Sperm sexing in Nili-Ravi buffalo through modified swim up: Validation using SYBR® green real-time PCR

•Modified swim up could be an effective method for separating X and Y bearing Nili Ravi buffalo bull sperm.•Significantly higher proportion of X bearing sperm were recovered from lower fraction, while significantly higher proportion of Y bearing sperm were separated from upper fraction of the supernatant.•Sperm separation through modified swim up did not compromise sperm quality after separation and after freeze thawing.•Sperm sexing through modified swim up method has been corroborated by SYBR green real time PCR.

Sperm sexing through flow-sorting technology is relatively expensive, requires considerable technical support and is actually not practicable in many developing countries. The aim of this study was to investigate the feasibility of producing enriched pools of X or Y chromosome-bearing sperm by a modified swim-up method. For this purpose semen was collected from five mature Nili-Ravi buffalo bulls for a period of six weeks. The qualifying ejaculates were divided into two aliquots for further processing through modified swim-up or control (untreated). After processing, semen was cryopreserved in tris citric acid extender using standard techniques. Semen quality was assessed at pre dilution, post dilution and post thawing. Validation of technique was done by using SYBR® green real time PCR using two sets of primers, PLP and SRY for X and Y chromosome of buffalo genes, respectively. Sperm recovery rates, pre freeze and post thaw sperm quality were found significantly higher in X chromosome bearing sperm fraction than Y chromosome bearing fraction and control. Mean fold relative expression of X bearing sperm was significantly higher (4-5 fold) in X chromosome bearing fraction of supernatant than Y chromosome bearing fraction (0.06 fold), similarly mean fold relative expression of Y chromosome bearing sperm was significantly higher in Y chromosome bearing fraction (4 fold) of supernatant than X chromosome bearing fraction (0.15 fold) compared to control (1.00). In conclusion, a modified swim up method proved to be an effective method for Nili-Ravi buffalo sperm sexing as validated by real time PCR.