Development of a buffer system for dialysis of bovine spermatozoa before freezing. I. Effect of zwitterion buffers.
The effect of N-TRIS(hydroxymethyl)methyl-2-aminoetane sulfonic acid (TES); N,N BIS (2 hydroxvethyl)-2 aminoethane sulfonic acid (BES), N-2(hydroxyethyl)piperazine-N-2-ethane sulfonic acid (HEPES), morpholinopropane sulfonic acid (MOPS), and piperazine-N-N-BIS(2-ethane sulfonic acid (PIPES) solutions on dialyzed semen was studied. Each was titrated to pH 7.0 with TRIS-(hydroxymethyl)-amino methane (TRIS) solution and the osmotic pressure was adjusted to between 320 to 325 mOsm/kg. The new solutions were identified as TEST, BEST, HEPEST, MOPST and PIPEST, respectively. The solutions were used 1) alone, 2) in a composite with equal parts (V/V) of each solution and 3) in a 1:1 (V/V) combination with isosmotic trisodium citrate solution. Later, TRIS and sodium hydroxide (NaOH) were compared as titration bases for piperazine-N-N-BIS (2-ethane sulfonic acid) (PIPES) and N-Tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES). Ejaculates were diluted 1:10 (V/V) in extenders containing buffer, 20% egg yolk and 5% glycerol (V/V). The samples were dialyzed (1:50) during cooling for a period of 2 h. Each sample was dialyzed against the same buffer system containing 5% glycerol without egg yolk and later it was frozen in pellets. The treatments were evaluated by observation of sperm motility in fresh and thawed semen samples. The latter were also analyzed by electronic count of cells that passed through the Sephadex column. Sperm survival was higher in PIPEST (PIPES titrated with TRIS) or the composite buffer, and the inclusion of 50% sodium citrate (Na citrate) improved significantly (P<0.05) sperm motility in fresh and frozen-thawed semen samples. There was no difference (P>0.05) between the titration bases. In the second experiment, sperm survival was superior in extenders containing PIPEST (P<0.05) than in those containing TEST independently of the inclusion of Na citrate.
Garcia
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MA
Department of Animal Science University of Minnesota Saint Paul, MN 55108 USA.
Graham
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EF