Fractionation of X and Y Chromosome-Bearing Bovine Spermatozoa through Sugar Gradients for Sex Predetermination in Dairy Cattle

• Published in: International journal of bioscience, biochemistry, bioinformatics (IJBBB) biochemistry, bioinformatics (IJBBB), 2012-05, Vol.2 (3), p.203-206
• Main Authors: Kanesharatnam, Nilani, Eswaramohan, Thampoe, Balasubramaniam, Kandiah
• Format: Article
• Language: English
• ISSN: 2010-3638; 2010-3638
• DOI: 10.7763/IJBBB.2012.V2.101

Mankind has been interested in sex preselectionsince ancient times. It can be achieved either by sexing of earlyembryos or by separation of X and Y chromosome-bearingspermatozoa, but new separation techniques with betteraccuracy and low costs are necessary. The objective of thisstudy was to fractionate X and Y-bearing bovine sperm usingsucrose gradient. A discontinuous sucrose density gradient wasprepared by layering successive decreasing sucrose solutionupon one another. Finally 20 mu L semen sample was loaded onthe top layer. Then it was centrifuged at 500 x g for 12 minutesat room temperature. After elution of fractions andcentrifugation (at 700 x g for 5 minutes), sperm viability andacrosome integrity were assessed by using 0.4% Trypan Blueand 0.75% Giemsa stain. Other part of the pellet was stainedwith 2% orcein red for 30 minutes to obtain sets ofchromosomes. Repeated measures analysis of variance(ANOVA) with Bonferroni's multiple comparison test wasperformed to compare the percentages of female sperms atevery layer. Results have shown that means of percentage of Xchromosomes increased from layer 1 (15.55 plus or minus 2.939 %), layer 2(14.0 plus or minus 3.055%), layer 3(26.33 plus or minus 0.881%) to layer 4 (31.85 plus or minus 5.186), but there is a statistically significant difference betweenlayer 2 and layer 4 (P