Structural and functional analysis of the active cow rumen's microbial community provides a catalogue of genes and microbes participating in the deconstruction of cardoon biomass.

BACKGROUND: Ruminal microbial communities enriched on lignocellulosic biomass have shown considerable promise for the discovery of microorganisms and enzymes involved in digesting cell wall compounds, a key bottleneck in the development of second-generation biofuels and bioproducts, enabling a circular bioeconomy. Cardoon (Cynara cardunculus) is a promising inedible energy crop for current and future cellulosic biorefineries and the emerging bioenergy and bioproducts industries. The rumen microbiome can be considered an anaerobic "bioreactor", where the resident microbiota carry out the depolymerization and hydrolysis of plant cell wall polysaccharides (PCWPs) through the catalytic action of fibrolytic enzymes. In this context, the rumen microbiota represents a potential source of microbes and fibrolytic enzymes suitable for biofuel production from feedstocks. In this study, metatranscriptomic and 16S rRNA sequencing were used to profile the microbiome and to investigate the genetic features within the microbial community adherent to the fiber fractions of the rumen content and to the residue of cardoon biomass incubated in the rumen of cannulated cows. RESULTS: The metatranscriptome of the cardoon and rumen fibre-adherent microbial communities were dissected in their functional and taxonomic components. From a functional point of view, transcripts involved in the methanogenesis from CO2 and H2, and from methanol were over-represented in the cardoon-adherent microbial community and were affiliated with the Methanobrevibacter and Methanosphaera of the Euryarchaeota phylum. Transcripts encoding glycoside hydrolases (GHs), carbohydrate-binding modules (CBMs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), and glycoside transferases (GTs) accounted for 1.5% (6,957) of the total RNA coding transcripts and were taxonomically affiliated to major rumen fibrolytic microbes, such as Oscillospiraceae, Fibrobacteraceae, Neocallimastigaceae, Prevotellaceae, Lachnospiraceae, and Treponemataceae. The comparison of the expression profile between cardoon and rumen fiber-adherent microbial communities highlighted that specific fibrolytic enzymes were potentially responsible for the breakdown of cardoon PCWPs, which was driven by specific taxa, mainly Ruminococcus, Treponema, and Neocallimastigaceae. CONCLUSIONS: Analysis of 16S rRNA and metatranscriptomic sequencing data revealed that the cow rumen microbiome harbors a repertoire of new enzymes capable of degrading PCWPs. Our results demonstrate the feasibility of using metatranscriptomics of enriched microbial RNA as a potential approach for accelerating the discovery of novel cellulolytic enzymes that could be harnessed for biotechnology. This research contributes a relevant perspective towards degrading cellulosic biomass and providing an economical route to the production of advanced biofuels and high-value bioproducts.

Nutritional values of forage-legume-based silages and protein concentrates for growing pigs.

In organic pig production systems, one of the main challenges is to meet the demand for resources rich in protein. Among the resources available, temperate green plants, such as forage legumes, are potential sources of energy and protein. The aim of the study was to determine the nutritional value of silages (S) from the whole plant of lucerne (L) and red clover (R) and protein pastes (PPs) obtained from L and R leaves. In a first trial, 30 pigs were used in a factorial design to determine the total tract digestibility (TTD) of dietary nutrients and energy in five dietary treatments. The control group was fed a control diet (C1). The lucerne silage (LS) and red clover silage (RS) groups were fed a 78%:22% mixture (on a DM basis) of the C1 diet and LS or RS. The lucerne protein paste (LPP) and the red clover protein paste (RPP) groups were fed an 81%:19% mixture (on a DM basis) of the C1 diet and LPP or RPP. In the second trial, five pigs were used in a 5 × 5 Latin square design to evaluate the standardised ileal digestibility (SID) of amino acids (AAs) in the four legume products. The control diet (C2) was formulated with casein as the sole protein source. The LS and RS groups were fed an 85%:15% mixture (on a DM basis) of the C2 diet and LS or RS. The LPP and RPP groups were fed an 80%:20% mixture (on a DM basis) of the C2 diet and LPP or RPP. Regardless of the plant species, silages obtained from L and R leaves contained less AA and more fibre than protein pastes. While the fresh forages contained the same percentage of protein N in total N (63.6%), lucerne lost more protein N during ensiling than red clover (-75.5 vs -33.8%). The calculated TTD coefficient of energy was higher in silages than in protein pastes and lower in R than in L products (72.8, 71.5, 67.7, and 61.3 for LS, RS, LPP and RPP, respectively). The SID of total essential AA was higher in LPP than in RPP (87.2 vs 79.2%) whereas it was lower in LS than in RS (33.2 vs 56.8%). The lower SID values in silages were explained by the protein degradation during the ensiling process and a high proportion of AA linked to the NDF fraction. The results of the present study show that protein pastes obtained from lucerne and red clover are valuable protein sources for pig. In contrast, legume silages have to be considered as an energy source rather than a protein source.

Unravelling error sources in miniaturized NIR spectroscopic measurements: The case study of forages.

The use of miniaturized NIR spectrometers is spreading over the scientific literature with a particular focus on developing methods as rapid and easy-to-use as possible and following the philosophy of green analytical chemistry. Several applications and studies are typically presented by comparing results obtained with benchtop instrumentation even when the analytical strategies are substantially different. Indeed, analytical applications that include the use of miniaturized instrumentation are subject to several sources of variability that need to be known at the time of method development. In this study, different statistical strategies were employed to understand the features and limitations of handheld NIR instruments. Because of the high interest in real applications, a common type of hygroscopic powder sample was selected: forages. A step-by-step methodology is presented to statistically address the different issues to consider in order to obtain realistic models when using miniaturized NIR spectrometers. We demonstrate how a careful evaluation of the sources of variability related to an experiment can help in the understanding of the system under study in order to obtain a more reliable development of the method and consciously choose the analytical parameters and strategies of analysis. The results were also compared with those achieved on the same dataset from a benchtop system in order to provide references analogous with those in the literature.

Study on the Effect of Ensiling Process and Ruminal Digestion on the Synthesis and Release of Cyclopropane Fatty Acids in Cow Feeding.

Cyclopropane fatty acids (CPFA) were found in milk fat from cows fed maize silage and suggested to be synthesized by lactic acid bacteria during ensiling. This study aimed to elucidate some gaps of knowledge about the microbial synthesis of CPFA, to strengthen the current authentication method based on their detection in cheese fat and performed for Parmigiano Reggiano (UNI11650), whose Specifications forbid the use of silage. CPFA were screened in different ensiled cows' feeding by gas chromatography-mass spectrometry, and the effect of feed ingredients and ruminal digestion on CPFA microbial production were further examined by in vitro tests. Results showed that solely the environmental conditions developed in silos for specific plant materials (e.g., maize) are essential for the bacterial synthesis of CPFA, whereas rumen activity did not affect CPFA levels in feeds. This supports the suitability of using CPFA as biomarkers of a crop silage-based diet forbidden by certain PDO feedstock regulations.

A heritable subset of the core rumen microbiome dictates dairy cow productivity and emissions.

A 1000-cow study across four European countries was undertaken to understand to what extent ruminant microbiomes can be controlled by the host animal and to identify characteristics of the host rumen microbiome axis that determine productivity and methane emissions. A core rumen microbiome, phylogenetically linked and with a preserved hierarchical structure, was identified. A 39-member subset of the core formed hubs in co-occurrence networks linking microbiome structure to host genetics and phenotype (methane emissions, rumen and blood metabolites, and milk production efficiency). These phenotypes can be predicted from the core microbiome using machine learning algorithms. The heritable core microbes, therefore, present primary targets for rumen manipulation toward sustainable and environmentally friendly agriculture.

Innate immune responses to metabolic stress can be detected in rumen fluids.

Many production diseases of dairy cows are related to digestive troubles. The rumen subacute acidosis is the most relevant one, albeit not easily recognized. Recent studies suggest that forestomachs can perform regulatory actions at both regional and systemic levels, since forestomach walls express immune receptors and cytokines, and the rumen liquor is infiltrated by leukocytes. Therefore, the rumen fluid could be conveniently collected for investigating metabolic production diseases. Thus, we investigated the origin of the leukocytes of the rumen fluid and demonstrated that they partly derive from saliva. Next, we carried out a field survey of innate immunity in rumen fluids of 128 cows from 12 dairy farms, along with clinical inspections, assessment of milk yield, rumen pH, volatile fatty acids (VFA) and major inflammo-metabolic parameters. Significant statistical correlations were found between immune markers in rumen fluids and biochemical parameters. A significant negative correlation was found in rumen between CD45 gene expression (leukocyte infiltration) and pH level. B cells were the most frequent mononuclear leukocyte population in the rumen liquor and their infiltration was negatively affected by low ruminal pH and high concentrations of VFA. Moreover, total Ig and IgM in rumen fluids were negatively correlated with ruminal pH and positively correlated with uremia. Our data suggest that forestomach immune responses could be directed to "dangers" arising within the forestomach environment. The immune markers could integrate consolidated diagnostic parameters (e.g. rumen pH) and contribute to robust, early diagnosis of tricky digestive troubles of cattle.

Improving the prediction ability of FT-MIR spectroscopy to assess titratable acidity in cow's milk.

This study investigated the potential application of Fourier transform mid-infrared spectroscopy (FT-MIR) for the determination of titratable acidity (TA) in cow's milk. The prediction model was developed on 201 samples collected from cows in early and late lactation, and was successively used to predict TA on samples collected from cows in early lactation and in samples with high somatic cell count. The root mean square error of cross-validation of the model by using external validation dataset was 0.09 °Soxhlet-Henkel/50 mL. Applying the model on milk samples from cows in early lactation or with high somatic cell count, the root mean square error of prediction was 0.163 °Soxhlet-Henkel/50 mL, with a RER and RPD of 23.9 and 5.1, respectively. Our results seem to indicate that FT-MIR can be used in individual milk samples to accurately predict TA, and has the potential to be adopted to measure routinely the TA of milk.

Effect of DNA extraction and sample preservation method on rumen bacterial population.

The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile.

Plasma homocysteine concentration, C677T MTHFR genotype, and 844ins68bp CBS genotype in young adults with spontaneous cervical artery dissection and atherothrombotic stroke.

BACKGROUND AND PURPOSE: The role of mild hyperhomocysteinemia as a risk factor for cerebral ischemia may depend on stroke subtype. To test this hypothesis, we undertook a prospective case-control study of a group of patients with spontaneous cervical artery dissection (sCAD), a group of patients with atherothrombotic stroke (non-CAD), and a group of control subjects. METHODS: Fasting total plasma homocysteine (tHcy) concentration, C677T MTHFR genotype, and 844ins68bp CBS genotype were determined in 25 patients with sCAD, 31 patients <45 years of age with non-CAD ischemic stroke, and 36 control subjects. Biochemical data in the patient groups were obtained within the first 72 hours of stroke onset. RESULTS: Median tHcy levels were significantly higher in patients with sCAD (13.2 micromol/L; range, 7 to 32.8 micromol/L) compared with control subjects (8.9 micromol/L; range, 5 to 17.3 micromol/L; 95% CI, 1.05 to 1.52; P=0.006). Cases with tHcy concentration above the cutoff level of 12 micromol/L were significantly more represented in the group of patients with sCAD compared with control subjects (64% versus 13.9%; 95% CI, 2.25 to 44.23; P=0.003); a significant association between the MTHFR TT genotype and sCAD was also observed (36% versus 11.1%; 95% CI, 1.10 to 19.23; P=0.045). No significant difference in tHcy levels and in the prevalence of thermolabile MTHFR was found between patients with non-CAD ischemic stroke and control subjects and between patients with sCAD and non-CAD ischemic stroke. The distribution of the 844ins68bp CBS genotype and the prevalence of subjects carrying both the TT MTHFR and 844ins68bp CBS genotypes were not significantly different among the 3 groups. CONCLUSIONS: Our results are consistent with the hypothesis that increased plasma homocysteine levels and the TT MTHFR genotype may represent risk factors for sCAD. In contrast, their role in atherothrombotic strokes remains a contentious issue.