Canine sperm cryopreservation is increasingly used in assisted reproduction, yet sperm freezability in individual dogs or ejaculates remains difficult to predict. This study investigated whether circulating anti-Müllerian hormone (AMH) and insulin-like peptide 3 (INSL3), markers of Sertoli and Leydig cell function, are associated with semen quality and cryotolerance in dogs. Semen was evaluated before and after cryopreservation, and blood plasma AMH and INSL3 concentrations were measured by ELISA. AMH was associated with lower post-thaw total and progressive motility and greater total motility decline (Benjamini–Hochberg-adjusted p = 0.002, 0.004, and 0.013, respectively). In regression analysis, AMH predicted total motility decline (R2 = 0.317, p < 0.001), although the model accounted for a modest 31.7% of the observed variation. An AMH threshold of 19.9 ng/mL identified pronounced post-thaw motility decline (>30% total and >20% progressive) with an area under the curve (AUC) of 0.860 (95% CI: 0.733–0.987; p = 0.002). INSL3 showed nominal associations with the multiple anomaly index and, in a subset of 31 dogs, with post-thaw plasma membrane integrity, mitochondrial membrane potential, and plasma membrane integrity decline after freezing (p = 0.002–0.033), but none remained significant after correction. Exploratory ROC analysis suggested an INSL3 cutoff of 235.3 pg/mL for discriminating ≥15% membrane integrity decline (AUC = 0.791; 95% CI: 0.623–0.958; p = 0.001). In conclusion, AMH showed the strongest associations with post-thaw motility, whereas the INSL3 findings remain exploratory.
Embryo recovery (ER) is a major determinant of efficiency in equine embryo-transfer programs and may be influenced by biological, management, and technical factors. This retrospective multicenter study aimed to identify factors associated with positive ER and to describe the embryo-flushing (EF) techniques used in three commercial embryo-transfer programs. We hypothesized that factors associated with ER would differ among clinics because of differences in mare populations, semen types, management practices, and flushing systems. The study included 2861 EF cycles: 465 cycles from 196 mares in Clinic 1, 661 cycles from 265 mares in Clinic 2, and 1735 cycles from 277 mares in Clinic 3. Factors associated with positive ER were evaluated separately for each clinic using multivariable generalized linear mixed models, with mare identity included as a random intercept. Additional univariable analyses were used to examine selected factors individually. Overall ER was 55.7%, 51.4%, and 73.5% in Clinics 1, 2, and 3, respectively. In the multivariable analyses, none of the evaluated fixed effects were significantly associated with ER in Clinic 1. In Clinic 2, semen type was the only significant fixed effect, with greater odds of ER using chilled than frozen semen (OR = 2.07, 95% CI: 1.37–3.11; p < 0.001). Clinic 3 used fresh semen exclusively, and donor mare age was the only significant fixed effect, with the odds of ER decreasing by 9% for each additional year of age (OR = 0.91, 95% CI: 0.88–0.94; p < 0.001). Although the overall effect of season phase was not significant in Clinic 3, ER was lower during mid-season than early season (OR = 0.67, 95% CI: 0.46–0.99; p = 0.043). Mare identity had a significant random effect in all three clinics (p ≤ 0.05), indicating substantial between-mare variability. Repeated EF within the same season did not reduce ER in any clinic, including among mares undergoing four or more cycles. In the additional analyses, multiple ovulation was associated with higher ER in Clinics 1 and 3 but not in Clinic 2. In Clinic 2, experienced operators showed a tendency toward higher ER than less experienced operators (52.4% vs. 37.7%; p = 0.06). In conclusion, factors associated with ER differed among clinical settings. The consistent mare effect, the absence of a decline in ER with repeated flushing, and the potential influence of operator experience emphasize the importance of individual mare fertility and clinic-specific management when evaluating the efficiency of commercial embryo-transfer programs.
This study evaluated the effects of supplementing bovine in vitro maturation medium with 2-oxothiazolidine-4-carboxylate (OTC), a cysteine precursor, on oocyte quality, intracellular oxidative status, embryo development, and gene expression in oocytes and derived blastocysts. Oocytes were matured in TCM-199 maturation medium supplemented with 0 (control), 1, 2, or 4 mM OTC, followed by in vitro fertilization and embryo culture. Nuclear maturation, intracellular reactive oxygen species (ROS), glutathione (GSH) levels, embryo development, blastocyst quality, and expression of genes associated with developmental competence were evaluated.OTC supplementation significantly reduced intracellular ROS levels in all treated groups compared with the control. Intracellular GSH concentrations remained unchanged. OTC supplementation reduced the proportion of oocytes reaching the MII stage, whereas cleavage and blastocyst formation rates were not significantly affected in the separate set of oocytes subjected to IVF. Blastocysts derived from oocytes matured with 1 and 2 mM OTC contained significantly higher total cell numbers than controls. Gene expression analyses demonstrated concentration-dependent changes in selected oocyte and blastocyst transcripts.In conclusion, OTC exerted concentration-dependent effects on bovine oocyte maturation and subsequent embryo characteristics. OTC reduced intracellular ROS without altering total intracellular GSH, while 1–2 mM OTC increased blastocyst total cell number without increasing blastocyst yield. However, OTC supplementation reduced MII-stage maturation, and its effects on selected oocyte and blastocyst transcripts varied with concentration. These findings demonstrate endpoint-specific responses to OTC supplementation and support further investigation of its effects during bovine IVM.
