Biological resource banking is becoming important for endangered species conservation. A series of experiments were conducted to address issues concerning collection and utilization of biomaterials from Gulf Coast Native (GCN) sheep (model species) (Ovis aries) and Eland antelope (Taurotragus oryx).
In the first experiment, two ejaculates were collected 10 minutes apart from each of five rams three times a week for three weeks to maximize output and minimize handling time. Semen volume, concentration and total number of spermatozoa were significantly greater in first ejaculates, whereas pre-cooled, cooled and post-thaw motility, as well as sperm survival, were greater in second ejaculates.
The second experiment was designed to develop a practical method for freezing skin biopsies for tissue culture. Cell survival was enhanced by an equilibration time of at least 15 minutes in biological media (containing blood or serum) as compared to non-biological medium (containing PVP).
Then, the possibility of using semen and cooled milk as sources of somatic cells (SC) for in vitro culture was evaluated. Fresh, cooled and cryopreserved semen from rams and fresh eland semen was cultured and although SC plated, proliferation was low. From milk, cell attachment was observed in 92% of the samples, whereas only 38% proliferated in culture.
Therefore, the ensuing experiment was focused on increasing in vitro proliferation of semen-derived SC by developing (1) a Percoll gradient technique to separate SC from spermatozoa before culture and (2) a method for co-culture of isolated SC with inactivated mouse fibroblasts. Proliferation was significantly increased by co-culture, but contact between the semen-derived SC and feeder cells was not necessary.
Finally, intergeneric nuclear transfer (igNT) of semen-derived eland epithelial cells into bovine cytoplasts showed that these cells could direct early embryonic development up to the 8-cell stage. Determination of BrdU-incorporation and evaluation of nuclear status revealed that initial remodeling of the eland nucleus was similar to that of bovine NT embryos within the first 16 hours post-activation; however, after 84 hours, only 13% of interspecies embryos had cycling nuclei in their blastomeres. Future improvements in the technology should eventually allow cloned offspring to be produced from semen-derived somatic cells.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-04052006-105548 |
| Date | 06 April 2006 |
| Creators | Nel-Themaat, Liesl |
| Contributors | Charles E. Pope, John W. Lynn, Kenneth R. Bondioli, James H. Wandersee, Martha C. Gomez, Robert A. Godke |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-04052006-105548/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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