Page 6 - IJA2014v4n09

International Journal of Aquaculture, 2014, Vol.4, No.09 55- 63
http://ija.sophiapublisher.com
57
fish Biology Laboratory for DNA Extraction, PCR
amplification and agarose gel electrophoresis.
1.2 Feeding of the prawns
Reproductive performance is affected by the nutrient
availability of feeds supplied for culture. In order to
get good response from the prawns stocked in the
ponds, a good quality supplementary feed (CP 9043)
was given daily. The protein content of the feed was
more than 35%. Feed was applied at 3-4% of the total
body weight of the stocked prawns. Feed was supplied
twice a day: at evening (usually at 7 PM) and at
midnight (11 PM of night).
1.3 Monitoring to check brood readiness
The prawns were checked at fortnightly interval by
netting to monitor the breeding readiness. After 4
months of stocking, eggs were found in the egg
chambers of female prawns. During brood stock
development, the eggs were yellow in color and it
changed from yellow to orange and finally became
brown as the development progress within. Before
hatching, the eggs became grayish black.
1.4 Brood collection for seed production
The mature berried females were collected separately
from the ponds by gentle netting. At this stage, the
mean body weight of the prawns ranged between 45 to
80 gm. The collected broods were soaked in 5 ppm
formalin solution for 10 minutes for disinfection as
well as to avoid microbial or parasitic contamination
and then the broods were washed well by freshwater.
The disinfected fresh broods were then transferred to
the Prawn Hatchery. The ripe females were kept in the
separate brood stocking tanks of the hatchery in
accordance with different sex ratios by marking the
tanks. Uncooked rice was fed to the broods in the
brood stocking tanks.
1.5 Hatching of eggs and larvae collection
Berried females were kept in the hatching tanks with
blower for getting proper oxygen. After 2-3 days, larvae
were hatched out. Immediately after hatching, larvae
were collected by a disinfected scoop net and the
collected larvae were shifted in the larvae rearing tanks.
1.6 Feeding and rearing of the larvae
Live artemia was given to the larvae as live feed.
Before supplying the artemia to the larvae rearing tank,
the artemia were treated with 5 ppm formalin solution
for 5 minutes and then washed with 15 ppt saline
water. For the first 7 days only live feed (artemia) was
given to the larvae. After 7 days, custard was given
with artemia. The ingredients of custard included:
powdered milk, egg, multivitamin tablet, white flour,
agar powder, tetracycline tablet, vitamin-C tablet and
cod liver oil tablet. Larvae were reared in 12-15 ppt
water and salinity was maintained constantly at this
level. Water of the larvae rearing tanks exchanged
once daily. Bleaching water was used to wash the
floor of the hatchery daily to avoid any type of
microbial contamination.
1.7 Genomic DNA extraction from larvae
Genomic DNA of the prawn PL was extracted from
tissue by using AccuPrep® Genomic DNA extraction
kit (Bioneer, Korea) following the procedure of Chand
et al., (2005). By using the DNA extraction protocol,
genomic DNA was isolated from 80 different prawn
post larvae (PL). There were 4 experimental groups
and 20 samples were taken from each of the
experimental groups. Extracted DNA was then
preserved at -20ºC until use.
1.8 Primer selection
Five commercially available decamer primers of
random sequence (Operon Technologies, Inc.,
Alameda, CA, USA) were selected for the RAPD
analysis of giant freshwater prawn post larvae
produced from different sex ratios. The 5 different
primers were OPA 1, OPA3, OPA4, OPA9 and OPA10.
Presence of bands for each primer was confirmed on
the basis of intensity and resolution of the bands.
Name of the selected primers, DNA sequence and G-C
content have been presented in the Table 1.
Table 1 Sequence of 5 different primers used for RAPD analysis of prawn
Primer Nucleotide Length Sequence (5’ to 3’) Annealing Temperature TA (℃)
OPA1 10mer CAG GCC CTT C 34
OPA3 10mer AGT CAG CCA C 32
OPA4 10mer AAT CGG GCT G 32
OPA9 10mer GGG TAA CGC C 34
OPA10 10mer GTG ATC GCA G 32