ADVANCE AQUACULTURE PRODUCTION THROUGH REFINING AND TRANSFERRING TECHNOLOGY OF COMMERCIAL-SCALE PRODUCTION OF MONOSEX YELLOW PERCH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1026266
• Grant No.: 2021-70007-34785
• Cumulative Award Amt.: $300,000.00
• Proposal No.: 2020-05416
• Project Start Date: Sep 1, 2021
• Project End Date: Aug 31, 2024
• Grant Year: 2021
• Program Code: [AQUA]- Aquaculture Research

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

Performing Department
Animal Sciences

Non Technical Summary
Yellow perch (YP) females grow significantly faster and reach larger body sizes than males and amonosex cultureholds a great potential as a method to increase the efficiency and profitability of perchfood and recreational aquaculture by improving growth rate, and eliminating the problem of prolific reproduction, precocious maturity and their consequences. The overall goal of this project is to advance the North Central Region(NCR) and Great Lakes Region (GLR)yellow perchaquaculture for which a foundation of knowledge and activity currently exists but where significant barriers remain. To achieve the goal, we will pursue the following objectives:1) refine a technology of commercial-scale production of genetically fast-growing monosex female YP populations;2) transfer the technology of producing fast-growing monosex seed (fertilized "eyed-eggs" and fry) in YP to industry through workshops and hands-on training;3) promote monosex aquaculture and related training by delivering monosex fish to fish farms and performing on-farm and on-station demonstrations of genetically all-female YP populations vs. mixed-sex groups in the NCR and GLR;4) disseminate demonstration results to aquaculture industry and to end-user customers via fact sheets, scientific publications, and an on-farm field day.By the end of the project, we will expect to provide fertilized "eyed eggs" tofingerling producers and train them to produce monosex seed/fry in their own farms or hatcheries for dissemination of improved fingerlings to commercial grow-out producers.Theoutcomes will enable us or farmers to produce and use high quality fast-growing monosex fingerlings. Therefore, this will benefit fish farmers by increasing the efficiency and profitability of yellow perch aquaculture production in the U.S.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
303	3719	1080	100%

Knowledge Area
303 - Genetic Improvement of Animals;

Subject Of Investigation
3719 - Other cultured finfish;

Field Of Science
1080 - Genetics;

Keywords

monosex

farm demonstration

technology

transfer

yellow perch

Goals / Objectives
The overall goal of the proposed project is to advance the North Central Region (NCR) and Great Lakes Region (GLR) yellow perch (YP) aquaculture for which a foundation of knowledge and activity currently exists but where significant barriers remain. To achieve the goal, we will pursue the following objectives:Refine a technology of commercial-scale production of genetically fast-growing monosex female YP populations.Transfer the technology of producing fast-growing monosex seed (fertilized "eyed-eggs" and fry) in YP to industry through workshops and hands-on training.Promote monosex aquaculture and related training by delivering monosex fish to fish farms and performing on-farm and on-station demonstrations of genetically all-female YP populations vs. mixed-sex groups in the NCR and GLR.Disseminate demonstration results to aquaculture industry and to end-user customers via fact sheets, scientific publications, and an on-farm field day.

Project Methods
For Objective1 and 2 (Yr 1), we will refine monosex production technology by crossing neo-males/females with normal genetically improved superior strains to produce large-scale populations of fast-growing all-female YP, and nurse them in replicated ponds and tank systems using live-feeding technology to compare their survival vs. mixed-sex stocks. In the same time, we will conduct workshops and hands-on training on production of monosex seed (fertilized "eyed eggs" and fry) in this species for farmers.For Objective. 3 (Yr 1-2), we will conduct demonstration of the monosex populations of YP vs. local mixed-sex populations in two selected demonstration farms that attended the training at different latitudes in the GLR and train farmers how to raise monosex fish. A demonstration will be also conducted in flow-through/recirculating aquaculture systems (RAS) in a research station at Piketon. For the on-farm pond demonstration, the demonstration sites will conduct replicated tests of the monosex populations vs. mixed-sex groups using two types of rearing methods: 1) one site will rear monosex populations and mixed-sex groups in separate ponds, each having two replicates; 2) another site will raise the genetically monosex populations and mixed-sex groups communally in two ponds. This communal rearing is necessary in order to avoid environmental effects (pond environment, different density and feeding ration resulting from differential survival, and water quality et al), and to get solid results. Molecular markers will be used to assign both groups to their family of origin for trait comparisons using methods previously published by our lab. For the on-station RAS test, we will have different temperature/photoperiod regimes: constant temperature, ambient temperature, each having a constant photoperiod and an ambient photoperiod, to test whether constant temperature and constant photoperiod treatment can further improve growth and production of monosex fish by limiting sexual maturation. Fish in each treatment will be tagged with Visible Implant Elastomer (VIE) color tags and communally raised at/in the same density/environment for an accurate comparison. The on-farm pond demonstration trials and on-station RAS test will be conducted for one year; or a full cycle (16 months), if a year no-cost extension is granted.For Objective. 4, at the end of the demonstrations, fish will be harvested, and data, including survival, growth rate, fillet yield, feed conversion ratio, coefficient of variation, and sex ratio (for mixed-sex groups), will be compared between the all-female group and each mixed-sex group of normal mixed-sex population. On-farm field days will be held to disseminate project results at harvest of ponds. Project results will be disseminated to the aquaculture industry and to end-users via fact sheets, scientific publications, and website media. We will also conduct economic analysis using the harvest data. By the end of the project, we will expect to provide cryopreserved sperm or fertilized "eyed eggs" to 2-4 fingerling producers and train them to produce monosex seed/fry in their own farms or hatcheries for dissemination of improved fingerlings to commercial grow-out producers or farmers.

Progress 09/01/21 to 08/31/24

Outputs
Target Audience:Aquaculture and aquaponic farmers and producers in the North Central Region and Great Lake Region. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided two research assistants and a graduate student with opportunities for training and professional development in the fish genotyping, fish breeding, production of monosex fish and all-female fry. How have the results been disseminated to communities of interest?We held a workshop disseminating and showing the monosex technology in yellow perch to the aquaculture industry and producers. We have disseminated some of our experimental results to aquaculture communities via five journal publications and four conference or workshop presentations. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? For Objective 1, we have refined monosex production technology by crossing neo-males with normal genetically improved superior females to produce large-scale populations of fast-growing all-female yellow perch. For further refining monosex technology in yellow perch, we completed and published the following studies: 1) Transcriptome profile of sex-related gene expression and effects of cyp19a1 siRNA- silencing on sex differentiation and determination in yellow perch. The findings from this study provide valuable insights for future research into the underlying mechanisms involved and hold significant promise for sex control in aquaculture and the result was published in the journal Aquaculture (2024: 590). 2) Hormonal effects on sex differentiation and growth in juvenile yellow perch. This paper was published by Fish Physiology and Biochemistry in 2022 (DOI:10.1007/s10695-021-01038-0). 3) The effect of density on sex differentiation, stress, and related gene expression. This paper was published by PLoS One (2022: 17 (5)). 4) Effects of dmrt1 siRNA-silencing on sex differentiation and determination in yellow perch. A manuscript from this study is under review by the journal Aquaculture Reports. For Objective 2, A workshop was conducted to teach and show participants a technology for commercial scale production of monosex yellow perch, including how to breed monosex fish with neomale perch and rear monosex fry. We also conducted more than 40 one-on-one counseling and training services to transfer the technology and information of producing fast-growing monosex fish in yellow perch to industry and end-user customers. For Objective 3, we have completed farm and pond demonstration of the monosex populations of yellow perch vs. local mixed-sex fish using both separate rearing and communal rearing, which is necessary to avoid environmental effects (pond environment, different density and feeding ration resulting from differential survival, and water quality etc.) and to get solid results. For the separate rearing, selected monosex fish and a local mixed population were reared in four separate ponds, each having two replicates. At the end of 16-month experiment, monosex population grew 78.84% faster than regular males, 10.34% faster than regular females, and 39.39% faster than regular male and female together. For the communal rearing, the genetically monosex populations and mixed sex groups were raised for 16 months in two ponds and molecular markers were used to assign both groups to their family of origin for trait comparisons using methods previously published by our lab. The results showed that monosex population grew 90.8% faster than regular males, 39.1% faster than regular females, and 54.9% faster than regular male and female together in the communal pond. For the tank test, we conducted experiments in different temperature/photoperiod regimes: constant temperature with a constant photoperiod (0D: 24L) and an ambient natural photoperiod, and ambient temperature with an ambient natural photoperiod to test whether constant temperature and constant photoperiod treatment can further improve growth and production of monosex fish by limiting sexual maturation. Fish in each treatment were tagged with Visible Implant Elastomer (VIE) color tags and communally raised in the same environment for an accurate comparison. The results showed that monosex population grew 53.35%, 51.78% and 171.70% faster than regular mixed-sex group in the treatment of constant temperature with a constant 24h light photoperiod, constant temperature with an ambient natural photoperiod, and ambient temperature with an ambient natural photoperiod, respectively. For Objective 4, we held a bus tour and 16 field and facility tours to disseminate project and monosex fish development information to more than 600 clients with aquaculture and agriculture industry, end-user customers, and communities of interest. We also disseminated the monosex technology in yellow perch to additional 261 aquaculture producers through workshops, meetings and one-on-one counseling services. Meanwhile, we have disseminated our experimental results to aquaculture communities via five journal publications and four conference or workshop presentations.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Xie, D. K., H. P. Wang, R. Othman, H. Yao, P. OBryant, D. J. Rapp, and Y. J., Hong. 2024. Transcriptome profile of sex-related gene expression and effects of cyp19a1 siRNA-silencing on sex differentiation and determination in yellow perch. Aquaculture 590.

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Aquaculture and aquaponic farmers and producers in the North Central Region and Great Lake Region. Changes/Problems:Recentwinters werewarm, which affected the gonadal development of yellow perch What opportunities for training and professional development has the project provided?The project has provided two research assistants and one graduate student with opportunities for training and professional development in the fish genotyping, fish breeding, production of monosex fish and all-female fry. How have the results been disseminated to communities of interest?We held a workshop disseminating and showingthe monosextechnology in yellow perch to the aquaculture industry and producers.We have disseminatedsome of our experimental results to aquaculture communitesviafourscientific publications. What do you plan to do during the next reporting period to accomplish the goals?We planto complete the demonstration experiment of rearing the selected monosex fish and a local mixed population in four separate ponds, each having two replicates, and test whether constant temperature and constant photoperiod treatment can further improve growth and production of monosex fish in tank system by limiting sexual maturation. We will disseminate our final results to aquaculture industryand end-users throughscientific publications and/or factsheets.

Impacts
What was accomplished under these goals? For Objective 1, we have refined monosex production technology by crossing neo-maleswith normal genetically improved superior females to produce large-scale populations of fast-growing all-female yellow perch, and nurse them in replicated ponds. For further refining monosex technology in yellow perch, we completed and published the following studies. The effects of cyp19a1 siRNA-silencing on sex differentiation and determination in yellow perch: In this study, we found 1) the siRNA interference resulted in an incomplete masculinization and hermaphroditism of all-female monosex yellow perch, displaying the presence of male spermatocytes along with female oocytes; 2) the gonad weight and GSIs of the cyp19a1 group were significantly lower than those in the control groups, suggesting that the cyp19a1-siRNA interference might have led to a trend of male type of gonadal development; 3) the expression level of the dmrt1 along with cyp19a1 in the treatment group was significantly upregulated compared to the control groups, suggesting the correlation roles between these two genes in the regulation of sex development. The findings provide valuable insights for future research into the underlying mechanisms involved and hold significant promise for sex control in aquaculture. The manuscript was submitted to the journal Aquaculture. Hormonal effect on sex differentiation and growth in juveniles: We completed a study on the effect of methyltestosterone (MT) on sex differentiation and growth in juvenile yellow perch. The identification of the labile period of sex determination that is manipulated by MT dosage and age of treatment in this study would be a valuable progression toward optimizing commercially viable regimes for commercial scale production of all-female yellow perch for the aquaculture industry. This paper was published by Fish Physiology and Biochemistry in 2022: DOI:10.1007/s10695-021-01038-0 The effect of density on sex differentiation, stress, and related gene expression: An experiment was completed to evaluate the effects of density on sex differentiation, sexual dimorphism, stress, and related gene expression. In this study, we found that the stocking density did not influence the sexual determination of yellow perch. However, high density is a chronic stress that has adverse effects on fish well-being, growth, survival, expression of stress-related genes, and mobilization of liver reserve. A stocking density of 1,000 fish/m3 could be recommended to reduce stress, increase survival, and provide a better state of well-being and growth performance for rearing management of juvenile yellow perch for farmers.This paper was published by PLoS One in 2022: e0267904. https://doi.org/10.1371/journal.pone.0267904 For Objective 2, A workshop was conducted to teach and show participants a technology for commercial scale production of monosex yellow perch, including how to breed monosex fish with neomale perch and rear monosex fry. For Objective 3, we have completed the Year 2 demonstration of the monosex populations of YP vs. local mixed-sex populations in field ponds. The genetically monosex populations and mixed sex groups were raised for 16 months in two ponds. For the communal rearing, which is necessary to avoid environmental effects (pond environment, different density and feeding ration resulting from differential survival, and water quality et al) and to get solid results, molecular markers were used to assign both groups to their family of origin for trait comparisons using methods previously published by our lab. A local mixed-sex population was raised in separate ponds also for a double comparison. The results showed that monosex population grew 90.8% faster than regular males, 39.1% faster than regular females, and 54.9% faster than regular male and female together in the communal pond; and the monosex perch group in the communal pond grew 83.2%, 9.9% and 32.2% faster respectively than the regular males, regular females, and the mixed sex group in the separate pond. We are rearing the selected monosex fish and a local mixed population in four separate ponds, each having two replicates, for further comparison. For Objective 4, We have disseminatedsome of our experimental results to aquaculture communites and end-users through fourscientific publications. We alsodisseminatedthe monosextechnology in yellow perch to the aquaculture industry and producers visa workshop.

Publications

• Type: Books Status: Published Year Published: 2023 Citation: Piferrer F. and H.P. Wang. 2023. Epigenetics in Aquaculture. Wiley-Blackwell. https://www.wiley.com/en-us/Epigenetics+in+Aquaculture-p-9781119821915
• Type: Book Chapters Status: Published Year Published: 2023 Citation: Wang, H.P., and Z.G. Shen. 2023. The Potential Role of Epigenetics in Aquaculture: Insights from Different Taxa to Diverse Teleosts. In: Epigenetics in Aquaculture (Piferrer F. & H.P. Wang Ed.). Wiley-Blackwell.
• Type: Book Chapters Status: Published Year Published: 2023 Citation: Shen, Z.G., Y. Yu, and H.P. Wang. 2023. Environmental Epigenetics in Fish: Response to Climate Change Stressors. In: Epigenetics in Aquaculture (Piferrer F. & H.P. Wang Ed.). Wiley-Blackwell.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Han-Ping Wang, Paul OBryant, Dean Rapp and Hong Yao, 2023. Monosex production technology in food fish. Atlantic seafood technology conference, May 16-17, Miami, Florida.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Othman R, Wang H-P, Elabd H, Xie D-K, Yao H, O'Bryant P, et al. 2022. The effect of density on sex differentiation, sexual dimorphism, stress, and related gene expression in yellow perch. PLoS ONE 17(5): e0267904. https//doi.org/10.1371/journal.pone.0267904
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Rafidah Othman, Xiao?Jun Ron, Hong Yao, Paul OBryant, Dean Rapp, Jing?Chen Pei, Hong?Juan Wu, and Han?Ping Wang. 2022. The effect of methyltestosterone (MT) on sex differentiation and growth in juvenile yellow perch (Perca flavescens). Fish Physiol Biochem, https://doi.org/10.1007/s10695-021-01038-0.

Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Aquaculture and aquaponic farmers in the North Central Region and Great Lake Region. Changes/Problems:The last winter was warm, which affected the gonadal development of yellow perch What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?We will disseminate the project results to aquaculture industry and communities next year or at the end of this project What do you plan to do during the next reporting period to accomplish the goals?We planed tocompleteYear 2demonstration of the monosex populations of YP vs. local mixed-sex populationsin thecommunal rearing ponds.Wewill rear monosex populations and mixed-sexgroups in separate ponds, each having two replicates, for the demonstration of the monosex populations of YP vs. local mixed-sex populations. We planed toconduct workshops and hands-on training on production of monosex seed (fertilized "eyed eggs" and fry) in yellow perchfor farmers. We expect to request for oneyear no-cost extension to complete the project.

Impacts
What was accomplished under these goals? For Objective 1 and 2, we haverefined monosex production technology by crossing neo-males/females with normal genetically improved superior strains to produce large-scale populations of fast-growing all-female yellow perch (YP), and nurse them inreplicated ponds. For furtherrefining monosex technology in yellow perch, we completed and published the following studies Hormonal effect on sex differentiation and growth in juvenile:We completed a study on effect of methyltestosterone (MT) on sex differentiation and growth in juvenile yellow perch. The identification of the labile period of sex determination that is manipulated by MT dosage and age of treatment in this study would be a valuable progression toward optimizing commercially viableregimes for commercial scale production of all-female yellow perch for aquaculture industry. This paper was published by Fish Physiology and Biochemistry in 2022:https://doi.org/10.1007/s10695-021-01038-0. The effect of density on sex differentiation, stress, and related gene expression:An experiment was completed to evaluate the effects of density on sex differentiation, sexual dimorphism, stress, and related gene expression. In this study, we found that the stocking density did not influence the sexual determination of yellow perch. However, high density is a chronic stress that has adverse effects on fish wellbeing, growth, survival, expression of stress-related genes, and mobilization of liver reserve. A stocking density of 1,000 fish/m3could be recommended to reduce stress, increase survival, and provide a better state of wellbeing and growth performance for rearing management of juvenile yellow perch for farmers.This paper waspublished by PLOS One in 2022: e0267904. https//doi.org/ 10.1371/journal.pone.0267904. Proteomic profile and characteristics of skeletal muscle of the fast-growing perch:We completed a study on comparing skeletal muscle proteomic profiles, histochemical characteristics, and expression levels of myogenic regulatory factors between fast- vs. slow-growing yellow perch and identified the proteins that might play a crucial role in the muscle growth. The identified proteins could be useful markers of muscle growth to improve the growth performance in yellow perch. The study was published in Scientific Reports in 2021: https://doi.org/10.1038/s41598-021-95817-7 For Objective3, we havecompleted Year 1demonstration of the monosex populations of YP vs. local mixed-sex populations in ponds.The genetically monosex populations and mixed-sexgroups were raised communally in two ponds. This communal rearing is necessary in order to avoid environmental effects (pondenvironment, different density and feeding ration resulting from differential survival, and water quality et al), and to get solidresults. Molecular markers are beingused to assign both groups to their family of origin for trait comparisons using methodspreviously published by our lab. This family identification process will be completed by the February of 2023.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Othman R, Wang H-P, Elabd H, Xie D-K, Yao H, O'Bryant P, et al. 2022. The effect of density on sex differentiation, sexual dimorphism, stress, and related gene expression in yellow perch. PLoS ONE 17(5): e0267904. https//doi.org/ 10.1371/journal.pone.0267904
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Rafidah Othman � Xiao-Jun Ron � Hong Yao � Paul O. Bryant � Dean Rapp � Han-Ping Wang. 2021. The effect of methyltestosterone (MT) on sex differentiation and growth in juvenile yellow perch (Perca flavescens). Fish Physiol Biochem https://doi.org/10.1007/s10695-021-01038-0
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Wang H. P., Z. G. Shen, H. Yao, Paul OBryant, and Dean Rapp. 2021. Integrated sequencing uncovers genomic regulation and epigenomic modification of sexual size dimorphism in yellow perch. In: Proceedings of the World Aquaculture Society Conference, https://www.was.org/Meeting/Program/PaperDetail/157485. August 11-14, 2021, San, Antonio, Texas.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Yao, H., H.P. Wang, Paul OBryant, and Dean Rapp. 2021. Large-scale production and commercialization of monosex yellow perch. In: Proceedings of the World Aquaculture Society Conference, https://www.was.org/Meeting/Program/PaperDetail/157525. August 11-14, 2021, San, Antonio, Texas.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Karolina Kwasek Young Min Choi, Hanping Wang, Kichoon Lee, John Mark Reddish & Macdonald Wick. 2021. Proteomic profle and morphological characteristics of skeletal muscle from the fast and slow growing yellow perch (Perca favescens). Scientifc Reports 11:16272. https://doi.org/10.1038/s41598-021-95817-7.