The ruminal microbiome, comprising large numbers of bacteria, ciliate protozoa, archaea and fungi, responds to diet and dietary additives in a complex way. The aim of this study was to investigate the benefits of increasing the depth of the community analysis in describing and explaining responses to dietary changes. Quantitative PCR, ssu rRNA amplicon based taxa composition, diversity and co-occurrence network analyses were applied to ruminal digesta samples obtained from four multiparous Nordic Red dairy cows fitted with rumen cannulae. The cows received diets with forage:concentrate ratio either 35:65 (diet H) or 65:35 (L), supplemented or not with sunflower oil (SO) (0 or 50 g/kg diet dry matter), supplied in a 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments and four 35-day periods. Digesta samples were collected on days 22 and 24 and combined. QPCR provided a broad picture in which a large fall in the abundance of fungi was seen with SO in the H but not the L diet. Amplicon sequencing showed higher community diversity indices in L as compared to H diets and revealed diet specific taxa abundance changes, highlighting large differences in protozoal and fungal composition. Methanobrevibacter ruminantium and Mbb. gottschalkii dominated archaeal communities, and their abundance correlated negatively with each other. Co-occurrence network analysis provided evidence that no microbial domain played a more central role in network formation, that some minor-abundance taxa were at nodes of highest centrality, and that microbial interactions were diet specific. Networks added new dimensions to our understanding of the diet effect on rumen microbial community interactions.

The biohydrogenation theory of milk fat depression (MFD) attributes decreases in milk fat in cows to the formation of specific fatty acids (FA) in the rumen. Trans-10, cis-12-CLA is the only biohydrogenation intermediate known to inhibit milk fat synthesis, but it is uncertain if increased ruminal synthesis is the sole explanation of MFD. Four lactating cows were used in a 4×4 Latin square with a 2×2 factorial arrangement of treatments and 35-d experimental periods to evaluate the effect of diets formulated to cause differences in ruminal lipid metabolism and milk fat synthesis on the flow of FA and dimethyl acetal at the omasum. Treatments comprised total mixed rations based on grass silage with a forage:concentrate ratio of 35:65 or 65:35 containing 0 or 50 g/kg sunflower oil (SO). Supplementing the high-concentrate diet with SO lowered milk fat synthesis from -20·2 to -31·9 % relative to other treatments. Decreases in milk fat were accompanied by alterations in ruminal biohydrogenation favouring the trans-10 pathway and an increase in the formation of specific intermediates including trans-4 to trans-10-18 : 1, trans-8, trans-10-CLA, trans-9, cis-11-CLA and trans-10, cis-15-18 : 2. Flow of trans-10, cis-12-CLA at the omasum was greater on high- than low-concentrate diets but unaffected by SO. In conclusion, ruminal trans-10, cis-12-CLA formation was not increased on a diet causing MFD suggesting that other biohydrogenation intermediates or additional mechanisms contribute to the regulation of fat synthesis in the bovine mammary gland.

The effects of supplementing high- or low-concentrate diets with sunflower oil (SO) on rumen fermentation, nutrient utilization, and ruminal methane (CH4) emissions in lactating cows were examined. Four multiparous Nordic Red dairy cows fitted with rumen cannulae were used in a 4 × 4 Latin square with a 2 × 2 factorial arrangement of treatments and 35-d periods. Experimental treatments comprised iso-nitrogenous total mixed rations based on grass silage with forage to concentrate ratio of 65:35 or 35:65 supplemented with 0 or 50 g/kg diet DM of SO. Apparent ruminal OM and starch digestibility was greater (P < 0.05) with high- than low-concentrate diets but was unaffected by SO. Inclusion of SO in high-concentrate diet decreased (P ≤ 0.05) apparent total tract OM, fiber, and GE, and apparent ruminal fiber digestibility. High-concentrate diets and SO shifted (P < 0.05) fiber digestion from rumen to the hindgut. High-concentrate diet resulted in a lower rumen pH and elevated total rumen VFA concentration compared with low-concentrate diet, whereas SO increased rumen pH and decreased rumen VFA concentration when included in high-, but not low-concentrate diet (P < 0.05 for interaction). High-concentrate diet reduced rumen ammonia-N (P < 0.01) and molar proportion of acetate to propionate (P < 0.01), and decreased (P < 0.05) ruminal CH4 emissions when expressed as g/d or g/kg OM digested in the rumen. With both low- and high-concentrate diets, SO reduced (P < 0.05) daily emissions of CH4 as g/d or g/kg OM digested in the rumen, but SO reduced CH4 emissions expressed as g/kg OM intake, OM digested in total digestive tract, energy-corrected milk or % of GE intake only with low-concentrate diet (P ≤ 0.05 for interaction). In conclusion, replacing grass silage with concentrates led to a reduction in daily ruminal CH4 emissions that were accompanied by a shift in rumen fermentation toward the synthesis of propionate, and decreases in rumen pH and fiber digestion. Sunflower oil was effective in reducing daily CH4 emissions in lactating cows which was accompanied by a noticeable lower feed intake with high- but not low-concentrate diet. Overall the effects of SO and greater proportion of concentrates in the diet on daily CH4 emissions were additive but the additivity declined or vanished when different indices of CH4 emission intensity were considered. Consequently, SO was more effective in reducing CH4 emissions when low-concentrate diet was fed.

Diet-induced milk fat depression (MFD) in lactating cows has been attributed to alterations in ruminal lipid metabolism leading to the formation of specific fatty acid (FA) biohydrogenation intermediates that directly inhibit milk fat synthesis. However, the mechanisms responsible for decreased lipid synthesis in the mammary gland over time are not well defined. The aim of this study was to evaluate the effect of diet on milk FA composition and milk fat production over time, especially during MFD, and explore the associations between MFD and FA biohydrogenation intermediates in omasal digesta and milk. Four lactating Finnish Ayrshire cows used in a 4 × 4 Latin square with a 2 × 2 factorial arrangement of treatments and 35-d experimental periods were fed diets formulated to cause differences in ruminal and mammary lipid metabolism. Treatments consisted of an iso-nitrogenous total mixed ration based on grass silage with a forage to concentrate ratio of 65:35 or 35:65 without added oil, or with sunflower oil at 50 g/ kg of diet dry matter. The high-concentrate diet with sunflower oil (HSO) induced a 2-stage drop in milk fat synthesis that was accompanied by specific temporal changes in the milk FA composition. The MFD on HSO was associated especially with trans-10 18:1 and also with trans-9,cis-11 conjugated linoleic acid (CLA) in milk and omasal digesta across all diets and was accompanied by the appearance of trans-10,cis-15 18:2. Trans-10,cis-12 CLA was increased in HSO, but milk fat secretion was not associated with omasal or milk trans-10,cis-12 CLA. The temporal changes in milk fat content and yield and milk FA composition reflect the shift from the predominant ruminal biohydrogenation pathway to an alternative pathway. The ambiguous role of trans-10,cis-12 CLA suggests that trans-10 18:1, trans-9,cis-11 CLA and trans-10,cis-15 18:2 or additional mechanisms contributed to the diet-induced MFD in lactating cows.

Four multiparous dairy cows were used in a 4 × 4 Latin square to examine how starch level and oil mixture impact dry matter (DM) intake and digestibility, milk yield and composition, rumen fermentation, ruminal methane (CH4) emissions, and microbial diversity. Experimental treatments comprised high (HS) or low (LS) levels of starch containing 0 or 30 g of a mixture of sunflower and fish oils (2:1 w/w) per kg diet DM (LSO and HSO, respectively). Intake of DM did not differ between cows fed LS and HS diets while oil supplementation reduced DM intake. Dietary treatments did not affect milk and energy corrected milk yields. There was a tendency to have a lower milk fat concentration due to HSO compared with other treatments. Both high starch level and oil supplementation increased digestibility of gross energy. Cows receiving HS diets had higher levels of total rumen VFA while acetate was lower than LS without any differences in rumen pH, or ruminal CH4 emissions. Although dietary oil supplementation had no impact on rumen fermentation, decreased CH4 emissions (g/day and g/kg milk) were observed with a concomitant increase in Anoplodinium-Diplodinium sp. and Epidinium sp. but a decrease in Christensenellaceae, Ruminococcus sp., Methanobrevibacter ruminantium and Mbb. gottschalkii clades.

Introduction. Methane is an important atmospheric greenhouse gas. Around 500-600 Tg of CH 4 /yr is emitted globally. Of this, about 74% is biologically generated almost exclusively by methanogens that belong to the phylum Euryarchaeota and grouped under six orders. Ruminant methanogens generate 13-19% of global methane output which is the largest of anthropogenic emissions. Among the rumen methanogens, Rumen Cluster 'C' (RCC), an unclassified and uncultured archaeal clade, represents 15.8-80.9% of rumen archaea (Janssen and Kirs, 2008), which has the potential to contribute significantly to ruminant derived methane. Phylogenetically, RCC distantly affiliate to a non-methanogenic order-Thermoplasmatales. Analysis of 16S rDNA identified members of RCC in diverse habitats including digestive tracts of insects, birds, cattle, pigs, camels, macropod marsupials and man. However, their position as methanogens was unresolved because of a lack of pure cultures, despite methane producing, non-axenic archaeal cultures being enriched from these sites. Here, we report the isolation and characterisation of the first methanogen from the RCC clade, strain DOK-1 in pure culture, that in turn could help understand the role of this large group in the rumen. We propose the nomenclature Methanoplasma gallocaecorum strain DOK-1, for this novel methanogen. Material and methods Caecal contents from broiler chickens were anaerobically pooled and inoculated into 10 ml Balch tubes containing anaerobic BRN enrichment media (Rea et al., 2007). Methylotrophic methanogens were enriched by adding methanol, mono-, di-and trimethylamine HCl. Cultures were grown at 39 o C under 150 kPa of 80:20 H 2 :CO 2 and monitored for CH 4 production by GC and sub-cultured every 3-5 days for several transfers. Cultures of a 10-fold serial dilution with CH 4 production at the highest dilution were further purified by antibiotics. Methanogens were also monitored by phase-contrast microscopy and a qPCR using mcrA and total bacterial primers. Finally, a persistent bi-culture consisting of a methanogen and a bacterium was obtained. Isolation was achieved by inoculation of serial dilutions of cultures into agarose containing solid media. Several, single colonies were picked and cultured in liquid media to determine purity. Genomic DNA from these cultures were amplified using archaeal 16S primers 86F-1340R; direct sequencing of PCR products confirmed sequence alignment with Thermoplasmatales affiliated novel methanogens. Nutritional and physiological requirements and morphologic features of the isolate were studied; cells in late exponential phase were freeze-dried, fixed and analysed by transmission electron microscopy. Results and Conclusion Cells did not fluoresce under UV and appeared round (cocci) by both phase contrast and TEM microscopy measuring~650-1200 nm in diameter. They are non-motile and non-flagellated with a fragile proteinaceous cell wall with no detectable S-layer. EM showed a delicate cell wall, a simple plasma membrane and a nondescript cytoplasm ...