Anti-Müllerian hormone (AMH) is used as a marker of follicle population numbers and potential fertility in several species including horses but limited data exist across the lifespan. No one has decreased ovarian reserve experimentally to investigate whether a corresponding, quantitative decrease in AMH results. Concentrations of AMH across the lifespan were compiled from 1101 equine females sampled from birth to >33 years of age. Young and old mares (averaging 6 and 19 years) were hemi-ovariectomized and circulating AMH was assessed before and daily thereafter for 15 days. The remaining ovary was removed later and blood was drawn again before and after this second surgery for AMH determination. Polynomial regression analysis and analysis of mares grouped by 5-year intervals of age demonstrated AMH concentrations to be higher in mares aged 5–10 and 10–15 years than 0–5 years of age and lower in mares after 20 years of age. There was high variability in AMH concentrations among neonatal fillies, some of which had concentrations typical of males. Hemi-ovariectomy was followed by a decrease of AMH, almost exactly halving concentrations in intact mares. Concentrations of AMH had returned to intact levels in old mares before complete ovariectomy, as if exhibiting ovarian compensatory hypertrophy, but recovery of AMH was not evident in young mares. AMH may reflect ovarian senescence in mares after 20 years of age but is too variable to do so in the first two decades of life. The ovarian endocrine response to hemi-ovariectomy in mares appears to change with age.

Studies in mares have examined serum inhibin concentrations using immuno-assays unable to distinguish dimeric inhibin-A from inhibin-B isoforms. Inhibin-A and inhibin-B immuno-assays were used to investigate concentrations in cyclic mares, young and old (6 vs 19 years old, respectively) mares following hemi-ovariectomy, mares during pregnancy and in mares with confirmed granulosa cell tumors (GCTs). Mares with inter-ovulatory intervals of 26 days had ovulatory peaks of inhibin-A averaging 80 pg/mL with a mid-cycle nadir of 5 pg/mL. Inhibin-A and inhibin-B concentrations were highly correlated (r = + 0.79, P < 0.01) though peak and nadir concentrations of inhibin-B were not significantly different. However, the ratio of inhibin-A to inhibin-B (A/B) changed significantly through the cycle, highest at ovulation and <1 (more inhibin-B than -A) at mid-cycle. Two mares with grossly extended inter-ovulatory intervals demonstrated mid-cycle inhibin-A (and inhibin-B) excursions suggestive of follicular waves. Follicle-stimulating hormone was negatively correlated with inhibin-A and -B concentrations in all 6 mares. Hemi-ovariectomy in young mares resulted in a significant decrease in inhibin-A and inhibin-B concentrations one day later (P < 0.05) but older mares did not, suggesting a possible extra-ovarian source(s) of these hormones. Both inhibin isoforms dropped to very low levels during pregnancy (P < 0.0001), inhibin-A (P < 0.0001) more rapidly than -B (P < 0.05), so that inhibin-B became the predominant measured form throughout most of gestation (P < 0.05). Mares with confirmed GCTs had elevated inhibin-B concentrations more reliably than inhibin-A but neither inhibin-A or -B was correlated with anti-Müllerian hormone concentrations. Collectively, concentrations of inhibin-A and -B were aligned with physiological events in healthy mares, though more pronounced cyclic changes were seen with inhibin-A. Inhibin-B concentrations were significantly associated with GCTs (P < 0.01), inhibin-A concentrations were not. While both inhibin-A and -B concentrations track physiological events such as cyclic follicular activity, only inhibin-B concentrations effectively signal ovarian neoplasia in mares.

Several factors can interfere with sperm cryopreservation resistance, especially the genetic factors and those related to the plasma membrane composition of the sperm and seminal plasma. However, it is still unclear if the same factors that confer freezing resistance will perform the same role during the cooling process. Thus, the aim of this study was to determine the relation between the resistance to freezing and cooling processes in stallions. Two ejaculates from each of 75 stallions were used. All animals showed good quality of fresh semen (total motility higher than 60% and plasma membrane integrity higher than 50%). After collection, the semen was diluted 1 : 1 with commercial skim milk-based extender (Botu-SemenTM, Botupharma, Brazil) and then a part was designed to cooling and the another to freezing. The cooled semen was divided into 2 groups: Group PS, in which the semen was diluted with Botu-SemenTM at a concentration of 50 × 106 sperm mL–1, and Group SPS, which was subjected to a centrifugation at 600 × g for 10 min and resuspended with Botu-SemenTM at 50 × 106 sperm mL–1. Semen samples from both groups were placed in the same cooling passive system for a period of 24 h/5°C. To accomplish the freezing process, the semen sample was subjected to centrifugation at 600 × g for 10 min. The supernatant was discarded, and the pellet was re-suspended in a Botu-CrioTM. The straws were frozen according to the manufacture. The sperm parameters from fresh semen, cooled semen for 24 h with and without seminal plasma, and frozen semen were evaluated for kinetics by computer-assisted semen analysis and for plasma membrane integrity (IMP%) by epi-fluorescence microscopy. The animals were classified in relation to their resistance to cooling and freezing processes as follow: “bad coolers” – reduction in sperm total motility and in plasma membrane integrity higher than 35% after 24 h of cooling in samples with seminal plasma; “good coolers” – reduction in sperm total motility and in plasma membrane integrity lower than 35% after 24 h of cooling in samples with seminal plasma; “bad freezer” – sperm total motility lower than 40% and progressive motility lower than 20% in seminal sample after thawing; “good freezer” – sperm total motility higher than 60% and progressive motility higher than 30% in seminal sample after thawing. The comparison between the resistance to cooling and freezing processes was performed by Fisher's exact test. The level of significance was 5%. No difference (P < 0.05) between the resistance to cooling and freezing processes was observed. The percentage of stallions “good freezer” and “good cooler” was 54%, “good freezer” and “bad cooler” was 22.6%, “bad freezer” and “good cooler” was 12%, and “bad freezer” and “bad cooler” was 10.6%. Within stallions classified as “good freezer” and “bad cooler,” 52.9% also were “good cooler” when the seminal plasma was removed before the cooling process, and 47.1% remained as “bad cooler.” The result of this study demonstrates that there is a strong relation between the resistance to cooling and freezing processes in stallions. In stallions categorized as “bad cooler,” the seminal plasma presents a major influence on the quality and longevity of cooled semen.

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