Effect of high dietary zinc oxide on the caecal and faecal short-chain fatty acids and tissue zinc and copper concentration in pigs is reversible after withdrawal of the high zinc

ORIGINAL ARTICLE

Effect of high dietary zinc oxide on the caecal and faecal short-chain fatty acids and tissue zinc and copper concentration in pigsis reversible after withdrawal of the high zinc oxide from the dietP. Janczyk1, K. B€using2, B. Dobenecker3, K. N€ockler1 and A. Zeyner4

1 Unit for Molecular Diagnostics, Genetics and Pathogen Characterisation, Department of Biological Safety, Federal Institute for Risk Assessment

Berlin, Germany

2 Chair of Nutrition Physiology and Animal Nutrition, Faculty of Agricultural and Environmental Sciences, University of Rostock, Rostock, Germany

3 Animal Nutrition and Dietetics, Department of Veterinary Science, Ludwig-Maximilians-University Munich, Oberschleißheim, Germany, and

4 Group Animal Nutrition, Institute of Agricultural and Nutritional Sciences, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany

Summary

Zinc oxide (ZnO) used in high (‘pharmacological’) levels to prevent diarrhoea in pigs is assumed to reduce copper

(Cu) in tissues and inhibits large intestinal microbial fermentation. To test it, German Landrace pigs were weaned

on d28 of age and fed diets containing either 100 (LowZinc, LZn, n = 10) or 3100 mg ZnO/kg (HighZinc, HZn,

n = 10). The mixed feed (13.0 MJ ME, 18.5% crude protein) was based on wheat, barley, soya bean meal and

maize. After 4 weeks, the HZn group was further fed 100 mg ZnO/kg for another 2 weeks. Caecal contents, fae-

ces and tissues were collected after 4 weeks (n = 5 and n = 10 respectively) and 6 weeks (n = 5 and n = 5 respec-

tively). Faeces and caecal content were analysed for dry matter (DM), pH, ammonia, lactic acid (LA) and short-

chain fatty acids (SCFA) on native water basis. ANOVA was performed to elucidate significant differences at

p < 0.05. No diarrhoea occurred. After 4 weeks, the caecal contents’ pH increased (p < 0.001) and butyric

(p < 0.05) and valeric acid (p < 0.01) decreased in the HZn group in comparison with LZn. In faeces, a decrease

of acetic (p = 0.009), butyric (p = 0.007) and valeric acid (p = 0.046), as well as reduced acetic:propionic acid (A:

P) ratio (p = 0.025) was observed in the HZn group in comparison with LZn. Faecal ammonia decreased in HZn

(p = 0.018). No differences (p > 0.05) were recorded in caecal contents after 6 weeks. In faeces, acetic acid

remained lower in the HZn group in comparison with LZn (p = 0.006), as did the A:P ratio (p = 0.004). Zn con-

centration in liver, kidneys and ribs, and Cu concentrations in kidneys increased in HZn. Withdrawal of ZnO

resulted in reversibility of the changes. The effect on butyric acid should be discussed critically regarding the

energetic support for the enterocytes. High Zn and Cu tissue concentrations should be considered by pet food

producers.

Keywords intestinal microbial activity, pig, short-chain fatty acids, zinc oxide, copper

Correspondence Pawel Janczyk, Karl-Heinrich-Ulrichs-Strasse 8A, 10787 Berlin, Germany. Tel: +49-1708095390; Fax: +49-32221007198;

E-mail: [email protected]

Received: 30 January 2014; accepted: 27 June 2014

Introduction

Zinc (Zn) is a component of over 300 enzymes in

the mammalian cells and thus an essential micro-

nutrient (Poulsen and Larsen, 1995; Terr�es et al.,

2001). Growing pigs (up to 20 kg body weight)

require approximately 100 mg Zn/kg dry matter

(DM) of feed, and later approximately 80 mg Zn/kg

DM of feed (Gesellschaft f€ur Ern€ahrungsphysiologie,

2006). Because the availability of Zn occurring

naturally in feedstuffs may often be reduced by

many factors (Smith et al., 1962; Pond et al., 1985;

Poulsen and Carlson, 2001), supplementation of

diets with Zn by way of both organic and inorganic

sources has become a standard (Richards et al.,

2010). Furthermore, supplementation of dietary

zinc oxide (ZnO) at high levels (2000–3000 mg/kg)

has been used as prophylactic measure against post-

weaning diarrhoea and to improve growth perfor-

mance of weaning pigs (Poulsen, 1995; Wang et al.,

2010). Next to its direct effect on the Zn homeosta-

sis in the host, it is believed that ZnO acts locally

in the intestinal lumen affecting the microbiome

(Pieper et al., 2011), but its exact growth-promoting

Journal of Animal Physiology and Animal Nutrition 99 (Suppl. 1) (2015) 13-22 © 2015 Blackwell Verlag GmbH 13

DOI: 10.1111/jpn.12307

and antidiarrhoea mode of action still remains

unclear.

Enteric bacteria can be inhibited by increased Zn

concentrations (Surjawidjaja et al., 2004), and effects

on the composition of ileal core microbiome have

been reported (Vahjen et al., 2011). High concentra-

tions of Zn were shown to affect the functionality and

the microbial community of soils (Bewley and Sto-

tzky, 1983). So far there are little data on functional

changes of the large intestinal microbiome caused by

increased ZnO levels in the diet of pigs (Højberg et al.,

2005), but no data were found on their reversibility

after removal of the excess of ZnO. Few studies inves-

tigated the concentrations of Zn in the tissues after

feeding high amounts of ZnO (Hahn and Baker, 1993;

Poulsen, 1995; Shell and Kornegay, 1996; Jensen-Wa-

eren et al., 1998; Case and Carlson, 2002; Rincker

et al., 2005). However, no data could be found for tis-

sue concentrations of Zn when ZnO was fed in high

amounts for a period of time with the following reduc-

tion of ZnO level in the diet.

Excessive Zn in the diet is made responsible for a

reduction of the copper (Cu) bioavailability (Hill et al.,

1983). Increased dietary ZnO could therefore result in

a decrease of Cu in tissues of weaned pigs. Tissue

(especially edible as muscles) concentration of Zn and

Cu is important for the calculations of dietary intake

of these micronutrients, both for humans and espe-

cially for pets, as most of the animal by-products such

as liver and kidney are taken for pet food production.

This study aimed to evaluate the effect of a with-

drawal period for 2 weeks following the 4 weeks

high-Zn nursery period with large intestinal pH,

short-chain fatty acids, lactate and ammonia, as well

as tissue Zn and Cu concentrations as the response cri-

teria.

Material and methods

Animals and treatment

Twenty German Landrace piglets of both sexes were

weaned at 28 days of age (8.5 � 1.0 kg body weight),

transported to the experimental facility and allocated

to pens, two pigs each (male and female). The piglets

received a commercial creep feed from 2 weeks of age

(Turbostart, Trede & von Pein, Itzehoe, Germany) that

contained 19.0% of crude protein, 7.5% of crude fat,

3.0% of crude fibre, 5.5% of crude ash, 0.7% Ca,

0.6% P, 0.25% Na, 1.5% Lys, 0,5% Met and 14.8 MJ

ME/kg. The feed containing the following supple-

ments (calculated values): 22 500 I.U. Vitamin A

(E672), 2000 I.U. Vitamin D (E671), 150 mg Fe (Fe-

II-sulphate), 1.0 mg I (potassium iodide), 0.3 mg Co

(alcalic Co-II-carbonate), 150 mg Cu (Cu- II-sul-

phate), 70 mg Mn (Mn-III-oxide), 0.3 mg Se (sodium

selenite), 100 mg Zn (Zn sulphate). The study was

planned and performed before the ban of the use of

Co as supplement for pigs in the EU (European Com-

munity, 2013).

Five pens formed one group. Wheat–barley–soya–maize diet (Table 1) was prepared to contain 150 mg

Zn/kg diet that is the maximal allowed level of Zn

according to the EU legislation at that time (European

Community, 2003), by adding 100 mg of ZnO per kg

of diet (CAS: 1314-13-2; Sigma-Aldrich Chemie

GmbH, Germany). This feed was fed to the low-zinc

(LZn) group (control). The experimental group

received the same diet, but the Zn level was increased

by adding 3100 mg ZnO/kg – high zinc group (HZn).

Table 1 Composition of basal and experimental diets used in the study

Item [g as fed] LZn HZn

Wheat 384 384

Barley 303 303

Soya bean meal 234 234

Maize meal 10 7

Calcium carbonate 18 18

Monocalcium phosphate 18 18

Mineral mix* 13 13

Salt 2 2

Lysine-HCl 2 2

Methionine 1 1

Soybean oil 15 15

ZnO 0.1 3.1

Total 1000 1000

Nutrients (as calculated)

Dry matter [%] 87.9 87.9

Metabolisable energy [MJ] 13.0 13.0

Crude protein [%] 18.5 18.5

Starch [%] 37.6 37.6

Fibre [%] 3.5 3.5

Crude fat [%] 3.4 3.4

Crude ash [%] 8.1 8.1

Lysine [%] 1.15 1.15

Methionine [%] 0.35 0.35

Methionine + Cysteine [%] 0.7 0.7

[mg/kg DM] as analysed

Zn 179 2353

Cu 33 31

Fe 308 308

*Mineral mix contained per kg: semolina bran (36%), NaCl (33.6%), MgO

(10.5%), vit. A (600 000 IU), vit. D3 (120 000 IU), vit. E as alpha-tocoph-

erol acetate (8000 mg), vit. K3 as menadione-NaHSO3 (300 mg), vit. B1

as thamin-HCl (250 mg), vit. B2 as riboflavine (250 mg), vit. B6 as pyri-

doxol-HCl (400 mg), vit. B12 (2000 lg), nicotin acid (2500 mg), folic acid

(100 mg), biotine (25 000 lg), Ca-D-panthotenate (1000 mg), choline-Cl

(80 000 mg), MnO (6000 mg), FeCO3 (5000 mg), CuSO4 9 5H2O

(1000 mg), CoSO4 9 7H2O (30 mg), Ca(IO3)2 (45 mg), Na2SeO3 (35 mg).

Journal of Animal Physiology and Animal Nutrition © 2015 Blackwell Verlag GmbH14

Dietary zinc and intestinal microbial activity in pigs P. Janczyk et al.

To investigate the reversibility of potential changes

caused by the high content of ZnO, after 4 weeks of

treatment, the HZn group received the same feed as

LZn for another 2 weeks. Feed was fed in meal form

(88% DM) and offered mixed with some water twice

daily for an hour as semi ad libitum, to avoid refusals.

Water was provided ad libitum via nipple drinkers.

Ambient temperature was kept at 25 � 1 °C for the

first 4 weeks, and then reduced to 22 � 1 °C, with

humidity 30–55% and light regime of 12 h light and

12 h darkness. Pigs were weighed once a week before

the morning feeding. Feed intake was recorded daily

on dry matter basis for each pen. Average daily gain

(ADG), average daily feed intake (ADFI) and feed con-

version ratio (FCR) were calculated.

Faeces were collected after 4 and 6 weeks of treat-

ment, directly from the rectum, and kept cooled on

ice until further processing. At these time-points, five

pigs from each group were euthanized by overdose of

pentobarbital under general azaperon (Stresnil; Jans-

sen Animal Health, Neuss, Germany) and ketamine

(Ketamin 10%; Bremer Pharma GmbH, Warburg,

Germany) anaesthesia; the feed was withdrawn 2 h

before the euthanasia. Total digesta from caecum was

collected. The whole liver, both kidneys, left ham and

X-XIIth ribs were dissected for analysis of Zn and Cu

concentrations.

The animal experiment was approved by the local

authority (Landesamt f€ur Gesundheit und Soziales,

LAGeSo, Berlin) under the accession number G 0349/

09.

Analysis of digesta and faeces

Faeces and caecal digesta were analysed for DM, pH,

ammonia, lactic acid (LA) and short-chain fatty acids

(SCFA), namely acetic, butyric, propionic, valeric, and

capronic acid and their isoforms. DM was determined

by freezing fresh digesta or faeces at �20 °C with final

lyophilisation. For other analyses, fresh samples were

diluted 1:3 with sterile distilled water (20 g sample,

60 ml water) and homogenised in BagMixer (Inter-

science, Saint Nom, France) at maximum speed for

30 s. Forty-five millilitre of the homogenate was

transferred into 50 ml-Falcon tube and pH was mea-

sured using Digital-pH-Meter 646 (Knick, Berlin,

Germany). Further, the homogenates were centri-

fuged at 4000 g for 10 min, at 20 °C. Supernatant was

collected and stored at �20 °C before further analyses.

Within the thawed samples, the contents of LA and

SCFA were analysed by HPLC and gas chromatogra-

phy, respectively, as described (Hackl et al., 2010).

The content of ammonia was determined by the

modified microdiffusion method (Voigt and Steger,

1967). All concentrations were calculated as mmol of

substance per litre of native water content in caecum

digesta/faeces according to Zeyner et al. (2004). The

ratio of acetic to propionic acid (A:P) was calculated.

Zinc and copper analysis

Dissected organs, ham and ribs were weighed and ho-

mogenised using a kitchen mill (Moulinex, SEB S.A.,

Ecully Cedex, France). The homogenised tissues were

put into aluminium bowls and frozen at �20 °C. Theywere subsequently lyophilised, packed hermetically

and sent to the analytic laboratory.

The determination of Zn and Cu in the homogen-

ised and lyophilised tissues was performed in an acety-

lene flame using atomic absorption spectrometry

(PerkinElmer Inc., Waltham, Massachusetts, USA)

after wet hydrolysis with HNO3 in a microwave (Mile-

stone Inc., Shelton, Connecticut, USA).

Statistical analysis

Levene test was applied for testing the homogeneity

of variance of all traits. Effect of different ZnO levels

on the body weight of the pigs was analysed as analy-

sis of variance (ANOVA) with repeated measures with

pig as experimental unit. The effect of ZnO on DM,

pH, ammonia, LA and SCFA, as well as on tissue Zn

and Cu concentrations, were tested by t-test to eluci-

date which differences were responsible for the

observed effects. The calculations were performed

using SPSS for Windows version 12.0.2 (The Appache

Software Foundation; IBM Corp., Armonk, NY, USA).

Mean values with pooled standard error of the mean

(pSEM) are provided in the tables. Differences for the

traits within groups and between both time-points

were analysed performing also a t-test. When t-test

was performed, the Bonferroni correction was applied.

Differences between mean trait values were consid-

ered significant at p < 0.05. Tendency for differences

was considered at 0.05 < p ≤ 0.1.

Results

The mean analysed Zn concentration in the feed was

180 mg/kg DM (160 mg/kg feed) in LZn and

2400 mg/kg DM (2250 mg/kg feed) in HZn (Table 1).

The Cu concentration in the feed was analysed to be

33 and 31 mg/kg DM in LZn and HZn respectively

(Table 1).

All piglets were in good condition throughout

the time of the treatment; no diarrhoea occurred.

Journal of Animal Physiology and Animal Nutrition © 2015 Blackwell Verlag GmbH 15

P. Janczyk et al. Dietary zinc and intestinal microbial activity in pigs

There was no effect of ZnO on the body weight. How-

ever, the pigs from HZn weighed numerically more

than pigs from LZn from d53 onwards (Table 2). Dur-

ing the 2nd and 3rd week, the HZn gained more than

LZn (p < 0.05). ADFI was higher in the HZn during

the 2nd week (p < 0.05). The FCR was higher in HZn

during the 1st week and lower in the HZn during the

3rd and 5th week (Table 2).

After 4 weeks of the experimental period, pH of the

caecal digesta was greater in the HZn in comparison

with LZn (p < 0.001). Whereas (iso-)butyric and vale-

ric acid decreased in the HZn (p < 0.05), propionic

acid tended to decrease (0.05 < p < 0.1) but lactic and

acetic acid were not affected there (p > 0.1). The con-

tents of DM and ammonia were neither affected

(p > 0.1). A weak negative correlation was calculated

between the pH and the total SCFA in caecum

(r2 = �0.461), and the strongest correlation was

observed between the butyric acid concentration and

pH (r2 = �0.572). In faeces, acetic and (iso-)butyric,

(iso-) valeric acid decreased in the HZn (p ≤ 0.01).

Faecal propionic acid concentration was not affected

(p > 0.1). The A:P ratio in caecum was greater in HZn

in comparison with LZn (p < 0.05). In faeces, an

opposite was observed – A:P ratio was lower in HZn

(p < 0.05). Faecal ammonia concentration decreased

in HZn (p < 0.05). No correlation between the total

SCFA and pH was detected (r2 = 0.147). All traits are

summarised in Table 3.

After the change of diet in the HZn group to the

feed administered to the LZn group and feeding the

animals for two consecutive weeks, no differences

were recorded in the caecal digesta between the

groups. In faeces, again, acetic acid was lower in the

HZn in comparison with LZn (p < 0.01). Similar to the

results obtained after 4 weeks, in faeces, the A:P ratio

was lower in HZn in comparison with LZn (p < 0.01).

No further differences were recorded (Table 4).

Comparison of the data obtained after 4 and

6 weeks of feeding within the groups revealed no dif-

ferences for LZn except for isobutyric acid, which

decreased in caecal digesta in the LZn group

(p < 0.05). The change of the diet from HZn to LZn

and feeding it for consecutive 2 weeks resulted in

decrease of caecal pH and increase of native water

concentrations of propionic and butyric acid

(p < 0.01), and tendencies for increased acetic acid

(p = 0.076), valeric acid (p = 0.061) and decreased A:

P ratio (p = 0.093). In faeces, after the change from

HZn to LZn, an increase of concentrations of iso- and

butyric acid, and valeric acid was recorded (p < 0.05),

as well as a tendency for an increase of ammonia con-

centration (p = 0.095).

The detailed results of the tissue Zn and Cu mea-

surements are provided in Table 5. ZnO treatment

had an effect on the Zn concentration of liver, kidneys

and bone (rib) (p < 0.05), but no effect on the con-

centration in muscle tissue was measured. The phar-

macological level of ZnO (HZn) fed for 4 weeks after

weaning resulted in a fourfold increase of the Zn con-

centration in liver, a threefold increase of Zn in kid-

neys and an almost twofold increase of Zn in bones in

comparison with LZn (p < 0.05). After reduction of

the ZnO in the feed from HZn to LZn level and consec-

utive feeding for 2 weeks, almost half the Zn concen-

trations could be observed in liver and kidneys

compared to the animals of the HZn group after

Table 2 Body weight, average daily gain (ADG), average daily feed

intake (ADFI) based on dry matter, and feed conversion ratio (FCR) of

pigs fed different zinc oxide levels in the diet for 6 weeks

Trait* LZn† HZn† SEM p-value††

Body weight [kg]

At weaning 8.48 8.51 0.21 0.952

After week 1 10.15 10.19 0.27 0.944

After week 2 11.81 12.17 0.35 0.625

After week 3 14.82 16.02 0.47 0.210

After week 4 18.44 19.46 0.57 0.385

After week 5 22.12 23.44 1.07 0.570

After week 6 28.04 29.36 1.36 0.654

ADG [g]

Week 1 200 171 11 0.224

Week 2 237 282 11 0.033

Week 3 430 550 28 0.022

Week 4 517 491 28 0.676

Week 5 648 657 23 0.859

Week 6 833 919 86 0.670

ADFI [g]

Week 1 240 270 9 0.080

Week 2 360 449 18 0.005

Week 3 579 635 19 0.152

Week 4 787 811 21 0.600

Week 5 926 1025 44 0.315

Week 6 1184 1332 81 0.419

FCR

Week 1 1.21 1.62 0.10 0.031

Week 2 1.52 1.59 0.04 0.385

Week 3 1.35 1.17 0.04 0.012

Week 4 1.54 1.68 0.06 0.289

Week 5 1.43 1.56 0.03 0.013

Week 6 1.42 1.49 0.05 0.556

SEM, standard error of the mean.

*ADG was calculated using pig as experimental unit. ADFI and FCR were

calculated using pen as experimental unit.

†LZn received diet containing 100 mg ZnO/kg diet for 6 weeks; HZn

group received diet with 3100 mg ZnO/kg diet for 4 weeks, then the

same diet as LZn group for another 2 weeks.

††statistically significant differences at P < 0.05 presented in bold.



4 weeks of feeding (p ≤ 0.01). Despite this reduction

in the Zn tissue concentration in the HZn group, these

concentrations remained greater than in the LZn

(p < 0.01). The reduction of Zn in the diet did not

affected concentrations in bones.

An effect was observed of the ZnO amount in the

diet on the Cu concentration in kidneys but not in

muscle, liver and bones (Table 5). After 4 weeks of

feeding, the HZn diet an almost fourfold increase of

the Cu concentration in the kidneys was recorded

Table 3 Dry matter of digesta and pH, lactic and short-chain fatty acids in native water of caecal digesta and faeces of piglets fed different levels of

zinc oxide in the diet from weaning for 4 weeks

Trait

Caecum

p value†

Faeces

p valueLZn* HZn pSEM LZn HZn pSEM

DM [%] 15.4 15.6 0.68 0.914 25.7 25.0 0.67 0.604

pH 5.48b 6.62a 0.20 0.000 6.89 7.08 0.09 0.310

LA mmol/l 76.27 87.54 21.65 0.822 u.d.l. 19.0 4.96 –

AcetA 258.65 255.33 14.72 0.918 418.9a 297.1b 22.79 0.004

PropA 156.83 121.08 10.20 0.076 195.0 162.4 11.52 0.163

isoButA 11.61a 3.99b 1.86 0.029 19.5a 12.0b 1.38 0.003

ButA 80.24a 31.72b 10.44 0.009 100.9a 60.6b 7.56 0.004

iValerA 1.55 1.78 0.21 0.610 23.2a 15.1b 1.70 0.012

ValerA 14.47a 4.00b 2.18 0.005 28.9a 15.5b 2.73 0.010

nCapronA u.d.l. 0.63 – – 5.3 1.7 1.57 0.468

A:P 1.65b 2.11a 0.10 0.006 2.22a 1.85b 0.08 0.025

NH3 mmol/l 45.87 36.08 4.15 0.261 202.1a 136.2b 14.46 0.018

DM, dry matter; LA, lactic acid; AcetA, acetic acid; PropA, propionic acid; isoButA, iso-butyric acid; ButA, butyric acid; iValerA, iso-valeric acid; ValerA,

valeric acid; nCapronA, n-capron acid; A:P, acetic to propionic acid ratio; P:A, propionic to acetic acid ratio; NH3, ammonia; pSEM, pooled standard

error of the mean; u.d.l., under detection limit of 0.1 mmol/l.

Significant differences within row (superscript lower case letters) (p < 0.05).

*Pigs from LZn group received diet containing 100 mg ZnO/kg diet for 6 weeks; HZn group received diet with 3100 mg ZnO/kg for 4 weeks, then the

same diet as LZn for another 2 weeks.

†statistically significant differences at P < 0.05 presented in bold.

Table 4 Dry matter of digesta and pH, lactic and short-chain fatty acids in native water of caecal digesta and faeces of piglets fed different levels of

zinc oxide in the diet from weaning for 6 weeks

Trait

Caecum

p value†

Faeces

p valueLZn* HZn pSEM LZn HZn pSEM

DM % 16.3 16.0 0.71 0.857 28.1 31.2 1. 84 0.435

pH 5.52 5.71 0.11 0.390 7.11 7.05 0.06 0.596

LA mmol/l 110.1 31.9 21.80 0.077 u.d.l. u.d.l. – –

AcetA 291.6 299.1 8.77 0.695 404.3a 333.9b 14.75 0.006

PropA 188.7 182.2 9.54 0.757 176.4 198.9 12.48 0.398

isoButA 4.6 2.2 0.75 0.119 21.0 18.0 1.75 0.427

ButA 77.0 68.5 6.11 0.518 92.4 90.6 4.39 0.852

iValerA 1.3 1.1 0.18 0.588 25.6 20.1 2.97 0.380

ValerA 11.4 10.7 1.74 0.862 22.2 25.5 2.26 0.487

nCapronA u.d.l. 0.8 – – 3.3 2.1 0.66 0.490

A:P 1.57 1.70 0.11 0.562 2.30a 1.74b 0.12 0.004

NH3 mmol/l 42.5 31.0 5.27 0.319 193.5 209.8 28.04 0.790

DM, dry matter; LA, lactic acid; AcetA, acetic acid; PropA, propionic acid; isoButA, iso-butyric acid; ButA, butyric acid; iValerA, iso-valeric acid; ValerA,

valeric acid; nCapronA, n-capron acid; A:P, acetic to propionic acid ratio; P:A, propionic to acetic acid ratio; NH3, ammonia; pSEM, pooled standard

error of the mean; u.d.l., under detection limit of 0.1 mmol/l.

Significant differences within row (superscript lower case letters) (p < 0.05).

*Pigs from LZn group received diet containing 100 mg ZnO/kg diet for 6 weeks; HZn group received diet with 3100 mg ZnO/kg for 4 weeks, then the

same diet as LZn for another 2 weeks.




(p < 0.05). After the change from HZn to LZn feeding,

a twofold reduction of the Cu concentration in kid-

neys was measured. A time effect on Cu in liver was

observed as its level was reduced 6 weeks after the

treatment in comparison with 4 weeks in LZn

(p < 0.05). There was also a reduction of Cu in the

liver after reduction of the Zn supply in diet (change

from HZn to LZn) (p < 0.05).

Discussion

The analyses of the diet revealed higher levels of Zn

than calculated, possibly because of higher content of

Zn in the original feedstuffs. However, the analysis of

the minerals in feed depends on the reached level of

homogeneity of dispersion of the supplements and the

analytical method itself. The analytical margins allow

16% difference (Verband Deutscher Landwirtschaftli-

cher Untersuchungs- und Forschungsanstalten, 2012);

thus, the Zn content in the LZn feed remained within

the ranges allowed in the EU at that time (EU, 2003).

Most studies on effects of ZnO were performed for

up to 4 weeks after weaning and included high num-

ber of replications to obtain high statistical power. In

the present study, the performance data were not the

primary issue and only few replicates per group were

available, even if the number was enough to perform

statistical calculations. Nevertheless, the results

remain in concordance to other studies showing (even

if not significant) improvement of pigs’ performance

after feeding the weaned piglets with high doses of

ZnO (Hollis et al., 2005).

There is evidence of antimicrobial activity of Zn in vi-

tro (Surjawidjaja et al., 2004). As it is speculated that

the mode of action of high levels of ZnO on pig perfor-

mance is due to a modification of the intestinal mi-

crobiome (Højberg et al., 2005; Pieper et al., 2011;

Vahjen et al., 2011), we investigated in this study the

effects on caecal and faecal chemical parameters

indicating microbial activity. Indeed, changes in the

end products of the microbial fermentation were

recorded here.

ZnO is an insoluble molecule at neutral pH, but dis-

sociates into Zn++ ions at low pH (2–3) in the stomach,

being available for the host for a short time in the ion-

ised form. The Zn++ further bind to different dietary

components present in the gut lumen (Starke et al.,

2014). Zn bound to amino acids may increase its avail-

ability for the host, but also for the intestinal microbi-

ome. High dietary Zn in the ingesta stimulates

upregulation of intestinal Zn transporter ZnT1 respon-

sible for the transport of Zn from enterocytes to the

extracellular matrix, and downregulates the ZIP4,

responsible for transport of Zn from the intestinal

lumen into the enterocytes (Martin et al., 2013a),

protecting the organism from excess of this ion. The

intestinal Zn uptake can also occur through the extra-

cellular pathway by diffusion, when the Zn concentra-

tion in the intestinal lumen increases (Menard and

Cousins, 1983). However, most of the Zn from dietary

ZnO remains unabsorbed and reaches the large intes-

tine, where it can reach concentrations up to 8–10 g/

kg (data from this study published by Bratz et al.,

2012).

In our study, pH in the caecum increased when

3100 mg ZnO/kg diet was fed. No changes in lactic,

acetic and propionic acid concentrations were

observed, but butyric acid reached almost 1/3 of the

Table 5 Zn and Cu concentrations in liver, kidney, muscle (ham) and bone (rib) of pigs fed different levels of Zn (as ZnO) in the diet

4 weeks 6 weeks

LZn* HZn SEM p value† (LZn vs. HZn, 4 weeks) LZn* HZn SEM p value† (LZn vs. HZn, 6 weeks)

Zn [mg/kg DM]

Liver 234.0 855.1A 119.7 0.001 213.3 462.1B 48.6 0.002

Kidney 122.3 372.7A 49.8 0.003 124.4 162.0B 7.2 0.001

Muscle 69.6 66.7 1.9 0.472 66.3 61.3 1.9 0.208

Bone 154.9 273.4 26.4 0.013 159.8 241.2 14.5 <0.001

Cu [mg/kg DM]

Liver 38.14A 42.14A 1.7 0.255 30.78B 32.52B 2.4 0.734

Kidney 38.82A 119.16A 19.7 0.031 31.36B 58.42B 5.7 0.006

Muscle 3.42 2.58 0.3 0.115 4.00 3.56 0.2 0.235

Bone 3.56 3.48 0.3 0.908 3.78 3.48 0.2 0.446

SEM, standard error of the mean.

A,B – mean values within one dietary group (i.e. LZn or HZn) lacking same superscript differ between the two time-points (p < 0.05).

*LZn group received the diet with 100 mg ZnO/kg diet for 6 weeks after weaning; HZn group received diet with 3100 mg ZnO/kg diet for 4 weeks, then

100 mg ZnO/kg (as LZn) for another 2 weeks.




concentration measured in the LZn group. After

decreasing the ZnO concentration in the diet from

3100 mg ZnO/kg to 100 mg ZnO/kg, the situation

reversed, and the pH decreased in the HZn group. In a

similar study, it was shown that the same dietary ZnO

concentration had no effect on jejunal brush border

enzyme activity (Martin et al., 2013b). Thus, no

increased mucosal absorption of butyric acid would be

expected, and the observed changes were due to

decreased bacterial activity. The reduction of the buty-

ric acid was probably caused by a reduction in the

population of butyrate-producing bacteria, which uti-

lise lactic and acetic acid. Even though the bacterial

composition was not analysed in this study, evidence

for this hypothesis was provided by Pieper et al.

(2011) who showed a Zn-dependent reduction of clos-

tridial cluster XIVa, based on quantitative PCR analy-

sis of ileal digesta of pigs fed 2500 mg Zn/kg diet.

These clostridia are present in the intestinal lumen

with increasing concentrations in the distal intestine.

They belong to the main butyric acid producers in the

large intestine.

Similar to the present findings, Højberg et al.

(2005) and Starke et al. (2014) reported a decrease of

SCFA in the intestinal contents of pigs fed 2500 mg

Zn/kg diet. The lower butyric acid in the large intes-

tine could be considered negative for the host as buty-

ric acid is an important energy source for colonocytes

and supports colonic mucosal health (Henningsson

et al., 2001). This is to speculate whether long-term

feeding of high dietary ZnO would result in increased

sensibility of the colonic mucosa to pathogens or

commensals.

High level of dietary ZnO resulted in a decrease of

ammonia suggesting reduced protein degradation in

the colon. On the other hand, reduced ammonia

could implicate higher binding of nitrogen for bacte-

rial synthesis. However, in face of the reduced SCFA,

reduced bacterial protein degradation resulting from

reduced bacterial activity seems to be a more applica-

ble explanation. This phenomenon was reversible. At

all, the faecal microbiome from animals from LZn rep-

resented a higher activity. The A:P ratio in this group

indicates a well balanced community (Marchaim and

Krause, 1993). In opposite, the A:P ratio in faeces of

the HZn decreased and reduction of Zn in the diet did

not reduce this parameter. As there were no changes

in propionic acid concentration, this would addition-

ally indicate a shift in faecal microbial population and

a decrease or inhibition of acetogenic bacteria in the

presence of ZnO overload in conjunction with a sup-

port of propionate-producing bacteria. Furthermore,

there is evidence on toxicity of ZnO against yeasts

in vitro (Kasemets et al., 2009) and it cannot be

excluded that a large intestinal yeast population was

also suppressed. Even though the interplay between

intestinal yeasts and bacteria is still poorly understood,

a negative correlation to enterobacteria and a positive

correlation to lactobacilli were observed in weaned

pigs (Urubschurov et al., 2011). Considering the

report of Vahjen et al. (2011), who observed an

increase of enterobacteria and their diversity when

high ZnO levels were used in the diet, a decrease of

intestinal yeasts would be expected in the HZn group

in this study, but this should be examined in further

investigations.

Lactic acid is the main product of carbohydrate fer-

mentation performed by lactic acid bacteria (LAB)

such as streptococci and lactobacilli and is an energy

source for other bacteria for synthesis of acetate, pro-

pionate and butyrate. Lactate concentration in caecal

digesta after 4 weeks of treatment was not affected,

but 2 weeks later it tended to be greater in the LZn.

This would indicate that the large intestinal microbi-

ome in the LZn was less affected and more stable than

in the HZn. In the HZn, an adaptation of the microbi-

ome to the decreased Zn level was expected and it

remains unclear why the lactic acid concentration was

not comparable to LZn. Starke et al. (2014) showed a

strong negative correlation between lactobacilli and

Zn ions throughout the intestine, with species-specific

differences. Thus, it would be possible that these bac-

teria need long time to recover after being suppressed

by very high Zn concentrations for few weeks. Fur-

ther, higher lactic acid utilisation by other bacteria

would be possible.

Acetic acid absorbed from the large intestine is uti-

lised by the pig as energy source and can be used for

fat synthesis (Latymer et al., 1991). So its higher pro-

duction may have a positive effect on the performance

of growing pigs. Propionic acid is the main SCFA for

glucose synthesis in the liver. Moreover, there is evi-

dence on inhibiting fatty acids metabolism with low-

ering of plasma cholesterol by propionic acid and for

its antimicrobial and anti-inflammatory activity (Al-

Lahham et al., 2010). Thus, the intestinal SCFA status

observed in pigs from the LZn would provide better

benefit for the host than the one observed in the HZn

after 4 weeks of feeding the high dietary ZnO. It is a

hypothesis, but this benefit does not have to result in

higher weight of the animals but would rather

improve the immune system and its reaction to patho-

gens or stress.

Zn is excreted via urine at a constant, very low rate

(1–3% of total Zn excretion) independently of Zn

body equilibrium (Windisch and Kirchgessner, 1999),



and a large part of the absorbed and endogenic Zn is

re-entering the gut via bile and intestinal excretion

(Poulsen and Larsen, 1995). In this study approxi-

mately four times higher Zn concentrations were

observed in the livers of the animals of the HZn group.

Shell and Kornegay (1996) recorded a 2.3-fold and

7.3-fold increase of Zn levels in livers of piglets fed

2000 or 3000 mg/kg ZnO, respectively, whereas these

authors observed no increase of Zn in livers at

1000 mg ZnO/kg diet. Case and Carlson (2002)

reported threefold increase of liver Zn in nursery pigs

(weaned at 17–24 days of age) after 4 weeks of feed-

ing 3000 mg ZnO/kg diet. Jensen-Waeren et al.

(1998) reported a 4.5-fold increase of liver Zn concen-

tration in piglets fed 2500 mg ZnO/kg diet, and a simi-

lar observation was also reported by Mart�ınez et al.

(2004). In a study of Rincker et al. (2005), a 6.5-fold

increase of liver Zn was observed when nursery pigs

were fed 2000 mg Zn/kg diet as ZnO for 14 days.

Thus, the results from this study confirm previous

reports about Zn concentrations in liver after feeding

pharmacological amounts of ZnO to weaning pigs.

Kidney Zn concentrations in the present study

(threefold increase in the HZn group) were higher

than those reported previously by Jensen-Waeren

et al. (1998) who found a 2.1-fold increase after feed-

ing 2500 mg ZnO/kg diet and Mart�ınez et al. (2004)

who reported approximately twofold increase after

feeding 2500 mg ZnO/kg diet, as well as Shell and

Kornegay (1996) who reported a 1.3 and 1.6-fold

increase for 2000 and 3000 mg ZnO/kg diet respec-

tively. Also Case and Carlson (2002) reported 1.4-fold

increase of kidney Zn when piglets were fed 3000 mg

ZnO/kg diet for 28 days after weaning (at 17–24 days

of age).

As Zn requirements vary with the diet, climate con-

ditions, or stress (Chasapis et al., 2012), environmen-

tal conditions (season, light, temperature) applied in

each study, as well as the age of the animals and dura-

tion of feeding, might have influenced the Zn concen-

tration in kidneys. This might be a reason for an

opposite report provided by Rincker et al. (2005),

who observed almost fourfold reduction of Zn in kid-

neys in nursery pigs (weaned at 16–20 days of age)

fed 2000 mg Zn/kg diet as ZnO for 14 days.

In agreement with the study of Shell and Kornegay

(1996), in this study a 1.6-fold increase of Zn concen-

trations in the rib bones was recorded. Bones are an

important location for long-term storage of many

minerals. Reduction of dietary ZnO from 3100 to

100 mg/kg diet for 2 weeks showed no effect on bone

Zn concentration, but a significant reduction of its lev-

els in livers and kidneys. This proves the functioning

of different tissues as short or long-term buffers to

varying intake levels of this trace element to prevent

acute toxicity or deficiency (Hill and Link, 2009;

Chasapis et al., 2012).

Concentration of Zn in muscle tissue was unaffected

by dietary Zn concentration, as already observed pre-

viously (Shell and Kornegay, 1996; Jensen-Waeren

et al., 1998).

High dietary Zn concentrations are stimulating the

expression of intestinal mucosal metallothioneins

(Carlson et al., 1999; Mart�ınez et al., 2004; Martin

et al., 2013a). As they possess high affinity to bind Cu,

it was hypothesised that the reduction of Cu in tissues

could occur due to high amounts of dietary Zn (Hill

et al., 1983). This initial postulation seems to be

wrong. In contrast, in the present study, an almost

fourfold increase of Cu in kidneys was observed after

Zn supplementation, without changes in rib bone and

muscle Cu, remaining in agreement with the report of

Mart�ınez et al. (2004). Shell and Kornegay (1996)

reported a 1.5- to twofold increase of kidney Cu when

2000–3000 mg ZnO/kg diet were fed; and Jensen-Wa-

eren et al. (1998) reported a three times higher Cu

concentration in kidneys. Similarly, Rincker et al.

(2005) reported 2.6-fold increase of kidney Cu after

feeding 2000 mg Zn/kg diet as ZnO for 14 days.

Although up to date, it is speculative and needs fur-

ther investigation but probably the increased metallo-

thioneins in the gut wall and/or stronger binding of

Cu were fast transferred to liver and kidneys. In con-

trast to the present results, no change in liver Cu con-

centration was reported by Mart�ınez et al. (2004)

after feeding weaned pigs with 2500 mg ZnO/kg diet

for 14 days.

Reversibility of the observed effects on Zn and

Cu was observed after withdrawal of the high die-

tary ZnO. However, the reduction of the Zn and Cu

concentrations in the different tissues is quite slow.

Two weeks of withdrawal of the high dietary Zn

were not enough to fully diminish the observed dif-

ferences.

Conclusions

The present study provides evidence on influence of

dietary ZnO on large intestinal microbial metabolism.

High concentrations of ZnO in the diet (3100 mg

ZnO/kg diet) affected the function of the large intesti-

nal microbiome in a reversible way. The lower con-

centration of the short-chain fatty acids should be

discussed critically regarding their value for the host.

Furthermore, the effect of high dietary ZnO on

increased liver and kidney Zn and Cu concentrations



was reversible after the withdrawal of the ZnO. The

tissue Zn and Cu concentrations should be taken

into consideration both by human and by pet food

industry.

Acknowledgements

The authors thank Enno Luge and the team of Dr.

Stefanie Banneke from the Research Institute for Risk

Assessment, Berlin for the excellent animal care and

technical support during the experiment. We thank

Dr. Robert Pieper from the Veterinary Faculty of the

Freie Universit€at Berlin for composition of the feeds.

We also thank Mrs. Sabine Bremer from the Univer-

sity of Rostock, Chair for Nutrition Physiology and

Animal Nutrition for performing the chemical analy-

ses. The study was in part funded by the German

Research Foundation (Deutsche Forschungs-gemein-

schaft, DFG) within the Collaborative Research Group

(SFB, Sonderforschungsbereich) 852/1 ‘Nutrition and

intestinal microbiota – host interactions in the pig’.

The authors are solely responsible for the data and do

not represent any opinion of neither the DFG nor

other public or commercial entity.

Conflicts of interests

Authors have no conflicts of interests.

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Effect of high dietary zinc oxide on the caecal and faecal short-chain fatty acids and tissue zinc and copper concentration in pigs is reversible after withdrawal of the high zinc