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The swine leukocyte antigen (SLA) haplotype B is associated with increased penetrance of the tumor traits in Sinclair swine cutaneous melanoma (SSCM). We established a series of SinclairxHanford swine crosses to facilitate genetic mapping of the tumor-associated loci. In this study, the SLA diversity in the founding animals was characterized for effective selection of maximum tumor penetrance in the pedigrees. Using the sequence-based typing (SBT) method we identified a total of 29 alleles at five polymorphic SLA loci (SLA-1, SLA-3, SLA-2, DRB1 and DQB1) representing six class I and five class II haplotypes. We subsequently developed a rapid PCR-based typing assay using sequence-specific primers (PCR-SSP) to efficiently follow the SLA types of the crossbred progeny. In a total of 469 animals we identified three crossovers within the class I region and three between the class I and class II regions, which corresponded to recombination frequencies of 0.39% and 0.56%, respectively. We also confirmed the presence of two expressed SLA-1 loci in three of the class I haplotypes and were able to determine the relative chromosomal arrangement of the duplicated loci in two haplotypes. This study furthers our understanding of the allelic architecture and polymorphism of the SLA system and will facilitate the mapping of loci associated with the expression of SSCM., (Copyright 2009 Elsevier Ltd. All rights reserved.)

This paper reports the quantitative analysis of the historical database of a herd of Sinclair swine affected by cutaneous malignant melanoma. The herd was under partial and non-systematic selection for melanoma susceptibility (animals having at least one tumour during the first 6 weeks of life). Weighted selection differentials for the number of tumours at birth and the number of tumours at 6 weeks were generally positive and between -0.43 and 4.76 tumours for the number of tumours at 6 weeks. Estimates of the heritability for number of tumours at birth and at 6 weeks using 1934 animals were 0.27 (+/-0.03) and 0.25 (+/-0.03), respectively. The estimate of the genetic correlation between these two traits was 0.95 (+/-0.03). Genetic trends were positive for the number of tumours at birth and at 6 weeks. In spite of positive selection differentials and a moderate heritability, there was a negative phenotypic trend in the number of tumours. Natural selection might be acting in a direction opposite to artificial selection in the Sinclair herd. The slopes of the regression of the number of tumours at birth, at 6 weeks, and melanoma susceptibility on individual inbreeding coefficients were non-significant, indicating no evidence of dominance. The number of live-born pigs was lower in litters from parents susceptible to the disease (p < 0.01).

Recently we proposed the hypothesis that extensive telomeric association of chromosomes is an early manifestation of cell death and asked whether there are extensive telomeric associations present in metaphases of the spontaneously regressing Sinclair swine cutaneous melanoma (SSCM). Our results indicate that early passage SSCMs, in the accelerated growth phase, do not show telomeric associations but do have numerical and other specific structural abnormalities. However, the same melanoma cell lines at late passages or melanomas obtained from middle- and old-aged Sinclair swine show extensive telomeric associations in the form of dicentric, multicentric, and ring configurations. Such abnormal structures are present mostly in metaphases that are hyperploids. Increasing frequencies of apoptotic bodies were also observed in higher passage tumor cell lines obtained from younger animals or in melanomas obtained from older animals. The polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay shows no detectable telomerase activity in any of these regressing swine melanoma cell lines, neither in normal swine skin fibroblasts nor in nevi. However, the fetal swine (i.e., non-regressing) melanoma cells show telomerase activity. Fluorescence in situ hybridization (FISH) results using the commercially available human telomeric repeat DNA probe indicate a reduction of telomeric signals in metaphase and interphase cells of regressing melanomas. From these observations we conclude that spontaneous regression of SSCM is associated with the loss of telomerase activity and a reduction of telomeric repeats that results in the formation of multicentric and ring configurations. Such abnormal chromosome configurations are lost, following the breakage-fusion-bridge-cycles, and result in extensive DNA fragmentation, as shown by laddering experiments, and, finally, cell death.

Adrenocortical responsiveness to turning stress was examined in wild, reproductively-active olive ridley sea turtles (Lepidochelys olivacea) in relation to their mass nesting (arribada) behavior. We hypothesized that the high sensitivity threshold (HST) observed in ovipositing sea turtles is associated with a diminished sensitivity of the hypothalamo-pituitary-adrenal (HPA) axis to stressful stimuli in arribada females. We tested this hypothesis by determining whether arribada females exhibited an increased activation threshold of the HPA axis to an imposed stressor (turning stress). Mean basal corticosterone (B) and glucose levels were below 1.0 ng/ml and 60 mg/dl, respectively. Basal B remained unchanged throughout a 24-hr period in basking females. Most animals responded to turning stress with elevated mean B levels (up to 6.5 ng/ml after 6 hr) and no increase in circulating glucose. Nearly 50% of females (and none of the males) were refractory to the stimulation. Males exhibited the most rapid response, with B levels significantly elevated by 20 min over basal levels. Among females, arribada and solitary nesters exhibited a slower rate of response than basking, non-nesting animals. These results demonstrate that olive ridleys exhibit stress-induced changes in circulating B which are slower than those observed in most reptilian and in mammalian, avian, and piscine species. Furthermore, the presence of refractory females and the relatively slower increase in B in arribada and solitary nesters indicate a hyporesponsiveness of the HPA axis to turning stress in nesting olive ridleys. The hyporesponsiveness may be part of a mechanism to facilitate arribada nesting. J. Exp. Zool. 284:652-662, 1999., (Copyright 1999 Wiley-Liss, Inc.)

The seasonal reproductive cycle of the Kemp's ridley sea turtle (Lepidochelys kempi) was studied under seminatural conditions at the Cayman Turtle Farm, Grand Cayman, British West Indies, from June 1987 to July 1988. Male L. kempi displayed a prenuptial rise in serum testosterone 4 to 5 months prior to the mating period (March). Male testosterone then declined sharply during the mating period. Female L. kempi also displayed a prenuptial rise in serum testosterone, estradiol, and total calcium 4 to 6 months prior to the mating period (March). Female testosterone and estradiol declined during the nesting period (April to July) immediately following the mating period (March). Elevated levels in female estradiol and total calcium corresponded with the period of vitellogenesis as determined from gel electrophoresis and ultrasonography. Serum thyroxine also fluctuated seasonally with elevated levels observed in females associated with the period of vitellogenesis. L. kempi displayed a distinct seasonal reproductive cycle in captivity. Nesting in the captive study group corresponded with nesting in the wild population at Rancho Nuevo, Mexico (April to July). Female endocrine cycles during the nesting period were similar to those observed in the wild population.

The spontaneous regression of melanoma in Sinclair miniature swine involves the replacement of tumours by pigmented cells, hitherto interpreted as pigment-laden macrophages (PLMs). We hypothesized that these residual cells are terminally differentiated melanoma cells, not monocyte-derived macrophages. Swine melanoma explants with no regression were transplanted into severe combined immunodeficient (SCID) mice. Harvested transplant sites were examined by routine light and electron microscopy techniques. Paraffin sections were also stained with Hoeschst dye and examined by fluorescence microscopy. All but one site had completely regressed and were replaced by PLM-like cells. Hoeschst staining indicated they were of swine, not mouse, origin. The ultrastructural features of the single, partially regressed lesion demonstrated many premelanosomes in these cells. We conclude that tumour differentiation is an important mechanism of regression in the Sinclair swine melanoma model.

Selectively-bred Sinclair miniature swine exhibit a high incidence of congenital malignant melanoma which either proves fatal (10-15% of tumor-bearing piglets) or spontaneously regresses with a biphasic immunological phenomenon (85-90%) and no recurrence of malignancy. Mononuclear leukocytes were isolated from cutaneous melanomas and peripheral blood specimens collected from melanomatous (tumor-bearing) Sinclair swine during second-phase regression, and from peripheral blood specimens collected from non-melanomatous (tumor-free) Sinclair swine and control Hanford swine. Leukocyte identities were determined with single- and dual-parameter indirect immunofluorescence assays via flow cytometry. Assays for the specific surface antigens CD45, CD2, CD4, CD8, CD1, MHC class II, and N1 were employed to develop immunophenotypic profiles within the gated lymphocyte clusters from each TIL and PBL suspension. Significantly more CD8+ T-lymphocytes were identified in TIL suspensions than in peripheral blood leukocyte (PBL) suspensions (P < and = 0.05), regardless of breed or tumor status. Conversely, PBL suspensions contained significantly higher percentages of CD4+ T-lymphocytes than the levels found in TIL suspensions (P < and = 0.05). Virtually all TIL were MHC class II+, whereas the percentages of PBL expressing this antigen were markedly lower (P < and = 0.05). The percentages of T-lymphocytes co-expressing CD4 and CD8, a normal subset unique to swine, were generally consistent in all TIL and PBL suspensions examined. The results of this study have firmly established the immunophenotypic identities of cells associated with the second-phase regression phenomenon of this melanoma and have identified specific variations in the leukocyte profiles of the respective TIL and PBL suspensions.

The role of apoptosis in the spontaneous regression of Sinclair swine melanoma was investigated in vitro with swine melanoma cell lines. Growth characteristics and sensitivity to cycloheximide-induced apoptosis were determined in melanoma cell lines derived from tumours that were progressing or undergoing regression in vivo. In contrast to cell lines derived from progressing tumours, those derived from regressing tumours showed induction of apoptosis; this phenomenon was dependent on dose but independent of cell growth stage in culture. Chromatin condensation, cell shrinkage, and fragmentation into apoptotic bodies, as well as DNA fragmentation into large kilobase fragments, occurred in cell lines from regressing tumours but not from progressing tumours. These findings suggest that swine melanoma cells are inherently more sensitive to cell death during tumour regression. The apoptosis-sensitive and resistant cell lines will be important for further studies of the roles of cell signalling pathways and gene expression in tumour regression.

Background: Detailed histopathologic studies of melanomas occurring in neonatal Sinclair miniature swine have demonstrated a remarkable similarity to human melanoma. A significant difference is the predictable, complete regression of primary and metastatic tumors that occurs in all animals by early adulthood (1 to 2 years). Prior histopathologic descriptions of regression in this model have been incomplete with regard to the time of onset and chronologic sequence of events. This lack of data makes it difficult to plan studies of regression mechanisms especially when requiring the harvesting of tumor tissue., Experimental Design: By routine histologic methods, 94 tumors from 46 piglets were evaluated for the degree of regression, presence of pigment-laden macrophages, and presence of lymphocytes. One or more punch biopsies were performed on 51 tumors before excision, for a total of 256 biopsies., Results: Regression took place in two phases. The first phase began during the 4th week after birth; was preceded by a rapid, massive infiltration of pigment-laden macrophages; and was most active during the 2nd month. Significant numbers of lymphocytes were rarely seen in tumors during this phase of regression. In the vast majority of tumors, this initial regression activity was followed by regrowth of residual tumor usually appearing as emerging clones (intralesional transformation). The second phase of regression was characterized by asymmetrically distributed lymphocytic infiltration of the residual melanoma, and progressive regression of tumor over several months. Significant numbers of lymphocytes were not present in the majority of the tumors until the beginning of the 4th month., Conclusions: We conclude that regression of melanoma in this animal model is a complex event in which the immune system participates differentially during the natural history of the disease.

Mixed lymphocyte culture and serological typing of NIH and Sinclair miniature swine indicate that the two herds share a common SLA haplotype. NIH haplotype a (International Haplotype H10) appears identical to Sinclair haplotype B, which has significant effects on the penetrance of Sinclair swine cutaneous malignant melanoma (SSCM). Offspring of crosses between melanoma-bearing Sinclair swine homozygous for the B haplotype and non-melanoma NIH aa swine have tumour incidence identical to Sinclair melanoma BB x Sinclair non-melanoma BB offspring. Our results provide further support for the involvement of the swine leucocyte antigen (SLA) complex in the inheritance of SSCM, and identify a new source of non-tumour animals that have all of the genes for SSCM except those at the tumour-initiator locus.

Changes in serum concentrations of gonadotropins and gonadal steroids during the periovulatory period were monitored in green, Chelonia mydas, and loggerhead, Caretta caretta, sea turtles. Turtles were from natural populations that nest on a coral island on the Great Barrier Reef. After nesting, each turtle was transferred to a holding tank and held for a maximum of 8 days. A time series of blood samples was obtained from each of five sea turtles (three C. mydas and two C. caretta) starting immediately after nesting and then at approximately 12-hr intervals until the time of release. Prior to release back into the ocean, each turtle was examined by laparoscopy to verify that ovulation had occurred. Serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (PRO), and testosterone (T) in both species exhibited significant changes during this period. Surges of FSH, LH, and PRO were evident within approximately 20 to 50 hr after each turtle had nested. The significant change in FSH concentration during the periovulatory period is the first such report for a reptile. Coincident with maximal concentrations of FSH, LH, and PRO was a decline in T concentrations in both species. Estradiol-17 beta concentrations were near or below assay sensitivity in the C. mydas, whereas those in the C. caretta were detectable but exhibited no significant changes. The dynamic changes in FSH, LH, PRO, and T concentrations are consistent with the hypothesis that these hormones facilitate specific physiological events during ovulation and egg production.

Cutaneous malignant melanoma of Sinclair Swine (SSCM) is a heritable, congenital neoplasm which either proves fatal to the neonatal animal or undergoes spontaneous regression. Four SSCM cell lines, UISO-SSCM-433, UISO-SSCM-438, UISO-SSCM-5052, and UISO-SSCM-8093, were derived from biopsy specimens of primary tumors removed from swine at 26, 8, and 8 weeks of age, and 15 weeks gestation, respectively. Morphologic features, DOPA oxidase staining, and abnormal karyotype were suggestive of malignant melanoma. Each cell line was morphologically heterogeneous in culture with dendritic, spindle- and cuboidal-shaped cells. Pigmented melanosomes and DOPA oxidase activity were present in all cell lines at passages 20-22. UISO-SSCM-433 and UISO-SSCM-5052 contained hypodiploid and hypotetraploid sublines whereas UISO-SSCM-438 and UISO-SSCM-8093 were hypodiploid and hypotetraploid, respectively. At later passages, all cell lines presented evolutionary, karyotypic changes; the same chromosomes were involved in the alterations, however. Chromosomes 2, 6, 13, and 14 were the most affected, exhibiting numerical and structural alterations in all four cell lines. Despite the presence of multiple chromosomal anomalies in all cell lines, each with a unique set of chromosomal markers, clonal growth was not detected in soft agar, nor were any of the lines tumorigenic following s.c. inoculation in athymic mice. This suggests that the loss of malignant potential in SSCM may be inherent.

Epidermal growth factor (EGF) receptors were detected in plasma membrane preparations of equine hoof wall laminar tissue at concentrations comparable to that of equine liver. Scatchard analysis of the equilibrium binding data suggested the presence of two classes of EGF binding sites in most of the controls (plasma membranes from clinically normal horses); a high-affinity class and a more numerous low-affinity class. The dissociation constant of the low-affinity class of EGF-specific receptors (KD = 1 x 10(-9)M) is in reasonable agreement with other values established for the EGF receptor. The variability between individual estimates for the KD of the high-affinity receptor class precluded an accurate estimate for those sites. A possible explanation is discussed. The high-affinity binding sites were uniformly absent in plasma membranes prepared from horses affected by chronic laminitis. Autoradiographic analysis localised the EGF receptors primarily to the secondary epidermal laminae, with an apparent greater density over the proliferative basal keratinocytes. Little label was associated with the dermal or the keratinised primary epidermal laminae. Tissue from horses with chronic laminitis had EGF receptors located uniformly over the hyperplastic epidermal keratinocytes. These data suggest that an EGF-mediated response may be involved in the hyperproliferative response that is characteristic of chronic laminitis.

Initial results (Cancer Res., 48: 1708-1711, 1988) suggest that ovariectomy alters the proliferation of congenital exophytic melanoma in Sinclair swine. In order to provide a phenotypic basis for this effect, histopathological staging of 375 exophytic melanomas from 236 intact male and female Sinclair swine was compared with 114 lesions from 51 gonadectomized (6 weeks of age) and 90 lesions from castrated swine receiving s.c. silastic implants of estradiol during the first year of life. A rapid progression from actively proliferating tumor cells (Stages I-III) to regressive lesions (Stages IV-V) was virtually complete by 16 weeks of age in intact swine of both sexes. Bilateral ovariectomy reduced (P less than 0.02) tumor volume over time compared with intact animals. Replacement of estradiol in gilts increased tumor volume to that in intact animals. In contrast, neither bilateral orchidectomy nor estradiol replacement altered tumor volume in boars. Ovariectomy significantly reduced the tumor macrophage, keratinocyte, and fibroblast invasion that normally replaces tumor cells and expands tumor volume (Stages IV and V) with increasing age. Chronic exposure to estradiol reversed this process. Orchidectomy and estradiol replacement did not significantly affect histopathological stage in boars with increasing age. A significant number (20 of 41; 49%) of Stage III lesions (greater than 75% tumor cells) in swine of both sexes contained low but reproducibly measurable amounts of receptor for estrogen determined radiometrically and by enzyme-linked immunosorbent assay. These results suggest that reproductive steroids influence the natural history of these heritable lesions, and that the effect may be via alteration of host immune status.

Adult male loggerhead sea turtles, Caretta caretta, exhibited a "prenuptial" spermatogenic cycle that was coincident with increased concentrations of serum testosterone (T). Serum T was high during the months when migration and mating have been recorded for males. In contrast to females, males appear to be annual breeders. Nine reproductively active female C. caretta (as verified through laparoscopy) were tagged with sonic transmitters and were repeatedly bled prior to migration. Four months prior to the nesting season, the ovaries of reproductively active females had hundreds of vitellogenic follicles of approximately 1.5 cm in diameter (i.e., half the size of ovulatory follicles). Approximately 4-6 weeks prior to migration from feeding grounds to mating and nesting areas, serum estradiol-17 beta (E2) concentrations increased significantly and remained high for approximately 4 weeks, suggesting a period of increased vitellogenesis. During a 1- to 2-week period prior to migration, serum E2 decreased significantly, while serum T concentrations increased (at least) until the time of migration. Serum T, E2, and progesterone (PRO) were elevated during nesting if a turtle was going to nest again during that nesting season. During the last nesting of a season, turtles had low serum concentrations of T, E2, and Pro. The prenuptial pattern of gonadal recrudescence and gonadal steroid production in both male and female C. caretta contrasts with those of many temperate freshwater turtles, and this type of reproductive pattern may have been facilitated by adaptation to a tropical marine environment.

A heterologous radioreceptor binding assay (RRA) has been developed capable of detecting nanogram amounts of epidermal growth factor (EGF) receptor-binding activity in equine urine. The binding parameters of [125I]mEGF (murine EGF) to EGF receptors on equine plasma membranes are in good agreement with values from other EGF-RRA systems. The dissociation constant estimated from equilibrium methods (KD = 4 X 10(-10) M) is in reasonable agreement with that determined from the rate constants (KD = 6 X 10(-10) M) and is in good agreement with values determined in other species. The assay is specific for equine EGF (eEGF) receptor-binding activity and capable of detecting less than 0.34 nM eEGF receptor-binding activity in urine. Equine EGF receptor-binding activity in equine urine form adult horses varied widely between samples (8.5 +/- 6.5 nM). This variability was somewhat reduced when values were adjusted for dilutional effects using urine creatinine as an indicator (3.6 +/- 2.0 nanomoles/g creatinine). No significant differences were demonstrated between the means of EGF binding activity concentrations in clinically normal horses and horses affected by chronic laminitis.

Raynaud's phenomenon (RP) and equine laminitis in the horse are medical enigmas. Clinical and scientific data were compared to evaluate the degree of similarity that exists between these two peripheral vascular diseases. Data indicate that certain pathologic and pharmacologic aspects seem to have common features. Some of the correlations maybe due simply to both diseases having ischemia of the distal digits as a pathologic component. The exact etiology of the ischemia is not known for either disease. The results of this study suggest the hypothesis that RP and laminitis are the same disease in different species. This hypothesis can be tested more efficiently when the pathophysiology of both conditions is better documented. It is possible that comparative studies will promote advances in the understanding of both RP and laminitis. The fact that equine laminitis can be experimentally induced is of potential value in such future studies.

Cutaneous malignant melanoma of Sinclair swine (SSCM) is an inherited neoplasm present at birth in a majority of affected animals. We have characterized the swine leucocyte antigen (SLA) complex of the Sinclair herd at Texas A & M University using a one-way mixed lymphocyte test and found that one particular haplotype, arbitrarily identified as haplotype B, is associated with the expression of SSCM. Only a single dose of the B haplotype is required for a dominant allele at a 'tumour initiator' locus to be fully penetrant. In addition, swine homozygous for haplotype B develop more primary tumours between birth and weaning than those heterozygous for the B haplotype. Taken together, these findings indicate that tumour initiation, in utero, and expression between birth and weaning may involve different mechanisms.

It has been proposed that gonadotropin-releasing hormone (GnRH) stimulates Ca2+ entry by activation of voltage-independent, receptor-mediated Ca2+ channels in the rat gonadotroph. Little work has been done on the role of calcium in GnRH-induced luteinizing hormone (LH) release in species other than the rat. Therefore, this study was done to compare the effects of agents that alter Ca2+ or Na+ entry on LH release from calf anterior pituitary primary cells in culture. GnRH (100 ng/ml), Ca2+ ionophore A23187 (2.5 microM), and the depolarizing agent ouabain (0.1-10 microM) all produced significant increases (P less than 0.05) in LH release; these effects were significantly reduced when the cells were preincubated with the organic Ca2+ channel blockers nifedipine (1-10 microM) and verapamil (1-10 microM) and with Co2+ (0.01-1 mM). The effect of ouabain was inhibited by tetrodotoxin (TTX; 1-10 nM) as well as by nifedipine at 0.1-10 microM. In contrast to its effect on rat pituitary LH release, TTX significantly inhibited GnRH-stimulated LH release at 1-100 nM. These results suggest that GnRH-induced LH release may employ Ca2+ as a second messenger in bovine gonadotrophs and support recent speculation that GnRH-induced Ca2+ mobilization may in part be voltage dependent.

Serial physiological responses were examined for 150 min from captive collared peccaries during immobilization with ketamine hydrochloride. Rectal temperatures decreased significantly (P less than 0.01) during anesthesia. Serum concentrations of total proteins, albumin, cholesterol, alanine aminotransferase, and calcium declined significantly (P less than 0.05) during the first 45 min post-immobilization before stabilizing. Concentrations of lactate dehydrogenase and alkaline phosphatase in sera showed similar but nonsignificant (P greater than 0.05) trends. Inorganic phosphorus and aspartate aminotransferase concentrations increased significantly (P less than 0.05) throughout the trial. Concentrations of serum glucose and glucocorticoid during the immobilization period were highly variable between individuals. Serum electrolytes, urea nitrogen, creatinine, gammaglutamyl transferase and progesterone were not significantly (P greater than 0.05) affected by immobilization. Elevations in serum testosterone were noted. Results indicated appropriate sampling times relative to immobilization for assay of particular serum biochemicals and steroid hormones during investigations of the physiology of the collared peccary.

Responses of commercial laying hens to 16 management systems were examined for 10 laying periods of 28 days each. Twelve cage treatments consisted of housing three, four, or five hens in deep and shallow cages of different dimensions which provided .035 and .046 m2/hen. Four floor treatments housed 35 hens or 32 hens and three roosters at densities of .094 or .373 m2/bird, in two replicated pens each. Quantitative data were collected simultaneously for 23 production, physiological, and behavioral characteristics throughout the study. When comparing all caged with floor pen hens, caged hens had better (P less than .05) egg production rates (76.3 vs. 73.9%), gained more weight, had better feed efficiency, and had greater egg and egg shell weights than floor hens. All floor pen hens had higher (P less than .01) viability (98.9 vs. 95.0%), higher (P less than .01) plasma corticosterone levels (595.0 vs. 445.4 pg/ml), a greater (P less than .01) response to adrenocorticotropin (ACTH) challenges, and lower (P less than .01) antibody titers to Salmonella pullorum challenges than all caged hens. Caged hens preened, stood, crouched, and feather pecked more than floor hens, while floor hens drank and moved about more than caged hens. This study attempted to quantify production, physiological, and behavioral traits, all on the same flock of hens, in order to separate stressful from nonstressful management environments. Integration of all measurements indicates that properly managed caged hens were subjected to significantly fewer stressors than laying hens housed in floor pens, although the hens' well-being in the two environments could not be quantitatively compared.

A study was performed to determine the possible role of calmodulin (CaM) in regulating gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) release from the bovine pituitary using three structurally unrelated calmodulin antagonists. Primary calf anterior pituitary cell cultures (3 X 10(5)/well) were treated with either LH secretogogue (GnRH, 100 ng/ml; A23187, 2.5 microM; theophylline, 1 mM; prostaglandin E2, 1 microM; estradiol, 25 ng/ml; or KCl, 25 mM; final concentrations) or secretogogue plus CaM inhibitor in 1 ml Hanks' balanced salt solution plus 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid for 6 h. Significant (P less than 0.01) inhibition of GnRH- and A23187-stimulated LH release was obtained with calmidazolium (CMZ; 1-10 nM) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7; 1-10 microM). Both CMZ (10 nM) and W-7 (10 microM) significantly reduced (P less than 0.05) LH release by all of the other agents tested as well. In contrast, trifluoperazine (TFP; 0.1-100 microM) had no effect against most of the secretogogues tested. These results suggest that GnRH-stimulated LH release is in part a Ca2+-CaM-dependent process and may implicate a common CaM-dependent mechanism for LH release in general. The data also demonstrate a marked dichotomy in response between the phenothiazine, TFP, and other CaM antagonists in the calf pituitary.

In a study of the role of predisposing factors and gonadal hormones in the buller syndrome, it was found that weather, amount of space available to each steer, and entry weights of the steers were not associated with increased occurrence of bulling; however, the type and timing of administration of the hormonal implant, the number of steers in a pen, and the manner in which the steers were grouped after arrival at the feedlot were found to influence the incidence of the syndrome. Serum concentrations of estradiol and testosterone in the buller steers were found to be lower during bulling than after a short period of isolation and apparent recovery from the syndrome. It was concluded that the occurrence of bulling is related to the use of hormonal implants and certain feedlot management procedures but that it is apparently not related to increased serum estrogen or testosterone concentrations.

Pigmented tumors resembling cutaneous melanoma were first reported in Sinclair miniature swine in 1967. Since that time, carefully planned breeding has established that this is an inherited malignancy the natural history of which mimics human cutaneous melanoma in a number of ways. Because of these characteristics, miniature swine melanoma appears to be an effective model with which to investigate the mechanisms influencing initiation, growth, and progression of human melanoma. This investigation characterized histologically the cutaneous melanoma in miniature swine and compared the findings with human neoplasm. Primary cutaneous melanoma in swine has been reclassified and standardized according to the classifications currently in vogue in human melanoma. Our results suggest that the condition in miniature swine is histologically similar to that in humans. These observations will provide a basis for interpretation of the results derived in the biologic studies performed in this model.

Miniature Sinclair swine bearing cutaneous melanomas and nonmelanoma bearing littermate controls were tested for lymphocyte blastogenic response in a microculture assay. Lymphocytes were assayed for 3H-thymidine incorporation following stimulation with concanavalin A, phytohemagglutinin or pokeweed mitogen. Treatment with all mitogens revealed a significantly (p less than 0.05) reduced proliferative capacity in melanoma swine as compared to littermate controls. These results lend further support for the use of miniature Sinclair swine as a research model for human melanoma.

Forty Santa Gertrudis bulls were used to examine relationships among scrotal circumference, seminal quality, libido, and fertility [assessed as the percent pregnant of estrous females (PE rate) and the percent pregnant of females mated (PM rate)]. These bulls were selected from 220 two year old bulls to represent variations in scrotal circumference and seminal quality. Each of the 40 bulls were exposed to 100 cyclic Santa Gertrudis heifers for a 4-day (96 hr) breeding period. The number of estrous females available to each bull varied from 12 to 27. A breeding soundness examination (BSE) was conducted on each bull approximately 45 days prior to the 4-day breeding period and immediately after the 4-day breeding period. The three components of the BSE scroe (scrotal circumference, spermatozoal abnormalities and spermatozoal motility) were not significantly correlated with PE rate or PM rate at either evaluation. There was no significant correlation between PM rate and scrotal circumference; however, each of the 4 bulls having a scrotal circumference less than 30 cm had a PM rate below 31%. Relationships between seminal quality and PM rate were unclear and differed between the two evaluations. There was a trend for bulls having poor seminal quality at the first evaluation to improve by the second evaluation. Consequently, fluctuations in seminal quality between evaluations is one possible explanation for low correlations between seminal parameters and PM rate. Libido (number mated/number in estrus x 100) was positively correlated (r = 0.44) with PE rate. Using a stepwise regression procedure, the independent variables accounting for the most variation in PE rate (dependent variable) included libido, secondary spermatozoal abnormalities, and BSE score (r(2) = 0.44). Results of this study indicate that current methods of fertility evaluation did not accurately predict the fertility of individual Santa Gertrudis bulls as measured by PE rate and PM rate during a 4 day breeding period.

Metabolic and hormonal responses of eight adult male collared peccaries (Tayassu tajacu) to an ad libitum diet intake, or 25% of an ad libitum intake, were examined. Blood samples for hematological, serum-biochemical and hormonal profiles were collected at three week intervals during the nine week experiment starting 4 August 1983. Males fed on the restricted diet lost an average of 26% of their body weight during the trial, compared to a slight weight gain for those fed ad libitum. Characteristics of the red and white blood cell populations were not influenced by diet intake, with the exception of mean corpuscular volume, which was consistently lower amongst males fed on the restricted diet. Restricted food intake resulted in significantly elevated serum values for urea nitrogen, urea nitrogen:creatinine, urea index, alpha globulin:beta globulin, gamma globulin:albumin, nonesterified fatty acids, alkaline phosphatase and lactate dehydrogenase isozymes (LD1 and LD2). Restricted food intake resulted in significantly lowered serum values for total alpha globulin, alpha-1 globulin, total beta globulin, beta-1 globulin, beta-2 globulin, glucose, triglycerides, calcium, magnesium, sodium, chloride, copper and triiodothyronine. Serum levels of creatinine, total protein, albumin, alpha-2 globulin, uric acid, total bilirubin, cholesterol, aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase, lactate dehydrogenase, phosphorus, calcium:phosphorus, potassium, iron, zinc and thyroxine were unaffected by diet intake level. Semen evaluation indicated spermatogenesis was not affected by dietary restriction despite reductions in scrotal circumference and ejaculate gel volume. Serum testosterone levels were significantly lower among males fed on the restricted diet after nine weeks. These data suggest male libido might be depressed during poor range conditions, while maintenance of spermatogenesis might permit them to take immediate advantage of improved range conditions. Blood analysis of metabolic and hormonal function can provide useful information for predicting the adult male's nutritional and reproductive condition.

Blood samples and testicular measurements were obtained from 4-8 captive adult collared peccaries monthly for 18 months and from wild adult males during summer (N = 16) and winter (N = 22) seasons. Serum concentrations of testosterone were determined by radioimmunoassay. Semen samples were collected monthly by electroejaculation from captive males for 1 year. Serum testosterone concentrations and testicular measurements varied in a low-amplitude circannual pattern, with maximum mean testosterone concentrations in fall and winter (1150-1400 pg/ml) and minimum values in summer (500-700 pg/ml). Circannual rhythms appeared to be related to dominance. Serum testosterone levels in wild males generally were lower than in captive males, although this difference was not significant (P greater than 0.05). Semen characteristics did not exhibit a circannual rhythm. These results suggest that the male peccary remains reproductively fertile throughout the year, yet may undergo a facultative summer quiescence influenced by ambient temperature and social factors.

We have analyzed the association between host hormonal status and growth rate of congenital exophytic melanomas of Sinclair swine. The growth of multiple exophytic lesions during the first year of life of intact males, orchiectomized males, intact females, and ovariectomized females was quantitated using a proliferative index which assigned a numerical value to fixed increments of gains or losses in tumor volume. The proliferative index from 6 wk (gonadectomy at 6 wk) to 52 wk of age of each treatment group was statistically increased from 0 (P less than 0.01) except that of gonadectomized females. The proliferative indices from lesions in gonadectomized females were significantly lower than those from intact males, intact females, and gonadectomized males. A total of 93 exophytic tumors from 63 swine were biopsied and histopathologically staged according to the degree of progression or regression and analyzed as a function of animal age. There were no Stage I and only two Stage II lesions at the time biopsies were taken. Twenty-six of 32 (81.2%) Stage III tumors were found in swine of both sexes less than 26 wk of age of which 71.8% were found less than 10 wk of age, while 20 of 29 (68.9%) were Stage IV, and only 3 of 32 (9%) were Stage V lesions present in this age group (P less than 0.001, chi 2). Only 20.7% of Stage IV tumors were present prior to 6 wk of age. Preliminary results suggest that castration of either sex also altered tumor histopathology. Our data suggest that a reduction in gonadal steroid secretion was associated with a decrease in exophytic tumor growth rate and regression in animals of both sexes during the first year of life in Sinclair swine. The effect in female swine was due to significant reduction in the proliferative index over the first 6 mo of age.

Serum testosterone and estradiol 17-beta concentrations, and serum testosterone-to-estradiol ratio were evaluated in 15 dogs (greater than or equal to 5 years old) with perineal hernia (9 sexually intact males and 6 castrated males) and in 9 clinically normal sexually intact male dogs greater than or equal to 5 years old. There was no significant difference in serum testosterone-to-estradiol ratio between sexually intact male dogs with perineal hernia and clinically normal sexually intact male dogs. In castrated dogs with perineal hernia, serum testosterone concentration and testosterone-to-estradiol ratio were significantly (P less than 0.05) lower, compared with those values in sexually intact dogs with perineal hernia and in clinically normal sexually intact male dogs. There was no significant difference in serum estradiol 17-beta concentration among sexually intact male dogs with perineal hernia, castrated dogs with perineal hernia, and clinically normal sexually intact male dogs. Serum testosterone and estradiol 17-beta concentrations in dogs with perineal hernia did not differ from those values in clinically normal male dogs of the same age. Castration cannot be recommended for the treatment of perineal hernia unless a castration-responsive contributing factor such as prostatomegaly is identified, unless the pelvic diaphragm of dogs with perineal hernia has high sensitivity to normal or low serum testosterone and estradiol 17-beta concentrations, or unless there is documentation that other androgens and/or estrogens are involved.

The fertilization rate and incidence of embryonic loss in virgin Brahman crossbred heifers between d 3 and 35 postinsemination (d 0 = first day of estrus) were determined. One hundred fifty-five virgin heifers, maintained in a dry lot, received approximately 22 Mcal.head-1.d-1 and were allotted to one of three groups (d 3, 16 or 35) according to body weight at the time of estrous detection. Seven empty ruptured zona pellucidae (ERZP) were recovered from five heifers in the d 3 group. It was impossible to determine whether the ERZP originated from fertilized or unfertilized ova and whether they were ovulated at the estrus immediately preceding or during a previous cycle. Consequently, the fertilization rate (d 3) was calculated to be 80 or 93% depending upon whether the ERZP were included or deleted from the calculations, respectively. Because of the large number of ERZP recovered, a second study was conducted with an additional 21 virgin Brahman crossbred heifers from which ova were recovered on d 3 postinsemination. The results of the two studies were similar. The percentage of heifers with an embryo on d 16 was 78 (10% had degenerating embryos and 12% no embryos), and the percentage pregnant at d 35 was 72. The conclusions suggested from this study depend upon the classification of the ERZP. If the ERZP are designated as ova ovulated during a previous cycle or ova damage in the collection process and are deleted from the calculations, the fertilization rate is high (93%), and embryonic loss apparently occurs between d 3 and 35 (P less than .95). However, if the ERZP are classified as ova ovulated at the immediately preceding estrus, unavailable for further embryonic development, and are included in the calculations, the fertilization rate is comparatively low (80%). In the latter case, the primary loss of potential embryos occurs before d 3, and the loss after d 3 is negligible (P greater than .05).

This study was undertaken to determine if fertility could be improved by increasing the interval from foaling to breeding. Forty-two mares, not bred during normal post-partum oestrus, were injected with a prostaglandin analogue on Day 6 or 7 following ovulation. Mares were mated artifically with antibiotic-treated semen during the resulting oestrus and, if necessary, for the following 4 cycles. Their fertility was compared, by cycles/pregnancy and rate of fetal loss, to mares bred by the same methods on 86 normal post-partum oestrous periods. The interval from foaling to the onset of breeding and the duration of oestrus were longer in the injected mares than the intervals observed in the untreated mares. Both groups averaged 1.3 breeding periods/pregnancy. The rate of fetal loss was not significantly different between the groups. Increasing the interval from foaling to breeding did not improve fertility.

Plasma corticosterone was measured as a stress indicator of laying hens in different management conditions. Laying hens housed in cages and floor pens were sampled repeatedly in the morning and the afternoon. Plasma corticosterone concentrations of afternoon samples of hens from floor treatments were significantly higher than those from cage treatments, but morning values were not different. These results suggest that management systems, producing subtle adverse conditions for laying hens, are associated with elevated baseline corticosterone levels during the nadir of the diurnal response, whereas peak values may not be affected.

Arginine vasotocin (AVT) and neurophysin (NP) levels were measured by radioimmunoassay in two species of sea turtle, the olive ridley, Lepidochelys olivacea, and the loggerhead, Caretta caretta, during the brief period of nesting and oviposition. In both species, AVT was low in animals which were not reproductively active. AVT was also low at the time animals emerged from the surf to nest, but increased significantly during oviposition and then declined as the animals returned to the water. NP increased in concert with AVT, also reaching highest levels during oviposition. In both species, however, NP levels remained elevated over prenesting levels at the time of return to the water. These findings are consistent with the hypothesis that an AVT-neurophysin complex is released from the neurohypophysis during nesting, and that AVT is a physiological regulator of oviducal contractions in sea turtles.

Epidermal growth factor (EGF) receptor binding kinetics and EGF-mediated stimulation of DNA synthesis and cellular proliferation were studied in cultured vascular smooth muscle cells (VSMC) from the equine thoracic aorta. Binding studies, using murine 125I-labeled EGF, indicate the presence of a single class of high-affinity binding sites (apparent KD = 2.8 X 10(-11) M), with an estimated maximal binding capacity of 5,800 sites/cell. EGF stimulated [3H]thymidine uptake in confluent quiescent monolayers in a dose-dependent fashion, half-maximal stimulation occurring at 7.5 X 10(-11) M. Likewise, EGF-mediated cellular proliferation was dose dependent (50% effective dose = 5 X 10(-11) M) under reduced serum concentrations. Equine VSMC contain specific receptors for EGF, and EGF can stimulate DNA synthesis and proliferation in these cultured cells, which suggests that EGF may participate in the proliferative changes observed in equine distal digital peripheral vascular disease.

Genetic studies of familial human cutaneous malignant melanoma have failed to support a single mode of inheritance. To eliminate the complexities of genetic heterogeneity, we have turned to appropriate animal models to gain insights into possible genetic mechanisms that may be applicable to the human. Cutaneous malignant melanoma of Sinclair miniature swine is an inherited malignancy with many of the histopathological characteristics of human melanoma. The actual mode of inheritance of the melanoma has not been determined. Recently, we initiated experiments to characterize the swine major histocompatibility complex in melanoma- and nonmelanoma-bearing animals. These experiments led to the discovery of two loci that are involved in the expression of exophytic melanomas. The first locus lies within the swine major histocompatibility complex where one particular haplotype produces a phenotype in which the effects of a second locus are fully penetrant. The second locus segregates independently of the major histocompatibility complex. The melanoma-producing allele at this second locus is inherited in the heterozygous state and requires a somatic mutation of the normal allele to initiate tumor development.

Sixteen pregnant collared peccaries were assigned to four experimental diets representing two levels of crude protein and two levels of digestible energy. Serum levels of progesterone, estradiol-17 beta (E2), and glucocorticoids were measured by radioimmunoassay. There was no significant dietary effect (P greater than 0.05) associated with any hormone during gestation. Progesterone did not differ between days 11 and 140 of gestation (X +/- SE = 36.48 +/- 1.11 ng/ml, N = 72), began to decline during the last week prepartum, and continued to decline within 24 hr of birth. Glucocorticoid concentrations remained level throughout gestation (X +/- SE = 6.57 +/- 0.45 microgram/dl, N = 74). E2 levels were low during the first 90 days of gestation, rose significantly (P less than 0.001) from 10.11 +/- 1.73 pg/ml (X +/- SE, N = 8) at Days 81-90 gestation to 49.07 +/- 12.87 pg/ml (N = 3) at 2-4 days prepartum (Days 141-147 of gestation), and declined rapidly to baseline levels within 24 hr of farrowing. Litter size had no effect on progesterone or E2 concentrations. There was no significant dietary effect on litter size or gestation length. During lactation, glucocorticoids and E2 remained stable and did not differ by diet, while progesterone concentrations appeared to be affected by diet. Individuals on the high energy-high protein diet returned to normal ovarian function before animals on the other three diets. Results indicated that the pregnant collared peccary can maintain a reproductive steroid environment that allows for fetal development in the face of moderate caloric or protein restriction and that the lactating peccary can undergo a postpartum ovulation in the presence of good nutrition.