Applying real-time quantitative PCR to diagnosis of freemartin in Holstein cattle by quantifying SRY gene: A comparison experiment


Abstract

Background. Freemartinism is a condition generally occurring in the female offspring of dizygotic twins in a mixed-sex pregnancy. Most domestic animals such as bovine, sheep, goat and pig in this condition are sterile, but still about 15% of bovine heterosexual twin females are fertile. However, farmers in China culled almost all bovine fertile heterosexual twin females directly due to lack of practical diagnostic approach, resulting in substantial economic and genetic-material losses both for Chinese dairy and beef industry.

Methods. In this study, a comparative test, including qualitative detection of SRY gene by polymerase chain reaction (PCR), quantitative detection of relative content of SRY by real-time quantitative polymerase chain reaction (qPCR), and quantitative detection of H-Y antigen, was conducted to confirm the most accurate diagnosis for freemartin. Twelve Holstein heterosexual twin females were selected for this study, with three normal Holstein bulls and three normal Holstein cows served as positive and negative control, respectively.

Results. PCR results showed that three heterosexual twin females were absent of SRY gene whereas only two of them were subsequently verified as fertile. The qPCR results showed that relative content of SRY was over 14.2% in freemartin and below 0.41% in fertile heterosexual twin females. The H-Y antigen test showed no significant numerical differences between freemartin and fertile heterosexual twin female.

Discussion. Our results show that relative content of SRY quantified by qPCR, rather than qualitative detection of SRY gene by PCR or quantitative detection of H-Y antigen, is the most accurate method for diagnosis of freemartin in Holstein cattle. To the authors’ knowledge, this is the first time we applied qPCR to diagnosing freemartin by quantifying SRY gene and got the relative SRY content of each freemartin and fertile heterosexual twin female. We declare for the first time that it is the content of SRY gene, rather than its presence or absence, that determines the fertility of a heterosexual twin female.

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