In vitro evaluation of zwitterion buffers in diluents for freezing ram spermatozoa.

Diluents based on the zwitterion buffers Tes, Hepes and Pipes titrated to pH 7.0 with NaOH or Tris were compared with Tris-citrate diluents by assessment of post-thawing motility and acrosome integrity of frozen ram spermatozoa. Varying buffer osmolalities between 300 and 360 mosmol had no effect but there was a decrease (P < 0.001) in motility between 360 and 420 mosmol. There was a quadratic effect (P < 0.001) on motility of increasing glucose concentration in the diluent (30, 85, 140, 195 and 250 mM) with a maximum at 85 mM. At this sugar concentration, motility was higher (P < 0.001) for diluents containing glucose and fructose than lactose, sucrose or trehalose. Inclusion of egg yolk (optimum at 13.5% v/v; P < 0.05), centrifugation of the diluents (P < 0.01), use of low dilution rates (3 or 6-fold; P < 0.05) and freezing in pellets rather than minitubes or straws (P < 0.001) improved both the motility and acrosome integrity of spermatozoa in the zwitterion diluents. It is concluded that zwitterion buffers are superior to Tris-citrate in ram semen freezing diluents.