142 POSSIBLE BENEFIT OF INTRACYTOPLASMIC SPERM INJECTION ON THE EFFICIENCY OF EMBRYO PRODUCTION IN AGED COWS

Abstract

Reduction in oocyte quality is a major factor responsible for declining fertility with age. The abnormal fertilization rate of oocytes from aged cows was reportedly higher than that of oocytes from young cows (Iwata et al. 2011. Reprod. Fertil. Dev. 23, 424–432). We hypothesised that assisted fertilization by intracytoplasmic sperm injection (ICSI) might improve the developmental abilities of oocytes collected from aged females. The aims of the study were (1) to determine the effect of maternal aging on the ability of bovine oocytes to undergo cytoplasmic maturation, fertilization, and further embryo development; and (2) to determine whether ICSI would improve the efficiency of embryo production in aged cows. Cows aged 30 to 50 months or >120 months were defined as young or aged, respectively. Cumulus-oocyte complexes were harvested from abattoir-derived ovaries of young (40 ± 7 months, n = 89) and aged (136 ± 12 months, n = 55) Holstein cows and matured for 23 h in TCM-199 supplemented with 5% fetal bovine serum (FBS) at 38.5°C under 5% CO₂ with saturated humidity. Then, surrounding cumulus cells were removed, and cortical granules in the oocyte were stained with Lens culinaris–fluorescein isothiocyanate to evaluate the cytoplasmic maturation. Matured oocytes were inseminated by IVF or ICSI. At 15 h post-insemination, the numbers of pronuclei were determined to evaluate the fertilization rates. Presumptive IVF- or ICSI-derived zygotes were cultured for 5 days in CR1aa medium with 2% FBS and subsequently in USU6 with 5% FBS for 3 days at 38.5°C in 5% O₂, 5% CO₂, and 90% N₂ with saturated humidity. Chromosome numbers of blastocysts were counted to evaluate the effect of maternal aging on ploidy. All experiments were performed with more than 4 independent runs, and data were analysed using chi-square tests. The distribution of matured oocytes into different cortical granule classes was affected by age, with a significantly lower (P < 0.01) proportion of class III (mature cytoplasm) oocytes from aged cows (29%) compared with those from young cows (57%). Although fertilization rates following IVF did not differ between the groups, the proportion of abnormal fertilization (more than 2 pronuclei) was 32% in the aged group: higher than in the young group (15%; P < 0.01). The rates of cleaved embryos following IVF were the same among groups, but the rate of development to the blastocyst stage of oocytes from aged cows (38%) was significantly (P < 0.05) lower than in those from young cows (52%). Moreover, the proportion of diploid blastocysts with 2 sets of chromosomes (2n = 60) was lower (47%) in the aged than in the young groups (75%; P < 0.05). However, in the ICSI embryos, the rates of development to the blastocyst stage did not differ significantly between groups (young 36%; aged 43%). Thus, maternal aging might impair the cytoplasmic maturation of bovine oocytes, which could be associated with abnormal fertilization or low developmental competence. Our results also indicate possible beneficial effects of ICSI on the efficiency of embryo production in aged cows.