138 EFFECT OF SEMINAL PLASMA ON CHILLING AND FREEZING OF CANINE SPERMATOZOA

Abstract

Seminal plasma (SP) is usually removed from semen that is to be cryopreserved. However, some reports indicate that SP has beneficial effects on spermatozoa during chilling and freezing (Barrios et al. 2000 Biol. Reprod. 63, 1531–1537; Moore et al. 2005 Theriogenology 63, 2372–2381; Vadnais et al. 2005 Anim. Reprod. Sci. 87, 121–132). The purpose of this study was to determine the effect on sperm survival of adding SP to the extender before cooling and freezing canine spermatozoa. In replicate experiments, ejaculates obtained from 4 healthy dogs (3–4 years old) of various breeds were pooled and centrifuged at 300g for 10 min at 25°C; the supernatant of seminal plasma was decanted. Spermatozoa were suspended in egg yolk-Tris (EYT) buffer. The study comprised 2 experiments: Exp. 1: Sperm were suspended in EYT extender containing 0%, 20%, 50%, 80%, or 100% SP, and were slowly cooled to 4°C for 2 h or held at 25°C as controls. Exp. 2: Sperm samples, each of which contained 1 × 10⁸ sperm mL^-1, were assigned to 5 groups to be frozen. In group 1, sperm in EYT + 20% SP were cooled to 4°C, diluted to contain final concentrations of 5% glycerol + 10% SP in EYT, and then frozen. In the 4 other groups, sperm in EYT alone were first cooled slowly to 4°C, then diluted to contain 5% glycerol plus 0%, 20%, 40%, or 50% SP in EYT, and then frozen. Spermatozoa were frozen at 25°C min in plastic straws that were suspended above liquid nitrogen and thawed in water at 38°C for 30 s. Sperm survival was assayed by determining progressive motility and integrity of plasma and acrosome membranes. Progressive motility was determined by microscopic examination at 400× magnification. Membrane integrity was assessed by use of a double fluorescent dye, and acrosome integrity by staining sperm with Pisum sativum agglutinin. The results of the first experiment showed that 20%, 50%, 80%, or 100% SP did not improve motility, membrane integrity, or acrosome integrity of spermatozoa chilled to 4°C compared to those chilled without SP (P > 0.05). Survival of spermatozoa suspended in EYT + 20% SP and maintained at 25°C was significantly higher than for those that were chilled (P < 0.05). The results of the second experiment showed that spermatozoa suspended in EYT + 20% SP and then diluted at 4°C to contain 5% glycerol + 10% SP exhibited the highest progressive motility and membrane integrity after being frozen and thawed (P < 0.05). In summary, although seminal plasma did not affect spermatozoa that were only chilled, addition of seminal plasma did significantly improve survival of canine spermatozoa that were frozen and thawed.