274 SIMILARITY OF EMBRYOS PRODUCED BY OVUM PICK-UP AND IN VITRO FERTILIZATION IN IDENTICAL TWIN CATTLE

Abstract

The present study was designed to assess the similarity of follicular development, oocyte quality, and their subsequent development on ovum pick-up (OPU)-IVF in identical twin cattle. Four pairs of identical twin Japanese black cows (A, B pairs at 5 years old and C, D pairs at 3 years old) were kept under the same feeding and environmental conditions. OPU was performed for these cows once a week for seven continuous weeks. OPU was done by using a 7.5-MHz linear transducer with needle (17 G, 530-mm length) connected to an ultrasound scanner (SSD-1200, ALOKA, Tokyo, Japan). Oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. To assess the development, collected COCs were cultured for 20 h in TCM-199 supplemented with 5% calf serum (CS) in a microdroplet (volume was adjusted to 5 μL/oocyte) at 38.5°C under atmosphere of 5% CO₂ in air. After maturation, the COCs were inseminated with frozen-thawed semen collected from the same ejaculation of a single bull. The fertilization was performed with BO solution as described by Imai et al (J. Vet. Med. Sci., 2002, 64(10), 887–891). The zygotes were then cultured in CR1aa supplemented with 5% CS under the same condition of maturation for nine days. Embryo development was assessed by the cleavage rate on Day 2 and the blastocyst production rate on Days 7 to 9 (insemination day = Day 0). Blastocysts were classified according to the IETS creteria. Data were analyzed by ANOVA. A total 56 sessions of OPU were performed in this study. The overall mean number of developing follicles (larger than 2 mm in diameter), collected oocytes, and produced blastocysts were 30.3 ± 9.2, 20.1 ± 9.2 and 6.3 ± 3.8 (mean ± SD) per session, respectively. The mean number of developing follicles on the day of OPU were significantly different between B and D pairs (38.6 ± 7.5 and 21.9 ± 6.5, P < 0.01); however, no significant difference was found within each twin. In oocyte quality, C and D pairs were significantly higher grade than the A pair. The percentages of cleaved oocytes and embryos developed to the blastocyst stage (34 ± 16, 27 ± 10, 41 ± 17 and 39 ± 24) showed no differences among 4 pairs and within each twin. However, the percentage of Grade 1 blastocyst of B pair was significantly lower (P < 0.01) than that of other pairs, and C pair was significantly higher (P < 0.05) than that of A and D pairs (67 ± 25, 41 ± 22, 93 ± 10 and 71 ± 25; A, B, C and D pairs, respectively). There was no significant difference within twins. These results show little statistical variation between cows of the same genetic background in the production of embryos in vitro.