Stallion semen quality after different pre-sorting treatments

Abstract

Interest in flow-cytometrically sex-sorted stallion sperm has been increasing over the years but the commercial application of this process has been limited primarily because of the low fertilizing ability of frozenthawed, sex-sorted stallion sperm. The aim of this study was to investigate the effect of different pre-sorting treatments on sorted stallion sperm quality before and after freezing. Four ejaculates from each of two stallions were collected one week apart. Each ejaculate was diluted (1:1) in Kenney’s modified Tyrode’s medium (KMT) and divided into three different treatments: simple dilution (D), cushioned centrifugation (CC) and single layer density gradient centrifugation (CG) performed simultaneously (300 x g 20 min). For CC, 1 mL of a cushion solution (Cushion Fluid, Minitube, Germany) was placed at the bottom of a 50 mL glass tube; for CG a 70% gradient solution was prepared using a silica particle solution (RediGrad, GE Healthcare Life Sciences) diluted with a calciumfree buffer. Diluted semen (D) and sperm pellets after centrifugation (CC, CG) were diluted in KMT to 100 million sperm/mL and stained with 89mM Hoechst-33342 and