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

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 (n=3), 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 fluorescent-based sperm counter (NucleoCounter SP100TM, ChemoMetec, Allerød, Denmark) to determine sperm concentration and total sperm number (TN). A mixed model ANOVA procedure was used for analysis (SAS Institute Inc., Cary, NC, USA). Results were considered significant at the P<0.05 level. TMOT was higher in the 70 and 80% SPS compared to all other treatments. Sperm velocity (VCL) was lower in T80 compared to all other treatments except T70 and C. Percent viable sperm was similar among treatments C and T40-80. Percent acrosome intact (AI) sperm was higher in T70-80 than C, but similar to T20-60. The percent sperm with normal DNA was similar for C and T30-80; but higher in C and T60-80 than T10-20. Recovery rates for T10-50 were similar; T80 was lower than T10-60, but similar to T70. Percent morphologically normal sperm was higher in T10-30 and T50-80 than C. Single-layer centrifugation using silica particle solutions to separate normal from abnormal sperm are commonly used at an 80% SPS concentration. In this study, sperm quality and total sperm number were maximized at gradients other than 80% depending on the variable measured. While an 80% SPS concentration, in many but not all treatments, recovers a higher percentage of quality sperm, the TN of quality sperm is not maximized. These results suggest that an improvement in the total number of quality sperm recovered could be achieved in lesser SPS concentrations.