Effects of antibiotic substitutes on growth, intestinal microvillus morphology and M cells in pigs electron microscopy study

Background: Misuse of antibiotics in feed seriously affects intestinal tract health of pigs. The feed strategy of alternative feed additives is essential for the pig industry. A single-factor test was conducted to study the effects of antibiotics in feed, glucose oxidase (GOD), and bacterial direct-fed microbials on pig growth and the morphology of M cells the small intestine. Methods: A total of 160 40-d-old weaning piglets of the DLY (Duroc × Large White × Yorkshire) breed were randomly divided into 4 treatments with 4 replicates of 10 pigs each. Dietary treatments were control (commercial basal diet with no additive) and quantitative antibiotics (0.5 kg/t), bacterial direct-fed microbials, GOD (0.5 kg/t), and microbials + GOD added to the basal diet. The preliminary trial period was 7 days, and the formal test period was 35 days. Results: The results showed that the combination of microbials and GOD signicantly increased the average daily gain of pigs (ADG) and reduced the ratio of feed intake to gain (F/G) of pigs. The results of intestinal histology by electron microscopy suggested that antibiotics in feed seriously destroyed the morphology of intestinal microvilli and M cells, perturbed the intestinal microora in nursery pigs. The bacterial direct-fed microbials were most benecial to small intestine health, which is characterized by complete microvilli, compact and orderly arrangement of villi, and rich intestinal microbes. GOD changed the morphology of M cells in the small intestine, enhanced phagocytosis, but damaged the intestinal mucosa in a certain degree. The combined use of bacterial direct-fed microbials and GOD repaired the intestinal mucosa injury caused by GOD. Conclusions: In this experiment, the microbials and GOD as feed additives could replace antibiotics and improved the growth and immune performance of nursery pigs. The bacterial direct-fed microbials were benecial to intestinal health. The excessive use of antibiotics seriously affected the development and health of the small intestine. Our results provide a theoretical basis for the application of bacterial direct-fed microbials and GOD in pig production.


Background
Antibiotics are widely used in human health and veterinary practices, and the use of antibiotics bene ts the rate and e ciency of body weight gain, reduces mortality and morbidity, and improves health and economic bene ts in pigs [1]. Antibiotics have been administered to agricultural animals for disease prevention and growth promotion for decades. However, by altering the composition and functions of the microbiota, they can also produce long-lasting deleterious effects in the host. The emergence of multidrug-resistant pathogens raises concerns about the common, and at times inappropriate, use of antimicrobial agents [2]. In-feed antibiotics have been forbidden for use in swine and livestock production in many countries around the world. Modulation by natural feed supplements as alternatives to in-feed antibiotics has been successfully practiced, the most important being probiotics, prebiotics, bacteriocins, organic acids, enzymes, bioactive phytochemicals, and antimicrobial peptides [3][4][5]. The role of these additives and their potential use in managing gut health and function in newly weaned pigs has been reviewed extensively [6,7]. GOD has been widely used in the feed production industry and has been reported to have the best antagonistic effect against different food-borne pathogens, such as Salmonella infantis, Staphylococcus aureus, Clostridium perfringens, Bacillus cereus, Campylobacter jejuni and Listeria monocytogenes [8]. Supplementation of GOD in the diet promoted the growth of nursery pigs [9]. Lysozyme improved the growth performance and altered the small intestinal villus morphology in nursery pigs [4]. The dietary supplementation of microbials (a mixture of several bene cial microorganisms) increased the growth performance of birds at an early age, stimulated the immune response, and improved the ileal morphology of broiler chickens [5]. Using both prebiotics and probiotics in the diet of pigs from weaning to nishing improved the feed-conversion ratio [10]. So far, few results on the effect of feed additive on the ultra-microstructure of the intestinal villi of pigs have been reported.
Microfold cells (M cells) are enigmatic intestinal epithelial cells residing in the follicle-associated epithelium covering gut-associated lymphoid tissue follicles [11]. They have unique morphological characteristics, including irregular brush borders, reduced microvilli, and basolateral pockets containing mononuclear phagocytes and lymphocytes [12]. M cells initiate mucosal immune responses through the uptake and transcytosis of luminal microbial antigens, which are important for maintaining intestinal homeostasis [13]. It is generally known that antibiotics and probiotics can affect microbials in the intestinal tract. Do feed additives such as antibiotics and probiotics affect the morphology of M cells?
This question remains unclear.
Therefore, the present study aims to investigate the in uence of antibiotic substitutes on growth, intestinal microvillus morphology and M cells in pigs.

Animals and management
A total of 160 healthy weaned piglets (Duroc×Landrace×Large White) with similar weights (9.67 +0.13) kg were selected as test animals, which were provided by the Pig Breeding Farm of Shandong Huayu Group. The experiment was conducted at Huayu breeding farm in Guangrao County, Dongying City, Shandong Province, and the experimental pigs were fed at the same stationary pig house. Preparations for cleaning and disinfection of the pigsty were made before the start of the experiment. After the test started, the feed was given at 8 a.m. daily, and the pigs were given ad libitum access to feed and water. Piggery hygiene, pig immunization, insect repellent and other feeding management practices strictly followed the routine procedures of the pig farm. Page 4/28 Experimental design A single-factor design was applied to study the effects of antibiotic substitutes on growth, intestinal microvillus morphology and M cells in pigs. There was no significant difference in starting body weight between treatments (P > 0.05). A total of 160 weaning piglets (Duroc × Large White × Yorkshire) aged 40 days were randomly divided into 4 treatments with 4 replicates of 10 pigs each. Dietary treatments included 1, control (commercial basal diet with no additive; diet composition and nutrition level are illustrated in Table 1); 2, basal diet plus antibiotics (main ingredients: 50% gentamicin, 0.19 kg/t; 10% clomiphene, 0.42 kg/t; 10% bacitracin zinc, 0.44 kg/t); 3, basal diet plus GOD (0.5 kg/t, 3 000 U/g, from KRVAB Bio-tech group of Beijing); and 4, basal diet plus bacterial direct-fed microbials (Lactobacillus, 3.0 ×10 9 cfu/g, Bacillus subtilis 1.4 ×10 10 cfu/g, Bacillus licheniformis 1.3 ×10 10 cfu/g; commercial name: Kofulai, from KRVAB Bio-tech group of Beijing) and GOD.
The preliminary trial period was 7 days, and the formal test period was 35 days.

Growth indicators
The quantities of feed and residues in the pig house were recorded every week to calculate the average daily feed intake (ADFI) of each pig. The lean weight of every pig was individually measured every two weeks to calculate the average daily gain (ADG). The feed to gain (F/G) ratio was calculated.

Statistics and Analysis
All data were represented as the mean ± SEM. Statistical analysis was performed by the one-way ANOVA procedure of SAS 9.1. Differences among treatments were compared using Duncan's test and considered statistically significant at P < 0.05.

Effects of different antibiotic substitutes on the growth performance of nursery pigs
The statistical results of the growth performance of the nursery pigs are shown in Table 2.
The results showed that there were significant differences in ADG, ADFI and F/G among treatments (P < 0.01). Compared with the control, the addition of antibiotics and the antibiotic substitutes in the diet significantly increased the ADG and ADFI (P < 0.05) of the nursing pigs. In comparison with the ADG (0.43 kg/d) and F/G (2.19) of antibiotic treatment, the ADG of the GOD and the microbial + GOD treatments was significantly increased, with increases of 23% and 30%, respectively (P < 0.05), but F/G was significantly decreased (by 17% in both groups). Based on the analysis of each index of growth performance, the growth performance of pigs in the group of microbials + GOD was the best in the nursery stage by ADG (0.56 kg/d), ADFI (1.02 kg/d) and F/G (1.82). Effects of different antibiotic substitutes on the intestinal microvilli of nursery pigs Representative microvillus morphology of the ileum and jejunum is illustrated in Figure 1 and Supplementing with GOD also damaged the intestinal mucosa microvilli to a certain degree ( Figure 2B). In contrast, the damage to the villus barriers of the intestine from diets containing antibiotics were relieved significantly after adding microbials to the diet. The combined use of microbials and GOD made intestinal microvilli denser ( Figure 1D) and longer ( Figure 4J).       Interestingly, the combined use of GOD and microbials strengthened lipid mobilization and resulted in many lipid droplets in M cells and adjacent epithelial cells (Figure 8). There were large phagocytic fusion vesicles in the superficial cytoplasm of M cells ( Figure 8F, H) and endosomes in the deep cytoplasm ( Figure 8D).

Discussion
The use of antibiotics in feed is being forbidden in the animal farm industry, and the development of new feeding strategies to substitute antibiotics and stimulate gut development and health in weaned pigs is essential for the long-term sustainability of the pig industry. This article thoroughly studied the ultramicrostructure of intestinal villi and M cells using electron microscopy and found that antibiotics in feed seriously impacted the morphology of both, while a substitute of antibiotics, bacterial direct-fed microbials, effectively improved the side effects of antibiotic additives on the microvillus micro ora in nursery pigs.
It is well known that antibiotics in feed improve health and economic bene ts and disrupt the gastrointestinal microbiota [2]. This paper provides visual ultra-microstructural/morphologic evidence of the in uence of antibiotics on intestinal microvilli and micro ora by electron microscopy. Earlier research has shown that feed additives affect intestinal villus morphology at the optical microscope level, comprising the length and width of the villi and the depth of the crypt [14][15][16]. In our experiment, feeding antibiotics obviously induced damage to microvilli and obviously decreased the count of microbes in the intestinal tract. The damage to microvilli induced by antibiotics is likely associated with mucosal inflammation induced by antibiotics [17,18]. It was interesting that, even in the normal control, bacilli were rare on the folds and the surface of intestinal villi. Adding microbials directly signi cantly increased the micro ora and bacterial counts in the small intestine. This suggests that the long use of antibiotics in feed on pig farms has had an effect on the microbiota in the intestinal tract of pigs. This problem deserves attention. Our results also show that microbials could be an alternative to antibiotics in feed.
In this paper, treatment with GOD improved the growth performance of pigs. This is consistent with the reports in broiler chickens [19,20]. Although treatment with GOD promoted the growth of pigs, intestinal microvillus injury can also be induced by the product of GOD, hydrogen peroxide. Because GOD speci cally catalyzes the oxidation of β-D-glucose to gluconic acid and hydrogen peroxide [21], the latter has been evidenced to induce intestinal mucosal damage in a closed circulating intestinal loop [22]. This indicates that the additive dosage is noteworthy. Combination usage of bacterial direct-fed microbials and GOD effectively improved microvillus damage repair. This suggests that feeding direct microbials was bene cial to the health of the intestinal tract in pigs. In support of this hypothesis, B. subtilis-based probiotic supplementation in uenced gut barrier integrity through increased tight junction gene expression in broilers [23]. It is worth noting that directly fed GOD changed the microfold morphology of M cells in the intestinal tract, showing a long papillary uplift structure in the apical membrane and gap junction between M cells in the side face. As far as we know, this has rarely been reported. Only Wang et al. [20] reported that dietary treatment with GOD had bene cial effects on the expression of intestinal tight junction genes in broilers. This result indicates that directly fed GOD can intensify the phagocytic function of M cells. The relevant mechanisms need to be further identi ed.
Interestingly, many lipid droplets were observed in M cells and intestinal epithelial cells in the microbial group. Why did many lipid droplets appear in M cells and intestinal epithelial cells after treatment with microbials? What role do they play? These questions are not yet clear. Data have shown that lipid droplets are organelles involved in lipid metabolism and the production of in ammatory mediators [24].
We speculate that they are involved in the mobilization of lipids or in immunomodulation. Because research suggests that probiotic yeast has the ability to prevent in ammation by promoting proin ammatory immune function and increasing the production of short-chain fatty acids [25].  Effects of antibiotics and substitutes on microvilli in the intestinal epithelium of nursery pigs under scanning electron microscopy.

Figure 2
Page 19/28 Effects of antibiotics and substitutes on villus development in the intestine of nursery pigs. Note: Top three rows are scanning electron micrographs, and the last row is transmission electron micrographs. The arrows indicate lesions of microvilli. Bar: for scanning electron micrographs, 10 µm for the left two columns, 1 µm for the right column; for row D, 5 µm for the left, 2 µm for the right.    a gap junction between M cells, the black arrow indicates an endocytosed cell fragment, and the arrow head indicates a particle to be phagocytosed; Bar: 5 µm for the left column, 1 µm for the right columns.