Dairy products as sources of methanogens for humans

Methanogens are detected in human gut from the first moments of life and there is a diversification of methanogens during infancy. However, the sources of acquisition of methanogens are not well elucidated. We therefore investigated 56 dairy products as potential sources of methanogens by applying molecular biology. In the presence of negative controls, we obtained an overall prevalence of methanogens in 85.7% (48/56) of samples by real-time PCR. Further PCR-sequencing identified 73.2% (41/56) of Methanobrevibacter smithii. We also found for the first time in dairy products 1.8% (1/56) of Methanobrevibacter oralis, 7.1% (4/56) of Methanobrevibacter millerae, 1.8% (1/56) of Methanobrevibacter ruminantium, 1.8% (1/56) of Methanocorpusculum sp. We observed a significant presence (p-value=0.001) of methanogens in fermented dairy products compared to unfermented dairy products. This study gives credit to the fact that dairy products could be considered as a source of methanogens for humans, especially for children.

The Ability of Hop Extracts to Reduce the Methane Production of Methanobrevibacter ruminantium

Background. Methane emissions from agriculture are responsible for over 40% of the world’s greenhouse gas emissions. In the past, antibiotics were used to control methane production by animals, but concerns over the emergence and spread of antibiotic-resistant bacteria to humans have prompted a search for alternative approaches. Hops are the flowers of the hop plant Humulus lupulus. They have been used to feed cattle for many years and are known to contain antibacterial compounds, and their extracts have been shown to kill members of the Mycobacterium spp including Mycobacterium bovis, the causative agent of bovine tuberculosis as well as a number of human pathogens. In this study, hop extracts were studied for their ability to inhibit methane production from Methanobrevibacter ruminantium, a major methane-producing archaeon found in the rumen of cattle. Methods. Methanobrevibacter ruminantium M1T (DSM 1093) was grown at 37°C for 30 days, and the amount of methane produced at different time points during this period was measured using gas chromatography. The archaeon was exposed to commercial hop extracts (tetra-hydro-iso-alpha acid and beta acid) and to aqueous hop extracts of a range of hop variants, and their effect on methane production was determined. Results. All of the extracts reduced the level of methane production of M. ruminantium over the 30-day period compared to the negative control (sterile distilled water). The commercial hop extracts were the most effective at inhibiting methane production over the course of the experiment in contrast to the aqueous extracts, which showed a gradual reduction of inhibition with time. Conclusions. Hops contain compounds which inhibit methane production. Given that hops can be safely fed to cattle, this raises the possibility of rationally designing a feed strategy which could reduce greenhouse gas emissions and protect against bovine tuberculosis. This study recommends that further research be undertaken to further identifying bioactive components from hops and their efficacy against a range of archaea.

Factors influencing Euryarchaeal gut methanogens distribution in dairy cattle in smallholding farms

Guts of ruminants contain symbiotic domains (Eubacteria, Archaea and Eukarya) that aid in the breakdown of consumed carbohydrates from plants to simple molecules that can be absorbed into the ruminant’s bloodstream. Methanogenesis occurs during the gut fermentation and methane gas is released in the final step of biomass degradation from the fermentation chambers. The Archaea that play a major role critical for methane emissions are methanogens and are found freely in the ruminants’ gut. Methane production from ruminants has attracted global attention due to their input on the Green House Gases effect, contribution to global warming and negative effects on farmers’ productivity. The objective of this study was to determine the factors contributing to the methanogens’ gut distribution in dairy cows from smallholder farms using next generation sequencing techniques. A total of 48 samples from smallholding dairy farms were used during this study and were collected from Kenya (Kiambu county) and Tanzania (Lushoto and Rungwe). The collected data samples from the experimental animals were from both the rumen fluid (6) and fecal (42). Samples were analyzed using metagenomic approaches and statistical analysis was undertaken using IBM SPSS statistics software version 28.0.0.0. Results showed that the gut site along the gastrointestinal tract and the feeding regime significantly contributed to the distribution and presence of various methanogenic species (P<0.1). The herd and the genotype had no statistical effect. A total of 12 families were identified. The family Methanobacteriaceae was identified with the leading number (8) of the methanogenic species. A third of the identified families showed presence for at least two methanogenic species with Methanobrevibacter ruminantium being abundant. For proper curbing mechanisms, efforts to reduce methane release should be channeled to the whole gastrointestinal tract and advanced studies carried out on any potential interspecies presence facilitation and/or elimination.

Effects of cofD gene knock-out on the methanogenesis of Methanobrevibacter ruminantium

This study aimed to investigate the effects of cofD gene knock-out on the synthesis of coenzyme F420 and production of methane in Methanobrevibacter ruminantium (M. ruminantium). The experiment successfully constructed a cofD gene knock-out M. ruminantium via homologous recombination technology. The results showed that the logarithmic phase of mutant M. ruminantium (12 h) was lower than the wild-type (24 h). The maximum biomass and specific growth rate of mutant M. ruminantium were significantly lower (P < 0.05) than those of wild-type, and the maximum biomass of mutant M. ruminantium was approximately half of the wild-type; meanwhile, the proliferation was reduced. The synthesis amount of coenzyme F420 of M. ruminantium was significantly decreased (P < 0.05) after the cofD gene knock-out. Moreover, the maximum amount of H2 consumed and CH4 produced by mutant were 14 and 2% of wild-type M. ruminantium respectively. In conclusion, cofD gene knock-out induced the decreased growth rate and reproductive ability of M. ruminantium. Subsequently, the synthesis of coenzyme F420 was decreased. Ultimately, the production capacity of CH4 in M. ruminantium was reduced. Our research provides evidence that cofD gene plays an indispensable role in the regulation of coenzyme F420 synthesis and CH4 production in M. ruminantium.

Effects of long-acting, broad spectra anthelmintic treatments on the rumen microbial community compositions of grazing sheep

Anthelmintic treatment of adult ewes is widely practiced to remove parasite burdens in the expectation of increased ruminant productivity. However, the broad activity spectra of many anthelmintic compounds raises the possibility of impacts on the rumen microbiota. To investigate this, 300 grazing ewes were allocated to treatment groups that included a 100-day controlled release capsule (CRC) containing albendazole and abamectin, a long-acting moxidectin injection (LAI), and a non-treated control group (CON). Rumen bacterial, archaeal and protozoal communities at day 0 were analysed to identify 36 sheep per treatment with similar starting compositions. Microbiota profiles, including those for the rumen fungi, were then generated for the selected sheep at days 0, 35 and 77. The CRC treatment significantly impacted the archaeal community, and was associated with increased relative abundances of Methanobrevibacter ruminantium, Methanosphaera sp. ISO3-F5, and Methanomassiliicoccaceae Group 12 sp. ISO4-H5 compared to the control group. In contrast, the LAI treatment increased the relative abundances of members of the Veillonellaceae and resulted in minor changes to the bacterial and fungal communities by day 77. Overall, the anthelmintic treatments resulted in few, but highly significant, changes to the rumen microbiota composition.

Inoculation with rumen fluid in early life accelerates the rumen microbial development and favours the weaning process in goats

Background Newborn ruminants possess an underdeveloped rumen which is colonized by microorganisms acquired from adult animals and the surrounding environment. This microbial transfer can be limited in dairy systems in which newborns are separated from their dams at birth. This study explores whether the direct inoculation of fresh or autoclaved rumen fluid from adult goats to newborn kids has a beneficial effect on rumen microbial development and function. Results Repetitive inoculation of young kids with fresh rumen fluid from adult goats adapted to forage (RFF) or concentrate diets (RFC) accelerated microbial colonization of the rumen during the pre-weaning period leading to high protozoal numbers, a greater diversity of bacterial (+ 234 OTUs), methanogens (+ 6 OTUs) and protozoal communities (+ 25 OTUs) than observed in control kids (CTL) without inoculation. This inoculation also increased the size of the core bacterial and methanogens community and the abundance of key rumen bacteria (Ruminococcaceae, Fibrobacteres, Veillonellaceae, Rikenellaceae, Tenericutes), methanogens (Methanobrevibacter ruminantium, Methanomicrobium mobile and Group 9), anaerobic fungi (Piromyces and Orpinomyces) and protozoal taxa (Enoploplastron, Diplodinium, Polyplastron, Ophryoscolex, Isotricha and Dasytricha) before weaning whereas CTL kids remained protozoa-free through the study. Most of these taxa were positively correlated with indicators of the rumen microbiological and physiological development (higher forage and concentrate intakes and animal growth during the post-weaning period) favoring the weaning process in RFF and RFC kids in comparison to CTL kids. Some of these microbiological differences tended to decrease during the post-weaning period, although RFF and RFC kids retained a more complex and matured rumen microbial ecosystem than CTL kids. Inoculation with autoclaved rumen fluid promoted lower development of the bacterial and protozoal communities during the pre-weaning period than using fresh inocula, but it favored a more rapid microbial development during the post-weaning than observed for CTL kids. Conclusions This study demonstrated that inoculation of young ruminants with fresh rumen fluid from adult animals accelerated the rumen microbial colonization which was associated with an earlier rumen functional development. This strategy facilitated a smoother transition from milk to solid feed favoring the animal performance during post-weaning and minimizing stress.

Functional Prediction and Assignment of Methanobrevibacter ruminantium M1 Operome Using a Combined Bioinformatics Approach

Methanobrevibacter ruminantium M1 (MRU) is a rod-shaped rumen methanogen with the ability to use H2 and CO2, and formate as substrates for methane formation in the ruminants. Enteric methane emitted from this organism can also be influential to the loss of dietary energy in ruminants and humans. To date, there is no successful technology to reduce methane due to a lack of knowledge on its molecular machinery and 73% conserved hypothetical proteins (HPs; operome) whose functions are still not ascertained perceptively. To address this issue, we have predicted and assigned a precise function to HPs and categorize them as metabolic enzymes, binding proteins, and transport proteins using a combined bioinformatics approach. The results of our study show that 257 (34%) HPs have well-defined functions and contributed essential roles in its growth physiology and host adaptation. The genome-neighborhood analysis identified 6 operon-like clusters such as hsp, TRAM, dsr, cbs and cas, which are responsible for protein folding, sudden heat-shock, host defense, and protection against the toxicities in the rumen. The functions predicted from MRU operome comprised of 96 metabolic enzymes with 17 metabolic subsystems, 31 transcriptional regulators, 23 transport, and 11 binding proteins. Functional annotation of its operome is thus more imperative to unravel the molecular and cellular machinery at the systems-level. The functional assignment of its operome would advance strategies to develop new anti-methanogenic targets to mitigate methane production. Hence, our approach provides new insight into the understanding of its growth physiology and lifestyle in the ruminants and also to reduce anthropogenic greenhouse gas emissions worldwide.

Inoculation with rumen fluid in early life accelerates the rumen microbial development and favours the weaning process in goats

Background: Newborn ruminants possess an underdeveloped rumen which is colonized by microorganisms acquired from adult animals and the surrounding environment. This microbial transfer can be limited in dairy systems in which newborns are separated from their dams at birth. This study explores whether the direct inoculation of fresh or autoclaved rumen fluid from adult goats to newborn kids has a beneficial effect on rumen microbial development and function.Results: Repetitive inoculation of young kids with fresh rumen fluid from adult goats adapted to forage (RFF) or concentrate diets (RFC) accelerated microbial colonization of the rumen during the pre-weaning period leading to high protozoal numbers, a greater diversity of bacterial (+234 OTUs), methanogens (+6 OTUs) and protozoal communities (+25 OTUs) than observed in control kids (CTL) without inoculation. This inoculation also increased the size of the core bacterial and methanogens community and the abundance of key rumen bacteria (Ruminococcaceae, Fibrobacteres, Veillonellaceae, Rikenellaceae, Tenericutes), methanogens (Methanobrevibacter ruminantium, Methanomicrobium mobile and Group 9), anaerobic fungi (Piromyces and Orpinomyces) and protozoal taxa (Enoploplastron, Diplodinium, Polyplastron, Ophryoscolex, Isotricha and Dasytricha) before weaning whereas CTL kids remained protozoa-free through the study. Most of these taxa were positively correlated with indicators of the rumen microbiological and physiological development (higher forage and concentrate intakes and animal growth during the post-weaning period) favouring the weaning process in RFF and RFC kids in comparison to CTL kids. Some of these microbiological differences tended to decrease during the post-weaning period, although RFF and RFC kids retained a more complex and matured rumen microbial ecosystem than CTL kids. Inoculation with autoclaved rumen fluid promoted lower development of the bacterial and protozoal communities during the pre-weaning period than using fresh inocula, but it favoured a more rapid microbial development during the post-weaning than observed for CTL kids.Conclusions: This study demonstrated that inoculation of young ruminants with fresh rumen fluid from adult animals accelerated the rumen microbial colonization which was associated with an earlier rumen functional development. This strategy facilitated a smoother transition from milk to solid feed favoring the animal performance during post-weaning and minimizing stress.

PSXII-6 Supplementation of Angus crossbred steers with avian-derived polyclonal antibody preparations against the ruminal methanogen Methanobrevibacter ruminantium M1 does not alter in vivo methane emissions

An experiment was conducted to evaluate the effect of supplementing twenty Angus crossbred steers with avian-derived polyclonal antibody preparations (PAP) against the ruminal methanogen Methanobrevibacter ruminantium M1 on in vivo methane production, using the sulfur hexafluoride (SF6) tracer technique (Johnson et al., 1994). Steers were fed chopped bermudagrass hay (BGH) ad libitum and 0.7 kg d-1 of corn gluten feed. The experiment followed a change-over design. Treatments were 1) supplementation of 3 mL d-1 of PAP against M. ruminantium M1 (PAP-M1), and 2) supplementation of 3 mL d-1 of a non-immunized egg product (CON). Individual BGH intake was recorded using an electronic radio-frequency monitoring system (GrowSafe System Ltd., Airdrie, Alberta, Canada). There was a 14-d adaptation period to the feeding regime, with no PAP supplementation, followed by an 18-d treatment period. Steers were dosed with brass permeation tubes with a known release rate of SF6 on d 7 of treatment period. Enteric methane emissions were sampled from d 13 to 18 of the treatment period, into N-rinsed pre-evacuated U-shaped polyvinyl chloride canisters (2 L) through a capillary tube. Methane emissions were averaged per animal within period. Data were analyzed as a change-over design using a model with fixed effects of order, period, and treatment and random effect of steer within order. Dry matter intake (DMI) was not different (P = 0.44) between treatments. Methane emissions, expressed as grams per day (P = 0.86), as grams per kilogram of DMI (P = 0.78), or in terms of methane emission factors (Ym, P = 0.78) were not different between PAP-M1 and CON treatments. Supplementation of steers with PAP against M. ruminantium M1 did not decrease enteric methane emissions. Based on preliminary ex situ trials, evaluation of different doses and combinations of PAP against other methanogenic species warrant further investigation.