Sharma, MridulaChaudhary, Deeksha2019-02-062019-02-062018-08http://krishikosh.egranth.ac.in/handle/1/5810093875The present study was conducted to observe the effect of percoll density gradient method on X-sperm enrichment and quality of semen. It was performed at Semen Production Centre, CVASc, GBPUAT, Pantnagar, Uttarakhand. Ejaculates were collected by AV method from Crossbred and Sahiwal bulls. Xsperm enrichment was done by percoll density gradient method. Three types of gradients were prepared i.e. 7 layers (70-10%), 7 layers (80-20%) and 3 layers (30%, 505 and 70%). Semen (1ml or 3ml) was placed over the gradient in conical centrifuge tube. Centrifugation was done at 750 xg (22-24°C) for 15 min. Thus, Raman spectroscopy was performed in different groups. Those were fresh semen (Control), 7 layers (70%) (G III), 7 layer (80%) (G IV) and 3 layer (70%) (G V). The pellets obtained were diluted in PBS or EYC medium. Raman peaks (DNA specific) were not much clear for PBS diluted though intensities were highest for 7 layer (70%). Thus, quantification of X-sperms in enriched EYC diluted semen was done by calculating and comparing area at 785, 1095 and 1581 cm-1. There was 26-30% and nearly 17-20% X-sperm enrichment in 7 layer (70%) percoll centrifugation compared to control in Crossbred and Sahiwal, respectively. pH of percoll density gradient centrifuged (supernatant) decreased significantly (P<0.05) in Crossbred semen. Progressive motility of percoll density gradient centrifuged (supernatant) decreased significantly (P<0.05) in Crossbred semen. Live spermatozoa (%) of enriched semen (pellet) were increased in Crossbred and Sahiwal semen. Acrosome integrity was not affected in all the groups in Crossbred bull whereas intact sperm decreased significantly (P<0.05) in percoll density gradient centrifuged supernatant of Sahiwal semen. HOST responsive sperms were not affected in all the groups of Crossbrd and Sahiwal semen. X-sperms enrichment of 29.9% and 20.48% in Crossbred and Sahiwal semen was achieved by percoll density gradient method (7layer 70%). Semen quality of enriched semen was also not affected. Thus semen enriched by percoll density gradient method (7 layer 70%) can be used to increase female calves birth after A.I.ennullThesis