158 EFFECTS OF APOTRANSFERRIN ON IN VITRO MATURATION OF CUMULUS–OOCYTES COMPLEXES (COCs) IN HANWOO, KOREAN NATIVE COWS

Abstract

For in vitro production of embryos, animal sera have been used as energy sources, maturation promoters, vitamins, growth factors, and antioxidative compounds. However, the sera had risk of virus and mycoplasma infections which could result in too big offspring and cause dystocia in ovine and bovine. Apotransferrin (apo-Tf) is a component of mammalian sera and has played a role as an antioxidant in media. A study was conducted to investigate the effects of apo-Tf on in vitro maturation of cumulus-oocytes complexes (COCs) in Hanwoo, Korean native cows. Ovaries were collected from a slaughterhouse and COCs were taken from 2-6-mm antral follicles. The collected COCs were washed three times with 0.1M polyvinyl alcohol (PVA)-TCM199 and matured in 0, 1, 10, or 100 ¼g/mL apo-Tf with TCM-199 at 39°C, 5% CO₂, 95% air for 6, 12, or 24 h. Mature COCs were fertilized with frozen-thawed Korean native cattle semen treated with BO medium (Brackett and Oliphants 1975 Biol. Reprod. 12, 260-274) containing 5 mM caffeine and 1 ¼g/mL heparin for 8 h and developed to the blastocyst stage in 5% FBS and 0.3% BSA in TCM199-IVMD (IFP, Japan). To evaluate the morphology of nuclear types, the matured COCs were fixed in 1:3 acetic acid-ethanol for 30 s and stained with 3% basic Fuchsin. IVM and IVF were replicated three times. All of the results were analyzed by ANOVA using the STATVIEW program. The maturation rates of control were 34.2%, 37.3%, and 45.8% for 6, 12, and 24 h, respectively. There were no differences among the concentrations of apo-Tf, and nuclear types at 78.3-87.0% GVBD for 6 h, 82.8-91.3% MI for 12 h, and 88.9-100.0% MII for 24 h, with 1, 10, and 100 ¼g/mL apo-Tf, respectively. Conversely, there was significant difference between 1 µg/mL and 10 ¼g/mL in terms of cleavage rates, although the others did not vary significantly (P < 0.05). There were significant differences among the concentrations of apo-Tf for blastocyst formation (P < 0.05). Blastocysts matured with 1, 10, and 100 ¼g/mL apo-TF and developed in 5% FBS and 0.3% BSA in TCM199-IVMD showed rates of 8.8-21.6%, 9.4-35.3%, and 9.1-19.1%, respectively. The control groups developed to the blastocyst stage showed rates of 8.6%, 10.8%, and 10.5% in 5% FBS and 0.3% BSA in TCM199-IVMD, respectively. These results suggest that apo-Tf is an important factor for the in vitro maturation and in vitro development of bovine COCs.