Changes in the Rumen Microbiota of Cows in Response to Dietary Supplementation with Nitrate, Linseed, and Saponin Alone or in Combination

Methane emissions associated with ruminant livestock production are an important contributor to global greenhouse gas emissions (1). Rumen methanogenesis is a naturally occurring process that involves methanogenic archaea consuming hydrogen to reduce carbon dioxide. Hydrogen and carbon dioxide production occurs during feed fermentation by bacteria, protozoa, and fungi; hydrogen availability is a limiting factor for methane production. In addition, there is a significant linear relationship between protozoan concentration in the rumen and methane emissions (2). Among the measures that have been undertaken to reduce methane production by ruminants, diet composition and inclusion of feed additives have received the most attention (3). Among them, nitrate added to ruminants’ diets consistently and persistently lowers methane emissions (4). Linseed oil, which is rich in linoleic acid, has proven to be one of the most efficient lipid sources used in methane mitigation strategies (4). Saponins are natural phytogenic feed additives used to improve animal feeding and production characteristics (5). Theoretically, these three additives lead to decreased methane production via different modes of action. Nitrate is an alternative electron acceptor, as its reduction competes with methane production for hydrogen (6). Additionally, nitrate or its reduced forms might be toxic to rumen methanogens and protozoa (7), but this effect was not systematically reported (8, 9). Lipids from linseed (and fats in general) added to diets replace a proportion of dietary carbohydrates and, as rumen microbes do not ferment them, less hydrogen is produced. Protozoal numbers have been reported to decrease with supplementary linseed oil (8, 10), although this effect was not always observed (11). Saponins can reduce methanogenesis by a toxic effect on rumen protozoa (5), but in vivo results indicate otherwise, as rumen microbes can deglycosylate and, thus, inactivate saponins (12).

Based on available information, we hypothesized that linseed oil and saponins would mainly affect hydrogen production (by a toxic effect on protozoa or by providing alternative substrates for rumen fermentation) and nitrate would mainly modulate hydrogen consumption pathways (by providing an alternative hydrogen sink). We performed amplicon-type sequencing analysis of rumen contents, sampled during two previous studies (8, 13); the first one reported the effect of linseed, nitrate, and linseed plus nitrate supplementation on enteric methane production; tea saponin replaced linseed in the second one. The primary aim of the current study was to search for changes in rumen microbiota structure and methanogenic activity that could explain observed reductions in methane emissions.

Minor but significant changes induced by treatment can be masked by spurious between-group differences unrelated to the treatment but rather to the host animal, the diet, or sample management. Moreover, it is not unusual to find reports on nitrate and fatty acid supplementation where methane decreased in a similar way, but effects on rumen microbiota were contrasting (14–17). On the other hand, it was recently shown that combination of microbial data from multiple sets of hosts with supposed similar microbiota should increase specificity and allow identification of causal microbes (18). Therefore, we took advantage of the data available from two independent studies, analyzed it separately but by following the same procedures, and made an integrated interpretation. Our secondary objective was to try to find clues to explain inconsistency in results from published studies.

(This article was submitted to an online preprint archive [19].)

Eight nonlactating dairy cows were randomly allocated to two 2-by-2 factorial designs. In study 1, dietary treatments consisted of control (CTL) diet, supplemented alternatively with linseed oil (LIN), nitrate (NIT), and linseed plus nitrate (LIN+NIT); in study 2, tea saponin (TEA) replaced linseed oil. In order to achieve adequate statistical power, the statistical model for both studies included cow as random effect, and fixed effects were experimental period and the following: (i) in study 1, linseed (CTL and NIT versus LIN and LIN+NIT), nitrate (CTL and LIN versus NIT and LIN+NIT), and their interaction, termed linseed×nitrate (or lin×nit), and (ii) in study 2, saponin (CTL and NIT versus TEA and TEA+NIT), nitrate (CTL and TEA versus NIT and TEA+NIT), and their interaction, saponin×nitrate (sap×nit). Throughout the text, linseed, nitrate, and saponin will refer to diet contrasts detailed above.

In study 1, compared to CTL, dietary treatments LIN, NIT, and LIN+NIT decreased methane production (g/day) by 22%, 29%, and 33%, respectively, and methane yield (g/kg of dry matter intake [DMI]) by 25%, 29%, and 32% (8). In study 2, NIT and TEA+NIT decreased methane production by 42% and 34% and methane yield by 36% and 29%, respectively, compared to CTL (13). TEA alone had no effect on methane production or on volatile fatty acid (VFA) profiles.

In both studies, Bacteroidales and Clostridiales were the dominant bacterial orders and accounted for more than 88% of the classified reads, regardless of the dietary treatment (see Fig. S1 in the supplemental material). Sequences affiliated with the Methanobrevibacter genus accounted for 80% of all archaeal sequences in both studies, followed by Methanosphaera, unclassified methanogens, and three Methanomassiliicoccaceae genera (Fig. S1). In both studies, Piromyces represented more than 60% of rumen fungi, followed by Orpinomyces and Caecomyces. Dietary treatments did not affect fungal community composition or its structure (Fig. S2), and we are not going to discuss it further.

Guyader et al. (8) showed that combining dietary strategies acting theoretically on hydrogen production (lipids) and consumption (nitrate) can have an additive effect on methane reduction. In a second study, they confirmed the antimethanogenic potential of nitrate supplementation but observed no effect of tea saponin on methane production (13). These studies were conducted simultaneously; cows were selected at random from the same experimental herd and were randomly allocated to a study. Given the consistency of results for methane production and fermentation patterns reported in the two articles of Guyader et al. (8, 13), we decided to analyze the rumen microbiota from both studies at the same time (from DNA extraction up to statistical tests). Although linseed and nitrate have a medium to high potential methane-mitigating effect (the effect of saponins being less reproductive) (20), microbial data are scarce and inconsistent between studies. This could be explained by different methodologies for rumen sample collection, conservation, and nucleic acid extraction, as well as on how data were obtained and analyzed (21, 22). Thus, second, we compiled the microbial data in order to get insight into the mode of action of nitrate on the rumen microbial ecosystem.

To this aim, we first checked that the microbiota of the two groups of cows was comparable; hence, we performed a detailed analysis of microbial community structure and composition in rumen contents sampled during the period when CTL diet was fed to each animal. No major differences in bacterial communities were observed, except a nonsignificant shift in the Bacteroidales/Clostridiales ratio, which is known to vary widely across individual animals (23). However, we observed numerical differences in the relative abundance of Entodinium (60% in study 1 versus 46% in study 2), which is consistent with enumeration results reported previously (5.71 and 5.38 log₁₀ cells/ml in study 1 and study 2, respectively [8, 13]), showing more abundant ciliate populations in cows from study 1.

In study 1, dietary supplementation with linseed increased the relative abundance of Selenomonadales. This is in accordance with our previous work exploring the effects of linseed plus nitrate on rumen microbiota (24) in bulls, where we reported increased numbers in sequences affiliated with three Selenomonas genera and one unclassified Selenomonadales genus. As these microbes are potential nitrate reducers (25), we hypothesized that their growth was supported by the higher nitrate availability, but the present study suggests that it is a linseed effect. Oleic acid (representing, on average, 20% of linseed oil fatty acids) stimulated the growth of Selenomonas ruminantium in pure cultures (26). However, for in vivo studies, results are contrasting: Selenomonas was among the genera explaining differences in bacterial community structure between lambs fed a linseed diet and those fed a control diet (27), but there was no change in Selenomonas abundance when cows were fed sunflower oil (30% oleic acid) (23). Members of the Selenomonadales order are also known to reduce succinate to propionate, which is in agreement with a higher molar proportion of propionate in the rumen of cows fed linseed (8). Linseed supplementation also increased abundance of uncultured Bacteroidetes, and the Bacteroidales S27-7 family was negatively correlated with methane production and yield. On the other hand, linseed diets were characterized by decreased abundance of Ruminococcaceae, which is in agreement with previous findings that fatty acids are toxic to these cellulolytic microbes (23, 26, 28). We observed no effect on rumen protozoan numbers (8) and diversity, although Dasytricha correlated negatively with methane emissions and positively with the Bacteroidales S27-7 family. Linseed oil supplementation also had no effect on the abundance or diversity of the rumen methanogenic community. In accordance with previous results (9, 24), the antimethanogenic potential of linseed oil fatty acids was not related to archaeal numbers in the rumen but rather to a lower metabolic activity of these microbes, which could be explained by lower availability of hydrogen.

Adding tea saponins to the diet had no effect on microbial numbers or on diversity. This is consistent with the lack of changes in methane production or VFA profiles reported by Guyader et al. (13). The efficacy of saponins in suppressing methane production varies considerably depending on the chemical structure, source, dose, and diet (29). Saponins have been reported to inhibit rumen protozoa (5) and, thus, limit hydrogen production in the rumen. However, in our previous work (13) and the study of Ramírez-Restrepo et al. (30), adding tea saponins to ruminants’ diets had the opposite effect on protozoan numbers. Saponins break down the membrane of protozoa by interacting with their sterols. However, rumen microbes can degrade the sugar moiety of saponins, rendering them inactive. To improve the antiprotozoal effect of saponins, changing their chemical structure and, thus, protecting them from microbial degradation, was recently proposed (12).

Nitrate supplementation induced changes in the relative abundance of CowPI-generated functional profiles of bacteria, although metabolic pathways were affected in a dissimilar way between studies. This was expected, as predictions are based on 16S rRNA gene data and multivariate analysis of OTU tables also show differences between studies. In study 1, multivariate analysis revealed that nitrate supplementation altered bacterial and archaeal communities. However, in study 2, NMDS and PERMANOVA results were less conclusive, although reductions of methane emissions and changes in fermentation parameters were comparable between experiments. Nevertheless, both studies pinpointed a limited number of taxa associated with decreased methane emissions in nitrate-fed cows. Nitrate supplementation increased the abundance of Coriobacteriales and Burkholderiales orders, which contain taxa with known nitrate-reducing activity (31–33). This coincides with predicted higher nitrogen metabolism functions and is in accordance with the numerically higher nitrite concentrations measured by Guyader et al. (8, 13) in nitrate-fed cows. Also, cows not fed nitrate presented an enhanced cellulolytic community, which is in accordance with our previous results showing a toxic effect on Ruminococcaceae in animals fed linseed plus nitrate diets (24). Ruminococcus flavefaciens and Ruminococcus albus populations decreased in the rumen of goats when nitrate was added to the diet (25). An in vitro study (34) showed that the growth of these two cellulolytic bacteria was inhibited by nitrite at a level of 3 mmol/liter, but measured nitrite levels in our studies rarely exceeded 0.08 mmol/liter (8, 13). Lower concentrations could still be toxic, as another study showed that the specific growth rate of R. flavefaciens, but not R. albus, was decreased by less than 0.03 mmol/liter of nitrate (35). Marais et al. (35) also argued that nitrite inhibits electron transport systems (R. flavefaciens), so bacteria not possessing an electron transport system (R. albus) are less affected. R. flavefaciens and R. albus are the only cultured Ruminococcus species able to degrade cellulose (36), making them an important part of a functional rumen ecosystem. In vitro, R. albus produces acetate, hydrogen, and carbon dioxide, and its metabolic activity is stimulated by the presence of methanogens (37). Thus, reducing Ruminococcaceae numbers by nitrate supplementation would decrease the amount of hydrogen produced, which could indirectly reduce methane production. This conclusion is also supported by the decreased expression levels of the methanogenic mcrA gene, which has been shown to correlate with methane emissions (24, 38, 39). However, Ruminococcaceae are an important group of bacteria inhabiting the rumen and are able to degrade plant cell wall polysaccharides into metabolizable energy. This implies that inhibition of the rumen fibrolytic community decreases fiber degradation. In the present studies, nitrate supplementation did not affect total tract digestibility (8, 13), but linseed tended to reduce fiber digestibility (8).

We also observed a strong positive correlation between unclassified Methanomassiliicoccaceae and methane production when cows were fed nitrate-supplemented diets. Veneman et al. (9) also reported an increase in the abundance of Methanomassiliicoccaceae-related methanogens in the rumen of nitrate-fed animals. Methanomassiliicoccaceae are obligate hydrogen-dependent methylotrophic methanogens (40), whereas most of the other rumen methanogens perform methanogenesis from hydrogen and carbon dioxide. They are part of a unique methanogen order with a characteristic set of genes involved in the methanogenesis pathway (40). It is likely that their particular physiology confers on them a competitive advantage when the activity of other methanogens is affected in a nitrate/nitrite-enriched environment.

We conducted this study to understand how the rumen microbial ecosystem responds to dietary methane mitigation by linseed, saponin, and nitrate supplementation alone or in combination. We hypothesized that adding linseed or saponins to the diet reduces hydrogen production by a toxic effect on rumen protozoa and by replacing dietary carbohydrates with nonfermentable fatty acids; additionally, we were expecting that nitrate supplementation would redirect hydrogen consumption toward nitrate reduction rather than methanogenesis. Changes in the rumen microbial ecosystem were monitored using archaeon-, bacterium-, eukaryote-, and fungus-specific primers targeting either 16S or 18S rRNA genes and ITS1. Our sequencing strategy allowed us to accurately draw the parallel between changes in methane emissions and microbiota structure. Our study showed that linseed oil decreases methane emissions by reducing the number of hydrogen producers (cellulolytic Ruminococcaceae) and by stimulating propionate producers (Selenomonas), thereby diverting hydrogen from methanogenesis. Nitrate supplementation favored the development of nitrate-reducing bacteria (Coriobacteriales and Burkholderiales) and had a negative effect on cellulolytic Ruminococcaceae; as a consequence, nitrate supplementation also significantly affected methanogen community structure and activity. In contrast, we did not show any shifts in rumen microbiota structure and activity due to dietary supplementation with tea saponins.

In a secondary aim of our work, we capitalized on data available from two independent studies, expecting to draw relevant conclusions. It is common that studies exploring microbial mechanisms of the same methane abatement strategy come to dissimilar conclusions. Authors generally argue that these differences are due to differences in diet, animal species, physiologic stage, and different sample processing or bioinformatics pipelines. In the present work, we minimized the impact of study design on data interpretation, despite some inconsistent results being observed for nitrate-supplemented diets from study 1 and study 2. Nitrate reduced methanogen activity and stimulated nitrate-reducing bacterial populations in both studies. Similarly, Ruminococcaceae-related OTUs characterized nitrate-free diets in both studies. In contrast, multivariate analysis showed that nitrate altered bacterial and archaeal communities in study 1, whereas only a moderate effect on bacteria was observed in study 2. In both experiments, each experimental period lasted 5 weeks. It is possible that microbiota shifted as a result of imposed dietary treatments and did not completely migrate back to the initial state. In a massive rumen contents exchange study, Weimer et al. (41) found that cows almost completely reconstructed their microbiota in 3 weeks, with a complete return to its original host-specific state in 9 weeks. However, pH and VFA profiles returned to the original values much more quickly, within 1 day. We could argue that changes induced by nitrate supplementation were at the level of microbe function rather than species composition. This is supported by the fact that reductions in methane emissions and shifts in VFA profiles were comparable between studies. A metatranscriptomic approach will be more fruitful to further explore microbial mechanisms of methane mitigation using linseed and/or nitrate.

The experiments were conducted at the animal facilities of INRA Herbipôle Unit (Saint-Genès Champanelle, France). All procedures involving animals were conducted in accordance with the French Ministry of Agriculture guidelines for animal research and all applicable European guidelines and regulations on animal experimentation. The experiments were approved by the Auvergne Regional Ethics Committee for Animal Experimentation, approval number CE50-12.

J.G. was the recipient of an INRA-Région Auvergne Ph.D. scholarship. C.S. acknowledges receipt of a postdoctoral fellowship from Fundación Alfonso Martín Escudero (Madrid, Spain). We also thank NEOVIA for financial support.

We thank the INRA personnel of the Experimental Unit Herbipôle (L. Mouly, D. Roux, S. Rudel, and V. Tate) for taking care of the animals and of the UMR Herbivores (L. Genestoux and D. Graviou) for their help in sampling and performing laboratory analysis. We are grateful to the INRA MIGALE bioinformatics platform (http://migale.jouy.inra.fr) for providing computational resources. We thank David Marsh for checking and amending our English. We are grateful to Richard Dewhurst for constructive criticism of the manuscript.

Authors made the following contributions: conception or design of the work, M.P., J.G., M.S., and D.M.; data collection, M.P., J.G., M.S., C.S., and A.R.S.; data analysis and interpretation, M.P., J.G., M.S., C.S., A.R.S., C.G., C.M., D.M., and A.B.; drafting the article, M.P.; critical revision of the article, J.G., M.S., C.S., A.R.S., C.G., C.M., D.M., and A.B.; and final approval of the version to be published, M.P., J.G., M.S., C.S., A.R.S., C.G., C.M., D.M., and A.B. We have no competing financial interests to declare.