Isolation, identification and immunogenic characterization of secretory proteins of embryos and in blood circulation during estrus and in early pregnant buffalo

The aim of the study was to probe possibility of using that early secretory proteins from oocytes and embryos as a biomarker for detection/diagnosis of pregnancy in buffalo. The present study was conducted to analyze and characterize the secretory proteins of oocytes and IVF embryos of buffalo and search for these proteins in blood circulation of estrus and early pregnant buffalo. Oocytes were collected from ovaries of slaughtered buffalo and matured in vitro in maturation medium for 24 h. Fertilization was performed in BO medium with 24 h matured oocytes and capacitated spermatozoa. Cleaved oocytes were washed and cultured in mSOF till embryos developed to morula. The average 42.3% cleavage and 42.1% morula stage embryos were obtained. Morula stage embryos and 18 h matured oocytes were cultured in PVA medium without serum and BSA for its secretory proteins. Antisera were raised in rabbits against the secretory proteins of oocytes and embryos. Nine secretory proteins97, 97, 75, 66, 43, 30, 29, 27 and 20 kDa on SDS-PAGE were found in both oocytes and embryos when stained with silver nitrate. Three proteins of oocytes and embryos, viz. 66, 43 and 29 kDa were found in western blot using oocytes antisera without pre-treatment with non-pregnant serum and one protein 29 kDa with pre-treatment of oocyte antisera with non-pregnant serum. Similarly, 4 protein bands of embryos, viz. 75, 66, 43, and 29 kDa and 3 protein bands of oocytes, viz. 66, 43 and 27 kDa were found in Western blot using embryos antisera without pre-treatment with non-pregnant serum and 2 proteins bands 75 and 29 kDa of embryos and 1 protein 29 kDa of oocytes with pre-treatment of embryos antisera with non-pregnant serum. These 2 proteins of 75 and 29 kDa were also observed in estrus and pregnant serum and their absence in non-pregnant serum of buffalo. It could be concluded that 9 secretory proteins of oocyte and embryo approximetely MW97, 97, 75, 66, 43, 30, 29, 27 and 20 kDa were obtained in buffalo. Two of these proteins 75 and 29 kDa have pregnancy specificity as they were also present in blood circulation of estrus and pregnant buffaloes. These two proteins could be used as a biomarker for prediction/ diagnosis of pregnancy of buffaloes in future. To our knowledge, there has been no report to date that has evaluated of secretory proteins of oocytes and IVF embryos in buffalo.