Effects of medium-chain fatty acid supplementation on performance and rumen fermentation of lactating Holstein dairy cows

Medium-chain fatty acids (MCFAs) have antimicrobial properties and cause negative or positive effects on animal performance depending on its dosage. We hypothesized that MCFA supplementation at a lower dose (i.e., 0.05–0.2% of dietary DM) would increase rumen pH and milk production without decreasing nutrient digestibility which is typically observed with the higher inclusion rates (i.e., >1% of dietary DM). The objective of this study was to evaluate the effects of MCFA supplementation at a lower dose on productivity, plasma energy metabolite concentrations, apparent total tract nutrient digestibility, rumen fermentation, and rumen microbial profile of lactating dairy cows. Thirty (n = 8 primiparous, n = 22 multiparous) Holstein cows in mid-lactation (637 ± 68.5 kg of initial BW, 98.5 ± 27.4 d in milk; mean ± standard deviation) were used in a crossover design with two 28-d periods. The MCFA supplement, consisted of 25% MCFA (containing 32% C8:0, 21% C10:0, 47% C12:0 on DM basis) and 75% carrier ingredients, was fed at 0.25% of dietary DM replacing dry ground corn in control (CON). Total inclusion of MCFA was 0.063% of dietary DM. No differences were observed in DM intake, apparent total tract nutrient digestibility and BW change between MCFA and CON. Milk and milk component yields did not differ between treatment groups. The MCFA supplementation tended to have higher minimum rumen pH (5.66 vs. 5.54), and decreased daily fluctuation range of rumen pH (1.17 vs. 1.40) compared to CON. However, the duration of acidosis (pH < 5.8, min/d) did not differ between treatment groups and ruminal total volatile fatty acid concentration and its profile did not differ between treatment groups. For rumen microbiota, the Chao1 index of bacterial community tended to be lower (10.9 vs. 11.6) whereas the Shannon index did not differ (0.91 vs. 0.93) in MCFA compared to CON, and both indices did not differ for archaeal and protozoan communities between treatment groups. The relative abundance of Methanobrevibacter gottschalkii increased when supplemented with MCFA (5.14 vs. 4.92%). These results suggest that supplementation of MCFA at 0.063% dietary DM may not affect overall animal performance or total tract nutrient digestibility, but decrease the daily range of pH and the bacterial richness in the rumen.     

Microbiome analysis of dairy cows fed pasture or total mixed ration diets

Understanding rumen microbial ecology is essential for the development of feed systems designed to improve livestock productivity, health and for methane mitigation strategies from cattle. Although rumen microbial communities have been studied previously, few studies have applied next-generation sequencing technologies to that ecosystem. The aim of this study was to characterize changes in microbial community structure arising from feeding dairy cows two widely used diets: pasture and total mixed ration (TMR). Bacterial, archaeal and protozoal communities were characterized by terminal restriction fragment length polymorphism of the amplified SSU rRNA gene and statistical analysis showed that bacterial and archaeal communities were significantly affected by diet, whereas no effect was observed for the protozoal community. Deep amplicon sequencing of the 16S rRNA gene revealed significant differences in the bacterial communities between the diets and between rumen solid and liquid content. At the family level, some important groups of rumen bacteria were clearly associated with specific diets, including the higher abundance of the Fibrobacteraceae in TMR solid samples and members of the propionate-producing Veillonelaceae in pasture samples. This study will be relevant to the study of rumen microbial ecology and livestock feed management.     

Ruminal methanogenesis as influenced by individual fatty acids supplemented to complete ruminant diets

The objective of the present study was to investigate the effects of seven different pure fatty acids on rumen fermentation using the rumen simulation technique (RUSITEC). The fatty acids were supplied to a complete ruminant diet at a proportion of 50 g x kg(-1) dietary dry matter and compared with an unsupplemented control. Methane release and methanogenic counts were suppressed by the fatty acids C12 : 0, C14 : 0 and C18 : 2 whereas C8 : 0, C10 : 0, C16 : 0 and C18 : 0 showed no corresponding effects. Apart from C12 : 0 and C18 : 2, C8 : 0 and C10 : 0 also adversely affected ciliate protozoa suggesting independence from the methane-suppressing effect of medium-chain fatty acids (MCFA). Although MCFA but not C18 : 2 reduced ruminal fibre degradation, the influence on other fermentation traits remained low. In conclusion, the supply of certain fatty acids to ruminant diets seems to have the potential to reduce methane release.     

Methanogen community structure in the rumens of farmed sheep, cattle and red deer fed different diets

Development of inhibitors and vaccines that mitigate rumen-derived methane by targeting methanogens relies on knowledge of the methanogens present. We investigated the composition of archaeal communities in the rumens of farmed sheep (Ovis aries), cattle (Bos taurus) and red deer (Cervus elaphus) using denaturing gradient gel electrophoresis (DGGE) to generate fingerprints of archaeal 16S rRNA genes. The total archaeal communities were relatively constant across species and diets, and were less variable and less diverse than bacterial communities. There were diet- and ruminant-species-based differences in archaeal community structure, but the same dominant archaea were present in all rumens. These were members of three coherent clades: species related to Methanobrevibacter ruminantium and Methanobrevibacter olleyae; species related to Methanobrevibacter gottschalkii, Methanobrevibacter thaueri and Methanobrevibacter millerae; and species of the genus Methanosphaera. Members of an archaeal group of unknown physiology, designated rumen cluster C (RCC), were also present. RCC-specific DGGE, clone library analysis and quantitative real-time PCR showed that their 16S rRNA gene sequences were very diverse and made up an average of 26.5% of the total archaea. RCC sequences were not readily detected in the DGGE patterns of total archaeal 16S rRNA genes because no single sequence type was abundant enough to form dominant bands.     

Specificity and sensitivity of eubacterial primers utilized for molecular profiling of bacteria within complex microbial ecosystems

Efficient profiling of eubacterial diversity within complex communities requires that primers are specific for eubacterial 16S rRNA. Specificity of published primers against eubacterial and archaeal 16S rRNA as well as protozoal and fungal 18S rRNA was assessed in silico. The specificity and sensitivity of the V3 and V6–V8 (F968gc and R1401) Denaturing Gradient Gel Electrophoresis (DGGE) primers was subsequently verified using rumen-derived samples. An assessment of the effects of employing touchdown PCR cycling conditions was also made. For DGGE profiling of eubacteria within rumen samples, primers F968gc and R1401 proved the most specific and sensitive providing that touchdown PCR is not used.     

Molecular diversity analysis of rumen methanogenic Archaea from goat in eastern China by DGGE methods using different primer pairs

Aims:  To screen a pair of primers suitable for denaturing gradient gel electrophoretic (DGGE) analysis of ruminal methanogenic Archaea and to detect the archaeal communities in the rumen of goat.
Methods and Results:  Nine primer pairs for 16S rDNA of methanogenic Archaea , including six for directed polymerase chain reaction (PCR) and three for nested PCR were first evaluated by PCR amplification of the total DNA from rumen fluids and bacteria. The DGGE analysis of rumen fluids was then conducted with three primer sets (344fGC/915r, 1106fGC/1378r and 519f/915rGC) of the nine pairs tested. Good separation and quality of patterns were obtained in DGGE analysis with primer pairs 1106fGC/1378r and 519f/915rGC. A total of 40 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic Archaea while primer pair 519f/915rGC had better amplification ranges than the other two primer pairs.
Conclusions:  The procedure of DGGE analysis with primer pair 519f/915rGC was more suitable for investigating methanogenic archaeal community in the rumen. The dominant methanogenic Archaea in the rumen of goat was Methanobrevibacter sp. and an unidentified methanogenic Archaea .
Significance and Impact of the Study:  One pair of primers suitable for DGGE analysis of ruminal methanogenic Archaea was obtained and the molecular diversity of ruminal methanogenic Archaea in goat was investigated by PCR-DGGE.     

Effect of fat type and lysophosphatidylcholine addition to broiler diets on performance,apparent digestibility of fatty acids,and apparent metabolizable energy content

A completely randomized design study with a 3 × 2 factorial arrangement was conducted to evaluate the effects of three different fat sources (soybean oil, tallow, and poultry fat) with or without emulsifier supplementation on performance, coefficient of total tract apparent digestibility (CTTAD) of fatty acids, and apparent metabolizable energy (AME) content in broiler chickens. Two hundred and fifty-two one-day-old male Arbor Acres broiler chickens were randomly divided into 6 different treatments: (T1) basal diet containing soybean oil without lysophosphatidylcholine (LPC) supplementation, (T2) basal diet containing soybean oil with LPC supplementation, (T3) basal diet containing tallow without LPC supplementation, (T4) basal diet containing tallow with LPC supplementation, (T5) basal diet containing poultry fat without LPC supplementation, and (T6) basal diet containing poultry fat with LPC supplementation. Body weight gains from broiler chicks fed diets containing tallow were lower (P<0.05) than the body weight gains from chicks that were fed diets containing soybean oil or poultry fat in both the starter and grower periods. Birds fed diets containing tallow had the highest FCR (P<0.05), followed by the birds that were fed diets containing poultry fat, and soybean oil. The CTTAD of C16:0, C18:2, and C18:3n3 was greater (P<0.05) for broilers fed diets containing soybean oil than for those fed diets containing tallow or poultry fat in the starter period. The addition of LPC increased (P<0.05) body weight gain of broiler chickens in the starter period and the AME of the diets in the grower period, and tended to reduce FCR (P=0.072) in the starter period. LPC supplementation increased (P<0.05) the CTTAD of C16:0, C18:1n7 and C18:1n9 in the starter period, and of C18:2, and C18:3n3 in the grower period (P<0.05). There were no significant interactions between fat sources and the addition of LPC. These data indicated that LPC supplementation can improve body weight gain of broiler chickens in the starter period. This effect may be associated with an increase of CTTAD of FA due to LPC activity.     

The effect of dietary concentrate and soya oil inclusion on microbial diversity in the rumen of cattle

Aims: Methane emissions from ruminants are a significant contributor to global greenhouse gas production. The aim of this study was to examine the effect of diet on microbial communities in the rumen of steers. Methods and Results: The effects of dietary alteration (50 : 50 vs 90 : 10 concentrate–forage ratio, and inclusion of soya oil) on methanogenic and bacterial communities in the rumen of steers were examined using molecular fingerprinting techniques (T‐RFLP and automated ribosomal intergenic spacer analysis) and real‐time PCR. Bacterial diversity was greatly affected by diet, whereas methanogen diversity was not. However, methanogen abundance was significantly reduced (P = 0·009) in high concentrate–forage diets and in the presence of soya oil (6%). In a parallel study, reduced methane emissions were observed with these diets. Conclusions: The greater effect of dietary alteration on bacterial community in the rumen compared with the methanogen community may reflect the impact of substrate availability on the rumen bacterial community. This resulted in altered rumen volatile fatty acid profiles and had a downstream effect on methanogen abundance, but not diversity. Significance and Impact of the Study: Understanding how rumen microbial communities contribute to methane production and how these microbes are influenced by diet is essential for the rational design of methane mitigation strategies from livestock.     

Fermentation of plant cell walls by ruminal bacteria, protozoa and fungi and their interaction with fibre particle size

The objective of the experiment was to evaluate the contribution of various ruminal microbial groups to the fermentation of cell walls of corn stover with different particle sizes based on ruminal gas production in vitro. Physical, chemical, and antibiotical methods were used to differentiate groups of bacteria, protozoa and fungi in rumen fluid, offering following rumen microbial groups: whole rumen fluid (WRF), bacterial (B), protozoal (P), fungal (F), bacterial plus protozoal (B + P), bacterial plus fungal (B + F), protozoal plus fungal (P + F), and negative control (CON). Cell walls from corn stover were ground and ball milled to produce two different particle sizes. The results showed that digestion of the cell walls was undertaken by the interaction among ruminal bacteria, protozoa and fungi, and such co-actions seemed to fail alternation by one of three microbial groups or any combinations. However, B + P group showed a significant contribution to the degradation of milled cell walls, and B + F group revealed a great synergy effect on the ground cell walls degradation. Particle size of cell walls also had a considerable influence on their fermentation extent instead of the fermentative patterns by various rumen microbial groups.     

Impact of High-Concentrate Feeding and Low Ruminal pH on Methanogens and Protozoa in the Rumen of Dairy Cows

Non-lactating dairy cattle were transitioned to a high-concentrate diet to investigate the effect of ruminal pH suppression, commonly found in dairy cattle, on the density, diversity, and community structure of rumen methanogens, as well as the density of rumen protozoa. Four ruminally cannulated cows were fed a hay diet and transitioned to a 65% grain and 35% hay diet. The cattle were maintained on an high-concentrate diet for 3 weeks before the transition back to an hay diet, which was fed for an additional 3 weeks. Rumen fluid and solids and fecal samples were obtained prior to feeding during weeks 0 (hay), 1, and 3 (high-concentrate), and 4 and 6 (hay). Subacute ruminal acidosis was induced during week 1. During week 3 of the experiment, there was a significant increase in the number of protozoa present in the rumen fluid (P = 0.049) and rumen solids (P = 0.004), and a significant reduction in protozoa in the rumen fluid in week 6 (P = 0.003). No significant effect of diet on density of rumen methanogens was found in any samples, as determined by real-time PCR. Clone libraries were constructed for weeks 0, 3, and 6, and the methanogen diversity of week 3 was found to differ from week 6. Week 3 was also found to have a significantly altered methanogen community structure, compared to the other weeks. Twenty-two unique 16S rRNA phylotypes were identified, three of which were found only during high-concentrate feeding, three were found during both phases of hay feeding, and seven were found in all three clone libraries. The genus Methanobrevibacter comprised 99% of the clones present. The rumen fluid at weeks 0, 3, and 6 of all the animals was found to contain a type A protozoal population. Ultimately, high-concentrate feeding did not significantly affect the density of rumen methanogens, but did alter methanogen diversity and community structure, as well as protozoal density within the rumen of nonlactating dairy cattle. Therefore, it may be necessary to monitor the rumen methanogen and protozoal communities of dairy cattle susceptible to depressed pH when methane abatement strategies are being investigated.     

Buccal Swabbing as a Noninvasive Method To Determine Bacterial,Archaeal, and Eukaryotic Microbial Community Structures in the Rumen

Analysis of rumen microbial community structure based on small-subunit rRNA marker genes in metagenomic DNA samples provides important insights into the dominant taxa present in the rumen and allows assessment of community differences between individuals or in response to treatments applied to ruminants. However, natural animal-to-animal variation in rumen microbial community composition can limit the power of a study considerably, especially when only subtle differences are expected between treatment groups. Thus, trials with large numbers of animals may be necessary to overcome this variation. Because ruminants pass large amounts of rumen material to their oral cavities when they chew their cud, oral samples may contain good representations of the rumen microbiota and be useful in lieu of rumen samples to study rumen microbial communities. We compared bacterial, archaeal, and eukaryotic community structures in DNAs extracted from buccal swabs to those in DNAs from samples collected directly from the rumen by use of a stomach tube for sheep on four different diets. After bioinformatic depletion of potential oral taxa from libraries of samples collected via buccal swabs, bacterial communities showed significant clustering by diet (R = 0.37; analysis of similarity [ANOSIM]) rather than by sampling method (R = 0.07). Archaeal, ciliate protozoal, and anaerobic fungal communities also showed significant clustering by diet rather than by sampling method, even without adjustment for potentially orally associated microorganisms. These findings indicate that buccal swabs may in future allow quick and noninvasive sampling for analysis of rumen microbial communities in large numbers of ruminants.     

Evaluation of the effects of different diets on microbiome diversity and fatty acid composition of rumen liquor in dairy goat

Fat supplementation plays an important role in defining milk fatty acids (FA) composition of ruminant products. The use of sources rich in linoleic and α-linolenic acid favors the accumulation of conjugated linoleic acids isomers, increasing the healthy properties of milk. Ruminal microbiota plays a pivotal role in defining milk FA composition, and its profile is affected by diet composition. The aim of this study was to investigate the responses of rumen FA production and microbial structure to hemp or linseed supplementation in diets of dairy goats. Ruminal microbiota composition was determined by 16S amplicon sequencing, whereas FA composition was obtained by gas-chromatography technique. In all, 18 pluriparous Alpine goats fed the same pre-treatment diet for 40±7 days were, then, arranged to three dietary treatments consisting of control, linseed and hemp seeds supplemented diets. Independently from sampling time and diets, bacterial community of ruminal fluid was dominated by Bacteroidetes (about 61.2%) and Firmicutes (24.2%) with a high abundance of Prevotellaceae (41.0%) and Veillonellaceae (9.4%) and a low presence of Ruminococcaceae (5.0%) and Lachnospiraceae (4.3%). Linseed supplementation affected ruminal bacteria population, with a significant reduction of biodiversity; in particular, relative abundance of Prevotella was reduced (−12.0%), whereas that of Succinivibrio and Fibrobacter was increased (+50.0% and +75.0%, respectively). No statistically significant differences were found among the average relative abundance of archaeal genera between each dietary group. Moreover, the addition of linseed and hemp seed induced significant changes in FA concentration in the rumen, as a consequence of shift from C18 : 2n-6 to C18 : 3n-3 biohydrogenation pathway. Furthermore, dimethylacetal composition was affected by fat supplementation, as consequence of ruminal bacteria population modification. Finally, the association study between the rumen FA profile and the bacterial microbiome revealed that Fibrobacteriaceae is the bacterial family showing the highest and significant correlation with FA involved in the biohydrogenation pathway of C18 : 3n-3.     

The structure of the bacterial and archaeal community in a biogas digester as revealed by denaturing gradient gel electrophoresis and 16S rDNA sequencing analysis

Aims:  To identify the bacterial and archaeal composition in a mesophilic biogas digester treating pig manure and to compare the consistency of two 16S rDNA-based methods to investigate the microbial structure.
Methods and results:  Sixty-nine bacterial operational taxonomic units (OTU) and 25 archaeal OTU were identified by sequencing two 16S rDNA clone libraries. Most bacterial OTU were identified as phyla of Firmicutes (47·2% of total clones), Bacteroides (35·4%) and Spirochaetes (13·2%). Methanoculleus bourgensis (29·0%), Methanosarcina barkeri (27·4%) and Methanospirillum hungatei (10·8%) were the dominant methanogens. Only 9% of bacterial and 20% of archaeal OTU matched cultured isolates at a similarity index of ≥97%. About 78% of the dominant bacterial (with abundance >3%) and 83% of archaeal OTU were recovered from the denaturing gradient gel electrophoresis (DGGE) bands of V3 regions in 16S rDNAs.
Conclusions:  In the digester, most bacterial and archaeal species were uncultured; bacteria belonging to Firmicutes , Bacteroides and Spirochaetes seem to take charge of cellulolysis, proteolysis, acidogenesis, sulfur-reducing and homoacetogenesis; the most methanogens were typical hydrogenotrophic or hydrogenotrophic/aceticlastic; DGGE profiles reflected the dominant microbiota.
Significance and Impact of the Study:  This study gave a first insight of the overall microbial structure in a rural biogas digester and also indicated DGGE was useful in displaying its dominant microbiota.     

Comparative evaluation of rumen‐protected fat,coconut oil and various oilseeds supplemented to fattening bulls

The effects of five different dietary fat supplements on fatty acid composition and oxidative stability of subcutaneous and kidney fat were evaluated in 36 Brown Swiss bulls and compared to a low fat diet in a monofactorial design. The following fat supplements were provided as additional fat at 30 g per kg feed dry matter: crystalline rumen‐protected fat, coconut oil, and three types of crushed whole oilseeds (rapeseed, sunflower seed and linseed). Adipose tissues reflected differences (P < 0.05) in dietary fatty acid composition although to a lower extent. Using protected fat, which contained elevated levels of trans fatty acids, and sunflower seed, containing a high proportion of linoleic acid, significantly increased C18:1 trans fatty acid proportion in the adipose tissues. The use of sunflower seed increased conjugated linoleic acid. The oilseeds resulted in lower amounts of C16:0 in favour of C18:0. Except for linseed, all fat supplemented groups improved oxidative stability of adipose tissues as compared with control. This was explained by lower proportions of unsaturated fatty acids in adipose tissue (protected fat), by elevated α‐tocopherol contents (rapeseed, sunflower seed) or by a combination of both (coconut oil). Fat colour remained unaffected by treatments. Compared to other fat supplements oilseeds, especially sunflower seed and rapeseed, can therefore be recommended to be fed to bulls in order to increase the proportions of C18 unsaturated fatty acids in adipose tissues and to maintain or improve oxidative stability.     

Flaxseed supplementation decreases methanogenic gene abundance in the rumen of dairy cows

The objective of this study was to investigate the effects of a flaxseed-supplemented diet on archaeal abundance and gene expression of methanogens in the rumen of dairy cows. In all, 11 non-lactating dairy cows were randomly divided into two groups: group A (five cows) and B (six cows). The two diets fed were: (1) the control diet, a conventional dry cow ration; and (2) the flaxseed-supplemented diet, the conventional dry cow ration adjusted with 12.16% ground flaxseed incorporated into the total mixed ration. A cross-over experiment was performed with the two groups of cows fed the two different diets for five 21-day periods, which included the first adaptation period followed by two treatment and two wash out periods. At the end of each feeding period, rumen fluid samples were collected via rumenocentesis and DNA was extracted. Quantitative PCR was utilized to analyze the gene abundance of 16S ribosomal RNA (16S rRNA) targeting the ruminal archaea population and the mcrA gene coding for methyl coenzyme-M reductase subunit A, a terminal enzyme in the methanogenesis pathway. Results demonstrated a 49% reduction of 16S rRNA and 50% reduction of mcrA gene abundances in the rumen of dairy cows fed the flaxseed-supplemented diet in comparison with those fed the control diet. This shows flaxseed supplementation effectively decreases the methanogenic population in the rumen. Future studies will focus on the mechanisms for such reduction in the rumen of dairy cattle, as well as the relationship between methanogenic gene expression and methane production.     

Relative contributions of bacteria, protozoa, and fungi to in vitro degradation of orchard grass cell walls and their interactions

To assess the relative contributions of microbial groups (bacteria, protozoa, and fungi) in rumen fluids to the overall process of plant cell wall digestion in the rumen, representatives of these groups were selected by physical and chemical treatments of whole rumen fluid and used to construct an artificial rumen ecosystem. Physical treatments involved homogenization, centrifugation, filtration, and heat sterilization. Chemical treatments involved the addition of antibiotics and various chemicals to rumen fluid. To evaluate the potential activity and relative contribution to degradation of cell walls by specific microbial groups, the following fractions were prepared: a positive system (whole ruminal fluid), a bacterial (B) system, a protozoal (P) system, a fungal (F) system, and a negative system (cell-free rumen fluid). To assess the interactions between specific microbial fractions, mixed cultures (B+P, B+F, and P+F systems) were also assigned. Patterns of degradation due to the various treatments resulted in three distinct groups of data based on the degradation rate of cell wall material and on cell wall-degrading enzyme activities. The order of degradation was as follows: positive and F systems > B system > negative and P systems. Therefore, fungal activity was responsible for most of the cell wall degradation. Cell wall degradation by the anaerobic bacterial fraction was significantly less than by the fungal fraction, and the protozoal fraction failed to grow under the conditions used. In general, in the mixed culture systems the coculture systems demonstrated a decrease in cellulolysis compared with that of the monoculture systems. When one microbial fraction was associated with another microbial fraction, two types of results were obtained. The protozoal fraction inhibited cellulolysis of cell wall material by both the bacterial and the fungal fractions, while in the coculture between the bacterial fraction and the fungal fraction a synergistic interaction was detected.     

Effects of Changes in Feed Level, Starvation, and Level of Feed After Starvation Upon the Concentration of Rumen Protozoa in the Ovine

Four rumen fistulated sheep were used in five experiments to investigate the effect of feed level upon the concentration of rumen ciliate protozoa. The sheep were fed once daily 650 g of a pelleted diet composed of corn cobs, 45%; alfalfa meal, 35%; oats, 12.5%; cane molasses, 5%; urea, 0.4%; and vitamins and minerals, 2%. The concentration of protozoa reached minimum and maximum values at 5 and 22.5 h after feeding, respectively. Thus, to estimate apparent generation rates, concentrations of protozoa were determined at 5 and 20 h postfeeding. Apparent generation rate/h = natural log of ([concentration of protozoa at 20 h divided by concentration at 5 h] divided by the time interval, [T20 to T5]). Alteration of the feed to protozoa ratio by starvation and by changing the level of feed (200 to 900 g/day) showed that as the ratio of feed to protozoa increased, generation rate increased. Measurements of liquid turnover rates in the rumen showed that turnover rate decreased as feed level decreased. Turnover rate was near zero when the sheep were starved. Small quantities of soluble substrates, added directly to the rumen of starved sheep, maintained the protozoal population when rumen turnover was minimal. Furthermore, as rumen turnover rate increased with increased levels of feed, the effect of substrate on maintaining the protozoal population was negated. Thus, at high feed levels, turnover rate may be the dominant factor controlling the establishment and concentration of protozoa in the rumen.     

Effect of phenotypic residual feed intake and dietary forage content on the rumen microbial community of beef cattle

Feed-efficient animals have lower production costs and reduced environmental impact. Given that rumen microbial fermentation plays a pivotal role in host nutrition, the premise that rumen microbiota may contribute to host feed efficiency is gaining momentum. Since diet is a major factor in determining rumen community structure and fermentation patterns, we investigated the effect of divergence in phenotypic residual feed intake (RFI) on ruminal community structure of beef cattle across two contrasting diets. PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were performed to profile the rumen bacterial population and to quantify the ruminal populations of Entodinium spp., protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus, Prevotella brevis, the genus Prevotella, and fungi in 14 low (efficient)- and 14 high (inefficient)-RFI animals offered a low-energy, high-forage diet, followed by a high-energy, low-forage diet. Canonical correspondence and Spearman correlation analyses were used to investigate associations between physiological variables and rumen microbial structure and specific microbial populations, respectively. The effect of RFI on bacterial profiles was influenced by diet, with the association between RFI group and PCR-DGGE profiles stronger for the higher forage diet. qPCR showed that Prevotella abundance was higher (P < 0.0001) in inefficient animals. A higher (P < 0.0001) abundance of Entodinium and Prevotella spp. and a lower (P < 0.0001) abundance of Fibrobacter succinogenes were observed when animals were offered the low-forage diet. Thus, differences in the ruminal microflora may contribute to host feed efficiency, although this effect may also be modulated by the diet offered.     

Postinoculation protozoan establishment and association patterns of methanogenic archaea in the ovine rumen

Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.