Introduction
Methane (CH4) is a greenhouse gas that contributes substantially to climate change. The livestock sector is estimated to contribute up to 0.18 of total global anthropogenic greenhouse gas emissions (Steinfeld et al., Reference Steinfeld, Gerber, Wassenaar, Castel, Rosales and de Haan2006). The application of specific feeds and feeding strategies are among the most promising ways to mitigate CH4 formation during its synthesis in the rumen. Depending on the dosage or composition, plants can have positive or negative effects on digestive processes in herbivores. Plant secondary metabolites (PSM) such as saponins, phenols and essential oils play a central role in this respect (Waghorn, Reference Waghorn2008; Bodas et al., Reference Bodas, Prieto, García-González, Andrés, Giráldez and López2012). Apart from CH4, there are other harmful emissions from animal excretions, particularly urinary N, as it is emitted easily as ammonia or nitrates. Urinary N excretion is related closely to ruminal ammonia concentration which, therefore, is indicative of the N emission potential in diets (Dijkstra et al., Reference Dijkstra, Oenema, Van Groenigen, Spek, van Vuuren and Bannink2013).
There have been some extensive in vitro screenings of plant supplements, testing their CH4 and ammonia mitigating properties, for example with plants from natural grasslands (Banik et al., Reference Banik, Durmic, Erskine, Ghamkhar and Revell2013; Macheboeuf et al., Reference Macheboeuf, Coudert, Bergeault, Lalière and Niderkorn2014; Niderkorn and Macheboeuf, Reference Niderkorn and Macheboeuf2014). In the ‘Rumen Up’ project (https://www.abdn.ac.uk/research/rumen-up/report), 34 plants or plant extracts out of 500 were identified as being able to diminish CH4 production by >15% in vitro when given as additives without affecting ruminal fermentation negatively. Individual results regarding digestibility, methane, ammonia, total gas or short-chain fatty acid (SCFA) production were made available in peer-reviewed publications for only 14 of these 34 promising plants (Selje et al., Reference Selje, Hoffmann, Muetzel, Ningrat, Wallace and Becker2007; Bodas et al., Reference Bodas, López, Fernández, García-González, Rodríguez, Wallace and González2008).
In general, the list of effective, indigenous plants in the temperate climate zone that have a high or at least moderately high feeding value is still rather limited. Of particular interest are those plants that are affordable and available mostly in bulk so that implementation in animal feeding practice is feasible. Several plants growing naturally in grasslands, especially those in the legume family, are known to be rich in PSM and have the potential to mitigate CH4 and ammonia (Tavendale et al., Reference Tavendale, Meagher, Park-Ng, Waghorn and Attwood2005; Williams et al., Reference Williams, Eun, MacAdam, Young, Fellner and Min2011). However, in terms of PSM contents, forage from woody plants such as shrubs and trees might be more promising than herbaceous forages. In addition, these woody plant species typically do not compete directly with crops used for human food and have a particular ecological value as habitats for numerous animal and plant species. Nevertheless, forage from shrubs and trees can have greater lignification than herbaceous plants and thus offer a lower feeding value (Hummel et al., Reference Hummel, Südekum, Streich and Clauss2006). Another potential drawback is that PSM content and composition as well as nutrient contents may vary between seasons and habitats (Palo et al., Reference Palo, Sunnerheim and Theander1985; Sauter and Wellenkamp, Reference Sauter and Wellenkamp1998).
Therefore, the aim of the present study was to screen a variety of woody plants for their magnitude of mitigation of ruminal CH4 and ammonia when added to a considerable extent to a good quality total mixed ration (TMR). Besides describing fermentation, the microbial count and indicators of the feeding value of diets supplemented with the test plant additives (contents of net energy of lactation (NEL) and the utilizable crude protein at the duodenum (uCP)), were evaluated. Two lots from each test plant material were tested separately to account for the variability of natural plant material in phenol and nutrient content and composition.
Materials and methods
Test plant material
In the present study, 18 test plant materials derived from 16 plant species were chosen for in vitro incubation with the Hohenheim gas test (HGT) method (Table 1). Thirteen of the plants were selected from a list of 34 plant materials in the ‘Rumen Up’ report (Results section 3.5, page 3.46, available from https://www.abdn.ac.uk/research/rumen-up/documents/pdfs/3_results.pdf). A decrease of >15% CH4 production in vitro was observed when these plant materials were given as additives and no negative effect on ruminal fermentation (digestibility, total gas and SCFA production) was reported. The selection criteria in the current study were: (1) plants growing naturally under temperate climate conditions, (2) respective plant parts that are inexpensive and available in bulk and (3) plants that do not directly compete with the production of human food.
Details of the experimental plant materials
A, Alfred Galke GmbH, Bad Grund, Germany; B, Berg pharmacy, Zurich, Switzerland; C, own collection; D, St. Peter pharmacy, Zurich, Switzerland.
The following plant materials were tested: leaves of Arctostaphylos uva-ursi (bearberry), Betula pendula (silver birch), Castanea sativa (sweet chestnut), Corylus avellana (hazel), Populus tremula (aspen), Ribes nigrum (blackcurrant), Salix caprea (goat willow), Vitis vinifera and V. vinifera rubra (grape vine), aerial part of Epilobium angustifolium (fireweed), Geum urbanum (wood avens), Lotus corniculatus (birdsfoot trefoil) and Symphytum officinale (comfrey), fruit of Aesculus hippocastanum (horse-chestnut) and Prunus spinosa (blackthorn; entire fruit, kernel and pulp) and root of Paeonia alba (peony). Leaves of C. sativa (sweet chestnut) were used as a positive control due to their previously demonstrated mitigating effect on methane and ammonia formation (Jayanegara et al., Reference Jayanegara, Marquardt, Kreuzer and Leiber2011). The aerial part of L. corniculatus was tested because of contradicting reports in regard to CH4 and ammonia mitigation potential (no effect: Macheboeuf et al., Reference Macheboeuf, Coudert, Bergeault, Lalière and Niderkorn2014; Niderkorn and Macheboeuf Reference Niderkorn and Macheboeuf2014; mitigating effect: Tavendale et al., Reference Tavendale, Meagher, Park-Ng, Waghorn and Attwood2005; Williams et al., Reference Williams, Eun, MacAdam, Young, Fellner and Min2011). As summarized in Table 2, for nine test plant materials, no publications regarding effects on ruminal fermentation were available and for the remaining seven materials, investigations were incomplete in terms of the variables aimed at in the present study. The large variety of incubation methods used among the plant materials in the literature cited above and the missing results regarding variables of interest for the present study were reasons to include these plants. In particular the uCP and NEL contents of the plant materials were never reported in other studies, with the exception of uCP for L. corniculatus (Scharenberg et al., Reference Scharenberg, Arrigo, Gutzwiller, Soliva, Wyss, Kreuzer and Dohme2007). Thus, the results of the present study contribute considerably to the description of their feeding value. A TMR without plant additive served as the negative control.
Overview of in vitro studies describing methane, ammonia, SCFA or digestibility results with the plant species tested in the present study
DM, dry matter; NEL, net energy for lactation; uCP, utilizable crude protein.
Based on a search in Web of Science using ‘plant name’, ‘ammonia’, ‘methane’ and ‘fermentation’ as key words for the period from 1990 to 2018.
The test plant material was purchased from Alfred Galke GmbH (Bad Grund, Germany), Berg pharmacy (Zurich, Switzerland) and St. Peter pharmacy (Zurich, Switzerland), except for S. caprea which could not be purchased (Table 1). As stated by the suppliers, two lots of each type of plant material were collected in the countries and during the seasons indicated (Table 1), dried to constant weight at 35 °C in a drying oven and cut to a size of 4–6 mm. Two lots of each type of plant material were purchased from different suppliers, with the exception of L. corniculatus, P. alba and P. spinosa. The two lots each of B. pendula, G. urbanum and V. vinifera rubra were grown in different countries. The two lots each of C. avellana, E. angustifolium and R. nigrum were grown in the same countries but harvested in different months of the year. The two lots of all other plant materials were grown in the same countries at unspecified harvest time or harvested in the same month of the year. Leaves of S. caprea were collected in the form of autumn foliage in the area of Baden-Baden, Germany, and dried at 50 °C for 48 h. For this plant only one lot was available.
Incubation mode
The HGT method was performed as outlined by Menke and Steingass (Reference Menke and Steingass1988). For incubation, modified 100 ml glass syringes with two outlets, one for fluid and one for gas sampling, were used as described in Soliva and Hess (Reference Soliva, Hess, Makkar and Vercoe2007). All test materials were ground with a centrifugal mill (Model ZM1, Retsch GmbH, Haan, Germany) to pass through a 1 mm sieve. Material from the test plants (40 mg dry matter [DM]) was added to 200 mg DM TMR (167 mg test plant/g total substrate). This proportion was chosen as it was considered realistic for inclusion as a supplement to a complete ruminant diet and because a comparable level has been tested in similar studies (Selje et al., Reference Selje, Hoffmann, Muetzel, Ningrat, Wallace and Becker2007; Bodas et al., Reference Bodas, López, Fernández, García-González, Rodríguez, Wallace and González2008), as well.
The TMR (basal diet) was composed of maize silage, grass silage (mixed sward, ryegrass dominated), grass hay (mixed sward, balanced) and concentrate (0.50 : 0.25 : 0.10 : 0.15). The concentrate DM consisted of rapeseed cake (500 g/kg), soybean meal (170 g/kg), maize gluten (150 g/kg), wheat (130 g/kg), calcium phosphate (30 g/kg) and limestone (20 g/kg). Rumen fluid was taken from a fistulated, lactating Brown Swiss cow (Cantonal Veterinary Office approval number ZH 38/14) that was fed on second-cut grass hay and, additionally, a mineral supplement (Mineralsalz UFA 195; UFA AG, Herzogenbuchsee, Switzerland). On six occasions distributed across 6 weeks, rumen fluid was collected before morning feeding. It was transported in a pre-heated thermos flask to the laboratory. Within 1 h after collection, rumen fluid was strained through four layers of gauze and added to a buffer solution in a 1 : 2 ratio according to the protocol of Menke and Steingass (Reference Menke and Steingass1988).
In the next step, 30 ml of rumen fluid–buffer mixture were filled into syringes, which already contained the feed. Each of the two lots from the 17 plant materials was incubated in six HGT runs adding up to 12 observations (two lots × six runs). For S. caprea, only six observations (one lot × six runs) were available. Two syringes each per run were comprised of HGT standard hay, standard concentrate as well as HGT standard uCP (all material obtained from the Institute of Animal Nutrition, University of Hohenheim, Stuttgart, Germany). Additionally, each run included a duplicate of blanks (syringes without feed) and two syringes with just the basal diet as the negative control, adding up to 270 incubations in total.
Measurements, sampling and laboratory analysis
The incubation lasted for 24 h at 39 °C in an incubator with integrated rotor. After 24 h, the fermentation gas volume was recorded from the calibrated scale printed onto the syringes, and the fermentation was terminated by removing the incubation fluid from the syringes while the gas phase remained inside. Measurements of pH and ammonia in the incubation fluid were performed with a potentiometer directly after collection (pH: model 632; ammonia: model 713; Metrohm, Herisau, Switzerland), which was equipped with appropriate glass electrodes (pH: 6.0204.100; ammonia: 6.0506.100; Metrohm, Herisau, Switzerland). Fermentation gas samples of 150 µl were taken from the incubation syringes and injected using a gas-tight Hamilton syringe (Hamilton AG, Bonaduz, Switzerland) into a gas chromatograph (6890N, Agilent Technologies, Wilmington, DE, USA) equipped with a thermal conductivity detector. Concentrations of CH4 and carbon dioxide were analysed with this detector. Four millilitres of incubation fluid were centrifuged, and 2 ml of supernatant were conserved at −20 °C for later SCFA analysis. This analysis was performed on a HPLC (La Chrom, L-7000 series, Hitachi Ltd., Tokyo, Japan) equipped with an ultraviolet (UV) detector. For protozoal counting, the incubation fluid was preserved with a 60 ml formaldehyde/l solution (Sigma-Aldrich, Buchs, Switzerland) in a ratio of 1 : 1. Samples for bacteria counting were diluted 1 : 100 in a 40 ml of formaldehyde/l solution. A Bürker counting chamber (Blau Brand, Wertheim, Germany) with depth of 0.1 mm was used to count protozoa, and a Neubauer improved counting chamber (Blau Brand) with a depth of 0.02 mm was used for bacterial counting.
The chemical composition of test plant materials and the basal diet were determined according to AOAC (1997). DM and total ash (TA) were analysed by a TGA-701 furnace (Leco Corporation, St. Joseph, Michigan, USA; AOAC index no. 942.05), and organic matter (OM) was calculated as DM minus TA. The neutral detergent fibre (NDF) was assessed in a Fibertec System M 1020 Hot Extractor and a 1021 Cold Extractor (Tecator, Högamäs, Sweden) with the use of a heat-stable α-amylase, but without sodium sulphite (Van Soest et al., Reference Van Soest, Robertson and Lewis1991). The acid detergent fibre (ADF) and acid detergent lignin (ADL) were determined using the aforementioned Fibertec apparatus according to the protocol of Van Soest et al. (Reference Van Soest, Robertson and Lewis1991). The NDF and ADF values were expressed without residual ash.
A Soxhlet extractor was used to determine ether extract (EE) (Extraction System B-811, Büchi, Flawil, Switzerland; AOAC index no. 963.15) and nitrogen (N) contents were measured with a C/N analyser (TruMac CN, Leco Corporation, St. Josephs, Michigan, USA; AOAC index no. 968.06). Crude protein (CP) was calculated as 6.25 × N. All samples were analysed in duplicate, except for ADF and ADL, which were analysed in triplicate. Analyses of phenolic fractions were performed according to Makkar (Reference Makkar2003). Extraction for the subsequent measurements was done twice using a 700 ml acetone/l solution. Total phenols (TP) and non-tannin phenols (NTP) were analysed with the Folin–Ciocalteu method, but, in contrast to Makkar (Reference Makkar2003), the results were expressed as gallic acid equivalents. This was because the calibration of the UV-spectrophotometer (Shimadzu UV-160A; Shimadzu Corporation, Kyoto, Japan) was done with a gallic acid solution. To calculate the total tannin (TT) contents of the samples, the NTP were subtracted from the TP. The determination of the condensed tannins (CT) was performed with the butanol-HCl-iron method (Makkar, Reference Makkar2003) and the contents were given as leucocyanidin equivalents. The hydrolysable tannins (HT) were calculated as the difference between TT and CT.
Calculations and statistical analysis
Total gas amounts produced by the treatment diets were calculated by the difference between gas production from the blanks and the gas amount in the incubated samples. Afterwards, the average value of the gas amounts expected and produced by the standard hay (expected: 49.61 ml gas/200 mg DM; 24 h incubation) and concentrate (expected: 65.18 ml gas/200 mg DM; 24 h incubation) was used to adjust the gas formation data from each run separately before calculating the NEL values. The lower amount of fermentable OM provided by the basal diet alone (200 mg) when compared with the test diets (200 + 40 mg) was accounted for by adjusting the basal diet values of all quantitative variables to an amount of 240 mg. The values actually measured were specified as 200 mg DM of basal diet in brackets in the tables. For the relationship of CH4 to SCFA, the molar amount of CH4 was calculated by assuming a 20 °C temperature and an atmospheric pressure of 960 Pa.
In vitro OM digestibility (IVOMD) and NEL were calculated according to the protocol of Menke and Steingass (Reference Menke and Steingass1988) by:
$$\eqalign{{\rm IVOMD\,(mg/g)} & = 148.8 + 8.893 \times {\rm GP\,(ml/200\,mg\,DM)} \cr &\quad + 0.448 \times {\rm CP\,(mg/g)} + 0.651 \times {\rm TA\,(mg/g)};}$$
$$\eqalign{{\rm N}{\rm E}_{\rm L}\,({\rm kJ/g\,DM}) & = 1.64 + 0.0269\,{\rm GP\,(ml/200\,mg\,DM)} \cr &\quad + 0.00078\,{\rm G}{\rm P}^2\,({\rm ml/200\,mg\,DM}) \cr &\quad + 0.0051\,{\rm CP\,(mg/g)} + 0.01325\,{\rm EE\,(mg/g)}}$$where GP = adjusted gas production after 24 h of incubation. The content of uCP was estimated according to Steingass and Südekum (Reference Steingass and Südekum2013) by:
$$\eqalign{{\rm uCP\,(mg/g\,DM)} & = {\rm (N}{\rm H}_{\rm 3}{\hyphen }{\rm N}_{{\rm blank}}({\rm mg}) + {\rm N}_{{\rm sample}}\,{\rm (mg)}\ndash \cr &{\rm N}{\rm H}_{\rm 3} {\hyphen} {\rm N}_{{\rm sample}}\,{\rm (mg))}/{\hbox{weight of sample (mg DM)}} \cr &\quad \times 6.25 \times 1000}$$where Nsample = N originating from the incubated diet and NH3-Nblank and NH3-Nsample = ammonia-N measured in the blank and the incubated sample, respectively. The uCP values were corrected by the respective standard (183 mg uCP/g DM; 24 h incubation).
The data were analysed with the MIXED procedure of SAS version 9.4 (SAS Institute, Carry, NC, USA) with the Tukey–Kramer adjustment. The plant material (including the basal diet alone) was defined as fixed effect (n = 19), and incubation run (n = 6) was defined as random effect. The basal diet was replicated twice per run. Single incubations of each of the two different lots from the same plant material were repeated six times (runs). These six values per plant material were obtained over a period of 6 weeks where the presence of clear differences in the donor animal's rumen fluid was likely. Thus, the number of observations was n = 12 (two lots × six runs) per test plant material, except for S. caprea, where only six observations (one lot × six runs) were available. For B. pendula and the basal diet, only 10 and 11 total observations were available, respectively. Calculations for uCP were only possible in four runs due to the lack of the uCP standard in the first run and values out of the standard range (183 ± 18 mg uCP/g DM) in the last run. Differences among the Tukey–Kramer adjusted means were considered to be significant at P < 0.05 and as trends at 0.05 ⩽ P < 0.10. In a separate statistical evaluation, an indication of the presence or absence of lot differences in the data from the HGT experiment was tested as far as it was possible in the frame of the current set-up. This was accomplished by performing a simple Student's t test for every fermentation variable and each individual lot of test plant material (except for S. caprea, where only one lot was available).
Results
The content of OM (g/kg DM) for the test plant material lots ranged from 816 for the aerial part of S. officinale to 981 for the kernel of P. spinosa (Table 3). The difference was smaller in CP, with 145 g/kg DM between the highest (R. nigrum leaves) and lowest (P. spinosa fruit pulp) values. Extremely small amounts of EE were found in both lots of P. alba roots, L. corniculatus aerial part and the fruit pulp of P. spinosa. In contrast, P. spinosa kernels showed nearly ten times more EE (about 100 g/kg DM). Kernels of P. spinosa contained the most NDF (673 and 693 g/kg DM), while roots of P. alba contained the least (183 and 164 g/kg DM). The aerial part of E. angustifolium showed the largest difference (71 g NDF/kg DM) between the two lots. Contents of ADF (g/kg DM) were highest in S. caprea leaves (627) and lowest in P. alba root lots (99 and 106). The same plants also showed contrasting ADL, with on average 419 v. 1–5 g/kg DM, respectively. Among all plant materials, the leaves of A. uva-ursi clearly showed the highest contents of TP, NTP, TT and HT, all >80 g/kg DM. Kernels of P. spinosa had the lowest TP, TT and HT contents, all <10 g/kg DM. The plant material from A. hippocastanum (fruit), P. alba (root), P. spinosa (entire fruit, kernel, pulp) and S. caprea (leaves) were the only materials with <10 g NTP/kg DM. The highest content (g/kg DM) of CT (65.4) was found in the leaves of C. avellana; it was 500 and 260 times higher than the contents found in the basal diet (0.13) and in the aerial part of S. officinale (0.25), respectively.
Chemical composition (g/kg DM) of the basal diet and the experimental plant materials
Values are means of duplicate or triplicate analysis.
The pH of the incubation fluid always ranged between 6.7 and 6.9. The addition of different test plant materials to the basal diet caused a large variation in the magnitude of effects compared with incubation of the basal diet alone (Tables 4 and 5). All plant materials reduced ammonia concentrations (P < 0.05) by up to 36% compared with the basal diet as sole substrate. The bacteria count was affected (P < 0.05) by some test plant materials. The count was high with the leaves of V. vinifera rubra and low with the leaves of P. tremula. Protozoa counts remained unaffected by the supplements compared with the basal diet (value adjusted to 240 mg DM incubated). All plant supplementation resulted in a decrease (P < 0.05) in total SCFA concentration. The molar proportion of acetate in total SCFA was influenced by the plant additives (P < 0.05), but only with the addition of leaves from B. pendula and C. sativa was the molar proportion lower compared with the basal diet as sole substrate.
Fermentation characteristics as measured in the incubation fluid after incubation with the basal diet as sole substrate or supplemented with the experimental plants at a ratio of 5 : 1 (basal diet : plant)
C2, acetate; C3, propionate; C4, butyrate; C5, valerate; C2/C3, acetate/propionate ratio.
Least-square means of six incubation runs × 2 plant material lots.
Values in bold differ (P < 0.05) from basal diet as sole substrate.
a Plants ordered according to declining amount of CH4 relative to dOM (cf. Table 5).
b Variables where two values are given: values outside of brackets are least-square means from data adjusted to 240 mg DM equivalent to the other treatments. Values in brackets give least-square means from data actually measured with 200 mg DM of the basal diet.
Effects of the plant species incubated at a ratio of 1 : 5 with the basal diet on gas production, digestibility, as well as calculated contents of NEL and uCP in DM
dOM, digestible organic matter; CH4, methane; CO2, carbon dioxide; IVOMD, in vitro organic matter digestibility; SCFA, short-chain fatty acids.
Least-square means of six incubation runs × 2 plant material lots.
Values in bold differ (P < 0.05) from basal diet as sole substrate.
a Plants ordered according to declining amount of CH4 relative to dOM.
b Variables where two values are given: values outside of brackets are least-square means from data adjusted to 240 mg DM equivalent to the other treatments. Values in brackets give least-square means from data actually measured with 200 mg DM of the basal diet.
Propionate proportions of total SCFA in the incubation fluid were increased (P < 0.05) by nine of the plant supplements compared with the control. The acetate-to-propionate ratio was highest at 3.74 : 1 for the A. uva-ursi leaves and lowest for the C. sativa leaves at 3.27 : 1. It was reduced (P < 0.05) by nine of the plant supplements compared with the basal diet alone. Butyrate proportion was slightly reduced (P < 0.05) with the aerial part of S. officinale, and the iso-butyrate proportion was not influenced by any of the plant supplements. Molar proportions of valerate were decreased (P < 0.05) only by B. pendula leaves, and only the leaves of B. pendula and P. tremula increased the iso-valerate (P < 0.05).
Compared with the basal diet alone, total gas production of the incubated diets was increased (P < 0.05) only by supplementing with the root of P. alba (Table 5). In contrast, with leaves of P. tremula, the aerial part of E. angustifolium, leaves of C. sativa and the kernel of P. spinosa, the total gas amount was reduced (P < 0.05) between 4 and 8 ml/24 h. The remaining 14 plant additives did not affect the total gas produced compared with the basal diet alone. Accordingly, IVOMD and the amount of OM digested in 24 h declined (P < 0.05) with additions of P. tremula, E. angustifolium, C. sativa and kernels of P. spinosa. The amount of CH4 produced per unit of digestible OM (dOM) was reduced (P < 0.05) by 13 of the plant additives compared with the control. This was to an extent of 0.05 to 0.20 of the basal diet alone.
The methane yield per unit of dietary DM and absolute CH4 production in ml/24 h (adjusted to 240 mg of diet DM) showed similar results. C. sativa leaves exhibited the greatest CH4 mitigating effect, and the kernels of P. spinosa exhibited the smallest effect. The carbon dioxide per unit of feed DM was 183 ml/g in the basal diet, and it was lowered (P < 0.05) up to 25 ml/g DM by the aerial part of E. angustifolium, the leaves of C. sativa, and the kernels of P. spinosa. It was increased (P < 0.05) by the root of P. alba (+20 ml/g DM).
The same plant supplements were similarly effective (P < 0.05) in changing the amount of carbon dioxide per 24 h. The CH4-to-carbon dioxide ratio was reduced (P < 0.05) by ten of the plant supplements, with the highest decrease of 30 ml CH4/l of carbon dioxide in the case of C. sativa leaves. Only a few test plant materials affected the CH4-to-SCFA ratio. The ratio increased with the root of P. alba and the whole fruit of P. spinosa and decreased with the leaves of C. sativa. Adding five of the test plant materials reduced (P < 0.05) the NEL content of the diet to a degree ranging from 0.41 (P. tremula leaves) to 0.77 (C. sativa leaves) kJ/g of feed DM. Only one, the root of P. alba, increased (P < 0.05) the NEL content by 0.55 kJ/g of feed DM. The uCP content (g/kg DM) was reduced (P < 0.05) by only one plant supplement, the kernel of P. spinosa. Adding the leaves of R. nigrum, C. avellana and the aerial part of E. angustifolium even increased the uCP content by about 12 g/kg DM.
The lot effect was only significant (P < 0.05) with the kernel of P. spinosa for protozoal numbers and iso-valerate, with the pulp of P. spinosa for iso-valerate, with the leaves of R. nigrum for iso-butyrate, and with the leaves of V. vinifera rubra for total gas.
Discussion
Lot effects
The accuracy and repeatability of the results of plant screenings for feeding value and mitigation properties also depend on the recovery of the effects in other lots of the same plant material. Various factors may change the composition of woody plants and influence their digestibility. Palo et al. (Reference Palo, Sunnerheim and Theander1985) showed that the content of phenolic acids in fine twigs of B. pendula varies between April and July. At the same time, there were changes in the contents of CP (8–16%) and NDF (46–54%). Palo et al. (Reference Palo, Sunnerheim and Theander1985) and Sauter and Wellenkamp (Reference Sauter and Wellenkamp1998) showed how the harvest period could have an effect on the composition of the plant material. Nour et al. (Reference Nour, Trandafir and Cosmulescu2014) noted clear differences in leaves of R. nigrum in phenolic and mineral content among different cultivars in a sampling period between June and August: the highest phenolic contents were found in mid-June, followed by a decrease until August. Variability in woody plants seems to be particularly large in species that are very rich in PSM. Accordingly, tropical shrubs differed substantially in contents of nutrients and CT, as well as in their efficiency to affect ruminal fermentation and methanogenesis when grown on different soils, in varying climates and under different fertilization treatments (Tiemann et al., Reference Tiemann, Franco, Peters, Frossard, Kreuzer, Lascano and Hess2009, Reference Tiemann, Franco, Ramírez, Kreuzer, Lascano and Hess2010) or when originating from different cultivars (Bekele et al., Reference Bekele, Clément, Kreuzer and Soliva2009). In vitro studies have often used only one lot per plant material screened. In the present study, the two lots of every plant material differed in harvest time, country of harvest or supplier (with the exception of L. corniculatus, P. alba and P. spinosa), as well as in nutrient and PSM contents. Significant lot effects were only found in five out of a total of 391 combinations investigated (17 test plant materials with two lots × 23 variables), the lot effects were considered negligible in the present set-up and plant material. The use of two lots per plant material cannot represent the entire natural variation of the plant material, but is nevertheless an approach to take it into account.
In vitro fermentation, feed nutritive value and potential side-effects
The use of materials from trees and shrubs as forage for ruminants is often limited by a high prevalence of PSM (Papanastasis et al., Reference Papanastasis, Yiakoulaki, Decandia and Dini-Papanastasi2008). At the same time, woody plants are frequently underestimated for their nutritive value. The present in vitro screening provided several traits indicative of the feeding value of the test plant materials and the total diet. The high feeding value of the supplemented diets was indicated by variables demonstrating the presence of an intensive ruminal nutrient degradation, such as total gas production, high IVOMD and NEL contents.
All plant materials, except for kernels of P. spinosa and leaves of S. caprea, increased total SCFA when compared with the value measured with 200 mg DM of the basal diet, resulting from the fermentation of the additional organic material (200 + 40 mg). When comparing the amount of total SCFA found with the basal diet (adjusted to 240 mg DM) and those with the added test plant material, findings indicated that the plant materials were indeed less fermentable than the basal diet. Nevertheless, IVOMD was similarly high for many of the plant additives compared with the basal diet as sole substrate. The plant materials causing the lowest total SCFA concentration also exhibited the lowest IVOMD and total gas production.
In the present study, plant materials with the highest ADL content had the comparably lowest feeding value; these were kernels of P. spinosa and leaves of S. caprea. With these materials, the effect of tannins on digestibility could have been masked by the effect of ADL. The leaves of C. sativa and P. tremula (the former rich in tannins) likewise showed a low feeding value. The latter is in agreement with Jayanegara et al. (Reference Jayanegara, Leiber and Kreuzer2012), who described a decrease of total SCFA with an increasing dietary tannin level, and Tavendale et al. (Reference Tavendale, Meagher, Park-Ng, Waghorn and Attwood2005) found inverse relationships of CT concentrations with SCFA. Adding these four plant materials to the basal diet also eventually resulted in low NEL contents. The comparably low feeding value of C. sativa leaves was determined earlier (Jayanegara et al., Reference Jayanegara, Marquardt, Kreuzer and Leiber2011). The content of uCP was reduced only with the addition of the kernels of P. spinosa. This finding could have derived from the decrease in IVOMD and thus in fermentable OM needed for synthesis of microbial protein. Adding plant materials which increased the uCP (leaves of R. nigrum and C. avellana, aerial part of E. angustifolium) compared with the basal diet was accompanied by a decrease in protein degradation to ammonia thus resulting in more rumen-bypass protein. The digestibility of L. corniculatus found in earlier studies ranged from 62 to 79% for DM (Chen et al., Reference Chen, Zhao, Fu, Ma, Qian, Abibuli, Yang, Abula, Xu and Aniwaer2011; Banik et al., Reference Banik, Durmic, Erskine, Ghamkhar and Revell2013, incubated as plant alone) and 54% for OM (Williams et al., Reference Williams, Eun, MacAdam, Young, Fellner and Min2011) when including the plant at 400 g/kg DM. The leaves of V. vinifera were described by Kamalak (Reference Kamalak2005) and Peiretti et al. (Reference Peiretti, Masoero and Tassone2017) as plants with a high nutritive value for ruminant feeding. When the leaves of V. vinifera (green and red) were added to the basal diet in the present study, the IVOMD and uCP values were not affected. This supported the findings of the aforementioned studies.
The current results show that the majority of plant materials chosen for the present screening study are quite well-suited as feed supplements from a net energy point of view. With the root of P. alba, the total gas amount and dOM were even increased. It seems that the root of P. alba even exceeded the feeding value of the basal diet. Bodas et al. (Reference Bodas, López, Fernández, García-González, Rodríguez, Wallace and González2008) tested six promising plants (C. pycnocephalus, P. tremula, P. avium, Q. robur, R. nobile and S. caprea) from the original 500 samples of the Rumen Up project in more detail for their effect on digestibility, total gas, methane and SCFA production in vitro, and concluded that leaves (and the little stem) of P. tremula were more promising than leaves (and the little stem) of S. caprea. This can be only partially confirmed by the present results of the leaves of P. tremula and S. caprea. In addition, woody plants may have favourable and unfavourable effects not obvious from in vitro rumen fermentation studies. Accordingly, incorporating woody plants or plants rich in PSM, including some of the plant materials tested in the present study in animal diets might be advantageous to health and performance (Waghorn, Reference Waghorn2008). For example, extracts from woody plants (Rubus fructicosus, Quercus robur and C. avellana) had an inhibitory effect on gastrointestinal nematodes in vitro (Paolini et al., Reference Paolini, Fouraste and Hoste2004). A study by Marley et al. (Reference Marley, Cook, Keatinge, Barrett and Lampkin2003) showed that faecal egg counts of helminth parasites were reduced in lambs grazing on L. corniculatus. With regard to performance, feeding L. corniculatus increased the milk yield in dairy cows (Woodward et al., Reference Woodward, Laboyrie and Jansen2000, Reference Woodward, Waghorn and Laboyre2004). However, feed with a high HT content can be toxic to the animal (Waghorn, Reference Waghorn2008). Therefore, the leaves of A. uva-ursi and C. sativa must be used carefully in animal nutrition, although they may still be applicable after sufficient adaptation (Waghorn, Reference Waghorn2008). Pyrrolizidine alkaloids, present in S. officinale (Stickel and Seitz, Reference Stickel and Seitz2000), and amygdalin, occurring in the kernel of P. spinosa (Kumarasamy et al., Reference Kumarasamy, Cox, Jaspars, Nahar and Sarker2003), may also be toxic after ingestion. Great care is required when these materials are used in animal nutrition.
Ammonia mitigation potential of the test plant materials
Compared with the ammonia value from the basal diet alone, the reduction in ruminal ammonia by >3 mmol/l that was caused by 15 of the 18 plant supplements (value calculated for 240 mg DM incubated weight) is consistent with the numerical decrease in bacterial numbers. A. hippocastanum fruit, A. uva-ursi leaves, C. avellana leaves, C. sativa leaves, E. angustifolium aerial part and P. alba root were particularly effective, as they caused a decrease of at least 4 mmol ammonia/l. Bacteria are major producers of ammonia from dietary protein, and tannins can inhibit the growth of rumen bacteria by binding to bacterial cells (Molan et al., Reference Molan, Attwood, Min and McNabb2001). The tannins may reduce the export or activity of microbial enzymes or inhibit the separation of cells after division. Other studies found a significant decrease in bacterial growth with extracts from C. avellana leaves (Oliveira et al., Reference Oliveira, Sousa, Valentão, Andrade, Ferreira, Ferreres, Bento, Seabra, Estevinho and Pereira2007) and E. angustifolium herb (Rauha et al., Reference Rauha, Remes, Heinonen, Hopia, Kähkönen, Kujala, Pihlaja, Vuorela and Vuorela2000), which might be explained by the usage of extracts. Selje et al. (Reference Selje, Hoffmann, Muetzel, Ningrat, Wallace and Becker2007) tested eight promising plants of the original 500 samples from the Rumen Up project in more detail for their ruminal protein degradation effect in vitro. Among these, A. uva-ursi also inhibited proteolysis. Macheboeuf et al. (Reference Macheboeuf, Coudert, Bergeault, Lalière and Niderkorn2014) and Niderkorn and Macheboeuf (Reference Niderkorn and Macheboeuf2014) also found ammonia-reducing effects with E. angustifolium. Besides their antimicrobial effects, tannins, especially CT, form non-soluble tannin–protein complexes, thereby preventing ruminal degradation of feed proteins by the microbes (Kumar and Singh, Reference Kumar and Singh1984). The three plant materials (the aerial part of L. corniculatus, the kernel of P. spinosa and the leaves of S. caprea) with the lowest effect on ammonia formation (i.e. reduction of <3 mmol/l) had CT and TT contents of <2 and <7 g/kg DM, respectively.
Methane mitigation potential of the test plant materials
Supplementing feed with plants rich in PSM is a promising strategy to reduce CH4 emissions from ruminants (Jayanegara et al., Reference Jayanegara, Leiber and Kreuzer2012; Hristov et al., Reference Hristov, Oh, Firkins, Dijkstra, Kebreab, Waghorn, Makkar, Adesogan, Yang, Lee, Gerber, Henderson and Tricarico2013). The mode of action of tannin and NTP is their toxicity to some rumen microorganisms, e.g. by inhibiting their growth as outlined in the preceding section. In the present study, 13 of the 18 plant materials decreased CH4 per unit of digestible OM compared with the basal diet without supplements. Relating CH4 to DM supply and carbon dioxide produced also pointed towards a similar result. All these measures consider that CH4 mitigation is only useful in relation to nutrients and energy supplied to the animal. The CH4 mitigating effect of the plant materials used in the present study was occasionally less pronounced than that indicated by Bodas et al. (Reference Bodas, López, Fernández, García-González, Rodríguez, Wallace and González2008) as part of the Rumen Up report, although those authors used a lower dosage (91 g/kg DM). One reason might be the difference in the basal diet, because mainly forage (alfalfa hay) with a high CH4 formation potential was used by Bodas et al. (Reference Bodas, López, Fernández, García-González, Rodríguez, Wallace and González2008) as part of the Rumen Up project. The present study used a mixed diet containing concentrate and maize silage. In the Rumen Up report, S. caprea, P. tremula and C. avellana were described to reduce CH4 by more than 0.25 of total, and the root of P. alba even reduced it by 0.54 of total.
In the present study, such a reduction was not found for the root of P. alba; it even slightly increased the CH4 formation per unit of DM or digestible OM when added to the basal diet. Variation in results of plant material between studies might be attributed to different collection periods, drying methods or storage durations. Also, the leaves of S. caprea only moderately reduced CH4 when compared with the basal diet. In the present case, this material constituted fallen autumn leaves collected during winter. The biological degradation until collection obviously resulted in the highest contents of lignified fibre (ADF and ADL) of all plant materials investigated, whereas very few phenols were recovered. Among test plant materials, C. sativa leaves (positive control) were the most efficient in CH4 mitigation, consistent with findings from Bhatta et al. (Reference Bhatta, Uyeno, Tajima, Takenaka, Yabumoto, Nonaka, Enishi and Kurihara2009) and Jayanegara et al. (Reference Jayanegara, Marquardt, Kreuzer and Leiber2011). However, C. sativa leaves also suppressed total gas and CO2 production, showing that a part of the mitigation effect originated in a general inhibition of ruminal fermentation and, consequently, the feeding value was low. The C. sativa leaves had the highest HT content of all plant materials. The addition of C. avellana leaves and E. angustifolium aerial part to the basal diet reduced CH4 per digestible OM by >5 ml/g. In the case of C. avellana, total gas and CO2 formation was not affected. In the case of E. angustifolium, these indicators of fermentation intensity were depressed, as with C. sativa.
In a feeding experiment with sheep, Wang et al. (Reference Wang, Terranova, Kreuzer, Marquardt, Eggerschwiler and Schwarm2018) demonstrated that C. avellana leaves also have a clear methane-mitigating effect in vivo. As a sole substrate, L. corniculatus has been reported to reduce CH4 emissions in vivo (Pinares-Patiño et al., Reference Pinares-Patiño, Ulyatt, Waghorn, Lassey, Barry, Holmes and Johnson2003; Woodward et al., Reference Woodward, Waghorn and Laboyre2004) and in vitro (Williams et al., Reference Williams, Eun, MacAdam, Young, Fellner and Min2011). However, in the present study, the addition of L. corniculatus aerial part at levels of 167 mg/g had no CH4 mitigating effect in vitro. In the screenings of Macheboeuf et al. (Reference Macheboeuf, Coudert, Bergeault, Lalière and Niderkorn2014) and Niderkorn and Macheboeuf (Reference Niderkorn and Macheboeuf2014), L. corniculatus was not effective in reducing CH4.
Protozoa are involved in ruminal CH4 formation. Because none of the plant materials decreased protozoal counts, the CH4 mitigating effects most likely resulted from other influences on fermentation. In line with the current findings, the meta-analysis of Jayanegara et al. (Reference Jayanegara, Leiber and Kreuzer2012) revealed no relationship of dietary tannins and protozoal counts in vitro. In the Rumen Up report, B. pendula, G. urbanum, P. tremula and S. officinale were described to reduce the bacteriolytic activity of protozoa. Therefore, it can be speculated that the plant materials in the present study might have affected the activity of protozoa at unchanged total numbers.
In the present study, there was a shift towards a higher proportion of propionate with nine of the additives, while the acetate and butyrate proportions were not influenced by almost all of the plant additives. Consequently, the acetate-to-propionate ratio was decreased by these nine plant materials. In this variable, C. sativa leaves and P. alba root caused the highest decrease. However, among the materials decreasing the acetate-to-propionate ratio, unexpectedly only B. pendula (leaves), C. sativa (leaves), P. spinosa (kernel) and S. officinale (aerial part) also mitigated the CH4 formation. Consequently, in the present study, the change in the SCFA profile was not the only reason for the observed decrease in CH4 formation.
A higher potential to reduce methanogenesis in plant extracts containing both HT and CT than in those containing only HT was reported by Bhatta et al. (Reference Bhatta, Uyeno, Tajima, Takenaka, Yabumoto, Nonaka, Enishi and Kurihara2009). However, this was not the case in the present study, as plant materials with high or low HT proportions were effective in mitigating CH4 formation (highest ratios of HT to CT contents: 31 : 1 in C. sativa leaves and 1 : 15 in C. avellana leaves). There seems to be a clear general linear relationship between tannin (CT, HT or CT + HT) content in feed and methanogenesis, as quantified by Jayanegara et al. (Reference Jayanegara, Leiber and Kreuzer2012) in a meta-analysis. Accordingly, the plant materials in the present study, which had a notable TT content (⩾40 g/kg), showed substantial CH4 mitigation (i.e. the leaves of C. avellana, C. sativa and R. nigrum or the aerial part of G. urbanum). In the present study, even dietary tannin contents below the threshold (20 g tannins/kg, in vitro and in vivo) described in the meta-analysis by Jayanegara et al. (Reference Jayanegara, Leiber and Kreuzer2012) were effective in CH4 mitigation. The suggestion by Tavendale et al. (Reference Tavendale, Meagher, Park-Ng, Waghorn and Attwood2005) that CT concentrations below about 80 g/kg DM can reduce CH4 without inhibiting fermentation rate were also confirmed through the examples from the leaves of B. pendula, C. avellana, R. nigrum and V. vinifera and the aerial part of G. urbanum which caused no decrease in total gas production.
Conclusions
Methane formation per unit of digestible OM was reduced by 13 out of the 18 plant materials compared with the basal diet, and all materials decreased ammonia formation. The majority of the mostly woody plant supplements tested had no adverse effect on in vitro digestibility. Further in vitro studies should include the most promising and feasible plant materials as the leaves of B. pendula, C. avellana, R. nigrum and V. vinifera and the aerial part of G. urbanum at varying dosages and as sole substrates. Finally, experiments with live animals need to evaluate the palatability of the plant materials, confirm their mitigating effects and evaluate effects on production.
Author ORCIDs
A. Schwarm http://orcid.org/0000-0002-5750-2111.
Acknowledgements
The authors are grateful to M. Wolter, C. Kunz, M. Mergani and P. Stirnemann for their contribution to the laboratory analyses.
Financial support
The present study was funded by ETH Zurich Research Grant number ETH-49 15-1.
Conflict of interest
None.
Ethical standards
The current study is covered by Cantonal Veterinary Office approval number ZH 38/14.
