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Effect of condensed tannin supplementation on in vivo nutrient digestibilities and energy values of concentrates in sheep
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Effect of condensed tannin supplementation on in vivo nutrient digestibilities and energy values of concentrates in sheep Katrin Gerlacha, Martin Priesb, Karl-Heinz Südekuma, a b
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Institute of Animal Science, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany Chamber of Agriculture of North Rhine-Westphalia, Ostinghausen, 59505 Bad Sassendorf, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords: Digestibility Secondary plant compound Sheep Tannin
The objective of this study was to evaluate the effect of supplemented condensed tannins (CT) from the bark of the Black Wattle tree (Acacia mearnsii) on in vivo nutrient digestibility and energy values measured with sheep under standardized conditions. A commercial A. mearnsii extract (containing 0.203 g CT/g dry matter (DM)) was mixed into a pelleted concentrate. Four treatments consisting of grass hay and concentrate were investigated, containing different concentrations of the CT-rich extract (CON, without CT; CT1, CT3 and CT5, with 1, 3 and 5% CT-rich extract in ration DM), resulting in CT concentrations of 0, 2.03, 6.19 and 10.2 g/kg DM, respectively. In a 21-day period, nutrient digestibility of the concentrates was determined by difference with wethers (n = 4 per treatment, German Blackheaded Mutton) in metabolism crates following a standardized procedure. The organic matter digestibility of the concentrates was unaffected by CT1 and decreased strongly with CT3 (−21%) and CT5 (−28%; P < .05). Digestibility of fibre fractions was already reduced with CT1 (P < .05), representing a very low level of CT supplementation. The concentration of metabolizable energy of the concentrates estimated from digestible nutrients decreased strongly (-25%) from 12.9 (CON) to 9.7 MJ/kg DM (CT5) (P < .05). In conclusion, CT supplementation from A. mearnsii to rations of sheep reduced nutrient digestibilities at much lower levels than previously reported for CT from other sources (e.g., forage legumes).
1. Introduction In ruminant nutrition, the use of secondary plant compounds like condensed tannins (CT) obtained from mostly tropical trees and shrubs as well as from forage legumes has gained some importance in research. The literature about CT with both beneficial and adverse function in ruminants according to their concentration and chemical structure, is vast and often conflicting (Piluzza et al., 2014). Increasing CT concentrations in ruminant rations elevated the amount of undegraded feed protein leaving the rumen, most likely caused by decreased rates of degradation by rumen microorganisms and reduced growth rate of proteolytic bacterial species (Min et al., 2003). Furthermore, CT have improved bodyweight gain, wool production and reproductive efficiency in sheep and reduced the impact of gastro-intestinal parasitism, as reviewed by Waghorn (2008). However, CT in ruminant rations have also been reported to cause a reduction in nutrient digestibility (Frutos et al., 2004a; Silanikove et al., 1994), create negative post-ingestive feedback (Silanikove et al., 1996) and impair animal performance (Grainger et al., 2009). For temperate forages, CT concentrations of 20–45 g/kg dry matter
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(DM) are generally seen as having beneficial effects on ruminant production (Min et al., 2003). However, a more specific contemplation seems necessary. When forages are fed as a sole diet, the CT in birdsfoot trefoil (Lotus corniculatus) have been beneficial for ruminant production, but the CT in sainfoin (Onobrychis ssp.), sulla (Hedysarum coronarium) and big trefoil (L. pedunculatus) do not appear to benefit productivity except for mitigating the impact of parasites (Waghorn, 2008). There is also a considerable variability in concentration and chemical structure of CT especially in forage legumes such that the mode of action and clear effects are difficult to predict (Mueller-Harvey, 2006). As an alternative to forage legumes, industrial products containing defined amounts of tannins (e.g., made from the bark of the Black Wattle tree (Acacia mearnsii)) have been used for supplementation of ruminant rations. However, also with those more standardized products the effects on ruminant performance are equivocal such that there is still a lack of knowledge on clear dose-effect results of CT extracts on ruminants. The aim of this study was to investigate the influence of different levels of a supplemented commercial product rich in CT from A. mearnsii on in vivo nutrient digestibility and energy value of
Corresponding author. E-mail address: [email protected] (K.-H. Südekum).
https://doi.org/10.1016/j.smallrumres.2018.01.017 Received 28 May 2017; Received in revised form 26 January 2018; Accepted 27 January 2018 0921-4488/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Gerlach, K., Small Ruminant Research (2018), https://doi.org/10.1016/j.smallrumres.2018.01.017
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fibre assayed with heat-stable amylase and expressed exclusive residual ash (aNDFom) and acid detergent fibre expressed exclusive residual ash (ADFom) were determined following methods 6.1.1, 6.5.1 and 6.5.2., respectively. Starch was analysed following method 7.2.1 (VDLUFA, 2012). The determination of enzyme-soluble organic matter (ESOM) was done according to method 6.6.1. The Hohenheim gas test (HGT; method 25.1) was conducted for measuring the 24 h in vitro gas production (GP, ml/200 mg DM). The analysis of total phenol and total tannin concentration in the Weibull Black product was conducted using the Folin method (Makkar, 2000) and the concentration of CT was determined with the HCl-butanol method (Terrill et al., 1992). The concentration of metabolizable energy (ME) of the grass hay and the concentrates was calculated according to following equations: For grass hay (GfE, 2008): ME (MJ/kg DM) = 5.51 + 0.0828 × ESOM − 0.00522 × ash + 0.02507 × EE − 0.00392 × ADFom; all expressed as g/kg DM; For concentrate (GfE, 2009): ME (MJ/kg DM) = 7.17 − 0.01171 × ash + 0.00712 × CP + 0.01657 × EE + 0.00200 × starch − 0.00202 × ADFom + 0.06463 × GP (ml/200 mg DM); all expressed as g/kg DM unless stated. Using the amounts and chemical compositions of feed and faeces, nutrient digestibilities were calculated. The digestibilties of the concentrates were calculated as difference between digestibility of hay and digestibility of hay + concentrate. The digestibilities of the proximate constituents were then taken to calculate ME concentration of the concentrate according to GfE (2001): ME (MJ/kg DM) = 0.0312 × DEE + 0.0136 × DCF + 0.0147 × (DOM − DCL − DCF) + 0.00234 × CP, where DEE is digestible ether extract, DCF is digestible crude fibre and DOM is digestible organic matter (all expressed as g/kg DM). The NEL values were estimated from ME according to Weißbach et al. (1996): NEL = ME [0.46 + 12.38 × ME/(1000 − ash)].
concentrates under standardized conditions in sheep. We hypothesized that addition of CT in moderate concentrations of up to 30 g/kg DM would not negatively affect nutrient digestibility in sheep. 2. Material and methods 2.1. Animals and experimental design In June 2013, a digestibility trial with sheep was conducted at the Experimental and Educational Centre for Agriculture ‘Haus Riswick’, Chamber of Agriculture of North Rhine Westphalia, Kleve, Germany (51° 47′ 18N, 6° 8′ 19E) to study the effect of CT supplementation on nutrient digestibility and energy value of concentrates supplemented with CT. The digestibility trial was accompanied by a long-term feeding trial (169 days) with dairy cows that was conducted in a free-stall dairy barn at the same centre. Here, the same CT product was used to study the effect of CT supplementation on performance and N use efficiency in dairy cows (Gerlach et al., 2018) and the effects on gaseous emissions (CH4 and NH3) on dairy barn level (Schmithausen, 2017). The CT product (commercially available) used in both studies was an extract rich in CT made from the bark of A. mearnsii (declared concentration of CT of 0.725 g/g DM, Weibull Black, TANAC S.A., Montenegro, Brazil) and was mixed into the commercially produced pelleted concentrates during processing. The digestibility of the concentrates supplemented with different amounts of CT-rich extract was measured with four male wethers (German Blackheaded Mutton) per treatment according to GfE (1991). As it is not possible to feed concentrates as a single feed, their digestibility was determined by difference by feeding them together with a forage of known digestibility and deducting the estimated effect of the latter in the calculations as described by Schneider and Flatt (1975) and GfE (1991). Rations consisted of chopped grass hay and the different concentrates. The proportions were chosen to achieve concentrations of the CT-rich extract of 0 (CON), 1% (CT1), 3% (CT3) of ration DM in accordance with the dairy cow trial; additionally, a treatment with 5% CT-rich extract (CT5) of ration DM was tested. According to GfE (1991) one group received a ration consisting of grass hay only to determine the digestibility and feeding value of the hay. Composition of the different concentrates is given in Table 1 and the composition of the grass hay as well as of rations including the CT concentration is presented in Table 2. Lower concentrations of crude protein (CP), ether extract (EE), starch and fibre fractions in the supplemented concentrates were caused by the addition of the CT-rich extract (dilution effect). Each ration was offered to four wethers in two meals per day. A 14-d adaptation period was followed by a 7-d collection period where animals were kept in metabolism crates and all faeces and feed refusals were collected on a daily basis and aliquots (20% of daily amount) were stored at −18 °C. Samples of the hay and the concentrates were collected daily and a cumulative sample was added up for analyses. Samples were stored at −20 °C until analysis for chemical composition and estimation of energy value. At the end of each collection period composite samples of ration ingredients as well as of faeces spanning the entire 7-d period were prepared, freeze dried and analysed chemically.
2.3. Statistical analyses Statistical analyses were conducted using SAS 9.3. Data of the digestibility trial with sheep were analysed using one-way ANOVA. The Tukeys Honestly Significant Difference test (Tukey-HSD) was applied for post hoc comparisons. For all analyses, differences were considered significant with P ≤ .05. 3. Results When measured with the Folin method (Makkar, 2000), the total phenol concentration in the Weibull Black product was 0.630 g/g DM and the total tannin concentration was 0.568 g/g DM. Therefore, the total tannin concentrations in rations were 5.68, 17.3 and 28.6 g/kg DM for CT1, CT3 and CT5, respectively. The concentration of CT determined with the HCl-butanol method (Terrill et al., 1992) was 0.203 g/g DM. The low CT concentration in the extract resulted in dietary CT concentrations of 2.03, 6.19 and 10.2 g/kg DM for CT1, CT3 and CT5, respectively (Table 2). The digestibility trial with wethers could be conducted without health problems, decreases in feed intake or changes in texture of faeces. Supplementation of the CT-rich extract did not cause feed avoidance or other irregularities in feeding behaviour. In Table 3, the nutrient digestibilities and energy values of the concentrates estimated from digestible nutrients are presented. The in vivo digestibility of organic matter of the concentrates was unaffected by CT1 and decreased strongly with CT3 (−21%) and CT5 (-28%; P < .05). The decrease was even more pronounced for digestibility of aNDFom, but with a large variation among animals. For aNDFom and ADFom, the digestibility was already reduced with CT1 (P < .05), whereas other constituents were only affected with CT3 and CT5. The ME concentration of the concentrates estimated from digestibility of proximate constituents decreased markedly (−25%) from 12.9 to 9.7 MJ/kg DM (P < .05), for
2.2. Chemical analyses and calculations Analysis of the chemical composition of the ration components and faeces samples was done by the Landwirtschaftliche Kommunikationsund Service GmbH (Lichtenwalde, Germany). The DM concentration of faeces was determined daily using a two-step procedure involving predrying samples at 60 °C, followed by oven-drying at 105 °C. Proximate analyses were done according to VDLUFA (2012) and method numbers are given below. Ash, EE and CP were analysed using methods 8.1, 5.1 and 4.1.1. The concentrations of crude fibre (CF), neutral detergent 2
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Table 1 Ingredient and chemical composition of the concentrates used in the digestibility trial (expressed as g/kg dry matter (DM) unless stated otherwise) with sheep in control (CON) and experimental group supplemented with 1% (CT1), 3% (CT3) or 5% in ration DM (CT5) condensed tannin extract.a
Ingredients (g/kg) Rapeseed meal Maize grain Wheat grain Vegetable fat Molasses Urea Tannin extractb Chemical composition DM (g/kg) Ash Crude protein Ether extract Crude fibre Starch aNDFom ADFom GP (ml/200 mg DM) ESOM MEb (MJ/kg DM) NEL (MJ/kg DM)
Concentrate CON
Concentrate CT1
Concentrate CT3
Concentrate CT5
561 249 125 25 31 9 –
546 243 121 24 30 8 27
518 230 115 23 29 8 76
493 219 110 22 27 8 121
890 64 287 61 96 306 246 120 52.2 825 13.2 8.39
900 64 283 61 92 290 232 124 50.6 826 13.0 8.25
899 68 266 60 90 245 232 129 47.4 816 12.6 7.87
899 62 291 49 83 253 190 115 42.7 833 12.4 7.70
aNDFom = NDF assayed with heat-stable amylase and expressed exclusive residual ash; ADFom = ADF expressed exclusive residual ash; GP = 24 h gas production; ESOM = enzymesoluble organic matter; ME = metabolizable energy; NEL = net energy for lactation. a Acacia mearnsii tannin extract (Weibull Black, Tanac S.A., Montenegro, Brazil). b Estimated according to GfE (2009).
As expected, formation of tannin-protein-complexes seems to have occurred in this study. It is suggested that binding to CT and building of complexes persisted also postruminally such that digestibility of supplemented concentrates decreased. Not only nutrient degradation in the rumen might be affected by dietary CT but also digestion in the small and even large intestine. As summarized by Frutos et al. (2004a) decreased nutrient absorption from the intestine might be caused by persistence of tannin-protein complexes in the intestine which failed to dissociate in the abomasum, formation of tannin-digestive enzyme complexes or new tannin-dietary protein complexes, or changes in the intestinal absorption due to the interaction of tannins with intestinal mucosa. Furthermore, it can be assumed that rapeseed meal which was a main ingredient of the concentrates contains considerable amounts of protein that is bound to the cell wall which is only available for the ruminant after microbial degradation and fermentation of the fibre. Because of the limited fermentation in the rumen the protein may not have been digested postruminally. Ruminants can only benefit from CT when the increases in protein flow from the rumen exceed the reduction in the absorption of amino acids from the intestine (Waghorn, 1996). With drastically decreased total-tract digestibility of OM and CP this requirement is not met. The aNDFom digestibilities decreased to an even larger extent than that of the other feed fractions; therefore it can be assumed that not only protein but also fibre fractions were bound to tannins resulting in decreased degradability. This is in agreement with Henke et al. (2016) who observed strongly reduced digestibilities of OM and, even more pronounced, fibre fractions in dairy cows after addition of a Quebracho CT extract. As summarized by Frutos et al. (2004a) tannins mainly exert their effects on proteins, however, they also have effects on carbohydrates, namely hemicellulose, cellulose, starch and pectins. Different studies have shown that fibre degradation in the rumen can be drastically reduced in animals that consume tannin-rich feeds (Barry and McNabb, 1999; Staerfl et al., 2012). For example, digestibility of NDF decreased from 55.2 to 32.5% when a CT-rich extract from A. mearnsii (30 g/kg DM) was added to maize silage-based rations fed to five-month-old fattening bulls (Staerfl et al., 2012). However, with 11-month-old bulls offered the same ration, no effect of CT treatment occurred (NDF digestibility 54.4 vs. 45.1%, for control and treatment, P > .05). Orlandi et al. (2015) reported that not only the
CON and CT5, respectively. The in vitro GP of rations with 1, 3 and 5% CT supplementation was reduced by 3.0, 9.2 and 18.2% which exceeds the CT concentration in the ration. The ESOM, as a method based on enzymatic procedures, was not affected by addition of CT. 4. Discussion The CT concentrations in the extract were lower than stated but on a comparable level with Kozloski et al. (2012) and Cenci et al. (2007), whereas Carulla et al. (2005) using the same and Grainger et al. (2009) using a different A. mearnsii extract stated much higher CT concentrations (0.615 and 0.603 g/g DM). It shows that CT concentrations are variable even within the same commercial product and analysis before use in feeding trials is necessary. Not only the product concentration of supplemented extracts but also the CT concentrations and methods used for the determination have to be stated for a better comparison of trials and understanding of results. Results suggest that the CT supplementation negatively impacted in vivo nutrient digestibility and microbial activity measured in the in vitro system HGT, whereas the in vitro enzymatic degradability (ESOM) was not affected. Plant extracts containing CT are able to impair ruminal growth of bacteria strains that are involved in proteolysis (Jones et al., 1994). The decreased GP is in coincidence with results presented by Zeitz and Kreuzer (2013) where in vitro GP was reduced by 8.1% and volume of methane by 14.0%, when 30 g A. mearnsii extract/kg DM was added to a ration consisting of grass hay, barley grain and soybean meal in comparison to a control ration without CT. The in vivo digestibility of OM in concentrates measured with wethers was drastically reduced (82.2% for control vs. 58.9% for CT5) suggesting a strong impairment of ruminal microbial digestion and fermentation. Waghorn (2008) summarized that the impact of CT on intestinal function is not completely understood in ruminants but it is assumed that tannins inhibit the capability of endogenous enzymes to split proteins into peptides and amino acids, and also impede their absorption. Endogenous enzymatic activity exceeds requirements for proteolysis, but CT bound to either bacterial surfaces or to proteins may reduce the enzymatic access and activity (Waghorn, 2008). 3
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Table 2 Composition of rations used in the digestibility trial for sheep in control (CON) and experimental group supplemented with 1% (CT1), 3% (CT3) or 5% in ration dry matter (DM) (CT5) condensed tannin (CT) extract.a
Amount of hay (g/d) Amount of concentrate (g/d) Type of concentrate CT extract in ration (g/kg DM) Total tannins in ration (g/kg DM) CT in ration (g/kg DM) DM (g/kg) Crude ash (g/kg DM) Crude protein (g/kg DM) Ether extract (g/kg DM) Crude fibre (g/kg DM) aNDFom (g/kg DM) ADFom (g/kg DM) ME (MJ/kg DM)b
Hay
CON
CT1
CT3
CT5
1000 – – – – – 872 88.0 109 14 268 567 303 8.1
400 600 Concentrate CON – – – 883 73.6 216 42 165 374 193 11.2
650 350 Concentrate CT1 10.0 5.68 2.03 882 79.6 170 30 206 450 240 9.8
640 360 Concentrate CT3 30.5 17.3 6.19 882 80.8 166 31 204 446 240 9.7
630 370 Concentrate CT5 50.4 28.6 10.2 882 78.4 176 27 200 428 233 9.7
aNDFom = NDF assayed with heat-stable amylase and expressed exclusive residual ash; ADFom = ADF expressed exclusive residual ash; ME = metabolizable energy. a Acacia mearnsii tannin extract (Weibull Black, Tanac S.A., Montenegro, Brazil). b Estimated based on chemical composition and enzyme-soluble organic matter (grass hay; GfE, 2008) or in vitro gas production (concentrate; GfE, 2009).
Surprisingly, there was a large variation among wethers resulting in high standard errors of mean digestibility values, especially for aNDFom. Commonly these kinds of digestibility trials have a small variation between animals and a high repeatability (Spiekers et al., 2006). Apparently there were high individual differences between animals in the adaptability to CT and the ability of the digestive tract to dissolve the CT complexes, at least for the specific CT source tested here. Similar observations were made by Staerfl et al. (2012) who supplemented bulls with A. mearnsii extract and described a high standard error of the results that was especially due to the high variation between animals within treatment group. Obviously, when the digestibility trial with sheep is compared with the feeding trial with dairy cows (Gerlach et al., 2018), the CT supplementation exerted more pronounced effects on sheep than on dairy cows. In dairy cows, only minor changes (small shift in N excretion from urine to faeces, reduced milk protein yield) occurred when animals were supplemented with CT3 for a period of 63 days whereas milk yield and other production variables were unaffected. These results are somehow surprising, as recent research by Bueno et al. (2015) using in vitro gas production techniques and different ruminant species as inoculum donors has shown that CT had greater effects in large ruminants than in small ruminants. They hypothesized that there is less microbial adaptation to CT in grazing bovids compared to other species. As cattle are grazers they do not consume a lot of tanniniferous plants in their natural or typical rations (Bueno et al., 2015). Therefore they do not have as strong a genetic capability to ferment plant material containing secondary compounds as ruminants commonly consuming more browse (Van Soest, 1994). Also Frutos et al. (2004b) showed that CT reduced in vitro GP, with differences depending on the inoculum donor (sheep, goats, cows and deer, given the same feed). Authors stated that ruminants are known to differ in their capacity to tolerate or degrade plant secondary metabolites and effects of tannins on in vitro incubations might differ when rumen fluid is derived from animals that consume tannin-rich diets seldom or regularly. Possibly not only origin and composition of the rumen fluid but also species-related factors like function and structure of the digestive tract impact the mechanism and effects of tannins such that, for the special case of CT, transfer of experimental results from one ruminant species to the other has to be examined. Rumen microbes can adapt to tanniniferous diets by increasing the proportion of tannin-resistant bacteria in the rumen and therefore mitigating the inhibitory effects of these secondary plant compounds (Smith et al., 2005). Adaptive changes in the rumen might be another possible explanation for the diverse result in sheep and cattle in this study. The digestibility trial was conducted according to the
apparent and true digestibility of N compounds by steers but also totaltract OM and aNDF digestibility tended to linearly decrease at incremental tannin extract inclusion levels (CT from A. mearnsii; 9, 18 and 27 g/kg DM). The tendency for reduced OM digestibility suggests that, with increasing CT concentrations, there was a decrease in the supply of digestible energy. The effect on ME concentration of rations calculated from digestible nutrients was even more pronounced in the current study, with a decrease in ME of 25% when comparing CON and CT5. Similar strong effects were observed by Kozloski et al. (2012) who infused an A. mearnsii extract intraruminally at dosages of 0, 20, 40, 60 g/ kg DM to wethers offered ryegrass for ad libitum intake. Intake and digestibility of OM, NDF and N [(N intake (g/d) – faecal neutral detergent insoluble N (g/d))/N intake (g/d)] were linearly reduced at increasing levels of tannin infusions. However, reduced fibre digestibilities with rations containing CT can also be caused by analytical difficulties as the detergent extraction techniques might not predict or determine the in vivo digestibility of cell-wall constituents accurately which was shown to be due to the presence of tannin-protein complexes as artefacts in fibre fractions, especially in samples of faeces (Makkar, 2003). The supplemented CT exerted a strong negative impact on OM digestibility in sheep, although CT concentrations were lower than critical values reported in literature where negative impacts are commonly only expected at CT concentrations exceeding 55 g/kg DM (Beauchemin et al., 2007; Grainger et al., 2009; Min et al., 2003). They did not even match reported concentrations (e.g. Min et al. (2003); 20–45 g CT/kg DM) of being beneficial in forages (reduced protein degradation in the rumen, increased milk production, wool growth and lambing percentage, reduced bloat risk and internal parasites). These assumptions are based on recommendations originating from feeding trials with Lotus species and may not be applicable to other CT sources (Mueller-Harvey, 2006). Obviously, a more detailed analysis of CT structure is needed to better understand diverging effects of CT supplementation. Furthermore also the ration that is supplemented with CT might influence the functionality of the tannins: Waghorn (2008) summarized that the impact of CT upon ruminant performance will depend on the amount and astringency in the diet, animal nutrient requirements and other dietary components such that a clear, easy relation between CT in rations and animal performance hardly exists. In this regard it has to be considered that CT-supplemented rations had slightly higher concentrations of fibre fractions than CON which might also have caused a small decline in digestibility. However, the CTsupplemented rations (CT1, CT3 and CT5) had nearly equal concentrations of fibre fractions such that the observed reduction in digestibility can be related to the CT. 4
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Table 3 In vivo digestibilities (%; n = 4 per treatment) and energy values of grass hay (GH), control (CON) and experimental concentrate (containing 1%, 3% and 5% condensed tannin (CT) extract1 in ration DM) measured in sheep. GH Mean Organic matter 69.1 Crude protein 62.4 Ether extract 27.6 Crude fibre 73.1 aNDFom 73.2 ADFom 70.3 Energy value (MJ/kg DM) 8.1 ME2 NEL 4.73
CON
CT1
CT3
CT5
P value
SEM
Mean
SEM
Mean
SEM
Mean
SEM
Mean
SEM
0.78 1.18 3.74 0.67 0.94 0.78
a
82.3 83.3a 93.3 37.7a 55.0a 26.7a
0.58 0.89 0.86 1.56 2.47 0.76
a
77.6 78.0a 96.4 23.9a,b 35.4b 4.4b
1.96 2.23 1.71 6.23 5.78 4.23
b
65.1 69.6b 92.3 1.3c 15.9c 0.0b
1.28 0.56 5.54 1.33 4.57 0.0
b
59.0 66.4b 97.0 7.2b 1.8c 0.0b
2.56 1.60 2.79 7.28 1.80 0.0
< .05 < .05 ns < .05 < .05 < .05
0.10 0.07
12.9a 7.98a
0.07 0.06
12.3a 7.51a
0.27 0.21
10.6b 6.28b
0.09 0.01
9.68c 5.63c
0.31 0.22
< .05 < .05
ADFom = ADF expressed exclusive residual ash; aNDFom = NDF assayed with heat-stable amylase and expressed exclusive residual ash; ESOM = enzyme-soluble organic matter; ME = metabolizable energy; NEL = net energy for lactation. a,b,c Means for CON, CT1, CT3 and CT5 in a row with different superscripts are different (P < .05). 1 Acacia mearnsii tannin extract (Weibull Black, Tanac S.A., Montenegro, Brazil). 2 Estimated based on digestible nutrients: ME (MJ) = 0.0312 + DEE + 0.0136 × DCF + 0.0147 × (DOM − DCL − DCF) + 0.00234 × CP (where DCL is digestible (dig.) ether extract, DF is dig. crude fibre and DOM is dig. organic matter, all in g/kg DM; GfE, 2001), conversion into NEL (MJ) according to Weißbach et al. (1996).
Acknowledgements
recommendation (GfE, 1991) over a period of 21 days while the CT3 period in dairy cows lasted 63 days, giving the rumen microbes more time to adapt. Some kind of adaptation taking place in the digestive tract of ruminants when exposed to tannin-containing feed was also observed by Wischer et al. (2014): Plant extracts rich in chestnut tannin temporarily affected processes in the rumen of sheep but did not alter methane release in long-term measurements (190 days). However, nutrient digestibility was decreased for the whole period (Wischer et al., 2014). In literature, only few studies exist addressing the effect of adaptation time on impact of CT supplementation. There is evidence of reaction between tannins and the saliva obtained from browsing goats and sheep, as reviewed by Alonso-Díaz et al. (2010). They state that saliva rich in proline or other tannin-binding proteins might be one of the main adaptive mechanisms expressed by ruminant species against tannins. Ben Salem et al. (2005) recommended a minimum of 24 days as adaptation period for feeding and digestibility trials using tanninrich feeds for ruminants. However, this was the time needed to stabilize intake of the tannin-rich (A. cyanophylla Lidl. leaves + concentrate) diet when offered to lambs while an adaptation in digestive processes (e.g., improvement in OM digestibility) was not observed.
The study was financially supported by Landwirtschaftliche Rentenbank (Z-20039/-7). We thank Ludger Stevens and the team of the Experimental and Educational Centre for Agriculture ‘Haus Riswick’ for excellent support of the digestibility trial and sample collection. Jun.-Prof. Dr. Uta Dickhöfer (University of Hohenheim, Stuttgart) is acknowledged for determination of total phenol, total tannins and condensed tannins. References Alonso-Díaz, M.A., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Hoste, H., 2010. Tannins in tropical tree fodders fed to small ruminants: a friendly foe? Small Rumin. Res. 89, 164–173. Barry, T.N., McNabb, W.C., 1999. The implications of condensed tannins on the nutritive value of temperate forages fed to ruminants. Br. J. Nutr. 81, 263–272. Beauchemin, K.A., McGinn, S.M., Martinez, T.F., McAllister, T.A., 2007. Use of condensed tannin extract from quebracho trees to reduce methane emissions from cattle. J. Anim. Sci. 85, 1990–1996. Ben Salem, H., Nefzaoui, A., Makkar, H.P.S., Hochlef, H., Ben Salem, I., Ben Salem, L., 2005. Effect of early experience and adaptation period on voluntary intake digestion, and growth in Barbarine lambs given tannin-containing (Acacia cyanophylla Lindl. foliage) or tannin-free (oaten hay) diets. Anim. Feed Sci. Technol. 122, 59–77. Bueno, I.C.S., Brandi, R.A., Franzolin, R., Benetel, G., Fagundes, G.M., Abdalla, A.L., Louvandini, H., Muir, J.P., 2015. In vitro methane production and tolerance to condensed tannins in five ruminant species. Anim. Feed Sci. Technol. 205, 1–9. Carulla, J.E., Kreuzer, M., Machmüller, A., Hess, H.D., 2005. Supplementation of Acacia mearnsii tannins decreases methanogenesis and urinary nitrogen in forage-fed sheep. Aust. J. Agric. Res. 56, 961–970. Cenci, F., Louvandini, H., McManus, C., DelĺPorto, A., Costa, D., Araújo S. d. Minho, A., Abdalla, A., 2007. Effects of condensed tannin from Acacia mearnsii on sheep infected naturally with gastrointestinal helminthes. Vet. Parasitol. 144, 132–137. Frutos, P., Hervas, G., Giráldez, F.J., Mantecón, A., 2004a. Review. Tannins and ruminant nutrition. Span. J. Agric. Res. 2, 191–202. Frutos, P., Hervás, G., Giráldez, F.J., Mantecón, A., 2004b. An in vitro study on the ability of polyethylene glycol to inhibit the effect of quebracho tannins and tannic acid on rumen fermentation in sheep goats, cows, and deer. Aust. J. Agric. Res. 55, 1125–1132. Gerlach, K., Pries, M., Tholen, E., Schmithausen, A.J., Büscher, W., Südekum, K.-H., 2018. Effect of condensed tannins in rations of lactating dairy cows on production variables and nitrogen use efficiency. Animal. http://dx.doi.org/10.1017/ S1751731117003639. GfE, 1991. Leitlinien für die Bestimmung der Verdaulichkeit von Rohnährstoffen an Wiederkäuern. J. Anim. Physiol. Anim. Nutr. 65, 229–234. GfE, 2001. Empfehlungen zur Energie- und Nährstoffversorgung der Milchkühe und Aufzuchtrinder. DLG-Verlags-GmbH, Frankfurt a.M., Germany. GfE, 2008. New equations for predicting metabolisable energy of grass and maize products for ruminants. Proc. Soc. Nutr. Physiol. 17, 191–197. GfE, 2009. New equations for predicting metabolisable energy of compound feeds for cattle. Proc. Soc. Nutr. Physiol. 18, 143–146. Grainger, C., Clarke, T., Auldist, M.J., Beauchemin, K.A., McGinn, S.M., Waghorn, G.C., Eckard, R.J., 2009. Potential use of Acacia mearnsii condensed tannins to reduce methane emissions and nitrogen excretion from grazing dairy cows. Can. J. Anim. Sci.
5. Conclusions Supplementation of a CT-rich extract exerted a strong reducing effect on in vivo nutrient digestibility and energy value of concentrates in sheep, even at low concentrations. When focussing on improving the protein value of ruminant rations or decreasing methane emission the possibility of lowered total-tract digestibility and, thus, energy value of the ration, has to be taken into account. The CT concentration in A. mearnsii extracts seems to be very variable (even for the same commercial product) and was much lower than stated. Chemical analysis before use in research and practical on-farm feeding is necessary. Different ruminant species (e.g., sheep and cattle) might react differently when fed with CT which should be examined in further studies, also the effect of different durations of digestibility trials with supplemented CT. Different sources of CT act differently and, e.g., L. corniculatus recommendations might not be applicable for other CT sources.
Conflict of interest The authors have declared that no competing interests exist.
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poly (ethylene glycol) on intake and digestion of tannin-containing leaves (Ceratonia siliqua) by sheep. J. Agric. Food Chem. 42, 2844–2847. Silanikove, N., Gilboa, N., Nir, I., Perevolotsky, A., Nitsan, Z., 1996. Effect of a daily supplementation of polyethylene glycol on intake and digestion of tannin-containing leaves (Quercus calliprinos, Pistacia lentiscus, and Ceratonia siliqua) by goats. J. Agric. Food Chem. 44, 199–205. Smith, A.H., Zoetendal, E., Mackie, R.I., 2005. Bacterial mechanisms to overcome inhibitory effects of dietary tannins. Microb. Ecol. 50, 197–205. Spiekers, H., Menke, A., Pfeffer, E., 2006. Ringversuch zur Bestimmung der Verdaulichkeiten der Rohnährstoffe von Heu, Milchleistungsfutter und Palmkernexpeller an Hammeln. Züchtungskde 78, 384–398. Staerfl, S.M., Zeitz, J.O., Kreuzer, M., Soliva, C.R., 2012. Methane conversion rate of bulls fattened on grass or maize silage as compared with the IPCC default values, and the long-term methane mitigation efficiency of adding acacia tannin garlic, maca and lupine. Agric. Ecosyst. Environ. 148, 111–120. Terrill, T., Rowan, A., Douglas, G., Barry, T., 1992. Determination of extractable and bound condensed tannin concentrations in forage plants, protein concentrate meals and cereal grains. J. Sci. Food Agric. 58, 321–329. VDLUFA, 2012. VDLUFA-Methodenbuch, Bd. III. Die Chemische Untersuchung von Futtermitteln. VDLUFA-Verlag, Darmstadt, Germany. Van Soest, P.J., 1994. Nutritional Ecology of the Ruminant, 2nd ed. Cornell University Press, Ithaca, N.Y. Waghorn, G., 1996. Condensed tannins and nutrient absorption from the small intestine. Proc. Can. Soc. Anim. Sci. 175–194. Waghorn, G., 2008. Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production – progress and challenges. Anim. Feed Sci. Technol. 147, 116–139. Weißbach, F., Schmidt, L., Kuhla, S., 1996. Vereinfachtes Verfahren zur Berechnung der NEL aus der umsetzbaren Energie. Proc. Soc. Nutr. Physiol. 5, 117. Wischer, G., Greiling, A., Boguhn, J., Steingass, H., Schollenberger, M., Hartung, K., Rodehutscord, M., 2014. Effects of long-term supplementation of chestnut and valonea extracts on methane release, digestibility and nitrogen excretion in sheep. Animal 8, 938–948. Zeitz, J.O., Kreuzer, M., 2013. Effect of fermented charcoal on in vitro ruminal fermentation and its interaction with condensed tannins. Proc. Soc. Nutr. Physiol. 22, 141.
89, 241–251. Henke, A., Dickhoefer, U., Westreicher-Kristen, E., Knappstein, K., Molkentin, J., Hasler, M., Susenbeth, A., 2016. Effect of dietary Quebracho tannin extract on feed intake, digestibility, excretion of urinary purine derivatives and milk production in dairy cows. Arch. Anim. Nutr. 1–17. Jones, G.A., McAllister, T.A., Muir, A.D., Cheng, K.-J., 1994. Effects of sainfoin (Onobrychis viciifolia Scop.) condensed tannins on growth and proteolysis by four strains of ruminal bacteria. Appl. Environ. Microbiol. 60, 1374–1378. Kozloski, G.V., Härter, C.J., Hentz, F., de Ávila, S.C., Orlandi, T., Stefanello, C.M., 2012. Intake, digestibility and nutrients supply to wethers fed ryegrass and intraruminally infused with levels of Acacia mearnsii tannin extract. Small Rumin. Res. 106, 125–130. Makkar, H.P.S., 2000. Quantification of Tannins in Tree Foliage-A Laboratory Manual. A joint FAO-IAEA working document, Vienna, Austria, pp. 1–31. Makkar, H.P.S., 2003. Effects and fate of tannins in ruminant animals adaptation to tannins, and strategies to overcome detrimental effects of feeding tannin-rich feeds. Small Rumin. Res. 49, 241–256. Min, B., Barry, T., Attwood, G., McNabb, W., 2003. The effect of condensed tannins on the nutrition and health of ruminants fed fresh temperate forages: a review. Anim. Feed Sci. Technol. 106, 3–19. Mueller-Harvey, I., 2006. Unravelling the conundrum of tannins in animal nutrition and health. J. Sci. Food Agric. 86, 2010–2037. Orlandi, T., Kozloski, G.V., Alves, T.P., Mesquita, F.R., Ávila, S.C., 2015. Digestibility, ruminal fermentation and duodenal flux of amino acids in steers fed grass forage plus concentrate containing increasing levels of Acacia mearnsii tannin extract. Anim. Feed Sci. Technol. 210, 37–45. Piluzza, G., Sulas, L., Bullitta, S., 2014. Tannins in forage plants and their role in animal husbandry and environmental sustainability: a review. Grass Forage Sci. 69, 32–48. Schmithausen, A.J., 2017. On-Farm Research to Quantify Trace Gas Emissions from Dairy Production. Forschungsbericht Agrartechnik des Arbeitskreises Forschung und Lehre der Max-Eyth-Gesellschaft Agrartechnik im VDI (VDI-MEG), Nr. 583. ISSN 09316264. Schneider, B.H., Flatt, W.P., 1975. The Evaluation of Feeds Through Digestibility Experiments. University of Georgia Press, Athens. Silanikove, N., Nitsan, Z., Perevolotsky, A., 1994. Effect of a daily supplementation of
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Effect of condensed tannin supplementation on in vivo nutrient digestibilities and energy values of concentrates in sheep Katrin Gerlacha, Martin Priesb, Karl-Heinz Südekuma, a b
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Institute of Animal Science, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany Chamber of Agriculture of North Rhine-Westphalia, Ostinghausen, 59505 Bad Sassendorf, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords: Digestibility Secondary plant compound Sheep Tannin
The objective of this study was to evaluate the effect of supplemented condensed tannins (CT) from the bark of the Black Wattle tree (Acacia mearnsii) on in vivo nutrient digestibility and energy values measured with sheep under standardized conditions. A commercial A. mearnsii extract (containing 0.203 g CT/g dry matter (DM)) was mixed into a pelleted concentrate. Four treatments consisting of grass hay and concentrate were investigated, containing different concentrations of the CT-rich extract (CON, without CT; CT1, CT3 and CT5, with 1, 3 and 5% CT-rich extract in ration DM), resulting in CT concentrations of 0, 2.03, 6.19 and 10.2 g/kg DM, respectively. In a 21-day period, nutrient digestibility of the concentrates was determined by difference with wethers (n = 4 per treatment, German Blackheaded Mutton) in metabolism crates following a standardized procedure. The organic matter digestibility of the concentrates was unaffected by CT1 and decreased strongly with CT3 (−21%) and CT5 (−28%; P < .05). Digestibility of fibre fractions was already reduced with CT1 (P < .05), representing a very low level of CT supplementation. The concentration of metabolizable energy of the concentrates estimated from digestible nutrients decreased strongly (-25%) from 12.9 (CON) to 9.7 MJ/kg DM (CT5) (P < .05). In conclusion, CT supplementation from A. mearnsii to rations of sheep reduced nutrient digestibilities at much lower levels than previously reported for CT from other sources (e.g., forage legumes).
1. Introduction In ruminant nutrition, the use of secondary plant compounds like condensed tannins (CT) obtained from mostly tropical trees and shrubs as well as from forage legumes has gained some importance in research. The literature about CT with both beneficial and adverse function in ruminants according to their concentration and chemical structure, is vast and often conflicting (Piluzza et al., 2014). Increasing CT concentrations in ruminant rations elevated the amount of undegraded feed protein leaving the rumen, most likely caused by decreased rates of degradation by rumen microorganisms and reduced growth rate of proteolytic bacterial species (Min et al., 2003). Furthermore, CT have improved bodyweight gain, wool production and reproductive efficiency in sheep and reduced the impact of gastro-intestinal parasitism, as reviewed by Waghorn (2008). However, CT in ruminant rations have also been reported to cause a reduction in nutrient digestibility (Frutos et al., 2004a; Silanikove et al., 1994), create negative post-ingestive feedback (Silanikove et al., 1996) and impair animal performance (Grainger et al., 2009). For temperate forages, CT concentrations of 20–45 g/kg dry matter
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(DM) are generally seen as having beneficial effects on ruminant production (Min et al., 2003). However, a more specific contemplation seems necessary. When forages are fed as a sole diet, the CT in birdsfoot trefoil (Lotus corniculatus) have been beneficial for ruminant production, but the CT in sainfoin (Onobrychis ssp.), sulla (Hedysarum coronarium) and big trefoil (L. pedunculatus) do not appear to benefit productivity except for mitigating the impact of parasites (Waghorn, 2008). There is also a considerable variability in concentration and chemical structure of CT especially in forage legumes such that the mode of action and clear effects are difficult to predict (Mueller-Harvey, 2006). As an alternative to forage legumes, industrial products containing defined amounts of tannins (e.g., made from the bark of the Black Wattle tree (Acacia mearnsii)) have been used for supplementation of ruminant rations. However, also with those more standardized products the effects on ruminant performance are equivocal such that there is still a lack of knowledge on clear dose-effect results of CT extracts on ruminants. The aim of this study was to investigate the influence of different levels of a supplemented commercial product rich in CT from A. mearnsii on in vivo nutrient digestibility and energy value of
Corresponding author. E-mail address: [email protected] (K.-H. Südekum).
https://doi.org/10.1016/j.smallrumres.2018.01.017 Received 28 May 2017; Received in revised form 26 January 2018; Accepted 27 January 2018 0921-4488/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Gerlach, K., Small Ruminant Research (2018), https://doi.org/10.1016/j.smallrumres.2018.01.017
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fibre assayed with heat-stable amylase and expressed exclusive residual ash (aNDFom) and acid detergent fibre expressed exclusive residual ash (ADFom) were determined following methods 6.1.1, 6.5.1 and 6.5.2., respectively. Starch was analysed following method 7.2.1 (VDLUFA, 2012). The determination of enzyme-soluble organic matter (ESOM) was done according to method 6.6.1. The Hohenheim gas test (HGT; method 25.1) was conducted for measuring the 24 h in vitro gas production (GP, ml/200 mg DM). The analysis of total phenol and total tannin concentration in the Weibull Black product was conducted using the Folin method (Makkar, 2000) and the concentration of CT was determined with the HCl-butanol method (Terrill et al., 1992). The concentration of metabolizable energy (ME) of the grass hay and the concentrates was calculated according to following equations: For grass hay (GfE, 2008): ME (MJ/kg DM) = 5.51 + 0.0828 × ESOM − 0.00522 × ash + 0.02507 × EE − 0.00392 × ADFom; all expressed as g/kg DM; For concentrate (GfE, 2009): ME (MJ/kg DM) = 7.17 − 0.01171 × ash + 0.00712 × CP + 0.01657 × EE + 0.00200 × starch − 0.00202 × ADFom + 0.06463 × GP (ml/200 mg DM); all expressed as g/kg DM unless stated. Using the amounts and chemical compositions of feed and faeces, nutrient digestibilities were calculated. The digestibilties of the concentrates were calculated as difference between digestibility of hay and digestibility of hay + concentrate. The digestibilities of the proximate constituents were then taken to calculate ME concentration of the concentrate according to GfE (2001): ME (MJ/kg DM) = 0.0312 × DEE + 0.0136 × DCF + 0.0147 × (DOM − DCL − DCF) + 0.00234 × CP, where DEE is digestible ether extract, DCF is digestible crude fibre and DOM is digestible organic matter (all expressed as g/kg DM). The NEL values were estimated from ME according to Weißbach et al. (1996): NEL = ME [0.46 + 12.38 × ME/(1000 − ash)].
concentrates under standardized conditions in sheep. We hypothesized that addition of CT in moderate concentrations of up to 30 g/kg DM would not negatively affect nutrient digestibility in sheep. 2. Material and methods 2.1. Animals and experimental design In June 2013, a digestibility trial with sheep was conducted at the Experimental and Educational Centre for Agriculture ‘Haus Riswick’, Chamber of Agriculture of North Rhine Westphalia, Kleve, Germany (51° 47′ 18N, 6° 8′ 19E) to study the effect of CT supplementation on nutrient digestibility and energy value of concentrates supplemented with CT. The digestibility trial was accompanied by a long-term feeding trial (169 days) with dairy cows that was conducted in a free-stall dairy barn at the same centre. Here, the same CT product was used to study the effect of CT supplementation on performance and N use efficiency in dairy cows (Gerlach et al., 2018) and the effects on gaseous emissions (CH4 and NH3) on dairy barn level (Schmithausen, 2017). The CT product (commercially available) used in both studies was an extract rich in CT made from the bark of A. mearnsii (declared concentration of CT of 0.725 g/g DM, Weibull Black, TANAC S.A., Montenegro, Brazil) and was mixed into the commercially produced pelleted concentrates during processing. The digestibility of the concentrates supplemented with different amounts of CT-rich extract was measured with four male wethers (German Blackheaded Mutton) per treatment according to GfE (1991). As it is not possible to feed concentrates as a single feed, their digestibility was determined by difference by feeding them together with a forage of known digestibility and deducting the estimated effect of the latter in the calculations as described by Schneider and Flatt (1975) and GfE (1991). Rations consisted of chopped grass hay and the different concentrates. The proportions were chosen to achieve concentrations of the CT-rich extract of 0 (CON), 1% (CT1), 3% (CT3) of ration DM in accordance with the dairy cow trial; additionally, a treatment with 5% CT-rich extract (CT5) of ration DM was tested. According to GfE (1991) one group received a ration consisting of grass hay only to determine the digestibility and feeding value of the hay. Composition of the different concentrates is given in Table 1 and the composition of the grass hay as well as of rations including the CT concentration is presented in Table 2. Lower concentrations of crude protein (CP), ether extract (EE), starch and fibre fractions in the supplemented concentrates were caused by the addition of the CT-rich extract (dilution effect). Each ration was offered to four wethers in two meals per day. A 14-d adaptation period was followed by a 7-d collection period where animals were kept in metabolism crates and all faeces and feed refusals were collected on a daily basis and aliquots (20% of daily amount) were stored at −18 °C. Samples of the hay and the concentrates were collected daily and a cumulative sample was added up for analyses. Samples were stored at −20 °C until analysis for chemical composition and estimation of energy value. At the end of each collection period composite samples of ration ingredients as well as of faeces spanning the entire 7-d period were prepared, freeze dried and analysed chemically.
2.3. Statistical analyses Statistical analyses were conducted using SAS 9.3. Data of the digestibility trial with sheep were analysed using one-way ANOVA. The Tukeys Honestly Significant Difference test (Tukey-HSD) was applied for post hoc comparisons. For all analyses, differences were considered significant with P ≤ .05. 3. Results When measured with the Folin method (Makkar, 2000), the total phenol concentration in the Weibull Black product was 0.630 g/g DM and the total tannin concentration was 0.568 g/g DM. Therefore, the total tannin concentrations in rations were 5.68, 17.3 and 28.6 g/kg DM for CT1, CT3 and CT5, respectively. The concentration of CT determined with the HCl-butanol method (Terrill et al., 1992) was 0.203 g/g DM. The low CT concentration in the extract resulted in dietary CT concentrations of 2.03, 6.19 and 10.2 g/kg DM for CT1, CT3 and CT5, respectively (Table 2). The digestibility trial with wethers could be conducted without health problems, decreases in feed intake or changes in texture of faeces. Supplementation of the CT-rich extract did not cause feed avoidance or other irregularities in feeding behaviour. In Table 3, the nutrient digestibilities and energy values of the concentrates estimated from digestible nutrients are presented. The in vivo digestibility of organic matter of the concentrates was unaffected by CT1 and decreased strongly with CT3 (−21%) and CT5 (-28%; P < .05). The decrease was even more pronounced for digestibility of aNDFom, but with a large variation among animals. For aNDFom and ADFom, the digestibility was already reduced with CT1 (P < .05), whereas other constituents were only affected with CT3 and CT5. The ME concentration of the concentrates estimated from digestibility of proximate constituents decreased markedly (−25%) from 12.9 to 9.7 MJ/kg DM (P < .05), for
2.2. Chemical analyses and calculations Analysis of the chemical composition of the ration components and faeces samples was done by the Landwirtschaftliche Kommunikationsund Service GmbH (Lichtenwalde, Germany). The DM concentration of faeces was determined daily using a two-step procedure involving predrying samples at 60 °C, followed by oven-drying at 105 °C. Proximate analyses were done according to VDLUFA (2012) and method numbers are given below. Ash, EE and CP were analysed using methods 8.1, 5.1 and 4.1.1. The concentrations of crude fibre (CF), neutral detergent 2
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Table 1 Ingredient and chemical composition of the concentrates used in the digestibility trial (expressed as g/kg dry matter (DM) unless stated otherwise) with sheep in control (CON) and experimental group supplemented with 1% (CT1), 3% (CT3) or 5% in ration DM (CT5) condensed tannin extract.a
Ingredients (g/kg) Rapeseed meal Maize grain Wheat grain Vegetable fat Molasses Urea Tannin extractb Chemical composition DM (g/kg) Ash Crude protein Ether extract Crude fibre Starch aNDFom ADFom GP (ml/200 mg DM) ESOM MEb (MJ/kg DM) NEL (MJ/kg DM)
Concentrate CON
Concentrate CT1
Concentrate CT3
Concentrate CT5
561 249 125 25 31 9 –
546 243 121 24 30 8 27
518 230 115 23 29 8 76
493 219 110 22 27 8 121
890 64 287 61 96 306 246 120 52.2 825 13.2 8.39
900 64 283 61 92 290 232 124 50.6 826 13.0 8.25
899 68 266 60 90 245 232 129 47.4 816 12.6 7.87
899 62 291 49 83 253 190 115 42.7 833 12.4 7.70
aNDFom = NDF assayed with heat-stable amylase and expressed exclusive residual ash; ADFom = ADF expressed exclusive residual ash; GP = 24 h gas production; ESOM = enzymesoluble organic matter; ME = metabolizable energy; NEL = net energy for lactation. a Acacia mearnsii tannin extract (Weibull Black, Tanac S.A., Montenegro, Brazil). b Estimated according to GfE (2009).
As expected, formation of tannin-protein-complexes seems to have occurred in this study. It is suggested that binding to CT and building of complexes persisted also postruminally such that digestibility of supplemented concentrates decreased. Not only nutrient degradation in the rumen might be affected by dietary CT but also digestion in the small and even large intestine. As summarized by Frutos et al. (2004a) decreased nutrient absorption from the intestine might be caused by persistence of tannin-protein complexes in the intestine which failed to dissociate in the abomasum, formation of tannin-digestive enzyme complexes or new tannin-dietary protein complexes, or changes in the intestinal absorption due to the interaction of tannins with intestinal mucosa. Furthermore, it can be assumed that rapeseed meal which was a main ingredient of the concentrates contains considerable amounts of protein that is bound to the cell wall which is only available for the ruminant after microbial degradation and fermentation of the fibre. Because of the limited fermentation in the rumen the protein may not have been digested postruminally. Ruminants can only benefit from CT when the increases in protein flow from the rumen exceed the reduction in the absorption of amino acids from the intestine (Waghorn, 1996). With drastically decreased total-tract digestibility of OM and CP this requirement is not met. The aNDFom digestibilities decreased to an even larger extent than that of the other feed fractions; therefore it can be assumed that not only protein but also fibre fractions were bound to tannins resulting in decreased degradability. This is in agreement with Henke et al. (2016) who observed strongly reduced digestibilities of OM and, even more pronounced, fibre fractions in dairy cows after addition of a Quebracho CT extract. As summarized by Frutos et al. (2004a) tannins mainly exert their effects on proteins, however, they also have effects on carbohydrates, namely hemicellulose, cellulose, starch and pectins. Different studies have shown that fibre degradation in the rumen can be drastically reduced in animals that consume tannin-rich feeds (Barry and McNabb, 1999; Staerfl et al., 2012). For example, digestibility of NDF decreased from 55.2 to 32.5% when a CT-rich extract from A. mearnsii (30 g/kg DM) was added to maize silage-based rations fed to five-month-old fattening bulls (Staerfl et al., 2012). However, with 11-month-old bulls offered the same ration, no effect of CT treatment occurred (NDF digestibility 54.4 vs. 45.1%, for control and treatment, P > .05). Orlandi et al. (2015) reported that not only the
CON and CT5, respectively. The in vitro GP of rations with 1, 3 and 5% CT supplementation was reduced by 3.0, 9.2 and 18.2% which exceeds the CT concentration in the ration. The ESOM, as a method based on enzymatic procedures, was not affected by addition of CT. 4. Discussion The CT concentrations in the extract were lower than stated but on a comparable level with Kozloski et al. (2012) and Cenci et al. (2007), whereas Carulla et al. (2005) using the same and Grainger et al. (2009) using a different A. mearnsii extract stated much higher CT concentrations (0.615 and 0.603 g/g DM). It shows that CT concentrations are variable even within the same commercial product and analysis before use in feeding trials is necessary. Not only the product concentration of supplemented extracts but also the CT concentrations and methods used for the determination have to be stated for a better comparison of trials and understanding of results. Results suggest that the CT supplementation negatively impacted in vivo nutrient digestibility and microbial activity measured in the in vitro system HGT, whereas the in vitro enzymatic degradability (ESOM) was not affected. Plant extracts containing CT are able to impair ruminal growth of bacteria strains that are involved in proteolysis (Jones et al., 1994). The decreased GP is in coincidence with results presented by Zeitz and Kreuzer (2013) where in vitro GP was reduced by 8.1% and volume of methane by 14.0%, when 30 g A. mearnsii extract/kg DM was added to a ration consisting of grass hay, barley grain and soybean meal in comparison to a control ration without CT. The in vivo digestibility of OM in concentrates measured with wethers was drastically reduced (82.2% for control vs. 58.9% for CT5) suggesting a strong impairment of ruminal microbial digestion and fermentation. Waghorn (2008) summarized that the impact of CT on intestinal function is not completely understood in ruminants but it is assumed that tannins inhibit the capability of endogenous enzymes to split proteins into peptides and amino acids, and also impede their absorption. Endogenous enzymatic activity exceeds requirements for proteolysis, but CT bound to either bacterial surfaces or to proteins may reduce the enzymatic access and activity (Waghorn, 2008). 3
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Table 2 Composition of rations used in the digestibility trial for sheep in control (CON) and experimental group supplemented with 1% (CT1), 3% (CT3) or 5% in ration dry matter (DM) (CT5) condensed tannin (CT) extract.a
Amount of hay (g/d) Amount of concentrate (g/d) Type of concentrate CT extract in ration (g/kg DM) Total tannins in ration (g/kg DM) CT in ration (g/kg DM) DM (g/kg) Crude ash (g/kg DM) Crude protein (g/kg DM) Ether extract (g/kg DM) Crude fibre (g/kg DM) aNDFom (g/kg DM) ADFom (g/kg DM) ME (MJ/kg DM)b
Hay
CON
CT1
CT3
CT5
1000 – – – – – 872 88.0 109 14 268 567 303 8.1
400 600 Concentrate CON – – – 883 73.6 216 42 165 374 193 11.2
650 350 Concentrate CT1 10.0 5.68 2.03 882 79.6 170 30 206 450 240 9.8
640 360 Concentrate CT3 30.5 17.3 6.19 882 80.8 166 31 204 446 240 9.7
630 370 Concentrate CT5 50.4 28.6 10.2 882 78.4 176 27 200 428 233 9.7
aNDFom = NDF assayed with heat-stable amylase and expressed exclusive residual ash; ADFom = ADF expressed exclusive residual ash; ME = metabolizable energy. a Acacia mearnsii tannin extract (Weibull Black, Tanac S.A., Montenegro, Brazil). b Estimated based on chemical composition and enzyme-soluble organic matter (grass hay; GfE, 2008) or in vitro gas production (concentrate; GfE, 2009).
Surprisingly, there was a large variation among wethers resulting in high standard errors of mean digestibility values, especially for aNDFom. Commonly these kinds of digestibility trials have a small variation between animals and a high repeatability (Spiekers et al., 2006). Apparently there were high individual differences between animals in the adaptability to CT and the ability of the digestive tract to dissolve the CT complexes, at least for the specific CT source tested here. Similar observations were made by Staerfl et al. (2012) who supplemented bulls with A. mearnsii extract and described a high standard error of the results that was especially due to the high variation between animals within treatment group. Obviously, when the digestibility trial with sheep is compared with the feeding trial with dairy cows (Gerlach et al., 2018), the CT supplementation exerted more pronounced effects on sheep than on dairy cows. In dairy cows, only minor changes (small shift in N excretion from urine to faeces, reduced milk protein yield) occurred when animals were supplemented with CT3 for a period of 63 days whereas milk yield and other production variables were unaffected. These results are somehow surprising, as recent research by Bueno et al. (2015) using in vitro gas production techniques and different ruminant species as inoculum donors has shown that CT had greater effects in large ruminants than in small ruminants. They hypothesized that there is less microbial adaptation to CT in grazing bovids compared to other species. As cattle are grazers they do not consume a lot of tanniniferous plants in their natural or typical rations (Bueno et al., 2015). Therefore they do not have as strong a genetic capability to ferment plant material containing secondary compounds as ruminants commonly consuming more browse (Van Soest, 1994). Also Frutos et al. (2004b) showed that CT reduced in vitro GP, with differences depending on the inoculum donor (sheep, goats, cows and deer, given the same feed). Authors stated that ruminants are known to differ in their capacity to tolerate or degrade plant secondary metabolites and effects of tannins on in vitro incubations might differ when rumen fluid is derived from animals that consume tannin-rich diets seldom or regularly. Possibly not only origin and composition of the rumen fluid but also species-related factors like function and structure of the digestive tract impact the mechanism and effects of tannins such that, for the special case of CT, transfer of experimental results from one ruminant species to the other has to be examined. Rumen microbes can adapt to tanniniferous diets by increasing the proportion of tannin-resistant bacteria in the rumen and therefore mitigating the inhibitory effects of these secondary plant compounds (Smith et al., 2005). Adaptive changes in the rumen might be another possible explanation for the diverse result in sheep and cattle in this study. The digestibility trial was conducted according to the
apparent and true digestibility of N compounds by steers but also totaltract OM and aNDF digestibility tended to linearly decrease at incremental tannin extract inclusion levels (CT from A. mearnsii; 9, 18 and 27 g/kg DM). The tendency for reduced OM digestibility suggests that, with increasing CT concentrations, there was a decrease in the supply of digestible energy. The effect on ME concentration of rations calculated from digestible nutrients was even more pronounced in the current study, with a decrease in ME of 25% when comparing CON and CT5. Similar strong effects were observed by Kozloski et al. (2012) who infused an A. mearnsii extract intraruminally at dosages of 0, 20, 40, 60 g/ kg DM to wethers offered ryegrass for ad libitum intake. Intake and digestibility of OM, NDF and N [(N intake (g/d) – faecal neutral detergent insoluble N (g/d))/N intake (g/d)] were linearly reduced at increasing levels of tannin infusions. However, reduced fibre digestibilities with rations containing CT can also be caused by analytical difficulties as the detergent extraction techniques might not predict or determine the in vivo digestibility of cell-wall constituents accurately which was shown to be due to the presence of tannin-protein complexes as artefacts in fibre fractions, especially in samples of faeces (Makkar, 2003). The supplemented CT exerted a strong negative impact on OM digestibility in sheep, although CT concentrations were lower than critical values reported in literature where negative impacts are commonly only expected at CT concentrations exceeding 55 g/kg DM (Beauchemin et al., 2007; Grainger et al., 2009; Min et al., 2003). They did not even match reported concentrations (e.g. Min et al. (2003); 20–45 g CT/kg DM) of being beneficial in forages (reduced protein degradation in the rumen, increased milk production, wool growth and lambing percentage, reduced bloat risk and internal parasites). These assumptions are based on recommendations originating from feeding trials with Lotus species and may not be applicable to other CT sources (Mueller-Harvey, 2006). Obviously, a more detailed analysis of CT structure is needed to better understand diverging effects of CT supplementation. Furthermore also the ration that is supplemented with CT might influence the functionality of the tannins: Waghorn (2008) summarized that the impact of CT upon ruminant performance will depend on the amount and astringency in the diet, animal nutrient requirements and other dietary components such that a clear, easy relation between CT in rations and animal performance hardly exists. In this regard it has to be considered that CT-supplemented rations had slightly higher concentrations of fibre fractions than CON which might also have caused a small decline in digestibility. However, the CTsupplemented rations (CT1, CT3 and CT5) had nearly equal concentrations of fibre fractions such that the observed reduction in digestibility can be related to the CT. 4
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Table 3 In vivo digestibilities (%; n = 4 per treatment) and energy values of grass hay (GH), control (CON) and experimental concentrate (containing 1%, 3% and 5% condensed tannin (CT) extract1 in ration DM) measured in sheep. GH Mean Organic matter 69.1 Crude protein 62.4 Ether extract 27.6 Crude fibre 73.1 aNDFom 73.2 ADFom 70.3 Energy value (MJ/kg DM) 8.1 ME2 NEL 4.73
CON
CT1
CT3
CT5
P value
SEM
Mean
SEM
Mean
SEM
Mean
SEM
Mean
SEM
0.78 1.18 3.74 0.67 0.94 0.78
a
82.3 83.3a 93.3 37.7a 55.0a 26.7a
0.58 0.89 0.86 1.56 2.47 0.76
a
77.6 78.0a 96.4 23.9a,b 35.4b 4.4b
1.96 2.23 1.71 6.23 5.78 4.23
b
65.1 69.6b 92.3 1.3c 15.9c 0.0b
1.28 0.56 5.54 1.33 4.57 0.0
b
59.0 66.4b 97.0 7.2b 1.8c 0.0b
2.56 1.60 2.79 7.28 1.80 0.0
< .05 < .05 ns < .05 < .05 < .05
0.10 0.07
12.9a 7.98a
0.07 0.06
12.3a 7.51a
0.27 0.21
10.6b 6.28b
0.09 0.01
9.68c 5.63c
0.31 0.22
< .05 < .05
ADFom = ADF expressed exclusive residual ash; aNDFom = NDF assayed with heat-stable amylase and expressed exclusive residual ash; ESOM = enzyme-soluble organic matter; ME = metabolizable energy; NEL = net energy for lactation. a,b,c Means for CON, CT1, CT3 and CT5 in a row with different superscripts are different (P < .05). 1 Acacia mearnsii tannin extract (Weibull Black, Tanac S.A., Montenegro, Brazil). 2 Estimated based on digestible nutrients: ME (MJ) = 0.0312 + DEE + 0.0136 × DCF + 0.0147 × (DOM − DCL − DCF) + 0.00234 × CP (where DCL is digestible (dig.) ether extract, DF is dig. crude fibre and DOM is dig. organic matter, all in g/kg DM; GfE, 2001), conversion into NEL (MJ) according to Weißbach et al. (1996).
Acknowledgements
recommendation (GfE, 1991) over a period of 21 days while the CT3 period in dairy cows lasted 63 days, giving the rumen microbes more time to adapt. Some kind of adaptation taking place in the digestive tract of ruminants when exposed to tannin-containing feed was also observed by Wischer et al. (2014): Plant extracts rich in chestnut tannin temporarily affected processes in the rumen of sheep but did not alter methane release in long-term measurements (190 days). However, nutrient digestibility was decreased for the whole period (Wischer et al., 2014). In literature, only few studies exist addressing the effect of adaptation time on impact of CT supplementation. There is evidence of reaction between tannins and the saliva obtained from browsing goats and sheep, as reviewed by Alonso-Díaz et al. (2010). They state that saliva rich in proline or other tannin-binding proteins might be one of the main adaptive mechanisms expressed by ruminant species against tannins. Ben Salem et al. (2005) recommended a minimum of 24 days as adaptation period for feeding and digestibility trials using tanninrich feeds for ruminants. However, this was the time needed to stabilize intake of the tannin-rich (A. cyanophylla Lidl. leaves + concentrate) diet when offered to lambs while an adaptation in digestive processes (e.g., improvement in OM digestibility) was not observed.
The study was financially supported by Landwirtschaftliche Rentenbank (Z-20039/-7). We thank Ludger Stevens and the team of the Experimental and Educational Centre for Agriculture ‘Haus Riswick’ for excellent support of the digestibility trial and sample collection. Jun.-Prof. Dr. Uta Dickhöfer (University of Hohenheim, Stuttgart) is acknowledged for determination of total phenol, total tannins and condensed tannins. References Alonso-Díaz, M.A., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Hoste, H., 2010. Tannins in tropical tree fodders fed to small ruminants: a friendly foe? Small Rumin. Res. 89, 164–173. Barry, T.N., McNabb, W.C., 1999. The implications of condensed tannins on the nutritive value of temperate forages fed to ruminants. Br. J. Nutr. 81, 263–272. Beauchemin, K.A., McGinn, S.M., Martinez, T.F., McAllister, T.A., 2007. Use of condensed tannin extract from quebracho trees to reduce methane emissions from cattle. J. Anim. Sci. 85, 1990–1996. Ben Salem, H., Nefzaoui, A., Makkar, H.P.S., Hochlef, H., Ben Salem, I., Ben Salem, L., 2005. Effect of early experience and adaptation period on voluntary intake digestion, and growth in Barbarine lambs given tannin-containing (Acacia cyanophylla Lindl. foliage) or tannin-free (oaten hay) diets. Anim. Feed Sci. Technol. 122, 59–77. Bueno, I.C.S., Brandi, R.A., Franzolin, R., Benetel, G., Fagundes, G.M., Abdalla, A.L., Louvandini, H., Muir, J.P., 2015. In vitro methane production and tolerance to condensed tannins in five ruminant species. Anim. Feed Sci. Technol. 205, 1–9. Carulla, J.E., Kreuzer, M., Machmüller, A., Hess, H.D., 2005. Supplementation of Acacia mearnsii tannins decreases methanogenesis and urinary nitrogen in forage-fed sheep. Aust. J. Agric. Res. 56, 961–970. Cenci, F., Louvandini, H., McManus, C., DelĺPorto, A., Costa, D., Araújo S. d. Minho, A., Abdalla, A., 2007. Effects of condensed tannin from Acacia mearnsii on sheep infected naturally with gastrointestinal helminthes. Vet. Parasitol. 144, 132–137. Frutos, P., Hervas, G., Giráldez, F.J., Mantecón, A., 2004a. Review. Tannins and ruminant nutrition. Span. J. Agric. Res. 2, 191–202. Frutos, P., Hervás, G., Giráldez, F.J., Mantecón, A., 2004b. An in vitro study on the ability of polyethylene glycol to inhibit the effect of quebracho tannins and tannic acid on rumen fermentation in sheep goats, cows, and deer. Aust. J. Agric. Res. 55, 1125–1132. Gerlach, K., Pries, M., Tholen, E., Schmithausen, A.J., Büscher, W., Südekum, K.-H., 2018. Effect of condensed tannins in rations of lactating dairy cows on production variables and nitrogen use efficiency. Animal. http://dx.doi.org/10.1017/ S1751731117003639. GfE, 1991. Leitlinien für die Bestimmung der Verdaulichkeit von Rohnährstoffen an Wiederkäuern. J. Anim. Physiol. Anim. Nutr. 65, 229–234. GfE, 2001. Empfehlungen zur Energie- und Nährstoffversorgung der Milchkühe und Aufzuchtrinder. DLG-Verlags-GmbH, Frankfurt a.M., Germany. GfE, 2008. New equations for predicting metabolisable energy of grass and maize products for ruminants. Proc. Soc. Nutr. Physiol. 17, 191–197. GfE, 2009. New equations for predicting metabolisable energy of compound feeds for cattle. Proc. Soc. Nutr. Physiol. 18, 143–146. Grainger, C., Clarke, T., Auldist, M.J., Beauchemin, K.A., McGinn, S.M., Waghorn, G.C., Eckard, R.J., 2009. Potential use of Acacia mearnsii condensed tannins to reduce methane emissions and nitrogen excretion from grazing dairy cows. Can. J. Anim. Sci.
5. Conclusions Supplementation of a CT-rich extract exerted a strong reducing effect on in vivo nutrient digestibility and energy value of concentrates in sheep, even at low concentrations. When focussing on improving the protein value of ruminant rations or decreasing methane emission the possibility of lowered total-tract digestibility and, thus, energy value of the ration, has to be taken into account. The CT concentration in A. mearnsii extracts seems to be very variable (even for the same commercial product) and was much lower than stated. Chemical analysis before use in research and practical on-farm feeding is necessary. Different ruminant species (e.g., sheep and cattle) might react differently when fed with CT which should be examined in further studies, also the effect of different durations of digestibility trials with supplemented CT. Different sources of CT act differently and, e.g., L. corniculatus recommendations might not be applicable for other CT sources.
Conflict of interest The authors have declared that no competing interests exist.
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