SURVIVAL OF RAM SPERMATOZOA AT HIGH DILUTION - PROTECTIVE EFFECT OF SIMPLE CONSTITUENTS OF CULTURE MEDIA AS COMPARED WITH SEMINAL PLASMA

During incubation of ram spermatozoa at 1 x 10(7) cells mL(-1) or less in a simple HEPES-buffered saline medium, high levels of cell death were detected using propidium iodide as a probe of viability (membrane integrity): some 70% of the cells died during 3 h incubation at 37 degrees C. Because the conditions of incubation were similar to those encountered during manipulations for in vitro fertilization, this phenomenon was investigated further. If ram spermatozoa were diluted in an equivalent sucrose-based medium, or if the saline medium was supplemented with 10% seminal plasma, survival was greatly improved (only 5-15% died during a 3-h incubation at 37 degrees C); the protective effect of seminal plasma resided in a 5-10 kDa fraction. Sperm death in the basal saline medium was strongly dependent on cell concentration below 5 x 10(7) spermatozoa mL(-1) whereas little effect of concentration was seen in the sucrose medium or in the presence of seminal plasma. The presence of Ca2+ (2 mM), EGTA (1 mM) or mercaptoethanol (1 mM) enhanced sperm survival in saline medium, but no effect was gained by replacing NaCl with KCl, and neither BSA nor fetal calf serum were beneficial. However, when a combination of pyruvate (1 mM), lactate (21.7 mM), Mg2+ (0.4 mM), phosphate (0.3 mM) and Ca2+ (2 mM) was included in the saline medium (to render it similar to Tyrode's medium), cell survival was greatly improved (12% died during the 3-h incubation). Nevertheless, although ram spermatozoa survived well after semen dilution in HEPES-buffered Tyrode's medium, dilution after washing through a Percoll system based on this medium caused considerable sperm death. Spermatozoa washed through the sucrose-based medium, on the other hand, could be diluted in HEPES-buffered Tyrode's medium with minimal cell death. We conclude that ejaculated ram spermatozoa tend to die following washing and high dilution in simple isotonic saline-based media. Survival in defined media can be maximized by washing through a sucrose-based medium and incubating in media containing combinations of low molecular weight components such as lactate, pyruvate, Ca2+, Mg2+, and phosphate.