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.
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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
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