https://orcid.org/0000-0003-3320-2381
ABSTRACT
This study was carried out to investigate the effect of different iron sources on haemoglobin and myoglobin (Mb) synthesis, and the mRNA expression of the key genes related to iron metabolism in the skeletal muscle of piglets. Forty-eight piglets were assigned into four treatments including control group, ferrous sulphate (FeSO4) group, ferrous glycinate (Fe-Gly) group and amino acid-Fe(II)-chelator complexes group. The results showed that iron supplementation increased Mb concentration in muscles (P < 0.05). In addition, iron supplementation increased hephaestin (HEPH) expression in the longissimus dorsi muscle (P < 0.1), decreased transferrin receptor 1 (TFR1) expression in the longissimus dorsi muscle (P < 0.05) and increased lipocalin 2 (Lcn2) expression in gastrocnemius muscle, respectively (P < 0.05). In summary, compared with high-dose inorganic iron, low-dose organic iron supplements have the same effects in haemoglobin and myoglobin biosynthesis, and mobilize iron in muscles into circulation.
Introduction
Iron can synthesize haemoglobin and myoglobin (Mb), which are essential for animals (Philip et al., Citation2020). Haemoglobin is essential in transporting and storing oxygen (Wu et al., Citation2020) and Mb can protect the muscle from oxidative damage. Iron deficiency could impair haemoglobin production (Edison et al., Citation2008) and Mb synthesis (Hagler et al., Citation1981).
As the most widely distributed tissue in the body, the skeletal muscle is an important site to regulate whole body metabolism (Hargreaves & Cameron-Smith, Citation2002). Previous studies found iron could stimulate red muscle fibre production (Ponnampalam et al., Citation2019; Stugiewicz et al., Citation2016) and skeletal muscle protein synthesis (Higashida et al., Citation2020). Iron deficiency can decrease protein synthesis in the skeletal muscle (Higashida et al., Citation2021) and promote the atrophy of skeletal myocytes (Kobak et al., Citation2018). Therefore, normal iron metabolism in muscle could be a potential therapeutic option for muscle wasting diseases (Wyart et al., Citation2022).
In animal production, ferrous sulphate (FeSO4) is usually supplemented as the standard inorganic iron source (Zhuo et al., Citation2019). However, more studies found that inorganic iron had poor bioavailability than organic iron (Fang et al., Citation2013; Ma et al., Citation2012; Zhuo et al., Citation2014). Zhuo et al. (Citation2014) found that ferrous glycinate (Fe-Gly) could be absorbed more and utilized faster than FeSO4 (Zhuo et al., Citation2014). The mechanism might be due to the differences in both absorption, transport and utilization processes (Zhuo et al., Citation2016). In addition, amino acid-Fe(II)-chelator complexes have been proposed as a superior iron supplement for increasing iron absorption in recent years (Zhao et al., Citation2021a).
Some important genes related to the iron metabolism had higher expression in the skeletal muscle (Polonifi et al., Citation2010). In these genes, divalent metal-ion transporter (DMT) −1 is the major iron transporter (Yanatori & Kishi, Citation2019), transferrin receptor (TFR) 1 can regulate iron entry into cells (Gammella et al., Citation2017; Zhang et al., Citation2020), and hemochromatosis (HFE) influences iron homeostasis in cells (Enns, Citation2006). Therefore, in this study, our objective was to investigate whether the low-dose organic iron had the same or higher bioavailability on haemoglobin and Mb synthesis, and the mRNA expression of the genes is key to iron metabolism than the high dose inorganic iron in the skeletal muscle of piglets.
Materials and methods
Experimental design
Forty-eight piglets (Duroc × Large White × Landrace, barrows, 9.39 ± 1.55 kg, 40 ± 2 d) from different littermates were assigned into four treatments including control group, a basal diet without iron supplemented in mineral premix; the FeSO4 group, the basal diet supplemented with FeSO4 to provide 100 mg supplemental Fe/kg dry matter (DM); the Fe-Gly group, the basal diet supplemented with Fe-Gly to provide 80 mg supplemental Fe/kg DM and the amino acid-Fe(II)-chelator complexes group, the basal diet supplemented with amino acid-Fe(II)-chelator complexes to provide 30 mg supplemental Fe/kg DM. There were six pens for each treatment, and each pen had two piglets. The basal diet was corn-soybean diets and prepared to meet or exceed NRC (Citation2012) nutrient requirement (NRC, Citation2012). All piglets were allowed ad libitum access to water and feed during a 28-day experimental study after the four adaptation days. The Animal Care and Use Committee of Wuhan Polytechnic University (No. WHPU202111028, Wuhan, China) approved the animal use protocol for this research.
Blood collection
After 12-h of fasting, blood samples were collected into 10-mL heparinized vacuum tubes on day 28. Haematological parameters in blood were analysed by automated haematology analysers (ADVIA 2120i, Siemens Healthcare Diagnostics Inc, USA).
Meat colour
On day 28, after the piglets were slaughtered, the gastrocnemius muscle under the semitendinosus and biceps flexor cruris (left hind leg) and longissimus dorsi muscle was removed. The colour parameters of meat were presented in the CIE L∗a∗b∗ colour scale, using the colour meat instrument (OPTO-STAR, German) with illuminant D65 and 50 mm viewing port. Results were expressed as L∗ (lightness), a∗ (redness) and b∗ (yellowness). Then the muscles were frozen in liquid nitrogen immediately and stored in −80°C for further analysis.
Mb concentration in muscle
The Mb concentration in muscles was measured using a commercial ELISA kit (Nanjing Jiancheng Biotechnology Co, Ltd, China) according to the manufacturer’s instruction. Briefly, muscle homogenates were mixed with tbiotin-labelled antigens and incubated at 37°C for 30 min. Then avidin–horseradish peroxidase was added. The absorbance was assessed at 450 nm using a commercial microplate reader (SpectraMax M2, Molecular Devices, USA).
DNA, RNA and protein contents in muscle
The muscle samples were homogenized in ice-cold PBS EDTA (0.05 M – Na3PO4, 2.0 M – NaCl, 2 × 10−3 M – EDTA, pH 7.4) using a 1:10 (w/v) ratio. The protein concentration of muscle homogenates was determined according to the method proposed by Lowry et al. (Citation1951) and using bovine serum albumin as the standard. Muscle DNA content and RNA level were evaluated by a fluorometric assay (Labarca & Paigen, Citation1980) and ultraviolet absorption at 232 and 260 nm (Storch et al., Citation2003), respectively.
Real-time qPCR
The gene expression in muscle was measured by real-time qPCR according to the published method (Kang et al., Citation2020). Briefly, total RNA in muscle was isolated by the Trizol reagent (#9108, TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China). cDNA was obtained using a commercial kit (#RR047A, TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China). Then real-time PCR assay was carried out using a SYBR® Premix Ex TaqTM (Tli RNaseH Plus) qPCR kit (#RR420A, TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China). The PCR cycling conditions were 95°C × 30 s, followed by 40 cycles of 95°C × 5 s and 60°C × 34 s. The sequence of the forward and reverse primers is shown in . The mRNA expressions relative to housekeeping gene (β-actin) were calculated (Livak & Schmittgen, Citation2001).
Table 1. Primer sequences used for real-time PCRTable Footnote1.
Statistical analysis
The experimental data were analysed by ANOVA with SPSS 17.0 software. Differences among treatments were determined by Duncan’s multiple range tests. All data were expressed as means ± SE. The statistical significance level for all analyses was set at P < 0.05.
Results
Blood cell parameters
As shown in , compared with the control group, FeSO4 and Fe-Gly supplementation increased the mean corpuscular volume (MCV) and the mean cell haemoglobin concentration (MCHC) (P < 0.05). Iron supplementation increased the mean corpuscular haemoglobin (MCH) (P < 0.05).
Table 2. Effects of different iron sources on hematological parameters in blood in pigs at 28 d1,2.
Meat colour
The meat colour results are shown in . Iron supplements in diet had no effect on the colour parameters (L*, a*, b*) of longissimus dorsi muscle and gastrocnemius muscle, respectively (P > 0.05).
Table 3. Effects of different iron sources on meat colour in pigs at 28 dTable Footnote1.
Mb concentration in muscle
The total Mb concentration in both longissimus dorsi and gastrocnemius muscles showed an increase after different iron supplements in diet (P < 0.05) ().
Table 4. Effects of different iron sources on myoglobin (Mb) content in muscles in pigs at 28 d1,2.
DNA, RNA, and protein contents in muscle
DNA、RNA and protein contents in muscles are shown in . Muscle protein and DNA concentrations are important indexes for muscle mass or muscle protein metabolism (Smith et al., Citation2011). Their ratio can be a sensitive measure for muscle protein mass (Crossland et al., Citation2008). In this study, we found iron supplements in diet had no effect on the protein contents, RNA / DNA ratio and protein/DNA ratio in muscles (P > 0.05).
Table 5. Effects of different iron sources on the protein, DNA and RNA contents in muscles in pigs at 28 d1,2.
Muscle mRNA expression analysis
As shown in , iron supplements had a tendency to decrease DMT-1 and hemojuvelin (HJV) expression in both the longissimus dorsi muscle and gastrocnemius muscle, and to increase hephaestin (HEPH) expression in longissimus dorsi muscle (P < 0.1). Compared with FeSO4 and Fe-Gly, amino acid-Fe(II)-chelator complexes reduced ferroportin (Fpn) 1 expression in the longissimus dorsi muscle, however, its supplementation increased Fpn1 expression in the gastrocnemius muscle (P < 0.05). Fe-Gly increased lipocalin (LCN) 2 and reduced TFR 2 expression in the longissimus dorsi muscle (P < 0.05). Iron supplementation decreased TFR1 expression in the longissimus dorsi muscle, and increased LCN2 expression in the gastrocnemius muscle, respectively (P < 0.05). In addition, FeSO4 reduced TFR1 expression in the gastrocnemius muscle (P < 0.05).
Table 6. Effects of different iron sources on genes expression related to iron metabolism in muscles in pigs at 28 d1,2,3.
Discussion
MCV, MCH and MCHC are three main red blood cell indices. They relate to the amount of haemoglobin in red blood cell (Sarma, Citation1990). Name et al. (Citation2018) found iron bisglycinate chelate could increase MCV and MCH levels and thought iron bisglycinate chelate had a great efficacy in increasing iron stores (Name et al., Citation2018). In this study, we found iron supplementation increased these indexes, which suggested that organic iron has the same effectiveness in haemoglobin biosynthesis as the inorganic iron.
Meat colour is the first visual sensory index of meat quality. Porcine meat colour varies at different stages of growth or the type of muscles, its scores were higher in pigs during the early growth stage (Yu et al., Citation2017). In addition, different iron sources have an effect on the meat colour. Behroozlak et al. (Citation2021) found FeSO4 increased L* value of breast meat in broilers during their earlier growth stage (Behroozlak et al., Citation2021). However, ferric chloride led to the pale colour and a reduction in redness in chicken breast meat (Zhang et al., Citation2021). Moreover, the meat colour is highly correlated with Mb concentration (Suman & Joseph, Citation2013), which could be regarded as an additional oxygen radical scavenger (Mates et al., Citation1999). Many factors, such as dietary treatment, have impact on Mb content in muscle (Li et al., Citation2013; Pogorzelska-Nowicka et al., Citation2018). In the current study, we found iron supplementation had no effect on the meat colour in both the longissimus dorsi muscle and gastrocnemius muscle, however, its supplementation increased Mb content in muscle, which indicated the low-dose organic iron could have the same effect on protecting muscle from oxidative damage as the high-dose inorganic iron.
As the important indexes for muscle mass or muscle protein metabolism, both muscle protein and DNA concentrations (Smith et al., Citation2011) were measured in the current study. In addition, the protein/DNA ratio can be used to measure muscle protein concentration (Carlson, Citation1973) and cell size (Petersson et al., Citation1995), and RNA/DNA ratio can be a measure of synthetic capacity of the cell (Li et al., Citation2010). In the current study, we found different iron sources had no effect on muscle protein content, as well as on the capacity for protein synthesis in muscle cells.
DMT-1 contributes to non-heme iron uptake in most types of cells (Yanatori & Kishi, Citation2019). FeSO4 overload could significantly increase DMT-1 expression in human umbilical vein endothelial cells, which suggested that the cells might attempt to reduce intracellular iron intake (Zhao et al., Citation2021b). TFR1 can regulate iron entry into cells, higher TFR1 expression is in response to iron deficiency (Gammella et al., Citation2017; Zhang et al., Citation2020), which causes newborn piglets being acutely susceptible to porcine epidemic diarrhea virus (Zhang et al., Citation2020). Whereas Fpn1 exports iron into the circulation. Excess iron can be exported from the cell via Fpn1 (Gao et al., Citation2019). Fpn gene knockout mice showed that iron was retained within enterocytes, which stimulated iron deficiency anaemia in mice (Donovan et al., Citation2005). HEPH is necessary for effective iron transport from intestinal enterocytes into the circulation and well expressed in the skeletal muscle (Polonifi et al., Citation2010). In the intestine of copper-deficient mice, the decreased hephaestin activity causes systemic iron deficiency (Chen et al., Citation2006). In the current study, iron supplementation in diets had a tendency to decrease DMT-1 expression and to increase HEPH expression in muscle, respectively. Amino acid-Fe(II)-chelator complexes supplementation increased Fpn1 expression and reduced TFR1 expression in muscle, which indicated that iron supplementation could reduce iron entry into muscle cells and stimulate iron export from muscle cells. Amino acid-Fe(II)-chelator complexes, compared with FeSO4 and Fe-Gly, had a greater efficacy in mobilizing iron in muscles into the circulation.
Lcn2 is a critical iron regulatory protein as well as the part of innate immune defenses against bacterial infection (Bhusal et al., Citation2021). Its high level is associated with the increased cell proliferation (Santiago-Sánchez et al., Citation2020). In addition, Lcn2 could modulate cytosolic iron levels and reduce oxidative stress (Xiao et al., Citation2017). In this study, iron supplementation increased Lcn2 expression, suggested that iron supplementation could stimulate muscle cells proliferation and have a positive effect to protect muscle cells against oxidative stress, which was in accordance with our Mb results in muscles.
Conclusions
In summary, compared with high-dose inorganic iron, low-dose organic iron supplements have the same effects in haemoglobin and myoglobin biosynthesis, and mobilizing iron in muscles into circulation.
Author contributions
All authors were involved in study design and implementation, data acquisition, analysis and interpretation. PK and LY wrote the manuscript. All authors read and approved the final version.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Additional information
Funding
References
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