Preliminary Results on Cryopreservation of Alligator Gar ( Atractosteus spatula ) Sperm
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Preliminary Results on Preliminary Results on Cryopreservation of Alligator Cryopreservation of Alligator Gar (Gar (Atractosteus spatulaAtractosteus spatula) )
SpermSpermJaclyn Zelko & Carlos Echevarría
Warm Springs National Fish HatcheryWarm Springs, GA
Ricky CampbellPvt. John Allen NFHTupelo, MS
William Wayman and Bill BouthillierWarm Springs Fish Technology Center
Warm Springs, GA
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AcknowledgementsPvt. John Allen National Fish HatcheryTupelo, Mississippi
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Acknowledgements
Fish Technology CenterWarm 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
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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
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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
Cryopreservation
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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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Extended sperm was mixed with cryoprotectant
Materials & Methods
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 40°C water bath for 8 seconds
Photo: USFWSPhoto: 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
0
20
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Perc
ent (
%)
5% DMSO10% DMSO 5%Methanol
10%Methanol
Treatment
Evaluation of Two Cryoprotectants
EquilibrationMotilityPost-ThawMotility
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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
Alligator Gar Male #309 Cryoprotectant - Methanol
75 75
60
757060
75 75
50
7575 75
40
60
0
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5% Methanol 10% Methanol
Cryoprotectant Treatment
Mot
ility
(%)
Initial Motility
1:2 Equilibration
1:2 Post-Thaw
1:5 Equilibration
1:5 Post-Thaw
1:10 Equilibration
1:10 Post-Thaw
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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
48-Hour % Fertilization
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DMSO 5% DMSO 10% Methanol5%
Methanol10%
DMSO 5% DMSO 10% Methanol5%
Methanol10%
None
Male 268 Male 309 Male 616ControlMale / Treatment
% F
ertil
izat
ion
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Collection techniquesShort-term storage Cryo effectivenessFertilization trials
Photo: USFWS
Future Research Needs