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

摘要

The objective of this study was to determine effects of supplementing Tris-based semen extenderswith either cholesterol-loaded cyclodextrin (CLC) or 7-dehydrocholesterol loaded cyclodextrin (7-DCLC) plustrehalose (T) for cryopreservation of ram semen. Semen was collected with an artificial vagina from five Merinorams (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 or3.5 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 cmabove liquid nitrogen for 12 minutes, then plunged into liquid nitrogen. After thawing, a computer-aided semenanalyzer system (CASA) was used to assess motility, whereas plasma membrane and acrosome integrity (PMAI) andhigh mitochondrial membrane potential (HMMP) were assessed with flow cytometry. Sperm supplemented with2.5 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. Spermwith 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 wasin the 2.5 mg 7-DCLC + T group; otherwise, there were no significant differences among groups for any otherCASA end point. In conclusion, adding CLC + T to a tris-based extender optimized quality of frozen-thawed ramsemen. Therefore, extenders including CLC + T have potential to improve quality of frozen-thawed ram sperm.