Development and application of bovine in vitro fertilization
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Improved understanding of in vitro fertilization, in vitro oocyte maturation, and embryo culture will allow laboratory production of bovine blastocysts to become a well-established procedure. However, data related to production of bovine blastocysts in chemically defined media, i.e., no component of animal-origin included, are limited. Bovine blastocyst production in chemically defined media is an important technology that remains in need of improvement. The objective of this study was to examine the influence of growth factors or cytokines supplemented to embryo culture media on bovine embryonic development. A better means for handling of ovaries to maximize the efficiency of this procedure was also evaluated. Experimentation was also conducted to assess the feasibility of using bovine spermatozoa as a gene transfer vector to produce transgenic bovine embryos through in vitro fertilization or through intracytoplasmic sperm injection. Results presented here demonstrated that the way in which bovine ovaries were handled had a great impact on developmental competence of the harvested oocytes. Two hours of postmortem delay prior to oocyte aspiration benefited in vitro bovine embryo production. An effectect of adding leukemia inhibitory factor to embryo culture media was shown to increase numbers of inner cell mass nuclei without improving blastocyst yields or survival after cryopreservation. Also, this treatment stimulated in vitro hatching of blastocysts from their surrounding zonae pellucidae. Both epidermal growth factor and insulin-like growth factor-I improved rates of blastocyst development (from inseminated oocytes) when added to defined embryo culture media at a concentration of 5 and 50 ng/mL, respectively. No significant interaction resulting from combined treatment with those growth factors was detected. Insulin-like growth factor-I also increased numbers of inner cell mass nuclei and reduced numbers of DNA fragmented nuclei while epidermal growth factor had no effect either on numbers of inner cell mass nuclei or numbers of DNA fragmented nuclei when compared to untreated controls. Only bovine epididymal spermatozoa, not ejaculated spermatozoa, possess an ability to take up exogenous DNA. However, in contrast to recent findings with murine spermatozoa, there remains a barrier in efforts to adopt this approach for bovine spermatozoa as gene transfer vectors either in conjunction with in vitro fertilization or intracytoplasmic sperm injection. This body of research has contributed to a better understanding of requirements for laboratory production of bovine blastocysts and provides encouragement for realistic optimization of this reproductive biotechnology.