Production of pig embryos following microinjection of somatic cells and sperm

The thesis investigated embryonic development through micromanipulation techniques in pigs. To investigate embryonic development, intracytoplasmic sperm injection (ICSI) and nuclear transfer (NT) research focused on strategies that help to achieve the goals of healthy and normal offspring. ^ For parthenogenetic activation electrical activation was used and optimized to apply NT and ICSI processes. We found that a single 30 μsec pulse of 2.2 kV/cm DC followed by culturing in post-activation medium with cytochalasin B for 5 h was effective for parthenogenetic activation and blastocyst formation of porcine oocytes. This activation regimen was adopted in nuclear transfer experiments and produced 4 live cloned pigs. ^ This thesis provides direct evidence that porcine oocytes can be fertilized by injection of frozen-thawed spermatozoa. In addition, electrical activation is beneficial to enhance normal fertilization and preimplantation development of porcine oocytes following ICSI. ^ We developed a whole cell injection technique for nuclear transfer and produced 4 live cloned piglets. This protocol should be of particular interest because it can cut down the time oocytes are manipulated during nuclear transfer and result in higher pregnancy rates.