334 EFFECT OF A GROWTH MEDIUM DURING IVM ON EMBRYO DEVELOPMENT OF PREPUBERTAL EWE OOCYTES

The aim of this study was to assess the effect of an in vitro growth medium (De Wu et al. 2006 Biol. Rep. 75, 547-554) in prepubertal ewe oocytes selected by the brilliant cresyl blue (BCB) test. Prepubertal ewe oocytes were recovered by slicing ovaries of slaughtered animals and immediately exposed during 1 h to 13 μM BCB and classified according to their cytoplasm coloration (Rodriguez-Gonzalez E et al. 2002 Theri- ogenology 57(5), 1397-1409): BCB+ (blue cytoplasm, hypothetically grown oocytes) and BCB- (uncolored cytoplasm, hypothetically growing oocytes). Uncolored oocytes (BCB-) were matured using three culture media: growth medium (GM: TCM-199, 0.04 μg mL-1 FSH, 0.04 μg mL-1 LH, 0.004 μg mL-1 estradiol, 100 μg mL-1 ascorbic acid, and 5 μL mL-1 ITS: insulin transferrin selenium), conventional maturation medium (CM: TCM-199, 10 μg mL-1 FSH, 10 μg mL-1 LH and 1 μg mL-1 estradiol) and modified maturation medium (MM: CM with the addition of 100 μg mL-1 ascorbic acid and 5 μL mL-1 ITS). Oocytes were matured in GM for 12 h and then separated into 2 groups, CM (GM+CM) and MM (GM+MM) for another 12 h of maturation. Two extra groups of BCB- oocytes were directly cultured for 24 h in CM or MM media (BCB-/CM and BCB-/MM). Colored oocytes (BCB+) and a control group (oocytes not exposed to BCB) were matured for 24 h in CM. All groups were cultured at 38.5°C and 5% CO2 in humidified atmosphere. Fertilization took place in SOF medium supplemented with 10% of estrous sheep serum during 20 h with a sperm concentration of 1 × 106 spermatozoa/mL. Presumptive zygotes were cultured for 8 days in SOF with 10% FCS at 38.5°C, 5% CO2 and 5% O2. Results are shown in Table 1. The percentage of morula plus blastocyst obtained from BCB - oocytes was significantly increased in oocytes exposed to growth medium (containing ITS, ascorbic acid and low hormone concentrations; groups GM+CM and GM+MM) for the first 12 h. An increasing tendency has also been observed in blastocyst yield in these two groups. Regarding maturation rate, no differences were found in all groups (data not shown). In conclusion, as De Wu et al. (2006) showed in prepubertal gilts, we also achieved some improvements in embryo development of growing oocytes when the first 12 h of maturation took place in a growth medium. However, embryo developmental potential of BCB- oocytes is still lower compared with that of BCB+ oocytes. Table 1.Effect of GM on embryo development of BCB- oocytes Grant sponsor Spanish Ministry of Science and Innovation.Code: AGL2007-60227-CO2-01