Source: UNIVERSITY OF MAINE submitted to
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Apr 1, 2015
Project End Date
Mar 31, 2018
Grant Year
Project Director
Hamlin, H.
Recipient Organization
ORONO,ME 04469
Performing Department
School of Marine Sciences
Non Technical Summary
The US currently imports 91% of its seafood, resulting in a seafood trade deficit of more than 10 billion dollars; this is the greatest agricultural trade deficit in the US. Aquaculture currently accounts for approximately 50% of US seafood imports, with Atlantic salmon (Salmo salar) being the second leading import. In New England, where the majority of Atlantic salmon are produced in the US, commercial aquaculture production has dropped more than 35% since 2000. This is due in large part to reduced embryo survival, which has become a considerable challenge for hatchery operations. Egg production is directed largely by hormones, and factors that alter hormone production can have serious consequences for embryo growth and survival. At the time of spawning, female Atlantic salmon that produce batches of eggs with high survival have different hormone profiles than salmon that produces batches of eggs with low survival. In order to understand what these differences mean, as well as develop a framework to understand the mechanisms involved, more work is needed to understand the endocrine processes that underlie reproduction. Specifically, we need to understand seasonal reproductive hormone profiles for North American strains of adult female Atlantic salmon, including 11-ketotestosterone, un understudied but important androgen. Because whole animal studies are time consuming and costly, tissue and cell culture models for this salmon strain need to be refined to complement whole animal experimentation. This information can then be used to design future studies to address causes of embryo mortality.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
0810 - Finfish;

Field Of Science
1020 - Physiology;
Goals / Objectives
The goal of this project is to increase our understanding of Atlantic salmon endocrine function, both in-vivo adn in-vitro, which is necessary to develop appropriate experimental designs for future funding.Specific objectives include:1) Determine seasonal sex steroid cycles for North American strains of commercially cultured Atlantic salmon?2) Develop a cell culture model for Atlantic salmon ovarian cells
Project Methods
Year 1Experimental Design: A system of 12 tanks (2 m3 tank volume) holding 11 adult female Atlantic salmon (≈ 3-kg ea.) per tank will be constructed (Fig. 3). Eleven females/tank (22 females/tmt) was chosen based on a power analysis, taking into account an alpha of 0.05, a standard deviation of 107 (based on our preliminary data), a difference to detect of 20% and a power of 80%. Tanks will also house 2 males each to use for spawning purposes. Each treatment will contain 2 replicate tanks. Blood samples of fish in treatment A will be taken monthly; fish in treatment B will be sampled every 3 months; fish in treatment C will be sampled at 6 and again at 12 months; and fish in treatment D will be sampled only at 12 months. The purpose of treatments B-D is to determine whether repeated blood sampling affects plasma hormone concentrations. Plasma samples will be extracted with di-ethyl ether and analyzed for E2, T and 11-KT concentration using a solid phase radio-immunoassay(23). All hormones have been validated for use in this assay by ensuring parallelism of serially diluted plasma samples with the standard curve. When ready to spawn, females will be strip spawned and milt from a mixture of 3-5 males will be added to each female's eggs. The same milt mixture will be combined with each batch of eggs to reduce male influence. Embryos will then be incubated at 10°C until hatch, and percent embryo survival will be calculated.Year 2Two different in-vitro assays will be developed and optimized for Atlantic salmon, which will include the culture of ovarian follicles, as well as the development of primary cell culture lines of Atlantic salmon ovarian tissues.Ovarian tissue culture Ovarian follicle tissue will be cultured according to methods previously established for Atlantic salmon (Baltic strain) by Lee at al, (2006)(27). Briefly, ovarian tissue (five to ten follicles) will be dissected from the ovary and incubated in chilled Cortland's solution in 24-well tissue culture plates at 10°C for 18-h. Either T or 17α-hydroxyprogesterone (17P) will be added (in ethanol) to achieve a concentration of 100ng/ml. Other studies have shown a clear dose response to these steroid substrates, suggesting that substrate is a limiting factor for in-vitro steroid production by the follicles(27). The hormone production of follicle tissue will be quantified by RIA.Primary cell culture The ovarian follicular cell culture for Atlantic salmon will be prepared according to protocols established for zebrafish(28). Briefly, salmon ovaries will be removed and placed into 60% Leibovitz L-15 media, and the follicles separated and cultured for 6 days in M199 + 10% fetal bovine serum under 10°C and 5% CO2 to increase cell yield. Tissues will then be washed in culture media and digested with 0.25% trypsin in phosphate buffered saline at 10°C for 15 minutes. Cells will then be centrifuged and filtered with a 40 μM cell strainer to remove follicular debris, and cultured in 24-well culture plates at a density of 100,000 cells/ml per well for 24 h. Studies using other fish species found it necessary to modify this method to optimize assay performance(29), so it is expected that this method will need to be optimized for Atlantic salmon. Performance of ovarian follicle cells on hormone production can then be assessed in the culture medium using a radioimmunoassay. Culture media containing hormones will need to be validated for use in the RIA to verify results. Primary cell culture offers several advantages over tissue culture in that the response of specific cell types (theca and granulosa cells) can be determined following cell separation(28,30), allowing for greater utility in ascertaining mechanisms(31)

Progress 04/01/15 to 03/31/18

Target Audience:The target audience reached during this project includes numerous commercial producers, federal and state salmonid hatchery managers and members of the public through a discussion at an annual salmonid hatchery roundtable as well as a national conference. In addition, a number of undergraduates assisted with monthly sampling, increasing their research skills and interest in agricultural science. A portion of this project became an undergraduate senior thesis, and this project formed two chapters of a Ph.D. dissertation. Further, two new tank-based commercial recirculating aquaculture salmon operations are coming to Maine, and this information has been shared with them. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In addition to serving as the basis for a Ph.D. student's dissertation, 4 undergraduates have assisted with various components of the project. These students have learned how to condition and spawn salmon, observe and describe embryonic development, collect blood samples, run lab assays, and analyze data. An undergraduate presented her work at an annual University of Maine undergraduate conference. I have discussed this work with commercial stakeholders that are initiating construction of two new facilities here in Maine. This work has forged a shared interest and I will continue to work on projects that benefit the advancement of Maine aquaculture. How have the results been disseminated to communities of interest?Results have been disseminated via conference presentations, in-person meetings with stakeholders (described above), and outreach displays describing our work that will remain in the Aquaculture Research Center and will be a part of facility tours. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

What was accomplished under these goals? Objective 1: Determine seasonal sex steroid cycles for North American strains of commercially cultured Atlantic salmon. We have successfully completed all of our stated aims for this objective. We collected monthly blood samples of female Atlantic salmon broodstock reared in an indoor recirculating aquaculture system. Samples were analyzed for plasma 17B-estradiol (E2) and 11-ketotestosterone (11-KT), and seasonal hormone profiles have been established. Plasma E2 peaks in September, while plasma 11KT peaks in October, with spawning occurring primarily in November. Further, hormone profiles of females that were sampled monthly, were not significantly different from those sampled every three months, or those sampled only at the beginning and end of the project. Assessments of oocytes also do not indicate that monthly sampling negatively affects development. Taken together, these data indicate that monthly sampling does not adversely affect the reproductive and endocrine endpoints measured in this study, an important determination for experimental planning. In addition to the completion of our stated aims, we collected samples of ovarian fluid and ovulated oocytes at the time of spawning to determine concentrations of E2 and 11-KT. We found that plasma and egg steroids did not correlate, but that ovarian fluid and egg steroids correlated strongly. These findings suggest that the follicle is a key source of egg sex steroid concentrations, but that plasma hormones do not necessarily reflect follicle production. Objective 2: Develop a cell culture model for Atlantic salmon ovarian cells. We were able to successfully culture ovarian follicle tissue and produce concentrations of hormones (E2 and 11KT) detectable by commercial assay kits (Cayman Chemical). We determined that commercially available Minimal Essential Media (MEM) with Earle's salts and fetal bovine serum plus gentamicin was an effective culture media, but contained appreciable concentrations of E2 as well as androgens (T), which varied somewhat by batch, albeit at lower concentrations than produced by the follicles. Hyclone MEM/EBSS, which is a purified and charcoal filtered medium, contained far lower hormone concentrations, but they werestill detectable. By using this medium, and subtracting the Hyclone MEM/EBSS hormone concentrations, we have developed an effective follicle culture protocol. However, we continue our efforts to develop or source an effective media that is free of detectable hormones. Samples of ovarian tissue for cell culture were collected at two different timepoints, in mid and late stages of oocyte development and vitellogenesis. In both stages, we were able to obtain granulosa cell growth and confluence, however, cells grown from mid stage ovaries were far more successful in terms of cell abundance and confluence. However, despite trying many different medias and conditions, we were not able to achieve a high enough concentration of hormone to be appreciably above media background concentrations. We continue our efforts to establish an effective protocol, however, the follicle culture protocol described previously can be employed for studies necessitating in-vitro or ex-vivo culture.


  • Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Thayer, L.R., Tudor, M.S., Conlin, S., Hamlin, H.J, Annual hormone profiles and the influence of blood sampling in the North American strain of female Atlantic salmon, Salmo salar L. broodstock, in a recirculating aquaculture system. Submitted to Aquaculture, June 2018.
  • Type: Journal Articles Status: Other Year Published: 2018 Citation: Thayer, L.R., Tudor, M.S., Hamlin, H.J. Correlations of plasma, ovarian fluid and egg sex steroids, at the time of spawning in Atlantic salmon, Salmo salar L. To be submitted to Aquaculture Research in July 2018.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Hamlin, H.J., Thayer, L.R., Maternal endocrine status and embryo survival in Atlantic salmon (Salmo salar L.). Eastern Fish Health Workshop, April 2018.

Progress 04/01/16 to 03/31/17

Target Audience:The target audience reached during this reporting period includes numerous commercial producers, federal and state salmonid hatchery managers and members of the public through a discussion at an annual salmonid hatchery roundtable. In addition, a number of undergraduates assisted with monthly sampling, increasing their research skills and interest in agricultural science. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project supported a Ph.D. student, who will graduate this summer and thereafter enter the workforce. In addition, several graduate students and undergraduates participated in monthly sampling and have gained valuable skills including fish blood sampling, proper fish handling and anesthetization, and sample processing. How have the results been disseminated to communities of interest?This work was presented to attendees at a local salmonid hatchery roundtable. Attendees included commercial producers, state and federal hatchery managers and the public. What do you plan to do during the next reporting period to accomplish the goals?Our focus for the next reporting period is to complete Objective 2: Develop a cell culture model for Atlantic salmon ovarian cells. We intend to collect ovarian samples from July-October to continue our work developing a cell culture line for ovarian cells. We are currently attempting to locate a culture medium that itself does not contain detectable concentrations of sex steroids, or refine a method to strip bovine serum of hormones.

What was accomplished under these goals? Objective 1: Determine seasonal sex steroid cycles for North American strains of commercially cultured Atlantic salmon This objective has been completed and all our stated aims have been accomplished. The previous report detailed the successful spawning of broodstock donated to us by a local USDA facility. Blood samples of female broodstock were obtained according to our proposal protocols (monthly, bi-monthly and end sampling). Fish were back on feed within two weeks of spawning, and continued to feed despite the stress of routine sampling. Plasma 17B-estradiol and 11-ketotestosterone was assessed at the end of the experiment for all samples, and seasonal reproductive hormone profiles have been established. The stress of monthly sampling did not appear to alter the reproductive hormone profiles compared to animals sampled bi-monthly or those sampled only at the beginning and end of the trial. Images of the gonads have been taken to assess developmental stage, and anecdotally it appears the monthly sampling did not contribute to atresia or a cessation of oocyte development. Beyond our stated goals, we have taken samples of ovarian fluid and eggs to determine patterns of steroid deposition into the egg. Two manuscripts are currently being drafted. Objective 2: Develop a cell culture model for Atlantic salmon ovarian cells The majority of this work will take place this summer and fall, and no new work on this objective has been accomplished beyond what was reported in the last progress report. We have made contact with commercial producers to collect ovarian samples, and anticipate no problems with sample collection.


    Progress 04/01/15 to 03/31/16

    Target Audience:The target audience reached by our efforts includes industry, students, and indirectly, seafood consumers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research will be included as part of LeeAnne Thayer's Ph.D. dissertation, and has allowed her to liaise with industry partners more closely than she might have otherwise. She and our technician have been working with an undergraduate student to collect additional data from this project to fulfill a senior research project, that we feel will be publishable. In addition, everyone from my research group, which includes two Ph.D. students, a M.S. student, two technicians, and an undergraduate student participate in monthly sampling, which has allowed all of us to gain experience spawning salmon, handling the fish, taking blood samples and processing the blood. How have the results been disseminated to communities of interest?We have maintained communication with administrators at the USDA, and continue to liaise with stakeholders at Cooke Aquaculture, a leading producer of Atlantic salmon. Once we have collected and analyzed our data, we hope to present our work at future symposia (both local and national). In addition, we have given tours of our experimental room to various members of the public, legislators, and researchers from a number of institutions. What do you plan to do during the next reporting period to accomplish the goals?We will continue to collect monthly blood samples, and will analyze these samples in August. We will collect tissue samples for both cell and tissue culture in August to begin this portion of our work.

    What was accomplished under these goals? Following system construction, 60 broodstock females and 11 males were transported from the USDA NCWMAC facility in Franklin, ME, to the Aquaculture Research Center at the University of Maine in September of 2015. Fish were transported in a shipping container on a flatbed truck via Cooke Aquaculture, and water was monitored for oxygen content continuously throughout the 1.5 hour trip. Each fish held a passive integrated transponder (PIT) tag which identified individuals. Fish were anesthetized prior to transport, weighed, and initial blood samples were taken prior to transport. A total of five fish (7%) were lost during transport, or shortly thereafter, which is within the 10% transport loss that we anticipated. Fish were eating pelleted food within a week of transport. Blood samples continue to be collected according to the sampling regime detailed in our grant proposal. During our monthly blood sampling in November, fish were checked for spawning readiness, and were spawned if eggs freely flowed from the vent with only moderate abdominal message. Milt from at least five males was pooled prior to fertilizing the eggs, and these same five males were used for all spawnings, to reduce variation in male contribution. All fish spawned between November and December, and we were able to collect eggs from 34 out of 55 females (62%), which is higher than the industry average of 50%. Embryos were placed into Heath trays with individual partitions to separate embryos by female. The embryos are light sensitive, therefore, the Heath tray system was covered to prevent light penetration, and a small number of embryos from each female was placed in an end rack to allow for visual inspection of development, without disturbing the remaining embryos. A headlamp with red spectrum light was used during visual inspections. Embryos were prophylactically treated for fungus with formaldehyde weekly, in accordance with industry practice. Embryos from the November spawns began to reach the eyed stage in January (approximately 6 weeks post-fertilization). We waited for three days following observation of the first eyed embryos to ensure all viable embryos had reached that stage. Half of the embryos from each tray were then removed and manually counted for percent eyed embryos, with non-eyed embryos considered non-viable. Microscope dissection of a portion of non-eyed embryos confirmed non-viability. The remaining embryos were allowed to hatch and percent alevins (newly hatched salmon larvae) was determined. Approximately 46% of the embryos were viable, which is close to the industry average of 50%. Blood samples will be analyzed at the end of year 1 for hormone analysis. In summary, we have made great progress, and our accomplishments align with or exceed the objectives set forth in the grant application. Additional accomplishments: Although scheduled for year two, a preliminary experiment was performed using ovarian tissue from maturing Atlantic salmon in order to determine the optimal conditions for establishing primary ovarian cells in culture. Based on current literature with other fish species, the objective was to obtain ovarian tissue in mid- vitellogenesis for producing primary ovarian cells. Female Atlantic salmon were selected from a pool of this year's broodstock that were maturing but did not spawn. Five female salmon were euthanized and ovarian tissue was collected and examined. Ovarian tissue from females that had not ovulated was extracted and processed from each female. Ovarian tissue was washed in sterile PBS, connective tissue removed and tissue placed in HBSS for transport back to the laboratory. Once in the laboratory, enzymatic cell disaggregation was performed by repeated trypsinization. Debris was filtered from cells, cells dispersed in 25cm2 cell culture flasks with appropriate culture medium and placed at 16C with 5% CO2. Levels of cellular confluence were monitored and recorded. For this experiment, cells were monitored until 100% confluence which was achieved for each flask. The experiment was very successful in establishing monolayers of primary ovarian cells and allowed for optimization of steps with cell disaggregation and cell dispersal. During the course of monitoring embryo development, we sought published pictures of embryo stages throughout development, and realized only drawings exist for Atlantic salmon. Therefore, we took the opportunity to take samples for imaging, and have begun constructing high quality images using image stacking. An image stack combines a group of pictures taken throughout the focal length of the embryo, and then combines the images, removing all areas that are out of focus, with the end result being a complete and very crisp image of the whole embryo. In addition, we've collected samples for clearing and staining to describe the development of cartilage and bone in the embryos, and are image stacking these pictures as well. This image collection and assessment will comprise the senior project of one of our undergraduate students.