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.

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.

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.

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.

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.

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.

Molecular identification and risk factor analysis of the first Lumpy skin disease outbreak in cattle in Mongolia.

Lumpy skin disease (LSD) is a transboundary viral infectious disease in cattle caused by a Capripoxvirus. LSD has been recently introduced in some Asian countries. However, in Mongolia, no report of LSD is publicly available. We clinically examined LSD symptoms in 1,034 cattle from 4 soum (district) in Dornod province in Mongolia. Sixty-one cattle of them were confirmed with symptoms of LSD and then viral P32 gene was detected by a PCR. The overall prevalence of LSD in cattle was 5.9%. Females odds ratios (OR)=2.27 than males, adults (>2.5-years-old, OR=3.68) than young (1-2.5-years-old) and calves (<1-year-old) were at higher risks for LSD cases in Mongolia, while locations near the tube well and pond water are major risk areas for viral transmission due to density of insects often is high. For virus isolation, skin nodule tissue samples of 4 cattle located in four distinct soums were used for viral propagation using the MDBK cell line. Internal terminal repeat region and RPO30 gene of 4 Mongolian isolates were amplified and sequenced. In the phylogenetic trees, Mongolian LSDVs (2021) were clustered together with the Chinese (2020) and Vietnamese isolates (2020). This is the first report alarming the LSD outbreak in Mongolia that was confirmed by our study. The newly isolated viruses would be a useful base for developing diagnostic tools and inactivated vaccine technology. A large-scale study of LSD is next priority for establishing successful control strategy of further disease outbreak.

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.

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.