Malaysian Society of Animal Production 73 Verification of Caprine Sexed-separated Spermatozoa by Real Time PCR

Abstract Sexed spermatozoa could contribute to increasing profitability of the dairy and beef industries worldwide by producing offspring of predetermined sex. Various methods have been designed to separate sperm population into X-and Y-chromosome bearing spermatozoa. In this study, the caprine semen sample from Savanna (n=2) and Boer (n=4) breeds were sex-separated using free flow electrophoresis. In order to determine the efficiency of sex-separation procedure, a rapid and accurate technique real time PCR (qPCR) was used to validate the X-and Y-chromosome bearing spermatozoa following separation. The primers and probes were developed to detect the X-and Y-chromosome bearing spermatozoa of Capra hircus based on ZFX and SRY genes, respectively, using TaqMan qPCR assay. The amplification of ZFX and SRY genes produced a single fragment of 93 bp and 137 bp, respectively. The primers used in separated spermatozoa by free-flow electrophoresis technique showed that the mean ratio of X-and Y-chromosomes bearing spermatozoa from the cathodic and anodic fractions were 36:64 and 44:56, respectively, compared to unseparated semen was 51:49. Thus, these primers can be used to validate the gene copy of X-and Y-chromosome bearing spermatozoa in caprine semen