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).
Table 2 Effects of different antibiotic substitutes on the growth performance of nursery pigs
Items
|
ADG(kg/d)
|
ADFI(kg/d)
|
F/G
|
Control
|
0.38±0.01d
|
0.86±0.01c
|
2.29±0.09a
|
Antibiotic
|
0.43±0.01c
|
0.95±0.08b
|
2.19±0.23ab
|
GOD
|
0.53±0.01a
|
0.96±0.04b
|
1.82±0.10c
|
Microbials+GOD
|
0.56±0.03a
|
1.02±0.04a
|
1.82±0.06c
|
P-value
|
< 0.001
|
< 0.001
|
0.009
|
Note: ADG, average daily gain; ADFI, average daily feed intake; F/G, the ratio of feed to gain, indicating feeding efficiency. Feed was calculated based on dry weight. The same letters to the right of data in the same row denote no significant difference (P > 0.05), while different letters indicate significant differences (P < 0.05). The same holds in the following table.
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 Figure 4. The results from scanning and transmission electron microscopy revealed that the addition of antibiotics to feed resulted in a large area of microvillus shedding in the jejunum and ileum (Figure 2A) and structural destruction of epithelial cells (Figure 2D), especially in the ileum, compared with the control group. Epithelial cells on the surface of the intestinal villus were not intact and exhibited histological lesions. There were many hollow holes on the surface of intestinal epithelial cells (Figure 1B). Transmission electron microscopy revealed that intestinal villus epithelial cells had lateral junctions with adjacent cells (Figure 4A, D). Antibiotics caused microvillus degradation (Figure 4B, C, G, H). 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).
Figure 1. Effects of antibiotics and substitutes on microvilli in the intestinal epithelium of nursery pigs under scanning electron microscopy.
Figure 2. 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.
Intestinal microflora was also observed in the fold and surface of villi. Only a small number of cocci adsorbed among the villous folds, much fewer than in other treatments (Figure 3). This indicates that only a small number of drug-resistant cocci remained in the intestinal tract. Interestingly, under all treatments, including the basal diet, bacilli were rarely found on the surface of intestinal microvilli. Only after microbial treatment there were a small number of brevibacteria observed between the villus folds. The combination treatment with microbials and GOD significantly increased the number and richness of microorganisms between the microvilli of the jejunum (Figure 3). This showed that the abuse of antibiotics and additives destroyed the normal intestinal tract flora of pigs.
Figure 3. Effects of feed additives on the number of bacteria in the intestine of nursery pigs.
Note: A, control; B, antibiotics; C, GOD; D, microbials +GOD. The arrows indicate Bacillus brevis. Bar: 10 µm.
Figure 4. Effects of antibiotics and substitutes on microvilli in the intestinal epithelium of nursery pigs under transmission electron microscopy.
Note: The white arrow indicates the gap junction between cells. Bar: 1 µm.
Effects of different antibiotic substitutes on M cells in the intestinal epithelium of nursery pigs
Scanning electron microscopy revealed that M cells in antibiotics adding group had fewer microvilli or lacked normal microvilli on their apical plasma membrane and instead had short fold-like structures, showing a concave shape compared with normal villus epithelial cells (Figure 5A). Adding antibiotics destroyed the microvilli of intestinal epithelial cells, so it was difficult to identify which cells were M cells; we only observed M-like cells (Figure 5B). Treatment with GOD stimulated the development of short fold-like structures of M cells, resulting in papillary uplift structures on the apical surface of M cells, enhanced phagocytosis, and even bacterial-like particles adsorbed on the protrusion of M cells (Figure 5C). Combination treatment with GOD and microbials also promoted the growth of fold-like structures of M cells compared with that of the normal diet. The phenomenon that particles were captured on the protrusion of M cells was also observed.
Figure 5. Effects of antibiotics and substitutes on M cells in the intestinal epithelium of nursery pigs under scanning electron microscopy.
Note: The triangle indicates M cells. Bar: 10 µm for the left 1st column, 5 µm for the left 2nd column, 1 µm for the right columns.
Transmission electron microscopy observations showed that normal M cells lacked microvilli or had fewer and untidy, unhealthy microvilli (Figure 6A, C, E). The basal plasma membrane was deeply invaginated to form a large sac-like structure, the so-called M-cell pocket, where dendritic cells and/or lymphocytes could move in and reside. M cells possessed tight junctions and desmosomes that contacted adjacent columnar cells and lymphocytes (Figure 6D, Figure. 7E, F, Figure 8B, G, white arrow). In the cytoplasm, phagocytic endosomes and secondary lysosomes were observed (Figure 6E, F). Treatment with antibiotics resulted in organelle vacuolation and microvillus degradation and fragmentation that made the subcellular structure unrecognizable (Figure 6G, H).
Figure 6. Ultramicrostructure of M cells in the intestinal epithelium of nursery pigs under transmission electron microscopy.
Note: M indicates M cell; Ly, lysosome; mit, mitochondria; ER, endoplasmic reticulum; L, lymphocyte. The white arrow indicates a desmosome between lymphocytes and M cells, and the black arrow indicates an endocytosed cell fragment. Bar: 5 µm for the left column, 1 µm for the right columns beside 7-H (2 µm).
The addition of GOD changed the morphology of M cells in the small intestine, causing irregular microvillus uplifting on the apical surface of M cells and a stronger capture and phagocytosis capacity (Figure 7C, D), and cell fragments appeared in the deep cytoplasm (Figure 7G). In superficial cytoplasm, more phagocytic endosomes and secondary lysosomes were observed (Figure 6E, F). Multiple adjacent M cells had gap junctions between them (Figure 7E, F).
Figure 7. Effects of GOD on M cells in the intestinal epithelial cells of nursery pigs under transmission electron microscopy.
Note: M indicates M cell; Ly, lysosome; E, endosome; L, lymphocyte; the white arrow indicates 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.
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).
Figure 8. Effects of the combination of microbials and GOD on M cells in the intestinal epithelial cells of nursery pigs under transmission electron microscopy.
Note: M indicates M cell; Ly, lysosome; E, endosome; L, lymphocyte; the white arrow indicates gap junction; the black arrow indicates a lipid droplet. Bar: 5 µm for the left column, 1 µm for the right columns beside part B (2 µm).