APPLICATION OF GERMPLASM PRESERVATION ON BREEDING PROGRAMS FOR MOLLUSCAN SHELLFISH AQUACULTURE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1005105
• Project Start Date: Nov 1, 2014
• Project End Date: Oct 31, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
Forest Resources and Conservation

Non Technical Summary
Cryopreservation is a technique to preserve genetic materials in perpetuity, and germplasm cryopreservation includes preservation of gametes (sperm and eggs or oocytes), embryos, or larvae. This technology has been used for human artificial reproduction as a widely used clinical treatment for infertility, and for livestock this technology has been used as powerful tools for breeding programs and developed into a huge industry worldwide. Generally, germplasm cryopreservation is useful for preservation of natural resources to maintain the biodiversity and conserve endangered species. Formolluscan shellfish, cryopreservation is a useful tool for preservation of the superior traits selected or created from breeding practices,and also can be used as an integrity part in genetic approaches for creation of inbred or mutant lines such as self-fertilization in oysters andassistaquaculture hatchery practice for seed production.The preserved germplasm can function as a reservoir to meet the need of gametes for hybridization and regular fertilization practice, and can save the cost for space and maintenance of certain broodstock.

Animal Health Component

10%

Research Effort Categories

Basic

50%

Applied

10%

Developmental

40%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
303	0811	1080	100%

Knowledge Area
303 - Genetic Improvement of Animals;

Subject Of Investigation
0811 - Shellfish;

Field Of Science
1080 - Genetics;

Keywords

molluscan shellfish

germplasm

cryopreservation

Goals / Objectives
The goal of this project is to apply germplasm preservation technology on breeding programs for molluscan shellfish aquaculture. Targeted species will include the hard clam Mercenaria mercenaria and eastern oyster Crassostrea virginica. This is a new project for the program of Molluscan Shellfish Aquaculture Production and Restoration. The objectives are to:1. Develop techniques for germplasm cryopreservation of the hard clam by using current aquaculture stocks.2. Apply sperm cryopreservation technology in the eastern oyster for production of self-fertilized inbred lines.3. Establish quality control assays for fresh and post-thaw sperm by microscopic evaluation for motility and use of flow cytometry (FCM) for measuring plasma membrane, mitochondrial, and acrosome integrity.

Project Methods
METHODSObjective 1 - Develop techniques for germplasm cryopreservation of the hard clam Hard clam broodstock from Cedar Key aquaculture hatcheries will be used for this research. For this objective, sperm, oocytes, embryos, and larvae will be cryopreserved.Gamete collection:Gametes will be collected from thermally-induced spawns, a common practice in clam aquaculture. Males and females will be spawned separately: sperm will be collected as dry as possible to maintain high concentration by removing males (when spawning starts) to a small container for dry sperm release; and, oocytes will be collected by filtering the oocyte suspension (within 30 min after spawning starts) through a 15-um screen, and re-suspending in fresh seawater. Embryos and larvae will be collected at different stages after mixing oocytes and sperm at a proper ratio (to be decided by experiment trials). Clams used for gamete collection will be weighed and measured, and a pieces of gill and adduct muscle will be flash frozen by directly plunge in liquid nitrogen for possible future DNA analysis.1. Container types:French straws (0.5 ml) will be used for all of the experiments to facilitate the sample biosecurity, high-throughput processing, freezing and thawing, and efficiency of storage space.2. Freezing processThis is a new laboratory. No large equipment such as a programmable freezer is available at this time. A cryokit made from Styrofoam boxes will be used for the freezing process. The cooling rates will be pre-determined by measuring the temperature with a digital thermocouple, and cooling rates will be controlled by the size of floating boat (to be used for holding the samples to be frozen) and the liquid nitrogen amount.3. Development of protocolsBased on the pathway for development of cryopreservation protocols, we will perform the following series of experiments for systematic evaluation of each step: (1) evaluation of fertilization capacity of gametes with time, temperature, sperm-egg ratio; (2) toxicity evaluation by exposure gametes, embryos, or larvae to commonly used cryoprotectants for bivalves at concentrations 5, 10, 15 and 20%; (3) equilibration time evaluation with the cryoprotectants selected for up to 1 hour; (4) evaluation of cooling rates ranging from -0.5°C to -40°C/min; (5) evaluation of sample volume and concentration; (6) evaluation of thawing temperature, and (7) evaluation of the post-thaw survival. For these experiments, fertilization, D-larvae hatching level, or spat harvest level (after metamorphosis) will be used as parameters to estimate the survival based on the materials to be cryopreserved.Objective 2 - Apply sperm cryopreservation technology in the eastern oyster for production of self-fertilized inbred lines. Non-lethal sperm collection and cryopreservation protocols have been established. Further, the feasibility of the approach for production of self-fertilized offspring by using cryopreserved sperm has been proven with the harvest of confirmed self-fertilization larvae. Therefore, for this study, the goal will be to produce confirmed spats (beyond metamorphosis), and culture to spat stage.Procedures will be:Sperm samples will be collected individually from 500 males by the non-lethal method (biopsy or natural spawning) and cryopreserved by using established protocols. Oysters will be collected from the cultured broodstock in the Gulf region or certain populations from the current selective breeding programs such as the disease-resistant one from Louisiana State University.After gonad biopsy, oysters will be cultured, and survival will be monitored.In the spawning season of the years after sampling, sex of each oyster will be examined, and eggs from sex-reversed females will be collected following standard hatchery procedures and fertilized with cryopreserved sperm from this same individual.Fertilization will be determined by counting the developing embryos divided by the total number of eggs used for fertilization. Fertilized eggs will be cultured for larval (spat) harvest by following regular hatchery practice, and offspring will be analyzed with microsatellite markers selected for pedigree confirmation (Yang, et al., 2014).Objective 3 - Establish quality control assays for fresh and post-thaw sperm by microscopic evaluation for motility and use of flow cytometry (FCM) for measuring plasma membrane, mitochondrial, and acrosome integrity.For each sample, quality monitoring will be processed for fresh sperm (before cryopreservation) and post-thaw sperm. The procedures will be:1) Motility estimationMotility is the most direct index for sperm quality. For each sample, motility will be estimation by using a microscope with a magnification of x 100. Sperm motility activation will be performed by using sea water at the same osmolality as that in the culture system or environment. Sperm from hard clam and eastern oyster can be motile for several hours, therefore short videos made with a high-speed camera will be recorded for analyses of velocity, amplitude of lateral head displacement (ALH, μm), and beat cross frequency (BCF, Hz) using free software ImageJ or commercially available software.2) Flow cytometry analysisFor plasma membrane integrity, established protocols will be followed (Paniagua-Chavez, et al., 2006; Daly and Tiersch, 2012). Briefly, sperm samples at a contraction of 1 x 106 cells/ml will be stained with a commercial live/dead sperm kit (Invitrogen, L7011, Eugene, Oregon) for 10 min with 100nM SYBR green14 and 12µM propidium iodide (PI). SYBR 14 is a membrane-permeate nucleonic acid stain and the PI is a conventional dead cell stain, both dye labels DNA. Fluorescent of SYBR 14-stained cells was detected after a 530 ± 15 nm band-pass filter and that of PI-stained cells was detected after a > 670 nm long-pass filter. Sperm stained with SYBR 14 alone were separated with the cells stained with PI, and were named as "membrane-intact cells". For this analysis, the sperm membrane integrity is calculated as the percentage of membrane-intact cells out of the total cells stained with PI and SYBR14.For mitochondria analysis, MitoTracker™green FM (30-200 mM) will be used for staining the sperm cells at a concentration of 1 x 106 cells/ml for 30 min to evaluate the mitochondria membrane potential and other MitoTracker stains (MitoTracker Orange CM-H2TMRos and MitoTracker Red CM-H2XRos) will be used to stain sperm samples to monitor the oxidation status of cells (Cottet-Rousselle, et al., 2011). Protocols will be developed by evaluating sperm concentration, volume together with stain concentration and time. The protocols will be used for monitoring the sperm quality during the process of freezing.Analysis of acrosome will be another useful parameter for sperm quality evaluation because mollusk sperm possess acrosome (Gosling, 2003). Lectin, a specific acrosome binding, will be used for protocol development (Martinez-Pastor, et al., 2010) together with conjugated fluorescein isothiocyanate (FITC) and combined with the viability dyes PI (Uhler, et al., 1993). The protocols will be used for monitoring sperm quality during the process of freezing.Data collection and statistics Data will be analyzed by use of statistical software (SYSTAT version 13, Systat Software, Inc., San Jose, California). Normality of data will be tested before performing analyses, and percentage data will be Arcsin transformed. For most analyses it is anticipated that t-test, Chi-square, ANOVA, and correlation analyses will be used for data analysis.

Progress 11/01/14 to 10/31/19

Outputs
Target Audience:Target audiences are members of the scientific community, aquaculture industry farmers, country extension agents, and administration agencies involvingwith aquaculture and fishery management, natural resourcebiodiversity, and genetic resourceconservation. Changes/Problems:No significant changes will be made. What opportunities for training and professional development has the project provided?One OPT biologist, one undergraduate student, and one summer intern were trained in this program with this reporting period. Training activities include scientific research activities, such as procedure demonstration and laboratory techniques, and extension activities, such as on-site visit and extension publications. Professional development activities includes supervisory special course, online-course, and participation of academic conferences. How have the results been disseminated to communities of interest?Dissemination of research updates were made through oral or poster presentation in the conferences, extension workshops, onsite demonstration, field trip visit, extension publications, graduate student thesis, and peer-reviewed journal publications. What do you plan to do during the next reporting period to accomplish the goals?This is the final report of this HATCH project.

Impacts
What was accomplished under these goals? Improvement of trochophore larval cryopreservation in hard clams has been conducted, and a preliminary protocol was generated through evaluating cryoprotectant acute toxicity, cooling rate, and thawing rate. With this protocol, the post-thaw trochophore larvae survival was 23 ± 14%, and the post-thaw larvae developed to D-stage was 27 ± 14%. The result was publication in one peer-review journal. This is the first report on larval cryopreservation in the hard clam and would have application for genetics breeding and seed production. Sperm cryopreservation initiated in hard clams was continued with focus on solving the sperm agglutination. The effects of different cryomedium were designed for this purpose. In addition, an onsite cooling approach using shipping dewar was developed to compare the effects of spawning time on sperm agglutination. Effects of sugar addition on oyster sperm cryopreservation was studied to improve the oyster sperm cryopreservation protocol, and will be applied on an oyster genetic breeding program.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhang X, Shi J, Sun Y, Habib Y J, Yang H, Zhang Z and Wang Y, 2019. Integrative transcriptome analysis and discovery of genes involving in immune response of hypoxia/thermal challenges in the small abalone Haliotis diversicolor. Fish & Shellfish Immunology. 84, 609-626.
• Type: Websites Status: Published Year Published: 2019 Citation: Yang H, Guo X and Scarpa J, 2019. Oyster Tetraploid Induction and Establishment of Breeding Stocks for All-Triploid Seed Production. https://edis.ifas.ufl.edu/fa215. (peer reviewed).
• Type: Websites Status: Published Year Published: 2018 Citation: Yang H, Simon N(g) and Sturmer L, 2018. Production and Performance of Triploid Oysters for Aquaculture. http://edis.ifas.ufl.edu/fa208. (peer reviewed).

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Target audiences are members of the scientific community, aquaculture industry farmers, country extension agents, and administration agencies involvingwith aquaculture and fishery management, natural resourcebiodiversity, and genetic resourceconservation.? Changes/Problems:No significant changes will be made. ? What opportunities for training and professional development has the project provided?One PhD graduate students were trained in this program within this reporting period. Additionally, undergraduate students were trained in this program with one as summer intern in 2018 and another as student worker in 2018. Training activities include scientific research activities, such as procedure demonstration and laboratory techniques, and extension activities, such as on-site visit and extension publications. Professional development activities includes supervisory special course, online-course, and participation of academic conferences. How have the results been disseminated to communities of interest?Dissemination of research updates were made through oral or poster presentation in the conferences, extension workshops, onsite demonstration, field trip visit, extension publications, graduate student thesis, and peer-reviewed journal publications. What do you plan to do during the next reporting period to accomplish the goals?In next reporting period, the goal to this project will be to: 1) continue to improve the larval cryopreservation technique to increase the post-thaw survival, and 2) apple the larval cryopreservation technique on potential commercial use.

Impacts
What was accomplished under these goals? 1) Improvement of trochophore larval cryopreservation in hard clams has been conducted, and a preliminary protocol was generated through evaluating cryoprotectant acute toxicity, cooling rate, and thawing rate. With this protocol, the post-thaw trochophore larvae survival was 23 ± 14%, and the post-thaw larvae developed to D-stage was 27 ± 14%. The result was publication in one peer-review journal. This is the first report on larval cryopreservation in the hard clam and would have application for genetics breeding and seed production. 2) Sperm cryopreservation initiated in hard clams was continued with focus on solving the post-thaw sperm agglutination. Experiments to compare the effect of sperm at different time after spawning on cryopreservation were designed. An onsite cooling approach using shipping dewar was quantified for the cooling rate readings. Further investigation is ongoing. 3) Effects of sugar addition on oyster sperm cryopreservation was studied and the manuscript for the results was is in process of preparation. This study will be useful in oyster aquaculture for preserving valuable germplasm and hybrid breeding. ? 4) With the research updates for sperm and trochophore larval cryopreservation, one proposal was developed and submitted to the USDA AFRI foundational program to seek financial support to continue the research in hard clam germplasm cryopreservation for improvement of shellfish aquaculture.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Simon, N.A., Yang, H., 2018. Cryopreservation of trochophore larvae from the hard clam Mercenaria mercenaria: Evaluation of the cryoprotectant toxicity, cooling rate and thawing temperature. Aquaculture Research. 49, 2869-2880.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Yang, H., Hu, E., Buchanan, J.T., Tiersch, T.R., 2018. A strategy for sperm cryopreservation of Atlantic Salmon, Salmo salar, for remote commercial-scale high-throughput processing. Journal of the World Aquaculture Society. 49, 96-112.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Matthews, J.L., Murphy, J.M., Carmichael, C., Yang, H., Tiersch, T., Westerfield, M., Varga, Z.M., 2018. Changes to Extender, Cryoprotective Medium, and In Vitro Fertilization Improve Zebrafish Sperm Cryopreservation. Zebrafish. 15, 279-290.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Simon, N., Yang, H., 2018. Effects of Sugar Additions on Trochophore Larvae Cryopreservation of the Hard Clam Mercenaria mercenaria, The 38th Annual Meeting of the Florida Chapter American Fisheries Society Haines City, FLorida. April 11-13, 2018.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Simon, N., Yang, H., 2018. Effects of Cooling Rates on Trochophore Larvae Cryopreservation of the Hard Clam Mercenaria mercenaria, 148th Annual Meeting of the American Fisheries Society, Atlantic City, New Jersey from August 19-23, 2018.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Target audiences are members of the scientific community, aquaculture industry farmers, and administration agencies involvingwith aquaculture and fishery management, natural resourcebiodiversity, and genetic resourceconservation. Changes/Problems:No significant changes will be made. What opportunities for training and professional development has the project provided?Two Master graduate students were trained in this program and they graduated and obtained Master degree in August 2017. Additionally, one undergraduate student was trained in this program as summer intern in 2017. Training activities include scientific research activities, such as procedure demonstration, laboratory techniques, experimental designation, and data collection, and extension activities, such as on-site visit of industry farms, extension publications, and knowledge dissemination about shellfish seed production and triploid-tetraploid technology. Professional development activities includes supervisory special course, online-course, and participation of academic conferences. How have the results been disseminated to communities of interest?Dissemination of research updates were made through oral or poster presentation in academic conferences, extension workshops, onsite demonstration, field trip visit, extension publications, graduate student thesis, and peer-reviewed journal publications. What do you plan to do during the next reporting period to accomplish the goals?In next reporting period, I am planning to work on: 1) improving the techniques for larval cryopreservation to increase the survival, 2) development of the potential application of the larval cryopreservation technique for commercialization use, and 3) extending the larval cryopreservation technique to oyster triploid and tetraploid technology.

Impacts
What was accomplished under these goals? Sperm cryopreservation was initiated in hard clams. Experiments for sperm collection and transportation in hard clams indicated that sperm collected through natural spawning can reach a concentration of over 4 x 10^8 cell/ml for cryopreservation. If not, centrifugation at 2000 rpm for 5 min could allow sperm cells concentrated without affecting quality. Evaluation of cryoprotectant toxicity showed that dimethyl sulfoxide (DMSO), propylene glycol (PG) (5 and 10%) showed the least toxicity on fresh sperm within 15 min exposure time. However, after cryopreservation, post-thaw sperm showed a unique phenomenon - agglutination, this affected the post-thaw motility. Further investigation is ongoing. Trochophore larval cryopreservation in hard clams has been conducted, and a preliminary protocol was generated through evaluating cryoprotectant acute toxicity, cooling rate and thawing rate. DMSO and propylene glycol at 5% and 10% showed the least toxicity to trochophore larvae regardless of exposure time. With DMSO or propylene glycol (5% and 10%) as cryoprotectants, cooling rates did not show significant effects on post-thaw viability. Thawing temperature had varied effects without apparent trends. With the basic protocol, post-thaw trochophore larvae survived (23 ± 14%), and developed to D-stage (27 ± 14%). This is the first report on larval cryopreservation in the hard clam, and would have application for genetics breeding and seed production. One manuscript was submitted for publication. Effects of sugar addition on oyster sperm cryopreservation was studied. Different from the results in most previous publications, no improvement on post-thaw viability was identified with sugar addition (glucose, sucrose, fructose, or trehalose at 0.2 M or 0.5 M). The evaluation included the effects of sugar addition on fresh sperm motility, cooling rate, and thawing rate. One manuscript is in process of preparation. This study will be useful in oyster aquaculture for preserving valuable germplasm and hybrid breeding. Improvement of trochophore larval cryopreservation in hard clams was ongoing by addition of sugars. Protocols for sperm quality analysis and ploidy determination by using flow cytometer were established. A neutral red staining method was developed for analysis of live/dead of trochophore larvae in hard clams. The optimal staining concentration and time were determined as 50 mg/L for 1 hour based on the comparison of counted staining results and known live/dead percentage.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: H. Yang. 2017. Application of germplasm preservation in breeding programs for molluscan shellfish aquaculture and restoration. Bulletin of Japan Fisheries Research and Education Agency. 45, 15-20.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: E. Heenkenda, 2017. Genetic and Phenotypic Identification of Mercenaria mercenaria, Mercenaria campechiensis and Their Hybrids. University of Florida, Gainesville, FL, pp. 71.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: N. Simon, 2017. Cryopreservation of Trochophore Larvae in the Hard Clam. University of Florida, Gainesville, FL, pp. 98.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Target audiences are the scientific community, aquaculture industry farmers, and administration agencies involvingwith aquaculture and fishery management, natural resourcebiodiversity, and genetic resourceconservation. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students and one undergraduate student were trained in this program for scientific research activities, laboratory techniques, and basic shellfish biology and aquaculture. Participation of academic conferences were made for presenting the research updates. Extension activities including on-site visit and project collaboration were made for knowledge dissemination about shellfish aquaculture and application of all-triploid oyster production. How have the results been disseminated to communities of interest?Dissemination of research updates were made through oral or poster presentation in academic conferences, extension workshops, onsite demonstration, field trip visit, extension publications, and peer-reviewed journal publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Wild hard clam populations were collected from different geographical locations along the U.S. east coast for development of molecular tools to distinguish the southern and norther species. Experiments for sperm collection and transportation in hard clams indicated that sperm collected through natural spawning can reach a concentration of over 2 x 108 cell/ml for cryopreservation. If not, centrifugation at 2000 rpm for 5 min could allow sperm cells concentrated without affecting quality. Effects of sugar addition on sperm cryopreservation was studied in eastern oysters, the results showed no improvement on post-thaw viability with addition of glucose, sucrose, fructose, or trehalose at 0.2 M or 0.5 M. Larval cryopreservation in hard clams indicated that dimethyl sulfoxide (DMSO) and propylene glycol had less toxicity at concentrations of 5 and 10% (v/v) for fresh larvae. Analysis of sperm quality, ploidy determination, and other cellular characterizations were established by using flow cytometry.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Hu, E., R. Cuevas-Uribe, H. Yang, R. Sanderson, A. O. Gill, H. Daniels & T. R. Tiersch. 2016. High-throughput Cryopreservation of Sperm from Sex-reversed Southern Flounder, Paralichthys lethostigma. Journal of the World Aquaculture Society 47:555-565.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Yang, H., J. Daly & T. R. Tiersch. 2016. Determination of Sperm Concentration Using Flow Cytometry with Simultaneous Analysis of Sperm Plasma Membrane Integrity in Zebrafish Danio rerio. Cytometry Part A 89a:350-356.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Yang, H., J. Daly, C. Carmichael, J. Matthews, Z. M. Varga & T. Tiersch. 2016. A Procedure-Spanning Analysis of Plasma Membrane Integrity for Assessment of Cell Viability in Sperm Cryopreservation of Zebrafish Danio rerio. Zebrafish 13:144-151.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Heenkenda, E. & H. Yang. 2016. Development of Effective Method for Recognition of Mercenaria mercenaria, Mercenaria campechiensis and Their Hybrids for Clam Breeding and Aquaculture In: The Florida Chapter of the American Fisheries Society 2016 Symposium. Haines City, FL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Yang, H. 2016. Germplasm Preservation for Molluscan Shellfish Aquaculture and Restoration. In: The 53rd Annual Meeting of the Society for Cryobiology. Ottawa, Canada.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Simon, N. & H. Yang. 2016. Germplasm cryopreservation techniques in the eastern oyster Crassostrea virginica. In: The Florida Chapter of the American Fisheries Society 2016 Symposium. Haines City, FL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Liu, Y., L. Torres, H. Yang & T. R. Tiersch. 2016. Addressing quality control for development of sperm repositories of problematic fish species. In: Triennial American Aquaculture Las Vegas, Nevada.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Yang, H., E. Hu, L. Torres, S. K. Allen & T. R. Tiersch. 2016. Cryopreservation of sperm from tetraploid eastern oyster Crassostrea virginica. In: Triennial American Aquaculture Las Vegas, Nevada.

Progress 11/01/14 to 09/30/15

Outputs
Target Audience:Target audiences are members of the scientific community, aquaculture industry farmers, and administration agencies involving with aquaculture and fishery management, natural resourcebiodiversity, and genetic resourceconservation. Changes/Problems:No changes were made. What opportunities for training and professional development has the project provided?Two graduate students and one undergraduate student were trained in this program for scientific research activities, laboratory techniques and basic aquaculture methods. Demonstration of oyster dissection spawning and artificial fertilization was provided for industry growers for all-triploid production through onsite visit. Participation of academic conferences and technical workshops (oyster reef restoration and aquaculture water quality) were made for presenting the research updates of this program. How have the results been disseminated to communities of interest?The dissemination of research results and updates were made through oral or poster presentation in academic conferences, extension workshops, onsite demonstration, field trip visit, extension publications, and peer-reviewed journal publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1) Experiments for sperm collection, transportation, and quality analysis were initiated for the hard clam, and will be continued in next spawning season. 2) By using the protocol for sperm cryopreservation developed in diploid eastern oysters sperm from tetraploid oysters were cryopreserved and preliminary data were collected. This research will be continued as long as tetraploid oysters are available. In addition, wild oyster populations in Florida were collected for continue research to improve the protocols for oyster germplasm cryopreservation and production of self-fertilization inbred lines. 3) One portable flow cytometry was equipped in the laboratory to support the research for analysis of gamete quality, ploidy determination and other cellular characterization such as calcium ion transportation for sperm motility activation.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Yang H, Wang Y, Guo X, Tiersch TR (2015) Production of inbred larvae through self-fertilization using oocytes and cryopreserved sperm from the same individuals after sex reversal in eastern oyster Crassostrea virginica. Aquaculture Research, 46, 2153-2165.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Yang H, Daly J, Tiersch TR (2016) Determination of sperm concentration using flow cytometry with simultaneous analysis of sperm plasma membrane integrity in zebrafish Danio rerio. Cytometry Part A,
• Type: Websites Status: Awaiting Publication Year Published: 2015 Citation: Yang H, Sturmer, LN, Baker, S (2015) Molluscan Shellfish Aquaculture and Production. http://edis.ifas.ufl.edu/
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Yang H, Sturmer LN (2015) Potential Application of Germplasm Preservation for Breeding Programs of Molluscan Shellfish Aquaculture. In: National Shellfisheries Association 107th Annual Meeting, Monterey Bay, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Yang H (2015) Potential Application of Germplasm Preservation in Breeding Programs for Molluscan Shellfish Aquaculture and Restoration. In: The 43rd United States - Japan Natural Resources Scientific Symposium: Evaluation of the Impact of Bredding Prganisms on the Ecosystem and Aquacutlrue Industry, Nagasaki University, Nagasaki, Japan.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Liu Y, Yang H, Tiersch TR (2015) Ionic Activiation of Sperm Motiltiy in an Endangered Viviparous Fish. In: American Aquaculture, New Orleans, LA
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Matthews J, Carmichael C, Yang H, A G, Torres L, Murphy J, Tiersch T, Westerfield M, Varga ZM (2015) Optimized Cryopreservation and Thawing Methods for Community and Resource Center Use. In: The 9th European Zebrafish Meeting, Oslo, Norway.