T. Yamanouchi

14 ENHANCEMENT OF THE EARLY RECOVERY OF OVARY AND UTERUS FUNCTIONS BY TREATMENT OF eCG AND POVIDONE-IODINE IN POSTPARTUM JAPANESE BLACK CATTLE

The aim of this study was to evaluate the effect of administration of eCG and intrauterine infusion of povidone-iodine to promote the early recovery of ovary and uterus functions in early stage after delivery on postpartum beef cows. Thirty-three postpartum Japanese Black cows without retention of placenta were used for this investigation. After delivery, cows nursed colostrums to calves for several hours in a pen, and then were separated from calves and returned into a barn managed in a group. In experimental group (n=14), on Day 10 (Day 0=day of parturition), an intramuscular administration of 500IU of eCG and infusion of 30mL of 2% povidone-iodine into the uterus body were conducted. The untreated cows delivered in the same period were compared as a control group (n=19). Cows were inseminated artificially using commercial Japanese Black frozen-thawed semen when standing oestrus was detected until Day 90. Pregnancy diagnosis was performed 30 days after insemination by ultrasonography. For all of the experimental group and 8 cows in the control group, the diameter of follicles >8mm and the diameter of cross-section between endometrium in uterus of the implanted side at the point of ~2cm from bifurcation of the uterus were measured using an ultrasound scanning machine connected to a 7.5-MHz convex probe. This monitoring was carried out every 10 days from Day 10 until the day of first oestrus (= insemination) or until Day 80 for non-returned oestrus cows. Data were analysed using Fishers exact test and ANOVA. The ratio of returned oestrus by Day 90 was 100% (14/14) in experimental group and 89.5% (17/19) in control group. The first oestrus day postpartum in the experimental group and the control group was the almost same: 52.9±12.1 (24-88) days and 57.2±19.9 (29-78) days, respectively. The conception rate until Day 90 tended to be higher (P=0.07) in the experimental group (78.6%, 11/14) than in the control group (47.4%, 9/19). The days inseminated to cows that were conceived, was almost same between the experimental group (57.5±18.6, 24-87 d) and the control group (55.4±12.9, 36-78 d). The ratio of cows observed follicles more than 8mm, was higher in the experimental group than control group on Day 50 (90.9% v. 50.0%; P=0.09) and Day 60 (66.7% v. 0%; P<0.05), respectively. The diameter of uterus tended to be lesser in the experimental group than in the control group on Day 30 (16.9±2.8mm v. 19.2±2.8mm; P=0.09), and that regressed linearly to Day 30 in the experimental group however that was prolonged until Day 40 (16.4±1.7mm) in the control group. These results indicate that administration of eCG and uterus infusion of povidone-iodine at an early stage postpartum may promote the early recovery of ovary and uterus functions in beef cows.

143 EFFECT OF FSH OR EPIDERMAL-GROWTH-FACTOR-LIKE PEPTIDE SUPPLEMENTATION TO MATURATION MEDIUM ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES DERIVED FROM FULLY DEVELOPED FOLLICLES INDUCED BY SUPER-STIMULATION

Bovine oocytes obtained by ovum-pick-up (OPU) following follicle growth treatment (FGT) have improved quality and competence (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). However, the effect of the presence of FSH or epidermal growth factor (EGF) like peptide during in vitro maturation (IVM) on the developmental competence of FGT oocytes has not been well known. This study was undertaken to examine the developmental competence of FGT oocytes following IVM in the presence of FSH (recombinant human FSH) or EGF-like peptide (amphiregulin; Areg) and IVF. Japanese Black cows (n=17) were used as donors. Five days after arbitrary OPU (opu group), follicles ≥8mm in diameter were aspirated again, a controlled internal drug release (CIDR) was inserted into the vagina, and then pFSH was injected twice a day from the evening of Day 6 to the morning of Day 10 with decreasing doses (total of 20 AU; 4, 4, 3, 3, 2, 2, 1, 1 AU/day). On the evening of Day 8, PGF2α (0.5mg of cloprostenol) was administered. On Day 11, oocytes were aspirated from follicles with ≥5mm in diameter of the treated donors by OPU (fgt group). The cumulus-oocyte complexes (COC) were cultured in the absence (opu-cont and fgt-cont groups) or presence of 0.1IUmL-1 FSH (opu-fsh and fgt-fsh groups) or 100ngmL-1 Areg (opu-areg and fgt-areg groups) in IVM medium (mTCM199 containing 5mgmL-1 BSA) for 20 to 22h (1 COC/5µL, total of 162-171 COC per group), and then co-cultured with 3×106 sperm/mL for 6h. The presumptive zygotes were continued to culture in mCR1aa supplemented with 5% newborn calf serum for 216h (1 zygote/5µL) using micro-well culture dishes (Dai-Nippon-Print). When repeating this opu-fgt session in the same cow, an interval at least for 50 days was kept, and the session was performed 28 times. Statistical analysis was carried out by Mann-Whitneys U-test (between opu and fgt groups) or Steel-Dwass test after Kruskal-Wallis test (among all groups). The number of follicles ≥5mm increased in the fgt than opu group (17.8v. 2.9; P<0.01). The number of COC collected was not different between the opu and fgt groups (23.1v. 19.6; P>0.05). The blastocyst formation rate was higher in the fgt than opu group (36.9v. 23.1%; P<0.01). Within 6 groups, the blastocyst formation rate was higher in the fgt-fsh (43.3%; P<0.01) and fgt-areg (39.5%; P<0.05) groups than the opu-cont (16.3%) group. The rate in the fgt-fsh group was also higher than that in the opu-fsh group (43.3v. 18.7%; P<0.01). These results suggested that FGT improved the developmental competence of bovine oocytes, probably through improving the ability of the COC to react against FSH/Areg.

159 RELATIONSHIP BETWEEN CLEAVAGE PATTERNS OF FIRST CELL CYCLE AND POST-TRANSFER VIABILITY IN BOVINE EMBRYOS OBTAINED BY OVUM PICKUP AND IN VITRO FERTILIZATION

More than 300 000 embryos have been transferred all over the world (Stroud 2010 IETS Newsl. 27(4), 11–21). We have reported that embryos that showed the abnormal cleavage pattern at the first cell division can develop to the blastocyst stage (Somfai et al. 2010 J. Reprod. Dev. 56, 200–207). However, we have limited knowledge about the consequences of the pattern of first embryonic cleavage on their post-transfer developmental competence. The present study was conducted to determine the developmental competence of bovine blastocysts showing different cleavage patterns at their first cell division. Cumulus–oocyte complexes were collected by ovum pickup from Japanese Black cows and were subjected to in vitro maturation and IVF as reported previously (Imai et al. 2006 J. Reprod. Dev. 52, S19–S29 suppl). Inseminated oocytes were cultured in CR1aa medium supplemented with 5% calf serum covered by mineral oil at 38.5°C in 5% CO2 in air with micro-droplets or 5% CO2, 5% O2 and 90% N2. The kinetics of embryo development were analysed by time-lapse cinematography for 168 h after IVF by using a Cultured Cell Monitoring System (CCM-M1.4ZS, Astec, Fukuoka, Japan). A total of 673 photographs of each embryo were taken (1 photograph in every 15 min) during in vitro culture. Image stacks were analysed by the CCM-M1.4 software. Embryos were classified in 5 groups according to the pattern of first cleavage as normal cleavage (NC), direct cleavage from 1 cell to 3 to 4 blastomeres (3–4BL), unequal blastomeres (UB), multiple fragments (MF) and protrusion formation (PT). Blastocysts developing from each group were transferred into the ipsilateral uterine horn of each synchronized recipient on Day 7 or 8 after oestrus. Data on conception at Day 60, abortion and delivery were then recorded. Data were analysed by chi-square test and Students t-test. In total, 43 embryos were transferred, 17 conceptions (39.5%) were established and 16 recipients (94.1%) were delivered. Only 1 abortion was detected at Day 223 in the NC group. The highest conception rate was observed in the NC group (55%, n = 20) and the 3–4BL (n = 12), UB (n = 6) and PT (n = 3) groups showed similar conception rates of 33.3% (1 implanted embryo belonged to 2 classes in UB and PT) and none of the embryos derived from the MF group (n = 3) could cause conception. There was a significant difference (P < 0.05) in conception rates between the NC group and totals of each of the other cleavage groups. No significant difference was found in gestation lengths and birth weights between the NC group (282.2 ± 4.4 days, 30.6 ± 3.8 kg, respectively) and totals of each of the other cleavage groups (282.8 ± 5.3 days, 30.3 ± 1.9 kg, respectively). These results indicate that embryos showing abnormal cleavage patterns at first cell division can develop to normal calves with normal gestation lengths and birth weights; however, their post-transfer viability is lower than for NC embryos. This work was supported by the Research and Development Program for New Bio-industry Initiatives.