Fertilization experiment
For fertilization, pooled eggs from six mature females were used
to determine fertilization rates. Egg samples (about 1000 eggs)
were inseminated in dry Petri dishes with fresh sperm or frozen
sperm immediately after thawing at spermatozoa:egg ratio of
1 105:1 [5]. Thawed sperm was added over the eggs and gently
mixed before activation with 20 ml of fertilization solution (3 g
urea and 4 g NaCl in 1 L distilled water). After fertilization the eggs
were stirred for 1 h and at the end of the stirring they were treated
with tannic acid (0.5 g/L) for 30 s to remove the stickiness of eggs.
Then the eggs were rinsed with hatchery water and gently transferred
to labelled Zuger glass incubators with running water
(24 C). When the fertilized eggs developed to 4-cell stage, the fertilization
rate (number of 4-cell stage embryos/number of total
eggs) was determined under a stereomicroscope at 20-fold magnification.
Fertilization experiments were performed in 3 replicates