SYBR® Green quantitative PCR for sex determination of bovine spermatozoa

Spermatozoa sexing technology in cattle breeding is being done by separation of x- and y- chromosome bearing spermatozoa using flow-sorting technology. however, the sexing technique needed to be validated to ensure the accuracy of the technology. a technique to determine the sex of bovine spermatozoa using sybr® green real-time quantitative pcr (qpcr) was developed. two sets of primers, zfx and sry were designed specifically to x- and y- chromosome bovine genes respectively. plasmid was inserted with zfx and sry gene fragment separately to create standard curves that ranged from 3.0 x 102 to 3.0 x 106 copies. the standards generated linear relationship with regression coefficient r^2 = 0.984 for zfx and r^2 = 0.996 for sry. both standards of zfx and sry showed melting peak at temperature of 83 ºc and 85 ºc respectively. real-time qpcr of bovine spermatozoa dna samples and cloned plasmid zfx and sry genes for creating standard samples were performed simultaneously. the percentages of unsexed x- and y- chromosome-bearing spermatozoa did not differ much from the 1:1 as reported in unsexed spermatozoa population. therefore, the highly sensitive and fast method of real time pcr is a suitable technique for quantitating x- and y- chromosome-bearing spermatozoa in semen samples.