Effect of warming regimen on canine epididymal spermatozoa following vitrification using a coconut water extender supplemented with egg yolk

Abstract Background Preservation of canine genetic material through epididymal sperm recovery and vitrification has gained increasing importance in breeding programs and germplasm conservation. The warming procedure represents a critical step affecting post-vitrification sperm survival and functionality. This study evaluated the effect of two warming temperatures on the quality of vitrified canine epididymal spermatozoa. Methods Epididymal spermatozoa were collected from twelve clinically healthy dogs undergoing routine orchiectomy. Samples were vitrified using a coconut water-based extender supplemented with egg yolk and subsequently warmed at either 37 °C or 42 °C for 2 min. Sperm quality was evaluated before vitrification and after warming by assessment of motility, viability, morphology, and kinematic parameters using computer-assisted sperm analysis (CASA). Results Vitrification significantly reduced sperm quality compared with fresh samples. However, warming at 37 °C resulted in significantly higher total motility, viability, and percentage of morphologically normal spermatozoa compared with warming at 42 °C. Several kinematic parameters were also better preserved following warming at 37 °C. Conclusions The warming protocol significantly influences post-vitrification quality of canine epididymal spermatozoa. Warming at 37 °C for 2 min provided superior preservation of sperm function compared with warming at 42 °C and may represent a practical approach for canine germplasm banking and assisted reproduction.