Sodium Humate And Glutamine Combined Supplementation Alleviate Diarrhea of Weaned Calves Via Alter Intestinal Microbiota And Metabolites

Background: Weaning is one of the most stressful periods that cause gastrointestinal tract dysfunction and diarrhea in calves. HNa and Gln were reported to exert benecial effects on promoting growth performance, decreasing diarrhea incidence, and modulating intestinal microbiota in animals. Therefore, this study investigated the effect of HNa and Gln combined supplementation on growth performance, diarrhea incidence, serum parameters, intestinal microbiome, and metabolites of weaned calves. Results: In Exp. 1, 40 calves at 51±3 days of age with similar body weight (66.82±4.31 kg) were randomly assigned to 4 treatments fed with a basal diet (NC group), and a basal diet supplemented with 100 mL of 1%, 3%, or 5% HNa+1% Gln, twice daily, respectively. In a 21-day trial, calves on the 5% HNa+1% Gln group had higher ADG and lower fecal score and diarrhea incidence than those in the control group (P < 0.05). In Exp. 2, 20 calves at 51±3 days of age with similar body weight (69.37±6.28 kg) were randomly assigned to 2 treatments fed with a basal diet (NC group) and a basal diet supplemented with 100 mL of 5% HNa+1% Gln, twice daily (H+G group, the dose was obtained from Exp. 1). In a 21-day trial, calves supplemented with HNa and Gln had higher nal BW and ADG, serum IgG concentration and GSH-Px and T-AOC activities, but lower fecal score, diarrhea incidence, as well as serum DAO, D-lac, TNF-α, and MDA concentrations compared to NC group (P < 0.05). Analysis of intestinal microbiota indicated that supplemented with HNa and Gln signicantly increased the abundance of phyla Firmicutes and genus of Bidobacterium, Lactobacillus, Olsenella, Ruminiclostridium 9, Howardella, and uncultured organism, whereas the abundance of phyla Bacteroidetes, genus of Helicobacter and Lachnoclostridium were decreased as compared with NC group. Moreover, untargeted metabolomics analysis revealed that supplemented with HNa and Gln altered 18 metabolites and enriched 6 KEGG pathways (primary fatty acid biosynthesis) compared to the NC group. Conclusions: This study showed that combined supplemented with HNa and Gln could decrease diarrhea of weaned calves, which may be associated with improved intestinal microbial ecology and altered metabolism prole.


Background
Weaning is one of the most stressful periods in calve life, which can cause gastrointestinal tract dysfunction and diarrhea [1]. High diarrhea incidence in weaned calves is the main cause of growth retardation and death, which seriously affects the welfare of calves and causes serious economic losses in the dairy industry [2]. Antibiotics have long been used in calves as growth promoters and therapeutic agents for diarrhea, but overuse of antibiotics resulted in development of antibiotic resistance and negative public health outcomes [3]. Previous studies indicated that intestinal microbiota plays an essential role in intestinal morphology, nutrient absorption, immunity response, and host health [4].
Meanwhile, intestinal microbiota participates in many metabolic activities of the host, such as amino acid and vitamin synthesis, and lipid and bile acid metabolism [5]. Studies have shown that weaning can signi cantly alter intestinal microbiota and metabolism in pigs, resulting in increased diarrhea incidence and growth retarded [6]. Thus, searching for a suitable alternative to modulate intestinal microbiota and metabolites of calves at weaning stages thus improving intestinal health has gained more and more attention worldwide.
Humic acids (HAs), which are derived from the decomposition and transformation of decaying organic matter in the soil, are natural organic bioactive agents. As a type of HAs, sodium humate (HNa) has antimicrobial, antioxidant, anti-in ammatory, and antidiarrheal activities [7]. It has been reported that HAs and HNa were allowed to use in animals for dyspepsia, diarrhea, and acute intoxication [8,9]. Wang et al.
[8] con rmed the bene cial effects of HNa supplementation in nishing pigs. The growth-promoting e cacy of HNa was also con rmed in broilers [11]. Glutamine (Gln) which could maintain intestinal integrity and prevent bacterial translocation is a major fuel source for rapidly dividing cells including enterocytes, macrophages and lymphocytes [10]. The bene ts of Gln supplementation on improving growth performance, repairing intestinal epithelium, enhancing nutrient digestion and absorption, activating the immune system, and modulating intestinal microbiota have been observed in rats [12], broilers [13], weaning piglets [14] and calves [15]. Furthermore, our previous study found that supplemention with HNa and Gln effectively improved the growth performance and decreased diarrhea incidence in weaned calves. Based on the studies above, we hypothesized that HNa and Gln combined supplementation might have therapeutic potential on calf diarrhea by modulating intestinal microbiota and metabolites in weaned calves.
In the present study, intestinal microbiota sequencing and fecal untargeted metabolomics were integrated to investigate the bene cial effects of HNa and Gln combined supplementation on weaned calves. Furthermore, the correlation of intestinal microbiota, metabolites and growth performance, diarrhea incidence, and serum parameters were also evaluated.

Materials And Methods
The experimental protocol was approved by the Ethics Committee of Northeast Agricultural University (Harbin, China). The study was conducted at Harbin Modern Farming (Harbin, China).

Exp. 1
Animals, diets, and experimental design A total of 40 Holstein calves at 51 ± 3 days of age with similar body weight (66.82 ± 4.31 kg) were fed 5 L of milk replacer until weaning (58 d of age), twice daily at 08:30 am and 4:30 pm. The milk replacer used in this study contained lactose ≥ 40%, CP ≥ 22%, crude fat ≥ 19%, water ≤ 4.0%, ash ≤ 8.0%, and ber ≤ 0.3%. All calves had free access to water and starter during the entire experimental period. The ingredients and chemical composition of the starter are shown in Table 1. All of the calves were randomly assigned to 4 treatments (n = 10): (1) NC (basal diet), (2) 1% H + G (basal diet supplemented with 100 mL of 1% HNa + 1% Gln, twice daily), (3) 3% H + G (basal diet supplemented with 100 mL of 3% HNa + 1% Gln, twice daily), and (4) 5% H + G (basal diet supplemented with 100 mL of 5% HNa + 1% Gln, twice daily). In the present study, 0, 1, 3, or 5 g of HNa and 1 g of Gln were mixed with 100 mL of milk replacer or water, respectively. The HNa and Gln were administered to each calf from a bottle before feeding (milk replacer or starter) at 08:30 a.m and 4:30 p.m. The trial lasted for 21 days.
Growth performance, fecal score, and diarrhea incidence The calves were weighed individually at the start and the end of the experimental period, and feed consumption per calves was recorded daily to calculate average daily feed intake (ADFI), average daily gain (ADG), and the ratio of feed to gain (F: G).
The fecal scores were monitored daily before the morning feeding according to the method of Renaud et al. [16]. Fresh feces were scored by consistency: 0 = rm; 1 = loose or moderate consistency; 2 = very loose or mild diarrhea; and 3 = watery or profuse diarrhea. Diarrhea was de ned as fecal scores ≥ 2 occurring for 2 or more consecutive days. The diarrhea incidence was calculated according to Renaud

Intestinal microbiota analysis
On day 73, fecal samples were collected from each calf before the morning feeding using sterile tubes, and then immediately frozen at liquid nitrogen until microbiota analysis. Total genome DNA from each digested sample was extracted using cetyltriethylammnonium bromide (CTAB) method, and then the integrity of extracted DNAs was detected by 1% agarose gel. The V3-V4 regions of the bacterial 16S rDNA gene were ampli ed by speci c primers: 341F (5′-CCTACGGGNGGCWGCAG − 3′) and 806R (5′-GGACTACHVGGGTATCTAAT − 3′) with the following procedures: initial denaturation at 98℃ for 1 min, followed by 30 cycles of denaturation at 98℃ for 10 s, annealing at 50℃ for 30 s, and elongation at 72℃ for 60 s, nally 72℃ for 5 min. PCR products were detected by 1% agarose gel and puri ed with and Agilent Bioanalyzer 2100 system. After the library was quali ed, it was sequenced by Illumina HiSeq (Illumina, San Diego, USA).
Paired-end reads from the original DNA fragments were merged using FLASH, and the sequences analysis was performed by UPARSE software package using the UPARSE-OTU and UPARSE-OTUref algorithms. In-house Perl scripts were used to analyze alpha (within samples) and beta (among samples) diversity, and PCoA (principal coordinates analysis) of weighted unifrac was generated in R project Vegan package (version 2.5.3). Sequences with ≥ 97% similarity were assigned to the same OTUs. The statistical signi cance of comparison in bacterial community composition between the two groups was assessed using Student's t-test, and STAMP software was utilized to con rm differences in the abundances of individual taxonomy between the two groups. The gradient was 85% B for 1 min and was linearly reduced to 65% in 11 min, and then was reduced to 40% in 0.1 min and kept for 4 min, and then increased to 85% in 0.1 min, with a 5 min. The column temperature was 25°C, and the ow rate was 0.3 mL/min. A 2 µL aliquot of each sample was injected. The ESI source conditions were set as follows: Ion Source Gas1 (Gas1) as 60, Ion Source Gas2 (Gas2) as 60, curtain gas (CUR) as 30, source temperature: 600℃, IonSpray Voltage Floating (ISVF) ± 5500 V. In MS only acquisition, the instrument was set to acquire over the m/z range 60-1000 Da, and the accumulation time for TOF MS scan was set at 0.20 s/spectra. In auto MS/MS acquisition, the instrument was set to acquire over the m/z range 25-1000 Da, and the accumulation time for product ion scan was set at 0.05 s/spectra. The product ion scan is acquired using information dependent acquisition (IDA) with high sensitivity mode selected. The parameters were set as follows: the collision energy (CE) was xed at 35 V with ± 15 eV; declustering potential (DP), 60 V (+) and − 60 V (−); exclude isotopes within 4 Da, candidate ions to monitor per cycle: 10.
The collected data were used to identify the structure of metabolites using self-built MetDDA and LipDDA methods (Shanghai Applied Protein Technology Co. Ltd). The original data were converted into mzXML format by ProteoWizard MSConvert, and then the XCMS program was used for peak alignment, retention time correction, and peak area extraction. In the extracted ion features, only the variables having more than 50% of the nonzero measurement values in at least one group were kept. The metabolite structure identi cation was based on accurate mass matching (< 25 ppm) and secondary spectrum matching methods and search of the laboratory's self-built commercial database (Shanghai Applied Protein Technology Co. Ltd).
After normalized to total peak intensity, the processed data were analyzed by R package (ropls), where it was subjected to multivariate data analysis, including partial least-squares discriminant analysis (PLS-DA) and orthogonal partial least-squares discriminant analysis (OPLS-DA). The 7-fold cross-validation and response permutation testing were used to evaluate the robustness of the model.

Statistical analysis
Individual calves served as the experimental unit. For the growth performance in Exp.1, data were analyzed by one-way ANOVA using SPSS 20.0 software (SPSS Inc., IBM, Chicago, USA). The differences among treatments were evaluated using Turkey's test. For the growth performance, fecal score, and serum cytokines concentration and antioxidant capacity in Exp.2, data were analyzed using the independent sample t-test of the SPSS. Results are presented as mean ± standard error of the mean (SEM) except for the diarrhea incidence. Differences were considered signi cant at P < 0.05. The correlation analysis among intestinal microbiota, metabolites, ADG, fecal score and serum parameters was estimated by Spearman's correlation coe cient. Correlations were considered signi cantly different at r > 0.50 or r < − 0.50, P < 0.05.

Results
Growth performance and diarrhea incidence (Exp. 1) As shown in Table 2, the initial BW, nal BW, ADFI, and F: G of calves were similar among treatments (P > 0.05). However, the ADG of calves in 3% H + G and 5% H + G was higher (P < 0.05) than NC group. The decreased fecal scores and diarrhea incidence were signi cantly associated with the HNa and Gln supplementation. The diarrhea incidence of calves in the NC, 1% H + G, 3% H + G, and 5% H + G group was 25.81%, 17.08%, 21.42%, and 15.41%. Data were shown as means ± SEM (n = 10).
Growth performance and diarrhea incidence (Exp. 2) As shown in Table 3, compared with NC group, the calves in H + G group had greater nal BW (P = 0.026) and ADG (P = 0.003). No signi cant differences in F: G and ADFI were observed among treatments. In addition, the calves in H + G group had lower fecal scores (P = 0.001) and diarrhea incidence than calves in NC group. Data were shown as means ± SEM (n = 10).

Serum parameters (Exp. 2)
The concentration of serum DAO and D-lac are shown in Fig. 1A and 1B. Compared with NC group, supplemented with HNa and Gln signi cantly decreased the concentration of serum DAO and D-lac (P < 0.05). As shown in Table 4, compared with NC group, supplemented with HNa and Gln increased the IgG (P = 0.018) concentration, as well as activities of GSH-Px (P = 0.003) and T-AOC (P = 0.005) in the serum of calves. Furthermore, lower concentrations of serum TNF-α (P = 0.047) and MDA (P = 0.002) were observed in the H + G group compared with the NC group. There was no signi cant difference among treatments in serum IgA, IgM, IL-6, and T-SOD concentration (P > 0.05). Data were shown as means ± SEM (n = 10).

Analysis of intestinal microbiota in weaned calves (Exp. 2)
In the microbiome study, 5,116,836 effective tags were acquired after ltering the data quality, with an average number of 255,842 tags per sample. Based on the 97% identity level, these sequences were decomposed into 1,912 operational taxonomic units (OTUs), while 1,035 and 884 speci c OTUs were observed in H + G and NC groups, respectively (Fig. 2B). The Chao1, Ace, Shannon, and Simpson indexes associated with bacterial richness and diversity were similar among groups ( Fig. 2A). The principal coordinate analysis (PCoA) plots showed an overlap of partial samples between NC and H + G groups.

Analysis of metabolic pro ling in weaned calves (Exp. 2)
In the present study, the untargeted metabolomics analysis was generated based on fecal samples by ultra-high performance liquid chromatography-quadrupole time-of-ight mass spectrometry (UPLC- Metabolites with VIP values > 1.0 and P-value < 0.05 were considered signi cantly change. As shown in Table 5, a total of 18 (10 positive ion mode and 8 negative ion mode) signi cantly changed metabolites in fecal samples of weaned calves were detected among NC and H + G groups according tom ultivariate statistical analysis. Additionally, there were 16 (1-Palmitoylglycerol, Terbutaline, 3-Aminobutanoic acid, Leu-Ala, Thr-Arg, Nitrosobenzene, 1-Palmitoyl-sn-glycero-3-phosphocholine, Tyr-Met, N6-Methyladenine, Oleic acid, Sebacic acid, 13(S)-HODE, D-Mannose, Nname, cis-9,10-Epoxystearic acid, cis-9-Palmitoleic acid, and L-Malic acid) signi cantly upregulated metabolites and 2 (2-Methylbenzoic acid and N-Acetyl-Dlactosamine) signi cantly downregulated metabolites in the H + G group compared to NC group. To organize and cluster the signi cantly different metabolites, two-way hierarchical cluster analysis was performed for comparisons between NC and H + G groups under positive ion mode (Fig. 6A) and negative ion mode (Fig. 6B), which indicated that the metabolites were highly differentiated among the groups.
To reveal the underlying mechanism, these changed metabolites were further performed by KEGG enrichment analysis. The data of pathway analysis re ected that there were 6 signi cant enriched pathways of metabolites in weaned calves supplemented with HNa and Gln, which included: 1) Fatty acid biosynthesis, 2) Proximal tubule bicarbonate reclamation, 3) C-type lectin receptor signaling pathway, 4) PPAR signaling pathway, 5) Lysosome, 6) Renal cell carcinoma (Fig. 6C).

Discussion
Diarrhea, which is one of the most prevalent diseases of weaned calves, could result in growth retardation, reduced feed utilization, and increased mortality [17]. The ban on antibiotic growth promotion (AGP) has further intensi ed the search for suitable alternatives. Thus, seeking some alternatives for antibiotics to maintain the health status and decrease diarrhea incidence of weaned calves is necessary.
In the present study, the ADG of weaned calves was signi cantly improved by HNa and Gln combined supplementation. Another interesting discovery is that the diarrhea incidence and fecal score were dramatically decreased in weaned calves supplemented with HNa and Gln. This was consistent with the previous report in piglets [18], broilers [19], and growth-retarded yaks [20].
Calve diarrhea induced by weaning is associated with impaired intestinal epithelial barrier [21]. DAO and D-lac can be used as useful biomarkers for monitoring the integrity of intestinal mucosa barrier [22,23].
When the intestinal barrier function is impaired, the serum levels of DAO and D-lac will increase. In the present study, weaned calves supplemented with HNa and Gln signi cantly decreased the concentration of serum DAO and D-lac. The results are similar to Yasar et al. [24], who reported that dietary supplementation with HAs decreased serum D-lac concentration in rats. In addition, the positive effects of dietary Gln supplementation on intestinal barrier function have been reported in broilers [25], piglets [26], and growth-retarded yaks [20].
To the best of our knowledge, some investigators have indicated that HNa or Gln could boost immunity by improving host antioxidant function and immunoglobulin concentration [27][28][29]. Weaning is one of the most severe early-life stresses for calves which could induce in ammatory response, oxidative stress, intestinal dysfunction, and diarrhea. In our study, we found that calves in NC group had higher diarrhea incidence and concentration of TNF-α and MDA in the serum, nevertheless, these indicators were lower in H + G group, and serum concentration of IgG, GSH-Px, and T-AOC were increased, which indicated that HNa and Gln supplementation may alleviate diarrhea caused by weaning via improving the antioxidant and anti-in ammatory capacity of calves. Consistent with our results, previous research found that dietary supplementation with HAs improved serum activities of T-SOD and GSH-Px, but decreased serum MDA levels in juvenile broilers [30]. Rensburg and Constance [31] indicated that HAs could inhibit the release of pro-in ammatory cytokines by inhibiting the activation of classical in ammatory pathways.
Moreover, the anti-in ammatory and immunomodulatory activities of Gln have been widely reported. For example, Zhou et al. [32] indicated that intravenously administered Gln increased the concentration of serum IgA and IgG, intestinal mucosal sIgA in early-weaned calves. Ma et al. [20] demonstrated that dietary supplementation with Gln signi cantly decreased the mRNA expression of IL-1β and TNF-α in growth-retarded yaks.
It is well known that intestinal microbiota plays an important role in nutrient utilization, intestinal morphology and immunity [33][34][35]. To clarify the mechanism of bene cial effects of HNa and Gln supplementation in weaned calves, the 16S rRNA sequencing and untargeted LC − MS metabolomics analysis were performed. From the results of phylum analysis, we found that the intestinal microbiota was dominated by Firmicutes and Bacteroidetes, and supplemented with HNa and Gln signi cantly increased the abundance of Firmicute but decreased the abundance of Bacteroidetes of weaned calves, which is consistent with previous studies conducted by Zhang et al. [6]. Firmicutes are considered as one of the producers of short-chain fatty acid, and more e cient in promoting nutrition absorption than Bacteroidetes [36]. At the genus level, weaned calves supplemented with HNa and Gln had higher abundance of Bi dobacterium, Lactobacillus, Olsenella, Ruminiclostridium 9, Howardella, and uncultured organism but lower abundance of Helicobacter and Lachnoclostridium as compared with NC group. It is well known, Bi dobacterium and Lactobacillus were probiotics, which have excellent e cacy in reducing gastrointestinal infections [33,37]. The results of the present study indicated that supplemented with HNa and Gln could increase the abundance of probiotics in intestinal microbiota. In addition, Spearman's correlation analysis also indicated that Bi dobacterium, Lactobacillus, and Olsenella were positively correlated with ADG, serum IgG level and T-AOC, while negatively correlated with fecal score and concentration of serum DAO, D-lac, TNF-α, and MDA. This observation might explain that supplemented with HNa and Gln could improve growth performance, anti-in ammatory, and antioxidative status and alleviate diarrhea of weaned calves via increasing the abundance of bene cial intestinal microbiota.
In addition, feces metabolites may also re ect the physiological status of calves [38,39]. Currently, LC-MS based metabolomics analyses are being increasingly performed to explore the alteration of metabolites [40]. Based on that, we examined the effect of HNa and Gln combined supplementation on metabolites of weaned calves. The results showed that the levels of fatty acid metabolites (Oleic acid, Sebacic acid, 1-Palmitoylglycerol, Nname,cis-9,10-Epoxystearic acid, cis-9-Palmitoleic acid), amino acid metabolites (3-Aminobutanoic acid, Leu-Ala, Thr-Arg, Tyr-Met), and carbohydrates metabolites (D-Mannose) were signi cantly up-regulated by HNa and Gln inclusion. Furthermore, the results of KEGG enrichments suggested that weaned calves supplemented with HNa and Gln primarily up-regulated the fatty acid biosynthesis pathway. Surprisingly, Spearman's correlation analysis found that up-regulated metabolites were positively correlated with increased bene cial intestinal microbiota. For example, Bi dobacterium was positively correlated with Leu-Ala and Sebacic acid, Lactobacillus was positively correlated with 3-Aminobutanoic acid and Oleic acid, Olsenella was positively correlated with 1-Palmitoylglycerol and Oleic acid. This indicated that the improved growth performance and decreased diarrhea incidence may be attributed to the modulatory role of HNa and Gln on intestinal microbiota and metabolites.
An interesting result obtained by Spearman's correlation analysis was that Oleic acid, Sebacic acid, and 1-Palmitoylglycerol was negatively correlated with the fecal score, while, Bi dobacterium, Lactobacillus, Olsenella, Ruminiclostridium 9, and Howardella was positively correlated with Oleic acid, Sebacic acid, and 1-Palmitoylglycerol, which indicated that decreased diarrhea incidence is closely related to increased abundance of bene cial intestinal microbiota and altered metabolites. This might provide a new evidence for the mechanism of decreased diarrhea of weaned calves supplemented with HNa and Gln. Currently, information on the mechanism of HNa and Gln supplementation on the intestinal health of weaned calves is limited, further investigations required to be conducted.

Conclusion
Weaned calves supplemented with HNa and Gln had a higher ADG, antioxidant status and intestinal barrier function thereby lower diarrhea incidence. Moreover, Analysis of intestinal microbiota and metabolic pro le revealed that weaned calves supplemented with HNa and Gln increased the relative abundance of intestinal bene cial microbiota and enriched many lipid metabolites and KEGG pathway of fatty acid biosynthesis. These ndings provide a better understanding of the mechanism of decreasing diarrhea of HNa and Gln supplementation, which could further provide useful information for developing an effective and safe non-antibiotics alternative in the dairy calve industry to prevent and treatment calves diarrhea.