Micropterus salmoides feed and application thereof

Abstract

The invention provides a largemouth bass feed and application thereof. The largemouth bass feed comprises 0.5 * 10 < 9 >-1.2 * 10 < 9 > CFU / g of bacillus velezensis; the feed is applied to breeding of micropterus salmoides, the feeding rate is 2-4% / day, and the breeding temperature is 25-27 DEG C; the pH (Potential of Hydrogen) is 6.8 to 7.6; according to the micropterus salmoides feed, the use amount of antibiotics in aquaculture can be reduced, diseases in the micropterus salmoides culture process can be prevented, the physique and yield of micropterus salmoides can be enhanced, drug resistance risks, environment and food residues and toxic and side effects are avoided, the development requirements of green aquaculture are completely met, and a new technical path is provided for prevention and control of bacterial diseases of aquatic products.

Description

Technical Field

[0001] This invention relates to a feed for largemouth bass and its application, belonging to the field of aquaculture technology. Background Technology

[0002] With the rapid development of aquaculture towards intensification and high density, coupled with factors such as the deterioration of the aquaculture environment and the frequent use of chemical drugs, bacterial diseases in aquatic animals are frequent. Typical pathogens are characterized by a wide host range, strong pathogenicity, and wide spread, often leading to large-scale mortality of farmed organisms and causing serious economic losses, posing a severe challenge to the sustainable development of aquaculture.

[0003] However, the control of bacterial diseases in aquaculture still relies primarily on traditional methods such as antibiotics and chemical disinfectants. While these methods can suppress or kill pathogens and quickly control the spread of diseases in the short term, the long-term use of these methods has led to a series of increasingly prominent problems, specifically: increased drug resistance in pathogens, imbalance of the aquaculture environment's microecology, and drug residues and food safety risks. Therefore, developing efficient, green, and residue-free antibiotic alternatives has significant practical and industrial value.

[0004] Biological control technologies based on the theory of microecological balance, especially the application of antagonistic probiotics, are considered one of the most promising antibiotic alternatives. However, overall research is still in its early stages, particularly in the screening and application of aquatic animal-specific antagonistic probiotics, where progress is slow and the research foundation is weak. The main bottlenecks are: most reported studies focus on single pathogens or specific hosts, lacking screening for broad-spectrum antagonistic strains against multiple hosts and pathogens; the number of antagonistic probiotic strains screened is small and their functions are limited; most studies remain at the stage of laboratory antibacterial activity verification, lacking systematic research on the large-scale fermentation process, formulation technology, and field application effects of the strains, making it difficult for the strains to move from the laboratory to industrial application. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the first objective of this invention is to provide a largemouth bass feed that can reduce the amount of antibiotics used in aquaculture, prevent diseases during largemouth bass farming, and enhance the health and yield of largemouth bass. The second objective of this invention is to provide an application of the above-mentioned largemouth bass feed, which, when applied to the farming of largemouth bass, has no risk of drug resistance, no environmental or food residues, and no toxic side effects, fully meeting the development needs of green aquaculture and providing a new technical path for the prevention and control of bacterial diseases in aquatic products.

[0006] The first objective of this invention can be achieved by adopting the following technical solution: a largemouth bass feed, the feed comprising 0.5 × 10⁻⁶ Bacillus bellii. 9-1.2×10 9 CFU / g.

[0007] Furthermore, the feed includes 1×10⁶ Bacillus belye. 9 CFU / g.

[0008] Furthermore, the *Bacillus belyssus* is *Bacillus belyssus* GY1, an existing strain disclosed in patent CN202411750165.0, and its 16S rDNA gene sequence is shown in SEQ ID NO.1.

[0009] Furthermore, the feed is pelleted feed.

[0010] The second objective of this invention can be achieved by adopting the following technical solution: an application of largemouth bass feed, wherein the largemouth bass feed is applied to the farming of largemouth bass; the feed includes 0.5 × 10⁻⁶ Bacillus bellii. 9 -1.2×10 9 CFU / g.

[0011] Furthermore, the largemouth bass are fed with feed at a rate of 2-4% per day.

[0012] Furthermore, the culture temperature is 25-27℃; the pH is 6.8-7.6.

[0013] Furthermore, the largemouth bass were fed with largemouth bass feed twice a day.

[0014] Furthermore, the stocking density is 4-5 L / fish.

[0015] Furthermore, the dissolved oxygen in the aquaculture water was 7-8 mg / L; and the ammonia nitrogen was 0.1-0.2 mg / L.

[0016] The plan mentions largemouth bass ( Micropterus salmoides California bass, also known as California perch.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The largemouth bass feed of this invention can reduce the amount of antibiotics used in aquaculture, prevent diseases in the largemouth bass farming process, and enhance the physique and yield of largemouth bass. 2. The application of this invention is to raise largemouth bass with largemouth bass feed. It is the first application of Bacillus vesalis in the breeding process of largemouth bass juveniles. It has a good immune protection effect, especially against Aeromonas hydrophila and Nocardia in largemouth bass. 3. The application of the largemouth bass feed of this invention has core advantages such as no risk of drug resistance, no environmental and food residues, and no toxic side effects, which fully meet the development needs of green aquaculture and provide a new technical path for the prevention and control of bacterial diseases in aquatic products. Attached Figure Description

[0018] Figures 1-3 The histological morphology of largemouth bass intestines was shown in the control group, intermittent feeding group, and continuous feeding group, respectively. Figures 4-6 The histological morphology of the livers of largemouth bass in the control group, intermittent feeding group, and continuous feeding group are shown respectively. Figure 7 A comparative illustration of intestinal villus length; Figure 8 A comparative illustration of intestinal villus width; Figure 9 A comparative illustration of intestinal muscle layer thickness; Figure 10 A comparative illustration of intestinal crypt depth; Figure 11 A comparison chart showing the contrast between the velour and the filament ratio; Figures 12-21 The activities of total protein (TP), globulin (GLB), total complement (CH50), lysozyme (LYZ), amylase (AMS), alkaline phosphatase (AKP), pepsin, SOD, albumin (ALB), and lipase in the serum of largemouth bass were measured. Figures 22-29 In the liver GPX SOD, Grpr2, Nrfr2, ACP, AKP, Keap1, TGF-β The relative expression level; Figures 30-38 Head kidney α-SMA, myd88a, IL34, Fas, Casp8, Casp9, IL8, TNF-α, TGF-β The relative expression level; Figure 39 A comparison of the phylum-level composition and relative abundance of the gut microbiota; Figure 40 A comparison of bacterial genus-level composition and relative abundance of the gut microbiota in juvenile largemouth bass; Figure 41 Venn diagram of gut microbiota derived from weighted UniFrac and unweighted UniFrac distances. Detailed Implementation

[0019] The present invention will now be further described with reference to the accompanying drawings and specific embodiments: Example 1: Largemouth bass fry underwent acclimatization training, being fed a basic diet twice daily for two weeks. Approximately 30% v / v of water was changed daily in the aquariums, and the water temperature was adjusted to the optimal level for fry growth. Water quality parameters were monitored regularly daily, maintaining stable and suitable rearing conditions: temperature: 26.0±1.0℃; pH: 6.8-7.6; dissolved oxygen: 7.5±0.3mg / L; ammonia nitrogen: 0.19mg / L; nitrite: 0.01mg / L. After the acclimatization period, 270 largemouth bass (mean ± SD; 7.0±0.40g) were distributed into nine fiberglass aquariums (140L, 30 fish per tank). During the 15-week experiment, they were fed twice daily (at 8:30 and 17:30) at a feeding rate of approximately 3% per day.

[0020] The continuous feeding group was fed the experimental diet (containing 1×10⁻⁶ oz) throughout the entire experiment. 9 The diet contained CFU / g Bacillus belyssus GY1, approximately 50wt% crude protein, and approximately 12wt% crude ash. The intermittent feeding group was fed the experimental diet for 2 weeks (containing 1×10...). 9 Feed containing CFU / g Bacillus berberis GY1, approximately 50wt% crude protein, and approximately 12wt% crude ash, and a regular basal diet for 2 weeks, alternating between the two diets.

[0021] The control group was fed a standard basal diet throughout the entire experiment.

[0022] Test method: 1) For 15 weeks, before sampling (after 24 hours of starvation), fish were anesthetized with 100 mg / L of ethyl 3-aminobenzoate methanesulfonate (MS-222; Sigma, USA). All largemouth bass were counted and weighed. Final body weight (FBW), survival rate (SR), feed conversion ratio (FCR), weight gain (WGR), and specific growth rate (SGR) were then calculated.

[0023] 2) Blood and tissue sample collection: After 15 weeks of feeding, three fish were randomly selected from each replicate tank using a 1 mL sterile syringe for tail vein blood collection. The blood was placed in 1.5 mL centrifuge tubes and stored overnight at 4°C. The blood was centrifuged (1200 g, 4°C for 10 min), and the collected serum was stored at -80°C for subsequent biochemical analysis.

[0024] Liver and proximal intestine were randomly collected from three largemouth bass in each group for intestinal tissue morphology examination. Six fish were randomly selected from each replicate within each group. The intestines were aseptically dissected, and the intestinal contents were flash-frozen in liquid nitrogen and then stored at -80°C for intestinal microbial diversity analysis. Head kidney and liver tissue were dissected, flash-frozen in liquid nitrogen, and stored at -80°C for gene expression analysis.

[0025] 3) Determination of serum immunological and biochemical indicators: Serum samples were collected using commercially available kits provided by Nanjing Jiancheng Biotechnology Research Institute, China. Biochemical indicators such as immunoglobulin M, acid phosphatase, and alkaline phosphatase were determined strictly according to the manufacturer's instructions. Serum amylase, pancreatic enzyme, and lipase activities were measured using colorimetric methods.

[0026] 4) Total RNA was extracted from the liver and head kidney of largemouth bass using Trizol reagent (Beijing Transgenic Biotechnology Co., Ltd., Beijing, China) according to the manufacturer's instructions. RNA quality and content were analyzed by agarose gel electrophoresis (1%) and spectrophotometry (NanoDrop 2000, Thermo Science), with all samples showing the best absorbance ratios (260 / 280 > 1.8 and 260 / 230 > 1.8). First-strand cDNA was synthesized in RT using a PrimeScrip RT-PCR Kit (Takara, Kusatsu, Japan) according to the manufacturer's instructions and stored at -20°C for subsequent analysis. qRT-PCR analysis was performed according to the methods described above.

[0027] Gut microbiota sequencing was performed in accordance with detailed descriptions in existing studies. Total genomic DNA was extracted from fecal samples of fish intestines using the EZNA Fecal DNA Kit (Omega Bio-Tek Inc., USA), strictly following the company's protocols. DNA purity and concentration were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Science, Waltham, Massachusetts, USA). The 16S rDNA variable region was amplified using 341F:CCTACGGGNGGCWGCAG (SEQ ID NO.2); 806R:GGACTACHVGGTATCTAAT (SEQ ID NO.3). The purified PCR products were sequenced using an Illumina Hiseq 2500 sequencing system. Library sequencing was performed by Novogene Biotech Co., Ltd. (Beijing, China).

[0028] 5) The protective efficacy against Nocardia and Aeromonas hydrophila was assessed. 0.2 mL of a 1.0 × 10⁻⁶ solution was injected intraperitoneally into each tank of 15 largemouth bass. 7 Aeromonas hydrophila solution at cfu / mL was administered. 0.2 mL of a 1.0 × 10⁻⁶ cfu / mL solution was injected intraperitoneally into each tank of 15 largemouth bass. 7 Nocardia cfu / mL culture was administered. The cumulative mortality rate (CM) for each replicate group was observed and recorded daily for 7 days post-challenge, and the relative survival rate was calculated using the following formula: CM (%) = 100 (total mortality rate per treatment / total number of fish accepted the challenge); Relative survival rate (%) = 100[1 - (mortality rate of the feeding group / mortality rate of the control group)].

[0029] result: After a 15-week feeding trial, compared with the control group, the continuous feeding group significantly increased the fish's body weight, specific growth rate, and survival rate, and significantly reduced feed conversion ratio (P<0.05). On the other hand, the intermittent feeding group also significantly reduced feed conversion ratio and increased the survival rate of juvenile largemouth bass (P<0.05), although body weight and specific growth rate were improved, but not significantly (P>0.05).

[0030] Table 1. Effects of feeding methods on the growth performance of largemouth bass

[0031] Figures 1-3 The histological morphology of largemouth bass intestines was shown in the control group, intermittent feeding group, and continuous feeding group, respectively. Figures 4-6 The images show the histological morphology of the livers of largemouth bass from the control group, intermittent feeding group, and continuous feeding group, respectively. Microscopic photographs and measurements of the sections after HE staining analysis revealed that feeding largemouth bass with this diet had a significant impact on the target organs of the largemouth bass. Figures 7-11 As shown, compared with the control group, the intestinal villus length of the largemouth bass in the intermittent feeding group was significantly reduced, but the intestinal villus width and intestinal muscle layer thickness were significantly increased. While the intestinal villus length and width in the continuous feeding group were higher than those in the control group, the differences were not statistically significant (P>0.05), but the muscle layer thickness was significantly higher. Furthermore, compared with the control group, the intestinal crypt depth in both the continuous feeding and intermittent feeding groups was significantly lower, and the villus-crypt ratio in the continuous feeding group was significantly higher than that in both the control and intermittent feeding groups.

[0032] At the end of the 15-week breeding experiment, such as Figures 12-21 As shown, compared with the control group, the intermittent feeding group significantly increased the levels of serum albumin and lipase in largemouth bass (P<0.05). The levels of total protein (TP), globulin (GLB), total complement (CH50), lysozyme (LYZ), amylase (AMS), alkaline phosphatase (AKP), pepsin, and SOD in the serum of largemouth bass were also increased, but not significantly (P>0.05). Compared with the control group, the continuous feeding group showed significantly increased levels of serum albumin (ALB), total protein, globulin, total complement, lysozyme, lipase, amylase, alkaline phosphatase, pepsin, and SOD in largemouth bass (P<0.05).

[0033] like Figures 22-29 As shown, genes in the liver of largemouth bass GPX The expression level of [the substance] was upregulated in both the intermittent feeding group and the continuous feeding group, with the highest expression level in the continuous feeding group, significantly higher than that in the control group and the intermittent feeding group. [In the liver] SOD Gene expression levels were upregulated after feeding largemouth bass with a specific diet, with the highest expression levels observed in the continuously fed group, significantly higher than the control group. Grpr2, Nrfr2, ACP Expression was significantly upregulated in the intermittent feeding group, while in the continuous feeding group, although the expression level was slightly higher than that in the control group, it did not reach a significant level. Compared with the control group, AKP Expression was upregulated in both the intermittent feeding group and the continuous feeding group, and in the liver of the continuous feeding group... AKP The expression level of [gene] was significantly higher than that of the control group. Keap1 The expression levels in both the intermittent feeding group and the control group did not change significantly compared to the control group. (Gene) TGF-β The expression level was upregulated in both the intermittent feeding group and the continuous feeding group, and was significantly higher than that in the control group.

[0034] After feeding largemouth bass, if Figures 30-38 As shown, α-SMA expression in the head kidney of largemouth bass was significantly downregulated in the intermittent feeding group, and significantly lower than in the control and continuous feeding groups. The expression of genes myd88a, IL34, Fas, Casp8, and Casp9 was significantly upregulated in the continuous feeding group, and significantly higher than in the intermittent feeding group. The expression level of IL8 in the head kidney was significantly upregulated in both the intermittent and continuous feeding groups, with the highest expression level in the continuous feeding group, significantly higher than in the control and intermittent feeding groups. The expression levels of TNF-α and TGF-β in the head kidney were both upregulated after the addition of *Bacillus belyssus*, with the highest expression level in the continuous feeding group, significantly higher than in the control group.

[0035] 107,370, 101,560, and 104,311 raw reads were obtained from 18 gut microbiota samples of largemouth bass (six replicates each in the control, intermittent feeding, and continuous feeding groups). After data quality filtering, an average of 104,123, 98,141, and 101,210 clean reads were obtained in the control, intermittent feeding, and continuous feeding groups, respectively. Similarly, after filtering chimeras, subsequent analysis of gut clean reads in largemouth bass yielded an average of 100,638, 95,586, and 95,488 effective reads, respectively. There were no significant differences in reads between the feeding groups and the control group. P<0.05 ).

[0036] Cluster analysis was used to obtain the OTU and α diversity indices for each group, including the community diversity (Shannon and Simpson) h and richness (Chao1 and ACE) indices of the gut microbiota of largemouth bass after the addition of Bacillus belye to the feed, as shown in Table 2.

[0037] Table 2 Operational Taxonomic Units (OTUs), α-diversity and richness indices

[0038] In summary, except for the Simpson diversity index, other alpha diversity indices, including Chao1, ACE, and Shannon, showed no significant differences among all groups (P<0.05). The Simpson diversity index showed a significant difference between the intermittent and continuous feeding groups. The commodity coverage for the control, intermittent, and continuous feeding groups was 0.9995±0.0005, 0.9997±0.0005, and 0.9995±0.0005, respectively, effectively capturing most of the microbial diversity in the treated samples. The classification similarity of the sequencing results was 97%. Taxonomically, the obtained sequences were distributed across 31 phyla, 71 classes, 147 orders, 241 families, 529 genera, and 289 species of bacteria. Figure 39 This study compared the phylum-level composition and relative abundance of the gut microbiota in juvenile largemouth bass fed a diet. At the phylum level, the most prevalent phyla were Firmicutes, Proteobacteria, and Actinobacteria, with Firmicutes being the most abundant. Compared to the control group, the relative abundance of Actinobacteria, Planctomycetes, and Verrucous Microbes was highest in the continuously fed group. Furthermore, compared to the control group, the relative abundance of Bacteroidetes, Patellae, Archaea, and Chlorocybeta decreased after the addition of *Bacillus belyssae*.

[0039] Figure 40 This study compares the bacterial genus-level composition and relative abundance of the gut microbiota in juvenile largemouth bass fed a diet. The most prevalent bacteria observed at the genus level were *Mycoplasma*, *Citrobacter*, and *Bacillus*. Compared to the control group, the intermittent feeding group showed higher relative abundances of *Mycoplasma*, *Citrobacter*, and *Lactococcus*. Compared to the intermittent feeding group, the continuous feeding group showed higher abundances of *Bacillus*, *Citrobacter*, and *Citrobacter*.

[0040] Figure 41 Venn diagram of gut microbiota derived from weighted UniFrac and unweighted UniFrac distances for the continuously fed group.

[0041] Seven days after challenge with *Aeromonas hydrophila*, feeding largemouth bass with a diet effectively improved the survival rate of the fish. The cumulative mortality rates of the control group, intermittent feeding group, and continuous feeding group were 26.67%, 26.67%, and 13.33%, respectively. The relative survival rate (%) was highest in the continuous feeding group, at 50.19%. Seven days after challenge with *Nocardia*, feeding largemouth bass with a diet effectively improved the survival rate of the fish. The cumulative mortality rates of the control group, intermittent feeding group, and continuous feeding group were 100%, 26.67%, and 20.00%, respectively. The relative survival rate (%) was highest in the continuous feeding group, at 80.00%, and 73.33% in the intermittent feeding group.

[0042] This is the first application of Bacillus vesicles GY1 in the rearing of juvenile largemouth bass in largemouth bass feed, showing particularly good immunoprotective effects against Aeromonas hydrophila and Nocardia. The principle is that Bacillus vesicles competes with Aeromonas hydrophila and Nocardia, and Bacillus vesicles can gain a competitive advantage, thus exhibiting a significant protective effect.

[0043] Largemouth bass feed can inhibit the growth of pathogens by secreting antibacterial substances (such as bacteriocins, organic acids, and ferrophiles), or reduce the colonization ability of pathogens in the breeding environment and the host's intestines through nutrient competition and space occupation, thereby increasing the diversity of intestinal microbiota and keeping the concentration of pathogens below the pathogenic threshold. On the other hand, it can also improve the disease resistance and growth performance of farmed animals by regulating the structure of the host's intestinal flora and activating the host's non-specific immunity (such as increasing lysozyme activity and enhancing phagocytic cell function).

[0044] For those skilled in the art, various other corresponding changes and modifications can be made based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of this invention.

Claims

1. A feed for largemouth bass, characterized in that, The feed contains 0.5 × 10⁻⁶ Bacillus vesiculosus. 9 -1.2×10 9 CFU / g.

2. The largemouth bass feed as described in claim 1, characterized in that, The feed contains 1×10 Bacillus vesiculus. 9 CFU / g.

3. The largemouth bass feed as described in claim 1, characterized in that, The Bacillus belesii is Bacillus belesii GY1.

4. The largemouth bass feed as described in claim 1, characterized in that, The feed is pelleted feed.

5. An application of a largemouth bass feed, characterized in that, Largemouth bass feed was applied to the farming of largemouth bass; the feed included 0.5 × 10⁻⁶ Bacillus belysinus. 9 -1.2×10 9 CFU / g.

6. The application of the largemouth bass feed as described in claim 5, characterized in that, The aforementioned farming involves feeding largemouth bass with feed at a rate of 2-4% per day.

7. The application of the largemouth bass feed as described in claim 5, characterized in that, The culture temperature is 25-27℃; the pH is 6.8-7.

6.

8. The application of the largemouth bass feed as described in claim 5, characterized in that, Feed the largemouth bass with largemouth bass feed twice a day.

9. The application of the largemouth bass feed as described in claim 5, characterized in that, The stocking density is 4-5 L / fish.

10. The application of the largemouth bass feed as described in claim 5, characterized in that, The dissolved oxygen in the aquaculture water is 7-8 mg / L; the ammonia nitrogen is 0.1-0.2 mg / L.

Images