Source: UNIVERSITY OF MAINE submitted to NRP
ESTABLISHING METRICS OF BROODSTOCK AND OFFSPRING QUALITY IN ATLANTIC SALMON AQUACULTURE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
Reporting Frequency
Annual
Accession No.
1028336
Grant No.
2022-67016-37226
Cumulative Award Amt.
$600,000.00
Proposal No.
2021-06794
Multistate No.
(N/A)
Project Start Date
Jul 1, 2022
Project End Date
Jun 30, 2027
Grant Year
2022
Program Code
[A1211]- Animal Health and Production and Animal Products: Animal Reproduction
Recipient Organization
UNIVERSITY OF MAINE
(N/A)
ORONO,ME 04469
Performing Department
School of Marine Sciences
Non Technical Summary
The ability to identify the optimal fish for breeding (termed broodstock), that will result in high performing offspring is a cornerstone of any sustainable aquaculture industry. This project seeks to identify characteristics in broodstock or their offspring that will predict offspring quality and future performance, allowing farmers to optimize broodstock selection, and better manage their stocks. Therefore, the objectives of this project are to 1) develop metrics that will identify broodstock that will have a high likelihood of producing high quality offspring and 2) identify metrics that predict survival and growth of embryos and juveniles to inform hatchery managers of what growth models to plan for. These metrics can be used for the eventual development of on-site tools farm managers can use to identify the best fish to use for breeding, and to predict the growth performance of offspring. This project will increase our understanding of biological factors underlying reproductive performance, and could improve the economic viability of the Atlantic salmon aquaculture industry.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30137991020100%
Goals / Objectives
Aim 1: Examine the metabolome and transcriptome of broodstock female salmon and determine their ability to predict broodstock and offspring quality.Broodstock selection and the control of egg quality is a critical aspect of any sustainable finfish aquaculture industry. Increasing our ability to select optimally performing broodstock, and improve our understanding of the regulation of reproductive processes will have significant improvements across the production process.Aim 2: Examine the metabolome and transcriptome of adult male Atlantic salmon and determine their ability to predict early maturation.Early male maturation results in a significant financial loss to the industry, as these males must be culled often after prolonged periods of culture (>1yr). The mechanisms of early maturation are poorly understood, and the ability to cull these males as early in the process as possible would result in significant cost savings. Moreover, there is an urgent need to increase our understanding of the factors governing the shift from normal to precocious maturation.Aim 3: Examine the utility of morphometrics and behavior as predictive indicators of cohort quality in embryos or yolk-sac fry.The ability to predict offspring performance as early in the process as possible (e.g. as embryos or young fry) would enable hatchery managers to more efficiently cull poor performing batches, and more accurately plan for the sale of surplus eggs. Further, the identification of biomarkers of quality may increase our understanding of the mechanisms underpinning performance.
Project Methods
Methods:Experiment 1: Examine the metabolome and transcriptome of broodstock female Atlantic salmonand use AI/machine learning to predict quality and progeny success.Fish from both years of collections will be combined into either the high embryo survival rate (n = 20 for each tissue) or low survival rate (n = 20 for each tissue) with comparison to cull (n=20, ten from each sampling year) for metabolomic analysis. Metadata (including morphometrics) for all individual fish will be collected for AI analysis in conjunction with metabolic profiles.?Three-year-old, female Atlantic salmon from the USDA NCWMAC will besampled at spawning (n = 150/yr). Using the established protocols from USDA, fish areanesthetized in tricaine methane-sulfonate (MS-222) for 15 min and removed from the holdingtank for sampling. Mucus sampling is conducted using NIST validated methods. Samples from year one collections that are not earmarked for broodstock quality will be run through the standard NIST protocols for new matrix assessment to validate ovarian fluid as a viable sample type for NMR metabolomics.Automatic machine learning (Auto-ML) approaches will also be leveraged to search foroptimal classifiers and preprocessing steps (feature engineering and potentially dimensionalityreduction) to yield the best possible classifiers given the data. Models examined will include, butare not limited to: (deep) neural networks, tree-based models (gradient-boosted trees, randomforests), partial least squares and other projection methods, kernel-based support vectorclassifiers, and ensemble approaches. These will be trained to predict female quality as anoutput, given the NMR input. Prediction accuracies and confusion matrices are two keyperformance indicators, but the inner workings of the model may also reveal decision pathwaysthat are critical to understanding the connection between the metabolome and progeny success.To this end, we will then leverage SHAP (SHapley Additive eXplanations) to provide a scientificinterpretation and inspection of these black-box models to uncover any connection to underlyingbiomarkers.Transcriptomic Analysis:RNA will be isolated from mucus samples with a Qiagen RNeasy Mini kit, including optionalon-column DNase I treatment. RNA quality and quantity will be quantifiedspectrophotometrically and samples will be stored at -80C. Samples will be sent to theHudsonAlpha Institute for Biotechnology Genomic services lab, or a related facility, for librarypreparation and sequencing.Experiment 2: Examine the metabolome and transcriptome of male Atlantic salmon and useAI/machine learning to predict early maturation.In year one of this study, mucus and plasma will be collected from 120 two-year-old males.These fish are pit tagged and can be followed to the following year. In year two of this study,during a cull event, mucus and plasma from 30 precocious males and 30 normal males will besampled, specifically targeting precious males that were sampled in year 1. Metabolomicanalysis will be conducted on all two-year old samples, and the cull event samples fromindividuals sampled in year 1 (n = 20 for each tissue type (mucus and plasma, treatment, andtimepoint meaning a total of 160 samples from 40 individuals). Remaining samples from bothtime points will be used to generate QC pools as outlined in the broodstock comparison.Mucus and plasma samples will follow the same protocols as in experiment 1.Metabolic signatures in blood plasma indicative of early maturation are expected to correlate todistinctive markers within the more practical in-field sample collection (skin mucus).Experiment 3: Examine a suite of morphometrics in developing salmon embryos and yolk-sacfry to determine their predictive capacity for survival and future growth performance.Image stacking:To create images of optimal clarity for analysis, embryos and larvae will be placed in glass petridishes inside a 3D muted lightbox. This will prevent light shine that may obscure image detail. A high resolution digital camera with macro lens will be used in conjunction with a StackShot macrorail package.Morphometric measurements:Two embryonic stages that will be targeted for these analyses are stage t72 (mid somitogenesis)and the development of pigmented eyes. The egg will be oriented such that the embryo is centered in the vertical plane. Measurements will be taken from the median line to the edge of the circumorbital region, and from the median line to the midline of the eye on both right and left sides. For each measurement, midline asymmetry (Ma) will be calculated as the absolute difference between the distance from the midline to the measured parameter for both the right (R) and left (L) sides, using the following formula Ma= R-L .

Progress 07/01/24 to 06/30/25

Outputs
Target Audience:The primary audience for this research includesAtlantic salmon producers, with a specific focus on those involved inbroodstock management and reproduction. The work is also intended to supportaquaculture industry professionals, including hatchery managers, production specialists, and technical advisors who are directly engaged in improving reproductive performance and early life-stage success in salmon farming. We also aim to reachaquaculture researchersandtechnical staffworking in fish physiology and production efficiency, as well asuniversity and extension personnelwho provide training, technical assistance, and knowledge transfer to commercial operators. Our findings are relevant tofederal and state agenciesinvolved in managing aquaculture development and supporting domestic seafood production. Information generated by this project contributes to decision-making in areas such as broodstock selection, spawning practices, and larval rearing protocols. Lastly, the research outcomes are shared withindustry partnersandcommercialsupplierswhose work supports the reproductive success and overall performance of farmed salmon. Changes/Problems:During Year 2 of the project, we encountered unforeseen challenges related to broodstock availability and sampling access at the USDA facility, which led to delays in progress onAim 2. These logistical issues impacted our ability to collect a complete set of samples from adult male Atlantic salmon within the originally intended timeframe. Despite these setbacks, we were able to collect a foundational set of samples, including heart tissue, skin mucus, and plasma, that will enable us to begin analyses in the upcoming year. With a new graduate student joining the project this fall and sample material already in hand, we are confident that we can realign our efforts and regain momentum on this aim. At this time, no changes to the overall research design are anticipated. However, we remain attentive to potential future constraints and will proactively communicate with program officers if challenges arise that may impact our ability to fully meet the objectives of Aim 2 What opportunities for training and professional development has the project provided?This project has provided substantial opportunities for training and professional development across multiple levels of the research team. The principal investigator, postdoctoral researcher, and graduate student have actively participated in national and regional scientific conferences, where they presented research findings, engaged in peer networking, and received valuable feedback from both academic and industry experts. These experiences not only advanced individual professional growth but also helped refine the project's scientific approach based on emerging insights in aquaculture and fish physiology. Team members have also gained hands-on experience through regular collaboration with the USDA National Cold Water Marine Aquaculture Center (Franklin, ME), where they have contributed to broodstock management, sampling protocols, and hatchery operations. This partnership has enhanced their understanding of large-scale aquaculture production systems and provided real-world context for the project's scientific aims. Additionally, the research team has developed professional relationships with commercial aquaculture producers, allowing them to observe current industry practices and identify opportunities for future research translation and innovation. These interactions have been critical for aligning research objectives with industry needs and increasing the project's relevance and potential impact. Beyond technical training, the project has offered experience in scientific communication (through manuscript and abstract preparation), research design, sample management, and data interpretation. The postdoctoral researcher has mentored undergraduate assistants in basic lab techniques, and the graduate student has received training in experimental design, fish husbandry, and biomarker discovery. These layered learning experiences are helping build the next generation of professionals in aquaculture science. How have the results been disseminated to communities of interest?Project results have been actively shared with both scientific and industry communities. Members of the research team have presented findings at national conferences, including poster and oral presentations, with accompanying abstracts published in the respective proceedings. These venues provided valuable opportunities to share data with peers, gather expert feedback, and raise awareness of the project's objectives and outcomes within the broader aquaculture and fisheries science communities. In addition to academic dissemination, we have maintained direct communication withcommercial aquaculture producers, sharing updates on relevant findings, particularly those related to broodstock quality, early maturation, and egg viability. These informal knowledge exchanges have supported practical decision-making and helped ensure our research remains closely aligned with industry needs. Together, these efforts have helped bridge the gap between research and application, fostering collaboration across academic, government, and commercial sectors. Plans are in place to expand dissemination through peer-reviewed publications and targeted outreach to producer networks as additional results become available. What do you plan to do during the next reporting period to accomplish the goals?During the upcoming reporting period, we will continue building on the progress made under each of the project's three aims. ForAim 1, we plan to complete data analysis and prepare manuscripts based on our metabolomics work with ovarian fluid samples. This includes finalizing correlations between metabolite profiles and reproductive performance to identify potential non-lethal biomarkers of female broodstock quality. ForAim 2, we will expand our sampling of adult male Atlantic salmon and begin analyzing the stored tissue, plasma, and mucus samples collected this year. These efforts will focus on identifying metabolomic and transcriptomic indicators associated with early sexual maturation. The addition of a new graduate student this fall will increase our capacity to advance this aim significantly, particularly in terms of laboratory processing and data interpretation. UnderAim 3, we will continue our time-series analysis of broodstock and offspring, incorporating new data from the ongoing cardiac morphology studies. We also plan to complete egg quality assays (lipid, protein, and mineral content) and integrate these findings into thedynamic energy budget (DEB) modelcurrently in development, which will help quantify early energy use and growth potential in yolk-sac fry. Importantly,two publications are currently being draftedthat highlight early findings from this aim and lay the groundwork for future validation and application in hatchery settings. Across all aims, we will maintain active collaboration with USDA partners and commercial producers, and we plan to disseminate emerging results through scientific meetings and extension channels. These combined efforts will help move our research from discovery toward practical applications for the aquaculture industry.

Impacts
What was accomplished under these goals? Aim 1: Examine the metabolome and transcriptome of broodstock female salmon and determine their ability to predict broodstock and offspring quality. Objective: This year, our team made significant progress on Aim 1: to examine the chemical makeup (metabolome) and gene activity (transcriptome) of female Atlantic salmon used for breeding, and to evaluate whether these biological signatures can help predict which fish will produce the healthiest eggs and strongest offspring. Why It Matters: Over the past 25 years, egg hatch rates in North American salmon hatcheries have dropped sharply--from around 80% to between 35-40%. While current breeding programs focus on selecting fish that grow quickly, those growth traits don't always align with strong reproductive performance. Identifying new indicators of broodstock quality would allow hatcheries to select fish more effectively, improve reproduction rates, and reduce waste--offering substantial economic and production benefits to U.S. aquaculture. What We Did: To investigate this issue, we focused on analyzingovarian fluid, a naturally occurring fluid that surrounds the eggs during spawning. This fluid is easy to collect without harming the fish and may contain important chemical signals about the female's reproductive status. Over the past year, we: Collected ovarian fluid samplesfrom 50 Atlantic salmon females during the spawning season. Tested seven different laboratory techniquesto determine the most effective way to process these samples for chemical analysis using nuclear magnetic resonance (NMR) technology. These methods included variations such as ultrafiltration, protein precipitation, lyophilization (freeze-drying), and combinations of these techniques. Evaluated each methodfor its reliability, repeatability, ease of use, and ability to detect a broad range of small molecules (metabolites). Confirmed that ovarian fluid contains a rich and measurable profile of chemical compounds, many of which differ from one female to another. Created a framework for linking these chemical profiles to egg and embryo quality in future work, setting the stage for the development of practical "biomarker" tests that could help hatchery managers select the best female broodstock early and non-invasively. Aim 2:Examine the metabolome and transcriptome of adult male Atlantic salmon and determine their ability to predict early maturation Despite unexpected delays in access and sampling at the USDA facility during the second year of the grant, we made meaningful progress toward understanding early maturation in male Atlantic salmon. Over the reporting period, we successfully collected samples from male broodstock, including measurements of body size and samples of heart tissue, skin mucus, and blood plasma. These materials will allow us to examine both the chemical (metabolome) and genetic (transcriptome) profiles of males. In addition, we sampled juvenile salmon from the same cohort, and are preserving those samples for future comparisons with individuals that later exhibit signs of early sexual maturation. To further support this research, a new graduate student dedicated specifically to male salmon development will join the team this fall, strengthening our capacity to complete this objective in the next project year. Aim 3: Examine the utility of morphometrics and behavior as predictive indicators of cohort quality in embryos or yolk-sac fry. Objective: Aim 3 focused on identifying whether physical traits (like body shape and organ development) and early behaviors in salmon embryos and fry (newly hatched fish still nourished by their yolk sac) can help predict which fish will thrive. The goal is to find simple, reliable signs of quality and health (biomarkers) that farmers and hatchery managers can use as a predictive tool. Why This Matters: Raising healthy salmon from egg to harvest depends on many factors, including genetics, nutrition, and how well the young fish develop. Being able to identify high-performing individuals early could save time, reduce losses, and improve outcomes across the industry. One promising area of focus isheart development, as the heart plays a critical role in how well a fish can grow, survive stress, and perform under changing conditions. What We Accomplished: Over the past year, our team has made substantial progress through a combination of laboratory experiments, long-term data collection, and biological modeling: Dynamic Energy Budget (DEB) Model Development:We are using detailed physical measurements (morphometrics) and oxygen consumption data to build a model that describes how young salmon use energy to grow and develop. Thisdynamic energy budgetmodel helps us understand how much energy goes toward essential processes like metabolism and development under different conditions. In simple terms, it's a way to "balance the books" on how a young fish spends its energy--much like a household budget tracks income and expenses. Thermal Performance Studies:By analyzing how embryos respond to different water temperatures, we discovered important differences in how some fish can tolerate temperature extremes, as can sometimes be seen in indoor, recirculating aquaculture systems.These insights help us link early-life traits to later-life performance, and the results are being prepared for peer-reviewed publication. Broodstock Time Series Study:We launched an extensive, ongoing study tracking a large group of female broodstock through their reproductive cycle (oogenesis). We are collecting multiple measurements--such as size, body condition, and egg quality indicators--to identify traits that reliably predict the quality of their offspring. Nutritional Egg Quality Analysis:We are analyzing the eggs for key nutritional components, including fat content, protein levels, and mineral content. This information is being added to existing datasets to improve the accuracy of our models and help identify what makes a high-quality egg. Cardiac Morphology and Performance Indicators: We have found thatheart structure (morphology), particularly in early development, shows strong promise as a predictor of overall fish health and performance. Ongoing studies are measuringheart development in both female and male fish during reproduction, including detailed analysis of how the heart changes over time (remodeling) and how this may relate to gamete (egg and sperm) quality. A special focus is being placed on whethercertain heart traits in malesmight help predictearly sexual maturation, a trait that can impact production value and management practices in salmon farming. Why the Heart? As highlighted in recent scientific reviews, the heart is a highly adaptable organ in fish and plays a vital role in determining how well a fish can perform, especially under challenging conditions like temperature changes or low oxygen. Unlike most organs, the fish heart shows remarkableplasticity--it can change in structure and function based on environmental demands. This makes it a powerful early indicator of long-term performance. Impact and Federal Investment Value: This year's work under Aim 3 has combinedcutting-edge physiological research, large-scale data collection, andpractical applicationsfor hatchery operations. We have laid the groundwork for predictive tools that could help producers identify top-performing fish from the earliest life stages--improving yields, reducing costs, and making breeding programs more efficient and science-driven.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Bair, H., Morefield, R., Hamlin, H.J. Establishing predictive metrics for Atlantic salmon Salmo salar Broodstock and offspring. World Aquaculture Society, March 6-10, 2025, New Orleans, LA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Bair, H., Morefield, R., Hamlin, H.J. A glimpse: What can early metabolism amongst S. salar families tell us. World Aquaculture Society, March 6-10, 2025, New Orleans, LA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Morefield, R., Hamlin, H.J., Bair, H. Developmental biomarkers in Atlantic salmon Salmo salar for prediction of favorable traits in domesticated lineages: growth, survival, and temperature tolerance. World Aquaculture Society, March 6-10, 2025, New Orleans, LA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Casu, F., Schock, T., Bayless, A., Mahynski N., and Boggs A. NMR-Based metabolomics and machine learning for reproductive biomarker discovery in Atlantic salmon Salmo salar. World Aquaculture Society, March 6-10, 2025, New Orleans, LA.


Progress 07/01/23 to 06/30/24

Outputs
Target Audience:The target audience for this research primarily includes aquaculture farmers, aquaculture scientists, and policymakers who focus on improving aquacultural practices, sustainability, and food security. Additionally, our target audience encompasses educators and extension agents who disseminate aquaculture knowledge, and commercial producers of Atlantic salmon. The research also aims to benefit consumers by promoting safe, nutritious, and affordable food options, and to support environmentalists interested in conservation and sustainable farming practices. This diverse audience is united by their vested interest in advancing aquaculture innovation, productivity, and resilience. Changes/Problems:An unexpected fish loss at the USDA facility in Franklin, ME delayed work for Aim 2, but we hope to continue that work this fall. What opportunities for training and professional development has the project provided?PI Hamlin, her graduate student, and postdoc, have had regular training meetings with our collaborator at NIST, Nathan Mahynski, that is skilled in methods to apply machine learning and AI to understand the predictive utility of our data. These are valuable analysis skills that will translate beyond this project. This grant has also provided opportunities for the PhD student working on this project to report her findings at a national research meeting. This work forms the basis of her dissertation, and contributes to workforce development. She has also learned valuable research tools that will become part of her research toolkit for her future research career. Both the postdoc and PhD student assisted with the commercial fish spawning that provided the embryos for use in this project. These interactions with commercial stakeholders forge professional contacts that increase their professional networks, and potential prospects for future employment. How have the results been disseminated to communities of interest?This work has been presented at multiple national conferences, and has been communicated through conversations with local producers. The conferences targeted for this work is heavily attended by fish producers across the world, and has provided opportunities for us to connect with a number of local producers. One paper has been accepted with minor revision, and has recently been resubmitted, so we anticipate reporting on this publication next reporting period. What do you plan to do during the next reporting period to accomplish the goals?Over summer and early fall we will be analyzing datacollected over the previous fall/winter. These data will drive to some degree our sampling plans for this fall. We currently intend to sample fish (skin mucus) for Aim 2 this fall, as well as conduct behavior trials using ethovision to investigate behavioral biomarkers of performance in parr.

Impacts
What was accomplished under these goals? Aim 1: Examine the metabolome and transcriptome of broodstock female salmon and determine their ability to predict broodstock and offspring quality. Methods development for three different tissue matrices have been developed over the reporting period, including plasma, skin mucus, and ovarian fluid. In collaboration with our group at the University of Maine, the USDA, and collaborators at NIST, skin mucus, plasma, and ovarian fluid were collected from broodstock female Atlantic salmon at the USDA-ARS National Cold Water Marine Aquaculture Center (NCWMAC) in Franklin, ME. NIST has analyzed these samples for untargeted metabolomics using nuclear magnetic resonance spectroscopy to identify biomarkers of reproductive success by directly comparing high performers ( >70% embryo survival)with low performers (<70% embryo survival). Work is ongoing to refine methods to reduce interference with blood contamination. Following statistical analyses with various machine learning pipelines, 15 purported influential metabolites have been identified which include various amino acids, organic acids, polyamines, and carbohydrates. Our group at UMaine has analyzed plasma, skin mucus, and ovarian fluid for steroid hormones using LC-MS-MS (steroidomics), and we are currently in the process of analyzing these data using machine learning pipelines to examine their predictive utility for both broodstock and cohort performance. Aim 3: Examine the utility of morphometrics and behavior as predictive indicators of cohort quality in embryos or yolk-sac fry. Similar to year 1, fertilized eggs of Atlantic salmon were collected following a seasonal spawn and were transported to our heath tray incubation systems at the University of Maine. Embryos were evenly split into two separate tray systems, one intended for regular sampling, and the other remained untouched apart from routine monitoring. It is common practice in commercial culture to avoid disturbing developing embryos as it is thought this will lead to increased mortality. Samples of the embryos were taken daily over the ~7 week incubation period to determine developmental timelines related to mortality events (i.e. what stage of development are the embryos most prone to mortality). Morphometrics (measurements of size and shape) were taken routinely over the incubation period, as well as for newly hatched yolk sac larvae (alevin). We then took measurements of parr (1+ years old) from the cohorts we measured inyear 1. Interestingly, and counter to expectations, we found that smaller eggs resulted in significantly larger parr. This phenomenon has also been observed in another salmonid, steelhead trout. This led us to ask questions related to the predictive utility of markers of metabolism.The most common ways to measure metabolism in fish is through respiration, and heart rate. We are currently in the process of analyzing dataof embryo respiration rates, tracked using Pyroscience oxygen probes inerted into 25ml air tight scintillation vials from embryos sampled this past spawning season. We also tracked the heart rates of embryos that were placed in a temperature ramp within an Isotemp refrigerated bath circulator. Heart rate increases withtemperature,untilthe heart rate begins to becomeerratic (i.e. when the temperature begins to exceed the embryo's tolerance). Two regression lines are then calculated, one for the consistent heart rate increase, and anotherwhen it becomes erratic, and the intersection is termed the Arrhenius Break Temperature (ABT). The ABT is an indicator of thermal and metabolic tolerance. Respiration and ABT data will be analyzed over the summer, and the same cohorts of fish will be followed to determine the predictive utility of these data.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Hamlin, H.J., Bair, H., Moorefield, R., Peterson, B., Legacki, E., Burr, G., Zohar, Y., Miller, J., Stubblefield, J., Firkus, T. Improving reproductive efficiency for Atlantic salmon grown in recirculating aquaculture systems. RASTECH, June 2024, Charlotte NC.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Hamlin, H.J., Bair, H., Morefield, R, Casu, F., Schock, T., Mahynski, N., Legacki, E., Peterson, B., Identifying biomarkers to improve reproductive performance in Atlantic salmon (Salmo salar). Aquaculture America 2024, February 18-21, 2024, San Antonio, TX.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Hamlin, H.J., Bair, H., Peterson, B., Burr, G., Legacki, E., Zohar, Y., Miller, J., Stubblefield, J., Firkus, T., Improving year-round egg production for Atlantic salmon grown in RAS. RASTECH, April, 2023, Orlando, FL.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Bair, H., Morefield, R., Hamlin, H.J., Establishing predictive metrics for Atlantic salmon Salmo salar broodstock and offspring. Aquaculture America 2024, February 18-21, 2024, San Antonio, TX.


Progress 07/01/22 to 06/30/23

Outputs
Target Audience:Target audiences reached during this reporting period includes industry professionals, federal and state agencies, and the public at large (via conference presentations and in-person meetings). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has allowed for networking / partnering with other institutions, such as the USDA and University of Maine faculty members. USDA staff members with specialties in reproductive endocrinology and nutrition, as well as staff technicians have been essential in the data collection, field work, and data dissemination. Working with the USDA has provided professional development opportunities through field work; harvesting Atlantic salmon in Machiasport at the Cooke facility to obtain carcass weight metrics for the selective breeding program. Completing field work for the USDA's annual spawns has shed light on industry processing methods; data collection, stripping, fertilizing, blood collection, mucus collection, and the collaborative teamwork required to successfully complete industry tasks. This project has fostered connections to be made with faculty and graduate students in different disciplines at the University of Maine. Dr. Emma Perry, an electron microscopist, has provided professional development in offering imaging guidance and ideas regarding the morphometric work with small embryos and alevin. Working with Emma has allowed familiarity to be gained in areas of microscopy, fixative, and histological techniques. Images have been taken with a scanning electron microscope, where imaging shows surface and cell composition in great detail. Additionally, conversations with University of Maine's developmental biologist Dr. Jared Talbot have allowed professional development through training with zebrafish standard operating procedures. Developmental biology exposure has offered the opportunity for methodologies to be integrated into our project scope to better understand embryo mortality, and identify indicators of success early on in the aquaculture process. How have the results been disseminated to communities of interest?Results were shared at a two day graduate student symposium held May 8th-9th at the Darling Marine Center, Walpole, Maine. This was a professional development retreat hosted by the University of Maine's School of Marine Sciences. The audience consisted of faculty and fellow SMS graduate students. The poster presented was titled; "Establishing predictive metrics of Atlantic salmon". Results outlining the eye up rates and mortality in comparison to the USDA's breeding values were presented, showing no correlation. The poster results elucidated the embryo mortality issue within the industry from 2008-present. Emphasis on the breeding values was presented to amplify the idea that selecting for growth and disease resistance, leaves the breeding program with an energy trade-off, that may be apparent with the lack of optimum embryo survival. Progress displaying images of eggs and alevin for morphometric study were presented. Future direction of investigating the genome of eggs through transcriptomic analyses was explained. The utility of morphometrics and fluctuating asymmetry as a predictive metric of embryo quality was disseminated to classmates within graduate school courses SMS 691(Marine science seminar), and SMS 500 (Marine Biology) in the form of elevator pitches, and a project proposal presentation. Results for Aim 1 were presented at a national conference, heavily attended by industry personnel? Hamlin, H.J., Bair, H., Peterson, B., Burr, G., Legacki, E., Zohar, Y., Miller, J., Stubblefield, J., Firkus, T., Improving year-round egg production for Atlantic salmon grown in RAS. RASTECH, April, 2023, Orlando, FL. What do you plan to do during the next reporting period to accomplish the goals?Aim 1; In addition to continuation of mucus swabs, ovarian fluid, and blood sampling for omics analysis, we have planned to take metabolomic samples of embryos, and yolk-sac fry in sync with the developmental experiment time points. Samples of eggs amounting to 0.1 g will be collected at multiple time points: prior to fertilization, post fertilization, and every other day until reaching yolk-sac fry stage. Using the liquid chromatography mass spectrometry (LC-MS-MS) we will run samples to gain understanding of the present sex steroids and activity at each stage of development. Correlation of steroids to batch quality will be key to understanding biological intricacies throughout development, and pose the opportunity to be incorporated as predictive metrics of future offspring performance. Aim 2 - We intend to focus on this aim in year 3. Aim 3; we plan to collect eggs from the USDA's 2023 spawning broodstock at the Cooke Bingham Maine facility. The collection will follow suit with last year; eggs will be raised at the DRL, University of Maine campus. Experimental design will ensure equal representation of all EBV's (control, low, medium, and high) for proper replication. The 2023 egg experiment will maintain incubation temperatures at industry standard to make findings applicable in production settings. This fall we will integrate developmental biology aspects to the incubation portion of the experiment allowing for in depth examination of the morphometrics changes that take place from egg, embryo, to yolk-sac fry. We plan to assess morphology during cleavage, gastrulation, and assess the relationship of morphology to survival to aid in understanding the quality of egg batches. Integrating developmental biology into the experimental design will be helpful in understanding early signs of batch quality (growth, survival) that can be incorporated into predictive indicators for future performance of cohorts. Morphometric measurements of total length, yolk diameter, yolk volume, eye diameter, will continue to be recorded for individuals.This 2023 spawn collection we Intend to take a bulk of measurements on live individuals, as opposed to preserved individuals to maintain biological integrity of a living individual. Measurements and analysis with a developmental biology lens will propel our understanding of mechanisms influencing early performance, and ultimately can be incorporated into predictive biomarkers early in the production process.

Impacts
What was accomplished under these goals? Aim 1: Samples (n = 120) of eggs, skin mucus, and ovarian fluid were take in the fall 2022 spawning season from the USDA's National Coldwater Marine Aquaculture Center (NCMAC), Franklin, Maine and were shipped to NIST for metabolome assessment. NIST personnel are currently processing these samples. Plasma samples were validated for LC-MS-MS hormone assessment, and samples from the fall 2022 spawning season will be processed in the summer of 2023. Aim 2: Nothing to report Aim 3: Fifty three batches of eggs were collected. The batches of eggs were collected from mid-November 2022 to mid-December 2022 and incubated on The University of Maine's campus until hatching. Samples were collected throughout the incubation period when eye pigmentation became present (eye-up) and when the organism hatched from the chorion and was attached to the yolk-sac, at alevin stage. A total of 496 eyed eggs were preserved, and 466 post hatch alevin in a 10% formalin preservative. Samples have been imaged with a Nikon DSFi3 camera and the NIS elements imaging software through the Nikon basic research package. This software has extended depth focus (EDF) which allows the fine focus to stack 3 dimensional depths into a resulting 2d image. To grasp batch morphometrics 2 images at the eyed stage have been taken; 1 of each eyed egg individual to measure size attributes: egg area.The second image of an individual was taken with the chorion removed to gain understanding of qualitative attributes; eyes, notochord, and deformities that may exist. At the alevin stage 5 images have been taken for each individual. One image was taken of the left whole organism, and another of the right whole organism to record right and left eye diameters, total length, head depth. Images 3 and 4 were taken from the left and right of the yolk being removed. Image 5 was taken of the individual ventrally to see down the notochord from above to record fluctuating asymmetry metrics from notochord to left and right features, such as eyes, nares. Measurements have been taken through the software, and maintained in metadata to be compared to cohort parent weight, and growth metrics from the USDA. Eye up rates for batches of eggs raised at the University of Maine were calculated. Our overall eye-up was very low averaging about 20% survival, synonymous to what was seen in industry. Fifteen of the batches ranged in eye up from 1-10% and 16 batches ranged from 11%-80%, where a majority lied within the 25% eye up range.The remaining 22 batches showed zero eye up. The estimated breeding values (EBV) which is an average of the mother and father's breeding values were averaged and determined to be low, medium, or high. The values of batch EBV show no correlation to the percent embryo survival. Once all images and measurements are recorded for all batches further analysis will reveal correlation to growth and survival when comparing eggs raised at NCMAC to our morphometric findings.

Publications