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An Intersection Of Science & Art: Vitrification Approaches And Open-Fabricated Tools For The Biomedical Model Sea Hare, Aplysia Californica, Allyssa M. Oune
An Intersection Of Science & Art: Vitrification Approaches And Open-Fabricated Tools For The Biomedical Model Sea Hare, Aplysia Californica, Allyssa M. Oune
LSU Master's Theses
The California sea hare (Aplysia californica) is an important biomedical model for molecular neurobiology, electrophysiology, learning, and memory due to their well-mapped and large neurons and well-characterized learning capabilities. The National Resource for Aplysia (NRA, University of Miami) maintains large stocks of live animals and relies on regular shipments of wild-caught individuals to maintain genetic diversity. This is labor and cost-intensive, and environmental changes could alter the availability of wild animals increasing the need to preserve this genetic resource. One solution is vitrification, ultra-fast cooling which produces an amorphous glass that minimizes damage to cells. Aplysia californica presents …
A Modular Open-Technology Device To Measure And Adjust Concentration Of Sperm Samples For Cryopreservation, Nikolas C. Zuchowicz
A Modular Open-Technology Device To Measure And Adjust Concentration Of Sperm Samples For Cryopreservation, Nikolas C. Zuchowicz
LSU Master's Theses
Repositories for aquatic germplasm can safeguard the genetic diversity of species of interest to aquaculture, research, and conservation. The development of such repositories is impeded by a lack of standardization both within laboratories and across the research community. Protocols for cryopreservation are often developed ad hoc and without close attention to variables, such as sperm concentration, that strongly affect the success and consistency of cryopreservation. The wide dissemination and use of specialized tools and devices can improve processing reliability, provide data logging, produce custom hardware to address unique problems, and save costs, time, and labor. The goal of the present …
Vitrification Of Immature And Mature Bovine Oocytes, Paige T. Hardin
Vitrification Of Immature And Mature Bovine Oocytes, Paige T. Hardin
LSU Master's Theses
Vitrification is the latest technique used in cryopreservation, the ability to utilize this method with oocytes and embryos has become a valuable system. Vitrification has been successful with bovine embryos and oocytes but is far from optimal. Following cryopreservation storage discarding embryos can cause ethical issues, and mature oocytes have fragile organelles that can be detrimentally affected by ice crystal formation during freezing. Immature oocytes have not formed some of these temperature sensitive microfilaments and can circumvent these detrimental effects. The common intracellular cryoprotective agents are dimethyl sulfoxide, glycerol and ethylene glycol. Different combination of these agents have been reported …
Vitrification Of Equine Expanded Blastocysts, Fabian Andres Diaz
Vitrification Of Equine Expanded Blastocysts, Fabian Andres Diaz
LSU Master's Theses
The cryopreservation of equine expanded blastocysts (>300 um) has been largely unsuccessful primarily due to the low permeability to cryoprotectants and the large size of the equine embryo. Mechanical alternatives may provide means to overcome the capsule barrier and the relative large embryo size. In this regard, multiple experiments were performed in this study to evaluate different approaches of capsule puncture and blastocoele fluid extraction with the objective to develop a cryopreservation protocol for Day 8 equine expanded blastocysts. In the first experiment, twenty-four Day 8 expanded blastocysts were exposed to standard equine embryo vitrification solutions following one- or …
Development And Permeability Of Equine Blastocysts, Brittany Reshel Scott
Development And Permeability Of Equine Blastocysts, Brittany Reshel Scott
LSU Master's Theses
Equine embryo cryopreservation is unsuccessful in larger, more easily collected, day-7 embryos. It is imperative that methods to successfully cryopreserve large equine embryos or develop reliable methods to determine embryo size before collection. Therefore the objectives for this study were to quantify the amount of tritiated glycerol that would permeate various sizes of equine embryos and to determine if circulating progesterone concentration was correlated with in utero embryo size. Mean embryo diameter (± SEM) across treatments (1.4M and 3.4M tritiated glycerol) was 696.5µm ± 108.6µm and 925.9 µm ± 214.1µm, respectively and were not different (P=0.44). The percent permeation for …
Cryopreservation Of White-Tail Deer Epididymal Sperm For Artificial Insemination, Jesse Ray Saenz
Cryopreservation Of White-Tail Deer Epididymal Sperm For Artificial Insemination, Jesse Ray Saenz
LSU Master's Theses
The ability to cryopreserve epididymal sperm from mature postmortem bucks has long been of interest to both wildlife conservationists and deer ranchers. Increased understanding of the cryobiology of epididymal sperm from a non domestic species, such as White-tail deer, could aid in development of future protocols to assist in the preservation of endangered species. In Experiment I, results showed that after cooling postmortem bull testes for 22 hours, no significant difference was noted between sperm parameters of epididymal sperm collected at room temperature or at a cool enviorment. In Experiment II, it was shown that White-tail deer sperm could be …
Effects Of Cryopreservation And Constituents Of Semen Extenders On Mitochondrial Function Of Bull Spermatozoa, Abdulhakeem Hashim Eljarah
Effects Of Cryopreservation And Constituents Of Semen Extenders On Mitochondrial Function Of Bull Spermatozoa, Abdulhakeem Hashim Eljarah
LSU Doctoral Dissertations
This study investigated the effects of semen extender constituents and cryopreservation on bovine spermatozoal mitochondrial function. Three yearling Holstein bulls were used. Two ejaculates per bull were collected and pooled on a weekly basis for five weeks and extended in four treatments: 1) sodium citrate egg yolk extender with antibiotics (lincomycin, spectinomycin, gentamicin and tylosin); 2) ¡°1¡± with glycerol; 3) ¡°2¡± without antibiotics; and 4) ¡°1¡± without antibiotics. Each was divided into portions for analyses before freezing and after cryopreservation. The pre-freeze and thawed semen were transferred to a 37¡ãC water bath, the same assays were performed. In experiment 1, …
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. …
Analysis Of U.S. Aquacultural Producer Preferences For Genetic Improvement And Cryopreservation, Brian Paul Boever
Analysis Of U.S. Aquacultural Producer Preferences For Genetic Improvement And Cryopreservation, Brian Paul Boever
LSU Master's Theses
Aquaculture industries in the U.S. generate $1 billion in farm-level sales. Genetic improvement of fish stocks may be a way to increase the market share of aquaculture within the U.S. seafood market as well as the market share within the world market. This study evaluates the preferences, beliefs, and opinions of aquaculture producers across the U.S. about topics such as cryopreservation, genetic improvement, and the future of the aquaculture industry. Willingness-to-pay values for specific genetic improvements by aquaculture grow-out producers were elicited. A national survey of aquaculture producers was used to elicit the information used in the analysis. The survey …
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.
Cryopreservation Of Bovine And Caprine Oocytes By Vitrification, Sabrina Marie Luster
Cryopreservation Of Bovine And Caprine Oocytes By Vitrification, Sabrina Marie Luster
LSU Master's Theses
Cryopreservation of animal oocytes will permit germplasm of valuable or unique females to be preserved for extended times. The objective of this research was to derive a procedure to cryopreserve bovine oocytes by vitrification to be used as recipients for somatic cell nuclear transfer (SCNT). Caprine oocytes vitrified by the same procedure were assayed by cytological examination of microtubules. In the first two of three experiments, bovine oocytes matured in vitro were vitrified in a mixture of ethylene glycol (EG), dimethylsulfoxide (Me2SO) and trehalose, and then subjected to in vitro fertilization (IVF) or SCNT. For vitrification, oocytes were first exposed …
Preservation Of Sperm Harvested From The Rat, Caprine, Equine, And Bovine Epididymis, Aida Nioma James
Preservation Of Sperm Harvested From The Rat, Caprine, Equine, And Bovine Epididymis, Aida Nioma James
LSU Doctoral Dissertations
The interest in preserving endangered species has increased the amount of attention lent to the recovery of functional sperm from the epididymides of deceased males (Foote, 2000). Postmortem specimens have a finite time period before decomposition affects functionality. Determination of this “window of opportunity” to harvest and preserve epididymal sperm would be beneficial for further research in sperm preservation and assisted reproductive technologies. The objective of these studies was to determine 1) the “window of opportunity” to collect viable rat, caprine, equine and bovine epididymal sperm, 2) if epididymal sperm collected could be cryopreserved, 3) to test two common cryoprotectants …
Survival Of Canine Epididymal Sperm Under Cooled And Frozen-Thawed Conditions, Karla Stilley
Survival Of Canine Epididymal Sperm Under Cooled And Frozen-Thawed Conditions, Karla Stilley
LSU Master's Theses
The objective of the first experiment was to determine the effects of storage at 22„aC vs. 4„aC on the motility and percentage of membrane-intact sperm (%MIS) of epididymal mouse sperm. Testicles were allocated into 22„aC or 4„aC treatment groups and stored for 24 or 48 hours. Additional testicles were allocated into the 4„aC treatment for storage for 72 or 96 hours. Sperm was collected and analyzed at each time point. Storage at 22„aC lowered motility and %MIS (P<0.05) when compared with control sperm and 4„aC sperm at both the 24 and 48 hours. Motility and %MIS of 4„aC sperm did not decrease when compared with the control until after 72 hours of storage. The second experiment evaluated the effects of 22„aC vs. 4„aC storage for 24 and 48 hours on epididymal dog sperm. Motility and %MIS of the 22„aC sperm was lower than that of the 4„aC sperm and the control (P<0.05) at 24 and 48 hours. Motility of the 4„aC sperm was lower than the control at 24 and 48 hours (P<0.05), however %MIS was not lower than the control until 48 hours. The third experiment tested the effect of cryopreservation on epididymal dog sperm. Sperm was frozen immediately (A), after 48 hours at 4„aC in liquid (B) or after 48 hours at 4„aC of the whole testicle (C). Both pre-freeze (PF) and post-thaw (PT) motility and %MIS of B and C were lower than A (P<0.05). PT values were lower than PF values in all treatments (P<0.05). PT motility of B was lower than C (0 vs. 35.0¡Ó3.6%). Storage at 4„aC allows collection of motile epididymal mouse and dog sperm for several days after death. Dog testicles can be refrigerated for 2 days and epididymal sperm frozen with PT motility and %MIS of 35 and 62%.