【作者】 张涛；

【导师】 毛胜勇；

【作者基本信息】 南京农业大学 ， 动物营养与饲料科学， 2021， 硕士

【摘要】 亚急性瘤胃酸中毒(subacute ruminal acidosis,SARA)是反刍动物养殖过程中一类常见的营养代谢疾病。以往研究表明,反刍动物对SARA的耐受性普遍存在个体差异。然而,以往的研究重在描述日粮形态或动物采食行为对SARA耐受性的影响,对造成SARA耐受性差异的生理因素尚不清楚。因此,本研究以奶牛为对象,从瘤胃微生物-瘤胃代谢物-瘤胃上皮角度探究奶牛发生SARA耐受性不同的机制,同时描述了两组奶牛的血浆及乳代谢物的组成差异。主要内容如下:1.SARA耐受性不同奶牛的瘤胃发酵参数和瘤胃上皮形态及功能研究本试验旨在探究对SARA耐受性不同奶牛的瘤胃发酵参数、瘤胃上皮形态及其功能差异。选取12头装有永久性瘤胃瘘管的泌乳中期荷斯坦奶牛,饲喂精粗比为4:6的日粮,整个试验期为35天,并在第34和35天采集样品。根据检测的瘤胃p H值的高低,将奶牛分为SARA易感组(SUS,n=4)和SARA耐受组(TOL,n=4)。采集瘤胃液用于测定瘤胃p H和VFA浓度,采集瘤胃上皮乳头用于上皮形态及功能研究,收集奶样用于检测乳成分。结果显示,与TOL组比较,SUS组奶牛瘤胃p H(P<0.05)显著降低,而两组奶牛的干物质采食量、产奶量及乳成分均没有差异(P>0.05)。瘤胃发酵参数测定结果显示,SUS组瘤胃内TVFA、丙酸、丁酸、戊酸浓度及丁酸比例显著高于TOL组(P<0.05)。瘤胃上皮形态学测定及功能分析结果表明,SUS组奶牛瘤胃上皮棘突层和基底层厚度增厚(P<0.05)。SUS组H~+转运载体NHE1、NHE2、NHE3和调节胞内p H的v H+ATPase和Na+/K+ATPase的表达量显著升高(P<0.05),而参与VFA吸收的PAT1、MCT4和DRA基因表达量显著降低(P<0.05);瘤胃VFA代谢相关的基因定量结果表明,SUS组PDHA 1和SREBP-2的表达量显著升高(P<0.05),而HMGCL-2的表达量显著降低(P<0.05);对调控瘤胃上皮细胞增殖和凋亡的基因分析显示,SUS组CDK 2、CDK6和Cyclin D1、Bad及Caspase-9基因表达量极显著高于TOL组(P<0.001)。综上表明,奶牛对SARA的耐受性差异与干物质采食量无关,SARA易感奶牛瘤胃上皮细胞对游离脂肪酸吸收速率降低,而对非游离离脂肪酸的吸收速率增加,并一定程度上造成瘤胃内VFA的累积,引起瘤胃p H下降,增加了奶牛患SARA的风险。2.SARA耐受性不同奶牛的瘤胃菌群差异研究本章旨在探究对SARA耐受性不同奶牛的瘤胃菌群差异。试验设计同第一部分,试验期第35天晨饲前采集瘤胃内容物,并提取瘤胃微生物DNA,用于16S r RNA和18S r RNA检测。基于16S r RNA的高通量测序结果表明,SUS和TOL两组奶牛的瘤胃细菌群落明显区分开(PCo A,P<0.05),而Alpha多样性指数均没有差异(P>0.05)。与TOL组相比,在属水平上,SUS组奶牛瘤胃微生物区系中Papillibacter和unclassified Family＿XIII的相对丰度显著降低。在OTU水平上,SUS组瘤胃微生物区系中15个OTU的相对丰度显著升高(P<0.05),其中9个OTU属于Prevotella菌属,且OTU239、OTU156及OTU146的丰度与瘤胃p H呈显著负相关(r<-0.8,P<0.05),OTU239和OTU146丰度与丙酸浓度呈显著正相关(r>0.8,P<0.05)。此外,有20个OTU的相对丰度显著降低(P<0.05)。基于18S r RNA的高通量测序结果显示,两组奶牛的瘤胃原虫群落的丰富度(OTU numbers)、均匀度(shannon)及Chao 1指数均没有差异(P>0.05),SUS组和TOL组的瘤胃原虫在属水平及OTUs水平均没有显著性差异(P>0.05)。综上所述,SUS奶牛瘤胃中主要淀粉降解菌-Prevotella菌属部分OTUs丰度的升高,加速日粮中淀粉多糖的降解,可能是导致瘤胃VFA浓度升高、瘤胃p H降低的重要因素。3.SARA耐受性不同奶牛的瘤胃、血浆及乳代谢组差异研究本章旨在探究对SARA耐受性不同奶牛的瘤胃、血浆及乳中代谢物组成差异,并解释其与SARA耐受性的潜在联系。试验设计同第一部分,分别采集晨饲前瘤胃液、早晚乳样及晨饲后6 h的血液用于代谢组学分析。基于LC-MS代谢组学分析显示,瘤胃液中共鉴定出195种代谢产物,其中135种瘤胃代谢物的含量在两组间有显著差异(P<0.05)。与TOL组相比,SUS组瘤胃中126种代谢物含量显著升高(P<0.05),这些差异代谢物主要为氨基酸、脂质、有机酸、糖类、生物胺等物质;而9种代谢物含量显著降低(P<0.05),如硬脂酸、肌苷和9-HETE等。差异代谢物通路富集分析结果显示,与氨基酸代谢和生物合成、嘌呤代谢、三羧酸循环和丁酸代谢等有关的10条代谢通路得到显著富集(Impact>0.1,P<0.05)。血浆代谢组分析结果显示,两组奶牛血浆中共鉴定出208种代谢物。与TOL组比较,SUS组血浆中MG(18:0/0:0/0:0)、9-HODE、12(13)Ep-9-KODE、Niacinamide、Isovalerylcarnitine、Phosphocreatine、L-Glutamic acid等7种化合物的含量显著升高(P<0.05),而L-Phenylalanine的含量显著降低(P<0.05)。通路富集分析结果显示,与苯丙氨酸、酪氨酸和色氨酸生物合成、D-谷氨酰胺和D-谷氨酸代谢、苯丙氨酸代谢、烟酸和烟酰胺代谢及精氨酸生物合成等有关的6条通路得到显著富集(Impact>0.1,P<0.05)。基于乳代谢组分析结果,乳中共鉴定出188种小分子代谢物,其中16个代谢物含量显著差异(P<0.05)。与TOL组比较,除1-Stearoylglycerophosphoserine和Inosine 2’,3’-cyclic phosphate外,其余14种物质含量在SUS组显著降低。代谢物富集通路结果显示,乳中甘油磷脂代谢通路显著富集(Impact>0.1,P<0.05)。上述结果表明,SUS组奶牛瘤胃生理代谢明显更为活跃,其中丙酸代谢及氨基酸代谢等多种代谢活动明显增强,并增加了瘤胃VFA的积累和生物胺等促炎因子的释放,导致患SARA风险性升高。此外,SUS组乳中甘油磷酯代谢减弱,暗示其乳脂合成能力可能较低。综上所述,本研究发现,泌乳奶牛对SARA的耐受性普遍存在个体差异,且该差异与奶牛的干物质采食量无关。在本研究中,SARA的耐受性不同的原因主要有:SUS组奶牛瘤胃Prevotella菌属中部分OTUs丰度的增加可促进日粮中淀粉多糖的降解,加速瘤胃丙酸、丁酸及乳酸等有机酸的积累,从而导致瘤胃p H下降;其次,SUS组瘤胃上皮中吸收游离脂肪酸的PAT1、MCT4和DRA表达量下调,而H~+转运载体NHE1、NHE2、NHE3和调节胞内p H的v H+ATPase和Na+/K+ATPase的表达量显著上调,表明SUS奶牛对游离脂肪酸吸收降低,而对非游离脂肪酸的吸收增加,间接导致瘤胃中VFA的积累。此外,SUS奶牛瘤胃微生物的氨基酸合成及代谢明显增强,生物胺等致炎物质的生成数量增多,这些物质一方面可能损害瘤胃上皮屏障正常功能,另一方面潜在的增加了奶牛发生SARA的风险。更多还原

【Abstract】 Subacute ruminal acidosis（SARA）is a common metabolic disease in ruminant.Previous studies have shown that a wide variation in tolerance to SARA exists among ruminant animals.However,most of the research conducted on the variation in SARA tolerance focused on the factors related to diet and feeding behavior,and the physiological factors which remain unclear.In this study,we performed an integrated analysis of rumen microbiota,ruminal epithelium in lactation cow with variation in SARA tolerance.Besides,we also compared the difference in plasma and milk metabolome between the two groups of cows.1.Comparision of the rumen fermentation parameters and ruminal epithelial morphology and function in dairy cows with variation in SARA toleranceThe objective of this study is to investigate the rumen fermentation parameters,rumen epithelial morphology,and function in dairy cows with variation in SARA tolerance.Twelve multiparous ruminally cannulated mid-lactating Holstein cows were housed in individual tie-stalls and fed a diet with a concentrate to forage ratio of 4:6throughout the experimental period.The experimental period was 35 days,and samples were collected on the 34^th and 35^th days.Based on the ruminal p H,cows with relatively lower and higher mean ruminal p H was assigned to the susceptible（SUS,n=4）and tolerant（TOL,n=4）groups.The ruminal p H and VFA concentrations of rumen fluid were measured,while the rumen papilla was collected through the rumen fistula and analyzed for morphology and function.Then,50 m L milk was collected for composition analysis.The results showed that the ruminal p H was lower in the SUS group（P<0.05）compared with the TOL group,while no significance difference in the DMI,milk yield and component were observed between two groups（P>0.05）.Rumen fermentation parameters analysis showed that the concentrations of TVFA,propionate,butyrate,and valerate were higher in the SUS group（P<0.05）than those in TOL group.The examination of ruminal epithelial morphology and function revealed that the thickness in the spinous process and basal layer of ruminal epithelium were higher in the SUS group.In the SUS group,the expression levels of PAT1,MCT4,and DRA genes involved in VFA absorption were significantly lower（P<0.05）compared with the TOL group,while the NHE1,NHE2,NHE3,v H⁺ATPase,and Na⁺/K⁺ATPase expression were higher in the SUS group than those in TOL group（P<0.05）.However,the SUS group had a significantly lower expression of HMGCL-2 than that in the TOL group.Compared with the TOL group,the expression levels of cell involved in cell proliferation and apoptosis such as the CDK2,CDK6,Cyclin D1,Bad and Caspase-9were significantly higher in the SUS group（P<0.05）.In conclusion,the dairy cow has a wide variation in SARA tolerance which is independent of dry matter intake and milk performance.In SUS cows,the ability to absorb free fatty acid by ruminal epithelial cell was relatively weak,while the ability of undisintegrated fatty acid absorption was higher.These results lead to accumulation of VFA in the rumen,thereby decreasing the ruminal p H and increasing the risk of SARA in the dairy cows.2.Study on the difference in rumen microbiota of dairy cows with variation in SARA toleranceThe purpose of this chapter was to explore the difference in rumen microbiota of dairy cows with variation in SARA tolerance.Rumen contents were collected through rumen fistulas before morning feeding at 35 d,and microbial DNA was extracted and used for 16S r RNA and 18S r RNA sequencing.The results of 16S r RNA gene sequencing showed that the bacterial communities were clearly separated in the two groups（PCo A,P<0.05）,while the alpha diversity indexes showed no difference（P>0.05）.Compared with the TOL group,at the genus level,the relative abundance of Papillibacter and unclassified Family＿XIII were significantly higher in the SUS group（P<0.05）.At the OTUs level,abundance of 15 OTUs were significantly higher in the SUS group（P<0.05）,and 9 OTUs among them belonged to Prevotella.Moreover,the abundance of OTU239,OTU156,and OTU146 were negatively correlated with ruminal p H（r<-0.8,P<0.05）,while OTU239 and OTU146 were positively correlated with propionate concentration（r>0.8,P<0.05）.In addition,compared with the TOL group,the relative abundance of 20 OTUs were significantly lower in the SUS group（P<0.05）.The result of 18S r RNA genus sequencing revealed that protozoal communities were not distinct separation between the two groups（P>0.05）,and there was also no difference in Alpha diversity indexes（P>0.05）.The results of statistical analysis showed that no significant difference was observed in the composition between the two group（P>0.05）.In summary,the increased abundance of OTUs belong to the genus Prevotella,the dominant starch-degrading bacteria in the rumen of SUS dairy cows,promotes the degradation of starch polysaccharides in the diet,which may be an important factor leading to the increase of rumen VFA concentration and the decrease of ruminal p H.3.Study on the difference in rumen,plasma,and milk metabolome of dairy cows with variation in SARA toleranceThis chapter aims to explore the differences in the rumen,plasma,and milk metabolites in dairy cows with different levels of SARA tolerance,and to explain the potential association among them.Ruminal fluid was collected before morning feeding.Milk samples were collected in the morning and evening,while the blood samples were collected at 6 h after morning feeding.The rumen fluid,milk,and blood samples were further used for metabolomics analysis.Based on the results of ruminal LC-MC metabolomics,a total of 195 metabolites were identified in the two groups,and 135metabolites were significantly different among of them（P<0.05）.Compared with the TOL group,126 differential compounds were high in the SUS group（P<0.05）,which mainly including amino acids,lipids,organic acids,sugars,biogenic amines and other substances.Meanwhile,9 metabolites were low in the SUS group,such as stearic acid,inosine,and 9-HETE.According to the pathway topology analysis,10 metabolic pathways were significantly enriched by differential metabolites（Impact>0.1,P<0.05）,including amino acid metabolism and biosynthesis,purine metabolism,tricarboxylic acid cycle and butyrate metabolism,etc.Analysis of plasma metabolome showed that 208 metabolites were identified in two groups.In SUS group,the levels of MG（18:0/0:0/0:0）,9-HODE,12（13）Ep-9-KODE,niacinamide,Isovalerylcarnitine,phosphocreatine,and L-glutamic acid were increased（P<0.05）,while the L-Phenylalanine level was lower（P<0.05）.The result of pathway topology analysis indicated that 6 metabolic pathways related to phenylalanine,tyrosine and tryptophan biosynthesis,D-glutamine and D-glutamate metabolism,phenylalanine metabolism,nicotinic and nicotinamide metabolism,and arginine biosynthesis were significantly enriched（Impact>0.1,P<0.05）.Milk metabolome analysis revealed that 188metabolites were identified,and 16 metabolites were significantly different among them（P<0.05）.Compared with the TOL group,excepted the levels of 1-Stearoylglycerophosphoserine and Inosine 2’,3’-cyclic phosphate were higher,14metabolites were significantly lower in the SUS group.The pathway topology analysis showed that glycerolipid metabolism was enriched（P<0.05）.In conclusion,the rumen physiological metabolism was significantly stronger in the SUS group,such as propionate metabolism and amino acid metabolism.This will promote the accumulation of VFA and increase the pro-inflammatory factors released in the rumen,such as biogenic amines.Consequently,it could lead to a higher risk of SARA.In addition,the plasma and milk metabolome of cows had a less affected by ruminal metabolites changes.In summary,we found that there was widely individual difference in the SARA tolerance of lacating cows,and it was independent of dry matter intake.In the present study,the main reasons for the variation in tolerance of SARA was that:The increased abundance of some OTUs in the SUS group which belong to Prevotella dpromote the degradation of starch polysaccharides in the rumen,which may accelerate the accumulation of organic acids in the rumen,such as propionate,butyrate,and lactate,and lead to a low ruminal p H.Moreover,the expression levels of PAT1,MCT4,and DRA genes involved in non-free VFA absorption were significantly lower in the SUS group,while the expression of H⁺transporters NHE1,NHE2,NHE3,v H⁺ATPase,and Na⁺/K⁺ATPase were higher.These results indicate tha the absorption of free VFA by SUS cows is relatively lower,and the absorption of non-free fatty acids was higher,which indirectly leads to the accumulation of VFA in the rumen.In addition,the amino acids synthesis and metabolism of rumen microbes were stronger in the SUS group,and these resulted in a higher level of biogenic anmines.A high concentration of biogenic amines may lead to the injury of the barrier function of rumen epithelium and potentially increase the risk of SARA in lactation cows.更多还原