Bovine embryo microinjection, culture, microsurgery, and DNA analysis by the polymerase chain reaction technique

The first experiment was conducted to determine the optimum in vitro culture system for one-cell bovine embryos. Subsequent experiments compared bisection and biopsy for acquisition of cellular material from bovine morulae for DNA amplification by the polymerase chain reaction technique (PCR), and evaluated the use of DNA microinjection, in vitro culture, morula bisection, and PCR for production and selection of transgenic bovine preimplantation embryos. In vivo fertilized one-cell bovine embryos were cultured in TCM-199 (n=46), co-cultured with bovine oviductal epithelial cells (OEC; n=38), or in explanted immature mouse oviducts (n=54). Of the embryos that cleaved once, 52.6, and 30.4 and 0.0% developed to morulae/blastocysts after culture in OEC, TCM-199, and explanted mouse oviducts. Mean cell number for embryos cultured in OEC (24.5) was higher than for embryos cultured in TCM-199 (12.8) or in explanted mouse oviducts (5.9; P<.05).

Bovine morulae were subjected to bisection (n=44; 20 to 30 cells) or biopsy (n=50; 8 to 12 cells) to assess embryo development in vitro and compare the efficiency of PCR amplification of an endogenous 18S rRNA. Mean development scores (l1=degenerate, 2=morula, 3=blastocyst) and mean cell number after microsurgery and 48 h of culture did not differ between treatments (P>.05; 2.4 ± .1 and 41.8 ± 2.5 versus 2.3 ± .1 and 48.8 ± 2.9, respectively). Frequency of the 18S rRNA amplifications was similar (P>.05) for demi-morulae (78%; 32/41) and biopsies (81%; 39/48).

In the third experiment, in vivo fertilized one- (n=155), two- (n=57) and four-cell (n=62) bovine embryos were collected for pronuclear and nuclear DNA microinjection. Approximately 70% of the embryos were injected with DNA and 30% served as controls. Injection did not affect (P>.05) mean development scores after 144 h of cultured in TCM-199 with OEC. Sixty-five (34%) of the DNA injected embryos developed to morulae and were bisected. Injected DNA was amplified by PCR in 29% (19/65) of the demi-morulae. Frequency of DNA detection was more frequent (P<.01) in morulae injected at the 1-cell stage (50%: 16/32) than at the 2-cell (10%; 1/10) or 4-cell (9%: 2/23) stage. Production and selection of transgenic bovine morulae was most successful when one-cell bovine embryos were microinjected.