page 124 of 155
-------
Final July 2015
(2) Gross Figures
g 130
/
a
/
4 •
if*
\
\
\
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.
Page 125 of 155
-------
Final July 2015
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.
Page 126 of 155
-------
Final July 2015
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.
Page 127 of 155
-------
Final July 2015
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.]
Page 128 of 155
-------
Final July 2015
(3) Histologic Figures
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 uin.
Figure 138. Higher magnification of the preceding figure to illustrate the thyroid gland (TG) and thymus (TH). Bar =
100 uni. Figure 139. Thyroid gland ('T(i) and parathyroid gland (PC i) from a 2-week-old chick. Thymus and
parathyroid gland may or may not be present in the thyroid sections. Bar = 50 am.
Page 129 of 155
-------
Final July 2015
Fig 140
P CV.
fffi
Jtf&V vv'V 2 & -? ,~ *. '*¦ ¦*, *
«g'SU i^«s •$»•&«t| " •• *ij»'
i $ * * ^3 V %&/ J
.1*1 *•' -'V: VS?
®» : j '"' - ' v? ^ •'* ' * ' -""' a if >'
-* WBSEXSei1^ *v *
ift li 1$ • m£X ¦ V* & JiP • -/ §
^», w $ v<
RHh ——
-
Figure 140. Section of liver from a hatchling qiiail. 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 [xm.
Page 130 of 155
-------
Final July 2015
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 11 T j. Bar = 500 Jiffi,
Page 131 of 155
-------
Final July 2015
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 |im.
Page 132 of 155
-------
Final July 2015
Fig 143
Hflr?Np
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 urn.
Page 133 of 155
-------
Final July 2015
Fig 144
* /»"'
V-. - B ..a* *• %>¦:* j
i t - .~ »v«j
~ • ¦¦.'" •»'a y. «l
-V "•%
- ; ... «* -., • -
^ .V ¦ V a . ^'LNv ,v- -'Tr ? f J[ i^umV -»¦
"^^u|V v^V ® ^
¦VI. . * MM
~ ;' El*, ;•%'*>
.. • • - •
C3
'~ fff;
Ail '
v , M *' . •-
¦ • : -•• ¦ .V, ¦¦'
^ m . j»- * /Lr. ^Fw '/
.,. wri8&'.,<& A*
-: •"- ^ ^ ^•-• ¦rJT$y '
' vj) -'Si »• ".-* 1-A /c- @ 0 ' «->V«v
tK ««*£. £JFfr £ 0 •) « '+ « . ' ¦»« 9* \ v.• >*». \ *•.• ¦¦. ¦ .f %
i• . •« •'< ••¦ .a- • • - • •
A*$» 'Ml
'•iVv - « "''•' ->.* • - '* ' i"< « • I" ?' • • - '•
Fiaure 144. Hioh maenification ni adrenal eland from a hatrhlin" male. Difterentiatino cortical tissue I'C'l from the
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 |tm.
Page 134 of 155
-------
Final July 2015
Fig 145
» */* y, v " •„? .
—
• w * i. .. A ^ ~ r. /j
* •
mj .«sr ' •
* V, ~ J tSfci * •
Figure 145, High magnification of ntesonephric kidney from a hatchling male. Renal tubules (T) and rudimentary
glomerulus-like structures (RG) are evident. Bar = 25 pro.
Page 135 of 155
-------
Final July 2015
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), mesoitephric kidney remnants (MK), and left and right testes (T). Bar = 250 ftm.
Fig 147
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 Jim.
Page 136 of 155
-------
Final July 2015
Figure 148. Higher magnification of the boxed area in the preceding figure to illustrate the relationship
between the ureter (metunephric duct) (U) and the Wolffian (mesonephric) duct (W). In males, the bilateral
Wolffian ducts will eventually become the left and right ductus deferens I in females, these ducts attenuate
and become non-functional). Bar = 25 Jim.
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 jim.
Page 137 of 155
-------
Final July 2015
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), ntesonephric 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 jtm.
Figure 151, Fligher 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 inn.
Page 138 of 155
-------
Final July 2015
-
Oc
' * - • 0 . ,¦•.'•¦•* ft. .^\.
.: ;. \*jwsr-v . ..-• ' • *;i '¦¦
••• ». .• ..•» ,'¦• . * •• > <¦'¦• ¦... • \ *•
nn
* »¦
Fig 152
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 (im.
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 jutt.
Page 139 of 155
-------
Final July 2015
Figure 154. Higher magnification of the boxed area in the preceding figure to illustrate the relationship
between the ureter (metunephric duct) (U), the Wolffian (mesonephric) duct (W), and the MilUerian
(paramesonepliric) duct (M). The Mtlllerian 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
Miillerian duct is normally present. In hatchling males, the Mtlllerian duct degenerates and is not normally
evident at this stage (conversely, Wolffian ducts can be found in both males and females). Bar = 50 um.
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 MilUerian duct (M). Bar = 500 tun.
Page 140 of 155
-------
Final July 2015
Figure 156. Midline sagittal section through the cloacal region of a hatchling quail. Cranial is to the left, and caudal is
to the right. StracUires 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 ofFabricius and cloacal glands are relatively
underdeveloped at this stage, and there is little difference between the cloacal glands of males and females. Bar = 500
pttt.
Page 141 of 155
-------
Final July 2015
Fisiure 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 tun.
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 Jim,
Page 142 of 155
-------
Final July 2015
Figure 159. Higher magnification of the cloacal glands (CG) from Eig. 156. The glands are relatively
underdeveloped in both male and female hatchlings. Bar =100 urn.
Figure 160. Higher magnification of the rudimentary cloacal glands (CG) from the preceding figure. Bar =
25 mn.
Page 143 of 155
-------
Final July 2015
(h) References.
(1) AdkinsE.K. (1975) Hormonal basis of sexual differentiation in the Japanese quail.
Journal of Comparative Physiology and Psychology, 89:61-71.
(2) Aire, T.A. (1979) The epididymal region of the Japanese quail (Coturnix coturnix
japonica). ActaAnatomica, 103:305-312.
(3) Altinok I. and Capkin E. (2007) Histopathology of rainbow trout exposed to
sublethal concentrations of methiocarb or endosulfan. Toxicologic Pathology,
35(3):405-410.
(4) Andersen H.R., Vinggaard A.M., Rasmussen T.H., Gjermandsen I.M, and
Bonefeld-J0rgensen E.C. (2002) Effects of currently used pesticides in assays for
estrogenicity, androgenicity, and aromatase activity in vitro. Toxicology and
Applied Pharmacology, 179(1): 1-12.
(5) Aste N., Balthazart J., Absil P., Grossmann R., Mulhbauer E., Vigilietti-Panzica C.,
and Panzica G.C. (1998) Anatomical and neurochemical definition of the nucleus
of the stria terminalis in Japanese quail (Coturnix japonica). Journal of
Comparative Neurology, 396:141-157.
(6) Ata A., Hatipoglu F.S., Yildiz-Gulay O., and Gulay M.S. (2007) Protective role of
ascorbic acid on subacute sperm toxicity in male New Zealand white rabbits treated
with endosulfan. Drug and Chemica Toxicology, 30(3): 181-195.
(7) Ballesteros M.L., Bianchi G.E., Carranza M., and Bistoni M.A. (2007) Endosulfan
acute toxicity and histomorphological alterations in Jenynsia multidentata
(Anablepidae, Cyprinodontiformes). Journal of Environmental Science and Health
PartB, 42(4):351-357.
(8) Balthazart J., Schumacher M., and Malacarne G. (1984) Relative potencies of
testosterone and 5a-dihydrotestosterone on crowing and cloacal gland growth in the
Japanese quail (Coturnix coturnix japonica). Journal of Endocrinology, 100:19-23.
(9) Balthazart J. and Schumacher M. (2005) Organization and activation of behavior
in quail: Role of testosterone metabolism. Journal of Experimental Zoology,
232(3):595-604.
(10) Bellaris R. and Osmond M. (2005) The Atlas of Chick Development, Second
Edition. Elsevier, Amsterdam, 470 pages.
(11) Berg C., Halldin K., Fridolfsson A-K., Brandt I., and Brunstrom B. (1999) The
avian egg as a test system for endocrine disrupters: effects of diethylstilbestrol and
ethynylestradiol on sex organ development. The Science of the Total Environment,
233(l-3):57-66.
Page 144 of 155
-------
Final July 2015
(12) Berg C., Holm L., Brandt I., and Brunstrom B. (2001) Anatomical and histological
changes in the oviducts of Japanese quail, Coturnix japonica, after embryonic
exposure to ethynyloestradiol. Reproduction, 121:155-165.
(13) Biswas A., Ranganatha O.S., Mohan J., and Sastry K.V. (2007) Relationship of
cloacal gland with testes, testosterone and fertility in different lines of male
Japanese quail. Animal Reproduction Science, 97(l-2):94-102.
(14) Brunstrom B., Axelsson J., and Halldin K. (2003) Effects of endocrine modulators
on sex differentiation in birds. Ecotoxicology, 12(l-4):287-95.
(15) Brunstrom B., Axelsson J., Mattsson A., Halldin K. (2009) Effects of estrogens on
sex differentiation in Japanese quail and chicken. General and Comparative
Endocrinology, 163 (1-2): 97-103.
(16) Budras K.D. and Sauer T. (1975) Morphology of the epididymis of the cock
{Gallus domesticus) and its effect upon the steroid sex hormone synthesis. I.
Ontogenesis, morphology and distribution of the epididymis. Anatomy and
Embryology, 148:175-196.
(17) Budras K-D. and Sauer T. (1975) Morphology of the epididymis of the cock
{Gallus domesticus) and its effect upon the steroid sex hormone synthesis. Anatomy
and Embryology, 148(2):197-213.
(18) CallebautM. (1973) Correlation between germinal vesicle and oocyte
development in the adult Japanese quail (Coturnix coturnix japonica). A
cytochemical and autoradiographic study. Journal of Embryology and
Experimental Morphology, 29(1): 145-157.
(19) Choudhary N., Sharma M., Verma P., and Joshi S.C. (2003) Hepato and
nephrotoxicity in rat exposed to endosulfan. Journal of Environmental Biology,
24(3):305-308.
(20) Coil W.H. and Wetherbee D.K. (1959) Observations on the cloacal gland of the
Eurasian quail, Coturnix coturnix. The Ohio Journal of Science, 59(5):268-270.
(21) Crissman J.W., Goodman D.G., Hildebrandt P.K., Maronpot R.R., Prater D.A.,
Riley J.H., Seaman W.J., and Thake D.C. (2004) Best Practices Guideline:
Toxicologic Histopathology. Toxicol Pathol. 32:126-131.
(22) D'Herde K. and Roels F. (1993) Experimental modification of vitellogenesis in
Japanese quail by trypan blue in vivo. Reproduction Nutrition Development,
33:597-608.
Page 145 of 155
-------
Final July 2015
(23) Dalsenter P.R., de Araujo S.L., de Assis H.C., Andrade A.J., and Dallegrave E.
(2003) Pre and postnatal exposure to endosulfan in Wistar rats. Human &
Experimental Toxicology, 22(4): 171 -175.
(24) Dutta H.M., Misquitta D., and Khan S. (2006) The effects of endosulfan on the
testes of bluegill fish, Lepomis macrochirus: a histopathological study. Archives of
Environmental Contamination and Toxicology, 51(1): 149-156.
(25) Eroschenko, V.P. and Wilson, W.O. (1974) Histological changes in the regressing
reproductive organs of sexually mature male and female Japanese quail. Biology of
Reproduction, 11:168-179.
(26) Eroschenko V.P. and Wilson W.O. (1975) Cellular changes in the gonads, livers
and adrenal glands of Japanese quail as affected by the insecticide Kepone.
Toxicology and Applied Pharmacology, 31(3):491-504.
(27) Eustis S.L, Boorman G.A, Harada T., and Popp J.A. (1990) Liver. In: Pathology of
the Fischer Rat: Reference and Atlas. Academic Press, San Diego, p. 78.
(28) Fitzgerald T.C. (1969) The Coturnix Quail: Anatomy and Histology. The Iowa
State University Press, Ames, Iowa, 306 pages.
(29) Fransson-Steen R., Flodstrom S., and Warngard L. (1992) The insecticide
endosulfan and its two stereoisomers promote the growth of altered hepatic foci in
rats. Carcinogenesis, 13(12):2299-2303.
(30) Fry D.M. and Toone C.K. (1981) DDT-induced feminization of gull embryos.
Science, 213(4510):922-924.
(31) Fujimoto T., Ukeshima A., and Kiyofuji R. (1975) The origin, migration and
morphology of the primordial germ cells in the chick embryo. The Anatomical
Record, 185(2): 139-153.
(32) Gasc J-M., Stumpf W., and Sar M. (1978) Estrogen target sites in the cloacal
region of female and male chick embryos. Cell and Tissue Research, 193:457-462.
(33) Glidersleeve R.P., Tilson H.A., and Mitchell C.L. (2005) Injection of
diethylstilbestrol on the first day of incubation affects morphology of sex glands
and reproductive behavior of Japanese quail. Experimental Teratology, 31(1): 101-
109.
(34) Glover C.N., Petri D., Tollefsen K-E., Jorum N., Handy R.D., and Berntssen
M.H.G. (2007) Assessing the sensitivity of Atlantic salmon (Salmo salaf) to
dietary endosulfan exposure using tissue biochemistry and histology. Aquatic
Toxicology, 84(3):346-355.
Page 146 of 155
-------
Final July 2015
(35) Green J.W., Springer T.A., Saulnier A.N., Swintek J. (2014) Statistical analysis of
histopathology endpoints. Environ Toxicol Chem. 33(5): 1108-1116.
(36) Groenendijk-Huijbers M.M. (1962) The cranio-caudal regression of the right
Miillerian duct in the chick embryo as studied by castration experiments and
estrogen treatment. Anatomical Record, 142:9-19.
(37) Grote K., Niemann L., Selzsam B., Haider W., Gericke C., Herzler M., and
Chahoud I. (2008) Epoxiconazole causes changes in testicular histology and sperm
production in the Japanese quail (Coturnix coturnix japonica). Environmental
Toxicology & Chemistry, 27(11):2368-2374.
(38) Gunawardana V.K. (1977) Stages of spermatids in the domestic fowl: a light
microscope study using Araldite sections. Journal of Anatomy, 123:351-360.
(39) Haffen K., Scheib D., Guichard A. and Cedard L. (1975) Prolonged sexual
bipotentiality of the embryonic quail testis and relation with sex steroid
biosynthesis. General and Comparative Endocrinology, 26:70-78.
(40) Halldin K., Holm L., Ridderstrale Y., and Brunstrom B. (2003) Reproductive
impairment in Japanese quail (Coturnix japonica) after in ovo exposure to
o,p'-DDT. Archives of Toxicology, 77(2): 116-122.
(41) Halldin K., Axelsson J., and Brunstrom B. (2005) Effects of endocrine modulators
on sexual differentiation and reproductive function in male Japanese quail. Brain
Research Bulletin, 65(3):211-218.
(42) Hamilton T.H. (1961) Studies on the physiology of urogenital differentiation in the
chick embryo. The Journal of Experimental Zoology, 146:265-274.
(43) Harada T., Enomoto A., Boorman G.A., and Maronpot R.R. (1999) Liver and
gallbladder. In: Pathology of the Mouse, Maronpot RR, ed., Cache River Press,
Vienna, IL, p. 139.
(44) Holdway D.A., Hefferman J., and Smith A. (2008) Multigeneration assessment of
nonylphenol and endosulfan using a model Australian freshwater fish, melanotaenia
fluviatilis. Environmental Toxicology, 23(2):253-262.
(45) Huss D., Poynter G., and Lansford R. (2008) Japanese quail (Coturnix japonica)
as a laboratory animal model. Lab Animal, 37(11):513-519.
(46) Hutson J.M., Ikawa H., and Donahoe P.K. (1982) Estrogen inhibition of Miillerian
inhibiting substance in the chick embryo. Journal of Pediatric Surgery, 17:453-
458.
Page 147 of 155
-------
Final July 2015
(47) Kaku A., Chang C., Tamura T., Okamoto T., and Yoshimura Y. (1993)
Immunolocalization of androgen receptor in the cloacal gland of male Japanese
quail (Coturnix coturnix japonica). Japanese Poultry Science, 30:413-418.
(48) Kalender Y., Kalender S., Uzunhisarcikli M., Ogutcu A., Agikgoz F., and Durak D.
(2004) Effects of endosulfan on B cells of Langerhans islets in rat pancreas.
Toxicology, 200(2-3):205-211.
(49) Kiss A., Jurani M., and Kvaltinova Z. (1983) Distribution of catecholamines in
individual hypothalamic nuclei of Japanese quail. Journal of Endocrinology,
99:217-NP.
(50) Kohler P., Grimley P.M., and O'Malley B.W. (1969) Estrogen-induced
cytodifferentiation of the ovalbumin-secreting glands of the chick oviduct. Journal
of Cell Biology, 40:8-27.
(51) KondoK. (1959) Breed differences in the response of the male reproductive
organs of chicken embryos to estrogen. Journal of Experimental Zoology, 141:1-
14.
(52) KondoK. (1963) The effect of estrogen treatment on male embryos from
reciprocal crosses between breeds of fowl with high and low response. Journal of
Experimental Zoology, 154:329-337.
(53) Kondo, K. (2005) Breed differences in the response of the male reproductive
organs of chicken embryos to estrogen. Journal of Experimental Zoology,
141(1): 1-14.
(54) Koyanagi F, Masuda S., and Nishiyama H. (1986) Feminization of the male left
gonad of chicks by injection of estrogen during incubation, especially on the
interrelations between doses and morphological and histological changes.
Proceedings of Faculty of Agriculture - Kyushu Tokai University, 5:61-69.
(55) Kuwana T. (1993) Migration of avian primordial germ cells toward the gonadal
anlage. Development, Growth & Differentiation, 35(3):237-243.
(56) Lin M. and Jones R.C. (1990) Spatial arrangement of the stages of the cycle of the
seminiferous epithelium in the Japanese quail, Coturnix coturnix japonica. Journal
of Reproduction and Fertility, 90:361-367.
(57) Lin M. and Jones R.C. (1992) Renewal and proliferation of spermatogonia during
spermatogenesis in the Japanese quail, Coturnix coturnix japonica. Cell and Tissue
Research, 267:591-601.
(58) Lin M. and Jones R.C. (1993) Spermiogenesis and spermiation in the Japanese
quail {Coturnix coturnix japonica). Journal of Anatomy, 183:525-535.
Page 148 of 155
-------
Final July 2015
(59) Lin M., Jones R.C., and Blackshaw A.W. (1990) The cycle of the seminiferous
epithelium in the Japanese quail (Coturnix coturnix japonica) and estimation of its
duration. Journal of Reproduction and Fertility, 88:481-490.
(60) Lutz-Ostertag Y. and Kantelip J.P. (1971) Sterilizing action of endosulfan
(Thiodan) (organochlorinated insecticide) on the gonads of chick and quail embryos
in vivo and in vitro. Comptes Rendus des Seances de la Societe de Biologie et de
ses Filiales, 165(4):844-848.
(61) MacLaughlin D.T., Hutson J.M., and Donahoe P.K. (1983) Specific estradiol
binding in embryonic Miillerian ducts: a potential modulator of regression in the
male and female chick. Journal of Endocrinology, 113(1):141-145.
(62) Maeda T. and Yoshimura Y. (2002a) Effects of ethynylestradiol injection into
maternal Japanese quail (Coturnix japonica) on male reproductive function of the
F1 generation. Journal of Poultry Science, 39:310-315.
(63) Maeda T. and Yoshimura Y. (2002b) Effects of ethynylestradiol administration on
sperm motility and reproductive function in male Japanese quail (Coturnix
japonica). Journal of Poultry Science, 39:27-33.
(64) Massa R., Davies D.T., and Bottoni L. (1980) Cloacal gland of the Japanese quail:
androgen dependence and metabolism of testosterone. Journal of Endocrinology,
84:223-230.
(65) Masuda S. and Koyanagi F. (1998) Effect of estrogen on sexual differentiation of
the medial preoptic area in chicken. Japanese Poultry Science, 35:245-248.
(66) Masuda S. and Koyanagi F. (2001) Changes in the ovotestes transformed by
exogenous estrogen during incubation in the male chicken after hatching. Japanese
Poultry Science, 38:J28-J35.
(67) Maraud R., Vergnaud O., Castet M.C., and Stoll R. (1975) Influence of an
antiandrogen, cyprotereon acetate, on the morphogenesis of the epididymis in the
chick. Comptes Rendus des Seances de la Societe de Biologie et de ses Filiales,
169:541-543.
(68) Masuda S. and Koyanagi F. (2002) Effects of exogenous estrogen on the
differentiation and development of the right oviduct in female chickens. Japanese
Poultry Science, 39:1-7.
(69) McFarland L.Z., Warner R.L., Wilson W.O., and Mather F.B. (1968) The cloacal
gland complex of the Japanese quail. Cellular and Molecular Life Sciences
(CMLS), 24(9): 941-943.
(70) McLelland J. (1991) A Color Atlas of Avian Anatomy. W.B. Saunders Company,
Philadelphia, 127 pages.
Page 149 of 155
-------
Final July 2015
(71) Mezes M., Surai P., Salyi G., Speake B.K., Gaal T., and Maldjian A. (1997)
Nutritional metabolic diseases of poultry and disorders of the biological antioxidant
defense system. Acta Veterinaria Hungarica, 45(3):349-360.
(72) Moreng G.R., Cain J.R., and Warner R.L. (1980) The effect of adrenal glands on
ovulation in Japanese quail (Coturnix coturnix japonica). Poultry Science,
59(12):2760-2764.
(73) Morton D., Sellers R.S., Barale-Thomas E., Bolon B., George C., Hardisty J.F.,
Irizarry A., McKay J.S., Odin M., Teranishi M. (2010) Recommendations for
pathology peer review. Toxicologic Pathology, 38(7): 1118-1127.
(74) Niemann L., Selzsam B., Haider W., Gericke C., and Chahoud I. (2004) Effects of
vinclozolin on spermatogenesis and reproductive success in the Japanese quail
(iCoturnix coturnix japonica). Archives of Environmental Contamination and
Toxicology, 46(4):528-533.
(75) Oliveira-Filho E.C., Grisolia C.K., and Paumgartten F.J. (2009) Effects of
endosulfan and ethanol on the reproduction of the snail Biomphalaria tenagophila:
a multigeneration study. Chemosphere, 75(3):398-404.
(76) Pageaux J.F., Laugier C., Pal D. and Pacheco H. (1984) Development of the
oviduct in quail during sexual maturation in relation to plasma concentrations of
oestradiol and progesterone. Journal of Endocrinology, 100:167-173.
(77) Panzica G.C., Vigilietti-Panzica C., and Balthazart J. (1996) The sexually
dimorphic medial preoptic nucleus of quail: a key brain area mediating steroid
action on male sexual behavior. Frontiers in Neuroendocrinology, 17:51-125.
(78) Panzica G., Vigilietti-Panzica C., and Balthazart J. (2001) Sexual dimorphism in
the neuronal circuits of the quail preoptic and limbic regions. Microscopy Research
and Technique, 54:364-374.
(79) Perrin F.M.R., Stacey S., Burgess A.M.C., and Mittwoch U. (1995) A quantitative
investigation of gonadal feminization by diethylstilbestrol of genetically male
embryos of the quail Coturnix coturnix japonica. Journal of Reproduction and
Fertility, 103:223-226.
(80) Presibella K.M., Kita D.H., Carneiro C.B., Andrade A.J., and Dalsenter P.R.
(2005) Reproductive evaluation of two pesticides combined (deltamethrin and
endosulfan) in female rats. Reproductive Toxicology, 20(1):95-101.
(81) Quinn M.J., Lavoie E.T., and Ottinger M.A. (2006) Reproductive toxicity of
trenbolone acetate in embryonically exposed Japanese quail. Chemosphere,
66(7): 1191-1196.
Page 150 of 155
-------
Final July 2015
(82) Razia S., Soda K., Yasuda K., Tamotsu S., and Oishi T. (2005) Effects of estrogen
(17 beta-estradiol) and p-nonylphenol on the development of immune organs in
male Japanese quail. Environmental Science, 12(2):99-110.
(83) Riddell C. (1987) Avian Histopathology. American Association of Avian
Pathologists, Saskatoon, 152 pages.
(84) Rissman E.F., Ascenzi M., Johnson P. and Adkins-Regan A. (1984) Effects of
embryonic treatment with oestradiol benzoate on reproductive morphology,
ovulation and oviposition and plasma LH concentrations in female quail. Journal
of Reproduction and Fertility, 71:411-417.
(85) Ro S. and Kondo K. (1977a) Inhibitory effect of exogenous estrogen on the
testicular size of the embryo in the Japanese quail. Japanese Journal of
Zootechnical Science, 48:13-21.
(86) Ro S. and Kondo K. (1977b) Female like gonadal patterns of the male embryos of
Japanese quail with a high dose of exogenous estrogen to early stage of incubation.
Japanese Journal of Zootechnical Science, 48:325-330.
(87) Ro S. and Kondo K. (1977c) Variations in feminization of the gonads of male
embryos given various amounts of estrogen on day 4 of incubation in the Japanese
quail. Japanese Journal of Zootechnical Science, 48: 766-771.
(88) Ro S. and Kondo K. (1979) The effects of prenatal treatment of exogenous
estrogen on the postnatal development of the sexual organs and reproductive ability
in the male Japanese quail. Japanese Journal of Zootechnical Science, 50(11): 821 -
832.
(89) Rosenstrauch A., Weil S., Degen A. A., and Friedlander M. (1998) Leydig cell
functional structure and plasma androgen level during the decline in fertility in
aging roosters. General and Comparative Endocrinology, 109:251-258.
(90) Ruffins S.W., Martin M., Keough L., Truong S., Fraser S.E., Jacobs R.E., and
Lansford R. (2007) Digital three-dimensional atlas of quail development using
high-resolution MRI. TSW Development and Embryology, 2:47-59.
(91) Satterlee D.G. and Marin R.H. (2004) Photoperiod-induced changes in cloacal
gland physiology and testes weight in male Japanese quail selected for divergent
adrenocortical responsiveness. Poultry Science, 83(6): 1003-1010.
(92) Sayler A. and Wolfson A. (1967) Avian pineal gland: progonadotropic response in
the Japanese quail. Science, 158(3807):1478-1479.
(93) Scheib D. (1983) Effects and role of estrogens in avian gonadal differentiation.
Differentiation (Supplement), 23:87-92.
Page 151 of 155
-------
Final July 2015
(94) Scheib D., Guichard A., Mignot T.M., and Reyss-Brion M. (1985) Early sex
differences in hormonal potentialities of gonads from quail embryos with a sex-
linked pigmentation marker: an in vitro radioimmunoassay study. General and
Comparative Endocrinology, 60(2):266-72.
(95) Scheib D. and Reyss-Brion M. (1979) Feminization of the quail by early
diethylstilbestrol treatment: histoenzymelogical investigations on steroid
dehydrogenases in the gonads. Archives d'Anatomie Microscopique et de
Morphologie Experimental, 68:85-98.
(96) Seiwert C.M. and Adkins-Regan E. (1998) The foam production system of the
male Japanese quail: characterization of structure and function. Brain, Behavior
and Evolution, 52:61-80.
(97) Shackelford C.C., Long G., Wolf J., Okerberg C., and Herbert R. (2002)
Qualitative and Quantitative Analysis of Nonneoplastic Lesions in Toxicology
Studies. Toxicol Pathol. 30:93-96.
(98) Silva M.H. and Gammon D. (2009) An assessment of the developmental,
reproductive, and neurotoxicity of endosulfan. Birth Defects Research. Part B,
Developmental and Reproductive Toxicology, 86(1): 1-28.
(99) Singh S. and Chaturvedi C.M. (2008) Changes in vasotocin immunoreactivity of
paraventricular nuclei and adrenal function of Japanese quail in relation to different
phases of sexual development. Domest Anim Endocrinol, 34(3):
293-300.
(100) SinhaN., NarayanR., ShankerR., and SaxenaD.K. (1995) Endosulfan-induced
biochemical changes in the testis of rats. Vet Hum Toxicol, 37(6):547-549.
(101) Singh N.D., Sharma A.K., Dwivedi P., Patil R.D., and Kumar M. (2008)
Experimentally induced citrinin and endosulfan toxicity in pregnant Wistar rats:
histopathological alterations in liver and kidneys of fetuses. Journal of Applied
Toxicology, 28(7):901-907.
(102) Smith P.H. (1974) Pancreatic islets of the Coturnix quail. A light and electron
microscopic study with special reference to the islet organ of the splenic lobe.
AnatatomicalRecord, 178(3): 567-585.
(103) The Society of Toxicologic Pathologists (1991). Peer review in toxicologic
pathology: some recommendations. Toxicologic Pathology, 19,290-292.
(104) The Society of Toxicologic Pathologists (1997). Documentation of pathology
peer review. Position of the Society of Toxicologic Pathologists. Toxicologic
Pathology, 25 (6), 655.
Page 152 of 155
-------
Final July 2015
(105) Tixier-Vidal A., Follet B.K., and Farner D.S. (1968) The anterior pituitary of the
Japanese quail, Coturnix coturnix japonica. Cell and Tissue Research, 92(4):610-
635.
(106) Ukeshima A. and Fujimoto T. (1991) A fine morphological study of germ cells
in symmetrically developing right and left ovaries of the chick. Anatomical
Record, 230:378-386.
(107) Ukeshima A. (1994) Abandonment of germ cells in the embryonic chick ovary:
TEM and SEM studies. Anatomical Record, 240:261-266.
(108) Ukeshima A. (2003) Ultrastructure of the ovarian germ cells in the quail
embryos, with special reference to the oocytes. Okajimas Folia Anatomica,
Japan, 80(4): 85-92.
(109) Van Nassauw L., CallebautM. (1991) Structural and immunohistochemical
aspects of the postovulatory follicle in Japanese quail. Anatomical Record,
229(l):27-30.
(110) Vigilietti-Panzica C., Panzica G.C., Fiori M.G., Calcagni M., Anselmetti G.C.,
and Balthazart J. (1986) A sexually dimorphic nucleus in the quail preoptic area.
Neuroscience Letters, 64:129-134.
(111) Wakamatsu M., Yamagata T., Namikawa T., and Mizutani M. (2000) Hereditary
persistent right oviduct in the chicken PNP/DO line. Poultry Science, 79(8): 1075-
1081.
(112) Weng Q., Saita E., Watanabe G., Takahashi S., Sedqyar M., Suzuki A.K., Taneda
S., and Taya K. (2007) Effect of methimazole-induced hypothyroidism on
adrenal and gonadal functions in male Japanese quail {Coturnix japonica).
Journal of Reproductive Development, 53(6):1335-1341.
(113) Willey J.B. and Krone P.H. (2001) Effects of endosulfan and nonylphenol on the
primordial germ cell population in pre-larval zebrafish embryos. Aquatic
Toxicology, 54(1-2): 113-123.
(114) Wilson W.O. (1972) A review of the physiology of coturnix (Japanese quail).
World's Poultry Science Journal, 28:413-429.
(115) Wirth E.F., Lund S.A., Fulton M.H., and Scott G.I. (2002) Reproductive
alterations in adult grass shrimp, Palaemonetespugio, following sublethal,
chronic endosulfan exposure. Aquatic Toxicology, 59(l-2):93-99.
(116) Yamamoto S., Tamate H., and Itikawa O. (1967) Morphological studies on the
sexual maturation in the male Japanese quail (Coturnix coturnix japonica). II.
The germ cell types and cellular associations during spermatogenesis. Tohoku
Journal of Agricultural Research, 18:27-37.
Page 153 of 155
-------
Final July 2015
(117) Yoshimura Y. and Kawai, H. (2002) Structures and androgen receptor
localization in the testes and epididymis of Japanese quail hatched from the eggs
exposed to diethylstilbestrol. Journal of Reproduction and Development, 48:79-
85.
(118) Yoshimura Y., Tamura Y., and Nishikori M. (2000) Effects of diethylstilbestrol
intake during growing phase on the reproductive organs in Japanese quail
(Coturnix japonica). Japanese Poultry Science, 37: 323-333.
(119) Yoshimura Y., Chowdhury V.S., Fujita M., Maeda T., and Obitsu T. (2002)
Effects of Nonylphenol Injection into Maternal Japanese Quail (Coturnix
japonica) on the Female Reproductive Functions of F1 Generation. Journal of
Poultry Science, 39:266-273.
Page 154 of 155
------- |