芦丁对奶牛瘤胃体外发酵参数、微生物区系、乳酸代谢产物含量及酶活性的影响

Effects of Rutin on Rumen Fermentation Parameters,Microflora,Lactic Acid Metabolite Contents and Enzyme Activities of Dairy Cows in Vitro

本试验旨在研究高精料底物条件下添加芦丁对奶牛瘤胃体外发酵参数、微生物区系、乳酸代谢产物含量及酶活性的影响。采用体外批次培养法及单因素试验设计,以精粗比为7∶3的0.7 g混合精料(玉米∶豆粕=4∶1)和0.3 g混合干草(苜蓿∶燕麦=1∶1)为发酵底物,按芦丁的添加水平[0(对照)、1.5%、3.0%和4.5%]分为4组,每组设3个平行;同时,选取4头体况相近的健康荷斯坦奶牛,晨饲前采集瘤胃液,制备人工瘤胃培养液,于39℃下进行厌氧发酵,并分别于培养3、6、9、12、18和24 h时取样测定相应指标。结果表明:1)在高精料底物条件下,瘤胃发酵液pH<5.80的时间长达6 h,表明成功建立了亚急性瘤胃酸中毒(SARA)模型;且与对照组相比,添加1.5%和3.0%芦丁能显著提高18~24 h瘤胃发酵液pH(P<0.05),显著降低24 h丙酸、异丁酸、戊酸及乳酸含量(P<0.05),显著降低24 h甲烷产量(P<0.05)。多项指标综合指数(MFAEI)结果表明,芦丁添加水平为4.5%时,对瘤胃液体外发酵参数产生了正组合效应,具有较好的体外发酵效果。2)与对照组相比,添加1.5%和3.0%芦丁能显著降低6~12 h产甲烷菌、6 h乳酸杆菌和6~12 h牛链球菌数量(P<0.05),添加3.0%芦丁能显著降低6~24 h乳酸杆菌(P<0.05),而添加4.5%芦丁能显著提高12 h反刍兽月形单胞菌和埃氏巨型球菌数量(P<0.05)。3)与对照组相比,添加1.5%芦丁能显著降低12 h丙酮酸含量(P<0.05),添加1.5%和4.5%芦丁能显著降低6 h乳酸脱氢酶(LDH)、丙酮酸激酶(PK)及淀粉酶(AMS)活性(P<0.05)。4)与对照组相比,添加芦丁能显著降低6~12 h脂多糖(LPS)含量(P<0.05)。综上所述,在高精料条件下添加芦丁能改善奶牛体外发酵,抑制甲烷产生,以4.5%为适宜添加水平;且芦丁能通过降低LPS含量、抑制乳酸产生菌和乳酸代谢相关酶活性减少乳酸产生,减缓瘤胃发酵液pH的下降,缓解高精料诱导的SARA。

This experiment was conducted to investigate the effects of rutin supplementation on rumen fermentation parameters,microflora,lactic acid metabolite contents and enzyme activities of dairy cows in vitro under the condition of high-concentrate substrate.Using in vitro batch culture method and single factor experimental design,0.7 g mixed concentrate(corn∶soybean meal=4∶1)and 0.3 g mixed hay(alfalfa∶oat=1∶1)with concentrate to roughage ratio of 7∶3 were used as fermentation substrates,and were divided into 4 groups according to the supplemental levels of rutin[0(control),1.5%,3.0% and 4.5%],and each group had 3 parallels.At the same time,the rumen fluid was collected from 4 healthy Holstein dairy cows with similar body conditions before morning feeding to prepare the artificial rumen culture medium,which was subjected to anaerobic fermentation at 39℃,and the corresponding indices were measured at 3,6,9,12,18 and 24 h,respectively.The results showed as follows:1)under the condition of high-concentrate substrate,the rumen fermentation fluid pH<5.80 lasted for 6 h,indicating that subacute rumen acidosis(SARA)model was successfully established.Moreover,compared with the control group,the supplementation of 1.5%and 3.0%rutin significantly increased the pH of rumen fermentation fluid at 18 to 24 h(P<0.05),significantly decreased the contents of propionic acid,isobutyric acid,valeric acid and lactic acid at 24 h(P<0.05),and significantly decreased the methane production at 24 h(P<0.05).The results of multiple-factor associative effects index(MFAEI)showed that the rutin supplemental level at 4.5%had a positive combined effect on rumen fluid fermentation parameters,and had a good in vitro fermentation effect.2)Compared with the control group,the supplementation of 1.5%and 3.0%rutin significantly decreased the numbers of Methanogens at 6 to 12 h,Lactobacillus at 6 h and Streptococcus bovis at 6 to 12 h(P<0.05),the supplementation of 3.0%rutin significantly decreased the Lactobacillus number at 6 to 24 h(P<0.05),and the supplementation of 4.5%rutin significantly increased the numbers of Selenomonas ruminantium and Megacoccus escheriensis at 12 h(P<0.05).3)Compared with the control group,the supplementation of 1.5%rutin significantly decreased the pyruvate content at 12 h(P<0.05),and the supplementation of 1.5%and 4.5%rutin significantly decreased the activities of lactate dehydrogenase(LDH),pyruvate kinase(PK)and amylase(AMS)at 6 h(P<0.05).4)Compared with the control group,the rutin supplementation significantly decreased the lipopolysaccharide(LPS)content at 6 to 12 h(P<0.05).In conclusion,the rutin supplementation can improve the in vitro fermentation of dairy cows and inhibit the methane production under the condition of high-concentrate,and the appropriate supplemental level is 4.5%.In addition,the rutin can reduce the lactic acid production by reducing LPS content,inhibiting lactic acid producing bacteria and lactic acid metabolization-related enzyme activities,slow down the decrease of the rumen fermentation fluid pH,and relieve the SARA induced by high concentrate.