Non-suppurative and necrotizing testicular arteritis in the male reproductive organs of a heavy draft horse.

Equine testicular arteritis commonly occurs as a consequence of the migration of nematode larvae or equine arteritis virus (EAV) infection. However, testicular arteritis without evidence of these infections has been reported, and the underlying pathogenesis remains unclear. We encountered testicular arteritis without evidence of nematode or EAV infection in a 3-year-old male heavy draft horse with scrotal enlargement. Grossly, the volume of the pampiniform plexus was markedly increased due to edema. Histologically, non-suppurative and necrotizing testicular arteritis, characterized by lymphocyte infiltration and fibrinoid necrosis of the arterial walls, was diffusely observed in the spermatic cord, pampiniform plexus (most severe), testis, and epididymis. We were unable to identify the cause of arteritis, such as a viral infection or autoimmune abnormality.

Use of Transabdominal Ultrasound and Maternal Hormone Testing for the Prenatal Monitoring of Equine Fetal Enlarged Bladder.

Ultrasound examination during late gestation is one of the best methods for monitoring potential pregnancy risks. Enlarged bladder is a urological disorder rarely observed in equine fetuses. This clinical case report aimed to present a case illustrating the development of equine fetal enlarged bladder using transabdominal ultrasound examinations and maternal hormone evaluation during gestation. An 8-year-old Hokkaido native pony was impregnated by embryo transfer, and at 215 days of gestation, abnormalities of the fetal bladder were detected. The bladder volume increased with gestational age, and a second bladder was observed at 257 days of gestation. No abnormalities were observed in the fetal kidneys. Moreover, the maternal plasma progesterone concentration was measured throughout the gestation period. The progesterone concentration was elevated from 36 weeks of gestation until parturition. At 363 days of gestation, parturition induction was conducted, and a foal successfully delivered. This case report is the first to describe the development of equine fetal enlarged bladder and record the corresponding ultrasound and hormone profiles.

First Kiso pony foal produced via transfer of long-distance shipped fresh embryo to Hokkaido native pony.

Japanese native horses, which consists of 8 breeds, are threatened with extinction. Embryo transfer (ET) is used to reproduce endangered animals in various mammalian species. We aimed to perform ET using native ponies from Kiso and Hokkaido as donors and recipients, respectively. ET operation included long-distance transport of non-cryopreserved embryos from Nagano Prefecture to Hokkaido. Embryos were transported 1500 km over 9 h in a container maintained at 22°C. After transferring two embryos to two recipients, one mare delivered a healthy live foal. These results demonstrated that reciprocal ET with long-distance transportation of fresh embryos between the isolated breeds may allow for the proliferation of Japanese native horses.

Plasma activin A concentrations during late gestation in Thoroughbred mares with abnormal pregnancies.

Late-term fetal loss in horses is a major problem in the horse-breeding industry globally. Abnormal pregnancies should be diagnosed as early as possible to prevent abortions and other gestational problems. According to our previous longitudinal study in healthy pregnant mares, the plasma activin A concentration increases as pregnancy progresses. The aim of the present study was to compare plasma activin A concentrations in healthy pregnant Thoroughbred mares (n=40) with those in pregnant mares that suffered fetal loss or showed abnormal symptoms (n=30) during late gestation. This field study found that plasma activin A concentrations were higher in the abnormal group (pregnancy loss, red bag delivery, premature udder development, and vaginal discharge) than the normal group (P < 0.001; cutoff value: ≥ 138.2 pg/mL; sensitivity, 74.4%; specificity, 77.5%). More specifically, plasma activin A concentrations in the "symptom" and "abnormal delivery" subgroups were higher than those in gestational-age-matched normal groups (P < 0.001). Nevertheless, the plasma activin A concentration in the "normal delivery" subgroup was not different from that in the "abnormal delivery" subgroup in samples collected within 10 days before delivery. In conclusion, this study is the first to demonstrate a significantly earlier increase in plasma activin A concentration in abnormal pregnancies of Thoroughbred mares during late gestation.

Ultrasonographic examination of equine fetal growth parameters throughout gestation in pony for Equine-Assisted Therapy.

Equine-Assisted Therapy (EAT) is gaining popularity. Ultrasound examination is used to decrease the abortion rate in horses. In this study, to monitor fetal well-being throughout the gestation for EAT, we measured fetal heart rate (FHR), fetal eye orbit (FEO), fetal gonad length (FGL), fetal kidney length (FKL), and the combined thickness of the uterus and placenta (CTUP) by ultrasonography in pony mares. Additionally, we measured the plasma progesterone (P4) and estradiol (E2) concentrations in pregnant horses using enzyme immunoassay. The FGL peaked at week 32 and then decreased to term, and a strong correlation (r=0.72, P<0.001) between the FGL and E2 concentration was observed. A strong correlation with gestational age was detected among the FEO (r=0.96, P<0.001), FKL (r=0.85, P<0.001), and CTUP (r=0.96, P<0.001). The P4 concentration peaked at week 10, decreased to low levels (below 5 ng/ml), and peaked before parturition. In conclusion, this study provides information on fetal growth throughout gestation in pony mares for EAT. In addition, it revealed the relationship between ultrasonographic profile and plasma hormone concentrations during gestation.

Estrogens Regulate Placental Angiogenesis in Horses.

A sufficient vascular network within the feto-maternal interface is necessary for placental function. Several pregnancy abnormalities have been associated with abnormal vascular formations in the placenta. We hypothesized that growth and expansion of the placental vascular network in the equine (Equus caballus) placenta is regulated by estrogens (estrogen family hormones), a hormone with a high circulating concentration during equine gestation. Administration of letrozole, a potent and specific inhibitor of aromatase, during the first trimester (D30 to D118), decreased circulatory estrone sulfate concentrations, increased circulatory testosterone and androstenedione concentrations, and tended to reduce the weight of the fetus (p < 0.1). Moreover, the gene expression of CYP17A1 was increased, and the expression of androgen receptor was decreased in the D120 chorioallantois (CA) of letrozole-treated mares in comparison to that of the control mares. We also found that at D120, the number of vessels tended to decrease in the CAs with letrozole treatment (p = 0.07). In addition, expression of a subset of angiogenic genes, such as ANGPT1, VEGF, and NOS2, were altered in the CAs of letrozole-treated mares. We further demonstrated that 17ß-estradiol increases the expression of ANGPT1 and VEGF and increases the angiogenic activity of equine endothelial cells in vitro. Our results from the estrogen-suppressed group demonstrated an impaired placental vascular network, suggesting an estrogen-dependent vasculogenesis in the equine CA during the first trimester.

Circulating activin A during equine gestation and immunolocalization of its receptors system in utero-placental tissues and fetal gonads.

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.

Influence of exercise and emotional stresses on secretion of prolactin and growth hormone in Thoroughbred horses.

The secretion of prolactin and growth hormone in response to exercise and emotional stresses was investigated in Thoroughbreds. Two experiments were performed: one with loading of only exercise stress and one with simultaneous loading of exercise and emotional stresses. Exercise stress was loaded in 4 steps using a treadmill for horses: pre-exercise period (5 min), walking period (6.5 min), galloping period (3 min), and cooling down period (10 min). Emotional stress was loaded by showing a loud video of an audience at a racetrack during the walking period. The results clearly demonstrated that exercise stress rapidly increased the secretion of prolactin and growth hormone and that secretion of them persisted for a specific period after the exercise. In addition, emotional stress promoted prolactin secretion.

Case report: The case of a 17 kg ovarian granulosa cell tumor in a Breton draft mare.

Granulosa cell tumor (GCT) is a benign tumor which affects the mare's ovaries. In this report, a case of unilateral GCT in an ovary, which weighed 17.04 kg, of a 9-year-old Breton draft mare is described. A transrectal ultrasonography exam revealed a unilateral multi-cystic enlarged ovary. Laparoscopic ovariectomy was difficult due to enlargement of blood vessels in the ovarian broad ligament. The mare was necropsied, and the pathological changes in the GCT-affected ovary and unaffected ovary were evaluated. The ovarian mass in the GCT-affected ovary had a cribriform pattern and was positive for anti-Müllerian hormone (AMH) and its receptor (AMHR2). The contralateral ovary showed no follicular development and was negative for AMH. AMHR2 was positively expressed in stromal cells. The AMH concentration in plasma was 4,210 ng/ml. This is the first report showing the presence of AMH (2,210 ng/ml) in ascites fluid, and it also shows that laparoscopic ovariectomy might not be suitable for larger ovaries affected by a GCT. Ultrasonographic, endocrine, and histopathological analyses were helpful for making a definitive diagnosis of GCT in this mare.

Evaluating the use of piezo manipulator, laser or their combination for blastocoel cavity puncture to improve cryopreservation outcomes of large equine embryos.

The main difficulty of large equine embryo cryopreservation is the replacement of blastocoel fluid with cryoprotectant solution. The objective of this study was to improve the cryopreservation of large equine embryos with PMAP and/or LAP. Embryos were collected via the non-surgical transcervical procedure and divided into three groups based on their size (A ≤ 300 µm, 300 µm300 µm). However, more research is required to find the best method for embryos ≥700 µm.

Presence of surfactant proteins in the uteri and placentae of pregnant mares.

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.

Effect of prostaglandin F2α administration on uterine polymorphonuclear neutrophil counts in Japanese heavy draft horses.

The objective of this study was to examine the effect of prostaglandin F2α (dinoprost) and oxytocin administration on uterine polymorphonuclear neutrophil counts in the Japanese heavy draft mare. To compare polymorphonuclear neutrophil (PMN) counts in the endometrium, a total of 162 samples were collected from 54 estruses of 47 mares (before ovulation, day 0, and day 2) using a double-guarded cytology brush. Dinoprost (PG; 5 mg) was administered intramuscularly (i.m.) only once, on day 0, whereas oxytocin (OT; 20 U i.m.) was administered three times at 12-hr intervals starting on day 0. The plasma progesterone concentrations from days 0 to 14 were not different between the non-treatment (CON), PG treatment, and OT treatment groups. The PMN counts increased in all the groups from before administration to day 0 (CON, 0.90 to 3.55; PG, 1.20 to 8.45; and OT, 0.70 to 1.70; P=0.0014, 0.0046, and 0.0073, respectively). There was a significant decrease in PMNs from day 0 to day 2 only in the PG group (P=0.0073). The pregnancy rate was not different among the CON (12/18), PG (14/18), and OT (10/18) groups. The results of this study indicate that dinoprost can reduce uterine polymorphonuclear neutrophil counts.

Equine nonneoplastic abnormal ovary in a draft mare with high serum anti-Müllerian hormone: a case study.

We performed a standing hand-assisted laparoscopic ovariectomy in a draft mare that presented with high serum anti-Müllerian hormone (AMH) level and had an enlarged single cystic ovary. Histopathological examination revealed no tumor cell proliferation in the ovary, but the presence of a large ovarian cyst was confirmed. In the diagnosis of abnormal ovaries in mares, a comprehensive assessment should be performed, including the monitoring of ovarian morphology and biomarkers over time, to determine the disease prognosis and treatment plan. The case of this mare with a nonneoplastic abnormal ovary and increased serum AMH level was rare. We suggest that standing hand-assisted laparoscopic ovariectomy is useful for the removal of large ovaries in draft mares.

Serum concentrations and testicular expressions of insulin-like peptide 3 and Anti-Müllerian hormone in normal and cryptorchid male horses.

Insulin-like peptide 3 (INSL3) is an important hormone for testicular descent during embryonic development and a factor for assessing functional status of Leydig cells of testes, but there is limited number of equine studies. Anti-Müllerian hormone (AMH) is a useful diagnostic marker for cryptorchidism in horses. This study aimed to compare serum concentrations and testicular expression intensity of INSL3 and AMH in intact and cryptorchid male horses. Serum INSL3 concentrations in intact (n = 9; mean ± SEM, 19.9 ± 5.9 ng/mL) and noncastrated unilateral cryptorchid (UC) male horses (n = 16; mean ± SEM, 16.8 ± 4.1 ng/mL) were higher compared with hemicastrated unilateral cryptorchid (HCUC) male horses (n = 9; mean ± SEM, 3.8 ± 0.7 ng/mL) (P < 0.05). And serum INSL3 in bilateral cryptorchid (BC) male horses (n = 4; 1.9 ± 0.4; mean ± SEM, ng/mL) were lower compared with intact male horses (P < 0.05). Serum AMH concentrations in BC male horses (n = 3; mean ± SEM, 30.6 ± 4.8 ng/mL) were higher compared with intact male horses (n = 5; mean ± SEM, 12.2 ± 3.9 ng/mL) (P < 0.05). Immunostaining of scrotal and cryptorchid testis showed that Sertoli cells were positive for AMH, and Leydig cells were positive for INSL3. Staining intensity of AMH was higher in cryptorchid testis than in scrotal testis (P < 0.05). Furthermore, AMH expression intensity was higher in abdominal testis than in inguinal testis (P < 0.05). Immunostaining intensity of INSL3 in the testis was positively correlated with serum INSL3 (r, 0.7; P < 0.01), seminiferous tubule area (r, 0.727; P < 0.01), and Johnsen score for spermatogenesis (r, 0.604; P < 0.05), whereas immunostaining intensity of AMH in the testis was negatively correlated with seminiferous tubule area (r, -0.814; P < 0.01) and Johnsen score for spermatogenesis (r, -0.807; P < 0.01). Our findings suggested that AMH is a good biomarker for diagnosing cryptorchidism in male horses, in addition to INSL3 values to assess the testis of intact and cryptorchid male horses.

Colostral and foal serum immunoglobulin G levels and associations with perinatal abnormalities in heavy draft horses in Japan.

The purpose of this study was to elucidate the colostral and foal serum immunoglobulin G (IgG) concentration values in heavy draft horses in Japan and to examine the effects of peripartum mare condition on colostral immunity. Colostrum was obtained 1 hr after foaling (pre-suckling; n=178). Blood was collected from the jugular vein of the foals (n=147) at 24 to 48 hr after birth. The foaling statuses of 73 mares were recorded. The average colostral IgG concentration was 10,540 ± 3,190 mg/dl (median=10,928; range 1,434-17,514 mg/dl). The average serum IgG concentration obtained from neonatal foals 24 to 48 hr after birth was 1,750 ± 919 mg/dl (median=1,890; range 0-3,510 mg/dl). Although colostral IgG did not differ between the normal foaling mare (n=59) and dystocial mare (n=14), foal serum IgG was lower in foals born in dystocia than in foals in normal foaling (P<0.05). This study demonstrates reference values for colostral and foal serum IgG specific to heavy draft horses in Japan and suggests that dystocia may interfere with the acquisition of colostral immunity in neonatal foals.

Birth of first foals through embryo transfer after artificial insemination using frozen semen in Japan.

Until now, there have been no reports of foals born through embryo transfer after artificial insemination using frozen semen in Japan. The aims of this study were to develop a riding crossbred horse and evaluate the prospects of embryo transfer technology in multiplying horse population. In both donor and recipient mares, luteolysis was induced by the administration of 0.1 mg Cloprostenol to synchronize the onset of estrus, and ovulation was induced by administering 2000 IU human chorionic gonadotropin (hCG) or 0.75 mg Deslorelin. Frozen semen from an Irish Connemara pony stallion was used to breed a Hokkaido native pony mare by deep-horn artificial insemination (dose, 400 × 106 sperm). A non-surgical technique was used to collect embryos from the donor mare at day 7 post-ovulation and transfer them transcervically into the uterus of recipient mares (n = 4) immediately after collection. Weekly blood samples were collected from the recipients throughout pregnancy. A total of four embryos were recovered from seven collection attempts (57% recovery) from a donor mare in a single breeding season. Three of the four transferred embryos maintained successful pregnancy and delivered a healthy live foal (75% birth). A normal progesterone profile was observed throughout gestation in recipient mares. In conclusion, for the first time, to the best of our knowledge, this study describes the birth of foals through non-surgical transcervical embryo transfer in Japan after artificial insemination using frozen semen. We expect that this new crossbreed (Connemara pony × Hokkaido native pony) will be a good riding breed.

Age related and seasonal changes of plasma concentrations of insulin-like peptide 3 and testosterone from birth to early-puberty in Thoroughbred male horses.

The peripheral blood concentrations of insulin-like peptide 3 (INSL3) have been detected in many mammalian species, but the level of INSL3 in horse remains unknown. The objectives were to develop a time-resolved fluorescence immunoassay (TRFIA) to detect INSL3 concentrations from horse blood as well as to determine the age-related and seasonal changes of plasma concentrations of INSL3 and testosterone from birth to early-puberty in Thoroughbred male horse (n = 11). Monthly blood sample and measurement of body weight, height, chest and cannon bone size were done from birth until 16 mo. The TRFIA and EIA were used to measure plasma concentrations of INSL3 and testosterone, respectively. An increase in mean body weight, height, chest and cannon bone size was observed throughout the study. The monthly blood sampling revealed an increase in mean plasma INSL3 concentrations up to 2 mo, followed by a decreasing and increasing pattern until the end of experiment at 16 mo. A high testosterone level was detected at birth followed by a sharp decrease to basal level within 1 mo, maintained low level up to10 mo before a gradual rise until 16 mo. In case of seasonality, there was no difference in mean plasma INSL3 concentrations between breeding (March to September) and non-breeding (October to February) seasons, whereas a higher (P < 0.001) mean plasma testosterone concentrations in the second breeding season compared to non-breeding season was observed. In age categorized group, an increase (P < 0.01) in mean plasma INSL3 concentrations was noticed at pre-puberty (1-12 mo) and early-puberty (13-16 mo) compared to birth, but a lower (P < 0.001) mean plasma testosterone concentrations was observed at pre-puberty compared to birth and early-puberty. In conclusion, a TRFIA was developed to measure INSL3 levels in horse. An increase in plasma concentrations of INSL3 and testosterone were observed with the advancement of age, whereas for testosterone a very lower level was detected at the non-breeding season than in the second breeding season after birth in Thoroughbred male horse. The INSL3 secretions seemed independent of seasonal influence, at least before puberty.

Successful embryo transfer from Hokkaido native pony after artificial insemination with frozen semen.

There has been no report of equine embryo transfer in Japan for the last 24 years. Our objective was to establish an effective protocol for embryo transfer in domestic horse. A Hokkaido native pony was bred by deep-horn artificial insemination with frozen semen from a Connemara pony. Embryo collection was performed using a non-surgical method on day 7. Two embryos were obtained from three flushes (67% recovery) and were transferred fresh into crossbred recipient mares. Both recipient mares were diagnosed pregnant (100% pregnancy rate) 5 days after embryo transfer and had normal progesterone levels until the end of the observation on day 35 of gestation. This is the first successful embryo transfer in Japan by artificial insemination using frozen semen. Embryo transfer technology could be extremely useful in improving the productivity of Japanese domestic and special riding horses.

Expression of activin receptors in the equine uteroplacental tissue: an immunohistochemical analysis.

Activin is secreted from equine uterine glands and plays important roles in establishment and maintenance of pregnancy in mares. This study aimed to localize activin receptors (ActRs) IA/B and IIA/B using immunohistochemistry in the uteroplacental tissues of seven pregnant Thoroughbred mares. At the time of tissue collection, the mares were at the following days of pregnancy: 88, 120, 161, 269, 290, 313, and 335 days. We fixed the uteroplacental tissues in 4% paraformaldehyde and obtained serial sections that were subsequently stained for analysis. All four isoforms of ActR were expressed in the uteroplacental tissues, including the endometrial epithelium, uterine glands, trophoblasts, and myometrium, throughout pregnancy. Our results suggested the potential role of activin in the uteroplacental tissues.

Combined thickness of the uterus and placenta and ultrasonographic examinations of uteroplacental tissues in normal pregnancy, placentitis, and abnormal parturitions in heavy draft horses.

The combined thickness of the uterus and placenta (CTUP) and ultrasonographic images of uteroplacental tissues were investigated in 35 pregnant heavy draft horses in Months 7-12 of pregnancy. The mares were divided into three groups: those pathologically diagnosed as placentitis (placentitis group, n=3); those who had abortion, premature birth, or fetal malformation (abnormal group, n=7); and those who had no abnormal findings (normal group, n=25). In the normal group, CTUP increased as pregnancy progressed from Months 7 (median, 7.08 mm; range, 5.68-11.27) to 12 (13.31 mm; 7.44-16.31 mm) (P<0.05) and was higher than those reported previously in Thoroughbred, quarter, and American paint horses. Values of CTUP greater than the 75th percentile of the normal group from Months 7 (7.54 mm) to 12 (15.19 mm) were detected in 100% of the placentitis group (3/3) and in 86% of the abnormal group (6/7). Ultrasonographic images showing placental separation were obtained in 67% of the placentitis group (2/3), 29% of the abnormal group (2/7), and 20% of the normal group (5/25). Pathological placental edema and ultrasonographic images showing uteroplacental roughness or distinguishability were observed even in the normal group. These findings suggest that increased CTUP and placental separation would reflect placentitis and abnormal pregnancies and may help to detect them in heavy draft horses.