1. Introduction
Oxidative stress occurs when cellular reactive oxygen species (ROS) overwhelms the endogenous antioxidant defense capacity. Thus, redox homeostasis is not maintained, which is suggested to influence the development of the metabolic syndrome and neurodegenerative disorders [
1]. At weaning, piglets are subjected to a number of stressors, which elevates pigs’ exposure to oxidative stress conditions [
2]. Weaning is one of the most stressful events in a neonate’s life, which contributes to low feed intake, weight loss, diarrhea, intestinal dysfunction, and atrophy [
3,
4]. Antibiotics have its merits in increased body weight, decreased mortality and morbidity, and reduction in the occurrence of subclinical diseases. The subtherapeutic use of antibiotics, which has been used as a supplement to feed for a long period to solve postweaning problems, has turned into a global practice [
5,
6]. However, antibiotic residues in livestock products and the emergence of antibiotics resistant bacterial strains have remained a burning question in the world, and a number of countries have banned the use of subtherapeutic antibiotics in diet [
7]. Therefore, alternatives to antibiotics for the alleviation and treatment of weaning stress are urgently needed. As the most preferred and effective alternatives for antibiotics in animal feeding, the beneficial effects of flavonoid-containing plant extracts and probiotics, namely antioxidative and intestinal health effects, have been shown in an increasing number of studies [
8,
9].
Flavonoids are part of the polyphenol class of phytonutrients and each type of flavonoid carries its own distinct set of actions and benefits. In recent years, a considerable number of studies focused on the effect of flavonoids, such as soy isoflavone, mulberry leaf flavonoids, or tengcha flavonoids, in improving the growth performance and health of livestock [
10,
11,
12]. Tartary buckwheat, one of the major species of pseudocereals of the genus
Fagopyrum in the
polygonaceae family, is a popular nutritional food which has been reported to contain a great abundance of amino acids, vitamins, and flavonoids [
13,
14]. As plant-derived natural molecules, tartary buckwheat flavonoids have attracted research interest. Studies using the model animal exhibited a myriad of biological activities, such as antidiabetic [
15,
16], antioxidative, anti-hypertensive [
17], and anti-inflammatory properties [
14].
Lactobacillus plantarum (
L. plantarum) is considered as probiotic which can compete with harmful gut flora colonization, maintain the gut integrity and stimulate the immune system of the host to increase the resistance to infectious agents [
18,
19]. Yang et al. (2014) reported that the supplementation of
L. plantarum improved pig’s performance and effectively prevented diarrhoea by improving the function of the intestinal barrier in early life [
20]. The cytological study showed that simultaneous application of
L. plantarum and chlorogenic acid resulted in a protective effect against LpS-induced inflammation and oxidative stress in intestinal epithelial cells [
21].
To the best of our knowledge, no study has investigated the application of tartary buckwheat flavonoids in the postweaning swine and the synergistic effect of flavonoids and probiotics has rarely been studied. The aim of the present study was therefore to examine the effect of dietary supplementation with tartary buckwheat flavonoids or L. plantarum, or their combinations on the growth performance, nutrient digestibility, antioxidant capacity and microbial diversity of weaned piglets.
2. Materials and Methods
This research was conducted at the Fang Shan pig breeding farm, Beijing, China (latitude 40.23′ N, longitude 116.60′ E). The Chinese Academy of Agricultural Sciences Animal Ethics Committee approved the experimental protocol, and all the methods conducted in this experiment were in accordance with humane animal care and handling procedures (AEC-CAAS-2017-02).
2.1. Animals, Diets and Management
The tartary buckwheat flavonoids selected in this research were purchased from MidWest bio-technology col., Ltd, Beijing, China. The crops for buckwheat flavonoids production were collected from the Sichuan province. According to the manufacturing company recommendations, the level of inclusion of tartary buckwheat flavonoids was 85% and the other 15% consisted of vitamin, microelement, and little rutin. The gradient addition test indicated the optimums doses for the growth performance was 40 mg/kg of diet. Fifty 35-day-old weaned piglets (Large White × Landrace) with an initial bodyweight of 7.85 ± 0.67 kg were randomly assigned to five treatments with 10 piglets per treatment, and the piglets were housed individually. The treatments were as follows: (1) Negative control (NC): Piglets were fed with basal diets; (2) positive control (pC): Piglets were fed with basal diets supplemented with 40 mg/kg of colistin sulfate (Zhongnongxing Feed SCI. & Tech. Co., Ltd, Beijing, China); (3)
L. plantarum (Lp): Piglets were fed with basal diets supplemented with 10
9 CFU/kg of
L. plantarum (JN560899.1); (4) tartary buckwheat flavonoids (BF): Piglets were fed with basal diets supplemented with 40 mg/kg of tartary buckwheat flavonoids; and (5)
L. plantarum and tartary buckwheat flavonoids (LB): Piglets were fed basal diets supplemented with 10
9 CFU/kg of
L. plantarum and 40 mg/kg of tartary buckwheat flavonoids. The basal diet was formulated following NRC (2012) recommendations to meet the nutrient requirements of weaned piglets and free of antibiotics or other additives (
Table 1). The feed and water were available
ad libitum during the experimental period. Feed offered and refusals and body weight were recorded at the beginning and end of the experiment to calculate the feed consumption, average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency (F:G ratio). The trial lasted 28 days.
2.2. Digestibility Trial and Chemical Analyses
Piglets were fed diets mixed with 0.1% titanium dioxide (TiO2) as exogenous indigestible marker to determine apparent digestibility of nutrients during the last 10 days of the trial. After four days of adaptation, freshly voided feces were collected by grab sampling from the pen floors of six randomly selected piglets of each treatment for six further days. All diets and fecal samples were stored immediately at −20 °C until analysis. Feces were then thawed, homogenized, subsampled, dried in an air-forced oven (Model FC-610, Advantec, Toyo Seisakusho Co. Ltd., Tokyo, Japan) at 65 °C for 48 h, and ground into 1-mm particles in a centrifugal mill (model ZM200; Retsch GmbH, Haan, Germany) for chemical analyses.
The feces and diets were analyzed for dry matter (DM, method 930.15; AOAC 1990), crude protein (Cp, 6.25 × N; method 984.13; AOAC 1990), and gross energy (GE) using an automatic adiabatic oxygen bomb calorimeter (C200; IKA Works Inc., Staufen, Germany). The contents of ash (method 942.05), ether extract (EE; method 920.39), phosphorus (p, method 965.17), and calcium (Ca, method 968.08) of the feces and diets were conducted according to the methods of AOAC (1990). The TiO
2 content was measured by the method of Myers et al. (2004) [
22]. The apparent digestibility of GE, DM, OM, Cp, EE, Ca, and p were calculated using the marker concentration of feces relative to feed by the indicator method [
23].
2.3. Blood Profiles
By the end of the trail, blood samples were collected via anterior vena cava puncture into vacuum tubes from the same piglets, which provided the fecal samples for measuring the concentration of SOD, GSH-px, CAT, and the concentration of malondialdehyde (MDA) after piglets fasted for 3 h. Blood samples were centrifuged for 15 min at 3400 rcf and the serums were removed to 1.5-mL plastic tubes and stored at −20 °C. The activity of SOD, GSH-px, and CAT and the concentration of MDA were analyzed with commercial kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) according to the manufacturer’s protocols. Blood urea nitrogen (BUN), glucose, and immunoglobulin were analyzed by a biochemical auto analyzer (Hitachi automatic biochemical analyzer 7600, Tokyo, Japan) using commercial kits.
2.4. DNA Extraction, pCR Amplification of 16S rRNA and Illumina Hiseq Sequencing
Fecal samples were collected from each pen via rectal massage and microbial DNA was extracted using a commercially available kits (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s protocols. The universal primers 341F (5′-barcode-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACCVGGGTATCTAAT-3′) were used to obtain the pCR amplicons for paired-end sequencing on an Illumina HiSeq platform. The amplification and 16S rRNA gene high-throughput sequencing were performed in Realbio Genomics Institute (Shanghai, China).
2.5. Processing of Sequencing Data
The raw Illumina fastq files were subjected to a quality control procedure using UpARSE. After quality control and filtering chimeras, the assembled sequences were clustered to generate operational taxonomic units (OTUs) at a 97% identity threshold using USEARCH. The representative sequence of each OTU was assigned to a taxonomic level in the RDp database using the RDp classifier with a confidence threshold of 80%. The α diversity index, including Chao1, Shannon, and Simpson were calculated with Mothur. The sequencing data obtained in this study were deposited in the NCBI Sequence Read Archive (SRA) under accession numbers SRR6334389 to SRR6334415.
2.6. Statistical Analysis
The relative abundances of communities do not fit normal distribution, and arcsine transformation function was performed before analyses. All data were analyzed by one-way ANOVA using SAS (version 9.1, SAS Institute, Inc., Cary, NC, USA; 2004). Statistical differences among the means of the treatments were compared using the Duncan’s Multiple Range Test. Treatment differences with p < 0.05 were considered statistically significant and 0.05 ≤ p < 0.10 were designated as a tendency.
4. Discussion
In the current study, the supplementation of tartary buckwheat flavonoids significantly increased the ADG, which is in accordance with the reports of Zhao et al. [
24] that dietary tartary buckwheat flavonoids increased the bodyweight and ADG of lamb. Similarly, flavonoids from alfalfa and
Allium mongolicum Regel increased the growth performance of growing rabbits, geese, and sheep, respectively [
25,
26]. Furthermore, previous studies reported that the similar structure of flavonoids to estradiol, which regulates the secretion of growth hormone, can accelerate protein synthesis or stimulate IGF-1 to promote the body weight gain [
27,
28,
29,
30]. However, previous studies showed the inconsistent result of the effect of probiotics on growth performance. Le et al. (2016) reported that feeding fermented wheat with
L. reuteri did not affect the growth performance of weaned piglets, while Cheng reported that the daily weight gain of weaned piglets was improved by a diet supplemented with
L. plantarum [
31]. In this research, supplementation with
L. plantarum had no significant effect on the growth performance of weaning piglets. The composition of diets, strains, and addition amount might explain the contradictory results on the use of
L. plantarum in weaned piglets.
Previous studies have confirmed that enhanced digestibility of nutrients could promote the absorption and improve the growth performance of livestock. In the present study, the supplementation of colistin sulfate,
L. plantarum, and tartary buckwheat flavonoids increased the digestibility of GE, DM, OM, Cp, EE, Ca, and p, which is in accordance with the study of Meng et al. (2010), who reported that supplementation of
Clostridium butyricum endospore complex had beneficial effects on apparent total tract digestibility of finishing piglets [
32]. Studies in weaning piglets indicated that dietary supplementation with probiotics improved the growth performance and nutrition digestibility [
33]. The promoting digestion function of probiotics might attribute to the increased secretion and activity of digestive enzymes [
34]. Compared with
L. plantarum or tartary buckwheat flavonoids fed to piglets alone, dietary supplementation of the combination of
L. plantarum and tartary buckwheat flavonoids significantly improved the digestibility of GE, DM. and p. Previous studies have verified the combined beneficial effects of probiotics in broilers [
35], weaning rabbits [
36], and growing piglets [
37]. However, further research is needed to investigate the possible mechanism of combined effects.
Serum parameters represent an integrated index of nutrient supply in relation to the utilization of nutrients. BUN is a marker of the protein metabolism and dietary amino acid balance which reflects the N utilization in the animal [
38]. In the current study, the increased concentration of BUN indicated that supplementation of tartary buckwheat flavonoids increased the protein utilization, which might be the possible cause of improved growth performance of the piglets. This effect warrants future research. Extensive studies have provided a wealth of information on the modulation of immune ability of flavonoids [
39]. In this research, the supplementation of tartary buckwheat flavonoids increased the concentration of IgA, IgG, and IgM of the weaned piglets, which is in accordance with a number of in vitro studies reporting the role of flavonoids in the regulation of immune response [
40]. The role of flavonoid in the modulation of immune system is substantiated by the data from this research.
Oxidative stress is recognized as an imbalance between reactive oxygen species (ROS) level and antioxidant mechanism activity and is considered one of the primary determinants of aging and carcass quality [
41]. Increasing experimental evidence indicated that probiotics and flavonoids exert beneficial antioxidant effects [
42]. In the present study, dietary supplementation of
L. plantarum significantly increased the activities of SOD, GSH-px, and CAT, which is inconsistent with previous observations that
L. plantarum markedly elevated the gene expression of several antioxidant genes such as GR, GSH-px, and SOD by Nrf2-mediated signal pathway [
43,
44]. Suzuki et al. (2013) identified two compounds that demonstrated 2, 2-diphenyl-1-picrylhydrazy (DppH) radical scavenging activity from cultures of
L. plantarum, which might be the underlying antioxidant mechanism of
L. plantarum [
41]. Compared with the control group, the supplementation of tartary buckwheat flavonoids improved the activity of SOD and GSH-px in this research. However, the activity of antioxidant enzymes was significantly lower than that of the
L. plantarum and combination treatments. The antioxidant activity can be attributed to the various bioactive compounds present in buckwheat, such as the total flavonoid content, which was positively correlated to the antioxidant activity of the tartary buckwheat [
45]. Supplementation with buckwheat honey also resulted in strong DppH radical scavenging activity and potential ferric reducing antioxidant activity, and enhanced hepatic antioxidant enzymes such as SOD and GSH-px [
41]. The discrepancy might attribute to the level of inclusion of tartary buckwheat flavonoids, which needs further research.
Previous studies revealed that correlation of antioxidant status and gut microbiota of the mouse and HT-29 cell model were supplemented with
L. plantarum, and studies of flavonoid metabolism indicated that intestinal bacteria are deeply involved in the interaction between metabolites and intestinal microbiota [
35,
42,
46]. In the present study, HiSeq sequencing of 16S rRNA was used to evaluate the changes in the fecal bacteria community of weaned piglets. The diversity indices and observed species were increased in the LB group compared with the other groups, suggesting that the combination of
L. plantarum and tartary buckwheat flavonoids increased the intestinal community diversity in weaned piglets and a synergistic effect of
L. plantarum and tartary buckwheat flavonoids on microbial diversity existed. In the current study, the community richness index of Chao and Observed species significantly decreased in the pC group compared with all other groups, suggesting that the addition of colistin sulfate decreased the intestinal microbial richness in weaned piglets, and the antimicrobial activity of antibiotics might be one of the reasons [
47].
We found that
Firmicutes, Bacteroidetes, and
Proteobacteria were the dominant phyla among the groups, which was consistent with the studies of Ley et al. [
48] and de Oliveira et al. [
49], who reported that
Firmicutes and
Bacteroidetes were the most numerically dominant phyla in the microbiome of terrestrial mammals. The treatments shared 114 genera, and the 19 most abundant genera (the relative abundance of genera representing more than 1% of the five libraries) were present in all samples across the different treatments. The relative abundance of the genera from this shared community varied considerably among the groups.
Megasphaera sp. is a normal inhabitant in the rumen of cattle and sheep and is also found in the feces and intestine of humans and piglets [
50]. In the current study, the genus
Megasphaera was significantly more abundant in the pC group than in the Lp and BF groups. Shetty et al. reported that
Megasphaera has several mechanisms for protection against oxidative stress and harbor multidrug resistance efflux pumps and genes that confer resistance to specific antibiotics, which might account for the growth of
Megasphaera [
51].
Selenomonas species are colonizers of the digestive system where they act at the interface between health and disease [
52]. In the current study, the genus
Selenomonas was significantly more abundant in the Lp group compared with the other four groups. Owing to their fastidious and incomplete known growth requirements, a large number of
Selenomonas was not cultured and the mechanism of more abundant in
L. plantarum treatment needs further research.