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 w ere detected using propidium iodide as a probe of viability (membrane integrity): some 70% of the cells died during 3 h incubation at 37 deg rees C. Because the conditions of incubation were similar to those enc ountered during manipulations for in vitro fertilization, this phenome non was investigated further. If ram spermatozoa were diluted in an eq uivalent sucrose-based medium, or if the saline medium was supplemente d with 10% seminal plasma, survival was greatly improved (only 5-15% d ied 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 bas al 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 p resence of Ca2+ (2 mM), EGTA (1 mM) or mercaptoethanol (1 mM) enhanced sperm survival in saline medium, but no effect was gained by replacin g NaCl with KCl, and neither BSA nor fetal calf serum were beneficial. However, when a combination of pyruvate (1 mM), lactate (21.7 mM), Mg 2+ (0.4 mM), phosphate (0.3 mM) and Ca2+ (2 mM) was included in the sa line medium (to render it similar to Tyrode's medium), cell survival w as 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 sy stem 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 medi um and incubating in media containing combinations of low molecular we ight components such as lactate, pyruvate, Ca2+, Mg2+, and phosphate.