Carol-Anne Duthie

Bovine Host Genetic Variation Influences Rumen Microbial Methane Production with Best Selection Criterion for Low Methane Emitting and Efficiently Feed Converting Hosts Based on Metagenomic Gene Abundance.

Methane produced by methanogenic archaea in ruminants contributes significantly to anthropogenic greenhouse gas emissions. The host genetic link controlling microbial methane production is unknown and appropriate genetic selection strategies are not developed. We used sire progeny group differences to estimate the host genetic influence on rumen microbial methane production in a factorial experiment consisting of crossbred breed types and diets. Rumen metagenomic profiling was undertaken to investigate links between microbial genes and methane emissions or feed conversion efficiency. Sire progeny groups differed significantly in their methane emissions measured in respiration chambers. Ranking of the sire progeny groups based on methane emissions or relative archaeal abundance was consistent overall and within diet, suggesting that archaeal abundance in ruminal digesta is under host genetic control and can be used to genetically select animals without measuring methane directly. In the metagenomic analysis of rumen contents, we identified 3970 microbial genes of which 20 and 49 genes were significantly associated with methane emissions and feed conversion efficiency respectively. These explained 81% and 86% of the respective variation and were clustered in distinct functional gene networks. Methanogenesis genes (e.g. mcrA and fmdB) were associated with methane emissions, whilst host-microbiome cross talk genes (e.g. TSTA3 and FucI) were associated with feed conversion efficiency. These results strengthen the idea that the host animal controls its own microbiota to a significant extent and open up the implementation of effective breeding strategies using rumen microbial gene abundance as a predictor for difficult-to-measure traits on a large number of hosts. Generally, the results provide a proof of principle to use the relative abundance of microbial genes in the gastrointestinal tract of different species to predict their influence on traits e.g. human metabolism, health and behaviour, as well as to understand the genetic link between host and microbiome.

Hydrogen and methane emissions from beef cattle and their rumen microbial community vary with diet, time after feeding and genotype.

The aims of the present study were to quantify hydrogen (H2) and methane (CH4) emissions from beef cattle under different dietary conditions and to assess how cattle genotype and rumen microbial community affected these emissions. A total of thirty-six Aberdeen Angus-sired (AAx) and thirty-six Limousin-sired (LIMx) steers were fed two diets with forage:concentrate ratios (DM basis) of either 8:92 (concentrate) or 52:48 (mixed). Each diet was fed to eighteen animals of each genotype. Methane (CH4) and H2 emissions were measured individually in indirect respiration chambers. H2 emissions (mmol/min) varied greatly throughout the day, being highest after feed consumption, and averaged about 0·10 mol H2/mol CH4. Higher H2 emissions (mol/kg DM intake) were recorded in steers fed the mixed diet. Higher CH4 emissions (mol/d and mol/kg DM intake) were recorded in steers fed the mixed diet (P< 0·001); the AAx steers produced more CH4 on a daily basis (mol/d, P< 0·05) but not on a DM intake basis (mol/kg DM intake). Archaea (P= 0·002) and protozoa (P< 0·001) were found to be more abundant and total bacteria (P< 0·001) less abundant (P< 0·001) on feeding the mixed diet. The relative abundance of Clostridium cluster IV was found to be greater (P< 0·001) and that of cluster XIVa (P= 0·025) lower on feeding the mixed diet. The relative abundance of Bacteroides plus Prevotella was greater (P= 0·018) and that of Clostridium cluster IV lower (P= 0·031) in the LIMx steers. There were no significant relationships between H2 emissions and microbial abundance. In conclusion, the rate of H2 production immediately after feeding may lead to transient overloading of methanogenic archaea capacity to use H2, resulting in peaks in H2 emissions from beef cattle.

Nitrogen excretion at different stages of growth and its association with production traits in growing pigs

The objectives of this study were to determine nitrogen loss at different stages of growth and during the entire growing period and to investigate the associations between nitrogen excretion and production traits in growing pigs. Data from 315 pigs of an F(2) population which originated from crossing Pietrain sires with a commercial dam line were used. Nitrogen retention was derived from protein retention as measured using the deuterium dilution technique during different stages of growth (60 to 90 kg, 90 to 120 kg, and 120 to 140 kg). Pigs were fed ad libitum with 2 pelleted diets containing 17% (60 to 90 kg) and 16.5% (90 to 120 and 120 to 140 kg) CP. Average daily nitrogen excretion (ADNE) within each stage of growth was calculated on the basis of the accumulated difference between average daily nitrogen intake (ADNI) and average daily nitrogen retention (ADNR). Least ADNE, nitrogen excretion per BW gain (NEWG) and total nitrogen excretion (TNE) were observed during growth from 60 to 90 kg. In contrast, the greatest ADNE, NEWG, and TNE were found during growth from 120 to 140 kg. Statistical analyses indicated that gender, housing type, the ryanodine receptor 1 (RYR1) gene, and batch influenced nitrogen excretion (P < 0.05), but the degree and direction of influences differed between growth stages. Gender differences showed that gilts excreted less nitrogen than barrows (P < 0.05), which was associated with decreased feed conversion ratio (FCR; feed:gain) and lipid:protein gain ratio. Single-housed pigs showed reduced nitrogen excretion compared with group-housed pigs (P < 0.05). In comparison to other genotypes, pigs carrying genotype NN (homozygous normal) at the RYR1 locus had the least nitrogen excretion (P < 0.05) at all stages of growth except from 60 to 90 kg. The residual correlations indicated that NEWG and TNE have large positive correlations with FCR (r = 0.99 and 0.91, respectively) and moderate negative correlations with ADG (r = -0.53 and -0.48, respectively), for the entire growing period. Improvement in FCR, increase in ADG and reduction in lipid:protein gain ratio by 1 phenotypic SD reduced TNE per pig by 709 g, 307 g, and 211 g, respectively, over the entire growing period. The results indicate that nitrogen excretion changes substantially during growth, and it can be reduced most effectively by improvement of feed efficiency and to a lesser extent through the improvement of BW gain or body composition or both.

Effectiveness of nitrate addition and increased oil content as methane mitigation strategies for beef cattle fed two contrasting basal diets

The objectives of this study were to investigate the effects of (1) the addition of nitrate and (2) an increase in dietary oil on methane (CH4) and hydrogen (H2) emissions from 2 breeds (cross-bred Charolais and purebred Luing) of finishing beef cattle receiving 2 contrasting basal diets consisting (grams per kilogram DM) of 500:500 (Mixed) and 80:920 (Concentrate) forage to concentrate ratios. Within each basal diet there were 3 treatments: (i) control treatments (mixed-CTL and concentrate-CTL) contained rapeseed meal as the protein source, which was replaced with either (ii) calcium nitrate (mixed-NIT and concentrate-NIT) supplying 21.5 g nitrate/kg DM, or (iii) rapeseed cake (mixed-RSC and concentrate-RSC) to increase dietary oil from 27 (CTL) to 53 g/kg DM (RSC). Following adaption to diets, CH4 and H2 emissions were measured on 1 occasion from each of the 76 steers over a 13-wk period. Dry matter intakes tended (P = 0.051) to be greater for the concentrate diet than the mixed diet; however, when expressed as grams DMI per kilogram BW, there was no difference between diets (P = 0.41). Dry matter intakes for NIT or RSC did not differ from CTL. Steers fed a concentrate diet produced less CH4 and H2 than those fed a mixed diet (P < 0.001). Molar proportions of acetate (P < 0.001) and butyrate (P < 0.01) were lower and propionate (P < 0.001) and valerate (P < 0.05) higher in the rumen fluid from steers fed the concentrate diet. For the mixed diet, CH4 yield (grams per kilogram DMI) was decreased by 17% when nitrate was added (P < 0.01), while H2 yield increased by 160% (P < 0.001). The addition of RSC to the mixed diet decreased CH4 yield by 7.5% (P = 0.18). However, for the concentrate diet neither addition of nitrate (P = 0.65) nor increasing dietary oil content (P = 0.46) decreased CH4 yield compared to concentrate-CTL. Molar proportions of acetate were higher (P < 0.001) and those of propionate lower (P < 0.01) in rumen fluid from NIT treatments compared to respective CTL treatments. Overall, reductions in CH4 emissions from adding nitrate or increasing the oil content of the mixed diet were similar to those expected from previous reports. However, the lack of an effect of these mitigation strategies when used with high concentrate diets has not been previously reported. This study shows that the effect of CH4 mitigation strategies is basal diet-dependent.

Evaluation of the laser methane detector to estimate methane emissions from ewes and steers.

The laser methane detector (LMD) has been proposed as a method to characterize enteric methane (CH4) emissions from animals in a natural environment. To validate LMD use, its CH4 outputs (LMD-CH4), were compared against CH4 measured with respiration chambers (chamber-CH4). The LMD was used to measure CH4 concentration (µL/L) in the exhaled air of 24 lactating ewes and 72 finishing steers. In ewes, LMD was used on 1 d for each ewe, for 2-min periods at 5 hourly observation periods (P1 to P5, respectively) after feeding. In steers fed either low- or high-concentrate diets, LMD was used once daily for a 4-min period for 3 d. The week after LMD-CH4 measurement, ewes or steers entered respiration chambers to quantify daily CH4 output (g/d). The LMD outputs consisted of periodic events of high CH4 concentrations superimposed on a background of oscillating lower CH4 concentrations. The high CH4 events were attributed to eructation and the lower background CH4 to respiration. After fitting a double normal distribution to the data set, a threshold of 99% of probability of the lower distribution was used to separate respiration from eructation events. The correlation between mean LMD-CH4 and chamber-CH4 was not high, and only improved correlations were observed after data were separated in 2 levels. In ewes, a model with LMD and DMI (adjusted R(2) = 0.92) improved the relationship between DMI and chamber-CH4 alone (adjusted R(2) = 0.79) and between LMD and chamber-CH4 alone (adjusted R(2) = 0.86). In both experiments, chamber-CH4 was best explained by models with length of eructation events (time) and maximum values of CH4 concentration during respiration events (µL/L; P < 0.01). Correlation between methods differed between observation periods, indicating the best results of the LMD were observed from 3 to 5 h after feeding. Given the short time and ease of use of LMD, there is potential for its commercial application and field-based studies. Although good indicators of quantity of CH4 were obtained with respiration and eructation CH4, the method needed to separate the data into high and low levels of CH4 was not simple to apply in practice. Further assessment of the LMD should be performed in relation to animal feeding behavior and physiology to validate assumptions of eructation and respiration levels, and other sources of variation should be tested (i.e., micrometeorology) to better investigate its potential application for CH4 testing in outdoor conditions.

Impact of adding nitrate or increasing the lipid content of two contrasting diets on blood methaemoglobin and performance of two breeds of finishing beef steers.

Adding nitrate to the diet or increasing the concentration of dietary lipid are effective strategies for reducing enteric methane emissions. This study investigated their effect on health and performance of finishing beef cattle. The experiment was a two×two×three factorial design comprising two breeds (CHX, crossbred Charolais; LU, Luing); two basal diets consisting of (g/kg dry matter (DM), forage to concentrate ratios) 520 : 480 (Mixed) or 84 : 916 (Concentrate); and three treatments: (i) control with rapeseed meal as the main protein source replaced with either (ii) calcium nitrate (18 g nitrate/kg diet DM) or (iii) rapeseed cake (RSC, increasing acid hydrolysed ether extract from 25 to 48 g/kg diet DM). Steers (n=84) were allocated to each of the six basal diet×treatments in equal numbers of each breed with feed offered ad libitum. Blood methaemoglobin (MetHb) concentrations (marker for nitrate poisoning) were monitored throughout the study in steers receiving nitrate. After dietary adaptation over 28 days, individual animal intake, performance and feed efficiency were recorded for a test period of 56 days. Blood MetHb concentrations were low and similar up to 14 g nitrate/kg diet DM but increased when nitrate increased to 18 g nitrate/kg diet DM (P0.05). Neither basal diet nor treatment affected carcass quality (P>0.05), but CHX steers achieved a greater killing out proportion (P<0.001) than LU steers. Thus, adding nitrate to the diet or increasing the level of dietary lipid through the use of cold-pressed RSC, did not adversely affect health or performance of finishing beef steers when used within the diets studied.

The rumen microbiome as a reservoir of antimicrobial resistance and pathogenicity genes is directly affected by diet in beef cattle

BackgroundThe emergence and spread of antimicrobial resistance is the most urgent current threat to human and animal health. An improved understanding of the abundance of antimicrobial resistance genes and genes associated with microbial colonisation and pathogenicity in the animal gut will have a major role in reducing the contribution of animal production to this problem. Here, the influence of diet on the ruminal resistome and abundance of pathogenicity genes was assessed in ruminal digesta samples taken from 50 antibiotic-free beef cattle, comprising four cattle breeds receiving two diets containing different proportions of concentrate.ResultsTwo hundred and four genes associated with antimicrobial resistance (AMR), colonisation, communication or pathogenicity functions were identified from 4966 metagenomic genes using KEGG identification. Both the diversity and abundance of these genes were higher in concentrate-fed animals. Chloramphenicol and microcin resistance genes were dominant in samples from forage-fed animals (P < 0.001), while aminoglycoside and streptomycin resistances were enriched in concentrate-fed animals. The concentrate-based diet also increased the relative abundance of Proteobacteria, which includes many animal and zoonotic pathogens. A high ratio of Proteobacteria to (Firmicutes + Bacteroidetes) was confirmed as a good indicator for rumen dysbiosis, with eight cases all from concentrate-fed animals. Finally, network analysis demonstrated that the resistance/pathogenicity genes are potentially useful as biomarkers for health risk assessment of the ruminal microbiome.ConclusionsDiet has important effects on the complement of AMR genes in the rumen microbial community, with potential implications for human and animal health.

Changes in feed intake during isolation stress in respiration chambers may impact methane emissions assessment

Respiration chambers are considered the ‘gold standard’ technique for measuring in vivo methane (CH4) emissions in live animals. However, the imposed isolation required may alter feeding behaviour and intake, which ultimately impact CH4 emissions. The aim of this study was to assess the impact of isolation within respiration chambers on feed intake and CH4 emissions with two different diets and breeds of beef cattle. In addition, a routine stressor (transport) was used to examine the relationship between individual stress responsiveness and changes in feed intake during isolation. Eighty-four steers (castrated males) (569 ± 5.7 kg bodyweight, BW) were divided into two groups and each group fed with one of two basal diets consisting of (g/kg dry matter, DM) either 50 : 50 (Mixed) or 8 : 92 (Concentrate) forage to concentrate ratios. Within each basal diet there were three supplementation treatments: (1) control, (2) calcium nitrate, and (3) rapeseed cake. The stress biomarkers plasma cortisol, creatine kinase (CK), and free fatty acids (FFA) were determined before (0 h) and after (30 min, 3 h, 6 h and 9 h) a 30-min journey, when steers were transported to the respiration chamber facilities. Methane emissions were measured over a 3-day period using individual respiration chambers. Dry matter intake (DMI) was assessed within the group-housed pens (4 weeks before entry to training pen), in the training pens and the chambers. Cortisol, FFA and CK increased (P < 0.05) after transport confirming a stress response. DMI (g/kg BW) decreased (P < 0.001) during isolation in the training pens (14.7 ± 0.28) and the chambers (14.3 ± 0.26) compared with that of the same animals in the group pens (16.8 ± 0.23). DMI during isolation decreased more in those animals which had an increased (P < 0.05) stress response during transport as measured by cortisol, FFA and CK. With the Mixed diet, the decline in DMI was estimated to result in an increase in CH4 (g/kg DMI) (r = 0.25, P = 0.001), which did not occur with the Concentrate diet. According to the results of this experiment, the stress associated with isolation reduces the DMI resulting in an increase in g CH4/kg DMI in fibrous diets. Habituation to isolation needs refinement in order to reduce the impact of stress on intake and therefore achieve more accurate estimates of CH4 emissions. Alternatively, modelling CH4 estimations according to behavioural and physiological changes associated with isolation stress would improve accuracy of CH4 estimations.

Identification, Comparison, and Validation of Robust Rumen Microbial Biomarkers for Methane Emissions Using Diverse Bos Taurus Breeds and Basal Diets

Previous shotgun metagenomic analyses of ruminal digesta identified some microbial information that might be useful as biomarkers to select cattle that emit less methane (CH4), which is a potent greenhouse gas. It is known that methane production (g/kgDMI) and to an extent the microbial community is heritable and therefore biomarkers can offer a method of selecting cattle for low methane emitting phenotypes. In this study a wider range of Bos Taurus cattle, varying in breed and diet, was investigated to determine microbial communities and genetic markers associated with high/low CH4 emissions. Digesta samples were taken from 50 beef cattle, comprising four cattle breeds, receiving two basal diets containing different proportions of concentrate and also including feed additives (nitrate or lipid), that may influence methane emissions. A combination of partial least square analysis and network analysis enabled the identification of the most significant and robust biomarkers of CH4 emissions (VIP > 0.8) across diets and breeds when comparing all potential biomarkers together. Genes associated with the hydrogenotrophic methanogenesis pathway converting carbon dioxide to methane, provided the dominant biomarkers of CH4 emissions and methanogens were the microbial populations most closely correlated with CH4 emissions and identified by metagenomics. Moreover, these genes grouped together as confirmed by network analysis for each independent experiment and when combined. Finally, the genes involved in the methane synthesis pathway explained a higher proportion of variation in CH4 emissions by PLS analysis compared to phylogenetic parameters or functional genes. These results confirmed the reproducibility of the analysis and the advantage to use these genes as robust biomarkers of CH4 emissions. Volatile fatty acid concentrations and ratios were significantly correlated with CH4, but these factors were not identified as robust enough for predictive purposes. Moreover, the methanotrophic Methylomonas genus was found to be negatively correlated with CH4. Finally, this study confirmed the importance of using robust and applicable biomarkers from the microbiome as a proxy of CH4 emissions across diverse production systems and environments.

The effect of dietary addition of nitrate or increase in lipid concentrations, alone or in combination, on performance and methane emissions of beef cattle

Adding nitrate to or increasing the concentration of lipid in the diet are established strategies for reducing enteric methane (CH4) emissions, but their effectiveness when used in combination has been largely unexplored. This study investigated the effect of dietary nitrate and increased lipid included alone or together on CH4 emissions and performance traits of finishing beef cattle. The experiment was a 2×4 factorial design comprising two breeds (cross-bred Aberdeen Angus (AAx) and cross-bred Limousin (LIMx) steers) and four dietary treatments (each based on 550 g forage : 450 g concentrate/kg dry matter (DM)). The four dietary treatments were assigned according to a 2×2 factorial design where the control treatment contained rapeseed meal as the main protein source, which was replaced either with nitrate (21.5 g nitrate/kg DM); maize distillers dark grains (MDDG, which increased diet ether extract from 24 to 37 g/kg DM) or both nitrate and MDDG. Steers (n=20/dietary treatment) were allocated to each of the four treatments in equal numbers of each breed with feed offered ad libitum. After 28 days adaptation to dietary treatments, individual animal intake, performance and feed efficiency were recorded for 56 days. Thereafter, CH4 emissions were measured over 13 weeks (six steers/week). Increasing dietary lipid did not adversely affect animal performance and showed no interactions with dietary nitrate. In contrast, addition of nitrate to diets resulted in poorer live-weight gain (P<0.01) and increased feed conversion ratio (P<0.05) compared with diets not containing nitrate. Daily CH4 output was lower (P<0.001) on nitrate-containing diets but increasing dietary lipid resulted in only a non-significant reduction in CH4. There were no interactions associated with CH4 emissions between dietary nitrate and lipid. Cross-bred Aberdeen Angus steers achieved greater live-weight gains (P<0.01), but had greater DM intakes (P<0.001), greater fat depth (P<0.01) and poorer residual feed intakes (P<0.01) than LIMx steers. Cross-bred Aberdeen Angus steers had higher daily CH4 outputs (P<0.001) but emitted less CH4 per kilogram DM intake than LIMx steers (P<0.05). In conclusion, inclusion of nitrate reduced CH4 emissions in growing beef cattle although the efficacy of nitrate was less than in previous work. When increased dietary lipid and nitrate inclusion were combined there was no evidence of an interaction between treatments and therefore combining different nutritional treatments to mitigate CH4 emissions could be a useful means of achieving reductions in CH4 while minimising any adverse effects.