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

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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
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Acknowledgements

• Pvt. John Allen National Fish Hatchery
• Tupelo, Mississippi

Photo USFWS
Photo USFWS
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Acknowledgements
Photo Tennessee Wildlife Resources Agency
Photo USFWS

• Fish Technology Center
• Warm Springs, Georgia

• Fisheries Management Division
• Nashville, Tennessee

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Background Life History

• Historical range waters within the Mississippi
  River basin in Tennessee

Obion River
Photo USGS
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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
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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
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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

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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

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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

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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

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Materials Methods

• Extended sperm was mixed with cryoprotectant

• Equilibrated for 4 minutes

• Ten 0.5-mL straws per treatment

• Frozen in shipping dewar

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Cryopreservation

• Cryopreserved sperm were stored for 48 days in
  liquid nitrogen
• Straws were thawed by placing in a 40C water
  bath for 8 seconds

Photo USFWS
Photo USFWS
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2005 Efforts

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

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2005 Results based on 1 male
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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)

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2006 Results based on 1 male
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2007 Efforts

• Collection technique changed

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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

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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

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2007 Efforts Fertilization Trial
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Future Research Needs
Collection techniques Short-term storage Cryo
effectiveness Fertilization trials
Photo USFWS