# Replacing hexane with 2-methyloxolane for defatting soybean meal fed to dairy cows: Effects on dairy performance and nitrogen partitioning

Valentin Menoury, Anne Ferlay, Cécile Ginane, Gonzalo Cantalapiedra-Hijar, Sophie Lemosquet, Donato Andueza, Gwenaël Leday, J. Engel, Pierre Noziere

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## ► To cite this version:

Valentin Menoury, Anne Ferlay, Cécile Ginane, Gonzalo Cantalapiedra-Hijar, Sophie Lemosquet, et al. Replacing hexane with 2-methyloxolane for defatting soybean meal fed to dairy cows: Effects on dairy performance and nitrogen partitioning. Journal of Dairy Science, 2025, 108 (6), pp.5904-5928. 10.3168/jds.2024-25860. hal-05109811

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# Replacing hexane with 2-methyloxolane for defatting soybean meal fed to dairy cows: Effects on dairy performance and nitrogen partitioning

V. Menoury,<fnref n="1" /> Ⓓ A. Ferlay,<fnref n="1" /> Ⓓ C. Ginane,<fnref n="1" /> Ⓓ G. Cantalapiedra-Hijar,<fnref n="1" /> Ⓓ S. Lemosquet,<fnref n="2" /> Ⓓ D. Andueza,<fnref n="1" />
G. G. R. Leday,<fnref n="3" /> Ⓓ J. Engel,<fnref n="3" /> and P. Nozière<fnref n="1" />* Ⓓ

## ABSTRACT

2-Methyloxolane (MeOx) is a promising candidate for replacing hexane as a solvent for defatting soybean meal (SBM). However, the use of MeOx requires adjustments of the oil extraction process that may affect the nutritive value of SBM. This study aims to ensure that process modifications due to the use of MeOx do not affect the protein value of SBM for dairy cows. Sixteen primiparous Holstein dairy cows were included in a 4 × 4 Latin square design experiment. The 4 dietary treatments consisted of hexane-defatted SBM, MeOx-defatted SBM, or a combination in various proportions: 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). The diets contained 16% SBM and were iso-CP. Feed, rumen fluid, blood, urine, feces, and milk samples were collected. The traits measured were indicators of ruminal protein degradation (NH₃ concentration and branched-chain volatile fatty acid proportion in the rumen fluid), N metabolism (plasma concentrations of essential AA, 1-methylhistidine, and 3-methylhistidine), N partitioning and N use efficiency (N use efficiency for milk production, N isotopic discrimination between plasma and diet), and milk quality (protein and fat contents, N fractions, hexane and MeOx residues). Statistical analyses used difference and equivalence tests. Replacing hexane-defatted SBM with MeOx-defatted SBM resulted in likely equivalent dairy performance, N partitioning, and N use efficiency for milk production, although some indicators suggested slight rumen and digestive alterations that may have subtly affected whole-body N metabolism. A subset of 6 cows was selected for analysis of solvent residues in milk. Even though hexane-defatted SBM contained

only 11 mg/kg DM of n-hexane residues, 5 out of 6 milk samples from cows fed HEX presented detectable n-hexane residues, whereas only 1 out of 6 milk samples from cows fed 100MeOx presented detectable n-hexane residues. Therefore, n-hexane residues in milk tended to be detected more often when cows were fed hexane-defatted SBM compared with MeOx-defatted SBM. The MeOx-defatted SBM contained 977 mg/kg DM of MeOx residues, but only 2 out of 6 milk samples from cows fed 100MeOx and 0 out of 6 milk samples from cows fed HEX presented detectable MeOx residues. These results raise prospects for using MeOx as a hexane substitute for defatting SBM fed to dairy cows, although further studies are needed to confirm the slight differences observed here in N metabolism.

**Key words:** solvent extraction, nitrogen use efficiency, feeding behavior, equivalence test

## INTRODUCTION

Soybean meal (SBM), containing 438 to 585 g of CP/kg DM (INRA CIRAD AFZ, 2024), is used as a protein supplement in diets fed to dairy cows. In 2015, 36% of all SBM fed to livestock in France was consumed by dairy and dual-purpose cattle, followed by broilers (29%) and laying hens (11%; Groupement d'intérêt scientifique Avenir Élevages, 2024). The SBM is produced by extracting oil from soybean flakes using mechanical pressure or solvent extraction (Newkirk, 2010). Solvent extraction enables a greater oil extraction yield, resulting in a crude fat content of 0.5% to 1.5% of DM in SBM (Demarco and Gibon, 2020; Rapinel et al., 2020).

Extraction-grade hexane, a mixture of n-hexane and its isomers, is the only solvent used for defatting meals at industrial scale (Fine et al., 2013; Demarco and Gibon, 2020). In this paper, we use “hexane” as a simplified name for “extraction-grade hexane.” The use of hexane poses concerns for human health and the environment, as hexane is product of petroleum distillation and n-hexane is neurotoxic, suspected to be reprotoxic, and toxic to

Received November 13, 2024.

Accepted March 11, 2025.

*Corresponding author: pierre.noziere@inrae.fr

The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-25. Nonstandard abbreviations are available in the Notes.

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

<footnote n="2">²PEGASE, INRAE, Institut Agro, 35590 Saint-Gilles, France</footnote>

<footnote n="3">³Biometris, Wageningen University & Research, 6700AA, Wageningen, the Netherlands</footnote>
aquatic life (ANSES, 2014; INRS, 2019; ECHA, 2022). Furthermore, European Food Safety Authority (EFSA) has recently identified the need to re-evaluate the safety of the use of hexane as an extraction solvent for food (EFSA et al., 2024). 2-Methyloxolane (MeOx) makes a potentially good candidate for replacing hexane as it presents several advantages: It has good extractive performance and a safer toxicological profile than n-hexane, can be produced from plant biomass, and has already been approved for use as a food processing aid in the European Union (Rapinel et al., 2020; EFSA Panel on Food Contact Materials, Enzymes and Processing Aids et al., 2022; European Union, 2023).

However, the use of MeOx requires some adjustments of the SBM production process. The MeOx has a boiling point of 71°C (azeotrope with water) to 80°C (dry meOx) versus 66 to 70°C for hexane, which means that the solvent oil extraction step may be performed at a slightly higher temperature (i.e., 65°C for MeOx instead of 55°C for hexane; Rapinel et al., 2020). Following the extraction step, the solvent is separated from SBM by evaporation during the desolventization step. In addition, MeOx also has a higher enthalpy of vaporization than n-hexane (i.e., 364 kJ/kg for MeOx vs. 334 kJ/kg for n-hexane; Rapinel et al., 2020). The desolventization step therefore requires a greater amount of heat to evaporate the solvent (i.e., 80°C for MeOx vs. 70°C for n-hexane; Sicaire et al., 2015). This higher heating when defatting SBM with MeOx may promote Maillard reactions between AA and reducing carbohydrates (Helou et al., 2014). Maillard reaction products can reduce the rumen CP degradability (Manzocchi et al., 2023). This may increase the flow of rumen undegradable protein to the duodenum and result in a greater N intake partitioning toward milk N. Using MeOx instead of hexane as defatting solvent may therefore affect the nutritive value of the resulting SBM.

Despite the desolventization step, hexane-defatted SBM contains up to 250 mg/kg DM of hexane residues at exit from the processing plant (Demarco and Gibon, 2020). These residues partly evaporate during transport and storage (Bauer et al., 2009), but some may still be present in the SBM offered to livestock. Hexane is lipophilic and MeOx is mostly lipophilic (Rapinel et al., 2020), and so any of their residues in SBM may transfer to the lipid phase of the milk. Hexane residues have already been detected in human milk (Pellizzari et al., 1982). However, to our knowledge, the transfer of solvent residues from SBM to animal products has never been studied.

The aim of this study was to evaluate the effects of process modifications required for replacing hexane with MeOx on the value of SBM as a protein-rich feed for dairy cows and on the prevalence of solvent residues in cow milk. We investigated whether replacing hexane-

defatted SBM with MeOx-defatted SBM has effects on (1) the prevalence of milk containing hexane and MeOx residues, (2) dairy performance, and (3) N partitioning by dairy cows. We hypothesized that (1) hexane residues would transfer from SBM into milk more often than MeOx residues, (2) milk yield and milk protein profile would be equivalent between cows fed hexane-defatted SBM or MeOx-defatted SBM, (3) N partitioning would be equivalent between cows fed hexane-defatted SBM and MeOx-defatted SBM.

## MATERIALS AND METHODS

The experiment was led from November 2021 to March 2022 at the French National Research Institute for Agriculture, Food and Environment (INRAE) Herbipôle experimental facility (Theix, France; https://doi.org/10.15454/1.5572318050509348E12), after securing ethical approval from the French Ministry of Higher Education and Research and the local Auvergne-region Ethical Committee for Animal Experimentation (APAFIS no. 30891-2017062616304407 v14).

### Animals, Housing, Experimental Design

The experiment used 18 midlactation primiparous Holstein dairy cows, 4 of which were rumen-cannulated. Cows were housed indoors in a freestall barn and milked twice a day, at 0800 h and 1530 h. Health events and related veterinary treatments were recorded.

A palatability test was conducted during a 3-wk pre-experimental phase to ensure that cows were willing to ingest the MeOx-defatted SBM. The 18 cows were fed either hexane-defatted SBM or MeOx-defatted SBM, and we measured SBM intake and cows' behavioral responses to SBM. Intake of both SBM was equivalent, and feeding behavior was likely equivalent (Supplemental File S1, see Notes).

After this pre-experimental phase, a 12-wk experimental phase was started to evaluate dairy performance and N partitioning. At the start of the experimental phase, 16 of the 18 cows used in the pre-experimental phase were selected and allocated into 4 groups of 4 animals. Allocation was done to best balance the groups according to DIM (71 ± 20.5 d, mean ± SD), cannulation status (i.e., one rumen-cannulated cow per group), DMI (19.0 ± 1.72 kg/d), milk yield (26.9 ± 3.98 kg/d), milk true protein content (28.5 ± 1.84 g/kg), milk fat content (35.7 ± 3.18 g/kg), and BW (627 ± 45.1 kg) measured during the pre-experimental phase. The experiment was designed as a Latin square with 4 groups, 4 periods, and 4 dietary treatments. Each period lasted 21 d: 14 d of adaptation followed by 7 d of measurements. 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 extra cows received 50% hexane-defatted SBM plus 50% MeOx-defatted SBM and were used as substitutes to replace any experimental cows that reached an endpoint and had to be removed from the study. One of these extra 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. Diet transitions between periods lasted 3 d.

## Soybean Meal

A single batch of dehulled soybean flakes was used to produce both hexane-defatted SBM and MeOx-defatted SBM. The production was carried out in 2 runs on the same 10 t/d extraction plant (New Holland Extraction Ltd., Barrow-upon-Humber, UK) equipped with a continuous immersion extractor, a desolventizer-toaster, and a dryer-cooler. The plant was designed for hexane extraction, and was adapted to run on either hexane or MeOx. The hexane-defatted SBM was produced in the summer of 2021. The MeOx-defatted SBM was produced im-

mediately after the plant modification in October 2021, during the very first production run with MeOx. This first run allowed the production of MeOx-defatted SBM containing 100 to 1,000 mg/kg DM solvent residues. The batch with the highest MeOx residues (i.e., 977.4 mg/kg DM; Table 1) was used in this experiment to test a “worst case” scenario for the MeOx residues in SBM. The proximate composition, concentrations of Maillard reaction products and solvent residues, and in situ CP degradability characteristics of hexane-defatted SBM and MeOx-defatted SBM are presented in Table 1.

## Diets

The diet contained (on a DM basis): 44% corn silage, 15% grass silage, 7% hay, 3% straw, 10% ground corn grain, 1% vitamins and minerals, 16% SBM, and 4% production concentrate (Table 2). All ingredients except the SBM and production concentrate were distributed as a partial mixed ration (PMR) containing 84 g CP/kg DM. The proximate composition of the PMR and individual feed ingredients are presented in Supplemental Table S1 (see Notes). The PMR and SBM were distributed at 1000 h and 1600 h, with the SBM being distributed on top of the PMR. Production concentrate was fed at the milking parlor, with 0.5 kg distributed at each milking. Diets were formulated to be iso-CP (163 g of CP/kg DM),

<table><caption>**Table 1.** Proximate composition, concentrations of solvent residues and Maillard reaction products, and in situ CP degradability of hexane-defatted and 2-methyloxolane-defatted soybean meals</caption><thead><tr><th>Item</th><th>Hexane-defatted soybean meal</th><th>MeOx<sup>1</sup>-defatted soybean meal</th><th>SEM difference<sup>2</sup></th><th>P value<sup>3</sup></th></tr></thead><tbody><tr><td>Proximate composition (g/kg DM)</td><td></td><td></td><td></td><td></td></tr><tr><td>OM</td><td>932</td><td>932</td><td>—</td><td>—</td></tr><tr><td>CP</td><td>555</td><td>552</td><td>—</td><td>—</td></tr><tr><td>NDF</td><td>83</td><td>115</td><td>—</td><td>—</td></tr><tr><td>Ether extract</td><td>26</td><td>22</td><td>—</td><td>—</td></tr><tr><td>Solvent residues (mg/kg DM)</td><td></td><td></td><td></td><td></td></tr><tr><td>N-hexane</td><td>11.2</td><td>0.0</td><td>—</td><td>—</td></tr><tr><td>2-methyloxolane</td><td>4.4</td><td>977.4</td><td>—</td><td>—</td></tr><tr><td>Maillard reaction products<sup>4</sup></td><td></td><td></td><td></td><td></td></tr><tr><td>Furosine (mg/kg DM)</td><td>95.5</td><td>1,509.5</td><td>—</td><td>—</td></tr><tr><td>CML<sup>5</sup> (mg/kg DM)</td><td>47.6</td><td>57.7</td><td>—</td><td>—</td></tr><tr><td>Lysine (g/kg DM)</td><td>35.0</td><td>40.7</td><td>—</td><td>—</td></tr><tr><td>In situ CP degradability<sup>6</sup> (%, except if stated otherwise)</td><td></td><td></td><td></td><td></td></tr><tr><td>ED<sub>6</sub><sup>7</sup></td><td>73.4</td><td>73.8</td><td>0.28</td><td>0.38</td></tr><tr><td>Soluble CP fraction</td><td>35.3</td><td>25.3</td><td>0.74</td><td>0.005</td></tr><tr><td>Potentially degradable CP fraction</td><td>64.4</td><td>74.5</td><td>0.99</td><td>0.009</td></tr><tr><td>Degradation rate of the potentially degradable CP fraction (%/h)</td><td>17.1</td><td>13.6</td><td>1.82</td><td>0.20</td></tr><tr><td>Lag time (h)</td><td>3.4</td><td>0.9</td><td>0.29</td><td>0.014</td></tr></tbody></table>

<footnote n="12">¹²-Methyloxolane.</footnote>

<footnote n="2">²Standard error of the mean difference for in situ CP degradability measurement.</footnote>

<footnote n="3">³P-value from paired t-test of difference for in situ CP degradability measurement.</footnote>

<footnote n="4">⁴Maillard reaction products were quantified in SBM sampled at the factory.</footnote>

<footnote n="5">⁵Nε-carboxymethyl-lysine.</footnote>

<footnote n="6">⁶Measured according to INRA (2018) in a separate experiment using 3 rumen-cannulated cows.</footnote>

<footnote n="7">⁷Theoretical CP degradability assuming a passage rate of 6%/h.</footnote>
<table><caption>**Table 2.** Ingredients, proximate composition, and nutritive value of the diets ingested by dairy cows during the experimental phase</caption><thead><tr><th rowspan="2">Item</th><th colspan="4">Diet<fnref n="1" /></th></tr><tr><th>HEX</th><th>33MeOx</th><th>67MeOx</th><th>100MeOx</th></tr></thead><tbody><tr><td colspan="5">Diet ingredients (% of DM)</td></tr><tr><td>Corn silage</td><td>44.2</td><td>44.5</td><td>44.5</td><td>44.6</td></tr><tr><td>Grass silage</td><td>14.7</td><td>14.8</td><td>14.8</td><td>14.9</td></tr><tr><td>Hay</td><td>6.9</td><td>6.9</td><td>6.9</td><td>6.9</td></tr><tr><td>Straw</td><td>2.9</td><td>3.0</td><td>3.0</td><td>3.0</td></tr><tr><td>Ground corn grain</td><td>9.8</td><td>9.9</td><td>9.9</td><td>9.9</td></tr><tr><td>Vitamins and minerals<fnref n="2" /></td><td>1.2</td><td>0.9</td><td>1.0</td><td>0.9</td></tr><tr><td>Hexane-defatted soybean meal</td><td>16.1</td><td>10.6</td><td>5.3</td><td>0.0</td></tr><tr><td>2-methyloxolane-defatted soybean meal</td><td>0.0</td><td>5.1</td><td>10.3</td><td>15.6</td></tr><tr><td>Production concentrate<fnref n="3" /></td><td>4.2</td><td>4.3</td><td>4.3</td><td>4.2</td></tr><tr><td colspan="5">Proximate composition (g/kg DM)</td></tr><tr><td>OM</td><td>933</td><td>932</td><td>933</td><td>932</td></tr><tr><td>CP</td><td>166</td><td>163</td><td>162</td><td>162</td></tr><tr><td>NDF</td><td>418</td><td>418</td><td>416</td><td>417</td></tr><tr><td>Starch</td><td>204</td><td>202</td><td>204</td><td>202</td></tr><tr><td>Ether extract</td><td>27</td><td>27</td><td>27</td><td>27</td></tr><tr><td colspan="5">Nutritive value<fnref n="4" /> (INRA, 2018)</td></tr><tr><td>PDI<fnref n="2" /> (g/kg DM)</td><td>93.7</td><td>92.8</td><td>92.3</td><td>92.3</td></tr><tr><td>PDIA<fnref n="5" /> (g/kg DM)</td><td>44.8</td><td>43.9</td><td>43.5</td><td>43.6</td></tr><tr><td>RPB<fnref n="7" /> (g/kg DM)</td><td>21.3</td><td>20.1</td><td>19.4</td><td>19.2</td></tr><tr><td>Net energy (UFL)<fnref n="8" />/kg DM</td><td>0.92</td><td>0.92</td><td>0.91</td><td>0.91</td></tr><tr><td>PDI:net energy (g PDI/UFL)</td><td>102</td><td>101</td><td>101</td><td>102</td></tr></tbody></table>

iso-protein truly digestible in the intestine (PDI; INRA, 2018; 92.8 g of PDI/kg DM) and iso-net energy (0.91 unité fourragère lait [UFL]/kg DM; 1 UFL = 7.37 MJ of NEL; INRA, 2018). Individual feed refusals were removed and weighed every day before the morning meal. Feed was distributed in individual feeding troughs accessible to single cows identified by their wireless collar. Feed was offered ad libitum, with a target of 5% of feed refusals on a DM basis. The amount of feed distributed was adjusted individually twice a week. Water and salt were available ad libitum.

## Sampling and Measurements

### SBM Sampling Before the Start of the Experiment.
After the 2 production runs, hexane-defatted SBM and

MeOx-defatted SBM were sampled at the factory for determination of furosine, Nε-carboxymethyl-lysine (CML), and total lysine concentrations. Before the start of the experiment, the 2 SBM were also sampled to measure in situ CP degradability. This measurement was performed in a separate experiment conducted in April 2022 at the INRAE Herbipôle experimental facility (Theix, France; https://doi.org/10.15454/1.5572318050509348E12; ethical approval APAFIS no. 30891-2017062616304407 v14). The experiment was conducted with 3 dry Holstein cows, each equipped with a rumen cannula according to the experimental and laboratory procedures described in INRA (2018). The cows were housed indoors in individual tiestalls and fed 10 kg DM/d of a diet containing, on a DM basis, 70% hay, 25% production concentrate, 5% SBM, and 200 g/d of

<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">²Vitamins and minerals mix containing 3.5% phosphorus, 20.0% calcium, 4.5% magnesium, 3.5% sodium, 400,000 UI/kg vitamin A, 12,000 UI/kg vitamin D₃, 1,800 UI/kg vitamin E, 6,000 mg/kg zinc oxide, 3,500 mg/kg manganese(II) oxide, 1,300 mg/kg copper(II) sulfate pentahydrate, 100 mg/kg calcium iodate anhydrous, 40 mg/kg cobalt(II) carbonate, 30 mg/kg sodium selenite.</footnote>

<footnote n="3">³Concentrate containing, on a DM basis, 30% sugar beet pulp, 22.7% wheat, 20% barley, 15% rapeseed meal, 7.9% soybean meal, 1.5% sugarcane molasses, 1% dicalcium phosphate, 0.5% magnesium oxide, 0.5% NaCl, 0.15% flavor mix, 9,975 UI/kg vitamin A, 1,395 UI/kg vitamin D₃, 15 UI/kg vitamin E, 3.3 UI/kg vitamin B₁, 90 mg/kg zinc oxide, 60 mg/kg manganese(II) oxide, 15 mg/kg copper(II) sulfate pentahydrate, 0.75 mg/kg calcium iodate anhydrous, 0.26 mg/kg cobalt(II) carbonate, 0.25 mg/kg sodium selenite.</footnote>

<footnote n="4">⁴Calculated for a feeding level of 3.1 kg DM/100 kg BW.</footnote>

<footnote n="5">⁵PDI = protein truly digestible in the intestine.</footnote>

<footnote n="6">⁶PDIA = protein truly digestible in the intestine from feed origin.</footnote>

<footnote n="7">⁷RPB = rumen protein balance.</footnote>

<footnote n="8">⁸UFL = NEL, expressed in unité fourragère lait; 1 UFL = 7.37 MJ of NEL.</footnote>
minerals and vitamins. The diet was distributed at 0815 and 1615 h.

**Feed Intake and Biochemical Composition.** From d 15 to 21, daily individual DMI was calculated from the difference between feed distributed and feed refusals. The DM content (103°C, 24 h in a ventilated oven) was measured on d 15 to 21 for offered PMR, on d 17, 19, and 21 for corn and grass silages, on d 17 for other offered ingredients, and on d 16 to 21 for individual feed refusals above 2 kg. These samples collected for DM analysis were also pooled per period before biochemical analyses. The pooling of feed samples per period was done by sampling 200 g on a wet matter basis of feed materials from each sampling day. The pooling of feed refusals per period was done by sampling 5% on a wet matter basis of feed refusals from each sampling day.

**Animal Characteristics.** The BW of each cow was measured twice a day at exit from the milking parlor. The BCS was evaluated by 2 graders before the experimental phase and 3 d after the end of each experimental period, on a scale from 0 (i.e., lean) to 5 (i.e., fat) with 0.25 increments.

**Rumen Fluid.** Rumen fluid was sampled via oral stomach tubing at 5 h after the morning meal on d 18 and 21 of each experimental period. Rumen fluid pH was measured before filtration onto a 250-µm nylon filter. For $NH_3$ assay, the filtrate was diluted (1:0.1, vol:vol) with 5% phosphoric acid. For VFA assay, the filtrate was diluted (0.8:0.5, vol:vol) in a deproteinizing solution (0.4% wt/vol crotonic acid and 2% wt/vol metaphosphoric acid in a 0.5 N HCl solution). For protozoa counts, the filtrate was and diluted (1:1, vol:vol) in a saline solution (0.6 g of methyl-green, 8 g of NaCl, 100 mL of formaldehyde at 35%, 900 mL of $H_2O$) according to Ogimoto and Imai (1981). Before VFA assay, samples from the 2 d of measurement were pooled per cow. Rumen fluid was also sampled through the cannula of rumen-cannulated cows at 0, 1, 2.5, 5, and 8 h after the morning meal on d 18 and 20 for pH and $NH_3$ assays. All samples for $NH_3$ and VFA assays were stored at -20°C until analyses, and samples for protozoa counts were stored in the dark at room temperature.

**Plasma.** Blood was collected at the coccygeal vessel into 2 lithium-heparin-coated tubes (9-mL tubes, ref. 455084, Greiner, Austria) on d 20. After collection, the tubes were gently inverted several times and stored on ice until centrifugation (3,500 × g, 15 min, 4°C). Waiting time between sampling and centrifugation was ~30 min. Plasma was stored at -80°C until analyses.

**Milk Yield and Composition.** 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 true protein, fat, lactose, and urea contents and SCC. On d20, the morning milk was collected and stored at 4°C for determination of milk N fractions: i.e., total N, NPN, and soluble N concentrations. Concentrations of solvent residues (i.e., 2-methylloxolane and n-hexane and its isomers 2-methylpentane and 3-methylpentane) were measured on a pooled sample from morning and afternoon milk collected on d 20, on a subset of 6 cows: 3 cows receiving HEX in period 1 and 100MeOx in period 4, and 3 cows receiving 100MeOx in period 1 and HEX in period 4. Milk was stored at -20°C until solvent analysis.

**Urine and Feces.** Spot samples of urine and feces were collected before the morning meal and before milking for most cows, to estimate daily N excretions and apparent digestibility. Urine was sampled on d 18 and 20 and feces were sampled on d 19 and 21. Urine was centrifuged (3,500 × g, 10 min at room temperature) and diluted to 1:10 using ultrapure water. Urine and feces samples were quickly put into storage at -20°C until analyses.

## Laboratory Analyses

**Feed, Refusals, and Feces.** Wet feed (i.e., corn and grass silages, offered PMR), refusals, and feces samples were dried in a ventilated oven at 60°C for 72 h (UL 80, Memmert, Germany). Feed (i.e., oven-dried samples, as well as offered SBM, ground corn grain, hay, and straw) and feces were ground using a hammer mill with a 1-mm screen (BJL8500-2, Boisson, France) before analyses. Ash concentrations of feed and feces samples were measured after ashing at 550°C for 6 h in a muffle furnace (Heraeus, Germany) according to European Union (2022b). Feed N concentration was measured using the Dumas method (Rapid N Cube, Elementar, Germany) according to ISO (2008). Feed NDF concentration was measured according to Van Soest et al. (1991) using the fiber bag technique for forages (Fibersac, Humeau; results expressed on a with-ash basis) and glass crucibles for concentrates (Fibertec, Foss; results expressed on an ash-free basis) on samples treated with heat-stable $\alpha$-amylase (Ankom, or Novozymes) and pronase from *Streptomyces griseus* (Roche). Feed ether extract concentration was measured after HCl hydrolysis (Hydrotherm, Gerhardt) using petroleum ether with a Randall extractor (Extractor E816-H, Buchi) according to procedure B described in European Union, 2022b. Pepsin-cellulase digestibility was measured via a sequence of pepsin-acid (Pepsin 2000 FIP [Fédération International Pharmaceutique] U/g, Merck), acid, and cellulase (cellulase Onozuka R-10) hydrolyzes steps according to Aufrère and Michalet-Doreau (1988). The CML, furosine, and total lysine concentrations were measured using HPLC (Accela HPLC system, Thermo
Electron) coupled to triple quadrupole ion trap tandem MS (TSQ Quantum Ultra, Thermo Electron) after acid hydrolysis for furosine (Nogueira Silva Lima et al., 2023) and after reduction and acid hydrolysis for CML and total lysine (Niquet-Léridon and Tessier, 2011). N-hexane and its isomers (2-methylpentane and 3-methylpentane) and MeOx residues in SBM were quantified according to the method described in ISO (1987) modified using a mix of n-heptane and tetrahydrofuran as internal standard. Briefly, 0.5 g of SBM was introduced into a sealed glass vial with 2 mL of water and 20 µL of internal standard. Then, the sample was ultrasonicated for 30 min, vortexed, and analyzed using headspace-GC (MPS2, Gerstel, and 6890A, Agilent) coupled to MS (5973N, Agilent). The limits of quantification of solvent residues in SBM were 10 mg/kg. Near-infrared spectrometry (NIRS) was used on feces samples to estimate their N concentration as well as the apparent digestibility of DM (DMd) in the diet. The characteristics of the model used to predict diet DMd are the following: n = 380, mean = 0.719 g/g, minimum = 0.600 g/g, maximum = 0.770 g/g, standard error of prediction (SEP) = 0.0167 g/g, and coefficient of determination in external validation (R²V) = 0.80. This model was developed using data from experiments performed in Europe, including INRAE's Herbipôle experimental facility (Theix, France), and including diets similar to the ones of the present study. The characteristics of the model used to predict feces N concentration are the following: n = 764, mean = 2.491 g N/kg DM, minimum = 1.309 g N/kg DM, maximum = 4.367 g N/kg DM, standard error of cross validation = 0.0689 g N/kg DM, and R²V = 0.98. This model was developed using data from experiments performed in Europe, including INRAE's Herbipôle experimental facility of Theix (France), and including diets similar to the ones of the present study. In addition, the NIRS predictions for feces N concentration was validated by measuring N concentration using the Dumas method (Rapid N Cube, Elementar; ISO, 2008) on a subset of 29 feces samples chosen according to the method proposed by Shenk and Westerhaus (1991). The predicted and measured values were compared, resulting in a R²V of 0.96, a SEP of 0.058 g of N/kg DM, and a bias of 0.002 g of N/kg DM. Feed samples were further ground using an ultra-centrifugal abrasion mill combined with a 0.5-mm screen (Cyclotec CT193, Foss, Denmark). The δ¹⁵N enrichment in feed ground to 0.5 mm was measured with an elemental analyzer (EA Vario Isotope Cube, Elementar) coupled with an isotope ratio MS system (IRMS isoprime visION, Elementar). For starch measurement, samples of feed ground to 0.5 mm were first hydrolyzed with amyloglucosidase from Aspergillus niger (EC 3.2.1.3, Sigma-Aldrich) according to NF V18-121 (AFNOR, 1997). Then starch was measured

using photometry (λ = 340 nm, Lambda25, PerkinElmer) and a glucose kit (D-Glucose, R-Biopharm) based on the hexokinase method (Schmidt, 1961).

**Rumen Fluid.** Ammonia concentration in the rumen fluid was determined using Berthelot's reaction and spectrophotometry (λ = 630 nm, Infinite 200, Tecan) according to Weatherburn (1967). Volatile fatty acids were quantified via GC (Clarus 580, PerkinElmer) according to Jouany (1982). Protozoa in rumen fluid were counted in a Neubauer chamber under a microscope (Ogimoto and Imai, 1981) and each sample was evaluated in duplicate. We differentiated 4 categories of protozoa: small entodinomorphs (i.e., <100 µm), large entodinomorphs (i.e., >100 µm), isotrichs, and dasytrichs.

**Milk, Plasma, and Urine.** Milk true protein, fat, lactose, and urea contents were determined using infrared spectrometry (Milkoscan, Foss) at Agrolabs (Aurillac). Milk somatic cells were counted using epifluorescence. Total N and NPN concentrations were measured on morning milk sampled on d 20 of each period following ISO and IDF (2014) for total N and ISO (2016) for NPN. Milk soluble N concentration was measured after acid precipitation of caseins, filtration, and measurement of N content of the filtrate (ISO and IDF, 2004) using the Kjeldahl method (ISO and IDF, 2014). N-hexane and its isomers (2-methylpentane and 3-methylpentane) and MeOx residues in milk were quantified by introducing 1 g of milk with 5 mL of dimethyl sulfoxide (10% vol/ vol) and 10 µL of internal standard (mix of n-heptane and tetrahydrofuran) in a sealed glass vial. The sample was then analyzed using the same technique as above for SBM. The limits of quantification of solvent residues in milk were 10 ng/g. A Konelab Arena 20 XT analyzer (Thermo Fisher Scientific) was used to measure plasma and urine urea concentrations (λ = 340 nm, urea kit 981818 based on urease/glutamate dehydrogenase reactions; Roch-Ramel, 1967; Thermo Fisher Scientific) and urine creatinine (λ = 510 nm, creatinine kit 981811 based on Jaffé (1886); Thermo Fisher Scientific). Plasma AA, 1-methylhistidine (N-π-methylhistidine, Chemical Abstract Service [CAS] No. 368-16-1), and 3-methylhistidine (N-τ-methylhistidine, CAS No. 332-80-9) were quantified by ultra-performance liquid chromatography-MS (Waters Acquity UPLC System, Waters Corporation) as described by Haque et al. (2012). The δ¹⁵N enrichment in plasma was measured using the same method as described above for feed.

## Calculations

The in situ CP degradability was calculated with a lag time using the model of Dhanoa (1988). The degradation data were fitted to the model using PROC NLIN
procedure from SAS software version 9 (SAS Institute Inc.). It allowed to estimate the soluble CP fraction, the potentially degradable CP fraction, the degradation rate of the potentially degradable CP fraction, and the lag time. The theoretical CP degradability assuming a passage rate of 6%/h was calculated using the equation of Denham et al. (1989). Nitrogen intake was calculated from the amounts of offered and refused diet components and their N concentrations. The PMR intake was calculated as the difference between offered PMR and PMR refusals. The PMR refusals were calculated from N concentration in offered PMR, offered SBM, and feed refusals:

$$ \text{PMR refusals}_{(\text{kg DM/d})} = \text{Refusals}_{(\text{kg DM/d})} \times \frac{N_{\text{SBM(g/kg DM)}} - N_{\text{refusals(g/kg DM)}}}{N_{\text{SBM(g/kg DM)}} - N_{\text{PMR(g/kg DM)}}} $$

The SBM intake was then calculated as the difference between offered SBM and SBM refusals, taking SBM refusals as the difference between total feed refusals and PMR refusals. As proposed by Hall (2023), the ECM yield was calculated from the milk energy equation proposed by NASEM (2021) and considering a standardized milk containing 40 g of fat/kg, 31 g of true protein/kg, 48 g of lactose/kg, and 742.6 kcal/kg:

$$ \text{ECM yield}_{(\text{kg/d})} = \text{MY}_{(\text{kg/d})} \times \left[ \begin{array}{l} 9.29 \times \text{ Milk fat}_{(\text{g/kg})} + 5.85 \\ \times \text{ Milk true protein}_{(\text{g/kg})} \\ + 3.95 \times \text{ Milk lactose}_{(\text{g/kg})} \end{array} \right] \div 742.6 $$

Milk N casein concentration was calculated as the difference between milk total N concentration and milk soluble N concentration. Milk soluble protein N concentration was calculated as the difference between milk soluble N and milk NPN. Daily N secretion in milk was calculated assuming a 15.7% N concentration in milk true protein and a 5% NPN concentration in total N (Cerbulis and Farrell, 1975). The assumed 5% milk NPN concentration was preferred to measured NPN concentration, as NPN was only measured in morning milk samples:

$$ \text{Milk N}_{(\text{g/d})} = \text{Milk yield}_{(\text{kg/d})} \times \frac{\text{Milk true protein}_{(\text{g/kg})}}{6.38} \times \frac{1}{0.95} $$

Urine N excretion was estimated from urine ureic N and creatinine concentrations (Supplemental File S2, see Notes):

$$ N \text{ urine}_{(\text{g/d})} = \left[ \begin{array}{l} 0.862 + 0.539 \times \frac{\text{Urine ureic N}_{(\text{g/L})}}{\text{Urine creatinine}_{(\text{mmol/L})}} \\ \times \text{BW}_{(\text{kg})}^{0.75} \end{array} \right] $$

Fecal N excretion was estimated from measured DMI and estimated diet DMd and feces N concentration:

$$ N \text{ feces}_{(\text{g/d})} = \text{DMI}_{(\text{kg DM/d})} \times \left[ 1 - \text{DMd}_{(\text{g/g})} \right] \times N_{\text{feces(g/kg DM)}} $$

Nitrogen use efficiency for milk production (g/g) was calculated as N secretion in milk divided by N intake. Nitrogen balance (g/d) was calculated as the difference between N intake and N secretion or excretion in milk, urine, and feces. The N isotopic discrimination between plasma and diet, $\Delta^{15}\text{N}$ (‰), was calculated as the difference between plasma and diet $^{15}\text{N}$ abundance. Net energy balance and efficiency of PDI use were calculated according to INRA (2018).

## 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 least squares means) ± SEM, unless stated otherwise.

**Data Exclusion.** One rumen-cannulated cow mediated twice 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. The data from these cows was removed from the dataset for all response variables except the rumen kinetics parameters, which were only measured on rumen-cannulated cows. 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. Eleven out of 453 cow × period × day datapoints were excluded for BW, which is sensitive to measurement errors when a cow walks too fast across the weighing scale. Seven out
of 144 cow × period × day datapoints were excluded for milk fat content, which is sensitive to sampling errors due to heterogeneity in milk fat content. One datapoint that was excluded for milk fat content was also excluded for milk true protein and lactose content. These concomitant abnormalities were thought to be caused by a sampling issue. For VFA analyses in rumen fluid sampled through oral stomach tubing, 2 cow × period datapoints were excluded from the dataset due to an abnormal VFA profile (i.e., 24 to 26 mol/100 mol total VFA of acetate and 30 to 31 mol/100 mol total VFA of butyrate) and abnormally low total VFA concentration (i.e., 54 to 57 mM). These abnormalities were thought to be caused by laboratory assay issues.

**In Situ CP Degradability.** The theoretical CP degradability assuming a passage rate of 6%/h, the soluble CP fraction, the potentially degradable CP fraction, the degradation rate of the potentially degradable CP fraction, and the lag time differences between hexane-defatted SBM and MeOx-defatted SBM were analyzed using paired t-tests (t.test function in the stats 4.4.1 package).

**Solvent Residues in Milk.** Solvent residues in milk were analyzed as binomial variables, the 2 outputs being “0” for “absence” and “1” for “presence” of solvent residues in the milk. Two χ² tests of homogeneity were run (chisq.test function in the stats 4.4.1 package).

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

$$Y_{dpc} = \mu + \text{diet}_d + \text{period}_p + \text{cow}_c + \varepsilon_{dpc},$$

where $Y_{dpc}$ is the response for the cth cow fed the dth diet during the pth period, $\mu$ is the overall mean response, $\text{diet}_d$ is the fixed effect of the dth diet, $\text{period}_p$ is the fixed effect of the pth period, $\text{cow}_c$ is the random effect of the cth cow, and $\varepsilon_{dpc}$ is the residual error term. For milk SCC and protozoa counts, a generalized linear mixed model (glmmTMB function in the glmmTMB 1.1.9 package) was used with a negative binomial error distribution, a logc link function, a REML fit, and zero-inflation when necessary (i.e., for dasytricha count). For rumen kinetics analyses, a linear mixed model (lme function in the nlme 3.1-164 package) was fitted using REML and continuous autoregressive covariance structure to account for repeated measures:

$$Y_{dpsc} = \mu + \text{diet}_d + \text{period}_p + \text{sampling time}_s + \text{diet} \times \text{sampling time}_{ds} + \text{cow}_c + \varepsilon_{dpsc},$$

where $Y_{dpsc}$ 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, $\text{diet}_d$ is the fixed effect of the dth diet, $\text{period}_p$ is the fixed effect of the pth period, sampling time is the fixed effect of the sth sampling time, diet is the fixed interaction between the dth diet and the sth sampling time, $\text{cow}_c$ is the random effect of the cth cow, and $\varepsilon_{dpsc}$ 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 (function Anova from car 3.1-2 package), we investigated pairwise differences between experimental diets and HEX (functions emmeans in emmeans 1.10.4 package, function pairs in graphics 4.4.1 package, and function confint from stats 4.4.1 package). We adjusted the $\alpha$ 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 $\alpha_1$ risk level, (H₂) 67MeOx versus HEX tested at an $\alpha_2$ risk level, and (H₃) 33MeOx versus HEX tested at an $\alpha_3$ risk level (Figure 1A). These hypotheses were chosen in this order on the grounds that, assuming a linear response, $H_1$ is expected to be easier to prove than $H_3$. Comparisons were considered significant at $P \le \alpha_i$ and tendencies at $\alpha_i < P \le 2 \times \alpha_i$. 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 $P \le 0.05$ and tendencies at $0.05 < P \le 0.10$.

**Post Hoc Analyses for Equivalence Tests.** Post hoc analyses were performed to test for equivalence among the experimental diets (100MeOx, 67MeOx, and 33MeOx) and the control diet (HEX). The equivalence interval was defined as $\pm 5\%$ 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 $100 - (2 \times \alpha_i)\%$ 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, we used a similar fallback multiple testing procedure but with reversed a priori ordering of hypotheses: (H₁) 33MeOx versus HEX tested at an $\alpha_1$ risk level, (H₂) 67MeOx versus HEX tested at an $\alpha_2$ risk level, and (H₃) 100MeOx versus HEX tested at an $\alpha_3$ risk level (Figure 1B). These hypotheses were chosen in this order on the grounds that, assuming a linear response, $H_1$ is expected to be easier to prove than $H_3$. A simplified EFSA terminology was used to draw conclusions about equivalence (EFSA Panel on GMO, 2010; Figure 2).

## RESULTS

### Difference Tests

**SBM Characteristics.** The in situ CP degradation measurements showed that the soluble CP fraction and
A

<figure><img src="image_3.png" /></figure>

B

<figure><img src="image_4.png" /></figure>

**Figure 1.** Fallback multiple testing procedure for post hoc analysis of difference tests (A) and equivalence tests (B) during the experimental phase (Tamhane and Gou, 2018). $H_i$ = i-th hypothesis; $H_{i-1}$: i - 1th hypothesis; $\alpha_i$ = risk level for the i-th hypothesis; $\alpha_{i-1}$ = risk level for the i-1th hypothesis; $w_i$ = weight on the i-th hypothesis; 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.

the lag time were lower with MeOx-defatted SBM compared with hexane-defatted SBM (P = 0.005 and P = 0.014, respectively; Table 1), whereas the potentially degradable CP fraction was higher (P = 0.009). We did not find evidence that the degradation rate of the potentially degradable CP fraction (P = 0.20) and the theoretical CP degradability assuming a passage rate of 6%/h (P = 0.38) differed between MeOx-defatted SBM and hexane-defatted SBM. The concentrations of furosine and CML were numerically greater in MeOx-defatted SBM than in hexane-defatted SBM. The MeOx-defatted SBM contained 977.4 mg/kg DM of MeOx residues, whereas the hexane-defatted SBM

contained 11.2 mg/kg DM of n-hexane and 4.4 mg/kg DM of MeOx residues.

**Diet Composition and Feed Intake.** Soybean meal intake was negatively linearly related to the proportion of MeOx-defatted SBM in the diet (P = 0.034, Table 3). This was in line with the total diet CP concentration, which was numerically reduced by 3 to 4 g/kg DM in experimental diets compared with HEX (Table 2). The PDI-to-net energy ratio was also negatively linearly related to the proportion of MeOx-defatted SBM in the diet (P = 0.010, Table 3), and was 1 g of PDI/UFL lower in all experimental diets compared with HEX (100MeOx vs. HEX: P = 0.007, $\alpha_i$ = 0.017; 67MeOx vs. HEX:
<figure><img src="image_5.png" /></figure>

<table><thead><tr><th>Equivalence category</th><th>EFSA terminology</th><th>Simplified terminology</th></tr></thead><tbody><tr><td>(i)</td><td>Equivalence</td><td>Equivalent</td></tr><tr><td>(ii)</td><td>Equivalence is more likely than absence of equivalence</td><td>Likely equivalent</td></tr><tr><td>(iii)</td><td>Equivalence is less likely than absence of equivalence</td><td>Unlikely equivalent</td></tr><tr><td>(iv)</td><td>Non-equivalence</td><td>Not equivalent</td></tr></tbody></table>

**Figure 2.** Simplification of the EFSA terminology used for concluding on equivalence tests (adapted from EFSA Panel on GMO, 2010). 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.

$P = 0.011$, $α₂ = 0.033$; 33MeOx vs. HEX: $P = 0.011$, $α₃ = 0.05$).

**Solvents in Milk.** Milk residues of n-hexane, 2-methylpentane, 3-methylpentane, and MeOx were not quantified above the limit of quantification of 10 ng/g, but n-hexane and MeOx were detected at concentrations below 10 ng/g. Residues of n-hexane were detected in 5 out of 6 milk samples from cows fed HEX and in 1 out of 6 milk samples from cows fed 100MeOx. Thus, n-hexane residues tended to be detected more often in milk from cows fed HEX than in milk from cows fed

100MeOx ($P = 0.083$, data not shown). Residues of MeOx were detected in 2 out of 6 milk samples from cows fed 100MeOx, but were not detected in any of the 6 milk samples from cows fed HEX. However, we did not find evidence that the presence of MeOx residues in milk differed between cows fed 100MeOx and cows fed HEX ($P = 0.44$, data not shown).

**Dairy Performance and Milk Composition.** We did not find evidence of differences between diets in dairy performance and milk composition ($P > 0.05$), except for milk NPN-to-total N ratio, which tended to be positively
<table><caption>**Table 3.** Dry matter intake, BW, BCS, milk yield and composition, and energy and protein intake in cows fed hexane-defatted or 2-methyloxolane-defatted soybean meal</caption><thead><tr><th rowspan="2">Item</th><th colspan="4">Diet<fnref n="1" /></th><th rowspan="2">SEM<fnref n="2" /></th><th rowspan="2">P diet</th><th colspan="2">P contrasts</th></tr><tr><th>HEX</th><th>33MeOx</th><th>67MeOx</th><th>100MeOx</th><th>L<fnref n="3" /></th><th>Q<fnref n="4" /></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>DMI partial mixed ration (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>DMI soybean meal (kg DM/d)</td><td>3.3</td><td>3.2</td><td>3.2</td><td>3.2</td><td>0.09</td><td>0.063</td><td>0.034</td><td>0.12</td></tr><tr><td>BW (kg)</td><td>654</td><td>657</td><td>654</td><td>654</td><td>11.6</td><td>0.86</td><td>0.78</td><td>0.69</td></tr><tr><td>BCS (point)</td><td>2.20</td><td>2.22</td><td>2.19</td><td>2.17</td><td>0.151</td><td>0.94</td><td>0.67</td><td>0.76</td></tr><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>ECM<fnref n="3" /> yield (kg/d)</td><td>25.5</td><td>24.8</td><td>25.4</td><td>24.9</td><td>0.90</td><td>0.39</td><td>0.43</td><td>0.78</td></tr><tr><td>Milk true protein content (g/kg)</td><td>30.5</td><td>30.5</td><td>30.6</td><td>30.7</td><td>0.57</td><td>0.75</td><td>0.28</td><td>0.93</td></tr><tr><td>Milk true protein yield (g/d)</td><td>793</td><td>776</td><td>782</td><td>779</td><td>27.5</td><td>0.66</td><td>0.46</td><td>0.52</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 lactose content (g/kg)</td><td>50.4</td><td>50.5</td><td>50.5</td><td>50.5</td><td>0.45</td><td>0.85</td><td>0.41</td><td>0.80</td></tr><tr><td>Milk lactose yield (g/d)</td><td>1,316</td><td>1,286</td><td>1,296</td><td>1,286</td><td>47.5</td><td>0.51</td><td>0.27</td><td>0.55</td></tr><tr><td>Milk casein N:total N (g/100 g)</td><td>75.6</td><td>75.3</td><td>74.6</td><td>75.6</td><td>0.89</td><td>0.81</td><td>0.90</td><td>0.44</td></tr><tr><td>Milk soluble protein N:total N (g/100 g)</td><td>19.8</td><td>19.7</td><td>20.2</td><td>19.2</td><td>0.85</td><td>0.89</td><td>0.79</td><td>0.62</td></tr><tr><td>Milk NPN:total N (g/100 g)</td><td>4.65</td><td>5.08</td><td>5.21</td><td>5.11</td><td>0.213</td><td>0.17</td><td>0.082</td><td>0.17</td></tr><tr><td>Milk urea (mg/L)</td><td>332</td><td>325</td><td>335</td><td>342</td><td>12.5</td><td>0.64</td><td>0.35</td><td>0.44</td></tr><tr><td>Milk SCC (log<sub>e</sub> cells/mL)</td><td>11.0</td><td>10.9</td><td>10.9</td><td>11.2</td><td>0.31</td><td>0.73</td><td>0.48</td><td>0.38</td></tr><tr><td>Net energy intake (UFL<sup>6</sup>/d)</td><td>19.0</td><td>18.6</td><td>18.5</td><td>18.6</td><td>0.45</td><td>0.51</td><td>0.21</td><td>0.42</td></tr><tr><td>Net energy balance (UFL/d)</td><td>1.24</td><td>1.23</td><td>0.86</td><td>1.15</td><td>0.336</td><td>0.72</td><td>0.59</td><td>0.57</td></tr><tr><td>PDI<sup>7</sup> intake (g PDI/d)</td><td>1,943</td><td>1,890</td><td>1,879</td><td>1,882</td><td>56.2</td><td>0.31</td><td>0.12</td><td>0.32</td></tr><tr><td>PDI efficiency (g/100 g)</td><td>66</td><td>67</td><td>67</td><td>67</td><td>0.6</td><td>0.39</td><td>0.089</td><td>0.91</td></tr><tr><td>PDI:net energy (g PDI/UFL)</td><td>102</td><td>101<sup>a</sup></td><td>101<sup>a</sup></td><td>101<sup>a</sup></td><td>0.63</td><td>0.018</td><td>0.010</td><td>0.091</td></tr></tbody></table>

linearly related to the proportion of MeOx-defatted SBM in the diet (P = 0.082).

**Nitrogen Partitioning.** Nitrogen intake tended to be negatively linearly related to MeOx-defatted SBM pro-

portion (P = 0.061) and fecal N excretion was negatively linearly related to MeOx-defatted SBM proportion (P = 0.038, Table 4). Diet DMd estimated by NIRS on fecal samples was also negatively linearly related to MeOx-

<table><caption>**Table 4.** Partition of N in cows fed hexane-defatted or 2-methyloxolane-defatted soybean meal</caption><thead><tr><th rowspan="2">Item</th><th colspan="4">Diet<fnref n="1" /></th><th rowspan="2">SEM<fnref n="2" /></th><th rowspan="2">P diet</th><th colspan="2">P contrasts</th></tr><tr><th>HEX</th><th>33MeOx</th><th>67MeOx</th><th>100MeOx</th><th>L<fnref n="3" /></th><th>Q<fnref n="4" /></th></tr></thead><tbody><tr><td>N intake (g/d)</td><td>547</td><td>532</td><td>528</td><td>529</td><td>14.3</td><td>0.18</td><td>0.061</td><td>0.27</td></tr><tr><td>N milk (g/d)</td><td>131</td><td>128</td><td>128</td><td>129</td><td>4.5</td><td>0.67</td><td>0.46</td><td>0.52</td></tr><tr><td>Diet DM digestibility (g/100 g)</td><td>70.9</td><td>70.2</td><td>70.0</td><td>70.1</td><td>0.28</td><td>0.059</td><td>0.023</td><td>0.14</td></tr><tr><td>N feces (g/d)</td><td>178</td><td>175</td><td>170</td><td>172</td><td>5.5</td><td>0.11</td><td>0.038</td><td>0.30</td></tr><tr><td>N urine (g/d)</td><td>178</td><td>175</td><td>178</td><td>176</td><td>2.9</td><td>0.49</td><td>0.62</td><td>0.76</td></tr><tr><td>N balance (g/d)</td><td>60.1</td><td>55.0</td><td>51.8</td><td>53.0</td><td>7.00</td><td>0.59</td><td>0.24</td><td>0.50</td></tr><tr><td>N milk:N intake (g/100 g)</td><td>23.9</td><td>24.0</td><td>24.4</td><td>24.3</td><td>0.55</td><td>0.71</td><td>0.29</td><td>0.85</td></tr><tr><td>N feces:N intake (g/100 g)</td><td>32.5</td><td>32.9</td><td>32.1</td><td>32.5</td><td>0.42</td><td>0.43</td><td>0.61</td><td>0.92</td></tr><tr><td>N urine:N intake (g/100 g)</td><td>33.0</td><td>33.1</td><td>34.0</td><td>33.5</td><td>1.10</td><td>0.36</td><td>0.23</td><td>0.42</td></tr></tbody></table>

<footnote>ªThe value for this diet was significantly different from the value obtained with the control diet HEX (P < 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">²Highest SEM.</footnote>

<footnote n="3">³Linear contrast for diet effect.</footnote>

<footnote n="4">⁴Quadratic contrast for diet effect.</footnote>

<footnote n="5">⁵ECM calculated as proposed by Hall (2023) with the milk energy equation proposed by NASEM (2021).</footnote>

<footnote n="6">⁶UFL = NEL, expressed in unité fourragère lait; 1 UFL = 7.37 MJ of NEL.</footnote>

<footnote n="7">⁷PDI = protein truly digestible in the intestine.</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">²Highest SEM.</footnote>

<footnote n="3">³Linear contrast for diet effect.</footnote>

<footnote n="4">⁴Quadratic contrast for diet effect.</footnote>
defatted SBM proportion (P = 0.023) and was numerically lower by 0.7 to 0.9 percentage points with experimental diets compared with HEX.

**Rumen Fermentation Traits.** We did not find evidence of differences among diets in rumen fluid pH, NH₃, and total VFA concentration, or proportion of branched-chain VFA out of total VFA at 5 h after the morning meal (P > 0.05; Table 5). Only valerate proportion tended to be quadratically related to MeOx-defatted SBM proportion (P = 0.060), and was reduced by 0.11 mol/100 mol of total VFA when cows were fed 67MeOx compared with HEX (P = 0.006; α₂ = 0.017). We did not find evidence of differences between diets in rumen fluid NH₃ concentration and pH from 0 to 8 h after the morning meal (P > 0.05; Figure 3 and Supplemental Figure S1, see Notes). Large entodinomorph count was positively linearly related to MeOx-defatted SBM proportion (P = 0.004) and was significantly higher (+0.47 log<sub>e</sub> cells/mL, i.e., +5,790 cells/mL) with 100MeOx compared with HEX (P = 0.010, α₁ = 0.017). Large entodinomorph count also tended to be higher (+0.38 log<sub>e</sub> cells/mL; i.e., +4,540 cells/mL) with 67MeOx compared with HEX (P = 0.034, α₂ = 0.033). Small entodinomorph and isotrich counts tended to be

<figure><img src="image_6.png" /><figcaption>**Figure 3.** Evolution of rumen fluid ammonia (NH₃) concentration over time in cows fed hexane-defatted or 2-methyloxolane-defatted soybean meal (sampling through rumen cannula). Confidence intervals around estimated means represent SEM. 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.</figcaption></figure>

<table><caption>**Table 5.** Rumen fluid composition in cows fed hexane-defatted or 2-methyloxolane-defatted soybean meal (sampling through oral stomach tubing at 5 h after the morning meal)</caption><thead><tr><th rowspan="2">Item</th><th colspan="4">Diet<fnref n="1" /></th><th rowspan="2">SEM<fnref n="2" /></th><th rowspan="2">P diet</th><th colspan="2">P contrasts</th></tr><tr><th>HEX</th><th>33MeOx</th><th>67MeOx</th><th>100MeOx</th><th>L<fnref n="3" /></th><th>Q<fnref n="4" /></th></tr></thead><tbody><tr><td>pH</td><td>6.49</td><td>6.52</td><td>6.48</td><td>6.53</td><td>0.036</td><td>0.57</td><td>0.51</td><td>0.77</td></tr><tr><td>NH₃ (mg/L)</td><td>140</td><td>136</td><td>142</td><td>146</td><td>8.2</td><td>0.78</td><td>0.43</td><td>0.58</td></tr><tr><td>Total VFA (mM)</td><td>177</td><td>169</td><td>181</td><td>172</td><td>5.6</td><td>0.32</td><td>0.94</td><td>0.99</td></tr><tr><td>VFA (mol/100 mol total VFA)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Acetate</td><td>65.1</td><td>65.3</td><td>66.0</td><td>65.5</td><td>0.56</td><td>0.52</td><td>0.29</td><td>0.43</td></tr><tr><td>Propionate</td><td>18.3</td><td>18.8</td><td>18.5</td><td>18.6</td><td>0.38</td><td>0.74</td><td>0.69</td><td>0.58</td></tr><tr><td>Butyrate</td><td>12.1</td><td>11.5</td><td>11.3</td><td>11.5</td><td>0.42</td><td>0.41</td><td>0.18</td><td>0.31</td></tr><tr><td>Valerate</td><td>1.29</td><td>1.25</td><td>1.18ª</td><td>1.24</td><td>0.031</td><td>0.048</td><td>0.063</td><td>0.060</td></tr><tr><td>Caproate</td><td>0.71</td><td>0.66</td><td>0.64</td><td>0.69</td><td>0.041</td><td>0.48</td><td>0.56</td><td>0.15</td></tr><tr><td>Branched-chain VFA</td><td>2.57</td><td>2.52</td><td>2.38</td><td>2.51</td><td>0.091</td><td>0.27</td><td>0.26</td><td>0.18</td></tr><tr><td>Iso-butyrate</td><td>0.90</td><td>0.89</td><td>0.85</td><td>0.88</td><td>0.030</td><td>0.46</td><td>0.36</td><td>0.30</td></tr><tr><td>Iso-valerate</td><td>1.67</td><td>1.63</td><td>1.53</td><td>1.62</td><td>0.065</td><td>0.24</td><td>0.24</td><td>0.16</td></tr><tr><td>Acetate:propionate (mol/mol)</td><td>3.60</td><td>3.50</td><td>3.59</td><td>3.54</td><td>0.093</td><td>0.75</td><td>0.81</td><td>0.69</td></tr><tr><td>Total protozoa count (log<sub>e</sub> cells/mL)</td><td>12.00</td><td>11.91</td><td>11.86</td><td>11.80</td><td>0.150</td><td>0.58</td><td>0.17</td><td>0.89</td></tr><tr><td>Entodinomorph count</td><td>11.98</td><td>11.89</td><td>11.84</td><td>11.77</td><td>0.153</td><td>0.56</td><td>0.16</td><td>0.92</td></tr><tr><td>Small entodinomorph count (&lt;100 µm)</td><td>11.91</td><td>11.81</td><td>11.72</td><td>11.63</td><td>0.163</td><td>0.32</td><td>0.062</td><td>0.96</td></tr><tr><td>Large entodinomorph count (&gt;100 µm)</td><td>9.18</td><td>9.31</td><td>9.56ª</td><td>9.65ª</td><td>0.187</td><td>0.031</td><td>0.004</td><td>0.86</td></tr><tr><td>Holotrich count</td><td>8.09</td><td>7.88</td><td>8.16</td><td>8.21</td><td>0.189</td><td>0.37</td><td>0.33</td><td>0.37</td></tr><tr><td>Isotrich count</td><td>7.80</td><td>7.66</td><td>7.95</td><td>8.13</td><td>0.175</td><td>0.14</td><td>0.062</td><td>0.29</td></tr><tr><td>Dasytrich count</td><td>6.63</td><td>6.04</td><td>6.13</td><td>5.95</td><td>0.401</td><td>0.31</td><td>0.12</td><td>0.48</td></tr></tbody></table>

<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">²Highest SEM.</footnote>

<footnote n="3">³Linear contrast for diet effect.</footnote>

<footnote n="4">⁴Quadratic contrast for diet effect.</footnote>
negatively linearly related to MeOx-defatted SBM proportion (P = 0.062).

**Plasma Parameters.** We did not find evidence of differences between diets in plasma AA, AA derivatives, and urea concentrations (P > 0.05; Table 6). The TAA, EAA, group 1 AA, group 2 AA, Ile, and Val concentrations tended to be quadratically related to the proportion of MeOx-defatted SBM in the diet (0.098 < P < 0.066). The Δ¹⁵N was positively linearly related to MeOx-defatted SBM proportion (P < 0.001) and was higher for 100MeOx compared with HEX (+0.20‰, P < 0.001, α₁ = 0.017). The Δ¹⁵N also tended to be higher for 67MeOx compared with HEX (+0.08‰, P = 0.043, α₂ = 0.033).

## Equivalence Tests

**Feed Intake and Animal Characteristics.** The PMR, SBM, and total diet DMI, and BCS were all likely equiv-

alent between all experimental diets and HEX (Figure 4 and Supplemental Figures S2 and S3, see Notes). The BW was equivalent between all experimental diets and HEX.

**Dairy Performance and Milk Composition.** Milk yield, ECM yield, milk true protein yield, milk fat content and yield, milk lactose yield, and milk urea content were likely equivalent between 100MeOx and HEX (Figure 4). Milk true protein content and lactose content were equivalent between 100MeOx and HEX. Milk casein N-to-total N ratio was equivalent between 100MeOx and HEX and milk soluble protein N-to-total N was likely equivalent between 100MeOx and HEX. However, milk NPN-to-total N ratio was unlikely equivalent and was 0.46 g/100 g higher for 100MeOx compared with HEX. Net energy and PDI intake were likely equivalent between 100MeOx and HEX. However, net energy balance was unlikely equivalent and was 0.09 UFL/d lower

<table><caption>**Table 6.** Plasma concentrations of AA, AA derivatives and urea, and N isotopic discrimination between plasma and diet in cows fed hexane-defatted or 2-methyloxolane-defatted soybean meal</caption><thead><tr><th rowspan="2">Item (µmol/L unless stated otherwise)</th><th colspan="4">Diet<fnref n="1" /></th><th rowspan="2">SEM<fnref n="2" /></th><th rowspan="2">P diet</th><th colspan="2">P contrasts</th></tr><tr><th>HEX</th><th>33MeOx</th><th>67MeOx</th><th>100MeOx</th><th>L<fnref n="3" /></th><th>Q<fnref n="4" /></th></tr></thead><tbody><tr><td>TAA<fnref n="5" /></td><td>2,291</td><td>2,383</td><td>2,329</td><td>2,255</td><td>54.2</td><td>0.29</td><td>0.44</td><td>0.091</td></tr><tr><td>Group 1 AA<fnref n="6" /></td><td>194</td><td>208</td><td>194</td><td>188</td><td>6.7</td><td>0.12</td><td>0.24</td><td>0.098</td></tr><tr><td>Group 2 AA<fnref n="7" /></td><td>757</td><td>801</td><td>774</td><td>743</td><td>30.4</td><td>0.23</td><td>0.45</td><td>0.073</td></tr><tr><td>BCAA<fnref n="8" /></td><td>508</td><td>534</td><td>510</td><td>491</td><td>21.8</td><td>0.18</td><td>0.22</td><td>0.10</td></tr><tr><td>NEAA<fnref n="9" /></td><td>1,385</td><td>1,425</td><td>1,407</td><td>1,371</td><td>31.6</td><td>0.59</td><td>0.65</td><td>0.21</td></tr><tr><td>EAA</td><td>906</td><td>959</td><td>923</td><td>889</td><td>33.9</td><td>0.20</td><td>0.39</td><td>0.072</td></tr><tr><td>Arg</td><td>75</td><td>85</td><td>76</td><td>77</td><td>3.8</td><td>0.078</td><td>0.77</td><td>0.15</td></tr><tr><td>His</td><td>54</td><td>57</td><td>54</td><td>52</td><td>1.9</td><td>0.17</td><td>0.13</td><td>0.13</td></tr><tr><td>Ile</td><td>118</td><td>126</td><td>119</td><td>116</td><td>5.0</td><td>0.14</td><td>0.39</td><td>0.066</td></tr><tr><td>Leu</td><td>140</td><td>145</td><td>136</td><td>132</td><td>6.4</td><td>0.18</td><td>0.10</td><td>0.26</td></tr><tr><td>Lys</td><td>79</td><td>87</td><td>85</td><td>85</td><td>4.6</td><td>0.28</td><td>0.28</td><td>0.18</td></tr><tr><td>Met</td><td>19</td><td>21</td><td>19</td><td>19</td><td>1.0</td><td>0.60</td><td>0.70</td><td>0.50</td></tr><tr><td>Phe</td><td>47</td><td>49</td><td>46</td><td>45</td><td>1.9</td><td>0.27</td><td>0.17</td><td>0.34</td></tr><tr><td>Thr</td><td>95</td><td>95</td><td>104</td><td>91</td><td>6.1</td><td>0.15</td><td>0.75</td><td>0.11</td></tr><tr><td>Trp</td><td>29</td><td>32</td><td>29</td><td>30</td><td>1.3</td><td>0.23</td><td>0.91</td><td>0.18</td></tr><tr><td>Val</td><td>251</td><td>262</td><td>254</td><td>244</td><td>11.1</td><td>0.22</td><td>0.30</td><td>0.081</td></tr><tr><td>AA derivatives</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>1-methylhistidine<fnref n="10" /></td><td>5.8</td><td>5.8</td><td>6.0</td><td>6.3</td><td>0.39</td><td>0.60</td><td>0.19</td><td>0.74</td></tr><tr><td>3-methylhistidine<fnref n="11" /></td><td>5.6</td><td>5.4</td><td>5.3</td><td>5.6</td><td>0.30</td><td>0.75</td><td>0.96</td><td>0.29</td></tr><tr><td>Urea (g/L)</td><td>0.28</td><td>0.28</td><td>0.28</td><td>0.29</td><td>0.016</td><td>0.91</td><td>0.70</td><td>0.57</td></tr><tr><td>Δ¹⁵N (‰)<fnref n="12" /></td><td>2.30</td><td>2.37</td><td>2.38³</td><td>2.49³</td><td>0.074</td><td>&lt;0.001</td><td>&lt;0.001</td><td>0.43</td></tr></tbody></table>

<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">²Highest SEM.</footnote>

<footnote n="3">³Linear contrast for diet effect.</footnote>

<footnote n="4">⁴Quadratic contrast for diet effect.</footnote>

<footnote n="5">⁵TAA = EAA + NEAA.</footnote>

<footnote n="6">⁶Group 1 AA = His + Met + Phe + Tyr + Trp (Mepham, 1982; Lapierre et al., 2012).</footnote>

<footnote n="7">⁷Group 2 AA = Arg + Ile + Leu + Lys + Thr + Val (Mepham, 1982; Lapierre et al., 2012).</footnote>

<footnote n="8">⁸Branched-chain AA = Ile + Leu + Val.</footnote>

<footnote n="9">⁹NEAA = Ala + Asn + Asp + Gln + Glu + Gly + Pro + Ser + Try.</footnote>

<footnote n="10">¹⁰1-methylhistidine stands for N-π-methylhistidine (CAS No. 368-16-1).</footnote>

<footnote n="11">¹¹3-methylhistidine stands for N-τ-methylhistidine (CAS No. 332-80-9).</footnote>

<footnote n="12">¹²N isotopic discrimination between plasma and diet.</footnote>
<figure><img src="image_7.png" /><figcaption>**Figure 4.** (A) Scaled difference between estimated marginal means of 100MeOx and HEX (100MeOx minus HEX) for DMI, BW, BCS, milk yield, milk composition, energy, and protein intake during the experimental phase. In green: equivalence margins and equivalence interval. In black or red: estimated difference and its confidence interval. 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). (B) Scaled difference between estimated marginal means of 100MeOx and HEX (100MeOx minus HEX) for milk SCC during the experimental phase. Analyses were performed on the $log_c$ 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 $log_c$ 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; PMR = partial mixed ration; ECM calculated as proposed by Hall (2023) with the milk energy equation proposed by NASEM (2021); UFL = NEL expressed in unité fourragère lait (1 UFL = 7.37 MJ of NEL); PDI = protein truly digestible in the intestine.</figcaption></figure>

for 100MeOx compared with HEX. The PDI efficiency and ratio of PDI to net energy were equivalent between 100MeOx and HEX. Milk SCC was unlikely equivalent between 100MeOx and HEX and was 12,500 cells/mL higher for 100MeOx compared with HEX.

Comparisons between 67MeOx or 33MeOx and HEX found the same conclusions except for milk SCC, milk fat content, and net energy balance. Milk SCC was unlikely equivalent to HEX, toward lower counts for 67MeOx compared with HEX (–4,000 cells/mL), but likely equivalent between 33MeOx and HEX (Supplemental Figures

S2 and S3). Milk fat content was unlikely equivalent (+2.2 g/kg) for 67MeOx compared with HEX. Net energy balance was likely equivalent between 33MeOx and HEX.

**Nitrogen Partitioning.** Nitrogen intake, milk N secretion, and N intake partitioning toward milk N were likely equivalent between 100MeOx and HEX (Figure 5). Fecal N excretion was likely equivalent and N intake partitioning toward fecal N was equivalent between 100MeOx and HEX. Urine N excretion was equivalent between 100MeOx and HEX and N intake partitioning toward
<figure><img src="image_8.png" /><figcaption>**Figure 5.** Scaled difference between estimated marginal means of 100MeOx and HEX (100MeOx minus HEX) for N partitioning during the experimental phase. In green: equivalence margins and equivalence interval. In black or red: estimated difference and its confidence interval. 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.</figcaption></figure>

urinary N was likely equivalent between 100MeOx and HEX. The N balance was unlikely equivalent and was 7.1 g/d lower for 100MeOx compared with HEX.

Comparisons between 67MeOx or 33MeOx and HEX found the same conclusions, except for the following variables for 33MeOx compared with HEX: N intake partitioning toward milk and urine were equivalent and daily urinary N excretion was likely equivalent (Supplementary Figures S4 and S5, see Notes).

**Rumen Fermentation Traits.** At 5 h after the morning meal, rumen fluid pH was equivalent and rumen fluid $NH_3$ concentration was likely equivalent between 100MeOx and HEX (Figure 6). Total VFA concentration, and the proportions of isobutyrate, isovalerate, and branched-chain VFA (i.e., sum of isobutyrate and isovalerate) out of total VFA in rumen fluid were likely equivalent between 100MeOx and HEX. Total protozoa count was unlikely equivalent between 100MeOx and HEX, toward

<figure><img src="image_9.png" /><figcaption>**Figure 6.** Scaled difference between estimated marginal means of 100MeOx and HEX (100MeOx minus HEX) for rumen fluid composition during the experimental phase (sampling through oral stomach tubing at 5 h after the morning meal). In green: equivalence margins and equivalence interval. In black or red: estimated difference and its confidence interval. 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.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Figure 7.** Scaled difference between estimated marginal means of 100MeOx and HEX (100MeOx minus HEX) for rumen fluid protozoa counts during the experimental phase (sampling through oral stomach tubing at 5 h after the morning meal). Analyses were performed on the $\log_e$ scale and back-transformed for graphical representation. In green: equivalence margins and equivalence interval. In black or red: estimated difference and its confidence interval. 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 on the $\log_e$ 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.</figcaption></figure>

lower count (–30,100 cells/mL) for 100MeOx (Figure 7). This lower count was driven by fewer entodinomorphs (unlikely equivalent, –30,500 cells/mL), which was due to fewer small entodinomorphs (unlikely equivalent, –37,000 cells/mL) but more large entodinomorphs (not equivalent, +5,790 cells/mL). Holotrich count was also unlikely equivalent between 100MeOx and HEX, but toward higher count for 100MeOx (+491 cells/mL) due to more isotrichs (unlikely equivalent, +956 cells/mL) but fewer dasytrichs (unlikely equivalent, –370 cells/mL).

Conclusions regarding VFA were similar between 33MeOx and HEX but differed slightly between 67MeOx and HEX, as proportions of isobutyrate (–0.05 mol/100 mol total VFA), isovalerate (–0.14 mol/100 mol total VFA) and branched-chain VFA (–0.19 mol/100 mol total VFA) were unlikely equivalent (Supplemental Figures S6 and S7, see Notes). Regarding protozoa, the large entodinomorphs count was unlikely equivalent between 67MeOx or 33MeOx and HEX, toward higher counts for 67MeOx (+4,450 cells/mL; Supplemental Figure S8, see Notes) and for 33MeOx (+1,320 cells/mL; Supplemental Figure S9, see Notes). Holotrich and isotrich counts were unlikely equivalent between 33MeOx and HEX, toward lower counts for 33MeOx (–624 and –331 cells/mL respectively).

**Plasma Parameters.** Plasma urea, total AA, EAA, NEAA, group 1 AA (His + Met + Phe + Tyr + Trp; Mepham, 1982; Lapierre et al., 2012), group 2 AA (Arg + Ile + Leu + Lys + Thr + Val; Mepham, 1982; Lapierre et al., 2012), branched-chain AA (BCAA, Ile + Leu + Val), and 3-methylhistidine concentrations were likely equivalent between 100MeOx and HEX (Figure 8). Regarding individual EAA, only plasma Leu (–8 µmol/L), Lys (+6 µmol/L), and Thr (–5 µmol/L) concentrations were unlikely equivalent between 100MeOx and HEX. Plasma 1-methylhisitidine concentration was unlikely

equivalent between 100MeOx and HEX (+0.5 µmol/L). The $\Delta^{15}$N was unlikely equivalent, and toward higher values (+0.20‰) for 100MeOx compared with HEX.

For 67MeOx and 33MeOx compared with HEX, differing conclusions are presented below (Supplemental Figure S10 and S11, see Notes). For individual EAA, plasma Leu concentration was likely equivalent for 67MeOx compared with HEX, whereas Thr was unlikely equivalent for 67MeOx compared with HEX (+9 µmol/L). Comparisons between 33MeOx and HEX showed that EAA (+53 µmol/L), group 1 AA (+14 µmol/L), group 2 AA (+44 µmol/L), and BCAA (+27 µmol/L) were all unlikely equivalent and toward higher concentrations for 33MeOx. Concerning individual EAA, comparisons between 33MeOx and HEX showed that plasma Leu concentration was likely equivalent whereas plasma Arg (+10 µmol/L), Ile (+9 µmol/L), Met (+1 µmol/L), and Trp (+3 µmol/L) concentrations were unlikely equivalent. Plasma 1-methylhistidine concentration and $\Delta^{15}$N were likely equivalent for both 67MeOx and 33MeOx compared with HEX.

## DISCUSSION

Overall, replacing hexane with MeOx as defatting solvent led to SBM that allowed likely equivalent dairy performance. However, some unlikely equivalent outcomes (i.e., branched-chain VFA in rumen fluid, total protozoa count in rumen fluid, plasma Lys concentration, $\Delta^{15}$N, net energy balance, and N balance) suggest minor alterations at rumen and digestive level that may have led to slightly affected whole-body N metabolism in favor of the hexane-defatted SBM. In terms of food safety, residues of both n-hexane and MeOx were detected in the milk of the cows, but not at quantifiable levels. Replacing hexane by MeOx logically tended to reduce the frequency of n-hexane residues detection in the milk. However, we did
<figure><img src="image_11.png" /><figcaption>**Figure 8.** Scaled difference between estimated marginal means of 100MeOx and HEX (100MeOx minus HEX) for plasma concentrations of AA, AA derivatives, and urea and N isotopic discrimination between plasma and diet (Δ¹⁵N) during the experimental phase. In green: equivalence margins and equivalence interval. In black or red: estimated difference and its confidence interval. 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; TAA = EAA + NEAA; Group 1 AA = His + Met + Phe + Tyr + Trp (Mepham, 1982; Lapierre et al., 2012); Group 2 AA = Arg + Ile + Leu + Lys + Thr + Val (Mepham, 1982; Lapierre et al., 2012); Branched-chain AA = Ile + Leu + Val; NEAA = Ala + Asn + Asp + Gln + Glu + Gly + Pro + Ser + Try; 1-methylhistidine stands for N-π-methylhistidine (CAS No. 368-16-1); 3-methylhistidine stands for N-τ-methylhistidine (CAS No. 332-80-9).</figcaption></figure>

not find evidence that the presence of MeOx residues in
milk differed when cows were fed MeOx-defatted SBM
instead of hexane-defatted SBM.

Effects of Using 2-Methyloxolane as Defatting
Solvent on SBM Characteristics and Palatability

Maillard Reaction Products Indicating More Intense
Processing of 2-Methyloxolane-Defatted SBM. Fu-
rosine and CML are products of the Maillard reaction,

a nonenzymatic browning reaction between an amino
group (e.g., in AA, peptides, proteins) and a reducing
carbohydrate, which can be promoted by heating (Mar-
tins et al., 2000; Hellwig and Henle, 2014; Helou et al.,
2014). More specifically, furosine is an Amadori product
formed during the early stage of the Maillard reaction,
whereas CML is formed during the advanced stage of the
Maillard reaction (Erbersdobler and Somoza, 2007). The
greater concentrations of furosine, and to a lesser extent
CML, in MeOx-defatted SBM agree with MeOx-defatted
SBM undergoing a more intensive heat processing leading to more Maillard reactions, which is consistent with differences in physicochemical properties between MeOx and hexane. The MeOx has a boiling point of 71°C (azeotrope with water) to 80°C (dry MeOx) compared with 66 to 70°C for hexane (Rapinel et al., 2020). This greater boiling point for MeOx allows for higher temperatures at the extraction stage compared with hexane (Rapinel et al., 2020). In addition to the higher boiling point, MeOx also has a greater enthalpy of vaporization (364 kJ/kg for MeOx vs. 334 kJ/kg for n-hexane), and thus requires more heat input at the desolventizing stage compared with hexane (Rapinel et al., 2020). Taken together, these greater extraction temperatures and heat requirements for desolventization with MeOx may have triggered more Maillard reactions. However, the overall process appeared to be relatively low-intensity given the CML concentrations obtained here (i.e., 47.6 to 57.7 mg/kg DM) compared with the value of 123 mg/kg DM (Nozière et al., 2022) reported in another experiment. The MeOx-defatted SBM was produced during the first MeOx production runs in a plant originally designed and optimized for hexane extraction. Further optimization of the process may reduce the differences observed here between the 2 SBM.

**Quantification of Solvent Residues in SBM.** The levels of n-hexane residues in hexane-defatted SBM were very low (11 mg/kg DM). Out of crushing factories, SBM usually contains between 300 to 500 mg/kg hexane residues, of which 200 mg/kg evaporate during storage of the meal (Bauer et al., 2009), resulting in a meal that contains 100 to 300 mg/kg when fed to animals. A more recent paper cited a target of ≤250 mg/kg hexane residues in SBM (Demarco and Gibon, 2020). This indicates that the concentration of n-hexane residues in SBM is probably greater in commercial settings than in the current study. In contrast, the level of MeOx residues found here in MeOx-defatted SBM was high (977 mg/kg DM) and near the threshold of 1,000 mg/kg above which chemical impurities resulting from processing aids should be declared in the catalog of feed (European Union, 2022a). This level of MeOx residues may thus be considered a worst-case scenario for solvent contamination of SBM, but it also highlights a need to further improve the meal desolventization process to recover more solvent. The hexane-defatted SBM was very slightly contaminated with MeOx (4 mg/kg DM). This contamination may have happened during storage of the SBM, which were stored next to each other in a barn, or else during sampling, as the same shovel was used for sampling the hexane-defatted SBM and the MeOx-defatted SBM. However, the amounts of MeOx in hexane-defatted SBM were largely inferior to the amounts of MeOx residues in the MeOx-defatted SBM (977 mg/kg DM), and so this very

slight contamination unlikely affected the results and conclusions of the current study.

**SBM Palatability.** Despite a linear decrease in SBM intake with increasing proportion of MeOx-defatted SBM in the diet, the SBM intake was only 0.1 kg DM/d (i.e., 3% of SBM intake) lower with 100MeOx compared with HEX. Moreover, the equivalence test showed likely equivalent SBM intake for all experimental diets compared with HEX. Also, likely equivalent palatability was concluded from the trial conducted during pre-experimental phase and designed to study the palatability of MeOx-defatted SBM compared with hexane-defatted SBM (Supplemental File S1). Therefore, the palatability of MeOx-defatted SBM and hexane-defatted SBM was likely equivalent.

## Effects of Using 2-Methyloxolane-Defatted SBM on Solvent Transfer from Feed to Milk

**Transfer of n-Hexane and 2-Methyloxolane from SBM to Milk.** Residues of hexane in milk have already been detected in a study that aimed to identify environmental pollutants in human milk (Pellizzari et al., 1982). However, to our knowledge, this study is the first to evaluate transfers of solvent used for defatting meals from the feed to the milk. Residues of n-hexane were detected in 5 out of 6 milk samples from cows fed HEX, whereas the level of n-hexane residues in SBM was very low (11 mg/kg DM of n-hexane). In contrast, MeOx residues were only detected in 2 out of 6 milk samples from 100MeOx-fed cows, whereas the level of MeOx residues levels in MeOx-defatted SBM was high (977 mg/kg DM). Thus, MeOx appears to have a lower transfer potential from feed to milk compared with n-hexane.

**Detection of n-Hexane Traces in the Milk of Cows Fed 2-Methyloxolane-Defatted SBM.** Unexpectedly, n-hexane residues were found in 1 out of 6 milk samples from cows fed 100MeOx. This may be explained by a too-short wash-out period between weeks of measurement in successive experimental periods (i.e., 2 wk). The contamination may also have come from the production concentrate, which contained 15% rapeseed meal and 7.9% SBM, but this seems unlikely given that the intake of these meals came to only 200 g DM/d. Another explanation may be an accumulation of n-hexane residues ingested before the experiment (e.g., plant-based milk replacer ingested when the cow was still a calf, concentrate containing defatted meals ingested when the cow was a heifer, and so on) or n-hexane residues ingested during previous periods of the experiment that accumulated in body fat reserves. This hypothesis is supported by the greater octanol-water partition coefficient (indicating preferential partitioning of the solvent toward octanol or water) of n-hexane compared with MeOx (4 vs. 1.85
respectively), which points to a greater potential for n-hexane to bioaccumulate (Claux et al., 2021). Body fat reserve turnover may thus lead to the release of n-hexane residues into the blood and, subsequently, into the milk.

*No Traces of 2-Methyloxolane in the Milk of Cows Fed Hexane-Defatted SBM.* Despite slight contamination of hexane-defatted SBM with MeOx (as discussed above), MeOx residues were never found in the milk of cows fed HEX. This reinforces the assertion that the very slight potential contamination of hexane-defatted SBM unlikely affected the results and conclusions of the current study.

## Effects of Using 2-Methyloxolane-Defatted SBM on Dairy Cow Nutrition and Performance

**Ruminal Protein Degradation.** In agreement with the greater furosin and CML concentrations in MeOx-defatted SBM compared with hexane-defatted SBM, in situ CP degradation measurements showed that the soluble CP fraction was lower with MeOx-defatted SBM compared with hexane-defatted SBM, whereas the potentially degradable CP fraction was higher. However, due to a shorter lag time for MeOx-defatted SBM compared with hexane-defatted SBM, we did not find evidence for differences in theoretical CP degradability assuming a passage rate of 6%/h. Similarly, previous research with raw and extruded lupine and fava bean did not find evidence that increasing CML concentration beyond 20 mg/ kg DM affected theoretical CP degradability (Nozière et al., 2022). Despite these differences in CP ruminal degradation kinetics, we did not find evidence for a diet effect on the timing of the peak of rumen fluid NH₃ concentration. Additionally, NH₃ concentration at 5 h after the morning meal was likely equivalent between all diets and HEX. However, other measurements suggested that protein degradation by rumen microorganisms may have been slightly reduced, as indicated by unlikely equivalent proportion of branched-chain VFA, toward lower proportion for 67MeOx than HEX, and equivalent but linear decrease of diet DMd with increasing MeOx-defatted SBM proportion in the diet. Indeed, ruminal branched-chain VFA (i.e., isovalerate, isocaproate, and their sum) originate from the proteolysis of BCAA by rumen microorganisms (Andries et al., 1987) and are thus indicators of ruminal protein degradation. However, the numerical difference in proportion of branched-chain VFA in rumen fluid between 67MeOx and HEX was small, and proportion of branched-chain VFA was likely equivalent to HEX with both 33MeOx and 100MeOx. Taken together, the different kinetics of ruminal CP degradation, unlikely equivalent toward lower ruminal branched-chain VFA proportion, and linear decrease of diet DMd with increasing MeOx-defatted SBM proportion in the diet may have

driven slightly lower microbial protein synthesis with 67MeOx and 100MeOx compared with HEX.

*Nitrogen Absorption and Plasma AA Profile.* Analysis of plasma AA concentrations never found strict equivalence, because the 100 – (2 × αᵢ)‰ CI around estimated differences were always larger than the equivalence interval. With the exception of Leu and Thr in 100MeOx versus HEX, whenever unlikely equivalence was stated, the experimental diets always led to greater plasma AA concentrations, although the differences were never significant. Plasma Lys concentration was always unlikely equivalent toward higher concentrations for experimental diets compared with HEX. The plasma concentration of an AA results from a balance between AA absorption at the duodenum and protein synthesis for milk, growth, and nonproductive functions (INRA, 2018). Given that needs for growth and nonproductive functions are expected to be similar across diets, and that milk true protein secretion was likely equivalent between all experimental diets and HEX, we can reasonably assume that the unlikely equivalence of plasma Lys concentration (i.e., toward greater Lys concentration with experimental diets compared with HEX) was linked to greater Lys absorption at the duodenum with experimental diets compared with HEX. This greater Lys absorption at the duodenum with MeOx may be due to the greater furosin and CML concentrations in the SBM, as mild Maillard reactions increase AA availability at the duodenum due to protection from proteolysis in the rumen, as suggested by certain measurements for 67MeOx and 100MeOx, and a reversal of protection in the abomasum (Mendowski et al., 2019). Lysine has a free amino group on its side chain, which makes it sensitive to Maillard reactions (Erbersdobler and Somoza, 2007). However, milk true protein yield was equivalent between experimental diets and HEX, which indicated that the extra plasma Lys was not used for milk true protein synthesis. This was most likely due to Met digestible in the intestine (MetDI) being more limiting for milk true protein synthesis than the Lys digestible in the intestine (LysDI). Using the AA profile of feed ingredients from feed tables (INRA CIRAD AFZ, 2024), calculated diet LysDI and MetDI concentrations were 6.9% of PDI and 1.9% of PDI, respectively (data not shown), whereas the recommendations for LysDI and MetDI are 7.0% and 2.4% of PDI, respectively (INRA, 2018). The MetDI was therefore more limiting than LysDI in the current study.

**Dairy Performance and Milk Composition.** Overall, dairy performance and milk composition were likely equivalent with all experimental diets compared with HEX, with the exception of milk fat content, milk SCC, and milk NPN-to-total N ratio. Milk fat content was unlikely equivalent toward greater values with 67MeOx compared with HEX but was likely equivalent to HEX
with all other diets. The numeric increase in milk fat content with 67MeOx is likely due to a milk concentration effect, with likely equivalent but numerically lower milk yield for 67MeOx compared with HEX (–0.5 kg/d). Milk SCC was unlikely equivalent between 100MeOx and HEX, toward greater count for 100MeOx. However, the EMM of milk SCC was low for all diets (i.e., $53 \times 10^3$ to $70 \times 10^3$ cells/mL when $\log_e$ counts are back-transformed to the original scale) according to the review of Dufour et al. (2011), who reported that low-SCC herds were defined by milk SCC <150 $\times 10^3$ to <264 $\times 10^3$ cells/mL. The NPN-to-total N ratio was also unlikely equivalent toward greater values for all experimental diets compared with HEX and tended to be positively linearly related to the MeOx-defatted SBM proportion in the diet. The NPN fraction contains mainly urea, which had a likely equivalent concentration between experimental diets and HEX, but also other N molecules including AA, peptides, creatine, creatinine, ammonia, and purine derivatives such as uric acid and allantoin (Journet et al., 1975; DePeters and Ferguson, 1992). In this study, milk urea was likely equivalent in all experimental diets compared with HEX. Tendency for greater milk NPN fraction may be explained by greater concentrations of other NPN molecules, such as purine derivatives (including uric acid which represents 2%–4% of milk NPN; Journet et al., 1975; DePeters and Ferguson, 1992). Purine derivatives originate from rumen microbes’ nucleic acids as well as endogenous turnover of nucleic acids in tissues (Gonzalez-Ronquillo et al., 2003; Tas and Susenbeth, 2007). Thus, if there was no dietary treatment effect on the growth efficiency of rumen microbes, then the increased concentration of purine derivatives in milk may originate from a greater endogenous supply, provided by an increased muscle turnover rate for instance.

### Nitrogen Partitioning and Nitrogen Use Efficiency.

Equivalence or likely equivalence was declared for all variables related to N partitioning except N balance, which was unlikely equivalent and toward lower values in all experimental diets compared with HEX. However, as expected for midlactation primiparous dairy cows, N balance was positive for all experimental diets. Similarly, net energy balance was positive but unlikely equivalent and toward lower values in 67MeOx and 100MeOx compared with HEX. These lower yet positive N and net energy balances were driven by likely equivalent yet numerically lower N and net energy intake in experimental diets compared with HEX. These slightly lower N and net energy balances may also be due to a subtle change in muscle protein accretion. This hypothesis is supported by $\Delta^{15}$N, which was declared unlikely equivalent and significantly greater for 100MeOx compared with HEX. The $\Delta^{15}$N has been proposed as a proxy for N use efficiency in ruminants (Cantalapiedra-Hijar et al.,

2015). However, N use efficiency for milk production was likely equivalent between all experimental diets and HEX. Thus, differences in $\Delta^{15}$N cannot be explained by variations in N use efficiency for milk production but may instead originate from metabolism changes in other N pools, such as skeletal-muscle protein reserves as reported in growing beef cattle (Guarnido-Lopez et al., 2022). For instance, milk and plasma $\Delta^{15}$N were shown to increase with body protein mobilization in dairy cows (Chen et al., 2020; Correa-Luna et al., 2022). Furthermore, plasma 1-methylhistidine, which results from catabolism of the dipeptide anserine present in muscle, was unlikely equivalent toward greater concentration in HEX compared with 100MeOx, which possibly indicates mobilization of free AA and small peptides from skeletal muscle (Siachos et al., 2024). However, we believe that substantial body protein mobilization is improbable because the cows were in mid lactation, and plasma 3-methylhisitidine concentration, which is usually used as a marker of whole-body myofibrillar protein breakdown (Sadri et al., 2023), was likely equivalent between experimental diets and HEX. Rather, a small change in muscle protein accretion when cows were fed MeOx-defatted SBM may explain the greater $\Delta^{15}$N.

### Equivalence Testing

### Usefulness of the Equivalence Test Approach.
To our knowledge, this study is the first to use equivalence tests in the context of research on livestock nutrition. Equivalence tests are well-established statistical tools used for regulatory composition assessments on genetically modified feeds in the European Union, but they are not required for regulatory toxicological and nutritional assessments of these same feeds (EFSA GMO Panel Working Group on Animal Feeding Trials, 2008; European Union, 2013). However, these tests have been applied in toxicity studies on rodents fed genetically modified feed (van der Voet et al., 2017), and the EFSA Panel on GMO (2010) states that equivalence tests could be used for animal feeding studies. Unlike traditional difference tests, which only indicate a failure to show a difference when results are nonsignificant, equivalence tests can demonstrate equivalence between dietary treatments. Nonsignificant results from difference tests do not provide evidence of equivalence between treatments (Bello and Renter, 2018).

### Limitations of Equivalence Tests.
The most important limit to the equivalence approach lies in the definition of equivalence margins, which affect the conclusion of the equivalence test. Although large equivalence margins make equivalence easier to prove, strict equivalence margins make equivalence more difficult to prove. Equivalence margins should be fixed a priori and can
be defined by expertise based on previous knowledge of interindividual variability for each response variable in comparable situations (van der Voet et al., 2019). Nonetheless, here, for homogeneity reasons, we set a priori equivalence margins to ± 5% of the EMM of the control HEX diet for all response variables. Equivalence tests are common practice in pharmacokinetics: equivalence margins of ± 20% are used for bioequivalence studies, and equivalence margins ≤10% are recommended for dissolution tests (FDA, 2001; EMEA CHMP, 2010). The threshold of ± 5% used here might therefore be considered strict in comparison, thus resulting in equivalence being more difficult to prove. Also, equivalence tests were based on pairwise comparisons. Although we corrected for multiplicity using an a priori assumption of linearity in the response, more complex patterns (e.g., quadratic response) were not considered. This is a limitation of equivalence tests when analyzing data from dose-response designed experiments. Another limitation is that, for proportion data, we did not use logit models because equivalence margins of ± 5% are hard to translate into the logit scale.

*Advantages of Graphing the Results of Equivalence Tests.* The graphical representation of the scaled differences presented here (Figures 4 through 8) and adapted from van der Voet et al. (2019) allows to quickly draw conclusions on a large number of variables while highlighting the uncertainty of the estimation. This graphical representation also allows readers to easily draw their own conclusions according to their own equivalence margins, although the correction for multiple testing using a fallback procedure may be slightly different. Indeed, because the data are scaled to the equivalence margin, ± x on the scaled unit corresponds to equivalence margins of ± 5x% of the EMM of HEX. Notably, if we had used less-strict equivalence margins of ± 10% of the EMM of HEX, then the tests would have concluded equivalence or likely equivalence between experimental diets and the reference HEX on all variables except rumen fluid protozoa counts, plasma Arg, Lys, and Trp concentrations, milk SCC, milk NPN-to-total N ratio, and net energy and N balances.

*Comparisons of Results from Equivalence and Difference Tests.* The choice between equivalence and difference tests must be driven by the research question. In the context of our study, the equivalence tests seemed more adequate to investigate whether MeOx-defatted SBM can replace hexane-defatted SBM without affecting dairy performance and N partitioning. However, difference tests provided additional information regarding (1) the presence of significant pairwise differences between treatments, and (2) the presence of significant linear or quadratic response patterns. The application of equivalence tests in this study yielded 4 possible outcomes:

equivalence, likely equivalence, unlikely equivalence, or nonequivalence. Conversely, difference tests yielded 2 possible outcomes: either the difference (i.e., pairwise difference or linear or quadratic response pattern) is significant or there is no evidence for a difference. In the context of our study, the combination of outcomes from equivalence and difference tests yielded 7 situations. When we established equivalence (e.g., milk true protein content) without demonstrating a significant difference, equivalence and difference tests yielded consistent results that allowed us to conclude equivalence. When we established likely equivalence (e.g., milk yield) without demonstrating a significant difference, equivalence and difference tests also yielded consistent results that allowed us to conclude likely equivalence, although the magnitude of the difference may be greater than the a priori-defined equivalence margins. Conversely, when we established nonequivalence while demonstrating a significant difference (e.g., large entodinomorph count), equivalence and difference tests yielded consistent results that allowed us to conclude a difference. When we established unlikely equivalence while demonstrating a significant difference (e.g., Δ¹⁵N), equivalence and difference tests also yielded consistent results that allowed us to conclude a difference, although the magnitude of the effect may be lower than the a priori-defined equivalence margins. When we established equivalence while demonstrating a significant difference (e.g., diet DMd), equivalence and difference tests seemed to yield inconsistent results. However, we could conclude that although the diet has a significant effect on the response variable, the magnitude of this effect is smaller than the a priori-defined equivalence margins. When we established likely equivalence while demonstrating a significant difference (e.g., SBM intake), equivalence and difference tests also seemed to yield inconsistent results. However, we could conclude that although the diet has a significant effect on the response variable, the magnitude of this effect is likely smaller than the a priori-defined equivalence margins. Eventually, when we established unlikely equivalence without demonstrating a significant difference (e.g., milk SCC, N balance), equivalence and difference tests yielded inconsistent results and weak conclusions. This scenario resulted from 2 possible causes: (1) a low statistical power leading to large margins of error around estimated difference, or (2) overly strict a priori-defined equivalence margins, as discussed previously. If the response variable is of primary interest, further study is needed to draw firmer conclusions.

## CONCLUSIONS

Through a statistical approach that is innovative in the field of livestock nutrition, this study showed that
that hexane-defatted SBM and MeOx-defatted SBM had likely equivalent palatability for dairy cows. Replacing hexane by MeOx for defatting SBM afforded likely equivalent dairy performance, N partitioning, and N use efficiency for milk production. However, some unlikely equivalences between MeOx-defatted SBM and hexane-defatted SBM suggest slight alterations at rumen and digestive level that may, in turn, have subtly affected the whole-body N metabolism. Interestingly, replacing hexane by MeOx as defatting solvent tended to reduce the frequency of detection of n-hexane residues in milk. Further studies are needed to draw firmer conclusions on certain indicators, such as N metabolism and milk fat content.

## 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) for conducting the experiment, collecting samples, and taking care of the animals. We also thank C. Labonne, L. Genestoux, I. Constant, L. Pluchino, Y. Radouani, F. Anglard, L. Salis, A. Torrent, D. Bany, C. Moreau, A. Le Morvan, G. Maxin (Université Clermont Auvergne, INRAE, VetAgroSup, UMR Herbivores, Theix, Saint-Genès-Champanelle, France) and L. Le Normand and C. Mustière (PEGASE, INRAE, Institut Agro, Saint-Gilles, France) for their help with behavioral observations, sampling, and laboratory analyses. C. Chantelauze (Université Clermont Auvergne, INRAE, VetAgroSup, UMR Herbivores) performed the analyses of the natural abundance of stable nitrogen isotopes at the French metaboHUB infrastructure (ANR-INBS-0010, UCA, INRAE, UNH, Plateforme d'Exploration du Métabolisme, MetaboHUB Clermont, Clermont-Ferrand, France). C. García-Vázquez (Université Clermont Auvergne, INRAE, VetAgroSup, UMR Herbivores) performed the laboratory analysis of urinary ureic N and creatinine that enabled us to develop the prediction equation for urinary N excretion. Supplemental material for this article is available at https://doi.org/10.5281/zenodo.13919971. 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:** $\Delta^{15}$N = N isotopic discrimination between plasma and diet; 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; BCAA = branched-chain AA; CAS = Chemical Abstract Service; CML = Nε-carboxymethyl-lysine; DMd = apparent digestibility of DM; EFSA = European Food Safety Authority; EM = equivalence margin; EMM = estimated marginal mean; HEX = control diet, 100% hexane-defatted SBM; INRAE = French National Research Institute for Agriculture, Food and Environment; LysDI = Lys digestible in the intestine; MetDI = Met digestible in the intestine; MeOx = 2-methyloxolane; NIRS = near-infrared spectrometry; PDI = protein truly digestible in the intestine; PMR = partial mixed ration; R⁵V = coefficient of determination in external validation; SBM = soybean meal; SEP = standard error of prediction; UFL = NEL, expressed in unité fourragère lait (1 UFL = 7.37 MJ of NEL).

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ORCIDS

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

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

C. Ginane, https://orcid.org/0000-0001-9059-6001

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

S. Lemosquet, https://orcid.org/0000-0002-1077-1285

G. G. R. Leday, https://orcid.org/0000-0002-3753-4060

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