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

SpermSperm

Jaclyn ZelkoU.S. Fish & Wildlife Service

Warm Springs, Georgia

Ricky Campbell

Tupelo, Mississippi

Carlos Echevarria

Warm Springs, Georgia

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

Process - Chemicals

Cryoprotectants = ChemicalAdded 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 cellsincrease the internal

osmolality of the cell.

Osmolality = total dissolved salts in a liquid

Process - Chemicals

Limit exposure time to minimize damage but allow chemicals to enter cells

Cryoprotectants = Toxic to cells!

Process – FreezingSperm + Cryoprotectant

Process – FreezingLoading Straws

Process – FreezingLoading Straws

Process – FreezingStraws, Goblets, Canes

Process – FreezingShipping Dewars

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****

Advantages of CryopreservationPreservation 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

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

Program at PJANFH

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

Repository - Study Objectives

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2005 Efforts

Sperm were collected from only available male

Initial motility was 95%

Extended sperm with a modified Hanks’ balanced salt solution

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2005 Efforts

Evaluated two cryoprotectants

Dimethyl sulfoxide and Methanol

Evaluated two concentrations (5%, 10%)

Extended sperm was mixed with cryoprotectant

Equilibrated for 4 minutes

Ten 0.5-mL straws per treatment

Frozen in shipping dewar (40 straws)

2005 Efforts

Photo: USFWS

Photo: USFWS

2005 Efforts

Cryopreserved sperm were stored for 48 days in liquid nitrogen

Straws were thawed in a 40°C water bath for 8 seconds

2005 - Results

0102030405060708090

100

Perc

en

t (%

)

5%DMSO

10%DMSO

5%Methanol

10%Methanol

Treatment

Equilibration Motility

Post-Thaw Motility

2006 Efforts

Sperm were collected from three males

Initial motilities 10 - 75%

Sperm frozen from 1 male (120 straws)

Used 3 concentrations of Extender

Four cryoprotectant treatments

2006 - Results2006 Alligator Gar Male #1 Cryoprotectant - Methanol

75 75

60

7570

60

75 75

50

7575 75

40

60

0

20

40

60

80

100

5% Methanol 10% Methanol

Cryoprotectant Treatment

Mo

tili

ty (

%)

Initial Motility

1:2 Equilibration

1:2 Post-Thaw

1:5 Equilibration

1:5 Post-Thaw

1:10 Equilibration

1:10 Post-Thaw

Future Efforts

Collection techniques

Short-term storage

Cryo effectiveness

Fertilization trials

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