Ramnath VADARSH. N2019-07-272019-07-272015http://krishikosh.egranth.ac.in/handle/1/5810117315Binder of sperm (BSP) proteins, constituted by three acidic proteins; BSP1, BSP3 and BSP5, form major fertility-associated gelatin-binding proteins in bull seminal plasma. These proteins play crucial role in fertilization and hence are regarded as markers of fertility. The present study was aimed at isolation of these proteins from seminal plasma of Vechur and crossbred bulls and characterize the same by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Semen was collected from ten Vechur bulls and three crossbred bulls and centrifuged to separate seminal plasma. Proteins in seminal plasma was precipitated by adding cold ethanol, recovered by centrifugation, dissolved in ammonium bicarbonate and lyophilized. The gelatin-binding proteins were isolated from crude proteins by gelatin-agarose affinity chromatography and were pooled, precipitated and lyophilized. SDS-PAGE of gelatin bound proteins revealed presence of three bands, two of which had molecular weight (MW) in the range of 15 to 17 kDa and one at 30 kDa. The same proteins on 2-D PAGE revealed presence of three thick spot at molecular weight between 14 and 20 kDa and a series of faint spots at MW 30 kDa, and all the spots exhibited acidic pI. The results indicated that Vechur and crossbred seminal plasma contain an average of 77.3 to 106 mg/mL and 84.6 to 104.5 mg/mL total proteins respectively and an average of 54.6 percent and 43.9 percent gelatin-binding proteins respectively. These proteins exhibited gelatin-binding property, elution characteristics, abundance in seminal plasma SDS-PAGE pattern and 2-D PAGE characteristics similar to BSP proteins reported elsewhere.ennullThesis