Date of Award

1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

School of Animal Science

First Advisor

Robert A. Godke

Abstract

To improve the efficiency of the culture system for bovine in vitro fertilization (IVF), several factors affecting the success of in vitro maturation (IVM), IVF and in vitro culture (IVC) of bovine oocytes were studied. A simple and efficient procedure has been developed for IVM/IVF/IVC of bovine follicular oocytes, which consistently produced 45 to 50% morulae and blastocysts during the treatment of over 6,000 oocytes. Ovary holding temperatures of $\rm 0\sp\circ C,\ 18\sp\circ C\ and\ 25\sp\circ C$ were found to affect the rate of maturation, fertilization and cleavage (P $<$ 0.05). When ovaries were held at 25$\sp\circ$C for 3 h, 20% of the oocytes developed to blastocysts and hatched in vitro, indicating that this temperature is suitable for holding ovaries during transportation. The highest cleavage rate (79%) was obtained when oocytes were matured and fertilized both at 39$\sp\circ$C in 5% CO$\sb2$. Fertilization rate was impaired with a lowered incubation temperature (37$\sp\circ$C) and CO$\sb2$ level (2.5%). Cumulus cells were shown to be important and necessary for IVM and acquisition of competence for full embryonic development. Cumulus cell removal before IVM, before IVF or 7 h after IVF reduced the rates of maturation, fertilization and development at all embryo stages evaluated (P $<$.05). Cumulus cell removal at 20 h after fertilization resulted in a rate of development similar to control embryos (P $>$.05). Co-culture enhanced the development of IVF-derived embryos. More embryos developed to blastocysts and hatched when co-cultured with bovine cumulus cells (BCC), bovine oviduct epithelial cells (BOEC) and BCC plus BOEC cells than in medium alone (P $<$.05). A simple serum-free medium (CZB) supported more IVF-derived early embryos to the morula stage than a complex serum-containing medium (TCM-199). However, fewer embryos hatched in CZB medium than in serum-containing TCM-199 (P $<$.05). Among growth factors evaluated, arachidonic acid (AA) had a significant stimulatory effect on embryo development (P $<$.05). The optimal AA concentration was 50 ng/ml; a high concentration (500 ng/ml) was toxic. Bovine IVM/IVF/IVC embryos were successfully cryopreserved, thawed and transferred into recipient animals. Nine clinical pregnancies were established with the birth of five normal calves. This success indicates that viable bovine embryos can be produced in the laboratory.

Pages

190

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