Journal of Animal Science, 2022, 100, 1–12 https://doi.org/10.1093/jas/skac224 Advance access publication 20 June 2022 Non Ruminant Nutrition How copper can impact pig growth: comparing the effect of copper sulfate and monovalent copper oxide on oxidative status, inflammation, gene abundance, and microbial modulation as potential mechanisms of action Asal Forouzandeh,†, Laia Blavi,†,1, Jose Francisco Pérez,†, Matilde D’Angelo,† Francesc González-Solé,† Alessandra Monteiro,‡ Hans H. Stein,|| and David Solà-Oriol†, † Animal Nutrition and Welfare Service (SNiBA), Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, Bellaterra, Spain ‡ Animine, Annecy, France || Division of Nutritional Sciences, University of Illinois, Urbana, IL, USA Corresponding author: lblavi@asn-nutrition.es 1 Abstract The beneficial effect of elevated concentrations of copper (Cu) on growth performance of pigs has been already demonstrated; however, their mechanism of action is not fully discovered. The objective of the present experiment was to investigate the effects of including Cu from copper sulfate (CuSO4) or monovalent copper oxide (Cu2O) in the diet of growing pigs on oxidative stress, inflammation, gene abundance, and microbial modulation. We used 120 pigs with initial body weight (BW) of 11.5 ± 0.98 kg in 2 blocks of 60 pigs, 3 dietary treatments, 5 pigs per pen, and 4 replicate pens per treatment within each block for a total of 8 pens per treatment. Dietary treatments included the negative control (NC) diet containing 20 mg Cu/kg and 2 diets in which 250 mg Cu/kg from CuSO4 or Cu2O was added to the NC. On day 28, serum samples were collected from one pig per pen and this pig was then euthanized to obtain liver samples for the analysis of oxidative stress markers (Cu/Zn superoxide dismutase, glutathione peroxidase, and malondialdehyde, MDA). Serum samples were analyzed for cytokines. Jejunum tissue and colon content were collected and used for transcriptomic analyses and microbial characterization, respectively. Results indicated that there were greater (P < 0.05) MDA levels in the liver of pigs fed the diet with 250 mg/kg CuSO4 than in pigs fed the other diets. The serum concentration of tumor necro- sis factor-alpha was greater (P < 0.05) in pigs fed diets containing CuSO4 compared with pigs fed the NC diet or the diet with 250 mg Cu/kg from Cu2O. Pigs fed diets containing CuSO4 or Cu2O had a greater (P < 0.05) abundance of genes related to the intestinal barrier function and nutrient transport, but a lower (P < 0.05) abundance of pro-inflammatory genes compared with pigs fed the NC diet. Supplementing diets with CuSO4 or Cu2O also increased (P < 0.05) the abundance of Lachnospiraceae and Peptostreptococcaceae families and reduced (P < 0.05) the abundance of the Rikenellaceae family, Campylobacter, and Streptococcus genera in the colon of pigs. In conclusion, adding 250 mg/kg of Cu from CuSO4 or Cu2O regulates genes abundance in charge of the immune system and growth, and promotes changes in the intestinal microbiota; however, Cu2O induces less systemic oxidation and inflammation compared with CuSO4. Lay Summary Copper is a nonrenewable mineral resource that is essential for all biological organisms. After banning the antibiotics, copper has received considerable attention due to its antimicrobial properties that improve performance in animals when fed over the minimum requirement. The present study evaluated two sources of Cu (copper sulfate and monovalent copper oxide) compared with a nonsupplemented diet and the likely mechanism of action which leads to improved pig performance. Pigs fed high concentrations of copper sulfate showed increased liver oxidation and inflammatory indicators in the blood. Elevated concentrations of Cu improved intestinal epithelial barrier function, modulation of inflamma- tory responses, increased beneficial microbes, and reduced pathogens in the gut. Therefore, supplementation of high levels of Cu appears to be effective in promoting pig growth, but therapeutic doses of Cu sulfate increase the inflammatory response. Key words: copper sulfate, gene abundance, monovalent copper oxide, microbiota, oxidative status, swine Abbreviations: BF, barrier function; EH, enzymes and hormones; GSH-Px, glutathione peroxidase; IR, immune response; MDA, malondialdehyde; NT, nutrient transport; RNA, ribonucleic acid; ST, stress; SOD, Cu/Zn superoxide dismutase Introduction droxylase, lysyl oxidase, and copper-zinc superoxide dis- Copper (Cu) is part of many enzymes related to biologi- mutase (Cu, Zn-SOD; Gaetke 2003). The beneficial effect cal processes required for growth, development, and of adding Cu in quantities that exceed the assumed require- maintenance, such as cytochrome c oxidase, tyrosinase, ment for maximizing the growth of pigs has been demon- p-hydroxyphenyl pyruvate hydrolase, dopamine beta-hy- strated (Cromwell et al., 1989; Hill et al., 2000), and the Received February 25, 2022 Accepted June 17, 2022. © The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 2 Journal of Animal Science, 2022, Vol. 100, No. 9 prevalence of diarrhea is also reduced if diets containing Animal management and husbandry Cu above the requirement are fed (Pérez et al., 2011; Espi- A total of 120 growing pigs (60 barrows and 60 gilts) with nosa et al., 2017). These effects can be attributed to anti- average initial body weight (BW) of 11.5 ± 0.98 kg were allot- microbial properties (Hill et al., 2000; Pang et al., 2009) ted to a randomized complete block design with 2 blocks of and improved fat digestibility (Luo and Dove, 1996; Espi- 60 pigs with the weaning group being the blocking factor. nosa et al., 2021) of diets containing high Cu doses. How- There were 3 dietary treatments, 5 pigs per pen (half pens ever, excessive Cu may exert a toxic effect leading to cell with 2 males and 3 females and the other half with 3 males death (Nawaz et al., 2006). Ozcelik et al. (2003) observed and 2 females), and 4 replicate pens per treatment in each that excess Cu in the organism can cause cellular damage block. Thus, there were a total of 8 replicate pens per treat- through the formation of free radicals, which induce oxi- ment in the experiment. Pigs were housed in pens with fully dative stress (Gaetke, 2003), changes in lipid profile, and slatted floors and a dry feeder, and a nipple drinker was hepatic dysfunction (Sarkar et al., 2011). installed in each pen. Dietary Cu may be present in the oxidized cupric form (Cu2+), or the reduced cuprous form (Cu+; Linder and Haze- gh-Azam, 1996). Although Cu can be provided in many differ- Diets and feeding ent forms, Cu sulfate pentahydrate (CuSO4·5H2O) is widely Three diets based on corn and soybean meal were for- used in diets for pigs due to its relatively low cost and high mulated (Table 1). Diet contained 500 units of phytase solubility in water and acidic solvents (Pang and Applegate, per kilogram (Quantum Blue, AB Vista Feed Ingredients, 2006; Park and Kim, 2016). A new source of Cu, monovalent copper oxide (Cu2O; Animine, Annecy, France), provided at up to 250 mg/kg diet results in improved growth performance Table 1. Ingredient composition and nutrient content of the control diet in the experiment as fed-basis1 and lower accumulation of Cu in the liver of broiler chickens (Hamdi et al., 2018; Forouzandeh et al., 2021) and weanling Item Control diet pigs (Bikker et al., 2017; Blavi et al., 2021) compared with animals fed Cu sulfate. However, no data are demonstrat- Ingredients, % ing how Cu2O impacts oxidative stress of pigs and limited Ground corn 59.75 information is available on the effects of Cu2O on immune Soybean meal, 48% CP 26.00 function, intestinal gene abundance, and the microbial popu- Dried whey 5.00 lation in the hindgut of growing pigs. To shed light on these issues, we used previously collected samples from phase one Fish meal 3.00 of a recently published study (Blavi et al., 2021). The previous Soybean oil 3.00 study revealed that at the end of phase one, therapeutic doses Ground limestone 0.86 of Cu (either from Cu2O or CuSO4) were effective in improv- Dicalcium phosphate, 19% P 0.55 ing pig growth, but addition of Cu from Cu2O reduced Cu Lysine HCL, 78% Lys 0.35 accumulation in the liver and spleen compared with CuSO4 dl-Met, 98% Met 0.09 addition. From this phase, pigs fed 250 mg Cu/kg from Cu2O Threonine, 98% Thr 0.10 tended to have greater ADG compared with pigs fed 250 mg Salt 0.40 Cu/kg from CuSO4. Therefore, we hypothesized that dietary Cu2O may result Vitamin-mineral premix 2 0.30 in less oxidative stress and inflammation than CuSO4. It was Phytase premix3 0.10 also hypothesized that the addition of 250 mg/kg of Cu in Titanium dioxide 0.50 diets for growing pigs modulates gene abundance and micro- Copper, mg/kg 20.00 bial population. Consequently, the objective of this exper- Analyzed composition, % iment was to determine the effect of Cu2O and CuSO4 on Dry matter 87.19 oxidative stress, immune function, gene abundance, and gut Ash 4.07 microbiota of growing pigs. Crude protein 17.88 Lys 1.38 Materials and Methods Ether extract 5.50 The Institutional Animal Care and Use Committee at the 1 Two additional diets were formulated by adding 250 mg/kg of Cu from University of Illinois, USA, reviewed and approved the pro- copper sulfate pentahydrate (25% Cu) or 250 mg/kg of Cu from copper (I) tocol for the animal part of the experiment. The experiment oxide (75% Cu) to the control diet. The two copper sources were added at was a collaborative project between the University of Illi- the expense of ground corn. 2 Provided the following quantities of vitamins and micro minerals per nois, Urbana–Champaign, IL and Universitat Autònoma de kilogram of complete diet: Vitamin A as retinyl acetate, 11,136 IU; vitamin Barcelona, Bellaterra, Spain. The animal part of the experi- D3 as cholecalciferol, 2,208 IU; vitamin E as DL-alpha tocopheryl acetate, ment was conducted at the University of Illinois, whereas the 66 IU; vitamin K as menadione dimethylprimidinol bisulfite, 1.42 mg; thiamin as thiamine mononitrate, 0.24 mg; riboflavin, 6.59 mg; pyridoxine sample analysis was conducted at Universitat Autònoma de as pyridoxine hydrochloride, 0.24 mg; vitamin B12, 0.03 mg; D-pantothenic Barcelona. Pigs used in the experiment were the offspring of acid as D-calcium pantothenate, 23.5 mg; niacin, 44.1 mg; folic acid, 1.59 mg; biotin, 0.44 mg; Cu, 10 mg as copper sulfate and 10 mg copper L 359 boars mated to Camborough females (Pig Improve- chloride; Fe, 126 mg as ferrous sulfate; I, 1.26 mg as ethylenediamine ment Company, Hendersonville, TN). Data for growth per- dihydriodide; Mn, 60.2 mg as manganese sulfate; Se, 0.15 mg as sodium formance, carcass characteristics, bone mineralization, and selenite and 0.15 mg selenium yeast; and Zn, 125.1 mg as zinc sulfate. 3 Phytase premix was prepared by mixing 900 g ground corn and 100 g organ accumulation of Cu have been published (Blavi et al., Quantum Blue 5000 G (AB Vista Feed Ingredients, Marlborough, UK) to 2021). provide 500 phytase units per kilogram complete diet. Forouzandeh et al. 3 Malborough, UK). Dietary treatments consisted of the neg- recommended by the manufacturer and were read on a ative control diet (NC) with 20 mg/kg of added Cu, and Luminex 200 (Luminex Co., TX). two diets in which 250 mg Cu/kg from either CuSO4 or Cu2O was added to the NC diet. Pigs were fed experimen- Gene abundance analysis tal diets for 28 d and had ad libitum access to feed and water throughout the experiment. Diets were prepared in a Jejunum tissues were analyzed to determine the abundance meal form and were formulated to meet current estimates of 56 intestinal genes classified into 5 groups related to bar- for nutrient requirements for growing pigs (NRC, 2012). rier function (BF), immune response (IR), nutrient transport The analyzed composition values of the experimental diets (NT), gut enzyme/hormone (EH), and stress (ST) using an for DM, Ash, CP, AA, AEE, Ca, P, Cu, and Zn are described Open Array Real-Time PCR Platform (Applied Biosystems, in Blavi et al. (2021). Waltham, MA) as explained by González-Solé et al. (2020). Sample collection Microbiota 16S rRNA gene analysis On the last day of the experiment, a blood sample was col- Library preparation and sequencing lected from the jugular vein via vena-puncture of the pig in Bacterial DNA was recovered from 250 mg of colon digesta each pen that had a BW closest to the pen average (4 barrows following the manufacturer’s instructions with the commer- and 4 gilts per treatment). Samples were collected in two eth- cial MagMAX CORE Nucleic Acid Purification Kit 500RXN ylenediaminetetraacetic acid (EDTA) vacutainers (BD Diag- (Thermo Fisher, Barcelona, Spain). Mock community DNA nostics, Franklin Lakes, NJ). To recover serum, vacutainers was involved as a control (Zymobiomics Microbial Com- were centrifuged at 700 × g at 4 °C for 13 min. One set of munity DNA). Samples were amplified using specific primers serum samples was stored at −20 °C until analyzed for cyto- to the V3-V4 regions of the 16S rRNA DNA (V3-V4-For- kines and chemokines. The other set of serum samples was ward 5ʹ-TCGTCGGCAGCGTCAGATGTGTATAAGAG- stored at −80 °C until analyzed for malondialdehyde (MDA), ACAGCCTACGGGNGGCWGCAG-3ʹ, V3-V4-Reverse glutathione peroxidase (GSH-Px), and Cu/Zn superoxide dis- 5ʹGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG- mutase (SOD) activity. GACTACHVGGGTATCTAATCC-3ʹ) (Klindworth et al., Pigs from which blood samples were collected were killed 2013). The library preparation was performed in Microomics to obtain liver, jejunum, and colon digesta. Liver samples Systems SL (Barcelona, Spain). were collected and immediately placed in liquid nitrogen and stored at −80 °C for analysis of GSH-Px activity and MDA. Jejunum tissue was collected from the mid-jejunum Amplicon sequences processing and analysis (30 cm of the middle of the jejunum) by cutting pieces of Forward and reverse reads of raw demultiplexed samples 3 to 4 cm and removing the digesta. Jejunum tissues were were processed following the methods and pipelines in thoroughly washed in ice-cold phosphate-buffered saline QIIME2 version 2019.4 with defaulting parameters unless solution and placed into 2-mL RNAse-free vials to analyze indicated (Bolyen et al., 2019). DADA2 was used for qual- gene abundance. Jejunum samples were frozen and stored ity filtering, denoising, pair-end merging, and amplicon at −80 °C immediately after collection. Colon content was sequence variant calling (ASV, i.e., phylotypes) using qiime also collected from the spiral colon approximately 50 cm dada2 denoise-paired method (Callahan et al., 2016). Q20 from the cecum. The colonic digesta were analyzed for was used as a quality threshold to define read sizes for microbiota. trimming before merging (parameters: --p-trunc-len-f and --p-trunc- len-r). Reads were truncated at the place when Oxidative stress marker and antioxidant analysis the 75th percentile Phred score felt below Q20 for both Serum MDA was measured with a thiobarbituric acid reactive forward and reverse reads. After quality filtering steps, the substances assay kit (Cayman Chemical, USA). Activities of average sample size of reads was resolved and phylotypes SOD and GSH-Px in serum were determined by spectrometry were detected. ASVs were aligned using the qiime alignment following instructions of Ransod and Ransel kits, respectively mafft method (Katoh and Standley, 2013). The alignment (Randox, County Antrim, UK). All the samples were analyzed was used to generate a tree and to calculate phylogenetic in duplicate. relations between ASVs using qiime phylogeny FastTree One gram of liver was mixed with 4 mL of sucrose buffer method (Price et al., 2010). To even sample sizes for the (0.32 M) and homogenized with a tissue homogenizer and diversity analysis using qiime diversity core-metrics-phy- was immediately placed on ice. The content was then added logenetic pipeline, ASV tables were subsampled without to a centrifuge tube and centrifuged at 3,000 × g at 4 °C for replacement. The sample with the smallest size was dis- 15 min. The supernatant was filtered with a double paper fil- carded to take advantage of the sequencing depth of the ter and used to assess GSH-Px activity and MDA with the dataset. Afterward, subsampling to the next lowest sam- same method as used for serum samples. ple size was used for each comparison. Unweighted and weighted Unifrac distances were calculated to compare Multiplex immunoassay community structures (Lozupone et al., 2011). Taxonomic The following cytokines and chemokines were analyzed in assignment of ASVs was performed using a Bayesian Clas- serum: interferon alfa (INF-α), interferon-gamma (IFN-γ), sifier trained with Silva V4 database (i.e., 99% OTUs data- interleukin 10 (IL-10), interleukin 1 beta (IL-1β), interleu- base) using the qiime feature-classifier classify-sklearn kin 4 (IL-4), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor method (Pedregosa et al., 2011). Unifrac distance matrices necrosis factor-alpha (TNF-α), and interleukin (IL-12) using and ASV tables were used to calculate principal coordinates the 9-Plex Porcine ProcartaPlex Panel 1 (ThermoFisher Sci- and construct ordination plots using the R software pack- entific, EXP090-60829-901). All samples were analyzed as age version 3.6.0 (http://www.R-project.org). 4 Journal of Animal Science, 2022, Vol. 100, No. 9 Calculations and statistical analyses Table 2. Mean plus/minus standard deviation values of oxidative stress markers (SOD and GSH-Px) and malondialdehyde concentration at the Normality of residuals was verified using the UNIVARIATE end of the experiment in the serum and liver of pigs fed control diet and procedure (SAS Inst. Inc., Cary, NC) and outliers were identi- diets with 250 mg Cu/kg from CuSO4 or Cu2O1 fied using PROC ROBUSTREG of SAS and removed. Oxida- tive stress markers, MDA, and cytokines were analyzed as a Item NC2 CuSO4 Cu2O randomized complete block design, using the PROC MIXED of SAS with a model that included treatment as the main effect Serum and block as a random effect. Mean values were calculated  MDA, µM 9.13 ± 1.06 9.47 ± 1.57 10.43 ± 2.53 using the LSMeans statement. The pen was the experimental  SOD, U/L 0.22 ± 0.00 0.20 ± 0.02 0.24 ± 0.06 unit for all analysis. An alpha value of 0.05 was used to assess  GSH-Px, U/L 3919 ± 197.98 3646 ± 350.07 3626 ± 502.34 significance among means and tendencies were considered at Liver 0.05 ≤ P < 0.10.  MDA, µM 38.19 ± 5.65b 42.96 ± 3.20a 37.38 ± 1.35b Gene abundance data were analyzed using the Ther-  GSH-Px, 1.12 ± 0.07y 1.13 ± 0.05y 1.19 ± 0.08x moFisher Cloud software 1.0 (Applied Biosystems, Waltham, U/mg prot MA) applying the 2−ΔΔCt method for relative quantification and using the sample with the lowest abundance as a calibra- 1 Data are shown as the mean ± SD with 8 observations per treatment. tor. Some parameters were adjusted: maximum Crt allowed 2 NC: negative control; CuSO4: 250 mg Cu/kg from CuSO4; Cu2O: 250 mg was 26, AMP score < 1.240, Cq confidence > 0.8, and max- Cu/kg from Cu2O. a,b Means with different subscripts within a row differ (P < 0.05). imum standard deviation allowed between duplicates were x,y Means with different subscripts within a row differ (P < 0.10). fixed at <0.38. Relative quantification values were checked for normalization by a log10 transform, and all the statisti- cal analysis was performed with R 3.4.3 software (R Devel- tended to be lower in pigs fed 250 mg Cu/kg from CuSO4 opment Core Team, 2013) and Bioconductor (Gentleman et or Cu2O compared with those fed the NC (Figure 1). Pigs al., 2004). One-way ANOVA and Benjamini-Hochberg false fed 250 mg Cu/kg from CuSO4 tended to have greater (P < discovery rate (FDR Q-value) to control multiple P-values 0.10) IL-1β and had greater (P < 0.05) TNF-α concentra- were calculated (Benjamini and Hochberg, 1995) giving an tion than pigs fed the diet containing 250 mg Cu/kg from upper bound of the expected proportion of false significant Cu2O or the NC. tests, that is, false significant treatment differences in mean Gene abundance abundance levels between treatments. For microbiota, Alpha and Beta diversity were analyzed using the Vegan package A heatmap was constructed to observe overall similarities and taxa differences with the MetagenomeSeq package in among gene abundance profiles (Figure 2). A total of 47 genes open-source software RStudio v.3.5.1. Alpha diversity was were successfully amplified. calculated with raw counts based on Simpson, Shannon, and Twenty-six genes involved in diverse physiological func- Inverse-Simpson estimators. Beta diversity was evaluated by tions responded to the exposed diets (P < 0.05, Q < 0.10, multivariate ANOVA based on dissimilarities through envfit Table 3). The CLDN15, MUC2, and TFF3 genes from the BF and adonis functions. Finally, differential abundance anal- group, TLR2 gene from the IR group, SLC39A4/ZIP4, and ysis was performed with taxa relative abundances under a SLC5A1/SGLT1 from the NT group, DAO1, IGF1R, and zero-inflated log-normal mixture model after normalization SOD2 gene from the EH group had a higher abundance in with Cumulative Sum Scaling. P-values were corrected by pigs fed 250 mg Cu/kg treatments compared with those fed FDR with the metagenomeseq package (Paulson et al., 2017). the NC diet (P < 0.05). Pigs fed 250 mg Cu/kg from CuSO4 Statistical differences among the treatments were identified had a lower (P < 0.0.05) abundance of CCL20 (IR), and SI at P-values under 0.05 for the ANOVA and Tukey’s analysis, (EH) than pigs fed the NC diet, and a higher (P < 0.05) abun- and Q-values under 0.1 for the FDR. dance of SLC16A1/MCT1 gene (NT) than pigs fed the NC diet or the diet with 250 mg Cu/kg from Cu2O. However, pigs fed the diet containing 250 mg Cu/kg from Cu2O had a Results lower abundance of ZO1 (BF) compared with pigs fed NC or the diet containing 250 mg Cu/kg from CuSO4, and these Oxidative stress markers and malondialdehyde pigs also had a lower (P < 0.05) abundance of HSPA4 and concentrations IFNGR1 (IR), but an elevated (P < 0.05) abundance of PYY Malondialdehyde, SOD, and GSH-Px in the serum were not (EH) compared with pigs fed NC. different among dietary treatments (Table 2). However, pigs fed 250 mg Cu/kg from CuSO4 had greater (P < 0.05) MDA Microbiota 16S rRNA gene analysis in the liver compared with pigs fed the NC diet or the diet For an assessment of the effects of Cu supplementation on containing 250 mg Cu/kg from Cu2O. The GSH-Px concen- large intestinal microbiota, the 16S rRNA of colon microbi- tration tended to be greater (P <0.10) in the liver of pigs fed ota was determined. Alpha (Shannon, Simpson, and Inverse 250 mg Cu/kg from Cu2O compared with pigs fed the other Simpson index) and beta diversity metrics were used to esti- diets. mate diversity among microbial communities. There were no differences in alpha diversity indices (P > 0.1, data not Cytokine concentration shown), and beta diversity among experimental treatments Among the studied cytokines, IL-4, IL-6, and IL-10 were not was not different either (PENVFIT = 0.4, data not shown). detected because the values were below the standard curve. In the colon microbiota, 17 operational taxonomic units No differences were observed among treatments for representing the phyla Actinobacteria, Bacteroidetes, Chla- IFN-α, IL-8, and IL-12, but serum concentration of IFN-γ mydiae, Cyanobacteria, Deferribacteres, Epsilonbacteraeota, Forouzandeh et al. 5 Figure 1. Cytokine concentration in the serum of pigs fed control diet and diets with 250 mg Cu/kg from CuSO4 or Cu2O. Data are means of 8 observations per treatment. Error bars show the standard deviation. PROC MIXED significance, * indicates P < 0.1 and ** indicates P < 0.05. Euryarchaeota, Fibrobacteres, Firmicutes, Kiritimatiellae- fold increase), and Peptostreptococcaceae (1.31 fold increase) ota, Lentisphaerae, Patescibacteria, Planctomycetes, Pro- families, and Holdemanella (0.34 fold increase) genera com- teobacteria, Spirochaetes, Synergistetes, and Tenericutes pared with pigs fed a diet not supplemented with Cu. Pigs were identified (Figure 3a). Firmicutes and Bacteroidetes fed 250 mg Cu/kg from CuSO4 had lower (P < 0.05) levels were present at a mean relative abundance of ≥30% (aver- of Rikenellaceae (0.3 fold decrease), Christensenellaceae age 52% and 30% respectively), followed by Spirochaetes (0.79 fold decrease) families, and Campylobacter (0.82 fold (average 3.40%) and Tenericutes (average 3%). The major- decrease), and Streptococcus (1.57 fold decrease) genera than ity of Firmicutes phylum corresponded to Ruminococcaceae the NC. (average 26.8%), Erysipelotrichaceae (average 8.2%), Lach- Supplementing 250 mg Cu/kg from Cu2O to the diet nospiraceae (average 6.2%), Acidaminococcaceae (average increased (P < 0.05) relative abundance of Lachnospir- 3.8%), Veillonellaceae (average 3.6%), and Christensenel- aceae (0.31 fold increase), Spirochaetaceae (1.90 fold laceae (0.8-1.4%). The majority of Bacteroidetes phylum increase), and Peptostreptococcaceae (0.97 fold increase) corresponded to Prevotellaceae (average 16.8%), Rikenel- families compared with pigs fed the non-supplemented laceae (5.7%–8.1%), Muribaculaceae (average 5.2%), and diet. In contrast, Cu2O supplementation decreased (P < most Spirochaetes belonged to the Spirochaetaceae family 0.05) the relative abundance of the Rikenellaceae family (1.5%–5.5%), as illustrated in Figure 3b. (0.51 fold decrease), and genera like Holdemanella (0.9 The following families were different (Figure 4) among fold decrease), Campylobacter (1.28 fold decrease), and dietary treatments: Rikenellaceae, Erysipelotrichaceae, Lach- Streptococcus (0.58 fold decrease) compared with pigs fed nospiraceae, Spirochaetaceae, Christensenellaceae, and Pep- the NC diet. tostreptococcaceae. Some significances at the genus level, i.e., Pigs fed the diet with 250 mg Cu/kg from CuSO4 tended Holdemanella, Campylobacter, and Streptococcus have been to have increased abundance of Rikenellaceae (0.22 fold also observed. increase, P = 0.088) and Erysipelotrichaceae (0.22 fold Pigs fed 250 mg Cu/kg from CuSO4 had a greater (P < 0.05) increase, P = 0.068) families, and increased (P < 0.05) the relative abundance of Erysipelotrichaceae (0.28 fold increase), abundance of Holdemanella (1.24 fold increase) genera, com- Lachnospiraceae (0.26 fold increase), Spirochaetaceae (1.1 pared with pigs fed 250 mg Cu/kg from Cu2O. 6 Journal of Animal Science, 2022, Vol. 100, No. 9 Figure 2. Heatmap representing the most abundant genes (n = 47) related to barrier function (BR), immune response (IR), nutrient transport (NT), enzyme/hormone (EH), and stress (ST). The color gradient represents the relative gene abundance. Rows represent the results of each gene shown on the y-axis. Columns represent tissue samples (n = 24) related to each treatment: negative control (NC), 250 mg Cu/kg from CuSO4, and 250 mg Cu/kg Cu2O. Discussion to its cation-selective channel property (Tamura et al., 2011). MUC2 is the key component of the mucus gel layer in the This work was part of a larger study that has been previ- gut mucosa, which is secreted by goblet cells (Li, 2011) and ously published (Blavi et al., 2021) and is recommended as protects against luminal viral infection as an essential struc- complementary information. The previous study reported tural component of the intestinal mucin layer (Deplancke increased BW by supplementation of diets with 250 mg/kg of and Gaskins, 2001). TFF3 is important for repairing the Cu, specifically Cu2O, starting from the end of phase one. At intestinal mucosa (Liu et al., 2021). Therefore, the observa- the same time, pigs fed 250 mg Cu/kg from Cu2O had lower tion that therapeutic doses of Cu increased the abundance Cu concentration in the liver and spleen compared with those of CLDN15, MUC2, and TFF3 may indicate that dietary Cu fed 250 mg Cu/kg from CuSO4. Therefore, we decided to take may help improve intestinal barrier function. Upregulation advantage of the previously collected samples at the end of of TFF3 and CLDN15 coincides with better growth perfor- phase one to elucidate the differences between the Cu sources mance in weanling pigs (González-Solé et al., 2020). and the NC. One of the roles of Cu is aiding the improvement of ani- mals’ innate and acquired immunological functions (Pro- Effects of copper on the jejunum gene abundance haska and Failla, 1993). The immune system is triggered The integrated intestinal barrier is critical for epithelial cell when it is exposed to pathogenic or nonpathogenic antigens, function as well as for preventing harmful microorganisms resulting in the release of cytokines such as TNF-α, IL-1, from passing through the mucosa (Hu et al., 2012). CLDN15 and IL-6 (Al-Sadi et al., 2009). The reduced abundance of is a tight junction barrier protein (Tamura et al., 2008), the immune response proteins CXCL2, IFN-γ, IL-6, IL-8, which is important for small intestinal sugar absorption due and TGF-β1 that was observed in pigs fed diets containing Forouzandeh et al. 7 Table 3. Relative gene abundance differences of jejunum between pigs fed control diet and diets with 250 mg Cu/kg from CuSO4 or Cu2O1 Function Genes2 NC3 CuSO4 Cu2O Contrast Statistic4 P-value Q-value (FDR) Barrier function CLDN15 1b 2.30a 2.39a 10.535 0.001 0.003 MUC2 1 b 2.83 a 2.23a 11.597 < 0.001 0.002 OCLN 1a 0.75b 0.72b 5.604 0.012 0.030 TFF3 1b 4.74a 4.34a 30.806 < 0.001 < 0.001 ZO1 1 a 0.72 b 0.63c 33.595 < 0.001 < 0.001 Immune response CCL20 1a 0.31b 0.56ab 12.355 0.041 0.071 CXCL2 1a 0.53b 0.54b 8.514 0.025 0.050 HSPA4 1a 0.87ab 0.73b 28.923 0.012 0.030 IFN-γ 1a 0.42b 0.54b 11.792 0.030 0.057 IFNGR1 1a 0.87ab 0.80b 3.801 0.044 0.074 lL-6 1a 0.55b 0.33b 6.152 0.001 0.003 IL-8 1 a 0.48 b 0.45 b 4.530 0.022 0.047 TGF-β1 1a 0.83b 0.79b 10.668 0.017 0.040 TLR2 1b 1.93a 1.65a 3.722 0.018 0.040 Nutrient transport SLC16A1/MCT1 1 b 1.23 a 0.90b 4.412 0.002 0.006 SLC30A1/ZnT1 1a 0.54b 0.51b 5.546 < 0.001 < 0.001 SLC39A4/ZIP4 1b 1.43a 1.84a 4.262 0.001 0.005 SLC5A1/SGLT1 1 b 2.11 a 2.79a 3.625 < 0.001 0.001 Enzyme/Hormone ANPEP 1a 0.39b 0.34b 4.626 < 0.001 0.002 CCK 1a 0.69b 0.64b 10.469 0.002 0.006 DAO1 1b 3.60a 3.64a 4.953 < 0.001 0.000 HNMT 1a 0.74b 0.70b 4.925 < 0.001 0.002 IGF1R 1 b 1.41 a 1.44 a 8.580 0.040 0.071 PYY 1b 1.31ab 1.84a 23.337 0.023 0.048 SI 1a 0.56b 0.78ab 9.335 0.008 0.024 SOD2 1 b 1.93 a 1.50a 14.961 0.001 0.003 1 Data are means of 8 observations per treatment. Gene abundance values are presented as ratios of cycle relative threshold value for each gene normalized to that of the reference sample. 2 CLDN15: claudin 15; MUC2: mucin 2; OCLN: occludin; TFF3: trefoil factor 3; ZO1: zonula occludens 1; CCL20: chemokine (C-C motif) ligand 20; CXCL2: chemokine (C-X-C motif) ligand 2; HSPA4: heat shock protein 70; INF-γ: interferon gamma; IFNGR1: interferon gamma receptor 1; IL-6: interleukin 6; IL-8: interleukin 8; TGF-β1: transforming growth factor beta 1; TLR2: toll-like receptor 2; SLC16A1/MCT1: monocarboxylate transporter 1; SLC30A1/ZnT1: solute carrier family 30 (zinc transporter) member 1; SLC39A4/ZIP4: solute carrier family 39 (zinc transporter) member 4; SLC5A1/ SGLT1: solute carrier family 5 (sodium/glucose cotransporter) member 1; ANPEP: aminopeptidase-N; CCK: cholecystokinin; DAO1: diamine oxidase; HNMT: histamine N-methyltransferase; IGF1R: insulin-like growth factor 1 receptor; PYY: peptide tyrosine tyrosine; SI: sucrase-isomaltase; SOD2: superoxide dismutase 3 NC: negative control; CuSO4: 250 mg Cu/kg from CuSO4; Cu2O: 250 mg Cu/kg from Cu2O. 4 Contrast statistic expresses the variability comparison between the experimental diet and the residual variability within diets. a-b Means with different subscripts within a row differ (P < 0.05) therapeutic levels of Cu (CuSO4 or Cu2O) also indicates an The fact that supplementation of diets with 250 mg/kg of improved immune response. CXCL2 is a chemoattractant Cu upregulated the abundance of SLC5A1/SGLT1 may indi- for neutrophils (Kielian et al., 2001) and is important for the cate that pigs fed the diets containing therapeutic levels of inflammatory response and growth regulation (Kim et al., Cu may have had improved absorption of glucose. SLC5A1/ 2010). Pro-inflammatory cytokines including IFN-γ, IL-6, SGLT1 is the main intestinal glucose transporter (Röder et and IL-8 enhance intestinal epithelial permeability and pro- al., 2014) that transports glucose into the enterocytes from voke a pathologic opening of the intestinal tight junction bar- the lumen. rier (Al-Sadi et al., 2009). The reduced mucosal IFN-γ, IL-6, The downregulation of CCK and upregulation of PYY and IL-8 in the presence of high levels of Cu indicate that was observed for pigs fed diets with elevated concentra- therapeutic levels of Cu can minimize intestinal inflammation, tions of Cu. CCK is a brain-gut peptide (Strader and Woods, thus minimizing tissue damage and reducing gut permeability. 2005) that inhibit feed intake and satiety (Moran and These results agree with Song et al. (2013) who also showed Kinzig, 2004), so a reduction of CCK may have the opposite reduced mucosal IL-6 and TNF-α and improved mucosal effect. The hormone PYY is primarily produced and released barrier integrity of weanling pigs fed a diet with increased by endocrine L-cells in the distal region of the gastrointesti- Cu. The reduced concentration of the anti-inflammatory nal tract in response to feed intake (Karhunen et al., 2008). cytokine TGF-β1, which protects against intestinal inflamma- Because PYY mediates ileal and colonic brakes, which slow tion (Howe et al., 2005), with Cu supplementation may be a down gastric emptying and stimulate digestive processes result of a low concentration of pathogens and low intestinal to promote nutrition absorption (Pironi et al., 1993) while inflammation in the pigs used in this experiment. reducing appetite (Sleeth et al., 2010). Although we only 8 Journal of Animal Science, 2022, Vol. 100, No. 9 pathogenic organism in the gut (Xia et al., 2004) and reduc- ing susceptibility to disease due to its antimicrobial effect. In fact, one of the possible mechanisms by which Cu may pro- mote growth in animals is restricting the growth of microbes in the intestinal tract (Espinosa et al., 2019) and increasing nutrient absorption (Villagómez-Estrada et al., 2020). Members of the Lachnospiraceae family produce butyric acid via fermentation of polysaccharides (Quan et al., 2018), and its abundance is positively correlated with energy metab- olism (Zhang et al., 2019) and improved feed efficiency in pigs (Yang et al., 2017; Quan et al., 2018) and poultry (Stanley et al., 2016). The role of the Spirochaetaceae family is not com- pletely clear, but some of its members have greater relative abundance in low residual feed intake (RFI) pigs compared with high RFI (McCormack et al., 2017). The Peptostrepto- coccaceae family is present in a higher proportion in the gut microbiota of healthy animals than of animals experiencing dysbiosis of the intestinal microbiota (Fan et al., 2017). There- fore, the fact that at the family level, the relative abundance of Lachnospiraceae, and Peptostreptococcaceae increased with high levels of CuSO4 or Cu2O, and the relative abundance of Spirochaetaceae increased with high levels of Cu2O, may indi- Figure 3. Relative abundance (%) of phyla (a), and families (b) present in the colon microbiota of pigs fed control diet and diets with 250 mg Cu/ cate that changes in the microbiota contributed to a greater kg from CuSO4 or Cu2O. The rest of the taxonomic groups are pooled growth performance of pigs fed the Cu-supplemented diets. together (those representing less than a mean of 1% of phyla and However, the implication of the reduced concentration of the families). family Christensenellaceae in feces from pigs fed diets with high concentrations of Cu is less certain because the relation- ship between the abundance of Christensenellaceae and gut analyzed the gene expression at the end of phase one, long- health remains unclear. term regulation of these hormones may be one of the rea- An increase in the concentration of favorable families in the sons behind the increased feed intake in pigs fed the diet gut may result in a reduction of potentially pathogenic organ- containing Cu2O throughout the experiment (Blavi et al., isms. The reduced abundance of Rikenellaceae and Campy- 2021). lobacter and Streptococcus, therefore, may result in reduced Insulin-like growth factor 1 (IGF1) has many growth-pro- intestinal disease as has been demonstrated in mice (He et al., moting and metabolic activities (Froesch, 1985) and is an 2019). essential regulator of intestinal cell growth and differentia- Overall, the increase in beneficial families and the reduc- tion (Jones and Clemmons, 1995). Increased abundance of tion of pathogens in the gastrointestinal tract of pigs fed diets IGF1 and IGF1R in piglet small intestine enhances growth with elevated concentrations of Cu possibly improved intes- performance (Li et al., 2006). The upregulation of IGF1R tinal nutrient absorption and contributed to the increased that was observed in pigs fed diets with added Cu, there- growth performance observed in the experiment (Blavi et fore, might have been associated with the increased growth al., 2021). The reduced concentration of pathogens also sup- performance of these pigs. The downregulation of digestive ports the lower abundance of immune response genes that enzyme SI, which was caused by CuSO4, but not by Cu2O, were observed in pigs fed the diets with therapeutic doses may have restricted the absorption of glucose and free AA. of Cu. This observation agrees with similar modulations of Feeding high levels of Cu may result in an increased abun- the microbiota in pigs fed a diet containing 160 mg/kg of dance of antioxidant-related mRNA SOD2 (Huo et al., Cu from CuSO4 or Cu hydroxychloride, which resulted in 2021; Li et al., 2021), and a reduced SOD level is likely to improved growth performance (Villagómez-Estrada et al., result in an increased ROS level. Therefore, the upregula- 2020). The observation that Cu2O seemed to be more efficient tion of SOD2 that was observed in pigs fed the diets with than CuSO4 in terms of reducing the growth of Rikenella- 250 mg/kg of Cu may be considered an anti-inflammatory ceae family and Holdemanella genus may indicate that Cu2O response by Cu. promotes intestinal health to a greater degree than CuSO4. Overall, the intestinal gene abundance data from the exper- Similar observations were reported from a study with broiler iment indicate that the improved growth performance by pigs chickens (Forouzandeh et al., 2021). fed diets with therapeutic levels of Cu (Blavi et al., 2021), maybe a result of improvement in intestinal epithelial barrier function, modulation of immunological and inflammatory Effects of copper on oxidation and inflammation responses, and increased feed intake and nutrient absorption. Although Cu plays a pivotal role for some key enzymes, the addition of therapeutic levels of Cu (150–250 mg Cu/kg) Effects of copper on the colon microbiota profile results in oxidation and catalyzing of hydroxyl radical forms Therapeutic levels of Cu alter the intestinal microbiota in (Gaetke, 2003). One of the metabolic products of lipid per- poultry (Pang et al., 2009; Forouzandeh et al., 2021) and oxides is MDA, which is generated by oxygen-free radicals in swine (Wang et al., 2012; Villagómez-Estrada et al., 2020). tissues (Zhan et al., 2006). A high concentration of MDA is Copper supplementation may act by reducing the total an indicator of oxidation and has been identified in the liver Forouzandeh et al. 9 Figure 4. Colonic bacterial families and genera whose abundance significantly differs between pigs fed control diet and diets with 250 mg Cu/kg from CuSO4 or Cu2O. Data are means of 8 observations per treatment. Error bars show the standard deviation. ANOVA significance, *indicates P < 0.1, and ** P < 0.05. (Pu et al., 2016) and duodenal mucosa (Huang et al., 2015) some extent agrees with the tendency in reduction of the con- of pigs fed diets with high Cu concentrations. The observa- centration of the antioxidant enzyme GSH-Px. These results tion that liver MDA in pigs fed the diet with 250 mg Cu/ are consistent with the fact that there is greater prooxidant kg from CuSO4 was approximately 12% greater than in the activity in the liver of broiler chickens and pigs fed a diet liver of pigs fed NC or the diet with Cu2O supplementation to containing CuSO4 compared with those fed a diet containing 10 Journal of Animal Science, 2022, Vol. 100, No. 9 tribasic Cu chloride (Miles et al., 1998; Luo et al., 2005; Fry Literature Cited et al., 2012). The chemical characteristics of Cu could be one Al-Sadi, R., M. Boivin, and T. Ma. 2009. Mechanism of cytokine mod- of the reasons behind this difference. 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Dietary Cu increased the concentrations of microbial on intestinal microbial concentration and apparent ileal and to- populations that are favorable to growth, immunity, and gut tal tract digestibility of energy and nutrients by growing pigs1. J. health. However, CuSO4, but not Cu2O, increased liver oxida- Anim. Sci. 97:4904–4911. doi:10.1093/jas/skz340. tion and biomarkers for inflammation in serum. Espinosa, C. D., R. S. Fry, J. L. Usry, and H. H. Stein. 2017. Copper hydroxychloride improves growth performance and reduces di- arrhea frequency of weanling pigs fed a corn–soybean meal diet Acknowledgments but does not change apparent total tract digestibility of energy and acid hydrolyzed ether extract. J. Anim. Sci. 95:5447. doi:10.2527/ The financial support from Animine, Annecy, France, is great- jas2017.1702. ly appreciated. This work was also supported by Agéncia de Espinosa, C. D., R. S. Fry, J. L. Usry, and H. H. Stein. 2021. Copper Gestió d’Ajusts Universitaris i de Recerca de la Generalitat de hydroxychloride improves gain to feed ratio in pigs, but this is not Catalunya (PhD grant 2019FI_B 00282). David Solà-Oriol is due to improved true total tract digestibility of acid hydrolyzed supported by the UAB-Banco de Santander Talent Program. ether extract. Anim. Feed Sci. Technol. 274:114839. doi:10.1016/j. anifeedsci.2021.114839. Fan, P., P. Liu, P. Song, X. Chen, and X. Ma. 2017. Moderate dietary Conflict of Interest Statement protein restriction alters the composition of gut microbiota and improves ileal barrier function in adult pig model. Sci. Rep. 7:1–12. Alessandra Monteiro is an employee at Animine, Annecy, doi:10.1038/srep43412. France, a company that has commercial interests in the min- Forouzandeh, A., L. Blavi, N. Abdelli, D. Melo-Duran, A. Vidal, M. eral nutrition of food-producing animals. Animine adheres to Rodríguez, A. Monteiro, J. Pérez, L. Darwich, and D. 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