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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.

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.

Measurement of progestines and estrogens in maternal plasma has been advocated as an indicator of foeto-placental function. The goals of the present study were as follows: to determine progestin and estrogen concentrations in mares with normal and abnormal pregnancies during late gestation, evaluate the diagnostic value of hormone concentrations, and investigate the relationship between hormone concentrations after treatment and the survival of foals. Blood samples were collected monthly from 459 pregnant mares between Day 201 of gestation and foaling. Progestin and estrogen concentrations were measured using a time-resolved fluoroimmunoassay with anti-progesterone and anti-17β-estradiol antibodies. The cutoff values of hormone concentrations that best discriminated between healthy foals and foal loss groups were determined using receiver-operating characteristic (ROC) curve analysis. Mares with compromised pregnancies are treated with progestins, tocolytics and antibiotics according to clinical signs, ultrasonographic examination and hormonal test. We investigated the relationship between the restoration of hormone concentrations after treatment and the survival rate of the foals of 62 compromised mares. Maternal serum progestin and estrogen concentrations on Days 241-320 of gestation differed significantly between healthy foals and foal loss of each group. Predictions of pregnancy outcomes were based on ROC curve analysis that established cutoff values of serum progestin and estrogen concentrations for each 20-day interval during gestation. All mares with improved concentrations of progestins or estrogens after treatment delivered healthy foals. When hormone concentrations got worse after treatment, the rate of loss was higher when treatment was administered during Days 201-290 of gestation than after Day 290. Mares with high progestin and low estrogen concentrations were likely to deliver aborted/dead foals during mid-to-late gestation. These results suggest that measurement of progestin and estrogen concentrations may be useful for diagnosing abnormal pregnancy and implementing early treatment strategies.

The objective was to assess the optimal procedure for real-time, three-dimensional (3D) ultrasound (US) imaging for assessing the equine fetus during the first half of gestation and the possibility of using 3D US imaging of the equine fetus in clinical applications. Seventeen pregnant mares were examined by 3D US between Days 35 and 180 of gestation. Abdominal and endo-vaginal real-time 3D transducers used in human medicine were used for transrectal and transvaginal examinations, respectively. Images were recorded by both 3D stationary and real-time movies. In a comparison of four methods, transrectal examination with a bulb-shaped abdominal 3D transducer enabled the equine fetus to be clearly visualized, and did not require sedation of the mare. Therefore, this approach was the most suitable procedure for examining equine fetuses during the first half of gestation. Each scan required only a few seconds and an entire examination took