1. Introduction
The Food and Agricultural Organization of United Nations reported that the increasing demand of protein with high biological value (e.g., from meat and milk) will lead to an expansion of livestock production, which, in turn, will lead to increasing environmental problems. In particular, ruminants are the main contributors of methane emissions in the atmosphere [
1]. Rumen can be considered a fermenter in which many bacterial species cooperate for the transformation of the nutrients ingested by the host animal. During the fermentation of dietary fibre, hydrogen is produced. Its accumulation in rumen liquor can have toxic effects on the microbiota. Hydrogen is removed by the synthesis of methane that is emitted during animal eructation [
2]. For this reason, several feeding strategies were developed to lower methane emissions and to improve the environmental sustainability of ruminant production systems [
3].
Methane emissions from ruminants can be efficiently lowered by polyphenols, but the decrease of methanogenesis is often associated with a lower fibre degradability and, consequently, to a reduced production of acetate [
4,
5,
6,
7]. Chestnut tannins (CHT) were effective in decreasing methanogenesis without compromising acetate production [
8,
9]. Chestnut tannins are hydrolysable polyphenols composed by several fractions, including vescalagin (VES) and gallic acid (GAL), which account for nearly 20% and 6% of total tannins, respectively [
10].
Data reported in literature showed that the use of CHT in ruminant feeding might result in changes in the profile of rumen microbial community [
9,
11,
12]. Vescalagin is the main active component present in CHT [
13], while GAL is quickly released in rumen liquor (RL) by hydrolysis of CHT [
14]. These compounds were studied in medicine and veterinary science for their potential use as antimicrobials [
15]. Indeed, Quideau et al. [
13] studied several nonahydroxyterphenoyl-containing C-glycosidic ellagitannins (i.e., castalagin and VES from
Castanea spp.) and found that VES was the most effective against both acyclovir-susceptible and acyclovir-resistant
herpes simplex virus strains, showing a 50% inhibitory concentration (IC
50) at 0.04 nM. Panizzi et al. [
16] demonstrated the efficacy of GAL as antimicrobial against
Staphylococcus aureus,
Bacillus cereus,
Pseudomonas aeruginosa,
Escherichia coli,
Saccharomyces cerevisiae and
Candida albicans.
In this in vitro study, the effects of CHT, VES and GAL on neutral detergent fibre (NDF) degradability, microbial community profile and dimethyl acetals (DMA) composition of rumen liquor were evaluated, in order to assess the effect of the phytocomplex (i.e., CHT) versus the effect of the main individual components of CHT.
4. Discussion
The inclusion of polyphenols in ruminant diets can modulate the diversity and activity of rumen microorganisms, the nutrient degradability and rumen methanogenesis [
36]. In the present trial, the inclusion of CHT did not affect the degradability of dietary NDF compared to the control. The low value of NDF
deg in all fermenters suggested that the conditions of in vitro trials were not optimal for the evaluation of fibre degradability. As a consequence, data related to NDF
deg have to be considered only as qualitative markers (i.e., referred to the control). Several authors reported an increase in acetic acid concentration in RL from dairy ewes fed diets supplemented with 10–30 g/kg DM intake (DMI) [
8] or 16 g/kg DMI [
9] of CHT extract, demonstrating that this kind of polyphenol had no detrimental effect on the activity of cellulolytic bacteria. In contrast, Zimmer and Cordesse [
37] found that CHT decreased in vivo apparent organic matter (OM) digestibility in ewes and goats. Tabacco et al. [
38] confirmed that lucerne ensiled with CHT reduced in vitro OM digestibility by 5%. The percentage of tannins included in the diet and the kind of polyphenol certainly play an important role and could explain the inconsistency of many data reported in the literature. In the present experiment, the CHT inclusion was about 1.6 g/100 g of DM, lower than in the trials cited above.
The ability of polyphenols in lowering methane emissions is usually a consequence of a decreasing NDF degradation, which, in turn, leads to a reduction of acetate synthesis and, eventually, to a decrease in the availability of electron donors for the methanogens [
2]. Hence, the reduction in methane production is often linked to a decrease in NDF rumen degradability. The findings of the present study confirmed that CHT had no detrimental effects on NDF
deg and the activity of cellulolytic bacteria. Since several authors reported that CHT dietary supplementation was been associated with a lowering in methane production [
7,
8], the administration of CHT in ruminant feeding could be an interesting perspective to increase the environmental sustainability of livestock farming without affecting feed efficiency.
Tannins can modulate the rumen microbiota, inhibit microbial enzymes or complex cell wall components [
39]. Commonly, condensed tannins decrease the rumen degradation of fibre because they induce important changes in the microbial community, while the effect of hydrolysable tannins is usually milder [
40]. Chestnut tannin extract has been reported to be less effective on the growth of cellulolytic bacteria than condensed tannins, such as quebracho or mimosa [
11,
12,
40,
41]. A plausible explanation for this behaviour could be related to the higher grade of depolymerization of hydrolysable tannins in rumen compared to condensed tannins [
42]. Recently, in an in vivo trial with fistulated sheep, Costa et al. [
40] compared mimosa tannins and CHT. The authors observed a reduced growth of cellulolytic bacteria with condensed tannins compared to hydrolysable tannins. Moreover, Liu et al. [
8] found that in vitro acetate production was not affected by CHT, confirming that this kind of tannin extract did not influence the activity of cellulolytic bacteria. This result is consistent with our microbiological findings that show only marginal changes in the composition of the microbial community.
To better understand the role of single components of CHT, the effect of VES and GAL on NDF degradability was evaluated. Vescalagin and GAL reduced NDF degradability. However, no considerable changes in the composition of the microbial community were observed compared to diet C. These findings can be explained considering the possibility that VES and GAL could i) inhibit the activity of the microorganisms; ii) inhibit the enzymes involved in NDF degradation; or iii) complex the fibre, thus making it unavailable [
43,
44]. Since the microbial community was characterized by DNA extraction and HTS of the 16S rRNA gene, it is not possible to discern between active and inactive microorganisms in the present trial. Indeed, a recent comparison of the results obtained by RNA amplicon sequencing and DNA amplicon sequencing showed a different relative abundance of the main bacterial phyla such as
Bacteroidetes (22.7 ± 8.1% and 50.3 ±8.7%, respectively) and
Proteobacteria (46.3 ± 14.3% and 4.3 ± 8.5%, respectively) [
45].
To improve knowledge on the effects of CHT, VES and GAL on rumen microbial communities, the DMA profile was investigated, and a metataxonomic approach was exploited.
Dimethyl acetals are derived from the plasmalogen lipids of bacterial membranes, and their composition is similar to fatty acid profile showing odd, even, saturated and unsaturated chains from C12 to C18. Their variation is strongly linked to the ability of bacteria to be resilient to environmental changes, modifying their membrane fluidity as a defence strategy [
46,
47]. Alves et al. [
25] showed that DMA could be an efficient tool as microbial condition marker.
In our trial, DMA 15:0
iso increased and it could be symptomatic of adaptation of cellulolytic bacteria to the stimuli induced by CHT, VES, and GAL present in RL. Similar considerations may be applied to DMA 17:0
ante for amylolytic bacteria. Our data are consistent with the findings reported by Costa et al. [
40], since the inclusion of CHT, or of its components, in the diet was able to affect the concentration of DMA 13:0, 14:0
iso, 16:1 and 18:0. Moreover, the presence of DMA 18:1
trans-11 may be considered a marker of the incorporation in the structural lipids of the biohydrogenation intermediates, as previously reported also by Alves et al. [
25].
Our study showed diet-specific correlations among several plasmalogen derivatives and microbial genera, confirming the importance of DMA composition as a tool of microbial characterization in a specific environmental condition.
Metataxonomic analysis of RL inoculated with CHT, VES, and GAL showed that only the genera
Prevotella,
Paraprevotella,
Succinivibrio, and
Treponema were detected in all conditions. In a previous study, Henderson et al. [
48] described a “core bacterial microbiome”, that was composed by key microorganisms detected in all samples:
Prevotella,
Butyrivibrio,
Ruminococcus, unclassified members of the families
Lachnospiraceae and
Ruminococcaceae, as well as unclassified
Bacteroidales and
Clostridiales. In another study, the genera
Paraprevotella,
Succinivibrio,
Treponema,
Fibrobacter, and
Oscillibacter, in addition to the above mentioned
Prevotella,
Butyrivibrio and
Ruminococcus were identified as core genera in rumen bacterial communities of pre-ruminant dairy calves, cows and beef steers [
49]. The comparison of our results with the core microbiome described in previous studies [
48,
49] highlighted the importance of the genus
Prevotella in the rumen’s ecology since this genus is involved in the utilization of hemicellulose [
49,
50]. Moreover, in our study, members of the genus
Oscillibacter were not detected in the whole dataset. Since changes in the detected microorganisms in different studies can be due to the use of different primer sets, to exclude this hypothesis, the 16S rRNA gene sequences of different strains of
Oscillibacter were retrieved from GenBank [
51] (accession numbers NR_118156.1, HM626173.1, NR_074793.2) and aligned to the primer sequences. The alignment confirmed that our primer set was able to target also the genus
Oscillibacter. A possible reason of the differences observed between this in vitro study and the core rumen communities studied in vivo by Henderson et al. [
48] and by Wu et al. [
49] can be the absence of microorganisms-host interactions (e.g., absence of immune system) that characterized the in vitro trials. However, the overall behaviour can be considered reliable since in vitro experiments are widely used [
7,
23,
52].
Regarding the microbial genera affected by CHT, VES, and GAL (i.e.,
Anaerovibrio,
Arcobacter,
Bibersteinia,
Escherichia/Shigella,
Pseudobutyrivibrio,
Streptococcus,
Treponema), little information is available in literature. The relative abundance of
Streptococcus spp. increased in diet T, in accordance with Costa et al. [
40], who showed a higher abundance of this genus in RL from ewes fed CHT compared to RL from ewes fed mimosa tannins or a mix of CHT extract and mimosa. Furthermore, our metataxonomic data showed that diet T increased the relative abundance of the genus
Anaerovibrio, which was negatively affected by polyphenols from olive oil pomace in a previous study [
28].
The reason for the different responses of bacteria to tannins is unclear, and factors such as dose, type of tannin and purity of the extract contribute to explain the conflicting results reported in the literature.