Occurrence of 2-aminoethylphosphonic acid in feeds, ruminal bacteria and duodenal digesta from defaunated sheep

A quantitative method of analysis for 2-aminoethylphosphonic acid (AEP) was developed using reverse-phase HPLC. The detection limit for AEP was 15 nM, and the detector response (peak area) was linear from AEP levels up to 100 microM (R = .99). Mean recovery of AEP added to strained ruminal fluid from faunated sheep was 98.2%. When AEP was added to a fermentation mixture at a concentration of 22.6 micrograms/ml, 78% disappeared during a 24-h incubation. 2-Aminoethylphosphonic acid was readily detected in preparations of mixed ruminal ciliate protozoa as well as in mixed and pure strains of ruminal bacteria, feedstuffs, and ruminal fluid and duodenal digesta from defaunated sheep. The occurrence of AEP in feed and bacterial hydrolysates was confirmed by organic phosphorus analyses. The concentration of AEP in mixed ruminal protozoa was three times greater than its concentration in mixed ruminal bacteria (4,304 vs 1,383 micrograms/g DM, respectively). The AEP values for pure ruminal bacterial cultures ranged from 733 micrograms/g DM in Bacteroides succinogenes B21a to 1,166 micrograms/g DM in Butyrivibrio fibrisolvens H17c. Ruminal fluid and duodenal digesta from defaunated sheep contained AEP concentrations of 30 micrograms/ml and 90 micrograms/g DM, respectively. The concentration of AEP in feedstuffs ranged from 25 micrograms/g DM in wheat straw to 263 micrograms/g DM in oats. Because AEP occurrence is not limited to ruminal ciliate protozoa, it is of little value as a marker for protozoal presence in or passage out of the rumen.     

In vitro bacterial growth and in vivo ruminal microbiota populations associated with bloat in steers grazing wheat forage

The role of ruminal bacteria in the frothy bloat complex common to cattle grazing winter wheat has not been previously determined. Two experiments, one in vitro and another in vivo, were designed to elucidate the effects of fresh wheat forage on bacterial growth, biofilm complexes, rumen fermentation end products, rumen bacterial diversity, and bloat potential. In Exp. 1, 6 strains of ruminal bacteria (Streptococcus bovis strain 26, Prevotella ruminicola strain 23, Eubacterium ruminantium B1C23, Ruminococcus albus SY3, Fibrobacter succinogenes ssp. S85, and Ruminococcus flavefaciens C94) were used in vitro to determine the effect of soluble plant protein from winter wheat forage on specific bacterial growth rate, biofilm complexes, VFA, and ruminal H2 and CH4 in mono or coculture with Methanobrevibacter smithii. The specific growth rate in plant protein medium containing soluble plant protein (3.27% nitrogen) was measured during a 24-h incubation at 39 degrees C in Hungate tubes under a CO2 gas phase. A monoculture of M. smithii was grown similarly, except under H2:CO2 (1:1), in a basal methanogen growth medium supplemented likewise with soluble plant protein. In Exp. 2, 6 ruminally cannulated steers grazing wheat forage were used to evaluate the influence of bloat on the production of biofilm complexes, ruminal microbial biodiversity patterns, and ruminal fluid protein fractions. In Exp. 1, cultures of R. albus (P < 0.01) and R. flavefaciens (P < 0.05) produced the most H2 among strains and resulted in greater (P < 0.01) CH4 production when cocultured with M. smithii than other coculture combinations. Cultures of S. bovis and E. ruminantium + M. smithii produced the most biofilm mass among strains. In Exp. 2, when diets changed from bermudagrass hay to wheat forage, biofilm production increased (P < 0.01). Biofilm production, concentrations of whole ruminal content (P < 0.01), and cheesecloth filtrate protein fractions (P < 0.05) in the ruminal fluid were greater on d 50 for bloated than for nonbloated steers when grazing wheat forage. The molecular analysis of the 16S rDNA showed that 2 different ruminal microbiota populations developed between bloated and nonbloated animals grazing wheat forage. Bloat in cattle grazing wheat pastures may be caused by increased production of biofilm, resulting from a diet-influenced switch in the rumen bacterial population.     

Effect of ethanol on nitrate and nitrite reduction and methanogenesis in the ruminal microbiota

The effect of ethanol on nitrate and nitrite reduction was examined by conducting in vitro experiments with mixed ruminal microbes. The addition of ethanol to cultures of mixed ruminal microbes stimulated nitrate reduction, and, to a greater extent, nitrite reduction, which resulted in a decrease in nitrite accumulation. However, known nitrate‐reducing ruminal bacteria, such as Selenomonas ruminantium, Veillonella parvula and Wolinella succinogenes, were unable to utilize ethanol directly as an electron donor for nitrate reduction. No nitrate‐reducing bacterium capable of utilizing ethanol was found in the rumen of goats. However, when mixed ethanol‐utilizing, hydrogen gas (H₂)‐producing bacteria (Ruminococcus albus and Ruminococcus flavefaciens) were added to the culture of the mixed nitrate‐reducing bacteria described above, nitrate and nitrite reduction was observed. These results suggest that the nitrate‐reducing bacteria utilized the H₂ that was produced from ethanol oxidation by the ethanol‐utilizing bacteria as an electron donor. It is conceivable that the stimulation of nitrate and nitrite reduction by ethanol, observed in the culture of mixed ruminal microbes, was a result of electron transfer from ethanol to nitrate, and nitrite through H₂, that is, ‘interspecies hydrogen transfer’ from ethanol‐metabolizing bacteria to nitrate‐reducing bacteria. Thus, the addition of ethanol to high‐nitrate diets may be effective for preventing nitrate poisoning. Furthermore, methane production was reduced to less than one‐third by the addition of mixed nitrate‐reducing bacteria to the co‐culture of mixed methanogens with mixed ethanol‐utilizing bacteria incubated in a medium containing ethanol and nitrate. Therefore, the addition of ethanol and nitrate may decrease methanogenesis without suppressing overall fermentation in the rumen.     

Effects of the acid-tolerant engineered bacterial strain Megasphaera elsdenii H6F32 on ruminal pH and the lactic acid concentration of simulated rumen acidosis in vitro

The aim of this study was to examine the effects of the acid-tolerant engineered bacterial strain Megasphaera elsdenii H6F32 (M. elsdenii H6F32) on ruminal pH and the lactic acid concentrations in simulated rumen acidosis conditions in vitro. A mixed culture of ruminal bacteria, buffer, and primarily degradable substrates was inoculated with equal numbers of M. elsdenii H6 or M. elsdenii H6F32. The pH and lactic acid concentrations in the mixed culture were determined at 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 h of incubation. Acid-tolerant M. elsdenii H6F32 reduced the accumulation of lactic acid and increased the pH value. These results indicate that acid-tolerant M. elsdenii H6F32 could be a potential candidate for preventing rumen acidosis.     

The effect of replacing corn with glycerol on ruminal bacteria in continuous culture fermenters

The effects of substituting corn with glycerol on DNA concentration of selected ruminal bacteria were investigated using continuous fermenters. Four continuous culture fermenters were used in a 4 × 4 Latin Square design with four 10 days consecutive periods. Treatment diets (60:40 forage to concentrate) were fed at 45 g/day dry matter (DM) in three equal portions. Glycerol (0.995 g/g glycerol) was used to replace corn in a grain mix at proportions of 0% (T0; control), 15% (T15), 30% (T30) and 45% (T45). On day 10 of each period, samples were collected from each fermenter 3 h after the morning feeding and analysed for volatile fatty acid and bacterial DNA concentration. Glycerol substitution was related to significantly higher butyrate, valerate and isovalerate concentrations. Compared with the T0 diet, acetate concentration was significantly lower with the T30 and T45 diets whilst propionate concentration was higher only with the T45 diet. The DNA concentrations for Butyrivibrio fibrisolvens and Selenomonas ruminantium decreased with the T30 and T45 diets compared with the T0 diet. No differences in the DNA concentrations for Ruminococcus albus and Succinivibrio dextrinosolvens amongst diets were observed. The findings show that substituting 15% of the dietary corn with glycerol had no substantive effects on fermentation processing or ruminal bacteria. Higher substitution levels, however, may adversely affect ruminal bacteria and negatively impact acetate production.     

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。     

Effects of concentrate‐to‐forage ratios and 2‐methylbutyrate supplementation on ruminal fermentation,bacteria abundance and urinary excretion of purine derivatives in Chinese Simmental steers

This study evaluated the effects of dietary concentrate levels and 2‐methylbutyrate (2MB ) supplementation on performance, ruminal fermentation, bacteria abundance, microbial enzyme activity and urinary excretion of purine derivatives (PD ) in steers. Eight ruminally cannulated Simmental steers (12 months of age; 389 ± 3.7 kg of body weight) were used in a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement. Moderate‐concentrate (400 g/kg diet [MC ]) or high‐concentrate (600 g/kg diet [HC ]) diets were fed with or without 2MB (0 g/day [2MB ?] or 15.0 g/day [2MB +]). Dry matter intake and average daily gain increased, but feed conversion ratio decreased with the HC diet or 2MB supplementation. Ruminal pH decreased, but total volatile fatty acid increased with the HC diet or 2MB supplementation. Molar proportion of acetate and acetate‐to‐propionate ratio decreased with the HC diet, but increased with 2MB supplementation. Propionate molar proportion and ruminal NH ₃‐N content increased with the HC diet, but decreased with 2MB supplementation. Neutral detergent fibre degradability decreased with the HC diet, but increased with 2MB supplementation. Crude protein degradability increased with the HC diet or 2MB supplementation. Abundance of Ruminococcus albus , Ruminococcus flavefaciens , Fibrobacter succinogenes and Bufyrivibrio fibrisolvens as well as activities of carboxymethyl cellulase, cellobiase, xylanase and pectinase decreased with the HC diet, but increased with 2MB supplementation. However, abundance of Prevotella ruminicola and Ruminobacter amylophilus as well as activities of α‐amylase and protease increased with the HC diet or 2MB supplementation. Total PD excretion also increased with the HC diet or 2MB supplementation. The results suggested that growth performance, ruminal fermentation, CP degradability and total PD excretion increased with increasing dietary concentrate level from 40% to 60% or 2MB supplementation. The observed diet × 2MB interaction indicated that supplementation of 2MB was more efficacious for improving growth performance, ruminal fermentation and total PD excretion with promoted ruminal bacteria abundance and enzyme activity in the MC diet than in the HC diet.     

Effects of protein concentration and degradability on performance, ruminal fermentation, and nitrogen metabolism in rapidly growing heifers fed high-concentrate diets from 100 to 230 kg body weight

Twenty crossbred heifers (101 +/- 4.5 kg BW) were used to examine the effects of protein concentration and degradability on performance, ruminal fermentation, nutrient digestion, N balance, and urinary excretion of purine derivatives. Heifers were offered concentrate and barley straw for ad libitum consumption. Two protein concentrations (17 vs 14%, DM basis) and two protein sources differing in ruminal degradability (58 vs 42% of CP for soybean meal and treated soybean meal, respectively) were tested. The experiment was divided into four consecutive 28-d periods to evaluate the age (period) effect. Increasing protein concentration and degradability did not improve ADG or intake (P > .05). The increase in urinary N excretion (P < .001) in heifers fed 17% CP suggests that N was in excess of requirements. When the low-degradable protein source was supplemented and(or) CP concentration was low, ruminal NH3 N concentrations fell below 5 mg/100 mL. Urinary excretion of purine derivatives was not affected (P > .05) by protein concentration and degradability, suggesting that in high-concentrate diets NH3 N concentration was not limiting microbial growth. Total VFA concentration decreased (P < .001) and the acetate:propionate ratio increased (P < .01) with advancing period, suggesting an increase in ruminal absorption capacity and an increase in fiber fermentation. The decrease in ruminal NH3 N concentration in the last period suggests a greater use of NH3 N by microorganisms. This hypothesis is supported by the increase (P < .001) in urinary excretion of allantoin and estimated duodenal flows of purine bases and microbial protein with advancing period. Reducing CP concentration and increasing ruminal undegradable protein supply did not affect animal performance or estimated duodenal flow of microbial protein in rapidly growing heifers fed high-concentrate diets.     

不同长链脂肪酸组合对体外瘤胃细菌发酵和群体结构的影响

本试验旨在研究不同长链脂肪酸组合对体外培养瘤胃细菌发酵和群体结构的影响。以3头瘤胃瘘管奶牛提供瘤胃液,对照(A)组底物含5%脂肪酸钙,试验组培养底物中硬脂酸、油酸、亚油酸和亚麻酸的含量分别为1.5%、1.0%、0.5%和1.5%(B组),1.5%、1.0%、1.5%和1.0%(C组),1.0%、1.5%、1.5%和0.5%(D组)以及1.5%、0.5%、0.5%和1.0%(E组)。在培养后0、3、6、12、18、24 h采集培养液,测定p H、氨氮浓度和瘤胃细菌含量。结果表明:1)培养液p H在组间的差异不显著(P0.05);C组的培养液氨氮浓度显著高于B、D组(P0.05)。2)除白色瘤胃球菌,其他菌属含量在组间存在显著差异(P0.05)。其中琥珀酸拟杆菌、生黄瘤胃球菌、蛋白溶解梭菌和嗜淀粉瘤胃杆菌含量在B组较高;C组溶纤维丁酸弧菌、埃氏巨球菌、降解淀粉瘤胃球菌以及瘤胃总细菌含量显著高于其他各组(P0.05)。培养液埃氏巨球菌含量最高,为优势菌。综合得出,脂肪酸组合对瘤胃总细菌和大部分细菌种属含量有显著影响,这与发酵模式有关。     

Effects of the ionophore tetronasin on nitrogen metabolism by ruminal microorganisms in vitro

The effects of tetronasin on ruminal protein metabolism were investigated in vitro using ruminal fluid from cattle receiving tetronasin in the diet, ovine ruminal fluid from animals not receiving tetronasin and pure cultures of proteolytic ruminal bacteria. Ruminal fluid from cattle receiving tetronasin in a predominantly barley diet had lower proteolytic (76% of control, P less than .10) and deaminative (58% of control, P less than .05) activities than controls after 42 d. The effect of deamination disappeared after 84 d, although the proteolytic activity remained lower (P less than .10) than that of controls. When tetronasin was added in vitro to ruminal fluid from sheep not receiving the ionophore, proteolytic activity (14C-labeled casein hydrolysis) was unaffected, but the rate of ammonia production from amino acids was decreased by 87% (P less than .01). Oligopeptide breakdown was inhibited to a lesser extent (21%, P less than .05). Dipeptidase activity (dialanine hydrolysis) was not affected. The addition of tetronasin to cultures of the ruminal bacteria Ruminobacter amylophilus and Bacteroides ruminicola had no influence on their protease, deaminase or dipeptidase activities. However, when the bacteria were adapted to grow in the presence of tetronasin, deamination of amino acids was severely inhibited (87 to 100%, P less than .01), even when tetronasin was absent from the incubation mixture. Tetronasin had no effect on the proteolytic activity of adapted cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of forage source on ruminal microbial nitrogen metabolism and carbohydrate digestion in continuous culture

Eight single-flow, continuous culture fermentors were used in Exp. 1 to study the effects of forage source on ruminal bacterial N metabolism and carbohydrate digestion. Forages included alfalfa, cicer milkvetch, birdsfoot trefoil and sainfoin with respective CP concentrations of 26.0, 28.7, 26.3 and 20.0%. Each forage provided 100% of the substrate for microbial metabolism and supplied 2.6 g N/d. Ammonia-N, protein degradation and efficiency of ruminal bacterial protein synthesis were lowest (P less than .05) for sainfoin. Protein degradation and efficiency of bacterial protein synthesis were higher (P less than .05) for birdsfoot trefoil than for alfalfa. Effluent flow of amino acids was highest (P less than .05) for sainfoin. Total nonstructural carbohydrate digestion tended to be highest for sainfoin and birdsfoot trefoil, whereas structural carbohydrate digestion was highest (P less than .05) for alfalfa and cicer milkvetch. In Exp. 2, mixed diets were supplied to dual-flow, continuous culture fermentors with alfalfa, cicer milkvetch, birdsfoot trefoil and sainfoin contributing 85% of the total dietary CP. Each diet contained approximately 12.9% CP. Ammonia-N concentration in the effluent and CP degradation tended to be lowest with the sainfoin diet and highest with the birdsfoot trefoil diet. Effluent flow of amino acids tended to be highest with the cicer milkvetch diet and lowest with the alfalfa and birdsfoot trefoil diet. Total structural and nonstructural carbohydrate digestion was not different (P greater than .05) among forages. Results from these experiments indicate that bacterial degradation of protein was lower for sainfoin than for alfalfa. Birdsfoot trefoil and cicer milkvetch appear to be comparable to alfalfa with regard to metabolism of N and carbohydrates by ruminal bacteria.     

Comparison of direct and indirect tests for small intestinal bacterial overgrowth and antibiotic-responsive diarrhea in dogs

Controversy exists over the diagnosis of idiopathic small intestinal bacterial overgrowth (SIBO) in dogs and some clinicians use the term antibiotic-responsive diarrhea (ARD) in preference. However, whether such terms are interchangeable is not clear. To examine the relationship between duodenal bacterial numbers and a clinical response to antibiotics, SIBO and ARD were defined by nonoverlapping criteria. Quantitative duodenal juice bacteriology and indirect serum biochemical tests were used to assess small intestinal bacterial populations in 30 dogs with gastrointestinal disorders, including 9 with ARD. Serum total unconjugated bile acid (TUBA) concentrations were measured in all dogs, serum folate and cobalamin concentrations were measured in 29 of 30 dogs, and quantitative culture of duodenal juice was performed in 22 of 30 dogs. Serum TUBA concentrations also were measured in samples from 38 control dogs. Twenty of 22 affected (clinical) dogs in which quantitative bacteriology was performed were classified as having SIBO (>10(5) colony-forming units of total bacteria per milliliter of duodenal juice), but bacterial numbers did not differ significantly between dogs with ARD and dogs with other disorders. Increased folate (19/29), decreased cobalamin (16/ 29), or a combination (9/29) were common, but increased TUBA concentrations were documented in only 5 of 30 clinical dogs. Again, no significant differences were observed between dogs with ARD and those with other disorders, and a similar proportion (5/38) of controls had abnormally high TUBA concentrations. Finally, no significant differences were noted when duodenal bacteriology and TUBA concentrations were assessed before and during antibiotic therapy. These results question the utility of quantitative duodenal juice bacteriology and indirect biochemical marker tests for SIBO in the investigation of canine gastrointestinal disorders.     

Interaction of rumen bacteria as assumed by colonization patterns on untreated and alkali‐treated rice straw

Colonization patterns of representative rumen bacteria were compared between untreated rice straw (UTS) and sodium hydroxide‐treated rice straw (SHTS). UTS and SHTS were incubated in the rumen of sheep for 10 min, 1, 2, 6, 12, 24, 48 and 96 h using the nylon bag method. The population sizes of 13 representative bacterial species or groups were quantified by real‐time PCR. The total bacterial population size (abundance) was similar in both UTS and SHTS. Fibrobacter succinogenes showed a higher population size compared to other fibrolytic species and was detected at a higher level in SHTS (3.7%) than in UTS (2.6%). Ruminococcus albus and Ruminococcus flavefaciens were also detected at higher levels in SHTS (0.15% and 0.29%) than in UTS (0.03% and 0.18%). Population sizes of non‐fibrolytic species, such as Selenomonas ruminantium, Anaerovibrio lipolytica and Succinivibrio dextrinosolvens were higher in UTS than in SHTS. Coefficient of determination (r²) on population changes between bacterial species or groups were higher in UTS than in SHTS, suggesting the necessity of stronger bacterial interactions for UTS digestion. Therefore, not only colonization of fibrolytic species, but also synergistic interactions between different bacterial species may be key to the ruminal digestion of rice straw.     

日粮补充异丁酸对犊牛生长性能、瘤胃发酵和纤维分解菌菌群的影响

为探索异丁酸对犊牛生长性能、瘤胃发酵、纤维分解菌菌群和酶活性的影响,试验选用15日龄荷斯坦犊牛36头,随机分成4组,对照组、低水平异丁酸组(LIB)、中水平异丁酸组(MIB)和高水平异丁酸组(HIB)分别饲喂异丁酸0、3、6和9g·d^-1,预饲15d,分别于60日龄(断奶)和90日龄测定体重和采集瘤胃液进行分析。结果表明,MIB组和HIB组犊牛干物质采食量、日增重和经济效益显著高于对照组(P<0.05)。90日龄时HIB组犊牛瘤胃pH值显著低于对照组和LIB组(P<0.05)。MIB和HIB组瘤胃总挥发性脂肪酸和乙酸浓度显著高于对照组和LIB组(P<0.05);MIB和HIB组犊牛60日龄时瘤胃乙酸/丙酸显著高于对照组(P<0.05),90日龄时显著高于对照组和LIB组(P<0.05)。60和90日龄时MIB和HIB组瘤胃异丁酸均显著高于对照组(P<0.05)。60日龄时MIB和HIB组木聚糖酶和α-淀粉酶显著高于对照组(P<0.05),MIB和HIB组纤维二糖酶、果胶酶、溶纤维丁酸弧菌、黄色瘤胃球菌和产琥珀酸丝状杆菌显著高于对照组和LIB组(P<0.05);90日龄时MIB和HIB组各种瘤胃酶活力和纤维分解菌均显著高于对照组和LIB组(P<0.05)。结果显示,日粮补充异丁酸促进了犊牛瘤胃纤维素分解菌的生长,提高了纤维素分解酶的活性,进而促进了犊牛瘤胃发酵和生长发育。由于MIB与HIB之间各指标差异不显著,兼顾经济效益,在本试验条件下异丁酸的最佳添加量为6.0g·d^-1。     

Effect of incremental levels of fish oil supplementation on specific bacterial populations in bovine ruminal fluid

The objective of this study was to determine the effects of incremental replacement of dietary linoleic acid by >20-carbon polyunsaturated fatty acids (PUFA) on changes in population of ruminal micro-organisms associated with fibre digestion and biohydrogenation using real-time PCR of bacterial 16S rRNA sequences. Four beef steers with ruminal cannulas were randomly assigned to control (CK, 65:35 forage to concentrate), CK with 3% sunflower oil plus 1% fish oil (S3F1), 2.5% sunflower oil plus 1.5% fish oil (S2.5F1.5) or 2% sunflower oil plus 2% fish oil (S2F2) in a 4 × 4 Latin square design with 21-day periods. Ruminal fluid was collected on day 15 of each period. Compared with CK, oil addition led to lower ruminal acetate and butyrate but greater propionate concentration. DNA copy number of Anaerovibrio lipolytica in ruminal fluid was greater with oil (average 5.38 vs. 3.62 × 10(5) DNA copy number), particularly with S2F2 relative to CK. Fibrobacter succinogenes and Butyrivibrio fibrisolvens DNA copy number decreased by 74% (1.06 vs. 4.01 × 10(5)) and 39% (5.16 vs. 8.42 × 10(7)) in response to S2F2 compared with CK. DNA copy numbers of Ruminococcus flavefaciens and Ruminococcus albus were not affected by incremental fish oil. Results suggest that greater availability of PUFA with >20 carbons (i.e. eicosapentaenoic acid and docosahexaenoic acid) promoted changes in bacterial populations that are relevant for fibre digestion and biohydrogenation.     

Endogenous fraction and urinary recovery of purine derivatives obtained by different methods in Nellore cattle

Two experiments were conducted to assess the endogenous fraction of purine derivative (PD) excretion, urinary recovery, and intestinal digestibility of purines in Nellore heifers. For both experiments, 8 Nellore heifers fitted with ruminal and abomasal cannulas were allocated to two 4 × 4 Latin squares. The diets were based on corn silage and concentrate (60 and 40% DM basis, respectively); feces and urine samples were obtained by total collection, and abomasal DM flow was estimated using indigestible NDF as an internal marker. In Exp. I, 4 of the 8 heifers (BW 258 ± 20 kg) were also fitted with ileal cannula. The planned treatments were 4 different DMI: 1.2, 1.6, 2.0, and 2.4% of BW (DM basis). The endogenous losses and purine recovery as urinary PD were estimated using linear regression between daily urinary PD excretion (Y) and daily abomasal flow of purine bases (X), expressed in millimoles per kilogram of BW(0.75). In Exp. II, the same 8 Nellore heifers (BW of 296 ± 15 kg) were fed at 1.37% BW (DM basis). The treatments were the infusion of purines (RNA from torula yeast, type VI, Sigma) into the abomasum in increasing amounts (0, 33, 66, and 100 mmol/d). All statistical analyses were performed using the PROC MIXED procedure in SAS. In Exp. I, the DMI range was 1.16 to 1.84% of BW and did not affect (P > 0.05) the apparent RNA digestibility in the small intestine, which had a mean of 75.6%, and a true digestibility of 93.0%. The mean ratio of the N-RNA to the total-N in the ruminal bacteria was 0.137. The daily urinary PD excretion (Y, mmol/kg of BW(0.75)) was a function of RNA flow in the abomasum (X, mmol/kg of BW(0.75)): Y = 0.860X + 0.460, where 0.860 and 0.460 were the PD recovery of purines and the endogenous fraction (in mmol/kg of BW(0.75)), respectively. In Exp. II, the daily urinary PD excretion was a function of RNA flow in the abomasum: Y = 0.741X + 0.301, where 0.741 and 0.301 were the recovery of PD in urine of infused purines and the endogenous losses (in mmol/kg of BW(0.75)), respectively. In conclusion, our data suggest that in Nellore heifers the respective values of endogenous PD excretion (mmol/kg of BW(0.75)), urinary recovery of the purines absorbed in the abomasum, and true digestibility of RNA in the small intestine were 0.30, 0.80, and 0.93.     

内蒙古白绒山羊瘤胃中内纤毛虫及三种纤维分解菌的定量研究

试验采用荧光定量PCR技术,在不同精粗比日粮条件下对内蒙古白绒山羊瘤胃内容物中内纤毛虫和3种主要纤维分解菌进行定量研究。结果表明:日粮中粗料比例增加,内纤毛虫(Ento-dinium)的数量降低,组间差异不显著(P>0.05);日粮中粗料比例增加,产琥珀酸丝状杆菌(Fibrobacter succinogenes)、黄色瘤胃球菌(Ruminococcus flavefaciens)和白色瘤胃球菌(Ruminococcus albus)的数量均增加,组间差异不显著(P>0.05)。     

Effect of dietary protein level on nitrogen metabolism in the growing bovine: II. Diffusion into and utilization of endogenous urea nitrogen in the rumen

Six Angus heifer calves (234 kg) were assigned to either a high (HP; 126.1 g N/d) or low (LP; 66.5 g N/d) protein intake to evaluate ruminal criteria associated with movement of blood urea-N (BUN)-derived NH3-N from the rumen wall into interior ruminal digesta. Calves received 4.8 kg DM/d of diets containing 30% cottonseed hulls and 70% cornsoybean meal in equal portions at 4-h intervals. Following single i.v. injections of 15N-urea, ruminal fluid was collected serially for 4 h postinjection from digesta located adjacent to the rumen wall (wall-proximate digesta; WPD) and from the center of the rumen digesta mass after manual agitation (center mixed digesta; CMD). Mean ruminal NH3-N (RAN) concentrations were higher (P less than .05) for HP than for LP, but were not affected (P greater than .05) by digesta sampling site. Ruminal urease activity was higher (P less than .05) for LP than for HP and tended (P = .14) to be higher for WPD than for CMD. Area under the 15N enrichment curve (AUC) ratios between sampling sites (WPD/CMD x 100) for RAN were greater (P less than .05) for LP than for HP. However, AUC ratios for bacterial N were not affected (P greater than .05) by protein level. Whereas BUN-derived 15NH3 appeared to thoroughly equilibrate with RAN in interior ruminal digesta with HP, there appeared to be a declining enrichment gradient for RAN from the rumen wall to the interior ruminal digesta with LP. Data are interpreted to suggest that bacteria at or near the rumen wall may preferentially utilize some BUN-derived NH3-N entering through the rumen wall in calves fed LP diets.     

Quantitative comparisons of select cultured and uncultured microbial populations in the rumen of cattle fed different diets

ABSTRACT: BACKGROUND: The number and diversity of uncultured ruminal bacterial and archaeal species revealed by 16S rRNA gene (rrs) sequences greatly exceeds that of cultured bacteria and archaea. However, the significance of uncultured microbes remains undetermined. The objective of this study was to assess the numeric importance of select uncultured bacteria and cultured bacteria and the impact of diets and microenvironments within cow rumen in a comparative manner. RESULTS: Liquid and adherent fractions were obtained from the rumen of Jersey cattle fed hay alone and Holstein cattle fed hay plus grain. The populations of cultured and uncultured bacteria present in each fraction were quantified using specific real-time PCR assays. The population of total bacteria was similar between fractions or diets, while total archaea was numerically higher in the hay-fed Jersey cattle than in the hay-grain-fed Holstein cattle. The population of the genus Prevotella was about one log smaller than that of total bacteria. The populations of Fibrobacter succinogenes, Ruminococcus flavefaciens, the genus Butyrivibrio, and R. albus was at least one log smaller than that of genus Prevotella. Four of the six uncultured bacteria quantified were as abundant as F. succinogenes, R. flavefaciens and the genus Butyrivibrio. In addition, the populations of several uncultured bacteria were significantly higher in the adherent fractions than in the liquid fractions. These uncultured bacteria may be associated with fiber degradation. CONCLUSIONS: Some uncultured bacteria are as abundant as those of major cultured bacteria in the rumen. Uncultured bacteria may have important contribution to ruminal fermentation. Population dynamic studies of uncultured bacteria in a comparative manner can help reveal their ecological features and importance to rumen functions.     

Effects of level of feeding and ruminally undegraded protein on ruminal bacterial protein synthesis, escape of dietary protein, intestinal amino acid profile, and performance of dairy cows.

Six cannulated lactating cows were used in two replicated, concurrently run 3 x 3 Latin square experiment to study the interaction between level of feeding and diets differing in ruminally undegraded protein (RUP) on bacterial protein synthesis, ruminal escape of dietary protein, and flow of total and individual amino acids (AA) to the small intestine. Treatments consisted of three diets formulated to contain 69 g (HL), 53 g (HH), and 48 g (LL) of RUP per kilogram of DM, respectively. Measurements were made in early lactation, at high feeding level (19.3 kg DM/d), and repeated at late lactation (9.8 kg DM/d, low feeding level) with the same animals and diets. Decreasing feed intake increased (P < .05) the apparent digestibility of OM, NDF, and ADF in the rumen and the total tract, decreased (P < .05) ruminal liquid and particulate passage rate and total ruminal VFA concentration, and increased ruminal pH and ammonia concentration. Decreased level of intake reduced the (P < .05) efficiency of bacterial N synthesis (28.1 vs 23.7 g bacterial N/kg OM truly digested in the rumen) and decreased (P < .05) ruminal protein degradation rate measured with an in situ method. Duodenal flow of nonammonia nitrogen (NAN), and total AA were highest (P < .05) for the HL diet and lowest (P < .05) for the LL diet at the high feeding level. However, at the low feeding level, diet composition did not affect the amount of NAN or total AA passing to the small intestine. Diet HL increased the proportion of Met, His (P < .05), and Arg (P < .07) in the duodenal digesta at both feeding levels. When purines were used to calculate bacterial N synthesis, no differences between diets were detected. However, when diaminopimelic acid was used, highest bacterial N synthesis was detected for diet HH at the high feeding level. Diet HL supported the highest (P < .05) milk protein production at the high feeding level, and the highest (P < .05) milk protein content at the low feeding level. In conclusion, level of feeding and amount of RUP altered the amount and composition of AA presented to the cows.