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

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

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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enne

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life

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Background – Life History Historical range: waters within the

Mississippi River basin in Tennessee

Obion River

Photo: USGS

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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ncy

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

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

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

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

Extended sperm was mixed with cryoprotectant

Materials & Methods

Equilibrated for 4 minutes

Ten 0.5-mL straws per treatment

Frozen in shipping dewar

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

2005 Efforts Collected sperm from 1 male Initial motility 95% Evaluated two cryoprotectants

Dimethyl sulfoxide and Methanol

Evaluated two concentrations (5%, 10%)

2005 – Results **based on 1 male

0

20

40

60

80

100

Perc

ent (

%)

5% DMSO10% DMSO 5%Methanol

10%Methanol

Treatment

Evaluation of Two Cryoprotectants

EquilibrationMotilityPost-ThawMotility

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)

2006 – Results **based on 1 male

Alligator Gar Male #309 Cryoprotectant - Methanol

75 75

60

757060

75 75

50

7575 75

40

60

0

20

40

60

80

100

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

2007 Efforts Collection technique changed

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

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

2007 Efforts – Fertilization Trial

48-Hour % Fertilization

0

10

20

30

40

50

60

70

80

90

DMSO 5% DMSO 10% Methanol5%

Methanol10%

DMSO 5% DMSO 10% Methanol5%

Methanol10%

None

Male 268 Male 309 Male 616ControlMale / Treatment

% F

ertil

izat

ion

Collection techniquesShort-term storage Cryo effectivenessFertilization trials

Photo: USFWS

Future Research Needs