Introduction
As one of the essential micronutrients, iron is required for growth, development, and many physiological processes. Intestinal iron absorption is an important process for maintaining body iron level within the optimal physiological range.(Reference Fuqua, Vulpe and Anderson1) Dietary iron is absorbed primarily in small intestine, and plays a crucial role in modulating intestinal development, epithelial maturation, and cell proliferation.(Reference Song, Christova and Perusini2,Reference Radulescu, Brookes and Salgueiro3) Previous studies have found that iron promoted intestinal development and epithelial maturation in piglets.(Reference Radulescu, Brookes and Salgueiro3,Reference Zhou, Dong and Wan4)
In animal production, ferrous sulfate (FeSO4) is usually supplemented as the standard inorganic iron source.(Reference Pu, Li and Xiong5) However, ferrous salts can cause free-radical-mediated mucosal damage, even if they can rapidly correct the iron deficiency.(Reference Zhuo, Yu and Li6) In addition, FeSO4 has poor bioavailability,(Reference Goddard, McIntyre and Scott7) and unabsorbable iron is excreted in feces. Therefore, considering the environmental impact caused by pig manure iron, alternative iron should be evaluated in order to improve iron utilization.
Many studies have reported that organic iron has better bioavailability than inorganic iron.(Reference Zhu, Yang and Fan8,Reference Ma, Sun and Zhou9) Zhuo et al.(Reference Zhuo, Fang and Yue10) found that ferrous glycinate (Fe-Gly) was absorbed more efficiently and utilized faster than FeSO4. Lin et al.(Reference Lin, Shu and Fu11) reported that Fe-Gly improved the bioavailability and antioxidant capacity of iron and reduced iron output of faeces. The mechanism may be due to the differences in absorption, transport and utilization processes.(Reference Zhuo, Fang and Hu12) In addition, amino acid-Fe-chelator complexes have been proposed as a superior iron supplement for improving iron absorption and bioavailability.(Reference Wu, Yang and Sun13) Therefore, the low-dose organic iron may have the same or higher efficiency than the high-dose inorganic iron in animal production.
Iron supplementation is necessary to prevent anaemia in piglets because of their special physiological condition.(Reference Ding, Yu and Feng14,Reference Sutherland15) The current study aimed to investigate the effects of different iron sources on growth performance and small intestinal morphology, development, and proliferation in weaned piglets, and to explore whether the low-dose organic iron had the same or higher bioavailability on growth performance and intestine development than the high-dose inorganic iron in the weanling piglets.
Materials and methods
Experimental design
The animal use protocol for this research was approved by Wuhan Polytechnic University Institutional Animal Care and Use Committee (Wuhan, China). Two hundred and forty piglets (Duroc × Large White × Landrace, 9.52 ± 1.60 kg, 40 ± 2 d) were randomly assigned to four treatments: (1) control group, a basal diet without iron supplemented in mineral premix; (2) FeSO4 group (basal diet with iron compensation by FeSO4.H2O, FeSO4.H2O was bought from Lomon Corporation, purity 91.3%), 100 mg Fe/kg dry matter (DM); (3) Fe-Gly (Fe-Gly was kindly provided by DeBon Bio-Tech Co., Ltd. Fe≥17.0%) group, 80 mg Fe/kg DM; (4) amino acid-Fe(II)-chelator complexes (the complex was kindly provided by DeBon Bio-Tech Co., Ltd., Fe≥15.0%) group, 30 mg Fe/kg DM. There were four pens for each treatment, and each pen had fifteen piglets. The basal diet was corn-soybean diet and prepared to meet or exceed NRC(16) nutrient requirement (Table 1). All piglets were allowed ad libitum access to water and feed during a 28-day experimental study. The ambient temperature was maintained at 22∼25 ℃ and the living environment was in accordance with animal welfare guidelines. The investigators monitored animals twice daily. Health was monitored by weight (once weekly), food and water intake, and general assessment of animal activity.
Growth performance
Feed consumption was measured every day during the entire experimental time, and body weight was individually measured at the beginning of the trial, and at the end of experiment to calculate average daily gain (ADG), average daily feed intake (ADFI), and feed: gain ratio (F/G) in order to assess growth performance.
Sample collection
On day 28, six healthy piglets were selected from each treatment group, with their body weights approximating the group mean. Then blood samples were collected via the jugular vein into 10-mL vacuum tubes on d 14 and d 28, respectively. The blood samples were centrifuged at 3000 × g for 10 min to collect serum, then frozen at −80℃ until further analysis. Blood haemoglobin was analyzed (#V-52D, diluted solution for animal blood cell analysis, #V-52DIFF, haemolytic agents for animal blood cell analysis, Shenzhen Mindray Animal Medical Technology Co., Ltd, Shenzhen, China) using a fully automated blood analyzer (ADVIA 2120i, Siemens, Germany). Serum iron concentration (#A039-1-1, serum iron assay kit), total iron binding capacity (TIBC, #A040-1-1, total iron binding capacity assay kit) were analyzed by the commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Serum hepcidin level was determined by using a commercially available porcine ELISA assay kit (#RX500303P, pig hepcidin quantitative detection kit, Ruixin Biotechnology Co., Ltd, Quanzhou, China). After blood collection, pigs were slaughtered under anaesthesia with an intravenous injection of pentobarbital sodium (50 mg/kg BW). Duodenal and jejunal mucosa were collected, frozen in liquid nitrogen, and then stored in a freezer at –80°C for further analysis.
Intestinal morphology
Approximately 0.5 cm of the second centimetre of each small intestinal section (duodenum and jejunum) was isolated, rinsed with sterile PBS, and fixed overnight with 10% formalin. Fixed tissues were embedded in paraffin, sectioned (5 μm), and stained with haematoxylin and eosin (H&E) for morphologic examinations. The lengths of villous height and crypt depth were measured by using Olympus software.
DNA, RNA and protein contents in intestinal mucosa
The duodenum and jejunum mucosa were homogenized with a tissue homogenizer (PT-3100D, Kinematica, Switzerland). Protein concentration of mucosa homogenates was determined using a detergent-compatible protein assay (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as standards.(Reference Lowry, Rosebrough and Farr17) Mucosa DNA content was evaluated by a fluorometric assay.(Reference Labarca and Paigen18) Mucosa RNA content was measured according to the previous study.(Reference Johnson and Chandler19) Briefly, the mucosa homogenate was mixed with perchloric acid and centrifuged. Then the precipitates were dissolved in potassium hydroxide (KOH). The absorbances of the supernatants at 232 and 260 nm were recorded and the micrograms of RNA per millilitre were calculated according to the formula.(Reference Munro and Fleck20)
Disaccharidase activities in intestine
The disaccharidase activities in intestinal mucosa homogenates were determined by the commercial assay kits (#A082-1-1, lactase assay kit; #A082-2-1, sucrase assay kit; #A082-3-1, maltase assay kit, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The absorbances were measured spectrophotometrically at 505 nm.
Real-time PCR
The gene expression was measured by real-time PCR. Briefly, total RNA was isolated by the Trizol reagent (#9108, TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China), and cDNA was synthesized using PrimeScript® RT reagent kit (#RR047A, TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China). Real-time PCR assay was carried out on an ABI 7500 Real-Time PCR System (Applied Biosystems, Life Technologies) using a SYBR® Premix Ex TaqTM (Tli RNaseH Plus) qPCR kit (#RR420A, TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China). The PCR cycling conditions were 95ºC × 30 s, followed by 40 cycles of 95ºC × 5 s and 60ºC × 34 s. The forward and reverse primers for the target genes were designed with Primer Premier 6.0 and synthesized by TaKaRa Biotechnology (Dalian, China, Table 2). The mRNA expression relative to housekeeping gene (GAPDH) was calculated according to the 2−△△CT method.(Reference Livak and Schmittgen21)
Table 1. Ingredient composition of diet (as fed basis)
a A compound acidifier including lactic acid and phosphoric acid, provided by Wuhan Fanhua Biotechnology Company, Wuhan, China.
b A compound mould inhibitor including calcium propionate, fumaric acid, fumaric acid monoethyl ester and sodium diacetate, provided by Sichuan Minsheng Pharmaceutical Co., Ltd, Chengdu, China.
c A compound sweetener including saccharin sodium and disodium 5’-guanylate, provided by Wuhan Fanhua Biotechnology Company, Wuhan, China.
d The premix provided the following amounts per kilogram of complete diet: retinol acetate, 2700 μg; cholecalciferol, 62.5 μg; dl-α-tocopheryl acetate, 20 mg; menadione, 3 mg; vitamin B12, 18 μg; riboflavin, 4 mg; niacin, 40 mg; pantothenic acid, 15 mg; choline chloride, 400 mg; folic acid, 700 μg; thiamine, 1.5 mg; pyridoxine, 3 mg; biotin, 100 μg; Zn, 80 mg (ZnSO4·7H2O); Mn, 20 mg (MnSO4·5H2O); Cu, 25 mg (CuSO4·5H2O); I, 0.48 mg (KI); Se, 0.36 mg (Na2SeO3·5H2O).
e Calculated.
f Analyzed.
Protein abundance analysis by Western blot
Quantification of protein expression in intestinal mucosa was performed as previously described.(Reference Liu, Chen and Odle22,Reference Liu, Chen and Odle23) Briefly, extracted proteins were quantified, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were then transferred to polyvinylidene difluoride membranes (Millipore). After blocking, membranes were incubated with primary antibodies at 4℃ overnight, followed by three 15-minute washes with PBS containing 0.1% Tween-20. The primary antibodies included rabbit anti-claudin-1 (#51-9000, Invitrogen), mouse anti-occludin (#ab31721, Abcam), mouse anti-zonula occluden-1 (ZO-1, #33-1500, invitrogen), and the secondary antibodies included goat anti-rabbit IgG-HRP (#ANT020, Wuhan Antejie Biotechnology Co., Ltd) and goat anti-mouse IgG-HRP (#ANT019, Wuhan Antejie Biotechnology Co., Ltd). The bands were analyzed by densitometry using GeneTools software (Syngene), and the abundance of each target protein was expressed as the target protein: β-actin ratio.
Statistical analysis
The experimental data were analyzed by one-way ANOVA with SPSS 17.0 software. If the differences were significant, Duncan’s multiple comparisons were further used to determine the specific differences among experimental groups. All data were expressed as means ± SE. Differences were considered to be significant if P < 0.05.
Results
Growth performance
Growth performance is shown in Table 3. Compared with the control group, iron supplementation increased ADFI throughout the experimental period (P < 0.05). Diets supplemented with Fe-Gly or amino acid-Fe(II)-chelator complexes increased ADG (P < 0.05).
Table 2. Primer sequences used for real-time PCR *
* ZO-1, zonula occluden-1; PCNA, proliferating cell nuclear antigen; Lgr5, leucine rich repeat containing G protein-coupled receptor 5; Olfm, olfactomedin; Bmi1, B cell-specific moloney murine leukaemia virus integration site 1; Lyz, lysozyme; ChgA, chromogranin A ; Muc2, mucins.
Blood haemoglobin and serum iron concentration, total iron binding capacity, transferrin saturation, and hepcidin concentration
As shown in Table 4, iron supplementation had no effect on blood haemoglobin concentration, serum iron concentration, TIBC, TSAT and hepcidin concentration in serum on d 14. On d 28, Fe-Gly and amino acid-Fe(II)-chelator complexes supplementation decreased serum iron concentration and TSAT (P < 0.05), and FeSO4 and Fe-Gly supplementation reduced TIBC (P < 0.05).
Table 3. Effects of different iron sources on growth performance in pigs during 1–28 days *
* Values are means ±SE, n = 4 per treatment.
a-c Values in the same row with different superscript letters are significantly different (P < 0.05).
Intestinal morphology
As shown in Table 5, regardless of iron sources, iron supplementation decreased villus height, and FeSO4 and amino acid-Fe(II)-chelator complexes supplementation reduced crypt depth in duodenum (P < 0.05). However, amino acid-Fe(II)-chelator complexes supplementation increased villus height in jejunum (P < 0.05). Both FeSO4 and Fe-Gly decreased crypt depth in jejunum (P < 0.05).
Table 4. Effects of different iron sources on blood haemoglobin concentration, serum iron concentration, total iron-binding capacity (TIBC), transferrin saturation and hepcidin concentration in pigs at 14 and 28 days *
* Values are means ±SE, n = 6 per treatment.
a-c Values in the same row with different superscript letters are significantly different (P < 0.05).
Protein, RNA and DNA levels
Protein, RNA and DNA levels in intestine are shown in Table 6. Amino acid-Fe(II)-chelator complexes supplementation increased protein to DNA ratio in duodenum (P < 0.05), and increased protein content, RNA to DNA ratio, and protein to DNA ratio in jejunum (P < 0.05).
Table 5. Effects of different iron sources on intestinal morphology in pigs at 28 d *
* Values are means ±SE, n = 6 per treatment.
a-c Values in the same row with different superscript letters are significantly different (P < 0.05).
Disaccharidase activities in intestine
Disaccharidase activities in intestine are shown in Table 7. Amino acid-Fe(II)-chelator complexes decreased maltase activity in both duodenum and jejunum, and Fe-Gly increased sucrase activity in duodenum (P < 0.05). FeSO4 increased lactase and sucrase activities in jejunum (P < 0.05).
Table 6. Effects of different iron sources on the protein, DNA and RNA contents in intestine of pigs at 28 d *
* Values are means ±SE, n = 6 per treatment.
a, b Values in the same row with different superscript letters are significantly different (P < 0.05).
mRNA expression of intestinal epithelial barrier function, and cell proliferation related genes
As shown in Table 8, compared with control group, both FeSO4 and Fe-Gly supplementation increased Ki67 mRNA expression, and Fe-Gly supplementation increased leucine rich repeat containing G protein-coupled receptor 5 (Lgr5) mRNA expression in duodenum (P < 0.05). In jejunum, amino acid-Fe(II)-chelator complexes increased claudin-1 mRNA expression (P < 0.05), and both amino acid-Fe(II)-chelator complexes and Fe-Gly increased Lgr5 mRNA expression (P < 0.05), Fe-Gly increased Lysozyme (Lyz) mRNA expression, and FeSO4 and amino acid-Fe(II)-chelator complexes increased Villin mRNA expression (P < 0.05).
Table 7. Effects of different iron sources on the disaccharidase activities in intestine of pigs at 28 d *
* Values are means ±SE, n = 6 per treatment.
a-c Values in the same row with different superscript letters are significantly different (P < 0.05).
Table 8. Effects of different iron sources on intestinal epithelial barrier function and proliferation-related genes expression in pigs at 28 d *
* ZO-1, zonula occluden-1; PCNA, proliferating cell nuclear antigen; Lgr5, leucine rich repeat containing G protein-coupled receptor 5; Olfm, olfactomedin; Bmi1, B cell-specific moloney murine leukaemia virus integration site 1; Lyz, lysozyme; ChgA, chromogranin A; Muc2, mucins
Values are means ±SE, n = 6 per treatment.
a-c Values in the same row with different superscript letters are significantly different (P < 0.05).
Protein expression of claudin-1, occludin, and ZO-1 in intestine
As shown in Figure 1 and 2, different iron sources supplementation had no effect on protein expression of claudin-1 and occludin in duodenum. FeSO4 and amino acid-Fe(II)-chelator complexes increased protein expression of ZO-1 in duodenum (P < 0.05). However, protein expression of claudin-1, occludin, and ZO-1 in jejunum had no difference among these treatments.
Figure 1. The effects of different iron sources on protein expression of claudin-1, occludin, and ZO-1 in duodenum in the weaned piglets. Values were means (n = 6), with their standard errors represented by vertical bars. () Control group, a basal diet without iron supplemented in mineral premix; () ferrous sulfate (FeSO4) group, 100 mg Fe/kg dry matter (DM); () ferrous glycinate (Fe-Gly) group, 80 mg Fe/kg DM; () amino acid-Fe(II)-chelator complexes group, 30 mg Fe/kg DM.
Figure 2. The effects of different iron sources on protein expression of claudin-1, occludin, and ZO-1 in jejunum in the weaned piglets. Values were means (n = 6), with their standard errors represented by vertical bars. () Control group, a basal diet without iron supplemented in mineral premix; () ferrous sulfate (FeSO4) group, 100 mg Fe/kg dry matter (DM); () ferrous glycinate (Fe-Gly) group, 80 mg Fe/kg DM; () amino acid-Fe(II)-chelator complexes group, 30 mg Fe/kg DM.
Discussion
Iron is an essential micronutrient and required for growth, development, and many physiological processes. Increasing dietary iron can improve growth performance in piglets.(Reference Williams, Woodworth and DeRouchey24) In this study, the iron content in the basal diet was 153.92 mg/kg. However, animals exhibit limited ability to utilize dietary iron.(Reference Liu, Li and Su25) Consequently, the iron in basal diet is typically negligible in practical production. FeSO4 is usually supplemented as the standard inorganic iron source,(Reference Zhuo, Yu and Li6) and its supplementation exhibits a dose-dependent increase in ADG in piglets(Reference Rincker, Hil and Link26) and growing-finishing pigs.(Reference Deng, Wang and Wang27) Previous studies have demonstrated that organic iron exhibits superior bioavailability compared to inorganic iron.(Reference Ma, Sun and Zhou9,Reference Zhuo, Fang and Yue10,Reference Fang, Zhuo and Fang28) Furthermore, amino acid-Fe-chelator complexes have been proposed as superior iron supplements due to their potentially higher biological utilization efficiency. Therefore, in this study, two types of organic iron compounds with different doses were selected to investigate: (1) whether low-dose organic iron could achieve comparable or superior efficacy to high-dose inorganic iron, and (2) whether low-dose amino acid-Fe-chelator complexes could exhibit bioavailability equivalent to high-dose Fe-Gly. In this study, we found both Fe-Gly and amino acid-Fe(II)-chelator complexes increased ADG and tended to decrease F/G. However, no significant differences were observed between two organic iron sources. Our results suggest that organic iron sources including both Fe-Gly and amino acid-Fe(II)-chelator complexes were more effective in improving growth performance in weanling pigs compared to inorganic iron. Notably, lower-dose amino acid-Fe(II)-chelator complexes achieved comparable efficacy to Fe-Gly in enhancing growth performance.
TIBC and TSAT are important biochemical markers of body iron status. Low TSAT level is an indicator of iron deficiency.(Reference Kasvosve and Delanghe29,Reference Ambrosy, Fitzpatrick and Tabada30) However, elevated TSAT can damage cells and tissues through oxidative stress.(Reference McCord31) Zhou et al.(Reference Zhuo, Fang and Hu12) found that Fe-Gly increased TSAT in SD rats. However, inconsistent with this report, we found that Fe-Gly supplementation decreased TSAT and serum iron concentration compared to FeSO4. Moreover, we found Fe-Gly and amino acid-Fe(II)-chelator complexes decreased serum iron concentration, but this result might not mean iron deficiency. Nemeth and Ganz(Reference Nemeth and Ganz32) reported serum iron concentration was low despite adequate iron stores. The results of growth performance in this study also imply this inference.
Hepcidin is the central regulator of iron homeostasis in the body. The production of hepcidin in hepatocytes can be greatly stimulated by plasma iron and iron stores.(Reference Ganz33) Then, the increased hepcidin can inversely lead to decreased iron absorption and release of iron from stores. In this study, in consistent with the results of the decreased serum iron concentration, organic iron had a tendency to increase serum hepcidin concentration, which indicated that organic iron had better bioavailability to be absorbed easily, then stimulated hepcidin production to decrease serum iron concentration.
Villus height and crypt depth is closely related to primary function of digestion and absorption of nutrients in small intestine.(Reference Xie, Liu and Wang34) Moderate FeSO4 can increase duodenal villus height and the colonic crypt depth.(Reference Deng, Wang and Wang27) Zhuo et al.(Reference Zhuo, Yu and Li6) found diet supplement with iron significantly increased villus height, and Fe-Gly exhibited better bioavailability than FeSO4. However, contrary to the above results, we found that villus height in duodenum was higher in control group than other groups. Wayhs et al. (Reference Wayhs, Patrício and Amancio35) reported that iron deficiency had a compensatory intestinal mechanism to increase intestinal villus height to improve iron absorption. Therefore, the higher villus height in the control group might result from the iron deficiency, which stimulated the compensatory intestinal mechanism to increase iron absorption.
Iron can stimulate DNA and protein synthesis.(Reference Higashida, Inoue and Nakai36,Reference Gozzelino and Arosio37) Protein/DNA ratio can be a measure of protein concentration(Reference Petersson, Hultman and Andersson38) and cell size.(Reference Enesco and Leblond39) RNA/DNA ratio provides a measure of synthetic capacity of cell and indicates the capacity of protein synthesis.(Reference Li, Zlabek and Grabic40) Higher RNA/DNA ratio shows the increased ability to synthesize intracellular RNA and protein.(Reference Moretti, Nordi and Lima41) Higashida et al.(Reference Higashida, Inoue and Nakai36) found iron was essential to protein synthesis in skeletal muscle, and its deficiency was associated with muscle weakness. In addition, impaired protein synthesis resulting from iron deficiency in pups limited their ability to produce antibodies. Our results showed that amino acid-Fe(II)-chelator complexes had a greater efficacy in increasing protein synthesis in intestine. However, Yamauchi et al. (Reference Yamauchi, Adjei and Ameho43). found that protein deficiency was associated with decreased intestinal villous height, whereas in our study, amino acid-Fe(II)-chelator complexes increased protein synthesis but reduced villus height. The mechanism requires further investigation.
Disaccharidases are crucial for the intestinal function. Both iron deficiency and iron excess decrease disaccharidase activities.(Reference West and Oates44,Reference Vir, Kaur and Mahmood45) Many studies reported that iron deficiency decreased the activity of disaccharidases,(Reference West and Oates44,Reference Vieira, Galvão and Fernandes46) especially lactase,(Reference Buts, Delacroix and Dekeyser47,Reference Sriratanaban and Thayer48) which might result from the reduced ability to synthesize lactase in enterocytes.(Reference Gawde, Patel and Rege49) The present study found that amino acid-Fe(II)-chelator complexes reduced maltase activity in intestine. However, the performance data, as well as the increased protein/DNA ratio suggested that iron supplemented by amino acid-Fe(II)-chelator complexes was enough in this study.
Claudin-1, occludin, and ZO-1 are tight junction proteins, which can maintain the intestinal epithelial barrier function. Iron can enhance intestinal barrier function by increasing the colonic expressions of occludin and claudin-1.(Reference Sun, Yu and Luo50) Sun et al.(Reference Liang, Xiong and Kong51) found Fe-Gly supplementation increased the abundance of tight-junction protein ZO-1 in jejunum in weaned piglets. However, overload ferric citrate impairs intestinal barrier function evidenced by the reduced tight junction proteins.(Reference Luo, Lao and Huang52) Wu et al. (Reference Wu, Wei and Guo53) also found iron overload decreased the levels of ZO-1 and Occludin. In this study, the increased mRNA expression of claudin-1 and ZO-1 in jejunum after amino acid-Fe(II)-chelator complexes supplemented suggested that it could have a positive role to form a barrier in small intestine and prevent pathogen invasion. In our study, the protective effects of amino acid-Fe(II)-chelator complex supplementation on intestinal integrity may be closely related to protein synthesis capacity in the intestine.
Ki-67 protein is a cell proliferation-associated nuclear marker.(Reference Shi, Zhang and Han54) The number of Ki67-positive cells is usually in line with the proliferation of gastrointestinal epithelial cells.(Reference Zhou, Qin and Xiong55) Lgr5 is a marker of active stem cells in the small intestine and colon.(Reference Barker, van Es and Kuipers56) B cell-specific moloney murine leukaemia virus integration site 1 (Bmi1) is adult stem cell gene. Precious study found heat stress inhibited the intestinal epithelial cell proliferation and stem cell expansion by down-regulating the expression of Ki67, Lgr5, and Bmi1.(Reference Zhou, Huang and Zhu57) Zhou et al.(Reference Zhou, Qin and Xiong55) found FeSO4 decreased the expression of Lgr5 in jejunum. However, our results showed that Fe-Gly had a better effect on activating stem cells in the small intestine. In addition, Lyz can protect intestinal epithelium against bacterial infection(Reference Bel, Pendse and Wang58) and enhance intestinal functions and gut microflora of piglets.(Reference Xiong, Zhou and Liu59) In swine feed, it can be supplemented as the alternative to growth-promoting subtherapeutic antibiotic.(Reference Oliver and Wells60) Villin is the marker of absorptive cells,(Reference Chong, Youn and Shin61) its expression can assess recovery of the intestinal absorptive surface area.(Reference Araújo, Lazzarotto and Aquino62) In this study, we found that organic iron improved and maintained jejunal epithelial barrier and absorption function, as evidenced by increased mRNA expressions of Lyz, Villin, claudin-1, and ZO-1 in the jejunum. These results were consistent with the imoroved growth performance observed in piglets, suggesting that this enhancement may result from enhanced intestinal absorption capacity and barrier function.
Conclusion
In summary, organic iron is more effective than FeSO4 in improving performance, and has a positive effect on intestinal health in the weanling piglets. Low-dose organic iron may replace high-dose inorganic iron in piglet diet.
Acknowledgments
This research was financially supported by the Project of the National Natural Science Foundation of China (No. U22A20517), Hubei Key Laboratory of Animal Nutrition and Feed Science (ANFS202308).
Authors’ contributions
The authors’ contributions are as follows: Y. L., P. K., and G. S. designed research; Y. L., P. K., G. S., J. F., Q. L., D. G., B. S., Q. C., K. Q., Y. K., D. W., Q.W., and H. Z. conducted research. Y. L. P. K. and G. S. analyzed data and wrote the paper. Y. L. had primary responsibility for final content.
Competing interests
The author(s) declare that they have no conflict of interest.
Ethics statement
All animal and experimental procedures were in agreement with the National Research Council Guide for the Care and Use of Laboratory Animals and approved by the Wuhan Polytechnic University Institutional Animal Care and Use Committee.
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