Cesar S. Pinares-Patiño

Methane yield phenotypes linked to differential gene expression in the sheep rumen microbiome

Ruminant livestock represent the single largest anthropogenic source of the potent greenhouse gas methane, which is generated by methanogenic archaea residing in ruminant digestive tracts. While differences between individual animals of the same breed in the amount of methane produced have been observed, the basis for this variation remains to be elucidated. To explore the mechanistic basis of this methane production, we measured methane yields from 22 sheep, which revealed that methane yields are a reproducible, quantitative trait. Deep metagenomic and metatranscriptomic sequencing demonstrated a similar abundance of methanogens and methanogenesis pathway genes in high and low methane emitters. However, transcription of methanogenesis pathway genes was substantially increased in sheep with high methane yields. These results identify a discrete set of rumen methanogens whose methanogenesis pathway transcription profiles correlate with methane yields and provide new targets for CH4 mitigation at the levels of microbiota composition and transcriptional regulation.

Strategies to reduce methane emissions from farmed ruminants grazing on pasture

Methane emissions from livestock are a significant contributor to greenhouse gas emissions and have become a focus of research activities, especially in countries where agriculture is a major economic sector. Understanding the complexity of the rumen microbiota, including methane-producing Archaea, is in its infancy. There are currently no robust, reproducible and economically viable methods for reducing methane emissions from ruminants grazing on pasture and novel innovative strategies to diminish methane output from livestock are required. In this review, current approaches towards mitigation of methane in pastoral farming are summarised. Research strategies based on vaccination, enzyme inhibitors, phage, homoacetogens, defaunation, feed supplements, and animal selection are reviewed. Many approaches are currently being investigated, and it is likely that more than one strategy will be required to enable pastoral farming to lower its emissions of methane significantly. Different strategies may be suitable for different farming practices and systems.

Heritability estimates of methane emissions from sheep

The objective of this study was to determine the genetic parameters of methane (CH4) emissions and their genetic correlations with key production traits. The trial measured the CH4 emissions, at 5-min intervals, from 1225 sheep placed in respiration chambers for 2 days, with repeat measurements 2 weeks later for another 2 days. They were fed in the chambers, based on live weight, a pelleted lucerne ration at 2.0 times estimated maintenance requirements. Methane outputs were calculated for g CH4/day and g CH4/kg dry matter intake (DMI) for each of the 4 days. Single trait models were used to obtain estimates of heritability and repeatability. Heritability of g CH4/day was 0.29 ± 0.05, and for g CH4/kg DMI 0.13 ± 0.03. Repeatability between measurements 14 days apart were 0.55 ± 0.02 and 0.26 ± 0.02, for the two traits. The genetic and phenotypic correlations of CH4 outputs with various production traits (weaning weight, live weight at 8 months of age, dag score, muscle depth and fleece weight at 12 months of age) measured in the first year of life, were estimated using bivariate models. With the exception of fleece weight, correlations were weak and not significantly different from zero for the g CH4/kg DMI trait. For fleece weight the phenotypic and genetic correlation estimates were −0.08 ± 0.03 and −0.32 ± 0.11 suggesting a low economically favourable relationship. These results indicate that there is genetic variation between animals for CH4 emission traits even after adjustment for feed intake and that these traits are repeatable. Current work includes the establishment of selection lines from these animals to investigate the physiological, microbial and anatomical changes, coupled with investigations into shorter and alternative CH4 emission measurement and breeding value estimation techniques; including genomic selection.

Two different bacterial community types are linked with the low-methane emission trait in sheep.

The potent greenhouse gas methane (CH4) is produced in the rumens of ruminant animals from hydrogen produced during microbial degradation of ingested feed. The natural animal-to-animal variation in the amount of CH4 emitted and the heritability of this trait offer a means for reducing CH4 emissions by selecting low-CH4 emitting animals for breeding. We demonstrate that differences in rumen microbial community structure are linked to high and low CH4 emissions in sheep. Bacterial community structures in 236 rumen samples from 118 high- and low-CH4 emitting sheep formed gradual transitions between three ruminotypes. Two of these (Q and S) were linked to significantly lower CH4 yields (14.4 and 13.6 g CH4/kg dry matter intake [DMI], respectively) than the third type (H; 15.9 g CH4/kg DMI; p<0.001). Low-CH4 ruminotype Q was associated with a significantly lower ruminal acetate to propionate ratio (3.7±0.4) than S (4.4±0.7; p<0.001) and H (4.3±0.5; p<0.001), and harbored high relative abundances of the propionate-producing Quinella ovalis. Low-CH4 ruminotype S was characterized by lactate- and succinate-producing Fibrobacter spp., Kandleria vitulina, Olsenella spp., Prevotella bryantii, and Sharpea azabuensis. High-CH4 ruminotype H had higher relative abundances of species belonging to Ruminococcus, other Ruminococcaceae, Lachnospiraceae, Catabacteriaceae, Coprococcus, other Clostridiales, Prevotella, other Bacteroidales, and Alphaproteobacteria, many of which are known to form significant amounts of hydrogen. We hypothesize that lower CH4 yields are the result of bacterial communities that ferment ingested feed to relatively less hydrogen, which results in less CH4 being formed.

Natural variation in methane emission of sheep fed on a lucerne pellet diet is unrelated to rumen ciliate community type

Only limited information is available on the roles of different rumen ciliate community types, first described by Eadie in 1962, in enteric methane (CH4) formation by their ruminant hosts. If the different types were differentially associated with CH4 formation, then ciliate community typing could be used to identify naturally high and low CH4-emitting animals. Here we measured the CH4 yields [g CH4 (kg feed dry matter intake, DMI)(-1)] of 118 sheep fed a standard pelleted lucerne diet at two different times, at least 2 weeks apart. There were significant differences (P < 2.2 × 10(-16), Wilcoxon rank sum test) in the CH4 yields (± sd) from sheep selected as high [16.7 ± 1.5 g CH4 (kg DMI)(-1)] and low emitters [13.3 ± 1.5 g CH4 (kg DMI)(-1)]. A rumen sample was collected after each of the two measurements, and ciliate composition was analysed using barcoded 454 Titanium pyrosequencing of 18S rRNA genes. The genera found, in order of mean relative abundance, were Epidinium, Entodinium, Dasytricha, Eudiplodinium, Polyplastron, Isotricha and Anoplodinium-Diplodinium, none of which was significantly correlated with the CH4 emissions ranking associated with the rumen sample. Ciliate communities naturally assembled into four types (A, AB, B and O), characterized by the presence and absence of key genera. There was no difference in CH4 yield between sheep that harboured different ciliate community types, suggesting that these did not underlie the natural variation in CH4 yields. Further research is needed to unravel the nature of interactions between ciliate protozoa and other rumen micro-organisms, which may ultimately lead to contrasting CH4 emission phenotypes.

Extending the Collection Duration of Breath Samples for Enteric Methane Emission Estimation Using the SF6 Tracer Technique

Simple Summary Extended sample collection for the SF6 tracer technique is desirable for extensive grazing systems. Breath samples from eight cows were collected while lucerne silage was fed to achieve fixed intakes among the cows. Samples were collected over a 10-day period, using either apparatuses used in New Zealand (NZL) or Argentina (ARG), and either daily, over two consecutive 5-day periods or over a 10-day period (in duplicate). The NZL system had a greater sampling success and more consistent CH4 emission estimates than the ARG system, with no differences in mean emissions among sample collection periods. This study showed that extended sample collection is feasible, but definitive evaluation under grazing situation is required before a decision on recommendation can be made. Abstract The daily sample collection protocol of the sulphur hexafluoride (SF6) tracer technique for the estimation of methane (CH4) emissions from ruminants may not be practical under extensive grazing systems. Here, under controlled conditions, we evaluated extended periods of sampling as an alternative to daily sample collections. Eight rumen-fistulated cows were housed and fed lucerne silage to achieve common daily feed intakes of 6.4 kg dry matter per cow. Following SF6 permeation tube dosing, eight sampling lines were fitted to the breath collection harness, so that a common gas mix was available to each line. Half of the lines collected samples into PVC yokes using a modified capillary system as commonly used in New Zealand (NZL), and half collected samples into stainless steel cylinders using a ball-bearing flow restrictor as used in Argentina (ARG), all within a 10-day time frame, either daily, across two consecutive 5-day periods or across one 10-day period (in duplicate). The NZL system had greater sampling success (97.3 vs. 79.5%) and yielded more consistent CH4 emission estimates than the ARG system. Emission estimates from NZL daily, NZL 5-day and NZL 10-day samplings were 114, 110 and 111 g d−1, respectively. Extended sample collection protocol may be feasible, but definitive evaluation of this alternative as well as sample collection systems is required under grazing situations before a decision on recommendation can be made.

Methane emission from sheep is related to concentrations of rumen volatile fatty acids

Globally, ruminants are the most important source of emission of methane (CH4). Animal-to-animal variation in CH4 emission has genetic basis (Pinares-Patino et al., 2011), hence offering a potential mitigation avenue through animal breeding. However, for this approach to progress to practical application a rapid and reliable method of ranking animals for their CH4 emissions is required. Microbial fermentation of feed in the rumen produces volatile fatty acids (VFA), hydrogen (H2), carbon dioxide (CO2), ammonia and heat. A last step in the process is the reduction of CO2 to CH4 by Archaea using H2 as a source of energy. Formation of both acetic and butyric acids is accompanied by the production of H2 and CO2, whereas propionic production involves a net uptake of H2, hence VFA profiles may be used to predict CH4 emission rates (Benchaar et al., 2001). This controlled study conducted with sheep explored the relationship between rumen VFA and CH4 emission.

Genetic parameters of methane emissions determined using portable accumulation chambers in lambs and ewes grazing pasture and genetic correlations with emissions determined in respiration chambers1

Abstract Methane (CH4) emission traits were previously found to be heritable and repeatable in sheep fed alfalfa pellets in respiration chambers (RC). More rapid screening methods are, however, required to increase genetic progress and to provide a cost-effective method to the farming industry for maintaining the generation of breeding values in the future. The objective of the current study was to determine CH4 and carbon dioxide (CO2) emissions using several 1-h portable accumulation chamber (PAC) measurements from lambs and again as ewes while grazing ryegrass-based pasture. Many animals with PAC measurements were also measured in RC while fed alfalfa pellets at 2.0 × maintenance metabolizable energy requirements (MEm). Heritability estimates from mixed models for CH4 and CO2 production (g/d) were 0.19 and 0.16, respectively, when measured using PAC with lambs; 0.20 and 0.27, respectively, when measured using PAC with ewes; and 0.23 and 0.34, respectively, when measured using RC with lambs. For measured gas traits, repeatabilities of measurements collected 14 d apart ranged from 0.33 to 0.55 for PAC (combined lambs and ewes) and were greater at 0.65 to 0.76 for the same traits measured using RC. Genetic correlations (rg) between PAC in lambs and ewes were 0.99 for CH4, 0.93 for CH4 + CO2, and 0.85 for CH4/(CH4 + CO2), suggesting that CH4 emissions in lambs and ewes are the same trait. Genetic correlations between PAC and RC measurements were lower, at 0.62 to 0.67 for CH4 and 0.41 to 0.42 for CH4 + CO2, likely reflecting different environmental conditions associated with the protocols used with the 2 measurement methods. The CH4/(CH4 + CO2) ratio was the most similar genetic trait measured using PAC (both lambs and ewes, 63% and 66% selection efficiency, respectively) compared with CH4 yield (g/kg DMI) measured using RC. These results suggest that PAC measurements have considerable value as a rapid low-cost method to estimate breeding values for CH4 emissions in sheep.

Across-Experiment Transcriptomics of Sheep Rumen Identifies Expression of Lipid/Oxo-Acid Metabolism and Muscle Cell Junction Genes Associated With Variation in Methane-Related Phenotypes

Ruminants are significant contributors to the livestock generated component of the greenhouse gas, methane (CH4). The CH4 is primarily produced by the rumen microbes. Although the composition of the diet and animal intake amount have the largest effect on CH4 production and yield (CH4 production/dry matter intake, DMI), the host also influences CH4 yield. Shorter rumen feed mean retention time (MRT) is associated with higher dry matter intake and lower CH4 yield, but the molecular mechanism(s) by which the host affects CH4 production remain unclear. We integrated rumen wall transcriptome data and CH4 phenotypes from two independent experiments conducted with sheep in Australia (AUS, n = 62) and New Zealand (NZ, n = 24). The inclusion of the AUS data validated the previously identified clusters and gene sets representing rumen epithelial, metabolic and muscular functions. In addition, the expression of the cell cycle genes as a group was consistently positively correlated with acetate and butyrate concentrations (p < 0.05, based on AUS and NZ data together). The expression of a group of metabolic genes showed positive correlations in both AUS and NZ datasets with CH4 production (p < 0.05) and yield (p < 0.01). These genes encode key enzymes in the ketone body synthesis pathway and included members of the poorly characterized aldo-keto reductase 1C (AKR1C) family. Several AKR1C family genes appear to have ruminant specific evolution patterns, supporting their specialized roles in the ruminants. Combining differential gene expression in the rumen wall muscle of the shortest and longest MRT AUS animals (no data available for the NZ animals) with correlation and network analysis, we identified a set of rumen muscle genes involved in cell junctions as potential regulators of MRT, presumably by influencing contraction rates of the smooth muscle component of the rumen wall. Higher rumen expression of these genes, including SYNPO (synaptopodin, p < 0.01) and NEXN (nexilin, p < 0.05), was associated with lower CH4 yield in both AUS and NZ datasets. Unlike the metabolic genes, the variations in the expression of which may reflect the availability of rumen metabolites, the muscle genes are currently our best candidates for causal genes that influence CH4 yield.