Indonesian

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