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The weaning process in pig production is commonly associated to low feed intake, slow growth rate and increased morbidity of piglets. This study evaluates a nutritional intervention consisting of supplementing with a fish oil product rich in mono- and diglycerides (FOMG) and containing 0.069 g/g of ω-3, to improve animal health and productive performance during the post-weaning period. In a preliminary experiment (experiment 1), a total of 136 piglets were randomly distributed in two groups (4 pens per group) to evaluate the productive effects of dietary supplementation with FOMG at 15 g/kg (T15) in substitution of lard (T0) as fat source during the post-weaning period in a commercial farm. Besides, in experiment 2 a total of 72 weaned piglets were fed on a control diet (T0) or supplemented with 15 (T15) or 30 g FOMG/kg (T30) in substitution of sunflower oil, with 6 pens of 4 piglets per treatment. In experiment 2 growth and intake were weekly controlled, and blood was sampled on days 14 and 34. At day 35 post-weaning, 6 piglets per treatment were euthanized to study the microbial fermentation and the ileal and caecal bacterial community by 16 S amplicon sequencing. Results indicated that piglets fed T15 diet tended to have a higher growth gain during the post-weaning period in experiment 1 (P = 0.067). This increased growth was partially explained by a greater feed intake (0.14 higher) but also due to improved animal health as showed by the lower proportion of neutrophils (P = 0.006), blood cortisol (P = 0.098) and morbidity (P < 0.05) in experiment 2. Treatment T15 also tended (P = 0.064) to promote a higher volatile fatty acids (VFA) concentration at the ileum, which could be compatible with a higher nutrient absorption and a subsequent lower VFA concentration in the hindgut (P < 0.001). Moreover, FOMG supplementation at T15 exerted modulatory effects on the gut microbiota promoting a shift in the bacterial community structure, lower diversity (Richness index, P < 0.05) and a trend (P = 0.076) for a higher butyrate proportion at the ileum, together with a lower (P < 0.05) and most favourable Firmicutes-to-Bacteroidetes ratio at the caecum. On the contrary, T30 diet promoted less beneficial effects than T15. These findings indicated that supplementation of piglets with FOMG at a level of 15 g/kg represents a suitable strategy to improve pig performance and gut health during the post-weaning period.

Enteric methane (CH4) emissions from sheep contribute to global greenhouse gas emissions from livestock. However, as already available for dairy and beef cattle, empirical models are needed to predict CH4 emissions from sheep for accounting purposes. The objectives of this study were to: 1) collate an intercontinental database of enteric CH4 emissions from individual sheep; 2) identify the key variables for predicting enteric sheep CH4 absolute production (g/d per animal) and yield [g/kg dry matter intake (DMI)] and their respective relationships; and 3) develop and cross-validate global equations as well as the potential need for age-, diet-, or climatic region-specific equations. The refined intercontinental database included 2,135 individual animal data from 13 countries. Linear CH4 prediction models were developed by incrementally adding variables. A universal CH4 production equation using only DMI led to a root mean square prediction error (RMSPE, % of observed mean) of 25.4% and an RMSPE-standard deviation ratio (RSR) of 0.69. Universal equations that, in addition to DMI, also included body weight (DMI + BW), and organic matter digestibility (DMI + OMD + BW) improved the prediction performance further (RSR, 0.62 and 0.60), whereas diet composition variables had negligible effects. These universal equations had lower prediction error than the extant IPCC 2019 equations. Developing age-specific models for adult sheep (>1-year-old) including DMI alone (RSR = 0.66) or in combination with rumen propionate molar proportion (for research of more refined purposes) substantially improved prediction performance (RSR = 0.57) on a smaller dataset. On the contrary, for young sheep (, Journal of Cleaner Production, 384, ISSN:0959-6526

Ruminants are born with an undeveloped physical, metabolic, and microbial rumen. Rumen development is limited under artificial rearing systems when newborn animals are separated from the dam, fed on milk replacer, and weaned at an early age. This study aims to evaluate the effects of early-life inoculation of young ruminants with rumen fluid from adult animals. Eighty newborn goat kids were randomly allocated to 1 of 4 experimental treatments and inoculated daily from d 1 to wk 11 with autoclaved rumen fluid (AUT), fresh rumen fluid obtained from adult goats fed either a forage diet (RFF) or concentrate-rich diet (RFC), or absence of inoculation (CTL). Goat kids were artificially reared with ad libitum access to milk replacer, starter concentrate, and forage hay. Blood was sampled weekly and rumen microbial fermentation was monitored at 5 (preweaning), 7 (weaning), and 9 wk of age (postweaning). Results indicated that inoculation with fresh rumen fluid accelerated the rumen microbial and fermentative development before weaning. As a result, RFC and RFF animals had higher solid feed intake (+73%), rumen concentrations of ammonia-N (+26%), total volatile fatty acids (+46%), butyrate (+50%), and plasma β-hydroxybutyrate (+48%), and lower milk intake (−6%) than CTL and AUT animals at wk 5. Inoculation with fresh inoculum also promoted early rumen colonization by a complex and abundant protozoal community, whereas CTL animals remained protozoa free. Although all kids experienced moderate growth retardation during 1 wk after weaning, inoculation with fresh rumen fluid favored the weaning process, leading to 2.2 times higher weight gain than CTL and AUT animals during wk 8. Some of these advantages were retained during the postweaning period and RFF and RFC animals showed higher forage intake (up to +44%) than CTL and AUT animals with no detrimental effects on feed digestibility or stress levels. The superior microbial load of RFC compared with RFF inoculum tended to provide further improvements in terms of forage intake, plasma β-hydroxybutyrate, and rumen protozoa, whereas AUT inoculation provided minor (if any) advantages with respect to CTL animals. Although no differences were noted on animal growth, this study suggests that early life inoculation of goat kids with rumen microbiota can represent an effective strategy to accelerate the rumen development, facilitating a smooth transition from milk to solid feed and to the potential implementation of early weaning strategies., This study was funded by the Spanish government through the project AGL2017-86938-R and the Training Program for Academics grant, Madrid, Spain (FPU16/01981).

Ruminants are able to produce large quantities of saliva which enter into the rumen. Although previous research has indicated that salivary immunoglobulins can partially modulate the rumen microbial activity, the role of the salivary components other than ions on the rumen microbial ecosystem has not been thoroughly investigated in ruminants. A total of 16 semi-continuous in vitro cultures were used to incubate rumen fluid from 4 donor goats inoculated with autoclaved saliva (AUT) as negative control, saliva from the same rumen fluid donor (OWN) as positive control, and either GOAT or SHEEP saliva as experimental interventions. Fermentation was monitored throughout the 7 days of incubation and the prokaryotic communities and metabolome were analysed at day 7 of incubation. Characterization of the salivas used prior to incubation showed a high degree of individual variability in terms of the salivary metabolites and proteins, including immunoglobulins. The prokaryotic community composition in AUT incubators was the most divergent across treatments, suggesting a modulatory effect of active salivary components, which were not affected in the other treatments (OWN, GOAT and SHEEP). The differences across treatments in microbial diversity were mostly caused by a greater abundance of Proteobacteria and Rikenellacea and lower of Prevotellaceae, a key rumen bacterium with greater abundance in GOAT and SHEEP treatments. These results suggest that specific salivary components contribute to host-associated role in selecting the rumen commensal microbiota and its activity.

The research about the role of saliva in ruminants has been mainly focused on its buffering capacity together with facilitation of the rumination process. However, the role of salivary bioactive components on modulating the activity of the rumen microbiota has been neglected until recently. This study developed an in vitro approach to assess the impact of different components in saliva on rumen microbial fermentation. Four different salivary fractions were prepared from four goats: (i) non-filtrated saliva (NFS), (ii) filtrated through 0.25 µm to remove microorganisms and large particles (FS1), (iii) centrifuged through a 30 kDa filter to remove large proteins, (FS2), and (iv) autoclaved saliva (AS) to keep only the minerals. Two experiments were conducted in 24 h batch culture incubations with 6 ml of total volume consisting of 2 ml of rumen fluid and 4 ml of saliva/buffer mix. In Experiment 1, the effect of increasing the proportion of saliva (either NFS or FS1) in the solution (0%, 16%, 33% and 50% of the total volume) was evaluated. Treatment FS1 promoted greater total volatile fatty acids (VFA) (+8.4%) and butyrate molar proportion (+2.8%) but lower NH-N concentrations than NFS fraction. Replacing the bicarbonate buffer solution by increasing proportions of saliva resulted in higher NH-N, total VFA (+8.0%) and propionate molar proportion (+11%). Experiment 2 addressed the effect of the different fractions of saliva (NFS, FS1, FS2 and AS). Saliva fractions led to higher total VFA and NH-N concentrations than non-saliva incubations, which suggests that the presence of some salivary elements enhanced rumen microbial activity. Fraction FS1 promoted a higher concentration of total VFA (+7.8%) than the other three fractions, and higher propionate (+26%) than NFS and AS. This agrees with findings from Experiment 1 and supports that ‘microbe-free saliva’, in which large salivary proteins are maintained, boosts rumen fermentation. Our results show the usefulness of this in vitro approach and suggest that different salivary components can modulate rumen microbial fermentation, although the specific metabolites and effects they cause need further research., This work was financially supported by the Spanish Research Agency (AEI) through the project AGL2017-86938-R and the Training Program for Academics Grant, Madrid, Spain (FPU16/01981)

BACKGROUND: Meeting the energy and nitrogen (N) requirements of high-performing ruminants at the same time as avoiding digestive disturbances (i.e. rumen acidosis) is a key priority in ruminant nutrition. The present study evaluated the effect of a cereal ammoniation treatment, in which barley grains are combined with urea and enzymes that catalyze the conversion of urea to ammonia to optimize rumen function. Twelve rumen cannulated sheep were randomly divided into two groups and fed a diet containing 60% of ammoniated barley (AMM) or untreated barley supplemented with urea (CTL) to investigate the impact on rumen fermentation and feed utilization. RESULTS: AMM had higher total N content and effective rumen degradable N than untreated barely. AMM sheep had a consistently higher rumen pH throughout the day (6.31 versus 6.03) and tended to have a lower post-prandial ammonia peak and higher acetate molar proportion (+5.1%) than CTL sheep. The rumen environment in AMM sheep favored the colonization and utilization of agro-industrial by-products (i.e. orange pulp) by the rumen microbes leading to a higher feed degradability. AMM sheep also had higher total tract apparent N digestibility (+21.7%) and urinary excretion of purine derivatives (+34%), suggesting a higher N uptake and microbial protein synthesis than CTL sheep. CONCLUSION: The inclusion of AMM in the diet of ruminants represents a valid strategy for maintaining rumen pH within a physiological range and improving N utilization by the rumen microbes, which could have positive effects on the health and productivity of animals in intensive production systems. These findings warrant further studies under conventional farm conditions. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., This work was supported by the European Union´s Horizon 2020 research and innovation program under grant agreement No818368 (MASTER) and Harbro Ltd.

Background The knowledge about blood circulating microbiome and its functional relevance in healthy individuals remains limited. An assessment of changes in the circulating microbiome was performed by sequencing peripheral blood mononuclear cells (PBMC) bacterial DNA from goats supplemented or not in early life with rumen liquid transplantation. Results Most of the bacterial DNA associated to PBMC was identified predominantly as Proteobacteria (55%) followed by Firmicutes (24%), Bacteroidetes (11%) and Actinobacteria (8%). The predominant genera found in PBMC samples were Pseudomonas, Prevotella, Sphingomonas, Acinetobacter, Corynebacterium and Ruminococcus. Other genera such as Butyrivibrivio, Bifidobacterium, Dorea and Coprococcus were also present in lower proportions. Several species known as blood pathogens or others involved in gut homeostasis such as Faecalibacterium prausnitzii were also identified. However, the PBMC microbiome phylum composition differed from that in the colon of goats (P ≤ 0.001), where Firmicutes was the predominant phylum (83%). Although, rumen liquid administration in early-life altered bacterial community structure and increased Tlr5 expression (P = 0.020) in colon pointing to higher bacterial translocation, less than 8% of OTUs in colon were also observed in PBMCs. Conclusions Data suggest that in physiological conditions, PBMC microbiome differs from and is not affected by colon gut microbiota in small ruminants. Although, further studies with larger number of animals and covering other animal tissues are required, results point to a common circulating bacterial profile on mammals being phylum Proteobacteria, and genera Pseudomonas and Prevotella the most abundants. All suggest that PBMC microbiome in healthy ruminants could be implicated in homeostatic condition. This study expands our knowledge about PBMC microbiome contribution to health in farm animals.

This work was partially supported by the Spanish Research Agency (Ref. AGL2017-86938-R).

The implementation of Precision Livestock Farming (PLF) concepts has been pointed out as an indirect strategy that could potentially help mitigating the environmental impacts of livestock production systems. To date, few studies have focused on analyzing specifically the relationship among PLF adoption and environmental performance, so sustainability benefits have not yet been quantified for many technologies. Moreover, studies evaluating the environmental impact of dairy production have traditionally focused on cattle, and when exploring sheep or goats, they have often involved extensive, low-productive systems, providing an incomplete picture of the sector. In this study we apply life cycle assessment (LCA) to analyze the environmental impact associated to intensive dairy goat production, and to explore the influence of adopting a smart-farming PLF platform on the environmental performance of a group of dairy goat farms in Spain. The PLF-platform relies on systematic on-farm monitoring of individual animal data, coupled with big data processing and interpretation, which supports farmers to take adequate -and timely-farm management decisions. In order to capture its influence, two different periods were analyzed in five selected farms: a baseline year just before innovation was implemented (2014) and four years after (2018), when most of the effect of improved management was reflected. Results after the PLF-platform implementation showed significant reductions (−11%) in greenhouse gas emissions and similar trends in other impact categories (9–16% reductions). The PLF platform provided a precise monitoring of the productivity, genetic merit and physiological state of each animal, allowing adequate criteria for a number of decision-making processes, such as selecting animals for breeding, replacement or culling. This optimization led to an increase in the genetic selection progress, ultimately reflected on milk productivity. Moreover, a reduction of unproductive periods such as first partum age or dry period length was often achieved. As a result of this general improvement, the efficiency of resource usage in relation to milk (and meat) production was increased, with positive effect on the environmental performance. Production of 1 kg of fat and protein corrected milk (FPCM) resulted in 1.53–1.71 kg CO eq. Results in other impacts categories were also in a similar range than previously reported values for highly productive dairy systems, including dairy cattle, which stresses the important role that small ruminant farming can play on environmentally sustainable livestock production, particularly in the Mediterranean context., This research is supported by the Spanish Government through María de Maeztu excellence accreditation 2018–2022 (Ref. MDM-2017-0714) and by the Basque Government through the BERC 2018–2021 program. This work was also supported by the Horizon2020 SFS-01c-2015 project entitled “Innovation of sustainable sheep and goat production in Europe (iSAGE)” (grant number 679302). Alejandro Belanche and Agustin del Prado are funded by the Ramon y Cajal program from the Spanish Ministry of Sciences and Innovation (RYC2019-027764 and RYC-2017-22143, respectively).

Ruminant agriculture suffers from inefficient capture of forage protein and consequential release of N pollutants to land. This is due to proteolysis in the rumen catalyzed by both microbial but initially endogenous plant proteases. Plant breeding‐based solutions are sought to minimize these negative environmental impacts. The aim of this study was to perform an integrated study of rumen N metabolism using semi‐continuous rumen simulation fermenters (Rusitec) to explore the extent to which swards containing Festulolium populations (interspecific hybrids between Lolium and Festuca grass species) with decreased rates of endogenous protein degradation conferred advantageous protein utilization in comparison with a National Listed perennial ryegrass. An in vitro experiment was conducted using three Festulolium hybrids (Lolium perenne × Festuca arundinacea var. glaucescens, LpFg; Lolium perenne × Festuca mairei, LpFm; and Lolium multiflorum × Festuca arundinacea var. glaucescens, LmFg) and a Lolium perenne, Lp control. LpFm and LmFg demonstrated significantly lower plant‐mediated proteolysis than the control. Fresh forage was incubated in Rusitec with rumen fluid from four donor cows. Feed disappearance and production of gas, methane, and volatile fatty acids were similar across cultivars. Whereas no differences in microbial protein synthesis were noted across treatments during early fermentation (0–6 hr after feeding), an increased microbial N flow in LpFm (+30%) and LmFg hybrids (+41%) was observed during late fermentation (6–24 hr after feeding), with higher overall microbial N flows (+13.5% and + 20.2%, respectively) compared with the control (Lp). We propose an underpinning mechanism involving the partitioning of amino acid catabolism toward branched‐chain amino acids and microbial protein synthesis in grasses with slow plant‐mediated proteolysis instead of accumulation of rumen ammonia in grasses with fast plant‐mediated proteolysis. These observations indicate the potential of Festulolium hybrids with a slow plant‐mediated proteolysis trait to improve the efficiency of capture of forage protein and decrease the release of N pollutants onto the land., Excessive protein loss from fresh forage feeds is a problem for ruminant agriculture. Festulolium hybrids identified with lower endogenous induced proteolysis in response to rumen conditions also demonstrated improved N flows in rumen‐simulating fermenters and decreased ammonia production with no negative impact on microbial protein synthesis. These hybrids offer considerable potential for decreasing the environmental impact of ruminant agriculture.

There is an increasing pressure to identify feed additives which increase productivity or decrease methane emissions. This paper aims to elucidate the effects of supplementing a specific essential oils blend Agolin&reg, Ruminant on the productivity of dairy cows in comparison to non-treated animals. A total of 23 in vivo studies were identified in which Agolin was supplemented at 1 g/d per cow, then a meta-analysis was performed to determine the response ratio on milk yield, rumen fermentation, methane emissions and health. Results indicated that an adaptation period of at least 4 weeks of treatment is required. Whereas short-term studies showed minor and inconsistent effects of Agolin, long-term studies (&gt, 4 weeks of treatment) revealed that Agolin supplementation increases milk yield (+3.6%), fat and protein corrected milk (+4.1%) and feed efficiency (+4.4%) without further changes in milk composition and feed intake. Long-term treatment also decreased methane production per day (&minus, 8.8%), per dry matter intake (&minus, 12.9%) and per fat and protein corrected milk yield (&minus, 9.9%) without changes in rumen fermentation pattern. In conclusion, despite the mode of action is still unclear and the small number of studies considered, these findings show that Agolin represents an encouraging alternative to improve productivity in dairy cows.

Condensed tannins are polyphenolic compounds which can exert beneficial effects in ruminants. They have the ability to bind proteins and decrease their degradation. They have also been reported to reduce methanogenesis and improve ruminants performances. The present work aimed to study the effect of purified condensed tannins from Acacia horrida extract on fermentation parameters, gas and methane production. In this context, seven substrates were selected: four Acacia species (Acacia pycnantha, Acacia dealbata, Acacia horrida and Acacia cyanophylla) and three plant by-products (date palm leaves, grenade peel and artichoke stems). Biological activity of tannins was evaluated by the incubation of the substrates in vitro with Polyethylene glycol (PEG). Tannins decreased concentration of all fermentation parameters (gas (p < 0.0001), methane (CH4) (p= 0.0983), ammonia (N-NH3) (p= 0.0382), Volatile Fatty Acids (VFA) (p= 0.0009), acetate (p 100 g/kg DM) and moderate cell-wall components. CT content was comprised between 120.5 and 680.4 g/kg DM in plants and between 23.1 and 170.4 g/kg DM in plant by-products. A. horrida presented the highest biological activity. Thus, their CT were extracted and purified on a sephadex LH-20 column. Purified tannins from A. horrida were incubated with two different forages alfalfa hay and barley straw at three different concentrations: 50, 100, 150 mg/g DM. Results showed that purified tannins from A. horrida had no effect on fermentation parameters (P > 0.05). The effects of A. horrida tannins may be strongly linked to their structure and their molecular weight more than to their concentration. For this reason, it is interesting that this work may be completed by physical characterization of these tannins.

BACKGROUND: The rumen microbiota has been used as inoculum for in vitro studies and as a probiotic to improve productivity in young animals. However, great variability across studies has been noted depending on the inoculum considered. The present study aims to assess the relevance of different factors (microbial fraction, collection time, donor animal diet, fermentation substrate and inoculum preservation method) to maximize the rumen inoculum activity and set the standards for further in vitro and in vivo applications. RESULTS: Rumen inoculum sampled at 3 h after feeding led to greater microbial growth and activity [+12% volatile fatty acid (VFA), +17% ammonia] compared to before feeding. Similar results were noted when rumen liquid or rumen content were used as inocula. Rumen inoculum adapted to concentrate diets increased microbial activity (+19% VFA) independently of the substrate used in vitro. Freezing-thawing the inoculum, in comparison to fresh inoculum, decreased microbial activity (−14% VFA, −96% ammonia), anaerobic fungi and protozoa, with holotrichs protozoa being particularly vulnerable. Inoculum lyophilization had a stronger negative effect on microbial activity (−51% VFA) and delayed re-activation of the microbes, leading to lower levels of methanogens and anaerobic fungi, as well as almost complete wipe out of rumen protozoa. CONCLUSIONS: Fresh rumen fluid sampled at 3 h after feeding from donor animals that were fed concentrate diets should be chosen when the aim is to provide the most diverse and active rumen microbial inoculum. © 2018 Society of Chemical Industry., This study was funded by the Economy and Competitiveness Spanish Ministry (Ref. AGL2017-86938-R) and the CSIC (PIE201640E045)

Increasing productivity is a key target in ruminant science which requires better understanding of the rumen microbiota. This study investigated how maternal versus artificial rearing shapes the rumen microbiota using 24 sets of triplet lambs. Lambs within each sibling set were randomly assigned to natural rearing on the ewe (NN); ewe colostrum for 24 h followed by artificial milk feeding (NA); and colostrum alternative and artificial milk feeding (AA). Maternal colostrum feeding enhanced VFA production at weaning but not thereafter. At weaning, lambs reared on milk replacer had no rumen protozoa and lower microbial diversity, whereas natural rearing accelerated the rumen microbial development and facilitated the transition to solid diet. Differences in the rumen prokaryotic communities disappear later in life when all lambs were grouped on the same pasture up to 23 weeks of age. However, NN animals retained higher fungal diversity and abundances of Piromyces, Feramyces and Diplodiniinae protozoa as well as higher feed digestibility (+4%) and animal growth (+6.5%) during the grazing period. Nevertheless, no correlations were found between rumen microbiota and productive outcomes. These findings suggest that the early life nutritional intervention determine the initial rumen microbial community, but the persistence of these effects later in life is weak., This work was supported by the European Regional Development Fund Program through the Welsh Government (WISE Network) and BBSRC strategic fund (BBS/E/ W/10964A01).

The European small ruminants (i.e. sheep and goats) farming sector (ESRS) provides economic, social and environmental benefits to society, but is also one of the most vulnerable livestock sectors in Europe. This sector has diverse livestock species, breeds, production systems and products, which makes difficult to have a clear vision of its challenges through using conventional analyses. A multi-stakeholder and multi-step approach, including 90 surveys, was used to identify and assess the main challenges for the sustainability of the ESRS to prioritize actions. These challenges and actions were identified by ESRS experts including farmers, cooperatives, breeding associations, advisers and researchers of six EU countries and Turkey. From the 30 identified challenges, the most relevant were economy-related challenges such as ‘uncertainty of meat and milk prices’, ‘volatility of commodity prices’, ‘low farm income’, ‘high subsidy dependency’ and ‘uncertainty in future changes in subsidies’ resulting in ‘a sector not attractive to young farmers’. Most of these challenges were beyond the farmer's control and perceived as difficult to address. Challenges were prioritized using an index, calculated by multiplying the relevance and the feasibility to address measures. The identified challenges had a similar priority index across the whole sector with small differences across livestock species (sheep vs goats), type of products (meat vs dairy) and intensification levels (intensive vs semi-intensive vs extensive). The priorities were different, however, between socio-geographical regions (Southern vs Central Europe). Some of the top prioritized challenges were linked to aspects related to the production systems (‘low promotion of local breeds’ and ‘slow adaptability of high producing breeds’) and market practices (‘unfair trade/lack of traceability’). The majority of the priority challenges, however, were associated with a deficient knowledge or training at farm level (‘poor business management training’, ‘lack of professionalization’, ‘slow adoption of innovations’), academia (‘researchers do not address real problems’) and society as a whole (‘low consumer education in local products’, ‘low social knowledge about farming’, ‘poor recognition of farming public services’). Thus, improved collaboration among the different stakeholders across the food chain with special implication of farmers, associations of producers, academia and governments is needed to facilitate knowledge exchange and capacity building. These actions can contribute to make ESRS economically more sustainable and to adapt the production systems and policy to the current and future societal needs in a more region-contextualized framework., This study was supported by the EU Horizon 2020 Research and In-novation Action (RIA) through the project‘Innovation for sustainablesheep and Goat production in Europe (iSAGE)’under grant agreement 679302

Festulolium is an interspecific hybrid between Lolium and Festuca species which combines the superior forage quality characteristics of ryegrass with fescue’s ability to grow in harsh environments. The aim of this study was to perform an integrated study of rumen nitrogen metabolism by using rumen simulation to explore the extent to which swards containing Festulolium populations previously shown to have decreased rates of endogenous protein degradation can confer advantageous protein utilisation in comparison with a currently market leading diploid ryegrass. An in vitro experiment was therefore conducted using three Festulolium hybrids: L. perenne × F. glaucescens(LpFg); L. perenne × F. mairei(LpFm); and L. multiflorum × F. glaucescens(LmFg) and Lolium perenne (Lp) as a control. Festulolium hybrids had higher conversion ratios of nitrogen into microbial protein than Lolium perenne thus suggesting greater potential to improve ruminal nitrogen use efficiency in a fresh feeding system. This would help to increase production efficiency as well as decreasing the environmental impact of livestock agriculture.

Background Eight natural products from animal, unicellular algae, brown seaweed and plant origins were chosen according to their theoretical antimicrobial activity: Diatomaceous earths (DE), insoluble chitosan (ICHI), soluble chitosan (CHI), seaweed meal (SWM), Ascophyllum nodosum (ASC), Laminaria digitata (LAM), neem oil (NOIL) and an ivy fruit extract rich in saponins (IVY). Dose-response incubations were conducted to determine their effect on rumen fermentation pattern and gas production, while their anti-protozoal activity was tested using (14) C-labelled bacteria. Results DE, SWM, NOIL and ICHI had very small effects on rumen function when used at inclusion rate up to 2 g L(-1) . ASC had anti-protozoal effects (up to -23%) promoting a decrease in gas production and methanogenesis (-15%). LAM increased VFA production (+7%) and shifted from butyrate to acetate. CHI also shifted fermentation towards propionate production and lower methane (-23%) and protozoal activity (-56%). IVY decreased protozoal activity (-39%) and ammonia concentration (-56%), as well as increased feed fermentation (+11% VFA concentration) and shifted from acetate to propionate production. Conclusions ASC, LAM, CHI and IVY showed promising potential in vitro as feed additives to improve rumen function, thus more research is needed to investigate their mode of action in the rumen microbial ecosystem. © 2015 Society of Chemical Industry.

The aim of this work was to elucidate the best conditions for preparing rumen microbiome as pro and prebiotic by the analysis of fermentation parameters resulting from in vitro incubations (trials 1 and 2), for subsequent use in in vivo trial (trial 3) consisting of an inoculation of newborn goat kids with the ruminal microbiota developed in the in vitro experiment. This study used a 2×3×2×2 factorial design to evaluate the effects of two diets (forage and concentrate), three buffers (6.80, 6.25 y 5.75), two sample times (0 vs 3h) and two ruminal fractions (liquid vs whole content) of the ruminal content. Eight Murciano-Granadina goats were used as a donor of the ruminal content; each four animals were assigned to one group: FOR received 100% forage (based on 50% alfalfa and 50% oat hay) and CON fed with concentrate and forage (80:20) with the same forage composition. In the in vivo experiment, seventy four goat kids were randomly distributed into 4 identical groups with 16 and 4 animals (4x4) per group and experimental group respectively: CTL (control group without any inoculation), PRE (group inoculated with ruminal liquor autoclaved), LRC (probiotic group inoculated with ruminal liquor of animals fed concentrate diet) and LRF (probiotic group inoculated with ruminal liquor of animals fed forage diet). Experimental groups had similar average birth weight and the same number of males and females. Newborn goats were daily inoculated with fresh rumen fluid obtained from the same donors used in in vitro experiment during the first 12 weeks of life. The feed intake (milk, concentrate and forage), body weight and rumen fermentation were studied during the same inoculation period. In the first trial, both diet (FOR vs CON) and buffer were studied; the result showed that independently of the diet, the buffer which generates higher pH promotes higher fermentation activity in terms of VFAs (P=0.001), ammonia (P=0.005), fermentable organic matter (P= 0.006) and gas production (P

Increasing the efficiency of utilization of fresh and preserved forage is a key target for ruminant science. Vitamin E is often used as additive to improve product quality but its impact of the rumen function is unknown. This study investigated the successional microbial colonization of ryegrass (GRA) vs. ryegrass hay (HAY) in presence of zero or 50 IU/d supplementary vitamin E, using a rumen simulation technique. A holistic approach was used to link the dynamics of feed degradation with the structure of the liquid-associated (LAB) and solid-associated bacteria (SAB). Results showed that forage colonization by SAB was a tri-phasic process highly affected by the forage conservation method: Early colonization (0-2 h after feeding) by rumen microbes was 2× faster for GRA than HAY diets and dominated by Lactobacillus and Prevotella which promoted increased levels of lactate (+56%) and ammonia (+18%). HAY diets had lower DM degradation (-72%) during this interval being Streptococcus particularly abundant. During secondary colonization (4-8 h) the SAB community increased in size and decreased in diversity as the secondary colonizers took over (Pseudobutyrivibrio) promoting the biggest differences in the metabolomics profile between diets. Secondary colonization was 3× slower for HAY vs. GRA diets, but this delay was compensated by a greater bacterial diversity (+197 OTUs) and network complexity resulting in similar feed degradations. Tertiary colonization (> 8 h) consisted of a slowdown in the colonization process and simplification of the bacterial network. This slowdown was less evident for HAY diets which had higher levels of tertiary colonizers (Butyrivibrio and Ruminococcus) and may explain the higher DM degradation (+52%) during this interval. The LAB community was particularly active during the early fermentation of GRA and during the late fermentation for HAY diets indicating that the availability of nutrients in the liquid phase reflects the dynamics of feed degradation. Vitamin E supplementation had minor effects but promoted a simplification of the LAB community and a slight acceleration in the SAB colonization sequence which could explain the higher DM degradation during the secondary colonization. Our findings suggest that when possible, grass should be fed instead of hay, in order to accelerate feed utilization by rumen microbes., This work has been supported by the BBRSC (BBS/E/W/10964A01) and by the EU Regional Development Fund Program through the Welsh Government (WISE Network). Donation of vitamin E and concentrate was much appreciated (Wynnstay and Celtic Pride Ltd., United Kingdom).

Protozoa-associated methanogens (PAM) are considered one of the most active communities in the rumen methanogenesis. This experiment investigated whether methanogens are sequestrated within rumen protozoa, and structural differences between rumen free-living methanogens and PAM. Rumen protozoa were harvested from totally faunated sheep, and six protozoal fractions (plus free-living microorganisms) were generated by sequential filtration. Holotrich-monofaunated sheep were also used to investigate the holotrich-associated methanogens. Protozoal size determined the number of PAM as big protozoa had 1.73.3 times more methanogen DNA than smaller protozoa, but also more endosymbiotic bacteria (2.2- to 3.5-fold times). Thus, similar abundance of methanogens with respect to total bacteria were observed across all protozoal fractions and free-living microorganisms, suggesting that methanogens are not accumulated within rumen protozoa in a greater proportion to that observed in the rumen as a whole. All rumen methanogen communities had similar diversity (22.2 ± 3.4 TRFs). Free-living methanogens composed a conserved community (67% similarity within treatment) in the rumen with similar diversity but different structures than PAM (P< 0.05). On the contrary, PAM constituted a more variable community (48% similarity), which differed between holotrich and total protozoa (P< 0.001). Thus, PAM constitutes a community, which requires further investigation as part of methane mitigation strategies. This work was supported by the Commission of the European Communities (REDNEX project FP7-KBBE-2007-1) and the Welsh Government.

Rumen function is generally suboptimal leading to losses in methane and nitrogen. Analysis of the rumen microbiome is thus important to understanding the underlying microbial activity under different feeding strategies. This study investigated the effect of forage conservation method and vitamin E supplementation on rumen function using a rumen simulation technique. Ryegrass (GRA) or ryegrass hay (HAY) was supplemented with 20% concentrate containing zero or 50 IU/d vitamin E, as α-tocopheryl acetate, according to a 2 × 2 factorial design. The forage conservation method did not substantially change the nutrient composition but had a profound impact on the structure and diversity of the rumen microbiome. HAY diets promoted a more complex bacterial community (+38 OTUs) dominated by Firmicutes. This bacterial adaptation, together with increased rumen protozoa levels and methanogen diversity, was associated with greater fiber disappearance (+12%) in HAY diets, but also with greater rumen true N degradability (+7%) than GRA diets. HAY diets also had a higher metabolic H recovery and methane production (+35%) suggesting more efficient inter-species H transfer between bacteria, protozoa and methanogens. Contrarily, GRA diets promoted more simplified methanogen and bacterial communities, which were dominated by Bacteroidetes and Lactobacillus, thus lactate formation may have acted as an alternative H sink in GRA diets. Moreover the structure of the bacterial community with GRA diets was highly correlated with N utilization, and GRA diets promoted greater bacterial growth and microbial protein synthesis (+16%), as well as a more efficient microbial protein synthesis (+22%). A dose-response experiment using batch cultures revealed that vitamin E supplementation increased rumen fermentation in terms of total VFA and gas production, with protozoal activity higher when supplying α-tocopheryl acetate vs. α-tocopherol. Moreover, α-tocopheryl acetate promoted a small increase in feed degradability (+8%), possibly as a result of its antioxidant properties which led to higher bacterial and protozoal levels. Vitamin E supplementation also modified the levels of some methanogen species indicating that they may be particularly sensitive to oxidative stresses. Our findings suggested that when possible, grass should be fed instead of grass hay, in order to improve rumen function and to decrease the environmental impact of livestock agriculture.

There is an increasing need to identify alternative feeds for livestock that do not compete with foods for humans. Seaweed might provide such a resource, but there is limited information available on its value as an animal feed. Here we use a multi-omics approach to investigate the value of two brown seaweeds, Ascophyllum nodosum (ASC) and Laminaria digitata (LAM), as alternative feeds for ruminants. These seaweeds were supplemented at 5% inclusion rate into a control diet (CON) in a rumen simulation fermenter. The seaweeds had no substantial effect on rumen fermentation, feed degradability or methane emissions. Concentrations of total bacteria, anaerobic fungi, biodiversity indices and abundances of the main bacterial and methanogen genera were also unaffected. However, species-specific effects of brown seaweed on the rumen function were noted: ASC promoted a substantial decrease in N degradability (−24%) due to its high phlorotannins content. Canonical correspondence analysis of the bacterial community revealed that low N availability led to a change in the structure of the bacterial community. ASC also decreased the concentration of Escherichia coli O157:H7 post-inoculation. In contrast, LAM which has a much lower phlorotannin content did not cause detrimental effects on N degradability nor modified the structure of the bacterial community in comparison to CON. This adaptation of the microbial community to LAM diets led to a greater microbial ability to digest xylan (+70%) and carboxy-methyl-cellulose (+41%). These differences among brown seaweeds resulted in greater microbial protein synthesis (+15%) and non-ammonia N flow (+11%) in LAM than in ASC diets and thus should led to a greater amino acid supply to the intestine of the animal. In conclusion, it was demonstrated that incorporation of brown seaweed into the diet can be considered as a suitable nutritional strategy for ruminants; however, special care must be taken with those seaweeds with high phlorotannin concentrations to prevent detrimental effects on N metabolism. This study highlights the value of combining fermentation and enzyme activity data with molecular characterization of the rumen microbiome in evaluating novel feeds for ruminants. Further experiments are required to determine the maximum seaweed inclusion rate tolerated by rumen microbes.

Bacterial predation by protozoa has the most deleterious effect on the efficiency of N use within the rumen, but differences in activity among protozoal groups are not completely understood. Two in vitro experiments were conducted to identify the protozoal groups more closely related with rumen N metabolism. Rumen protozoa were harvested from cattle and 7 protozoal fractions were generated immediately after sampling by filtration through different nylon meshes at 39 °C, under a CO(2) atmosphere to maintain their activity. Protozoa were incubated with (14)C-labeled bacteria to determine their bacterial breakdown capacity, according to the amount of acid-soluble radioactivity released. Epidinium tended to codistribute with Isotricha and Entodinium with Dasytricha; therefore, their activity was calculated together. This study demonstrated that big Diplodiniinae had the greatest activity per cell (100 ng bacterial CP per protozoa and hour), followed by Epidinium plus Isotricha (36.4), small Diplodiniinae (34.2), and Entodinium plus Dasytricha (14.8), respectively. However, the activity per unit of protozoal volume seemed to vary, depending on the protozoal taxonomy. Small Diplodiniinae had the greatest activity per volume (325 ng bacterial CP per protozoal mm(3) and hour), followed by big Diplodiniinae (154), Entodinium plus Dasytricha (104), and Entodinium plus Dasytricha (25.6). A second experiment was conducted using rumen fluid from holotrich-monofaunated sheep. This showed that holotrich protozoa had a limited bacterial breakdown capacity per cell (Isotricha 9.44 and Dasytricha 5.81 ng bacterial CP per protozoa and hour) and per protozoal volume (5.97 and 76.9 ng bacterial CP per protozoal mm(3) and hour, respectively). Therefore, our findings indicated that a typical protozoal population (10(6) total protozoa/mL composed by Entodinium sp. 88%, Epidinium sp. 7%, and other species 4%) is able to break down ~17% of available rumen bacteria every hour. Entodinium sp. is responsible for most of this bacterial breakdown (70 to 75%), followed by Epidinium sp. (16 to 24%), big Diplodiniinae (4 to 6%), and small Diplodiniinae (2 to 6%), whereas holotrich protozoa have a negligible activity (Dasytricha sp. 0.6 to 1.2% and Isotricha sp. 0.2 to 0.5%). This in vitro information must be carefully interpreted, but it can be used to indicate which protozoal groups should be suppressed to improve microbial protein synthesis in vivo.

Accurate estimates of microbial synthesis in the rumen are vital to optimize ruminant nutrition. Liquid- (LAB) and solid-associated bacterial fractions (SAB) harvested from the rumen are generally considered as microbial references when microbial yield is calculated; however, factors that determine their composition are not completely understood. The aim of this study was to evaluate the effect of diet and absence or presence of rumen protozoa on the rumen microbial community. It was hypothesized that these treatments could modify the composition and representativeness of LAB and SAB. Twenty twin lambs (Ovis aries) were used; one-half of the twins were kept protozoa-free, and each respective twin sibling was faunated. At 6 mo of age, 5 animals from each group were randomly allocated to the experimental diets consisting of either alfalfa hay as the sole diet, or 50:50 mixed with ground barley grain. After 15 d of adaptation to the diet, animals were euthanized, rumen and abomasum contents were sampled, and LAB and SAB isolated. The presence of protozoa buffered the effect of diet on the rumen bacterial population. Faunated animals fed alfalfa hay had a greater abundance of F. succinogenes, anaerobic fungi and methanogens, as well as an enhanced rumen bacterial diversity. Cellulolytic bacteria were more abundant in SAB, whereas the abomasal abundance of most of the microorganisms studied was closer to those values observed in LAB. Rumen and abomasal samples showed similar bacterial DNA concentrations, but the fungal and protozoal DNA concentration in the abomasum was only 69% and 13% of that observed in the rumen, respectively, suggesting fungal and protozoal sequestration in the rumen or possible preferential degradation of fungal and protozoal DNA in the abomasum, or both. In conclusion, absence of protozoa and type of diet extensively modified the chemical composition of LAB and SAB as a consequence of changes in the microbial composition of these fractions.

International audience; Balancing energy and nitrogen in the rumen is a key to both profitability and environmental sustainability. Four dairy cows were used in a Latin square experimental design to investigate the effect of severe nitrogen underfeeding (110 vs. 80% of requirements) and the type of carbohydrate consumed [neutral detergent fiber rich (FIB) vs. starch rich (STA)] on the rumen ecosystem. These dietary treatments modified both rumen fermentation and microbial populations. Compared with STA diets, consumption of FIB diets increased bacterial and fungal diversity in the rumen and also increased the concentrations of cellulolytic microorganisms, including protozoa (+38%), anaerobic fungi (+59%), and methanogens (+27%). This microbial adaptation to fiber utilization led to similar digestibility values for the 2 carbohydrate sources and was accompanied by a shift in the rumen fermentation patterns; when the FIB diets were consumed, the cows had greater ruminal pH, ammonia concentrations, and molar proportions of acetate and propionate compared with when they consumed the STA diets. Certain rumen microorganisms were sensitive to a shortage of nitrogen; rumen concentrations of ammonia were 49% lower when the low-protein (LP) diets were consumed as were total bacteria (-13%), anaerobic fungi (-28%), methanogens (-27%), protozoa (-19%), cellulolytic bacteria, and microbial diversity compared with when the high-protein (HP) diets were consumed. As a result, the digestibility of the LP diets was less than that of the HP diets. These findings demonstrated that the rumen microbial ecosystem is directly linked to the rumen fermentation pattern and, to some extent, to the efficiency of diet utilization by dairy cattle.

Belanche, A., Abecia, L., Holtrop, G., Guada, J. A., Castrillo, C., de la Fuente, G., Balcells, J. (2011). Study of the effect of presence or absence of protozoa on rumen fermentation and microbial protein contribution to the chyme. Journal of Animal Science, 89, (12), 4163-4174. IMPF: 02.09 RONO: 00

Two groups of 5 lambs were euthanized at the weaning (T45) and fattening stages (T90) to evaluate the use of microbial ribosomal DNA (rDNA) sequences as potential microbial markers in relation to purine bases (PB) as a conventional marker. Both microbial markers originated similar microbial N concentrations (mg/g of DM), although T45 showed decreased values compared with the T90 group when either PB or rDNA were considered (P = 0.02). The survival of microbial rDNA was determined in 3 digestive sites (omasum, abomasum, and duodenum), but no substantial differences were observed, indicating that rDNA maintains the molecular stability along the sampling sites analyzed. Contrarily PB concentration increased successively along the digestive tract (P < 0.05), likely as a consequence of the endogenous PB secretion. Undegraded milk PB may also explain the overestimation of the microbial N concentration (2.8 times greater) using PB than rDNA sequences. Abomasum was the sampling site where the best agreement between PB and rDNA estimations was observed. Protozoal N concentration was irrelevant in T45 animals, although substantial in T90 lambs (18% of microbial N). In conclusion, bacterial 16S and protozoal 18S rDNA sequences may persist through the gastric digestive tract and their utilization as a highly specific microbial marker should not be neglected.

Summary This study examined the reticulo-rumen characteristics of the microbial community and its fermentative characteristics in milk-fed, at weaning and finished lambs in a conventional fattening system. Five lambs were assigned to each of three groups: milk-fed lambs slaughtered at 30 days (T30), weaned lambs slaughtered at 45 days (T45) and ‘finished lambs’ slaughtered at 90 days (T90). At slaughter, rumen size, fermentation parameters (pH, volatile fatty acids and microbial enzyme activity) and protozoal counts were recorded. Quantitative PCR was used to quantify the genes encoding 16S and 18S ribosomal DNA of the rumen bacterial and protozoal populations, respectively, and the sequential colonization of the rumen by cellulolytic (Ruminococcus albus, Ruminococcus flavefaciens) and amylolytic (Prevotella ruminicola, Streptococcus bovis) bacteria, and protozoa (Entodinium sp.). Denaturing gradient gel electrophoresis was used to study the development of rumen microbiota biodiversity. Intake of solid food before weaning caused a significant increase in rumen weight (p

Eight natural products from animal, unicellular algae, brown seaweed and plant origins were chosen according to their theoretical antimicrobial activity: Diatomaceous earths (DE), insoluble chitosan (ICHI), soluble chitosan (CHI), seaweed meal (SWM), Ascophyllum nodosum (ASC), Laminaria digitata (LAM), neem oil (NOIL) and an ivy fruit extract rich in saponins (IVY). Dose-response incubations were conducted to determine their effect on rumen fermentation pattern and gas production, while their anti-protozoal activity was tested using (14) C-labelled bacteria.DE, SWM, NOIL and ICHI had very small effects on rumen function when used at inclusion rate up to 2 g L(-1) . ASC had anti-protozoal effects (up to -23%) promoting a decrease in gas production and methanogenesis (-15%). LAM increased VFA production (+7%) and shifted from butyrate to acetate. CHI also shifted fermentation towards propionate production and lower methane (-23%) and protozoal activity (-56%). IVY decreased protozoal activity (-39%) and ammonia concentration (-56%), as well as increased feed fermentation (+11% VFA concentration) and shifted from acetate to propionate production.ASC, LAM, CHI and IVY showed promising potential in vitro as feed additives to improve rumen function, thus more research is needed to investigate their mode of action in the rumen microbial ecosystem. © 2015 Society of Chemical Industry.

Rumen methanogenesis represents an energy waste for the ruminant and an important source of greenhouse gas; thus, integrated studies are needed to fully understand this process. Eight fauna-free sheep were used to investigate the effect of successive inoculation with holotrich protozoa then with total fauna on rumen methanogenesis. Holotrichs inoculation neither altered rumen fermentation rate nor diet digestibility, but increased concentrations of acetate (+15%), butyrate (+57%), anaerobic fungi (+0.82 log), methanogens (+0.41 log) and methanogenesis (+54%). Further inoculation with total fauna increased rumen concentrations of protozoa (+1.0 log), bacteria (+0.29 log), anaerobic fungi (+0.78 log), VFA (+8%), ammonia and fibre digestibility (+17%) without affecting levels of methanogens or methanogenesis. Rumen methanogens population was fairly stable in terms of structure and diversity, while the bacterial community was highly affected by the treatments. Inoculation with holotrich protozoa increased bacterial diversity. Further inoculation with total fauna lowered bacterial diversity but increased concentrations of certain propionate and lactate-producing bacteria, suggesting that alternative H2 sinks could be relevant. This experiment suggests that holotrich protozoa have a greater impact on rumen methanogenesis than entodiniomorphids. Thus, further research is warranted to understand the effect of holotrich protozoa on methane formation and evaluate their elimination from the rumen as a potential methane mitigation strategy., Methane emissions from ruminants represent a concern of global magnitude. Understanding the interactions of different rumen protozoa with bacteria and methanogens is vital to develop adequate methane mitigation strategies.

The development of next generation sequencing has challenged the use of other molecular fingerprinting methods used to study microbial diversity. We analysed the bacterial diversity in the rumen of defaunated sheep following the introduction of different protozoal populations, using both next generation sequencing (NGS: Ion Torrent PGM) and terminal restriction fragment length polymorphism (T-RFLP). Although absolute number differed, there was a high correlation between NGS and T-RFLP in terms of richness and diversity with R values of 0.836 and 0.781 for richness and Shannon-Wiener index, respectively. Dendrograms for both datasets were also highly correlated (Mantel test = 0.742). Eighteen OTUs and ten genera were significantly impacted by the addition of rumen protozoa, with an increase in the relative abundance of Prevotella, Bacteroides and Ruminobacter, related to an increase in free ammonia levels in the rumen. Our findings suggest that classic fingerprinting methods are still valuable tools to study microbial diversity and structure in complex environments but that NGS techniques now provide cost effect alternatives that provide a far greater level of information on the individual members of the microbial population. This work was supported by the Commission of the European Communities (Rednex project FP7-KBBE-2007-1) and the Welsh Government.