This study evaluated procedures to improve culture and cryopreservation of in vitro produced bovine embryos. Successful in vitro development of early embryos is crucial for areas of biotechnology such as transgenics and cloning. The limiting factor in culturing in vitro matured and fertilized (IVM-IVF) embryos is the media in which the embryos develop in vitro. An optimal culture system which would support development equivalent to that found in vivo has yet to be found. If such a system were developed, studies could be conducted to elucidate the factors responsible for normal embryonic development and then apply these factors toward the development of defined media. The mitogenic capabilities of bovine (BVH) and rabbit (RVH) vitreous humor were tested on the in vitro development of IVM-IVF bovine embryos. Embryos were cultured from day 2, 3, 4, 5 or 6 until day 8 in different concentrations of BVH/RVH. Bovine embryos are inconsistent in their ability to develop in these fluids until the late morulae or early blastocyst stage. The activity of vitreous humor is not species-specific as bovine embryos developed in RVH as well or better than BVH. Blastocyst development was not improved with culture in BVH or RVH, although there is evidence to suggest that RVH enhances mean cell number in developing embryos. Delaying culture in BVH or RVH did improve mean cell number and blastocyst development. A simple and reliable method was developed to cryopreserve in vitro produced bovine embryos. The developmental capability of IVM-IVF derived morulae/early blastocysts was evaluated following different methods of diluting cryoprotectants after vitrification. One-step 0.1M or 0.3M sucrose dilution was optimal for removing the high concentrations of intracellular cryoprotectants in the vitrification medium as post-treatment survival was no different than culture controls. Step-wise dilution/gradual rehydration of vitrified embryos does not improve development following vitrification. Freezing and vitrification are equivalent for in vitro development of IVM-IVF bovine embryos following cryopreservation, although blastocyst development is approximately 1/2 the rate of their culture controls. These data are the first to indicate that IVM-IVF generated bovine embryos can be cryopreserved by vitrification.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-8417 |
| Date | 01 January 1992 |
| Creators | Dobrinsky, John Reid |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
Page generated in 0.0167 seconds