Performing Department
(N/A)
Non Technical Summary
Our long-term goal is to develop xenogenesis (method of reproduction in which successive generations differ from each other) resulting in xenogens (an organism comprised of elements typically foreign to its species) to increase efficiency of reproduction for catfish hybridization. Our overall objective is to improve xenogenesis efficiency in catfish by increasing colonization of stem cells, developing early detection techniques to identify xenogenic individuals, and evaluation of long-term reproductive performance of xenogenic catfish.Currently, channel catfish female X blue catfish male hybrid catfish embryos are produced by hormone-induced ovulation and hand stripping to obtain the eggs followed by the sacrifice of blue catfish males to obtain sperm. Artificial fertilization is then used to produce the embryos. This is labor intensive and 6 years are required for the blue catfish males to reach sexual maturity for a singel use. Xenogenesis would be advantageous and protocols have been developed that should allow commercialization with the optimization that will result from the current project.For this optimization, we plan to improve purification and propagation of donor stem cells and compare the effectiveness of injection of fresh mixed donor cells with injection of pure cultured cells for producing xenogenicindividuals, to compare surgical biopsy with DNA analysis and ELISA for donor proteins found in the circulatory system for early detection of xenogenic individuals and to evaluate the multiple year reproductive performance and longevity of xenogenic catfish and the capacity of males to mate multiple females in the same year.Because of the performance of hybrid catfish, a positive outcome will lead to greater output of hybrid catfish embryos. This will lead to greater production, efficiency, and sustainability in the catfish industry, and enhance the quality of rural life. Other powerful genetic and conservation applications are possible.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Our long-term goal is to develop xenogenesis (method of reproduction in which successive generations differ from each other) resulting in xenogens (an organism comprised of elements typically foreign to its species) to increase the efficiency of reproduction for catfish hybridization, and secondarily for other applications such as to increase the efficiency of reproduction in difficult to spawn species such as blue catfish.Our overall objective is to improve xenogenesis efficiency in catfish by increasing colonization of stem cells, developing early detection techniques to identify xenogenic individuals, and evaluation of the long-term reproductive performance of xenogenic catfish.Specific objectives:1) Develop technology to purify and propagate donor stem cells and compare the effectiveness of injection of fresh mixed donor cells with injection of pure cultured cells for producing xenogenic individuals2) Compare surgical biopsy with DNA analysis and ELISA for donor proteins found in the circulatory system for early detection of xenogenic individuals3) Evaluate the multiple-year reproductive performance and longevity of xenogenic catfish and the capacity of males to mate multiple females in the same year
Project Methods
Immature testes or oogonia will be collected from blue catfish euthanized with 300 ppm tricaine methanesulfonate (MS-222) and disassociated.The fresh cell isolate stored on ice or in short-term cultureuntil use. Purification and propagation of stem cells will be accomplished by evaluating variables found by Shikina et al. (2008), Ryu et al. (2016), and Takahashi et al. (2020) that affected the successful purification and propagation of stem cells for rainbow trout and Siberian sturgeon.Differential platingwill be used to purify the stem cells.Blue catfish-specific germ cell and SSC gene expression will be measured using RT-PCR of sycp3, neurogenin3, Smad5, and Kit genes. Mixed fresh cells and purified cultured stem cells will be compared for colonization of donor cells.Rate of xenogenesis will be determined utilizing blue catfish and channel catfish-specific DNA markers and by determining fertility.Surgical biopsy with DNA analysis will be compared toELISA for donor proteins found in the circulatory system for early detection of xenogenic individuals.The multiple year reproductive performance and longevity of xenogenic catfish and the capacity of males to mate multiple females in the same year will be evaluated. Xenogenic males will be mated with normal channel catfish females and white catfish xenogenic males will be mated with white catfish xenogenic females.Parameters evaluated will include spawning percentage, fertilization rate, fecundity, hatching % and fry output (fry/kg female body weight and fry/male).Body weight of females before spawning, male body weight, egg number, fertility at 36 hr, estimated hatch 12 hr before hatching, hatch %, and fry/kg will be determined for each pairing. Total hatched fry will be determined for each treatment group.Males that spawn will be given an opportunity to spawn a second female. After spawning is exhausted or the spawning season ends, the fish will be returned to earthen ponds at 2,400 fish/ha to prepare for spawning the next year. At the beginning of each spawning season, the fish will be harvested, survival determined, and attrition surmised. Then the spawning procedures above will be repeated.Fish that produce egg masses that do not hatch will be sacrificed, their gonads examined, and blue catfish, channel catfish and white catfish fst and hamp genetic markers assayed to determine if the fish were xenogenic triploids or normal triploids.