246 FOLLICULAR DEVELOPMENT AND OOCYTE QUALITY AFTER OVUM PICKUP IN DONOR COWS

Abstract

The present study was designed to assess the renewal of follicular development and oocyte quality after ovum pickup (OPU) in Holstein dry cows. Cows were kept under the same feeding and environmental conditions. In Experiment 1, follicle aspiration (more than 2 mm) by OPU using a 7.5 MHz linear transducer with needle (cova needle, Misawa Medical, Tokyo, Japan) connected to an ultrasound scanner (SSD-1200, ALOKA, Tokyo, Japan) was performed in four cows. After OPU ovaries were observed from Day 4 (Day 0 = the day of OPU) to Day 11 to assess the number of follicles that developed. In Experiment 2, two sessions of OPU (n = 11) were performed with a 7-day interval between to assess the quality of developing follicles and oocytes. Oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. To assess the developmental competence of oocytes, collected cumulus-oocyte complexes (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. (2002 J. Vet. Med. Sci. 64(10), 887–891). The putative zygotes were then cultured in CR1aa supplemented with 5% CS under the same conditions as maturation culture for nine days. Embryo development was assessed by the cleavage rate on Day 2 and the blastocyst formation rate on Days 7 to 9 (the day of insemination = Day 0). Data were analyzed by ANOVA or Student's t-test. In Experiment 1, the mean number of developing follicles (larger than 2 mm in diameter) were increased from Day 4 to Day 11 (Day 4: 19.8 ± 10.0, Day 7: 32.5 ± 9.5; Day 11: 39.5 ± 10.7 (mean ± SD), respectively, P < 0.05). In Experiment 2, the mean number of developing follicles and collected oocytes on the day of OPU were significantly (P < 0.05) different between the first and second sessions (54.2 ± 12.4 and 40.8 ± 12.7, 45.7 ± 20.2 and 27.7 ± 8.7, respectively). The percentage of Grade 1 and 2 oocytes for the first session was significantly lower (P < 0.05) than those for the second session (59.1 ± 8.4 and 69.0 ± 11.8), and no significant differences were found within cleavage and blastocyst rates. The mean numbers of blastocysts obtained per session were 14.2 ± 8.9 and 9.7 ± 6.3 in the first and second sessions, respectively. These results indicate that populations of follicles were increased till Day 11 after OPU, and proportion of normal oocytes were increased in the renewal follicles.