50 PRE-MATURATION OF BOVINE OOCYTES SUBMITTED TO NUCLEAR TRANSFER: EFFECTS ON IN VIVO DEVELOPMENT

Abstract

In vitro embryo production by somatic cell nuclear transfer (SCNT) still presents low efficiency and blastocyst production rates are around 20%. Pre-maturation with cell cycle inhibitors is one alternative that has been studied to improve oocyte competence for use in in vitro production systems. The neurotrophin brain-derived neurotrophic factor (BDNF) has been reported to improve oocyte maturation. The aim of this work was to optimize the in vitro pre-maturation culture of bovine oocytes and its use in SCNT. Oocytes that were submitted to meiosis block before IVM (BL group) were cultured in TCM-199 medium supplemented with 10 ng mL-1 BDNF and 10 μM butyrolactone I for 24 h and then matured in IVM medium (TCM-199, 10% FCS, 0.5 μg mL-1 FSH, 5.0 μg mL-1 LH, 2.0 mM pyruvate, and 50 μg mL-1 gentamicin). Control oocytes (control group) were matured in IVM medium. After 19 h of IVM, oocytes from both groups were denuded with 0.2% hyarulonidase, enucleated, and reconstructed. Reconstructed embryos were chemically activated with ionomycin (5 min) and 6-DMAP (3h) and cultured in vitro in SOF medium for 7 or 8 days. Statistical analyses were performed by using BIOSTATS v.4.0 software. In vitro development variables [1st polar body (PB), fusion, cleavage, and blastocyst rates on Days 7 and 8] and in vivo development rates on Days 35, 60, 90, and 120 of pregnancy were analyzed by chi-square test. Total cell numbers and cells with fragmented DNA were analyzed by ANOVA. A level of 5% significance was considered. Extrusion of 1st PB (BL: n = 693; 69.3% and control: n = 639; 63.5%) and fusion rates (BL: n = 397; 79.2% and control: n = 345; 72.9%) were higher (P < 0.05) in the BL group. There were no differences between treatments for cleavage rates (BL: n = 268; 67.5% and control: n = 228; 66.1%) or blastocyst rates on Day 7 (BL: n = 77; 19.4% and control: n = 69; 20.0%) and Day 8 (BL: n = 81; 20.4% and control: n = 73; 21.2%). Cloned blastocysts from both groups were submitted to TUNEL reaction (Day 8 blastocysts, n = 15 for BL and control groups) for DNA fragmentation analysis or were transferred to synchronized recipients (Day 7 blastocysts, n = 26 and n = 28 for BL and control groups, respectively) for in vivo development analysis. No differences were observed (P > 0.05) between BL and control groups for total cell numbers (n = 127 and n = 138, respectively) and cells with fragmented DNA (0.0209 and 0.0188, respectively). Pregnancy rates at 35 (BL: n = 5; 19.2% and control: n = 9; 32.1%), 60 (BL: n = 3; 11.5 and control: n = 3; 10.7), 90 (BL: n = 3; 11.5 and control; n = 3; 10.7), and 120 days (BL: n = 3; 11.5 and control: n = 3; 10.7) also did not differ (P > 0.05) between treatments. In conclusion, pre-maturation enhanced 1st PB extrusion and fusion rates of oocytes submitted to SCNT, and moreover, it was able to establish pregnancies until 120 days, similarly to the control group. Financial support: FAPESP and CNPQ, Brazil.