PARENT SESSION
Oocyte Development
93
EFFECT OF RECIPIENT OOCYTE METABOLISM ON DEVELOPMENT OF OVINE NUCLEAR TRANSFER EMBRYOS.
Bormann, Charles1, Swain, Jason1, Clifford, Alicia1, Gourley, Dennis2, Krisher, Rebecca1, 1 2
ABSTRACT- Successful cytoplasmic maturation of recipient oocytes is required for development of nuclear transfer embryos. The objective of this study was to examine the effect of recipient oocyte metabolism on the development of cloned embryos. Oocytes were randomly allocated to three maturation treatments in TCM199 based medium; cibracon blue (CB), pyroline carboxylic acid (PC) and control (C). Matured oocytes from each treatment were assessed for glucose metabolism via glycolysis (D-[3H]-glucose) and pentose phosphate pathway (PPP; D-[1-C14]-glucose). Remaining oocytes were used as recipients for nuclear transfer or parthenogenetically activated as developmental controls. Reconstructed and activated oocytes were cultured in Gardner's G1.2/G2.2 medium. Cloned embryos were laparoscopically transferred to recipient ewes on day 5 of culture, while remaining embryos were assessed for development and cell number on day 6. Data were analyzed using ANOVA and Fisher's LSD Multiple-Comparison Test. Glycolytic activity (pmol/oocyte/3h ± SEM) of oocytes after maturation in C was 2.5 ± 0.21, in CB 2.35 ± 0.15 and in PC 1.98 ± 0.16. Activity of PPP was 0.65 ± 0.09 in C, 0.76 ± 0.16 in CB and 0.7 ± 0.1 in PC. The percentage of glucose utilized via PPP of total glucose utilized was 20.6 in C, 24.4 in CB and 26.1 in PC. Activated oocytes in CB had significantly more hatched blastocysts than C and PC (13.0 ± 2.7, 7.2 ± 0.02 and 5.6 ± 1.8, respectively). No differences were found in cell number of activated blastocysts (C, 89.0 ± 5.2; CB, 135.8 ± 28.2; PC, 80.6 ± 5.7). Blastocyst development of cloned embryos in CB (23.0 ± 3.9) and PC (24.4 ± 5.9) was significanlty greater than C (10.7 ± 3.6. There were significant differences in cloned cell numbers between CB (58.3 ± 8.8) and C (37.6 ± 4.0). Transfer of C embryos resulted in 1 of 2, CB 2 of 3 and PC 0 of 1 ewes pregnant at 30 d. These results suggest manipulation of recipient oocyte glucose metabolism, when used in nuclear transfer procedures, may increase viability and developmental competence of oocytes. These results further demonstrate that metabolism is an important and independent component of cytoplasmic maturation.
KEY WORDS: Ovine, Clone, Metabolism, Oocytes