Ways to improve zinc bioavailability
Schlegel Patrick, Agroscope Liebefeld-Posieux ALP, 1725 Posieux, Switzerland
Jondreville Catherine, INRA, Nancy Université, USC340 Animal et Fonctionnalités des Produits Animaux, 54505 Vandoeuvre-les-Nancy, France
E-mail: patrick.schlegel@alp.admin.ch
Summary
Zinc is a heavy metal, an essential trace element and a non renewable resource. Dietary factors reducing Zn bioavailability in monogastrics are mainly identified as phytate, non starch polysaccharides and excessive dietary contents of Fe and Cu. Dietary phytate is the major limiting factor for Zn bioavailability in rats, broilers and piglets and non starch polysaccharides play their negative role in broilers. In cereal and oilseed diets, phytate antagonism principally concerns native Zn, already bound to phytates. Data strongly suggests that various levels of plant phytates do not interact with supplemented Zn in broilers and in piglets. In diets with added sodium phytate the relative bioavailability of an organic Zn source may be improved, but in the presence of plant phytate, their relative bioavailability is not different in broilers and piglets. Ways to improve Zn bioavailability in broilers and piglets therefore need to be focused on native Zn. This, basically by removing plant phytates with the use of feed components low in phytic P, by hydrolyzing phytates with microbial phytase and by lowering digestive pH with acids. These actions are more pronounced in piglets than in broilers, most probably due to their respective digestive conditions. The pH in the gizzard is lower than in the stomach, which allows broilers to dissociate, at least partially, zinc-phytates complexes. In the end, the potential to improve Zn bioavailability in piglets is large, but seems limited in broilers.
Keywords: zinc, phytate, bioavailability, broiler, piglets
1 Introduction
The global demand for meat production is expected to increase by 70% from 1999 to 2030 (FAO, 2002). Fastest development is foreseen in broiler production. The future animal production should aim for a sustainable intensification and for further improvements in the efficient use of limiting resources. An efficient use of Zn in animal nutrition is encouraged in this respect, as it is one of the few heavy metals, which is simultaneously an essential trace mineral for living animals. As a transition metal, Zn presents a high affinity for complexing with ligands, such as proteins which involve this element to numerous structural and physiological functions in the metabolism. Zinc contents of feed components, their bioavailability and feed intake are susceptible to vary greatly. This leads to the application of comfortable safety margins, when compared to recommended dietary Zn levels in poultry and pigs. A better understanding of such variability will allow the feed industry to fine tune Zn supplementation levels.
The aim of this paper is to review the major dietary factors affecting Zn bioavailability in monogastrics and to suggest ways for its improvement.
2 Dietary factors reducing zinc bioavailability in monogastrics
Zinc bioavailability can be defined as the maximal degree of ingested Zn, utilized for the biological, physiological and storage functions by a healthy animal. As the metabolic use of Zn is relatively high, Zn bioavailability is mainly limited by its absorption. Zn absorbability can be altered when its intra-luminal solubility is reduced or when competition with plethoric levels of other minerals are ingested.
2.1 Reaction with feed components to form insoluble complexes: phytates and fibers When Zn is supplemented into a purified diet with levels not reaching the homeostatic Zn regulation capacity of rats, its true absorption reached levels close to 100% (Weigand and Kirchgessner, 1980). Such a diet does not contain any complexing agent known to interact with dietary Zn, suggesting that the supplemented zinc is completely soluble at absorption sites. When sodium phytate is gradually supplemented (0 to 9 g / kg diet), true Zn absorbability in adult rats was progressively reduces from 55 to 0% (Windisch and Kirchgessner, 1999a). This molecule contains sodium (Na) bonds which are easily dissociated in aqueous solutions. Stronger cations, such as Zn replace Na to form insoluble phytate – Zn complexes (Davies and Nightingale, 1975). These findings illustrate the strong antagonistic effect of supplemented sodium phytates on Zn absorbability, thus reducing Zn bioavailability.
The addition of Ca in form of chloride did not alter Zn bioavailability in rats fed a phytate free diet. However when supplemented into a diet containing sodium phytate, Ca addition depressed Zn bioavailability (Oberleas et al., 1966).
In poultry and swine nutrition, it is difficult to avoid the presence of phytates as they are the main storage forms of P in seeds. Diets based on corn and soybean meal generally contain between 2,0 and 2,5 g phytic P / kg. Zinc content in feed components from plant origin is positively correlated to the phytic P content, with ~10 mg of Zn to 1 g phytic P (Revy et al., 2003). Rodrigues-Filho et al. (2005) determined that two out of the three identified phytate molecules from wheat grains contain Zn (Figure 1).
Figure 1 Molecular structure of Na3Mn5 * (C6H6O24P6)OH * 9H2O and of Na3Zn(II)5 * (C6H6O24P6)OH * 9H2O. Arrow show either Mn or Zn atoms. H atoms are not represented
(from Rodrigues-Filho et al., 2005).
The negative effect of Ca on Zn bioavailability observed in diets with added sodium phytate seems however very limited or even inexistent when conventional diets (Ca up to 11 g / kg) are fed to broilers and piglets (Windisch and Kirchgessner, 1995; Larsen and Sandström, 1993).
Diets rich with non starch polysaccharides (NSP) reduce Zn bioavailability in broilers and in piglets. Newton et al. (1983) used wheat bran to increase dietary fibers in piglets. As wheat bran is rich in phytic P, the observed negative effect of dietary fiber on Zn bioavailability in pigs cannot be definitively attributed to fibers. In broilers, van der Aar et al. (1983) assumed that the addition of dietary fiber would increase the intestinal mucosa (rich in Zn content) turnover through an abrasive effect, thus leading to increased Zn loss. Mohanna and Nys (1999c) used arabic gum to increase the digestive viscosity in broilers. This product is not known to directly interfere with mineral bioavailability. These authors measured a reduced Zn bioavailability with increased digestive viscosity. Feed components rich in NSP, such as barley or wheat may therefore reduce Zn bioavailability in broilers, not only due to their phytic P content but also due to an increased digestive viscosity.
2.2 Mineral competition at absorption site
An excessive supply of dietary Fe reduced Zn bioavailability as these two elements are probably absorbed with the same cellular membrane transporters, such as Zip 14 (Hansen et al., 2009). An excessive supply of dietary Cu reduces Zn bioavailability as these two elements bind to metallothionein in the enterocyte. In the body, both elements are transported to the liver mainly bound to albumin. An excess of Cu may therefore limit cell storage capacity and transport in the bloodstream for Zn (Jondreville et al., 2002). Dietary Fe and / or Cu levels needed to impair Zn bioavailability in broilers and in piglets have not been assessed.
3 Ways to improve zinc bioavailability
3.1 The use of enzymes
Microbial phytase hydrolyzes up to respectively 35 and 50% of the dietary phytates in poultry and pigs (Selle and Ravindran, 2007). Through this hydrolysis Zn is liberated from phytate.
The addition of microbial phytase (500 FTU) increased Zn solubility in the piglet stomach, but not significantly in the broiler gizzard (Schlegel et al., 2010). Jondreville et al. (2005;
2007) have estimated that 500 FTU from microbial phytase added into corn soybean meal diets permits to replace an equivalent amount of respectively 32 and 5 mg Zn / kg from ZnSO4 in pigs and in broilers. The dietary addition of NSP-hydrolyzing enzymes improves nutrient availability for broilers fed NSP rich diets and may as suggested by Mohanna and Nys (1999c) also be beneficial for Zn bioavailability.
3.2 The use of feed components low in phytate contents
When Zn unsupplemented diets are considered, femoral Zn content of pigs was improved when soybean meal was replaced with skim milk powder (Ashida et al., 2000). Veum et al.
(2009) did not measure any difference on tibia ash and Zn contents of pigs fed either a diet containing a genetically modified low-phytate barley or a wild type barley (respectively 1,3 and 2,3 g phytic P / kg), with equal Zn contents. However, the same parameters did not change either with gradual dietary Zn addition. Data from our latest experiment (Schlegel et al., 2010) show that soluble Zn contents in the stomach were equal between piglets fed a diet low in phytate and in native Zn (1,3 g phytic P / kg diet; 25 mg Zn / kg diet) and a corn soybean meal diet rich in phytate and native Zn (2,3 g phytic P / kg diet; 38 mg / kg diet).
Pigs fed the low phytate diet (also lower in Zn) had higher bone Zn contents (+ 44%) than pigs fed the phytate rich diet. In broilers, Linares et al. (2007) conducted a similar study to Veum et al. (2009) and measured an improved bone Zn content (+ 217%) when chicks were fed the diet containing the low phytate barley (0,03 compared to 1,43 g phytic P / kg diet).
The antagonism of phytate on native Zn availability was assessed using the molar phytate / Zn ratio adjusted with the estimates of either Selle and Ravindran (2007) or Jondreville et al.
(2007) within the linear response of bone Zn. Figure 2 shows bone Zn contents from broiler and weaned piglet experiments (INRA experiments and wild type barley by Linares et al., 2009) dependent from the most suitable corrected phytate / Zn molar ratio. The weak correlation between the adjusted phytate antagonism and bone Zn in broilers and the stronger correlation of the adjusted (Jondreville et al., 2007) dietary native Zn content (Bone Zn [mg / kg DM] = 0.65 (P > 0.1) + 3.15 (P = 0.001)* adjusted dietary Zn [mg / kg], R2 = 59.4, r.m.s.e
= 21.8) indicates that native dietary Zn bioavailability for broilers is independent from dietary phytate.
Figure 2 Response of bone Zn content on the dietary adjusted molar phytate / Zn ratio in broilers (left) and piglets (right) fed cereal based diets unsupplemented with Zn. (from Mohanna et Nys, 1999a ; 1999b ; Revy et al., 2002 ; 2004 ; 2006 ; Jondreville et al., 2005 ; 2007 ; Linares et al., 2007 ; Schlegel et al., 2010).
35 30 25 20 15 10 5 0 200
150
100
50
0
A djusted molar phytate / Zn ratio (Jondreville et al., 2007)
Bone Zn [mg / kg DM]
S 30.7924
R-Sq 19.1%
Bone Zn = 180.7 (P = 0.001) - 3.258 (P = 0.1) * adjusted ratio
35 30 25 20 15 10 5 0 100
80
60
40
20
0
Adjusted molar phytate / Zn ratio (Selle and Ranvindran, 2007)
Bone Zn [mg / kg DM]
S 6.28941 R-Sq 89.3%
Bone Zn = 103.7 (P < 0.001) - 2.694 (P < 0.001) * Adjusted ratio
3.3 The supplementation of highly bioavailable zinc sources
Organic Zn sources are generally considered as more bioavailable than inorganic sources, such as oxides or sulfates. In organic Zn sources, the metal is sequestered by carbohydrates (commonly named polysaccharides) or bound to molecules containing nitrogen such as amino acids, peptides or proteins (commonly named complexes, chelates or proteinates). Several categories are defined by the Association of American Feed Control Officials and the European legislation. The defined categories are not compatible between the two organisms.
They are based on chemical properties, but most do not include any analytical method to verify them. Most organic Zn sources available on the market are therefore considered as organic based on the chemical formula used for production. Methods to identify bonds between Zn and ligand(s) and to measure their integrity in aqueous solutions are necessary to know the Zn fraction of a product effectively in organic form, when marketed and when ingested.
To identify organic Zn sources, X-ray diffraction was proposed (Hynes and Kelly, 1995).
Oguey et al. (2008) have identified a zinc glycinate and reported that the entire Zn was bound to glycine.
To measure the integrity or the binding strength of an organic Zn source in a aqueous media, the soluble Zn fraction was analyzed mainly by three methods: ultra filtration which consists in filtering the soluble Zn fraction through defined molecular sizes and to measure Zn and N contents in the filtrate; gel permeation chromatography which consists in the assumption that bound Zn elutes earlier from the column than Zn ions because of its larger molecular size;
polarography which is based on the necessary energy needed to draw a current through mercury amalgated Zn. With ultra filtration, Helle and Kampf (2009) reported that more than 85% of the soluble Zn and between 5 and 90% of the soluble N were smaller than a 1000 Dalton, indicating that soluble N and soluble Zn were dissociated when not found in comparable proportions after ultra filtration. With polarography, Leach et al. (1997) reported that the non ionized soluble Zn fraction, representing the organic Zn fraction was between 1 and 35% of the tested products. Using gel permeation chromatography, Cao et al. (2000) reported that Zn from all the products they tested was dissociated with pH between 2 and 5.
These authors also reported that, respectively 6 and 10 to 13% of the Zn from sources with respectively amino acids and peptides or proteins as ligands, was considered as organic. Cao et al. (2000) and Huang et al. (2009) determined the product integrity from organic Zn
sources (polarography) and their Zn bioavailability relative to ZnSO4 from broiler tibia Zn.
The combined results suggest that there is no correlation (R2 = 0,26) between Zn source integrity and Zn bioavailability.
Last but not least, Vacchina et al. (2009) identified and measured the bond integrity of zinc glycinate in solution. The results indicate that the proportion of free glycine remains stable between pH 7 and 4, but increases progressively from pH 4 to 2.
It is suggested that Zn from organic sources is protected by the ligand from reacting with feed antagonists, such as phytate to form insoluble complexes. Zinc from organic sources is absorbed by the intestinal cells as an ion, or it is also suggested that Zn is absorbed intact thanks to a second absorption pathway related to the ligand.
Using in vitro experiments with intestinal segments from rats, broilers or pigs, Zn uptake by intestinal cells was improved when using organic sources compared to salts (Ashmead et al., 1985; Hill et al., 1987a). However, other experiments on rats failed to show this effect (Hill et al., 1987b ; Hempe et Cousins (1989). Hill et al. (1987a) measured an identical Zn transfer from intestinal pig or broiler cells to the serosal side with an organic source compared to a salt. Beutler et al. (1998) demonstrated that intestinal cells absorb Zn and methionine independently from each other when using an organic source (zinc methionine). This data suggests that Zn from organic sources may be better taken up by the intestinal cell. However, no data on organic Zn provides evidence for an improved Zn transfer to the serosal side, meaning that an increased intestinal Zn absorption, thanks to a second pathway, is not proven.
In vivo, we measured the net fluxes of Zn in 65Zn labeled growing rats fed a purified Zn diet containing 8 g / kg of sodium phytate (Schlegel and Windisch, 2006). Zinc sources were ZnSO4 (0, 10 and 55 mg Zn / kg diet) and the previously identified (Oguey et al., 2008;
Vacchina et al., 2009) Zn glycinate (0, 10 mg Zn / kg diet). Relative to ZnSO4, rats fed Zn glycinate numerically improved apparent and true Zn absorption (respectively +30% and +17%) and significantly improved Zn retention (+ 30%). Expressed as percent of ingested Zn, all Zn fluxes, except urinary Zn, were significantly improved with rats fed Zn glycinate. This data suggests that the amino acid protected Zn, at least partially, from complexing with added sodium phytate in the digestive tract. As endogenous Zn losses (faecal and urine) were not different between sources, the additionally absorbed Zn from Zn glycinate could be used by the metabolism. The same Zn sources (ZnSO4 and Zn glycinate) were supplemented (0, 15 mg Zn / kg) to three corn soybean meal basal diets varying in their native Zn (25, 38 and 38 mg / kg), phytic P (1,3, 2,3 and 2,3 g / kg) and phytase activity (27, 201 and 688 FTU / kg).
Diets were fed to one day old broilers and weaned piglets during 20 days (Schlegel et al., 2010). In both species, Zn status (plasma Zn and bone Zn) was improved with supplemental Zn, irrespective of Zn source and basal diet. There was no interaction between basal diet and supplemented Zn suggesting that there was no antagonistic effect from plant phytate on supplemented Zn in either specie. The potential benefit of Zn glycinate in avoiding a complexation with dietary phytates, as previously shown on rats fed sodium phytate (Schlegel and Windisch, 2006) was not demonstrated in diets containing various levels of plant phytates. The relative bioavailability of organic Zn sources was recently assessed in broilers and weaned piglets using a meta-analysis technique (Schlegel et al., unpublished). No significant difference between organic and inorganic sources was calculated within the dietary Zn range presenting a dose-response (Table 1).
All this data suggests that the potential of organic Zn sources compared to inorganic Zn sources cannot be expressed in broiler and piglet diets, because there is no antagonism between supplemented Zn and dietary plant phytates and because there is no clear indication that organic sources are absorbed intact into the bloodstream.
Table 1 Relative bioavailability of organic Zn sources in broilers and weaned piglets.
Parameter
RBV 1) P-value 2) RBV 1) P-value 2)
Gain to feed 106 > 0.05 - -
Plasma Zn 93 > 0.05 92 > 0.05
Plasma AP activity - - 94 > 0.05 1) RBV: Relative bioavailability. Relative diff erence of Liver Zn - - 97 > 0.05 response slope betw een organic and inorganic source.
Bone Zn 110 > 0.05 100 > 0.05 2) Dif ference of the linear ef fects betw een Absorbed Zn - - 117 > 0.05 inorganic and organic Zn sources.
Piglet Broiler
4 Differences between broilers and piglets
As said earlier, Zn absorbability is dependent from pH conditions in the digestive tract. Based on our combined broiler and piglet experiment (Schlegel et al., 2010), Zn solubility (Znsol) is dependent from gizzard / stomach pH with following regression: Znsol [%] = 225.9 - 75.53 * pH + 6.571 * pH2; R2 = 0.62 ; P < 0.001 ; n = 81). Zn solubility was not dependent from intestinal pH. Bone Zn was dependent from Znsol in gizzard / stomach: Bone Zn [mg / kg DM] = - 12.60 + 9.199 * Znsol - 0.1027 * Znsol2; R2 = 0.67; P < 0.001 ; n = 81). According to Ellis et al. (1982) Zn-phytates complexes dissociate as soon as pH is decreased down to 4.
Thus the low pH in gizzard (~3,5) allows zinc-phytates complex to dissociate, even in the absence of phytase, whereas, in piglet’s stomach, where the pH is higher (~4,5), phytates must be hydrolyzed by phytase before Zn can be released. This phenomenon would explain why microbial phytase (500 FTU / kg diet) is eight times less efficient in improving Zn bioavailability in broilers compared to piglets (Jondreville et al., 2005; 2007). This phenomenon would also explain the beneficial effects of the dietary addition of organic acids on Zn bioavailability in piglets (Höhler et Pallauf 1993; Roth et al., 1998), but not in broilers (Brenes et al., 2003). This phenomenon would result in a physiologically higher Zn bioavailability in chickens than in piglets, explaining the lower dietary Zn requirements of chickens than piglets. In the end, the potential to improve native Zn bioavailability in pigs is large, whereas in poultry, it is rather limited.
5 Conclusions
Ways to improve dietary Zn bioavailability in broilers are lower than in piglets, as the birds are able, at least partially, to separate Zn from phytates in the gizzard. In broilers, improvements in Zn bioavailability remain possible with the dietary addition of microbial phytase. In piglets, however, the same actions are highly efficient to improve Zn bioavailability. As plant phytates are bound with strong cations such as native Zn or Mn, data suggest that the bioavailability of supplemented Zn is not negatively affected by plant phytate.
A soluble Zn source, such as ZnSO4 is therefore highly bioavailable in broilers and piglets.
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