Louisiana Agricultural Experiment Station research is conducted through approved projects that meet specific criteria. Each project is assigned a CRIS (Current Research Information System) number that is used to track the progress of the research and to report the project status to the Louisiana Agricultural Experiment Station and USDA. CRIS project reports from other states are available from the online archives in Washington, D.C. The current projects for the Aquaculture Research Station are listed below. Details of each project are available by clicking on the project number.
LAB 93865 - Improving Growth and Feed Efficiency in Warmwater Aquaculture by Dr. Robert Reigh
NON-TECHNICAL SUMMARY: Anti-phospholipase A2 (aPLA2) has growth promoting effects in some fishes. Its effects on channel catfish are not known. The coppernose bluegill has potential to become a new aquacultural food commodity, but cost-effective production methods for this species have not been developed. This project will determine if adding aPLA2 to channel catfish diets increases catfish growth. The project will also measure the effectiveness of different stocking densities on the growth and production cost of coppernose bluegill raised to a size of 0.5 kg in ponds.
OBJECTIVES: #1. Determine the efficacy of anti-phospholipase A2 (aPLA2) as a growth promoter for channel catfish. A laboratory trial will be conducted at the Louisiana State University Agricultural Center under controlled environment in replicated aquaria. Fish will be fed diets containing various levels of aPLA2 once daily to apparent satiation. The purpose of this trial is to determine the effects of aPLA2 on weight gain, feed efficiency, body composition, and to evaluate the biological and economic value the aPLA2 as a potential additive for channel catfish diets. Pond production trials may follow if laboratory results warrant. #5. Develop cost-effective methods for the production of coppernose bluegill as a new aquacultural food commodity. A series of experiments will be conducted in replicated earthen ponds at the Louisiana State University Agricultural Center to determine (1) appropriate stocking densities for the production of food-size (500 g) coppernose bluegill within a one-year growing period, (2) appropriate feeding rates for intensively cultured coppernose bluegill, (3) diet cost associated with the production of food-size coppernose bluegill, and (4) processing characteristics (e.g., dressing percentage and fillet yield) and body composition of coppernose bluegill raised to a weight of approximately 500 g. Results will be used to develop recommendations on stocking densities and feeding rates that are appropriate for the production of food-size coppernose bluegill as an aquacultural commodity.
APPROACH: In Objective 1, experimental diets containing graded levels of anti-phospholipase A2 (aPLA2) will be used in a laboratory feeding trial to determine the effectiveness of aPLA2 as a growth promoter for juvenile channel catfish. In Objective 5, stocking densities and feeding rates will be evaluated in a pond feeding trial with coppernose bluegill to determine the optimum density for cost-effective production of food size (0.5 kg) coppernose bluegill during a single, 8-month growing season.
PROGRESS: 2007/01 TO 2007/12
OUTPUTS: The project's first production season ran from early spring through the winter of 2007, so no outputs to target audiences were available in 2007. PARTICIPANTS: Participants included Dr. M. Li, Research Professor, Delta Research and Extension Center, Mississippi Agricultural and Forestry Experiment Station; M. Williams, research associate, Aquaculture Research Station, Louisiana State University Agricultural Center; two graduate students (C. Gothreaux and B. Patro); and several undergraduate student workers. TARGET AUDIENCES: Target audiences of the project are members of the scientific community and aquaculturists interested in production of coppernose bluegill as a food commodity or for use as a sport fish in recreational ponds. These audiences will be reached through peer-reviewed publications, trade publications, presentations at scientific meetings and industry/trade meetings, and in-person consultations. Two graduate students and several undergraduate students have gained knowledge of, and experience with, the development of aquacultural production practices for coppernose bluegill as a result of their work on this project. PROJECT MODIFICATIONS: None in 2007. In 2008, coppernose bluegill will be stocked in floating cages to eliminate bird predation and to prevent spawning in the ponds, which compromised the results of the 2007 production trial.
IMPACT: 2007/01 TO 2007/12
The first year of coppernose bluegill production was completed in late 2007. Survival of fish among all treatment groups was low. It is likely that this was due, at least in part, to predation by cormorants that overwinter in high numbers in the vicinity of the Aquaculture Research Station until late spring every year; however, the actual cause of poor fish survival in 2007 is not known. Natural spawning of coppernose bluegill also occurred in about one-quarter of the ponds during the 2007 production season, as evidenced by the presence of young-of-year fish and numerous nest cavities in the bottoms of some ponds when they were drained. These problems caused high variability in population densities among ponds, which were not treatment related and which prevented accurate measurement of the treatment (i.e., stocking density) effects that were being evaluated in the 2007 production trial. In 2008, we will stock the fish into two, 1-m x 2-m floating cages in each pond to protect them from predatory birds. Also, we anticipate that the cage culture approach will eliminate spawning by preventing fish from accessing the pond bottom. We expect that these measures will solve the problems we observed in 2007, which will allow us to obtain accurate information on the production characteristics of cage-cultured coppernose bluegill and evaluate the feasibility of producing this species as an aquacultural commodity.
LAB 93891 - Improving Production Efficiency in Procambarid Crawfish Aquaculture by Dr. Robert Romaire
NON-TECHNICAL SUMMARY: The red swamp crawfish and the white river crawfish are a valuable seafood resource in Louisiana, representing the only large-scale, commercially successful crustacean aquaculture enterprise in North America, with over 130,000 acres, 1,200 farmers, and an estimated economic impact in Louisiana that exceeds $250 million. The extensive method of crawfish cultivation, although profitable for many producers, presents a challenge because yield and quality (size) is not consistent from year to year. A significant contributor to production variability appears to be reproduction. Field evidence indicates some crawfish crop failures may be associated with crawfish exposure to poor water quality or accidental introductions of ecotoxicants. Crawfish harvested early in the production season receive the highest prices because of the low availability of crawfish at that time. For many producers, early season production of crawfish is critical to profitability. Flooding ponds prior to the currently recommended flooding date of early October may have potential to increase the supply of crawfish during low production months. Bait and labor are among the highest expenses in crawfish aquaculture, and more than any other management factor, they can most easily be managed by the producer to maximize harvest and minimize cost. This project addresses several priority research areas of crawfish aquaculture identified by LSU AgCenter aquaculture researchers, extension specialists and crawfish producers. These areas include assessment of the effects of climatic and physicochemical stressors on reproduction, survival, growth, and production; evaluation of the potential to produce early season and out-of-season crawfish when prices are highest; and refinement of harvesting practices that increase efficiency by reducing labor and bait cost. The anticipated impact of this project is that improved management guidelines and management alternatives for commercial freshwater crawfish aquaculture producers in Louisiana will be developed and extended to the crawfish farming community in outreach programs to improve their production efficiency and increase profitability of their farming operations.
OBJECTIVES: The goal of this project to provide improved management guidelines and management alternatives to commercial freshwater crawfish aquaculture producers in Louisiana in order to improve production efficiency and increase profitability of their farming operations. The objectives of this project include assessment of the effects of climatic and physicochemical stressors on reproduction, survival, growth and production of crawfish; evaluation of the potential to produce early season and out-of-season crawfish when supply is low and prices are highest; and to refine crawfish harvesting strategies to reduce production cost, with emphasis on determining crawfish movement patterns to traps as influenced by bait type and water temperature. Expected deliverables from this project include improved management procedures for crawfish producers that will be conveyed via technical scientific journal articles to the scientific community, and extension publications and newsletters on crawfish aquaculture best management practices that minimize production cost and increase profitability while minimizing any negative effects on the environment. New findings from this project will be dissemination at crawfish producer meetings and workshops, and via web-based delivery systems from the Louisiana State University AgCenter public website.
APPROACH: The effects of stress on reproduction will be simulated by exposing mature females to stressing factors including chronic hypoxia and food deprivation. Mature females will be placed in artificial burrows and survival, time to spawning, spawning percentage, and egg hatch determined. Pond soils will be analyzed for texture and permeability, and chemical composition of soil and water will be analyzed for mineral content. Historical data on precipitation, air temperature, and soil moisture will be analyzed with principal-components analysis to determine factor scores, which will be regressed on crawfish reproductive indices to determine the relationship between environmental factors and crawfish reproduction. Juvenile red swamp and white river crawfish will be exposed to four oxygen concentrations (10, 20, 50 and 90% of oxygen saturation) at each of two water temperatures (20 and 28 C) for up to 120 days. Response variables to be measured on each species of crawfish will include growth rate, survival distribution, percent survival, inter-molt time duration, and number of molts. The effects of water temperature on growth and molting frequency of red swamp and white river crawfish will be determined by exposing juveniles (10-12 mm TL) to 10, 15, 20, 25, and 30 C for up to 120 days. The acute and chronic effects of selected toxicants on red swamp and white river crawfish will be determined in the laboratory and in field mesocosms. The 96-h LC50 will be determined for hydrogen sulfide and selected agricultural pesticides. Experimental ponds will be randomly assigned to the following flooding regimes: 1 May, 1 June, and 1 July, 1 August, 15 August, 1 September, 15 September, and 1 October, to assess the potential for out-of-season and early-season crawfish production. Crawfish will be sampled monthly to monitor growth and relative density. Crawfish will be harvested with baited traps. Data will be analyzed with the analysis of variance to determine the effects of flooding date on crawfish yield and size at harvest. The capture efficiency of various trap mesh x trap density combinations will be evaluated from crawfish marking and recapture experiments. Marked market-size crawfish will be released into large experimental ponds, and the crawfish will be harvested 2 or 3 days per week after either 24 or 48 hour trap soak times. The number of recaptured marked crawfish, and the overall number of crawfish caught will be recorded at each harvesting event. Sequential crawfish mark and recapture samplings will provide data on the portion of harvestable size crawfish standing stock that is removed (harvest efficiency) as a function of mesh size, trap density, bait type, and water temperature. Findings of these research activities will be delivered at professional meetings, and presentations will be made to crawfish farmers in Louisiana at educational training meetings and workshops.
LAB 93892 - Development and Evaluation of Production Methods for Promising Native Aquatic Species by Dr. Greg Lutz
NON-TECHNICAL SUMMARY: Louisiana has historically supported one of the most diverse aquaculture industries in the nation, including species and products such as crawfish, catfish, alligators, oysters, tilapia, baitfish, hybrid striped bass, redfish, soft shell crawfish and crabs, various ornamental fishes, baby turtles, and a variety of freshwater game fishes. However, several sectors of Louisiana aquaculture production have been in decline in recent years. Many additional aquatic species could be grown in Louisiana, including a number of species that occur naturally in Louisiana's coastal and inland waters. New species, both marine and freshwater, are continually being evaluated and adapted for aquaculture production throughout the world. Many candidate species present specific problems that complicate commercial development, but once basic life history and production requirements have been worked out, most remaining constraints relate to marketing, financing or regulatory considerations. There are many opportunities to utilize native aquatic species, especially as alternatives to exotic species, whether to fill specific roles in polyculture strategies or as monoculture crops for human consumption, bait use or stocking in private waters. Progress in developing culture methods for these species should bolster existing aquaculture industries in Louisiana and potentially result in new industries.
OBJECTIVES: The goal of the project is to capitalize on the potential of native aquatic species to create or enhance economically viable aquaculture industries in Louisiana and elsewhere. Objectives are to eliminate barriers to the enhancement of native aquatic species culture by defining production characteristics, adapting production systems and addressing potential adverse ecological impacts, and to generate data that will identify economic and marketing opportunities and constraints associated with culture of native aquatic species. The proposed work will generate much-needed baseline data and production recommendations, as well as captive stocks, to allow researchers and producers the opportunity to evaluate the feasibility of native aquatic species for bait, polyculture, or direct consumption. Results and products will be disseminated to the academic community, regional stakeholders and would-be producers via presentations, direct consultation, and educational workshops and materials.
APPROACH: Key questions will involve establishing biologically and economically optimum environments for reproduction and grow-out. A number of outdoor tanks (nine tanks of approximately 6 m diameter by 1 m depth, and 40 tanks of approximately 2.5 m diameter by 1 m depth) will be utilized as mesocosms for spawning, nursery culture and grow-out trials. It is anticipated that small ponds will also be used with fresh or brackish water for maturation studies as required. An enclosed room for environmental induction of spawning will be utilized for certain species, such as Atlantic croaker and Freshwater Drum. Individual project components will be evaluated directly as relates to attainment of captive spawning, successful larval culture, and similar metrics. As culture methods are developed for species with potential for commercial importance they will be presented to stakeholders through a variety of means, but impact will ultimately be determined by the extent to which new economic activity is generated through the culture and commercialization of these native aquatic species.
LAB 93904 - Spawning, Germplasm, and Genetic Improvement in Aquatic Species by Dr. Terry Tiersch
NON-TECHNICAL SUMMARY: Worldwide, more than 200 species of finfish and shellfish are cultured. Within the United States, aquaculture has become an established sector in agriculture over the past 20 years. Various species are cultured commercially in Louisiana, including channel catfish and the hybrid cross with blue catfish, red drum, striped bass and its hybrids, crawfish, eastern oyster, and alligators. Aquaculture has matured into several major industries in the United States, with strong demand for these products. These industries provide employment and alternative income opportunities for farmers and rural communities devastated in Louisiana from the hurricanes of 2005, provide an increased and reliable supply of fish and shellfish products, and assist in reducing the U. S. trade deficit. However, production aquaculture, including that in Louisiana, has recently been presented with significant challenges due to rising costs of feed and fuel, and increasing global competition. This has created urgent needs for increased efficiency, new product forms, and eventually a shift from production to other sectors of aquaculture. This proposal addresses these needs by providing a structure for genetic improvement to increase efficiency, laying the groundwork for creating new markets in germplasm, and enabling a shift from farm production to creation and marketing of genetic improvement. Most research in aquaculture has been directed toward production, nutrition, disease control, and water quality. Less emphasis has been placed on genetic improvement. Important traits such as growth rate, disease resistance, and processing characteristics can be improved and make aquatic animals more profitable to raise. Because of increased production costs, genetic improvement will be necessary to maintain and increase profit in commercial aquaculture. Genetic research offers solutions to problems encountered in aquaculture. For example, the development of improved stocks is considered to be of prime importance to the catfish and oyster industries. Significant gains have been realized in the poultry and livestock industries through genetic research, and considerable potential exists for improvement in aquatic species, given that in many cases, commercial producers are using unselected wild stocks for production. The work in this project is intended to address the problem of inefficient or slow progress in genetic improvement. This research would form a 5-year project aimed at creation of interlocking sets of methods to accelerate genetic improvement by enhancing broodstock selection and conditioning, reproduction, and the storage and recovery of superior genetic material in aquatic species. This would assist seed stock producers by increasing control of broodstock management, and would assist aquaculture industries overall by allowing faster development and increased availability of improved stocks.
OBJECTIVES: This project is intended to establish new capacity for genetic improvement in aquatic species. Production of improved animals through selective breeding would be realized through application of this work by industry or the public sector. The objectives are to: 1) Establish, refine, optimize, and adapt induced spawning and specialized breeding techniques such as hybridization and chromosome set manipulation for genetic improvement. 2) Establish, refine, optimize, and adapt gamete quality evaluation, cryopreservation techniques, and germplasm storage. 3) Establish, refine, optimize, and adapt the use of geothermal water for broodstock conditioning, and the use of ultrasound for evaluation of gonadal condition. The expected outputs are activities including conducting and analyzing experiments; assessment of new technologies; facilitating adoption in the industry of improved methods, and teaching and mentoring of undergraduate and graduate students. We expect to organize and participate in events such as symposia and conferences addressing this work, and to provide training for researchers and producers. We expect to produce products including new fundamental and applied knowledge; new uses for equipment; collaborations fostered by the project; physical collections of animal germplasm, and establish new methods and techniques. We expect to disseminate these outputs through interactions with cooperators, researchers, and stakeholders, and by publication of results in various outlets.
APPROACH: Spawning will be performed using groups of mature females that will be induced to undergo final gonadal maturation and ovulation by injection of gonadotropic hormones. Fish will be monitored after injection until stripping of eggs. Parameters including size, quantity, and color of eggs, ease of stripping, presence of blood or mucus, and spawning latency will be evaluated. Embryos will be incubated in tanks with recirculated water. Sperm motility and concentration will be used to determine the optimal ratio of sperm for fertilization of eggs. These methods will be used for crossbreeding and for production of interspecific hybridization, facilitated by use of cryopreserved sperm. With control of temperature and timing after fertilization, these treatments can be used to manipulate the number and sources of chromosome sets in the offspring. Refrigerated storage will be studied to extend the working lifetime of gametes. We will shift from cryopreservation research to high-throughput (commercial scale) application. Sperm will be collected, diluted in extender solutions, and frozen with different cryoprotectants in a computer-controlled freezer. Cryopreservation will be evaluated by sperm motility and fertilization trials. Samples will be collected to expand the germplasm repository at the Aquaculture Research Station. Records will be maintained on origins and traits, and procedures will be developed to process samples from large numbers of males. We will work with commercial cooperators to scale-up techniques for transport, refrigerated storage, and cryopreservation of sperm. We will use automated equipment to process large numbers of plastic straws for cryopreservation. For reproductive conditioning we will use a geothermal well to deliver heated water to 0.12-acre ponds. We will use mixed-sex stocking to maximize egg production, and will also use all-female stocking. Ponds will be heated during January through April, and after 1 week, the ponds will be checked every 3 days for males preparing spawning sites or the presence of eggs. Unheated control ponds will be stocked and monitored in the same way. Spawns will be brought into the hatchery and be incubated and hatched using standard methods. Floating cages will be placed in ponds to hold broodstock males and females separately. These fish will be evaluated for induced spawning in the laboratory. We will improve control and replication by utilizing outdoor pools. The use of greenhouse enclosures will be evaluated to reduce costs. We will study ultrasound monitoring of ovarian maturation to enable quantitative classification of female broodstock for induced spawning. We will establish and refine methods to use this technology to rapidly screen large numbers of females for induced spawning in commercial settings. Ultimately we seek to shift commercial application to all-female systems to maximize egg and fry production, with the sperm component provided by use of cryopreserved samples from superior males.
LAB 93936 - Understanding and Improving Reproductive Biology of Cultured Aquatic Species by Dr. Chris Green
NON-TECHNICAL SUMMARY: Objective 1. The current research needs of each potential baitfish species are primarily centered on reproductive success, practical propagation techniques, and economic feasibility. Development of practical aquaculture techniques involving appropriate sex ratios, stocking densities, salinity and fecundity will be needed for the fat sleeper and other potential species. Environmental parameters during reproductive periods will be replicated experimentally with reproductive output quantified to identify ideal spawning conditions. To better understand the effects of critical environmental variables on basic reproductive endocrinology, steroids involved in gonadal maturation and gonad histology will be examined in males and females. Objective 2. Indices of condition as they relate to post stocking survival and reproduction have not been established for several species. Mortality of both invertebrate and vertebrate aquaculture species through disease is the most significant factor contributing to economic loss within the aquaculture industry (Meyer 1991). Quantification of stressors at critical periods could be developed in tandem with reproductive conditional indices with crawfish used as a model decapod crustacean. Several novel methods for quantifying stress and individual health have been developed from research on lobsters (Evans and Jones 1999). Although many of these measures can be assessed quickly and are relatively inexpensive, there is a clear need to research these biomarkers in crawfish and relate their responses to survival and reproduction (Adegboye et al. 1972; Paterson et al. 1999). Objective 3. Examinations evaluating male hormone profiles and spermatogenesis of aquaculture species will provide a better understanding of the mechanisms needed to assist in spermatozoa maturation, whether through hormonal, nutritional, or environmental manipulations. The development of baseline data to demonstrate the coordination of sex hormones and testes maturation with respect to natural and altered temperature regimes would represent a great advance in optimizing the conditioning of male channel catfish and blue catfish. By developing an understanding of the events within these species, managers could alter production time to better utilize males as an important reproductive resource. Altering temperature regimes within the period of natural spermatogenesis will assist in managing both channel catfish and blue catfish males for coordination of spawning and improvement of reproductive capacity among these fishes and their hybrids. It is also possible that future research on hybrid production could utilize exogenous manipulations of the hypothalamic-pituitary-gonadal axis to optimize male gamete maturation and streamline hybrid catfish production.
OBJECTIVES: 1. Describe/evaluate critical parameters that influence reproductive success of marine baitfish and other aquatic species to develop practical propagation techniques. 2. Describe mechanisms by which reproduction and maintenance of homeostasis are coordinated in order to improve both reproduction and survival in crustaceans. 3. Improve methods for broodstock conditioning by manipulation of biotic and abiotic factors to maximize reproductive efficiency and improvements in gamete quality for warmwater finfish.
APPROACH: The development of an aquaculture industry centered on the culture of marine baitfish represents a possible new economic market within Louisiana and the surrounding coastal region and has been identified by the LAES and LA SeaGrant as a priority and as such, considerable research will be needed in order to accomplish this goal. At the moment, marine baitfish are limited to collections from the wild and many species preferred by anglers are only seasonally available.There are a number of candidate species that have potential as marine baitfish such as fat sleeper Dormitator maculatus and cocaho minnow Fundulus grandis. From an understanding of propagation techniques and reproductive physiology, research could move towards refinement of parameters necessary to hatch viable fry and raise juveniles for the eventual establishment of a marine baitfish industry within the state. The ontogeny of digestive enzymes in larval stages of these fishes could reveal dietary limitations at the first instance of exogenous feeding. As an additional advantage, the techniques and materials developed with these investigations can be implemented to further understand digestive physiology and egg-yolk utilization in other aquaculture species. Stress is an ever present attribute of animal culture. Indices of condition and reproduction are needed to better elucidate its effects on animals within aquaculture operations. Some physiological responses to stress in both vertebrate and invertebrate aquatic organisms are similar, such as changes in plasma glucose, lactate, protein, and osmolality. Several of these variables could be measured via handheld devices and therefore be refined for use at aquaculture facilities as independent measures of condition. Multiple assays used together to form an index of condition could be utilized for several taxa. Specifically, crawfish could be used as a model for such an index in decapod crustaceans. It would be possible to expand or modify the index to accommodate numerous species. Efficiency in reproduction of several aquaculture animals is a limiting factor that contributes to a shortage of animals to be reared and available for market. It is therefore imperative to increase the sustainability of aquatic species production, beginning with the efficiency of broodfish reproduction. The role of the endocrine system can be examined in order to observe critical variables in coordinating gonadal development and egg maturation. Reproductive investment in terms of egg composition has been implicated in fry survival and could be investigated as a management strategy for several brood fish species. Hormone production as it relates to gonadal maturation is not well understood in several aquaculture species and understanding these systems would be critical in later improvement of gamete quality through changes in feed components, temperature and photoperiod manipulations, and therapeutant treatments.
LAB 93954 - Preservation and Improvement of Genetic Resources of Fish and Shellfish by Dr. Huiping Yang
NON-TECHNICAL SUMMARY: Aquarium fishes, characterized by small body size (< 5 cm), account for the bulk of the ornamental market within aquaculture, and are the major fish models for biomedical research. Currently, the most widely used fish models are zebrafish (Danio rerio), medaka (Oryzias latipes), and live-bearing fishes of the genus Xiphophorus, which are also major species in the ornamental trade market. With the development of genomic research and extensive studies using these fish models, tens of thousands of specific strains and lines have been created, and are currently housed worldwide as live animals in resource centers. In addition, there are a number of new technologies becoming available, such as generation of transgenic reporter lines, knock-out of genes, viral and transposon based insertions, and enhancer or gene trap strategies. Thus, in the near future the number and variety of these model strains will rapidly increase. Preservation of the genetic resources of these and other valuable fishes presents significant and urgent challenges. Gamete or embryo cryopreservation is an approach to address these challenges. Triploid oysters possess three sets of chromosomes in their body cells rather than the normal two sets possessed by diploids. Compared to diploids, triploid oysters possess desirable production traits such as superior growth, reproductive sterility, and improved meat quality during the summer months. Tetraploids are organisms that possess four sets of chromosomes in their body cells, and are valuable because of their capability of production of 100% triploid by crossing with normal diploids. Therefore, tetraploid production and sperm cryopreservation are key approaches for the application of triploid-tetraploid in the oyster industry.
OBJECTIVES: This state project includes two major research goals: 1) establishment of sperm repositories for aquarium fishes, especially zebrafish (Danio rerio), medaka (Oryzias latipes) and Xiphophorus fishes, which are important biomedical research models, and 2) production of tetraploid eastern oysters Crassostrea virginica and protocol development for tetraploid sperm cryopreservation. Based on the current research status, the objectives of this state project will be: 1) Standardization of the protocol for sperm cryopreservation in the zebrafish, medaka, and Xiphophorus fishes. 2) Evaluation of sperm quality, including use of flow cytometry (FCM) for measuring membrane integrity and mitochondrial integrity, use of computer-assisted sperm analysis (CASA) for quantifying sperm motility, velocity and trajectory, and detection of the changes in protein profiling in sperm after cryopreservation. 3) Development of high-throughput methods for rapid processing and cryopreservation of samples including the use of automated or semi-automated sample handling systems for straw labeling, filling, and freezing in conjunction with an integrated database. 4) Production of tetraploid eastern oysters, and development of protocols for sperm cryopreservation of tetraploid oysters for future establishment of an oyster sperm repository for the triploid oyster industry in Louisiana.
APPROACH: Objective 1. Standardization of the protocol for sperm cryopreservation in zebrafish, medaka, and Xiphophorus fishes: The process will include standardization of each step within the protocols, especially for sperm collection, estimation of sperm concentration, rules and agreements for labeling and coding, and refrigerated storage and sample transport among locations. Objective 2. Evaluation of sperm quality: Flow cytometry (FCM) will be used to measure membrane integrity and mitochondrial integrity, computer-assisted sperm analysis (CASA) will be used to quantify motility characteristics such as progressive motility, velocity, and moving trajectory. The methodology for these two analyses is already established, and these two instruments are now available in the laboratory. For each fish species, strain, or line, the quality monitoring by FCM and CASA will be processed for fresh sperm (before cryopreservation) and post-thaw sperm, and the correlations among the tested quality factors and sperm motility or fertility will be analyzed, and possible predictive biomarkers will be selected and tested when a positive relationship occurs. Objective 3. Develop high-throughput methods for rapid processing and cryopreservation of samples including the use of automated or semi-automated sample handling systems for straw labeling, filling, and freezing in conjunction with an integrated database: To develop the high-throughput method for rapid processing and cryopreservation of samples, a series of technologies need to be identified. For each species, systematic evaluations will be performed to optimize the conditions for: 1) sample shipping and refrigerator storage; 2) dilution of the sperm sample in extender; 3) materials and sizing for packaging of samples; 4) sample loading volume in each packaging straw; 5) maximum use of thawed samples for fertilization; 6) standardization of cryopreservation protocol; and 7) analysis of correlations among the tested factors. The problems involved in each step will be identified. A semi-automated sample handling system (MAPI, Cryo Bio System) will be used for straw labeling, filling, and freezing. A high bio-security straw made of new materials will be used for packing samples. Objective 4. Develop reliable protocols for cryopreservation of tetraploid eastern oyster Crassostrea virginica, and test the high-throughput protocol for establishment of a large-scale tetraploid oyster sperm repository for the triploid oyster industry in Louisiana: The approaches for developing cryopreservation protocol for tetraploid sperm will be: 1)Estimation of sperm motility activation; 2)Toxicity of cryoprotectants to sperm cells; 3)Estimation of cooling rate for sperm cryopreservation; and 4)Fertility estimation of cryopreserved sperm. Data Collection and Statistics: Data will be analyzed by use of statistical software (SYSTAT version 12, Systat Software, Inc., Chicago, IL). Normality of data will be tested before performing analyses, and percentage data will be arcsin transformed before analysis. For most analyses, it is anticipated that T test, Chi-square, ANOVA, and correlation analyses will be used for data analysis.