213 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS IN THE PRESENCE OF GROWTH HORMONE, INSULIN-LIKE GROWTH FACTOR-1, AND INSULIN IN OOCYTE MATURATION AND EMBRYO CULTURE MEDIA

Abstract

The addition of hormones and growth factors to bovine IVM and IVC media has been reported to affect early embryonic development by enhancing the blastocyst formation rate and quality of embryos produced. The purpose of this study was to investigate the influence of adding growth hormone (GH), insulin-like growth factor-1 (IGF-1), and insulin to IVM and IVC media. Blastocyst production rate and blastocyst quality, as verified by the number of cells with DNA fragmentation, were evaluated. Ovaries from an abattoir were transported to the laboratory and COC were selected and cultured in IVM medium 199 (Earle's salts, Sigma, St. Louis, MO, USA), 10% fetal calf serum (Sigma), 50 µg mL^–1 of sodium pyruvate, 1 µg mL^–1 of estradiol (Sigma), 50 µg mL^–1 of hCG (Profasi hp^®, 5000 IU, Serono Inc., Rockland, MA, USA), 5 µg mL^–1 of FSH (Folltropin^®, Vetrepharm, Ontario, Canada), and 75 µg mL^–1 of gentamicin sulfate for 24 h. After IVF (18 h), zygotes were partially denuded and transferred to IVC medium HTF (HTF^®, Irvine Scientific, Santa Ana, CA, USA) and BME (BME^®, Sigma), in a 1:1 proportion (HTF:BME), 0.6% BSA (Sigma), 0.01% myoinositol (Sigma), and 75 µg mL^–1 of gentamicin sulfate, at 38.5°C, in a humidified atmosphere of 5% CO₂ in air, supplemented with 10% fetal calf serum at Day 3 of culture. Three different experiments were performed. The first and second experiments were analyzed using the chi-square test (P < 0.05). The third experiment was analyzed with the general linear model of SAS^® (SAS Institute Inc., Cary, NC, USA) and the Tukey test (P < 0.1). In the first experiment, oocytes were cultured in IVM medium supplemented with GH (10 ng mL^–1), IGF-1 (100 ng mL^–1), insulin (1 µg mL^–1), or all 3 combined. In the second experiment, IVC medium was supplemented with GH, IGF-1, insulin, or all 3 combined (same concentrations as above). In the third experiment, the quality of the embryos produced in the first 2 experiments was determined by the percentage of cells with DNA fragmentation. After 96 h of culture, embryos were stained with orange acridin (100 µg mL^–1) and propidium iodide (100 µg mL^–1) and slides were evaluated by fluorescence microscopy (450 to 490 nm). Rates of blastocyst production (blastocysts/oocytes) in the first experiment (29, 28, 28, 26, and 28% for control, GH, IGF-1, insulin, or all 3 combined, respectively) and in the second experiment (35, 35, 36, 35, and 31%) were not statistically different among the groups. In the third experiment, the addition of GH, IGF-1, or insulin to IVM medium did not affect the DNA fragmentation rate (11, 5, 2, 12, and 12%). However, the addition of insulin to IVC medium led to a higher DNA fragmentation rate (24%), when compared with the other groups (11, 10, 6, and 8% for control, GH, IGF-1, and all 3 combined). The addition of GH or IGF-1 to bovine IVM and IVC media did not affect the blastocyst production rate or the quality of embryos produced. The quality of embryos cultured in the presence of insulin was negatively affected.