167THE USE OF BULL ROUND SPERMATIDS FOR PRODUCING RECONSTRUCTED EMBRYOS

Abstract

Recently, sperm has been used as a vector to carry exogenous genes for the production of transgenic animals. However, the success in cattle is low, due to deficiencies in oocyte activation and sperm decondensation caused by high disulphide bond (S=S) content in mature sperm. This study was carried out to develop an effective method for producing transgenic animals with round spermatids (RS). Two methods of embryo production—electric fusion (EC) or intracytoplasmic injection (IC)—and three activation treatments were compared. RS were isolated from bull testes by Percoll density gradients (20, 35, 40, 45 and 90%). Fusion between ooplast and RS was performed with a single DC electric pulse delivered by BTX 200 (1.0KVcm−1, 45μs) in 0.28M mannitol solution supplemented with 100μM CaCl2 and 100μMMgCl2. MII oocytes were divided into three activation groups for EC and IC methods, respectively. In Group 1, oocytes were activated with ionomycin (5μM, 5min) prior to EC/IC. In Group 2, oocytes were activated with ionomycin (5μM, 5min) after EC/IC. In Group 3, oocytes were activated twice with ionomycin, before and after EC/IC. All eggs were then incubated in cycloheximide (10μgmL−1) for 5h and cultured in CR1aa medium to Day 8. The rates of cleavage and blastocyst development were evaluated on Days 2 and 8, respectively. All experiments were performed as 5 replicates, and statistical differences were analyzed with one-way ANOVA (P<0.05). In IC, cleavage rates were significantly higher (P<0.05) in Group 3 (99/123, 80.5%) compared to Groups 1 and 2 (31/68, 45.6% and 45/70, 64.3%, respectively). Blastocyst development rates were also significantly higher (P<0.05) in Group 3 (15/123, 12.2%) compared to Group 2 (1/70, 1.4%), but did not differ compared to Group 1 (4/68, 5.9%). In EC, the cleavage rates in all three activation treatment groups remained low (43–45%), and no blastocysts developed. These results suggest that intracytoplasmic RS injection combined with repeated ionomycin activation is more efficient than electric fusion for producing developmentally competent embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 300012-05-3-SB010 and Cho-a Pharm. Co. LTD.]