Effect of Condensed Tannins on Nitrogen Distribution and Metabolome after Aerobic Exposure of Sainfoin Silage

Round 1

Reviewer 1 Report

Very interesting paper with real application possibilities. Bacterial community structure of sainfoin silage is clearly presented. Interesting correlation between bacteria and metabolites after aerobic exposure of sainfoin silage was found. The content of the paper is well organized. English level is acceptable.

Some important papers are missing and should be added to the Introduction and/or Discussion.

Lorenz et al. (2010) investigated the effect of wilting, silage additive, PEG treatment and tannin content on the distribution of N between different fractions after ensiling of three different sainfoin (Onobrychis viciifolia) varieties. GRASS AND FORAGE SCIENCE, 65(2), 175-184. 10.1111/j.1365-2494.2010.00736.x

As ensiling causes protein breakdown and elevated levels of buffer soluble N (BSN), the Authors studied the distribution of N before and after ensiling sainfoin. Three varieties of sainfoin were either direct-cut and frozen directly or wilted and frozen before later ensiling in mini-silos with and without acidification with Promyr (PM; an acidifying commercial mixture of propionic and formic acid) and with or without polyethylene glycol (PEG). Extractable tannins (ET) and protein-bound tannins (PBT) were measured with an HCl/butanol method in an attempt to correlate tannin levels to N fractions. The sainfoin silages showed good ensiling characteristics and had relatively high concentrations of undegraded protein. The effect of wilting on BSN levels (g/kg N) was dependent on sainfoin variety (P < 0 center dot 001). PEG increased and PM decreased the level of BSN in the silages (P < 0 center dot 001). PM treatment also produced less non-protein N and ammonia-N (P < 0 center dot 05) as compared with no additive. Addition of PEG to the silage increased the BSN-proportion 1 center dot 8- and 2 center dot 6-fold for both DM stages. A strong tannin-protein binding effect is, therefore, confirmed in sainfoin. However, correlations between tannin levels (ET and PBT) and BSN were poor in the (non-PEG) silages, indicating either that the HCl/butanol method is unsuitable for measuring tannin in silages or that qualitative attributes of tannins are more relevant than quantitative. The HCl/butanol method seems therefore not to be useful to predict degradation of protein in sainfoin silages.

Maras et al. (2000) investigated polyphenols, condensed tannins, and other natural products in Onobrychis viciifolia (Sainfoin). JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 48(8), 3440-3447. 10.1021/jf000388h.

An acetone/water extract of the fodder legume Onobrychis viciifolia afforded arbutin, kaempferol, quercetin, rutin, afzelin, the branched quercetin-3-(2(G)-rhamnosylrutinoside), the amino acid L-tryptophan, the inositol (+)-pinitol, and relatively high concentrations of sucrase (ca. 35% of extractable material). Acid-catalyzed cleavage of the condensed tannins with phloroglucinol afforded catechin, epicatechin and gallocatechin as the terminal and extender units, but epigallocatechin was only present in extender units. The condensed tannins in O. viciifolia presumably consist of hetero- and homopolymers containing both procyanidin and prodelphinidin units. Comparison of data from the present study and the literature suggests that sainfoin tannins have a highly variable composition with cis:trans ratios rangin

Lees et al. (1995) measured content of CONDENSED TANNINS IN SAINFOIN .2. OCCURRENCE AND CHANGES DURING LEAF DEVELOPMENT. CANADIAN JOURNAL OF BOTANY-REVUE CANADIENNE DE BOTANIQUE, 73(10), 1540-1547. 10.1139/b95-167

A histological study examined condensed tannin (CT) formation in plant tissue samples taken from the meristematic area of very young sainfoin (Onobrychis viciifolia Scop.) seedlings and from leaflets sampled at various stages of development in mature plants growing in the greenhouse. Light and electron microscopy revealed no CT in the seedling meristem and leaf primordia, but CTs were seen very early in leaf development forming first in the vacuoles of discrete cells of the abaxial subepidermal layer when the leaflets were recognizable, but still folded. Immature leaflets collected from the growing point of a mature sainfoin plant show similar CT formation with the abaxial cell vacuoles filled with CT when the new leaves have reached the 90 degrees-fold stage. As the leaflets unfold and mature, CTs begin to appear in the vacuoles of small, but discrete cells in the adaxial subepidermal layer while the tannin-containing cells in the abaxial subepidermal layer begin to lose CT. The CT continues to increase in the adaxial layer until typical enlarged tannin idioblasts or sacs are observed at full leaflet expansion and maturity. By this stage, the vacuoles in the abaxial layer are almost empty. In senescing leaflet samples collected from the leaf rachis attached to the last and second to last node near the base of the plant, the cells in both subepidermal layers have lost the majority of the CT that was originally formed. At senescence all tannin-containing cells appear as empty shells. We speculate that a finite amount of CT is formed in the two subepidermal layers of new leaves at different stages of early leaf development, does not increase during the mature phase, and is catabolized in older leaves and during senescence.

Hatew et al. (2015) looked at diversity of condensed tannin structures affects rumen in vitro methane production in sainfoin (Onobrychis viciifolia) accessions. GRASS AND FORAGE SCIENCE. 70(3), 474-490. 10.1111/gfs.12125

Sainfoin is a non-bloating temperate forage legume with a moderate-to-high condensed tannin (CT) content. This study investigated whether the diversity of sainfoin accessions in terms of CT structures and contents could be related to rumen in vitro gas and methane (CH4) production and fermentation characteristics. The aim was to identify promising accessions for future investigations. Accessions differed (P<00001) in terms of total gas and CH4 productions. Fermentation kinetics (i.e. parameters describing the shape of the gas production curve and half-time gas production) for CH4 production were influenced by accession (P0038), but not by PEG. Accession, PEG and time affected (P<0001) CH4 production, but accession and PEG interaction showed only a tendency (P=008). Increase in CH4 due to PEG addition was not related to CT content. Further analysis of the relationships among multiple traits (nutritional composition, CT structure and CH4 production) using principal component analysis (PCA) based on optimally weighted variables revealed differences among accessions. The first two principal component axes, PC1 (576%) and PC2 (184%), explained 760% of the total variation among accessions. Loading of biplots derived from both PCAs made it possible to establish a relationship between the ratio of prodelphinidin:procyanidin (PD:PC) tannins and CH4 production in some accessions. The PD:PC ratio seems to be an important source of variation that is negatively related to CH4 production. These results suggested that sainfoin accessions collected from across the world exhibited substantial variation in terms of their effects on rumen in vitro CH4 production, revealing some promising accessions for future investigations.

Aufrere et al. (2013) mixed sainfoin and lucerne to improve the feed value of legumes fed to sheep by the effect of condensed tannins. ANIMAL, 7(1), 82-92. 10.1017/S1751731112001097

The aim of this study was to investigate whether the use of sainfoin-based condensed tannins (CT) enhances feed value when given with tannin-free legumes (lucerne) to sheep. The experiments were conducted with fresh sainfoin and lucerne harvested at two stages (vegetative stage as compared with early flowering) in the first growth cycle. Fresh sainfoin and lucerne forages were combined in ratios of 100 : 0, 75 : 25, 25 : 75 and 0 : 100 (denoted S100, S75, S25 and S0, respectively). Voluntary intake, organic matter digestibility (OMD) and nitrogen (N) retention were measured in sheep fed the different sainfoin and lucerne mixtures. Loss of dry matter (DM) and N from polyester bags suspended in the rumen, abomasum and small intestine (SI) was also measured using rumen-fistulated sheep and intestinally fistulated sheep. The CT content in sainfoin (S100) decreased with increasing percentage of lucerne in the mixture (mean value from 58 g/kg DM for S100 to 18 g/kg DM for S25) and with growth stage (S100: 64 to 52 g/kg DM). OMD did not differ between different sainfoin/lucerne mixture ratios. Sainfoin and lucerne had an associative effect (significant quadratic contrast) on voluntary intake, N intake, total-tract N digestibility, N in faeces and urine (g/g N intake) and N retained (g/g N intake). Compared with lucerne mixtures (S0 and S25), high-sainfoin-content mixtures (S100 and S75) increased the in situ estimates of forage N escaping from the rumen (from 0.162, 0.188 for S0 and S25 to 0.257, 0.287 for S75 and S100) but decreased forage N intestinal digestibility (from 0.496, 0.446 for S0 and S25 to 0.469, 0.335 for S75 and S100). The amount of forage N disappearing from the bags in the SI (per g forage N) was the highest for high-sainfoin mixtures (from 0.082, 0.108 for S100 and S75 to 0.056, 0.058 for S25 and S0, P < 0.001). Rumen juice total N (tN) and ammonia N (NH3-N) values were the lowest in the high-sainfoin diet (mean tN 0.166 mg/g in S100 as compared with 0.514 mg/g in S0; mean NH3-N 0.104 mg/g in S100 as compared with 0.333 mg/g in S0, P < 0.001).

Author Response

Thanks for you advice, we added some sentences in our manuscript according to you recommendation. Followed as:

Sentence 1; “The capacity of tannins bind with protein attributed to hydrogen bonds formed between protein residues and its hydroxyl group”.

Sentence 2:" CT from sainfoin contain both procyanidin (catechin/epicatechin) and prodelphinidin (gallocatechin/epigallocatechin) units, which all had 2 or 3 hydroxyl group at B-ring, indicate that could be better bind with protein."

Sentence 3:"The strong tannin-protein binding effect was observed result from the level of buffer soluble N (BSN) were decreased due to CT in sainfoin silage."

Reviewer 2 Report

This is an interesting study on the effect of condensed tannins on nitrogen distribution, and metabolome after aerobic exposure to sainfoin silage. Designing the experiment is scientific and justified. There is a logical flow in the paper. The statistical analysis is also done very well and the model chosen for the data analysis is appropriate for the type of data generated in the study. The table (a minor correction is needed) and diagram are clear and easy to understand and the conclusion is justified.

However, in Table 1.,  Superscripts a-c are missing in the SOLP (CK & PEG) rows and also in the NDIP (PEG) row. Please check the data and define it appropriately.  

Author Response

Respond for Q1: thanks for you advice. We added superscripts a-c in Table 1