Source: AUBURN UNIVERSITY submitted to NRP
INCREASING COLONIZATION OF STEM CELLS AND EARLY DETECTION OF XENOGENESIS IN CATFISH WITH LONG TERM REPRODUCTIVE PERFORMANCE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030459
Grant No.
2023-67016-39786
Cumulative Award Amt.
$650,000.00
Proposal No.
2022-07879
Multistate No.
(N/A)
Project Start Date
May 1, 2023
Project End Date
Apr 30, 2027
Grant Year
2023
Program Code
[A1211]- Animal Health and Production and Animal Products: Animal Reproduction
Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849
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%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3010210103030%
3030210108030%
3010210108130%
3040210104010%
Knowledge Area
303 - Genetic Improvement of Animals; 304 - Animal Genome; 301 - Reproductive Performance of Animals;

Subject Of Investigation
0210 - Water resources;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology; 1081 - Breeding; 1080 - Genetics;
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.

Progress 05/01/24 to 04/30/25

Outputs
Target Audience:scientists, farmers, students, biotechnologists, the public Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?8 graduate students, 2 undergraduate students, and 3 post-docs received training How have the results been disseminated to communities of interest?Results were disseminated via manuscripts, seminars and presentations at conference as well as personal communication with farmers and biotechnologist What do you plan to do during the next reporting period to accomplish the goals?We will continue working on all objectives.

Impacts
What was accomplished under these goals? The maintenance of spermatogonial stem cells was investigated in this work using short-term culture prior to transplantation. The SSCs were incubated with and without 5% CO2 at 24-30°C temperature range and with 0-75uM of Y-27632, a specific inhibitor of Rho kinase (ROCK I). Spermatogonia 72 hour in vitro survival was best at 28°C, utilizing 50-75 uM ROCK I with 5% CO2 (p=0.001) for 72 hours.Good progress was made towards long term culture of catfish stem cells. One-hundred % of catfish biopsied with surgery to determine if they were xenogenic survived. Several treatments, mostly those involved with utilizing primordial germ cells or low numbers of spermatogonial stem cells to produce the original xenogens had low or no success in producing donor progeny. However, implantation of 80,000 donor gonadal cells on days 3- 5 post-hatch to create xenogenic brood stock resulted in 80.0%% of these spawns producing donor progeny. Putative xenogenic brood stock produced by injecting stem cells 0-2 days post hatch did not become gravid. The fecundity, and hatch percentages and fry/kg of spawns producing donor progeny were not different (P>0.05) than spawns producing host progeny. Xenogenic white catfish produced both channel catfish and blue catfish progeny, and xenogenic channel catfish produced blue catfish progeny. Spawning rates and fry output of normal channel catfish females mated with xenogenic males was the same as control matings. In the previous year, no xenogenic males repeat spawned but in the current year almost half of xenogenic males that spawned once repeat spawned.

Publications

  • Type: Other Journal Articles Status: Published Year Published: 2025 Citation: Pottle, K.B., D. U. Hettiarachchi, M. Shang, B. Su, J. Al-Armanazi, J. Wang, M. Soman, H. Dilawar, I. A.E. Butts and R. A. Dunham. 2025. Impacts of host, cell density, and timing of injection on efficiency for xenogen production in ictalurid catfish. Marine Biotechnology:27:90 https://doi.org/10.1007/s10126-025-10466-5


Progress 05/01/23 to 04/30/24

Outputs
Target Audience:scientists, farmers, students, biotechnologist Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?8 graduate students, 2 undergraduates and 3 post docs received training. How have the results been disseminated to communities of interest?Papers have been written and are in review, papers published, presentations and posters have been presented at symposia and campus student competitions. What do you plan to do during the next reporting period to accomplish the goals?We will continue addressing the objectives

Impacts
What was accomplished under these goals? The present study compared effectiveness of fresh vs. cultured oogonial (OSCs) and spermatogonial (SSCs) stem cells for transplantation. Triploid channel catfish fry were injected at 5 days post-hatch (DPH) with PKH26 labelled fresh or cultured OSCs or SSCs. Growth and survival of recipient fish were assessed at 45 and 90 DPH, while donor cell colonization was quantified using PKH26. PCR and fluorescence images were used to determine percent xenogens. No significant difference in fry growth were observed between fresh and cultured treatments at 45 and 90 DPH. However, fluorescence imaging revealed significantly higher cluster area in cultured treatments compared to fresh treatments. Cluster areas significantly increased from 45 to 90 DPH in both fresh and cultured treatments. Cell area at 45 DPH was significantly higher in cultured treatments than fresh treatments, while no difference was detected at 90 DPH. PCR analyses revealed a higher proportion of xenogens in recipients injected with cultured cells (85.7%) compared to fresh cells (83.3%). Our findings demonstrate that cultured stem cells perform comparably to fresh stem cells, offering a promising approach for future cell transplantation. The effect of density of unsorted gonadal cells (80,000, 100,000, or 120,000 cells/fry) from blue catfish (BGCs) injected into triploid channel catfish surrogatesand BGCs or channel catfish (CGCs) into triploid white catfish (Ameiurus catus) surrogates on proliferation and colonization rates in surrogates injected at 4-, 5-, or 6-days post-hatch (DPH) was evaluated. At 45 and 90 DPH, survival and size of surrogates, and colonization/proliferation of donor cells (cell area <150 μm2 and cluster area >150 μm2) were evaluated. Survival and size of all surrogate species were not impacted by cell density or donor. All surrogate species injected with 100,000 cells/fry had larger cluster cell areas than those injected with 80,000 cells/fry. White catfish surrogates with BGCs and CGCs had larger cell areas when injected with 100,000 cells/fry than those injected with 80,000 cells/fry. Both cell and cluster area increased by 90 DPH for all surrogates. PCR and PKH26 red fluorescence analysis confirmed that >89% and >86% of surrogates were positive xenogens at 45 and 90 DPH, respectively. No surrogate type or donor was superior to the others regarding colonization and proliferation, survival or growth, thus, channel catfish or white catfish were equally effective surrogates. Potential advantages of white catfish are small size, early sexual maturity, and spawning early in the season. These findings enhance the efficiency of germ cell transplantation for commercial hybrid catfish production.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Hettiarachchi, D.U., V. N. Alston, L. Bern, J. Al-Armanazi, B. Su, M. Shang, J. Wang, D. Xing, S. Li., M. K. Litvak, R. A. Dunham, I.A.E. Butts. 2024. Advancing Aquaculture: Production of xenogenic catfish by transplanting blue catfish (Ictalurus furcatus) and channel catfish (I. punctatus) stem cells into white catfish (Ameiurus catus) triploid fry. Plos One. https://doi.org/10.1371/journal.pone.0302687
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: 2038. Hettiarachchi, D. U., V. N. Alston, L. Bern, B. Su, M. Shang, J. Wang, D. Xing, S. Li, M. K. Litvak, R. A. Dunham and I. A.E. Butts. 2023. Maximizing colonization and proliferation of blue catfish (Ictalurus furcatus) donor stem cells for the creation of xenogenic catfish: Identifying the best host age of triploid channel catfish (I. punctatus). Aquaculture. https://doi.org/10.1016/j.aquaculture.2023.739400
  • Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: Alston, V. 2024. Genetic biotechnology to improve reproduction of North American catfish for aquaculture, genetic enhancement and genetic conservation. Doctoral Dissertation. Auburn University, AL.
  • Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: Pottle, K. 2024. Effects of cell number, host, and timing of injection on efficiency of xenogen production in ictalurid catfish. M. S. Thesis. Auburn University, AL
  • Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: Bern. L. 2024. Gene editing for growth enhancement in channel catfish, Ictalurus punctatus, and xenogenesis in common carp, Cyprinus carpio to produce blue catfish, I. furcatus, sperm. M. S. Thesis. Auburn University, AL