SMZ residual characteristics
In this study, the food intake of fish performed well, the feed coefficient at 4th week showed no significant difference. Various nutrients in the feed were digested and absorbed by the fish intestinal epithelial cells [3, 18]. The feed ingredients also contained amount of SMZ (0.019 µg/kg). In NS group, the SMZ residual in fish muscles was below the maximum limited value (MRLs value, 100 µg/kg) stipulated in the 235 announcement of the Ministry of Agriculture (Fig. 1a). And in other groups, the SMZ residual in fish muscles and intestinal contents reached the highest value in the 4th week, and the residual dose of SMZ in each group was closely related to the feed exposure (Fig. 1a, b). After returning to normal feed in the 6th -8th week, the residual SMZ in the fish intestinal contents were still at a high level, which may release into environment through fish excretion activity.
The SMZ residual in the aquaculture system mainly comes from the excretion of fish and the release of SMZ from the unconsumed bait . The SMZ concentration in NS group were in the range of 0.14–1.31 µg/L. And in other groups, the SMZ concentration increased rapidly from the start of aquaculture and reached a maximum in the 6th week, and then decreased rapidly, and the growth rate in the first 4 weeks was higher than that in the 4th to 6th weeks. At the 8th week, there was no difference in the residual of SMZ in the aquaculture waters of each group (Fig. 1c). These results shows that with SMZ feed, the SMZ residual in the aquaculture water will increase due to the presence of SMZ; after stopping SMZ feed, the content of SMZ in the aquaculture water environment will decrease rapidly. The sediment located at the bottom of the culture system, and there is a large amount of fish excrement and bait accumulation . And low oxygen and low temperature condition at bottom was not conducive to the degradation of SMZ . Therefore, the SMZ residues are still at a high level at the end of aquaculture period. The SMZ residues in the sediments of each treatment group were also positively correlated with the amount of feed antibiotics.
O. niloticus growth performance
Growth performance is one of the most important aspects to fish farmers because it affects production and economic benefit in the aquaculture. LS and MS feeding group promoted growth of O. niloticus by reducind feed coefficient in LS and MS group (Fig. 2c). This result most likely occurred because the appropriate amount SMZ improved the activity of intestinal epithelial cells and promoted the digestion and absorption of nutrients [22, 23]. Antibiotics reduced the negative effects of anti-nutritional factors in feed. This finding agrees with those obtained in previous studies on an appropriate amount of antibiotics can promote the growth of farmed aquatic products [23–25]. For the exposure of O. niloticus to SMZ feed significantly retarded growth performance and weight gain in HS group (P < 0.05; Fig. 2a, b, Table 1). This result is in line with excessive antibiotics may inhibit the growth of farmed aquatic products . This finding may account for the excessive SMZ produced toxicity and impaired normal life activities of O. niloticus.
The liver is an accumulation organ for fish body fat. The fat in the fish liver mainly comes from the conversion and synthesis of excess protein, carbohydrates and absorption of fat in the feed . The O. niloticus exposed to SMZ had fatter liver and higher viscera than control fish with the increased number of fat particles (P < 0.05; Fig. 3). These results indicated that, exposured to LS and MS concentration of SMZ have positive effects on fish growth performance, feed coefficient, except for HS diet. Exposure of O. niloticus to SMZ also increased the content of TG in liver (P < 0.05; Fig. 2d). These finding agree with those obtained in previous studies on several animals including mice and rainbow trout [28, 29], which showed that penicillin and erythromycin exposure greatly improved the fat accumulation may be limited fish energy metabolism and growth performance, this is most likely the reasons why the weight gain of O. niloticus in this experiment is LS, MS > NS > HS.
The SCFAs contents
SCFAs are the products of intestinal flora metabolizing and fermenting nutrients in feed. The contents of 7 SCFAs in the intestinal contents of O. niloticus in each treatment group were quantitatively analyzed. Only acetic acid, propionic acid, butyric acid, isovaleric acid, valeric acid and caproic acid were detected among all samples, isobutyric acid was not detected. The total content of 6 SCFAs in the intestinal contents of tilapia is between 45.11 µg/g − 68.92 µg/g (Fig. 4a). And the percentage content of SCFAs in all samples are mainly acetic acid (accounting for 90.45%-94.44%), propionic acid (2.88%-4.38%), butyric acid (0.76%-1.49%), isovaleric acid (0.31%-0.89%), valeric acid (0.33%-0.42%) and caproic acid (0.65%-3.15%), respectively (Fig. 4b). In animal intestines, Bacteroides mainly produces acetic acid and propionic acid and Phylum Firmicutes can produce butyric acid , while acetic acid and propionic acid not only provide energy for cells but also participate in energy metabolism in the liver , and butyric acid can regenerate mucosal cells in the intestine [32, 33]. The results of SCFAs showed that LS and MS diet would increase the content of SCFAs, and the main increase was propionic acid and butyric acid; HS group would reduce the content of SCFAs, and the main reduction was acetic acid. The content of SCFAs in the intestinal of O. niloticus in the SMZ treatment group was: LS, MS > NS > HS. These explanation supported the results that proper SMZ was fed to promote growth and excessive SMZ inhibited growth.
Intestinal flora diversity
The intestinal flora of O. niloticus has been established 20–60 days after hatching . 16 s high-throughput sequencing was used to obtain bacterial data for each treatment group. Alpha diversity index analysis showed that all the community richness index (Sobs, Ace and Chao1) were decreased with the increase of SMZ dose, and the Sobs index in HS group was significantly lower than that of NS group (P < 0.05). As for community evenness index (Shannon and Simpson), Shannon index diminished with increasing dose of SMZ, and Simpson index increased with increasing dose of SMZ. These results suggested that exposure to SMZ impair species abundance, biological abundance and intestinal flora biodiversity of O. niloticus (Table. 2). SMZ reduces the biodiversity of the intestinal flora, which was consistent with the results of other antibiotic feeding and breeding experiments . PCoA was used to explore the similarity or difference of sample community composition. Based on the sample OUT level, the binary-jaccard was used to calculate the distance between the samples and to explore the influence of SMZ on the structure of the intestinal flora. The SMZ group (LS, MS, and HS) were significantly separated from the NS group (Fig. 5), the distance with NS group increased with the increase of the SMZ dose. These results indicated that the structure of intestines flora were altered in O. niloticus treated with SMZ feed.
The intestinal flora composition and function
Based on the annotation of OUT, the influence of SMZ on the intestinal flora composition and function from the perspective of community phylogeny was clarified, the species consumption of each group in the classification level (Phylum, class, and Genus) were shown in Fig. 6.
The main dominant species in the tested group were Fusobacteria, Proteobacteria, Cyanobacteria and Actinobacteria at the phylum level, which were basically consistent with the results of He’s study , which indicating that SMZ reduced the biological diversity and changed the structure of the tilapia intestinal flora, and effected the number and proportion of dominant bacteria, but the main dominant bacteria at the phylum level was not changed. Fusobacteria in MS and HS group were much higher than NS and LS group, Proteobacteria in NS group was highest. Cyanobacteria in LS and MS group were higher than NS and HS group (Fig. 6a). These results implying that SMZ would affect the relative abundance of major dominant species.
The main dominant species were Fusobacteriia, Alphaproteobacteria, Cyanobacteria and Unclassified-p-proteobacteria at the class level (Fig. 6b), and were Cetobacterium in Fusobacteria and norank-c-cyanobacteria in Cyanobacteria at the genus level (Fig. 6c). These results showed that the abundance of Fusobacteria was positively correlated with the exposure dose. Species composition analysis showed that SMZ can inhibit the growth of Fusobacterium in the intestine of O. niloticus. Fusobacterium have obvious advantages in all samples, which may be related to their phylogenetic and phenotypic advantages . Proper SMZ promotes its growth, which may be related to the large number of Fusobacterium and the characteristics of broad-spectrum antibacterial SMZ. The Cyanobacteria was main distributed in the aquaculture water, and the abundance was related to the nutrient content of the input (mainly feed) and the eutrophication of the aquaculture environment . The Cyanobacteria in all tested tilapia intestine came from the same species; Actinomycetes are an important component of the freshwater plankton bacterial community , the Actinomycetes in the intestine of tilapia were also from the same species. This is most likely related to the culture environment (feed nutrition, SMZ and temperature).The intestinal flora is the most important group to maintain the intestinal microenvironment. The greater the number and uniformity of the intestinal flora, the stronger the resistance of the intestinal flora, micro ecology is also more stable . Obviously, the addition of SMZ changed the balance of intestinal flora in O. niloticus.