A focus for vitrification research in the literature has been on
penetrating cryoprotectants with relatively low critical warming
rates. The theory has been that solutions incorporating these solvents
would avoid recrystalization at lower and hopefully less toxic
concentrations. Unfortunately these cryoprotectants tend to be quite toxic
even at low concentrations.
An alternative strategy would be to cast the net wider to include
more consideration of cryoprotectants with exceptionally low toxicity,
rather than the lowest critical warming rates. A clear standout in this
regard is trimethylene glycol. It is the only cryoprotectant which gave no
evidence of any toxic effect at the maximum dosage tested (3.1 M), as
measured by rabbit spermatozoa mobility. (Incubation of spermatozoa was
for 30 minutes at 20 C.) DMSO earned second place with a maximum non toxic
concentration of 2.17 M. Ethylene glycol was third at 1.55 M. Lactamide,
acetamide, and propionamide tied for fourth at 0.93 M. Butyramide,
dimethylformamide, glycerol, and malomide proved to be yet more
Glycerol has been used in vitrification solutions. The critical
warming rate for trimethylene glycol is about the same as for glycerol
(see Cryobiology 40: 151-158 2000). I suspect that replacing glycerol in
vitrification solutions with trimethylene glycol would reduce
vitrification solution toxicity significantly.
Note: The medline abstract below unfortunately hides
trimethylene glycol's exceptional property.
Cryoprotective effects of
some amides on rabbit spermatozoa.
Journal of Reproduction & Fertility. 60(1):247-52, 1980 Sep.
Semen was diluted 1:9 with egg yolk-citrate medium containing 0.31--3.1 M
(final concentration) formamide, butyramide, acetamide, propionamide,
dimethylformamide, lactamide, malomide, ethylene glycol, trimethylene glycol,
dimethylsulphoxide (DMSO) or glycerol. After 30 min incubation at 20 degrees
C, sperm motility was superior in hypertonic solutions of acetamide,
lactamide, dimethylsulphoxide, trimethylene glycol and ethylene glycol.
Some of these compounds were added to semen diluted 1:2 in
an isotonic egg-yolk-glucose-lactose-raffinose solution and frozen by the
pellet method. Relatively good survival of motility was obtained in 1.0
M-DMSO, -lactamide or -acetamide. Dimethylformamide (0.5 M), ethylene glycol
(0.5--1.5 M), trimethylene glycol (1.5 M) and propionamide (0.75 M) also gave
some protection. Insemination of does with semen frozen and
thawed with 1.0 M-DMSO, -lactamide or acetamide gave fertilization rates of
68--88%, and 84% (38/45) of does gave birth to an average of 5.3 young.
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