XENOGENESIS TO IMPROVE ARTIFICIAL SPAWNING OF REPRODUCTIVELY DIFFICULT CATFISH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1005731
• Grant No.: 2015-67015-23285
• Cumulative Award Amt.: $450,000.00
• Proposal No.: 2014-05151
• Project Start Date: Mar 15, 2015
• Project End Date: Mar 14, 2019
• Grant Year: 2015
• 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
School of Fisheries

Non Technical Summary
Production of hybrid catfish with the current technology is labor intensive and requires more skill than most hatcheries possess. Genetic improvement of blue catfish is hindered by their erratic spawning. By using stem cell technology coupled with we should be able to produce channel catfish possessing blue catfish sperm or eggs. Then they could be mated naturally with each other or normal channel catfish to more easily produce either blue catfish or hybrid catfish. This could also be done with white catfish with the advantage of the early maturity of white catfish. The specific objectives are 1) to determine which is more effective, the channel catfish system or the white catfish system and 2) to determine which is more effective for producing these fish, introducing the stem cells a few hours after fertilization, at hatch or in young fish. We expect to produce channel catish that when mated together produce hybrid catfish or blue catfish, and to produce white catfish that when mated together produce hybrid catfish or blue catfish. The impact is that this research will make it much easier and cheaper to produce hybrid catfish. This will help rescue and grow a US catfish industry that is stressed by foreign imports. Success will return the catfish industry to its previous status, a multi-billion dollar industry.

Animal Health Component

40%

Research Effort Categories

Basic

60%

Applied

40%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3710	1080	100%

Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3710 - Catfish;

Field Of Science
1080 - Genetics;

Keywords

catfish

hybrid

reproduction

stem cell

xenogenesis

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 efficiency of reproduction for catfish hybridization and to increase efficiency of reproduction in difficult to spawn species such as blue catfish, Ictalurus furcatus. To accomplish this, our overall research objective is to isolate embryonic stem (ES) cells, spermatogonia A (spermatogonial stem cells, SSCs), and successfully accomplish interspecific transplantation from a diploid donor catfish into a triploid host catfish (surrogacy), which will later produce diploid offspring from the donor. The two primary objectives of the project are: 1) to compare triploid channel catfish, Ictalurus punctatus, and triploid white catfish, Ameirus catus, for effectiveness as xenogenic hosts to produce diploid hybrid catfish and blue catfish, and 2) compare the efficacy (ability to produce reproductively functional xenogens) of introducing the stem cells into the host at the blastula, hatchling and sub-adult stages. The hypotheses are 1) diploid stem cells derived from testes of one catfish species can colonize in a triploid host of a second catfish species resulting in a xenogen capable of producing gametes of the donor species, 2) xenogens can be produced at different life stages, 3) xenogens will exhibit normal reproductive behavior and fertility and 4) white catfish xenogens will reproduce at younger ages than channel catfish xenogens.

Project Methods
Stem cells will be isolated and purified with density gradient centrifugation. Triploid hosts will be generated using hydrostatic pressure. The diploid donor stem cells will be transferred to the triploid hosts at blastula, sac fry and juvenile stage by injection. Xenogenic individuals will be identified by ploidy analysis, PCR and sexual development. Putative xenogenic individuals will be mated together and with controls to confirm fertility and sucessful stem cell transfer. Progeny will be identified with PCR, mtDNA analysis and morphological analysis to confirm that the expected type of catfish was produced and xenogenesis was accomplished.

Progress 03/15/15 to 03/14/19

Outputs
Target Audience:farmers, scientists, graduate students, biotech firms Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?five graduate students were provided training How have the results been disseminated to communities of interest?Updates were given at scientific conferences and in seminars at research institutions and universities. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We were able to produce white catfish xenogens, but they were unable to spawn at one year of age.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Shang, M., B. Su, D. A. Perera, A. Alsaqufi, E. A. Lipke, S. Cek, D. A Dunn, Z. Qin, E. Peatman and R. A. Dunham. 2018. Testicular germ-line cell identification, isolation and transplantation in two North American catfish species. Fish Physiology and Biochemistry. doi: 10.1007/s10695-018-0467-3.

Progress 03/15/17 to 03/14/18

Outputs
Target Audience:scientists and students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?two graduate students, a research assistant and a post doc received training How have the results been disseminated to communities of interest?Presentations were made to students and faculty at Auburn University and to scientists from around the world at Aquaculure America. What do you plan to do during the next reporting period to accomplish the goals?Major emphasis will be placed upon conducting spawning experiments to determine the most effective means of xenogenesis.

Impacts
What was accomplished under these goals? The primary effort was to prepare brood stock for mating experiments. However, we were able to overcome the spawning problems with white catfish. Also, for the first time we were able to produce xenogenic white catfsh and are growing them for spawning.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Ramjie Odin, 1st Place: Research Focus Area B: Efficient Food Animal Systems and Research Focus Area C: Integrated Food and Health Systems: Poster title: Xenogenesis to improve artificial spawning of reproductively difficult catfish. 2017 College of Agriculture Graduate Student Research Poster Showcase Winners Ramjie Odin, Top overall score for all categories: Poster title: Xenogenesis to improve artificial spawning of reproductively difficult catfish. 2017 College of Agriculture Graduate Student Research Poster Showcase Winners
• Type: Journal Articles Status: Published Year Published: 2017 Citation: �ek ?, Shang M, Perera DA, Su B, Dunham RA. 2016. Bal?k k�k h�creleri: S?n?fland?rma, kaynaklar?, karakteristik �zellikleri ve uygulama alanlar?. Stem cells: classification, resources, characteristics and application areas. LimnoFish. 2(2):107-119. doi: 10.17216/LimnoFish-5000182817
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Shang, M., B. Su, D. A. Perera, A. Alsaqufi, E. A. Lipke, S. Cek, D. A Dunn, Z. Qin, E. Peatman and R. A. Dunham. 2018. Testicular germ-line cell identification, isolation and transplantation in two North American catfish species. Fish Physiology and Biochemistry. DOI: 10.1007/s10695-018-0467-3

Progress 03/15/16 to 03/14/17

Outputs
Target Audience:Scientists, graduate students, farmers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?2 graduate students received training. How have the results been disseminated to communities of interest?The results have been disseminated via meetings with biotechnology firms, through publcation in scientific journals, departmental seminars and at scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?Additional breeding experiments will be conducted. The white catfish portion of the objectives has been unsuccessful because of spawning problems and more effort will be made in this area.

Impacts
What was accomplished under these goals? Primary findings during this year were that the stem cell number introduced to fry and blastula are critical for colonization and xenogeness. We were able to demonstrate that the stem cells are important for both the formation of somatic gonadal tissue as well as the gametes. Diploid donor cells are able to outcompete triploid host cells for formation gonadal tissue if the stem cells are introduced to hatching or earlier.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Perera, D. A., A. Alsaqufi, M. Shang, D. C. Wade, B. Su, A. Elaswad, M. Fobes, R. Beam, G. Garcia, D. A. Dunn, E. A. Lipke, and R. A. Dunham. 2016. Xenogenesis-production of channel catfish, Ictalurus punctatus, x blue catfish I. furcatus, hybrid progeny by fertilization of channel catfish eggs with sperm from triploid channel catfish males with transplanted blue catfish germ cells. North American Journal of Aquaculture. 79:61-74. DOI: 10.1080/15222055.2016.1221008
• Type: Other Status: Published Year Published: 2016 Citation: Odin, R., K. Vo, A. Alsaqufi, D. Perera, S. Mei, B. Su, S. Dong, G. Qin, A. Elaswad, K. Khalil, E. Lipke, E. Peatman and R. A. Dunham. 2016. Production of channel catfish Ictalurus punctatus x blue catfish I. furcatus hybrid progeny using xenogenesis.Meeting abstracts. Aquaculture America 2016. Las Vegas, NV. World Aquaculture Society.
• Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Alsaqufi, A. S. 2015. Approaches to improve production and performance of channel catfish (Ictalurus punctatus) female x blue catfish (Ictalurus furcatus) male hybrid catfish. Doctoral Dissertation. Auburn University, AL.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Shang, M. B. Su, E. A. Lipke, D. A. Perera, C. Li, Z. Qin, Y. Li, D. A. Dunn, S. Cek, E. Peatman and R. A. Dunham. 2015. Spermatogonial stem cells specific marker identification in channel catfish, Ictalurus punctatus and blue catfish, I. furcatus. Fish Physiology and Biochemistry. DOI 10.1007/s10695-015-016-1

Progress 03/15/15 to 03/14/16

Outputs
Target Audience:scientists, students and farmers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Several graduate students received training and one made multiple scientific presentations. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest through presentations at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?We have generated more fish for testing and will increase replication.

Impacts
What was accomplished under these goals? Xenogenic triploid channel catfish males that had blue catfish stem cells introduced as sub-adults produced very little sperm. However, their sexual behavior and sperm production appeared to be increasing annually. One male that was unable to naturally fertilize an egg mass was sacrificed and his sperm manually extracted. The sperm was used to fertilize eggs from a channel catfish female. The sperm from this male was able to fertilize 11 of 1,000 eggs, and 7 of these embryos survived until they reached the fingerling stage. All were channel catfish female- blue catfish male hybrids based upon external and internal morphology, and upon nuclear and mtDNA analysis. This was the first time that 100% hybrid progeny were produced using xenogenesis for any fish. The following spawning season one xenogenic channel catfish male that had received blue catfish stem cells as a sub-adult was able to fertilize a naturally spawned egg mass, but the fertility was extremely low. However, 4 spawns were produced semi-naturally from xenogenic triploid channel catfish males that received the stem cells as fry. Small egg masses were produced, two with high fertility and two with low fertility. The progeny were xenogenically produced hybrids. Relatively, large scale fertilization of channel catfish eggs by xenogenic of hybrids was demonstrated for the first time. These results show that it is feasible to alter and improve the spawning technology for producing channel catfish X blue catfish interspecific hybrids. We were also able to produce xenogenic catfish from cryopreserved stem cells. These combinations of results open the possibility of many breeding and conservation applications for catfish using xenogenesi. To fully utilized the demonstrated feasibility, the production technique to make xenogenic brood stock and the colonization rate of the introduced stem cells will need to be improved.

Publications