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Although equine gestation is unique from the standpoint of fetal gonadal enlargement and regression, the activator of this process is still unknown. The present study aimed to show a possible role of activin during equine gestation. In the first experiment, weekly plasma samples from six pregnant mares were used to measure activin A. In the second experiment, eight pregnant mares carrying female (gestational days 110, 140, 180, and 270) and male fetuses (gestational days 120, 180, 225, and 314) were used for immunohistochemistry of activin receptors (IA, IB, IIA, IIB), and their intracellular mediators (Smad2, Smad3, Smad4). Activin A levels in maternal circulation remained low until fourth weeks of gestation, thereafter, started to increase, and peaked first at 11 weeks of gestation. The second significant peak was observed on the day of parturition. Activin receptors type IA, IB, IIA, and IIB were immunostained in interstitial and germ cells of fetal ovaries and testes along with utero-placental tissues. Smad2, Smad3, and Smad4 were also immunolocalized in all these organs. These results demonstrated the activin-producing capacity of utero-placental tissues, and also evidenced the existence of activin receptors and functional signaling molecules in these organs. The first increment in circulating activin A in maternal circulation coinciding with the timing of initiation of fetal gonadal enlargement suggests that activin from the utero-placental tissues may have a stimulatory role in fetal gonad enlargement and utero-placental development in mares, whereas the second peak could be important to follicular development in the maternal ovary for foal heat.

Immunohistochemical investigations of the expression of surfactant protein A (SP-A) and surfactant protein D (SP-D) in the uterine and placental tissues of 13 pregnant mares were performed using anti-horse monoclonal primary antibodies. Strong positive reactions for both SP-A and SP-D were observed in the trophoblasts in the microcotyledons of the placentae at 182 to 314 days of gestation; in uterine glandular epithelial cells, faint-to-weak reactions were observed during gestation. This study describes, for the first time, the changes in the SP-A and SP-D expression levels in the endometrium of mares during gestation; the SP-A and SP-D expression levels increased after the second trimester of gestation.

In 2017, two Thoroughbred stallions, A and B in Farms A and B, respectively, in Hokkaido in Japan showed clinical signs of equine coital exanthema (ECE). In 2020, stallion C in Farm B showed clinical signs of ECE. Eighteen mares were mated within five days before stallion A developed ECE. Ten mares that mated within 3 days before onset showed clinical signs of ECE on the external genitalia. Equine herpesvirus 3 (EHV-3) was isolated from vaginal swabs from three mares that mated within 2 days before onset. Swabs from 12 mares that mated within 4 days before onset were real-time PCR (rPCR)-positive and nine of those mares had an increased EHV-3 antibody titer. The three stallions were administered valaciclovir orally and topical acyclovir ointment was applied. Treatment started on the next day after onset in stallion A and on the day of onset in stallions B and C. EHV-3 was firstly isolated from penis swabs of stallions A and B before treatment and from penis swabs of stallion C 2 days after treatment. EHV-3 was not isolated after 8, 5 and 8 days from onset in stallions A, B and C, respectively. However, swabs were rPCR-positive for at least 12, 9 and 15 days after onset of stallions A, B and C, respectively. EHV-3 was excreted from the stallions at least within 4 days before the onset of ECE, and acyclovir treatment resulted in the termination of excretion within 8 days after onset.

An equine foamy virus (EFV) was isolated for the first time in Japan from peripheral blood mononuclear cells of a broodmare that showed wobbler syndrome after surgery for intestinal volvulus and the isolate was designated as EFVeca_LM. Complete nucleotide sequences of EFVeca_LM were determined. Nucleotide sequence analysis of the long terminal repeat (LTR) region, gag, pol, env, tas, and bel2 genes revealed that EFVeca_LM and the EFV reference strain had 97.2% to 99.1% identities. For a sero-epidemiological survey, indirect immunofluorescent antibody tests were carried out using EFVeca_LM-infected cells as an antigen against 166 sera of horses in five farms collected in 2001 to 2002 and 293 sera of horses in eight farms collected in 2014 to 2016 in Hokkaido, Japan. All of the farms had EFV antibody-positive horses, and average positive rates were 24.6% in sera obtained in 2001 to 2002 and 25.6% in sera obtained in 2014 to 2016 from broodmare farms. The positive rate in a stallion farm (Farm A) in 2002 was 10.7%, and the positive rates in two stallion farms, Farms A and B, in 2015 were 40.9% and 13.3%, respectively. The results suggested that EFV infection is maintained widely in horses in Japan.

Aortic rupture is a well recognized cause of sudden death in thoroughbred horses. Some microscopic lesions, such as those caused by cystic medial necrosis and medionecrosis, can lead to aortic rupture. However, these microscopic lesions are also observed in normal horses. On the other hand, a previous study of aortic rupture suggested that underlying elastin and collagen deposition disorders might be associated with aortic rupture. Therefore, the purpose of this study was to compare the structural components of the tunica media of the aortic arch, which is composed of elastin, collagen, smooth muscle cells and mucopolysaccharides (MPS), in fetal to mature thoroughbred horses. The percentage area of elastin was greatest in the young horses and subsequently decreased with aging. The percentage area of collagen increased with aging, and the elderly horses (aged ≥20) exhibited significantly higher percentage areas of collagen than the young horses. The percentage area of smooth muscle cells did not change with age. The percentage area of MPS was inversely proportional to the percentage area of elastin. The fetuses exhibited a markedly larger percentage area of MPS than the mature horses. We concluded that the medial changes seen in the aortic arch, which included a reduction in the amount of elastin and increases in the amounts of collagen and MPS, were age-related variations.

In the spring of 2015, two stallions reared in Farms A and B in Hokkaido in Japan showed symptoms of equine coital exanthema. Equine herpesvirus 3 (EHV-3) was isolated from penis swab samples of both stallions, and the isolates from each stallion in Farms A and B were designated as SS-1 and YS-1 strains, respectively. BamHI restriction profiles of SS-1 and Japanese reference strain Iwate-1 were indistinguishable, but the BamHI-A fragment of YS-1 was larger than those of SS-1 and Iwate-1 by 1.9 kbp because of the lack of two BamHI sites. Nucleotide sequence analyses of glycoprotein G (gG), gB, gC and VP13/14 coding regions revealed that SS-1 and YS-1 had 99.77% to 100% identities to each other. These results suggested that the origins of SS-1 and YS-1 were different. For a sero-epidemiological survey, serum neutralizing tests using SS-1 against 319 sera of horses from eight farms in Hokkaido were conducted. Six of the eight farms were EHV-3 antibody-positive, and positive rates ranged from 2.6% to 17.6%. To determine the infection time of four EHV-3 antibody-positive horses, a retrospective study was conducted. Infection time of the four horses was in the breeding season, and re-infection or reactivation of latently infected EHV-3 might have occurred in one horse. However, these four horses had never shown any clinical symptoms. The results suggested that several EHV-3 strains are distributed in Japan and that infection is maintained widely in horses without clinical symptoms.

Equine coital exanthema (ECE) has been reported in many countries, but equine herpesvirus 3 (EHV-3) has been isolated only once in Japan. In 2015, symptoms of ECE were found, and EHV-3 was isolated in two stallions. Valacyclovir, an anti-herpesvirus agent, was administered orally. The stallions rested from mating for more than two weeks, causing enormous financial losses because of their high fees. This is the first study in which valacyclovir was administered for ECE. Though valacyclovir treatment did not shorten the duration of healing, the affected area did not expand after administration of valacyclovir. Valacyclovir therefore seems to be effective for suppression of EHV-3 infection. Further investigation about the administration protocol might be required.

A novel chemiluminescent enzyme immunoassay system, PATHFAST, for the measurement of estradiol in horses was evaluated. The concentrations of estradiol in the whole blood and serum of mares were measured using PATHFAST and the estradiol concentrations measured by PATHFAST were compared with those measured by a time-resolved fluoro-immunoassay (FIA). To monitor physiological changes, serum estradiol concentrations in mares were measured using PATHFAST throughout the gestation period. The serum estradiol concentrations correlated highly with those in whole blood samples. The serum concentrations of estradiol measured by PATHFAST also correlated well with FIA. Circulating estradiol increased during mid-gestation and high levels of serum estradiol were maintained in late gestation, followed by an abrupt decline to term. These results demonstrate the utility of PATHFAST in equine clinics as an accurate diagnostic tool for the rapid assay of estradiol within 26 min using unextracted whole blood.

Testosterone (T) concentration is a useful indicator of reproductive function in male animals. However, T concentration is not usually measured in veterinary clinics, partly due to the unavailability of reliable and rapid assays for animal samples. In this study, a rapid chemiluminescent enzyme immunoassay system (CLEIA system) that was developed for the measurement of T concentration in humans use was validated for stallion blood samples. First, serum T concentrations were measured using the CLEIA system and compared with those measured by a fluoroimmunoassay that has been validated for use in stallions. The serum T concentrations measured by the two methods were highly correlated (r = 0.9865, n = 56). Second, to validate the use of whole blood as assay samples, T concentrations in whole blood and in the serum were measured by the CLEIA system. T concentrations in both samples were highly correlated (r = 0.9665, n = 64). Finally, to evaluate the practical value of the CLEIA system in clinical settings, T concentrations were measured in three stallions with reproductive abnormalities after the administration of human chorionic gonadotropin (hCG). Two stallions with small or absent testes in the scrotum showed an increase in T production in response to hCG administration and one stallion with seminoma did not. In conclusion, the CLEIA system was found to be a rapid and reliable tool for measuring T concentrations in stallions and may improve reproductive management in clinical settings and in breeding studs.

Evaluation of a new chemiluminescent enzyme immunoassay, the PATHFAST assay system (PATHFAST), for measurement of circulating progesterone in mares was performed. Five mares at the mid-luteal stage were administrated a single i.m. injection of prostaglandin F2α analog (PGF2α; cloprostenol 250 μg/ml), and then blood samples were collected from the jugular vein at 0, 15, 30 and 45 min, at one-hour intervals until 24 and at 48 hr via a catheter in the jugular vein. To monitor the physiological changes in circulating progesterone in mares after induced luteolysis, concentrations of progesterone in whole blood and serum samples were measured by PATHFAST. In addition, concentrations of progesterone in serum samples measured by PATHFAST were compared with those measured by radioimmunoassay (RIA) and enzyme immunoassay (EIA). Using PATHFAST, the serum concentrations of progesterone in mares correlated highly with those of whole blood samples (r=0.9672, n=88). The serum concentrations of progesterone as measured by PATHFAST correlated well with RIA (r=0.9654, n=88) and EIA (r=0.9323, n=112). An abrupt decline in circulating progesterone in whole blood samples was observed within 2 hr (50%), followed by a gradual decline until 48 hr later. The results for progesterone in whole blood samples correlated highly with those in serum samples, and the declining pattern paralleled that of the serum samples. These results demonstrated that PATHFAST is useful in the equine clinic as an accurate diagnostic tool for rapid assay of progesterone within 26 min, using unextracted whole blood.

The aim of present study was to clarify the post-natal profile of follicle-stimulating hormone (FSH), luteinizing hormone (LH), immunoreactive (ir)-inhibin, progesterone, testosterone, and estradiol-17β, and their relationships in Thoroughbred colts. Six hundred and thirty-six colts were used for the study. Single plasma samples from each animal were harvested from the blood drawn through jugular venipuncture. The subjects were born with high amounts of progesterone, testosterone, and estradiol-17β, all of which dropped significantly and remained at lower levels till the end of 6 months. FSH decreased transiently after birth until day 12 and then gradually increased to peak at day 100 which then maintained in lesser levels towards the end of the studied period. LH was highest during birth which decreased until day 26 and then increased slowly to sub-birth levels up to day 90. Animals were born with high amounts of ir-inhibin. It dropped slowly and halved by day 20 and then decreased towards rest of the studied period. The increase in FSH is negatively correlated with the declining ir-inhibin levels. The early increase in FSH can be the indication of early post-natal maturation of the hypothalamic pituitary testicular axis that ultimately might be responsible for priming the testes for future development.

Changes in follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, immunoreactive(ir)-inhibin, testosterone, estradiol-17β, and insulin-like growth factor (IGF)-I in Thoroughbred stallions along with changes in prolactin secretion in geldings were studied. The correlations of day-length with changes in the concentrations of these hormones were also studied. Five stallions and thirteen geldings were employed to draw blood samples in monthly basis and radioimmunoassay was performed to measure these hormones. All hormones showed a seasonal pattern, the levels being highest during the breeding season and lowest during the winter months. Most of the hormones were at their highest concentration during the month of April, the mid of spring in northern hemisphere. The concentration of circulating IGF-I also demonstrated seasonality, the peak lying on the month of April. The plasma concentration of prolactin also increased during the breeding season. This phenomenon was similar both in stallions and geldings although geldings had lower concentration than that of stallions. The changes in concentration of prolactin in stallions and geldings correlated more towards the day-length than towards the temperature. These results clearly indicate the seasonality of pituitary and gonadal hormones of Thoroughbred stallions, the activity being highest during the month of April and May of the breeding season.

To clarify the physiological changes of sperm morphology in active Thoroughbred stallions during the breeding season, we examined the dismount semen collected from the penile urethra immediately after service. The spermatozoa were analyzed for relationships between the morphology and the stallion's age or the number of services. Seasonal variation was apparent in the rate of the sperm tail abnormalities, spermatozoa with cytoplasmic droplets, appearance of medusa cells, and sperm head length. Area and width of the sperm head correlated negatively with age (P

Objective—To evaluate the histologic diversity of epidermal laminae in hooves from horses without clinical signs of laminitis. Sample Population—Formalin-fixed samples of stratum internum obtained from the mid region of the dorsal aspect of the hoof wall from the forelimbs of 35Thoroughbred cadavers (including foals [n = 9], yearlings [5], 2 year olds [6], racing horses [5], and mares [10]). Procedures—Paraffin-embedded laminar tissues were stained with H&E for the evaluation of architectural variety of primary epidermal laminae (PEL) and secondary epidermal laminae (SEL). For detection of cytokeratin (CK) expression in epidermal laminae, immunohisto-chemical staining was performed by use of anti-CK14 and anti-CK8.12 antibodies. Results—The morphology of the PEL, SEL, and tips of PEL was classified into 3, 5, and 3 patterns, respectively. Differences in the predominant type of SEL depended on their location with respect to the laminar interface. In SEL attached to the sides of PEL, the basal cells were immunoreactive to CK14 and CK8.12, which was interpreted as a normal pattern. In some SEL at the tips of PEL, the suprabasal cells expressed CK14, CK8.12, or both, which constituted a hyperplastic pattern. Conclusions and Clinical Relevance—The histologic diversity of epidermal laminae from hooves of Thoroughbreds was attributable to the combined morphology of PEL and SEL. Detection of hyperplastic changes in the laminar interface does not justify a diagnosis of laminitis because such changes can develop independent of clinical disease. The classification system used here should aid investigators in making a more accurate histologic evaluation of laminae.

The objective of this study was to investigate the cellular immunolocalization of inhibin α and inhibin/activin (βA and βB) subunits and steroidogenic enzymes in neonatal equine ovaries. The ovaries were obtained from two equine neonates of 3 days and 18 days of age. The sections of ovarian tissues were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin α, inhibin/activin βA and inhibin/activin βB and bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3β-hydroxysteroid dehydrogenase (3βHSD), porcine testicular 17α-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). Inhibin α and inhibin/activin βA subunits, but not inhibin/activin βB subunits were expressed in the ovarian interstitial cells of equine neonates. P450scc and P450c17 were also identified in the ovarian interstitial cells and P450arom with weak immunostaining. However, 3βHSD was not found in the ovarian interstitial cells of the equine neonates. These results suggest that equine neonatal ovaries have the ability to synthesize inhibin A and steroid hormones, which may play some important physiological roles in the developing ovaries of equine neonates.

The changes in testicular and pituitary functions in response to human chorionic gonadotropin (hCG) in the breeding and non-breeding season were investigated and compared in the Thoroughbred stallion. Five mature Thoroughbred stallions ranging in ages from 7 to 21 years were injected in May (breeding season) and October (non-breeding season). All animals received an intramuscular injection of 5,000 IU hCG in the experiments. Peripheral blood samples were collected in heparinized tubes from the jugular vein for hormonal assays just before the injection (Day 0) and at a daily interval for five days following the injections (Days 1, 2, 3, 4 and 5). Basal levels of immunoreactive (ir) inhibin, testosterone, estradiol-17β, FSH and LH were higher in the breeding season than in the non-breeding season. There were significant differences in estradiol-17β, FSH and LH between the two seasons. Ir-inhibin exhibited significant increases on Day 2 after the hCG treatment in the non-breeding season, though there was no change in the breeding season. Plasma levels of testosterone showed remarkable increases after the hCG injection in both seasons. The peak levels of plasma testosterone were observed on Days 2 and 3 in the breeding and non-breeding seasons. Plasma estradiol-17β was significantly higher than the basal level on Day 3 in the non-breeding season, whereas there was no change in the breeding season. Plasma levels of FSH declined from Day I to Day 3, then recovered to the basal level (Day 0) on Day 4 after hCG treatment in the breeding season, whereas there was no change in the non-breeding season. Circulating LH showed a significant decrease on Day 3 compared to the based level in the breeding season, but no significant change in the non-breeding season after a treatment of hCG. A significant negative correlation was observed between testosterone and FSH in the breeding season. In conclusion, hCG treatment stimulates secretion of testicular hormones and these testicular hormones temporarily suppress secretion of gonadotropins from the pituitary gland. These results also suggested that Leydig cells of thoroughbred stallion testes have LH receptors in the non-breeding season, the same as in the breeding season.

To elucidate the function of inhibin/activin subunits in developmental fetal adrenal glands, we investigated immunolocalization of inhibin α and inhibin/activin (βA and βB) subunits protein in the equine fetal adrenal glands during mid to late gestation. Fetal adrenal glands of three equines were obtained at 217, 225 and 235 days of gestation. The sections of fetal adrenal glands were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin α, inhibin/activin βA and inhibin/activin βB. Inhibin/activin βB was identified in cortex cells of fetal adrenal glands throughout 217-235 days of gestation, but inhibin α and inhibin/activin βA were not observed in fetal adrenal glands in this period. These results suggest that equine fetal adrenal glands have the ability to synthesize activin B but not inhibins and activin A. Also, inhibin/activin βB may play a regulatory role in developing equine fetal adrenal glands.

To elucidate the relationship between steroidogenic hormones and developing adrenal glands, we investigated the immunolocalization of steroidogenic enzymes in equine fetal adrenal glands during mid-late gestation. Fetal adrenal glands were obtained from three horses at 217, 225 and 235 days of gestation. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17) and human placental aromatase cytochrome P450 (P450arom). Histologically, cortex and medulla cells were clearly observed in the three fetal adrenal gland tissue samples. P450scc and P450c17 were identified in cortex cells close to medulla cells and in some medulla cells in the fetal adrenal glands. P450arom was present in both cortex and medulla cells in the fetal adrenal glands. However, 3betaHSD was not found in any of the equine fetal adrenal gland tissue samples. These results suggest that equine fetal adrenal glands have the ability to synthesize androgen and estrogen, which may play an important physiological role in the development of equine fetal adrenal glands.

The relationship among circulating levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), immunoreactive (ir) inhibin, progesterone, testosterone and estradiol-17β were investigated in fillies from birth to 6 months of age. Six hundred ninety-four Thoroughbred female foals were used for evaluating the changes in these hormones. Blood samples were collected by jugular venipuncture for hormonal assays. The results show that after birth, progesterone, testosterone and estradiol-17β levels rapidly drop within 48 hr, and remain at this lower level until six months of age. On the other hand, plasma concentrations of ir-inhibin begin to decrease at 3 days after birth, falling by more than half in the first 20 days after birth, and then continue to slowly decline until six months of age. Circulating levels of FSH show a small increase within 48 hr after birth, then decline to the lowest levels at 30 days after birth, followed by a rise to more or less stable levels at six months of age, whereas LH concentrations rapidly decrease after birth, dropping to a lower level at 10 days after birth, then remain at the lower value until six months of age. These results demonstrate that, after birth, in a filly, there is a notable decline in steroid hormones which then remain at these relatively stable lower levels until six months of age. The steady increase in basal FSH negatively correlates with the decline in circulating inhibin. This slow increase in the basal levels of FSH may stimulate follicular development during the early stages of puberty in a filly.

We isolated a variant equine herpesvirus-1 (EHV-1), strain 5089, from the lung of a dead neonatal foal in Japan and characterized the biological nature of the virus. The virus spread in cultured cells mainly by cell-to-cell infection, unlike wild-type EHV-1, which spreads efficiently as a cell-free virus. The virus titer in cultured supernatant and the intracellular virus titer were low compared to those of wild-type EHV-1. Heparin treatment of the virus had no effect on viral infectivity in cell culture. Glycoprotein C (gC) was not detected by Western blotting and fluorescent antibody tests in 5089 virions and 5089-infected cells, respectively. RT-PCR analysis revealed that the expression level of 5089 gC mRNA was reduced considerably compared to that of wild-type EHV-1. Sequencing analysis of the 5089 gC coding region showed a point mutation in the promoter region of the gC open reading frame. However, the mutation did not affect the promoter activity. These results suggested that the lack of gC in 5089 virions might be one of the reasons for spread of the virus by cell-to-cell infection and that gC mRNA expression might not be activated efficiently due to factors other than the mutation in the gC promoter region.

To clarify the source of inhibins in equine female fetuses, concentrations of immunoreactive (ir-) inhibin, inhibin pro-alphaC, and inhibin A in both fetal and maternal circulation and in fetal ovaries were measured. In addition, the localization of inhibin alpha and inhibin/activin beta(A), and beta(B) subunits and the expression of inhibin alpha(A) and inhibin/activin beta(A) subunit mRNA in fetal ovaries were investigated using immunohistochemistry and in situ hybridization. Concentrations of circulating ir-inhibin, inhibin pro-alphaC, and inhibin A were remarkably more elevated in the fetal than in the maternal circulation between Days 100 and 250 of gestation. Fetal ovaries contained large amounts of ir-inhibin, inhibin pro-alphaC, and inhibin A. In contrast, these inhibin forms were undetectable in both the maternal ovaries and placenta. The inhibin alpha and inhibin/activin beta(A) and beta(B) subunit proteins were localized to enlarged interstitial cells of the equine fetal ovary. Expression of inhibin alpha and inhibin/activin beta(A) subunit mRNAs were also observed in the interstitial cells. We conclude that the main source of large amounts of inhibins in fetal circulation is interstitial cells of fetal ovary and is not of maternal origin. Furthermore, these inhibins may play some important physiological roles in the development of gonads in the equine fetus.

Control of ovulation in mares obviously has many advantages in a breeding program. Two pony mares were challenged against synthetic Gonadotropin Releasing Hormone (GnRH) agonist (Deslorelin) for six consecutive cycles. Clinical and endocrinological findings after treatment (n=6) with GnRH agonist were compared with those of the control findings before treatment (n=3) of the same mares. The time interval from administration of the GnRH agonist till ovulation (43.5 ± 4.0 hr) and the length of the estrous cycle (19.5 ± 1.0 days) at the time of treatment were significantly (p

To clarify the clinical findings in mares with granulosa theca cell tumor (GTCT) and to determine reproductive activity after surgery, 16 mares with GTCT were studied. The GTCTs were found in 10.4 ± 0.9 year olds. The clinical signs manifested by the affected animals varied, and the mares showed abnormal behavior, that is, anestrus (n=5), continuous estrus (n=8) and stallion-like behavior (n=3), but most of the affected mares (11/16) were sterile for long periods (1-4 years), and in four animals GTCTs were detected at the post-partum period. In 11 of the 16 animals, the ovary containing tumor was enlarged on the right side, whereas the contralateral ovary was atrophied in all the 16 animals. Mares in which the affected ovary was surgically removed in the non-breeding season (n=8) had estrus 2.6 months earlier and became pregnant 2.9 months earlier than those operated on (n=8) in the breeding season, by resuming the reproductive function in the remaining ovary. All three mares that died from hemorrhage after the surgery were operated on in the breeding season. From these results we confirmed that GTCT is related to sterility for long periods in mares and that surgical removal of the affected ovary is effective in restoring reproductive function. And it is also suggested that the affected ovary should be removed in the non-breeding season.

Localization of inhibin subunits and steroidogenic enzymes in the various-sized antral follicles of the mare was examined. Antral follicles of four sizes (1, 2.5, 4.5 and 6 cm in diameter) were collected from the ovary and subjected to histological analysis. Inhibin α-subunit mRNA was localized in granulosa cells of follicles of all sizes, and inhibin βA-subunit mRNA was confined to granulosa cells of the 1-4.5-cm follicles. Inhibin α-subunit protein was found in granulosa cells of follicles of all classes, and theca cells of the 6-cm follicle. Inhibin βA- and βB-subunit proteins were found in granulosa cells of the 1-4.5-cm follicles, and theca cells of the 2.5-6-cm follicles despite the absence of inhibin/activin subunit mRNAs in theca cells. This suggests that inhibin and/or activin regulates the function of theca cells in a paracrine fashion. Aromatase protein was confined to granulosa cells of the 2.5- and 4.5-cm follicles, and to theca cells of the 4.5-cm follicle. Strong staining for aromatase protein was noted in the 4.5-cm follicle as compared to the 2.5-cm follicle. Positive immunostaing for 3β-hydroxysteroid dehydrogenase was observed in granulosa cells of the 2.5-6-cm follicles, and in theca cells of the 4.5- and 6-cm follicles. These data suggest that inhibin is predominantly secreted from 1-4.5-cm antral follicles and estradiol is predominantly secreted from antral follicles of about 4.5-cm in diameter. Further, it is suggested that progesterone is the predominant hormone secreted form the preovulatory follicle in the mare.

軽種馬における受胎性の向上を図る目的で,繁殖用サラブレッド種雌馬339例について交配時の血中プロジェステロン(P)およびエストラジオール17β(E2)濃度を測定するとともに,受胎の有無を調べた.その結果,339例は以下の4群,すなわちI群: P>1ng/mlでE2>20pg/ml (39例),II群: P>1ng/mlでE2≦20pg/ml (29例),III群: P≦lng/mlでE2>20pg/ml (201例),IV群: P≦1ng/mlでE2≦20pg/ml(70例)に区分された.群内の受胎率は,III群が52.7%で最も高く,ついでIV群が41.4%,IIが30.8%であり,I群は17.2%で最も低かった.また,交配時に高P濃度を示した9頭の雌馬を対象に,周排卵期の血中PおよびE2濃度の経日的な変化と受胎率を調べたところ,血中E2濃度およびE2/P値が雌馬の受胎性の良否に関与していることが示唆された.

To clarify the endocrinological characteristics of mares with granulosa-theca cell tumor (GTCT), 2 mares with GTCT and a long estrous period were investigated. Before removal of the GTCT in both cases, the concentrations of testosterone, immunoreactive (ir-) inhibin and follicle stimulating hormone (FSH) in the peripheral blood were higher than those of normal mares at the follicular phase, whereas the plasma concentrations of progesterone were constantly low. In Case 1 mare, the plasma concentrations of testosterone, ir-inhibin and follicle stimulating hormone (FSH) changed to significantly high levels. In Case 2 mare, plasma concentrations of all hormones except for progesterone changed to significantly high levels. After surgical removal of the GTCT, the circulating concentration of ir-inhibin decreased rapidly to the basal level. The ir-inhibin levels in GTCT tumor fluid were similar to the follicular fluid in healthy follicles 3.5-5.0 cm in diameter. Concentrations of estradiol in the GTCT tumor fluid were low, but testosterone levels were higher than those in healthy follicles 3.5-5.0 cm in diameter. These results suggest that high levels of circulating inhibin in addition to high levels of testosterone and low levels of progesterone may be used as a clinical sign in the diagnosis of GTCT in mares.

Inhibin pro-αC is a precursor form of inhibin α-subunit. A large amount of inhibin pro-αC is secreted from testes in male animals, and it is considered to have an important role in the physiology of male reproduction. The purpose of this study was to determine the relationship between inhibin pro-αC and reproductive ability in the mature stallion. Peripheral blood samples were collected from normal stallions and anabolic steroid (nandrolone decanoate) treated stallions. Plasma concentrations of inhibin pro-αC were measured by an enzyme linked immunosorbent assay system. Circulating concentrations of inhibin pro-αC in normal stallions showed clear seasonal changes and the highest concentrations were observed in the breeding season (from March to June). After treatment with anabolic steroid, peripheral inhibin pro-αC levels rapidly decreased and reached significantly lower levels compared to the pre-treatment value (p

A 20-year-old thoroughbred mare had a mass in the right kidney. The mass was encapsulated with fibrous capsule and composed of variably-sized papillary projections lined by a single layer of flattened to cuboidal neoplastic epithelial cells with no cytological and nuclear atypia. Immunohistochemically, the neoplastic cells were broadly positive for cytokeratin AE1/AE3 and granular staining for alpha-1-antitrypsin was focally detected; this immunohistochemical property was similar to that of the normal distal nephron. From these results, this case was diagnosed as papillary renal adenoma of distal nephron differentiation.

To determine the source of circulating inhibin and estradiol-17beta during the estrous cycle in mares, the cellular localization of the inhibin alpha, betaA, and betaB subunits and aromatase in the ovary was determined by immunohistochemistry. Concentrations of immunoreactive (ir-) inhibin, estradiol-17beta, progesterone, LH, and FSH in peripheral blood were also measured during the estrous cycle in mares. Immunohistochemically, inhibin alpha subunits were localized in the granulosa cells of small and large follicles and in the theca interna cells of large follicles, whereas inhibin betaA and betaB subunits were localized in the granulosa cells and in the theca interna cells of large follicles. On the other hand, aromatase was restricted to only the granulosa cells of large follicles. Plasma ir-inhibin concentrations began to increase 9 days before ovulation; they remained high until 2 days before ovulation, after which they decreased when the LH surge was initiated. Thereafter, a further sharp rise in circulating ir-inhibin concentrations occurred during the process of ovulation, followed by a second abrupt decline. After the decline, plasma concentrations of ir-inhibin remained low during the luteal phase. Plasma estradiol-17beta concentrations followed a profile similar to that of ir-inhibin, except during ovulation, and these two hormones were positively correlated throughout the estrous cycle. Plasma FSH concentrations were inversely related to ir-inhibin and estradiol-17beta. These findings suggest that the dimeric inhibin is mainly secreted by the granulosa cells and the theca cells of large follicles; granulosa cells of small follicles may secrete inhibin alpha subunit, and estradiol-17beta is secreted by the granulosa cells of only large follicles in mares.

The cellular localization of inhibin a, bA, and bB subunits, 3b-hydroxysteroid dehydrogenase (3b-HSD), and cytochrome P450 aromatase (aromatase) in stallion testes was investigated. In addition, detailed seasonal changes in circulating immunoreactive (ir)-inhibin were investigated in correlation with testosterone, estradiol, LH, and FSH. Inhibin a subunit-positive staining was observed in Sertoli cells, and more clearly positive staining was noted in Leydig cells. Inhibin bA and bB subunits were also stained in both types of cells. Immunoreactivity of 3b-HSD and aromatase was confined to the Leydig cells. There was no seasonal effect on the percentage of the areas within seminiferous tubules and interstitial tissues that stained positive for the inhibin a subunit. The highest plasma concentrations of ir-inhibin were observed in the breeding season, and the lowest levels were noted during the nonbreeding season. The circulating concentrations of ir-inhibin, steroid hormones, and gonadotropins were positively correlated with each other throughout the 2 years studied. The presence of the inhibin a and b subunits in Leydig cells and Sertoli cells in the equine testis suggests that these cells may secrete dimetric (bioactive) inhibin in circulation of stallions, and that the circulating ir-inhibin may be a useful indicator of the testicular function of stallions.

Summary To examine the physiological role of inhibin in the stallion, a heterologous radioimmunoassay (RIA) based on a bovine RIA was validated and used to measure immunoreactive (ir)-inhibin concentrations in plasma and testicular homogenates. The bioactivity of equine testicular inhibin was also examined using an assay for suppression of FSH secretion from rat anterior pituitary cells. In addition, to identify the cell responsible for secreting testicular inhibin, the localisation of inhibin in the testis was investigated by an immunohistochemical method using a polyclonal antibody against (Tyr30)-porcine inhibin α(1–30) NH2. In the RIA, parallel dose response curves were obtained for the bovine inhibin standard and serial dilutions of stallion plasma and equine testicular homogenates. Parallel FSH inhibition curves were also observed for the bovine inhibin standard and serial dilutions of equine testicular homogenates in the bioassay. The inhibition of FSH secretion from rat pituitary cells by equine testicular homogenates was neutralised by an antiserum against bovine inhibin in vitro. Plasma concentrations of ir-inhibin, testosterone and oestradiol-17s in stallions decreased abruptly after bilateral gonadectomy and FSH and LH concentrations in the plasma subsequently increased. Therefore, circulating inhibin in the stallion appeared to be largely of testicular origin. The histochemical results showed for the first time that strong immunopositive staining for inhibin occurred in the Leydig cells of the testes. Sertoli cells were also stained by the inhibin antibody but the reaction was weaker than that in Leydig cells. These results indicate clearly that both Leydig and Sertoli cells are potential sources of testicular inhibin in the stallion. A clear increase in plasma ir-inhibin concentrations was observed during the natural breeding season. Similar seasonal changes in the plasma concentrations of testicular steroid hormones and pituitary gonadotrophins occurred throughout the year. In conclusion, the testes appear to be the main source of inhibin, and testicular inhibin is secreted by Leydig and Sertoli cells in stallions. The positive correlations between plasma ir-inhibin and testicular activity during both the breeding and nonbreeding seasons indicate that plasma irinhibin is a useful indicator of reproductive activity in the stallion.

A morphometric study of gonadal interstitial cells in 16 equine fetuses, ranging in age from 120 to 330 days of fetal age, was done to assess the possible relationship of fetal gonads to high plasma concentrations of immunoreactive inhibin in pregnant mares during the second half of pregnancy with its peak at about 280 days of gestation, which has been reported recently. Interstitial cells of fetal gonads underwent progressive hypertrophy to reach a maximum size around 250 days of gestation, and subsequent degenerative changes in these cells resulted in a rapid decrease in the volume density and size of the interstitial cells. Thus, the morphometric change in the interstitial cells was almost synchronous with high concentrations of immunoreactive inhibin in the plasma of pregnant mares. This observation, taken in conjunction with the fact that inhibin α-chain (1-30) was detected immunohistochemically in the cytoplasm of the interstitial cells, appears to provide morphometric evidence that the interstitial cells in fetal gonads are closely related to inhibin production.

Serum amyloid A protein (SAA) was isolated from equine acute-phase serum by repeating Sephadex G-75 gel filtration 3 times. Quantitative measurement of equine SAA was performed by the single radial immunodiffusion technique with rabbit anti-equine SAA serum. In clinically normal horses, the SAA concentration remained relatively high from immediately after birth up to 1 week of age. After this the concentration showed periodic fluctiation in the range of approximately 13 to 30 micrograms/ml. The mean (+/- SD) concentration of SAA in foals (or = 12 months old) and in adult horses (or = 18 months old) was 19.37 +/- 9.41 and 21.53 +/- 9.81 micrograms/ml, respectively. In mares during the perinatal period, the SAA concentration remained stable and within the normal range for 4 months before parturition. After foaling, it increased quickly and reached a peak value of 136.78 +/- 56.74 micrograms/ml on day 3 postpartum, and then began to decrease at 2 weeks postpartum returning to within the normal range by 1 month postpartum. In horses with experimentally induced inflammation, the SAA concentration increased quickly, and reached the highest value, approximately 4 to 20 times higher than pre-treatment values, on day 2 after treatment. It then returned to the base line values within 10 days to 4 weeks, concurrent with the disappearance of local inflammatory signs. The SAA concentration was very high in most horses with clinical signs of inflammation. It was concluded from these data that equine SAA was a sensitive acute-phase reactive protein which increased in the early phase of various acute inflammations.

A 22-year-old Thoroughbred stallion had severe left jugular thrombophlebitis. Macroscopic and microscopic examinations revealed extension of the lesions from a penetrating ulcer on the left buccal mucosa to the underlying muscle and local vein, and sequentially to the left jugular vein. This was a rare case of equine jugular thrombophlebitis caused by direct extension of infection from a traumatic oral lesion.

Haptoglobin (Hp) was isolated from equine serum by ammonium sulphate precipitation, anion-exchange chromatography and gel filtration. Equine Hp which migrated to the alpha 2-globulin region in electrophoresis, contained 2 fractions with molecular weights (NW) of 108,000 and 105,000, and each fraction consisting of 2 subunits. Quantitative measurement of Hp in equine serum was performed by the single radial immunodiffusion method using anti-equine Hp serum. In clinically normal horses, the highest concentration of serum Hp was found in newborn foals and a high value was maintained until 12 months of age. The concentration then decreased with age. Normal Hp values were 5.25 +/- 2.36 mg/ml in foals (less than or equal to 12 months old), 2.19 +/- 1.54 mg/ml in adult horses (greater than or equal to 18 months old) and 3.62 +/- 0.81 mg/ml in all horses. Serum Hp concentration in mares during the perinatal period in comparison with the normal adult female was high for 4 months pre-partum, a passing increase at delivery, and then decreased at 2 weeks post-partum returning to normal within 1 month of delivery. In horses with experimentally-induced inflammation, serum Hp concentration began to increase immediately after treatment and reached the highest value, 1.5 to 9 times higher than those of pre-treatment at 2 to 5 days, then decreased within 4 weeks. It was also elevated in most cases of horses with clinically inflammatory signs.

The serum concentration of C-reactive protein (CRP) in horses was measured by single radial immunodiffusion (SRID) using rabbit anti-equine CRP serum to evaluate the significance of CRP as an acute-phase reactive protein. In serum samples of clinically normal newborn thoroughbred foals before being given colostrum, serum CRP was not detectable (less than or equal to 1 microgram/ml). The serum CRP concentration was found to increase quickly and reach a peak level of approximately 14.1 micrograms/ml in 12-month-old horses. Then, it decreased gradually to reach a low value of 5.4 micrograms/ml in the 4-year-old. In horses over 5-year-old, serum CRP concentration was stable at values of 7 to 8 micrograms/ml. In mares during the peri-natal period, serum CRP concentration decreased at 2 months before delivery, showed moderate changes just before and after delivery, and then increased to relatively high level by 2 months after delivery. The concentration of serum CRP was found to be increased at 24 hours after experimentally inflammatory stimuli in adult horses, and reached peak values of 3 to 6 times as high as the base line values at 3 to 5 days. Serum CRP concentration returned to base line values by 2 to 3 weeks after treatments. It is clear from these data that CRP is one of the acute-phase reactive proteins in horses.

The morphology of spermatozoa of modern Thoroughbred stallions in Japan was investigated during the breeding season. A total of 299 semen samples were collected from the penises of 16 stallions immediately after service. The rate of abnormalities in sperm heads and tails, spermatozoa with cytoplasmic droplets and slides with medusa cells to total observed slides in each stallion were 3.9 +/- 2.1%, 11.5 +/- 5.9%, 2.4 +/- 2.6% and 20.1%, respectively. The values for the area, length, width and aspect ratio of the stallion sperm head were 12.54 +/- 1.34 microm(2), 5.93 +/- 0.40 microm, 2.69 +/- 0.21 microm and 0.46 +/- 0.05, respectively. With the exception of medusa cells, the features were significantly different among the stallions (P0.05).

To clarify the endocrinological characteristics of the mares with granulosa theca cell tumor (GTCT), peripheral plasma samples from the 6 mares affected with GTCT were collected before and after the surgical removal of the affected ovary. Concentrations of testosterone (T), follicle stimulating hormone (FSH), luteinizing hormone (LH), immunoreactive-inhibin (ir-INH), progesterone (P) and estradiol-17beta (E(2)) in the plasma samples were measured by radioimmunoassay. Before removal of GTCT in all cases, the concentrations of T were significantly higher than those of normal mares at the breeding and non-breeding seasons, whereas plasma concentrations of FSH, LH, ir-INH, P and E(2) were lower. After surgical removal of the affected ovary, the circulatory concentrations of T was declined, but the concentrations of other hormones were constantly low as compared with those of normal mares. The present study suggests that 1) the source of higher T may be due to the abnormal follicles in ovary of GTCT, 2) in the case of GTCT the elevated level of T is observed due to the lack of aromatase, and 3) the high level of T is a typical characteristics for GTCT in mares. It is also suggested 4) due to the elevated levels of T the concentrations of gonadotropins may be suppressed.

To clarify the source of inhibins in equine female fetuses, concentrations of immunoreactive (ir-) inhibin, inhibin pro-alphaC, and inhibin A in both fetal and maternal circulation and in fetal ovaries were measured. In addition, the localization of inhibin alpha and inhibin/activin beta(A), and beta(B) subunits and the expression of inhibin alpha(A) and inhibin/activin beta(A) subunit mRNA in fetal ovaries were investigated using immunohistochemistry and in situ hybridization. Concentrations of circulating ir-inhibin, inhibin pro-alphaC, and inhibin A were remarkably more elevated in the fetal than in the maternal circulation between Days 100 and 250 of gestation. Fetal ovaries contained large amounts of ir-inhibin, inhibin pro-alphaC, and inhibin A. In contrast, these inhibin forms were undetectable in both the maternal ovaries and placenta. The inhibin alpha and inhibin/activin beta(A) and beta(B) subunit proteins were localized to enlarged interstitial cells of the equine fetal ovary. Expression of inhibin alpha and inhibin/activin beta(A) subunit mRNAs were also observed in the interstitial cells. We conclude that the main source of large amounts of inhibins in fetal circulation is interstitial cells of fetal ovary and is not of maternal origin. Furthermore, these inhibins may play some important physiological roles in the development of gonads in the equine fetus.

Immunolocalization of the inhibin (a) and inhibin/activin (beta3A and betaB) subunit proteins in equine fetal testes was investigated to determine the ability of the fetal testis to produce inhibins at 120, 150, 200, and 250 days of gestation. In addition, concentrations of immunoreactive (ir)-inhibin, inhibin pro-aC, and inhibin A in both the maternal and fetal circulation were measured. It was found that plasma concentrations of ir-inhibin, inhibin pro-alphaC, and inhibin A were much higher (P.05) in the fetal than in the maternal circulation at any stage of gestation examined. Similarly, while fetal testicular homogenate contained increased amounts of inhibins, the inhibins were undetectable in homogenates of maternal ovaries and placentae. At 120 days of gestation, all 3 subunit proteins were localized to the interstitial cells, while the immunoreactivity for the inhibin/activin 3B subunit protein was also observed in Sertoli cells. The intensity of immunoreactivity for the 3 subunit proteins in interstitial cells increased as pregnancy advanced to day 200, and, at this stage, immunoreactivity for the inhibin alpha subunit protein was observed in the fetal testes in a pattern consistent with localization in Sertoli cells. Thus, the inhibin/activin betaA subunit protein was confined to interstitial cells during the gestational periods examined. We conclude that equine fetal testes secrete large amounts of inhibins, including dimeric inhibin A and possibly other dimeric forms, such as inhibin B and activins, into the fetal circulation. These results suggest that these proteins may play some important roles in the development of fetal testes during gestation.

To investigate the prevalence of diseases in the Borna disease virus (BDV) antibody positive race horses, we undertook seroepidemiological studies of BDV infection on 125 culled race horses in Hokkaido, Japan. The serological study disclosed the presence of antibodies only to BDV-p40 or -p24 in 19.2% (24/125) and 3.2% (4/125) of culled horses, respectively. Antibodies to both BDV-p40 and -p24 were found in 24.0% (30/125) of these horses. Of particular note was the finding that locomotorium disorders were detectable at a significantly higher rate in BDV antibody positive horses than that in the seronegative horses. These results imply that BDV infection may possibly contribute to an increase in the incidence rate of locomotorium disorders in race horses.