Purification of cattle oviduct specific proteins and their effect on in vitro embryo development

摘要

The purpose of this study was to determine the effect of oviduct specific proteins as a media supplement for in vitro embryo development in cattle. The proteins were extracted from oviducts of cows and precipitated by ammonium sulfate (30%, 40%, 50% and 60%) followed by dialysis in 50 mM Tris-HCl (pH 7.0) buffer. The dialyzed proteins were fractionated into acidic, basic and neutral fractions using SP sephadex cation exchange and DEAE sephadex anion exchange column chromatography respectively. Cow oviduct specific proteins (cOSPs) constituting all the extracted proteins were used as media supplement in three different concentrations (10, 50 and 100 mu g/ml) for in vitro maturation, fertilization and culture (IVMFC) of cow oocytes. Acidic, basic and neutral (unbound) fractions were also used as media supplement in three different concentrations (10, 30 and 50 mu g/ml) for IVMFC. Cumulus oocytes complexes were collected from slaughterhouse ovaries, washed thoroughly and cultured in maturation media for 24 h in 5% CO2 at 38.5 degrees C with maximum humidity. In vitro matured oocytes were co-incubated with in vitro capacitated sperm in Fert-BO media at 38.5 degrees C for 18 h in 5% CO2. The fertilized oocytes were washed and cultured in embryo development media for cleavage. After 40-42 h cleavage was observed and embryos were put in the replacement media for further development. The cleavage rates (%) for cOSPs were observed as 68.24 +/- 2.46, 69.28 +/- 2.05, 61.77 +/- 0.93 and 42.62 +/- 1.31 at concentrations of 0, 10, 50 and 100 mu g/ml respectively. Rates of blastocyst stage development were 14.49 +/- 3.61, 21.17 +/- 2.77, 14.66 +/- 1.06 and 11.98 +/- 1.84. These results indicate that addition of cOSP at10 mu g/ml increased blastocyst formation as compared to other concentrations (0, 50 and 100 mu g/ml). Although acidic, basic and neutral fractions seemed to have no major effect on cleavage rate, but both acidic and neutral fraction of oviduct specific proteins improved the cleavage rate at 30 mu g/ml concentration and basic fraction improved the blastocyst formation at 10 mu g/ml concentration