Early Diagnostics of Freemartinism in Polish Holstein-Friesian Female Calves

SIMPLE SUMMARY: Freemartinism is the most common type of gender developmental disorder, resulting in infertility of heifers from multiple-sex twin pregnancies. The frequency of this syndrome is related to the frequency of multiple pregnancies, the number of which has significantly increased in dairy cattle populations (HF). Therefore, rapid diagnostics is necessary to enable early elimination of heifers with freemartinism from breeding. The aim of the study was to compare and identify the best method for early identification of freemartinism. The use of cytogenetic and molecular methods (PCR, short tandem repeats (STRs), real-time PCR) allowed us to conclude that molecular methods are more effective and guarantee fast and precise diagnosis. An additional advantage of molecular methods is the easy way to collect test material, which can be frozen, unlike blood samples for cytogenetic analysis, which must be fresh and delivered within 24 h to the laboratory, which generates further costs. ABSTRACT: Freemartinism in females born from heterosexual multiple pregnancies is characterized by the presence of XX/XY cell lines due to the formation of a shared blood system by anastomoses between fetal membranes of co–twins and leads to disturbed development of the reproductive system, including infertility. The aim of this study was to estimate the most precise and effective diagnostic method, especially useful for early identification of freemartinism in young female calves. The cytomolecular evaluation results of 24 Holstein-Friesian heifers from heterosexual twins was verified by molecular techniques: PCR, short tandem repeats (STRs), and relative quantitative PCR. The molecular analyses have been found to be a more efficient testing strategy, with a higher diagnostic success rate than karyotype evaluation. In 21 heifers, leucocyte chimerism determined by the 60, XX/60, XY karyotype was revealed—the proportion of the 60, XY male cell line in individual animals was in the range of 4–66%. In three cases, a normal karyotype 60, XX was identified, which indicates that anastomoses did not occur in 12.5% of studied twins and suggests that these potentially fertile heifers can be qualified for further breeding. The precise and early identification of freemartinism can be the basis for guidelines and selection recommendations concerning the reproductive performance of heifers born from heterosexual multiple pregnancies.