Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon

The feeding trial in section 4 was performed according to Norwegian (FOR-2015-06 - 18-761) and European legislation (Directive 2010/63/EU). 

1. Evaluation of Zn chemical species in the soluble fraction of an Atlantic salmon feed using a SEC-ICP-MS method

1. Extraction buffer (100 mM Tris-HCl, pH 8.5)
   1. Prepare the extraction buffer by dissolving an appropriate amount of tris(hydroxymethyl)aminomethane to reach the desired ionic strength (100 mM) in ultrapure H₂O.
   2. Adjust the pH of the solution to pH 8.5 with HCl solution, monitoring the pH change with a pH meter.
2. Preparation of feed samples
   NOTE: The feed sample used was formulated based on commercial feed for Atlantic salmon, containing protein sources mainly from plant-based ingredients (i.e., approximately 5% fish protein, 10% fish oil, 68% plant-based protein and 12% plant-based oil). Zinc sulphate was supplemented to the feed.
   1. Grind the feed sample by hand using a pestle and a mortar.
   2. Sieve the feed sample to ensure that the extraction is performed in a feed fraction with similar particle size (from 850 µm to 1.12 mm).
   3. Proceed to perform the Zn extraction.
3. Zinc extraction from a feed sample
   1. Weigh approximately 0.5 g of feed in triplicate into 15 mL conical tubes.
   2. Add the extraction buffer (5 mL of 100 mM Tris-HCl, pH 8.5) to the samples.
   3. Extract the samples in a rotator (20 rpm) at 4 °C for 24 h.
   4. Separate the soluble and non-soluble fractions by centrifugation for 10 min at 3000 x g.
   5. Use a 0.45 µm disposable syringe filter to filter the soluble fraction.
   6. Transfer the filtered samples to clean tubes.
   7. Perform the Zn speciation analysis in the soluble fractions using SEC-ICP-MS, as described in step 1.6.
      NOTE: A summary of the procedure for Zn extraction from a feed sample is described in Figure 2.
4. Mobile phase solution (50 mM Tris-HCl + 3% MeOH, pH 7.5)
   1. Prepare the mobile phase solution dissolving 6.057 g of tris(hydroxymethyl)aminomethane in 1 L of 3% MeOH solution (v/v).
   2. Adjust the pH of the solution to pH to 7.5 with HCl solution, monitoring the pH change with a pH meter.
   3. Filter the mobile phase solution through a 0.45 μm membrane filter.
5. Molecular weight calibration of the SEC column separation range
   1. Calibrate the separation range by performing a molecular weight calibration.
      NOTE: In this study thyroglobulin (660 kDa), Zn/Cu superoxide dismutase (32 kDa), myoglobin (17 kDa) and vitamin B12 (1.36 kDa) were used.
   2. Prepare each of the standards with a known concentration in ultrapure H₂O.
   3. Prepare a high-performance liquid chromatography (HPLC) vial by adding 250 µL of standard to a vial.
   4. Load the vials with standards to the sequence run of the samples.
   5. Run the molecular weight calibration at the beginning and at the end of the analytical sequence, monitoring ¹²⁷I (thyroglobulin), ⁶⁶Zn (Zn/Cu superoxide dismutase), ⁵⁷Fe (myoglobin) and ⁵⁹Co (vitamin B12).
      NOTE: The molecular weight calibration is simultaneously performed with the Zn speciation analysis.
6. Zinc speciation analysis using SEC-ICP-MS
   NOTE: The Zn speciation analysis by the SEC-ICP-MS method was developed based on principles described elsewhere^10,11 and further optimization was performed for the analysis of an Atlantic salmon feed⁷.
   1. Perform the Zn speciation analysis on the soluble fractions by using a size exclusion chromatography (SEC) column and an HPLC coupled with inductively coupled plasma mass spectroscopy (ICP-MS).
   2. Prepare an HPLC vial by adding 250 µL of soluble fraction to a vial.
   3. Before analysis, spike all the samples with 0.5 µL of vitamin B12. This step allows to correct for retention times shifts, monitoring ⁵⁹Co.
   4. Dilute the soluble fraction with extraction buffer (100 mM Tris-HCl, pH 8.5) and adjust to a final volume of 1 mL.
   5. Prepare the sequence run of the samples in random order.
   6. Tune the ICP-MS according to manufacturer’s instructions.
   7. Follow the instrument settings for the HPLC and ICP-MS performing the Zn speciation analysis (see Table 1).

2. In vitro solubility of supplemented Zn in Atlantic salmon feed

NOTE: The feed sample used was formulated based on commercial feed for Atlantic salmon, containing protein sources mainly from plant-based ingredients (i.e., approximately 5% fish meal, 10% fish oil, 68% plant-based ingredients and 12% vegetable oil).

1. Grind the Atlantic salmon feed samples for 10 s at 3000 rpm using a knife mill and store at 4 °C until further analysis.
2. Weigh ~0.2 g of the feed samples ground in step 2.1 and add Zn radiotracer (⁶⁵Zn) of known specific activity in a 5 mL volume sample tube (with a cap).
   CAUTION: This procedure must be performed inside a radionuclide suite. The person performing this step should be trained and certified to handle radio isotopes. The safety and precautionary measures advised by the radiation safety administration of the institute must be strictly followed.
3. Then prepare the freshwater intestinal luminal buffer solution as described below.
   1. For salt solution A, weigh 11.65 g of NaNO₃, 0.55 g of KNO₃ and 0.4 g of MgSO₄. Dissolve the salts in ultrapure H₂O and adjust to a final volume of 60 mL.
   2. For salt solution B, weigh 0.31 g of Ca(NO₃)₂·₄H₂O. Dissolve the salts in ultrapure H₂O and adjust to a final volume of 10 mL.
   3. Use 500 mM HEPES stock solution as salt solution C.
   4. For salt solution D, weigh 1.2 g of MgCl₂, dissolve the salt in ultrapure H₂O and adjust to a final volume of 20 mL.
   5. For salt solution E, weigh 0.9 g of MgSO₄, dissolve the salt in ultrapure H₂O and adjust to a final volume of 20 mL.
   6. Prepare pyruvate solution by dissolving 0.55 g of CH₃COCOONa in 10 mL of ultrapure H₂O.
   7. Dissolve 0.9 g of galactose (C₆H₁₂O₆) in ultrapure H₂O.
   8. Dissolve well using a magnetic stirrer and sterilize salt solutions A, B, D and E by autoclaving, and solution C, pyruvate and galactose by filtration through a 200 µm syringe filter.
   9. After preparation of the different stock solutions, to prepare 100 mL of working solution of the buffer, mix the above prepared solutions in the following proportion: 6.8 mL of salt solution A, 4.14 mL of B, 5 mL of C, 2.5 mL of D, 1.5 mL of E, and 1.14 mL each of pyruvate and galactose. Make up the volume to 100 mL using deionized water.
      NOTE: The above buffer will now represent the ionic composition of the intestinal lumen found in freshwater salmonids.
4. Prepare six other aliquots of the buffer described in step 2.6 and add one of the following amino acids (cysteine, methionine, glycine, histidine, lysine and arginine) to reach a final molar concentration of 5 mM. 
5. Add the freshwater intestinal luminal buffer (reaction volume = 3 mL; pH 7.4) to the feed sample.
6. Repeat step 2.5 with the buffers described in 2.4 (in the presence of different amino acids at 5 mM concentration).
7. Close the tubes and allow them to spin in a rotary spinner for 30 min at 25 rpm.
8. Separate the soluble and non-soluble fractions by centrifuging for 10 min at 1157 x g.
9. Use a gamma teller to measure the counts per minute (cpm) of ⁶⁵Zn in the soluble and non-soluble fractions.
10. Calculate the proportion of the radio isotopes of Zn (⁶⁵Zn) present in the soluble and non-soluble fractions.

3. Evaluation of Zn chemical species uptake using an in vitro intestinal model (RTgutGC)

1. RTgutGC cells culture
   NOTE: All working materials used in this step should be sterile.
   1. Revive the frozen RTgutGC cells gently in a water bath set at 20 °C.
   2. Gently pipette out the solution containing the cells and suspend them in 10 mL of L15 medium containing 10% fetal bovine serum (FBS).
      NOTE: 10% FBS is only used for reviving the frozen cells. For subsequent passage, FBS is used at 5%. The composition of FBS can vary between batches, so it is advisable to buy and stock as much as required from a single batch to avoid inter-batch variations in the serum composition.
   3. Add the cell suspension to 75 cm² cell culture flasks and incubate in an incubator set at 19 °C under normal atmosphere.
   4. Check the cells and when confluent (80% confluence, assess visually by examining the density of the cell surface under a microscope), split the cells to new flasks (subsequent passage) or harvest to use in experiments.
      NOTE: An example of the RTgutGC cells 1 h and 1 week after seeding to the cell culture flasks is shown in Figure 3.
2. Cell harvest and preparing for exposure experiments
   1. Wash cells twice with 1 mL ethylenediaminetetraacetic acid (EDTA) solution. After each wash, siphon out the EDTA solution using a sterile suction tube.
   2. Treat the cells with trypsin (0.7 mL of trypsin, in 0.25% in phosphate-buffered saline [PBS]).
   3. Gently rotate the flask at acute angles to spread the trypsin all along the surface of the flask.
   4. Continue rotation for 2 min, while the cells detach.
   5. After gently rotating for 2 min, add 10 mL of L15/FBS medium to neutralize trypsin.
   6. Decant the resulting cell suspension into a conical bottom centrifuge tube using a sterile pipette and centrifuge for 3 min at 130 x g.
   7. Determine the density of the harvested cells by manual counting using hemocytometer.
   8. Add required volume of L15/FBS medium to attain a cell density of 5 x 10⁴ cells/mL.
   9. Seed the cells onto 24-well plates by pipetting 1 mL of cell suspension per well to attain the final cell density of 5 x 10⁴ cells/well.
      NOTE: Preferably use multi-dispensing pipettes to minimize variation and reduce time.
   10. Place the seeded plates in an incubator under normal atmosphere at 19 °C for 48 h prior to experiments.
       NOTE: Trypsin to be stored at -20 °C; EDTA solution and L15/FBS media at 4 °C. Adjust temperature of all working solutions and media to 19 °C just before use.
3. Preparation of exposure media
   NOTE: This step has to be done under the fume hood under aseptic and sterile conditions.
   1. Prepare L15/ex by mixing 6.8 mL of salt solution A (step 2.3.1), 1.14 mL of B (step 2.3.2), 5 mL of C (step 2.3.3), and 1.14 mL each of pyruvate (step 2.3.6) and galactose (step 2.3.7). Make up the volume to 100 mL using sterile cell culture-grade distilled water.
   2. Prepare FW by mixing 6.8 mL of salt solution A (2.3.1), 4.14 mL of B (2.3.2), 5 mL of C (2.3.3), 2.5 mL of D (2.3.4), 1.5 mL of E (2.3.5), and 1.14 mL each of pyruvate (2.3.6) and galactose (2.3.7). Make up the volume to 100 mL using sterile, cell culture-grade distilled water.
   3. Quantify the concentrations of ions in the exposure media using ICP-MS as described elsewhere¹².
      NOTE: Ionic concentrations analyzed in the media preparations are presented in Table 2.
4. Zinc (⁶⁵Zn) influx assays
   1. Seed the RTgutGC cells onto 24-well plates (5 x 10⁴ cells/well) in complete L15/FBS medium.
   2. Incubate for 48 h in an incubator with normal atmosphere at 19 °C.
   3. Adjust all experimental media preparations to pH 7.4 using 0.5 M NaOH in the presence or absence of L-methionine (L-Met) or DL-methionine (DL-Met) at 2 mM concentration.
      NOTE: The pH adjustment of the buffers described in 3.4.3 needs to be done freshly before treatment of the cells in step 3.4.5.
   4. After the completion of the incubation period, remove the medium from the wells, and rinse thoroughly with PBS.
   5. Add the pH-adjusted FW experimental media, and allow to acclimatize for 20 min.
   6. Expose the RTgutGC cells to nominal concentrations of 3.07, 6.14, 12.27 and 24.55 µM ⁶⁵Zn(II) (as ZnCl₂; ~4 kBq/mL) in the media described in step 3.4.3.
   7. Immediately after, keep the cells in the incubator at 19 °C for 15 min.
   8. After the 15 min incubation is over, aspirate the culture supernatant and remove from the well.
   9. Rinse the cells with ice cold FW medium (with 200 µM Zn, pH 7.4) and then quench by adding the 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) buffer (pH 7.4) for 5 min, to get rid of any adsorbed ⁶⁵Zn(II).
   10. Expose the cells to the above media preparations in the presence or absence of 10 mM 2-Aminobicyclo [2.2.1] heptane-2-carboxylic acid (BCH), an amino acid transport inhibitor.
   11. After the exposure period of 15 min, repeat steps 3.4.8 and 3.4.9.
   12. The RtgutGC cells will be adhered to the bottom of the wells as a monolayer. Digest the cells by using 0.2% hot sodium dodecyl sulfate (SDS) detergent (100 µL/well).
       NOTE: The SDS solution needs to be placed for 1 h in a water bath set to 90 ˚C prior to use.
   13. Aspirate and recover the cell digestate into a 1.5 mL tube.
   14. Measure radioactivity of the cell digests using a gamma counter.
       NOTE: The counts per minute (cpm) needs to be corrected for radioactive decay, background activity and are subjected to specific activity calculations following the formulae described by Glover and Hogstrand¹³.
   15. To quantify the protein concentration of the cells, homogenize the cells with 500 µL of 0.5 M NaOH.
   16. Use a Bradford assay kit to measure the protein concentration in the cell sample, with bovine serum albumin (BSA) as the standard.
       NOTE: Once the protein concentration is quantified, the rate of Zn uptake by RTgutGC cells can be expressed as pmoles Zn min^-1 mg^-1 protein.

4. Apparent availability of dietary Zn in Atlantic salmon (Salmo salar)

NOTE: The Atlantic salmon feeds were formulated based on commercial feeds, containing protein sources mainly from plant-based ingredients (i.e., approximately 5% fish protein, 10% fish oil, 68% plant-based protein and 12% plant oil). Two feeds were supplemented with an inorganic source (Zn sulphate) or an organic source (Zn chelate of glycine) to achieve a Zn concentration of 150 mg/kg of feed. In addition, Yttrium oxide (feed grade) was added to the feed at 0.01% as the inert marker to enable calculation of apparent availability coefficient.

1. Acclimatize the Atlantic salmon (SalmoBreed strain, age 1+ years, mixed-sex groups) in their respective tanks until the fish are used to the experimental conditions.
2. Assess the acclimation of the Atlantic salmon by monitoring their daily feed intake.
   NOTE: This trial was performed in triplicate tanks, thus a total of six tanks were used. During the feeding trial the water temperature was 11.9 ± 0.3 °C and dissolved oxygen saturation was 101 ± 5%.
3. Feed the fish with experimental feeds for 11 days.
4. Euthanize the fish by overdose using 6 mL of tricaine methanesulphonate stock solution per liter of water.
5. Collect a pooled sample of feces from the fish from the same tank into a plate by striping from the ventral fin to anus.
6. Remove the feces from the plate with a spatula into a 50 mL conical tube and immediately store the samples at -20 °C.
   NOTE: The samples were kept at -20 °C until further analysis.
7. Freeze dry the feces samples for 72 h at -80 °C.
8. Manually homogenize manually the feces sample into a fine powder using a pestle and mortar.
9. Determine the concentration of Zn and Yttrium in the feed and feces samples using an ICP-MS (as described elsewhere⁹).
10. Determine apparent availability coefficient (AAC, %) using the following formula: