103 OVIDUCTAL SECRETORY PROTEINS AS MEDIA SUPPLEMENT FOR IN VITRO EMBRYO DEVELOPMENT IN CATTLE

Abstract

The objective of this study was to determine the effect of oviducal secretory proteins (cOSP) as a media supplement on in vitro embryo development in cattle. Oviducal secretory proteins were collected from slaughterhouse oviducts by repeated freeze–thaw process and purified by ammonium sulfate precipitation (30%, 40%, 50% and 60%) followed by dialysis in 50 mM tris-HCl (pH 7.0) buffer. Dialyzed products were further purified by SP Sephadex cation exchange column and diethylaminoethyl cellulose (DEAE) anion exchange column, extensively washed and eluted by 50 mM tris-HCl (pH 7.0) containing 1.5 M NaCl. Both bound and unbound proteins were collected separately, dialyzed in phosphate buffer saline and quantified. Presence of protein was confirmed by running sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and OSP bands between ∼66 kD to ∼97 kD were found. The pooled purified cOSP were used as a media supplement in 3 different concentrations (0, 10, 50 and 100 μg mL^–1) for in vitro production (n = 3) of cattle embryos. Cumulus–oocyte complexes (n = 370) were collected from slaughterhouse ovaries, washed thoroughly and cultured in maturation media for 24 h in 5% CO₂ at 38.5°C with maximum humidity. In vitro-matured oocytes were fertilized with in vitro capacitated sperm in Fert-BO media at 38.5°C in 5% CO₂. After 16 to 18 h, oocytes were washed and cultured in embryo development media for cleavage. After 40 to 42 h, cleavage was observed and embryos were transferred into the replacement media for further development. A total of 68.42%, 69.31%, 61.82% and 41.67% cleavage rate and 15.38%, 21.31%, 14.70% and 15.0% blastocyst rate was observed at concentrations of 0, 10, 50 and 100 μg mL^–1, respectively. These results indicate that addition of cOSP at 10 μg mL^–1 increased blastocyst formation significantly (P < 0.05) compared with 0, 50 and 100 μg mL^–1 and increased cleavage rate significantly (P < 0.05) compared with 50 and 100 μg mL^–1.

The authors acknowledge sincere thanks to the Director, Joint Director (Research), National Dairy Research Institute, Karnal and Incharge, Eastern Regional Station, National Dairy Research Institute, Kalyani, for providing the necessary facilities to carry out the work.