2.3. Sampling
At the end of the experimental period, fish weighed 163–186 g,
with no significant differences between groups (Pérez-Jiménez et al.,
in press). Fish feeding was discontinued 24 h before sampling and
three animals per tank (nine per treatment) were randomly sampled
and killed with a sharp blow in the head. The digestive tract was
excised and divided into four sections: stomach, pyloric caeca and
anterior and posterior intestine. It was checked that therewas no food
in any portion. All digestive sectionswere immediately frozen in liquid
nitrogen, and stored at −80 °C. Each part was homogenized in nine
volumes of ice-cold 100 mM Tris–HCl buffer containing 0.1 mM EDTA
and 0.1% (v/v) Triton X-100, pH 7.8. All procedureswere performed on
ice. Homogenates were centrifuged at 30,000 g for 30 min at 4 °C and
the resultant supernatants were kept in aliquots and stored at−80 °C
for further digestive enzyme assays.
2.3. Sampling
At the end of the experimental period, fish weighed 163–186 g,
with no significant differences between groups (Pérez-Jiménez et al.,
in press). Fish feeding was discontinued 24 h before sampling and
three animals per tank (nine per treatment) were randomly sampled
and killed with a sharp blow in the head. The digestive tract was
excised and divided into four sections: stomach, pyloric caeca and
anterior and posterior intestine. It was checked that therewas no food
in any portion. All digestive sectionswere immediately frozen in liquid
nitrogen, and stored at −80 °C. Each part was homogenized in nine
volumes of ice-cold 100 mM Tris–HCl buffer containing 0.1 mM EDTA
and 0.1% (v/v) Triton X-100, pH 7.8. All procedureswere performed on
ice. Homogenates were centrifuged at 30,000 g for 30 min at 4 °C and
the resultant supernatants were kept in aliquots and stored at−80 °C
for further digestive enzyme assays.
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