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

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

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

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

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

Severity grading should usually use the following system:
   * NR (not remarkable)
   * Grade 1 (minimal)
   * Grade 2 (mild)
   * Grade 3 (moderate)
   * Grade 4 (severe)

Findings that are not present are not graded (i.e., there is no Grade 0); however, if there are no findings for an entire tissue section, that section is scored as NR (not remarkable).

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

                     *       Discrete: these are changes that could be readily counted.  Examples include atretic follicles, oocytes in the testis, and clusters of apoptotic cells.

                     *       Spatial: these are changes that could be quantified by area measurements.  Includes lesions that are typically classified as focal, multifocal, coalescing, or diffuse.  Specific examples include granulomatous inflammation and tissue necrosis.

                     *       Global: these are generalized changes that would usually require more sophisticated measurement techniques for quantification.  Examples include increased hepatocyte basophilia, thyroid follicular cell hypertrophy, or quantitative alterations in cell populations.

Listed below are general guidelines for the use of a severity grading system, with examples of how the system could be applied to each of the above categories.  Please understand that the terms Discrete, Spatial, and Global are used for illustrative purposes only; it is not intended that these terms be incorporated into any diagnosis or grade.  It should be stressed that the examples below should be modified as needed for each particular type of change (diagnosis).

Grade 1:
         *       Discrete change example: 0 to 2 occurrences per microscopic field, or 1 to 2 occurrences per tissue section.

         *       Spatial change example: the change occupies a miniscule area of either a specific tissue type or the entire tissue section.

         *       Global change example: the least perceptible alteration relative to control animals or prior experience.

Grade 2:
         *       Discrete change example: 3 to 5 occurrences per microscopic field or tissue section.

         *       Spatial change example: the change occupies a larger area than Grade 1, but still less than or equal to 25% of either a specific tissue type or the tissue section.

         *       Global change example: the alteration is easily appreciated, but still not dramatic.

Grade 3:
         *       Discrete change example: 6 to 8 occurrences per microscopic field or tissue section.

         *       Spatial change example: the change occupies more than 25% but less than or equal to 50% of either a specific tissue type or the entire tissue section.

         *       Global change example: the alteration is dramatic, but a more pronounced alteration can be envisioned.

Grade 4:
         *       Discrete change example: 9 or more occurrences per microscopic field or tissue section.

         *       Spatial change example: the change occupies more than 50% of either a specific tissue type or the entire tissue section.

         *       Global change example: essentially, the most pronounced imaginable alteration.

At least some of the histomorphologic changes that have been associated with EDCs in wildlife are considered to be exacerbations of "normal", physiologic findings.  Whenever possible, the severity of a given change should be scored relative to the severity of the same change in concurrent control animals.  For each important (i.e., treatment-associated) finding, the severity scoring criteria should be stated in the Materials and Methods section of the pathology narrative report.  By convention, it is recommended that severity grading should not be influenced by the estimated physiologic importance of the change.  For example, the presence of two oocytes in the testis should not be graded as "severe", even if the pathologist considers this finding to be highly significant in terms of endocrine modulation.  The reason is that estimating the physiologic importance adds a further layer of subjectivity to the findings that complicates interlaboratory results comparisons.

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

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

      (5)  Data Interpretation.  Once the microscopic examinations have been completed and statistical analyses have been performed on the resulting data, the pathologist interprets the histopathologic findings.  The initial task is to determine which, if any, of the recorded findings are related to administration of the test article, and which are not.  The goal is to classify each type of recorded finding (i.e., diagnosis) into one of three categories: 1) Treatment-related, 2) Potentially treatment-related, and 3) Non-treatment-related.  Criteria for these determinations are listed below.

      (i)  Determining Relationship to Treatment.  A weight-of-evidence (WOE) approach is used to determine if a particular finding should be considered treatment-related.  Such evidence may include any or all of the following as available:

         *       Differences between groups of control and treated animals in terms of lesion prevalence and severity, utilizing statistical analytical results to test for significance as warranted.

         *       Ancillary data from the current study, involving information such as behavioral observations, liver and body weights, genotypic sex, time-to-metamorphosis, and age at sacrifice.

         *       Results from other submitted or pending agency studies.

         *       The at-large scientific literature, giving greater weight to studies in which the quality of the research can be established and is considered superior.

         *       Overall biological, physiological, and toxicological plausibility.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

             (9)  Atlas of Histopathologic Findings

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(e)  Fixative Formulations

                           Modified Davidson's Fixative
                            Formaldehyde (37-40%)	220 ml
                             Glacial acetic acid		115 ml
                              95% Ethyl alcohol		330 ml
                              Distilled water			335 ml

                   Davidson's Fixative (included for comparison)
                            Formaldehyde (37-40%)	200 ml
                                  Glycerol			100 ml
                             Glacial acetic acid		100 ml
                              Absolute alcohol		300 ml
                              Distilled water			300 ml

(f)  Automatic Processor Schedule
     The following is an example automated processor schedule that can be used to create paraffin-embedded sections of most types of quail tissues.

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

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

      (1)  Specimen Sampling and Processing

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

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

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

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

                                    Fig 130
                                    Fig 130
                                    Fig 129
                                    Fig 129

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

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

                                    Fig 132
                                    Fig 132
T
T
A
A
K
K
K
K
K
K
K
K
T
T
A
A
T
T
K
K
K
K
T
T
                                    Fig 133
                                    Fig 133

                                       A
A
A
A
O
O
K
K
K
K
O
O
K
K
K
K
K
K
K
K
                                    Fig 135
                                    Fig 135
                                    Fig 134
                                    Fig 134

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

                            Fig<PHONE_NUMBER>3137713
                            Fig<PHONE_NUMBER>3137713

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

      (3)  Histologic Figures

                                       T
T
E
E
SV
SV
                                    Fig 137
                                    Fig 137

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

                                      PG
PG
TH
TH
TH
TH
TG
TG
                                    Fig 138
                                    Fig 138
                                    Fig 139
                                    Fig 139

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

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

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

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

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

                                    Fig 143
                                    Fig 143

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

                                    Fig 144

                                    Fig 144

M
M
C
C

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

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

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

                                      Kcr
Kcr
SV
SV
T
T
MK
MK
SC
SC
T
T
MK
MK
Kcr
Kcr
                                    Fig 146
                                    Fig 146

Figure 146.  Transverse section through the proximal segment of the posterior carcass, at a more caudal level than in Fig. 141.  Structures evident in this image include the spinal cord (SC), spinal vertebra (SV), cranial kidney lobes (Kcr), mesonephric kidney remnants (MK), and left and right testes (T).  Bar = 250 um.

                                      Km
Km
Km
Km
SC
SC
SC
SC
NC
NC
                                    Fig 147
                                    Fig 147
SV
SV

Figure 147.  Transverse section through the middle segment of the posterior carcass of a hatchling male.  Structures evident in this image include the notochord (NC), spinal cord (SC), spinal vertebra (SV), and middle kidney lobes (Km).  The testes and adrenal glands are not present at this sectioning level.  The box indicates the position of the next figure.  Bar = 500 um.

                                       W
W
U
U
                                    Fig 148
                                    Fig 148

Figure 148.  Higher magnification of the boxed area in the preceding figure to illustrate the relationship between the ureter (metanephric duct) (U) and the Wolffian (mesonephric) duct (W).  In males, the bilateral Wolffian ducts will eventually become the left and right ductus deferens (in females, these ducts attenuate and become non-functional).  Bar = 25 um.

                                       W
W
U
U
Kca
Kca
SC
SC
SV
SV
Kca
Kca
W
W
U
U
                                    Fig 149
                                    Fig 149
Kca
Kca
Kca
Kca

Figure 149.  Transverse section through the distal segment of the posterior carcass of a hatchling male.  Structures evident in this image include the spinal cord (SC), spinal vertebra (SV), caudal kidney lobes (Kca), ureters (U), and Wolffian ducts (W).  Bar = 250 um.
                                      MK
MK
SV
SV
SC
SC
SV
SV
Kcr
Kcr
MK
MK
AG
AG
O
O
AG
AG
                                    Fig 150
                                    Fig 150
Kcr
Kcr

Figure 150.  Transverse section through the proximal segment of the posterior carcass of a hatchling female.  Structures evident in this image include the spinal cord (SC), spinal vertebra (SV), cranial kidney lobes (Kca), adrenal glands (A), mesonephric kidney remnants (MK), and ovary (O).  Note that the ovary is a flattened structure compared to the testis (Fig. 146), and that normally only the left ovary is present.  Bar = 500 um.

                                      Om
Om
Oc
Oc
AG
AG
                                    Fig 151
                                    Fig 151

Figure 151.  Higher magnification of the adrenal gland (AG) and ovary from the preceding figure.  In this image, the ovarian cortex (Oc) is paler than the medulla (Om), and the two layers are divided by an irregular border.  Bar = 100 um.

                                    Fig 152
                                    Fig 152
Om
Om
Oc
Oc
OC
OC
Om
Om

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

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

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

                                    Fig 154
                                    Fig 154
M
M
W
W
U
U

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

                                    Fig 155
                                    Fig 155
M
M
SV
SV
Kca
Kca
SC
SC
Kca
Kca

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

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

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

                                    Fig 157
                                    Fig 157

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

                                    Fig 158
                                    Fig 158

Figure 158.  Higher magnification of the bursa of Fabricius from the preceding figure.  The spherical bursal follicles appear to be populated predominantly by epithelial cells rather than lymphocytes.  It is not uncommon to see cells undergoing mitotic division and a few necrotic/apoptotic cells in the centers of follicles.  Bar = 25 um.

                                    Fig 159
                                    Fig 159
CG
CG

Figure 159.  Higher magnification of the cloacal glands (CG) from Fig. 156.  The glands are relatively underdeveloped in both male and female hatchlings.  Bar = 100 um.

                                    Fig 160
                                    Fig 160

Figure 160.  Higher magnification of the rudimentary cloacal glands (CG) from the preceding figure.  Bar = 25 um.

(h)	References.

   (1)            Adkins E.K.  (1975)  Hormonal basis of sexual differentiation in the Japanese quail.  Journal of Comparative Physiology and Psychology, 89:61-71.

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