NATIONAL ANIMAL GERMPLASM PROGRAM (NAGP)
Location: Plant And Animal Genetic Resources Preservation Research Unit
Title: Effects of alternative cryoprotectants, diluents, straw size and cholesterol addition on cryopreserved rooster sperm
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 29, 2012
Publication Date: July 29, 2012
Citation: Tarvis, K.M., Purdy, P.H., Graham, J.K. 2012. Effects of alternative cryoprotectants, diluents, straw size and cholesterol addition on cryopreserved rooster sperm. Meeting Abstract. International Congress on Animal Reproduction, Vancouver, BC, Canada, July 29-August 2, 2012.
Rooster sperm do not cryopreserve well. This is due in part to osmotic changes that sperm undergo during addition and removal of cryoprotectant (CPA), as well as membrane damage during the membrane phase transition from fluid at 37 degrees C to gel at low temperatures. When a CPA is removed from sperm, it induces a transient osmotic gradient across the plasma membrane as CPA permeability is lower than that of water. An alternative CPA, methylacetamide (MA), with a lower molecular weight than glycerol (GLY) permeates the plasma membrane more quickly than GLY, potentially decreasing osmotic damage. To decrease membrane damage induced by the membrane phase transition, cholesterol can be added to plasma membranes, increasing membrane fluidity at lower temperatures. Rooster sperm were collected from several birds, pooled and diluted to 1 billion cells/mL in either a trehalose-based diluent (TD) or glutamate-based diluent (GD). In experiment 1, sperm were diluted 1:1 at 5ºC with either 18% GLY or 18% MA resulting in a 9% CPA final concentration. Sperm were packaged in 0.25-mL or 0.5-mL straws and frozen in LN vapor. Motility analyses were conducted using CASA for sperm thawed in a 5 degrees C water bath and diluted 1:10 in GD or TD containing 10% BSA. Data for all experiments were analyzed by ANOVA and means separated using Student-Newman-Keuls multiple comparison test. Higher motility rates were seen for treatments packaged in 0.5-mL straws (P<0.05). In addition, for sperm frozen in 0.5-mL straws, cells frozen in TD exhibited higher motility (>60%) than sperm frozen in GD (46-53%). In experiment 2, cholesterol was added to sperm using cholesterol-loaded cyclodextrins (CLCs) at 0.5, 1, 2 and 3 mg/mL after initial sperm dilution and incubating at 5°C for 30 min. Sperm were then diluted 1:1 with either 18% GLY or 18% MA in either GD or TD, packaged in 0.25-mL straws and frozen. Addition of CLCs did not improve sperm post-thaw motility rates (P>0.05). To determine if MA exposure is detrimental to sperm, in experiment 3, sperm were exposed to 9% MA at 5 degrees C in TD for 0, 2.5, 5 and 10 min prior to freezing. MA exposure time prior to cryopreservation did not affect post-thaw sperm motility rates (P>0.05). In experiment 4, sperm were left with 9% MA for 0, 5, 10, 15, 20, 30, 45 and 60 min after thawing, before analysis. Exposure to MA for up to 1 hr post-thaw did not affect sperm motility rate (P>0.05). In conclusion, altering sperm membrane composition by adding CLCs did not improve post-thaw motility of rooster sperm. Sperm frozen in TD with 9% MA or 9% GLY in 0.5-mL straws protected the cells from cryodamage most effectively, and exposure to MA prior to or after freezing did not affect sperm motility. Future experiments will determine the fertilizing capacity of sperm frozen using MA as the cryoprotectant.