As outlined in the experimental methods, we maintained
zebrafish in 0.5% ethanol concentration for 9 weeks (hereafter
referred to as chronic alcohol exposure), and then assessed its effect
on fecundity. To assess the effect of chronic alcohol exposure on
zebrafish reproductive capacity, we set up zebrafish breeding
among the four experimental groups as follows: both partners from
control fish groups, only male partner alcohol-exposed, only female
partner alcohol-exposed, both partners alcohol-exposed. The
number of total eggs laid down in each experimental combination,
from four breedings set up over the period of 1 month were taken
for counting. Eggs obtained from different breedings were counted
manually for six breeding sets. The average of six breedings was
taken as a direct measure of fecundity. Our data show a drastic
decrease in fecundity, when either one or both of the partners were
taken from a chronic alcohol exposure group. Ethanol-exposed
males showed more severe effects of chronic ethanol exposure on
fecundity than exposed females (Fig. 2); similarly, a decrease in
fecundity is observed when both partners were taken from a
chronic alcohol exposure group as compared to a control group.We
observed more severe effects of chronic alcohol exposure on
fecundity, when ethanol-exposed parents were bred in the presence
of 0.5% ethanol in water (Fig. S1). However, control fish also
showed a significant drop in fecundity in such breeding conditions.
These results show that chronic alcohol exposure dramatically
decreases fecundity and negatively affects reproductive capacity of
both male and female zebrafish subjects.
Alcohol withdrawal restores fecundity in alcohol-exposed subjects
Next, we asked whether a 9-week withdrawal regime can
alleviate the consequences of chronic alcohol exposure. To test
this, alcohol exposure was terminated in each of the alcoholexposed
groups and zebrafish were maintained in an alcoholfree
medium for 9 weeks. After this withdrawal program, fish
were allowed to breed in the same four combinations and the
total numbers of eggs produced from the four breeding sets were
counted. The average number of eggs produced from these
breeding sets showed no difference in fecundity in alcoholexposed
fish as compared to the controls (Fig. 3). We found
complete restoration of fecundity to normalcy in all alcoholwithdrawal
groups. Each breeding combination, containing
either one or both of the parents from an alcohol-withdrawal
group, yielded an average number of eggs comparable to the
control experiment. These findings indicate that loss of fecundity,
including the possible toxic effects of long-term exposure to
alcohol, are fully reversible upon withdrawal.