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Effect of mineral source and mannan oligosaccharide supplements on zinc and copper digestibility in growing pigs a
b
a
Alexandre Lebel , J. Jacques Matte & Frédéric Guay a
Département des sciences animals, Université Laval Québec, Québec, Canada G1V 0A6 b
Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, Sherbrooke, Québec, Canada J1M 0C8 Published online: 02 Sep 2014.
To cite this article: Alexandre Lebel, J. Jacques Matte & Frédéric Guay (2014) Effect of mineral source and mannan oligosaccharide supplements on zinc and copper digestibility in growing pigs, Archives of Animal Nutrition, 68:5, 370-384, DOI: 10.1080/1745039X.2014.954357 To link to this article: http://dx.doi.org/10.1080/1745039X.2014.954357
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Archives of Animal Nutrition, 2014 Vol. 68, No. 5, 370–384, http://dx.doi.org/10.1080/1745039X.2014.954357
Effect of mineral source and mannan oligosaccharide supplements on zinc and copper digestibility in growing pigs Alexandre Lebela, J. Jacques Matteb and Frédéric Guaya*
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a Département des sciences animals, Université Laval Québec, Québec, Canada G1V 0A6; bDairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, Sherbrooke, Québec, Canada J1M 0C8
(Received 26 May 2014; accepted 9 August 2014) This study aimed to compare the effects of organic (proteinate) and inorganic (sulphate) copper (Cu) and zinc (Zn) supplements, in presence or absence of a mannan oligosaccharide (MOS) supplement, on mineral solubility and digestibility in pigs. Twenty-eight barrows (25 ± 4 kg) assigned randomly to four treatment groups were fed a corn-wheat-soya bean meal diet with 10 mg/kg of Cu and 100 mg/kg of Zn supplied as organic or inorganic supplement, and supplemented or not with 0.1% MOS. After an adaptation period, total faeces and urine were collected for a period of 6–7 days. Pigs were then euthanatised and digesta from ileum and caecum were collected. Apparent digestibility was calculated in ileum and caecum using titanium dioxide. The organic mineral supplement improved total (faecal) digestibility and retained/ ingested ratio of Cu (p < 0.05) while reducing apparent digestibility of Zn in the ileum (p < 0.05) without effect on total digestibility of Zn. Solubilities of Cu and Zn in liquid fraction of ileum and caecum were not affected by mineral sources. Although MOS supplement increased Cu solubility in the ileum (p < 0.05), it had no effect on digestibility of Zn and Cu in ileum, caecum and faeces, retained/ingested ratio of Zn and Cu, or pH and volatile fatty acid concentration in ileal and caecal digesta. In conclusion, organic mineral supplement improved total digestibility and retained/ ingested ratio of Cu in pigs but this cannot be attributed to its solubility in ileal and caecal digesta. The MOS supplement did not interfere with digestibility or dietary utilisation of Zn and Cu in pigs fed above the Zn and Cu requirements. Keywords: copper; digestibility; oligosaccharides; organometallic compounds; pigs; zinc
1. Introduction Inorganic mineral supplements such as zinc (Zn) sulphate or oxide and copper (Cu) sulphate have long been added to swine diets. These trace elements dissolve in the digestive tract and release free ions that can bind to organic molecules (e.g. phytates, fibre) present in ingested matter, leaving the minerals difficult to absorb (Wapnir 1998; Camara and Amaro 2003). Moreover, as the pH increases in the small intestine, Zn and Cu are trapped in insoluble hydroxide precipitates (Powell et al. 1999). Chelating of dietary trace minerals is claimed to prevent the formation of insoluble complexes in the digestive tract, increase transfer from the intestinal lumen to epithelial cells and hence, improve intestinal absorption (Mullan and D’Souza 2005). Dietary supplements of Zn and Cu chelated with organic molecules such as amino acids (e.g. lysine, methionine), polysaccharides or partially hydrolysed proteins are nowadays available in swine *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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nutrition. The efficiency of replacing inorganic Zn or Cu with organic sources is controversial. Numerous studies have reported little benefit in terms of swine performance (Jondreville et al. 2002; Revy et al. 2003; Schlegel et al. 2013), whereas others indicate that using chelated forms can reduce mineral excretion in pigs (Lee et al. 2001; Creech et al. 2004; Korniewicz et al. 2007; Huang, Yoo, et al. 2010; Huang, Zhou, et al. 2010). In fact, mechanisms involved in absorption of Zn and Cu from dietary organic complexes (e.g. increased solubility or others) have not been established yet. This lack of knowledge is critical for a reliable understanding of these inconsistent responses. Non-digestible oligosaccharides (NDO) like inulin and other fructo-oligosaccharides (FOS) are known to improve digestibility (i.e. absorption) of calcium, magnesium, Zn and Cu in rats (Delzenne et al. 1995; Lopez et al. 2000; Mineo et al. 2004; Coudray et al. 2006) but no such effects have been reported for pigs (Houdijk et al. 1999, 2002; Jolliff and Mahan 2012). Mannan oligosaccharides (MOS) are known to bind bacteria like Escherichia coli and Salmonella spp. and to prevent the proliferation of these pathogens at the mucosal surface of the intestine in weanling pigs (Halas and Nochta 2012). In contrast to FOS, MOS have been shown to improve the digestibility of calcium in weanling piglets (Nochta et al. 2010). This beneficial effect may be due to a reduction in pH of the intestinal contents (Halas and Nochta 2012) along with a decrease in mineral precipitation. To the best of our knowledge the effect of MOS on intestinal absorption of Zn and Cu has never been investigated. The objective of the present study was to evaluate the effects of MOS on the apparent digestibility of Zn and Cu in growing pigs fed organic or inorganic supplement of these minerals. The solubility of Zn and Cu in the intestinal contents was also measured.
2. Materials and methods 2.1. Animals and experimental conditions Twenty-eight barrows (Landrace × Yorkshire) weighing on average 25 ± 4 kg were moved from the nursery of a commercial farm to a controlled-environment room with individual pens (1 m × 2 m) for 6 days and then transferred to metabolic cages for total collection of faeces and urine. Room temperature was maintained at 20°C. The animals had unlimited access to water and feed (approximately five times the level of maintenance metabolic energy of 440 kJ/kg of BW0.75). Refusals were weighed and harvested daily in order to determine feed intake. All animal procedures were conducted according to the guidelines set by the Canadian Council of Animal Care (2009).
2.2. Dietary treatments Animals were randomly distributed into seven repetitions based on initial weight. Within each repetition, pigs were distributed into factorial treatments (2 × 2) with organic or inorganic dietary mineral supplementation and presence or absence of MOS (Table 1). The level of mineral supplementation was 10 mg/kg of Cu and 100 mg/kg of Zn, provided as sulphate (inorganic) or as a protein complex (organic) (Bioplex®, Alltech, Nicholasville, KY, USA). These dietary levels of Cu and Zn are higher than those recommended by National Research Council (2012) but they are representative of those used in practice as recommended by van Heugten (2010) and, as such, are more appropriate to study a realistic interaction with NDO feed additives. MOS was supplemented at 0.1% with Bio-Mos® (Alltech, Nicholasville, KY, USA). Titanium dioxide (TiO2) was
372 Table 1.
A. Lebel et al. Ingredient composition of the experimental diets (as-fed basis).
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Inorganic mineral source
Ingredient [g/kg diet] Wheat Corn Soya bean meal Ground limestone Dicalcium phosphate Salt Lysine HCl L-threonine DL-methionine Vitamins* Minerals# ZnSO4 (inorganic) CuSO4 (inorganic) Zn proteinate (organic)$♦ Cu proteinate (organic)$♦ Mannan oligosaccharide$◊ Titanium dioxide Chemical composition, calculated† [g/kg] Crude protein Calcium Total phosphorus Chemical composition, analysed [mg/kg] Total copper Total zinc
Organic mineral source
0% MOS♦
0.1% MOS
0% MOS
0.1% MOS
150.00 615.71 200.00 12.00 8.50 3.50 3.00 0.50 0.30 2.50 2.50 0.45 0.04 – – – 1.00
150.00 614.71 200.00 12.00 8.50 3.50 3.00 0.50 0.30 2.50 2.50 0.45 0.04 – – 1.00 1.00
150.00 615.43 200.00 12.00 8.50 3.50 3.00 0.50 0.30 2.50 2.50 – – 0.67 0.10 – 1.00
150.00 614.43 200.00 12.00 8.50 3.50 3.00 0.50 0.30 2.50 2.50 – – 0.67 0.10 1.00 1.00
160.0 6.9 5.1
160.0 6.9 5.1
160.0 6.9 5.1
160.0 6.9 5.1
25.0 143.6
25.0 144.0
23.7 137.0
26.7 151.4
Notes: ♦MOS, Mannan oligosaccharide; *Contribution per kilogram of complete feed (DM): vitamin A palmitate 5000 IU, vitamin D3 1000 IU, vitamin E acetate 22.5 mg, menadione sodium bisulphite 3.75 mg, thiamine HCl 10 mg, riboflavin 4.5 mg, niacin 20.0 mg, calcium pantothenate 25.0 mg, pyridoxine HCl 1.5 mg, biotin 0.2 mg, choline bitartrate 375 mg, vitamin B12 25.0 µg; #Contribution per kilogram of complete feed: iron citrate 100 mg, potassium iodide 0.28 mg, manganese carbonate 46 mg, sodium selenite 0.30 mg; $Alltech Inc. Nicholasville, KY, USA, ♦Bioplex Zn and Bioplex Cu, respectively, composition: 29% crude protein, 0.5% ether extract, 35% ash, acid detergent fibre 0.78%, amino acid profile: lysine 100, histidine 42, isoleucine 62, leucine 113, methionine 21, phenylalanine 80, threonine 53, valine 77, tryptophan 19; ◊Bio-MOS, composition: crude protein 31%, ether extract 4%, ash 6%, acid detergent fibre 9%. †Nutritional composition based on Sauvant et al. (2004).
added (0.1%) to all four diets as an external marker for evaluation of mineral apparent digestibility in the ileum, caecum and faeces.
2.3. Collection of samples for measurement of mineral absorption After 2 days of adaptation to the metabolic cages, all faeces and urine produced were collected twice daily for a period of 6 or 7 days. The cage floors were fitted with funnels for urine collection. Faeces were frozen at −20°C without delay, as was 10% of the urine. At the end of the collection period, all frozen faeces were thawed, mixed thoroughly and frozen again until analysis. Frozen urine was treated likewise.
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2.4. Euthanasia and sampling of digesta The animals had access to feed until 2 hours before euthanasia. They were given an intramuscular injection of a mixture of ketamine (Ketalean 20 mg/kg BW, Vétoquinol Canada Inc., Lavaltrie, Québec, Canada) and xylazine (Xylamax 5 mg/kg BW, Vétoquinol Canada Inc., Lavaltrie, Québec, Canada). Sodium pentobarbital (Euthanyl: 120 mg/kg BW, Vétoquinol Canada Inc., Lavaltrie, Québec, Canada) was administered to the anaesthetised animals via an ear vein. The abdomen was then opened and the entire intestine removed (from the duodenum to the colon). The ileum (the last metre of the small intestine) and the caecum were isolated using plastic strips and then separated. The digesta in each segment was collected and three types of digesta samples were prepared. One portion was acidified with HCl (1 mol/l) in order to minimise volatilisation of volatile fatty acids (VFA) and was immediately frozen at −20°C with a second non-acidified portion. A third portion was maintained on ice for pH determination after which it was centrifuged at 2000 g (4°C) for 20 min and the supernatant was frozen for dissolved mineral analysis as described below.
2.5. Analytical procedures The pH of intestinal contents diluted 1:1 with distilled water was measured directly using a pH probe and meter (Check-Mite, Buffalo, NY, USA). Analyses of total Zn and Cu were performed using Association of Official Analytical Chemists (2005) method 985.01. Briefly, 1 g of feed or freeze-dried digesta was placed in a refractory oven at 500°C for at least 8 h. The ash was hydrated with 10 drops of distilled water and 4 ml of HNO3 (1 mol/l) were added. This mixture was evaporated on a hot plate at 100–120°C and then placed in the refractory oven for an additional 8 h. The cooled ash was dissolved in 10 ml of HNO3. Dissolved Cu and Zn were determined after a dilution with HNO3 at ratios of 1:5 and 1:10 for caecal and ileal samples, respectively. Urine was mixed 1:5 with diluted HNO3. Mineral concentration was measured using an inductively coupled plasma (ICP) spectroscope (Optima 4300, Perkin Elmer, Wellesley, MA, USA). Titanium dioxide was determined according to the method of Jagger et al. (1992) along with the procedures proposed by Short et al. (1996). Briefly, lyophilised digesta or faeces or dry feed was incinerated at 500°C. The sample was mixed with H2SO4 and boiled gently for 1 h, then filtered on Whatman 541 paper (Buckinghamshire, UK). The filtrate was mixed with 30% hydrogen peroxide. Absorbance at 410 nm was measured using a Varioskan spectrophotometer (Thermo Electron Corporation, Vantaa, Finland) and compared to a standard curve. VFA were measured using HPLC (Waters Corporation, Milford, MA, USA) according to the method of Canale et al. (1984). VFA were detected by ultraviolet visible detector at 210 nm.
2.6. Calculations and statistical analysis Apparent digestibility based on the TiO2 marker was calculated using the equation proposed by Adeola (2001): TiO2 feed ½%Mineralsample ½mg=kg 100 Apparent digestibility ½% ¼ 100 TiO2 sample ½%Mineralfeed ½mg=kg
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Treatment effects on feed intake, body weight gain, on mineral absorption, deposition, excretion and concentration, and on pH and VFA concentrations were analysed according to a complete block design with initial body weight as criteria in a 2 × 2 factorial arrangement of treatments using the SAS MIXED procedure (SAS Institute Inc., Cary, NC, USA). The statistical model included the treatment effect (mineral source and MOS) and their interactions. Differences were considered significant when p < 0.05 and indicative of a tendency when p < 0.1. Pearson correlation coefficients were determined using the SAS Correlation procedure.
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3. Results Whatever the studied variables, there was no significant interaction between sources of minerals and MOS supplementation. During this experiment, no animal has to be discarded, no health problem was observed and no particular veterinary intervention was required. 3.1. Daily Cu and Zn balance The average Cu and Zn balances over the experimental period are presented in Table 2. Feed intake was increased for diets with the organic mineral supplement (p < 0.05) and, as a tendency, for those with the MOS supplement (p < 0.10). No treatment effect was observed on daily body weight gain but the final body weight tended to be greater in pigs fed organic mineral supplement (p < 0.08) or its combination with MOS supplement (Mineral source × MOS, p < 0.10). Daily absorption and retention of Cu and Zn were greater in pigs given the organic mineral supplement (p < 0.05) or the MOS supplement (p < 0.05). Excretion of Cu in faeces tended to be reduced (p < 0.09) in organic mineral treatments. This reduction was associated with increased apparent digestibility and retained/ingested ratio of Cu (p < 0.01). Urinary excretion of Cu tended to increase in MOS supplemented treatments (p < 0.06). Neither the mineral source nor the MOS supplement had any significant effect on the apparent digestibility or retained/ingested ratio of Zn but urinary excretion of Zn was greater with organic mineral supplementation (p < 0.05). 3.2. Apparent digestibility of Zn and Cu in the ileum, caecum and faeces The apparent digestibility of minerals in different parts of the gastrointestinal tract is presented in Table 3. As shown with the total faecal collection procedure (Table 2), estimations of total apparent digestibility of Cu with TiO2 marker were also improved in pigs receiving the organic mineral supplement (p < 0.05). However, this is not reflected in separate parts of the digestive tract. For Zn, apparent digestibility in ileum was decreased in pigs fed the organic supplement (p < 0.05) whereas it was only a trend (p < 0.08) in caecum. Globally, the mineral source had no significant effect on apparent digestibility of Zn in faeces. There was no significant effect of the MOS supplement on apparent digestibility of Zn and Cu measured in the ileum, caecum or faeces. 3.3. VFA, pH and mineral solubility in ileum and caecum There was no effect of the mineral source and supplement of MOS on the dry matter content of digesta in the ileum or caecum (Table 4). The dietary supplement of MOS
30.1 36.5 2141 1047 53.6 27.6 25.4 49.2 0.87 25.1 47.8 307.5 152.7 154.4 51.0 1.87 152.9 50.3
30.8 37.5 1994 1104
49.3 27.7 21.6 43.1 0.38 21.3 42.3
285.9 146.7 139.3 48.7 1.71 137.5 48.1
307.2 146.2 162.0 52.6 2.08 158.8 51.7
53.1 21.9 31.2 58.6 0.54 30.7 57.6
30.6 37.5 2242 1140
362.2 160.6 200.6 54.8 2.33 198.3 54.2
63.4 25.8 37.6 58.7 0.79 36.9 57.5
32.1 39.4 2380 1196
12.6 12.0 11.4 2.9 0.18 11.4 2.1
2.5 2.2 2.1 3.3 0.18 2.5 3.3
0.7 0.8 0.086 0.057
SEM*
0.033 NS 0.040 NS 0.033 0.044 NS
0.045 0.090 0.001 0.003 NS 0.001 0.001
NS† 0.080 0.050 NS
Mineral source
0.042 NS 0.050 NS NS 0.050 NS
0.049 NS 0.042 NS 0.059 0.041 NS
NS NS 0.096 NS
MOS
p-Value
NS NS NS NS NS NS NS
NS NS NS NS NS NS NS
NS 0.095 NS NS
Mineral × MOS
Note: ‡Initial and final body weights were evaluated before pigs were transferred in metabolic crates and at the end of collection period, respectively; *SEM, Standard error of the mean, n = 7 for each treatment, faeces and urine were collected on 6 or 7 days; †NS, Not significant (p > 0.10).
Initial body weight [kg]‡ Final body weight [kg]‡ Feed intake [g/d] Body weight gain [g/d] Copper Ingested [mg/d] In faeces [mg/d] Absorbed [mg/d] Apparent digestibility [%] In urine [mg/d] Retained [mg/d] Retained/ingested ratio [%] Zinc Ingested [mg/d] In faeces [mg/d] Absorbed [mg/d] Apparent digestibility [%] In urine [mg/d] Retained [mg/d] Retained/ingested ratio [%]
0.1% MOS
0% MOS
0% MOS
0.1% MOS
Organic mineral source
Inorganic mineral source
Table 2. Effect of organic or inorganic mineral supplementation, with or without mannan oligosaccharide (MOS) supplementation, on feed intake, body weight gain and copper and zinc balance in growing pigs.
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Table 3. Effect of organic or inorganic mineral supplementation, with or without mannan oligosaccharide (MOS) supplementation, on apparent digestibility [%]# of copper and zinc in growing pigs.
Ileum Copper Zinc Caecum Copper Zinc Faeces Copper Zinc
Inorganic mineral source
Organic mineral source
0% MOS
0.1% MOS
0% MOS
0.1% MOS
SEM*
Mineral source
MOS
Mineral × MOS
37.9 30.0
24.4 24.5
36.2 −0.1
25.8 9.4
7.1 10.0
NS† 0.021
NS NS
NS NS
29.6 59.0
32.5 62.0
29.7 53.1
39.5 58.0
4.9 2.6
NS 0.080
NS NS
NS NS
48.7 55.3
54.3 56.7
59.7 50.6
58.4 55.3
2.5 2.9
0.041 NS
NS NS
NS NS
p-Value
Note: #Using the calculation of Adeola (2001) and TiO2 as an external marker; *SEM, Standard error of the mean, n = 7 for each treatment; †NS, Not significant (p > 0.10).
reduced the concentration and proportion of dissolved Cu in the ileum (p < 0.05), while it had no effect on dissolved Zn. In caecum, neither the mineral source nor the addition of MOS had any effect on Cu or Zn solubility, although MOS did increase the total concentration of Zn (p < 0.05). Values of dissolved mineral concentrations were not correlated with those of apparent digestibility of Cu and Zn in the ileum and caecum (Cuileum: r = 0.053, p = 0.819; Cucaecum: r = 0.160, p = 0.455; Znileum: r = –0.180, p = 0.422; Zncaecum: r = −0.099, p = 0.632). Finally, no treatment effect was observed on pH or VFA concentrations either in ileum or caecum (Table 5).
4. Discussion Feed intake was greater in pigs fed the MOS supplement but it was only a tendency. This is in line with results on weaned piglets where MOS have shown positive effects on feed intake and growth performance but the response was transient for about two weeks postweaning and faded out thereafter (Miguel et al. 2004). The greater feed intake and higher final body weight in pigs fed organic mineral supplements were in contrast with recent results obtained by Jolliff and Mahan (2013) and Martin et al. (2011), who did not report any effect of mineral source (Cu-peptide vs. Cu sulphate) on feed intake and growth performance in growing pigs when diets contained about 15 mg/kg of Cu and 150 mg/kg of Zn. For Cu, it is known that feed intake and average daily gain in growing pigs may be increased with high supplementations of 125 to 200 mg/kg but usually not at concentrations like those used in the present experiment (about 25 mg/kg) (review by Jondreville et al. 2002). It is unlikely that other components than Cu and Zn which differed between trace mineral premixes might have had an impact on feed intake and then on growth performance as the relative contribution of these differences for the whole content of major nutrients of the diet was negligible (Table 1). Along with the effect of organic mineral and MOS supplements on feed intake, there was also an increase of Cu and Zn retention and the final body weight of these pigs tended to be greater by 10% as compared to those fed the inorganic mineral supplement. This
14.6 11.6
0.117 35.9 21.9 0.072 6.9 4.4 0.584 233.6 18.6 0.244 52.3 9.8
14.9 13.5
0.087 66.1 27.6
0.073 7.9 5.0
0.608 172.2 9.8
0.218 43.3 8.2
0.1% MOS
0.225 49.4 9.9
0.818 153.1 10.9
0.062 7.0 5.2
0.094 56.1 24.9
14.2 12.5
0% MOS
0.267 56.9 6.9
0.727 321.8 17.4
0.066 7.2 5.0
0.084 24.8 13.5
14.1 12.5
0.1% MOS
Organic mineral source
0.015 11.5 1.9
0.091 79.9 6.2
0.006 1.5 0.9
0.016 9.5 3.8
1.1 0.6
SEM*
NS NS NS
NS NS NS
NS NS NS
NS NS NS
NS† NS
Mineral source
0.045 NS NS
NS NS NS
NS NS NS
NS 0.006 0.040
NS NS
MOS
p-Value
NS NS NS
NS NS NS
NS NS NS
NS NS NS
NS NS
Mineral × MOS
Note: *SEM, Standard error of the mean, n = 7 for each treatment; †NS, Not significant (p > 0.10); #On dry matter basis; $Per cent of dissolved mineral for 100 g of fresh digesta.
Dry matter [%] Ileum Caecum Copper Ileum Total# [mg/g] Dissolved [µmol/l] Dissolved$ [%] Caecum Total# [mg/g] Dissolved [µmol/l] Dissolved$ [%] Zinc Ileum Total# [mg/g] Dissolved [µmol/l] Dissolved$ [%] Caecum Total# [mg/g] Dissolved [µmol/l] Dissolved$ [%]
0% MOS
Inorganic mineral source
Table 4. Effect of organic or inorganic mineral supplementation, with or without mannan oligosaccharide (MOS) supplementation on dry matter content, zinc and copper total concentration and solubility in the caecum and ileum of growing pigs.
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Table 5. Effect of organic or inorganic mineral supplementation, with or without mannan oligosaccharide (MOS) supplementation, on volatile fatty acid concentration and pH in the caecum and ileum of growing pigs.†
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Inorganic mineral source
pH Ileum Caecum VFA# [mmol/l] Ileum Lactic acid Acetic acid Propionic acid Isobutyric acid Butyric acid Total Caecum Lactic acid Acetic acid Propionic acid Isobutyric acid Butyric acid Isovaleric acid Valeric acid Total
Organic mineral source
0% MOS
0.1% MOS
0% MOS
0.1% MOS
SEM*
7.2 5.9
6.9 5.9
6.9 5.8
7.0 5.9
0.1 0.1
25.2 3.69 0.81 0.15 0.86 30.7
14.1 2.89 0.64 0.24 0.40 18.3
23.6 3.66 0.78 0.15 1.00 29.3
20.7 3.92 1.06 0.12 0.72 26.6
7.2 1.35 0.27 0.03 0.28 9.0
0.68 57.9 37.0 1.89 11.4 0.33 3.26 112.6
0.52 61.7 36.7 1.01 12.2 0.27 3.06 115.5
0.22 60.8 39.5 1.67 13.3 0.26 3.72 119.5
1.68 62.1 37.8 1.03 11.9 0.26 3.05 117.8
0.60 3.2 1.9 0.45 1.7 0.06 0.45 6.2
Note: †All parameters in this table were not significantly modified by mineral source or MOS supplementation (p > 0.10); *SEM, Standard error of the mean, n = 7 for each treatment; #Per litre of fresh digesta.
higher retention suggests that, within the actual daily intake of Cu and Zn above estimated requirements (NRC 2012), the homoeostatic regulation of Zn and Cu was not clearly affecting the absorption and excretion of minerals to limit the mineral retention (van den Berghe and Klomp 2009; King 2010). Such increase of Zn and Cu retentions was reported in previous studies with pigs using comparable daily mineral intakes for Zn (Wedekind et al. 1994; Revy et al. 2002; Veum et al. 2014) and for Cu (Adeola 1995; Veum et al. 2004). Finally, it is also possible that these effects on feed intake and body weight might not be of biological significance because the present experiment was not designed, in terms of sampling size and experimental conditions, to assess effects on growth performance.
4.1. Intestinal fate of Cu The results from present study showed that pigs fed a diet supplemented with organic minerals had a better apparent digestibility and retention of Cu, and tended to excrete less Cu in faeces. This is consistent with reports from Lee et al. (2001), Creech et al. (2004), Huang, Yoo, et al. (2010), Huang, Zhou, et al. (2010) and Korniewicz et al. (2007) showing that total apparent digestibility of Cu was increased and faecal concentrations were lower in weanling and growing pigs fed an organic source (amino acid chelate) of this mineral. However, this is in contrast with Apgar and Kornegay (1996) who did not
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report any effect in Cu digestibility and retention in finishing pigs fed diets containing CuSO4 or Cu-lysine at a Cu concentration of 200 mg/kg. A similar lack of treatment effect was observed by Jolliff and Mahan (2013) using 15 mg/kg of Cu provided as CuSO4 or Cu-peptide complex. It is important to note that the present higher digestibility of Cu was obtained when animals were fed above the estimated requirement (NRC 2012). As mentioned previously, the homoeostatic regulation would normally limit the absorption or stimulate biliary excretion when the animals are fed beyond the need to minimise the retention of mineral. Veum et al. (2009) found that the apparent digestibility of Cu for weanlings was reduced linearly (35.4–19.8%) for dietary concentrations ranging from 8 to 16 mg/kg, suggesting a down regulation of the intestinal absorption or stimulation of biliary excretion. However, this reduction may be explained by a linear increase of feed intake in pig fed diet containing 8 to 16 mg/kg of Cu. Moreover, Cao and Chavez (1995) in pregnant sows noted no change in the apparent Cu digestibility of diets containing 2 and 12 mg/kg of Cu. In growing pigs, Veum et al. (2004) have also observed no significant effect on the apparent digestibility of Cu for diets containing between 30 and 130 mg/kg of Cu. Although it cannot be excluded that the homoeostatic control could have interfered with the assessment of digestibility Cu from different forms (organic and inorganic), the present data in pigs showed that within the range of concentrations used in the present study, the effects of dietary Cu concentration on the apparent Cu digestibility may be limited. This assumption for Cu could also applied to Zn as a recent meta-analysis (Schlegel et al. 2013) has been revealed that the dietary Zn content did not affect the apparent digestibility of Zn in pigs for the diets containing between 40 and 140 mg/kg. One hypothesis raised to explain a greater total tract apparent digestibility of dietary Cu provided as an organic form is related to the dissolution step of the mineral before it reaches the absorption sites located on the brush border of the small intestine (Wapnir 1998). The solubility of Cu in the digesta was not influenced by the type of dietary minerals in ileum or caecum. The global ileal concentration of dissolved Cu in the present study, at 45.7 µmol/l, was close to that measured in growing pigs (31.6 µmol/l) in a study conducted by Dintzis et al. (1995) with a diet containing 13 mg/kg of Cu. To our knowledge, Km (Michaelis constant) values for Cu in intestinal mucosa have never been reported in pigs. However, if values are comparable to those reported for rat (Varada et al. 1993) and mice (Bronner and Yost 1985) at 10–13 and 4.3 µmol/l, respectively, the present amount of dissolved Cu, varying from 24 to 66 µmol/l, was unlikely a limiting factor for the active uptake of Cu in the intestinal mucosa. This is supported by the fact that the lack of treatment effect on solubility of Cu in ileal and caecal digesta was not reflected on total tract apparent digestibility where the response was greater with the organic source of minerals. Such observation combined with the lack of correlation between dissolved concentrations of Cu and apparent digestibility in the ileum, caecum and faeces suggest that the increased apparent digestibility of Cu provided in organic form was due to other factors more related to intestinal mucosa than to luminal contents. As proposed by Aldridge et al. (2007) and Tastet et al. (2010), the improved absorption capacity of organic Cu complexes could be attributed to other route of absorption, as Peptide transporter-1 (PEPT1), or to better interaction between Cu supplement and intestinal mucosa. It is noteworthy that the present lack of relationship between the solubility of Cu and digestibility were observed with pigs fed above the estimated requirements (NRC 2012). It is possible that the soluble proportion of Cu and thus the relation with apparent digestibility were different with animals fed according to the estimated requirements (van den Berg et al. 1994).
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The lack of relationship between solubility of Cu and intestinal absorption is further supported by the positive effect of MOS on the solubility of Cu in the ileal content without corresponding effects on apparent digestibility in ileum, caecum or faeces. In addition, the reduced global concentrations of dissolved Cu observed in caecal compared to ileal digesta was not associated to corresponding differences in apparent digestibility of Cu in these intestinal segments. In fact, the apparent digestibility of Cu was close in these two parts of digestive tract (31.1% vs. 32.8% for ileum and caecum, respectively) suggesting that the responses of dietary mineral sources on total tract apparent digestibility is related to post-ileo-caecal effects. In fact, our results suggest that there would be absorption of Cu in the colon, as the apparent digestibility of Cu increased from 31.1% and 34.6% in the caecum for the inorganic and organic source, respectively, to 51.5% and 59.1% in the faeces. Although data on the Cu absorption by the colon are not available for the pigs, Turner et al. (1987) noted that in sheep, Cu could be absorbed from the colon but not from the caecum. Moreover, the Cu influx transporter 1 (Ctr1) protein was identified in human colonic epithelium (Holzer et al. 2006). For absorption of Cu-peptide via PEPT1 transporter, the mRNA of this transporter is expressed in the colonic mucosa of miniature pigs (Nosworthy et al. 2013). Further investigations are necessary to better assess the importance of the colon for Cu absorption. 4.2. Intestinal fate of Zn No major treatment effect was noted for apparent digestibility and retained/ingested ratio of Zn. Although a decrease of apparent digestibility was noted in the ileum for the organic mineral source, this result was not supported by data obtained from the caecum and faeces, or during the balance study. This lack of effect of organic Zn supplement on apparent digestibility of Zn is in agreement with results obtained by Revy et al. (2002, 2004), Paulicks et al. (2011), Schlegel et al. (2010) and Jolliff and Mahan (2013). In addition, no treatment effect was noted for dissolved Zn in the ileum or the caecum. Schlegel et al. (2010) obtained similar results in jejunal digesta of weanling piglets fed diets containing 38 mg/kg of Zn and supplemented with Zn sulphate or Zn-glycine. The dissolved Zn concentrations observed in the ileum were close (219 vs. 143 µmol/l) to the values obtained by Dintzis et al. (1995) for a diet containing 163 mg/kg of Zn. These concentrations were also higher than the Km value (67 µmol/l) reported for the mucosa of the small intestine in pigs (Blakeborough and Salter 1987), suggesting that the active enterocyte transport system of Zn was likely saturated, regardless the source of Zn when pigs fed above the Zn requirements at about 140 mg/kg. Nevertheless, the apparent digestibility of Zn in intestinal segments was smaller in pigs fed organic mineral supplement and this effect was particularly marked in the ileum where values were lower than 10%. As the present ileal samples represented a pool of the entire terminal metre of the small intestine, it is possible that values were not representative of the apparent digestibility of Zn at the far end portion of the ileum. In rats, the absorption capacity of the intestinal mucosa is not homogenous throughout the small intestine being greater in the proximal and distal portions than in the medial (Condomina et al. 2002; Yasuno et al. 2012). Such information is not available in pigs but if such partitioning of absorption capacity also occurs in pigs, these present apparent digestibility values might be an underestimation of the real digestibility at the terminal ileum. Such hypothesis of a gradual increase of absorption capacity towards the far end of ileum would be coherent with the important absorption of Zn observed further in the caecum. In rats, however, studies suggest that the caecum contributes little to Zn absorption (Hara et al. 2000).
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Besides changes of absorption capacity along the intestinal tract, ileal and caecal digestibility of Zn was reduced (or tended to be reduced) with dietary organic minerals. Such effect was not related to luminal dissolved levels of Zn in these intestinal segments. Moreover, the response did not persist until the end of the digestive tract, for faecal values. Such results suggest that mucosal factors related to intestinal mucosa interfere with the treatment effects in the colon but the information to assess the importance of this mechanism for Zn metabolism is lacking.
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4.3. MOS and its intestinal effects No effect of the MOS supplement was observed on intestinal pH, VFA concentration, apparent digestibility and retained/ingested ratio of Cu or Zn in the present study. In the case of pH and VFA concentration, this is in contrast with previous results obtained from studies of piglets (Halas and Nochta 2012). It is possible that the duration of present experimental period could have limited the effect of MOS on the gut flora (Houdijk et al. 1999). Houdijk et al. (1999) noted that pigs might require more than two weeks to adapt to dietary NDO and affect these two variables. However, for apparent digestibility or retention of Cu and Zn, the present absence of MOS effects is in agreement with Houdijk et al. (1999) using FOS dietary supplements (1.35%) and trans-galacto-oligosaccharides (0.8%) in growing pigs. 5. Conclusions In conclusion, the organic mineral supplement increased the apparent digestibility and retained/ingested ratio of Cu, but this effect cannot be attributed to changes in Cu solubility in ileal or caecal digesta in growing pigs fed above the requirement of Cu (25 mg/kg). For Zn, in spite of a reduction in apparent digestibility in ileum and caecum with the organic mineral supplement, the total tract digestibility and the retained/ingested ratio of Zn were similar between mineral sources provided above the requirement (140 mg/kg). The organic mineral and MOS supplements also increased feed intake which led to an increase in retention of Cu and Zn. The dietary supplement of MOS did not interfere with the mineral sources and had no main effect on the intestinal environment (pH and VFA concentration) or the apparent digestibility of Zn and Cu in growing pigs. Funding Financial support for this work was provided by a grant from the Natural Science and Engineering Research Council (NSERC) of Canada awarded to F. Guay and by a studentship from Alltech Inc., Nicholasville, KY, USA, awarded to A. Lebel.
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Archives of Animal Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gaan20
Effect of mineral source and mannan oligosaccharide supplements on zinc and copper digestibility in growing pigs a
b
a
Alexandre Lebel , J. Jacques Matte & Frédéric Guay a
Département des sciences animals, Université Laval Québec, Québec, Canada G1V 0A6 b
Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, Sherbrooke, Québec, Canada J1M 0C8 Published online: 02 Sep 2014.
To cite this article: Alexandre Lebel, J. Jacques Matte & Frédéric Guay (2014) Effect of mineral source and mannan oligosaccharide supplements on zinc and copper digestibility in growing pigs, Archives of Animal Nutrition, 68:5, 370-384, DOI: 10.1080/1745039X.2014.954357 To link to this article: http://dx.doi.org/10.1080/1745039X.2014.954357
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Archives of Animal Nutrition, 2014 Vol. 68, No. 5, 370–384, http://dx.doi.org/10.1080/1745039X.2014.954357
Effect of mineral source and mannan oligosaccharide supplements on zinc and copper digestibility in growing pigs Alexandre Lebela, J. Jacques Matteb and Frédéric Guaya*
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a Département des sciences animals, Université Laval Québec, Québec, Canada G1V 0A6; bDairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, Sherbrooke, Québec, Canada J1M 0C8
(Received 26 May 2014; accepted 9 August 2014) This study aimed to compare the effects of organic (proteinate) and inorganic (sulphate) copper (Cu) and zinc (Zn) supplements, in presence or absence of a mannan oligosaccharide (MOS) supplement, on mineral solubility and digestibility in pigs. Twenty-eight barrows (25 ± 4 kg) assigned randomly to four treatment groups were fed a corn-wheat-soya bean meal diet with 10 mg/kg of Cu and 100 mg/kg of Zn supplied as organic or inorganic supplement, and supplemented or not with 0.1% MOS. After an adaptation period, total faeces and urine were collected for a period of 6–7 days. Pigs were then euthanatised and digesta from ileum and caecum were collected. Apparent digestibility was calculated in ileum and caecum using titanium dioxide. The organic mineral supplement improved total (faecal) digestibility and retained/ ingested ratio of Cu (p < 0.05) while reducing apparent digestibility of Zn in the ileum (p < 0.05) without effect on total digestibility of Zn. Solubilities of Cu and Zn in liquid fraction of ileum and caecum were not affected by mineral sources. Although MOS supplement increased Cu solubility in the ileum (p < 0.05), it had no effect on digestibility of Zn and Cu in ileum, caecum and faeces, retained/ingested ratio of Zn and Cu, or pH and volatile fatty acid concentration in ileal and caecal digesta. In conclusion, organic mineral supplement improved total digestibility and retained/ ingested ratio of Cu in pigs but this cannot be attributed to its solubility in ileal and caecal digesta. The MOS supplement did not interfere with digestibility or dietary utilisation of Zn and Cu in pigs fed above the Zn and Cu requirements. Keywords: copper; digestibility; oligosaccharides; organometallic compounds; pigs; zinc
1. Introduction Inorganic mineral supplements such as zinc (Zn) sulphate or oxide and copper (Cu) sulphate have long been added to swine diets. These trace elements dissolve in the digestive tract and release free ions that can bind to organic molecules (e.g. phytates, fibre) present in ingested matter, leaving the minerals difficult to absorb (Wapnir 1998; Camara and Amaro 2003). Moreover, as the pH increases in the small intestine, Zn and Cu are trapped in insoluble hydroxide precipitates (Powell et al. 1999). Chelating of dietary trace minerals is claimed to prevent the formation of insoluble complexes in the digestive tract, increase transfer from the intestinal lumen to epithelial cells and hence, improve intestinal absorption (Mullan and D’Souza 2005). Dietary supplements of Zn and Cu chelated with organic molecules such as amino acids (e.g. lysine, methionine), polysaccharides or partially hydrolysed proteins are nowadays available in swine *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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nutrition. The efficiency of replacing inorganic Zn or Cu with organic sources is controversial. Numerous studies have reported little benefit in terms of swine performance (Jondreville et al. 2002; Revy et al. 2003; Schlegel et al. 2013), whereas others indicate that using chelated forms can reduce mineral excretion in pigs (Lee et al. 2001; Creech et al. 2004; Korniewicz et al. 2007; Huang, Yoo, et al. 2010; Huang, Zhou, et al. 2010). In fact, mechanisms involved in absorption of Zn and Cu from dietary organic complexes (e.g. increased solubility or others) have not been established yet. This lack of knowledge is critical for a reliable understanding of these inconsistent responses. Non-digestible oligosaccharides (NDO) like inulin and other fructo-oligosaccharides (FOS) are known to improve digestibility (i.e. absorption) of calcium, magnesium, Zn and Cu in rats (Delzenne et al. 1995; Lopez et al. 2000; Mineo et al. 2004; Coudray et al. 2006) but no such effects have been reported for pigs (Houdijk et al. 1999, 2002; Jolliff and Mahan 2012). Mannan oligosaccharides (MOS) are known to bind bacteria like Escherichia coli and Salmonella spp. and to prevent the proliferation of these pathogens at the mucosal surface of the intestine in weanling pigs (Halas and Nochta 2012). In contrast to FOS, MOS have been shown to improve the digestibility of calcium in weanling piglets (Nochta et al. 2010). This beneficial effect may be due to a reduction in pH of the intestinal contents (Halas and Nochta 2012) along with a decrease in mineral precipitation. To the best of our knowledge the effect of MOS on intestinal absorption of Zn and Cu has never been investigated. The objective of the present study was to evaluate the effects of MOS on the apparent digestibility of Zn and Cu in growing pigs fed organic or inorganic supplement of these minerals. The solubility of Zn and Cu in the intestinal contents was also measured.
2. Materials and methods 2.1. Animals and experimental conditions Twenty-eight barrows (Landrace × Yorkshire) weighing on average 25 ± 4 kg were moved from the nursery of a commercial farm to a controlled-environment room with individual pens (1 m × 2 m) for 6 days and then transferred to metabolic cages for total collection of faeces and urine. Room temperature was maintained at 20°C. The animals had unlimited access to water and feed (approximately five times the level of maintenance metabolic energy of 440 kJ/kg of BW0.75). Refusals were weighed and harvested daily in order to determine feed intake. All animal procedures were conducted according to the guidelines set by the Canadian Council of Animal Care (2009).
2.2. Dietary treatments Animals were randomly distributed into seven repetitions based on initial weight. Within each repetition, pigs were distributed into factorial treatments (2 × 2) with organic or inorganic dietary mineral supplementation and presence or absence of MOS (Table 1). The level of mineral supplementation was 10 mg/kg of Cu and 100 mg/kg of Zn, provided as sulphate (inorganic) or as a protein complex (organic) (Bioplex®, Alltech, Nicholasville, KY, USA). These dietary levels of Cu and Zn are higher than those recommended by National Research Council (2012) but they are representative of those used in practice as recommended by van Heugten (2010) and, as such, are more appropriate to study a realistic interaction with NDO feed additives. MOS was supplemented at 0.1% with Bio-Mos® (Alltech, Nicholasville, KY, USA). Titanium dioxide (TiO2) was
372 Table 1.
A. Lebel et al. Ingredient composition of the experimental diets (as-fed basis).
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Inorganic mineral source
Ingredient [g/kg diet] Wheat Corn Soya bean meal Ground limestone Dicalcium phosphate Salt Lysine HCl L-threonine DL-methionine Vitamins* Minerals# ZnSO4 (inorganic) CuSO4 (inorganic) Zn proteinate (organic)$♦ Cu proteinate (organic)$♦ Mannan oligosaccharide$◊ Titanium dioxide Chemical composition, calculated† [g/kg] Crude protein Calcium Total phosphorus Chemical composition, analysed [mg/kg] Total copper Total zinc
Organic mineral source
0% MOS♦
0.1% MOS
0% MOS
0.1% MOS
150.00 615.71 200.00 12.00 8.50 3.50 3.00 0.50 0.30 2.50 2.50 0.45 0.04 – – – 1.00
150.00 614.71 200.00 12.00 8.50 3.50 3.00 0.50 0.30 2.50 2.50 0.45 0.04 – – 1.00 1.00
150.00 615.43 200.00 12.00 8.50 3.50 3.00 0.50 0.30 2.50 2.50 – – 0.67 0.10 – 1.00
150.00 614.43 200.00 12.00 8.50 3.50 3.00 0.50 0.30 2.50 2.50 – – 0.67 0.10 1.00 1.00
160.0 6.9 5.1
160.0 6.9 5.1
160.0 6.9 5.1
160.0 6.9 5.1
25.0 143.6
25.0 144.0
23.7 137.0
26.7 151.4
Notes: ♦MOS, Mannan oligosaccharide; *Contribution per kilogram of complete feed (DM): vitamin A palmitate 5000 IU, vitamin D3 1000 IU, vitamin E acetate 22.5 mg, menadione sodium bisulphite 3.75 mg, thiamine HCl 10 mg, riboflavin 4.5 mg, niacin 20.0 mg, calcium pantothenate 25.0 mg, pyridoxine HCl 1.5 mg, biotin 0.2 mg, choline bitartrate 375 mg, vitamin B12 25.0 µg; #Contribution per kilogram of complete feed: iron citrate 100 mg, potassium iodide 0.28 mg, manganese carbonate 46 mg, sodium selenite 0.30 mg; $Alltech Inc. Nicholasville, KY, USA, ♦Bioplex Zn and Bioplex Cu, respectively, composition: 29% crude protein, 0.5% ether extract, 35% ash, acid detergent fibre 0.78%, amino acid profile: lysine 100, histidine 42, isoleucine 62, leucine 113, methionine 21, phenylalanine 80, threonine 53, valine 77, tryptophan 19; ◊Bio-MOS, composition: crude protein 31%, ether extract 4%, ash 6%, acid detergent fibre 9%. †Nutritional composition based on Sauvant et al. (2004).
added (0.1%) to all four diets as an external marker for evaluation of mineral apparent digestibility in the ileum, caecum and faeces.
2.3. Collection of samples for measurement of mineral absorption After 2 days of adaptation to the metabolic cages, all faeces and urine produced were collected twice daily for a period of 6 or 7 days. The cage floors were fitted with funnels for urine collection. Faeces were frozen at −20°C without delay, as was 10% of the urine. At the end of the collection period, all frozen faeces were thawed, mixed thoroughly and frozen again until analysis. Frozen urine was treated likewise.
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2.4. Euthanasia and sampling of digesta The animals had access to feed until 2 hours before euthanasia. They were given an intramuscular injection of a mixture of ketamine (Ketalean 20 mg/kg BW, Vétoquinol Canada Inc., Lavaltrie, Québec, Canada) and xylazine (Xylamax 5 mg/kg BW, Vétoquinol Canada Inc., Lavaltrie, Québec, Canada). Sodium pentobarbital (Euthanyl: 120 mg/kg BW, Vétoquinol Canada Inc., Lavaltrie, Québec, Canada) was administered to the anaesthetised animals via an ear vein. The abdomen was then opened and the entire intestine removed (from the duodenum to the colon). The ileum (the last metre of the small intestine) and the caecum were isolated using plastic strips and then separated. The digesta in each segment was collected and three types of digesta samples were prepared. One portion was acidified with HCl (1 mol/l) in order to minimise volatilisation of volatile fatty acids (VFA) and was immediately frozen at −20°C with a second non-acidified portion. A third portion was maintained on ice for pH determination after which it was centrifuged at 2000 g (4°C) for 20 min and the supernatant was frozen for dissolved mineral analysis as described below.
2.5. Analytical procedures The pH of intestinal contents diluted 1:1 with distilled water was measured directly using a pH probe and meter (Check-Mite, Buffalo, NY, USA). Analyses of total Zn and Cu were performed using Association of Official Analytical Chemists (2005) method 985.01. Briefly, 1 g of feed or freeze-dried digesta was placed in a refractory oven at 500°C for at least 8 h. The ash was hydrated with 10 drops of distilled water and 4 ml of HNO3 (1 mol/l) were added. This mixture was evaporated on a hot plate at 100–120°C and then placed in the refractory oven for an additional 8 h. The cooled ash was dissolved in 10 ml of HNO3. Dissolved Cu and Zn were determined after a dilution with HNO3 at ratios of 1:5 and 1:10 for caecal and ileal samples, respectively. Urine was mixed 1:5 with diluted HNO3. Mineral concentration was measured using an inductively coupled plasma (ICP) spectroscope (Optima 4300, Perkin Elmer, Wellesley, MA, USA). Titanium dioxide was determined according to the method of Jagger et al. (1992) along with the procedures proposed by Short et al. (1996). Briefly, lyophilised digesta or faeces or dry feed was incinerated at 500°C. The sample was mixed with H2SO4 and boiled gently for 1 h, then filtered on Whatman 541 paper (Buckinghamshire, UK). The filtrate was mixed with 30% hydrogen peroxide. Absorbance at 410 nm was measured using a Varioskan spectrophotometer (Thermo Electron Corporation, Vantaa, Finland) and compared to a standard curve. VFA were measured using HPLC (Waters Corporation, Milford, MA, USA) according to the method of Canale et al. (1984). VFA were detected by ultraviolet visible detector at 210 nm.
2.6. Calculations and statistical analysis Apparent digestibility based on the TiO2 marker was calculated using the equation proposed by Adeola (2001): TiO2 feed ½%Mineralsample ½mg=kg 100 Apparent digestibility ½% ¼ 100 TiO2 sample ½%Mineralfeed ½mg=kg
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Treatment effects on feed intake, body weight gain, on mineral absorption, deposition, excretion and concentration, and on pH and VFA concentrations were analysed according to a complete block design with initial body weight as criteria in a 2 × 2 factorial arrangement of treatments using the SAS MIXED procedure (SAS Institute Inc., Cary, NC, USA). The statistical model included the treatment effect (mineral source and MOS) and their interactions. Differences were considered significant when p < 0.05 and indicative of a tendency when p < 0.1. Pearson correlation coefficients were determined using the SAS Correlation procedure.
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3. Results Whatever the studied variables, there was no significant interaction between sources of minerals and MOS supplementation. During this experiment, no animal has to be discarded, no health problem was observed and no particular veterinary intervention was required. 3.1. Daily Cu and Zn balance The average Cu and Zn balances over the experimental period are presented in Table 2. Feed intake was increased for diets with the organic mineral supplement (p < 0.05) and, as a tendency, for those with the MOS supplement (p < 0.10). No treatment effect was observed on daily body weight gain but the final body weight tended to be greater in pigs fed organic mineral supplement (p < 0.08) or its combination with MOS supplement (Mineral source × MOS, p < 0.10). Daily absorption and retention of Cu and Zn were greater in pigs given the organic mineral supplement (p < 0.05) or the MOS supplement (p < 0.05). Excretion of Cu in faeces tended to be reduced (p < 0.09) in organic mineral treatments. This reduction was associated with increased apparent digestibility and retained/ingested ratio of Cu (p < 0.01). Urinary excretion of Cu tended to increase in MOS supplemented treatments (p < 0.06). Neither the mineral source nor the MOS supplement had any significant effect on the apparent digestibility or retained/ingested ratio of Zn but urinary excretion of Zn was greater with organic mineral supplementation (p < 0.05). 3.2. Apparent digestibility of Zn and Cu in the ileum, caecum and faeces The apparent digestibility of minerals in different parts of the gastrointestinal tract is presented in Table 3. As shown with the total faecal collection procedure (Table 2), estimations of total apparent digestibility of Cu with TiO2 marker were also improved in pigs receiving the organic mineral supplement (p < 0.05). However, this is not reflected in separate parts of the digestive tract. For Zn, apparent digestibility in ileum was decreased in pigs fed the organic supplement (p < 0.05) whereas it was only a trend (p < 0.08) in caecum. Globally, the mineral source had no significant effect on apparent digestibility of Zn in faeces. There was no significant effect of the MOS supplement on apparent digestibility of Zn and Cu measured in the ileum, caecum or faeces. 3.3. VFA, pH and mineral solubility in ileum and caecum There was no effect of the mineral source and supplement of MOS on the dry matter content of digesta in the ileum or caecum (Table 4). The dietary supplement of MOS
30.1 36.5 2141 1047 53.6 27.6 25.4 49.2 0.87 25.1 47.8 307.5 152.7 154.4 51.0 1.87 152.9 50.3
30.8 37.5 1994 1104
49.3 27.7 21.6 43.1 0.38 21.3 42.3
285.9 146.7 139.3 48.7 1.71 137.5 48.1
307.2 146.2 162.0 52.6 2.08 158.8 51.7
53.1 21.9 31.2 58.6 0.54 30.7 57.6
30.6 37.5 2242 1140
362.2 160.6 200.6 54.8 2.33 198.3 54.2
63.4 25.8 37.6 58.7 0.79 36.9 57.5
32.1 39.4 2380 1196
12.6 12.0 11.4 2.9 0.18 11.4 2.1
2.5 2.2 2.1 3.3 0.18 2.5 3.3
0.7 0.8 0.086 0.057
SEM*
0.033 NS 0.040 NS 0.033 0.044 NS
0.045 0.090 0.001 0.003 NS 0.001 0.001
NS† 0.080 0.050 NS
Mineral source
0.042 NS 0.050 NS NS 0.050 NS
0.049 NS 0.042 NS 0.059 0.041 NS
NS NS 0.096 NS
MOS
p-Value
NS NS NS NS NS NS NS
NS NS NS NS NS NS NS
NS 0.095 NS NS
Mineral × MOS
Note: ‡Initial and final body weights were evaluated before pigs were transferred in metabolic crates and at the end of collection period, respectively; *SEM, Standard error of the mean, n = 7 for each treatment, faeces and urine were collected on 6 or 7 days; †NS, Not significant (p > 0.10).
Initial body weight [kg]‡ Final body weight [kg]‡ Feed intake [g/d] Body weight gain [g/d] Copper Ingested [mg/d] In faeces [mg/d] Absorbed [mg/d] Apparent digestibility [%] In urine [mg/d] Retained [mg/d] Retained/ingested ratio [%] Zinc Ingested [mg/d] In faeces [mg/d] Absorbed [mg/d] Apparent digestibility [%] In urine [mg/d] Retained [mg/d] Retained/ingested ratio [%]
0.1% MOS
0% MOS
0% MOS
0.1% MOS
Organic mineral source
Inorganic mineral source
Table 2. Effect of organic or inorganic mineral supplementation, with or without mannan oligosaccharide (MOS) supplementation, on feed intake, body weight gain and copper and zinc balance in growing pigs.
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Table 3. Effect of organic or inorganic mineral supplementation, with or without mannan oligosaccharide (MOS) supplementation, on apparent digestibility [%]# of copper and zinc in growing pigs.
Ileum Copper Zinc Caecum Copper Zinc Faeces Copper Zinc
Inorganic mineral source
Organic mineral source
0% MOS
0.1% MOS
0% MOS
0.1% MOS
SEM*
Mineral source
MOS
Mineral × MOS
37.9 30.0
24.4 24.5
36.2 −0.1
25.8 9.4
7.1 10.0
NS† 0.021
NS NS
NS NS
29.6 59.0
32.5 62.0
29.7 53.1
39.5 58.0
4.9 2.6
NS 0.080
NS NS
NS NS
48.7 55.3
54.3 56.7
59.7 50.6
58.4 55.3
2.5 2.9
0.041 NS
NS NS
NS NS
p-Value
Note: #Using the calculation of Adeola (2001) and TiO2 as an external marker; *SEM, Standard error of the mean, n = 7 for each treatment; †NS, Not significant (p > 0.10).
reduced the concentration and proportion of dissolved Cu in the ileum (p < 0.05), while it had no effect on dissolved Zn. In caecum, neither the mineral source nor the addition of MOS had any effect on Cu or Zn solubility, although MOS did increase the total concentration of Zn (p < 0.05). Values of dissolved mineral concentrations were not correlated with those of apparent digestibility of Cu and Zn in the ileum and caecum (Cuileum: r = 0.053, p = 0.819; Cucaecum: r = 0.160, p = 0.455; Znileum: r = –0.180, p = 0.422; Zncaecum: r = −0.099, p = 0.632). Finally, no treatment effect was observed on pH or VFA concentrations either in ileum or caecum (Table 5).
4. Discussion Feed intake was greater in pigs fed the MOS supplement but it was only a tendency. This is in line with results on weaned piglets where MOS have shown positive effects on feed intake and growth performance but the response was transient for about two weeks postweaning and faded out thereafter (Miguel et al. 2004). The greater feed intake and higher final body weight in pigs fed organic mineral supplements were in contrast with recent results obtained by Jolliff and Mahan (2013) and Martin et al. (2011), who did not report any effect of mineral source (Cu-peptide vs. Cu sulphate) on feed intake and growth performance in growing pigs when diets contained about 15 mg/kg of Cu and 150 mg/kg of Zn. For Cu, it is known that feed intake and average daily gain in growing pigs may be increased with high supplementations of 125 to 200 mg/kg but usually not at concentrations like those used in the present experiment (about 25 mg/kg) (review by Jondreville et al. 2002). It is unlikely that other components than Cu and Zn which differed between trace mineral premixes might have had an impact on feed intake and then on growth performance as the relative contribution of these differences for the whole content of major nutrients of the diet was negligible (Table 1). Along with the effect of organic mineral and MOS supplements on feed intake, there was also an increase of Cu and Zn retention and the final body weight of these pigs tended to be greater by 10% as compared to those fed the inorganic mineral supplement. This
14.6 11.6
0.117 35.9 21.9 0.072 6.9 4.4 0.584 233.6 18.6 0.244 52.3 9.8
14.9 13.5
0.087 66.1 27.6
0.073 7.9 5.0
0.608 172.2 9.8
0.218 43.3 8.2
0.1% MOS
0.225 49.4 9.9
0.818 153.1 10.9
0.062 7.0 5.2
0.094 56.1 24.9
14.2 12.5
0% MOS
0.267 56.9 6.9
0.727 321.8 17.4
0.066 7.2 5.0
0.084 24.8 13.5
14.1 12.5
0.1% MOS
Organic mineral source
0.015 11.5 1.9
0.091 79.9 6.2
0.006 1.5 0.9
0.016 9.5 3.8
1.1 0.6
SEM*
NS NS NS
NS NS NS
NS NS NS
NS NS NS
NS† NS
Mineral source
0.045 NS NS
NS NS NS
NS NS NS
NS 0.006 0.040
NS NS
MOS
p-Value
NS NS NS
NS NS NS
NS NS NS
NS NS NS
NS NS
Mineral × MOS
Note: *SEM, Standard error of the mean, n = 7 for each treatment; †NS, Not significant (p > 0.10); #On dry matter basis; $Per cent of dissolved mineral for 100 g of fresh digesta.
Dry matter [%] Ileum Caecum Copper Ileum Total# [mg/g] Dissolved [µmol/l] Dissolved$ [%] Caecum Total# [mg/g] Dissolved [µmol/l] Dissolved$ [%] Zinc Ileum Total# [mg/g] Dissolved [µmol/l] Dissolved$ [%] Caecum Total# [mg/g] Dissolved [µmol/l] Dissolved$ [%]
0% MOS
Inorganic mineral source
Table 4. Effect of organic or inorganic mineral supplementation, with or without mannan oligosaccharide (MOS) supplementation on dry matter content, zinc and copper total concentration and solubility in the caecum and ileum of growing pigs.
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Table 5. Effect of organic or inorganic mineral supplementation, with or without mannan oligosaccharide (MOS) supplementation, on volatile fatty acid concentration and pH in the caecum and ileum of growing pigs.†
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Inorganic mineral source
pH Ileum Caecum VFA# [mmol/l] Ileum Lactic acid Acetic acid Propionic acid Isobutyric acid Butyric acid Total Caecum Lactic acid Acetic acid Propionic acid Isobutyric acid Butyric acid Isovaleric acid Valeric acid Total
Organic mineral source
0% MOS
0.1% MOS
0% MOS
0.1% MOS
SEM*
7.2 5.9
6.9 5.9
6.9 5.8
7.0 5.9
0.1 0.1
25.2 3.69 0.81 0.15 0.86 30.7
14.1 2.89 0.64 0.24 0.40 18.3
23.6 3.66 0.78 0.15 1.00 29.3
20.7 3.92 1.06 0.12 0.72 26.6
7.2 1.35 0.27 0.03 0.28 9.0
0.68 57.9 37.0 1.89 11.4 0.33 3.26 112.6
0.52 61.7 36.7 1.01 12.2 0.27 3.06 115.5
0.22 60.8 39.5 1.67 13.3 0.26 3.72 119.5
1.68 62.1 37.8 1.03 11.9 0.26 3.05 117.8
0.60 3.2 1.9 0.45 1.7 0.06 0.45 6.2
Note: †All parameters in this table were not significantly modified by mineral source or MOS supplementation (p > 0.10); *SEM, Standard error of the mean, n = 7 for each treatment; #Per litre of fresh digesta.
higher retention suggests that, within the actual daily intake of Cu and Zn above estimated requirements (NRC 2012), the homoeostatic regulation of Zn and Cu was not clearly affecting the absorption and excretion of minerals to limit the mineral retention (van den Berghe and Klomp 2009; King 2010). Such increase of Zn and Cu retentions was reported in previous studies with pigs using comparable daily mineral intakes for Zn (Wedekind et al. 1994; Revy et al. 2002; Veum et al. 2014) and for Cu (Adeola 1995; Veum et al. 2004). Finally, it is also possible that these effects on feed intake and body weight might not be of biological significance because the present experiment was not designed, in terms of sampling size and experimental conditions, to assess effects on growth performance.
4.1. Intestinal fate of Cu The results from present study showed that pigs fed a diet supplemented with organic minerals had a better apparent digestibility and retention of Cu, and tended to excrete less Cu in faeces. This is consistent with reports from Lee et al. (2001), Creech et al. (2004), Huang, Yoo, et al. (2010), Huang, Zhou, et al. (2010) and Korniewicz et al. (2007) showing that total apparent digestibility of Cu was increased and faecal concentrations were lower in weanling and growing pigs fed an organic source (amino acid chelate) of this mineral. However, this is in contrast with Apgar and Kornegay (1996) who did not
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report any effect in Cu digestibility and retention in finishing pigs fed diets containing CuSO4 or Cu-lysine at a Cu concentration of 200 mg/kg. A similar lack of treatment effect was observed by Jolliff and Mahan (2013) using 15 mg/kg of Cu provided as CuSO4 or Cu-peptide complex. It is important to note that the present higher digestibility of Cu was obtained when animals were fed above the estimated requirement (NRC 2012). As mentioned previously, the homoeostatic regulation would normally limit the absorption or stimulate biliary excretion when the animals are fed beyond the need to minimise the retention of mineral. Veum et al. (2009) found that the apparent digestibility of Cu for weanlings was reduced linearly (35.4–19.8%) for dietary concentrations ranging from 8 to 16 mg/kg, suggesting a down regulation of the intestinal absorption or stimulation of biliary excretion. However, this reduction may be explained by a linear increase of feed intake in pig fed diet containing 8 to 16 mg/kg of Cu. Moreover, Cao and Chavez (1995) in pregnant sows noted no change in the apparent Cu digestibility of diets containing 2 and 12 mg/kg of Cu. In growing pigs, Veum et al. (2004) have also observed no significant effect on the apparent digestibility of Cu for diets containing between 30 and 130 mg/kg of Cu. Although it cannot be excluded that the homoeostatic control could have interfered with the assessment of digestibility Cu from different forms (organic and inorganic), the present data in pigs showed that within the range of concentrations used in the present study, the effects of dietary Cu concentration on the apparent Cu digestibility may be limited. This assumption for Cu could also applied to Zn as a recent meta-analysis (Schlegel et al. 2013) has been revealed that the dietary Zn content did not affect the apparent digestibility of Zn in pigs for the diets containing between 40 and 140 mg/kg. One hypothesis raised to explain a greater total tract apparent digestibility of dietary Cu provided as an organic form is related to the dissolution step of the mineral before it reaches the absorption sites located on the brush border of the small intestine (Wapnir 1998). The solubility of Cu in the digesta was not influenced by the type of dietary minerals in ileum or caecum. The global ileal concentration of dissolved Cu in the present study, at 45.7 µmol/l, was close to that measured in growing pigs (31.6 µmol/l) in a study conducted by Dintzis et al. (1995) with a diet containing 13 mg/kg of Cu. To our knowledge, Km (Michaelis constant) values for Cu in intestinal mucosa have never been reported in pigs. However, if values are comparable to those reported for rat (Varada et al. 1993) and mice (Bronner and Yost 1985) at 10–13 and 4.3 µmol/l, respectively, the present amount of dissolved Cu, varying from 24 to 66 µmol/l, was unlikely a limiting factor for the active uptake of Cu in the intestinal mucosa. This is supported by the fact that the lack of treatment effect on solubility of Cu in ileal and caecal digesta was not reflected on total tract apparent digestibility where the response was greater with the organic source of minerals. Such observation combined with the lack of correlation between dissolved concentrations of Cu and apparent digestibility in the ileum, caecum and faeces suggest that the increased apparent digestibility of Cu provided in organic form was due to other factors more related to intestinal mucosa than to luminal contents. As proposed by Aldridge et al. (2007) and Tastet et al. (2010), the improved absorption capacity of organic Cu complexes could be attributed to other route of absorption, as Peptide transporter-1 (PEPT1), or to better interaction between Cu supplement and intestinal mucosa. It is noteworthy that the present lack of relationship between the solubility of Cu and digestibility were observed with pigs fed above the estimated requirements (NRC 2012). It is possible that the soluble proportion of Cu and thus the relation with apparent digestibility were different with animals fed according to the estimated requirements (van den Berg et al. 1994).
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The lack of relationship between solubility of Cu and intestinal absorption is further supported by the positive effect of MOS on the solubility of Cu in the ileal content without corresponding effects on apparent digestibility in ileum, caecum or faeces. In addition, the reduced global concentrations of dissolved Cu observed in caecal compared to ileal digesta was not associated to corresponding differences in apparent digestibility of Cu in these intestinal segments. In fact, the apparent digestibility of Cu was close in these two parts of digestive tract (31.1% vs. 32.8% for ileum and caecum, respectively) suggesting that the responses of dietary mineral sources on total tract apparent digestibility is related to post-ileo-caecal effects. In fact, our results suggest that there would be absorption of Cu in the colon, as the apparent digestibility of Cu increased from 31.1% and 34.6% in the caecum for the inorganic and organic source, respectively, to 51.5% and 59.1% in the faeces. Although data on the Cu absorption by the colon are not available for the pigs, Turner et al. (1987) noted that in sheep, Cu could be absorbed from the colon but not from the caecum. Moreover, the Cu influx transporter 1 (Ctr1) protein was identified in human colonic epithelium (Holzer et al. 2006). For absorption of Cu-peptide via PEPT1 transporter, the mRNA of this transporter is expressed in the colonic mucosa of miniature pigs (Nosworthy et al. 2013). Further investigations are necessary to better assess the importance of the colon for Cu absorption. 4.2. Intestinal fate of Zn No major treatment effect was noted for apparent digestibility and retained/ingested ratio of Zn. Although a decrease of apparent digestibility was noted in the ileum for the organic mineral source, this result was not supported by data obtained from the caecum and faeces, or during the balance study. This lack of effect of organic Zn supplement on apparent digestibility of Zn is in agreement with results obtained by Revy et al. (2002, 2004), Paulicks et al. (2011), Schlegel et al. (2010) and Jolliff and Mahan (2013). In addition, no treatment effect was noted for dissolved Zn in the ileum or the caecum. Schlegel et al. (2010) obtained similar results in jejunal digesta of weanling piglets fed diets containing 38 mg/kg of Zn and supplemented with Zn sulphate or Zn-glycine. The dissolved Zn concentrations observed in the ileum were close (219 vs. 143 µmol/l) to the values obtained by Dintzis et al. (1995) for a diet containing 163 mg/kg of Zn. These concentrations were also higher than the Km value (67 µmol/l) reported for the mucosa of the small intestine in pigs (Blakeborough and Salter 1987), suggesting that the active enterocyte transport system of Zn was likely saturated, regardless the source of Zn when pigs fed above the Zn requirements at about 140 mg/kg. Nevertheless, the apparent digestibility of Zn in intestinal segments was smaller in pigs fed organic mineral supplement and this effect was particularly marked in the ileum where values were lower than 10%. As the present ileal samples represented a pool of the entire terminal metre of the small intestine, it is possible that values were not representative of the apparent digestibility of Zn at the far end portion of the ileum. In rats, the absorption capacity of the intestinal mucosa is not homogenous throughout the small intestine being greater in the proximal and distal portions than in the medial (Condomina et al. 2002; Yasuno et al. 2012). Such information is not available in pigs but if such partitioning of absorption capacity also occurs in pigs, these present apparent digestibility values might be an underestimation of the real digestibility at the terminal ileum. Such hypothesis of a gradual increase of absorption capacity towards the far end of ileum would be coherent with the important absorption of Zn observed further in the caecum. In rats, however, studies suggest that the caecum contributes little to Zn absorption (Hara et al. 2000).
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Besides changes of absorption capacity along the intestinal tract, ileal and caecal digestibility of Zn was reduced (or tended to be reduced) with dietary organic minerals. Such effect was not related to luminal dissolved levels of Zn in these intestinal segments. Moreover, the response did not persist until the end of the digestive tract, for faecal values. Such results suggest that mucosal factors related to intestinal mucosa interfere with the treatment effects in the colon but the information to assess the importance of this mechanism for Zn metabolism is lacking.
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4.3. MOS and its intestinal effects No effect of the MOS supplement was observed on intestinal pH, VFA concentration, apparent digestibility and retained/ingested ratio of Cu or Zn in the present study. In the case of pH and VFA concentration, this is in contrast with previous results obtained from studies of piglets (Halas and Nochta 2012). It is possible that the duration of present experimental period could have limited the effect of MOS on the gut flora (Houdijk et al. 1999). Houdijk et al. (1999) noted that pigs might require more than two weeks to adapt to dietary NDO and affect these two variables. However, for apparent digestibility or retention of Cu and Zn, the present absence of MOS effects is in agreement with Houdijk et al. (1999) using FOS dietary supplements (1.35%) and trans-galacto-oligosaccharides (0.8%) in growing pigs. 5. Conclusions In conclusion, the organic mineral supplement increased the apparent digestibility and retained/ingested ratio of Cu, but this effect cannot be attributed to changes in Cu solubility in ileal or caecal digesta in growing pigs fed above the requirement of Cu (25 mg/kg). For Zn, in spite of a reduction in apparent digestibility in ileum and caecum with the organic mineral supplement, the total tract digestibility and the retained/ingested ratio of Zn were similar between mineral sources provided above the requirement (140 mg/kg). The organic mineral and MOS supplements also increased feed intake which led to an increase in retention of Cu and Zn. The dietary supplement of MOS did not interfere with the mineral sources and had no main effect on the intestinal environment (pH and VFA concentration) or the apparent digestibility of Zn and Cu in growing pigs. Funding Financial support for this work was provided by a grant from the Natural Science and Engineering Research Council (NSERC) of Canada awarded to F. Guay and by a studentship from Alltech Inc., Nicholasville, KY, USA, awarded to A. Lebel.
References Adeola O. 1995. Digestive utilization of minerals by weanling pigs fed copper- and phytasesupplemented diets. Can J Anim Sci. 75:603–610. Adeola O. 2001. Digestion and balance techniques in pigs. In: Lewis AJ, Southern LL, editors. Swine nutrition. Boca Raton (FL): CRC Press; p. 904–909. Aldridge BE, Saddaris KL, Radcliffe JS. 2007. Copper can be absorbed as a Cu-peptide chelate through the PepT1 transporter in the jejunum of weanling pigs. J Anim Sci. 87:154. Apgar GA, Kornegay ET. 1996. Mineral balance of finishing pigs fed copper sulfate or copperlysine complex has any growth-stimulating levels. J Anim Sci. 74:1594–1600.
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