Does Dietary Mitigation of Enteric Methane Production Affect Rumen Function and Animal Productivity in Dairy Cows?

It has been suggested that the rumen microbiome and rumen function might be disrupted if methane production in the rumen is decreased. Furthermore concerns have been voiced that geography and management might influence the underlying microbial population and hence the response of the rumen to mitigation strategies. Here we report the effect of the dietary additives: linseed oil and nitrate on methane emissions, rumen fermentation, and the rumen microbiome in two experiments from New Zealand (Dairy 1) and the UK (Dairy 2). Dairy 1 was a randomized block design with 18 multiparous lactating cows. Dairy 2 was a complete replicated 3 x 3 Latin Square using 6 rumen cannulated, lactating dairy cows. Treatments consisted of a control total mixed ration (TMR), supplementation with linseed oil (4% of feed DM) and supplementation with nitrate (2% of feed DM) in both experiments. Methane emissions were measured in open circuit respiration chambers and rumen samples were analyzed for rumen fermentation parameters and microbial population structure using qPCR and next generation sequencing (NGS). Supplementation with nitrate, but not linseed oil, decreased methane yield (g/kg DMI; P<0.02) and increased hydrogen (P<0.03) emissions in both experiments. Furthermore, the effect of nitrate on gaseous emissions was accompanied by an increased rumen acetate to propionate ratio and consistent changes in the rumen microbial populations including a decreased abundance of the main genus Prevotella and a decrease in archaeal mcrA (log₁₀ copies/ g rumen DM content). These results demonstrate that methane emissions can be significantly decreased with nitrate supplementation with only minor, but consistent, effects on the rumen microbial population and its function, with no evidence that the response to dietary additives differed due to geography and different underlying microbial populations.     

Diet supplementation with cinnamon oil,cinnamaldehyde, or monensin does not reduce enteric methane production of dairy cows

This study was conducted to evaluate the effect of dietary addition of cinnamon oil (CIN), cinnamaldehyde (CDH), or monensin (MON) on enteric methane (CH₄) emission in dairy cows. Eight multiparous lactating Holstein cows fitted with ruminal cannulas were used in a replicated 4×4 Latin square design (28-day periods). Cows were fed (ad libitum) a total mixed ration ((TMR); 60 : 40 forage : concentrate ratio, on a dry matter (DM) basis) not supplemented (CTL), or supplemented with CIN (50 mg/kg DM intake), CDH (50 mg/kg DM intake), or monensin (24 mg/kg of DM intake). Dry matter intake (DMI), nutrient digestibility, N retention, and milk performance were measured over 6 consecutive days. Ruminal degradability of the basal diet (with no additive) was assessed using in sacco incubations (0, 2, 4, 8, 16, 24, 48, 72 and 96 h). Ruminal fermentation characteristics (pH, volatile fatty acids (VFA), and ammonia (NH₃)) and protozoa were determined over 2 days. Enteric CH₄ emissions were measured over 6 consecutive days using the sulfur hexafluoride (SF₆) tracer gas technique. Adding CIN, CDH or MON to the diet had no effects on DMI, N retention, in sacco ruminal degradation and nutrient digestibility of the diet. Ruminal fermentation characteristics and protozoa numbers were not modified by including the feed additives in the diet. Enteric CH₄ emission and CH₄ energy losses averaged 491 g/day and 6.59% of gross energy intake, respectively, and were not affected by adding CIN, CDH or MON to the diet. Results of this study indicate that CIN, CDH and MON are not viable CH₄ mitigation strategies in dairy cows.     

Rumen enzyme profile and fermentation characteristics in sheep as affected by treatment with sodium lauryl sulfate as defaunating agent and presence of ciliate protozoa

The efficiency of sodium lauryl sulfate as a defaunating agent and effect of rumen protozoa on nutrient utilization, fermentation characteristics and enzyme profile were evaluated in adult sheep maintained on a mixed ration containing 65:35% Pala (Ziziphus numularia) leaf: concentrate. Twenty-one adult Malpura sheep divided into three equal groups (DF, RF and F) were either defaunated by oral administration of sodium lauryl sulfate at the rate of 8 g/100 kg body weight (DF), or defaunated and again refaunated (RF), or maintained faunated (F). Daily dry matter intake was similar in defaunated, refaunated and faunated sheep. However, digestibility of cell wall and cell wall contents (NDF, ADF and cellulose) were lower (P < 0.01) in defaunated than refaunated and faunated sheep. Irrespective of the presence or absence of rumen protozoa, daily intake of DCP and DE were similar in the three experimental groups. Even with similar DM, DCP and DE intake, N-retention, blood glucose level, ruminal concentration of total VFA and total-N were higher (P < 0.01), while rumen pH and NH₃-N concentration were lower (P < 0.01) in defaunated sheep. Ruminal activity of amylase, xylanase, protease and urease enzymes were not influenced by presence or absence of ciliate protozoa. However, carboxymethyl cellulase enzyme activity was lower (P < 0.01) in the rumen of defaunated sheep. The total and differential counts of rumen protozoa were similar in refaunated and faunated sheep indicating lack of residual toxic effect of sodium lauryl sulfate. It is concluded that absence of ciliate protozoa increased ruminal TVFA, total-N with lower NH₃-N concentration and fibre digestibility in sheep. Moreover, sodium lauryl sulfate was fully effective for complete removal of rumen ciliate protozoa and successfully defaunated the sheep.     

Effect of disodium fumarate on microbial abundance,ruminal fermentation and methane emission in goats under different forage : concentrate ratios

This study investigated the effects of disodium fumarate (DF) on methane emission, ruminal fermentation and microbial abundance in goats under different forage (F) : concentrate (C) ratios and fed according to maintenance requirements. Four ruminally fistulated, castrated male goats were used in a 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments and the main factors being the F : C ratios (41 : 59 or 58 : 42) and DF supplementation (0 or 10 g/day). DF reduced methane production (P < 0.05) on average by 11.9%, irrespective of the F : C ratio. The concentrations of total volatile fatty acids, acetate and propionate were greater in the rumen of goats supplemented with DF (P < 0.05), whereas the abundance of methanogens was lower (P < 0.05). In high-forage diets, the abundance of Selenomonas ruminantium, a fumarate-reducing bacterium, was greater in the rumen of goats supplemented with DF. The abundance of fungi, protozoa, Ruminococus flavefaciens and Fibrobacter succinogenes were not affected by the addition of DF. Variable F : C ratios affected the abundance of methanogens, fungi and R. flavefaciens (P < 0.05), but did not affect methane emission. The result implied that DF had a beneficial effect on the in vivo rumen fermentation of the goats fed diets with different F : C ratios and that this effect were not a direct action on anaerobic fungi, protozoa and fibrolytic bacteria, the generally recognized fiber-degrading and hydrogen-producing microorganisms, but due to the stimulation of fumarate-reducing bacteria and the depression of methanogens.     

Dietary starch and rhubarb supplement increase ruminal dissolved hydrogen without altering rumen fermentation and methane emissions in goats

Hydrogen is an important intermediate that is produced during carbohydrate fermentation to volatile fatty acid and utilized by methanogens to produce methane in the rumen. Ruminal volatile fatty acid and dissolved methane concentrations are more than 500 times greater than dissolved hydrogen concentration. Therefore, we hypothesized that dissolved hydrogen might have a higher sensitivity in response to dietary changes compared with volatile fatty acid and dissolved methane. Using goats, we investigated the effects of increasing dietary starch content (maize replaced with wheat bran) and supplementing with rhubarb rhizomes and roots on the relationships among dissolved hydrogen, dissolved methane and other fermentation end products. The study was conducted in a replicated 4×4 Latin square with a 2×2 factorial arrangement of four treatments: two starch levels (220 v. 320 g/kg dry matter (DM)), without and with rhubarb supplement (0% v. 2.8% of total mixed ration). Increased dietary starch and rhubarb supplementation did not alter volatile fatty acid concentrations or methane emissions in terms of g/day, g/g DM intake and g/g organic matter digested. However, goats fed the high-starch diet had greater dissolved hydrogen (P=0.005) and relative abundance of Selenomonas ruminantium (P<0.01), and lower (P=0.02) copy number of protozoa than those fed the low-starch diet. Rhubarb increased ruminal dissolved H₂ (P=0.03) and total volatile fatty acid concentration (P<0.001), but decreased copies of bacteria (P=0.002). In conclusion, dissolved hydrogen appears to be more sensitive to dietary changes with starch content and rhubarb supplementation, when compared with volatile fatty acid concentrations and methane production.     

Methane production and diurnal variation measured in dairy cows and predicted from fermentation pattern and nutrient or carbon flow

Many feeding trials have been conducted to quantify enteric methane (CH₄) production in ruminants. Although a relationship between diet composition, rumen fermentation and CH₄ production is generally accepted, the efforts to quantify this relationship within the same experiment remain scarce. In the present study, a data set was compiled from the results of three intensive respiration chamber trials with lactating rumen and intestinal fistulated Holstein cows, including measurements of rumen and intestinal digestion, rumen fermentation parameters and CH₄ production. Two approaches were used to calculate CH₄ from observations: (1) a rumen organic matter (OM) balance was derived from OM intake and duodenal organic matter flow (DOM) distinguishing various nutrients and (2) a rumen carbon balance was derived from carbon intake and duodenal carbon flow (DCARB). Duodenal flow was corrected for endogenous matter, and contribution of fermentation in the large intestine was accounted for. Hydrogen (H₂) arising from fermentation was calculated using the fermentation pattern measured in rumen fluid. CH₄ was calculated from H₂ production corrected for H₂ use with biohydrogenation of fatty acids. The DOM model overestimated CH₄/kg dry matter intake (DMI) by 6.1% (R²=0.36) and the DCARB model underestimated CH₄/kg DMI by 0.4% (R²=0.43). A stepwise regression of the difference between measured and calculated daily CH₄ production was conducted to examine explanations for the deviance. Dietary carbohydrate composition and rumen carbohydrate digestion were the main sources of inaccuracies for both models. Furthermore, differences were related to rumen ammonia concentration with the DOM model and to rumen pH and dietary fat with the DCARB model. Adding these parameters to the models and performing a multiple regression against observed daily CH₄ production resulted in R² of 0.66 and 0.72 for DOM and DCARB models, respectively. The diurnal pattern of CH₄ production followed that of rumen volatile fatty acid (VFA) concentration and the CH₄ to CO₂ production ratio, but was inverse to rumen pH and the rumen hydrogen balance calculated from 4×(acetate+butyrate)/2×(propionate+valerate). In conclusion, the amount of feed fermented was the most important factor determining variations in CH₄ production between animals, diets and during the day. Interactions between feed components, VFA absorption rates and variation between animals seemed to be factors that were complicating the accurate prediction of CH₄. Using a ruminal carbon balance appeared to predict CH₄ production just as well as calculations based on rumen digestion of individual nutrients.     

Protozoa populations are ecosystem engineers that shape prokaryotic community structure and function of the rumen microbial ecosystem

Unicellular eukaryotes are an integral part of many microbial ecosystems where they interact with their surrounding prokaryotic community—either as predators or as mutualists. Within the rumen, one of the most complex host-associated microbial habitats, ciliate protozoa represent the main micro-eukaryotes, accounting for up to 50% of the microbial biomass. Nonetheless, the extent of the ecological effect of protozoa on the microbial community and on the rumen metabolic output remains largely understudied. To assess the role of protozoa on the rumen ecosystem, we established an in-vitro system in which distinct protozoa sub-communities were introduced to the native rumen prokaryotic community. We show that the different protozoa communities exert a strong and differential impact on the composition of the prokaryotic community, as well as its function including methane production. Furthermore, the presence of protozoa increases prokaryotic diversity with a differential effect on specific bacterial populations such as Gammaproteobacteria, Prevotella and Treponema. Our results suggest that protozoa contribute to the maintenance of prokaryotic diversity in the rumen possibly by mitigating the effect of competitive exclusion between bacterial taxa. Our findings put forward the rumen protozoa populations as potentially important ecosystem engineers for future microbiome modulation strategies.Subject terms: Microbial ecology, Food webs     

Use of short-term breath measures to estimate daily methane production by cattle

Methods to measure enteric methane (CH₄) emissions from individual ruminants in their production environment are required to validate emission inventories and verify mitigation claims. Estimates of daily methane production (DMP) based on consolidated short-term emission measurements are developing, but method verification is required. Two cattle experiments were undertaken to test the hypothesis that DMP estimated by averaging multiple short-term breath measures of methane emission rate did not differ from DMP measured in respiration chambers (RC). Short-term emission rates were obtained from a GreenFeed Emissions Monitoring (GEM) unit, which measured emission rate while cattle consumed a dispensed supplement. In experiment 1 (Expt. 1), four non-lactating cattle (LW=518 kg) were adapted for 18 days then measured for six consecutive periods. Each period consisted of 2 days of ad libitum intake and GEM emission measurement followed by 1 day in the RC. A prototype GEM unit releasing water as an attractant (GEM water) was also evaluated in Expt. 1. Experiment 2 (Expt. 2) was a larger study based on similar design with 10 cattle (LW=365 kg), adapted for 21 days and GEM measurement was extended to 3 days in each of the six periods. In Expt. 1, there was no difference in DMP estimated by the GEM unit relative to the RC (209.7 v. 215.1 g CH₄/day) and no difference between these methods in methane yield (MY, 22.7 v. 23.7 g CH₄/kg of dry matter intake, DMI). In Expt. 2, the correlation between GEM and RC measures of DMP and MY were assessed using 95% confidence intervals, with no difference in DMP or MY between methods and high correlations between GEM and RC measures for DMP (r=0.85; 215 v. 198 g CH₄/day SEM=3.0) and for MY (r=0.60; 23.8 v. 22.1 g CH₄/kg DMI SEM=0.42). When data from both experiments was combined neither DMP nor MY differed between GEM- and RC-based measures (P>0.05). GEM water-based estimates of DMP and MY were lower than RC and GEM (P<0.05). Cattle accessed the GEM water unit with similar frequency to the GEM unit (2.8 v. 3.5 times/day, respectively) but eructation frequency was reduced from 1.31 times/min (GEM) to once every 2.6 min (GEM water). These studies confirm the hypothesis that DMP estimated by averaging multiple short-term breath measures of methane emission rate using GEM does not differ from measures of DMP obtained from RCs. Further, combining many short-term measures of methane production rate during supplement consumption provides an estimate of DMP, which can be usefully applied in estimating MY.     

The use of direct-fed microbials for mitigation of ruminant methane emissions: a review

Concerns about the environmental effect and the economic burden of methane (CH₄) emissions from ruminants are driving the search for ways to mitigate rumen methanogenesis. The use of direct-fed microbials (DFM) is one possible option to decrease CH₄ emission from ruminants. Direct-fed microbials are already used in ruminants mainly to increase productivity and to improve health, and are readily accepted by producers and consumers alike. However, studies on the use of DFM as rumen CH₄ mitigants are scarce. A few studies using Saccharomyces cerevisiae have shown a CH₄-decreasing effect but, to date, there has not been a systematic exploration of DFM as modulators of rumen methanogenesis. In this review, we explored biochemical pathways competing with methanogenesis that, potentially, could be modulated by the use of DFM. Pathways involving the redirection of H₂ away from methanogenesis and pathways producing less H₂ during feed fermentation are the preferred options. Propionate formation is an example of the latter option that in addition to decrease CH₄ formation increases the retention of energy from the diet. Homoacetogenesis is a pathway using H₂ to produce acetate, however up to now no acetogen has been shown to efficiently compete with methanogens in the rumen. Nitrate and sulphate reduction are pathways competing with methanogenesis, but the availability of these substances in the rumen is limited. Although there were studies using nitrate and sulphate as chemical additives, use of DFM for improving these processes and decrease the accumulation of toxic metabolites needs to be explored more. There are some other pathways such as methanotrophy and capnophily or modes of action such as inhibition of methanogens that theoretically could be provided by DFM and affect methanogenesis. We conclude that DFM is a promising alternative for rumen methane mitigation that should be further explored for their practical usage.     

Dietary fat sources affect feed intake,digestibility, rumen microbial populations,energy partition and methane emissions in different beef cattle genotypes

The mitigation of enteric methane emission in beef cattle production is important for reducing feed energy loss and increasing environmental sustainability. The main objective of this study was to evaluate the effect of different oilseeds included in fermented total mixed rations (whole soyabean seed (SBS, control), whole kapok seed (KPS) and cracked oil palm fruit (OPF)) on feed intake, digestibility, rumen microbial populations, energy partition and methane emissions in different cattle genotypes (Charolais crossbred v. Japanese Black crossbred). Three Charolais crossbred and three Japanese Black crossbred bulls were studied in a replicated 3×3 Latin square experimental design; genotypes were analysed in separate squares including three periods of 21 days each and three dietary oilseed treatments fed ad libitum. The cattle were placed in a metabolic cage equipped with a ventilated head box respiration system for evaluating digestibility and energy balance. As compared with Charolais crossbred individuals, Japanese Black crossbred bulls showed consistently lower dry matter intake (15.5%, P<0.01), metabolisable energy (ME) intake (13.8%, P<0.05), ME requirement for maintenance (10.3%; 386 v. 430 kJ/kg metabolic BW, respectively), faeces energy loss (19.2%, P<0.001) and enteric methane emissions (18.5%, P<0.001). However, these two genotypes did not differ in energy retention (ER) (P=0.80). Among the three dietary oilseed treatments, OPF exhibited higher NDF intake (P<0.01) and digestibility (P<0.01), which was associated with a larger (P<0.05) total number of bacteria in the rumen. In addition, the OPF diet contributed to higher ME intake and ER than that of the KPS diet, whereas the SBS diet presented intermediate values (P<0.05). The methane conversion factor of these crossbreds was not significantly affected by genotype (P>0.05) or diet (P>0.05) under the experimental conditions and ranged from 5.8% to 6.0% of gross energy intake. This value is lower than that reported by the Intergovernmental Panel on Climate Change (6.5%) for cattle fed with low-quality crop residues or by-products. Thus, our results imply that the Japanese Black crossbred cattle consume less feed and emits less enteric methane than the Charolais crossbred does, mainly owing to its lower ME requirement for maintenance. The OPF diet could be used to replace SBS for high beef production, although further studies are required to evaluate their application across a wide range of beef production systems.     

Network analysis and functional estimation of the microbiome reveal the effects of cashew nut shell liquid feeding on methanogen behaviour in the rumen

The effects of cashew nut shell liquid (CNSL) feeding on the methane (CH₄) emission and the ruminal microbiome of Lai Sind beef cattle were investigated. Changes in the methane production and rumen microbiome by CNSL feeding were monitored by a respiration chamber and 16S rRNA gene amplicon sequencing respectively. The results demonstrated that CNSL feeding mitigated 20.2%–23.4% of the CH₄ emission in vivo without apparent adverse effects on feed intake and feed digestibility. The rumen fluid analysis revealed a significant increase in the proportion of propionate in the total short-chain fatty acids. The relative abundance of methanogen (order Methanobacteriales) decreased significantly, indicating the direct inhibitory effect of CNSL on methanogens. The predicted function of the rumen microbiome indicated that carbohydrate and lipid metabolisms including propionate production were upregulated by CNSL feeding, whereas CH₄ metabolism was downregulated. A network analysis revealed that methanogen changed its partner bacteria after CNSL feeding. The δ¹³C of CH₄ ranged from −74.2‰ to −66.6‰ with significant fluctuation by CNSL feeding, in agreement with the shift of the rumen microbiome. Our findings demonstrate that CNSL feeding can mitigate the CH₄ emission from local cattle production systems in South-East Asia by modifying the rumen microbiome and its function.     

Diversity of rumen microbiota using metagenome sequencing and methane yield in Indian sheep fed on straw and concentrate diet

An in vivo study aiming to investigate the rumen methanogens community structure was conducted in Mandya sheep fed on straw and concentrate diet. The ruminal fluid samples were collected and processed for unravelling the rumen microbiota and methanogens diversity. Further, the daily enteric methane emission and methane yield was also quantified using the SF₆ tracer technique. Results indicated that the Bacteroidetes (～57%) and Firmicutes (25%) were two prominent affiliates of the bacterial community. Archaea represented about 2.5% of the ruminal microbiota. Methanobacteriales affiliated methanogens were the most prevalent in sheep rumen. The study inveterate that the ruminal archaea community in sheep is composed of 9 genera and 18 species. Methanobrevibacter represented the largest genus of the archaeome, while methylotrophs genera constituted only 13% of the community. Methanobrevibacter gottschalkii was the prominent methanogen, and Methaobrevibacter ruminantium distributed at a lower frequency (～2.5%). Among Methanomassiliicoccales, Group 12 sp. ISO4-H5 constituted the most considerable fraction (～11%). KEGG reference pathway for methane metabolism indicated the formation of methane through hydrogenotrophic and methylotrophic pathways, whereas the acetoclastic pathway was not functional in sheep. The enteric methane emission and methane yield was 19.7 g/d and 20.8 g/kg DMI, respectively. Various species of Methanobrevibacter were differently correlated, and the distribution of hydrogenotrophic methanogens mainly explained the variability in methane yield between the individual sheep. It can be inferred from the study that the hydrogenotrophic methanogens dominate the rumen archaeal community in sheep and methylotrophic/aceticlastic methanogens represent a minor fraction of the community. Further studies are warranted for establishing the metabolic association between the prevalent hydrogenotrophs and methylotrophs to identify the key reaction for reducing methane emission.     

Accumulation of Reserve Carbohydrate by Rumen Protozoa and Bacteria in Competition for Glucose

The aim of this study was to determine if rumen protozoa could form large amounts of reserve carbohydrate compared to the amounts formed by bacteria when competing for glucose in batch cultures. We separated large protozoa and small bacteria from rumen fluid by filtration and centrifugation, recombined equal protein masses of each group into one mixture, and subsequently harvested (reseparated) these groups at intervals after glucose dosing. This method allowed us to monitor reserve carbohydrate accumulation of protozoa and bacteria individually. When mixtures were dosed with a moderate concentration of glucose (4.62 or 5 mM) (n = 2 each), protozoa accumulated large amounts of reserve carbohydrate; 58.7% (standard error of the mean [SEM], 2.2%) glucose carbon was recovered from protozoal reserve carbohydrate at time of peak reserve carbohydrate concentrations. Only 1.7% (SEM, 2.2%) was recovered in bacterial reserve carbohydrate, which was less than that for protozoa (P < 0.001). When provided a high concentration of glucose (20 mM) (n = 4 each), 24.1% (SEM, 2.2%) of glucose carbon was recovered from protozoal reserve carbohydrate, which was still higher (P = 0.001) than the 5.0% (SEM, 2.2%) glucose carbon recovered from bacterial reserve carbohydrate. Our novel competition experiments directly demonstrate that mixed protozoa can sequester sugar away from bacteria by accumulating reserve carbohydrate, giving protozoa a competitive advantage and stabilizing fermentation in the rumen. Similar experiments could be used to investigate the importance of starch sequestration.     

Assessing the impact of rumen microbial communities on methane emissions and production traits in Holstein cows in a tropical climate

The evaluation of how the gut microbiota affects both methane emissions and animal production is necessary in order to achieve methane mitigation without production losses. Toward this goal, the aim of this study was to correlate the rumen microbial communities (bacteria, archaea, and fungi) of high (HP), medium (MP), and low milk producing (LP), as well as dry (DC), Holstein dairy cows in an actual tropical production system with methane emissions and animal production traits. Overall, DC cows emitted more methane, followed by MP, HP and LP cows, although HP and LP cow emissions were similar. Using next-generation sequencing, it was found that bacteria affiliated with Christensenellaceae, Mogibacteriaceae, S24-7, Butyrivibrio, Schwartzia, and Treponema were negatively correlated with methane emissions and showed positive correlations with digestible dry matter intake (dDMI) and digestible organic matter intake (dOMI). Similar findings were observed for archaea in the genus Methanosphaera. The bacterial groups Coriobacteriaceae, RFP12, and Clostridium were negatively correlated with methane, but did not correlate with dDMI and dOMI. For anaerobic fungal communities, no significant correlations with methane or animal production traits were found. Based on these findings, it is suggested that manipulation of the abundances of these microbial taxa may be useful for modulating methane emissions without negatively affecting animal production.     

Effect of coconut oil and defaunation treatment on methanogenesis in sheep

The present study was conducted to evaluate in vivo the role of rumen ciliate protozoa with respect to the methane-suppressing effect of coconut oil. Three sheep were subjected to a 2 x 2 factorial design comprising two types of dietary lipids (50 g x kg(-1) coconut oil vs. 50 g x kg(-1) rumen-protected fat) and defaunation treatment (with vs. without). Due to the defaunation treatment, which reduced the rumen ciliate protozoa population by 94% on average, total tract fibre degradation was reduced but not the methane production. Feeding coconut oil significantly reduced daily methane release without negatively affecting the total tract nutrient digestion. Compared with the rumen-protected fat diet, coconut oil did not alter the energy retention of the animals. There was no interaction between coconut oil feeding and defaunation treatment in methane production. An interaction occurred in the concentration of methanogens in the rumen fluid, with the significantly highest values occurring when the animals received the coconut oil diet and were subjected to the defaunation treatment. Possible explanations for the apparent inconsistency between the amount of methane produced and the concentration of methane-producing microbes are discussed. Generally, the present data illustrate that a depression of the concentration of ciliate protozoa or methanogens in rumen fluid cannot be used as a reliable indicator for the success of a strategy to mitigate methane emission in vivo. The methane-suppressing effect of coconut oil seems to be mediated through a changed metabolic activity and/or composition of the rumen methanogenic population.     

Effects of protozoa on Methane production in rumen and hindgut of calves around time of weaning

Effects of the presence or absence of ciliate protozoa on methanogenesis in the rumen and hindgut were investigated in young calves during a 7-week period. Ten Holstein calves, aged 7 days, were divided in two groups (n = 5) and fed an increasing amount of a commercial milk replacer and small amounts of a calves starter. One group was inoculated with ciliate fauna on two occasions, week 5 and 6, while the second remained ciliate-free. The absence of protozoa in the rumen decreased rumen empty weight ( ? 23%, P < 0.01), and rumen pool size of N ( ? 36%, P < 0.01) and crude fat ( ? 37%, P < 0.05). Rumen bacteria of non-faunated calves contained a higher proportion of total amino acid-N per 16 g N ( + 3%, P < 0.01) and D-alanine-N per 16 g N ( + 13%, P < 0.05) compared to faunated calves. Further results contain a reference for a higher bacterial mass in the ciliate-free rumen with an increased number of bacteria adherent to rumen mucosa. The CH₄ production in the rumen increased exponentially with the increase in protozoa population size (R² = 0.68). In presence of 46 · 10⁴ protozoa per ml rumen fluid, the in vitro CH₄ production of rumen fluid per mol total VFA was about 34% higher in faunated than in non-faunated calves (P < 0.001). Hydrogen (2H) recovery of rumen fermentation was positively correlated (R² = 0.55) to the CH₄ production rate. Methanogens were attached on rumen mucosa. Methanogenesis, induced by rumen mucosa attached bacteria, was stimulated by ruminal protozoa. In the absence of protozoa in the rumen, the acetate - propionate ratio and butyrate proportion of VFA were reduced. In vivo in the absence of protozoa not only the whole animal CH₄ production ( ? 30%, P < 0.05) but also the digestibility of carbohydrates ( ? 4%, P < 0.05) was reduced. Thereby no difference was observed in the intake of ME per kg DM between the groups. In conclusion, the methanogenesis in the rumen, but not in hindgut, is associated with the development of the ruminal protozoa population. The level of methanogenesis (mol/mol VFA) in the hindgut amounts to 20% of the ruminal methanogenesis.     

Rumen methanogen and protozoal communities of Tibetan sheep and Gansu Alpine Finewool sheep grazing on the Qinghai–Tibetan Plateau,China

Background

Tibetan sheep (TS) and Gansu Alpine Finewool sheep (GS) are both important plateau sheep raised and fed on the harsh Qinghai–Tibetan Plateau, China. Rumen methanogen and protozoal communities of plateau sheep are affected by their hosts and living environments, and play important roles in ruminant nutrition and greenhouse gas production. However, the characteristics, differences, and associations of these communities remain largely uncharacterized.

Results

The rumen methanogen and protozoal communities of plateau sheep were investigated by 16S/18S rRNA gene clone libraries. The predominant methanogen order in both sheep species was Methanobacteriales followed by Methanomassiliicoccales, which is consistent with those seen in global ruminants. However, the most dominant species was Methanobrevibacter millerae rather than Methanobrevibacter gottschalkii seen in most ruminants. Compared with GS and other ruminants, TS have more exclusive operational taxonomic units and a lower proportion (64.5%) of Methanobrevibacter. The protozoa were divided into Entodiniomorphida and Vestibuliferida, including nine genera and 15 species. The proportion of holotrich protozoa was much lower (1.1%) in TS than ordinary sheep. The most predominant genus was Entodinium (70.0%) in TS and Enoploplastron (48.8%) in GS, while the most common species was Entodinium furca monolobum (43.9%) and Enoploplastron triloricatum (45.0%) in TS and GS, respectively; Entodinium longinucleatum (22.8%) was only observed in TS. LIBSHUFF analysis indicated that the methanogen communities of TS were significantly different from those of GS, but no significant differences were found in protozoal communities.

Conclusion

Plateau sheep have coevolved with unique rumen methanogen and protozoal communities to adapt to harsh plateau environments. Moreover, the host appears to have a greater influence on rumen methanogen communities than on rumen protozoal communities. The observed associations of methanogens and protozoa, together with the findings of previous studies on methane emissions from ruminant livestock, revealed that the lower proportion of Methanobrevibacter and holotrich protozoa may be responsible for the lower methane emission of TS. These findings facilitate our understanding of the rumen microbial ecosystem in plateau sheep, and could help the development of new strategies to manipulate rumen microbes to improve productivity and reduce the emission of greenhouse gases.

     

Feed digestion,rumen fermentation and blood biochemical constituents in Malpura rams fed a complete feed-block diet with the inclusion of tree leaves

This experiment assayed the influence of the inclusion of dried Azardirachta indica, Albizzia lebbek or Ailanthus excelsa leaves in pearl millet stover-based complete feed block diets on feed intake, nutrient utilization, rumen fermentation characteristics, ciliate protozoa population and blood biochemical constituents in adult Malpura sheep. Complete feed blocks were formulated to have roughage-to-concentrate ratio of 70:30. Pearl millet stover (PMS) was used as basal roughage; 30 parts of pearl millet stover was replaced with dried leaves either of Azardirachta indica (NL), Albizzia lebbek (SL) or Ailanthus excelsa (AL). Twelve hogget Malpura rams, divided into four equal groups, were offered one of the four dietary treatments. A feeding-cum-metabolic trial was conducted to assess nutrient utilization. Rumen liquor samples were collected at 0, 3, 6, 12, 18 and 24 h post-feeding to assess rumen fermentation pattern and ciliate protozoa population. Inclusion of dried leaves in PMS-based diets improved CP and DCP content. Dietary DCP was low (P < 0.01) in PMS (8.52%) compared to tree leaves (9.77–11.59%) diets. AL and NL diets had higher (P < 0.05) DCP than the SL diet. The inclusion of tree leaves did not influence organic matter, crude protein or cellulose digestibility, but depressed dry matter, NDF, ADF and energy digestibility. DE content was also lower in tree leave diets. Inclusion of tree leaves improved CP and DCP intake, but DE intake and nitrogen utilization did not change. The pH of rumen liquor (SRL) was low (6.99, P < 0.05), but total nitrogen (52.9 mg/dl SRL) and NH₃-nitrogen (9.34 mg/dl SRL) concentrations were higher (P < 0.01) in the AL diet. TVFA concentrations and ciliate protozoa population were similar on the four diets. Animals in the four groups had the desired concentration of rumen metabolites required for fibrous diets. Complete feed-block feeding provided a constant nutrient supply to rumen microbes that optimise rumen fermentation. Blood biochemical constituents did not change due to the inclusion of tree leaves. Therefore, tree leaves can be included with roughage-based feeding to improve the protein nutrition status of ruminants. Further studies are required to assess the negative influence of tree leaves on digestibility.     

Effect of wheat hay particle size and replacement of wheat hay with wheat silage on rumen pH,rumination and digestibility in ruminally cannulated non-lactating cows

This study examined the effects on intake, diurnal rumen pH changes, rumination and digestibility of feeding ruminally cannulated non-lactating cows in a Latin square design (four cows×four periods) with four total mixed rations (TMRs) typical for lactating cows. TMRs were based on: long wheat hay or short wheat hay, wheat silage or wheat silage+1.5% NaHCO₃ buffer, as the sole roughage source (30% of TMR dry matter (DM)). The level of physically effective NDF remaining above the 8 mm screen (peNDF) was similar in the long hay and silage-based TMRs (9.45% to 9.64% of DM) and lower in the short hay TMR (7.47% of DM). The four TMRs were offered individually at 95% of ad libitum intake to avoid orts within 24 h. Cows fed long hay consumed less DM than the short hay and silage groups (9.6 v. 10.5 and 10.8 kg/day, respectively) and sorted against large hay particles at 12 h post-feeding. Under the limitations of this study (non-lactating cows fed at restricted intake) short hay TMR prevented sorting within 12 h post-feeding, encouraged rumination per kg peNDF ingested, and had higher average rumen pH (6.24), whereas preventing sub acute ruminal acidosis (SARA, defined as pH<5.8 for at least 5 h/day). In contrast, the long hay and silage-based groups were under SARA. In vitro methane production of rumen fluid was higher in the hay-fed cows than in their silage-fed counterparts, and in all treatments lower at 1 h pre-feeding than at 6 h post-feeding. In vivo DM and NDF digestibility were similar for the short hay and silage TMRs, and higher than those of the long hay TMR. Under the conditions of this study, addition of 1.5% buffer to the wheat silage TMR had no effect on intake, rumen pH, creation of SARA and digestibility.     

Influence of natural magnesium sources on the in vitro fermentation and protozoan population in the rumen fluid collected from sheep

The objective of this experiment was to determine the effect of two types of caustic calcinated magnesite (caustic magnesite (CM) and Agromag (AG)) upon the end products of in vitro fermentation (total gas, methane, total and individual fatty acids, and VFA) and protozoan population in the rumen fluid collected from sheep. Both magnesium additives (CM and AG) as natural products in the dose of 0.01 g were added to the fermentation bottles containing rumen inoculum from sheep and different substrates. Meadow hay (MH), wheat straw (WS), amorphous cellulose (AC) and barley grain (BG) were used as substrates and incubated with the buffered rumen fluid using an in vitro gas measuring technique during 72 h of incubation. The rumen protozoa, Entodinium spp., Trichostomatids and large Entodiniomorphids and the total protozoan concentration were counted after 24 h of incubation. The methane production was significantly decreased with CM or AG, respectively, by 58 or 62% (MH), by 65% (WS), by 52% (AC) and by 58% (BG). The total VFA concentration was significantly lower compared to control for CM plus MH, WS, AC, BG and AG plus WS. The total VFA concentration was significantly higher compared to control for AG plus AC. The effect of the both additives on ciliate population was not uniform and depended on the substrates used and protozoan type. Ciliate population was significantly increased in Entodinium spp. (AG plus BG) and Diploplastron affinae (CM or AG plus BG) compared to control. Tested additives significantly decreased population of Entodinium spp. (AG plus MH or AC), Dasytricha ruminantium (AG plus AC), Ophryoscolex c. tricoronatus, Eremoplastron dilobum and Polyplastron multivesiculatum (CM or AG plus BG). It can be concluded that both natural magnesium sources influenced rumen fermentation patterns and protozoan population in vitro depending on the type of the substrate used; therefore, the relative efficacy of individual tested additive cannot be determined from these experiments. In vivo experiments are required in future.