Doctor of Philosophy (PhD)
School of Animal Science
The primary lines of defense in preventing any exotic species from becoming endangered or extinct should be the protection of their habitat and from poaching. For wildlife conservationists, these primary measures are often not feasible, and thus cannot be the only means to prevent extinction. The objective of this research was to identify a semen extender that contained little to no egg yolk and could still effectively freeze domestic cat epididymal spermatozoa, for the purpose to sort spermatozoa into X and Y, using flow cytometry. In the first experiment we compared a TesT (Tes + Tris) 20% egg yolk extender (control) to a modified human sperm preservation medium (HSPM) and a Tris citrate extender that contained bovine serum albumin (TCBSA) to compare their effectiveness of preserving feline epididymal spermatozoa in relation to control spermatozoa. Results from this one experiment showed no significant difference among any of the treatments when measuring membrane integrity or acrosomal status. There was significant difference when comparing the pre-cool motility value between the control and the human sperm preservation medium extender (HSPM), but this difference was not detected in either of the post-thaw evaluations. In the second experiment, an attempt was made to determine what concentration could egg yolk be reduced to, and still exhibit cryoprotection. Three egg yolk concentrations (10%, 5% and 2%) were evaluated. It was determined that even at the lowest concentration, 2% egg yolk was not significantly different from the 10% or the 5% when comparing motility, membrane integrity and acrosomal status. In the third series of experiments, a TesT 2% egg yolk extender was evaluated to determine if exposure of the spermatozoa, before and after freezing, to 0nM, 1nM and 5nM of pentoxifylline (a motility stimulant) would have an effect on the post-thaw parameters measured. It was determined that there was no significant in post-thaw sperm motility, membrane integrity or acrosomal status when the epididymal sperm were exposed to the pentoxifylline both before and after freezing. In the last series of experiments TesT 2% egg yolk extender was compared with the BioXCell® and Biolife® extenders (neither containing egg yolk, and predominantly used for cool liquid sperm storage) on their effectiveness to maintain feline epididymal spermatozoa at 4°C for 72 hours. Although no significant difference was noted, the TesT 2% egg yolk and the BioXCell® extenders showed the most promising results for cooled liquid storage up to 72 hours. These experiments have shown that viable epididymal cat sperm can be collected from the epididymides of castrated toms, cryopreserved in extenders that contain little to no egg yolk and thawed, resulting in acceptable post-thaw values sufficient enough for IVF, possibly for AI and most certainly for ICSI. Finally, sperm were frozen in the TesT extender with and without egg yolk and frozen from room temperature and after cooling to 4°C. The TesT without egg yolk, frozen from room temperature, consistently had lower sperm motility and membrane integrity then sperm frozen in one or both of the extenders frozen after cooling to 4°C. There was no difference between the two TesT extenders frozen after cooling to 4°C for any of the parameters measured.
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Saenz, Jesse Ray, "Cryopreservation of Domestic Cat Epididymal Spermatozoa" (2015). LSU Doctoral Dissertations. 158.