Papel de las actividades superóxido dismutasa y catalasa en la ...
Papel de las actividades superóxido dismutasa y catalasa en la ...
Papel de las actividades superóxido dismutasa y catalasa en la ...
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2.2. Fish and experim<strong>en</strong>tal <strong>de</strong>sign<br />
S<strong>en</strong>egalese sole specim<strong>en</strong>s (Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup, 1838) of 15-30 g weight,<br />
were kept in four 300 l seawater tanks (75 fish per tank), 36 ‰ salinity, at 22 ºC and fed<br />
with a commercial pellet diet (Gemma 0.5, Skreeting, Trouw España, Nutreco, Burgos,<br />
Spain). Specim<strong>en</strong>s were sacrificed by overdose of c<strong>la</strong>ve oil.<br />
Experim<strong>en</strong>tal diets were prepared in the <strong>la</strong>boratory from the commercial pellet<br />
diet. The diet preparation was carried out with alginate (0.5% kg -1 ) and 50 mM calcium<br />
chlori<strong>de</strong> (0.4% kg -1 ) to facilitate the lyophilized bacteria incorporation to the pellet.<br />
Alginate and Ca Cl 2 were sprayed into the feed slowly, mixing with the required amount<br />
of lyophilized bacteria (10 9 cfu g -1 ).<br />
Thus, fish in each tank received one of four differ<strong>en</strong>t diets: a commercial diet<br />
supplem<strong>en</strong>ted with Pdp11 (10 9 cfu g -1 ) (diet A); the same diet supplem<strong>en</strong>ted with Pdp13<br />
(10 9 cfu g -1 ) (diet B); a diet consisting of the commercial diet (control without alginate)<br />
(diet C); and, finally, the fourth group of fish received a diet supplem<strong>en</strong>ted with alginate<br />
and calcium chlori<strong>de</strong> (control with alginate) (diet D). Fish were fed at a rate of 15 g dry<br />
diet Kg -1 biomass (1.5 %) per day for 60 days. The biomass of the fish in each aquarium<br />
was measured at the beginning of the experim<strong>en</strong>t and daily ratio was adjusted<br />
accordingly. No mortality was observed during the experim<strong>en</strong>t.<br />
Sampling was carried out at the <strong>en</strong>d of the feeding trial, day 60, for the chall<strong>en</strong>ge<br />
test and study of intestinal microbiota. Samples for measurem<strong>en</strong>t of the respiratory burst<br />
activity were tak<strong>en</strong> at the middle of the trial and at the <strong>en</strong>d, at day 30 and 60,<br />
respectively.<br />
2.3. Iso<strong>la</strong>tion of head kidney phagocytes<br />
The influ<strong>en</strong>ce of the treatm<strong>en</strong>t on respiratory burst activity was tested in<br />
phagocytes iso<strong>la</strong>ted from the kidney of soles following the technique <strong>de</strong>scribed by<br />
Secombes [45]. Briefly, the kidney was removed aseptically and pushed through a 100<br />
μm nylon mesh with Leibovitz medium (L-15) containing 2% foetal calf serum (FCS,<br />
Sigma), 1% p<strong>en</strong>icillin-streptomycin (Sigma), 0.1 % (5 mg ml -1 ) g<strong>en</strong>tamicine (Sigma)<br />
(P/S/G) and 10 U heparine ml -1 . This cell susp<strong>en</strong>sion was <strong>la</strong>yered on a 30 to 51%<br />
Percoll (Amersham) gradi<strong>en</strong>t and c<strong>en</strong>trifuged at 600 × g for 30 min. Th<strong>en</strong>, the bands<br />
5