Development of SYBR green chemistry based real time PCR for validation of sperm sex ratio in buffalo bull semen

Title: Development of SYBR green chemistry based real time PCR for validation of sperm sex ratio in buffalo bull semen

Authors: R Kumari, V Kumar, K Batra, A Kumar, N Bansal, A Dalal, S Maan and T Nanda

Source: Ruminant Science (2019)-8(1):39-44

Cite this reference as: Kumari R, Kumar V, Batra K, Kumar A, Bansal N, Dalal A, Maan S and Nanda T (2019). Development of SYBR green chemistry based real time PCR for validation of sperm sex ratio in buffalo bull semen. Ruminant Science 8(1):39-44.


The present study aimed at the development of cost-effective technology for the evaluation of sperm sex ratio in buffalo bull semen. SYBR Green-based Real-Time PCR chemistry was utilized for the development of an assay for sex ratio determination from buffalo bull semen.  Two sets of primers were designed for the amplification of PLP(X chromosome-specific gene) and SRY(Y chromosome-specific gene) genes.  Amplified PLP and SRY gene fragment were cloned in plasmid pJET1.2vector. Standard curves were generated for determination of PCR amplification efficiency. The standards generated a linear relationship with regression coefficient r2=0.996 for PLP and r2=0.993 for SRY. Our results show an insignificant difference in semen sex ratio in unsorted semen (51.7±0.52% X and 49.6±0.60% Y). For the validation of this method, repeatability and reproducibility were tested. The low mean values of CV achieved in repeatability and reproducibility assays demonstrated the high reliability of this new approach for quantifications of sexual chromosome content in semen samples.


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