Interactions among pre-cooling, cryoprotectant, cooling, and thawing for sperm cryopreservation in rhesus monkeys

Abstract

The present study evaluated the interactions among pre-cooling, cryoprotectant, cooling, and thawing for rhesus monkey sperm using a four-way factorial design. Specifically, pre-cooling and thawing were evaluated for two conditions: slow vs. fast. Cooling was evaluated at four rates of 5, 29, 200, and 400 °C/min. The types of cryoprotectant involved combinations of egg yolk and glycerol, egg yolk and ethylene glycol, and egg yolk alone without permeable cryoprotectants or buffer alone with glycerol but without egg yolk. Our findings showed strong interactions among cryoprotectants, cooling, and thawing rates, but not pre-cooling rate, on post-thaw motility and forward progression. The optimal combination of cooling and thawing for maximum post-thaw survival depended on the types of cryoprotectant. When glycerol was used as a permeable cryoprotectant in the presence of egg yolk, slow thawing yielded similar success as fast thawing in some males. However, when glycerol was replaced with ethylene glycol for the same treatment, post-thaw motility was significantly lower in samples that were thawed slowly than those that were thawed rapidly. In the absence of permeable cryoprotectant but the presence of egg yolk, fast cooling was always favorable. On the contrary, in the absence of egg yolk but the presence of permeable cryoprotectant (glycerol), post-thaw motility was significantly reduced especially when samples were thawed slowly. Generally, fast thawing was superior to slow thawing regardless of the types of cryoprotectant or cooling rates, and glycerol in the presence of egg yolk yielded the highest post-thaw motility in all treatment groups.

Optimization of Ethylene Glycol Concentrations, Freezing Rates and Holding Times in Liquid Nitrogen Vapor for Cryopreservation of Rhesus Macaque (Macaca mulatta) Sperm

“…A previous study on rhesus sperm cryopreservation with EG showed that 2 and 4% EG (corresponding to 0.28 and 0.56 M, respectively) were optimal for preserving rhesus sperm motility [2]. However, in our study, a low level (0.175 and 0.35 M) of EG was insufficient to preserve rhesus sperm motility compared with 0.7 M EG.…”

“…The optimal Gly concentration for rhesus sperm cryopreservation is 5% (0.7 M) [20], which is consistent with the results for cynomolgus macaque sperm cryopreservation [19]. In addition to Gly, other types of penetrating CPAs, including dimethyl sulfoxide (DMSO), propylene glycol (PG) and ethylene glycol (EG), have been applied for cryopreservation of nonhuman primate sperm [2,3,7,11,20]. However, study of the fundamental cryobiology of rhesus macaque sperm indicated that EG is the most appropriate CPA for cryopreservation of rhesus macaque sperm due to its higher permeability coefficient compared with Gly, DMSO and PG [1].…”

“…In previous studies, rhesus or cynomolgus sperm diluted with sperm freezing medium usually were frozen by holding the straws with sperm in LN 2 vapor for either 10 min [2,11,21] or 15 min [3] before plunging them into LN 2 for storage. However, the effect of the holding time in LN 2 on the cryosurvival of sperm is still unknown.…”

“…The optimal freezing rate for sperm cryosurvival should be low enough to avoid IIF but fast enough to minimize the solution effects. In the conventional freezing method, rhesus sperm are usually frozen in LN 2 vapor in a Styrofoam box after being loaded into cryostraws [2,5,10,16,20,21]. Therefore, the variations in penetrating CPA concentrations in sperm freezing medium and the freezing rates resulting from varied distances between cryostraws and the LN 2 surface affect the cryosurvival of rhesus sperm.…”

Applications and optimization of cryopreservation technologies to cellular therapeutics

“…It has been alternatively argued that slow warming may be beneficial to allow time for osmotic re-equilibration processes to take place [25] when the highly shrunken cells start to encounter more liquid water as the ice matrix melts at high subzero temperatures. In some experiments on particular cell types, the impact of slow versus fast warming was either not significant or was related to the particular CPA used [44]. This raises another potential warming factor -exposure of the cells to toxic high CPA concentrations at high subzero temperatures during slow warming.…”