207 EFFECT OF REPEAT HORMONE STIMULATION ON THE YIELD AND IN VITRO DEVELOPMENT OF JUVENILE CALF OOCYTES

Juvenile in vitro embryo transfer can markedly reduce animal generation intervals. The purpose of this study was to investigate the ovarian response of juvenile calves and in vitro oocyte developmental capacity after superstimulation. Experiments on calves were performed in accordance with the Animal Welfare Regulations. A total of 36 donor juvenile calves on standard nutrition and in a disease-free environment, were selected from the breeding farm of the Beijing Dairy Cattle Center. At 60 days of age, calves were randomly assigned into three groups of four calves each, replicated three times. On day 1, Group 1 received a progesterone vaginal insert (CIDR, 300 mg per device); Group 2 received a CIDR and 0.5 mg oestrogen benzoate (China); Group 3 received a CIDR, 0.5 mg oestrogen benzoate, and 50 mg progesterone (China). Then, calves were injected with FSH (Folltropin-V, Bioniche Animal Health, Belleville, ON, Canada) twice daily on days 5 (40 mg/40 mg) and 6 (30 mg/30 mg) at 12 h intervals. Cumulus–oocyte complexes (COCs) were recovered from the superstimulated calves 12 to 14 h after the final FSH treatment. COCs were considered usable unless they were damaged or had expanded cumulus layers. Usable COCs were matured in vitro for 24 h in maturation medium consisting of TCM199, 10% FBS, 10 μg mL–1 FSH, 1 μg mL–1 LH, 1 μg mL–1 E2–17β, 100 IU mL–1 penicillin, 100 μg mL–1 streptomycin, with (+Cys) or without (–Cys) 100 μM Cysteamine. Each calf oocyte was cultured in one well. The final concentration added to each fertilization drop was 5 × 106 sperm mL–1. Sperm and oocytes were co-cultured in IVF-100 medium (BO liquid+10 μg mL–1 heparin, Japan) at 38.5°C, 5% CO2 and a saturated humidity for 6 to 8 h. Blastocyst production rates were determined after 7 and 8 d of in vitro culture in CR1aa medium without the addition of cysteamine. Differences among treatments in each experiment were determined by one-way ANOVA and a multiple range test. Superstimulatory results indicated that more follicles were aspirated (63.2 per calf) and more usable oocytes were recovered (48.0 per calf) in Group 1 than in the other two groups (Group 2–45.2 and 31.8, respectively; Group 3–35.4 and 28.3, respectively; P < 0.05). No difference was observed between Groups 2 and 3. Superstimulation of calves twice at 30 day intervals in Group 2 (n = 12) did not affect the number of follicles or usable oocytes (overall, 44.2 and 28.0 per calf). Maturation rates (86.5% v. 85.0%, respectively) and cleavage rates (84.4% v. 80.0%, respectively) did not differ whether cysteamine was not (–Cys; n = 318) or was (+Cys; n = 330) added to the maturation medium. However, the blastocyst rate differed significantly (12.9% v. 35.2%, respectively; P < 0.01). This study established a protocol for the superstimulation of juvenile calves with an average of 48 oocytes obtained per calf. Superstimulation and surgical oocyte recovery twice at an interval of 30 days had no adverse effect on follicle development or oocyte recovery. The novelty of this research is that the blastocyst production rate of calf oocytes (35.2%) in maturation medium supplemented with cysteamine was similar to that reported in the cow.

187 DIFFERENTIAL EXPRESSIONS OF GROWTH AND DIFFERENTIATION FACTOR 9 AND BONE MORPHOGENETIC PROTEIN 15 GENES IN OVARIES OF THE CALF AND COW

It has been reported that prepubertal calf oocytes are less developmentally competent than those obtained from cows. The bone morphogenetic protein (BMP) family of proteins regulate folliculogenesis and the ovulation rate in mammals. Of the members of the BMP family, growth and differentiation factor 9 (GDF9) and BMP15 are oocyte-derived proteins that play critical roles in granulosa cell proliferation and differentiation. In the present study, we characterised the gene expression of bovine GDF9 and BMP15 in calf and adult cow ovaries. The ovaries obtained from 4 calves at 9 to 11 months old and 4 cows at 4 to 6 years old. For quantitative real-time RT-PCR (qPCR), cumulus–oocyte complexes (COC) and mural granulosa cells were collected by aspiration of follicles 2 to 5 mm in diameter from an ovary from each animal. Ovaries from the other side were used for in situ hybridization. The COC and mural granulosa cells were separated and cultured for 22 h according to the protocol for oocyte maturation. Total RNA was isolated from denuded oocytes, cumulus cells, and mural granulosa cells. Bovine glyceraldehyde-3-phosphate dehydrogenase was used to normalise qPCR efficiency. For in situ hybridization, the collected ovaries were immediately fixed with 4% formaldehyde-PBS and embedded in a paraffin block. In situ hybridization was carried out with digoxigenin-labelled RNA probes. We confirmed there was no contamination of oocytes in the collected cumulus and mural granulosa cells by determining the mRNA expression of germ cell–oocyte markers (ZAR1 and VASA). Two, 16, 7, and 10 COC were collected from the ovary on one side of each calf, and 14, 22, 29, and 33 COC were collected from an ovary of each adult cow. Two COC from the calves could not be used for qPCR analysis. Both GDF9 and BMP15 mRNA were detected in oocytes and cumulus cells at the end of maturation culture, whereas only GDF9 mRNA was detected in mural granulosa cells. Quantitative PCR detection revealed that BMP15 and GDF9 mRNA expression of the cumulus cells from adult ovaries was significantly greater than that from calf ovaries. The expression of GDF9 mRNA was significantly greater in calf oocytes than in oocytes from cows. However, BMP15 mRNA expression in the oocytes of calf and adult ovaries was not significantly different. In mural granulosa cells, the intensities of GDF9 mRNA expression were not significantly different between calves and cows. These qPCR results were also ascertained by in situ hybridization. In conclusion, we clarified that the characteristics of bovine GDF9 and BMP15 mRNA expression in oocytes and cumulus cells were different between calves and cows. Our results indicate the possibility that the calf oocytes were less developmentally competent because of excess GDF9 on the oocytes, deficiencies of GDF9 and BMP15 on the cumulus cells, or both.

124 TAXOL™ COULD PROMOTE EMBRYO DEVELOPMENT OF BOVINE OOCYTES VITRIFIED BY OPS

Stabilizing the cytoskeleton system during vitrification could be beneficial for improving post-thawed survival and subsequent development of vitrified oocytes. Taxol™, paclitaxel, is a microtubule stabilizer that has been found to improve development competence of vitrified mouse and human oocytes. The objective of this work was to study the effect of a Taxol pretreatment before OPS vitrification on the post-thaw cow and calf oocyte development. Oocytes were aspirated from slaughterhouse-derived ovaries and matured in TCM-199. Oocytes were randomly assigned to one of 3 experimental groups: (1) control oocytes matured in vitro for 24 h, (2) oocytes matured for 22 h and vitrified by the OPS method (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58), and (3) oocytes matured for 22 h and vitrified by OPS method with 1 µM Taxol. OPS and Taxol–OPS oocytes were transferred back into the maturation dishes and matured for 2 additional h before being subjected to fertilization. Fertilization was performed using frozen–thawed Percoll-selected sperm. At 22 h after insemination, presumptive zygotes were pipetted and then cultured in drops of 25 µL SOF medium and 5% fetal calf serum under paraffin oil at 38.5°C in 5% CO2, 5% O2, 90% N2, and maximum humidity. The Taxol–OPS group provided a significantly higher cleavage rate than the OPS group in cows (41.9% and 34.0%, respectively) or in calves (33.7% and 23.5%, respectively). However, cleavage rate in the experimental groups was significantly lower than in the control group (78.3% and 69.7% for cow and calf control groups, respectively). Blastocyst yield was also higher for the Taxol–OPS group (3.2%) than the OPS group (0%) in cow oocytes. There was no blastocyst development when calf oocytes were vitrified with or without Taxol pretreatment. As expected, cow and calf vitrification groups triggered a significantly lower blastocyst yield when compared with their control (26.7% and 14.9% for cow and calf control groups, respectively). In conclusion, this study showed that supplementation of 1 µM Taxol could promote embryo development after thawing. Further research is indicated to clarify the function of Taxol and its optimal concentration in order to improve the rate of embryo development. Table 1. Effect of Taxol pretreatment on development of cow and calf oocytes vitrified by OPS

340 IN VITRO MATURATION OF PREPUBERTAL BOVINE OOCYTES ON A GRANULOSA CELL MONOLAYER FROM ADULT ANIMALS IMPROVES THEIR DEVELOPMENTAL COMPETENCE

Oocytes from prepubertal calves have a decreased developmental competence compared with oocytes from adult animals. The goal of this study was to improve the developmental competence of juvenile oocytes by maturation on granulosa cell (GC) monolayers from adult animals. Oocytes were recovered by ovum pickup (OPU) from 48 Holstein Friesian calves at 7-8 months of age and 18 adult cows. Animals received intramuscular injections of 60 mg FSH 48 h prior to each OPU session. Follicles were punctured twice per week in six consecutive OPU sessions. Cumulus oocyte complexes (COCs) recovered from calves were divided into three quality groups (classes I-III) and were then randomly distributed into three maturation groups: COCs were matured for 24 h on either GC or fibroblasts or without co-culture. Cow oocytes were matured without co-culture. TCM-199 supplemented with BSA (0.1%), hCG (5 IU/mL), and eCG (10 IU/mL) served as the medium in all groups. After maturation, all COCs were fertilized in vitro; after 18 h, presumptive zygotes were cultured in SOF+BSA for 8 days (37�C, 5% CO2). On Day 3, cleavage rates and, on Day 8, blastocyst rates were determined. The relative mRNA abundance of the following transcripts, critically involved in early embryonic development was determined: growth differentiation factor-9 (GDF-9), heat shock protein 70 (Hsp-70), and glucose transporter-3 (Glut-3). Single immature and matured oocytes (for GDF-9 and Hsp-70) and 8-16-cell embryos and expanded blastocysts (for Hsp-70 and Glut-3) from calves and cows were examined by semiquantitative RT-PCR. Cleavage and blastocyst rates were similar in oocytes derived from cows and calves matured on GC (74.3% vs. 70.0% and 22.3% vs. 22.3%, respectively), but were significantly higher (P < 0.05; one way ANOVA, Student-Newman-Keuls Method) than in the group without co-culture on fibroblasts (55.2% vs. 53.6% and 11.7% vs. 5.5%, respectively). GDF-9 expression was similar in immature calf and cow oocytes. After maturation, a significant decrease in GDF-9 expression was observed in calf oocytes. Matured cow oocytes showed a significantly higher mRNA abundance of GDF-9 than matured calf oocytes. The relative abundance of Hsp-70 was decreased in matured oocytes of all groups. Expanded blastocysts derived from adult oocytes expressed Hsp-70 significantly higher than blastocysts derived from oocytes of the control calves. The relative abundance of Glut-3 mRNA was similar in 8-16 cell embryos and expanded blastocysts in all groups. Overall, mRNA expression pattern for Hsp-70 and Glut-3 in blastocysts from GC matured oocytes were similar to that of cow blastocysts. Results indicate that maturation of juvenile calf oocytes on granulosa cells from adult animals improves their developmental competence. These findings provide clues toward identification of factors critically involved in acquiring full developmental capacity at puberty.