INFLUENCE OF TIME OF GENE MICROINJECTION ON DEVELOPMENT AND DNA DETECTION FREQUENCY IN BOVINE EMBRYOS

The effect of DNA microinjection at various times after in vitro insem ination on DNA detection and survival rates of bovine embryos was inve stigated. Oocytes were inseminated 24 h after maturation with frozen/t hawed semen prepared with a Percoll separation procedure. At 11, 15 an d 19 h after insemination, embryos were centrifuged to visualize pronu clei and microinjected with a murine whey acidic protein-human protein C genomic DNA construct. After culture for 7 days on Buffalo Rat Live r cells, embryos were assessed for stage of development and assayed fo r the presence of the transgene by polymerase chain reaction. Of zygot es in the 11 h after insemination treatment, 16% (25/152) of non-injec ted and 7% (11/161) of injected embryos developed to the morula or bla stocyst stage. Comparable development of non-injected and injected emb ryos treated at 15 h after insemination was 15% (23/158) and 4% (6/159 ) and treated at 19 h after insemination was 14% (23/162) and 1% (1/16 5), respectively. Development of injected embryos was greater (p < 0.0 5) when injection was performed at 11 h after insemination compared to 19 h after insemination. Development of non-injected embryos was grea ter (p < 0.01) than that of injected embryos. There was no difference in transgene detection frequency in embryos of all developmental state s between treatments (53% at 11; 50% at 15; 48% at 19 h after insemina tion). Injected embryos testing positive for the presence of the trans gene exhibited increased development over negative embryos (p < 0.01). Greater development efficiencies can be obtained in microinjected bov ine embryos when injection is performed early in pronuclear formation.