Text: Bread Clip Knowledge
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«——【Preamble】】 ——»
Intensive aquaculture typically uses chemicals and antibiotics to prevent and control diseases caused by bacteria, fungi, viruses and parasites. However, food safety problems caused by antibiotics are becoming more and more serious.
Therefore, the search for safe and effective alternatives to antibiotics is important for the sustainable development of aquaculture.
Probiotics have the advantages of safety, effectiveness, no residue and no pollution, and probiotics such as bacillus, lactococcus, yeast, enterococci and lactobacillus can affect the intestinal microbial community structure of aquatic animals through in vivo colonization.
Improve digestion, absorption and metabolic function, enhance immunity level and prevent diseases, thereby promoting the growth of fish and shrimp, and ensuring the healthy breeding of aquatic animals.
However, a scientific research base in Beijing found that Lactobacillus can improve the intestinal microecological environment, enhance the barrier layer function of intestinal epithelial cells, improve the immune function of animals, etc., and play a role in animal production.
Adding a single lactobacillus or prebiotics containing lactobacilli to the feed can improve the survival rate of aquatic animals, promote their growth, and increase the number of intestinal beneficial bacteria.
Red crayfish belongs to the order Decapodae, Crayfishidae and Slippery Crayfish, which has the advantages of miscellaneous diet, fast growth, high yield, wide environmental tolerance, delicate meat, delicious taste, high meat yield and strong ornamentality.
In this study, this study was conducted to explore the effects of lactobacillus added to the diet on its growth performance and intestinal microbial community structure, so as to provide a reference for the healthy cultivation of crayfish.
«——【Materials and Methods·】——»
Lactobacilli were the species that were retained after screening (viable count was 1.8×109 CFU/mL). The strains were activated and expanded in nutrient broth (NB) medium and nutrient agar (NA) medium to obtain active strains for follow-up experiments.
Red crayfish are self-propagated shrimp fry in the laboratory. A total of 600 healthy and disease-free shrimp seedlings with initial weight (0.55±0.05) g were randomly divided into 4 groups, with 3 replicates in each group and 50 shrimp seedlings in each replicate.
The basic feed is freshwater crayfish compound feed produced by Shanghai Hongma Feed Co., Ltd., with a nutritional level of 32% crude protein, 5% crude fat, 1.3% lysine and 1.0% total phosphorus.
Lactobacillus in PBS with a viable number of 1.8×107 CFU/mL was added to the basic feed of red crayfish, and the addition amounts were 0 (CA group), 1.8×105 (TB group), 1.8×106 (TC group) and 1.8×107 CFU/g (TD group), respectively, and the experimental period was 45 days.
The breeding test was carried out in the aquatic aquaculture training center of Rizhao Vocational and Technical College, in the circulating water system.
Each breeding tank is 1 repeat, the shelter is placed in the breeding tank, the breeding water is aerated tap water, the water temperature is 25~26 °C, the dissolved oxygen is greater than 5mg/L, feeding 2 times a day (7:00, 17:00), after feeding for 30min, siphon out the manure and residual bait. Observe the growth, health status and molting of shrimp every day.
PCR products were detected by electrophoresis using agarose gels at a concentration of 2%, PCR products were recovered by agarose gel at a concentration of 1× TAE at a concentration of 2%, and purified using the GeneJET Gel Recovery Kit.
The off-the-shelf data (RawPE) obtained by IlluminaNovaSeq sequencing was spliced and QC to obtain Cleanags, and then chimeric filtering was performed to obtain valid data for subsequent analysis.
The experimental data were analyzed by SPSS21 statistical analysis program software, and Duncan's method was used for multiple comparison. The results were expressed as "mean ± standard error", and P<0.05 indicated significant differences.
«——【Results and Analysis·】——»
The effect of lactobacilli on the growth performance and feed efficiency of red crayfish can be seen from Table 1, and the survival rate of red crayfish is 83.33%~85.33%.
The final weight, weight gain rate and specific growth rate of crayfish in TB group and TC group were significantly higher than those in CA group (P<0.05), and the feed coefficient of crayfish in TC group was significantly lower than that in CA group (P<0.05).
Compared with the TC group, the growth performance of crayfish in the TD group was not further improved.
Based on the IlluminaNova sequencing system and data analysis, an average of 82,540 tags were measured per sample, and an average of 76,811 valid data were obtained after quality testing, with a total of more than 51,646 valid data and an effective quality control rate of more than 67.24%.
Species annotation is done on the database after sorting. A total of 1166 OTUs were annotated to the species level, and sample dilution curves were created based on the Chao1 index, as shown in Figure 1.
As can be seen from Figure 1, with the continuous increase of the number of sequencing, each sampling curve reached a plateau, indicating that the sequencing depth can basically cover all microbial species in all test samples.
The average coverage of the sequencing of each sample was above 0.996, which also indicated that the sequencing basically included all the microbial species in the test samples.
As can be seen from Figure 2, on the 15th day of the trial, the Shannon and Simpson indices of the TB group and the TC group were significantly higher than those of the CA group, that is, the uniformity of the number of intestinal microbiota and species diversity in the TB group and the TC group was significantly better than that of the CA group.
On the 45th day of the experiment, the Shannon and Simpson indices of each experimental group were higher than those of the CA group, that is, the biodiversity and evenness of the experimental group were significantly better than those of the CA group.
As can be seen from Figure 3, the main intestinal functional bacteria of red crayfish are composed of Actinomycetes, Bacteroidetes, Firmicutes and Proteobacteria.
变形菌门几乎在所有组中占据优势地位，其次是厚壁菌门。 由图4可知，红螯螯虾肠道优势菌科主要是红杆菌科（Rhodobacteraceae）和气单胞菌科（Aeromonadaceae）。
由图5可知，红螯螯虾肠道优势菌属为副球菌属（Paracoccus）和气单胞菌属（Aeromonas）。
At the phylum level, the relative abundance of Actinobacteriota in the TB and TC groups was significantly higher than that in the CA group, and the relative abundance of Actinobacteriota in the TD group was also increased.
At the family level, compared with the CA group, the relative abundance of Rhodobacteraceae in the experimental group increased, and the relative abundance of Aeromonadaceae decreased.
At the genus level, the relative abundance of Paracoccus in the experimental group was higher than that in the CA group, and the relative abundance of Aeromonas was decreased.
Compared with the CA group, the relative abundance of Aeromonas was increased in the TC group and decreased in the TB and TD groups.
At the genus level, the relative abundance of Paracoccus in the TB and TC groups decreased compared with the CA group, the relative abundance of Paracoccus in the TD group increased, the relative abundance of Aeromonas in the TB and TD groups decreased compared with the CA group, and the relative abundance of Aeromonas in the TC group increased.
At the phylum level, compared with the CA group, the relative abundance of Actinomycetes in the TB group and the TC group decreased, the relative abundance of Actinomycetes in the TD group increased, the relative abundance of Actinomycetes in the TC group and the TD group increased, and the relative abundance of Proteobacteria in the experimental group increased.
At the family level, the relative abundance of Rhodobacterium decreased in the TB group and increased in the TC and TD groups compared with the CA group.
Compared with the CA group, the relative abundance of Aeromonas in the experimental group was increased. At the genus level, the relative abundance of Paracoccus spp. was significantly reduced in the TB group compared to the CA group.
The relative abundance of Paracoccus in TC group and TD group increased, the relative abundance of Aeromonas in TB group increased, and the relative abundance of Aeromonas in TC group and TD group was significantly increased.
«——【Discussion·】——»
Effect of Lactobacillus on Growth Performance and Feed Efficiency of Red CrayfishProbiotics can improve the nutritional value of feed by changing the host or environmental microbial community.
Pathways such as enhancing the host's response to disease or improving the breeding environment have a beneficial effect on the host. Growth performance is one of the main indicators to evaluate the effect of probiotics.
Lactic acid bacteria can significantly improve the growth performance of tilapia, black sea bream, carp, salmon and largemouth bass, improve feed utilization, enhance antioxidant and immunity, and improve intestinal health.
In this experiment, the addition of 1.8×106 CFU/g Lactobacillus to the feed could significantly increase the final weight, weight gain rate and specific growth rate of red crayfish, and reduce the feed coefficient.
Studies have shown that the addition of 1.5×107 CFU/g Lactococcus lactis to feed significantly improved the growth performance and digestive enzyme activity of hepatopancreatic tissue.
The results showed that the addition of 0.5% Lactobacillus plantarum and 1.0×1010CFU/kg of different lactic acid bacteria to the feed could promote the growth of Penaeus vannamei.
The prebiotic effects of lactobacilli on growth can be explained as the production of hydrolytic enzymes (including palkinases and proteases) and vitamins (such as VB12), eliminating the effects of harmful substances in feed, stimulating the secretion of digestive enzymes, and maintaining intestinal flora, thereby improving nutrient absorption and promoting growth.
Effect of Lactobacillus on the intestinal microbial diversity of red crayfishThe intestinal microbiota has an important impact on the growth and development, digestion and immunity of animals, and is usually used as a comprehensive indicator of the health status of aquatic animals.
The composition of intestinal microorganisms in fish and shrimp is affected by various factors such as developmental stage, water environment, bait composition and feeding strategy, and the change of microbial structure will indirectly affect the health of fish and shrimp.
According to the experimental scientists, the addition of lactobacillus to the feed significantly changed the intestinal microbiota structure of red crayfish, and the diversity and abundance of intestinal flora of red crayfish increased when 1.8×105 CFU/g and 1.8×107 CFU/g were added.
Studies have also shown that the addition of 1.0×1010 CFU/kg of Lactobacillus acidophilus, Enterococcus faecalis and Bacillus coagulans in feed significantly improved the composition and abundance of intestinal microbial communities in Penaeus vannamei.
At the phylum level, Proteobacteria, Firmicutes and Bacteroidetes were the dominant intestinal flora of shrimp. At the class level, α-Proteobacteria, erysipelas and Flavobacterium were the dominant groups.
The intestinal colonization of Chinese mitten crabs in ponds was poor, and short-term application of probiotics could significantly change the β-diversity of microbial communities and promote the butyrate metabolism of intestinal flora.
Bacillus subtilis significantly increased the abundance and diversity of intestinal microbiota in bullfrogs.
The core bacteria in the intestinal flora of Penaeus vannamei include Bacteroides, Proteus, Firmicutes, and Actinobacterium, which is the main bacteria in the intestinal tract of shrimp.
Lactobacillus supplementation could significantly increase the relative abundance of dominant bacteria such as Actinomycetes, Bacteroidetes, Firmicutes and Proteobacteria in the digestive tract of red crayfish, among which Proteobacteria was the main intestinal flora.
Bacteroides, firmicutes, and actinomycetes play an important role in maintaining the physiology of host health by influencing metabolic activity, fermenting indigestible carbohydrates in the host to produce short-chain fatty acids.
Actinobacillus is a beneficial bacterium capable of producing phenol oxidase, which secretes antibacterial, antioxidants, and mucins to protect the host's normal intestinal function.
Lactobacillus plantarum was added to the feed to breed salmon, and the diversity analysis showed that at the phylum level, the intestinal flora was dominated by Proteobacteria, Firmicutes and Cyanobacteria.
Some studies have found that the addition of Lactococcus lactis to the feed can significantly increase the abundance of intestinal beneficial bacteria and reduce the abundance of potential pathogenic bacteria in red crayfish.
For example, some studies have found that the addition of microbial preparations can significantly improve the biodiversity and abundance of bacteria in the intestinal tract of crayfish, the dominant intestinal flora is Proteobacteria, the relative abundance of Firmicutes is increased from 6.10% to 14.21%, and the number of harmful bacteria Vibrio is reduced from 10.11% to 4.68%, and the intestinal flora tends to be rich and stable.
In addition, similar results have been obtained by adding Bacillus subtilis, 3% photosynthetic bacteria, 0.1% compound bacteria and lactococcus spp. to the feed.
The addition of lactobacilli to the feed can improve the health of red crayfish during the farming process, which is conducive to growth and development.
After the addition of lactobacilli to the diet, the abundance of Rhodobacterae belonging to the class α-Proteobacteria was greater in the colonizing microbiota of red crayfish, while the relative abundance of Aeromonas was reduced.
Rhodobacterium strains are able to produce polyhydroxybutyrate, which has the potential to promote the growth and development of fish and shrimp, and Rosebacterium marinium has been used as a beneficial bacterium in aquaculture.
Aeromonas is ubiquitous in the intestinal tract of aquatic organisms and is a normal component of digestive tract bacteria, which is a conditional pathogen that can cause host infection and in severe cases, host death.