Molecular verification of sperm sexing method with BSA (Bovine Serum Albumin) column

Abstract

Kaiin EM, Gunawan M, Octaviana S, Nuswantara S. 2017. Molecular verification of sperm sexing method with BSA (Bovine Serum Albumin) column. Pros Sem Nas Masy Biodiv Indon 3: 241-245. Determining the sex of cattle was a strategic step in the development of artificial insemination technology. Separation of sperm carriers female sex chromosome (X) or male sex chromosome (Y ) methods with a BSA column has produced successful sex-matched calves after Artificial Insemination (AI) by 76% to 89%. The aim of this study was to verify the results of sperm separation with a BSA column method based on molecular technique. Simmental sperm was separated with 5% BSA column (X sperm) and 10% BSA column (Y sperm). Sperm quality was tested microscopically with parameters: motility, viability, sperm abnormalities and intact membrane plasma (IMP). DNA of sperm from each column was extracted using spin-column method and was then amplified using the Polymerase Chain Reaction (PCR) with the designed primer of the primary genes of Sex-Determining Region Y (SRY) located in the region of the Y chromosome and autosomal GADPH (Glyceraldehyde 3- phosphate dehydrogenase) gene in the area of HMG Box (High Mobility Group Box). The results showed that the X and Y sperm motility was 60-70 %, X sperm viability was 76,9-80,1%, whereas Y sperm was 75,5-77,7 %. X sperm abnormality was 6,4-7,6% and Y sperm was 5,2-5,5%. The results showed that sperm separation with 10% BSA and non sexed sperm (control) revealed two molecular bands that were verified as SRY gene (318 bp) and GADPH gene (415 bp). This indicates that the 10% BSA column contained more Y sperm than X sperm. In 5% BSA column only one band was formed in gel electrophoresis at 415 bp (GAPDH), it showed that only contain X sperm. The methods to separated bull sperm (sexing) using BSA column 5 % and 10% can be verified molecularly by duplex PCR to distinguish between X chromosome and Y chromosome in bull sperm