Preliminary Results on Cryopreservation of Alligator Gar ( Atractosteus spatula ) Sperm

1. Preliminary Results on Cryopreservation of Alligator Gar (Atractosteus spatula) Sperm Jaclyn Zelko & Carlos Echevarría Warm Springs National Fish Hatchery Warm Springs, GA Ricky Campbell Pvt. John Allen NFH Tupelo, MS William Wayman and Bill Bouthillier Warm Springs Fish Technology Center Warm Springs, GA

2. Acknowledgements Pvt. John Allen National Fish Hatchery Tupelo, Mississippi Photo: USFWS Photo: USFWS

3. Acknowledgements Fish Technology Center Warm Springs, Georgia Photo: Tennessee Wildlife Resources Agency Photo: USFWS Fisheries Management Division Nashville, Tennessee

4. Background – Life History • Historical range: waters within the Mississippi River basin in Tennessee Obion River Photo: USGS

5. Background – Life History • Currently listed by Tennessee as a species in-need-of-management • Spawning: April through June • Females lay large, sticky eggs Photo: USFWS Photo: USFWS

6. Enhancement & Restoration • Tennessee Wildlife Resources Agency initiated an enhancement and restoration program • Program Goal: stocking alligator gar within their historic range in the West Tennessee Mississippi River Basin to establish a sport fishery when population abundance and structure allows Photo: Tennessee Wildlife Resources Agency

7. 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

8. 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

9. Cryopreservation Program at Pvt. John Allen NFH • Alleviate the problem of obtaining ripe members of both sexes at the same time • Have more management options during spawning • Program initiated in 2005

10. 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

11. Materials & Methods • Extended sperm was mixed with cryoprotectant • Equilibrated for 4 minutes • Ten 0.5-mL straws per treatment • Frozen in shipping dewar

12. Cryopreservation • Cryopreserved sperm were stored for 48 days in liquid nitrogen • Straws were thawed by placing in a 40°C water bath for 8 seconds Photo: USFWS Photo: USFWS

13. 2005 Efforts • Collected sperm from 1 male • Initial motility 95% • Evaluated two cryoprotectants • Dimethyl sulfoxide and Methanol • Evaluated two concentrations (5%, 10%)

14. 2005 – Results **based on 1 male

15. 2006 Efforts • Collected sperm from 3 males • Initial motility 10 - 75% • Used 3 Extender (HBSS-S) concentrations at four cryoprotectant treatments • Sperm frozen from 1 male (120 straws)

16. 2006 – Results **based on 1 male

17. 2007 Efforts • Collection technique changed

18. 2007 Efforts - Cryopreservation • Collected sperm from 3 males • Initial motility 50 - 95% • Sperm frozen from 2 males (240 straws) using same treatments for 2006 • Attempted a fertilization trial

19. 2007 Efforts – Fertilization Trial • Unable to distinguish any division in 5-hour samples due to bleaching • Data extracted from 48-hr samples • Only one male per study – no replication

20. 2007 Efforts – Fertilization Trial

21. Future Research Needs Collection techniques Short-term storage Cryo effectiveness Fertilization trials Photo: USFWS