Recovery rate and sperm quality after centrifugation of stallion sperm in different gradient concentrations

Abstract

Single-layer sperm centrifugation (SLC) is commonly used in the horse industry to select sperm of high quality for artificial insemination or ICSI techniques. Most current protocols use 80% silica particle solution (SPS). It has not been determined whether this SPS concentration optimizes both sperm quality and recovery. The objective of this study was to determine recovery rate and sperm quality after SLC in varying SPS concentrations to determine the total number and quality of sperm recovered. For each replicate (n1⁄43), ejaculates from each of three stallions were pooled. Treatments included eight SPS concentrations (10,20,30,40,50,60,70,80%) and a control (C) sample. Treatments were prepared using a silica particle solution (RediGrad, GE Healthcare Life Sciences) diluted with a calcium-free TALP buffer. Following simple centrifugation (400 x g for 20 min) using a cushion solution (Minitube, Germany) the sperm pellet on top of the cushion fluid was extended to 500 million sperm/mL and 1mL was layered on top of each SPS solution and centrifuged (200 x g for 30 min). The SPS was removed and the pellet resuspended to approximately 1 mL in extender (Heitland, Eq Vet J 28:47,1996). Treatments were evaluated using 1) CASA for total sperm motility (TMOT),2) flow cytometry to evaluate DNA quality (Sperm Chromatin Structure Assay), and viability/acrosome integrity (Pisum sativum agglutinin/propidium iodide), 3) DIC microscopy to evaluate sperm morphology, and 4) a