SBIR-STTR Award

The channel catfish industry must rapidly improve efficiency. Production of hybrid catfish can increase efficiency. This proposal is intended to improve hybrid production. Four objectives: 1)optimize methods of collecting and handling blue catfish sperm; 2)adapt protocols to accommodate use of equipment developed for mammals;3)evaluate the quality of fresh and thawed sperm in the laboratory,and 4)evaluate the quality of thawed sperm in the hatchery.With the commercialscale availability of blue catfish sperm, development of new markets for genetic improvement and germplasm become possible. Production of channel catfish is an important regional and national market.Between 2001 and 2007 the number of fingerlings produced by the four major producing regions (Louisiana, Alabama, Arkansas, and Mississippi) shrank by approximately 22% during this time,due to reduced profit margins.The hybrid of channel catfish (female) x blue catfish Ictalurus furcatus (male)is a superior production animal with fast growth,superior disease resistance,and high carcass yield and could dramatically contribute towards increased efficiency and profitability in the catfish industry as it rebuilds. However, channel and blue catfish do not readily hybridize naturally and can only be produced by a labor-intensive process of manually collecting and fertilizing eggs with sperm (termed controlled reproduction or artificial spawning),Current technology allows collection of commercial quantities of channel catfish eggs, but catfish males must be killed to obtain sperm.Seasonal differences in temperature regimes are not conducive to transfer of valuable blue catfish genetic material between hatcheries because of differences in seasonal thermal profiles. Artificial spawning represents the future of genetic improvement for the catfish industry. Sperm cryopreservation is a crucial part of artificial spawning. Sperm cryopreservation, or the storage of sperm cells in liquid nitrogen (-196C), bridges the gap between the availability of ripe channel catfish eggs and blue catfish sperm for artificial spawning to produce hybrids. Our goal is to build directly on previous and ongoing research to make hybrid catfish widely available to farmers by overcoming barriers to commercialization of sperm cryopreservation for blue catfish and other aquatic species, maximizing the quality of males, and by developing production markets for sperm. This will be accomplished during Phase I through refinement of sperm collection protocols, adoption of commercialscale straw handling and cryopreservation technology, laboratory evaluation of gamete quality, and hatchery evaluation of gamete quality, and during Phase II through improving male quality and developing markets for frozen sperm and hybrid production. We believe that blue catfish sperm production and distribution through cryopreservation and shipping could evolve into a separate enterprise within the catfish industry, and that the increased availability of sperm will facilitate business development by enabling producers to focus on fingerling production for growout. OBJECTIVES: Our goal is to build directly on previous and ongoing research to overcome barriers to commercialization of blue catfish sperm cryopreservation for production of hybrid catfish fingerlings. The use of automated straw-filling equipment and bulk freezing apparatus is a powerful means for providing a weakened aquaculture sector with vital services. The proposed research represents the core of a process by which blue catfish males can be evaluated, processed, and used for commercial-scale production of hybrids. The specific technical objectives are to: 1) optimize methods of collecting and handling blue catfish sperm; 2) adapt protocols to accommodate use of equipment developed for mammals; 3) evaluate the quality of fresh and thawed sperm in the laboratory, and 4) evaluate the quality of thawed sperm in the hatchery. APPROACH: Task 1. Dilution of sperm suspensions. Sperm will be collected according to standard practices of the BLC hatchery and Dr. Tiersch's laboratory. Task 2. Purification of diluted sperm.To facilitate accurate cell quantification and to increase the longevity of sperm suspensions, we will evaluate the use of density gradient centrifugation to separate sperm cells from specimens prepared from tissue homogenates that contain somatic and other immune cells.Task 3. Establish methods of measuring sperm concentration. Task 4. Refrigerated storage. We will compare use of 50-mL centrifuge tubes, 250-mL beakers, and plastic storage bags as containers for refrigerated sperm storage. Objective 2: Technology Adaptation. To compare cooling profiles, sperm of multiple males will be collected as described for Task 1.Sperm will be added to French straws and CBS straws using the MAPI automated apparatus.Straws will be added to the bulk-freezing apparatus, and chamber cooled at 20,30,or 40C per minute. Task 6. Evaluate cryoprotectants. Sperm suspensions will be prepared in HBSS with 5, 10, or 15 percent MeOH, and sperm will be exposed to the cryoprotectant for at least 10 min prior to cryopreservation. Straws will be cooled to -80C, plunged into liquid nitrogen, and held in the liquid phase for at least 2 hours before thawing. Straws will be thawed by immersion in a 40C water bath for 8 seconds.After straws are thawed as described above, the sperm will be transferred to an equal volume of fresh HBSS, and motility will be estimated using darkfield microscopy and CASA. Task 7. Determine optimal chamber cooling rate. Cooling rate is an important parameter. Cooling rates will be compared by preparing sperm as described for Task 1, exposing them to MeOH (Task 6), adding them to CBS straws, and cooling at 20C,30C,and 40C per minute. Objective 3: Laboratory Evaluation of Sperm Quality The use of CASA to measure sperm motility objectively is our ultimate goal, and we will use Phase I as a transition period from the exclusive use of darkfield microscopy to laboratory use of CASA for quality assessment and control. Objective 4. Hatchery Evaluation of Sperm Quality. Eggs of multiple females will be fertilized with thawed blue catfish sperm. Task 14. Evaluate optimal sperm-to-egg ratios. Different sperm-to-egg ratios will be determined for fresh and thawed sperm of blue catfish males with eggs of channel catfish females

Our long-range goal is to cryopreserve blue catfish sperm reliably at a commercial scale in a cost-effective manner for high-throughput production of catfish hybrids. This effort could allow farmers to specialize in germplasm only, or hybrid fingerling production only, thereby diversifying the industry and enhancing economic opportunities for catfish producers throughout rural southeastern region of the United States, a region that is impoverished and economically suppressed. By ensuring a reliable supply of blue catfish sperm for the production of hybrids, the industry can continue to be profitable amid rising prices for fuel and feeds, thereby maintaining food security for the nation. Furthermore, our industry will continue to compete effectively in a market that has seen an influx of cheap and sometimes low quality imports. OBJECTIVES: Our goal is to build directly on previous and ongoing research to make hybrid catfish widely available to farmers by overcoming barriers to commercialization of sperm cryopreservation for blue catfish and other aquatic species, maximizing the quality of males, and by developing production markets. Baxter Land Company's (BLC) product is cryopreserved sperm for use in producing hybrid (female channel catfish crossed with male blue catfish) catfish. The farm raised catfish industry is the largest aquaculture industry in the United States and is a major contributor to the economy of the Southeast. Hybrid catfish has been well documented as a superior fish for culture by the catfish industry. The availability of sexually mature blue catfish males is the single most limiting factor in the production of hybrid catfish. The use of cryopreserved sperm dates back to the 1940's and has become commonplace for many agriculture species. Expertise and automated processing equipment developed for mammalian species are available at Louisiana State University Agricultural Center (LSUAC) for this work and has been adapted for use with fish sperm. With the commercial-scale availability of blue catfish sperm, production of hybrid catfish will increase and new markets for genetic improvement and germplasm become possible. Baxter Land Co. has a surplus inventory of blue catfish and has the pond space to increase production. Once they are assured that they can preserve enough sperm to protect their hybrid fingerling customers, they will be able to cryopreserve additional sperm for sale to hatcheries that want to produce hybrids but have no mature blue males. These hatcheries would have the additional benefits of having no acreage tied up in blue catfish production, no additional labor to daily harvest and prepare the fresh sperm, and need no vat space in the hatchery to hold the males. They would contract with Baxter during the winter for the quantity they anticipated using the next spring. If Baxter had enough inventory he would deliver the order as needed to the customer. Baxter would process additional sperm that spring with any surplus being available for the next spring. If the customer was having a better than expected year he could buy additional sperm or, if he had surplus he could return it to Baxter for a small restocking fee. The customer would have one less thing to worry about with a guaranteed supply of a known quality product delivered in a timely manner. APPROACH: The hybrid of channel catfish females and blue catfish males is a highly desirable production animal because of its superior growth rate (Yant et al. 1975), disease resistance (Dunham and Smitherman 1987), tolerance to low dissolved oxygen (Dunham et al. 1983), ease of harvest (Dunham et al. 1986), and high dress-out percentage (Brooks et al. 1982). However, channel and blue catfish do not readily hybridize in ponds using standard production techniques such as pond spawning (Tave and Smitherman 1982). The only reliable method of producing this hybrid is through artificial spawning, in which eggs are collected ("stripped") from females that have been injected with hormones that encourage ovulation, and sperm are collected from males whose testes have been surgically removed. This is severely labor intensive and requires considerable expertise with respect to handling of males and females. Prior research efforts by the Baxter Land Company (BLC), in cooperation with university and USDA researchers, have been aimed at increasing the efficiency and effectiveness of artificial spawning to offset the labor and technical requirements to produce hybrid catfish. To date, methods have been successfully implemented by which unfertilized channel catfish eggs can be collected at a commercial scale. Catfish females can be induced to yield unfertilized eggs for hybrid production in the absence of male stimulation, by injecting them with carp pituitary extract or synthetic leuteinizing hormone releasing hormone (LHRHa) (Busch and Steeby 1990) and holding them in all female groups (Bates and Tiersch 1998). ). From the product view, besides the advantage of barcodes, CBS straws use heat sealing at both ends, while traditional French straw use powder to plug both ends. Heat sealing provides more safety to the samples inside. However the heat sealing creates a storage problem: Daisy goblets were needed for storing heat-sealed CBS straws, and each Daisy goblet can hold 121 straws; while either Daisy goblets or plastic goblets on metal canes can be used to hold French straws. The metal canes and goblets required less room when samples were shipped. Thus both of these straws had their advantages and disadvantages, but CBS straws had a much higher biosecurity capability, which will be a major concern for building germplasm preservation capabilities. Thus, overall, after weighing the variables we concluded that the higher quality, more biosecure CBS straw is a better choice, despite the higher supply costs, because these costs are small in comparison to the overall costs for hybrid production. We propose to quantify the economics of cryopreservation in more detail in Phase 2