Effects of exogenous fibrolytic enzymes on in vitro ruminal fermentation of substrates with different forage:concentrate ratios

Batch cultures of mixed rumen micro-organisms were used to study the effects of three fibrolytic enzymes (xylanase from Trichoderma viride (XYL) and fibrolytic enzymes from Aspergillus niger (ASP) and Trichoderma longibrachiatum (TR)) on the fermentation of three substrates composed of grass hay:concentrate in the proportions (dry matter (DM) basis) of 0.7:0.3 (HF), 0.5:0.5 (MF) and 0.3:0.7 (LF). Enzymes were characterized for xylanase, endoglucanase, exoglucanase and amylase activities, and were supplied at rates of 40 and 80 enzymatic units/g substrate DM. In 8 h incubations, all enzymes increased (P=0.048 to P<0.001) the true degradability of substrate DM and the production of acetate, propionate, total volatile fatty acids (VFA) and gas. After 24 h incubation, some of the observed effects disappeared, but all enzymes still increased (P=0.028 to P<0.001) the degradability of substrate acid detergent fibre and the production of acetate, propionate and total VFA. For all enzymes, the effects on ruminal variables were less marked at 24 than at 8 h of incubation. Only few significant (P=0.044 to P=0.001) enzyme × substrate interactions were detected, although the magnitude of the response for each substrate varied with the enzyme. When considering the amount of organic matter apparently fermented (OMAF) and the methane:OMAF ratio as main variables, TR80 produced the greatest increase in OMAF (17.0%) for HF substrate, with ASP80 and TR40 having similar values (11.1 and 12.6%), and XYL and ASP40 showing no effects (P>0.05). A decrease (P<0.05) of methane:OMAF ratio was only found for TR80 at 8 h (17.4%). All enzymes, with the exception of ASP40, increased (P<0.05) OMAF at 8 h for MF substrate (11.3–25.4%), TR80 showing the greatest response. After 24 h of incubation, both doses of XYL and TR increased (P<0.05) OMAF (mean value 8.2%) and decreased methane:OMAF ratio (mean value 9.5%). All enzymes increased significantly OMAF with LF substrate at 8 h (7.5–19.9%), but after 24 h no effect (P>0.05) was detected on OMAF and methane:OMAF ratio. In general, few differences were detected between both doses of enzymes, which indicate than the used enzymes would be effective in enhancing ruminal degradation of substrates at a dose lower than 80 enzymatic units/g substrate DM.     

Effects of inclusion of neutral detergent soluble fibre sources in diets varying in forage particle size on feed intake, digestive processes, and performance of mid-lactation Holstein cows

The aim of this study was to evaluate effects of partial replacement of neutral detergent soluble fibre (NDSF) for starch in diets varying in particle size (PS) of alfalfa hay on chewing activities, ruminal fermentation, nutrient digestibility and performance in mid-lactation dairy cows. Eight multiparous Holstein cows (146 ± 6.0 d in milk; 36.7 ± 2.57 kg milk/d) were used in a replicated 4 × 4 Latin square design with four 21 d periods with the last 7 d for data collection. The experiment was a 2 × 2 factorial arrangement with 2 levels of NDSF (low = 85 g/kg or high = 130 g/kg diet dry matter) each combined with 2 PS (short = 20 mm or long = 40 mm) of alfalfa hay. Results show that forage PS alone, or in combination with NDSF inclusion, had no effect on dry matter (DM) intake. Although total chewing, eating and ruminating times were not affected by treatments, eating time per kg of neutral detergent fibre (NDF) ingested was higher in long versus short alfalfa hay-based diets (P<0.05). Feeding long forage PS increased sorting of the diet against particles >19 mm, and in favor of those <8 mm (P<0.05). Feeding diets high in NDSF lowered DM intake (P<0.05), but increased apparent digestibility of all nutrients including NDF (P<0.05) independent of forage PS. Ruminal pH and concentrations of total volatile fatty acids were unaffected by dietary treatments, however the proportion of butyrate was higher in ruminal fluid of cows fed high NDSF diets (P<0.05). Changes in milk composition included lower milk crude protein content in high NDSF diets and higher lactose content for short hay-based diets (P<0.05). That milk yield and milk energy output were similar in low versus high NDSF diets suggests that high NDSF-fed cows had higher energy efficiency due to lower DM intake. Results suggest that, independent of forage PS, NDSF sources can be successfully included to partly replace starchy grains in diets exceeding minimum fibre recommendations.     

Effects of fibre concentration of diets consisting of hay and slowly degradable concentrate on ruminal fermentation and digesta particle size in mid-lactation dairy cows

The objective of this study was to investigate the intestinal microbiota of growing kittens fed moderate- or high-protein diets using DNA-based qualitative and quantitative techniques. Kittens were weaned to a high-protein (HP; n = 7) or moderate-protein (MP; n = 10) diet at 8 weeks of age. Fresh faecal samples were collected at 8, 12, and 16 weeks of age. DNA was extracted and quantitative PCR used to quantify Bifidobacterium, Lactobacillus, Clostridium perfringens, and Escherichia coli concentrations. Denaturing gradient gel electrophoresis was performed to create a dendrogram and unrooted trees using Bionumerics 5.0 to identify similarity due to litter, age, or diet. Kittens fed HP had lower (p = 0.02) Bifidobacteria and Lactobacillus counts than MP-fed kittens. E. coli was lower (p = 0.02) in HP-fed kittens and tended to be affected by age (p = 0.09). Kittens were clustered by litter at 8 weeks of age, and then clustered by diet at 12 and 16 weeks of age. Our data suggest that faecal microbiota of growing kittens change after weaning and that dietary protein concentration affects E. coli, Bifidobacterium, and Lactobacillus populations. The relevance of these data in terms of intestinal health and disease remain to be determined and justifies further study.     

Evaluation of the effects of dietary particle fractions on fermentation profile and concentration of microbiota in the rumen of dairy cows fed grass silage-based diets

The study evaluated the effects of three different theoretical particle lengths (TPL) of grass silage on the distribution of particle fractions of the diet and the resulting effects on fermentation profile and concentrations of protozoa and mixed bacterial mass in the rumen of three lactating Holstein cows fed total mixed rations (45% grass silage, 5% grass hay and 50% concentrate) ad libitum. Decreasing TPL of grass silage (long, medium, short) reduced particles retained on the 19-mm sieve of the Penn State Particle Separator, while particle fractions from 8 mm to 19 mm and smaller than 8 mm were increased. Different TPL did not affect pH and the concentration of volatile fatty acids in the rumen. However, lowering the TPL from long to medium increased significantly the bicarbonate concentration, acetate proportion and protozoal number in the rumen, whereas the proportion of bacterial protein in ruminal digesta and its amino acid concentration were significantly increased by the short TPL. For the current feeding conditions, it can be concluded that increasing the fraction of particles between 8 and 19 mm and probably even the fraction below 8 mm by decreasing TPL of grass silage do not adversely affect rumen conditions and can be beneficial in terms of optimising concentration and activity of ruminal microbiota in high-yielding dairy cows.     

Effect of the proportions of neutral detergent fibre and starch, and their degradation rates, on in vitro ruminal fermentation

Effects of proportions of neutral detergent fibre (aNDFom) and starch, as well as their degradation rates, on rumen fermentation were tested using an in vitro rumen simulation system (SIMCO). The in vitro system was designed to simulate selective particle retention and had an average fluid volume of 1150 ml with a liquid dilution rate of approximately 0.07 h⁻¹. Two types of hay (aNDFom sources) and two types of starch were each included at two different levels in the diet and were examined in an experiment following a 2×2×2 factorial arrangement of treatments (eight diet combinations). The hay was either late-cut timothy (Phleum pretense L.) or early cut meadow grass (Poa pratensis L.), with ruminal in situ aNDFom digestion rates of 0.03–0.04 and 0.07–0.08 h⁻¹, respectively. The two starch types were raw (R) and cooked (C) potato starch with previously determined in vitro ruminal digestion rates of 0.04 and 0.20 h⁻¹, respectively. The starch levels were 300 and 600 g/kg diet dry matter (DM) with the remaining being hay (282–682 g/kg DM) and peptone (14–111 g/kg DM). The aNDFom level varied among the diets with different starch levels and hay types. The peptone acted as a source of peptides and, together with ammonia salts from buffer, was used to balance the N contents of the diets. The feeding level for each of the eight vessels was 28 g DM/d. Two 10-day simulations were made with the system. The average pH was higher (P<0.05) for all treatments with raw potato starch (6.19) versus cooked starch (6.07). Protozoa scores, on a qualitative scale, declined faster at the higher starch level. The aNDFom digestibility was, as expected, higher (P<0.001) for meadow hay (0.57) than timothy (0.32), and was also higher (P<0.001) at the lower starch level (0.54) versus the higher (0.35). Microbial protein production efficiency (mg microbial N/g organic matter truly digested) was higher for the faster degrading aNDFom (P<0.01) and starch (P<0.05) sources, but was not affected by starch level. Cooked starch resulted in a lower acetate proportion (449 mmol/mol versus 591 mmol/mol VFA; P<0.001) but higher proportions of propionate (297 mmol/mol versus 236 mmol/mol VFA; P<0.001), and butyrate (169 mmol/mol versus 127 mmol/mol VFA; P<0.01). Butyrate increased with starch level (127 mmol/mol versus 169 mmol/mol VFA; P<0.01), and was also higher for meadow hay versus timothy (168 mmol/mol versus 128 mmol/mol VFA; P<0.01). Interactions between the treatments demonstrate that the response in VFA pattern to starch level is dependent on starch and aNDFom sources. Substrates such as starch and aNDFom are fermented differently depending on their rates of ruminal degradation.     

Effect of the level of dry matter and protein and degradation rate of starch on in vitro ruminal fermentation

Effects of starch type, feeding level and level of a mixed N source upon rumen fermentation were examined using an in vitro rumen simulation system (i.e., SIMCO) with a fluid volume of 1100 ml and liquid dilution rate of approximately 0.07/h. Two sources of starch, two feeding levels and two levels of N were examined in an experiment following a 2 × 2 × 2 factorial arrangement of treatments. The starch sources were raw slowly degrading (R), and cooked fast degrading (C), potato starch and constituted 450 g/kg diet dry matter (DM). The remaining was supplied in the form of a grass hay mixture. The feeding levels were 20 (DM20) and 40 (DM40) g DM/d and the diet N level was either low (N1) or high (N2), using peptone and ammonia salt additions in the buffer. Two simulations of 10 days each were completed. The amount of bicarbonate in buffer was varied according to feeding level, and pH (average 6.3) did not differ between treatments. An increased degradation rate of starch (R vs. C) depressed neutral detergent fibre (aNDFom) digestibility (0.46 vs. 0.36) and organic matter true digestibility (OMTD; 0.73 vs. 0.68), but there were no other treatment effects on digestibility. The VFA production efficiency (average 0.47 g VFA/g OMTD) was not affected by the main treatments, although an interaction between starch type and feeding level occurred. A decline in protozoa rating over the 10 day simulations was more apparent at the lower feeding level (DM20), indicating poor growth conditions for protozoa. Higher microbial efficiency (11.0 vs. 8.9 mg microbial N/g OMTD) and a higher proportion of propionate (272 vs. 207 mmol/mol VFA) occurred at DM20 compared to DM40. Increased degradation rate of starch (i.e., R vs. C) resulted in an improved microbial N efficiency (8.8 vs. 11.2 mg/g OMTD) and an increase in the proportion of propionate (226 vs. 253 mmol/mol VFA) at the expense of acetate (610 vs. 591 mmol/mol VFA). Increasing the level of N (i.e., N1 vs. N2) improved the microbial N efficiency (9.2 vs. 10.7 mg/g OMTD) and increased the proportion of propionate (230 vs. 249 mmol/mol VFA) and butyrate (100 vs. 119 mmol/mol VFA) at the expense of acetate (629 vs. 572 mmol/mol VFA). Interactions between starch type and N level upon VFA patterns were apparent. Results support earlier findings in that carbohydrate degradation rate is an important factor determining microbial growth rates and VFA distribution.     

Effects of Bacillus subtilis natto on milk production,rumen fermentation and ruminal microbiome of dairy cows

Two experiments were conducted to evaluate the effects of Bacillus subtilis natto, which was initially isolated from fermented soybeans on milk production, rumen fermentation and ruminal microbiome in dairy cows. In Experiment 1, 36 early lactation Chinese Holstein dairy cows (56 ± 23 days in milk) were randomly assigned to three groups: Control, cows were fed total mixed ration (TMR); BSNLOW, TMR plus 0.5 × 10¹¹ colony-forming units (cfu) of B. subtilis natto/cow per day; and BSNHIGH, TMR plus 1.0 × 10¹¹ cfu of B. subtilis natto/cow per day. During the 70-day treatment period, daily milk production and daily milk composition were determined in individual cows. The results showed that supplementing dairy cows with 0.5 × 10¹¹ and 1.0 × 10¹¹ cfu of B. subtilis natto linearly increased (P < 0.01) milk production (25.2 and 26.4 kg/day v. 23.0 kg/day), 4% fat-corrected milk (27.3 and 28.1 kg/day v. 24.2 kg/day), energy-corrected milk (27.3 and 28.2 kg/day v. 24.2 kg/day), as well as milk fat (1.01 and 1.03 kg/day v. 0.88 kg/day), protein (0.77 and 0.82 kg/day v. 0.69 kg/day) and lactose yield (1.16 and 1.22 kg/day v. 1.06 kg/day) but decreased milk somatic cell counts (SCC) by 3.4% to 5.5% (P < 0.01) in BSNLOW and BSNHIGH treatments compared with Control. In Experiment 2, four rumen-cannulated dairy cows were fed the basal diet from 1 to 7 days (pre-trial period) and rumen samples were collected on days 6 and 7; the same cows then were fed 1.0 × 10¹¹ cfu/day B. subtilis natto from days 8 to 21 (trial period) and rumen samples were collected on days 20 and 21. B. subtilis natto was discontinued from days 22 to 28 (post-trial period) and rumen samples were collected on days 27 and 28. Compared with the pre- and post-periods, ruminal pH decreased by 2.7% to 3.0% during the trial period (P < 0.01), whereas ammonia nitrogen (NH₃-N), total volatile fatty acids and molar proportion of propionate (P < 0.01) and valerate (P < 0.05) increased. Molar proportion of acetate decreased and the acetate to propionate ratio was lower (P < 0.01) during the trial period. However, no differences for 24-h in sacco dry matter digestibility were detected among different periods (treatments) though NDF digestibility was reduced in the trial and post-trial periods (P < 0.01). Compared with pre-trial period, total ruminal bacteria, proteolytic and amylolytic bacteria in rumen enumerated by culture methods increased by 15.0%, 16.2% and 11.7%, respectively (P < 0.01) but protozoa decreased to 5.35 log₁₀ cfu/ml (P < 0.01) during the trial period. These results demonstrate that B. subtilis natto improves milk production and milk components yield, decreases SCC and promotes the growth of total ruminal bacteria, proteolytic and amylolytic bacteria, which indicate that B. subtilis natto has potential to be applied as a probiotic for dairy cows.     

溶菌酶对瘤胃体外发酵、甲烷生成及微生物菌群结构的影响

【目的】通过体外静态模拟瘤胃发酵法研究溶菌酶对瘤胃发酵、甲烷生成及微生物菌群结构的影响。【方法】采用单因素多水平试验设计,溶菌酶添加水平分别为0(L-0,对照组)、0.1 mg/100 m L(L-0.1)、1 mg/100 m L(L-1)、10 mg/100 m L(L-10)和100 mg/100 m L(L-100),定时测定产气量和甲烷产量,培养24 h后,发酵液用于发酵参数和微生物菌群数量的q PCR测定,其中L-0、L-1和L-100三个组发酵液同时进行16S r RNA基因Illumina高通量测序。【结果】与对照组相比,低剂量溶菌酶添加(L-0.1组)不影响甲烷产量、氨氮浓度、干物质消失率、有机物消失率和总挥发性脂肪酸等瘤胃发酵参数(P0.05);随着剂量提高,L-1处理组甲烷产量、氨氮浓度显著降低(P0.05),丙酸浓度显著增加(P0.05),并且干物质消失率、有机物消失率和总挥发性脂肪酸不受影响(P0.05);而较高剂量组(L-10和L-100组)虽然甲烷产量显著降低,丙酸浓度显著增加(P0.05),但干物质消失率和有机物消失率也显著降低(P0.05)。q PCR结果显示高剂量组(L-100组)总菌、原虫、甲烷菌数量与对照组相比显著降低(P0.05),而L-0.1、L-1和L-10组总菌、真菌和原虫数量与对照组相比均无显著变化(P0.05)。高通量测序主成分分析(PCA)显示对照组与溶菌酶添加组间瘤胃细菌组成的明显区分,说明添加溶菌酶显著改变了瘤胃细菌菌群结构。溶菌酶通过增加月形单胞菌和琥珀酸弧菌等丙酸生成菌的相对丰度,使更多的氢被用于生成丙酸,导致甲烷产量降低;溶菌酶可抑制普雷沃氏菌和拟杆菌属等蛋白降解菌的生长,进而减少蛋白质过度降解,降低氨氮浓度。【结论】添加适宜浓度(1 mg/100 m L)的溶菌酶可通过调控瘤胃微生态改变瘤胃发酵模式,降低瘤胃甲烷和氨的生成,短期内并不影响饲料消化。     

Effects of isobutyrate on rumen fermentation, lactation performance and plasma characteristics in dairy cows

The objective of this study was to evaluate effects of isobutyrate supplementation on rumen fermentation, lactation performance and plasma characteristics of dairy cows. Twenty multiparous second filial generation (F2) cows of a cross between Chinese Jinnan Yellow and Holstein cows at 148 ± 4.5 days in milk and 22.3 ± 0.81 kg milk production were used in a replicated 4 × 4 Latin square experiment. The treatments were: control (without isobutyrate), low (LIB), medium (MIB) and high (HIB) isobutyrate supplementation of 20, 40 and 60 g per cow per day, respectively. Experimental periods were 30 days with 15 d of adaptation and 15 d of data collection. Dry matter (DM) intake was not affected by increasing isobutyrate supplementation, but milk yields were highest for the 40 g/d isobutyrate supplementation level, where proportion of milk fat, true protein and lactose were minimized. Ruminal pH (6.38–6.24) and ammonia N (13.8–11.1 mg/100 ml) were linearly (P<0.01) decreased, whereas total VFA concentration (124–131 mM) increased at a decreasing rate with increasing isobutyrate supplementation. The ratio of acetate to propionate increased linearly (P<0.01) from 2.77 to 4.43 as isobutyrate supplementation increased due to the increase in acetate production and decrease in propionate production. Digestibilities of OM in the total tract increased linearly (P<0.01) as isobutyrate supplementation increased, digestibilities of DM and EE were highest for the 40 g/d isobutyrate supplementation level, digestibilities of CP, aNDF and ADF increased at a decreasing rate with increasing isobutyrate supplementation. Plasma concentrations of glucose and growth hormone linearly (P<0.03) increased, whereas concentrations of non-esterified fatty acids linearly (P<0.01) decreased. Results indicate that supplementation of this diet with isobutyrate changed the rumen fermentation pattern towards acetate production, improved digestion and modified plasma concentrations of glucose and growth hormone. This suggests that isobutyrate stimulated digestive microorganisms or enzymes in a dose-dependent manner with the optimum isobutyrate dose at about 40 g per cow per day in terms of improved digestion.     

Effects of essential oils on in vitro ruminal fermentation and ammonia release

Ruminal inoculum enriched with particle-associated microorganisms was collected from two lactating dairy cows fed an alfalfa hay/cereal silage/concentrate diet 1 h before feeding and used to evaluate effects of essential oils (EO) on ruminal fermentation in short-term in vitro incubations. Ruminal ammonia N was labeled with ¹⁵N and native and hydrolyzed casein were provided as sources of amino acids. Forty EO were tested at 10 and 100 mg/l final medium concentration. Monensin-Na, and sodium laurate were also incubated at 5 and 2000 mg/l, respectively. Compared with blanks (i.e., no addition of EO), sodium laurate increased medium pH and a number of EO reduced medium pH. Both sodium laurate and monensin reduced ammonia concentrations compared to the blank. Only one of the tested EO (i.e., Caraway) slightly reduced ammonia concentration, by 8%, compared with the blank. Monensin and sodium laurate resulted in higher (i.e., 9–34%, monensin, and 29–47%, sodium laurate) ¹⁵N enrichment of ammonia N, an indication of reduced deamination of amino acids in these treatments versus the blank. Several EO (i.e., FrankMyrrh, Gardenia, Hibiscus, Eucaliptus, and Peppermint) had similar effects, but of a smaller magnitude (i.e., 5–12%). Some EO increased medium total VFA concentration, primarily through an increase in acetate concentration. Overall, effects of EO on fermentation were subtle, and it is unlikely that these moderate in vitro effects would correspond to any substantive impact on ruminal fermentation in vivo.     

Effects of expander-treating a barley-based concentrate on ruminal fermentation, bacterial N synthesis, escape of dietary N, and performance of dairy cows

Three primiparous dairy cows in early lactation with cannulas in rumen, duodenum and ileum were used in a 3×3 Latin square design to study effects of expander treatment of a barley-based concentrate. The concentrate was either pelleted at 75–80°C or expander treated at 125–130°C prior to pelleting. The diets consisted of 6.7 kg DM of grass silage and 10 kg DM of (1) 100% pelleted, (2) 50% pelleted and 50% expanded or (3) 100% expanded concentrate. The diets were offered as a mixed ration in four equal meals daily. Ruminal fermentation, bacterial N synthesis, duodenal, ileal and faecal flow of nutrients, and animal performance were monitored. Expander treatment numerically increased ruminal digestion of starch, which explained the observed increase in ruminal VFA concentration and the lowered ruminal pH (P<0.05). The proportion of butyrate in rumen liquid increased, whereas the proportion of propionate decreased in the expanded compared to the pelleted treatment (P<0.05). Expander treatment tended to increase rumen volume and rumen NDF pool size. Ruminal digestion of NDF was numerically lower in the expanded than in the pelleted treatment. No differences in bacterial N synthesis or efficiency of synthesis were observed among treatments. Expander treatment numerically increased the duodenal flow of non-ammonia N (NAN) and amino acid N (AAN), and seemed to increase the flow of non-ammonia non-bacterial N (NANBN) to the duodenum to a similar extent as was indicated by nylon bag studies. Milk production and milk fat and protein content were increased by the expander treatment (P<0.05), indicating that expander treatment increased the supply of nutrients for milk production.     

多糖类益生元的筛选及凝结芽孢杆菌体外发酵特性北大核心

【目的】探究9种多糖对凝结芽孢杆菌(Bacillus coagulans)的增殖、产酶特性的影响。【方法】将凝结芽孢杆菌分别添加至菊粉多糖(inulin polysaccharide)、刺五加多糖(Eleutherococcus senticosus polysaccharide)、壳寡糖(chitosan oligosaccharide)、防风多糖(Saposhnikovia divaricata polysaccharide)、低聚木糖(xylo-oligosaccharide)、黄芪多糖(Astragalus polysaccharide)、甘露糖(D-mannose)、白术多糖(Atractylodes macrocephala Koidz polysaccharide)和玉屏风多糖(Yu Ping Feng polysaccharide)为唯一碳源的培养基中,通过菌株生长、酶活性及其体外厌氧发酵等作为指标,筛选出最优益生元。【结果】凝结芽孢杆菌能很好地利用防风多糖、黄芪多糖、白术多糖和玉屏风多糖;添加量为4%的防风多糖和白术多糖,pH差值差异最大,蛋白酶活性差异显著(P<0.05)。体外发酵乳酸活性和总蛋白酶活性均提高,4%白术多糖的乳酸和总蛋白酶活性差异显著(P<0.05);肠道内容物发酵液16S rRNA基因高通量测序结果表明,与对照组比较,添加黄芪多糖、防风多糖、甘露糖3种益生元发酵凝结芽孢杆菌显著降低了气单胞菌(Aeromonas)、α-变形菌(α-Proteobacteria)、链球菌(Streptococcus)、志贺氏杆菌属(Shigella)等致病菌的相对丰度,提高了乳杆菌(Lactobacillus)、厚壁菌门(Firmicutes)、乳酸乳球菌(Lactococcus lactis)、产酸杆菌(Acidobacteria)的相对丰度。【结论】凝结芽孢杆菌发酵4%白术多糖具有较好的产酶性能与益生特性,二者协同发酵添加至饲料中具有较好的发展潜力。     

乳酸链球菌素对瘤胃体外发酵、甲烷生成及功能菌群数量的影响北大核心CSCD

【目的】旨在通过体外静态模拟瘤胃发酵法研究乳酸链球菌素(NI)对瘤胃发酵、甲烷生成及功能菌群数量的影响。【方法】以不添加任何添加剂处理做阴性对照(NC),以莫能菌素(MON,5μmol/L)做阳性对照,试验组NI添加水平分别为3(NI-3)、9(NI-9)和27 mg/100 m L(NI-27),每个处理4个重复,分别于培养后的0、3、6、9、12、24 h测定产气量和甲烷产量。培养24 h后,采集发酵液样品,用于发酵参数和菌群数量的测定。【结果】与NC组相比,添加NI和MON均能显著降低产气量和甲烷产量(P<0.05);添加NI对pH值、干物质消失率(DMD)和有机物消失率(OMD)无显著影响(P>0.05);NI-9处理组与NC组相比氨态氮浓度显著降低(P<0.05),而NI-3和NI-27组氨氮浓度没有显著变化(P>0.05);相比而言,MON处理组DMD、OMD和氨氮浓度与NC组相比均显著降低(P<0.05),而pH值与其他各处理组相比没有差异(P>0.05);与NC组相比,NI各处理组和MON组乙酸浓度及乙丙比均显著降低(P<0.05),丙酸浓度显著提高(P<0.05)。功能菌方面,qPCR结果显示添加NI和MON对总菌和拟杆菌门数量均无显著影响(P>0.05);与NC相比,添加NI对原虫、甲烷菌、真菌和厚壁菌门数量均无显著影响(P>0.05),而MON组原虫、甲烷菌、真菌和厚壁菌门数量显著降低(P<0.05);NI和MON处理均显著提高了硫还原菌和C.aminophilum数量(P<0.05),但C.sticklandii数量不受影响(P>0.05)。【结论】添加适宜浓度的NI可降低瘤胃甲烷与氨的生成,但并不影响饲料消化,这种发酵模式的改变可能与瘤胃功能菌群数量与多样性的变化密切相关。     

Effects of the level of feed intake and ergot contaminated concentrate on ruminal fermentation and on physiological parameters in cows

The aim of the present study was to examine the effects of ergot contaminated feed concentrate at differing levels of feed intake on ruminal fermentation, and on various physiological parameters of dairy cows. Twelve double fistulated (in the rumen and the proximal duodenum) Holstein Friesian cows were fed either a control diet (on a dry matter (DM) base: 60% maize silage, 40% concentrate) or a diet containing ergot alkaloids (concentrate contained 2.25% ergot resulting in an ergot alkaloid concentration of the daily ration between 505 and 620 (μg/kg DM) over a period of four weeks. Daily feed amounts were adjusted to the current performance which resulted in a dry matter intake (DMI) variation between 6.0 and 18.5 kg/day. The resulting ergot alkaloid intake varied between 4.1 and 16.3 (μg/kg body weight when the ergot contaminated concentrate was fed. Concentrations of isovalerate, propionate and ammonia nitrogen in the rumen fluid were significantly influenced by ergot feeding, and the amount of ruminally undegraded protein, as well as the fermentation of neutral detergent fibre, tended to increase with the ergot supplementation at higher levels of feed intake, which might indicate a shift in the microbial population. Other parameters of ruminal fermentation such as ruminai pH, fermented organic matter as a percentage of intake, or the amount of non-ammonia nitrogen measured at the duodenum were not significantly influenced by ergot feeding. The activities of liver enzymes (aspartate aminotransferase, γ-glutamyltransferase, glutamate dehydrogenase, creatine kinase) in the serum were not affected by ergot feeding. The rectal measured body temperature of the cows significantly increased after ergot administration (p=0.019). Thus, body temperature can be regarded as a sensitive parameter to indicate ergot exposure of dairy cows.     

The influence of addition of gallic acid, tannic acid, or quebracho tannins to alfalfa hay on in vitro rumen fermentation and microbial protein synthesis

Tannins may reduce rumen degradability of protein, increase the proportion of feed protein reaching the lower digestive tract for enzymatic digestion and thereby increase the efficiency of protein utilization. The objective was to assess the effects of different types and levels of tannins on rumen in vitro gas production and its kinetics, in vitro true degradability (IVTD) and rumen degradability of protein (IVRDP), and microbial protein synthesis by incubating alfalfa (Medicago sativa L.) hay in buffered rumen fluid. Alfalfa was incubated in buffered rumen fluid with and without the addition of different levels of gallic acid (GA), quebracho tannin (QT), or tannic acid (TA). Tannins at the lower inclusion levels had minimal effects on fermentation products compared to the higher levels. Addition of QT and TA reduced ammonium-N (NH₄⁺-N) concentration. Addition of QT at 20, 40, and 60 g/kg DM decreased NH₄⁺-N by 2, 7, and 12% compared with control whereas addition of TA reduced NH₄⁺-N by 5, 6, and 12% when added at 20, 40 and 60 g/kg DM, respectively. In experiment 2, addition of QT at 50, 100, and 150 g/kg DM, resulted in reduction of NH₄⁺-N by 12, 30, and 51%, respectively, compared with the control. Addition of TA at 50, 100, and 150 g/kd DM reduced NH₄⁺-N by 14, 26, and 47% compared with control. Inclusion of QT at 50, 100, 150 DM reduced IVRDP by 13, 30, and 36% compared with control whereas at these levels of inclusion, TA resulted in reduction of IVRDP by 14, 25, and 48%. Rate of gas production decreased (P<0.001), while asymptotic gas production increased (P<0.0001) with increasing level of GA and TA. Quebracho tannin decreased (P<0.0001) both the rate and asymptotic of gas production. Gallic acid had a positive effect on fermentation as indicated by increased gas production and total short chain fatty acids (SCFA) production. Quebracho tannin decreased 24 h gas production, IVTD, and total SCFA production. Acetate to propionate ratio increased with the addition of GA and but decreased when QT was added. Addition of tannins did not markedly increase total purines but numerical values tended to be higher in the presence of tannins compared with the control. Efficiency of microbial growth was lower in the presence of GA and unaltered by TA, but higher in the presence of QT compared with the control. The effect of tannins on rumen fermentation and protein degradation varied with type and level of tannins. In vivo studies will be conducted to validate the in vitro results.     

Evaluation of the effects of dietary particle fractions on fermentation profile and concentration of microbiota in the rumen of dairy cows fed grass silage-based diets

The study evaluated the effects of three different theoretical particle lengths (TPL) of grass silage on the distribution of particle fractions of the diet and the resulting effects on fermentation profile and concentrations of protozoa and mixed bacterial mass in the rumen of three lactating Holstein cows fed total mixed rations (45% grass silage, 5% grass hay and 50% concentrate) ad libitum. Decreasing TPL of grass silage (long, medium, short) reduced particles retained on the 19-mm sieve of the Penn State Particle Separator, while particle fractions from 8 mm to 19 mm and smaller than 8 mm were increased. Different TPL did not affect pH and the concentration of volatile fatty acids in the rumen. However, lowering the TPL from long to medium increased significantly the bicarbonate concentration, acetate proportion and protozoal number in the rumen, whereas the proportion of bacterial protein in ruminal digesta and its amino acid concentration were significantly increased by the short TPL. For the current feeding conditions, it can be concluded that increasing the fraction of particles between 8 and 19 mm and probably even the fraction below 8 mm by decreasing TPL of grass silage do not adversely affect rumen conditions and can be beneficial in terms of optimising concentration and activity of ruminal microbiota in high-yielding dairy cows.     

Effects of fibre concentration of diets consisting of hay and slowly degradable concentrate on ruminal fermentation and digesta particle size in mid-lactation dairy cows

Four multiparous ruminally cannulated Holstein cows (mean bodyweight [BW] 615 kg) in mid-lactation (103 days in milk and 32 kg milk x d(-1) at start of the experiment) were used in an one-factorial experiment to evaluate the effects of fibre level (19, 24, 28, 32 and 39% physically effective NDF [peNDF] in dry matter [DM]) in diets consisting of hay and slowly degradable concentrate on rumen fermentation patterns and digesta particle size, under a constant intake level (146 g DM x kg(-0.75). The different fibre concentrations in the diet were achieved by adjusting the hay to concentrate ratio. The above-mentioned levels of peNDF corresponded to 70, 60, 50, 40 and 25% concentrate in diet DM, respectively, and followed the lactation curve of the cows. The ruminal pH was positively and linearly correlated to the percentage of fibre (peNDF, NDF or CF) in ration DM with R2 of 0.76-0.88 (p < 0.001) for solid digesta (particle-associated rumen fluid, PARL), and R2 of 0.26-0.29 (p < 0.05) for fluid digesta (free rumen liquid, FRL). The lowest fibre level in the diet (19% peNDF) or the highest level of concentrate (70% on DM basis) caused pH values lower than 6.0 at almost all sampling times only in PARL but not in FRL, and significantly increased the proportion of large particles in rumen digesta, which in turn was reflected by a depression of fibre digestibility. A level of 24% peNDF or 60% concentrate in the diet maintained the ruminal pH higher than 6.0 and 5.8 in FRL and PARL, respectively. Therefore, the inclusion of more than 60% slowly degradable concentrate in dairy cows diets fed approximately 18 kg DM x d(-1) is discouraged. Based on the response of ruminal solid digesta to dietary fibre, it can be concluded that the recommendations of feeding a structural value > or =1 per kg DM (De Brabander et al. 1999) underestimated, and 400 g CF per 100 kg BW (Hoffmann 1990) overestimated the evaluation of structural effectiveness of the present diet.     

A new in vitro system to study the effect of liquid phase turnover and pH on microbial fermentation of concentrate diets for ruminants

A simple and inexpensive in vitro feed evaluation system which estimates microbial fermentation by measuring gas production and allows for adjustment of liquid dilution rate has been design. Working conditions, in terms of substrate and inoculum proportions (experiment 1, EXP 1) and the liquid replacement schedule (experiment 2, EXP 2) were initially set. In EXP 1, three substrate concentrations (10, 20 and 30 mg/ml: S1, S2 and S3) and three inoculum proportions (0.10, 0.20 and 0.30; I10, I20 and I30) were assayed in a 3 × 3 factorial design, in three incubations of 24 h. Gas production (ml/g organic matter) with S1 was the highest, avoiding to be in excess for microbial colonisation and activity and inoculum I20 limited inoculum contribution by self-fermentation; they were hence considered for further trials. In EXP 2, two liquid replacement schedules (every hour from 0 to 12 h and every 3 h from 12 to 24 h, HF, and every 2 h from 0 to 12 h and every 4 h from 12 to 24 h, LF) were compared with a non-replacement (NR) treatment, using three bottles per treatment. No differences among treatments were detected, and thus the LF schedule was chosen because it was easier to carry out. Once these working conditions were adjusted in EXP 1 and 2, the effect of fluid turnover and incubation pH on the extent of microbial fermentation in a mixed diet (experiment 3, EXP 3) was studied in two incubations. Two liquid turnover rates (0.06 and 0.10 h⁻¹) were compared with a non-replacement treatment at two different pH (6.8 and 6.1) in a 3 × 2 factorial design. Liquid outflow increased gas production, dry matter disappearance (DMd) and total volatile fatty acid (VFA) concentration at both pH (P>0.01), but protozoal numbers were unaffected. Liquid replacement at pH 6.8 did not affect the VFA profile, but the acetate proportion diminished (P=0.04) and the propionate increased (P<0.001), with replacement at pH 6.1. Increasing the fluid turnover rate from 0.06 to 0.10 h⁻¹ reduced gas production from 8 to 20 h of incubation (P<0.05) but it did not alter the VFA profile. The volume of gas produced decreased (P<0.05) with incubation pH, as did DMd (P=0.04) and protozoal (P=0.03) and total VFA (P=0.002) concentration. The acetate proportion was lower (P<0.001) and that of propionate and butyrate was higher (P<0.001) at pH 6.1 than at pH 6.8. Both liquid turnover and pH must be taken into consideration for in vitro evaluation of mixed and concentrate diets for ruminants and this system provides a simple and inexpensive method for doing this.     

Effects of feeding level and NDF content of grass-clover silages on chewing activity,fecal particle size and NDF digestibility in dairy heifers

The objective of this study was to assess effects of feed intake and NDF content of highly digestible grass-clover silage on chewing behavior, fecal particle size distribution and apparent digestibility in restrictively fed heifers. Four grass-clover silages (Lolium perenne, Trifolium pratense and Trifolium repens) were harvested in 2009 at different regrowth stages, resulting in silages with NDF contents of 312, 360, 371 and 446 g/kg dry matter (DM), respectively. Four rumen-fistulated Jersey heifers (343±32 kg BW) were fed silage at 90% of ad libitum levels in a 4×4 Latin square design, replicated with further restricted feeding levels (50%, 60%, 70% or 80% of ad libitum) in a balanced 4×4×4 Greco-Latin square design. Eating activity was estimated from test meal observations, while rumination activity was estimated from jaw movements logged by a jaw recorder system. Total tract digestibility was estimated from chromic oxide marker and fecal spot sampling, and fecal particle size distribution in washed and freeze-dried particulate DM was determined by dry sieving (2.36, 1.0, 0.5, 0.212 and 0.106 mm, and bottom bowl). Higher NDF content of silage stimulated longer eating time per kg DM intake (P<0.001), while reduced feeding level caused a reduction in eating time per kg DM intake (P<0.001) and NDF (P<0.001). Rumination time per kg DM intake (P<0.01) increased with reduced feeding level, with less effect of feeding level at lower NDF contents (P<0.01) and more rumination with greater NDF content (P<0.01). Relative to NDF intake, rumination time increased with greater NDF content (P<0.01), at a higher rate with reduced feeding level (P<0.05). Digestibility of potentially digestible NDF (DNDF) decreased with greater NDF content (P<0.001) and increased with reduced feeding level (P<0.05). Increasing NDF content resulted in more particulate DM in feces (g/kg DM; P<0.05) and larger mean particle size (P<0.001). In conclusion, feeding heifers with grass-clover silages of decreasing NDF content increased chewing time relative to NDF intake, reduced mean fecal particle size, and increased DNDF digestibility. Restricting feeding level made heifers eat for a shorter time period while rumination and total chewing was increased, causing the ratio between eating and rumination time to decrease with lower intake of forage fiber. Particle size reduction and digestibility depended mostly on changes in NDF content, especially the indigestible NDF content.