How temperature affects equine semen: refrigeration versus cryopreservation. A simple method to select high quality spermatozoa

Cooled and frozen equine semen shows a reduction in fertility, compared to fresh one. In this study, cooled and frozen-thawed equine spermatozoa were compared and analyzed for plasma and acrosomal membrane integrity and mitochondrial membrane potential, combining three fluorescent probes: H258, CTC, JC-1 with a micro-spectrofluorimetric analysis (Quanticell equipped with a digital system for color images acquisition). Total and progressive motility, average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) were measured by CASA system. We employed an innovative approach to study the reproductive potential of the male gamete subjected to cooling protocols for semen storage. In fact, we evaluated the modifications of equine sperm physiology induced by temperature during cooling and freezing treatments looking at the modifications of different functional sperm characteristics by a simultaneous analysis of different sperm markers with the aim of selecting those that are the most efficient signs for sperm fertility. We identified the mitochondrial membrane potential because it provides useful information on equine sperm quality strictly correlated with fertility. We consider it a useful marker for sperm fertility to be used as a guide to select high-quality semen to be employed in equine breeding farmers.