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.

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