206 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND SUPERSTIMULATION ON IN VITRO EMBRYO PRODUCTION

Abstract

We previously reported that follicular wave synchronization, by removal of the dominant follicle on Day 5 after ovum pickup (OPU), was effective in increasing oocyte quality in the developing follicles (Imai et al. 2006 32th Annual Conference of the IETS, poster presentation no. 277). The current study was designed to examine the effect of superstimulatory treatment to induce subsequent follicular wave synchronization on embryo production by OPU and IVM-IVF-IVC in Holstein dry cows. Cows were reared under the same feeding and environmental conditions, and 2 OPU sessions were conducted in each cow. In the first session, OPU was performed in 8 cows on arbitrary days of the estrous cycle by 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). Follicles larger than 8 mm in diameter were then aspirated and a CIDR was inserted on Day 5 (the day of first OPU session = Day 0). Cows then received 30 mg of FSH (Antrin-R10; Kawasaki Mitaka Pharmaceutical Co., Tokyo, Japan) twice a day from Days 7 to 10 in decreasing doses (6, 6, 4, 4, 3, 3, 2, 2 mg) by i.m. injection. Cloprostenol (PGF; Clopromate C; Sumitomo Pharmaceuticals Co., Tokyo, Japan; 0.75 mg) was administered in the morning of Day 9 (third day of superstimulation). The second OPU session was performed 48 h after PGF administration (Day 11), and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Collected oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. Grades 1 and 2 COC were matured, fertilized, and cultured as described by Imai et al. [2006 J. Reprod. Dev. 52(Suppl.), S19–S29]. Embryo development was assessed by the cleavage rate on Day 2 and by the blastocyst formation rate on Days 7 to 8 (the day of insemination = Day 0). Data were analyzed by Student's t-test. There were no differences in the mean (± SD) number of aspirated follicles or collected oocytes between the first (32.5 ± 6.8 and 26.0 ± 12.7, respectively) and second (29.3 ± 10.4 and 19.0 ± 9.4, respectively) OPU sessions (P > 0.1). The percentage of Grade 1 and 2 oocytes for the second OPU session (90.5 ± 13.8%) was significantly higher (P < 0.01) than for the first OPU session (63.1 ± 6.3%), and significant differences were found for cleavage (79.4 ± 14.1, 61.8 ± 25.1, P < 0.01) and blastocyst rates (68.1 ± 16.7, 24.2 ± 22.3, P < 0.001) between sessions. The mean numbers of blastocysts obtained per session were 4.3 ± 2.9 and 12.8 ± 8.7 in the first and second sessions, respectively (P < 0.01). These results indicate that superstimulatory treatment and subsequent follicular wave synchronization were effective on in vitro embryo production by increasing the oocyte quality.