Performing Department
Aquaculture Research Station
Non Technical Summary
This project aims to apply the germplasm preservation techniques on genetic improvement of fish and shellfish. The fish to be studied include mostly aquarium fishes, especially zebrafish Danio rerio and Xiphophorus fishes, which are also important biomedical research models, and shellfish, including eastern oyster, Crassostrea virginica, which is a major species along the Gulf coast of Louisiana. The objectives will be to:1) Evaluate existing protocols for sperm cryopreservation in zebrafish, in parallel, to standardize baseline aspects for high-throughput pathways. Currently there are a large variety of protocols for sperm cryopreservation in the community. Therefore it is necessary to evaluate the current existing protocols in parallel to generate standards in each aspect of the freezing process. This would enhance the application of sperm cryopreservation in more and more research fields.2) Continue to apply sperm quality analysis to fresh and post-thaw sperm; including use of flow cytometry (FCM) for measuring membrane integrity and mitochondrial integrity and use of computer-assisted sperm analysis (CASA) for quantifying sperm motility and velocity. An understanding of sperm quality needs to be developed for assessing the susceptibility of spermatozoa to damage during cryopreservation, such as membrane integrity. Estimation of sperm quality before cryopreservation provides an opportunity to predict the outcome of sperm cryopreservation.3) Develop basic strategies for establishment of sperm repositories for Xiphophorus fishes, with streamlining of cryopreservation processing and use of pooled samples. After development of protocols for sperm cryopreservation and artificial insemination in four Xiphophorus species, the next target is to establish a sperm repository for germplasm preservation. Currently, germplasm resource programs have been established mostly for domestic livestock species. Few reports address establishment of fish germplasm repositories. Based on previous established germplasm repositories for livestock and the reproductive characteristics of Xiphophorus fishes, basic approaches for establishment of sperm repositories need to be developed.Apply sperm cryopreservation to oyster genetic improvement and breeding programs, such as production of self-fertilized, inbred lines of oysters for genome research and sperm cryopreservation of tetraploids. Inbreeding can be used to create broodstocks with genetic homogeneity. Heterosis can result from crossing of inbred lines, and is an extremely useful approach for genetic improvement. Inbred animal lines are difficult to produce and usually require many generations of sib-mating or backcrossing. Eastern oysters can change sex during the spawning season, mostly beginning life as males and changing into females as they age. This characteristic allows for fertilization of oocytes with cryopreserved sperm from the same oyster, after natural sex change, and enables creation of self-fertilized inbred lines. Triploid-tetraploid breeding technology is another promising avenue for the oyster industry. Tetraploids are valuable because of their capability to produce 100% triploid oysters by crossing with normal diploids. Sperm cryopreservation can be applied to triploid-tetraploid oyster programs to preserve sperm or larvae of valuable tetraploid oysters, and can extend the commercialization of triploid-tetraploid technology in the oyster industry through sale of frozen sperm from tetraploids, providing cost savings for maintaining tetraploids and protection of intellectual property.
Animal Health Component
0%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
The goal of this project is to apply the germplasm preservation techniques on genetic improvement of fish and shellfish. The objectives of this project will be:1) Evaluate existing protocols for sperm cryopreservation in zebrafish, in parallel, to standardize baseline aspects for high-throughput pathways;2) Continue to apply sperm quality analysis to fresh and post-thaw sperm; including use of flow cytometry (FCM) for measuring membrane integrity and mitochondrial integrity and use of computer-assisted sperm analysis (CASA) for quantifying sperm motility and velocity.3) Develop basic strategies for establishment of sperm repositories for Xiphophorus fishes, with streamlining of cryopreservation processing and use of pooled samples;4) Apply sperm cryopreservation to oyster genetic improvement and breeding programs, such as production of self-fertilized, inbred lines of oysters for genome research and sperm cryopreservation of tetraploids.
Project Methods
Objective 1 - Evaluate existing protocols for sperm cryopreservation in zebrafish, in parallel, to standardize baseline aspects for high-throughput pathways1) Sperm collection:Crushing of dissected testis andstripping will be used for sperm collection. 2) Container types:In previous publications, the containers used for packaging sperm samples to freeze varied largely, including cryovials and capillaries of varied types and volumes, and 0.25-µl French straws. Considering factors such as sample biosecurity, volume to recover after thawing, convenience of the freezing and thawing process, efficiency of storage space, and the working habits in the community, two containers - 2-ml cryovials and 0.25-ml French straws - will be used in parallel for all of the evaluations.3) Protocols for evaluationBased on the protocol published previously, protocols which use different buffer systems including Hanks' balanced salt solution (HBSS), Ginsburg with milk, and raffinose will be evaluated. The aspects to be evaluated will include equilibration time, sample volume, cooling rate, and post-thaw fertilization.Objective 2 - Continue to apply sperm quality analysis to fresh and post-thaw sperm; including use of flow cytometry (FCM) for measuring membrane integrity and mitochondrial integrity and use of computer-assisted sperm analysis (CASA) for quantifying sperm motility and velocity.Flow cytometry will be used to measure membrane integrity and mitochondrial integrity, and computer-assisted sperm analysis (CASA) will be used to quantify motility characteristics such as progressive motility, velocity, and moving trajectory. The methodology for these analyses is already established, and the required instruments are now available in the laboratory. For each fish species, strain, or line, quality monitoring by FCM and CASA will be processed for fresh sperm (before cryopreservation) and post-thaw sperm, and correlations among the tested quality factors and sperm motility or fertility will be analyzed. Possible predictive biomarkers will be selected and tested when a positive relationship occurs.Objective 3 - Develop basic strategies for establishment of sperm repositories for Xiphophorus fishes with streamlining of cryopreservation processing and use of pooled samples1) Sampling and processing. At a minimum, the major purpose of a sperm repository would be to enable reconstitution of species, strains or breeds, including specific mutated genes or chromosomes, or whole genomes, or populations. Depending on the type of reproduction and breeding efficiency, a strategy involving the following aspects should be developed. A. Development of breeding strategies for regeneration of target broods with cryopreserved sperm. A specific breeding plan should be in place, and females necessary for the breeding plan need to be identified. For example, the specific strain of X. maculatus with one chromosome mutated can be recovered through the use of a single population of wild-type females. Thus, maintenance of live females can be eliminated for most strains that have been cryopreserved.B. Identification of the minimum capacity of frozen samples necessary for regeneration of viable strains. This can be calculated based on breeding outputs, fecundity of females, and the fertility of cryopreserved sperm especially when calculated on a per-sperm basis.C. Identification of the number of individuals and their genetic relationship necessary for accession. For domestic livestock, a target of 50 unrelated animals (i.e. effective population size) has been recommended for reconstitution of particular populations to control the occurrence of inbreeding. For Xiphophorus fishes, the populations are mostly lines inbred for many generations. Therefore, consideration of the number of individuals necessary for a sperm repository should provide sufficient germplasm for recovery of live young based on the fertilization success of each individual, especially given the limited availability of sperm sample. Future work should address cryopreservation for non-lethal collection of stripped sperm to enable multiple collections for individual males.D. Thoughtful selection of packaging containers and labeling formats for frozen samples to ensure integrity of biosecurity protocols, efficient storage and use, and easy inventory. Currently 0.25-ml French straws are used for sample packaging and storage.E. Assurance of quality control of sperm samples during collection, before freezing during storage and transport (if needed), and after thawing. Motility, sperm cell structure (such as plasma membrane integrity), and fertility are the most important quality control indices. A comprehensive quality control program would include screening of individual animals before entering the repository, and would assure the quality of cryopreserved sperm.F. Standardization of procedures including sample labeling, packaging, sealing, sorting for storage, thawing, and use can assure the uniformity and reliability of the repository.2) Information systems. A sperm repository should plan to preserve and store genetic materials for decades. Therefore, a flexible and comprehensive database is necessary to manage the cryopreserved samples. The database should include the following components:G. Genetic data associated with the animals providing germplasm, such as species, line or strain, pedigree, phenotypes, and genotypes.H. Biological data including body length, body weight, testis weight, and phenotypes.I. Inventory data associated with frozen samples in the repository, such as the date of processing, sperm concentration and volume, sperm quality, straw numbers, labeling codes, storage location, and dispositions.J. Data linkage among germplasm samples with other related materials such as body tissues or cells saved for DNA and RNA analysis.Consideration also should be given to integrating comprehensive germplasm repository facilities with other end users (such as research laboratories) in order to constitute a network-based repository system. This will place greater emphasis on standardization across all aspects.Objective 4 - Apply sperm cryopreservation to oyster genetic improvement and breeding programs, such as production of self-fertilized inbred lines of oysters for genome research and sperm cryopreservation of tetraploids To produce self-fertilized oysters, sperm samples will be collected by the non-lethal method (biopsy) and cryopreserved by using established protocols in our laboratory. After biopsy sampling, male oysters will be cultured to monitor sex reversal. In the second year, eggs from sex-reversed females will be collected following standard hatchery procedures and fertilized with cryopreserved sperm from this same individual. Fertilization will be determined by counting the developing embryos divided by the total number of eggs used for fertilization. Fertilized eggs will be cultured for larval (spat) harvest by following regular hatchery practice, and offspring will be analyzed with microsatellite markers for pedigree confirmation.Development of protocols for sperm cryopreservation from tetraploid oysters will be performed based on the protocol for diploid oysters. Different extenders with different osmolalities will be tested for sperm motility activation; toxicity of different cryoprotectants will be tested at several equilibration times; and cooling rates will be screened across a temperature range (5-40 ºC/min) in relation to selected cryoprotectants. Motility, fertility, and survival of larva and spat will be collected for evaluation.