Bacterial direct-fed microbials fail to reduce methane emissions in primiparous lactating dairy cows

Direct-fed microbials(DFM) are considered as a promising technique to improve animal productivity without affecting animal health or harming the environment.The potential of three bacterial DFM to reduce methane(CH4)emissions,modulate ruminal fermentation,milk production and composition of primiparous dairy cows was examined in this study.As previous reports have shown that DFM respond differently to different diets,two contrasting diets were used in this study.Eight lactating primiparous cows were randomly divided into two groups that were fed a corn silage-based,high-starch diet(HSD) or a grass silage-based,high-fiber diet(HFD).Cows in each dietary group were randomly assigned to four treatments in a 4 × 4 Latin square design.The bacterial DFM used were selected for their proven CH4-reducing effect in vitro.Treatments included control(without DFM) and 3 DFM treatments: Propionibacterium freudenreichii 53-W(2.9 × 10^10 colony forming units(CFU)/cow per day),Lactobacillus pentosus D31(3.6 × 10^11 CFU/cow per day) and Lactobacillus bulgaricus D1(4.6 × 10^10 CFU/cow per day).Each experimental period included 4 weeks of treatment and 1 week of wash-out,with measures performed in the fourth week of the treatment period.Enteric CH4 emissions were measured during 3 consecutive days using respiration chambers.Rumen samples were collected for ruminal fermentation parameters and quantitative microbial analyses.Milk samples were collected for composition analysis.Body weight of cows were recorded at the end of each treatment period.Irrespective of diet,no mitigating effect of DFM was observed on CH4 emissions in dairy cows.In contrast,Propionibacterium increased CH4 intensity by 27%(g CH4/kg milk) in cows fed HSD.There was no effect of DFM on other fermentation parameters and on bacterial,archaeal and protozoal numbers.Similarly,the effect of DFM on milk fatty acid composition was negligible.Propionibacterium and L.pentosus DFM tended to increase body weight gain with HSD.We conclude that,contrary to the effect previously observed in vitro,bacterial DFM Propionibacterium freudenreichii 53-W,Lactobacillus pentosus D31 and Lactobacillus bulgaricus D1 did not alter ruminal fermentation and failed to reduce CH4 emissions in lactating primiparous cows fed high-starch or high-fiber diets.

Implication and challenges of direct-fed microbial supplementation to improve ruminant production and health

Direct-fed microbials(DFMs)are feed additives containing live naturally existing microbes that can benefit animals’health and production performance.Due to the banned or strictly limited prophylactic and growth promoting usage of antibiotics,DFMs have been considered as one of antimicrobial alternatives in livestock industry.Microorganisms used as DFMs for ruminants usually consist of bacteria including lactic acid producing bacteria,lactic acid utilizing bacteria and other bacterial groups,and fungi containing Saccharomyces and Aspergillus.To date,the available DFMs for ruminants have been largely based on their effects on improving the feed efficiency and ruminant productivity through enhancing the rumen function such as stabilizing ruminal pH,promoting ruminal fermentation and feed digestion.Recent research has shown emerging evidence that the DFMs may improve performance and health in young ruminants,however,these positive outcomes were not consistent among studies and the modes of action have not been clearly defined.This review summarizes the DFM studies conducted in ruminants in the last decade,aiming to provide the new knowledge on DFM supplementation strategies for various ruminant production stages,and to identify what are the potential barriers and challenges for current ruminant industry to adopt the DFMs.Overall literature research indicates that DFMs have the potential to mitigate ruminal acidosis,improve immune response and gut health,increase productivity(growth and milk production),and reduce methane emissions or fecal shedding of pathogens.More research is needed to explore the mode of action of specific DFMs in the gut of ruminants,and the optimal supplementation strategies to promote the development and efficiency of DFM products for ruminants.

Isolation, screening and identifi cation of Bacillus spp. as direct-fed microbial candidates for aflatoxin B_1 biodegradation

Objective: To evaluate the ability of Bacillus spp. as direct-fed microbials(DFM) to biodegrade al atoxin B1(AFB1) by using an in vitro digestive model simulating in vivo conditions.Methods: Sixty-nine Bacillus isolates were obtained from intestines, and soil samples were screened by using a selective media method against 0.25 and 1.00 μg/m L of AFB1 in modii ed Czapek-Dox medium. Plates were incubated at 37 °C and observed every two days for two weeks. Physiological properties of the three Bacillus spp. candidates were characterized biochemically and by 16 S r RNA sequence analyzes for identii cation. Tolerance to acidic p H, osmotic concentrations of Na Cl, bile salts were tested, and antimicrobial sensitivity proi les were also determined. Bacillus candidates were individually sporulated by using a solid fermentation method and combined. Spores were incorporated into 1 of 3 experimental feed groups: 1) Negative control group, with unmedicated starter broiler feed without AFB1; 2) Positive control group, with negative control feed contaminated with 0.01% AFB1; 3) DFM treated group, with positive control feed supplemented with 109 spores/g. After digestion time(3:15 h), supernatants and digesta were collected for high-performance liquid chromatography l uorescence detection analysis by triplicate.Results: Three out of those sixty-nine DFM candidates showed ability to biodegrade AFB1 in vitro based on growth as well as reduction of l uorescence and area of clearance around each colony in modii ed Czapek-Dox medium which was clearly visible under day light after 48 h of evaluation. Analysis of 16S-DNA identii ed the strains as Bacillus amyloliquefaciens, Bacillus megaterium and Bacillus subtilis. The three Bacillus strains were tolerant to acidic conditions(p H 2.0), tolerant to a high osmotic pressure(Na Cl at 6.5%), and were able to tolerate 0.037% bile salts after 24 h of incubation. No signii cant dif erences(P > 0.05) were observed in the concentrations of AFB1 in neither the supernatants nor digesta samples evaluated by highperformance liquid chromatography with l uorescence detection between positive control or DFM treated groups. Conclusions: In vitro digestion time was not enough to confirm biodegradation of AFB1. Further studies to evaluate the possible biodegradation ef ects of the BacillusDFM when continuously administered in experimentally contaminated feed with AFB1, are in progress.

Prospective use of bacteriocinogenic Pediococcus pentosaceus as direct-fed microbial having methane reducing potential

Direct-fed microbials（DFM）, generally regarded as safe status, are successfully used in improving rumen ecology, gastro-intestinal health, feed efficiency, milk production and growth rate in ruminants. On the other hand, methanogenesis in rumen, which accounts for a significant loss of ruminant energy and increased greenhouse gas in environment, is of great concern, therefore, use of DFM for improving productivity without compromising the animal health and ecological sustainability is encouraged. The present study was conducted to investigate the methane reducing potential of bacteriocinogenic strain Pediococcus pentosaceus-34. Since, the culture showed no hemolysis on blood agar and DNase activity, hence, it was considered to be avirulent in nature, a prerequisite for any DFM. The culture also showed tolerance to pH 5.0 for 24 h with 0.5% organic acid mixture, whereas when given a shock for 2 h at different p H and organic acids concentrations, it showed growth at pH 3.0 and 4.0 with 0.1 and 1.0% organic acids, respectively, as having good animal probiotics attributes. The total gas production was significantly（P〈0.05） higher in live pedicoccal culture（LPC） and dead pedicoccal culture（DPC） both with wheat straw, when compared to the control. In sugarcane bagasse, gas production was significantly lower（P〈0.05） with LPC compared to the control and DPC both. Methane was reduced by the inclusion of LPC in sugarcane bagasse（0.07 mL CH4 mg–1 dry matter digestibility） with no effect on other rumen fermentation parameters. However, with wheat straw and LPC total gas, in vitro dry matter digestibility, total volatile fatty acids increased significantly but no reduction in methane production was observed in comparison to the control. Therefore, further research is warranted in this direction, if the bacteriocinogenic strains can be used as DFM for ruminants to improve the ruminant productivity.

Effect of Direct-Fed Microbial Supplementation on Pathogenic <i>Escherichia coli</i>Fecal Shedding, Live Performance, and Carcass Characteristics in Feedlot Steers

Three experiments were conducted to evaluate direct-fed microbial (DFM) supplementation on live performance, carcass characteristics, and fecal shedding of E. coli in feedlot steers. In Exp. 1, 400 steers (BW = 348 kg) were assigned to treatments: CON = lactose carrier only, BOV = P. freudenreichii (NP24) + L. acidophilus (NP51), BOVD = P. freudenreichii (NP24) + L. acidophilus (NP51), and COMB = BOV fed for the first 101 d on feed, followed by BOVD for the final 28 d prior to harvest. In Exp. 2 (n = 1800;BW = 354 kg) and Exp. 3 (n = 112;BW = 397 kg), steers were utilized in a randomized complete block design and assigned to DFM treatments using low dose and high dose, respectively. Fecal samples were collected prior to harvest and analyzed for E. coli serogroups. In Exp. 1, DFM reduced (P < 0.01) the concentration of E. coli O157. Prevalence of O157 was reduced by BOVD supplementation in Exp. 2 and 3 (P < 0.01 and P = 0.08, respectively), and concentration of E. coli O157 in positive samples was reduced in both experiments where enumeration was performed (P ≤ 0.02). Weighted mean differences across the three experiments were equal to a 33% reduction in the prevalence of E. coli O157:H7 in BOVD treated cattle. A significant reduction in prevalence of O26, O45, O103, and O121 was observed in Exp. 2 (P ≤ 0.03). These results indicate that high levels of L. acidophilus (NP51) may represent an effective pre-harvest food safety intervention to reduce fecal shedding of several E. coli serogroups.

Direct-fed microbes: A tool for improving the utilization of low quality roughages in ruminants

For many years, ruminant nutritionists and microbiologists have been interested in manipulating the microbial ecosystem of the rumen to improve production efficiency of different ruminant species. Removal and restriction of antibiotics subtherapeutic uses from ruminant diets has amplified interest in improving nutrient utilization and animal performance and search for more safe alternatives. Some bacterial and fungal microorganisms as a direct-fed microbial（DFM） can be the most suitable solutions. Microorganisms that are commonly used in DFM for ruminants may be classified mainly as lactic acid producing bacteria（LAB）, lactic acid utilizing bacteria（LUB）, or other microorganism＇s species like Lactobacillus, Bifidobacterium, Enterococcus, Streptococcus, Bacillus, Propionibacterium, Megasphaera elsdenii and Prevotellabryantii, in addition to some fungal species of yeast such as Saccharomyces and Aspergillus. A definitive mode of action for bacterial or fungal DFM has not been established; although a variety of mechanisms have been suggested. Bacterial DFM potentially moderate rumen conditions, and improve weight gain and feed efficiency. Fungal DFM may reduce harmful oxygen from the rumen, prevent excess lactate production, increase feed digestibility, and alter rumen fermentation patterns. DFM may also compete with and inhibit the growth of pathogens, immune system modulation, and modulate microbial balance in the gastrointestinal tract. Improved dry matter intake, milk yield, fat corrected milk yield and milk fat content were obtained with DFM administration. However, the response to DFM is not constant; depending on dosages, feeding times and frequencies, and strains of DFM. Nonetheless, recent studies have supported the positive effects of DFM on ruminant performance.

Effect of Yeast Culture and Direct-Fed Microbes on the Growth Performance and Rumen Fermentation of Weaner Lambs

The effects of yeast culture and directfed microbes on the growth performance of weaner lambs was examined. Thirty-two Hu lambs with inihtial weight of 22.20 （ ±0.75 ） kg were randomly assigned to one of four treatments： basal diet without additive （control）, added with yeast culture at 15 g/head/d （YEC）, YEC plus Bacillus licheniformis preparation at 2. 3 g/head/d （YBL） or plus Clostridium butyricum preparation at 2. 3 g/head/d （YCB）. The feeding trial lasted 75 d with 15 d for adaptation. Feed intake was not influenced （P 〉0. 05） by treatment. Average daily gain of growing lambs was 102, 114, 90, and 89 g/d in control, YEC, YBL, and YCB, respectively, with no significant difference （P 〉 0.05） among treatments, but the carcass weight of YEC lambs was significantly higher （P 〈 0.05） than that of other treatments. Total volatile fatty acids and acetate to propionate ratio in the rumen were unaffected, although the butyric acid concentration was higher （ P 〈 0.05 ） in the ru men fluid of YCB lambs compared with YEC lambs and slightly higher （ P 〉 0. 05 ） than in controls and YBL lambs. Solid-associated fungi population relative to total rumen bacteria 16S ribosomal DNA was significantly lower （ P 〈 0. 05 ） in YBL lambs （3.55） compared with those on YCB （23.12）. There was little difference in blood glucose and plasma urea-N concentrations among the treatments. Blood concentrations of creatinine and globulin were significantly higher （P 〈0.05） in YBL lambs, compared with the control and YEC-fed animals, and no difference with YCB lambs. Total protein and triglycerides in blood were significantly （P 〈 0.05） higher in YBL lambs, compared with controls. These serum biochemical parameters suggest that treatment increased amounts of absorbable protein but not efficiency of protein utilization and in YBL and YCB lambs. The results indicated that yeast culture improve growth performance, while little advantage could be expected from combining yeast culture with either the B. licheniformis preparation or C. butyricum preparation. More research using lower doses of B. licheniformis prepara- tion or C. butyricum preparation in combination with yeast culture is warranted.

DFM菌株生理特性的测试以及摇床发酵条件的优化

以研究适合工业化生产的直接饲用微生物 (DirectFeedMicrobials,DFM )为目的 ,通过对 5株细菌的生理特性的测定结果 ,显示出这几株菌具有良好的性状 ,有助于改善目前饲料添加剂领域存在的问题。实验还对菌种的摇床发酵条件进行了正交优化 ,证明了 3株菌可以利用廉价的培养基实现高密度发酵[1] 。另外菌株利用玉米秸秆和麦秸秆的实验为进一步降低发酵的成本开辟了方向。

Role of live microbial feed supplements with reference to anaerobic fungi in ruminant productivity: A review

To keep the concept of a safe food supply to the consumers, animal feed industries world over are showing an increasing interest in the direct-fed microbials（DFM） for improved animal performance in terms of growth or productivity. This becomes all the more essential in a situation, where a number of the residues of antibiotics and/or other growth stimulants reach in milk and meat with a number of associated potential risks for the consumers. Hence, in the absence of growth stimulants, a positive manipulation of the rumen microbial ecosystem to enhance the feedstuff utilization for improved production efficiency by ruminants has become of much interest to the researchers and entrepreneurs. A few genera of live microbes（i.e., bacteria, fungi and yeasts in different types of formulations from paste to powder） are infrequently used as DFM for the domestic ruminants. These DFM products are live microbial feed supplements containing naturally occurring microbes in the rumen. Among different DFM possibilities, anaerobic rumen fungi（ARF） based additives have been found to improve ruminant productivity consistently during feeding trials. Administration of ARF during the few trials conducted, led to the increased weight gain, milk production, and total tract digestibility of feed components in ruminants. Anaerobic fungi in the rumen display very strong cell-wall degrading cellulolytic and xylanolytic activities through rhizoid development, resulting in the physical disruption of feed structure paving the way for bacterial action. Significant improvements in the fiber digestibility were found to coincide with increases in ARF in the rumen indicating their role. Most of the researches based on DFM have indicated a positive response in nutrient digestion and methane reducing potential during in vivo and/or in vitro supplementation of ARF as DFM. Therefore, DFM especially ARF will gain popularity but it is necessary that all the strains are thoroughly studied for their beneficial properties to have a confirmed ‘generally regarded as safe＇ status for ruminants.

瘤胃源粪臭素降解菌的分离鉴定及其降解特性研究

【目的】旨在从绵羊瘤胃中分离粪臭素降解菌,并评估其粪臭素降解能力和生长性能,以期开发适用于反刍动物降臭的直接饲喂微生物。【方法】以绵羊瘤胃液为分离来源,使用含有粪臭素的MSM培养基进行富集和分离,通过细菌菌落形态观察进行初步分类;应用16S rRNA基因扩增、测序及系统发育分析进行物种鉴定;绘制菌株生长曲线,通过HPLC技术测定粪臭素降解曲线。【结果】从绵羊瘤胃液中分离出25株粪臭素降解菌,经菌落形态鉴定选出11株代表菌株进行后续研究。物种鉴定结果显示,MSML2和MSML6属于枯草芽孢杆菌(Bacillus subtilis),MSML4、MSML5、MSML7和MSML10属于阿氏普里斯特氏菌(Priestia aryabhattai),MSML3和MSML11属于污染伯克霍尔德氏菌(Burkholderia contaminans),MSML1、MSML8和MSML9属于成都假单胞菌(Pseudomonas chengduensis)。其中,MSML5生长速度最快,在约12 h后进入稳定期,稳定期菌体浓度最高;MSML3和MSML8在前16 h生长缓慢,32 h后进入稳定期。在粪臭素降解方面,MSML2粪臭素降解效率最高,48 h内的降解率达到23.03%,其次是MSML7、MSML8和MSML10,降解率均超过20%。【结论】成功从绵羊瘤胃中分离获得25株粪臭素降解菌,涉及4个物种,首次报道了具备粪臭素降解能力的枯草芽孢杆菌、阿氏普里斯特氏菌和成都假单胞菌,为直接饲喂反刍动物的菌剂开发提供了宝贵的菌株资源。

日粮添加可直接饲喂的微生物对仔猪生产性能的影响

试验选用 35～ 40日龄的杜长大三元杂交仔猪 72头 ,随机分成 2组 ,分别设为对照组和试验组。每组 3个重复 ,每个重复 12头猪。对照组饲喂基础日粮 ,试验组在对照组的基础上添加 0 .1%可直接饲喂微生物。研究结果表明 ,对照组日增重 417.2 6g ,试验组日增重为 436 .6 9g;试验组提高日增重 11.13% ,两组间差异显著 (P <0 .0 5 )。对照组和试验组的饲料转化率分别为 1.82和 1.71;试验组比对照组提高 6 .0 4% ,差异不显著 (P >0 .0 5 )。经济效益分析表明 ,试验组每头仔猪可多收入 10 .0 3元。这说明添加可直接饲喂微生物可提高仔猪生产性能和养猪收益。

日粮添加可直接饲喂微生物对生长肥育猪生产性能的影响

试验选用 12 0头 80日龄 ,体重约 2 5kg的三元杂交 (杜×长×大 )商品代瘦肉型生长肥育猪 ,随机分为 2组 ,分别设为对照组和试验组 ,每组两个重复 ,每个重复 30头猪 ,对照组饲喂基础日粮 ,试验组饲料在对照组日粮基础上添加可直接饲喂微生物。经过 31d的饲养试验 ,结果表明 :①试验组猪只的日增重比对照组提高 6 .0 7% (P <0 .0 5 ) ,料肉比比对照组提高 3.2 8% (P >0 .0 5 ) ;②经济效益 ,试验组猪只提高了 8.11% ;③试验组猪只的腹泻率较对照组降低 5 .6 7%。

Non-antibiotic feed additives in diets for pigs:A review

A number of feed additives are marketed to assist in boosting the pigs' immune system, regulate gut microbiota, and reduce negative impacts of weaning and other environmental challenges.The most commonly used feed additives include acidifiers, zinc and copper, prebiotics, direct-fed microbials, yeast products, nucleotides, and plant extracts.Inclusion of pharmacological levels of zinc and copper, certain acidifiers, and several plant extracts have been reported to result in improved pig performance or improved immune function of pigs.It is also possible that use of prebiotics, direct-fed microbials, yeast,and nucleotides may have positive impacts on pig performance, but results have been less consistent and there is a need for more research in this area.