153 HEPARIN-BINDING OVIDUCTAL SECRETORY PROTEINS AS A MEDIUM SUPPLEMENT FOR IN VITRO EMBRYO PRODUCTION IN CATTLE

Abstract

The purpose of this study was to determine the effect of heparin-binding oviducal secretory proteins as a media supplement on IVF and embryo development in cattle. The native proteins were isolated from oviducts collected from a slaughterhouse by repeated freeze-thawing and precipitated by ammonium sulfate (60%) followed by dialysis overnight in 10 mM phosphate buffer solution containing 1 mM PMSF at pH 7.0 at 4°C. The dialyzed product was then passed through a high-performance liquid chromatography system with a HiTrap™ heparin prepacked column (GE Healthcare, Piscataway, NJ) equilibrated with 10 mM phosphate buffer containing 1 mM EDTA. The collected protein fractions [heparin-binding protein (HBP), heparin-unbinding protein (HUBP)] and total protein (TP) were run on SDS-PAGE. The SDS-PAGE profile of the eluted HBP fraction contained 5 major protein bands visible between approximately 66 to 97 kDa, the profile of the HUBP fraction contained 2 bands nearer to approximately 66 kDa and in TP, all 7 bands were visible between approximately 60 to 95 kDa. All 3 fractions (i.e. HBP, HUBP and TP) were used in 3 different concentrations (0, 1, 10 and 30 μg mL^–1) for in vitro maturation, sperm preparation, IVF and in vitro culture of cleaved embryos. Cumulus–oocyte complexes were collected from slaughterhouse ovaries, washed 4 to 5 times and cultured in maturation media for 24 h in a 5% CO₂ incubator at 38.5°C with maximum humidity. In vitro-matured oocytes were co-incubated with capacitated sperm in Fert-BO media at 38.5°C in 5% CO₂ in air with maximum humidity. After 18 h, oocytes were washed thoroughly 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. In the HBP group, overall cleavage rates (%) were 63 ± 3.0, 53 ± 2.6, 65 ± 4.7 and 43 ± 0.6 and blastocyst formation (%) was 17 ± 1.6, 23 ± 2.5, 25 ± 2.2 and 24 ± 1.7 in 0, 1, 10 and 30 μg mL^–1 concentrations, respectively. However, in the HUBP group, the overall cleavage rates (%) were 66 ± 4.3, 56 ± 2.0, 60 ± 2.7 and 32 ± 0.8 and blastocyst formation (%) was 18 ± 1.1, 25 ± 1.8, 24 ± 2.2 and 14 ± 0.8 in 0, 1, 10 and 30 μg mL^–1 concentrations, respectively. In the TP group, cleavage rates (%) were 67 ± 4.9, 60 ± 4.1, 73 ± 3.2 and 41 ± 1.6 and blastocyst formation (%) was 19 ± 1.1, 18 ± 1.9, 27 ± 2.2 and 20 ± 1.6 in 0, 1, 10 and 30 μg mL^–1 concentrations, respectively. These results indicate that 10 μg mL^–1 of the total oviductal secretory protein fraction used as a media supplement significantly increased (P < 0.01) the cleavage rate and blastocyst formation as compared with the HBP and HUBP fractions.

The authors sincerely acknowledge the director and joint director (R), National Dairy Research Institute, Karnal and Incharge, Eastern Regional Station, National Dairy Research Institute, Kalyani, India, for providing the facilities to carry out the work.