Blood plasma anti-Müllerian hormone and insulin-like peptide 3 levels in relation to canine semen freezability.

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.