Improved in vitro procedure for maintaining stock cultures of three genera of rumen protozoa

To maintain stock cultures of rumen protozoa, studies were initiated to explore possible methods for keeping the protozoa viable without feeding every day. Cultures of Entodinium caudatum, Epidinium caudatum, Enoploplastron triloricatum, and Entodinium exiguum were used to study the effect of not feeding for 1 or 2 d. The study lasted 88 d, and although bacterial concentrations decreased when cultures were not fed for 2 d (over the weekend), they recovered quickly with subsequent daily feedings. The exception was Enoploplastron triloricatum, which showed a gradual decline over the entire study. Addition of streptomycin to the media had little effect on maintaining bacterial concentrations in all cultures except Entodinium caudatum, in which the overall mean concentration was greater (P < 0.01). No differences in pH or bacterial concentrations were found between cultures fed daily and those held without feed for 2 d, with or without streptomycin. For maintaining protozoal cultures (10-mL volumes) over a long period without feeding on weekends, the following schedule is proposed: transfer and feed 0.12 mL of 1.5% ground wheat-1% orchardgrass on Monday; feed 0.12 mL of 1.5% ground wheat-1% orchardgrass on Tuesday, Wednesday, and Thursday; transfer and feed 0.5 mL of 1.5% ground wheat-1% orchardgrass on Friday; do not feed on Saturday and Sunday.     

The community structure and microbial linkage of rumen protozoa and methanogens in response to the addition of tea seed saponins in the diet of beef cattle

Background: This study investigated changes in rumen protozoal and methanogenic communities, along with the correlations among microbial taxa and methane(CH_4) production of six Belmont Red Composite beef steers fed tea seed saponins(TSS). Animals were fed in three consecutive feeding periods, a high-grain basal diet for 14 d(BD period) then a period of progressive addition of TSS to the basal diet up to 30 g/d for 20 d(TSS period), followed by the basal diet for 13 d without TSS(BDP post-control period).Results: The study found that TSS supplementation decreased the amount of the protozoal genus Entodinium and increased Polyplastron and Eudiplodinium genera. During BDP period, the protozoa community of steers did not return to the protozoal profiles observed in BD period, with higher proportions of Metadinium and Eudiplodinium and lower Isotricha. The addition of TSS was found to change the structure of methanogen community at the subgenus level by decreasing the abundance of methanogens in the SGMT clade and increasing the abundance of methanogens in the RO clade. The correlation analysis indicated that the abundance of SGMT clade methanogens were positively correlated with Isotricha, and Isotricha genus and SGMT clade methanogens were positively correlated with CH_4 production. While RO clade were positively correlated with the proportion of Metadinium genus, which was negatively correlated with CH_4 emission.Conclusions: These results suggest that different genera of rumen protozoa ciliates appear to be selectively inhibited by TSS, and the change in methanogen community at the subgenus level may be due to the mutualistic relationships between methanogens and rumen ciliates.     

瘤胃纤毛虫对甲烷菌和甲烷产量的影响

反刍动物体内甲烷的产生是瘤胃内发酵能量损失的主要原因 ,减少甲烷的产生对提高饲料能量利用率和改善环境有重要意义。为调节和控制瘤胃内甲烷产量 ,需要对甲烷产生的代谢机制进行探讨。研究表明 ,甲烷菌与瘤胃纤毛虫关系密切 ,本文从甲烷菌和纤毛虫两者的关系入手对瘤胃甲烷的产生进行探讨     

瘤胃甲烷生成过程中微生物之间的相互关系

反刍动物瘤胃含有甲烷菌,是主要的甲烷(methane,CH4)排放源,甲烷菌利用饲料发酵过程中生成的氢和CO2作为主要的电子受体和供体通过还原反应生成甲烷,在瘤胃功能性生态生境(niche)中发挥重要作用.除甲烷菌以外,其他瘤胃微生物也影响甲烷产量.瘤胃微生物总体来说可分为产氢微生物(hydrogenogens)和耗氢...     

Rumen ciliate faunae of water buffalo (Bubalus bubalis) and goat (Capra hircus) in Nepal

Rumen ciliate composition of river-type water buffalo and goat in Nepal was surveyed. As the result of survey, 13 genera representing 52 species and 20 formae of the ciliates were identified. Of them 13 genera with 44 species and 9 formae were found from the water buffalo and 8 genera with 21 species and 12 formae from the goat. The present paper shows the first report of Hsiungella triciliata, Entodinium brevispinum, E. convexum, E. javanicum, E. rectangulatum f. rectangulatum, E. rectangulatum f. lobosospinosum, Diplodinium nanum, D. psittaceum, D. sinhalicum and Ostracodinium quadrivesiculatum from water buffalo and Epidinium ecaudatum f. parvicaudatum from goat.     

Growth and morphologic response of rumen methanogenic archaea and bacteria to cashew nut shell liquid and its alkylphenol components

The growth and morphology of rumen methanogenic archaea (15 strains of 10 species in 5 genera, including 7 strains newly isolated in the present study) and bacteria (14 species in 12 genera) were investigated using unsupplemented in vitro pure cultures and cultures supplemented with cashew nut shell liquid (CNSL) and its phenolic compound components, anti-methanogenic agents for ruminant animals. Growth of most of the methanogens tested was inhibited by CNSL and alkylphenols at different concentrations ranging from 1.56 to 12.5 μg/ml. Of the alkylphenols tested, anacardic acid exhibited the most potent growth inhibition. Three gram-negative bacterial species involved in propionate production were resistant to CNSL and alkylphenols (>50 μg/ml). All the methanogens and bacteria that were sensitive to CNSL and alkylphenols exhibited altered morphology; disruption of the cell surface was notable, possibly due to surfactant activity of the tested materials. Cells division was inhibited in some organisms, with cell elongation and unclear septum formation observed. These results indicate that CNSL and alkylphenols, particularly anacardic acid, inhibit both rumen bacteria and methanogens in a selective manner, which could help mitigate rumen methane generation.     

Rumen ciliate protozoal fauna of reindeer in Inner Mongolia, China

Rumen ciliate species and composition were surveyed on the reindeer kept in Inner Mongolia, China. As a result of survey, all the reindeer had the same 18 species of 8 genera of the ciliates in their rumen. Appearance of Entodinium parvum is the first record in this host species. Of the species detected, 15 species including Entodinium anteronucleatum, E. bicornutum, Enoploplastron confluens and Epidinium gigas which have been detected only from reindeer were common with those in the reindeer kept in the other areas, such as Alaska, Russia and Finland, indicating that every rumen ciliate faunae in reindeer has high similarity to each other, and suggesting that these hosts have been isolated from the other ruminants for long time and established characteristic ciliate fauna. The ciliate density was estimated as 1.1-2.5 x 10 (6)/ml, which value was fairly higher than those in domestic ruminants. Surface structures of characteristic species were observed by SEM.     

瘤胃产甲烷菌与其他微生物间的氢传递及其调控研究进展

在反刍动物瘤胃中产甲烷菌生成甲烷既造成能量的浪费又产生大量温室气体,因此减少瘤胃甲烷生成与排放是提升生产效率与维持可持续发展的要求。瘤胃内的产甲烷菌通过共生、黏附和伴生模式,分别从原虫、细菌和真菌中摄取氢,保证氢营养型甲烷生成途径的顺利进行。抑杀原虫和产氢细菌、竞争性结合氢和阻断氢生成甲烷是基于氢调控抑制甲烷生成的途径。由于瘤胃微生物的冗余和互作,降甲烷的同时,瘤胃中饲料消化可能受到抑制,且单一的氢调控往往会诱发瘤胃的适应,瘤胃的降甲烷效果仅能短时间维持。为此,需从瘤胃微生物整体出发,通过多种氢调控机制的添加剂联用及间歇饲喂、幼龄反刍动物瘤胃早期调控、甲烷生成途径关键酶调控等的综合应用,实现更优的甲烷减排。     

Long-term defaunation increases the abundance of cellulolytic ruminococci and methanogens but does not affect the bacterial and methanogen diversity in the rumen of sheep

Protozoa are commensal eukaryotes in the rumen of herbivores. Protozoa are large producers of hydrogen, which is utilized by methanogenic archaea to produce methane, a greenhouse gas. The removal of protozoa from the rumen (defaunation) decreases methanogenesis, but also negatively affects fiber digestion, which is the main function of the rumen. The aim of this study was to examine the effect of long-term defaunation on the structure of the microbiota and particularly methanogenic archaea and fibrolytic bacteria to better understand the microbial mechanisms responsible for the decrease in methanogenesis and fibrolysis. The trial was conducted in 5 adult sheep subjected successively to long-term defaunation (2 yr), refaunation (12 wk), and short-term defaunation (10 wk). Methanogens were enumerated by quantitative PCR targeting the rrs (16S ribosomal RNA subunit) and mcrA (methyl coenzyme-M reductase) genes. The rrs gene was used to quantify the 3 major culturable rumen cellulolytic bacterial species (i.e., Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) and total bacteria. Bacterial and methanogen diversity was also examined by PCR-DGGE (PCR-denaturing gradient gel electrophoresis) analysis targeting the rrs and mcrA genes, respectively. Total rumen bacterial density estimated as rrs copies per gram of DM of rumen content increased in response to long- and short-term defaunation (+1 log, P < 0.001), but without noticeable shifts in diversity. Defaunation increased the rrs copies per gram of DM of rumen content of R. albus and R. flavefaciens (+2 log, P < 0 0.001), but did not affect that of F. succinogenes. Despite a 20% reduction in methane emission in the 2 defaunated periods, the mcrA and rrs copies of methanogens per gram of DM of rumen content increased (+1 log, P < 0.001) in the absence of protozoa, whereas the diversity of the dominant methanogenic community was not modified. This study shows no major difference between long- and short-term defaunation in abundance and diversity of bacteria and archaea. It also provides evidence that monitoring the abundance and diversity of methanogens is not sufficient to comprehend the microbial mechanisms leading to a reduction in methane emissions by ruminants. This study also reports for the first time in sheep a selective effect of defaunation on the abundance of cellulolytic bacterial species.     

High methanogenic potential of sucrose compared with starch at high ruminal pH

The role of ruminal pH with respect to the expression of a differentiation in the methanogenic potential of easily fermentable carbohydrates was determined using the rumen simulation technique. The target pH at 21 h after feed supply was set to 6.0 and 7.0 through a specific buffer management. A basal diet was supplemented with crystalline sucrose or steamflaked maize starch. With sucrose instead of starch, the decline in fermenter fluid pH, 4 h after introducing new feed, was more pronounced at both buffer levels, and degradability of all nutrients, including fibre was higher too. At low pH, molar propionate proportion was higher with sucrose. Methane quantity depended on the combination of pH and source of easily fermentable carbohydrate as sucrose, compared with starch, enhanced methanogenesis (+40%), but only at the high pH. The concomitant increase in organic matter degradability with sucrose at high target pH did not completely explain this phenomenon, as the effects on methane were still significant when related to apparently fermented organic matter. However, differences caused by pH and carbohydrate type decreased when methane was related to degraded fibre, suggesting that there was a mutual supportive effect of high pH and sucrose on fibre degradation.     

Quantitative analysis of ruminal methanogenic microbial populations in beef cattle divergent in phenotypic residual feed intake (RFI) offered contrasting diets

Background

Methane (CH₄) emissions in cattle are an undesirable end product of rumen methanogenic fermentative activity as they are associated not only with negative environmental impacts but also with reduced host feed efficiency. The aim of this study was to quantify total and specific rumen microbial methanogenic populations in beef cattle divergently selected for residual feed intake (RFI) while offered (i) a low energy high forage (HF) diet followed by (ii) a high energy low forage (LF) diet. Ruminal fluid was collected from 14 high (H) and 14 low (L) RFI animals across both dietary periods. Quantitative real time PCR (qRT-PCR) analysis was conducted to quantify the abundance of total and specific rumen methanogenic microbes. Spearman correlation analysis was used to investigate the association between the relative abundance of methanogens and animal performance, rumen fermentation variables and diet digestibility.

Results

Abundance of methanogens, did not differ between RFI phenotypes. However, relative abundance of total and specific methanogen species was affected (P < 0.05) by diet type, with greater abundance observed while animals were offered the LF compared to the HF diet.

Conclusions

These findings suggest that differences in abundance of specific rumen methanogen species may not contribute to variation in CH₄ emissions between efficient and inefficient animals, however dietary manipulation can influence the abundance of total and specific methanogen species.     

2008-2009年反刍动物营养研究进展 Ⅱ.外源添加剂对瘤胃发酵的调控

作者综述了2009年在ADSA-ASAS大会和CNKI、PubMed等数据库中外源添加剂(离子载体、酶制剂、植物提取物、饲用微生物)对瘤胃发酵调控的相关文献39篇。离子载体如莫能菌素主要通过促进瘤胃丙酸的产生,抑制生物氢化菌,促进长链脂肪酸在瘤胃内发生不完全氢化等瘤胃调控作用,从而缓解反刍动物的能量负平衡,减少甲烷排放,降低乳中脂肪含量。酶制剂(包括纤溶酶和淀粉酶等)在饲喂前加入日粮中能够免受瘤胃蛋白酶的降解,并保持酶活性,促进饲料的消化。植物提取物中皂角甙通过抑制原虫生长降低甲烷的排放,动物长期采食富含单宁的植物可产生单宁耐受菌,并且单宁的植物提取物可影响瘤胃的生物氢化作用,植物精油对瘤胃的调控作用包括减少淀粉和蛋白的降解,以及通过对某种瘤胃微生物的选择性作用抑制氨氮的降解。饲用微生物(主要有酵母菌、乳酸菌、芽孢杆菌等)能增加营养物质消化率,改变瘤胃发酵模式,增加瘤胃微生物菌群,但其益生效果与泌乳阶段、日粮类型、环境条件有关。     

Effects of tea saponins on rumen microbiota,rumen fermentation,methane production and growth performance—a review

Reducing methane emission from ruminant animals has implications not only for global environmental protection but also for efficient animal production. Tea saponins (TS) extracted from seeds, leaves or roots of tea plant are pentacyclic triterpenes. They have a lasting antiprotozoal effect, but little effect on the methanogen population in sheep. There was no significant correlation between the protozoa counts and methanogens. The TS decreased methanogen activity. It seems that TS influenced the activity of the methanogens indirectly via the depressed ciliate protozoal population. The TS addition decreased fungal population in the medium containing rumen liquor in in vitro fermentation, but no such effect was observed in the rumen liquor of sheep fed TS. Tea saponins had a minor effect on the pattern of rumen fermentation and hence on nutrient digestion. When added at 3 g/day in diets, TS could improve daily weight gain and feed efficiency in goats. No positive associative effect existed between TS and disodium fumarate or soybean oil on methane suppression. Inclusion of TS in diets may be an effective way for improving feed efficiency in ruminants.     

Rumen methanogens and mitigation of methane emission by anti-methanogenic compounds and substances

Methanogenic archaea reside primarily in the rumen and the lower segments of the intestines of ruminants, where they utilize the reducing equivalents derived from rumen fermentation to reduce carbon dioxide, formic acid, or methylamines to methane(CH_4). Research on methanogens in the rumen has attracted great interest in the last decade because CH_4 emission from ruminants contributes to global greenhouse gas emission and represents a loss of feed energy. Some DNA-based phylogenetic studies have depicted a diverse and dynamic community of methanogens in the rumen. In the past decade, researchers have focused on elucidating the underpinning that determines and affects the diversity, composition, structure, and dynamics of methanogen community of the rumen. Concurrently, many researchers have attempted to develop and evaluate interventions to mitigate enteric CH_4 emission. Although much work has been done using plant secondary metabolites, other approaches such as using nitrate and 3-nitrooxy propanol have also yielded promising results. Most of these antimethanogenic compounds or substances often show inconsistent results among studies and also lead to adverse effects on feed intake and digestion and other aspects of rumen fermentation when fed at doses high enough to achieve effective mitigation. This review provides a brief overview of the rumen methanogens and then an appraisal of most of the antimethanogenic compounds and substances that have been evaluated both in vitro and in vivo. Knowledge gaps and future research needs are also discussed with a focus on methanogens and methane mitigation.     

Analysis of methanogens in the bovine rumen by polymerase chain reaction single-strand conformation polymorphism

The polymerase chain reaction single‐strand conformation polymorphism (PCR‐SSCP) method reported by Schwieger and Tebbe (1998) was used to analyze the diversity of methanogens inhabiting the rumen. Partial 16S rRNA gene fragments were amplified from DNA extracted from rumen contents by PCR with archaea‐specific primers, Ar1000F and Ar1500R, or methanogen‐specific primers, M301F and M915R, with one primer phosphorylated at the 5′ end. The amplified DNA fragments were analyzed by SSCP gel electrophoresis after the phosphorylated strands of the PCR products were digested with λ exonuclease. When we analyzed samples collected from the six Holstein cows used in a previous study, in which cows were given feed with or without α‐cyclodextrin‐horseradish oil complex (CD‐HR), nine and six bands were identified in the profiles generated by PCR products amplified with archaea‐specific and methanogen‐specific primers, respectively. While dendrogram analysis based on SSCP gel profiles found that the methanogens from each rumen showed a particular composition of methanogens, the profiles of the methanogens isolated from two of three cows fed with CD‐HR fell into the same branch in the dendrogram constructed from the profiles. Therefore, this study demonstrates the potential of the PCR‐SSCP method in the methanogenic community analysis of the rumen and in investigating changes in the methanogenic community due to the addition of CD‐HR to the rumen.     

Digestibility, methane production and nitrogen balance in sheep fed ensiled or fresh mixtures of sorghum–soybean forage

The feeding value of a mixture of sorghum and soybeans plants, either fresh or ensiled, was evaluated with sheep. Sorghum and soybeans were harvested during the Cuban rainy season and ensiled in a ratio of 0.6/0.4 (w/w, as feed) with molasses and a bacterial inoculant. Silos were opened between 162 and 182 d post ensiling during the Cuban dry season and silages were fed to six pelibuey sheep (including two fistulated). Six other sheep (also including two fistulated) were fed sorghum and soybean in the same proportion, but freshly harvested during the dry season. The experiment lasted 21 d (14 d adaptation and 7 d data collection period). Silage quality parameters included pH, ammonia, lactate, and short chain fatty acids (SCFA). Further, both fresh and ensiled diets were offered to study the rumen fermentation characteristics (pH, ammonia, lactate, SCFA, in situ degradability and methane), duodenal flow of microbial protein (assessed through urinary purine derivatives secretion), fecal degradability of nutrients and urinary N excretion. From these measurements ME value and degradable CP supply at the small intestine (DCPSI) were estimated. Silage was of excellent quality. Compared to fresh forage feeding silage increased molar propionate proportion and rumen microbial protein synthesis and reduced methane emission. Fresh forage showed lower rumen degradability and total digestibility. Further, the higher ME concentration (11.2 vs. 10.3 MJ/kg DM) and DCPSI (84.7 vs. 56.1 g/kg DM) of the silage would allow a higher milk production or daily gain as compared with fresh forage available during the Cuban dry season.     

Sulphur availability and microbial fermentation in the fauna-free rumen.

The concentration of H2S and rate of feed digestion, VFA and methane production in the rumen were measured in sulphur deficient (-S) and S supplemented (+S) sheep while fauna-free (-P) and after refaunation (+P). The concentration of rumen H2S was increased by S supplementation but was not affected by the presence of protozoa. When fauna-free, -S sheep had reduced rates of feed digestion (30 v 48%/d), acetate synthesis (35 v 115 g/d) and methane synthesis (0.4 v 7.4 g C/d) compared to +S sheep and up to 36% (v/v) of total rumen gas was H2. When these sheep were given a single dose of 8.8 g Na2SO4, the proportion of methane increased and the proportion of H2 in rumen gas decreased. After refaunation of sheep, their rates of feed digestion, VFA production and methane production were higher and were not affected by the level of S. From this differential response to S deficiency by -P and +P sheep, it is concluded rumen protozoa increase the availability of S to other rumen microbes. This is probably because they degrade protein through to peptides and amino acids.     

大蒜素生理功能及在反刍动物上的应用

大蒜素是一种从大蒜中提取的含硫化合物，具有安全性高、无残留、不产生耐药性、抗菌谱广、无配伍禁忌等优点。大蒜素的生理功能包括降低血糖和血压、抗氧化、增强免疫力和抑制有害细菌等，并且在动物生产中发挥着维持动物肠道健康、改善肠道菌群、调节脂肪沉积等作用。作为饲料添加剂的大蒜素主要为化学合成类型。首先合成二烯丙基二硫醚，再通过氧化反应合成大蒜素，在实验室中大蒜素还可以通过过氧化氢、过氧邻苯二甲酸镁或氯代苯甲酸氧化二烯丙基二硫化物来合成。在实际生产中，大蒜素对反刍动物有促生长、提高饲料消化率、改善瘤胃发酵等诸多功能。大蒜素还可以通过抑制瘤胃内产甲烷菌活性和减少产甲烷菌数量来降低瘤胃甲烷产量。作者主要介绍了大蒜素的合成、理化特性及其生理功能，并阐述了在反刍动物中应用大蒜素的研究成果，以期为大蒜素在反刍动物生产中的应用提供参考。     

Physiological Function of Allicin and Its Application in Ruminants

Allicin is a sulfur compound extracted from garlic,which has the advantages of high safety,no residue,no drug resistance,wide antibacterial spectrum,no incompatibility and so on.The physiological functions of allicin include reducing blood sugar and blood pressure,anti-oxidation,enhancing immunity and inhibiting harmful bacteria,and plays a role in maintaining animal intestinal health,improving intestinal flora and regulating fat deposition in animal production.Allicin,as a feed additive,is mainly of chemical synthesis type.Diallyl disulfide is synthesized at first,and then allicin is synthesized by oxidation reaction.Allicin can also be synthesized by hydrogen peroxide,magnesium peroxy phthalate or chlorobenzoic acid oxidizing diallyl disulfide in the laboratory.In actual production,allicin can promote the growth of ruminants,improve feed digestibility,improve rumen fermentation.Allicin can also reduce rumen methane production by inhibiting the activity of methanogenic bacteria in the rumen and reducing the number of methanogenic bacteria.The authors mainly introduced the synthesis,physical and chemical properties and physiological functions of allicin,and expounded the research results and prospects of the application of allicin in ruminants,in order to provide reference for the better application of allicin in ruminant production.     

蛋白质来源对瘤胃细菌和原虫群体结构的影响

以4只瘘管山羊为试验动物,采用4×4拉丁方设计,使用克隆测序、遗传指纹以及细胞显微计数等技术,研究以羽毛粉(A)、玉米蛋白粉(B)、豆粕(C)和鱼粉(D)4种蛋白补充料分别配合的混合日粮对瘤胃细菌和原虫群体结构的影响。结果表明:细菌的R.flavefaciens、R.bromii、R-7、Roseburia faecalis、Uncultured T33H60F48、Uncultured GRANT42及1个尚不能定性的类群在组间存在显著差异(P<0.05);原虫的内毛虫、前毛虫、头毛虫、等毛虫4个类群在处理间差异显著(P<0.05)。综上可见,蛋白质源对瘤胃细菌和原虫类群结构有显著影响。