AGRICULTURAL AND FOOD SCIENCE Agricultural and Food Science (2025) 34: 125–140 125 https://doi.org/10.23986/afsci.149470 In vitro evaluation of agro-industrial by-products in diets for cattle Sophie J. Krizsan1, Marcia Franco2, Marketta Rinne2, Mohammad Ramin3 and Farhang Fatehi4 1Inland Norway University of Applied Sciences, Faculty of Applied Ecology, Agricultural Sciences and Biotechnology, Department of Agricultural Sciences, Campus Blæstad, 2322 Hamar, Norway 2Natural Resources Institute Finland (Luke), FI-31600 Jokioinen, Finland 3Swedish University of Agricultural Sciences, Department of Applied Animal Science and Welfare, Umeå, Sweden 4University of Tehran, Campus of Agricultural and Natural Resources, Department of Animal Science, 31587-77871, Karaj, Tehran, Iran e-mail: sophie.krizsan@inn.no The objective of this in vitro batch culture study was to evaluate various agro-industrial by-products as feeds for cattle. In Experiment 1, a basal diet composed of grass silage and barley grain was replaced by additional barley grain, palm kernel cake (PKC), beet molasses (M), wheat bran (WB), or sugar beet pulp (SBP) at two levels of dietary supplementation. In Experiment 2, soybean meal (SBM) was compared with heat-treated rapeseed meal, dried distillers´ grains (DDG), rapeseed cake (RSC), and rapeseed meal (RSM) at two levels of dietary protein concentra- tion in diets based on grass silage supplemented with barley grain or SBP. Propionate and branched chain volatile fatty acids decreased, and CH4 production increased when energy by-products of higher fibre content replaced bar- ley grain. Diets incorporating PKC and WB were less fermentable, while M and SBP did not alter digestibility when replacing barley. Generally, incremental levels of protein in Experiment 2 linearly increased digestibility, utilisable crude protein (uCP), isobutyrate and valerate in vitro. Utilisable crude protein increased for all by-products that replaced SBM, except for RSM in diets including SBP. There was a positive linear effect of by-product level on uCP and valerate, and a negative linear effect for acetate. Diet digestibility was equivalent for DDG and SBM in diets composed with barley as well as SBP. Overall, by-products provided more uCP than diets supplemented with SBM. However, the intestinal digestibility of uCP of the different feeds can vary. In vivo production experiments are needed to ful- ly assess the potential of in vitro evaluated by-products as widely applicable alternative feeds in diets for cattle. Key words: barley, by-products, methane, protein, soybean meal, utilisable crude protein Introduction The growing worldwide population, which is predicted to reach 11 billion people by the next century, will demand a secure and increased global food supply. Despite the increased food production in the past 50 years, not every- one has access to sufficient protein and energy within their diet (FAO et al. 2020). Additionally, food consumption and food trade patterns within developed countries have evolved, mainly towards an increased consumption of high-value foods with a larger environmental footprint, such as meat and refined dairy products. Currently, the environmental issues which are primarily raised within ruminant livestock production systems are related to great- er use of agrochemicals and greenhouse gas emissions thereby contributing to global warming (Cederberg et al. 2019). This issue has launched an aim across several countries in Northern Europe to ensure, not only increased, but also sustainable production of food. It is likely that despite climate change, Northern Europe will also in the future have access to the most important natural resources such as agricultural arable land, pastures and water for food production (Olesen et al. 2011). Dairy foods have a beneficial nutrient composition and hold important characteristics such as hypotensive and muscle protein synthesis stimulation effects (Givens 2020). This suggests an important role from dairy farming systems in future food production. Food production based on grass-fed ruminants can become more sustainable by developing grasslands through better management, feed evaluation, and improved varieties and through the implementation of efficient envi- ronmental goals (including CH4 mitigation strategies), and targeted supplementation of forage-based diets with human-inedible resources (e.g. agro-industrial by-products) to improve human-edible output in the production (Ertl et al. 2015b, 2016, Whelan et al. 2017, Krizsan et al. 2021). To utilize the genetic potential for milk production by modern dairy cows, grass-based diets are supplemented with high energy and protein supplements to increase feed and nutrient intake. Many by-products from the agricultural and food industries can be used in dairy cow diets. However, few studies have been conducted where by-products relevant to Nordic conditions have been evaluated in wider comparisons simultaneously. In vivo studies are both expensive and laborious, but use of an automated in vitro gas production technique enables a large comparative screening of suitable by-products in cattle diets. Received 4 November 2024 / Accepted 2 June 2025 The Scientific Agricultural Society of Finland ©This is an open access article under the CC BY 4.0 S.J. Krizsan et al. 126 Additionally, there has recently been significant progress in the development of the automated gas in vitro tech- nique, which enables a complete evaluation of ruminant digestion (Huhtanen et al. 2008), CH4 production (Ramin and Huhtanen 2012) and estimates of utilisable crude protein (uCP) (Edmunds et al. 2012). The primary objective of this in vitro study was to compare a wide variety of agro-industrial by-products, that are readily available in Northern Europe for inclusion in ruminant diets. The use of agro-industrial by-products in diets to ruminants seems particularly suitable due to a generally high fibre content. There is a limited ability of monogastric livestock species to digest fibre supporting an improved feed utilisation when agro-industrial by- products are fed to ruminants. We hypothesized that currently and commonly used human-edible barley grain and soybean meal (SBM) can be replaced by agro-industrial by-products with no or limited food use in grass silage-based diets for dairy cows. The objective of this study was to evaluate the effect of several agro-industrial by-products as feed supplements in grass silage-based diets fed to dairy cows on digestibility and fermentation, feed value traits (e.g. uCP), and production of CH4. Materials and methods The animals used for rumen fluid collection in this experiment were registered and cared for according to the guidelines approved by the Animal Care and Use Committee of Swedish University of Agricultural Sciences. The experiment was also carried out in accordance with the laws and regulations controlling experiments using live animals in Sweden. Experimental samples and diets To effectively evaluate energy and protein by-products, this study was conducted as two separate experiments. In the first experiment, energy by-products were evaluated (Experiment 1). In Experiment 1 diets were composed from grass silage and barley grain in a ratio of 700:300 g kg-1 of dietary dry matter (DM). The basal diet consist- ing of grass silage and barley grain was replaced with additional barley grain, palm kernel cake (PKC), beet molas- ses (M), wheat bran (WB), or sugar beet pulp (SBP) to inclusions of two levels of 200 and 400 g kg-1 of diet DM. Replacements were made to ensure that the grass silage to barley grain ratio remained constant across all diets (i.e. forage to barley grain ratio was 700:300, 560:240 and 420:180 of control diet and of the two inclusion levels of by-products) except when additional barley grain were used as replacement. Protein by-products were evalu- ated in the second experiment (Experiment 2). The diets used as controls were grass silage:barley grain and grass silage:SBP using a ratio 600:400 g kg-1 of dietary DM. Soybean meal (SBM) was used as the conventional crude protein (CP) source and was replaced with heat-treated rapeseed meal (Expro®; AAK Sweden AB, Karlshamn, Sweden), dried distillers´ grains with solubles (DDG) (AgrodrankTM90; Lantmännen Agroetanol AB, Norrköping, Sweden), rapeseed cake (RSC) or rapeseed meal (RSM). Inclusions of protein by-products were made at two levels of CP in the diets to increase the CP concentration by 20 g kg-1 of diet DM per increment, aiming to give dietary CP concentration of 146, 166 and 186, and 126, 146 and 166 g kg-1 of diet DM for barley grain and SBP based diets, respectively. Replacements were made so that all treatments had the same grass silage:barley grain or grass silage:SBP ratio across all experimental diets in line with the composition of diets in Experiment 1. The grass silage was harvested from a primary growth of a timothy (Phelum pratense) ley. The grass silage was har- vested from a primary growth of a timothy (Phleum pratense) ley and ensiled using an acid-based additive (Pro- myr XR 630, Perstorp, Sweden), which was applied at a rate of 3.5 l tonne-1. The grass was ensiled and stored in a bunker silo after wilting overnigh in the field. Samples of the agro-industrial by-products were provided by the feed manufacturer AB Västerbottens Fodercentral in Umeå, Sweden, if otherwise not stated. In vitro and in situ incubations Two rumen cannulated lactating Swedish Red cows fed ad libitum, a total mixed ration composed of 600 g kg-1 grass silage and 400 g kg-1 concentrate on DM basis, were used to collect rumen fluid for the in vitro incubations. Rumen fluid was collected from the same cows for all in vitro incubations at 2 h after morning feeding. Cows were separated individually by gates in alleys next to the collection area at the milking parlor. The rumen content was collected through the fistulas by grab samples from top to middle regions of the rumen (with vivid fermentation). The collected rumen content was squeezed and the fluid from each cow was strained separately through a dou- ble layer of cheesecloth into pre-heated (39 °C) insulated steel bottles that had been previously flushed with CO2. Agricultural and Food Science (2025) 34: 125–140 127 One thermos of almost 1500 ml of rumen fluid per cow was collected before each in vitro incubation and then immediately brought to the laboratory. In the laboratory, rumen fluid from the two cows was blended carefully in equal parts and filtered through four layers of cheesecloth into a bottle kept in a water bath at 39 °C and un- der constant CO2 saturation. Prior to the incubation of Experiment 2 the rumen fluid was pre-incubated for 3 h with a carbohydrate mixture according to the procedure described by Gidlund et al. (2018). In this procedure, a mixture of maltose (3.2 g), starch (1.6 g), xylose (1.6 g), pectin (1.6 g), and NaHCO3 (2.8 g) dissolved in buffer as described by Menke and Steingaß (1988) was added to the rumen fluid, which was stirred for 10 min at 39 °C. After 30 min, the top layer of foam was removed with a vacuum pump and the stirrer was turned on again. The rumen fluid was then incubated at 39°C under a constant flush of CO2 for an additional 2.5 h. Following pre-incubation, the rumen fluid was mixed with a low-N bicarbonate buffer (20:80 vol/vol), micro (0.24 ml in buffer solution) and macro minerals (482.35 ml in buffer solution) and resazurin (2.47 ml in buffer solution) according to the procedure described by Gidlund et al. (2018). Diets of 1000 mg DM and 500 mg DM for Experiment 1 and 2, respectively, were previously weighed directly in 250-ml serum bottles (Schott, Mainz, Germany), which were flushed with CO2 prior to incubation. All diets were incubated in 60 ml of the buffered rumen fluid for 48 h. Incubations were conducted at 39 °C in 36 bottles con- tinually agitated in three separate water baths throughout the entire incubation. All diets were incubated in three consecutive runs, resulting in three replicates per diet, and treatments were randomly allocated between differ- ent in vitro flasks. All runs included triplicate bottles with blanks divided equally in the three separate water baths. The fully automated gas in vitro system used in both experiments was a custom made device obtained from Wa- gening University in the Netherlands and have been described earlier by Ramin (2013) and by Gidlund (2017). The same two cows were used for a 288 h in situ incubation according to Huhtanen et al. (1994) to determine the concentration of indigestible neutral detergent fibre (iNDF) in all feed samples. Samples of 2 (±0.1) g were weighed in polyester bags of 11 µm pore size (07-11/5 Sefar Petex; Sefar AG, Heiden, Switzerland) as described by Krizsan et al. (2015). All samples were incubated in duplicates. Sample collection and calculations Gas production was automatically recorded and corrected to normal atmospheric pressure (101.3 kPa; Cone et al. 1996). Mean blank gas production within run was subtracted from the sample gas production. Digestion rate was calculated from the cumulative gas production curve of each replicated experimental diet and predicted based on digestibility using a dynamic mechanistic rumen model, as described by Huhtanen et al. (2008). A 2-pool Gompertz model was fitted to the cumulative gas production profile and parameter estimates were used in a dy- namic mechanistic rumen model (Huhtanen et al. 2008) to determine digestibility of potentially digestible DM. The digestibility equation by Allen and Mertens (1988) was solved for digestion rate using a rumen residence time of 50 h distributed between the two compartments in a ratio of 40:60. In Experiment 1, gas samples were drawn from each bottle using a gas tight syringe (Hamilton, Bonaduz, Switzer- land) at 2, 4, 8, 24, 32 and 48 h of incubation, while only at 24 and 48 h of incubation in Experiment 2. Methane production was calculated as described by Ramin and Huhtanen (2012). In brief, concentration of CH4 gas was de- termined by injecting 0.2 ml of collected gas into a star 3400 (CX series) gas chromatograph (Varian Chromatog- raphy, Palo Alto, CA, USA) equipped with a thermal conductivity detector. Calibration gas of a mixture of CO2 and CH4 (100 mmol mol-1) prepared by AGA Gas AB (Sundbyberg, Sweden) was used. Mean blank gas production within run was subtracted from sample gas production. In vivo predicted CH4 production was calculated as: CH4 (ml) = 265 (ml) x CH4 concentration (ml l-1) + total gas production (ml) x CH4 concentration (ml l-1) x 0.55 where CH4 represents cumulative CH4 produced at a given time point, 265 is the total headspace volume, and 0.55 is the ratio of CH4 emissions in the outflow gas from the in vitro system. A mean retention time of 50 h (20 h in the first compartment and 30 h in the second compartment), corresponding to the maintenance level of feed intake, was used in model simulations of in vivo predicted CH4 in Experiment 1. Liquid samples of 0.6 ml for NH3-N analysis were taken from each bottle using a plastic syringe at 24 and 48 h of incubation in Experiment 1, and at 8, 16, 24, and 30 h after incubation in Experiment 2. The liquid samples were preserved with 0.024 ml of 18 M H2SO4. Utilisable CP at 16 h was calculated as described by Edmunds et al. (2012): S.J. Krizsan et al. 128 uCP (g kg-1) = (NH3-Nblank + Nsample – NH3-Nsample) / (sample weight [mg DM]) × 6.25 × 1000 In both experiments, samples of 0.6 ml of rumen fluid were collected at 48 h of incubation from the bottles and immediately stored at –20 °C until being processed for volatile fatty acid (VFA) determination. Total VFA produc- tion was calculated as a sum of individual VFA by subtracting the mean value of blank within run and multiplying it with 60 ml (the volume of buffered rumen fluid). After 48 h incubation, all flasks were removed from the baths and placed into crushed ice to prevent fermentation. In vitro incubation residues were quantitatively transferred to the same 11-μm polyester bags that were used for the in situ incubation. In vitro true organic matter digestibility (TOMD) was determined for all diets in all runs by analyzing the ash-free NDF concentrations in the residues after the 48 h incubations and subtracting that from the amount of OM at the start of incubation according to Van Soest (1992). Mean blank true in vitro digestibility within run was subtracted from the sample in vitro TOMD. Chemical analyses All feed and by-product samples were dried at 60 °C for 48 h and were ground through a 1.0-mm screen using a cutting mill Retsch SM 2000 (Retsch GmbH, Haan, Germany) prior to chemical analysis and in vitro incubations. Samples for in situ incubations were ground with mortar and pestle, and manually sieved through a 2.5-mm sieve. Residual moisture of all feed samples was determined by oven drying for 16 h at 105 °C. Ash concentration was determined by ignition of the dried samples at 500 °C for 4 h. The samples, and in vitro and in situ residues were analyzed for NDF including sodium sulphite and heat stable α-amylase (Mertens et al. 2002) in an ANKOM200 Fiber Analyzer (Ankom Technology Corp., Macedon, NY, USA). Values of NDF and iNDF were expressed on an ash-free basis. Concentration of N was determined by Kjeldahl digestion of 1.0 g sample in 12 M H2SO4 using Foss Tecator Kjeltabs Cu (Höganäs, Sweden) in a Block Digestion 28 system (SEAL Analytical Ltd., Mequon, WI, USA) with deter- mination of total N by continuous flow analysis using an Auto Analyzer 3 (SEAL Analytical Ltd., Mequon, WI, USA). Crude protein (CP) was determined from N concentrations multiplied by 6.25. Individual VFA concentrations in rumen fluid samples were determined using a Waters Alliance 2795 UPLC system as described by Puhakka et al. (2016), and NH3 according to the method provided by the SEAL Analytical (Method nr G-102-93 multitest MT7) using an Auto Analyzer 3 (SEAL Analytical Ltd., Mequon, WI, USA). The protein by-products were analyzed in a commercial laboratory (Dairy One, Ithaca, NY, USA) for Cornell protein fractions according to Higgs et al. (2015), and for neutral and acid detergent insoluble CP. Statistical analysis Data were analyzed using the GLM procedure (SAS Inc. 2002–2003, Release 9.2; SAS Inst. Inc., Cary, NC, USA) of SAS with a model correcting for the fixed effects of run and experimental diet. Orthogonal polynomial contrasts were used to evaluate linear and quadratic responses to level of by-products, and to compare by-product diets with the control diet in both experiments (barley vs. by-products or SBM vs. by-products). p-values less than 0.05 were considered statistically significant and less than 0.10 indicated a tendency for significance. Results Experiment 1 Chemical composition of experimental feed ingredients for Experiment 1 is presented in Table 1. In situ iNDF values indicated a potential digestibility of the NDF fraction ranging between 705 and 920 g kg-1 for energy by- products (PKC and SBP, respectively) compared with 824 g kg-1 for barley. For protein by-products this ranged between 563 and 781 g kg-1 (RSM and DDG, respectively) compared with 975 g kg-1 for SBM. Crude protein concentrations were higher in PKC and WB compared with barley, while both M and SBP displayed lower concen- trations. Further, non-fibre carbohydrate concentrations were much higher for M and SBP compared to PKC and WB. Dietary feed composition of incubated experimental diets in Experiment 1 is given in Appendix 1. Agricultural and Food Science (2025) 34: 125–140 129 Measurements derived from the gas in vitro incubation of the basal diet (grass silage and barley), and with the replacements of barley and by-product feed ingredients at two levels of inclusion are presented in Table 2. In vitro TOMD was lower (p≤ 0.02) in diets including PKC and WB compared with barley. Ammonia-N measured in buff- ered rumen fluid at 8 h after start of the incubation was lower (p≤ 0.01) for M and SBP compared with barley, and lower at 24 h for M compared to barley. Furthermore, NH3-N at 24 h decreased (p≤ 0.02) with increased barley and by-product inclusion. There was a quadratic increase (p= 0.04) in total VFA production with the replacement of basal diet with bar- ley and by-product feed ingredients indicating that PKC, WB and SBP diets were most fermentable at the 200 g kg-1 inclusion level. All by-product ingredients resulted in a fermentation with higher (p< 0.01) molar proportion of acetate than diets with barley, which was also reflected in less (p≤ 0.05) propionate except for diets with WB inclusion. There was a linear decrease (p< 0.01) in propionate and increase (p< 0.01) in butyrate with increased dietary inclusion levels of PKC. All by-product ingredients induced a fermentation that was lower (p< 0.01) in molar proportion of butyrate when compared with diets including barley, except with PKC that had a higher (p= 0.03) molar proportion. Further, molar proportions of branched-chain VFA increased (p≤ 0.03) quadratically giving the highest molar proportion at 200 g kg-1 diet DM inclusion. The by-products PKC, M and SBP were lower (p≤ 0.03) in molar proportion of isobutyric acid than barley. Additionally, M and SBP were also lower (p< 0.01) in molar proportion of isovaleric acid compared with barley. Molar proportions of valeric acid was lower (p< 0.01) in M and SBP than in diets including barley. Molar proportions of caproic acid increased (p< 0.01) linearly with increased barley and PKC inclusion level. All by-product ingredients were lower (p≤ 0.04) in molar proportion of caproic acid, except PKC which was higher (p< 0.01), than diets with barley. Predicted CH4 production increased (p< 0.01) quadratically with higher dietary supplementation of barley and by- product ingredients. The by-products PKC and WB gave less (p< 0.01) in vivo predicted CH4 than diets including barley, while M inclusion increased (p< 0.01) in vivo predicted CH4 compared with barley. Table 1. Chemical composition of experimental dietary ingredients (g kg-1 DM unless otherwise stated) By-products Energy (Experiment 1) Protein (Experiment 2) Item Silage1 Silage2 Barley SBP3 SBM PKC M WB SBP4 Expro® DDG RSC RSM DM, g kg-1 259 255 779 917 854 922 718 896 917 906 877 921 911 OM 919 842 972 848 925 948 877 936 924 837 827 859 840 CP 143 157 129 78 496 179 101 139 79 387 315 378 392 NDF 552 611 239 339 237 606 NA 487 339 322 288 251 270 NFC5 200 286 582 578 688 88 773 267 495 584 589 670 641 iNDF 85 102 42 30 6 179 NA 106 27 129 63 109 118 1Grass silage used in Experiment 1; 2Grass silage used in Experiment 2; 3Sugar beet pulp used in basal diet in Experiment 2; 4Sugar beet pulp used as by-product in Experiment 1; 5Calculated using tabulated values of ether extracts for all feeds and for NDF for molasses from NRC (2001) and Alimon (2004). SBM: soybean meal; PKC: palm kernel cake; M: beet molasses; WB: wheat bran; SBP: sugar beet pulp; Expro®: heat-treated RSM; DDG: dried distillers’ grains; RSC: rapeseed cake; RSM: rapeseed meal; NA: not analysed; DM: dry matter; OM: organic matter; CP: crude protein; NDF: neutral detergent fibre; NFC: non-fibre carbohydrate; iNDF: indigestible neutral detergent fibre S.J. Krizsan et al. 130 Table 2. Measurements derived from the automated gas in vitro system of basal diet (grass silage and barley) replaced in two levels of diet dry matter (DM) with barley (B), palm kernel cake (PKC), beet molasses (M), wheat bran (WB) and sugar beet pulp (SBP) (Experiment 1) Item Basal diet 200 g kg-1 diet DM 400 g kg-1 diet DM p-value1 B PKC M WB SBP B PKC M WB SBP SEM C1 C2 C3 C4 Lin Quad TOMD, g kg-1 862 867 829 869 829 888 878 823 911 837 867 15.0 <0.01 0.25 0.02 0.73 0.83 0.57 NH3-N8, mg l-1 247 407 329 210 377 219 313 385 153 426 215 53.0 0.96 <0.01 0.44 0.01 0.49 0.35 NH3-N24, mg l-1 555 455 542 657 583 433 364 274 527 303 525 70.0 0.98 0.02 0.64 0.33 0.02 0.26 Total VFA, mmoles 1.95 2.24 2.12 2.20 2.12 2.58 2.22 1.82 2.25 1.90 2.24 0.150 0.10 0.99 0.16 0.24 0.78 0.04 Molar proportions, mmoles mole-1 Acetate 601 587 598 595 595 615 581 598 600 601 623 4.5 <0.01 <0.01 <0.01 <0.01 0.91 0.38 Propionate 242 236 220 238 232 228 230 201 244 227 224 3.4 <0.01 0.03 0.38 0.06 <0.01 0.24 Butyrate 117 132 136 128 127 116 144 151 122 128 116 2.4 0.03 <0.01 <0.01 <0.01 <0.01 0.06 Isobutyric acid 9.2 10.8 9.9 8.6 11.0 9.9 10.7 9.5 7.3 10.7 8.6 0.45 0.03 <0.01 0.81 <0.01 0.69 0.03 Isovaleric acid 7.4 9.0 8.1 6.7 9.2 7.7 8.5 7.6 5.1 8.7 6.6 0.50 0.10 <0.01 0.66 <0.01 0.44 0.04 Valeric acid 19.3 21.0 20.4 19.2 20.8 19.0 20.9 20.5 17.6 20.3 18.0 0.49 0.31 <0.01 0.41 <001 0.83 0.07 Caproic acid 4.0 5.1 7.5 4.6 4.6 4.6 5.6 12.0 4.4 4.6 4.7 0.32 <0.01 0.02 0.03 0.04 <0.01 0.60 kd, h -1 0.117 0.104 0.101 0.117 0.097 0.118 0.119 0.091 0.139 0.102 0.145 0.0100 0.12 0.12 0.24 0.06 0.54 0.14 CH4 2 ml g-1 DM 35.3 39.0 36.1 40.5 38.1 40.0 40.9 36.7 43.5 36.3 41.8 0.53 <0.01 <0.01 <0.01 0.09 <0.01 <0.01 SEM = standard error of mean; TOMD = true organic matter digestibility, NH3-N8 = ammonia N in sampled rumen fluid 8 h after start of incubation; NH3-N24 = ammonia N in sampled rumen fluid 24 h after start of incubation; Total VFA = volatile fatty acids (sum of all individual acids); kd = diet digestion rate. 1C1 = B vs. PKC; C2 = B vs. M; C3 = B vs. WB; C4 = B vs. SBP; Lin = linear effect of supplementary inclusion level (significant if at least one treatment display a linear effect); Quad = quadratic effect of supplementary inclusion level (significant if at least one treatment display a quadratic effect); 2Predicted CH4 in vivo Agricultural and Food Science (2025) 34: 125–140 131 Experiment 2 In Experiment 2, levels of CP in the by-products ranged between 315 and 392 g kg-1 DM (DDG and RSM) compared to 496 g CP kg-1 DM in SBM (Table 3). Inclusions of protein by-products were made at two levels of CP in the diets, which resulted in dietary CP of 146, 166, and 186 g kg-1 DM, respectively, for diets based on silage and barley, and dietary CP of 126, 146 and 166 g kg-1 DM, respectively, in diets based on silage and beet fibre. Dietary feed com- position of incubated experimental diets in Experiment 2 is given in Appendix 2. Table 3 reports the protein fractions according to Higgs et al. (2015), and neutral detergent in soluble CP (NDICP) and acid detergent insoluble CP (ADICP) of SBM and protein by-products. There were some differences in protein fractions among the different supplements. Dried distillers´ grains were the highest in ammonia (A1) and indigest- ible protein (C), while SBM and Expro were clearly higher in insoluble true protein (B1). Moreover, RSC and RSM were the supplements highest in soluble true protein (A2). The differences in fibre bound protein fractions were relatively small between the protein supplements, but SBM and DDG were highest in ADICP among the feeds. This rendered the smallest value for DDG of the difference between NDICP and ADICP. The effect of two inclusion levels of protein by-products replacing SBM on digestibility, uCP, fermentation parameters, and CH4 production in diets based on grass silage supplemented with barley grain or SBP are provided in Tables 4 and 5, respectively. The results indicated that higher dietary CP concentration linearly increased (p= 0.03) TOMD in diets composed of barley and SBM. All by-product inclusions except DDG decreased (p≤ 0.05) TOMD compared to SBM, except for RSC in the diet based on SBP. Generally, uCP increased linearly (p< 0.01) with increased CP concentration of the diets. All by-product inclusions gave higher (p< 0.01) estimates of uCP than SBM, except for RSM in the diet based on SBP. Molar proportions of acetate decreased (p< 0.01) linearly with increased dietary CP concentration in diets composed of barley and SBP. Notably, DDG generated less (p< 0.01) acetate and butyrate, and more (p< 0.01) propionate than SBM in diets with both barley and beet fibre. Expro and RSM also resulted in higher (p≤ 0.03) propionate than SBM in diets with beet fibre. Molar proportions of butyrate and valerate increased linearly (p≤ 0.05) with higher dietary CP concentration in diets with barley and SBP. In diets with barley, isobutyrate also increased (p< 0.01) linearly with increased dietary CP concentration. All by-products gave more (p≤ 0.03) valerate than SBM in diets with barley, but only with Expro and DDG when diets were composed with SBP. In diets with barley, DDG and RSC gave less (p≤ 0.01) CH4. In diets with SBP, Expro, DDG and RSM produced less (p≤ 0.04) CH4 compared with diets with SBM. Table 3. Protein fractions (g kg-1 CP) according to Higgs et al. (2015), and neutral and acid detergent insoluble crude protein of soybean meal and protein by-products (g kg-1 DM) Item SBM1 Expro DDG RSC RSM Cornell protein fractions2 A1 55 100 307 145 131 A2 2 27 35 152 212 B1 673 600 243 431 344 B2 128 164 123 180 213 C 142 109 291 92 100 Fibre-bound protein fractions3 NDICP 127 104 127 102 122 ADICP 67 42 89 35 39 NDICP – ADICP 60 62 38 67 83 1SBM= soybean meal; Expro®= heat-treated RSM; DDG = dried distillers’ grains; RSC = rapeseed cake; RSM = rapeseed meal; 2A1 = ammonia; A2 = soluble true protein; B1 = insoluble true protein; B2 = fibre-bound protein; C = indigestible protein; 3NDICP = neutral detergent insoluble crude protein; ADICP = acid detergent insoluble crude protein S.J. Krizsan et al. 132 CP = crude protein; Expro® = heat-treated rapeseed meal; DDG = dried distillers’ grains; RSC = rapeseed cake; RSM = rapeseed meal; SEM = standard error of mean; TOMD = true organic matter digestibility; uCP = utilisable crude protein; Total VFA = volatile fatty acids (sum of all individual acids); kd = diet digestion rate. 1C1 = SBM vs. Expro; C2 = SBM vs. DG; C3 = SBM vs. RSC; C4 = SBM vs. RSM; Lin = linear effect of supplementary inclusion level (significant if at least one treatment display a linear effect). 2Quadratic term was not significant. Table 4. Effect of increasing level of agro-industrial by-products replacing soybean meal (SBM) on digestibility, estimated utilisable crude protein, fermentation parameters and CH4 production in diets based on silage and barley (Experiment 2) Basal diet 146 g CP kg-1 DM Diets 166 g CP kg-1 DM Diets 186 g CP kg-1 DM p-value1,2 Item SBM Expro DDG RSC RSM SBM Expro DDG RSC RSM SEM C1 C2 C3 C4 Lin TOMD, g kg-1 849 856 849 864 852 857 869 849 859 855 853 3.3 <0.01 0.60 0.01 0.02 0.03 uCP, g kg-1 DM 150 158 164 165 161 163 167 179 182 172 171 0.8 <0.01 <0.01 <0.01 <0.01 <0.01 Total VFA, mmole 3.54 3.54 3.28 3.48 3.58 3.62 3.58 3.57 3.43 3.27 3.34 0.118 0.28 0.37 0.27 0.52 0.37 Molar proportions, mmoles mole-1 Acetate 591 590 586 585 594 591 589 587 579 584 590 1.9 0.15 <0.01 0.89 0.66 0.01 Propionate 228 226 229 233 221 228 227 227 241 227 224 1.9 0.47 <0.01 0.18 0.71 0.41 Butyrate 111 111 107 105 110 109 108 109 102 107 108 1.1 0.20 <0.01 0.42 0.30 <0.01 Isobutyrate 19 20 20 19 20 19 20 20 20 21 20 0.4 0.94 0.12 0.33 0.91 <0.01 Valerate 27 27 29 31 29 28 28 31 31 32 30 0.5 <0.01 <0.01 <0.01 0.03 <0.01 Isovalerate 25 26 28 27 26 25 27 26 27 29 28 1.1 0.60 0.90 0.50 0.91 0.10 kd, h -1 0.073 0.073 0.072 0.074 0.077 0.077 0.077 0.068 0.077 0.077 0.082 0.0028 0.09 0.80 0.42 0.10 0.26 CH4, ml g-1 DM 53.5 53.3 52.1 50.9 49.6 51.8 53.9 51.4 49.8 51.9 52.9 1.06 0.09 <0.01 0.01 0.24 0.33 Agricultural and Food Science (2025) 34: 125–140 133 Table 5. Effect of increasing level of agro-industrial by-products replacing soybean meal (SBM) on digestibility, estimated utilisable crude protein, fermentation parameters and CH4 production in diets based on silage and beet fibre (Experiment 2) Basal diet 126 g CP kg-1 DM Diets 146 g CP kg-1 DM Diets 166 g CP kg-1 DM p-value1,2 Item SBM Expro DDG RSC RSM SBM Expro DDG RSC RSM SEM C1 C2 C3 C4 Lin TOMD, g kg-1 841 850 837 856 838 839 855 845 846 846 842 6.0 0.05 0.85 0.09 0.05 0.33 uCP, g kg-1 DM 140 151 152 155 152 151 158 167 171 162 162 1.0 <0.01 <0.01 <0.01 0.08 <0.01 Total VFA, mmole 3.56 3.73 3.41 3.50 3.73 3.45 3.89 3.68 3.58 3.60 3.74 0.158 0.11 0.09 0.37 0.18 0.08 Molar proportions, mmoles mole-1 Acetate 633 628 622 623 627 623 623 619 613 620 622 2.6 0.07 <0.01 0.45 0.28 <0.01 Propionate 230 222 228 231 222 228 224 225 238 228 226 1.6 0.03 <0.01 0.17 0.02 0.70 Butyrate 81 85 84 80 85 83 86 86 81 83 86 1.3 0.50 <0.01 0.20 0.34 0.05 Isobutyrate 17 18 19 18 18 18 18 19 17 18 18 0.7 0.24 0.28 0.86 0.70 0.32 Valerate 22 24 26 26 25 24 25 28 27 26 26 0.6 <0.01 0.01 0.30 0.54 <0.01 Isovalerate 24 23 26 23 23 25 24 23 24 25 22 1.1 0.30 0.94 0.58 0.95 0.73 kd, h -1 0.076 0.077 0.074 0.081 0.079 0.077 0.078 0.077 0.082 0.088 0.083 0.0030 0.54 0.24 0.06 0.43 0.05 CH4, ml g-1 DM 52.8 52.1 52.0 50.9 49.8 51.9 55.6 51.6 51.8 54.9 51.1 1.10 0.04 0.01 0.14 0.02 0.36 CP = crude protein; Expro® = heat-treated rapeseed meal; DDG = dried distillers’ grains; RSC = rapeseed cake; RSM = rapeseed meal; SEM = standard error of mean; TOMD = true organic matter digestibility; uCP = utilisable crude protein; Total VFA = volatile fatty acids (sum of all individual acids); kd = diet digestion rate. 1C1 = SBM vs. Expro; C2 = SBM vs. DDG; C3 = SBM vs. RSC; C4 = SBM vs. RSM; Lin = linear effect of supplementary inclusion level (significant if at least one treatment displays a linear effect). 2Quadratic term was not significant, except for propionate (p < 0.01) and CH4 production (p = 0.02). S.J. Krizsan et al. 134 Discussion The aim of the current in vitro study was to explore the various effects of ingredients included in whole diets, because ruminants are fed multiple ingredients that interact and complement each other. Therefore, single feeds were not incubated in vitro, but dietary composition was restricted to include a single by-product to elucidate the most suitable dietary by-product inclusions. Since by-products originate from agricultural crops and human food processing industries, their chemical composition varies markedly between, as well as potentially within by- products (Rinne et al. 2014, García-Rodríguez et al. 2019). Feeds used in this study originated only from one batch of that particular by-product, and to obtain a reliable feed value, a manufacturer should consistently repeat analyses to monitor batch-to-batch uniformity of the product. However, all current feeds were within the typical range of that particular feed material when compared with published values in feed tables, e.g. Feedipedia (2025), Luke (2025) and NorFor (2025), and the products were obtained from commercial sources so that they represent the options available for practical farms. Experiment 1 The energy by-products used in this study originated from food milling or extraction processes that generated fibrous residues except for molasses. Wheat bran and SBP are the most widely used nonforage fibre sources (NFFS) derived from agro-industries that are used in Swedish ruminant production systems (Swedish Board of Agricul- ture 2022) while PKC is imported by feed industry to be included in commercial concentrate mixtures. The feeds chosen for the experiment showed a wide range in their fibre content and quality most clearly described by their iNDF content, which was highest in PKC, intemediate in WB and lowest in SBP, while M did not contain any fibre. Starch content of the samples was not analysed as the study focused on by-products low in starch concentration, but in general, starch is a key dietary component in commercial concentrate feeds to ruminants. Increasing diet fibre content via by-product addition increased acetate production in the incubation medium, and CH4 production (per g DM) decreased in line with the lower TOMD. All energy by-products in this study ex- hibited a higher proportion of acetate when replacing barley, but PKC decreased molar proportion of propionate and increased that of butyrate. Only M increased molar proportion of propionate, while WB and SBP induced no change. Ertl et al. (2015a) observed a lower acetate to propionate ratio in vitro for a diet supplemented with by- -products compared with a control concentrate mixture and attributed this to more easily fermentable fibre in the by-products. This was assumed to stimulate propionate formation and may be beneficial, particularly during early lactation, through improved energy efficiency. Molasses was clearly highest in NFC of all energy by-products included in this study. The higher NFC in M and SBP can also explain the lower NH3 in Experiment 1, which is also in line with the lower CP concentration in these energy by-products. Ertl et al. (2015b) completely replaced cereals and pulses with agro-industrial by-products in diets to organic cows, which generated a slightly lower energy and CP concentration in the diet, but did not affect any production traits except milk urea concentration that was decreased when cows were fed by-products. Based on the results obtained by Ertl et al. (2015b), Ertl et al. (2016) conducted an experiment where cereals and field beans were replaced with wheat bran and SBP without any additional protein supplementation. They concluded that no pro- duction traits were affected, but that the results were too limited to be considered valid for post-peak lactating cows and that there were trade-offs in the use of nonforage fibre sources (NFFS) and current efficiency criteria of dairy production. Moreover, Guinguina et al. (2021) reduced starch content in the diets of early lactating cows from 170 to 53 g kg-1 DM by replacing cereals with fibrous by-products. In accordance with Dann et al. (2014) and the in vitro results of PKC and WB in this study, TOMD decreased, but also digestibility of NDF, neutral detergent solubles, and CP when fed with by-products. There were no effects on DM intake or any production traits, but daily enteric CH4 production decreased by 10% (Guinguina et al. 2021). Replacement of barley with NFFS has been reported to result in greater milk yield, an effect explained by an im- proved silage DM intake (e.g. Huhtanen 1993) or with no change in intake (Huhtanen 1987). It has been specula- ted that greater milk yield with fibrous supplements, despite a lower intake of energy, may be related to positive associative effects from a combination of different carbohydrate sources compared with barley (Huhtanen 1991). Carbohydrates fermented at different rates compared with barley can improve microbial protein synthesis in grass silage-based diets (Huhtanen 1987). Additionally, the CP content of NFFS is sometimes higher than that of barley, which could explain a general increase in milk yield (Huhtanen 1993). Dann et al. (2014) partly replaced ground corn with wheat middlings and beet pulp to evaluate the effect of increasing fibre content of the diets from 342 to 380 g kg-1 DM for dairy cows without any effects on production traits except a slight decrease in milk urea. Agricultural and Food Science (2025) 34: 125–140 135 The high fibre diet also decreased TOMD and increased the molar proportion of butyrate in rumen fluid. In accordance with Dann et al. (2014), the more fibrous by-products, i.e., PKC and WB in this study decreased in vitro TOMD. The lower digestibilities of PKC and WB compared with barley are in line with their high NDF and iNDF concentrations. Generally, the variability in feed value can be assumed to be greater if individual by-products are used rather than mixtures consisting of several by-products. Huhtanen (1993) and Huhtanen et al. (1995) suggested that variable production responses in dairy cows fed NFFS supplements can be explained by the rumen fermentation profile. This is in line with the differences observed in in vitro fermentation when barley was replaced by different agro- industrial by-products in this study. Ruminal branched-chain VFA (i.e. isobutyric and isovaleric acid) and valeric and caproic acid primarily originate from degradation of dietary protein (Tedeschi et al. 2000), and in the current study they generally decreased in diets containing by-products compared with diets supplemented only with bar- ley, especially for molasses and SBP. Changes in NH3-N in buffered rumen fluid, particularly for M, can be difficult to explain and can, in addition to diet degradation, be a result of degradation of feed particles from the rumen fluid medium, or at later time points, be due to microbial lysis and degradation. Experiment 2 Several in vivo studies have demonstrated that SBM can be successfully replaced with RSM in grass silage-based diets for dairy cows without compromising production (e.g. Shingfield et al. 2003, Huhtanen et al. 2011, Martineau et al. 2013, Gidlund et al. 2015). Although the Finnish ruminant livestock sector has not used soya bean-based feeds since 2018 (Rinne et al. 2023), there is still a substantial import of soybean meals to Northern Europe. Farmers are also exposed to fluctuations in feed prices on the global market, and there is a low marginal efficiency of SBM of just 10% in milk production (Huhtanen et al. 2011). However, the national supply of RSM may be insufficient under Nordic conditions. For example, in Sweden, RSM comprises 74% of the agro-industrial by-products fed to ruminants and imported RSM comprises 17% of the total amount of agro-industrial by-products used in rumi- nant production systems, while there is a surplus of DDG being exported. Further, according to feed companies, imported SBM comprises 88% of the total SBM used for cattle in Sweden (Swedish Board of Agriculture 2022). Increasing cultivation of rapeseed will likely not meet the demand of protein feed to livestock in Northern Europe, due to the necessity for other crops before rapeseed can return to the crop rotation. Oilseeds (such as rapeseed or turnip rape) are valuable crops in grain-dominated crop rotations but should not occur more than every five to six years, as they carry risks especially for fungal diseases (Bernes and Gustavsson 2016). All protein supplements evaluated in this study were within a CP concentration ranging from 315 g kg-1 DM in DDG to 392 g kg-1 DM in RSM, and DDG differed from the other protein by-products because it did not decrease TOMD, which can be explained by its relatively low proportion of iNDF compared with the other by-products. DDG also showed the highest uCP concentration of the protein feeds evaluated. Dairy cow studies have however shown reduced concentration of milk protein and total tract CP digestibility of DDG compared to PKC and RSM (Karlsson et al. 2018, Pang et al. 2018). Pang et al. (2018) explained the reduction in CP digestibility by a potential heat damage of the protein in DDG during the drying process. Gaillard et al. (2017) gradually replaced SBM and canola cake with DDG to dairy cows, and observed a negative effect on both milk yield and protein with increased inclusion of DDG. Supplementation with DDG affected rumen fermentation differently than other protein feeds by increasing the proportion of propionate and decreasing that of butyrate with a subsequent decrease in CH4 production. A reason for this could be that propionate and CH4 production requires H2, and since propionate production increased, less hydrogen was available for CH4 production. There is a high correlation between CH4 production and digestibility (Ramin and Huhtanen 2013) and this was evident in the RSM diet, where NDF digestibility decreased, TOMD tended to decrease, and CH4 production also decreased. The same pattern was also observed when SBM was replaced by RSC in diets based on silage and barley, in accordance with the results by Jentsch et al. (2007). By-products provided more uCP in comparison to SBM, which could be explained by the higher proportions of ammonia and soluble true protein (fractions A1 and A2; Table 2), which may supply a greater amount of substrate to produce microbial protein flowing to the duodenum. The A1 fraction was particularly high in DDG potentially explaining the good evaluation as protein supplement in vitro. Further, DDG having a greater proportion of indigestible protein (both C fraction and ADICP) than all other protein supplements is in line with the reported decrease in CP digestibility by Karlsson et al. (2018) and Pang et al. (2018). Finally, the subtraction of the ADICP from NDICP was shown to be greater in RSC and RSM than in SBM, and it may contribute to more potentially available fermentable substrate for ruminal bacteria to produce microbial protein. Generally, reduced digestibility S.J. Krizsan et al. 136 was more prominent in the diets based on silage and barley than the diets based on silage and SBP. This could be explained by a better rumen environment when digestible fibre from the beet-based feed replaced the starch in barley (Huhtanen 1993). The increase in uCP in response to incremental levels of dietary CP concentration suggested that all by-products qualified as potential protein feed sources to ruminants. A high uCP level, defined as the sum of microbial protein (MP) and rumen undegraded protein (RUP) (Edmunds et al. 2012), indicates that there is a higher proportion of utilizable protein substrate available in the duodenum. In the in vitro uCP estimation, RUP and MP are simultane- ously estimated and cannot be differentiated. According to Edmunds et al. (2012), validation using in vivo data is recommended, and based on the evaluation of Gidlund et al. (2018), the uCP method ranked the feeds similarly as in vivo data measuring the flow of protein into duodenum. Although uCP was used, it is not possible to differ- entiate between RUP and MP, and it is likely that Expro, RSC, and RSM have a relatively high proportion of RUP due to decreased TOMD when those by-products replaced SBM in this in vitro evaluation. On top of total amount of amino acids entering the small intestine, the profile also plays a role and there are e.g., indications that rape- seed based feeds may provide a more balanced amino acid profile to dairy cows than those based on soya bean protein (Rinne et al. 2015). Conclusions When comparing the energy by-products, replacing barley with M and SBP in grass silage-based diets did not decrease diet TOMD in buffered rumen fluid in vitro. However, both M and SBP inclusion changed rumen fermen- tation profile towards more acetate and less butyrate. Inclusion of a mixture of NFFS and M in a concentrate is more likely to not induce changes in the rumen fermentation profile. In the protein by-product experiment, DDG had the same digestibility performance as SBM in both diets. Diets with DDG decreased acetate and butyrate, while propionate proportion increased compared with diets with SBM. Utilisable CP was, on average, higher in diets based on silage and barley than silage and SBP. Overall, by-products provided more uCP than diets supplemented with SBM. However, the amino acid profile and intestinal digest- ibility of uCP of the different feeds can vary and is necessary to fully evaluate the true protein value of the diet. 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Dietary feed composition in Experiment 1 Diets1 Basal diet First supplementation level Second supplementation level Dietary composition B PKC M WB SBP B PKC M WB SBP Grass silage 695 554 554 560 561 559 420 420 420 425 421 Barley (B) 305 446 243 246 246 245 580 184 184 186 185 Palm kernel cake (PKC) - 203 - - - 396 - - - Molasses (M) - - 194 - - - 395 - - Wheat bran (WB) - - - 193 - - - 389 - Sugar beet pulp (SBP) - - - - 196 - - - 395 Chemical composition Dry matter, g kg-1 418 491 520 476 510 516 561 617 536 604 536 Organic matter 935 943 938 924 935 933 950 940 912 935 916 Crude protein 139 137 147 131 139 127 135 155 124 139 130 Neutral detergent fibre 457 412 487 368 462 433 370 516 276 468 182 Appendix 2. Dietary feed composition in Experiment 2 (g kg-1 DM if not otherwise stated) Diets1 Basal diet First supplementation level Second supplementation level Dietary composition SBM Expro DDG RSC RSM SBM Expro DDG RSC RSM Grass silage 614 577 563 538 561 564 540 511 462 508 513 Barley 386 362 353 337 352 354 339 321 290 319 322 Soybean meal (SBM) - 61 - - - - 121 - - - - Expro - - 84 - - - - 169 - - - Dried distillers´grains (DDG) - - - 125 - - - - 248 - - Rapeseed cake (RSC) - - - - 86 - - - - 173 - Rapeseed meal (RSM) - - - - - 82 - - - - 165 Chemical composition Dry matter, g kg-1 457 481 495 509 497 494 505 533 561 537 532 Organic matter 874 877 871 868 873 871 880 868 862 871 868 Crude protein 146 167 166 167 166 166 188 187 188 186 187 Neutral detergent fibre 467 454 455 445 449 451 440 443 423 430 435 1 The diets used as controls were grass silage:barley grain. Soybean meal (SBM) was used as the conventional crude protein (CP) source and was replaced with heat-treated rapeseed meal (Expro®; AAK Sweden AB, Karlshamn, Sweden), dried distillers´ grains with solubles (DDG) (AgrodrankTM90; Lantmännen Agroetanol AB, Norrköping, Sweden), rapeseed cake (RSC) or rapeseed meal (RSM). Inclusions of protein by- products were made at two levels of CP in the diets to increase the CP concentration by 20 g kg-1 of diet dry matter per increment, aiming to give dietary CP concentration of 146, 166 and 186 g kg-1 of diet dry matter. S.J. Krizsan et al. 140 Appendix 3. Diets1 Basal diet First supplementation level Second supplementation level Dietary composition SBM Expro DDG RSC RSM SBM Expro DDG RSC RSM Grass silage 605 571 559 539 557 560 537 512 473 510 515 Beet fibre 395 373 365 352 364 365 351 334 308 333 336 Soybean meal (SBM) - 56 - - - - 113 - - - - Expro - - 76 - - - - 153 - - - Dried distillers´grains (DDG) - - - 109 - - - - 219 - - Rapeseed cake (RSC) - - - - 79 - - - - 158 - Rapeseed meal (RSM) - - - - - 75 - - - - 149 Chemical composition Dry matter, g kg-1 516 536 546 556 548 546 555 576 595 580 575 Organic matter 844 849 843 842 845 844 853 843 840 846 843 Crude protein 126 147 146 147 146 146 168 166 167 166 166 Neutral detergent fibre 504 489 490 480 484 486 474 476 457 464 469 1The diets used as controls were grass silage:sugar beet pulp. Soybean meal (SBM) was used as the conventional crude protein (CP) source and was replaced with heat-treated rapeseed meal (Expro®; AAK Sweden AB, Karlshamn, Sweden), dried distillers´ grains with solubles (DDG) (AgrodrankTM90; Lantmännen Agroetanol AB, Norrköping, Sweden), rapeseed cake (RSC) or rapeseed meal (RSM). Inclusions of protein by- products were made at two levels of CP in the diets to increase the CP concentration by 20 g kg-1 of diet DM per increment, aiming to give dietary CP concentration of 126, 146 and 166 g kg-1 of diet dry matter. In vitro evaluation of agro-industrial by-products in diets for cattle Introduction Materials and methods Experimental samples and diets In vitro and in situ incubations Sample collection and calculations Chemical analyses Statistical analysis Results Experiment 1 Experiment 2 Discussion Experiment 1 Experiment 2 Conclusions Acknowledgement References