The anelastic deformation, resulting from partial reversal of {10 The hexagonal closed-packed (HCP) crystal structure of magnesium (Mg) provides only two independent basal slip systems , while t strains ,8. Theseondition , and theg alloys ,12,13, mg alloys , Mg–Zn ag alloys ,15, Mg–Gg alloys , Mg–Al ag alloys , AZ31  at RT of about 22 °C (295 K) and at elevated temperatures of 50 °C (323 K), 100 °C (373 K), and 150 °C (423 K). Temperature was allowed to equilibrate for 5–10 min before testing. To accurately measure the temperature, a K-type thermocouple was bent and inserted between the grips of the machine, so that it was contacting the specimen. Temperature variations of 1–2 °C were observed during testing.All specimens used in this study are dog-bone shaped with a cylindrical cross-section diameter of 5.6 mm and a 36 mm parallel section in the gauge length, as shown in The cyclic tension loading–unloading tests were strain-controlled, loaded to a predetermined strain, unloaded to zero stress, and then reloaded again following the International Standards Organization (ISO) standard . All allMicrostructures were characterised by a scanning electron microscopy (SEM) equipped with an energy dispersive X-ray (EDX) spectroscopy and an electron backscatter diffraction (EBSD). EDX line scan was conducted to measure the Al solute concentration level in the α-Mg matrix. EBSD data were collected at 20 kV with a 0.5 μm step size to reveal the deformation twins. For each sample, three locations were analysed. Samples for microstructural analysis were polished with 600 and 2400 SiC papers followed by 3 and 1 μm alcohol-based diamond suspensions and were finished by a 0.06 μm colloidal silica suspension.A typical cyclic loading–unloading tensile curve is shown in Similar cycles for as-cast AE44 and AZ91 at RT and 150 °C are shown in ae, defined in sec, in The anelastic strain ; higher solute level was not investigated in . HoweverIn Mg–Al–RE alloy AE44, T5 ageing may also influence the anelastic deformation, as T5-aged AE44 consistently shows smaller maximum anelasticity, regardless of the temperatures. Although AE44 and T5-aged AE44 have similar intermetallic phases, there was a formation of Al–Mn nanoscale precipitates after T5 ageing, as reported recently . These p17Al12 has a measurable effect on the work hardening [Note that it is more complex to compare between Mg–Al–RE and Mg–Al alloys due to microstructural difference, as shown in ardening ,68 and tardening . It is, 0.2, leaves only a very small fraction of permanent plastic strain (<0.1%) due to the reversible anelastic component of Mg and Mg alloys, as illustrated in The yield strength of materials is generally measured by offsetting the linear elastic modulus, E to some amount of permanent plastic strain. The amount of permanent plastic strain can range from 0.1% for ferrous to 0.5% for non-ferrous materials , and 0.2sec  activity.3. In Mg–Al–RE alloy, T5-aged AE44 consistently exhibits smaller maximum anelasticity compared to as-cast AE44. T5 ageing forms Al–Mn nanoscale precipitates, and these precipitates may decrease twin-boundary mobility, making partial reversal of twinning more difficult.sec methods, which account for the anelastic effect, are proposed to improve the accuracy of the yield strength measurement for die-cast magnesium alloys.4. Anelasticity observed in Mg alloys leads to inaccuracy in yield strength measurement using the conventional 0.2% offset method. Higher offset strain or lower E


The cambuci is a native Brazilian fruit from the Atlantic Forest biome. A soft and astringent pulp, a green color, and a sweet aroma are its main characteristics. Classical food quality attributes  and the metabolic profile from ten accessions from three different locations were analyzed herein by analytical methods  and nuclear magnetic resonance spectroscopy. Concerning sugar content, sucrose was the predominant compound, with glucose and fructose alternating in second, depending on the accession. Citric acid was the most relevant acid, followed by shikimic and quinic acids in quite variable amounts. These three main acids vary in amounts for each accession. Ascorbic acid content emerges as an important quality attribute and makes this fruit nutritionally attractive, due to values comparable to those contained in citric fruits. The main amino acids identified in cambuci were glutamic acid individually or in comprising the tripeptide glutathione . The quality diversity of the evaluated accessions suggests the potentiality of cambuci use in future breeding programs.  Campomanesia phaea) is a native Brazilian Atlantic Rainforest fruit. It is an endangered species that naturally occurs in a mountainous relief area, named Serra do Mar, mainly in the state of São Paulo and in smaller numbers in the states of Rio de Janeiro and Minas Gerais. Belonging to the Myrtaceae botanical family, cambuci contain a fleshy and soft pulp when ripe, a thin skin and persistent green coloration. All cambuci parts  are edible, resulting in high yields and the total use of this fruit. It also presents a sweet and attractive aroma with a peculiar astringent taste. At room temperature, cambuci postharvest longevity extends only for up to 3 to 4 days, which may limit its in natura use. In this regard, the fruits are often frozen, which allows them to be used in preparations for an extended period of time. Despite the viability of frozen cambuci, its current use is restricted to the local population, who consumes it prepared as juices, ice creams, and jellies. Nowadays, cambuci fruit does not have an international demand; however, its commercial products should be stimulated.Cambuci  (23° 23’ 09” S 45° 39’ 43” O), Mogi das Cruzes  (23° 31’ 22” S 46° 11’ 16” O), and Salesópolis  (23° 31’ 55” S 45° 50’ 45” O), located in a region known as Serra do Mar. Paraibuna and Salesópolis are neighboring municipalities, but climatically different according to the Köppen-Geiger classification. Paraibuna is located within the metropolitan region of São Paulo and is classified as a temperate oceanic climate (Cfb), while Salesópolis is located in Paraíba valley and the northern coast of the São Paulo region, presenting a humid subtropical climate (Cfa). Mogi das Cruzes is part of the metropolitan region of São Paulo and Alto Tietê and is inserted in a humid climate territory with monsoon influence (Cwa). The Cfa and Cfb climates are humid, while the Cwa presents a dry season during the winter. In the summer, Cfa and Cwa reaches temperatures of over 22 °C, in contrast with CFb, where this temperature is not reached. Therefore, despite the proximity of the fruit sampling areas, temperature may influence fruit quality. Typically, the cambuci harvest is carried out in the summer–autumn seasons in the southern hemisphere, from February to June.Forty-four fruits collected from each accession were used in the present study, totaling four hundred and forty fruits. The samples were then transported to the Laboratory of Postharvest of the Horticultural Products, at the Luiz de Queiroz Superior School of Agriculture (ESALQ-USP) and to Embrapa Instrumentation, where they were frozen at −20 °C.Fresh fruit masses (g), were determined using an analytical balance. Fruit heights and diameters were measured using a digital caliper and expressed as millimeters (mm). Total soluble solid contents (SSCs) were determined by using method 932.12 [Eppendorf MiniSpin 11 mm diameter) at 14,000 rpm for 30 min. The supernatant juices were then separated from the fibers and kept at 4 °C (~25 mL). The 50 juice samples (quintuplicates of the ten accessions) were divided in two analytical replicates, yielding 100 NMR samples. The maximum quantification errors between the two NMR analyses were lower than 15%. A total of 450 µL of the supernatants were solubilized in 450 µL of deuterium oxide phosphate buffer  containing 0.050% w/w of sodium 3-trimethylsilyl-2,2,3,3-d4-propionate  and 0.02% m/v of sodium azide. The pH of each sample was adjusted to 3.00 ± 0.02 using microliter amounts of 1 M NaOH or 1 M HCl solutions. Subsequently, 550 µL were transferred to a 5 mm NMR tube. Pulps of each fruit (~50 g) was individually crushed using a mixer and centrifuged . The TMSP-d4 signal was calibrated at δ 0.00. The 1D spectra were assigned using the literature and databases values, in which overlap signals of each compounds were deconvoluted using the software Chenomix NMR Suite 8.4 , then the identity of compound was confirmed using the 2D NMR experiments 1H JRES, 1H-13C HSQC, and 1H-1H COSY were performed on selected samples, by the correlated signals between vicinal hydrogens (COSY), the correlated signals between carbon and correspondent hydrogen (HSQC), and J-coupling constant between protons of a specific signal (JRES). Metabolite peaks were integrated and quantified relative to the “Electronic to Access In Vivo Concentration 2” (ERETIC2) signal, referenced using a 2 mM sucrose standard and the fixed RG used for the samples. It was used the absolute area of integrals, molar mass, and the number of hydrogens of the respective NMR signal of each compound in the identified peaks to calculate concentration in mM, and then their respective mg 100 g−1. NMR spectra were obtained at 298 K using a 14 T Bruker Avance III spectrometer , 5 mm PABBO probe head with gradients, automated tuning and matching accessory (ATMATM), BCU-I for temperature regulation and a Sample-Xpress sample changer. A protocol  was first established for a QC sample . Then, this protocol was run in fully automatic mode using Bruker routines  for each sample. Spectra were acquired for proton NMR using the NOESY-presaturation pulse sequence (Bruker 1D noesygppr1d pulse sequence), 64 k data points, spectral width of 20.0276 ppm, acquisition time of 2.726 s, recycle delay 4s), dummy scans of 4, accumulation of 256 transients, mixing time of 0.005 s, and fixed receiver gain (45.2). FIDs were multiplied by a 0.3 Hz exponential multiplication function prior to the Fourier transformation. Phase and baseline corrections were carried out using the TopSpin1H-NMR spectra data, from 0 to 8.5 ppm, were aligned using the Icoshift algorithm [Arabidopsis thaliana  (KEGG), PubChem Compound, Chemical Entities of Biological Interest (ChEBI), Japan Chemical Substance Dictionary Web (NIKKAJI), and Chemical Abstracts Service (CAS). The top altered pathways were identified and built according to the potential functional analysis, using the MetaboAnalyst 4.0 platform [The lgorithm , followelgorithm  (sample lgorithm  and R solgorithm . Metabolplatform .p < 0.05). The coefficient of variation (CV) is the ratio of the standard deviation to the means.Data were subjected to an analysis of variance and mean comparisons were performed using the Tukey test  and Statistica v.7 software .The physicochemical parameters of the investigated cambucis reflected accession diversity, as expected, since cambuci have not yet been selected in genetic breeding programs . The aveThe average SSC of cambuci was 10.8 Brix, ranging from 7.8 to 14.0 Brix. These values are similar to the SSC observed in other chemical characterization studies in cambuci, which reported values between 7.3 to 13.3 Brix and 5.1 to 11.0 Brix . TA valuFor comparison, in a study evaluating the quality of passion fruits, 9.1%, 11.6% and 12.3% of soluble solids were reported for yellow, purple and orange fruits, respectively . SimilarWhen the SSC/TA ratio is considered as a quality attribute, each fruit will present its own scale, based on sugar and acid contents, as well as other minority components. The SSC/TA ratio can be used as a discriminating quality criterion in fruit, as described previously for peaches and nectarines . When 39−1 DW [Usually, cambuci fruits present the following values of other physical and chemical properties: moisture content 86–90%, pH 2–3, fibers 0.1–0.2%, total lipids 0.02–0.05%, proteins 0.02–0.03%, carbohydrate 0.9 g Kg-1, ash content 0.02%, and mineral profile comprising nitrogen 0.01–0.03, phosphorus 0.01, potassium 0.1, calcium 0.001–0.005, magnesium 0.01, sulfur 0.01, boron 0.1, zinc 0.1, iron 0.2–0.3, manganese 0.02–0.1, copper 0.02–0.04, sodium 0.3–0.6 g kg−1 DW . These p−1 DW . HoweverA typical cambuci NMR spectrum of 0.9–8.5 ppm is presented in 1H NMR spectrum of a sample with database, and confirmation with the interpretation of 1H-1H COSY NMR, 1H-13C HSQC NMR spectrum, and 1H JRES NMR spectrum as showed in The characteristic polyphenol region containing numerous signals is also exhibited, which may be associated to the presence of proanthocyanidins, such as ellagitannins. These characteristic polyphenol signals have also been reported for plums and peaches . The comThe NMR analysis in the present study was possible once careful sample preparation with pH control of the extracts and mathematical alignment of peaks was employed to prevent some compounds which are susceptible to interactions from affecting their chemical shifts and to keep the reproducibility. Additionally, the resolution was guaranteed by the carefully adjusted parameters such as calibrated pulse at 90° and the respective power to suppress water residual, correspond receiver gain (intensity), and the number of points.Eretic2 method with metabolites and sample information  conditions, NMR spectra can provide direct quantitative information since the signal intensity of each resonance in the NMR spectrum is directly proportional to the number of equivalent nuclei responsible for that signal, or in other words, is directly proportional to the molar amount of the detected isotope ,25. Thusanalysis , we usedanalysis ,27. CareSucrose, citric acid, and glutathione represented the main cambuci pulp constituents, comprising 61% of the total amount of the polar metabolites in this fruit .−1 of sucrose, 1.7–2.5 g 100 g−1 of glucose and 1.6–2.9 g 100 g−1 of fructose [Total sugar content  represented 40% of all pulp metabolites. A balance between sugars is characteristic of each fruit species and decisively affects the quality perception experienced by consumers. Sucrose was the predominant sugar, although accession comparisons indicate a sucrose content variation of up to 3.9-fold . Sucrosefructose  indicate−1 to 51.2 mg 100 g−1  and grapefruit (1190–2100 mg 100 g−1), and is significantly higher than in apples (30–50 mg 100 g−1) [−1 in apples and 84.4 mg 100 g−1 in jostaberries [−1. The richest quinic acid cambuci accession contained 466.9 mg 100 g−1 (accession 4)  . Typicalaberries . Neverthaberries . Accessission 4) . Quinic,ectively . Quinic Accession 6 and 8 contained the highest amounts of polyphenols, while accession 3 contained the lowest . A paper−1, ranging from 26 mg 100 g−1 to 103 mg 100 g−1 (1 were described [−1), mandarins (16.2–31.6 mg 100 g−1) and lemons (39.6–42.3 mg 100 g−1) [−1, sufficient to meet cellular needs and reduce human health risks, such as cardiovascular and neurodegenerative diseases, cancer and stroke [Ascorbic acid (vitamin C), a natural antioxidant that prevents the action of free radicals, is an important organic acid in cambuci, averaging 47 mg 100 g 100 g−1 . These v100 g−1) . The recd stroke . This fiDistinguishing sugar, organic acid and phenolic compound levels can contribute to the quality differentiation of fruits produced from different plants and aid in future efforts to breed cambucis. This type of differentiation has been previously reported for other cultivars, such as apples . Amino acids’ relevance in plants arises from their role as central regulators of plant growth and responses to environmental signals, along with being agents that influence human food nutritional quality . Eleven Other amino acids and related compounds exhibiting a high positive correlation (>0.9) comprised gamma-aminobutyric acid (GABA) vs. isoleucine, GABA vs. valine, GABA vs. leucine, GABA vs. asparagine, isoleucine vs. valine, isoleucine versus leucine, isoleucine vs. asparagine, valine vs. leucine, and valine vs. asparagine. Amino acids in fruits are not only linked to protein constitution, but also contribute to organoleptic fruit qualities. For example, in tomatoes, the taste described as “umami” is directly associated to glutamic acid levels . Amino aThe sugar and organic acid ratio (S/OA) ranged between 0.5 and 2.1, indicating both sweet and acidic fruits. The S/OA ratio was also correlated to the SSC/TA ratio, in which 0.5 (S/OA) corresponded to 3 (SSC/TA) and 2.1 (S/OA) to 8 (SSC/TA) . The useFurthermore, a lower sugar content was detected in accession 4, and lower organic acid and polyphenols, in accession 3. Accessions 3, 4, and 6 were obtained from the same city, under similar environmental conditions, which emphasizes possible genetic differences among producing plants.Accessions 3 and 4 stood out for their visible separation tendency while accessions 01 and 02, from Paraibuna-SP, showed high similarity A. NevertTherefore, the fruits could not be clearly separated by their municipality of origin. A range of 66% to 100% similarity in fruit pulp constituents was found by correlating parameters reported in this study C.The metabolic pathway analysis indicated a total of 36 pathways . The topp values and impact values, this relevance can be further restricted to five metabolisms, namely (1), (2), (4), (5), and (6), with significant p values <0.05 (−log p > 3.15).When assessing the top 12 relevant pathways, only seven exhibited a pathway impact value higher than 0.1, as follows: (1) alanine, aspartate and glutamate metabolism, (2) starch and sucrose metabolism, (3) glutathione metabolism, (4) citrate cycle (TCA cycle), (5) butanoate metabolism, (6) glycine, serine and threonine metabolism, and (7) inositol phosphate metabolism, with impact values of 0.77, 0.53, 0.40, 0.16, 0.14, 0.12, and 0.10, respectively. Based on both their The alanine, aspartate and glutamate metabolism is the most relevant for cambucis, associated to their fundamental involvement as a central molecule of the amino acid metabolism in higher plants  Table 6. Amino ad-glucose and d-fructose are the main starch and sucrose metabolism components  in cambuci is related to γ-aminobutyric acid , also derived from glutamic acid . Glutami+ levels . In addi+ levels . Some amino acids are synthesized as a consequence of the association between GABA production and the C and N metabolism, both under both normal and stress conditions ,65, suchThis work reports, for the first time in the literature, the metabolite profile of cambucis measured by the NMR technique, focusing mostly on sugars, acids, amino acids and total polyphenols. This is also the first detailed description of amino acid composition in cambuci, adding that research addressing its sugars, acids and polyphenols are also scarce.Sucrose is the predominant sugar, while glucose and fructose rank second, depending on the cambuci accession. Citric acid is the most common acid in cambuci, followed by shikimic and quinic acids in widely variable amounts. Cambuci contain ascorbic acid at levels comparable to citrus fruits, which make this fruit a rich source of this compound. Glutathione and glutamic acid are noteworthy among amino acids and their related compounds. According to the diverse metabolic profile described herein for cambuci, different sensorial experiences can be achieved by consuming this fruit. The quality richness described in the present study for the ten evaluated cambuci accessions offer valuable insights into future breeding programs and possible domestication actions for this fruit, leading to the possibilities of products of food, cosmetics and pharmaceutical industries.


Clostridium perfringens in cecum. NE infection significantly reduced body weight gain, thymus index, lymphocyte proliferation activity in blood and ileum, villus height and V/C in jejunum, Th cells% and Mucin2 gene expression in ileum. Dietary APS supplementation significantly increased body weight, feed intake, proportion of immune cells , lymphocyte proliferation activity, V/C in jejunum, and ZO-1 gene expression in ileum. Dietary APS supplementation significantly reduced FCR and mortality rate, Th17/Treg, Th17%, intestinal pathology scores, intestinal inflammatory cytokine gene expression levels, and the number of Clostridium perfringens in cecum. In addition, broilers challenged with NE significantly increased Staphylococcus and Turicibacter and reduced α diversity of microbiota in ileum. Dietary APS supplementation significantly increased α diversity, Romboutsia, Halomonas, propionic acid, butyric acid, formononetin, taurine, cholic acid and equol and downregulated uric acid, L-arginine and serotonin in ileum. Spearman’s correlation analysis revealed that Romboutsia, Turicibacter, Staphylocpccus, Halomonas, Streptococcus, Escherichia-Shigella, Prevotella, uric acid, L-arginine, jerivne, sodium cholate and cholic acid were related to inflammation and Th17/Treg balance. In conclusion, APS alleviated intestinal inflammation in broilers challenged with NE probably by regulating intestinal immune, Th17/Treg balance, as well as intestinal microbiota and metabolites.This study aimed to investigate the effects of dietary astragalus polysaccharide (APS) supplementation on the immune function, gut microbiota and metabolism of broiler chickens challenged with necrotic enteritis (NE). Two hundred forty Arbor Acres broiler chicks (one day old) were randomly assigned using a 2 × 2 factorial arrangement into two groups fed different levels of dietary APS (0 or 200 ppm of diet) and two disease challenge groups . The results showed that NE infection significantly increased FCR, mortality rate, Th17/Treg , NO, lysozyme activity and IL-1β in blood, intestinal immune cell proportion and activity , intestinal inflammatory cytokines  gene expression levels, and the number of  Clostridium perfringens. The mortality rate of necrotizing enteritis is high, and the mortality rate can be as high as 50% when an acute disease occurs, especially when it occurs in chicks, while chronic necrotizing enteritis will significantly reduce the growth performance of chickens, causing intestinal ulcers, erosions, and other adverse effects, seriously affecting the health of poultry and causing serious economic losses to the poultry farming industry (Necrotic enteritis (NE) is an enterotoxic disease caused by type A or type C industry , 2.Escherichia coli significantly increase the proportions of Th17 and Treg cells in the spleen and intestinal mucosal lymph nodes, upregulate the mRNA and protein expression of RORα in the intestinal mucosal lymph nodes, and downregulate the mRNA and protein expression of Foxp3  and regulatory T cells (Treg) are two closely related T cell types. They play key roles in promoting the immune response and suppressing immunity, respectively. Th17 cells and Tregs coordinate and balance to maintain the normal immune function of animals. Previous studies have found that IL-1β is significantly increased when intestinal inflammation occurs in mice and mediates chronic intestinal inflammation by promoting the accumulation of IL-17A-secreting innate lymphoid cells and Th17 cells . Studiesof Foxp3 . In addiof Foxp3 . Chicken tonsils . Th17/TrChickens with healthy intestines can not only improve the digestion and absorption rate of feed nutrients, but also resist the invasion of intestinal pathogenic bacteria and reduce economic losses caused by disease deaths and complications, which are the key factors to ensure good growth performance of chickens . FactorsAstragalus polysaccharide (APS) can regulate the immune function of the body. With immune-enhancing and anti-inflammatory properties, it has a unique two-way regulatory ability. Studies have found that the addition of 4 mg/kg APS to the diet has an immunomodulatory effect on broilers challenged by LPS and can reverse the negative effects of LPS. This includes decreases in daily weight gain and feed intake and decreases in the number of intraepithelial lymphocytes and villus height in the jejunum. APS can also reduce the feed conversion rate, serum NO levels and the heterophilic cell/lymphocyte (H: L) ratio . StudiesFirmicutes and Lactobacteriaceae  Bge. var. mongholicus (Bge.) Hsiao. The purity of APS is 80%. The molecular weight of APS was determined by LC-MS, ranging from 8 to 160,000 Daltons. According to the best dosage recommended by Beijing Centre Biology Co., Ltd., the added dosage of APS in this study was 200ppm.The APS used in this study was purchased from Beijing Centre Biology Co., Ltd. (Beijing). The APS was extracted from the Two hundred forty Arbor Acres broiler chicks (one day old) were randomly assigned using a 2 × 2 factorial arrangement into two groups fed different levels of dietary APS (0 or 200 ppm of diet) and two disease challenge groups . According to the recommendations of the National Research Council (NRC 2012), drug-free corn-soybean meal diets should be formulated to meet or exceed the nutritional requirements of broilers Table 1.Clostridium perfringens CVCC2030 at a concentration of 1×109 CFU/mL . Broilers in the control group were force-fed the same volume (1 mL) of sterilized thioglycolate fluid medium  at the same time point.A broiler subclinical NE model was constructed based on previous research experience . The metAt 13, 25, and 31 days of age, feed and broilers were weighed in cages to calculate the average daily feed intake (ADFI), average daily gain (ADG), feed conversion rate (FCR) and mortality rate. All chicken coops were checked every day for broiler deaths.At 2 days and 8 days after infection, namely, 25 and 31 days of age, 6 healthy chickens were randomly selected for each treatment (1 for each replicate). After weighing, the chickens were euthanized by intravenous injection of 50 mg/kg body weight sodium pentobarbital solution under the wings. The thymus, spleen and bursa of fabricius were collected and weighed. Immune organ index (g/kg) = immune organ weight (g)/body weight (kg).g for 30 min at room temperature, the white flocculent material on the interface between the plasma and the lymphocyte separation medium was transferred to a clean tube using a sterile transfer pipette. The lymphocyte suspension was washed 3 times with RPMI 1640  incomplete culture medium and then resuspended in 2 mL of RPMI 1640 complete culture medium supplemented with 5% (vol/vol) fetal calf serum, 0.5% penicillin , 0.5% streptomycin , and 1% N-(2-hydroxyethyl)-piperazine-N-2-ethanesulfonic acid . The live cells were detected using the Trypan blue dye exclusion technique and a microscope . The cell suspensions were diluted to a final concentration of 1 × 107 cells/mL in RPMI 1640 medium for subsequent analysis.The isolation of peripheral blood mononuclear cells (PBMC) was conducted as previously described  using deSeparation solution was prepared by adding 5% FBS , 1 mmol/L DTT  and 10 mmol/L HEPES  to D-Hank’s solution without calcium magnesium phenol red . Digestion solution was prepared by adding 5% FBS , 0.25% pancreatin , 0.1% collagenase I  and 100 U/mL DNAse I  and incubating at 37°C for 5 min.For cleaning, 1 g of the anterior ileum (1 cm after the yolk antrum) was cut out. All samples were the same weight. The intestinal tube was cut longitudinally along the mesentery side. The small intestine was turned so that the mucosa faced outward, after which it was rinsed gently in Hank’s until the chyme was completely rinsed, and it was then cut into approximately 0.5 cm intestinal tissue fragments laterally. For separation, 5 ml of separation solution was added to a 50-ml centrifuge tube, the tube was placed in a constant temperature shaking box and shaken at 37°C (250 r/min) for 15 minutes and vortexed for 30 seconds, and the intestinal tissue fragments were then filtered through a 200-mesh cell sieve, after which the intestinal tissue fragments were collected in a 50-ml centrifuge tube. To chop the intestinal tissue fragments, they were moved to a petri dish and ophthalmic scissors were used to cut the tissue 100 times into a muddy shape. Five millilitres of digestion solution was then added to a 50-ml centrifuge tube, and the samples were placed in a constant temperature shaking box and shaken (250 r/min) at 37°C for 30–45 minutes. The samples were then vortexed for 30 seconds, the intestinal tissue fragments were filtered through a 300-mesh (48 μm) cell sieve, and the filtrate was collected in a sterile 7 ml centrifuge tube and centrifuges at 4°C (400 x g or 3000 r/min) 10 min . The supernatant was discarded, and the pellet was resuspended in 2 ml of RPMI 1640 . Samples were then centrifuged (400 x g or 3000 r/min) at 4°C for 10 min, the supernatant was discarded, and the pellet was resuspend in 2 ml of RPMI 1640. The organ tissue lymphocyte separation solution was extracted by differential centrifugation (2500 r/min), washed repeatedly and resuspended, and finally resuspended and cultured with complete RPMI 1640. According to the method of the organ lymphocyte extraction kit, Ficoll-Paque Plus  was used for density gradient centrifugation, and the subsequent method was the same as that of peripheral blood lymphocyte separation.6 cells/mL. Two microliters of cell activation stimulator , was added , and the cells were incubated in a CO2 incubator at 37°C for 6 hours. The cells were then centrifuged at 350 x g for 5 minutes, the supernatant was discarded, and the activated cells were collected. Then, 2.5 mL of cell staining buffer  was added to the cell pellet, mixed by vortexing or pipetting, and centrifuged at 350 x g for 5 minutes. The supernatant was then discarded, and the cells were collected by centrifugation again and resuspended in complete RPMI-1640, after which they were ready for analysis to detect the surface markers or intracellular IL-17 expression of the cells.For the intracellular staining of IL-17A, the volume of the isolated ileal lymphocytes was adjusted to 1 ml, and the concentration was adjusted to 2 x 102 incubator  at 39°C, MTT was added to each well at a final concentration of 5 mg/mL. The cells were incubated for an additional 4 h, and then, 100 μL of 10% sodium dodecyl sulfate dissolved in 0.04 mol/L HCl solution was added to each well to lyse the cells and solubilize the MTT crystals. Finally, the absorbance value of each sample was determined using an automated ELISA reader  at 570 nm. The stimulation index (SI) for each sample was calculated based on the following formula:A 3--2,5-diphenyltetrazolium bromide  assay was used to determine the peripheral blood and ileum lymphocyte proliferation response. Briefly, 100 μL of the PBMCs suspension and 100 μL of RPMI 1640 in the absence or presence of 90 μg/mL concanavalin A  or 50 μg/mL lipopolysaccharide  were added to a 96-well microtiter plate . The cultures were set up in triplicate. After a 68-h incubation in a 5% CO6/ml with RPMI 1640 medium. Then, 100 μL of cell suspension was incubated in a 96-well cell culture plate for 2 hours (with 3 replicate wells), the supernatant was discarded, and 200 μL/well of 0.1% neutral red solution  was added, and the cells were further cultured for 2 h. The supernatant was then discarded, and any remaining neutral red was washed away with PBS (3 times). Cell lysate was added at 200 μl/well (ethanol:acetic acid=1:1), kept in the dark at 4°C for 12 h, and the OD value was measured at 550 nm.The phagocytic activity levels of peripheral blood and ileal mononuclear lymphocytes were measured by the neutral red assay method. In short, peripheral blood PBMCs were collected, and the number of cells was adjusted to 2×10® 700 (8200-27), mouse anti-chicken CD4-APC (8210-11), mouse anti-chicken CD8α-Pacific Blue™ (8220-26), mouse anti-chicken monocyte/macrophage-PE (8420-09), mouse anti-chicken CD4-Pacific Blue™ (8210-26) , human anti-chicken CD25-Alexa Fluor® 647  and rat anti-mouse IL-17A-PE  in PBS (pH 7.2) were used. A volume of 100 μL of PBMCs (2×106 cells) was added to a 1.0-mL Eppendorf tube, and 25 μL of diluted primary monoclonal antibody (1:100 dilution) and negative isotype control IgG (mouse IgG1κ)-SPRD, mouse IgG1κ-FITC and mouse IgG1κ-R-PE) were used for staining. After incubating for 45 minutes at room temperature, the cells were washed twice with cold PBS and centrifuged at 1,800 x g for 30 minutes to remove unbound primary antibody. A total of 300 μL of haemolysin solution diluted in PBS (1:25) was added to each tube. Finally, the cells were washed twice, and the final volume was 500 μL. Five-color flow cytometry analysis was performed using a Coulter XL  at Xiyuan Hospital of Traditional Chinese Medicine, China Academy of Chinese Medical Sciences. Then, the percentages of CD3+ T, CD4+ T, CD8+ T, CD25+ T, IL-17A+ T and monocyte/macrophage cells in PBMCs and LPL were calculated.As mentioned earlier , 20, floA commercial ELISA kit  was used to determine the serum levels of IL-1β, IL-4, IL-10, IL-17 and IFN-γ. According to the instructions, a chicken IgG ELISA kit  was used to determine the level of IgG in the serum. Serum IgG levels were determined using a commercial ELISA kit  according to the protocol recommended by the manufacturer. Serum NO and lysozyme activities were measured using commercial ELISA kits . Ileal mucosa sIgA was measured using a commercial ELISA kit .According to a method previously described, the intestinal lesion score was determined: the lesions were observed, and the lesion scores were evaluated .Fixed intestinal tissue was dehydrated and embedded in paraffin. Tissue sections (thickness of 4 μm) were stained with haematoxylin and eosin . Observed by a Leica microscope , 10 intact intestinal villi were randomly selected for each slice. Image-ProPlus (version 6.0) software was used to measure the height of each intestinal villus and its corresponding crypt depth and to calculate the ratio of the two. The height of the villi is defined as the vertical distance from the tip of the villi to the villi-crypt junction, and the crypt depth is the vertical distance from the villi-crypt junction to the base of the crypt.The fixed jejunum tissue samples were dehydrated and embedded in paraffin. A serial microtome  was used to slice the embedded tissue into thin slices (4 μm) and mount it on a polylysine-coated glass slide  following haematoxylin-eosin (HE) staining. Histopathological examination was performed by optical microscopy and a pathological image analysis system . The pathology of the jejunum was determined according to the following criteria: (1) inflammation (0~3 points): 0=no inflammatory cell infiltration; 1=mild inflammatory cell infiltration; 2=moderate inflammatory cell infiltration; and 3=severe inflammatory cell infiltration. (2) Damage (0~3 points): 0=no damage; 1=damage to the mucosal layer; 2=damage to the mucosal layer and submucosa; 3=transparent and disappearance of the cell wall. (3) Crypt damage (0~4 points): 0=no damage to crypts; 1 = 1/3 crypts disappeared; 2 = 2/3 crypts disappeared; 3=only the epithelial surface was intact; 4=crypts and all the epithelium on the surface disappeared. The sum of the three scores is the final intestinal pathology score.® Premix Ex Taq™ II (Tli RNaseH Plus), with GAPDH as the internal reference, and the results are expressed as 2 (-△△CT).At 31 days of age, molecular samples of the jejunum and ileum were quick-frozen in liquid nitrogen and then transferred to a -80°C low-temperature freezer to determine the expression levels of immune function-related genes in the spleen and ileum. TRIzol reagent was used to extract total jejunum RNA, and a NanoDrop ultra-micro-calculation protein analyzer was used to determine the quality and concentration of RNA. The reagent kit used in the reverse transcription step was the PrimeScript™ RT reagent Kit with gDNA Eraser  from Takara. The cDNA obtained after reverse transcription was subjected to real-time fluorescent quantitative PCR on an ABI 7500 real-time fluorescent quantitative PCR instrument with the primers shown in 2, 103, 104, 105, 106, 107, 108. One hundred microlitres of the solution from each dilution tube was inoculated on the corresponding selective medium, and the plate was spread until the solution was dry on the medium. After culturing under the corresponding conditions, 30–300 colonies were selected for bacterial count. Among them, Clostridium perfringens was selected on tryptone-sulfite-cycloserine (TSC) medium  and cultured under anaerobic conditions at 37°C. Plates were counted after 24 hours. Lactic acid bacteria were counted on MRS agar medium , and the culture conditions were 5% CO2 and 37°C for 24 hours. The logarithm of the number of bacteria per gram of caecal content (log10 CFU/g) was used to express the results.Under aseptic conditions, caecum specimens from broiler chickens 2 and 8 days after infection were collected, quickly frozen in liquid nitrogen and stored at -20°C for caecal bacterial count. The specific method was as follows: the caecum was placed on ice (approximately 4°C) to thaw, and 0.3 g was weighed on a balance on a clean bench and placed in a 5-ml sterile centrifuge tube. Then, 2.7 ml of sterile saline was added for a 10-fold dilution, and the sample was shaken and mixed on a micro shaker and allowed to stand for 10 minutes. Then, 0.3 ml of the supernatant was moved to a sterile centrifuge tube, and sterile saline was used to perform gradient dilutions of 10Bacterial DNA was extracted from ileal digesta with a QIAamp DNA Stool Mini Kit  according to the manufacturer’s protocol. The concentrations of DNA extracts were measured on a NanoDrop 2000 spectrophotometer . The V4 region of the bacterial 16S rRNA gene was amplified with the barcoded primer pair 515F/806R  according to previously described methods . After avia ANOSIM using R (v2.15.3). PICRUSt analysis was used to predict the functional potential of bacteria communities (Raw tags were generated by merging paired-end reads using FLASH software (v1.2.7) . Highquamunities . OTUs wemunities .Ileal chyme (0.5 g) was weighed in a 10-mL plastic centrifuge tube, 8 ml of deionized water was added, and the samples were ultrasonically shaken for 30 minutes and centrifuged at 8000 rpm at 4°C for 10 minutes. The supernatant was diluted ten times and filtered with a 0.22-μm filter. Twenty-five microlitres of the filtrate was collected, and an ICS-3000 high-performance ion chromatograph  was used to detect SCFAs and lactic acid by conductivity. Organic acids were separated under gradient conditions in an AS11 analytical column (250 mm×4 mm) and a AG11 guard column: the gradient used potassium hydroxide as the carrier, 0–5 min, 0.8–1.5 mM; 5–10 min, 1.5–2.5 mM, 10–15 min, 2.5 mM, flow rate 1.0 mL/min.The sample detection and data analysis were all completed by Tianjin Nuohe Zhiyuan Technology Co., Ltd. The metabolite extraction process was as follows: (1) To 100 μL of ileum content, 350 μL of extract  was added, and then 20 μL of internal standard L-2-chlorophenylalanine  was added. The samples were vortexed and mixed for 30 seconds and then sonicated under ice-water bath conditions for 10 min. (2) The samples were incubated at -20°C for 1 h and then centrifuged at 13,000 rpm for 15 min at 4°C. (3) After carefully removing 400μL of supernatant to a 1.5 mL centrifuge tube, the extract was then dried in a vacuum concentrator. (4) Then, 100 μL of extract  was added to the dried extract for reconstitution, and the samples were vortexed for 30 s and ultrasonicated for 10 min in an ice water bath. (5) The samples were then centrifuged at 12,000 rpm at 4°C for15 min. (6) Finally, 60 μL of supernatant was carefully removed and placed in a 2-mL injection bottle, and 10 μL of each sample was mixed into a quality control sample, while 60 μL was taken for computer testing.The system was used to analyze the metabolome components of the ileum content using an Agilent 1290 ultrahigh-performance liquid chromatograph in a series ABSciexTripleTOF6600 high-resolution mass spectrometer. The column used was an ACQUITYUPLCBEHAmide column (1.7 μm2.1*100 mm) purchased from Waters. The mobile phase conditions were 25 mM ammonium acetate and 25 mM ammonia solution (A) and 100% acetonitrile (B). The gradient items for the analysis of the ileal contents sample were: 0–0.5 min, 5%A, 95%B; 0.5–7 min, 5–35%A, 95–65%B; 7–8 min, 35–60%A, 65–40%B; 8–9 min, 60%A, 40%B; 9–9.1 min, 60–5% A, 40–95%B; 9.1–12 min, 5%A, 95%B; the flow rate was 0.5 mL/min, and the injection volume was 2 μL. The mass spectrometry conditions were as follows: AB6600TripleTOF mass spectrometer, which can perform primary and secondary mass spectrometry data based on the IDA function under the control of the software . In each data acquisition cycle, the strongest signals with greater than 100 molecules are screened out. The ions were collected corresponding to the secondary mass spectrum data. Bombardment energy: 35 eV, 15 secondary spectra every 50 ms. The ESI ion source parameters were set as follows: atomization pressure (GS1): 60 Pa, auxiliary pressure: 60 Pa, air curtain pressure: 30 Pa, temperature: 550°C, and spray voltage: 5500 V (positive ion mode) or -4500 V (negative ion mode). QC samples with RSD<30% were screened, and a feature yield rate>80% was required to ensure good system stability.The data were first converted to mzXML format using MSconventer, and XCMS (XCMS1.41.0) was used for peak search, peak alignment and other data processing. Then, the data processing and matching of substance identification were performed, and the xcms4dda and xcms4lipid developed by Nuovo Zhiyuan based on XCMS were used. The program and self-built library were processed, minfrac was set to 0.5, and the cut-off was set to 0.8. First, the secondary data were screened. The screening principle is that as long as one forward and one reverse signal is identified, the peak is retained. Second, the peaks of the first- and second-level data are matched, that is, the peaks of the first-level data corresponding to the second-level data are found, and matching is performed according to mztolerance ± 25 ppm. Data analysis included three parts: basic data analysis and personalized data analysis. The goal of basic data analysis is to perform univariate analysis (UVA) and multivariate analysis (MVA) on the qualitative and quantitative results of the metabolome to screen for metabolites with significant differences. Univariate statistical analysis includes data preprocessing , Student’s t-test and analysis of variance. Multivariate statistical analysis includes principal component analysis (PCA) and partial least squares regression analysis , with differential compound screening and identification. Personalized data analysis is a series of bioinformatics analyses on metabolites with significant differences on the basis of basic data analysis, including KEGG annotation analysis of different metabolites and metabolic pathway analysis.P < 0.05 was considered significant, and P values between 0.05 and 0.10 were classified as trends.SPSS 20.0 software was used to perform the statistical analysis on each group of data. The GLM process was used for statistical analysis. When the interaction was significant, one-way analysis was used, and Duncan’s multiple comparison analysis was used for differences between treatments. The spearman rank correlation coefficient was used for the evaluation of the correlation analysis of the variables and microbes in the broiler chickens.P < 0.05) and significantly increased FCR on d 13–25 and d 25–31 (P < 0.05). Compared with the control group, dietary APS supplementation alleviated the negative impact of NE on growth performance, significantly reduced the FCR on d 13–25 and the mortality rate on d 13–31 (P < 0.05), and significantly increased the average weight on d 31 and the d 25–31 feed intake (P < 0.05). There was a tendency to increase body weight gain on d 25–31 (0.05 < P < 0.1). Compared with the unchallenged birds, NE-challenged birds had a significantly reduced d 25 thymus index (P < 0.05). Compared with the NE group, APS supplementation significantly reduced the spleen index (P < 0.05). Compared with the control group, APS supplementation significantly increased the bursa index (P < 0.05).P < 0.05). There was a tendency to improve the phagocytic function of monocytes (0.05 < P < 0.1). Compared with the unchallenged birds, NE-challenged birds had significantly reduced proliferation activity of B lymphocytes in peripheral blood on d 25 (P < 0.05), while dietary APS supplementation increased the proliferation activity of B lymphocytes in peripheral blood on d 25 (P < 0.05).P < 0.05). There was a tendency to increase the number of d 25 peripheral blood mononuclear macrophages and decrease the proportion of Treg cells (0.05 < P < 0.1). Compared with the control group, dietary APS supplementation significantly increased the proportion of T cells in peripheral blood on d 25 (P < 0.05) and significantly reduced the Th17/Treg cells at 31 days of age (P < 0.05). Compared with the unchallenged birds, NE-challenged birds had significantly reduced proliferation activity of T lymphocytes in peripheral blood on d 25 (P < 0.05), while dietary APS supplementation increased the proliferation activity of T lymphocytes in peripheral blood on d 25 (P < 0.05). Compared with the unchallenged birds, NE-challenged birds had significantly increased d 25 serum cytokine  and d 31 serum cytokine  levels (P < 0.05). Compared with the control group, dietary APS supplementation significantly reduced d 25 serum cytokines  and d 31 IL-17/IL-10 levels (P < 0.05) and increased d 31 IL-1β and IL-10 levels (P < 0.05). There was a tendency to increase the ratios of d 25 peripheral blood mononuclear macrophages and d 31 Treg cells (0.05 < P < 0.1).As shown in P < 0.05). Compared with the control group, dietary APS supplementation significantly reduced the scores of d 25 jejunum and d 31 duodenum, jejunum, and ileum and total score of the intestine (P < 0.05). Compared with the unchallenged birds, NE-challenged birds significantly improved the inflammatory cell infiltration and pathological score of the jejunum of d 25 and d 31 broilers and the degree of jejunum injury at d 25 (P < 0.05). Compared with the control group, dietary APS supplementation significantly reduced the inflammatory cell infiltration in the jejunum of d 31 broilers and had a tendency to decrease the pathological score of the d 31 jejunum (P < 0.05). Compared with the unchallenged birds, NE-challenged birds had significantly reduced d 25 jejunum villus height and d 31 V/C (P < 0.05) and a significantly increased d 31 jejunal crypt depth (P < 0.05). Compared with the control group, dietary APS supplementation significantly reduced the depth of jejunal crypts in broilers at d 31 (P < 0.05) and significantly increased the V/C (P < 0.05). Compared with the control group, dietary APS supplementation significantly increased the mRNA expression of ZO-1 in ileum on d 31 (P < 0.05).iNOS, IgA and pIgR mRNA levels (P < 0.05) and significantly reduced Mucin-2 mRNA levels in jejunum and ileum at d 31 (P < 0.05). Compared with unchallenged birds, NE-challenged birds had significantly reduced proliferation activity of B lymphocytes in the ileum at d 25 (P < 0.05) and significantly improved the phagocytic function of monocytes at d 25 (P < 0.05). Compared with the control group, dietary APS supplementation improved the proliferation activity of B lymphocytes in the ileum at d 25 (P < 0.05). Compared with the unchallenged birds, NE-challenged birds had significantly reduced sIgA levels in the ileal mucosa on d 25 (P < 0.05).As shown in P < 0.05) and significantly reduced the proportion of d 25 Th cells (P < 0.05). There was a tendency to increase the proportion of d 25 Th17 cells (0.05 < P < 0.1). Compared with the control group, dietary APS supplementation significantly reduced the proportion of Th17 cells in the ileum at d 25 (P < 0.05) and significantly increased the proportion of Tc and Treg cells in the ileum at d 31 (P < 0.05). There was a tendency to decrease Th17/Treg cells in the ileum at d 25 (0.05 <P <0.1). Compared with unchallenged birds, NE-challenged birds had significantly reduced proliferation activity of T lymphocytes in the ileum at d 25 (P < 0.05) at d 25. Compared with the control group, dietary APS supplementation improved the proliferation activity of T lymphocytes in the ileum at d 25 (P < 0.05). Compared with the unchallenged birds, NE-challenged birds had significantly increased TLR2, IRAK4, NF-κB, IFN-γ, TNF-α, TGF-β1, IL-6, IL-10, RORα and Foxp3 mRNA levels (P < 0.05) in jejunum on d 31. There was a tendency to increase IL-17F and IL-17F/IL-10 expression (0.05 < P < 0.1) in jejunum. Compared with the control group, dietary APS supplementation significantly reduced the mRNA levels of TNF-α, IL-17F, and Foxp3 (P < 0.05) in jejunum. There was a tendency to downregulate IL-6 and RORα (0.05 <P <0.1) in jejunum. A strong interaction between dietary APS and NE infection was observed in relation to TLR2, IFN-γ, TNF-α, IL-6, IL-10, RORα and Foxp3 (P < 0.05) in jejunum. Compared with the unchallenged birds, NE-challenged birds had significantly increased NLRP3, TLR2, IRAK4, NF-κB and IFN-γ mRNA levels (P < 0.05) in ileum on d 31. There was a tendency to increase RORα (0.05 < P < 0.1) in ileum. Compared with the control group, dietary APS supplementation significantly reduced mRNA levels of IL-4 and IL-17F/IL-10 (P < 0.05) and significantly increased AhR and ZO-1 mRNA levels (P < 0.05) in ileum on d 31. There was a tendency to increase Foxp3 and decrease IL-17F (0.05 < P < 0.1) in ileum. A strong interaction between dietary APS and NE infection was observed in relation to IFN-γ, IL-17F and IL-22 (P < 0.05) in ileum.As shown in As shown in P < 0.05). Dietary APS supplementation significantly increased the observed_species at d 25 (P < 0.05). NE and APS supplementation had significant interaction effects on the d 31 Shannon and Simpson indices (P < 0.05). Dietary APS supplementation significantly increased the alpha diversity of unchallenged broilers but had no significant effect on NE-challenged broilers.Lactobacillus and d 25 Staphylococcus and decreased the relative abundance of d 25 Escherichia-Shigella, d 25 Prevotella, d 25 Halomonas, and d 31 Escherichia-Shigella.Escherichia-Shigella, d 25 Streptococcus, d 25 Staphylococcus, d 25 Halomonas, d 31 Escherichia-Shigella, d 31 Romboutsia and d 31 Streptococcus and decreased the relative abundance of d 25 and d 31 Lactobacillus.Dietary APS supplementation increased the relative abundance of d 25 Lactobacillus and d 31 Turicibacter (P < 0.05). The addition of APS to broiler diets significantly increased d 25 Staphylococcus, d 25 Halomonas and d 31 Romboutsia (P < 0.05) and significantly reduced d 25 Lactobacillus and d 31 Lactobacillus (P < 0.05).As shown in Clostridium perfringens numbers in the caecum at d 31 (P < 0.05). Compared with the control group, dietary APS supplementation significantly reduced the number of Clostridium perfringens in the caecum at d 31 (P < 0.05).To ensure the reliability of the data, the influence of factors such as the sample preparation process and instrument instability must be minimized. In the process of detection and analysis, three QC samples are used to monitor and ensure the stability of the instrument, after which the missing values are simulated in the original data to standardize the data. After obtaining the sorted data, we perform partial least squares discrimination analysis (PLS-DA). Orthogonal partial least squares-discriminant analysis was performed on the data, and the PLS-DA model of the APS supplementation group versus the control group was obtained. As shown in P < 0.05, 35 different metabolites were identified in positive ion mode  of the first principal component of the PLS-DA model was greater than 1 and ion mode Table 3.P < 0.05 in the enrichment analysis and an influence value greater than 0.1 in the topological analysis. As shown in The metabolites of differences between groups were entered into MetaboAnalyst, the database corresponding to chickens was used to perform enrichment analysis on metabolic pathways, and the corresponding P values were calculated. Through topological and enrichment analyses of the metabolic pathways where the differential metabolites are located, the metabolic pathways are screened in depth to identify the metabolic pathways that are more relevant to the experimental treatment. The screening conditions were P < 0.05). There was a tendency to increase isobutyric acid and caproic acid (0.05 < P <0.1).To explore the relationships between the intestinal microbiota, intestinal metabolites and apparent immune indicators in broiler chickens, based on the abovementioned ileal microbiome and metabolomics data, we subsequently performed a spearman correlation analysis Figure 9Turicibacter, Romboutsia, propionic acid, butyric acid, formononetin, S-equol, and cholic acid and was negatively correlated with Lactobacillus, Halomonas, Prevotella, uric acid, and L-arginine (P < 0.05). The proportion of Th17 cells in peripheral blood was positively correlated with Lactobacillus, Halomonas, Prevotella, Jerrivne, sodium cholate and cholic acid and negatively correlated with Turicibacter and Romboutsia (P < 0.05). The proportion of Treg cells in peripheral blood was positively correlated with Lactobacillus, jervine, sodium cholate, 7-ketodeoxycholic acid and cholic acid and negatively correlated with Turicibacter and Romboutsia (P < 0.05). The peripheral blood Th17/Treg ratio was positively correlated with uric acid and L-arginine (P < 0.05) and negatively correlated with 7-ketodeoxycholic acid, glycoursodeoxycholic acid and taurine (P < 0.05). Serum IL-1β was positively correlated with propionic acid, formononetin, jervine, sodium cholate, 7-ketodeoxycholic acid, cholic acid, glycoursodeoxycholic acid, taurine and milbemycin A4 oxime (P < 0.05) and negatively correlated with Escherichia-Shigella, Prevotella, uric acid and L-arginine (P < 0.05). Serum IL-17 was positively correlated with Turicibacter, Romboutsia, propionic acid, 7-ketodeoxycholic acid, glycoursodeoxycholic acid and milbemycin A4 oxime (P < 0.05) and negatively correlated with Staphylococcus, Halomonas, Streptococcus, uric acid and L-arginine (P < 0.05). Serum IL-17/IL-10 was positively correlated with Romboutsia and equol (P < 0.05) and negatively correlated with Staphylococcus, Halomonas and Streptococcus (P < 0.05). Peripheral blood T cell proliferation activity was positively correlated with Halomonas, Escherichia-Shigella, Prevotella and milbemycin A4 oxime (P < 0.05) and negatively correlated with Lactobacillus (P < 0.05). Peripheral blood B cell proliferation activity was positively correlated with Halomonas, Prevotella and milbemycin A4 oxime (P < 0.05) and negatively correlated with Turicibacter and uric acid (P < 0.05). Lactobacillus was positively correlated with serotonin and negatively correlated with propionic acid and S-equol (P < 0.05). As shown in Romboutsia (P < 0.05). The depth of jejunal crypts was positively correlated with equol (P < 0.05). Jejunal TNF-α gene expression was positively correlated with equol and DL-lysine and negatively correlated with jervine, 7-ketodeoxycholic acid and glycoursodeoxycholic acid (P < 0.05). The expression of the IL-6 gene in the jejunum was negatively correlated with taurine (P < 0.05). The expression of the IL-17F gene in the jejunum was negatively correlated with 7-ketodeoxycholic acid and glycoursodeoxycholic acid (P < 0.05). The proportion of Th cells in the ileum was positively correlated with Lactobacillus, Halomonas and Prevotella (P < 0.05) and negatively correlated with Turicibacter, Romboutsia and milbemycin A4 oxime (P < 0.05). The proportion of Tc cells in the ileum was positively correlated with Romboutsia, formononetin and milbemycin A4 oxime (P < 0.05) and negatively correlated with Prevotella and L-arginine (P < 0.05). The proportion of Th17 cells in the ileum was positively correlated with Turicibacter, Romboutsia, propionic acid and formononetin (P < 0.05) and negatively correlated with Halomonas, Escherichia-Shigella, Prevotella, uric acid and L-arginine (P < 0.05). The proportion of Treg cells in the ileum was positively correlated with Turicibacter, Romboutsia, propionic acid and formononetin (P < 0.05) and negatively correlated with Lactobacillus, Halomonas, Escherichia-Shigella, Prevotella, uric acid and L-arginine (P < 0.05). The ratio of Th17/Treg cells in the ileum was positively correlated with Lactobacillus, Halomonas, and Prevotella (P < 0.05) and negatively correlated with Turicibacter and Romboutsia (P < 0.05). The expression of TLR2 and NF-κB genes in the ileum was positively correlated with S-equol (P < 0.05). The expression of the iNOS gene in the ileum was positively correlated with Turicibacter and negatively correlated with L-arginine (P < 0.05). IgA gene expression in the ileum was positively correlated with 7-ketodeoxycholic acid (P < 0.05). Ileal ZO-1 gene expression was negatively correlated with serotonin (P < 0.05). In addition, Lactobacillus was positively correlated with serotonin and negatively correlated with propionic acid and S-equol (P < 0.05). Turicibacter was positively correlated with formononetin, sodium cholate and cholic acid (P < 0.05) and negatively correlated with L-arginine (P < 0.05). Romboutsia was positively correlated with propionic acid, butyric acid, formononetin, S-equol, glycitein, sodium cholate and cholic acid (P < 0.05) and negatively correlated with uric acid and serotonin (P < 0.05). The results show that the immunomodulatory effect of APS is associated with a specifically altered microbe-metabolic axis.As shown in Clostridium perfringens. This disease often occurs in broilers at 2–5 weeks of age, with a mortality rate of approximately 2%-10%, with a maximum of 50%. Geier et al.  is an enterotoxic disease caused by r et al.  showed tntestine , 31, thintestine , 33 but ntestine , 35. APSFirst, we tested the systemic immune response and found that NE infection reduced the broiler thymus index, increased peripheral blood NO levels and lysozyme activity, increased the Th17 cell ratio and Th17/Treg cell ratio, and increased the production of the proinflammatory cytokines IL-17 and Th17/IL-10. At the same time, other inflammatory cytokines, such as IL-1β and IFN-γ, and the anti-inflammatory cytokine IL-4 were increased, reducing the proliferation capacities of T and B lymphocytes in the preinfection period, indicating that NE Infection of broilers caused systemic inflammation and simultaneously suppressed systemic cellular immune function. Previous studies have found that NE-challenged broilers significantly upregulate the expression of splenic inflammatory cytokine genes, causing systemic inflammation , 37. It Clostridium perfringens in the caecum were significantly increased, and the structure of the jejunum was severely damaged. The decrease in sIgA in the ileal mucosa indicated that the NE model was successfully established, the intestinal mucosa had a strong inflammatory response and some mucosal immune functions were impaired. These results are consistent with previous studies, indicating that NE infection causes inflammation of the intestinal mucosa inflammatory response and simultaneously damages the intestinal barrier structure and increases the permeability of the intestine. Pathogens and proinflammatory cytokines pass through the intestinal barrier and into the blood, which induces the occurrence of systemic inflammation -induced colitis increases and is significantly positively correlated with DSS treatment  play an important role in intestinal physiology. According to reports, SCFAs, especially butyrate, can be used as an energy source for colonic epithelial cells . In addiBacteroides polymorpha, Bacteroides fragilis) in the murine intestinal bile acid pool regulates the number of RORγ+ regulatory T cells in the colon. In SPF mice with insufficient diet and nutrition, the intestinal bile acid pool was restored (replenishing a specific combination of primary or secondary bile acids), colon RORγ+ Tregs were increased through the bile acid-VDR axis, and the susceptibility to colitis was reduced . This data can be found here: The animal study was reviewed and approved by China Agricultural University Animal Care and Use Committee .BS designed and performed experiments, analyzed data, and wrote the paper. PL and SY performed some experiments, and analyzed some data. YL and MG analyzed some 16SrRNA sequencing data. HL analyzed some metabolome data. ZL performed some experiments. YG initiated the study, designed animal experiments, analyzed data, and wrote the paper. All authors contributed to the article and approved the submitted version.This study was funded by the China Agriculture Research System program (CARS-41-G11).Author HL is employed by Beijing Centre Biology Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hippodamia variegata is one of the most important predatory natural enemies of aphids. Recently, transcriptome and genome sequencings of H. variegata facilitate the gene functional studies. However, there has been rare investigation on the detection of stably expressed reference genes in H. variegata. In the current study, by using five analytical tools , eight candidate reference genes, namely, Actin, EF1α, RPL7, RPL18, RPS23, Tubulin-α, Tubulin-β, and TufA, were evaluated under four experimental conditions including developmental stages, tissues, temperatures, and diets. As a result, a specific set of reference genes were recommended for each experimental condition. These findings will help to improve the accuracy and reliability of RT-qPCR data, and lay a foundation for further exploration on the gene function of H. variegata.Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is an accurate and convenient technique for quantifying expression levels of the target genes. Selection of the appropriate reference gene is of the vital importance for RT-qPCR analysis.  The reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is one of the most sensitive, accurate, and convenient methods for detecting quantitative analysis of gene expression in biological samples . For RT-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S3 (RPS3), 18S ribosomal RNA (18S), 25S ribosomal RNA (25S), tubulin, translation elongation factor 1-alpha (EF1α), and others have been used extensively for RT-qPCR analysis  is one of the most important natural enemies of aphids infesting various crops in many countries , ribosomal protein L7 (RPL7), ribosomal protein L18 (RPL18), ribosomal protein L23 (RPL23), tubulin alpha (Tubulin-α), tubulin beta (Tubulin-β), and elongation factor Tu (TufA), have been selected as candidate reference genes for H. variegata. The expression stabilities of these genes were assessed with respect to different developmental stages, tissues, temperatures, and diet treatments using five different statistical algorithms. The five statistical algorithms are Delta Ct method (olfactory co-receptor (Orco) of H. variegata was assayed to verify the reference genes. The results will serve as a guide for gene expression studies in H. variegata.ountries . The staaxyridis , Henosepmaculata , Coccinepunctata , and Hipnvergens  have beet method , geNorm t method , NormFint method , BestKeet method , and Reft method , which iH. variegata were obtained from Langfang Experimental Station of Chinese Academy of Agricultural Sciences, Hebei Province, China. Larvae and adults were maintained in plastic containers (8 × 8 × 11 cm) with Myzus persicae as a food source. The colonies were reared under conditions of 25 ± 1°C, 65 ± 10% relative humidity, and a 14:10-h light/dark photoperiod.The N = 30).Samples at developmental stages including eggs (50 eggs), first-instar larvae , second-instar larvae , third-instar larvae , fourth-instar larvae , pupae , and adults  were selected as different treatment groups. Moreover, different tissues including head, thorax, abdomen, leg, and wing were dissected and harvested from adult females or males at 2 to 3 days , nymphs and adults of M. persicae, and canola pollen separately after 12 h of starvation  following the manufacturer’s instructions. RNA purity was checked on a NanoDrop spectrophotometer . Each sample of 2.0 μg total RNA was used to synthesize single-stranded cDNA using the FastQuant RT kit .H. variegata transcriptome and genome data (unpublished data). The reference genes from other insect species in GenBank were downloaded from NCBI1 queried individually to H. variegata transcriptome and genome using the TBLASTN program with a permissive E-value cutoff of 10–5 to get the hits. And then, each of the queried hits was compared back against non-redundant database of NCBI by the BLASTX program (E < 10–5) to determine whether the original sequence was one of the hits. All the selected reference genes are listed in The reciprocal BLAST hits approach was used to screen reference genes from H. variegata. Primer parameters were as follows: optimal temperature 60 ± 2°C, GC content 40–60%, and 18–24 bp in length. The amplification size was between 100 and 200 bp  according to sequences obtained from our recently sequenced transcriptomes and genome for d 200 bp .Orco gene. PCR amplifications were performed in 50-μl reactions containing 25 μl 2 × Phanta Max Master Mix, 2 μl of each primer (10 μM each), 4 μl first-strand cDNA, and 17 μl ddH2O. The PCR parameters were as follows: one cycle of 94°C for 3 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; a final cycle of 72°C for 10 min. PCR products were purified and cloned into the pcloneEZ-Blunt TOPO vector  for sequencing confirmation.Gene-specific primers  were des2), fivefold dilution series of cDNA  were used as templates to construct a standard curve of each gene. Also, the homologous RT-qPCR efficiencies (E) were calculated according to the equation: E = (10[–1/slope] - 1) × 100.The RT-qPCR measurements were performed on an ABI Prism 7500 system . The reaction system contained a mixture of 12.5 μl 2 × SuperReal PreMix Plus , 0.75 μl of each primer (10 μM), 1 μg sample cDNA, 0.6 μl 50 × ROX Reference Dye, and proper volume of RNase-free water. PCR cycling parameters were as follows: 95°C for 15 min, followed by 40 cycles of 95°C for 10 s, and cooled to 60°C for 32 s. Then, the PCR products were heated to 95°C for 15 s, cooled to 60°C for 1 min, heated to 95°C for 30 s, and cooled to 60°C for 15 s to measure the dissociation curves. To determine the amplification efficiency (E) and the correlation coefficients  were used to evaluate the stability of each reference gene. The Delta Ct method was used to select the optimal reference gene . geNorm nce gene . The Besnce gene . Finallynce gene .Orco is highly conserved among insect species and is essential for localization of ORs in olfactory sensory neuron (ORN) dendrites and reception of odor signals  and the least stable reference genes (Tubulin-α and Tubulin-β). RT-qPCR analysis was performed as described previously. Data were calculated using the 2–△△Ct method (Orco) has been normalized with the CT-value average of two best and least stable reference genes. All the experiments were performed in three replications, and the results are expressed as means ± SD.The  signals . Thus, Ot method  and the 2) values of 0.9851–1 , the expression pattern of Orco in various tissues were similar. The expression level of Orco was as follows: head > leg > wing > abdomen > thorax. However, the expression level of Orco in the head was almost 600-fold higher than in the leg when the two most stable reference genes were used for normalization (Tubulin-α and Tubulin-β) were used for normalization, the Orco expression in the head was tremendously high. The calculated relative expression of Orco in head with two unstable genes was almost 10 times higher than that of normalized with two stable genes . To further investigate the biological roles of any specific gene in H. variegata, quantitative gene expression through RT-qPCR is essential. Following the “Minimum Information for Publication of Quantitative Real-Time PCR Experiments” (MIQE) guideline, reference genes with high stability are necessary  (Cydia pommnella (Lepidoptera: Tortricidae) (Aphis gossypii (Hemiptera: Aphididae) (Locusta migratoria manilensis (Orthoptera: Acrididae)  , Cydia pricidae) , Aphis ghididae) , Locustarididae) , and H. nvergens . The ICGrst rank .RPL18 encodes a ribosomal protein that is a component of the 60S subunit and belongs to the L18E family of ribosomal proteins     , Anastreritidae) , and Cimmicidae) . Consist website .RPS23 has also been reported as the most appropriate reference gene in insects  of olfactory sensilla, distributed on the head (mainly expressed in antennae and mouthparts), legs, wings, and abdomen . The relative expression level of OBP20, normalized with the stable reference genes (RPL32 and RPL27), was approximately threefold higher in the head than when normalized with two unstable reference genes (ACT and βACT)  . Under tThe original contributions presented in the study are included in the article/JX and AK conceived and designed the research. JX and CY conducted the experiments. TL and XL analyzed the data. JX and YZ wrote the article. All authors have read and agreed to the published version of the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


KRC) meat helps differentiate KRC from mainstream chicken. We aimed to investigate the effects of β-alanine and L-histidine supplementation on the carnosine synthesis in and quality and secondary structure of proteins in slow-growing KRC meat. Four hundred 21-day-old female KRC were used, and a completely randomized design was applied. The chickens were divided into 4 experimental groups: basal diet (A), basal diet supplemented with 1.0% β-alanine (B), 0.5% L-histidine (C), and 1.0% β-alanine combined with 0.5% L-histidine (D). Each group consisted of 5 replicates (20 chickens per replicate). On d 70, 2 chickens per replicate were slaughtered, and the levels of carnosine, anserine, and thiobarbituric acid reactive substances were analyzed. Biochemical changes were monitored using synchrotron radiation-based Fourier transform infrared microspectroscopy; 5 chickens per replicate were slaughtered, and the meat quality was analyzed. Statistical analysis was performed using ANOVA and principal component analysis (PCA). Group D chickens exhibited the highest carnosine meat content, followed by those in groups B and C. However, amino acid supplementation did not affect anserine content and growth performance. Higher carnosine levels correlated with increasing pH45 min and decreasing drip loss, cooking loss, shear force, and lipid oxidation. PCA revealed that supplementation with only β-alanine or L-histidine was related to increased content of β-sheets, β-turns, and aliphatic bending groups and decreased content of α-helix groups. This study is the first to report such findings in slow-growing chicken. Our findings suggest that KRC can synthesize the highest carnosine levels after both β-alanine and L-histidine supplementation. Higher carnosine contents do not adversely affect meat quality, improve meat texture, and alter the secondary structures of proteins. The molecular mechanism underlying carnosine synthesis in chickens needs further study to better understand and reveal markers that facilitate the development of nutrient selection programs.Carnosine enrichment of slow-growing Korat chicken ( KRC) is a crossbreed between Thai indigenous Leung Hang Khao sires and Suranaree University of Technology synthetic breed dams. The main purpose of the crossbreeding was to provide an alternative breed to promote the occupation of smallholder farmers in Thailand and Southeast Asia. Korat chicken is categorized as a slow-growing chicken . 45 min, drip loss, and cooking loss. Dietary β-alanine supplementation reduces the shear force of the meat in broiler chicks  spectrometry has been used to study meat quality in terms of biochemical changes (SR-FTIR) microspectroscopy is a powerful and sensitive approach for detecting the vibrations of molecules that provide information about protein secondary structure, lipids, and glycogen. This technique can be used to analyze samples at the micron level or micro-sample areas  to increase carnosine levels in KRC breast meat. The experiments were approved by the Ethics Committee on Animal Use of the Suranaree University of Technology, Nakhon Ratchasima, Thailand (document ID: U1-02631-2559).Female KRC produced in the Suranaree University of Technology farm were used for the study. When the chickens were 21 days old, they were randomly assigned to 4 experimental diet groups using a completely randomized design with 5 replicates per group and 20 chickens per replicate. The mean and SD of the chicken body weight was approximately 266.04 ± 3.03 g.The 4 experimental diets, all of which were formulated based on the National Research Council nutrient recommendations (1994), were as follows: basal diet (A), basal diet supplemented with 1.0% β-alanine (B), 0.5% L-histidine (C), and 1.0% β-alanine combined with 0.5% L-histidine (D). The ingredient and nutrient compositions of the experimental diet for growers (22–42 days) and finishers (43–70 days) are shown in 2. Food and water were provided ad libitum. The vaccination program was performed as per guidelines set by the Department of Livestock Development, Bangkok, Thailand.The chickens were raised in an open house, and the stocking density was 8 birds/mThe chickens were weighed, feed intake data were collected weekly, and data were used to calculate the body weight gain, average daily feed intake, average daily gain, and feed conversion ratio at 21, 42, and 70 d.M. pectoralis major) was packed in a vacuum bag and frozen at −80°C. Samples were analyzed for carnosine, anserine, and TBARS. The other portion (10 g) was kept in a zip-lock bag, stored at 4°C for 24 h, and used for biochemical analysis.On d 70, 10 chickens per group (2 chickens/ replicate) were randomly tagged and stunned by chloroform. Then chickens were slaughtered by decapitation and bled. Approximately 10 g of breast meat  was measured using an electronic pH meter  at 45 min and 24 h postmortem. The pH was measured 3 times in the same area, and the probe was washed with ultrapure water between different sample measurements.Drip loss. At 24 h postmortem, drip loss was calculated using the method establish by Cooking loss. Cooking loss was determined using the method described by 3 from the same location and weighed. Samples were cooked in a water bath at 80°C until the core temperature reached 71°C. The samples were then cooled at room temperature for 3 h and weighed. The percentage of cooking loss was calculated using the following equation: Cooking loss (%) = [/initial weight] × 100%.Shear force. The shear force of cooked samples (2 × 1 × 1 cm3) was determined following the method of g and 4°C for 10 min. One milliliter of the supernatant was added to 1 mL of 0.02 M thiobarbituric acid, and incubated at 95°C for 20 min. Subsequently, the sample was cooled at 5°C for 5 min, and the absorbance was measured at 532 nm using a BioTek Epoch Microplate Spectrophotometer . Malonaldehyde bis   was used as a standard.Breast meat stored at −80°C for 5 mo was used for TBARS measurements according to the method described by g at 4°C for 10 min. Supernatants were filtered through a 0.45-μm syringe filter. The filtrate (250 µL) was mixed with 750 µL of acetonitrile and stored at 4°C for 20 min. Then, the sample was centrifuged at 10,000 rpm at 4°C for 10 min, filtered through a 0.2-μm syringe filter, and stored at −20°C until use. Carnosine and anserine were separated on an Atlantis HILIC silica column  equipped with high-performance liquid chromatography . Mobile phase A containing 0.65 mM ammonium acetate in 75% acetonitrile at pH 5.5 and mobile phase B containing 4.55 mM ammonium acetate in 30% acetonitrile at pH 5.5 were used. The separation conditions were determined by the linear gradient of phase B from 0 to 100% for 13 min at 1 mL/min. Twenty microliters of the sample obtained after the addition of acetonitrile were injected. Dipeptides were detected at a wavelength of 210 nm. Quantification was performed using the external standard carnosine and anserine  at 25°C.The carnosine and anserine contents were determined using the method of After chilling, 10 breast meat samples from each group were cross-sectionally cut into a muscle fiber size of 1 × 1 cm and placed into an aluminum foil block. The frozen tissues were cut using a cryostat  at a thickness of 7 µm and placed on the IR window , then placed in a vacuum desiccator for 2 to 3 d before the SR-FTIR measurement.−1. The FTIR spectra were obtained in the transmission mode, collecting 64 scans with a 10 × 10 µm aperture size at a resolution of 6 cm−1 over a measurement range from 4,000 to 800 cm−1. Each group comprising 400 spectra (20 spectra × 2 muscles replicates × 10 chickens) were processed using the OPUS 7.5 software (Bruker Optics Ltd.).Two muscle sections of each chicken were subjected to spectra analysis. FTIR spectra were measured at BL4.1 Infrared spectroscopy & Imaging, Synchrotron Light Research Institute, using the SR-FTIR spectrometer with a synchrotron light source in the mid-IR region. Spectra were collected on a Bruker FTIR spectrometer  coupled to a Bruker Hyperion 2000-IR Microscope  with a 36x objective, coupled to an MCT detector cooled with liquid nitrogen covering a measurement range from 4,000 to 800 cm−1, including lipid, amide I, amide II, CH-binding of lipid, amide III, and glycogen.The integral areas were determined using second-derivative processing at the spectral regions from 3,000 to 900 cm−1), α-helix , β-sheet , β-turn , and antiparallel  regions based on Gaussian and Lorentzian functions.Curve fitting of amide I was determined using the original spectra after preprocessing to calculate the integral areas of amide I  was used to identify the biochemicals in the spectral ranges from 3,000 to 2,800 cm−1 and 1,800 to 900 cm−1. All spectral data were preprocessed using the Savitzky-Golay algorithm for second derivative transformations at 13 smoothing points and normalized with extended multiplicative signal correction using the Unscrambler X Multivariate Data Analysis software . The 400 spectra were averaged into 20 spectra per treatment, and outliers were removed until 5 spectra per treatment for groups or clusters were obtained using PCA. The bi-plot correlation was used to represent the clustered differentiation of data, and related variables were recalculated using the two-dimensional scatter plot of PCA with the predominant spectral range. The high loading SR-FTIR spectra were selected for multivariate analysis with dipeptide content and physicochemical properties. Data for all variables were weighted using an SD weighting process, and the relationship between variables was investigated using PCA bi-plot correlation.Principal component analysis  was used for statistical analysis.ANOVA was used to analyze the effect of experimental diets on growth performance, meat quality, TBARS, dipeptide content, the ratio of integral area, and secondary structure ratio. Significant differences between the means of the treatments were determined using Tukey's multiple tests. A P < 0.05). The highest level of carnosine was found in group D, with a 52.8% increase compared to that of the control group. However, amino acid supplementation had no significant effect on the anserine content (P > 0.05).The carnosine and anserine contents in KRC breast meat are presented in The results revealed that KRC could synthesize carnosine at approximately 2.76 mg/g. Carnosine synthesis can be improved when the chickens are fed carnosine synthase substrates. The carnosine content in the meat of chickens in groups B and C, supplemented with only β-alanine or L-histidine, respectively, was higher than that in the control group . Regarding β-alanine supplementation, our results align with many previous studies . As demoThe highest carnosine content was found when the diet was supplemented with both amino acids, consistent with the hypothesis that both amino acids are limiting amino acids for carnosine synthesis . This reWhen compared with the other groups, a significant difference in the anserine content was not found in the L-histidine supplementation group or the combined amino acid supplementation group. These results may be explained by the fact that anserine can be synthesized via 2 pathways: carnosine methylation  or 3-metP > 0.05; KRC performance was expected to improve when the diet was supplemented with carnosine synthase substrates; however, contrary to our expectation, the results showed no significant difference in performance, including feed intake, body weight gain, average daily feed intake, average daily gain, body weight, and feed conversion ratio, among the different groups in each growth phase (ROS) levels and antioxidant activity related to environmental factors  and animal's characteristics . Increase in ROS can decrease productive performance , whereas but led to an increase in pH45 min, the ability to retain water in meat and a decrease in the shear force (P < 0.05). The results, shown in Amino acid supplementation did not affect pH45 min). When an animal dies, carnosine removes lactic acid and regulates Ca2+ discharge from the sarcoplasmic reticulum, reducing ATP consumption during postmortem glycolytic metabolism  was the lowest in group D . The integral area of amide I in group D was lower than that in the control group. In contrast, those of groups B and C were not significantly different compared with that of the control or group D. Regarding amide II, the integral areas of the control, B, and D groups were higher than that of group C (P < 0.05). In addition to lipid, amide III, CH bending, and glycogen/carbohydrate showed nonsignificant differences (P > 0.05). These results suggested that the supplemented amino acids strongly affected the change in the secondary structure of muscle proteins.The percentages of integration of each biomolecule in The secondary structure of the protein in the amide I region is composed of β-sheet, α-helix, β-turn, and antiparallel, and their integral areas are presented in −1, 1,635 cm−1, 1,529 cm−1, and 1,255 cm−1), β-turn , doublet due to the 2 protonated tautomers of histidine , amide II , and CH and CH2 aliphatic bending  groups. Groups A and C were positively correlated with α-helix , tyrosine , and CH and CH2 aliphatic bending groups . The reference SR-FTIR bands with high correlation loading in KRC breast meat are shown in The PCA score plot of the chicken meat biomolecule spectra is shown in As demonstrated by these results, the carnosine content can affect the quality of the meat related to its texture and water retention ability, which is consistent with previous studies . However45 min, amide II, cooking loss, TBARS, and wavenumber 1,664 cm−1, 1,650 cm−1, 1,635 cm−1, 1,587 cm−1, 1,540 cm−1, 1,529 cm−1, 1,519 cm−1, 1,457 cm−1, 1,448 cm−1, 1,365 cm−1, and 1,255 cm−1) showed a significant correlation with the 4 experimental groups with a variance greater than 50%. Group A (−1), tyrosine , and CH and CH2 aliphatic bending groups  , β-turn , and CH and CH2 aliphatic bending groups . Group C was positively correlated with α-helix  and CH and CH2 aliphatic bending groups . In group D, a positive correlation was found with the carnosine content, pH45 min, and doublet due to the 2 protonated histidine tautomers .The score plot and correlation loading that expresses the relationship between SR-FTIR spectra and physicochemical results are shown in From the results, 2 interesting points need to be discussed. The first point is that carnosine levels can affect the physiological properties of meat by changing biomolecules in meat, as demonstrated using SR-FTIR. The second point is that β-alanine or L-histidine supplementation may decrease meat quality because it affects the increase in cooking loss and levels of TBARS, as demonstrated by the results, which aligned with those shown in 45 min, leading to a better maintenance of pH45 min. Simultaneously, the TBARS value decreased, resulting in an improved water-holding ability of muscle fibers. Interestingly, supplementation with both amino acids also increased the integral area to 1,587 cm−1, representing the binding of carnosine to Cu2+  can increase pH to Cu2+ . This fo to Cu2+ . Cong etLOC107051274, ACSBG1, and CAPNS2 and downregulated genes MYO7B, MYBPH, SERPINH1, and PGAM1 may be related to meat tenderness in Korat chicken. However, it is still unclear how these genes change the protein secondary structure in this study. Further studies are required to clarify the molecular mechanism involved in meat quality.Supplementation with either β-alanine or L-histidine has adverse effects on the physiological properties of meat, such as an increase in cooking loss and TBARS. The results may be caused by the increase in β-sheet, β-turn, and aliphatic bending groups. Residual amino acids from carnosine synthesis, free amino acids, might induce the oxidative modification of proteins . Protein45 min can be improved. In addition, the results suggest that single β-alanine or L-histidine supplementation may negatively affect physiological properties, confirmed by the increase in β-sheet, β-turn, and aliphatic bending groups. However, we used only one level of both amino acids. Further studies are required to examine the optimum level of supplemented amino acids and genetics involved with carnosine synthesis.This study confirmed that the carnosine content could be increased by amino acid supplementation (substrates of carnosine synthesis) in slow-growing KRC meat. Supplementation cannot improve the performance of chickens because of the ability of carnosine to resist oxidative stress. Furthermore, the highest carnosine synthesis was observed with supplementation of both β-alanine and L-histidine. Moreover, the water retention ability of muscle cells and pH


The purpose of this study is to functionally characterize the attB in S. pneumoniae strains with different genetic backgrounds and in other bacterial species, and to investigate the presence of Tn5253 attB site into bacterial genomes.Tn5253-carryng transconjugants obtained in S. pneumoniae strains with different genetic backgrounds and in other bacterial species, namely Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes, and Enterococcus faecalis showed that: (i) Tn5253 integrates in rbgA of S. pneumoniae and in orthologous rbgA genes of other bacterial species, (ii) integration occurs always downstream of a 11-bp sequence conserved among streptococcal and enterococcal hosts, (iii) length of the attB site corresponds to length of the duplication after Tn5253 integration, (iv) attB duplication restores rbgA CDS, (v) Tn5253 produced circular forms containing the attTn site at a concentration ranging between 2.0 × 10−5 to 1.2 × 10−2 copies per chromosome depending on bacterial species and strain, (vi) reconstitution of attB sites occurred at 3.7 × 10−5 to 1.7 × 10−2 copies per chromosome. A database search of complete microbial genomes using Tn5253 attB as a probe showed that (i) thirteen attB variants were present in the 85 complete pneumococcal genomes, (ii) in 75 pneumococcal genomes (88.3 %), the attB site was 83 or 84 nucleotides in length, while in 10 (11.7 %) it was 41 nucleotides, (iii) in other 19 bacterial species attB was located in orthologous rbgA genes and its size ranged between 17 and 84 nucleotides, (iv) the 11-bp sequence, which correspond to the last 11 nucleotides of attB sites, is conserved among the different bacterial species and can be considered the core of the Tn5253 integration site.Analysis of representative Tn5253 attB integration site combined with genome analysis contributed to elucidating the potential of Tn5253 horizontal gene transfer among different bacterial species.A functional characterization of the TnThe online version contains supplementary material available at 10.1186/s13100-021-00253-z. Tn5253, once integrated into the chromosome, was flanked by the attL site, identical to attB, and the attR site, identical to attTn. Pneumococcal mobilome analysis showed the frequent presence of Tn5253-like elements in multidrug-resistant S. pneumoniae strains and the maintenance of the element in all derivative isolates [5253 integration site, by analyzing attB in Tn5253-carrying transconjugants obtained in S. pneumoniae strains with different genetic backgrounds and in strains belonging to other bacterial species. We then investigated the presence of the Tn5253 attB site into the complete microbial genomes available in public databases.The acquisition of new genetic material by horizontal gene transfer (HGT) significantly drives bacterial genome evolution and is mediated by Mobile Genetic Elements (MGEs). The term “mobilome” is used to indicate the entire set of MGEs of the microbiome. MGEs ar of ICEs . Conjugaromosome , while ofic site –26. We pectively –29. Tn52romosome . The ICEisolates –34. In t5253-carrying transconjugants were obtained in S. pneumoniae with different genetic backgrounds, namely TIGR4, A66 and SP18-BS74 [5253-chromosome junction fragments showed that: (i) Tn5253 integration occurred at a specific integration site (attB) located in rbgA gene of the pneumococcal chromosome [attL was identical to attB and (iii) attR was identical to attTn, as already described for D39 and its derivative strains [attB sites among these pneumococcal strains were not identical, with their size varying from 41 nucleotides (variant attB13 in SP18-BS74) to 83 nucleotides  . However, these results did not correlate with the conjugation frequency, which was 6.1 × 10−6, indicating that the frequency of circularization is not the only limiting factor of the conjugation process. Neither circular forms nor reconstituted attB of Tn5253 could be detected in the TIGR4 background , correlating with the absence of conjugal transfer (<9.9 × 10−8). Analysis of Tn5253 integration, in pneumococci with different genetic backgrounds, revealed that the element always integrates downstream of nucleotide position 20 of rbgA coding sequence (CDS)  at strains , and tham BM6001 . attL settB site . To inveassembly . Integra5253 functional analysis to streptococci and enterococci characterizing Tn5253 circular forms and integration sites in the transconjugants obtained in S. agalactiae H36B, S. pyogenes SF370, S. gordonii V288, E. faecalis OG1SS and JH2-2 backgrounds  attL was identical to attB regardless of the bacterial strain harbouring the element and attR was identical to attTn suggesting a polarization of Tn5253 integration process, (ii) integration always occurs downstream of a 11-bp conserved sequence, namely the last 11 nucleotides of attB sites, (iii) length of the attB site corresponds to length of the duplication after Tn5253 integration, (iv) attB site duplication restores rbgA CDS. It is worth to note that in E. faecalis, attB duplication modifies the rbgA predicted gene product  and S. pyogenes (3.0 × 10−5 copies per chromosome), while no circular forms were detected in E. faecalis JH2-2 genetic background (<2.7 × 10−7 copies per chromosome)  to 1.7 × 10−2  copies per chromosome. In E. faecalis, Tn5253 excision and circularization are strain dependent: a representative transconjugant obtained in OG1SS background produced circular forms and reconstituted attB site at 1.4 × 10−4 and 6.8 × 10−3 copies per chromosome, respectively; transconjugant FR50, obtained in JH2-2 background, produced reconstituted attB site at a frequency of 1.7 × 10−2 copies per chromosome but did not produce circular forms (<2.7 × 10−7). Conjugation frequency was lower than circularization frequency in all the tested strains except in S. pyogenes FR68. Many other factors are likely to be important in the conjugation process such as the expression of a capsular polysaccharide [We then extended Tn5253 attB among pneumococci was carried out. The database of 85 complete S. pneumoniae genomes (accessed in August 2021) was interrogated by using as a query the 83-bp attB. Sequence homology analysis identified thirteen allelic variants of attB.  it was 41 nucleotides. The 83-bp attB variant 1 is the most frequent variant, carried by 38 genome strains , including D39 strain, its rough derivative R6, and the classical type 3 Avery’s strain A66. Variant 2 is carried by TIGR4 and other 8 genome strains (10.6 %) and contains two nucleotide substitutions. Variant 13 is harboured by G54 and other 9 genome strains (11.7 %) and contains only the last 41 nucleotides of variant 1. In addition, SP18-BS74, whose draft genome is available, also harbours variant 13. Variants 5 and 7 are carried by 7 strains (8.2 %), variant 4 by 5 strains (5.8 %), variants 6 and 9 by 2 strains (2.4 %). The remaining 5 variants  were found in only in one strain. In thirteen pneumococcal genomes, carrying the attB variants 1, 2, 7, 11 and 12, Tn5253-like elements were integrated into the pneumococcal chromosome, resulting in the duplication of the attB site.To integrate biological data, a genome-wide investigation of Tn5253 attB site in 18 other bacterial species, including the functionally characterized S. agalactiae, S. pyogenes,S. gordonii, hosts . Homology search identified the Tn5253 attB site in S. pneumoniae and other streptococcal and enterococcal species and found that: (i) during conjugal transfer, Tn5253 integrated in S. pneumoniae rbgA gene or in the orthologous rbgA genes of the other bacterial hosts, (ii) Tn5253 produced circular forms containing the attTn site and the frequency was species- and strain-dependent, (iii) reconstitution of attB site was species- and strain-dependent. Through a DNA homology search conducted in the complete microbial genome database, we also found that: (i) thirteen allelic variants of the Tn5253 attB site were present in the complete S. pneumoniae genomes and their size ranged from 41 to 84 nucleotides, (ii) in other bacterial species, Tn5253 attB is located in orthologous rbgA genes with a size ranging between 17 and 84 nucleotides. Tn5253 integration, in the original S. pneumoniae host, as in the other streptococcal and enterococcal hosts, occurs always downstream of a 11-bp conserved sequence located in the rbgA CDS. Genome analysis revealed that the 11 nucleotides, corresponding to the last 11 nucleotides of the attB sites, are conserved also among the attB sites of other bacteria and can be considered the core of the integration site. In conclusion, even if a huge number of bacterial genomes is available, an in-silico analysis and a functional characterization of the mobilome is reported only in few cases. In this work, a functional characterization of the Tn5253 attB integration site, combined with genome sequence analysis, contributed to elucidating the potential of Tn5253 horizontal gene transfer among different bacterial species.In the present paper we conducted a functional characterization of Tn5253 and HB565, a streptomycin resistance derivative of type 3 Avery strain A66 [Bacterial strains and their relevant properties are reported in Table rain A66 , 39, 40,590 about 0.2, roughly corresponding to 5 × 108 CFU/ml) and centrifuged at 11,000 x g for 2 min. Pneumococcal lysates were obtained by using lysis solution  as already reported [E. faecalis) or mutanolysin  was added at a final concentration of 1 mg/ml or 20 µg/ml respectively and mixtures were incubated at 37 °C for 1 h. Protoplasts were centrifuged at 3,000 x g for 15 min, resuspended in 100 µl of dH2O, heated at 85 °C for 5 min and kept on ice until use.Bacterial cultures (1 ml) were harvested in exponential phase  and the KAPA SYBR FAST qPCR kit Master Mix Universal (2X) (Kapa Biosystems) were used for Real Time PCR experiments according to the protocol extensively described . Quantifectively . Reconstreported .https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC.Homology searches of the databases available at the National Center for Biotechnology Information were conducted using the Microbial Nucleotide BLAST , selecting the complete genomes database. Default parameters were used and only alignments with significant e-values were considered. We built a stand-alone database containing only genomes of interest to be searched with BLAST software to confirm the results. Sequence analysis was carried out with BioEdit 7.2.5 .com/). Multiple DNA sequence alignments were performed using Clustal Omega  relative to circular forms (CI), reconstitution of attB site, and chromosomal reference gene (gyrB) quantification are reported. Standard curves, slope and intercept values are reported.