围产期奶牛瘤胃微生物区系的变化及微生态制剂的调控作用
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摘要
奶牛处于分娩前三周至分娩后三周这段时间称为围产期。分娩前1周奶牛的采食量下降30%，产后5周内摄食不能满足泌乳所需的能量，这经常会导致能量负平衡，严重的能量负平衡可引发酮病和脂肪肝。瘤胃中乙酸、丙酸和丁酸等挥发性脂酸的比率与瘤胃中微生物的种类和数量密切相关。正常状态下，瘤胃中的碳水化合物被微生物发酵生成挥发性脂肪酸。反刍动物所需的葡萄糖90%是通过糖异生获得，其中50-60%是由丙酸通过糖异生生成。因此，研究围产期奶牛瘤胃微生物区系（尤其是丙酸生成优势菌）的变化及调控对于防治酮病和脂肪肝等围产期能量负平衡性疾病有着重要的意义。本实验通过末端限制性片段长度多态性分析（T-RFLP）技术和荧光定量PCR技术对围产期奶牛瘤胃微生物区系进行分析，并运用瘤胃微生态理论、细菌代谢工程策略，以丙酸生成菌和酵母复合调控瘤胃丙酸生成为技术核心，研制针对围产期奶牛能量负平衡的微生态制剂，为防治围产期奶牛能量负平衡开辟一条新的途径。
连续检测围产期健康奶牛和酮病奶牛瘤胃微生物区系证明，围产期健康奶牛从高粗日粮转换成高精日粮时，瘤胃中乳酸生成菌--牛链球菌和乳酸杆菌的数量显著增加，但丙酸生成菌--反刍月形单胞菌和埃氏巨型球菌的数量显著减少，乳酸不能充分分解利用，导致挥发性脂酸浓度降低，生糖先质丙酸供给不足，促进了能量负平衡和酮病疾病的发生。
酵母菌与反刍月形单胞菌+埃氏巨型球菌在体外复合培养表明，以啤酒酵母、产朊假丝酵母和伯顿毕赤酵母这三种酵母组合利用乳酸生成丙酸的能力最强，可促进反刍月形单胞菌、埃氏巨型球菌生成丙酸的能力，增加培养基中丙酸的浓度。
围产期健康奶牛和酮病奶牛灌服复合微生态制剂后，可以促进瘤胃反刍兽月形单胞菌、埃氏巨型球菌、产琥珀酸丝状杆菌、纤维素分解菌和厌氧菌的生长和繁殖，抑制乳酸杆菌和牛链球菌的生长；增加瘤胃内挥发性脂肪酸浓度；降低分娩后健康奶牛和酮病奶牛血液中β-羟丁酸浓度，升高血液中葡萄糖浓度。
健康奶牛灌服复合瘤胃微生态制剂后，临床检查、血液生化指标分析未见异常，说明该微生态制剂安全、无毒、可靠。初步证实该微生态制剂可缓解围产期奶牛的能量负平衡。
The transition period of a dairy cow is defined as the period from three weeks priorto calving to three weeks after calving. Feed consumption during the week beforecalving declines 30%, while feed intake during the first five weeks of lactation isinsufficient to meet the increasing energy demands of lactation. During this time,energy output in the form of milk exceeds energy input in the form of feed, which leadsto negative energy balance (NEB) or ketosis. The ratio of acetic acid, propionic acidand butyric acid generated in the rumen is affected by the type of forage, and the speciesand quantity of rumen bacteria. In the normal state, carbohydrates are fermented by avariety of bacteria in the rumen, and transformed into volatile fatty acids by thecorresponding enzymes. In ruminant animals, approximately 90% of the glucose issupplied by gluconeogenesis, with 50-60％of this being derived from propionate. It isimportant to investigate the relationship between composition of the bacterialcommunity and concentration of VFAs in the rumen during the transition period, or inthe case of ketosis, in dairy cows to identify strategies that might alleviate or avoidNEB and ketosis through regulation of microbial fermentation in the rumen. Inpresent study we analyzed the rumen microbiome composition of cows in the transitionperiod, cows with ketosis and non-perinatal cows using terminal restriction fragmentlength polymorphism (TRFLP) analysis of 16S rRNA genes and Quantitative PCR. Andbacteria metabolic engineering strategy and the change of rumen function are used asthe theoretical basis, and with the compound of propionic bacteria and yeast as thecore technology. This will open up a new way for the prevention and control for NEBin transition period cows.
This test proved that transition and ketosis cows into a high-grain from alow-gtain diet, the amount of latic-producing baceria—lactobacillus and streptococcusbovis were significantly increase in the rumen, but the amount of propionic-ptoducingbaceria—selenomonas ruminantium and megasphaera elsdeniim were significantlyreduce in the rumen, lactic acid can not be fully catabolism, resulting in lowerconcentration of VFAs to promote a negative energy balance and ketosis disease.
Screening the optimal yeast combination from Saccharomyces cerevisiae, Candida utilis, Burton pichia pastorios with Selenomonas ruminantium andMegasphaera elsdenii, a rumimal compound probiotics for negative energy balance intransition period cows was developed successfully. The probiotics could promote theuse of lactic acid by Saccharomyces cerevisiae, Candida utilis, Burton pichiapastorios combined with Selenomonas ruminantium and Megasphaera elsdenii, andincreased the concentration of propionic acid in vitro.
When administered with the probiotics to the transition period cows with NEB orketosis, increased the ruminal amount of Selenomonas ruminantium, Megasphaeraelsdeniim, Fibrobacter succinogenes, cellulose decomposition bacteria and anaerobicbacteria; decreased the amount of Lactobacillus and Streptococcus bovis. Increasedthe concentration of VFAs in rumen; decreased the concentration of BHBA in serumof post-partum and ketiosis cows; increased the concentration of glucose in serum ofpost-partum and ketiosis cows.
The health cows which were administered with the composite ruminal probioticsdisplayed no change of the blood biochemical index and clinical manifestation,indicating that the composite ruminal probiotics is safe，non-toxic and reliable fortransition period cows.
连续检测围产期健康奶牛和酮病奶牛瘤胃微生物区系证明，围产期健康奶牛从高粗日粮转换成高精日粮时，瘤胃中乳酸生成菌--牛链球菌和乳酸杆菌的数量显著增加，但丙酸生成菌--反刍月形单胞菌和埃氏巨型球菌的数量显著减少，乳酸不能充分分解利用，导致挥发性脂酸浓度降低，生糖先质丙酸供给不足，促进了能量负平衡和酮病疾病的发生。
酵母菌与反刍月形单胞菌+埃氏巨型球菌在体外复合培养表明，以啤酒酵母、产朊假丝酵母和伯顿毕赤酵母这三种酵母组合利用乳酸生成丙酸的能力最强，可促进反刍月形单胞菌、埃氏巨型球菌生成丙酸的能力，增加培养基中丙酸的浓度。
围产期健康奶牛和酮病奶牛灌服复合微生态制剂后，可以促进瘤胃反刍兽月形单胞菌、埃氏巨型球菌、产琥珀酸丝状杆菌、纤维素分解菌和厌氧菌的生长和繁殖，抑制乳酸杆菌和牛链球菌的生长；增加瘤胃内挥发性脂肪酸浓度；降低分娩后健康奶牛和酮病奶牛血液中β-羟丁酸浓度，升高血液中葡萄糖浓度。
健康奶牛灌服复合瘤胃微生态制剂后，临床检查、血液生化指标分析未见异常，说明该微生态制剂安全、无毒、可靠。初步证实该微生态制剂可缓解围产期奶牛的能量负平衡。
The transition period of a dairy cow is defined as the period from three weeks priorto calving to three weeks after calving. Feed consumption during the week beforecalving declines 30%, while feed intake during the first five weeks of lactation isinsufficient to meet the increasing energy demands of lactation. During this time,energy output in the form of milk exceeds energy input in the form of feed, which leadsto negative energy balance (NEB) or ketosis. The ratio of acetic acid, propionic acidand butyric acid generated in the rumen is affected by the type of forage, and the speciesand quantity of rumen bacteria. In the normal state, carbohydrates are fermented by avariety of bacteria in the rumen, and transformed into volatile fatty acids by thecorresponding enzymes. In ruminant animals, approximately 90% of the glucose issupplied by gluconeogenesis, with 50-60％of this being derived from propionate. It isimportant to investigate the relationship between composition of the bacterialcommunity and concentration of VFAs in the rumen during the transition period, or inthe case of ketosis, in dairy cows to identify strategies that might alleviate or avoidNEB and ketosis through regulation of microbial fermentation in the rumen. Inpresent study we analyzed the rumen microbiome composition of cows in the transitionperiod, cows with ketosis and non-perinatal cows using terminal restriction fragmentlength polymorphism (TRFLP) analysis of 16S rRNA genes and Quantitative PCR. Andbacteria metabolic engineering strategy and the change of rumen function are used asthe theoretical basis, and with the compound of propionic bacteria and yeast as thecore technology. This will open up a new way for the prevention and control for NEBin transition period cows.
This test proved that transition and ketosis cows into a high-grain from alow-gtain diet, the amount of latic-producing baceria—lactobacillus and streptococcusbovis were significantly increase in the rumen, but the amount of propionic-ptoducingbaceria—selenomonas ruminantium and megasphaera elsdeniim were significantlyreduce in the rumen, lactic acid can not be fully catabolism, resulting in lowerconcentration of VFAs to promote a negative energy balance and ketosis disease.
Screening the optimal yeast combination from Saccharomyces cerevisiae, Candida utilis, Burton pichia pastorios with Selenomonas ruminantium andMegasphaera elsdenii, a rumimal compound probiotics for negative energy balance intransition period cows was developed successfully. The probiotics could promote theuse of lactic acid by Saccharomyces cerevisiae, Candida utilis, Burton pichiapastorios combined with Selenomonas ruminantium and Megasphaera elsdenii, andincreased the concentration of propionic acid in vitro.
When administered with the probiotics to the transition period cows with NEB orketosis, increased the ruminal amount of Selenomonas ruminantium, Megasphaeraelsdeniim, Fibrobacter succinogenes, cellulose decomposition bacteria and anaerobicbacteria; decreased the amount of Lactobacillus and Streptococcus bovis. Increasedthe concentration of VFAs in rumen; decreased the concentration of BHBA in serumof post-partum and ketiosis cows; increased the concentration of glucose in serum ofpost-partum and ketiosis cows.
The health cows which were administered with the composite ruminal probioticsdisplayed no change of the blood biochemical index and clinical manifestation,indicating that the composite ruminal probiotics is safe，non-toxic and reliable fortransition period cows.
引文
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