299 DEVELOPMENTAL COMPETENCE OF TRANSGENIC BOVINE EMBRYOS RECONSTRUCTED BY NUCLEAR TRANSFER USING MEIOSIS-BLOCKED OOCYTES

Abstract

Recent progress in animal cloning by nuclear transfer (NT) has made it feasible to produce transgenic animals using genetically modified cell lines. Healthy cells and competent oocytes are needed to maximize the number of transgenic calves produced. Oocyte maturation plays a central role in oocyte competence. Prematuration inducing meiosis block is, therefore, a possible tool in transgenesis, because it allows further optimization of oocyte maturation protocols. In field conditions, it is not always possible to precisely control timing between oocyte collection and NT procedures. Therefore, temporarily and reversibly blocking maturation may also be used as a strategy to optimize cloning protocols. The aim of this study was to analyze the developmental competence of embryos reconstructed by NT using cells modified genetically as nuclei donors and oocytes submitted, or not, to meiosis block as cytoplasts. The hypothesis was that blocking meiosis does not alter the embryonic developmental physiology nor production of transgenic cloned bovine blastocysts. Ovaries were collected at a slaughterhouse and follicles between 3 and 6 mm were aspirated. Oocytes were divided into 2 groups. The first group (CTR, n = 145) was matured in vitro (IVM) with TCM-199 supplemented with 10% fetal calf serum, 5.0 µg mL^–1 of LH, 0.5 µg mL^–1 of FSH, 0.2 mm pyruvate, and 10 µg mL^–1 of gentamicin for 18 h at 38.5°C under 5% CO₂ in a humidified atmosphere. The second group (BL, n = 153) had meiosis blocked by in vitro culture with TCM-199 supplemented with 0.2 mm pyruvate, 10 µg mL^–1 of gentamicin, and 10 µm butyrolactone I for 24 h, and then matured in vitro for 18 h. Parthenogenetic embryos resulted from meiosis-blocked and non-blocked oocytes were used as controls for the respective groups of NT embryos. After IVM, oocytes from both groups were reconstructed using bovine fetal fibroblasts transduced previously with a lentivirus and expressing the green fluorescent protein gene. The effect of treatment on fusion rates in cloned embryos, cleavage rates on Day 2, and blastocyst rates on Day 7 of in vitro culture of cloned and parthenogenetic embryos from CTR and BL groups were analyzed by chi-square test at 5% significance. Meiosis block did not affect fusion rates (n = 68, 46.90% and n = 86, 56.21% for CTR and BL cloned groups, respectively). Cleavage rates did not differ between cloned groups (n = 43, 63.24% and n = 49, 56.98% for CTR and BL groups) or between parthenogenetic groups (n = 15, 50% and n = 21, 70% for CTR and BL groups). Also, no difference was observed in blastocyst rates between cloned groups (n = 5, 7.35% and n = 6, 6.98% for CTR and BL groups) and between parthenogenetic groups (n = 11, 36.67% and n = 9, 30% for CTR and BL groups). It was concluded that meiosis block does not affect embryo development to the blastocyst stage. It is suggested that temporarily blocking meiosis may be a useful strategy to optimize NT protocols.

FAPESP, Brazil.