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Background  Freemartinism generally occurs in female offspring of dizygotic twins in a mixed-sex pregnancy. Most bovine heterosexual twin females are freemartins. However, about 10% of bovine heterosexual twin females are fertile. Farmers mostly cull bovine fertile heterosexual twin females due to the lack of a practical diagnostic approach. Culling of such animals results in economic and genetic-material losses both for 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 performed to establish the most accurate diagnosis for freemartin. Twelve Holstein heterosexual twin females were used in this study, while three normal Holstein bulls and three normal Holstein cows were used as a positive and negative control, respectively.    Results  Polymerase chain reaction results revealed that  SRY  gene were absent in three heterosexual twin females and only two of them were verified as fertile in later age. The qPCR results showed that relative content of  SRY  was more than 14.2% in freemartins and below 0.41% in fertile heterosexual twin females. The H-Y antigen test showed no significant numerical difference between freemartin and fertile heterosexual twin female.    Discussion  Our results show that relative content of  SRY  quantified by qPCR is a better detection method for diagnosis of freemartin in Holstein cattle as compare to qualitative detection of  SRY  gene by PCR or quantitative detection of H-Y antigen. To the authors’ knowledge, this is the first time we applied qPCR to diagnosing freemartin by quantifying  SRY  gene and got relative  SRY  content of each freemartin and fertile heterosexual twin female. We concluded that low-level of  SRY  would not influence fertility of bovine heterosexual twin female.

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