Biosynthesis of arginine from citrulline and related compounds by mixed ruminal bacteria, protozoa and their mixture in vitro

Biosynthesis of arginine (Arg) from citrulline (Cit), ornithine (Orn), proline (Pro), and 5-aminovaleric acid (5AV) by mixed rumen bacteria (B), protozoa (P), and their mixture (BP) was quantitatively investigated in an in vitro system from the standpoint of protein nutrition in ruminants. Rumen microorganisms, collected from ruminally fistulated goats, were anaerobically incubated with or without 1 mmol/L each of substrates at 39°C for 12 h. Arginine and other related compounds, produced in both supernatants and acid-hydrolyzates of microorganisms in B, P, and BP suspensions, were analyzed by high-performance liquid chromatography. Arginine production from Cit in BP, when expressed with a unit of 'μmol/g microbial nitrogen', was approximately 70% and 94% higher than that in B and P, respectively, in a 12-h incubation period. In the case of Orn, the values were approximately 30% and 75%. Both rumen bacteria and protozoa could produce Cit and Orn from Pro, so it is assumed that they can produce Arg from Pro. Rumen protozoa were unable to degrade 5AV and it was the final product in the metabolism of Cit, Orn and Pro in P suspension. A trace amount of Orn and Pro produced from 5AV in B and BP suspensions indicated that the reversible reaction of 5AV formation was performed only by rumen bacteria. This is the first quantitative report on Arg biosynthesis from its precursors by rumen microorganisms.     

Production of aromatic amino acids and related compounds from p-hydroxyphenylacetic acid by rumen microorganisms in vitro

Suspensions of mixed rumen bacteria (B), protozoa (P), and mixed rumen microorganisms (BP) prepared from rumen contents of fistulated goats were anaerobically incubated with 1 mM p‐hydroxyphenylacetic acid (HPA) at 39°C for 24 h. Tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp) and other related compounds in both supernatants and hydrolyzates of microbial cells in all incubations were analyzed by HPLC. Large amounts of Tyr (32.1, 42.7 and 36.1% of disappeared HPA in B, P and BP, respectively) were produced from HPA during a 12 h incubation period. The formation of Tyr in P (178.6 µmol/g MN) was 1.5 and 2 times higher than in B and BP, respectively. Phe (7–11% of the disappeared HPA) and Trp (3–6% of the disappeared HPA) were also synthesized from HPA in B, P, and BP. Phe synthesis in P (46.3 µmol/g MN) was 1.7 times higher than in B but, in contrast, Trp synthesis in B, was 1.6 times higher than in P. The metabolites p‐hydroxyphenylpyruvic acid (in the range of 5–14% of disappeared HPA), phenylacetic acid (1–11%), p‐hydroxybenzoic acid (3–7%) and benzoic acid (1–6%) were produced from HPA in B, P and BP. Phenylpropionic acid (6% of the disappeared HPA) was produced only in B and BP.     

In vitro metabolism of tyrosine by rumen bacteria, protozoa and their mixture

An anaerobic in vitro study of tyrosine (Tyr) metabolism by rumen bacteria (B), protozoa (P) and their mixture (BP) was conducted using an HPLC analytical method to obtain some basic and systematic information on aromatic amino acid metabolism in the rumen. Most of the Tyr (> 96%) disappeared after 12 h incubation in B and BP, but only 58% in P. Tyr was converted mainly to p‐hydroxyphenylacetic acid (HPA) in all microbial suspensions. About 45% of disappeared Tyr in B and P, and about 35% in BP were converted to HPA. An appreciable amount of phenylalanine (Phe), 13 and 3% of disappeared Tyr, and a small amount of tryptophan (Trp), 8 and 1% of disappeared Tyr, were also produced from Tyr by rumen bacteria and protozoa, respectively. Small amounts of p‐hydroxyphenylpyruvic acid (about 4 and 6% of disappeared Tyr) were produced from Tyr in B and P, respectively. A moderately large amount of phenylacetic acid (14% of disappeared Tyr) was produced from Tyr in P which was 1.9 times higher than that in B. Phenylpropionic acid and p‐hydroxybenzoic acid were produced only in B and BP. It was concluded that the Tyr degradation ability of rumen bacteria was about 1.5 times higher than that of rumen protozoa. Degraded Tyr mainly produces HPA and then two other aromatic amino acids, Phe and Trp, which are considered essential amino acids for ruminants. Therefore, it is speculated that the requirement for Phe and Trp in ruminants may be partially fulfilled if Tyr is sufficiently supplied in rations.     

Quantitative studies of the in vitro production of pipecolic acid by rumen protozoa and its degradation by rumen bacteria

An in vitro study was conducted to quantitatively investigate the metabolism of pipecolic acid (Pip), a neuromodulator, by mixed rumen bacteria (B), mixed rumen protozoa (P), a combination of B and P (BP), species‐enriched rumen protozoal suspension (Polyplastron sp., Diploplastron sp., entodinia and Entodinium caudatum) and pure cultures of several isolates of rumen bacteria (Prevetolla bryantii, Prevetolla albensis, Streptococcus bovis, Veillonella parvula, Megasphaera elsdenii and Ruminococcus albus). Only P produced Pip from L‐lysine (1.0 mmol/L L‐Lys) at a rate of 83.5 ± 1.6 µmol/L/h and even in BP, Pip was produced from L‐Lys by P and increased at a rate of 31.2 ± 3.8 µmol/L/h. Pip production by P was highest when the substrate (L‐Lys) concentration was 6 mmol/L and then the rate was 580 ± 36 µmol/L/h. Pipecolic acid production by P suspension enriched with different species of protozoa showed that Polyplastron sp. had the highest Pip production rate of 0.907 ± 0.092 µmol/L/mg protozoal protein per h, and Diploplastron sp. had the lowest rate of 0.55 ± 0.13 µmol/L/mg protozoal protein per h. The addition of D‐Lys (1.0 mmol/L) as a substrate to the P suspension revealed that P were also able to produce Pip from D‐Lys, though at a lower rate (1/3) compared with L‐Lys (1.0 mmol/L), suggesting the presence of epimerases in P. It was confirmed that B were unable to produce Pip from L‐ or D‐Lys. Only B degraded Pip (1.0 mmol/L) after a lag phase at a rate of 56.0 ± 1.5 µmol/L/h. The B suspension was able to degrade D‐Lys, though the products were not identified. Pip degradation by pure culture of some species of rumen bacteria showed that P. bryantii and R. albus had the highest rate followed by P. albensis, S. bovis and M. elsdenii with a low rate of Pip degradation. Veillonella parvula showed no ability to degrade Pip. The results suggest that a fairly large proportion of rumen‐produced Pip is likely to be absorbed by the host animal before degradation by rumen bacteria.     

Urea cycle metabolism: effects of supplemental ornithine or citrulline on performance, tissue amino acid concentrations and enzymatic activity in young pigs fed arginine-deficient diets

Two experiments were conducted with young pigs to determine the efficacy of ornithine (Orn) or citrulline (Cit) as precursors of arginine (Arg). In Exp. 1, pigs were individually fed an Arg-deficient, semipurified diet (.18% Arg) supplemented with .3% Arg or an equimolar quantity of Orn or Cit. Supplemental Arg or Cit increased rate and efficiency of weight gain, but Orn addition was without effect. Free Arg in plasma 3 h post-prandial was increased by addition of either Arg or Cit to the basal diet. Liver Arg was elevated by dietary addition of Arg, Orn or Cit; kidney Arg and Orn were elevated only in pigs receiving supplemental Cit. Arginine or Cit addition to the diet increased Arg concentration in muscle tissue, but muscle Orn was unresponsive to any of the supplements fed. In Exp. 2, pigs were again fed the Arg-deficient, semipurified diet supplemented with .3% Arg or four times an isomolar quantity of ornithine. Arginine addition to the diet increased weight gain and feed efficiency, while Orn supplementation was without effect. Plasma Orn was increased by excess Orn, while plasma Cit was unaffected by supplemental Arg or Orn. Moreover, excess Orn increased free Orn and proline (Pro) in liver, kidney and muscle. Free Cit, however, increased only in liver from feeding excess Orn. In addition, excess Orn decreased both plasma ammonia and free glutamine (Gln) concentration in brain. Arginase activity was roughly 10, 40 and 100 times greater in hepatic tissue than in renal cortex, renal medulla or intestinal mucosa, respectively, while hepatic ornithine transcarbamoylase (OTC) activity was about 15 times greater than the activity present in mucosa tissue. Renal OTC activity was too low to be accurately measured.     

绵羊瘤胃细菌、原虫蛋白质分解代谢相关酶活力及谷氨酸脱氢酶体系的米氏常数值

本试验旨在研究绵羊瘤胃细菌、原虫蛋白质分解代谢相关酶活力及谷氨酸脱氢酶体系的米氏常数(Km)值,为解释绵羊瘤胃细菌、原虫蛋白质分解代谢特征提供酶学依据。选用6只1岁左右安装永久性瘤胃瘘管的中国美利奴(新疆型)绵羊[平均体重为(32.00±1.36)kg],饲喂精粗比为30∶70的饲粮,依次采集饲喂前(0 h)和饲喂后1.5、3.0、6.0、9.0、12.0 h 6个时间点的瘤胃液,重复采集3次。分离和制备细菌、原虫破碎液,分别测定相关酶活力及谷氨酸脱氢酶体系的Km值。结果显示:1)绵羊瘤胃细菌、原虫破碎液中蛋白酶、谷丙转氨酶、谷草转氨酶和谷氨酸脱氢酶的活力随饲喂时间的延长均呈现先升高后降低的动态变化规律,总体在饲喂后1.5 h达到峰值;谷氨酸和氨含量也呈现相似的变化规律。原虫破碎液中参与蛋白质分解代谢的这4种酶的活力在各时间点均极显著高于细菌(P0.01)。2)原虫破碎液谷氨酸含量极显著高于细菌(P0.01);原虫破碎液氨含量在1.5、6.0、9.0和12.0 h显著或极显著高于细菌(P0.05或P0.01)。3)绵羊瘤胃细菌、原虫谷氨酸脱氢酶对烟酰胺腺嘌呤二核苷酸(NAD)的Km值分别为2.60×10~(-7)、1.48×10~(-7)mol/L;细菌、原虫谷氨酸脱氢酶对谷氨酸的Km值分别为8.41×10~(-6)、4.91×10~(-6)mol/L;细菌、原虫谷氨酸脱氢酶对还原型烟酰胺腺嘌呤二核苷酸(NADH)的Km值分别为3.80×10~(-8)、2.70×10~(-8)mol/L;细菌、原虫谷氨酸脱氢酶对α-酮戊二酸的Km值分别为1.16×10~(-6)、2.07×10~(-6)mol/L;细菌、原虫谷氨酸脱氢酶对氨的Km值分别为2.97×10~(-5)、1.40×10~(-5)mol/L。结果提示,总体上,绵羊瘤胃细菌、原虫中蛋白酶、谷氨酸脱氢酶、谷丙转氨酶、谷草转氨酶的活力在饲喂后1.5 h达到峰值,之后逐渐降低;绵羊瘤胃原虫中蛋白酶、谷丙转氨酶、谷草转氨酶和谷氨酸脱氢酶的活力均极显著高于细菌,原虫中蛋白质分解代谢更旺盛;瘤胃原虫中不仅存在谷氨酸转氨机制,还可能存在利用氨重新合成氨基酸的机制。     

蛋白质源对山羊瘤胃微生物蛋白AA组成的影响

以4只瘘管山羊作为试验动物,研究羽毛粉(A)、玉米蛋白粉(B)、豆粕(C)和鱼粉(D)等蛋白质源配合的混合日粮对瘤胃微生物蛋白AA组成模式的影响规律。采用4×4拉丁方设计进行试验。结果表明:部分种类AA的含量在原虫和细菌区系间和同一区系内不同蛋白质处理间差异显著。原虫的Val比例高于细菌的,而细菌的Lys则高于原虫。细菌的Arg,原虫的Met、Leu和His在蛋白质处理间差异显著。综上可见,瘤胃微生物AA组成是因蛋白质来源而变化的。     

不同外源寡糖对崂山奶山羊瘤胃微生物区系的影响

本试验旨在研究不同外源寡糖对奶山羊瘤胃微生物区系的影响。试验选用 ６只平均体重为（３２．８０±２．４５）ｋｇ、装有永久性瘤胃瘘管的崂山奶山羊,采用分期分组试验设计。各组羊饲喂基础饲粮（对照组）以及分别在基础饲粮中添加 １％甘露寡糖（ＭＯＳ组）、半乳甘露寡糖（ＧＭＯＳ组）、寡木糖（ＸＯＳ组）、低聚异麦芽糖（ＩＭＯ组）、果寡糖（ＦＯＳ组）的试验饲粮。共进行４期试验,每期 ２３ｄ,其中预试期 １４ｄ,正试期 ９ｄ。于晨饲前（０ｈ）,晨饲后 ２、４、６、８ｈ以及晚饲前（晨饲后 １０ｈ）采集瘤胃液,测定瘤胃液中细菌总数以及纤维素分解菌、原虫和真菌数量。结果表明：与对照组相比,试验组瘤胃液细菌总数趋于增加,仅晨饲前 ＭＯＳ组显著提高（Ｐ＜０．０５）;试验组瘤胃液纤维素分解菌数量有所提高,其中 ＭＯＳ组（０ｈ）、ＧＭＯＳ组（０、２、４ｈ）、ＦＯＳ组（０、２、４ｈ）显著高于对照组（Ｐ＜０．０５）;试验组瘤胃液原虫数量有所提高,其中 ＭＯＳ组（２和 ４ｈ）、ＸＯＳ组（４ｈ）显著高于对照组（Ｐ＜０．０５）;试验组瘤胃液真菌数量有所提高,其中ＧＭＯＳ组和 ＦＯＳ组（０ｈ）极显著高于对照组（Ｐ＜０．０１）,ＭＯＳ组（２ｈ）、ＩＭＯ组（２ｈ）、ＦＯＳ组（２ｈ）、ＭＯＳ组（４ｈ）、ＦＯＳ组（６ｈ）显著高于对照组（Ｐ＜０．０５）。细菌总数、纤维素分解菌和原虫数量呈先下降后上升趋势,真菌数量呈先上升后下降趋势。本试验条件下,添加不同外源寡糖对瘤胃微生物影响有所差异,其中甘露寡糖、半乳甘露寡糖和果寡糖对瘤胃液纤维素分解菌、原虫和真菌的增殖作用较明显。     

发酵玉米蛋白粉对奶牛瘤胃体外发酵特性及微生物菌群的影响

本试验旨在研究全混合日粮（TMR）中添加发酵玉米蛋白粉（fermented corn gluten meal,FCGM）对奶牛瘤胃体外发酵特性及微生物菌群的影响。选用3头体重（600±25）kg,安装永久性瘤胃瘘管的荷斯坦奶牛作为瘤胃液供体,发酵底物为TMR,分为对照组和3个试验组,各组分别在发酵液中添加0、0.3、0.6、0.9 g/L FCGM（干物质基础）,每个处理3个重复。记录体外发酵12、24、36和48 h产气量,测定体外发酵12、24和48 h发酵液pH、体外干物质消失率（IVDMD）、纤维素酶活性、氨态氮（NH₃-N）、挥发性脂肪酸（VFA）和菌体蛋白浓度,并测定体外发酵24 h发酵液中瘤胃微生物菌群相对丰度。结果显示：①添加不同水平FCGM组的体外产气量（除12 h外）、慢速产气部分、潜在产气部分和有效产气速率均显著或极显著高于对照组（P < 0.05;P < 0.01）;②与对照组相比,添加不同水平FCGM处理组的发酵液pH显著或极显著低于对照组,纤维素酶活性、菌体蛋白、挥发性脂肪酸、氨态氮含量和体外干物质消失率均显著或极显著升高（P < 0.05;P < 0.01）,且0.9 g/L FCGM组达到最高。③添加0.6和0.9 g/L FCGM组发酵液中白色瘤胃球菌、黄色瘤胃球菌、产琥珀酸丝状杆菌、牛链球菌、普雷沃氏菌、溶纤维丁酸弧菌、嗜淀粉瘤胃杆菌、真菌和原虫相对丰度均显著高于对照组（P < 0.05）,且0.9 g/L FCGM组达到最高,而产甲烷菌相对丰度显著低于对照组（P < 0.05）,且0.9 g/L FCGM组达到最低。综上所述,TMR中添加FCGM可提高体外发酵产气量,增加发酵液内纤维素酶活性、VFA、NH3-N及菌体蛋白含量,提高瘤胃内某些纤维降解菌、蛋白降解菌、淀粉降解菌、真菌和原虫相对丰度,降低产甲烷菌相对丰度,调节瘤胃微生物菌群结构,改善瘤胃发酵,其中以添加0.9 g/L FCGM为宜。     

不同瘤胃细菌培养物添加水平对奶牛瘤胃发酵及BCP含量的影响

本文旨在研究不同剂量的瘤胃细菌培养物对荷斯坦奶牛瘤胃发酵及菌体蛋白(BCP)含量的影响。试验采用单因素试验设计,选用4头装有永久性瘤胃瘘管,体况相近的荷斯坦奶牛,取其瘤胃液混匀,添加不同剂量的瘤胃细菌培养物进行体外培养,分别为:对照组(0 CUF/g),试验Ⅰ组(添加量为3%,4.4×108CUF/g),试验Ⅱ组(添加量为5%,7.8×108CUF/g),试验Ⅲ组(添加量为10%,1.2×109CUF/g),每组分别设2、4、8、12、24、36、48 h 7个指标测定时间点,每个时间点3个重复。结果表明:(1)添加瘤胃细菌培养物使瘤胃液pH有所降低,但始终在6.5～7.0,对瘤胃内环境无不良影响;(2)添加3%的瘤胃细菌培养物显著降低氨态氮(NH3-N)的含量(P<0.05),显著增加了培养液BCP的浓度(P<0.05);(3)添加3%的瘤胃细菌培养物可一定程度增加总挥发性脂肪酸(TVFA)和乙酸浓度,但对其他各挥发性脂肪酸及乙酸/丙酸比值无显著影响。由此可见,添加3%剂量的瘤胃细菌培养物可改变瘤胃的发酵环境,提高BCP浓度。     

Fatty acid composition of ruminal bacteria and protozoa,and effect of defaunation on fatty acid profile in the rumen with special reference to conjugated linoleic acid in cattle

Objectives of this study were to compare fatty acid (FA) composition of ruminal bacterial (B) and protozoal (P) cells, and to investigate effect of protozoa on FA profile in the rumen of cattle. Three cows were used to prepare ruminal B and P cells. Four faunated and three defaunated cattle (half‐siblings) were used to study effect of protozoa on ruminal FA profile. Proportions of C16:0 and C18:0 in total fatty acids in B cells were 20.7% and 37.4%, whereas those in P cells were 33.4% and 11.6%, respectively. Proportions of trans‐vaccenic acid (VA) and cis‐9, trans‐11 conjugated linoleic acid (CLA) in B cells were 3.9% and 1.0%, and those in P cells were 5.5% and 1.6%, respectively, being higher in P cells. Proportions of C18:1, C18:2 and C18:3 in P cells were two to three times higher than in B cells. Proportions of unsaturated fatty acids, VA and CLA in B cells of faunated cattle were higher than those of defaunated. VA and CLA in the ruminal fluid of faunated were also 1.6 to 2.5 times higher than those of defaunated. This tendency was similar for cell‐free fraction of ruminal fluid. These results indicate that protozoa contribute greatly in VA and CLA production in the rumen.     

饲粮中燕麦干草含量对绵羊瘤胃液pH及微生物区系的影响

本试验旨在研究饲粮燕麦干草含量对绵羊瘤胃液p H及微生物区系的影响。选取9只体况和体重[(70.32±2.14)kg]相近、装有永久性瘘管的德国美利奴与蒙古羊杂种公羊,采用3×3拉丁方设计,随机分为3组,每组3只,各组分别采用全株玉米青贮、全株玉米青贮+燕麦干草(1∶1)(混合组)、燕麦干草为粗饲料。饲粮精粗比34.50∶65.50。进行3期饲养试验,每期20 d,15 d预试期,5 d采样期。采集饲喂前(0 h)和饲喂后1、3、5和7 h的瘤胃液,测定p H,采用实时定量PCR方法测定微生物相对含量。结果表明:1)全株玉米青贮组的瘤胃液p H在1、5 h均显著低于燕麦干草组(P0.05),在3 h极显著低于混合组(P0.01);2)混合组和燕麦干草组瘤胃液真菌的相对含量在0 h均极显著高于全株玉米青贮组(P0.01),燕麦干草组在5 h真菌相对含量显著高于全株玉米青贮组(P0.05);3)混合组原虫的相对含量在1、5 h显著低于全株玉米青贮组(P0.05);4)饲喂后5 h,混合组和燕麦干草组的纤维分解菌相对含量均较高,其中燕麦干草组黄色瘤胃球菌相对含量显著高于全株玉米青贮组(P0.05),白色瘤胃球菌和产琥珀酸丝状杆菌的相对含量极显著高于全株玉米青贮组(P0.01)。综上所述,在精粗比为34.50∶65.50的饲粮中采用全株玉米青贮+燕麦干草(1∶1)的粗饲料,有利于维持绵羊瘤胃内环境的稳态及瘤胃微生物的生长,白色瘤胃球菌和产琥珀酸丝状杆菌为优势菌。     

用荧光标记细菌法研究丝兰提取物对瘤胃原虫对细菌吞噬的影响

采用荧光染色标记细菌(fluorescence labeled bacteria,FLB)技术,研究山羊瘤胃中原虫对细菌的吞噬速率.试验选用3头装有永久瘘管的徐淮山羊作为瘤胃液供体,采用体外培养法研究在精粗比为1:9的情况下,探讨不同丝兰提取物水平(A组0 mg/L、B组35 mg/L、C组70 mg/L)对瘤胃原虫吞噬细菌速率的影响.荧光镜检与回归分析结果表明:原虫吞噬细菌的速率:A组:339.9 cells/(cell·h);B组:314.7 cells/(cell·h);C组:339.9 cells/(cell·h).结果表明荧光标记细菌技术能够应用于瘤胃原虫吞噬细菌速率的研究.[动物营养学报,2009,21(3):417-422][中文全文见<动物营养学报>网站(www.ChinaJAN.com)中文版2009年21卷3期]     

Effects of protozoa on methane production in rumen and hindgut of calves around time of weaning.

Effects of the presence or absence of ciliate protozoa on methanogenesis in the rumen and hindgut were investigated in young calves during a 7-week period. Ten Holstein calves, aged 7 days, were divided in two groups (n = 5) and fed an increasing amount of a commercial milk replacer and small amounts of a calves starter. One group was inoculated with ciliate fauna on two occasions, week 5 and 6, while the second remained ciliate-free. The absence of protozoa in the rumen decreased rumen empty weight (-23%, P < 0.01), and rumen pool size of N (-36%, P < 0.01) and crude fat (-37%, P < 0.05). Rumen bacteria of non-faunated calves contained a higher proportion of total amino acid-N per 16 g N (+3%, P < 0.01) and D-alanine-N per 16 g N (+13%, P < 0.05) compared to faunated calves. Further results contain a reference for a higher bacterial mass in the ciliate-free rumen with an increased number of bacteria adherent to rumen mucosa. The CH4 production in the rumen increased exponentially with the increase in protozoa population size (R2 = 0.68). In presence of 46 x 10(4) protozoa per ml rumen fluid, the in vitro CH4 production of rumen fluid per mol total VFA was about 34% higher in faunated than in non-faunated calves (P < 0.001). Hydrogen (2H) recovery of rumen fermentation was positively correlated (R2 = 0.55) to the CH4 production rate. Methanogens were attached on rumen mucosa. Methanogenesis, induced by rumen mucosa attached bacteria, was stimulated by ruminal protozoa. In the absence of protozoa in the rumen, the acetate-propionate ratio and butyrate proportion of VFA were reduced. In vivo, in the absence of protozoa not only the whole animal CH4 production (-30%, P < 0.05) but also the digestibility of carbohydrates (-4%, P < 0.05) was reduced. Thereby no difference was observed in the intake of ME per kg DM between the groups. In conclusion, the methanogenesis in the rumen, but not in hindgut, is associated with the development of the ruminal protozoa population. The level of methanogenesis (mol/mol VFA) in the hindgut amounts to 20% of the ruminal methanogenesis.     

不同纤维素与淀粉比率等氮纯化底物瘤胃发酵微生物蛋白质合成量

采用持续动态瘤胃模拟装置 (RSI)研究了 6种不同纤维素和淀粉比率 (C S) :0 .14、0 .44、0 .94、1.92、4.75、15 .76,等氮 ( 14.2 2 % ,风干物质基础 )纯化底物养分瘤胃发酵微生物组分及微生物蛋白质合成量。结果显示 :微生物有机物中氮的含量MN MOM在 5 5 0 %～ 6 64 %之间 ;微生物有机物中RNA含量 (RNA MOM ,% )不受日粮C S影响(P >0 0 5 ) ,仅发现微生物氮含量受到日粮碳水化合物比率影响 (P <0 0 5 )。 72小时发酵后 ,瘤胃微生物蛋白质合成效率与底物纤维素和淀粉比率成乘幂负相关关系 :MN RDN =0 .764 9(C S) - 0 .1 0 6 3,n =6,R =- 0 .93 1;MN FOM(g kg) =2 3 .88(C S) - 0 .0 988,n =6,R =- 0 .881。结果表明 ,在瘤胃可利用氮相同条件下 ,微生物蛋白合成效率取决于适宜的底物结构性碳水化合物和非结构性碳水化合物比率     

Contribution of different rumen microbial groups to gas,short‐chain fatty acid and ammonium production from different diets—an approach in an in vitro fermentation system

In this study, the relative contribution of different microbial groups to ruminal metabolism was investigated for different diets. The rumen microbial cultures included whole rumen fluid, fungi + protozoa, bacteria + protozoa, protozoa and bacteria + fungi and were established by physical and chemical methods. Gas production, short‐chain fatty acid (SCFA) and ammonium production were measured at 24 hr in in vitro incubations using the Hohenheim gas test (HGT) procedure. Seven donor animal diets with different concentrate‐to‐roughage ratios (C:R: 10:90, 30:70, 50:50, 70:30, 70:30BC (BC = NaHCO₃), 90:10 and 90:10BC) and five HGT diets (C:R: 10:90, 30:70, 50:50, 70:30 and 90:10) were formulated. Incubations in the HGT were always based on inoculum from sheep diets with the respective C:R ratio. Gas and ammonium production increased (p < 0.001) as a result of a gradual increase in concentrate proportion of the diets. In general, SCFA production followed the same trend. Whole rumen fluid and bacteria + fungi produced approximately 50% higher gas volume than protozoa and fungi + protozoa fractions, whereas gas production with bacteria + protozoa was at an intermediate level. Coculture of protozoa either with bacteria or with fungi produced more ammonium. Populations without bacteria were characterized by a particularly high acetate/propionate ratio. Although an interaction between microbial group and diet was observed for several variables, no clear direction could be established. Manipulating rumen fluid by selectively suppressing specific rumen microbial groups may be a helpful tool in elucidating their role in nutrient degradation and turnover in vitro.     

Effects of protozoa on the antioxidant activity in the ruminal fluid and blood plasma of cattle

The objective of this study was to investigate the antioxidant status in ruminal fluid and blood plasma among three faunated and two defaunated (protozoa‐free) cattle (average bodyweight of 225 kg), fed hay plus concentrate. The extra cellular antioxidant activity of the mixed protozoa and bacteria suspensions were also studied in vitro. The antioxidant activity was detected by means of the free radical scavenging ability. The activity (units/microbial nitrogen) of the protozoal suspension increased from 59 (0 h) to 318 (18 h), and decreased to 40 (24 h) during incubation. The activity of the bacterial suspension also increased from 111 (0 h) to 644 (18 h), and decreased to 533 (24 h). The antioxidant activity (units/mL, U/mL) in the ruminal fluid of faunated (ranging from 116 to 254) and defaunated (ranging from 66 to 110) cattle was increased after 2 h and decreased after 5 h of feeding, being significantly higher in the faunated cattle. The antioxidant activity of blood plasma (U/mL) ranged from 248 to 316 in the faunated and 121–170 in the defaunated cattle during 0–5 h after feeding, being significantly higher in the faunated cattle. Therefore, defaunation possibly causes a decrease in the antioxidant level in the ruminal fluid, and may impair the health and performance of ruminants through an oxidant–antioxidant imbalance.     

不同添加水平多聚甲醛对绵羊瘤胃微生物数量及消化酶活性的影响

将5只体重(55.6±3.2)kg、2～3岁、装置了永久性瘤胃瘘管的小尾寒羊公羊,按5×5拉丁方设计,在饲喂相同日粮条件下,在5期试验中分别添喂0、150、300、450和600 mg/kg多聚甲醛,测定瘤胃液pH,氨态氮及挥发性脂肪酸浓度,内纤维素酶、外纤维素酶、纤维二糖酶、木聚糖酶、果胶酶、淀粉酶和蛋白酶活性,并进行原虫、细菌显微镜分类和真菌PCR计数,以研究不同添加剂量多聚甲醛对绵羊瘤胃微生物及消化酶活性的影响。结果表明,与对照组相比,添喂150、300、450和600 mg/kg多聚甲醛,绵羊的干物质自由采食量分别增加2.3%(P>0.05)、22.1%(P<0.01)、7.5%(P<0.01)和-6.6%(P<0.01);瘤胃液原虫总数分别减少14.2%、62.5%、72.6%和85.3%(P<0.01);而细菌总数分别增加35.8%、93.8%、-6.8%和-24.2%(P<0.01);真菌拷贝数分别增加2.0%(P>0.05)、5.4%(P>0.05)、14.3%(P<0.01)和26.8%(P<0.01);氨态氮分别降低9.0%、12.0%、14.9%和20.9%(P<0.01);挥发性脂肪酸分别提高2.4%(P>0.05)、-0.3%(P>0.05)、-1.3%(P>0.05)和-10.3%(P<0.01);内纤维素酶活性分别提高6.5%、22.4%、-10.2%和-19.2%(P<0.01);外纤维素酶活性分别提高3.4%(P>0.05)、17.5%(P<0.01)、-18.0%(P<0.01)和-25.7%(P<0.01);纤维二糖酶活性分别提高8.3%、23.7%、-24.9%和-31.8%(P<0.01);淀粉酶活性分别提高2.8%、8.6%、16.0%和23.3%(P<0.01);果胶酶活性分别降低11.8%、12.1%、11.2%和11.1%(P<0.01);蛋白酶活性分别降低10.9%、30.4%、49.6%和56.2%(P<0.01);而木聚糖酶活性没有显著变化(P>0.05)。综合试验结果,多聚甲醛添喂剂量为300 mg/kg时,绵羊的自由采食量最高,此时瘤胃液原虫数量减少而细菌总数最多、纤维素酶活性最高。     

Microbial population in the rumen of swamp buffalo (Bubalus bubalis) as influenced by coconut oil and mangosteen peel supplementation

Four, rumen fistulated swamp buffalo bulls were used to study microbial populations in the rumen when supplemented with coconut oil and mangosteen peel. Animals were randomly assigned to a 4 × 4 Latin square design. Four treatments were un‐supplemented (Control), supplementation with coconut oil at 50 g/kg (CO5), supplementation with mangosteen peel at 30 g/kg (MP3) and supplementation with CO5 and MP3 (COM), of total DM intake. Animals received concentrate at 10 g/kg of BW, and rice straw was given ad libitum. Abundance of total bacteria was increased by CO5 supplementation, whereas populations of protozoa and Fibrobacter succinogenes were reduced by CO5 and COM supplementation. Dietary supplementation did not affect methanogen, Ruminococcus flavefaciens or Ruminococcus albus abundances. Dietary treatments changed denaturing gradient gel electrophoresis (DGGE) band patterns of methanogens and protozoa when compared with the control group, especially when supplemented with MP3. Supplementation of COM resulted in the greatest difference in pattern of DGGE bands for total bacteria compared with the control. Coconut oil and mangosteen peel supplementation resulted in changing of rumen microbial abundances and communities; however, combination of them could be more benefit to improve rumen fermentation of swamp buffalo fed on rice straw.     

ORIGINAL ARTICLE: Description of development of rumen ecosystem by PCR assay in milk‐fed,weaned and finished lambs in an intensive fattening system

This study examined the reticulo‐rumen characteristics of the microbial community and its fermentative characteristics in milk‐fed, at weaning and finished lambs in a conventional fattening system. Five lambs were assigned to each of three groups: milk‐fed lambs slaughtered at 30 days (T30), weaned lambs slaughtered at 45 days (T45) and ‘finished lambs’ slaughtered at 90 days (T90). At slaughter, rumen size, fermentation parameters (pH, volatile fatty acids and microbial enzyme activity) and protozoal counts were recorded. Quantitative PCR was used to quantify the genes encoding 16S and 18S ribosomal DNA of the rumen bacterial and protozoal populations, respectively, and the sequential colonization of the rumen by cellulolytic (Ruminococcus albus, Ruminococcus flavefaciens) and amylolytic (Prevotella ruminicola, Streptococcus bovis) bacteria, and protozoa (Entodinium sp.). Denaturing gradient gel electrophoresis was used to study the development of rumen microbiota biodiversity. Intake of solid food before weaning caused a significant increase in rumen weight (p < 0.0001) and bacterial DNA (p < 0.05) and volatile fatty acid analysis concentration (p < 0.01), whereas pH declined. In milk‐fed lambs, cellulolytic bacteria were evident after 30 days. Thereafter, in the 45‐day and 90‐day groups, the proportions of R. flavefaciens decreased and R. albus increased. Amylolytic bacteria were present in milk‐fed lambs; the proportion of P. ruminicola increased in fattening lambs and S. bovis was the least abundant species. Protozoal concentrations were irregular; milk‐fed lambs had a significant number of protozoa species from Entodinium and subfamily Isotrichiidae, but they disappeared at weaning. Lamb rumen were refaunated in some individuals at 90 days (Entodinium and subfamily Diplodiniinae spp.), although individual concentrations were variable.