Factors affecting in vivo viability of DNA-injected bovine blastocysts produced in vitro

In vitro matured and fertilized bovine ova were microinjected with pBL1, which consisted of the bovine beta-casein gene promoter, human lactoferrin cDNA and SV40 polyadenylation signal. Of the 2931 zygotes injected, 2505 (85.5%) survived 1 h after DNA injection and were cultured in 50-microl drops of CR1aa medium containing 3 mg/ml BSA under mineral oil at 39 degrees C, 5% CO2 in air. Cleaved (2- to 8-cell) embryos were selected at approximately 48 h after DNA injection and then cultured further in 50-microl drops of CR1aa medium supplemented with 10% (v/v) FBS. Blastocysts were classified into 4 quality grades and 3 developmental stages by morphological criteria. Then all but poor quality blastocysts were nonsurgically transferred to the uterus of heifers 7 to 8 d after natural estrus. Following transfer, the recipients were observed for signs of estrus, and pregnancy was confirmed by palpation per rectum at approximately 60 d of gestation. Although 72.0% (1804/2505 ) of the DNA-injected zygotes reached 2- to 8-cell stages only 5.2% (131/2505) developed to blastocysts. A total of 75 DNA-injected, in vitro cultured blastocysts were transferred to 59 recipients. When 2 blastocysts were transferred to a single recipient, only the better quality embryo was counted. The overall pregnancy rate was 30.5% (18/59 ) and reflected 1) an apparent correlation between the quality of embryos and the pregnancy rate. However, the difference was not statistically significant. 2) expanded blastocysts had a higher pregnancy rate (50.0%, 11/22 ) than early (13.3%, 2 15 ) or mid (22.7%, 5/22 ) blastocysts with a significant difference between expanded and early blastocysts (P < 0.05). 3) the pregnancy rate of DNA-injected blastocysts was higher when they were transferred at Day 7 (34.5%, 10/29 ) or 8 (36.8%, 7/19 ) than at Day 6 (9.0%, 1/11 ). The results indicate that the developmental stage of DNA-injected bovine embryos may be one of contributing factors in improving the pregnancy rate after transfer, although the effects of the quality and culture period of the embryos may not be inconsequential.