Eucommia ulmoides Flavones as Potential Alternatives to Antibiotic Growth Promoters in a Low-Protein Diet Improve Growth Performance and Intestinal Health in Weaning Piglets

Simple Summary Antibiotic resistance is a growing threat to the effective treatment of bacterial infections in both humans and animals, making the treatment of patients and livestock more difficult or even impossible. Eucommia ulmoides flavones (EUF), which were extracted from Eucommia ulmoides leaf, have been shown strong antioxidant properties and the inhibition of pro-inflammation in our previous studies. We found that EUF could promote growth performance, improve intestinal health, and reduce colonization of coliform bacteria and diarrhea index in weanling piglets; and this suggests that EUF may be a potential alternative to in-feed antibiotic growth promoter in pig husbandry. Abstract Eucommia ulmoides flavones (EUF) have been demonstrated to attenuate the inflammation and oxidative stress of piglets. This study aimed to test whether EUF could be used as an alternative antibiotic growth promoter to support growth performance and maintain intestinal health in weanling piglets. Weaned piglets (n = 480) were assigned into three groups and fed with a low-protein basal diet (NC), or supplementation with antibiotics (PC) or 0.01% EUF (EUF). Blood, intestinal contents, and intestine were collected on days 15 and 35 after weaning. The results showed the PC and EUF supplementations increased (p < 0.05) body weight on day 35, average daily gain and gain: feed ratio from day 15 to day 35 and day 0 to day 35, whereas decreased (p < 0.05) the diarrhea index of weanling piglets. EUF treatment increased (p < 0.05) jejunal villus height: crypt depth ratio, jejunal and ileal villus height, and population of ileal lactic acid bacteria on day 15 but decreased (p < 0.05) the population of ileal coliform bacteria on day 15 and day 35. These findings indicated the EUF, as the potential alternative to in-feed antibiotic growth promoter, could improve growth performance and intestinal morphology, and decrease colonization of coliform bacteria and diarrhea index in weanling piglets.

intestinal homeostasis alternation, including decreased tight junction protein expressions and infection resistance (12). It has been reported that EUF could improve microbial balance and reduce the response of in ammation (13)(14)(15), which may help to enhance the host defense and physical barrier function . Our group has also showed that EUF alleviate the oxidative stress induced by diquat in piglets by reducing the growth performance impairment, pro-in ammatory cytokines secretion and intestinal barrier dysfunction (5,16). Meanwhile, we recently report the EUF could modulate the NF-E2-related factor 2 (Nrf2) signaling pathway in the intestine to mitigate the oxidative stress of piglets (17). The Nrf2 pathway not only involves in antioxidant by regulating the mRNA levels of antioxidant enzymes, but also enhances the intestinal barrier integrity through increasing the expression of tight junction proteins (18), which may suggest that EUF has positive impact on intestinal barrier integrity. The improved functional gut immunity and integrity are vital to reduce the permeability for viable pathogens and pathogen colonization in the gut (19,20). These bene t effects may enable it to be an effective antibiotic alternative to promote animal growth in animal husbandry. Therefore, the present study was conducted to investigate the effect of EUF as antibiotic alternative in a low-protein diet on growth performance and intestinal health of weaning piglets. The low-protein diet was used in this study to promote gut health and maintain the normal digestion and absorption capacities of enterocytes without impairing the growth performance of piglets in piglets (21). Growth performance, serum biochemical parameters, intestinal morphology and microbiota composition were monitored so as to provide the scienti c basis for the application of EUF in antibiotics-free diets in swine production.

Methods
The animal experiments were approved by the Institutional Animal Care and Use Committee of Hunan Agricultural University, Hunan, China. The animal protocol was approved by Institutional Animal Care and Use Committee (IACUC No. 20190056).

Intestinal Morphology Evaluation
The jejunal and ileal morphology were analyzed using hemotoxylin eosin staining according to Wang et al.(23). The segments of the jejunum and ileum xed in 4 % formaldehyde were used to determine morphology using hematoxylin-eosin staining. After dehydration, embedding, sectioning, and staining, villous height and crypt depth were measured with computer-assisted microscopy (Micrometrics TM; Nikon ECLIPSE E200, Tokyo, Japan). Villous height and crypt depth, counts were measured by Image-Pro Plus software, Version 6.0 on images at 40-fold magni cation in ve randomly selected elds.

Microbiota Composition Analysis
Bacteria counting were performed according to the previous studies (7,24). 0.2 g of ileal and colonic contents were weighed and immediately diluted with 9 mL of 0.9% sterilizing saline and homogenized.
Then, 10-fold dilutions of homogenate were performed (ranging from 10 −1 to 10 −8 ) and then cultivated onto MacConkey Agar Medium for the enumeration of Escherichia coli, and GM17 Medium for the enumeration of Lactic acid bacteria. The GM17 medium were then incubated for 48 h at 30 °C under anaerobic conditions, while the MacConkey agar plates were incubated for 24 h at 37 °C. The coliform bacteria and lactic acid bacteria colonies were counted immediately after removal from the incubator. Values were reported as log10 colony-forming units per gram.
DNA was extracted from ileul and colonic contents with the Tiangen stool mini kit (TianGen) according to the instructions of the manufacturer. DNA concentration was determined by spectrophotometry (Nanodrop). The DNA obtained from the intestinal luminal content was used as the template to analyze intestinal bacteria by qRT-PCR. Primers (Lactobacillus spp. were synthesized according to the previous study (25). Relative expression of genes in the treatment group was normalized to the values for the NC.

Statistical Analysis
All the results were analyzed using the SPSS v.23 software package (SPSS Inc. Chicago, IL, USA). Data are expressed as mean ± SEM. The differences among treatments were evaluated using one-way ANOVA with Tukey's test, following by the Kruskal-Wallis test when data were normally distributed. Probability values P < 0.05 were taken to indicate statistical signi cance.

Results
Growth Performance and Diarrhea Index The average daily gain, average daily feed intake, and gain : feed ratio were showed in Fig. 1 (Fig. 3). There was no difference in determined microbiota in the colon among three treatments on d 15 and 35 (P > 0.05).

Intestinal Morphology
The representative images and the results of intestinal morphology were presented in Fig. 4 and

Discussion
The present study demonstrated that dietary supplementation with EUF in a low-protein and antibioticfree diet improved growth performance and intestinal morphology, decreased the colonization of coliform bacteria and diarrhea index in weaning piglets. A low-protein diet was used as basal diet, which could improve intestinal health. Some metabolites from protein fermentation in hindgut could damage the intestinal barrier function (26). Pluske et al. (27) suggests that the fermentation of undigested protein and amino acids might be important factors contributing to the post-weaning diarrhea. While reducing the dietary protein levels could help to alleviate the diarrhea, as well as mitigate the alternation of intestinal morphology induced by pathogenic bacteria (21,28). Thus, in this study low protein diet was used as basal diet to maintain the normal absorption and digestion capacity of enterocytes and improve gut health (29,30).
Ideal antibiotic alternatives should have the same bene cial effects of antibiotic growth promoters, ensuring optimum animal performance and nutrient availability (10). Similar increases in body weight, average daily gain and feed e cacy between the piglets of antibiotic positive control and EUF treatment were obtained in our present study. In addition, serum TP level and ALP activity could re ect the body protein metabolic stasis (31) and the alteration of soft tissue membrane permeability (32,33), respectively. EUF supplementation promoted serum TP content and ALP activity compared to antibiotic positive control, which in a certain extent showing the increased nutrient e ciency and development of piglets and their organ (34). It is important to develop an alternative strategy to stimulate innate immune response and limit the infections in livestock, and subsequently decrease the use of antibiotics (35,36).
IgG is a feature of immune cell maturation and plays a critical role in defensing against infection via the direct neutralization of toxins and microbes (37)(38)(39). In our study, serum IgG concentrations were enhanced by dietary supplementation with EUF, which is also consistent with previous published report that EUF exerts immunomodulatory activities by modifying the production of cytokines in vivo (16) and regulating the NF-κB pathway in vitro (40). Accordingly, EUF may have great potential as alternative antibiotics to improve immunity and protect piglets from pathogen infection by mediating the NF-κB pathway and partly depending on its antioxidative capacity.
The proposed mechanism of promoting growth effects of a practical alternative may be involved in modulating the gut microbiome (9,10,41). Weaning stress induces the population of pathogenic Escherichia coli to proliferate to exceed those of other bacterial populations, which is associated with many diseases after weaning (42). Removing antibiotics from the diet will inevitably lead to a further increase in the number of microorganisms (43). In current study, antibiotic free dietary supplementation with EUF signi cantly increased the population of lactic acid bacteria and decreased coliform bacteria in ileal content. Various stress factors expose to piglets could lead to microbial imbalance due to the increased Enterobacteriaceae and decreased lactic acid bacteria counts resulting in infectious diarrhea (44,45). Lactic acid bacteria is most commonly used probiotic in livestock, and predominant at the early stage of pig gut micro ora construction (46). Increased lactic acid bacteria can reduce fecal pH and ammonia nitrogen levels (44,47), as well as prevent colonization of pathogenic organism colonization, therefore improve the natural microbiome and gut health (41). Lots of bioactive antimicrobial chemical forms, including phenolic acids, quinones, avonoids, tannins and alkaloids, have been identi ed and been used in animal nutrition (9,10). However, due to their complex compositions and the potential for multiple sites of action, the mechanisms of antibacterial activity are not fully understood. One of the mechanisms of inhibitory action is involved that hydroxyl groups in phenolic compounds interact with the cell membrane of bacteria to disrupt membrane structures and cause the leakage of cellular components (48).
Recent studies have explored that antibiotic exposure early in life has long-term consequences on intestinal homeostasis and epithelial barrier function (49,50). In addition to antibacterial activity, the effect of EUF on the intestinal barrier function was investigated in present study. Although we did not detect the tight junction protein expression and intestinal permeability, the signi cantly increased villus height : crypt depth ratio in jejunum and villus height in jejunal and ileal mucosa were observed in EUF supplemented group. In our previous study, EUF improves the morphology structure and barrier function of intestine in piglets challenged by diquat exhibiting higher intestinal villus height and lower serum concentrations of D-lactate and diamine oxidase (16). The intestinal barrier is the rst line of defense against pathogen attachment to and invasion of epithelial cells (51). The effect of EUF on intestinal barrier function may bene t its ability to enhance host defense against microbial infections (10,52). It should be mentioned that the e cacy of EUF supplementation on piglets were inconsistent between d 15 and d 35. The post-weaning diarrhoea, the dynamic restoration of intestinal barrier and the functional rearrangement of bacterial community in piglets are mainly occurred during rst two weeks after weaning (53,54), thereafter the microbiota population and intestinal barrier are relatively stable and maturational (55). Our results showed that the dietary EUF intervention has more optimal impact on piglet health and growth in d 0~15 than d 15~35 after weaning. It might be explained that the intestine of piglet during d 0~15 post-weaning is more susceptible to the infections (23,53) and has more urgent needs to restore the intestinal homeostasis and bacterial balance, while the mechanism remains unknown.

Conclusion
In summary, avones extracted from Eucommia ulmoides leaf have shown the positive impact on growth, immunity and microbial homeostasis in weanling piglets fed a low-protein diet. The results indicated that EUF improved the growth performance, maintained the intestinal barrier morphology, enhanced the serum immunoglobulin G level, as well as reduced the colonization of coliform bacteria and diarrhea index in weaning piglets. These ndings can contribute to exploration of EUF as potential alternative to in-feed antibiotic growth promoter to against the microbe infection in swine production although further studies are needed to further explain the mechanism.