Impact of zinc caproate supplementation on growth performance, intestinal health, anti-inflammatory activity, and Zn homeostasis in weaned piglets challenged with Escherichia coli K88

Impact of zinc caproate supplementation on growth performance, intestinal health, anti-inflammatory activity, and Zn homeostasis in weaned piglets challenged with Escherichia coli K88 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Impact of zinc caproate supplementation on growth performance, intestinal health, anti-inflammatory activity, and Zn homeostasis in weaned piglets challenged with Escherichia coli K88 Jilong Xu, Hanzhen Qiao, Liping Gan, Peng Wang, Yifeng Zhao, Zetian Lei, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5194232/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Mar, 2025 Read the published version in Journal of Animal Science and Biotechnology → Version 1 posted 5 You are reading this latest preprint version Abstract Background Enterotoxigenic Escherichia coli (ETEC) is one of the primary causes of diarrhea in piglets, creating substantial economic losses in the swine farming industry worldwide. This study aimed to investigate the impact of zinc caproate (ZnCA) on the intestinal health, growth performance, inflammatory status, and Zn homeostasis of weaned piglets challenged with ETEC K88. In total, 48 weaned piglets (Duroc × Landrace × Yorkshire, 7.78 ± 0.19 kg, 28 d) were selected for a 21-d experiment. Each experimental treatment consisted of six replicate pens with two piglets each. The treatment conditions were as follows: 1) a basal diet ( CON ), 2) a basal diet + ETEC K88 ( NC ), 3) a basal diet + 2500 mg/kg Zn oxide (ZnO) + ETEC K88 ( PC ), and 4) a basal diet + 1600 mg/kg ZnCA + ETEC K88 ( ZnCA ). Results The addition of 1600 mg/kg ZnCA to the diet of post-weaning piglets effectively enhanced growth performance and nutrient digestibility and reduced the incidence of diarrhea and inflammatory reactions caused by ETEC K88 infection. These therapeutic effects were comparable to those of pharmacological doses of ZnO. In terms of improving intestinal health and Zn homeostasis in post-weaning piglets challenged with ETEC K88, the effectiveness of 1600 mg/kg ZnCA surpassed that of pharmacological doses of ZnO. Conclusions Overall, under the experimental conditions of this study, ZnCA exhibited the potential to reduce the pharmacological dosage of ZnO while improving intestinal health and Zn homeostasis in weaned piglets. ZnCA pharmacological doses of ZnO ETEC K88 weaned pigs AGPs antibiotic growth promoters Figures Figure 1 Background Post-weaning diarrhea (PWD) frequently occurs in weaned piglets and is primarily the result of incomplete intestinal development and suboptimal feeding conditions on farms. This condition can become fatal, causing significant financial losses in the swine industry [ 1 ]. Enterotoxigenic Escherichia coli (ETEC) is the leading pathogenic cause of post-weaning colibacillosis, a common type of PWD. Globally, the predominant strain associated with PWD in piglets is ETEC K88, which typically infects these animals through the oral route [ 2 ]. The initial stages of ETEC infection involve the colonization of the intestinal epithelium and the secretion of enterotoxins. ETEC colonizes the intestinal epithelium by leveraging membrane adhesins to adhere to the intestinal mucosa and binding to glycoprotein receptors on the brush border of intestinal cells. Subsequently, it secretes enterotoxins (including heat-labile and heat-stable enterotoxins), disrupts the electrolyte balance in the intestines, and ultimately causes diarrhea [ 3 , 4 ]. Historically, antibiotic growth promoters (AGPs) and pharmacological doses of zinc oxide (ZnO, 2500 mg/kg) have been widely utilized in piglet feed to either prevent or treat PWD and enhance growth performance [ 5 , 6 ]. However, serious concerns have emerged regarding the misuse of AGPs and ZnO, since these chemicals can promote bacterial resistance, lead to drug residues in animal products, and cause environmental pollution [ 7 , 8 ]. Therefore, the European Union and China have imposed bans on the use of AGPs and restricted the dosage of ZnO in animal feed [ 9 ]. At present, in the European Union and China, the maximum permissible dosages of ZnO in animal feed are 150 mg/kg and 1600 mg/kg, respectively, during the first two weeks following weaning [ 10 ]. It is becoming increasingly evident that ZnO may fail to meet the production demands of the swine farming industry in the near future [ 11 ]. Consequently, there is an urgent need to identify ZnO substitutes for this purpose. So far, a range of Zn-containing molecules with the potential to treat PWD have been studied extensively. These include inorganic Zn compounds like ZnO nanoparticles (Nano-ZnO) [ 12 ] and tetrabasic Zn chloride [ 13 ] as well as organic Zn compounds such as Zn-amino acid complexes [ 14 ], Zn-polysaccharide complexes [ 15 ], and certain coated Zn products [ 16 , 17 ]. While these agents show promise in lowering the bioactive concentration of ZnO and mitigating its environmental impact, the doses employed fail to meet the European Commission's mandate of 150 mg/kg of total Zn in complete feed [ 18 ]. Medium-chain fatty acids (MCFAs), which are promising alternatives to antibiotics, play various roles in piglet nutrition. For instance, they can enhance growth performance, boost immunity, promote the growth of beneficial intestinal microbes, and maintain intestinal homeostasis [ 19 , 20 ]. MCFAs have the potential to reduce the acid-binding capacity in the intestines, thereby improving feed digestibility in weaned piglets [ 21 ]. Most MCFAs and their derivatives are generally considered safe in feed and food products and are widely used in various industries, including the daily chemicals, food, and pharmaceutical sectors [ 22 ]. Recently, studies have proposed that the synthesis of organic Zn compounds through the combination of caproic acid (CA) and Zn could potentially enhance the bioavailability of Zn, thus offering a novel avenue for effectively managing PWD and ETEC K88 infections. In our previous study, ZnCA was successfully synthesized using a solvothermal method and demonstrated remarkable antibacterial activity against ETEC K88 in vitro [ 23 ]. Consequently, the aim of the present study was to examine the impact of dietary supplementation with ZnCA on growth performance, inflammatory status, intestinal health, and Zn homeostasis in weaned piglets exposed to ETEC K88. Materials and Methods Experimental materials ZnO (75% Zn) was obtained from Shijiazhuang Hanying Feed Co., Ltd. (Shijiazhuang, China). Titanium dioxide (TiO 2 ) was procured from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). ZnCA, a Zn complex composed of ZnO and CA (21.91% Zn), was synthesized in our laboratory at the College of Bioengineering, Henan University of Technology. Animals, experimental design, and housing Forty-eight piglets (Duroc × Landrace × Yorkshire) aged 28 d (body weight [BW] = 7.78 ± 0.19 kg) were selected for a 21-d experiment. A randomized complete design consisting of four treatment conditions was employed (N = 6 pens/treatment). Groups were matched based on BW (with weaning BW balanced across pens) and sex. Each experimental group consisted of six replicate pens, with two piglets in each pen. The control group (CON) and negative control group (NC) received a basal diet (Table 1 ), while the positive group (PC) received the basal diet supplemented with pharmacological doses of ZnO (2500 mg/kg Zn). Meanwhile, the ZnCA group (ZnCA) received a basal diet supplemented with 1500 mg/kg Zn (ZnCA). All diets exceeded the nutritional recommendations proposed by the National Research Council [ 24 ]. Each pen, measuring 1.30 m × 0.5 m, was furnished with a single-side feeder and nipple drinker. The ambient temperature within the facility was initially set at 30℃ and subsequently reduced at a rate of 1.5℃ per week. The relative humidity was at 50%, and the animals had unrestricted access to feed and water throughout the 21-d study period. Table 1 Composition and nutrient content of the basal diet (as-fed basis, %) Ingredients Content (%) Corn 54.55 Soybean meal 46% 25 Extrusion soybean 10 Fish meal 5 Whey powder 2 Soybean oil 1 Dicalcium phosphate 0.8 Limestone 0.75 L-Lysine (98%) 0.3 NaCl 0.3 TiO 2 0.2 Mineral premix 1 0.08 Vitamin mix 2 0.02 Total 100 Analyzed nutrient composition Gross energy, MJ/kg 19.15 Dry matter, % 88.29 Crude protein, % 22.05 Zn, mg/kg 96.48 Calcium, % 0.80 Total phosphorus, % 0.65 Calculated nutrient composition 3 Metabolic energy, MJ/kg 13.31 Digestible energy, MJ/kg 14.60 L-Lysine, % 1.523 Methionine, % 0.398 1 Premix supplied per kilogram of complete diet: Cu, 20 mg; Fe, 104 mg; Mn, 12 mg; Zn 64 mg; I, 0.8 mg; Se, 0.4 mg. 2 Premix supplied per kilogram of complete diet: Vitamin A, 6,450 IU; Vitamin D 3 , 1,520 IU; Vitamin E, 39.53 IU; Vitamin K 3 , 2 mg; Vitamin B 1 , 1.61 mg; Vitamin B 2 , 5 mg; Vitamin B 6 , 2.55 mg; Vitamin B 12 , 20 µg; D-biotin, 120 µg; Niacin, 19.6 mg; D-pantothenic acid, 12.06 mg; Folic acid, 0.99 mg; Ethoxyquin, 0.1 mg. 3 Nutrient levels were calculated according to the guidelines of the National Research Council (NRC, 2012) ETEC K88 challenge To guarantee that the piglets were satiated and bacterial colonization could occur effectively, feeding was halted at 9 p.m. on the day preceding inoculation and resumed 30 min prior to inoculation. On days 8, 9, and 10 post-weaning, piglets in the NC, PC, and ZnCA groups received 10 mL of an oral ETEC K88 (O149: K91: K88ac; toxins LT, STa, and STb) suspension (3×10 10 CFU/mL) twice daily (9 a.m. and 3 p.m.). Conversely, piglets in the CON group received 10 mL of normal saline orally. Growth performance and diarrhea scores The BW was measured on the first and last day of the trial. Meanwhile, feed consumption was accurately recorded in each pen at 9 a.m. Growth performance was evaluated based on the feed efficiency (G:F) using the average daily gain (ADG) and average daily feed intake (ADFI) data. Furthermore, fecal consistency was assessed daily, throughout the study, by trained personnel. These assessments were based on visual examination and followed the grading system established by Atarashi et al. [ 25 ], as follows: 0, normal feces; 1, moist or soft feces; 2, thick liquid feces or mild diarrhea; and 3, watery feces and severe diarrhea. Sample collection During this study, 1 kg samples of the feed from each treatment group were individually obtained and preserved at -20℃. On days 19, 20, and 21, fecal samples were collected through rectal stimulation and preserved at -20°C for subsequent analysis. Prior to slaughter (day 21), blood samples were randomly collected from the jugular vein of one piglet per pen via venipuncture and added to Vacutainer coagulation tubes. All blood specimens were allowed to clot at ambient temperature; subsequently, they were centrifuged at 3500 × g and 4°C for 15 min. The serum was collected and placed in trace element-free tubes before freezing at -80℃ for subsequent analysis. On day 21, the piglets from which blood samples had been collected were selected and euthanized. The weights of the heart, liver, spleen, and kidneys were calculated after slaughter. Samples of hair, the longus dorsi muscle, the right lateral lobe of the liver, the jejunum, and the right kidney were obtained for Zn analysis. The tissues were washed with phosphate-buffered saline (PBS), rapidly frozen in liquid nitrogen, and preserved at -80°C. The small intestine was separated into three distinct regions: the duodenum, jejunum, and ileum. Tissues from the central portions of the duodenum, jejunum, and ileum were collected and washed with PBS after removing the intestinal contents. Histological samples, measuring 0.5 cm, were fixed in a 4% paraformaldehyde solution for 24 h before further morphological examination. Mucosal scrapings were gently collected from the remaining jejunal tissue using a sterile scalpel. The contents of the jejunum were also collected and immediately immersed in liquid nitrogen for preservation at -80°C before subsequent analysis. Intestinal histomorphometry Tissue samples from the duodenum, jejunum, and ileum — which had been preserved in 4% paraformaldehyde — were dehydrated, cleared, and embedded in paraffin. Following this, the samples were sliced into 4-µm sections and stained using hematoxylin and eosin (H&E). Morphological observations and measurements, with a special emphasis on villus height and crypt depth, were carried out using a light microscope (RVL-100-G, Echo Global Logistics, Inc., California, USA). Serum analyses The enzymatic activity of alkaline phosphatase (AKP), glutamic-pyruvic transaminase (ALT/GPT), glutamic-oxaloacetic transaminase (AST/GOT), and diamine oxidase (DAO) and the levels of nitric oxide (NO) and D-lactic acid (D-LA) in the serum were accurately measured using biochemical methods and commercial kits, strictly adhering to the manufacturer’s instructions. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were quantified using the enzyme-linked immunosorbent assay (ELISA) technique. Additionally, a sandwich ELISA kit was employed to detect the endotoxin content in the serum. Both the biochemical reagents and ELISA kits were sourced from Nanjing Jiancheng Technology Co., Ltd. (Nanjing, China). Nutrient digestibility Samples of feed and feces were dried at 65°C for 72 h and then ground to a powder, which was passed through a 1-mm sieve. This processed powder was used to analyze the apparent total tract digestibility (ATTD) of nutrients in the feed. The apparent total tract digestibility (ATTD) of the nutrients — including crude protein (CP), dry matter (DM), and gross energy (GE) — was accurately assessed by utilizing the indigestible marker method, with 0.2% TiO 2 employed as an exogenous indicator. The DM and CP content of both the feces and feed were meticulously analyzed using methods 930.15 and 990.03, respectively, outlined by the Association of Official Analytical Chemists [ 26 ]. Meanwhile, the gross energy (GE) values were determined by employing an adiabatic bomb calorimeter (Kalorimeter C6000 prozesso, IKA, Staufen, Germany). The elemental content of Zn and titanium (Ti) was determined using the AOAC method 985.0 and a spectroscope (Optima 5300 DV ICP-OES, PerkinElmer, MA, USA). The ATTD of the nutrients was then computed based on the following formula.: Zn status The concentrations of Zn were accurately determined in various samples, including the serum, feed, feces, hair, longus dorsi muscle, jejunum, liver, and kidney. Prior to analysis, all samples underwent wet digestion with a mixture of nitric acid and perchloric acid (4:1). Subsequently, the samples were diluted with ultra-pure H 2 O and examined using a spectroscope (Optima 5300 DV ICP-OES, PerkinElmer, MA, USA). The procedure involved weighing 0.5 g of lyophilized solid samples or 0.5 mL of liquid samples and then mixing them with 12 mL of nitric acid and 3 mL of perchloric acid. The samples were then digested in an adjustable electric furnace at 120°C for 0.5 h, 180°C for 24 h, and 220°C until the mixtures became colorless and transparent. For Zn analysis, the liver, feed, and fecal samples were diluted with ultrapure water to reach a final volume of 25 mL. Meanwhile, the longissimus dorsi, kidney, jejunum, and hair samples were diluted to a final volume of 10 mL, and serum samples were diluted to a final volume of 5 mL. RNA extraction, cDNA synthesis, and real-time quantitative polymerase chain reaction (RT-qPCR) Total RNA was isolated from 50 mg of kidney, liver, and jejunum mucosa tissues using the Freezol reagent (Vazyme, Nanjing, China). The mRNA concentrations were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized using a qPCR kit (Vazyme, Nanjing, China). RT-qPCR was conducted in a reaction volume of 20 µL, employing the SYBR qPCR Master Mix (Vazyme, Nanjing, China), on a quantitative fluorescence PCR instrument (Analytik Jena, Jena, Germany). The thermocycler protocol consisted of an initial denaturation step at 95℃ for 30 s, followed by 40 cycles of denaturation at 95℃ for 3 s and annealing/extension at 60℃ for 30 s. Melting curve analysis was conducted to assess the specificity of the amplified fragments using Dissociation Curves v1.0 software (PE Applied Biosystems). Each experimental sample was assayed using four technical replicates. The reference gene was glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ), and the relative mRNA expression of the genes of interest was assessed using the 2- ΔΔCt method. Here, the ΔCt value represents the difference between the Ct values of the target genes and the housekeeping gene. All primers, designed using sequences obtained from the National Center for Biotechnology Information (NCBI) database, are listed in Table S1 . Statistical analyses Each pen served as the experimental unit for assessing variations in growth performance and fecal scores among the piglets. Conversely, individual piglets were considered as the experimental units for analyzing intestinal morphology, serum parameters, nutrient digestibility, and mRNA expression levels. Experimental data were analyzed with a one-way ANOVA by utilizing the Statistics Analysis System (SAS, SAS Institute, Inc., version 9.4, Cary, NC), and Tukey’s test was employed for post hoc analysis. All experimental outcomes were reported as the mean ± standard error of the mean (SEM). Significance was determined at P < 0.05, while a trend was noted when 0.05 < P < 0.1. Results Growth performance, fecal scores, and organ index The growth performance and fecal scores of the weaned piglets are summarized in Table 2 . Initially, the BW of piglets was comparable across all treatment groups. Over the 3-week period following weaning, piglets from the ZnCA group exhibited a significantly higher final BW ( P = 0.021), ADG ( P = 0.026), and G:F ratio ( P = 0.026) than those from the NC group. Interestingly, there were no notable variances among the other treatment groups ( P > 0.05). Furthermore, no obvious variations in the ADFI were observed among the treatment groups. Table 2 Effects of dietary ZnCA supplementation on growth performance and fecal scores in piglets 1 Parameters Dietary treatments 2 SEM 3 P -value CON NC PC ZnCA Initial BW, kg 7.76 7.77 7.85 7.74 0.194 0.998 Final BW, kg 14.58 b 13.15 b 14.92 b 15.63 a 0.317 0.031 ADG, g 346 ab 273 b 361 ab 398 a 15.687 0.026 ADFI, g 620 557 646 660 23.259 0.429 G:F 0.28 ab 0.25 b 0.28 ab 0.30 a 0.006 0.043 Diarrhea score 0.79 ab 0.99 a 0.58 b 0.58 b 0.059 0.024 1 Data represent the mean of six replicate pens per treatment. 2 CON: basal diet + saline solution; NC: basal diet + ETEC K88 challenge; PC: basal diet + 2500 mg/kg of Zn (ZnO) + ETEC K88 challenge; ZnCA: basal diet + 1600 mg/kg of Zn (ZnCA) + ETEC K88 challenge. 3 SEM: standard error of the mean. a, b Means with different superscripts within the same row showed a significant difference ( P < 0.05). BW, body weight; ADG, average daily gain; ADFI, average daily feed intake; G:F, gain-to-feed ratio. Both the ZnCA ( P = 0.042) and PC ( P = 0.040) groups had significantly lower fecal scores than the NC group. However, no significant difference was noted between piglets in the CON group and those in the other treatment groups. The impact of dietary ZnCA supplementation on the organ indexes of piglets is presented in Table S2 . The weights of the kidneys, spleen, heart, and liver remained unaffected by the dietary treatments ( P > 0.05). Intestinal health (intestinal histomorphometry, ATTD of nutrients, and gut barrier) As depicted in Fig. 1 and Table 3 , there was no statistically significant variance in duodenum morphology across the four groups ( P > 0.05). In both the ileum and the jejunum, the ZnCA group demonstrated a higher villus height and villus/crypt ratio as well as a lower crypt depth than the CON and NC groups ( P < 0.05). The PC group demonstrated an increased villus/crypt ratio in the jejunum and villus height in the ileum along with a decreased crypt depth in the jejunum when compared with the CON and NC groups ( P < 0.05). Notably, the NC group exhibited a significantly lower villus/crypt ratio ( P = 0.033) and higher villus height in the jejunum ( P = 0.020) than the CON group. Table 3 Effects of dietary ZnCA supplementation on intestinal histomorphometry in piglets 1 Duodenum Parameters Dietary treatments 2 SEM 3 P -value CON NC PC ZnCA Villus height, µm 318.46 296.76 315.67 309.89 4.442 0.428 Crypt depth, µm 184.89 179.11 178.93 176.72 3.322 0.872 Villus/crypt ratio 1.72 1.68 1.79 1.79 0.024 0.316 Jejunum Villus height, µm 301.57 b 284.73 b 307.03 b 371.83 a 4.678 < 0.001 Crypt depth, µm 128.77 a 131.15 a 101.19 b 109.69 b 2.069 < 0.001 Villus/crypt ratio 1.56 c 1.42 d 2.05 b 2.48 a 0.045 < 0.001 Ileum Villus height, µm 250.12 c 230.74 d 270.65 b 289.66 a 2.876 < 0.001 Crypt depth, µm 109.34 ab 100.05 b 110.79 ab 118.85 a 1.808 0.003 Villus/crypt ratio 2.33 b 2.34 b 2.46 ab 2.51 a 0.020 0.001 1 Data represent the mean of six replicate pens per treatment. 2 CON: basal diet + saline solution; NC: basal diet + ETEC K88 challenge; PC: basal diet + 2500 mg/kg of Zn (ZnO) + ETEC K88 challenge; ZnCA: basal diet + 1600 mg/kg of Zn (ZnCA) + ETEC K88 challenge. 3 SEM: standard error of the mean. a−d Means with different superscripts within the same row showed a significant difference ( P 0.05). The addition of ZnO and ZnCA increased the digestibility of DM and GE ( P < 0.05). Moreover, the digestibility of crude protein in the NC group was notably lower than that in the CON group ( P = 0.001). Table 4 Effects of dietary ZnCA supplementation on the ATTD of nutrients in piglets 1 ATTD, % Dietary treatments 2 SEM 3 P -value CON NC PC ZnCA Dry matter 75.357 b 75.273 b 79.6583 a 79.710 a 0.763 0.029 Crude protein 68.837 a 67.083 b 69.817 a 69.818 a 0.267 < 0.001 Gross energy 71.718 b 66.866 b 77.332 a 77.3214 a 1.088 < 0.001 1 Data represent the mean of six replicate pens per treatment. 2 CON: basal diet + saline solution; NC: basal diet + ETEC K88 challenge; PC: basal diet + 2500 mg/kg of Zn (ZnO) + ETEC K88 challenge; ZnCA: basal diet + 1600 mg/kg of Zn (ZnCA) + ETEC K88 challenge. 3 SEM: standard error of the mean. a, b Means with different superscripts within the same row showed a significant difference ( P < 0.05). ATTD, apparent total tract digestibility. The serum concentrations of D-LA, DAO, and endotoxin — which are indicators of intestinal permeability — were assayed (Table 5 ). No significant differences in the serum levels of DAO were observed among the treatment groups ( P > 0.05). However, the levels of D-LA and endotoxin were significantly higher in the CON group than in the other experimental groups ( P 0.05). Meanwhile, in comparison to the PC group, the ZnCA group demonstrated reduced serum levels of D-LA and endotoxin ( P < 0.05). Table 5 Effects of dietary ZnCA supplementation on intestinal barrier function in piglets 1 Serum indicators of intestinal barrier function Parameters Dietary treatments 2 SEM 3 P -value CON NC PC ZnCA D-LA, µmol/mL 3.51 bc 4.86 a 4.02 b 3.22 c 0.159 < 0.001 DAO, U/L 152.549 129.79 146.67 150.16 5.908 0.547 Endotoxin, ng/mL 316.14 c 535.15 a 439.38 b 313.79 c 20.616 < 0.001 Expression of intestinal barrier-related genes in the jejunum mucosa ZO-1 1.058 a 0.76 b 0.930 ab 1.116 a 0.039 0.007 MUC-2 1.029 bc 1.237 ab 0.884 c 1.363 a 0.043 < 0.001 Occludin 1.018 b 1.141 b 1.093 b 1.516 a 0.047 < 0.001 Claudin-1 1.122 b 0.225 c 1.203 b 2.811 a 0.104 < 0.001 Claudin-2 1.021 b 1.449 a 1.086 b 1.125 b 0.028 < 0.001 1 Data represent the mean of six replicate pens per treatment. 2 CON: basal diet + saline solution; NC: basal diet + ETEC K88 challenge; PC: basal diet + 2500 mg/kg of Zn (ZnO) + ETEC K88 challenge; ZnCA: basal diet + 1600 mg/kg of Zn (ZnCA) + ETEC K88 challenge. 3 SEM: standard error of the mean. a−c Means with different superscripts within the same row showed a significant difference ( P < 0.05). D-LA, D-lactic acid; DAO, diamine oxidase; ZO-1 , zonula occludens-1; MUC-2 , mucin 2. The relative mRNA levels of tight junction (TJ) proteins within the jejunum were also examined (Table 5 ). Notably, ZnCA supplementation significantly upregulated the mRNA levels of mucin 2 ( MUC-2 ), Occludin , and Claudin-1 in the jejunum. Interestingly, no notable differences in the mRNA levels of TJ proteins were detected between the CON and PC groups ( P > 0.05). However, the NC group exhibited lower mRNA levels of Claudin-1 and ZO-1 , as well as higher mRNA levels of Claudin-2 ( P > 0.05). Anti-inflammatory activity The anti-inflammatory activity of dietary ZnCA supplementation is detailed in Table 6 . Initially, we assessed serum liver function markers, including AKP, AST/GOT, and ALT/GPT. However, no notable variances were detected in the serum levels of AST/GOT and ALT/GPT across all groups ( P > 0.05). Table 6 Effects of dietary ZnCA supplementation on anti-inflammatory activity in piglets 1 Serum liver function Parameters Dietary treatments 2 SEM 3 P -value CON NC PC ZnCA AKP, U/L 33.76 ab 19.29 c 43.44 a 32.52 b 1.762 < 0.001 AST/GOT, U/L 15.91 10.60 14.40 12.80 1.017 0.289 ALT/GPT, U/L 25.84 27.10 24.09 29.36 1.101 0.408 Serum cytokines IL-6, pg/mL 111.93 b 158.29 a 122.79 b 108.47 b 4.866 < 0.001 IL-1 β , pg/mL 301.68 c 437.28 a 387.02 ab 312.13 bc 14.741 < 0.001 TNF- α , pg/mL 58.68 c 109.99 a 66.64 bc 83.55 b 4.942 < 0.001 NO, µmol/L 106.26 ab 130.12 a 77.51 b 77.11 b 7.018 0.007 Inflammatory factors in the jejunum mucosa IL-6 1.123 bc 2.376 a 1.170 b 0.5719 c 0.109 < 0.001 IL-1β 1.018 b 2.391 a 0.956 b 1.018 b 0.092 < 0.001 TNF-α 1.053 b 4.008 a 1.461 b 1.740 b 0.222 < 0.001 iNOS 1.087 c 3.328 a 2.092 b 1.250 c 0.140 < 0.001 Inflammatory factors in the liver IL-6 1.068 b 2.280 a 1.106 b 0.923 b 0.112 < 0.001 IL-1β 1.084 b 3.599 a 1.652 b 1.116 b 0.172 < 0.001 TNF-α 1.032 b 3.133 a 1.092 b 1.136 b 0.155 < 0.001 iNOS 1.161 b 3.413 a 1.844 b 1.083 b 0.158 < 0.001 1 Data represent the mean of six replicate pens per treatment. 2 CON: basal diet + saline solution; NC: basal diet + ETEC K88 challenge; PC: basal diet + 2500 mg/kg of Zn (ZnO) + ETEC K88 challenge; ZnCA: basal diet + 1600 mg/kg of Zn (ZnCA) + ETEC K88 challenge. 3 SEM: standard error of the mean. a−c Means with different superscripts within the same row showed a significant difference ( P < 0.05). IL-6, interleukin-6; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; NO, nitric oxide; AKP, alkaline phosphatase; AST/GOT, aspartate aminotransferase; ALT/GPT, alanine aminotransferase; iNOS, inducible nitric oxide synthase. Interestingly, the serum levels of IL-6, NO, IL-1β, and TNF-α were notably decreased after the NC treatment ( P 0.05). When compared to CON treatment, PC treatment produced higher serum concentrations of IL-1β ( P 0.05). Consistent with the serum cytokine levels, the mRNA levels of inflammatory cytokines — such as IL-6 , iNOS , TNF-α , and IL-1β — were markedly elevated in the jejunum mucosa and liver following NC treatment ( P 0.05). Interestingly, the PC group demonstrated elevated mRNA levels of IL-6 and iNOS when compared with the ZnCA group ( P < 0.05). Zn homeostasis The impact of dietary ZnCA supplementation on the homeostasis of trace metals in piglets is shown in Table 7 . Notably, a positive correlation was observed between the Zn concentration in the feces, liver, and kidney and the Zn concentration in the feed ( P < 0.05). However, no notable differences in serum Zn concentrations and the Zn concentrations in the longissimus muscle and hair were observed between the ZnCA group and the other three treatment groups ( P > 0.05). Notably, the Zn concentration in the jejunum was lower in the NC group than in the other treatment groups ( P > 0.05). Table 7 Effects of dietary ZnCA supplementation on the homeostasis of trace metals in piglets 1 Zn concentration Parameters 4 Dietary treatments 2 SEM 3 P -value CON NC PC ZnCA Feed, mg/kg 128.93 c 128.93 c 2508.30 a 1607.71 b 306.27 < 0.001 Feces, g/kg 1.644 c 1.548 c 24.815 a 15.748 b 2.251 < 0.001 Serum, mg/L 2.842 b 3.208 b 5.558 a 4.781 ab 0.332 0.003 Liver, mg/kg 201.91 c 197.78 c 1346.57 a 907.44 b 108.26 < 0.001 Kidney, mg/kg 109.36 c 114.62 c 396.31 a 202.94 b 29.088 < 0.001 Jejunum, mg/kg 196.66 a 130.00 b 199.64 a 215.89 a 8.604 < 0.001 Longissimus muscle, mg/kg 73.608 77.103 73.627 74.052 1.683 0.878 Hair, mg/kg 1192.88 1086.23 1092.47 1090.28 23.001 0.298 Cu concentration Feed, mg/kg 24.053 24.053 23.750 24.337 0.602 0.759 Feces, mg/kg 272.37 b 246.84 b 377.08 a 285.32 b 11.334 < 0.001 Serum, mg/L 3.161 4.054 3.257 3.552 0.138 0.085 Liver, mg/kg 24.700 ab 31.117 a 19.183 b 24.754 ab 1.165 0.001 Kidney, mg/kg 40.650 bc 32.792 c 121.895 a 75.102 b 8.577 < 0.001 Jejunum, mg/kg 18.192 a 13.658 bd 17.029 ab 11.613 cd 0.758 0.002 Longissimus muscle, mg/kg 7.879 a 7.250 ab 5.329 b 5.613 ab 0.351 0.014 Hair, mg/kg 8.618 a 7.521 b 7.137 b 7.691 ab 0.164 0.004 Fe concentration DM of feed, mg/kg 309.67 309.67 323.29 322.94 2.927 0.137 DM of feces, mg/kg 2933.61 b 2954.32 b 4163.78 a 3405.58 ab 153.12 0.005 Serum, mg/L 12.283 12.987 11.904 10.627 0.351 0.106 Liver, mg/kg 135.59 130.53 148.78 144.33 7.524 0.884 Kidney, mg/kg 136.11 131.19 135.38 134.88 4.182 0.986 Jejunum, mg/kg 228.90 a 198.54 ab 181.01 b 174.77 b 7.055 0.019 Longissimus muscle, mg/kg 157.38 a 125.35 ab 110.55 b 93.56 b 6.294 < 0.001 Hair, mg/kg 615.78 471.93 648.56 523.88 41.08 0.421 Mn concentration Feed, mg/kg 43.034 43.034 47.247 45.890 0.798 0.139 Feces, mg/kg 443.87 442.88 536.42 470.94 16.183 0.132 Liver, mg/kg 6.313 5.746 4.501 5.375 0.332 0.284 Kidney, mg/kg 6.542 4.985 5.330 4.815 0.264 0.077 Hair, mg/kg 18.275 17.275 17.333 16.521 0.862 0.925 1 Data represent the mean of six replicate pens per treatment. 2 CON: basal diet + saline solution; NC: basal diet + ETEC K88 challenge; PC: basal diet + 2500 mg/kg of Zn (ZnO) + ETEC K88 challenge; ZnCA: basal diet + 1600 mg/kg of Zn (ZnCA) + ETEC K88 challenge 3 SEM: standard error of the mean. 4 With the exception of the serum, the levels in all other samples were calculated based on DM values. a−d Means with different superscripts within the same row showed a significant difference ( P 0.05). Interestingly, the PC group exhibited higher levels of Cu and Fe in the feces than the other treatment groups ( P < 0.05). A trend towards increased serum Cu concentrations was also detected in the NC treatment group ( P = 0.085). In comparison to the CON group, the PC group exhibited elevated levels of Cu in the kidneys and reduced levels of Cu in the longissimus muscle and hair ( P < 0.05). Additionally, the NC group demonstrated higher hepatic levels of Cu ( P 0.05). Notably, the ZnCA treatment group had lower Cu levels in the jejunum than the CON and PC treatment groups ( P 0.05). The CON group exhibited higher Fe levels in the jejunum and longissimus muscle than the PC and ZnCA groups ( P 0.05), the Mn concentration in the kidneys tended to show variations ( P = 0.077). The relative mRNA levels of Zn/iron-regulated transporter-like 5 ( ZIP5 ) remained unaffected ( P > 0.161) in the kidneys, liver, and jejunum mucosa, as presented in Table 8 . Meanwhile, the mRNA expression of ZIP4 in the jejunum mucosa was altered in both the NC and PC groups ( P < 0.001). Notably, the NC group exhibited the highest relative mRNA levels of ZIP14 in the jejunum mucosa and liver ( P 0.05). However, the PC group demonstrated the highest mRNA levels of ZNT1 across all these three tissues ( P < 0.05). The ZnCA group exhibited similar mRNA levels of ZNT1 in the jejunum mucosa as the PC group, but the hepatic and renal expression levels of this gene were lower in the ZnCA group ( P < 0.05). Furthermore, significant variances in the mRNA expression of metallothionein 1 ( MT1 ), MT2 , and MT3 were observed among the four dietary treatments ( P < 0.05). Specifically, the PC treatment group showed the highest levels of these genes, with the ZnCA group following closely behind. In contrast, the expression of these genes in the kidneys, liver, and jejunum mucosa was lower in the CON and NC treatment groups ( P 0.05). Table 8 Effects of dietary ZnCA supplementation on the relative mRNA levels of specific Zn transporters in piglets 1 Jejunum mucosa Genes Dietary treatments 2 SEM 3 P -value CON NC PC ZnCA ZIP4 1.039 b 2.626 a 0.197 c 0.609 bc 0.142 < 0.001 ZIP5 1.060 1.105 1.064 1.065 0.043 0.984 ZIP14 1.145 b 2.628 a 1.734 b 0.904 b 0.142 < 0.001 ZNT1 1.048 b 0.892 b 1.273 a 1.401 a 0.044 < 0.001 MT1 1.042 c 0.513 c 158.632 a 57.965 b 9.174 < 0.001 MT2 1.037 c 0.957 c 102.363 a 53.122 b 3.962 < 0.001 MT3 1.359 b 1.196 b 747.351 a 103.034 b 35.545 < 0.001 Liver ZIP5 0.989 1.046 1.059 1.052 0.392 0.929 ZIP8 1.204 1.413 1.376 1.001 0.074 0.160 ZIP14 1.009 b 2.233 a 1.150 b 1.018 b 0.095 < 0.001 ZNT1 1.193 c 1.274 c 3.251 a 2.198 b 0.117 < 0.001 MT1 1.055 c 0.618 c 35.232 a 14.245 b 1.505 < 0.001 MT2 1.050 c 1.531 c 21.362 a 8.880 b 1.044 < 0.001 MT3 1.284 c 0.935 c 284.354 a 66.097 b 17.453 < 0.001 Kidney ZIP5 1.057 1.297 1.030 1.050 0.041 0.161 ZNT1 1.071 b 1.212 b 2.011 a 1.062 b 0.085 < 0.001 MT1 1.265 c 1.754 c 34.989 a 8.809 b 1.535 < 0.001 MT2 1.142 c 1.518 c 22.805 a 7.588 b 1.089 < 0.001 MT3 1.033 c 1.132 c 7.297 a 4.652 b 0.372 < 0.001 1 Data represent the mean of six replicate pens per treatment. 2 CON: basal diet + saline solution; NC: basal diet + ETEC K88 challenge; PC: basal diet + 2500 mg/kg of Zn (ZnO) + ETEC K88 challenge; ZnCA: basal diet + 1600 mg/kg of Zn (ZnCA) + ETEC K88 challenge. 3 SEM: standard error of the mean. a−c Means with different superscripts within the same row showed a significant difference ( P < 0.05). ZIP4 , Zn/iron-regulated transporter-like 4; ZIP5 , Zn/iron-regulated transporter-like 5; ZIP8 , Zn/iron-regulated transporter-like 8; ZIP14 , Zn/iron-regulated transporter-like 14; MT1 , metallothionein 1; MT2 , metallothionein 2; MT3 , metallothionein 3; ZNT1 , Zn transporter 1. Discussion Due to the combined stress of weaning, incomplete intestinal development, and the influence of the rearing environment, piglets frequently develop PWD. This condition can lead to diarrhea, decreased feed intake, growth retardation, and even mortality among weaned piglets [ 27 ]. In context of the dual challenges arising from the ban on AGPs and the limitations imposed upon Zn supplementation, organic Zn has emerged as a promising solution for PWD management [ 28 ]. The impact of pharmacological doses of ZnO on nutrient digestibility, diarrhea incidence, and growth performance in weaned piglets has been studied extensively [ 29 – 31 ]. In the current study, dietary ZnO supplementation (2500 mg/kg) significantly reduced the diarrhea score in weaned piglets challenged with ETEC K88, but it did not improve their growth performance. However, these findings contrast with those reported by Lei and Kim [ 17 ], who observed that pharmacological levels of ZnO enhance growth performance in weaned piglets exposed to ETEC K88. This discrepancy could arise because Lei and Kim administered a high-Zn diet to the piglets for 21 d prior to the ETEC K88 challenge. As expected, in the present study, piglets who received a diet containing an additional 1600 mg/kg of Zn in the form of ZnCA exhibited a higher ADG and G:F ratio as well as a lower fecal score after the ETEC K88 challenge than the piglets who were fed a basal diet. These results confirmed that incorporating a small amount of ZnCA into the diet can promote growth and reduce diarrhea in weaned piglets, offering results comparable to those achieved with pharmacological doses of ZnO. After weaning and exposure to ETEC K88, piglets often experience alterations in the structure and functionality of the intestinal tract. These changes primarily manifest in the form of villus atrophy and crypt hyperplasia [ 32 ]. Previous research has established that pharmacological ZnO supplements can improve the intestinal structure in weaned piglets [ 16 , 33 , 34 ]. In accordance with these results, our findings demonstrated that both 2500 mg/kg of conventional ZnO and 1600 mg/kg of ZnCA can improve the ratio of villus height to crypt depth and reduce crypt depth in the jejunum. Moreover, ZnCA supplementation was observed to enhance villus height and the villus/crypt ratio in the ileum, while also decreasing crypt depth. Meanwhile, ZnO supplementation also increased the villus/crypt ratio in the ileum. Notably, 1600 mg/kg ZnCA improved the villus/crypt ratio in the jejunum to a greater degree than the pharmacological doses of ZnO, indicating a potential enhancement of absorption capacity in the intestine following ZnCA supplementation. The villi of the small intestine play a key role in nutrient absorption. Hence, PWD typically reduces the nutrient absorption capacity in affected animals [ 32 ]. In this study, piglets challenged with ETEC K88 and treated with ZnCA exhibited an improvement in intestinal structure, resulting in the increased ATTD of DM, CP, and GE. Similar findings have also been reported by Lei and Kim [ 17 ]. This increase in the digestibility of DM, CP, and GE observed in ZnCA-treated piglets suggests that improved nutrient digestibility is at least partly responsible for enhanced growth performance in these animals [ 17 ]. This potential increase in the rate of nutrient digestion can be attributed to improvements in intestinal tract structure [ 35 ]. Furthermore, previous studies conducted by Hedemann et al. [ 36 ] and Hu et al. [ 37 ] indicate that pharmacological doses of ZnO can enhance the activity of digestive enzymes in the intestines of weaned piglets. Therefore, increased enzyme activity may also contribute to the accelerated rate of nutrient digestion observed in these piglets. The onset of PWD is intricately linked to intestinal permeability [ 38 , 39 ]. TJ proteins — such as Occludin, Claudin-1, and ZO-1 — play an important role in regulating the permeability of intestinal epithelial cells and maintaining barrier function [ 40 ]. In this study, we observed that infection with ETEC significantly reduced the mRNA levels of Claudin-1 and ZO-1 in the jejunal mucosa of weaned piglets. However, supplementation with ZnCA could alleviate this damage and partially increase the mRNA expression of TJ proteins. Consistent with our findings, Xie et al. [ 41 ] discovered that the addition of polysaccharide-Zn complexes to the diet can upregulate the mRNA expression of TJ proteins. Claudin-2, a pore-forming protein, is known to disrupt the TJ barrier [ 42 ]. Notably, in our study, the mRNA levels of Claudin-2 exhibited an inverse trend compared to those of Claudin-1 , echoing the similar antagonistic effects reported by Jung et al. [ 43 ]. As reliable indicators of intestinal permeability, the levels of D-LA, DAO, and endotoxin provide key insights into intestinal barrier function [ 44 ]. An investigation conducted by Xu et al. [ 45 ] revealed that supplementation with 1500 mg/kg of ZnO can effectively reduce DAO and endotoxin levels in the serum. In our study, we observed that ETEC K88 could significantly upregulate the serum concentrations of D-LA and endotoxin. However, pharmacological doses of ZnO and 1600 mg/kg of ZnCA could mitigate these elevations, with ZnCA demonstrating superior efficacy. These findings suggest that ZnCA, which also reduces the required amount of Zn supplementation, enhances intestinal health in weaned piglets challenged with ETEC K88 through various mechanisms. These mechanisms include improvements in intestinal morphology, augmented nutrient digestibility, and reinforcement of intestinal barrier function. Notably, the efficacy of ZnCA surpasses that of pharmacological ZnO doses in achieving these benefits. After weaning, piglets frequently experience inflammation due to dietary transitions and infections caused by pathogens such as ETEC [ 46 ]. AKP plays a crucial role in detoxification and anti-inflammatory processes following ETEC infections [ 47 ]. In this study, exposure to ETEC K88 in weaned piglets led to decreased levels of AST/GOT and ALT/GPT and significantly suppressed AKP activity in the serum. Conversely, Zn supplementation could enhance AKP activity, likely because AKP is a Zn-containing metalloenzyme and Zn augments its functionality [ 48 ]. Comparable findings have been documented by Liu et al. [ 49 ]. Our previous research demonstrated the anti-inflammatory effects of Zn laurate in mice infected with ETEC K88 [ 50 ]. Therefore, in the present study, we further explored the impact of ZnCA on inflammatory markers in post-weaned piglets challenged with ETEC K88. Akin to pharmacological doses of ZnO, ZnCA notably decreased the mRNA levels of iNOS , IL-6 , TNF-α , and IL-1β in the liver and jejunum mucosa, as well as the levels of NO, IL-6, TNF-α, and IL-1β in the serum of weaned piglets challenged with ETEC K88. Evidence suggests that MCFAs possess immunomodulatory properties and may improve the overall health of weaned piglets [ 51 ]. Consequently, the anti-inflammatory effects of ZnCA on weaned piglets could be attributed to the synergistic actions of MCFAs and Zn. So far, studies on organic and inorganic Zn in weaned piglets have explored the mechanisms of Zn metabolism and the anti-inflammatory effects of Zn independent of each other. Previous studies suggest that Zn can modulate the activity of inflammatory pathways [ 52 ]. These inflammatory processes can, in turn, influence the expression of Zn transporters [ 53 ]. Hence, one key objective of the present study was to examine the relationship between the anti-inflammatory activity of ZnCA and Zn metabolism following ZnCA administration, offering insights into their interrelationships. ZIP4, which is a Zn transporter, is predominantly expressed in the brush border of the intestines and facilitates the absorption of Zn from the intestinal lumen [ 54 ]. In this study, the mRNA levels of ZIP4 were found to be the highest in the NC treatment group and the lowest in the PC treatment group. The downregulation of ZIP4 in the intestinal mucosa of piglets in the PC and ZnCA groups can be attributed to the mechanism of Zn absorption at saturation [ 55 ], wherein the rate of intestinal absorption is inversely correlated with the intake [ 56 ]. Conversely, the overexpression of ZIP4 mRNA in the NC group may be due to the pathogenic effects of ETEC K88 on the intestines, resulting in decreased Zn absorption and a compensatory upregulation of ZIP4 . Nevertheless, alterations in absorption rates do not necessarily reflect changes in total nutrient absorption [ 57 ]. Despite the decreased absorption rate, the Zn content in the jejunum of the PC and ZnCA treatment groups remained significantly higher than that in the jejunum of the CON group. Conversely, the jejunum Zn content in the NC group continued to remain the lowest among all the groups, consistent with previous findings [ 58 ]. ZIP5 , which is integral for Zn uptake from the bloodstream, did not exhibit altered expression levels in the small intestinal mucosa, liver, and kidneys in the present study. This contradicts the results reported by Dalto et al. [ 59 ], and these differences are likely due to variations in the feeding duration between the two studies. ZIP8 transcription is directly modulated by the NF-κB signaling pathway [ 60 ]. However, our findings revealed no notable changes in ZIP8 mRNA expression in the liver under the different treatment conditions, possibly due to the tissue-specific nature of this mRNA [ 61 ]. Unlike ZIP8, ZIP14 can suppress NF-κB signaling via a negative feedback loop [ 62 ]. In the present study, ZIP14 mRNA levels showed a significant increase in the NC group, in contrast to the downregulation of ZIP14 in the PC and ZnCA groups. This suggests that ZIP14 expression may primarily be governed by inflammatory factors and not Zn regulation [ 63 ]. ZNT1 is a basolateral membrane transporter for Zn in enterocytes. The mRNA expression of ZNT1 is positively correlated with tissue Zn levels. Following excessive Zn intake, ZNT1 expression increases to facilitate Zn excretion. Therefore, it is evident that ZNT1 expression is primarily regulated by Zn levels. Additionally, Nishito and Kambe [ 64 ] delineated the cooperative roles of ZNT1 and MT in maintaining cellular Zn homeostasis. In the present study, we observed similar trends in MT1 , MT2 , and MT3 expression, consistent with the changes in ZNT1 expression. Wang et al. [ 65 ] reported that the kidneys play a crucial role in Zn excretion when dietary Zn intake becomes high. However, in the present study, the mRNA levels of ZNT1 in the kidneys of weaned piglets subjected to ZnCA treatment were not significantly different from those in the CON group. Consequently, unlike the administration of 2500 mg/kg of ZnO, supplementation with 1600 mg/kg of ZnCA appeared to have minimal impact on Zn homeostasis in the kidneys. The body maintains Zn homeostasis through both a rapid exchange pool (encompassing the serum, intestines, liver, and kidneys) and a slow exchange pool (including skeletal muscles, bones, and hair) [ 66 ]. Our findings revealed that ZnO and ZnCA primarily influence the Zn content within the tissues encompassing the rapid exchange pool. Notably, even under relatively limited Zn supplementation, jejunum Zn levels were comparable between ZnCA treatment and ZnO treatment. These findings pointed to a higher absorption rate for organic Zn than for inorganic Zn, echoing the findings reported by Oh et al. [ 28 ]. Nevertheless, the regulation of Zn homeostasis can affect the metabolism of other minerals, such as Cu and Fe. Matte et al. [ 67 ] observed that an increase in dietary Zn intake can reduce Cu efflux from intestinal cells. Consistent with these findings, in the PC group, the concentration of Cu was elevated in the jejunum but reduced in the liver and serum. Additionally, our study revealed that high-Zn diets can enhance the excretion of Cu and Fe. This conclusion was corroborated by the findings reported by Foligné et al. [ 68 ]. Elevations in Zn content likely contribute to this phenomenon by upregulating the expression of MT [ 69 ], which can bind to Fe and Cu and thereby limit their transport into the blood and liver [ 70 ]. Ultimately, the Cu and Fe bound to MT can be eliminated via the shedding of intestinal cells [ 59 ]. Notably, supplementation with 1600 mg/kg ZnCA only decreased duodenal Cu and longissimus dorsi Fe levels in weaned piglets, indicating that ZnCA had a significantly lower metabolic impact on Cu and Fe than pharmacological levels of ZnO. Finally, neither dietary manipulation nor toxin exposure was found to produce any discernible effect on Mn metabolism in this study, suggesting that high Zn supplementation may not alter Mn metabolism. Conclusions The findings of the current study demonstrate that incorporating 1600 mg/kg of ZnCA into the diet of weaned piglets can lead to improved growth performance and nutrient digestibility, while also reducing the incidence of diarrhea and inflammatory responses triggered by ETEC K88 infection. These beneficial effects are comparable to those achieved with pharmacological doses of ZnO. Furthermore, in terms of improving intestinal health and Zn homeostasis, the efficacy of 1600 mg/kg ZnCA surpasses that of pharmacological doses of ZnO in weaned piglets challenged with ETEC K88. Pharmacological doses of ZnO not only increase Zn excretion but also disrupt the absorption of copper and iron. However, our study revealed that the ZnCA (1600 mg/kg) can achieve reduced Zn substitution while still enhancing Zn deposition in weaned piglets. Our future studies will involve a deeper exploration of the impact of lower ZnCA doses on weaned piglets. Given the current restrictions regarding the pharmacological dosage of ZnO, this study offers valuable insights for reducing Zn excretion and presents a novel approach towards managing PWD and developing antibiotic alternatives for weaned piglets. Abbreviations ZnCA, zinc caproate; ZnO, zinc oxide; PWD, post-weaning diarrhea; ETEC, Escherichia coli ; MCFAs, medium-chain fatty acids; CA, caproic acid; G:F, feed efficiency; ADG, average daily gain; ADFI, average daily feed intake; AKP, alkaline phosphatase; ALT/GPT, glutamic-pyruvic transaminase; AST/GOT, glutamic-oxaloacetic transaminase; DAO, diamine oxidase; NO, nitric oxide; D-LA, D-lactic acid; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; IL-1β, interleukin-1β; ATTD, apparent total tract digestibility; CP, crude protein; DM, dry matter; GE, gross energy; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ZO-1, zonula occludens-1; MUC-2, mucin 2; iNOS, inducible nitric oxide synthase; ZIP4, Zn/iron-regulated transporter-like 4; ZIP5, Zn/iron-regulated transporter-like 5; ZIP8, Zn/iron-regulated transporter-like 8; ZIP14, Zn/iron-regulated transporter-like 14; MT1, metallothionein 1; MT2, metallothionein 2; MT3, metallothionein 3; ZNT1, Zn transporter 1. Declarations Acknowledgements Not applicable. Authors’ contributions †Jilong Xu and Hanzhen Qiao equally contributed to this work: conceptualization, investigation, data curation, writing-original draft; Liping Gan: investigation, project administration; Peng Wang: methodology, resources; Yifeng Zhao: validation, visualization; Zetian Lei: conceptualization, project administration; Yixuan Chou: supervision, project administration; Chenrui Hou: conceptualization; Mengqi Li: methodology; Jinrong Wang: conceptualization, project administration, writing - review & editing, funding acquisition. Funding This study was funded by the National Key Research and Development Program of China (2021YFD1300300), the Innovation Fund of Henan University of Technology (2020ZKCJ25), and the Cultivation Programme for Young Backbone Teachers in Henan University of Technology (21421204). Availability of data and materials The datasets used in the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate All animal experimental protocols employed in this study were thoroughly evaluated and authorized by the Animal Protection and Use Committee of Henan University of Technology, Zhengzhou, China, in strict accordance with the Chinese Guidelines for Animal Welfare. The research was carried out at the Henan Agricultural University Teaching Experimental Field located in Xuchang, China. Consent for publication Not applicable. Competing interests The authors declare that they have no conflict of interest. Author details College of Bioengineering, Henan University of Technology, Lianhua Street 100, Zhengzhou 450001, China; Email: [email protected] References Castro J, Barros MM, Araújo D, Campos AM, Oliveira R, Silva S, et al. Swine enteric colibacillosis: Current treatment avenues and future directions. 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Zinc oxide-montmorillonite hybrid influences diarrhea, intestinal mucosal integrity, and digestive enzyme activity in weaned pigs. Biol Trace Elem Res. 2012;149(2):190-196. Fan P, Tan Y, Jin K, Lin C, Xia S, Han B, et al. Supplemental lipoic acid relieves post-weaning diarrhoea by decreasing intestinal permeability in rats. J Anim Physiol Anim Nutr. 2017;101(1):136-146. Huang C, Song P, Fan P, Hou C, Thacker P, Ma X. Dietary sodium butyrate decreases postweaning diarrhea by modulating intestinal permeability and changing the bacterial communities in weaned piglets. J Nutr. 2015;145(12):2774-2780. Suzuki T. Regulation of intestinal epithelial permeability by tight junctions. Cell Mol Life Sci. 2013;70(4):631-659. Xie C, Zhang Y, Niu K, Liang X, Wang H, Shan J, et al. Enteromorpha polysaccharide-zinc replacing prophylactic antibiotics contributes to improving gut health of weaned piglets. Anim Nutr. 2021;7(3):641-649. Venugopal S, Anwer S, Szászi K. Claudin-2: Roles beyond permeability functions. Int J Mol Sci. 2019;20(22):5655. Jung H, Jun KH, Jung JH, Chin HM, Park WB. The expression of claudin-1, claudin-2, claudin-3, and claudin-4 in gastric cancer tissue. J Surg Res. 2011;167(2):185-191. Wen ZS, Tang Z, Ma L, Zhu TL, Wang YM, Xiang XW, et al. Protective effect of low molecular weight seleno-aminopolysaccharide on the intestinal mucosal oxidative damage. Mar Drugs. 2019;17(1):64. Xu T, Ma X, Zhou X, Qian M, Yang Z, Cao P, et al. Coated tannin supplementation improves growth performance, nutrients digestibility, and intestinal function in weaned piglets. J Anim Sci. 2022;100(5):88. Upadhaya SD, Kim IH. The impact of weaning stress on gut health and the mechanistic aspects of several feed additives contributing to improved gut health function in weanling piglets-A review. Animals. 2021;11(8):33-36. Dong Y, Xia Y, Yin J, Zhou D, Sang Y, Yan S, et al. Optimization, characteristics, and functions of alkaline phosphatase from escherichia coli. Front Microbiol. 2021;12:761189. Min YN, Liu FX, Qi X, Ji S, Ma SX, Liu X, et al. Effects of methionine hydroxyl analog chelated zinc on laying performance, eggshell quality, eggshell mineral deposition, and activities of zn-containing enzymes in aged laying hens. Poultry Sci. 2018;97(10):3587-3593. Liu FF, Azad M, Li ZH, Li J, Mo KB, Ni HJ. Zinc supplementation forms influenced zinc absorption and accumulation in piglets. Animals. 2020;11(1):36. Chen Q, Wang P, Wang J, Xu J, Liu C, Qiao H, et al. Zinc laurate protects against intestinal barrier dysfunction and inflammation induced by Etec in a mice model. Nutrients. 2022;15(1):54. Jackman JA, Boyd RD, Elrod CC. Medium-chain fatty acids and monoglycerides as feed additives for pig production: Towards gut health improvement and feed pathogen mitigation. J Anim Sci Biotechnol. 2020;11:44. Colomar-Carando N, Meseguer A, Company-Garrido I, Jutz S, Herrera-Fernández V, Olvera A, et al. Zip6 transporter is an essential component of the lymphocyte activation machinery. J Immunol. 2019;202(2):441-450. Liu MJ, Bao S, Gálvez-Peralta M, Pyle CJ, Rudawsky AC, Pavlovicz RE, et al. Zip8 regulates host defense through zinc-mediated inhibition of nf-κb. Cell Rep. 2013;3(2):386-400. Andrews GK. Regulation and function of zip4, the acrodermatitis enteropathica gene. Biochem Soc Trans. 2008;36(6):1242-1246. Lönnerdal B. Dietary factors influencing zinc absorption. J Nutr. 2000;130(5):1378-1383. Dalto DB, Audet I, Matte JJ. Impact of dietary zinc: Copper ratio on the postprandial net portal appearance of these minerals in pigs. J Anim Sci. 2019;97(9):3938-3946. Coppen DE, Davies NT. Studies on the effects of dietary zinc dose on 65zn absorption in vivo and on the effects of zn status on 65zn absorption and body loss in young rats. Brit J Nutr. 1987;57(1):35-44. Pieper R, Martin L, Schunter N, Villodre TC, Weise C, Klopfleisch R, et al. Impact of high dietary zinc on zinc accumulation, enzyme activity and proteomic profiles in the pancreas of piglets. J Trace Elem Med Biol. 2015;30:30-36. Dalto DB, Audet I, Roy C, Novais AK, Deschêne K, Goulet K, et al. Effects of dietary zinc oxide levels on the metabolism of zinc and copper in weaned pigs. J Anim Sci. 2023;101:55. Liu MJ, Bao S, Gálvez-Peralta M, Pyle CJ, Rudawsky AC, Pavlovicz RE, et al. Zip8 regulates host defense through zinc-mediated inhibition of nf-κb. Cell Rep. 2013;3(2):386-400. Troche C, Aydemir TB, Cousins RJ. Zinc transporter slc39a14 regulates inflammatory signaling associated with hypertrophic adiposity. Am J Physiol-Endoc M. 2016;310(4):258-268. Aydemir TB, Troche C, Kim J, Kim MH, Teran OY, Leeuwenburgh C, et al. Aging amplifies multiple phenotypic defects in mice with zinc transporter zip14 (slc39a14) deletion. Exp Gerontol. 2016;85:88-94. Aydemir TB, Chang SM, Guthrie GJ, Maki AB, Ryu MS, Karabiyik A, et al. Zinc transporter zip14 functions in hepatic zinc, iron and glucose homeostasis during the innate immune response (endotoxemia). PLoS One. 2012;7(10):e48679. Nishito Y, Kambe T. Zinc transporter 1 (znt1) expression on the cell surface is elaborately controlled by cellular zinc levels. J Biol Chem. 2019;294(43):15686-15697. Wang F, Kim BE, Petris MJ, Eide DJ. The mammalian zip5 protein is a zinc transporter that localizes to the basolateral surface of polarized cells. J Biol Chem. 2004;279(49):51433-51441. Hess SY, Peerson JM, King JC, Brown KH. Use of serum zinc concentration as an indicator of population zinc status. Food Nutr Bull. 2007;28(3):403-429. Matte JJ, Girard CL, Guay F. Intestinal fate of dietary zinc and copper: Postprandial net fluxes of these trace elements in portal vein of pigs. J Trace Elem Med Biol. 2017;44:65-70. Foligné B, George F, Standaert A, Garat A, Poiret S, Peucelle V, et al. High-dose dietary supplementation with zinc prevents gut inflammation: Investigation of the role of metallothioneins and beyond by transcriptomic and metagenomic studies. Faseb J. 2020;34(9):12615-12633. Martínez MM, Link JE, Hill GM. Dietary pharmacological or excess zinc and phytase effects on tissue mineral concentrations, metallothionein, and apparent mineral retention in the newly weaned pig. Biol Trace Elem Res. 2005;105(3):97-115. Irato P, Santon A, Ossi E, Albergoni V. Interactions between metals in rat liver and kidney: Localization of metallothionein. Histochem J. 2001;33(2):79-86. Supplementary Files coverart.tif supplementarytables.docx Cite Share Download PDF Status: Published Journal Publication published 14 Mar, 2025 Read the published version in Journal of Animal Science and Biotechnology → Version 1 posted Editorial decision: Major revision 30 Oct, 2024 Reviewers agreed at journal 11 Oct, 2024 Reviewers invited by journal 10 Oct, 2024 Editor assigned by journal 07 Oct, 2024 First submitted to journal 05 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5194232","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":364781760,"identity":"09b63ffc-554c-43e6-a6eb-a15de4499f93","order_by":0,"name":"Jilong Xu","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-8623-1608","institution":"Henan University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Jilong","middleName":"","lastName":"Xu","suffix":""},{"id":364781761,"identity":"35fda6fc-f5da-488a-826b-fd0acd2499f9","order_by":1,"name":"Hanzhen Qiao","email":"","orcid":"","institution":"Henan University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Hanzhen","middleName":"","lastName":"Qiao","suffix":""},{"id":364781762,"identity":"f191c9ce-dea6-4ab7-9905-58f42fdec648","order_by":2,"name":"Liping Gan","email":"","orcid":"","institution":"Henan University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Liping","middleName":"","lastName":"Gan","suffix":""},{"id":364781763,"identity":"ed5e793e-6abe-4dfa-ac53-0bc249b5bcd8","order_by":3,"name":"Peng Wang","email":"","orcid":"","institution":"Henan University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Wang","suffix":""},{"id":364781764,"identity":"27a57183-a40e-47fc-83c3-f60a066308c9","order_by":4,"name":"Yifeng Zhao","email":"","orcid":"","institution":"Henan University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yifeng","middleName":"","lastName":"Zhao","suffix":""},{"id":364781765,"identity":"92392220-9c16-4324-b617-0ec13fed6422","order_by":5,"name":"Zetian Lei","email":"","orcid":"","institution":"Henan University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Zetian","middleName":"","lastName":"Lei","suffix":""},{"id":364781766,"identity":"59be793f-3d0f-436f-8394-1a374d2d60fc","order_by":6,"name":"Yixuan Chou","email":"","orcid":"","institution":"Henan University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yixuan","middleName":"","lastName":"Chou","suffix":""},{"id":364781767,"identity":"3c6202a1-2dae-429b-becf-737c977c621a","order_by":7,"name":"Chenrui Hou","email":"","orcid":"","institution":"Henan University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Chenrui","middleName":"","lastName":"Hou","suffix":""},{"id":364781768,"identity":"5e7ff27b-1e47-4dc2-b961-7eef646c0d91","order_by":8,"name":"Mengqi Li","email":"","orcid":"","institution":"Henan University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Mengqi","middleName":"","lastName":"Li","suffix":""},{"id":364781769,"identity":"9dc4b8ca-af5f-42bf-a6b8-e22ffea01266","order_by":9,"name":"Jinrong Wang","email":"","orcid":"https://orcid.org/0000-0002-1450-1784","institution":"Henan University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Jinrong","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-10-02 17:04:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5194232/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5194232/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40104-025-01172-2","type":"published","date":"2025-03-14T15:58:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66534353,"identity":"df87495c-ca14-407b-b5e8-34a8b2e0ceee","added_by":"auto","created_at":"2024-10-14 06:45:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":209370975,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of dietary ZnCA supplementation on intestinal morphology in piglets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll intestinal tissue samples were examined using H\u0026amp;E staining, and images were captured at a magnification of 40× using an optical microscope.\u0026nbsp;CON: basal diet + saline solution; NC: basal diet + ETEC K88 challenge; PC: basal diet + 2500 mg/kg of Zn (ZnO) + ETEC K88 challenge; ZnCA: basal diet + 1600 mg/kg of Zn (ZnCA) + ETEC K88 challenge.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5194232/v1/cc073ce65ad2a2b0681bcbc2.png"},{"id":78689166,"identity":"bf0ecf89-e584-42e9-b887-15cdc4bacb67","added_by":"auto","created_at":"2025-03-17 16:12:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":70854043,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5194232/v1/6a2c043f-a273-4ad3-ad28-fbe5e6e492d2.pdf"},{"id":66534351,"identity":"6eddb72e-56aa-446d-9270-ac7ca9bd2f67","added_by":"auto","created_at":"2024-10-14 06:45:16","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13253776,"visible":true,"origin":"","legend":"","description":"","filename":"coverart.tif","url":"https://assets-eu.researchsquare.com/files/rs-5194232/v1/8ab70bdd4f503cdf48019eb1.tif"},{"id":66534350,"identity":"94b51f62-ce29-4c68-a7a1-922cb07b3dfe","added_by":"auto","created_at":"2024-10-14 06:45:16","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":69918,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarytables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5194232/v1/854674dfad407f18d6ee41a8.docx"}],"financialInterests":"","formattedTitle":"Impact of zinc caproate supplementation on growth performance, intestinal health, anti-inflammatory activity, and Zn homeostasis in weaned piglets challenged with Escherichia coli K88","fulltext":[{"header":"Background","content":"\u003cp\u003ePost-weaning diarrhea (PWD) frequently occurs in weaned piglets and is primarily the result of incomplete intestinal development and suboptimal feeding conditions on farms. This condition can become fatal, causing significant financial losses in the swine industry [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Enterotoxigenic \u003cem\u003eEscherichia coli\u003c/em\u003e (ETEC) is the leading pathogenic cause of post-weaning colibacillosis, a common type of PWD. Globally, the predominant strain associated with PWD in piglets is ETEC K88, which typically infects these animals through the oral route [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The initial stages of ETEC infection involve the colonization of the intestinal epithelium and the secretion of enterotoxins. ETEC colonizes the intestinal epithelium by leveraging membrane adhesins to adhere to the intestinal mucosa and binding to glycoprotein receptors on the brush border of intestinal cells. Subsequently, it secretes enterotoxins (including heat-labile and heat-stable enterotoxins), disrupts the electrolyte balance in the intestines, and ultimately causes diarrhea [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHistorically, antibiotic growth promoters (AGPs) and pharmacological doses of zinc oxide (ZnO, 2500 mg/kg) have been widely utilized in piglet feed to either prevent or treat PWD and enhance growth performance [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, serious concerns have emerged regarding the misuse of AGPs and ZnO, since these chemicals can promote bacterial resistance, lead to drug residues in animal products, and cause environmental pollution [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, the European Union and China have imposed bans on the use of AGPs and restricted the dosage of ZnO in animal feed [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. At present, in the European Union and China, the maximum permissible dosages of ZnO in animal feed are 150 mg/kg and 1600 mg/kg, respectively, during the first two weeks following weaning [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is becoming increasingly evident that ZnO may fail to meet the production demands of the swine farming industry in the near future [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Consequently, there is an urgent need to identify ZnO substitutes for this purpose. So far, a range of Zn-containing molecules with the potential to treat PWD have been studied extensively. These include inorganic Zn compounds like ZnO nanoparticles (Nano-ZnO) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and tetrabasic Zn chloride [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] as well as organic Zn compounds such as Zn-amino acid complexes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], Zn-polysaccharide complexes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and certain coated Zn products [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. While these agents show promise in lowering the bioactive concentration of ZnO and mitigating its environmental impact, the doses employed fail to meet the European Commission's mandate of 150 mg/kg of total Zn in complete feed [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMedium-chain fatty acids (MCFAs), which are promising alternatives to antibiotics, play various roles in piglet nutrition. For instance, they can enhance growth performance, boost immunity, promote the growth of beneficial intestinal microbes, and maintain intestinal homeostasis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. MCFAs have the potential to reduce the acid-binding capacity in the intestines, thereby improving feed digestibility in weaned piglets [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Most MCFAs and their derivatives are generally considered safe in feed and food products and are widely used in various industries, including the daily chemicals, food, and pharmaceutical sectors [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Recently, studies have proposed that the synthesis of organic Zn compounds through the combination of caproic acid (CA) and Zn could potentially enhance the bioavailability of Zn, thus offering a novel avenue for effectively managing PWD and ETEC K88 infections.\u003c/p\u003e \u003cp\u003eIn our previous study, ZnCA was successfully synthesized using a solvothermal method and demonstrated remarkable antibacterial activity against ETEC K88 \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Consequently, the aim of the present study was to examine the impact of dietary supplementation with ZnCA on growth performance, inflammatory status, intestinal health, and Zn homeostasis in weaned piglets exposed to ETEC K88.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eExperimental materials\u003c/h2\u003e\n \u003cp\u003eZnO (75% Zn) was obtained from Shijiazhuang Hanying Feed Co., Ltd. (Shijiazhuang, China). Titanium dioxide (TiO\u003csub\u003e2\u003c/sub\u003e) was procured from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). ZnCA, a Zn complex composed of ZnO and CA (21.91% Zn), was synthesized in our laboratory at the College of Bioengineering, Henan University of Technology.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eAnimals, experimental design, and housing\u003c/h3\u003e\n\u003cp\u003eForty-eight piglets (Duroc \u0026times; Landrace \u0026times; Yorkshire) aged 28 d (body weight [BW]\u0026thinsp;=\u0026thinsp;7.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 kg) were selected for a 21-d experiment. A randomized complete design consisting of four treatment conditions was employed (N\u0026thinsp;=\u0026thinsp;6 pens/treatment). Groups were matched based on BW (with weaning BW balanced across pens) and sex. Each experimental group consisted of six replicate pens, with two piglets in each pen. The control group (CON) and negative control group (NC) received a basal diet (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), while the positive group (PC) received the basal diet supplemented with pharmacological doses of ZnO (2500 mg/kg Zn). Meanwhile, the ZnCA group (ZnCA) received a basal diet supplemented with 1500 mg/kg Zn (ZnCA). All diets exceeded the nutritional recommendations proposed by the National Research Council [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Each pen, measuring 1.30 m \u0026times; 0.5 m, was furnished with a single-side feeder and nipple drinker. The ambient temperature within the facility was initially set at 30℃ and subsequently reduced at a rate of 1.5℃ per week. The relative humidity was at 50%, and the animals had unrestricted access to feed and water throughout the 21-d study period. \u0026nbsp;\u003c/p\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eComposition and nutrient content of the basal diet (as-fed basis, %)\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIngredients\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eContent (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCorn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSoybean meal 46%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eExtrusion soybean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFish meal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWhey powder\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSoybean oil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDicalcium phosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLimestone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL-Lysine (98%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMineral premix\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVitamin mix\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eAnalyzed nutrient composition\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGross energy, MJ/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDry matter, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrude protein, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZn, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCalcium, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal phosphorus, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCalculated nutrient composition\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMetabolic energy, MJ/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDigestible energy, MJ/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL-Lysine, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.523\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMethionine, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.398\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\"\u003e\u003csup\u003e1\u003c/sup\u003e Premix supplied per kilogram of complete diet: Cu, 20 mg; Fe, 104 mg; Mn, 12 mg; Zn 64 mg; I, 0.8 mg; Se, 0.4 mg.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\"\u003e\u003csup\u003e2\u003c/sup\u003e Premix supplied per kilogram of complete diet: Vitamin A, 6,450 IU; Vitamin D\u003csub\u003e3\u003c/sub\u003e, 1,520 IU; Vitamin E, 39.53 IU; Vitamin K\u003csub\u003e3\u003c/sub\u003e, 2 mg; Vitamin B\u003csub\u003e1\u003c/sub\u003e, 1.61 mg; Vitamin B\u003csub\u003e2\u003c/sub\u003e, 5 mg; Vitamin B\u003csub\u003e6\u003c/sub\u003e, 2.55 mg; Vitamin B\u003csub\u003e12\u003c/sub\u003e, 20 \u0026micro;g; D-biotin, 120 \u0026micro;g; Niacin, 19.6 mg; D-pantothenic acid, 12.06 mg; Folic acid, 0.99 mg; Ethoxyquin, 0.1 mg.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\"\u003e\u003csup\u003e3\u003c/sup\u003e Nutrient levels were calculated according to the guidelines of the National Research Council (NRC, 2012)\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eETEC K88 challenge\u003c/h3\u003e\n\u003cp\u003eTo guarantee that the piglets were satiated and bacterial colonization could occur effectively, feeding was halted at 9 p.m. on the day preceding inoculation and resumed 30 min prior to inoculation. On days 8, 9, and 10 post-weaning, piglets in the NC, PC, and ZnCA groups received 10 mL of an oral ETEC K88 (O149: K91: K88ac; toxins LT, STa, and STb) suspension (3\u0026times;10\u003csup\u003e10\u003c/sup\u003e CFU/mL) twice daily (9 a.m. and 3 p.m.). Conversely, piglets in the CON group received 10 mL of normal saline orally.\u003c/p\u003e\n\u003ch3\u003eGrowth performance and diarrhea scores\u003c/h3\u003e\n\u003cp\u003eThe BW was measured on the first and last day of the trial. Meanwhile, feed consumption was accurately recorded in each pen at 9 a.m. Growth performance was evaluated based on the feed efficiency (G:F) using the average daily gain (ADG) and average daily feed intake (ADFI) data. Furthermore, fecal consistency was assessed daily, throughout the study, by trained personnel. These assessments were based on visual examination and followed the grading system established by Atarashi \u003cem\u003eet al.\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e], as follows: 0, normal feces; 1, moist or soft feces; 2, thick liquid feces or mild diarrhea; and 3, watery feces and severe diarrhea.\u003c/p\u003e\n\u003ch3\u003eSample collection\u003c/h3\u003e\n\u003cp\u003eDuring this study, 1 kg samples of the feed from each treatment group were individually obtained and preserved at -20℃. On days 19, 20, and 21, fecal samples were collected through rectal stimulation and preserved at -20\u0026deg;C for subsequent analysis. Prior to slaughter (day 21), blood samples were randomly collected from the jugular vein of one piglet per pen via venipuncture and added to Vacutainer coagulation tubes. All blood specimens were allowed to clot at ambient temperature; subsequently, they were centrifuged at 3500 \u0026times;\u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C for 15 min. The serum was collected and placed in trace element-free tubes before freezing at -80℃ for subsequent analysis. On day 21, the piglets from which blood samples had been collected were selected and euthanized. The weights of the heart, liver, spleen, and kidneys were calculated after slaughter. Samples of hair, the longus dorsi muscle, the right lateral lobe of the liver, the jejunum, and the right kidney were obtained for Zn analysis. The tissues were washed with phosphate-buffered saline (PBS), rapidly frozen in liquid nitrogen, and preserved at -80\u0026deg;C. The small intestine was separated into three distinct regions: the duodenum, jejunum, and ileum. Tissues from the central portions of the duodenum, jejunum, and ileum were collected and washed with PBS after removing the intestinal contents. Histological samples, measuring 0.5 cm, were fixed in a 4% paraformaldehyde solution for 24 h before further morphological examination. Mucosal scrapings were gently collected from the remaining jejunal tissue using a sterile scalpel. The contents of the jejunum were also collected and immediately immersed in liquid nitrogen for preservation at -80\u0026deg;C before subsequent analysis.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eIntestinal histomorphometry\u003c/h2\u003e\n \u003cp\u003eTissue samples from the duodenum, jejunum, and ileum \u0026mdash; which had been preserved in 4% paraformaldehyde \u0026mdash; were dehydrated, cleared, and embedded in paraffin. Following this, the samples were sliced into 4-\u0026micro;m sections and stained using hematoxylin and eosin (H\u0026amp;E). Morphological observations and measurements, with a special emphasis on villus height and crypt depth, were carried out using a light microscope (RVL-100-G, Echo Global Logistics, Inc., California, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSerum analyses\u003c/h3\u003e\n\u003cp\u003eThe enzymatic activity of alkaline phosphatase (AKP), glutamic-pyruvic transaminase (ALT/GPT), glutamic-oxaloacetic transaminase (AST/GOT), and diamine oxidase (DAO) and the levels of nitric oxide (NO) and D-lactic acid (D-LA) in the serum were accurately measured using biochemical methods and commercial kits, strictly adhering to the manufacturer\u0026rsquo;s instructions. The levels of tumor necrosis factor-\u0026alpha; (TNF-\u0026alpha;), interleukin-6 (IL-6), and interleukin-1\u0026beta; (IL-1\u0026beta;) were quantified using the enzyme-linked immunosorbent assay (ELISA) technique. Additionally, a sandwich ELISA kit was employed to detect the endotoxin content in the serum. Both the biochemical reagents and ELISA kits were sourced from Nanjing Jiancheng Technology Co., Ltd. (Nanjing, China).\u003c/p\u003e\n\u003ch3\u003eNutrient digestibility\u003c/h3\u003e\n\u003cp\u003eSamples of feed and feces were dried at 65\u0026deg;C for 72 h and then ground to a powder, which was passed through a 1-mm sieve. This processed powder was used to analyze the apparent total tract digestibility (ATTD) of nutrients in the feed. The apparent total tract digestibility (ATTD) of the nutrients \u0026mdash; including crude protein (CP), dry matter (DM), and gross energy (GE) \u0026mdash; was accurately assessed by utilizing the indigestible marker method, with 0.2% TiO\u003csub\u003e2\u003c/sub\u003e employed as an exogenous indicator. The DM and CP content of both the feces and feed were meticulously analyzed using methods 930.15 and 990.03, respectively, outlined by the Association of Official Analytical Chemists [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. Meanwhile, the gross energy (GE) values were determined by employing an adiabatic bomb calorimeter (Kalorimeter C6000 prozesso, IKA, Staufen, Germany). The elemental content of Zn and titanium (Ti) was determined using the AOAC method 985.0 and a spectroscope (Optima 5300 DV ICP-OES, PerkinElmer, MA, USA). The ATTD of the nutrients was then computed based on the following formula.:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1728887224.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eZn status\u003c/h2\u003e\n \u003cp\u003eThe concentrations of Zn were accurately determined in various samples, including the serum, feed, feces, hair, longus dorsi muscle, jejunum, liver, and kidney. Prior to analysis, all samples underwent wet digestion with a mixture of nitric acid and perchloric acid (4:1). Subsequently, the samples were diluted with ultra-pure H\u003csub\u003e2\u003c/sub\u003eO and examined using a spectroscope (Optima 5300 DV ICP-OES, PerkinElmer, MA, USA). The procedure involved weighing 0.5 g of lyophilized solid samples or 0.5 mL of liquid samples and then mixing them with 12 mL of nitric acid and 3 mL of perchloric acid. The samples were then digested in an adjustable electric furnace at 120\u0026deg;C for 0.5 h, 180\u0026deg;C for 24 h, and 220\u0026deg;C until the mixtures became colorless and transparent. For Zn analysis, the liver, feed, and fecal samples were diluted with ultrapure water to reach a final volume of 25 mL. Meanwhile, the longissimus dorsi, kidney, jejunum, and hair samples were diluted to a final volume of 10 mL, and serum samples were diluted to a final volume of 5 mL.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA extraction, cDNA synthesis, and real-time quantitative polymerase chain reaction (RT-qPCR)\u003c/h2\u003e\n \u003cp\u003eTotal RNA was isolated from 50 mg of kidney, liver, and jejunum mucosa tissues using the Freezol reagent (Vazyme, Nanjing, China). The mRNA concentrations were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized using a qPCR kit (Vazyme, Nanjing, China). RT-qPCR was conducted in a reaction volume of 20 \u0026micro;L, employing the SYBR qPCR Master Mix (Vazyme, Nanjing, China), on a quantitative fluorescence PCR instrument (Analytik Jena, Jena, Germany). The thermocycler protocol consisted of an initial denaturation step at 95℃ for 30 s, followed by 40 cycles of denaturation at 95℃ for 3 s and annealing/extension at 60℃ for 30 s. Melting curve analysis was conducted to assess the specificity of the amplified fragments using Dissociation Curves v1.0 software (PE Applied Biosystems). Each experimental sample was assayed using four technical replicates. The reference gene was glyceraldehyde 3-phosphate dehydrogenase (\u003cem\u003eGAPDH\u003c/em\u003e), and the relative mRNA expression of the genes of interest was assessed using the 2-\u003csup\u003e\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method. Here, the \u0026Delta;Ct value represents the difference between the Ct values of the target genes and the housekeeping gene. All primers, designed using sequences obtained from the National Center for Biotechnology Information (NCBI) database, are listed in \u003cstrong\u003eTable S1\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analyses\u003c/h2\u003e\n \u003cp\u003eEach pen served as the experimental unit for assessing variations in growth performance and fecal scores among the piglets. Conversely, individual piglets were considered as the experimental units for analyzing intestinal morphology, serum parameters, nutrient digestibility, and mRNA expression levels. Experimental data were analyzed with a one-way ANOVA by utilizing the Statistics Analysis System (SAS, SAS Institute, Inc., version 9.4, Cary, NC), and Tukey\u0026rsquo;s test was employed for post hoc analysis. All experimental outcomes were reported as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Significance was determined at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, while a trend was noted when 0.05\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.1.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eGrowth performance, fecal scores, and organ index\u003c/h2\u003e\n \u003cp\u003eThe growth performance and fecal scores of the weaned piglets are summarized in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Initially, the BW of piglets was comparable across all treatment groups. Over the 3-week period following weaning, piglets from the ZnCA group exhibited a significantly higher final BW (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.021), ADG (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026), and G:F ratio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026) than those from the NC group. Interestingly, there were no notable variances among the other treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Furthermore, no obvious variations in the ADFI were observed among the treatment groups.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of dietary ZnCA supplementation on growth performance and fecal scores in piglets\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eDietary treatments\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSEM\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCON\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZnCA\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInitial BW, kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.998\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFinal BW, kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.58\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.92\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.317\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADG, g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e346\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e273\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e361\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e398\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.687\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.026\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADFI, g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e620\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e557\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e646\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e660\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.429\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG:F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.28\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.28\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.043\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDiarrhea score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.79\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.58\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.58\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.059\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.024\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e1\u003c/sup\u003e Data represent the mean of six replicate pens per treatment.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e2\u003c/sup\u003e CON: basal diet\u0026thinsp;+\u0026thinsp;saline solution; NC: basal diet\u0026thinsp;+\u0026thinsp;ETEC K88 challenge; PC: basal diet\u0026thinsp;+\u0026thinsp;2500 mg/kg of Zn (ZnO)\u0026thinsp;+\u0026thinsp;ETEC K88 challenge; ZnCA: basal diet\u0026thinsp;+\u0026thinsp;1600 mg/kg of Zn (ZnCA)\u0026thinsp;+\u0026thinsp;ETEC K88 challenge.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e3\u003c/sup\u003e SEM: standard error of the mean.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003ea, b\u003c/sup\u003e Means with different superscripts within the same row showed a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eBW, body weight; ADG, average daily gain; ADFI, average daily feed intake; G:F, gain-to-feed ratio.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eBoth the ZnCA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.042) and PC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.040) groups had significantly lower fecal scores than the NC group. However, no significant difference was noted between piglets in the CON group and those in the other treatment groups.\u003c/p\u003e\n \u003cp\u003eThe impact of dietary ZnCA supplementation on the organ indexes of piglets is presented in \u003cstrong\u003eTable S2\u003c/strong\u003e. The weights of the kidneys, spleen, heart, and liver remained unaffected by the dietary treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eIntestinal health (intestinal histomorphometry, ATTD of nutrients, and gut barrier)\u003c/h2\u003e\n \u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, there was no statistically significant variance in duodenum morphology across the four groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In both the ileum and the jejunum, the ZnCA group demonstrated a higher villus height and villus/crypt ratio as well as a lower crypt depth than the CON and NC groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The PC group demonstrated an increased villus/crypt ratio in the jejunum and villus height in the ileum along with a decreased crypt depth in the jejunum when compared with the CON and NC groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, the NC group exhibited a significantly lower villus/crypt ratio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.033) and higher villus height in the jejunum (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.020) than the CON group.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of dietary ZnCA supplementation on intestinal histomorphometry in piglets\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003eDuodenum\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eDietary treatments\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSEM\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCON\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZnCA\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVillus height, \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e318.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e296.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e315.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e309.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.442\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.428\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrypt depth, \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e184.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e179.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e178.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e176.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.322\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.872\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVillus/crypt ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.316\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eJejunum\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVillus height, \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e301.57\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e284.73\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e307.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e371.83\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.678\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrypt depth, \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e131.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e101.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e109.69\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.069\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVillus/crypt ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.56\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.42\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.045\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eIleum\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVillus height, \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e250.12\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e230.74\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e270.65\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e289.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.876\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrypt depth, \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e109.34\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e110.79\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e118.85\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.808\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVillus/crypt ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.46\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e1\u003c/sup\u003e Data represent the mean of six replicate pens per treatment.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e2\u003c/sup\u003e CON: basal diet\u0026thinsp;+\u0026thinsp;saline solution; NC: basal diet\u0026thinsp;+\u0026thinsp;ETEC K88 challenge; PC: basal diet\u0026thinsp;+\u0026thinsp;2500 mg/kg of Zn (ZnO)\u0026thinsp;+\u0026thinsp;ETEC K88 challenge; ZnCA: basal diet\u0026thinsp;+\u0026thinsp;1600 mg/kg of Zn (ZnCA)\u0026thinsp;+\u0026thinsp;ETEC K88 challenge.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e3\u003c/sup\u003e SEM: standard error of the mean.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u0026minus;d\u003c/sup\u003e Means with different superscripts within the same row showed a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe ATTD of nutrients in piglets is summarized in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. There were no statistically significant differences detected between the PC and ZnCA groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The addition of ZnO and ZnCA increased the digestibility of DM and GE (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, the digestibility of crude protein in the NC group was notably lower than that in the CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of dietary ZnCA supplementation on the ATTD of nutrients in piglets\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eATTD, %\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eDietary treatments\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSEM\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCON\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZnCA\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDry matter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75.357\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75.273\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.6583\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.710\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.763\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.029\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrude protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.837\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.083\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69.817\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69.818\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGross energy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71.718\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.866\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.332\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.3214\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.088\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e1\u003c/sup\u003e Data represent the mean of six replicate pens per treatment.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e2\u003c/sup\u003e CON: basal diet\u0026thinsp;+\u0026thinsp;saline solution; NC: basal diet\u0026thinsp;+\u0026thinsp;ETEC K88 challenge; PC: basal diet\u0026thinsp;+\u0026thinsp;2500 mg/kg of Zn (ZnO)\u0026thinsp;+\u0026thinsp;ETEC K88 challenge; ZnCA: basal diet\u0026thinsp;+\u0026thinsp;1600 mg/kg of Zn (ZnCA)\u0026thinsp;+\u0026thinsp;ETEC K88 challenge.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e3\u003c/sup\u003e SEM: standard error of the mean.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003ea, b\u003c/sup\u003e Means with different superscripts within the same row showed a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eATTD, apparent total tract digestibility.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe serum concentrations of D-LA, DAO, and endotoxin \u0026mdash; which are indicators of intestinal permeability \u0026mdash; were assayed (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). No significant differences in the serum levels of DAO were observed among the treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, the levels of D-LA and endotoxin were significantly higher in the CON group than in the other experimental groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Interestingly, there was no notable distinction in serum D-LA and endotoxin levels between the CON and ZnCA groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Meanwhile, in comparison to the PC group, the ZnCA group demonstrated reduced serum levels of D-LA and endotoxin (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of dietary ZnCA supplementation on intestinal barrier function in piglets\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003eSerum indicators of intestinal barrier function\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eDietary treatments\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSEM\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCON\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZnCA\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD-LA, \u0026micro;mol/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.51\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.22\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDAO, U/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e152.549\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e129.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e146.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.908\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.547\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEndotoxin, ng/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e316.14\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e535.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e439.38\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e313.79\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.616\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eExpression of intestinal barrier-related genes in the jejunum mucosa\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZO-1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.058\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.76\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.930\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.116\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.039\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMUC-2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.029\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.237\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.884\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.363\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.043\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eOccludin\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.018\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.141\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.093\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.516\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.047\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eClaudin-1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.122\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.225\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.203\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.811\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.104\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eClaudin-2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.021\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.449\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.086\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.125\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e1\u003c/sup\u003e Data represent the mean of six replicate pens per treatment.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e2\u003c/sup\u003e CON: basal diet\u0026thinsp;+\u0026thinsp;saline solution; NC: basal diet\u0026thinsp;+\u0026thinsp;ETEC K88 challenge; PC: basal diet\u0026thinsp;+\u0026thinsp;2500 mg/kg of Zn (ZnO)\u0026thinsp;+\u0026thinsp;ETEC K88 challenge; ZnCA: basal diet\u0026thinsp;+\u0026thinsp;1600 mg/kg of Zn (ZnCA)\u0026thinsp;+\u0026thinsp;ETEC K88 challenge.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e3\u003c/sup\u003e SEM: standard error of the mean.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u0026minus;c\u003c/sup\u003e Means with different superscripts within the same row showed a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eD-LA, D-lactic acid; DAO, diamine oxidase; \u003cem\u003eZO-1\u003c/em\u003e, zonula occludens-1; \u003cem\u003eMUC-2\u003c/em\u003e, mucin 2.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe relative mRNA levels of tight junction (TJ) proteins within the jejunum were also examined (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Notably, ZnCA supplementation significantly upregulated the mRNA levels of mucin 2 (\u003cem\u003eMUC-2\u003c/em\u003e), \u003cem\u003eOccludin\u003c/em\u003e, and \u003cem\u003eClaudin-1\u003c/em\u003e in the jejunum. Interestingly, no notable differences in the mRNA levels of TJ proteins were detected between the CON and PC groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, the NC group exhibited lower mRNA levels of \u003cem\u003eClaudin-1\u003c/em\u003e and \u003cem\u003eZO-1\u003c/em\u003e, as well as higher mRNA levels of \u003cem\u003eClaudin-2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eAnti-inflammatory activity\u003c/h2\u003e\n \u003cp\u003eThe anti-inflammatory activity of dietary ZnCA supplementation is detailed in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. Initially, we assessed serum liver function markers, including AKP, AST/GOT, and ALT/GPT. However, no notable variances were detected in the serum levels of AST/GOT and ALT/GPT across all groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of dietary ZnCA supplementation on anti-inflammatory activity in piglets\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003eSerum liver function\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eDietary treatments\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSEM\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCON\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZnCA\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAKP, U/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.76\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.29\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.52\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.762\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAST/GOT, U/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.289\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eALT/GPT, U/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.408\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eSerum cytokines\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIL-6, pg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e111.93\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e158.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e122.79\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e108.47\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.866\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIL-1\u003cem\u003e\u0026beta;\u003c/em\u003e, pg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e301.68\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e437.28\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e387.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e312.13\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.741\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTNF-\u003cem\u003e\u0026alpha;\u003c/em\u003e, pg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58.68\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e109.99\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.64\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.55\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.942\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNO, \u0026micro;mol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e106.26\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e130.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.51\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eInflammatory factors in the jejunum mucosa\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eIL-6\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.123\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.376\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.170\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5719\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eIL-1\u0026beta;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.018\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.391\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.956\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.018\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.092\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eTNF-\u0026alpha;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.053\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.008\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.461\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.740\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.222\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eiNOS\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.087\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.328\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.092\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.250\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.140\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eInflammatory factors in the liver\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eIL-6\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.068\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.280\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.106\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.923\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eIL-1\u0026beta;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.084\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.599\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.652\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.116\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eTNF-\u0026alpha;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.032\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.133\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.092\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.136\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eiNOS\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.161\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.413\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.844\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.083\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e1\u003c/sup\u003e Data represent the mean of six replicate pens per treatment.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e2\u003c/sup\u003e CON: basal diet\u0026thinsp;+\u0026thinsp;saline solution; NC: basal diet\u0026thinsp;+\u0026thinsp;ETEC K88 challenge; PC: basal diet\u0026thinsp;+\u0026thinsp;2500 mg/kg of Zn (ZnO)\u0026thinsp;+\u0026thinsp;ETEC K88 challenge; ZnCA: basal diet\u0026thinsp;+\u0026thinsp;1600 mg/kg of Zn (ZnCA)\u0026thinsp;+\u0026thinsp;ETEC K88 challenge.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e3\u003c/sup\u003e SEM: standard error of the mean.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u0026minus;c\u003c/sup\u003e Means with different superscripts within the same row showed a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eIL-6, interleukin-6; IL-1\u0026beta;, interleukin-1\u0026beta;; TNF-\u0026alpha;, tumor necrosis factor-\u0026alpha;; NO, nitric oxide; AKP, alkaline phosphatase; AST/GOT, aspartate aminotransferase; ALT/GPT, alanine aminotransferase; iNOS, inducible nitric oxide synthase.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eInterestingly, the serum levels of IL-6, NO, IL-1\u0026beta;, and TNF-\u0026alpha; were notably decreased after the NC treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, notable differences in the serum levels of the cytokines IL-6, NO, and IL-1\u0026beta; were observed between the CON and ZnCA groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). When compared to CON treatment, PC treatment produced higher serum concentrations of IL-1\u0026beta; (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but did not alter those of IL-6, TNF-\u0026alpha;, and NO (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eConsistent with the serum cytokine levels, the mRNA levels of inflammatory cytokines \u0026mdash; such as \u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eiNOS\u003c/em\u003e, \u003cem\u003eTNF-\u0026alpha;\u003c/em\u003e, and \u003cem\u003eIL-1\u0026beta;\u003c/em\u003e \u0026mdash; were markedly elevated in the jejunum mucosa and liver following NC treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, no notable differences in inflammatory factor mRNA levels in both the jejunum mucosa and the liver were observed between the CON and ZnCA groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Interestingly, the PC group demonstrated elevated mRNA levels of \u003cem\u003eIL-6\u003c/em\u003e and \u003cem\u003eiNOS\u003c/em\u003e when compared with the ZnCA group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eZn homeostasis\u003c/h2\u003e\n \u003cp\u003eThe impact of dietary ZnCA supplementation on the homeostasis of trace metals in piglets is shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e. Notably, a positive correlation was observed between the Zn concentration in the feces, liver, and kidney and the Zn concentration in the feed (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, no notable differences in serum Zn concentrations and the Zn concentrations in the longissimus muscle and hair were observed between the ZnCA group and the other three treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Notably, the Zn concentration in the jejunum was lower in the NC group than in the other treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab7\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of dietary ZnCA supplementation on the homeostasis of trace metals in piglets\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003eZn concentration\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eParameters\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eDietary treatments\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSEM\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCON\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZnCA\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFeed, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128.93\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128.93\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2508.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1607.71\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e306.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFeces, g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.644\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.548\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.815\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.748\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.251\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerum, mg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.842\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.208\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.558\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.781\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.332\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLiver, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e201.91\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e197.78\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1346.57\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e907.44\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e108.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKidney, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e109.36\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e114.62\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e396.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e202.94\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.088\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJejunum, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e196.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e130.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e199.64\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e215.89\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.604\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLongissimus muscle, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73.608\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73.627\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e74.052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.683\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.878\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHair, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1192.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1086.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1092.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1090.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.298\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eCu concentration\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFeed, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.053\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.053\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.337\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.602\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.759\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFeces, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e272.37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e246.84\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e377.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e285.32\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.334\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerum, mg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.161\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.054\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.257\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.552\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.138\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.085\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLiver, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.700\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.117\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.183\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.754\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKidney, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.650\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.792\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e121.895\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75.102\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.577\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJejunum, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.192\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.658\u003csup\u003ebd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.029\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.613\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.758\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLongissimus muscle, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.879\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.250\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.329\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.613\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.351\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHair, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.618\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.521\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.137\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.691\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eFe concentration\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDM of feed, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e309.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e309.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e323.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e322.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.927\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.137\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDM of feces, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2933.61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2954.32\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4163.78\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3405.58\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e153.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerum, mg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.283\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.987\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.904\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.627\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.351\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.106\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLiver, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e135.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e130.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e148.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e144.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.524\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.884\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKidney, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e136.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e131.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e135.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e134.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.986\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJejunum, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e228.90\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e198.54\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e181.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e174.77\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.055\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.019\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLongissimus muscle, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e157.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e125.35\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e110.55\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.56\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.294\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHair, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e615.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e471.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e648.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e523.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.421\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eMn concentration\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFeed, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.034\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.034\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.247\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.890\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.798\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.139\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFeces, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e443.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e442.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e536.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e470.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.183\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.132\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLiver, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.313\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.746\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.501\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.332\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.284\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKidney, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.542\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.985\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.330\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.815\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.264\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.077\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHair, mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.275\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.275\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.333\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.521\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.862\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.925\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e1\u003c/sup\u003e Data represent the mean of six replicate pens per treatment.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e2\u003c/sup\u003e CON: basal diet\u0026thinsp;+\u0026thinsp;saline solution; NC: basal diet\u0026thinsp;+\u0026thinsp;ETEC K88 challenge; PC: basal diet\u0026thinsp;+\u0026thinsp;2500 mg/kg of Zn (ZnO)\u0026thinsp;+\u0026thinsp;ETEC K88 challenge; ZnCA: basal diet\u0026thinsp;+\u0026thinsp;1600 mg/kg of Zn (ZnCA)\u0026thinsp;+\u0026thinsp;ETEC K88 challenge\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e3\u003c/sup\u003e SEM: standard error of the mean.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e4\u003c/sup\u003e With the exception of the serum, the levels in all other samples were calculated based on DM values.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u0026minus;d\u003c/sup\u003e Means with different superscripts within the same row showed a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThere were no notable variances in the Cu and Fe concentrations of the feed among the groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Interestingly, the PC group exhibited higher levels of Cu and Fe in the feces than the other treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A trend towards increased serum Cu concentrations was also detected in the NC treatment group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.085). In comparison to the CON group, the PC group exhibited elevated levels of Cu in the kidneys and reduced levels of Cu in the longissimus muscle and hair (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, the NC group demonstrated higher hepatic levels of Cu (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There were no notable differences in the Cu concentrations in the liver, kidney, longissimus muscle, and hair between the ZnCA and CON treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Notably, the ZnCA treatment group had lower Cu levels in the jejunum than the CON and PC treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eFurthermore, no notable variances were detected in the Fe concentrations of the liver, kidneys, and hair (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The CON group exhibited higher Fe levels in the jejunum and longissimus muscle than the PC and ZnCA groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Although the Mn concentration of the feed, feces, liver, and hair was comparable across the various treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), the Mn concentration in the kidneys tended to show variations (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.077).\u003c/p\u003e\n \u003cp\u003eThe relative mRNA levels of Zn/iron-regulated transporter-like 5 (\u003cem\u003eZIP5\u003c/em\u003e) remained unaffected (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.161) in the kidneys, liver, and jejunum mucosa, as presented in Table \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e. Meanwhile, the mRNA expression of \u003cem\u003eZIP4\u003c/em\u003e in the jejunum mucosa was altered in both the NC and PC groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Notably, the NC group exhibited the highest relative mRNA levels of \u003cem\u003eZIP14\u003c/em\u003e in the jejunum mucosa and liver (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). With regard to the mRNA expression of Zn transporter 1 (\u003cem\u003eZNT1\u003c/em\u003e) in the jejunum mucosa, liver, and kidney, no significant variances were detected between the CON and NC treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, the PC group demonstrated the highest mRNA levels of \u003cem\u003eZNT1\u003c/em\u003e across all these three tissues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The ZnCA group exhibited similar mRNA levels of \u003cem\u003eZNT1\u003c/em\u003e in the jejunum mucosa as the PC group, but the hepatic and renal expression levels of this gene were lower in the ZnCA group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, significant variances in the mRNA expression of metallothionein 1 (\u003cem\u003eMT1\u003c/em\u003e), \u003cem\u003eMT2\u003c/em\u003e, and \u003cem\u003eMT3\u003c/em\u003e were observed among the four dietary treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Specifically, the PC treatment group showed the highest levels of these genes, with the ZnCA group following closely behind. In contrast, the expression of these genes in the kidneys, liver, and jejunum mucosa was lower in the CON and NC treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Finally, no notable variations were observed in the mRNA expression of \u003cem\u003eZIP8\u003c/em\u003e in the liver tissue among the four treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cdiv\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab10\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of dietary ZnCA supplementation on the relative mRNA levels of specific Zn transporters in piglets\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003eJejunum mucosa\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eGenes\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eDietary treatments\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSEM\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCON\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZnCA\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZIP4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.039\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.626\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.197\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.609\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZIP5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.060\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.065\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.043\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.984\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZIP14\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.145\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.628\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.734\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.904\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZNT1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.048\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.892\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.273\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.401\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMT1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.042\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.513\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e158.632\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57.965\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.174\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.037\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.957\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e102.363\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.122\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.962\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMT3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.359\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.196\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e747.351\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e103.034\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.545\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eLiver\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZIP5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.989\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.059\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.392\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.929\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZIP8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.204\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.413\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.376\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.074\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.160\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZIP14\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.009\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.233\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.150\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.018\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.095\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZNT1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.193\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.274\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.251\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.198\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.117\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMT1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.055\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.618\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.232\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.245\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.505\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.050\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.531\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.362\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.880\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMT3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.284\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.935\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e284.354\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.097\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.453\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eKidney\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZIP5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.057\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.297\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.050\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.041\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.161\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZNT1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.071\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.212\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.011\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.062\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.085\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMT1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.265\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.754\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.989\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.809\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.535\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.142\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.518\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.805\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.588\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.089\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMT3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.033\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.132\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.297\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.652\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.372\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e1\u003c/sup\u003e Data represent the mean of six replicate pens per treatment.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e2\u003c/sup\u003e CON: basal diet\u0026thinsp;+\u0026thinsp;saline solution; NC: basal diet\u0026thinsp;+\u0026thinsp;ETEC K88 challenge; PC: basal diet\u0026thinsp;+\u0026thinsp;2500 mg/kg of Zn (ZnO)\u0026thinsp;+\u0026thinsp;ETEC K88 challenge; ZnCA: basal diet\u0026thinsp;+\u0026thinsp;1600 mg/kg of Zn (ZnCA)\u0026thinsp;+\u0026thinsp;ETEC K88 challenge.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e3\u003c/sup\u003e SEM: standard error of the mean.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u0026minus;c\u003c/sup\u003e Means with different superscripts within the same row showed a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003cem\u003eZIP4\u003c/em\u003e, Zn/iron-regulated transporter-like 4; \u003cem\u003eZIP5\u003c/em\u003e, Zn/iron-regulated transporter-like 5; \u003cem\u003eZIP8\u003c/em\u003e, Zn/iron-regulated transporter-like 8; \u003cem\u003eZIP14\u003c/em\u003e, Zn/iron-regulated transporter-like 14; \u003cem\u003eMT1\u003c/em\u003e, metallothionein 1; \u003cem\u003eMT2\u003c/em\u003e, metallothionein 2; \u003cem\u003eMT3\u003c/em\u003e, metallothionein 3; \u003cem\u003eZNT1\u003c/em\u003e, Zn transporter 1.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDue to the combined stress of weaning, incomplete intestinal development, and the influence of the rearing environment, piglets frequently develop PWD. This condition can lead to diarrhea, decreased feed intake, growth retardation, and even mortality among weaned piglets [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In context of the dual challenges arising from the ban on AGPs and the limitations imposed upon Zn supplementation, organic Zn has emerged as a promising solution for PWD management [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe impact of pharmacological doses of ZnO on nutrient digestibility, diarrhea incidence, and growth performance in weaned piglets has been studied extensively [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In the current study, dietary ZnO supplementation (2500 mg/kg) significantly reduced the diarrhea score in weaned piglets challenged with ETEC K88, but it did not improve their growth performance. However, these findings contrast with those reported by Lei and Kim [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], who observed that pharmacological levels of ZnO enhance growth performance in weaned piglets exposed to ETEC K88. This discrepancy could arise because Lei and Kim administered a high-Zn diet to the piglets for 21 d prior to the ETEC K88 challenge.\u003c/p\u003e \u003cp\u003eAs expected, in the present study, piglets who received a diet containing an additional 1600 mg/kg of Zn in the form of ZnCA exhibited a higher ADG and G:F ratio as well as a lower fecal score after the ETEC K88 challenge than the piglets who were fed a basal diet. These results confirmed that incorporating a small amount of ZnCA into the diet can promote growth and reduce diarrhea in weaned piglets, offering results comparable to those achieved with pharmacological doses of ZnO.\u003c/p\u003e \u003cp\u003eAfter weaning and exposure to ETEC K88, piglets often experience alterations in the structure and functionality of the intestinal tract. These changes primarily manifest in the form of villus atrophy and crypt hyperplasia [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Previous research has established that pharmacological ZnO supplements can improve the intestinal structure in weaned piglets [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In accordance with these results, our findings demonstrated that both 2500 mg/kg of conventional ZnO and 1600 mg/kg of ZnCA can improve the ratio of villus height to crypt depth and reduce crypt depth in the jejunum. Moreover, ZnCA supplementation was observed to enhance villus height and the villus/crypt ratio in the ileum, while also decreasing crypt depth. Meanwhile, ZnO supplementation also increased the villus/crypt ratio in the ileum. Notably, 1600 mg/kg ZnCA improved the villus/crypt ratio in the jejunum to a greater degree than the pharmacological doses of ZnO, indicating a potential enhancement of absorption capacity in the intestine following ZnCA supplementation.\u003c/p\u003e \u003cp\u003eThe villi of the small intestine play a key role in nutrient absorption. Hence, PWD typically reduces the nutrient absorption capacity in affected animals [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In this study, piglets challenged with ETEC K88 and treated with ZnCA exhibited an improvement in intestinal structure, resulting in the increased ATTD of DM, CP, and GE. Similar findings have also been reported by Lei and Kim [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This increase in the digestibility of DM, CP, and GE observed in ZnCA-treated piglets suggests that improved nutrient digestibility is at least partly responsible for enhanced growth performance in these animals [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This potential increase in the rate of nutrient digestion can be attributed to improvements in intestinal tract structure [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, previous studies conducted by Hedemann et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and Hu et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] indicate that pharmacological doses of ZnO can enhance the activity of digestive enzymes in the intestines of weaned piglets. Therefore, increased enzyme activity may also contribute to the accelerated rate of nutrient digestion observed in these piglets.\u003c/p\u003e \u003cp\u003eThe onset of PWD is intricately linked to intestinal permeability [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. TJ proteins \u0026mdash; such as Occludin, Claudin-1, and ZO-1 \u0026mdash; play an important role in regulating the permeability of intestinal epithelial cells and maintaining barrier function [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In this study, we observed that infection with ETEC significantly reduced the mRNA levels of \u003cem\u003eClaudin-1\u003c/em\u003e and \u003cem\u003eZO-1\u003c/em\u003e in the jejunal mucosa of weaned piglets. However, supplementation with ZnCA could alleviate this damage and partially increase the mRNA expression of TJ proteins. Consistent with our findings, Xie et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] discovered that the addition of polysaccharide-Zn complexes to the diet can upregulate the mRNA expression of TJ proteins.\u003c/p\u003e \u003cp\u003eClaudin-2, a pore-forming protein, is known to disrupt the TJ barrier [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Notably, in our study, the mRNA levels of \u003cem\u003eClaudin-2\u003c/em\u003e exhibited an inverse trend compared to those of \u003cem\u003eClaudin-1\u003c/em\u003e, echoing the similar antagonistic effects reported by Jung et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs reliable indicators of intestinal permeability, the levels of D-LA, DAO, and endotoxin provide key insights into intestinal barrier function [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. An investigation conducted by Xu et al. [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] revealed that supplementation with 1500 mg/kg of ZnO can effectively reduce DAO and endotoxin levels in the serum. In our study, we observed that ETEC K88 could significantly upregulate the serum concentrations of D-LA and endotoxin. However, pharmacological doses of ZnO and 1600 mg/kg of ZnCA could mitigate these elevations, with ZnCA demonstrating superior efficacy. These findings suggest that ZnCA, which also reduces the required amount of Zn supplementation, enhances intestinal health in weaned piglets challenged with ETEC K88 through various mechanisms. These mechanisms include improvements in intestinal morphology, augmented nutrient digestibility, and reinforcement of intestinal barrier function. Notably, the efficacy of ZnCA surpasses that of pharmacological ZnO doses in achieving these benefits.\u003c/p\u003e \u003cp\u003eAfter weaning, piglets frequently experience inflammation due to dietary transitions and infections caused by pathogens such as ETEC [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. AKP plays a crucial role in detoxification and anti-inflammatory processes following ETEC infections [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In this study, exposure to ETEC K88 in weaned piglets led to decreased levels of AST/GOT and ALT/GPT and significantly suppressed AKP activity in the serum. Conversely, Zn supplementation could enhance AKP activity, likely because AKP is a Zn-containing metalloenzyme and Zn augments its functionality [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Comparable findings have been documented by Liu et al. [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Our previous research demonstrated the anti-inflammatory effects of Zn laurate in mice infected with ETEC K88 [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Therefore, in the present study, we further explored the impact of ZnCA on inflammatory markers in post-weaned piglets challenged with ETEC K88. Akin to pharmacological doses of ZnO, ZnCA notably decreased the mRNA levels of \u003cem\u003eiNOS\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eTNF-α\u003c/em\u003e, and \u003cem\u003eIL-1β\u003c/em\u003e in the liver and jejunum mucosa, as well as the levels of NO, IL-6, TNF-α, and IL-1β in the serum of weaned piglets challenged with ETEC K88. Evidence suggests that MCFAs possess immunomodulatory properties and may improve the overall health of weaned piglets [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Consequently, the anti-inflammatory effects of ZnCA on weaned piglets could be attributed to the synergistic actions of MCFAs and Zn.\u003c/p\u003e \u003cp\u003eSo far, studies on organic and inorganic Zn in weaned piglets have explored the mechanisms of Zn metabolism and the anti-inflammatory effects of Zn independent of each other. Previous studies suggest that Zn can modulate the activity of inflammatory pathways [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. These inflammatory processes can, in turn, influence the expression of Zn transporters [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Hence, one key objective of the present study was to examine the relationship between the anti-inflammatory activity of ZnCA and Zn metabolism following ZnCA administration, offering insights into their interrelationships.\u003c/p\u003e \u003cp\u003eZIP4, which is a Zn transporter, is predominantly expressed in the brush border of the intestines and facilitates the absorption of Zn from the intestinal lumen [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In this study, the mRNA levels of \u003cem\u003eZIP4\u003c/em\u003e were found to be the highest in the NC treatment group and the lowest in the PC treatment group. The downregulation of \u003cem\u003eZIP4\u003c/em\u003e in the intestinal mucosa of piglets in the PC and ZnCA groups can be attributed to the mechanism of Zn absorption at saturation [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], wherein the rate of intestinal absorption is inversely correlated with the intake [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Conversely, the overexpression of \u003cem\u003eZIP4\u003c/em\u003e mRNA in the NC group may be due to the pathogenic effects of ETEC K88 on the intestines, resulting in decreased Zn absorption and a compensatory upregulation of \u003cem\u003eZIP4\u003c/em\u003e. Nevertheless, alterations in absorption rates do not necessarily reflect changes in total nutrient absorption [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Despite the decreased absorption rate, the Zn content in the jejunum of the PC and ZnCA treatment groups remained significantly higher than that in the jejunum of the CON group. Conversely, the jejunum Zn content in the NC group continued to remain the lowest among all the groups, consistent with previous findings [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eZIP5\u003c/em\u003e, which is integral for Zn uptake from the bloodstream, did not exhibit altered expression levels in the small intestinal mucosa, liver, and kidneys in the present study. This contradicts the results reported by Dalto et al. [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], and these differences are likely due to variations in the feeding duration between the two studies.\u003c/p\u003e \u003cp\u003e \u003cem\u003eZIP8\u003c/em\u003e transcription is directly modulated by the NF-κB signaling pathway [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. However, our findings revealed no notable changes in \u003cem\u003eZIP8\u003c/em\u003e mRNA expression in the liver under the different treatment conditions, possibly due to the tissue-specific nature of this mRNA [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Unlike ZIP8, ZIP14 can suppress NF-κB signaling via a negative feedback loop [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. In the present study, \u003cem\u003eZIP14\u003c/em\u003e mRNA levels showed a significant increase in the NC group, in contrast to the downregulation of \u003cem\u003eZIP14\u003c/em\u003e in the PC and ZnCA groups. This suggests that \u003cem\u003eZIP14\u003c/em\u003e expression may primarily be governed by inflammatory factors and not Zn regulation [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eZNT1 is a basolateral membrane transporter for Zn in enterocytes. The mRNA expression of \u003cem\u003eZNT1\u003c/em\u003e is positively correlated with tissue Zn levels. Following excessive Zn intake, \u003cem\u003eZNT1\u003c/em\u003e expression increases to facilitate Zn excretion. Therefore, it is evident that \u003cem\u003eZNT1\u003c/em\u003e expression is primarily regulated by Zn levels. Additionally, Nishito and Kambe [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] delineated the cooperative roles of ZNT1 and MT in maintaining cellular Zn homeostasis. In the present study, we observed similar trends in \u003cem\u003eMT1\u003c/em\u003e, \u003cem\u003eMT2\u003c/em\u003e, and \u003cem\u003eMT3\u003c/em\u003e expression, consistent with the changes in \u003cem\u003eZNT1\u003c/em\u003e expression. Wang et al. [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] reported that the kidneys play a crucial role in Zn excretion when dietary Zn intake becomes high. However, in the present study, the mRNA levels of \u003cem\u003eZNT1\u003c/em\u003e in the kidneys of weaned piglets subjected to ZnCA treatment were not significantly different from those in the CON group. Consequently, unlike the administration of 2500 mg/kg of ZnO, supplementation with 1600 mg/kg of ZnCA appeared to have minimal impact on Zn homeostasis in the kidneys.\u003c/p\u003e \u003cp\u003eThe body maintains Zn homeostasis through both a rapid exchange pool (encompassing the serum, intestines, liver, and kidneys) and a slow exchange pool (including skeletal muscles, bones, and hair) [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Our findings revealed that ZnO and ZnCA primarily influence the Zn content within the tissues encompassing the rapid exchange pool. Notably, even under relatively limited Zn supplementation, jejunum Zn levels were comparable between ZnCA treatment and ZnO treatment. These findings pointed to a higher absorption rate for organic Zn than for inorganic Zn, echoing the findings reported by Oh et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Nevertheless, the regulation of Zn homeostasis can affect the metabolism of other minerals, such as Cu and Fe. Matte et al. [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] observed that an increase in dietary Zn intake can reduce Cu efflux from intestinal cells. Consistent with these findings, in the PC group, the concentration of Cu was elevated in the jejunum but reduced in the liver and serum. Additionally, our study revealed that high-Zn diets can enhance the excretion of Cu and Fe. This conclusion was corroborated by the findings reported by Folign\u0026eacute; et al. [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Elevations in Zn content likely contribute to this phenomenon by upregulating the expression of MT [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e], which can bind to Fe and Cu and thereby limit their transport into the blood and liver [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Ultimately, the Cu and Fe bound to MT can be eliminated via the shedding of intestinal cells [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Notably, supplementation with 1600 mg/kg ZnCA only decreased duodenal Cu and longissimus dorsi Fe levels in weaned piglets, indicating that ZnCA had a significantly lower metabolic impact on Cu and Fe than pharmacological levels of ZnO. Finally, neither dietary manipulation nor toxin exposure was found to produce any discernible effect on Mn metabolism in this study, suggesting that high Zn supplementation may not alter Mn metabolism.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe findings of the current study demonstrate that incorporating 1600 mg/kg of ZnCA into the diet of weaned piglets can lead to improved growth performance and nutrient digestibility, while also reducing the incidence of diarrhea and inflammatory responses triggered by ETEC K88 infection. These beneficial effects are comparable to those achieved with pharmacological doses of ZnO. Furthermore, in terms of improving intestinal health and Zn homeostasis, the efficacy of 1600 mg/kg ZnCA surpasses that of pharmacological doses of ZnO in weaned piglets challenged with ETEC K88.\u003c/p\u003e \u003cp\u003ePharmacological doses of ZnO not only increase Zn excretion but also disrupt the absorption of copper and iron. However, our study revealed that the ZnCA (1600 mg/kg) can achieve reduced Zn substitution while still enhancing Zn deposition in weaned piglets. Our future studies will involve a deeper exploration of the impact of lower ZnCA doses on weaned piglets. Given the current restrictions regarding the pharmacological dosage of ZnO, this study offers valuable insights for reducing Zn excretion and presents a novel approach towards managing PWD and developing antibiotic alternatives for weaned piglets.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eZnCA, zinc caproate; ZnO, zinc oxide; PWD, post-weaning diarrhea; ETEC, \u003cem\u003eEscherichia coli\u003c/em\u003e; MCFAs, medium-chain fatty acids; CA, caproic acid; G:F, feed efficiency; ADG, average daily gain; ADFI, average daily feed intake; AKP, alkaline phosphatase; ALT/GPT, glutamic-pyruvic transaminase; AST/GOT, glutamic-oxaloacetic transaminase; DAO, diamine oxidase; NO, nitric oxide; D-LA, D-lactic acid; TNF-\u0026alpha;, tumor necrosis factor-\u0026alpha;; IL-6, interleukin-6; IL-1\u0026beta;, interleukin-1\u0026beta;; ATTD, apparent total tract digestibility; CP, crude protein; DM, dry matter; GE, gross energy; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ZO-1, zonula occludens-1; MUC-2, mucin 2; iNOS, inducible nitric oxide synthase; ZIP4, Zn/iron-regulated transporter-like 4; ZIP5, Zn/iron-regulated transporter-like 5; ZIP8, Zn/iron-regulated transporter-like 8; ZIP14, Zn/iron-regulated transporter-like 14; MT1, metallothionein 1; MT2, metallothionein 2; MT3, metallothionein 3; ZNT1, Zn transporter 1.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026dagger;Jilong Xu and Hanzhen Qiao equally contributed to this work:\u0026nbsp;conceptualization, investigation, data curation, writing-original draft;\u0026nbsp;Liping Gan:\u0026nbsp;investigation, project administration;\u0026nbsp;Peng Wang:\u0026nbsp;methodology, resources;\u0026nbsp;Yifeng Zhao:\u0026nbsp;validation, visualization;\u0026nbsp;Zetian Lei:\u0026nbsp;conceptualization, project administration;\u0026nbsp;Yixuan Chou:\u0026nbsp;supervision, project administration;\u0026nbsp;Chenrui Hou:\u0026nbsp;conceptualization;\u0026nbsp;Mengqi Li:\u0026nbsp;methodology;\u0026nbsp;Jinrong Wang:\u0026nbsp;conceptualization, project administration, writing - review \u0026amp; editing, funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the National Key Research and Development Program of China (2021YFD1300300), the Innovation Fund of Henan University of Technology (2020ZKCJ25), and the Cultivation Programme for Young Backbone Teachers in Henan University of Technology (21421204).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used in the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experimental protocols employed in this study were thoroughly evaluated and authorized by the Animal Protection and Use Committee of Henan University of Technology, Zhengzhou, China, in strict accordance with the Chinese Guidelines for Animal Welfare. The research was carried out at the Henan Agricultural University Teaching Experimental Field located in Xuchang, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollege of Bioengineering, Henan University of Technology, Lianhua Street 100, Zhengzhou 450001, China; Email: [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCastro J, Barros MM, Ara\u0026uacute;jo D, Campos AM, Oliveira R, Silva S, et al. Swine enteric colibacillosis: Current treatment avenues and future directions. 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Histochem J. 2001;33(2):79-86.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-animal-science-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jasb","sideBox":"Learn more about [Journal of Animal Science and Biotechnology](http://jasbsci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jasb/default.aspx","title":"Journal of Animal Science and Biotechnology","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ZnCA, pharmacological doses of ZnO, ETEC K88, weaned pigs, AGPs, antibiotic growth promoters","lastPublishedDoi":"10.21203/rs.3.rs-5194232/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5194232/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEnterotoxigenic \u003cem\u003eEscherichia coli\u003c/em\u003e (ETEC) is one of the primary causes of diarrhea in piglets, creating substantial economic losses in the swine farming industry worldwide. This study aimed to investigate the impact of zinc caproate (ZnCA) on the intestinal health, growth performance, inflammatory status, and Zn homeostasis of weaned piglets challenged with ETEC K88. In total, 48 weaned piglets (Duroc \u0026times; Landrace \u0026times; Yorkshire, 7.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 kg, 28 d) were selected for a 21-d experiment. Each experimental treatment consisted of six replicate pens with two piglets each. The treatment conditions were as follows: 1) a basal diet (\u003cb\u003eCON\u003c/b\u003e), 2) a basal diet\u0026thinsp;+\u0026thinsp;ETEC K88 (\u003cb\u003eNC\u003c/b\u003e), 3) a basal diet\u0026thinsp;+\u0026thinsp;2500 mg/kg Zn oxide (ZnO)\u0026thinsp;+\u0026thinsp;ETEC K88 (\u003cb\u003ePC\u003c/b\u003e), and 4) a basal diet\u0026thinsp;+\u0026thinsp;1600 mg/kg ZnCA\u0026thinsp;+\u0026thinsp;ETEC K88 (\u003cb\u003eZnCA\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe addition of 1600 mg/kg ZnCA to the diet of post-weaning piglets effectively enhanced growth performance and nutrient digestibility and reduced the incidence of diarrhea and inflammatory reactions caused by ETEC K88 infection. These therapeutic effects were comparable to those of pharmacological doses of ZnO. In terms of improving intestinal health and Zn homeostasis in post-weaning piglets challenged with ETEC K88, the effectiveness of 1600 mg/kg ZnCA surpassed that of pharmacological doses of ZnO.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOverall, under the experimental conditions of this study, ZnCA exhibited the potential to reduce the pharmacological dosage of ZnO while improving intestinal health and Zn homeostasis in weaned piglets.\u003c/p\u003e","manuscriptTitle":"Impact of zinc caproate supplementation on growth performance, intestinal health, anti-inflammatory activity, and Zn homeostasis in weaned piglets challenged with Escherichia coli K88","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-14 06:45:11","doi":"10.21203/rs.3.rs-5194232/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2024-10-30T04:55:04+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-10-11T09:06:28+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-11T03:33:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-07T11:17:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Animal Science and Biotechnology","date":"2024-10-05T08:18:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-animal-science-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jasb","sideBox":"Learn more about [Journal of Animal Science and Biotechnology](http://jasbsci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jasb/default.aspx","title":"Journal of Animal Science and Biotechnology","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ac09dde6-7e34-4e2e-947d-2ed3f5dd7d64","owner":[],"postedDate":"October 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-17T16:05:51+00:00","versionOfRecord":{"articleIdentity":"rs-5194232","link":"https://doi.org/10.1186/s40104-025-01172-2","journal":{"identity":"journal-of-animal-science-and-biotechnology","isVorOnly":false,"title":"Journal of Animal Science and Biotechnology"},"publishedOn":"2025-03-14 15:58:53","publishedOnDateReadable":"March 14th, 2025"},"versionCreatedAt":"2024-10-14 06:45:11","video":"","vorDoi":"10.1186/s40104-025-01172-2","vorDoiUrl":"https://doi.org/10.1186/s40104-025-01172-2","workflowStages":[]},"version":"v1","identity":"rs-5194232","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5194232","identity":"rs-5194232","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}