Effect of Different Levels of Glycerol and Cholesterol‐Loaded Cyclodextrin on Cryosurvival of Ram Spermatozoa

Abstract

This study was conducted to determine the optimum level of glycerol and cholesterol-loaded cyclodextrin (CLC) in a Tris-based diluent for cryopreservation of ram spermatozoa. Ram semen was treated with 0, 1.5, 3 or 4.5 mg CLC/120 × 10(6) cells in Tris-based diluents containing 3, 5 or 7% glycerol in a factorial arrangement 3 × 4 and frozen in liquid nitrogen vapour. Sperm motility, viability (eosin-nigrosin staining) and functional membrane integrity (hypo-osmotic swelling test) were assessed immediately after thawing (0 h) and subsequently after 3 and 6 h at 37°C. There was an interaction between CLC and glycerol on the functional membrane integrity (p < 0.05). In the presence of 3% glycerol, the highest functional membrane integrity (32.2%) was found in the spermatozoa treated with 1.5 mg CLC/120 × 10(6) sperm. Post-thaw sperm motility was highest in 1.5 mg CLC immediately after thawing (40.5%) and after 3-h (30.6%) incubation at 37°C (p < 0.05). Viability of spermatozoa was higher in all CLC treatments than in the untreated samples, and it was highest (33.9%) in the spermatozoa treated with 1.5 mg CLC (p < 0.05). These data indicate that the addition of cholesterol to sperm membranes by 1.5 mg CLC/120 × 10(6) cells may allow the use of a lower concentration of glycerol (3%), which is sufficient to mitigate the detrimental effects of freezing and thawing.

Substitution of egg yolk by a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing medium of equine sperm

“…Measuring cell volume changes, Glazar and colleagues showed that the addition of CDC complex in the extender increased the permeability of stallion spermatozoa to cryoprotectants [12]. Additionally, the CDC complex can form a protective sheath against water exchange [4] and can regulate the cholesterol content and thickness of membrane, which may help spermatozoa to tolerate volume changes [22]. Therefore our study confirms the protective role of cyclodextrincholesterol complex in a commercial extender for the freezing of sperm stallion.…”

ADDITIVES USED IN EXTENDERS TO IMPROVE THE FREEZABILITY OF RAM SEMEN IN RECENT YEARS: a mini review

“…Moreover, in two trials comparing methyl formamide (MF) and ethylene glycol (EG) with glycerol (G) it has been observed that glycerol gave more cryosurvival quality [44] than MF and EG additions [33], however, the nearest results to glycerol (5 %) were provided in the sample concentrated with EG 3 % (Table). On the other hand, the addition of 3 % glycerol ve 1.5 mg CLC/120½10 6 spermatozoa combination to freezing diluent was more amendatory compared to other groups, despite glycerol decreased the viability [45]. o t е.…”

“…Some alternative substances have been tested, as alone or combined by, with/without various glycerol concentrations in extenders for improving freezability of ram semen [33,44,45].…”

Effect of cyclodextrins, cholesterol and vitamin E and their complexation on cryopreserved epididymal ram semen

“…In Ram particularly, cyclodextrins-cholesterol complex has been reported to improve motility, viability and membrane integrity (Ahmad et al, 2013;Awad, 2011;Mocé et al, 2010;Motamedi-Mojdehi et al, 2014). Cyclodextrins are oligosaccharides with an internal hydrophobic cavity which forms inclusion complexes with various hydrophobic guest molecules, including cholesterol and lipophilic vitamins, and an external hydrophilic face increasing their solubility in semen extenders (Dodziuk, 2006;López-Nicolás et al, 2012).…”