Eucommia Ulmoides Flavones as Antibiotic Alternatives in a Low-protein Diet Improve Growth Performance and Intestinal Health in Weaning Piglets

Eucommia ulmoides flavones (EUF), have been demonstrated to attenuate the inflammation and oxidative stress of piglets. This study was designed to test whether EUF could be as an antibiotic alternative to support growth performance and maintain intestinal health in weanling-piglets. Weaned-piglets (n = 480) were assigned into 3 groups and fed with a low-protein basal diet (NC), or supplementation with antibiotics (PC) or 0.1‰ EUF (EUF). Blood, intestinal contents and intestine were collected on d-15 and d-35, respectively. The results showed that the body weight on d-35, average daily gain and gain : feed from d 15 to d 35 and d 0 to d 35 in piglets of PC and EUF treatments were higher than ( p < 0.05) these in NC treatment, whereas PC and EUF treatments decreased ( p < 0.05) the diarrhea index of weanling piglets. Dietary supplementation of EUF significantly enhanced ( p < 0.05) the serum concentrations of total protein, alanine transaminase and the immunoglobulin G on d-15 of piglets compared to piglets in NC treatment. EUF supplementation increased ( p < 0.05) the jejunal and ileal villus height and the population of ileal lactic acid bacteria on d-15 but remarkable decreased ( p < 0.05) the population of ileal coliform bacteria on d-15 and d-35.


Results
The results showed that the body weight on d-35, average daily gain and gain : feed from d 15  China stipulate that medicated feed additives will be prohibited for using in animal feed in 2020 [1].
Antibiotics free diets have become a necessity in livestock and poultry industry. The withdrawal of antibiotic use will lead to lower quality and yield of animals or death, more serious disease outbreaks, then result in greater use of antibiotics for therapeutic purposes [2]. Therefore, new antibiotic alternative strategies are needed to guarantee animal health and yield growth. It has been well documented that some antibiotic alternatives and feed additives, such as plant extracts, organic 3 acids, microecologics, antimicrobial peptides, could promote the animal development and enhance the intestinal health [3][4][5][6].
Of the various alternative, plant extract is one of the most readily available and safe choice that being investigated [7,8]. The Eucommia ulmoides flavones (EUF), which are bioactive phytochemicals derived from the leaves of Eucommia ulmoides, have been demonstrated to improve the antioxidative function by reacting with free radicals [9], inhibit the growth of bacteria and reduce the response of inflammation in previous studies [10][11][12]. Our group has showed that EUF alleviate the oxidative stress induced by diquat in piglets by reducing the growth performance impairment, pro-inflammatory cytokines secretion and intestinal barrier dysfunction [3,13]. Meanwhile, we recently reported the NF-E2-related factor 2 (Nrf2) signaling pathway played an important role in EUF-regulating oxidative stress in the intestine of piglets [14]. The Nrf2 pathway not only involved in antioxidant by regulating the mRNA levels of antioxidant enzymes, but also enhancing the intestinal barrier integrity through increasing the expression of tight junction proteins [15]. The improved functional gut immunity and integrity were vital to reduce the permeability for viable pathogens and pathogen colonization in the gut. These benefit 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 because it was benefit to relieve the nutritional burden of excess dietary protein and alleviate intestinal dysfunction and diseases [16]. Growth performance, serum biochemical parameters, intestinal morphology and microbiota composition were monitored so as to provide the scientific 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.

Animal Protocol and Dietary Treatment
A total of 480 piglets (Duroc × Landrace × Large Yorkshire) weaned at 25-d of age were randomly 4 assigned to 3 groups (8 pens per group and 20 piglets per pen) as follows: (1) Negative Control (NC), low-protein basal diet no antibiotics included; (2) Positive control (PC), low-protein Basal diet + antibiotics (75 ppm quinocetone, 20 ppm virginomycin and 50 ppm aureomycin); (3) EUF treatment (EUF), low-protein Basal diet + 0.1‰ EUF. The low-protein basal diet was formulated in two phases (phase 1, days 0-15; phase 2, days 15-35) according to the nutrient requirements for weanling piglets (NRC, 2012) and the previous studies [17] (Table 1). EUF powder contained 83.61% total flavones was prepared at the department of medicine, Jishou University (Jishou, Hunan, China), which has been used in the previous study by Yuan et al [13]. Additives were added to the negative control diet at the expense of corn.
The piglets were housed in an environmentally controlled nursery with hard plastic slatted flooring, and had free access to drinking water. Piglets were fed their respective diets 3 times per day at 8:00, 13:00 and 18:00 for a 35-d period. On the morning at d 15 and 35, 24 piglets (1 piglet per pen) were randomly selected and blood samples were obtained aseptically from the jugular vein at 2 h after feeding. Serum samples were obtained by centrifugation at 2000 × g for 10 min at 4 o C and then immediately stored at -80 o C for further analysis. Piglets were anesthetized with sodium pentobarbital and killed by jugular puncture. And the intestine samples were collected after rinsing thoroughly with ice-cold physiological saline solution. About 2 cm segments of the jejunum and ileum were cut and fixed in 4% formaldehyde for observation of the morphology of intestinal mucosa. Ileal and colonic contents were collected for bacteria counting and DNA extraction.

Growth Performance, Diarrhea Index
Body weight and feed intake were measured at day 15 and 35. Average daily gain, average daily feed intake and gain : feed ratio were calculated. The number of pigs with diarrhea was recorded every day. Diarrhea index (%) was calculated as the number of diarrhea piglets × diarrhea days / the total number of piglets × experiment days.

Intestinal Morphology Evaluation
The jejunal and ileal morphology were analyzed using hemotoxylin eosin staining according to Xiao et 5 al. [18]. Villous height and crypt depth were measured with computer-assisted microscopy (Micrometrics TM; Nikon ECLIPSE E200, Tokyo, Japan).

Microbiota Composition Analysis
Bacteria counting were performed according to the previous studies [5,19] were synthesized according to the previous study [20]. Relative expression of genes in the treatment group was normalized to the values for the NC.

Statistical Analysis
All data were subjected to ANOVA analysis using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA).
The differences among treatments were evaluated using Tukey's test. Probability values p < 0.05 6 were taken to indicate statistical significance.

Growth Performance and Diarrhea Index
The average daily gain, average daily feed intake, and gain : feed were showed in Figure 1 (Table 2).

Microbiota Composition in Ileum and Colon
In the ileum, EUF addition increased the number of Lactic acid bacteria on d 15 but decreased the number of Coliform bacteria on d 15 and 35 compared with NC treatment (p < 0.05). And no significant difference was observed in the numbers of Lactic acid bacteria and Coliform bacteriabetween PC and EUF treatments on d15 and 35 (p > 0.05) ( Table 3)

Intestinal Morphology
Dietary supplementation with EUF increased (p < 0.05) the jejunal and ileal villus height on d 15 compared to these in NC treatment. No difference was observed on villus height and crypt depth in jejunum and ileum between PC and EUF treatments on d 15 and 35 (p > 0.05) ( Table 4).

Discussion
The present study demonstrated that dietary supplementation with EUF in a low-protein and antibiotic-free 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. Growing evidences have showed that feeding a lowprotein in post-weaning period decreased the cost of feed while effectively relieving the nutritional burden of excess dietary protein by decreasing hindgut microbial protein fermentation [16,21].
Although reducing the dietary protein content and removing antibiotics influence the growth performance and intestinal microbial structure [22,23], the low-protein diets is an efficient way for antibiotic alternative.
Ideal antibiotic alternatives should have the same beneficial effects of antibiotic growth promoters, ensuring optimum animal performance and nutrient availability [8]. Similar increases in body weight, average daily gain and feed efficacy between the piglets of antibiotic positive control and EUF treatment were obtained in our present study. In addition, EUF supplementation promoted serum TP content and ALP activity, which in a certain extent showing the increased nutrient efficiency [24]. 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 [25,26]. 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 [27][28][29]. Dietary supplementation with EUF was shown to enhance the serum IgG concentration in current study. Results from our previous published report also demonstrated that EUF exerts immunomodulatory activities by modifying the production of cytokines in vivo [13] and regulating the NF-κB pathway in vitro [30].
The proposed mechanism of promoting growth effects of a practical alternative may be involved in 8 modulating the gut microbiome [7,8,31]. 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 [32]. Removing antibiotics from the diet will inevitably lead to a further increase in the number of microorganisms [33]. In current study, antibiotic free dietary supplementation with EUF significantly increased the population of lactic acid bacteria and decreased coliform bacteria in ileal content. Various stress factors exposed to piglets could lead to microbial imbalance due to the increased Enterobacteriaceae and decreased lactic acid bacteria counts