Detection and Quantification of X and Y sperms in Enriched Sahiwal Semen diluted in PBS using Raman Spectroscopy

Abstract

The present study was undertaken to enrich X-sperms in Sahiwal bull semen by percoll density gradient method and to detect X and Y sperms in enriched semen by Raman spectroscopy. The experiment was performed at Semen Production Centre, CVASc, GBPUAT, Pantnagar, Uttarakhand. Ejaculates were collected by AV method from Sahiwal bull. X-sperm enrichment was done by the Percoll density gradient method. Three types of gradients were prepared i.e. Group I: Seven layers (70-10%), Group II: Seven layers (80-20%) and Group III: Three layers (30%, 50% and 70%). Semen (1ml or 3ml) was placed over the gradient in a conical centrifuge tube. Centrifugation was done at 750 g (22-24°C) for 15 min. The pel-lets thus obtained were diluted in PBS medium. Raman spectroscopy was performed in different groups viz. fresh semen (Control), and three treatment groups. Raman peaks (DNA specific) were not much clear for PBS diluted semen samples though intensities were highest for seven layers (70-10%) among all groups. Thus, semen enriched by the percoll-density gradient method (7 layers i.e. 70-10%) can be used to increase female calf birth after A.I.