Ruminal distribution of the cellulolytic bacterium Fibrobacter succinogenes in relation to its phylogenetic grouping

To investigate the ecological importance of the cellulolytic bacterium Fibrobacter succinogenes in fiber digestion, ruminal distribution of F. succinogenes was determined in relation to its phylogenetic grouping. Rumen digesta from wethers and steers fed orchardgrass hay, rice straw or fresh orchardgrass were employed as the materials for the analyses. Orchardgrass hay stem incubated in the rumen was also used. By using total DNA extracted from these materials, population sizes of total F. succinogenes and of four different phylogenetic groups of this species were quantitated through competitive polymerase chain reaction (PCR), and restriction fragment length polymorphism analysis of PCR products targeted the bacterial 16S rDNA. Rumen digesta and ruminally incubated hay stems had a reasonably high population size of F. succinogenes (×10^7?8/g) that was composed of strains belonging to the phylogenetic groups 1 and 3. The relative abundance of each group was different among the samples; group 1 dominated on the ruminally incubated hay stem and in the rumen of wethers fed fresh orchardgrass, while group 3 was major in the rumen of wethers and steers on hay diet. These results suggest that there could be phenotypic differences among the phylogenetic groups of F. succinogenes, and group 1 dominating on hay stem might contribute to rumen fiber digestion more than the other groups.     

Determination of bacteria constituting ruminal fibrolytic consortia developed on orchard grass hay stem

To determine the relationship between Fibrobacter succinogenes and other rumen bacteria, the bacterial community structure on fiber was analyzed by using two different materials. These were ruminally incubated orchard grass hay stems without and with preincubation with F. succinogenes (natural and artificial consortia, respectively). The natural consortium mainly consisted of Firmicutes (56.6%) and Bacteroidetes (33.1%), while the artificial consortium showed a significantly higher proportion of Firmicutes (85.5%) and a lower proportion of Bacteroidetes (4.6%). At species or genus level, Butyrivibrio fibrisolvens, the U2 group, Ruminococcus albus and Lachnospiraceae incertae sedis made up a higher proportion in the artificial consortium. The most dominant bacterial group was the Butyrivibrio‐Pseudobutyrivibrio‐Lachnospiraceae incertae sedis group, which accounted for 19.7% in the natural and 29.5% in the artificial consortium. Within the genus Butyrivibrio, the phylogenetic groups SA and VA2 and phylogeny‐undefined Butyribivrio, but not VA1, were detected at high frequency in the artificial consortium. These results suggest that ecological and possibly functional relationships exist in the rumen among F. succinogenes, a subset of B. fibrisolvens, the U2 group, R. albus and Lachnospiraceae incertae sedis.     

Detection and identification of rumen bacteria constituting a fibrolytic consortium dominated by Fibrobacter succinogenes

A fibrolytic consortium, dominated by the rumen cellulolytic bacterium Fibrobacter succinogenes, was artificially constructed on hay stems to detect and identify rumen bacteria that can potentially interact with F. succinogenes . Consortium-bacterial members were determined by DGGE and sequencing analysis targeted bacterial 16S rDNA. An artificial consortium was formed in a 2-step incubation of hay stems; the first step with group 1, 2 or 3 F. succinogenes strains, the second step with rumen fluid. After consortium formation, morphologically different bacteria were observed in association with F. succinogenes . DGGE exhibited more than 30 bands, the pattern of which depended on the F. succinogenes group. Sequencing suggested that Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis , Clostridium sp., F. succinogenes group 2, Prevotella ruminicola and unclassified Bacteroides were prominent in the group 1 consortium and that Treponema bryantii , B. fibrisolvens , Acinetobacter sp, and Wolinella succinogenes were prominent in the group 2 consortium. However, in the group 3 consortium, F. succinogenes -like bacteria were microscopically undetectable, whereas cellulolytic Ruminococcus albus and F. succinogenes group 1 were prominent, suggesting that the group 3 cannot be a core member of this consortium. This study is the first attempt to identify bacterial members of a fibrolytic consortium dominated by a specific bacterium.     

Validation and application of real-time polymerase chain reaction assays for representative rumen bacteria

Real‐time polymerase chain reaction (PCR) assays for 11 representative rumen bacterial species were validated. The sensitivity was tested by using the serially diluted target 16S rDNA from respective bacterial species. The recovery of the target DNA and the assay reproducibility were determined using DNA from rumen fluid spiked with different quantities of the target. Minimum detection levels for the target were 10–100 copies in pure culture. The recovery of the added target ranged from 82.4 to 116.6%. The intra‐ and inter‐assay variations of each assay were <9.4 and <12.6%, respectively. Therefore, the real‐time PCR assays evaluated in the present study are considered to be sufficiently reliable for monitoring all 11 bacterial species in the rumen. The assays were then applied to the monitoring of the bacterial species attached to ruminally incubated rice straw. Among the monitored fibrolytic species, Fibrobacter succinogenes was found to be the most dominant, accounting for 2.61% of total bacteria after 24 h incubation. Selenomonas ruminantium and Streptococcus bovis, non‐fibrolytics, were detected on the rice straw at 8.96% and 1.16% of total bacteria, respectively. Such high levels of non‐fibrolytics on the plant fiber suggest a synergistic relationship between fibrolytics and non‐fibrolytics.     

Metagenomic profiles of the rumen microbiota during the transition period in low‐yield and high‐yield dairy cows

We investigated potential relationships between rumen microbiota and milk production in dairy cows during the transition period. Twelve dairy cows were divided into a low‐yield (LY) or high‐yield (HY) group based on their milk yield. Rumen samples were taken from dairy cows at 3 weeks before parturition, and at 4, 8, and 12 weeks after parturition. 16S rDNA‐based metagenomic analysis showed that diversities of rumen microbiota in both groups were similar and the number of operational taxonomic units (OTUs) was lower in the postpartum than prepartum period in both groups. The abundance of Bacteroidetes and ratio of Bacteroidetes:Firmicutes was higher in the HY than the LY group. OTUs assigned to Prevotella bryantii, Fibrobacter succinogenes, Ruminococcus albus, Butyrivibrio fibrisolvens, and Succinivibrio sp. were abundant in the HY group. These OTUs were significantly related to the propionate molar proportion of rumen fluids in the HY group. OTUs assigned to Lachnospiraceae, Bifidobacterium sp. and Saccharofermentans were dominant in the LY group. Predictive functional profiling revealed that abundance of gene families involved in amino acid and vitamin metabolism was higher in the HY than the LY group. These results suggest that the community structure and fermentation products of rumen microbiota could be associated with milk production of dairy cows.     

Ruminal fermentation and microbial ecology of buffaloes and cattle fed the same diet

Although buffaloes and cattle are ruminants, their digestive capabilities and rumen microbial compositions are considered to be different. The purpose of this study was to compare the rumen microbial ecology of crossbred water buffaloes and cattle that were fed the same diet. Cattle exhibited a higher fermentation rate than buffaloes. Methane production and methanogen density were lower in buffaloes. Phylogenetic analysis of Fibrobacter succinogenes‐specific 16S ribosomal RNA gene clone library showed that the diversity of groups within a species was significantly different (P < 0.05) between buffalo and cattle and most of the clones were affiliated with group 2 of the species. Population densities of F. succinogenes, Ruminococcus albus and R. flavefaciens were higher until 6 h post‐feeding in cattle; however, buffaloes exhibited different traits. The population of anaerobic fungi decreased at 3 h in cattle compared to buffaloes and was similar at 0 h and 6 h. The diversity profiles of bacteria and fungi were similar in the two species. The present study showed that the profiles of the fermentation process, microbial population and diversity were similar in crossbred water buffaloes and crossbred cattle.     

Effect of non‐starch‐polysaccharide‐degrading enzymes as feed additive on the rumen bacterial population in non‐lactating cows quantified by real‐time PCR

The effects of non‐starch‐polysaccharide‐degrading enzymes, added to a maize silage‐ and grass silage‐based total mixed ration (TMR) at least 14 h before feeding, on the rumen bacterial population were investigated. Six non‐lactating Holstein Friesian cows were allocated to three treatment groups using a duplicate 3 × 3 Latin square design with three 31‐day periods (29 days of adaptation and 2 days of sampling). Treatments were control TMR [69% forage and 31% concentrates on a dry matter (DM) basis] or TMR with 13.8 or 27.7 ml/kg of feed DM of Roxazyme G2 liquid with activities (U/ml enzyme preparation) of xylanase 260 000, β‐glucanase 180 000 and cellulase 8000 (DSM Nutritional Products, Basel, Switzerland). The concentrations of 16S rDNA of Anaerovibrio lipolytica, Fibrobacter succinogenes, Prevotella ruminicola, Ruminococcus flavefaciens, Selenomonas ruminantium and Treponema bryantii, and their relative percentage of total bacteria in rumen samples obtained before feeding and 3 and 7 h after feeding and from two rumen fractions were determined using real‐time PCR. Sampling time had only little influence, but bacterial numbers and the composition of the population differed between the transition layer between rumen fluid and the fibre mat (fraction A) and the rumen fluid (fraction B) highlighting the importance to standardize sampling. The 16S rDNA copies of total bacteria and the six bacterial species as well as the population composition were mainly unaffected by the high levels of exogenous enzymes supplemented at all sampling times and in both rumen fractions. Occasionally, the percentages of the non‐fibrolytic species P. ruminicola and A. lipolytica changed in response to enzyme supplementation. Some increases in the potential degradability of the diet and decreases in lag time which occurred collaterally indicate that other factors than changes in numbers of non‐particle‐associated bacteria are mainly responsible for the effects of exogenous enzymes.     

Preliminary study of the effects of condensed barley distillers soluble on rumen fermentation and plasma metabolites in Japanese Black cows

In Japan, condensed barley distillers soluble (CBDS) is a widely known liquor byproduct that contains a high level of protein and is used as a supplementary protein feed for cattle. The present study evaluated the effects of CBDS feed on rumen fermentation and plasma metabolites in Japanese Black cows. Applying a replicated 3 × 3 Latin square design, nine cows were offered CBDS and hay (CBDS‐t), soy bean meal and hay (Soybean‐t) and only hay (Hay‐t) over 35 days. We collected ruminal fluid and plasma just before feeding and at 3 h after feeding. The concentrations of propionate and butyrate in the rumen before feeding were lower in the CBDS‐t than in the Soybean‐t group (P < 0.05). However, after 3 h, the concentrations were higher in the CBDS‐t than in the Soybean‐t and Hay‐t groups (P < 0.05). Although, there were no differences in the compositions (% mol) of propionate and butyrate in the rumen and the concentration of plasma β‐hydroxybutyric acid before feeding between treatments, after 3 h they were significantly higher in the CBDS‐t than in the Soybean‐t and Hay‐t groups (P < 0.05). These results indicate that feeding CBDS promotes rumen fermentation and butyrate metabolism.     

The effect of fibre source on the numbers of some fibre-degrading bacteria of Arabian camel’s (Camelus dromedarius) foregut origin

The total bacterial community of Fibrobacter succinogenes and Ruminococcus flavefaciens in fibre-enriched culture of the foregut contents of 12 adult feral camels (Camelus dromedaries) fed on native vegetation in Australia was investigated using quantitative PCR. Foregut contents were collected postmortem, pooled and filtered before divided into two fractions. One fraction was used for extraction of DNA, while the other fraction was inoculated straight away into BM 10 contained filter paper (FP), cotton thread (CT) or neutral detergent fibre (NDF) as the sole carbohydrate sources in Hungate tubes. The tubes were incubated anaerobically at 39 °C for 1 week. After a near complete degradation of the FP and CT and extensive turbidity in the NDF, media subculturing was carried out into fresh media tubes. This was repeated twice before genomic DNA was extracted and used for quantification of bacteria. Using an absolute quantification method, the numbers of cells in 1 ml of each sample ranged from 4.07?×?10⁶ to 2.73?×?10⁹ for total bacteria, 1.34?×?10³ to 2.17?×?10⁵ for F. succinogenes and 5.78?×?10¹ to 3.53?×?10⁴ for R. flavefaciens. The mean cell number of F. succinogenes was highest in the FP enrichment medium at approximately 107-fold, whereas for the R. flavefaciens targeted primer, the NDF enrichment media had the highest mean cell number at approximately 4-fold when compared to the rumen content. The data presented here provide evidence of fibre type preference by the two main fibre-degrading bacteria and would help us understand the interaction between fibre type and fibre-degrading microorganisms, which has ramification on camel nutrition at different seasons and environments.     

Interaction of rumen bacteria as assumed by colonization patterns on untreated and alkali‐treated rice straw

Colonization patterns of representative rumen bacteria were compared between untreated rice straw (UTS) and sodium hydroxide‐treated rice straw (SHTS). UTS and SHTS were incubated in the rumen of sheep for 10 min, 1, 2, 6, 12, 24, 48 and 96 h using the nylon bag method. The population sizes of 13 representative bacterial species or groups were quantified by real‐time PCR. The total bacterial population size (abundance) was similar in both UTS and SHTS. Fibrobacter succinogenes showed a higher population size compared to other fibrolytic species and was detected at a higher level in SHTS (3.7%) than in UTS (2.6%). Ruminococcus albus and Ruminococcus flavefaciens were also detected at higher levels in SHTS (0.15% and 0.29%) than in UTS (0.03% and 0.18%). Population sizes of non‐fibrolytic species, such as Selenomonas ruminantium, Anaerovibrio lipolytica and Succinivibrio dextrinosolvens were higher in UTS than in SHTS. Coefficient of determination (r²) on population changes between bacterial species or groups were higher in UTS than in SHTS, suggesting the necessity of stronger bacterial interactions for UTS digestion. Therefore, not only colonization of fibrolytic species, but also synergistic interactions between different bacterial species may be key to the ruminal digestion of rice straw.     

Use of bean husk as an easily digestible fiber source for activating the fibrolytic rumen bacterium Fibrobacter succinogenes and rice straw digestion

A series of in sacco and in vitro studies were carried out to evaluate bean husks for activation of fibrolytic rumen bacteria and rice straw digestion. First, lablab bean husk, chickpea husk and rice straw were suspended in the rumen of sheep to analyze the bacterial consortium developed on each fiber source. Known members of fiber‐associating bacteria were found on both lablab bean husk and rice straw, but some of these bacteria were lacking on chickpea husk. Second, a pure culture study was carried out using six strains of Fibrobacter succinogenes. Both husks stimulated the growth of all tested strains, including a strain that did not grow on rice straw. The strain OS128 that showed the highest growth on rice straw displayed even higher growth on lablab bean husk without a time lag. Finally, two‐step incubations were carried out to determine whether prior incubation of rumen fluid with husks stimulates subsequent rice straw digestion. Higher digestibility of rice straw was recorded in the second‐round incubation following the first incubation with bean husks. These results suggest that the tested bean husks improve the digestion of rice straw by activating fibrolytic F. succinogenes and other associated bacteria.     

Phylogenetic analysis of hindgut microbiota in Hokkaido native horses compared to light horses

The analysis of 16S rDNA sequence of bacteria in feces of Hokkaido native horses and light horses were performed to compare the hindgut microbiota between the two breeds. One hundred and four bacterial 16S rDNA clones (57 clones from four native horses and 47 clones from two light horses) were obtained. Only four sequences (3.8% of total sequences) showed 97% or more similarity to known species. The sequences were mainly affiliated with Cytophaga–Flavobacter–Bacteroides and low GC Gram‐positive bacteria (LGCGP). Proportion of LGCGP was higher in light horses. Other phyla including Verrucomicrobia, Spirochaetes and Archaea were detected only for native horses, suggesting high diversity of microbiota in native horses. In LGCGP, clusters related to known cellulolytic species were found only for native horses, while a cluster related to soluble sugar‐utilizing species was detected only for light horses. The library composition‐comparing software LIBSHUFF showed significant (P < 0.05) difference of fecal microbiota between the horse breeds. The number of Fibrobacter succinogenes‐related sequence and the frequency of detection of novel groups were found to be higher in native horses by selective amplification analysis. The results suggest that genetic diversity and population size of the F. succinogenes group are higher in the hindgut of native horses.     

Monitoring in a complex world — seeking slow variables,a scaled focus,and speedier learning

The nutritive value of four subtropical grasses (Panicum maximum, Anthephora pubescens, Digitaria eriantha and Chloris gayana) standing hay were compared in terms of qualitative intake and partial digestibility by sheep. The species differed significantly in terms of diet quality selected by sheep grazing the standing hay. The rumen ammonia nitrogen (NH₃-N), total volatile fatty acid and propionic acid concentrations of sheep grazing P. maximum and A. pubescens were higher than those sheep grazing D. eriantha and C. gayana standing hay. Organic matter intake (OMI) (g kg^?1 W^0.75 d^?1), nitrogen intake (g d^?1), digesta flow, the total N flow, NH₃-N flow, non-ammonia nitrogen (NAN) flow and NAN disappearance (g d^?1) in the ileum were higher for sheep grazing P. maximum than for those grazing the other standing hays. The organic matter disappearance in the stomach and small intestine of sheep grazing P. maximum and D. eriantha standing hay was higher than for those sheep grazing either A. pubescens or C. gayana standing hay. The NAN flow/N intake were the highest for sheep grazing P. maximum and A. pubescens compared to C. gayana. The NAN digestibility was, however, not significantly different among the four species. The standing hays (except for C. gayana) seemed to have the capacity to meet the N requirement of the sheep for production, but the OMI (g kg^?1 W^0.75 d^?1) was not sufficient to support maintenance requirement of the sheep.     

Rumen development and growth of Balouchi lambs offered alfalfa hay pre- and post-weaning

The consumption of solid feed is essential for successful transition from a pre-ruminant to a functional digestive tract. Lambs fed starter rations containing highly fermentable carbohydrates often experience dramatic changes in concentrations of rumen and blood metabolites. The optimal amount of roughage required in the diet of pre-ruminant animals is still unclear. The objective of this study was to determine the effect of feeding alfalfa hay on performance and rumen development in young Balouchi lambs. In a completely randomized design, 30 lambs were fed one of three experimental diets consisting of a control, without alfalfa hay (C), a diet containing 7.5% alfalfa hay (A1), and a diet containing 15% alfalfa hay (A2). Lambs fed A1 and A2 diets had lower dry matter intake during the pre-weaning period (P?P?=?0.02), but feed conversion ratio and average daily gain were not affected by feeding alfalfa hay. Concentration of beta-hydroxybutyric acid was higher in C compared with the A1 and A2 groups (P?P?=?0.04) and increased thickness of muscular layer (P?=?0.05). We concluded that including 15% alfalfa hay in the starter diet could reduce thickness of the keratinization layer and increase muscularity of rumen wall without adverse effects on growth and performance of newborn lambs.     

Minimum length of the adaptation and collection period in digestibility trials with sheep fed ad libitum only forage or forage plus concentrate

Two in vivo digestibility trials with sheep were conducted to identify the minimum period length of feeding a new diet to obtain reproducible values of nutritional variables onward and the minimum length of collection period as to obtain maximal precision for each variable. Trial 1 was conducted with ten Polwarth male sheep (34 ± 5 kg body weight (BW)) throughout three 21‐day periods, in a completely randomized two‐way crossover design. The animals were divided into two groups (Group A and B, n = 5 per group) which were fed ad libitum with a sequence of the following diets throughout the periods: Group A: hay – hay plus concentrate – hay; Group B: hay plus concentrate – hay – hay plus concentrate. The concentrate was included in a proportion of 0.33 of the total diet. The intake, and the faecal and urinary excretion were measured daily throughout the experiment. For evaluating rumen fermentation variables, in Trial 2 four Santa Inês male sheep (65 ± 5 kg BW) fitted with ruminal cannula were used. The animals were randomly divided into two groups (n = 2 per group), and the trial was conducted through four 21 days experimental period, in a three‐way crossover design, using experimental diets and feeding management similar to Trial 1. The results indicated that, even though no clear or consistent steady‐state condition was identified for rumen fermentation or urinary excretion variables, the adaptation period for measuring OM digestibility in in vivo trials with sheep fed ad libitum where the diet shifts from one of only hay to another containing concentrate, or vice‐versa, should be at least 12 days long. Moreover, although no precision improvement was obtained by increasing the collection period above 1 day for measuring OM digestibility, the minimal length of collection period should be 4 days for measuring faecal excretion variables and 7 days for measuring urinary excretion variables.     

Evaluation of intraperitoneal and incisional lidocaine or bupivacaine for analgesia following ovariohysterectomy in the dog

Objective To determine if intraperitoneal (IP) and incisional (SC) lidocaine or bupivacaine provide analgesia following ovariohysterectomy (OHE). Study Design Prospective, randomized, controlled, blinded clinical trial. Animals Thirty dogs presenting to the Veterinary Teaching Hospital for elective OHE. Methods Dogs were pre‐medicated with acepromazine and butorphanol, induced with thiopental and maintained with isoflurane. They were randomly assigned to three groups: 10 received 8.8 mg kg^?1 2% lidocaine with epinephrine IP (LID); 10 received 4.4 mg kg^?1 0.75% bupivacaine IP (BUP); and 10 received 0.9% saline IP (SAL) upon completion of OHE. All IP doses were standardized to 0.88 mL kg^?1 with saline. An additional 2 mL of undiluted solution was placed SC prior to incisional closure. Dogs were scored at 0.5, 1, 2, 3, 6, 8 and 18 hours post‐extubation by one observer. Dogs were evaluated using a visual analogue scale (VAS) for pain and sedation, and a composite pain scale (CPS) that included physiologic and behavioral variables. Dogs were treated with 0.22 mg kg^?1 butorphanol + acepromazine if their VAS (pain) score was >50. Parametric variables were analyzed using Student's t‐test or repeated measures anova as appropriate. Non‐parametric variables were analyzed by χ²‐test. Results There were no significant differences in age, weight, incision length, surgery time, anesthesia time, or total thiopental dose among groups. Peak post‐surgical pain scores for all groups occurred at 0.5 hours and returned to baseline by 18 hours. Dogs in the BUP group had significantly lower VAS‐pain scores overall than dogs in the SAL group. Seven out of 10 dogs in the SAL group, 4/10 in the LID group and 2/10 in the BUP group were treated with supplemental acepromazine and butorphanol. No differences between groups were detected with the CPS. No adverse side‐effects were observed. Conclusions and clinical relevance Our findings support the use of IP and SC bupivacaine for post‐operative analgesia following OHE in the dog.     

Influences of dietary regimens on microbial content in gastrointestinal tracts of meat goats

This experiment was conducted to evaluate the effects of dietary treatments on microbial loads and pH of gastrointestinal tract contents in meat goats, as well as the concentration of volatile fatty acid (VFA) in the rumen. Crossbred (Boer x Spanish) goats (n = 36; BW = 17.7 kg) were assigned randomly to one of three experimental diets (n = 12/diet or 3 pens/treatment) for 90 days:alfalfa (Medicago sativa) hay alone (AH-diet); 18% CP concentrate alone (C-diet); or, a combined diet (AHC-diet), consisting of the AH-diet for the first 45 days, followed by 45 days of the C-diet. After evisceration, pH values of rumen liquor and colon digesta were immediately measured from each animal, as well as aseptically collected rumen liquor and rectal samples to determine the microbial loads. Collected rumen liquor was also prepared for volatile fatty acid (VFA) contents. Feeding meat goats with alfalfa hay alone had higher (P < 0.05) rumen (7.17) and colon (7.10) pH compared with those fed either the concentrate alone or combined-diet. Although the acetate content was high in the AH-fed group (66.3 mM) compared to the AHC-diet group (34.6 mM), no significant differences were found in the total VFA contents in rumen liquor among the goats fed three different dietary regimens. Total plate counts were not significantly different among goats fed the experimental diets in the rumen or rectal samples. Escherichia coli counts in the rectal samples were lower (P < 0.05) in the AH-diet group (6.43 log₁₀ CFU/g) compared with the C-diet (8.21 log₁₀ CFU/g) or AHC-diet (8.40 log₁₀ CFU/g) groups. However, no significant differences were found in the E. coli counts of rumen samples from goats fed the experimental diets. The mean (± SEM) rumen E. coli counts were 1.38, 1.65, and 2.51 ± 0.560 log₁₀ CFU/g in the AH-, C-, and AHC-diet groups, respectively. The results indicate that feeding hay alone may decrease the fecal shedding of E. coli in meat goats with increasing the rumen and colon pH.     

The mcrA gene and 16S rRNA gene in the phylogenetic analysis of methanogens in the rumen of faunated and unfaunated cattle

The influence of rumen protozoa on the composition of rumen methanogens was studied by using seven growing Holstein cattle divided into two groups: four faunated and three unfaunated. 16S ribosomal RNA gene (rDNA) and methyl coenzyme‐M reductase (MCR) α subunit (mcrA) gene clonal libraries were constructed. The results of each analysis showed that Methanobacteriales was dominant in the rumen of both groups. By mcrA gene analysis, 22.1% of unfaunated clones were classified into unfaunated group 1, which was not detected from faunated cattle. The 16S rRNA gene analysis showed that the number of operational taxonomic units was higher in unfaunated than faunated cattle, suggesting the diversity of methanogens tended to be higher by the removal of protozoa. The results of the LIBSHUFF program indicated that the 16S rRNA gene and mcrA gene clone libraries for the faunated group differed from those for the unfaunated group (P = 0.001). It was suggested that the presence of protozoa strongly affected the composition of rumen methanogens.     

The effect of dietary Chlorella vulgaris supplementation on micro‐organism community,enzyme activities and fatty acid profile in the rumen liquid of goats

Microalgae might be considered as an alternative source of fat and/or protein for ruminant's diets. However, changes in populations of ruminal micro‐organisms associated with biohydrogenation process, methane and ammonia production in response to microalgae dietary supplementation have not been well characterized. Thus, 16 cross‐bred goats were divided into two groups. Each goat of both groups was fed individually with alfalfa hay and concentrates separately. The concentrates of the control group had no microalgae while those of the treated group were supplemented with 10 g lyophilized Chlorella vulgaris/kg concentrate (chlor). On the 30th experimental day, samples of rumen fluid were collected for microbial DNA extraction, fatty acid profile and enzyme activity analyses. The results showed that the chlor diet compared with the control increased significantly the populations of Methanosphaera stadtmanae, Methanobrevibacter ruminantium and Methanogens bacteria and protozoa in the rumen of goats. A significant reduction in the cellulase activity and in the abundance of Ruminococcus albus, and a significant increase in the protease activity and in the abundance of Clostridium sticklandii in the rumen liquid of goats fed with the chlor diet, compared with the control, were found. Chlorella vulgaris supplementation promoted the formation of trans C_18:1, trans‐11 C_18:1 and monounsaturated fatty acids (MUFA), while the proportions of C_18:0 and long‐chain fatty acids (LCFA) reduced significantly in the rumen liquid of goats. This shift in ruminal biohydrogenation pathway was accompanied by a significant increase in Butyrivibrio fibrisolvens trans C_18:1‐producing bacteria. In conclusion, the supplementation of diets with microalgae needs further investigation because it enhances the populations of methane‐producing bacteria and protozoa.