# Replacing hexane with 2-methyloxolane for defatting soybean meal fed to dairy cows: Effects on ruminal and milk fatty acid profiles and health indicators

V. Menoury, P. Nozière, C. Delavaud, Y. Farizon, A. Ferlay

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V. Menoury, P. Nozière, C. Delavaud, Y. Farizon, A. Ferlay. Replacing hexane with 2-methyloxolane for defatting soybean meal fed to dairy cows: Effects on ruminal and milk fatty acid profiles and health indicators. Journal of Dairy Science, 2026, 109 (3), pp.2344-2367. 10.3168/jds.2025-27215. hal-05520340

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# Replacing hexane with 2-methyloxolane for defatting soybean meal fed to dairy cows: Effects on ruminal and milk fatty acid profiles and health indicators

V. Menoury,<fnref n="1" /> Ⓓ P. Nozière,<fnref n="1" /> Ⓓ C. Delavaud,<fnref n="1" /> Ⓓ Y. Farizon,<fnref n="2" /> Ⓓ and A. Ferlay<fnref n="1" />*

## ABSTRACT

Replacing hexane with 2-methyloxolane (MeOx) for defatting soybean meal (SBM) requires adaptations of the SBM production process. These modifications may increase the concentration of Maillard reaction products and reduce the residual oil content in MeOx-defatted SBM compared with hexane-defatted SBM. In addition, despite desolventization, solvent residues may still be present in the SBM when fed to livestock. This study aims to ensure that the replacement of hexane with MeOx for defatting SBM does not affect the ruminal and milk fatty acid profiles nor the liver activity, liver functionality, and inflammatory status in dairy cows. A 4 × 4 Latin square design experiment was conducted with 16 primiparous dairy cows that received 4 dietary treatments: 100% hexane-defatted SBM (control diet, HEX), 67% hexane-defatted SBM plus 33% MeOx-defatted SBM (33MeOx), 33% hexane-defatted SBM plus 67% MeOx-defatted SBM (67MeOx), and 100% MeOx-defatted SBM (100MeOx). Diets contained 16% SBM on a DM basis and were iso-CP and iso-net energy. We collected feed, ruminal content, blood, and milk samples. We measured traits related to lipid digestion in the rumen and secretion in milk (feed, ruminal, and milk fatty acid profiles), energy metabolism (plasma acetate, BHB, nonesterified fatty acids, and glucose concentrations, as well as C isotopic discrimination between plasma and diet), liver integrity and functionality (plasma enzyme activities and serum albumin and plasma total bilirubin concentrations), and inflammatory status (blood cell counts and plasma cytokine concentrations). We used difference and equivalence tests for statistical analyses. Replacing HEX with 100MeOx resulted in likely equivalent milk fat content, fat yield, and major fatty acid profile. Stearyl-CoA desaturase activity in the mammary gland, indicated by

the C14:1 *cis*-9-to-C14:0 ratio, was negatively linearly related to the proportion of MeOx-defatted SBM in the diet. We did not find evidence of strict equivalence between 100MeOx and HEX in ruminal fatty acid profile. However, only minor differences were observed. Plasma γ-glutamyltransferase, aspartate aminotransferase, and alanine aminotransferase activities, as well as total bilirubin concentration, were unlikely equivalent toward greater values with 100MeOx compared with HEX, suggesting slight changes in liver integrity and functionality with 100MeOx. The overall inflammatory status of dairy cows was unlikely equivalent between 100MeOx and HEX. However, significant differences were limited to blood basophil count and plasma chemokine C-C motif ligand 4 concentration that were negatively linearly related to the proportion of MeOx-defatted SBM in the diet, and plasma chemokine C-X-C motif ligand 8 concentration that was quadratically related to the proportion of MeOx-defatted SBM in the diet. Altogether, these results indicate that the replacement of hexane-defatted SBM with MeOx-defatted SBM in the diet of dairy cows resulted in likely equivalent lipid secretion in milk despite a slight reduction in stearyl-CoA desaturase activity in the mammary gland with MeOx-defatted SBM. In the context of this study (i.e., short-term exposure, high level of MeOx, low level of hexane), neither hexane-defatted SBM nor MeOx-defatted SBM impaired health, but slight changes in some indicators of liver integrity, liver functionality, and inflammatory profile may warrant further investigation (e.g., long-term exposure).

**Key words:** solvent extraction, hepatic function, cytokine, equivalence test

## INTRODUCTION

Soybean oil extraction, using mechanical pressing or solvent extraction (Newkirk, 2010), yields oil and soybean meal (SBM). In France, during the 2021 to 2022 production campaign, 3.4 Mt of SBM were used as feed, including 1.0 Mt for dairy cattle (Terres Univia [Paris,

<footnote n="1">¹Université Clermont Auvergne, INRAE, VetAgro Sup, UMR Herbivores, Route de Theix, 63122 Saint-Genès-Champanelle, France</footnote>

<footnote n="2">²GenPhySE, Université de Toulouse, INRAE, ENVT, 31326 Castanet Tolosan, France</footnote>

<footnote>Received July 8, 2025.
Accepted November 3, 2025.
*Corresponding author: anne.ferlay@inrae.fr</footnote>
France], personal communication, based on Oriflaam, Campagne 2021-2022).

Hexane (i.e, technical hexane), containing mainly n-hexane and its isomers (INRS, 2025), is the most widely used solvent for defatting oilseed meals at a large industrial scale (Fine et al., 2013; Demarco and Gibon, 2020). Its low cost and physical properties make hexane a good solvent for oil extraction (Rapinel et al., 2020). Nonetheless, hexane is sourced from nonrenewable petroleum resources and n-hexane is toxic to health and the environment (ANSES, 2014; ECHA, 2025a; INRS, 2025). Recently, the need to re-evaluate the safety of hexane use in the food sector has been highlighted by the European Food Safety Authority (EFSA et al., 2024).

Among alternative solvents, 2-methyloxolane (MeOx) may be a good candidate for replacing hexane because it is biobased, it offers good extraction performance, and it has a safer toxicological profile than n-hexane (Rapinel et al., 2020; EFSA Panel on Food Contact Materials, Enzymes and Processing Aids et al., 2022; ECHA, 2025b). Furthermore, MeOx has already been authorized for use as a food processing aid in the European Union, Australia, and New Zealand (European Union, 2023; Australia New Zealand Food Standard Code, 2025).

The use of MeOx requires some adjustments of the soybean crushing process (Sicaire et al., 2015; Rapinel et al., 2020), which can result in a slightly more defatted SBM (Claux et al., 2021; Bartier et al., 2024), may promote Maillard reactions between AA and reducing carbohydrates (Helou et al., 2014), and is likely to change the type of solvent residues present in the SBM fed to livestock (Bauer et al., 2009; Demarco and Gibon, 2020).

Our previous study based on both equivalence and difference statistical tests demonstrated that hexane-defatted SBM and MeOx-defatted SBM had likely equivalent palatability and afforded likely equivalent dairy performance, N partitioning, and N use efficiency (Menoury et al., 2025). However, N digestion and metabolism may have been slightly altered as indicated by unlikely equivalent ruminal fluid branched-chain VFA proportion toward lower proportion with MeOx-defatted SBM, and unlikely equivalent plasma Lys concentration and N isotopic discrimination between plasma and diet toward greater values with MeOx-defatted SBM (Menoury et al., 2025). Residues of both solvents in milk were detected below the quantification threshold of 10 ng/g (Menoury et al., 2025). Replacing hexane-defatted SBM with MeOx-defatted SBM tended to reduce the frequency of detection of n-hexane residues in milk, and we did not find evidence of more frequent MeOx residues in milk when cows were fed MeOx-defatted SBM compared with hexane-defatted SBM (Menoury et al., 2025).

As a continuation of this first study, we suggest that a slightly lower ether extract content of MeOx-defatted

SBM compared with hexane-defatted SBM (Claux et al., 2021; Bartier et al., 2024) may reduce the intake of total fatty acids (FA) and major FA in SBM (C18:2c9,c12 and C18:1c9 [c = cis]; INRA et al., 2024). Feeding excluded SBM (8.64% to 10.9% of ether extract) instead of solvent-extracted SBM (1.32% to 1.82% of ether extract) has been shown to result in a lower proportion of SFA and greater proportions of MUFA, PUFA, and FA ≥18 carbon atoms in milk (Giallongo et al., 2015; Harper et al., 2019). In addition, non-enzymatic browning, including Maillard reactions, has been suggested as a potential inhibitor of ruminal biohydrogenation, thus increasing the flow of UFA to the duodenum (Toral et al., 2024). If these UFA are digestible in the intestine and used by the mammary gland for milk fat synthesis, the proportion of UFA in milk may increase (Ferlay et al., 2017; Bernard et al., 2018). However, to our knowledge, no study has investigated the effects of Maillard reactions on FA digestibility. In addition, the efficacy of non-enzymatic browning to inhibit ruminal biohydrogenation may be limited (Toral et al., 2024). Overall, the effects of slight differences in ether extract and Maillard reaction products contents between hexane-defatted SBM and MeOx-defatted SBM on ruminal and milk FA profiles are yet to be determined.

Solvent residues may be present in SBM offered to livestock (Bauer et al., 2009; Demarco and Gibon, 2020). On the one hand, n-hexane is absorbed, metabolized in the liver into several metabolites, and excreted unchanged in exhaled air and as metabolites in urine (EPA, 2005; ANSES, 2014; ATSDR, 2025). In rodents, n-hexane had effects on liver-to-BW ratio (Cavender et al., 1984), on liver cell damage and indicators of liver integrity and functionality (Goel et al., 1982), and on differential blood cell counts (Bouakkaz et al., 2018), but this was not always shown (API, 1978; Cavender et al., 1984; NTP, 1991). On the other hand, MeOx is absorbed, metabolized, and excreted unchanged or as CO₂ in exhaled air and as metabolites in urine (Henderson et al., 2007). High levels of MeOx administration to rats increased liver weight, centrilobular hepatocyte hypertrophy, and serum cholesterol concentration (Parris et al., 2017), but there were no evidence of adverse effects on indicators of liver integrity, liver functionality, and differential blood cell count (Parris et al., 2017; EFSA Panel on Food Contact Materials, Enzymes and Processing Aids et al., 2022). Overall, the effects of n-hexane on liver health and inflammation remain unclear, and MeOx was not shown to impair liver health or to affect differential blood cell counts. However, only a limited number of studies investigated these effects.

The aim of this study was to test the hypotheses that (1) ruminal and milk FA profiles would be equivalent between cows fed hexane-defatted SBM or MeOx-defatted
SBM, and (2) liver integrity, liver functionality, and inflammatory status would be equivalent between cows fed hexane-defatted SBM or MeOx-defatted SBM.

# MATERIALS AND METHODS

## Experimental Design and Diets

This experiment was conducted from November 2021 to March 2022 at the French National Research Institute for Agriculture, Food and Environment (INRAE) Herbipôle experimental facility (Theix, France). It received ethical authorization from the French Ministry of Higher Education and Research and the Ethical Committee for Animal Experimentation in Auvergne (APAFIS no. 30891-2017062616304407 v14). The experimental design was described in Menoury et al. (2025). Briefly, 16 mid-lactation primiparous Holstein dairy cows, including 4 rumen-cannulated cows (i.e., 1 per group), were allocated to 4 groups of 4 animals in an experiment designed as a Latin square with 4 groups, 4 periods, and 4 dietary treatments. The 4 dietary treatments contained different proportions of hexane-defatted SBM and MeOx-defatted SBM: (1) 100% hexane-defatted SBM (the control diet, HEX), (2) 67% hexane-defatted SBM plus 33% MeOx-defatted SBM (33MeOx), (3) 33% hexane-defatted SBM plus 67% MeOx-defatted SBM (67MeOx), and (4) 100% MeOx-defatted SBM (100MeOx). Two additional non-cannulated cows received 50% hexane-defatted SBM plus 50% MeOx-defatted SBM and could be used as substitutes. One of these additional noncannulated cows was used from period 2 onward to replace a rumen-cannulated cow medicated 3 times due to drastic appetite loss following oral stomach tubing performed in period 1. Each period lasted 21 d: 14 d of adaptation and 7 d of measurements, with diet transition of 3 d between periods.

## Soybean Meal

As described in Menoury et al. (2025), the 2 SBM were produced in the same extraction plant (New Holland Extraction Ltd., Barrow-upon-Humber, UK) using the same batch of dehulled soybean flakes. A first production run in the summer of 2021 enabled the production of hexane-defatted SBM containing 11.2 mg/kg DM of n-hexane. The hexane-defatted SBM also contained 4.4 mg/kg DM of MeOx residues. This slight contamination may have happened during storage or sampling of SBM (Menoury et al., 2025). The plant was then adapted to run on MeOx (95.5% MeOx and 4.5% water), and MeOx-defatted SBM was produced in October 2021. We selected the batch of MeOx-defatted SBM with the highest MeOx residue concentration (i.e., 977.4 mg/kg DM) to test a worst-case scenario for MeOx residues in SBM.

The proximate composition and Maillard reaction products concentrations in the 2 SBM were already presented in Menoury et al. (2025). Briefly, the hexane-defatted SBM contained 555 ± 3.4 g of CP/kg DM, 83 ± 5.5 g of NDF/kg DM, 26 ± 1.9 g of ether extract/kg DM (mean ± SD, n = 5 with one pooled sample per period during the experimental phase and one pooled sample from the pre-experimental phase). The hexane-defatted SBM also contained 95.5 mg of furosine/kg DM and 47.6 mg of Nε-carboxymethyl-lysine/kg DM (n = 1, one SBM sample collected at the extraction plant). The MeOx-defatted SBM contained 552 ± 13.6 g of CP/kg DM, 115 ± 35.0 g of NDF/kg DM, 22 ± 2.1 g of ether extract/kg DM (mean ± SD, n = 5 with one pooled sample per period during the experimental phase and one pooled sample from the pre-experimental phase). The MeOx-defatted SBM also contained 1,509.5 mg of furosine/kg DM and 57.7 mg of Nε-carboxymethyl-lysine/kg DM (n = 1, one SBM sample collected at the extraction plant). The FA content and profile of the 2 SBM are presented in Table 1.

## Diets

The ingested diets contained (on a DM basis): 44% ± 0.6% corn silage, 15% ± 0.2% grass silage, 6.9% ± 0.10% hay, 3.0% ± 0.04% straw, 10% ± 0.1% ground corn grain, 1.0% ± 0.01% vitamins and minerals, 16% ± 0.9% SBM, and 4.2% ± 0.49% production concentrate (mean ± SD, n = 61; Menoury et al., 2025). The proximate composition, Maillard reaction products, and nutritive value of the diets were already presented in Menoury et al. (2025). Briefly, ingested diets contained 27 ± 1.8 g of ether extract/kg DM and were formulated to be iso-CP (163 ± 7.4 g of CP/kg DM, mean ± SD, n = 61), iso-protein truly digestible in the intestine (PDI; INRA, 2018; 92.8 ± 1.90 g of PDI/kg DM, mean ± SD, n = 61) and iso-net energy (0.91 ± 0.015 unité fourragère lait [UFL]/kg DM, mean ± SD, n = 61; 1 UFL = 7.37 MJ of NEL, INRA, 2018). The FA content and profile of the ingested diets, as well as calculated exposure of dairy cows to solvents are presented in Table 2. Exposure of dairy cows to n-hexane originating from experimental SBM ranged from 0.000 mg/kg BW per day for 100MeOx to 0.057 mg/kg BW per day for HEX. Exposure to MeOx originating from experimental SBM ranged from 0.023 mg/kg BW per day for HEX to 4.958 mg/kg BW per day for 100MeOx.

## Sampling and Measurements

**Feed Intake and Biochemical Composition, and Animal Characteristics.** The measurement of feed intake, the sampling of feed ingredients for biochemical analyses, and the measurement of BW were described in Menoury et al. (2025).
<table><caption>**Table 1.** Fatty acid content and profile of hexane-defatted and 2-methyloxolane-defatted soybean meals<fnref n="1" /></caption><thead><tr><th>Item</th><th>Hexane-defatted<br>soybean meal</th><th>MeOx<sup>2</sup>-defatted<br>soybean meal</th></tr></thead><tbody><tr><td>Total fatty acids (g/kg DM)</td><td>16.9 (1.59)</td><td>14.2 (1.43)</td></tr><tr><td>C16:0</td><td>2.94 (0.253)</td><td>2.41 (0.238)</td></tr><tr><td>C18:0</td><td>0.80 (0.069)</td><td>0.73 (0.076)</td></tr><tr><td>C18:1c9</td><td>2.93 (0.296)</td><td>2.55 (0.271)</td></tr><tr><td>C18:2c9,c12</td><td>8.61 (0.811)</td><td>7.10 (0.733)</td></tr><tr><td>C18:3c9,c12,c15</td><td>0.99 (0.108)</td><td>0.81 (0.085)</td></tr><tr><td>Other fatty acids</td><td>0.60 (0.055)</td><td>0.56 (0.042)</td></tr><tr><td>Fatty acid profile (g/100 g total fatty acids)</td><td></td><td></td></tr><tr><td>C16:0</td><td>17.4 (0.23)</td><td>17.1 (0.07)</td></tr><tr><td>C18:0</td><td>4.74 (0.058)</td><td>5.15 (0.020)</td></tr><tr><td>C18:1c9</td><td>17.4 (0.25)</td><td>18.0 (0.13)</td></tr><tr><td>C18:2c9,c12</td><td>51.0 (0.11)</td><td>50.2 (0.14)</td></tr><tr><td>C18:3c9,c12,c15</td><td>5.88 (0.118)</td><td>5.69 (0.069)</td></tr><tr><td>Other fatty acids</td><td>3.59 (0.037)</td><td>3.96 (0.288)</td></tr></tbody></table>

**Ruminal Content.** Ruminal content was sampled via the cannula of the rumen-cannulated cows at 0 and 5 h after the morning meal (i.e., at 17 and 5 h after the last meal, respectively) on d 20. Five samples of approximately 100 g were collected in 5 different sites in the rumen, to ensure representativeness, without draining the fluid. The 500 g of ruminal content were mixed by hand and an aliquot of approximately 125 g was stored at -20°C until freeze-drying and analysis of FA.

**Plasma.** Blood was collected on d 20 before the morning meal at the coccygeal vessels into 1 tube with silica clot activator, 2 lithium-heparin-coated tubes, and 4 K₃EDTA-coated tubes (9-mL tubes, ref. 455092, 455084,

and 455036, Greiner). After collection, the lithium-heparin and K₃EDTA-coated tubes were gently inverted several times and stored on ice. One K₃EDTA-coated tube was used for blood cell counts, and 2 lithium-heparin and 3 K₃EDTA-coated tubes were centrifuged (3,500 × g, 15 min, 4°C). Waiting time between sampling and centrifugation was ~30 min. Tubes without anticoagulant were gently inverted several times and maintained at room temperature for 2 h before centrifugation (3,500 × g, 15 min, 4°C). Serum and plasma were stored at -20°C (total protein, albumin, glucose, acetate, BHB, nonesterified FA (NEFA), and total bilirubin) or -80°C (alanine aminotransferase (ALT), aspartate aminotransferase

<table><caption>**Table 2.** Fatty acid content and profile of diets ingested by dairy cows, as well as solvent exposure of dairy cows<fnref n="1" /></caption><thead><tr><th rowspan="2">Item</th><th colspan="4">Diet<sup>2</sup></th></tr><tr><th>HEX</th><th>33MeOx</th><th>67MeOx</th><th>100MeOx</th></tr></thead><tbody><tr><td>Total fatty acids (g/kg DM)</td><td>17.0 (1.77)</td><td>16.7 (1.64)</td><td>16.8 (1.77)</td><td>16.4 (1.58)</td></tr><tr><td>C16:0</td><td>3.14 (0.327)</td><td>3.07 (0.301)</td><td>3.09 (0.329)</td><td>3.02 (0.293)</td></tr><tr><td>C18:0</td><td>0.49 (0.057)</td><td>0.48 (0.052)</td><td>0.48 (0.052)</td><td>0.48 (0.045)</td></tr><tr><td>C18:1c9</td><td>2.91 (0.639)</td><td>2.85 (0.621)</td><td>2.87 (0.630)</td><td>2.80 (0.590)</td></tr><tr><td>C18:2c9,c12</td><td>6.85 (1.079)</td><td>6.67 (1.022)</td><td>6.69 (1.052)</td><td>6.51 (0.952)</td></tr><tr><td>C18:3c9,c12,c15</td><td>2.48 (0.759)</td><td>2.47 (0.788)</td><td>2.51 (0.774)</td><td>2.45 (0.797)</td></tr><tr><td>Other fatty acids</td><td>1.11 (0.089)</td><td>1.11 (0.091)</td><td>1.11 (0.096)</td><td>1.10 (0.096)</td></tr><tr><td>Fatty acid profile (g/100 g total fatty acids)</td><td></td><td></td><td></td><td></td></tr><tr><td>C16:0</td><td>18.5 (0.21)</td><td>18.5 (0.19)</td><td>18.4 (0.18)</td><td>18.4 (0.15)</td></tr><tr><td>C18:0</td><td>2.90 (0.161)</td><td>2.90 (0.150)</td><td>2.89 (0.136)</td><td>2.91 (0.131)</td></tr><tr><td>C18:1c9</td><td>17.0 (2.59)</td><td>17.0 (2.67)</td><td>17.0 (2.66)</td><td>17.0 (2.68)</td></tr><tr><td>C18:2c9,c12</td><td>40.2 (3.23)</td><td>39.9 (3.26)</td><td>39.8 (3.19)</td><td>39.7 (3.13)</td></tr><tr><td>C18:3c9,c12,c15</td><td>14.9 (5.56)</td><td>15.1 (5.69)</td><td>15.2 (5.58)</td><td>15.2 (5.61)</td></tr><tr><td>Other fatty acids</td><td>6.59 (0.585)</td><td>6.68 (0.608)</td><td>6.63 (0.575)</td><td>6.76 (0.606)</td></tr><tr><td>Solvent exposure<sup>3</sup> (mg/kg BW per day)</td><td></td><td></td><td></td><td></td></tr><tr><td>n-Hexane</td><td>0.057 (0.0199)</td><td>0.038 (0.0127)</td><td>0.019 (0.0067)</td><td>0.000 (0.0000)</td></tr><tr><td>2-Methyloxolane</td><td>0.023 (0.0059)</td><td>1.614 (0.3449)</td><td>3.154 (0.6192)</td><td>4.958 (0.7331)</td></tr></tbody></table>

<footnote n="1">¹Mean (SD), with 1 pooled sample per period during the experimental phase (n = 4).</footnote>

<footnote n="2">²2-Methyloxolane.</footnote>

<footnote n="1">¹Mean (SD), with n = 16 for HEX and n = 15 for other dietary treatments.</footnote>

<footnote n="2">²HEX = 100% hexane-defatted soybean meal; 33MeOx = 67% hexane-defatted plus 33% 2-methyloxolane-defatted soybean meal; 67MeOx = 33% hexane-defatted plus 67% 2-methyloxolane-defatted soybean meal; 100MeOx = 100% 2-methyloxolane-defatted soybean meal.</footnote>

<footnote n="3">³Solvent exposure from hexane-defatted soybean meal and 2-methyloxolane-defatted soybean meal.</footnote>
(AST), γ-glutamyltransferase (γGT), alkaline phosphatase (ALP), and cytokines) until analyses. In period 4, the blood samples intended for blood cell counts were lost during shipment. Therefore, cows were maintained on their experimental diets for an additional week and new blood samples were collected on d 27 for blood cell counts.

**Milk Yield, Fat Content, and FA Profile.** Milk yield was recorded at each milking using a milk meter. On d 18 and 20 at the morning and afternoon milkings, milk samples were collected and stored in 2-bromo-2-nitropropane-1,3-diol at 4°C for determination of milk fat. On d 20 at the morning and afternoon milkings, milk samples were collected without preservatives and stored at -20°C until freeze-drying and analysis of FA.

## Laboratory Analyses

**Feed and Refusals.** The proximate composition of offered feed and refusals was analyzed as described in Menoury et al. (2025). The δ¹³C enrichment was measured in feed ground to 0.5 mm (Cyclotec CT193, Foss) using an elemental analyzer (EA Vario Isotope Cube, Elementar) coupled with an isotope ratio mass spectrometry system (isoprime visION, Elementar). Internal standards (i.e., glutamic acid) were included in every 5 samples to correct for possible variations in the raw values determined by the mass spectrometer. Typical replicate measurement errors for these reference materials were ± 0.1‰. Results are expressed according to the following equation:

$$ \delta^{13}\text{C} = \left[ \left( \frac{R_{\text{sample}}}{R_{\text{standard}}} \right) - 1 \right] \times 1,000, $$

where Rsample and Rstandard are the C isotope ratio between the heavier isotope and the lighter isotope (¹³C:¹²C) for the sample being analyzed and the internationally defined standard (i.e., Vienna Peecee Belemnite), respectively (Chen et al., 2020). Freeze-dried (MUT PCCPLS1.5 001, Cryotec) maize silage, grass silage, hay, straw, and offered partial mixed ration (PMR) samples, along with raw hexane-defatted SBM, MeOx-defatted SBM, and production concentrate samples were ground using a hammer mill equipped with a 1-mm screen (BJL8500-2, Boisson). The FA in offered feed samples were extracted and methylated in situ using the one-step extraction-transesterification procedure described by Sukhija and Palmquist (1988), modified by Loor et al. (2004), and using hexane as solvent and 0.5 mL of C23:0 in a chloroform solution (0.4 mg/L) as internal standard. Analysis of FA methyl esters was performed with an Agilent 7890A gas chromatograph equipped with a 7693A autosampler and

a flame ionization detector (Agilent Technologies), using a 100 m × 0.25 mm fused-silica capillary column (CP-Sil 88, 0.20 µm film thickness, Agilent Technologies). We used the program described by Loor et al. (2004) and modified using 0.6 µL of sample, a 50:1 split ratio, an injector temperature of 255°C, a detector temperature of 260°C, and a constant injector pressure of 150 kPa. Peaks were identified by comparison with commercial standards (NCP463, Nu-Chek Prep Inc.; Supelco37, Supelco Inc.) and according to their order of elution. Chromatograms were integrated using the software Azur (version 6.1.0.0, Antelia).

**Ruminal Content.** Ruminal content samples were freeze-dried (MUT PCCPLS1.5 001, Cryotec) and ground using an ultra-centrifugal abrasion mill equipped with a 0.5-mm screen (Cyclotec CT193, Foss). The FA in 100 mg of freeze-dried ruminal content and 200 µL of C19:0 in a methylene chloride solution (72332, Sigma-Aldrich; 4 g/L; used as internal standard) were extracted and methylated in situ using the one-step extraction-transesterification procedure described by Sukhija and Palmquist (1988), modified by Jenkins (2010), and using hexane as solvent. Analysis of FA methyl esters was performed using the 2 programs described by Troegeler-Meynadier et al. (2014) with an Agilent 7890N gas chromatograph equipped with a model 7683B autosampler and a flame ionization detector (Agilent Technologies), using a 100 m × 0.25 mm fused-silica capillary column (CP-Sil 88, 0.20 µm film thickness, Agilent Technologies). Peaks were identified by comparison with commercial standards (20-0400-9, 20-0600-9, 20-0700-9, 20-0800-9, 20-0900-9, 20-1000-9, 20-1100-9, 20-1200-9, 20-1300-9, 20-1400-9, 20-1500-9, 20-1600-9, 20-1700-9, 20-1800-9, 21-1211-7, 21-1312-7, 21-1413-7, 21-1615-7, 21-1716-7, 21-1210-7, 21-1412-7, 21-1614-7, 20-1401-9, 20-1601-9, 20-1701-9, 20-1810-13, 20-1813-9, 20-1801-13, 20-2001-9, 20-1802-13, 20-1823-7, 20-1826-7, 20-1825-7, 20-1824-7, 20-1803-9, 20-1900-13, Larodan) and according to their order of elution. Chromatograms were integrated using the software Peak Simple (Peak Simple Data System, version 4.89, SRI).

**Blood, Serum, and Plasma.** Blood was analyzed with the Sysmex XT-2000Vet (Sysmex) automated hematology analyzer. Erythrocytes and platelets were counted in a fixed known volume of blood using impedance measurement. Hematocrit was simultaneously calculated using the erythrocyte pulse height detection method. Blood hemoglobin concentration was determined using cyanide-free sodium lauryl sulfate and spectrophotometry (λ = 555 nm). Erythrocyte mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were calculated from blood hemoglobin concentration, hematocrit, and erythrocyte
count. The erythrocyte distribution width coefficient of variation was calculated from the erythrocyte size distribution curve. Fluorescence flow cytometry using a semiconductor laser beam (λ = 633 nm) was used to count reticulocytes and leukocytes (i.e., lymphocytes, monocytes, neutrophils, basophils, and eosinophils). The δ¹³C enrichment in 10 µL of lithium-heparin plasma was measured using the method described above for feed. A Konelab Arena 20 XT analyzer (Thermo Fisher Scientific) was used for K₃EDTA plasma analysis of glucose (λ = 510 nm, kit 981379 based on the oxidation of glucose to gluconic acid and reduction of O₂ to H₂O₂ in the presence of glucose oxidase, followed by H₂O₂ reaction with phenol and 4-aminoantipyrine in the presence of peroxidase to form quinoneimine and water; Trinder, 1969; Thermo Fisher Scientific), acetate (λ = 340 nm, kit 984356 based on the formation of NADH + H⁺ when acetate is in the presence of coenzyme A, ATP, NAD⁺, acetyl-CoA synthetase, citrate synthetase, and malate dehydrogenase, Thermo Fisher Scientific), NEFA [λ = 546 nm, kit NEFA-HR(2) based on the reaction of NEFA with coenzyme A in the presence of ATP and acyl-CoA synthetase to form acyl-CoA, followed by acyl-CoA oxidation to *t*-2,3-enoyl-CoA (*t* = *trans*) and O₂ reduction to H₂O₂ in the presence of acyl-CoA oxidase, followed by H₂O₂ reaction with 4-amino-antipyrine and 3-methyl-N-ethyl-N-(β-hydroxyethyl)-aniline in the presence of peroxidase to form a blue purple pigment, Fujifilm Wako Chemicals Europe GmbH], BHB (λ = 540 nm, kit 984325 based on the oxidation of BHB to acetoacetate and reduction of NAD⁺ to NADH + H⁺ in the presence of D-3-hydroxybutyric dehydrogenase, followed by the oxidation of NADH to NAD⁺ and reduction of oxidized nitro blue tetrazolium in the presence of diaphorase, Thermo Fisher Scientific), and total bilirubin (λ = 510 nm, kit 981897 based on modified Mallow-Evelyn method consisting in indirect bilirubin liberation by tenside and HCl followed by total bilirubin coupling with p-nitrobenzenediazonium salts forming azobilirubin, Thermo Fisher Scientific). The same analyzer was used with lithium-heparin plasma to determine ALT (λ = 340 nm, kit 981769 based on the reactions described by Schumann et al. 2002a; Thermo Fisher Scientific), AST (λ = 340 nm, kit 981771 based on the reactions described by Schumann et al., 2002b; Thermo Fisher Scientific), ALP (λ = 405 nm, kit 981832 based on reaction no. 1 described by Schumann et al., 2011; Thermo Fisher Scientific), and γGT (λ = 405 nm, kit 981377 based on the reactions described by Schumann et al., 2002c; Thermo Fisher Scientific). A Daytona+ analyzer (Randox) was used with serum samples to determine total protein (kit TP 8336 based on the Biuret method, Randox) and albumin (kit AB 8301 based on the bromocresol green method as described by Doumas et al., 1971; Randox). The cytokines IL-1α (CV<sub>intra-assay</sub>

= 4.2%), IL-1β (CV<sub>intra-assay</sub> = 19.0%), IL-6 (CV<sub>intra-assay</sub> = 30.2%), IL-10 (CV<sub>intra-assay</sub> = 3.0%), tumoral necrosis factor α (TNFα, CV<sub>intra-assay</sub> = 4.2%), vascular endothelial growth factor A (VEGF-A, CV<sub>intra-assay</sub> = 1.9%), chemokine C-C motif ligand 2 (CCL2 or monocyte chemoattractant protein 1 or MCP-1, CV<sub>intra-assay</sub> = 2.6%), chemokine C-C motif ligand 3 (CCL3 or macrophage inflammatory protein 1α or MIP-1α, CV<sub>intra-assay</sub> = 2.6%), chemokine C-C motif ligand 4 (CCL4 or macrophage inflammatory protein 1β or MIP-1β, CV<sub>intra-assay</sub> = 4.5%), and chemokine C-X-C motif ligand 8 (CXCL8 or IL-8, CV<sub>intra-assay</sub> = 0.8%) were measured in K₃EDTA plasma using MILLIPLEX Bovine Cytokine/Chemokine Magnetic Bead Panel 1 (BCYT1-33K-10, Merck) according to the manufacturer's instructions. The assay was carried out on a 96-well plate containing background, 7 standards, 2 quality controls, 3 high and low home-made controls, and plasma samples. The plate was analyzed on a Luminex 200TM (Bio-Rad Laboratories, Hercules) equipped with the Bio-Plex Manager software version 6.0 (Bio-Rad Laboratories, Hercules).

**Milk.** Milk fat was determined using infrared spectrometry (Milkoscan, FOSS) at Agrolabs (Aurillac). Before FA analysis, morning and afternoon milk samples were freeze-dried (MUT PCCPLS1.5 001, Cryotec) and pooled per cow × period according to milk fat yield. The FA in 100 mg of freeze-dried milk were extracted and methylated in situ using the one-step extraction-trans-esterification procedure described by Lerch et al. (2012) and modified using heptane instead of hexane as solvent. Analysis of FA methyl esters was performed with the same material used for feed samples with the program described by Lerch et al. (2012) and modified using an injector temperature of 255°C, a detector temperature of 260°C, and a constant injector pressure of 150 kPa. Peaks were identified by comparison with commercial standards (NCP463, Nu-Chek Prep Inc.; Supelco37, Supelco Inc.; O5632, Sigma-Aldrich) and according to their order of elution. Chromatograms were integrated using the software Azur (version 6.1.0.0, Antelia). Correction factors for short-chain FA (C4:0 to C10:0) were estimated using a reference milk of known composition.

## Calculations

The PMR, hexane-defatted SBM, and MeOx-defatted SBM intakes were calculated as described in Menoury et al. (2025). Intake of individual FA was calculated from the DM intake and individual FA content of PMR, hexane-defatted SBM, MeOx-defatted SBM, and production concentrate. Exposures of dairy cows to n-hexane and MeOx from experimental SBM (i.e., hexane-defatted SBM and MeOx-defatted SBM), expressed in mg/kg BW per day, were calculated as the product of SBM DMI and
solvent concentration in SBM, divided by the BW. Calculated ruminal biohydrogenation indices were the C18:1t10-to-C18:1t11 ratio, the extent of C18:1c9, C18:2c9, c12, and C18:2c9, c12, c15 biohydrogenation, and biohydrogenation completeness (according to Oliveira et al., 2016, and Alves et al., 2017). Serum globulin concentration was calculated from the difference between serum total protein and albumin concentrations (Bertoni et al., 2008). De novo FA were calculated as the sum of FA with less than 16 C atoms (including odd- and branched-chain FA), mixed FA was calculated as the sum of FA with exactly 16 C atoms, and preformed FA was calculated as the sum of FA with more than 16 C atoms. The mammary gland stearyl-CoA desaturase (SCD) activity index was calculated as the ratio of C14:1c9-to-C14:0 in milk, as this ratio is considered the best indicator of the mammary gland SCD activity (Bernard et al., 2008). The C isotopic discrimination between plasma and diet, Δ¹³C (‰), was calculated as the difference between plasma and diet ¹³C abundance (as for C isotopic discrimination between plasma and milk in Chen et al., 2020). The Δ¹³C is used as a proxy of adipose tissue mobilization, as demonstrated for early lactation dairy cows (Chen et al., 2020).

## Statistical Analyses

Statistical analyses were performed using R software version 4.4.1 (R Core Team, 2024). Results are expressed as estimated marginal means (EMM; another term for LSM) ± SEM, unless stated otherwise. We used difference and equivalence tests in parallel, as these approaches are complementary. Equivalence tests were used to test hypotheses of equivalence between treatments through pairwise comparisons, whereas difference tests provided additional information on pairwise differences between treatments and on linear and quadratic response patterns (Menoury et al., 2025).

**Data Exclusion.** One rumen-cannulated cow mediated 3 times following the oral stomach tubing in period 1 was replaced by a noncannulated substitute cow from period 2 onward. Another rumen-cannulated cow experienced a technical problem with the door for accessing the feeding trough in period 1, and lameness in period 4. Data from these cows were removed from the dataset for all response variables except the ruminal kinetics, which were only measured on rumen-cannulated cows. Platelet count results were not reported because clumps have been reported in 66% of blood samples, leading to underestimated platelet counts. Due to high proportions of datapoints below the quantification threshold for IL-1β (74% of datapoints) and IL-6 (59% of datapoints), data from these 2 cytokines were not analyzed. Outliers were visually identified by graphing the data (ggplot function in the ggplot2 3.5.1 package) and descriptive statistics

(stats 4.4.1 package). These analyses were performed at the cow × period × day level. When outlier values were considered biologically impossible, by visualizing the kinetics of the response variables, the datapoints were excluded. Seven out of 144 cow × period × day datapoints were excluded for milk fat content, which is sensitive to sampling errors due to heterogeneity of milk fat content. The 7 datapoints excluded ranged from 44.2 to 56.3 g of fat/kg of milk.

**Mixed Models.** A linear mixed model (lmer function in the lme4 1.1–35.5 package) was fitted with REML:

$$Y_{dpc} = \mu + diet_d + period_p + cow_c + \varepsilon_{dpc},$$

where $Y_{dpc}$ is the response for the cth cow fed the dth diet during the pth period, $\mu$ is the overall mean response, $diet_d$ is the fixed effect of the dth diet, $period_p$ is the fixed effect of the pth period, $cow_c$ is the random effect of the cth cow, and $\varepsilon_{dpc}$ is the residual error term. For blood cell counts, a generalized linear mixed model (glmmTMB function in the glmmTMB 1.1.9 package) was used with a negative binomial error distribution, a $log_e$ link function, and a restricted maximum likelihood fit. For plasma concentrations of NEFA, $\gamma$GT, VEGF-A, CCL4, and CXCL8, residuals were not normally distributed when using a linear mixed model. Therefore, a generalized linear mixed model (glmmTMB function in the glmmTMB 1.1.9 package) was used with a Gaussian error distribution, a $log_e$ link function, and a REML fit. For ruminal kinetics analyses, a linear mixed model (lmer function in the nlme 3.1–164 package) was fitted using REML and continuous autoregressive covariance structure to account for repeated measures:

$$Y_{dpse} = \mu + diet_d + period_p + sampling\ time_s + diet \times sampling\ time_{ds} + cow_c + \varepsilon_{dpse},$$

where $Y_{dpse}$ is the response for the cth cow at the sth sampling time fed the dth diet during the pth period, $\mu$ is the overall mean response, $diet_d$ is the fixed effect of the dth diet, $period_p$ is the fixed effect of the pth period, $sampling\ time_s$ is the fixed effect of the sth sampling time, $diet \times sampling\ time_{ds}$ is the fixed interaction between the dth diet and the sth sampling time, $cow_c$ is the random effect of the cth cow, and $\varepsilon_{dpse}$ is the residual error term.

**Post Hoc Analyses for Difference Tests.** When the effect of the dietary treatment was significant ($P \le 0.05$) according to ANOVA (Anova function in the car 3.1–2 package or anova function in the stats 4.4.1 package), we investigated pairwise differences between experimental diets and HEX (emmeans function in the emmeans 1.10.4 package, pairs function in the graphics 4.4.1 package, and confint function in the stats 4.4.1 package). We adjusted
<table><caption>**Table 3.** Dry matter intake and fatty acid intake in dairy cows fed hexane-defatted or 2-methyloxolane-defatted soybean meal<fnref n="1" /></caption><thead><tr><th rowspan="2">Item</th><th colspan="4">Diet<sup>2</sup></th><th rowspan="2">SEM<sup>4</sup></th><th rowspan="2">P diet</th><th colspan="2">P contrast<sup>3</sup></th></tr><tr><th>HEX</th><th>33MeOx</th><th>67MeOx</th><th>100MeOx</th><th>L</th><th>Q</th></tr></thead><tbody><tr><td>DMI (kg DM/d)</td><td>20.7</td><td>20.4</td><td>20.3</td><td>20.5</td><td>0.57</td><td>0.76</td><td>0.49</td><td>0.44</td></tr><tr><td>Partial mixed ration DMI (kg DM/d)</td><td>16.5</td><td>16.3</td><td>16.3</td><td>16.4</td><td>0.48</td><td>0.91</td><td>0.70</td><td>0.55</td></tr><tr><td>Soybean meal DMI (kg DM/d)</td><td>3.33</td><td>3.19</td><td>3.18</td><td>3.19</td><td>0.091</td><td>0.063</td><td>0.034</td><td>0.12</td></tr><tr><td>Total fatty acid intake (g/d)</td><td>351</td><td>342</td><td>340</td><td>337</td><td>9.5</td><td>0.18</td><td>0.041</td><td>0.49</td></tr><tr><td>C16:0</td><td>64.8</td><td>63.1</td><td>62.7</td><td>62.2</td><td>1.75</td><td>0.17</td><td>0.038</td><td>0.50</td></tr><tr><td>C18:0</td><td>10.2</td><td>9.9</td><td>9.8</td><td>9.8</td><td>0.28</td><td>0.18</td><td>0.046</td><td>0.43</td></tr><tr><td>C18:1c9</td><td>60.0</td><td>58.5</td><td>58.5</td><td>57.7</td><td>1.66</td><td>0.35</td><td>0.10</td><td>0.70</td></tr><tr><td>C18:2c9,c12</td><td>141</td><td>137</td><td>136</td><td>134</td><td>3.9</td><td>0.077</td><td>0.013</td><td>0.57</td></tr><tr><td>C18:3c9,c12,c15</td><td>51.9</td><td>50.7</td><td>50.2</td><td>50.8</td><td>1.43</td><td>0.43</td><td>0.26</td><td>0.23</td></tr><tr><td>Other fatty acids</td><td>23.0</td><td>22.7</td><td>22.6</td><td>22.6</td><td>0.63</td><td>0.80</td><td>0.42</td><td>0.58</td></tr></tbody></table>

the α value to correct for the multiplicity of comparisons using the fallback multiple testing procedure (Tamhane and Gou, 2018) with the following a priori ordering of hypotheses: (H₁) 100MeOx versus HEX tested at an α₁ risk level, (H₂) 67MeOx versus HEX tested at an α₂ risk level, and (H₃) 33MeOx versus HEX tested at an α₃ risk level (as presented in Menoury et al., 2025). These hypotheses were chosen in this order on the grounds that, assuming a linear response, H₁ is expected to be easier to prove than H₃. Comparisons were considered significant at *P* ≤ αᵢ and as tendencies at αᵢ < *P* ≤ 2 × αᵢ. We also investigated linear and quadratic contrasts for the effect of dietary treatment (contrast function in the emmeans 1.10.4 package). Contrasts were considered significant at *P* ≤ 0.05 and as tendencies at 0.05 < *P* ≤ 0.10.

**Post Hoc Analyses for Equivalence Tests.** Post hoc analyses were performed to test the equivalence between the experimental diets (i.e., 100MeOx, 67MeOx, and 33MeOx) and the control diet (i.e., HEX). The equivalence interval was defined as ± 5% of the EMM of the control diet (i.e., HEX). We computed estimates for the difference between the EMM of the experimental diets and HEX, and their 100 – (2 × αᵢ)% CI (emmeans function in the emmeans 1.10.4 package, pairs function in the graphics 4.4.1 package, and confint function in the stats 4.4.1 package). Compared with the post hoc analyses for difference tests, here we used a similar fallback multiple testing procedure but with reversed a priori ordering of hypotheses: (H₁) 33MeOx versus HEX tested at an α₁ risk level, (H₂) 67MeOx versus HEX tested at an α₂ risk level, and (H₃) 100MeOx versus HEX tested at an α₃ risk level (as presented in Menoury et al., 2025). These hypotheses were chosen in this order on the grounds that, assuming a linear response, H₁ is expected to be easier to prove than

H₃. A simplified terminology based on the work of the EFSA Panel on Genetically Modified Organisms (2010) was used to draw conclusions about equivalence (see Figure 1 in Menoury et al., 2025).

## RESULTS

### Difference Tests

**Intake and Animal Characteristics.** We did not find evidence of differences between diets in total DMI and PMR DMI, but SBM DMI and intakes of total and several major FA (i.e., C16:0, C18:0, C18:2c9,c12) were negatively linearly related to the proportion of MeOx-defatted SBM in the diet (*P* ≤ 0.046, Table 3).

**Ruminal FA Profile.** We did not find evidence of significant diet × time interactions for individual FA proportions (*P* > 0.05, Supplemental Table S1, see Notes), sums of FA proportions, and ruminal biohydrogenation indices (*P* > 0.05, Table 4). For most individual FA proportions and sums of FA proportions in the rumen, we did not find evidence of differences between diets (*P* > 0.05). Exceptions are C9:0 and C18:1t12 proportions, which were quadratically related to the proportion of MeOx-defatted SBM (*P* ≤ 0.030), and C18:1c11 proportion which was 0.09 g/100 g total FA greater for 33MeOx compared with HEX (*P* = 0.007, α₃ = 0.017). We did not find evidence of differences between diets for ruminal biohydrogenation indices (*P* > 0.05).

**Plasma Indicators of Energy Metabolism.** We did not find evidence of differences between diets in plasma glucose, acetate, and BHB concentrations, and in Δ¹³C (*P* > 0.05, Table 5). Plasma NEFA concentration was positively linearly related to the proportion of MeOx-

<footnote n="1">¹Data for DMI are reproduced from Menoury et al. (2025; creative commons BY license).</footnote>

<footnote n="2">²HEX = 100% hexane-defatted soybean meal; 33MeOx = 67% hexane-defatted plus 33% 2-methyloxolane-defatted soybean meal; 67MeOx = 33% hexane-defatted plus 67% 2-methyloxolane-defatted soybean meal; 100MeOx = 100% 2-methyloxolane-defatted soybean meal.</footnote>

<footnote n="3">³L = linear contrast for diet effect; Q = quadratic contrast for diet effect.</footnote>

<footnote n="4">⁴Highest SEM.</footnote>
<table><caption>**Table 4.** Sums of fatty acid (FA) in the rumen and ruminal biohydrogenation indices in dairy cows fed hexane-defatted or 2-methyloxolane-defatted soybean meal (sampling through ruminal cannula)</caption><thead><tr><th rowspan="2">Item (g/100 g total FA, except if stated otherwise)</th><th colspan="4">Diet¹</th><th rowspan="2">SEM</th><th rowspan="2">P diet</th><th rowspan="2">P time</th><th rowspan="2">P diet × time</th><th colspan="2">P contrast²</th></tr><tr><th>HEX</th><th>33MeOx</th><th>67MeOx</th><th>100MeOx</th><th>L</th><th>Q</th></tr></thead><tbody><tr><td>Total FA (g/kgDM)</td><td>24.9</td><td>23.3</td><td>26.6</td><td>22.7</td><td>1.15</td><td>0.014</td><td>0.73</td><td>0.71</td><td>0.42</td><td>0.23</td></tr><tr><td>Σ SFA</td><td>68.2</td><td>66.9</td><td>68.8</td><td>65.4</td><td>1.43</td><td>0.12</td><td>&lt;0.001</td><td>0.40</td><td>0.21</td><td>0.35</td></tr><tr><td>Σ Even-chain FA</td><td>62.6</td><td>61.0</td><td>63.1</td><td>59.5<sup>A</sup></td><td>1.56</td><td>0.029</td><td>&lt;0.001</td><td>0.34</td><td>0.11</td><td>0.30</td></tr><tr><td>Σ Odd- and branched-chain FA</td><td>5.58</td><td>5.89</td><td>5.70</td><td>5.97</td><td>0.422</td><td>0.80</td><td>0.011</td><td>0.90</td><td>0.49</td><td>0.95</td></tr><tr><td>Σ Odd-chain FA</td><td>2.50</td><td>2.61</td><td>2.43</td><td>2.52</td><td>0.174</td><td>0.83</td><td>0.23</td><td>0.73</td><td>0.85</td><td>0.97</td></tr><tr><td>Σ Branched-chain FA</td><td>3.08</td><td>3.29</td><td>3.27</td><td>3.45</td><td>0.275</td><td>0.68</td><td>0.003</td><td>0.89</td><td>0.27</td><td>0.95</td></tr><tr><td>Σ Iso branched-chain FA</td><td>1.43</td><td>1.48</td><td>1.50</td><td>1.55</td><td>0.122</td><td>0.84</td><td>0.003</td><td>0.93</td><td>0.38</td><td>0.98</td></tr><tr><td>Σ Anteiso branched-chain FA</td><td>1.65</td><td>1.80</td><td>1.77</td><td>1.90</td><td>0.155</td><td>0.56</td><td>0.005</td><td>0.83</td><td>0.22</td><td>0.93</td></tr><tr><td>Σ MUFA</td><td>10.9</td><td>11.5</td><td>10.7</td><td>11.6</td><td>0.45</td><td>0.30</td><td>&lt;0.001</td><td>0.43</td><td>0.48</td><td>0.69</td></tr><tr><td>Σ Cis MUFA</td><td>4.67</td><td>5.40</td><td>4.47</td><td>5.57</td><td>0.437</td><td>0.091</td><td>&lt;0.001</td><td>0.34</td><td>0.29</td><td>0.62</td></tr><tr><td>Σ Trans MUFA</td><td>6.25</td><td>6.10</td><td>6.24</td><td>6.04</td><td>0.168</td><td>0.39</td><td>&lt;0.001</td><td>0.66</td><td>0.30</td><td>0.83</td></tr><tr><td>Σ PUFA</td><td>7.22</td><td>8.41</td><td>6.65</td><td>8.85</td><td>0.860</td><td>0.093</td><td>&lt;0.001</td><td>0.26</td><td>0.33</td><td>0.48</td></tr><tr><td>Σ ω-6 FA</td><td>5.12</td><td>6.15</td><td>4.66</td><td>6.39</td><td>0.673</td><td>0.086</td><td>&lt;0.001</td><td>0.30</td><td>0.36</td><td>0.54</td></tr><tr><td>Σ ω-3 FA</td><td>1.45</td><td>1.59</td><td>1.26</td><td>1.75</td><td>0.174</td><td>0.13</td><td>&lt;0.001</td><td>0.13</td><td>0.41</td><td>0.28</td></tr><tr><td>Σ CLA<sup>3</sup></td><td>0.38</td><td>0.38</td><td>0.46</td><td>0.40</td><td>0.057</td><td>0.68</td><td>0.69</td><td>0.087</td><td>0.60</td><td>0.62</td></tr><tr><td>Σ Trans FA</td><td>6.91</td><td>6.77</td><td>6.96</td><td>6.75</td><td>0.181</td><td>0.50</td><td>&lt;0.001</td><td>0.43</td><td>0.59</td><td>0.76</td></tr><tr><td>Σ Unidentified FA</td><td>13.7</td><td>13.2</td><td>13.8</td><td>14.1</td><td>0.38</td><td>0.27</td><td>0.54</td><td>0.68</td><td>0.21</td><td>0.28</td></tr><tr><td>C18:1t10-to-C18:1t11 (×10<sup>-1</sup> g/g)</td><td>1.81</td><td>1.76</td><td>1.84</td><td>1.96</td><td>0.176</td><td>0.69</td><td>0.006</td><td>0.37</td><td>0.34</td><td>0.48</td></tr><tr><td colspan="11"><strong>Biohydrogenation (%)</strong></td></tr><tr><td>C18:1c9</td><td>78.6</td><td>75.3</td><td>79.9</td><td>73.2</td><td>2.75</td><td>0.21</td><td>&lt;0.001</td><td>0.49</td><td>0.28</td><td>0.49</td></tr><tr><td>C18:2c9,c12</td><td>87.2</td><td>84.7</td><td>88.3</td><td>83.6</td><td>1.81</td><td>0.16</td><td>&lt;0.001</td><td>0.39</td><td>0.31</td><td>0.49</td></tr><tr><td>C18:3c9,c12,c15</td><td>89.5</td><td>87.8</td><td>90.6</td><td>87.7</td><td>1.14</td><td>0.14</td><td>&lt;0.001</td><td>0.10</td><td>0.55</td><td>0.53</td></tr><tr><td>Biohydrogenation completeness (%)</td><td>69.2</td><td>67.1</td><td>69.8</td><td>66.1</td><td>2.03</td><td>0.18</td><td>&lt;0.001</td><td>0.57</td><td>0.26</td><td>0.54</td></tr></tbody></table>

AThe value for this diet tended to be different from the value obtained with the control diet HEX ($\alpha_i < P \le 2 \times \alpha_i$).

¹HEX = 100% hexane-defatted soybean meal; 33MeOx = 67% hexane-defatted plus 33% 2-methyloxolane-defatted soybean meal; 67MeOx = 33% hexane-defatted plus 67% 2-methyloxolane-defatted soybean meal; 100MeOx = 100% 2-methyloxolane-defatted soybean meal.

²L = linear contrast for diet effect; Q = quadratic contrast for diet effect.

³Conjugated linoleic acid.

defatted SBM ($P = 0.005$) and greater for 100MeOx compared with HEX (+0.044 mM, back-transformed from the $\log_e$ scale, $P = 0.002$, $\alpha_1 = 0.017$).

**Milk Fat Content and FA Profile.** We did not find evidence of differences between diets in milk yield, milk fat content, and milk fat yield ($P > 0.05$, Table 6 and reported in Menoury et al., 2025). Among major FA ($\ge 0.10$ g/100 g total FA), C18:1t6 + C18:1t7 + C18:1t8 proportion was negatively linearly related to the propor-

tion of MeOx-defatted SBM ($P = 0.032$). Proportions of C14:1c9, C18:0, and C20:0 were quadratically related to the proportion of MeOx-defatted SBM ($P \le 0.038$), and C14:1c9 was 0.12 g/100 g total FA lower for 67MeOx compared with HEX ($P = 0.002$, $\alpha_2 = 0.017$). The C16:1c9 proportion was also 0.09 g/100 g total FA lower for 67MeOx compared with HEX ($P = 0.016$, $\alpha_2 = 0.017$). We did not find evidence of differences between diets for other major milk FA proportions ($P > 0.05$). Minor FA

<table><caption>**Table 5.** Plasma metabolites and C isotopic discrimination between plasma and diet (Δ¹³C) in dairy cows fed hexane-defatted or 2-methyloxolane-defatted soybean meal</caption><thead><tr><th rowspan="2">Item</th><th colspan="4">Diet¹</th><th rowspan="2">SEM³</th><th rowspan="2">P diet</th><th colspan="2">P contrast²</th></tr><tr><th>HEX</th><th>33MeOx</th><th>67MeOx</th><th>100MeOx</th><th>L</th><th>Q</th></tr></thead><tbody><tr><td>Glucose (g/L)</td><td>0.72</td><td>0.71</td><td>0.71</td><td>0.72</td><td>0.015</td><td>0.73</td><td>0.63</td><td>0.31</td></tr><tr><td>Acetate (mM)</td><td>1.35</td><td>1.22</td><td>1.17</td><td>1.22</td><td>0.081</td><td>0.29</td><td>0.15</td><td>0.22</td></tr><tr><td>BHB (mM)</td><td>0.73</td><td>0.67</td><td>0.68</td><td>0.71</td><td>0.039</td><td>0.36</td><td>0.59</td><td>0.098</td></tr><tr><td>Nonesterified fatty acid (log<sub>e</sub> mM)</td><td>-2.71</td><td>-2.40</td><td>-2.44</td><td>-2.20<sup>a</sup></td><td>0.168</td><td>0.004</td><td>0.005</td><td>0.71</td></tr><tr><td>Δ¹³C<sup>4</sup> (‰)</td><td>1.03</td><td>1.09</td><td>1.03</td><td>1.07</td><td>0.054</td><td>0.32</td><td>0.66</td><td>0.74</td></tr></tbody></table>

aThe value for this diet was significantly different from the value obtained with the control diet HEX ($P \le \alpha_i$).

¹HEX = 100% hexane-defatted soybean meal; 33MeOx = 67% hexane-defatted plus 33% 2-methyloxolane-defatted soybean meal; 67MeOx = 33% hexane-defatted plus 67% 2-methyloxolane-defatted soybean meal; 100MeOx = 100% 2-methyloxolane-defatted soybean meal.

²L = linear contrast for diet effect; Q = quadratic contrast for diet effect.

³Highest SEM.

⁴C isotopic discrimination between plasma and diet.
<table><caption>**Table 6.** Milk yield, fat content and yield, and major fatty acids (≥0.10 g/100 g total fatty acids) in dairy cows fed hexane-defatted or 2-methyloxolane-defatted soybean meal<fnref n="1" /></caption><thead><tr><th rowspan="2">Item</th><th colspan="4">Diet<sup>2</sup></th><th rowspan="2">SEM<sup>4</sup></th><th rowspan="2">P diet</th><th colspan="2">P contrast<sup>3</sup></th></tr><tr><th>HEX</th><th>33MeOx</th><th>67MeOx</th><th>100MeOx</th><th>L</th><th>Q</th></tr></thead><tbody><tr><td>Milk yield (kg/d)</td><td>26.1</td><td>25.5</td><td>25.7</td><td>25.5</td><td>0.93</td><td>0.30</td><td>0.16</td><td>0.44</td></tr><tr><td>Milk fat content (g/kg)</td><td>36.6</td><td>37.1</td><td>38.8</td><td>37.3</td><td>0.86</td><td>0.084</td><td>0.17</td><td>0.12</td></tr><tr><td>Milk fat yield (g/d)</td><td>958</td><td>945</td><td>991</td><td>950</td><td>39.1</td><td>0.33</td><td>0.79</td><td>0.49</td></tr><tr><td>Milk major fatty acids (g/100 g total fatty acids)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>C4:0</td><td>2.92</td><td>2.91</td><td>2.78</td><td>2.92</td><td>0.105</td><td>0.64</td><td>0.71</td><td>0.41</td></tr><tr><td>C6:0</td><td>2.29</td><td>2.29</td><td>2.23</td><td>2.29</td><td>0.059</td><td>0.73</td><td>0.77</td><td>0.55</td></tr><tr><td>C8:0</td><td>1.39</td><td>1.38</td><td>1.40</td><td>1.40</td><td>0.028</td><td>0.92</td><td>0.66</td><td>0.66</td></tr><tr><td>C10:0</td><td>3.21</td><td>3.16</td><td>3.26</td><td>3.28</td><td>0.107</td><td>0.44</td><td>0.23</td><td>0.53</td></tr><tr><td>C10:1c9</td><td>0.33</td><td>0.32</td><td>0.31</td><td>0.32</td><td>0.010</td><td>0.095</td><td>0.084</td><td>0.098</td></tr><tr><td>C12:0 + C11:1c9</td><td>3.74</td><td>3.65</td><td>3.76</td><td>3.84</td><td>0.132</td><td>0.42</td><td>0.24</td><td>0.30</td></tr><tr><td>C12:1c9 + C13:0</td><td>0.11</td><td>0.10</td><td>0.10</td><td>0.10</td><td>0.007</td><td>0.88</td><td>0.87</td><td>0.70</td></tr><tr><td>C14:0</td><td>12.2</td><td>12.2</td><td>12.2</td><td>12.4</td><td>0.23</td><td>0.86</td><td>0.58</td><td>0.54</td></tr><tr><td>Iso C14:0</td><td>0.19</td><td>0.19</td><td>0.18</td><td>0.18</td><td>0.011</td><td>0.79</td><td>0.62</td><td>0.95</td></tr><tr><td>C14:1c9</td><td>1.17</td><td>1.11</td><td>1.05<sup>a</sup></td><td>1.10</td><td>0.056</td><td>0.019</td><td>0.025</td><td>0.038</td></tr><tr><td>C15:0</td><td>1.26</td><td>1.25</td><td>1.23</td><td>1.23</td><td>0.030</td><td>0.73</td><td>0.28</td><td>0.84</td></tr><tr><td>Iso C15:0</td><td>0.29</td><td>0.29</td><td>0.29</td><td>0.30</td><td>0.007</td><td>0.51</td><td>0.14</td><td>0.78</td></tr><tr><td>Anteiso C15:0</td><td>0.55</td><td>0.55</td><td>0.55</td><td>0.54</td><td>0.014</td><td>0.51</td><td>0.18</td><td>0.73</td></tr><tr><td>C16:0</td><td>32.1</td><td>32.1</td><td>32.0</td><td>31.9</td><td>0.70</td><td>0.98</td><td>0.68</td><td>0.99</td></tr><tr><td>Iso C16:0</td><td>0.44</td><td>0.45</td><td>0.44</td><td>0.43</td><td>0.023</td><td>0.82</td><td>0.59</td><td>0.54</td></tr><tr><td>C16:1c7</td><td>0.15</td><td>0.16</td><td>0.15</td><td>0.15</td><td>0.006</td><td>0.49</td><td>0.46</td><td>0.71</td></tr><tr><td>C16:1c9</td><td>1.41</td><td>1.42</td><td>1.33<sup>a</sup></td><td>1.40</td><td>0.056</td><td>0.037</td><td>0.21</td><td>0.20</td></tr><tr><td>C17:0</td><td>0.77</td><td>0.77</td><td>0.77</td><td>0.78</td><td>0.013</td><td>0.57</td><td>0.38</td><td>0.32</td></tr><tr><td>Iso C17:0 + C16:1t9</td><td>0.38</td><td>0.40</td><td>0.39</td><td>0.39</td><td>0.009</td><td>0.35</td><td>0.30</td><td>0.20</td></tr><tr><td>Anteiso C17:0</td><td>0.48</td><td>0.50</td><td>0.49</td><td>0.48</td><td>0.014</td><td>0.67</td><td>0.85</td><td>0.26</td></tr><tr><td>C17:1c9</td><td>0.22</td><td>0.23</td><td>0.22</td><td>0.24</td><td>0.011</td><td>0.63</td><td>0.40</td><td>0.57</td></tr><tr><td>C18:0</td><td>8.92</td><td>9.15</td><td>9.45</td><td>8.82</td><td>0.293</td><td>0.058</td><td>0.99</td><td>0.015</td></tr><tr><td>C18:1t6 + C18:1t7 + C18:1t8</td><td>0.20</td><td>0.19</td><td>0.19</td><td>0.18</td><td>0.006</td><td>0.092</td><td>0.032</td><td>0.32</td></tr><tr><td>C18:1c9 + C18:1t13 + C18:1t14</td><td>17.5</td><td>17.5</td><td>17.4</td><td>17.6</td><td>0.49</td><td>0.98</td><td>0.82</td><td>0.74</td></tr><tr><td>C18:1t9</td><td>0.16</td><td>0.16</td><td>0.16</td><td>0.16</td><td>0.005</td><td>0.94</td><td>0.56</td><td>0.82</td></tr><tr><td>C18:1t10</td><td>0.22</td><td>0.22</td><td>0.22</td><td>0.21</td><td>0.008</td><td>0.36</td><td>0.24</td><td>0.43</td></tr><tr><td>C18:1c11</td><td>0.46</td><td>0.47</td><td>0.48</td><td>0.50</td><td>0.026</td><td>0.46</td><td>0.11</td><td>0.98</td></tr><tr><td>C18:1t11</td><td>0.71</td><td>0.74</td><td>0.71</td><td>0.67</td><td>0.038</td><td>0.31</td><td>0.19</td><td>0.18</td></tr><tr><td>C18:1c12</td><td>0.20</td><td>0.19</td><td>0.20</td><td>0.20</td><td>0.008</td><td>0.58</td><td>0.33</td><td>0.67</td></tr><tr><td>C18:1t12 + C18:1c6</td><td>0.21</td><td>0.21</td><td>0.22</td><td>0.21</td><td>0.008</td><td>0.36</td><td>0.64</td><td>0.48</td></tr><tr><td>C18:1c15 + C19:0</td><td>0.13</td><td>0.13</td><td>0.14</td><td>0.13</td><td>0.004</td><td>0.087</td><td>0.91</td><td>0.19</td></tr><tr><td>C18:1t16 + C18:1c14</td><td>0.22</td><td>0.22</td><td>0.23</td><td>0.21</td><td>0.008</td><td>0.16</td><td>0.69</td><td>0.066</td></tr><tr><td>C18:2c9,c12</td><td>1.72</td><td>1.63</td><td>1.74</td><td>1.83</td><td>0.103</td><td>0.61</td><td>0.36</td><td>0.39</td></tr><tr><td>C18:2c9,t13 + C18:2c10,t14</td><td>0.13</td><td>0.13</td><td>0.13</td><td>0.13</td><td>0.006</td><td>0.79</td><td>0.39</td><td>0.60</td></tr><tr><td>C18:2c9,t11 + C18:2t7,c9 + C18:2t8,c10</td><td>0.34</td><td>0.32</td><td>0.32</td><td>0.34</td><td>0.034</td><td>0.91</td><td>0.92</td><td>0.48</td></tr><tr><td>C18:3c9,c12,c15 + C20:1c11</td><td>0.31</td><td>0.28</td><td>0.32</td><td>0.33</td><td>0.024</td><td>0.61</td><td>0.49</td><td>0.46</td></tr><tr><td>C20:0</td><td>0.16</td><td>0.16</td><td>0.17</td><td>0.15</td><td>0.005</td><td>0.16</td><td>0.90</td><td>0.037</td></tr><tr><td>C20:1c9</td><td>0.13</td><td>0.13</td><td>0.13</td><td>0.13</td><td>0.005</td><td>0.91</td><td>0.65</td><td>0.97</td></tr><tr><td>C20:4c5,c8,c11,c14</td><td>0.20</td><td>0.17</td><td>0.17</td><td>0.18</td><td>0.017</td><td>0.71</td><td>0.68</td><td>0.30</td></tr></tbody></table>

(<0.10 g/100 g total FA) are presented in Supplemental Table S2 (see Notes). For sums of FA proportions, we did not find evidence for differences among diets (P > 0.05, Table 7). However, the ratio of C14:1c9 to C14:0 was negatively linearly related to the proportion of MeOx-defatted SBM (P = 0.006) and 0.96 × 10⁻<fnref n="2" /> g/g lower for 67MeOx compared with HEX (P = 0.001, α₂ = 0.017).

**Indicators of Liver Integrity and Functionality.** We did not find evidence of differences between diets in se-

rum total protein, globulin, and albumin concentrations, plasma total bilirubin concentration, and plasma ALT, AST, and ALP activities (P > 0.05, Table 8). Plasma γGT activity was positively linearly related to the proportion of MeOx-defatted SBM (P = 0.040), with greater activity for 67MeOx compared with HEX (+8.0 IU/L, back-transformed from the log<sub>c</sub> scale, P = 0.011, α₂ = 0.017).

**Inflammatory Status.** We did not find evidence of differences between diets in blood cell counts, blood hemo-

<footnote>ªThe value for this diet was significantly different from the value obtained with the control diet HEX (P ≤ αᵢ).</footnote>

<footnote n="1">¹Data for milk yield, milk fat content, and milk fat yield are reproduced from Menoury et al. (2025; creative commons BY license).</footnote>

<footnote n="2">²HEX = 100% hexane-defatted soybean meal; 33MeOx = 67% hexane-defatted plus 33% 2-methyloxolane-defatted soybean meal; 67MeOx = 33% hexane-defatted plus 67% 2-methyloxolane-defatted soybean meal; 100MeOx = 100% 2-methyloxolane-defatted soybean meal.</footnote>

<footnote n="3">³L = linear contrast for diet effect; Q = quadratic contrast for diet effect.</footnote>

<footnote n="4">⁴Highest SEM.</footnote>
<table><caption>**Table 7.** Sums of fatty acids (FA) and C14:1c9-to-C14:0 ratio in milk in dairy cows fed hexane-defatted or 2-methyloxolane-defatted soybean meal</caption><thead><tr><th rowspan="2">Item (g/100 g total FA, unless stated otherwise)</th><th colspan="4">Diet<fnref n="1" /></th><th rowspan="2">SEM<fnref n="3" /></th><th rowspan="2">P diet</th><th colspan="2">P contrast<fnref n="2" /></th></tr><tr><th>HEX</th><th>33MeOx</th><th>67MeOx</th><th>100MeOx</th><th>L</th><th>Q</th></tr></thead><tbody><tr><td>Σ SFA</td><td>71.4</td><td>71.5</td><td>71.6</td><td>71.4</td><td>0.71</td><td>0.99</td><td>1.00</td><td>0.80</td></tr><tr><td>Σ Even-chain FA</td><td>67.2</td><td>67.2</td><td>67.4</td><td>67.1</td><td>0.72</td><td>0.99</td><td>0.96</td><td>0.81</td></tr><tr><td>Σ Odd- and branched-chain FA</td><td>4.29</td><td>4.31</td><td>4.28</td><td>4.27</td><td>0.086</td><td>0.85</td><td>0.51</td><td>0.71</td></tr><tr><td>Σ Odd-chain FA</td><td>2.22</td><td>2.20</td><td>2.19</td><td>2.20</td><td>0.044</td><td>0.82</td><td>0.44</td><td>0.61</td></tr><tr><td>Σ Branched-chain FA</td><td>2.07</td><td>2.11</td><td>2.09</td><td>2.07</td><td>0.063</td><td>0.79</td><td>0.83</td><td>0.39</td></tr><tr><td>Σ Iso branched-chain FA</td><td>1.02</td><td>1.05</td><td>1.03</td><td>1.03</td><td>0.041</td><td>0.86</td><td>0.96</td><td>0.60</td></tr><tr><td>Σ Anteiso branched-chain FA</td><td>1.05</td><td>1.06</td><td>1.06</td><td>1.04</td><td>0.027</td><td>0.76</td><td>0.69</td><td>0.31</td></tr><tr><td>Σ MUFA</td><td>23.7</td><td>23.6</td><td>23.4</td><td>23.7</td><td>0.59</td><td>0.97</td><td>0.97</td><td>0.74</td></tr><tr><td>Σ Cis MUFA</td><td>21.9</td><td>21.8</td><td>21.6</td><td>22.0</td><td>0.56</td><td>0.93</td><td>0.92</td><td>0.63</td></tr><tr><td>Σ Trans MUFA</td><td>0.80</td><td>1.82</td><td>1.81</td><td>1.71</td><td>0.066</td><td>0.21</td><td>0.13</td><td>0.13</td></tr><tr><td>Σ PUFA</td><td>3.19</td><td>3.03</td><td>3.20</td><td>3.29</td><td>0.198</td><td>0.82</td><td>0.58</td><td>0.52</td></tr><tr><td>Σ ω-6 FA</td><td>2.12</td><td>2.00</td><td>2.11</td><td>2.21</td><td>0.126</td><td>0.69</td><td>0.48</td><td>0.38</td></tr><tr><td>Σ ω-3 FA</td><td>0.43</td><td>0.38</td><td>0.44</td><td>0.44</td><td>0.036</td><td>0.60</td><td>0.50</td><td>0.49</td></tr><tr><td>Σ CLA<fnref n="4" /></td><td>0.39</td><td>0.36</td><td>0.37</td><td>0.39</td><td>0.035</td><td>0.89</td><td>0.98</td><td>0.46</td></tr><tr><td>Σ Trans FA</td><td>2.44</td><td>2.47</td><td>2.46</td><td>2.34</td><td>0.092</td><td>0.50</td><td>0.30</td><td>0.27</td></tr><tr><td>Σ De novo FA<fnref n="5" /></td><td>29.9</td><td>29.7</td><td>29.6</td><td>30.1</td><td>0.47</td><td>0.70</td><td>0.78</td><td>0.26</td></tr><tr><td>Σ Mixed FA<fnref n="6" /></td><td>34.2</td><td>34.2</td><td>34.0</td><td>34.0</td><td>0.71</td><td>0.95</td><td>0.60</td><td>1.00</td></tr><tr><td>Σ Preformed FA<fnref n="7" /></td><td>34.4</td><td>34.4</td><td>34.8</td><td>34.5</td><td>0.73</td><td>0.96</td><td>0.79</td><td>0.77</td></tr><tr><td>Σ Co-eluted FA</td><td>0.65</td><td>0.65</td><td>0.65</td><td>0.64</td><td>0.017</td><td>0.88</td><td>0.51</td><td>0.64</td></tr><tr><td>Σ Unidentified FA</td><td>1.06</td><td>1.22</td><td>1.12</td><td>0.99</td><td>0.062</td><td>0.027</td><td>0.16</td><td>0.010</td></tr><tr><td>Desaturation index<br>C14:1c9-to-C14:0 (×10<sup>-2</sup> g/g)</td><td>9.59</td><td>9.18</td><td>8.63ª</td><td>8.97ª</td><td>0.504</td><td>0.006</td><td>0.006</td><td>0.051</td></tr></tbody></table>

globin concentration, hematocrit, and erythrocyte characteristics (P > 0.05, Table 8), except for basophil count, which was negatively linearly related to the proportion of MeOx-defatted SBM (P = 0.024). We did not find evidence of differences between diets in plasma IL-1α, IL-10, TNFα, VEGF-A, CCL3, and CCL2 concentrations (P > 0.05). Plasma CCL4 concentration was negatively linearly related to the proportion of MeOx-defatted SBM (P = 0.011). Plasma CXCL8 concentration was quadratically related to the proportion of MeOx-defatted SBM (P = 0.003) and lower for 33MeOx compared with HEX (–169 pg/mL, back-transformed from the log<sub>e</sub> scale, P = 0.002, α₃ = 0.017).

## Equivalence Tests

To simplify the presentation of the results of equivalence tests, we reported only the results from the comparison of the 2 extreme treatments (i.e., 100MeOx vs. HEX). Results from the comparisons of 67MeOx versus HEX and 33MeOx versus HEX are presented in Supplemental Figures S1 to S18 (see Notes).

**Intake and Animal Characteristics.** Total DMI, PMR DMI, SBM DMI, total FA intake, and major FA intakes were likely equivalent between 100MeOx and HEX, except for C18:2c9,c12, in which intake was unlikely equivalent toward lower value for 100MeOx compared with HEX (–7 g/d, Figure 1).

**Ruminal FA Profile.** Proportions of individual FA in the rumen were either likely equivalent or unlikely equivalent between 100MeOx and HEX, but we did not find evidence of strict equivalence (Supplemental Figure S3). Briefly, ruminal concentration of total FA and proportions of odd- and branched-chain FA, MUFA, and PUFA were unlikely equivalent between 100MeOx and HEX (–2.3 mg/kg DM, and +0.39, +0.7, and +1.63 g/100 g total FA, respectively, Figure 2). Proportions of SFA, even-chain FA, and trans FA were likely equivalent between 100MeOx and HEX. Regarding ruminal biohydrogenation indices, the C18:1t10-to-C18:1t11 ratio and biohydrogenation extent of C18:1c9 were unlikely equivalent between 100MeOx and HEX (+0.15 × 10⁻<fnref n="1" /> g/g and –5.4 points of percentage, respectively). The biohydrogenation extents of C18:1c9,c12 and C18:3c9,c12

<footnote>ªThe value for this diet was significantly different from the value obtained with the control diet HEX (P ≤ αᵢ).</footnote>

<footnote>AThe value for this diet tended to be different from the value obtained with the control diet HEX (αᵢ < P ≤ 2 × αᵢ).</footnote>

<footnote n="1">¹HEX = 100% hexane-defatted soybean meal; 33MeOx = 67% hexane-defatted plus 33% 2-methyloxolane-defatted soybean meal; 67MeOx = 33% hexane-defatted plus 67% 2-methyloxolane-defatted soybean meal; 100MeOx = 100% 2-methyloxolane-defatted soybean meal.</footnote>

<footnote n="2">²L = linear contrast for diet effect; Q = quadratic contrast for diet effect.</footnote>

<footnote n="3">³Highest SEM.</footnote>

<footnote n="4">⁴Conjugated linoleic acid.</footnote>

<footnote n="5">⁵FA < 16 C atoms.</footnote>

<footnote n="6">⁶FA with exactly 16 C atoms.</footnote>

<footnote n="7">⁷FA > 16 C atoms.</footnote>
<table><caption>**Table 8.** Blood chemistry, hematology, and immunology in dairy cows fed hexane-defatted or 2-methyloxolane-defatted soybean meal</caption><thead><tr><th rowspan="2">Item</th><th colspan="4">Diet<sup>1</sup></th><th rowspan="2">SEM<sup>3</sup></th><th rowspan="2">P diet</th><th colspan="2">P contrast<sup>2</sup></th></tr><tr><th>HEX</th><th>33MeOx</th><th>67MeOx</th><th>100MeOx</th><th>L</th><th>Q</th></tr></thead><tbody><tr><td>Chemistry<sup>4</sup></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Total protein (g/L)</td><td>76.5</td><td>77.0</td><td>77.7</td><td>76.5</td><td>1.09</td><td>0.23</td><td>0.78</td><td>0.078</td></tr><tr><td>Globulin</td><td>44.6</td><td>45.3</td><td>45.8</td><td>44.8</td><td>1.15</td><td>0.27</td><td>0.67</td><td>0.067</td></tr><tr><td>Albumin</td><td>31.9</td><td>31.6</td><td>32.0</td><td>31.7</td><td>0.41</td><td>0.73</td><td>0.81</td><td>0.98</td></tr><tr><td>Total bilirubin (µmol/L)</td><td>1.38</td><td>1.53</td><td>1.38</td><td>1.50</td><td>0.101</td><td>0.40</td><td>0.53</td><td>0.86</td></tr><tr><td>ALT (IU/L)</td><td>15.9</td><td>17.7</td><td>20.5</td><td>18.7</td><td>2.09</td><td>0.19</td><td>0.098</td><td>0.25</td></tr><tr><td>AST (IU/L)</td><td>66.3</td><td>74.7</td><td>70.8</td><td>69.8</td><td>7.71</td><td>0.67</td><td>0.76</td><td>0.34</td></tr><tr><td>γGT (log<sub>e</sub> IU/L)</td><td>3.23</td><td>3.28</td><td>3.51<sup>a</sup></td><td>3.40</td><td>0.131</td><td>0.030</td><td>0.040</td><td>0.36</td></tr><tr><td>ALP (IU/L)</td><td>65.2</td><td>63.1</td><td>64.2</td><td>65.1</td><td>4.06</td><td>0.83</td><td>0.91</td><td>0.41</td></tr><tr><td>Blood cell counts (log<sub>e</sub> cells/µL)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Erythrocyte</td><td>15.7</td><td>15.7</td><td>15.8</td><td>15.7</td><td>0.02</td><td>0.21</td><td>0.34</td><td>0.28</td></tr><tr><td>Reticulocyte</td><td>8.47</td><td>8.52</td><td>8.32</td><td>8.44</td><td>0.132</td><td>0.46</td><td>0.47</td><td>0.67</td></tr><tr><td>Leukocyte</td><td>9.10</td><td>9.10</td><td>9.05</td><td>9.08</td><td>0.032</td><td>0.32</td><td>0.21</td><td>0.49</td></tr><tr><td>Lymphocyte</td><td>8.11</td><td>8.10</td><td>8.06</td><td>8.04</td><td>0.058</td><td>0.46</td><td>0.11</td><td>0.95</td></tr><tr><td>Monocyte</td><td>6.68</td><td>6.66</td><td>6.63</td><td>6.60</td><td>0.063</td><td>0.64</td><td>0.20</td><td>0.89</td></tr><tr><td>Neutrophil</td><td>8.31</td><td>8.31</td><td>8.27</td><td>8.31</td><td>0.060</td><td>0.89</td><td>0.81</td><td>0.70</td></tr><tr><td>Eosinophil</td><td>6.52</td><td>6.51</td><td>6.39</td><td>6.58</td><td>0.112</td><td>0.53</td><td>0.87</td><td>0.29</td></tr><tr><td>Basophil</td><td>4.27</td><td>4.23</td><td>4.23</td><td>4.01</td><td>0.117</td><td>0.087</td><td>0.024</td><td>0.23</td></tr><tr><td>Blood hemoglobin concentration (g/L)</td><td>108</td><td>107</td><td>108</td><td>106</td><td>1.8</td><td>0.30</td><td>0.16</td><td>0.48</td></tr><tr><td>Hematocrit (%)</td><td>32.6</td><td>32.4</td><td>32.9</td><td>32.1</td><td>0.56</td><td>0.40</td><td>0.48</td><td>0.41</td></tr><tr><td>Erythrocyte characteristics<sup>5</sup></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MCV (fL)</td><td>47.4</td><td>47.4</td><td>47.4</td><td>47.6</td><td>0.67</td><td>0.77</td><td>0.31</td><td>0.74</td></tr><tr><td>MCH (pg)</td><td>15.7</td><td>15.7</td><td>15.6</td><td>15.7</td><td>0.24</td><td>0.57</td><td>0.40</td><td>0.50</td></tr><tr><td>MCHC (%)</td><td>33.2</td><td>33.1</td><td>32.9</td><td>33.0</td><td>0.30</td><td>0.35</td><td>0.13</td><td>0.66</td></tr><tr><td>RDW-CV (%)</td><td>22.1</td><td>22.0</td><td>21.9</td><td>21.9</td><td>0.39</td><td>0.72</td><td>0.27</td><td>0.80</td></tr><tr><td>Cytokines<sup>6</sup> (pg/mL, except if stated otherwise)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>IL-1α</td><td>39.3</td><td>41.2</td><td>42.1</td><td>41.1</td><td>9.50</td><td>0.72</td><td>0.44</td><td>0.42</td></tr><tr><td>IL-10</td><td>234</td><td>257</td><td>257</td><td>253</td><td>55.3</td><td>0.66</td><td>0.40</td><td>0.40</td></tr><tr><td>TNFα</td><td>4,855</td><td>5,442</td><td>5,682</td><td>5,450</td><td>1,513.4</td><td>0.50</td><td>0.26</td><td>0.31</td></tr><tr><td>VEGF-A (log<sub>e</sub> pg/mL)</td><td>4.24</td><td>4.10</td><td>4.18</td><td>4.17</td><td>0.167</td><td>0.44</td><td>0.66</td><td>0.32</td></tr><tr><td>CCL2</td><td>596</td><td>587</td><td>615</td><td>615</td><td>76.1</td><td>0.77</td><td>0.40</td><td>0.83</td></tr><tr><td>CCL3</td><td>187</td><td>198</td><td>195</td><td>200</td><td>33.6</td><td>0.71</td><td>0.34</td><td>0.76</td></tr><tr><td>CCL4 (log<sub>e</sub> pg/mL)</td><td>4.64</td><td>4.56</td><td>4.51</td><td>4.40</td><td>0.127</td><td>0.073</td><td>0.011</td><td>0.81</td></tr><tr><td>CXCL8 (log<sub>e</sub> pg/mL)</td><td>6.19</td><td>5.76<sup>a</sup></td><td>5.94</td><td>6.12</td><td>0.174</td><td>0.009</td><td>0.96</td><td>0.003</td></tr></tbody></table>

<sup>a</sup>The value for this diet was significantly different from the value obtained with the control diet HEX (P ≤ α<sub>i</sub>).

<sup>1</sup>HEX = 100% hexane-defatted soybean meal; 33MeOx = 67% hexane-defatted plus 33% 2-methyloxolane-defatted soybean meal; 67MeOx = 33% hexane-defatted plus 67% 2-methyloxolane-defatted soybean meal; 100MeOx = 100% 2-methyloxolane-defatted soybean meal.

<sup>2</sup>L = linear contrast for diet effect; Q = quadratic contrast for diet effect.

<sup>3</sup>Highest SEM.

<sup>4</sup>ALT = alanine aminotransferase; AST = aspartate aminotransferase; γGT = γ-glutamyltransferase; ALP = alkaline phosphatase.

<sup>5</sup>MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; RDW-CV = erythrocyte distribution width – coefficient of variation.

<sup>6</sup>TNF-α = tumor necrosis factor α; VEGF-A = vascular endothelial growth factor A; CCL2 = chemokine C-C motif ligand 2 or monocyte chemoattractant protein 1; CCL3 = chemokine C-C motif ligand 3 or macrophage inflammatory protein 1α; CCL4 = chemokine C-C motif ligand 4 or macrophage inflammatory protein 1β; CXCL8 = chemokine C-X-C motif ligand 8 or IL-8.

c15, and the biohydrogenation completeness were likely equivalent between 100MeOx and HEX.

**Plasma Indicators of Energy Metabolism.** Comparison of 100MeOx with HEX indicated equivalent plasma glucose concentration and likely equivalent plasma BHB concentration and Δ<sup>13</sup>C (Figure 3). However, plasma acetate concentration was unlikely equivalent toward lower concentration for 100MeOx compared with HEX (–0.13 mM), whereas plasma NEFA concentration was not equivalent toward greater concentration for 100MeOx compared with HEX (+0.044 mM, back-transformed from the log<sub>e</sub> scale).

**Milk Fat Content and FA Profile.** Milk yield, milk fat content, and milk fat yield were likely equivalent between 100MeOx and HEX (Figure 4 and reported in Menoury et al., 2025). Major FA proportions (≥0.10 g/100 g total FA) were mostly declared equivalent or likely equivalent when 100MeOx was compared with HEX. Exceptions were milk proportions of C14:1c9, C18:1t6 + C18:1t7 + C18:1t8, C18:1t11, and C20:4c5,c8,c11,c14 (–0.07, –0.02, –0.04, and –0.01 g/100 g total FA, respectively) that were unlikely equivalent toward lower proportions for 100MeOx compared with HEX, and proportions of C17:1c9, C18:1c11, and C18:2c9,c12 (+0.01, +0.04, and
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Scaled difference between estimated marginal means of 100MeOx and HEX (100MeOx minus HEX) for DMI and fatty acid intake. In green: equivalence margins and equivalence interval. In black or red: estimated difference and its 100% (2 × α) % confidence interval (error bars). Points within the equivalence interval are black, points outside the equivalence interval are red. Values on the left: estimated marginal mean of 100MeOx and estimated marginal mean of HEX, SEM. Values on the right: estimated difference ± margin of error of estimated difference, equivalence margin (EM). HEX = 100% hexane-defatted soybean meal; 100MeOx = 100% 2-methyloxolane-defatted soybean meal; PMR = partial mixed ration. Data for DMI are reproduced from Menoury et al. (2025; creative commons BY license).</figcaption></figure>

+0.10 g/100 g total FA, respectively) that were unlikely equivalent toward greater proportions for 100MeOx compared with HEX. Milk minor (<0.10 g/100 g total FA) and unidentified FA proportions are presented in Supplemental Figure S12. The sums of FA proportions were equivalent between 100MeOx and HEX for SFA, even-chain FA, odd- and branched-chain FA, odd-chain FA, branched-chain FA, de novo FA, and mixed FA proportions (Figure 5). Other sums of FA proportions (e.g., *iso* branched-chain FA, *anteiso* branched-chain FA, MUFA, PUFA, *trans* FA, preformed FA) were likely equivalent between 100MeOx and HEX. The C14:1c9-to-C14:0 ratio was unlikely equivalent toward lower value for 100MeOx compared with HEX (−0.62 × 10⁻² g/g).

**Indicators of Liver Integrity and Functionality.** Serum total protein, globulin, and albumin concentrations were equivalent and plasma ALP activity was likely equivalent between 100MeOx and HEX (Figure 6). However, plasma ALT (+2.8 IU/mL), AST (+3.5 IU/mL), and γGT (+4.7 IU/mL, back-transformed from the log~e~ scale) activities and total bilirubin concentration (+0.13 µmol/ mL) were unlikely equivalent toward greater values for 100MeOx compared with HEX.

**Inflammatory Status.** Blood hemoglobin concentration, hematocrit, erythrocyte count, and erythrocyte characteristics were equivalent, and reticulocyte, leukocyte, and neutrophil counts were likely equivalent between 100MeOx and HEX (Figure 6). However, blood counts of lymphocytes, monocytes, eosinophils, and basophils were unlikely equivalent for 100MeOx compared with HEX (−203, −56, +44, and −16 cells/µL, respectively, back-transformed from the log~e~ scale). Among

cytokines, plasma IL-1α and CCL2 concentrations were likely equivalent between 100MeOx and HEX. However, plasma IL-10, TNFα, and CCL3 concentrations were unlikely equivalent toward greater concentrations for 100MeOx compared with HEX (+20, +595, and +13 pg/ mL, respectively). Plasma VEGF-A, CCL4, and CXCL8 concentrations were unlikely equivalent toward lower concentrations for 100MeOx compared with HEX (−4.8, −21.7, and −31 pg/mL, respectively, back-transformed from the log~e~ scale).

## DISCUSSION

This study used the complementarity between difference and equivalence testing approaches, previously discussed by Menoury et al. (2025), to evaluate the effects of replacing hexane-defatted SBM with MeOx-defatted SBM on ruminal and milk FA profiles and health indicators. Milk fat content and yield, and most of the milk major FA proportions were likely equivalent between cows fed 100MeOx and HEX, despite a small linear reduction of the mammary gland SCD activity index (i.e., C14:1c9-to-C14:0 ratio) with increasing MeOx-defatted SBM proportion in the diet. Although we could not prove equivalence of the ruminal FA profiles between cows fed 100MeOx and HEX, we found only few evidence of differences in ruminal FA profile when replacing hexane-defatted SBM with MeOx-defatted SBM. Plasma NEFA concentration was unlikely equivalent toward greater values for 100MeOx compared with HEX and positively linearly related to the proportion of MeOx-defatted SBM. Regarding liver integrity and functionality, plasma ALT,
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Scaled difference between estimated marginal means of 100MeOx and HEX (100MeOx minus HEX) for sums of fatty acids (FA) in the rumen and ruminal biohydrogenation indices (sampling through ruminal cannula). In green: equivalence margins and equivalence interval. In black or red: estimated difference and its 100 − (2 × αᵢ)% confidence interval (error bars). Points within the equivalence interval are black, points outside the equivalence interval are red. Values on the left: estimated marginal mean of 100MeOx and estimated marginal mean of HEX, SEM. Values on the right: estimated difference ± margin of error of estimated difference, equivalence margin (EM). HEX = 100% hexane-defatted soybean meal; 100MeOx = 100% 2-methyloxolane-defatted soybean meal; CLA = conjugated linoleic acid.</figcaption></figure>

AST, and γGT activities, as well as plasma total bilirubin concentration, were unlikely equivalent toward greater values for 100MeOx compared with HEX. Plasma γGT activity was also positively linearly related to the proportion of MeOx-defatted SBM. Regarding inflammation, lymphocyte, monocyte, eosinophil, and basophil blood counts, as well as plasma concentration of most cytokines, were unlikely equivalent between cows fed 100MeOx compared with HEX. Nonetheless, only blood basophil count and plasma CCL4 concentration were negatively linearly related to the proportion of MeOx-defatted SBM, and plasma CXCL8 concentration was quadratically related to the proportion of MeOx-defatted SBM.

## Solvent Residues and FA Profile of SBM

**Solvent Residues.** Although the hexane-defatted SBM contained 11.2 mg/kg DM of n-hexane and 4.4 mg/kg DM of MeOx residues, the MeOx-defatted SBM contained no hexane and 977.4 mg/kg DM of MeOx residues (Menoury et al., 2025). This high level of MeOx residues in MeOx-defatted SBM, close to the threshold for declaration in the catalog of feed (European Union, 2022) and higher than usual values for solvent residue concentration measured out of the crushing factory (≤250 to 500 mg/kg; Bauer et al., 2009; Demarco and Gibon, 2020), enabled to test for a worst-case scenario for solvent residues in SBM, as discussed by Menoury et al. (2025). Im-
<figure><img src="image_4.png" /></figure>

**Figure 3.** Scaled difference between estimated marginal means of 100MeOx and HEX (100MeOx minus HEX) for plasma metabolites and C isotopic discrimination between plasma and diet (Δ¹³C). In green: equivalence margins and equivalence interval. In black or red: estimated difference and its 100% (2 × αᵢ) % confidence interval (error bars). Points within the equivalence interval are black, points outside the equivalence interval are red. (A) Analyses were performed on the natural scale. Values on the left: estimated marginal mean of 100MeOx and estimated marginal mean of HEX, SEM. Values on the right: estimated difference ± margin of error of estimated difference, equivalence margin (EM). (B) Analyses were performed on the logₑ scale and back-transformed for graphical representation. Values on the left: estimated marginal mean of 100MeOx and estimated marginal mean of HEX, SEM on the logₑ scale. Values on the right: estimated difference and its confidence interval, equivalence margin (EM). HEX = 100% hexane-defatted soybean meal; 100MeOx = 100% 2-methyloxolane-defatted soybean meal; Δ¹³C: C isotopic discrimination between plasma and diet.

provements in the production process during subsequent production runs with MeOx already produced oilseed meals containing 100 to 400 mg/kg of MeOx residues (EcoXtract [Dunkirk, France], personal communication, 2025).

**FA Profile and Intake.** The use of MeOx enabled the production of defatted SBM having a similar FA profile, with ~51% C18:2c9,c12, ~18% C18:1c9, and ~17% C16:0 (g/100 g of total FA). The similarity in FA profile was expected as SBM were produced from the same batch of dehulled soybean flakes and previous studies showed similar FA profiles for soybean oil extracted with hexane and MeOx (Claux et al., 2021; Bartier et al., 2024). However, the MeOx-defatted SBM contained 16% (i.e., 2.7 g/kg DM) less total FA than hexane-defatted SBM. This agrees with greater oil extraction yield and lower residual oil content in SBM obtained by MeOx compared with hexane (Claux et al., 2021; Bartier et al., 2024). The greater defatting of SBM using MeOx compared with hexane was explained by the greater ability of MeOx compared with hexane to extract polar compounds such as phospholipids (Claux et al., 2021) and by the ability of MeOx to extract an oil fraction inaccessible to hexane (Bartier et al., 2024). The former may be related to better solvent diffusivity in the matrix due to partial miscibility of MeOx with water (Rapinel et al., 2020; Bartier et al.,

2024). The C18:2c9,c12 intake was unlikely equivalent for 100MeOx compared with HEX, toward lower intake for 100MeOx. This can be explained by the lower total FA concentration in MeOx-defatted SBM compared with hexane-defatted SBM and the linear decrease in SBM intake with increasing MeOx-defatted SBM proportion, despite SBM intake being likely equivalent between all experimental diets and HEX.

## Effects of Using 2-Methyloxolane as Defatting Solvent on Ruminal and Milk FA Profiles

**Ruminal FA Profile.** For most individual FA proportions and sums of FA proportions in the rumen, we did not have sufficient evidence to prove either difference or strict equivalence between experimental diets and HEX. This is probably due to a low statistical power, as ruminal FA were measured only on a subset of 4 rumen-cannulated cows, and to overly strict a priori defined equivalence margins (i.e., ± 5% of the EMM of HEX). For example, minor FA (<0.10% of total FA) were studied with equivalence margins lower than 0.005% of total FA (i.e., 5% × 0.10% of total FA). We want to note that the quantification of C6:0 to C10:0 may have been underestimated due to potential volatilization of short-chain FA during storage, freeze-drying, extraction, and methylation. There-
<figure><img src="image_5.png" /><figcaption>**Figure 4.** Scaled difference between estimated marginal means of 100MeOx and HEX (100MeOx minus HEX) for milk yield, fat content and yield, and major fatty acids (≥0.10 g/100 g total fatty acids, FA). In green: equivalence margins and equivalence interval. In black or red: estimated difference and its 100 – (2 × αᵢ)% confidence interval (error bars). Points within the equivalence interval are black, points outside the equivalence interval are red. Values on the left: estimated marginal mean of 100MeOx and estimated marginal mean of HEX, SEM. Values on the right: estimated difference ± margin of error of estimated difference, equivalence margin (EM). HEX = 100% hexane-defatted soybean meal; 100MeOx = 100% 2-methyloxolane-defatted soybean meal. Data for milk yield, milk fat content, and milk fat yield are reproduced from Menoury et al. (2025; creative commons BY license).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 5.** Scaled difference between estimated marginal means of 100MeOx and HEX (100MeOx minus HEX) for sums of fatty acids (FA) and C14:1c9-to-C14:0 ratio in milk. In green: equivalence margins and equivalence interval. In black or red: estimated difference and its $100 - (2 \times \alpha_i)\%$ confidence interval (error bars). Points within the equivalence interval are black, points outside the equivalence interval are red. Values on the left: estimated marginal mean of 100MeOx and estimated marginal mean of HEX, SEM. Values on the right: estimated difference ± margin of error of estimated difference, equivalence margin (EM). HEX = 100% hexane-defatted soybean meal; 100MeOx = 100% 2-methyloxolane-defatted soybean meal; CLA = conjugated linoleic acid; De novo FA = FA < 16 C atoms; Mixed FA = FA with exactly 16 C atoms; Preformed FA = FA > 16 C atoms.</figcaption></figure>

fore, results related to ruminal short-chain FA should be interpreted with caution.

**Ruminal Biohydrogenation.** Heat processing was likely more intense with MeOx compared with hexane as indicated by numerically greater furosine (i.e., early Maillard reaction product) and Nε-carboxymethyl-lysine (i.e., advanced Maillard reaction product) concentrations in MeOx-defatted SBM (Menoury et al., 2025). The use of nonenzymatic browning, including Maillard reactions, has been proposed to protect PUFA from ruminal biohydrogenation, although its efficacy seems limited (Toral et al., 2024). The C18:1t10-to-C18:1t11 ratio and biohydrogenation extent of C18:1c9 were unlikely equivalent between 100MeOx and HEX, but there was no evidence of differences among diets. In addition, biohydrogena-

tion extent of C18:2c9,c12 and C18:3c9,c12,c15, as well as biohydrogenation completeness were likely equivalent between 100MeOx and HEX. Milk FA are also indicators of ruminal biohydrogenation, as ruminal biohydrogenation products can be directly secreted in milk or desaturated by the mammary gland SCD before secretion (Chilliard et al., 2007). Unlikely equivalence between 100MeOx and HEX together with significant difference was only reported for C18:1t6 + C18:1t7 + C18:1t8, which are minor intermediates of C18:1c9, C18:2c9,c12 and C18:3c9,c12,c15 ruminal biohydrogenation (Toral et al., 2024). As a whole, indicators in milk seem to confirm a limited efficacy of Maillard reactions to protect PUFA from ruminal biohydrogenation. However, this could not be confirmed or unconfirmed at the ruminal level. As dis-
<figure><img src="image_7.png" /><figcaption>**Figure 6.** Scaled difference between estimated marginal means of 100MeOx and HEX (100MeOx minus HEX) for blood chemistry, hematology, and immunology. In green: equivalence margins and equivalence interval. In black or red: estimated difference and its 100 – (2 × α) % confidence interval (error bars). Points within the equivalence interval are black, points outside the equivalence interval are red. Values on the left: estimated marginal mean of 100MeOx and estimated marginal mean of HEX, SEM. Values on the right: estimated difference ± margin of error of estimated difference, equivalence margin (EM). For variables indicated with *: (1) analyses were performed on the log<sub>e</sub> scale and back-transformed for graphical representation, (2) values on the left: estimated marginal mean of 100MeOx and estimated marginal mean of HEX, SEM on the log<sub>e</sub> scale, and (3) values on the right: estimated difference and its confidence interval, equivalence margin (EM). HEX = 100% hexane-defatted soybean meal; 100MeOx = 100% 2-methyloxolane-defatted soybean meal; ALT = alanine aminotransferase; AST = aspartate aminotransferase; γGT = γ-glutamyltransferase; ALP = alkaline phosphatase. MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; RDW-CV = erythrocyte distribution width – coefficient of variation; TNFα = tumoral necrosis factor α; VEGF-A = vascular endothelial growth factor A; CCL2 = chemokine C–C motif ligand 2 or monocyte chemoattractant protein 1; CCL3 = chemokine C–C motif ligand 3 or macrophage inflammatory protein 1α; CCL4 = chemokine C–C motif ligand 4 or macrophage inflammatory protein 1β; CXCL8 = chemokine C–X–C motif ligand 8 or IL-8.</figcaption></figure>
cussed by Menoury et al. (2025), an overall low-intense heating process applied to the SBM used in the current study compared with the SBM used by Nozière et al. (2022) may have contributed to the absence of evidence of differences in ruminal biohydrogenation between dietary treatments.

**Milk FA Profile and Mammary Gland SCD Activity Index.** The milk FA profile was likely equivalent between 100MeOx and HEX. The mammary gland SCD activity index (i.e., C14:1c9-to-C14:0 ratio) was, however, negatively linearly related to MeOx-defatted SBM proportion in the diet and reduced by 10% (i.e., -0.96 x 10⁻² g/g) with 67MeOx compared with HEX. This reduction was associated with a quadratic response of C14:1c9 proportion in milk to MeOx-defatted SBM proportion in the diet, and corroborated by the reduced C16:1c9 proportion in milk with 67MeOx compared with HEX. Previously, reduction of the mammary gland SCD activity index through dietary factors has been associated with a down-regulation of gene expression (e.g., abomasal and intra-venous infusion of C18:2t10,c12 reduced SCD1 mRNA abundance in the mammary gland although this effect was not always found; Baumgard et al., 2002; Gervais et al., 2009; Harvatine et al., 2018), or by interference with the enzyme's mode of action (e.g., ruminal infusion of Co-EDTA downregulated mammary gland SCD activity without evidence of reduced SCD1 mRNA and SCD protein abundance; Shingfield et al., 2008; Leskinen et al., 2016). Soybean oil reduced mammary gland SCD1 mRNA expression of SCD1 compared with rapeseed and linseed oils, although no evidence of an effect on the C14:1c9-to-C14:0 ratio was reported (Jacobs et al., 2011). Discrepancy with the current study reporting a negative linear relationship between the C14:1c9-to-C14:0 ratio and MeOx-defatted SBM, that was more defatted than hexane-defatted SBM, may originate from the amount of oil supplemented (i.e., the diet contained 53 g/kg ether extract in the study from Jacobs et al., 2011, versus 27 g/kg ether extract in the current study) and the form of oil supplementation (i.e., soybean oil versus SBM). The mechanisms of action of MeOx-defatted SBM on SCD activity index warrant further investigation.

**Plasma NEFA.** Plasma NEFA concentration was positively linearly related to the proportion of MeOx-defatted SBM and slightly greater in cows fed 100MeOx compared with HEX (+0.044 mM, back-transformed from the log~e~ scale). Greater plasma NEFA concentration may originate from metabolic modifications in the adipose tissue (e.g., greater lipolysis of triglycerides or lower esterification of NEFA), in the liver (e.g., lower NEFA uptake before esterification to triglycerides, partial oxidation into ketone bodies, or complete oxidation), or in peripheral tissues such as the mammary gland (e.g., greater NEFA uptake for milk fat synthesis; Bell, 1995;

Drackley, 1999). At the start of the experiment cows were primiparous, 36 ± 2.1 (mean ± SD) months old, and reaching midlactation (i.e., 71 ± 20.5 DIM). Given their age (i.e., <40 mo old), cows had energetic requirements for growth (INRA, 2018). However, age was balanced across diets. Adipose tissue mobilization appears unlikely, because the net energy balance was positive, although slightly lower for 100MeOx compared with HEX (-0.09 UFL/d, Menoury et al., 2025), and EMM for plasma NEFA concentration were lower than those observed in lactating dairy cows experiencing negative energy balance during sequential feed restriction (Pires et al., 2025). Additionally, plasma BHB concentration and milk fat yield were likely equivalent between 100MeOx and HEX. Altogether, these findings suggest that the greater plasma NEFA concentration in cows fed 100MeOx may not originate from greater lipolysis of triglycerides in the adipose tissue nor lower plasma NEFA uptake for oxidation to ketone bodies in the liver or milk fat synthesis in the mammary gland. Instead, it may reflect lower plasma NEFA uptake for esterification in the adipose tissue or lower esterification or complete oxidation in the liver.

## Effects of Using 2-Methyloxolane as Defatting Solvent on Health Indicators

### Indicators of Liver Integrity and Functionality.

Plasma ALT and AST activities may indicate hepatocyte damage, plasma γGT and ALP activities may indicate cholestasis, and plasma albumin and cholesterol concentrations are indicators of liver functionality (Bionaz et al., 2007; Russell and Roussel, 2007; Bertoni and Trevisi, 2013). Despite ALT, AST, and γGT activities in plasma being unlikely equivalent toward greater activity with 100MeOx compared with HEX, the majority of individual cow x period values remained within or below reference intervals (Dubreuil and Lapierre, 1997; Cozzi et al., 2011; Premi et al., 2021; Cornell University College of Veterinary Medicine, 2025). An exception was observed for 4 datapoints, including 3 from the same cow, where plasma γGT activity ranged from 67 to 157 IU/L and exceeded the upper limit of reference intervals (37 to 44 IU/L; Cozzi et al., 2011; Premi et al., 2021). This cow had leverage on the statistical results, as its removal from the dataset changed conclusions to likely equivalent γGT activity in the plasma between 100MeOx and HEX, and absence of evidence of positive linear relationship of plasma γGT activity with MeOx-defatted SBM proportion (P = 0.80, data not shown). Nonetheless, the data from this cow were kept in the statistical analysis because other liver health indicators were within reference intervals and no health nor experimental problem was reported during the pre-experimental and experimental phases (i.e., 3 and 12 wk, respectively).
Despite likely equivalence between 100MeOx and HEX, the EMM for plasma albumin concentration for all diets were on average 1.2 g/L lower than reference intervals for bovine (Cozzi et al., 2011; Cornell University College of Veterinary Medicine, 2025) that may indicate an overall low liver synthesis of albumin across the 4 dietary treatments. Also, unlikely equivalent toward increased concentration of total bilirubin in plasma for 100MeOx compared with HEX may indicate impaired liver function with 100MeOx, because unconjugated bilirubin is conjugated by hepatocytes before excretion in the bile (Tennant, 1997). However, we did not find evidence of difference in plasma total bilirubin concentration between experimental diets and HEX. Overall, replacing hexane-defatted SBM by MeOx-defatted SBM has unclear effects on liver integrity and functionality, as plasma γGT results were leveraged by one cow, and the unlikely equivalent outcomes on AST, ALT, and total bilirubin were not corroborated by evidence of differences. Further studies, such as long-term feeding trials, are required to study the chronic effect of the type of SBM on liver integrity and functionality.

**Inflammatory Status.** Blood hemoglobin concentration, hematocrit, and erythrocyte characteristics were equivalent between 100MeOx and HEX and the individual cow × period values were generally within reference intervals. Although 20% of mean corpuscular hemoglobin concentrations were lower than the reference interval for mid-lactating cows (i.e., 32.4%; Herman et al., 2018), only 1 value was lower than the reference interval for adult cows (i.e., 29.7% vs. 30.0%; LabOniris [Nantes, France], personal communication). Most indicators of inflammation were unlikely equivalent between 100MeOx and HEX but largest variations, toward reduced values for 100MeOx, were observed for blood basophil count and CCL4 plasma concentration. Furthermore, difference tests were only significant for blood basophil count (i.e., negatively linearly related to MeOx-defatted SBM proportion) and plasma concentrations of CCL4 (i.e., negatively linearly related to MeOx-defatted SBM proportion) and CXCL8 (i.e., quadratically related to MeOx-defatted SBM proportion, with reduced concentration for 33MeOx compared with HEX). Basophils are the rarest leukocytes in healthy ruminants (George et al., 2010; Herman et al., 2018) and are involved in allergic and inflammatory processes (Jones and Allison, 2007). Although 15% of individual cow × period blood basophil counts were above the reference interval for adult cows (i.e., 100 cells/mL; Herman et al., 2018), only 1 was above the reference interval of the laboratory for adult cows (i.e., 230 vs. 200 cells/mL; LabOniris [Nantes, France], personal communication). Overall, these individual values suggest that health of cows was not impaired although the linear decrease of blood basophil count with

increasing MeOx-defatted SBM proportion remains to be explained. Many leukocyte types (e.g., monocytes, B and T lymphocytes, neutrophils, dendritic cells, mastocytes, natural killer cells) produce CCL4, a chemoattractant for monocytes, dendritic cells, and T lymphocytes, and a regulator of transendothelial migration of monocyte, T lymphocytes, immature dendritic cells, and natural killer cells (Gangur et al., 2002; Menten et al., 2002). However, no effect of CCL4 on basophil was reported in vitro (Menten et al., 2002). Therefore, the linear decreases in basophil count and CCL4 concentration with increasing MeOx-defatted SBM proportion are not expected to be directly related. On the other hand, the proinflammatory chemokine CXCL8 produced by activated monocytes, macrophages, smooth muscle cells, epithelial cells, and endothelial cells is active on neutrophils, basophils, and T lymphocytes (Mathelier-Fusade et al., 1990; Gangur et al., 2002). However, although plasma CXCL8 concentration was quadratically related to the proportion of MeOx-defatted SBM and lower with 33MeOx compared with HEX, there was no evidence of quadratic effects for blood neutrophil, basophil, and lymphocyte counts. These counts were also likely equivalent between 33MeOx and HEX. Overall, 3 wk after the beginning of the dietary treatment, the inflammatory profile was unlikely equivalent between cows fed 100MeOx and HEX. However, only few differences were detected on blood basophil count and plasma CCL4 and CXCL8 concentrations. Because inflammatory responses can occur rapidly following the beginning of the dietary treatment, further studies may investigate the short-term kinetics of the inflammatory reaction around the beginning of the dietary treatment distribution. In addition, given the small changes observed on inflammatory status and liver integrity and functionality, further studies may consider evaluating the positive acute phase proteins (e.g., serum amyloid A, haptoglobin, LPS-binding protein) which are at the crossroad between liver functionality and inflammatory response.

## CONCLUSIONS

Overall, 100MeOx and HEX resulted in likely equivalent milk fat content, yield, and major milk FA profile, although a slight linear decrease in the mammary gland SCD activity index was observed with increasing proportion of MeOx-defatted SBM in the diet. We did not find evidence of strictly equivalent ruminal FA profile, but only minor differences were observed. Some indicators suggested slight changes in liver integrity and functionality with 100MeOx compared with HEX, but these effects remain unclear. The inflammatory profile was unlikely equivalent between cows fed 100MeOx and HEX, but significant differences were limited to blood basophil
count and plasma CCL4 and CXCL8 concentrations. However, neither hexane-defatted SBM nor MeOx-defatted SBM impaired health. Future work may consider short-term study of the inflammatory response but also long-term trial to evaluate potential chronic effects. This study represented a worst-case scenario regarding MeOx residues in SBM. Reducing residue levels may mitigate the slight changes observed in liver integrity, liver functionality, and inflammatory status.

## NOTES

This work was part of the EcoXtract Protéines project, a partnership between INRAE (Paris, France), Avignon Université (Avignon, France), and EcoXtract (Dunkirk, France), with funding from the French Agency for Ecological Transition (ADEME, Angers, France). The authors thank F. Fournier, D. Roux, J. Bourdassol, V. Tate, and F. Rosa (INRAE, Herbipôle, Saint-Genès-Champanelle, France [https://doi.org/10.15454/1.5572318050509348E12]) for conducting the experiment, collecting samples, and taking care of the animals. We also thank C. Labonne, L. Genestoux, I. Constant, L. Pluchino, and E. Tixier (INRAE, UMR Herbivores, Saint-Genès-Champanelle, France) for their help with sampling and laboratory analyses. C. Chantelauze (INRAE, UMR Herbivores, Saint-Genès-Champanelle, France) performed the analyses of the natural abundance of stable carbon isotopes at the French metaboHUB infrastructure (ANR-INBS-0010, UCA, INRAE, UNH, Plateforme d'Exploration du Métabolisme, MetaboHUB Clermont, Clermont-Ferrand, France). We thank G. R. R. Leday and J. Engel (Biometris, Wageningen University & Research, Wageningen, the Netherlands) for their assistance with modeling and conducting of equivalence tests. We also thank G. Cantalapiedra-Hijar and E. E. Jorge Smeding (INRAE, UMR Herbivores, Saint-Genès-Champanelle, France), as well as G. Foucras (IHAP, Université de Toulouse, INRAE, ENVT, Toulouse, France), for their assistance with data analysis and interpretation. Supplemental material for this article is available at https://doi.org/10.5281/zenodo.15838679. During the preparation of this work, the authors used ChatGPT (OpenAI) in order to assist with R coding. After using ChatGPT (OpenAI), the authors reviewed and edited the content as needed and take full responsibility for the content of the publication. Ethical approval for this study was obtained from the French Ministry of Higher Education and Research and the local Auvergne-region Ethical Committee for Animal Experimentation (APAFIS no. 30891-2017062616304407 v14). The authors have not stated any conflicts of interest.

**Nonstandard abbreviations used:** 33MeOx = 67% hexane-defatted SBM plus 33% MeOx-defatted SBM; 67MeOx = 33% hexane-defatted SBM plus 67% MeOx-defatted SBM; 100MeOx = 100% MeOx-defatted SBM; Δ¹³C = C isotopic discrimination between plasma and diet; γGT = γ-glutamyltransferase; ALP = alkaline phosphatase; ALT = alanine aminotransferase; AST = aspartate aminotransferase; c = cis; CCL2 = chemokine C–C motif ligand 2 or monocyte chemoattractant protein 1; CCL3 = chemokine C–C motif ligand 3 or macrophage inflammatory protein 1α; CCL4 = chemokine C–C motif ligand 4 or macrophage inflammatory protein 1β; CXCL8 = chemokine C–X–C motif ligand 8 or IL-8; EM = equivalence margin; EMM = estimated marginal means; FA = fatty acid; HEX = control diet, 100% hexane-defatted SBM; L = linear contrast for diet effect; MeOx = 2-methyloxolane; NEFA = nonesterified FA; PDI = iso-protein truly digestible in the intestine; PMR = partial mixed ration; Q = quadratic contrast for diet effect; SBM = soybean meal; SCD = stearyl-CoA desaturase; *t* = trans; UFL = NEL, expressed in unité fourragère lait (1 UFL = 7.37 MJ of NEL); VEGF-A = vascular endothelial growth factor A.

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ORCIDS

V. Menoury, https://orcid.org/0009-0008-1616-9333

P. Nozière, https://orcid.org/0000-0003-1727-8984

C. Delavaud, https://orcid.org/0000-0002-3256-2230

Y. Farizon, https://orcid.org/0000-0002-7128-8633

A. Ferlay, https://orcid.org/0000-0002-0651-792X