Source: UNIVERSITY OF MARYLAND BALTIMORE COUNTY submitted to NRP
PRODUCTION OF REPRODUCTIVELY STERILE RAINBOW TROUT FOR ENVIRONMENTALLY-RESPONSIBLE AND ECONOMICALLY-SUSTAINABLE US AQUACULTURE INDUSTRY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1013857
Grant No.
2017-70007-27177
Cumulative Award Amt.
$320,984.00
Proposal No.
2017-04465
Multistate No.
(N/A)
Project Start Date
Sep 1, 2017
Project End Date
Aug 31, 2021
Grant Year
2017
Program Code
[AQUA]- Aquaculture Research
Recipient Organization
UNIVERSITY OF MARYLAND BALTIMORE COUNTY
1000 HILLTOP CIRCLE
BALTIMORE,MD 21250
Performing Department
Dept. of Marine Biotechnology
Non Technical Summary
Aquaculture is becoming increasingly important to resolve the current and projected shortfalls in aquatic food production. Optimization of aquaculture methods will be necessary to maximize cost-effective fish production and minimize ecological impact. One approach to increasing aquaculture production to meet the demand of seafood supply is through the use of selectively bred (non-native species in some cases) and, eventually, genetically-modified fish that exhibit enhanced growth characteristics and/or disease resistance. However, these fish may escape from captivity, propagate and/or interbreed with wild stocks, subsequently changing the genetic composition of populations or causing species extinction. Because intensifying aquaculture activity is essential for securing our future seafood supply, it is imperative that highly effective containment methods are available to prevent escaped aquaculture fish from propagation in the natural environment. We developed a fish sterilization technology that utilizes bath-immersion to administer transient gene silencers, against deadend gene, into the eggs, which disrupts primordial germ cell development and results in the production of reproductively sterile fish. Promisingly, it has also been shown to induce sterility in salmonids. The goal of this proposal is to optimize parameters and establish the best possible conditions for applying this bath-immersion sterilization strategy to rainbow trout and study growth performance of these sterile fish. The expected achievement will promote environmentally-responsible aquaculture development, public awareness of containment issues, and garner the attention of stakeholders and investors in the aquaculture industry. This goal will be achieved by 1) Optimize dnd-MO-Vivo post-fertilization immersion protocol for large scale production to determine the most economical conditions that achieve 100% sterility induction; 2) Establish pre-fertilization immersion protocols that may further reduce production costs, and optimize treatment parameters and conditions to achieve 100% sterility induction; 3) Compare growth related performance traits among sterilized diploid, fertile diploid, and triploid fish.The expected successful outcomes of rainbow trout work will further advance this sterilization technology, with future expected applications to other important aquaculture species. This achievement will have significant impacts on environmental management, cost-reduction and rights-protection, promote public awareness of containment issues, and garner the attention of stakeholders and investors in the aquaculture industry, which will further promote environmentally-responsible aquaculture development. Since sterility is inducible by bath-immersion, our method makes it convenient to produce a fertile broodstock population (i.e., skip the treatment) when required. In addition, the ease and safety of this approach means it can easily be transferred to small producers, which further guarantees the immediate advancement to promote US aquaculture production in the short term.
Animal Health Component
80%
Research Effort Categories
Basic
10%
Applied
80%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3083711105040%
3053711102040%
3073711108120%
Knowledge Area
305 - Animal Physiological Processes; 308 - Improved Animal Products (Before Harvest); 307 - Animal Management Systems;

Subject Of Investigation
3711 - Trout;

Field Of Science
1081 - Breeding; 1020 - Physiology; 1050 - Developmental biology;
Goals / Objectives
Fish farming has become increasingly important as a means to meet the growing demand for aquatic food production. Increased reliance on aquaculture production will also require implementation of commercial fish farming methods that are environmentally sustainable. Without minimizing environmental risk generated from fish farming activities, the development of aquaculture will be impeded. One of the most serious ecological impacts is the threat of genetic contamination and ecological imbalance posed by escaped farmed fish that are selectively bred to a large degree, non-native and/or (eventually) genetically-engineered. It is crucial that efficient, cost-effective methods of containment are developed. In addition, many farmed fish attain sexual maturity before reaching market size. Sexual maturation is associated with a substantial decrease in somatic growth due to the diversion of energy into development of the gonads. The period of intensive gonadal growth during sexual maturation also results in deterioration of flesh quality and an increase in susceptibility to stress and disease. Sterilization minimizes energy input toward gonadal growth while enhancing muscle (flesh) development and promoting fish health. Furthermore, sterility is a means for producers to protect their valuable, genetically-selected strains from unauthorized propagation. Equally important to the ability to sterilize a fish is the impact that sterilization approach has on expression of production traits for which that species or strain was desired for culture, or for which selective breeding programs made major investments to improve.We have developed a fish sterilization technology that utilizes bath-immersion to administer transient "gene silencing" Morpholino oligomer (MO) that transiently blocked the expression of Dnd (dnd-MO), an essential protein for PGC development. Of particular interest, we discovered that a molecular transporter, also known as Vivo, can effectively carry dnd-MO across the chorion, enter the embryo and reach the target cells, PGCs, which led to the elimination of germ cells and resulted in the development of reproductively sterile fish. Promisingly, it has also been shown to induce sterility in salmonids. The goal of this proposal is to optimize parameters and establish the best possible conditions for applying a bath-immersion sterilization strategy to rainbow trout. The best possible conditions are defined as the shortest immersion time and lowest concentration of dnd-MO-Vivo that can achieve 100% sterility. Rainbow trout has been one of the major freshwater aquaculture species for several hundred years and it is cultured globally using well-established systems. In the past 6 decades, production of rainbow trout has increased exponentially. Since 1980, rainbow trout production has grown more than 5-fold, peaking at 878,000 tons in 2012. The United States is one of the ten top-producing countries with 113.1 million tons of production in 2015. Although many aspects of trout farming are highly advanced, research and development teams are continuously attempting to increase production efficiency and market profit by selecting genetically superior lines that are fast-growing and disease resistant including lines we developed at NCCCWA for the rainbow trout industry. However, these well-selected fish may escape from captivity, propagate and/or interbreed with wild stocks, subsequently changing the genetic composition of wild populations or potentially displacing endemic species and causing species extinction. It is imperative that highly effective containment methods are available to prevent aquaculture escapees from propagation in the natural environment. Moreover, the early sexual maturation (before market size) of male rainbow trout reduces the growth and flesh quality of male rainbow trout, due to the diversion of energy into development of the testes. Thus, mono-sex female progeny, which exhibit late sex maturation, are commonly used for rainbow trout aquaculture. To produce all-female progeny, genetic (XX) females are fed with diets that contain 17α methyl-testosterone (MT), which sex-reverses the genetic females into phenotypic males that produce all-X sperm. The sperm is used to fertilize eggs to produce all-female (XX) progeny. MT is an endocrine disrupter at very low (ppb) levels and can disrupt the normal functions of the reproductive systems of humans and animals. Although the amount of MT used by the rainbow trout industry is very small because only a limited number of males for breeding are treated as fry, is easily contained before reaching the environment, and is not used on the marketed fish, the use of steroids in the production cycle is often resisted by the public affecting market value of the product.The goal of this proposed research will be achieved by three specific objectives: 1) Optimize the rainbow trout dnd-MO-Vivo post-fertilization immersion protocol for large scale production to determine the most economical conditions that achieve 100% sterility induction. 2) Establish pre-fertilization immersion protocols that may further reduce production costs, and optimize treatment parameters and conditions to achieve 100% sterility induction. 3) Compare growth related performance traits among dnd-MO sterilized diploid female (2N-S), fertile diploid female (2N), and triploid female fish (3N).
Project Methods
The goal of this proposal is to optimize the bath-immersion sterilization technology for application to rainbow trout to demonstrate the feasibility of applying this technology to aquaculture species. The goal of this proposed research will be achieved through the following three specific objectives:1) Optimize the rainbow trout dnd-MO-Vivo post-fertilization immersion protocol for large scale production to determine the most economical conditions that achieve 100% sterility induction. We will initially compare the level of the disruption of PGC development in embryos that are exposed to five different conditions, including 2 different control groups and 3 experimental groups that will be treated with different concentrations of salmonid dnd-MO-Vivo. The two control groups will be water-only control and the immersion with 40 µM control MO-Vivo for 24 hours and then 20 µM for 24 hours. The immersion conditions of three experimental groups with salmonid dnd-MO-Vivo are 1) 50 µM for 24 hours and then 25 µM for 24 hours, 2) 40 µM for 24 hours and then 20 µM for 24 hours and 3) 30 µM for 24 hours and then 15 µM for 24 hours. Duplicates of each treatment will be conducted; oxygen will be injected into each bottle twice a day. After 48-hour immersion at 4 ºC on a 60 rpm orbital shaker and then incubated to hatch at 5-6 °C in a Heath tray hatching incubator.At the eyed stage and just before hatching, 24 embryos will be sampled from each group and processed for subsequent analysis of PGC development by whole-mount in situ hybridization using rainbow trout vasa probes. Additionally, 16 embryos per group will be collected for qPCR analysis for measuring the levels of germ cell marker genes, dnd, nonos3 and vasa. The survival rate and normality of the embryos/larvae in each treatment will be documented and compared. Results obtained from whole-mount in situ, germ cell marker gene qPCR will be used for statistical analysis and as references for optimizing the bath-immersion condition. At 8 months, 16 fish from each group will be dissected and gonadal tissue will be collected for histological analysis. The results of gonadal histology will be used for comparisons with the results obtained from whole-mount in situ, germ cell marker gene qPCR. Fish groups in which molecular markers show possible sterility will be identified and raised to 16-24 months old to evaluate their gonadal development.2) Establish pre-fertilization immersion protocols that may further reduce production costs, and optimize treatment conditions to achieve 100% sterility induction. To continue the optimization of the bath-immersion technology, we will investigate the possibility of administering dnd-MO-Vivo into fish eggs before fertilization and water hardening. Therefore, pre-fertilization immersion protocols will be developed and compared with the post-fertilization immersion (Objective 1). Similar to the Objective 1, two control groups will be conducted under the same conditions, except the system water will be replaced with ovarian fluid and modified Cortland medium for pre-fertilization immersion. Because the egg chorion is more permeable before fertilization, the concentration of salmonid dnd-MO-Vivo can be reduced. Accordingly, three experimental groups will be conducted using the immersion conditions of 1) 30 µM for 24 hours and then 15 µM for 24 hours, 2) 20 µM for 24 hours and then 10 µM for 24 hours, 3) 10 µM for 48 hours. Unfertilized rainbow trout eggs will be immersed with control MO-Vivo or salmonid dnd-MO-Vivo that are described above. After immersion, the eggs will be fertilized as described above. The fertilized eggs will be set for hatching and sampling at the eyed stage, immediately before hatching, and 8 months old. Samples will be collected for whole-mount in situ, qPCR and gonadal histology analyses following the experimental procedures that are described in Objective1, and results will be used for statistical analysis and as references for optimizing the pre-fertilization bath-immersion conditions.3) Compare growth related performance traits among dnd-MO sterilized diploid female (2N-S), fertile diploid female (2N), and triploid female fish (3N).Triploid induction and verification: Triploid induction will be achieved by applying 633 kg/cm2 (9000 psi) for 8 min starting at 30 min post-fertilization at 10.0 ± 0.3 °C. Ploidy levels in dispersed cells from eyed-stage embryonic tissues will be determined based on DNA content using focusing flow cytometer and Becton Dickinson Cycle TEST PLUS reagent kits. Ten embryos per 3N family will be examined to confirm polyploidy induction success. In addition, gonads will be examined for all tagged fish by the end this study, to confirm sex and that gonad development was disrupted in 3N and 2N-S fish, and identify non-maturing 2N fish. If females with developing ovaries are identified in the 3N group, red blood cells will be used to confirm triploidy.Design and Procedures: The fish will be produced and reared at NCCCWA. Animals from an NCCCWA selective breeding program aimed at improving fillet yield are available for this research. In spring of 2018, after two generations of selection for fillet yield, 20 females and 20 males will be selected for breeding based on family estimated breeding values (EBV) for fillet yield at 14 months post-hatching and 13 month body weight with the aim of maximizing genetic diversity for both traits. Single-sire X single-dam matings will be conducted and sires and dams will be used for only one mating. Each family will be treated to yield 2N-S (based on the best approach available at the time), 2N and 3N offspring. At hatching, 10 fish from each 2N-S family will be examined for germ cell depletion as described for objective 1. Ten families will be selected at swim-up for the study based on EBVs, triploid induction rate, sterilization rate, and a minimum of 100 fish per treatment available for rearing. Total tank biomass will be determined monthly. At approximately 4 months post-hatch, 50 fish per unit or tank will be anesthetized and given unique identification by tagging with a passive integrated transponder intraperitoneally. Each treatment, 2N-S, 2N, and 3N will be divided among 4 tanks each, with 10 individuals from each of the 10 families represented in each of the tanks. Individual BW and FL will be measured monthly starting at this sorting using a Biomark pit tag integrated Data Collection System (DCS). Fish will be photographed at 13 months of age and these photographs will be inspected. At 14 months, two fish per family per tank will be removed at random for determining fillet yield.Statistical analyses: All data including BW, FL, CF, fillet yield data will be checked for outliers by first identifying records less or greater than 3 SD compared to the respective family mean, and then evaluating each outlier datum against the corresponding BW, FL, and CF measurement and serial data for the respective fish. Body weight data recorded before tagging will be analyzed separately by month using PROC MIXED. Post-tagging BW, FL, and CF data will be analyzed separately by week of measurement using PROC MIXED. Fillet yield will be conducted for only one time point. Models will include fixed effects of treatment type (three levels), family (ten levels), and tank (twelve levels). The SATTERTHWAITE option will be used to compute the denominator degrees of freedom for the tests of fixed effects. The Fisher scoring algorithm will be used as the optimization technique. The preliminary model will include fixed effects of treatment type, family, tank, and the interaction between family and treatment type as a random effect. In all analyses, non-significant effects (P > 0.05) will be sequentially removed from the models.

Progress 09/01/17 to 08/31/21

Outputs
Target Audience:This project aims to develop a rainbow trout sterilization technique and make it readily available for trials and applications in large-scale commercial production. Therefore, the primary target audience for our outreach efforts will be aquaculture industry stakeholders, including producers and growers and companies interested in rainbow trout aquaculture. We have been working with Troutlodge (the biggest trout seed producer in the US) to apply this fish sterilization technology commercially. This approach indirectly leverages USDA/NIFA funds by attracting the aquaculture industry, which will promote the more widespread use of the technology. Unfortunately, due to the COVID19 pandemic, our Aquaculture Research Center (ARC) tours for public audiences have been suspended in the past year. Therefore, our outreach effort for the lay public was minimal. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this funding period, one postdoc, one research associate, and one graduate student were trained in fish science and biotechnology. Also, the results generated from the proposed work were communicated and discussed with researchers, aquaculture companies, biotechnology companies, and public audiences through facility tours and the IMET virtual open house and video media, IMET Minute: Dr. Ten-Tsao Wong-Why is it important to create sterile fish? In addition, the development and results were incorporated into teaching materials for Marine and Environmental Biotechnology. How have the results been disseminated to communities of interest?To disseminate information and results from the proposed research, we participated in and organized an IMET Open-House event and conducted Aquaculture Research Center (ARC) tours for public audiences and aquaculture industry stakeholders, including investors and biotech companies. Below is an abbreviated listing of outreach and education efforts related to this project (in reverse chronological order), including tours, presentations, and face-to-face interactions with industry representatives, academics, students and teachers, the general public, and other targeted audiences. Wong, Ten-Tsao and Zohar, Yonathan. "Sterility induction by disrupting early germ cell development". Aquaculture America 2020, Sterility in Aquaculture special session, Honolulu, HI, Feb 9-12. Wong, Ten-Tsao and Zohar, Yonathan. "Induced sterility to block salmon maturation". Aquaculture America 2020, Honolulu, HI, USA Feb 9-12. Luckenbach, J.A., Hayman, E.S., Fairgrieve, W.T., Wong, Ten-Tsao, Zohar, Y., Huynh, T.B. Investigation of approaches for reproductive sterilization of sablefish Anoplopoma fimbria. Aquaculture America 2020, Sterility in Aquaculture special session, Honolulu, HI, Feb 9-12. Wong, Ten-Tsao. "Reproductively Sterile Fish for Environmentally-Responsible Aquaculture". Aoshan Forum on Progress in Biotechnology for Marine and Fisheries, Pilot National Laboratory for Marine Science and Technology Qingdao, China. 09/16- 20, 2019 Xu, Lan, Peng, Kuan-Chieh, Zohar, Yonathan and Wong, Ten-Tsao. "Developing Technologies to Induce Reproductive Sterility in Eastern Oysters without Chromosome Set Manipulation". Aoshan Forum on Progress in Biotechnology for Marine and Fisheries, Pilot National Laboratory for Marine Science and Technology Qingdao, China. 09/16-20, 2019 Xu, Lan, Peng, Kuan-Chieh, Zohar, Yonathan and Wong, Ten-Tsao. "Developing Technologies to Induce Reproductive Sterility in Eastern Oysters without Chromosome Set Manipulation". IMBC 2019, Shizuoka city, Shizuoka, Japan. 09/09 -13, 2019. Peng, Kuan-Chieh, Zohar, Yonathan and Wong, Ten-Tsao. "Sterilizing fish by gene knockdown techniques". IMBC 2019, Shizuoka city, Shizuoka, Japan. 09/09 -13, 2019 Wong, Ten-Tsao. "Reproductive sterility induction by disrupting primordial germ cell development.". South Ehime Fisheries Research Center, Ehime University, Ehime, Japan. 09/13/20191. Aug 29, 2019. Talbot County Board of Development July 15, 2019. Sea Grant Aquaculture workshop (25 participants). May 5, 2018. IMET Open House. Facility tour and general discussion of IMET research for the lay public. Approximately 550 non-science visitors participated. Nov 7-9, 2019. Fish 2.0 Workshop (30 participants) July 19, 2018. Fisheries AquaTec, Ltd. Facility tour for 3 senior company officials and technical discussion of the USDA funded work to date July 10, 2018. National Fisheries Institute. Facility tour for 2 senior institute officials, Educators/PR. Facility tour for NOAA Sea Grant-sponsored Educators (18 participants). June 29, 2018. Kepley BioSystems. Facility tour for investment group including a company rep from Kepley BioSystems, which is a biotechnology company based in North Carolina May 1, 2018. IMET Open House. Facility tour and general discussion of IMET research for the lay public. Approximately 250 non-science visitors were provided an overview of the USDA-funded project Dec 2, 2017. Goucher College. Educational tour for class (15 participants, students, and teachers) October 30, 2017. Towson High (Maryland) School. Facility tour for 40 high school students. October 17, 2017. AquaInvest. Facility tour for 2 AquaInvest representatives. AquaInvest is a marine science/marine biotechnology investment operation. Oct 11, 2017. Intrexon. Facility tour for a biotechnology company (3 participants). What do you plan to do during the next reporting period to accomplish the goals?Due to the COVID19 pandemic, the progress of our research plan was delayed, and this project was ended on Aug. 31st, 2021. In the coming years, we will continue optimizing the dnd-MO-Vivo pre-fertilization immersion protocol using a defined medium to achieve a higher survival rate and sterility induction and produce sterile rainbow trout. Once this condition is achieved, we will apply for a grant for a growth performance study of sterile rainbow trout.

Impacts
What was accomplished under these goals? This project aims to develop a sterilization technique for rainbow trout and make it readily available for trial and application in large-scale commercial production. Therefore, the primary target audience for our outreach efforts will be aquaculture industry stakeholders, including producers and growers and companies interested in rainbow trout aquaculture. We worked with Troutlodge to commercially apply this fish sterilization technology through all the grant-funded years. This approach indirectly leverages USDA/NIFA funds by attracting the aquaculture industry, which will promote the more widespread use of the technology. For the lay public. We conducted Aquaculture Research Center (ARC) tours in-person and virtual (due to COVID19 pandemic) IMET Open-House events for public audiences. The goal of this proposed research was completed or achieved through the following three specific objectives: 1) Optimize the rainbow trout dnd-MO-Vivo post-fertilization immersion protocol for large-scale production to determine the most economical conditions that achieve 100% sterility induction. We have identified a salmonid dnd-MO-Vivo that could disrupt PGC development and generate sterile rainbow trout. This specific objective aims to optimize this sterilization technology to be applied to the large-scale production of sterile rainbow trout. We initially compared the immersion condition using a lower concentration of dnd-MO-Vivo (10, 20, and 30 uM) and found the sterility induction was 10% or less in these conditions. We then started the immersion with higher concentrations (30, 40, and 50 uM) and investigated the sterility induction under these 3 conditions. To enhance the uptake of dnd-MO-Vivo, we utilized an orbital shaker to constantly mix eggs and dnd-MO-Vivo and found that, unlike fertilized zebrafish eggs, shaking fertilized rainbow trout eggs caused high mortality. Even in the control groups that had no dnd-MO-Vivo, the shaken control eggs have a survival rate of 10-15% of the unshaken control eggs. Our results suggested that post-fertilization immersion is not suitable for rainbow trout eggs that need a steadier environment during early development. 2) Establish pre-fertilization immersion protocols to reduce production costs further and optimize treatment conditions to achieve 100% sterility induction. Water hardening after fertilization results in a change of chorion characteristics that leads to the increase of chorion strength but the decrease of permeability. To optimize the bath-immersion technology, we investigated the possibility of administering dnd-MO-Vivo into fish eggs before fertilization and water hardening. As such, the bath immersion was conducted using the unfertilized eggs with either 10 or 20 uM of dnd-MO-Vivo. We found a very low survival rate in all immersion conditions that used 20 uM of dnd-MO-Vivo. Some higher survival rates with 40% sterility induction were identified in 10uM dnd-MO-Vivo treated groups. We also noticed that in addition to the concentration of dnd-MO-Vivo, different medium components also affected the survival rate of treated embryos. We hence initiated the comparison and investigation of the embryos immersed in various media. Among 10 media that have been tested, we identified two media that can be used for pre-fertilization immersion. One medium enhanced dnd-MO-Vivo uptake and achieved over 80% sterility induction. This medium contains 50% of ovarian fluid (OVF). The other medium was modified from an L-15 medium and achieved 50% sterility induction. Therefore, we conducted 3 optimization trials focusing on OVF containing medium at USDA ARS USDA/NCCCWA, West Virginia. We tested the various percentage of OVF (35% -65%) and hoped to increase sterility induction efficiency. However, high mortality occurred among these trials. After comparing with other experiments, we concluded that the iodine disinfection procedure and hard water appearing in NCCCWA's system were the reasons for the high mortality. We did not conduct an iodine disinfection procedure when establishing this immersion procedure in a laboratory setting. Since Vivo enhances chorion permeability, the iodine was much easier to get into the eggs and caused mortality when eggs were disinfected immediately after fertilization. These treated eggs also suffered in a hard water condition resulted in high mortality. Recently, we were able to resolve this challenge by delaying the disinfection procedure and using artificial water containing 0.2 ppt sea salt in deionized water for egg hatching. At the same time, we also realized that OVF was not consistent from batch to batch or among females. This inconsistency of OVF makes it very difficult in SOP establishments to commercialize this bath-immersion sterility induction technology. The modified L-15 medium containing defined components would be better for establishing a commercial SOP for this bath immersion technology. Due to the government shutdown in January 2019 and then the COVID-19 pandemic in 2020 and 2021, the facility shut down, or reduced operation capacity in our facility and NCCCWA (25% operation capacity during COVID pandemic), our experimental plan was disrupted, and we could not further carry out medium optimization or analyze some other treated fish. 3) Compare growth-related performance traits among dnd-MO sterilized diploid female (2N-S), fertile diploid female (2N), and triploid female fish (3N). We hypothesize growth-related traits will be similar between 2N-S and 2N females, and both will generally be superior to 3N females. Because the results obtained from Objectives 1 and 2 indicated that pre-fertilization immersion might be more effective for sterile fish generation, we initiated the production of sterile trout using pre-fertilization immersion. Since the pre-fertilization medium was not optimized and the survival rate was low and different among different families after the immersion. The number of sterile trout for Objective 3 in tall the trials was very limited. Due to the government shutdown in January 2019 (rainbow trout spawning season at USDA/NCCCWA, West Virginia) and then the COVID-19 pandemic in 2020 and 2021. We missed many opportunities to optimize our protocols to produce sterile fish for objective 3. We were able to have a small-scale growth study that consisted of 40 fertile females and 40 sterile females for more than 9 months at our ARC facility. So far, we did see the growth difference between these two groups.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 1. Wong, Ten-Tsao and Zohar, Y. 2020. Sterility in aquaculture - advances, performance, impacts: Sterility Induction by Disrupting Early Germ Cell Development. World Aquaculture Magazine | June 2020 | Volume - 51 Issue- 2
  • Type: Other Status: Published Year Published: 2020 Citation: 1. Wong, Ten-Tsao and Zohar, Y. Methods of Agent Delivery into Eggs and Embryos of Egg-producing Aquatic Animals for Drug Screening, Agent Toxicity Assay and Production of Infertile Fish. U.S. Patent No. 10,709,119 issued on Jul 14, 2020
  • Type: Other Status: Published Year Published: 2018 Citation: 2. Zohar, Y. and Wong, Ten-Tsao. Method of producing infertile fish and egg-producing aquatic animals and of delivering compounds into eggs and embryos. U.S. Patent No. 9,999,208 issued on Jun 19, 2018.
  • Type: Book Chapters Status: Published Year Published: 2018 Citation: Wong Ten-Tsao and Zohar Y (2018) Reproductive Technology (Non-human/Non-primate): Sex Control and Sterilization in Fish. Encyclopedia of Reproduction (Second Edition), ed Skinner MK (Academic Press, Oxford), pp 796-801.


Progress 09/01/19 to 08/31/20

Outputs
Target Audience:This project aims to develop a sterilization technique for rainbow trout and to make it readily available for trial and application in large-scale commercial production. The primary target audience for our outreach efforts will therefore be aquaculture industry stakeholders, including producers and growers, and companies interested in rainbow trout aquaculture. In this, we continued to work with Troutlodge (the biggest trout seed producer in the US) for the possible commercial application of this fish sterilization technology. This approach indirectly leverages USDA/NIFA funds by attracting the aquaculture industry, which will promote the more widespread use of the technology. For the lay public, we conducted several Aquaculture Research Center (ARC) tours for public audiences before the COVID19 pandemic. We also carried out an IMET Open-House event in virtual formate. Changes/Problems:No major changes in the 3rd year research. The progress was delayed due to COVID19 pandemic. What opportunities for training and professional development has the project provided?During this funding period, one postdoc, one research associate, and one graduate student were trained in fish science and biotechnology. How have the results been disseminated to communities of interest?To disseminate information and results from the proposed research, we participated and organized the IMET virtual Open-House event and conducted Aquaculture Research Center (ARC) tours and a video, IMET Minute: Dr. Ten-Tsao Wong Why is it important to create sterile fish? for public audiences and aquaculture industry stakeholders including investors and biotech companies. including tours, conference presentations, seminar, and face-to-face interactions with industry representatives, academics, students and teachers, the general public, and other targeted audiences. In addition, the development and results were incorporated into teaching materials for a Marine and Environmental Biotechnology. Below is an abbreviated listing of outreach and education efforts related to this project. Wong, Ten-Tsao and Zohar, Yonathan. "Sterility induction by disrupting early germ cell development". Aquaculture America 2020, Sterility in Aquaculture special session, Honolulu, HI, Feb 9-12. Wong, Ten-Tsao and Zohar, Yonathan. "Induced sterility to block salmon maturation". Aquaculture America 2020, Honolulu, HI, USA Feb 9-12. Luckenbach, J.A., Hayman, E.S., Fairgrieve, W.T., Wong, Ten-Tsao, Zohar, Y., Huynh, T.B. Investigation of approaches for reproductive sterilization of sablefish Anoplopoma fimbria. Aquaculture America 2020, Sterility in Aquaculture special session, Honolulu, HI, Feb 9-12. Wong, Ten-Tsao. "Reproductively Sterile Fish for Environmentally-Responsible Aquaculture". Aoshan Forum on Progress in Biotechnology for Marine and Fisheries, Pilot National Laboratory for Marine Science and Technology Qingdao, China. 09/16-20, 2019 Xu, Lan, Peng, Kuan-Chieh, Zohar, Yonathan and Wong, Ten-Tsao. "Developing Technologies to Induce Reproductive Sterility in Eastern Oysters without Chromosome Set Manipulation". Aoshan Forum on Progress in Biotechnology for Marine and Fisheries, Pilot National Laboratory for Marine Science and Technology Qingdao, China. 09/16-20, 2019 Xu, Lan, Peng, Kuan-Chieh, Zohar, Yonathan and Wong, Ten-Tsao. "Developing Technologies to Induce Reproductive Sterility in Eastern Oysters without Chromosome Set Manipulation". IMBC 2019, Shizuoka city, Shizuoka, Japan. 09/09 -13, 2019. Peng, Kuan-Chieh, Zohar, Yonathan and Wong, Ten-Tsao. "Sterilizing fish by gene knockdown techniques". IMBC 2019, Shizuoka city, Shizuoka, Japan. 09/09 -13, 2019 Wong, Ten-Tsao. "Reproductive sterility induction by disrupting primordial germ cell development.". South Ehime Fisheries Research Center, Ehime University, Ehime, Japan. 09/13/2019 What do you plan to do during the next reporting period to accomplish the goals?Due to the COVID19 pandemic, the progress of our 3rd-year research plan was delayed. In the coming years, we will continue the optimization of dnd-MO-Vivo pre-fertilization immersion protocol (Objective 2) to achieve the higher survival rate and sterility induction and produce sterile rainbow trout for growth performance study (Objective 3).

Impacts
What was accomplished under these goals? This project aims to develop a sterilization technique for rainbow trout and to make it readily available for trial and application in large-scale commercial production. The primary target audience for our outreach efforts will therefore be aquaculture industry stakeholders, including producers and growers, and companies interested in rainbow trout aquaculture. this year, we continued to work with Troutlodge for the possible commercial application of this fish sterilization technology. This approach indirectly leverages USDA/NIFA funds by attracting the aquaculture industry, which will promote the more widespread use of the technology. For the lay public. We conducted Aquaculture Research Center (ARC) tours and a virtual (due to COVID19 pandemic) IMET Open-House event for public audiences. The goal of this proposed research will be achieved through the following three specific objectives: 1) Optimize the rainbow trout dnd-MO-Vivo post-fertilization immersion protocol for large scale production to determine the most economical conditions that achieve 100% sterility induction. (100% completion) We have identified a salmonid dnd-MO-Vivo that was able to disrupt PGC development and generate sterile rainbow trout. The goal of this specific objective is to optimize this sterilization technology to be applied to the large-scale production of sterile rainbow trout. We initially compared the immersion condition using a lower concentration of dnd-MO-Vivo (10, 20 and 30 uM) and found the sterility induction was 10% or less in these conditions. We then started the immersion with higher concentration (30, 40 and 50 uM) and investigate the sterility induction under these 3 conditions. To enhance the uptake of dnd-MO-Vivo, we utilized an orbital shaker to constantly mix eggs and dnd-MO-Vivo and found that unlike fertilized zebrafish eggs, shaking fertilized rainbow trout eggs caused high mortality. Even in the control groups that had no dnd-MO-Vivo, the shaken control eggs have a survival rate of 10-15% of the unshaken control eggs. Post-fertilization immersion is not a suitable method for rainbow trout eggs that need to have a steadier environment during early development. Our results suggested that post-fertilization immersion is not a suitable method for rainbow trout eggs that need to have a steadier environment during early development. 2) Establish pre-fertilization immersion protocols that may further reduce production costs and optimize treatment conditions to achieve 100% sterility induction (80% completion). Water hardening after fertilization results in a change of chorion characteristics that leads to the increase of chorion strength but the decrease of permeability. To continue the optimization of the bath-immersion technology, we investigated the possibility of administering dnd-MO-Vivo into fish eggs before fertilization and water hardening. As such, the bath immersion was conducted using the unfertilized eggs with either 10 or 20 uM of dnd-MO-Vivo. We found very low survival rate in all immersion conditions that used 20 uM of dnd-MO-Vivo. Some higher survival rate with 40% sterility induction were identified in 10uM dnd-MO-Vivo treated groups. We also noticed that in addition to the concentration of dnd-MO-Vivo, different medium components also affected the survival rate of treated embryos. We hence initiated the comparison and investigation of the embryos that are immersed in different media. Among 10 media that have been tested, we identified two media that can be used for pre-fertilization immersion. These two media have been used to produce sterile fish. In this year, we also tested the immersion temperature (4 °C or 8 °C) with the combination of different immersion media. Due to COVID-19 pandemic and facility shutdown, we were not able to further analyze these treated fish. 3) Compare growth related performance traits among dnd-MO sterilized diploid female (2N-S), fertile diploid female (2N), and triploid female fish (3N) (30% completion). We hypothesize growth related traits will be similar between 2N-S and 2N females, and both will be generally superior to 3N females. Because the results obtained from Objectives 1 and 2 indicated that pre-fertilization immersion may be more effective for sterile fish generation, we initiated the production of sterile trout using pre-fertilization immersion. Since the pre-fertilization medium was not optimized and the survival rate was low and different among different families after the immersion. The number of sterile trout for Objective 3 in the first and 2nd trials was very limited. Due to the government shutdown in January, 2019 (the only once per year rainbow trout spawning season at USDA/NCCCWA, West Virginia). We missed lots of opportunities for optimizing our protocols to produce sterile fish for the objective 3. In this year, we initiated several treatments in February, 2020 and planned to use these fish for growth study. However, due to COVID19 pandemic and facility shutdown, the progress of the objective 3 has hence been delayed.

Publications


    Progress 09/01/18 to 08/31/19

    Outputs
    Target Audience:This project aims to develop a sterilization technique for rainbow trout and to make it readily available for trial and application in a large-scale commercial production. The primary target audience for our outreach efforts will therefore be aquaculture industry stakeholders, including producers and growers, and companies interested in rainbow trout aquaculture. In the 2nd year, we reached out to trout seed producer, Troutlodge for the possible commercial application of this fish sterilization technology. This approach indirectly leverages USDA/NIFA funds by attracting aquaculture industry, which will promote more widespread use of the technology. For the lay public. We conducted IMET Open-House event and Aquaculture Research Center (ARC) tours for public audiences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this funding period, one research associate, one graduate student and one undergraduate student were trained in fish science and biotechnology. Also, the results generated from the proposed work were communicated and discussed with researchers, aquaculture companies, biotechnology companies and public audiences through facility tours and the IMET openhouse. In addition, the development and results were incorporated into teaching materials for a Marine and Environmental Biotechnology. The conceptual aspects of The technology was also included in a facility tour coordinated by our local Maryland Sea Grant Education Specialist. How have the results been disseminated to communities of interest?To disseminate information and results from the proposed research, we participated and organized IMET Open-House event and conducted Aquaculture Research Center (ARC) tours for public audiences and aquaculture industry stakeholders including investors and biotech companies. Below is an abbreviated listing of outreach and education efforts related to this project in late 2018 and 2019 (in reverse chronological order), including tours, presentations and face-to-face interactions with industry representatives, academics, students and teachers, the general public, and other targeted audiences. 1. Aug 29, 2019. Talbot County Board ofDevelopment 2. July 15, 2019. Sea Grant Aquaculture workshop (25 participants). 3. May 5, 2018. IMET Open House. Facility tour and general discussion of IMET research for the lay public. Approximately 550 non-science visitors participated. 4. Nov 7-9, 2019. Fish 2.0 Workshop (30 participants) What do you plan to do during the next reporting period to accomplish the goals?Due to the federal government shutdown, the progress of our 2nd year research plan was delayed. In the coming years, we will continue the optimization of dnd-MO-Vivo pre-fertilization immersion protocol (Objective 2) to achieve the higher survival rate and sterility induction and produce sterile rainbow trout for growth performance study (Objective 3).

    Impacts
    What was accomplished under these goals? This project aims to develop a sterilization technique for rainbow trout and to make it readily available for trial and application in a large-scale commercial production. The primary target audience for our outreach efforts will therefore be aquaculture industry stakeholders, including producers and growers, and companies interested in rainbow trout aquaculture. In the 2nd year, we reached out to trout seed producer, Troutlodge for the possible commercial application of this fish sterilization technology. This approach indirectly leverages USDA/NIFA funds by attracting aquaculture industry, which will promote more widespread use of the technology. For the lay public. We conducted IMET Open-House event and Aquaculture Research Center (ARC) tours for public audiences. The goal of this proposed research will be achieved through the following three specific objectives: 1) Optimize the rainbow trout dnd-MO-Vivo post-fertilization immersion protocol for large scale production to determine the most economical conditions that achieve 100% sterility induction. (70% completion) We have identified a salmonid dnd-MO-Vivo that was able to disrupt PGC development and generate sterile rainbow trout. The goal of this specific objective is to optimize this sterilization technology to be applied to a large-scale production of sterile rainbow trout. We initially compared the immersion condition using lower concentration of dnd-MO-Vivo (10, 20 and 30 uM) and found the sterility induction was 10% or less in these conditions. We then started the immersion with higher concentration (30, 40 and 50 uM) and investigate the sterility induction under these 3 conditions. To enhance the uptake of dnd-MO-Vivo, we utilized an orbital shaker to constantly mix eggs and dnd-MO-Vivo and found that unlike fertilized zebrafish eggs, shaking fertilized rainbow trout eggs caused high mortality. Even in the control groups that had no dnd-MO-Vivo, the shaken control eggs have a survival rate of 10-15% of the unshaken control eggs. Post-fertilization immersion is likely not going to work for rainbow trout eggs that need to have a steadier environment during early development. The mixing of fertilized eggs and dnd-Mo-Vivo by orbital shaking may cause high mortality. 2) Establish pre-fertilization immersion protocols that may further reduce production costs and optimize treatment conditions to achieve 100% sterility induction (70% completion). Water hardening after fertilization results in a change of chorion characteristics that leads to the increase of chorion strength but the decrease of permeability. To continue the optimization of the bath-immersion technology, we investigated the possibility of administering dnd-MO-Vivo into fish eggs before fertilization and water hardening. As such, the bath immersion was conducted using the unfertilized eggs with either 10 or 20 uM of dnd-MO-Vivo. We found very low survival rate in all immersion conditions that used 20 uM of dnd-MO-Vivo. Some higher survival rate with 40% sterility induction were identified in 10uM dnd-MO-Vivo treated groups. We also noticed that in addition to the concentration of dnd-MO-Vivo, different medium components also affected the survival rate of treated embryos. We hence initiated the comparison and investigationof the embryos that are immersed in different media. Among 10 media that have been tested, we identified two media that can be used for pre-fertilization immersion. These two media have been used to produce sterile fish. 3) Compare growth related performance traits among dnd-MO sterilized diploid female (2N-S), fertile diploid female (2N), and triploid female fish (3N) (20% completion). We hypothesize growth related traits will be similar between 2N-S and 2N females, and both will be generally superior to 3N females. Because the results obtained from Objectives 1 and 2 indicated that pre-fertilization immersion may be more effective for sterile fish generation, we initiated the production of sterile trout using pre-fertilization immersion. Since the pre-fertilization medium was not optimized and the survival rate was low and different among different families after the immersion. The number of sterile trout for Objective 3 in the first and 2nd trials was very limited. Due to the government shutdown in the January of this year (the only once per year rainbow trout spawning season at USDA/NCCCWA, West Virginia). We missed lots of opportunities for optimizing our protocols to produce sterile fish for the objective 3. The progress of the objective 3 has hence been delayed.

    Publications

    • Type: Book Chapters Status: Published Year Published: 2018 Citation: Wong Ten-Tsao and Zohar Y (2018) Reproductive Technology (Non-human/Non-primate): Sex Control and Sterilization in Fish. Encyclopedia of Reproduction (Second Edition), ed Skinner MK (Academic Press, Oxford), pp 796-801.
    • Type: Other Status: Published Year Published: 2018 Citation: Zohar, Y. and Wong, Ten-Tsao. Method of producing infertile fish and egg-producing aquatic animals and of delivering compounds into eggs and embryos. U.S. Patent No. 9,999,208 issued on 06/19, 2018.


    Progress 09/01/17 to 08/31/18

    Outputs
    Target Audience:This project aims to develop a sterilization technique for rainbow trout and to make it readily available for trial and application in large-scale commercial production in the short term. The primary target audience for our outreach efforts will therefore be aquaculture industry stakeholders including producers and growers, and companies interested in rainbow trout aquaculture. In the first year, we reached out to trout grower Riverence/Evaqua, and trout seed producer, Troutlodge for the possible commercial application of this fish sterilization technology. This approach indirectly leverages USDA/NIFA funds by attracting industry-based funding and investment, which will promote more widespread use of the technology. For the lay public. we conducted IMET Open-House event and Aquaculture Research Center (ARC) tours for public audiences. Changes/Problems: Our major problem was the low survival rate in pre-fertilization immersion treatments. We also noticed that in addition to the concentration of dnd-MO-Vivo, different medium components also affected the survival rate. We hence initiated the comparison and investigation of survival rate of the embryos that are immersed in different medium (6 different media). Once the best medium condition is identified, we will use this condition for dnd-MO-Vivo pre-fertilization immersion. What opportunities for training and professional development has the project provided?During this funding period, one research associate, one graduate student and one undergraduate student were trained in fish science and biotechnology. Also, the results generated from the proposed work were communicated and discussed with scientists, aquaculture companies and biotechnology companies through facility tours. In addition, the development and results were incorporated into teaching materials for a Marine and Environmental Biotechnology. The conceptual aspects of the technology were also included in a facility tour coordinated by our local Maryland Sea Grant Education Specialist. How have the results been disseminated to communities of interest?To disseminate information and results from proposed research, we participated and organized IMET Open-House event and conducted Aquaculture Research Center (ARC) tours for public audiences and aquaculture industry stakeholders including investors and biotech companies. Below is an abbreviated listing of outreach and education efforts related to this project in late 2017 and 2018 (in reverse chronological order), including tours, presentations and face-to-face interactions with industry representatives, academics, students and teachers, the general public, and other targeted audiences. 1. July 19, 2018. Fisheries AquaTec, Ltd. Facility tour for 3 senior company officials and technical discussion of the USDA-funded work to date 2. July 10, 2018. National Fisheries Institute. Facility tour for 2 senior institute officials, Educators/PR. Facility tour for NOAA Sea Grant-sponsored Educators (18 participants). 3. June 29, 2018. Kepley BioSystems. Facility tour for investment group including a company rep from Kepley BioSystems, which is a biotechnology company based in North Carolina 4. May 1, 2018. IMET Open House. Facility tour and general discussion of IMET research for the lay public. Approximately 250 non-science visitors were provided an overview of the USDA-funded project 5. Dec 2, 2017. Goucher College. Educational tour for class (15 participants, students and teachers) 6. October 30, 2017. Towson High (Maryland) School. Facility tour for 40 high school students. 7. October 17, 2017. AquaInvest. Facility tour for 2 AquaInvest representatives. AquaInvest is a marine science/marine biotechnology investment operation. 8. Oct 11, 2017. Intrexon. Facility tour for a biotechnology company (3 participants). What do you plan to do during the next reporting period to accomplish the goals?Our research plan for the coming years is to continue the optimization of dnd-MO-Vivo post-fertilization immersion protocol (Objective 1) to investigate the effect of higher concentration (40, 50 uM) of dnd-MO-Vivo in sterility induction. We will also completed the optimization of immersion medium for prefertilization immersion (Objective 2) to achieve the higher survival rate. Once the optimal pre-fertilization immersion medium is obtained, we will initiate the 2nd trial to produce sterile trout for growth performance study (Objective 3).

    Impacts
    What was accomplished under these goals? The goal of this proposed research will be achieved through the following three specific objectives: 1) Optimize the rainbow trout dnd-MO-Vivo post-fertilization immersion protocol for large scale production to determine the most economical conditions that achieve 100% sterility induction. (40% completion) We have identified an effective salmonid dnd-MO-Vivo that was able to disrupt PGC development and generate sterile rainbow trout. The goal of this specific objective is to optimize this sterilization technology to be applied to large-scale production of sterile rainbow trout. We initially compared the immersion condition using lower concentration of dnd-MO-Vivo (10, 20 and 30 uM) and found the sterility induction were 10% or less in these conditions. We will start the immersion with higher concentration (40 and 50 uM) and investigate the sterility induction under these 2 conditions. 2) Establish pre-fertilization immersion protocols that may further reduce production costs and optimize treatment conditions to achieve 100% sterility induction (50% completion). Water hardening after fertilization results in a change of chorion characteristics that leads to the increase of chorion strength but the decrease of permeability. To continue the optimization of the bath-immersion technology, we investigated the possibility of administering dnd-MO-Vivo into fish eggs before fertilization and water hardening. As such, the bath immersion was conducted using unfertilized eggs with either 10 or 20 uM of dnd-MO-Vivo. We found very low survival rate in all immersion conditions that used 20 uM of dnd-MO-Vivo. Some higher survival rate with 40% sterility induction were identified in early stage (6-month old) of treated fish . We also noticed that in addition to the concentration of dnd-MO-Vivo, different medium components also affected the survival rate of treated embryos. We hence initiated the comparison and investigation of survival rate of the embryos that are immersed in different medium (6 different media). Once the best medium condition is identified, we will use this condition for dnd-MO-Vivo pre-fertilization immersion. 3) Compare growth related performance traits among dnd-MO sterilized diploid female (2N-S), fertile diploid female (2N), and triploid female fish (3N) (10-20% completion). We hypothesize growth related traits will be similar between 2N-S and 2N females, and both will be generally superior to 3N females. Because the results obtained from Objectives 1 and 2 indicated that pre-fertilization immersion maybe more effective for sterile fish generation, we initiated the production of sterile trout using pre-fertilization immersion. Since the pre-fertilization medium was not optimized and the survival rate was low and different among different families after the immersion. The number of sterile trout for Objective 3 in the first trial was very limited. We have initiated the optimization of pre-fertilization immersion medium (see above). Once the optimal immersion medium is obtained, we will start 2nd trial of sterile fish production.

    Publications