Early Diagnosis of 60, XX/XY Chimerism in Cattle using RT-PCR

Background: Freemartinism is a special kind of cell chimerism occuring in female calf born co-twin with male calf. It is one of the most common forms of intersexuality found in cattle, arises as a consequence of vascular anastamosis between fetuses of different genders. Freemartinism causes alterations in the development of female reproductive organs and sterility. However, there were reports of fertile heterosexual twins also. Hence, the present study aimed at early diagnosis of 60, XX/XY chimerism in heterosexual pregnancies to prevent the unnecessary economic losses and preserve valuable genetic material. Methods: Chromosomal profile of female and male calves were analyzed through short term lymphocyte culture technique. The gene expression levels of ZFX and SRY genes were determined through SYBR green real-time PCR. Result: Both female and male co-twins were found to possess 60, XX/60, XY chimerism, yielding the PCR products of both the male (SRY) and female (ZFX) specific genes. A real-time PCR revealed higher levels of ZFX gene expression in female (81.66 per cent) and male (88.35 per cent) calves than SRY gene. Combination of karyotyping and real-time PCR gave a confirmative diagnosis of freemartinism, suggesting the early diagnosis soon after birth.