48 EFFECT OF TREHALOSE ADDITION ON IN VITRO VIABILITY OF COOLED RAM SPERMATOZOA

Abstract

Refrigeration of ovine spermatozoa is a valuable tool for genetic progress. Sugars, in particular disaccharides, have been shown to have positive effects in semen extender, providing energy substrate during cell incubation, decreasing osmotic pressure, causing cell dehydration and ultimately reducing cold shock and cryodamage. Several authors have reported changes in plasma membrane protein organisation and membrane fluidity during cooling. Trehalose, a non-reducing disaccharide has been associated with stabilisation of cellular proteins and interaction with membrane phospholipids due to its ability to replace water at the membrane-solution interface. Many studies have confirmed the beneficial effect of the addition of trehalose during cryopreservation at –196°C; however, its effects during refrigeration (5–7°C) have not been reported. Ejaculates form four fertile Corriedale rams were collected by electroejaculation during the ovine reproductive season in the Southern Hemisphere (May–August); the study was conducted in 12 replicates. After initial quality assessment, ejaculates were pooled into heterospermic samples, diluted in Tris-buffered extender and separated into treatments consisting of (a) no refrigeration (kept at room temperature 22–25°C), (b) refrigeration (cooling rate of 2°C/3 min to reach a final temperature of 5–7°C), or (c) refrigeration with the addition of 150 mOsm trehalose. Samples were evaluated for total motility, progressive motility, hyposmotic swelling test (HOST) and eosin Y (1 step, WHO protocol) live/dead stain at 0, 6, 12, 24 and 48 h post-refrigeration. Results were analysed using 1-way ANOVA with Tukey post-hoc multiple comparison test. Compared with refrigeration without the addition of disaccharides, the refrigeration in presence of trehalose significantly increased (P < 0.05) total motility at 48 h (32 ± 0.9 vs 50 ± 1.1) and progressive motility at 24 (40 ± 0.9 vs 65 ± 0.6) and 48 h (0 vs 38 ± 1.0) post-refrigeration. Trehalose also decreased (P < 0.05) the percentage of damaged spermatozoa as evidenced by HOST and live/dead stain after 12 (42 ± 1.1 vs 29 ± 0.7), 24 (52 ± 0.9 vs 37 ± 0.6) and 48 h (74 ± 0.8 vs 60 ± 1.0) of refrigeration. Although no statistical difference was detected, there was a trend of trehalose to improve total and progressive motility parameters at the initiation of treatments (0 h). In addition, spermatozoa refrigerated in the presence of trehalose was the only treatment group to retain progressive motility after 48 h of storage. Our results indicate that trehalose could be used to extend the in vitro viability of refrigerated ovine spermatozoa for up to 48 h. Further studies are needed to assess the in vivo fertility (pregnancy rate) of refrigerated spermatozoa in the presence of this non-permeating cryoprotectant sugar.