202 EFFECT OF OSTEOPONTIN ON IN VITRO EMBRYO DEVELOPMENT IN CATTLE

Abstract

Osteopontin (OPN) is an acidic single-chain phosphorylated glycoprotein found both in the oviduct fluid (ODF) and oviductal epithelium in cattle, which is believed to facilitate fertilization. It was recently reported that addition of a rabbit polyclonal immunoglobulin G antibody against purified bovine milk OPN with sperm oocytes, bovine oocytes, or both decreased (P < 0.05) fertilization compared with the in vitro-fertilized control (Goncalves et al. 2007 Theriogenology 67, 468–74). The aim of the present work was to determine the effect of in vitro addition of OPN to the fertilization medium on both cleavage and postfertilization embryo development in cattle. In the first experiment, in vitro-matured oocytes were fertilized in modified TALP medium in the presence of 0.0, 0.1, 1.0, or 10 µg mL^–1 of OPN. In a second experiment, matured oocytes were in vitro-fertilized in modified TALP medium in the presence of 0.0, 10, 20, or 40 µg mL^–1 of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull previously tested for IVF. After 20 to 22 h of coincubation at 39°C, 5% in CO₂ in air, presumptive zygotes were vortexed to remove cumulus cells, washed, and transferred, 30 to 50 per well, into 400 µL of SOF modified medium. Zygotes were incubated in a humidified mixture of 5% CO₂, 7% O₂, and 88% N₂ in air at a temperature of 39°C. On Day 7 of development (Day 0 = day of insemination), cleavage and development rates into transferable embryos (TE)–tight morulae (TM) and blastocysts (Bl) of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by a chi-square test. In experiment 1, numerically higher percentages of TM–Bl (29.5, 29.5, 30.5, and 37.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 µg mL^–1 of OPN; P = 0.25) and Bl (28.6, 27.5, 29.1, and 36.7, respectively, in the control group and in the groups with 0.1, 1, and 10 µg mL^–1 of OPN; P = 0.24) were observed with 10 µg mL^–1 of OPN. In experiment 2, significantly more cleavage (80.0 v. 71.3%; P < 0.05) and higher percentages of TM–Bl (44.6 v. 34.5%; P < 0.05) and Bl (39.2 v. 30.6%; P = 0.06) were observed with 10 µg mL^–1 of OPN v. the control. Combined analysis from both experiments showed an overall effect of 10 µg mL^–1 of OPN v. the control in the percentages of TM–Bl and Bl (respectively, 41.1 v. 33.3% and 37.7 v. 30.5%; P < 0.05). These results indicate that it is possible to improve the efficiency of bovine in vitro embryo production by adding the oviductal protein OPN.