ARISA analysis of ruminal bacterial community dynamics in lactating dairy cows during the feeding cycle

The bovine rumen undergoes substantial changes in environmental conditions during the animal's feeding cycle, but the effects of these changes on microbial populations have not been examined systematically. Two dairy cows fed a mixed forage/concentrate ration at 12 h intervals over 4 feeding cycles displayed substantial changes in ruminal pH and volatile fatty acid (VFA) concentrations. Automated ribosomal intergenic spacer analysis (ARISA) of solid- and liquid-associated bacterial populations in samples collected at 2, 4, 6, 9, and 12 h after feeding revealed a high degree of bacterial diversity. A total of 155 different amplicon lengths (ALs) were detected across all 83 samples, and 11–74 detected per sample. A substantial proportion (11%) of the ALs was detected in one cow but not in the other. The proportions of ALs that were detected only in the liquid phase or the solid phase were 13.5% and 1.9%, respectively. Correspondence analysis indicated that bacterial community composition differed between cows and between solid or liquid phases, but overall the solid-associated population displayed less change in composition within and across feeding cycles. The data support the notion that cows fed the same diets can have substantial differences in bacterial community composition, and that the solids-associated (biofilm) communities display greater stability than do associated planktonic communities.     

Utilization of individual cellodextrins by three predominant ruminal cellulolytic bacteria.

Growth of the ruminal bacteria Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and R. albus 7 followed Monod kinetics with respect to concentrations of individual pure cellodextrins (cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose). Under the conditions tested, R. flavefaciens FD-1 possesses the greatest capacity to compete for low concentrations of these cellodextrins.     

Comparative studies of the composition of bacterial microbiota associated with the ruminal content,ruminal epithelium and in the faeces of lactating dairy cows

The objective of this research was to compare the composition of bacterial microbiota associated with the ruminal content (RC), ruminal epithelium (RE) and faeces of Holstein dairy cows. The RC, RE and faecal samples were collected from six Holstein dairy cows when the animals were slaughtered. Community compositions of bacterial 16S rRNA genes from RC, RE and faeces were determined using a MiSeq sequencing platform with bacterial‐targeting universal primers 338F and 806R. UniFrac analysis revealed that the bacterial communities of RC, RE and faeces were clearly separated from each other. Statistically significant dissimilarities were observed between RC and faeces (P = 0.002), between RC and RE (P = 0.003), and between RE and faeces (P = 0.001). A assignment of sequences to taxa showed that the abundance of the predominant phyla Bacteroidetes was lower in RE than in RC, while a significant higher (P < 0.01) abundance of Proteobacteria was present in RE than in RC. When compared with the RC, the abundance of Firmicutes and Verrucomicrobia was higher in faeces, and RC contained a greater abundance of Bacteroidetes and Tenericutes. A higher proportions of Butyrivibrio and Campylobacter dominated RE as compared to RC. The faecal microbiota was less diverse than RC and dominated by genera Turicibacter and Clostridium. In general, these findings clearly demonstrated the striking compositional differences among RC, RE and faeces, indicating that bacterial communities are specific and adapted to the harbouring environment.     

Antibiosis between ruminal bacteria and ruminal fungi

Cellulose digestion, bacterial numbers, and fungal numbers were monitored over time in vitro by using a purified cellulose medium with and without antibiotics (penicillin and streptomycin). All fermentations were inoculated with a 1:10 dilution of whole rumen contents (WRC). Without antibiotics, cellulose digestion was higher (P < 0.01) at 24, 30, 48, and 72 h; fungi had almost disappeared by 24 h, while bacterial concentrations increased over 100-fold in 24 h and then decreased gradually up to 72 h. In those fermentations with added antibiotics, fungal concentrations increased 4-fold by 30 h and up to 42-fold at 72 h; bacterial concentrations were markedly reduced by 24 h and remained low through 72 h. Similar results were obtained with ground alfalfa as a substrate. In further studies, the in vitro fermentation of purified cellulose without antibiotics was stopped after 18 to 20 h, and the microbial population was killed by autoclaving. Antibiotics were added to half of the tubes, and all tubes were reinoculated with WRC. After 72 h, extensive cellulose digestion had occurred in those tubes without antibiotics, as compared to very low cellulose digestion with added antibiotics. The extent of this inhibition was found to increase in proportion to the length of the initial fermentation period, suggesting the production of a heat-stable inhibitory factor or factors. The inhibitory activity was present in rumen fluid, could be extracted from lyophilized rumen fluid (LRF) with water, and was stable in response to proteolytic enzymes. In addition, the water-extracted residue of LRF was found to contain growth factor activity for rumen fungi in vitro.     

Bacteriocins of ruminal bacteria

Similar sequences of distribution of structural genes encoding enterocin A (isolated from the ruminal strainE. faecium BC25) and enterolysin A (isolated from the ruminal amylolytic strainS. bovis II/I) were demonstrated by PCR using oligonucleotide primers specific for these bacteriocins within the ruminal enterococcal and streptococcal strains. Variable occurrence of these bacteriocins was found within the populations of Gram-positive ruminal cocci. An erratum to this article is available at .     

Effects of alfalfa silage storage structure and roasting corn on ruminal digestion and microbial CP synthesis in lactating dairy cows

The objective of this experiment was to quantify the effects of unroasted or roasted ground-shelled corn (GSC), when fed with alfalfa ensiled in bag, bunker, or O2-limiting tower silos on ruminal digestion and microbial CP synthesis in lactating dairy cows. The roasted corn was heat-treated in a propane-fired roasting system. Alfalfa was harvested as second cutting from fields with regrowth of the same maturity. A portion of each field was allotted to each silo. The diets with 3 × 2 factorial arrangement of treatments were fed to six multiparous rumen-cannulated Holstein cows in a cyclic change-over design with five 21-day periods. Experimental diets were comparable and averaged (on dry matter (DM) basis): 410 g/kg alfalfa silage (AS), 150 g/kg corn silage, 350 g/kg GSC, 50 g/kg soybean meal, 40 g/kg roasted soybeans, 177 g/kg CP, 264 g/kg NDF and 250 g/kg starch. Nutrient flow was quantified by the omasal sampling technique with use of three markers (Co, Yb and indigestible NDF). Continuous infusion of 10% atom excess (15NH4)2SO4 was used to label microbial CP. None of the interactions between storage structure of dietary AS and corn type were significant. DM intake was not different among dietary treatments, averaging 24.5 kg/day across diets. Means of ADF digested in the rumen for cows fed diets with AS from bag, bunker and O2-limiting tower silo were 2.1, 1.7 and 2.1 kg/day, respectively, and was lower in cows fed AS from the bunker silo. This response may partly be a reflection of the higher intake of ADF by cows fed AS ensiled in the O2-limiting tower silo compared with the bunker. There was a slightly greater supply of fermentable substrates for cows fed diets with roasted compared with unroasted GSC. The small increases in yield of milk protein and lactose observed in the previous production trial in cows fed diets containing roasted corn may have occurred because of greater supply of fermentable substrates.     

Inducible bacteriophages from ruminal bacteria

The incidence of temperate bacteriophage in a wide range of ruminal bacteria was investigated by means of induction with mitomycin C. Supernatant liquid from treated cultures was examined for phagelike particles by using transmission electron microscopy. Of 38 ruminal bacteria studied, nine organisms (23.7%) representing five genera (Eubacteria, Bacteroides, Butyrivibrio, Ruminococcus, and Streptococcus) produced phagelike particles. Filamentous particles from Butyrivibrio fibrisolvens are the first of this morphological type reported from ruminal bacteria. All of the other particles obtained possessed polyhedral heads and long, noncontractile tails (group B-type phage). The limited range of morphological types produced by mitomycin C induction cannot yet account for the much wider range of types found in ruminal contents by direct examination. The presence of viral genetic material in a significant percentage of the bacteria tested, as well as in a range of different genera, indicates that viral genetic material may be a normal constituent of the genome of appreciable numbers of ruminal bacteria.     

High-concentrate diets based on forages harvested at different maturity stages affect ruminal synthesis of B vitamins in lactating dairy cows

Effects of plant maturity on apparent ruminal synthesis and post-ruminal supply of B vitamins were evaluated in two feeding trials. Diets containing alfalfa (Trial 1) or orchardgrass (Trial 2) silages harvested either (1) early cut, less mature (EC) or (2) late cut, more mature (LC) as the sole forage were offered to ruminally and duodenally cannulated lactating Holstein cows in crossover design experiments. In Trial 1, conducted with 16 cows (569±43 kg of empty BW (ruminal content removed) and 43.7±8.6 kg/day of 3.5% fat-corrected milk yield; mean±SD) in two 17-day treatment periods, both diets provided ~22% forage NDF and 27% total NDF, and the forage-to-concentrate ratios were 53 : 47 and 42 : 58 for EC and LC, respectively. In Trial 2, conducted with 13 cows (588±55 kg of empty BW and 43.7±7.7 kg/day of 3.5% fat-corrected milk yield; mean±SD) in two 18-day treatment periods, both diets provided ~25% forage NDF and 31% total NDF; the forage-to-concentrate ratios were 58 : 42 and 46 : 54 for EC and LC, respectively. Thiamin, riboflavin, niacin, vitamin B₆, folates and vitamin B₁₂ were measured in feed and duodenal content. Apparent ruminal synthesis was calculated as the duodenal flow minus the intake. Diets based on EC alfalfa decreased the amounts of thiamin, niacin and folates reaching the duodenum, whereas diets based on EC orchardgrass increased riboflavin duodenal flow. Daily apparent ruminal synthesis of thiamin, riboflavin, niacin and vitamin B₆ were correlated negatively with their intake, suggesting a microbial regulation of their concentration in the rumen. Vitamin B₁₂ apparent ruminal synthesis was correlated negatively with total volatile fatty acids concentration, but positively with ruminal pH and microbial N duodenal flow.     

Superoxide dismutase in ruminal bacteria

Of 13 species of anaerobic ruminal bacteria examined, 11 were found to contain measurable levels of superoxide dismutase activity. Four of five other strict anaerobic species studied for comparison were found to contain superoxide dismutase activity.     

Managing the dominant follicle in lactating dairy cows

Reproductive efficiency is not optimal in high-producing dairy cows. Although many aspects of ovarian follicular growth in cows are similar to those observed in heifers, there are numerous specific differences in follicular development that may be linked with changes in reproductive physiology in high-producing lactating dairy cows. These include: 1) reduced circulating estradiol (E2) concentrations near estrus, 2) ovulation of follicles that are larger than the optimal size, 3) increased double ovulation and twinning, and 4) increased incidence of anovulation with a distinctive pattern of follicle growth in anovular dairy cows. The first three changes become more dramatic as milk production increases, although anovulation has not generally been associated with level of milk production. To overcome reproductive inefficiencies in dairy cows, reproductive management programs have been developed to synchronize ovulation and enable the use of timed AI in lactating dairy cows. Effective regulation of the CL, follicles, and hormonal environment during each part of the protocol is critical for optimizing these programs. This review discusses the distinct aspects of follicular development in lactating dairy cows and the methodologies that have been utilized in the past two decades in order to manage the dominant follicle during synchronization of ovulation and timed AI programs.     

Managing the dominant follicle in lactating dairy cows

Reproductive efficiency is not optimal in high-producing dairy cows. Although many aspects of ovarian follicular growth in cows are similar to those observed in heifers, there are numerous specific differences in follicular development that may be linked with changes in reproductive physiology in high-producing lactating dairy cows. These include: 1) reduced circulating estradiol (E2) concentrations near estrus, 2) ovulation of follicles that are larger than the optimal size, 3) increased double ovulation and twinning, and 4) increased incidence of anovulation with a distinctive pattern of follicle growth in anovular dairy cows. The first three changes become more dramatic as milk production increases, although anovulation has not generally been associated with level of milk production. To overcome reproductive inefficiencies in dairy cows, reproductive management programs have been developed to synchronize ovulation and enable the use of timed AI in lactating dairy cows. Effective regulation of the CL, follicles, and hormonal environment during each part of the protocol is critical for optimizing these programs. This review discusses the distinct aspects of follicular development in lactating dairy cows and the methodologies that have been utilized in the past two decades in order to manage the dominant follicle during synchronization of ovulation and timed AI programs.     

Supplementing lactating dairy cows with a vitamin B12 precursor, 5,6-dimethylbenzimidazole,increases the apparent ruminal synthesis of vitamin B12

Cobalamin (CBL), the biologically active form of vitamin B₁₂, and its analogs, are produced by bacteria only if cobalt supply is adequate. The analogs differ generally by the nucleotide moiety of the molecule. In CBL, 5,6-dimethylbenzimidazole (5,6-DMB) is the base in the nucleotide moiety. The present study aimed to determine if a supplement of 5,6-DMB could increase utilization of dietary cobalt for synthesis of CBL and change ruminal fermentation, nutrient digestibility, omasal flow of nutrients and ruminal protozoa counts. Eight ruminally cannulated multiparous Holstein cows (mean±standard deviation=238±21 days in milk and 736±47 kg of BW) were used in a crossover design. Cows were randomly assigned to a daily supplement of a gelatin capsule containing 1.5 g of 5,6-DMB via the rumen cannula or no supplement. Each period lasted 29 days and consisted of 21 days for treatment adaptation and 8 days for data and samples collection. Five corrinoids, CBL and four cobamides were detected in the total mixed ration and the omasal digesta from both treatments. The dietary supplement of 5,6-DMB increased (P=0.02) apparent ruminal synthesis of CBL from 14.6 to 19.6 (s.e.m. 0.8) mg/day but had no effect (P>0.1) on apparent ruminal synthesis of the four analogs. The supplement of 5,6-DMB had no effect (P>0.1) on milk production and composition, or on protozoal count, ruminal pH and concentrations of volatile fatty acids and ammonia nitrogen in rumen content. The supplement had also no effect (P>0.1) on intake, omasal flow and apparent ruminal digestibility of dry matter, organic matter, NDF, ADF and nitrogenous fractions. Plasma concentration of CBL was not affected by treatments (P=0.98). Providing a preformed part of the CBL molecule, that is, 5,6-DMB, increased by 34% the apparent ruminal synthesis of CBL by ruminal bacteria but had no effect on ruminal fermentation or protozoa count and it was not sufficient to increase plasma concentrations of the vitamin. Even though the efficiency of cobalt utilization for apparent synthesis of CBL was increased from 2.0% to 2.7% by the 5,6-DMB supplement, this improved efficiency was still very low. Further research is needed to identify the factors affecting efficiency of utilization of cobalt for synthesis of CBL by the bacterial populations in rumen.     

Trace mineral source influences digestion,ruminal fermentation,and ruminal copper,zinc, and manganese distribution in steers fed a diet suitable for lactating dairy cows

High solubility of certain trace minerals (TM) in the rumen can alter nutrient digestibility and fermentation. The objectives of the present studies were to determine the effects of TM source on 1) nutrient digestibility and ruminal fermentation, 2) concentrations of soluble Cu, Zn, and Mn in the rumen following a pulse dose of TM, and 3) Cu, Zn, and Mn binding strength on ruminal digesta using dialysis against a chelating agent in steers fed a diet formulated to meet the requirements of a high producing dairy cow. Twelve Angus steers fitted with ruminal cannulae were adapted to a diet balanced with nutrient concentrations similar to a diet for a high producing lactating dairy cow for 21 d. Steers were then randomly assigned to dietary treatments consisting of 10 mg Cu, 40 mg Mn, and 60 mg Zn/kg DM from either sulfate (STM), hydroxychloride (HTM) or complexed trace minerals (CTM). The experimental design did not include a negative control (no supplemental Cu, Mn, or Zn) because the basal diet did not meet the National Research Council requirement for Cu and Zn. Copper, Mn, and Zn are also generally supplemented to lactating dairy cow diets at concentrations approximating those supplied in the present study. Following a 14-d adaptation period, total fecal output was collected for 5-d. Following the fecal collection period, rumen fluid was collected for Volatile fatty acid (VFA) parameters. On the following day, the same diet was provided for 14 d, without supplemental Cu, Zn, and Mn. This period served as a wash-out period. A pulse dose of 100, 400, and 600 mg of Cu, Zn, Mn, respectively, from either STM, HTM, or CTM, was administered via ruminal cannulae to the steers on day 15. Over a 24-h period ruminal samples were obtained every 2-h. Following centrifugation, the supernatant was analyzed for Cu, Mn, and Zn. Ruminal solid digesta samples from times 0, 12, and 24 h after bolus dosing were exposed to dialysis against Tris-EDTA. Digestibility of NDF and ADF were lesser in STM vs. HTM and vs. CTM supplemented steers. Steers receiving HTM and CTM had greater total VFA concentrations than STM, and molar proportions of individual VFA were not affected by treatment. Ruminal soluble Cu and Zn concentrations were greater post dosing in STM and CTM supplemented steers at 2, 4, and 6 h for Cu and 4, 6, 8, 10 and 12 h for Zn when compared to HTM supplemented steers. The release of Cu and Zn from ruminal solid digesta following dialysis against Tris-EDTA at 12 and 24 h postdosing was greater for steers receiving HTM compared to those receiving STM or CTM. Results indicate trace mineral source impacts: 1) how tightly bound Cu and Zn are to ruminal solid digesta; 2) fiber digestion; 3) and ruminal total VFA concentrations.     

Inducible bacteriophages from ruminal bacteria.

The incidence of temperate bacteriophage in a wide range of ruminal bacteria was investigated by means of induction with mitomycin C. Supernatant liquid from treated cultures was examined for phagelike particles by using transmission electron microscopy. Of 38 ruminal bacteria studied, nine organisms (23.7%) representing five genera (Eubacteria, Bacteroides, Butyrivibrio, Ruminococcus, and Streptococcus) produced phagelike particles. Filamentous particles from Butyrivibrio fibrisolvens are the first of this morphological type reported from ruminal bacteria. All of the other particles obtained possessed polyhedral heads and long, noncontractile tails (group B-type phage). The limited range of morphological types produced by mitomycin C induction cannot yet account for the much wider range of types found in ruminal contents by direct examination. The presence of viral genetic material in a significant percentage of the bacteria tested, as well as in a range of different genera, indicates that viral genetic material may be a normal constituent of the genome of appreciable numbers of ruminal bacteria.     

Superoxide dismutase in ruminal bacteria.

Of 13 species of anaerobic ruminal bacteria examined, 11 were found to contain measurable levels of superoxide dismutase activity. Four of five other strict anaerobic species studied for comparison were found to contain superoxide dismutase activity.     

GATC-specific restriction-modification systems in ruminal bacteria

The GATC-specific restriction and modification activities were analyzed in 11 major bacterial representatives of ruminal microflora. Modification phenotype was observed in 13 out of 40 ruminal strains. MboI isoschizomeric restriction endonucleases were detected in 10 bacterial strains tested; three strains lacked any detectable corresponding endonuclease activity. The only examined strain of Mitsuokella multi-acida was found to possess a different type of endonuclease activity. This is the first report on restriction activity in ruminal treponemes M. multiacida and Megasphaera elsdenii.     

Diet-dependent shifts in ruminal butyrate-producing bacteria

The influence of a host's diet on Butyrivibrio and Pseudobutyrivibrio populations was investigated by competitive PCR. Specific primers were designed and competitive PCRs developed for both groups. Results (from 4 cows with different diets) suggested that high-fiber intake essentially increases the Butyrivibrio amounts in the rumen, whereas high-energy food additives lead to its suppression. The Pseudobutyrivibrio concentration also changed during the experiment but without any significant relation to the host's diet.     

Effect of phenolic monomers on ruminal bacteria

Ruminal bacteria were subjected to a series of phenolic compounds in various concentrations to acquire fundamental information on the influence on growth and the potential limits to forage utilization by phenolic monomers. Ruminococcus albus 7, Ruminococcus flavefaciens FD-1, Butyrivibrio fibrisolvens 49, and Lachnospira multiparus D-32 were tested against 1, 5, and 10 mM concentrations of sinapic acid, syringaldehyde, syringic acid, ferulic acid, vanillin, vanillic acid, p-coumaric acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and hydrocinnamic acid. Responses were variable and dependent on the phenolic compound and microbial species. Compounds especially toxic (i.e., resulting in poor growth, effect on several species, dose-related response) were p-coumaric acid and p-hydroxybenzaldehyde, and adaptation to the toxins did not occur after three 24-h periods. Syringic, p-hydroxybenzoic, and hydrocinnamic acids stimulated growth of all four species and also stimulated filter paper degradation by R. flavefaciens. None of the stimulatory compounds supported microbial growth in the absence of carbohydrates. In vitro dry matter digestibility of cellulose (Solka-Floc) was not stimulated by any of the phenolic compounds (10 mM), but the cinnamic acids and benzoic aldehydes (10 mM) reduced (P less than 0.05) digestion by the mixed population in ruminal fluid. Growth of R. flavefaciens in the presence of p-hydroxybenzoic acid (10 mM) or p-coumaric acid (5 mM) resulted in recognizable alterations in cell ultrastructure. Both phenolics caused a reduction in cell size (P less than 0.05), and p-coumaric acid caused a reduction in capsular size (P less than 0.05) and produced occasional pleomorphic cells.     

Intracellular pH of acid-tolerant ruminal bacteria.

Acid-tolerant ruminal bacteria (Bacteroides ruminicola B1(4), Selenomonas ruminantium HD4, Streptococcus bovis JB1, Megasphaera elsdenii B159, and strain F) allowed their intracellular pH to decline as a function of extracellular pH and did not generate a large pH gradient across the cell membrane until the extracellular pH was low (less than 5.2). This decline in intracellular pH prevented an accumulation of volatile fatty acid anions inside the cells.