Aliquots of pooled ejaculates from Holstein bulls were diluted to 1 x 10⁸ sperm/ml in diluents containing 50 mM buffer, 50 mM fructose, NaCl, and either 0% or 20% yolk (290 mOsm, pH 7.0, at 25°C). Lipid droplets and high-density lipoproteins were precipitated from yolk of eggs of Gallus domesticus by diluting whole yolk to 40% (v/v) in distilled water and centrifuging. Sperm viability, based on percent intact acrosomes (PIAs), and pH of the incubating medium were measured on each sample after 0, 4, and 8 h of incubation at 37°C immediately postdilution and after storage for 1 day at 4°C.

During 37°C incubation on the day of semen collection, acrosomal maintenance was comparable in diluents containing 20% yolk. PIAs were higher in HEPES- and PIPES-than Tris-buffered diluents in the absence of yolk. Regardless of the buffer, PIAs were extremely low in yolk-free diluents after 4°C storage. However, in 20% yolk diluents, maintenance of PIAs during 37°C incubation was better with HEPES and PIPES than with Tris. Buffering capacity increased with Tris, HEPES, and PIPES, respectively.

Sperm cholesterol (C) and phospholipids (P) were analyzed on aliquots of 5 x 10⁸ sperm after 0 and 3 h at 37°C and at 3 h after cooling at 4°C in PIPES-buffered diluents. C or P did not differ between 0% and 20% yolk diluents at 37°C while C and P were lower for sperm cooled in 0% vs 20% yolk diluents. The C/P ratio of bovine sperm was 0.5 and did not differ between sperm incubated or cooled in 0% or 20% yolk diluents. These results indicate that low density lipoproteins from yolk do not alter the C/P ratio of bovine spermatozoa during in vitro aging at 4°C or 37°C.