Received: 4 May 2020 | Revised: 6 July 2020 | Accepted: 7 August 2020 DOI: 10.1111/jpn.13447 ORIGINAL ARTICLE Effects of two zinc supplementation levels and two zinc and copper sources with different solubility characteristics on the growth performance, carcass characteristics and digestibility of growing-finishing pigs Sandra Villagómez-Estrada1 | José Francisco Pérez1 | Sandra van Kuijk2 | Diego Melo-Durán1 | Razzagh Karimirad1,3 | David Solà-Oriol1 1 Animal Nutrition and Welfare Service, Department of Animal and Food Science, Abstract Universitat Autonòma de Barcelona, The present study was conducted to evaluate the effect of two Zn supplemented lev- Bellaterra, Spain 2 els and two Zn and Cu sources (sulphate and hydroxychloride) on growing-finishing Trouw Nutrition, Research and Development Department, Amersfoort, The pigs. An in vitro study and an in vivo study were conducted. In the in vitro study, Netherlands Zn solubility from each source at different Zn supplementation levels was evalu- 3 Department of Animal Science, Lorestan University, Khorramabad, Iran ated, as well as the phytic phosphorus (PP) solubility derived from the interaction or not with phytic acid at similar conditions to those found in digestive tract. The most Correspondence David Solà-Oriol, Animal Nutrition and critical interaction of Zn with phytic acid was at pH 6.5 and with Zn sulphate, result- Welfare Service (SNIBA), Department ing in the reduction in PP solubility. In the in vivo experiment, a total of 444 pigs of Animal and Food Science, Universitat Autonòma de Barcelona, Bellaterra, Spain. ([Duroc × Landrace]×Pietrain; initial BW: 18.7 ± 0.20 kg) were allotted to 36 pens in a Email: david.sola@uab.cat randomized complete block design (2 × 2) factorial arrangement with two Zn and Cu sources and two Zn supplemental levels (20 and 80 mg/kg). The Cu supplementation was fixed at 15 mg/kg for all diets. There was no effect of the interaction between mineral source × Zn level or Zn level on growth performance or carcass characteris- tics (p > .10). Apparent total digestibility of Zn and Cu along with carcass yield was higher for pigs fed hydroxychloride than pigs fed the sulphate counterparts (p < .05). Feeding low levels of Zn decreased Zn (45.5%; p < .0001) and Cu(18.5%; p = .018) faecal excretion. In conclusion, under commercial conditions, feeding growing-finish- ing pigs with Zn levels below those established by the European Union regulation did not affect growth performance and carcass characteristics. Reducing dietary mineral (Zn and Cu) diet content resulted in a lower faecal mineral excretion. Pigs fed sul- phate minerals had an improved performance during grower period, while pigs fed hydroxychloride minerals showed an improved performance during finishing period and a greater carcass yield and mineral digestibility than those fed sulphates. KEYWORDS carcass characteristics, copper and zinc, digestibility, growing pigs, solubility, trace mineral sources This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Journal of Animal Physiology and Animal Nutrition published by Wiley-VCH GmbH J Anim Physiol Anim Nutr. 2021;105:59–71.  wileyonlinelibrary.com/journal/jpn | 59 60 | VILLAGÓMEZ-ESTRADA et al. 1 | I NTRO D U C TI O N sulphates, hydroxychloride mineral sources (55% Zn; 54% Cu) have a crystalline structure formed by covalent bonds located between Trace minerals play an essential physiological role for the normal the soluble metal ion, multiple hydroxyl groups and the chloride ions growth and health of animals. For instance, Zn and Cu are involved (Zhang & Guo, 2007), with low solubility in water but high solubil- in the synthesis of more than 300 enzymes and proteins, immune ity in acid solutions (Cao, Henry, Ammerman, Miles, & Littell, 2000). system activation and bone development, among other functions This makes them less reactive with other components in the diet (Suttle, 2010). The National Research Council (NRC) (2012) estab- (Lu et al., 2010; Luo et al., 2005). The difference between hydroxy- lished a total requirement of Zn (50–60 mg/kg feed) and Cu (3–4 mg/ chloride mineral sources and chelated or organic counterparts is that kg feed) for pigs from 25 to 135 kg body weight (BW). European in- the coordinate covalent bond is located between the metal ions and stitutions such as FEDNA (2013) and INRA (1989) have also emitted organic molecules (e.g. amino acids, peptides) (AAFCO, 2002). recommendations for the total feed content of Zn (80 and 100 mg/ In the present study, we hypothesized that due to the chemi- kg feed) and Cu (8 and 10 mg/kg feed) in growing-finishing pig diets. cal properties of the trace mineral sources the productive perfor- However, in non-European Union (EU) countries (e.g. United States, mance of grower-finisher pigs fed hydroxychloride minerals would China, Brazil), the swine industry supplements grower-finisher diets be higher than those fed sulphates when Zn and Cu doses are lower at higher levels than NRC requirements, such as in United States the than those established by the EU regulations. In order to test the hy- supplementation is approximately 1.6 times for Zn and 25.8 times for pothesis, a preliminary in vitro assay and a successive in vivo exper- Cu (Flohr et al., 2016), usually to promote growth (Coble et al., 2017; iment were conducted. To further understand the Zn solubility from Liu et al., 2018) and prevent intestinal dysbiosis (Højberg, Canibe, each source and the degree of interaction with phytic acid, as the Poulsen, Hedemann, & Jensen, 2005; Liu et al., 2018). At high sup- major binding molecule present in swine diets, the in vitro assay was plemental, and therefore total dietary levels, trace minerals are performed at several Zn concentrations (50, 100, 200 and 300 mg barely absorbed, while may interact with other nutrients (Pang & Zn/L) from each source and under similar conditions to those found Applegate, 2006; Walk, Wilcock, & Magowan, 2015) or with the ac- throughout the gastrointestinal tract (pH 2.5, 4.5 and 6.5). Likewise, tivity of other additives, such as phytase (Akter, Graham, & Iji, 2015; the phytic phosphorus (PP) solubility was evaluated. An early simi- Augspurger, Spencer, Webel, & Baker, 2004). Moreover, due to the lar in vitro assay was performed to assess the Cu solubility charac- efficient homoeostasis systems, when these minerals are supple- teristics from both mineral sources (Villagómez-Estrada, Pérez, van mented over the animals’ requirements, the mechanisms of absorp- Kuijk, et al., 2020). The in vivo experiment was aimed to evaluate the tion are reduced while increasing the endogenous secretions, mainly effect of two supplemented Zn levels (20 mg/kg as a low level and to intestinal pathways (pancreatic and bile secretions) (Goff, 2018; 80 mg/kg as a nutritional level) as well as the effect of two Zn and Spears, 1996; Suttle, 2010). Therefore, large amounts of trace min- Cu sources (sulphate and hydroxychloride) on growth performance, erals are excreted in manure. Subsequent applications of manure to carcass characteristics, tissue mineral content and mineral apparent the soil may increase the ecotoxicity of plants, microorganisms and total tract digestibility in grower-finisher pigs reared under commer- soil, which is a major environmental concern (EFSA FEEDAP, 2016). cial conditions. The supplemental level of Cu was fixed at 15 mg/kg Likewise, there is an increasing public health concern due to the as- for all diets. sociation of the great dietary, and therefore environmental, Zn and Cu contents with the increasing microbial resistance rates to antibi- otic agents by co-selection, as well as the possibility of transferring 2 | M ATE R I A L S A N D M E TH O DS of such resistant bacteria from food-producing animals to humans such as farmers, veterinarians and consumers (Ciesinski et al., 2018; All animal experimentation procedures were approved by the Ethics Yazdankhah, Rudi, & Bernhoft, 2014). To prevent environmental and Committee of the Universitat Autònoma de Barcelona in compliance public health concerns, dietary trace mineral levels can be reduced with the EU guidelines for the care and use of animals in research without affecting the productive performance of pigs. Therefore, EU (European Parliament, 2010). countries have established maximum total Zn (120 mg/kg) (European Commission, 2016) and Cu (25 mg/kg) (European Commission, 2018) levels for grower-finisher pigs. 2.1 | In vitro solubility test Sulphate mineral sources (35% Zn; 25% Cu) are the most used mineral sources in the animal feed industry due to their high solu- The methodology for in vitro test is described in an early in vitro bility and relatively low cost compared to hydroxychloride and or- test (Villagómez-Estrada, Pérez, van Kuijk, et al., 2020) performed ganic bound mineral sources. Sulphates are characterized by having to assess the Cu solubility characteristics from both mineral sources. a labile molecular bond that connects the metal ion with the sul- Briefly, the solubility of Zn sulphate, Zn hydroxychloride and the phate group, allowing high solubility in water and acid solutions (Guo concentration of soluble PP were measured in duplicate. Zn from et al., 2001; Park & Kim, 2016). Therefore, sulphates are frequently each source was added at concentrations of 50 mg/L, 100 mg/L, used as a reference point for comparing bioavailability of different 200 mg/L and 300 mg/L in 200 mM glycine buffer (pH 2.5) (Merck, mineral sources (Guo et al., 2001; Park & Kim, 2016). Alternatively to Germany) and 200 mM sodium acetate buffer (pH 4.5 and 6.5) VILLAGÓMEZ-ESTRADA et al. | 61 (Merck, Germany). Zn was mixed with 20 ml of buffer with and with- TA B L E 1 Composition of the basal diet for the three phases, as out 2.9 mM phytate (Merck, Germany), incubated at 41°C in a shak- fed-basisa ing water bath for 1 hr and filtered through 42-μm Whatman filter Ingredients, % Pre-growing Growing Finishing paper. The solubility of Zn and PP was analysed with inductively Maize 30.00 32.00 32.00 coupled, plasma-optical emission spectroscopy (ICP-OES, model Barley 21.34 13.68 14.65 Optima 4300DV, Perkin-Elmer Inc.; Waltham; MA, US). The solubil- Wheat 15.01 32.00 32.00 ity of PP was expressed as mg/L, whereas the solubility of Zn was Soya bean meal 47% CP 13.37 12.64 11.99 calculated using the following equation: [ ] Extruded soya beans 5.00 - - Mineral Solubility, % = (Soluble mineral) ∕ (Total mineral) × 100 Canola meal - 5.00 5.00 Carob bean pulp 4.00 - - 2.2 | In vivo experiment Wheat middlings 4.00 - - Sugar beet pulp 2.50 - - 2.2.1 | Animals and Housing Lard 1.64 1.77 1.69 DL-Methionine 0.11 0.07 0.04 The experiment was performed on a commercial farm in Catalonia, L-Lysine 0.45 0.49 0.46 Spain, from February to June with a single batch of pigs. At the end of L-Threonine 0.16 0.17 0.15 the nursery phase (63 ± 1.1 d of age), 444 pigs ([Duroc × Landrace] L-Valine - 0.02 - ×Pietrain) purchased from a different commercial farm and with an initial average BW of 18.7 ± 0.20 kg were used in a 105-d study. L-Tryptophan - 0.02 0.01 Pigs were weaned at 28 ± 1.1 d of age and nursed during 35 d under Monocalcium phosphate 0.86 0.24 0.21 commercial conditions and fed the same commercial pre-starter and Calcium carbonate 0.66 0.97 0.91 starter diets. Equal numbers of boars and gilts were ear tag identified Sodium bicarbonate 0.15 0.13 - and blocked according to sex and initial BW (heavy: 21.7 ± 1.4 kg Salt 0.35 0.40 0.49 and light: 15.7 ± 1.9 kg). Pigs were randomly distributed into four Vitamin-premix 0.40 0.40 0.40 experimental diet groups in 36 pens (9 replicate pens/treatment). nucleousb Pigs were allocated to 12 large pens (10.2 m2) of 15 animals and 24 Calculated composition smaller pens (7.5 m2) of 11 animals. Each pen had a fully slatted floor NE, kcal/kg 2,375 2,400 2,400 and was equipped with a commercial non-lidded hopper and a nipple CP 16.0 15.8 15.5 drinker to provide ad libitum access to feed and water. The facility Ether extract 4.7 4.0 3.9 was environmentally controlled (temperature and ventilation rate) Ca 0.60 0.65 0.62 using thermostatically controlled heaters and exhaust fans. Total P 0.56 0.42 0.42 Dig P 0.35 0.26 0.26 2.2.2 | Experimental design and dietary treatments Analysed composition CP 15.8 15.5 15.5 Three-phase diets (Table 1) were formulated to meet or exceed Ether extract 4.3 3.4 3.6 nutrient requirements (NRC, 2012): the pre-grower phase from d NDF 13.0 11.9 12.4 1 to 21, the grower phase from d 21 to 84 and the finisher phase Ash 4.6 5.5 4.1 from d 84 to 105. Four experimental diets were prepared accord- Ca 0.70 0.78 0.66 ing to a 2 × 2 factorial arrangement, with two Zn and Cu sources P 0.59 0.45 0.40 (sulphate and hydroxychloride) and two Zn supplementation levels a (low: 20 mg/kg, and nutritional: 80 mg/kg). The supplementation Pre-grower phase diets were fed from d 0 to 21, grower phase diets from d 21 to 84 and the finisher phase diets from d 84 to 105. level of Cu was fixed at 15 mg/kg for all diets. To achieve these sup- b Provided per kg of feed: vitamin A (acetate): 7,150 IU; vitamin D3 plemental levels, the total amount of Zn sulphate monohydrate (35%; (cholecalciferol): 1,320 IU; vitamin E: 25 IU; vitamin K3: 1.10 mg; Pintaluba, Reus, Spain) added was 228.6 g/ton in the nutritional diet, vitamin B1: 0.88 mg; vitamin B2: 2.75 mg; vitamin B6: 1.10 mg; vitamin and 57.1 g/ton in the low diet. The supplementary rate of Cu sulphate B12: 0.02 mg; vitamin B3: 16.5 mg; acid D-pantothenic acid: 8.80 mg; biotin: 0.01mg; Fe (sulphate): 60 mg; Mn (oxide): 50 mg;; I: 0.55 mg; pentahydrate (25%; Pintaluba, Reus, Spain) was 60.0 g/ton for both Se (sodium): 0.27 mg. Butylhydroxytoluene: 0,09 mg; sepiolite: sulphate diets. The supplementary amount of hydroxychloride Zn 3.46 mg; phytase: 500 FTU (Axtra® PHY, Danisco Animal Nutrition, (55%, IntelliBond Z; Trouw Nutrition, the Netherlands) was 145.5 g/ Marlborough, UK); glucanase: 1,500 VU and xylanase: 1,100 VU ton in nutritional diet and 36.7 g/ton in low diet. The supplemen- (Rovabio Excel AP, Adisseo, France). tal rate of Cu (54%, IntelliBond C, Trouw Nutrition, the Netherlands) 62 | VILLAGÓMEZ-ESTRADA et al. in both hydroxychloride diets was 27.8 g/ton. Phytase was added analysed using the Ankom nylon bag technique (Ankom 200 fiber at 500 FTU per kg of complete feed (Axtra® PHY, Danisco Animal Analyzer, Ankom Technology, Macedon, NY). Nutrition, Marlborough, UK). A vitamin-premix without Zn and Cu Liver and faeces were dried in a forced-air oven at 102ºC for was prepared. For each dietary treatment, Zn and Cu products were 12 hr, until constant weight, and then milled at 0.5 mm. Bone ash pre-mixed with 25 kg of basal diet before being incorporated directly determination protocol was based on the methodology described into the mixer during the feed manufacturing process. Finisher phase by Brenes et al., (2003). Briefly, front toe was dissected, and the IV diets were supplemented with titanium dioxide (0.5%) as an indigesti- metacarpal was autoclaved for 30 min at 121ºC to remove all the ble marker. In order to avoid cross-contamination with elements from adherent tissues, including cartilage. Subsequently, bone was ov- previous productions, feed was manufactured in an appropriate rank en-dried for 12 hr at 102°C and then soaked in acetone under a order starting with the lower concentrations to be included in the chemical hood for 48 hr to extract fat. After this period, the bone diet. All diets were offered in mash form. Composite samples (1 kg) was again oven-dried for 12 hr at 102ºC and weighted before being were collected during the bagging process and were representative broken in the middle to be ashed overnight at 550ºC in a muffle of each experimental treatment. Antibiotics or feed additives with furnace. Systematically after the incineration process, bone was antimicrobial properties were not included in the diets. again weighed, finely ground with mortar, sieved to a particle size of 0.5 mm and homogeneously mixed before mineral analysis. Bone ash content was calculated based on fat-free dried bone, and the mineral 2.2.3 | Experimental procedures and sampling content is expressed on bone ash basis. Prior mineral analysis by ICP-OES samples of feed, faeces, liver Individual pig weights and feed leftovers were recorded at the end and bone ash were digested with concentrated nitric acid (HNO3, of each feed phase. From these data, average daily gain (ADG), aver- 65%) in a microwave oven (model Ultrawave, Milestone Srl, Sorisole, age daily feed intake (ADFI) and gain: feed (G:F) were calculated. In Italy). Samples of feed and faeces (0.5 g) were digested with 5 ml of order to reach the market slaughter weight (110 kg), pigs were reared HNO3 and 0.2 ml of hydrofluoric acid (HF; 40%) at 240ºC for 30 min. for 25 additional days with the same dietary treatment. During this For samples of liver, 0.25 g was digested with 5 ml of HNO3 at 220ºC period, only the individual BW of pigs was recorded one day before for 15 min; for bone ash samples, 0.20 g was digested with 4 ml of slaughter. HNO3 at 220ºC for 15 min. Serum samples (0.25 ml) were diluted At the end of pre-growing phase, one pig per pen was se- in 1:20 volume ratio with 5 ml of a solution constituted by 0.05% lected based on the mean BW within the pen (median) to take ethylenediaminetetraacetic acid (EDTA) and 0.5% of ammonia (NH3). blood samples by jugular puncture. The same pig was sampled Immediately after digestion or dilution, samples were analysed by at the end of growing and finisher phases and during slaughter ICP-OES. All samples were analysed for Zn and Cu content. process. Blood samples for determining Zn and Cu were collected The coefficient of apparent total tract digestibility (CATTD) of into 10-mL vacutainer tubes (BD Vacutainer®, Z, BD-Plymouth, trace minerals was calculated as follow: UK) that were free of detectable Zn. Serum was obtained after centrifugation (3,000 × g for 15 min) and immediately frozen at [ ] CATTD = 1 − (TiD × NuF) ∕ (TiF × NuD) −20ºC. At d 105, faecal samples were collected from three pigs per pen through digital stimulation, pooled and immediately fro- where TiD and TiF represent the concentrations of the non-di- zen at −20ºC. At the slaughterhouse, all animals were electrically gestible marker in the diet and faeces, respectively; NuD and stunned and killed by exsanguination. Data on the carcass charac- NuF represent the nutrient concentrations in the diet and faeces teristics were collected using an Autofom (SFK, Herlev, Denmark). respectively. During the slaughter process, samples of the medial right lobe of the liver (excluding the gall bladder) and right front toe were col- lected from the selected pig per pen, after weighing the hot car- 2.3 | Statistical analysis cass and immediately frozen at −20ºC until analysis. Data were analysed as a randomized complete block design using the MIXED procedure for mixed linear models of SAS (version 9.4, SAS 2.2.4 | Chemical analysis Institute; Cary, USA). The model included the fixed effects of min- eral source, Zn level and their interaction (source × level). Moreover, Samples of feed were milled at 0.5 mm before chemical and min- the block of BW category and sex were considered as random ef- eral analysis. Analytical determinations of diets were performed ac- fects. Pen was considered the experimental unit for the perfor- cording to the AOAC International, (2005) methods for dry matter mance parameters. The carcass characteristics and mineral tissue (Method 934.01), the Dumas Method for crude protein (Method content were analysed with individual pig as the experimental unit. 968.06), traditional Soxhlet extraction for ether extract (Method The normality and homoscedasticity of data were examined using 920.39) and ash (Method 942.05). Neutral detergent fibre was Shapiro–Wilk test and assessing the normal plot before statistical VILLAGÓMEZ-ESTRADA et al. | 63 analysis. When the two-way interaction between mineral source and (6.5), the mean solubility of Zn hydroxychloride decreased from 73% Zn level was not significant, the interaction was removed from the to 6% in the presence of phytic acid, while the mean solubility of Zn model and the data re-analysed for main factors. Therefore, the main sulphate decreased from 99% to 6% (Figure 1b,d). effects are discussed for responses in which the interaction was not Overall, soluble PP decreased as the pH solution and the Zn significant. Significantly different means were separated using Tukey dose increased, regardless of the source (pH 2.5 = 424 mg/L; pH adjust. Significance was determined at a probability of p ≤ .05, and 4.5 = 371 mg/L; and pH 6.5 = 312 mg/L; Figure 1c,d). At pH 2.5 tendencies were considered when the p-value was between > 0.05 and 4.5, the concentration of soluble PP was similar for both sources and < 0.10. (Figure 1c). However, at pH 6.5, increasing levels of Zn as sulphate, but not as hydroxychloride, notably reduced the PP soluble content (298 versus 326 mg/L; Figure 1d). 3 | R E S U LT S 3.1 | In vitro solubility test 3.2 | In vivo experiment The results of Zn solubility sources and their interaction with phytic 3.2.1 | Growth performance and carcass acid are shown in Figure 1. Overall, the presence of phytic acid de- characteristics creased the mean Zn solubility as pH increased (from 89% at pH 2.5 to 6% at pH 6.5; Figure 1), regardless of the source. At pH 2.5, The analysed mineral content in feed is shown in Table 2. The ex- the mean solubility of Zn, as an average of the four concentrations, pected differences between low and nutritional levels of Zn in the was 100% and 94.7% for Zn hydroxychloride and Zn sulphate re- diets were achieved with both sulphate and hydroxychloride mineral spectively (Data not shown). The presence of phytic acid at pH 2.5 sources. The Cu content in all diets was within the expected values. reduced the mean solubility of Zn from 97% to 89%, regardless of There was no effect of the interaction between mineral source the source or dose (Data not shown). At pH 4.5, the presence of and Zn supplemented level for any of the growth performance re- phytic acid decreased the Zn solubility as the Zn dose increased in sponses (p > .10). Therefore, data were re-analysed and only the both sources (Figure 1c). Phytic acid reduced the Zn hydroxychlo- main effects of mineral source and Zn supplemented level are pre- ride solubility from 100% (without phytic acid) to 43% at pH 4.5 sented in Table 3. The Zn supplementation level was not observed to (Figure 1a,c), whereas the mean Zn solubility of sulphate decreased have any effect on the growth performance during the experimental from 98% (without phytic acid) to 55% (Figure 1a,c). At intestinal pH periods (p > .10; Table 3). At the end of the grower period, pigs fed F I G U R E 1 Effects of Zn source and level on Zn solubility and soluble phytic phosphorus content at without (a and b) and with (c and d) phytic acid addition at pH 4.5 (a and c) and pH 6.5 (b and d) respectively 64 | VILLAGÓMEZ-ESTRADA et al. TA B L E 2 Analysed trace mineral content in experimental diets Finisher, Pre-grower, mg/kg Grower, mg/kg mg/kg Added Zn, Added Cu, Zn and Cu source mg/kg mg/kg Zn Cu Zn Cu Zn Cu Sulphate 20 15 54.1 22.0 73.4 25.4 60.7 17.5 80 15 116.6 30.8 117.6 19.8 115.2 20.5 Hydroxychloride 20 15 50.7 19.6 90.0 22.7 62.0 15.4 80 15 109.6 20.0 130.0 21.7 97.6 15.7 Zn and Cu sulphate mineral source had a higher ADG (743 versus Table 4). Likewise, increasing dietary levels of Zn increased the Cu 713 g; p = .006) and G:F (0.524 versus 0.485; p = .027) than those content in the serum and liver (p < .01). Pigs fed sulphate trace min- fed Zn and Cu hydroxychloride mineral source. However, at the end erals tended to store higher levels of Cu in the liver than those fed of the finisher period pigs fed hydroxychloride mineral source diets hydroxychloride minerals (p = .090). All treatments showed low lev- tended to have a higher ADG than those fed the sulphate counter- els (<20 mg/kg) of Cu storage in the metacarpal bone, below the parts (774 versus 728 g; p = .088). No signs of Zn deficiency, like ICP-OES detection limit. parakeratosis or decreased growth, were observed. No mineral source × Zn supplemented level interaction was observed for any of the carcass characteristics (p > .10; data not shown). Likewise, no 4 | D I S CU S S I O N effect of dietary Zn level was observed (p > .10). Pigs fed Zn and Cu hydroxychloride source had a higher carcass yield percentage (p < 4.1 | Growing-finishing pig responses to low dietary .0001; Table 3) than those fed Zn and Cu sulphate mineral source. Zn levels The current estimates recommended by European institutions of 3.2.2 | Apparent total tract digestibility and faecal animal nutrition as well as NRC values for Zn and Cu are considered mineral excretion the minimum requirements for the normal growth of pigs (25–135 kg BW). Contrary to the strategy followed for amino acid, N, Ca and P A two-way interaction between mineral source × Zn supplemented requirements, which are generated by a modelling approach, the Zn level was observed in the CATTD of Zn and Cu (p < .05; Figure 2). and Cu requirements are derived from a critical evaluation of nutri- Pigs fed Zn hydroxychloride at 80 mg/kg had a greater CATTD of ent requirement studies (NRC, 2012). In accordance with our study, Zn (0.39 versus 0.33) and Cu (0.41 versus 0.08) than those fed Zn previous studies conducted in growing-finishing pigs have also shown sulphate at 80 mg/kg, being the nutritional supplemental levels of no differences in growth performance and carcass characteristics Zn from both mineral sources intermediate. between growing-finishing pigs fed diets with low or non-supple- A two-way interaction mineral source × Zn supplemented level mentary levels of Zn and those fed Zn levels similar or up to NRC was also observed for Cu faecal excretion (p = .011; Figure 3d). Pigs requirements (Cemin, Woodworth, et al., 2019; Creech et al., 2004; fed Zn sulphate at nutritional supplemental levels excreted higher Gowanlock, Mahan, Jolliff, Moeller, & Hill, 2013; Paboeuf, Nys, & quantities of Cu than those fed Zn sulphate at low supplemental lev- Corlouer, 2000) even under a restricted floor space allowance (Holen, els (159.6 versus 107.1 mg/kg), whereas pigs fed Zn hydroxychloride Rambo, Hilbrands, & Johnston, 2018). In general, the requirements at both nutritional and low supplemental levels were intermediate for most of the nutrients (as percentage of the diet), including trace (112.1 and 114.4 mg/kg respectively). Feeding diets with lower sup- minerals, decrease as the BW of the pig increases (NRC, 2012). For plemental levels of Zn decreased the Zn (p < .0001) and Cu (p = .018) instance, higher levels of Zn (100 mg/kg feed) and Cu (6 mg/kg feed) excretion by 45.5% and 18.5% respectively (Figure 3c and 3d). are needed for pigs from 5 to 11 kg BW compared to growing pigs (NRC, 2012). The lack of growth response between these two supple- mented levels of Zn in the present study could be attributed to (a) the 3.2.3 | Mineral tissue content absence of intestinal and immunological challenges experienced by growing-finishing pigs. Unlike newly weaned pigs, growing-finishing There was no effect of the interaction between mineral source × Zn pigs have a mature immune system and intestinal microbiome (Wang supplemented level in any of the tissue mineral content except for et al., 2019), which is already adapted to solid feed and other stress Cu content in liver (Table 4). Pigs fed Zn sulphate at 80 mg/kg had factors such as the environmental conditions. (b) The greater intake a higher Cu content (92.0 mg/kg) than pigs with the other experi- capacity of growing-finishing pigs may allow satisfying their Zn re- mental diets (p < .0001; Figure 3b), while no differences in Zn liver quirement as more feed is consumed, although the bioavailability of content were observed (p > .10; Figure 3a). Feeding diets with Zn at natural minerals in the cereals and grains of their diet is low or not 80 mg/kg increased liver and the metacarpal content of Zn (p < .05; completely known. (c) The addition of phytase (in the present study VILLAGÓMEZ-ESTRADA et al. | 65 at 500 FTU) may have increased Zn bioavailability and minimized <0.0001 Carcass yield, % 0.232 0.237 0.158 0.106 other dietary antagonisms of phytic acid with other minerals and di- 70.04 70.22 70.40 70.57 gestive enzymes (Revy, Jondreville, Dourmad, & Nys, 2004; Schlegel, Sauvant, & Jondreville, 2013). (d) The supplementation of high-fibre Carcass characteristicsb diets with carbohydrase enzymes (i.e. glucanase and xylanase) may Data are the average value of n = 444 pigs for the two-way interaction, whereas for the main effects of source and level are the average value of n = 222 pigs. CCW = cold carcass weight. Lean meat, Growth performance and carcass characteristics of pigs fed diets with two Zn and Cu sources (sulphate and hydroxychloride) at two Zn levels (20 and 80 mg/kg) have enhanced the nutrient digestibility, improved intestinal bar- 0.359 0.725 0.816 0.351 0.165 67.08 67.26 67.13 66.96 rier integrity and modulated the intestinal microbiota, consequently, % supporting growth performance (Torres-Pitarch et al., 2020). (e) The 2.022 complex homoeostasis body system of Zn and Cu may also play an 2.039 0.138 0.479 0.199 CCW, 74.02 74.82 73.96 74.76 important role in supporting the growth performance of pigs, par- kg ticularly in those supplemented with a low Zn level, mobilizing miner- als from the storage sites in case of being necessary (Suttle, 2010). d 0–105 Data are the average value of n = 9 replicate pens for the two-way interaction, whereas for the main effects of source and level are means of 18 replicate pens. 0.0083 0.0081 0.409 0.607 0.428 0.421 0.108 0.415 0.714 Nevertheless, it is known that pigs fed with Zn-deficient diets usually G:F manifest several subclinical and clinical signs such as decreased water and feed intake, growth depression, parakeratosis, hair loss and dis- 0.325 0.312 0.629 49.40 48.72 d 0–105 ADFI, g 1735.2 1686.5 1734.6 1687.1 coloration. Among these, parakeratosis is the pathognomonic clinical sign and may mark the end point in response to a long-term insuf- ficient supply of Zn (Brugger & Windisch, 2016). Since in the current 0.251 0.206 0.281 d 0–105 21.63 21.67 707.2 716.5 706.8 716.1 study, none of the pigs showed signs of Zn deficiency, and no differ- ences were observed in the long-term growth performance between d 85–105 Zn dietary levels, it could be suggested that feeding pigs with these 0.443 0.088 0.579 31.26 30.95 758.2 728.4 773.7 743.9 low levels did not cause a mineral-deficit challenge for the animals. It is not easy to assess the essential trace mineral status of animals, such as Zn and Cu, because the minerals are involved in 0.611 0.745 0.006 20.54 20.61 22–84 725.2 730.5 743.0 712.6 many physiological functions and their metabolism is complex and d not totally understood (Suttle, 2010). Once absorbed Cu from in- 0.575 0.363 0.784 ADG, g 29.37 29.52 d 0–21 testine is primarily stored in liver and thereafter distributed, bind 614.8 617.3 607.4 609.9 to ceruloplasmin, to other tissues such as kidneys, brain, skele- ton and skeletal muscle, but in less proportion (Linder et al., 1998; Roberts & Sarkar, 2008). Therefore, liver is considered the main 0.448 5.036 5.032 0.326 0.319 94.06 93.03 92.89 93.91 d 105 organ responsible for Cu homoeostasis (Goff, 2018). The low Cu values detected in metacarpal bone in the present study may be attributed to the predilection of body for storing greater amounts 0.608 4.584 0.086 of Cu in liver than in bone. Generally, the inclusion of pharma- 0.828 4.579 76.40 77.81 78.59 77.18 d 84 cological doses of Zn has been associated with a decrease in Cu bioavailability (Revy et al., 2004). It is likely that, in response to a Growth performancea dietary Zn overload, more metallothionein (MT) proteins are ex- 0.645 3.653 0.841 3.655 0.978 31.80 31.44 pressed in the enterocytes, which instead bind Cu, reducing its 31.63 31.61 d 21 availability to be absorbed (Goff, 2018; Revy et al., 2004). Others have been proposed a competition between Zn and Cu for the BW, kg 3.049 0.790 0.750 0.696 3.051 18.83 18.66 18.87 18.62 use of the divalent metal transporter 1 (DMT1) (Lutsenko, Barnes, d0 Bartee, & Dmitriev, 2007); however, it is also known that both Zn and Cu have their own specific transporters, which are con- Zn level, mg/ sidered the main pathways used to cross the apical membrane (Goff, 2018). Since in the present study, the exposure level of Zn 20 80 kg was within the nutritional range, the Cu long-term storage was not impaired but instead improved as pigs were fed nutritional lev- els of Zn, suggesting a greater mineral absorption as previously Zn and Cu Source Source × Level Hydroxychloride reported (Carlson et al., 2004). Regarding Zn storage in body, as expected, increasing Zn levels in the diets resulted in higher Zn TA B L E 3 Source content in target tissues such as liver and bone. The main stor- Sulphate Level p-value ages sites of Zn are the liver, muscle and bone (Revy, Jondreville, SEM SEM Dourmad, & Nys, 2003). However, it should be considered that b a 66 | VILLAGÓMEZ-ESTRADA et al. F I G U R E 2 Coefficient of apparent total tract digestibility (CATTD) of Zn (a) and Cu (b) at the end of finisher phase (d 105) of pigs fed diets with two Zn and Cu sources (sulphate and hydroxychloride) at two Zn levels (20 and 80 mg/kg). Data are means of 9 replicate pens for the two-way interaction (3 pigs per replicate pen were sampled and pooled). a-c: values with different letters differ significantly for the two- way source × level (p < .05) F I G U R E 3 Liver trace mineral accumulation (a-b) and faecal excretion (c-d) of pigs fed diets with two Zn and Cu sources (sulphate and hydroxychloride) at two Zn levels (20 and 80 mg/kg). Data are means of 9 replicate pens for the two-way interaction (1 pig for liver and 3 pooled pig for faeces were sampled per replicate pen). a-b: values with different letters differ significantly for the two-way source × level (p < .05) under dietary Zn deficiency the Zn stored in bone cannot be easily additional biomarkers should be considered to confirm that these mobilized (McDowell, 2003), contrary to others internal pools as low dietary supplies match the pigs’ metabolic requirements, es- liver (Blaabjerg & Poulsen, 2017); therefore, a suitable content of pecially under stressing or poor sanitary conditions in which the dietary Zn is necessary. In the present study, although pigs fed Zn requirements for optimizing the immune function may be a low Zn level (20 versus 80 mg/kg) showed lower Zn content in higher than those for growth (Klasing, 2001). the liver, no differences were observed in growth performance be- Reducing dietary Zn levels resulted in a reduction in Zn and Cu tween dietary levels. Nonetheless, complementary studies using faecal excretion of 45.5% and 18.5% respectively. In agreement with VILLAGÓMEZ-ESTRADA et al. | 67 TA B L E 4 Tissue mineral content of pigs fed diets with two Zn and Cu sources (sulphate and hydroxychloride) at two Zn levels (20 and 80 mg/kg) Trace mineral contenta Zn Cu Liver, mg/kg Serum, mg/L (d Liver, mg/kg Zn and Cu Source Zn level, mg/kg Serum, mg/L (d 105) DM Boneb , mg/kg 105) DM Sulphate 1.07 207.0 219.0 2.02 69.79 Hydroxychloride 1.14 190.7 213.9 1.84 62.50 SEM 0.064 16.53 7.83 0.136 2.947 20 1.10 173.9 204.9 1.84 54.79 80 1.10 223.8 227.9 2.05 77.50 SEM 0.065 16.30 7.62 0.136 2.947 p-value Source 0.102 0.226 0.635 0.600 0.090 Level 0.977 0.002 0.038 0.010 <0.0001 Source × Level 0.241 0.124 0.912 0.317 <0.0001 a Data are of the average values of n = 9 replicate pens for the two-way interaction, whereas for the main effects of source and level are the average values of n = 18 replicate pens (1 pig per pen was sampled). b Bone ash basis. Cu-detected values are lower than 0.02 mg/g by ICP-OES. our results, previous studies with pigs have reported that Zn and counterparts. The opposed was observed in the finisher feed period, Cu excretion could be reduced without negative effects on growth where pigs fed hydroxychloride mineral sources diets showed an performance, for instance, by 50% by reducing micro-mineral sup- improved performance than those fed the sulphate sources. Similar plementation from commercially utilized levels (Zn 150–100 and Cu effects were described in a meta-analysis performed by van Kuijk, 25–15 mg/kg, respectively) to 25 and 5 mg/kg respectively (Creech Jacobs, Smits, and Han, (2019). The authors reported that the sup- et al., 2004; Liu et al., 2016; Paboeuf et al., 2000; Van Heugten, plementation of growing-finishing pig diets with Zn hydroxychloride O’Quinn, Funderburke, Flowers, & Spears, 2004). The absorption (80 mg/kg) resulted in a 3.9% improvement in G:F and ADG, dur- and excretion of Zn and Cu are strictly regulated through homoeo- ing the last feed phase, compared to Zn sulphate source. It is likely static control mechanisms, and when these minerals are supple- that these improvements, during the last period of pig growth, might mented over the animals’ requirements, the excess is not absorbed be related to improvements in carcass characteristics, as a greater or is endogenously secreted (Goff, 2018; Spears, 1996), ending up in carcass yield in the present study. In agreement, results from previ- manure. There are increasing environmental and public health con- ous studies reported that the supplementation of growing-finishing cerns about the swine industry due to its contribution to contam- pig diets with Zn hydroxychloride (50, 100 and 150 mg/Zn kg) pro- inating soils and ground water with high Zn and Cu levels (Wuana moted greater hot carcass weight (p = .041) (Carpenter et al., 2016), & Okieimen, 2011) and possibly spreading antibiotic resistance by higher carcass yield (p = .017) and a tendency for greater hot carcass co-selection (Yazdankhah et al., 2014). Therefore, feeding trace min- weight (p = .058) (Cemin, Carpenter, et al., 2019) compared with the erals to animals as precisely as possible is a potential approach for Zn sulphate supplementation. Likewise, the supplementation of Zn reducing these environmental and public health risks. The present (80 mg/kg) as hydroxychloride mineral source resulted in a higher study found that feeding growing pigs with Zn levels under those lean meat percentage (p = .001) compared with Zn sulphate supple- established by the EU regulation did not affect growth performance mentation (van Kuijk et al., 2019). Similar improvements on carcass and carcass characteristics, and also resulted in lower faecal mineral composition were reported in a broiler chicken study with the sup- (Zn and Cu) excretion. plementation of Zn and Cu hydroxychloride (Olukosi, van Kuijk, & Han, 2018). One proposed mechanism of action of hydroxychloride minerals is the influence in the ratio of protein to fat deposition (van 4.2 | Growing-finishing pig responses to different Kuijk et al., 2019). Nevertheless, Coble et al., (2017) reported no dif- Zn and Cu sources ference in carcass characteristics when pig diets were supplemented with 75 or 150 mg Cu/kg from a sulphate or hydroxychloride mineral Interestingly, at the end of the grower feed period, pigs fed diets source. supplemented with Zn and Cu as sulphate mineral source had higher Although the metabolic action of Zn and Cu from different growth performance than those fed diets with the hydroxychloride sources is not completely understood, results from previous studies 68 | VILLAGÓMEZ-ESTRADA et al. reported different beneficial effects of hydroxychloride minerals on fed with a mineral source of Cu sulphate at nutritional levels of Zn, animal health, which may influence the beneficial productive results. the homoeostasis mechanism reduced the absorption of Cu from the For instance, the supplementation of weanling pig diets with Cu hy- intestine by increasing the sequestration of Cu in MT, while increas- droxychloride decreased the oxidative stress in the duodenum (Fry ing hepatobiliary excretion through the bile into the intestine and, et al., 2012; Huang et al., 2015) and increased the serum activity of consequently, in the faeces (Bremner, 1998; Gaetke & Chow, 2003). SOD and ceruloplasmin (Zheng et al., 2018) compared to Cu sulphate. It should be noted that the Cu excreted into bile is poorly available Likewise, the supplementation of Cu and Zn hydroxychloride tended to for its reabsorption (Roberts & Sarkar, 2008). Further comprehen- increase serum GPx activity in weaned pigs (Villagómez-Estrada, Pérez, sive studies involving several complementary analyses need to be Darwich, et al., 2020). Although the absorption and retention rates of performed to elucidate the action mechanisms for hydroxychloride trace mineral sources were not measured, a greater apparent total tract minerals and to understand the trace mineral metabolism better. digestibility was observed for Zn and Cu hydroxychloride, suggesting In conclusion, feeding growing-finishing pigs with Zn levels below a better absorption, and therefore less faecal excretion, compared to those established by the European Union regulation did not affect sulphates. The last could also be related to the solubility character- growth performance and carcass characteristics in the present con- istics observed throughout the in vitro study and the improvements ditions. Moreover, reducing dietary mineral (Zn and Cu) content re- observed at the end of finisher phase and in carcass characteristics, sulted in a lower faecal mineral excretion, which makes it one of the despite their lower growth performance during grower period. possible approaches for reducing the associated environmental and The results of the present in vitro study of Zn, as well as our public health risks. The supplementation of pig diets with hydroxy- previous in vitro study of Cu (Villagómez-Estrada, Pérez, van Kuijk, chloride minerals resulted in higher apparent digestibility of Zn and et al., 2020), reported that sulphates have a greater range of solu- Cu and carcass yield compared with diets supplemented with the bility than hydroxychloride, depending on pH, making them more sulphate counterparts. Further studies with complementary physi- prone to interact with other components of the diet such as phytic ological biomarkers are necessary to confirm that these low dietary acid. Indeed, the mean Cu solubility for sulphate mineral source re- levels match the pigs’ metabolic requirements. ported was of 98% in the whole range of pH levels (from 2.5 to 6.5), whereas the hydroxychloride Cu mean solubility decreased drasti- cally from 100% at pH 2.5 and 4.5 to 9% at pH 6.5. The addition of AC K N OW L E D G E M E N T S phytic acid resulted in a decrease in Cu solubility of both sources, being most critical at pH 6.5 and with Cu sulphate than with Cu hy- The authors gratefully acknowledge the support of the Secretaria droxychloride, which also resulted in a decreased solubility of PP de Educación Superior, Ciencia, Tecnología e Innovación de Ecuador (Villagómez-Estrada, Pérez, van Kuijk, et al., 2020), similar to that (SENESCYT) for the provision of a pre-doctoral scholarship (CZ03- observed in the present Zn in vitro study. In this sense, different 000367-2018). The authors are also grateful to the Servei d´Anàlisi studies of bioavailability in pigs and broiler chickens have reported Química of Universitat Autònoma de Barcelona for the chemical a slightly higher bioavailability of Zn hydroxychloride (111%) com- service. pared with Zn sulphate (Batal et al., 2001) but greater than Zn oxide (159.3%) (Mavromichalis, Webel, Parr, & Baker, 2001; Zhang C O N FL I C T O F I N T E R E S T & Guo, 2007). Similar relative bioavailability of Cu hydroxychloride The authors declare no conflict of interest. compared with Cu sulphate has been reported in pigs (112%) and poultry (109%) (Luo et al., 2005; Miles, O’Keefe, Henry, Ammerman, A N I M A L W E L FA R E S TAT E M E N T & Luo, 1998). Nevertheless, in the present study no differences be- The authors confirm that they have followed European Union tween mineral sources in Zn and Cu tissue content were observed, standards for the protection of animals used for scientific purposes except for the Cu content in liver. Certainly, the mechanisms of the (European Parliament, 2010). influence on metabolism of Zn and Cu of both trace mineral sources need further research, especially because after a long-term sup- ORCID plementation of Cu (15 mg/kg), pigs fed Zn sulphate at nutritional Sandra Villagómez-Estrada https://orcid. levels had a higher Cu content in the liver than the other experimen- org/0000-0003-3086-3256 tal groups and even higher than the normal range (19 to 56 mg/kg; José Francisco Pérez https://orcid.org/0000-0001-8853-8945 Puls, 1994). Moreover, besides this greater liver Cu accumulation, a Sandra van Kuijk https://orcid.org/0000-0002-1264-9857 reduced Cu apparent digestibility and a higher Cu faecal excretion Diego Melo-Durán https://orcid.org/0000-0003-3944-1746 were also observed in those pigs. The complex homoeostasis pro- David Solà-Oriol https://orcid.org/0000-0001-8365-340X cess of Cu is aimed to ensure a constant and sufficient supply of the micronutrient while simultaneously avoiding excess body levels REFERENCES (Scheiber, Dringen, & Mercer, 2013). In this sense, it is possible that AAFCO (2002). Official Publication of the Association of American Feed to counteract the excessive accumulation of Cu in the liver in pigs Control Officials. OxfordIndiana, US. VILLAGÓMEZ-ESTRADA et al. | 69 Akter, M., Graham, H., & Iji, P. A. (2015). Interactions between phytase concentration and source (inorganic vs. chelated) on performance, and different dietary minerals in in vitro systems. 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