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

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

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

Cryoprotectants = Toxic to cells! Process - Chemicals Cryoprotectants = Toxic to cells! Limit exposure time to minimize damage but allow chemicals to enter cells

Sperm + Cryoprotectant Process – Freezing Sperm + Cryoprotectant

Process – Freezing Loading Straws

Process – Freezing Loading Straws

Process – Freezing Straws, Goblets, Canes

Process – Freezing Shipping Dewars

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****

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

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

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

2005 Efforts Sperm were collected from only available male Initial motility was 95% Extended sperm with a modified Hanks’ balanced salt solution Photo: USFWS

2005 Efforts Evaluated two cryoprotectants Dimethyl sulfoxide and Methanol Evaluated two concentrations (5%, 10%) Photo: USFWS Photo: USFWS

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)

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

2005 - Results

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 - Results

Future Efforts Collection techniques Short-term storage Cryo effectiveness Fertilization trials Photo: www.aimlowproductions.com

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