Life Sciences Commons^™

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 …

The Art Of Amphibian Conservation: Linking In-Situ And Ex-Situ Populations Of Endangered Species Through Genome Banking, Isabella Joann Burger

Theses and Dissertations

Limited breeding success in captive breeding programs has necessitated the development of assisted reproductive technologies (ART) to preserve and increase genetic variation and population numbers of both captive and wild amphibian groups. ART has been shown to be successful in numerous anuran species, and current studies focus on the application of ART in ex-situ populations. The focus of this project is to show that linking in-situ and ex-situ amphibian populations through sperm cryopreservation, genome banking, and in-vitro fertilization is possible, with the goal of increasing gene diversity throughout groups in order to produce self-sustaining, wild populations in the future. Specific …

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 …

Investigation Of Techniques And Their Application For The Cryopreservation Of Algal Species, Jazmine Leija

Theses and Dissertations

An alternative source for petroleum-based crude oils are algae derived biofuels. Fossil fuels are harmful for the environment, expensive and becoming scarce. There has been an increase in research on environmentally sustainable energy, using lipids derived from microalgae, which can be converted into biofuel. The use of microalgae as a source for biofuels has a lot of benefits including decreasing greenhouse gas emissions, rapid fuel production, absorption of carbon dioxide, and production of a renewable source of energy.

Algae cryopreservation aids in the maintenance of the best algae strains selected for producing lipids for biofuels. Cryopreservation will help minimize genetic …

Functionality Of Red Blood Cells After Cryo-Preservation., Charles Andrew Elder

College of Arts & Sciences Senior Honors Theses

One of the most common medical procedures performed in US hospitals is blood transfusions. Unfortunately, the red blood cells (RBCs) for transfusion have a limited shelf life after donation due to detrimental storage effects on morphological and biochemical properties. Inspired by nature, I am developing a biomimetics approach to preserve RBCs for long-term storage using compounds that occur in animals that have developed a natural propensity to survive in a frozen or desiccated state for decades. Trehalose was employed as a cryoprotective agent when added to the extracellular freezing solution. The highest percent of RBCs with intact membranes after freezing …

Lipid Quantification And Cryopreservation Of In Vitro Produced Jersey Cattle Embryos, Katherine A. Rhodes-Long

Master's Theses

Cryopreservation of in vivo derived Jersey bovine embryos have resulted in a 10% lower pregnancy rate compared to other dairy breeds. Poor embryo survival after cryopreservation has been partially attributed to the high lipid content of Jersey embryos. In vitro-produced (IVP) bovine embryos have darker cytoplasm than their in vivo-derived counterparts because of higher lipid accumulation. High lipid accumulation is associated with impaired embryo quality. Forskolin is an adenylate cyclase activator that regulates cAMP levels in cells and has been shown to induce lipolysis in IVP embryos. L-carnitine is required for transport of fatty acids from the intermembrane space of …

Metabolic Regulation And Cryotolerance Of In Vitro-Produced Holstein Embryos, Melissa Ann Roberts

Master's Theses

In vitro production and transfer of embryos has become a common practice within the dairy industry to efficiently breed superior animals and meet the consumption demand of the growing population. Cyropreservation is necessary for the application of commercialized embryo transfer, however, in vitro-produced embryos show morphological and physiological defects which negatively impact their ability to withstand cryopreservation in comparison to their in vivo counterparts. These artifacts result from culture conditions that cause stress to the embryo during development, leading to an accumulation of intracellular lipids, mitochondrial dysfunction, and ultimately poor ability to withstand freezing and thawing. The objective of …

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 …

Modulation Of Cell-Matrix Interaction For Cryopreservation Of Engineered Tissue, Angela Christine Seawright

Open Access Theses

Long term preservation of functional engineered tissues can significantly advance tissue engineering industry and regenerative medicine. Several preservation techniques have been proposed and investigated for this purpose, and cryopreservation is a leading candidate. While tissues are cryopreserved, ice forms in both the extracellular and intracellular spaces and causes freezing-induced spatiotemporal deformation of the tissue. During this process the cells undergo dehydration by the freezing-induced osmotic pressure difference and mechanical deformation, transmitted through cell-extracellular matrix adhesions. However, the significance and interaction of these cellular level transport and mechanics processes are not well understood. Therefore, this study aims to establish mechanistic understanding …

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 …

Open Pulled Straw (Ops) Vitrification Of Mus Musculus Morula And Blastocyst Survival In Two Common Cryopreservation Medias, Gina Marie Cirimele

Animal Science

The objectives of this study were to: (1) determine an optimal cryoprotectant of mice embryos; and (2) determine whether morula or blastocyst stage is ideal for vitrification in both medias. One experiment was performed using two different medias for vitrification with open pulled straws (OPS) with morulae and blastocysts. In the first protocol, we called V, embryos were exposed to 10% ethylene glycol (EG) for 5 minutes, then 40% ethylene glycol and 0.6 M galactose for about 30 seconds, loaded into an OPS, and plunged into liquid nitrogen. In the second protocol, we called VG, embryos were exposed to 7.5% …

Efficiency Of Two Cryopreservation Methods Using Direct In-Straw Rehydration After Repeated Vitrification Of Mouse Embryos, Ashley Morgan Payne

Animal Science

Experiments were conducted to determine which of two direct in-straw rehydration methods was optimal for obtaining high survival of mouse embryos after repeated vitrification. The first vitrification method compared was a one-step design, designated “House method,” where embryos were equilibrated in 3.5 M ethylene glycol for 3 min before rapid plunge vitrification in 7 M ethylene glycol/0.5 M glucose/18% w/v Ficoll 70. The second method was the commercially available BoviPRO Embryo Vitrification Kit ^TM(MiniTube of America, Verona, WI), which first exposed embryos to 1.4 M glycerol for 5 min, then 1.4 M glycerol/3.6 M ethylene glycol for 5 min, …

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 …

The Development And Analysis Of A Closed System Of Vitrification For Mammalian Embryos, Jennifer Graves-Herring

All Dissertations

Embryo cryopreservation is an integral part of assisted reproduction because it allows for future use of these embryos. Cryopreservation occurs when there are supernumerary embryos or when an embryo transfer cannot be performed.
There are two main methods to cryopreserve embryos. The most recent is vitrification, which uses high concentrations of cryoprotectants, a short time to cool and avoids ice crystals. The 'gold standard' is the slow-cool method, which uses low concentrations of cryoprotectants, a long time to cool embryos and produces extracellular ice crystals.
Prior to introducing vitrification as part of the human cryopreservation regime, it is important that …

Role Of Calcium And Nitric Oxide Synthase (Nos) In Brain Mitochondrial Dysfunction, Vidya Nag Nukala

Theses and Dissertations--Neuroscience

Mitochondria are essential for promoting cell survival and growth through aerobic metabolism and energy production. Mitochondrial function is typically analyzed using mitochondria freshly isolated from tissues and cells because they yield tightly coupled mitochondria, whereas those from frozen tissue can consist of broken mitochondria and membrane fragments. A method, utilizing a well-characterized cryoprotectant such as dimethyl sulfoxide (DMSO), is described. Such mitochondria show preserved structure and function that presents us with a possible strategy to considerably expand the time-frame and the range of biochemical, molecular and metabolic studies that can be performed without the constraints of mitochondrial longevity ex vivo …

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

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

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

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

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

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

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

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%.