FACTORS AFFECTING IN-VIVO VIABILITY OF DNA-INJECTED BOVINE BLASTOCYSTS PRODUCED IN-VITRO

In vitro matured and fertilized bovine ova were microinjected with pBL 1, which consisted of the bovine beta-casein gene promoter, human lact oferrin cDNA and SV40 polyadenylation signal. Of the 2931 zygotes inje cted, 2505 (85.5%) survived 1 h after DNA injection and were cultured in 50-mu l 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 wer e selected at approximately 48 h after DNA injection and then cultured further in 50-mu l drops of CR1aa medium supplemented with 10% (v/v) FBS. Blastocysts were classified into 4 quality grades and 3 developme ntal stages by morphological criteria. Then all but poor quality blast ocysts were nonsurgically transferred to the uterus of heifers 7 to 8 d after natural estrus. Following transfer, the recipients were observ ed for signs of estrus, and pregnancy was confirmed by palpation per r ectum at approximately 60 d of gestation. Although 72.0% (1804/2505) o f 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 c ultured blastocysts were transferred to 59 recipients. When 2 blastocy sts were transferred to a single recipient, only the better quality em bryo was counted. The overall pregnancy rate was 30.5% (18/59) and ref lected 1) an apparent correlation between the quality of embryos and t he pregnancy rate. However, the difference was not statistically signi ficant. 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 th ey 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 t he quality and culture period of the embryos may not be inconsequentia l.