Unexpected Cu and Zn speciation patterns in the broiler feed-animal-excreta system revealed by XAS spectroscopy.

Trace minerals such as copper (Cu) and zinc (Zn) are animal nutrition supplements necessary for livestock health and breeding performance, yet they also have environmental impacts via animal excretion. Here we investigated changes in Cu and Zn speciation from the feed additive to the broiler excreta stages. The aim of this study was to assess whether different Cu and Zn feed additives induce different Cu and Zn speciation patterns, and to determine the extent to which this speciation is preserved throughout the feed-animal-excreta system. Synchrotron-based X-ray absorption spectroscopy (XAS) was used for this investigation. The principal findings were: (i) in feed, Cu and Zn speciation changed rapidly from the feed additive signature (Cu and Zn oxides or Cu and Zn sulfates) to Cu and Zn organic complexes (Cu phytate and Zn phytate). (ii) in the digestive tract, we showed that Cu and Zn phytate were major Cu and Zn species; Cu sulfide and Zn amorphous phosphate species were detected but remained minor species. (iii) in fresh excreta, Cu sulfide and Zn amorphous phosphate were major species. These results should help to: (i) enhance the design of future research studies comparing different feed additive performances; (ii) assess Cu and Zn bioavailability in the digestive tract; (iii) gain further insight into the fate of Cu and Zn in cultivated soils when poultry manure is used as fertilizer.

Limited impacts of high doses of dietary copper on the gut bacterial metal resistome explain negligible co-selection of antibiotic resistance.

High dietary intake of Cu has previously been linked to the selection of Cu resistance and co-selection of antibiotic resistance in specific gut bacteria. Based on a novel HT-qPCR metal resistance gene chip as combined with 16S rRNA gene amplicon sequencing and phenotypic resistance typing of Escherichia coli isolates, we here report the impacts of two contrasting Cu-based feed additives on the swine gut bacterial metal resistome and community assembly. DNA was extracted from fecal samples (n = 80) collected at day 26 and 116 of the experiment from 200 pigs allotted to five dietary treatments: negative control (NC) diet with 20 µg CuSO4 g-1 and four diets added 125 or 250 µg CuSO4 g-1 feed or 125 or 250 µg Cu2O g-1 feed to the NC diet. Dietary Cu supplementation reduced the relative abundance of Lactobacillus, but it had negligible impacts on bacterial community composition relative to the gut microbiome maturation effect (time). The relative importance of different bacterial community assembly processes was not markedly affected by the dietary Cu treatments, and differences in swine gut metal resistome composition could be explained primarily by differences in bacterial community composition rather than by dietary Cu treatments. High dietary Cu intake (250 µg Cu g-1) selected for phenotypic Cu resistance in E. coli isolates, but surprisingly it did not result in increased prevalence of the Cu resistance genes targeted by the HT-qPCR chip. In conclusion, the lacking impacts of dietary Cu on the gut bacterial metal resistome explain results from a previous study showing that even high therapeutic doses of dietary Cu did not cause co-selection of antibiotic resistance genes and mobile genetic elements known to harbor these genes.

Impacts of dietary copper on the swine gut microbiome and antibiotic resistome.

Restrictions on antibiotic growth promoters have prompted livestock producers to use alternative growth promoters, and dietary copper (Cu) supplementation is currently being widely used in pig production. However, elevated doses of dietary Cu constitute a risk for co-selection of antibiotic resistance and the risk may depend on the type of Cu-based feed additives being used. We here report the first controlled experiment investigating the impact of two contrasting Cu-based feed additives on the overall swine gut microbiome and antibiotic resistome. DNA was extracted from fecal samples (n = 96) collected at four time points during 116 days from 120 pigs allotted to three dietary treatments: control, divalent copper sulfate (CuSO4; 250 µg Cu g-1 feed), and monovalent copper oxide (Cu2O; 250 µg Cu g-1 feed). Bacterial community composition, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) were assessed, and bioavailable Cu ([Cu]bio) was determined using whole-cell bacterial bioreporters. Cu supplementation to feed increased total Cu concentrations ([Cu]total) and [Cu]bio in feces 8-10 fold and at least 670-1000 fold, respectively, but with no significant differences between the two Cu sources. The swine gut microbiome harbored highly abundant and diverse ARGs and MGEs irrespective of the treatments throughout the experiment. Microbiomes differed significantly between pig growth stages and tended to converge over time, but only minor changes in the bacterial community composition and resistome could be linked to Cu supplementation. A significant correlation between bacterial community composition (i.e., bacterial taxa present) and ARG prevalence patterns were observed by Procrustes analysis. Overall, results of the experiment did not provide evidence for Cu-induced co-selection of ARGs or MGEs even at a Cu concentration level exceeding the maximal permitted level for pig diets in the EU (25 to 150 µg Cu g-1 feed depending on pig age).

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.

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 necrosis 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.

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 inflammatory 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.

Strategies of inorganic and organic trace mineral supplementation in gestating hyperprolific sow diets: effects on the offspring performance and fetal programming.

The aim of the present study was to evaluate the effect of trace mineral nutrition on sow performance, mineral content, and intestinal gene expression of neonate piglets when inorganic mineral sources (ITM) were partially replaced by their organic mineral (OTM) counterparts. At 35 d postmating, under commercial conditions, a total of 240 hyperprolific multiparous sows were allocated into three experimental diets: 1) ITM: with Zn, Cu, and Mn at 80, 15, and 60 mg/kg, respectively; 2) partial replacement trace mineral source (Replace): with a 30 % replacement of ITM by OTM, resulting in ITM + OTM supplementation of Zn (56 + 24 mg/kg), Cu (10.5 + 4.5 mg/kg), and Mn (42 + 18 mg/kg); and 3) Reduce and replace mineral source (R&R): reducing a 50% of the ITM source of Zn (40 + 24 mg/kg), Cu (7.5 + 4.5 mg/kg), and Mn (30 + 18 mg/kg). At farrowing, 40 piglets were selected, based on birth weight (light: <800 g, and average: >1,200 g), for sampling. Since the present study aimed to reflect results under commercial conditions, it was difficult to get an equal parity number between the experimental diets. Overall, no differences between experimental diets on sow reproductive performance were observed. Light piglets had a lower mineral content (P < 0.05) and a downregulation of several genes (P < 0.10) involved in physiological functions compared with their average littermates. Neonate piglets born from Replace sows had an upregulation of genes involved in functions like immunity and gut barrier, compared with those born from ITM sows (P < 0.10), particularly in light piglets. In conclusion, the partial replacement of ITM by their OTM counterparts represents an alternative to the totally inorganic supplementation with improvements on neonate piglet gene expression, particularly in the smallest piglets of the litter. The lower trace mineral storage together with the greater downregulation of gut health genes exposed the immaturity and vulnerability of small piglets.