Comparison of Two Commercial Extenders for Refrigerated Stallion Semen: Effects on Sperm Quality over 48 h

Refrigerated Stallion Semen Extenders

Abstract

Artificial insemination in horses relies on effective semen preservation to maintain sperm function during storage and transport, particularly for refrigerated semen used in national and international shipment within 24 to 48 h. This study compared the effect of two commercial extenders, INRA 96® and Equiplus®, on the quality of refrigerated stallion semen. Semen from ten stallions of proven fertility was collected and diluted to a final concentration of 25 × 10⁶ spermatozoa/mL in each extender. Samples were evaluated at 0, 24, and 48 h for total motility (TM) and progressive motility (PM) using computer-assisted sperm analysis (CASA), and for functional parameters using flow cytometry, including viability, mitochondrial membrane potential (MMP) and acrosome status. Sperm motility decreased over time after dilution with both extenders; however, TM and PM were significantly higher in samples diluted with INRA 96® at 48 h (p < 0.05), with no significant differences at 0 or 24 h (p > 0.05). Sperm viability declined during storage but remained significantly higher in samples diluted with INRA 96® at 48 h (p < 0.05). No significant differences were observed in MMP between extenders at any time point (p > 0.05). Acrosome damage differed between extenders, being significantly higher in samples diluted with Equiplus® at 48 h (p < 0.05), with no significant changes over time within each extender (p > 0.05). These results suggest that INRA 96® may be associated with improved preservation of motility and viability, and reduced acrosome damage during refrigerated storage for up to 48 h.