Effect of Supplementation Yeast Fermentation Products on Growth Performance and Intestinal Health of Weaned Piglets Challenged With Salmonella Typhimurium

Background: This study investigated the effects of piglets with dietary supplementation yeast fermentation product (YFP) on growth performance, immune status and intestinal inammation after a Salmonella typhimurium challenge. Twenty-four weaned piglets were assigned to four treatments including: non-challenge control (Con); Salmonella typhimurium-challenged control (ST); ST + 0.2% YFP (0.2% YFP); and ST + 0.4% YFP (0.4% YFP). All piglets were challenged twice with Salmonella typhimurium. All of them were killed at 7 th day after the second challenge to obtain plasma and intestine for analysis. Results: 0.4% YFP increased average daily gain (ADG) and duodenal villus height and villus height/ crypt depth (P < 0.05) and decreased feed-gain ratio (P < 0.05) after Salmonella typhimurium challenge compared with ST group. The 0.4% YFP decreased the elevating concentrations IL-1b and IL-6 (P < 0.01) induced by Salmonella typhimurium and increased the concentration of IL-10 (P < 0.05) in plasma compared with ST group. Furthermore, YFP inuenced the apoptosis related mRNA levels of Bax, Bcl-2 and caspase 3 (P < 0.05) and increased intestinal occludin protein expression (P < 0.05). 0.4% YFP down-regulated the mRNA expressions of TLR4, MyD88, IRAK1, TRAF6 and NFkB (P < 0.05) and decreased the mRNA expression of IL-6, TNFa (P < 0.05) and increased the IL-10 (P < 0.01) in duodenum compared with ST group. In addition, 0.4% YFP decreased the phosphorylation levels of p38 MAPK and ERK1/2 (P < 0.01). Conclusions: Dietary 0.4% yeast fermentation product supplementation had positive effects on growth performance and intestinal barrier function and reduced intestinal inammation of weaned piglets challenged with Salmonella


Background
Due to the risk of emergence of resistant bacteria and resistance in humans, China has already banned the use of antibiotics in feed from 2020, though amounts of studies have reported that the use of antibiotics can improve growth performance and protect intestinal infections of weaned piglets [1][2][3].
Therefore, increasing attention has focused on searching for alternatives to antibiotics.
The effects of dietary supplementation with yeast fermentation products on the improvement of intestinal health could therefore be of great importance in pigs. Yeast fermentation products contain amounts of bioactive compounds including nucleotides, nutritional metabolites and cell wall polysaccharides (speci cally β-glucan and mannan). Several studies have investigated the impact of yeast cell wall components on immune function of weaning piglets [4][5][6][7]. It has been reported that yeast β-glucans alleviated the elevated of pro-in ammation cytokines and enhanced the production of antiin ammatory cytokines in LPS or Escherichia coli challenged piglets [8,9]. Dietary supplementation yeast-derived mannans could improve piglets' weight gain and enhanced immune function [10]. Since yeast fermentation products are bene cial to the growth of piglets, it has been considered to be a potential in-feed antibiotic alternative.
However, how the in uence of yeast fermentation products on intestinal health on the condition of Salmonella typhimurium challenge is unclear and the underlying mechanism is not documented. Therefore, this study focused on indicating how yeast fermentation products improved growth performance and intestinal health for piglets challenged with Salmonella typhimurium. Our study will provide potentiality to nd alternatives to antibiotics.

Animals and experimental design
A total of 24 weaned male piglets (Duroc ⋅ Yorkshire ⋅ Landrace) with an initial average body weight of were housed at Laboratory Animal Center of Nanjing Agricultural University. The piglets used did not receive Salmonella typhimurium vaccines, antibiotic injections or antibiotics in feed. All piglets used in this study were susceptible to Salmonella typhimurium. Piglets were housed in pens (pen size: 1.2 m ⋅ 1 m) in an environmental controlled nursery building. Each pen was equipped with a feeder, a nipple drinker and plastic-covered expanded metal oors. The room temperature was maintained at 25 ± 1℃ throughout the study. The piglets had free access to feed and water. The yeast fermentation products are provided by Cargill Company ( XPC, Diamond V, Cedar Rapids, IA, USA).
The experiment lasted for 5 weeks, including 21 days before and 14 days after the rst Salmonella typhimurium challenge. All piglets were randomly allotted to four dietary treatments (n = 6) based on the initial body weight included: (1) Negative control (Con): control diet, without salmonella typhimurium challenge; (2) Positive control (ST): control diet, with salmonella typhimurium challenge; (3) 0.2% YFP: control diet plus 2 g/kg feed, with salmonella typhimurium challenge; (4) 0.4% YFP: control diet plus 4 g/kg feed, with salmonella typhimurium challenge ( Table 1). The experimental diets were fed to pigs through the study duration. After 21 days experimental diets feeding, the piglets of the last three groups orally inoculated with 5 mL suspension containing 10 9 CFU of Salmonella typhimurium. A week later, the second challenge was performed the same as the rst. Body weight of per piglet was recorded at days 0, 7, 14, 21 and 35 and feed consumption per pen was recorded every day of the experiment to calculate the average daily gain (ADG), average daily feed intake (ADFI) and feed e ciency (F:G) from d 1 to 21 (prechallenge) and d 21 to 35 (post-challenge).

Sample collections
All the piglets were sacri ced by exsanguination at the end of the study. Blood samples were collected into heparinized tubes. Plasma was separated by centrifugation at 3000 rpm/min for 15 min and stored at -20 °C until analysis. Once dead, the abdomen was immediately opened, and the intestinal tract was excised. Duodenum segments (approximately 8 cm in length) were opened longitudinally and gently ushed with sterile saline to remove the contents. Duodenal mucosa samples were collected by lightly scraping with sterile glass slides on rice, immediately frozen in liquid nitrogen and stored at -80 °C for analysis. 3-cm sections from the duodenum (to about 10 cm distal to the pylorus) were collected and xed in 4% paraformaldehyde for intestinal morphology analysis.

Intestinal morphology analysis
Duodenal segments were removed from the stationary solution and then were embedded in para n. Sections (3 µm) were stained with hematoxylin and eosin (HE) to investigate architecture of the duodenum. Stained slices were scanned with the Pannoramic SCAN II and images were captured with 3DHISTECH software (3DHISTECH Ltd. Budapest, Hungary).

Plasma in ammatory cytokine concentration analysis
Plasma IL-1β, IL-6 and IL-10 concentrations were measured using the ELISA kits suitable for porcine IL-1β, IL-6 and IL-10 (Jiangsu MEIMIAN Industrial Co. LTD, China), respectively, according to the manufacture's protocol. Plasma concentrations of IL-1β, IL-6 and IL-10 were calculated from the standard curve and expressed as ng/L.

Total RNA isolation and real-time PCR
Total RNA was isolated from 30 mg frozen duodenal mucosa with 1 mL TRIzol (Sangon Biotech, Shanghai, China) and reverse-transcribed according to the manufacturer's protocol (Vazyme Biotech, Nanjing, China). Diluted cDNA (2 µL, 1:25) was used as template for real-time PCR that was performed on a real-time PCR system (Mx3000P, Stratagene, USA). Moreover, GAPDH was chosen as a reference gene to normalize the mRNA abundance of target genes. All primers were synthesized by and listed in Table 2.
The 2 −ΔΔCT method was used to analyze real-time PCR data.

Statistical analysis
All data were checked for normality using exploratory analysis. Data were analyzed using SPSS 21.0 for Windows (SPSS Inc., Chicago, IL, USA). All data were analyzed using One-Way ANOVA with diet (YFP) as a xed factor. The piglets were recognized as a statistical unit. The values were presented as the means ± SEM with signi cance at P < 0.05.

Growth performance
Salmonella typhimurium administration tended (P = 0.09) to decrease the average daily gain (ADG) compared with Con group, yet 0.4% YFP supplement was signi cantly (P < 0.05) increased compared with ST group. The ratio of feed and gain (F/G) was observed to signi cantly increase with Salmonella typhimurium challenged compared with control group and it reversed by 0.4% YFP (P < 0.05). No signi cant changes were observed in ADG or F/G between control and YFP supplement groups before Salmonella typhimurium treatment (Table 3).

Intestinal morphology
Compared with the Con, the duodenum villus height and villus height/crypt depth were signi cantly lower (P < 0.01) in the ST group; however, the crypt depth was not affected by Salmonella typhimurium. The villus height and villus height/crypt depth were reversed to the control levels by 0.2% or 0.4% YFP exposure (Fig. 2).

Intestinal apoptosis-related gene abundances and protein expression
The relative mRNA abundances of Bcl2-associated X protein (Bax) (P < 0.01) and cysteinyl aspartate speci c protease 3 (caspase 3) (P < 0.001) were up-regulated by Salmonella typhimurium treatments; however, they were down-regulated by 0.4% YFP addition. B-cell lymphoma-2 (Bcl-2) ( P < 0.05) mRNA abundance was down-regulated by Salmonella typhimurium treatments, 0.4% YFP addition up-regulated the mRNA abundance (Fig. 4A). Furthermore, Bax protein expression in the ST groups was signi cantly higher (P < 0.05) than the control group, but 0.2% or 0.4% YFP addition retrieved it (Fig. 4B). These results deduced that YFP could alleviate the intestine mucous apoptosis induced by Salmonella typhimurium.

Intestinal MAPK signaling pathway expression
Compared with the Con piglets, the phosphorylation levels of ERK (Fig. 7A) and p38 (Fig. 7B) were signi cantly increased (P < 0.01) in the Salmonella typhimurium-challenged piglets. Meanwhile, 0.2 % and 0.4% YFP all signi cantly inhibited the activation of the key proteins in the MAPK pathway compared with ST group.

Discussion
Yeast fermentation product is used widely in the livestock industry. The potential mechanisms of action of yeast products that maintains an advantageous intestinal environment [12] by regulating microbial ecology and elevates intestinal immunity through preventing pathogenic bacteria from intestinal epithelial cells, consequently improving growth performance have been reported [13,14]. In this study, during the pre-challenge period, supplementation of 0.2% and 0.4% YFP had no signi cant effect on growth performance of piglets compared with the control. However, during the Salmonella typhimurium challenge period, 0.4% YFP signi cantly enhanced ADG compared with ST group. In general, the present study indicates that the relatively high dose of YFP offered protection against Salmonella typhimurium and that is bene cial to enhance growth performance of piglets during Salmonella typhimurium infection.
The following discussion was focused on the 0.4% YFP effects after Salmonella typhimurium infection.
Intestinal morphology can be used as an indicator for intestinal health [15]. To response in ammation induced by pathogens, deeper crypts exhibited faster cellular turnover allowing renewal of villus as needed [16]. In the current study, dietary YFP supplementation improved intestinal histomorphology in the infectious piglets. Salmonella typhimurium challenge induced signi cantly deeper crypt and shorter villus in the duodenum, the same observations reported challenged with Salmonella typhimurium in piglets. In the challenged piglets, however, the dietary YFP supplementation remarkably increased villus height and decreased crypt depth, which contributed signi cantly to enhance V/C ratio, indicating the effective nutrient absorption capability and the decreased F/G.
Tight junctions among intestinal epithelial cells, which protect the body from intestinal pathogens, in uence intestinal mucosal barrier function to a great extent [17]. Occludin, as the major integral proteins, form the continuous tight junction strands [18]. In the current study, YFP supplementation signi cantly increased occludin expression in duodenum, which is expected to improve intestinal mucosal barrier function. Our results demonstrated that YFP kept intestinal integrity and barrier function partially by elevating intestinal tight junction protein expression.
Currently, apoptosis is derived by the expression of Bax, Bcl-2 and caspase 3 [19]. In this study, the mRNA abundances of Bax and caspase 3 were signi cantly up-regulated, and Bax protein expression level was also increased in the ST group, demonstrating that ST induced intestinal epithelial cell apoptosis, but both mRNA abundances and Bax protein expression were down-regulated with supplementation YFP. Our ndings showed that YFP might protect intestinal barrier function by inhibiting apoptosis pathway.
LPS induces in ammation in pigs by stimulating the production of cytokines such as IL-1, IL-6 and TNFα, which are considered as endogenous mediators of in ammation [20]. IL-10 is an important antiin ammatory cytokine which is required for protection in the regulation of intestine homeostasis during host defense [21,22]. HSP70 also plays a signi cant role on intestinal in ammation response. The concentrations of plasma IL-1β, IL-6 and IL-10 were measured as indicators of systemic pro-and antiin ammation responses, respectively. In the present study, the increased plasma IL-1β and IL-6 concentrations indicated successful establishment of the Salmonella typhimurium challenge model. And Salmonella typhimurium caused an increased of intestinal HSP70 protein expression. Piglets receiving YFP had less concentration of plasma IL-1β and IL-6 and higher concentration of IL-10 than ST group, hence, implying that YFP lowered pro-in ammation of the immune system. The overproduction of proin ammatory cytokines is associated with anorexia, which may explain the observed reduction in ADG in Salmonella typhimurium-challenged piglets in our study [23,24]. Meanwhile, the evidence that lower mRNA expression of IL-1β, IL-6 and TNFα, and higher IL-10 expression compared to the ST piglets in duodenum indicated that dietary supplementation with YFP contributed to bene cial immunoregulatory responses.
Toll like receptor 4 (TLR4) is the rst identi ed of TLRs family, which recognizes the LPS component of Gram negative bacteria on the cell surface [25]. The activation of TLR4 signaling pathway activates members of the MAPK family including p38 and ERK1/2, leading to numerous pro-in ammatory cytokine genes translation [26]. TLR4 and MAPK signaling are of great signi cance in intestinal in ammation [27,28]. In the present study, ST challenge increased mRNA expression of TLR4 and its downstream signals  IRAK1, TRAF6 and NFκB. YFP supplementation decreased TLR4, MyD88, IRAK1, TRAF6 and NFκB mRNA  expression and TLR4 protein, p38 phosphorylation and ERK1/2 phosphorylation. Duan et al. [29] demonstrated that mannan oligosaccharide (a component of YFP) supplementation could enhance intestinal mucosal immune competence and suppress intestinal in ammation by decreasing the contents of pro-in ammatory cytokines. Sun et al. [30] demonstrated that saccharomyces cerevisiae polysaccharide (a component of YFP) could decrease the pro-in ammation mediators of IL-1β and IL-6 at protein and mRNA levels by inhibiting MAPK activity in mice with DSS-induced colitis, which were consistent with our results.

Conclusion
Dietary yeast fermentation products supplementation could suppress Salmonella typhimurium hazards. And salmonella typhimurium-challenged piglets fed diets supplementation with 0.4% yeast fermentation products exhibited better growth performance possibly by alleviating intestinal in ammation and apoptosis to improve intestinal health. Effect of YFP supplementation on mRNA and protein expression of in ammation-related signaling molecules after Salmonella typhimurium challenge in piglets. β-actin were used for normalization for relative TLR4 expression. The values are presented as mean ± SEM. *P < 0.05, **P < 0.01

Figure 6
Effect of YFP supplementation on mRNA expression of in ammation cytokines in duodenum after Salmonella typhimurium challenge in piglets. The values are presented as mean ± SEM. *P < 0.05, **P < 0.01