Dairy ruminants often experience physiological disturbances during the post-weaning, including suboptimal rumen fermentation due to an underdeveloped rumen microbiota, and diarrhoea associated with intestinal inflammation and oxidative stress. This study explored six commercial feed additives with a potential dual mode of action to optimise both rumen fermentation and intestinal health in a post-weaning scenario. The experimental treatments included dietary supplementation with saponins (SAP), tannins (TAN), lauric acid (LAU), fish oil (FIO), high-unsaturated olein (HUO) and high-saturated olein (HSO). Additives were tested at four doses (0, 100, 300 and 600 mg/L) with four experimental replicates. Effects on rumen fermentation were evaluated in batch cultures using inocula from dairy lambs and a high concentrate substrate, while intestinal health responses were evaluated in Caco-2 cells. Saponins (p = 0.01) and tannins (p < 0.01) linearly decreased rumen ammonia-N concentration in batch cultures. In Caco-2 cells, saponins improved metabolic activity (p = 0.04), whereas tannins reduced pro-inflammatory IL-6 concentration (p < 0.01), suggesting beneficial effects on intestinal health. Lipid sources exerted minimal effects on rumen fermentation and induced intestinal inflammation when supplemented at high doses. Overall, the physiological constrains of dairy ruminants during the post-weaning period, such as an immature rumen microbiota, absence of protozoa and low rumen pH, may limit the efficacy of additives in improving rumen fermentation. Nevertheless, saponins and tannins showed potential to reduce rumen proteolysis and support intestinal health during this critical developmental stage. Further in vivo research is required to validate these findings.

Young dairy ruminants often have limited rumen fermentative capacity, which can impair adaptation to solid diet during weaning. This study evaluated the effects of ruminant colostrum supplementation to enhance in vitro rumen fermentation at weaning. A batch culture system using rumen inoculum from artificially reared lambs was incubated with ovine, bovine, or reconstituted bovine colostrum at 4 and 20 mL/L. Sequential colostrum fractionation was used to identify the main functional components. Whole colostrum supplementation at high doses reduced ruminal pH (p < 0.001) and increased gas production (+18%), ammonia-N (+41%) and butyrate proportion (+10%) across colostrum sources, indicating enhanced microbial fermentation. Responses were dose-dependent and consistent across colostrum sources. Lipid removal had minimal effects, whereas depletion of high-molecular-weight molecules, mostly IgG, markedly reduced gas production and protein degradation. Removal of medium-size proteins such as lactoferrin produced minor changes. Residual activity in low-molecular-weight fractions was retained likely associated with the presence of fermentable substrates and bioactive compounds. These findings indicate that colostrum may exhibit both nutritional and modulatory effects on in vitro rumen fermentation, with IgG identified as a key component. These findings support the preservation of IgG in colostrum-derived products to enhance rumen fermentation, although further in vivo validation is required to evaluate the effects in weaned ruminants.

Improving efficiency and reducing environmental impacts pose challenges to Mediterranean livestock systems, with few viable alternatives to conventional forages. This study evaluated cucumber-straw silage (SIL) as an alternative forage to oat hay (HAY) for lamb production using integrated in vitro, in vivo, and life-cycle assessment approaches. An in vitro fermentation study was conducted by progressively replacing concentrate with SIL or HAY, and rumen fermentation, and CH4 production were measured over 144 h. Subsequently, a 12-week in vivo trial was conducted on a commercial farm with 450 lactating Segureña ewes and 610 lambs to assess productive performance and metabolic status. In vitro experiment followed a factorial design (two forage types × four inclusion levels) within a completely randomized design, while in vivo experiment applied a completely randomized design. In vitro results showed that a 50% inclusion of SIL maintained rumen fermentation activity comparable to HAY in terms of gas production and volatile fatty acid concentrations; moreover, SIL shifted fermentation toward higher butyrate (+13.3%; P = 0.001) and lower CH4 emissions (-17.3%, interaction P = 0.007). In vivo, SIL supported comparable animal performance to HAY, with no significant differences in lamb growth (210 vs. 215 g/day), ewe body condition score, or health, as blood metabolites remained within physiological ranges. A cradle-to-farm-gate life cycle assessment, following ISO-14044 standards and applying two allocation approaches for cucumber waste, revealed that under a zero-allocation scenario (treating waste as burden-free), greenhouse gas emissions averaged 9.50 CO2-eq/kg liveweight (95% CI: 8.29-13.6) compared with 11.6 (95% CI: 10.1-15.9) for HAY, alongside reductions in land occupation of 50%, and water consumption of 70%. Therefore, SIL presents an effective strategy to valorise horticultural waste while maintaining productivity and potentially improving the environmental performance of Mediterranean lamb systems. Further validation across diverse management conditions and by-product sources is recommended.

Evolution has enabled ruminants to develop a complex rumen microbiota that aids in the digestion of fibrous feeds. This study examines whether promoting a highly diverse rumen microbiota during early life continues to offer long-term benefits in modern dairy production systems, where young ruminants are reared without exposure to adult ruminants and are fed high-concentrate diets. A total of 36 newborn goat kids were divided in 4 groups. During the first 10 weeks of age, animals were daily inoculated with autoclaved rumen fluid (AUT), fresh rumen fluid from adult goats fed forage (RFF), or concentrate diet (RFC), or received no inoculation (CTL). At 29 weeks of age, following an 18-week wash out period, the animals were shifted from a full-forage to a high-concentrate diet to assess their ability to adapt and digest this later diet. Results revealed that early life inoculation with fresh rumen fluid had a lasting effect on the rumen microbiota, promoting higher bacterial (+93 OTUs), methanogens (+5 OTUs) and protozoal diversity (+23 OTUs), whereas CTL animals remained protozoa-free. This superior microbial complexity accelerated the adaptation to high-concentrate diets, decreased digestive disorders (rumen acidosis and diarrhoea) and increased BW gain. Once adapted to the diet, inoculated animals exhibited higher rumen VFA concentration (+16%), blood glucose (+28%), rumen papillae width (+43%) and increased expression of rumen epithelium genes involved in the cell proliferation (Cyclin 1), VFA absorption (MCCT1) and VFA metabolism (HMGCL), suggesting an enhanced energy uptake capacity. Inoculation with autoclaved rumen fluid as source of VFA had lower long-term effects compared to fresh inocula. No differences across treatments were noted for feed digestibility, N excretion, and microbial protein synthesis. In conclusion, promoting greater rumen microbial diversity is a desirable strategy to prevent digestive disorders during the adaptation process to high-concentrate diets, having minor effects once the animals are adapted to this diet.

Despite the increasing interest in developing antimethanogenic additives to reduce enteric methane (CH4) emissions and the extensive research conducted over the last decades, the global livestock industry has a very limited number of antimethanogenic feed additives (AMFA) available that can deliver substantial reduction, and they have generally not reached the market yet. This work provides technical recommendations and guidelines for conducting tests intended to screen the potential to reduce, directly or indirectly, enteric CH4 of compounds before they can be further assessed in in vivo conditions. The steps involved in this work cover the discovery, isolation, and identification of compounds capable of affecting CH4 production by rumen microbes, followed by in vitro laboratory testing of potential candidates. The finding of new bioactive compounds as AMFA can be based on 2 approaches: empirical and mechanistic. The empirical approach involves obtaining and screening compounds present in databases and repositories that potentially possess the desired effect but have not yet been tested, screening natural sources of secondary compounds such as plants, fungi, and algae for their antimethanogenic effects, or examining compounds with antimethanogenic effect on microbes in other research domains outside the rumen. In contrast, the mechanistic approach is the theoretical process of discovery new bioactive compounds based on existing knowledge of a biological target or process. The in vitro methodologies reviewed include examining effects at the subcellular level, in single pure cultures of methanogens and examining in more complex mixed rumen microbial populations. Simple in vitro methodologies (subcellular assessments and batch culture) allow testing a large number of compounds, whereas more complex systems simulating the rumen microbial ecosystem can test a limited number of candidates but provide better insight about the antimethanogenic efficacy. This work collated the main advantages, limitations, and technical recommendations associated with each step and methodology use during the identification and screening of AMFA candidates.

This study assessed the effects of using a total mixed ration (TMR), compared to distributing the concentrate and the forage separately, on the productive performance of beef cattle fed high-concentrate diets and on rumen fermentation characteristics, including methane production. Eighteen rumen-fistulated beef calves were fed a concentrate plus pelleted barley straw separately (CS; n = 9) or as TMR (n = 9). Average daily gain and intake of TMR and of straw and concentrate with the CS treatment were recorded. Digestibility was estimated using ashes insoluble in hydrochloric acid as a marker. Rumen liquid and gas samples were obtained, and the abundance of total bacteria, methanogens, protozoa, and anaerobic fungi was analysed. Animals were slaughtered at 500 kg live weight (LW), and dressing percentage and carcass classification were obtained. Another 49 animals for each treatment were used on a commercial farm to exclusively assess animal performance. The treatment (CS or TMR) did not significantly affect (p > 0.05) final LW, average daily gain, carcass dressing percentage, dry matter or digestible organic matter intake, digestibility values, production of methane and carbon dioxide, and abundance of microorganisms in the rumen. However, animals fed TMR had lower rumen pH (5.87 vs. 5.58, p = 0.041 for CS and TMR), suggesting a higher risk of experiencing rumen acidosis than those fed straw and concentrate separately. In conclusion, using a total mixed ration does not seem a suitable strategy to reduce methane production or prevent rumen acidosis in feedlot cattle.

Horticultural production in the Mediterranean basin generates considerable quantities of byproducts, which, if not valorised, can pose environmental problems. This study evaluated the potential of cucumber-straw silage as an alternative forage for sheep. Eighteen Segurena sheep (10 months old; 30 +/- 0.4 kg BW) were allocated to two experimental forages: oats hay (CTL, 985 g/kg DM) or cucumber-straw silage (SIL, 15 g/kg DM). The SIL forage contained more NDF (780 vs. 516 g/kg DM) and less CP (64.9 vs. 82.9 g/kg DM) than CTL. The trial included one week of adaptation with ad libitum feeding followed by two weeks of restricted intake. Measurements during the last week included digestibility, N balance, rumen fermentation, and blood metabolites. Results indicated that health status remained unaffected, and DMI was similar under ad libitum feeding. Under restricted intake, SIL-fed sheep displayed slightly higher DMI (+12 %), greater digestible energy intake (+33 %), and higher ruminal acetate (+10 %) and butyrate (+40 %), accompanied by increased blood beta-hydroxybutyrate concentrations (+89 %). Despite lower CP intake, SIL-fed animals exhibited higher ruminal iso-acid proportions and blood urea N, suggesting enhanced proteolysis and urea recycling. These compensatory mechanisms resulted in similar ruminal NH3-N levels and microbial protein synthesis across treatments, although N retention and efficiency were significantly lower in the SIL group. In conclusion, SIL represents a feasible energy source for sheep, although its limited protein content may require further N supply in high-yielding ruminants. This silage valorisation could reduce food waste and improve circular agriculture and sustainability in livestock production.

BACKGROUND:Weaning represents a significant digestive challenge for dairy ruminants due to their underdeveloped rumen microbiota resulting from early separation from their dams shortly after birth. This study aimed to optimize the weaning process by evaluating six commercial feed additives, including four blends of essential oils (BEOs), a probiotic (live yeast) and a prebiotic (pectin). The effects on rumen fermentation were evaluated using batch cultures with rumen inocula collected at the post-weaning period from artificially reared lambs, whereas the effects on gut health indicators were assessed using Caco-2 cell monolayers. RESULTS:Most tested additives showed minor benefits on rumen fermentation, even at higher doses (600 mg L-1). A BEO containing turmeric, thymol and yeast cell wall promoted a shift towards propionic fermentation, whereas supplementation with pectin decreased NH3-N concentrations. Conversely, all four BEOs positively influenced intestinal health, resulting in improved Caco-2 cell viability and lower levels of pro-inflammatory interleukin-6. CONCLUSION:Modulating rumen fermentation during the post-weaning in artificially reared lambs is challenging due to their immature rumen microbiota, absence of protozoa, and low rumen pH. Nevertheless, this in vitro study suggests that certain BEOs may enhance intestinal health by improving cell viability and reducing inflammation. Further in vivo studies are needed to confirm these findings. © 2025 Society of Chemical Industry.

In Spain, a large number of unweaned calves from northern Europe's dairy industry are transported for intensive beef production, which could pose health risks around weaning due to the separation from their mothers. This study evaluated the impact of including different feed additives in starter concentrate on blood parameters and rumen functional development in 112 calves at the growing farm. We hypothesized that feed additives could enhance rumen function and mitigate health risks associated with transportation and intensive management. The treatments tested, against a control diet (CTL), included: blend of essential oils from plant extracts (EO), yeast-based products (SYN) and a mix of yeast probiotics, oregano-based essential oil and sodium butyrate (MIX). Each treatment was administered during the preweaning stage, with all calves transitioning to the MIX diet postweaning. In the experiment, blood and rumen samples were collected before weaning (8 weeks old) and two months after weaning (18 weeks old) for rumen fermentation and microbial population analyses. Calves were weighed upon arrival (3 weeks), at weaning (10 weeks) and two months postweaning (18 weeks) to assess performance. Results from the experiment showed that rumen fermentation profiles remained stable with regard to volatile fatty acids (VFA) concentrations and pH, indicating effective solid feed consumption and microbial activity before weaning. Postweaning, all feed additives treatments improved rumen fermentation by increasing total VFA and lowering pH, though body weight gains remained unaffected. Concentrations of bacteria and archaea increased compared to pre- weaning levels, protozoa were absent, and anaerobic fungi did not become established until 2 months postweaning. In conclusion, feed additives provided preweaning improved rumen development, although they did not increase productivity. The treatment should be applied preweaning, highlighting the importance of this particular window of time.

The objective of this study was to assess the effect of using ground and pelleted straw, instead of straw in the long form, on feed utilization by and methane production from feedlot cattle. Eighteen Montbeliarde cross bred male beef calves (358 +/- 3,8 kg and 251 +/- 0,5 days) were assigned to two treatments (9 animals per treatment) in a Randomized Complete Block Design: large straw (LS), and ground (6 mm sieve) and pelleted (8 mm granule diameter) straw (PS), both offered ad libitum. All animals were fitted in the dorsal sac of the rumen with a 15 mm internal diameter permanent cannula, and consumed the same concentrate ad libitum. Average daily gain, and intake of straw and concentrate, were recorded through a sixteen-week period. Digestibility was estimated using chromic oxide as marker, and rumen liquid and gas samples were obtained at different times of the day in two occasions during the experimental period to characterize rumen fermentation. Abundance of total bacteria, methanogens, protozoa and anaerobic fungi was also analysed. All animals were slaughtered at a target LW of 500 kg, and dressing percentage and carcass classification were obtained from the slaughterhouse. All data were analysed using the PROC MIXED of SAS. The results showed that the particle size of straw had not a significant effect (P > 0.05) on final live weight (LW), average daily gain, carcass dressing percentage, intake of straw, concentrate, dry matter or digestible organic matter, digestibility values, production of methane (CH4) and carbon dioxide (CO2), and abundance of microorganisms in the rumen. However, animals fed pelleted straw had lower rumen pH (5.97 vs 6.43, P = 0,0423), and tended to have lower rumen protozoal concentration (P = 0.0826), suggesting a higher risk of experiencing rumen acidosis than those fed long straw. In conclusion, reducing the particle size of straw does not seem a suitable strategy to reduce methane production or prevent rumen acidosis in feedlot cattle.

This study evaluated the effects of supplementing pre-weaned lambs with live yeast (Saccharomyces cerevisiae CNCM I-1077; 3 x 10<^>9 CFU/kg DM) on rumen fermentation, blood metabolites, gut development, and performance. Forty-six male lambs were randomly assigned to two treatments in a completely randomised design: a control group (CTL) fed a starter concentrate, and a yeast group (YST) receiving the same concentrate supplemented with live yeast. Both groups had ad libitum access to diets and barley straw from two weeks of age until weaning at eight weeks. Rumen fluid and blood samples were collected at 7 and 10 weeks to assess fermentation parameters and metabolites, and rumen tissue samples were collected at slaughter to evaluate papillae development. Rumen fermentative activity increased from 7 to 10 weeks, with higher concentrations of volatile fatty acids (VFA), lactate, and ammonia-N (p<0.001). YST lambs showed higher rumen concentrations of protozoa (p-0.007) and anaerobic fungi (p-0.009), and a 56% increase in total VFA concentration pre-weaning and slightly enhanced concentrate intake. Blood metabolites remained within a physiological range, and YST lambs showed higher b-hydroxybutyrate (p<0.001) and total protein (p-0.054) than CTL lambs. Yeast also reduced parakeratosis scores (p<0.001) and increased papillae width (p-0.020). However, growth performance was not significantly affected. These results suggest that live yeast supplementation during the pre-weaning period supports microbial colonisation and rumen papillae development, facilitating a smoother transition from liquid to solid feeding and improved rumen function post-weaning.

An in vitro and an in vivo study were conducted to investigate the effects of a blend of cinnamaldehyde, eugenol, and capsicum oleoresin (CEC) on rumen fermentation parameters, animal performance, and methane (CH4) emissions in dairy cows. Continuous culture fermenters (CCF) were utilized to test one of two treatments: (1) CON; no supplementation and (2) CEC supplemented at 0.0125 g/d. The basal diet consisted of grass hay and concentrate (50:50). Supplementation with CEC increased (p < 0.01) total volatile fatty acids (VFA; mM) and decreased (p = 0.02) CH4 concentration compared with CON in vitro. Additionally, protozoa abundance tended (p = 0.07) to decrease in CEC compared with CON. The in vivo experiment utilized forty Holstein-Friesian dairy cows (32% primiparous and 68% multiparous) averaging 163 ± 48 days in milk (DIM) and 38 ± 6.2 kg/d of milk yield (MY). Cows were blocked by parity and randomly assigned to one of two treatments: (1) CON; no supplementation and (2) CEC supplemented at 1.2 g/cow/d. The basal diet consisted of grass hay and concentrate (40:60). Individual CH4 emissions were recorded using the sniffer technique. Dry matter intake (DMI) and eating rate were increased (p < 0.01; 3.6% and 5.2%, respectively), while feed efficiency decreased (p < 0.05) in CEC compared with CON. Additionally, CEC decreased (p = 0.02) CH4 yield by 16.4% and tended to reduce daily CH4 production (p = 0.09) and CH4 intensity (p = 0.08) by 13.4% and 14.0%, respectively. Supplementing CEC decreased CH4 concentration in vitro and CH4 yield in vivo without negatively impacting performance parameters.

This publication aims to provide guidelines of the knowledge required and the potential research to be conducted in order to understand the mode of action of antimethanogenic feed additives (AMFA). In the first part of the paper, we classify AMFA into 4 categories according to their mode of action: (1) lowering dihydrogen (H2) production; (2) inhibiting methanogens; (3) promoting alternative H2-incorporating pathways; and (4) oxidizing methane (CH4). The second part of the paper presents questions that guide the research to identify the mode of action of an AMFA on the rumen CH4 production from 5 different perspectives: (1) microbiology; (2) cell and molecular biochemistry; (3) microbial ecology; (4) animal metabolism; and (5) cross-cutting aspects. Recommendations are provided to address various research questions within each perspective, along with examples of how aspects of the mode of action of AMFA have been elucidated before. In summary, this paper offers timely and comprehensive guidelines to better understand and reveal the mode of action of current and emerging AMFA.

There is a need for rigorous and scientifically-based testing standards for existing and new enteric methane mitigation technologies, including antimethanogenic feed additives (AMFA). The current review provides guidelines for conducting and analyzing data from experiments with ruminants intended to test the antimethanogenic and production effects of feed additives. Recommendations include study design and statistical analysis of the data, dietary effects, associative effect of AMFA with other mitigation strategies, appropriate methods for measuring methane emissions, production and physiological responses to AMFA, and their effects on animal health and product quality. Animal experiments should be planned based on clear hypotheses, and experimental designs must be chosen to best answer the scientific questions asked, with pre-experimental power analysis and robust post-experimental statistical analyses being important requisites. Long-term studies for evaluating AMFA are currently lacking and are highly needed. Experimental conditions should be representative of the production system of interest, so results and conclusions are applicable and practical. Methane-mitigating effects of AMFA may be combined with other mitigation strategies to explore additivity and synergism, as well as trade-offs, including relevant manure emissions, and these need to be studied in appropriately designed experiments. Methane emissions can be successfully measured, and efficacy of AMFA determined, using respiration chambers, the sulfur hexafluoride method, and the GreenFeed system. Other techniques, such as hood and face masks, can also be used in short-term studies, ensuring they do not significantly affect feed intake, feeding behavior, and animal production. For the success of an AMFA, it is critically important that representative animal production data are collected, analyzed, and reported. In addition, evaluating the effects of AMFA on nutrient digestibility, animal physiology, animal health and reproduction, product quality, and how AMFA interact with nutrient composition of the diet is necessary and should be conducted at various stages of the evaluation process. The authors emphasize that enteric methane mitigation claims should not be made until the efficacy of AMFA is confirmed in animal studies designed and conducted considering the guidelines provided herein.

The safety of novel proteins is routinely evaluated in various regulated areas of the food and feed chain, including genetically modified (GM) crops and novel foods (NFs). This project aimed to map the food and feed products containing protein from the main GM crops, relevant food categories falling under the NF Regulation, and unconventional feed, together with their production processes and to discuss the effect of the mapped processes on the safety of the corresponding novel proteins. A scoping literature review (1,325 documents included), an open online survey and a stakeholder workshop were the basis to build up the mappings for products and processes, also including operational conditions for each processing step. In the case of crops, the information gathered also helped identify more than 40 products, and the corresponding production processes, not included in the OECD consensus documents for compositional considerations of GM crops. Moreover, a systematic literature review (154 documents included), carried out within the project, assisted in the identification of the available evidence on the impact of processing on protein safety. Overall, certain processes, such as thermal treatments, fermentation, or enzymatic hydrolysis, significantly enhanced protein digestibility across various food/feed matrices. Similarly, fermentation, ensiling, and extraction processes have been shown to improve nutritional properties in various products. The data collected seemed to indicate that heating can effectively reduce the activity of NEPs from GM crops and that heating and enzymatic hydrolysis can reduce IgE reactivity for certain proteins and operational conditions. However, exceptions to these trends were also reported in the literature, and in certain cases (e.g., impact on gut microbiota), the evidence gathered was insufficient to draw substantiated conclusions. This project also contributed to identify existing knowledge gaps and research needs towards regulatory risk assessment of food and feed products containing protein.

Many strategies for mitigating enteric methane (CH4) emissions in ruminants have focused on suppressing the activity of rumen methanogens, but this often leads to excess dihydrogen (H2) accumulation in the rumen, which is subsequently expelled and represents a potential energy loss. We hypothesized that phloroglucinol could act as a H2 acceptor when rumen methanogenesis is inhibited and be potentially transformed into beneficial compounds for the animal. Eight adult goats were randomly assigned to a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments: two levels of Asparagopsis taxiformis as CH4 inhibitor [0 vs. 5g/kg on a dry matter (DM) basis; AT- and AT+, respectively] and two levels of phloroglucinol as alternative H2 acceptor (0 vs. 20g/kg DM, PG- and PG+, respectively). Therefore, four dietary treatments were considered: i) basal diet (AT-PG-); ii) A. taxiformis alone (AT+PG-); iii) phloroglucinol alone (AT-PG+); and iv) the combination of A. taxiformis and phloroglucinol (AT+PG+). Animals were fed a maintenance diet with a 70:30 forage-to-concentrate ratio. After 10 d of adaptation to the diet, enteric gas emissions were measured in respiration chambers during 3 d prior to rumen content sampling on d 14. Dietary supplementation with A. taxiformis decreased CH4 production (-33.9%) and increased H2 emissions (+3465%), along with greater rumen propionate concentration. In contrast, phloroglucinol supplementation alone did not impact CH4 emissions or the rumen concentration of the main microbial groups but substantially increased acetate molar proportion (+10.2%) which could act as an alternative H2 acceptor. Moreover, when A. taxiformis was combined with phloroglucinol, it resulted in a decrease in H2 emissions (-68.1%). However, this decrease in H2 emissions was not fully explained by the increase in the acetate as phloroglucinol led to an increase in acetate both when methanogenesis was inhibited and when it was not. These findings suggest that the rumen fermentation of phloroglucinol may capture some of the additional H2 arising from the inhibition of methanogenesis by A. taxiformis through pathways other than acetate formation. Moreover, H2 emissions were not eliminated and most of the decrease occurred during the post-prandial stage, suggesting that the efficiency of H2 redirection could be further improved.

The rumen represents a dynamic microbial ecosystem where fermentation metabolites and microbial concentrations change over time in response to dietary changes. The integration of microbial genomic knowledge and dynamic modelling can enhance our system-level understanding of rumen ecosystem’s function. However, such an integration between dynamic models and rumen microbiota data is lacking. The objective of this work was to integrate rumen microbiota time series determined by 16S rRNA gene amplicon sequencing into a dynamic modelling framework to link microbial data to the dynamics of the volatile fatty acids (VFA) production during fermentation. For that, we used the theory of state observers to develop a model that estimates the dynamics of VFA from the data of microbial functional proxies associated with the specific production of each VFA. We determined the microbial proxies using CowPi to infer the functional potential of the rumen microbiota and extrapolate their functional modules from KEGG (Kyoto Encyclopedia of Genes and Genomes). The approach was challenged using data from an in vitro RUSITEC experiment and from an in vivo experiment with four cows. The model performance was evaluated by the coefficient of variation of the root mean square error (CRMSE). For the in vitro case study, the mean CVRMSE were 9.8% for acetate, 14% for butyrate and 14.5% for propionate. For the in vivo case study, the mean CVRMSE were 16.4% for acetate, 15.8% for butyrate and 19.8% for propionate. The mean CVRMSE for the VFA molar fractions were 3.1% for acetate, 3.8% for butyrate and 8.9% for propionate. Ours results show the promising application of state observers integrated with microbiota time series data for predicting rumen microbial metabolism.

The effects of dietary inclusion of silver nanoparticles (NanoAg, <100 nm diameter) on growth performance, gut microbiota, and silver tissue retention was assessed in broilers from 1 to 42 days of age. A total of 870 1-day-old male broilers (Ross 308) were weighted and distributed in 36 floor pens (n = 24) in an environmentally controlled room. The feeding program consisted of two periods (1-21 days and 22 to 42 days of age) and two experimental diets with the same ingredient composition including 2 g kaolin/kg with or without adsorbed NanoAg (10 mg/g kaolin). The experimental design was completely randomized, and the effects of the length of the period in which the birds received NanoAg (none, Ag0; from 1 to 21 days, Ag21; or from 1 to 35 days, Ag35) on growth performance, biodiversity of digestive microbiota and silver retention in body tissues were studied. A common feed without NanoAg was provided to all pens from 36 days onwards. At 21 and 42 days of, one random bird per pen (n = 12) was slaughtered and cecal samples were collected from 9 birds per treatment randomly selected to analyse gut microbiota. Besides, samples of liver and breast muscle were collected to determine silver tissue retention. From 1 to 21 days of age, NanoAg supplementation tended to improve feed conversion ratio (FCR, P = 0.070). From 22 to 35 days of age, FCR tended to be lower (P = 0.072) and average daily gain (ADG) was greater (P < 0.001) in broilers fed Ag21 and Ag35 than in those fed Ag0. Cumulatively, ADG was greater (P < 0.001) for Ag21 and Ag35 than for Ag0, but FCR was unaffected. Caecal microbiota was affected by age of birds, but dietary supplementation with NanoAg did not modify bacterial community structure, diversity and taxa distribution in the caecum neither at 21 nor at 42 days. At 21 days of age, silver retention in liver was 0.591 mg/kg dry tissue in all broilers supplemented with NanoAg. However, at 42 days of age, silver retention in the liver was only detected in two (0.139 mg/kg, n = 2) and 8 (0.183 mg/kg, n = 8) birds fed Ag21 and Ag35, respectively. Silver retention in muscle was exclusively detected at 42 days in two birds (0.124 mg/kg, n = 2) fed Ag35. Irrespective of the supplementation period, NanoAg increased ADG from 1 to 42 d. No silver retention was detected in the liver nor in breast muscle after 21 days of the end of treatment. However, traces of silver might remain in the liver 7 days after removal of NanoAg. Silver supplementation might be a promising strategy to improve growth performance in broilers without expecting any changes in the gut microbiota nor tissue retention after 21 days of NanoAg withdrawal.