Cholesterol-loaded cyclodextrin plus trehalose improves quality of frozen-thawed ram sperm

Abstract

The objective of this study was to determine effects of supplementing Tris-based semen extenders with either cholesterol-loaded cyclodextrin (CLC) or 7-dehydrocholesterol loaded cyclodextrin (7-DCLC) plus trehalose (T) for cryopreservation of ram semen. Semen was collected with an artificial vagina from five Merino rams (2-3 years of age) during the non-breeding season. Ejaculates were pooled, divided into eight equal portions, diluted with a standard Tris-based extender containing: no additive (control); T (50 mM); or T (50 mM) + 1.5, 2.5 or 3.5 mg of either 7-DCLC or CLC. Semen was chilled from 37°C to 4°C, placed in 0.25 ml French straws, held 5 cm above liquid nitrogen for 12 minutes, then plunged into liquid nitrogen. After thawing, a computer-aided semen analyzer system (CASA) was used to assess motility, whereas plasma membrane and acrosome integrity (PMAI) and high mitochondrial membrane potential (HMMP) were assessed with flow cytometry. Sperm supplemented with 2.5 mg and 3.5 mg CLC + T had the highest (P < 0.05) total and progressive motility (65.2 ± 4.7 and 19.0 ± 1.0% respectively, mean ± SEM), albeit with no significant differences from sperm with 1.5 or 3.5 mg CLC + T. Sperm with 2.5 mg CLC + T had the highest (P < 0.05) PMAI (59.3%; not different from 3.5 mg CLC + T) and highest (P < 0.05) HMMP (64.6%; not different from 1.5 or 3.5 mg CLC + T). The lowest ALH value, 2.8 ± 0.3 µm was in the 2.5 mg 7-DCLC + T group; otherwise, there were no significant differences among groups for any other CASA end point. In conclusion, adding CLC + T to a tris-based extender optimized quality of frozen-thawed ram semen. Therefore, extenders including CLC + T have potential to improve quality of frozen-thawed ram sperm.

Keywords

• 7-dehydrocholesterol
• semen cryopreservation
• computer-assisted semen analyzer
• flow cytometry