325 INSULIN-LIKE GROWTH FACTOR-I CONCENTRATIONS IN OVIDUCT FLUID OF SUPEROVULATED EWES DURING THE PERI-OVULAR PERIOD

This study examined the concentrations of insulin-like growth factor-1 (IGF-1) in oviduct fluid during the peri-ovular period as a reference for the establishment of optimal in vitro culture conditions for sheep embryos. Six mature ewes (4–5 years, 58–67 kg) of comparable body condition were fed a standard diet for two weeks before the start of fluid collection. Ewes were superovulated using conventional treatment involving a progestagen, FSH, and GnRH treatment. Oviducts were catheterized four days (which is sufficient time to recover from surgery) before collection of oviductal fluid, which started one day (Day 1) before the time of ovulation (Day 0) and continued until five days later (Day 5). Oviductal fluid was acidified by diluting into 0.8 M acetic acid/0.2 M trimethylamine, pH 2.8, mixed, and incubated to dissociate IGFs from IGF-binding proteins (IGFBPs). Following incubation, acidified fluid was centrifuged at 10,000g through a 0.1-mm Micro-spin centrifuge filter; the filtrate transferred to glass high-performance liquid chromatography (HPLC) vials. IGFs and IGFBPs were separated from one another by high-performance size-exclusion liquid chromatography using a Protein-Pak 125 column (Waters Corporation, Milford, MA, USA) and 0.2 M acetic acid, 0.05 M trimethylamine, pH 2.8, at a flow rate of 1 mL/min. Oviductal fluid IGF-I was collected in a single 2-mL fraction directly from the HPLC and its concentration measured by an IGF-I-specific enzyme-linked immunosorbent assay (Diagnostic Systems Laboratories, Inc. Webster, TX, USA). The data were analyzed by analysis of variance. The non-superovulation group had significantly higher concentrations of oviductal IGF-I compared with the superovulation group. In the superovulated group, there was, however, a significant effect of day on the oviductal fluid IGF-I concentration (P < 0.01) such that the concentrations of IGF-I first increased for three days and then decreased for the remaining four days. In the non-superovulation group, there was no significant two-way interaction between ovulation and day. It can be concluded that the levels of IGF-I increase over time and then decrease. Authors express thanks to the help of Jenn Skye and Hemish Turretfield Research Station SA.