A G R I C U LT U R A L A N D F O O D S C I E N C E Agricultural and Food Science (2022) 31: 260–281 260 https://doi.org/10.23986/afsci.115262 Chemical treatment of straw for ruminant feeding with NaOH or urea – investigative steps via practical application under current European Union conditions Siriwan D. Martens1, Vicki Wildner1,2, Johanna Schulze1,2, Wolfram Richardt3, Jörg M. Greef4, Annette Zeyner2 and Olaf Steinhöfel1,2 1Department of Animal Husbandry, Saxon State Office for Environment, Agriculture and Geology (LfULG), Am Park 3, 04886 Köllitsch, Germany 2Institute of Agricultural and Nutritional Sciences, Martin Luther University Halle-Wittenberg, Theodor-Lieser-Str. 11, 06120 Halle (Saale), Germany 3Landwirtschaftliche Kommunikations- und Servicegesellschaft mbH (LKS), August-Bebel-Str. 6, 09577 Niederwiesa, Germany 4Institute for Crop and Soil Science, Julius Kühn-Institut (JKI), Bundesallee 58, 38116 Braunschweig, Germany e-mail: siriwan.martens@smekul.sachsen.de Weather extremes in parts of Europe have led to a renewed search for alternative feeds for ruminants. Cereal straw presents one source of fibre, which is hard to digest due to its lignin-carbohydrate complexes. Chemical and bio- logical treatments have been investigated to improve digestibility. Here, the applicability of alkaline treatments for farming conditions under EU legislation and their efficacy were checked. Thus, we tested caustic soda (60, 120 g kg-1straw) and urea (15, 30, 45, 60 g kg-1straw without and with urease addition) applications both at laboratory scale and using a mixer-wagon. The nutritive value was evaluated analyzing chemical parameters including fibre compo- nents and estimating in vitro digestibility. The in vitro digestibility indicated by gas production, enzymatically soluble substrate and neutral detergent fibre digestibility (30h) was highest for the NaOH treatments, which did not differ by dose. Remoistening the straw to 600 g DM kg-1 was a precondition for the effectiveness of both treatments. Ure- ase addition enhanced the intended ammonification when urea was applied at ≥ 30 g kg-1. An ambient temperature for urea treatment ≥ 25 °C was necessary and had to be maintained for at least 14 d post treatment. The determi- nation of crude ash in NaOH treated feeds by the standard procedure and time overestimated the mineral fraction and had to be modified. This systematic approach provides guidance for feasible straw treatments for EU farmers. However, trials for feed acceptance and in vivo digestibility are needed to demonstrate the real effect in animals. Key words: wheat straw, urease, fibre components, in vitro digestibility, gas production Introduction Lack of roughage for ruminants due to drought in parts of Europe in 2018 and 2019 has reactivated the search for fibre sources other than “traditional” forages such as grass. Cereal straw is widely available throughout the year in Europe. The continent had a share of almost 35 % of the world wheat straw production with 213 million tonnes in 2019 (calculated with a grain to straw ratio of 1:0.8; FAO 2021). However, the main obstacle for extensive straw use in animal feeding is its low digestibility due to the high lignin content (Jung 1989, Adesogan et al. 2019). The plant cell wall is a very complex construction. Cellulose, hemicellulose and lignin are interlinked (Lee et al. 2014). By special treatments these bonds can be loosened so that cellulose and hemicellulose are more accessi- ble to the rumen microflora (Jung and Deetz 1993). The objective of physical, chemical and biological treatments, alone or in combination, is either to increase feed intake, digestibility or both, especially for ruminants (Flachowsky 1987). In contrast to the more recent developments aimed at bioethanol production from straw (Yoswathana et al. 2010), this does not include the conversion of cellulose to sugar prior to the rumen. In the 1980s intensive research efforts were directed towards chemical treatment of straw in Germany (Fla- chowsky 1987, Ochrimenko et al. 1987, Schneider et al. 1987). Research and implementation worldwide have been reported (e.g. Owen et al. 2012, Adesogan et al. 2019). Even though some of those approaches were prom- ising, under the current environmental regulations of the EU, the way NaOH or NH 3 were applied in the past is not now feasible. For example, NaOH was predominantly washed out of the straw resulting in the production of contaminated residual water as a by-product. Ammonia was often applied in a gaseous form contributing to gas Received 4 March 2022 / Accepted 7 December 2022 The Scientific Agricultural Society of Finland ©This is an open access article under the CC BY 4.012 S.D. Martens et al. 261 emissions. In the European Union Register of Feed Additives, NaOH is currently listed in the Annex I (released on 21 January 2021) as an acidity regulator for cats, dogs and ornamental fish (Code 1j524) (EC 2021). However, in the sense of the specific use here, it can be defined as a processing aid (EC 2003). On the other hand, feed grade urea is registered for ruminants (EC 2012). The first objective of these trials was to test alternative methods of ap- plying NaOH and urea with regard to their applicability on farm. A secondary objective was to identify simple and inexpensive laboratory indicators for in vitro digestibility to allow for a screening of a range of treatments. It was hypothesized that dosage, the humidity of the straw, and in the case of urea also storage temperature, duration and pressure plus the use of an external urease would contribute to the increase of in vitro digestibility of cereal straw when treated with either NaOH or urea . Material and methods Winter wheat straw was harvested in July 2018 in Köllitsch, Northern Saxony, Germany, with excellent sensory quality because of preceding dry weather conditions and stored as square bales in a dry environment. Trial 1 – NaOH addition During the first half of 2019, caustic soda (NaOH microbeads, technical grade, WHC GmbH, Hilgertshausen, Germa- ny) was dissolved in tap water to obtain the respective concentrations and masses to be added to the dry wheat straw, following the Sodagrain process of Orskov et al. 1979. Personal protection measures recommended by the manufacturer such as protective gloves and eyewear as well as sufficient fresh air circulation were followed. In June 2019, the following treatments were applied, partly derived from Flachowsky et al. 1984 and Block et al. 1985, in triplicates: 1. Control (air dry straw, ~880 g DM kg-1) 2. 60 g NaOH kg-1 air-dry straw + H 2 O to remoisten the straw to 600 g kg-1 dry matter (DM) 3. 60 g NaOH kg-1 air-dry straw + H 2 O to remoisten the straw to 450 g kg-1 DM 4. 120 g NaOH kg-1 air-dry straw + H 2 O to remoisten the straw to 600 g kg-1 DM 5. 120 g NaOH kg-1 air-dry straw + H 2 O to remoisten the straw to 450 g kg-1 DM The dissolved NaOH was added to 1.0 kg of chopped straw (2 − 5 cm) in a concrete mixer (120 l, 550 W, 230 V; CM120L, HORNBACH Baumarkt AG, Bornheim, Germany). The straw was mixed for 5 min. The pH and DM con- tent at 105 °C (for > 12 h) were determined. The straw was filled into plastic bags (120 l, 69 cm × 107 cm, poly- ethylene), which were kept open. Each treatment was stored for either 2 or 7 d at ambient temperature and then frozen to −20 °C for further analysis. Weight was determined before and after storage. Trial 2 – Urea addition with loose or compact storage In March 2020, in a second approach, urea (feed grade, 90 %)(SALVANA Tiernahrung GmbH, Klein Offenseth-Spar- rieshoop) without or with urease (MAXAMMON®, Harbro Ltd, Turriff, Aberdeenshire, UK) was applied. The ure- ase product contained a mixture of extruded soybeans, pea meal and a by-product of Aspergillus niger. For each treatment, a self-propelled diet feeder (SILOKING, 12 m³, 2006, SILOKING Mayer Maschinenbau GmbH, Tittmon- ing, Germany) was filled with 100 kg air dry wheat straw (~880 g DM kg-1). The dry ingredients were added first, followed by water. The following treatments were applied: 1. 1.5 kg urea 100 kg-1 air-dry straw, the straw remoistened to 800 g DM kg-1 2. 1.5 kg urea + 0.5 kg Maxammon per 100 kg air-dry straw, the straw remoistened to 800 g DM kg-1 (following the recommendations of the Maxammon manufacturer) 3. 1.5 kg urea 100 kg-1 air-dry straw, the straw remoistened to 600 g DM kg-1 4. 1.5 kg urea + 0.5 kg Maxammon per 100 kg air dry straw, the straw remoistened to 600 g DM kg-1 Agricultural and Food Science (2022) 31: 260–281 262 The straw was subsequently mixed thoroughly for 5 min. The mixture was divided in two parts: one part was loose- ly (L) filled into plastic bags (120 l, 69 cm × 107 cm, polyethylene), closed with a rubber ring (around 6 and 8 kg treated straw per bag for higher and lower DM), and the other part was compacted (C) by feet in drums of 120 l and closed with an airtight lid (around 14 and 19 kg treated straw drum-1 for higher and lower DM). A data logger for temperature (TG-4080; Gemini Data Loggers Ltd, Chichester, UK) was enclosed in each of the triplicates. The straw was stored at about 25 °C for 14 d. Trial 3 – Increasing urea addition with two storage temperatures This trial resembled Trial 2. However, we remoistened the straw to 500 g kg-1 DM in June 2020. The treatments were applied in laboratory scale by dissolving the ingredients in the liquid first and then applied to the air-dried straw, which was mixed by hand, wearing chemical protection gloves: 1. 33 g urea (90%) kg-1 air-dried straw, dissolved in water 2. As 1), +5 g Maxammon kg-1 air-dried straw 3. 50 g urea (90%) kg-1 air-dried straw, dissolved in water 4. As 3), +5 g Maxammon kg-1 air-dried straw 5. 67 g urea (90%) kg-1 air-dried straw, dissolved in water 6. As 5), +5 g Maxammon kg-1 air-dried straw About 1 kg of treated straw was packed into side seal bags (50cm × 30 cm, 90 μm, polyethylene) in triplicates and closed using rubber rings. The straw was stored at about 25 °C and 40 °C, respectively for 14 d and 28 d respectively, resulting in 24 treat- ments in total. Sufficient ventilation was provided, especially at opening of the treatments. An overview of the chemical treatments is given in Table 1. Chemical analysis Samples of untreated and treated straw were analyzed for DM (at 105 °C for 12 h), crude ash, neutral detergent fibre assayed with a heat stable amylase and expressed exclusive of residual ash (aNDFom), acid detergent fibre expressed exclusive of residual ash (ADFom), acid detergent lignin (ADL), crude fat (EE), crude protein (CP), enzy- matically soluble organic substance (ELOS), gas production according to the Hohenheim Gas Test (HFT). Analytical methods of the Association of German Agricultural Analytic Research Institutes (VDLUFA 1976) were applied (for the principle of the methods and further references see Appendix 1). The parameter aNDFom digestibility was de- termined after 30 h (NDFD 30h ). The latter is using the DAISY II-Incubator® (ANKOM Technology, Macedon NY, USA) applying the In Vitro True Digestibility Procedure suggested by ANKOM implemented by the Landwirtschaftliche Kommunikations GmbH Lichtenwalde (LKS) (LKS FMUAA 223, Rev. 02/02/2018) (ANKOM 2006, Goeser and Combs 2009). Standard time for NDFD determination by LKS is 30 h. As extraordinary high crude ash values were deter- mined in NaOH treated samples at the beginning applying the standard procedure for 3 h, subsequently different time periods for ashing were tested for 15 selected samples treated with NaOH. Wheat straw treated with 120 g NaOH kg-1DM with varying amounts of water and storage duration were subjected to different ashing times in Table 1. Chemical treatments in the different trials Trial 1 Trial 2 Trial 3 Reagent Substance NaOH Urea (feed grade) Urea (feed grade) Application rate 60; 120 g kg-1 straw 15 g kg-1 straw ± urease 30; 45; 60 g kg-1 straw ± urease Straw Target DM (g kg-1) 600; 450 600; 800 500 Storage Ambient °C 22 °C 25 °C 25; 40 °C Sampling days after treatment 2; 7 14 14; 28 S.D. Martens et al. 263 a muffle furnace and compared: 3 h, 13 h, 17.5 h and 24 h. Additionally a combination of ashing +NH 4 NO 3 (solution of 20 % w/v as described in VDLUFA III, 8.1 [paragraph 7.1]) was applied after 6 h and 9 h in the muffle furnace. As one aim was to identify simple analytical parameters, which have a high correlation to in vitro digestibility of straw, the following were calculated: digestible aNDFom (g kg-1DM) = NDFD 30h (in %)/100 × aNDFom (g kg-1 DM), indigestible aNDFom = aNDFom (g kg-1DM) – digestible aNDFom (g kg-1 DM), Non-fibre carbohydrates (NFC) = (1000 − [aNDFom+CP+EE+ash]), when urea was applied NFC = 1000 − ash − EE − aNDFom − (CP − CP u + U) where CP u is the CP from urea and U is the urea content (Hall 2000, Detmann and Valadares Filho 2010), N share in urea at 950 g kg-1 DM = 0.45, i.e. each kg of urea = 2.81 kg CP u ), cellulose = (ADFom − ADL), hemicellulose = (aNDFom − ADFom), the ratio ADL/ADFom as indicator of degree of lignification (Zeyner 1995). For comparison with the literature, the indicators total digestible nutrients (TDN), rel- ative forage quality (RFQ) and estimated DM intake (DMI) have been calculated and are presented in the appen- dix (Tables A1–A8). In the urea treated samples NH 3 -N of total N (VDLUFA III, 4.8.1) was determined. Non-starch polysaccharides (NSP) were analyzed by the Julius Kühn-Institut in Braunschweig (AOAC 2000). The BfUL (State owned company for Environment and Agriculture, Nossen) determined minerals and trace elements according to DIN EN 15510 (DIN 2017) using ICP-OES (inductively coupled plasma optical emission spectrometry). Statistical analysis The effects of the NaOH treatments on chemical composition and digestibility in Trial 1 were analyzed using the model: Y ijk = µ + NaOHconc i + DM j + NaOHconc × DM ij + ε ij where µ = general mean, i = 1, 2 (60 g, 120 g NaOH kg-1straw), j = 1, 2 (450, 600 g DM kg-1 straw), ε ij = residual error The effects of the urea treatments on chemical composition and digestibility in Trials 2 and 3 were analyzed using the models: Trial 2: Y ijk = µ + DM i + Max j + Comp k + DM * Max ij + DM * Comp ik + Max × Comp jk + DM × Max × Comp ijk + ε ijk where µ = general mean, i = 1, 2 (600, 800 g DM kg-1 straw), j = 1, 2 (0, 5 g Maxammon kg-1 straw), k = 1, 2 (com- pact, loose), ε ijk = residual error Trial 3: Y ijk = µ + Urea i + Max j + Temp k + Per l + Urea × Max ij + ε ijkl where µ = general mean, i = 1, 2, 3 (30, 45, 60 g urea kg-1 straw), j = 1, 2 (0, 5 g Maxammon kg-1), k = 1, 2 (25, 40 °C), l = 1, 2 (14, 28 d), ε ijkl = residual error Variance analysis using the univariate and multivariate procedures was performed for the treatments after the respective storage time, while the posthoc Tukey test included the untreated straw. The Pearson correlation was calculated. The software IBM® SPSS® Statistics (Version 19, SPSS, Inc., IBM Company©) was used. Agricultural and Food Science (2022) 31: 260–281 264 Results Crude ash determination when NaOH is used Figure 1 shows the crude ash concentrations at different times of ashing for straw samples, which were all treat- ed with 120 g NaOH kg-1. The shorter the ashing period in the muffle furnace, the higher the standard deviations within replicates (n = 3) of a treatment. With increasing time both the variation within a treatment and the vari- ation between treatments diminished apart from a decreasing absolute value for crude ash. Trial 1 – NaOH addition Results for chemical composition are presented in Table 2. Values of the different storage duration (2 d and 7 d) were averaged because of their similarity. The pH varied between 11 and 12 depending on the NaOH application rate. In order not to confuse the replacement of organic matter by sodium with the possible effect of NaOH, the fibre components including the cell wall carbohydrates and NFC are given in g kg-1organic matter (OM). The crude ash almost doubled and tripled when adding 60 or 120 g NaOH kg-1straw respectively. While aNDFom decreased with increasing NaOH concentration, ADFom increased compared to the control. ADL was lower with the highest application rate at 600 g DM kg-1. Thus the cellulose: ADL ratio increased likewise (further calculated parameters see Appendix, Table A1). All three parameters for in vitro digestibility (NDFD 30h , HFT, ELOS) were higher compared to the control. There was no difference in these parameters between the NaOH treatments. The different pro- portions of the fibre fractions (cellulose/ADL, ADL/ADFom) of the high NaOH treatment were significantly higher or lower respectively than the control. The main cell wall carbohydrate monomers which were analyzed, namely glucose and xylose, did not change with treatments. Only the minor sugar monomers arabinose and mannose increased with NaOH addition. The xylose:arabinose ratio lowered (7.3 vs. 6.2 vs. 5.9 with 0, 60 and 120 g NaOH kg-1 DM). OM losses were highest with high moisture (450 g kg-1) and the lower NaOH application rate (60 g kg-1), and amounted only one quarter with the high application rate and 600 g kg-1 DM (Table 2). When correlating the different indicators of digestibility with each other (HFT, ELOS, NDFD 30h ) the correlation co- efficient R was < 0.2 with p > 0.1 whith regard to the NaOH treatments alone (Appendix, Fig. A1). Main and significant differences in the mineral composition were present depending on the NaOH addition, i.e. 31 g vs. 62 g Na kg-1 DM for the 60 and 120 g treatment respectively while untreated straw contained about 0.2 g Na kg-1 DM. Fig. 1. Crude ash concentrations in different NaOH treated samples following increasing ashing times at 550 °C (a–e, all 120 g NaOH kg-1; a: 450 g DM kg-1, 2 d storage; b: 600 g DM kg-1, 2 d; c: 600 g DM kg-1, 7d; d: like a [other batch]; e: 450 g DM kg-1, 7 d). Error bars show the standard deviation. S.D. Martens et al. 265 Trial 2 – Urea addition with loose or compact storage When monitoring the temperature of the urea treated straw (15 g kg-1) stored in drums and bags at 25 °C ambi- ent, a small peak was recognized during the second day, especially in the loose treatments with 600 g kg-1 DM. The treatment without Maxammon peaked after 25 h with 5.8 K above ambient, while the treatment with Maxam- mon peaked only after 36 h with 7.9 K above ambient. The treatments with 800 g kg-1 DM adapted to the ambient temperature (25 °C), and the compact lower dry matter treatments remained below 29 °C. The chemical compo- sition of the urea treated straw is presented in Table 3. After 14 d the pH was highest in the high DM treatments while it was more differentiated in the lower dry matter treatments with approximately 8.4 without and 8.9 with Maxammon treatment. There was a significant influence of the urease treatment after 14 d on almost all param- eters, with DM playing the second most important role while compaction was subordinated (detailed results in the Appendix, Table A2). Ether extract content decreased with urease application. The aNDFom and ADFom val- ues of the treatments with urease was similar to the original straw while they were lower with the urea only ad- ditive treatment. ADL content did not change significantly compared to the original straw. Gas production and ELOS increased with urea application without urease. In general, those nutritional quality parameters were highest with 600 g kg-1 DM without Maxammon. Also, the DM losses were highest with this treatment (43 g kg-1 on aver- age). NH 3 -N was negatively correlated to pH after 14 d of storage in the range of pH 8.5–9.4 (R = –0.68, p < 0.001). Table 2. Chemical composition and in vitro digestibility of NaOH treated wheat straw (2 and 7 d storage duration resumed) (Trial 1) g NaOH kg-1 0 (untreated) 60 60 120 120 SEM Treatment effects (Significance level) Target DM [g kg-1] 900 450 600 450 600 n 11 12 7 11 7 NaOH DM NaOH x DM pH 6.20c 10.7b 10.8b 12.1a 12.3a 0.12 *** n.s. n.s. DM [g kg-1] 906a 432e 593c 462d 614b 2.5 *** *** n.s. Crude ash [g kg-1 DM] 79c 140b 151b 215a 230a 6.0 *** n.s. n.s. Crude protein [g kg-1 OM] 38.8 45.8 47.3 44.6 42.8 1.29 n.s. n.s. n.s. Ether extract [g kg-1 OM] 9.28c 16.3b 13.1bc 24.8a 25.3a 0.82 *** n.s. n.s. aNDFom [g kg-1 OM] 832a 765b 740b 681c 694c 6.3 *** n.s. n.s. ADFom [g kg-1 OM] 468b 521a 514a 531a 529a 4.6 n.s. n.s. n.s. ADL [g kg-1 OM] 62.7a 64.5a 54.9a 45.1ab 30.2b 2.77 *** * n.s. NFC [g kg-1 OM] 120b 165ab 179a 148ab 160ab 6.6 n.s. n.s. n.s. Ratios dig/indig aNDFom 0.46b 1.22a 1.06a 1.55a 1.42a 0.080 * n.s. n.s. Cellulose/ADL 6.55c 7.23c 8.66bc 14.1ab 19.9a 0.90 *** o n.s. ADL/ADFom 0.134a 0.124a 0.106ab 0.084bc 0.056c 0.0048 * * n.s. NDFD 30h [g kg-1 NDF] 310b 533a 512a 591a 572a 13.5 * n.s. n.s. HFT [ml 200 mg-1 DM] 32.1b 43.1a 41.6a 42.8a 41.4a 0.24 n.s. ** n.s. ELOS [g kg-1 DM] 346b 516a 496a 494a 511a 5.9 n.s. n.s. n.s. Polysaccharides [g kg-1 OM] (n = 3) n.s. n.s. Arabinose 29.3b 37.2a 37.1a 38.5a 38.7a 0.51 n.s. n.s. n.s. Xylose 215 233 225 225 229 1.9 n.s. n.s. n.s. Mannose 1.70c 2.55b 2.61b 3.33a 3.27a 0.087 *** n.s. n.s. Galactose 7.03 7.11 7.25 7.68 7.82 0.122 * n.s. n.s. Glucose 248 268 269 258 254 2.8 * n.s. n.s. Losses (n = 6) n.s. DM losses [g kg-1] 137a 94.4ab 51.5b 31.0b 11.67 *** o n.s. OM losses [g kg-1] 118a 80.4ab 40.7bc 24.4c 10.03 *** o n.s. DM = dry matter; aNDFom = neutral detergent fibre analyzed with heat-stable amylase and expressed without residual ash; ADFom = acid detergent fibre expressed without residual ash; ADL = acid detergent lignin; NFC = non-fibre carbohydrates; NDFD 30h = aNDFom in vitro digestibility in 30 h; dig/indig = digestible/indigestible; HFT = Hohenheim Feed value Test; ELOS = enzymatically soluble organic substance; SEM = standard error of the mean. Variance analysis excluding the untreated control (n.s. = not significant, o p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001). Treatments with different letters are significantly different (Tukey test, p < 0.05). A gricultural and Food Science (2022) 31: 260–281 266 Table 3. Chemical composition and in vitro digestibility of urea treated wheat straw (15 g urea kg-1) at different DM levels, after 14 d of storage (compact and loose resumed) (Trial 2) Target DM [g kg-1] 880# 600 600 800 800 SEM Treatment effects (Significance level) Maxammon [g kg-1] 0 0 5 0 5 Max. DM Comp. Max. × DM Max. × Comp. DM × Comp. Max. × DM × Comp. storage [d] 0 14 14 14 14 n 3 6 6 6 6 pH 6.74d 8.46c 8.90b 9.33a 9.32a 0.012 *** *** n.s. *** n.s. ** n.s. DM [g kg-1] 877a 623d 607e 796c 815b 0.4 o *** * *** *** ** o Crude ash [g kg-1 DM] 73.9a 63.8cd 70.0b 61.6d 64.7c 0.24 *** *** n.s. ** n.s. n.s. n.s. Crude protein [g kg-1 DM] 34.7c 79.0a 62.3b 75.7a 67.3b 0.87 *** n.s. n.s. * n.s. n.s. n.s. Ether extract [g kg-1 DM] 17.4ab 20.4a 12.5c 16.3b 12.0c 0.31 *** ** n.s. * n.s. * * aNDFom [g kg-1 DM] 793a 746c 791a 775b 802a 1.1 *** *** o ** n.s. o n.s. ADFom [g kg-1 DM] 475a 440c 486a 455b 475a 1.7 *** n.s. n.s. ** n.s. n.s. n.s. ADL [g kg-1 DM] 60.7abc 57.5bc 61.0ab 53.1c 68.3a 0.87 *** n.s. n.s. ** n.s. n.s. n.s. NFC [g kg-1 DM] 81.3c 116a 91.1bc 98.9b 80.6c 1.19 *** *** n.s. n.s. n.s. n.s. n.s. NH 3 -N [g kg-1 N] 17.1e 29.8b 37.4a 19.5c 17.6d 1.60 *** *** n.s. *** n.s. * n.s. In vitro digestibility NDFD 30h [g kg-1 NDF] 328 360 328 361 318 1.9 *** n.s. ** n.s. *** ** ** HFT [ml 200 mg-1 DM] 33.8c 39.6a 33.6c 35.5b 32.8c 0.122 *** *** n.s. *** ** n.s. n.s. ELOS [g kg-1 DM] 354c 459a 340c 404b 358c 1.7 *** *** n.s. *** o n.s. n.s. DM losses [g kg-1] 43.3a 8.53b 15.1b 2.18b 0.192 *** *** n.s. * ** n.s. n.s. # = no urea; DM = dry matter; aNDFom = neutral detergent fibre analysed with heat-stable amylase and expressed without residual ash; ADFom = acid detergent fibre expressed without residual ash; ADL = acid detergent lignin; NFC = non-fibre carbohydrates; NDFD 30h = aNDFom in vitro digestibility in 30 h; HFT = Hohenheim Feed value Test; ELOS = enzymatically soluble organic substance; SEM = standard error of the mean. Variance analysis excluding the untreated control (n.s. = not significant, o p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001). Treatments with different letters are significantly different (Tukey test, p < 0.05). S.D. Martens et al. 267 Trial 3 – Increasing urea addition with two storage temperatures The components pH and crude protein were ranked according to urea concentration, especially the latter increas- ing with rising urea addition, while aNDFom content decreased at the highest urea dosage in contrast to NFC con- tent (Table 4). The urease application had the strongest impact on gas production (Table 4; Appendix, Table A6). The Appendix (Tables A3−A5) gives more details according to temperature and storage time. In all three urea levels (30, 45, 60 g kg-1) the pH increased immediately after urease was added in contrast to the treatments without Maxammon (Appendix, Tables A3–A5, 0 d of storage). The same was true for the NH 3 -N con- centration, which rose when urease was applied; it further increased during storage. In most of the cases, final NH 3 -N concentration in the straw was lower when stored at 40 °C while CP was higher (significant effect of tem- perature, Table 4; Appendix, Tables A3−A6). The fibre fractions ADFom and ADL did not differ significantly among treatments. A significant increase in ELOS compared to the original straw and to 30 g urea only was achieved with the highest urea dosage plus urease. NDFD 30h did not increase significantly compared to the original straw (Table 4). In general, longer storage times increased NDFD 30h (significant effect of period, Table 4; Appendix, Table 4. Chemical composition and in-vitro digestibility of urea treated wheat straw (30, 45, 60 g kg-1) after ≥ 14 d of storage (14 & 28 d storage period and 25 & 40 °C storage temperature resumed) (Trial 3) Urea [g kg-1] 0 (untreated) 30 30 45 45 60 60 Treatment effects (Significance level)Maxammon [g kg-1] 0 0 5 0 5 0 5 n 15 12 12 12 12 12 12 SEM Urea Max Urea x Max Temp Per pH 6.45c 8.51b 8.57ab 8.61ab 8.74a 8.61ab 8.77a 0.020 *** *** n.s. *** ** DM [g kg-1] 895a 529b 508b 515b 503b 521b 505b 3.8 n.s. o n.s. o n.s. Crude ash [g kg-1 DM] 77.4b 79.7ab 81.9a 80.9ab 78.5ab 79.4ab 79.9ab 0.44 n.s. n.s. o ** n.s. Crude protein [g kg-1 DM] 35.6d 79.7c 78.5c 106b 89.2bc 143a 113b 3.6 *** *** *** *** o Ether extract [g kg-1 DM] 10.6 b 13.2ab 13.6a 13.3ab 12.7ab 13.2ab 13.2ab 0.11 n.s. n.s. n.s. n.s. n.s. aNDFom [g kg-1 DM] 779 a 774ab 772abc 758bcd 777a 753d 755cd 1.9 *** * * o n.s. ADFom [g kg-1 DM] 451 472 470 476 486 456 477 2.0 ** ** * n.s. ** ADL [g kg-1 DM] 61.6 60.3 59.5 59.8 63.0 60.6 55.9 1.02 n.s. n.s. n.s. * n.s. NFC [g kg-1 DM] 97.4 c 108bc 108bc 124ab 124ab 120bc 149a 3.0 *** ** ** *** ** NH 3 -N [g kg-1 N] 27.2 c 470ab 497ab 465ab 558a 379b 517a 13.8 ** *** ** *** * In vitro digestibility NDFD 30h [g kg-1 NDF] 303ab 299b 303ab 316ab 334ab 308ab 343a 4.5 * * n.s. * *** HFT [ml 200 mg-1 DM] 32.9cd 33.6bc 35.1ab 33.9bc 36.5a 31.3d 36.8a 0.30 * *** *** n.s. *** ELOS [g kg-1 DM] 347 b 353b 375ab 372ab 374ab 377ab 396a 3.5 * * n.s. * n.s. DM losses [g kg-1] 17.1 -35.8 -6.6 -13.7 20.3 -15.1 6.88 n.s. * n.s. n.s. o Per =storage period; Temp = storage temperature; Max = Maxammon; DM = dry matter; aNDFom = neutral detergent fibre analysed with heat-stable amylase and expressed without residual ash; ADFom = acid detergent fibre expressed without residual ash; ADL = acid detergent lignin; NFC = non-fibre carbohydrates; NDFD 30h = aNDFom in vitro digestibility in 30 h; HFT = Hohenheim Feed value Test; ELOS = enzymatically soluble organic substance; SEM = standard error of the mean. Variance analysis excluding the untreated control (n.s. = not significant, o p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001). Treatments with different letters are significantly different (Tukey test, p < 0.05). Agricultural and Food Science (2022) 31: 260–281 268 Table A6). This corresponded also to the ratio of digestible/indigestible aNDFom. The result was more defined with gas production (HFT), which was lowest immediately after applying urea and significantly higher than the original straw in the treatments with 45 and 60 g urea plus urease (Table 4; Appendix, Tables A4–A6). The ratio of cellu- lose/ADL was highest with 60 g urea plus urease and stored for 28 d at 25 °C. At the same time, it corresponded to the lowest ADL/ADF ratio (Appendix, Table A5). The correlation of digestibility indicators with each other (HFT, ELOS, NDFD 30h , TDN, DMI, RFQ) was at the highest ≥ 0.5 with RFQ on HFT and digestible NDF on HFT (Appendix, Fig. A2) (with coefficients of determination R = 0.59 at maximum whether in- or excluding the control treatment [0 g urea]), whereupon all correlations were significant. Table A7 (Appendix) gives an overview of the potential digestibility values with the different treatment groups. Graphically it can be viewed in the supplemental figures A1 and A2. The NDFD 30h and the ratio of digestible/indi- gestible NDF are higher with NaOH, and similar between urea and control. The NaOH treatment showed a much higher variability in the ratio of cellulose/ADL than urea and control treatment, while the mean was slightly higher. Gas production (HFT) and ELOS were highest with NaOH, while urea treatment only gave a slightly higher mean than the control. Discussion Protocol for crude ash determination Caustic soda reacts with carbon dioxide from the air: 2 NaOH + CO 2 → Na 2 CO 3 + H 2 O. Pure sodium carbonate is very water soluble, but has a high melting point of 851 °C. That is probably the cause of the difficulties in deter- mining the crude ash by the standard method of 3 h at 550 °C. As a result of our comparison of different ashing times without or with ammonium nitrate a safe protocol for NaOH treated straw apparently is either ashing for 24 h at 550 °C or for 9 h plus an application of a solution of NH 4 NO 3 . Chemical composition of NaOH treated straw (Trial 1 – NaOH addition) It can be assumed that NaOH is washed out in the process of NDF and ADF analysis and that the values are conse- quently actually “exclusive of residual ash” as declared. The increase in ADFom while aNDFom decreased means a reduction of hemicellulose and a rise in cellulose. The reduction of hemicellulose was also observed with urea (Harada et al. 1999, Vadiveloo and Fadel 2009) and after white-rot fungal treatment (Nayan et al. 2018). It was probably partly solubilized (Canale et al. 1990). The main component of hemicellulose in monocotyledons are xy- lans. These are long chains of xylose with side branches of arabinose and uronic acid, among others (Sauermost and Freudig 1999). Cellulose consists of a linear chain of several glucose units (Klemm et al. 1998a, Klemm et al. 1998b). However, it is not quite clear why ADF increased and how NaOH could have caused it. Although arabinose has been found in ADF (overview see Südekum 1994), compared to the magnitude of the difference between ADF and NDF here, the rise in arabinose (aldopentose) and mannose (aldohexose) concentrations measured in our samples are negligible. On the other hand, the xylose/arabinose ratio was improved with rising NaOH dos- age. It may improve microbial digestion in the rumen (Agbagla-Dohnani et al. 2012). According to Zeyner (1995), the ratio of ADL/ADFom (and its common logarithm) is an indicator of the lignification of a forage and thus of its digestibility. Similarly, this may also apply to cellulose/ADL (Nayan et al. 2018). According to this, the high NaOH treatment should result in the highest NDF digestibility and gas production. However, NDFD30h did not increase linearly in this experiment, and this supports with the mediocre correlations between the concentrations of chemi- cal components and organic matter digestibility found by Galvao et al. 2008. Nevertheless, the ratio of digestible/ indigestible NDF doubled and tripled compared to the original straw. The in vitro digestibility of aNDFom, the gas production and the enzymatically soluble organic matter increased by adding NaOH independent of the dose applied, and a poor correlation (R < 0.1) was found between those pa- rameters when the NaOH treated samples were considered exclusively. This shows that an alignment with in vivo values is essential in order to classify the values obtained in the laboratory. Trial 2 – Urea addition with loose or compact storage The contents of crude protein, NH 3 -N and the pH changed as expected. At the low level of application (15 g urea kg-1), the ratio between fibre components did not change significantly. However, ether extract content declined S.D. Martens et al. 269 with urea + urease. That might be explained by the fact that the cuticle waxy layer, which is a component of crude fat (Sun and Tomkinson 2003) was broken down as observed in a trial with rice straw (Shen et al. 1999). In our case this might have been enhanced by the urease. This may also contribute to a higher digestibility (Shen et al. 1999). However, the effect could not be observed in Trial 3. In vitro parameters for digestibility (HFT, ELOS) apart from NDFD30h were highest at 600 g kg-1 DM without Max- ammon before and after storage (Appendix, Table A2). The immediate change was surprising as a storage time of at least 14 d is recommended for urea application (Ochrimenko et al. 1987). Only NDFD30h improved with time in this treatment. The high DM and application of Maxammon was unfavorable for the potential digestibility at the low urea dosage of 15 g kg-1. Trial 3 – Increasing urea addition with two storage temperatures In contrast to the trial with only 15 g urea kg-1, this trial showed a positive effect of urease with increasing urea dosage (≥ 45 g urea kg-1 DM) on gas production. Urease has an accelerating effect to increase organic matter digestibility (Jayasuriya and Pearce 1983). Urease is an enzyme naturally occurring in many plants, bacteria, fungi and algae. According to earlier findings, soybean, which was also an ingredient of the product used in the Trials 2 and 3, seems to be an adequate source of urease for effective application in cereal straw (Jayasuriya and Pearce 1983, Khan et al. 1999). When comparing the absolute values of in vitro digestibility across the two trials (Trial 2 and 3), no clear urea dose-effect relationship was observed. As mediocre correlations between different indicators for potential digest- ibility were found, in vivo trials have to confirm the feeding potential. The figures of DM-losses are inconsistent. Probably the DM determination at 105 °C with highly volatile components (NH 3 at pH > 5.0) is the cause of the error (Weissbach and Kuhla 1995). The principle of straw treatment with urea is based on the reaction H 2 N-CO-NH 2 + H 2 O -> 2NH 3 + CO 2 , which is catalyzed by urease. Overall, approximately 50% of the total nitrogen was present in the form of ammonia after storage. The treatments stored at 40 °C maintained a higher CP content than the ones at 25 °C, while the ammonia-N portion in the DM was similar. Ammonia can be utilized by the ruminal microflora as non-protein nitrogen. However, the volatility of NH 3 is enhanced with increasing pH. General considerations and comparison of the treatments with urea and NaOH The true NDFD of the different treatments may have varied more widely than in the results presented with only 30 h incubation. Krämer et al. (2012) determined the indigestible NDF in forages after 288 h in situ in cows (i.e. 10 times longer incubation time). The real retention time in the rumen depends on the whole ration including its structure. For a first screening the 30 h incubation might have been sufficient. NaOH seems to give a clear advantage over urea when comparing the in vitro digestibility parameters NDFD 30h , HFT and ELOS to the untreated control. In a palatability trial with sheep, there was no difference in the DM intake of either NaOH or urea treated barley straw (60 g or 40 g kg-1) as the only feed component after three weeks of adap- tation (data not shown). In a feeding trial with sheep with the same straw as component of a total mixed ration the NaOH treatment increased the in vivo fibre digestibility in contrast to the urea treatment (Bachmann et al. 2022). The high content of sodium has to be considered in the formulation of the ration for cattle when feeding NaOH treated straw. The daily requirement of sodium of a dairy cow giving 30–40 kg milk per day can be met by an in- take of 31–38 g Na per cow per day (700 kg live weight) (Kirchgessner et al. 2014). Therefore, that 1 kg of soda straw (60 g NaOH kg-1 DM) is already sufficient to meet the daily sodium requirement of a cow. Thus a sufficient quantity of drinking water has to be provided to facilitate the endogenous regulation and excretion of surplus cations to prevent nephritis (Suttle 2010). Furthermore, the alkaline pH should be neutralized by combining the straw with acidic feeds such as silages. As a general recommendation, alkali-treated feeds should not be used for transition cows because they may increase the incidence of milk fever (Suttle 2010). Conclusions A moisture content of 400 g kg-1 clearly contributed to the effectivity of both NaOH and urea treatments. While there was a dose-response relationship with urea, doubling the NaOH concentration at an already elevated level did not further increase in vitro digestibility. The differences in pressure produced manually were marginal. Agricultural and Food Science (2022) 31: 260–281 270 Storage temperature played a subordinate role while an extended storage period increased gas production and NDFD 30h with the urea application. Urease addition was only advantageous in terms of increased gas production and NDFD 30h when applied at urea dosages ≥ 30 g kg-1. Treatment of wheat straw with 60 g kg-1 NaOH or ≥ 45 g kg-1 urea at 600 or 500 g DM kg-1 respectively increased the in vitro digestibility. Urea treated straw had to be stored for at least two weeks (preferably 4) at ≥ 25 °C am- bient. Studies on voluntary feed intake and in vivo digestibility are needed to confirm the expected benefit in ru- minant animals. 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Agricultural and Food Science (2022) 31: 260–281 272 Appendix Appendix 1: Analytical methods VDLUFA III and calculation forage quality indicators Principles 8.1 Crude ash at 550 °C for at least 3 h 6.5.1 aNDFom adapted from Mertens 2002 6.5.2 ADFom adapted from Van Soest et al. 1991 6.5.3 ADL adapted from Goering and Van Soest 1970 5.1.1 EE extraction with petroleum ether, see also Mattsson 1978 4.1.2 CP combustion according to Dumas 6.6.1 ELOS using pepsin, HCl and cellulose, see also De Boever et al. 1986 25.1 HFT see Menke et al. 1979, Menke and Steingass 1987, Steingass and Menke 1986 4.8.1 NH3-N by microdiffusion, fresh sample extracted with water, compare Conway 1962, Conway and Byrne 1933 Calculations total digestible nutrients TDNgrass = (NFC1 × 0.98) + (CP × 0.87) + (FA × 0.97 × 2.25) + (NDF × 0.93 × (22.7 + 0.664 × NDFD30h) / 100) – 10 (in % of DM, FA fatty acids = ether extract – 1; equation for grass (Moore and Undersan- der 2002); NDFD48h replaced with NDFD30h), estimated dry matter intake (DMIgrass) = –2.318 + 0.442 × CP – 0.01 × CP2 – 0.0638 × TDN + 0.000922 × TDN2 + 0.18 × ADFom – 0.00196 × ADF2 – 0.00529 × CP × ADFom (for grass, Moore and Kunkle 1999), relative forage quality RFQ = (DMIgrass, % of BW) × TDNgrass, % of DM) / 1.23 (Undersander and Moore 2004). 1NFC Non-fiber carbohydrates = (1000 – [aNDFom+CP+EE+ash]), when urea was applied NFC = 1000 – ash – EE – aNDFom – (CP – CPu + U) where CPu is the CP from urea and U is the urea content (Hall 2000, Detmann and Valadares Filho 2010), N share in urea at 950 g kg-1 DM = 0.45, i.e. each kg of urea = 2.81 kg CPu) References Conway, E.J. 1962. 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Neutral Detergent Soluble Carbohydrates Nutritional Relevance and Analysis: A Laboratory Manual. University of Florida. 77 p. Mattsson, P. 1978. Crude fat determination in feedingstuffs: some studies of extraction and hydrolysis methods. Meddelande - Statens Lantbrukskemiska Laboratorium (Sweden) 49. Menke, K.H., Raab, L., Salewski, A., Steingass, H., Fritz, D. & Schneider, W. 1979. The estimation of the digestibility and metabo- lizable energy content of ruminant feedingstuffs from the gas production when they are incubated with rumen liquor in vitro. Journal of Agricultural Science 93: 217–222. https://doi.org/10.1017/S0021859600086305 Menke, K.H. & Steingass, H. 1987. Estimation of the energy feeding value from gas formation estimated in vitro with rumen fluid and from chemical analysis. 2. Regression equations. Ubersichten zur Tierernahrung 15: 59–93. Mertens, D.R. 2002. Gravimetric determination of amylase-treated neutral detergent fiber in feeds with refluxing in beakers or crucibles: collaborative study. Journal of AOAC International 85: 1217–1240. https://doi.org/10.1093/jaoac/85.6.1217 Moore, J.E. & Kunkle, W.E. 1999. Evaluation of equations for estimating voluntary intake of forages and forage-based diets. Jour- nal of Animal Science (Suppl. 1) 204. Agricultural and Food Science (2022) 31: 260–281 273 Appendix Moore, J.E. & Undersander, D.J. 2002. Relative forage quality: An alternative to relative feed value and quality index. Proceedings 13th Annual Florida Ruminant Nutrition Symposium. p. 16–32. https://animal.ifas.ufl.edu/apps/dairymedia/rns/2002/moore.pdf Steingass, H. & Menke, K.H. 1986. Estimation of the energy feeding value from gas formation estimated in vitro with rumen fluid and from chemical analysis. 1. Studies of the method. Übersichten zur Tierernährung 14: 251–270. Van Soest, P.J., Robertson, J.B. & Lewis, B.A. 1991. Methods for Dietary Fiber, Neutral Detergent Fiber, and Nonstarch Polysaccha- rides in Relation to Animal Nutrition. Journal of Dairy Science 74: 3583–3597. https://doi.org/10.3168/jds.S0022-0302(91)78551-2 Undersander, D. & Moore, J.E. 2004. Relative Forage Quality (RFQ) - Indexing legumes and grasses for forage quality. In: Proceed- ings, National Alfalfa Symposium, 13-15 December, 2004, San Diego, CA, UC Cooperative Extension. University of California. Da- vis. https://alfalfa.ucdavis.edu/+symposium/proceedings/2004/04-193.pdf Table A1. Chemical composition and in vitro digestibility of NaOH treated wheat straw (2 and 7 d storage duration resumed) (Trial 1) g NaOH kg-1 0 (untreated) 60 60 120 120 Target DM [g kg-1] 900 450 600 450 600 SEM Significance level n 11 12 7 11 7 NaOH DM NaOH x DM Calculated forage quality TDN [g kg-1 DM] 345c 437a 418ab 373bc 371bc 7.5 ** n.s. n.s. RFQ 37.2c 51.5a 48.8ab 41.5bc 39.2c 1.27 *** n.s. n.s. DMI [g kg-1 BW] 13.2 14.4 14.4 13.4 13.0 0.18 ** n.s. n.s. aNDFom = neutral detergent fiber analysed with heat-stable amylase and expressed without residual ash; ADFom = acid detergent fiber expressed without residual ash; ADL = acid detergent lignin; dig/indig = digestible/indigestible; TDN = total digestible nutrients; RFQ = relative forage quality; DMI = dry matter intake; BW = body weight; SEM = standard error of the mean. Variance analysis excluding the untreated control (n.s. = not significant, o p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001). Treatments with different letters are significantly different (Tukey test, p < 0.05). A gricultural and Food Science (2022) 31: 260–281 274 A ppendix Table A2. Chemical composition and in vitro digestibility of urea treated wheat straw (15 g urea kg-1) at different DM levels, compact and loose, after 0 and 14 d of storage (Trial 2) Target DM [g kg-1] 880 (no urea) 600 600 600 600 600 600 800 800 800 800 800 800 Maxammon [g kg-1 DM] 0 0 5 0 0 5 5 0 5 0 0 5 5 compaction L L L L C L C L L L C L C SEM storage [d] 0 0 0 14 14 14 14 0 0 14 14 14 14 14 n 3 3 3 3 3 3 3 3 3 3 3 3 3 pH 6.74e 7.24d 8.37c 8.45c 8.47c 8.85b 8.94b 8.37c 7.21d 9.37a 9.29a 9.38a 9.26a 0.076 DM [g kg-1] 877a 646e 602h 617g 630f 608h 607h 805c 814b 794d 797cd 818b 813b 19.9 Crude ash [g kg-1 DM] 73.9a 61.9e 70.1abc 63.8de 63.7e 70.6ab 69.3abcd 63.5cd 66.2bcde 61e 62.3e 64.6de 64.7cde 0.68 Crude protein [g kg-1 DM] 34.7f 91.8a 45.8f 79.7ab 78.3abc 61.6e 62.9de 76.1bcd 75.2bcde 78.2abc 73.2bcde 65.9cde 68.6bcde 1.59 Ether extract [g kg-1 DM] 17.4abc 18.5ab 18.3ab 21.1a 19.6a 12.5cde 12.6cde 17.1abcd 18.3 14.0bcde 18.6ab 12.3de 11.7e 0.79 aNDFom [g kg-1 DM] 793abc 753de 813a 747de 746e 790abc 791abc 779bc 796ab 780bc 771cd 806a 798ab 4.5 ADFom [g kg-1 DM] 475abcd 425f 490a 442ef 439ef 486a 487a 454cde 477abc 451de 460bcde 472abcd 478ab 4.0 ADL [g kg-1 DM] 60.7abc 44.6d 64.4abc 58.1abcd 57.0abcd 62.2abc 59.8abc 60.3abc 67.5ab 54.9bcd 51.2cd 65.7abc 70.9a 1.42 NFC [g kg-1 DM] 81.3defg 99.7abcd 63.8g 114ab 118a 91.9cdef 90.3cdef 91.9cdef 72.5fg 95bcde 103abc 77.6efg 83.6cdefg 2.90 NH 3 -N [g kg-1 N] 17.1h 107g 451a 295c 302c 369b 379b 137fg 155ef 202d 188d 175de 177de 16.83 Ratios dig/indig aNDFom 0.488bc 0.498bc 0.577ab 0.599a 0.527abc 0.461cd 0.516abc 0.481bc 0.515abc 0.605a 0.526abc 0.381d 0.561ab 0.0149 Cellulose/ADL 6.83abc 8.73a 6.65bc 6.61bc 6.72bc 6.85abc 7.18abc 6.52bc 6.08bc 7.21abc 7.98ab 6.25bc 5.76c 0.157 ADL/ADFom 0.128abc 0.105c 0.131abc 0.132abc 0.130abc 0.128abc 0.123abc 0.133abc 0.142a .122abc 0.111bc 0.139ab 0.148a 0.0027 Calculated forage quality TDN [g kg-1 DM] 354fg 410bc 376de 435a 421ab 370ef 382de 392cd 389cde 416ab 410bc 346g 391cde 5.9 RFQ 35.9h 62.5a 40.6fg 63.7a 61a 43.9ef 45.8de 53.9bc 49.8cd 58.6ab 55.5b 43.2ef 49.1cd 1.61 DMI [g kg-1 BW] 12.5f 18.8a 13.3f 18.0ab 17.8abc 14.6e 14.7e 16.9bcd 15.8de 17.3bc 16.6cd 15.4e 15.4e 0.28 In vitro digestibility NDFD30h [g kg-1 NDF] 328cd 332bcd 366abc 374ab 345abcd 316de 340abcd 325cd 338abcd 377a 344abcd 276e 359abc 6.65 HFT [ml 200 mg-1 DM] 33.8bcd 38.0a 35.0bc 39.2a 39.9a 34.0bcd 33.2cd 34.0bcd 32.8d 35.0bc 35.9b 33.8d 32.4d 0.56 ELOS [g kg-1 DM] 354cd 463a 344d 450a 468a 341d 338d 389bc 361cd 403b 406b 358cd 358cd 8.7 DM losses [g kg-1] 55.9a 30.8ab 2.81c 14.2bc 19.7bc 10.5bc 0.825a 3.53c 3.95 DM = dry matter; aNDFom = neutral detergent fiber analysed with heat-stable amylase and expressed without residual ash; ADFom = acid detergent fiber expressed without residual ash; ADL = acid detergent lignin; NFC = non-fiber carbohydrates; dig/indig = digestible/indigestible; TDN = total digestible nutrients; RFQ = relative forage quality; DMI = dry matter intake; LW = live weight; NDFD30h = aNDFom in vitro digestibility in 30 h; HFT = Hohenheim Feed value Test; ELOS = enzymatically soluble organic substance. Treatments with different letters are significantly different (Tukey test, p < 0.05). Agricultural and Food Science (2022) 31: 260–281 275 Appendix Table A3. Chemical composition and in vitro digestibility of urea treated wheat straw (30 g urea kg-1) at different temperatures and for varying storage duration (Trial 3) Urea [g kg-1 DM] 0 30 30 30 30 30 30 30 30 30 30 Maxammon [g kg-1 DM] 0 0 0 0 0 0 5 5 5 5 5 Nominal temperature [°C] 25 25 40 40 25 25 40 40 Storage [d] 0 0 14 28 14 28 0 14 28 14 28 SEM n 15 3 3 3 3 3 3 3 3 3 3 (30g urea) pH 6.45b 6.05b 8.60a 8.48a 8.53a 8.42a 8.13a 8.68a 8.63a 8.46a 8.51a 0.141 DM [g kg-1] 895a 521b 518b 526b 530b 545b 513b 506b 497b 508b 522b 4.9 Crude ash [g kg-1 DM] 77.4 76.2 78.7 81.1 78.7 80.2 79.4 85.7 83.8 80 78.1 0.719 Crude protein [g kg-1 DM] 35.6e 133a 63.7d 72.0cd 92.6b 90.6b 125a 68.7cd 78.3bcd 81.7bcd 85.5bc 4.15 Ether extract [g kg-1 DM] 10.6 12.9 13.4 12.8 13.6 13.2 12.8 13.3 14.4 13.4 13.2 0.18 aNDFom [g kg-1 DM] 779 765 781 776 770 769 761 776 763 775 775 2.2 ADFom [g kg-1 DM] 459 451 467 464 486 471 441 456 473 473 478 2.9 ADL [g kg-1 DM] 61.6 56.9 66.7 55.2 56.3 63.2 67 53.3 56.6 67.7 60.5 1.52 NFC [g kg-1 DM] 97.4ab 66.9b 118a 113a 99.4ab 101ab 76.4ab 111a 115a 104ab 102ab 3.4 NH 3 -N [g kg-1 N] 27.2f 15.8f 563a 524ab 374d 421cd 146e 555a 528ab 440bcd 466bc 32.7 Ratios dig/indig aNDFom 0.440 0.378 0.415 0.451 0.393 0.448 0.438 0.443 0.471 0.360 0.477 0.0105 Cellulose/ADL 6.55 7.05 6.03 7.41 7.71 6.53 5.66 7.66 7.5 6.01 7.07 0.199 ADL/ADFom 0.134 0.126 0.143 0.119 0.116 0.134 0.152 0.117 0.119 0.143 0.126 0.0033 Calculated forage quality TDN [g kg-1 DM] 338b 379ab 385a 392a 382a 394a 393a 389a 402a 369ab 400a 2.6 RFQ 35.3b 50.8a 48.7a 51.8a 48.6a 53.1a 56.4a 51.1a 53.1a 48.5a 52.4a 0.67 DMI [g kg-1 BW] 12.8b 16.5a 15.6ab 16.3a 15.6ab 16.5a 17.6a 16.2a 16.2a 16.1a 16.1a 1.46 In vitro digestibility NDFD30h [g kg-1 NDF] 303 274 293 310 281 310 305 314 320 262 322 5.3 HFT [ml 200 mg-1 DM] 32.9b 27.6c 33.7ab 34.4ab 32.3b 34ab 28.0c 33.9ab 34.3ab 36.0a 36.2a 0.56 ELOS [g kg-1 DM] 347 353 353 351 360 347 376 361 359 392 380 4.4 DM losses [g kg-1] 58.2 9.6 2.77 -2.71 -4.87 1.51 -21.4 -74.6 9.237 DM = dry matter; aNDFom = neutral detergent fiber analysed with heat-stable amylase and expressed without residual ash; ADFom = acid detergent fiber expressed without residual ash; ADL = acid detergent lignin; NFC = non-fiber carbohydrates; dig/indig = digestible/indigestible; TDN = total digestible nutrients; RFQ = relative forage quality; DMI = dry matter intake; LW = live weight; NDFD30h = aNDFom in vitro digestibility in 30 h; HFT = Hohenheim Feed value Test; ELOS = enzymatically soluble organic substance. Treatments with different letters are significantly different (Tukey test, p < 0.05). Agricultural and Food Science (2022) 31: 260–281 276 Appendix Table A4. Chemical composition and in-vitro digestibility of urea treated wheat straw (45 g urea kg-1) at different temperatures and for varying storage duration (Trial 3) Urea [g kg-1 DM] 0 45 45 45 45 45 45 45 45 45 45 Maxammon [g kg-1 DM] 0 0 0 0 0 0 5 5 5 5 5 Nominal temperature [°C] 25 25 40 40 25 25 40 40 Storage [d] 0 0 14 28 14 28 0 14 28 14 28 SEM n 15 3 3 3 3 3 3 3 3 3 3 (45 g urea) pH 6.45b 6.06b 8.79a 8.60a 8.49a 8.55a 8.51a 8.85a 8.73a 8.76a 8.60a 0.153 DM [g kg-1] 895a 508b 510b 511b 526b 515b 514b 489b 513b 505b 508b 5.6 Crude ash [g kg-1 DM] 77.4 72.7 82 82.2 79.2 80.1 74.9 79.1 76.9 81.2 76.7 0.84 Crude protein [g kg-1 DM] 35.6f 184a 87.9de 80.1de 132b 1222bc 167a 75.9e 83.2de 101cd 96.8de 6.7 Ether extract [g kg-1 DM] 10.6 12.3 13.3 13.3 13.5 13 13.5 12.6 12.2 13.2 12.6 0.17 aNDFom [g kg-1 DM] 779 759 765 751 755 760 770 786 780 774 768 2.7 ADFom [g kg-1 DM] 459 442 465 484 472 484 476 481 492 481 490 3.6 ADL [g kg-1 DM] 61.6 62 58.5 55 61.9 63.7 64.8 52.1 70 70 60.1 1.59 NFC [g kg-1 DM] 97.4b 54.2c 133ab 155a 101b 107b 56.5c 128ab 129ab 112b 128ab 5.94 NH 3 -N [g kg-1 N] 27.2d 11.7d 565a 584a 314cd 398bc 182d 608a 588a 505ab 530ab 36.50 Ratios dig/indig aNDFom 0.440ab 0.461ab 0.488ab 0.479ab 0.412b 0.477ab 0.381b 0.476ab 0.520ab 0.422ab 0.599a 0.0130 Cellulose/ADL 6.55 6.18 6.94 8.09 6.68 6.68 6.35 8.63 6.10 5.91 7.41 0.260 ADL/ADFom 0.134 0.14 0.126 0.115 0.131 0.132 0.136 0.108 0.142 0.145 0.123 0.0035 Calculated forage quality TDN [g kg-1 DM] 338c 426ab 430ab 437ab 417ab 429ab 402b 417ab 433ab 409b 455a 3.3 RFQ 35.3c 42.0bc 60.1a 57.6a 52.4ab 53.9ab 36.6c 54.5ab 55.6a 53.7ab 59.8a 1.49 DMI [g kg-1 BW] 12.8bcd 12.1cd 17.2a 16.2a 15.5abc 15.4abc 11.2d 16.0ab 15.8ab 16.1ab 16.2a 0.37 In vitro digestibility NDFD30h [g kg-1 NDF] 303ab 316ab 327ab 324ab 290b 323ab 276b 322ab 342ab 296ab 374a 6.1 HFT [ml 200 mg-1 DM] 32.9c 26.9d 33.5c 35.3bc 32.0c 34.7bc 27.4d 35.0bc 34.5bc 37.1ab 39.3a 0.72 ELOS [g kg-1 DM] 347 385 364 378 372 372 345 345 357 380 405 5.9 DM losses [g kg-1] 5.55 -10.2 5.82 -27.6 -27.6 -3.33 -2.20 -21.7 8.754 DM = dry matter; aNDFom = neutral detergent fiber analysed with heat-stable amylase and expressed without residual ash; ADFom = acid detergent fiber expressed without residual ash; ADL = acid detergent lignin; NFC = non-fiber carbohydrates; dig/indig = digestible/indigestible; TDN = total digestible nutrients; RFQ = relative forage quality; DMI = dry matter intake; LW = live weight; NDFD30h = aNDFom in vitro digestibility in 30 h; HFT = Hohenheim Feed value Test; ELOS = enzymatically soluble organic substance. Treatments with different letters are significantly different (Tukey test, p < 0.05). Agricultural and Food Science (2022) 31: 260–281 277 Appendix Table A5. Chemical composition and in-vitro digestibility of urea treated wheat straw (60 g urea kg-1) at different temperatures and for varying storage duration (Trial 3) Urea [g kg-1 DM] 0 60 60 60 60 60 60 60 60 60 60 Maxammon [g kg-1 DM] 0 0 0 0 0 0 5 5 5 5 5 Nominal temperature [°C] 25 25 40 40 25 25 40 40 Storage [d] 0 0 14 28 14 28 0 14 28 14 28 SEM n 15 3 3 3 3 3 3 3 3 3 3 (60 g urea) pH 6.45c 6.20c 8.75a 8.87a 8.40a 8.43a 7.57b 8.91a 8.76a 8.73a 8.67a 0.151 DM [g kg-1] 895a 533b 498b 517b 520b 549b 505b 495b 500b 502b 524b 6.1 Crude ash [g kg-1 DM] 77.4ab 72.4b 78.5ab 81.6ab 79.8ab 77.9ab 77.2ab 84.9a 79.9ab 77.9ab 76.7ab 0.74 Crude protein [g kg-1 DM] 35.6e 197ab 128cd 95.1d 187ab 164bc 208a 104d 95.6d 125cd 126cd 7.99 Ether extract [g kg-1 DM] 10.6 13.8 13.5 13.7 11.9 13.6 15.9 13.6 13.4 12.3 13.3 0.29 aNDFom [g kg-1 DM] 779 752 751 763 744 754 745 759 758 763 739 2.4 ADFom [g kg-1 DM] 459 445 457 471 437 459 442 463 482 469 494 3.7 ADL [g kg-1 DM] 61.6 57.0 58.1 62.3 58.2 63.6 49 52.5 47.6 62.4 61.0 1.50 NFC [g kg-1 DM] 97.4cde 73.1e 138abc 155a 86.7de 99.8bcde 62.8e 148ab 162a 130abcd 153a 7.01 NH 3 -N [g kg-1 N] 27.2d 11.9d 483a 569a 185d 278bc 153cd 574a 579a 424ab 490a 37.12 Ratios dig/indig aNDFom 0.440 0.459 0.460 0.525 0.389 0.429 0.418 0.534 0.546 0.461 0.567 0.0166 Cellulose/ ADL 6.55ab 6.90ab 6.89ab 6.60ab 6.53ab 6.25b 8.10ab 7.84ab 10.4a 6.61ab 7.12ab 0.345 ADL/ADFom 0.134 0.128 0.127 0.132 0.133 0.139 0.111 0.113 0.099 0.133 0.124 0.0033 Calculated forage quality TDN [g kg-1 DM] 338b 456a 458a 466a 437a 445a 447a 466a 478a 449a 488a 4.17 RFQ 35.3cd 35.8cd 64.3a 66.8a 41.7bcd 50.4abc 27.6d 68.7a 66.5a 60.9ab 62.0ab 2.99 DMI [g kg-1 BW] 12.8abcd 9.69cd 17.2ab 17.6a 11.7bcd 13.8abc 7.67d 18.1a 17.2ab 16.7ab 15.6ab 0.747 In vitro digestibility NDFD30h [g kg-1 NDF] 303 314 313 343 279 297 292 347 362 312 361 7.9 HFT [ml 200 mg-1 DM] 32.9bcd 25.1f 29.9de 34.1abc 29.2de 31.8cd 26.0ef 36.0ab 37.6a 36.2ab 37.7a 0.85 ELOS [g kg-1 DM] 347 363 381 359 376 394 350 385 375 405 408 5.5 DM losses [g kg-1] 72.3 3.73 46.3 -41.3 -16.3 -18.6 -5.4 -20.1 10.71 DM = dry matter; aNDFom = neutral detergent fiber analysed with heat-stable amylase and expressed without residual ash; ADFom = acid detergent fiber expressed without residual ash; ADL = acid detergent lignin; NFC = non-fiber carbohydrates; dig/indig = digestible/indigestible; TDN = total digestible nutrients; RFQ = relative forage quality; DMI = dry matter intake; LW = live weight; NDFD30h = aNDFom in vitro digestibility in 30 h; HFT = Hohenheim Feed value Test; ELOS = enzymatically soluble organic substance. Treatments with different letters are significantly different (Tukey test, p < 0.05). Agricultural and Food Science (2022) 31: 260–281 278 Appendix Table A6. Significance of the effect of the factors urea concentration (30, 45, 60 g kg-1), maxammon application (0, 5 g kg-1), storage period (14, 28 d) and temperature (25, 40 °C) on the nutritional composition of wheat straw (Trial 3) Significance level Urea Per Temp Max Urea×Max Urea×Temp Urea×Per Per×Max Per×Temp Max×Temp pH *** ** *** *** n.s. * n.s. n.s. n.s. o DM [g kg-1] n.s. n.s. o o n.s. n.s. n.s. n.s. n.s. n.s. Crude ash [g kg-1 DM] n.s. n.s. ** n.s. o n.s. n.s. * n.s. n.s. Crude protein [g kg-1 DM] *** o *** *** *** *** ** * n.s. *** Ether extract [g kg-1 DM] n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. aNDFom [g kg-1 DM] *** n.s. n.s. * * n.s. n.s. n.s. n.s. n.s. ADFom [g kg-1 DM] ** 0.001 n.s. *** ** o * n.s. n.s. n.s. ADL [g kg-1 DM] n.s. n.s. * n.s. n.s. n.s. n.s. n.s. n.s. o NFC [g kg-1 DM] *** ** *** ** ** * o n.s. n.s. *** NH 3 -N [g kg-1 N] ** * *** *** ** * n.s. n.s. o *** Ratios dig/indig aNDFom ** *** o ** n.s. n.s. n.s. n.s. * n.s. Cellulose/ADL n.s. n.s. * n.s. n.s. n.s. n.s. n.s. n.s. n.s. ADL/ADFom n.s. n.s. * n.s. n.s. n.s. n.s. n.s. n.s. n.s. Calculated forage quality TDN [g kg-1 DM] *** *** n.s. n.s. n.s. n.s. n.s. o n.s. n.s. RFQ *** n.s. ** o o ** n.s. n.s. n.s. o DMI [g kg-1 BW] n.s. n.s. ** o n.s. ** n.s. n.s. n.s. * In vitro digestibility NDFD30h [g kg-1 NDF] * ** * * n.s. n.s. n.s. n.s. o n.s. HFT [ml/200 mg DM] * *** n.s. *** *** o n.s. * n.s. *** ELOS [g kg-1 DM] * n.s. * n.s. n.s. n.s. n.s. n.s. n.s. o DM losses [g kg-1] n.s. o n.s. * n.s. n.s. n.s. n.s. n.s. n.s. Per = storage period; Temp = storage temperature; Max = Maxammon; DM = dry matter; aNDFom = neutral detergent fiber analysed with heat-stable amylase and expressed without residual ash; ADFom = acid detergent fiber expressed without residual ash; ADL = acid detergent lignin; NFC = non-fiber carbohydrates; dig/indig = digestible/indigestible; TDN = total digestible nutrients; RFQ = relative forage quality; DMI = dry matter intake; LW = live weight; NDFD30h = aNDFom in vitro digestibility in 30 h; HFT = Hohenheim Feed value Test; ELOS = enzymatically soluble organic substance. n.s. = not significant, o p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001 A gricultural and Food Science (2022) 31: 260–281 279 A ppendix Table A7. Ranges of potential digestibility indicators plus pH overall and in the different treatment groups (Trial 1, 2, 3) Treatment All Control Urea NaOH n Min Max Mean SEM n Min Max Mean SEM n Min Max Mean SEM n Min Max Mean SEM dig/indig aNDFom 189 0.268 2.584 0.652 0.030 17 0.286 0.674 0.452 0.028 133 0.268 0.655 0.479 0.007 39 0.658 2.584 1.325 0.080 Cellulose/ ADL 196 3.99 29.5 7.84 0.273 19 5.08 8.68 6.47 0.179 133 4.75 16.3 7.01 0.119 44 3.99 29.5 10.9 1.04 ADL/ADF 195 0.033 0.200 0.123 0.002 18 0.103 0.164 0.133 0.003 133 0.058 0.177 0.128 0.002 44 0.033 0.200 0.106 0.006 TDN (g kg-1 DM) 189 288 520 406 3.11 17 293 388 343 5.77 133 341 500 415 3.00 39 288 520 402 8.14 RFQ 189 17.4 75.3 49.7 0.770 17 28.5 44.2 35.7 1.22 133 17.4 75.3 52.7 0.851 39 29.3 68.9 45.7 1.38 DMI (g kg-1 LW) 189 4.55 19.2 15.0 0.172 17 10.7 15.9 12.8 0.349 133 4.55 19.2 15.6 0.212 39 12.2 16.4 13.9 0.172 NDFD 30h (g kg-1 NDF) 190 311 721 370 7.81 17 222 403 315 12.2 133 211 396 322 3.26 40 397 721 554 12.5 HFT (ml 200 mg-1 DM) 185 22.3 45.3 35.2 0.359 15 29.6 35.1 32.5 0.419 132 24.1 41.2 33.6 0.316 38 22,3 45,3 41.9 0.578 ELOS (g kg-1 DM) 184 286 581 400 4.72 15 323 382 348 5.19 131 311 473 378 3.16 38 286 581 499 8.12 pH 165 6.01 12.5 8.93 0.113 3 6.16 6.74 6.53 0.186 132 6.01 9.41 8.41 0.067 30 9.50 12.5 11.5 0.177 dig/indig aNDFom = digestible/indigestible neutral detergent fiber analyzed with heat-stable amylase and expressed without residual ash; ADL = acid detergent lignin; ADFom: acid detergent fiber expressed without residual ash; TDN = total digestible nutrients; RFQ = relative forage quality; DMI = dry matter intake; LW = live weight; NDFD30h = aNDFom in vitro digestibility in 30 h; HFT = Hohenheim Feed value Test; ELOS = enzymatically soluble organic substance; SEM = standard error of the mean. Agricultural and Food Science (2022) 31: 260–281 280 Appendix Fig. A1. Correlation matrix (Trial 1 – NaOH). The bigger the circle the higher the correlation. Blue – positive, red - negative correlation. aNDFom: neutral detergent fibre analysed with heat-stable amylase and expressed without residual ash; ADFom: acid detergent fibre expressed without residual ash; ADL: acid detergent lignin; NFC: non-fibre carbohydrates; NDFD: aNDFom in vitro digestibility in 30 h; HFT: Hohenheim Feed value Test; ELOS: enzymatically soluble organic substance; TDN: total digestible nutrients; RFQ: relative forage quality; DMI: dry matter intake Fig. A2. Correlation matrix (Trial 3 – Urea). The bigger the circle the higher the correlation. Blue – positive, red - negative correlation. aNDFom: neutral detergent fibre analysed with heat-stable amylase and expressed without residual ash; ADFom: acid detergent fibre expressed without residual ash; ADL: acid detergent lignin; NFC: non-fibre carbohydrates; NDFD: aNDFom in vitro digestibility in 30 h; HFT: Hohenheim Feed value Test; ELOS: enzymatically soluble organic substance; TDN: total digestible nutrients; RFQ: relative forage quality; DMI: dry matter intake Agricultural and Food Science (2022) 31: 260–281 281 Appendix Fig. A3. Box plots showing the range of different digestibility indicators for the three treatment groups (urea, NaOH, untreated). Top: ratio of digestible/indigestible aNDFom, cellulose/ADL, ADL/ADFom. Middle line: TDN total digestible nutrients, RFQ relative forage quality, DMI dry matter intake (% of body weight). Bottom: NDFD 30h aNDFom digestibility (%); HFT Hohenheim feed value test (ml 200 mg-1 DM); ELOS enzymatically soluble organic substance (% of DM) Chemical treatment of straw for ruminant feeding with NaOH orurea – investigative steps via practical application under currentEuropean Union conditions Introduction Material and methods Trial 1 – NaOH addition Trial 2 – Urea addition with loose or compact storage Trial 3 – Increasing urea addition with two storage temperatures Chemical analysis Statistical analysis Results Crude ash determination when NaOH is used Trial 1 – NaOH addition Trial 2 – Urea addition with loose or compact storage Trial 3 – Increasing urea addition with two storage temperatures Discussion Protocol for crude ash determinationCaustic soda reacts with carbon dioxide from the air: 2 NaOH + CO2 → Na2CO3 + H Chemical composition of NaOH treated straw (Trial 1 – NaOH addition) Trial 2 – Urea addition with loose or compact storage Trial 3 – Increasing urea addition with two storage temperatures General considerations and comparison of the treatments with urea and NaOH Conclusions Acknowledgements References Appendix Appendix 1: Analytical methods VDLUFA III and calculation forage quality indicators Table A1. Chemical composition and in vitro digestibility of NaOH treated wheat straw (2 and 7 d storage duration resumed) (Trial 1) Table A2. Chemical composition and in vitro digestibility of urea treated wheat straw (15 g urea kg-1) at different DM levels, compact and loose, after 0 and 14 d of storage (Trial 2) Table A3. Chemical composition and in vitro digestibility of urea treated wheat straw (30 g urea kg-1) at different temperatures andfor varying storage duration (Trial 3) Table A4. Chemical composition and in-vitro digestibility of urea treated wheat straw (45 g urea kg-1) at different temperatures and forvarying storage duration (Trial 3) Table A5. Chemical composition and in-vitro digestibility of urea treated wheat straw (60 g urea kg-1) at different temperatures andfor varying storage duration (Trial 3) Table A6. Significance of the effect of the factors urea concentration (30, 45, 60 g kg-1), maxammon application (0, 5 g kg-1), storageperiod (14, 28 d) and temperature (25, 40 °C) on the nutritional composition of wheat straw (Trial 3) Table A7. Ranges of potential digestibility indicators plus pH overall and in the different treatment groups (Trial 1, 2, 3) Fig. A1. Fig. A2. Fig. A3.