Title page for ETD etd-07132006-174133

Type of Document Dissertation
Author Guerrero, Carlos Andres
Author's Email Address cguerr1@lsu.edu
URN etd-07132006-174133
Title Cryopreservation and Intracytoplasmic Sperm Injection with Bovine Epididymal Spermatozoa
Degree Doctor of Philosophy (Ph.D.)
Department Animal Science (Animal, Dairy, & Poultry Sciences)
Advisory Committee
Advisor Name Title
Robert A. Godke Committee Chair
Jill Jenkins Committee Member
John Lynn Committee Member
Kenneth Bondioli Committee Member
Stanley Leibo Committee Member
Bruce Eilts Dean's Representative
  • epididymal spermatozoa
  • cryopreservation
  • seminal plasma
  • intracytoplasmic sperm injection
  • bovine
Date of Defense 2006-06-30
Availability unrestricted
Recently, interest in the preservation of epididymal sperm as a potential source of valuable genes for genome resource banks has escalated. The development of a successful protocol to recover and cryopreserve sperm harvested from the epididymides would salvage germplasm from genetically valuable males that are injured and can no longer mate or have unexpectedly died and can be used as a model for the preservation of male gametes from endangered species. In a series of experiments, epididymal sperm was successfully harvested, cryopreserved and used for intracytoplasmic sperm injection. In Experiment I, ethylene glycol was found to cause significantly (P<0.05) less osmotic damage to bovine sperm during a one step addition and/or removal at 4C as compared with glycerol in all concentrations evaluated. Furthermore, prolonged exposure (5 days at 4C) of ethylene glycol was found to be less toxic than glycerol to sperm. In Experiment II, it was demonstrated that glycerol was more effective than ethylene glycol in providing protection against freezing injury during the cryopreservation process in the concentrations evaluated. In Experiment III, it was demonstrated that epididymal sperm retrieval using seminal plasma is beneficial to enhance sperm overall and progressive motility characteristics and to protect it from morphological abnormalities derived from the freezing process. In Experiment IV, a one step dilution process for removal of glycerol from cryopreserved epididymal sperm was found to significantly affect plasma membrane integrity and mitochondrial function of sperm previously exposed to seminal plasma. However, seminal plasma exposure did not have any significant detrimental effect on acrosome integrity. Furthermore, it was demonstrated that the longevity and survivability in vitro during a 4-hour incubation period at 37C of post-thaw epididymal sperm exposed to seminal plasma prior to cryopreservation was not compromised when compared with the control extended sperm. In Experiment V, we have demonstrated that fertilization, blastocyst and fetal development could be achieved with cryopreserved bovine epididymal sperm by intracytoplasmic sperm injection (ICSI). To our knowledge, this is the first report in the United States and second in the world to use bovine epididymal sperm for ICSI. We achieved far markedly improved blastocyst rates over those results recently reported in the first study originating in Japan.
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