Background

Milk contains high levels of nutrients such as proteins,fatty acids,phospholipids,vitamins and minerals[1].Among these nutrients,milk fat plays an important role in determining the quality and energy composition of dairy products[2].Milk fat contains large amount of saturated fatty acids(SFAs)and unsaturated fatty acids(UFAs).Recently,evidence has shown that high levels of SFAs pose a potential risk to human health such as cardiovascular disease(CVD)[3].In contrast,there is epidemiological evidence suggesting that dietary monounsaturated fatty acids(MUFAs)and polyunsaturated fatty acids(PUFAs)have beneficial effects for preventing CVD by favorably affecting a number of risk factors for CVD,including plasma lipids and lipoproteins.For example,oleic acid has a protective effect against retinopathy[4].Similarly,eico-sapentaenoic acid(EPA)and docosahexaenoic acid(DHA)play beneficial roles in preventing diabetes,atherosclerosis and arthritis[5].Many strategies have been investigated to enhance the unsaturated fatty acid content of milk[6-8].

大学生在校期间容易受到外界不良因素干扰，影响其身心的健康发展。进行高校学生党建工作能够正确引导并促进大学生身心健康发展，从而为祖国培育更多的高素质人才。高校学生党建工作促进大学生身心健康发展，对大学生的成长成才有着至关重要的影响。高校通过开展学生党建工作能够为大学生提供实践平台，从而促使大学生在参加各种党员活动过程中受到党组织的感染，更好地发挥自我价值。[1]同时，高校学生党建工作能够丰富大学生业余生活，避免大学生过分沉迷于网络，帮助学生更好地规划自己的生活，促进自身的健康成长。

Milk triglycerides are derived from two sources:the biosynthesis of fatty acids and their subsequent esterification within the mammary gland and the uptake of lipids from plasma into the mammary gland[9].Fatty acids in the mammary gland can also be generated by two pathways:short-and medium-chain fatty acids(C4-C14)can be synthesized de novo under the control of several key factors and enzymes including SREBP-1(sterol regulatory element binding protein 1),ACACA(acetyl-coenzyme A carboxylase alpha),FAS(fatty acid synthase),and ACSS2(acyl-CoA synthetase short-chain family member 2).Long-chain fatty acids(C＞16)are taken up from plasma via transporters such as fatty acid binding protein(FABP),fatty acid transport protein(FATP)and cluster of differentiation 36(CD36)[10].Acetate and 3-hydroxybutyrate are the main sources of a de novo synthesis for short-and medium-chain FAs[11].These FAs in the mammary gland can be desaturated by stearoyl-CoA desaturase(SCD)and a synthesis of cis-9 unsaturated FA and can then be esterified to glycerol sequentially via glycerol-3phosphate acyl transferase(GPAT),acyl glycerol phosphate acyl transferase(AGPAT),and diacylglycerol acyltransferase(DGAT)[11];subsequently,the triglycerides are secreted into the milk as fat globules.The composition of milk fatty acids is highly variable and depends on several factors such as the animal breed,the lactating stage,the dietary lipids composition,the energy status,and the feeding regimes[12-14].High levels of FAs are mobilized from adipose tissue in early lactation to provide more nutrients for the milk synthesis[12,13].In adipose tissue,fatty acids of 18:1c9,16:0,and 18:0 account for nearly 90%of total FAs[14]that are released into the blood during the negative energy balance of early lactation.These FAs are taken up through blood into the mammary gland and ultimately inhibit the de novo synthesis of fatty acids[13,15].As a consequence,the concentration of short-and medium-chain FAs in milk is lower during the early lactation than the later lactation[13,16].However,it has been reported that during the early lactation,unsaturated fatty acids such as conjugated linoleic acid,linoleic acid,and omega-3 were enriched in the milk[17,18].

Most dairy animals in intensive production systems are fed high levels of grain to maximize energy intake and milk production.However,excessive amounts of non-structural carbohydrates and highly fermentable forage will lead to a rapid fermentation and the accumulation of organic acids in the rumen[19],which likely induces subacute ruminal acidosis(SARA)[19,20].Both acute and subacute ruminal acidosis can decrease the production of milk fat and cause milk fat depression and the shift of fatty acids profiles in lactating cows[20].To our knowledge,the effects of longterm(more than 4 mo)feeding of a high concentrate(HC)diet to lactating ruminants on the production and composition of milk fat and the relevant mechanisms behind milk fat alterations are still unknown.In this study,mid-lactating dairy goats were fed a HC diet for a long(19 wk)or short(4 wk)period.Milk fat and FAs profiles were measured and the relevant gene expression and DNA methylation in the mammary gland were evaluated to investigate the mechanisms underlying the changes in milk fat production and composition.

Methods

Animals and experimental procedures

医院拥挤不堪很容易加剧患者看病难的抱怨，国家卫健委早已启动改善医疗服务行动，希望激励医院主动采取措施。面对问题，朱鹏立和福建省立医院也有一些自己的尝试。

Seventeen healthy,mid-lactating goats(Guanzhong dairy goats,60±5 d of lactation)with an average initial body weight of 49.7±5.5 kg(mean±SD)and similar daily milk yield(1.18±0.13 kg/d)were selected and housed individually(square measure:3.0～3.2 m2)in a standard animal feeding house at Northwest A and F University(Shanxi,China).Prior to the experiment,all goats were allowed free access to a control diet containing a forage to concentrate ratio of 65:35 for 2 wk.Ingredients and chemical composition of the experimental diets were shown in the Additional file 1:Table S1.After dietary adaptation,goats were randomly assigned to three groups,and the daily milk yield in each group prior to the start of experiment did not show a significant difference as shown in Fig.1(P＞0.05).The control treatment was fed a low-concentrate diet(35%concentrate,n=5,LC)while there were two high concentrate treatments(65%concentrate,HC),one fed a high concentrate diet for a long period(19 wk,n=7,HL),and the other fed a high concentrate diet for a short period of time(4 wk,n=5,HS)after 15 wk of low concentrate diet.All goats were fed daily at 08:00 and 18:00,respectively,and milked twice daily before feeding.

Sample collection and assay

Fig.1　The changes of milk yield,milk fat and protein.a Daily milk yield(kg/d);b Milk fat content(%);c Milk protein content(%).The asterisk*indicates a significant difference between HL and LC control group

At the end of the experiment,goats were euthanized after overnight fasting.All goats were killed with neck vein injections of xylazine[0.5 mg/kg body weight;Xylosol;Ogris Pharme,Wels,Austria]and pentobarbital[50　mg/kg　bodyweight;Release;WDT,Garbsen,Germany].After euthanasia,a portion of mammary gland tissues were collected and immediately frozen in liquid nitrogen,and then used for total RNA,genomic DNA and protein extraction.

Milk fatty acid analysis

Milk samples were collected twice daily before feeding at 08:00 and 18:00,respectively.The samples of milk were mixed thoroughly,and one portion was stored at 4°C for milk fat and protein analysis,another was stored at-70°C for fatty acids composition analysis.The level of milk fatty acids was detected following the standard protocol of gas chromatography in Jiangsu Academy of Agricultural Sciences as previously described[21].

Fatty acid synthesis is a complex process including uptake,transport,synthesis and oxidation of fatty acids.With respect to the FAs uptake,LPL is an important enzyme in the mammary gland responsible for taking up long-chain fatty acids from albumin-bound fatty acids,lipoproteins,or　chylomicrons　[35].LPL　also　can hydrolyze triglyceride(TAG)in lipoprotein core and then deliver fatty acids to the mammary gland for milk fat synthesis[36].The HL goats showed a significant decrease of the LPL mRNA expression in the mammary gland compared with the LC group as reported previously[34].SREBP1 and PPAR γ are important transcription factors involved in milk fat synthesis[37,38]and the lipogenic genes including ACACA,FAS,ACSL1,and SCD are all target genes of SREBP1 in bovine mammary epithelial cells[39,40].Our results showed that the SREBP1 mRNA expression and its target downstream genes were significantly decreased in the mammary gland of the HS and HL groups compared with the LC group,while the PPARγ gene expression was not changed.Moreover,short-and medium-chain FAs(C4-C14),as well as some portions of the C16 FAs,are mainly synthesized de novo in the mammary gland[41].ACACA is the rate-limiting enzyme involved in the FAs de novo synthesis by controlling the production of short chain fatty acid(SCFA)and palmitic from acetate[42].Fatty acid synthase(FAS)is a multifunctional protein that can catalyze the majority of the enzymatic steps in the fatty acid synthesis[43].In the present study,we found that,in conjunction with the decrease in total milk fat percent and some specific FAs concentrations including C15:0,C17:0,C17:1,C18:1n-9c,C20:0 and C18:3n-3r,the mRNA expressions of the ACACA and FAS genes and the enzyme activity of FAS were significantly downregulated in the mammary gland of the HL goats.Therefore,it is reasonable to speculate that the decrease in the gene expression and enzyme activity responsible for the FAs synthesis in the mammary gland may contribute to milk fat depression in HL goats.

RNA extraction and real-time quantitative PCR

Total RNA was extracted from each dairy goat using TRIZOL reagent(Takara,Dalian,China)according to the manufacturer’s protocol,and total RNA concentration was then quantified by measuring the absorbance at 260 nm in a NanoDropND-1000 Spectrophotometer(Thermo Fisher Scientific,Madison,WI,USA).The quality of total RNA was also verified by electrophoresis.Then two micrograms of total RNA were treated with RNAse-Free DNase and reverse transcribed according to manufacturer’s instructions(Takara,Dalian,China).The qRT-PCR efficiency was determined with serial 4-fold cDNA　dilutions(1,1/4,1/16,1/64,1/256cDNA template),and the efficiency was high with values between 95 and 100%.Two microliter of diluted cDNA(1:16 vol/vol)was used for real-time PCR which was performed in Stratagene Mx3000P qPCR instrument(Agilent,California,USA).The information of primer sequence was shown in Table 1.The mRNA expression of 18S rRNA,β-actin and GAPDH genes was measured in the mammary gland tissues by real-time PCR,and GAPDH was finally used as a reference gene for normalization purpose due to the high efficiency and the stable expression in all samples.The method of 2-△△Ct was used to analyze the real-time PCR results and gene mRNA levels were expressed as the fold change relative to the mean value of control group[22].

Western blotting analysis

Milk fatty acid composition

Frozen mammary gland(100 mg)was minced and homogenized in 1 mL of ice-cold RIPA buffer(pH 8.0,50 mmol/L Tris,150 mmol/L NaCl,1.0%Triton X-100,0.5%　sodium deoxycholate,0.1%　SDS)containing a protease inhibitor cocktail(EDTA-free;50×Conc.)(Roche Applied Science,Penz-berg,Germany).Then the homogenates was centrifuged for 20 min at 12,000×g at 4°C.Protein concentrations were measured with a Pierce BCA Protein Assay Kit(No.23225,Thermo,USA).Sixty micrograms of protein extract from each sample were separated by electrophoresis in 7.5%or 10%SDS-PAGE,transferred onto nitrocellulose membrane(BioTrace,Pall Co,USA).After transferred,membranes were blocked for 2 h at room temperature in blocking buffer(3%albumin from bovine serum),then incubated overnight at 4°C with the following primary antibodies:rabbit anti-mouse α-Tublin(Bioworld,BS1699 1:500),goat anti-mouse ACSL1(santa cruz,sc-98,925,1:200),goat anti-human SCD(santa cruz sc-23,016,1:200).In western blot detection,α-tublin was used as the internal control.Then the blots were incubated with the rabbit anti-goat　horseradish　peroxidase　(HRP)-conjugated second antibody(E030130-01,Earth Ox,CA,1:10,000)or goat anti-rabbit HRP-conjugated second antibody(Bioworld,BS13278,1:10,000)for 2 h at 25°C,and the bound HRP activity was detected by use of VersaDoc Imaging System(Bio-Rad,CA,USA).

综上所述，南通范氏家族的家风在宏观方面体现出“万家忧乐尽关心”的民胞物与之情，在微观方面的表现也涵盖了温、良、恭、俭、让、孝、悌、忠、信等各个方面。这些南通范氏家族世代传承的美好品德，也是维护范氏家族声誉不坠的行为准则，更是范氏家族延续四百五十年之久的传家之宝。南通范氏并非簪缨望族，但由于对文正家风的恪守，对诗礼书香传家的践行，使范氏家族成为南通乃至江苏的文化世家，其鲁殿灵光的“家风”与“家学”对今天的教育体制具有诸多启示意义，对南通范氏“家风”的梳理与解读也有助于弘扬中华民族的传统家庭美德，为树立优良家风、构建和谐社会提供可资借鉴的模板。

DNA methylation assay

Genomic DNA was extracted from mammary gland tissues using a commercial kit(DP304,TIANGEN Biotech Co.,LTD.Beijing,China).In brief,100 mg mammary gland powder was incubated with 1 mL lysis buffer(pH 8.0,50 mmol/L Tris;100 mmol/L EDTA;100 mmol/L NaCl;1.0%SDS)containing phenol,chloroform and 50 μL proteinase K(10 mg/mL stock)at 55 °C for 2 h,and then centrifugated at 12,000×g for 5 min.About 500 μL supernatant was collected,and incubated with 500 μL isopropyl alcohol and 60 μL 3.0 mol/L sodium acetate(pH 5.2)at room temperature for 5 min,and then centrifugated at 12,000 rpm for 15 min.The pellet was washed twice with 70%ethanol,after drying the pellet was finally resuspended in TE buffer(pH 8.0,10 mmol/L Tris,1 mmol/L EDTA).Samples were incubated at 65°C in a shaking water bath for 1 h to ensure a good resuspension.

新生代农民工流动性强，像候鸟一样飞来飞去，高失业率是一大明显特征，加之失地农民可持续生计问题面临挑战。然而，失业保险除了保障失业者在失业期间的基本生活之外，还可以促进失业者重新就业，以妥善解决失业者的可持续生计问题。因此，规范企业雇主职能，加强相关法律制度，实施用工“合同制”，赋予农民工“以农村土地换城市生计”的权利，有效解决“三农问题”，推进新型城镇化进程。

Short-term feeding of a HC diet had minor effects on milk fat production and composition in lactating dairy goats.However,long-term feeding of a HC diet will induce milk fat depression and a FAs profile shift with lower MUFAs but higher SFAs.A downregulation of the gene expression involved in the process of lipid production and the upregulation of the DNA methylation in the mammary gland may contribute to the decrease in milk fat production in HL goats.

FAS enzyme activity assay

FAS enzyme activity in mammary gland tissues was measured by FAS activity Assay Kit(jiancheng BioEngineering Institute,Nanjing,China).

Table 1　PCR primer sequences of the target genes

Target genes　Primer sequences　GenBank accession No.　PCR products,bp LPL　F:5′-CAAGTCGCCTTTCTCCCGAT-3’　　　177 R:5′-CTGCAATCACACGGAGAGCTT-3’ACACA　F:5′-TATGACGGCAGCAGTTACACC-3’　　　201 R:5′-CACCTCGATCTCAGCATAGCAC-3’FAS　F:5′-CCCCAAGCTCTTTGACAACCG-3’　　　100 R:5′-CAGCTCCTTGTACACGTCACC-3’FABP3　F:5′-TGAGACCACGGCAGATGA-3’　　　230 R:5′-TAGCCCACTGGCAGAAGA-3’FABP4　F:5′-CATAAACTTAGATGAAGGTGCTC-3’　　　116 R:5′-CACCGTTCATGACACATTCCA-3’DGAT1　F:5′-AAGCCCTTCAAGGACATG-3’　　　100 R:5′-AGAGCCAGTAGAAGAAGATG-3’CPT1B　F:5′-CCAGCCACAGTTCATCGGTA-3’　　　103 R:5′-CAGTCTTCTCCTCGAACTCC-3’ACSS1　F:5′-TGAGCGACTGCGACTTCC-3’　　　223 R:5′-CGGCGGACTCCATACCTCT-3’ACSS2　F:5′-TGAGCCAGAGGAAACCA-3’　　　157 R:5′-ACACGCCAGCATAGCC-3’ACSL1　F:5′-CGCAGTGGCATCATTAG-3’　　　183 R:5′-CTCGGTCTGTCCGTAGC-3’SREBP1　F:5′-ACGCCATCGAGAAACGCTAC-3’　　　181 R:5′-GTGCGCAGACTCAGGTTCTC-3’PPARγ　F:5′-CCTTCACCACCGTTGACTTCT-3’　　　145 F:5′-GATACAGGCTCCACTTTGATTGC-3’CD36　F:5′-ATAATACTGCGGATGGA-3’　　　165 R:5′-TCTCAACGAAAGGTGG-3’LDLR　F:5′-GTGGCTGACACCAAAGGG-3’　　　175 R:5′-CGGTCACCAGCGAGTAAAT-3’SCD　F:5′-GTGGGTTGGCTGCTTG-3’　　　243 R:5′-CCAGGTGGCATTGAGC-3’FADS2　F:5′-GCATCGCCTGGTTCACT-3’　　　249 R:5′-GGAAGATGTTGGGTTTGG-3’GAPDH　F:5′-GGGTCATCTCTCTGCACCT-3’　　　176 R:5′-GGTCATAAGTCCCTCCACGA-3’

Statistical analysis

All data are presented as the mean±SEM.The data of milk yield and milk composition was analyzed for differences due to diet treatment,time effect,and their interaction by using PROC MIXED,SAS 9.3,(SAS Institute Inc.,Cary,NC,USA).The data of milk yield,milk fat and protein obtained before the beginning of the treatment was considered as a co-variable in the statistical analysis.The differences of parameters in fatty acids composition,gene and protein expression,enzyme activities in mammary gland were analyzed by using the post hoc analysis with the least significant difference test following ANOVA of SPSS 11.0.The 2-ΔΔCtmethod was applied to analyze the real-time PCR data.Differences were considered significant at P＜0.05,and 0.05＜P＜0.1 is considered as a tendency.Numbers of replicates used for statistics are noted in the Tables and Figures.

Results

Milk yield and production of milk protein and fat

As shown in Fig.1a-c,goats fed a high concentrate diet(HL and HS group)produced(P＜0.01)more milk thangoats fed a low concentrate diet(LC).Diet significantly affected the milk fat production(P＜0.05).The percentage of milk fat decreased in the HL group after 16 wk and was significantly different from the control group(LC)(P＜0.05),while the protein was not significantly affected by dietary treatment.

Table 2　PCR primer sequences using for MeDIP

Target genes　MeDIP primer sequence　GenBank accession No.　PCR products,bp FADS2　F:5′-GGCACTAATCCCAAGCAG-3’　XM_018043056.1　138 R:5′-GGAAACAATACAGGACCTCATA-3’ACSL1　F:5′-GGGAGGCGAGCAGAAAGA-3’　XM_018041881.1　142 R:5′-AACAGACGGTGGAGGGTG-3’ACSS1　F:5′-ACCTGAGAAGGGATGTGG-3’　XM_018057282.1　104 R:5′-AGAAGACTCAACGCAAACA-3’FAS　F:5′-TTGCCTAAAGTCAGTGTCG-3’　NM_001285629.1　169 R:5′-GCAGGTCAACCGCATAAC-3’ACACA　F:5′-GCTTTCTTCACCGAGGCT-3’　XM_018064168.1　173 R:5′-CGGAGGGTATCGCATTCA-3’ACSS2　F:5′-CCTCCCGTTCTGCTTTCC-3’　XM_005688483.3　132 R:5′-AGCCGTGCCTGGTGGTGTTG-3’SCD　F:5′-CCCCAGTGCCCATCCATTT-3’　NM_001285619.1　168 R:5′-TCCCTTTCTCCTCGGCTTCTC-3’

教师的教学方式单一是影响教学质量和教学效果的重要原因。很多教师在教学过程中往往采用“灌输式”的教学方式，只注重教师讲课的连贯性，忽视了与学生之间的交流和沟通，导致课堂教学气氛沉闷。此外，这种方式也忽视了实践性教学的重要性，没有将道德与法治课堂知识与生活结合，学生在理解时不够深入[1]。

The design of the study and collection,analysis,and interpretation of data were supported by the National Nature Science Foundation of China(project no.31572433)and the National Key R&D Program(2016YFD0501203),the Program for New Century Excellent Talents in University(NCET-13-0862)and the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD).

Funding

As shown in Fig.2,feeding a HC diet to lactating dairy goats down-regulated gene expression involved in milk fat production in the mammary gland.With respect to the fatty acid transport process,the mRNA expressions of the LPL,ACSL1,ACSS1 and ACSS2 genes were lower in the mammary gland of the HL goats(P＜0.05)and the CD36 and ACSS2 mRNA expressions were lower inthe HS group compared with the LC group(P＜0.05).The expressions of the genes involved in fatty acids synthesis and desaturation including SREBP-1,FAS,ACACA,SCD,FADS2,and DGAT1 were also downregulated in the HL goats(P＜0.05)and the mRNA expressions of ACACA,FADS2,SREBP-1,and CPT1B were lower in the HS group compared with the LC group(P ＜ 0.05).However,the levels of the PPAR-γ,LDLR,FABP-3 and FABP-4 mRNA expressions were not changed by feeding a HC diet(P＞0.05).

Table 3　Fatty acids composition in the milk fat

Values are means±SE.Mean values without common superscript(a,b)differ significantly among LC,HS and HL groups(P＜0.05)

Fatty acids　LC　HS　HL C4:0　0.78±0.05　0.79±0.06　0.91±0.08 C6:0　1.19±0.06　1.16±0.08　1.27±0.11 C8:0　1.45±0.08　1.38±0.10　1.48±0.14 C10:0　7.27±0.29　6.48±0.39　7.23±0.55 C11:0　0.13±0.03　0.09±0.02　0.09±0.01 C12:0　4.72±0.36　3.78±0.39　4.69±0.45 C13:0　0.09±0.01　0.07±0.02　0.10±0.03 C14:0　12.15±0.38　10.28±0.49　11.30±0.6 C14:1　0.24±0.02　0.22±0.08　0.21±0.04 C15:0　1.22±0.05a　0.91±0.13ab　0.85±0.06b C15:1　0.17±0.08　0.04±0.00　0.10±0.03 C16:0　33.16±1.90　33.89±0.49　38.19±1.8 C16:1　1.31±0.07　1.35±0.26　1.18±0.08 C17:0　0.93±0.02a　0.81±0.09ab　0.74±0.02b C17:1　0.29±0.05a　0.31±0.03ab　0.22±0.22b C18:0　7.01±0.69　7.29±1.13　6.32±0.57 C18:1n-9c　20.71±0.72a　22.80±1.06a　18.17±0.51b C18:2n-6 t　0.12±0.01　0.17±0.01　0.14±0.01 C18:2n-6c　1.96±0.12b　2.39±0.08a　1.93±0.05b C18:3n-3 r　0.22±0.02a　0.12±0.01b　0.10±0.00b C20:0　0.21±0.01a　0.17±0.01b　0.15±0.01b C20:4n-6　0.14±0.01b　0.18±0.01a　0.14±0.01b ΣSFA　68.39±1.03　67.10±1.35　73.34±0.65 UFA　25.26±0.94a　27.58±1.36a　22.21±0.60b ΣMUFA　22.79±0.83a　24.71±1.29a　19.89±0.58b ΣPUFA　2.47±0.13b　2.87±0.10a　2.32±0.05b

Fig.2　Genes expression involved in milk fat production in the mammary gland.a mRNA expression of genes involved in FAs up-take;b mRNA expression of genes regulating FAs synthesis and desaturation;c mRNA expression of genes for FAs activation and transport;d mRNA expression of genes involved in TG synthesis.Data are presented as mean±SE.Mean values without common superscript(a,b)differ significantly among LC,HS and HL groups(P＜0.05)

Protein expression and FAS enzyme activity

城乡一体化发展的过程中，城市居民和农民都是其中不可或缺的一部分，政府应该更好地利用各种资源，更为全面地规划和实施相关政策，从而促进城乡结构的合理优化，加快农村经济发展的步伐。政府应采取积极有效的方式来保护农民的土地财产权，专门制定详细的农民土地财产权制度，从而保障农民的合法权益和利益。

The levels of the ACSL1 and SCD protein expressions in the mammary gland were determined by western blotting.The results showed that the level of ACSL1 protein expression in the mammary gland was decreased by feeding a HC diet(P＜0.05),while the SCD protein expression was not altered(P＞0.05).The activity of the FAS enzyme was also decreased in the HL goats compared with the LC group(P＜0.05)and there was no significant difference between the HS and LC groups(P＞0.05)(Fig.3).

DNA methylation analysis

Due to a decrease in the gene expression involved in milk fat production in the mammary gland,the level of DNA methylation in the promoter regions of the encoding genes was measured using the MeDIP method.The results showed that the DNA methylation in the promoter regions of the SCD and ACACA genes was greater in the mammary gland of the HL goats compared with the LC group(P＜0.05).However,the levels of DNA methylation in the promoters of the ACSL1,ACSS1&2,FAS and FADS2 genes were not changed in the HC goats compared with the LC group(P＞0.05)(Fig.4).

Discussion

To meet the energy demand of high milk production,dairy animals are commonly fed a HC diet,which likely causes abnormal fermentation in the rumen and leads to metabolic disorders known as SARA.Previous studies have shown that 19%of early lactation and 26%of midlactation　cows　experienced　SARA　[23].Lactating animals suffering from SARA have increased risks for diarrhea,laminitis and inflammatory responses[24,25].This extensively-used feeding strategy ultimately leads to a decrease in milk quality with a lower quantity of milk fat[25-27].In this study,as found in practice,our results showed that milk production was significantly increased by feeding a HC diet in both the HL and HS groups compared with the LC group,while the percentage of milk fat was significantly lower in the HL group.A previous study also demonstrated that feeding high grain diets to lactating dairy cows led to milk fat depression with lower milk fat concentrations[28].

Fig.3　The level of the ACSL1 and SCD proteins expression and the FAS activity in the mammary gland.a Protein expression of ACSL1 and SCD;b FAS enzyme activity.Data are presented as mean±SE.Mean values without common superscript(a,b)differ significantly among LC,HS and HL groups(P＜0.05)

Fig.4　The level of the DNA Methylation in the promoter regions of the target genes.Data are presented as mean±SE.Mean values without common superscript(a,b)differ significantly among LC,HS and HL groups(P＜0.05)

Milk and dairy products contain fatty acids with high proportions of SFA and MUFA and small amounts of PUFA[29,30].Research has shown that humans consuming an excess of SFA have increased risk for coronary heart disease[31].In contrast,UFAs benefit human health[4,5].Chilliard et al.observed that high-quality alfalfa hay-based diets can increase the level of C18:3n-3 in the milk of dairy cows[32].However,cows fed a high starch diet experienced milk fat depression syndrome[28].A low milk fat content has even been suggested as a noninvasive indicator to identify cows with a greater risk for SARA[33].Previous studies have shown that lactating ruminants with SARA induced by feeding a HC diet did not experience altered concentrations of SFAs,PUFAs or MUFAs in the milk but had markedly decreased concentrations of C18:2n:6c,C18:3n3,and C20:3n6[27,34].Our results showed that the concentrations of C18:1n-9c,SFAs and MUFAs were not significantly changed by feeding a short-term HC diet in the HS group as reported in a previous study[27].However,it is very important to note that long-term feeding of a HC diet significantly decreased the percentage of milk fat and the concentrations of UFAs and MUFAs,while the concentration of SFAs exhibited an increasing tendency in the milk compared with the LC control goats.

木兰溪两岸地区风景优美，具有丰富的旅游资源，在对其进行开发的过程中，没有对其进行统筹开发，缺乏完善的顶层策划，缺乏对旅游资源合理的挖掘。同时，在建设基础设施的过程中，如防洪工程等，并没有将其同全域旅游规划以及城市建设发展规划进行充分融合，没有从全市的角度对木兰溪沿河景观进行规划，从而为提升木兰溪沿岸城市品位奠定基础，充分挖掘莆田市木兰溪的旅游资源，对其进行合理利用，进而带动一溪两岸风光带的建设发展，进而促进莆田市经济以及政治文化的稳定发展。

As a desaturase,SCD is an important enzyme for controlling the intracellular FAs composition by catalyzing the conversion of SFA into monounsaturated FAs[44,45].In this study,we found that the SCD gene expression was significantly downregulated in the HL group,which may be responsible for the decrease in C18:1n-9c and the MUFA concentration in the milk of HL goats.Long-chain fatty acids were activated by acyl-CoA synthetase via long-chain family member isoforms(ACSL)[46],while short-chain fatty acids were activated by acyl-CoA synthetase shortchain family members(ACSS)[47].ACSL1 is a predominant enzyme among the ACSL isoforms in the bovine mammary gland[48].CD36 is another important enzyme controlling the long chain FAs uptake working in conjunction with intracellular fatty acidbinding proteins(FABP)[49,50].ACSS1&2 mainly regulate the oxidative pathway of lipids and acetate activation,respectively[47,51].In the present study,our results showed that the ACSL1 protein expression was also significantly decreased in the HL group compared with the LC group.The ACSS2 mRNA expression was also markedly decreased in the HL goats.These results indicate a potential decrease in the milk fat synthesis in the mammary glands of HL goats compared with their LC counterparts.

Epigenetic modifications are involved in regulating gene transcription[52].DNA methylation is perhaps the most extensively studied epigenetic modification and plays an important role in the regulation of gene expression[53].For decades,methylation has been believed to play a crucial role in repressing gene expression through blocking the promoter region where the activating transcription factors should bind[54].With respect to the general downregulation of the gene expression involved in the milk fat synthesis in the mammary gland,the status of the DNA methylation in the promoter regions of the target genes has been analyzed as previously described[51,54].The results demonstrated that the methylation of the SCD and ACACA promoters was significantly increased in the HL group compared with the LC group,which was consistent with the decrease in their gene expression.Consistently,previous studies also showed that the methylation in the SCD promoter region was increased in dairy goats and cows fed a highconcentration diet[54].However,the methylation in the promoter regions of FAS,ACSL1,ACSS1,ACSS2,and FADS2 DNA was not changed by feeding a HC diet.We speculate that the modification of the DNA methylation was involved in regulating the milk fat depression in the HL goats.In contrast,the DNA methylation in the lipogenic genes promoter was not significantly altered in the mammary gland of the HS goats compared to the LC group.To date,no information has been provided for explaining the difference in the DNA methylation between the HS and HL groups.Moreover,the mechanisms behind the changes in DNA methylation in the HL goats still require further investigation.

Conclusions

The isolated genomic DNA was sonicated to produce random fragments with the size from 300 to 500 bp.The sonication condition was set up with the following parameters:output power 30 W,5 s pulse on and 5 s pulse off,10 cycles.Two microgram of sonicated genomic DNA was heat-denatured to produce singlestranded DNA,and the same portion of the undenatured DNA was stored as control(input)DNA.The methylated DNA fragments was immune-precipitated by the　mouse　monoclonalantibody　against5-methyl cytidine(ab10805,Abcam).Precleared Protein A/G Plus Agarose(Santa Cruz,Dallas,TX)was used to immuneprecipitate　the　antibody/DNA　complexes,and　the MeDIP DNA was purified.Forty nanogram of MeDIP DNA and control input DNA was used to amplify the ACACA,SCD,FAS,ACSL1,ACSS1&2,FADS2 promotor regions by real-time PCR with specific primers(Table 2).The primers were designed with the software of“Methyl Primer Express”using the specific promotor sequence enriched with CpG sites blasted from the website of “http://www.ensembl.org/index.html”.The ratios of the signals in the immunoprecipitated DNA vs input DNA were calculated as a measure for representing the relative enrichment of methylation in the particular sample.

Additional file

Additional file 1:Table S1.Ingredients and composition of the experimental diets(%).(DOCX 13 kb)

Abbreviations

ACACA:Acetyl-coenzyme A carboxylase alpha;ACSL1:Acyl-CoA synthetase long-chain family member 1;ACSS1:Acyl-CoA synthetase short-chain family member 1;ACSS2:Acyl-CoA synthetase short-chain family member 2;AGPAT:Acyl glycerol phosphate acyl transferase;CD36:Cluster of differentiation 36;CVD:Cardiovascular disease;DGAT:Diacylglycerol acyltransferase;DHA:Docosahexaenoic acid;EPA:Eico-sapentaenoic acid;FABP3:Fatty acids binding protein 3;FABP4:Fatty acids binding protein 4;FADS2:Fatty acid desaturase 2;FAS:Fatty acid synthase;GPAT:Glycerol-3 phosphate acyl transferase;LDLR:Low density lipoprotein receptor;LPL:Lipoprotein lipase;MUFA:Monounsaturated fatty acid;PPARγ:Peroxisome proliferator activated receptor gamma;PUFA:Polyunsaturated fatty acid;SARA:Subacute ruminal acidosis;SCD:Stearoyl-CoA desaturase;SCFA:Short chain fatty acid;SFA:Saturated fatty acid;UFA:Unsaturated fatty acid

Acknowledgements

现代工业中含重金属离子废液的不合格排放会严重污染土壤、水源，危害人们的身体健康，因此，对废液里的重金属离子进行合理处理十分重要[1-2]。目前，对重金属离子的处理方法[3]有离子交换法、吸附法、化学沉淀法、铁氧体法、膜分离法、浮选法等。其中，吸附法[4-5]是利用具有高比表面积的固体材料通过吸附作用去除废水中的重金属离子，操作简单、环保、有效。吸附剂的选择是影响吸附效果的重要因素，研究表明，成本低、来源广的农作物等生物质炭用作吸附剂对铅、铜、锌等众多重金属离子有较好的吸附效果，已成为吸附方向上的研究热点。

假设系统当中的各决策主体按照一定的比例来分配集中决策时的总利润，设制造商、零售商、物流服务集成商、物流服务提供商各自的利润比例分别为 λ1、 λ2、 λ3和 1-λ1-λ2-λ3， 且 0≤λ1≤1、 0≤λ2≤1、 0≤λ3≤1， 称为分配因子， 分配因子的大小在某种程度上反映了各决策主体讨价还价能力的大小。

The authors thank the National Nature Science Foundation of China(project no.31572433),the National Key Research and Development Program of China(2016YFD0501203),the Program for New Century Excellent Talents in University(NCET-13-0862)and the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)for supporting this study.

Gene expression involved in milk fat production

The composition of the milk fatty acids was analyzed and is shown in Table 3.Compared with the LC group,the HL group exhibited a lower level of unsaturated fatty acids(UFA)and monounsaturated fatty acids(MUFA)in the milk(P＜0.05),while there was no significant difference between the LC and HS groups(P＞0.05).The content of saturated fatty acids(SFA)in the milk showed a tendency to increase in the HL group compared with the LC group(0.05＜P＜0.1).Compared with LC,the concentration of C15:0(P＜0.01),C17:0(P＜0.01),C17:1(P＜0.01),C18:1n-9c(P＜0.05),C20:0(P＜0.01)and C18:3n-3r(P＜0.01)in the milk was markedly lower in the HL group and the concentrations of C20:0(P＜0.05)and C18:3n-3r(P＜0.01)were lower in the HS group.Moreover,the contents of C18:2n-6c(P＜0.05)and C20:4n-6(P＜0.05)in the milk were higher in the HS compared with the LC group.

Availability of data and materials

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

Authors’contributions

同时，网络文学、网络游戏等过去网络出版服务中的缺项和短板，近来年正在兴起，盛世阅读网、迅游等原创网络文学网络和游戏公司的崛起，对完善重庆数字出版的产业链，构建重庆数字出版生态圈有积极的作用。

堆载预压塑料排水板排水固结法在软土地基处理中的应用……………………………………………………… 刘华先（8-66）

PT:performed the experiment and drafted the manuscript.YL and XL:performed the experiment and analyzed the data.YN and RZ:contributed in experiment design and manuscript revision.JT and ST:carried out milk fatty acids analysis.CH and YG:conceived the idea,designed the experiment,and finalized the manuscript.All authors read and approved the final manuscript.

Ethics approval and consent to participate

The Institutional Animal Care and Use Committee(IACUC)of Nanjing Agricultural University approved all animal procedures.The“Guidelines on Ethical Treatment of Experimental Animals”(2006)No.Three hundred ninety eight by the Ministry of Science and Technology,“China and the Regulation regarding the Management and Treatment of Experimental Animals”(2008)No.Forty five by the Jiangsu Provincial People’s Government,was be strictly followed during the slaughter and sampling procedures.

Tennant河地区主要为管状(筒状)。矿床总体在深部的蚀变作用为钠质蚀变组合，在中浅部为钾质蚀变组合，而在浅部则为绢云母化和硅化；以磁铁矿为主的矿化以钠质蚀变为主，以赤铁矿为主的矿化以钾质蚀变为主。该区有经济价值的金-铜-铋矿化大多数与单个富磁铁矿或其相当的氧化矿相伴，呈现高的航磁异常，矿体地表露头以褐铁矿为主，深部以磁铁矿为主，高品位的金通常位于铁矿体底部，呈细脉-浸染状产出。铁矿的形状从近于圆形到筒状，长轴通常沿着EW向的裂隙，产出于元古代变质细粒沉积层系中，部分含有浸染磁铁矿。

2.2 急性缺血性脑卒中影响因素分析 血清脂蛋白相关磷脂酶A2水平升高、D-二聚体水平升高、高密度脂蛋白水平降低、高血压病史是急性缺血性脑卒中的独立影响因素(P<0.05)。见表2。

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

1Key Laboratory of Animal Physiology&Biochemistry,Nanjing Agricultural University,Nanjing 210095,People’s Republic of China.2College of Veterinary Medicine,Northwest A and F University,Yangling,Shannxi,China.

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