51 CHANGING ROOSTER SPERM MEMBRANES TO FACILITATE CRYOPRESERVATION

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. Some of 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 before freezing and when the CPA are removed from cells after thawing also cause membrane damage. To minimize this damage, low molecular weight CPA with high membrane permeability were tested to determine their effectiveness for cryopreserving sperm. Rooster semen was collected from several birds, pooled and diluted to 800 million sperm mL^–1 at 5°C in Lake's Low Temperature diluent (LLT). Sperm were treated with either LLC (0.25, 0.5, 1, 1.5, 2, 4 and 6 mg mL^–1) or CLC (0.5, 1 and 2 mg mL^–1) for 30 min. The sperm were diluted 1:1 with LLT containing 18% CPA, resulting in final CPA concentrations of 9%. The CPA tested were glycerol (G), methylacetamide (MA), dimethylformamide (DMF), methylformamide (MF) and ethylene glycol (EG). The sperm were frozen in liquid nitrogen vapor and stored in liquid nitrogen. Straws were thawed in 5°C water and sperm motility and membrane integrity analysed immediately. Sperm motility was measured using computer-assisted sperm analysis (CASA) and membrane integrity was analysed by flow cytometry using propidium iodide to detect cells with damaged membranes. Data were analysed by ANOVA and means separated using Student–Newman–Keuls multiple comparison test. Addition of LLC and CLC did improve sperm cryosurvival rates (P > 0.05). Using G as the CPA resulted in higher percentages of motile (54%) and viable (58%) sperm than MA (47 and 52%; P < 0.05), whereas DMF, EG and MF resulted in less than 45% motile cells (P < 0.05). In conclusion, altering sperm membrane composition using CLC and LLC 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 CPA.

We thank CSU-CVBMS for funding support.