Papel de las actividades superóxido dismutasa y catalasa en la ...

2.2. Fish and experimental design
Senegalese sole specimens (Solea senegalensis, Kaup, 1838) of 15-30 g weight,
were kept in four 300 l seawater tanks (75 fish per tank), 36 ‰ salinity, at 22 ºC and fed
with a commercial pellet diet (Gemma 0.5, Skreeting, Trouw España, Nutreco, Burgos,
Spain). Specimens were sacrificed by overdose of clave oil.
Experimental diets were prepared in the laboratory from the commercial pellet
diet. The diet preparation was carried out with alginate (0.5% kg -1 ) and 50 mM calcium
chloride (0.4% kg -1 ) to facilitate the lyophilized bacteria incorporation to the pellet.
Alginate and Ca Cl 2 were sprayed into the feed slowly, mixing with the required amount
of lyophilized bacteria (10 9 cfu g -1 ).
Thus, fish in each tank received one of four different diets: a commercial diet
supplemented with Pdp11 (10 9 cfu g -1 ) (diet A); the same diet supplemented with Pdp13
(10 9 cfu g -1 ) (diet B); a diet consisting of the commercial diet (control without alginate)
(diet C); and, finally, the fourth group of fish received a diet supplemented with alginate
and calcium chloride (control with alginate) (diet D). Fish were fed at a rate of 15 g dry
diet Kg -1 biomass (1.5 %) per day for 60 days. The biomass of the fish in each aquarium
was measured at the beginning of the experiment and daily ratio was adjusted
accordingly. No mortality was observed during the experiment.
Sampling was carried out at the end of the feeding trial, day 60, for the challenge
test and study of intestinal microbiota. Samples for measurement of the respiratory burst
activity were taken at the middle of the trial and at the end, at day 30 and 60,
respectively.
2.3. Isolation of head kidney phagocytes
The influence of the treatment on respiratory burst activity was tested in
phagocytes isolated from the kidney of soles following the technique described by
Secombes [45]. Briefly, the kidney was removed aseptically and pushed through a 100
μm nylon mesh with Leibovitz medium (L-15) containing 2% foetal calf serum (FCS,
Sigma), 1% penicillin-streptomycin (Sigma), 0.1 % (5 mg ml -1 ) gentamicine (Sigma)
(P/S/G) and 10 U heparine ml -1 . This cell suspension was layered on a 30 to 51%
Percoll (Amersham) gradient and centrifuged at 600 × g for 30 min. Then, the bands
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