Alternative to ZnO to establish balanced intestinal microbiota for weaning piglets.

A wide range of phytobiotic feed additives are available on the market claiming to have beneficial effects on the growth of the host animal and to promote the development of a balanced microflora. The present study investigated the effects of the phytobiotic-prebiotic mixture of curcumin, wheat germ, and chicory on the growth performance and on the intestinal microflora composition of weaning piglets. Post weaning diarrhea causes significant losses for the producers, most commonly it is prevented by feeding high doses of zinc oxide (ZnO). The effect of a phytobiotic-prebiotic feed additive (1 kg T-1) was compared to a positive control (3.1 kg T-1 ZnO) and to a negative control (no feed supplement) in an in vivo animal trial. There was no significant difference in the final body weight and average daily gain of the trial and positive control groups, and both groups showed significantly (P<0.05) better results than the negative control. The feed conversion ratio of the phytobiotic-prebiotic supplemented group was significantly improved (P<0.05) compared to both controls. Both phytobiotic-prebiotic mixture and ZnO were able to significantly reduce (P<0.05) the amount of coliforms after weaning, even though ZnO reduced the amount of coliforms more efficiently than the trial feed additive, it also reduced the amount of potentially beneficial bacteria. Metagenomic data also corroborated the above conclusion. In the trial and positive control groups, the relative abundance of Enterobacteriaceae decreased by 85 and 88% between 3 weeks and 6 weeks of age, while in the negative control group a slight increase occurred. Lactobacillaceae were more abundant in the trial group (29.98%) than in the positive (8.67%) or in the negative (22.45%) control groups at 6 weeks of age. In summary, this study demonstrated that a phytobiotic-prebiotic feed additive may be a real alternative to ZnO for the prevention of post weaning diarrhea and promote the development of a balanced gut system.

Introduction

Weaning of piglets is a complex process [1], where changes of the gut microbiota may cause digestive disorders, primarily caused Enterobacteriaceae that include pathogens such as Salmonella enterica [2, 3] and Escherichia coli (E. coli) [4-6]. For long time antibiotics or high levels of zinc oxide (ZnO) have been used as feed additives for the prevention and treatment of these post-weaning disorders in piglets [7]. Many studies proved that dietary supplementation with high levels of ZnO effectively suppressed the growth of E. coli and other coliform bacteria in weaning piglets [8-10]. However other studies showed adverse effects of high dietary ZnO, such as reducing the numbers of intestinal beneficial bacteria, or increasing environmental zinc emissions [11-13]. European legislation limits total dietary zinc to 150 mg kg-1 in piglet feed [14], and experts have already proposed to further reduce ZnO levels in swine diets [15]. As more and more countries are implementing restrictions on the use of medical ZnO levels in swine diets alternatives ought to be investigated. Many in vitro and in vivo studies suggested that phytobiotics and/or prebiotics could be alternatives to antibiotics and ZnO in animal diets [16-18].

Phytobiotics are natural, biologically active substances that are abundant in essential oils [19, 20]. Curcuma longa has long been used in traditional Asian culture, for its flavors and health benefits. It’s primary active substance is curcumin, a hydrophobic polyphenol [20]. In vitro and in vivo studies showed that curcumin has various biological properties including antioxidant, anti-inflammatory, and anti-carcinogenic effects [21-23]. Additionally, a variety of gastrointestinal disease models demonstrated the protective effects of curcumin on the intestinal mucosa barrier in humans and in animals [24-26]. These beneficial effects of curcumin fate it to be a favored ingredient in phytobiotic feed additives [20, 27].

Prebiotics, such as wheat germ and chicory, are non-digestible oligosaccharides that reach the intestine undigested and stimulate the growth of beneficial bacteria [28]. Matteuzzi et al. [29] showed that wheat germ has prebiotic effects due to its polysaccharide and raffinose content. These compounds resist digestion and reach the large intestine, where they affect the colonic microflora by promoting the growth of Bifidobacteria and Lactobacilli. Chicory contains large quantities of prebiotic compounds namely inulin and oligofructose [30]. These compounds promote the growth of intestinal beneficial bacteria, induce the production of pro- and anti-inflammatory cytokines, and reduce the diarrheal incidence in swine [30-32].

Post-weaning diarrhea is caused by disbalance of the intestinal microflora of piglets. ZnO has long been used to control the proliferation of E. coli and reduce weaning diarrhea, however legislation and ecological efforts require to reduce its use. Phyto- and prebiotic feed additives may promote the development of a balanced microbiota of piglets and may prevent diarrhea. Thus, the objective of this study was to evaluate the effect of such a feed additive on the intestinal microbiota composition and on the growth performance in weaned piglets, to assess its use as an alternative to ZnO.

Materials and methods

Animals, diets, and experimental design

The animal experiments were conducted in strict accordance with the guidelines of Hungarian Government decree No. 40/2013. (II. 14.) and were approved by the Research Institute for Animal Breeding, Nutrition and Meat Science (Herceghalom, Hungary, approval number: 27/3/2015). No piglets were killed or injured in the study, only fresh fecal samples were collected from the floor of the pen.

A total of 110 piglets (Hungarian Large White × Hungarian Landrace) × (Pietrain × Duroc), weaned at 28 ± 1 day of age were involved in two consecutive experiments in 2018 (E1; from 11 July 2018 to 3 October 2018, 12 weeks) and in 2019 (E2; from 7 October 2019 to 6 January 2020, 13 weeks). Both experiments were performed at Herceghalom, Hungary. Animals received a two-phase basal diet: a pre-starter diet from 2 weeks of age until 5 weeks of age; and a starter diet from 5 weeks of age until the end of the trial (S1 and S2 Tables). During the nursing period, piglets had access to standard pre-starter feed from the second week after birth. Feed supplementation (with the trial feed additive or ZnO) started at 3 weeks of age and continued until the end of the experiment (S1 Fig). No other feed additives or antibiotics were included in any of the diets. Each pen was equipped with a stainless-steel feeder and a nipple drinker with ad libitum access to feed and water throughout the experiment.

Experimental groups received different diets: negative control (NC)–basal diet; positive control (PC)–basal diet supplemented with 3.1 kg T-1 ZnO; and trial diet (T)–basal diet supplemented with 1 kg T-1 phytobiotic-prebiotic feed additive containing curcumin, wheat germ extract, and zinc-chelate of tartaric acid, spray dried on chicory pulp.

In the E1 experiment, 54 piglets were randomly allocated into two treatments (T and PC) with 5 replicates per treatment (4–6 piglets per replicates). In the E2 experiment, 56 piglets were randomly allocated into three treatments (T, PC and NC) with 4 replicates per treatment (4–6 piglets per replicate). In each pen, there was an equal number of male and female piglets. Piglets were weighed individually at weaning (day 28 ± 1 day) and at the end of the experimental period (12 or 13 weeks), and average daily gains (ADGs) were calculated. Feed intake was recorded on a pen basis daily, and average daily feed intake (ADFI) and feed conversion ratio (FCR) were calculated for all treatment groups. Piglets were observed daily for signs of diarrhea from the start of the application of the pre-starter diet (two weeks of age). The severity of diarrhea was assessed visually by using a fecal consistency scoring (0, normal; 1, soft feces; 2, mild diarrhea; 3, semi liquid diarrhea and 4, liquid diarrhea) as described by Jamalludeen et al. [33].

Sample collection and fecal microbiota composition by enumeration

One fecal sample per pen was collected at three time points during the E1 and E2 experimental periods: one week before weaning (3 weeks of age; before the start of feed supplementation), two weeks after the weaning (6 weeks of age) and at the end of the feeding trial (12 weeks of age). The three sampling time points (3, 6, and 12 weeks of age) are referred to as 3W, 6W, and 12W. Although the duration of the experiment was one week longer in E2, the fecal samples were collected at the same age in both cases (S1 Fig). The samples were collected during the peak hours of defecation in sterile fecal containers (Biolab Inc., Budapest, Hungary) from the pen floor. The floor was cleaned before and after sampling to avoid contamination.

The fecal samples were transported on ice to the laboratory, and processed immediately. The samples were homogenized with a sterile plastic spoon and small portions (4–5 g) of each sample were stored at -80 °C until DNA extraction and metagenomic analysis. Serial 10 –fold dilutions were prepared from the remaining portion of each sample in 1% Trypton saline (1 g L-1 of Trypton dissolved in 8.5 g L-1 of NaCl solution). One hundred μL from the 103 to 107 serial dilutions were plated in duplicate on different culture media: De Man, Rogosa and Sharpe Agar (MRS), Eosin Methylene Blue Agar (EMB), Nutrient Agar and Columbia Blood Agar media were used for lactic acid bacteria (LAB), coliform-, total aerobic and anaerobic bacteria, respectively. All media were purchased from Biolab Inc. (Budapest, Hungary). The inoculated agar plates were incubated at 37 ± 1 °C for 24–48 hours using aerobic or microaerophilic/anaerobic conditions (the latter for LAB and anaerobic bacteria). Number of colonies (colony forming unit, CFU) on the Petri dishes were counted in the range of 10–300 CFU plate-1, and the number of viable bacteria were calculated per gram of feces (CFU g-1 feces, wet weight). The results are presented as log10-transformed data.

DNA extraction and metagenomic analysis

The metagenomic analysis involved all fecal samples from the three treatments (T, PC, and NC) and three sampling time points (3W, 6W, and 12W) of the E2 experiment. DNA was extracted from the fecal samples (50 ± 1 mg) using a Quick-DNA Fecal/Soil Microbe Microprep Kit (ZYMO Research, CA, USA), the extraction procedure followed the manufacturer’s instructions. The yield and purity of the DNA extracts were quantified using an Implen Nanophotometer P300 (Implen GmbH, München, Germany). Purified DNA from four samples (by pens) per treatment per sampling time were pooled, and were used for sequence analysis. The abundance of the bacterial communities of the fecal samples were measured by high-throughput sequencing on an Illumina MiSeq platform by UD-GenoMed Ltd. (Debrecen, Hungary). Amplification of the V3-V4 region of the 16S rRNA gene was performed following the recommendations of the 16S Metagenomic Sequencing Library Preparation Guide (Illumina, San Diego, CA, USA) [34].

DNA was amplified by 25 PCR cycles starting from 12.5 ng DNA using the KAPA HiFi HotStart Ready Mix (KAPA Biosystems, Wilmington, MA, USA). Each PCR cycles consisted of denaturing at 95 °C for 30 sec, annealing at 55 °C for 30 sec and extension at 72 °C for 30 sec. Post-amplification quality control was performed by an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). MagSi-NGSPrep Plus (Magtivio BV, Limburg, Netherlands) magnetic beads were used to purify the amplicons. The Index PCR was run with the Nextera XT Index Kit (Illumina, San Diego, CA, USA) with 502, 503, 504, 701, 702, 703, 704, 705, and 706 index primers. KAPA HiFi Hot Start Ready Mix was used for the subsequent PCR reaction, with: 8 cycles of denaturing at 95 °C for 30 sec, annealing at 55 °C for 30 sec, and extension at 72 °C for 30 sec. Before the library quantification MagSi-NGSPrep Plus magnetic beads were used again to clean the PCR products. For the library validation, 1 μL of the diluted final library was run on a Bioanalyzer DNA 100 chip on an Agilent Bioanalyzer. Next, each library was normalized, pooled, and loaded onto an Illumina MiSeq platform for 2x250 bp paired-end sequencing (Illumina, San Diego, CA, USA).

The 16S rRNA gene paired-end amplicon reads were processed using the Frogs pipeline [35]. Briefly, forward and reverse reads were filtered and merged using VSEARCH [36] with the parameters: min amplicon size: 44; max amplicon size: 550; mismatch rate: 0.15. Merged sequences were clustered using Swarm [37]. Chimeric sequences were removed using remove_chimera.py of the Frogs pipeline. Taxonomic assignment was performed using BLAST [38] against SILVA_SSU_r132_March2018 database [39] for ribosomal small-subunit RNA.

Statistical analysis

Statistical analysis of piglet growth performances and CFU values were performed with R Statistical Software 4.0.4 [40]. Values were expressed as means ± standard deviation. For the statistical evaluation of growth parameters (body weight, ADFI, ADG, and FCR) and fecal bacterial enumeration, pens were used as the experimental unit (n = 4-6/pen for the growth parameters; n = 1/pen for the fecal bacterial enumeration). Differences between treatments were determined by one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test. In all tests, a P-value < 0.05 was considered to indicate statistical significance. The alpha diversity indices were calculated using functions in the vegan package of the R environment [41]. Multidimensional scaling analysis (MDS) was performed using the ‘plotly’ function of the R environment. Linear discriminant analysis (LDA) coupled with the effect size (LEfSe algorithm) was used to identify characteristic biomarkers for each age group based on the abundance values [42]. Z-scores were calculated to demonstrate the abundance of the taxonomic profiles in each sample with the formula of z = (x − μ)/σ, where x is the abundance of the taxonomic profiles in each sample, μ is the mean value of the abundances in all samples, and σ is the standard deviation of the abundances. The heatmap of the results was created with the R package pheatmap [43].

Results

Growth performance of piglets

The effect of feeding the phytobiotic-prebiotic feed additive (1 kg T-1) on the growth parameters of piglets was evaluated in two independent trials, and compared to a positive control group with 3.1 kg T-1 ZnO and a negative control group without any treatment (Table 1). There were no statistically significant differences in the body weight, ADFI, and ADG between T and PC groups in the E1 trial. FCR of PC was statically significantly higher than of the T group (P<0.05). In the E2 trial, both T and PC groups had statistically significantly higher body weights than the NC group (P<0.05), but there was no significant difference between body weights of the T and PC groups. In the E2 trial, the ADFI of the PC group was significantly higher than that of the T group or the NC group (P<0.05). In contrast, both the PC and T groups showed statistically significantly higher ADG than the NC group. The FCR of the T group was statically significantly lower than that of the PC or NC group. Overall, the growth parameters in the two experiments indicated that the phytobiotic-prebiotic feed additive performed equally or better than the PC group, and both the T and PC group performed significantly better than the NC group. Furthermore, fecal consistency scores varied between 0 to 1 through both experiments (S3 Table), hence no diarrhea was observed in any of the groups. These results show that from the production perspective the phytobiotic-prebiotic feed additive presents an alternative to ZnO for enabling the smooth weaning of piglets. Next, we sought to assess the effect of different treatments on the microbiome of the animals.

Table 1

Growth performance of piglets in the trial (T), positive control (PC), and negative control (NC) groups during the two experiments.

Item	E1_T	E1_PC	E2_T	E2_PC	E2_NC
Number of piglets	28	26	20	16	20
Weaning weight, kg	7.70±1.32	7.23±1.48	7.23±1.26	6.52±1.06	6.58±0.94
Final weight, kg	27.29±5.92	27.29±9.27	29.44±5.53B	29.81±4.72B	25.51±4.30A
ADFI, g/day	773±176	842±98	736±21A	879±42B	731±50A
ADG, g/day	363±103	371±153	352±82B	370±66B	300±59A
FCR	2.13±0.07a	2.27±0.13b	2.09±0.07A	2.38±0.09B	2.44±0.25B

Statistical significance between the groups was determined separately for the two experiments, based on the data measured at the end of the experiments. The experiment ended at days 56 and 63 post weaning for E1 and E2, respectively.

a-b Means with the different lowercase letters differ significantly (P<0.05) during the E1 experiment.

A-B Means with different capitals differ significantly (P<0.05) during the E2 experiment.

Fecal microbiota composition by enumeration

The microbiota composition was characterized by traditional cultivation-based methods from excreted feces (Table 2). The first fecal samples were collected during the nursing period, before the start of differential feeding (3W).

Table 2

Fecal microbiota composition of piglets at different ages and diets.

Samples	Treatments
	E1_T1	E1_PC1	E2_T2	E2_PC2	E2_NC2
	Total aerobic bacteria (log10 CFU g-1)
3W	8.70±0.33B	8.59±0.45	8.88±0.57B	9.00±0.15B	8.46±0.42AB
6W	7.88±0.32A	8.05±0.41	9.07±0.11B	8.98±0.11B	9.12±0.34B
12W	7.75±0.29A	7.63±1.38	7.86±0.31A	7.94±0.81A	8.16±0.40A
	Total coliform bacteria (log10 CFU g-1)
3W	8.15±0.07B	8.40±0.59B	8.80±0.53B	8.31±0.54C	8.01±0.62
6W	7.50±0.64B	7.63±0.82B	8.07±0.48AB	7.57±0.26B	7.85±0.36
12W	5.45±0.79A	5.82±0.79A	7.44±0.56Ab	6.64±0.22Aa	7.43±0.65b
	Total lactic acid bacteria (log10 CFU g-1)
3W	8.65±0.10Aa	8.96±0.08b	9.04±0.22Ab	8.17±0.33Aa	7.88±0.59Aa
6W	9.55±0.26C	9.17±0.40	9.42±0.10Bb	8.85±0.07ABa	9.27±0.19Bb
12W	9.06±0.25B	9.15±0.26	9.53±0.28B	9.16±0.37B	9.31±0.14B
	Total anaerobic bacteria (log10 CFU g-1)
3W	8.98±0.19A	8.99±0.20A	9.08±0.28Ab	9.09±0.18b	8.21±0.46Aa
6W	9.51±0.25B	9.46±0.27B	9.49±0.18B	9.29±0.25	9.33±0.16B
12W	9.38±0.22AB	9.70±0.45B	9.59±0.21B	9.31±0.27	9.40±0.33B

1For E1, the number of samples was n = 3 (3W) and n = 5 (6W and 12W).

2For E2, the number of samples was n = 3 (3W) and n = 4 (6W and 12W).

Absence of letters signify that there was no statistically significant difference between the results.

a-b Means within a row (different treatments) with different lowercase letters differ significantly (P<0.05). Statistical significance between the groups was determined separately for the two experiments.

A-C Means within a column (different sampling times) with different capitals differ significantly (P<0.05). Statistical significance between the groups was determined separately for the two experiments.

The amount of aerobic, coliform and anaerobic bacteria was indistinguishable in the trial and positive control groups in E1 at the first sampling, in contrast the amount of LAB was significantly lower (P<0.01) in the T group than in the PC group. The difference in the amount of LAB was also present in E2, but in this case, the T group was significantly higher (P<0.01) than the PC and NC groups. The initial count of anaerobic bacteria was around 1x109 CFU g-1 feces, except for E2_NC, where it was significantly lower (P<0.01). In the E1 experiment, there was no significant difference in the fecal microbiota composition between the T and PC groups at 6 and 12 weeks of age (two weeks after weaning and at the end of weaning period). In the E2 experiment, the amount of LAB in the 6W samples and the amount of coliforms in the 12W samples were significantly (P<0.05) lower in the PC group than in the T and NC groups.

The age of the animals appeared to have a greater influence on the microbiota composition than the different treatments. The mean number of aerobic bacteria decreased significantly (P<0.05) from 3 to 12 weeks of age. The decrease was continuous in E1, but in E2 higher CFU values were observed at 6W than at 3W, although the increase was not significant. The number of anaerobic bacteria increased during the experiments: between 3 and 6 weeks of age a significant (P<0.01) increase was observed in E1_PC, E2_T, and E2_NC, in the other cases the increase was not significant. The CFU counts of 6W and 12W samples hardly differed and the final anaerobic counts were between 2.04 × 109 and 5.01 × 109 CFU g-1 feces. The number of coliform bacteria was above 108 CFU g-1 at the first sampling (3W) and decreased continuously from 3W to 12W. The decrease in the amount of coliforms was significant (P<0.01) between the first and the last sampling in all groups except NC, where the decrease was not significant. In the E1 experiment, the decrease in the number of coliforms was greater than in E2. The change in the amount of LAB was similar to that of anaerobic bacteria. In general, the amount of LAB in the first samples was lower than in the 6W and 12W samples, the increase was significant in all groups except in E1_PC.

Fecal microbiota composition by sequencing

16S rDNA sequencing of all feces samples from the E2 trial characterized the fecal bacterial composition of the piglets and assessed the effects of different feed supplements. Isolated nucleic acid from four individual fecal samples was pooled separately for treatments and sampling times before sequencing. Samples were named based on sampling times and treatments. As the first samples (3W) were taken before the treatments started (S1 Fig), the effects of the treatments were assessed by comparing the samples taken at 6 and 12 weeks.

Illumina MiSeq sequencing generated 2,914,984 sequences from the nine pooled samples, one for each sampling time (3W, 6W, and 12 W) and each treatment (T, PC, and NC). The paired-end sequences, with expected length, were chimera filtered and grouped into clusters. All clusters containing less than 0.005% of all sequences were removed from further analysis. Finally, 1,334,221 sequences, grouped into 721 Operational Taxonomic Units (OTUs) were kept and identified based on 97% species similarity. The number of sequences of the individual samples ranged from 101,818 to 180,941 and contained 569 to 672 OTUs per sample. The average OTU number of the 3W samples was significantly lower compared to those of the 6W and 12W samples (S4 Table). All nine samples shared 320 OTUs and all OTUs were present at least in two different samples. Although there was no sample specific OTU, several unique OTUs were identified when comparing the treatments at 3, 6, and 12 weeks or the age-dependent groups (S2 Fig).

Taxonomic profiles

A total of 14 phyla, 19 classes, 30 orders, 62 families, 195 genera and 277 species were detected by sequencing. Firmicutes and Bacteroidetes were the two most abundant phyla in all samples with an average abundance of 75.47% and 17.13%, respectively (Fig 1). Proteobacteria was the third most abundant phylum in 8 samples, with an average abundance of 4.43%, but this was only the fourth most abundant phylum in NC_3W sample (1.35%). In this sample (NC_3W), the third most abundant phylum was Euryarchaeota (2.16%), which proved to be the fifth in all other samples (0.56% in average, but ranged from 0.02% to 1.44% in all other samples). The average abundance of all other identified phyla was less than 1%, except for Actinobacteria (1.46%). Five phyla (Cyanobacteria, Patescibacteria, Synergistetes, Verrucomicrobia and WSP-2) were represented only by one OTU, although these OTUs appeared in almost all samples. The phylogenetic classification of samples showed greater (mainly age-related) differences at lower taxonomy ranks. At family level, the five most abundant families and their average abundance were Lactobacillaceae (13.46%), Ruminococcaceae (12.33%), Prevotellaceae (11.71%), Lachnospiraceae (10.40%) and Clostridia 1 (8.69%), respectively. The average relative abundance of top five genera was 13.46%, 8.50%, 6.71%, 4.52% and 2.48% for Lactobacillus, Clostridium sensu stricto 1, Streptococcus, Prevotella 9 and Romboutsia, respectively.

Fig 1

Relative abundance of bacterial community of the nine fecal samples.

(A) Top 10 phyla. (B) Top 20 families.

Bacterial diversity

Alpha diversity indices showed the largest differences by age (S5 Table). The number of observed species (P<0.01) and the Shannon index (P<0.05) were significantly lower at 3 weeks than at 6 and 12 weeks. Chao1 (P<0.01) and ACE (P<0.001) indices showed significantly higher diversity at 6 weeks than at 3 and 12 weeks, but the Simpson indices did not differ significantly. The effect of the treatments was evaluated based on the merged data of the 6W and 12W samples, as the 3W samples were collected before the start of the use of feed additives. The differentially abundant taxa between different sampling times were confirmed with beta-diversity analysis, at 3W higher bacterial diversity was present than at 6W and 12W (S3 and S4 Figs).

Age-related differences

The main age-related changes at phylum level was a decrease of Proteobacteria (from 6.91% to 1.86%), and an increase of Bacteroidetes (from 11.58% to 21.98%). Interestingly, the largest change occurred in Fusobacteria, where the average relative abundance decreased more than 400-fold, yet the relative abundance remained lower than 0.2% except for T_3W (0.97%).

The decrease of Enterobacteriales (3.37% to 0.22%) and Pseudomonadales (2.35% to 0.40%) induced the changes of Proteobacteria. Proteobacteria were represented by 38 OTUs in 3W and 6W samples but only by 31 OTUs in 12W samples (mainly due to the decrease of Acinetobacter-related OTUs).

Although the amount of Bacteroidetes increased steadily with age, certain taxonomic subgroups changed differently. Sphingobacteriales (1 OTU) were only observed in 3W samples, and Flavobacteriales were 21-fold more abundant in 6W samples than in 3W and 12W samples. Most of the families belonging to Bacteroidales decreased continuously with age, however the relative abundance of Prevotellaceae (85 OTUs) increased so significantly (from 2.35% to 18.42%), that the whole phylum increased. According to the linear discriminant effect size analysis (LEfSe) one of the 3W related feature was Bacteroidaceae and 12W related was Prevotellaceae (Fig 2). Most of the other significant features related to Clostridiales (341 OTUs) and younger age (3W: Christensenellaceae, Family XI, some Ruminococcaceae and Romboutsia; 6W: Lachnospiraceae). Although the average relative abundance of Clostridia decreased (from 52.22% to 34.93%), the abundance of other Firmicutes bacteria increased: Negativicutes (from 2.68% to 8.26%), Erysipelotrichia (from 3.27% to 4.43%), Bacilli (from 18.67% to 26.38%). In the case of Bacilli, the abundance of Bacillales decreased (from 3.82% to 0.77%) mainly due to the changes in Planococcaceae (Lysinibacillus was a significant 3W related feature). The relative abundance of Lactobacillales increased (3W: 14.85%, 6W: 27.02%, 12W: 25.61%), but the two most abundant families of Lactobacillales changed differently: Lactobacillaceae (17 OTUs) were more abundant in 6W (20.37%) than in 3W (10.77%) and 12W (9.25%), while the relative abundance of Streptococcaceae (3 OTUs) increased continuously (from 1.34% to 16.00%).

Fig 2

Differentially abundant taxa among sampling times.

For the analysis of age dependent change the data of all treatments per age was merged. (A) Histogram of the results of LEfSe among 3, 6, and 12 weeks-age and their respective effect size; P values <0.05 considered significant. (B) Heatmap of the 30 most abundant families of bacteria at different ages. The color scale shows the Z-score of abundance of families within each group.

Treatment-related differences

The microbial composition of treatments was compared at 6 and 12 weeks. At 6 weeks of age, the microbial composition of the NC and T samples (at all taxonomic levels) was more similar to each other than the PC sample (Fig 3). In the PC sample three phyla (Fusobacteria, Actinobacteria and Proteobacteria) and several families of Firmicutes, Bacteroidetes and Actinobacteria were more abundant. Lactobacillaceae were more abundant in T (29.98%) than in NC (22.45%) and in PC (8.67%). Clostridiales were less abundant in T (26.62%) than in NC (35.14%) and in PC (35.21%). Enterobacteriaceae and E. coli were also more abundant in T (1.20%) than in PC (0.21%), but in both cases it was lower than before the application of the feed additives (T_3W: 7.81%; PC_3W: 1.75%), thus in both T and PC groups the relative abundance of Enterobacteriaceae decreased by 85% and 88% between weeks 3 and 6. In contrast a slight increase was observed in the NC group (from 0.55% at 3W to 0.74% at 6W).

Fig 3

Heatmap of (A) all identified phyla and (B) the 30 most abundant families of bacteria.

The color scale shows the Z-score of abundance of phyla and families within each group.

At 12 weeks of age, Firmicutes were approximately 7.5% more abundant in T than in the control samples (78.99% compared to 71.57% and 71.47%), while the relative abundances were lower in the next three most common phyla (Bacteroidetes, Proteobacteria and Actinobacteria) than in the control samples. Lactobacillaceae and Streptococcaceae were more abundant in T and in NC than in PC. Clostridiales were most abundant in PC (38.63%; NC: 32.90%, T: 33.25%). Enterobacteriaceae and E. coli were 0.32% and 0.31% in T and in NC, respectively, while they were only 0.04% in PC.

Discussion

Many studies claim important effects of feed additives on the intestinal microbiota, the production and physiological parameters in piglets and in vivo microbiological and metagenomic studies are rarely presented together. In our study the piglet rearing data was analysed with the results of the traditional culture-based microbiological and modern metagenomic analyzes. Changes due to the age and the general composition of the intestinal microbiota of the pigs have been thoroughly studied [44, 45]. and it was confirmed by our results, however we chose to focus primarily not on general but on treatment-dependent changes, with a particular focus on analyzing changes of microbiota caused by phytobiotic-prebiotic compound compared to the controls.

Our results show in two independent trials that there was no significant difference in the final body weight and ADG of the animals in the T groups receiving the feed additive in 1 kg T-1 compared to the PC groups receiving ZnO in 3.100 kg T-1 dosage. Furthermore, both groups showed a significantly (P<0.05) higher ADG than the NC group receiving no feed additive. The FCR value was significantly lower for the T groups in both experiments compared to the PC groups (P<0.05), consequently the T group receiving the feed supplement performed better than PC in terms of feed to weight conversion.

Similarly to our experiments, Xun et al. [20] reported that a high dose of curcumin (0.3 or 0.4 kg T-1) significantly reduced the FCR compared to non-treated group, but the final weight and ADG were not affected. Shi et al. [46] also noted that 0.3 kg T-1 curcumin and the combination of curcumin and piperine, in addition to several other beneficial effects, significantly lowered the FCR compared to non-treated group. Fermented wheat germ was reported to significantly enhance immune status and increase ADG [47]. No significant effects were reported for chicory for ADG, ADFI, and FCR compared to basal diet, although chicory did have a beneficial prebiotic effect [48, 49]. In our case, similar results were observed (FCR decreased and ADG increased only compared to the basal diet), as the additives were not used separately but together.

The beneficial and harmful effects of ZnO have been reported in numerous studies [50-52]. One of the best-known applications of ZnO in pig rearing is the prevention of post weaning diarrhea caused by E. coli [4, 5, 50], despite the fact that the antimicrobial effect of ZnO is not specific and has remarkable effects on porcine microbial populations [53]. For example, it can lower the amount of LAB [54, 55] and its antimicrobial effect is mainly addressed against Gram-positive rather than Gram-negative bacteria [56]. Our CFU counts confirmed both observations: ZnO significantly reduced the number of coliforms in both experiments (E1, P<0.001; E2, P<0.05), and the amount of LAB was also lower at 6 weeks in the ZnO fed PC group than in the T and NC groups (significantly in E2, P<0.05).

In the E1 experiment, PC and T treatments performed similarly in reducing the number of coliform bacteria as the average CFU counts were similar. In the E2 experiment, the feed additives performed slightly differently, even though after weaning (6W) the difference in the amount of coliforms was not significant (P>0.1), it became significantly higher in the T group compared to the PC group at 12 weeks (P<0.001). In conclusion the data indicate that the phytobiotic-prebiotic feed additive adequately controlled the number of coliform bacteria. The amount of LAB was significantly lower in the PC group than in the NC, while the CFU counts of LAB of the T group were tendentiously or significantly higher than in the PC and NC groups. In agreement with our results, ZnO has been reported to reduce LAB in numerous studies [50, 54, 55], while the active ingredients of the phytobiotic-prebiotic feed additive (curcumin, wheat germ and chicory) are known to promote the growth of LAB [48, 57].

In order to better understand the effect of the phytobiotic-prebiotic feed additive on the gut microbial community, the culture based broad microbial counts were complemented with the highly detailed metagenomic analysis of the fecal samples. The metagenomic analysis was performed in the E2 trial, in which the effect of the trial feed supplement on the intestinal microbiota community was compared with the effect of negative and positive controls. The same fecal samples were analyzed in both culture dependent and independent methods, but before the metagenomic analysis, the isolated DNA samples were pooled, therefore one sample represents the average data of the feces of four piglets. The effect of the treatments was evaluated based on the data of the 6W and 12W samples.

Based on alpha and beta diversity analysis, the microbial community composition varied mainly with age and not treatment. The 3W samples differed more by groups than the 6W or 12W samples, of the 721 identified OTUs, the 3W samples shared only 464, while the 6W and 12W shared 587 and 574 OTUs, respectively. The greater diversity of 3W samples may originate from dissimilar early life colonization [58], where the gut microbiome is thought to be shared at least partially from the parents, and it becomes relatively stable once the dense microbial population is established later in life [59]. Although the microbial communities within one age group are less different in 6W and 12 W, the Chao1, ACE and Shannon indices show the diversity to be significantly higher (P<0.05) than at 3W. The largest alpha diversity was observed at 6W when the ACE index was significantly higher (P<0.001) than at 3W or 12W, while Chao1 and Shannon indices representing the observed species were tendentiously higher than at 12W. Previous studies also found that the alpha diversity increased with age at the early life of piglets [44, 45].

In accordance with previous reports, the two most abundant phyla were Firmicutes and Bacteroidetes in all samples. Proteobacteria were the third most abundant except in one of the 3W samples, which is particularly interesting as at this age Proteobacteria is expected to be one of the dominant phyla. In this sample the relative abundance of Proteobacteria was 1.35% while in the other two 3W samples it was 10.70 and 8.68%, respectively. This may be due to different parental and environmental factors which significantly affect the pig microbiome, as it was reported previously [49, 59].

The trends observed in the culture- dependent microbiota analysis were confirmed by metagenomic data for Lactobacillaceae and Enterobacteriaceae. In agreement with the CFU changes during the E2 experiment, lower Enterobacteriaceae values were obtained for the PC group than for the T group (6W: 0.21% vs. 1.20%; 12W: 0.04% vs. 0.32%), and the relative abundance of the beneficial Lactobacillaceae was also lower at both ages (6W: 8.69% vs. 29.98%; 12W: 5.30% vs. 11.39%). Streptococcaceae showed less differences than Lactobacillaceae with higher relative abundance in the T group (17.92%) than the PC group (14.60%) at 12W. Like Lactobacillaceae, members of Streptococcaceae are known for their beneficial effects and has been generally considered to be health-promoting microbes [60]. In our case the 3rd most abundant OTU of all samples was Streptococcus gallolyticus which was reported previously to be an important candidate for improving porcine feed efficiency [59]. The relative abundance of Streptococcus gallolyticus was also higher in the T group (17.67%) than in the PC group (14.29%) at 12W. Previous studies revealed Christensenellaceae, members of Clostridiales, to be associated with health and increased feed efficiency [61, 62]. In our case Christensenellaceae were more abundant in the T group than in the PC group (6W: 0.42% vs. 0.20%; 12W: 0.94% vs. 0.71%). These potentially beneficial bacteria were the most abundant in the T group and more abundant in the NC group than in the PC group in general, indicating that the phytobiotic-prebiotic feed additive increased the amount of beneficial microbes. The decrease of LAB and other beneficial microbes was reported several times for ZnO treatment [50, 54, 55]. The metagenomic analysis concluded that the relative abundance of coliforms in the T group was higher than in the PC group after weaning (at 6W), nonetheless the decrease of coliforms compared to pre-weaning (3W) was similar (T: 85%, PC: 88% reduction). The decrease in coliforms combined with an increase in beneficial bacteria in the T group successfully controlled the post-weaning diarrhea. The results also indicate that the general antimicrobial effect of ZnO can be replaced by a more specific effect of natural feed additives for preventing post-weaning diarrhea.

Interestingly, some other families (Ruminococcaceae, Lachnospiraceae, Clostridiaceae 1) belonging to Clostridiales were less abundant in the T group compared to the PC group at 6W, however the differences became only marginal at 12W with the exception of Clostridiaceae 1 which were 3–4% more abundant in the PC group than in the T group. Prevotellaceae, a family of Bacteroidales, showed larger differences and it was also more abundant in the PC group compared to the T group (6W: 19.47% vs. 10.80%; 12W: 20.09% vs. 14.25%). Ruminococcaceae, Lachnospiraceae, and Prevotallaceae as well as Clostridiales and Bacteroidales were reported to be associated with better feed efficiency and healthier microbiome [60, 63, 64] as these bacteria may enable a more efficient energy harvesting through the fermentation of various polysaccharides and dietary proteins [65, 66].

Conclusion

This study compared the effects of curcumin, wheat germ, and chicory containing feed additive, ZnO and a basal diet on pig rearing and intestinal microbiome. The results demonstrate that the phytobiotic-prebiotic feed additive can be as effective as the commonly used ZnO in preventing post-weaning diarrhea and improve the feed efficiency of piglets. The feed additive was able to control the amount of potentially pathogenic bacteria and increase the number of health-promoting microbes (like Lactobacillaceae, Streptococcaceae and Christensenellaceae), unlike ZnO which have a non-specific general antimicrobial effect. The study used pharmaceutical levels of ZnO, 3100 mg kg-1 (3.1 kg T-1), while the current legislation only permits the use of 150 mg kg-1 in regular piglet feed which unlikely exerts the same effects as pharmacological levels of ZnO. Therefore, we believe, that such a phytobiotic-prebiotic feed additive may be a sustainable alternative to ZnO in piglet rearing.

Schematic design of the experiments.

The numbers (1–13) represent the average age of the animals in weeks. The dotted part represents the nursing period. The gray shaded part indicates the continuous monitoring of feed consumption. The colored areas indicate the type of feeds and treatments in the T and PC groups.

(TIF)

Click here for additional data file.

Venn diagram of OTU distribution among different treatments.

A, B and C: differences between treatments at 3, 6, and 12 weeks. D: age-related differences.

(TIF)

Click here for additional data file.

Beta-diversity of the nine fecal samples.

(A) Heatmap highlighting the Jaccard diversity index among the microbial populations. The higher the color intensity, the lower the similarity between the pairs. (B) Multi-dimensional scaling of the data set. The dots of green, blue, and purple represent 3, 6, and 12 weeks, respectively.

(TIF)

Click here for additional data file.

OTU abundance based heatmap of all samples.

(TIF)

Click here for additional data file.

The composition and nutritional parameters of pre-starter feeds.

(XLSX)

Click here for additional data file.

The composition and nutritional parameters of starter feeds.

(XLSX)

Click here for additional data file.

Fecal consistency scores according to Jamalludeen et al. [33] in E2.

(XLSX)

Click here for additional data file.

Base sequence information of all samples.

(XLSX)

Click here for additional data file.

Alfa diversity indices of all samples and grouped by age or treatment.

(XLSX)

Click here for additional data file.

21 Jan 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Saeed El-Ashram

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf  and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Additional Editor Comments (if provided): Please accept my apologies for the delay

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Use the term “diarrhea” only for your recommendation for the future studies, since your experimental design does not contain diarrhea treatment.

For discussion part, try to give explanation why your results are similar to the literature results, such as mechanism or physiological effects on bacterial functions. For example, phenol compound affects cell wall of gram-negative bacteria. Or the effect of poly saccharides or your study bioactive compounds etc….

Reviewer #2: The paper could be accepted for publication after minor revision.

1. Please correct typos in your MS.

2. Please format the tables in your MS to meet Plos standard. or upload tables using a seperate file.

Reviewer #3: The possibility of using phytobiotic-prebiotic feed additive to replace zinc oxide in weaned piglets was studied in this ms, which was great significance in production. But There are some major questions I would like to bring up in order to make the ms better for publication.

Major issues

1.There are a number of grammatical errors and instances of badly worded/constructed sentences. Please ask someone familiar with English language to help you rewrite this paper.

2. The effect of calculating the average value of all samples in Table 3.

3. How is a healthy intestinal flora defined? According to the decrease of harmful bacteria and the increase of beneficial bacteria? In fact, intestinal flora is a complex environment, which can not be judged as healthy only by relying on several bacteria.

4. Zinc Oxide instead of antibiotics was used to alleviate diarrhea. Which indicator was used to measure diarrhea change after Phytobiotic-prebiotic feed additive adding?

Minor issues

1. Line 25-the letter “P”, indicated statistical difference, should be in italics along the text.

2. Line 29- “though” and “but” should not be used together.

3. Line 42, 63 and 187- “that” should not be used after the comma.

4. Line 200, 202 and 206- “feces” instead of “faces”.

5. Line 259- “fourteen” is written as “14” that can match the latter better.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: PONE-D-2136256 Ghonimy letter.doc

Click here for additional data file.

22 Feb 2022

Response to reviewers:

Thank you for your comments and advice, we took it into focus when we have improved the manuscript. We respond to your review below:

General major changes:

- Language improvement: the language correctness of the manuscript has been checked.

- New result presentation: according to reviewer 1 and 3 we provided some new information about the monitoring of diarrhea. The fecal consistency scoring was assessed visually as described by Jamalludeen et al (2009). A new supplementary table was added too (S3 Tab). The original S3 Tab was renamed to S4 Tab (base sequence data).

- Table format change: the format of the tables in the manuscript has been changed to meet the requirements of PloS ONE. The original Table 3 was difficult to interpret (and understand) in this new format and was therefore transferred to the supporting materials section in its original format (S5 Tab).

- Significant restructuring of the manuscript for better comprehensibility. Rewriting of the introduction, discussion and conclusion, and minor changes throughout the text.

Response to individual reviewers:

Reviewer 1:

1. Use the term “diarrhea” only for your recommendation for the future studies, since your experimental design does not contain diarrhea treatment.

The manuscript was expanded with a new table showing the fecal scoring system used to monitor diarrhea during the experiments (S3 Tab). The applied method is detailed in “Materials and Methods” (see line 105-109) while results are summarized in “Results” (see lines 199-201).

2. L65, add the hypothesis and L 66-68, I suggest you using this rephrased paragraph

The objective of this study was to evaluate the effect of phyto- and pre-biotic feed additives (curcumin, wheat germ and chicory) as alternatives to ZnO on intestinal microbiota composition and growth performance in weaned piglets.

The introduction has been significantly redesigned and the aim of the study has been clarified. See lines L69-75, but the full introduction was reorganised or rewritten.

3. L 71, provide a figure describing your experimental design.

The design of the experiment is described in S1 Fig.

4. L 229, delete “with different”

It was corrected. See line L241.

5. L 58-61, it is already stated at introduction section.

According to your suggestion we rewrote and shortened the introductory part of the discussion section (see lines 358-366) and changed some other parts of the discussion. Additionally, some part of the original discussion was moved to introduction section.

6. L 363, write it in more simple way

The introductory part of the discussion has been reworked, see above (5.). See lines L358-366.

7. L 354-369, it is too long introduction till you started to explain your results, I suggest you summarizing it.

The introductory part of the discussion has been shortened and reworked, see above (5.). See lines L358-366.

8. L 377, explain why your results are in agreement with the literature.

This part of the discussion was reworked too. See lines 374-383.

9. For discussion part, try to give explanation why your results are similar to the literature results, such as mechanism or physiological effects on bacterial functions. For example, phenol compound affects cell wall of gram-negative bacteria. Or the effect of poly saccharides or your study bioactive compounds etc….

In this study we investigated the in vivo effect of a phyto- and pre-biotic feed additive containing a mixture of curcumin, wheat germ and chicory. Curcumin, the main bioactive substance of our feed additive, is reported as a strong antioxidant, anti-inflammatory, antibacterial, antifungal, and antiviral agent. Both wheat germ and chicory have been described as prebiotic material. These studies were mentioned in the introduction and in the discussion sections, which were also significantly rewritten (see lines 54-60; 65-68; 374-381; 402-403 and related citations: 20-27; 30-32; 46-49; 57-58).

In our current work, individual active ingredients such as the flavonoid and polyphenol content of curcumin or the effect of prebiotic additives on microbes were not planned to be demonstrated. The main novelty of our work was the use of the combination of these compounds and in this study the detailed in vitro was not investigated, therefore we decided not to detail the hypothetical approach in this study and only to report what our own results support.

10. L 481, summarize the conclusion stating the most important results based on your measurements, in addition to the future application or invention which may be used based on your results.

The conclusion was reworked based on your suggestions. See lines 471-481.

Reviewer 2:

1. Please correct typos in your MS.

The language correctness of the manuscript has been improved and checked.

2. Please format the tables in your MS to meet Plos standard. or upload tables using a seperate file.

The format of the tables in the manuscript has been changed to meet the requirements of PloS ONE. The original Table 3 was difficult to interpret (and understand) in this new format and was therefore transferred to the supporting materials section in its original format (S5 Tab).

Reviewer 3:

Major issue 1.: There are a number of grammatical errors and instances of badly worded/constructed sentences. Please ask someone familiar with English language to help you rewrite this paper.

The language correctness of the manuscript has been improved and checked.

Major issue 2.: The effect of calculating the average value of all samples in Table 3.

At the request of Reviewer 2, we changed the format of the tables and finally decided to include Table 3 as a supporting material (S5 Tab). The content of the table and its accompanying text was not affected by this change. Average values were calculated by age and by treatments. For treatments, only data at 6 and at 12 weeks were considered, as treatments had not been started until 3 weeks of age. Although calculating the average value may not be the most appropriate method to compare individual groups, we did not have the opportunity to sequence multiple samples, so comparing averages was the only way to determine differences within treatments and within age.

Major issue 3.: How is a healthy intestinal flora defined? According to the decrease of harmful bacteria and the increase of beneficial bacteria? In fact, intestinal flora is a complex environment, which can not be judged as healthy only by relying on several bacteria.

Based on your suggestion we renamed the term “healthy microbiota” to “balanced microbiota”. The intestinal microbiota is a complex community where not only microbes form relationships with each other, but it is also influenced in many ways by the host-intestinal microbiota interaction. Complete removal of (non-pathogenic) microbial groups, that are normally part of the intestinal microbiota, would presumably cause significant microbiota imbalance, and it is difficult to clearly describe some microbes as “beneficial” or “harmful”.

We believe that the main purpose of the phytobiotic-prebiotic feed additive is to maintain the balance of the microbiota. During our experiments with the continuous feeding of the additive we found the increase of the number of certain microbial groups (like lactic acid bacteria), which are traditionally called “beneficial”, although there is no doubt that the number of other microbes has decreased in parallel. The increase in the number of beneficial microbes and the results of the production parameters obtained during pig rearing together confirmed that the effect of the applied feed additive was beneficial.

Major issue 4.: Zinc Oxide instead of antibiotics was used to alleviate diarrhea. Which indicator was used to measure diarrhea change after Phytobiotic-prebiotic feed additive adding?

According to your suggestion we provided some new information about the monitoring of diarrhea. The fecal consistency scoring was assessed visually as described by Jamalludeen et al (2009). A new supplementary table was added too (S3 Tab). Diarrhea was not observed in any of the experiments and fecal consistency scores remained in between 0 to 1 values.

Minor issues

1. Line 25-the letter “P”, indicated statistical difference, should be in italics along the text.

2. Line 29- “though” and “but” should not be used together.

3. Line 42, 63 and 187- “that” should not be used after the comma.

4. Line 200, 202 and 206- “feces” instead of “faces”.

5. Line 259- “fourteen” is written as “14” that can match the latter better.

All listed issues have been corrected (or modified during grammatical revision).

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

4 Mar 2022

Alternative to ZnO to establish balanced intestinal microbiota for weaning piglets

PONE-D-21-36256R1

Dear Dr. Juhász Ákos ,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Saeed El-Ashram

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I suggest you these main lines in your conclusion section:

This study compared the effects of curcumin, wheat germ, and chicory feed additives on the growth performance and intestinal microflora composition of weaning piglets. Phytobiotic-prebiotic feed additives effectively prevented the post-weaning diarrhea compared to ZnO application. That potentially pathogenic bacteria abundance was decreased. The bioactive compounds extraction of these feed additives in form of therapeutic dietary powder may represent a sustainable management of piglets health in future.

Reviewer #2: (No Response)

Reviewer #3: Congratulations to all authors of this Manuscript. I think it is perfect enough to publish on PLOS ONE.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

9 Mar 2022

PONE-D-21-36256R1

Alternative to ZnO to establish balanced intestinal microbiota for weaning piglets

Dear Dr. Juhász:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Saeed El-Ashram

Academic Editor

PLOS ONE