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. 2019 Jul 30;97(9):3938–3946. doi: 10.1093/jas/skz238

Impact of dietary zinc:copper ratio on the postprandial net portal appearance of these minerals in pigs1

Danyel Bueno Dalto 1, Isabelle Audet 1, J Jacques Matte 1,✉
PMCID: PMC6736085  PMID: 31292635

Abstract

The present study compared the net intestinal absorption of zinc (Zn) and copper (Cu) after meals containing different dietary ratios among these trace elements. Ten 46-kg pigs were used in a cross-over design to assess the 10-h net portal-drained viscera (PDV) flux of serum Cu and Zn after ingestion of boluses containing ZnSO4 and CuSO4 in different Zn:Cu ratios (mg:mg): 120:20; 200:20; 120:8; and 200:8. Arterial Zn concentrations peaked within the first hour post-meal and responses were greater with 200 (0.9 to 1.8 mg/L) than with 120 mg (0.9 to 1.6 mg/L) of dietary Zn (dietary Zn × time, P = 0.05). Net PDV flux of Zn was greater (P = 0.02) with 200 than with 120 mg of dietary Zn and tended to be greater (P = 0.10) with 20 than with 8 mg of dietary Cu. The cumulative PDV appearance of Zn (% of dietary intake) was greater with 120 than 200 mg of dietary Zn from 8 h post-meal (P ≤ 0.04) and with 20 than 8 mg of dietary Cu from 7 h post-meal (P ≤ 0.05). At the end of the postprandial period (10 h), estimated PDV appearance of Zn was 16.0%, 18.4%, 12.0%, and 15.3% of Zn intake for 120:8, 120:20, 200:8, and 200:20 ratios, respectively. For Cu, irrespective of treatment, arterial values varied (P < 0.01) by less than 5% across postmeal times. Net PDV flux was not affected by treatments (P ≥ 0.12), but the value for ratio 120:20 was different from zero (P = 0.03). There was an interaction dietary Zn × dietary Cu on cumulative PDV appearance of Cu (% of dietary intake) at 30 min post-meal (P = 0.04) and thereafter at 3 h post-meal (P = 0.04). For the whole postprandial period (10 h), estimated PDV appearance of Cu was 61.9%, 42.1%, −17.1%, and 23.6% of Cu intake for 120:8, 120:20, 200:8, and 200:20 ratios, respectively. In conclusion, the present dietary amounts and ratios of Zn and Cu can affect the metabolic availability of both trace minerals for pigs. Ratios with 120 mg of dietary Zn maximized the postintestinal availability of both Zn and Cu.

Keywords: copper, dietary ratio, intestinal absorption, minerals, pigs, zinc

INTRODUCTION

Environmentally classified as heavy metals, trace minerals are essential nutrients in livestock diets (National Research Council [NRC], 2012). Besides their nutritional function, some trace minerals such as zinc (Zn) and copper (Cu) are also used at pharmacological levels in pigs’ diets due to their bacteriostatic actions (Heo et al., 2013).

In pigs, absorption of Zn and Cu range from 30% to 50% at nutritional levels (Lebel et al., 2014; Liu et al., 2014) and is inversely proportional to their intake (Jondreville et al., 2002; Revy et al., 2003). Therefore, a significant amount of dietary Zn and Cu fed to pigs is lost in manure with potentially negative environmental impacts (Jondreville et al., 2002; Revy et al., 2003). Although strategies to optimize absorption of these minerals are important for the pig production chain and environment, the limited knowledge on the gastrointestinal fate of dietary Zn and Cu in pigs has hampered advances in this matter.

In a recent study of this laboratory, Matte et al. (2017) measured the net portal-drained viscera (PDV) flux of Zn and Cu in pigs fed low vs. high dietary levels of organic or inorganic sources of these minerals. During the early postprandial period, greater net PDV flux of Zn was observed after ingestion of high dietary levels of Zn and Cu independently of source. For Cu, whereas net PDV flux was detected in pigs fed low dietary levels of Cu and Zn, no net PDV flux was detected for high dietary levels of both trace minerals, suggesting impairment of Cu absorption within the intestinal mucosa and/or metabolization in enterocytes. These minerals do not bind major common receptors on the intestinal mucosa (Cousins and McMahon, 2000; Wang et al., 2011) but share at least one intracellular transporter (metallothionein) that is known to be responsive to dietary Zn levels and preferentially saturated by Cu (Cousins, 1985). Matte et al. (2017) suggested that the unexpected blockage of net PDV Cu flux at high levels of dietary Cu supplementation would be associated with the concomitant high levels of dietary Zn. In that study, the 2 dietary levels of both trace minerals were given at a constant dietary Zn:Cu ratio of 10:1 (200:20 and 400:40). Therefore, it was hypothesized that, besides an optimum dietary Zn:Cu ratio (Jondreville et al., 2002), intestinal absorption of Cu would be affected by the absolute amount of dietary Zn.

In the present study, net PDV fluxes of Zn and Cu were compared in pigs fed diets with different Zn:Cu ratios. Such approach aims to determine the dietary Zn:Cu ratio that optimizes the intestinal absorption of both minerals and evaluate whether, under nutritional levels, dietary absolute amounts of Zn and Cu affect their mutual intestinal absorption.

MATERIALS AND METHODS

The experimental procedures followed the guidelines of the Canadian Council on Animal Care (2009) and were approved by the Institutional Animal Care Committee of the Sherbrooke Research and Development Centre (Quebec, Canada). All animals were cared for according to the recommended code of practice of the National Farm Animal Care Council (2014).

Animals, Surgeries, and Postoperatory Care

Fourteen Yorkshire-Landrace × Duroc gilts were selected at 30 kg of BW and fed ad libitum a conventional basal diet for growing pigs (Table 1) until surgery. Average BW at surgery was 45.6 ± 3.9 kg at 80.5 ± 4.9 d of age. The surgery procedure has been described by Hooda et al. (2009) and modified by Dalto et al. (2018). Briefly, a catheter was inserted in the portal vein at approximately 2.5 cm before its entry into the liver and an ultrasonic flow probe was installed around the portal vein 1.0 cm distal to the catheter. Another catheter was inserted through the carotid artery up to the junction between the carotid and subclavian arteries.

Table 1.

Composition of basal and semipurified diets

Ingredients Amount, %
Basal diet1
 Corn 56.3
 Soybean meal 48% 20.5
 Wheat 15.0
 Distillers dried grain with solubles 5.8
 Macro premix2 2.4
Semipurified diet3
 Cornstarch 71.4
 Casein 17.2
 Sucrose 11.4
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1Analyzed values: basal diet contained 17.8% protein, 0.73% Ca, 0.52% P, 140 mg/kg of Zn, 18 mg/kg of Cu, 479 mg/kg of Fe, 143 mg/kg Mn. Calculated ME content was 3,082 kcal.

2Provided per kilogram of diet: manganese as manganous oxide, 29 mg; zinc as zinc oxide,144 mg; iron as ferrous sulfate, 100 mg; copper as copper sulfate, 25 mg; iodine as calcium iodate, 1.0 mg; selenium as sodium selenite, 0.3 mg; vitamin A, 3,020 IU; vitamin D, 900 IU; vitamin E, 32 IU; vitamin K, 1.5 mg; thiamin, 2.0 mg; riboflavin, 3.5 mg; niacin, 20 mg; pantothenic acid, 15 mg; pyridoxine, 2.0 mg; biotin, 0.05 mg; folic acid, 0.5 mg; and vitamin B12, 0.02 mg.

3Analyzed values: 16.2% protein, <0.03% Ca, 0.07% P, 2.7 mg/kg of Zn, 1.3 mg/kg of Cu, 15 mg/kg Fe, 2 mg/kg Mn. Calculated ME content was 3,868 kcal/kg.

After surgery, animals were penned individually (1 m × 1.8 m) and fed the conventional growing-phase diet described above in a single daily meal according to their BW (1.0 kg/d until 50 kg BW; 1.2 kg/d from 50 to 60 kg BW; and 1.5 kg/d after 60 kg BW). Seven to 10 d after surgery, when animals have fully recovered appetite and normal growth rate, they were gradually adapted during 3 to 5 d to the metabolic cage (with free access to water) and to the consumption of a semipurified diet (up to 0.8 kg/d; Table 1) containing cornstarch (Produits Alimentaires Berthelet, Laval, QC, Canada), casein (C-7078, Sigma–Aldrich, St Louis, MO), and sucrose (S-8501, Sigma–Aldrich).

Treatments

The above-described semipurified diet was used to provide a feed matrix devoid of significant amounts of endogenous trace minerals. Daily amounts (based on a feed intake of 2 kg/d) of supplemental dietary Zn and Cu were based on EFSA (2014, 2016) or NRC (2012) recommendations. On experimental days, treatments were served in a bolus of 25 g of semipurified diet given just prior to the full meal (0.8 kg/d) of the same semipurified diet. According to a cross-over experimental design, each animal received 4 dietary treatments in a 2 × 2 factorial arrangement providing different Zn:Cu ratios: 120:20—120 mg of Zn and 20 mg of Cu; 200:20—200 mg Zn and 20 mg Cu; 120:8—120 mg of Zn and 8 mg of Cu; and 200:8—200 mg Zn and 8 mg Cu. Inorganic sources of these minerals (ZnSO4 and CuSO4; ref. nos. 307491 and 451657, Sigma–Aldrich) were chosen because of their stability, reliability, and purity in terms of basal composition.

Blood Collection and Analysis

On experimental days, pigs were moved to metabolic cages and blood samples (4 mL) were collected simultaneously from both catheters (artery and vein) 5 min before the oral bolus containing one of the 4 treatments, every 30 min for the first 2 h after bolus, and every hour for the following 8 h for a total of 10 postprandial hours. Portal blood flow was recorded continuously during 10 h using a flowmeter (Transonic 400-series; Ithaca, NY) and the PowerLab System (AD Instruments, Colorado Springs, CO). After each sampling period (10 h), pigs were offered the conventional growing-phase diet described above to complete their usual daily allowance according to their BW. Between experimental days (2 per week), animals were moved back to their respective pens and fed the conventional growing-phase diet described above.

Immediately after sampling, arterial and PDV blood were transferred from syringes into EDTA-treated tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) and trace element-free tubes BD Hemogard Closure (Vacutainer, Becton Dickinson). Packed cell volume was measured in duplicate on fresh PDV blood by microcentrifugation. Aliquots of arterial and PDV blood were frozen for hemoglobin determination according to the method of Drabkin (Manet, 1969). After at least 4-h standing, arterial and PDV blood were centrifuged at 1,800 × g for 10 min at 4 °C and serum was collected and frozen at −20 °C for Zn and Cu determination. Prior to analysis, serum samples were diluted 1:6 in 0.1 N HCl for Zn and 1:10 in ultra-purified H2O for Cu determination. Serum concentrations of both minerals were determined by flame atomic absorption spectrometry (AAnalyst 300 atomic absorption spectrometer, Perkin-Elmer Corp., Norwalk, CO) according to methods adapted from Arnaud et al. (1986) for Zn and from the standard analytical methods from Perkin-Elmer Instruments (2000) for Cu. Radiation sources were Lumina lamps specific for each mineral. An oxidizing air-acetylene flame was used at wavelengths of 213.9 nm for Zn and 324.8 nm for Cu with slit width of 0.7 for both minerals. Airflow (10 L/min) and acetylene flow (2 L/min) were similar for both minerals. Average intra- and interassay CV were 1.2% and 4.7% for Zn and 1.0% and 4.0% for Cu. For both minerals, a CV equal or smaller than 3% between duplicates was accepted for assessing the average value of a sample.

Calculations and Statistical Analysis

Out of the 14 surgically prepared pigs, portal catheters became inoperative before the first sampling day in 4 individuals that were eliminated from the trial. From the 40 postprandial serum profiles collected, 2 profiles from ratio 120:8 were incomplete (8 and 11 samples instead of 13) because of portal catheter malfunctioning during blood sampling. Net PDV fluxes of Zn and Cu were calculated for each sampling time as described by Girard et al. (2001). Positive net PDV flux indicates the release of the mineral from PDV, whereas negative net PDV flux indicates uptake of mineral by PDV. Arterial concentrations and net PDV fluxes of Zn and Cu recorded 5 min before the meal were used as basal levels (t0). Serum PDV flow (L/min) as well as serum arterial and PDV concentrations of Zn and Cu (mg/L), serum arterial and PDV fluxes of Zn and Cu (mg/min), portoarterial differences of Zn and Cu (µg/L), net PDV flux of Zn and Cu (µg/min), and cumulative serum Zn and Cu PDV appearance (expressed in % of dietary intake) were analyzed for each sampling time and across sampling times using the mixed procedure of SAS (SAS Inst., Inc., Cary, NC; Littell et al., 1996). The experimental design was a cross-over in which pigs, periods, dietary Zn levels, dietary Cu levels, and the interaction dietary Zn level × dietary Cu level were included in the model along with repeated measures (when appropriate) across sampling times (unequally spaced). For serum PDV flow, arterial and PDV concentrations, and serum arterial and PDV fluxes of Zn and Cu, the time effect covered the whole postprandial period (600 min). For portoarterial differences of Zn and Cu, net PDV fluxes of Zn and Cu, and cumulative serum Zn and Cu PDV appearance, t0 was removed for the statistical analysis. Because time intervals were unequally spaced, the following covariance structures were compared: space (power), space (Gaussian), space (exponential), space (linear), space (linear logarithmic), space (spherical), ante-dependence, and unstructured. For each variable, the best-fit statistic value was used as the criterion for the most relevant covariance structure. When the treatment effect was significant, multiple comparisons were performed using a t-test. Results are reported as least square means ± SEM. Differences were considered significant at P ≤ 0.05 and tendencies at 0.05 < P ≤ 0.10.

RESULTS AND DISCUSSION

Analytical concentrations of Zn and Cu in the semipurified diets and in premixes are presented in Table 1. The actual total intakes of Zn and Cu for each experimental meal (bolus + semipurified diet) corresponded to 123 and 21 mg for 120:20, 203 and 21 mg for 200:20, 123 and 9 mg for 120:8, and 202 and 9 mg for 200:8.

Portal-drained viscera serum flow was not affected by treatments (P ≥ 0.50; Table 2). Before the experimental meal (t0), average value was 0.76 ± 0.02 L/min. It rapidly increased to 1.01 ± 0.03 L/min after 30 min post-meal, remained almost constant until 90 min (1.05 ± 0.05 L/min), and decreased gradually thereafter to 0.75 ± 0.03 L/min at the end of the sampling period (time effect; P < 0.01; data not shown). These results are similar to those previously reported in growing pigs by Hooda et al. (2009, 2010) and Matte et al. (2010, 2012, 2017) using ultrasonic perivascular blood flow measurements.

Table 2.

Average portal-drained viscera (PDV) serum flow, Cu and Zn arterial serum concentration, portoarterial differences, and net PDV serum fluxes during 600 min post-meal according to factorial dietary treatments

Item/Zn:Cu ratios1 120:20 200:20 120:8 200:8 SEM P-value
Zn Cu Zn × Cu
PDV serum flow, L/min 0.85 0.86 0.89 0.89 0.05 0.92 0.50 0.88
Arterial serum Zn, mg/L 1.16 1.38 1.10 1.25 0.12 0.13 0.44 0.77
Zn portoarterial difference, µg/L 46.65 65.38 43.60 51.51 6.46 0.05 0.20 0.40
Net PDV flux of Zn, µg/min 37.93 54.59 34.87 41.45 4.63 0.02 0.10 0.29
Arterial serum Cu, mg/L 1.94 1.90 1.89 1.93 0.03 0.97 0.66 0.27
Cu portoarterial difference, µg/L2 14.28 7.56 10.29 −1.92 8.08 0.25 0.42 0.74
Net PDV flux of Cu, µg/min3 12.68 6.68 8.74 −3.50 5.62 0.12 0.23 0.59
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1Dietary treatments: 120:20, 120 mg Zn and 20 mg Cu; 200:20, 200 mg Zn and 20 mg Cu; 120:8, 120 mg Zn and 8 mg Cu; 200:8, 200 mg Zn and 8 mg Cu.

2Values were not different from zero (P ≥ 0.09).

3Only ratio 120:20 was different from zero (P = 0.03).

Zinc

Arterial serum Zn concentrations (Fig. 1) were greater with high dietary Zn from 60 min post-meal (interaction dietary Zn level × time; P = 0.05). Premeal values of 0.92 ± 0.03 mg/L for arterial Zn concentrations increased to maximum levels at the first postprandial hour (1.63 ± 0.12 mg/L) followed by a gradual decrease to 1.09 ± 0.04 mg/L by the end of the sampling period (time effect; P < 0.01). Similarly, Matte et al. (2017) have reported peaks in arterial Zn concentrations at 45 min after meals supplemented with different sources and levels of Zn in pigs. Although in the present study significant high levels (1.45 ± 0.07 mg/L for arterial serum) of Zn were observed earlier at 30 min post-meal (Fig. 1), it is likely that the true peak of Zn concentration would range between 45 and 60 min after the bolus. Such early peak of arterial Zn concentration after a meal strongly suggests that the absorption of this mineral takes place at the upper regions of the gut. Although the sites for Zn absorption have never been deeply studied in pigs, reports in rats have shown that duodenum absorbs approximately 55% of dietary Zn followed by ileum (18% to 30%; Van Campen and Mitchell, 1965; Davies, 1980). Interestingly, Martin et al. (2013) reported the presence of ZIP11 in the gastric mucosa of mice, an intestinal Zn transporter responding to dietary Zn. If this is the case in pigs, it appears possible that part of dietary Zn can be absorbed in the stomach because the mean digesta retention time and gastric emptying in pigs are as short as 1 and 3 h, respectively (Wilfart et al., 2007; Strathe et al., 2008) and the low pH of gastric secretions may increase Zn solubility and absorbability (Cousins, 1985). Nevertheless, it cannot be ruled out that the type of diet may be involved in variation of gastric emptying. Cuber et al. (1979, 1980) demonstrated that 33% of the ingested dry matter leaves the stomach within the first 15 min in pigs fed a semipurified diet similar to the present one. This could have carried part of dietary Zn to the proximal duodenum shortly after bolus. Further studies measuring directly the net gastric vein flux of Zn could bring relevant information on that matter.

Figure 1.

Figure 1.

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Average arterial serum Zn concentrations (mg/L) within 600 post-prandial minutes, presented as LS means ± SEM according to dietary zinc levels. Time effect (P < 0.01); Zn level × time interaction (P = 0.05).

Net PDV flux of Zn was greater (P = 0.02; Table 2) after intake of the highest dietary Zn. This result is in accordance with Matte et al. (2017) who evaluated different sources and levels of dietary Zn and Cu for pigs using this same surgical approach. However, specifically for ratio 200:20, the present value (54.59 µg/min) using ZnSO4 differ from that reported by Matte et al. (2017; 35.8 µg/min) using ZnO at similar dietary concentrations of Zn and Cu (200 and 20 mg). Among the most common sources of dietary Zn, ZnSO4 is generally recognized as more bioavailable than ZnO in pigs (Wedekind et al., 1994). In line with the above-mentioned comparison between net PDV flux of Zn in Matte et al. (2017) and the present results, the bioavailability of ZnO relative to ZnSO4 may vary from 39% to 88% (Revy et al., 2003). Interestingly, a tendency for a greater Zn net PDV flux (P = 0.10) was detected with the highest level of dietary Cu. To the best of our knowledge, this is the first report in pigs showing a positive effect of dietary Cu on Zn absorption. In rats, Jackson et al. (1981) reported that Cu supplementation in Zn-adequate diets reduce the amount of Zn within enterocytes and slightly increased Zn transfer to the carcass. It is known that the homeostasis of Zn within enterocytes is regulated by metallothionein (Davis and Cousins, 2000), and this metalloprotein constitutes the main intracellular pool of this mineral (Wang and Zhou, 2010). However, it has also been shown that metallothioneins preferentially binds Cu within enterocytes (Oestreicher and Cousins, 1985), following the Williams/Irving order of metals (Williams and Silva, 1997). Therefore, considering the distinct main transporters of Zn and Cu at the apical and basolateral regions of enterocytes (Cousins and McMahon, 2000; Wang et al., 2011) along with a common intracellular metalloprotein (metallothionein), a plausible explanation for the present results could be related to greater intracellular Cu levels preferentially binding metallothionein (at the expense of Zn), which would leave more free Zn available to be transported through the basolateral membrane into the portal system. For the present net PDV flux, no time effect or interactions between dietary Zn or Cu levels was detected (P ≥ 0.14). Values were different from zero during the whole experimental period (P < 0.01; data not shown), suggesting that Zn absorption takes place in multiple sites within the intestine. In fact, the presence of Zn transporters has been reported throughout the gastrointestinal system (Cousins, 2010), and although intestinal Zn absorption occurs mainly in the duodenum and ileum, jejunum and cecum/colon are also recognized as minor contributors (3.0% to 8.4%; Van Campen and Mitchell, 1965; Davies, 1980). Furthermore, it cannot be excluded that, as reported in humans (Cousins, 2010), the recycling of endogenous Zn in the intestinal lumen may also occur in pigs.

The cumulative PDV appearance of Zn (expressed as % of dietary intake) was affected by Cu levels after 420 min post-meal (P = 0.05) and by Zn levels and Cu levels from 480 min post-meal (P ≤ 0.04; data not shown). At the end of the experimental period (10 h), cumulative PDV appearance of Zn was affected by Zn levels (P = 0.03) and tended to be affected by Cu levels (P = 0.09), and all values were different from zero (P < 0.01). At the end of the postprandial period (10 h), estimated PDV appearance of Zn represented 16.0%, 18.4%, 12.0%, and 15.3% of Zn intake for 120:8, 120:20, 200:8, and 200:20 ratios, respectively (Fig. 3). Altogether, these last results reinforce the above-discussed positive impact of dietary Cu on Zn absorption. For Zn, considering that its absorption consists of a saturable and a nonsaturable component (Menard and Cousins, 1983; Steel and Cousins, 1985), the decrease in efficiency of absorption with the increase of dietary Zn levels is not surprising. In fact, according to Lonnerdal (2000), the efficiency of Zn absorption is inversely proportional to its intake probably because of the saturation of transport mechanism. Coppen and Davies (1987) suggested that a reduced efficiency of Zn absorption concomitant with greater dietary levels of Zn and enhanced amounts of total absorbed Zn, which is similar to the present results, would be consistent with an intestinal diffusion process. As mentioned previously for net PDV flux, the present cumulative values are greater than those observed by Matte et al. (2017), reinforcing that ZnSO4 is more efficiently absorbed than ZnO.

Figure 3.

Figure 3.

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Cumulative PDV appearance of Zn and Cu (expressed as % of dietary intake) after 600 post-prandial minutes, presented as LS means ± SEM. For zinc: Zn level effect (P = 0.03); tendency for Cu level effect (P = 0.09); values were different from zero (P < 0.01) for all treatments. For copper: no treatment effect (P ≥ 0.12); values were different from zero at 120 mg (P = 0.02), but not at 200 mg of Zn supplementation (P = 0.88); the value for ratio 120:8 tended to be different from zero (P = 0.06). Dietary treatments: 120:20, 120 mg Zn and 20 mg Cu; 200:20, 200 mg Zn and 20 mg Cu; 120:8, 120 mg Zn and 8 mg Cu; 200:8, 200 mg Zn and 8 mg Cu.

Copper

No treatment effect was detected for arterial (P ≥ 0.25; Table 2) serum Cu concentrations. Time effect (P < 0.01) was observed for arterial (Fig. 2) serum concentrations, but variations were numerically small at ≤3.1%. In fact, there was a gradual decrease from premeal values (1.93 ± 0.02 mg/L) to minimum values at 90 to 180 min (1.87 ± 0.02 mg/L) followed by a gradual increase to 1.92 ± 0.02 mg/L at the end of the sampling period. These results are in line with those from Matte et al. (2017). The absence of treatment effects and the small global postprandial variations on arterial serum Cu concentrations despite treatment effects on cumulative PDV appearance of Cu during the first 180 min post-meal (see below) suggest a tight homeostatic control of Cu metabolism at the systemic level. However, the absence of treatment effect for PDV serum Cu values (data not shown), in particular, suggests that a significant part of this homeostatic control takes place within the intestinal tissue. The decrease in arterial serum Cu within 90 min post-meal, independently of treatment, is compatible with a postprandial intestinal retention of dietary Cu. These results reinforce the above-mentioned hypothesis on the positive effect of dietary Cu supplementation levels on Zn absorption.

Figure 2.

Figure 2.

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Average arterial serum Cu concentrations (mg/L) within 600 post-prandial minutes, presented as LS means ± SEM across treatments. Time effect (P < 0.01).

Net PDV flux of Cu was not affected by dietary treatments (P ≥ 0.12; Table 2), and besides values for ratio 120:20 (P = 0.03), none of the other ratios were different from zero (P ≥ 0.14). The numerical value for ratio 200:20 (6.68 µg/min) is lower than that reported by Matte et al. (2017) at 18.1 µg/min. Both studies used the same dietary concentrations of Zn and Cu (200 and 20 mg), a similar Cu source (CuSO4) but different Zn sources, ZnSO4 vs. ZnO in Matte et al. (2017). As previously discussed, it is recognized that ZnSO4 is better absorbed than ZnO and this is confirmed by the greater net PDV flux of Zn in the present study compared with Matte et al. (2017; see above). This implies that a greater amount of Zn was transported within enterocytes. Considering that intracellular Zn concentrations are positively related to metallothionein levels (Davis and Cousins, 2000) and that metallothionein preferentially binds Cu over Zn within enterocytes impairing Cu efflux from cells (Oestreicher and Cousins, 1985), the better intestinal absorption of ZnSO4 may have negatively affected the present net PDV flux of Cu. Although no Zn level effect (P = 0.12) was observed on net PDV flux of Cu, average values for 120 mg treatments were different from zero (10.7 µg/min; P = 0.01), but not for 200 mg of Zn supplementation (1.6 µg/min; P = 0.69).

The cumulative PDV appearance of Cu (expressed as % of dietary intake) tended to be affected by dietary Zn level and by an interaction dietary Zn level × dietary Cu level at 30 min post-meal (0.06 ≤ P ≤ 0.10) and the last effect (interaction) was more pronounced (P = 0.04) after 180 min post-meal. Tendencies for a dietary Zn level effect (0.08 ≤ P ≤ 0.10) on cumulative PDV appearance of Cu were observed further from 480 min post-meal. At the end of the experimental period (600 min), no treatment effect was detected (P ≥ 0.12; Fig. 3) on cumulative PDV appearance of Cu and only values for ratio 120:8 tended to be different from zero (P = 0.06). For the whole postprandial period (10 h), estimated PDV appearance of Cu was 61.9%, 42.1%, −17.1%, and 23.6% of Cu intake for 120:8, 120:20, 200:8, and 200:20 ratios, respectively (Fig. 3). Similar to net PDV flux of Cu, although no Zn level effect (P = 0.12) was observed, average values of cumulative PDV appearance of Cu for treatments containing Zn at 120 mg were different from zero (52.0%; P = 0.02), but not for Zn at 200 mg (3.3%; P = 0.88). Interestingly, this apparent interference of supplemental Zn appeared more marked at lower levels of Cu supplementation. In fact, the increase of dietary Zn from 120 to 200 mg was followed by a decrease in cumulative PDV appearance of Cu of 18.5% within dietary Cu at 20 mg (120:20 vs. 200:20), whereas it corresponded to 79% within dietary Cu at 8 mg (120:8 vs. 200:8). This apparent impairment of Cu absorption by higher Zn levels is in line with Fosmire (1990) who reported that high Zn intake concomitant with low Cu intake increases the sequestration of Cu in enterocytes, which may eventually result in systemic Cu deficiency. In terms of impact of dietary Cu, Jondreville et al. (2002) reported that the bioavailability of Cu is normally inversely proportional to its intake. According to the present results, this was the case within treatments at 120 mg of dietary Zn (61.9% and 42.1% for 8 and 20 mg of dietary Cu), but not at 200 mg of dietary Zn (−17.1% and 23.6% for 8 and 20 mg of dietary Cu). It appears, therefore, that the level effect of dietary Cu on its absorption could be dependent on the level of dietary Zn.

Similar impairment of Cu absorption was observed by Matte et al. (2017) when high dietary Zn was supplemented. Those authors used different levels of Zn (200 or 400 mg) with the same ratio Zn:Cu (200:20 and 400:40). According to O’Dell (1985), Zn is the most critical trace mineral interfering with the absorption of Cu. In fact, as commented above, intracellular Zn concentrations raise metallothionein levels within enterocytes (Davis and Cousins, 2000) and these metalloproteins have greater binding affinity for Cu compared with Zn, causing intracellular sequestration of Cu and, consequently, impairing Cu efflux from cells (Oestreicher and Cousins, 1985). The present results may be an element of explanation for the greater concentration of Cu in the large intestine (Davin et al., 2012) and negative Cu digestibility ratios (Walk et al., 2015) after supplementation of weanling piglets with supranutritional dietary levels of Zn. The present results together with those of Matte et al. (2017) support the observations of Fosmire (1990) that not only the ratio between dietary Zn and Cu but also the absolute dietary amount of these minerals is crucial to optimize their absorption.

CONCLUSIONS

The present study confirmed the rapid absorption of Zn and the strict homeostatic control of Cu previously observed by this laboratory using the net PDV appearance approach. For Zn, the contribution of the gastric mucosa to the absorption of this mineral remains to be investigated. For Cu, the present study indicates that the intestinal tissue may play a crucial role in systemic Cu homeostasis.

The known negative effects of high dietary Zn levels on Cu availability were confirmed in the present study, emphasizing the potential risk of Cu deficiency under long-term supranutritional supplementation of Zn. Inversely, an unexpected positive effect of dietary Cu levels on Zn availability was demonstrated highlighting the importance of a dietary equilibrium between both minerals. In this sense, an optimum ratio among Zn and Cu was shown to improve both minerals’ absorption.

Within nutritional levels, ratio 120:8 which corresponds to the NRC (2012) recommendation appears to optimize Zn and Cu net postabsorptive availability.

Footnotes

1

The authors are grateful to the animal care team under the supervision of M. Turcotte. This study was supported by Agriculture and Agri-Food Canada core budget. There is no conflict of interest for any of the authors.

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