PARENT SESSION
Oocyte Development
87
EFFECTS OF GLUCOSE AND CULTURE MEDIA ON IN VITRO DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS.
Martins , Alicio1,2, Freitas, Camila1, Brackett, Benjamin2, Negrelli, Giuliano1, Lopes, Paula Helena1, 1 2
ABSTRACT- In spite of recent improvements in culture media intense efforts are underway to maximize bovine oocyte to blastocyst development in vitro. This study was conducted to test Medium 199 and m-SOF+NEA- based media with attention to effects of added glucose on in vitro production of bovine embryos. After washing and selection in Medium 199 with 2.2 mg/ml sodium bicarbonate, 50 
g/ml sodium pyruvate, 50 g/ml gentamicin sulfate, and 1 mg/ml polyvinyl alcohol (PVA), referred to here as basic-maturation medium (b-MM), oocytes (15/drop) were matured in b-MM without PVA (maturation medium = MM), but in presence of 1000 ng/ml FSH, 10 ng/ml EGF, and 15% FCS for 24 h. Sperm handling was in m-DM with 5 h insemination interval and 20 g/ml heparin for capacitation. Media were prepared without Hepes and incubations performed under a 5% CO2 atmosphere. Presumptive zygotes were cultured in 3 steps designated by h postinsemination, i.e., step 1 (5 to 72 h), step 2 (72 to 144 h) and step 3 (144 to 196 h) either in MM or m-SOF+NEA (without citrate or glutamine, with penicillin). Treatment groups I (step 1 in MM and steps 2 and 3 in fresh MM plus 5% FCS), II (steps 1, 2 and 3 in m-SOF+NEA without glucose, but steps 2 and 3 with 5% FCS), and III (same medium as II, but with glucose and 5% FCS in steps 2 and 3). Glucose concentration was 5.5 M, 0 M and 1.5 M, respectively for I, II and III. Cleavage rate (C, 72 h) and proportions of morulae (M, 144 h), blastocysts (B, 144 and 168 h), and expanded blastocysts (EB, 168 and 196 h) are reported based on numbers of oocytes matured. ANOVA and t- test were used to determine differences among groups. No differences were found among groups I, II and III, respectively for C (89.0, 87.3 and 85.8%), M (40.4, 27.3 and 34.5%) and B at 168 h (26.7, 15.3 and 20.9%). However, more (p<0.05) B developed by 144 h in groups II (28.7%) and III (20.9%) than for I (14.4%). Higher (p<0.05) percentages of EB were obtained for II at 168 and 196 h (20.7 and 26.0) than for I (2.1 and 6.2) and III (6.8 and 9.5). Data indicated that added glucose was not required for bovine embryo culture in vitro under these conditions. Embryonic development was faster and quality was improved in II (SOF-based, no added glucose) compared to I (Medium 199- based with 5.5 M glucose added). (Supported by FAPESP, Altagenetics-VRBV and Araçafrigo, SP, Brazil.)
KEY WORDS: culture media, in vitro culture, glucose, bovine