AQUACULTURE RESEARCH AND DEVELOPMENT IN OHIO AND POTENTIAL IMPACTS ON FISH PRODUCTION, INVASIVE SPECIES AND RECREATIONAL FISHERIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1004510
• Project Start Date: Oct 1, 2014
• Project End Date: Sep 30, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691

Performing Department
School of Natural Resources

Non Technical Summary
Aquaculture production of food fish and production of fish for recreational and sport-fishery purposes can become a part of agricultural growth in the state. It is known that techniques used in aquaculture such as artificial propagation and rearing of larvae, determination of nutritional requirement of larvae, juveniles and broodstock, genetic improvements, and diseases prevention have been utilized to enhance production efficiency and contribute to recreational fisheries in the United States.Considering that aquaculture is still the fastest growing sector of agriculture in the United States, we infer that economic returns from aquaculture in Ohio could be significantly improved by utilizing its potential to promote special, high technology input production and recreational fisheries in the state. Yellow perch, walleye, saugeye and eel have aquaculture potential and have been traditionally considered as significant components of recreational fisheries in Ohio.

Animal Health Component

30%

Research Effort Categories

Basic

30%

Applied

30%

Developmental

40%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	0810	1010	100%

Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
0810 - Finfish;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

aquaculture

yellow perch

asian carp

eel

fish nutrition

fish reproduction

fish genetics

Goals / Objectives
Objectives5.1.1 Perform nutritional studies over the two generations of yellow perch utilizing a "nutritional programming" approach and individual selection of the fastest growing females when provided with soybean meal-based (75% fish meal protein replacement) diets.5.1.2 Carry out meiotic gynogenesis in order to produce a pure line of genetically improved fish that have been selected to perform well on plant-based diets.5.1.3 Study the second generation of fish by monitoring growth and utilization efficiency of plant-based diets. 5.2.1 Characterize the microbial community of early ontogeny of both healthy yellow perch and yellow perch infected with 'columnaris' bacteria.5.2.2. Isolate and archive the bacteria that cause columnaris disease symptoms in the yellow perch in Lake Erie and our labs and evaluate bacteria isolated from healthy yellow perch as potential probiotics. 5.3.1 Characterize the most efficient water temperatures for embryo exposure to shock, shock duration, and determine at what time after fertilization the second meiotic and the first mitotic cell divisions take place in order to create triploid or tetraploid fish. Alongside the temperature and cold and heat shock experiments, hydrostatic pressure shock experiments will be carried out in order to compare the efficacy of all three methods.We will examine the survival, frequency of deformities, and ploidy of embryos (flow cytometry of cell suspension) and juveniles during the first 2 years of rearing (blood sampling).We will determine growth rate and gonad differentiation of diploids, polyploids, pure species, and hybrids in identical conditions when raised in captivity using standard techniques developed in our laboratory for cyprinid fishes.5.4.1 Measure gonad growth in hormonally treated American eels and establish reliable procedures for mass production of viable eel larvae through the initial exogenous feeding period.Characterize the status of vitamin B1, vitamin E, and lipids in the maturation cycle of female eel and establish reliable technologies for parenteral enrichment (implantation, microencapsulation) during vitellogenesis (ovarian growth).5.4.3 Establish intensive glass eel culture using formulated diets and establish the technology for the delivery of attractants and nutrients through controlled release from microencapsulated diets.Collaborate with fish farmers and ornamental fish producer (Coral Reef Systems Farm, New Albany, OH) on production of larval eel (endogenous-exogenous feeding transition) using a natural food chain production system and culture of glass eel to yearlings and 2 year old stocking material.5.4.4 Use degenerate phylogenetic sequence analyses to design PCR primer sequences to characterize the DNA sequences of thiamine and tocopherol transfer proteins. Use the knowledge of these sequences to quantify the temporal transcriptional events of the transporter genes during the maturation cycle of female eel related to the metabolism of vitamin B1 and vitamin E.5.4.5 Perform a cost-benefit analysis to determine profitability of eels culture and estimate markets and export volume/value for American farmers. Summarize our findings of reproductive capacity of eel for regulatory authorities (National Marine Fisheries Service, Fish and Wildlife Service) detailing implications for wild eel propagation, and long-term conservation.5.5.1 We will analyze the level of free amino acids in embryos and alevins of two dozen chinnok salmon from Lake Oahe (SD) and examine association with fish survival and growth during early life.

Project Methods
Methods6.1. Nutritional studies of yellow perch and selection of the strain of this species able to utilize efficiently plant protein-based diets. A nutritional programming experiment is currently in progress with broodstock yellow perch. It will serve as preliminary data for the proposed project in order to further improve yellow perch growth on plant-based diets. The project is distributed into four phases in which the fish diets will be changed in order to determine if a programming effect is occurring. In phase 1 of the project, fish (0.25 +0.01 g mean weight) were randomly distributed into 12 tanks with 20 fish per tank, three groups per diet. Four diets were formulated: 1) fishmeal as the major protein carrier (Control), 2) wheat gluten meal replacing 75% of the fish meal protein (W.G.) (Kwasek et al., 2012), and 3) two soybean meal varieties providing 75% replacement of fish meal protein (Soy A and Soy B). These diets were fed to the fish for two months. In Phase 2, fish from all four dietary groups were tagged and combined in triplicate to be raised in a "common garden" design for seven months. All of the fish were fed the same experimental fishmeal-based diet (F. Barrows, USFWS, Bozeman, MO). In Phase 3, which has just begun, also referred to as the "challenge" phase, all of the fish are being fed a soy-protein-based diet for seven months. The fish will continue to be raised using the same "common garden" design from phase two. In the current third phase, we are determining if nutritional programming is occurring in those fish that were originally fed soy protein as juveniles, as described in rainbow trout by Geurden et al. (2013). Selection will begin for the best performing fish within these groups for future broodstock. In Phase 4 of the experiment, the selected fish from all groups will be fed fishmeal-based diets for six months during gametogenesis in order to not compromise genetic impact on gamete quality (Vandeputte et al., 2002). The fishmeal diet, in the case of yellow perch, will not have an impact on somatic growth at this time (Kwasek et al., 2012). During winter months when the water temperature is below 10°C, gametogenesis proceeds based on mobilization of nutrients from the somatic portion within the body of the yellow perch. Preliminary results from the first phase showed a significantly depressed growth performance in the fish fed soy-based diets compared to the Control and W.G. diets. However, this trend was reversed in the second phase of the trial, and fish that had received soy-based feed gained a significantly higher percentage of weight than fish from the other two groups. Even though the soybean groups were gaining a larger percentage of weight, the data show that the mean weight of the fish within the groups is similar among the four original diet groups, with no significant difference. Since the fish in this above study will be bred in the proposed experiment, we hypothesize that a nutritional programming effect is occurring, and the fish that were originally fed a plant-based diet will over-perform controls when reintroduced to that diet again as adults. We will continue the nutritional programming regimen in the second generation (F2) of selected fish and compare to non-selected individuals.Asian carp gynogenesis and polyploidy6.5.1 Broodstock fish and gamete collectionBroodstock silver carp and bighead carp will be obtained with the help of Dr. Mark Gaikowski and transported to the isolation facility at The Ohio State University, School of Environment and Natural Resources (Kottman Hall). Fish will be obtained prior to the reproduction season in May-July 2015-2018, and maintained below 18oC until induced to spawn by hormonal injection of pituitary extracts (Aldridge et al. 1990) or human chorionic gonadotropin (Zhang et al., 2011). Water temperature will be increased to 23oC and ovulation will be expected in 10-16 h after injection, or 6-8 h after a second injection. Mature eggs will be collected and used in experiments after the time of first cleavage at 23oC is determined.Individual batches of eggs will be used to compare efficacy of 3 types of shock, cold (4-6oC), heat (40-42oC) and hydrostatic pressure (9,000-11,000 psi). Each experiment will include one control (diploid, or hybrid diploid), and two experimental groups: triploid (meiotic shock) and tetraploid (mitotic division shock). We will test several variations in order to obtain the optimal conditions for tetraploidy. For instance, cold shocks will be tested in three sets of experimental design, exposure to 4-6oC to be given during 8-10 or 10-12 min, and to be initiated at 1-30 min post-fertilization at 2 min intervals. These conditions have already been described as the most successful for meiotic and mitotic gynogen production of grass carp (Zhang et al. 2011). 6.5.2 Monitoring viability indicators of early embryonic stages We will be monitoring oxygen consumption and pH at 1 min intervals in mini-chambers of 1 mL (Loligo Systems, Tjele, Denmark) following cold and heat shock exposure. These measurements should be indicative of ova cell content leakages during the procedure and destruction of cell membrane integrity.6.5.3 UV irradiation and sperm inactivation Sperm of Asian carp or common carp (domesticated common carp obtained from Auburn University, Dr. Dunham) will be diluted with 2-3 parts of Moore's extender (Garcia-Abiado et al. 2001b), stored on ice and used within 1-2 hours. Diluted sperm with good motility will be poured on Petri dishes to form a layer of sperm 1mm thick, cooled on ice, and then exposed to UV irradiation with a short wave (254 nm) from a mineral light lamp (Model UVG-111, UVP, Inc., San Gabriel, CA), at 520 mW/cm2 as described in past experiments in our laboratory (Dabrowski et al. 2000; Rinchard et al. 2006).6.5.4 Ploidy analysis by flow cytometryTwenty hatched embryos per batch at the eyed stage will be fixed in 5% dimethylsulfoxide (DMSO) in isotonic solution and kept frozen at -20oC until the analysis. The embryonic tissues will be dissected from yolk in 0.75% sodium chloride solution following the detailed protocol used in past experiments in our laboratory (Garcia Abiado et al. 2001a, b). Flow cytometry analysis will be performed on a Coulter EPICS Elite flow cytometer (Beckman-Coulter, Inc. Miami, FL).6.5.5 Sexual differentiation and gonad developmentIn cyprinid fishes, primordial germ cells (PGCs) begin to proliferate and somatic cells actively multiply at 7-9 weeks after fertilization. At this time gonads can be clearly distinguished by histological examination. The head, tail and viscera will be removed from fish before being fixed in Bouin's solution. The vertebra and muscle tissue will be removed before the fish will be embedded in paraffin. Serial cross sections and staining will be performed as described earlier in our laboratory (Lin et al.1996; 1998; Dabrowski et al. 2000).6.5.6 Larval and juvenile rearingRearing techniques of larval silver carp and hybrids will follow protocols developed for Asian carp and common carp using a combination of live food and formulated feeds (Dabrowski et al. 1979; Dabrowski 1984; Kwasek et al. 2010). Our aquaculture laboratory is equipped with isolation rooms with recirculating, temperature controlled systems and excess water (10% daily) is drained into a chemically treated sewage water system.and other tissues were described recently (Kwasek et al. 2014).

Progress 10/01/14 to 09/30/19

Outputs
Target Audience:The PI and several students have attended the annual meeting of the World Aquaculture Society and American Fisheries Society each year from 2014 through 2019. These conferences have been held in New Orleans, Louisiana, Las Vegas, Nevada, and San Antonio, Texas. The conference is attended by industry professionals, farmers, scientists, and students from all over the world (2,000-3,5000 participants). While attending these meetings, both the PI and students have also given oral presentations on the research being conducted in the OSU lab. At the 2016 meeting, the PI organized a special "Percid Culture" information session, where 10 presentations were given covering a variety of research on percids, including 4 presentations by students in the OSU aquaculture laboratory. In 2016, the PI also attended the Wisconsin Aquaculture Association meeting and the annual North Central Regional Aquaculture Center (NCRAC) meeting to give oral presentations covering nutrition studies in percids. NCRAC is one of 5 regional aquaculture centers in the US, established by the US congress, and administered by the USDA. It's members include extension specialists, scientists, aquaculture professionals, students, and farmers. A graduate student also presented two research posters at the Allied Genetics Conference, 2016 in Orlando Florida, sharing results on a new system of rearing zebrafish with genetics researchers from across many disciplines. In 2017, both the PI and a graduate student presented work on soybean replacement of fishmeal, and in 2019, the PI presented work on producing all-female yellow perch through temperature manipulation at the NCRAC annual meeting. These conferences and meetings described above are where we are able to interact with diverse members of the aquaculture community, farmers and extension personnel, share our research directly, and identify the current needs and issues affecting the industry. Graduate students have also presented research at the OSU Hayes graduate research forum and the college (CFAES) research forum, sharing results with the student and faculty communities across various disciplines at OSU. In addition, we have published articles in scientific journals and conference publications which are available to the greater scientific community. The PI also teaches a graduate/undergraduate combined course Spring semester (ENR 5355), where OSU students receive hands-on instruction in the aquaculture lab as well as lecture-based learning. In addition, we have hosted several volunteer-based internships and paid positions for undergraduate and high school students in the lab, where they receive training on conducting scientific research and animal use practices. There have been several undergraduate students who have conducted independent research projects in the lab and have presented their results at the CFAES undergraduate research forum and the OSU Denman undergraduate research forum.We have also hosted several international scholars and post-doctoral fellows, as well as summer research opportunities for Veterinary students. The PI has also traveled internationally to various institutions in China, Malaysia, Czech Republic, and Bangladesh, over the reporting period, where he presented current research being conducted at OSU to fellow scientists, academics and students and formed new collaborations. Changes/Problems:The objectives which describe working with 'columnaris' bacteria and characterizing the microbial community of early ontogeny perch were not completed. The project was submitted to Ohio Seagrant, however, we did not receive funding and the scope of the work was too large to complete without sufficient financial support. Therefore, this project was replaced with work on developing triploid and gynogenetic walleye. What opportunities for training and professional development has the project provided?Multiple training and professional development activities have been conducted during the reported period. A total of 15 undergraduate students have completed volunteer-based internships in the aquaculture laboratory, and an additional 8 undergraduates have been employed as undergraduate research assistants. These internship and employment opportunities provide undergraduates with valuable experience conducting research, being a part of a research team, use of live animals in a laboratory setting, and knowledge of general fish husbandry and aquaculture practices. Undergraduates who have been members of our lab have gone on to become natural resources professionals and fishery industry personnel as well as gone on to attend graduate, medical, and veterinary school. Two undergraduates have conducted Honor's research projects in the last 5 years. These projects provided a first independent research experience for these student, as well as training in technical writing as they developed their Honor's theses. We have also had 12 international visiting scholars, professors, and post-doctoral fellows (Borlaug and Fulbright) come to the OSU aquaculture lab to take part in these projects and further their training in the aquaculture field. In addition, a high school student completed a summer internship, working on zebrafish genetics and rearing, and presented results to fellow students at a forum at the end of his summer experience. Undergraduates have also completed an independent study in the lab, working with graduate students on zebrafish nutrition, percid nutrition, and rainbow trout nutrition studies. Graduate and undergraduate students have had the opportunity to report their research at international, national, and university level conferences and forums. These meetings also provide the opportunity for students to network with aquaculture and natural resources professionals, scientists, and other students. Several projects in the lab are also being conducted in coordination with private industry and fish farmers, providing students with the unique opportunity to work directly with industry and aquaculturists over the course of the project, through final reporting. Projects have required that students travel to fish farms in Ohio, Kentucky, and Missouri, in order to collect broodstock, perform in vitro fertilization, and stock and monitor fish, requiring students to work closely with professionals in order to plan and coordinate research activities. Graduate and undergraduate students have also had the opportunity to further develop their technical writing and reporting skills through collaboration on grant proposals and project reporting through progress and final reports to state and federal agencies. How have the results been disseminated to communities of interest?The PI has traveled internationally to several countries in order to give invited lectures on the research being conducted at OSU. The PI as well as students have also presented research at various international, national, and university-wide conferences and forums during the reporting period. Examples include World Aquaculture Society and American Fisheries Society annual conferences, NCRAC annual meetings, Allied Genetics Conference, Ohio Aquaculture Association annual conference, CFAES Research Forum (OSU), and undergraduate research forums (OSU). Attendees to these conferences and forums include students and academics from across various disciplines, as well as industry representatives and professionals, extension specialists, scientists, and students from the aquaculture/fisheries field. Students and visiting scholars/post-docs have also given scholarly seminars within the School of Environment and Natural Resources at OSU. The Aquaculture lab has also hosted guided tours of the facilities as well as provided explanation of undergoing research for courses and student groups from various departments at OSU. Research in the lab also provides lecture material for the Introduction to Aquaculture course taught to undergraduate and graduate student by the PI during spring semesters. Additionally, as can be seen in the "Products" section of this report, the PI and lab group have been publishing peer-review articles in reputable journals, effectively disseminating the results of the research to the scientific and academic communities. Additional publications include summaries of research presented in conference proceedings. Several graduate and undergraduate students have also graduated during this reporting period, and their dissertations and theses have been published in the OSU Knowledge Bank. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Nutrition-Live food enrichment with polyunsaturated fatty acids (PUFA) and lipid-soluble antioxidant, tocopherol (vitamin E) is a common method to increase growth and survival of larval fish. We examined performance of yellow perch (YP) larvae fed live feeds enriched with PUFA and mixed-tocopherols. Treatments correlated with significant differences (α≤0.1) in fish survival, final weight, and growth rate. Data suggest α-T supplementation to live food PUFA enrichments is beneficial to culture of yp larvae, and 50% supplementation has no significant benefit compared to 25%. Replacement of fish meal and fish oil with plant-based ingredients in fish feeds is a promising solution to sustainability and profitability concerns in aquaculture. While conventional soybean oil has been extensively studied as lipid source, no studies evaluated high oleic acid soybean oil (HOSBO) that is rising in popularity with U.S. soy industry. We examined culture performance of rainbow trout (RT) fry fed experimental starter diets with conventional and HOSBO sources. Mean weight and feed conversion ratio (FCR) of fish were significantly different between diet groups through first 4 weeks of feeding (p<0.0001) but not at 6 and 8 weeks (p>0.1). Results suggest RT have reduced growth efficiency on HOSBO starter diets, but this may be an appropriate lipid source for grow-out of larger fish. We examined soybean-dependent nutritional programming, for growth and effects of antinutritional factors in YP. There were 3 phases. Diets were changed after a period of time to determine presence of programming effect based on early life diets. Phase 1 showed significantly depressed growth in fish fed soy-based diets compared to control diets. This trend was reversed in phase 2. Fish fed soy gained significantly more weight than fish from other two groups. In phase 3, fish originally fed soybean meal (phase 1), gained more weight than other groups. We hypothesize these juvenile fish indeed showed a soybean-dependent nutritional programming as adults. Genetic manipulation-Walleye propagation and stocking programs are a major focus of various fisheries management agencies; however, development of this species in the aquaculture industry has been limited. Induction of triploidy (3n) has been used as a sterilization method in many fish species and proved a valuable tool to preserve genetic integrity of natural stocks. We optimized production of 3n walleye and compared growth and survival to control (diploid) and hybrids. Pressure shock of 9000 psi produced highest rate of 3n (95%). Fish are undergoing grow-out, and survival and growth will be compared. We initiated production of gynogenetic (all-female) walleye, characterized by superior growth and delayed maturation compared to males. We attempted hormonal sex reversal to produce neomales via methyltestosterone (MT)-containing diet. Sex ratios of MT-treated gynogen and control fish will be examined histologically at the advanced stage of gametogenesis. We are developing basic understanding of sex differentiation in YP using hormonal treatment and environmental conditions to affect phenotypic sex in this species. Females grow faster and larger than males, resulting in higher aquaculture value. By producing neomales (genetic females, phenotypic males), we obtain solely X-sperm, and can produce all female fish. We aimed to determine efficiency of masculinization with MT and determined successful sex reversal in treated groups. We also saw presence of neomales in untreated control, most likely due to higher water temperature than natural conditions. We investigated possible effect of high temperature during sex differentiation as a method to produce neomales. Experiments are ongoing. We observed male-biased sex ratio in high temperature groups, while low-temperature siblings present normal mixed sex ratio, suggesting temperature manipulation may be effective method to produce neomales. Adult triploid zebrafish Danio rerio have previously been reported to be all male. To investigate the mechanism responsible we observed gonad development, and confirmed all adult triploids were male. Male triploids were able to induce oviposition by diploid females during natural spawning trials, but fertilization rates were low (1.0%) compared to diploid male siblings (67.4%), and all embryos were aneuploid. Additionally, triploids treated with estradiol remained all male, demonstrating triploidy is acting downstream of estrogen synthesis in the sex differentiation pathway. We also produced, for the first time, a mass group of gynogenetic (fatherless) zebrafish using UV-irradiated sperm of common carp) zebrafish that had growth comparable to controls. All gynogens which reached reproductive maturity were male. Fertility of gynogen males was found to be inferior to control males. Hybridization-Fertility, survival, and growth of Central American cichlid hybrids and backcrosses were investigated to address production in captivity and prediction of success in invading new environments (Texas, Florida). Little attention in scientific literature has been devoted to the fertility and viability of these hybrids that are frequently released or escape to the wild, threatening native fishes. We addressed reproduction, embryonic development, growth, maturation and fertility of red parrot cichlid, hybrid of redhead Mexican cichlid (Vieja synspilum) female and midas (Amphilophus citrinellum) male, compared to half siblings of each parental species. We did not observe differences in growth during juvenile stages. A feeding study with controls on acceptance of formulated commercial diets in comparison to live feed produced survival of 97+3% in all diet groups, allowing us to conclude this species can be fed formulated feeds instead of live food during early life. Rearing-Despite widespread use of zebrafish as a model organism, husbandry methods and nutrition have not been standardized. We characterize a protocol for rearing zebrafish using a recently developed combination of environmental parameters that yield rapid growth rates: high densities of live food organisms (marine rotifers and brine shrimp nauplii), low salinity, continuous light, and algal turbidity during larval and early juvenile stages (5-21 days post-fertilization) followed by transition to conventional rearing methods (dark/light photoperiod, freshwater). We assessed a range of larval fish densities and found densities of 12-22 fish/L gave maximum growth rate and growth rate declined as stocking density increased past this threshold. This protocol will significantly increase productivity in zebrafish facilities. We explored broostock transport, captive maintenance, analyzed biochemical body composition, and performed gonads histology, and evaluated effects of hormonal stimulation on eel morphology and gonad growth of American eel and identified the stock (river) with most advanced ovary development. Lipid and fatty acid characterization of fish tissues in hormonally induced eel was performed to gather preliminary data. Cryopreservation -We improved the effectiveness of simple cryopreservation techniques of YP sperm by examining different cryoprotectants (DMSO,MetOH) utilizing two simple cryopreservation methods. We determined that both methods with either cryoprotectant are efficient to cryopreserve YP sperm. Cryopreserved sperm using both methods and cryoprotectants and control fresh sperm were then used to fertilize YP eggs. Fertilization rate was significantly lower for cryopreserved sperm (28.2±9.7%) overall than for fresh control sperm of identical concentration (60.2±26.0%). We also evaluated growth of progenies resulting from cryopreserved and fresh (control) sperm. Survival and growth results from the rearing experiment suggest progeny from cryopreserved sperm do not experience growth suppression or higher mortality than progeny from fresh control sperm.

Publications

• Type: Books Status: Published Year Published: 2015 Citation: Kestemont, P. Dabrowski, K. and Summerfelt, R. 2015. Biology and Culture of Percid Fishes. Principles and Perspectives (Springer Berlin) 35 Chapters, 901 pages Monography on North American and European Percids.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Chen, K-Y., Ludsin, S.A., M..M. Corey, P.D. Collingsworth, M.K. Nims, J.W. Olesik, K. Dabrowski, J.J. van Tassell, and E.A. Marschall. 2017. Experimental and field evaluation of otholith strontium as a marker to discriminate between river-spawning populations of walleye in Lake Erie. Can.J.Fish.Aquat.Sci. 74: 693-701.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Kwasek, K., Rimoldi, S., Cattaneo, A.G., Parker, T., Dabrowski, K., Terova, G. 2017. The expression of hypoxia-inducible factor1-alfa gene is not affected by low-oxygen conditions in yellow perch (Perca flavescens) juveniles. Fish Physiol. Biochem. 43: 849-862.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Delomas, T., Dabrowski, K. 2017. The importance of controlling genetic variation  remarks on Appropriate rearing density in domesticated zebrafish to avoid masculinization: links with the stress response. J. Exp. Biol. 200: 4078.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Ostaszewska, T., Krajnik, K., Adamek-Urbanska, D., Kasprzak, R., Rzepkowska, M., Luczynski, M., Karczewska, A.T., Dabrowski, K. 2018 Effect of feeding strategy on digestive tract morphology and physiology of lake whitefish (Coregonus lavaretus). Aquaculture 497:32-41.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Dabrowski, K., Miller, M. 2018. Contested paradigm in raising zebrafish (Danio rerio). Zebrafish 15: 295-309.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Delomas, T.A., Dabrowski, K. 2018. Improved protocol for rapid zebrafish growth without reducing reproductive performance. Aquaculture Res. https://doi.org/10.1111/are.13912
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Delomas, T. Dabrowski, K. 2018. Effect of homozygosity on sex determination in zebrafish Danio rerio. J. Fish Biol. https://doi.org/10.1111/jfb.13836
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Delomas, T. Dabrowski, K. 2018. Why are triploid zebrafish all male? Molecular Reprod. Develop. 85: 612-621.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Dabrowski, K., M. Wojno, M. Miller, K. Kwasek, J.D. Grayson. 2018. Continued embryonic development, survival, and growth of walleye larvae following exposure to dewatering and storage in melting ice temperatures. North American J. Aquaculture 80: 404-410 .
• Type: Books Status: Published Year Published: 2015 Citation: Zaccone, G., Dabrowski, K., Hedrick, M.S., Fernandes, J.M.O., Icardo, J.M. 2015. Phylogeny, Anatomy and Physiology of Ancient Fishes. CRC Press, 297 pp.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Kemski, M.M., Rappleye, C.A., Dabrowski, K., Bruno, R.S., Wick, M. 2019. Transcriptomic response to soybean meal-based diets as the first formulated feed in juvenile yellow perch (Perca flavescens). Submitted to Scientific Reports
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Delomas, T. Dabrowski, K. 2018. Larval rearing of zebrafish at suboptimal temperatures. J.Thermal Biol. 74: 170-173.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Kemski, M., Wick., M., Dabrowski, K. 2018. Nutritional programming effects on growth and reproduction of broodstock and embryonic development of progeny in yellow perch (Perca flavescens) fed soybean meal based diets. Aquaculture 497: 452-461.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Miller, M., Kemski, M., Grayson, J.D., Towne, K., Dabrowski, K. 2018. Yellow perch sperm motility, cryopreservation, and viability of resulting larvae and juveniles. North American Journal of Aquaculture 80: 3-12.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Delomas, Thomas A.; Dabrowski, Konrad 2019. Effects of homozygosity on sex determination in zebrafish Danio rerio. JOURNAL OF FISH BIOLOGY 93, 6: 1178-1187.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Kamaszewski, Maciej; Ostaszewska, Teresa; Napora-Rutkowski, Lukasz, 2019. The role of dipeptide on fish growth and digestive enzyme activity modulation in common carp (Cyprinus carpio L.) . ANIMAL SCIENCE PAPERS AND REPORTS 37, 1: 75-85.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Hayes, J. 2017. The design and construction of an axenic system, and the survival, growth, and baseline microbial load of the rearing environment, food source and internal microbiota of a cichlid fish. The Ohio State Univeristy, School of Environment and Natural Reources, Honors Thesis.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience: I attended annual meeting of the North Central Regional Aquaculture Center in Kansas City, Missouri in February 9-10, 2018 and presented report on "Feeding of larval fish - environmental. genetic and nutritional factors of live feeds and/or formulated diets utilization" to researchers, academics, extension agents and fish farmers from the region, approximately 50 in attendance. I covered several aspects of digestive tract differentiation in stomach possessing (percids and salmonids) and stomachless (cyprinids) fish. Questions from attendees concentrated on live feeds availability and possible replacement with formulated, off-the shelf diets. In the most recent paper dealing with zebrafish (Dabrowski and Miller 2018) we outlined novel procedure to raise zebrafish from larvae to juveniles and transition to formulated feeds that is applicable to yellow perch and walleye. We attended World Aquaculture Society meeting in Las Vegas in February 19-22, and investigators and fish producers were in attendance. Overall at least 200 persons attended 3 presentations devoted to yellow perch nutrition, larvae rearing using enriched live food with fatty acids and vitamin E, as well maturation of triploid yellow perch and fecundity. The subject of one of the presentation was soybean-dependent nutritional programming and individual selection for growth and effects of anti-nutritional factors. In this experiment, the first generation of fish (F0) were nutritionally programmed and reproduced annually for three years to determine if their offspring had improved growth performance when given a fish meal or soybean meal-based diet as their first formulated feed. Nutritional programming of the F0 generation consisted of 4 phases; in phase 1 fish were separated into two groups, one group fed a fish meal (FM) based diet (control) and the other group fed a soybean meal-based (SBM) diet (75% replacement of FM protein) for 2 months. In Phase 2, fish were combined in triplicate tanks and all fed a FM diet for 9 months. In Phase 3, all fish were transitioned to a SBM diet for 7 months. In Phase 4, fish were fed FM diets for 6 months during winter months during gametogenesis. Results of the three spawning events over subsequent years continue to show improvement in growth performance, particularly for fish fed the SBM based diet as their initial feed. Results showed that differences among F1 generation and offspring groups as well as diet were significant (P<0.001). The interaction between all groups and diet was also significant (P<0.05). Overall, it was determined that crossing nutritionally programmed females and males allows for increased weight gain (%) as seen in 2016 and 2017 offspring. The size of juveniles when transitioned to the formulated feed is important, as 2017 was the largest weight at the time of stocking, which resulted in the best survival. Several farmers after presentation in this meeting volunteered to become participants in the study in the future. We have two publications in 2018 in North American Journal of Aquaculture. One was devoted to delayed hatching of walleye after storage on melting ice. I have already obtained correspondence from Minnesota. "I liked your recent article in the NAJA. I see that you were studying the possible effects of cold temperature exposure to 8 day old eyed eggs. Have you looked at temperature effect on pre eyed up eggs. We seem to have our greatest egg mortality between days 3-5 post fertilization at our New London State Fish Hatchery" (Brad Carlson, MN DNR, Spicer Area Fisheries Office). This new method will allow farmers and state hatcheries great flexibility in respect to timing of walleye hatching and synchronization of weather conditions, zooplankton availability in nursery ponds, and larval walleye stocking. In Las Vegas in February we also presented the work on triploidy in yellow perch. In this presentation we included the data on first production of functional neomale (XX genotype) and their progeny. We will be able to offer to fish farmers a means of all-female yellow perch production. This should increase production efficiency of this species by 30% and increase profitability by $550 per 1,000 marketable size fish. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students presented several reports to professional associations and aquaculture community at large (WAS, Las Vegas 2018) and established direct contact with industry representatives. Specifically, producers of yellow perch for pond stocking and food fish have historically fed fish with currently available trout diets but have questioned whether such diets are optimal for percids. We have established collaboration with the industry, Organic Technologies, Coshocton, OH that is funding portion of the research on utilization of ethyl esters processed from fish oil as replacement of high cost marine fish oils. One of the PhD student is directly collaborating with the sponsor. This study involve both replacement of fish oil in rainbow trout diets and enrichment of live feed organisms for larval fish feeding, yellow perch and ornamental fish species. These aspects of research on fish nutrition have been consistently asked the research community associated with North Central Aquaculture Center and in 2018 we were awarded research project entitled "Formulation and assessment of a new generation of starter diets for largemouth bass (Micropterus salmoides) and yellow perch (Perca flavescens) larvae". This project is in collaboration with T. Melman's Coral Reefs farm, New Albany, OH. Results will allow us to formulate the replacement for expensive ingredients and lower cost of fish diets for largemouth bass and yellow perch. All carried projects have a special emphasis on the extension objective, as we invite fish farmers community to visit our laboratories and develop research agenda that will be compatible with their needs. We will encourage wide participation in extension workshops on yellow perch and feed formulation and will present such diet recommendations at workshops. We will attend the World Aquaculture Society annual meeting in New Orleans in 2019 and 10 oral papers will be presented dealing with yellow perch nutrition, reproduction, larviculture, sex reversal, hybridization/gynogenesis of zebrafish and Central American cichlid fishes. The aim of this last task is to develop technology for production of ornamental fish that can be produced in recirculation systems in the North Central region. In 2018 two Ph.D students, Miss Megan Kemski and Thomas Delomas graduated. In 2018 we hosted three international scholars. Mrs. Nurulhuda Ahmad Fatan is the Senior Researcher in one of the largest international organizations in aquaculture and fisheries, WorldFish, was invited to our laboratory after receiving Borlaug Fellowship. Professor Atse Boua Celestin is a Fulbright Fellow from Oceanological Research Center in Abidjan, Ivory Coast. Professor Celestin also teaches aquaculture and fish reproduction courses at Felix Houphouët-Boigny University in Ivory Coast and advises graduate students.Ebtehel Hussein is a former Ph.D student of Professor Dabrowski, who has returned to The Ohio State University with the support of the Egyptian government as a postdoctoral fellow. She is currently a professor at the Manoufiya University in Shebin-el-Kom in Egypt. All three visiting scholars worked on utilization of fish waste in diets of zebrafish and effect of these feeds on reproduction efficiency and quality of progenies obtained. How have the results been disseminated to communities of interest?The PI invested considerable time to zebrafish model studies (Delomas, T. PhD student) and five peer-reviewed articles were published in 2018 in the reputable journals. Four more manuscripts on zebrafish genetics and hybridization are in preparation and one of them will described a new mutant of pearl danio (Danio albolineatus) that carries the same defect of the gene mpv17 that results in Roy Orbison appearance (loss of irridophores). This work as well as research on ornamental fish species both can provide significant input to new aquarium fish industry and medical research sing zebrafish as model species. We licensed new technology of production of Red parrot, cichlid fish hybrid and industry has expressed considerable interest in it. In Spring 2018 "Introduction to Aquaculture" was offered and 10 undergraduate students took part. Students worked on four different projects divided into teams. These research projects encompassed subject such as walleye reproduction and gynogenesis, wild zebrafish from Brahmaputra River (Bangladesh) reproduction and sex ratio, growth and morphology of parrot cichlid backcrosses. PI presented invited lectures at the International Workshop in Normal Hunan University, Changsha, Hunan, China, in December 2018. Letter of Intent , describing collaborative research project with Professor Shaojun Liu was submitted to NIFA-USDA. What do you plan to do during the next reporting period to accomplish the goals?In the following 2 years we will concentrate on NIFA funded research of invasive bighead carp that also represents considerable interest to aquaculture worldwide. We anticipate to continue research on gynogenetic walleye that were in part subjected to methyltestosterone treatment (masculinization). In 2019 we will examine some fish gonad histology and follow up maturation of these gynogens. We anticipate funding from the North Central Regional Aquaculture Center (NCRAC) where initial project reviews were very positive.

Impacts
What was accomplished under these goals? As the priority fish species for aquaculture in North Central region of US is yellow perch we focus on production of larval stages of this species. Specifically, with the aquaculture industry facing fish meal sustainability issues, plant based protein sources are becoming a requirement at higher inclusion levels in aquafeeds. One possible mechanism of adaptation for fish to utilize alternative protein sources in their diet is nutritional programming. The concept of nutritional programming can be defined as early nutritional events exerted during critical developmental stages that may result in changes expressed later in life affecting growth potential, health and metabolic status. By feeding juvenile fish a soybean meal-based diet as their first formulated feed, it is hypothesized that they will have enhanced growth performance when reintroduced to soybean meal later in life and these modifications will be passed on to from parental phenotypes to offspring. Live food enrichment with polyunsaturated fatty acids (PUFA) and lipid-soluble antioxidant, tocopherol (vitamin E) is a commonly used method for increasing the growth and survival of larval fish. This investigation examined the intensive culture performance of yellow perch larvae when fed live feeds enriched with PUFA and mixed-tocopherols preparation from local manufacturer, Organic Technologies (Coshocton, Ohio), of antioxidant supplement. The experiment encompassed the first 10 days of exogenous feeding by larvae on PUFA enriched rotifers (Brachionus plicatilis) and Artemia nauplii. Larvae were reared in a specialized recirculating system with nine 50-L conical tanks equipped with surface spray and greenwater (turbidity) conditions. The treatments correlated with significant differences (α≤0.1) in fish survival, final weight, and growth rate at the end of the feeding period. The α-T content of both live feeds and the fish were significantly influenced by treatment, with the 25% and 50% α-T groups having greater concentration than the PUFA control group (p<0.001). The only significant difference between the 25% and 50% α-T requirement groups was in yellow perch α-T concentration (p=0.038). These data suggest that α-T supplementation to live food PUFA enrichments is beneficial to the intensive culture of yellow perch larvae, and that 50% supplementation has no significant benefit to culture performance compared to 25% supplementation. Fertility, survival, and growth of Central American cichlid hybrids and their backcrosses were investigated to address both production in captivity and prediction of success in invading new environments (Texas, Florida). Surprisingly little attention in the scientific literature has been devoted to the fertility and viability of these high priced ornamental fish hybrids that frequently are released or escape to the wild. We addressed reproduction, embryonic development, growth, maturation and ferrtility of red parrot cichlid, hybrid between redhead Mexican cichlid (Vieja synspilum) females and midas (Amphilophus citrinellum) males. Juveniles began consuming exogenous live food after 7 days post-fertilization (dpf) at 28-30oC, prior to complete yolk absorption. Following 10 days of feeding with brine shrimp nauplii, fish were transitioned to formulated feeds at 28 dpf. We did not observe differences in growth of hybrids and Cs during juvenile stages. In parallel, a feeding study was carried out with Cs on the acceptance of the formulated commercial diets in comparison to live feed. Fish fed Artemia were significantly larger (0.29±0.03 g) than other treatments and differences were also significant among commercial diet groups. However, survival was 97+3% in all diet groups and so we conclude that in this species formulated feeds can completely replace live Artemia during early life. In 2017-2018 the original hybrid (red parrot) has a high survival rate and females reach maturity at one year old. The backcross has low viability (11+7% feeding; n=6), whereas the parrot/Mayan hybrid (PMH) has comparatively high viability (30% feeding), and a low rate of skeletal deformities. Juveniles were fed with rotifers and Artemia nauplii for the first 3 weeks and then transitioned to commercial feed, Otohime). Mean weight of PMH at 28 dpf (326+94 mg) exceeded that of parental species, red parrot hybrid (310+ 8 mg) and C. urophthalmus (Mayan cichlid) (291+24 mg). Despite widespread use of zebrafish Danio rerio (Hamilton) as a model organism for finfish aquaculture, genetics, and molecular biology, husbandry methods and nutrition have not been standardized. We characterize a protocol for rearing zebrafish using a recently developed combination of environmental parameters that yield rapid growth rates: high densities of live food organisms (marine rotifers and brine shrimp nauplii), low salinity, continuous light, and algal turbidity during larval and early juvenile stages (5 - 21 days post-fertilization, dpf) followed by a transition to conventional rearing methods (dark/light photoperiod, freshwater). We assessed a range of larval fish densities in a stagnant water system with continuous ad libitum feeding until 21dpf and periodic water changes. Fish stocking densities of 12 - 22 fish/L gave maximum growth rate and growth rate declined as stocking density increased past this threshold. Survival rates during 5-21 and 21-42dpf were 87±9 and 98±3%, respectively. Mean±SD weight and length were 64±9 mg and 19.4±1.0 mm at 21dpf and 245±28mg and 30.4±1.5mm at 42dpf. Fertility was assessed at 66±3dpf. Mean clutch size was 232±124 oocytes/female and fertilization rate was 72±16%. This protocol will significantly increase productivity in zebrafish facilities. Adult triploid zebrafish Danio rerio have previously been reported to be all male. This phenomenon has only been reported in one other fish species, the rosy bitterling Rhodeus ocellatus, despite triploidy being induced in numerous species. To investigate the mechanism responsible, we first produced triploid zebrafish and observed gonad development. Histological sections of juvenile triploid gonads showed that stage I oocytes were able to develop in the juvenile ovary, but no stage II or more advanced oocytes were found. All adult triploids were male. Male triploids were able to induce oviposition by diploid females during natural spawning trials, but fertilization rates were low (1.0 ± 3.1%) compared to diploid male siblings (67.4 ± 16.6%). Embryos produced by triploid sires were aneuploid with a mean ploidy of 2.44 ± 0.10n, demonstrating that triploid males produce aneuploid spermatozoa. After confirming that adult triploids are all male, we produced an additional batch of triploid zebrafish and exposed them (and a group of diploid siblings) to 100 ng/L estradiol (E2) from 5 - 28 dpf. E2 treated triploids and non-treated triploids were all male. Non-treated diploids were also all male, but E2 treated diploids were only 11% male. This demonstrates that triploidy is acting downstream of estrogen synthesis in the sex differentiation pathway to induce male development. Based on this and the observations of juvenile gonad development in triploids, we suggest that triploidy inhibits development of oocytes past stage I, and this causes female to male sex reversal.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Ostaszewska, T., Krajnik, K., Adamek-Urbanska, D., Kasprzak, R., Rzepkowska, M., Luczynski, M., Karczewska, A.T., Dabrowski, K. 2018 Effect of feeding strategy on digestive tract morphology and physiology of lake whitefish (Coregonus lavaretus). Aquaculture 497:32-41. Dabrowski, K., Miller, M. 2018. Contested paradigm in raising zebrafish (Danio rerio). Zebrafish 15: 295-309. Delomas, T.A., Dabrowski, K. 2018. Improved protocol for rapid zebrafish growth without reducing reproductive performance. Aquaculture Res. https://doi.org/10.1111/are.13912 Delomas, T. Dabrowski, K. 2018. Effect of homozygosity on sex determination in zebrafish Danio rerio. J. Fish Biol. https://doi.org/10.1111/jfb.13836 Delomas, T. Dabrowski, K. 2018. Why are triploid zebrafish all male? Molecular Reprod. Develop. 85: 612-621. Dabrowski, K., M. Wojno, M. Miller, K. Kwasek, J.D. Grayson. 2018. Continued embryonic development, survival, and growth of walleye larvae following exposure to dewatering and storage in melting ice temperatures. North American J. Aquaculture 80: 404-410 . Delomas, T. Dabrowski, K. 2018. Larval rearing of zebrafish at suboptimal temperatures. J.Thermal Biol. 74: 170-173. Kemski, M., Wick., M., Dabrowski, K. 2018. Nutritional programming effects on growth and reproduction of broodstock and embryonic development of progeny in yellow perch (Perca flavescens) fed soybean meal based diets. Aquaculture 497: 452-461. Miller, M., Kemski, M., Grayson, J.D., Towne, K., Dabrowski, K. 2018. Yellow perch sperm motility, cryopreservation, and viability of resulting larvae and juveniles. North American Journal of Aquaculture 80: 3-12.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:I attended annual meeting of the North Central Regional Aquaculture Center in Ames, Iowa in February 3-4, 2017 and presented report on "Probiotic studies in yellow perch" to researchers, academics, extension agents and fish farmers from the region, approximately 70 in attendance. Probiotics are chemical compounds, mostly of bacterial origin that affect growth and health of the host organisms. We addressed bacterial flora in the intestinal content of yellow perch cultured in outdoor ponds or indoor tanks in conditioned (practically sterile) city water. Work on yellow perch larvae included culture in high density using live zooplankton for 17 days. The average rate of survival through the entire experimental period was 32.0±7.6% and the swim bladder inflation rate was 35.8±20.6%. The average juvenile weight was 24.5±5.0 mg and average growth rate 29.4±1.6%/day. Fish were then subjected to treatments with isolated probiotic strains of bacteria and potential pathogenic bacteria isolates. Isolates from adult yellow perch were used to further characterize by heat shock challenge and determine their inhibitory potential against common fish pathogens, Vibrio anguillarum and Aeromonas salmonicida. Of the 8 isolates tested all but 3 isolates showed inhibition of Vibrio while there appears to be only a weak inhibition to Aeromonas. Feeding experiment was performed with yellow perch juveniles that included the following dietary treatments: commercial diet (control), commercial diet with yellow perch isolated probiotic, commercial diet with commercial probiotic, and yeast and krill based diet. Flavobacterium columnare (columnaris) challenge was performed final fish survival rates amounted to 93-100%. It appeared that fish we used were previously exposed to this pathogen. Studies need to be continued with pathogen-free yellow perch. We attended World Aquaculture Society meeting in San Antonio, TX in February 19-22, and investigators and fish producers were in attendance. Overall at least 200 persons attended 3 presentation devoted to yellow perch larvae rearing and nutrition. The topic of one of the presentation was soybean-dependent nutritional programming and individual selection for growth and effects of anti-nutritional factors. This experiment, already in the 4rd year of study, addressed several progenies from broodstock perch programmed over this period repeatedly. The response consists of 3 phases in which diets were changed after a period of a soybean meal-based (SBM) "programming" that occurred in early life, juvenile stage. In the first phase fish are "programmed "on soybean meal based diet and then, after a period of fish meal-based diet are entering a "challenge" phase, a soybean meal-based diet again. Results from the second progeny during the first phase (programming) showed no significant differences in weight gain (%) within groups fed fish meal (FM) or SBM. While juveniles fed SBM as the first formulated diet have significantly lower growth rate (%) than those fed FM, the progeny from the second spawning already shows a significant improvement in growth on SBM after two years of nutritional programming. Several farmers after presentation in this meeting volunteered to become participants in the study in the future. We have one publication in press in North American Journal of Aquaculture on yellow perch sperm cryopreservation and one manuscript on "nutritional programing" was just submitted to "Aquaculture". One Master dissertation was completed in December 2016 and three PhD dissertations will result from this work on yellow perch. We will publish our results in relevant peer-reviewed journals. These publications will allow the model developed in this project with yellow perch to be translated to other fish species. It is expected that these concepts will be adapted by aquaculturists throughout the U.S. We will attend the Aquaculture America conference in Las Vegas in February 2018 and present a series of presentations that will include the report on first production of functional neomale (XX genotype) and their progeny. We will be able to offer to fish farmers a means of all-female yellow perch production. This should increase production efficiency of this species by 30% and increase profitability by $550 per 1,000 marketable size fish. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students presented several reports to professional associations and aquaculture community at large (WAS, San Antonio 2017) and established direct contact with industry representatives. Specifically, producers of yellow perch for pond stocking and food fish have historically fed fish with currently available trout diets but have questioned whether such diets are optimal for percids. We have established collaboration with the industry, Organic Technologies, Coshocton, OH that is funding portion of the research on utilization of ethyl esters processed from fish oil as replacement of high cost marine fish oils. One of the PhD student is directly collaborating with the sponsor. This study involve both replacement of fish oil in rainbow trout diets and enrichment of live feed organisms for larval fish feeding, yellow perch and ornamental fish species. These aspects of research on fish nutrition have been consistently asked the research community associated with North Central Aquaculture Center. Results will allow to formulate the replacement for expensive ingredients and lower cost of fish diets for rainbow trout and yellow perch. All carried projects have a special emphasis on the extension objective, as we invite fish farmers community to visit our laboratories and develop research agenda that will be compatible with their needs. We will encourage wide participation in extension workshops on yellow perch and feed formulation and will present such diet recommendations at workshops. We will attend the World Aquaculture Society annual meeting in Las Vegas in 2018 and 10 oral papers will be presented dealing with yellow perch nutrition, reproduction, larviculture, sex reversal, hybridization/gynogenesis of zebrafish and Central American cichlid fishes. The aim of this last task is to develop technology for production of ornamental fish that can be produced in recirculation systems in the North Central region. How have the results been disseminated to communities of interest?The PI invested considerable time to zebrafish model studies (Delomas, T. PhD student) as well as research on ornamental fish species that both can provide significant input to new aquarium fish industry. We licensed new technology of production of Red parrot, cichlid fish hybrid and industry has expressed considerable interest in it. We are negotiating through OSU license office the submission of new mutant of aquarium fish, Pearl danio to Ximbio (UK company) that would post it on their website as a biorepository option. PI presented invited lecture at the International Conference "LARVI 2017" in Ghent, Belgium in September 2017 (200 participants in attendance). This review will be published in special issue of "Aquaculture" journal. Results on yellow perch and zebrafish research were presented in the Institute of Hydrobiology and Fisheries, Vodnany, Czech Republic, to approximately 100 students and faculty. What do you plan to do during the next reporting period to accomplish the goals?In the following 2 years we will concentrate on NIFA funded research of invasive bighead carp that also represents considerable interest to aquaculture worldwide. This research addresses production of tetraploid bighead carp and consequently crosses between tetraploid and diploid to obtain sterile triploids. One manuscript in currently in press and several manuscripts are in the review process or will be submitted shortly: Dabrowski, K. and Miller, M. 2017. Contested paradigm in raising zebrafish (Danio rerio). Zebrafish (second revision). Kemski, M., Wick, M., Dabrowski, K. 2017. Nutritional programming effects on growth and reproduction of broodstock and quality of offspring of yellow perch (Perca flavescens). Aquaculture (submitted). Delomas, T. , Dabrowski, K. 2017. New method to achieve rapid larval and juvenile zebrafish growth. Aquaculture (submitted). Miller, M., Kemski, M., Grayson, J., Towne, K. and Dabrowski, K. 2017. Yellow perch sperm motility, cryopreservation and viability of resulting larvae/juveniles. North Am. Journal of Aquacult. (in press).

Impacts
What was accomplished under these goals? As the priority fish species for aquaculture in North Central region of US is yellow perch we focus on production of larval stages of this species. Specifically, live food enrichment with polyunsaturated fatty acids (PUFA) is a commonly used technique for increasing the growth and survival of larval fish, but increased dietary PUFA can also be a major source of oxidative stress. This investigation examined the intensive culture performance of yellow perch larvae when fed live feeds enriched with PUFA and three levels of α-tocopherol (α-T) antioxidant supplementation. The experiment encompassed the first 10 days of exogenous feeding by larvae on PUFA enriched rotifers (Brachionus plicatilis) and Artemia nauplii. Larvae were reared in a specialized recirculating system with nine 50-L conical tanks equipped with surface spray and greenwater (turbidity) conditions. The treatments correlated with significant differences (α≤0.1) in fish survival, final weight, and growth rate at the end of the feeding period. The α-T content of both live feeds and the fish were significantly influenced by treatment, with the 25% and 50% α-T groups having greater concentration than the PUFA control group (p<0.001). The only significant difference between the 25% and 50% α-T requirement groups was in yellow perch α-T concentration (p=0.038). These data suggest that α-T supplementation to live food PUFA enrichments is beneficial to the intensive culture of yellow perch larvae, and that 50% supplementation has no significant benefit to culture performance compared to 25% supplementation. We addressed reproduction, embryonic development, and growth of red parrot cichlid (estimated world trade of $10 million), hybrid between redhead (Cichlasoma synspilum) and midas (Amphilophus citrinellum). We performed several spawnings of parental C. synspilum (Cs) and production of hybrids. Juveniles began consuming exogenous live food after 7 days post-fertilization (dpf) at 28-30oC, prior to complete yolk absorption. Following 10 days of feeding with brine shrimp nauplii, fish were transitioned to formulated feeds at 28 dpf. We did not observe differences in growth of hybrids and Cs during juvenile stages. In parallel, a feeding study was carried out with Cs on the acceptance of the formulated commercial diets in comparison to live feed. Cs progenies from one pair of parents were divided into 12 glass aquaria and fed one of three commercial starter feeds or Artemia nauplii for 21 days of growth. Fish fed Artemia were significantly larger (0.29±0.03 g) than other treatments and differences were also significant among commercial diet groups. However, survival was 97+3% in all diet groups and so we conclude that in this species formulated feeds can completely replace live Artemia during early life. Zebrafish have become a widely popular model organism. However, husbandry techniques, particularly in the early life stages, are poorly developed and lacking standardization. We described a novel larval culture protocol that results in high survival and rapid growth during the larval and juvenile stages. Larval fish were reared in static, saline water with constant light from 5-21 days post-fertilization (dpf). Turbidity was maintained with Nannochloropsis algae paste. For the first five days of feeding marine rotifers Brachionus plicatilis were maintained in the tank at a concentration of 200 rotifers/mL. At 11dpf, density of fish was decreased by 1/3. At 21dpf, 22 progenies were transferred to a freshwater recirculating system with a 13L:11D photoperiod. Fish were fed live Artemia nauplii supplemented with dry commercial feed. Once all fish were clearly identifiable as male or female, three pairs from three progenies were spawned. Sex ratio was determined in 16 progenies and the average percent male was 58±24%. These results demonstrated that the described protocol for larval rearing results in high survival and rapid growth without any negative impacts on fertility or sex differentiation. Using a novel larval rearing method, we produced gynogenetic (fatherless; using UV-irradiated sperm of common carp) zebrafish that had growth comparable to controls. All gynogens which reached reproductive maturity (n=46) were male. The fertility of gynogen males was first assessed by performing natural spawns; 60% of meiotic and 40% of mitotic gynogen males successfully fertilized oocytes compared to 100% of biparental males. In vitro fertilization (sperm collected by dissection and maceration of testes) was attempted with two meiotic and five presumed mitotic gynogens which failed to fertilize oocytes by natural spawns. Sperm from both meiotic and mitotic gynogens successfully fertilized oocytes in vitro. Fertilization rate was significantly lower among these fertile (in vitro) mitotic gynogens (50%±33) than in biparental controls; 75%±17). This is the first ever mass production of zebrafish gynogenes.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Chen, K-Y., Ludsin, S.A., M..M. Corey, P.D. Collingsworth, M.K. Nims, J.W. Olesik, K. Dabrowski, J.J. van Tassell, and E.A. Marschall. 2017. Experimental and field evaluation of otholith strontium as a marker to discriminate between river-spawning populations of walleye in Lake Erie. Can.J.Fish.Aquat.Sci. 74: 693-701. Kwasek, K., Rimoldi, S., Cattaneo, A.G., Parker, T., Dabrowski, K., Terova, G. 2017. The expression of hypoxia-inducible factor1-alfa gene is not affected by low-oxygen conditions in yellow perch (Perca flavescens) juveniles. Fish Physiol. Biochem. 43: 849-862. Delomas, T., Dabrowski, K. 2017. The importance of controlling genetic variation  remarks on Appropriate rearing density in domesticated zebrafish to avoid masculinization: links with the stress response. J. Exp. Biol. 200: 4078.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Diet formulations studies on fish meal replacement with soybean meal in yellow perch were presented during the Ohio Aquaculture Association annual meeting in January 29, 2016 and Wisconsin Aquaculture Association annual meeting in Milwaukee, March 10, 2016. In particular, these presentations introduced the topic of soybean-dependent nutritional programming, individual selection for growth and effects of anti-nutritional factors in yellow perch. This experiment, already in the 3rd year of study, addressed a multigenerational response and consists of 3 phases in which diets were changed after a period of "programming" that occurred in early life, juvenile stage. In the first phase fish are "programmed "on soybean meal based diet and then, after a period of fish meal-based diet are entering a "challenge" phase, a soybean meal-based diet. We observed that nutritional programming has a significant positive impact on the growth of yellow perch in the second generation and farmers in both meetings volunteered to become participants in the study. We attended the World Aquaculture Association conference in Las Vegas on February 28, 2016 and organized a special session on "Yellow perch aquaculture". Ten presentations were given on progress in yellow perch larvae and juvenile rearing, sex reversal, sperm cryopreservation, and hybridization with other percid species. We will publish our results in relevant peer-reviewed journals. These publications will allow the model developed in this project with yellow perch to be translated to other fish species. It is expected that these concepts will be adapted by aquaculturists throughout the U.S. We will attend the Aquaculture America conference in San Antonio in February 2017 and present a series of presentations that will include the report on first production of functional neomale (XX genotype) of yellow perch and we will be able to offer to fish farmers a means of all-female yellow perch production. This should increase production efficiency of this species by 30% and increase profitability by $550 per 1,000 marketable size fish. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students presented several reports to professional associations and the aquaculture community at large (WAS, Las Vegas) and established direct contacts with industry representatives. Specifically, producers of yellow perch for pond stocking and food fish have historically fed fish with currently available trout diets but have questioned whether such diets are optimal for percids. They have consistently asked the research community associated with North Central Aquaculture Center to develop a replacement low-cost diet for yellow perch. All carried projects have a special emphasis on the extension objective, as we invite the fish farming community to visit our laboratories and develop research agenda that will be compatible with their needs. We will encourage wide participation in extension workshops on yellow perch and feed formulation and will present such diet recommendations at workshops. We attended the World Aquaculture Society annual meeting in Las Vegas in 2016 and 8 oral papers were presented dealing with yellow perch nutrition, reproduction, larviculture, sex reversal, sperm cryopreservation and hybridization/gynogenesis/androgenesis with other percid fishes. The aim of this last task is to develop a model system for conservation of North American darters that will utilize a surrogate species (egg donor) and cryopreserved sperm to produce offspring via androgenesis. How have the results been disseminated to communities of interest?Dabrowski was invited speaker at the 2015 Annual Meeting of Animal Science Society of Mexico in Porto Vallarta and presented "Methionine requirements in fish - conventional and novel approaches using Atlantic salmon and common carp as a model". Principal Investigator has co-edited the book on "Biology and Culture of Percid Fishes" published in 2015 by Springer. He also co-authored 3 chapters in this book dealing with larval perch rearing, the effect of broodstock diets on yellow perch egg quality and muscle growth in yellow perch. Two manuscripts dealing with yellow perch larval rearing and yellow perch sperm cryopreservation were prepared and will be submitted to "North American Journal of Aquaculture" (December 2016). Dabrowski visited University of Mindanao, Philippines, and as a result of this collaboration popular paper was published in World Aquaculture in 2015 "Mindanao: An aquaculture haven in the Philippines". What do you plan to do during the next reporting period to accomplish the goals?We have submitted two research/extension proposals to the North Central Regional Aquaculture Center (USDA) that are under consideration for funding. The proposed research expands on large scale production of yellow perch neomales, cryopreservation of this sperm and transfer to fish farming community in the North Central Region. The second proposal deals with production of all-female hybrid walleye x sauger (saugeye) and if funded this new technology can be significantly advanced within the next 2 years. With the inclusion of larger percentages of soybean meal replacements in yellow perch diets, the effect of nutrition on reproductive performance and growth of F2 generation is underway. Our goal is to determine if replacing fishmeal with soybean meal protein would have an effect on fecundity, fertilization, larvae survival and growth. Juveniles obtained from individual females will be raised on soybean and control diets and compared based on nutritional history of their mothers. We plan to attend North Central Regional Aquaculture Center (USDA) annual meeting in Des Moines, Iowa in February 2017 where presentation will be given on progress on use of probiotics in yellow perch and results on possible production of axenic fish (germ-free). We will attend Aquaculture America in San Antonio, TX in February 2017 where 6 oral presentations will be delivered on yellow perch, cichlid hybrids and zebrafish.

Impacts
What was accomplished under these goals? The present study examined enrichment of live zooplanktonic organisms for yellow perch larvae feeding with polyunsaturated fatty acids (PUFA) supplemented with antioxidant, vitamin E. This experiment was carried out during the first 10 days of feeding by larvae on PUFA enriched rotifers and brine shrimp nauplii. Perch larvae were cultured in a specialized recirculating system with 50 L conical tanks equipped with surface spray and aeration via either a central air stone (Control 1) or a diffuser ring around the perimeter of the tanks (Control 2). The third treatment group had tanks with central air stones and α-tocopherol supplementation within the PUFA enrichment (Tocopherol). Temperature was maintained at 24.5±1.5?C and marine microalgae and evaporated salt were added to the system to maintain a turbidity of 5.5±0.6 NTU and a salinity of 2.7±0.4 ppt. The fatty acid and tocopherol composition of live feeds and fish were analyzed using gas chromatography and high-performance liquid chromatography, respectively. After 10 days of feeding, no significant differences (p≤0.05) in swim bladder inflation were found among treatments. The Tocopherol group had a higher rate of survival than the Control 2 group and a higher final weight and growth rate than the Control 1 and 2 groups. Two-way ANOVA also showed a significant difference in weight between fish with and without inflated swim bladders, but the interaction of swim bladder status and treatment group was not significant. These data suggest that PUFA enrichment with α-tocopherol supplementation provides the most advantageous larval diet for yellow perch. We are developing basic understanding of sex differentiation in yellow perch using hormonal treatment and environmental conditions to affect phenotypic sex in this species. The females of this species grow faster and to a larger final size than the males, resulting in their superior value in aquaculture. One option to obtain an all-female generation of this species is through sex reversal. By producing "neomales" (genetic females, but phenotypic males), we can obtain solely X-sperm, thus producing an entire generation of female fish. Masculinization is carried out with the use of 17α-methyltestosterone (MT), while the production of phenotypic females from genetic males is done using estradiol (E2). Previous authors have attempted to produce neomale yellow perch, but with only marginal success, likely due to the late timing of hormone exposure relative to sexual differentiation. The present study was conducted to determine efficiency of masculinization and feminization in fish less than 12 and 14 mm total length, and if growth differences are present following exposure to those hormones. The fish were exposed to the hormone via: MT-Diet (fed MT-enriched live Artemia nauplii for 21 days), MT-Immersion (subjected to MT bath on 4 occasions), or E2-Immersion (subjected to E2 bath on 4 occasions). Control fish were not exposed. Success of the sex reversal will be determined this December after several months of growth. At the conclusion of the hormone exposure phase, the fish were transferred to a recirculation system, while remaining separated by replicate. A 4-week feeding trial was conducted in a recirculation system containing 12 aquaria, 30 L each. Once the fish had acclimated to the new system and had accepted a formulated diet (21 days), all fish in each tank were weighed and the number was counted. The specific growth rate of fish exposed to either hormone by either means of delivery did not significantly differ from one another or the control (P=0.6091). These results indicate that exposure to sex reversal hormones does not have an impact on the growth of yellow perch following the exposure at early juvenile stage. We initiated a new area of research, utilization of the model fish species, zebrafish (Danio rerio) as the vehicle to address questions the aquaculture community is asking, but that cannot be answered using traditional, long lived aquaculture species. Larviculture methods utilized in this multi-generational study were based on an initial investigation of zebrafish and other cyprinid larvae, common carp and bighead carp, culture under continuous illumination. Zebrafish larvae, across all generations, were stocked to the experimental system at 5 dpf (days post fertilization) and fed live marine rotifers, Brachionus plicatilis. At 12 dpf, larvae were transitioned to and fed live brine shrimp (Artemia) nauplii for the duration of the experiment. Individuals were measured throughout the experiment and no significant difference in weight or length at 21 dpf was observed between generations 1-6. The study is ongoing and data for the seventh and eighth generations are currently beingcollected. Results suggest that zebrafish can be successfully grown under continuous illumination, and that doing so produces a higher growth rate and shorter generation time than the suggested 10 h dark, 14 h light regime. Based on the presented results, 8 generations of zebrafish can be produced inone year following this methodology, reducing the time required for multigenerational selection, a trait useful in various research fields.

Publications

• Type: Books Status: Published Year Published: 2015 Citation: Kestemont, P. Dabrowski, K. and Summerfelt, R. 2015. Biology and Culture of Percid Fishes. Principles and Perspectives (Springer Berlin) 35 Chapters, 901 pages Monography on North American and European Percids.
• Type: Books Status: Published Year Published: 2015 Citation: Zaccone, G., Dabrowski, K., Hedrick, M.S., Fernandes, J.M.O., Icardo, J.M. 2015. Phylogeny, Anatomy and Physiology of Ancient Fishes. CRC Press, 297 pp.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Results on diet formulations will be made available to feed mills in the U.S. via research reports, journal publications, presentations at selected aquaculture meetings, and face-to-face meetings with feed manufacturers. This project has a special emphasis on the extension objective. We will encourage wide participation in extension workshops on yellow perch and feed formulation and will present diet recommendations at such workshops. We attended Ohio Aquaculture Association meeting in 2015 and the Aquaculture America We plan to attend NCRAC annual meeting in Milwaukee, WI in March 2016 where presentation will be given on progress in yellow perch larvae and juveniles rearing, sex reversal and sperm cryopreservation. Results of this research will be presented at the annual meetings of the World Aquaculture Society, American Fisheries Society, and Fish Culture Section. Collaborations will include many researchers in the field in order to be as comprehensive and thorough as possible. We will seek input from industry by asking them to review drafts of publications and by these means also secure an expeditious way to implement our results into fish feed manufacturing. Presentations will be prepared and continuously updated with new findings. Information could potentially be shared via the internet at websites such as the NCRAC and OARDC. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Producers of yellow perch for pond stocking and food fish have historically fed fish with currently available trout diets but have questioned whether such diets are optimal for percids. They have consistently asked the research community to develop a replacement low-cost diet for yellow perch and to do so in a timely fashion. This project has a special emphasis on the extension objective. We will encourage wide participation in extension workshops on yellow perch and feed formulation and will present diet recommendations at such workshops. We attended Aquaculture America of the World Aquaculture Society annual meeting in New Orleans in February 2015 and 4 oral papers were presented dealing with yellow perch nutrition, bighead carp (cyprinid) larviculture, walleye embryos transport and bighead carp growth dependence on water temperature and hypoxia. How have the results been disseminated to communities of interest?The PI has co-edited the book on "Biology and Culture of Percid Fishes" published in 2015 by Springer. He co-authored 3 chapters in this book dealing with larval perch rearing, the effect of broodstock diets on yellow perch egg quality and muscle growth in yellow perch. Two manuscripts dealing with yellow perch larval rearing and yellow perch sperm cryopreservation are currently in preparation for submission to "North American Journal of Aquaculture" (January 2016). What do you plan to do during the next reporting period to accomplish the goals?With the inclusion of larger percentages of soybean meal replacements in fish diets, the effect of nutrition on reproductive performance is going to be analyzed in respect to different protein quality. This study will examine in the phase 3 both reproductive investment and progeny quality in yellow perch that have been nutritionally programmed on four different diets over the past two years. Our goal is to determine if replacing fishmeal with wheat gluten or soybean meal protein would have an effect on fecundity, fertilization, larvae survival and growth. Juveniles obtained from individual females will be raised on soybean and control diets and compared based on nutritional history of their mothers. We plan to attend North Central Regional Aquaculture Center (USDA) annual meeting in Milwaukee, WI in March 2016 where presentation will be given on progress in yellow perch "nutritional programming" in phase 3 of the study. The PI will organize special session during the World Aquaculture Society meeting in Las Vegas in February 2016 where 6 oral presentations will be delivered on yellow perch gynogenesis, sex reversal, larvae and juveniles rearing using live feeds enriched in vitamin E, ands perm cryopreservation.

Impacts
What was accomplished under these goals? The present study examined soybean-dependent nutritional programming, individual selection for growth and effects of anti-nutritional factors in yellow perch. This experiment consists of 3 phases in which diets were changed after a period of time in order to determine if a programming effect is occurring based on early life diets. In phase 1, juveniles were distributed into 12 tanks with 20 fish per tank, 3 groups per diet. Four diets were formulated and fed to fish for two months: 1) fishmeal (Control), 2) wheat gluten meal replacing 75% of the fish meal protein (WG), 3 and 4) two soybean meal varieties providing 75% replacement of fish meal protein (Soy A and Soy B). In phase 2, fish from all four dietary groups were fin-clipped, and combined in triplicate to be raised in a "common garden" design for 7 months. All of the fish were fed the same fishmeal-based diet. In phase 3, or the "challenge" phase, all of the fish are being fed a soy protein-based diet for 7 months. In this ongoing phase we are determining if nutritional programming has a significant impact on those fish that were originally fed soy protein as juveniles. Preliminary results from phase 1 showed a significantly depressed growth in the fish fed soy-based diets compared to the Control and WG diets. This trend was reversed in phase 2 and fish fed soy A and B gained significantly more weight than fish from the other two groups. In phase 3 we are observing that fish originally fed soybean meal (in phase 1), are gaining more weight than the other groups. We hypothesize that these fish indeed show a soybean-dependent nutritional programming as adults. We are developing basic aspects of yellow perch intensive culture and one of them is production of all-female progeny through gynogenesis and sex reversal. In order to secure efficient production of gynogenotes, we attempted to use heterologous sperm of other percids. As the literature on survival of yellow perch cross with walleye (Sander vitreus) (PxW) and yellow perch with logperch (Percina caproides) (PxL) is contradictory, we carried out several experiments to test the outcome of these crosses. Three spawnings of domesticated yellow perch and wild walleye were carried out between March and April by stripping gametes and in vitro fertilization. Control fertilization of perch x perch was also carried out. Logperch (Delaware Reservoir, OH) was used to fertilize yellow perch eggs on 3 occasions between April and May. Embryonic development was monitored in replicated baskets incubated in California trays or shallow troughs at 14-18oC. Fish were transferred 2-3 days after hatching and rotifers followed by brine shrimp nauplii were added several times during the day and were present and alive 24h. Food for zooplankton was also provided (algae) that maintained high turbidity (18+8 NTU). This phase was terminated after 10-12 days, and juveniles of perch x logperch hybrids transferred to a regular aquaria. Survival of PxW hybrids was not significantly different at the blastula stage (3-10 hpf), however hybrid survival decreased toward 6-7 days post fertilization. PxL hybrid survival decreased further near the time of hatching. Both hybrids showed notochord deformities, and PxW remained almost motionless, whereas PxL were very active swimmers after forced hatch. We conclude that walleye sperm can be used to induce gynogenesis in yellow perch, while hybridization with logperch requires further studies. PxL hybrids have been raised to juveniles of 4-5 cm body length by September (90DPF).

Publications