【作者】 张玲；

【导师】 罗玉衡；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2017， 硕士

【摘要】 本研究首先以BALB/c小鼠为研究对象,在其饲粮中短期添加可溶性日粮纤维(soluble dietary fiber,SDF)燕麦 β-葡聚糖、不可溶性日粮纤维(insoluble dietary fiber,IDF)微晶纤维素(Microcrystalline Cellulose,MCC)及二者混合物,考察这两种类型日粮纤维(dietaryfiber,DF)对小鼠粪便和结肠微生物群落结构的影响,并探索小鼠后肠特异性利用两种DF的核心菌群。其次以生长猪为研究对象,通过在体交叉试验,探讨猪后肠细菌和产甲烷菌群落分别对这两种纤维的动态响应过程和潜在互作关系。试验一高水平SDF和IDF对小鼠生长性能、器官指数及后肠微生物群落结构的影响采用单因子试验设计,将36只6周龄健康BABL/c雄性小鼠(17.95 ± 0.95 g)按体重无差异原则随机分为4组。对照组(C组)小鼠饲喂基础饲粮,不额外添加DF;微晶纤维素组(Microcrystalline Cellulose,MCC;M组)饲粮中添加20%MCC(纯度≥ 99%);β-葡聚糖组(G组)饲粮中添加28%燕麦β-葡聚糖(纯度为70%);混合组(GM组)饲粮中添加14%燕麦β-葡聚糖和10%MCC。各组饲粮营养水平保持基本一致。试验期为21天。结果表明:1.饲粮中添加高水平DF(绝对浓度约为20%)显著降低小鼠平均日采食量(ADFI,P<0.01),表现为C组>1M组>GM组>G组;各组小鼠体增重差异不显著(P>0.05)。GM组小鼠附睾脂肪垫重量显著高于G组和M组小鼠(P<0.05);2.G组和GM组小鼠结肠食糜乙酸含量有高于M组和C组的趋势(P<0.1),其中G组高于C组65%,高于GM组52%;G和GM组小鼠结肠中丙酸含量极显著高于M组和C组(P<0.01),其中G组高于C组75%,高于GM组71%。4.PCR-DGGE(变性凝胶梯度电泳)分析结果表明,第4天,G组小鼠后肠细菌多样性显著低于C组(P<0.05);第7天,G组小鼠粪便细菌多样性极显著低于其他组(P<0.01);第10天,M组小鼠粪便细菌多样性显著高于G组和 GM 组(P<0.01);5.高通量测序结果表明,G组小鼠结肠食糜中Bacteroidetes门相对丰度显著高于其余三组(P<0.01),M(P = 0.03)和GM组(P = 0.02)该菌相对丰度显著高于C组(12.9 ± 4.2%)。C组小鼠结肠食糜中Firmicutes门相对丰度最高(80.1 ±4.8%),G组小鼠结肠中该菌相对丰度(38.7± 14.8%)极显著低于其余组(P<0.01)。6.G组小鼠结肠中β-proteobacteria和γ-proteobacteria的相对丰度极显著高于其余组(P<0.01),但δ-proteobacteria的相对丰度极显著低于其余组(P<0.01)。G组小鼠结肠中Enterobacteriaceaae相对丰度极显著高于其余组(P<0.01)。7.小鼠结肠中与SDF利用有关的菌群以Bacteroids spp.、Oscillospira spp.、Prevotellaspp.和 Ruminococcus spp.为代表;与 IDF 利用有关的菌群以 Oscillospira spp.、Ruminococcusspp.、Desulfovibriospp.、Coprococcus spp.和 Bacteroides spp.为代表。8.qPCR分析表明,硫酸盐还原菌(SRB)的数量在G组小鼠结肠中显著低于其余组,而在M组和GM组小鼠结肠中最高(P<0.01),但4组小鼠结肠中产甲烷菌的mcrA基因绝对拷贝数无显著差异(P>0.05)。结果提示:小鼠后肠存在特异性利用SDF和IDF的核心菌群;后肠细菌发酵SDF产生更多SCFAs(尤其是丙酸和乙酸),可能通过直接供能和糖异生途径为机体平衡由采食量降低引起的体重和能量损失。试验二猪后肠微生物群落对日粮纤维类型转变的响应规律选择16头健康的DLY(杜洛克×长白×大约克)生长期阉公猪(40.38±1.19kg),按体重无差异原则随机分为2组。正式试验分为4个阶段,第一阶段为期1周,之后每个阶段持续3周。DF添加物同试验一。采用交叉试验设计:第一阶段所有猪只均饲喂基础饲粮;第二阶段处理1(T1)组饲粮中含7.14%燕麦β-葡聚糖,处理2(T2)组饲粮中含5%MCC;第三阶段T1组饲粮中的燕麦β-葡聚糖替换为5%MCC,T2组饲粮中的MCC替换为7.14%燕麦β-葡聚糖;第三阶段所有猪只均饲喂基础饲粮。日粮中燕麦β-葡聚糖和MCC的绝对浓度为5%。在第二、三、四阶段的第三周进行为期4天的消化试验。所有饲粮营养水平保持基本一致。试验期间每隔两天采集一次直肠内容物,换料时连续采集三天直肠内容物。结果表明:1.T2组由IDF饲粮转换为SDF饲粮后,试猪ADFI极显著低于T1组(P<0.01)。2.纤维类型显著影响粗蛋白(CP)、粗脂肪(EE)、粗灰分(Ash),粗纤维(CF)和酸性洗涤纤维(ADF)的表观消化率。与采食IDF饲粮的猪只相比,采食SDF饲粮的猪只CP、EE、Ash表观消化率显著降低(P<0.05),且CF和ADF表观消化率极显著升高(P<0.01)。3.PCR-DGGE分析表明,猪后肠细菌对SDF和IDF两种DF的实时动态响应过程呈现相反趋势。采食含SDF饲粮的猪只后肠细菌多样性呈降低趋势,与第 7 天(Shannon 指数为 3.51 ± 0.18)相比,第 28 天 Shannon 指数(3.32 ± 0.09)显著降低(P<0.01),β多样性指数呈上升趋势;采食含IDF饲粮的猪只后肠细菌多样性呈上升趋势,与第7天(3.25 ± 0.08)相比,第28天Shannon指数(3.57±0.10)显著升高(P<0.01),β多样性指数呈下降趋势。4.当饲粮纤维类型由IDF转变为SDF时,猪后肠细菌和产甲烷菌群落的响应更敏感,细菌多样性出现明显波动。第29、31、40和49天细菌Shannon指数分别为:3.62±0.12、3.12±0.09、3.47±0.06 和 3.15±0.09(P<0.01);产甲烷菌多样性呈现明显的先上升后下降趋势,d29,d31,d43,d49Shannon指数分别为 2.77 ±0.15、2.80 ±0.09、2.96 ±0.09 和 2.55 ±0.10(P<0.01)。5.高通量测序分析表明,采食含SDF饲粮的猪只后肠Firmicutes门细菌相对丰度从73%(d7)迅速升至82%(d8)之后逐渐降至68%(d22),Bacteroidetes相对丰度则表现出相反的变化趋势:从17%(d7)迅速降低6%(d8),之后升至19%(d22)。采食含IDF饲粮的猪只后肠Firmicutes相对丰度则由81.99%(d7)降至 63.33%(d22)然后略微回升(71.02%,d28)。6.LefSe(线性判别分析)结果表明,T1组第2阶段猪后肠差异菌群分属于Actinobacteria和Firmicutes门,且含有糖降解相关菌群,如Mitsuokella、Ruminococcaceae、Megasphaera等。T1组第3阶段含有的差异菌群种类较多,分属于 Actinobacteria、Firmicutes、Proteobacteria 和 Bacteroidetes 门,可鉴定的差异菌属包含Sharpea、Epulopisium、Clostridium等。与其余时间点样本相比,T1组第51天样本(试验第4阶段)中含有的差异菌群种类最多,分属于Lentisphaerae、Proteobacteria、Bacteroidetes、Proteobacteria Firmicutes 门;T2组试验第2阶段差异菌群分属于Baacteroidetes、Firmicutes和Chlamydia门,第3阶段差异菌群分属于Firmicutes和Spirochaetes门,而在试验第51天时,样本中的差异菌群分属于 Proteobacteria、Lentisphaerae、Verrucomicrobia、Bacteroidetes 和 Firmicutes 门。结果提示:猪后肠存在特异性利用SDF和IDF的菌群;当DF类型改变,与产甲烷菌相比,细菌群落结构变化更为剧烈;摄入SDF和IDF的先后顺序明显影响细菌和产甲烷菌群落结构;产甲烷菌群落结构的变化晚于细菌,暗示二者在不同类型DF的后肠发酵过程中存在复杂的交互饲喂关系;菌群结构的变化可直接影响其功能(如代谢产物SCFAs的变化),改变后肠发酵方式,间接影响DF的利用效率和宿主对营养物质的消化率。综上,小鼠和猪后肠均存在特异性利用两种DF的细菌群落;后肠微生物对DF类型改变的响应较为复杂,在此过程中产甲烷菌和细菌存在复杂的互作关系;高水平SDF在结肠内能被发酵产生更多SCFAs(尤其是丙酸),促进机体生长;研究结果可为动物生产上科学利用不同类型/来源的DF提供参考。更多还原

【Abstract】 In current study,the effect of different types of dietary fibers(DF),oat derived β-glucan(SDF),microcrystalline cellulose(MCC)and their mixture,to the microbial community structure in the feces and colonic digesta of BALB/c mice was first investigated.The core bacteria specifically utilize SDF and IDF in the hindgut of the mice were also explored.Next the dynamic response of bacteria and methanogens as well as their potential interaction in the hindgut of growing pigs was also discussed using the cross test of the two types of DF in vivo.Experiment 1 Effects of high-level dietary SDF and IDF on the growing performance,organ indexes and microbial community in the hindgut of BALB/c miceA total of 36 healthy BALB/c male mice(17.95 ± 0.95 g)were selected and randomly divided into 4 groups based on their body weight.Mice in control group were fed with basal diet(C,no non-feed-sourced fibers),while mice in the other three groups were fed with basal diet containing 28%oat derived β-glucan(G,the purity was 70%),20%MCC(M,the purity ≥ 99%),and 14%glucan and 10%MCC,respectively.The nutrient levels of diet for each group was consistent.The whole experiment lasted for 21 days.Results1.High level(20%)of DF significantly reduced the average feed intake(ADFI)of the mice(P<0.01),and the trend presented as follows:C>M>GM>G.The average weight gain(AWG)of mice in different groups showed no significant differences(P>0.05).Compared to group G and M,the epididymal fat index of mice in group GM showed significantly higher(P<0.05).2.The concentration of acetate in the colonic digesta of mice in group G and GM tended to be increased compared with those in group M and C(P<0.1),in which the concentration showed 65%higher in group G than that in group C and 52%higher than that in group GM.The concentration of propionate in the colonic digesta of mice in G and GM was significantly higher that that in group M and C(P<0.01),in which the concentration showed 75%higher in group G than that in group C and 71%higher than that in group GM.4.The result of PCR-DGGE(Denaturant Gel Gradient Electrophoresis)analysis showed that at day 4,the bacterial Shannon index of fecal samples from mice in group G was significantly lower than group C(P<0.05),while at day 7,this index of samples from group G was significantly lower than the other three groups(P<0.01).At day 10,the bacterial Shannon index of fecal samples from group M showed significantly higher than group G and GM(P<0.01).5.The relative abundance of Bacteroidetes in the colon of mice in group G was significantly higher than the other three groups(P<0.01),while the abundance of this phylum in the colon of mice in group M and GM(P = 0.03,0.02,respectively)was significantly higher than group C(12.9 ± 4.2%).The relative abundance of Firmicutes in the colon of mice in group C was highest(80.1 ± 4.8%),but it showed significantly lower in the colon of mice in group G(38.7 ± 14.8%)compared to other groups(P<0.01).6.The relative abundance of β-and γ-proteobacteria in the colon of mice in group G was significantly higher than other groups(P<0.01),while the abundance of 8-proteobacteria was significantly lower than others(P<0.01).The relative abundance of family Enterobacteriaceae was significantly lower than other groups(P<0.01).7.The bacterial genera associated with SDF utilization in the colon of mice were represented by Bacteroides spp.,Oscillospira spp.,Prevotella spp.and Ruminococcus spp..On the other hand,the bacterial genera associated with IDF utilization in the colon of mice were represented by Oscillospira spp.、Ruminococcus spp.、Desulfovibrio spp.、Coprococcus spp.and Bacteroides spp..8.The result of qPCR analysis showed that the number of SRB(Sulfate Reducing Bacteria)was significantly lower in the colon of mice in group G,while represented highest in the colon of mice in group M and GM(P<0.01).However,the absolute copy number of archaeal mcrA gene was not observed significantly changed among the different four groups(P>0.05).Our results suggested that there were core bacterial groups specifically using SDF and IDF in the hindgut of mice.Compared to IDF,more amount of short chain fatty acids(SCFAs),especially propionate and acetate,was produced through the fermentation of SDF by the bacteria in the hindgut,which in turn to possibility reduce the loss of body weight and energy induced by the decrease of ADFI by the direct supplement of energy or gluconeogenesis pathway.Experiment 2 The response of bacteria and methanogens in the hindgut of pig to the change of types of dietary fibersA total of 16 healthy and castrated growing DLY(Duroc×Landrace×Yorkshire)(40.38 ± 1.19 kg)pigs were selected and randomly divided into 2 groups according to their body weight.The whole experiment was divided into four periods.The first period lasted for one week and each of the next 3 periods lasted for 3 weeks.The supplementary DF were oat derived β-glucan(SDF)and MCC(IDF,same as experiment 1).A cross test design was used in current study.All of the animals were fed with basal diet at the first experimental period.During the second period,pigs in one group(T1)was fed with diet containing 7.14%oat beta-glucan,while those pigs in another group(T2)was fed with diet containing 5%MCC.During the third period,pigs in group T1 were fed with the diet containing 5%MCC and pigs in group T2 were fed with the diet containing 7.14%oat beta-glucan.All pigs were fed with basal diet(same as the first period)during the last period.A digestion trial lasted for 4 days was conducted at the end of each of the last three experimental periods.The nutrient levels of basal and all fibrous diets were similar.The rectal content of each pig was collected every two days,and the rectal content was continuously collected for 3 days when the diet was changed.Results1.When the IDF containing diet was changed into SDF containing diet,the ADFI of pigs in group T2 was significantly lower than groupTl(P<0.01).2.The apparent digestibility of crude protein(CP),crude fat(EE),crude ash(Ash),crude fiber(CF)and acid detergent fiber(ADF)was significantly affected by the types of DF.Compared with those pigs fed IDF containing diet,the apparent digestibility of CP,EE and Ash of pigs fed SDF containing diet was significantly decreased(P<0.05),while the apparent digestibility of CF and ADF showed significantly increased(P<0.01).3.The real-time response of bacteria in the hindgut of pigs showed opposite to dietary SDF and IDF.The change of bacterial diversity in the hindgut of pigs fed SDF containing diet showed a reducing tendency.Compared with samples collected at day 7(the Shannon index was 3.32 ± 0.09),the bacterial Shannon index of samples collected at day 28(Shannon index was 3.51 ± 0.18)was significantly decreased(P<0.01),while the βdiversity index of bacterial in the hindgut of these pigs showed increase.On the other hand,the change bacterial diversity in the hindgut of pigs fed IDF containing diet showed an increasing tendency.Compared with samples collected at day 7(3.25 ± 0.08),the bacterial Shannon index of samples collected at day 28(3.57± 0.10)was significantly increased(P<0.01),while the βdiversity index showed decreased.4.When the type of DF changed from IDF into SDF,the responses of bacteria and methanogens in the hindgut of pigs were more sensitive and the bacterial diversity fluctuated remarkably.The bacterial Shannon index of samples collected at day 29,31,40 and 49 was 3.62 ± 0.12,3.12 ± 0.09,3.47 ± 0.06 and 3.15 ± 0.09,respectively(P<0.01).The Shannon index of methanogens showed a remarkable increase during the early period and then decreased.The Shannon index of methanogens of samples collected at day 29,31,43 and 49 was 2.77 ± 0.15,2.80 ± 0.09,2.96 ± 0.09 and 2.55 ±0.10,respectively(P<0.01).5.The relative abundance of Firmicutes in the hindgut of pigs fed SDF containing diet was increased from 73%(d7)to 82%(d8)rapidly and then decreased to 68%(d22),while the relative abundance of Bacteroidetes showed an opposite change:rapidly decreased from 17%(d7)to 6%(d8)and then increased to 19%(d22).The relative abundance of Firmicutes in the hindgut of pigs fed IDF containing diet showed decreased from 81.99%(d7)to 63.33%(d22)and then slightly increased(71.02%,d28).6.LefSe analysis showed that the specific bacterial groups in the hindgut of pigs in T1 group at the second period belonged to Actinobacteria and Firmicutes,which also included polysaccharides-utilizing bacteria such as Mitsuokella,Ruminococcaceae and Megasphaera.More specific bacterial taxa belong to Actinobacteria,Firmicutes,Proteobacteria and Bacteroidetes were found in the hindgut of pigs in T1 group at the third period.The classified genera included Sharpea,Epulopiscium and Clostridium.The number of specific bacterial taxa of samples in the samples collected at day 51(the fourth period)was the highest compared to those samples collected at other times,and belonged to Proteobacteria,Lentisphaerae,Verrucomicrobia,Bacteroidetes and Firmicutes.The specific bacteria in the hindgut of pigs in T2 group at the second period belonged to Bacteroidetes,Firmicutes and Chlamydiae,which were identified belonging to Firmicutes and Spirochaetes at the third period.The specific bacteria in the samples collected at day 51 belonged to Proteobacteria,Lentisphaerae,Verrucomicrobia,Bacteroidetes and Firmicutes.Results above showed that there were bacteria specifically using SDF and IDF in the hindgut of pigs.When the types of DF changed,the alteration of bacterial community showed more drastically compared to methanogens.The community of methanogens in the hindgut of pigs was remarkably influenced by the order of ingestion of SDF and IDF.The change of methanogen community was observed later than bacteria,indicating a complex interaction between the two microbes during the fermentation of DF in the hindgut of the pigs.The alteration of microbial community can directly impact microbial function(such as the alteration of SCFAs),and in turn,induce the fermentation in the hindgut and indirectly influence the efficiency of DF utilization of the microbes as well as the nutrients digestibility of host.In summary,there are bacteria specifically using SDF and IDF in the hindgut of both mice and pigs.The response of microbes in the hindgut to the change of fiber types is complicated,in which methanogens and bacteria have a complex interaction.High-level of SDF can be fermented in the colon to produce more SCFAs(especially propionate)compared to IDF,promoting the growth of the animals.Results of current study provide a new thought for the better utilization of different types/sources of DF in animal production.更多还原