A Low-Cost Efficient Multiplex P CR for Prenatal Sex Determination in Bovine Fetus U sing Free Fetal DNA in Maternal Plasma

Background: In order to establish a reliable non-invasive method for sex determinationin a bovine fetus in a routine setting, the possibility of identifying specific sequence in thefetal X and Y-chromosomes has been evaluated in maternal plasma using conventionalmultiplex polymerase chain reaction (PCR) analysis. The aim of this study was to providea rapid and reliable method for sexing bovine fetuses.Materials and Methods: In this experimental study, peripheral blood samples were takenfrom 38 pregnant heifers with 8 to 38 weeks of gestation. DNA template was extractedby phenol-chloroform method from 350 μl maternal plasma. Two primer pairs for bovineamelogenin gene (bAML) and BC1.2 were used to amplify fragments from X and Ychromosomes. A multiplex PCR reaction has been optimized for amplification of 467bp and 341 bp fragments from X and Y bAML gene and a 190 bp fragment from BC1.2related to Y chromosome.Results: The 467 bp fragment was observed in all 38 samples. Both 341 and 190 bp fragmentswere detected only in 24 plasma samples from male calves. The sensitivity andspecificity of test were 100% with no false negative or false positive results.Conclusion: The results showed that phenol-chloroform method is a simple and suitablemethod for isolation of fetal DNA in maternal plasma. The multiplex PCR method is anavailable non-invasive approach which is cost efficient and reliable for sexing bovine fetuses.