Strategies to Reduce Use of Zinc Oxide by Modulating Gut Microbiota in Pigs

Zinc (Zn) plays an important role in the metabolism of swine and as such is an essential trace element for growing pigs, according to Nutriad. Applying a sound SANACORE EN strategy can make a real difference for producers that want to limit the use of zinc oxide in pig feeds.

Zinc deficiency may result in reduced feed intake and growth, parakeratosis, impaired wound healing, alopecia, thymus atrophy and impaired immune function.

Zinc recommendations for growing pigs vary among national institutes but decrease with increasing body weight from approximately 100 to 50mg per kg. In practice, Zn is often administered in excess to assure sufficient supply.

During the late 1980s, it was discovered that pharmacological concentrations (1,500 to 3,000ppm) of Zn oxide (ZnO) resulted in reduced diarrhoea and increased growth in weanling pigs (Poulsen, 1989; Sales, 2013). Unfortunately, as Zn is poorly absorbed, it becomes highly concentrated in manure. To minimise the risk of environmental pollution, European regulations have reduced the maximal Zn concentration authorised in pig diets. In the EU, the legal norm for Zn is 150mg per kg (ppm) of Zn in complete animal feed (EU Regulation 1334/2003). It should be noted that pig feed usually already contains levels of around 30 to 40ppm of Zn, as it is a natural component of grains in feed. This means that, on average, 110ppm of Zn can be added as an additive.

Since 2005, ZnO at pharmacological levels has been reallowed in some European countries as a means to reduce the use of antibacterial compounds in the period shortly after weaning. As in those countries, supplementation of ZnO at dosages higher than 150ppm Zn in complete feed falls under the Veterinary Medicines Regulations, the use of ZnO is only possible on veterinary prescription.

Mode of Action of ZnO

The mechanisms behind the beneficial effects of ZnO to prevent diarrhoea and promote growth in weaned piglets are not well understood.

Recent advances suggest that the effects of ZnO on piglet growth are achieved through multiple regulatory pathways (Li et al., 2010).

Growth-promoting effect
As Zn absorption after weaning is about 30 per cent lower than before weaning and bio-availability may be less than 20 per cent, administering higher levels of ZnO in feed will increase plasma Zn levels which is needed to meet the requirements of the piglets. Moreover, it has been shown that ZnO regulates secretion of brain-gut peptides that stimulate feed intake (Li et al., 2010).
Improved intestinal barrier function
ZnO improves intestinal structure and function, alleviating the intestinal damage caused by weaning, by favouring cell regeneration and stimulating epithelial growth. Barrier function is strengthened by increasing the expression of intestinal insulin-like growth factor-1 (IGF 1, regulates cell growth and development) and its receptors, in this way speeding up the recovery of the damage induced by the changed diet at weaning (Li et al., 2010). ZnO also strengthens the intestinal barrier function through reducing paracellular permeability. This effect is the consequence of the upregulation of tight junction proteins in the intestine (Zhang & Ghuo, 2009).
Immunomodulating effect
ZnO reduces the release of pro-inflammatory histamine, by inhibiting proliferation and activation of intestinal mast cells (Kim et al., 2012).
Reduced ion secretion
ZnO reduces secretion of ions to the intestinal lumen, thereby enhancing water resorption and preventing diarrhoea.
Effects on bacteria
Supplementation of weaning diets of piglets with ZnO during a short period of time at relatively high doses (2,500ppm) stabilizes intestinal microbiota and prevents attachment of pathogenic bacteria to the intestinal villi, which may prevent many problems associated with weaning diarrhoea.

Research on the in vitro sensitivity to ZnO of a range of reference strains of intestinal origin has shown that Zn sensitivity is very variable. Liedtke & Vahjen (2012) have concluded that we cannot assume a generalised antibacterial effect of ZnO as MIC values differ greatly among but also within different bacterial species.
Roselli et al (2003) demonstrated that ZnO reduces adhesion and invasion of ETEC in an enterocyte cell culture and that it recovers an optimal anti-inflammatory cytokine balance after ETEC infection. Also in in vivo studies with weaned piglets it was demonstrated that a diet with pharmacological concentrations of ZnO diminishes the paracellular permeability (Huang et al., 1999; Zhang & Guo, 2009) and prevents the translocation of (pathogenic) bacteria such as E. coli and Enterococcus spp. in the mesenteric lymph nodes of the small intestine (Huang et al., 1999).
High levels of Zn enhance stability and diversity of the microbiota after the change of diet in weaned piglets (Starke et al., 2012).

It is clear that the use of high levels of ZnO (1,500 to 3,000ppm) has many positive effects on piglet health and performance. However, such use of pharmacological ZnO dosages has potentially some negative consequences as well.

Downsides of Zn Usage

1. Zinc toxicity

Although ZnO promotes health and performance in piglets, Zn remains a heavy metal and is as such toxic to most living organisms, including pigs. Pharmacological usage of ZnO may benefit piglets post-weaning, but according to the US National Research Council (NRC), it may affect piglet health and performance (marked depression in feed intake) if used for longer periods. In countries where pharmaceutical concentrations of ZnO in weaning diets are allowed, so far no severe negative consequences for the health of the animal have been reported, provided the supplementation is done for short periods (maximum 14 days). After three to four 4 weeks of overdosing, health problems have been reported.

2. Nutritional interactions

High levels of Zn result in overproduction of metallothionein, an intestinal transporter protein that binds several heavy metals such as copper, iron, zinc and selenium. When higher levels of these transport proteins are present there is a risk for sub-deficiencies of some of these minerals. For example, reduced absorption of iron and copper (which has a role in iron transport) might lead to anaemia (Sandström, 2001).

Studies about interactions between Zn and phytase suggest that pharmacological dosages of Zn have a negative effect on phytase activity and consequently on phytate-phosphorus (P) liberation: by complex formation of Zn with P-phytate, the phosphorus cannot be released by the phytase. This implies a reduced efficacy in phytase functions, and possible phosphorus deficiency for affected animals (Lizardo, 2004).

3. Contamination by heavy metals

Impurities in commercial ZnO can be a considerable problem when Zn quality is not strictly controlled. For example, tissues contaminated during the post-weaning period with cadmium (known for of its organ toxicity), can result in elevated levels of cadmium in tissues at slaughter time due to its long half-life. A study from the French institute IFIP indicated that cadmium concentration in kidneys exceeds the regulatory limit (1mg per kg) for human consumption when pigs are fed contaminated diets (0.5mg cadmium per kg diet) between 42 and 160 days.

In Thailand, where pharmacological Zn dosages are used, a recent study on 214 pork kidneys showed that more than 25 per cent of pork kidneys contained cadmium concentrations exceeding the regulatory limits. Contaminated ZnO was most probably the major source of dietary cadmium.

4. Zinc and microbial resistance

Although bacterial sensitivity to Zn is not always defined, acquired Zn resistance does seem to occur. Little is known about this resistance.

Much more alarming, however, intensive usage of Zn in animal diets may favour the development of bacterial resistance against other antimicrobials. This can be caused by different mechanisms.

Bacteria regulate intracellular Zn concentration with a system of efflux pumps. These pumps can be specific to Zn or can evacuate other molecules such as antibiotics. High levels of Zn tend to increase their synthesis, and the use of ZnO at pharmacological dosages may reduce the sensitivity of bacteria to antibiotics.
A genetic coupling can also be observed, as genes of heavy metal resistance and those of antibiotic resistance are sometimes associated. Consequently, the selection of bacteria resistant to Zn leads to the co-selection of bacteria resistant to some antibiotics.

5. Environmental concerns

Zn as a heavy metal tends to accumulate in soil after Zn-rich manure from piggeries is applied to the fields. High levels of Zn in soil, and in water reservoirs due to run-off, are considered to be an environmental pollutant and health hazard.

Search for Alternatives: SANACORE®EN

Because of these negative aspects of ZnO usage, producers are looking to find a suitable replacement.

When developing an antimicrobial support product, Nutriad has focused on products with a similar mode of action as ZnO. This research has resulted in the development of SANACORE EN, a multifunctional product with a broad spectrum antibacterial approach. The aim of using the product is to reduce the use of ZnO and/or antimicrobials while supporting health and welfare of the animals, as well as to improve production results.

SANACORE EN is a product based on a balanced, well-researched formula of different active components. Each component has a specific mode of action and thanks to the specially developed production process of SANACORE EN, the various active components are delivered to the correct places in the intestine. A specifically designed coating, for example, ensures that butyrate, one of the important components in SANACORE EN, is delivered to the more distal regions of the intestine. This all results in the unique antimicrobial and gut empowering effect of SANACORE EN.

The different components in SANACORE EN have comparable effects as described above for ZnO:

Growth promoting effect
Butyrate is a strong stimulator of several intestinal growth factors in humans and animals, for example glucagon-like peptide-2 (GLP-2). There is a great number of GLP-2-induced effects in the intestine that promote growth and performance: decrease in gastric motility, enhancement of intestinal nutrient transport, stimulation of intestinal blood flow and increase in intestinal cell proliferation.
Improved intestinal barrier function
Besides providing epithelial cells with energy, butyrate markedly enhances proliferation, differentiation and maturation of enterocytes in the small intestine (Wang et al., 2005; Manzanilla et al., 2006; Sengupta et al., 2006). Through its influence on gene expression and protein synthesis, butyrate speeds up intestinal mucosa maturation during the development or repair after injury (Kotunia et al., 2004; Guilloteau et al., 2010). Butyrate also up-regulates tight junction proteins in the intestine and in this way decreases intestinal permeability (Guilloteau et al., 2010).
Immunomodulating effect
butyrate has been shown to reduce inflammation through its effects on several types of immune cells. It is for these anti-inflammatory properties that butyrate is used in human medicine in patients with Crohn’s disease, which is characterized by excessive intestinal inflammation.
Reduced ion secretion
Butyrate modulates ion absorption and may alleviate the severity of the diarrhoea (Vidyasagar & Ramakrishna, 2002; Binder, 2010).
Effect on bacteria

For the development of SANACORE EN several components with a distinct antibacterial spectrum were selected ensuring that their combination resulted in a broad antibacterial spectrum (Gram-negative and Gram-positive).
Butyrate also stimulates antimicrobial host defense peptides secretion in the avian GIT (Sunkara et al. 2011).
Studies done by Galfi and coworkers (Galfi et al., 1991) have shown that butyrate increases the number of intestinal lactic acid and lactobacilli in butyrate-fed pigs, while decreasing the number of coliforms and E. coli, hereby ensuring a healthier and stable intestinal microbiota.
A trial done at the University of Bologna (Bosi et al., 2009) looked into the possible impact of butyrate (coated and uncoated) on E. coli K88 (ETEC) infection in piglets. The piglets receiving the uncoated sodium butyrate showed a lower mortality rate (5.0 per cent) and a higher growth than the challenged control group. In the group receiving the coated sodium butyrate, none of the piglets died and the growth rate was even slightly higher in comparison with the piglets that were not challenged.
In studies of Boyen and co-workers it was shown that butyrate, when present in the intestinal tract, is able to alter virulence properties of Salmonella Typhimurium and decrease intestinal colonisation in pigs (Boyen et al., 2008). The mode of action of this activity of butyrate is hypothesized to be at least partially mediated through modulation of bacterial gene expression. Butyrate specifically down-regulates Salmonella Pathogenicity Island 1 (SPI-1) gene expression, hereby preventing invasion of intestinal epithelial cells, one of the important steps of Salmonella pathogenesis in the bird (Gantois et al., 2006). Van Immerseel and colleagues (2005) also demonstrated the importance of an effective coating in order to get significant reduction in Salmonella colonisation in the ceca and internal organs in vivo.

Recently, several field trials have indeed shown that the use of SANACORE EN allows for reduction in the use of ZnO in piglets in the post-weaning phase.

Filed Trials with SANACORE EN

In a first trial, 68 crossbred piglets (LD-LW × Pietrain), weaned at 28 days of age, were allocated to one of two dietary treatments based on live weight (see below).

The following dietary treatment groups were evaluated:

It is important to allow some time before relying completely on the effects of SANACORE EN: strengthening intestinal barrier function and shifting a microbiota in a stable, healthy direction takes some time. For this reason, ZnO was only left out in the starter phase.

Zootechnical parameters were measured at the end of pre-starter phase and starter phase, when piglets were 42 and 76 days old, respectively.

Performance parameters during the trial:

SANACORE EN inclusion in pre-starter feed (on top of ZnO), resulted in a significant increase in feed intake of 48 g per day and an increase in ADG of 10g per day. These results indicate that using the product on top of normal ZnO usage can proof to be beneficial.

SANACORE EN inclusion in starter feed (no ZnO) lead to higher performance in comparison with the control group (standard ZnO inclusion): a significant increase in feed intake with 56g per day, a significant increase in ADG of 94g per day and a reduced feed conversion ratio (0.29 lower).

When looking at the performance results throughout the complete trial period (pre-starter + starter period), SANACORE EN application in pre-starter (on top of ZnO) and starter (no ZnO) feeds, resulted in a significant increase in feed intake of 56g per day, a significant higher ADG of 71g per day, and lower FCR (-0.2) in comparison with a standard ZnO programme (3kg per tonnes throughout pre-starter and starter). At the end of the trial period at 76 days of age, piglets on the SANACORE EN weighed on average 3.6kg heavier than the piglets from the straight ZnO programme.

Faecal consistency was monitored throughout the trial period and no severe diarrhoea was observed in the piglets of both trial groups.

This study demonstrated the potential of SANACORE EN in weaning piglet diets with reduced levels of ZnO and instigated further field trials using the same strategy. As the goal of these field trials was to include larger numbers of animals, the set-up was done differently to the first trial in the sense that comparison was done over time, not in one trial (historical comparison).

Trial with 1,000 piglets per group, weaned at 28 days

In this field trial, feed intake and ADG was increased and FCR and mortality was decreased when using the SANACORE EN strategy.

Trial results from 3.750 sows, 19.000 piglets

Results of this trial show and increase in weight at 76 days (end of starter period) and lower mortality with SANACORE EN programme.

Trial results from 16,000 piglets per group

In the above field trial, a financial comparison was made taking into account all performance benefits but also the cost of SANACORE EN and of medication per piglet.

Next to the benefits of being able to reduce ZnO usage, applying the SANACORE EN programme resulted in a financial profit for the pig producer.

Summary

In conclusion, several field trials have proven the potential of SANACORE EN to be used in programmes with reduced ZnO levels. Not only does this programme diminish the dependency of ZnO, it also results in a financial profit, making it an economical solution.

The positive results seen can easily be explained when comparing the mode of action of SANACORE EN with that of ZnO.

Applying a sound SANACORE EN strategy can make a real difference for producers that want to limit the use of ZnO.

References

Binder, H.J. 2010. Role of colonic short-chain fatty acid transport in diarrhea. Annual review of physiology 72: 297-313.
Bosi, P., Messori, S., Nisi, I., Russo, D., Casini, L., Coloretti, F., Schwarzer, K. and Trevisi, P. 2009. Effect of different butyrate supplementations on growth and health of weaning pigs challenged or not with E. coli K88. Italian Journal of Animal Science, No2s. Proceedings of the 18th ASPA Congress, Palermo, June 9-12: 268-270.
Boyen, F., Haesebrouck, F., Vanparys, A., Volf, J., Mahu, M., Van Immerseel, F., Rychlik, I., Dewulf, J.,
Ducatelle, R. and Pasmans, F. 2008. Coated fatty acids alter virulence properties of Salmonella Typhimurium and decrease intestinal colonization of pigs. Veterinary microbiology 132(3-4):319-327.
Galfi, P., Neogradi, S. and Sakata, T. 1991. Effects of volatile fatty acids on the epithelial cell proliferation of digestive tract and its hormonal mediation. In “Physiological aspects of digestion and metabolism in ruminants”, Ed.: Tsuda, T., Sasaki, Y., and Kawashima, R., Academic Press, Orlando, Florida, 49-59.
Gantois, I., Ducatelle, R., Pasmans, F., Haesebrouck, F., Hautefort, I., Thompson, A., Hinton, J. C. and Van Immerseel, F. 2006. Butyrate specifically down-regulates salmonella pathogenicity island 1 gene expression. Applied and Environmental Microbiology 72(1): 946-949.
Guilloteau, P., L. Martin, L., Eeckhaut, V., Ducatelle, R., Zabielski, R. and Van Immerseel, F. 2010. From the gut to the peripheral tissues: the multiple effects of butyrate. Nutrition Research Reviews 23(2):366-384.
Huang, S.X., McFall, M., Cegielski, A.C., and Kirkwood, R.N. 1999. Effect of dietary zinc supplementation on Escherichia coli septicemia in weaned pigs. Swine Health and Production. 7(3):109-111.
Kim, J.C., Hansen, C.F., Mullan, B.P. and Pluske, J.R. 2012. Nutrition and pathology of weaner pigs: Nutritional strategies to support barrier function in the gastrointestinal tract. Animal Feed Science and Technology, 173(1-2)3-16.
Kotunia, A., Wolinski, J., Laubitz, D., Jurkowska, M., Rome, V., Guilloteau, P. and Zabielski, R. 2004. Effect of sodium butyrate on the small intestine development in neonatal piglets fed [correction of feed] by artificial sow. Journal of Physiology and Pharmacology. 55 Suppl 2:59-68.
Li, X.L., Dong, B., Li, D.F., Yin, J.D. 2010. Mechanisms involved in the growth promotion of weaned piglets by high-level zinc oxide. Journal of Animal Science and Biotechnology, 1:59-67.
Liedtke, J. and W. Vahjen 2012. In vitro antibacterial activity of zinc oxide on a broad range of reference strains of intestinal origin. Veterinary Microbiology 160(1-2):251-255.
Lizardo, R. 2004. Interaction between high dosed ZnO and phytase. Expert Talk Pig Progress, 2004.
Manzanilla, E.G. (2006). Effects of butyrate, avilamycin, and a plant extract combination on the intestinal equilibrium of early-weaned pigs. Journal of Animal Science 84(10):2743-2751.
Poulsen, H.H.D. (1989). Zinc oxide for weaned pigs. In: Proceedings of the 40th annual meeting of the European Association for Animal Production. EAAP Publications, Dublin. 265-266.
Roselli, M., Finamore, A., Garaguso, I., Britti, M. S. and Mengheri, E. 2003. Zinc oxide protects cultured enterocytes from the damage induced by Escherichia coli. Journal of Nutrition. 133(12):4077-4082.
Sales, J. 2013. Effects of pharmacological concentrations of dietary zinc oxide on growth of post-weaning pigs: a meta-analysis. Biol. Trace Elem. Res. 152(3):343-349.
Sandstrom, B. 2001. Micronutrient interactions: effects on absorption and bioavailability. British Journal of Nutrition. 85 Suppl 2:S181-185.
Sengupta, S., Muir J.G. and Gibson, P.R 2006. Does butyrate protect from colorectal cancer? Journal of Gastroenterology and Hepatology 21(1 Pt 2):209-218.
Starke, I., Vahjen, W., Zentek, J. 2012. Dietary zinc oxide leads to short and long term modification in the intestinal microbiota of piglets. XII International symposium on digestive physiology in pigs. May-June 2012, USA.
Sunkara, L.T., Achanta, M., Schreiber, N.B., Bommineni, Y.R., Dai, G., Jiang, W., Lamont, S., Lillehoj, H.S., Beker, A., Teeter, R.G. and Zhang, G. 2011. Butyrate enhances disease resistance of chickens by inducing antimicrobial host defense peptide gene expression. PloS one 6(11):e27225.
Vidyasagar, S. and Ramakrishna, B.S. 2002. Effects of butyrate on active sodium and chloride transport in rat and rabbit distal colon. Journal of Physiology 539(Pt 1):163-173.
Wang, J., Chen Y., Wang, Z., Dong, S. and Lai, Z. 2005. Effect of sodium butyrate on the structure of the small intestine mucous epithelium of weaning piglets. Chinese Journal of Veterinary Science and Technology 35(4):298-301.
Zhang, B. and Y. Guo (2009). Supplemental zinc reduced intestinal permeability by enhancing occludin and zonula occludens protein-1 (ZO-1) expression in weaning piglets. British Journal of Nutrition 102(5):687-693.