“…A rapid, sensitive, and inexpensive method for identification of freemartin at birth or early age becomes an urgent need to reduce unnecessary economic losses and to preserve important hereditary material, also to avoid the delivery of detrimental genetic materials by progeny ( Biswas et al, 2015 ). Many diagnostic methods have been established for identification of freemartin, such as measurement of vaginal length ( Khan & Foley, 1994 ), blood grouping test for degree of hemolysis ( Kästli & Hall, 1978 ), karyotype analysis for XX/XY chimera ( Dunn, Johnson & Quaas, 1981 ), polymerase chain reaction (PCR) or improved PCR techniques for detection of specific fragments located on the Y chromosome ( Hirayama et al, 2007 ; Ron et al, 2010 ; Ayalavaldovinos et al, 2014 ), fluorescence in situ hybridization technique for detection of Y chromosome ( Sohn et al, 2007 ; Villagómez & Pinton, 2008 ; Rubes et al, 2009 ), quantitative detection of hormones such as progesterone, estradiol, and anti-Müllerian hormone ( Rota et al, 2002 ; Cabianca et al, 2007 ; Remnant et al, 2014 ; Kitahara et al, 2018 ), and detection of H-Y antigen qualitatively ( Wachtel et al, 1980 ). Most of the methods are based on the assumption that freemartin contains XX/XY chimera and H-Y antigen, whereas fertile heterosexual twin female does not contain XX/XY chimera or H-Y antigen.…”