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Cryopreservation And Intracytoplasmic Sperm Injection With Bovine Epididymal Spermatozoa, Carlos Andres Guerrero
Cryopreservation And Intracytoplasmic Sperm Injection With Bovine Epididymal Spermatozoa, Carlos Andres Guerrero
LSU Doctoral Dissertations
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 4°C as compared with glycerol in all concentrations evaluated. Furthermore, prolonged exposure (5 days at 4°C) 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 37°C 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.
Vitrification And Dehydration For The Preservation Of Gametes, Allison E. Moisan
Vitrification And Dehydration For The Preservation Of Gametes, Allison E. Moisan
LSU Doctoral Dissertations
Of the 36 species of felines in the world, all except the domestic cat are listed as endangered or threatened. To preserve the genetic diversity of felines and other species, genome resource banks have been established. Due to limited availability of germ cells for research, studies must use models to optimize the techniques before they are applied to endangered species. In this study, preservation of oocytes and spermatozoa was examined using the bovine as a model for felines. In the first series of experiments, bovine and feline oocytes were dehydrated, vitrified, warmed and cultured to assess their ability to undergo …
Gamete And Cell Technologies For Use In Biological Resource Banking, Liesl Nel-Themaat
Gamete And Cell Technologies For Use In Biological Resource Banking, Liesl Nel-Themaat
LSU Doctoral Dissertations
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. …
Evaluation Of A Plasmid Delivery System For Production Of Gnrh And Ghrh In The Horse And Goat, William Andrew Storer
Evaluation Of A Plasmid Delivery System For Production Of Gnrh And Ghrh In The Horse And Goat, William Andrew Storer
LSU Doctoral Dissertations
The efficacy of a novel plasmid delivery system was assessed for long-term expression of gonadotropin releasing hormone (GnRH) and growth hormone releasing hormone (GHRH) in horses and goats. The efficacy of the technology was demonstrated using 3 novel plasmids: pSEAP [expressing secreted embryonic alkaline phosphatase (SEAP)], pGHRH (expressing GHRH), and pGnRH (expressing GnRH). Geldings were electroporated with a reporter plasmid expressing SEAP in 3 muscle sites. Expression of SEAP, measured from jugular plasma samples, indicated muscle specificity for uptake and expression of the plasmid. Concentrations of SEAP were greatest (P < 0.05) after pectoralis injection, which was chosen as the site for electroporation in subsequent studies. In a second experiment, stallions were electroporated with pGHRH or pSEAP to evaluate the effect of long-term GHRH treatment on the growth hormone (GH) axis and testicular function. Stallions treated with pGHRH had increased (P < 0.05) plasma concentrations of IGF-I, increased (P < 0.05) volume of accessory sex gland fluid, and increased (P < 0.05) number of normal spermatozoa in the ejaculate relative to controls. In the third experiment, stallions were electroporated with pGnRH or pSEAP to test the effects of GnRH on the reproductive axis. Treatment with pGnRH increased (P < 0.05) plasma testosterone concentrations to d 56 and increased (P < 0.01) the LH response to GnRH on d 21, but did not alter (P > 0.1) seminal characteristics evaluated after 36 d of treatment. In …