Title page for ETD etd-04052006-105548


Type of Document Dissertation
Author Nel-Themaat, Liesl
Author's Email Address lnel1@lsu.edu
URN etd-04052006-105548
Title Gamete and Cell Technologies for Use in Biological Resource Banking
Degree Doctor of Philosophy (Ph.D.)
Department Animal Science (Animal, Dairy, & Poultry Sciences)
Advisory Committee
Advisor Name Title
Robert A. Godke Committee Chair
Charles E. Pope Committee Member
John W. Lynn Committee Member
Kenneth R. Bondioli Committee Member
Martha C. Gomez Committee Member
James H. Wandersee Dean's Representative
Keywords
  • epithelial cell culture
  • cryopreservation
  • biological resource banking
  • nuclear transfer
  • gulf coast native sheep
  • eland
Date of Defense 2006-03-06
Availability unrestricted
Abstract
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.

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