Effects of Fermented Feed on the Growth Performance, Intestinal Function and Microbiota of Piglets Weaned at Different Age

Effects of Fermented Feed on the Growth Performance, Intestinal Function and Microbiota of Piglets Weaned at Different Age | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Effects of Fermented Feed on the Growth Performance, Intestinal Function and Microbiota of Piglets Weaned at Different Age Shuai Liu, Yunxia Xiong, Jingping Chen, Hao Xiao, Qiwen Wu, Xiaolu Wen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1063432/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Apr, 2022 Read the published version in Frontiers in Veterinary Science → Version 1 posted You are reading this latest preprint version Abstract BACKGROUND: The beneficial function of fermented feed in livestock industry has been widely investigated. However, little is known about the effects of fermented feed on different weaned-day piglets. This study aimed to investigate the effects of fermented diet on the growth performance, intestinal function and microbiota of piglets weaned at age of 21 days and 28 days. RESULTS: The results found that weaning on d 21 significantly increased ( P < 0.05) ADG, and ADFI (calculated based on wet weight and dry matter), while reduced ( P < 0.05) F: G, the activities of trypsin and lipase of jejunum and villus height of ileum, compared with 28-d weaning. The protein levels of Occludin, Claudin-1, ZO-1 of ileum in the groups weaning on d 21 were less ( P < 0.05) than the groups weaning on d 28. Moreover, dietary supplementation with fermented diet upregulated ( P < 0.05) Occludin, Claudin-1, ZO-1 proteins of ileum, compared with the groups treated with control diet both weaning on d 21 and d 28. In addition, dietary supplementation with fermented diet decreased ( P < 0.05) the relative abundance of Clostridia (class) and increased ( P < 0.05) Bacteroidia (class) level of cecal microbiota, compared with the groups treated with control diet both weaning on d 21 and d 28. However, supplementation with fermented diet did not affect the concentrations of short-chain fatty acids in the cecum ( P > 0.05). CONCLUSION: Therefore, our data suggest that feed digestibility is improved in piglets weaned at 21 days, but intestinal barrier function is weaker than in piglets weaned at 28 days. However, compared with feeding control diet, supplementation with fermented diet both improved feed conversion and intestinal barrier function of weaned piglets by modulating intestinal microbiota. Animal Science Fermented diet Weaning age Intestinal function Intestinal microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Weaning would harm the intestinal function of piglets and lead to food intake reduction, diarrhea risk increasing and even death in severe cases [1-2]. Weaning affects production performance. Jamil et al found that the average daily gain (ADG) and average daily feed intake (ADFI) of piglets increased linearly with weaning age increasing [3]. Pigs weaned at 14 days old grow slower, and the feed intake are reduced compared to those weaned at 24 days old [3]. Compared with weaning on 12 days, weaning on 20 days increased ADG [4]. Alison has pointed out that delaying weaning can improve feed digestibility [5]. Another study showed that the villi height of jejunum in weaned piglets was significantly lower than in unweaned piglets at the same age [6, 7]. According to Blecha et al, early weaning can cause passive immunity weakened in piglets [8]. The study of Janeen found that piglets weaned at 28 days of age were stronger in immunity and had more antibodies after exposure to the virus than piglets weaned at 14 days of age [9]. Weaning age affects piglet performance, immunity and intestinal function. Therefore, weaning age is an extremely important parameter that affects piglets' growth. Soybean meal is widely used in pig production as a plant-derived protein source. However, soybean is limited to piglets’ diet because of the anti-nutritional factors, such as soybean globulin, β-glycoprotein, trypsin inhibitor and non-starch polysaccharide, which can exacerbate weaning stress, increase diarrhea risk, and hinder the growth rate of piglets. Currently, the use of microbial fermented feed is a common method to reduce antinutritional factors in feed. The amounts of soybean antigenic proteins (β-conglycinin and glycinin) in feed was decreased after fermented by Bacillus subtilis [10]. Feeding fermented feed can improve the intestinal health of piglets, increase productivity and change the intestinal microbial structure [11]. Study have shown that mixed solid-state fermentation of Bacillus subtilis, Hansenula anomala and Lactobacillus casei can improve the nutrient digestion of piglets [12]. Another study showed that fermented feed can increase the concentration of butyric acid in the cecum of piglets [13]. On the other hand, fermented diet can provide probiotics and their metabolites, which improve the digestibility of feed and intestinal function of weaned piglets [14-16]. However, studies on the effects of fermented feed on piglets at different weaning days are still lacking. Therefore, the present study was conducted to investigate the effects of fermented feed, weaning age, and their interaction effect on the growth performance, intestinal function and microbiota of piglets, and to provide a new insight for pig production. Materials And Methods 2.1. Fermented feed preparation Bacillus subtilis , Saccharomyces cerevisiae , and Lactobacillus ( Lactobacillus casei and Lactobacillus plantarum ) were used to ferment feed materials (corn, soybean meal, soybean hull) in this study. Mixture of Bacillus subtilis and Saccharomyces cerevisiae are considered as bacteria A which was purchased from Prosyn (Haerbing, China), and the number of viable Bacillus subtilis is 7.2×10 9 CFU/g, the number of viable Saccharomyces cerevisiae is 3.1×10 9 CFU/g. Lactobacillus is considered as bacteria B which was purchased from Inner Mongolia Sci-Plus Biotech (Inner Mongolia, China), and the total amount of viable Lactobacillus casei + Lactobacillus plantarum is 5.1×10 9 CFU/g. Bacteria A and B were mixed at a ratio of 1:1 as fermentation strains. Fermented feed was prepared with 18.1 kg ingredients, 6.9 kg water and 25 g fermentation strains, and then fermented in the laboratory for 5 days. The ingredients used to ferment were mixed at a ration completely corresponding with the basal diet (Table 1). The contents of anti-nutritional factors in the materials before and after fermented are shown in Table 2. Fermented feed was blended with other ingredients before used as fermented diet. 2.2. Animals and Experimental Design As shown in Figure 1, a 2×2 two-factor design was adopted in this study. Six 21-day-old piglets (Duroc×Landrace×Yorkshire, body weight about 6.15±0.02 kg) were selected from the same litter of 20 sows, and 3 among them were randomly selected as a replicate and to be weaned, and the others were continued to suckle until 28 days old before weaning. As soon as weaned, the replicates were randomly allocated into two treatments, fed with basal diet as the control group or fermented diet as the fermented group, respectively. The feed trial was finished when the piglets were at the age of 42 days old. Piglets with approximate average BW were randomly selected from each replicate to slaughter and sample at the age of 28 days old and 42 days old, respectively. The basal diet (Table 1) was formulated according to NRC (2012) to meet the nutritional requirements of piglets about 7-11 kg [17] without high copper and zinc supplementation. The nutrient composition of the experiment diet before and after fermentation is shown in Table 3. During the whole experiment, the animals were free to access feed and water. Once the experiment finished, ADG, ADFI and feed to gain ratio (F: G) were calculated. 2.3. Sample Collection At the end of the experiment, after fasting overnight, all animals were sacrificed after i. m. injection with sodium pentobarbital (40 mg/kg BW). The digesta from the stomach, jejunum, ileum, cecum and colon were carefully collected and snap frozen in liquid nitrogen. Tissue samples from the proximal duodenum, middle jejunum and distal ileum were dissected, and one sample was immediately frozen in liquid nitrogen for subsequent protein expression analysis, and another sample was fixed with 4% paraformaldehyde for subsequent morphological analysis. 2.4. Measurement of Digestive Enzyme Activity The activities of trypsin, lipase and amylase were determined by commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The protein concentrations of sample were determined with BCA kit (Thermo Fisher, USA), and the final results were expressed as /mg protein. 2.5. Intestinal Histopathological Examination The intestinal morphology was examined according to the method of Gao et al [18]. Briefly, after paraffin embedded fixed, intestinal segments were cut into 5-μm sections. Then, the sections were dewaxed with xylene, hydrated with alcohol, and stained with hematoxylin and eosin (H&E). Images were obtained by using a fluorescent orthochromatic microscope (Haier, China). 10 bright fields were randomly selected for each section. The villus height, and crypt depth were measured using image-pro image processing software (Media Cybernetics, Rockville, MD). 2.6. Western Blotting After extracted with lysis buffer (Biosharp, Anhui, China), the protein concentrations of samples were determined with the BCA kit (Thermo Fisher, USA), and then proteins were separated by 10% SDS-PAGE, and transferred onto a nitrocellulose membrane (BIO-RAD, USA). After blocking with blocking buffer (Beyotime, Shanghai, China), the membranes were incubated with the corresponding primary antibody at 4°C for 12-16 h, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. Bands were detected using ECL PlusTM Western Blotting Substrate (CliNX, Shanghai, China). Band intensity was quantified using ImageJ software (ImageJ 1.52, National Institutes of Health, USA). Primary antibodies for β-actin (1:10,000), zonula occludens-1 (ZO-1, 1:250), Occludin (1:1,000), and Claudin-1(1:1,000) (Abcam, MA, USA) were used in this study. 2.7. Microbiota Profiling Total genomic DNA of cecal content samples was extracted by using the OMEGA Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA, #D5625-01) according to the manufacturer's instructions and store at -20°C. Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify the sample DNA, and the DNA quality was checked by 1.2% agarose gel electrophoresis. PCR amplification of the bacterial 16S rRNA genes V3-V4 regions was performed using forward primer 338F (5’-ACTCCTACGGGAGGCAGCA-3’) and the reverse primer 806R (5'-TCGGACTACHVGGGTWTCTAAT-3'). Sample-specific 7-bp barcodes were incorporated into the primers for multiplex sequencing. The PCR components contained 5 μL buffer (5×), 0.25 μL Fast pfu DNA Polymerase (5 U/μL), 2 μL dNTPs (2.5 mM), 1 μL Forward and Reverse primer (10 μM), 1 μL DNA Template, and 14.75 μL ddH 2 O. The PCR amplification program consisted of initial denaturation at 98 °C for 5 min, followed by 25 cycles of denaturation at 98 °C for 30 s, annealing at 53 °C for 30 s, elongation at 72 °C for 45 s, and then a final extension at 72 °C for 5 min. PCR products were recovered and quantified by Vazyme VAHTSTM DNA Clean Beads (V azyme, Nanjing, China) and the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA), respectively. Paired-end 2×250 bp sequencing was performed using the Illumina kit and the Illumina MiSeq platform (Shanghai, China). The bioinformatic analysis was conducted with QIIME2 pipeline. Briefly, the clean reads were got by de-primed, quality filtered, denoised, spliced and de-chimerism using DADA2 method from raw reads [19]. After merged into Amplicon Sequence Variants (ASVs) feature sequence, singletons ASVs removed, the ASVs was clustered using QIIME2's classify-sklearn algorithm [20] and then annotation using Naive Bayes classifier based on Greengenes database [21]. The Rarefaction method is used to normalize and classify the ASVs with a depth of 95% [22, 23]. Chao1 [24] index and Observed species index were used to characterize richness, and Shannon [25] index and Simpson [26] index were used to characterize evenness of the bacterial community. Principal component analysis (PCA) based on ASVs physical distance, and principal coordinate analysis (PCoA) based on unweighted Unifrac distance are used to display the distribution of samples. 2.8. Determination of Short-Chain Fatty Acids Short-chain fatty acids in the cecal content were determined according to the method of Xiong et al [27]. Briefly, samples of cecum contents were added to NaOH and caproic acid-d3, and centrifuged to get supernatant. The supernatant was mixed with water, and propyl/pyrimidine, and propyl chloroformate, and to derive for 5 min. Finally, the supernatant was extracted twice with hexane, and the final supernatant was dehydrated with Na 2 SO 4 The determination was carried out with gas chromatograph-mass spectrometer detector (7890A and 5975C inert XL EI/CI mass spectrometric detector, Agilent Technologies, Santa Clara, CA). 2.9. Statistical Analysis SPSS 25 software (IBM, USA) was used for statistical analysis. The effects of fermented diet, and weaning age, and their interaction effect were assessed by ANOVA using general linear model (GLM) procedure. Results are represented as means and pooled standard error, and P < 0.05 indicates significant differences. Results 3.1. Weaning days and fermented diet both affect the growth performance of piglets BW, ADG, ADFI (calculated based on wet weight and dry matter), and F: G are shown in Table 4. The BW on d 21 and d 28 were similar among the treatments. Dietary supplementation with fermented diet increased ( P < 0.05) BW on d 42, compared with the groups treated with control diet both weaning on d 21 and d 28. Weaning on d 21 significantly increased ( P < 0.05) ADG, and ADFI (calculated based on wet weight and dry matter), and reduced F: G, compared with 28-d weaning. Piglets fed fermented diet had higher ( P < 0.05) ADFI (calculated based on wet weight) during 28-42 days old, but F: G was lower ( P 0.05) on all indexes. 3.2. Weaning days and fermented diet both affect digestive enzyme activity The trypsin activity, lipase activity, and amylase activity of jejunum and ileum are shown in Figure 2. Compared with the groups treated with control diet both weaning on d 21 and d 28, dietary supplementation with fermented diet had no significant effect on the lipase activity of ileum ( P > 0.05). However, there was interaction effect between fermented diet and weaning age ( P 0.05) in trypsin activity and lipase activity of the jejunum between the groups treated with control diet and the groups supplemented with fermented diet among weaning on d 21 and 28. Compared with 28-d weaning, weaning on d 21 significantly decreased ( P < 0.05) trypsin activity and lipase activity of jejunum. Dietary supplementation with fermented diet increased ( P 0.05) on trypsin activity, lipase activity and amylase activity of jejunum. 3.3. Weaning days and fermented diet have no significant effect on intestinal morphology Sections of duodenum, jejunum and ileum in each group are shown in Figure 3A. The morphology of intestinal villi was normal in all groups. Villus height, crypt depth and villus height/crypt depth are shown in Figure 3B. The villus height, crypt depth and villus height/crypt of duodenum and jejunum were similar ( P > 0.05) among the treatments. Compared with 28-d weaning, weaning on d 21 significantly reduced ( P 0.05) crypt depth and villus height/crypt depth of ileum. Dietary supplementation with fermented diet did not affect ( P > 0.05), compared with the groups treated with control diet both weaning on d 21 and d 28. There was no interaction effect between fermented diet and weaning age ( P > 0.05) on all indexes. 3.4. Weaning days and fermented diet both affect the expression of tight junction proteins The relative expression of jejunal tight binding protein is shown in Figure 4A. Although the Occludin proteins of jejunum among the treatments was not affected ( P > 0.05) on d 21 and d 28, it is affected ( P < 0.05) by the interaction of fermented diet and weaning age. Weaning on d 21 significantly downregulated ( P < 0.05) Claudin-1 proteins of jejunum, compared with 28-d weaning. Dietary supplementation with fermented diet upregulated ( P 0.05) on Claudin-1 proteins of jejunum. The effect of weaning age and fermented diet on ZO-1 proteins is similar ( P > 0.05) to Claudin-1 proteins, but there is no interaction on ZO-1 proteins. The relative expression of ileum tight binding protein is shown in Figure 4B. Weaning on d 21 significantly downregulated ( P < 0.05) Occludin, Claudin-1, ZO-1 proteins of ileum, compared with 28-d weaning. Dietary supplementation with fermented diet upregulated ( P < 0.05) Occludin, Claudin-1, ZO-1 proteins of ileum, compared with the groups treated with control diet both weaning on d 21 and d 28. There was no interaction effect between fermented diet and weaning age ( P > 0.05) on Occludin and Claudin-1. There was interaction effect between fermented diet and weaning age ( P < 0.05) on ZO-1 proteins. 3.5. Weaning days and fermented diet both affect the richness of microbiota In this experiment, a total of 25 cecal content samples were collected for 16S rRNA sequencing. After removing low-quality sequences, 139,985 clean reads were clustered into 4,281 ASVs. The complexity of species diversity was estimated by diversity indexes (Shannon and Simpson) and richness indexes (observed species and Chao1). As shown in Figure 5 A, weaning days had no significant influence ( P > 0.05) on the Observed species, Chao1 index, Simpson index and Shannon index among the treatments. The Observed species, Simpson index and Shannon index of dietary supplementation with fermented diet were similar ( P > 0.05), compared with the groups treated with control diet both weaning on d 21 and d 28. Weaning on d 21 significantly reduced ( P 0.05) on all indexes. As displayed in Figure 5 B, PCA and PCoA plot indicated that both weaning age and fermented diet had an effect on the gut microbial structure of piglets. 3.6 Weaning days and fermented diet both affect intestinal microbial structure The top 20 bacteria at levels of phylum, class, order, family and genus were selected for analysis. As shown in Figure 6A at the phylum level, Bacteroidetes (phylum) and Firmicutes (phylum) were dominant in the gut which accounted for more than 90%. Weaning days had no significant effect ( P > 0.05) on the abundance of Bacteroidetes (phylum) and Firmicutes (phylum) among the treatments. Dietary supplementation with fermented diet increased ( P 0.05) between dietary supplementation with fermented diet and the groups treated with control diet both weaning on d 21 and d 28. As shown in Figure 6B-C at the class level and order level, both weaning on d 21 and d 28 did not affect ( P > 0.05) on the abundance of Clostridia (class), Bacteroidia (class), Clostridiales (order) and Bacteroidales (order). Dietary supplementation with fermented diet increased ( P < 0.05) the abundance of Bacteroidia (class) and Bacteroidales (order), reduced ( P < 0.05) the abundance of Clostridia (class) and Clostridiales (order), compared with the groups treated with control diet both weaning on d 21 and d 28. As shown in Figure 6D at the family level, both weaning on d 21 and d 28 did not affect ( P > 0.05) on the abundance of Clostridiaceae (family) and Porphyromonadaceae (family). Compared with the groups treated with control diet both weaning on d 21 and d 28, dietary supplementation with fermented diet reduced ( P < 0.05) the abundance of Clostridiaceae (family), and increased ( P 0.05) on the abundance of Parabacteroides (genus). Dietary supplementation with fermented diet increased ( P < 0.05) the abundance of Parabacteroides (genus), compared with the groups treated with control diet both weaning on d 21 and d 28. Weaning on d 21 significantly increased ( P 0.05) the abundance of Parabacteroides (genus) and Clostridium (genus), compared with the groups treated with control diet both weaning on d 21 and d 28. There was no interaction effect between fermented diet and weaning age ( P > 0.05) on all indexes. 3.7. Weaning days and fermented diet do not affect the short-chain fatty acids of cecum The short-chain fatty acid of cecal contents is shown in Table 5. Weaning day had no significant effect (P > 0.05) on the content of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid and Isovaleric acid in cecum among the treatments. Dietary supplementation with fermented diet did not affect ( P < 0.05) the content of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid and Isovaleric acid in cecum, compared with the groups treated with control diet both weaning on d 21 and d 28. There was no interaction effect between fermented diet and weaning age ( P > 0.05) on all indexes. Discussion Studies have showed that fermented feed can improve the growth performance of weaned piglets [12, 28, 29]. Fermented feed has attracted much attention in recent researches, whereas the weaning age was often neglected. Few studies have focused on the interaction effect between fermented feed and weaning age. The present study was conducted to investigate the effects of fermented feed on the growth performance, intestinal function and microbiota of piglets weaned at different age. In production, piglets are often weaned at 21 days of age. Jamile found that production performance of piglet improved linearly with increasing weaning days [30]. A study by Dinan [31] showed that whether the piglets weaned at 21 days old or weaned at 28 days old, there was no significant difference in body weight at 46 days old. Retardation of weaning age increases ADG in short term, but the increasing in body weight is offset within 14 days post weaning [31]. Although the 28-d weaned piglets had a higher ADG during the period from 21 to 28 days old, the ADG and ADFI during 28 to 42 days old decreased, and F: G increased. This implies that compared with 21-d weaning, postponing the weaning age to 28 days old, the feed intake reduction caused by the weaning stress has not been effectively relieved. The results of a previous study also showed that whether piglets weaned at 14, 21, or 28 days old, there was no significant difference in body weight when they reached 42 days old [32]. In this study, supplementation with fermented feed increased the ADG during 28-42 days of age, and the results are consistent with previous studies [33, 34]. Compared with the control diet group, the fermented diet group increased the ADFI, and decreased F/G. After fermented, the macromolecular substances in corn and soybean meal are decomposed into small molecular substances that are more conducive to the absorption for piglets, and improves the utilization rate of feed. Study have shown that the β-sheet structure in Bacillus subtilis fermented soybean meal is reduced by 43.2%, and the more easily absorbed random coil structure is increased by 49.9 % [35]. Few literatures have studied on the effects of fermented feed on digestive enzyme activity of weaned piglets. Fed with soybean meal fermented by Bacillus subtilis was reported to increase the total protease and trypsin in the duodenum and jejunum of piglets [36]. Inconsistently, in this study, feeding the fermented diet had no effect on the digestive enzymes in the stomach, jejunum and ileum. Different weaning age may be one main reason to explain the different results. In this paper, the feed trial was last for 22 days, and the test time may be too short for fermented feed to change the digestive tract enzyme activity of piglets. Differences in the fermentation bacterial strains and fermentation processes may be another reason. A study by Lei [32] have shown that as weaning age increases, the rate of fat absorption by piglets also increases. In this study, the jejunal lipase activity at 42 days old of 28-d weaned piglets was significantly higher than that of 21-d weaned piglets. The lipase activity increased along with the retardation of weaning age, and the digestion and absorption of fat promoted. In this study, 28-d weaning significantly increased the ileal villus height, but had no significant effect on the villus height/crypt depth. The small intestinal morphology of 21-d weaned piglets was damaged due to weaning stress, and manifested in the decreasing of intestinal villi height and crypt depth deeper. Study have shown that the small intestinal villus in 28-d weaned piglets shrink to the shortest on the 3rd day after weaning, and begins to recover on the 5th day [32]. Another study showed that the small intestine of piglets weaned at the age older than 28 days old recovery faster when suffering weaning stress [7]. Weaning age affect the recovery speed of the small intestine suffering weaning stress, but on the 14th day post weaning, the villus height of the small intestine can be restored to the length before weaning [2, 37], and that is also consistent with the results of our study. However, the fermented diet had no significant effect on the integrity of the small intestine. Moreover, the piglets used in this study are self-breeding and self-raising on our farm, with strict biological prevention and control, and transportation stress greatly reduced. Tight junction proteins such as Claudins, ZO-1 and Occludin are important components of the intestinal barrier [38], which selectively permeate nutrients and water, and block pathogens [39, 40]. Extracellular regulated protein kinases (ERK1/2) activation of ETS-like 1 transcription factor 1 (Elk-1) can lead to the upregulation of pro-inflammatory cytokines interferon gamma (IFN-γ) and tumor necrosis factor (TNF-α), which in turn damage the intestinal barrier functions and increase intestinal permeability [41-43]. Early weaning stress can lead to the activation of mitogen-activated protein kinase (MAPK) signaling pathway ERK1/2, resulting in an inflammatory response in the intestine. Study have shown that 14 days post weaning, the relative mRNA expression of Occludin, Claudin-1 and ZO-1 in the jejunal mucosa was droped [2], intestinal permeability was augmented, and the intestinal barrier functions were weakened. In this study, compared with piglets fed control diet, supplementation with fermented diet significantly increased the relative expression of Occludin, Claudin-1 and ZO-1 in the small intestine. The results indicates that fermented diet can improve the intestinal barrier functions of piglets. As for the weaning age, the intestinal barrier functions of 28-d weaned piglets is stronger, and there is a certain interaction effect between feed diet and weaning age. Another study showed that toll-like receptor 2 (TLR 2) enhances the expression of ZO-1 to maintain the integrity of the intestinal barrier by protein kinase C [44], and the toll-like receptor recognition may be affected by the regulation of intestinal microbiota [45]. In this study, both fermented diet and 28-d weaning improved the damage of the intestinal barrier functions, but the specific effect of that pathway needs to be further explored. Crosstalk exists between the intestinal microbiota and the host. Commensal microbes that inhabit the gut participate in digestion and absorption of nutrients, mediation of immunity system, and take part in the host’s metabolism via their metabolites. In turn, alterations of intestinal microbiota reflect the state of the host [46, 47]. Weaning stress causes volatility of gut microbiota in piglets [48, 49]. Study have shown that Fusobacterium (genus), Akkermansia (genus) , Clostridiales (genus), Deltaproteobacteria (genus), Selenomonadales (genus) in the intestine of post-weaned diarrheal piglets are increased compared to healthy weaned piglets. while Prevotella (genus), and Faecalibacterium (genus) decreased [50]. Bacteroidales (order) and Clostridiales (order) are the two most dominant orders involved in glycolysis in the large intestine [51, 52]. In this study, fermented diet reduced the relative abundance of Clostridiales (order) and increased the relative abundance of Bacteroidales (order). Fermented diet increased the relative abundance of beneficial bacteria, thereby improving the intestinal barrier functions. Fermentation of insoluble polysaccharide by microbiota in the hindgut yields short-chain fatty acids, which take part in improve intestinal health of weaned piglets [53]. However, in this study, the composition of the intestinal microbiota has been changed, and the alterations in short-chain fatty acids producing bacteria can also be found, but the contents of short-chain fatty acids in the intestine did not change significantly. Conclusion In summary, delaying the weaning time can improve feed conversion and intestinal barrier function. Supplementation with fermented diet significantly reduces F: G during 28 to 42 days of age in piglets, and improves the small intestinal barrier functions and cecal microbial structure. Supplementation with fermented diet improve feed efficiency, and intestinal barrier functions of weaned piglets by modulating intestinal microbiota. In addition, addition of fermented feed has a significant effect on the intestinal barrier of piglets at different weaning ages. Abbreviations BW：Body weight ADG：Average daily gain ADFI：Average daily feed intake F: G：Feed to gain ratio ZO-1：Zonula occluden-1 ERK1/2：Extracellular regulated protein kinases Elk-1：ETS-like 1 transcription factor 1 IFN-γ：Interferon gamma TNF-α：Tumor necrosis factor MAPK：Mitogen-activated protein kinase TLR 2：Toll-like receptor 2 Declarations Ethics approval and consent to participate All animal procedures used in the present study were approved by the Animal Care and Use Committee of Guangdong Academy of Agricultural Sciences and followed the Guidelines for the Care and Use of Animals for Research and Teaching (Authorization number GAASIAS-2016-017). Consent for publication All the authors read and agree to the content of this paper and its publication. Availability of data and materials The raw 16S rRNA sequences have been deposited in the NCBI SRA under BioProject PRJNA779532. The datasets generated for this study are available upon request to the corresponding author. Competing interests The authors declare no competing interest. Funding This study was supported by the China Agriculture Research System of MOF and MARA; Special fund for scientific innovation strategy-construction of high level Academy of Agriculture Science (R2018PY-JC001, R2020PY-JG009, R2017YJ-YB1004, R2018PY-QF001); the Project of Swine Innovation Team in Guangdong Modern Agricultural Research System (2021KJ126). Independent Research and Development Projects of Maoming Laboratory(2021ZZ003). Authors' contributions SL, YXX and JPC performed experiments, analyzed data, and wrote the manuscript. QWW and XLW performed experiments. HX, ZYJ and LW supervised the project, developed the study concept, and wrote and edited the manuscript. The authors read and approved the final manuscript. Acknowledgements We would like to thank the staff at our laboratory for their ongoing assistance. References Pluske JR, Hampson DJ, Williams IH. 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Cell Host Microbe. 2017;21(4):433-42. https://doi.org/10.1016/j.chom.2017.03.010. Donaldson GP, Lee SM, Mazmanian SK. Gut biogeography of the bacterial microbiota. Nat Rev Microbiol. 2016;14(1):20-32. https://doi.org/10.1038/nrmicro3552. Lordelo MM, Cunha LF, Freire J. Effects of dietary fibre source and enzyme supplementation on faecal apparent digestibility, short chain fatty acid production and activity of bacterial enzymes in the gut of piglets. Anim Feed Sci Tech. 2008;146(1-2):124-36. https://doi.org/10.1016/j.anifeedsci.2007.12.001. Tables Table 1. Ingredients and nutrient levels of basal diet (air-dry basis). Ingredients Content, % Nutrient levels 2 Corn 32.92 ME 3 , kcal/kg 3403.00 Expanded corn 15.00 CP 4 , % 19.00 Soybean meal 10.00 Ca, % 0.76 Soybean hulls 5.00 Total P, % 0.76 Extruded soybean 12.00 AP 5 , % 0.53 Soybean oil 1.50 SID 6 Lys, % 1.35 Fish meal 6.50 SID 6 Met＋Cys, % 0.74 Whey powder 11.00 SID 6 Thr, % 0.79 Lactose 1.00 SID 6 Trp, % 0.22 Sucrose 1.00 SID 6 Ile, % 0.63 Choline chloride (50%) 0.30 SID 6 Val, % 0.70 Salt 0.45 SID 6 Arg, % 0.97 Calcium hydrogen phosphate 0.70 SID 6 His, % 0.39 Calcium citrate 0.50 SID 6 Leu, % 1.24 Phytase 0.02 SID 6 Phe, % 0.69 L-Lys HCl 0.60 DL-Met 0.23 L-Thr 0.23 L-Trp 0.05 Premix 1 1.00 Total 100.00 1 Provided per kilogram of diet: 12,400 IU vitamin A, 2,800 IU vitamin D3, 30 mg vitamin E, 5 mg vitamin K3, 3 mg thiamin, 10 mg riboflavin, 40 mg niacin, 8 mg pyridoxine, 40 μg vitamin B12, 0.08 mg biotin, 15 mg pantothenic acid, 1 mg folic acid, 80 mg Zn, 120 mg Fe, 70 mg Mn, 16 mg Cu, 0.7 mg I, 0.3 mg Se. 2 Values were calculated according to NRC (2012) [17]. 3 ME, metabolic energy. 4 CP, crude protein. 5 AP, available phosphorous. 6 SID, standardized ileal digestible. Table 2. Contents of anti-nutritional factors (mg/g). Items Ingredients before fermented Ingredients after fermented Basal diet Fermented diet β-Conglycinin 166.51 67.57 108.31 51.02 Glycinin 233.66 63.45 129.69 44.14 Trypsin inhibitor factor 1.34 0.28 0.51 0.18 Table 3. Nutrient levels in before and after fermented Items Before fermented After fermented Water, % 10.83 27.96 CP 1 , % 18.53 19.68 GE 2 , MJ/kg 16.86 17.18 EE 3 , % 3.74 2.49 CF 4 , % 4.41 3.44 NDF 5 , % 4.53 2.45 ADF 6 , % 3.29 1.75 Ash, % 5.56 5.18 Calcium, % 1.69 1.63 Phosphorus, % 0.57 0.52 1 CP, crude protein. 2 GE, gross energy. 3 EE, ether extract. 4 CF, crude fiber. 5 NDF, neutral detergent fiber. 6 ADF，acid detergent fiber. The nutrient levels were determined value based on dry matter. Table 4. Effects of fermented diet and weaning age on the growth performance of piglets Items 21-d 28-d SEM P-value CD FD CD FD F W F*W BW at 21 days old, kg 6.17 6.15 6.15 BW at 28 days old, kg 7.01 7.07 8.71 8.80 1.05 0.338 0.125 0.102 BW at 42 days old, kg 11.54 12.21 11.69 12.36 0.91 0.021 0.603 0.997 ADG, g/d 323.6 363.25 212.04 230.09 73.61 0.009 < 0.001 0.900 1 ADFI, g/d 488.76 592.28 345.71 426.16 101.41 < 0.001 < 0.001 0.494 2 ADFI, g/d 444.34 422.88 314.28 304.28 75.96 0.259 < 0.001 0.679 F: G 1.45 1.19 1.59 1.31 0.24 < 0.001 0.041 0.872 21-d, weaning at 21 days old. 28-d, weaning at 28 days old. CD, the control diet. FD, the fermented diet. BW, body weight. ADG, average daily gain. ADFI, average daily feed intake. F: G, feed: gain ratio. F, the main effect of fermented diet. W, the main effect of weaning age. F*W, the interaction effect between fermented diet and weaning age. Data are expressed as mean and pooled SEM. 1 ADFI was calculated based on wet weight; 2 ADFI was calculated based on dry matter Table 5. Effects of fermented diet and weaning age on the concentrations of short-chain fatty acids in cecal content of piglets (mg/kg) Items 21-d 28-d SEM P-value CD FD CD FD F W F*W Acetic acid 1.83 7.21 3.36 3.97 1.37 0.298 0.763 0.405 Propionic acid 597.20 799.68 688.84 654.92 39.00 0.286 0.733 0.140 Butyric acid 273.34 396.92 310.20 308.38 26.01 0.255 0.624 0.241 Valeric Acid 56.98 93.36 76.82 85.30 8.70 0.218 0.742 0.438 Isobutyric acid 22.39 34.99 168.12 28.95 23.52 0.153 0.116 0.900 Isovaleric acid 29.03 39.46 31.46 36.08 2.74 0.194 0.933 0.609 21-d, weaning at 21 days old. 28-d, weaning at 28 days old. CD, the control diet. FD, the fermented diet. F, the main effect of fermented diet. W, the main effect of weaning age. F*W, the interaction effect between fermented diet and weaning age. Data are expressed as mean and pooled SEM. 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Six 21-day-old piglets with similar body weight were randomly selected from the same litter of 20 sows, and then 3 of them were randomly selected as a replicate and to be weaned immediately, and the others continue to suckle until 28 days old before weaning. As soon as weaned, the piglets were randomly allocated into two treatments, fed with basal diet as the control group and fermented diet as the fermented group, respectively. The experiment was finished at the age of 42 days old. 21d-CD, 21-day-old weaned piglets fed the control diet. 21d-FD, 21-day-old weaned piglets fed the fermented diet. 28d-CD, 28-day-old weaned piglets fed the control diet. 28d-FD, 28-day-old weaned piglet fed the fermented diet. The same as below.","description":"","filename":"fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-1063432/v1/3225a2f9cab8cb3798ca2ff9.png"},{"id":15555652,"identity":"c97e3dbe-9384-4c16-b4e2-64f210c1aa10","added_by":"auto","created_at":"2021-11-15 16:43:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":71000,"visible":true,"origin":"","legend":"Effects of fermented diet and weaning age on digestive enzyme activity. 21d-CD, 21-day-old weaned piglets fed the control diet. 21d-FD, 21-day-old weaned piglets fed the fermented diet. 28d-CD, 28-day-old weaned piglets fed the control diet. 28d-FD, 28-day-old weaned piglet fed the fermented diet. F, the main effect of fermented diet. W, the main effect of weaning age. F*W, the interaction effect between fermented diet and weaning age. Values are presented as the mean ± SEM.","description":"","filename":"fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-1063432/v1/f90809ce17f6da07a6cc6ad5.png"},{"id":15556069,"identity":"39751cca-b7dc-45f0-b7f8-f61a3a9e410e","added_by":"auto","created_at":"2021-11-15 16:46:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2109395,"visible":true,"origin":"","legend":"Hematoxylin-eosin staining photos of the duodenum, jejunum and ileum (magnification 200×). 21d-CD, 21-day-old weaned piglets fed the control diet. 21d-FD, 21-day-old weaned piglets fed the fermented diet. 28d-CD, 28-day-old weaned piglets fed the control diet. 28d-FD, 28-day-old weaned piglet fed the fermented diet. F, the main effect of fermented diet. W, the main effect of weaning age. F*W, the interaction effect between fermented diet and weaning age. Values are presented as the mean ± SEM.","description":"","filename":"fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-1063432/v1/68f2b133817e1568da6bbc65.png"},{"id":15555656,"identity":"d147d6c0-cc7c-4048-bf71-b97c98f3115e","added_by":"auto","created_at":"2021-11-15 16:43:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":122318,"visible":true,"origin":"","legend":"Effects of fermented diet and weaning age on the tight junction proteins expression in the small intestine of weaned piglets. The protein expression of Occludin, Claudin and ZO-1 in the jejunum (A) and ileum (B) were detected via western blotting, and the intensity of the bands was determined using Image J. 21d-CD, 21-day-old weaned piglets fed the control diet. 21d-FD, 21-day-old weaned piglets fed the fermented diet. 28d-CD, 28-day-old weaned piglets fed the control diet. 28d-FD, 28-day-old weaned piglet fed the fermented diet. F, the main effect of fermented diet. W, the main effect of weaning age. F*W, the interaction effect between fermented diet and weaning age. Values are presented as the mean ± SEM.","description":"","filename":"fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-1063432/v1/c41fcbfd604e685ddfd12eae.png"},{"id":15555654,"identity":"3af189f9-9d31-4f84-b49e-ae455f295cbc","added_by":"auto","created_at":"2021-11-15 16:43:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":90897,"visible":true,"origin":"","legend":"Effects of fermented diet and weaning age on the cecal microbial composition of piglets. 21d-CD, 21-day-old weaned piglets fed the control diet. 21d-FD, 21-day-old weaned piglets fed the fermented diet. 28d-CD, 28-day-old weaned piglets fed the control diet. 28d-FD, 28-day-old weaned piglet fed the fermented diet. F, the main effect of fermented diet. W, the main effect of weaning age. F*W, the interaction effect between fermented diet and weaning age. Values are presented as the mean ± SEM.","description":"","filename":"fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-1063432/v1/6bbbf1a4b9be181b472631c8.png"},{"id":15555655,"identity":"c1a20afd-ed10-4e89-8786-9547e63bb79e","added_by":"auto","created_at":"2021-11-15 16:43:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":119863,"visible":true,"origin":"","legend":"Composition of the top 20 bacteria at the level of phylum, family and genus. 21d-CD, 21-day-old weaned piglets fed the control diet. 21d-FD, 21-day-old weaned piglets fed the fermented diet. 28d-CD, 28-day-old weaned piglets fed the control diet. 28d-FD, 28-day-old weaned piglet fed the fermented diet. F, the main effect of fermented diet. W, the main effect of weaning age. F*W, the interaction effect between fermented diet and weaning age. Values are presented as the mean ± SEM.","description":"","filename":"fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-1063432/v1/38278d90b4ed7820c3a4798f.png"},{"id":20099196,"identity":"a382ac5f-1a02-49e8-bdae-0779fda5850d","added_by":"auto","created_at":"2022-04-08 06:57:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1684562,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1063432/v1/2b8658c2-24c6-4e86-8b34-0d6213500532.pdf"}],"financialInterests":"","formattedTitle":"Effects of Fermented Feed on the Growth Performance, Intestinal Function and Microbiota of Piglets Weaned at Different Age","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWeaning would harm the intestinal function of piglets and lead to food intake reduction, diarrhea risk increasing and even death in severe cases [1-2]. Weaning affects production performance. Jamil et al found that the average daily gain (ADG) and average daily feed intake (ADFI) of piglets increased linearly with weaning age increasing [3]. Pigs weaned at 14 days old grow slower, and the feed intake are reduced compared to those weaned at 24 days old [3]. Compared with weaning on 12 days, weaning on 20 days increased ADG [4]. Alison has pointed out that delaying weaning can improve feed digestibility [5]. Another study showed that the villi height of jejunum in weaned piglets was significantly lower than in unweaned piglets at the same age [6, 7]. According to Blecha et al, early weaning can cause passive immunity weakened in piglets [8]. The study of Janeen found that piglets weaned at 28 days of age were stronger in immunity and had more antibodies after exposure to the virus than piglets weaned at 14 days of age [9]. Weaning age affects piglet performance, immunity and intestinal function. Therefore, weaning age is an extremely important parameter that affects piglets\u0026apos; growth.\u003c/p\u003e\n\u003cp\u003eSoybean meal is widely used in pig production as a plant-derived protein source. However, soybean is limited to piglets\u0026rsquo; diet because of the anti-nutritional factors, such as soybean globulin, \u0026beta;-glycoprotein, trypsin inhibitor and non-starch polysaccharide, which can exacerbate weaning stress, increase diarrhea risk, and hinder the growth rate of piglets. Currently, the use of microbial fermented feed is a common method to reduce antinutritional factors in feed. The amounts of soybean antigenic proteins (\u0026beta;-conglycinin and glycinin) in feed was decreased after fermented by Bacillus subtilis [10]. Feeding fermented feed can improve the intestinal health of piglets, increase productivity and change the intestinal microbial structure [11]. Study have shown that mixed solid-state fermentation of Bacillus subtilis, Hansenula anomala and Lactobacillus casei can improve the nutrient digestion of piglets [12]. Another study showed that fermented feed can increase the concentration of butyric acid in the cecum of piglets [13]. On the other hand, fermented diet can provide probiotics and their metabolites, which improve the digestibility of feed and intestinal function of weaned piglets [14-16].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, studies on the effects of fermented feed on piglets at different weaning days are still lacking. Therefore, the present study was conducted to investigate the effects of fermented feed, weaning age, and their interaction effect on the growth performance, intestinal function and microbiota of piglets, and to provide a new insight for pig production.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e2.1. Fermented feed preparation\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, and \u003cem\u003eLactobacillus\u003c/em\u003e (\u003cem\u003eLactobacillus casei\u003c/em\u003e and \u003cem\u003eLactobacillus plantarum\u003c/em\u003e) were used to ferment feed materials (corn, soybean meal, soybean hull) in this study. Mixture of \u003cem\u003eBacillus subtilis\u003c/em\u003e and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e are considered as bacteria A which was purchased from Prosyn (Haerbing, China), and the number of viable \u003cem\u003eBacillus subtilis\u003c/em\u003e is 7.2\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/g, the number of viable \u003cem\u003eSaccharomyces cerevisiae\u0026nbsp;\u003c/em\u003eis 3.1\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/g. \u003cem\u003eLactobacillus\u003c/em\u003e is considered as bacteria B which was purchased from Inner Mongolia Sci-Plus Biotech (Inner Mongolia, China), and the total amount of viable \u003cem\u003eLactobacillus\u003c/em\u003e \u003cem\u003ecasei\u003c/em\u003e+\u003cem\u003eLactobacillus plantarum\u003c/em\u003e is 5.1\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/g. Bacteria A and B were mixed at a ratio of 1:1 as fermentation strains. Fermented feed was prepared with 18.1 kg ingredients, 6.9 kg water and 25 g fermentation strains, and then fermented in the laboratory for 5 days. The ingredients used to ferment were mixed at a ration completely corresponding with the basal diet (Table 1). The contents of anti-nutritional factors in the materials before and after fermented are shown in Table 2. Fermented feed was blended with other ingredients before used as fermented diet.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.2. Animals and Experimental Design\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 1, a 2\u0026times;2 two-factor design was adopted in this study. Six 21-day-old piglets (Duroc\u0026times;Landrace\u0026times;Yorkshire, body weight about 6.15\u0026plusmn;0.02 kg) were selected from the same litter of 20 sows, and 3 among them were randomly selected as a replicate and to be weaned, and the others were continued to suckle until 28 days old before weaning. As soon as weaned, the replicates were randomly allocated into two treatments, fed with basal diet as the control group or fermented diet as the fermented group, respectively. The feed trial was finished when the piglets were at the age of 42 days old. Piglets with approximate average BW were randomly selected from each replicate to slaughter and sample at the age of 28 days old and 42 days old, respectively. The basal diet (Table 1) was formulated according to NRC (2012) to meet the nutritional requirements of piglets about 7-11 kg\u0026nbsp;[17]\u0026nbsp;without high copper and zinc supplementation. The nutrient composition of the experiment diet before and after fermentation is shown in Table 3. During the whole experiment, the animals were free to access feed and water. Once the experiment finished, ADG, ADFI and feed to gain ratio (F: G) were calculated.\u003c/p\u003e\n\u003cp\u003e2.3. Sample Collection\u003c/p\u003e\n\u003cp\u003eAt the end of the experiment, after fasting overnight, all animals were sacrificed after i. m. injection with sodium pentobarbital (40 mg/kg BW). The digesta from the stomach, jejunum, ileum, cecum and colon were carefully collected and snap frozen in liquid nitrogen. Tissue samples from the proximal duodenum, middle jejunum and distal ileum were dissected, and one sample was immediately frozen in liquid nitrogen for subsequent protein expression analysis, and another sample was fixed with 4% paraformaldehyde for subsequent morphological analysis.\u003c/p\u003e\n\u003cp\u003e2.4. Measurement of Digestive Enzyme Activity\u003c/p\u003e\n\u003cp\u003eThe activities of trypsin, lipase and amylase were determined by commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The protein concentrations of sample were determined with BCA kit (Thermo Fisher, USA), and the final results were expressed as /mg protein.\u003c/p\u003e\n\u003cp\u003e2.5. Intestinal Histopathological Examination\u003c/p\u003e\n\u003cp\u003eThe intestinal morphology was examined according to the method of Gao et al\u0026nbsp;[18]. Briefly, after paraffin embedded fixed, intestinal segments were cut into 5-\u0026mu;m sections. Then, the sections were dewaxed with xylene, hydrated with alcohol, and stained with hematoxylin and eosin (H\u0026amp;E). Images were obtained by using a fluorescent orthochromatic microscope (Haier, China). 10 bright fields were randomly selected for each section. The villus height, and crypt depth were measured using image-pro image processing software (Media Cybernetics, Rockville, MD).\u003c/p\u003e\n\u003cp\u003e2.6. Western Blotting\u003c/p\u003e\n\u003cp\u003eAfter extracted with lysis buffer (Biosharp, Anhui, China), the protein concentrations of samples were determined with the BCA kit (Thermo Fisher, USA), and then proteins were separated by 10% SDS-PAGE, and transferred onto a nitrocellulose membrane (BIO-RAD, USA). After blocking with blocking buffer (Beyotime, Shanghai, China), the membranes were incubated with the corresponding primary antibody at 4\u0026deg;C for 12-16 h, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. Bands were detected using ECL PlusTM Western Blotting Substrate (CliNX, Shanghai, China). Band intensity was quantified using ImageJ software (ImageJ 1.52, National Institutes of Health, USA). Primary antibodies for \u0026beta;-actin (1:10,000), zonula occludens-1 (ZO-1, 1:250), Occludin (1:1,000), and Claudin-1(1:1,000) (Abcam, MA, USA) were used in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.7. Microbiota Profiling\u003c/p\u003e\n\u003cp\u003eTotal genomic DNA of cecal content samples was extracted by using the OMEGA Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA, #D5625-01) according to the manufacturer\u0026apos;s instructions and store at -20\u0026deg;C. Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify the sample DNA, and the DNA quality was checked by 1.2% agarose gel electrophoresis. PCR amplification of the bacterial 16S rRNA genes V3-V4 regions was performed using forward primer 338F (5\u0026rsquo;-ACTCCTACGGGAGGCAGCA-3\u0026rsquo;) and the reverse primer 806R (5\u0026apos;-TCGGACTACHVGGGTWTCTAAT-3\u0026apos;). Sample-specific 7-bp barcodes were incorporated into the primers for multiplex sequencing. The PCR components contained 5 \u0026mu;L buffer (5\u0026times;), 0.25 \u0026mu;L Fast pfu DNA Polymerase (5 U/\u0026mu;L), 2 \u0026mu;L dNTPs (2.5 mM), 1 \u0026mu;L Forward and Reverse primer (10 \u0026mu;M), 1 \u0026mu;L DNA Template, and 14.75 \u0026mu;L ddH\u003csub\u003e2\u003c/sub\u003eO. The PCR amplification program consisted of initial denaturation at 98 \u0026deg;C for 5 min, followed by 25 cycles of denaturation at 98 \u0026deg;C for 30 s, annealing at 53 \u0026deg;C for 30 s, elongation at 72 \u0026deg;C for 45 s, and then a final extension at 72 \u0026deg;C for 5 min. PCR products were recovered and quantified by Vazyme VAHTSTM DNA Clean Beads (V azyme, Nanjing, China) and the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA), respectively. Paired-end 2\u0026times;250 bp sequencing was performed using the Illumina kit and the Illumina MiSeq platform (Shanghai, China).\u003c/p\u003e\n\u003cp\u003eThe bioinformatic analysis was conducted with QIIME2 pipeline. Briefly, the clean reads were got by de-primed, quality filtered, denoised, spliced and de-chimerism using DADA2 method from raw reads\u0026nbsp;[19]. After merged into Amplicon Sequence Variants (ASVs) feature sequence, singletons ASVs removed, the ASVs was clustered using QIIME2\u0026apos;s classify-sklearn algorithm\u0026nbsp;[20]\u0026nbsp;and then annotation using Naive Bayes classifier based on Greengenes database\u0026nbsp;[21]. The Rarefaction method is used to normalize and classify the ASVs with a depth of 95%\u0026nbsp;[22, 23]. Chao1\u0026nbsp;[24]\u0026nbsp;index and Observed species index were used to characterize richness, and Shannon\u0026nbsp;[25]\u0026nbsp;index and Simpson\u0026nbsp;[26]\u0026nbsp;index were used to characterize evenness of the bacterial community. Principal component analysis (PCA) based on ASVs physical distance, and principal coordinate analysis (PCoA) based on unweighted Unifrac distance are used to display the distribution of samples.\u003c/p\u003e\n\u003cp\u003e2.8. Determination of Short-Chain Fatty Acids\u003c/p\u003e\n\u003cp\u003eShort-chain fatty acids in the cecal content were determined according to the method of Xiong et al\u0026nbsp;[27]. Briefly, samples of cecum contents were added to NaOH and caproic acid-d3, and centrifuged to get supernatant. The supernatant was mixed with water, and propyl/pyrimidine, and propyl chloroformate, and to derive for 5 min. Finally, the supernatant was extracted twice with hexane, and the final supernatant was dehydrated with Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e The determination was carried out with gas chromatograph-mass spectrometer detector (7890A and 5975C inert XL EI/CI mass spectrometric detector, Agilent Technologies, Santa Clara, CA).\u003c/p\u003e\n\u003cp\u003e2.9. Statistical Analysis\u003c/p\u003e\n\u003cp\u003eSPSS 25 software (IBM, USA) was used for statistical analysis. The effects of fermented diet, and weaning age, and their interaction effect were assessed by ANOVA using general linear model (GLM) procedure. Results are represented as means and pooled standard error, and \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 indicates significant differences.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e3.1. Weaning days and fermented diet both affect the growth performance of piglets\u003c/p\u003e\n\u003cp\u003eBW, ADG, ADFI (calculated based on wet weight and dry matter), and F: G\u0026nbsp;are shown in Table 4.\u0026nbsp;The BW on d 21 and d 28 were similar among the treatments. Dietary supplementation with fermented diet increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) BW on d 42, compared with the groups treated with control diet both weaning on d 21 and d 28. Weaning on d 21 significantly increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) ADG, and ADFI (calculated based on wet weight and dry matter), and reduced F: G, compared with 28-d weaning. Piglets fed fermented diet had higher (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) ADFI (calculated based on wet weight) during 28-42 days old, but F: G was lower (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), compared with piglets fed with the control diet. There was no interaction effect between fermented diet and weaning age (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) on all indexes.\u003c/p\u003e\n\u003cp\u003e3.2. Weaning days and fermented diet both affect digestive enzyme activity\u003c/p\u003e\n\u003cp\u003eThe trypsin activity, lipase activity, and amylase activity of jejunum and ileum are shown in Figure 2. Compared with the groups treated with control diet both weaning on d 21 and d 28, dietary supplementation with fermented diet had no significant effect on the lipase activity of ileum (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). However, there was interaction effect between fermented diet and weaning age (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) on lipase activity and amylase activity of ileum. There was no difference (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) in trypsin activity and lipase activity of the jejunum between the groups treated with control diet and the groups supplemented with fermented diet among weaning on d 21 and 28. Compared with 28-d weaning, weaning on d 21 significantly decreased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) trypsin activity and lipase activity of jejunum. Dietary supplementation with fermented diet increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) the amylase activity of jejunum, compared with the groups treated with control diet both weaning on d 21 and d 28. There was no interaction effect between fermented diet and weaning age (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) on trypsin activity, lipase activity and amylase activity of jejunum.\u003c/p\u003e\n\u003cp\u003e3.3. Weaning days and fermented diet have no significant effect on intestinal morphology\u003c/p\u003e\n\u003cp\u003eSections of duodenum, jejunum and ileum in each group are shown in Figure 3A. The morphology of intestinal villi was normal in all groups. Villus height, crypt depth and villus height/crypt depth are shown in Figure 3B. The villus height, crypt depth and villus height/crypt of duodenum and jejunum were similar (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) among the treatments. Compared with 28-d weaning, weaning on d 21 significantly reduced (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) villus height of ileum. However, weaning days did not affect (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) crypt depth and villus height/crypt depth of ileum. Dietary supplementation with fermented diet did not affect (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05), compared with the groups treated with control diet both weaning on d 21 and d 28. There was no interaction effect between fermented diet and weaning age (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) on all indexes.\u003c/p\u003e\n\u003cp\u003e3.4. Weaning days and fermented diet both affect the expression of tight junction proteins\u003c/p\u003e\n\u003cp\u003eThe relative expression of jejunal tight binding protein is shown in Figure 4A. Although the Occludin proteins of jejunum among the treatments was not affected (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) on d 21 and d 28, it is affected (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) by the interaction of fermented diet and weaning age. Weaning on d 21 significantly downregulated (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) Claudin-1 proteins of jejunum, compared with 28-d weaning. Dietary supplementation with fermented diet upregulated (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) Claudin-1 proteins of jejunum, compared with the groups treated with control diet both weaning on d 21 and d 28. There was interaction effect between fermented diet and weaning age (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) on Claudin-1 proteins of jejunum. The effect of weaning age and fermented diet on ZO-1 proteins is similar (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) to Claudin-1 proteins, but there is no interaction on ZO-1 proteins. The relative expression of ileum tight binding protein is shown in Figure 4B. Weaning on d 21 significantly downregulated (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) Occludin, Claudin-1, ZO-1 proteins of ileum, compared with 28-d weaning. Dietary supplementation with fermented diet upregulated (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) Occludin, Claudin-1, ZO-1 proteins of ileum, compared with the groups treated with control diet both weaning on d 21 and d 28. There was no interaction effect between fermented diet and weaning age (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) on Occludin and Claudin-1. There was interaction effect between fermented diet and weaning age (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) on ZO-1 proteins.\u003c/p\u003e\n\u003cp\u003e3.5. Weaning days and fermented diet both affect the richness of microbiota\u003c/p\u003e\n\u003cp\u003eIn this experiment, a total of 25 cecal content samples were collected for 16S rRNA sequencing. After removing low-quality sequences, 139,985 clean reads were clustered into 4,281 ASVs. The complexity of species diversity was estimated by diversity indexes (Shannon and Simpson) and richness indexes (observed species and Chao1). As shown in Figure 5 A, weaning days had no significant influence (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) on the Observed species, Chao1 index, Simpson index and Shannon index among the treatments. The Observed species, Simpson index and Shannon index of dietary supplementation with fermented diet were similar (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05), compared with the groups treated with control diet both weaning on d 21 and d 28. Weaning on d 21 significantly reduced (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) Chao-1 index compared with 28-d weaning. There was no interaction effect between fermented diet and weaning age (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) on all indexes. As displayed in Figure 5 B, PCA and PCoA plot indicated that both weaning age and fermented diet had an effect on the gut microbial structure of piglets.\u003c/p\u003e\n\u003cp\u003e3.6 Weaning days and fermented diet both affect intestinal microbial structure\u003c/p\u003e\n\u003cp\u003eThe top 20 bacteria at levels of phylum, class, order, family and genus were selected for analysis. As shown in Figure 6A at the phylum level, \u003cem\u003eBacteroidetes\u003c/em\u003e (phylum) and \u003cem\u003eFirmicutes\u003c/em\u003e (phylum) were dominant in the gut which accounted for more than 90%. Weaning days had no significant effect (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) on the abundance of \u003cem\u003eBacteroidetes\u003c/em\u003e (phylum) and \u003cem\u003eFirmicutes\u003c/em\u003e (phylum) among the treatments. Dietary supplementation with fermented diet increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) the abundance of \u003cem\u003eBacteroidetes\u003c/em\u003e (phylum), compared with the groups treated with control diet both weaning on d 21 and d 28. The abundance of \u003cem\u003eFirmicutes\u003c/em\u003e (phylum) was similar (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) between dietary supplementation with fermented diet and the groups treated with control diet both weaning on d 21 and d 28. As shown in Figure 6B-C at the class level and order level, both weaning on d 21 and d 28 did not affect (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) on the abundance of \u003cem\u003eClostridia\u003c/em\u003e (class), \u003cem\u003eBacteroidia\u003c/em\u003e (class), \u003cem\u003eClostridiales\u003c/em\u003e (order) and \u003cem\u003eBacteroidales\u003c/em\u003e (order). Dietary supplementation with fermented diet increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) the abundance of \u003cem\u003eBacteroidia\u003c/em\u003e (class) and \u003cem\u003eBacteroidales\u003c/em\u003e (order), reduced (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) the abundance of \u003cem\u003eClostridia\u003c/em\u003e (class) and \u003cem\u003eClostridiales\u003c/em\u003e (order), compared with the groups treated with control diet both weaning on d 21 and d 28. As shown in Figure 6D at the family level, both weaning on d 21 and d 28 did not affect (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) on the abundance of \u003cem\u003eClostridiaceae\u003c/em\u003e (family) and \u003cem\u003ePorphyromonadaceae\u003c/em\u003e (family). Compared with the groups treated with control diet both weaning on d 21 and d 28, dietary supplementation with fermented diet reduced (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) the abundance of \u003cem\u003eClostridiaceae\u003c/em\u003e (family), and increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) \u003cem\u003ePorphyromonadaceae\u003c/em\u003e (family). As shown in Figure 6E at the genes level, both weaning on d 21 and d 28 did not affect (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) on the abundance of \u003cem\u003eParabacteroides\u003c/em\u003e (genus). Dietary supplementation with fermented diet increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) the abundance of \u003cem\u003eParabacteroides\u003c/em\u003e (genus), compared with the groups treated with control diet both weaning on d 21 and d 28. Weaning on d 21 significantly increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) the abundance of \u003cem\u003e[prevotella]\u003c/em\u003e (genus) and \u003cem\u003eClostridium\u003c/em\u003e (genus), compared with 28-d weaning. Dietary supplementation with fermented diet did not affect (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) the abundance of \u003cem\u003eParabacteroides\u003c/em\u003e (genus) and \u003cem\u003eClostridium\u003c/em\u003e (genus), compared with the groups treated with control diet both weaning on d 21 and d 28. There was no interaction effect between fermented diet and weaning age (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) on all indexes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.7. Weaning days and fermented diet do not affect the short-chain fatty acids of cecum\u003c/p\u003e\n\u003cp\u003eThe short-chain fatty acid of cecal contents is shown in Table 5. Weaning day had no significant effect (P \u0026gt; 0.05) on the content of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid and Isovaleric acid in cecum among the treatments. Dietary supplementation with fermented diet did not affect (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) the content of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid and Isovaleric acid in cecum, compared with the groups treated with control diet both weaning on d 21 and d 28. There was no interaction effect between fermented diet and weaning age (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) on all indexes.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eStudies have showed that fermented feed can improve the growth performance of weaned piglets\u0026nbsp;[12, 28, 29]. Fermented feed has attracted much attention in recent researches, whereas the weaning age was often neglected. Few studies have focused on the interaction effect between fermented feed and weaning age. The present study was conducted to investigate the effects of\u0026nbsp;fermented\u0026nbsp;feed on the\u0026nbsp;growth\u0026nbsp;performance, intestinal function and microbiota of piglets weaned at different age.\u003c/p\u003e\n\u003cp\u003eIn production, piglets are often weaned at 21 days of age. Jamile found that production performance of piglet improved linearly with increasing weaning days\u0026nbsp;[30]. A study by Dinan\u0026nbsp;[31]\u0026nbsp;showed that whether the piglets weaned at 21 days old or weaned at 28 days old, there was no significant difference in body weight at 46 days old. Retardation of weaning age increases ADG in short term, but the increasing in body weight is offset within 14 days post weaning\u0026nbsp;[31]. Although the 28-d weaned piglets had a\u0026nbsp;higher\u0026nbsp;ADG during the period from 21 to 28 days\u0026nbsp;old, the ADG and ADFI during 28 to 42 days old decreased, and F: G increased. This implies that compared with 21-d weaning, postponing the weaning age to 28 days old, the feed intake reduction caused by the weaning stress has not been effectively relieved. The results of a previous study also showed that whether piglets weaned at 14, 21, or 28 days old, there was no significant difference in body weight when they reached 42 days old\u0026nbsp;\u0026nbsp;[32].\u003c/p\u003e\n\u003cp\u003eIn this study, supplementation with fermented feed increased the ADG during 28-42 days of age, and the results are consistent with previous studies\u0026nbsp;[33, 34]. Compared with the control diet group, the fermented diet group increased the ADFI, and decreased F/G. After fermented, the macromolecular substances in corn and soybean meal are decomposed into small molecular substances that are more conducive to the absorption for piglets,\u0026nbsp;and\u0026nbsp;improves the utilization rate of feed. Study have shown that the\u0026nbsp;\u0026beta;-sheet structure\u0026nbsp;in\u0026nbsp;\u003cem\u003eBacillus subtilis\u003c/em\u003e fermented soybean meal is reduced by 43.2%, and the more easily absorbed random coil structure is increased by 49.9 %\u0026nbsp;[35].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFew literatures have studied on the effects of fermented feed on digestive enzyme activity of weaned piglets. Fed with soybean meal fermented by Bacillus subtilis was reported to increase the total protease and trypsin in the duodenum and jejunum of piglets\u0026nbsp;[36]. Inconsistently, in this study, feeding the fermented diet had no effect on the digestive enzymes in the stomach, jejunum and ileum. Different weaning age may be one main reason to explain the different results. In this paper, the feed trial was last for 22 days, and the test time may be too short for fermented feed to change the digestive tract enzyme activity of piglets. Differences in the fermentation bacterial strains and fermentation processes may be another reason. A study by Lei\u0026nbsp;[32]\u0026nbsp;have shown that as weaning age increases, the rate of fat absorption by piglets also increases. In this study, the jejunal lipase activity at 42 days old of 28-d weaned piglets was significantly higher than that of 21-d weaned piglets. The lipase activity increased along with the retardation of weaning age, and the digestion and absorption of fat promoted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, 28-d weaning significantly increased the ileal villus height, but had no significant effect on the villus height/crypt depth. The small intestinal morphology of 21-d weaned piglets was damaged due to weaning stress, and manifested in the decreasing of intestinal villi height and crypt depth deeper. Study have shown that the small intestinal villus in 28-d weaned piglets shrink to the shortest on the 3rd day after weaning, and begins to recover on the 5th day\u0026nbsp;[32]. Another study showed that the small intestine of piglets weaned at the age older than 28 days old recovery faster when suffering weaning stress\u0026nbsp;[7]. Weaning age affect the recovery speed of the small intestine suffering weaning stress, but on the 14th day post weaning, the villus height of the small intestine can be restored to the length before weaning\u0026nbsp;[2, 37], and that is also consistent with the results of our study. However, the fermented diet had no significant effect on the integrity of the small intestine. Moreover, the piglets used in this study are self-breeding and self-raising on our farm, with strict biological prevention and control, and transportation stress greatly reduced.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTight junction proteins such as Claudins, ZO-1 and Occludin are important components of the intestinal barrier\u0026nbsp;[38], which selectively permeate nutrients and water, and block pathogens\u0026nbsp;[39, 40]. Extracellular regulated protein kinases (ERK1/2) activation of ETS-like 1 transcription\u0026nbsp;factor 1 (Elk-1) can lead to the upregulation of pro-inflammatory cytokines interferon gamma (IFN-\u0026gamma;) and tumor necrosis factor (TNF-\u0026alpha;), which in turn damage the intestinal barrier functions and increase intestinal permeability\u0026nbsp;[41-43]. Early weaning stress can lead to the activation of\u0026nbsp;mitogen-activated protein kinase (MAPK) signaling pathway ERK1/2, resulting in an inflammatory response in the intestine. Study have shown that 14 days post weaning, the relative mRNA expression of Occludin, Claudin-1 and ZO-1 in the jejunal mucosa was droped\u0026nbsp;[2], intestinal permeability was\u0026nbsp;augmented, and the intestinal barrier functions were weakened.\u0026nbsp;In this study, compared with piglets fed control\u0026nbsp;diet, supplementation with fermented\u0026nbsp;diet\u0026nbsp;significantly increased the relative expression of Occludin, Claudin-1 and ZO-1 in the small intestine. The results indicates that fermented\u0026nbsp;diet\u0026nbsp;can improve the intestinal barrier functions of piglets. As for the weaning age, the intestinal barrier functions of 28-d weaned piglets is stronger, and there is a certain interaction effect between feed diet and weaning\u0026nbsp;age. Another study showed that toll-like receptor 2 (TLR 2) enhances the expression of ZO-1 to maintain the integrity of the intestinal barrier by protein kinase C\u0026nbsp;[44], and the toll-like receptor recognition may be affected by the regulation of intestinal microbiota\u0026nbsp;[45].\u0026nbsp;In this study, both fermented diet and 28-d weaning improved the damage of the intestinal barrier functions, but the specific effect of that pathway needs to be further explored.\u003c/p\u003e\n\u003cp\u003eCrosstalk exists between the intestinal microbiota and the host. Commensal microbes that inhabit the gut participate in digestion and absorption of nutrients, mediation of immunity system, and take part in the host\u0026rsquo;s metabolism via\u0026nbsp;their metabolites. In turn, alterations of intestinal microbiota reflect the state of the host\u0026nbsp;[46,\u0026nbsp;47]. Weaning stress causes volatility of gut microbiota in piglets\u0026nbsp;[48, 49]. Study have shown that \u003cem\u003eFusobacterium\u0026nbsp;\u003c/em\u003e(genus), \u003cem\u003eAkkermansia\u0026nbsp;\u003c/em\u003e(genus)\u003cem\u003e,\u003c/em\u003e \u003cem\u003eClostridiales\u0026nbsp;\u003c/em\u003e(genus), \u003cem\u003eDeltaproteobacteria\u0026nbsp;\u003c/em\u003e(genus), \u003cem\u003eSelenomonadales\u0026nbsp;\u003c/em\u003e(genus) in the intestine of post-weaned diarrheal piglets\u0026nbsp;are\u0026nbsp;increased compared to healthy weaned piglets. while \u003cem\u003ePrevotella\u0026nbsp;\u003c/em\u003e(genus), and \u003cem\u003eFaecalibacterium\u0026nbsp;\u003c/em\u003e(genus) decreased\u0026nbsp;[50]. \u003cem\u003eBacteroidales\u0026nbsp;\u003c/em\u003e(order) and \u003cem\u003eClostridiales\u0026nbsp;\u003c/em\u003e(order) are the two most dominant orders involved in glycolysis in the large intestine\u0026nbsp;[51, 52]. In this study, fermented diet reduced the relative abundance of \u003cem\u003eClostridiales\u0026nbsp;\u003c/em\u003e(order) and increased the relative abundance of \u003cem\u003eBacteroidales\u0026nbsp;\u003c/em\u003e(order). Fermented diet increased the relative abundance of beneficial bacteria, thereby improving the intestinal barrier functions. Fermentation of insoluble\u0026nbsp;\u003ca href=\"javascript%3A;\"\u003epolysaccharide\u003c/a\u003e by microbiota in the hindgut yields short-chain fatty acids, which take part in improve intestinal health of weaned piglets [53]. However, in this study, the composition of the intestinal microbiota has been changed, and the alterations in short-chain fatty acids producing bacteria can also be found, but the contents of short-chain fatty acids in the intestine did not change significantly.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, delaying the weaning time can improve feed conversion and intestinal barrier function. Supplementation with fermented diet significantly reduces F: G during 28 to 42 days of age in piglets, and improves the small intestinal barrier functions and cecal microbial structure. Supplementation with\u0026nbsp;fermented diet improve feed efficiency, and intestinal barrier functions of weaned piglets by modulating intestinal microbiota.\u0026nbsp;In addition, addition of fermented feed has a significant effect on the intestinal barrier of piglets at different weaning ages.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBW：Body weight\u003c/p\u003e\n\u003cp\u003eADG：Average daily gain\u003c/p\u003e\n\u003cp\u003eADFI：Average daily feed intake\u003c/p\u003e\n\u003cp\u003eF: G：Feed to gain ratio\u003c/p\u003e\n\u003cp\u003eZO-1：Zonula occluden-1\u003c/p\u003e\n\u003cp\u003eERK1/2：Extracellular regulated protein kinases\u003c/p\u003e\n\u003cp\u003eElk-1：ETS-like 1 transcription factor 1\u003c/p\u003e\n\u003cp\u003eIFN-\u0026gamma;：Interferon gamma\u003c/p\u003e\n\u003cp\u003eTNF-\u0026alpha;：Tumor necrosis factor\u003c/p\u003e\n\u003cp\u003eMAPK：Mitogen-activated protein kinase\u003c/p\u003e\n\u003cp\u003eTLR 2：Toll-like receptor 2\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures used in the present study were approved by the Animal Care and Use Committee of Guangdong Academy of Agricultural Sciences and followed the Guidelines for the Care and Use of Animals for Research and Teaching (Authorization number GAASIAS-2016-017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors read and agree to the content of this paper and its publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw 16S rRNA sequences have been deposited in the NCBI SRA under BioProject PRJNA779532. The datasets generated for this study are available upon request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the China Agriculture Research System of MOF and MARA; Special fund for scientific innovation strategy-construction of high level Academy of Agriculture Science (R2018PY-JC001, R2020PY-JG009, R2017YJ-YB1004, R2018PY-QF001); the Project of Swine Innovation Team in Guangdong Modern Agricultural Research System (2021KJ126). Independent Research and Development Projects of Maoming Laboratory(2021ZZ003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSL, YXX and JPC performed experiments, analyzed data, and wrote the manuscript. QWW and XLW performed experiments. HX, ZYJ and LW supervised the project, developed the study concept, and wrote and edited the manuscript. The authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the staff at our laboratory for their ongoing assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePluske JR, Hampson DJ, Williams IH. Factors influencing the structure and function of the small intestine in the weaned pig: a review. Livestock production science. 1997;51(1-3):215-36. https://doi.org/10.1016/S0301-6226(97)00057-2.\u003c/li\u003e\n\u003cli\u003eHu CH, Xiao K, Luan ZS, Song J. Early weaning increases intestinal permeability, alters expression of cytokine and tight junction proteins, and activates mitogen-activated protein kinases in pigs. 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A mathematical theory of communication. The Bell system technical journal. 1948;27(3):379-423.\u003c/li\u003e\n\u003cli\u003eSimpson EH. Measurement of diversity. Nature. 1949;163(4148):688.\u003c/li\u003e\n\u003cli\u003eXiong Y, Yi H, Wu Q, Jiang Z, Wang L. Effects of acute heat stress on intestinal microbiota in grow‐finishing pigs, and associations with feed intake and serum profile. J Appl Microbiol. 2020;128(3):840-52. https://doi.org/10.1111/jam.14504.\u003c/li\u003e\n\u003cli\u003eZhu J, Gao M, Zhang R, Sun Z, Wang C, Yang F, et al. Effects of soybean meal fermented by l. Plantarum, b. Subtilis and s. Cerevisieae on growth, immune function and intestinal morphology in weaned piglets. Microb Cell Fact. 2017;16(1):1-10. https://doi.org/10.1186/s12934-017-0809-3.\u003c/li\u003e\n\u003cli\u003eZhang Y, Shi C, Wang C, Lu Z, Wang F, Feng J, et al. Effect of soybean meal fermented with bacillus subtilis bs12 on growth performance and small intestinal immune status of piglets. Food Agr Immunol. 2018;29(1):133-46. https://doi.org/10.1080/09540105.2017.1360258.\u003c/li\u003e\n\u003cli\u003eFaccin JE, Tokach MD, Allerson MW, Woodworth JC, DeRouchey JM, Dritz SS, et al. Relationship between weaning age and antibiotic usage on pig growth performance and mortality. J Anim Sci. 2020;98(12):a363. https://doi.org/10.1093/jas/skaa363.\u003c/li\u003e\n\u003cli\u003eTurpin DL, Langendijk P, Chen T, Pluske JR. Intermittent suckling in combination with an older weaning age improves growth, feed intake and aspects of gastrointestinal tract carbohydrate absorption in pigs after weaning. Animals. 2016;6(11):66. https://doi.org/10.3390/ani6110066.\u003c/li\u003e\n\u003cli\u003eLeibbrandt VD, Ewan RC, Speer VC, Zimmerman DR. Effect of weaning and age at weaning on baby pig performance. J Anim Sci. 1975;40(6):1077-80. https://doi.org/10.2527/jas1976.421289x.\u003c/li\u003e\n\u003cli\u003eYun HM, Nyachoti CM, Kim IH. Effect of dietary supplementation of fermented garlic by leuconostoc mesenteroides kccm35046, on growth performance, blood constituents, nutrient digestibility, fecal microflora, and fecal scores in sows and their piglets. Can J Anim Sci. 2018;99(2):349-56. https://doi.org/10.1139/cjas-2017-0203.\u003c/li\u003e\n\u003cli\u003eKim YG, Lohakare JD, Yun JH, Heo S, Chae BJ. Effect of feeding levels of microbial fermented soy protein on the growth performance, nutrient digestibility and intestinal morphology in weaned piglets. Asian Austral J Anim. 2007;20(3):399-404. https://doi.org/10.5713/ajas.2007.399.\u003c/li\u003e\n\u003cli\u003eZheng L, Li D, Li ZL, Kang LN, Jiang YY, Liu XY, et al. Effects of bacillus fermentation on the protein microstructure and anti‐nutritional factors of soybean meal. Lett Appl Microbiol. 2017;65(6):520-6. https://doi.org/10.1111/lam.12806.\u003c/li\u003e\n\u003cli\u003eFeng J, Liu X, Xu ZR, Lu YP, Liu YY. The effect of aspergillus oryzae fermented soybean meal on growth performance, digestibility of dietary components and activities of intestinal enzymes in weaned piglets. Anim Feed Sci Tech. 2007;134(3-4):295-303. https://doi.org/10.1016/j.anifeedsci.2006.10.004.\u003c/li\u003e\n\u003cli\u003eAl Masri S, H\u0026uuml;nigen H, Al Aiyan A, Rieger J, Zentek J, Richardson K, et al. Influence of age at weaning and feeding regimes on the postnatal morphology of the porcine small intestine. J Swine Health Prod. 2015;23(4):186-203..\u003c/li\u003e\n\u003cli\u003eSuzuki T. Regulation of intestinal epithelial permeability by tight junctions. Cell Mol Life Sci. 2013;70(4):631-59. https://doi.org/10.1007/s00018-012-1070-x.\u003c/li\u003e\n\u003cli\u003eDe Vos M, Huygelen V, Casteleyn C, Van Cruchten S, Van Ginneken C. Alternative models to study the intestinal barrier function of piglets. Alternatives to Laboratory Animals. 2012;40(6):P26-7. https://doi.org/10.1177/026119291204000619.\u003c/li\u003e\n\u003cli\u003eModina SC, Polito U, Rossi R, Corino C, Di Giancamillo A. Nutritional regulation of gut barrier integrity in weaning piglets. Animals. 2019;9(12):1045. https://doi.org/10.3390/ani9121045.\u003c/li\u003e\n\u003cli\u003eAl-Sadi R, Boivin M, Ma T. Mechanism of cytokine modulation of epithelial tight junction barrier. Frontiers in bioscience: a journal and virtual library. 2009;14(2765.\u003c/li\u003e\n\u003cli\u003eAl-Sadi R, Guo S, Ye D, Ma TY. TNf-\u0026alpha; modulation of intestinal epithelial tight junction barrier is regulated by ERK1/2 activation of Elk-1. The American journal of pathology. 2013;183(6):1871-84. https://doi.org/10.1016/j.ajpath.2013.09.001.\u003c/li\u003e\n\u003cli\u003ePatrick DM, Leone AK, Shellenberger JJ, Dudowicz KA, King JM. Proinflammatory cytokines tumor necrosis factor-\u0026alpha; and interferon-\u0026gamma; modulate epithelial barrier function in madin-darby canine kidney cells through mitogen activated protein kinase signaling. BMC physiology. 2006;6(1):1-15. https://doi.org/10.1186/1472-6793-6-2.\u003c/li\u003e\n\u003cli\u003eCario E, Gerken G, Podolsky DK. Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase c. Gastroenterology. 2004;127(1):224-38. https://doi.org/10.1053/j.gastro.2004.04.015.\u003c/li\u003e\n\u003cli\u003eRakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell. 2004;118(2):229-41. https://doi.org/10.1016/j.cell.2004.07.002.\u003c/li\u003e\n\u003cli\u003eRound JL, Mazmanian SK. The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol. 2009;9(5):313-23. https://doi.org/10.1038/nri2515.\u003c/li\u003e\n\u003cli\u003eGentile CL, Weir TL. The gut microbiota at the intersection of diet and human health. Science. 2018;362(6416):776-80.https://doi.org/10.1126/science.aau5812.\u003c/li\u003e\n\u003cli\u003eKubasova T, Davidova-Gerzova L, Babak V, Cejkova D, Montagne L, Le-Floc'H N, et al. Effects of host genetics and environmental conditions on fecal microbiota composition of pigs. Plos One. 2018;13(8):e201901. https://doi.org/10.1371/journal.pone.0201901.\u003c/li\u003e\n\u003cli\u003eDou S, Gadonna-Widehem P, Rome V, Hamoudi D, Rhazi L, Lakhal L, et al. Characterisation of early-life fecal microbiota in susceptible and healthy pigs to post-weaning diarrhoea. Plos One. 2017;12(1):e169851. https://doi.org/10.1371/journal.pone.0169851.\u003c/li\u003e\n\u003cli\u003eKarasova D, Crhanova M, Babak V, Jerabek M, Brzobohaty L, Matesova Z, et al. Development of piglet gut microbiota at the time of weaning influences development of postweaning diarrhea\u0026ndash;a field study. Res Vet Sci. 2021;135(59-65. https://doi.org/10.1016/j.rvsc.2020.12.022.\u003c/li\u003e\n\u003cli\u003eTropini C, Earle KA, Huang KC, Sonnenburg JL. The gut microbiome: connecting spatial organization to function. Cell Host Microbe. 2017;21(4):433-42. https://doi.org/10.1016/j.chom.2017.03.010.\u003c/li\u003e\n\u003cli\u003eDonaldson GP, Lee SM, Mazmanian SK. Gut biogeography of the bacterial microbiota. Nat Rev Microbiol. 2016;14(1):20-32. https://doi.org/10.1038/nrmicro3552.\u003c/li\u003e\n\u003cli\u003eLordelo MM, Cunha LF, Freire J. Effects of dietary fibre source and enzyme supplementation on faecal apparent digestibility, short chain fatty acid production and activity of bacterial enzymes in the gut of piglets. Anim Feed Sci Tech. 2008;146(1-2):124-36. https://doi.org/10.1016/j.anifeedsci.2007.12.001.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Ingredients and nutrient levels of basal diet (air-dry basis).\u003c/strong\u003e\u003c/p\u003e\n\u003ctable align=\"left\" border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIngredients\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e\u003cstrong\u003eContent, %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNutrient levels\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eCorn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e32.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eME\u003csup\u003e3\u003c/sup\u003e, kcal/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e3403.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eExpanded corn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e\u0026nbsp;15.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eCP\u003csup\u003e4\u003c/sup\u003e, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e19.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eSoybean meal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e10.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eCa, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eSoybean hulls\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e5.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eTotal P, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eExtruded soybean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e12.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eAP\u003csup\u003e5\u003c/sup\u003e, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eSoybean oil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e1.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eSID\u003csup\u003e6\u003c/sup\u003e Lys, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eFish meal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e6.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eSID\u003csup\u003e6\u003c/sup\u003e Met＋Cys, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eWhey powder\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e11.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eSID\u003csup\u003e6\u003c/sup\u003e Thr, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eLactose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eSID\u003csup\u003e6\u003c/sup\u003e Trp, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eSucrose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eSID\u003csup\u003e6\u003c/sup\u003e Ile, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eCholine chloride (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eSID\u003csup\u003e6\u003c/sup\u003e Val, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eSalt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eSID\u003csup\u003e6\u003c/sup\u003e Arg, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eCalcium hydrogen phosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eSID\u003csup\u003e6\u003c/sup\u003e His, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eCalcium citrate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eSID\u003csup\u003e6\u003c/sup\u003e Leu, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e1.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003ePhytase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eSID\u003csup\u003e6\u003c/sup\u003e Phe, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eL-Lys HCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eDL-Met\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eL-Thr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eL-Trp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003ePremix\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e100.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.584652862362972%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.24604141291108%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e\u003cbr\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Provided per kilogram of diet: 12,400 IU vitamin A, 2,800 IU vitamin D3, 30 mg vitamin E, 5 mg vitamin K3, 3 mg thiamin, 10 mg riboflavin, 40 mg niacin, 8 mg pyridoxine, 40 \u0026mu;g vitamin B12, 0.08 mg biotin, 15 mg pantothenic acid, 1 mg folic acid, 80 mg Zn, 120 mg Fe, 70 mg Mn, 16 mg Cu, 0.7 mg I, 0.3 mg Se. \u003csup\u003e2\u0026nbsp;\u003c/sup\u003eValues were calculated according to NRC (2012) [17]. \u003csup\u003e3\u0026nbsp;\u003c/sup\u003eME, metabolic energy. \u003csup\u003e4\u0026nbsp;\u003c/sup\u003eCP, crude protein. \u003csup\u003e5\u0026nbsp;\u003c/sup\u003eAP, available phosphorous. \u003csup\u003e6\u0026nbsp;\u003c/sup\u003eSID, standardized ileal digestible.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e \u003cstrong\u003eContents of anti-nutritional factors\u003c/strong\u003e \u003cstrong\u003e(mg/g).\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e\u003cstrong\u003eItems\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIngredients before fermented\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIngredients after fermented\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBasal diet\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFermented diet\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e\u0026beta;-Conglycinin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e166.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e67.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e108.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e51.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eGlycinin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e233.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e63.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e129.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e44.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eTrypsin inhibitor factor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Nutrient levels in before and after fermented\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.46113989637306%\"\u003e\n \u003cp\u003e\u003cstrong\u003eItems\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBefore fermented\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAfter fermented\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.46113989637306%\"\u003e\n \u003cp\u003eWater, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e10.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e27.96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.46113989637306%\"\u003e\n \u003cp\u003eCP\u003csup\u003e1\u003c/sup\u003e, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e18.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e19.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.46113989637306%\"\u003e\n \u003cp\u003eGE\u003csup\u003e2\u003c/sup\u003e, MJ/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e16.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e17.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.46113989637306%\"\u003e\n \u003cp\u003eEE\u003csup\u003e3\u003c/sup\u003e, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e3.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e2.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.46113989637306%\"\u003e\n \u003cp\u003eCF\u003csup\u003e4\u003c/sup\u003e, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e4.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e3.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.46113989637306%\"\u003e\n \u003cp\u003eNDF\u003csup\u003e5\u003c/sup\u003e, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e4.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e2.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.46113989637306%\"\u003e\n \u003cp\u003eADF\u003csup\u003e6\u003c/sup\u003e, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e3.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e1.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.46113989637306%\"\u003e\n \u003cp\u003eAsh, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e5.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e5.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.46113989637306%\"\u003e\n \u003cp\u003eCalcium, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e1.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e1.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.46113989637306%\"\u003e\n \u003cp\u003ePhosphorus, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.26943005181347%\"\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e1\u0026nbsp;\u003c/sup\u003eCP, crude protein. \u003csup\u003e2\u0026nbsp;\u003c/sup\u003eGE, gross energy. \u003csup\u003e3\u003c/sup\u003eEE, ether extract. \u003csup\u003e4\u0026nbsp;\u003c/sup\u003eCF, crude fiber. \u003csup\u003e5\u0026nbsp;\u003c/sup\u003eNDF, neutral detergent fiber. \u003csup\u003e6\u0026nbsp;\u003c/sup\u003eADF，acid detergent fiber. The nutrient levels were determined value based on dry matter.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4. Effects of fermented diet and weaning age on the growth performance of piglets\u003c/strong\u003e\u003c/p\u003e\n\u003ctable align=\"left\" border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"12.65397536394177%\"\u003e\n \u003cp\u003e\u003cstrong\u003eItems\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"24.63605823068309%\"\u003e\n \u003cp\u003e\u003cstrong\u003e21-d\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"29.56326987681971%\"\u003e\n \u003cp\u003e\u003cstrong\u003e28-d\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"7.502799552071669%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSEM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"25.643896976483763%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.92134831460674%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.97752808988764%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.53932584269663%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.53932584269663%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.938202247191011%\"\u003e\n \u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.938202247191011%\"\u003e\n \u003cp\u003e\u003cstrong\u003eW\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.146067415730338%\"\u003e\n \u003cp\u003e\u003cstrong\u003eF*W\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.668161434977579%\"\u003e\n \u003cp\u003eBW at 21 days old, kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.31390134529148%\"\u003e\n \u003cp\u003e6.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.349775784753364%\"\u003e\n \u003cp\u003e6.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"29.59641255605381%\"\u003e\n \u003cp\u003e6.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.511210762331839%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.52914798206278%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.52914798206278%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.502242152466367%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.668161434977579%\"\u003e\n \u003cp\u003eBW at 28 days old, kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.31390134529148%\"\u003e\n \u003cp\u003e7.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.349775784753364%\"\u003e\n \u003cp\u003e7.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.798206278026905%\"\u003e\n \u003cp\u003e8.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.798206278026905%\"\u003e\n \u003cp\u003e8.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.511210762331839%\"\u003e\n \u003cp\u003e1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.52914798206278%\"\u003e\n \u003cp\u003e0.338\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.52914798206278%\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.502242152466367%\"\u003e\n \u003cp\u003e0.102\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.668161434977579%\"\u003e\n \u003cp\u003eBW at 42 days old, kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.31390134529148%\"\u003e\n \u003cp\u003e11.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.349775784753364%\"\u003e\n \u003cp\u003e12.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.798206278026905%\"\u003e\n \u003cp\u003e11.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.798206278026905%\"\u003e\n \u003cp\u003e12.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.511210762331839%\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.52914798206278%\"\u003e\n \u003cp\u003e0.021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.52914798206278%\"\u003e\n \u003cp\u003e0.603\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.502242152466367%\"\u003e\n \u003cp\u003e0.997\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.668161434977579%\"\u003e\n \u003cp\u003eADG, g/d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.31390134529148%\"\u003e\n \u003cp\u003e323.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.349775784753364%\"\u003e\n \u003cp\u003e363.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.798206278026905%\"\u003e\n \u003cp\u003e212.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.798206278026905%\"\u003e\n \u003cp\u003e230.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.511210762331839%\"\u003e\n \u003cp\u003e73.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.52914798206278%\"\u003e\n \u003cp\u003e0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.52914798206278%\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.502242152466367%\"\u003e\n \u003cp\u003e0.900\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.668161434977579%\"\u003e\n \u003cp\u003e\u003csup\u003e1\u003c/sup\u003eADFI, g/d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.31390134529148%\"\u003e\n \u003cp\u003e488.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.349775784753364%\"\u003e\n \u003cp\u003e592.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.798206278026905%\"\u003e\n \u003cp\u003e345.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.798206278026905%\"\u003e\n \u003cp\u003e426.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.511210762331839%\"\u003e\n \u003cp\u003e101.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.52914798206278%\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.52914798206278%\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.502242152466367%\"\u003e\n \u003cp\u003e0.494\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.668161434977579%\"\u003e\n \u003cp\u003e\u003csup\u003e2\u003c/sup\u003eADFI, g/d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.31390134529148%\"\u003e\n \u003cp\u003e444.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.349775784753364%\"\u003e\n \u003cp\u003e422.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.798206278026905%\"\u003e\n \u003cp\u003e314.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.798206278026905%\"\u003e\n \u003cp\u003e304.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.511210762331839%\"\u003e\n \u003cp\u003e75.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.52914798206278%\"\u003e\n \u003cp\u003e0.259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.52914798206278%\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.502242152466367%\"\u003e\n \u003cp\u003e0.679\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.668161434977579%\"\u003e\n \u003cp\u003eF: G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.31390134529148%\"\u003e\n \u003cp\u003e1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.349775784753364%\"\u003e\n \u003cp\u003e1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.798206278026905%\"\u003e\n \u003cp\u003e1.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.798206278026905%\"\u003e\n \u003cp\u003e1.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.511210762331839%\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.52914798206278%\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.52914798206278%\"\u003e\n \u003cp\u003e0.041\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.502242152466367%\"\u003e\n \u003cp\u003e0.872\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e21-d, weaning at 21 days old. 28-d, weaning at 28 days old. CD, the control diet. FD, the fermented diet. BW, body weight. ADG, average daily gain. ADFI, average daily feed intake. F: G, feed: gain ratio. F, the main effect of fermented diet. W, the main effect of weaning age. F*W, the interaction effect between fermented diet and weaning age. Data are expressed as mean and pooled SEM. \u003csup\u003e1\u0026nbsp;\u003c/sup\u003eADFI was calculated based on wet weight; \u003csup\u003e2\u0026nbsp;\u003c/sup\u003eADFI was calculated based on dry matter\u003c/p\u003e\n\u003cp\u003e\u003cstrong id=\"isPasted\"\u003eTable 5. Effects of fermented diet and weaning age on the concentrations of short-chain fatty acids in cecal content of piglets (mg/kg)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable align=\"left\" border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"15.301724137931034%\"\u003e\n \u003cp\u003e\u003cstrong\u003eItems\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"24.56896551724138%\"\u003e\n \u003cp\u003e\u003cstrong\u003e21-d\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"28.017241379310345%\"\u003e\n \u003cp\u003e\u003cstrong\u003e28-d\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"7.435344827586207%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSEM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"24.676724137931036%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.687150837988828%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.156424581005588%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.156424581005588%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.156424581005588%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.240223463687151%\"\u003e\n \u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.240223463687151%\"\u003e\n \u003cp\u003e\u003cstrong\u003eW\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.363128491620111%\"\u003e\n \u003cp\u003e\u003cstrong\u003eF*W\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.318230852211435%\"\u003e\n \u003cp\u003eAcetic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.57173678532902%\"\u003e\n \u003cp\u003e1.83\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e7.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e3.36\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e3.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.44336569579288%\"\u003e\n \u003cp\u003e1.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.3646170442286945%\"\u003e\n \u003cp\u003e0.298\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.3646170442286945%\"\u003e\n \u003cp\u003e0.763\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.86623516720604%\"\u003e\n \u003cp\u003e0.405\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.318230852211435%\"\u003e\n \u003cp\u003ePropionic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.57173678532902%\"\u003e\n \u003cp\u003e597.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e799.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e688.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e654.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.44336569579288%\"\u003e\n \u003cp\u003e39.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.3646170442286945%\"\u003e\n \u003cp\u003e0.286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.3646170442286945%\"\u003e\n \u003cp\u003e0.733\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.86623516720604%\"\u003e\n \u003cp\u003e0.140\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.318230852211435%\"\u003e\n \u003cp\u003eButyric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.57173678532902%\"\u003e\n \u003cp\u003e273.34\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e396.92\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e310.20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e308.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.44336569579288%\"\u003e\n \u003cp\u003e26.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.3646170442286945%\"\u003e\n \u003cp\u003e0.255\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.3646170442286945%\"\u003e\n \u003cp\u003e0.624\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.86623516720604%\"\u003e\n \u003cp\u003e0.241\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.318230852211435%\"\u003e\n \u003cp\u003eValeric Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.57173678532902%\"\u003e\n \u003cp\u003e56.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e93.36\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e76.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e85.30\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.44336569579288%\"\u003e\n \u003cp\u003e8.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.3646170442286945%\"\u003e\n \u003cp\u003e0.218\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.3646170442286945%\"\u003e\n \u003cp\u003e0.742\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.86623516720604%\"\u003e\n \u003cp\u003e0.438\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.318230852211435%\"\u003e\n \u003cp\u003eIsobutyric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.57173678532902%\"\u003e\n \u003cp\u003e22.39\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e34.99\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e168.12\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e28.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.44336569579288%\"\u003e\n \u003cp\u003e23.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.3646170442286945%\"\u003e\n \u003cp\u003e0.153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.3646170442286945%\"\u003e\n \u003cp\u003e0.116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.86623516720604%\"\u003e\n \u003cp\u003e0.900\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.318230852211435%\"\u003e\n \u003cp\u003eIsovaleric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.57173678532902%\"\u003e\n \u003cp\u003e29.03\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e39.46\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e31.46\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.023732470334412%\"\u003e\n \u003cp\u003e36.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.44336569579288%\"\u003e\n \u003cp\u003e2.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.3646170442286945%\"\u003e\n \u003cp\u003e0.194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.3646170442286945%\"\u003e\n \u003cp\u003e0.933\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.86623516720604%\"\u003e\n \u003cp\u003e0.609\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e21-d, weaning at 21 days old. 28-d, weaning at 28 days old. CD, the control diet. FD, the fermented diet. F, the main effect of fermented diet. W, the main effect of weaning age. F*W, the interaction effect between fermented diet and weaning age. Data are expressed as mean and pooled SEM.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Fermented diet, Weaning age, Intestinal function, Intestinal microbiota","lastPublishedDoi":"10.21203/rs.3.rs-1063432/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1063432/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBACKGROUND: \u003c/strong\u003eThe beneficial function of fermented feed in livestock industry has been widely investigated. However, little is known about the effects of fermented feed on different weaned-day piglets. This study aimed to investigate the effects of fermented diet on the growth performance, intestinal function and microbiota of piglets weaned at age of 21 days and 28 days.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eRESULTS: \u003c/strong\u003e\u0026nbsp;The results found that weaning on d 21 significantly increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) ADG, and ADFI (calculated based on wet weight and dry matter), while reduced (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) F: G, the activities of trypsin and lipase of jejunum and villus height of ileum, compared with 28-d weaning. The protein levels of Occludin, Claudin-1, ZO-1 of ileum in the groups weaning on d 21 were less (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) than the groups weaning on d 28. Moreover, dietary supplementation with fermented diet upregulated (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) Occludin, Claudin-1, ZO-1 proteins of ileum, compared with the groups treated with control diet both weaning on d 21 and d 28. In addition, dietary supplementation with fermented diet decreased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) the relative abundance of \u003cem\u003eClostridia\u003c/em\u003e (class) and increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) \u003cem\u003eBacteroidia\u003c/em\u003e (class) level of cecal microbiota, compared with the groups treated with control diet both weaning on d 21 and d 28. However, supplementation with fermented diet did not affect the concentrations of short-chain fatty acids in the cecum (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCONCLUSION: \u003c/strong\u003eTherefore, our data suggest that feed digestibility is improved in piglets weaned at 21 days, but intestinal barrier function is weaker than in piglets weaned at 28 days. However, compared with feeding control diet, supplementation with fermented diet both improved feed conversion and intestinal barrier function of weaned piglets by modulating intestinal microbiota.\u003c/p\u003e","manuscriptTitle":"Effects of Fermented Feed on the Growth Performance, Intestinal Function and Microbiota of Piglets Weaned at Different Age","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-11-15 16:43:39","doi":"10.21203/rs.3.rs-1063432/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"172c3e0c-d9e0-4335-9821-afe40bfe6939","owner":[],"postedDate":"November 15th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":8511184,"name":"Animal Science"}],"tags":[],"updatedAt":"2022-04-08T06:57:54+00:00","versionOfRecord":{"articleIdentity":"rs-1063432","link":"https://doi.org/10.3389/fvets.2022.841762","journal":{"identity":"frontiers-in-veterinary-science","isVorOnly":true,"title":"Frontiers in Veterinary Science"},"publishedOn":"2022-04-08 06:57:54","publishedOnDateReadable":"April 8th, 2022"},"versionCreatedAt":"2021-11-15 16:43:39","video":"","vorDoi":"10.3389/fvets.2022.841762","vorDoiUrl":"https://doi.org/10.3389/fvets.2022.841762","workflowStages":[]},"version":"v1","identity":"rs-1063432","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1063432","identity":"rs-1063432","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}