13363 ABSTRACT Expanded basic research is needed to discover and develop wider selection of dietary additives that are eco- nomically feasible without compromising animal perfor- mance or health. The objective of this study was to evalu- ate the effect of 2 feed additives as methane-mitigating agents in milk production systems. Four multiparous Nordic Red dairy cows were assigned to a 4 × 4 Latin square experiment with four 28-d periods using respira- tion chambers. The control diet (CON) consisted of grass silage and dietary concentrates mixed at forage-to-con- centrate ratio of 65:35 on DM basis. The 3 experimental treatments consisted of the CON diet supplemented with 0.2% of biochar with fibrolytic enzymes and live yeast additive (BFE) or with 0.75% or 1.5% CaO2 on a DM basis (CaPe1 and CaPe2, respectively). Calcium perox- ide was included in the concentrate pellet; the mixture of biochar, fibrolytic enzymes and live yeast was added to the diet during TMR preparation; and diets were fed as TMR 4 times daily. Feeding BFE had minor effect on the parameters evaluated in the experiment. Feed- ing CaPe resulted in linear reductions in DM, OM, CP, ether extract (EE), NDF, and gross energy (GE) intake compared with CON. Yields of milk, ECM, fat, protein, lactose and TS decreased linearly, but milk composition and SCC were not affected. Apparent total-tract digest- ibilities of DM, OM, CP, EE, NDF, and GE decreased linearly, whereas excretion of Ca and P in feces increased linearly with increasing CaO2 level. We found that CaPe1 tended to decrease the molar proportion of acetate and increased that of propionate, whereas butyrate increased linearly. Dietary CaO2 inclusion decreased daily CH4 production (g/d) linearly by 15.0%, but CH4 yield (g/ kg DM or OM intake) and intensity (g/kg milk or ECM) were not affected. Hydrogen production (g/d) and yield (g/kg DMI) decreased at CaPe1 but plateaued at CaPe2. Feeding CaO2 increased richness of ciliate protozoa and influenced rumen bacteria and ciliate protozoa commu- nity structure. No such effect was observed on archaea or anaerobic fungi. The feed additives BFE and CaO2 were not effective CH4-mitigating agents under the conditions of the present experiment. Key words: grass silage, feed additive, greenhouse gases, microbiota, respiration chamber INTRODUCTION Ruminants have evolved a symbiotic relationship with anaerobic microbes to efficiently digest fibrous plants and convert them into milk and meat, contributing to human food security. However, a natural byproduct of this digestion process is enteric methane (CH4), which is a short-lived gas with atmospheric lifetime of ~12 yr (Wahlen, 1993). Because of the high global warming po- tential of CH4, decreasing the emissions of enteric CH4 from ruminant production is considered as one of the goals in strengthening the sustainability of the livestock sector. In Finland, CH4 emissions from dairy production represent ~2.5% of the national anthropogenic GHG emissions (Huhtanen et al., 2022), whereas global dairy sector’s share of GHG emissions is 2.7% (FAO, 2010). To date various enteric CH4 mitigation strategies have been evaluated. Among them, short-term strategies include modification of diets using feed additives that suppress CH4 production, whereas long-term strategies include genetic selection of livestock for improved effi- ciency (Hayes et al., 2013; Knapp et al., 2014) combined with precision management that supports animal health and longevity by ensuring that animals have the capacity to produce to their ultimate genetic potential by offering balanced diets (González et al., 2018). To reduce global CH4 emissions by at least 30% from 2020 levels by 2030 (Malley et al., 2023), the livestock sector should combine both short- and long-term strategies. Effects of calcium peroxide or biochar-enzyme feed additives on milk production, enteric methane emissions, and ruminal microbiota in Nordic Red dairy cows J. Vattulainen,1 A. R. Bayat,1 T. Stefański,1 M. Rinne,1 and I. Tapio2* 1Animal Nutrition, Production Systems, Natural Resources Institute Finland (Luke), FI-31600 Jokioinen, Finland 2Genomics and Breeding, Production Systems, Natural Resources Institute Finland (Luke), FI-31600 Jokioinen, Finland J. Dairy Sci. 108:13363–13380 https://doi.org/10.3168/jds.2025-27123 © 2025, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/). The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-25. Nonstandard abbreviations are available in the Notes. Received June 18, 2025. Accepted September 11, 2025. *Corresponding author: ilma.tapio@​luke​.fi https://orcid.org/0009-0001-6268-2014 https://orcid.org/0000-0002-4894-0662 https://orcid.org/0000-0001-5553-9941 https://orcid.org/0000-0001-6323-0661 https://orcid.org/0000-0002-0752-9551 https://adsa.org/jds-abbreviations-25 mailto:ilma.tapio@luke.fi 13364 Journal of Dairy Science Vol. 108 No. 12, 2025 Currently, only a limited number of effective feed ad- ditives targeted for reducing enteric CH4 are approved for food production (Hegarty et al., 2021). Thus, there is a need to expand basic research aiming to discover and develop a wider selection of additives that are economi- cally feasible and do not compromise animal performance or health (Morgavi et al., 2023). Oxidizing agents such as calcium peroxide (CaO2) are commonly used in agriculture, environmental res- toration, and pharmacy. During hydrolysis, CaO2 pro- duces calcium hydroxide, hydrogen peroxide, oxygen and water. Due to its oxygenation properties, CaO2 can be used in anaerobic digestors to create microaerobic conditions, where limited oxygen release can stimulate the growth of aerobic or facultatively aerobic microbes, positively affecting the hydrolysis of cellulose (Tsa- pekos et al., 2017). However, higher CaO2 doses can have a substantial inhibitory effect on methanogenesis (Wang et al., 2019). Recently, CaO2 was used as an antimethanogenic feed supplement in a dairy-beef bull experiment that demonstrated 16% to 27% reduction in daily CH4 production (Roskam at al., 2024). Other ox- ygen-releasing agents have been successfully tested for significant reductions of gaseous emissions from stored cattle manure slurry (Thorn et al., 2022; Connolly et al., 2023) and in an in vitro rumen simulation system (O’Donnell et al., 2024). These observations suggest that oxidizing agents could potentially be considered as versatile, economically feasible, and environmentally friendly dietary supplements to reduce CH4 emissions in the beef sector, but to our knowledge, no similar in vivo experiments on dairy cows have been published. Biochar has demonstrated beneficial impacts across different agricultural applications. When used as a feed additive for ruminants, biochar has shown potential to im- prove the production performance and health of animals (Nair et al., 2023) or to reduce enteric CH4 emissions in growing steers (Winders et al., 2019; Nair et al., 2023). Nevertheless, some studies demonstrated no effect of bio- char on animal performance, rumen fermentation, or CH4 emissions (Dittmann et al., 2024), indicating more re- search is needed on the subject. A combination of biochar with fibrolytic enzymes and live yeast (BFE) has shown positive effects on CH4 mitigation in vitro and milk pro- duction in vivo (personal communication, Branko Petruj- kic, GoBioFarm Ltd., Iisalmi, Finland). It is expected that the improved diet digestibility via fibrolytic enzyme and provision of optimal conditions for rumen microbiota via biochar could lead to increased milk production in addi- tion to the potential to reduce CH4 production, resulting in reduced CH4 intensity (g/kg milk). Therefore, BFE was chosen as an additive to be tested in this study. The objective of this study was to evaluate the effect of dietary supplementation with BFE and 2 levels of CaO2 on enteric CH4 production, milk production, rumen fer- mentation, and ruminal and fecal microbiota composition in lactating Nordic Red dairy cows. The hypothesis was that CaO2 will reduce enteric CH4 production linearly, and BFE will reduce enteric CH4 or affect milk produc- tion positively by improving nutrient digestibility. MATERIALS AND METHODS Animals, Experimental Design, and Diets The experiment was conducted at the experimental dairy barn of the Natural Resources Institute Finland (Luke; Jokioinen, Finland, 60°49′N, 23°28′E) from October 2023 to February 2024. The experiment was conducted accord- ing to Regional State Administrative Agency permission ESAVI/25708/2023 in accordance with the guidelines established by the European Community Council Direc- tive 2010/63/EU for animal experiments and complied with the ARRIVE guidelines (Kilkenny et al., 2010). Four healthy multiparous Nordic Red dairy cows (DIM 58 ± 9.2, BW 637 ± 59.3 kg, and milk yield 38.4 ± 2.6 kg/d at the beginning of the study) were selected based on their calving date, parity, BW, and milk yield and assigned to a 4 × 4 Latin square experiment, which consisted of four 28-d periods. Animals were randomly allocated to the experimental treatments and the treatment sequences were balanced to remove the carryover effects. The first 24 d of each period were used for dietary adaptation where cows were housed in a freestall barn with feed provided in controlled individual feed bins. Cows had constant access to water and salt blocks, were milked twice a day, and received concentrates in the milking parlor (in total 0.5 kg DM/d). During the sampling period (d 24–28), cows were kept in open-circuit respiratory chambers for gas exchange measurements and sample collection. In the chambers, cows were milked twice a day at 0700 and 1700 h. Experimental diets comprised grass silage and dietary concentrates. Grass silage was made from timothy-mead- ow fescue swards preserved with formic acid-based addi- tive (AIV 2 Plus Na, Eastman, Oulu, Finland; 5 L/tonne fresh grass). The control diet (CON) consisted of grass silage and dietary concentrates mixed at forage to concen- trate ratio of 65:35 on DM basis. For the 3 experimental treatments, the CON diet was supplemented with 0.2% of DiGestoChar (BFE; GoBioFarm Ltd., Iisalmi, Finland) or with either 0.75% (CaPe1) or 1.5% CaO2 (CaPe2), respectively; supplied by GlasPort Bio Ltd., Galway, Ire- land). Treatment doses were selected based on consulta- tion with additive providers and in vitro test results (data not presented) using total gas production measurements by Ankom modules (Ankom Technology, Macedon, NY) and analysis of methane concentration from gas collected after 6 and 24 h of fermentation. The doses used in the in Vattulainen et al.: CALCIUM PEROXIDE AND BIOCHAR FEED ADDITIVES Journal of Dairy Science Vol. 108 No. 12, 2025 13365 vitro trial were 1.35% and 2.25% for CaO2, and 0.1% and 0.2% for BFE. The CaO2 was included in the concentrate pellets, which were produced in one batch at the begin- ning of the experiment, whereas BFE was mixed with 3 kg of concentrate and added during TMR preparation. All diets were offered 4 times daily at 0700, 1300, 1700, and 1900 h as TMR. The CON and BFE diets were balanced for ME and MP concentrations and Ca:P ratio in line with Luke (2025) feeding recommendations. However, to balance the Ca:P ratio in the CaPe1 and CaPe2 diets, the forage-to-concentrate ratio was marginally adjusted due to the increasing mineral concentration caused by dietary CaO2 inclusion. Sugar beet pulp and rapeseed meal were kept constant in the diets to avoid any confounding effect from palatability and CP concentration with the treatment effect, respectively. The formulation and chemical compo- sition of the experimental diets are presented in Table 1. Sample Collection and Measurements Silage and concentrate samples were taken twice per week and analyzed for DM content throughout the experiment to maintain the forage-to-concentrate ratio. Representative silage and concentrate samples were taken during d 25 to 27 of each experimental period and kept at −20°C until chemical composition analysis. Leftovers were collected and weighed during d 25 to 27 of each experimental period, and representative samples were taken and analyzed for DM content before calcu- lating DM intake. Milk yield and feed intake recorded between d 24 and 28 of each period were used for statisti- cal analysis. In addition, milk samples of 30 mL were collected from 6 consecutive milkings during d 25 to 27, and samples were analyzed for fat, CP, lactose, and urea concentrations, as well as SCC (MilkoScan FT6000, Foss Electric, Hillerød, Denmark). Oxygen, carbon dioxide, and CH4 exchanges of the cows were measured over 4 d (starting at 1000 h on d 24 to 1000 h on d 28) using 4 open-circuit respiratory chambers (21.5 m3). The first day was used for acclimatization to the chamber environment, and gas measurements were re- corded over the subsequent 3-d period. The details of gas exchange measurements have been described previously (Bayat et al., 2022). Briefly, concentrations of the gases Vattulainen et al.: CALCIUM PEROXIDE AND BIOCHAR FEED ADDITIVES Table 1. Formulation of experimental diets and their chemical composition Item2 Treatment1   CON CaPe1 CaPe2 BFE Feed ingredient (g/kg DM)           Grass silage3 646 637 623 646   Barley 86.9 85.7 83.8 86.9   Oats 86.9 85.7 83.8 86.9   Molassed sugar beet pulp 70 70 70 70   Rapeseed meal 94.5 94.5 94.5 94.5   Calcium peroxide 0 7.5 15 0   Limestone4 6 0 0 6   Monocalcium phosphate 0 10 20 0   Mineral and vitamin premix5 10 10 10 10   Forage proportion 646 637 623 646   Forage proportion (excluding minerals) 656 655 652 656 Chemical composition (g/kg DM unless otherwise stated)   DM 511 519 530 511   OM 925 917 902 925   CP 148 147 144 148   EE 38 39 38 38   NDF 422 414 404 422   Forage NDF 327 322 315 327   GE (MJ/kg DM) 18.0 17.8 17.5 18.0   Ca:P ratio 1.90 1.70 1.82 1.89 1CON = control diet; CaPe1 = diet containing 7.5 g/kg calcium peroxide; CaPe2 = diet containing 15 g/kg calcium peroxide; BFE = control diet containing 2 g/kg DM DiGestoChar (GoBioFarm Ltd., Iisalmi, Finland). 2EE = ether extract; GE = gross energy; Ca:P = calcium to phosphorus ratio. 3Mean fermentation characteristics of the basal grass silage: pH, 4.15; in DM (g/kg) lactic acid, 41.7; acetic acid, 34.3; propionic acid, 0.73; butyric acid, 1.08; soluble N (g/kg of total N), 599; ammonium N (g/kg of total N), 62.9; gross energy (MJ/kg of DM), 18.1; D-value 688 ± 7.9 (g/kg DM). 4Total diet Ca:P ratio (including grass silage) was balanced based on Luke (2025) Ca and P values of the feed ingredients. 5Kalkitonkivennäinen, A-Rehu, Seinäjoki, Finland for experimental concentrates; Milking parlor mineral and vitamin premix was Lypsykivennäinen Tiineys+, Hankkija Oy, Riihimäki, Finland; LypsyMelli (Mg) offered ad libitum, Lantmännen Agro, Raisio, Finland. 13366 Journal of Dairy Science Vol. 108 No. 12, 2025 in the inlet and outlet air were measured by a computer- controlled system using dedicated analyzers (Oxymax, Columbus Instruments) with 3.5-min intervals for each chamber and the reference air. Gas analyzers were cali- brated using the authentic standard gases (AGA Ltd.) at the beginning of each measurement period. Air outflow for each chamber was measured by HFM-200 mass flow meter with a laminar flow element capable of measuring up to 3,000 L/min with an accuracy of <1% of full scale and repeatability of 0.05% of full scale (Teledyne Hast- ings Instruments, Hampton, VA). The air flow was set to 1,500 L/min, and the measured flow was corrected using an integrated thermometer and manometer to standard temperature and pressure (0°C and 101.325 kPa). A re- covery test of CO2 was performed on the chambers after the experiment. Continuous measured versus released CO2 (7.5 g/min) was compared for at least 75 min after reaching the steady-state condition. The gas recovery ratio was calculated as the ratio between measured and released CO2 multiplied by 100. The CO2 recovery was 100.5%, 98.7%, 94.9%, and 92.5% for chamber 1 to 4, respectively, or 96.7% ± 3.49% (mean ± SD). Each cow was kept in the same chamber during all periods to avoid potential confounding effects between the cow and the chamber in the statistical analysis. Thus, the small differ- ences in the recovery rates between chambers did not in- terfere with comparisons between the experimental diets. In the chambers, total feces and urine excretions were collected from d 25 to 28, and feces was weighed, thoroughly mixed, subsampled (5%, wt/wt), and stored at −20°C before chemical analysis for determination of nutrient digestibility. Urine was separated from feces via a lightweight harness and flexible tubing attached to the vulva and collected in plastic canisters containing 500 mL of 5 M sulfuric acid. Collection vessels were changed at 12-h intervals, and daily samples (5%, wt/wt) were taken and stored at −20°C. Feces and urine samples were pooled over sampling days to provide a representative sample for chemical analysis. The cows were milked inside the chambers using a can milking system (SAC, Kolding, Denmark). At 1000 h on d 28 of each experimental period, im- mediately after the cows left the respiratory chambers, samples of rumen liquid (0.5 L) were collected via the esophagus using a Ruminator device (Profs Products, Wittybreut, Germany). In case of saliva presence, con- taminated samples were discarded and repeated samples were obtained. Immediately after collection, rumen liquid pH was measured using a portable pH meter, and subsamples were taken for VFA and ammonia-N deter- mination and rumen microbiota analysis as described by Ahvenjärvi et al. (2024). Chemical Analysis Frozen feed, and fecal and urinary samples were thawed in room temperature. Feed and fecal samples were dried in a forced-air oven at 50°C and milled using a sample mill with a 1-mm screen (Sakomylly KT-120, Koneteol- lisuus Oy, Helsinki, Finland) before chemical analysis. Dry matter concentration of milled feed and fecal samples was determined based on weight loss in a forced-air oven at 105°C for 16 h, and grass silage DM concentration was corrected for the loss of volatile compounds (Huida et al., 1986). The official method of AOAC-942.05 (AOAC International, 2019) was used for analysis of ash concen- tration. Nitrogen concentration in fresh samples of urine was determined by the Kjeldahl method using CuSO4 as a catalyst, and for dry feed and fecal samples, the Dumas method was applied (Leco FP-428, Leco Corporation, St. Vattulainen et al.: CALCIUM PEROXIDE AND BIOCHAR FEED ADDITIVES Table 2. Feed and nutrient intakes of lactating dairy cows fed diets containing different feed additives Intake (kg/d unless otherwise stated) Treatment1 SEM P-value CON CaPe1 CaPe2 BFE Lin2 Quad3 CON vs. BFE Total DM 24.7 22.1 20.6 24.7 1.10 0.02 0.61 1.00 OM 22.9 20.2 18.6 22.9 0.99 0.01 0.64 1.00 CP 3.69 3.27 3.00 3.69 0.15 0.01 0.65 0.99 EE4 0.93 0.84 0.78 0.93 0.04 0.02 0.81 0.99 NDF 10.3 9.0 8.2 10.3 0.46 0.01 0.62 1.00 Starch 2.67 2.70 2.66 2.68 0.17 0.99 0.86 0.98 GE (MJ/d) 445 394 362 445 19.3 0.01 0.64 0.99 Ca (g/d) 179 236 334 179 19.7 <0.001 0.37 0.99 P (g/d) 94.5 139 183 94.7 9.72 <0.001 0.98 0.99 Ca:P 1.90 1.70 1.82 1.89 0.02 0.04 0.001 0.99 1CON = control diet; CaPe1 = diet containing 7.5 g/kg calcium peroxide; CaPe2 = diet containing 15 g/kg calcium peroxide; BFE = control diet containing 2 g/kg DM DiGestoChar (GoBioFarm Ltd., Iisalmi, Finland). 2Linear effect of CaO2 inclusion. 3Quadratic effect of CaO2 inclusion. 4EE = ether extract; GE = gross energy; Ca:P = calcium to phosphorus ratio. Journal of Dairy Science Vol. 108 No. 12, 2025 13367 Joseph, MI). Starch concentration in feed samples was determined according to Salo and Salmi (1968). The NDF concentration was analyzed in the presence of Na2SO3 (Van Soest et al., 1991) using Ankom 220 Fiber Analyzer (An- kom Technology, Macedon, NY) and presented ash-free. For samples containing starch, heat-stable α-amylase was used. Ether extract concentration in feed ingredients was determined according to AOAC official method 920.39 (AOAC International, 2019) and in feces after hydrolysis with 3 M HCl. Gross energy (GE) was determined using a Parr 6200 Oxygen Bomb Calorimeter (Parr Instrument Co, Moline, IL) with benzoic acid as a standard. In vitro OM digestibility of grass silage was determined based on pepsin-cellulase solubility according to Nousiainen et al. (2003). The concentration of digestible OM (D-value) in grass silage was estimated using the equation suggested by Huhtanen et al. (2006) for primary growth grass silage: D-value (g/kg of DM) = [1,000 − ash (g/kg of DM)] × [0.077 + 0.86 × OM solubility (g/g of OM)]. Volatile fatty acid concentrations in grass silage and rumen fluid were determined by GC as described by Huhtanen et al. (1998). Microbiota Analysis Total DNA was extracted from 0.5 mL of rumen liquid and 0.2 to 0.3 g of feces following a protocol described by Rius et al. (2012), with initial bead beating performed 3 times at 6 m/s × 1 min in FastPrep (MP Biomedicals, Irvine, CA). Bacterial communities were amplified using universal primers 515F and 806R for the 16S rRNA gene (Caporaso et al., 2011). Anaerobic fungi were determined using AGF-LSU-EnVs primers that code D2 region of the 28S rRNA gene (Young et al., 2022), and rumen ciliate protozoa were determined using P-SSU-316F and GIC758R primers targeting the 18S rRNA gene (Ishaq and Wright, 2014). The sequenc- ing libraries were prepared and sequenced at Edinburgh Genetics (Edinburgh, United Kingdom) on an Illumina Novaseq 6000 platform (500 cycles). Demultiplexing of sequences was performed by the sequencing provider. Sequence read quality control and removal of chimeric reads, as well as clustering of microbiota sequences into amplicon sequence variants (ASV), was performed us- ing QIIME v2 (Bolyen et al., 2019) following default settings in DADA2 (Callahan et al., 2016). The ASV with fewer than 10 reads in total or present in only one sample were removed. The reference databases used for taxonomical assignment of ASV were Silva 138.1 (Quast et al., 2013) for bacteria; RIM-DB (Seedorf et al., 2014) for archaea; the Kittelmann et al. (2015) pub- lished database for ciliate protozoa; and the AF_LSU v1.0 (https:​/​/​anaerobicfungi​.org) for anaerobic fungi. The fungal database was developed based on data previ- ously published by Hanafy et al. (2020). After quality control, the number of sequencing reads per sample was 196,288 to 279,727 for rumen bacteria, 15,116 to 24,387 for archaea, 61,530 to 176,091 for ciliate protozoa, and 150,375 to 615,173 for anaerobic fungi. Fecal samples resulted in 176,623 to 371,496 quality filtered reads for bacteria, 8,827 to 34,896 for archaea, and 263,137 to 469,982 for anaerobic fungi. The raw sequence reads were submitted to the National Center for Biotechnology Information Sequence Read Archive under BioProject PRJNA1220600. Vattulainen et al.: CALCIUM PEROXIDE AND BIOCHAR FEED ADDITIVES Table 3. Milk and ECM yield, milk composition, and milk production efficiency of lactating dairy cows fed diets containing different feed additives Item Treatment1 SEM P-value CON CaPe1 CaPe2 BFE Lin2 Quad3 CON vs. BFE Yield (kg/d)                   Milk 33.9 30.6 28.2 33.9 1.87 0.01 0.70 0.99   ECM 37.8 33.8 30.8 38.6 1.44 0.01 0.76 0.70   Fat 1.62 1.44 1.31 1.67 0.07 0.02 0.76 0.62   Protein 1.26 1.12 1.01 1.28 0.06 0.01 0.77 0.70   Lactose 1.55 1.39 1.29 1.54 0.10 0.01 0.65 0.93   TS 4.78 4.27 3.90 4.84 0.20 0.01 0.72 0.81 Concentration (g/kg)                   Fat 48.2 47.8 47.5 49.4 3.24 0.53 0.96 0.32   Protein 37.2 36.7 36.0 37.8 0.89 0.10 0.91 0.43   Lactose 45.6 45.4 45.5 45.5 0.75 0.37 0.31 0.46   TS 142 140 139 143 3.73 0.22 0.89 0.51   Urea (mg/100 mL) 16.9 16.8 15.9 16.9 1.00 0.26 0.52 0.94   SCC (1000/mL) 35.6 104 90.1 110 69.8 0.22 0.28 0.11 ECM/DMI 1.53 1.54 1.49 1.57 0.05 0.60 0.66 0.60 ECM/OMI 1.65 1.68 1.66 1.69 0.05 0.98 0.73 0.61 1CON = control diet; CaPe1 = diet containing 7.5 g/kg calcium peroxide; CaPe2 = diet containing 15 g/kg calcium peroxide; BFE = control diet containing 2 g/kg DM DiGestoChar (GoBioFarm Ltd., Iisalmi, Finland). 2Linear effect of CaO2 inclusion. 3Quadratic effect of CaO2 inclusion. https://anaerobicfungi.org 13368 Journal of Dairy Science Vol. 108 No. 12, 2025 Calculations and Statistical Analysis Total-tract apparent digestibility coefficients were calculated based on the difference between intake of a nutrient and its fecal output divided by the corresponding intake of the nutrient. Intake of ME was calculated as the difference between GE intake and energy excretion in feces, urine, and CH4. Energy loss as CH4 was calculated using the factor 55.24 kJ/g (Kriss, 1930). Energy cor- rected milk yield was calculated using milk fat, protein, and lactose yields according to Sjaunja et al. (1990), and energy secretion (MJ/d) in milk was calculated as 3.14 × ECM yield (kg/d). Heat production was calculated us- ing gas exchanges and urinary N output according to the equation suggested by Brouwer (1965). Nitrogen balance was calculated as the difference between N intake and N excretion in feces, urine, and milk, where milk N was calculated as milk CP/6.38. Statistical analyses were performed using PROC MIXED of SAS 9.4. (SAS institute Inc., Cary, NC), using period and treatment as fixed effects and cow as a random effect in the model. The treatment means were compared using contrasts as follows: CON ver- sus BFE, and linear and quadratic effects of CaO2 addition. The effects were considered statistically sig- nificant when P < 0.05 and tendencies were reported Vattulainen et al.: CALCIUM PEROXIDE AND BIOCHAR FEED ADDITIVES Table 4. Apparent total-tract digestibility and fecal Ca and P concentrations of lactating dairy cows fed diets containing different feed additives Item Treatment1 SEM P-value CON CaPe1 CaPe2 BFE Lin2 Quad3 CON vs. BFE Digestibility (g/kg or otherwise stated)4   DM 718 701 677 707 7.24 <0.001 0.61 0.13   OM 732 719 702 721 7.25 <0.01 0.62 0.09   CP 669 648 620 654 10.7 0.01 0.77 0.26   EE 610 598 556 605 11.3 <0.01 0.12 0.56   NDF 646 620 611 624 12.0 0.01 0.35 0.06   GE (kJ/MJ) 695 680 658 681 7.84 <0.001 0.51 0.07   Ca 299 298 286 262 23.9 0.65 0.82 0.23   P 374 319 320 332 29.0 0.08 0.26 0.15 Fecal concentration (g/kg DM)   Ca 18.1 25.3 35.6 18.3 1.38 <0.001 0.27 0.90   P 8.48 14.4 18.6 8.69 0.70 <0.001 0.22 0.78 1CON = control diet; CaPe1 = diet containing 7.5 g/kg calcium peroxide; CaPe2 = diet containing 15 g/kg calcium peroxide; BFE = control diet containing 2 g/kg DM DiGestoChar (GoBioFarm Ltd., Iisalmi, Finland). 2Linear effect of CaO2 inclusion. 3Quadratic effect of CaO2 inclusion. 4EE = ether extract; GE = gross energy. Table 5. Rumen fermentation characteristics of lactating dairy cows fed diets containing different feed additives Item Treatment1 SEM P-value CON CaPe1 CaPe2 BFE Lin2 Quad3 CON vs BFE pH 6.81 6.54 6.65 6.64 0.09 0.25 0.12 0.21 Ammonia-N (mM) 4.05 3.23 3.36 3.01 0.83 0.53 0.62 0.36 Total VFA (mM) 110 122 116 118 4.13 0.34 0.11 0.17 Molar proportion (mmol/mol)   Acetate 648 635 642 642 7.24 0.33 0.08 0.35   Propionate 182 189 176 188 5.49 0.17 0.03 0.22   Butyrate 122 126 130 124 3.39 0.03 0.75 0.55   Isobutyrate 7.18 6.42 7.26 6.78 0.25 0.72 0.01 0.11   Valerate 17.2 18.8 18.4 16.9 0.84 0.25 0.24 0.80   Isovalerate 11.4 11.0 13.8 10.7 1.29 0.06 0.14 0.52   Caproate 11.8 13.9 12.7 11.5 0.88 0.53 0.16 0.77 Acetate:​propionate 3.57 3.36 3.66 3.44 0.14 0.37 0.02 0.21 (Acetate + butyrate):propionate 4.25 4.03 4.40 4.10 0.16 0.22 0.03 0.26 1CON = control diet; CaPe1 = diet containing 7.5 g/kg calcium peroxide; CaPe2 = diet containing 15 g/kg calcium peroxide; BFE = control diet containing 2 g/kg DM DiGestoChar (GoBioFarm Ltd., Iisalmi, Finland). 2Linear effect of CaO2 inclusion. 3Quadratic effect of CaO2 inclusion. Journal of Dairy Science Vol. 108 No. 12, 2025 13369 when P < 0.1. Diurnal variations of enteric CH4 and hydrogen emissions were calculated using a mixed model including fixed effects of period, diet, time of day, interactions of period and diet with time of day, and random effect of cow. Time of day was considered as a repeated measurement. Bacterial community α-diversity of ruminal and fe- cal samples was estimated using Shannon diversity index and the richness (number of observed ASV) as implemented in the MicrobiotaProcess R package (Xu et al., 2023). The data were evenly subsampled to the lowest number of reads per sample in each dataset and significant differences in pairwise comparisons were estimated using nonparametric Wilcoxon test. To evalu- ate treatment effect on the changes in ruminal and fe- cal microbial community structure, between sample diversity was calculated as Bray–Curtis dissimilarities following Hellinger transformation and visualized us- ing principal coordinate analysis. The significance of groups was evaluated by distance-based permutational multivariate ANOVA (adonis) and defined at the P < 0.05 level after 999 permutations, as implemented in the vegan R package (Oksanen et al., 2020). Diet effect on individual microbial genera was evaluated using PROC MIXED as described previously. Before the analysis, all genera below 0.001% abundance in less than 25% of samples were filtered out, number of reads were log base transformed [log2 (x + 1)] and standardized by data centering. For easier interpretation of the results, mi- crobial genera significantly affected by treatment were converted back to compositional data and presented as relative abundances. RESULTS Feed Intake, Milk Production, and Diet Digestion The CaPe treatments linearly reduced (P < 0.05) DMI by 16.6% and OMI by 18.6% compared with CON, which was accompanied by linear decreases in CP, EE, NDF, and GE intakes (Table 2). However, the Ca:P ratio decreased linearly (P < 0.05), and intakes of Ca and P increased linearly (P < 0.001). Inclusion of BFE did not affect (P ≥ 0.98) DM and nutrient intakes. Yields of milk, ECM, fat, protein, lactose, and TS de- creased linearly (P < 0.05) due to CaPe, but milk compo- sition (fat, protein, lactose, TS, urea) and SCC were not affected (P ≥ 0.11; Table 3). Use of BFE did not affect (P ≥ 0.32) milk yield or composition. None of the dietary treatments affected (P ≥ 0.60) feed efficiency expressed as milk or ECM yield divided by DM or OM intake. Apparent total-tract digestibilities of DM, OM, CP, EE, NDF, and GE decreased linearly (P < 0.05) by CaPe treatment (Table 4). Apparent bioavailability of Ca was not affected by CaPe, but that of P tended to decline (P = 0.08). Excretion of both Ca and P in feces increased linearly (P < 0.001) in response to CaPe. We found no significant differences in DM, CP, EE, Ca, or P digest- ibilities or fecal concentration of Ca and P between CON and BFE (P ≥ 0.13), but a tendency for lower digestibil- Vattulainen et al.: CALCIUM PEROXIDE AND BIOCHAR FEED ADDITIVES Table 6. Methane, carbon dioxide, and hydrogen production of lactating dairy cows fed diets containing different feed additives Item Treatment1 SEM P-value CON CaPe1 CaPe2 BFE Lin2 Quad3 CON vs. BFE Methane                   g/d 492 431 419 489 19.7 0.01 0.21 0.88   g/kg DM intake 20.0 19.6 20.2 20.0 0.73 0.62 0.32 1.00   g/kg OM intake 21.6 21.4 22.5 21.6 0.79 0.20 0.27 1.00   g/kg OM digested 29.5 29.7 32.0 30.0 0.92 0.04 0.25 0.62   g/kg Milk 14.6 14.2 15.0 14.5 0.69 0.34 0.08 0.68   g/kg ECM 13.1 12.8 13.6 12.7 0.55 0.19 0.10 0.32   % of GE intake 6.17 6.12 6.42 6.17 0.22 0.20 0.27 0.98 Carbon dioxide                   g/d 13,638 14,487 14,920 12,866 701.1 0.23 0.81 0.46   g/kg DM intake 604 604 618 609 15.9 0.47 0.65 0.81   g/kg OM intake 653 659 685 658 17.2 0.14 0.55 0.06   g/kg OM digested 893 917 977 914 21.0 0.03 0.50 0.50   g/kg Milk 437 408 461 474 8.9 0.02 <0.001 <0.001   g/kg ECM 392 385 434 383 10.4 0.02 0.06 0.57 Hydrogen                   g/d 0.68 0.24 0.18 0.74 0.066 <0.001 0.03 0.55   g/kg DMI 0.03 0.01 0.01 0.03 0.00 <0.001 0.03 0.46 1CON = control diet; CaPe1 = diet containing 7.5 g/kg calcium peroxide; CaPe2 = diet containing 15 g/kg calcium peroxide; BFE = control diet containing 2 g/kg DM DiGestoChar (GoBioFarm Ltd., Iisalmi, Finland). 2Linear effect of CaO2 inclusion. 3Quadratic effect of CaO2 inclusion. 13370 Journal of Dairy Science Vol. 108 No. 12, 2025 ity for NDF, GE, and OM with BFE compared with CON (P ≤ 0.09) was observed. Rumen pH, ammonia-N and total VFA concentrations were not affected (P ≥ 0.11) by the treatments (Table 5). The first level of CaPe treatment tended to decrease the molar proportion of acetate (quadratic effect P = 0.08) and increased that of propionate (quadratic effect P < 0.05), whereas butyrate increased linearly (P < 0.05) in response to CaPe treatments. These changes resulted in significant quadratic effects in the lipogenic to glucogen- ic VFA ratios so that they were lowest at the first level of CaPe addition (P < 0.05). Addition of BFE did not affect molar proportions of ruminal VFA. Gas Production, Energy, and N Metabolism Dietary CaO2 inclusion decreased daily CH4 produc- tion (g/d) linearly (P < 0.01) by 15.0%, but CH4 yield (g/ kg DM or OM intake) and intensity (g/kg milk or ECM) were not affected (Table 6). We found no effect of CaPe treatment on CO2 production (g/d) or yield (g/kg DMI or OMI), but CO2 yield (g/kg OM digested) increased linearly (P < 0.05). The intensity of CO2 (g/milk) was lowest at the first level of CaPe addition (quadratic ef- fect P < 0.05). Hydrogen production (g/d) and yield (g/kg DMI) decreased at the first level of CaPe treatment but plateaued thereafter, resulting in significant quadratic effects of CaPe addition (P < 0.01). The only effect of BFE on gas production was a higher intensity of CO2 (g/ kg milk) compared with CON (P < 0.001). The diurnal variation in CH4 and hydrogen production is presented in Figures 1 and 2, respectively. Diurnal variation of CH4 tended (P = 0.10) to be affected by the interaction of diet and time of day. Hourly CH4 production was more constant for CON and BFE, whereas there were more fluctuations for CaPe treatments during feeding times (0700, 1300, 1700, and 1900 h). In general, CH4 pro- duction gradually increased during the daytime, peaking around the last feeding, and gradually declined afterward until feeding in the next morning. Hydrogen production was influenced by the interaction of diet and time of day (P < 0.01). Although H2 production was in general lower (P < 0.01) for CaPe treatments than CON and BFE, it had sharper peaks during feeding times, returning to its baseline shortly after feeding. The CaPe treatments linearly reduced (P < 0.05) GE and ME intakes by 18.7% (Table 7). The proportion of GE lost in feces was linearly increased (P < 0.01) by CaPe, whereas BFE tended (P = 0.07) to increase it. Pro- portions of GE excreted in urine, CH4, and milk were not (P ≥ 0.20) affected by the treatments. However, heat production as a proportion of GE intake tended to increase linearly (P = 0.09) due to CaPe. Intake of N de- creased linearly (P < 0.01) whereas N excretion in feces as a proportion of N intake increased linearly (P < 0.01) in response to CaPe. However, N excreted in urine and milk and N balance (g/d) were not affected (P ≥ 0.15) by treatments. Dietary inclusion of BFE did not affect (P ≥ 0.15) energy and N metabolism. Rumen Microbiota Dietary feed additives did not stipulate significant changes in bacterial α-diversity, but influenced bacterial community structure. Bray–Curtis dissimilarities demon- strated significant (adonis P = 0.03) differences between CON and CaPe2 treatments (Figure 3A). In comparison to CON, CaPe linearly reduced the relative abundances of Gastranaerophilales, Butyrivibrio, [Eubacterium] cellulosolvens and [Eubacterium] ruminantium groups, Rhodospirillales sp., and Succinivibrionaceae UCG- 002, but linearly increased Bacteroidales BS11, RF16 Vattulainen et al.: CALCIUM PEROXIDE AND BIOCHAR FEED ADDITIVES Figure 1. Diurnal variation of methane production of dairy cows fed control diet (CON) and diets containing 2 levels of calcium peroxide (CaPe1 and CaPe2) or biochar with fibrolytic enzymes and live yeast (BFE) additives. The P-values for diet, time, and interaction of diet and time were <0.01, < 0.01, and 0.10, respectively. Journal of Dairy Science Vol. 108 No. 12, 2025 13371 and p-251-o5 groups, Prevotellaceae sp., Prevotellaceae UCG-004, several members of the Firmicutes bacilli class (Anaeroplasma, Erysipelatoclostridiaceae UCG-004, Ery- sipelotrichaceae sp., Solobacterium, and RF39), several members from the Lachnospiraceae family (Coprococ- cus, Lachnospiraceae UCG-008, Marvinbryantia, Mory- ella), and Pirellulaceae p-1088-a5 gut group (Table 8). Archaeal community was dominated by Methanobre- vibacter gottschalkii clade (average 76.6%), followed by Methanosphaera sp. ISO3-F5 (10.4%), Methano- brevibacter ruminantium (8%), and Methanosphaera sp. (1.9%). Members from Methanomassiliicoccaceae family were observed at low abundances. Among them, Methanomassiliicoccus Group 10 sp. (1.5%) and Methanomassiliicoccus Group 12 sp. ISO4-H5 (0.7%) were the more abundant groups. Alpha diversity analy- sis showed a tendency (P = 0.053) for lower archaeal richness in CaPe1 treatment as compared with CON but similar trend was not observed in CON–CaPe2 or CON–BFE comparisons. In this experiment archaeal community structure was not influenced by the treat- ments (adonis P = 0.82; Figure 3B). At the genus level, the ciliate protozoan community was dominated by Epidinium (average 29.5%), Entodinium (26.9%) and Isotricha (25%). Less abundant genera were represented by Ostracodinium (7%), Eudiplodinium/Er- emoplastron (3.4%), Dasytricha (2.1%), Eudiplodinium (1.9%), and Anoplodinium/Diplodinium (1.7%). Alpha diversity analysis demonstrated a significant increase in richness in CaPe2 as compared with the CON diet (P = 0.029). No significant differences were observed in other pairwise comparisons. Also, Bray–Curtis dissimilarities showed CaPe2 tendency for treatment effect on ciliate protozoan community structure (adonis P = 0.056; Figure 3C). In comparison to CON, CaPe linearly increased the relative abundances of Entodinium sp., and Entodinium longinucleatum, but decreased Isotricha sp. The effect on Epidinium sp. was quadratic (P < 0.05), with CaPe1 increasing and CaPe2 decreasing abundance as compared with CON (Table 8). The total fungal community was dominated by Neo- callimastix (average 33.3%), Piromyces (28.3%), SK3 (11.7%) and Cecomyces (8.4%), followed by less abun- dant Buwchfawromyces (6%), Neocallimastigaceae sp. (5.7%), Orpinomyces (2.9%), and AL8 (2.1%). The remaining 6 genus level taxonomical groups were ob- served at minor relative abundance. Dietary treatments did not affect fungal α-diversity or fungal community structure (Figure 3D). Fecal Microbiota The fecal bacterial community was represented by 307 genera level taxonomical groups, but only 27 of them were detected at a relative abundance >1%. Among the more abundant genera from Bacteroidota phylum were Bacteroides (average 6.3%), Bacteroidales RF16 group (2%), members of Prevotellaceae family (8.2%; UCG-003, UCG-004, UCG-001, and Prevotella), and members from Rikenellaceae family (8.2%; Rikenella- ceae RC9 gut group, Alistipes). More abundant genera from the Firmicutes phylum were Christensenellaceae R-7 group (5.7%), Oscillospiraceae UCG-005 (4.8%), members of the Lachnospiraceae family (5.4%; unclas- sified sp., NK3A20 group, Acetitomaculum), [Eubac- terium] coprostanoligenes group (3.1%), Monoglobus (1.5%), Romboutsia (1.6%), RF39 (1.6%), and Rumi- nococcus (1.1%). Representatives of other phyla were Treponema (1.1%), Akkermansia (1.2%), and Olsenella (1.2%). Dietary treatments did not affect fecal bacte- rial α-diversity, but compared with CON, CaPe linearly reduced the relative abundance of Bacteroidales RF16 Vattulainen et al.: CALCIUM PEROXIDE AND BIOCHAR FEED ADDITIVES Figure 2. Diurnal variation of hydrogen production of dairy cows fed control diet (CON) and diets containing 2 levels of calcium peroxide (CaPe1 and CaPe2) or biochar with fibrolytic enzymes and live yeast (BFE) additives (P < 0.01 for diet, time, and interaction of diet and time). 13372 Journal of Dairy Science Vol. 108 No. 12, 2025 group, Erysipelatoclostridium, Lactobacillus, How- ardella, and Oscillospirales sp. On the other hand, CaPe linearly increased the relative abundance of Barnesiel- laceae sp., Bacteroidales F082, Catenisphaera, Solo- bacterium, [Eubacterium] hallii group, [Ruminococ- cus] gauvreauii group, Anaerovoracaceae sp., Family XIII UCG-001, and Rhodospirillales sp. (Table 9). The BFE diet had a significantly (P < 0.01) higher relative abundance of Paludibacter and Ruminococcaceae sp. as compared with CON. Dietary treatments did not induce changes in archaeal α-diversity or archaeal community structure. Fecal archaeal community was largely dominated by Mbb. gottschalkii clade (average 81.2%), followed by Metha- nosphaera sp. ISO3-F5 (6.8%), Mbb. ruminantium clade (5.2%), Methanocorpusculum sp. (4.1%), and Methano- sphaera sp. (1.9%). In addition Methanomassiliicoccus Group 10 sp., Group 12 sp. ISO4-H5, and Group 9 sp. ISO4-G1 were observed at the relative abundance <0.05%. Dietary treatments did not affect anaerobic fungi α-diversity and did not stimulate significant changes in community structure. Fecal anaerobic fungal com- munity was represented by the same genera as in the rumen, but with Piromyces (average 30.4%), Cecomy- ces (28.8%) and SK3 (18.4%) being the predominant groups. Less abundant fungi in feces were Neocallimas- tigaceae sp. (9.4%), Neocallimastix (6.9%), and AL8 (3.5%), whereas Buwchfawromyces and Orpinomyces decreased in abundance to <1% when compared with their abundance in rumen. DISCUSSION Effects of Calcium Peroxide Addition There is an urgent need to find effective CH4-mitigat- ing measures to improve the sustainability of ruminant production. The use of CaO2 has potential to manipulate the rumen environment, but limited data are available about its effects on enteric CH4 production and other ani- mal parameters. A recently published in vivo study using growing bulls (Roskam et al., 2024) and an in vitro study reported by Graham et al. (2025) showed positive pro- spective results. However, preliminary results from an in vivo trial using sheep failed to demonstrate significant reduction in enteric CH4 production (Pugh et al., 2024), which was also the case in our experiment. Although daily CH4 production decreased on CaPe treatments in our study, this was a reflection of reduced feed intake, and we found no differences between CON and CaPe treatments within CH4 yield (g/kg DMI or g/ kg OMI) or intensity (g/kg milk or g/kg ECM). Reduced feed intake was also linked to the negative effect of CaPe on milk production, as conversion of feed into milk (milk yield/DMI and ECM/DMI) remained similar as in CON. Our observations contrast with Roskam et al. (2024), who reported a reduction of 20% to 27% CH4 yield (g/ kg DMI) and 22% to 32% intensity (g CH4/ kg ADG) in beef cattle, mainly due to no negative CaO2 effect on DM intake in low (1.35% CaO2) and high (2.25% CaO2) treatments, where CaO2 was included into pelleted con- centrate, and no significant treatment effect on animal Vattulainen et al.: CALCIUM PEROXIDE AND BIOCHAR FEED ADDITIVES Table 7. Energy and nitrogen metabolism of lactating dairy cows fed diets containing different feed additives Item Treatment1 SEM P-value CON CaPe1 CaPe2 BFE Lin2 Quad3 CON vs. BFE Energy GE intake (MJ/d) 445 394 362 445 19.3 0.01 0.64 0.99 ME intake (MJ/d) 255 221 193 250 11.7 0.01 0.81 0.72 Proportion of energy intake (kJ/MJ)   Feces 305 320 342 319 7.8 <0.01 0.51 0.07   Urine 59.6 58.7 60.1 58.7 1.03 0.70 0.38 0.56   Methane 61.7 61.2 64.2 61.7 2.24 0.20 0.27 0.98   Milk 267 271 267 273 8.7 0.97 0.73 0.62   Heat 332 340 352 335 9.6 0.09 0.83 0.78 Milk energy/ME intake 470 480 500 490 20 0.16 0.97 0.36 Energy balance −10.7 −18.9 −31.2 −19.3 5.41 0.03 0.77 0.29 Nitrogen (N)                 N intake (g/d) 590 523 480 590 24.3 0.01 0.65 0.99 Proportion of N intake (g/kg)   Feces 331 352 380 346 10.7 <0.01 0.77 0.26   Urine 321 294 302 301 11.7 0.16 0.15 0.15   Milk 334 336 330 341 10.2 0.79 0.74 0.61 N balance (g/d) 8.79 10.5 −5.80 8.53 8.35 0.25 0.40 0.98 1CON = control diet; CaPe1 = diet containing 7.5 g/kg calcium peroxide; CaPe2 = diet containing 15 g/kg calcium peroxide; BFE = control diet containing 2 g/kg DM DiGestoChar (GoBioFarm Ltd., Iisalmi, Finland). 2Linear effect of CaO2 inclusion. 3Quadratic effect of CaO2 inclusion. Journal of Dairy Science Vol. 108 No. 12, 2025 13373 performance and efficiency traits. However, when a higher dose of CaO2 was fed as coarse ration, DM intake in beef cattle was significantly reduced. Several aspects of CaO2 feeding may affect these out- comes. The level of inclusion is one important factor. The concentrations used in the current dairy cow experiment were 0.75% and 1.50% of diet DM, whereas Roskam et al. (2024) used higher doses for growing bulls (1.35% and 2.25% of diet DM). However, in our study, the effect of CaO2 concentration on dairy animal responses was not linear for several parameters, and finding the optimal dose would require further experimentation. In general, a very high inclusion rate of CaO2 is not favorable due to diluting energy and nutrient concentrations of the diets and the need for adjustment of the Ca:P ratio. An imbal- ance in the Ca:P ratio can cause metabolic or skeletal disorders in dairy cows, which may be a challenge in formulation of commercial diets. The format and frequency of CaO2 administration may play a role in explaining its efficacy. Roskam et al. (2024) offered CaO2 as powder or in a pelleted form. The powdered form of CaO2 decreased DMI, but it was Vattulainen et al.: CALCIUM PEROXIDE AND BIOCHAR FEED ADDITIVES Figure 3. Rumen bacterial (A), archaeal (B), ciliate protozoan (C), and anaerobic fungal (D) principal coordinate analysis (PCoA) visualization of Bray–Curtis dissimilarities for dairy cows when offered dietary treatments: control diet (CON), biochar with fibrolytic enzymes and live yeast (BFE), and treatments containing 2 levels of calcium peroxide (CaPe1 and CaPe2), showing principal coordinates 1 (PCoA1) and 2 (PCoA2). 13374 Journal of Dairy Science Vol. 108 No. 12, 2025 not affected by the pelleted form. In our trial, CaO2 was included in a pelleted concentrate mixture, but it nev- ertheless negatively affected intake. Based on subjec- tive observations, the cows marginally sorted against the concentrate pellets containing CaO2, which can be interpreted as an indication of reduced palatability. The leftovers were only analyzed for DM content, and cal- culating the proportion of silage (88%, 79%, 75%, and Vattulainen et al.: CALCIUM PEROXIDE AND BIOCHAR FEED ADDITIVES Table 8. Rumen microbiota relative abundance (%) of lactating dairy cows fed diets containing different feed additives Item2 Treatment1 SEM P-value CON CaPe1 CaPe2 BFE Lin3 Quad4 CON vs. BFE Actinobacteriota                   Bifidobacteriaceae spp. 0.096 0.000 0.023 0.070 0.0300 0.10 0.02 0.87   Olsenella 2.17 2.33 1.73 2.25 0.477 0.03 0.07 0.92 Bacteroidota                   Bacteroidales BS11 gut group 0.102 0.113 0.134 0.113 0.0429 0.04 0.89 0.98   Bacteroidales RF16 group 1.886 2.189 3.523 1.839 0.446 0.02 0.56 0.95   Prevotellaceae spp. 0.656 0.893 1.69 0.766 0.1660 <0.001 0.26 0.25   Prevotella 20.4 20.9 17.0 20.4 0.8795 <0.01 0.05 0.74   Prevotellaceae UCG-004 0.220 0.244 0.391 0.244 0.0280 <0.01 0.18 0.38   Prevotellaceae YAB2003 group 1.27 1.38 0.427 1.23 0.3197 0.01 0.04 0.60   Bacteroidales p-251-o5 0.477 0.506 1.05 0.491 0.1921 <0.01 0.05 0.94 Cyanobacteria                   Gastranaerophilales 0.999 0.608 0.174 1.15 0.0721 <.0001 0.07 0.43 Fibrobacterota                   Fibrobacter 0.962 0.738 1.01 0.854 0.0799 0.59 0.01 0.25 Firmicutes                   Anaeroplasma 0.227 0.231 0.300 0.216 0.0212 0.01 0.31 0.95   Erysipelatoclostridiaceae UCG-004 0.409 0.441 0.697 0.384 0.0441 0.01 0.18 0.77   Solobacterium 0.107 0.152 0.220 0.114 0.0275 <0.01 0.67 0.66   Erysipelotrichaceae spp. 0.055 0.076 0.129 0.078 0.0131 <0.01 0.60 0.04   RF39 1.55 1.91 2.19 1.66 0.194 0.01 0.60 0.48   Clostridia UCG-014 1.49 1.68 0.917 1.49 0.2185 0.01 0.02 0.93   Defluviitaleaceae UCG-011 0.084 0.089 0.120 0.082 0.009 0.02 0.62 1.0   Butyrivibrio 0.514 0.421 0.322 0.439 0.0438 0.01 0.56 0.31   Coprococcus 0.092 0.104 0.136 0.087 0.0096 <0.01 0.72 0.87   Lachnospiraceae UCG-008 0.119 0.120 0.149 0.111 0.0102 0.02 0.38 0.59   Marvinbryantia 0.180 0.205 0.312 0.168 0.0237 <0.01 0.37 0.86   Moryella 0.342 0.343 0.549 0.349 0.0348 <0.01 0.07 0.76   [Eubacterium] cellulosolvens group 0.199 0.184 0.064 0.199 0.0522 0.02 0.17 0.83   [Eubacterium] ruminantium group 0.794 0.743 0.591 0.802 0.0613 0.03 0.34 0.99   [Ruminococcus] gauvreauii group 0.624 0.700 0.766 0.590 0.0554 <0.01 0.28 0.47   Oscillospiraceae spp. 0.087 0.085 0.124 0.097 0.0115 0.01 0.1 0.31   [Eubacterium] coprostanoligenes group 0.267 0.345 0.337 0.280 0.0216 0.01 0.02 0.39   Anaerovoracaceae spp. 0.094 0.109 0.134 0.082 0.0142 <0.01 1.0 0.23   Succiniclasticum 3.83 4.08 3.38 4.10 0.241 0.03 0.05 0.48 Planctomycetota                   Pirellulaceae p-1088-a5 gut group 0.157 0.266 0.355 0.194 0.0185 <0.001 0.15 0.06 Proteobacteria                   Rhodospirillales spp. 0.374 0.157 0.055 0.228 0.0645 <0.01 0.40 0.41   Succinivibrionaceae UCG-002 1.97 1.30 0.621 2.74 0.6548 0.01 0.83 0.39 Verrucomicrobiota                   WCHB1–41 1.17 0.885 1.44 1.01 0.2161 0.06 0.01 0.73 Trichostomatia                   Entodinium sp. 11.3 19.2 31.3 14.2 2.98 0.01 0.67 0.26   Entodinium longinucleatum 0.184 1.21 5.47 1.12 1.252 <0.01 0.12 0.05   Epidinium sp. 0.954 1.27 0.362 0.706 0.1492 <0.01 0.02 0.07   Epidinium sp. 32.0 35.8 19.8 27.2 5.23 0.01 0.01 0.56   Isotricha sp. 19.8 7.9 1.9 16.5 3.19 <0.001 0.48 0.46 Neocallimastigomycota                   Buwchfawromyces 9.77 5.61 0.83 7.70 5.196 0.05 0.65 0.34   Neocallimastigaceae sp. 5.09 5.04 7.25 5.32 1.984 0.03 0.26 0.73 1CON = control diet; CaPe1 = diet containing 7.5 g/kg calcium peroxide; CaPe2 = diet containing 15 g/kg calcium peroxide; BFE = control diet containing 2 g/kg DM DiGestoChar (GoBioFarm Ltd., Iisalmi, Finland). 2Only significantly (P < 0.05) affected microbial genera are presented. 3Linear effect of CaO2 inclusion. 4Quadratic effect of CaO2 inclusion. Journal of Dairy Science Vol. 108 No. 12, 2025 13375 86%) and concentrate in the leftovers based on silage, concentrate, and leftover DM contents for CON, CaPe1, CaPe2, and BFE, respectively, indicated marginal sorting against CaO2 containing concentrates. Different pelleting techniques and encapsulation of CaO2 could be evalu- ated in future trials to improve the palatability of CaO2- containing feeds. Unlike for some other CH4 mitigating agents, the high temperature and pressure associated with pelleting has no effect on the viability and function of CaO2 (Roskam et al., 2024). Another difference between the beef trial and our dairy experiment was feeding frequency. The beef cattle were fed CaO2-containing concentrate twice daily with a 7-h break in between the meals and separately from forage. In our experiment, the CaO2-containing concentrate pellets were mixed into a TMR and fed 4 times daily. Because CaO2 is metabolized in the rumen into H2O2 before breaking down to oxygen and water, it potentially creates oxidative stress to the rumen and to rumen microbes, which might depress appetite (De- meyer, 1982). In a beef trial (Roskam et al., 2024) spe- cial boluses were used to measure oxidation-reduction potential (ORP) and demonstrated that ORP increased 2 to 3 h after feeding in diets with CaO2 but returned to the control level before the afternoon feeding of CaO2 containing concentrates. Graham et al., (2025) showed that ORP remained elevated even 8 h after the addition of CaO2–containing feed in an in vitro system. There- fore, it is plausible to hypothesize that a constant sup- ply of CaPe in every meal as a component in the TMR in our experiment was keeping the rumen environment outside the optimal ORP level, with negative effects on appetite, feed intake, and digestibility. It is possible that administration of CaO2-containing concentrates at intervals greater than 5 h would provide better condi- tions for the rumen environment and should be tested in future experiments with dairy cattle. This would make CaO2 supplementation attractive for farms that are using separate concentrate feeding (e.g., in grazing systems), unlike 3-nitrooxypropanol (3-NOP), which needs to be administered continuously (Vattulainen et al., 2024) and is thus feasible only for farms using a TMR feeding. Links between elevated ORP values and reduced di- gestibility are supported by the currently available data. In our study, CaPe linearly decreased the digestibilities of DM, OM, CP, EE, NDF, and GE, similarly with the observations on reduced DM, OM, and NDF digest- ibilities in beef cattle (Roskam et al., 2024), and reduced Vattulainen et al.: CALCIUM PEROXIDE AND BIOCHAR FEED ADDITIVES Table 9. Fecal microbiota relative abundance (%) of lactating dairy cows fed diets containing different feed additives Item2 Treatment1 SEM P-value CON CaPe1 CaPe2 BFE Lin3 Quad4 CON vs. BFE   Actinobacteriota                   Parvibacter 0.261 0.126 0.199 0.315 0.0601 0.38 0.02 0.68 Bacteroidota                   Bacteroides 6.83 5.82 6.28 6.17 0.306 0.19 <0.001 0.01   Bacteroidales RF16 group 2.24 1.90 1.63 2.15 0.200 <0.001 0.98 0.52   Barnesiellaceae sp. 0.342 0.402 0.460 0.312 0.0279 0.01 0.68 0.19   Bacteroidales F082 0.245 0.292 0.433 0.254 0.0415 <0.01 0.68 0.49   Paludibacter 0.436 0.330 0.356 0.595 0.0334 0.04 0.10 <0.01   Paludibacteraceae sp. 0.301 0.397 0.257 0.315 0.0322 0.22 0.02 0.88   Prevotella 1.77 0.929 1.33 1.43 0.191 0.09 0.01 0.18 Firmicutes                   Erysipelatoclostridium 0.195 0.167 0.123 0.166 0.0205 <0.01 0.17 0.17   Catenisphaera 0.080 0.112 0.136 0.061 0.0130 0.03 0.45 0.48   Solobacterium 0.135 0.212 0.351 0.162 0.0384 <0.01 0.79 0.27   Lactobacillus 0.386 0.308 0.291 0.320 0.0255 0.02 0.57 0.11   Howardella 0.115 0.062 0.053 0.135 0.0126 <0.001 0.28 0.27   [Eubacterium] hallii group 0.172 0.231 0.325 0.215 0.0271 <0.01 0.90 0.13   [Ruminococcus] gauvreauii group 0.294 0.335 0.431 0.306 0.0538 0.01 0.58 0.80   Oscillospirales spp. 0.129 0.074 0.050 0.102 0.0283 0.03 0.84 0.55   Ruminococcaceae sp. 0.364 0.537 0.333 0.588 0.0898 0.87 0.07 0.05   Negativibacillus 0.277 0.276 0.226 0.266 0.0299 0.04 0.09 0.90   Anaerovoracaceae sp. 0.159 0.185 0.221 0.167 0.0181 0.01 0.53 0.42   Family XIII UCG-001 0.076 0.090 0.127 0.077 0.0139 0.01 0.90 0.57 Proteobacteria                   Rhodospirillales spp. 0.150 0.158 0.348 0.094 0.0404 0.03 0.20 0.28   1CON = control diet; CaPe1 = diet containing 7.5 g/kg calcium peroxide; CaPe2 = diet containing 15 g/kg calcium peroxide; BFE = control diet containing 2 g/kg DM DiGestoChar (GoBioFarm Ltd., Iisalmi, Finland). 2Only significantly (P < 0.05) affected bacterial genera are presented. 3Linear effect of CaO2 inclusion. 4Quadratic effect of CaO2 inclusion. 13376 Journal of Dairy Science Vol. 108 No. 12, 2025 NDF, OM, and CP digestibilities in an in vitro RUSITEC system (Graham et al., 2025). The effects of CaO2 on rumen fermentation were dose dependent. In this study, no significant increases in pH or reductions in ammonia-N or total VFA were observed, which is contrary to the observations in beef trial. Molar proportions of butyrate increased, and isovalerate tended to increase linearly, whereas propionate and isobutyr- ate behaved quadratically. We hypothesize that rumen microbial community structure and activity is sensitive to CaO2 concentration, where shifts in fermentation path- ways may occur due to different thresholds for CaO2. In this experiment, the supplementation of CaO2 led to a linear decrease in hydrogen production (g/d) and yield (g/kg DMI). The diurnal pattern of gas produc- tion demonstrated a hydrogen spike immediately after feeding, with reduced values in CaPe treatments. The same pattern was observed in the beef trial by Roskam et al. (2024), with a significant reduction by 32% to 36%. With other CH4-inhibiting feed additives, such as 3-NOP and Asparagopsis, the reduction of methano- genesis is linked with an increase in hydrogen produc- tion (van Gastelen et al., 2020, Romero et al., 2024). This indicates that CaO2 alters hydrogen pathways and might have alternative hydrogen sinks in rumen fer- mentation differently to 3-NOP. Longer chain saturated VFA synthesis is one of the potential sinks for extra hydrogen. Because only major VFA were measured in our trial, it would be interesting to elucidate the rumen metabolome to determine if longer chain fatty acids are present in CaO2 treatments. This topic would warrant further metabolome research. Effects of Biochar with Fibrolytic Enzyme and Live Yeast Addition In the current study, supplementation with 0.2% BFE did not affect diet digestion, animal performance, or CH4 production compared with CON. Biochar has been identified as a feed additive with CH4 mitigation poten- tial (Hegarty et al., 2021), but results are controversial. The variable responses to biochar supplementation may be attributed to differences in biochar source material, production methods, inclusion rates, and variations in basal diets (Schmidt et al., 2019). It is possible that the beneficial effect of biochar would be more pronounced with lower quality feeds (Erickson et al., 2011). In this study, the diets were based on high-quality grass silage (D-value 734 g/kg DM); therefore, at the tested inclusion rate, there might have been no suitable conditions for improvement in diet digestion, as visible in lack of BFE effect on rumen microbial composition, rumen fermenta- tion parameters, and production traits. In addition, NDF and GE digestibilities tended to decrease on the BFE supplemented diet, which contradicts findings in other studies (Winders et al., 2019; Ni et al., 2024). Even if CH4 mitigation could not be achieved, biochar may have other beneficial properties that warrant further research. Biochar can be explored as an electron mediator, as it can be temporarily oxidized or reduced by microbes (Schmidt et al., 2019). We hypothesize that biochar could be tested in combination with CaO2 to elucidate potential benefits in supporting rumen microbiome to adjust to elevated ORP values. Effects of Calcium Peroxide on Rumen and Fecal Microbiota We elucidated for the first time the dietary CaO2 effect on all 4 microbial groups in the rumen (bacteria, archaea, ciliate protozoa, and anaerobic fungi), and our results sug- gest that bacteria and ciliate protozoa were most affected. In the rumen environment, CaO2 reacts with water to produce calcium hydroxide and hydrogen peroxide as intermediate products. Calcium hydroxide, through reaction with carbon dioxide, can create calcium car- bonate, which in the acidic environment of the rumen will release calcium and carbonate ions. The released calcium could be absorbed and utilized by the cow for various physiological functions, such as milk synthesis, or the excess may be excreted in feces. Gut bacteria have specific enzymes to release dietary bound calcium, although addition of calcium to the diet, such as in the form of calcium propionate, had little effect on the ru- men bacterial community composition (Zhang et al., 2022). On the other hand, Liu et al. (2020) studied goats for gut microbiota responses to digestibility of different dietary calcium amounts and noted significant increase in relative abundance of Christensenellaceae R-7 group, Ruminococcus 2, Quinella, Ruminococcaceae UCG- 004, Mogibacterium, Family XIII UCG-002, or Rumino- coccaceae UCG-007 in the higher calcium digestibility group. All of these genus level groups were present also in our data, but most of them did not show significant abundance changes in response to CaPe treatments. Therefore, the role of the rumen microbiota in the utili- zation of calcium requires further investigation. The second intermediate compound, hydrogen perox- ide, can induce oxidative stress with damaging effects on host and microbial cells (Pang et al., 2024). Rumen microorganisms have antioxidant defense mechanisms in the form of enzymes that reduce or break down hy- drogen peroxide into water and oxygen (Mann et al., 2018). It is expected that the extra O2 introduced into the system may elevate ORP outside of the favorable range for the growth and activity of rumen anaerobic microorganisms, as has been observed in the beef trial using ORP measuring boluses (Roskam et al., 2024). Vattulainen et al.: CALCIUM PEROXIDE AND BIOCHAR FEED ADDITIVES Journal of Dairy Science Vol. 108 No. 12, 2025 13377 Rumen samples in our study were collected 3 h after the morning feeding and fall within the time frame of potentially elevated ORP, suggesting that we might have captured microbial ecosystem under oxidative stress conditions. Supplementation of CaPe increased abundance of several bacilli (Anaeroplasma, Erysipela- toclostridiaceae UCG-004, Erysipelotrichaceae sp., Solobacterium, RF39). Interestingly, the relative abun- dance of Bacillus was 15 times higher during anaerobic fermentation of waste activated sludge treated with CaO2 (Li et al., 2015), and various bacilli responded to CaO2 pretreatment in waste composting trial (Hu et al., 2025). RF39 and several members from Erysipelo- trichales are uncultured orders within the bacilli. They have been shown to populate the human gut (Nayfach et al., 2019), and metagenome sequencing demonstrates that many of these members have hydrogen peroxide catabolism genes (Wang et al., 2020). The observed in- crease in bacilli could reflect their enzymatic capacity to manage reactive oxygen species. Supplementation with CaO2 increased Entodinium abundance as compared with CON at the expense of sig- nificant decreases in Epidinium and Isotricha. Demeyer (1982) experimented with increasing amounts of CaO2 in sheep and demonstrated a defaunating effect of CaO2, with Entodinium reappearing as the first ciliate proto- zoa. A 3-fold increase in abundance of Entodinium in the CaPe2 treatment suggests its potential tolerance to reactive oxygen species. In general, ciliate protozoa are aerotolerant anaerobes. Experiments with Polyplastron multivesiculatum and Eudiplodinium maggii demonstrat- ed their ability to consume O2 up to the inhibitory thresh- old specific for each species (Ellis et al., 1989), whereas Entodinium caudatum and Epidinium caudatum could be cultured successfully in aerobic media supplemented with selected antioxidants (Park and Yu, 2018). Further- more, Park et al. (2021) sequenced the macronuclear genome of Entodinium caudatum and found presence of genes encoding oxygen-scavenging enzymes, indicating that Entodinium caudatum can tolerate O2. We hypothesize that ciliate protozoa could be respon- sible, at least partially, for the reduction in hydrogen production observed with CaPe treatments. Protozoa can produce hydrogen through hydrogenosomes, but their function can be affected by the physiological lev- els of O2 in the rumen. Prins and Prast (1973) showed that in the presence of small amounts of O2, production of hydrogen by Isotricha spp. decreased. Ellis et al. (1989) demonstrated that in the presence of O2, E. mag- gii produced 8 times more hydrogen, whereas similar exposure of P. multivesiculatum to O2 decreased hydro- gen by 18%. This is suggested to be due to the modula- tion of the hydrogen-evolving system either directly by O2 or by reactive oxygen species. In this experiment, methanogens were not signifi- cantly affected by CaO2, suggesting that the minor de- crease in CH4 production in CaPe treatments resulted from the overall decrease in feed digestion and reduced availability of methanogenic substrates. Hydrogen emission was reduced in the CaPe treatments, and therefore our prior hypothesis was to see Mbb. rumi- nantium at higher relative abundance, as it is suggested to thrive better under low levels of hydrogen partial pressure due to the absence of the methyl coenzyme-M reductase II (Mcr II) system in their genome (Morgavi et al., 2013). However, the average abundance of Mbb. gottschalkii reached 76%, whereas Mbb. ruminantium clade was only detected at ~8% abundance. A potential explanation could be that ORP more strongly affected the particular microbial interactions between Mbb. ruminantium and other bacteria or ciliate protozoa in- volved in hydrogen metabolism and transfer than in the case of Mbb. gottschalkii. We elucidated the effect of dietary CaO2 on the fecal microbiome due to the increased Ca and P concentrations in feces and reduced digestibility in rumen. Bacteroida- les RF16 group, Prevotella, Solobacterium, [Ruminococ- cus] gauvreauii group, and Rhodospirillales spp. were a few of the genera significantly affected by CaPe in both rumen and feces, but the remaining affected genera were low abundance and feces specific. Liu et al. (2020) observed diverse bacterial genera responding to digest- ibility of calcium across different parts of the gastroin- testinal tract in goats, supporting lack of overlap in our data. However, the function of these genera in the colon requires further investigation. CONCLUSIONS Calcium peroxide, especially when used at a higher level (1.5% DM), reduced feed intake and milk yield without affecting feed efficiency. Daily CH4 and CO2 production were reduced by both levels of CaO2 but CH4 and CO2 yields (g/kg DMI) or intensities (g/kg ECM) were not different from the control, indicating, that the reduction in intake was the main reason for reduced CH4 and CO2 emissions. Feeding CaO2 influ- enced rumen bacteria and ciliate protozoa communities more than archaea or anaerobic fungi. To overcome pal- atability issues, future research with dairy cows could test feeding CaO2 pelleted concentrates separately from silage instead of feeding them as TMR, by allowing a break of several hours between the CaO2-concentrate offerings. The biochar with fibrolytic enzyme and live yeast product at the inclusion rate of 0.2% did not influ- ence feed intake, milk and ECM yields, feed efficiency, CH4 and CO2 emissions, rumen fermentation, or gut microbiota composition. Vattulainen et al.: CALCIUM PEROXIDE AND BIOCHAR FEED ADDITIVES 13378 Journal of Dairy Science Vol. 108 No. 12, 2025 NOTES This experiment was a part of IRMA-project (Cli- mate smart feeding solutions for Finnish milk produc- tion sector), which was funded by Finnish Ministry of Agriculture and Forestry (Helsinki, Finland; grant no. VN/20638/2022-MMM-3). We thank CSC-IT Center for Science (Espoo, Finland) for computational resources. We also thank the companies GoBioFarm Ltd. (Iisalmi, Finland) and GlasPort Bio Ltd. (Galway, Ireland) for providing the feed additives used in the study. The experiment was conducted according to Regional State Administrative Agency permission ESAVI/25708/2023 in accordance with the guidelines established by the European Community Council Di- rective 2010/63/EU for animal experiments and com- plied with the ARRIVE guidelines. The authors have not stated any conflicts of interest. Nonstandard abbreviations used: 3-NOP = 3-nitro- oxypropanol; ASV = amplicon sequence variant; BFE = biochar with fibrolytic enzymes and live yeast; CaPe = calcium peroxide; CaPe1 = diet supplemented with 0.75% CaO2 on a DM basis; CaPe2 = diet supplemented with 1.5% CaO2 on a DM basis; CON = control diet; EE = ether extract; GE = gross energy; Lin = linear effect; ORP = oxidation-reduction potential; PCoA = principal coordinate analysis; Quad = quadratic effect. REFERENCES Ahvenjärvi, S., A. R. Bayat, M. Toivanen, P. Mäntysaari, and I. Tapio. 2024. 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