We studied the availability of solubility of micro-metals like zinc selenium with different chemical forms or sources, as we call it, in the context of changing feed composition. In a radionuclide suite, weigh out approximately 0.2 grams of ground Atlantic salmon feed samples, and mix it with a zinc radiotracer of known specific activity in a five milliliter volume sample tube. Prepare six other aliquots of the buffer and add one of the amino acids shown here to each to reach a final molar concentration of five millimolar.
Add freshwater intestinal luminal buffer to feed the sample. Next, add the freshwater intestinal luminal buffer to the buffer aliquots containing the amino acids. Close the tubes and spin them on a rotary spinner at 25 RPM for 30 minutes.
Then, centrifuge at 1, 157 x g for 10 minutes, to separate the soluble and non soluble fractions. Use a gamma teller to measure the counts per minute of the zinc radiotracer in the soluble and non soluble fractions. Wash the rainbow trout derived intestinal cell line twice with one milliliter of EDTA solution, and siphon out the solution after each wash using a pipette.
Treat the washed cells with 0.25%trypsin solution. Gently rotate the flask at acute angles for two minutes. Then add 10 milliliters of L-15 medium containing 5%FBS to neutralize the trypsin.
Using a sterile pipette, decant the resulting cell suspension into a conical bottom centrifuge tube. Centrifuge at 130 x G for three minutes. Then using a hemocytometer, determine the density of the harvested cells by manual counting.
Seed the cells onto 24 well plates at a cell density of 50, 000 cells per milliliter, per well, and incubate the plates at 19 degrees Celsius under a normal atmosphere for 48 hours prior to the experiments. After feeding the fish for 11 days and euthanizing them, collect a pooled sample of the fish's feces by stripping from the ventral fin to the anus and place it on a plate. Use a spatula to transfer the feces from the plate into a 50 milliliter conical tube.
Immediately store the samples at 20 degrees Celsius. In this study, the mineral availability of fish feed is assessed using a number of complimentary methods. Representative analysis of Atlantic salmon feed via the size-exclusion chromatography inductively-coupled plasma mass spectrometry method, provides data about the zinc chemical species found in the soluble fraction.
Five zinc containing peaks can be seen in this fraction, each with a different molecular weight. The zinc chemical species found in the soluble fraction can have different sources, because the feed used contains both marine-based and plant-based ingredients, and is supplemented. The molecular weight range of these species suggest that these compounds might be metalloproteins.
While the solubility of supplemented zinc 65 increases the presence of all the tested amino acids, higher solubility is found with histidine and lysine. Apical zinc uptake in Arteca GC cells is significantly influenced by the presence of L-Met or DL-Met at two millimolar concentrations. Furthermore, the impact of methionine on zinc uptake is negatively affected by the presence of BCH.
The main advantage of this protocol is we tried to combine different approaches in visual, analytical, and invivo studies, to compare and compliment each other when it comes to studying mineral availability.
