This article explains in detail a systematic approach to assess micro-mineral availability in Atlantic salmon. The methodology includes tools and models with increasing biological complexity: (1) chemical speciation analysis, (2) in vitro solubility, (3) uptake studies in cell lines, and (4) in vivo fish studies.
Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a systematic approach combining different methodologies to assess the availability of zinc (Zn) in Atlantic salmon (Salmo salar). Considering that several Zn chemical species can be present in an Atlantic salmon feed, it was hypothesised that Zn availability is influenced by the Zn chemical species present in the feed. Thus, in this study, the first protocol is about how to extract the different Zn chemical species from the feed and to analyze them by a size exclusion chromatography-inductively coupled plasma mass spectroscopy (SEC-ICP-MS) method. Subsequently, an in vitro method was developed to evaluate the solubility of dietary Zn in Atlantic salmon feeds. The third protocol describes the method to study the impact of changing Zn chemical species composition on the uptake of Zn in a fish intestinal epithelial model using a rainbow trout gut cell line (RTgutGC). Together, the findings from the in vitro methods were compared with an in vivo study examining the apparent availability of inorganic and organic sources of Zn supplemented to Atlantic salmon feeds. The results showed that several Zn chemical species can be found in feeds and the efficiency of an organic Zn source depends very much on the amino acid ligand used to chelate Zn. The findings of the in vitro methods had less correlation with that outcome of the in vivo study. Nevertheless, in vitro protocols described in this article provided crucial information regarding Zn availability and its assessment in fish feeds.
Fish meal and fish oil were traditionally used in Atlantic salmon feed. However, these ingredients are being increasingly replaced by plant-based ingredients1. The aforementioned shift in feed composition has resulted in low dietary availability and an increased need for improving mineral availability in Atlantic salmon feeds, especially zinc (Zn)2. The reduced availability might be a result of a change in the Zn level, Zn chemical species or/and antinutritional factors present in the feed matrix. In this scenario, a new array of additives generically considered as 'organic sources' have emerged with potential of being a better available source of dietary minerals to fish. Therefore, it is important to understand fundamental chemistry and physiology governing the availability of minerals and their sources to fish. Zinc is an essential trace element for all living organisms3. The role of Zn as a signaling molecule has been described at both the paracellular and intracellular level in fish4. In Atlantic salmon, Zn deficiency has been associated with skeletal abnormalities and reduced activity of various Zn metalloenzymes5,6.
This study describes a systematic approach to understand Zn availability by categorizing it into four different compartments of varied chemical and biological complexity. The methods involved are described in four sections, as can be seen in Figure 1: (1) evaluation of Zn chemical species in the soluble fraction of an Atlantic salmon feed using a size exclusion chromatography-inductively coupled plasma mass spectroscopy (SEC-ICP-MS) method7; (2) in vitro solubility of supplemented Zn in Atlantic salmon feed; (3) evaluation Zn chemical species uptake by in vitro intestinal model (RTgutGC)8; and (4) apparent availability of Zn in Atlantic salmon (Salmo salar)9. Similar protocols can be developed for other minerals (e.g., manganese, selenium, copper) of nutritional interest to aquaculture fish species.
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
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).
3. Evaluation of Zn chemical species uptake using an in vitro intestinal model (RTgutGC)
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.
Evaluation of Zn chemical species in the soluble fraction of an Atlantic salmon feed using a SEC-ICP-MS method
The SEC-ICP-MS method provides data about the Zn chemical species found in the soluble fraction of the Atlantic salmon feed. Figure 4 illustrates the chromatographic profile of Zn found in the soluble fraction. This chromatogram was obtained using the SEC-ICP-MS method. Five Zn containing peaks were found in the soluble fractions of the Atlantic salmon feed. Each peak has a different molecular weight; peak one (~ 600 kDa), peak two and peak three (from 32 to 17 kDa), peak four (from 17 to 1.36 kDa) and peak five (> 1.36 kDa). Peak four was the most abundant, followed by peak two, three, five and one, respectively. The Zn chemical species found in the soluble fraction can have different sources because the feed used contains both marine-based and plant-based ingredients, and supplemented form (i.e., Zn sulphate). The molecular weight range of the Zn chemical species suggested that these compounds might be metalloproteins.
In vitro solubility of supplemented Zn in Atlantic salmon feed
Solubility of supplemented 65Zn increased in the presence of amino acids. All the tested amino acids increased the solubility of supplemented 65Zn. Methionine, glycine, cysteine, histidine, and lysine improved 65Zn solubility; higher solubility was found with histidine and lysine (Figure 5).
Evaluation of Zn species uptake using an in vitro intestinal model (RTgutGC)
Apical zinc uptake in RTgutGC cells were significantly influenced by the presence of L-Met or DL-Met at 2 mM concentrations. Furthermore, the impact of methionine on Zn uptake in RTgutGC cells was negatively affected by the presence of BCH (an amino acid transport system blocker), when compared to cells untreated with BCH (Figure 6).
Apparent availability of dietary Zn in Atlantic salmon (Salmo salar)
In practical feeds for Atlantic salmon, apparent Zn availability was the same when supplementing with an inorganic source (Zn sulphate) or an organic source (Zn chelate of glycine). The estimated values for apparent availability of Zn (%, n = 3) in Atlantic salmon were 31% ± 12% when supplementing with an inorganic source (Zn sulphate) and 31% ± 3% when supplementing an organic source (Zn chelate of glycine).
Figure 1: A summary of the systematic approach to assess mineral availability using complementary methods. This approach was used to study zinc availability in Atlantic salmon, including Zn speciation, Zn solubility in intestinal environment, Zn uptake by intestinal cells and Zn apparent availability. Please click here to view a larger version of this figure.
Figure 2: A summary of the procedure for Zn extraction from a feed sample. Zinc is extracted from a feed sample using mild extraction conditions. The extraction is followed by Zn speciation analysis. Please click here to view a larger version of this figure.
Figure 3: An example of the RTgutGC cells 1 h (left) and 1 week (right) after seeding in the cell culture flasks. Please click here to view a larger version of this figure.
Figure 4: A chromatogram showing the Zn-containing peaks from the soluble fraction of Atlantic salmon feed and analyzed by SEC-ICP-MS. The three replicates are characterized by the blue, red and black lines. A molecular weight calibration was performed using thyroglobulin (660 kDa, monitoring 127I), Zn/Cu superoxide dismutase (32 kDa, monitoring 66Zn), myoglobin (17 kDa, monitoring 57Fe), vitamin B12 (1.36 kDa, monitoring 59Co); Peak 1 (P1): ~600 kDa, retention time (RT) 8.2 min; Peak 2+3 (P2+3): from 32 to 17 kDa, RT 14.2 + 15.3 min; Peak 4 (P4): from 17 to 1.36 kDa, RT 16.3 min; Peak 5 (P5): > 1.36 kDa, Rt 23.2 min. Please click here to view a larger version of this figure.
Figure 5: The impact of amino acids on the in vitro solubility of supplemented Zn in Atlantic salmon feed. Data are presented as mean ± SD (n = 3). Data were analyzed through one-way ANOVA, followed by Dunnet’s multiple comparison test, comparing the mean of each AA group with that of control group (No AA). The asterisks denote the level of significance of ANOVA (P-values < 0.05 (*), < 0.01 (**), < 0.001 (***) and < 0.0001 (****)). Please click here to view a larger version of this figure.
Figure 6: The influence of methionine and an amino acid transport inhibitor (2-Aminobicyclo [2.2.1] heptane-2-carboxylic acid, BCH, 10 mM). Data are presented as mean ± SD (n = 3). Data were analyzed through two-way ANOVA, followed by Tukey’s multiple comparison test with p < 0.05 level of significance. Post-hoc differences among groups are represented as superscript letter above the bars; bars with different superscripts are statistically different (p < 0.05). Please click here to view a larger version of this figure.
HPLC settings | |
Column | SEC column (30 cm x 7.8 mm, 5 µm particle size) + guard column (7 µm particle size) |
Calibration range | 1.0 × 104 - 5.0 × 105 Da |
Mobile phase | 50 mM Tris-HCl + 3% MeOH (pH 7.5) |
Flow rate | 0.7 mL min−1 |
Injection volume | 50 μL |
ICP–MS settings | |
Forward power | 1550 W |
Plasma gas flow | 15.0 L min−1 |
Carrier gas flow | 0.86 L min−1 |
Makeup gas flow | 0.34 L min−1 |
Dwell time | 0.1 s per isotope |
Isotopes monitored | 127I, 66Zn, 59Co, 57Fe |
Table 1. An overview of instrument settings for the HPLC and ICP-MS.
Chemical composition (mM) | L15/ex | Experimental medium (L15/FW) |
Sodium nitrate | 155 | 155 |
Potassium nitrate | 6.2 | 6.2 |
Magnesium sulfate | 3.8 | 19.5 |
Calcium nitrate | 1.5 | 5.4 |
HEPES | 5 | 5 |
Magnesium chloride | - | 15 |
Sodium pyruvate | 5.7 | 5.7 |
Galactose | 5.7 | 5.7 |
pH | 7.1 | 7.4 |
Ionic strength | 178 | 258 |
Ionic composition (mM) | ||
Calcium, Ca2+ * | 1.6 ± 0.1 | 5.3 ± 0.2 |
Magnesium, Mg2+ * | 3.9 ± 0.3 | 32.5 ± 0.7 |
Potassium, K+ * | 8.2 ± 1.2 | 8.6 ± 1.1 |
Sodium, Na+ * | 160 ± 3 | 157 ± 2 |
Nitrate, NO3- ** | 164 | 172.4 |
Sulfate, SO4- ** | 3.8 | 18.7 |
Chloride, Cl- ** | 1.5 | 31.5 |
Table 2. The chemical and ionic composition of the experimental media tested.
The intestinal absorption of Zn seems to be influenced by the chemical form of the Zn species13. In this regard, the use of the protocols described in this article allowed the sequentially study of the chemical and biological aspects underlying the 'availability' of Zn in Atlantic salmon.
This study reported the use of a Zn speciation analysis method. The SEC-ICP-MS method provided qualitative data concerning the molecular weight of Zn chemical species present in the soluble fraction of an Atlantic salmon feed. This was achieved by comparison of the retention times of the molecular weight calibration standards (i.e., thyroglobulin (660 kDa), Zn/Cu superoxide dismutase (32 kDa), myoglobin (17 kDa) and vitamin B12 (1.36 kDa)) with the retention times of Zn containing peaks. A challenge found in the Zn speciation analysis was the identification of the unknown Zn chemical species due to lack of analytical standards. In SEC, the separation of the molecules is based on their sizes relative to the pores in the stationary phase. In principle, larger molecules will travel faster, eluting first, and smaller molecules will travel slower, eluting later14. Consequently, each Zn containing peak might contain several compounds with similar molecular weight15. This also contributes to the challenge of identifying unknown Zn chemical species. Moreover, several mild extraction conditions were tested for extraction of Zn. The extracted Zn was low (~10%). Mild extraction conditions were applied to keep the Zn chemical species intact but this may have compromised the extraction efficiency7.
In the in vitro solubility assay, the solubility of supplemented Zn (as radio isotope 65ZnCl2) indicated that the amino acids, especially histidine and lysine, increased the solubility of Zn (Figure 5). Using feed samples directly for in vitro solubility assays under simulated gastrointestinal conditions is based on the knowledge that change in Zn speciation is pH dependent16. However, acidic conditions at the beginning of the GI tract, might result in some change in the speciation which might be irreversible (e.g., ZnO -> ZnCl2, in the presence of HCl under acidic conditions in the stomach). Nevertheless, the Zn source used here is ZnSO4 and the solubility of which was improved by amino acids in the medium. The next question to be answered was, can the increased solubility be translated to availability? The RTgutGC intestinal cell line was used to study this question. In the context of mineral nutrition in animals, the term 'availability' is hard to define and can be regulated differentially in the cells (in vitro) compared to an animal (in vivo). Hence, the term 'uptake' was used when it came to the in vitro evaluation using intestinal cell line. The cell line provided useful information on the Zn uptake mechanisms at the intestinal epithelium which is part of the complex regulatory process which govern mineral availability in animals. The RTgutGC cells elicited a better capacity for apical uptake of Zn in the presence of an amino acid (i.e., methionine; Figure 6). However, the apparent availability in vivo did not significantly differ between inorganic and organic Zn sources in Atlantic salmon. In the in vivo availability study, the Zn source comparison was made at dietary Zn levels well exceeding the known Zn requirements of Atlantic salmon17, total Zn concentration of 150 mg/kg feed. The differences in availability are better visualized when the dietary levels tested fall in the linear dynamic range before the animal reaches saturation. In the present in vivo study, it is possible that the Atlantic salmon were well saturated to observed difference in Zn absorption between sources used.
In summary, the first method provided qualitative information regarding different Zn chemical species found in the soluble fraction of an Atlantic salmon feed; the second method, in vitro solubility of supplemented Zn was improved in the presence of amino acid ligands; the third method confirmed that improved solubility by amino acids can improve uptake at intestinal epithelium; conversely, the fourth method failed to find differences in availability of Zn from inorganic or organic source to Atlantic salmon. To conclude, although not in alignment with the in vivo findings, the in vitro protocols did provide interesting insights into understanding the different components of the Zn availability.
This work was performed under the project APREMIA (Apparent availability and requirement of minerals in Atlantic salmon, grant no. 244490) funded by the Norwegian Research Council.
Name | Company | Catalog Number | Comments |
0.45 µm syringe filter | Sartorius | ||
0.45 μm membrane filter | Pall | ||
10 % fetal bovine serum | Eurobio | ||
1282 Compugamma Laboratory Gamma Counter | LKB Wallac | ||
24 well plates (Falcon, TPP microplates) | Thermo Fisher Scientific | 10048760 | |
2-aminobicyclo(2.2.1)heptane-2-carboxylic acid | Sigma Aldrich | A7902 | |
75 cm2 cell culture flasks (Falcon, TPP tissue culture flasks) | TPP Techno Plastic Products AG | 90075 | |
L-Arginine | Sigma Aldrich | A5006 | |
Bradford assay kit | Bio-Rad | 5000001 | |
Centrifuge | Eppendorf Centrifuge 5702 | ||
L-Cysteine | Sigma Aldrich | 30089 | |
DL-methionine | Alfa Aesar | 59-51-8 | |
D-methionine | Sigma Aldrich | M9375 | |
Experimental fish feeds | Skretting | ||
Glycine | Sigma Aldrich | 410225 | |
Guard column, TSKgel SWxl Type (7 μm particle size) | Tosoh | ||
L-Histidine | Sigma Aldrich | 53319 | |
HPLC coupled with a 7500ce ICP-MS | Agilent Technologies | ||
Hydrochloric acid | Emsure ACS, ISO, 37% w/w, Merck | 1.00317 | |
Knife mill | GM 300, Retsch Gmbh | ||
L-15 medium | Invitrogen/Gibco | 21083027 | |
L-methionine | Sigma Aldrich | M9625 | |
L-Lysine | Sigma Aldrich | 23128 | |
Methanol | LiChrosolv, HPLC grade, Merck | 1.06035 | |
Milli-Q water (18.2 MΩ cm) | EMD Millipore Corporation | ||
Myoglobin | Sigma Aldrich | M1882 | |
NexION 350D ICP-MS | Perkin Elmer | ||
Pasteur pipette | VWR | ||
pH meter | inoLab | ||
Phosphate-buffered saline (PBS) | Sigma Aldrich | 806552 | |
RTgutGC cells | Obtained in kind from Professor Dr. Kristin Schirmer, Dept. of Environmental Toxicology, Eawag, Swiss Federal Institute of Aquatic Science and Technology, Switzerland | ||
SEC column, TSKgel G3000SWxl | Tosoh | ||
Sieve stainless steel (850 μm - 1.12 mm) | Retsch | ||
Sodium dodecyl sulphate (SDS) | Sigma Aldrich | 436143 | |
Superoxide dismutase | Sigma Aldrich | S7571 | |
Thyroglobulin | Sigma Aldrich | T1001 | |
Tricaine methanesulphonate | PharmaQ | ||
Tris(hydroxymethyl)aminomethane | Sigma Aldrich | 252859 | |
Trypsin in 0.25% in phosphate-buffer saline | Biowest | L0910 | |
Versene EDTA solution | Invitrogen/Gibco | 15040-033 | |
Vitamin B12 | Sigma Aldrich | V2876 | |
Zinc chelate of glycine | Phytobiotics | ||
Zinc sulphate | Vilomix |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved