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

Feeding assays were carried out with soles of 80 g mean body weight, which
were randomly separated into six experimental groups, and stocked in six 250 l tanks
(20 fish per tank) with similar culture conditions to those described above.
The diet assayed was prepared in the laboratory from the commercial pellet diet
routinely used in fish farms (Skreeting, Trouw España, Nutreco, Burgos, Spain).
Briefly, the commercial pellet diet was crushed and mixed with tap water before adding
the lyophilized alga Porphyridium cruentum at the desired concentration (10 g kg -1 ), and
then made into pellets again. The re-made pellets were allowed to dry and stored at 4 ºC
until use.
The commercial pellet Sanostim (Skreeting, Trouw España, Nutreco, Burgos,
Spain), containing β-glucans, was used to test the response of sole phagocytes. Two
groups of fish received daily one of the different diets assayed: diet consisting of nonsupplemented
commercial diet (control group); diet composed of the commercial diet
containing immunostimulant Sanostim; and finally, a commercial diet supplemented
with lyophilized alga (1%). Fish were fed at a rate of 20 g dry diet kg -1 biomass (2 %)
per day for 4 weeks. The biomass of the fish in each aquarium was measured before the
experiment and daily ration, being adjusted accordingly. No mortality was observed
during the experiment.
2.5. Immunization assay
Two weeks after beginning the feeding trial, fish from one tank per treatment
were intraperitoneally inoculated with a bacterin of P. damselae subsp. piscicida. The
formalin-killed aqueous vaccine was prepared with a virulent strain of P. damselae
subsp. piscicida (Lg h411/01 ) isolated from diseased sole [16] according to the following
protocol. Briefly, bacteria were cultured on TSAs for 24 h and one colony was
inoculated in tubes containing 5 ml of TSBs. After 18 h incubation at 22 ºC, an aliquot
of the culture, 50 μl, was inoculated in flasks with 50 ml TSBs and incubated at 22ºC
with continuous shaking. After 18 h incubation the culture achieved O.D. 600 of 1.2.
The total bacterial number was counted, obtaining a bacterial concentration of 6 x 10 8
bacteria ml -1 . Then bacterial cells were killed by addition of formaldehyde to achieve
1% final concentration, and overnight incubation. Sterility tests were performed by
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