Progress 02/01/18 to 01/30/23
Outputs
Target Audience:Scientists, aquaculturists, fish farmers, feed manufacturers, comparative genomisists. Changes/Problems:Federal government shutdowns impacted several aspects of this project and delayed data collection, analysis, and dissemination. The following is a list of shutdowns and impacts: 1) the start of our first feeding trial (Dec 2018-Jan. 2019) was delayed pushingour broodstock feeding trial to the following spawing season - which meant we used a different genetically selected line than originaly outlined. 2) the COVID-19 pandemic shutdowns (Federal, University) starting March 2020 led to several delays in sampling and sample sample processing. With one year no-cost extension, we were able to catch up on sampling and data pipeline completion. Data analysis continues for this project after the award closure. One graduate student will continue to complete the analyses and submit publications by the end of 2023. What opportunities for training and professional development has the project provided?Coding, genomic analysis with non-model organisms, and open science training. Data management and sharing best practices. Basic science overlap with aquaculture industry and food/nutrition security. Over the entire project, 9 undergaduate students gained one-on-one training with the mentor and graduate students, learning basic molecular biology and cell biology techniques. How have the results been disseminated to communities of interest?1. Peer-reviewed publications in Journal of World Aquaculture Society (1 already published - DOI: 10.1111/jwas.12634; 1 additional to be submitted in 2023)and Comparative Biochem & Physiology (1 published - Doi:10.1016/j.cbpa.2019.03.010; 1 to be submitted in 2023). 2. Conference presentations at Plant & Animal Genomes (PAG, 2022, 2023, 2024), Aquaculture America/World Aquaculture Society Meetings (2019, 2021, 2022, 2023, 2024), International Congress on the Biology of Fish (2022). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1 - To identify how maternal dietary choline intake affects offpsring performance, we individually PIT tadded 36fish from each of six diesase resistant broodstock families (N = 216fish total) from the NCCCWA selective breeding program (2018 spawning season). These PIT tagged fish were reared to15 months age (July2019) when the broodstock were fed experimental/dietary diets:adequate choline (4,400 ppm), excess choline (8,800 ppm), and low choline (2,200 ppm). Fish began consuming dietary treatments in July 2019 just prior to rapid gonad growth, and continued through to spawning in January 2020. Fish were weighed bimonthly. Spawning took place and offpspring were tagged and reared under normal conditions and monitored for growth monthly. Offpsring from dietary treatment groups were sampled at 0 day post hatch adn 14 days post hatch. These samples were isolated and processedfor RNAseq and RRBS analyses to analyzeglobal gene expression effects and global methylome changes associated with maternal choline intake. All sequencing (RNAseq adn RRBS) have been completed, and 75% of data analysis has been completed. The pipelines for both RNAseq and RRBS data processing have been built and are available on GitHub(https://github.com/peggybiga/diet-epi-stress).Complete analysis of all data (RNAseq and RRBS) is still processing and manuscripts are in process. Objective 2 - To assess direct effects of choline on specific tissues, myocyte cultures from trout were isolated and treated with different levels of choline. The analysis of cell proliferation and myogenic specific gene expression changes are still ongoing. In 2019, trout eggs were injected with 15 ug or 30 ug choline chloride, or saline as a control. These treatments were injected into fertilized eggs from six separate male x female crosses, although only two crossess produced viable embryos with acceptable hatch rates. 91 fish from these two crosses were PIT tagged in August 2019, and comingled for grow-out. A larger injection study was planned in 2019, however a 35 government shutdown impacted the ability to accomplish this. A larger study was planned for Spring 2020, and again government shutdowns hindered the ability to complete this part of the study. A plan for 2023 is in place for choline injections to allow for the appropriate analyses.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Cleveland, B.M., T.D. Leeds, M.J. Picklo, C. Brentesen, J. Frost, and P.R. Biga. 2020. Supplementing rainbow trout (Oncorhynchus mykiss) broodstock diets with choline and methionine improves growth in offspring. Journal of the World Aquaculture Society. 1-16. Doi: 10.1111/jwas.12634
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Latimer, M.N., R.M. Reid, P.R. Biga, and B.M. Cleveland. 2019. Glucose regulates protein turnover and growth-related mechanisms in rainbow trout myogenic precursor cells. Comp. Biochem. Physiol. A. 232:91-97. Doi:10.1016/j.cbpa.2019.03.010. PMID30904682
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Biga, P.R., B.M. Cleveland, T.D. Leeds, and K. Freij. Supplementing rainbow trout broodstock diets with choline and methionine improves offspring growth. Aquaculture Canada and WAS North America 2020
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Biga, P.R., I. Seiliez, J-C, Gabillard, M.N. Latimer, and B.M. Cleveland. Methionine Restriction Diminishes Skeletal Muscle Cell Differentiation Through Epigenetic Mechanisms in Rainbow Trout, Oncorhynchus mykiss. World Aquaculture Society, February 2022. San Diego, CA, USA
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
K. Freij, Cleveland, and P.R. Biga. Improving rainbow trout Oncorhynchus mykiss growth performance through optimization of diet-epigenetic interactions. World Aquaculture Society, February 2022, San Diego, CA USA
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
K. Freij, S. Turkmen, B. Cleveland, and P.R. Biga. Improving rainbow trout Oncorhynchus mykiss growth performance through optimization of diet-epigenetic interactions. Plant and Animal Genome, 2021, Virtual
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
K. Freij, S. Turkmen, B. Cleveland, and P.R. Biga. Improving rainbow trout Oncorhynchus mykiss growth performance through optimization of diet-epigenetic interactions. Aquaculture America, 2023, New Orleans, LA
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Progress 02/01/21 to 01/31/22
Outputs
Target Audience:Scientists, aquaculturists will be reached at the World Aquaculture Society Meeting in February 2022. We will present 2 papers related to this project. Changes/Problems:We have an apporved no cost extension for one year due to shutdown constraints caused by the Covid-19 pademic. What opportunities for training and professional development has the project provided?Coding, genomics analyses with non-model organisms, and open science training. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Final data analyses will be complete and we will have idetnfied pathways and genes regulated by maternal choline intake, and we will compare those to methylated status on the genome to determine if maternal choline affects gene expression via methylation (corralative analyses). We will also show direct choline effects on muscle cells in vitro to determine what local effects choline has on growth and metabolism.
Impacts
What was accomplished under these goals?
Offpsring from broodstock fed supplemental choline were sampled at o days post hatch and at 14 days post hatch.These samples have all been isolated and RNAseq and RRBS analyses completed. We are now in teh process of validating the data analysis pipeline for both RNAseq and RRBS. We have a GitHub page where all code is available. We hope to complete analyses soon. Myocyte cultures from trout have been isolated and cultured with different levels of choline. We will be analyzing gene expression effects of choline direclty on myocytes soon.
Publications
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Progress 02/01/20 to 01/31/21
Outputs
Target Audience:Graduate and undergraduate students at UAB, as well as one post-doctoral scientist, were trained during the second year of reporting. Overall, 2 undergraduate students and 1 graduate student were trained. In addition, a post-doctoral scientist has joined the team. Training included fish biology, nutrition, basic molecular biology, and basic genomic science. Additionally, research presentations were given to stakeholders of the rainbow trout industry, including fish producers and feed manufacturers, academic and government researchers, and policy makers. Changes/Problems:Due to the COVID-19 pandemic, problems with continual sampling and egg injections persisted. We plan to repeat egg injections in Spring 2021. Also, general sample preparations and analyses have been slowed down due to the pandemic and having to moth-ball the UAB reserach lab during early pandemic reponses. New protocols and procedures are now in place and analyses should be completed only a few months behind. What opportunities for training and professional development has the project provided?One graduate student, one post doctoral trainee, and three undergraduate students have been trained in fish physiology, fish nutrition, and epigenetics. How have the results been disseminated to communities of interest?One peer-reviewed manuscript published. [Two additional manuscripts are in preparation.] What do you plan to do during the next reporting period to accomplish the goals? To analyze the direct effects of choline on cells, muscle cells with varying choline concentrations were cultured. Cell viability, proliferation/differentiation, and specific growth-related pathways will be analyzed in response to choline treatment. We are currently analyzing the effects of methionine restriction on muscle cell epigenome changes, and we will compare this to methionine supplementation. We will also analyze the effects of choline supplementation and restriction on the epigenome.
Impacts
What was accomplished under these goals?
continuedthrough January 2020 when spawning occurs. Fishwereweighed bimonthly. Similar to the initial study, there is currently no effect of dietary choline on broodstock body weight gain. This is expected; the benefit of choline supplementation was previously detected only in offspring. while the remaining were fertilized. Once eggs reached the eyeing stage, which is the point where embryos are significantly developed, a subsample was retained for DNA and transcriptomic analysis. A similar sample collection occurred after the eggs hatch and just prior to the fry first-feeding.Just after first-feeding thenumber of fry were reduced to 100 per family to avoid density effects on growth. Fish were PIT tagged once body weights reached 20 g. Tagged fish are comingled among three grow-out tanks and body weights and lengths are recorded bimonthly. To address objective 3, nothing to report on progress for this objective.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Cleveland, B.M., T.D. Leeds, M.J. Picklo, C. Brentesen, J. Frost, and P.R. Biga. Supplementing rainbow trout (Oncorhynchus mykiss) broodstock diets with choline and methionine improves growth in offspring. 2019. Journal of the World Aquaculture Society. 1-16. Doi: 10.1111/jwas.12634
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Progress 02/01/19 to 01/31/20
Outputs
Target Audience:Graduate and undergraduate students at UAB, as well as one post-doctoral scientist, were trained during the second year of reporting. Overall, 2 undergraduate students and 1 graduate student were trained. In addition, a post-doctoral scientist has joined the team. Training included fish biology, nutrition, basic molecular biology, and basic genomic science. Additionally, research presentations were given to stakeholders of the rainbow trout industry, including fish producers and feed manufacturers, academic and government researchers, and policy makers. Changes/Problems:This project is running one and a half years behind schedule since the 2017 spawning season did not produce sufficient numbers ofoffspring to enable a complete experimental design and the 2018 government shutdown affected productivity. No major changes are being made, outside of the timing for spawning and offspring rearing. What opportunities for training and professional development has the project provided?Three total university students have been trained in year two, including 1 undergraduate, 1 graduate, and 1 post-bachelorette students. One post-doctoral scientist is also being trained and assisting in mentoring. Training has included basic fish biology, molecular biology, and genomic/epigenomic sciences. How have the results been disseminated to communities of interest?Results from the preliminary studies were presented at Aquaculture America, 2018 and Aquaculture 2019, the annual National meeting of the World Aquaculture Society. This conference is attended by stakeholders of the rainbow trout industry, including fish producers and feed manufacturers, academic and government researchers, and policy makers. A manuscript detailing the preliminary study was published in the Journal of the World Aquaculture Society in 2019. (Cleveland et al., 2019, J World Aqua Soc. 50(3):1-16). In addition, in 2019 the US Trout Farmers Association held its annual meeting in Shepherdstown, WV, just 15 minutes from the NCCCWA. Part of the meeting was a tour through the NCCCWA facility; during this tour scientists discussed ongoing research efforts. The broodstock nutrition study supported by this grant was a prominent discussion topic and the project received positive feedback from these industry stakeholders. What do you plan to do during the next reporting period to accomplish the goals?Broodstock spawning will occur during the next reporting period and will represent major progress towards accomplishing objective 1. Eggs will be collected from each female rainbow trout on the experimental broodstock diets. Data regarding egg yield and egg size will be collected, in addition to broodstock body weight and carcass yield. A subset of the eggs will be retained for genomic DNA analysis while the remaining will be fertilized. Once eggs reach the eyeing stage, which is the point where embryos are significantly developed, a subsample will be retained for DNA and transcriptomic analysis. A similar sample collection will occur after the eggs hatch and just prior to the fry first-feeding. The remaining fry will begin the grow-out study when offspring body weights are analyzed bimonthly. Once fish reach ~20 g they will be PIT tagged and comingled for grow-out.Sicne To address objective 2, characterize how choline concentration in the rainbow trout eggs affects egg epigenome. Eggs from the 2020 spawning season (Jan - Feb 2019) will be injected with 0 µg, 15 µg, and 30 µg choline. Green eggs and eyed eggs will be analyzed using RRBS for genome methylome analyses. To address objective 3, to analyze the direct effects of choline on cells, liver cells with varying choline concentrations will begin in May 2020 through December 2020. Cell viability, proliferation/differentiation, and specific growth-related pathways will be analyzed in response to choline treatment. We are currently analyzing the effects of methionine restriction on muscle cell epigenome changes, and we will compare this to methionine supplementation. We will also analyze the effects of choline supplementation and restriction on the epigenome.
Impacts
What was accomplished under these goals?
To address objective 1, to identify how maternal dietary choline intake affects offspring performance, we are currently analyzing growth-related gene expression changes in muscle tissue from samples collected from our preliminary broodstock feeding study that started in 2014. We analyzing the expression of Myf5, IGF-2, MSTN-2a, and Myogenin using quantitative PCR analysis. Also in support of objective 1, we have begun the broodstock nutrition study with 36 PIT tagged fish from each of six disease resistant broodstock families (N = 216 fish total). Fish were distributed among 9 tanks (24 fish per tank) and assigned to one of three broodstock diet treatments in triplicate. As planned, the treatment diets represent three different choline levels: 1) low choline (2200 ppm), 2) adequate choline (4400 ppm), and 3) excess choline (8800 ppm). To achieve the appropriate choline levels, all dietary ingredients were analyzed for choline content and dietary formulations were modified accordingly to achieve the target levels. This effort was completed in collaboration with the USDA/ARS Alternative Feeds lab in Bozeman, MT. Fish began consuming dietary treatments in July, 2019 just prior to the period of rapid gonad growth, and will continue through January 2020 when spawning occurs. Fish have been and will continue to be weighed bimonthly. Similar to the initial study, there is currently no effect of dietary choline on broodstock body weight gain. This is expected; the benefit of choline supplementation was previously detected only in offspring. To address objective 2, characterize how choline concentration in the rainbow trout eggs affects fry performance, rainbow trout eggs from the 2019 spawning season were injected with 15 µg or 30 µg choline chloride, or saline as a control. These treatments were injected into fertilized eggs from six separate male x female crosses, although only two crosses produced viable embryos with acceptable hatch rates. Ninety-one fish from these two crosses were PIT tagged in August, 2019 and comingled for grow-out. Weights and lengths have been and will continue to be recorded bimonthly. Currently, body weights are similar among the three treatments, suggesting that the beneficial effects of broodstock choline supplementation are not simply an effect of increased choline deposition as a nutrient source to the developing embryo. Rather, it suggests an epigenetic effect transferred from the broodstock to the embryo, potentially through epigenetic modification. The original plan for this study was to inject much larger numbers of eggs to allow sampling of eyed eggs and fry. However, the five week government shutdown in 2019 occurred in the middle of spawning season, providing limited opportunity to inject the number of eggs required for the DNA analysis. Choline injections are planned for the 2020 spawning season to allow for the appropriate analysis. To address objective 3, we have outlined muscle and liver cell culture experiments to analyze the role of choline on tissue-level effects on gene expression and cellular metabolism. Muscle cultures are currently underway with focus on choline concertation gradients to determine range related to physiological response. We are measuring cell viability, proliferation/differentiation, and specific growth-related pathways in response to choline treatment.
Publications
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Progress 02/01/18 to 01/31/19
Outputs
Target Audience:Graduate and undergraduate students at UAB were trained during this first year of reporting. Overall, 3 undergraduate students and 2 graduate students were trained. Training included fish biology, nutrition, basic molecular biology, and basic genomic science. In addition, we presented our preliminary study findings to stakeholders of the rainbow trout industry, including fish producers and feed manufacturers, academic and government researchers, and policy makers were reached at the Aquaculture America meeting of the World Aquaculture Society in February 2018. Changes/Problems:This project is running one year behind schedule since the 2017 spawning season did not produce sufficient numbers of offspring to enable a complete experimental design. No major changes are being made, outside of the timing for spawning and offspring rearing. What opportunities for training and professional development has the project provided?Five total university students have been trained, including 3 undergraduate and 2 graduate students. Training has included basic fish biology, molecular biology, and genomic/epigenomic sciences. How have the results been disseminated to communities of interest?The results from the preliminary study were presented at Aquaculture America, 2018, the annual National meeting of the World Aquaculture Society. This conference is attended by stakeholders of the rainbow trout industry, including fish producers and feed manufacturers, academic and government researchers, and policy makers. What do you plan to do during the next reporting period to accomplish the goals?To address objective 1, determining choline concentration effectiveness on offspring performance, we have outlined our strategy for the broodstock feeding trial that will begin in June, 2019 using fish that are currently reared on a standard commercial feed. In January 2019 the feed ingredients used to produce experimental broodstock diets will be analyzed for choline content. The experimental formulations will be modified accordingly so that three diets with varying choline concentrations can be generated. In the spring of 2019, three experimental diets will be produced at a USDA/ARS lab in Bozeman, MT and shipped to/stored at the NCCCWA until the feeding trial begins in June, 2019. To address objective 2, characterize how choline concentration in the rainbow trout eggs affects fry performance, eggs from the 2019 spawning season (Jan - Feb 2019) will be injected with 0 µg, 15 µg, and 30 µg choline. A subset of injected eggs will be harvested prior to hatch to determine treatment effects on the embryo epigenome. The remaining eggs will be hatched and offspring will be tagged, comingled, and growth indices measured monthly for 12 months. To address objective 3, to analyze the direct effects of choline on cells, we will begin to culture muscle and liver cells with varying choline concentrations beginning in March 2019 through December 2019. Cell viability, proliferation/differentiation, and specific growth-related pathways will be analyzed in response to choline treatment. We are currently analyzing the effects of methionine restriction on muscle cell epigenome changes, and we will compare this to methionine supplementation. We will also analyze the effects of choline supplementation and restriction on the epigenome.
Impacts
What was accomplished under these goals?
To address objective 1, to identify how maternal dietary choline intake affects offspring performance, we are currently analyzing growth-related gene expression changes in muscle tissue from samples collected from our preliminary broodstock feeding study that started in 2014. We have designed primers and are now validating them for quantitative PCR analysis. Also in support of objective 1, we have individually PIT tagged 50 fish from each of six disease resistant broodstock families (N = 300 fish total) from the NCCCWA selective breeding program (2018 spawning season). These tagged fish will be reared until 15 months of age (June 2019) when broodstock dietary treatments will begin concurrent with onset of gonad development. This project is running one year behind schedule since the 2017 spawning season did not produce sufficient numbers of offspring to enable a complete experimental design. To address objective 2, characterize how choline concentration in the rainbow trout eggs affects fry performance, rainbow trout eggs from the 2018 spawning season were injected with choline at various concentrations to optimize the injection methodology and determine a suitable dose range for large-scale injections. Rainbow trout eggs were injected with 30 µg, 75 µg, and 150 µg choline chloride, or saline as a control. Eggs injected with the 75 and 150 µg doses failed to hatch while survival of the 30 µg treated-eggs was similar to the saline control. Based on these findings, eggs from the 2019 spawning study will be injected with 0 µg, 15 µg, and 30 µg choline.
Publications
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