preliminary results on cryopreservation of alligator gar (atractosteus spatula) sperm jaclyn zelko...
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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
Ph
oto
: U
SFW
S
2005 Efforts
Sperm were collected from only available male
Initial motility was 95%
Extended sperm with a modified Hanks’ balanced salt solution
Ph
oto
: U
SFW
S
Ph
oto
: U
SFW
S
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
Ph
oto
: w
ww
.aim
low
pro
duct
ions.
com
Photo: D. Franklin, Courtesy Dept. of Library Services, American Museum of Natural History