The inclusion of forages with diverse functional groups (e.g. legumes) in mixtures generally improves the yield stability in the grassland-based livestock production systems 21,22. Forages rich in tannins and other polyphenols provide even more benefits as they have been shown to decrease CH4 and N emissions from ruminant based production systems 23,24. Although, several studies have analyzed TRFs for their effect on animal performance and CH4 reduction potential, the results till date have been contrasting, making it difficult to adapt the forages on the large scale. The reason for these contrasting results is assumed to be at least partially on the environmental conditions during plant growth, as these can affect tannin synthesis, as well as on colorimetric tannin assays that do not identify tannin structural characteristics or other compounds with potential interactions18,25. In the present study, the comparative assessment of 17 cultivars from eight agronomically promising temperate forage species and across two harvests provides a novel insight of how the bioactive properties of these forages vary across the different species, and how stable these CH4 reduction properties are across different cultivars of the same species and across different harvests.
PA structural characteristics were found to be more stable than forage chemical composition across harvests
The CP levels in legumes are known to generally be in the range of 180 to 300 g/kg of DM and this mostly exceeds animal requirements (130-170 g/kg of DM)26. As expected, in the present study, the CP in the forages was higher in the second harvest where the plants were harvested at the vegetative stage compared to the first harvest, which can be explained by the much higher leaf shares, as leaves are richer in CP than stems27. Higher CP content increases the digestibility of the forage, as it is easily degraded by microbes28. Conversely, high NDF and ADF content in the forages is known to be an indicator of lower forage digestibility26. A meta-analysis by Maccarana, et al. 29 found that NDF content in the feed is negatively correlated to GP in the rumen. The NDF content in the feed can reduce the availability of rapidly fermentable carbohydrates leading to lower microbial activity in the rumen and hence a decrease in GP 30. Due to potential matrix effects, as well as because of known anti-inflammatory and antimicrobial properties that are beneficial for ruminant immunological system, flavonoid content of the cultivars was also determined (Olagaray and Bradford 2019). Assessing the effect of tannins on ruminants based only on their concentration has given rise to ambiguous results in the past as structural traits of tannins have been found to play an important role in determining their bioactivity12. Consequently, in addition to PA concentration, PA structural features were also analyzed. In the present study, these features were found to be stable across the harvests, and within the cultivars of the same species the differences were comparatively lower than the variability across the species. This indicates that the methane reduction properties would be stable, and independent of the harvest stage of the plant material.
Methane production from TRFs was consistently lower than lucerne in both the harvests
Numerous studies have demonstrated that TRFs reduced enteric fermentation in ruminants, resulting in lower GP and CH4 production 31. Across all the species with varying forage quality and plant metabolite composition, in the first harvest, the largest reductions in CH4 were observed in TRFs rich species which were sulla, big trefoil, sainfoin and salad burnet in comparison with lucerne. This could be attributed to the high TT concentration in these forages compared to lucerne. A study by Jayanegara, et al. 32 found that HTs are more potent compared to PAs. In the present study, however, this was not confirmed, as both the GP and CH4 production of PI 308861 (salad burnet) were found to be lower than those of Sudda (sulla) and Lot 29 (big trefoil). This was despite the higher TT and TP concentrations in salad burnet, which reaffirms the results from studies indicating that the extent of CH4 reduction from TRFs does not only depend on tannin concentration but also on the tannin source and structural features 33,34. The differences between PAs and HTs bioactivity is likely to be dependent on their fate in biological systems. Proanthocyanidins exist in the form of large polymers, enabling them to bind to the feed components strongly and they do not get easily degraded in the rumen. On the other hand, HTs have been shown to undergo hydrolysis, hence degrading to form low molecular weight phenolics in the rumen, and can thereby, lose part of their bioactivity 32,35. A general trend of higher GP and CH4 production, when plants were harvested at the vegetative stage (second harvest), was recorded compared to when they were harvested at the flowering stage (first harvest) which could be a result of lower tannin content in the plants in the second harvest. Furthermore, a lower content of instantly fermentable fractions, such as water soluble carbohydrates in the leaves at the flowering stage can result into lower GP36. Interestingly, the species with lower TP and TT content, such as ribwort plantain and chicory, were also found to reduce CH4, but it was not accompanied by reduction in GP. Additionally, it is important to look at the CH4 percentage (MP) in the total gas, as this can be used as an indicator of the potential CH4 emissions per unit of organic matter (OM) degraded. Due to the concomitant reductions in GP and CH4 from TRFs, their MP ranged between 21% and 26% and hence was comparable to that of lucerne (24%) (Supplementary, Table S3). Chicory and ribwort plantain (17-21%) had the lowest MP compared to the other tested species in this study. The reduction in the CH4 from these species was not accompanied by the reduction in GP indicating their higher potential for CH4 abatement in ruminants without negatively affecting ruminal fermentation
PEG treatment affirmed the antimethanogenic activity of TRFs, however no influence on methane percentage in total gas was observed
The inclusion of PEG as a tannin inactivating agent, enabled the quantification of the specific tannin-effect from different species on the ruminal fermentation in vitro. Polyethylene glycol can inactivate tannins by displacing proteins from tannin-protein complexes, and binding with tannins to form tannin-PEG complexes. Increased GP and CH4 production from TRFs under +PEG treatment could be a result of high availability of protein after being dissociated from tannin-protein complexes. The increased availability of N compounds that can no longer bind to tannins with the addition of PEG leads to more substrate for microbial degradation and higher microbial growth. This in turn results into increased ruminal fermentation and higher GP34,37. However, the enzymatic or auto-oxidation of the initial tannins or their hydrolysis products could also produce large bio-oligomers or polymers with tannin-like functions in the rumen or small intestine38. Consequently, making it difficult to assess the fate of tannins in biological systems. As expected, no differences in GP and CH4 production were observed in samples of ribwort plantain, lucerne and chicory, due to the absence of tannins in these species. Generally, the variation in the extent of GP and CH4 reduction from across the different TRFs under -PEG and +PEG treatments was in line with the variation in tannin concentration and composition. Accordingly, stronger PEG-treatment effect were observed in cultivars with higher tannin concentrations, which is in line with the studies by Basha, et al. 37 and Jayanegara, et al. 39.
Although, it is evident that tannins reduce CH4 emissions, the mode of action by which they reduce CH4 in ruminants is still not well understood. A study by Hassanat and Benchaar 40 found that the PA (acacia and quebracho) and HT (chestnut and valonea) extracts had a more pronounced effect on rumen methanogenesis compared to the substrate degradation 40. Tannins are found to modify the rumen microflora by either directly inhibiting methanogenic population or indirectly affecting methanogen-protozoa symbiosis by inhibiting protozoal population in rumen 8,18. In a study by O'Donovan and Brooker 41, PAs from acacia were able to change the morphology of Streptococcus bovis, Butyrivibrio fibrisolvens and Prevotella ruminicola, and, hence, affected their activity. Additionally, HTs and PAs from different plant sources were also found to decrease CH4 production by suppressing the methanogenic archaea and protozoal populations in vitro 42.
PA structural features were able to explain the variability in methane production from PA containing species but model was not improved
As mentioned above, not only the differences between HT and PA are relevant, but also their structural characteristics affect their potential bioactivity. Both mDP and PD% are among the most relevant features of PAs, determining their protein precipitation activity, which in turn is linked to anthelmintic and antimethanogenic bioactivity of PAs 12,43. Hence, in the present study, a stepwise regression was performed to analyze which forage parameters were best suited to explain the total gas and CH4 production. By identifying covariates as potential predictors, fermentation patterns and CH4 reduction potential could be estimated from plant traits alone. This would enable extrapolations to any other species, enabling the prediction of their antimethanogenic potential and digestibility from their chemical composition alone. However, the updated model (covariate-based model) with forage composition parameters as covariates did not improve the base model in terms of AIC value. Nevertheless, the conditional R2 for CH4 and GP remained the same and even increased for MP. This indicates that the overall updated model, including both fixed and random effects, was able to explain the variance of CH4 and GP in the present study for PA containing species. However, in order to predict the fermentation parameters, cultivar remains the better estimate compared to the sum of all covariables, indicating that due to the large number of factors with interconnected effects, the tannin source remains more relevant compared to the traits alone. This could also arise from the fact that in addition to PAs, plants may produce other bioactive PSMs such as terpenes, organic acids, saponins and others which can also influence the CH4 production from the substrate 7,44, yet were not quantified in this study. Furthermore, the influence of matrix effects between tannins and other PSMs on tannin bioactivity, remains an unexplored arena. Still, the model quality was sufficient to identify parameters which had a strong influence on the in vitro fermentation end-products. Particularly in case of CH4, the variables CP, PA as well as mDP were found to exhibit a negative relationship with CH4 production. This is in accordance with the study by Hatew, et al. 45, which observed that the polymer size (mDP) was an important determinant of in vitro CH4 and GP production from the substrate. With regards to MP, however, both PA and PD% were found to have limited influence as indicated by their near-zero coefficient and low marginal R2 (0.05). One solution to improve the predictive capabilities of CH4 reductions from tannin traits compared to forage quality traits might be the inclusion of tannin extracts in the in vitro incubation instead of the whole plant sample.
The tested forage species are promising alternatives as sustainable feedstock in livestock production systems
The results in the present study underpin that TRFs have high potential for their use in livestock production as their CP content across both harvests was comparable to lucerne. This was further demonstrated by the GP production in the presence of PEG, which did not vary significantly from lucerne. The highest treatment effect was observed when using sulla and big trefoil as a substrate. This indicates their higher antimethanogenic activity owing to high PA content and potent PA structural characteristics. Sulla is a highly palatable forage and its supplementation can reportedly reduce the dependence on proprietary anthelmintics 46. In the previous studies, supplementation of fresh fed big trefoil was found to reduce CH4 emissions by 26% (g CH4 kg−1 DM intake) compared to lucerne in sheep47. However, the nutritional value of sulla and big trefoil need to be evaluated before providing them as a substantial feed component. Reportedly, owing to the high tannin content, sulla and big trefoil can compromise N digestibility and microbial activity in rumen48. The concomitant reduction in GP and CH4 from TRFs in the present study, makes it essential to research on how these forages can be incorporated in animal feed and in which proportion, to exploit their beneficial effects. A study by Orlandi, et al. 49 found that even though total-tract N digestibility by PA inclusion decreased linearly with the increased concentration, there was a linear increase in N retention and the efficiency of N utilization was improved with the inclusion of tannin extracts of Acacia mearnsii until the concentration of 18 g/kg DM, implying that the positive impact of duodenal flux of amino acid supply can outweigh the reduction in protein digestibility when tannins are supplied at optimum levels49. Although more research is required to determine the relationship between PAs and post ruminal amino acid availability, it is well established that low to moderate PA concentrations leads to increased rumen undegraded protein and post ruminal amino acid flux which improves the N utilization efficiency in ruminants7. Tannin content in the plants is known to be plastic in nature, however, the stability of PA structural features across the different harvests is promising ground to include PA structural features for future structure-activity relationship studies of tannins.
Furthermore, this study also showed that underutilized forbs such as chicory and ribwort plantain have a high CH4 reduction potential despite being void of tannins. The CH4 reduction from these species did not result in a negative impact on GP when compared to the other tested species in this study. This is in line with an in vivo study by Niderkorn, et al. 50, which found that sheep produced 23% lower CH4 per kg DM intake with pure chicory compared to when fed with pure perennial ryegrass (Lolium perenne L., cv. AberAvon) which no significant differences in the DM digestibility. Additionally, a study by Navarrete, et al. 51, found that chicory is rich in inulin and sesquiterpene lactones and, ribwort plantain leaves are known to contain acteoside, aucubin, and catapol. These compounds are known to reduce net NH3 production during ruminal fermentation and acteoside was found to increase potential GP51. Both chicory and ribwort plantain are promising alternatives to produce high quality feed to overcome feed-deficits during dry seasons due to their drought tolerance, and their inclusion in grass-clover mixtures can enhance above- and below-ground primary production in temporary grasslands52,53. Furthermore, ribwort plantain and chicory are known to benefit animal health due to their antimicrobial and anthelmintic properties which could be attributed to the presence of the PSMs other than polyphenols which were not analyzed in this study24,54.