Cryogenic Preservation of Oyster Gametes to Improve Hawaii and West Coast Oyster Stocks

Final report for FW16-020

Project Type: Farmer/Rancher
Funds awarded in 2016: $25,000.00
Projected End Date: 03/31/2018
Grant Recipient: Goosepoint Oyster Co.
Region: Western
State: Washington
Principal Investigator:
David Nisbet
Goosepoint Oyster Co.
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Project Information

Abstract:

Cryogenics is a useful method for animal breeding and production. Relatively little work has been done with oysters as compared to mammalian livestock. Goosepoint Oyster Co. and its subsidiary, Hawaiian Shellfish LLC identified the ability to cryopreserve oyster gametes as a fundamental technique for advancing their selective breeding program. Not only is this important for the companies directly involved, Hawaiian Shellfish LLC provides oyster seed to over 20 companies who can also benefit from improved oyster stock. This work focused on developing capacity among hatchery operators to cryopreserve oyster gametes using new methods developed by Dr. Terrence Tiersch.

All startup and organizational activities have been completed, including the visit of Dr. Terrence Tiersch (Louisiana State University Agricultural Center) to provide training in the process of cryogenic preservation to the staff of the Pacific Aquaculture and Coastal Resources Center (PACRC) at the University of Hawaii Hilo and Hawaiian Shellfish, LLC.

Dr. Tiersch made his first visit in early July 1016 where he visited the Pacific Aquaculture and Coastal Resources Center (PACRC) at the University of Hawaii Hilo and Hawaiian Shellfish, and gave lectures on the cryogenic preservation process for mammals as well as oysters. Approximately 15 staff and students attended the lectures. He also advised on the materials and equipment that would be needed for the training visit by Bill Childress 9/30/2016. With these materials purchased, Hawaiian Shellfish and PACRC created the capacity for cryopreservation work to begin in Hawaii.

 

Bill Childress visited Hawaiian Shellfish LLC and trained PACRC and Hawaiian Shellfish staff on the use of his “Cryogenic Cooler Kit” for freezing and thawing oyster sperm. An innovative and important feature of the kit is the use of 3-D printing to enable anyone to make a rack that precisely controls cooling rates which eliminates the need for expensive instrumentation normally used for freezing sperm. Liquid nitrogen was added to the Styrofoam cooler, and his “Straw Rack” held twenty straws of liquid sperm that were extracted from male Pacific Oysters. Staff were also trained on safety measures and the mixing of cryopreservants to be added to the oyster sperm.

Project Objectives:

1. Develop reliable and replicable sperm collection, handling, and shipping methods at the three oyster farms in Washington, and the two Hawaii hatcheries. Dr. Tiersch will receive the material at the LSUAC Aquatic Germplasm and Genetic Resources Center, freeze the sperm, and use it in fertilization tests.

2. Test cryopreserved sperm in fertilization trials to assess post-thaw viability and enable estimation of the quantities of material that will be needed for production-scale use.

3. Evaluate the materials, equipment, and training needs for the hatcheries to be able to collect, freeze, and store sperm on-site in remote locations. This will take into account problems such as electrical failures, natural disasters and personnel turn over that affect remotely located farm facilities. A plan for future on-site capacity will be developed.

 

4. Contribute specimens of wild and selected oyster lines to the USDA National Animal Germplasm Program (http://www.ars.usda.gov/Research/docs.htm?docid=22314) as back-up reserves of germplasm.

 

5. Conduct outreach and training for hatchery and farm personnel, students and the wider aquaculture stakeholders.

 

Cooperators

Click linked name(s) to expand
  • Dr. Terrence Tiersch - Technical Advisor (Educator and Researcher)
  • Dr. Maria Haws - Technical Advisor (Educator and Researcher)
  • William Childress - Technical Advisor (Researcher)
  • Provan Crump (Researcher)
  • Brian Koval (Researcher)

Research

Materials and methods:

Material List for Cryopreservation of Oyster Gametes:

  • Taylor Wharton HC35 dewar (2 are needed) 
  • CX100 shipping dewar
  • Dewar roller base 
  • Shipping dewar case
  • Liquid Nitrogen transfer hose
  • Liquid Nitrogen protection gloves
  • Face shield
  • Disposable 3mL syringes 
  • 120MM Daisy Goblets
  • "Cryo Cooler"
  • Straw rack
  • Storage straws
  • Straw sealer
  • Centrifuge tubes
  • Methanol
  • Dimethyl sulfoxide (DMSO)
  • Scalpels
  • 1-10µL Pipette
  • 20-200µL Pipette
  • 100-1000µL Pipette
  • Serological pipet 
  • 1-10µL Pipette Tips
  • 20-200µL Pipette Tips 
  • 100-1000µL Pipette Tips
  • 1mL Serological Pipette
  • 5mL Serological Pipette
  • 10mL Serological Pipette
  • 50mL Centrifuge Tubes
  • 15mL Centrifuge Tubes
  • 1.5 Microcentrifuge Tubes
  • 1mL Syringe 
  • 3mL Syringe 
  • Kimwipe
  • 30cm general purpose forceps
  • 1-L Nalgene bottles
Research results and discussion:

Impacts

If successful, Cryogenic preservation can be crucial to preserving genetic stocks of West Coast Oysters. If disease or climate change were to suddenly impact areas where oysters are living, complete lines could be lost forever. If this were to happen however, the surviving oysters which may be resistant to these effects could be preserved and used in other effected areas to create resistant lines to keep the industry going. Cryogenics can also be used in selective breeding efforts. In the case of mammalian livestock, top performing sires and dams can be kept for long periods of time to repeatedly breed numerous cohorts of improved progeny.  In the case of oysters, the scenario is complicated by the fact that oysters can change from male to female, hence a desirable sire may be lost to a breeding program. Cryopreserved sperm allows for repeated use of a male even after it may have changed into a female.  Oysters also require “conditioning” in order to develop gametes and this is a time consuming and expensive endeavor, which can be made simpler if preserved sperm is available.

 

Accomplishments

 

We have succeeded in accomplishing Project Objective 1: Develop reliable and replicable sperm collection, handling, and shipping methods. Broodstock oysters were sent from Hawaiian Shellfish on the 5/22/2016 and the 9/14/2016 to LSU Aquatic Germplasm and Genetic Resource Center for cryopreservation experiments and the collection of genetic material. Additional shipments have been made in 2017 where sperm samples were collected and successfully frozen.

We have made progress on Project Objective #2: Test cryopreserved sperm at the hatcheries in fertilization trials to assess post-thaw viability and enable estimation of the quantities of material that will be needed for production-scale use. From our initial trials at LSU, the sperm from the male oysters were 50%-80% motile before cryopreservation with 5-25% motility after freezing and thawing. Although we have not had success fertilizing eggs with cryopreserved sperm, from our motility results, it would be safe to estimate that you would need to use at least between two and ten times more sperm to fertilize eggs than normal non-preserved sperm. Very little sperm is needed from a male oyster to fertilize eggs, so this increased amount is well within acceptable levels.

We have completed Project Objective 3: Evaluate the materials, equipment, and training needs for the hatcheries to collect, freeze, and store sperm on-site in remote locations. Materials list for our research is located above in Materials and Methods.

We have completed Objective #4, by contributing 15 specimens to the USDA National Animal Germplasm Repository during the week of October 23rd, 2017. These oysters were from selected lines from offspring of the Molluscan Broodstock Program at Oregon State University. This helps increase the diversity and number of oyster specimens at the Repository as this species was severely under-represented.

We have nearly completed Project Objective 5: Conduct outreach and training for hatchery and farm personnel, students and the wider aquaculture stakeholders. A draft of cryogenic freezing and thawing methods has been written and will be further refined based on future results of the cryogenic trials.  Photos and research data was collected during Dr. Tiersch and Bill Childress’ visits to be used for training purposes. Our final report and educational materials will be made available for students and stakeholders.

Participation Summary
15 Farmers participating in research

Educational & Outreach Activities

2 Consultations
2 On-farm demonstrations
2 Tours
3 Webinars / talks / presentations
5 Workshop field days

Participation Summary:

15 Farmers
8 Ag professionals participated
Education/outreach description:

Dr. Tiersch made his first visit in early July 1016 where he visited the Pacific Aquaculture and Coastal Resources Center (PACRC) at the University of Hawaii Hilo and Hawaiian Shellfish, and gave lectures on the cryogenic preservation process for mammals as well as oysters. Approximately 15 staff and students attended the lectures. He also advised on the materials and equipment that would be needed for the training visit by Bill Childress 9/30/2016. With these materials purchased, Hawaiian Shellfish and PACRC created the capacity for cryopreservation work to begin in Hawaii.

During Bill Childress' visit in September/October of 2016, approximately 10 students and staff received training on the use of his “Cryogenic Cooler Kit” for freezing and thawing oyster sperm. Video and pictures were taken during important parts of the process to be used to create training manuals and educational materials.

Learning Outcomes

20 Farmers reported changes in knowledge, attitudes, skills and/or awareness as a result of their participation

Project Outcomes

5 Farmers changed or adopted a practice
5 Farmers intend/plan to change their practice(s)
10 New working collaborations
Project outcomes:

This project has laid the foundation for a new field in the cryogenic preservation of oyster genetic materials. Much of the oyster genetic diversity of the West Coast could be lost without this technology if a disease outbreak or other natural disaster were to occur. If a population was lost in the wild, but contained useful traits to farmers, it could be used to create crosses between disease resistant oyster families. There has been similar work done in Tasmania after the Oyster Herpes Virus outbreak where the few surviving oysters were used to create new families as the parents had some resistance to the disease. Having stored genetic materials would ensure the industry could recover more quickly and less economic damage would be done. It also makes more sophisticated breeding strategies possible, e.g. back-crossing.

Success stories:

Although there is much work to be done in refining our protocols, we did have success freezing and thawing the preserved oyster sperm, which is very encouraging. 

On October 5th, 2016 we preserved samples of oyster sperm with an initial motility of 45%.

After being thawed at 40 C for 8 seconds we had the following results. Motility =28% post-thaw; second count: 5-10% motility;  third count: 4.7%  (counts are from different straws)

This shows that the genetic material was intact, the sperm was active again and could be used to fertilize eggs. We did not have success getting eggs to develop at this time.

On September 29th, 2017 William Childress performed similar tests with 3 males with initial motility ranging from 50 to 80%. Post thaw ranged from 5-25%. Straws were taken and sent to the USDA National Animal Germplasm Program where they were received and are now stored at the facility.

 

Recommendations:

Further study would be beneficial for the following areas: Freezing and thawing speeds, chemical concentrations for cryopreservant as well as sperm concentration, and more fertilization trials withpreserved material. The variation in the latter may have a seasonal component related to egg quality that should be explored.

During the training, Bill Childress noted that there were differences in success depending on sperm concentration, chemical concentration, and thawing rates.  From our 10/5/2016 experiments we noticed that sperm in higher concentrations survived better than sperm in low concentrations. That could be from an overabundance of cryopreservant chemicals, but more research and replication is needed.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.