Zinc methionine improves the lipid metabolism that is associated with the alteration of intestine mucosal proteomes and microbiota of newborn Holstein dairy calves

Abstract

Supplementation of diets with a modest amount of zinc methionine (ZM) has been documented to improve the growth performance and antioxidant function of dairy calves, but the underlying mechanisms remain elusive. In the present study, 16 dairy calves were allocated to a control (CON) group (calves consumed a basal diet) and a ZM group (calves ingested the basal diet and had an additional 455 mg ZM/day, corresponding to 80 mg zinc/day). The calves were fed these diets for 2 weeks, after which their serum antioxidant parameters, lipids, and jejunal mucosal proteome and microbiota were analyzed. The ZM group had lower levels of total cholesterol, triglycerides, and malondialdehyde but higher high‐density lipoprotein cholesterol and glutathione peroxidase activity in the serum. A total of 142 differentially expressed proteins in the intestinal mucosa between the CON and ZM groups had been identified (ZM upregulated 117 proteins and downregulated 25 proteins). In addition, the protein expression of acyl‐coenzyme A oxidase 1, fatty acid binding protein 2, and peroxisome proliferator‐activated receptor gamma was higher in the ZM group. 16S rRNA gene sequencing indicated beneficial microbes, such as Veillonellaceae, Akkermansia_muciniphila, and Bifidobacterium adolescentis, which were more abundant, whereas Acinetobacter lwoffii was less abundant in calf jejunal mucosa in the ZM group. Finally, the propionate, butyrate, and iso‐valerate concentrations in the jejunal digesta were greater in the ZM group than those in the CON group. Collectively, the present study shows that ZM supplementation improves the serum and intestine mucosal lipid metabolism associated with the alterations in mucosal fatty acids β‐oxidation and microbiota.

INTRODUCTION

Appropriate intestinal lipid metabolism is critical in newborn dairy calves because it affects fat digestion and intestinal health []. When lipid metabolism is dysregulated, lipid digestion and absorption is defective []. The excessive accumulation of lipid species in the intestine, including cholesterol, triglycerides, and long-chain fatty acids stimulates the generation of reactive oxygen species (ROS) [], resulting in intestinal mucosal injury and dysfunction, and therefore poses a threat to health [].

Zinc is a micronutrient that is necessary for a lot of biological processes in both animals and humans []. It has previously been shown that zinc affects lipid transportation and fatty acid oxidation [], and it is included in various health-promoting products [].

Zinc methionine (ZM), a chelate of inorganic zinc and methionine, is composed of zinc sulfate complexed with methionine in a 1:1:1 ratio of zinc, methionine, and the sulfate anion []. It has been demonstrated that early supplementation with ZM could reduce diarrhea incidence and promote the growth performance of dairy calves []. Furthermore, ZM also has beneficial effects on lipid metabolism and shows a high level of bioavailability []. However, the underlying mechanisms of its effects remain elusive.

The intestinal microbiota is a dynamic ecosystem made up of trillions of microorganisms; hence, its disruption is considered to affect intestinal health []. Furthermore, zinc has been shown to alter the intestinal microbiota composition []. Therefore, we hypothesized that ZM improves the antioxidant function of dairy calves through regulating intestinal lipid metabolism and the microbiota. The purpose of this study was to detect the effects of ZM supplementation on serum antioxidant parameters and lipid indices. Furthermore, 16S rRNA gene sequencing and the isobaric tags for relative and absolute quantification (iTRAQ) method were used to characterize the jejunal mucosal microbiome and proteome of the calves to further dissect the mechanisms of the antioxidant effects of ZM.

MATERIALS AND METHODS

The procedures had been approved by the Ethics Committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (Beijing, China), and calves were reared in accordance with the standards of the Institute of Animal Science, Chinese Academy of Agricultural Sciences Animal Care and Use Committee (Beijing, China).

Calves, diets, and experimental design

This study was performed at Beijing Sino Farm (Beijing, China). Sixteen newborn female Holstein dairy calves of equivalent body weight (initial BW, 39.5 ± 0.7 kg) were randomly assigned to two treatment groups (n = 8) using a random number generator (Microsoft Corp.). The two groups were a control group (CON, comprising calves fed raw milk and a starter diet) and a ZM group (comprising calves fed raw milk and a starter diet along with an additional 455 mg ZM/day [equal to 80 mg zinc/day]). The dose of ZM chosen was that used in previous studies []. The calves were fed these diets for a total of 14 days, during which period ZM was mixed with milk.

All the cows who underwent parturition were cared for by a trained individual, and the newborn calves were removed from their dams immediately and housed in individual pens (1.8 × 1.4 × 1.2 m) bedded with straw and enclosed with iron fences. Four liters of colostrum was administered to each calf within 1 h of birth. Only the colostrum with a concentration of IgG ≥50 mg/mL was fed to the newborn calves, which was measured with an optical refractometer (HT113ATC; Ht Tech Ltd Tianyuan Optical Instruments Co., Ltd.).

The calves were given 2 L raw milk from a bottle twice (at 08:30 and 16:00) in the first 3 days. From days 4–14, the calves were supplied 4 L raw milk twice daily (at 08:30 and 16:00) using a bucket. From days 1–14, the appropriate dose of ZM combined with 200 mL colostrum or raw milk were provided at 08:30 h, and then additional colostrum or raw milk was provided until all the ZM had been consumed. The raw milk contained mean percentages of 3.87% protein, 4.32% fat, 4.88% lactose, and 13.5% total solids. Fresh water and pelleted starter were provided by Beijing Sanyuan Seed Technology Co., Ltd., which were available to the calves between days 4 and 14. This starter comprised 89.5% dry matter, 20.0% crude protein, 2.75% ether extract, 10.0% acid detergent fiber, and 18.0% neutral detergent fiber. The initial zinc concentrations of milk and starter were 4.05 and 176 mg/kg dry matter, respectively.

Sampling and analysis

Milk samples were collected once a week during the experiment period. Every 50 mL of milk sample was added a preservative (Bronopol Tablet, D&F Control System, San Ranmon Inc.) and stored at −20°C. The fat, protein, lactose, and total solid contents of the milk were measured using infrared analysis (Foss MilkoScan 2000, Foss Food Technology Corp.).

Starter samples were taken once weekly, dried for 48 h at 65°C, and thereafter ground and passed through a screen of 1 mm. The dry matter content was analyzed by drying at 105°C for 4 h to a constant weight in an oven. The standard procedures of the Association of Official Analytical Chemists were used for the measurement of dry matter (method 930.15; []), ether extract (method 4.5.05; []), and crude protein (method 976.05; []). Previously described techniques were used to examine the contents of neutral detergent fiber and acid detergent fiber []. Inductively coupled plasma optical emission spectroscopy was used to evaluate the zinc concentrations of milk and starter.

Blood was sampled from each calf through the jugular vein with vacutainer tubes (BD Biosciences) before their morning feed on day 15. The serum samples were obtained by being centrifuged at 4°C, 3000 × g for 15 min using a high-speed centrifuge (Eppendorf 5810R; Eppendorf AG) and stored at −80°C until analyzed.

The calves were sacrificed by jugular puncture after inducing anesthesia at 08:30 on day 15 of the study. Jejunal mucosal samples were obtained by scraping onto sterile slides and then stored at −80°C until analyzed. Fresh jejunal digesta were taken and promptly frozen in liquid nitrogen for subsequent analysis of short-chain fatty acid (SCFA) concentrations.

Measurement of serum lipid indices

Assay kits were used to measure the serum triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) concentrations (Nanjing Jiancheng Bioengineering).

Measurement of serum antioxidants

The serum glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities as well as the malondialdehyde (MDA) concentration were measured using assay kits from Nanjing Jiancheng Bioengineering, in accordance with the manufacturer's instructions.

Jejunal mucosal proteome analysis

The iTRAQ method was used for proteomic analysis. The jejunal contents were diluted (1:10, w/v) in RIPA (Radio immunoprecipitation assay) lysis buffer (P0013B, Beyotime) containing protease inhibitors (1%, v/v, Sigma, P8340) and phosphatase inhibitors (1%, v/v, Sigma, P2850). The samples were homogenized three times on ice at 8500 g for 60 s before being centrifuged at 14,000 g for 15 min at 4°C. The supernatants were collected and stored at −80°C for subsequent analyses of iTRAQ and western blot. The proteins in the supernatants were digested with trypsin (Promega) overnight at 37°C, after which the peptides were dried by vacuum centrifugation and reconstituted in TEAB (Applied Biosystems). The iTRAQ tags (CON, tags 113, 114, and 115; ZM, tags 116, 117, and 118) and an iTRAQ reagent-8 plex kit (AB Sciex) were used to label the peptides. The labeled samples were cultured for 2 h at room temperature and then mixed and dried by vacuum centrifugation for subsequent analysis. Nano-reverse phase liquid chromatography (RPLC) was performed on the Eksigent nanoLC-Ultra^TM 2D System (AB Sciex). The samples were loaded onto a C18 nanoLC trap column (100 μm × 3 cm, 3 µm, 150 Å) and washed at 2 μL/min for 10 min using Nano-RPLC Buffer A (0.1% formic acid, 2% acetonitrile). An elution gradient of 5%–35% acetonitrile (0.1% formic acid), a 70-min gradient, and an analytical Chrom XP C18 column (75 μm × 15 cm, 3 μm, 120 Å) with a spray tip were used.

Data acquisition was conducted using the triple TOF 5600 system equipped with a Nanospray III source (AB Sciex) and a pulled quartz tip as an emitter (New Objectives). Data were collected utilizing ion spray voltage of 2.5 kV, 30 PSI curtain gas, 5 PSI nebulizer gas, and a 150°C interfacial heater temperature. Investigation over 250 ms scans were gathered, and up to 35 product ion scans were collected if the threshold of 150 counts per second (cps) for 2⁺–5⁺ charge states was exceeded. The entire cycle took 2.5 s to complete. The rolling collision energy setting was employed for all precursor ions for collision-induced dissociation. Half of the peak width (18 s) was chosen for dynamic exclusion. The precursors identified were then compared with the exclusion list.

Proteins were identified and quantified using Proteome Discoverer 1.4 (Thermo Fisher Scientific) and Mascot engine 2.2 (Matrix Science) against the Uniprot Bos taurus database (UniProt) with the following parameters: sample type, iTRAQ 8 plex (peptide tagged); cysteine alkylation, iodoacetamide; digestion, trypsin; instrument, triple TOF 5600; search effort, thorough; and user modified parameter files, no. A false discovery rate (FDR) of less than 5% was used to adjust the p-values. A protein was deemed to be differentially expressed if its fold change (FC) was >1.8 or <5/9 and its corrected p-value was <0.05. Blast2GO 3.3.5 was used for gene ontology (GO) mapping and annotation, and differentially expressed proteins were further examined and subjected to BLAST search of the Kyoto Encyclopedia of the Genes and Genomes (KEGG) database () to map them to pathways.

Western blot analysis

SDS-PAGE was used to separate the jejunal mucosal proteins before they were transferred to polyvinylidene difluoride membranes (Millipore). Prior to primary antibody incubation overnight at 4°C, the membranes were blocked with Tris-buffered saline containing Tween 20% and 5% skim milk for 2 h at room temperature. The following antibodies were used: acyl-coenzyme A oxidase 1 (ACOX1, 1:1000), fatty acid binding protein 2 (FABP2, 1:1000), and peroxisome proliferator-activated receptor gamma (PPAR-γ, 1:1000), which were provided by Proteintech Group and β-actin (1:5000), which was purchased from ImmunoWay Biotechnology Company. The blots were then treated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). ChemiDoc XRS (Bio-Rad) was used to identify immunoreactive proteins, and Image J (National Institutes of Health) was used to quantify band densities.

Analysis of the jejunal mucosal microbiota

Stainless steel beads were used to homogenize frozen tissues in a Mixer Mill MM 400. According to the manufacturer's recommendations, bacterial genomic DNA was extracted using a QIAamp® Fast DNA Stool Mini Kit (Qiagen). A Nanodrop 2000 (Thermo Fisher Scientific) was used to test the concentration and purity of DNA. The 16S ribosomal RNA genes' V3-V4 regions were amplified using the primers (F). ACTCCTACGGGAGGCAGCA and (R) TCGGACTACHVGGGTWTCTAAT. The PCR products were purified using a QIAquick® Gel Extraction Kit from Qiagen. 250 bp paired-end reads were produced after the library was sequenced using a Hiseq 2500 platform (Illumina). All the raw data involved in the present study had been uploaded to the NCBI Sequence read archive (SRA) under accession number PRJNA984622. Alpha-diversity (Chao1, Shannon, Simpson, and Observed species indices) was assessed using Student's t-test in SAS software (version 9.2; SAS Institute), and p-values were adjusted using an FDR of <5%. A corrected p of <0.05 was considered to represent statistical significance. The composition of the microbial community was visualized using principal coordinates analysis (PCoA) on the distance data. The structures of bacterial communities were contrasted using the Bray–Curtis distances method. The sample composition was examined using a PERMANOVA based on these distance matrices. The bacterial taxa present in each group were compared using the Mann–Whitney U test and linear discriminant analysis (LDA) effect size (LEfSe) analysis, and taxa with an LDA >2 and a p < 0.05 were considered to differ in their abundance.

SCFAs analysis in the jejunal digesta

The concentrations of the SCFAs acetate, propionate, butyrate, valerate, and isovalerate in the jejunal digesta were determined using gas chromatography []. Briefly, 0.5 g digesta and 8 mL of ultrapure water were combined in a screw-cap tube, and the mixture was sonicated for 30 min at 4°C and then centrifuged at 8000 g for 10 min. Subsequently, 1 mL of supernatant was diluted with ultrapure water (1:50), filtered through a 0.22 μm mesh, and injected into a Dionex ICS-3000 Ion Chromatography System (Dionex) for quantification.

Statistical analysis

Student's t-test was used to compare the groups in terms of SCFA concentrations, serum lipid indices, antioxidant parameters, and protein expression levels on SAS software (version 9.2). Spearman correlation analysis was conducted to evaluate the relationships between the jejunal microbiota, proteome, serum lipid indices, antioxidant parameters, and SCFA concentrations. p < 0.05 was considered to represent statistical significance.

RESULTS

Serum concentrations of lipid indices and antioxidant parameters

As shown in Figure , the serum TC and TG concentrations of calves in the ZM group were significantly lower than those of calves in the CON group (p < 0.01). However, the serum HDL concentrations of calves in the ZM group were higher than those of calves in the CON group (p < 0.01). As shown in Figure , ZM supplementation significantly increased the serum activity of GSH-Px (p < 0.05) but reduced the serum concentration of MDA in the calves (p < 0.01).

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Jejunal mucosal proteome

A total of 4819 proteins were identified using the iTRAQ proteomic analysis, and of these, 4466 were confidently identified as Unused >1.3 and Peptides >1. A total of 142 proteins were discovered to be differentially expressed in the jejunal mucosa of the CON and ZM groups (ZM upregulated 117 proteins and downregulated 25 proteins; Figure ). Proteins with FC > 1.8 or <5/9 and p < 0.05 were considered to be differentially expressed. Information regarding differentially expressed proteins is provided in supplementary file 1, involved in diverse biological processes. For example, copper transport protein (ATOX1) and sodium/potassium-transporting ATPase subunit beta (ATP1B3) were significantly upregulated in the ZM group, both of which are relevant to mineral absorption (p < 0.05). In addition, several differentially expressed proteins are engaged in other relevant biological processes, such as lipid transport and fatty acid oxidation. These differentially expressed proteins included ACOX1, SCP2 (Sterol carrier protein 2), ATOX1, RHOA (Transforming protein RhoA), APOA1 (Apolipoprotein A-I), APOC3 (Apolipoprotein C-III), SLC27A4 (Solute carrier family 27 member 4), and FABP2, and all of these were upregulated in the ZM group (p < 0.05).

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GO is a functional classification system for genes that was used to annotate the 142 differentially expressed proteins according to the second-level terms, including a cellular component, a biological process, and molecular function. As shown in Figure , with respect to the metabolic process, the differentially expressed proteins participate in lipid metabolic processes (22.0%), immune system processes (22.0%), lipid catabolic processes (11.4%), fatty acid metabolic processes (9.85%), glycerolipid catabolic processes (5.30%), and triglyceride catabolic processes (4.55%). In addition, approximately 50% of the differentially expressed proteins were found to be involved in single-organism metabolic processes. With respect to the cellular component, the categories included VLDL particles (3.79%), triglyceride-rich plasma lipoprotein particles (3.79%), HDL particles (3.03%), and intermediate-density lipoprotein particles (2.27%). With respect to molecular function, 31.6% of the differentially expressed proteins were classified as small molecule-binding and 12.8% as lipid-binding.

Furthermore, the differentially expressed proteins were mapped through the KEGG database. As shown in Figure , the KEGG pathways that significantly differed in their representation (p < 0.05) included the pyruvate metabolism, PPAR signaling pathway, fatty acid degradation, fatty acid metabolism, peroxisome, glycolysis/gluconeogenesis, mineral absorption, fat digestion and absorption, and fatty acid biosynthesis.

Expression of the proteins involved in lipid metabolism in the jejunal mucosa

The lipid metabolism-related proteins ACOX1, FABP2, and PPAR-γ were selected for validatory expression analysis by western blot out of the identified differentially expressed proteins. As presented in Figure , the jejunal mucosal expression of all three proteins was significantly greater in the ZM group (p < 0.05 or 0.01).

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Jejunal mucosal microbial profile

As shown in Figure , these two groups have no significant difference in alpha-diversity as measured by the Chao 1, Observed species, Simpson, and Shannon indices. The Bray–Curtis distances, obtained following PCoA analysis, revealed that no differences were observed in the microbial communities of the two groups (PERMANOVA; Figure ).

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The taxonomic composition of the CON and ZM groups is compared in Figure . At the phylum level, the jejunal mucosal microbiota was dominated by the Proteobacteria and Firmicutes, and the Actinobacteria and Bacteroidetes were also abundant. At the genus level, Sphingomonas, Lactobacillus, and Shigella were the most common in the jejunal mucosal microbiota of the calves. In particular, Lactobacillus and Sphingomonas accounted for large proportions of the jejunal mucosal microbes.

Mann–Whitney U test and LEfSe analysis were also employed to compare the principal taxa comprising the jejunal mucosal microbiota between the groups (Figure ). At the phylum level, the relative abundance of the Verrucomicrobiae was significantly increased by ZM supplementation (p < 0.05). At the family level, the relative abundance of the Verrucomicrobiaceae was higher, whereas that of the Bdellovibrionaceae was lower in the ZM group (p < 0.05). The ZM group showed higher abundances of Akkermansia, Mitsuokella, and Roseateles at the genus level but a lower abundance of Bdellovibrio (p < 0.05). Interestingly, the relative abundance of the species Akkermansia_muciniphila was significantly higher in the ZM group (p < 0.05). The ZM group also showed higher relative abundances of Mitsuokella_multacida, Bifidobacterium_adolescentis, and Leptothrix_ochracea (p < 0.05) and lower relative abundances of Sporosarcina_ginsengi, Acinetobacter_lwoffii, and Burkholderia_gladioli (p < 0.05).

SCFA content of the jejunal digesta

Dairy calves in the ZM group showed higher jejunal concentrations of propionate, butyrate, and isovalerate than those in the CON group (Figure , p < 0.05). However, there were no differences identified between the CON and ZM groups in the concentrations of the other SCFAs.

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Correlation analysis of the relationships between the jejunal mucosal microbiota and proteome, jejunal SCFA concentrations, serum lipid indices, and antioxidant parameters

As shown in Figure , the relative abundance of Akkermansia_muciniphila positively correlated with the expression of ACOX1 (R = 0.82, p < 0.05) and the concentrations of propionate (R = 0.72, p < 0.01) and isovalerate (R = 0.72, p < 0.01) in the jejunal digesta. In addition, the serum concentration of TG negatively correlated with the abundance of Akkermansia_muciniphila (R = 0.50, p < 0.05, Figure ). The relative abundance of Veillonellaceae positively correlated with the jejunal concentration of acetate (R = 0.57, p < 0.05) but negatively correlated with the serum MDA concentration (R = −0.55, p < 0.05). Furthermore, the expression of FABP2 positively correlated with the serum concentration of HDL (R = 0.94, p < 0.01, Figure ) and PPAR-γ expression negatively correlated with the serum TC concentration (R = −0.83, p < 0.05). Finally, ACOX1 expression negatively correlated with the serum MDA (R = −0.88, p < 0.05) and TC (R = −0.94, p < 0.01) concentrations.

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DISCUSSION

ZM is a highly bioavailable organic form of zinc that can be used to supplement the diet []. We have shown that ZM can improve the growth performance and serum antioxidant status of calves during the first 2 weeks of life [], but the mechanisms involved remain uncertain. To better understand the effects of ZM on the microbiota and host, for the first time, the effects of ZM on the jejunal mucosal proteome and microbial diversity of dairy calves were investigated in this study.

Previous research has demonstrated that lipid disorders and oxidative stress have a negative impact on the health of newborn calves []. Therefore, lipid indices and antioxidant property in the serum were analyzed to reflect the overall changes caused by ZM supplementation. TC and TG are lipid species that are present at high circulating concentrations in obesity, and HDL is the form of cholesterol that is transported back to the liver from the periphery []. Numerous studies have confirmed the significance of zinc for metabolism, and zinc deficiency leads to increases in TC and TG concentrations, hepatic fat deposition, and the development of metabolic disorder and obesity []. Conversely, zinc supplementation reduces the concentrations of TC, TG, and low-density lipoprotein cholesterol and increases that of HDL in hepatocytes []. Consistent with this, ZM reduced the serum TC and TG concentrations and increased that of HDL in the current investigation, which implies that ZM increases the clearance of lipids in calves. A negative lipid profile is closely associated with oxidative stress and poor antioxidant enzyme activity, and SOD and GSH-Px constitute a key component of the antioxidant system: SOD eliminates superoxide radicals and GSH-Px converts hydroperoxides to harmless derivatives []. Zinc has previously been shown to enhance GSH-Px activity and reduce the serum MDA concentrations in 14-day-old calves [], which is consistent with our results that ZM supplementation increases the plasma activity of GSH-Px and reduces the MDA concentration. A possible explanation might be that as a sulfur-containing amino acid, methionine is utilized as a thiol source for the production of glutathione []. MDA, as a product of lipid peroxidation causes oxidative stress in cells [], and the lower serum concentration of MDA implies less oxidative injury as a result of ZM supplementation, which is consistent with previous findings.

Most nutrients are absorbed in the small intestine, and zinc ions have their highest bioactivity and bioavailability in the jejunum []. Therefore, here, we evaluated how ZM and the host interacted in the jejunal mucosa. ACOX1 is the initiating and rate-limiting enzyme of the β-oxidation of long-chain fatty acids generating acetyl-CoA [], and the small intestine is the key site for fatty acid digestion and absorption. This is the first study, to our knowledge, to show that dietary supplementation with zinc increases ACOX1 expression in the intestinal mucosa of dairy calves. The excessive accumulation of long-chain saturated fatty acids contributes to obesity along with oxidative stress and apoptosis. Numerous studies have shown that zinc affects lipid metabolism, and our finding that ZM increases ACOX1 expression suggests that zinc increases β-oxidation in the intestine.

FABP2 transports long-chain fatty acids across the apical membrane of intestinal epithelial cells, after which they are oxidized or used to synthesize TG []. We found that ZM supplementation increases the expression of FABP2 in the intestinal mucosa, which is consistent with greater capacity for the mucosa to deliver fatty acids. However, previous studies have suggested that FABP2 represents a biomarker of obesity and high-fat diet-feeding increases the expression of FABP2 in the intestine [], which might represent an adaptive mechanism to accelerate the processing of fatty acids. The low serum TG concentration in ZM calves also implies a greater ability of the calves to metabolize fatty acids.

The transcription factor PPAR-γ is a member of the nuclear receptor superfamily that regulates processes including lipid metabolism []. Zinc affects the mRNA expression of peroxisome proliferator-activated receptors, the activation of which is important for insulin sensitivity, energy homeostasis, and lipid metabolism []. In particular, PPAR-γ is a key regulator of the absorption of long-chain fatty acids and the function of the intestinal barrier []. Thus, PPARγ knockout mice showed delayed uptake of lipids by enterocytes []. In this study, the expression of PPARγ was enhanced due to ZM supplementation, which might imply that greater ability of the mucosa to scavenge long-chain fatty acids would help reduce the inflammation caused by their excessive accumulation.

Owing to differences in the microenvironment, including those with respect to oxygen, nitrogen, and carbohydrate supply, the mucosal microbiota significantly differs from that of the lumen with respect to composition and function []. Mucosal microbes colonize the mucin glycans on the intestinal epithelial cells and produce metabolites that increase their capacity to influence digestion [], and therefore, the mucosal microbiota represents the first point of interaction with host metabolism []. Zinc supplementation, especially in the form of high doses of zinc oxide, is one of the most effective antidiarrheal supplements because of this metal's antimicrobial activity. However, there have been conflicting findings regarding whether zinc affects the diversity and composition of the microbiota. It has been reported that zinc oxide reduces the cecal microbiota diversity of the weaned piglets [], whereas a combination of a high amount of zinc oxide and antibiotics increases the microbial diversity of their ileal digesta []. In addition, other studies have shown that supplementation with various zinc sources fails to alter the richness or diversity of the intestinal microbiota []. This inconsistency might be the result of differences in dose or production methods. Coating, nanotechnology, and chelation substantially alters the bioactivity of zinc supplements []. In this study, supplementation with ZM did not change the alpha- or beta-diversity of the mucosal microbiota of the calves, which is similar to our previous study, where ZM supplementation had no effect on the microbial diversity of the rectal microbiota of 14-day-old calves []. Furthermore, these results suggest that ZM might not affect the intestinal microbial diversity of dairy calves during the first 2 weeks of life.

As discussed above, differences in their microenvironment explain the differences between the lumen and mucosal microbiota and the microbial profiles of each differ when exposed to the same diet []. Some mucosal microbes are closely involved in the regulation of intestinal homeostasis, which is mediated through direct interaction with intestinal epithelial cells []. Therefore, it is meaningful to characterize the microbial profile of calves being administered ZM. A previous study demonstrated that the Enterobacteriaceae and Lactobacillaceae have antidiarrheal effects and are more abundant in the feces of patients with acute diarrhea []. In the current study, the Lactobacillaceae and Enterobacteriaceae were the most abundant microbial families in calves being administered ZM, which is consistent with its capacity to reduce the diarrhea incidence of newborn calves.

Sphingomonas can utilize a wide range of carbohydrates, including arabinose, fucose, lactose, mannose, glucose, xylose, and polysaccharides, as energy sources []. The abundance of Sphingomonas positively correlates with body mass and small intestinal length [], suggesting that it might have growth-promoting effects. Verrucomicrobia is a phylum of gram-negative intestinal bacteria that was found to be more abundant in the ZM group. This taxon has the ability to degrade carbohydrates, including xylan, and produces SCFA, and therefore some species in this phylum, including Akkermansia_muciniphila, can regulate lipid metabolism. The Veillonellaceae are gram-negative, strictly anaerobic, and known to ferment organic acids, and especially lactate, to produce acetate, propionate, and fumarate. Indeed, a previous study demonstrated that the abundance of the Veillonellaceae has a positive correlation with the concentrations of acetate, propionate, butyrate, and valerate in the intestines of young pigs []. Consistent with the findings of previous studies, the high concentrations of SCFAs might be attributed to the greater abundance of the Veillonellaceae associated with ZM supplementation in this study.

Bdellovibrio is a genus of highly motile gram-negative bacteria with the ability to parasitize and kill pathogenic bacteria, such as Escherichia, Helicobacter, Salmonella, Legionella, and Shigella []. In the current study, the relative abundances of Bdellovibrionaceae and Bdellovibrio were lower in calves of the CON group. Similarly, the injection of Bdellovibrio bacteriovorus fights against an antibiotic-resistant pathogen Shigella flexneri in vivo, implying an improvement in antimicrobial function in the intestine []. Akkermansia muciniphila is an anaerobic, gram-negative, nonmotile bacterial species that colonizes the intestinal mucosa []. Because Akkermansia muciniphila can degrade mucin to generate SCFAs, it is now one of the most popular beneficial mucosally associated microorganisms []. An increasing number of research studies have revealed that Akkermansia muciniphila affects host energy homeostasis and lipid metabolism through the production of SCFAs []. Furthermore, the abundance of Akkermansia muciniphila is closely related to lipid metabolism. Akkermansia muciniphila administration has been shown to promote the clearance of TC and chylomicrons, thereby reducing acute lipid overload in the circulation []. Additionally, Akkermansia muciniphila is involved in the immunity of obese germ-free mice, which is at least in part mediated through SCFA production and oxidation []. Therefore, the low concentrations of TC and TG in ZM-supplemented calves might be the result of the greater abundance of Akkermansia muciniphila.

Bifidobacterium_adolescentis can ferment lactose as an energy source and proliferates in the milk fed to calves []. As a result, the Bifidobacterial genus generates acetate, lactate, and butyrate and improves intestinal homeostasis by relieving inflammation []. Acinetobacter_lwoffii is a gram-negative microbe that is an intestinal pathogen in humans and animals; it invades intestinal epithelial cells, is frequently resistant to multiple antibiotics, and can cause meningitis, septicemia, pneumonia, and gastroenteritis []. In this study, ZM supplementation reduced the abundance of Acinetobacter_lwoffii, implying that it can inhibit the growth of pathogens in calves [].

Acetate, propionate, butyrate, and isovalerate are the principal SCFAs produced by fermentation of carbohydrates in the intestine []. Besides providing energy, SCFAs also regulate the metabolic homeostasis of epithelial cells []. In the current study, the concentrations of propionate, butyrate, and isovalerate were increased by ZM supplementation, which might be attributable to the greater abundances of beneficial bacteria, such as the Veillonellaceae, Akkermansia_muciniphila, and Bifidobacterium_adolescentis. In the jejunum, the relative abundance of the Veillonellaceae positively correlated with the concentration of acetate and that of Akkermansia muciniphila positively correlated with the concentrations of isovalerate and propionate, which is consistent with greater microbial fermentation in the intestines of calves supplemented with ZM.

CONCLUSIONS

As shown in the present study, ZM supplementation improves the serum and intestinal mucosal lipid profiles of dairy calves, which are associated with alterations in intestinal fatty acid β-oxidation and the intestinal microbiota. Therefore, the results of the current study suggest that beneficial interactions between the microbiota and host might be promoted by ZM supplementation, and this provides new insights into the mechanisms of the beneficial effects of ZM supplementation in newborn dairy calves.

AUTHOR CONTRIBUTIONS

Xin Yu and Yeqianli Wo: Conceptualization; Visualization; Writing – original draft. Fengtao Ma: Investigation; Methodology; Data curation. Qiang Shan and Jingya Wei: Investigation and Resources. Peng Sun: Conceptualization; Supervision; Writing – review & editing; Project administration; Funding acquisition; Validation.

ACKNOWLEDGMENTS

This study was supported by the National Key Research and Development Program of China (2022YFD1300505; 2022YFD1301101), the earmarked fund for China Agriculture Research System (CARS-37), and the Agricultural Science and Technology Innovation Program (cxgc-ias-07, Beijing, China).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are openly available in NCBI Sequence read archive (SRA) under accession number PRJNA984622.

References

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