Background

In intensive swine husbandry systems,piglets commonly receive in-feed antibiotics during early life stage,mainly aiming to prevent outbreaks of respiratory and gastrointestinal tract(GIT)infections,and promote growth[1].However,their enormous and uncontrolled use has led to many resistant bacterial strains,causing detrimental effects in both humans and animals.In China,although the use of antibiotics in feed during weaning period of the pig is still a common practice,it has increasingly received safety concerns from consumers and thus the use of antibiotics in feed will be banned in the future.Thus,to better design alternatives to antibiotics for pigs,it is important to understand the post-term effect and the possible mode of action of the antibiotics.The immediate effects of antibiotic intervention on blood parameters[2],apparent nutrient digestibility[3],gut bacterial composition[4,5]and microbial metabolism[6,7]have been described,especially in newly weaned pigs.However,much less attention has been paid to antibiotics intervention in suckling and nursing stage and long-term(carry-over)effects on host metabolism and GIT microbiota after withdrawal of antibiotics.In pigs,early life antibiotic exposure affected pancreatic development and glucose metabolism 5 wk after antibiotic withdrawal[8].A recent study found piglets that received an subcutaneous injection with 0.1 mL tulathromycin(dosage 2.5 mg/kg body weight)on d 4 after birth showed decreased microbial diversity and an altered microbial composition in jejunal digesta on d 176,but had limited effect on d 56[9].Using mice models,previous studies also have indicated that low-dose antibiotic exposure in early life had a longterm effect on host metabolism and adiposity[10,11].These results suggest that alteration of gut microbes at early life may have long-lasting effect on subsequent host metabolism and gut microbiota.However,it is still unclear whether the early use of antibiotics in suckling and nursing stage have long-term impact on subsequent metabolism and gut microbes in pigs.

In addition to the early use of antibiotics,dietary protein level could also affect the gut health of the animal[12].In practice,low-protein amino acid-supplementation diets are used to decrease nitrogen excretion and feed costs.Our previous study found that low crude protein(low-CP)diet increased the counts of Bacteroidetes,Clostridium cluster IVand Clostridium cluster XIVa,decreased Escherichia coli counts,and decreased cadaverine,phenol and indole compounds concentrations[13,14],suggesting a beneficial effect of low-CP diet.Antibiotics could decrease the nutrient utilization by gut microbiota and increase those available for body metabolism[15].However,when fed low-CP diet,whether early antibiotic intervention affect body metabolism and fecal microbial fermentation profile in pigs remains unclear.

To test the hypothesis that early antibiotic intervention(EAI)(from d 7 to d 42)may have long-lasting effects on subsequent host metabolism and GIT microbes and microbial fermentation in pigs,the present study examined the blood parameters,apparent nutrient digestibility,and major bacterial taxonomic groups and microbial metabolism in feces on d 77(short-term)and d 185(long-term)in relation to different CP diets.

Methods

Animals and experimental design

4.人才不够。各试点省份税务系统公职律师人员普遍不多，且分布不均，一些地市税务系统仍旧没有合格的公职律师人选。此外，部分具备法律专业背景的干部缺乏法律服务工作经验，法律服务的技能和水平还有待提高。

A total of 18 litters crossbred(Duroc×Landrace×Large White)newborn piglets(212 piglets in total)were used in this study.The piglets on d 7 while with the sow were randomly allotted to two groups(n=9 litters)and offered creep feed(Additional file 1:Table S1)either without(control)or with a mixture of antibiotics(50 mg/kg oxytetracycline calcium,50 mg/kg kitasamycin,and 50 mg/kg olaquindox)(antibiotic).This mixture of antibiotics is commonly used as a growth promoter for piglets in commercial farms in China in order to ensure healthy transition around weaning.Oxytetracycline calcium is a broad spectrum antibiotic with antibacterial activity against gram-negative and gram-positive bacteria,and kitasamycin exhibits activity mainly against grampositive bacteria,whereas olaquindox exhibits antibacterial activity both gram-negative and gram-positive bacteria.The dosages for the antibiotics used were according to the dose limitation in Regulations of Feeding Drug Additives(announcement No.168)approved by Ministry of Agriculture,China.On d 23,the sows were removed from the piglets,while the piglets remained in the environmentalcontrolled nursery with 1.8×2.5 m pens fitted with a hard plastic fully slotted floor,and fed the same creep feed until d 42,which is a typical feeding program in a commercial farms in China.On d 42,the piglets within the control or antibiotic group were mixed,respectively,and then randomly allotted to 1 of 2 dietary treatments(Low-crude protein,Low-CP;Normal-crude protein,Normal-CP,respectively)according to their equal average body weight(BW)and gender,respectively,which generated 4 groups(Control-Low CP,Con-LP;Control-normal CP,Con-NP;Antibiotic-Low　CP,Ant-LP;Antibiotic-Normal CP,Ant-NP,respectively).There were 5 pens replicates per treatment group and 9 pigs per pen.The numbers of barrows and gilts were not equal on d 42,but spilt as evenly as possible across treatments(23 gilts and 22 barrows,22 gilts and 23 barrows,24 gilts and 21 barrows,and 23 gilts and 22 barrows for treatments Con-LP,Con-NP,Ant-LP,and Ant-NP,respectively).

The diets comprised a normal-CP diet(20%,18%,and 14%CP during d 42 to d 77,d 77 to d 120,and d 120 to d 185,respectively,supplemented with lysine,methionine,threonine,and tyrptophan)and a low-CP diet(16%,14%,and 10%CP during d 42 to d 77,d 77 to d 120,and d 120 to d 185,respectively,supplemented with four limiting AA mentioned above)(Table 1).The 2 diets were formulated to meet or exceed NRC[16]nutrient recommendations.Creep feed diets were fed as crumble,whereas normal-or low-CP diets were in a pelleted form.After d 42,the pigs were moved to a total confinement house with 2.5×3.0 m pens that had partial concrete slatted floors.During the experimental period,the pigs consumed feed ad libitum and feed residues were recorded daily.All pigs had free access to water via a low-pressure nipple drinker.The body weight(BW)of pigs was recorded at the beginning of the experiment and at the end of each period to determine average daily gain(ADG),average daily feed intake(ADFI),and the ratio of gain to feed(G:F).

Sample collection and preparation

The fecal samples were collected for 3 consecutive days,from d 75 to 77 and 183 to 185 of age,respectively,during the feeding period in the morning by rectal stimulation.Feces from each day were collected at least 3 pigs from each of the pen replicates and were pooled for each replicate to create composite samples.The feces were then frozen at-20°C until the analysis of microbial metabolites and dominant bacterial community,which was completed within 2 weeks after sample collection.

综上所述，在初中英语教学中对于学生写作能力的培养，是提高学生英语整体能力和展现个人英语水平的重要途径。因此，在初中英语教学中，深化对学生英语写作能力的培养，对于学生今后的发展具有非常深远的影响和意义。但是，对于学生写作能力的提升并不是一朝一夕就能够完成的，这就要求教师在教学实践中不断进行探索，创新教学模式，优化教学方法，根据学生的需求制定合理的教学方案，从而有效提升学生的写作能力。

On d 77(28.4±0.6 kg)and d 185(102.0±0.6 kg),when the experimental pigs were fasted for approximately 12 h,one median BW barrow from each pen was selected for blood sampling(n=5).Blood samples(10 mL)were collected from the jugular vein and serumwas obtained by centrifuging at 3000×g for 10 min at 4 °C.Thereafter,samples were stored at-20°C until chemical analysis.

Table 1　Ingredient and nutrient composition of experimental diets(%,as-fed basis)a

aThe pigs of control and antibiotic groups were divided to 1 of 2 treatment groups(normal vs.low CP diet)after d 42.LP low protein diet,NP normal protein diet bProvided per kilogram of diet:vitamin A,2.85 million IU;vitamin D3,0.6 million IU;vitamin E,67.50 IU;vitamin K3,750 mg;vitamin B1,750 mg;vitamin B2,1500 mg;vitamin B6,900 mg;vitamin B12,7.5 mg;nicotinic acid,7500 mg;folic acid,300 mg;calcium pantothenate,3750 mg;biotin,37.5 mg;vitamin B4,100 mg;antioxidants,15 mg;Cu(CuSO4.5H2O),60 mg;Fe(FeSO4.H2O),51.6 mg;Co(CoCl2),50 mg;Mn(MnSO4.H2O),25 mg;Zn(ZnSO4.H2O),21.2 mg;Zn(ZnO),6 mg;I(KI),3.9 mg;Se(Na2SeO3),3.0 mg;Carrier(Sepiolite)604.3 mg cCalculate values according to NRC[16] dME=metabolizable energy eSID AA=standardized ileal digestible AA fAnalytical results obtained according to AOAC[17]

Age range,d:Ingredient,%42 to 77　77 to 120　120 to 185 LP-16%　NP-20%　LP-14%　NP-18%　LP-10%　NP-14%Corn　68.00　63.70　68.90　58.00　82.40　71.10 Soybean meal　18.00　21.00　18.80　29.38　8.71　19.13 Wheat bran　-　-　6.00　8.00　2.00　4.00 Whey power　4.30　4.30　-　-　-　-Fishmeal　3.20　8.00　-　-　-　-Soybean oil　2.70　0.70　2.56　1.60　2.50　2.00 Dicalcium phosphate　0.85　0.10　0.78　0.65　0.90　0.80 Calcium carbonate　0.50　0.36　0.89　0.89　0.82　0.90 Sodium chloride　0.30　0.30　0.30　0.30　0.30　0.30 Vitamin mixtureb　0.20　0.20　0.20　0.20　0.20　0.20 Mineral mixtureb　0.80　0.80　0.80　0.80　0.80　0.80 L-Lysine　0.65　0.33　0.49　0.17　0.78　0.48 DL-Methionine　0.20　0.09　0.10　-　0.20　0.11 L-Threonine　0.24　0.10　0.15　0.01　0.30　0.15 L-Tryptophane　0.06　0.02　0.03　-　0.09　0.03 Calculated contentc,%MEd,MJ/kg　14.02　14.02　13.82　13.80　13.81　13.82 SID AAe,%Lysine　1.23　1.23　0.99　0.98　0.98　0.99 Methionine+Cysteine　0.68　0.68　0.56　0.56　0.60　0.56 Threonine　0.73　0.73　0.59　0.60　0.61　0.60 Trptophane　0.20　0.20　0.17　0.20　0.18　0.17 Analyzed nutrient compositionf,%Crude protein　16.63　20.11　14.03　18.02　10.18　14.06

Nutrient composition and apparent digestibility analysis

山药不同生长期对水分要求不同，发芽期需足够的底墒，保证正常出苗；山药块茎形成期，尤其是8月份的大生长期不能缺水，内部营养物质的积累多要水分的充足供应，但切忌漫灌或长期积水，否则山药极易腐烂。除了在播种时浇施好粪水补充山药发芽和苗期水份外，还要在7、8月间如遇干旱应适当浇水1-2次。

4.4 减少非护理工作，提供良好支持 对于一些不需要专业护理人员完成的工作可由其他人员完成，减少护理工作量［5］。发挥辅助部门的效用，减少护士非护理工作的时间，减轻护士的压力。发挥社会支持系统的作用，通过家庭、亲友、同事等的支持，缓解护士的身心压力。

Serum biochemical parameters

Serum total protein,albumin,glucose,urea,cholesterol,triglyceride,high density lipoprotein-cholesterol(HDLC),and low density lipoprotein-cholesterol(LDLC)were measured by the enzymatic colorimetric method using an AU2700 autoanalyzer(Olympus,Tokyo,Japan).Globulin concentration of individual serum samples was calculated by subtracting the amount of albumin from the total protein concentration.

DNA extraction and quantitative real-time PCR

The total bacterial DNA was extracted from fecal samples(0.3 g)using the bead-beating and phenol-chloroform extraction methods[20].The qPCR assay was performed on StepOnePlus™Real-Time PCR System(Life Technologies,Califormia,USA)with SYBR Premix Ex Taq dye(Takala,Bio,Ostu,Japan).The sequences of the selected targets and primers are listed in Table 2.The reaction mixture(20 μL)consist of 10.0 μL SYBR Green Supermix(Bio-Rad),0.4 μL ROX Reference Dye,2.0 μL of template DNA,6.8 μL of double distilled water,and 0.4 μL of each primer set.The standard curves of each bacterial group were generated with 10-fold serial dilutions of the 16S rRNA genes amplified from the respective target strains.Quantitative real-time PCR amplification was performed using the following conditions:initial denaturation program(95°C for 30 s),denaturation program repeated for 40 cycles(95 °C for 5 s,60 °C for 30 s),followed by the melting curve program(60-95°C with a heating rate of 0.1°C/s).The melting curves were checked after amplification to ensure single product amplification of consistent melting temperature.Quantification was performed in triplicate,and the mean Ct value was calculated.The results were expressed as log1016S ribosomal DNA gene copies/g feces.

Table 2　Primers used for quantification in this study

Target　Primer sequence 5′→3′　Reference Total bacteria　Forward:CGGTGAATACGTTCYCGG Reverse:GGWTACCTTGTTACGACTT[]Firmicutes　Forward:GGAGYATGTGGTTTAATTCGAAGCA　[]Reverse:AGCTGACGACAACCATGCAC Bacteroidetes　Forward:GGARCATGTGGTTTAATTCGATGAT　[]Reverse:AGCTGACGACAACCATGCAG Clostridium IV　Forward:GCACAAGCAGTGGAGT　]Reverse:CTTCCTCCGTTTTGTCAA Clostridium XIVaForward:CGGTACCTGACTAAGAAGC　[]Reverse:AGTTTYATTCTTGCGAACG E.coli　Forward:CATGCCGCGTGTATGAAGAA　]Reverse:CGGGTAACGTCAATGAGCAAA Lactobacillus　Forward:AGCAGTAGGGAATCTTCCA　]Reverse:ATTCCACCGCTACACATG

Measurement of fecal microbial metabolites

The short-chain fatty acid(SCFA),amines,ammonia,and phenolic and indolic compounds were selected as markers of GIT microbiota metabolism.The concentrations of SCFA were analyzed by gas chromatography as described previously[21],with slight modifications.Briefly,approximately 0.4 g of fecal samples were weighed into a 2-mL centrifuge tube,1.6 mL of double distilled water was added.The mixture was vortexed for 10 min until the material was homogenized and then centrifuged at 13,000×g for 10 min at 4 °C.A portion of 1 mL of the clear supernatant was transferred into a new tube,and then added 0.2 mL 25%(w/v)metaphosphoric acid.After homogenization,the mixture was frozen at-20°C and kept overnight to precipitate the proteins.After thawing,a portion of 100 μL internal standard(0.64%(w/v)crotonic acid solution)was added.The tubes were vortexed for 1 min and then centrifuged at 13,000×g for 10 min at 4 °C.The supernatant was filtered through a 0.22-μm syringe filter and then analyzed on an Aglient 7890B system with a flame ionization detector(Agilent Technologies Inc.).The following column conditions were used:nitrogen was used as the carrier gas with a flow rate of(17.68 mL/min);the oven,detector and injector port temperature were 130°C,250 °C,220 °C,respectively.These acids were identified by their specific retention times and the concentrations determined and expressed as umol/g.

另外，以上问题的存在反映出博物馆的外宣资料译介翻译没有得到足够的重视和规范化。红色旅游文化的展示体现了我们国家的历史形象，而不标准和不规范的翻译不仅有损国家形象，还影响中国红色文化的传播，难以达到文化交流的目的性。所以，相关部门应采取相对措施，进一步改善和加强外宣资料的翻译，提升翻译质量，为传递中国优秀文化树立起好形象。

Amine concentrations in feces were determined by high-performance liquid chromatography(HPLC)with a method according to Yang et al.[22].Briefly,1.5 g of feces were treated with 3 mL of 5%trichloroacetic acid,homogenized for 10 min and then centrifuged at 3600×g for 10 min at 4°C.The supernatant was mixed with an equal volume of n-hexane and vortexed for 5 min,the water phase(0.5 mL)was transferred into a new tube,and then added with 1.5 mL saturated Na2CO3,1 mL dansyl chloride,and 1 mL NaOH(2 mol/L).The mixed solution was heated at 60°C for 45 min,and then added with 100 μL ammonia(2.8%)to stop the reaction.The mixture was kept in the water bath until the acetone was vaporized under nitrogen at 40°C.Finally,the sample was extracted with 3 mL diethyl ether.The extracts were dried under nitrogen and then re-dissolved in acetonitrile.The mixture filtered through 0.22-μm syringe filter and then analyzed on an Aglient 1220 Infinity LC system with an UV detector(Agilent Technologies Inc.).

式中：Kw为纯水体积模量(约为2×109N/m2)；Sr为土体饱和度；pwo为计算深度海床的静水压力。

The ammonia concentration in feces was analyzed using UV spectrophotometer according to Chaney and Marbach[23].Phenolic and indolic compounds concentration was determined by HPLC as previously described[24].Briefly,0.1 g of fecal sample was treated with 1 mL acetonitrile,homogenized for 10 min and then frozen at-20°C for 20 min.Finally,the mixture was centrifuged at 3000×g for 10 min at 4 °C.The supernatant was filtered through a 0.22-μm syringe filter and analyzed for phenolic and indolic compounds(i.e.,pcresol,skatole,indole,and phenol)using HPLC with an UV detector(Agilent Technologies Inc.).

All data were analyzed using 2×2 factorial MIXED procedure of SAS for a randomized complete block design(SAS 9.2 Institute,Cary,NC,USA).The model included the fixed effect of antibiotic,protein level,associated twointeractions and the random errors of a pen or an individual pig.The ADG,ADFI,G:F,digestibility of nutrients,and fecal fermentation profile were evaluated using the pen as the experimental unit.Blood parameters were assessed using the individual pig as the experimental unit.Differences were considered significant at P≤0.05,and tendency was declared with 0.05＜P＜0.10.When a significant interaction among antibiotic and CP was observed,data were further analyzed by using a one-way ANOVA with Duncan’s multiple range tests.Differences at P＜0.05 were identified significant.

Statistical analysis

Results

Growth performance and apparent nutrient digestibility

Growth performance of the pigs was shown in Table 3.For ADG,there was an interaction(P＜0.05)between EAI and dietary CP treatment from d 77 to d 185 and d 42 to d 185.The EAI increased ADG(P＜0.05)from d 42 to d 185 in the low-CP diets but not in the normal-CP diets.Meanwhile,low-CP diet tended to reduce the ADFI(P=0.08)from d 42 to d 185,but G:F at each feeding period did not differ among treatments.

For apparent nutrient digestibility,no interactions between EAI and dietary CP treatment were observed(Table 4).On d 77,there was no difference of OM digestibility among treatments.The EAI significantly increased CP digestibility(P＜0.05)and tended to increase(P=0.06)DM digestibility.The low-CP diet reduced CP and crude fat digestibility(P＜0.05).On d 185,digestibility of crude fat was decreased(P＜0.001)when pigs fed the low-CP diet,but DM,CP,and OM digestibility were not affected by treatment.Overall,these results indicated that EAI had short-term effect on nutrient digestibility.

Table 3　Effects of early antibiotic intervention on growth performance of pigs with different protein level diets1

a,bMeans in the same row with different superscripts differ(P＜0.05) 1The commercial creep feed with or without in-feed antibiotics(50 mg/kg olaquindox,50 mg/kg oxytetracycline calcium,and 50 mg/kg kitasamycin)was fed to pig from d 7 to d 42.Thereafter,the Con and Ant group were further randomly assigned to provide a normal(20%,18%,14%CP from d 42 to d 77,d 77 to d 120,d 120 to d 185,respectively)or low CP diet(16%,14%,10%CP from d 42 to d 77,d 77 to d 120,d 120 to d 185,respectively),respectively 2The P values indicate main effects for antibiotic(Ant),protein level(CP)and their interaction(Ant x CP),respectively

Item　Low-CP　Normal-CP　SEM　P-value2 Con　Ant　Con　Ant　Ant　CP　Ant×CP 42 d BW,kg　12.23　12.20　12.28　12.25　0.07　0.851　0.743　0.981 ADG,kg/d d 42 to 77　0.50　0.52　0.51　0.51　0.02　0.686　0.972　0.786 d 77 to 185　0.78b　0.81ab　0.84a　0.83a　0.01　0.361　0.002　0.048 d 42 to 185　0.71b　0.74a　0.76a　0.75a　0.01　0.180　0.001　0.019 ADFI,kg/d d 42 to 77　0.72　0.72　0.78　0.78　0.02　0.945　0.140　0.993 d 77 to 185　2.24　2.36　2.44　2.54　0.06　0.349　0.119　0.891 d 42 to 185　1.85　1.94　2.02　2.09　0.05　0.368　0.089　0.895 G:F d 42 to 77　0.70　0.73　0.67　0.66　0.02　0.880　0.227　0.675 d 77 to 185　0.36　0.35　0.36　0.33　0.01　0.323　0.787　0.655 d 42 to 185　0.44　0.44　0.44　0.42　0.01　0.555　0.414　0.605

Table 4　Effects of early antibiotic intervention on total tract apparent digestibility of pigs with different protein level dietsa

DM dry matter,CP crude protein,OM organic matter aThe commercial creep feed with or without in-feed antibiotics(50 mg/kg olaquindox,50 mg/kg oxytetracycline calcium,and 50 mg/kg kitasamycin)was fed to pig from d 7 to d 42.Thereafter,the Con and Ant group were further randomly assigned to provide a normal(20%,18%,14%CP from d 42 to d 77,d 77 to d 120,d 120 to d 185,respectively)or low CP diet(16%,14%,10%CP from d 42 to d 77,d 77 to d 120,d 120 to d 185,respectively),respectively. 2The P values indicate main effects for antibiotic(Ant),protein level(CP)and their interaction(Ant x CP),respectively

Item,%　Low-CP　Normal-CP　SEM P-value2 Con　Ant　Con　Ant　Ant　CP　Ant×CP d 77 DM　75.24 76.73 74.81 80.52 0.98　0.065 0.368　0.263 CP　60.00 64.84 64.54 73.45 1.75　0.032 0.040　0.498 OM　79.86 79.65 78.31 83.32 0.88　0.173 0.538　0.140 Crude fat 44.61 48.44 53.96 56.88 1.34　0.136＜0.001 0.831 d 185 DM　77.12 72.61 79.24 71.82 1.67　0.103 0.843　0.666 CP　72.46 72.45 75.08 73.35 0.67　0.550 0.240　0.550 OM　82.93 81.67 82.14 80.54 0.51　0.198 0.372　0.867 Crude fat 50.52 48.48 57.72 56.74 1.34　0.320　　＜0.001 0.720

The level of serum parameters of animals reflects the body metabolic status according to their internal and external environments[26].EAI exerted an effect on body metabolism on d 77,such as a significant increase of the concentrations of albumin and glucose,especially in low-CP diets,but not in normal-CP diets.A previous studyalso　indicated thatpigs received therapeutic amoxicillin exposure at birth through postnatal d 14 increased glucose concentration at postnatal d 49[8].Additionally,we found that the antibiotics effect on serum biochemistry was diminished on d 185.Thus,antibiotic intervention showed short-term effect on body metabolism,but had no long-term effect.

To dissect whether EAI and dietary CP level affected the host metabolism,basic serum biochemistry was analyzed.On d 77,albumin,glucose,and urea concentrations in the serum revealed interactions(P＜0.05)between EAI and dietary CP treatments(Table 5).The EAI increased albumin and glucose concentrations in the low-CP diet,but increased urea concentration in the normal-CP diet.Meanwhile,EAI increased(P＜0.05)triglyceride concentration irrespective of dietary CP,and tended to increase concentration of total protein(P=0.08).The low-CP diet reduced(P＜0.05)concentrations of total protein and LDLC irrespective of EAI.On d 185,EAI also interacted with dietary CP on the concentrations of globulin and glucose in the serum(P＜0.05).The EAI decreased glucose concentration in normal-CP diet,but increased globulin concentration.Furthermore,low-CP diet reduced(P＜0.01)concentrations of urea,triglyceride,and HDLC irrespective of EAI.In general,these results indicated that EAI had short-term effects on serum parameters,and these effects may be influenced by dietary CP levels.

Table 5　Effects of early antibiotic intervention on serum biochemical indexes of pigs with different CP level diets1

HDLC,high density lipoprotein-cholesterol,LDLC,low density lipoprotein-cholesterol a-cMeans in the same row with different superscripts differ(P＜0.05) 1The commercial creep feed with or without in-feed antibiotics(50 mg/kg olaquindox,50 mg/kg oxytetracycline calcium,and 50 mg/kg kitasamycin)was fed to pig from d 7 to d 42.Thereafter,the Con and Ant group were further randomly assigned to provide a normal(20%,18%,14%CP from d 42 to d 77,d 77 to d 120,d 120 to d 185,respectively)or low CP diet(16%,14%,10%CP from d 42 to d 77,d 77 to d 120,d 120 to d 185,respectively),respectively.2The P values indicate main effects for antibiotic(Ant),protein level(CP)and their interaction(Ant x CP),respectively

Item　Low-CP　Normal-CP　SEM　P-value2 Con　Ant　Con　Ant　Ant　CP　Ant×CP d 77 Total protein,g/L　62.00　66.21　63.61　70.79　1.67　0.082　0.031　0.643 Albumin,g/L　17.73b　23.20a　18.04b　19.45b　0.66　　＜0.001　0.122　0.049 Globulin,g/L　44.27　43.01　45.57　51.34　1.73　0.553　0.178　0.342 Glucose,mmol/L　4.39c　5.58a　5.44ab　5.26b　0.14　0.004　0.189　0.002 Urea,mmol/L　2.12b　2.16b　2.46b　3.26a　0.12　0.003　　＜0.001　0.003 Cholesterol,mmol/L　1.87　1.89　1.97　2.09　0.05　0.890　0.223　0.080 Triglyceride,mmol/L　0.45　0.60　0.63　0.75　0.03　0.039　0.145　0.323 HDLC,mmol/L　0.77　0.81　0.84　0.88　0.02　0.132　0.651　0.241 LDLC,mmol/L　1.03　0.99　1.04　1.14　0.04　0.430　0.045　0.603 d 185 Total protein,g/L　66.62　65.14　65.26　69.34　1.45　0.378　0.342　0.072 Albumin,g/L　31.60　32.64　33.48　31.78　0.73　0.664　0.489　0.081 Globulin,g/L　35.02ab　32.50b　31.78b　37.56a　1.56　0.313　0.567　0.022 Glucose,mmol/L　4.54b　4.88b　5.54a　4.78b　0.12　0.245　0.021　0.011 Urea,mmol/L　4.38　4.12　5.02　6.04　0.26　0.352　0.004　0.134 Cholesterol,mmol/L　2.10　1.97　2.15　2.20　0.07　0.793　0.322　0.542 Triglyceride,mmol/L　0.46　0.45　0.61　0.57　0.03　0.684　0.020　0.743 HDLC,mmol/L　0.85　0.77　0.91　0.95　0.02　0.593　0.011　0.151 LDLC,mmol/L　0.99　0.99　1.01　0.91　0.04　0.572　0.712　0.574

Fecal bacterial populations

再者，教师的提问策略不当容易导致课堂沉默现象。通常表现为提问时的问题开放程度不够、多为封闭式问题；问题难易程度不当，学生不会回答；问题指向不明或范围过大，学生无从回答。

In order to evaluate the effect of EAI on the quantitative change of bacterial community in the feces,real-time PCR was performed on some major bacterial groups.As shown in Additional file 1:Figure S1,dietary treatments had no effects on fecal major bacterial population on d 77 and d 185,such as total bacteria,Firmicutes,Bacteroidetes,Clostridium cluster IV,and Clostridium cluster XIVa.However,on d 77(Fig.1a),EAI significantly decreased(P＜0.05)the counts of E.coli,and tended to reduce the counts of Lactobacillus(P=0.09).Meanwhile,the low-CP diet also reduced the count of E.coli(P＜0.05).On d 185(Fig.1b),pigs with low-CP diet tended　to　record　a　lowercountofLactobacillus(P=0.09).Overall,these results suggest that the effect of EAI and dietary CP levels on bacterial groups in the fecal samples was limited except for E.coli.

Fermentation metabolites in fecal contents

To understand whether EAI affect fecal fermentation profiles with different CP diets,fermentation metabolites were determined.For SCFA(Fig.1),EAI and low-CP diet did not affect fecal total SCFA,acetate,butyrate,isobutyrate,valerate,and isovalerate concentrations on d 77(Fig.1a),but the low-CP diet tended to reduce(P=0.088)propionate concentration.On d 185(Fig.1b),EAI did not change the SCFA concentrations among groups in the present study.However,the low-CP diet decreased(P＜0.05)total SCFA,acetate,　propionate,　isobutyrate,　and　isovalerate concentrations.

Ammonia was produced through amino acids deamination by bacteria.On d 77,EAI did not affect the concentration of ammonia(data not shown).The low-CP diet significantly decreased(P＜0.05)ammonia concentration in feces.On d 185,dietary treatment did not affect ammonia concentration.

The dry matter(DM;procedure 930.15;AOAC,2007),crude protein(CP;N×6.25;procedure 990.03;AOAC,2007),crude fat(procedure 2003.06;AOAC,2007),ash(procedure 942.05;AOAC,2007),contents of experimental diets and freeze-dried feces were analyzed according to the procedure of the Association of Official Analytical Chemists(AOAC)[17].The acid insoluble ash(AIA)concentrations were analyzed by the method of ISO(method no.5985;ISO,2003)[18].Total tract apparent digestibility of nutrients was calculated based on the content of AIA as a marker in feed and feces,as previously described[19].

老职工居民区，低矮破旧的老平房拆得差不多了，新建的都是五层楼的新房，类似于城里的保障房，有几栋正在建。迟恒看俩老头树荫石桌上下棋，正好一人要走，迟恒补了他的位。

Amines are formed from the decarboxylation of proteins,amino acids,and other nitrogenous compounds in the GIT.As shown in Fig.2,cadaverine and putrescine are the main amines.On d 77(Fig.2a),EAI significantly decreased(P＜0.05)the concentration of total amines,cadaverine,and increased(P＜0.05)the concentration of tryptamine.The low-CP diet significantly decreased(P＜0.05)cadaverine,tyramine,methylamine,and tryptamine concentrations.However,the concentrations ofputrescine,spermidine,and　sperminewerenot affected by dietary treatment.On d 185(Fig.2b),there was an interaction(P＜0.001)between EAI and dietary CP level for putrescine.The EAI reduced(P＜0.05)concentrations of putrescine in normal-CP diet,but increased its concentration of(P＜0.05)in low-CP diet.Additionally,EAI increased(P＜0.05)the concentrations of tryptamine and cadaverine.The low-CP diet decreased(P＜0.01)the concentrations of total amines,methylamine,tryptamine,putrescine,cadaverine,tyramine,and spermidine.

Fig.1　Effects of EAI on SCFAs concentrations in the feces of pigs with different CP levels diets.(Con-LP,;Ant-LP,;Con-NP,;Ant-NP,).a On d 77.b On d 185.EAI:early antibiotic intervention.Total SCFA,total short-chain fatty acid.The commercial creep feed with or without in-feed antibiotics(50 mg/kg olaquindox,50 mg/kg oxytetracycline calcium,and 50 mg/kg kitasamycin)was fed to pig from d 7 to d 42.Thereafter,the control and antibiotic group were further randomly assigned to provide a normal(20%,18%,14%CP from d 42 to d 77,d 77 to d 120,d 120 to d 185,respectively)or low CP diet(16%,14%,10%CP from d 42 to d 77,d 77 to d 120,d 120 to d 185,respectively),respectively.The P values indicate main effects for antibiotic(A),protein level(C)and their interaction(AC),respectively

Fig.2　Effects of EAI on amines concentrations in the feces of pigs with different CP levels diets.(Con-LP,;Ant-LP,;Con-NP,;Ant-NP,).a On d 77.b On d 185.EAI:early antibiotic intervention.The commercial creep feed with or without in-feed antibiotics(50 mg/kg olaquindox,50 mg/kg oxytetracycline calcium,and 50 mg/kg kitasamycin)was fed to pig from d 7 to d 42.Thereafter,the control and antibiotic group were further randomly assigned to provide a normal(20%,18%,14%CP from d 42 to d 77,d 77 to d 120,d 120 to d 185,respectively)or low CP diet(16,14,10%CP from d 42 to d 77,d 77 to d 120,d 120 to d 185,respectively),respectively.The P values indicate main effects for antibiotic(A),protein level(C)and their interaction(AC),respectively

At present,information on the effects of EAI on subsequent growth performance under different dietary CP levels is limited.Skinner et al.[25]reported that the use of antibiotics(therapeutic dose usage)in the nursery diets with normal-CP level appeared to reduce subsequent ADG of the pigs as compared to non-treated controls.In the present study,the antibiotics at low dosage did not have effect on subsequent ADG with normal-CP diet,though increased the subsequent ADG under low-CP diet.Thus,the antibiotic effect on subsequent growth performance may vary depending on the antibiotic dosage and the diet CP level.

在70-80年代，十队的公购粮一直保持不变。其中公粮为7415斤，这是生产队必须无偿上交的。购粮为14431斤[注]华杨大队：《广西壮族自治区农村人民公社一九七五年收益分配统计表》，高县档案馆藏，71/1/75/53。 ，价格为9.5元100斤。公购粮共需21846斤。1975年粮食总产为129727斤，由于丰收，多交了2000斤双超粮。据老农们介绍，上交的公购粮数额是按照土改时各队分得田亩的等级来计算。在上表中，公购粮每年占总粮的比分别是：24.0%，18.3%，22.0%，平均占两成左右。这对于一个山区生产队而言无疑是巨大的数额。

Discussion

Fig.3　Effects of EAI on phenolic and indole compounds concentrations in the feces of pigs with different CP levels diets.(Con-LP,;Ant-LP,;Con-NP,;Ant-NP,).a On d 77.b On d 185.EAI:early antibiotic intervention.The commercial creep feed with or without in-feed antibiotics(50 mg/kg olaquindox,50 mg/kg oxytetracycline calcium,and 50 mg/kg kitasamycin)was fed to pig from d 7 to d 42.Thereafter,the control and antibiotic group were further randomly assigned to provide a normal(20%,18%,14%CP from d 42 to d 77,d 77 to d 120,d 120 to d 185,respectively)or low CP diet(16,14,10%CP from d 42 to d 77,d 77 to d 120,d 120 to d 185,respectively),respectively.The P values indicate main effects for antibiotic(A),protein level(C)and their interaction(AC),respectively

By employing a whole growth stage continuous feeding strategy,the current experiment enabled to evaluate the short-(77 d)and long-term(185 d)effect of EAI(from d 7 to d 42)on pig blood parameters,apparent nutrient digestibility,microbial metabolism,and bacterial counts in feces with different dietary CP level.Our results suggested that the EAI had short-term effects on blood parameters and microbial fermentation products in pigs,and that these effects were influenced by dietary CP levels.

In the intestine,there are differences in microbiome between gut segments and between fecal samples and intestinal samples.Along the hindgut fermentation in the pig(from cecum to distal colon),proportionally carbohydrate fermentation decreases while proteolytic fermentation increases.However,fecal samples are easy to access and easy for practice,and also can partly,though not exactly,reflect the general microbial metabolism in the gut.Thus,fecal samples are commonly used in many researches for host metabolism in humans and animals.In the present study,we intended to reflect the general gut metabolism through fecal samples analysis and the results showed that an alteration in microbial fermentation profiles of amino acid by dietary treatment,indicating that EAI and CP level affected the gut microbial composition and function.This approach by using fecal analysis can provide a feasible way to study the effects of EAI and CP on fermentation profiles of microbial amino acid metabolism.

Effects of early antibiotic intervention on growth performance and blood parameters with different protein levels after antibiotic withdrawal

Phenolic and indolic compounds originate from the intestinal degradation of aromatic amino acids.As shown in Fig.3,there was an interaction(P＜0.05)between EAI and dietary CP level for skatole concentration on d 77(Fig.3a).The EAI significantly increased(P＜0.05)concentration of skatole in low-CP diets,but not in normal-CP diet.Furthermore,pigs with low-CP diet reduced the concentrations of p-cresol and indole(P＜0.05).There was no dietary treatment effect on phenol concentration.On d 185(Fig.3b),the EAI did not affect the concentrations of phenol,p-cresol,and skatole,but tended to reduce(P=0.08)indole concentration.The low-CP diet decreased(P＜0.05)the concentration of phenol and skatole but increased(P＜0.05)concentration of p-cresol.Collectively,these results indicated that EAI had a great impact on metabolites of protein fermentation in feces,but had little effect on carbohydrate fermentation.The low-CP diets reduced the protein fermentation products in feces on both d 77 and d 185.

(4)在数组O=[ot,r]4×r的第二行中，重新分配某可选刀具集数大于1的子批量工序的加工刀具，并按照FCFS规则生成刀具选择相邻解。

Serum biochemical measurements

ADFI:average daily feed intake;ADG:average daily gain;AIA:acid-insoluble ash;BCFA:branched-chain fatty acid;BW:body weight;CP:crude protein;DW:dry matter;EAI:early antibiotic intervention;G:F:the ratio of gain to feed;GIT:gastrointestinal bacteria;HDLC:high density lipoprotein cholesterol;LDLC:low density lipoprotein cholesterol;OM:organic matter;SCFA:short chain fatty acid

Antibiotics are usually considered the most cost-effective way to reduce pathogenic bacteria[1].Based on the microbiota analyses,we found that EAI had limited effect on the bacterial composition,only decreased the numbers of E.coli counts in the fecal samples irrespective of dietary CP levels on d 77,which suggested that EAI had limited long-term impact on fecal microbiota.Janczyk et al.[27]observed that piglets treated with amoxicillin(15 mg/kg body weight)by intramuscular administration at d 1 showed a decreased microbial diversity in colon on d 39.Collectively,these findings suggest that EAI less affected bacterial communities in feces.

EAI also affected profiles of biogenic amimes in the pig feces.The significant decrease of cadaverine and total amines concentrations suggested a decreased amino acid decarboxylation after EAI treatment.High concentration of cadaverine can exert detrimental effects on the host,such as inducing DNA damage and oxidative stress[28].Thus,the decrease of cadaverine and total amines concentration by EAI may exert a beneficial influence on the host.Coincidently,among the bacteria examined in the present study,E.coli species are a major proteinfermenting group[29].Our previous study indicated that the cadaverine concentration was positively correlated with the abundance of E.coli[12].Therefore,the decrease of cadaverine concentration by EAI may be due to the decrease in protein-fermenting bacteria counts(E.coli)and the subsequent reduction of the catabolism of precursor amino acid(lysine).EAI increased putrescine concentration in low-CP diet but decreased it in normal-CP diet.This results indicates that the effect of EAI on subsequent concentrations of amines may vary with CP level.Furthermore,we found that cadaverine concentration varied between d 77 and d 185.Cadaverine is derived from decarboxylation of lysine[30].In the current study,the CP level and lysine concentration in the diet was much higher on d 77 than 185 d,which may lead to the variation in cadaverine concentration across ages.

On the other hand,EAI also changed aromatic amino acid metabolism by the microbes in the GIT.Tryptophan,one of the aromatic amino acid,can be decarboxylated into tryptamine by GIT microbes[30,31].In the present study,EAI increased tryptamine concentration in the feces irrespective dietary CP levels on d 77 and d 185,suggesting that EAI had a continuous effect on aromatic amino acid metabolism.Tryptamine can stimulate the secretion of serotonin by enterochromaffin cells,and then regulate intestinal motility[32].Thus,an increase of tryptamine may affect the function of intestine.Additionally,EAI also increased skatole concentration on d 77,especially in low-CP diets.Skatole is produced from tryptophan catabolism by GIT bacteria[30].The increase of skatole concentration indicates that EAI also affected tryptophan catabolism in the gut.Collectively,combined with serum parameters and microbial fermentation profiles,the effects of EAI persisted to short-term after withdrawal,but weakened at finishing period of the pigs.

Low-CP diets affected host metabolism and fermentation profiles of microbial amino acid metabolism

In our study,low-CP dietary treatment affected the urea metabolism as reflected by the decreased urea concentration in serum both on d 77 and d 185,which has also been found in other reports[33,34].Urea is the main nitrogenous end product of amino acids catabolism in pigs.A decrease in serum urea level is indicative of a more efficient use of dietary nitrogen[35].Thus,our study may indicate that low-CP diet affected nitrogen metabolism in pigs.

Corresponding to the decrease in urea concentration in serum,low-CP diet markedly affected the microbial metabolism of amino acids in the intestine,as shown by the decrease in concentration of most amines,suggesting decreased microbial decarboxylation.Furthermore,low-CP diet decreased some phenolic and indole compounds concentrations.Indole and skatole are products of tryptophan metabolism originating from dietary and endogenous protein[31].Although the concentration of tryptophan in the normal-CP diet is equal to that of the low-CP diet in the current study,crystalline amino acid is quickly absorbed before reaching the hindgut,and then reduced the availability of substrates for microbial fermentation in the hindgut.Overall,these findings suggest　that　low-CP　diets　significantly　reduced　the microbial fermentation of protein or amino acids and the concentration of potentially harmful metabolites derived from microbial metabolism of feed-derived amino acids.Thus,the dietary nitrogen was used more efficiently with low level of potentially harmful metabolites in pigs fed low-CP amino acids-supplemented diets.

企业的网络品牌维护，是指企业针对外部环境的变化，在网络上开展的维护品牌形象、保持品牌市场地位和品牌价值的一系列活动。企业网络品牌维护有助于预防品牌危机，巩固网络品牌的市场地位。企业网络品牌维护的有效方法是建立舆情监控机制，网络舆情具有传播速度快、不可控制性等特点，企业可通过网络舆情监测机制的建设，强化舆论引导的能力，遇到对企业品牌不利的负面信息能够快速反应并开展相应的危机处理活动，消除品牌危机。

Low-CP diets altered the markers of microbial carbohydrate metabolism on d 185,such as decreased the concentrations of acetate,propionate,isobutyrate,isovalerate,and total SCFA,consistent with Le et al.[36],who observed that reducing dietary CP level from 15%to 12%led to a decrease in total SCFA,acetate,and propionate concentrations for finishing pigs.The concentration of SCFA produced mainly depends on the amount and composition of substrate and the type of microbes present in the GIT[37].Thus,in the present study,a decrease of SCFAs may be related with the altered metabolism activity of gut microbiota after low-CP diet.

Conclusions

In conclusion,combined with the changes in blood metabolites,digestibility,and microbial metabolite,our results showed that EAI(from d 7 to d 42)showed short-term effects on pigs,especially on the body and microbial metabolism.The effects of EAI varied between CP levels,which was characterized by the significant alteration of glucose and putrescine concentration.The low-CP diet significantly decreased the amino acids fermentation products in feces.

Additional file

Additional file 1:Table S1.Ingredients and chemical composition of creep feed diets.Figure S1.Effects of early antibiotic intervention on bacterial abundance in the feces of pigs with different CP levels diets.(Con-LP,White;Ant-LP,Light gray;Con-NP,Dark gray;Ant-NP,Black).A:On d 77.B:On d 185.The commercial creep feed with or without in-feed antibiotics(50 mg/kg olaquindox,50 mg/kg oxytetracycline calcium,and 50 mg/kg kitasamycin)was fed to pig from d 7 to d 42.Thereafter,the control and antibiotic group were further randomly assigned to provide a normal(20%,18%,14%CP from d 42 to d 77,d 77 to d 120,d 120 to d 185,respectively)or low CP diet(16%,14%,10%CP from d 42 to d 77,d 77 to d 120,d 120 to d 185,respectively),respectively.The P values indicate main effects for antibiotic(A),protein level(C)and their interaction(AC),respectively.(DOC 576 kb)

Abbreviations

Effects of early antibiotic intervention on fermentation profiles of microbial amino acid metabolism with different protein levels after antibiotic withdrawal

Acknowledgements

The authors sincerely thanks to Professor Sung Woo Kim of North Carolina State University for his critical discussion and reading during manuscript preparation.

Funding

This work was supported by National Key Basic Research Program of China(2013CB127300)and Natural Science Foundation of China(31430082).The funders had no role in the design of the study,collection,analysis,and interpretation of data and in writing of the manuscript.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article.

Authors’contributions

The author’contributions are as follows:WYZ and YS were in charge of the whole trial;MY and WYZ wrote the manuscript;CLM and YXY for animal feeding and care;KFY,MY and CJZ assisted with sampling and laboratory analyses.All authors read and approved the final manuscript.

Ethics approval

比如一天中午，我本来兴高采烈的，可妈妈说：“方舟，今天我发了八十八块钱，和你考的一样。”说完，又装作天真无邪的样子捂住嘴，好像说漏了嘴一样。我警惕地看了爸爸一眼，爸爸正在做饭，不知他听见没有。

The experimental proposals and procedures for the care and treatment of the pigs were approved by the Animal Care and Use Committee of Nanjing Agricultural University,in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

科学技术与意识形态的关系很早就进入到了西方学者的视野当中，不同的学者依据不同的理论对两者的关系进行了论述，大致产生了两种不同的学说理论——科学技术与意识形态对立和科学技术即是意识形态。正是这两种学说理论的对立、碰撞，为哈贝马斯的科学技术意识形态论奠定了丰富的理论基础。

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