Fatty acid composition of ruminal bacteria and protozoa, with emphasis on conjugated linoleic acid, vaccenic acid, and odd-chain and branched-chain fatty acids

Knowledge of the fatty acid profile of microbial lipids is of great nutritional importance to the animals and, subsequently, their products. This study was conducted to examine the fatty acid profiles of mixed rumen bacteria and protozoa. Bacterial and protozoal cells were isolated by differential centrifugation of rumen contents. The main fatty acids were palmitic (16:0) and stearic (18:0) in both the bacterial and protozoal fractions. Palmitic acid was 74% greater in the protozoal fatty acids than in the bacterial fatty acids, whereas bacteria had 2.25-times greater stearic acid (18:0) proportions compared with protozoa. The total odd-chain plus branched-chain fatty acids were 16.5% of bacterial fatty acids and 11.0% of protozoal fatty acids. The anteiso-17:0 proportions in bacterial and protozoal fatty acids were 1.4 and 2.9%, respectively. The most abundant trans-18:1 isomer, vaccenic acid (18:1 trans-11), was 6.6% of total fatty acids in protozoa and 2.0% of total fatty acids in bacteria. The cis-9, trans-11 CLA was 8.6-times greater in the protozoal fraction (1.32% of total fatty acids) than in the bacterial fraction (0.15%). These results suggest that the presence of protozoa in the rumen may increase the supply of CLA and other unsaturated fatty acids for lower gut absorption by ruminants.     

Changes in the amino acid levels in hydrolysates of bacteria adhering to the rumen in sheep during feeding with high and low nitrogen diets]

The effects of low and high nitrogen diets on amino acid levels were studied in hydrolyzates of ruminal bacteria adhered to four topographically different anatomic parts of the ruminal wall (dorsal, ventral and caudal parts as well as reticulum) in 18 sheep + of the Slovak Merino breed divided into three experimental groups. The epimural bacteria of the dorsal and ventral parts of the ovine rumen revealed the most sensitive reaction to the varying amounts of nitrogen ingested with the diet. In hydrolyzates of ruminal bacteria adhered to the dorsal and ventral epithelium, 15 and 14 amino acids were changing (Figs. 1, 2). In hydrolyzates of epimural bacteria, a sensitive reaction was observed in the following amino acids: alanine, histidine, thyroxin, arginine and proline (Tabs. I-IV). In all topographical and anatomical parts of the rumen, both alanine and histidine levels in hydrolyzates of epimural ruminal bacteria significantly increased with the diet with high-nitrogen content fed, but was falling in sheep fed with low-nitrogen diet. Changes in alanine concentrations may be explained by the fact that alanine forms a part of the mechanism for short-time storage of ammonia in bacterial cells (Bartos, 1987). The fact that alanine is in its lack deaminated to pyruvate (Havassy, 1976) is explained by significant fall in alanine contained in hydrolyzates of bacterial proteins when fed low nitrogen diets. Significant fall in alanine in shortage of amino acid bound nitrogen can be explained by the fact that under these conditions, the alanine skeleton is being incorporated in to 80% of amino acids synthetized de novo by ruminal bacteria (Syv?oja and Kreula, 1980). When sheep flock was fed the high-nitrogen diet, thyroxin and proline levels were significantly reduced in hydrolyzates of epimural bacteria from all parts of the rumen, while low-nitrogen diet significantly increased the concentrations of both given amino acids in comparison with the control. Bartos (1987) gives in his study the table containing weight representation of different amino acids in proteins of bacteria of the ruminal content compiled on the basis of data of several authors. These data principally correspond to the results of our measurements in hydrolyzates of epimural bacteria. The highest weight representation of amino acids in hydrolyzates of epimural bacteria was found for glutamic acid, aspartic acid and arginine, while the lowest ones were detected for thyroxin, proline and phenylalanine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Amino acid and fatty acid composition of microorganisms passing into the abomasum of sheep

The bacterial and protozoal fractions were isolated by a differential centrifugation method from daily samples of chyme passing into the abomasum from the forestomach of sheep. The mean values for the nitrogen content (percentage of dry matter) were 8.3 ± 0.2% for the bacterial fraction and 5.3 ± 0.2% for the protozoal fraction. They were not significantly affected by the different products, methionine, lysine or starch, added to the basal ration. The amino acid composition of the bacterial and protozoal proteins was relatively constant, though some slight variations in certain amino acids were observed in relation to the nature of the supplements.The total lipid content ranged from 5.42 to 8.85% of the dry matter for the bacterial fraction. Its mean value was 9.63% for the protozoal fraction. It was not affected significantly by the supplements. Free fatty acids dominate the lipid composition of bacteria and phospholipids dominate in that of protozoa. The principal fatty acids of protozoal lipids were palmitic acid (41.32%) and oleic acid (19.33%).     

Effect of thermal treatment of potato products on nutrient decomposition in the digestive tract of swine. 2. Nutrient content and bacterial activity in various parts of the intestine

Dry matter (DM), starch, crude protein (CP), amino acids (AA) and diaminopimelic acid (DAPA) were estimated in the contents of the digestive tract of slaughtered pigs, which were fed a basal diet with raw (rKSt) or with cooked (QuSt) potato starch. When fed rKSt, there was larger quantities of starch and DAPA in the content of the distal small intestine and also of DM, CP, AA and DAPA in the contents of caecum and proximal colon, compared to QuSt. The bacterial breakdown of the raw potato starch granules in the proximal large intestine is shown by microscopic examinations. It is suggested, that the quantity of bacterial protein excreted in the faeces did not allow to quantify the extent of the bacterial activity in the different segments of the digestive tract.     

Comparison of three markers for the determination of bacterial protein in terminal ileal digesta in the growing pig

The aim of the study was to compare three methods commonly used to determine the concentrations of bacterial protein in digesta collected from the terminal ileum of growing pigs that had been fed a casein‐based diet. The amounts of bacterial protein in terminal ileal digesta were determined using three different markers: 2.6‐diaminopimelic acid (DAPA) and the d ‐amino acids, d ‐aspartic acid (d ‐Asp) and d ‐glutamic acid (d ‐Glu). The effectiveness of each marker was compared against a control based on physical fractionation by centrifugation. The total bacterial protein concentrations derived from the markers d ‐Asp and d ‐Glu were significantly different (p = 0.05) to those calculated from DAPA and the control, but there was no difference between DAPA and the control. The percentage of bacterial nitrogen ranged from 40% to 52% dependent on the marker used. Bacterial protein expressed as a percentage of the total protein, ranged from 48% to 62%, a substantial proportion of which (12–28%) was derived from lysed bacterial cells. Statistical correlations between the estimation methods were low. Such poor correlation between the markers may be the result of random errors such as variance in the epimerization of the two d ‐amino acids during protein hydrolysis. DAPA was accepted as a reliable marker for determining microbial protein in ileal digesta.     

Amino acid production in the isolated and perfused rumen wall of sheep

The metabolism of amino acids and urea in an isolated perfused wall of sheep rumen was studied by the method of organ perfusion, using 3 kinds of perfusate. In experiments with semisynthetic perfusate containing urea (SPurea) the levels of lysine, valine, glutamic acid and alanine increased at a simultaneous decrease of levels of leucine, arginine, aspartic acid, threonine and methionine. In perfusions with the semisynthetic medium without urea (SP) the levels of lysine, valine, phenylalanine, isoleucine, glutamic acid, glycine and alanine increased, the level of threonine dropped. In perfusions with whole autologous blood (KP) the levels of valine, glutamic acid and alanine increased, the level of arginine decreased. The level of urea decreased sharply at using SPurea and KP, however, the equal cumulation of ammonia (200-400 mumol x 1(-1)) was found in all three types of perfusate after 2-hr perfusion. Moreover, in experiments with SP a low level of urea was found already after 5-min. of perfusion. The results of the experiments suggest that the rumen wall is capable of producing amino acids also without the contents of rumen, and that independently on the presence or absence of urea as a nitrogen source.     

Estimation of the proportion of bacterial nitrogen in canine feces using diaminopimelic acid as an internal bacterial marker

A bacterial marker can be used to determine the portion of fecal N that is of bacterial origin, as well as the effect of dietary factors on the bacterial N in feces of the dog. Two experiments were conducted to determine the efficacy of diaminopimelic acid (DAPA) and purines as bacterial markers in dogs. In Exp. 1, five adult female dogs were fed the same commercial diet. In Exp. 2, 50 dogs were fed one of four test diets (as-fed basis): a prebiotic-free control or diets containing either 1% chicory, 1% mannanoligosaccharide (MOS), or 1% chicory plus 1% MOS. Fresh feces were collected in both experiments and used to isolate a bacteria-rich sample (BRS) by differential centrifugation. In Exp. 1, the BRS had a N:purine ratio of 0.66 and N:DAPA ratio of 18.9. The CV for the N:purine ratio (20.7%) was much higher than that for the N:DAPA ratio (6.1%), indicating that DAPA resulted in a less variable estimate of fecal bacterial N. Using either marker, approximately 50% of the fecal N was estimated to be of bacterial origin. In Exp. 2, the N:DAPA ratio of the BRS did not differ (P = 0.14) among treatments. The BRS from dogs fed prebiotic-containing diets had treatment averages for N:DAPA ratios ranging from 16.9 to 18.5, whereas BRS from dogs fed the control diet had a ratio of 15.9. Averaged across all dogs, approximately 46% of fecal N was of bacterial origin. When calculating fecal bacterial concentrations using the average N:DAPA ratio for all dogs, little difference existed in the estimation compared with using individual values. The value resulting from use of the average ratio was approximately 13% higher than when using the individual ratios for dogs fed the control diet, which was due to the lower N:DAPA ratio for dogs fed the control diet compared with dogs fed the other treatments. Based on the consistency of the N:DAPA ratio of the BRS, DAPA seems to be a suitable marker for estimation of bacterial N in the feces of dogs.     

羊瘤胃乳酸菌的分离鉴定及抗逆性筛选

为开发羊源乳酸菌菌种资源,从健康成年散养山羊瘤胃内容物中分离乳酸菌,经生化试验、基因测序以及同源性分析、构建遗传进化树等方法对分离出的乳酸菌进行鉴定,并通过耐酸和耐胆盐试验筛选抗逆性良好的乳酸菌.结果表明:从散养山羊瘤胃内容物中分离出11株乳酸菌,将其编号为R1～R9、R11和R12;其中R1、R2为杜氏肠球菌,R3、...     

Several methods for determining total titers of free alpha-amino acids in body fluids

Comparisons were made among different variants of ninhydrin reaction to determine overall titres of free alpha amino acids in body fluids. Included were para-benzoquinone, 2,4,6-trinitrobenzene sulphonic acid, and 2,4-dinitro-1-fluorobenzene (DNFB). The best reproducibility of results was recorded from DNFB which can be used, as well, for analysis of milk or rumen juice.     

气相色谱测定羊瘤胃液中挥发性脂肪酸方法研究

建立了程序升温气相色谱法测定羊瘤胃液中的挥发性脂肪酸。样品经过滤离心,偏磷酸溶液酸化后,直接进样分析。结果表明:该方法实现了羊瘤胃液中乙酸、丙酸和正丁酸直接快速分离,方法的精密度、回收率较高。     

Elution of bacteria adhering to the epithelium in the rumen in sheep and their detection using electron microscopy

A method of preparing the pure concentrate of epithelium-adherent bacteria in sheep rumen was worked out and it was tested in 24 samples of the rumen wall, obtained from 12 slaughtered sheep. The purity of the bacterial eluate was checked by transmission electron microscopy and negative staining, followed by electron-microscopic evaluation. Besides bacterial cells no residues of feed, epithelium and other undesirable impurities were found out in the eluate. The percent yield of this method was determined by scanning electron microscopy and it made 93.3%. An isolated pure concentrate of the mixture of bacterial cultures, previously adhered to the rumen epithelium, was prepared which can be used for morphological and biochemical studies of this interesting group of rumen bacteria.     

Comparison of rumen bacteria distribution in original rumen digesta,rumen liquid and solid fractions in lactating Holstein cows

Background: Original rumen digesta, rumen liquid and solid fractions have been frequently used to assess the rumen bacterial community. However, bacterial profiles in rumen original digesta, liquid and solid fractions vary from each other and need to be better established.Methods: To compare bacterial profiles in each fraction, samples of rumen digesta from six cows fed either a high fiber diet(HFD) or a high energy diet(HED) were collected via rumen fistulas. Rumen digesta was then squeezed through four layers of cheesecloth to separate liquid and solid fractions. The bacterial profiles of rumen original digesta, liquid and solid fractions were analyzed with High-throughput sequencing technique.Results: Rumen bacterial diversity was mainly affected by diet and individual cow(P 0.05) rather than rumen fraction. Bias distributed bacteria were observed in solid and liquid fractions of rumen content using Venn diagram and LEf Se analysis. Fifteen out of 16 detected biomarkers(using LEf Se analysis) were found in liquid fraction, and these 15 biomarkers contributed the most to the bacterial differences among rumen content fractions.Conclusions: Similar results were found when using samples of original rumen digesta, rumen liquid or solid fractions to assess diversity of rumen bacteria; however, more attention should be draw onto bias distributed bacteria in different ruminal fractions, especially when liquid fraction has been used as a representative sample for rumen bacterial study.     

Passage of amino acids through the minimal wall in sheep]

Three sheep with a small isolated rumen (after Gridin et al., 1964) were studied for the passage of amino acids from the blood to the isolated rumen before feeding and 1, 2, 3, 4 and 5 hours after feeding. It was found that, on an average for all the time intervals mentioned above, the passage of glycine was the largest of all the amino acids studied (0.512 muMol per 100 ml), followed, in descending order, by lysine, alanine, glutamic acid, aspartic acid, serine, leucine, threonine, isoleucine, arginine, tyrosine, and phenylalanine (0.038 muMol per 100 ml). Before feeding and one hour after feeding, lysine shared the greatest proportion of all amino acids that had passed into the isolated rumen (0.565-0.43 muMol per 100 ml), followed, in descending order, by glycine, alanine, valine, aspartic acid, glutamic acid, leucine, serine, threonine, isoleucine, tyrosine, phenylalanine; arginine was, in this case, represented by the smallest proportion (0.042-0.030 muMol per 100 ml). It is inferred from the results that the amount of amino acids passing from the blood through the rumen wall changes with the time that has elapsed from feeding, and that before feeding this passage is more intensive than after feeding. These changes are held to be related with an increased passage of endogenous nitrogen to the rumen in the period of a relative deficiency of substances which are derived from the feed and are essential for maintaining homeostasis in the rumen.     

体外培养条件下不同长链脂肪酸对山羊瘤胃原虫群体结构和原虫吞噬细菌循环的影响

本试验旨在研究体外培养条件下不同长链脂肪酸对山羊瘤胃原虫群体结构和原虫吞噬细菌循环的影响。试验选取6种不同不饱和度的长链脂肪酸[分别为硬脂酸(A组)、油酸(B组)、亚油酸(C组)、α-亚麻油酸(D组)、花生四烯酸(E组)、二十碳五烯酸(F组),不饱和键个数从0~5依次递增],各脂肪酸的添加水平为底物的3%,进行体外培养。在体外培养10 h后测定原虫吞噬细菌速率,在体外培养24 h后测定细菌、原虫密度以及细菌蛋白质、原虫蛋白质含量。结果表明:除F组头毛虫属(Ophryoscolecinae)比例显著高于B和D组(P0.05)外,不同长链脂肪酸的添加对原虫中厚毛虫属(Dasytricharuminantium)、内纤毛虫属(Entodinium)、双毛虫属(Diplodiniinae)、等毛虫属(Isotrichidae)、前毛虫属(Epidinium)比例的影响均不显著(P0.05)。原虫密度以A组最高,并显著高于B组(P0.05);细菌密度以D组最高,并显著高于A、B、E、F组(P0.05)。原虫吞噬细菌速率A~F组依次为244.50、236.51、229.60、189.04、200.51、174.24 cells/(cell·h);另外,以D组的细菌周转率(0.68%)最低,且其细菌周转时间(146.92 h)最长。所估算的细菌蛋白质循环量以D[126.75 mg/(d·头)]和F组[131.63 mg/(d·头)]相对较低。因此,在本试验基础上认为,体外培养条件下α-亚麻油酸抑制山羊瘤胃原虫吞噬细菌效果较好。     

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。     

Changes in amino acid levels in bacteria adhering to rumen epithelium in sheep after oral administration of very low doses of mercury

The effect of low concentrations of mercury taken in the diet (4 mg per a head and day) on the amino acid composition in the proteins of rumen bacteria adhering to the dorsal and ventral parts of the rumen was studied in six sheep. Though the mercury did not influence the amino acid concentration in hydrolyzates of epimural bacteria, low-mercury doses caused significant changes of some amino acids, as compared with the control group of animals. The levels of alanine, histidine, threonine, serine and glycine were significantly decreased and at the same time the levels of proline, valine, isoleucine, tyrosine, lysine and phenylalanine were significantly increased. It was stated in our experiment that the levels of histidine, alanine and threonine in hydrolyzates of epimural bacteria of the rumen were significantly decreased both in the sheep given the balanced feed ration with additions of mercury and nitrogen (according to the standard) and in the sheep given a low-nitrogen diet. Similarly, the levels of proline, tyrosine and phenylalanine in the hydrolizates of epimural bacteria in rumen were significantly increased both with the low-nitrogen diet and in the normal -nitrogen diet with mercury supplement. The results suggest that unexplained disorders frequently occurring in the microbial synthesis in the rumen might be caused by long-continued administration of feed contaminated with extremely low concentrations of heavy metals. The problem deserves detailed investigation in future studies, oriented to explaining the biosynthesis disorders in rumen, reducing the production ability of ruminants.     

添加剂对紫花苜蓿青贮品质及小肠可吸收氨基酸的影响

为研究添加复合乳酸菌和葡萄籽单宁对紫花苜蓿(Medicago sativa L.)青贮品质及小肠可吸收氨基酸的影响，选用凋萎后二茬现蕾期紫花苜蓿为材料，设置对照(CK)，葡萄籽单宁(G)、复合乳酸菌(戊糖乳杆菌+戊糖片球菌+乳酸片球菌，L)和复合乳酸菌+葡萄籽单宁(LG)组，青贮60 d后测定其品质，并采用体外三步法测定其小肠可吸收氨基酸含量。结果表明：与CK组相比，G和LG组青贮饲料乳酸、乙酸、丁酸、总挥发性脂肪酸和氨态氮含量均显著降低(P<0.05)；L和G组粗蛋白含量显著提高(P<0.05)。与其他三组相比，G组青贮饲料的Ile，Leu，Lys，His，Arg，Tyr和总氨基酸含量显著提高，菌体蛋白以及小肠可吸收Ile，Lys，His，Arg，Ser和总氨基酸含量显著提高(P<0.05)。因此，添加葡萄籽单宁对紫花苜蓿青贮品质有一定改善作用，明显提高青贮饲料中Lys，His和Arg以及总氨基酸的小肠可吸收量。     

Amino acid profile of escaped feed protein after rumen incubation and their intestinal digestibility.

The crude protein content and amino acid profile of seven feedstuffs (linseed meal, maize gluten meal, rapeseed meal, rapeseed meal protected, soybean meal, fullfat soybean extruded and sunflower meal) were determined before and after ruminal incubation for 16 h in three bulls with large rumen cannulas. The intestinal disappearance of amino acids was measured using mobile bag technique. Ruminal incubation affected amino acid profile of all experimental feedstuffs. Crude protein degradation varied from 29.3% for maize gluten meal to 86.4% for rapeseed meal. A tendency towards increased disappearance was observed for glutamic acid, histidine, lysine and proline and decreased disappearance for branched-chain amino acids. The intestinal crude protein digestibility was higher than > 80%, except rapeseed meal (66.4%) and sunflower meal (77.8%). The least digestible individual amino acids were methionine and isoleucine in rapeseed meal, histidine and methionine in rapeseed meal protected and arginine in sunflower meal. In general, the lowest amino acid digestibilities were found in feedstuffs with the highest fibre content. The feedstuffs show that they have different potential for supplying of limiting amino acids. Of particular value are the feedstuffs with low crude protein degradability in the rumen and high intestinal digestibility of amino acids.     

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。     

Effect of dietary amino acids on in vitro rumen bacterial protein synthesis in buffaloes.

The effect of different ratios of urea to amino acid N at a fixed concentration of soluble sugars as energy source and varying levels of soluble sugars at optimum urea cell suspension was obtained from the rumen fluid of buffalo (Bubalus bubalis) calves fed on a growth ration. Under glucose fermentation, the bacterial protein content of the incubation mixture (I. M.) was increased to 3.91, 6.31 and 5.08 times the control value (urea alone) when 25, 50 and 75% of urea-N was replaced with amino acid N, respectively. With cellobiose, the corresponding increase was 4.06, 5.29 and 5.63 times. At 50% urea-N replacement with amino acid N (a ratio for maximum protein synthesis), the bacterial content was maximum when 1 g glucose or cellobiose per 100 ml of I. M. was added. Per cent incorporation of radioactivity from amino acids into bacterial protein was maximum at 25% amino acid N level with both the soluble sugar sources. The total amino acids incorporated into bacterial protein were, however, more at 50% than at 25% amino acid N level.