International Journal of Aquaculture, 2016, Vol.6, No.2, 1
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2.2 Bacterial suspensions
The lactic acid bacteria (LAB) were grown daily in 400 mL LAPTg broth (Raibaud et al., 1963) and incubated at
37
℃
in static conditions for 8 h. Bacterial cells were harvested by centrifugation at 3,000 g for 10 min at 4
℃
,
washed twice with sterile distilled water and suspended to obtain the required viable cells concentration.
2.3 Live food preparation
One L freshwater containing a suspension of 1 g of brine shrimp cysts
Artemia
sp., 15 g of NaCl and 2 g of
sodium bicarbonate was submitted to an incubation process under intense aeration and lightening for 24 h. Live
hatched nauplii were harvested from the bottom of the hatching device by sedimentation, filtered to obtain those
between 250 and 150 µm size and suspended in freshwater. Aliquots of the nauplii suspension previously obtained
were counted in order to obtain their approximate number and concentration of the solution (Torrentera and Tacon,
1989).
2.4 Fish reproduction
P. mesopotamicus
larvae were obtained by controlled reproduction from broodstock of the Instituto de Ictiologia
del Nordeste (Corrientes, Argentina). Spawning was induced by injection of pituitary extract from the of
Prochilodus lineatus
(5 and 1.5 mg pituitary gland kg
-1
of body weight in females and males, respectively)
according to Da Silva et al.(1988). The sexual gametes were obtained by the stripping technique, mixed
immediately in a bowl, suspended into fresh water for 5 minutes, washed twice and suspended at the desired
concentration (Gómez et al., 2014). Aliquots of 1 mL were counted in order to obtain an approximate
concentration of fecundated eggs.
2.5 Experimental design and sampling
Experimental units were settled as 5 L plastic fishbowls with a constant recirculation system and approximately
300 fecundated eggs. The formulae were administered in three different concentrations, named as: 4 (6x10
4
CFU
L-1), 7 (6x10
7
CFU L-1) and 10 (6x10
10
CFU L-1) of each strain. Also at different stages of the biological cycle
of larvae: E (from the time of fecundation of the eggs until the beginning of the exogenous feeding, 5 days), L
(from the beginning of the exogenous feeding until the end of the assay in the laboratory, from day 5 up to day 15)
and E&L (from the fecundation of the eggs until the end of the assay in laboratory, from day 0 up to day 15).
Control units (CTRL) were performed with no addition of bacteria.
During stage E the bacterial suspension was added directly to the fish bowls four times a day with previous stop of
water recirculation which was restarted one hour after. In stage L, bacteria were co-incubated with live food
during two hours previous to administration; this procedure was performed
ad libitum
four times a day. Water
recirculation was interrupted before alimentation and restored one hour after. Water quality determinations of pH,
dissolved oxygen and temperature were performed daily in each experimental unit.
2.6 Sampling
At day 15, larvae were counted and weighed to obtain values of survival, mean weight and biomass. To evaluate
the normal development, macroscopic evaluations were performed with binocular magnifier Optical Kyowa
model SDZ. For histological studies, 10 larvae per treatment were collected and anaesthetized by chilling on ice,
fixed in Bouin’s solution (saturated picric acid 3000 ml, formaldehyde 1000 ml, glacial acetic acid 200 ml) for 12
h, washed twice with alcohol 70° and maintained in this solution until processing. Samples were then routinely
processed for histology, stained with haematoxylin and eosin and analyzed by light microscopy using a Leica
DM500 microscope, a Leica ICC50 digital camera and the Leica Application Suite 3.4.1 image analysis system
(Culling, 1974).
The procedures and experimental protocols applied to the animals in this work were in accordance with the ethical
principles of animal experimentation, and approved according to protocol n.0019/14-2011-02204 and
