Doctor of Philosophy (PhD)


Animal Science (Animal, Dairy, and Poultry Sciences)

Document Type



Salvaging of epididymal sperm from injured or deceased animals allows for the propagation of favorable traits from endangered or genetically superior males. Techniques developed in domestic species can also serve as the foundation for the collection, cryopreservation and utilization of epididymal sperm in exotic breeds. The need to preserve and utilize epididymal sperm, in the most efficient manner, is of the utmost importance. In a series of experiments, ejaculated and epididymal sperm from the same four mature, fertile, Holstein bulls were collected, cryopreserved, cultured and used for in vitro fertilization. In Experiment I, epididymal sperm was found to have higher post-thaw motility compared to ejaculated sperm. During cryopreservation, the membrane permeability of ejaculated and epididymal sperm was found to be similar. In addition, the membrane permeability of both ejaculated and epididymal sperm was decreased by the inclusion of glycerol during freezing. The optimal cooling rate for ejaculated and epididymal sperm was determined to be between 50 and 60⁰C/min. In Experiment II, we demonstrated that following castration, circulating concentration of plasma cholesterol increased. In Experiment III, the percentage of post-thaw auto-acrosome reacted ejaculated sperm was found to be higher than epididymal sperm. During in vitro culture, the percentage of auto-acrosome reacted ejaculated sperm remained relatively stable compared with epididymal sperm that significantly increased over time. The percentage of capacitation significantly increased over time for ejaculated sperm but not epididymal sperm, which only slightly increased following 6-hours of culture. In Experiment IV, cryopreserved ejaculated Holstein bull sperm was unaffected by the inclusion of PIF to the culture medium. PIF was also unable to improve or inhibit the in vitro fertility of cryopreserved ejaculated bull sperm. In Experiment V, ejaculated sperm with heparin and epididymal sperm with and without heparin were similar in their ability to fertilize oocytes in vitro and in their cleavage rates compared with ejaculated sperm without heparin, which was significantly lower. The number of embryos developing to the 8-cell stage was higher for the epididymal sperm plus heparin group compared with epididymal sperm without heparin. The number of blastocyst was similar for both ejaculated and epididymal sperm when heparin was added to the fertilization medium, compared with when heparin was not included in the fertilization medium. In summary, epididymal sperm was better able to endure cryopreservation. Ejaculated and epididymal sperm also displayed different in vitro dynamics. However, in vitro fertility of ejaculated and epididymal sperm was found to be similar and the inclusion of heparin increases blastocyst development in vitro. This may be useful when using epididymal sperm for in vitro reproductive techniques.



Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Godke, Robert A.