170 IN VITRO EMBRYO PRODUCTION AND APOPTOSIS DETECTION USING BOVINE OOCYTES MATURED WITH IGF-I OR IGF-LongR3

Abstract

The aim of this study was to compare the differential effects of the addition of IGF-LongR3 or insulin-like growth factor-1 (IGF-1), during in vitro maturation (IVM) of bovine oocytes on embryonic development, by analysing embryonic viability and apoptosis. Ovaries collected at a slaughterhouse were transported in saline solution (NaCl 0.9%) at 38°C. Follicles of 2–8 mm of diameter were aspirated. Only cumulus-oocyte complexes (COC) with homogeneous cytoplasm, surrounded by at least 3 layers of compact cumulus cells were selected in Dulbecco’ modified PBS 0.3% polyvinyl alcohol (PVA) and transferred to TCM HEPES at 38°C. Maturation medium was composed by TCM199, 0.2 mM pyruvate, 1 μg mL^–1 FSH, 50 μg mL^–1 LH, 100 μg mL^–1 streptomycin sulfate, 100 UI mL^–1 penicillin, and 85 μg mL^–1 amikacin. Four experimental groups were determined using basic medium supplemented with fetal bovine serum (FBS; 10%), PVA (3 mg mL^–1 Polyvinylpyrrolidone), IGF-1 (3.100 ng mL^–1 insulin growth factor), LongR3-IGF (14.100 ng mL^–1 long-chain human insulin growth factor 1-r3) respectively. The IVM was performed in Petri dishes (35 × 15 mm) with 90-μL droplets of the respective media, covered with sterile mineral oil, in 5% CO₂ and 38.5°C temperature atmosphere for 22–24 h. The matured oocytes were fertilised with 2 × 10⁶ spermatozoa mL^–1 and incubated for 18 h. Embryos were denuded and cultivated in SOFaa medium (2.7 mM myoinositol, 0.2 mM pyruvate, 5 mg mL^–1 BSA, 100 μg mL^–1 streptomycin, 100 UI mL^–1 penicillin, 85 μg mL^–1 amikacin, and 25 μL mL^–1 of FBS). Six repetitions were performed. After 7 days, blastocyst formation was analysed and all blastocysts were submitted to the TUNEL reaction. (In Situ Cell Death Detection Kit with Fluorescein, Roche®, Mannheim, BW, Germany), according to the technique adapted by Paula-Lopes and Hansen (2002).Green nuclei fluorescence (fluorescein isothiocyanate; FITC) was considered with fragmented DNA. Data were analysed by ANOVA), and least-squares means was used to verify differences of means using the PROC GLIMMIX model of SAS statistical software package (SAS Institute, Cary, NC, USA). Cleavage rate was similar for all groups. There was no statistical difference (P > 0.05) between groups regarding to embryo production. Most of the blastocysts obtained at Day 7 of culture had good quality (grade I). However, a difference (P = 0.03) on the number of expanded blastocyst was found between FBS (20.83 ± 3.22) and PVA (10.18 ± 3.22) groups. Furthermore, IGF-1 (12.03 ± 1.60) group showed a higher (P = 0.04, P = 0.02) apoptosis rates compared to groups FBS (7.96 ± 1.29) and PVA (6.97 ± 1.48). In the present study, it was observed that the addition of IGF-1 or LongR3-IGF during IVM provided a similar embryo production compared to FBS. On the other hand, embryos obtained from oocytes maturated on medium with the addition of IGF-1 had a high number of cells with fragmented DNA, indicating a more apoptosis.