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Ruminant husbandry is a major source of anthropogenic greenhouse gases (GHG). Filling knowledge gaps and providing expert recommendation are important for defining future research priorities, improving methodologies and establishing science-based GHG mitigation solutions to government and non-governmental organisations, advisory/extension networks, and the ruminant livestock sector. The objectives of this review is to summarize published literature to provide a detailed assessment of the methodologies currently in use for measuring enteric methane (CH4) emission from individual animals under specific conditions, and give recommendations regarding their application. The methods described include respiration chambers and enclosures, sulphur hexafluoride tracer (SF6) technique, and techniques based on short-term measurements of gas concentrations in samples of exhaled air. This includes automated head chambers (e.g. the GreenFeed system), the use of carbon dioxide (CO2) as a marker, and (handheld) laser CH4 detection. Each of the techniques are compared and assessed on their capability and limitations, followed by methodology recommendations. It is concluded that there is no ‘one size fits all’ method for measuring CH4 emission by individual animals. Ultimately, the decision as to which method to use should be based on the experimental objectives and resources available. However, the need for high throughput methodology e.g. for screening large numbers of animals for genomic studies, does not justify the use of methods that are inaccurate. All CH4 measurement techniques are subject to experimental variation and random errors. Many sources of variation must be considered when measuring CH4 concentration in exhaled air samples without a quantitative or at least regular collection rate, or use of a marker to indicate (or adjust) for the proportion of exhaled CH4 sampled. Consideration of the number and timing of measurements relative to diurnal patterns of CH4 emission and respiratory exchange are important, as well as consideration of feeding patterns and associated patterns of rumen fermentation rate and other aspects of animal behaviour. Regardless of the method chosen, appropriate calibrations and recovery tests are required for both method establishment and routine operation. Successful and correct use of methods requires careful attention to detail, rigour, and routine self-assessment of the quality of the data they provide.

In vitro fermentation techniques (IVFT) have been widely used to evaluate the nutritive value of feeds for ruminants and in the last decade to assess the effect of different nutritional strategies on methane (CH4) production. However, many technical factors may influence the results obtained. The present review has been prepared by the ‘Global Network’ FACCE-JPI international research consortium to provide a critical evaluation of the main factors that need to be considered when designing, conducting and interpreting IVFT experiments that investigate nutritional strategies to mitigate CH4 emission from ruminants. Given the increasing and wide-scale use of IVFT, there is a need to critically review reports in the literature and establish what criteria are essential to the establishment and implementation of in vitro techniques. Key aspects considered include: i) donor animal species and number of animal used, ii) diet fed to donor animals, iii) collection and processing of rumen fluid as inoculum, iv) choice of substrate and incubation buffer, v) incubation procedures and CH4 measurements, vi) headspace gas composition and vii) comparability of in vitro and in vivo measurements. Based on an evaluation of experimental evidence, a set of technical recommendations are presented to harmonize IVFT for feed evaluation, assessment of rumen function and CH4 production.

Introduction: This study explores if promoting a complex rumen microbiota represents an advantage or a handicap in the current dairy production systems in which ruminants are artificially reared in absence of contact with adult animals and fed preserved monophyte forage., Methods: In order to promote a different rumen microbial diversity, a total of 36 newborn goat kids were artificially reared, divided in 4 groups and daily inoculated during 10 weeks with autoclaved rumen fluid (AUT), fresh rumen fluid from adult goats adapted to forage (RFF) or concentrate (RFC) diets, or absence of inoculation (CTL). At 6 months of age all animals were shifted to an oats hay diet to determine their ability to digest a low quality forage., Results and Discussion: Early life inoculation with fresh rumen fluid promoted an increase in the rumen overall microbial diversity which was detected later in life. As a result, at 6 months of age RFF and RFC animals had higher bacterial (+50 OTUs) and methanogens diversity (+4 OTUs) and the presence of a complex rumen protozoal community (+32 OTUs), whereas CTL animals remained protozoa-free. This superior rumen diversity and presence of rumen protozoa had beneficial effects on the energy metabolism allowing a faster adaptation to the forage diet, a higher forage digestion (+21% NDF digestibility) and an energetically favourable shift of the rumen fermentation pattern from acetate to butyrate (+92%) and propionate (+19%) production. These effects were associated with the presence of certain rumen bacterial taxa and a diverse protozoal community. On the contrary, the presence of rumen protozoa (mostly Entodinium ) had a negative impact on the N metabolism leading to a higher bacterial protein breakdown in the rumen and lower microbial protein flow to the host based on purine derivatives urinary excretion (-17% to -54%). The inoculation with autoclaved rumen fluid, as source of fermentation products but not viable microbes, had smaller effects than using fresh inoculum. These findings suggest that enhancing rumen microbial diversity represents a desirable attribute when ruminants are fed forages in which the N supply does not represent a limiting factor for the rumen microbiota., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Belanche, Palma-Hidalgo, Jiménez and Yáñez-Ruiz.)

Methane (CH 4 ) emissions from ruminants are of a significant environmental concern, necessitating accurate prediction for emission inventories. Existing models rely solely on dietary and host animal-related data, ignoring the predicting power of rumen microbiota, the source of CH 4 . To address this limitation, we developed novel CH 4 prediction models incorporating rumen microbes as predictors, alongside animal- and feed-related predictors using four statistical/machine learning (ML) methods. These include random forest combined with boosting (RF-B), least absolute shrinkage and selection operator (LASSO), generalized linear mixed model with LASSO (glmmLasso), and smoothly clipped absolute deviation (SCAD) implemented on linear mixed models. With a sheep dataset (218 observations) of both animal data and rumen microbiota data (relative sequence abundance of 330 genera of rumen bacteria, archaea, protozoa, and fungi), we developed linear mixed models to predict CH 4 production (g CH 4 /animal·d, ANIM-B models) and CH 4 yield (g CH 4 /kg of dry matter intake, DMI-B models). We also developed models solely based on animal-related data. Prediction performance was evaluated 200 times with random data splits, while fitting performance was assessed without data splitting. The inclusion of microbial predictors improved the models, as indicated by decreased root mean square prediction error (RMSPE) and mean absolute error (MAE), and increased Lin's concordance correlation coefficient (CCC). Both glmmLasso and SCAD reduced the Akaike information criterion (AIC) and Bayesian information criterion (BIC) for both the ANIM-B and the DMI-B models, while the other two ML methods had mixed outcomes. By balancing prediction performance and fitting performance, we obtained one ANIM-B model (containing 10 genera of bacteria and 3 animal data) fitted using glmmLasso and one DMI-B model (5 genera of bacteria and 1 animal datum) fitted using SCAD. This study highlights the importance of incorporating rumen microbiota data in CH 4 prediction models to enhance accuracy and robustness. Additionally, ML methods facilitate the selection of microbial predictors from high-dimensional metataxonomic data of the rumen microbiota without overfitting. Moreover, the identified microbial predictors can serve as biomarkers of CH 4 emissions from sheep, providing valuable insights for future research and mitigation strategies., (© 2023. The Author(s).)

Background: The red macroalgae Asparagopsis is an effective methanogenesis inhibitor due to the presence of halogenated methane (CH 4 ) analogues, primarily bromoform (CHBr 3 ). This study aimed to investigate the degradation process of CHBr 3 from A. taxiformis in the rumen and whether this process is diet-dependent. An in vitro batch culture system was used according to a 2 × 2 factorial design, assessing two A. taxiformis inclusion rates [0 (CTL) and 2% DM diet (AT)] and two diets [high-concentrate (HC) and high-forage diet (HF)]. Incubations lasted for 72 h and samples of headspace and fermentation liquid were taken at 0, 0.5, 1, 3, 6, 8, 12, 16, 24, 48 and 72 h to assess the pattern of degradation of CHBr 3 into dibromomethane (CH 2 Br 2 ) and fermentation parameters. Additionally, an in vitro experiment with pure cultures of seven methanogens strains (Methanobrevibacter smithii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, Methanosarcina barkeri, Methanobrevibacter millerae, Methanothermobacter wolfei and Methanobacterium mobile) was conducted to test the effects of increasing concentrations of CHBr 3 (0.4, 2, 10 and 50 µmol/L)., Results: The addition of AT significantly decreased CH 4 production (P = 0.002) and the acetate:propionate ratio (P = 0.003) during a 72-h incubation. The concentrations of CHBr 3 showed a rapid decrease with nearly 90% degraded within the first 3 h of incubation. On the contrary, CH 2 Br 2 concentration quickly increased during the first 6 h and then gradually decreased towards the end of the incubation. Neither CHBr 3 degradation nor CH 2 Br 2 synthesis were affected by the type of diet used as substrate, suggesting that the fermentation rate is not a driving factor involved in CHBr 3 degradation. The in vitro culture of methanogens showed a dose-response effect of CHBr 3 by inhibiting the growth of M. smithii, M. ruminantium, M. stadtmanae, M. barkeri, M. millerae, M. wolfei, and M. mobile., Conclusions: The present work demonstrated that CHBr 3 from A. taxiformis is quickly degraded to CH 2 Br 2 in the rumen and that the fermentation rate promoted by different diets is not a driving factor involved in CHBr 3 degradation., (© 2023. Chinese Association of Animal Science and Veterinary Medicine.)

Ruminants are able to produce large quantities of saliva which enter into the rumen and salivary components exert different physiological functions. 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. To investigate this modulatory activity, a total of 16 semi-continuous in vitro cultures with oats hay and concentrate were used to incubate rumen fluid from four donor goats with autoclaved saliva (AUT) as negative control, saliva from the same rumen fluid donor (OWN) as positive control, and either goat (GOAT) or sheep (SHEEP) saliva as experimental interventions. Fermentation was monitored throughout 7 days of incubation and the microbiome and metabolome were analysed at the end of this incubation by Next-Generation sequencing and liquid chromatography coupled with mass spectrometry, respectively. Characterisation of the proteome and metabolome of the different salivas used for the incubation showed a high inter-animal variability in terms of metabolites and proteins, including immunoglobulins. Incubation with AUT saliva promoted lower fermentative activity in terms of gas production (-9.4%) and highly divergent prokaryotic community in comparison with other treatments (OWN, GOAT and SHEEP) suggesting a modulatory effect derived from the presence of bioactive salivary components. Microbial alpha-diversity at amplicon sequence variant (ASV) level was unaffected by treatment. However, some differences were found in the microbial communities across treatments, which were mostly caused by a greater abundance of Proteobacteria and Rikenellacea in the AUT treatment and lower of Prevotellaceae. These bacteria, which are key in the rumen metabolism, had greater abundances in GOAT and SHEEP treatments. Incubation with GOAT saliva led to a lower protozoal concentration and propionate molar proportion indicating a capacity to modulate the rumen microbial ecosystem. The metabolomics analysis showed that the AUT samples were clustered apart from the rest indicating different metabolic pathways were promoted in this treatment. These results suggest that specific salivary components contribute to host-associated role in selecting the rumen commensal microbiota and its activity. These findings could open the possibility of developing new strategies to modulate the saliva composition as a way to manipulate the rumen function and activity., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Enhancing the ability of animals to convert feed into meat or milk by optimizing feed efficiency (FE) has become a priority in livestock research. Although untargeted metabolomics is increasingly used in this field and may improve our understanding of FE, no information in this regard is available in dairy ewes. This study was conducted to (1) discriminate sheep divergent for FE and (2) provide insights into the physiological mechanisms contributing to FE through high-throughput metabolomics. The ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) technique was applied to easily accessible animal fluids (plasma and milk) to assess whether their metabolome differs between high- and low-feed efficient lactating ewes (H-FE and L-FE groups, respectively; 8 animals/group). Blood and milk samples were collected on the last day of the 3-wk period used for FE estimation. A total of 793 features were detected in plasma and 334 in milk, with 100 and 38 of them, respectively, showing differences between H-FE and L-FE. The partial least-squares discriminant analysis separated both groups of animals regardless of the type of sample. Plasma allowed the detection of a greater number of differential features; however, results also supported the usefulness of milk, more easily accessible, to discriminate dairy sheep divergent for FE. Regarding pathway analysis, nitrogen metabolism (either anabolism or catabolism) seemed to play a central role in FE, with plasma and milk consistently indicating a great impact of AA metabolism. A potential influence of pathways related to energy/lipid metabolism on FE was also observed. The variable importance in the projection plot revealed 15 differential features in each matrix that contributed the most for the separation in H-FE and L-FE, such as l-proline and phosphatidylcholine 20:4e in plasma or l-pipecolic acid and phosphatidylethanolamine (18:2) in milk. Overall, untargeted metabolomics provided valuable information into metabolic pathways that may underlie FE in dairy ewes, with a special relevance of AA metabolism in determining this complex phenotype in the ovine. Further research is warranted to validate these findings., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Most mitigation strategies to reduce enteric methane (CH 4 ) production in the rumen induce an excess of rumen dihydrogen (H 2 ) that is expelled and consequently not redirected to the synthesis of metabolites that can be utilised by the ruminant. We hypothesised that phenolic compounds can be potential H 2 acceptors when added to the diet, as they can be degraded to compounds that may be beneficial for the animal, using part of the H 2 available when ruminal methanogenesis is inhibited. We performed four in vitro incubation experiments using rumen inoculum from Murciano-Granadina adult goats: Experiment 1 examined the inhibitory potential of Asparagopsis taxiformis (AT) at different concentrations (0, 1, 2, 3, 4 and 5% of the substrate on a DM basis) in 24 h incubations; Experiment 2 investigated the effect of a wide range of phenolic compounds (phenol, catechol, resorcinol, hydroquinone, pyrogallol, phloroglucinol, gallic acid and formic acid) at different doses (0, 2, 4, and 6 mM) on rumen fermentation for 24 h; Experiment 3 evaluated the combined effect of each phenolic compound at 6 mM with AT at 2% DM in sequential batch cultures for 5 days; and Experiment 4 examined the dose-response effect of phloroglucinol at different concentrations (0, 6, 16, 26 and 36 mM) combined with AT in sequential batch cultures for 5 days. Results from Experiment 1 confirmed that AT at 2% DM substantially inhibited CH 4 production while significantly increasing H 2 accumulation and decreasing the acetate:propionate ratio. Results from Experiment 2 showed that phenolic compounds did not negatively affect rumen fermentation at any dose. In Experiment 3, each phenolic compound at 6 mM combined with AT at 2% DM inhibited CH 4 production. Phloroglucinol numerically decreased H 2 accumulation and significantly increased total gas production (TGP), volatile fatty acid (VFA) production and the acetate:propionate ratio. In Experiment 4, phloroglucinol at increasing doses supplemented with AT at 2% DM significantly decreased H 2 accumulation and the abundances of archaea, protozoa and fungi abundances, and increased TGP, total VFA production and the acetate:propionate ratio in a dose-dependent way. In conclusion, combined treatment with AT and phloroglucinol was successful to mitigate CH 4 production while preventing the accumulation of H 2 , leading to an increase in acetate and total VFA production and therefore an improvement in rumen fermentation in goats., (Copyright © 2023. Published by Elsevier B.V.)

Manure nitrogen (N) from cattle contributes to nitrous oxide and ammonia emissions and nitrate leaching. Measurement of manure N outputs on dairy farms is laborious, expensive, and impractical at large scales; therefore, models are needed to predict N excreted in urine and feces. Building robust prediction models requires extensive data from animals under different management systems worldwide. Thus, the study objectives were (1) to collate an international database of N excretion in feces and urine based on individual lactating dairy cow data from different continents; (2) to determine the suitability of key variables for predicting fecal, urinary, and total manure N excretion; and (3) to develop robust and reliable N excretion prediction models based on individual data from lactating dairy cows consuming various diets. A raw data set was created based on 5,483 individual cow observations, with 5,420 fecal N excretion and 3,621 urine N excretion measurements collected from 162 in vivo experiments conducted by 22 research institutes mostly located in Europe (n = 14) and North America (n = 5). A sequential approach was taken in developing models with increasing complexity by incrementally adding variables that had a significant individual effect on fecal, urinary, or total manure N excretion. Nitrogen excretion was predicted by fitting linear mixed models including experiment as a random effect. Simple models requiring dry matter intake (DMI) or N intake performed better for predicting fecal N excretion than simple models using diet nutrient composition or milk performance parameters. Simple models based on N intake performed better for urinary and total manure N excretion than those based on DMI, but simple models using milk urea N (MUN) and N intake performed even better for urinary N excretion. The full model predicting fecal N excretion had similar performance to simple models based on DMI but included several independent variables (DMI, diet crude protein content, diet neutral detergent fiber content, milk protein), depending on the location, and had root mean square prediction errors as a fraction of the observed mean values of 19.1% for intercontinental, 19.8% for European, and 17.7% for North American data sets. Complex total manure N excretion models based on N intake and MUN led to prediction errors of about 13.0% to 14.0%, which were comparable to models based on N intake alone. Intercepts and slopes of variables in optimal prediction equations developed on intercontinental, European, and North American bases differed from each other, and therefore region-specific models are preferred to predict N excretion. In conclusion, region-specific models that include information on DMI or N intake and MUN are required for good prediction of fecal, urinary, and total manure N excretion. In absence of intake data, region-specific complex equations using easily and routinely measured variables to predict fecal, urinary, or total manure N excretion may be used, but these equations have lower performance than equations based on intake., (© 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

To meet the 1.5 °C target, methane (CH4) from ruminants must be reduced by 11 to 30% by 2030 and 24 to 47% by 2050 compared to 2010 levels. A meta-analysis identified strategies to decrease product-based (PB; CH4 per unit meat or milk) and absolute (ABS) enteric CH4 emissions while maintaining or increasing animal productivity (AP; weight gain or milk yield). Next, the potential of different adoption rates of one PB or one ABS strategy to contribute to the 1.5 °C target was estimated. The database included findings from 430 peer-reviewed studies, which reported 98 mitigation strategies that can be classified into three categories: animal and feed management, diet formulation, and rumen manipulation. A random-effects meta-analysis weighted by inverse variance was carried out. Three PB strategies—namely, increasing feeding level, decreasing grass maturity, and decreasing dietary forage-to-concentrate ratio—decreased CH4 per unit meat or milk by on average 12% and increased AP by a median of 17%. Five ABS strategies—namely CH4 inhibitors, tanniferous forages, electron sinks, oils and fats, and oilseeds—decreased daily methane by on average 21%. Globally, only 100% adoption of the most effective PB and ABS strategies can meet the 1.5 °C target by 2030 but not 2050, because mitigation effects are offset by projected increases in CH4 due to increasing milk and meat demand. Notably, by 2030 and 2050, low- and middle-income countries may not meet their contribution to the 1.5 °C target for this same reason, whereas high-income countries could meet their contributions due to only a minor projected increase in enteric CH4 emissions.

Newborn dairy ruminants are usually separated from their dams after birth and fed on milk replacer. This lack of contact with adult animals may hinder the rumen microbiological and physiological development. This study evaluates the effects of rearing newborn goat kids in contact with adult companions on the rumen development. Thirty-two newborn goat kids were randomly allocated to two experimental groups which were reared either in the absence (CTL) or in the presence of non-lactating adult goats (CMP) and weaned at 7 weeks of age. Blood and rumen samples were taken at 5, 7, and 9 weeks of age to evaluate blood metabolites and rumen microbial fermentation. Next-generation sequencing was carried out on rumen samples collected at 7 weeks of age. Results showed that CTL kids lacked rumen protozoa, whereas CMP kids had an abundant and complex protozoal community as well as higher methanogen abundance which positively correlated with the body weight and blood β-hydroxybutyrate as indicators of the physiological development. CMP kids also had a more diverse bacterial community (+132 ASVs) and a different structure of the bacterial and methanogen communities than CTL kids. The core rumen bacterial community in CMP animals had 53 more ASVs than that of CTL animals. Furthermore, the number of ASVs shared with the adult companions was over 4-fold higher in CMP kids than in CTL kids. Greater levels of early rumen colonizers Proteobacteria and Spirochaetes were found in CTL kids, while CMP kids had higher levels of Bacteroidetes and other less abundant taxa (Veillonellaceae, Cyanobacteria, and Selenomonas). These findings suggest that the presence of adult companions facilitated the rumen microbial development prior to weaning. This accelerated microbial development had no effect on the animal growth, but CMP animals presented higher rumen pH and butyrate (+45%) and ammonia concentrations than CTL kids, suggesting higher fibrolytic and proteolytic activities. CMP kids also had higher blood β-hydroxybutyrate (+79%) and lower blood glucose concentrations (-23%) at weaning, indicating an earlier metabolic development which could favor the transition from pre-ruminant to ruminant after the weaning process. Further research is needed to determine the effects of this intervention in more challenging farm conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Palma-Hidalgo, Yáñez-Ruiz, Jiménez, Martín-García and Belanche.)

3-Nitrooxypropanol (3-NOP) supplementation to cattle diets mitigates enteric CH 4 emissions and may also be economically beneficial at farm level. However, the wider rumen metabolic response to methanogenic inhibition by 3-NOP and the N O 2 - intermediary metabolite requires further exploration. Furthermore, N O 3 - supplementation potently decreases CH 4 emissions from cattle. The reduction of N O 3 - utilizes H 2 and yields N O 2 - , the latter of which may also inhibit rumen methanogens, although a different mode of action than for 3-NOP and its N O 2 - derivative was hypothesized. Our objective was to explore potential responses of the fermentative and methanogenic metabolism in the rumen to 3-NOP, N O 3 - and their metabolic derivatives using a dynamic mechanistic modeling approach. An extant mechanistic rumen fermentation model with state variables for carbohydrate substrates, bacteria and protozoa, gaseous and dissolved fermentation end products and methanogens was extended with a state variable of either 3-NOP or N O 3 - . Both new models were further extended with a N O 2 - state variable, with N O 2 - exerting methanogenic inhibition, although the modes of action of 3-NOP-derived and N O 3 - -derived N O 2 - are different. Feed composition and intake rate (twice daily feeding regime), and supplement inclusion were used as model inputs. Model parameters were estimated to experimental data collected from the literature. The extended 3-NOP and N O 3 - models both predicted a marked peak in H 2 emission shortly after feeding, the magnitude of which increased with higher doses of supplement inclusion. The H 2 emission rate appeared positively related to decreased acetate proportions and increased propionate and butyrate proportions. A decreased CH 4 emission rate was associated with 3-NOP and N O 3 - supplementation. Omission of the N O 2 - state variable from the 3-NOP model did not change the overall dynamics of H 2 and CH 4 emission and other metabolites. However, omitting the N O 2 - state variable from the N O 3 - model did substantially change the dynamics of H 2 and CH 4 emissions indicated by a decrease in both H 2 and CH 4 emission after feeding. Simulations do not point to a strong relationship between methanogenic inhibition and the rate of N O 3 - and N O 2 - formation upon 3-NOP supplementation, whereas the metabolic response to N O 3 - supplementation may largely depend on methanogenic inhibition by N O 2 - ., Competing Interests: MK is affiliated with DSM Nutritional products, which is the funder of the present study and patented 3-NOP. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 van Lingen, Fadel, Yáñez-Ruiz, Kindermann and Kebreab.)

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 3 -N concentrations than NFS fraction. Replacing the bicarbonate buffer solution by increasing proportions of saliva resulted in higher NH 3 -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 3 -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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Recent evidence suggests that changes in microbial colonization of the rumen prior to weaning may imprint the rumen microbiome and impact phenotypes later in life. We investigated how dietary manipulation from birth influences growth, methane production, and gastrointestinal microbial ecology. At birth, 18 female Holstein and Montbéliarde calves were randomly assigned to either treatment or control (CONT). Treatment was 3-nitrooxypropanol (3-NOP), an investigational anti-methanogenic compound that was administered daily from birth until three weeks post-weaning (week 14). Samples of rumen fluid and faecal content were collected at weeks 1, 4, 11, 14, 23, and 60 of life. Calves were tested for methane emissions using the GreenFeed system during the post-weaning period (week 11-23 and week 56-60 of life). Calf physiological parameters (BW, ADG and individual VFA) were similar across groups throughout the trial. Treated calves showed a persistent reduction in methane emissions (g CH 4 /d) throughout the post-weaning period up to at least 1 year of life, despite treatment ceasing three weeks post-weaning. Similarly, despite variability in the abundance of individual taxa across weeks, the rumen bacterial, archaeal and fungal structure differed between CONT and 3-NOP calves across all weeks, as visualised using sparse-PLS-DA. Similar separation was also observed in the faecal bacterial community. Interestingly, despite modest modifications to the abundance of rumen microbes, the reductive effect of 3-NOP on methane production persisted following cessation of the treatment period, perhaps indicating a differentiation of the ruminal microbial ecosystem or a host response triggered by the treatment in the early development phase.

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., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Newborn ruminants possess an underdeveloped rumen which is colonized by microorganisms acquired from adult animals and the surrounding environment. This microbial transfer can be limited in dairy systems in which newborns are separated from their dams at birth. This study explores whether the direct inoculation of fresh or autoclaved rumen fluid from adult goats to newborn kids has a beneficial effect on rumen microbial development and function., Results: Repetitive inoculation of young kids with fresh rumen fluid from adult goats adapted to forage (RFF) or concentrate diets (RFC) accelerated microbial colonization of the rumen during the pre-weaning period leading to high protozoal numbers, a greater diversity of bacterial (+ 234 OTUs), methanogens (+ 6 OTUs) and protozoal communities (+ 25 OTUs) than observed in control kids (CTL) without inoculation. This inoculation also increased the size of the core bacterial and methanogens community and the abundance of key rumen bacteria (Ruminococcaceae, Fibrobacteres, Veillonellaceae, Rikenellaceae, Tenericutes), methanogens (Methanobrevibacter ruminantium, Methanomicrobium mobile and Group 9), anaerobic fungi (Piromyces and Orpinomyces) and protozoal taxa (Enoploplastron, Diplodinium, Polyplastron, Ophryoscolex, Isotricha and Dasytricha) before weaning whereas CTL kids remained protozoa-free through the study. Most of these taxa were positively correlated with indicators of the rumen microbiological and physiological development (higher forage and concentrate intakes and animal growth during the post-weaning period) favoring the weaning process in RFF and RFC kids in comparison to CTL kids. Some of these microbiological differences tended to decrease during the post-weaning period, although RFF and RFC kids retained a more complex and matured rumen microbial ecosystem than CTL kids. Inoculation with autoclaved rumen fluid promoted lower development of the bacterial and protozoal communities during the pre-weaning period than using fresh inocula, but it favored a more rapid microbial development during the post-weaning than observed for CTL kids., Conclusions: This study demonstrated that inoculation of young ruminants with fresh rumen fluid from adult animals accelerated the rumen microbial colonization which was associated with an earlier rumen functional development. This strategy facilitated a smoother transition from milk to solid feed favoring the animal performance during post-weaning and minimizing stress.

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., (The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

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 ® 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 (>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 (-8.8%), per dry matter intake (-12.9%) and per fat and protein corrected milk yield (-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.

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., (© 2019 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The role of marine lipids as modulators of ruminal biohydrogenation of dietary unsaturated fatty acids may be explained by the effects of their n-3 polyunsaturated fatty acids (PUFA) on the bacterial community. However, the impact of individual PUFA has barely been examined, and it is uncertain which bacteria are truly involved in biohydrogenation. In addition, despite interspecies differences in rumen bacterial composition, we are not aware of any direct comparison of bovine and ovine responses to dietary PUFA. Therefore, rumen fluid from cannulated cattle and sheep were used as inocula to examine in vitro the effect of 20:5n-3 (EPA), 22:5n-3 (DPA), and 22:6n-3 (DHA) on the bacterial community. Amplicon 16 S rRNA sequencing suggested that EPA and DHA had a greater contribution to the action of marine lipids than DPA both in cattle and sheep. Certain effects were exclusive to each ruminant species, which underlines the complexity of rumen microbial responses to dietary fatty acids. Based on changes in bacterial abundance, Barnesiella, Prevotella, Paraprevotella, Hallela, Anaerovorax, Succiniclasticum, Ruminococcus and Ruminobacter may be involved in the ruminal response in biohydrogenation to the addition of marine lipids, but further research is necessary to confirm their actual role in ruminal lipid metabolism.

Nitrogen is a component of essential nutrients critical for the productivity of ruminants. If excreted in excess, N is also an important environmental pollutant contributing to acid deposition, eutrophication, human respiratory problems, and climate change. The complex microbial metabolic activity in the rumen and the effect on subsequent processes in the intestines and body tissues make the study of N metabolism in ruminants challenging compared with nonruminants. Therefore, using accurate and precise measurement techniques is imperative for obtaining reliable experimental results on N utilization by ruminants and evaluating the environmental impacts of N emission mitigation techniques. Changeover design experiments are as suitable as continuous ones for studying protein metabolism in ruminant animals, except when changes in body weight or carryover effects due to treatment are expected. Adaptation following a dietary change should be allowed for at least 2 (preferably 3) wk, and extended adaptation periods may be required if body pools can temporarily supply the nutrients studied. Dietary protein degradability in the rumen and intestines are feed characteristics determining the primary AA available to the host animal. They can be estimated using in situ, in vitro, or in vivo techniques with each having inherent advantages and disadvantages. Accurate, precise, and inexpensive laboratory assays for feed protein availability are still needed. Techniques used for direct determination of rumen microbial protein synthesis are laborious and expensive, and data variability can be unacceptably large; indirect approaches have not shown the level of accuracy required for widespread adoption. Techniques for studying postruminal digestion and absorption of nitrogenous compounds, urea recycling, and mammary AA metabolism are also laborious, expensive (especially the methods that use isotopes), and results can be variable, especially the methods based on measurements of digesta or blood flow. Volatile loss of N from feces and particularly urine can be substantial during collection, processing, and analysis of excreta, compromising the accuracy of measurements of total-tract N digestion and body N balance. In studying ruminant N metabolism, nutritionists should consider the longer term fate of manure N as well. Various techniques used to determine the effects of animal nutrition on total N, ammonia- or nitrous oxide-emitting potentials, as well as plant fertilizer value, of manure are available. Overall, methods to study ruminant N metabolism have been developed over 150 yr of animal nutrition research, but many of them are laborious and impractical for application on a large number of animals. The increasing environmental concerns associated with livestock production systems necessitate more accurate and reliable methods to determine manure N emissions in the context of feed composition and ruminant N metabolism., (The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

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., (© 2018 Society of Chemical Industry.)

Heavy metal concentrations of Cd, Cr, Cu, Fe, Ni, Zn, Co, and Pb were investigated in soft tissues of Mytilus galloprovincialis coming from an aquaculture farm in Cala Iris sea of AlHoceima. Mytilus galloprovincialis were collected monthly during the period January to December 2016. The seasonal variations were affected significantly the concentrations of metals (Cd, Fe and Cr) in M. galloprovincialis. The highest heavy metal concentrations were recorded in winter (0.89 mg/kg, 673.2 and 3.330 mg/kg; for Cd, Fe and Cr, respectively) and the lowest values were founded in summer for Cd (0.646 mg/kg), and in autumn for Fe (340.1 mg/kg) and Cr (1.959 mg/kg). A significant effect of seasons on metal concentrations can be attributed to a number of biological and environmental inter-related factors. Data from this study may provide information on the use of M. galloprovincialis as a bioindicator for heavy metals pollution in the Cala Iris Sea., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This work aimed to gain insight into the transition from milk to solid feeding at weaning combining genomics and metabolomics on rumen contents from goat kids treated with a methanogenic inhibitor (bromochloromethane, BCM). Sixteen goats giving birth to two kids were used. Eight does were treated (D+) with BCM after giving birth and over 2 months. One kid per doe in both groups was treated with BCM (k+) for 3 months while the other was untreated (k-). Rumen samples were collected from kids at weaning (W) and 1 (W + 1) and 4 (W + 4) months after and from does at weaning and subjected to 16S pyrosequencing and metabolomics analyses combining GC/LC-MS. Results from pyrosequencing showed a clear effect of age of kids, with more diverse bacterial community as solid feed becomes more important after weaning. A number of specific OTUs were significantly different as a result of BCM treatment of the kid at W while at W + 1 and W + 4 less OTUs were significantly changed. At W + 1, Prevotella was increased and Butyrivibrio decreased in BCM treated kids. At W + 4 only the effect of treating mothers resulted in significant changes in the abundance of some OTUs: Ruminococcus, Butyrivibrio and Prevotella . The analysis of the OTUs shared by different treatments revealed that kids at weaning had the largest number of unique OTUs compared with kids at W + 1 (137), W + 4 (238), and does (D) (23). D + k+ kids consistently shared more OTUs with mothers than the other three groups at the three sampling times. The metalobomic study identified 473 different metabolites. In does, lipid super pathway included the highest number of metabolites that were modified by BCM, while in kids all super-pathways were evenly affected. The metabolomic profile of samples from kids at W was different in composition as compared to W + 1 and W + 4, which may be directly ascribed to the process of rumen maturation and changes in the solid diet. This study shows the complexity of the bacterial community and metabolome in the rumen before weaning, which clearly differ from that after weaning and highlight the importance of the dam in transmitting the primary bacterial community after birth.

Bacteria firmly attached to the gastrointestinal epithelium during the pre-weaning phase may show a significant impact on nutrient processing, immunity parameters, health and feed efficiency of lambs during post-weaning phases. Thus, the aim of this study was to describe the differences in the ileal epimural microbiota (e.g. total bacteria, Prevotella spp., Bifidobacterium spp. and Lactobacillus spp.) of fattening lambs promoted by early feed restriction during the suckling phase trying to elucidate some of the underlying mechanisms behind changes in feed efficiency during the fattening period. A total of 24 Merino lambs (average BW 4.81±0.256 kg) were used, 12 of them (ad libitum, ADL) kept permanently in individual pens with their mothers, whereas the other 12 lambs were separated from their dams for 9 h each day to be exposed to milk restriction (RES). After weaning (BW=15 kg) all the animals were penned individually, offered the same complete pelleted diet (35 g/kg BW per day) and slaughtered at a BW of 27 kg. During the fattening period, reduced gain : feed ratio (0.320 v. 0.261, P<0.001) was observed for the RES group. Moreover, increments of Prevotella spp. were detected in the ileal epimural microbiota of RES lambs (P<0.05). There were also higher numbers of infiltrated lymphocytes (T and B cells) in the ileal lamina propria (P<0.05), a higher M-cell labelling intensity in ileal Peyer's patches domes (P<0.05) and a trend towards a thickening of the submucosa layer when compared with the ADL group (P=0.057). Some other immunological parameters, such as an increased immunoglobulin A (IgA) production (pg IgA/µg total protein) and increments in CD45+ cells were also observed in the ileum of RES group (P<0.05), whereas transforming growth factor β and toll-like receptor gene expression was reduced (P<0.05). In conclusion, early feed restriction during the suckling phase promoted changes in ileal epimural microbiota and several immunity parameters that could be related to differences in feed efficiency traits during the fattening period of Merino lambs.

The rumen is a complex ecosystem composed of anaerobic bacteria, protozoa, fungi, methanogenic archaea and phages. These microbes interact closely to breakdown plant material that cannot be digested by humans, whilst providing metabolic energy to the host and, in the case of archaea, producing methane. Consequently, ruminants produce meat and milk, which are rich in high-quality protein, vitamins and minerals, and therefore contribute to food security. As the world population is predicted to reach approximately 9.7 billion by 2050, an increase in ruminant production to satisfy global protein demand is necessary, despite limited land availability, and whilst ensuring environmental impact is minimized. Although challenging, these goals can be met, but depend on our understanding of the rumen microbiome. Attempts to manipulate the rumen microbiome to benefit global agricultural challenges have been ongoing for decades with limited success, mostly due to the lack of a detailed understanding of this microbiome and our limited ability to culture most of these microbes outside the rumen. The potential to manipulate the rumen microbiome and meet global livestock challenges through animal breeding and introduction of dietary interventions during early life have recently emerged as promising new technologies. Our inability to phenotype ruminants in a high-throughput manner has also hampered progress, although the recent increase in "omic" data may allow further development of mathematical models and rumen microbial gene biomarkers as proxies. Advances in computational tools, high-throughput sequencing technologies and cultivation-independent "omics" approaches continue to revolutionize our understanding of the rumen microbiome. This will ultimately provide the knowledge framework needed to solve current and future ruminant livestock challenges.

Enteric methane (CH 4 ) production from cattle contributes to global greenhouse gas emissions. Measurement of enteric CH 4 is complex, expensive, and impractical at large scales; therefore, models are commonly used to predict CH 4 production. However, building robust prediction models requires extensive data from animals under different management systems worldwide. The objectives of this study were to (1) collate a global database of enteric CH 4 production from individual lactating dairy cattle; (2) determine the availability of key variables for predicting enteric CH 4 production (g/day per cow), yield [g/kg dry matter intake (DMI)], and intensity (g/kg energy corrected milk) and their respective relationships; (3) develop intercontinental and regional models and cross-validate their performance; and (4) assess the trade-off between availability of on-farm inputs and CH 4 prediction accuracy. The intercontinental database covered Europe (EU), the United States (US), and Australia (AU). A sequential approach was taken by incrementally adding key variables to develop models with increasing complexity. Methane emissions were predicted by fitting linear mixed models. Within model categories, an intercontinental model with the most available independent variables performed best with root mean square prediction error (RMSPE) as a percentage of mean observed value of 16.6%, 14.7%, and 19.8% for intercontinental, EU, and United States regions, respectively. Less complex models requiring only DMI had predictive ability comparable to complex models. Enteric CH 4 production, yield, and intensity prediction models developed on an intercontinental basis had similar performance across regions, however, intercepts and slopes were different with implications for prediction. Revised CH 4 emission conversion factors for specific regions are required to improve CH 4 production estimates in national inventories. In conclusion, information on DMI is required for good prediction, and other factors such as dietary neutral detergent fiber (NDF) concentration, improve the prediction. For enteric CH 4 yield and intensity prediction, information on milk yield and composition is required for better estimation., (© 2018 John Wiley & Sons Ltd.)

Ruminant production systems are important contributors to anthropogenic methane (CH 4 ) emissions, but there are large uncertainties in national and global livestock CH 4 inventories. Sources of uncertainty in enteric CH 4 emissions include animal inventories, feed dry matter intake (DMI), ingredient and chemical composition of the diets, and CH 4 emission factors. There is also significant uncertainty associated with enteric CH 4 measurements. The most widely used techniques are respiration chambers, the sulfur hexafluoride (SF 6 ) tracer technique, and the automated head-chamber system (GreenFeed; C-Lock Inc., Rapid City, SD). All 3 methods have been successfully used in a large number of experiments with dairy or beef cattle in various environmental conditions, although studies that compare techniques have reported inconsistent results. Although different types of models have been developed to predict enteric CH 4 emissions, relatively simple empirical (statistical) models have been commonly used for inventory purposes because of their broad applicability and ease of use compared with more detailed empirical and process-based mechanistic models. However, extant empirical models used to predict enteric CH 4 emissions suffer from narrow spatial focus, limited observations, and limitations of the statistical technique used. Therefore, prediction models must be developed from robust data sets that can only be generated through collaboration of scientists across the world. To achieve high prediction accuracy, these data sets should encompass a wide range of diets and production systems within regions and globally. Overall, enteric CH 4 prediction models are based on various animal or feed characteristic inputs but are dominated by DMI in one form or another. As a result, accurate prediction of DMI is essential for accurate prediction of livestock CH 4 emissions. Analysis of a large data set of individual dairy cattle data showed that simplified enteric CH 4 prediction models based on DMI alone or DMI and limited feed- or animal-related inputs can predict average CH 4 emission with a similar accuracy to more complex empirical models. These simplified models can be reliably used for emission inventory purposes., (The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).)

Early postnatal nutrition may have a significant subsequent impact on metabolic disorders during the entire lifespan of lambs. The aim of the present study was to describe the changes in biochemical, immunological, hepatic, and ruminal parameters of fattening lambs derived from early feed restriction during the suckling phase. Twenty-four merino lambs (average body weight, BW, 4.81 ± 0.256 kg) were used, 12 of them were milk-fed ad libitum (ADL) remaining permanently with their dams, whereas the other 12 lambs (restricted, RES) were subjected to milk restriction. After weaning, all the lambs were fed 35 g/kg BW per day of the same complete pelleted diet to ensure no differences between groups in dry matter intake (603 vs. 607 g/day for ADL and RES lambs, respectively, p = 0.703), and were slaughtered at a BW of 27 kg. Biochemical profiles revealed higher concentrations of ceruloplasmin and low-density lipoproteins, whereas insulin concentration was lower in the RES lambs compared to the ADL group. Liver thiobarbituric acid reactive substances were lower in the RES lambs. No significant differences in ruminal or blood immunological parameters were found. In conclusion, early feed restriction promoted metabolic disorders not related to ruminal acidosis occurrence, which can compromise the health status during the fattening period of merino lambs.

The aim of this work was to evaluate the effect of feeding management during the first month of life (natural with the mother, NAT, or artificial with milk replacer, ART) on the rumen microbial colonization and the host innate immune response. Thirty pregnant goats carrying two fetuses were used. At birth one kid was taken immediately away from the doe and fed milk replacer (ART) while the other remained with the mother (NAT). Kids from groups received colostrum during first 2 days of life. Groups of four kids (from ART and NAT experimental groups) were slaughtered at 1, 3, 7, 14, 21 and 28 days of life. On the sampling day, after slaughtering, the rumen content was sampled and epithelial rumen tissue was collected. Pyrosequencing analyses of the bacterial community structure on samples collected at 3, 7, 14 and 28 days showed that both systems promoted significantly different colonization patterns (P = 0.001). Diversity indices increased with age and were higher in NAT feeding system. Lower mRNA abundance was detected in TLR2, TLR8 and TLR10 in days 3 and 5 compared to the other days (7, 14, 21 and 28). Only TLR5 showed a significantly different level of expression according to the feeding system, presenting higher mRNA abundances in ART kids. PGLYRP1 showed significantly higher abundance levels in days 3, 5 and 7, and then experienced a decline independently of the feeding system. These observations confirmed a highly diverse microbial colonisation from the first day of life in the undeveloped rumen, and show that the colonization pattern substantially differs between pre-ruminants reared under natural or artificial milk feeding systems. However, the rumen epithelial immune development does not differentially respond to distinct microbial colonization patterns.

Ruminants, such as cows, sheep, and goats, predominantly ferment in their rumen plant material to acetate, propionate, butyrate, CO2, and methane. Whereas the short fatty acids are absorbed and metabolized by the animals, the greenhouse gas methane escapes via eructation and breathing of the animals into the atmosphere. Along with the methane, up to 12% of the gross energy content of the feedstock is lost. Therefore, our recent report has raised interest in 3-nitrooxypropanol (3-NOP), which when added to the feed of ruminants in milligram amounts persistently reduces enteric methane emissions from livestock without apparent negative side effects [Hristov AN, et al. (2015) Proc Natl Acad Sci USA 112(34):10663-10668]. We now show with the aid of in silico, in vitro, and in vivo experiments that 3-NOP specifically targets methyl-coenzyme M reductase (MCR). The nickel enzyme, which is only active when its Ni ion is in the +1 oxidation state, catalyzes the methane-forming step in the rumen fermentation. Molecular docking suggested that 3-NOP preferably binds into the active site of MCR in a pose that places its reducible nitrate group in electron transfer distance to Ni(I). With purified MCR, we found that 3-NOP indeed inactivates MCR at micromolar concentrations by oxidation of its active site Ni(I). Concomitantly, the nitrate ester is reduced to nitrite, which also inactivates MCR at micromolar concentrations by oxidation of Ni(I). Using pure cultures, 3-NOP is demonstrated to inhibit growth of methanogenic archaea at concentrations that do not affect the growth of nonmethanogenic bacteria in the rumen.

The nutritional manipulations of the rumen microbiome to enhance productivity and health are rather limited by the resilience of the ecosystem once established in the mature rumen. Based on recent studies, it has been suggested that the microbial colonization that occurs soon after birth opens a possibility of manipulation with potential to produce lasting effects into adult life. This paper presents the state-of-the-art in relation to early life nutritional interventions by addressing three areas: the development of the rumen as an organ in regards to the nutrition of the new-born, the main factors that determine the microbial population that first colonizes and establishes in the rumen, and the key immunity players that contribute to shaping the commensal microbiota in the early stage of life to understand host-microbiome specificity. The development of the rumen epithelium and muscularization are differently affected by the nature of the diet and special care should be taken with regards to transition from liquid (milk) to solid feed. The rumen is quickly colonized by all type of microorganisms straight after birth and the colonization pattern may be influenced by several factors such as presence/absence of adult animals, the first solid diet provided, and the inclusion of compounds that prevent/facilitate the establishment of some microorganisms or the direct inoculation of specific strains. The results presented show how early life events may be related to the microbial community structure and/or the rumen activity in the animals post-weaning. This would create differences in adaptive capacity due to different early life experiences and leads to the idea of microbial programming. However, many elements need to be further studied such as: the most sensitive window of time for interventions, the best means to test long term effectiveness, the role of key microbial groups and host-immune regulations.

Study of the efficacy of methanogenesis inhibitors in the rumen has given inconsistent results, mainly due to poorly understood effects on the key microbial groups involved in pathways for methane (CH4) synthesis. The experiment described in this report was designed to assess the effect of propyl propane thiosulfinate (PTS), diallyl disulfide (DDS) and bromochloromethane (BCM) on rumen fermentation, methane production and microbial populations in continuous culture fermenters. No effects on total volatile fatty acids (VFA) were observed with PTS or DDS, but VFA were decreased with BCM. Amylase activity increased with BCM as compared with the other treatments. A decrease in methane production was observed with PTS (48%) and BCM (94%) as compared with control values. The concentration of methanogenic archaea decreased with BCM from day 4 onward and with PTS on days 4 and 8. Pyrosequencing analysis revealed that PTS and BCM decreased the relative abundance of Methanomicrobiales and increased that of Methanobrevibacter and Methanosphaera. The total concentration of bacteria was not modified by any treatment, although treatment with BCM increased the relative abundance of Prevotella and decreased that of Ruminococcus. These results suggest that the inhibition of methane production in the rumen by PTS and BCM is associated with a shift in archaeal biodiversity and changes in the bacterial community with BCM., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The objective of this work was to compare the biomass and community structure of bacteria, protozoa and archaea communities in samples of rumen and faeces of goats and to what extent the diet (alfalfa hay with or without supplemented oats) offered to them exert an influence. Four cannulated adult goats fistulated in the rumen were used in a cross over design experiment in two experimental periods of 26 days, consisting in 14 days of adaptation, 7 days of sampling rumen contents and 5 days of digestibility measurement. Bacterial, protozoa and archaeal biomass and the communities' structure was assessed by real time PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE), respectively. The numbers of archaea and bacteria in both rumen and faeces were higher and lower, respectively, in animals fed AH diet (P < 0.005). Contrary, protozoal numbers were not affected by the diet but were lower (P < 0.001) in faeces than in rumen. The analysis of the community structure revealed a consistently different population in structure in rumen and faeces for the three studied microbial groups and that supplementing alfalfa hay with oats led to a decrease in the similarity between sites in the rumen and faeces: similarity indexes for bacteria (57 and 27%), archaea (26 and 9%) and protozoa (62 and 22%) in animals fed AH and AHO diets, respectively., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The aim of this work was to study whether feeding a methanogen inhibitor from birth of goat kids and their does has an impact on the archaeal population colonizing the rumen and to what extent the impact persists later in life. Sixteen goats giving birth to two kids were used. Eight does were treated (D+) with bromochloromethane after giving birth and over 2 months. The other 8 goats were not treated (D-). One kid per doe in both groups was treated with bromochloromethane (k+) for 3 months while the other was untreated (k-), resulting in four experimental groups: D+/k+, D+/k-, D-/k+, and D-/k-. Rumen samples were collected from kids at weaning and 1 and 4 months after (3 and 6 months after birth) and from does at the end of the treating period (2 months). Pyrosequencing analyses showed a modified archaeal community composition colonizing the rumen of kids, although such effect did not persist entirely 4 months after; however, some less abundant groups remained different in treated and control animals. The different response on the archaeal community composition observed between offspring and adult goats suggests that the competition occurring in the developing rumen to occupy different niches offer potential for intervention.

The aim of this work was to investigate the effect of feeding ethyl-3-nitrooxy propionate (E3NP) and 3-nitrooxypropanol (3 NP), 2 recently developed compounds with potential antimethanogenic activity, in vitro and in vivo in nonlactating sheep on ruminal methane production, fermentation pattern, the abundance of major microbial groups, and feed degradability. Three experiments were conducted, 1 in vitro and 2 in vivo. The in vitro batch culture trial (experiment 1) tested 2 doses of E3NP and 3 NP (40 and 80 μL/L), which showed a substantial reduction of methane production (up to 95%) without affecting concentration of volatile fatty acids (VFA). The 2 in vivo trials were conducted over 16 d (experiment 2) and 30 d (experiment 3) to study their effects in sheep. In experiment 2, 6 adult nonpregnant sheep, with permanent rumen cannula and fed alfalfa hay and oats (60:40), were treated with E3NP at 2 doses (50 and 500 mg/animal per day). After 7, 14, and 15 d of treatment, methane emissions were recorded in respiration chambers and rumen fluid samples were collected for VFA analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. Methane production decreased by 29% compared with the control with the higher dose of E3NP on d 14 to 15. A decrease in the acetate:propionate ratio was observed without detrimental effects on dry matter intake. In experiment 3, 9 adult nonpregnant sheep, with permanent rumen cannula and fed with alfalfa hay and oats (60:40), were treated with E3NP or 3 NP at one dose (100mg/animal per day) over 30 d. On d 14 and d 29 to 30, methane emissions were recorded in respiration chambers. Rumen fluid samples were collected on d 29 and 30 for VFA analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. In addition, on d 22 and 23, samples of oats and alfalfa hay were incubated in the rumen of sheep to determine dry matter ruminal degradation over 24 and 48 h, respectively; no effect was observed (78.6, 78.3, and 78.8% of alfalfa and 74.2, 74.0, and 70.6% of oats in control, E3NP, and 3 NP groups, respectively). A reduction in methane production was observed for both additives at d 14 and d 29 to 30. In both treatments, the acetate:propionate ratio was significantly decreased. Likewise, total concentrations of the analyzed microbial groups in the rumen showed no difference among treatments and doses for both experiments. Both tested compounds showed promise as methane inhibitors in the rumen, with no detrimental effects on fermentation or intake, which would need to be confirmed in lactating animals., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Two in vitro and one in vivo experiments were conducted to investigate the effects of a selection of plant compounds on rumen fermentation, microbial concentration and methane emissions in goats. Treatments were: control (no additive), carvacrol (CAR), cinnamaldehyde (CIN), eugenol (EUG), propyl propane thiosulfinate (PTS), propyl propane thiosulfonate (PTSO), diallyl disulfide (DDS), a mixture (40 : 60) of PTS and PTSO (PTS+PTSO), and bromochloromethane (BCM) as positive control with proven antimethanogenic effectiveness. Four doses (40, 80, 160 and 320 µl/l) of the different compounds were incubated in vitro for 24 h in diluted rumen fluid from goats using two diets differing in starch and protein source within the concentrate (Experiment 1).The total gas production was linearly decreased (P<0.012) by all compounds, with the exception of EUG and PTS+PTSO (P≥ 0.366). Total volatile fatty-acid (VFA) concentration decreased (P≤ 0.018) only with PTS, PTSO and CAR, whereas the acetate:propionate ratio decreased (P≤ 0.002) with PTS, PTSO and BCM, and a tendency (P=0.064) was observed for DDS. On the basis of results from Experiment 1, two doses of PTS, CAR, CIN, BCM (160 and 320 µl/l), PTSO (40 and 160 µl/l) and DDS (80 and 320 µl/l) were further tested in vitro for 72 h (Experiment 2). The gas production kinetics were affected (P≤ 0.045) by all compounds, and digested NDF (DNDF) after 72 h of incubation was only linearly decreased (P≤ 0.004) by CAR and PTS. The addition of all compounds linearly decreased (P≤ 0.009) methane production, although the greatest reductions were observed for PTS (up to 96%), DDS (62%) and BCM (95%). No diet-dose interaction was observed. To further test the results obtained in vitro, two groups of 16 adult non-pregnant goats were used to study in vivo the effect of adding PTS (50, 100 and 200 mg/l rumen content per day) and BCM (50, 100 and 160 mg/l rumen content per day) during the 9 days on methane emissions (Experiment 3). The addition of PTS and BCM resulted in linear reductions (33% and 64%, respectively, P≤ 0.002) of methane production per unit of dry matter intake, which were lower than the maximum inhibition observed in vitro (87% and 96%, respectively). We conclude that applying the same doses in vivo as in vitro resulted in a proportional lower extent of methane decrease, and that PTS at 200 mg/l rumen content per day has the potential to reduce methane emissions in goats. Whether the reduction in methane emission observed in vivo persists over longer periods of treatments and improves feed conversion efficiency requires further research.

The growing interest in reducing methane (CH4) emissions from ruminants by dietary means is constrained by the complexity of the microbial community in the rumen of the adult animal. The aim of this work was to study whether intervention in early life of goat kids has an impact on methane emissions and the microbial ecosystem in the rumen and whether the effects persist postweaning. Sixteen doe goats giving birth to 2 kids each were randomly split into 2 experimental groups: 8 does were treated (D+) with bromochloromethane (BCM) after giving birth and over 2 mo, and the other 8 does were not treated (D-). In both groups of does, 1 kid per doe was treated with BCM (k+) for 3 mo, and the other was untreated (k-), resulting in 4 experimental groups: D+k+, D+k-, D-k+, and D-k-. Methane emissions were recorded, and ruminal samples were collected from kids at 2 mo of age (weaning, W) and 1 (W+1) and 4 (W+4) mo later. At W+1 mo, CH4 emissions by k+ kids were 52% and 59% less than untreated kids (in D+ and D- groups, respectively). However, at W+4 mo, only D+k+ kids remained lower (33%) emitters and exhibited greater daily BW gain (146 g/d) compared with the other 3 groups (121.8 g/d). The analysis of the archaeal community structure by Denaturing Gradient Gel Electrophoresis (DGGE)showed a strong effect of BCM treatment on does and kids that persisted only in D+k+ kids. The study showed that the application of BCM during early life of kids modified the archaeal population that colonized the rumen, which resulted in decreased CH4 emissions around weaning. The effect is influenced by the treatment applied to the doe and persisted 3 mo later in D+k+ kids.

Oxalic acid (OA) is a secondary compound occurring in a wide range of plants consumed by ruminants, especially in saline lands or in arid and semi-arid regions. However, its impact on the rumen microbial community and its changes over time, as well as the potential consequences on ruminal function, remain unknown. To examine this impact, five ewes fitted with a ruminal cannula and fed low-quality grass hay were dosed daily with 0.6 mmol of OA/kg body weight through the cannula for 14 days. On days 0 (before the start), 4, 7 and 14 of the administration period, samples of ruminal digesta were collected throughout the day (0, 3, 6 and 9 h after the morning feeding) for analysis of the bacterial community and fermentation parameters (pH, ammonia and volatile fatty acid (VFA) concentrations). In addition, two feedstuffs were incubated in situ using the nylon bag technique to estimate ruminal degradation. Terminal restriction fragment length polymorphism was employed to monitor the dynamics of total bacteria, and quantitative real-time PCR was used to investigate the abundance of the oxalate-degrading Oxalobacter formigenes. Neither pH nor total VFA concentrations were affected. Nevertheless, OA dosing altered molar proportions of most individual VFA and ammonia concentrations (P < 0.001). The dry matter disappearance of alfalfa hay was reduced on days 7 and 14 and that of barley straw only on day 7 (P < 0.01). These slight changes were related to others observed in the relative frequency of a number of terminal restriction fragments. Variations in the ruminal microbiota occurred rapidly with OA administration, which did not modify the bacterial diversity significantly but altered the structure of the community. However, many of these changes were reversed by the end of the experiment, with no significant differences between days 0 and 14 of dosing. These results suggest a rapid adaptation of the rumen bacterial community linked to the estimated increase in the abundance of O. formigenes (from 0.002% to 0.007% of oxc gene in relation to the total bacteria 16S rDNA; P < 0.01), which is assumed to be responsible for oxalate breakdown.

Methane is the most important anthropogenic contribution to climate change after carbon dioxide and represents a loss of feed energy for the animal, mainly for herbivorous species. However, our knowledge about the ecology of Archaea, the microbial group responsible for methane synthesis in the gut, is very poor. Moreover, it is well known that hindgut fermentation differs from rumen fermentation. The composition of archaeal communities in fermentation compartments of goats and rabbits were investigated using DGGE to generate fingerprints of archaeal 16S rRNA gene. Ruminal contents and faeces from five Murciano-Granadina goats and caecal contents of five commercial White New Zealand rabbits were compared. Diversity profile of methanogenic archaea was carried out by PCR-DGGE. Quantification of methanogenic archaea and the abundance relative to bacteria was determined by real-time PCR. Methanogenic archaeal species were relatively constant across species. Dendrogram from DGGE of the methanogen community showed one cluster for goat samples with two sub-clusters by type of sample (ruminal and faeces). In a second cluster, samples from rabbit were grouped. No differences were found either in richness or Shannon index as diversity indexes. Although the primer sets used was developed to investigate rumen methanogenic archaeal community, primers specificity did not affect the assessment of rabbit methanogen community structure. Rumen content showed the highest number or methanogenic archaea (log₁₀ 9.36), followed by faeces (log₁₀ 8.52) and showing rabbit caecum the lower values (log₁₀ 5.52). DGGE profile showed that pre-gastric and hindgut fermenters hold a very different methanogen community. Rabbits hold a microbial community of similar complexity than that in ruminants but less abundant, which agrees with the type of fermentation profile., (Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH.)

Several technologies have been tested to reduce enteric methanogenesis, but very few have been successfully used in practical conditions for livestock. Furthermore, the consequences of reduced rumen methane production on animal performance and milk quality are poorly understood. The aim of this work was to investigate the effect of feeding bromochloromethane (BCM), a halogenated aliphatic hydrocarbon with potential antimethanogenic activity, to dairy goats on rumen methane production, fermentation pattern, the abundance of major microbial groups, and on animal performance and milk composition. Eighteen goats were allocated to 2 experimental groups of 9 animals each: treated (BCM+) or not (BCM-) with 0.30 g of BCM/100 kg of body weight per day. The BCM was administered per os in 2 equal doses per day from parturition to 2 wk postweaning (10 wk). After weaning, methane emissions were recorded over 2 consecutive days (d 57 and 58 on treatment) in polycarbonate chambers. On d 59, individual rumen fluid samples were collected for volatile fatty acid (VFA) analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. On d 69 and 70, daily milk production was recorded and samples were collected for determination of fat, protein, lactose, casein, and total solids concentration by infrared spectrophotometry, and fatty acid composition by gas chromatography. Treatment with BCM reduced methane production by 33% (21.6 vs. 14.4 L/kg of DMI) compared with nontreated animals, although it did not affect the abundance of rumen bacteria, protozoa, and total methanogenic archaea. The observed improvement in the efficiency of digestive processes was accompanied by a 36% increase in milk yield, probably due to the more propionic type of rumen fermentation and an increase in VFA production. The increase in milk yield was not accompanied by any changes in the concentrations or yields of fat, protein, or lactose. Despite the substantial decrease in methane production, only minor changes in milk fatty acid profile were observed, suggesting that ruminal biohydrogenation pathways were not affected. Compounds that influence terminal biochemical pathways for methane production deserve further development for future application in the dairy goat sector., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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.

Eight single-flow continuous-culture fermenters were used in a completely randomized block design with a 2 × 4 factorial arrangement of treatments to investigate the effects of the feed-to-buffer ratio (F/B) on ruminal fermentation, the diversity and community structure of bacteria, nutrient digestibility, and N metabolism. Four diets with forage-to-concentrate ratios of 70:30 or 30:70 with alfalfa or grass hay as forage were supplied to fermenters twice per day at 2 different F/B (23.5 and 35 g of DM/L). The dilution rate was kept constant (5.3%) among all fermenters by infusing the same volume of buffer. An increase in the total volatile fatty acid (VFA) concentration and a decrease in the average pH were observed with an increased F/B. In addition, the molar proportions of all individual VFA found in fermenters differed, depending on the F/B. A terminal restriction fragment length polymorphism analysis showed that the community structure and diversity of bacteria were highly influenced by the F/B. Both diversity and the number of peaks in the electropherograms were lower in most fermenters receiving diets at a high F/B, whereas the similarity percentage of the bacterial communities across diets was higher as the F/B increased. Moreover, the high reduction of neutral detergent fiber digestibility (15.3% ± 3.65) in fermenters with high F/B suggested a pH-related decrease in the cellulolytic bacterial community as the F/B increased. The crude protein degradation found in fermenters receiving diets with a high F/B was lower compared with that from fermenters with a low F/B. The VFA concentration and purine bases flow response patterns to diets were similar to in vivo conditions only in the case of fermenters with a low F/B. The results suggested that the community structure and diversity of bacteria, as well as the in vitro fermentation parameters, may be affected by the F/B that is used, most likely through a pH effect. In addition, several fermentation parameters showed different response patterns to diets according to the F/B used. Therefore, the amount of feed supplied to single-flow continuous-culture fermenters in which pH is not under control should be carefully chosen according to the volume of buffer infused for the purpose of simulating ruminal fermentation., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The aim of this experiment was to study the persistence in time of bacterial and methanogenic archaeal communities that establish in the rumen of lambs fed two different diets at weaning. Twenty ewes with single lambs were used in two phases. In phase I, 10 lambs had access only to grass hay (H group). The other 10 lambs had free access to concentrate and grass hay (C group). After 20 weeks, five lambs from each group were slaughtered and rumen samples were kept for analysis. In phase II, the remaining lambs were grouped together and fed grass plus concentrate for 4 months and then slaughtered, and rumen samples were collected for analysis. Terminal restriction fragment length polymorphism analysis showed a different bacterial and methanogen population established in the rumen of H and C lambs in phase I. These differences disappeared for methanogens after both groups were fed the same diet for 4 months; however, the total bacterial community remained different for the H and C samples. Our results suggest that some of the differences in the microbial populations that establish in animals fed different diets at weaning persist in the long term, which provides the possibility of programming the microbial populations in the adult animal.

This study evaluated the effects of tannins on ruminal biohydrogenation (BH) due to shifts in the ruminal microbial environment in sheep. Thirteen lambs (45 days of age) were assigned to two dietary treatments: seven lambs were fed a barley-based concentrate (control group) while the other six lambs received the same concentrate with supplemental quebracho tannins (9.57% of dry matter). At 122 days of age, the lambs were slaughtered, and the ruminal contents were subjected to fatty acid analysis and sampled to quantify populations of Butyrivibrio fibrisolvens, which converts C(18:2) c9-c12 (linoleic acid [LA]) to C(18:2) c9-t11 (rumenic acid [RA]) and then RA to C(18:1) t11 (vaccenic acid [VA]); we also sampled for Butyrivibrio proteoclasticus, which converts VA to C(18:0) (stearic acid [SA]). Tannins increased (P < 0.005) VA in the rumen compared to the tannin-free diet. The concentration of SA was not affected by tannins. The SA/VA ratio was lower (P < 0.005) for the tannin-fed lambs than for the controls, suggesting that the last step of the BH process was inhibited by tannins. The B. proteoclasticus population was lower (-30.6%; P < 0.1), and B. fibrisolvens and protozoan populations were higher (+107% and +56.1%, respectively; P < 0.05) in the rumen of lambs fed the tannin-supplemented diet than in controls. These results suggest that quebracho tannins altered BH by changing ruminal microbial populations.

The potential of different legume seeds species, including recently new developed varieties (Vicia faba: a commercial variety and varieties Alameda, Palacio and Baraka; Lupinus angustifolius; Pisum sativum and Cicer arietinum: varieties Fardon and Zegri) as protein supplements to low quality forages was evaluated. First, the chemical composition, in vitro digestibility, in situ degradability and in vitro/in situ intestinal digestibility of legume seeds were determined. The chemical composition was rather similar within genus. Vicia faba beans contained more condensed tannins (35.8-56.4 g/kg DM) and less ether extract (12.8-9.5 g/kg DM) than the other legumes. The rumen degradability and intestinal digestibility of the undegraded protein in the rumen was very similar among the seeds, with exception of lupins, having a much higher degradation rate than the rest. Second, the fermentation characteristics of diets, which were based on two low quality forages (olive leaves and barley straw) and feed blocks without or with supplementation of legume seeds or soybean meal, were investigated by using single-flow continuous culture fermenters. In this trial, the fermentation parameters (ammonia, pH and volatile fatty acids), the microbial protein synthesis and the degradation of olive leaves and barley straw promoted by the different diets were studied. Compared to soybean meal, beans and peas showed similar suitability as protein supplements for sustaining in vitro fermentation of low-quality forages. However, our results suggest a significant interaction between the type of legume used as supplement and the type of forage used, which need to be further studied in vivo.

The effects of forage type and forage:concentrate ratio (F:C) on apparent nutrient digestibility, ruminal fermentation, and microbial growth were investigated in goats. A comparison between liquid (LAB) and solid (SAB)-associated bacteria to estimate microbial N flow (MNF) from urinary purine derivative excretion was also examined. Treatments were a 2 x 2 factorial arrangement of forage type (grass hay vs. alfalfa hay) and high vs. low F:C (70:30 and 30:70, respectively). Four ruminally cannulated goats were fed, at maintenance intake, 4 experimental diets according to a 4 x 4 Latin square design. High-concentrate diets resulted in greater (P < 0.001) nutrient digestibility except for ADF. However, CP digestibility increased (P < 0.001) only for the high-concentrate diets including grass hay. Likewise, N retention, ruminal NH(3)-N concentration, and urinary excretion of purine derivatives increased (P < 0.05) with increasing concentrate in animals fed diets based on grass hay (0.23 vs. 0.13 g of retained N/g of digested N, 30.1 vs. 12.9 mg of NH(3)-N/100 mL, and 11.5 vs. 8.40 mmol/d, respectively), but not (P > 0.05) when diets included alfalfa hay. Total protozoa numbers and holotricha proportion were greater and less (P < 0.001), respectively, in high- than in low-concentrate diets. The F:C affected (P < 0.001) ruminal pH but not total VFA concentration (P = 0.12). Ammonia-N concentration was similar (P = 0.13) over time, whereas pH, VFA concentration, and protozoa numbers differed (P < 0.001) among diets. Estimated MNF was strongly influenced by using either the purine bases:N ratio obtained in our experimental conditions or values reported in the literature for small ruminants. There was a F:C effect (P = 0.006) on MNF estimated from LAB but not from SAB. The effect of F:C shifting from 70:30 to 30:70 in goat diets depends on the type of forage used. The MNF measured in goats fed different diets was influenced by the bacterial pellet (LAB or SAB). In addition, the purine bases:N ratio values found were different from those reported in the literature, which underlines the need for these variables to be analyzed directly in pellets isolated from specific animals and experimental conditions.

The aim of this study was to compare, in goats and wethers, the nutritive utilization of diets including olive leaves (OL) and the possible detrimental effect of that by-product. Three different diets were studied: OL, OL plus polyethylene glycol (PEG) (OLP) and OL supplemented with barley [164 g/kg dry matter (DM)] and faba beans (59 g/kg DM) (OLSUP). Apparent digestibility of nutrients and energy and nitrogen balances were determined along with creatinine concentrations and alkaline phosphatase (ALP) activity in the serum. The apparent digestibility of OL was low and similar in goats and wethers (54.4% and 53.5%, 22.2% and 21.6% and, 47.7% and 46.6% for DM, crude protein and neutral detergent fibre in goats and wethers, respectively). The addition of PEG did not improve (p > 0.05) digestibility of OL, although a slightly beneficial effect on the digestion of structural carbohydrates was observed (up to 8.4% and 7.10% in goats and wethers, respectively). The supplementation of OL with barley and faba beans increased (p < 0.001) the apparent digestibility of nutrients and the energy value. The consumption of the OL led to high ALP activity in the serum. Despite the higher sensitivity of wethers to Cu levels, our results show a similar digestive use of OL by wethers and goats and suggest the need of further comparative investigations focusing on the effect of the presence of high Cu levels in the by-product on the animal health.