The rapidly growing nursery pig requires 80 – 100 mg/kg DM dietary Fe; however, commercial starter diets often exceed this requirement by up to five-fold. This is due to the fact that several swine ingredients contain high total Fe contents, including blood meal (3,000 mg/kg DM), dicalcium phosphate (10,000 mg Fe/kg DM), and limestone (3,500 mg Fe/kg DM). The role of dietary Fe supplementation in the exacerbation of intestinal inflammatory disease in humans has received considerable attention lately.¹ Excessive dietary Fe can trigger oxidative stress pathways that lead to intestinal injury through mechanisms that include lipid peroxidation, inflammation, and cellular membrane breakdown.^{2, 3} In addition, excess dietary Fe is available to opportunistic pathogens, potentially contributing to bacterial overgrowth. However, the effect of high Fe levels in swine nursery diets on gastrointestinal health has not been investigated previously. The objective of this study was to assess the effects of dietary Fe levels on post-weaning intestinal health and defense.

Material and Methods

Pigs and experimental diets. Twenty-four weaned male pigs averaging 21 d of age and 5.5 ± 0.4 kg BW were used in this study. All piglets were injected with 100 mg of Fe dextran at birth to prevent anemia while also minimizing body Fe stores prior to initiation of the trial. At weaning pigs were blocked by weight within litter and randomly assigned to one of these three treatments: (1) no supplemental Fe (low Fe; L-Fe), (2) 100 mg supplemental Fe/kg DM (adequate Fe; A-Fe), and (3) 500 mg supplemental Fe/kg DM (high Fe; H-Fe). The basal diet was formulated based on NRC recommendations to meet or exceed a pig’s requirement for all nutrients except Fe. The basal diet contained 20 mg Fe/kg DM (approximately 25 per cent of a young pig’s requirement for Fe), and supplemental Fe was provided as FeSO₄.

Pig feeding and management. Pigs were housed in pens of two pigs (four replicate pens per treatment) in an environmentally controlled nursery and provided ad libitum access to both feed and water for a 32-day period. At the end of the 32-day feeding trial, pigs were euthanized and each pig’s duodenum was harvested and immediately mounted on Ussing chambers for measurements of transepithelial electrical resistance (TER) and mucosal-to-serosal flux of the paracellular probe [3H]-mannitol, both sensitive measures of intestinal permeability or barrier function. Intestinal tissues were fixed in 10 per cent neutral buffered formalin and processed for routine histologic analysis and Fe staining. The lipid peroxidation product malondialdehyde (MDA) was assayed in intestinal tissues using a commercial ELISA kit.

Results

Compared with A-Fe diets, both L-Fe and H-Fe diets had a detrimental effect on intestinal barrier function in post-weaned pigs as indicated by reductions in intestinal TER (p<0.05) and elevated 3H-mannitol permeability (p<0.05) (Figure 1). Histological evaluation of duodenum from pigs fed the H-Fe diet revealed blunted villi and marked inflammatory cell infiltrate consisting predominantly of neutrophils (Figure 2). Fe staining of the intestinal mucosa from pigs fed H-Fe diets showed increased accumulation of Fe within e nterocytes and subepithelial lamina propria cells (Figure 3). Increased inflammatory cells were noted in duodenum from pigs fed the L-Fe diet but to a lesser extent compared with H-Fe diets. The lipid peroxidation product malondialdehyde (MDA) was elevated in H-Fe tissues, suggesting that lipid peroxidation may play an important role in mucosal injury induced by H-Fe diets (not shown).

Discussion

In the present study, we show that dietary Fe level can directly influence mucosal barrier health in the post-weaned pig. A marked inflammatory response was observed in intestinal tissues from pigs fed H-Fe diets and was characterized by predominantly neutrophilic infiltration. The mechanism of mucosal barrier dysfunction and inflammation induced by feeding H-Fe diets is currently unclear. Elevated levels of MDA in intestinal tissues from pigs fed H-Fe diets suggest that lipid peroxidation is likely contributing to intestinal barrier injury. In the Fenton reaction, Fe reacts with free radicals produced by activated neutrophils, resulting in the production of highly toxic free radicals. Oxidative stress can induce intestinal mucosal damage through several mechanisms, including increased intestinal and vascular permeability, lipid peroxidation and destruction of enterocyte membranes, neutrophil recruitment, and activation of NF-kB inflammatory signaling pathways.^{2, 4} In the present study, we also observed barrier dysfunction and mild intestinal inflammation in intestinal tissues from pigs fed the L-Fe diets. Given the anemic status of pigs fed L-Fe diets, it is plausible that intestinal ischemia may be a potential contributing factor to intestinal injury. However, histological analysis did not reveal any characteristic lesions associated with ischemic damage. More studies are needed to investigate the mechanisms of intestinal barrier injury triggered by low Fe status.

Summary

These data demonstrate that Fe levels in post-weaning diets have a profound influence on intestinal barrier health. Intestinal barrier function is compromised when Fe is deficient or in excess. Although the intestinal lesions and functional data in this study suggest a detrimental effect of H-Fe and L-Fe diets on GI mucosal health, the consequences of these findings with regards to animal health and intestinal defense against enteric challenges remain to be elucidated.

References

1. Erichsen K., Ulvik R.J., Nysaeter G., Johansen J., Ostborg J., Berstad A., Berge R.K. and Hausken T. 2005. Oral ferrous fumarate or intravenous iron sucrose for patients with inflammatory bowel disease. Scand J Gastroenterol. 40:1058-65.
2. Grisham M.B. and Granger D.N. 1988. Neutrophil-mediated mucosal injury. Role of reactive oxygen metabolites. Dig Dis Sci. 33:6S-15S.
3. McKenzie S.J., Baker M.S., Buffinton G.D. and Doe W.F. 1996. Evidence of oxidant-induced injury to epithelial cells during inflammatory bowel disease. J Clin Invest. 98:136-41.
4. Carrier J.C., Aghdassi E., Jeejeebhoy K. and Allard J.P. 2006. Exacerbation of dextran sulfate sodium-induced colitis by dietary iron supplementation: role of NF-kappaB. Int J Colorectal Dis. 21:381-7.

This study was conducted as part of a collaborative effort with Drs Jerry Spears and Steph Hansen (NCSU Animal Science). Parallel data from this study on the effects of iron level on trace mineral metabolism are published in Hansen et al., 2009 J Nutr. 139(8):1474-9.

Submitted by Dr Chad Stahl, Department of Animal Science, North Carolina State University.

October 2009