Dialysis addition of trehalose/glycerol cryoprotectant allows recovery of cryopreserved mouse spermatozoa with satisfactory fertilizing ability as assessed by yield of live young
Ka. Thompson et al., Dialysis addition of trehalose/glycerol cryoprotectant allows recovery of cryopreserved mouse spermatozoa with satisfactory fertilizing ability as assessed by yield of live young, J ANDROLOGY, 22(2), 2001, pp. 339-344
Mouse sperm cryopreservation provides a means for storing the genetic infor
mation in genetically modified mice (mutants, transgenics, and "knockouts")
in a cost- and space-effective manner. Sperm from this species are highly
sensitive to cryodamage, which has impeded their cryopreservation in the pa
st. The cryoprotectant used in this study was 6% glycerol (0.65 M) plus 7.5
% trehalose (0.22 M), which was added to a concentrated suspension of sperm
from B6SJLF1/J mice in bicarbonate-free buffer by dialysis to minimize osm
otic stress on the cells. Sperm suspensions were frozen in 0.25 mL straws a
nd stored in liquid N-2. Eggs were obtained from B6SJLF1/J superovulated fe
males. For in vitro fertilization (IVF), 15-25 muL of sperm suspension post
-thaw from one straw was added directly to each of three 1.5 mL drops of fe
rtilization medium containing 30 eggs each, for 3 replicates per experiment
. The fertilized eggs were scored for blastocyst formation, after which 12
blastocysts from each drop were implanted into pseudopregnant CD-1 females.
The number of live pups were then scored at birth. Ten experiments yielded
21.7 +/- 1.4 (SD) blastocysts per 30 eggs inseminated (72%) and 7.3 +/- 0.
4 (SD) live pups per 12 blastocysts implanted (61%). The overall yield of l
ive pups was 44 per 100 eggs inseminated (44%). This yield should be satisf
actory for maintaining a mouse strain through sperm cryostorage, with resta
rt of the strain through IVF and embryo transfer. The method should also pr
ovide improvement in human sperm cryopreservation, as human sperm are less
sensitive to cryodamage than are mouse sperm.