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Five Layered Optiprep based Density Gradient Model is a Promising Model for Enrichment of Viable X Chromosome Bearing Spermatozoa in Bubalus bubalis

Background: A reliable method for controlling the sex of farm animals has the potential to revolutionize dairy farming. This can only be achieved by use of a method capable of separating X and Y chromosome bearing spermatozoa at lower cost and causing no significant damages to sperm viability. Realizing significance of buffaloes in Indian dairy farming, present study was aimed to develop appropriate density gradient model for enrichment of X chromosome bearing spermatozoa population in semen of Murrah buffalo bull. Methods: Density gradient centrifugation (DGC) technique was employed for enriching X chromosome spermatozoa in buffalo bull semen using four gradient media viz., Percoll, Optiprep, Ficoll and Sucrose. Percentage enrichment of X chromosome bearing spermatozoa in all the DGC models was determined by SYBR green based Real Time PCR. Result: Our investigations revealed that number of layers and centrifugation speed (g) factors in a density gradient centrifugation model, have significant effect on the percentage enrichment of X chromosome content in semen samples.Three layers, four layers and five layers density gradient centrifugation models showed significant differences (P less than 0.05) in the sex ratio towards X spermatozoa population in semen (61.72%±0.81, 64.55%±0.26 and 67.31±0.33%) respectively. Density gradient centrifugation models with centrifugation speed (g) 200x g and 300x g also showed significant differences (P less than 0.05) in the sex ratio towards X spermatozoa population in semen (66.36%±0.25 and 62.69±0.22%) respectively. Out of all DGC models, Optiprep density gradient model with 5 layers and centrifugation at 200xg (O3) yielded maximum enrichment (72.4%±1.38). In conclusion, O3 poses to be a promising model for enrichment of X spermatozoa in buffalo bull semen.