author(s)
Manju G Preedaa, K Loganathasamy, V Leela and V Pandiyan

abstract
The present investigation has been undertaken to study the expression level of osteopontin (OPN) gene and its relationship with in vitro sperm characteristics in bovine semen. Fresh semen samples from 12 Jersey cross breed bulls were collected from organized breeding center by using artificial vagina. The initial concentration of semen sample was checked immediately before proceeding for RNA isolation. Motile sperms were isolated by the swim-up procedure using sperm-TALP medium. Total RNA from bull spermatozoa was extracted and first strand cDNA was synthesized by using commercial kits. Confirmation of OPN and GAPDH genes by conventional PCR product size 100 and 198 bp was performed. Fragment of 267 bp OPN gene containing real time PCR primers was cloned in to a plasmid vector and was used to construct standard curves for the absolute quantification of OPN gene. The initial copy numbers of OPN gene for all the 12 bull semen was calculated. Variations in levels of OPN gene transcripts (12,000 - 650, 00,000 copies) were found among 12 different bull semen samples. The results of absolute quantification assays were correlated (p<0.01) with the in vitro sperm characteristics such as sperm motility, viability, acrosomal integrity, plasma membrane integrity and mitochondrial membrane potential except sperm morphology. From this study, it is evident that ejaculated bull semen has variations in expression level of OPN gene among bull spermatozoa and expression levels of OPN gene have correlation with in vitro sperm characteristics.