175 DETECTION OF LIPID GRANULES ON IVF BLASTOCYSTS MATURED IN APO-TRANSFERRIN IN KOREAN NATIVE COWS (HANWOO)

Abstract

Apo-transferrin (apo-Tf) is found in mammalian sera and plays a role as an anti-oxidant in media. The objective was to investigate the effects of apo-Tf on in vitro maturation and development and to examine the characteristics of blastocysts derived from IVF production in the presence of apo-Tf in Korean native cows (Hanwoo). Ovaries were collected from a slaughterhouse and cumulus–oocyte complexes (COCs) were taken from 2–6-mm antral follicles. The collected COCs were washed 3 times with 0.1 M PVA-TCM-199 and matured in 10 µg mL^-1 apo-Tf with TCM-199 at 39°C, 5% CO₂, 95% air for 24 h. Matured COCs were fertilized with frozen–thawed Korean native cattle semen treated with BO medium containing 5 mM caffeine and 1 µg mL^-1 heparin for 8 h, and developed to the blastocyst stage in 5% FBS, 0.3% BSA in TCM-199, and IVMD (IFP, Japan). To detect the lipid granules, blastocysts were fixed with 10% formalin-PBS for 2 h and then 50% ethanol for 2 min. Staining of blastocysts was performed with 1% Sudan Black B in 70% ethanol for 2 min. IVM and IVF were replicated 3 times. The results of blastocyst formation were analyzed by chi-squared test. The maturation rate was 92.6% at Met II in 10 µg mL^-1 apo-Tf. The blastocyst formation was 22.5%, 38.4%, and 34.8% in 5% FBS (control), 0.3% BSA in TCM-199 (P < 0.05), and IVMD (P < 0.05), respectively. The lipid granules in blastocysts from the 5% FBS-TCM-199 group were larger than those in the 0.3% BSA-TCM-199 and IVMD groups. The results suggest that apo-Tf is an important factor for in vitro maturation and that FBS in culture medium affects lipid granule formation on bovine blastocysts derived from in vitro fertilization.