X - sperm enrichment in buffalo bull semen by percoll density gradient
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Date
2018-08
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
The present research work was conducted to investigate the effect of percoll density gradient method on quality of enriched semen and quantify the X-sperm enrichment by Raman spectroscopy in buffalo bull semen. Semen samples collected by artificial vagina method from murrah buffalo bull with mass motility ˃+3 and progressive motility ˃70% were considered for the study. Fresh semen was divided into5 groups. The groups were control (fresh semen), centrifuged semen (supernatant), centrifuged semen (pellet), 7 layer (70-10%) percoll gradient centrifuged semen (supernatant) and 7 layer (70-10%) percoll gradient centrifuged semen (pellet). The 7 layered percoll density gradient medium was composed of 70 -10% percoll gradients with dense layer at bottom and lighter layer at top. Both the centrifugations were done at 750RCF for 20 minutes at 24ºC. Thereafter, the groups were diluted in either PBS or EYC dilutor after centrifugation and were sent to UGC-DAE Consortium for Scientific Research, Indore for Raman spectroscopy to check the separation efficiency in buffalo bull semen.The samples which were sent had equal volume (400 μl) and equal no. of sperms (2 x 106) and Raman spectroscopy was done at 100X magnification using 25w power for 30sec at 473nm wavelength with wave number ranging from 500 to 1800 cm-1 to detect the best method for X sperm enrichment and also to estimate the percentage increment of X sperms by percoll density gradient method in buffalo bull semen. Results concluded that the 7 layered 70% percoll density gradient method is good for X sperm enrichment and showed enhanced results compared to control i.e. the X sperm in 7 layer 70% percoll pellet was having more than 50% sperm count in EYC diluted semen. So quality assessment for various seminal parameters like volume, pH, motility, live percentage, sperm abnormality, concentration, metabolic activity (MBRT), membrane integrity (HOST) and acrosomal integrity was done for the 7 layered 70% percoll density gradient to evaluate the effect of percoll density gradient on quality of semen. The concentration, progressive motility, pH and MBRT decreased significantly in all the groups with highest value (P<0.05) for percoll pellet after fresh semen and lowest for centrifuged supernatant. The time taken to reduce methylene blue in all the groups was significantly (P<0.05) higher compared to group І. The percent live sperms and HOST showed similar responses. The sperm abnormality was significantly higher for percoll supernatant followed by centrifuged supernatant, centrifuged pellet, percoll pellet and was lowest for fresh semen. There was no significant effect on percent of spermatozoa with partially damaged acrosomal membrane and fully damaged acrosomal membrane in all five groups however, partially damaged acrosomal membrane sperms were significantly (P<0.05) more than the fully damaged acrosomal membrane sperms. In conclusion 7 layered 70% percoll density gradient method is good in both X sperm enrichment and sperm quality and can be used to enhance female progeny production.
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