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U.S. Fish & Wildlife Service
Preliminary Results on Cryopreservation of Alligator Gar (Atractosteus spatula) Sperm Jaclyn Zelko U.S. Fish & Wildlife Service Warm Springs, Georgia Ricky Campbell Tupelo, Mississippi Carlos Echevarria Warm Springs, Georgia
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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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Cryoprotectants = Chemical
Process - Chemicals Cryoprotectants = Chemical Added 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 cells increase the internal osmolality of the cell. Osmolality = total dissolved salts in a liquid
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Cryoprotectants = Toxic to cells!
Process - Chemicals Cryoprotectants = Toxic to cells! Limit exposure time to minimize damage but allow chemicals to enter cells
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Sperm + Cryoprotectant
Process – Freezing Sperm + Cryoprotectant
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Process – Freezing Loading Straws
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Process – Freezing Loading Straws
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Process – Freezing Straws, Goblets, Canes
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Process – Freezing Shipping Dewars
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Same Process at Freezing BUT in Reverse Order
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****
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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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Program was initiated in 2005
Program at PJANFH 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
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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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2005 Efforts Sperm were collected from only available male
Initial motility was 95% Extended sperm with a modified Hanks’ balanced salt solution Photo: USFWS
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2005 Efforts Evaluated two cryoprotectants
Dimethyl sulfoxide and Methanol Evaluated two concentrations (5%, 10%) Photo: USFWS Photo: USFWS
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2005 Efforts Extended sperm was mixed with cryoprotectant
Equilibrated for 4 minutes Ten 0.5-mL straws per treatment Frozen in shipping dewar (40 straws)
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2005 Efforts Cryopreserved sperm were stored for 48 days in liquid nitrogen Straws were thawed in a 40°C water bath for 8 seconds Photo: USFWS Photo: USFWS
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Results
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2006 Efforts Sperm were collected from three males
Initial motilities % Sperm frozen from 1 male (120 straws) Used 3 concentrations of Extender Four cryoprotectant treatments
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Results
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Future Efforts Collection techniques Short-term storage
Cryo effectiveness Fertilization trials Photo:
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Photo: D. Franklin, Courtesy Dept
Photo: D. Franklin, Courtesy Dept. of Library Services, American Museum of Natural History
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