147 EFFECT OF CULTURE SYSTEM ON THE DEVELOPMENT OF IN VITRO-FERTILIZED OR DNA-INJECTED BOVINE EMBRYOS

Abstract

DNA microinjection has become the most widely applied method for gene transfer in mammals. However, the production of the transgenic bovine is relatively inefficient with the pronuclear microinjection technology. This experiment was designed to compare the two different in vitro production systems the serum-containing system (IVM, IVF, and IVC; TCM199, TALP, and CR1aa) and the Serum-free system (IVM, IVF, and IVC; IVMD101, IVF100 and, IVMD101), on the development of in vitro-fertilized embryos (Experiment 1) and DNA-microinjected embryos (Experiment 2). Korean Native Cow (KNC) ovaries were obtained from slaughterhouse and cumulus oocyte complexes (COCs) were aspirated from 2- to 8-mm follicles. In the serum-containing system, groups of 15 COCs were matured in TCM199 supplemented with 10% fetal calf serum (FBS), 1 μg/mL FSH, 10 μg/mL LH, and 1 μg/mL estradiol-17β for 18 h. In vitro matured oocytes were fertilized using frozen-thawed percoll separated spermatozoa (Day 0) in fer-TALP medium for 20 h. Presumptive zygotes were cultured in CR1aa medium supplemented with 0.3% BSA (before Day 3) or 10% FBS (after Day 3). In the serum-free system, groups of 15 COCs were matured in IVMD101 medium, fertilized in IVF100 medium and cultured in IVMD101 medium (Hochi et al. 2003 Theriogenology 59, 675–685). The DNA used for microinjection was a green fluorescent protein. The zygotes were centrifuged in TALP medium at 15,000g for 7 min, and then were microinjected into the pronucleus. All cultures were maintained in an incubator at 39°C, 5%CO₂ in air with maximum humidity. Data from three replicates were analyzed by chi-square test. In Experiment 1, there were no differences in the cleavage rates between treatments (71.8% v. 65.3%, respectively). The development rate to the 8-cell stage was significantly higher in the serum-free system than in the serum-containing system (P < 0.05; 47.2% v. 24.7%, respectively). However, the development rates to the blastocyst stage were not different (20.4% v. 16.0%, respectively). In Experiment 2, the development rates to the cleavage, 8-cell, and blastocyst stages were significantly higher in the serum-free system than in the serum-containing system (P < 0.05; 47.2, 25.0, and 5.6% vs. 16.5, 3.5, and 0%, respectively). The results of this study suggest that the serum-free system was not effective on the development of in vitro-fertilized embryos, but it was effective on the development of DNA-microinjected embryos.

This study is supported by Technology Development Program for Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea.