Temporal characterization of conditions that promote functional capacitation of stallion sperm

Abstract

Equine in vitro fertilization (IVF) has historically been challenged due to lack of conditions that promote functional capacitation of stallion sperm. Capacitation success is often attributed to prolonged sperm incubation requirements. Advances to equine IVF are hindered by incomplete characterization of conditions that promote the functional capacitation of stallion sperm over time. Herein, we describe conditions that promote the functional capacitation of fresh, cooled, and frozen-thawed stallion sperm isolated by Percoll, Swim-Up, or Microfluidics (VetMotl). Fresh (diluted sperm) and previously cooled stallion sperm held at 5°C for 24–48 hr were maintained in capacitating conditions at 38°C or ambient temperature for up to 22 hr to determine motility, tyrosine phosphorylation, and acrosome integrity at 4 hr intervals. Holding sperm at 38°C was detrimental to motility while fresh and previously cooled sperm maintained at ambient temperature displayed maximum capacitation at 4 hr compared to prolonged holding times, regardless of sperm isolation technique. Importantly, sperm evaluated at 4 hr displayed the highest acrosome integrity. The functional capacitation of fresh sperm maintained under these conditions was challenged in a heterologous bovine IVF system where oocyte activation was confirmed by pro-nuclear formation and polar body extrusion. Frozen-thawed stallion sperm was then applied to an equine IVF system where Microfluidic sperm selection proved superior to Swim-Up regarding sperm kinematics, capacitation and acrosome status. Fertilization potential was confirmed by pro-nuclear formation and embryo development. These findings provide valuable information to conditions that support functional capacitation of stallion sperm towards advancing equine in vitro embryo production. Data availability Data is provided within the manuscript or supplementary information files. All data are available upon reasonable request to the corresponding author.