286 THE EFFECT OF IN VITRO MATURATION MEDIUM ON CRYOSURVIVAL, CELL NUMBERS AND APOPTOTIC INDEXES OF BOVINE EMBRYOS

Abstract

The success of in vitro maturation (IVM) has a significant impact on the oocytes ability to develop to blastocyst stage. The quality of the produced blastocysts can be evaluated using staining techniques. The aims of this study were (a) to compare the effect of different IVM media on embryo production rates, and (b) to utilize differential (DF) and TUNEL staining to evaluate the quality and cryosurvival of the produced blastocysts. Abattoir-derived oocytes were randomly divided into the IVM groups: (1) M199 IVM (n = 2305): TCM-199 with glutamax-I (GIBCO, Paisley, UK), 0.25 mM Na-pyruvate, 100 IU mL⁻¹ penicillin, and 100 μg mL⁻¹ streptomycin; (2) FBS IVM (n = 2484): M199 IVM medium with hormones (10 μg mL⁻¹ LH, 2 μg mL⁻¹ FSH, and 100 μg mL⁻¹ β-estradiol), and 10% FBS (GIBCO, New Zealand); and (3) FAFBSA IVM (n = 2411): as group (2), but FBS was replaced with 4 mg mL⁻¹ fatty acid free albumin. Fertilized oocytes were denuded and cultured in modified SOFaaci + 4 mg mL⁻¹ FAFBSA in 5% O₂ (Holm P et al. 1999 Theriogenology 52, 683–700). Fresh, Grade I Day 7 blastocysts were stained with TUNEL (n = 114) or with DF (n = 149). In addition, 184 Grade I Day 7 blastocysts were frozen in AG Freeze (AB Technology, Pullman, WA, USA), thawed, and cultured for 24 h. The re-expanded embryos were stained with TUNEL (n = 96) or with DF (n = 88). Modified TUNEL protocol (Upstate, Lake Placid, NY, USA; Makarevich A and Markkula M 2002 Biol. Reprod. 66, 386–392) and DF staining protocol (Thouas G.A. et al. 2001 Reprod Biomed Online 3, 25–29) were used. The results of embryo cleavage and D7 embryo development are based on logistic regression models, and the results of proportion of ICM and apoptotic index on general linear mixed models.

After FBS IVM, 83.6% of the fertilized oocytes were estimated to cleave and 25.5% to develop to the blastocyst stage by Day 7. The estimations for embryo cleavage and Day 7 development rates were significantly lower in FAFBSA IVM and M199 IVM groups (P < 0.0001): 74.0% and 15.0% for the FAFBSA, and 76.1% and 8.8% for the M199, respectively. The re-expansion rates (%) after thawing were 86.5, 90.6, and 73.3 for the FBS, FAFBSA, and M199 IVM groups, respectively. Freezing reduced the ICM proportions and elevated the apoptotic indexes (P < 0.001). The rate of ICM reduction after freezing was not influenced by the IVM medium. There was a significant interaction between the apoptotic index and the IVM group (P = 0.04). The increase of the apoptotic index was smallest in FAFBSA IVM and greatest in M199 IVM. The results indicate that exclusion of serum from IVM medium results in lower embryo cleavage and development rates. Freezing reduced ICM and increased apoptotic index of Day 7 embryos in every IVM group studied. FAFBSA IVM seemed to produce embryos of better quality as evidenced by the smallest increase in the apoptotic index after freezing.