African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12496

Full Length Research Paper

Verification of X- and Y-spermatozoa separation by nested polymerase chain reaction (PCR), motility and membrane integrity in bovine

Abdul Malik1,2, Abd Wahid Haron2*, Rosnina Yusoff2, Muhammad Bukar2, Azhar Kasim3 and Moh Sabri4  
  1Department of Animal Science, Faculty of Agriculture, Islamic Kalimantan University, Banjarmasin, Indonesia. 2Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. 3Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. 4Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.  
Email: [email protected]

  •  Accepted: 29 July 2011
  •  Published: 28 December 2011

Abstract

The aim of the present study was to verify the presence of X- and Y-chromosome spermatozoa after separation with swimming speed using oestrus cows vagina mucus, Percoll discontinuous gradient (45 to 90%) and swim-up using TALP medium. The nested polymerase chain reaction (PCR) was used to determine X- and Y-chromosome bearing spermatozoa after separation. The primers for PCR were designed using amelogenin cDNA sequence with 329 and 266 bp for X- and Y-bearing chromosome spermatozoa, respectively. The motility was analyzed using computer assisted sperm analyser, whereas the membrane integrity was analyzed using hypo-osmotic swelling test (HOST). Results were confirm by the absence of single band, either for X- or Y-chromosome. Inversely, the double band indicating that the spermatozoa cannot be separated was observed. The percentage of X-chromosome bearing spermatozoa in the swimming speed using oestrus cows vagina mucus media, Percoll discontinuous gradient and swim up methods were 58.33, 44.33 and 50%, respectively. Statistically, both percentages were significantly different (P<0.001) as compared to the theoretical ratio (50:50). Spermatozoa motility, membrane integrity and concentration before and after separation were also significantly different (P<0.05). This study shows that although swimming speed using oestrus cow’s vagina mucus media may be used to separate X- and Y-chromosome bearing spermatozoa in bulls, the results however, require further investigation.

 

Key words: Spermatozoa separation, nested polymerase chain reaction (PCR), motility, membrane integrity.