Effect of maize silage to grass silage ratio and feed particle size on ruminal fermentation in vitro

The effect of the forage source on ruminal fermentation in vitro was investigated for fine (F) and coarse (C) milled diets, using a modified Hohenheim gas production test and a semi-continuous rumen simulation technique (Rusitec). It was hypothesised that the replacement of maize silage by grass silage might lead to associative effects and that interactions related to particle size variation could occur. Five diets with a maize silage to grass silage ratio of 100 : 0, 79 : 21, 52 : 48, 24 : 76 and 0 : 100 differed in their content of CP and carbohydrate fractions, as well as digestible crude nutrients, derived from a digestibility trial with wether sheep. For in vitro investigations, the diets were ground to pass a sieve of either 1 mm (F) or 4 mm (C) perforation. Cumulative gas production was recorded during 93 h of incubation and its capacity decreased with increasing proportion of grass silage in the diet. Across all diets, gas production was delayed in C treatments compared with F treatments. Degradation of crude nutrients and detergent fibre fractions was determined in a Rusitec system. Daily amounts of NH3-N and short-chain fatty acids (SCFA) were measured in the effluent. Degradation of organic matter (OM) and fibre fractions, as well as amounts of NH3-N, increased with stepwise replacement of maize silage by grass silage. Degradability of CP was unaffected by diet composition, as well as total SCFA production. In contrast to the results of the gas production test, degradation of OM and CP was higher in C than in F treatments, accompanied by higher amounts of NH3-N and SCFA. Interactions of silage ratio and particle size were rare. It was concluded that the stepwise replacement of maize silage by grass silage might lead to a linear response of most fermentation characteristics in vitro. This linear effect was also supported by total tract digestibility data. However, further investigations with silages of variable quality seem to be necessary.     

The effect of Lactobacillus buchneri on the fermentation, aerobic stability and ruminal degradability of maize silage

AIMS: To evaluate the effect of Lactobacillus buchneri, heterofermentative lactic acid bacteria (LAB), on the fermentation, aerobic stability and ruminal degradability of whole-crop maize silages under laboratory conditions. Two homofermentative LAB were tested for the purpose of comparison. METHODS AND RESULTS: Maize was harvested at early dent [290 g kg(-1) dry matter (DM)] and one-half milk line (355 g kg(-1) DM) stages. Both homofermentative LAB were applied at 1 x 10(5) CFU g(-1) of fresh forage. Lactobacillus buchneri was applied at 1 x 10(5), 5 x 10(5) and 1 x 10(6) CFU g(-1) of fresh forage. Silages with no additives served as control. After treatment, the chopped forages were ensiled in 1.5-l anaerobic jars. Three jars per treatment were sampled on day 60. After 60 days of storage, silages were subjected to an aerobic stability test lasting for 5 days, in which CO(2) production, as well as chemical and microbiological parameters, was measured to determine the extent of aerobic deterioration. Both homofermentative LAB increased the concentration of lactic acid and the numbers of yeasts, and decreased the concentration of acetic acid and impaired the aerobic stability of silages. In contrast, applying L. buchneri decreased the concentration of lactic acid and increased the concentration of acetic acid of the silages. Under aerobic conditions, silages treated with 5 x 10(5) and 1 x 10(6) CFU g(-1) of L. buchneri, had lower pH, CO(2) production and the numbers of yeasts than the silages treated with 1 x 10(5) CFU g(-1) of L. buchneri (P < 0.05). However, all doses of L. buchneri and both homofermentative LAB did not affect in situ rumen DM, organic matter and neutral detergent fibre degradability of the silages. CONCLUSIONS: Lactobacillus buchneri was very effective in protecting maize silages exposed to air under laboratory conditions. All doses of L. buchneri, especially 5 x 10(5) CFU g(-1) or more, markedly decreased the numbers of yeasts and improved the aerobic stability of silages. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of L. buchneri, as a silage inoculant, can improve the aerobic stability of maize silages by inhibition of yeast activity.     

Identification of Lactobacillus alimentarius and Lactobacillus farciminis with 16S-23S rDNA intergenic spacer region polymorphism and PCR amplification using species-specific oligonucleotide

AIMS: The restriction fragment length polymorphism (RFLP) method was used to differentiate Lactobacillus species having closely related identities in the 16S-23S rDNA intergenic spacer region (ISR). Species-specific primers for Lact. farciminis and Lact. alimentarius were designed and allowed rapid identification of these species. METHODS AND RESULTS: The 16S-23S rDNA spacer region was amplified by primers tAla and 23S/p10, then digested by HinfI and TaqI enzymes and analysed by electrophoresis. Digestion by HinfI was not sufficient to differentiate Lact. sakei, Lact. curvatus, Lact. farciminis, Lact. alimentarius, Lact. plantarum and Lact. paraplantarum. In contrast, digestion carried out by TaqI revealed five different patterns allowing these species to be distinguished, except for Lact. plantarum from Lact. paraplantarum. The 16S-23S rDNA spacer region of Lact. farciminis and Lact. alimentarius were amplified and then cloned into vector pCR(R)2.1 and sequenced. The DNA sequences obtained were analysed and species-specific primers were designed from these sequences. The specificity of these primers was positively demonstrated as no response was obtained for 14 other species tested. RESULTS AND CONCLUSIONS: The species-specific primers for Lact. farciminis and Lact. alimentarius were shown to be useful for identifying these species among other lactobacilli. The RFLP profile obtained upon digestion with HinfI and TaqI enzymes can be used to discriminate Lact. farciminis, Lact. alimentarius, Lact. sakei, Lact. curvatus and Lact. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: In this paper, we have established the first species-specific primer for PCR identification of Lact. farciminis and Lact. alimentarius. Both species-specific primer and RFLP, could be used as tools for rapid identification of lactobacilli up to species level.     

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.     

In vitro ruminal fermentation characteristics and utilisable CP supply of sainfoin and birdsfoot trefoil silages and their mixtures with other legumes

The extensive protein degradation occurring during ensiling decreases the nutritive value of silages, but this might be counteracted by tannins. Therefore, silages from two legume species containing condensed tannins (CT) – sainfoin (SF) and birdsfoot trefoil (two cultivars: birdsfoot trefoil, cv. Bull (BTB) and birdsfoot trefoil, cv. Polom) – were compared for their in vitro ruminal fermentation characteristics. The effect of combining them with two CT-free legume silages (lucerne (LU) and red clover (RC)) was also determined. The supply of duodenally utilisable CP (uCP) in the forages was emphasised. The legumes were each harvested from three field sites. After 24 h of wilting on the field, the legumes were ensiled in laboratory silos for 86 days. Proximate constituents, silage fermentation characteristics, CT content and CP fractions were determined. Subsequently, silage samples and 1 : 1 mixtures of the CT-containing and CT-free silages were incubated for 24 h in batch cultures using ruminal fluid and buffer (1 : 2, v/v). Each treatment was replicated six times in six runs. The effects on pH, ammonia and volatile fatty acid concentrations, protozoal counts, and total gas and methane production were determined. uCP content was calculated by considering the CP in the silage and the ammonia in the incubation fluid from treatments and blanks. Statistical evaluation compared data from single plants alone and together with that from the mixtures. Among treatments, SF silage contained the least CP and the most CT. The non-protein nitrogen content was lower, favouring neutral detergent soluble and insoluble protein fractions, in the SF and RC silages. Absolute uCP content was lowest in SF and SF mixtures, although the ratio to total CP was the highest. In comparison with LU, the ammonia concentration of the incubation fluid was lower for SF, RC and BTB and for the mixture of SF with LU. The total gas and methane production was similar among the treatments, and the total volatile fatty acid production was decreased with the CT-containing legumes. Protozoal count was increased with the mixtures containing LU and either SF or BTB compared with single LU. In conclusion, compared with the other legumes, SF and RC have similar advantages as they show limited proteolysis during ensiling. In addition, SF supplies more uCP relative to total CP. The CT-containing legumes also differed in their effect on ruminal fermentation and ammonia formation, probably because of their different CT contents. Thus, SF and its mixtures appear promising for improving the protein utilisation of ruminants.     

乳杆菌DM9811代谢产物对种植体周围炎龈下菌群的影响

目的从维护种植体周围菌群平衡和种植体远期固持率的角度出发,观察乳杆菌DM9811代谢产物对种植体周围炎龈下菌群的影响,探索一种适用人群广泛、较少或无副作用、疗效好、复发率低的新型生态治疗药物。方法将16例符合种植体周围炎的病例随意分为试验组（乳杆菌代谢产物含漱组）和对照组（纯净水含漱组）,分别于含漱前、含漱后3d、7d、30d进行微生物学检查。结果治疗后试验组牙龈卟啉单胞菌（P．g）、中间普氏菌（P．i）显著低于对照组（P〈0．05）;口腔链球菌、变形链球菌显著高于对照组（P〈0．05）：乳杆菌差异无显著性。结论乳杆菌DM9811代谢产物有抑制P．g、P．i,扶植口腔链球菌,从而调节种植体周菌群平衡的作用。     

一株植物乳酸杆菌的高密度发酵

目的研究一株植物乳酸杆菌的高密度发酵。方法通过单因素实验来确定此植物乳酸杆菌的最适生长温度、pH、碳源、氮源、缓冲盐等。结果最适生长温度是37℃、起始pH6.4、最适碳源为葡萄糖、最适氮源为牛肉膏,通过对缓冲盐的选择最终确定NaAc：CaCO3比为0.5：1.5;控制发酵条件,结合优化培养基,可使发酵后的培养液达到2.75×10^10CFU/ml。结论该缓冲盐能够有效的缓解发酵液pH的下降,延长对数生长期,使活菌数提高1倍,适合高密度发酵培养。     

Increasing sodium bicarbonate level in high-concentrate diets for heifers. I. Effects on intake, water consumption and ruminal fermentation

Four ruminally fistulated Holstein heifers (BW = 264 ± 12 kg) were used in a 4 × 4 Latin square design experiment to determine the effect of increasing levels of sodium bicarbonate (BICARB; 0%, 1.25%, 2.50% and 5%, on concentrate dry matter (DM) basis) on DM intake (DMI), water consumption and ruminal fermentation. Sampling was carried out in the last week of each four 21-day experimental periods. Heifers were offered concentrate (13.4 ± 0.04% crude protein (CP), 13.3 ± 0.44% NDF, 51.7 ± 0.97% starch) and barley straw once daily at 0830 h ad libitum. There was a linear decrease in concentrate DMI and a linear increase in straw DMI with increasing buffer level in the diet, resulting in a tendency towards a linear decrease in total DMI. Intake of concentrate was 6.89, 7.66, 6.72 and 5.72 ± 0.83 kg/day, whereas straw intakes were 0.73, 0.84, 0.94 and 1.06 ± 0.14 kg/day, for the 0%, 1.25%, 2.5% and 5% BICARB, respectively. Water consumption was not affected by treatments when expressed as l/day or percentage of BW, but increased linearly when expressed as l/kg of DMI. The percentage of total daily water drunk in the morning (from 0830 to 1230 h) increased linearly with the level of buffer. Mean ruminal pH and total area under the pH curve were not affected with increasing buffer level. The lowest daily pH (5.65 ± 0.09) was not affected by treatments. A quadratic tendency (P 0.10) was observed in the number of hours and the area under the pH curve in which ruminal pH was below 5.8, with high values only at the 0% BICARB. Additionally, increasing bicarbonate level caused a linear increase in the ruminal pH at 2 and 4 h after feeding. Daily average NH3 N (2.4 ± 0.9 mg N/100 ml) and total volatile fatty acids (VFA) (143 ± 12 mM) concentrations were not affected by treatments. Daily average molar proportion of propionate decreased linearly, and acetate proportion and the acetate-to-propionate ratio were increased with increasing buffer level in the diet. Molar percentage of butyrate, isobutyrate and isovalerate, and branched-chain VFA concentration increased linearly as the level of bicarbonate increased in the diet. Results indicate that high levels of BICARB to finishing heifers fed high-concentrate diets may result in a decreased DMI without significant effects on mean ruminal pH, which may affect animal performance. All individual VFA proportions, except valerate, were changed by the addition of bicarbonate.     

Effects of soybean oil and dietary copper levels on nutrient digestion,ruminal fermentation,enzyme activity,microflora and microbial protein synthesis in dairy bulls

ABSTRACT

The study evaluated the effects of soybean oil (SO) and dietary copper levels on nutrient digestion, ruminal fermentation, enzyme activity, microflora and microbial protein synthesis in dairy bulls. Eight Holstein rumen-cannulated bulls (14 ± 0.2 months of age and 326 ± 8.9 kg of body weight) were allocated into a replicated 4 × 4 Latin square design in a 2 × 2 factorial arrangement with factors being 0 or 40 g/kg dietary dry matter (DM) of SO and 0 or 7.68 mg/kg DM of Cu from copper sulphate (CS). The basal diet contained per kg DM 500 g of corn silage, 500 g of concentrate, 28 g of ether extract (EE) and 7.5 mg of Cu. The SO × CS interaction was significant (p < 0.05) for ruminal propionate proportion and acetate to propionate ratio. Dietary SO addition increased (p < 0.05) intake and total tract digestibility of EE but did not affect average daily gain (ADG) of bulls. Dietary CS addition did not affect nutrient intake but increased (p < 0.05) ADG and total tract digestibility of DM, organic matter, crude protein and neutral detergent fibre. Ruminal pH was not affected by treatments. Dietary SO addition did not affect ruminal total volatile fatty acids (VFA) concentration, decreased (p < 0.05) acetate proportion and ammonia N and increased (p < 0.05) propionate proportion. Dietary CS addition did not affect ammonia N, increased (p < 0.05) total VFA concentration and acetate proportion and decreased (p < 0.05) propionate proportion. Acetate to propionate ratio decreased (p < 0.05) with SO addition and increased (p < 0.05) with CS addition. Dietary SO addition decreased (p < 0.05) activity of carboxymethyl cellulase, cellobiase and xylanase as well as population of fungi, protozoa, methanogens, Ruminococcus albus and R. flavefaciens but increased (p < 0.05) α-amylase activity and population of Prevotella ruminicola and Ruminobacter amylophilus. Dietary CS addition increased (p < 0.05) activity of cellulolytic enzyme and protease as well as population of total bacteria, fungi, protozoa, methanogens, primary cellulolytic and proteolytic bacteria. Microbial protein synthesis was unchanged with SO addition but increased (p < 0.05) with CS addition. The results indicated that the addition of CS promoted nutrient digestion and ruminal fermentation by stimulating microbial growth and enzyme activity but did not relieve the negative effects of SO addition on ruminal fermentation in dairy bulls.     

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.     

高糖和氮源对鼠李糖乳杆菌(Lactobacillus rhamnosus)L-乳酸发酵的影响

鼠李糖乳杆菌经实验室耐高糖高酸选育,能够在高糖浓度下高效高产L-乳酸。以酵母粉为氮源和生长因子,葡萄糖初始浓度分别为120 g/L和146 g/L,摇瓶培养120h,L-乳酸产量分别为104g/L和117.5g/L,L-乳酸得率分别为86.7%和80.5%。高葡萄糖浓度对菌的生长和乳酸发酵有一定的抑制。增加接种量,在高糖浓度发酵条件下,可以缩短发酵时间,但对增加乳酸产量效果不明显。乳酸浓度对鼠李糖乳杆菌生长和产酸有显著的影响。初始乳酸浓度到达70g/L以上时,鼠李糖乳杆菌基本不生长和产酸,葡萄糖消耗也被抑制。酵母粉是鼠李糖乳杆菌的优良氮源,使用其它被测试的氮源菌体生长和产酸都有一定程度的下降。用廉价的黄豆粉并补充微量维生素液,替代培养基中的酵母粉,可以使产酸浓度和碳源得率得以基本维持。     

The effect of Lactobacillus buchneri and L. plantarum, applied at ensiling, on the ensiling fermentation and aerobic stability of wheat and sorghum silages

The effect of applying Lactobacillus buchneri (LB), alone or in combinations with L. plantarum (LP) and yeasts at ensiling, on the ensiling fermentation and aerobic stability of wheat and sorghum silages was studied under laboratory conditions. Treatments comprised LB, LP, yeasts, LB + yeasts, LP + yeasts, LB + LP and B-589 (a lactic acid bacterial strain isolated from wheat silage in Israel) alone. The treatments were also applied to sterilized aqueous extracts of wheat which were incubated at 30°C for 10 days. The pH of all treatments was below 4.0 already on day 4 of the experiment. Silages treated with LB had higher acetic acid concentrations than those treated with LP: 32–34 vs 16–18, and 28–34 vs 4–7 g kg⁻¹ in the experiments with wheat and sorghum, respectively. Similar results were obtained in wheat extracts. In the aqueous phase, marked differences in pH decrease were noticed among the treatments: 4.4 in LB, 6.0 in the yeast, and 3.7 in LP and B-589 (from day 3 and onwards). In both crops LB resulted in aerobically stable silages when applied alone or with LP and yeasts, whereas LP resulted in unstable silages upon aerobic exposure; the stability of the LB-treated silages is attributed to the higher acetic acid concentrations. The isolated strain (B-589) did not exhibit any advantage with regard to aerobic stability. Received 26 April 1999/ Accepted in revised form 05 July 1999     

乳杆菌Lactobacillus sp.lxp发酵高产L-乳酸研究

筛选得到一株乳杆菌Laetobaeillus sp．,进行发酵生产高浓度L-乳酸的研究。考察了种龄、接种量、温度和不同pH调节剂对乳酸发酵的影响。结果表明：最佳种子培养时间为15h;最佳接种量为15％;最适培养温度为42℃;与氨水和氢氧化钠相比,碳酸钙更适于作为发酵过程的pH调节刺;以葡萄糖为碳源,添加豆粕水解液和玉米浆作为辅料,2L罐培养120h,L-乳酸质量浓度可达202 g/L,糖转化率91．3％,L-乳酸占发酵液中总酸含量98％以上。     

Effects of exogenous fibrolytic enzymes on ruminal fermentation and digestibility of total mixed rations fed to lambs

This experiment was designed to study effects of exogenous fibrolytic enzymes on digestibility and ruminal fermentation of total mixed rations (TMR) with different forage:concentrate (F:C) ratios. The experimental design was a 6×6 Latin square with six ruminally cannulated Ramboulliet lambs (58 ± 7.4 kg body weight) used in a 3×2 factorial arrangement of treatments using three forage:concentrate (F:C) ratios (400:600, 500:500, 600:400 kg/kg) and two (0 or 2 g) levels of fibrolytic enzymes/kg TMR dry matter (DM). Exogenous fibrolytic enzymes increased the soluble fraction of DM (P<0.01) and ruminal in situ disappearance rate of DM and neutral detergent fibre (aNDF; P<0.001). However, there were no differences among treatments in DM and aNDF in vivo digestibility due to enzyme addition to the diet. Feed intake and N balance were also not affected by enzymes, and ruminal fermentation patterns were unchanged. Exogenous fibrolytic enzymes improved ruminal disappearance rates of DM and aNDF, but had no impact on lamb performance or ruminal fermentation.