【作者】 李斌；

【导师】 王雅春； 马云；

【作者基本信息】 宁夏大学 ， 畜牧学， 2025， 博士

【摘要】 青藏高原作为世界上海拔最高的牧业区,其低氧、低温、强紫外线辐射环境对奶牛健康、繁殖与生产性能构成了严峻挑战。引入的荷斯坦牛品种,因原产于温带低海拔地区,普遍在初生到后备牛阶段会普遍出现发育迟缓、免疫抑制甚至早期死亡等问题,严重影响了种群稳定性和牧场经济效益。瘤胃微生物作为奶牛的“第二套”基因组,其构成的复杂群落主导着宿主的营养获取和代谢稳态,可能在这场“生命对抗环境”过程中,发挥了重要作用。基于此,本研究以瘤胃微生物为核心,基于大规模前瞻性队列捕获表型动态特征,结合精细化小队列阐明调控机制,最后围绕并贯通“宿主基因型关键瘤胃微生物”轴,为高原奶业发展提供了新视角。主要研究内容与发现如下:1.聚焦荷斯坦牛后备牛在高原环境下的生存发育动态特征,对210头后备牛(母牛)进行1月、2月、3月、6月、12月纵序跟踪测定。结果显示,6月龄时体长指数、胸围指数、管围指数达到峰值,分别为114.20%、135.11%和20.23%;12月龄时体躯指数达到最大,为124.12%;总体表明,6月龄是荷斯坦牛后备牛在高原环境下体型比例大幅调整的关键窗口期。0-3月龄为死亡高风险期,达到29%,肺炎、腹泻和高原病是最主要的疾病干扰;使用随机森林模型发现,大血小板比率、单核细胞数等10项指标对宿主生存死亡结局具有重要作用。2.相关时序瘤胃微生态特征与宿主生存发育关联分析显示,菌群多样性随时序显著增加;菌群结构在6月龄趋于稳定,Firmicutes和Bacteroidota为门水平优势菌。存活牛瘤胃Lachnospirales、Succinivibrio、Christensenellaceae和Methanobrevibacter等有益菌丰度较高,而死亡牛中更为富集Anaerovorax、Muribaculaceae、Cloacibacillus和Megasphaera等潜在致病菌。功能预测分析显示,存活组的能量代谢、挥发性脂肪酸(VFAs)合成及炎症调节通路活性更高。3.进一步选择其中55头荷斯坦牛后备牛,细分发育阶段,测定7、14、21日龄及1、2、3、6、12月龄体尺体重,瘤胃VFAs、微生物宏基因组,血液常规指标和代谢组。分析显示,高原荷斯坦牛后备牛微生物定植呈现出阶段特异性模式:瘤胃微生物群落随发育进程多样性显著增加,6月龄后趋于稳定。早期富集具有免疫调节功能的类群(Akkermansia、Bacteroides),随着犊牛成熟,厌氧发酵菌群(Prevotella、Succiniclasticum)逐渐占据主导地位,增加驱动VFAs合成酶活性,支持快速增重,反映营养利用策略的阶段性调整。按生存结局(存活组vs死亡组)比较发现,死亡犊牛表现出微生物多样性降低、致病菌(Fusobacterium necrophorum、Porphyromonas canoris)富集,以及与氨基酸和能量代谢相关的血液代谢谱改变。功能注释分析强调了年龄依赖性富集的通路,包括碳水化合物降解、甲烷生成和免疫调节等,其中关键酶(如EC 3.2.1.4、EC 6.2.1.1)的活性在6-12月龄达到峰值。4.血液代谢组学分析发现氨基酸、碳水化合物和胆汁酸等代谢物随发育阶段波动,参与能量代谢和免疫调节。早期(7天至2个月)反映糖酵解依赖;断奶后VFAs成为主要能量来源,代谢模式趋向成熟。Cox回归分析鉴定出与生存显著相关的微生物和代谢生物标志物(Prevotella ruminicola和赤藓糖醇),可作为早期预警生物标志物。这些发现构建了高原犊牛微生物与代谢成熟的时序图谱,突显了宿主微生物代谢互作在犊牛适应高原胁迫因子中的关键作用,为在极端环境下提升奶牛生产力提供了可操作的见解。5.进一步聚焦于瘤胃微生物的遗传调控基础,基于丰度与Cox回归模型结果,选取前期研究中生长发育以及存活相关的35个重要瘤胃微生物,结合Illumina Bovine芯片数据进行遗传力估计与GWAS关联分析,探索宿主对菌群丰度的遗传调控能力。结果显示,6月龄及12月龄宿主遗传调控增强,Bifidobacterium、Succiniclasticum等菌属在特定阶段表现出高遗传力(h~2≈0.999–1.0),进一步关注宿主遗传调控的关键基因与通路发现Lachnoclostridium的GWAS分析识别出7个显著SNP和11个潜在位点,注释至DIRAS3和GNG12等基因,功能富集至GABA能突触、HIF1信号通路等,结合50头健康后背牛验证表明Lachnoclostridium可通过调控HIF1和LXRα信号介导早期生长发育和生存结局。研究结果为断奶及育成期精准微生态干预和遗传育种提供了理论依据,未来可通过多组学验证推动高原奶牛健康管理。综上所述,本研究基于系统的纵向采样与多组学整合,构建了高原荷斯坦牛后备牛“发育表型瘤胃微生物宿主遗传”健康调控框架,识别了多个与早期死亡相关的风险因子与保护因子,并提出了基于微生物代谢互作的预警模型及潜在遗传干预靶点。研究成果为高原地区奶牛种群的健康管理和分子育种提供了理论依据与关键路径支撑。更多还原

【Abstract】 The Qinghai Xizang Plateau,as the world’s highest altitude pastoral region,poses severe challenges to dairy cattle health,reproduction,and production performance due to its hypoxic,low temperature,and high ultraviolet radiation environment.Introduced Holstein cattle breeds,originating from temperate low altitude areas,commonly exhibit developmental retardation,immunosuppression,and early mortality during the period from birth to replacement heifer stage,severely impacting herd stability and farm economic benefits.As the"second"genome of dairy cattle,the rumen microbiota may play a crucial role in this"struggle against environmental stressors."Based on this,this study focused on the rumen microbiota.It employed a"large scale"prospective cohort to capture key dynamics,connected with refined small cohorts to elucidate regulatory mechanisms,and ultimately centered on the"host genotype/key rumen microbe"axis to provide a new perspective for the development of the high altitude dairy industry.The main research content and findings are as follows:Focusing on the survival and developmental dynamics of Holstein replacement heifers in the high altitude environment,210 replacement heifers(female)were longitudinally tracked and measured at 1,2,3,6,and 12 months of age.Results showed that body length index,chest girth index,and cannon bone circumference index peaked at 6 months of age,reaching 114.20%,135.11%,and 20.23%respectively.The body mass index reached its maximum at 12 months,at 124.12%.Collectively,this indicates that 6 months of age is a critical window period for significant proportional body size adjustments in Holstein replacement heifers adapting to the high altitude environment.The period of 0-3 months was a high risk period for mortality,reaching 29%,with pneumonia,diarrhea,and altitude sickness being the primary disease disturbances.Using a Random Forest model,10indicators,including large platelet ratio and monocyte count,were found to be significantly important for host survival/mortality outcomes.Correlation analysis between rumen microbiota characteristics(16S r RNA gene sequencing)and host survival/development revealed that microbial diversity significantly increased over time;microbial structure stabilized around 6 months of age,with Firmicutes and Bacteroidota being the dominant phyla.Surviving calves exhibited higher abundances of beneficial bacteria such as Lachnospirales,Succinivibrio,Christensenellaceae,and Methanobrevibacter in the rumen,while deceased calves were more enriched with potentially pathogenic bacteria like Anaerovorax,Muribaculaceae,Cloacibacillus,and Megasphaera.Functional prediction analysis showed that the surviving group had higher activity in pathways related to energy metabolism,VFAs synthesis,and inflammation regulation,indicating a consistent association between microbial functional potential and health outcomes.Further selecting 55 Holstein replacement heifers from the cohort,developmental stages were finely divided to measure body size and weight,rumen VFAs,microbial metagenomes,blood routine parameters,and metabolomes at 7,14,21 days and 1,2,3,6,and 12 months of age.Analysis showed that rumen microbial colonization in highaltitude Holstein replacement heifers exhibited stages pecific patterns:rumen microbial community diversity significantly increased with development,stabilizing after 6 months;immunomodulatory groups(e.g.,Akkermansia,Bacteroides)were enriched early,while as calves matured,anaerobic fermentative bacteria(e.g.,Prevotella,Succiniclasticum)gradually dominated,driving increased activity of VFAs synthesis enzymes,supporting rapid weight gain,and reflecting stages pecific adjustments in nutrient utilization strategies.Comparing survival outcomes(Survived group vs.Deceased group),deceased calves exhibited reduced microbial diversity,enrichment of pathogenic bacteria(e.g.,Fusobacterium necrophorum,Porphyromonas canoris),and altered blood metabolite profiles linked to amino acid and energy metabolism.Functional annotation highlighted age dependent enrichment of pathways involved in carbohydrate degradation,methane production,and immune regulation,with critical enzyme activities(e.g.,EC 3.2.1.4,EC6.2.1.1)peaking at 6–12 months.Blood metabolomics revealed fluctuations in metabolites such as amino acids,carbohydrates,and bile acids across developmental stages,participating in energy metabolism and immune regulation.Early stages(7 days to 2months)reflected dependence on glycolysis;after weaning,VFAs became the primary energy source,and the metabolic pattern trended towards maturity.Cox regression identified microbial and metabolic biomarkers significantly associated with survival(e.g.,Prevotella ruminicola,erythritol),which could serve as early warning biomarkers.These findings established a time resolved map of microbial and metabolic maturation in high altitude calves,highlighting the critical role of host microbe metabolic interactions in calves’adaptation to high altitude stressors and providing actionable insights for enhancing dairy productivity in extreme environments.Further focusing on the genetic regulatory basis of the rumen microbiota,35 important rumen microbes previously associated with growth/survival were selected.Combined with Illumina Bovine HD chip data,heritability estimation and GWAS association analysis were performed to explore the host’s genetic regulatory capacity over microbial abundance.Results showed enhanced host genetic regulation at 6 and 12 months.Genera such as Bifidobacterium and Succiniclasticum exhibited high heritability(h~2≈0.999–1.0)at specific stages.Further investigation into key host regulatory genes and pathways revealed that GWAS analysis for Lachnoclostridium identified 7 significant SNPs and 11 potential loci,annotated to genes such as DIRAS3 and GNG12.Functional enrichment pointed to pathways like GABA ergic synapse and HIF1 signaling pathway,suggesting that this genus may play a key role in early growth/survival outcomes by regulating HIF and LXRαmediating developmental stress responses.This provides a theoretical basis for precise microecological interventions during weaning and rearing periods,as well as for genetic breeding.Future multiomics validation can advance health management strategies for high altitude dairy cattle.In summary,based on systematic longitudinal sampling and multiomics integration,this study constructed a health regulation framework of"developmental phenotype rumen microorganism host genetics"for plateau Holstein heifers,identified multiple risk factors and protective factors related to early death,and proposed an early warning model and potential genetic intervention targets based on microbe metabolism interactions.The research results provide a theoretical basis and key path support for the health management and molecular breeding of dairy cow populations in plateau regions.更多还原