NL2036281B1 - Application of 5-hmf in feed additives and feed for promoting growth and improving immunity of seriola lalandi - Google Patents
Application of 5-hmf in feed additives and feed for promoting growth and improving immunity of seriola lalandi Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/116—Heterocyclic compounds
- A23K20/121—Heterocyclic compounds containing oxygen or sulfur as hetero atom
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- Life Sciences & Earth Sciences (AREA)
- Polymers & Plastics (AREA)
- Chemical & Material Sciences (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Birds (AREA)
- Marine Sciences & Fisheries (AREA)
- Insects & Arthropods (AREA)
- Fodder In General (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to an application of 5—HMF in feed additives for promoting growth and improving immunity of Seriola lalandi, falling in the field of feed additives. The feed additive contains 5—HMF, short for 5—hydroxymethyl furfural. The present invention. further provides functional feed, for Seriola lalandi. The feed contains 5—HMF at a weight ratio of O.25—l%. According to the present invention, the addition of 5—HMF in the feed can improve the intestinal morphology and microbial community structure, increase the proportion of beneficial bacteria in the digestive tract, significantly promoting the feeding and growth of Seriola lalandi; and at the same time, the antioxidant capacity and immunity of the liver of Seriola lalandi can be improved.
Description
P1934 /NL
APPLICATION OF 5-HMF IN FEED ADDITIVES AND FEED FOR PROMOTING
GROWTH AND IMPROVING IMMUNITY OF SERIOLA LALANDI
The present invention relates to the field of feed additives, and specifically relates to an application of 5-HMF in feed addi- tives and feed for promoting growth and improving immunity of Se- riola lalandi.
Background technology
Seriola lalandi, belongs to Perciformes, Carangidae, and Se- riola, and is an economic warm fish distributed in pelagic ocean globally with long-distance migration characteristic. The demand for Seriola lalandi is mounting globally since it grows fast with large size, and tastes better with flourished nutrients, and therefore, the Seriola lalandi has been a fine species for the far-reaching development of marine aquaculture in China. In recent years, such factors as intensive aquaculture and water contamina- tion lead to frequent occurrence of aquatic diseases, resulting in a loss of fishery industry economically. Antibiotics are commonly used to prevent diseases in aquaculture, but it is prone to damage of aquatic product quality, contaminated environment and even threatening of human health due to drug residual and improper use.
Many researchers are devoted to finding substances capable of re- placing antibiotics, and the promotion of green additives has be- come an inevitable trend in the development of aquaculture. In ad- dition, since there is no specified compound feed for Seriola la- landi commercially, it is urgently to develop specified high- efficiency compound feed and green additives for the development of the aquaculture factory. 5-hydroxymethyl furfural (5-HMF) is a furfural compound with furan nucleus structure produced by dehydration of monosaccharide compounds such as glucose under conditions of high temperature or weak acid, which is one of the typical products of caramelization and Melad reaction, and it is widely existed in food, plants and traditional Chinese medicine. Over recent years, the biological functions about 5-HMF have been gradually explored, mainly includ- ing antioxidant activity, improvement of hemorheology, and influ- ence on glycyrrhetinic acid metabolism. With the wide application of modern science and technology in the field of traditional Chi- nese medicine research, 5-HMF has been deeply studied and under- stood.
The problem to be solved in the present invention is to pro- vide an application of 5-HMF in feed additives and feed for pro- moting growth and improving immunity of Seriola lalandi. The 5- hydroxymethyl furfural (5-HMF) can promote feeding, growth and im- mune function of Seriola lalandi.
The present invention is realized by the following technical solutions.
An application of 5-HMF in feed additives for promoting growth and improving immunity of Seriola lalandi, where the feed additive contains 5-HMF.
The present invention further provides functional feed for
Seriola lalandi, where the feed contains 5-HMF, and a mass of add- ed 5-HMF is 0.25%-1% by weight of the feed.
Further, a mass of added 5-HMF is 0.5% by weight of the feed.
Compared with the prior art, the present invention has the following advantageous effects.
According to the present invention, the addition of 5-HMF in the feed can improve the intestinal morphology and microbial com- munity structure, increase the proportion of beneficial bacteria in digestive tract, thereby significantly promoting the feeding and growth (including weight gain ratio, specific growth ratio and condition factor) of Seriola lalandi; and at the same time, the antioxidant capacity and immunity of the liver of Seriola lalandi can be improved.
FIG. 1 is effects of different concentrations of 5-HMF added in feed additives on the intestinal morphology of Seriola lalandi (by HE staining).
The present invention is further described by reference to the accompanying drawings and examples below.
Example 1 Effects of different concentrations of 5-HMF on proliferation of Caco-2 cells 1. Cell activity experiment design and method
To determine an appropriate concentration of feed additives, toxic effects of different concentrations of 5-HMF on cells were investigated firstly. In the experiment, different concentrations of 5-HMF (0 mMol, 1.6 mMol, 3.2 mMol, 6.3 mMol) were selected to intervene Caco-2 cells for 24 h, and a cell viability was detected by an MTT method. A to-be-tested mother solution was prepared by adding 0.01 mg of to-be-tested sample into 1 ml of DEME cell cul- ture medium, and an effective concentration of 5-HMF in the mother solution was 6.3 mmol/L. The preparation and component concentra- tion of to-be-tested solution in each experimental group are as follows:
Table 1 Preparation and component concentration of to-be- tested solution in different experimental groups
Concentration gradient Concentration annotation Preparation proportion
Group MO 0 mMol DEME
Group M1 1.6 mMol 1 ml 3.2 mMol + 1 ml DMEM
Group M2 3.2 mMol 1 ml mother solution + 1 ml
DMEM
Group M3 6.3 mMol Mother solution
Note: the concentrations in groups Ml, M2, and M3 in the ex- periment correspond to the concentrations in groups M1, M2, and M3 set in the additive feeding experiment. 2. Experimental cell culture and passage
Caco-2 cells (human colorectal adenocarcinoma cells) were cultured in a T.; cell flask containing 5 mL of complete DMEM medi- um (containing 10% fetal bovine serum and 1% antibodies to peni- cillin and streptomycin) in a culture thermostat incubator at 37°C with 5% of CO:, and the medium was changed every 2 days.
Upon the cells grew to about 930% of the T:s cell flask, the passage was performed. Specifically, the original medium was sucked out of the flask, the cells were rinsed gently with 2 mL of sterile PBS (1x), and treated with pancreatin (containing 0.25%
EDTA) for 3 min at 37°C after the PBS being sucked out; 2 mL of complete o-MEM medium was added to neutralize and terminate the reaction after the above treatment; centrifugation being performed at 1000 r/min for 5 min before supernatant was discarded; after that, the cells were resuspended with 1 mL of complete «-MEM medi- um and gently blown well; and then 0.5 mL was taken and added to a new Tz; cell culture flask containing 4.5 mL of complete a-MEM me- dium for further culture in an incubator. 3. Effect of 5-HMF on viability of Caco-2 cells detected by
MTT assay
Caco-2 cells within 5-20 generations were selected and inocu- lated into a 96-well plate at an inoculum size of 1x10" per well for 24 h. After aspirating the original medium from the wells, 100 uL of medium containing different concentrations of to-be-tested substance (5-HMF) was added (to-be-tested substance was dissolved in a medium without fetal bovine serum) at concentrations of 0 ng/mL, 25 pg/mL, 50 pg/mL, 100 pg/mL, 200 pg/mL, and 400 pg/mL.
After incubation being performed in the incubator for 20 h, 10 pL of 5 mg/mL MTT was added to each well, and incubation was per- formed at 37°C for 4 h. After incubation, the medium in the wells was sucked out, 150 pL of LDMSO was added, and an absorbance was measured at 490 nm after shaking the plate for 15 min. The cell proliferation activity is calculated according to the following formula: cell proliferation activity = (sample A - blank A) / (control
A - blank A) x 100%.
In the formula, blank A represents an absorbance at a sample concentration of 0 pg/mL.
Serum for culture medium, etc. are obtained from Biologi- calIndustries (IsraelBeitHaemekCo., Ltd). MTT was from Sigma. Ca- co-2 cells were from the Institute of Biochemistry and Cell Biolo- gy, Chinese Academy of Sciences (Shanghai, China). 4. Results
As shown in Table 2, 5-HMF administration groups show signif- icant differences compared with the control group (P < 0.05), but the cell proliferation rate in group M3 added with the highest concentration is still greater than 80%, indicating that there is no obvious toxic effect on the cells, and the concentration is suitable to be used in the feed additive.
Table 2 Effects of different concentrations of 5-HMF on pro- liferation of Caco-2 cells
Experimental Group MO Group M1 Group M2 Group M3
Group
Cellproliferation 140.08" 0.90+0.05° 0.89+0.07° 0.85+0.08" rate % 5 Note: different lowercase letters of superscript indicate significant difference between different experimental groups (P < 0.05).
Note: the concentrations of groups Ml, M2, and M3 in the ex- periment correspond to the concentrations in groups M1, M2, and M3 set in the additive feeding experiment.
Example 2
The experiment was completed in a factory breeding workshop of Dalian Fugu Food Co., Ltd. from September to October 2021, and the test period was 42 d. The Seriola lalandi used in the experi- ment was one year old with uniform size and healthy body cultured in the company's offshore net box base, with an average body length of (17.35x0.51) cm and an average body mass of (82.331t2.75) dg.
Factory workshop breeding was carried out in a 3 m’ circular glass tank, and the bait fed was compound pellet feed (Hayashikane
Sangyo Co., Ltd, Japan). The temporary breeding is carried out for 7 d before the start of the experiment, during which, basal feed, i.e., the compound pellet feed (Hayashikane Sangyo Co., Ltd, Ja- pan), was fed to twice a day, at 2% of body weight at 8:00 and 16:00, respectively, and the experimental Seriola lalandi was fasted for 24 h prior to the start of the experiment. The experi- mental Seriola lalandi was randomly divided into 4 groups (group
MO, group Ml, group M2, group M3) with 3 replicates of 30 fish each. The feed for experimental groups was proposed to be added with different concentrations of 5-HMF, which was evenly covered on the surface of the feed by spraying, and low-temperature drying was carried out to attach the 5-HMF to the basal feed. Groups Ml,
M2, and M3 were fed with the experimental feed with the addition of 5-HMF at a mass ratio of 0.25%, 0.5%, and 1% to the basal feed, respectively. The feeding amount was 2-3% of body weight, and the breeding was conducted in running water with a daily water ex- change rate of 200-300%. The water environment conditions during the whole breeding process were that: water temperature was 13- 24°C, salinity was 31-32, and dissolved oxygen was > 7 mg/L.
At the end of the experiment, 6 Seriola lalandi were sampled from each parallel of the control and experimental groups, total- ing 18 Seriola lalandi were sampled from each experimental group.
After Seriola lalandi being anesthetized with MS-222, the body surface thereof was wiped with 75% alcohol cotton, and the fish body mass, body length, etc., were measured and recorded. Blood samples were collected from the caudal vein using a 2 ml syringe, and the blood was to be static at 4°C. The blood was centrifuged at 4000 r/min for 10 min, and the supernatant was taken and stored at -80°C for the determination of serum biochemical indexes. After dissection, liver and digestive tract (stomach, pyloric caecum, intestine) samples were taken, and the residual contents in the digestive tract were discharged, rinsed with pre-cooled sterilized saline and stored separately in liquid nitrogen. 1. Growth performance measurement
The growth parameters are calculated according to the follow- ing formulas:
Weight gain ratio (WGR, 3) = [(M — Wy) / MW] =x 100;
Specific growth ratio (SGR, %/d) = [(1lnk, - ln) / t] x 100; and
Condition factor (CF, g/cm’) = W. / L°.
In the above formulas: W, and W, represent average fish body mass (wet weight, g) at the beginning and end of the experiment, respectively; t represents the number of experiment days (d); and
L represents the body length (cm).
Effects of different concentrations of 5-HMF added in feed additives on growth performance of juvenile Seriola lalandi are shown in Table 3. As can be seen from Table 3, after 42 d of feed- ing, the weight gain ratio and specific growth ratio in groups M2 and M3 are significantly higher than those in group MO (P < 0.05), and the condition factor in group M2 is significantly higher than that in group MO (P < 0.05).
Table 3 Effects of different concentrations of 5-HMF added in feed additives on growth performance of juvenile Seriola lalandi
Growth index Group MO Group M1 Group M2 Group M3
End body mass/g 140.41+5.17° 142.48+6.04° 149.339.29" 150.02+6.7°
Weight grain ratio/% 70.54+6.27° 73.0617.34° 81.37+11.28° 82.2148.14°
Specific growth ratio (%/d} 1.27+0.09° 1.31+0.1° 1.41+0.15° 1.43+0.11°
Condition factor {g/cm’) ~~ 1.6840.17° 1.75+0.14% 1.80.11" 1.75+0.09%
Note: different lowercase letters of superscript indicate significant difference between different experimental groups (P < 0.05). 2. Determination of antioxidant capacity of liver
The antioxidant enzyme test kit used in the experiment was purchased from Nanjing Jiancheng Bioengineering Institute. The ac- tivities of glutathione peroxidase (GSH-PX), superoxide dismutase (SOD) and catalase (CAT) of liver were measured according to the specification of the kit, and the concentration of malondialdehyde (MAD) was measured.
Effects of different concentrations of 5-HMF added in feed additives on antioxidant capacity of liver of juvenile Seriola la- landi are shown in Table 4. The feed additive added with 5-HMF can particularly improve the antioxidant capacity of liver of juvenile
Seriola lalandi. Compared with the control group, GSH in experi- mental groups is significantly increased (P < 0.05), the level of
MAD in group MO is significantly higher than that in groups M2 and
M3 (P < 0.05), and SOD and CAT activities in groups M2 and M3 are significantly higher than those in group MO (P < 0.05).
Table 4 Effects of different concentrations of 5-HMF on anti- oxidant capacity of liver of juvenile Seriola lalandi
Project Group MO Group M1 Group M2 Group M3
Glutathione peroxidase (U/mL) ~~ 146.77+3.2° 175.5245.87° 227.46%1.79° 217.6142.69°
Superoxide dismutase (U/mL) 62.44+1.98° 64.53+2.06° 81.39+1.28" 81.67+2.69"
Catalase (U/mL) 3.5710.36° 4,010.36 6.55+0.35° 6.04+0.46°
Malondialdehyde (nmol/mL) 11.08+0.14° 10.5+0.25° 9+0.25° 8.83+0.58"
Note: different lowercase letters of superscript indicate significant difference between different experimental groups (P <
0.05). 3. Serological index determination
Activities of immunoglobulin M (IgM), lysozyme (LZM), alka- line phosphatase (AKP), glutamic-pyruvic transaminase (GPT), glu- tamic oxalacetic transaminase (GOT) and acid phosphatase (ACP) were detected using kits produced by Nanjing Jiancheng Bioengi- neering Institute, and the test method was performed in accordance with the instructions of the kits.
Effects of different concentrations of 5-HMF added in feed additives on serum immune index of juvenile Seriola lalandi are shown in Table 5. Compared with group MO, feeding 5-HMF with dif- ferent concentrations improves the immune ability of experimental
Seriola lalandi in different degrees. The results of immune index- es show that IgM and AKP are significantly higher in juvenile Se- riola lalandi in groups M2 and M3 than those in group MO (P < 0.05); the activities of LZM and ACP are significantly higher in group M2 than those in group MO (2 < 0.05); the GPT is signifi- cantly lower in group MO than that in the experimental group (P < 0.05); and the activities of GOT in groups Ml and M3 are signifi- cantly higher than those in group MO (P < 0.05).
Table 5 Effects of different concentrations of 5-HMF on serum immune index of juvenile Seriola lalandi
Project Group MO Group M1 Group M2 Group M3
Lysozyme (ug/ml) 29.17+0.76° 28.83+0.76° 31.83#1.53° 25.67+0.58°
Immunoglobulins (ug/ml) 974.76+31.24° 957.77+18.74° 1400.55+20.25° 1248.0157.58"
Alkaline phosphatase 1.53+0.33° 1.76+0.16° 2.440.18° 2.69+0.05° {king unit/100mi}
Glutamic-pyruvic trans- 9.43+1.18° 28.5540.76° 20.10+1.21° 24.10+1.02° aminase (U/L)
Glutamic oxalacetic 28.7013.29° 116.32425.64° 48.06+13.80° 117.77+2.51" transaminase (U/L)
Alkaline phosphatase 6.68£0.45° 7.69£0.27" 7.9810.41° 5.01+0.93° {king unit/100 mi}
Note: different lowercase letters of superscript indicate significant difference between different experimental groups (P < 0.05). 4. Intestinal histomorphological analysis
Intestinal samples fixed with Bouin solution were taken and subjected to 75%, 80%, 95%, and 100% gradient alcohol for dehydra- tion, xylene transparency, conventional paraffin embedding and sectioning (5 um thickness), hematoxylin-eosin (HE) staining fol- lowed by neutral gum sealing, and were placed under an OLYMPUS mi- croscope for observation, and the samples were recorded by taking photos. The thickness of intestinal muscular layer, height of vil- lus and the number of goblet cells were measured using Image J software.
Effects of different concentrations of 5-HMF added in feed additives on intestinal morphology index of juvenile Seriola la- landi are shown in Table 6, and results of HE staining are shown in FIG. 1. The thickness of muscular layer and the height of vil- lus in group M2 are significantly higher than those in group MO (P < 0.05); and there is no significant difference in the number of goblet cells between 5-HMF group and control group.
Table 6 Effects of different concentrations of 5-HMF added in feed additives on intestinal morphology of juvenile Seriola Jalan- di “Project GroupMO GroupMI GroupM2 GroupM3 © “Thicknessof muscular layer 174.766+5.641) 142,723+22.983° 208,074+13.8° 184.342+18.375" (um)
Height of villus ab . b 5 (um 831.494465.556" 783.558174.479° 921.252+94.104 833.655469.468" 5
Goblet cell 25.6213.03 24.7443.66 28.13+3.26 27.57+2.42 {number}
Note: different lowercase letters of superscript indicate significant difference between different experimental groups (P < 0.05). 5. Extraction and high-throughput sequencing for total DNA of microoganism
The genomic DNA of the collected samples was extracted from the samples using a DNA extraction kit (MagPure Soil DNA KF Kit), after which the concentration of DNA was detected and separated using Nano Drop 2000 and agarose gel electrophoresis. Genomic DNA was used as a template for PCR using the specific primer Tks Gflex
DNA Polymerase (Takara) with barcode, and primers 343F (5'-
TACGGRAGGCAGCAG-3') and 798R (5't-AGGGGTATCTAATCCT-3') were used to amplify the 16S V3-V4 region. After being qualified by detection of agarose gel electrophoresis, the amplified sequences were passed to Qingdao Ouyi Biotechnology Co. Ltd. for high-throughput sequencing through the Illumina MiSeq PE300 platform.
The diversity index of the microorganisms in the digestive tract of juvenile Seriola lalandi spiked with different concentra- tions of 5-HMF was analyzed, and the results are shown in Table 7.
The Coverage index indicates the sequencing depth of the samples and the coverage of the samples, and the Coverage index is above 0.99 in all 12 treatment groups, indicating a low probability of not being detected in the samples. Shannon index and Simpson index reflect species diversity of the community. In the case of same species richness, the greater the evenness of the species in the community, the greater the diversity of the community is consid- ered to be. The shannon index in the pyloric caecum of group M2 is significantly higher than that in other groups (2 < 0.05), while the simpson index is significantly lower than that in other groups (P < 0.05). Chao index reflects the species richness of the commu- nity in the sample, which simply refers to the number of species in the community without considering the abundance of each species in the community. The chaol index of the intestines in group M2 is significantly higher than that in the other groups (P < 0.05).
Table 7 Effects of different concentrations of 5-HMF on alpha diversity index of the digestive tract of juvenile Seriola lalandi
Group goads_coverage shannon simpson chaol
MOS 0.99310° 7.17910.106° 0.983+0.001° 1154.257132.398%
MOP 0.99410° 7.36910.239° 0.984+0.002% 1134.587+29.569°
MOG 0.99440 7.19010.014° 0.983+0.002° 1028.265+32.789°
M1S 0.99410° 7.092+0.010° 0.981+0.001° 1055.4988177.997°
MIP 0.994+0° 7.129+0.098° 0.981+0.001° 1012.155+29.525°
M1G 0.994+0.001° 7.176+0.118° 0.982+0.002° 1122.356+85.304"
M25 0.994+0% 7.159+0.098" 0.982+0.002° 1082.302+69.585°
M2P 0.9940" 7.126+0.153° 0.981+0.003° 1034.250+58.744°
M2G 0.993+0.001%° 7.660+0.386° 0.987+0.004° 1277.3624215.104
M3S 0.994+0°% 7.23810.035° 0.983+0.001° 1089.228+107.982"
M3P 0.994+0.001% 7.230£0.111° 0.983+0.001° 1104.301+71.102%
M3G 0.993+0.002°% 7.23810.197° 0.983+0.002° 1145.733+194.459%
Note: different lowercase letters of superscript indicate significant difference between different experimental groups (P < 0.05). eee enne] N
ADS AOP ADG AIS AIP AIG AS AP A2G A35 ABP AG
Groups (%) (%) (%} {%) (9%) (%) (%) {%) (%) {%) (%} (%) > ote
Bacteroidetes 47.67 45.22 45.63 4742 4921 49.03 47.28 46.93 4239 44.87 47.04 46.76 :
Firmicutes 32,09 3227 3424 316 3215 3234 3328 33.06 3372 3186 323 33.39 gro
Proteobacteria 10.7 1178 952 1033 0.48 316 875 92 1137 10.33 1051 9.41
Actinchacteric 5,06 539 572 5853 4,99 529 56 535 625 654 53 4.56 ups ee MOS ,
MOP
’ and MOG are stomach, pyloric caecum, and intestinal samples of the control group; M1S, M1P, and M1G are stomach, pyloric caecum, and intestinal samples added with 0.25% of 5-HMF; M2S, M2P, and M2G are stomach, pyloric caecum, and intestinal samples added with 0.5% of 5-HMF; and M335, M3P, and M3G are stomach, pyloric caecum, and intestinal samples added with 1% of 5-HMF.
Statistics on microbial phyla and relative abundance at the phylum level shows that there is no significant difference in the number of bacterial groups among the digestive tract samples of
Juvenile Seriola lalandi by adding different concentrations of 5-
HMF to the feed. The top five bacterial abundances in the diges- tive tract samples are Bacteroidetes, Firmicutes, Proteobacteria,
Actinobacteria and Desulfobacterota. The relative abundance of
Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria reaches more than 90% in each group, but the proportion varied among the groups.
or L68 184 187 221 155 175 205 225 207 293 2.34 194
Compylobocter 1.37 126 131 128 121 © 123 143 164 174 113 144
Deferribacteres 0 0 0 0 0 0 0 0 0 0 0 117
Other 145 224 165 162 141 242 181 179 255 173 138 1.35
Statistics on microbial phyla and relative abundance at the genus level shows that the addition of different concentrations of 5-HMF to the feed has a similar composition of the bacterial flora in the digestive tract of juvenile Seriola lalandi, mainly com- posed of Bacteroides, Muribaculaceae, Lachnoclostridium, Lactoba- cillus, Parabacteroides, Ileibacterium, Lachnospirace- ae NK4A136 group, Bifidobacterium, and Faecalibaculum and other components. Bacteroides, Muribaculaceae, Lachnoclostridium and
Lactobacillus are the common bacterial genus with high relative abundance in each group. The percentage of each bacterial genus is shown in Table 8.
Table 8 Composition of microflora in digestive tract of juve- nile Seriola lalandi
Note: groups MOS, MOP, and MOG are stomach, pyloric caecum, and intestinal samples of the control group; M15S, M1P, and M1G are stomach, pyloric caecum, and intestinal samples added with 0.25% of 5-HMF; M2S, M2P, and M2G are stomach, pyloric caecum, and in- testinal samples with 0.5% of 5-HMF; and M33, M3P, and M3G are stomach and pyloric caecum, and intestinal samples with 1% of 5-
HME. 6. Data statistics and analysis
The raw image data files obtained from high-throughput se- quencing were transformed into raw sequencing sequences in FASTQ format by base calling analysis, and valid sequences were obtained after a series of cutting, de-hybridization, splicing, quality control and chimera removal. Sequences were categorized into mul- tiple classifiable operational units (OTUs) based on sequence sim- ilarity, and sequences with 2 97% sequence similarity were catego- rized as one OTU unit. The sequence with the greatest abundance in each OTU was selected as representative sequences of the OTU using the QIIME software package, and all representative sequences were annotated against the Silva (versionl23) database. The species comparison annotation was performed using the RDP classifier soft-
ware, which retained the annotation results with confidence inter- vals greater than 0.7. Comparative analysis of KEGG pathways in- volved in microbiota genes was performed using tax4fun (0.3.1).
Data were processed using Excel 2016 and statistically ana- lyzed with SPSS26.0 software. The indicators of similar samples at different concentrations were analyzed for differences using paired t-tests. Different types of samples at the same concentra- tion were analyzed using One-way ANOVA. Duncan multiple compari- sons were made for differences between groups, and differences were considered to be significant at the significant difference level of P < 0.05. Values were expressed as "MeantStandard Devia- tion" (MeantS.D.}).
Functional prediction analysis of OTUs in the digestive tract flora of juvenile Seriola lalandi with different concentrations of 5-HMF was performed in the KEGG database, and functional pathways with significant intergroup differences were screened at the Level 3 level according to the Kruskal-Wallis algorithm (P < 0.05). The main signaling pathways involved in the genes of the flora in each tissue of the digestive tract were the same, but there were some differences in gene abundance. Based on the number of OTUs anno- tated in the digestive tract flora, it was found that the differ- ential pathways were mainly concentrated in carbohydrate metabo- lism, amino acid metabolism and energy metabolism, etc.
In conclusion, the addition of 0.5% 5-HMF experimental feed can improve the intestinal morphology and growth performance of
Seriola lalandi, and promote serum immunity indexes and liver an- tioxidant capacity. In terms of intestinal microbial changes in juvenile Seriola lalandi, the Firmicutes and Bacteroidetes were absolutely predominant in the intestinal flora of juvenile Seriola lalandi in each group and the intestinal flora of juvenile Seriola lalandi in each group had the same predominant bacterial genus.
Moreover, the major functions of genes involved in the digestive tract flora of juvenile Seriola lalandi were similar in all groups.
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