Development of High Accuracy Multiplex Real - time PCR Technique for Determining Sperm Sex Ratio in Dairy Bull Semen

Abstract

Sperm sex ratio determination using PCR - based technique is widely validated on precise of X - and Y - chromosome purity in sex - sorted bovine semen. The aim of this study was to develop a technique for estimating sperm sex ratio by using a multiplex polymerase chain reaction (multiplex real - time PCR). The X and Y chromosome - specific markers, bovine proteolipid protein (PLP) gene and sex - determining region Y (SRY), were simultaneously quantified in a single tube. To quantify DNA products, Taqman probe -labeled fluorescence was used in the reaction i.e., PLP probe labeled HEX dye, and SRY probe labeled FAM dye. The reference DNA of male peripheral white blood cell (WBC) was used as a template for constructing the standard curve, which contained an equal ratio between X - chromosome and Y - chromosome. The standard curve of both PLP and SRY genes showed two linear equations for calculating the proportion of X - and Y - sperm ratio. The WBC standard curve indicated an average straight - line slope as - 3.330, representing high - efficiency (99.66 %) of the reaction to synthesize new DNA amplicons. Nine samples of bull semen, Y - sorted semen (Y = 90 %), semen samples with Y ratio in 80 %, 70 %, 60 %, 50 %, 40 %, 30 %, 20 %, and X - sorted semen (Y = 10 %), were prepared and quantified by using the multiplex real - time PCR technique. Two standard techniques (singleplex real - time PCR technique and multiplex droplet digital PCR technique (ddPCR)) were amplified X - and Y - chromosome specific genes and compared with the developed technique. Each sample was run in triplicate and calculated in percentage of Y sperm ratio. Results obtained from those three assays were not significantly different. Therefore, the multiplex real - time PCR assay in this study was cost - effective, reduce time - consuming, and providing an accurate estimation method.