Background

Over the past 50 yr the intensification(improved housing,husbandry and nutrition)of the broiler industry and concurrent commercial genetic selection for growth,feed efficiency and yield has resulted broiler growth increases in excess of 400%[1],with broilers having the capacity to reach 2 kg of live weight within 35 d post hatch[2,3].At least 85%of production improvements has been attributed to genetic selection with meat production efficiency continually increasing by 2-3%per year through selective breeding programs alone[1,4].

能留住食客的味觉记忆，以皮薄、馅丰、汁多、味鲜、形美著称的南翔小笼馒头，在食材与制作技艺方面都很有讲究。小笼皮坯采用多种面粉调和制作而成，每两面粉制作10个小笼包，可见其皮之薄；馅则选用精腿肉，保持肉质之原味，用骨汤熬煮肉皮成冻，拌入馅内，取其鲜美、多汁不油腻的特点。技艺上，采用双杆擀皮，皮子中间厚、四周薄，保证汤水不会流出来，最终小笼馒头呈现宝塔型；每只小笼馒头的收口处打16个以上的褶，小巧精致、玲珑剔透。

Selection for feed efficiency is largely measured as feed conversion ratio(FCR),the amount of feed intake(FI)per unit bodyweight gain.In poultry systems,feed accounts for approximately 70%of total production costs[5].Selection for efficiency has resulted in an FCR decrease of over 50%over the past 5 decades,maintaining poultry as a cost efficient source of protein[1].Despite continued improvements,there still remains significant(＞10%)variation in performance traits,including feed efficiency,bodyweight and growth rate within broiler strains[6].This performance variation can result in an economic cost to both the producer and industry[7].For example,variation in live weight is problematic for modern automated processing plants which reject carcasses out of a relatively narrow weight range,thus requiring further handling and sorting,and hence can incur economic loss to the processor[7].

Maintenance of innate immunity and intestinal barrier function is one parameter thought to be nutritionally costly to the host,in which exasperated or diminished immune responses could lead to increased performance variation[8].Our previous study(Willson N-L,Nattrass GS,Hughes RJ,Forder REA,and Hynd PI,unpublished)compared high and low performing broilers to determine whether or not innate immune function could be consistently linked to the phenotypic expression of FCR.A candidate gene approach was used to determine whether functional changes in innate immune parameters could be consistently associated with high or low FCR,the results of which,there was no association.Variable expression in the pathogen recognition receptor Toll-like receptor 2(TLR2)and membrane protein CD36 also known as FAT/CD36,was however of interest,as both of which have been linked to each other and various roles in fatty acid metabolism.Lee and Hwang[9]have reported on links between fatty acids and TLR activation,with saturated fatty acids activating TLR2 and TLR4 signalling pathways and unsaturated fatty acids having an inhibitory effect on TLR-mediated signalling pathways and gene expression.TLR2 is known to form complexes in lipid rafts with CD36,[10],and CD36 has been described in facilitating TLR2 signalling,although the mechanism remains somewhat unclear[11].Furthermore CD36,is thought to promote the synthesis of triglycerides in adipocytes,the clearance of chylomicrons from plasma,as well as mediate lipid metabolism and fatty acid transport[12,13].Additionally,studies in broilers have found that CD36 has a novel role in the visceral fat deposition of male broilers,and indicated that avian fat deposition has spatial and sex specific differences[14].

将A、B、C 3组薄片样本放置于IEC/TS 61956推荐的杯状试验装置，杯状实验装置中装有质量分数为20%的氯化钠溶液。将杯状实验装置放置于恒温箱中，将恒温箱的温度设置为0 ℃。将杯状实验装置下电极接地，上电极施加高频高压(有效值7.5 kV，频率400 Hz)，在0 ℃恒温条件下进行加速水树老化[11-12]，如图2所示。A、B、C 3组薄片样本的老化时间分别为14 d、21 d、28 d。

Fat deposition in broilers has been an unfavourable consequence of selection for growth,particularly up until the 1970s,however there has been reductions in body fat content from 26.9%in the 1970s to 15.3%in commercial breeds in the past decade(see Tallentire et al.,[15]for review).Fat deposition is negatively linked to FCR,with observations that heavier chickens usually have a higher FCR and deposit a higher amount of fat[16].The major site for fat deposition in broilers is the abdominal fat pad,which is highly correlated to total carcass fat[16,17].Fat has been demonstrated to account for 15-18%of the total broiler bodyweight and is considered the most variable body component,with a coefficient of variation for the total body fat content between 15 and 20%,and higher again for abdominal fat,varying between 25 and 30%[18-21].Excess fat accumulation and the variation may be considered the net balance of dietary absorbed fat,the rate of fat synthesis(primarily hepatic lipogenesis),and fat catabolism[22].As obesity is correlated with chronic low grade inflammation in humans[23],and that exasperated or diminished immune responses can result in inflammation potentially leading to decreased growth performance of the host,including chickens[24],it was hypothesised that interactions between fatty acid metabolism and innate immunity may be associated with performance variations commonly seen within commercial broiler flocks.

To investigate whether innate immunity and fatty acid metabolism　are　contributing　to　flock　performance variation,we compared broiler and layer chicken strains that have been intensively selected for different traits;high carcass yield and growth efficiency for broilers,commercial egg production and egg efficiency for layers[25].This selection over the years has seen the two strains diverge for these traits,with the bodyweight of broilers being five times that of layers by 6 wk of age[26].The aim of the current experiment was to utilise broilers,layers,and a layer×broiler F1 cross to identify how genetic selection has influenced carcass lipid composition,key genes involved in fatty acid metabolism and select innate immune parameters to enable a better understanding of the biological factors underpinning feed efficiency,growth and performance variation.

Methods

All animal procedures were approved by the University of Adelaide Animal Ethics Committee(approval#S-2015-171)and the PIRSA Animal Ethics Committee(approval#24/15).

Birds and management

众所周知，学生到了高年级阶段，对语言的学习开始进入提高阶段，有了质的变化，不应局限于语言的表面，而应该接触语言的社会文化（语用修辞）的深层。文学语言既是“文化的载体”，也是传播文化的工具。作为教学目的通过对语言本身（语言结构——语音、语符、语义、语用）的分析，既可以明确作品的特色风格，也可以透视出语词、语句中所蕴含的丰富的社会文化意蕴，从而提高学生对语言的理解，提高运用语言的灵活度，全面提高学生的语言素质。

Conclusion

All birds were fed ad libitum(standard commercially available broiler starter diet,no in-feed antimicrobials or coccidiostats added),and had unrestricted access to water via nipple drinker lines.The three experimental groups were selected for their growth potential:Fast growing(broilers;n=50)moderate growing(F1 layer×broiler;n=50)and slow growing(layer strain;n=50).Bodyweight was recorded weekly.On d 0,-7,-14 and-28 post hatch,36 birds(n=12 birds/breed)were randomly selected and euthanised by cervical dislocation for subsequent sampling.

Total carcass lipid and total blood lipid composition

Eviscerated carcasses(fat pads left intact on carcass)were weighed and immediately frozen at-20°C.Whole carcasses were submerged into liquid nitrogen for 3 min,shattered with a mallet in zip lock bags to contain all fragments,and homogenised in a 1700 W blender.Sub samples of homogenate were aliquoted(10 mL)and stored at-20°C for analysis of total carcass lipid%and lipid composition.Total lipids were extracted at the Waite Lipid Analysis Service(WLAS),Waite Campus SA,using the methods of Folch[27].Fatty acid composition of tissues was determined and quantified using a Hewlett-Packard 6890 GC(CA,USA)equipped with flameionization　detection　and　acapillarycolumn(50 m×0.32 mm internal diameter)coated with 70%cyanopropyl polysilphenylene-siloxane with a film thickness of 0.25 μm(BPX-70,SGE,Victoria,Australia).Fatty acid transmethylation for fatty acid methyl ester(FAME)extraction,and gas chromatography analysis of FAME were run by the methods of Folch[28].Fatty acid peaks were identified by comparing the retention time of each peak against the retention times of a fatty acid standard of known composition.Each peak from a trace was expressed as the relative percentage of the total FAME in the sample.The detection limit of each fatty acid was 0.05%of total fatty acids.

Total blood fatty acids were measuring using the PUFA-coat dried blood spot(DBS)card,developed by the Waite Lipid Analysis Service(WLAS),Waite Campus SA.Samples were prepared by placing a drop of blood on PUFAcoat DBS card and dried at room temperature for 5 h.See Liu,Mühlhäusler[29]for full methods and validation of the PUFAcoat DBS card.In brief,lipids were extracted using a modified Folch method and FAME were extracted　into　heptaneforgaschromatography.A Hewlett-Packard 6890 GC(CA,USA)equipped with a BPX70 capillary column 50 m×0.32 mm,film thickness 0.25 μm(SGC Pty Ltd.,Victoria,Australia),programmed temperature vaporisation injector and a flame ionisation detector(FID)was used.The identification and quantification of FAME were achieved by comparing the retention times and peak area values of unknown samples to those of commercial lipid standards(Nu-Chek Prep Inc.,Elysian,MN,USA)using the Hewlett-Packard Chemstation data system.

RNA extraction,library preparation and sequencing

Blood was collected by cardiac puncture immediately following cervical dislocation.Blood smears were made by placing 1 drop of whole blood on the end of a Starfrost frosted slide(ProSci Tech).Slides were air-dried and fixed in 100%methanol for 1 min,feather side down.Slides were stained with Geisma-Wright stain on a Hema-Tek 2000.A total of 100 cells(Cell types;lymphocytes,heterophils,eosinophils,basophils and monocytes)were counted at a 40×magnification.Subsequent heterophil:lymphocyte ratios were determined.

Heterophil:Lymphocyte ratios

In total,18 liver samples from d 14 post hatch(broiler n=6,cross n=6,layer n=6)birds were randomly selected for RNA-sequencing.Total RNA was isolated using　an　RNeasyPlusMiniKit(Qiagen,Hilden,Germany).Approximately 80 mg of frozen(-80°C)liver tissue was homogenised in 2 mL of Trizol reagent(Invitrogen,Carlsbad,CA).Aliquots of the Trizol homogenate(1 mL)were combined with chloroform(200 μL)and centrifuged for 15 mins at 4°C.The upper aqueous phase(350 μL)was transferred to a gDNA eliminator spin column and centrifuged at＞8000× g(14,000 rpm)for 30 s.The flow through(300 μL)was collected and combined with 70%ethanol(300 μL)for transfer onto RNeasy columns.The remaining collection and wash steps were performed to the manufacturer’s specifications.RNA was eluted in 200 μL of RNA-free water.Purity and concentration was determined using UV spectrophotometry(Nanodrop 1000;Thermo Scienfic,Wilmington,DE).

Bodyweights were recorded for a 28-day grow out period(Table 2).Starting bodyweights(mean±SEM)at hatch(d 0)were significantly different between broiler n=56(44.4±0.4 g);cross n=57(42.5±.04 g;P=0.008)and layer line n=54(38.5±0.4 g;P＜0.001)males.Bodyweights remained significantly different between all three groups of birds for the remainder of the grow-out period(P＜0.001).

RNA sequence(RNA-seq)analysis

本文参考刘生龙等(2016)[12]的做法，选取50%以上高收入、50%以下低收入分位点作为分析依据。图2描述了扩招政策对城乡居民在不同收入分位点的影响。

Reads were returned in fastq format.Low-quality base calls were trimmed from the 3′end of reads with FastQC and adaptor sequences were trimmed from the 3′end of reads with Cutadapt.Hisat2[30]was used to map reads to the reference chicken genome Galgal5.0(ftp://ftp.ncbi.nlm.nih.gov/genomes/Gallus_gallus).　Duplicate reads were then removed.Stringtie[30]was used to define the transcripts from the read mappings for each sample,and to merge the transcript definitions for all samples.Transcripts were cleaned up using in-house scripts.The number of raw read counts were calculated for each transcript and sample using the function featureCounts of the R package Rsubread[31].Another R package,edgeR[32]was used to analyse differential gene expression using normalised counts per million transcripts(CPM)to correct for varying depth of sequence among samples.Differential expression of genes was considered significant at P＜0.05,and a false discovery rate of＜0.05,with any fold change considered.Transcript data were aggregated by gene.Genes where the maximum CPM was＜1 were removed.Twenty two candidate genes primarily involved in fatty acid metabolism were selected from the RNA-seq analysis for inclusion in this study(Table 1).

Statistical analysis

Data were analysed by one-way ANOVA in SPSS(IBM SPSS Statistics 22).Any data not normally distributed were logged(Log10)to normalise and analysed by one-way ANOVA.Statistical significance was accepted at P＜0.05 level after which Post Hoc tests were performed using TukeysHSDto differentiate between the three groups of birds at each sampling time point.

Results

Bodyweight data

RNA-Seq wascarriedout bythe ACRF Cancer Genomics Facility,Adelaide,SA.The sample quality was analysed on an Agilent Bio-analyser(minimum RIN requirement of 7)and sequencing libraries were made using 2 μL of total RNA.PolyA mRNA isolation was performed using oligo dT beads.Libraries were prepared using KAPA Library Quantification Kits for Ilumina platforms　(KAPABiosystems,Massachusetts,USA).2×100 nt sequencing was carried out on an Illumin HiSeq 2500 Sequencing System to generate a minimum depth of 25 million reads.

（三）地产行业杠杆率只升不降，与去杠杆政策背道而驰。上市公司2017年年报显示，A股房地产行业平均资产负债率已达到99%，即使剔除预收账款也接近75%，而且永续债、基金、信托等工具的广泛使用，使地产公司的真实负债率可能比表面上高许多。港股房地产公司的负债率多在85%-90%。房地产行业的高杠杆率虽与行业自身的特点有关，但也与当前国内去杠杆、防范金融风险等政策取向相背离。

Table 1　Candidate genes involved with fatty acid metabolism and select parameters of innate immunity

aNCBI accession number

Gene name　RNA target　Accession no.a ACACA　Acetyl-CoA Carboxylase　NM_205505.1 ACADL　Acyl-CoA dehydrogenase　NM_001006511.2 ACLY　ATP-Citrate-lyase　NM_001030540.1 ACSL1　Acyl-CoA synthetase　NM_001012578.1 APOA1　Apolipoprotein A1　NM_205525.4 APOC3　Apolipoprotein cIII　NM_001302127.1 CD36　FATCD36　NM_001030731.1 CPT1A　Carnitine palmitoyltransferase 1　NM_001012898.1 CPT2　Carnitine palmitoyltransferase 12　NM_001031287.2 FABP1　fatty acid binding protein 1　NM_204192.3 FADS6　　Δ6 desaturase　XM_426241.5 FASN　Fatty Acid Synthase　NM_205155.2 LPL　Lipoprotein Lipase　NM_205282.1 MDH1　Malate dehydrogenase　NM_001006395.2 ME1　Malic Enzyme 1　NM_204303.1 PPARA　peroxisome proliferator-activated receptor alpha NM_001001464.1 RXRA　Retinoic X receptor-α　XM_003642291.3 SCD　Stearoyl-CoA desaturase　NM_204890.1 TLR2A　Toll-Like Receptor 2　NM_001161650 TLR4　Toll-Like Receptor-4　NM_001030693 XBP1　X-box binding protein　NM_001006192 ERN1　Inositol-requiring kinase 1　NM_001285501.1

Organ weights

高职学校的优势在于校企对接，学校培养出的人才会直接输送到对应的企业中去，因此，高职学校的教育就必须要重视学生的实践能力。为了符合实践需求，高职学校的教材编写就必须要重视实践。传统的高职教材往往都是高深理论的讲解，对于实践操作过程中所需要的操作技巧以及需要重点注意的关键点并没有提及，因此，现代高职教材改革除了高深理论讲解外，还要告诉学生实际操作过程中的重点和难点。为了达到这个目的，在高职教材编写时要邀请高级技工和企业管理人员共同参与，以此确保高职教材培养出的人才能够符合社会需求。

Organ weights were expressed as a percentage of total bodyweight to account for growth differences between broilers,layers and the F1 cross(Fig.1).At d 0 and d 7 the layers had significantly lower relative liver weight percentages than the broiler and cross males(P=0.006 and P＜0.001 respectively).Liver weight as a percentageof bodyweight peaked at d 14 in the broilers,which were significantly different from both the cross and layer birds(P＜0.001;Fig.1a),whereas the cross and layer birds reached peak relative liver weights at d 7 post hatch.By d 28 post hatch there were no differences in relative liver weight(～2.9%of total bodyweight)between the three groups of birds(P=0.852).

Table 2　Weekly bodyweights(grams)for broiler,cross,and layer line males for d 7,-14,-21 and-28 post hatch

a-cMeans(±SEM)within the same column with different superscripts are significantly different(P＜0.05)

d 0　d 7　d 14　d 21　d 28 Broiler　44.4±0.4a　195±2a　560±8a　1153±22a　2102±35a Cross　42.5±0.4b　137±3b　311±8b　603±12b　1037±31b Layer　38.5±0.4c　84±1c　159±2c　261±3.82c　403±6c

Fig.1　Organ weights presented as a percentage of total bodyweight(±SEM)for broiler,cross and layer line males at d 0,d 7,d 14 and d 28 post hatch for:a)Liver,b)Heart,c)Spleen and d)Bursa.a-c Differing scripts within each time point are significantly different(P＜0.05)

The heart accounted for 0.85-1.08%of total bodyweight at both d 0 and d 7 with no significant differences(P=0.202 and P=0.611)between broiler,cross and layers birds at each time point respectively(Fig.1b).The relative weight of the layer’s hearts remained constant for the 28 d growth period,representing～1%of total bodyweight.The broilers had significantly lower relative heart weights than the layer and cross birds at d 14 and d 28 post hatch(P＜0.001).

Relative spleen weights were not different between any of the three groups at d 0(P=0.233;Fig 1c).Layers had significantly heavier relative spleen weights than broilers from d 7 post hatch onwards(P=0.004).The cross and layer spleen weights continued to increase in relative weight over the 28 d period,whereas the broilers reached their maximum relative spleen weight by d 14 post hatch.By d 28 post hatch broiler spleens accounted for 0.07%of total body weight whereas layer spleens accounted for 0.17%of total bodyweight(P＜0.001).

No significant differences were found in relative bursa weight between broilers,layers and cross birds at d 0(P=0.997;Fig.1d).Relative bursa weights peaked in broilers at d 14 post hatch,exhibiting a 0.04%increase from d 0-d 14(0.12%-0.16%)then reducing slightly by d 28 to 0.14%of total bodyweight.Relative weights of the bursa increased in layer and cross birds at all sample time points.The increases were most pronounced in the layer birds with the bursa significantly different from both the crossed and layer birds at both d 14(P＜0.001)and d 28(P＜0.001).At d 28 post hatch the bursa weights were 0.14%and 0.67%of total bodyweight for broilers and layers respectively.

Total carcass and total blood lipids

Total carcass fat(%)and subsequent fatty acid composition was evaluated on eviscerated homogenised carcasses and blood samples at d 14 post hatch only.Broilers(n=12)had significantly higher total carcass fat percentage(11.3%)than　the　cross(n　=　6,8.9%;P=0.017)and layer line males(n=12,7.7%;P=0.002;Fig.2).The cross and layer total body fat percentages were not significantly different(P=0.523).

The fatty acid composition of the carcasses varied indicating differential fatty acid metabolism(Table 3).The layers had higher levels of total saturated fatty acids(SFAs),followed by broilers,and then the cross,all significantly different(P=0.001).The broilers had higher levels of palmitic acid(C16),whereas the layers had higher levels of stearic acid(C18),indicating increased elongation of SFAs in the layers.The same SFA pattern was seen in the blood(Table 4).Total carcass monounsaturated fatty acids(MUFAs)were higher in the broilers and cross relative to the layers(P＜0.001),indicating increased elongation of MUFAs in the broilers and cross,this pattern also reflected in the blood.The cross and layers had significantly higher carcass percentages of polyunsaturated fatty acids(PUFAs),both omega-3 and omega-6.This was reflective both the n-6:n-3 ratio as well as the PUFA:SFA ratios between the three groups of birds.The composition of the serum and the composition of the carcass was generally the same for broilers,layers and the F1 crosses.

Fig.2　Mean±SEM Total carcass fat%for eviscerated homogenised carcasses for broilers(n=12),cross(n=6)and layer line(n=12)males at d 14 post hatch.a-b Differing scripts are statistically different(P＜0.05)

Heterophil:lymphocyte ratios

我国在一段时期中的经济发展理念是先发展，之后再进行治理，这种模式虽然能够取得一定的经济效益，但是这也在一定程度上对生态造成了破坏。而随着生态环境的进一步恶化、水土流失和土地荒漠化的问题更加突出。因此，应该采取一定的措施进行营林护林工程的建设。在十八大的会议上，我国提出了“五位一体”建设规划，其中比较重要的内容是绿色发展的理念，这个理念的提出充分体现了我国进行生态文明建设的决心和改善生态环境建设的目标。对林地进行管理的强化不仅能够提升林场的经济效益，同时还能在一定程度上实现林业规模的扩大，为实现生态文明和经济建设提供坚实的基础。

The relative weight of both the spleen and bursa continued to increase in the cross and layer birds from d 0 until d 28 post hatch.The broilers reached maximum relative spleen and bursa weights at d 14 and then decreased from there on in.There has been conflicting interpretation as to whether relative increased immune organ size equates to a better immune defence system.Once such study found that the size of the spleen for example was correlated with changes in body condition,and that size was elevated in individual birds in prime body condition[37].It could be argued that all of our birds were in good body condition,as there was no disease,parasite infection or mortality.Body condition as measure of fatness v leanness however,as used by Møller et al.,[37],would assume the layers and the F1 cross were in better relative condition than the broilers,and potentially reflective of the smaller immune organs.Additionally broilers have repeatedly been shown to be less responsive to immune challenges experimentally,and this has been attributed to a negative consequence of genetic selection[24].Although relative decreased lymphoid organ weights(%of bodyweight)were observed in the broilers compared to the cross and layers,there was no evidence to suggest that the broilers were compromised immunologically due to increases in fat deposition in an unchallenged experimental setting.Heterophil:lymphocyte ratios were not significantly different between any of the birds although there was a high level of variation between the individuals.The cross did appear to have a lower ratio,however this is more likely attributed to a lower number of samples and the high variation in individual birds than a significant trend.

1.2.2 样本量估算 通过查阅文献，样本量为自变量的10倍[5]，考虑到20%的失访率和抽样误差，样本量最终确定为160例。

The 22 candidate genes selected(Table 5)revealed that broilers(n=6)in comparison to layers(n=6)had significant hepatic upregulation of genes involved in lipid transport;APOA1(P=0.019),APOC3(P=0.003),lipogenesis;ACACA(P=0.001),ME1(P=0.022),FASN(P＜0.001),GPAM(P=0.001),MDH1(P＜0.001),SCD(P＜0.001),fatty acid transport;FABP1(P=0.001),ACLY(P＜0.001),and fatty acid oxidation;ACADL(P=0.003),CPT-2(P＜0.001),(Fig.4).An exception was the downregulation of FADS6(P=0.054)in broilers,a rate-limitingenzyme involved in the elongation of PUFAs.Broilers when compared to the cross(n=6)birds exhibited generalised upregulation of fatty acid metabolism,although not as pronounced as seen between broilers and layers.Significant hepatic upregulation for lipid transport;APOC3(P=0.029),lipogenesis;GPAM(P=0.035),MDH1(P＜0.001),fatty acid transport;FABP1(P=0.008),ACLY(P＜0.001),and fatty acid oxidation;ACADL(P=0.015),CPT-2(P=0.019)were observed for broilers.Layers and cross comparisons indicated no real differential expression in fatty acid metabolism between the groups,with the exception of down regulation of lipogenic gene SCD1(P=0.003)and fatty acid oxidation CPT-2(P＜0.001)and ACAA1(P＜0.001)genes.Layers in comparison to the cross also had upregulated expression of the transcription factor PPARA(P=0.047),a difference not seen elsewhere.

三要建立保障机制。努力改进基层警务工作条件，尤其要解决农村辖区大、人口多、警力不足、装备落后、办案经费紧张等问题。建立保障机制可以在一定程度解决基层警务工作者工作中出现的差别对待问题。正所谓“高薪养廉”提高基层警务工作者的福利待遇可以提高其在面对诱惑时的抵抗力，同时和监督机制一起可以有效提高其违规工作的风险，以此防止基层警务工作者不作为和乱作为的问题。

Table 3　Fatty acid composition(%of total identified fatty acids)in homogenised carcass samples for broiler(n=12),cross(n=6)and layer line males(n=12)fed the same commercial broiler diet formulation at d 14 post hatch

1Data are expressed as the percentage of identified fatty acids±Standard error of means(SEM) a-cMeans within the same row for each parameter with different superscripts are significantly different(P＜0.05)

Fatty acid　Broiler(n=12)　Cross(n=6)　Layer(n=12)　P-value Eviscerated carcass Total Carcass Fat%　11.3a　8.90b　7.56b　　＜0.001 Total SFA　37.7±0.3b　36.8±0.2c　38.6±0.2a　0.001 Palmitic acid C16　27.7±0.24a　25.9±0.19b　25.3±.25b　　＜0.001 Stearic acid C18　7.8±0.12c　8.4±0.15b　10.0±0.18a　　＜0.001 TFA　0.8±0.03b　0.9±0.05ab　1.0±0.06a　0.038 Total MUFA　49.5±0.27a　48.7±0.34a　44.0±0.41b　　＜0.001 Palmitoleic acid(C161n-7)　7.8±0.17a　6.2±0.27b　4.8±0.19c　　＜0.001 Oleic acid(C181n-9)　38.6±.27a　38.9±0.27a　35.8±0.19b　　＜0.001 Vaccenic acid(C181n-7)　2.7±0.07b　3.1±0.09a　3.0±.0.06a　0.003 Total PUFAn-3　1.5±0.01b　1.6±0.02b　1.9±0.05a　　＜0.001 α-Linolenic acid(C183n-3)　1.1±0.01　1.1±0.00　1.1±0.01　0.684 Eicosapentanoic acid(C225n-3)　0.1±0.0　0.1±0.0　0.1±0.0　-Docosahexanoic acid(C226n-3)　0.2±0.01c　0.3±0.02b　0.6±0.02a　　＜0.001 Total PUFAn-6　10.4±0.12c　12.0±.017b　14.5±0.32a　　＜0.001 Linoleic acid(C182n-6)　9.8±0.12c　11.0±0.13b　12.8±0.24a　　＜0.001 Arachidonic acid(C204n-6)　0.3±0.02c　0.6±0.03b　1.1±0.07a　　＜0.001 n-6:n-3 ratio　6.88c　7.42b　7.68a　　＜0.001(MUFA+PUFA):SFA　1.61ab　1.68a　1.57b　0.004 PUFA:SFA　0.31b　0.40a　0.43a　　＜0.001

Table 4　Fatty acid composition(%of total identified fatty acids)in PUFAcoat DBS blood spot samples for broiler,cross and layer line males fed the same commercial broiler diet formulation at d 14 post hatch

1Data are expressed as the percentage of identified fatty acids±Standard error of means(SEM) a-cMeans within the same row for each parameter with different superscripts are significantly different(P＜0.05)

Fatty acid　Broiler(n=12)　Cross(n=6)　Layer(n=10)　P-value Total SFA　43.7±0.7　43.05±0.3　46.0±1.2　0.107 Palmitic acid C16　24.±0.52　22.5±0.18　23.9±1.78　0.424 Stearic acid C18　14.9±0.39b　16.16±0.21ab　17.07±0.51a　0.004 TFA　0.85±0.03b　0.93±0.05b　1.1±0.06a　0.004 Total MUFA　33.55±0.33a　28.55±0.65b　23.63±0.60c　　＜0.001 Palmitoleic acid(C161n-7)　4.19±0.17a　2.55±0.07b　1.69±0.13c　　＜0.001 Oleic acid(C181n-9)　26.53±.25a　23.08±0.65b　19.36±0.50c　　＜0.001 Vaccenic acid(C181n-7)　1.96±0.05ab　2.11±0.06a　1.78±0.10b　0.036 Total PUFAn-3　2.84±0.13b　3.58±0.18a　3.66±0.25a　0.007 α-Linolenic(C18n-3)　0.69±0.02　0.71±0.03　0.63±0.03　0.189 Eicosapentanoic(C225n-3)　0.133±0.01　0.35±0.04　0.31±0.03　0.628 Docosahexanoic(C226n-3)　1.59±0.09b　2.2±0.14a　2.4±0.18a　0.001 Total PUFAn-6　19.06±0.43v　23.86±.059b　25.62±1.1a　　＜0.001 Linoleic(C182n-6)　16.38±0.36b　19.45±0.33a　19.72±0.72a　　＜0.001 Arachidonic(C204n-6)　1.26±0.05c　2.6±0.23b　4.06±0.35a　　＜0.001 n-6:n-3 ratio　6.82　6.68　7.14　0.418(MUFA+PUFA):SFA　1.27　1.30　1.18　0.071 PUFA:SFA　0.51b　0.64a　0.65a　0.002

Fig.3　Heterophil:Lymphocyte(H:L)ratios(±SD)for broilers(n=6),Cross(n=6)and layer line males(n=6)

Endoplasmic reticulum(ER)stress-related gene ERN1 was not differentially expressed between any of the three groups(P=0.67).XBP1 was found to be significantly upregulated in layers in comparison to both broilers(P=0.002)and crossed birds(P=0.007).Toll-like receptors TLR2 and TLR4 were not found to be differentially　expressed　between　any　ofthe　three　groups(P=0.951).

Pearson’s two-tailed correlations with individual bird bodyweights(Table 5),revealed 15 of the 22 genes were highly correlated with bodyweight at P＜0.01,2 genes correlated at P＜0.05 and 6 of the genes non-significant with bodyweight.The highest correlation was between malate dehydrogenase(MDH1)and bodyweight(r=　0.902;P＜0.001).

Discussion

The aim of this experiment was to elucidate how genetic selection has influenced carcass composition,fatty acid metabolism and select innate immune parameters.The objective was to further develop the understanding of factors which may be underpinning performance variation in modern broilers.Our previous experimental work did not provide sufficient phenotypic variation in feed conversion ratio within flock,thus it was decided toinvestigate birds with grossly different growth potentials;namely,broilers,layers and a layer×broiler F1 cross.Although samples were taken at multiple time points,d 14 was selected as the primary sampling date due to the rapid growth acceleration seen in broilers from 2 to 3 wk of age.By sampling at this time point it was hoped to capture physiological changes at the beginning of the growth acceleration to further understand broiler growth rates.

Table 5　Pearson correlation coefficient(r)of target gene against individual bodyweight(BW),and mean expression levels(CPM)of genes between broilers(n=6),cross(n=6)and layers(n=6)

1Pearson’s correlation coefficient of target gene against individual bodyweight of all three groups of birds(BW);*Sig at P ＜ 0.05,**Sig at P ＜ 0.01 2Relative direction of regulation:↑Broiler upregulated(broiler＞ cross＞ layer);↓Broiler downregulated(broiler＜ cross＜ layer) a-cMeans(± SEM)within the same row for each parameter with different superscripts are significantly different(P ＜ 0.05).Means values are counts per million(CPM)transcripts,to correct for varying sequence depth between individual samples

Gene name　r(Gene vs.BW)1　Broiler(n=6)　Cross(n=6)　Layer(n=6)　Regulation2 ACACA　0.695**　3135.4±118.6a　2918.5±101.4a　2367.5±135.4b　　↑ACADL　0.734**　751.3±27.8a　648.1±15.3b　620.9±23.4b　　↑ACLY　0.855**　3605.7±201.6a　2386.9±117.4b　1956.9±163.3b　　↑ACSL1　0.336　693.5±61.9　553.0±21.6　612.8±27.1 APOA1　0.639**　1519.0±107.2a　1348.5±42.2ab　1194.1±57.3b　　↑APOC3　0.736**　1859.5±131.2a　1472.7±63.2b　1307.6±77.4b　　↑CD36　0.593**　517.8±24.6b　580.4±15.1ab　596.5±15.8a　　↓CPT1A　0.044　244.5±37.1　247.6±15.5　233.5±10.0 CPT2　0.853**　224.7±8.4a　195.4±6.7b　151.8±4.3c　　↑FABP1　0.722**　998.8±96.5a　687.5±30.3b　606.7±38.4b　　↑FADS6　0.547*　109.6±8.3　130.4±11.7　145.1±9.1　　↓FASN　0.769**　10,794±755.5b　8475.9±480.1a　6486.9±559.1a　　↑LPL　0.600**　48.2±22.3b　90.1±9.1ab　117.9±9.6a　　↓MDH1　0.902**　667.0±28.0a　462.6±18.8b　386.7±12.3b　　↑ME1　0.601**　1045.0±127.5a　963.7±75.0ab　600.6±92.9b　　↑PPARA　0.376　447.1±17.3ab　434.6±17.1b　496.8±15.3a RXRA　0.012　65.9±2.9　63.4±2.8　64.6±3.0 SCD　0.817**　2785.2±130.0a　2322.6±81.9a　1413.6±233.1b　　↑TLR2A　0.041　21.1±1.3　20.9±1.9　20.4±1.7 TLR4　0.360　10.2±0.9　9.3±0.4　12.6±1.2 XBP1　0.620**　225.6±9.1b　231.4±9.9b　281.8±12.4a　　↓ERN1　0.578*　28.9±2.5　23.9±1.2　23.2±1.1　　↑

As expected the growth rates of the broiler progeny well exceeded those of the layer strain progeny.By d 14 the broilers were four times the weight of the layer strain males and twice the weight of the F1 cross.The total lipid carcass percentage of the broilers was higher than both the layers and the cross,which weren’t significantly different from each other,despite the cross being twice the weight of the layers.Interestingly multiple studies have shown that the dietary fatty acid composition is reflected in the fatty acid composition of the tissues and serum of broilers[33,34].Despite being raised in the same environmental conditions and fed the same diet,the fatty acid composition of the carcasses and blood spots differed between the three groups in this study,suggesting difference existed in fatty acid metabolism.The broilers had increased overall MUFA percentages,which would correlate with the significant upregulation of SCD1 which encodes the rate-limiting enzyme converting SFAs into MUFAs[35].Comparisons of the total SFA,MUFA and PUFAs revealed layers had higher n-6 and n-3 levels,indicating two possibilities,layer strains either have a higher physiological requirement for long chain PUFAs,or,layers are more efficient at converting available dietary linoleic and alpha-linolenic fatty acids to their long chain derivatives.The gene encoding the enzyme FADS6,which is rate limiting in the elongation of PUFAs,was found to be upregulated in the layers in comparison to the broilers which may support this concept.

对学困生的转化工作不是一朝一夕就可以完成的事，数学教师要有足够的耐心，细致有效的开展这项工作，既要帮助他们改变对数学学科的思想认识，同时还要激发他们学习数学的兴趣，树立学好数学的自信，养成良好的数学学习习惯，才能让他们热爱数学，学好数学。

Fig.4　Changes in hepatic gene expression associated with the PPARA signalling pathway and fatty acid metabolism between broilers(n=6)and layers(n=6).Red boxes indicate gene upregulation in broilers,green boxes indicate gene downregulation in broilers in comparison to layers

Whilst it may be anticipated that the increased fat deposition is due to either increased lipogenesis and/or a decrease in fatty acid β-oxidation,we saw a net overall increase in both lipogenesis and fatty acid β-oxidation genes in the broilers compared to layers or the F1 cross.Although this could be controlled by transcription factors regulating FA metabolism,such as the nuclear receptor PPARA,we found no evidence to support this.The higher metabolic activity may therefore be reflective of the weight of the liver at d 14 which was relatively larger than that of the layers expressed as a percentage of bodyweight.An early increase in liver mass has also been observed in multiple studies,including comparisons of modern broilers and heritage lines[2].In the current study the layer and crossed birds had reached their peak relative liver mass by d 7,however the broilers had higher relative weights at d 7 and reached their relative maximum weights at d 14 post hatch.By d 28 there were no differences in relative liver mass between the broilers,layers and their F1 cross.Schmidt et al.,[2]propose this early increase in liver mass could correspond to increased liver capacity required in early post hatch,and that a possible effect of selection may have shifted earlier maturation of the liver in modern broiler lines.The relative heart weights followed a similar pattern to the liver in that they were at their maximums in the first 2 wks post hatch.From d 14 onwards the broiler relative heart weights had significantly reduced when compared to the cross and layers.These findings are not surprising as the reduction in cardiac relative size and capacity has been well documented in broilers due to genetic selection for increased growth[2,18,36].

Additional to differential fatty acid metabolism,it was hypothesised that innate immune parameters may also be interacting with fatty acid metabolism ultimately influencing performance variation.Modern broilers are now considered obese relative to layer strains,so obesity-related pathologies such as inflammation and cellular stress may be anticipated to be increased in broilers.To test this hypothesis immune organ weights(spleen and bursa),heterophil:lymphocyte ratios,as well as Toll-like Receptors(TLR2a,TLR4),fatty acid translocase(CD36)and endoplasmic reticulum stress indicator genes(ERN1,XBP1)were included in the current study.

由图2可知，线缆SC1固定点间最近的距离为371 mm，最远的距离为834 mm。根据车钩缓冲器的伸缩量进行曲线模拟，可得到表2。

Gene expression

案例1：在“等比数列”一节教学时，可设计如下问题引入等比数列的概念：阿基里斯（希腊神话中的赛跑英雄）和乌龟赛跑，乌龟在前方1里处，阿基里斯的速度是乌龟的10倍，当他追到了1里，乌龟前进了里，当他追到里，乌龟前进了里，当他追到里，乌龟又前进了里……

Whilst short-term stress is of minimal consequence to broilers,long-term stress results in increased serum corticosterone,increased heterophil:lymphocyte ratios and altered protein,carbohydrate and lipid metabolism,and increased deposition of abdominal fat[38].This poses a question,could a broiler be chronically stressed at a cellular level,particularly with the reduction of organ weights relative to overall bodyweight as growth increases?To investigate whether there was any evidence of organelle stress occurring,two key ER stress indicators which initiate the unfolded protein response(UPR)were assessed,inositol-requiring kinase 1(ERN1)and xbox binding protein(XBP1).Saturated fatty acids have been shown to trigger the UPR response in hepatocytes and the UPR has been linked to lipid synthesis and breakdown[39].Despite the broiler,layer and F1 cross birds having differing SFA levels,no differences were found in the expression levels of ERN1,XBP1 however was found to be upregulated in the layers in comparison to both the broiler and cross.Given that ERN1 levels are showing no indication of ER stress,the differential expression of XBP1 may align with the suggestion that XBP1 functions as a mediator of hepatic lipogenesis,distinct from its function in ER stress and the UPR[40].It is thought to regulate the transcription of genes involved with fatty acid synthesis,including SCD1 and ACACA,with deletion of XBP1 resulting in decreased triglyceride,cholesterol and free fatty acids[40].It is difficult to conclude whether XBP1 is exhibiting a regulatory effect on lipogenesis in the layers however the aforementioned genes are not seen to be increased in the layers compared to the broilers or the cross.

Additional to organelle stress,Toll-Like receptors,including TLR2 and TLR4 have received attention for their roles in the development of obesity and insulin resistance,although the mechanisms by which they contribute still remain unclear.Mice lacking TLR2 and TLR4 genes do show however that TLRs are involved in the development of obesity[41].In macrophage cell cultures,saturated fatty acids,such as stearic acid and palmitic acid,have been shown to activate TLR2 and TLR4 signalling pathways,which consequently activates down steam proinflammatory pathways,Conversely,PUFAs,particularly n-3 s,have been shown to inhibit TLR2/4 expression,activation and downstream signalling[42].In our current study we found no differential expression of TLR2a in the avian liver in any of the three types of birds.Additionally we found no evidence in the expression levels of TLR4 to suggest that the differing fatty acid profiles of the birds was having an effect or interaction with the expression of TLR4 at d 14 post hatch.This was also the case for CD36,with the exception of a down regulation in the broilers in comparison to the layers.Given the biological diversity for the role of CD36,this likely does not translate into down regulated facilitation of fatty acid transport given the overall upregulation of fatty acid metabolism seen in the broilers.

In total,150 newly hatched male chicks(50 broiler strain,50 F1 layer×broiler cross,50 layer strain)were obtained from the HiChick Breeding Company Pty Ltd.,Bethel,South Australia.The cross progeny were produced by HiChick utilising their commercial breeding lines.Briefly,three Isa Brown roosters and 135 Isa Brown breeder hens were used to produce layer progeny,three broiler breeder roosters and 135 broiler breeder hens used to produce the broiler progeny,and three Isa Brown roosters and 135 broiler breeder hens used to produce the F1 layer×broiler cross.All progeny were produced via natural mating.The F1 cross was utilised as an intermediate growth phenotype against broiler and layer strain progeny.Chicks were separated by breed and placed 25 chicks/rearing pen in a temperature and climate controlled room at the SARDI PPPI Poultry Research Unit,Roseworthy Campus,The University of Adelaide.

The results indicate a total upregulation of fatty acid metabolism in broiler chickens when compared to an F1 cross and commercial layer strain.This increase is most likely as a result of genetic selection for growth,with the overall increase resulting in increased FA synthesis as well as β-oxidation in the liver.There was no evidence to suggest that at d 14 post hatch that the broilers are in a state of cellular hepatic stress or demonstrating changes in innate immunity parameters such as TLR2 and TLR4 expression.This is despite the broilers growing at four times the rate of the layers and with significant increases in fat%.Day 14 post hatch was selected to capture the physiological changes as the broiler growth acceleration begins.It is possible that the d 14 sample time point was too early in relation to fatty acid metabolism and innate immunity/cellular stress interactions to capture changes that may ultimately be driving performance.Analysis at additional time points in the grow out phase could better revel indicators of chronic stress as the organ weights continue to decrease by relative weight,contributing to metabolic stress and altering metabolism.

Acknowledgements

The cross birds appeared to have a lower number of heterophils and a higher number of lymphocytes than the broiler and layer birds,however no statistical differences were detected in the heterophil;lymphocyte ratios between broilers,layers or the F1 cross(Fig.3;P=0.203).The differences were likely reflective of the high individual variation in cell frequencies,which is reflected by the large standard error.In addition to the heterophils and lymphocytes,basophils(P=0.094),monocytes(P=0.773)and eosinophils(P=0.561)were also assessed however no significant differences were detected in the cell frequencies between any of the groups.

This research was conducted within the Australian Poultry Cooperative Research Centre,established and supported under the Australian Government’s Cooperative Research Centres Program.N-LW received a stipend from the University of Adelaide(F J Sandoz Scholarship)and the Australian Poultry Cooperative Research Centre for PhD studies.We would like to thank D.Schultz,M.Bowling and N.Heberle for assistance during the trial and the HiChick Breeding Company Pty Ltd.for providing the birds.

Funding

This work was financially supported by the Australian Poultry Cooperative Research Centre.The funder had no role in study design,data collection,and analysis.The funder read and approved the manuscript for publication.

Availability of data and materials

The data supporting the conclusions of this article are included within the article with the exception of the RNASeq data.The RNAseq datasets generated and/or analysed during the current study are available in the NCBI Sequence Read Archive(SRA)under BioProject PRJNA392351[https://www.ncbi.nlm.nih.gov/bioproject/PRJNA392351/].

X2和X3各有5个水平，将每个组合的编码值代入相应的双因子农艺效应函数，可得到对应的产量值(Y)，见表4。

Authors’contributions

N-LW,PH,RF,RH and GN designed the study.N-LW and RF were involved in performing the experiment and laboratory analysis.RT performed RNA-seq data analysis.N-LW wrote the manuscript,PH,RF and RH revised the manuscript.All authors read and approved the final manuscript.

Ethics approval

The experimental protocol used in this study,including animal management,housing,and slaughter procedures were by the University of Adelaide Animal Ethics Committee(approval#S-2015-171)and the PIRSA Animal Ethics committee(approval#24/15).

Consent for publication

Not applicable.

Competing interests

The authors declare they have no competing interests.

Author details

1School of Animal and Veterinary Sciences,The University of Adelaide,Roseworthy,SA 5371,Australia.2Davies Research Centre,School of Animal and Veterinary Sciences,The University of Adelaide,Roseworthy,SA 5371,Australia.3South Australian Research and Development Institute(SARDI),Livestock and Farming Systems,Roseworthy,SA 5371,Australia.4South Australian Research and Development Institute(SARDI),Pig and Poultry Production Institute,Roseworthy,SA 5371,Australia.5The Australian Poultry and Cooperative Research Centre,University of New England,PO Box U242,Armidale,NSW 2351,Australia.

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