Lipid deficient extender for bovine spermatozoa: its development and use in measuring freezing-induced lipid loss

To facilitate the measurement of lipid losses from spermatozoa due to freezing, three low-lipid seminal extenders containing lactose, bovine serum albumin, or soybean protein were evaluated as potential cryoprotectants. All extenders were formulated to have an osmotic pressure within the range of 270 to 330 mosmol and a pH of 6.8 to 7.0. Soybean protein (Promine-D) maintained the highest post-thaw motility of spermatozoa with similar survival for spermatozoa frozen in ampules and straws. The extender derived from testing several components consisted of Tris(hydroxymethyl) aminomethane (245 mM), and citric acid monohydrate (78mM), as the buffering compounds; and fructose (69 mM), glycerol (7% vol/vol), and Promine-D (1.5% wt/vol). Post-thaw sperm motility of approximately 40% was not different from the Tris-egg yolk control. Fertility of fresh rabbit semen treated with the extender was normal. After freeze-thawing, protected spermatozoa contained more lipid (1.61 versus 1.20 mug/10-6 sperm) and lost less glutamic oxaloacetic transaminase enzyme (102 versus 108 Karmen units) than when Promine-D was not incorporated. However, even with protection by soybean protein, spermatozoa lipid content decreased from 2.43 to 1.61 mug/10-6 sperm after one freeze-thawing. The lipid status of spermatozoa frozen and thawed in conventional bull seminal extenders containing large amounts of lipids is unknown.