1. U.S. Fish & Wildlife Service
Preliminary Results on Cryopreservation of Alligator Gar (Atractosteus spatula) Sperm
Jaclyn Zelko
U.S. Fish & Wildlife Service
Warm Springs, Georgia
Ricky Campbell
Tupelo, Mississippi
Carlos Echevarria
Warm Springs, Georgia

2. Cryopreservation
Definition: process in which living biological material is frozen, stored for a period of time, thawed, and remains viable
Various processes = complex and highly technical

3. Cryoprotectants = Chemical
Process - Chemicals
Cryoprotectants = Chemical
Added to the sperm to allow the sperm to survive the freezing and thawing processes
1. Non-permeating = cannot enter the cells stabilize and reduce injuries to the cell membrane.
2. Permeating = enter the cells increase the internal osmolality of the cell.
Osmolality = total dissolved salts in a liquid

4. Cryoprotectants = Toxic to cells!
Process - Chemicals
Cryoprotectants = Toxic to cells!
Limit exposure time to minimize damage but allow chemicals to enter cells

5. Sperm + Cryoprotectant
Process – Freezing
Sperm + Cryoprotectant

6. Process – Freezing
Loading Straws

7. Process – Freezing
Loading Straws

8. Process – Freezing
Straws, Goblets, Canes

9. Process – Freezing
Shipping Dewars

10. Same Process at Freezing BUT in Reverse Order
Process - Thawing
Same Process at Freezing BUT in Reverse Order
Straws are removed from dewar and placed in a 40°C water bath until liquid is thawed
Motility of sperm is determined at collection, equilibration, and post-thaw to measure effectiveness of procedure
****No fish eggs have been cryopreserved****

11. Advantages of Cryopreservation
Preservation of genetic stocks
Transfer of genes from wild to hatchery
Spawning of asynchronous populations
Better control of selective breeding
Prevent in-breeding
Transport over long distances

12. Program was initiated in 2005
Program at PJANFH
Main Objective: Alleviate the problem of obtaining ripe members of both sexes at the same time, have more management options during spawning season
Program was initiated in 2005

13. Repository - Study Objectives
Evaluate acute toxicity of two cryoprotectants to determine the maximum equilibration time
Evaluate various cryoprotectants, cryoprotectant concentrations and freezing rates
Evaluate fertilization rates of cryopreserved sperm to determine effectiveness of freezing procedure

14. 2005 Efforts Sperm were collected from only available male
Initial motility was 95%
Extended sperm with a modified Hanks’ balanced salt solution
Photo: USFWS

15. 2005 Efforts Evaluated two cryoprotectants
Dimethyl sulfoxide and Methanol
Evaluated two concentrations (5%, 10%)
Photo: USFWS
Photo: USFWS

16. 2005 Efforts Extended sperm was mixed with cryoprotectant
Equilibrated for 4 minutes
Ten 0.5-mL straws per treatment
Frozen in shipping dewar (40 straws)

17. 2005 Efforts
Cryopreserved sperm were stored for 48 days in liquid nitrogen
Straws were thawed in a 40°C water bath for 8 seconds
Photo: USFWS
Photo: USFWS

18. Results

19. 2006 Efforts Sperm were collected from three males
Initial motilities %
Sperm frozen from 1 male (120 straws)
Used 3 concentrations of Extender
Four cryoprotectant treatments

20. Results

21. Future Efforts Collection techniques Short-term storage
Cryo effectiveness
Fertilization trials
Photo:

22. Photo: D. Franklin, Courtesy Dept
Photo: D. Franklin, Courtesy Dept. of Library Services, American Museum of Natural History