# Use of 3-nitrooxypropanol in early lactation dairy cows fed a high forage total mixed ration: Effect on enteric methane emissions, performance, and milk carbon isotopic signature

C. Saro, Cécile Martin, G. Cantalapiedra-Hijar, M. Bouchon, Céline Chantelauze, Diego Morgavi

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C. Saro, Cécile Martin, G. Cantalapiedra-Hijar, M. Bouchon, Céline Chantelauze, et al., Use of 3-nitrooxypropanol in early lactation dairy cows fed a high forage total mixed ration: Effect on enteric methane emissions, performance, and milk carbon isotopic signature. Journal of Dairy Science, 2025, 10.3168/jds.2025-26843. hal-05408327

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# Use of 3-nitrooxypropanol in early lactation dairy cows fed a high forage total mixed ration: Effect on enteric methane emissions, performance, and milk carbon isotopic signature

C. Saro,<sup>1,2,3*</sup> Cécile Martin,<sup>1*</sup> G. Cantalapiedra-Hijar,<sup>1</sup> M. Bouchon,<sup>4</sup> Céline Chantelauze,<sup>1,5</sup> and Diego P. Morgavi<sup>1†</sup>

## ABSTRACT

The aim of this study was to test the effect of 3-nitrooxypropanol (3-NOP) on methane emissions, animal performance, milk composition, and rumen fermentation in early lactation dairy cows fed a forage-rich diet. A second objective was to assess the ¹³C isotopic signature of milk as a potential proxy for methane-emission status. Twenty-seven cows with similar BW and age in early lactation (7–11 DIM) and fed a 75% forage-based diet were selected and distributed to 2 balanced groups in a randomized block design. The treatment group (n = 13) received 3-NOP (60 mg/kg DM basis) in a TMR for 105 d and the control group (n = 14) received a placebo. The 3-NOP additive was included in a supplement formulated with propylene glycol and adsorbed on silicon dioxide. The control group received the same supplement without the additive. Individual daily methane emissions were quantified using the GreenFeed system throughout the study. Intake and milk production were recorded daily, and milk composition (fat, protein, lactose, urea) twice a week. Natural ¹³C abundance (δ¹³C) in milk and feed samples was determined using isotope-ratio MS coupled with elemental analysis for each cow at wk 3, 7, 11, and 15. Methane emissions, methane yield, and methane intensity were lower in the treatment group throughout the 105 d (on average –31% g/d, –24% g/kg DMI, and –30% g/kg ECM). By monitoring methane emissions throughout the day, we found that the main effect occurred after feeding, preventing the postprandial peak in methane emissions. Intake was reduced by ~7% with 3-NOP, whereas milk production was similar between groups

(34.7 kg ECM/d). Consequently, feed conversion efficiency in animals tended to increase with 3-NOP (1.39 vs. 1.48 kg milk/kg DMI on average). Milk composition did not vary between groups. Whereas no differences were found in δ¹³C for both dietary treatments, milk from treatment cows had a lower δ¹³C than the control group throughout the experimental period. The results of this work with medium-producing dairy cows confirm the efficacy of the additive on diets relatively high in NDF, which broadens its applicability to less intensive production systems. The results for milk δ¹³C, suggesting its potential as a qualitative proxy for methane emissions, merit further investigation, as it could be used in breeding and in monitoring, reporting, and validation systems.

**Key words:** 3-nitrooxypropanol, enteric methane, early lactation dairy cows, milk isotopic signature

## INTRODUCTION

Enteric methane emissions represent 46% of GHG produced globally by livestock, and it is mainly produced in the digestive tract of ruminants (FAO, 2023). Among available mitigation strategies, one of the most promising is the use of specific methane inhibitors, which can cut emissions by ~35% on average (Arndt et al., 2022). A specific inhibitor is the compound 3-nitrooxypropanol (3-NOP) that blocks the activity of the nickel enzyme methyl coenzyme-M reductase (Duin et al., 2016), an enzyme necessary to produce methane and exclusively present in methanogenic archaea. The 3-NOP has been proven efficient to reduce methane emissions in various ruminant production systems (Jayanegara et al., 2017; Orzuna-Orzuna et al., 2024). Moreover, there is a particular abundance of studies in dairy cows, where prediction models have been developed, as is highlighted in a meta-analysis by Kebreab et al. (2023). However,

<footnote n="1"><sup>1</sup>Université Clermont Auvergne, INRAE, VetAgro Sup, UMR Herbivores, 63112 Saint-Genès-Chamanelle, France</footnote>

<footnote n="2"><sup>2</sup>Departamento de Producción Animal, Universidad de León, Campus Vegazana s/n, 24071 León, Spain</footnote>

<footnote n="3"><sup>3</sup>Instituto de Ganadería de Montaña (CSIC-Universidad de León), Finca Marzanas, 24346 Grulleros, Spain</footnote>

<footnote n="4"><sup>4</sup>INRAE, UE1414 Herbipôle, F-63122 Saint-Genès-Chamanelle, France</footnote>

<footnote n="5"><sup>5</sup>Université Clermont Auvergne, INRAE, UNH, Plateforme d'Exploration du Métabolisme, MetaboHUB, Clermont, F-63000 Clermont-Ferrand, France</footnote>

<footnote>Received April 28, 2025.</footnote>

<footnote>Accepted October 3, 2025.</footnote>

<footnote>*These authors contributed equally to this work.</footnote>

<footnote>†Corresponding author: diego.morgavi@inrae.fr</footnote>
Saro et al.: METHANE INHIBITION AND MILK C-13 ISOTOPE SIGNATURE

the data used in these models do not cover all the dietary diversity observed in dairy farms. For instance, fiber content of the diet (NDF) has been identified as a key factor in the efficacy of 3-NOP, yet the number of studies with diets having levels of NDF higher than 400 g/kg of DM is still scarce (Kebreab et al., 2023).

There is an interest in the field of enteric methane for the validation and use of proxies for emissions that could be used on a large scale for breeding or management purposes (Negussie et al., 2017). In dairy cows, milk is an easily accessible matrix, and some milk components are explored as indicators of enteric methane emissions. The analysis of milk fatty acid composition (Bougouin et al., 2019) and the use of milk mid-infrared spectra (MIR) are the most developed and are already integrated into equations for predicting methane emissions (Vanlierde et al., 2021; Coppa et al., 2022; Massaro et al., 2024). Other milk metabolites that can be linked to microbial activity in the gastrointestinal tract, such as dimethylsulfoxide, dimethylsulfone, and citramalic acid, have also been shown to vary with methane emissions (Yanibada et al., 2021). Methane production in the rumen has the potential to affect the natural abundance of ¹³C (δ¹³C) in other body compartments, including milk. Methane produced in the rumen is extremely depleted in ¹³C compared with the ingested diet (Lopes et al., 2016; van Cleef et al., 2021). This is the consequence of the strong isotopic fractionation occurring in the rumen when carbon dioxide is reduced into methane (Botz et al., 1996). As a result, it can be expected that individuals with high emissions will naturally become enriched with ¹³C in other body pools, such as milk, as previously reported (Cantalapiedra-Hijar et al., 2021). The milk carbon isotope signature has not previously been tested as a proxy for enteric methane emissions. The experimental setup of this study, where cows fed the same diet either received or did not receive a methanogenic inhibitor, was ideal for testing this hypothesis and confirming the results of Cantalapiedra-Hijar et al. (2021).

The objective of this study was to assess the methane-
decreasing effects of 3-NOP in cows from a French
dairy system fed a diet rich in fiber representative of a
medium-mountain production area. Cows had heteroge-
neous production potential and were supplemented with
the additive from the beginning of the lactation up to 105
DIM. A second objective was to monitor the ¹³C isotopic
signature of milk and confirm it as a potential proxy for
the methane-emission status of dairy cows.

MATERIALS AND METHODS

The study was conducted at the animal facilities of
the Herbipôle Unit (Herbipôle, INRA, 2018), medium-
mountain ruminant experimental facility, https://doi

.org/10.15454/1.5572318050509348E12) in Marcenat,
France (45°18′N, 2°49′E). Procedures were evaluated
and approved by the French Ministry of Education and
Research (APAFIS #6685–2016090512592554V2) and
carried out in accordance with French and European
guidelines and regulations for animal experimentation.

Experimental Design and Animal Management

The study was conducted on early lactation Holstein
dairy cows for 105 d. It consisted of a randomized block
design with 28 multiparous dairy cows allocated to 2
groups of 14 cows (BW 688 ± 39.6 vs. 679 ± 56.1 kg;
age: 4.6 ± 0.88 vs. 5.1 ± 0.91 yr; number of lactations: 2.5
± 0.66 vs. 2.7 ± 0.73, means ± SD for control and 3-NOP,
respectively, at the start of the study). All cows were
in the same pen. A stratified block randomization was
applied with cows divided into 3 strata, so-called low-
(<24.5 kg/d, n = 4 in each group), medium- (≥24.5 kg/d
and ≤ 28.2 kg/d, n = 5 in control and 4 in 3-NOP), and
high- (>28.2 kg/d, n = 5 in each group) producing cows. A
cow in the 3-NOP group that was assigned to the medium
stratum was excluded from the study because it had lami-
nitis at the beginning of the experimental period. The cut-
offs were determined before the start of the experiment
based on previous lactations' information. After calving,
the average milk production on d 4, 5, and 6 of lactation
was calculated and used as verification of the assigned
stratification based on historic data. Cows were recruited
from the dairy herd and entered the study progressively
within 7 to 11 d after calving. All cows were recruited
within 3 mo. Animals were housed in a freestall barn and
had free access to water throughout the experiment. The
barn was opened at the sides for natural ventilation.

Diets and Feeding

Dairy cows were fed a TMR (75% forages and 25% concentrates) with 3-NOP (3-NOP group) or without (control group) throughout the study. The basal TMR ingredients and chemical composition are shown in Table 1. The 3-NOP additive was included in a supplement consisting of 10% of 3-NOP formulated with propylene glycol and adsorbed on silicon dioxide. Placebo supplement for control was formulated similar to the 3-NOP premix but without addition of the additive. These supplements were integrated in a premix consisting of ground corn, dry molasses, sunflower oil, and supplement (70%, 17%, 8%, and 5% on a DM basis). Premixes were prepared twice weekly using an 85 L kneading machine (Fonderies et Ateliers Loiselet, Vernouillet, France). Every day premixes were mixed with the TMR and distributed in the mangers using a Data Ranger (American Calan Inc., NH), which is a small 2 m³ mixer and feeder designed
Saro et al.: METHANE INHIBITION AND MILK C-13 ISOTOPE SIGNATURE

for research purposes and suited to the inclusion of additives in TMR. The targeted inclusion rate was 60 mg 3-NOP/kg feed on a DM basis for the treatment group, equivalent to 600 mg 3-NOP supplement/kg feed, or 600 mg placebo supplement without 3-NOP for the control group. The preparation was made first for the control and then for the treatment. The Data Ranger was then used to feed the remaining cows in the herd, ensuring that no 3-NOP residue remained for the following day.

Cows were fed daily in the morning at 1130 h and in the afternoon, mangers were topped up with TMR. The daily amount of TMR offered to the animals was determined at the beginning of the study and readjusted daily, if necessary, according to the refusals to allow animals to be fed ad libitum. Feed was offered through mangers controlling and recording daily individual feed intake (CRFI, Biocontrol, Norway). Fourteen mangers were available for the study, 7 for each diet. All 14 cows in the same diet group had access to 7 mangers containing the corresponding diet for the group. The mangers have a gate that opens when a cow's RFID ear tag is read and closes as soon as the cow leaves. The gate only opens for cows receiving the right diet, preventing other cows from eating from the wrong mangers. Every morning, leftover TMR from the previous day was removed from the mangers before adding fresh TMR.

## Intake and Feed Analysis

Total DMI was obtained by adding the daily amount of commercial concentrate effectively ingested at the GreenFeed methane-measurement system (GF; C-Lock Inc., Rapid City, SD, see below) to the daily TMR intake recorded by the mangers. The DM content of the TMR was determined 3 times a week and the concentrate once a week by oven drying (60°C for 72 h; Andueza et al., 2016). The TMR offered and refusals were sampled separately 3 times a week using the coning and quartering method (Horwitz, 1990; starting with ~10 kg until obtainion of ~250 g), pooled, and stored at -20°C. Samples from 4 consecutive weeks were combined to produce a monthly sample for chemical composition. Samples of the concentrate distributed in the GF were taken once a month and stored at room temperature before determination of chemical composition. All feed samples were dried (60°C for 72 h) and ground (1-mm screen, ZM 200 Retsch Mill, Haan, Germany) before determination of OM, CP, NDF, and ADF, starch, ether extract (EE), and gross energy (GE) content. The OM content was determined by ashing at 550°C for 6 h (method 942.05; Association of Official Analytical Chemists, 2005), and CP content was calculated as N content × 6.25, with total N analyzed according to the Dumas method (method 968.06; Association of Official Analytical Chemists,

<table><caption>**Table 1.** Ingredients and chemical composition (mean ± SD) of diets fed to cows</caption><thead><tr><th>Item</th><th>Control diet</th><th>3-NOP diet</th></tr></thead><tbody><tr><td>Ingredient (%DM)</td><td></td><td></td></tr><tr><td>Corn silage</td><td>52.1</td><td>52.1</td></tr><tr><td>Hay regrowth</td><td>11.0</td><td>11.0</td></tr><tr><td>Grass hay</td><td>5.5</td><td>5.5</td></tr><tr><td>Wheat straw</td><td>6.0</td><td>6.0</td></tr><tr><td>Concentrate</td><td>24.4</td><td>24.4</td></tr><tr><td>Mineral supplement</td><td>0.5</td><td>0.5</td></tr><tr><td>Premix</td><td>0.5</td><td>0.5</td></tr><tr><td>Chemical composition (% DM)</td><td></td><td></td></tr><tr><td>OM</td><td>93.6 ± 0.19</td><td>93.5 ± 0.20</td></tr><tr><td>CP</td><td>11.7 ± 0.54</td><td>11.8 ± 0.52</td></tr><tr><td>NDF</td><td>41.5 ± 1.08</td><td>40.4 ± 1.70</td></tr><tr><td>ADF</td><td>22.8 ± 0.86</td><td>22.1 ± 0.91</td></tr><tr><td>Starch</td><td>18.5 ± 2.22</td><td>18.1 ± 1.78</td></tr><tr><td>Fat</td><td>3.30 ± 0.22</td><td>3.43 ± 0.12</td></tr><tr><td>Gross energy (MJ/kg MS)</td><td>18.6 ± 0.09</td><td>18.6 ± 0.03</td></tr><tr><td>3-Nitrooxypropanol</td><td>0</td><td>60.3 ± 22.18<sup>1</sup></td></tr><tr><td>δ<sup>13</sup>C, ‰</td><td>-20.75</td><td>-20.76</td></tr></tbody></table>

2005). The NDF and ADF were determined according to Van Soest et al. (1991). Starch was analyzed using an enzymatic method (Faisant et al., 1995). Ether extract was determined after acid hydrolysis (method 954.02; Association of Official Analytical Chemists, 2005). Gross energy was analyzed by isoperibolic calorimetry (model C200, IKA, Staufen, Germany). For 3-NOP analysis in the TMR, 250 g samples were taken on 3 different days a week, pooled, thoroughly mixed by hand, and packed in reclosable polyethylene bags. These composite samples were immediately frozen at -20°C and sent frozen every week for analysis to DSM Nutritional Products (Kaiseraugst, Switzerland).

## Milk Yield and Composition, BW, and Reproductive Parameters

Cows were milked twice daily, with milk yield measured at each milking and recorded on a daily basis. Milk samples (20 mL) were collected from the morning and afternoon milking of each cow on 2 consecutive days per week and analyzed by LIAL Massif Central (Auril-lac, France) for fat, protein, lactose, and urea nitrogen using MilkoScan FT+ and SCC using Fossomatic FC, both from Foss Electric A/S (Hillerød, Denmark). Milk composition per day was calculated taking into consideration the proportion of milk collected in the morning and afternoon milking. Milk production was converted to ECM (kg/d) according to Sjaunja et al. (1990).

The δ¹³C in milk and TMR samples was determined by using an isotope-ratio mass spectrometer (Isoprime, VG Instruments, Manchester, UK) coupled to an elemental

<footnote n="1">¹The additive was added to the TMR following a standard procedure. The variation (SD value) is attributed to the heterogeneity of the TMR and sample preparation for analysis, as there was a similar day to day variability in methane emissions between control and 3-NOP cows.</footnote>
Saro et al.: METHANE INHIBITION AND MILK C-13 ISOTOPE SIGNATURE

analyzer (EA Isoprime, VG Instruments, Manchester, UK) on morning milk samples taken in wk 3, 7, 11, and 15. Further, ~10 mg of milk samples and 4 mg of the TMR samples were weighed for isotopic analysis. Cow BW data were collected twice daily at the exit of the milking parlor using an automated scale system (AWS100 Delaval scale system; France). Reproductive data on number of inseminations, days open, and calving interval were recorded during and after the study to determine a potential posteffect of the 3-NOP treatment on reproductive traits.

Energy balance was estimated as the difference between energy intake and the sum of energy for maintenance and milk production using the INRA (2018) NEL system, in which “unité fourragère lait” (UFL) is the NEL equivalent of 1 kg of standard air-dried barley.

## Methane and Carbon Dioxide Emissions

Methane and CO₂ emissions from dairy cows were individually estimated using 2 GF located in the pen that were indistinctly accessed by the dairy cows. The GF were calibrated to distribute 6 drops of 50 g every 30 s per animal and per visit with a minimum interval of 3 h between visits (i.e., 2.4 kg maximum of concentrate delivered per day and per cow). Filters were changed weekly, or as soon as the flow fell below 27 L/s. The nondispersive infrared sensor was automatically calibrated once daily (between 0330 and 0430 h) when the animals did not visit the GF, by injecting a calibration gas mixture with certified concentrations of methane (mean ± SE; 1,005 ± 0.71 ppm) and CO₂ (10,065 ± 0.71 ppm) in N₂ (Air Liquide, Paris). In addition, the CO₂ recovery rate of the systems was calculated monthly as the ratio between the quantity of CO₂ recovered in the exhaust air (i.e., CO₂ concentration determined by the nondispersive infrared sensor multiplied by air flow rate) and the quantity of CO₂ released into the system during 3 min in triplicate and determined by gravimetry. Throughout the study, the average flow rate for both GF was 36.0 ± 2.96 L/s, and the CO₂ recovery rate averaged 100.1% ± 6.26%. Daily number of visits was on average 3.2 ± 1.71 for control group and 3.1 ± 1.51 for treatment group (mean ± SD). Visits were evenly spread throughout the day, except for a decrease in frequency between 0300 and 0500 h and at milking times (0800 and 1500 h; Supplemental Figure S1, see Notes).

## Plasma and Rumen Parameters

At 5, 10, and 14 wk of lactation, blood samples were taken through jugular puncture into evacuated tubes containing EDTA (1.95 mg/mL; Terumo Europe NV, Leuven, Belgium) and centrifuged at 1,400 × g for 15 min

at 4°C. Plasma was conserved at -20°C until analysis for nonesterified fatty acids (NEFA), glucose, BHB, and urea using an automatic analyzer (Arena 20XT, Thermo Fisher Scientific, Cergy Pontoise, France) as described (Pires et al., 2019).

Rumen fluid (~250 mL) was sampled by stomach tubing at the end of the experimental period in wk 15, 2 h after feeding. The reason for sampling after feeding was to increase the likelihood of capturing rumen changes brought about by the additive, as it is known that antimethanogenic additives mainly affect the postprandial methane production peak (Guyader et al., 2015). Samples were immediately strained through a polyester monofilament fabric (250 µm aperture). For VFA analysis, 0.8 mL of filtrate was mixed with 0.5 mL of a 0.5 M HCl solution containing 2% (wt/vol) metaphosphoric acid and 0.4% (wt/vol) crotonic acid (used as internal standard; Guyader et al., 2015). For ammonia-nitrogen (NH₃-N) analysis, 1 mL of filtrate was mixed with 0.1 mL of 5% orthophosphoric acid (Guyader et al., 2015). Samples were stored at -20°C until analysis. Concentrations of VFA and NH₃-N were analyzed by GC with a flame ionization detector and by colorimetry, respectively (Morgavi et al., 2008).

## Statistical Analysis

All data were analyzed using the MIXED procedure of SAS (version 9.4, SAS Inst. Inc., Cary, NC). The GF dataset collected by C-Lock during the whole experiment (11,390 spots) was filtered to eliminate outliers before statistical analysis according to Arbre et al. (2016). The proportion of outliers was 2.23% for methane and CO₂ emissions. Gas spot data were averaged over 7-d periods (15 periods) to reach reliable and repeatable (R >0.7) measurements of daily gas emissions in g/d (Arbre et al., 2016). Data on DMI, BW, and milk production and composition were averaged by 7-d periods to coincide with gas emissions. The model included the fixed effects of treatment (n = 2), period (n = 15), stratum (n = 3), the interactions between treatment and period and between treatment and stratum; cow (n = 27) was the random effect. Milk yield in d 4, 5, and 6 of lactation was included as a covariate in the model for the analysis of milk yield. Kinetics in gaseous emissions (methane and CO₂) across the day were obtained by averaging spot measurements of the whole experiment over 1-h time slots of the day (i.e., emissions measured between 0 and 1 h, between 1 and 2 h, and so on) before being included in the statistical analyses. The model included the fixed effects of treatment (n = 2) and hour (n = 24) and the interaction between treatment and hour. Cow was the random effect. Data on blood parameters and isotopic signature of milk were analyzed with a model that included the fixed ef-
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<table><caption>**Table 2.** Effect of 3-NOP on enteric methane emissions, yield, and intensity in dairy cows<fnref n="1" />,<fnref n="2" /></caption><thead><tr><th rowspan="2">Item</th><th rowspan="2">Control</th><th rowspan="2">3-NOP</th><th rowspan="2">SEM</th><th colspan="5">P-value</th></tr><tr><th>Treat</th><th>Week</th><th>Stratum<fnref n="3" /></th><th>Treat × week</th><th>Treat × stratum</th></tr></thead><tbody><tr><td>CH<sub>4</sub> (g/d)</td><td>425</td><td>293</td><td>15.9</td><td>&lt;0.001</td><td>&lt;0.001</td><td>0.53</td><td>0.30</td><td>0.14</td></tr><tr><td>CO<sub>2</sub> (g/d)</td><td>13,298</td><td>12,782</td><td>276.4</td><td>0.20</td><td>&lt;0.001</td><td>0.73</td><td>0.87</td><td>0.78</td></tr><tr><td>CH<sub>4</sub> yield (g/kg DMI)</td><td>16.4</td><td>12.4</td><td>0.58</td><td>&lt;0.001</td><td>0.006</td><td>0.39</td><td>0.97</td><td>0.15</td></tr><tr><td>CH<sub>4</sub> intensity (g/kg milk)</td><td>12.1</td><td>8.4</td><td>0.50</td><td>&lt;0.001</td><td>&lt;0.001</td><td>0.11</td><td>0.53</td><td>0.45</td></tr><tr><td>CH<sub>4</sub> intensity ECM<sup>4</sup> (g/kg ECM)</td><td>12.2</td><td>8.6</td><td>0.50</td><td>&lt;0.001</td><td>&lt;0.001</td><td>0.20</td><td>0.11</td><td>0.44</td></tr><tr><td>CH<sub>4</sub> (% of GEI)</td><td>5.01</td><td>3.73</td><td>0.181</td><td>&lt;0.001</td><td>0.010</td><td>0.34</td><td>0.94</td><td>0.18</td></tr></tbody></table>

fects of treatment (n = 2), sampling time (n = 3), and stratum (n = 3), the interactions between treatment and sampling time and between treatment and stratum, and the random effect of the animal.

For data collected over time, the effect of period (n = 15), hour (n = 24), or sampling time (n = 3) was considered as a repeated measure, and the covariance structure was selected on the basis of the lowest Akaike criteria values. Heterogeneous compound symmetry covariance matrix was selected for the percentage of protein and lactose in milk, autoregressive for BW and Toeplitz for SCC. For the rest of the parameters, the structure selected was heterogeneous autoregressive.

Data are presented as LSM ± SEM. Differences were considered significant when $P < 0.05$ and a tendency was considered when $P \le 0.10$.

RESULTS

Methane emission (g/d) was 31% lower in the 3-NOP group compared with control ($P < 0.001$; Table 2). Similarly, methane intensity, either expressed as g/kg milk or as g/kg ECM, was reduced by 30.5% on average with the 3-NOP treatment. Methane yield (g/kg DMI) and methane conversion rate, expressed as the proportion of GE intake converted to methane, were also ~25% lower in cows fed the additive than those fed the control diet ($P < 0.001$). No interaction was observed between treatment and week or treatment and level of milk production (stratum), with differences in emissions between groups constant throughout the 105 d of the study (Supplemental Figure S2, see Notes). The lower methane emissions in treatment cows were observed throughout the day, but they were particularly marked after feeding (Figure 1). The antimethanogenic additive suppressed the postprandial increase in methane normally observed. In contrast, hourly $CO_2$ emissions were similar for both groups throughout the day.

Intake and milk production performance of dairy cows are shown in Table 3. Dry matter intake of dairy cows fed the 3-NOP diet was significantly lower (−1.7 kg DM on average; $P = 0.01$) than for cows fed the control diet. As expected, DMI differed between strata for both the 3-NOP and control groups; DMI was highest in the high milk producer's stratum and similar for the medium and low milk producers (mean 27.1, 23.6, and 24.0 kg/d DMI, respectively; $P < 0.001$). There was a numerical decrease in daily milk production for cows fed the 3-NOP diet. However, differences were not significant (−1.6 kg milk and −1.4 kg ECM in 3-NOP cows as compared with control cows). Milk composition (fat, protein, urea, SCC, fat yield, protein yield, and lactose yield) was similar between control and 3-NOP groups. Only the proportion of lactose differed between strata ($P = 0.028$), with high-producing cows having a lower proportion of lactose in milk than medium- and low-producing cows (on average 5.08%, 5.29%, and 5.21%, respectively).

There was a numerical increase in feed conversion efficiency of dairy cows receiving 3-NOP (1.39 vs. 1.48 kg milk produced/kg DMI on average for control and 3-NOP groups, respectively). Independent of treatment, feed efficiency tended ($P = 0.10$) to be higher in the group of medium producers as compared with high and low producers (on average 1.39, 1.53, and 1.39 kg milk produced per kg DMI for high, medium, and low, respectively). No statistical differences in BW were detected between groups at the end of the study, although cows on 3-NOP lost 1.83 kg and control cows gained 1.36 kg. Although the natural $^{13}$C abundance ($\delta^{13}$C) in both TMR was similar, milk $\delta^{13}$C was higher in the control group throughout the experimental period ($P < 0.001$; Table 4 shows the average value for samples taken at wk 3, 7, 11, and 15 of the study). There were changes in milk $\delta^{13}$C as the lactation progressed ($P < 0.001$). The differences between groups were larger in wk 3 but the control group

<footnote n="1">¹3-NOP cows received a ration containing 3-nitrooxypropanol at a dose of 60 mg/kg DM for 105 d and control cows received a placebo.</footnote>

<footnote n="2">²Treat = treatment.</footnote>

<footnote n="3">³Three strata assigned based on historic average milk production: low (<24.5 kg/d); medium (≥24.5 kg/d and ≤28.2 kg/d); and high (>28.2 kg/d).</footnote>

<footnote n="4">⁴ECM = milk yield (kg/d) × [38.3 × fat (%) × 10 + 24.2 × protein (%) × 10 + 16.54 × lactose (%) × 10 + 20.7]/3.140 (Sjaunja et al., 1990).</footnote>
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<figure><img src="image_3.png" /><figcaption>**Figure 1.** Average emission of CH₄ (A) and CO₂ (B) throughout the day in control (black square, ■) or 3-nitrooxypropanol treatment (black triangle, ▲) cows. Values are expressed in g/d; mean values and SD are shown for each time frame. Arrows indicate feeding time.</figcaption></figure>

always had greater values than the 3-NOP group at all sampling periods (Figure 2).

Rumen fermentation parameters are shown in Table 5. No difference in total VFA concentration was observed between groups and strata. Molar proportion of acetate decreased by 4% (*P* = 0.006) and that of butyrate and propionate increased by 1.7% and 2%, respectively (*P* ≤ 0.05), in treatment cows compared with the control. Subsequently, acetate-to-propionate ratio was lower in treatment cows (*P* = 0.042) for all strata. No difference

between groups and strata was observed for ammonia concentrations.

No differences were observed in blood parameters between control and treatment, although concentration of BHB was lower (*P* = 0.05, Table 6) in the treatment group. Concentrations of NEFA and BHB decreased with advancing DIM. Average NEFA concentration for all cows at wk 5 was 0.36 mmol/L, at wk 10 was 0.26 mmol/L, and at wk 14 was 0.18 mmol/L. Whereas, BHB concentration was 0.67, 0.50, and 0.48 mmol/L at wk
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<table><caption>**Table 3.** Effect of 3-NOP on intake, milk production and composition, and BW in dairy cows¹,²</caption><thead><tr><th rowspan="2">Item</th><th rowspan="2">Control</th><th rowspan="2">3-NOP</th><th rowspan="2">SEM</th><th colspan="5">P-value</th></tr><tr><th>Treat</th><th>Week</th><th>Stratum³</th><th>Treat × week</th><th>Treat × stratum</th></tr></thead><tbody><tr><td>DMI (kg/d)</td><td>25.7</td><td>24.0</td><td>0.44</td><td>0.014</td><td>&lt;0.001</td><td>&lt;0.001</td><td>0.64</td><td>0.61</td></tr><tr><td>Milk yield (kg milk/d)</td><td>36.4</td><td>34.8</td><td>1.18</td><td>0.34</td><td>&lt;0.001</td><td>0.27</td><td>0.73</td><td>0.59</td></tr><tr><td>ECM<sup>4</sup> (kg ECM/d)</td><td>35.3</td><td>33.9</td><td>1.08</td><td>0.35</td><td>&lt;0.001</td><td>0.39</td><td>0.51</td><td>0.93</td></tr><tr><td>Feed efficiency (kg milk/kg DMI)</td><td>1.39</td><td>1.48</td><td>0.042</td><td>0.13</td><td>&lt;0.001</td><td>0.10</td><td>0.022</td><td>0.92</td></tr><tr><td>Milk fat (%)</td><td>3.85</td><td>3.79</td><td>0.099</td><td>0.64</td><td>0.79</td><td>0.38</td><td>0.60</td><td>0.48</td></tr><tr><td>Milk fat yield (kg/d)</td><td>1.37</td><td>1.31</td><td>0.045</td><td>0.34</td><td>&lt;0.001</td><td>0.38</td><td>0.61</td><td>0.71</td></tr><tr><td>Milk protein (%)</td><td>3.15</td><td>3.15</td><td>0.078</td><td>0.96</td><td>&lt;0.001</td><td>0.57</td><td>0.14</td><td>0.66</td></tr><tr><td>Milk protein yield (kg/d)</td><td>1.13</td><td>1.09</td><td>0.035</td><td>0.42</td><td>0.002</td><td>0.36</td><td>0.64</td><td>0.93</td></tr><tr><td>Milk lactose (%)</td><td>5.21</td><td>5.18</td><td>0.048</td><td>0.64</td><td>0.007</td><td>0.028</td><td>0.80</td><td>0.74</td></tr><tr><td>Milk lactose yield (kg/d)</td><td>1.85</td><td>1.79</td><td>0.060</td><td>0.48</td><td>&lt;0.001</td><td>0.24</td><td>0.69</td><td>0.96</td></tr><tr><td>Milk urea (mg/L)</td><td>151.</td><td>160</td><td>5.7</td><td>0.31</td><td>&lt;0.001</td><td>0.21</td><td>0.94</td><td>0.53</td></tr><tr><td>SCC (×1,000)</td><td>113</td><td>175</td><td>61.3</td><td>0.48</td><td>0.072</td><td>0.64</td><td>0.63</td><td>0.36</td></tr><tr><td>Energy balance, UFL/d</td><td>1.47</td><td>0.69</td><td>0.415</td><td>0.17</td><td>&lt;0.01</td><td>0.47</td><td>0.39</td><td>0.44</td></tr><tr><td>BW (kg)</td><td>689</td><td>673</td><td>12.4</td><td>0.32</td><td>0.77</td><td>0.45</td><td>0.46</td><td>0.16</td></tr></tbody></table>

¹³-NOP cows received a ration containing 3-nitrooxypropanol at a dose of 60 mg/kg DM for 105 d and control cows received a placebo.

²Treat = treatment.

³Three strata assigned based on historic average milk production: low (<24.5 kg/d); medium (≥24.5 kg/d and ≤28.2 kg/d); and high (>28.2 kg/d).

⁴ECM = milk yield (kg/d) × [38.3 × fat (%) × 10 + 24.2 × protein (%) × 10 + 16.54 × lactose (%) × 10 + 20.7]/3.140 (Sjaunja et al., 1990).

5, 10, and 14, respectively. In contrast, concentrations of glucose increased from 0.61 g/L at wk 5 to 0.68 and 0.67 g/L at wk 10 and 14. Similarly, urea concentrations increased in later stages of lactation (0.11, 0.13, and 0.14 g/L at wk 5, 10, and 14, respectively).

No difference between control and treatment cows was observed in the reproductive traits evaluated that included the number of inseminations, days open, and calving interval (Supplemental Table S1, see Notes).

DISCUSSION

This study assessed the methane-decreasing effects of 3-NOP in lactating dairy cows from a representative temperate climate French dairy system, where cows are fed a high-fiber diet. This chemical feed additive has been shown to consistently reduce enteric methane emissions under different production systems and in various ruminant types, including dairy cows (Kebreab et al., 2023), beef cattle (Orzuna-Orzuna et al., 2024), and sheep (Martínez-Fernández et al., 2014). The compound

has received approval in the European Union and other countries, where it is marketed under the brand name Bo-vær (dsm-firmenich AG, Kaiseraugst, Switzerland). Despite the wealth of information already available on this inhibitor, there are gaps in knowledge for adapting its use in some production systems. It is known that the inhibitory effect of 3-NOP can be influenced by dose (Haisan et al., 2017; Orzuna-Orzuna et al., 2024), mode of inclusion in the diet (Van Wesemael et al., 2019; Costigan et al., 2024; Munoz et al., 2024), and diet composition (van Gastelen et al., 2022).

For diet composition, the available data clearly indicate that NDF content is an important determinant of the efficacy of the additive (Kebreab et al., 2023). However, most studies to date have used diets with NDF contents (mean ~330 g/kg DM) that are not representative of the medium-mountain areas in France and many other regions of the world where pedoclimatic and production conditions, including economics, result in diets with higher forage proportions and levels of NDF. In our study, NDF in the basal diet was ~410 g/kg DM, and methane emis-

<table><caption>**Table 4.** Effect of 3-NOP on natural ¹³C abundance in milk (δ¹³C<sub>milk</sub>) and carbon isotope discrimination between the animal and its diet (Δ¹³C<sub>milk-diet</sub>) in dairy cows¹,²</caption><thead><tr><th rowspan="2">Item</th><th rowspan="2">Control</th><th rowspan="2">3-NOP</th><th rowspan="2">SEM</th><th colspan="5">P-value</th></tr><tr><th>Treat</th><th>Stratum³</th><th>Time⁴</th><th>Treat × time</th><th>Treat × stratum</th></tr></thead><tbody><tr><td>δ¹³C<sub>milk</sub> (‰)</td><td>-19.5</td><td>-19.9</td><td>0.089</td><td>0.006</td><td>0.19</td><td>&lt;0.001</td><td>0.42</td><td>0.12</td></tr><tr><td>Δ¹³C<sub>milk-diet</sub> (‰)</td><td>1.25</td><td>0.86</td><td>0.095</td><td>0.008</td><td>0.03</td><td>&lt;0.001</td><td>0.58</td><td>0.11</td></tr></tbody></table>

¹³-NOP cows received a ration containing 3-nitrooxypropanol at a dose of 60 mg/kg DM for 105 d and control cows received a placebo.

²Treat = treatment.

³Three strata assigned based on historic average milk production: low (<24.5 kg/d); medium (≥24.5 kg/d and ≤28.2 kg/d); and high (>28.2 kg/d).

⁴Four sampling times taken at wk 3, 7, 11, and 15.
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<figure><img src="image_4.png" /><figcaption>**Figure 2.** Evolution across time of milk ¹³C natural abundance in dairy cows receiving (3-NOP; □) or not (control; ○) a treatment with 3-nitrooxypropanol at a dose of 60 mg/kg DM for 105 d (A). Intake (kg DMI/d; B) and methane production (g/d; C) for the same weeks are indicated for comparison. Pearson correlations coefficients obtained when milk ¹³C natural abundances were regressed within week against methane production and DMI ranged from 0.32 to 0.47 (3 significant weeks) and 0.26 to 0.57 (2 significant weeks), respectively (see Supplemental Figure S3).</figcaption></figure>

sions corresponded to values normally reported in the literature. When adjusting by the reduction in efficacy due to the high NDF content calculated in the meta-analysis of Kebreab et al. (2023), the predicted decrease in

methane yield (g/kg of DMI) was similar to the observed decrease (−23% vs. −24%). However, the observed decrease in methane emissions and methane intensities was greater (ca. −30% vs. −24%) than that predicted by the equations of Kebreab et al. (2023). The inhibition effect was constant throughout the study, which can be considered long-term, as it lasted for a third of the duration of a normal lactation and started in the first week after calving. Starting the study at the beginning of the lactation captures the most physiologically demanding aspect of the production cycle, which was one reason to recruit cows based on their potential milk production and block them into high, medium, and low producers. No marked differences in the effect of the additive were observed between these 3 production strata. However, it is noted that the gap between strata in daily milk production was small, at only 2 to 3 L, and that there were not enough cows in each stratum to detect differences unless they were considerable. Also, average production per cow at ~35 L/d was low compared with other studies on more intensive production systems, i.e., ~45 L/d reported by Hristov et al. (2015).

A numerical increase in feed efficiency of 0.09 kg milk per kg DMI was observed in this work. This improvement in feed efficiency was also reported in the meta-analysis of Martins et al. (2025). It will be important to do follow-up studies on the reproductive performance of a large number of cows, as if the improved efficiency is mainly explained by the lower DMI, energy balance, and reproduction may be affected in the long term. Positive indicators are that there were no changes in BW at the end of the study and, for the cows that remained in the herd, reproductive parameters such as days from calving to first service, calving to conception, and number of artificial inseminations were similar between the treatment and control groups. Reduced intake with 3-NOP in dairy cows was reported in the meta-analysis of Martins et al. (2025), although several studies reported no change (e.g., Hristov et al., 2015; Haisan et al., 2017; Yanibada et al., 2020). Reduced intake is more common in beef cattle (Orzuna-Orzuna et al., 2024). The disparity between cattle types may be due to, among other things, the type of basal diet and the ratio of feed intake relative to rumen volume with changes in the passage rate. More generally, reductions in intake have been explained by a negative feedback effect of propionate on satiety (van Gastelen et al., 2022), but in the present study, VFA concentration was similar between groups, and the propionate levels, although increased significantly, were well below those used experimentally to induce a reduction in DMI (Sheperd and Combs, 1998).
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<table><caption>**Table 5.** Effect of 3-NOP on ruminal VFA concentration and molar proportions, and ammonia concentration in dairy cows¹,²</caption><thead><tr><th rowspan="2">Item</th><th rowspan="2">Control</th><th rowspan="2">3-NOP</th><th rowspan="2">SEM</th><th colspan="3">P-value</th></tr><tr><th>Treat</th><th>Stratum³</th><th>Treat × stratum</th></tr></thead><tbody><tr><td>Total VFA (mmol/L)</td><td>167.0</td><td>156.5</td><td>7.17</td><td>0.31</td><td>0.70</td><td>0.13</td></tr><tr><td>Molar proportions (mol/100 mol)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Acetate</td><td>64.9</td><td>60.9</td><td>0.94</td><td>0.006</td><td>0.52</td><td>0.78</td></tr><tr><td>Propionate</td><td>19.0</td><td>21.0</td><td>0.69</td><td>0.055</td><td>0.58</td><td>0.60</td></tr><tr><td>Butyrate</td><td>12.3</td><td>14.0</td><td>0.45</td><td>0.013</td><td>0.21</td><td>0.38</td></tr><tr><td>Others⁴</td><td>3.8</td><td>4.1</td><td>0.14</td><td>0.11</td><td>0.18</td><td>0.18</td></tr><tr><td>Ac:Pr</td><td>3.5</td><td>3.0</td><td>0.15</td><td>0.042</td><td>0.65</td><td>0.70</td></tr><tr><td>NH₃ (mmol/L)</td><td>2.3</td><td>1.9</td><td>0.47</td><td>0.55</td><td>0.34</td><td>0.82</td></tr></tbody></table>

¹³-NOP cows received a ration containing 3-nitrooxypropanol at a dose of 60 mg/kg DM for 105 d and control cows received a placebo.

²Treat = treatment.

³Three strata assigned based on historic average milk production: low (<24.5 kg/d); medium (≥24.5 kg/d and ≤28.2 kg/d); and high (>28.2 kg/d).

⁴Sum of valerate, caproate, isovalerate, and isobutyrate.

Carbon Isotopic Signature of Milk

There are no publications on the use of ¹³C in milk as a biomarker for methane emissions in dairy cows. The stable isotope profile in cow milk reflects the one of the diet and is therefore proposed as a tool for authenticating the nature of the feeding regimen (Schneider et al., 2015). Carbon transfer from feed to milk is dynamic, with isotopic signatures reflecting feed intake within 12 h (Boutton et al., 1988; Braun et al., 2013). This transfer is modulated by metabolic activities at each step, the first being microbial fermentation in the rumen. A characteristic of methanogenesis is that it strongly favors the lighter C isotope (¹²C), resulting in rumen methane being extremely depleted in ¹³C (Levin et al., 1993; Gruen et al., 2018). The rationale is that cows with increased emissions of methane, which is depleted in ¹³C, will have an increased proportion of the heavier C isotope in other body matrices. Effectively, milk from control cows had higher δ¹³C than 3-NOP-treated cows. Because the cows in this study were fed the same diet, and considering the rapid turnover of dietary carbon in milk, it was reasoned

that this difference in isotopic signature indicates daily methane emissions. Although there is no record of the use of ¹³C in milk as a biomarker for methane, there is a report for methane ¹³C/¹²C ratio emitted from 3-NOP-treated cows with a minor δ¹³C depletion as compared to controls (Lopes et al., 2016). The authors attributed the changes to methanogen metabolism in the rumen.

Another potential interpretation that warrants careful consideration is the possibility that changes in milk δ¹³C across treatments may, to some extent, result from the slight variation in DMI between the 3-NOP and control groups. Milk δ¹³C has been shown to reflect the energy balance of dairy cows in early lactation when body fat is mobilized (Chen et al., 2020). Body fat is typically depleted in ¹³C relative to the diet (Tieszen et al., 1983). Therefore, the observed changes in milk δ¹³C across treatments may also reflect a less positive energy balance in the 3-NOP group compared with controls. In this regard, it was observed that the strongest correlation with δ¹³C was found for the energy balance measured during early lactation (wk 3; Supplemental Figure S3, see Notes), possibly indicating a combined effect of decreased methane

<table><caption>**Table 6.** Effect of 3-NOP on plasma concentration of nonesterified fatty acids (NEFA), BHB, glucose, and urea in dairy cows¹,²</caption><thead><tr><th rowspan="2">Item</th><th rowspan="2">Control</th><th rowspan="2">3-NOP</th><th rowspan="2">SEM</th><th colspan="5">P-value</th></tr><tr><th>Treat</th><th>Stratum³</th><th>Time⁴</th><th>Treat × sampling</th><th>Treat × stratum</th></tr></thead><tbody><tr><td>NEFA (mmol/L)</td><td>0.276</td><td>0.257</td><td>0.0352</td><td>0.71</td><td>0.41</td><td>0.011</td><td>0.53</td><td>0.21</td></tr><tr><td>BHB (mmol/L)</td><td>0.612</td><td>0.496</td><td>0.0563</td><td>0.050</td><td>0.29</td><td>0.013</td><td>0.53</td><td>0.60</td></tr><tr><td>Glucose (g/L)</td><td>0.653</td><td>0.654</td><td>0.0129</td><td>0.95</td><td>0.54</td><td>&lt;0.001</td><td>0.73</td><td>0.45</td></tr><tr><td>Urea (g/L)</td><td>0.129</td><td>0.127</td><td>0.0083</td><td>0.91</td><td>0.85</td><td>&lt;0.001</td><td>0.65</td><td>0.30</td></tr></tbody></table>

¹³-NOP cows received a ration containing 3-nitrooxypropanol at a dose of 60 mg/kg DM for 105 d and control cows received a placebo.

²Treat = treatment.

³Three strata assigned based on historic average milk production: low (<24.5 kg/d); medium (≥24.5 kg/d and ≤28.2 kg/d); and high (>28.2 kg/d).

⁴Sampling was carried out at 5, 10, and 14 wk of lactation.
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emissions and the contribution of body fat (¹³C depleted) to milk synthesis. Nevertheless, differences in BW and energy balance between groups were not significant.

Milk is an easily accessible biological fluid that has been studied as a potential biomarker of methane emissions (Negussie et al., 2017). Milk MIR spectrum is being used to predict methane production (Vanlierde et al., 2021). Also, the use of milk fatty acids has been explored, as their production has a direct link to rumen fermentation (Bougouin et al., 2019), and, additionally, some fatty acids are of microbial origin (i.e., odd and branched-chain fatty acids; Fievez et al., 2012). Of these, MIR has been the most studied because it is routinely used in milk recording programs and for milk payments. However, predictive models based on MIR or fatty acids failed to detect decreases in methane emissions induced by 3-NOP (Coppa et al., 2022), probably because they are based on major milk constituents that are highly influenced by diet (Yanibada et al., 2021). The potential of the carbon signature as a marker of enteric methane emissions should be validated by other studies, and its sensitivity assessed, before being tested in predictive models. In addition, the influence of other body pools, particularly fat mobilization and accretion during lactation, which can affect the carbon signature of milk (Chen et al., 2020), should also be considered. As the carbon signature of milk has to be adjusted by the carbon signature of diet, it should circumvent the confounding effect of diet observed in models based on MIR or milk fatty acids.

## CONCLUSIONS

The supplementation of 3-NOP to lactating dairy cows fed a forage-rich diet (~410 g/kg NDF) typical of medium-mountain areas effectively reduced methane production, methane yield, and methane intensity. Intake and milk yield also decreased, but the effect on the latter was not statistically significant, resulting in a tendency for improved feed conversion efficiency in supplemented cows. The study was carried out in the first third of the lactation when nutritional demands are higher, but no negative effect on BW was recorded despite the lower DMI. The carbon signature of milk as an indicator of enteric methane production in lactating dairy cows shown in this work is a promising alternative tool for selection of animals and for measurement, reporting, and verification that merits further exploration.

## NOTES

This work was partially funded by DSM-Firmenich. The DSM-Firmenich was not involved in data analysis and interpretation of results. The authors wish to thank

personnel at the Herbipôle Marcenat for animal care, and Dominique Graviou for laboratory technical support. Author contributions (CRediT statement)—CS: writing original draft, methodology, data curation, formal analysis, and investigation; CM: writing, validation, methodology, investigation, and funding acquisition; MB: investigation and data curation; GC: formal analysis and investigation; CCh: methodology; and DM: writing, methodology, investigation, and funding acquisition. Supplemental figures and table available at https://entrepot.recherche.data.gouv.fr/privateurl.xhtml?token=3c1b1516-0c8b-43e0-93cd-a55839516459 (private URL for reviewing). All authors contributed to reviewing and editing and approved the manuscript. Procedures were evaluated and approved by the French Ministry of Education and Research (APAFIS #6685-2016090512592554V2) and carried out in accordance with French and European guidelines and regulations for animal experimentation. The authors have not stated any conflicts of interest.

**Nonstandard abbreviations used:** 3-NOP = 3-nitrooxypropanol; EE = ether extract; GE = gross energy; GF = GreenFeed methane-measurement system; MIR = mid-infrared spectra; NEFA = nonesterified fatty acids; UFL = “unité fourragère lait”.

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ORCIDS

C. Saro, https://orcid.org/0000-0002-1887-4529

Cécile Martin, https://orcid.org/0000-0002-2265-2048

G. Cantalapiedra-Hijar, https://orcid.org/0000-0001-9486-8238

M. Bouchon, https://orcid.org/0000-0002-7175-7233

Diego P. Morgavi, https://orcid.org/0000-0002-3883-0937