Performing Department
(N/A)
Non Technical Summary
Currently, methods used to determine sex in aquacultured Russian sturgeons (Acipenser gueldenstaedtii) are laborious and applicable only for older fish (5-6+ years old) due to slow reproductive organ maturity. Value of its caviar is high (up to $2,000 per female fish) and thus there is a great need for the development of a more efficient caviar production process. A solution would allow Russian sturgeon farmers to raise monosex caviar-producing populations from early on, resulting in increased caviar production. We propose a comparison of 2 potential DNA-based diagnostic tests using 2 different primer sets and to apply the most reliable version in actual field conditions at the sturgeon farm. The main goal is to develop a rapid (8-10 h), non-invasive (swab DNA collection), and cost-effective ($7-10 reagent cost/fish) DNA-based test that will allow sex determination of juvenile (≤2 month old) Russian sturgeons. Successful application of this technology would allow U.S. sturgeon farmers to potentially double their caviar production and enhance the capacity of the United States aquaculture industry to contribute to domestic and global food security and economic growth. Additionally, successful validation of our proposed methodology would open up application opportunities to other species of sturgeon for production and conservation purposes.
Animal Health Component
0%
Research Effort Categories
Basic
10%
Applied
70%
Developmental
20%
Goals / Objectives
The main goal of this project is to develop a rapid (8-10 h), non-invasive (swab DNA collection), and cost-effective ($7-10 reagent cost/fish) DNA-based test that will allow sex determination of juvenile (≤2 month old) Russian sturgeon. Successful application of this technology as a long-term goal would allow U.S. Russian sturgeon farmers to potentially double their caviar production capacity (if there is a roughly equal distribution of males/females) by raising monosex populations. Additionally, successful validation of our proposed methodology would open up application opportunities to other species of sturgeon for production and conservation purposes. The overall objectives of the research are:1. To establish dedicated PCR-based DNA detection capability at CMAST, Morehead City, NC2. To validate VAC-1M male-specific Russian sturgeon DNA fragment together with AllWSex2/Ag49 primer set through a direct side-by-side comparison using DNA collected during ultrasound sex determination of mature sturgeons3. To develop a rapid DNA-based method for sex determination that may be applied to juvenile Russian sturgeons using non-invasive skin swabbing for the DNA samples collection
Project Methods
MethodsPureLink Genomic DNA Mini Kit (Invitrogen) will be used to isolate DNA from at least 24 individual caviar grains from geographically distant sources of Russian sturgeon. VAC-1M primers (previously identified unique and strictly male-specific DNA sequence from Russian sturgeons grown at Marshallberg Farm, Smyrna, North Carolina) will be tested for specificity side-by-side with the DNA from the Marshallberg Farm caviar. If found to be specific and similar for all three groups, VAC-1M-based primers set will be compared directly against AllWSex2/Ag49 multiplex primers using 96 sturgeon skin swab-derived DNA samples that will be collected during routine ultrasound as described below. If not, only AllWSex2/Ag49 multiplex primers will be used for comparing DNA-based sex determination results with the ultrasound method and to complete objective 3. Marshallberg Farm will allow us to collect DNA swab samples during their routine process of ultrasound sex determination for the sturgeons. Very similar to the experiments described in the preliminary results, at least four sets of 24 swab samples each (coinciding with the maximum tube capacity for the centrifuge rotor) will be collected from mature fish (5-7 years old). DNA will be isolated at the PD's lab located within a 30 min drive from the farm. For sex determination, our VAC-1M-based primers set (if successfully validated from caviar analyses) will be compared directly against AllWSex2/Ag49 multiplex primers to routine ultrasound results using an E-gel power snap electrophoresis system with the 48 samples E-gel format.After experimental validation of the DNA-based genetic markers, we plan to apply our DNA-based test to juvenile Russian sturgeons of up to 2 months in age (the actual minimum age/size still to be determined during the future experiments). As part of their regular business activities, Marshallberg Farm will obtain fertilized Russian sturgeon eggs. Marshallberg Farm will be responsible for fish hatching and young sturgeon handling and rearing activities/care. Overall, Marshallberg Farm will house more than 1,000 juvenile Russian sturgeons and make them available in order to conduct the proposed research. The test is designed to use a 96 well PCR plate format and can be potentially scaled up easily by utilizing additional resources if needed. We envision having 96 numbered, cube-shaped netting cages (with sides of around 12 inches) hooked inside and around the large tanks used for sturgeon farming. After swabbing, the juvenile sturgeons will be comfortably separated from each other within these cages until the end of the test (projected to be completed within 8-10 h). Samples transportation, DNA isolation, and PCR set up is estimated to last about 6-8 h plus at least 1 h 30 min for running and documenting results of two sets of 48 PCR samples using 48-well gels (two of the 48-wells E-gels should be used instead of one 96 wells gel that is also available, because of the need to achieve proper bands separation and ultimately producing more accurate results). After testing and relaying results to the farm, the respective female and male juvenile sturgeons can be easily relocated to different holding tanks. The process will be repeated every working day resulting in accurate sex determination of about 1,000 juvenile sturgeons over a 10 working day period using only one 96-well plate PCR format.Marshallberg Farm will provide standard care for all sturgeon fish that are subjects of this proposal until they are ready for typical ultrasound sex determination. They will have the unique opportunity to acquire all valuable information about growing monosex fish populations before deciding about the method of sturgeon sex determination that serves their needs best. We expect to have a total cost of reagents necessary to conduct this test to be within $7-10 range per DNA sample which appears very reasonable considering the fact that one female can produce caviar worth up to $2,000. Also, total time to be able to determine sex can be reduced from at least 5 years to around 2 months (at least 30 times reduction).EvaluationAchievement of specific project objectives will be monitored as follows:1. To establish dedicated PCR-based DNA detection capability at CMAST, Morehead City, NC - Successful completion of objective 1 will rely upon purchase and receipt of equipment and supplies for DNA analysis, and fish holding nets and hooks in a timely manner. This, along with hiring and training of a research associate, will allow us to move on to other objectives.2. To validate VAC-1M male-specific Russian sturgeon DNA fragment together with AllWSex2/Ag49 primer set through a direct side-by-side comparison using DNA collected during ultrasound sex determination of mature sturgeons - Objective 2 will consist of two main procedures. The first part will be to validate VAC-1M DNA fragment together with AllWSex2/Ag49 primer set using DNA collected from differently-sourced Russian sturgeon caviar grains. The second part (main idea) will be to validate primers based on the VAC-1M fragment (if found to be specific for all Russian sturgeons regardless of the origin) and AllWSex2/Ag49 primer set by comparing our DNA-based method vs. ultrasound during routine sex determination of mature Russian sturgeons. If VAC-1M-based primers set is deemed unsuitable for diagnostics with caviar, we will apply only the AllWSex2/Ag49 primer set to the larger number of samples (96 swab DNA samples). This primer set was already validated using a small sample size by us when working with mature Russian sturgeons. Similar to our preliminary results, we expect our DNA-based method of sex determination to reveal significant inaccuracies regarding use of the ultrasound method.3. To develop a rapid DNA-based method for sex determination that may be applied to juvenile Russian sturgeons using non-invasive skin swabbing for the DNA samples collection - Keys to achieving objective 3 will be to maintain/care for enough juvenile fish in the appropriate age range (up to 2 months) for DNA collection. We do not anticipate any other potential issues for objective 3 because up to 1,000 juvenile sturgeon fish can be easily tested within two weeks.EffortsWe plan to present our findings at a national aquaculture conference and publish manuscripts in high impact peer-reviewed journals. We also plan to publish articles related to the study in relevant trade magazines and digital repositories available to everyone, including the general public. Presentations and/or workshops conducted by those involved in this study will target stakeholders and interested parties. Once established and validated, this technology will be available to anyone in the sturgeon aquaculture industry. Priority will be placed on dissemination of information to other sturgeon farmers in the United States. It is important to support U.S. caviar producers who compete with imported caviar which makes up most of the caviar consumed in the U.S. A main goal of this proposal is not only to develop a DNA-based sex diagnostic test using Marshallberg Farm as a base for the test's practical validation, but also to serve as a potential hub providing either direct DNA testing service on a fee-for-service basis or proper training to anybody who is interested.