Efficiencies of the Cryoprotectants N-Methylacetamide, N-Methylformamide and Dimethyl Sulfoxide, and the Cell Protectants Trehalose and Hydroxyethyl Starch in Cryopreservation of Swine Sperm

Authors:

Osuga T¹*, Hasegawa T², Aoki M¹, Fujie M³, Nakane T² and Asai T⁴

DOI: 10.23880/nnoa-16000139

Volume 3, Issue 2

Abstract

Polar molecules protect against freeze-induced damage by interfering with the polarised water arrangement during ice nucleation inside cells. Insufficient protection from freeze-induced damage (acting during freezing) and cryopreservative-induced cytotoxicity (acting before and after freezing) causes cell death due to cryopreservation. Although dimethyl sulfoxide (DMSO) is a useful cryopreservative, the oxidation and lone pair electrons of the sulfinyl group cause cytotoxicity. The dipole moment of the sulfinyl group in DMSO and those of the amide groups in N-methylacetamide(NMA) and N-methylformamide (NMF) are similar. Furthermore, NMA and NMF retain the methyl group present in DMSO, which confers permeation ability to DMSO. Thus, using NMA and NMF rather than DMSO may attenuate cell death due to cryopreservation, both by ensuring cryopreservation action consisting (molecular polarity and cell permeation) and by reducing cytotoxicity both before freezing and after thawing. In this study, we examined the efficiencies of different cryoprotectants in swine sperm. Swine sperm were cryopreserved in six combined solutions of three cryopreserves, i.e. NMF (7.5% [w/v], 1286 mM), NMA (7.5% [w/v], 964mM), and DMSO (7.5% [w/v], 1056mM), and two cell protectants, i.e. trehalose (5.8% [w/v]) and hydroxyethyl starch (HES; 6% [w/v]). Addition of NMF and NMA resulted in similar increases in cell activity compared with that in DMSO-treated cells before freezing and at 5 and 30 min after thawing. The ratio of cell activity resulting from the addition of trehalose to that of HES was about 1.5 before freezing but ranged from 2 to 5 at 5 and 30 min after thawing. These results showed that NMF and NMA may be more effective cryopreservatives than DMSO and that trehalose may be a better cell protectant than HES.

Keywords:

Freeze-induced damage; Cryopreservative-induced cytotoxicity; Cell permeation; Cell protectant; Hydroxyethyl starch; Trehalose

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