X-Message-Number: 28153
Date: Fri, 30 Jun 2006 10:53:57 -0700 (PDT)
From: Doug Skrecky <>
Subject: propylene glycol/methanol solution found wanting

[The highest survival in previous research was with a glycerol/methanol
combination. For some mysterious reason, this was ignored, and research
continued with the inferior propylene glycol/methanol combination.]

Cryobiology. 2006 Jun 1; [Epub ahead of print]
Japanese flounder (Paralichthys olivaceus) embryos are difficult to
cryopreserve by vitrification.
  The first successful cryopreservation of fish embryos was reported in
the Japanese flounder by vitrification [Chen and Tian, Theriogenology,
63, 1207-1219, 2005]. Since very high concentrations of cryoprotectants
are needed for vitrification and fish embryos have a large volume,
Japanese flounder embryos must have low sensitivity to cryoprotectant
toxicity and high permeability to water and cryoprotectants. So, we
investigated the sensitivity and the permeability of Japanese flounder
embryos. In addition, we assessed the survival of flounder embryos after
vitrification with solutions containing methanol and propylene glycol,
following Chen and Tian's report. The embryos were relatively insensitive
to the toxicity of individual cryoprotectants at lower concentrations,
especially methanol and propylene glycol as their report. Although their
permeability to water and cryoprotectants could not be measured from
volume changes in cryoprotectant solutions, the embryos appeared to be
permeable to methanol but less permeable to DMSO, ethylene glycol, and
propylene glycol. Although vitrification solutions containing methanol
and propylene glycol, which were used in Chen and Tian's report, were
toxic to embryos, a small proportion of embryos did survived. However,
when vitrified with the vitrification solutions, no embryos survived
after warming. The embryos became opaque during cooling with liquid
nitrogen, indicating the formation of intracellular ice during
cooling. When embryos had been kept in vitrification solutions for 60min
after being treated with the vitrification solution, some remained
transparent during cooling, but became opaque during warming. This
suggests that dehydration and/or permeation by cryoprotectants were
insufficient for vitrification of the embryos even after they had been
over-treated with the vitrification solutions. Thus, Chen and Tian's
cryopreservation method lacks general application to Japanese flounder
embryos.

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