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United States Department of Agriculture

Agricultural Research Service

Research Project: NATIONAL ANIMAL GERMPLASM PROGRAM (NAGP) Title: Changing rooster sperm membranes to facilitate cryopreservation

Authors
item Graham, James -
item Tarvis, Kim -
item Purdy, Phil

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 20, 2011
Publication Date: January 10, 2012
Citation: Graham, J.K., Travis, K.M., Purdy, P.H. 2012. Changing rooster sperm membranes to facilitate cryopreservation. Meeting Abstract. International Embryo Transfer Society, Phoenix, AZ. January 10, 2012.

Technical Abstract: Cryopreservation damages rooster sperm membranes. Part of this damage is due to membrane transitioning from the fluid to the gel state as temperature is reduced. This damage may be prevented by increasing membrane fluidity at low temperatures by incorporating cholesterol or unsaturated lipids into the membrane. Different concentrations of cholesterol-loaded cyclodextrins (CLC) and lipid-loaded cyclodextrins (LLC) containing 1, 2-Dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-Dilinoleoyl-rac-glycerol and 1,2-Dilinolenoyl-sn-glycero-3-phosphocholine were added to rooster sperm to determine if they improved cryopreservation. Osmotic stresses when cryoprotectants (CPA) are added to the cells prior to freezing and when the CPAs are removed from cells after thawing also cause membrane damage. To minimize this damage, low molecular weight CPAs with high membrane permeability were used to determine their effectiveness for cryopreserving sperm. Rooster semen was collected from several birds, pooled, and diluted to a concentration of 800 million sperm at 5C in Lake’s Low Temperature diluent (LLT). Sperm were treated with either LLC (0.25, 0.5, 1, 1.5, 2, 4 & 6 mg/mL) or CLC (0.5, 1 & 2 mg/mL) for 30 min. The sperm were diluted 1:1 with LLT containing 18% CPA (glycerol (G), methylacetamide (MA), dimethylformamide (DMF), methylformamide (MF) or ethylene glycol (EG)) for a final concentration of 9%. The sperm were frozen in liquid nitrogen vapor and stored in liquid nitrogen. Straws were thawed in 5C water, and sperm motility and membrane integrity analyzed immediately. Addition of LLCs and CLCs did not increase the percentages of motile sperm in the samples, after cryopreservation (p>0.05). Using G as the CPA resulted in higher percentages of motile (54%) and viable (58%) sperm than MA (47%, 52%, p<0.05), while DMF, EG and MF resulted in less than 45% motile cells (p<0.05). In conclusion, altering sperm membrane composition using CLCs and LLCs did not improve post thaw motility or viability in rooster sperm. Although MA did not protect the rooster sperm from cryodamage as effectively as G, future assays will need to determine the fertilizing capacity of sperm frozen using these CPAs.

Last Modified: 11/27/2014
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