Cholesterol and Phospholipids of Bovine Spermatozoa. I. Selection of a PIPES-Buffered Diluent for Evaluating the Effect of Egg Yolk Lipoproteins on Sperm Cholesterol and Phospholipids1

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Abstract
The effect of egg yolk low-density lipoproteins on bovine sperm cholesterol [C] and phospholipids [P] was examined at 4 and 37.degree. C. Although Tris is a common buffer component of semen storage diluents, to minimize the .DELTA.pH with temperature, buffers varying in .DELTA.pKa/.degree. C (Tris, HEPES [4-(2-hydroxyethyl)-1-piperazine-N''-2-ethane sulfonic acid] and PIPES [piperazine-N,N''-bis(2-ethane sulfonic acid)]) were studied as diluent components. The .DELTA.pKa/.degree. C values are -0.031, -0.014, and -0.009 for Tris, HEPES and PIPES, respectively. Aliquots of pooled ejaculates from Holstein bulls were diluted to 1 .times. 103 sperm/ml in diluents containing 50 mM buffer, 50 mM fructose, NaCl and either 0% or 20% yolk (290 mOsm [milliosmole] pH 7.0, at 25.degree. C). Lipid droplets and high-density lipoproteins were precipitated from yolk of eggs of Gallus domesticus by diluting whole yolk to 40% (vol/vol) in distilled H2O and centrifuging. Sperm viability, based on percent intact acrosomes (PIA), and pH of the incubating medium were measured on each sample after 0, 4 and 8 h of incubation at 37.degree. C immediately postdilution and after storage for 1 day at 4.degree. C. During 37.degree. C incubation on the day of semen collection, acrosomal maintenance was comparable in diluents containing 20% yolk. PIA were higher in HEPES- and PIPES- than Tris-buffered diluents in the absence of yolk. Regardless of the buffer, PIA were extremely low in yolk-free diluents after 4.degree. C storage. In 20% yolk diluents, maintenance of PIA during 37.degree. C incubation was better with HEPES and PIPES than with Tris. Buffering capacity increased with Tris, HEPES and PIPES, respectively. Sperm C and P were analyzed on aliquots of 5 .times. 103 sperm after 0 and 3 h at 37.degree. C and at 3 h after cooling at 4.degree. C in PIPES-buffered diluents. C or P did not differ between 0% and 20% yolk diluents at 37.degree. C while C and P were lower for sperm cooled in 0% vs. 20% yolk diluents. The C/P ratio of bovine sperm was .apprx. 0.5 and did not differ between sperm incubated or cooled in 0% or 20% yolk diluents. Apparently, low density lipoproteins from yolk do not alter the C/P ratio of bovine spermatozoa during in vitro aging at 4.degree. C or 37.degree. C.