Sex typing of single bovine spermatozoa using nested multiplex PCR

Ying Ju Tan, Mahanem Mat Noor, Ainu-Husna MS Suhaimi, Wan-Somarny Wan Md. Zain


The development of cell separation technology by flow cytometry have brought the spermatozoa sexing technology to the commercial application. In order to assure the accuracy of bovine spermatozoa sexing by flow cytometry, this study aimed to develop a simple and reliable screening method for bovine single spermatozoon sex determination. In this study, detection of specifically-amplified X- and Y- chromosomal DNA were performed by combination of nested and multiplex polymerase chain reaction (PCR) using four sets of primers specific to bovine sex chromosome on Zinc Finger Protein X (ZFX) and Sex Determining Region Y (SRY) genes. PCR products produced a single distinct band either sized 474bp or 525bp confirming the present of X- or Y-chromosome bearing spermatozoa, respectively. This method is applicable to single copy of spermatozoa genomic sample and can be used to assure the accuracy and percentage of X and Y spermatozoa population after spermatozoa sexing by automated cell sorting.

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