Comparison of Cholesterol Carriers and Substitution of Fructose and Glycerol With Trehalose on Frozen/Thawed ATP Content, DNA Integrity and Kinematics Variables of Ram Spermatozoa

• Published in: Reproduction in domestic animals 2024-12, Vol.59 (12), p.e14748
• Main Authors: Koorehpaz, Kave, Eslami, Mohsen, Farrokhi-Ardabili, Farhad
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Animals; beta-Cyclodextrins - pharmacology; Cholesterol; Cryopreservation - veterinary; Cryoprotective Agents - pharmacology; DNA Damage - drug effects; Fructose - pharmacology; Glycerol - pharmacology; Male; Semen Preservation - methods; Semen Preservation - veterinary; Sheep - physiology; Sheep, Domestic; Spermatozoa - drug effects; Spermatozoa - physiology; Trehalose - pharmacology
• ISSN: 1439-0531; 1439-0531
• DOI: 10.1111/rda.14748

Current study was aimed to assess the β-cyclodextrin (βCD) and methyl-β-cyclodextrin (MβCD) on delivery of cholesterol, and substitution of fructose and glycerol with trehalose on the ram semen cryosurvival. Samples were collected, diluted with Tris-citric acid-LDL extender, pooled, and used. In experiment I, βCD and MβCD carriers were used and compared to deliver the cholesterol (at 0, 0.5, 1, 1.5, 2, and 4 mg/mL). In the experiment II, trehalose (0, 7, 14, 21, and 28 mM) was substituted with fructose (28, 21, 14, 7, 0 mM, respectively). In the experiment III, the best cholesterol/carrier dose groups from the first experiment, were selected to be evaluated with the fructose/trehalose (14/14 mM) combination compared to fructose (28 mM) alone. The concentration of glycerol in the above-mentioned experiments was set at 4.5%. In the experiment IV, the effect of lowering glycerol (4% vs. 4.5%) was assessed using selected cholesterol/carrier groups. Kinematics, chromatin integrity, ATP contents, malondialdehyde amounts and viability were evaluated. Cholesterol (especially at 1.5 and 2 mg/mL) improved the kinematics and ATP levels using both carriers. The optimised amounts of trehalose (14 mM)/fructose(14 mM) reduced peroxidation and DNA fragmentation levels. Co-administration of optimised levels of cholesterol with trehalose/fructose did not show extra beneficial effects compared to each of them. Trehalose could not protect the spermatozoa at lower amounts of glycerol (4% vs. 4.5%). In conclusion, either the optimised levels of cholesterol (using βCD or MβCD carriers) or substitution of half of the fructose with the trehalose alone could lead to improvement in quality of frozen/thawed ram semen.