Does rosemary aqueous extract improve buck semen cryopreservation?

Abstract

This study was conducted to investigate the effect of rosemary aqueous extract for protecting buck spermatozoa from freeze–thawing damages. In this study, a total of 32 ejaculates were collected from four Kordian bucks (8 ejaculates for each buck). On the day of semen collection, four ejaculates (one ejaculate for each buck) were pooled and diluted with egg yolk Tris-based extenders containing 0 (E0), 2 (E2), 4 (E4), and 6 (E6) percent of rosemary aqueous extract. After thawing, sperm motility (computer-assisted sperm motility analysis (CASA)), membrane integrity (eosin/nigrosin) and functionality (Hypoosmotic swelling (HOS) test), abnormal morphology, lipid peroxidation (malondialdehyde (MDA) production), capacitation status (Chlortetracycline (CTC)), mitochondrial activity (rhodamine-123) and apoptotic features (Annexin V/propidium iodide) were assessed. The results showed that E4 extender significantly improved total (45.71±1.49) and progressive (24.86±0.84) motility, functional membrane (47.18±1.37), and integrity (49.25±1.17) of post-thawed buck spermatozoa when compared to E0 (36.43±1.49, 18.43±0.84, 39.42±1.37 and 41.32±1.17, respectively) and E6 (30.43±1.49, 16.29±0.84, 34.54±1.37 and 36.39±1.17, respectively) extenders. Moreover, spermatozoa with abnormal morphology were significantly increased in E6 (22.5±1.07) compared to E2 (17.29±1.07) and E4 (16.11±1.07) extenders. The results showed that only E2 (1.69±0.08) extender significantly decreased the level of MDA formation when compared to E0 (2.04±0.08) and E4 (2.03±0.08) extender. In this study, different level of rosemary aqueous extract failed to alter the CTC staining patterns and the mitochondrial activity of post-thawed buck spermatozoa. Moreover, E4 extender significantly increased live (55.92±2.97) and decreased dead (22.64±3.37) spermatozoa compared to other extenders. In conclusion, addition of rosemary aqueous extract at level of 4% can improve post-thawed buck spermatozoa quality. More studies are required to reveal the active components in rosemary extract.