Forms and prevalence of intersexuality and effects of environmental contaminants on sexuality in cricket frogs (Acris crepitans).

Cricket frogs (Acris crepitans) from several different sites in Illinois were collected to assess the effects of environmental contamination on the prevalence of intersex gonads. Of 341 frogs collected in 1993, 1994, and 1995, 2.7% were intersex individuals. There was no statistically significant relationship between the chemical compounds detected and cricket frog intersexuality. However, there was an association approaching significance (p = 0.07) between the detection of atrazine and intersex individuals. A comparison of reference sites with sites that had point polychlorinated biphenyl (PCB) and polychlorinated dibenzofuran (PCDF) contamination revealed a significant relationship between sex-ratio reversal and contamination with PCBs and PCDFs. The sex ratio of juvenile frogs studied from three sites with PCB and PCDF point contamination favored males over females, which was the opposite of the sex ratio in control ponds (p = 0.0007). The statistically significant correlation between organochlorine contamination and sex-ratio reversal suggests PCBs and PCDFs can influence cricket frog sexual differentiation. The current study suggests that in cricket frogs, sex ratios and the prevalence of intersex gonads are altered by environmental contamination.

Changes in thyroid gland morphology after acute acrylamide exposure.

High exposure to the acrylamide monomer has been associated with neuropathy and neurotoxic effects. Chronic lower exposure causes endocrine disruption associated with thyroid, testicular, and mammary tumors. To investigate mechanisms of endocrine disruption, short-term, low-level oral dosing studies were conducted. Weanling female Fischer 344 rats were acclimatized for two weeks before dosing. Controls were given distilled water by gavage and rats in other groups were given acrylamide at doses of 2 mg/kg/day and 15 mg/kg/day for 2 or 7 days by gavage. Twenty-four h after the last dose, the rats were killed by decapitation. Trunk blood was collected for hormone analyses and tissues for histopathological examination. There were no toxicity-related deaths, no clinical signs of toxicity, and no significant difference in the mean body weight of animal groups. Histopathological examination of select tissues showed no lesions of pathologic significance. Plasma thyroxine (T4), thyroid stimulating hormone (TSH), prolactin (PRL), and pituitary TSH and PRL analyses did not reveal significant changes between control vs. treated rats. In the 7-day study, however, there was a slight dose-dependent increase in plasma T4 and a slight dose-dependent decrease in plasma TSH. Thyroid gland morphometry showed a significant (p < 0.05) decrease in the colloid area and a significant increase (p < 0.05) in the follicular cell height of treated rats as compared to controls. The follicular area shrinkage was similar in both studies. These results show a very early endocrine response to very low levels of toxic insult and opens other venues to further investigate the mechanisms of endocrine disruption by acrylamide.

Canine distemper epizootic in lions, tigers, and leopards in North America.

Canine distemper virus (CDV) infection occurred in captive leopards (Panthera pardus), tigers (Panthera tigris), lions (Panthera leo), and a jaguar (Panthera onca) in 1991 and 1992. An epizootic affected all 4 types of cats at the Wildlife Waystation, San Fernando, California, with 17 mortalities. CDV-infected raccoons were thought to be the source of infection in these cats. Two black leopards died at the Naibi Zoo, Coal Valley, Illinois, and 2 tigers died at the Shambala Preserve, Acton, California. Initial clinical signs were anorexia with gastrointestinal and/or respiratory disease followed by seizures. Canine distemper virus was isolated from 3 leopards, 3 tigers, and 3 lions that died or were euthanized when moribund. Monoclonal antibody testing identified the virus isolates as CDV. Gross and histopathologic findings were similar to those found in canids with distemper with a few exceptions. There were fewer lesions in the brain, and there was a pronounced type 2 cell proliferation in the lung, with inclusion bodies and CDV antigen demonstrated by immunohistology. Neutralizing antibody to CDV was found in high titers in serum from most animals but was absent or was found only in low titers in some cats that succumbed after CDV infection. There was a marked difference in neutralizing antibody titers when tests were done with different strains of CDV.

Besnoitiosis of the reproductive tract of a Blue Duiker (Cephalophus monticola).

A mature male Blue Duiker that had been born in the United States was submitted for necropsy examination following a brief illness. On histologic examination of the reproductive tract several Besnoitia cysts were found in the epididymis, prostate and bulbourethral gland. The lack of an inflammatory response or any negative effect on fertility, based on histologic evaluation and breeding history, is in contrast with the severe orchitis, epididymitis and infertility of besnoitiosis in cattle. This is the first report of an autochthonous infection of Besnoitia in the United States as well as the first report of besnoitiosis in a Blue Duiker.

Intrathoracic granular cell tumour in a dog.

Multiple granular cell tumours occurred diffusely throughout the pleura of an adult German Shepherd Dog. The growths were scattered over the surface of the lungs, mediastinum and diaphragm. Histologically, the masses consisted of plump cords of cells with eosinophilic, weakly PAS-positive cytoplasmic granules. Electron microscopically, the granules consisted of a pleomorphic population of electron-dense granules. The predilection site in the dog is the tongue and this is the first report of an intrathoracic, pleural site of granular cell tumours in the dog.

Pathology of the corpus luteum of cows.

The corpus luteum (CL) is a transient endocrine organ which can manifest a number of pathologic conditions such as cysts, inflammation, adhesions, dysfunction and neoplasia. Luteal and follicular cysts are the most commonly encountered abnormalities and need to be distinguished from cysts within a normal CL. Inflammatory lesions are also frequently encountered and can be caused by viral, bacterial, or iatrogenic causes. If inflammation is severe, adhesions and subfertility/infertility can result. Luteal dysfunction is a broad classification of another pathologic condition encountered in the cow. Generally this results in abnormal production of progesterone or abnormal luteal lifespan resulting in infertility. Neoplasms are relatively rare in the CL but include both primary and metastatic tumors. Understanding the pathologic conditions that occur within the CL will allow a more accurate clinical assessment of these very dynamic endocrine structures.

Retrospective studies on the measurements, karyotyping and pathology of reproductive organs of bovine freemartins.

A retrospective study of 217 cases of bovine freemartinism was made. Of these, 14 cases were diagnosed clinically, 141 were karyotyped and 62 were subjected to macroscopical examination of the reproductive tract. Of the 62 cases, 43 were assessed by measuring the reproductive organs (size and/or weight) and 41 were examined microscopically. Complete information on gross, histopathological and karyotyping analysis was available for only 10 animals. Freemartins showed reduced measurements of all reproductive organs. The mean lengths of vulva and vagina were 6.4 cm and 8.1 cm, respectively. Vesicular glands were observed in 30 freemartins. Histologically, these glands had more fibrous and less glandular tissue than the normal male vesicular glands. Uterine changes were variable, ranging from aplasia (two animals) to normal. Likewise, endometrial glands ranged from hypoplastic (decreased number and size) to normal. Glandular and myometrial cells showed prominent nuclei and less than normal cytoplasm, indicating a non-functional status. Gonadal size was reduced and an epididymis was present in seven animals. Rete ovarii were prominent and frequently occupied two-thirds of the gonad. Undifferentiated sex cords and abundant interstitial cells were salient features of the freemartin gonad. Oocytes were not observed. Ovarian follicles were seen in six cases. Karyotypic analyses demonstrated XY chimaerism in all freemartins.

Endotoxemia in pregnant cows: Comparisons of maternal and fetal effects utilizing the chronically catheterized fetus.

We utilized the chronically catheterized bovine fetus to compare maternal and fetal responses to maternal lipopolysaccharide (LPS) infusion. Our hypothesis was that LPS-induced abortion was primarily a maternal luteolytic event with minimal transplacental fetal exposure. Fetal tibial arteries, amniotic, allantoic cavities and maternal carotid arteries were catheterized. Three cows had patent catheters with viable fetuses (190 to 200 days of gestation) 1 week after operation and were included in the study. Following a 2-day maternal and fetal baseline, 0.5 mug Salmonella typhimurium LPS/kg was infused into a maternal jugular vein over a 2-hour period. Maternal and fetal responses were monitored clinically, biochemically and hormonally. The maternal response consisted of marked increases in plasma prostaglandin F(2alpha) metabolite (PGFM), tumor necrosis factor (TNF), ACTH and cortisol with a dramatic maternal leucopenia within 2 hours. Progesterone concentrations decreased within 7 hours (P<0.05). The LPS was rapidly cleared from maternal circulation and no transplacental exposure was detected in the fetuses. Fetal responses to maternal endotoxemia consisted of increased ACTH and cortisol concentrations with delayed increases in PGE(2); TNF did not change in fetal fluids following maternal endotoxemia. There was a fetal leucocytosis within 2 hours. The results indicate that the fetus does not appear to play a major role in the pathogenesis of LPS-induced abortions. However, the role of maternal TNF in endotoxin abortion requires further study.

Fixation and staining of planaria for histological study.

Fixation and staining of planaria can affect the interpretation of histopathological changes following their exposure to various agents. We assessed several fixation protocols with various stains in planaria to determine an optimal combination. Planaria were fixed in each of the following: 10% neutral buffered formalin, 2.5%, glutaraldehyde, Bouin's, Zenker's, 70% ethanol, and relaxant. In addition, planaria were fixed in relaxant and postfixed in each of the fixatives above. Paraffin embedded sections from each fixation protocol were stained with hematoxylin and eosin (H & E), toluidine blue, periodic acid-Schiff (PAS), or phosphotungstic acid-hematoxylin (PTAH). Relaxant fixed planaria were also stained with Steiner's, Holmes, trichrome, Giemsa, Grocott's methenamine silver (GMS) and antibodies for intermediate filaments (cytokeratin, vimentin and desmin). Relaxant and Zenker's gave the best fixation with minimal artifacts. Formalin, glutaraldehyde, and ethanol were unacceptable because they caused contortions of the body, crenation, and a darkly pigmented epidermis. Gastroderm could be differentiated from stroma best when stained with H & E, toluidine blue and PTAH. Other organ systems differentially stained included the epidermis, marginal adhesion gland, nervous tissue, and muscle. PAS, Steiner's, Holmes, trichrome and the intermediate filament stains were not useful for planaria staining. The most morphological information was obtained with relaxant fixative and a combination of sections stained with H & E and PTAH.

Growth and ovarian function of weanling and yearling beef heifers grazing endophyte-infected tall fescue pastures.

Growth and ovarian function of crossbred beef heifers grazing low and high endophyte-infected tall fescue pastures were studied for 2 successive years. In April of each year, 20 weanling and 20 yearling heifers were included in the study. All heifers were weighed at 28-d intervals for 112 d. Blood samples were collected from each heifer on Day 0 and +7 of each of five 28-d periods and analyzed for progesterone concentration. Heifers with progesterone concentrations >/= 1.5 ng/ml on either or both Day 0 and +7 were classified as having normal cyclic ovaries. High endophyte-infected fescue pastures adversely altered the ovarian activity (P < 0.05) of weanling heifers in both years. In each trial, average weight gains were lower (P < 0.05) in yearling and weanling heifers grazing the high endophyte-infected pastures than in heifers grazing low endophyte-infected pastures. In 1992, heifers were synchronized with PGF(2alpha) administered on Days 101 and 112. Blood samples were collected on 0, 4, 8 and 12 d after the second PGF(2alpha) injection for progesterone analysis. Heifers grazing high and low endophyte-infected pastures were pastured separately with 4 bulls each and were given heatmount detectors. At 96 h, less estrus activity was observed (P < 0.10; power=0.63) in weanling heifers grazing the high vs. low endophyte pastures although pregnancy rates were similar for all groups. Progesterone concentrations suggested that weanling heifers on the high endophyte pastures had a higher incidence of luteal dysfunction after PGF(2alpha) synchronization. In summary, high endophyte-infected pastures decreased growth in both weanling and yearling heifers, ovarian activity and luteal function were adversely altered in weanling heifers with subsequent decreased estrus response to estrus synchronization.

Modification by polychlorinated biphenyls (PCBs) of cadmium induced lesions in the planarian model, Dugesia dorotocephala.

The appearance of abnormal growths on the planarian, Dugesia dorotocephala, in response to cadmium with and without pre-exposure to L-buthionine-R, S-sulfoximine (BSO) and concurrent exposure to the polychlorinated biphenyls (PCBs) Aroclor 1254, PCB 28, PCB 110 or PCB 126 is described. Pigmented rose thorn (PRT) lesions were non-invasive and appeared in response to PCBs. Post-head (PH) lesions developed in up to 100% of the animals within 6-20 days post-dosing, progressed rather rapidly and were highly invasive. Round tail tip (RTT) lesions appeared in lower frequencies within 10-30 days, but progressed extremely rapidly resulting in tail loss within 48 h. We have referred to these types of lesions as "tumors", but they are not necessarily characteristic of vertebrate neoplasms. PCBs interacted with cadmium in a complex way, in some cases increasing total lesions and decreasing time-to-lesion and in other cases having the opposite effects. A three-factor (PCB, PCB dose, Cd dose) nested analysis of variance model was used to determine lesion rates in order to compare PCB potencies as potentiators or antagonists. The Aroclor mixture was always the least potent co-toxicant but appeared to be the most potent antagonist; the coplanar PCB 126 was the most potent co-toxicant. The complex response surfaces and the lack of stoichiometry in dose-response relationships indicate that multiple mechanisms are responsible for PH and RTT lesions in planarians. These results emphasize the complexity of PCB toxicities and suggest further studies to validate the planarian model as a screen for combinations or environmental mixtures which may have altered biological potency in other species.

Evaluation of marking materials for cutaneous surgical margins.

Microscopic evaluation of the margins of excised cutaneous neoplasms is of paramount importance for determining that neoplastic tissue does not extend to the excision borders. Dyes or pigments that indelibly mark the tissue should be more reliable than sectioning techniques or suture markers for identifying the surgical margins before and after tissue processing. We evaluated 5 pigments to select a material that could be rapidly applied by surgeons, and readily identified on histologic section by the pathologists. Twenty normal canine skin specimens were assigned to each of 5 groups. Each group was treated with artists' pigments in acetone, India ink in acetone, alcian blue, typists' correction fluid, or a commercially available marking kit. Ten specimens within each group were marked before formalin fixation, and 10 were marked after fixation. Application properties, fixation and processing properties, and microscopic characteristics were evaluated for each material. Application properties were acceptable for all marking materials on unfixed specimens, and for alcian blue, India ink in acetone, and correction fluid on fixed specimens. Fixation and processing properties were acceptable for all materials except correction fluid. All marking materials survived fixation and processing, and colors were readily visualized under the microscope. Microscopic characteristics were acceptable for alcian blue, India ink in acetone, and the commercial kit. Overall, alcian blue was the best marking material, with India ink in acetone and the commercial kit also acceptable. Correction fluid and artist' pigments in acetone were not acceptable because pigment fragmentation and incomplete tissue coverage hindered microscopic evaluation of resection margins.

Influence of diet on the hematology and serum biochemistry of zinc-intoxicated mallards.

Changes in hematological and serum biochemistry parameters in female zinc (Zn)-dosed farm-raised mallards (Anas platyrhynchos) fed four different diets were examined. Sixty ducks received an average dose of 0.97 g of Zn in the form of eight, 3.30-mm diameter shot pellets containing 98% Zn and 2% tin, and another 60 ducks were sham-dosed as controls. Fifteen ducks from each of the two dosing groups were assigned to one of four dietary treatments: corn only, corn with soil, commercial duck ration only, or commercial duck ration with soil. Shot-pellet dissolution rates ranged from 7 mg/Zn/day to 27 mg/Zn/day. Regardless of diet, the Zn dose resulted in mortality; incoordination; paralysis and anorexia; decreased body, liver, pancreas, gonad, and gizzard weight; increased kidney weight; and macroscopic lesions. Zn-dosed ducks had a lower mean erythrocyte packed cell volume (PCV), higher mean reticulocyte count, and a greater number of individuals with immature and/or abnormal erythrocytes, than did control mallards. Mean total leucocyte counts were higher in Zn-dosed ducks than in controls. Zn-dosed ducks that had soil available had higher leucocyte counts than those without soil. Zn-dosed ducks were characterized by a marked heterophilia and relative lymphopenia. In Zn-dosed ducks, the mean lymphocyte count was highest in those provided a commercial duck ration, and lowest in those fed corn. In control ducks, the mean lymphocyte count was highest in ducks fed corn, and lowest in those provided soil along with a commercial duck ration. Zn-dosed mallards had higher serum aspartate aminotransferase and amylase levels, and lower alkaline phosphatase activities than control ducks. Serum phosphorus and uric acid concentrations were higher, and calcium, glucose, and total protein levels lower, in Zn-dosed ducks than in control ducks. Diet did affect serum calcium, phosphorus, total protein, and uric acid concentrations. Differences in erythrocyte and leucocyte parameters, serum enzyme activities, and metabolite concentrations were associated with dose and diet effects. Diets high in protein and other organic matter and calcium and phosphorus did not prevent or substantially alleviate Zn toxicosis in farm-raised mallard ducks.

Effects of lead, iron, and bismuth alloy shot embedded in the breast muscles of game-farm mallards.

Effects of five lead (Pb), iron (Fe), or bismuth (Bi)/tin (Sn) alloy shot embedded in the breast muscles of game-farm mallards (Anas platyrhynchos) were studied from 28 March 1994 through 27 March 1995. We detected no differences in the mean survival times, mean hematocrits, or mean body weights among the three shot types. Connective tissue encapsulated Pb and Bi/Sn shot but only slight changes occurred in tissues surrounding the shot. Recovered Pb and Bi/Sn shot were essentially unchanged in appearance and weight. A thin zone of "oxide" surrounded Fe shot with a slight inflammatory response and a small amount of scarring adjacent to the embedded shot. Fe shot decreased slightly in weight while embedded. Bacterial infections were absent in all dosed ducks. Mean weights of kidneys, livers, and gonads did not vary by type of shot. Kidneys and livers of Bi-dosed ducks had higher concentrations of Bi than in Pb- and Fe-dosed ducks. Muscle and blood showed no differences in Bi concentrations among doses. We found no histological dose-related effects in kidneys, liver, and gonads from the embedded shot.

Bacterial endotoxemia and reproductive effects in ruminants.

Gram-negative bacterial infections are fairly common in domestic ruminants. They can cause an endotoxemia with a variety of systemic signs, including fever, leukopenia, and ruminal stasis. Reproductive effects of endotoxemia include alterations in the estrous cycle and inability to maintain pregnancy. Diagnosis of endotoxemia is based largely on clinical evaluation. Confirming the diagnosis of endotoxin-induced reproductive alterations also is complicated by the lag period between clinical signs and abortion or infertility.

Clinical implications of rhinoceros reproductive tract anatomy and histology.

Reproductive tracts or tissues from five male black rhinoceroses (Diceros bicornis), two male white rhinoceroses (Ceratotherium simum), two male one-horned Asian rhinoceroses (Rhinoceros unicornis), seven female black rhinoceroses, and six female white rhinoceroses from multiple institutions were examined to characterize their anatomy and histology. Some observations and measurements were obtained from in situ tracts of intact animals before or during necropsy. Formalin-fixed tissues were dissected and examined histologically. Retrospective reproductive data from each rhinoceros was obtained from the institutions of origin. Reproductive histology of these species was similar to that of other mammals. Male accessory gland structure varied among species, and the Asian rhinoceros epididymis was more loosely attached and had larger duct diameters than did the epididymides of the African species. Although histology was typically mammalian, rhinoceros reproductive morphology combined characteristics of several different mammals. Defining this unique morphology of rhinoceroses may help in understanding their reproductive physiology and will effect the development of appropriate reproductive techniques.

Effects of feed restriction on fertility in female rats.

BACKGROUND: Feed restriction with its resultant body weight loss impacts the rodent estrous cycle; however, the manifestation of these changes in a regulatory study design has not been documented. This study reports the effects of feed restriction in the context of an FDA regulatory submission. METHODS: Adult female rats (n = 20/group; weighing approximately 200 g each) were provided rodent chow ad lib (control) or at 20, 15, 10, or 7.5 g/rat/day (g/day) during a 2-week pre-mating phase, throughout the mating phase, and up to gestation day (GD) 7. On GD 8, all animals were provided ad lib feed until necropsy on GD 14. Estrous cyclicity, mating, and fertility parameters were evaluated. RESULTS: Ad lib rats consumed approximately 20 and 28 g/day during the pre-mating and gestation phases, respectively. All measured fertility parameters in the 20 g/day group were similar to control values. In the 15 g/day group, body weight was reduced by 16% at 2 weeks, prolonged diestrus occurred, and fertility was compromised due to reductions in corpora lutea. Within 2 weeks, mean body weight in groups receiving < or = 10 g/day was reduced by > or = 29% compared to ad lib values, and overt changes in estrous cyclicity, mating, and fertility occurred. The 7.5 g/day group was not sustainable beyond the pre-mating phase. CONCLUSIONS: For this study type, feed intake at < or = 50% ad lib values (< or = 10 g/day) was inadequate due to the magnitude and rapidity of body weight effects. Estrous parameters appeared slightly more sensitive than functional measures, as body weight changes of approximately 16% appeared near the threshold of changing routinely calculated estrous cycle parameters and were later associated with reduced fertility. In general, body weight differences of 10-15% by themselves were not adverse to normal reproduction (20 g/day).

Overview of male reproductive pathology.

An understanding of form and function is important for examination of the male reproductive tract. A basic understanding of spermatogenesis and hormonal function in the reproductive tract is essential for the pathologists in this evaluation. Gross and histologic reproductive changes need to be distinguished from normal variation and correlated with the reproductive status of the animal. This is especially important when correlating histologic changes with organ weight and other reproductive parameters, such as seminal analysis data. Sexual maturity of animals and tissue handing can impact interpretation. Sexual immaturity of preclinical safety animals can present challenges for accurate identification of compound-related changes. Likewise, proper handling of unfixed reproductive tissues and appropriate selection of a fixation protocol are important in avoiding artifacts that may interfere with the microscopic evaluation. The histopathology technician needs to recognize testicular landmarks that allow for correct orientation at trimming so the pathologist can assess not only the morphology of seminiferous tubules but also the outflow tract. For the most effective evaluation of the male reproductive tract, the testes and epididymides should be examined concurrently. Although the term "staging" is often used inappropriately, the pathologist should review testicular tissues in a "stage-aware" manner. This article reviews gross and histologic changes of the male reproductive tract as well as tissue orientation and fixation to assist in accurate interpretation of potential treatment-related changes in male reproduction.