X-Message-Number: 25667 Date: Sun, 6 Feb 2005 20:37:28 -0800 (PST) From: Doug Skrecky <> Subject: methanol key additive for vitrification solutions? [Although methanol is a very poor glass former, its addition can significantly reduce the toxicity of other cryoprotectants. The toxicity of cryoprotectants to flounder embryos measured by percent survival, after 60 minutes cold storage at -15 C are as follows: Base Cryoprotectant Additive 20% DMSO: 22.4% +5% methanol 62.7% 25% DMSO: 18.9 20% glycerol: 34.9 +5% methanol 69.5 25% glycerol: 24.8 20% methanol: 32.0 +5% glycerol 62.0 25% methanol: 30.0 20% propylene glycol: 57.7 +5% methanol 60.9 25% propylene glycol: 45.7 20% ethylene glycol: 0.0 +5% methanol 38.6 " +5% DMSO 2.2 " +5% glycerol 1.3 " +5% PG 1.3] Theriogenology. 2005 Feb;63(3):763-73 Toxicity and protective efficiency of cryoprotectants to flounder (Paralichthys olivaceus) embryos. With the purpose of finding an ideal cryoprotectant or combination of cryoprotectants in a suitable concentration for flounder (Paralichthys olivaceus) embryo cryopreservation, we tested the toxicities, at culture temperature (16 degrees C), of five most commonly used cryoprotectants-dimethylsulfoxide (Me(2)SO), glycerol, methanol (MeOH), 1,2-propylene glycol (PG) and ethylene glycol (EG). In addition, cryoprotective efficiency to flounder embryos of individual and combined cryoprotectants were tested at -15 degrees C for 60min. Five different concentrations of each of the five cryoprotectants and 20 different combinations of these cryoprotectants were tested for their protective efficiency. The results showed that the toxicity to flounder embryos of the five cryoprotectants are in the following sequence: PG < MeOH < Me(2)SO < glycerol < EG (P < 0.05); whereas the protective efficiency of each cryoprotectant, at -15 degrees C for a period of 60min, are in the following sequence: PG > Me(2)SO approximately MeOH approximately glycerol > EG (greater symbols mean P < 0.05, and approximate symbols mean P > 0.05). Methanol combined with any one of the other cryoprotectants gave the best protection, while ethylene glycol combined with any one of the other cryoprotectants gave the poorest protection at -15 degrees C. Toxicity effect was concentration dependent with the lowest concentration being the least toxic for all five cryoprotectants at 16 degrees C. For PG, MeOH and glycerol, 20% solutions gave the best protection at -15 degrees C; whereas a 15% solution of Me(2)SO, and a 10% solution of EG, gave the best protection at -15 degrees C. Cryo Letters. 2004 Nov-Dec;25(6):415-24 Studies on cryoprotectant toxicity to zebrafish (Danio rerio) oocytes. Cryopreservation of fish germ cells is an important measure in conservation of fish genetic material. Although investigations on cryopreservation of fish sperm and embryos have been carried out extensively, cryopreservation of fish oocytes has not been studied systematically. In the present study the toxicity of cryoprotectants to zebrafish (Danio rerio) oocytes was investigated. Commonly used cryoprotectants dimethyl sulfoxide (DMSO), methanol, ethylene glycol (EG), propylene glycol (PG), sucrose and glucose were studied. Stage III (vitellogenic), stage IV (maturation) and stage V (mature egg) zebrafish oocytes were incubated in Hank's medium containing different concentrations of cryoprotectants (0.25-4M) for 30 min at room temperature. Three different tests were used to assess oocyte viability: trypan blue (TB) staining, thiazolyl blue (MTT) staining and in vitro maturation followed by observation of germinal vesicle breakdown (GVBD). Results showed that the toxic effect of cryoprotectant on oocytes generally increased with increasing concentration. MTT test was shown to be the least sensitive testing method and gave poor correlation to subsequent GVBD results. Sensitivity of vital tests increases in the order of MTT, TB and GVBD. GVBD test showed that cryoprotectant toxicity to stage III zebrafish oocytes increased in the order of methanol, PG, DMSO, EG, glucose and sucrose. No Observed Effect Concentrations (NOECs) for stage III oocytes were 2M, 1M, 1M, 0.5M, less than 0.25M and less than 0.25M for methanol, PG, DMSO, EG, glucose and sucrose respectively. TB test also showed that the toxicity of tested cryoprotectants increased in the same order. The sensitivity of oocytes to cryoprotectants appeared to increase with development stage with stage V oocytes being the most sensitive. Indian J Physiol Pharmacol. 2003 Apr;47(2):207-11 Free radical changes in methanol toxicity. Role of free radicals in methanol toxicity was evaluated in methanol treated albino rats. Methanol intoxication increased lipid peroxidation and depleted the free radical scavenging enzyme systems. The free radical quenching effect of vitamin E protected the animals from methanol induced free radical damage. Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=25667