• mixtures additives
• RUSITEC
• microbial biomarkers

Ruminant livestock is under the spotlight as being mainly responsible for non-CO2 GHG emissions, namely methane and nitrous oxide (as indirect emissions). Despite the fact that other sectors than agriculture are responsible for a higher proportion of GHG, and the emissions from livestock would still be mainly produced by the food production system even without animal livestock, their impact has to be lowered to tackle climate change.
Various effective strategies have been proposed previously in this sense, but they could be not applicable in all contexts. Tannins and essential oils compounds (EOC) as additives have been previously described for their properties to mitigate ruminal methane and ammonia. Their combination might be even more efficient, as they have different modes of action on rumen pathways. The present project focused on the combined use of such active compounds, supplemented under different in vitro conditions, with the aim of reducing ruminal ammonia and enteric methane. Overall, the fermentative profile, microbial community, ruminal fatty acid profile and microbial biomarkers were considered to assess the efficacy of mixtures and propose their mechanism of action.
Firstly, an extensive in vitro study was carried out with a total of 48 supplemented treatments, of which mitigating properties were evaluated in comparison to the basal diet fermented alone (control). The supplemented treatments were: 12 single additives, containing 10 mg of EO or 20 mg of tannins/g diet, to establish their basal efficiency; 36 mixture additives containing 20 mg of tannins/g + 10-15 mg of EO. Quebracho (Q) and chestnut (C) tannins defined C, Q and C/Q groups of mixtures with EO blends, formulated with oregano, thyme and clove EO, citrus peel, carvacrol, thymol, eugenol, α-pinene, and bornyl acetate. Effects on rumen pH, protozoal count, and proportions of individual volatile fatty acids (VFA) were limited. The tannins extracts seemed to cause most of the mitigating effects by suppressing ammonia by up to 31% and methane yield by up to 15%, with the highest reductions obtained with tannins+EOC supplements. However, this occurred by contemporary affecting the feeding value of the substrate, as indicated by reductions in total VFA and in vitro organic matter digestibility.
Overall, six mixtures of C and Q groups were the most efficient. Among these, the additives named C-10 and Q-2 were selected for further evaluation in a semi-continuous system (RUSITEC), and in comparison, to non-supplemented treatments (negative control, NC) and a commercial additive (positive control, PC). The Rusitec experiment lasted 10 days in which the fermentative profile was evaluated throughout the whole period, whereas on rumen liquor of the last day bacterial community composition and fatty acid profile were analysed. Considering the last 5 days as the experimental period, no effect considered differed significantly. Ruminal methane was reduced more by C-10 (-11.9%) than Q-2 methane (-7.2% in comparison to NC, not significant). On the other hand, Q-2 was more effective in mitigating ammonia formation (-36.6%, P<0.001) than C-10 (-25.5%). The mitigation extents for both additives were like the properties of the commercial additive, however, only the PC effect was supported by a wide-extent modification of the microbial community. The protozoal count was drastically reduced by PC along with bacterial biodiversity in comparison to other treatments. The C-10 and NC differed less for bacterial abundances, total VFA, and fatty acid profile, suggesting a direct effect on methanogens by C-10. Slightly higher differences in the bacterial community were reported between Q-2 and NC but not enough to explain neither mitigating properties on ammonia nor the slight negative effect on fibre and crude protein degradability or VFA. Therefore, Q-2 was suggested to synergistically bond diet protein and directly inhibit ruminal hyper-ammonia producers. Adverse effects on fibre degradability might be explained by a not-evaluated decrease of ruminal fungi or unclassified fibrolytic bacteria, as it was supported by significantly lower content of certain iso-fatty acids, characteristics of cellulolytic bacteria in rumen liquor fermented with C-10 and Q-2. Apart from such difference, the fatty acid profile of rumen liquor did not differ between C-10, Q-2 and NC, despite an accumulation of C18 biohydrogenation products was expected. The lack of such accumulation might be related to long-term microbial adaptation strategies that might have occurred over ten days of daily additive supplementation, such as adaptative modifications of overall energy metabolism or cell morphology that might also occur under in vivo conditions. Moreover, the total content of plasmalogen (evaluated as DMA, g/kg of dried rumen fluid) and their proportion were not affected by the treatment (except for an accumulation of DMA C18:0 and C18:1c9 with Q-2 and C-10); thus, suggesting that the growth of microorganisms was not particularly inhibited by C-10 and Q-2 additives. Such outcomes are overall promising as the newly formulated result effective in mitigating ruminal ammonia and methane while preserving ruminal biodiversity and resiliency. Both mitigating properties and slight reductions in feeding values have to be evaluated with further studies in vivo.