# Oral-stomach sampling as an alternative to rumen canula for the inoculation of in vitro batch fermentation systems

Vincent Niderkorn, Sandra Point, Angélique Torrent, Pierre Nozière, Mathieu Silberberg, Anne Boudon

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## To cite this version:

Vincent Niderkorn, Sandra Point, Angélique Torrent, Pierre Nozière, Mathieu Silberberg, et al. Oral-stomach sampling as an alternative to rumen canula for the inoculation of in vitro batch fermentation systems. Animal Feed Science and Technology, 2025, 327, pp.116416. 10.1016/j.anifeedsci.2025.116416. hal-05100241

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# Oral-stomach sampling as an alternative to rumen canula for the inoculation of *in vitro* batch fermentation systems

Vincent Niderkorn<sup>a,d,*</sup>, Sandra Point<sup>b</sup>, Angelique Torrent<sup>a</sup>, Pierre Nozière<sup>a</sup>, Mathieu Silberberg<sup>a</sup>, Anne Boudon<sup>c</sup>

## ARTICLE INFO

**Keywords:**

ruminant research tools
*in vitro* rumen fermentation
rumen cannulation
oral-stomach sampling

## ABSTRACT

The standard method to collect rumen fluid (RF) serving as inoculum for *in vitro* rumen fermentation assays and using ruminally cannulated animals is less and less accepted in some countries, and oral-stomach sampling (OSS) could be an alternative that needs to be validated. The objective of this study was to compare the *in vitro* rumen fermentation parameters of a large set of substrates with contrasted fermentation profiles using inocula obtained by OSS and from different sampling sites in the rumen of cannulated dairy cows. Rumen fermentation assays were conducted using twelve different substrates (six forages and six total mixed rations) and three types of inoculum consisting in fresh RF sampled in the reticulum (RF<sub>ret</sub>), sampled both in the reticulum and the ventral sac (RF<sub>mix</sub>) and using OSS (RF<sub>tub</sub>) during three feeding periods creating variability in RF composition (control and acidogenic diets). There was a strong effect of substrate on all the parameters (P < 0.001) and a limited effect of the type of RF on the overall fermentability of substrates, particularly between RF<sub>mix</sub> (standard method) and RF<sub>tub</sub> that had similar values for dry matter (DM) and fiber degradabilities, gas production (including proportion of methane (CH<sub>4</sub>) in the gas produced), total volatile fatty acids (VFA) and ammonia (NH<sub>3</sub>). Above all, we did not detect any interaction between the substrate and the type of RF, highlighting the possibility to measure confidently relative differences among substrates or treatments. Overall, our results showed that fresh OSS can be a relevant alternative to the fresh RF sampling using cannulated animals. To keep OSS as a research tool on the long term, efforts should be continued to improve the standardization and the refinement of the OSS method.

**Abbreviations:** ADF, acid detergent fibre; Bic, bicarbonate; C, cocksfoot; Ca, calcium; CH<sub>4</sub>, methane; CP, crude protein; DM, dry matter; GP, gas production; IVADFD, in vitro acid detergent fibre degradability; IVDMD, in vitro dry matter degradability; IVNDFD, in vitro neutral detergent fibre degradability; MgO, magnesium oxide; MF, meadow fescue; NDF, neutral detergent fibre; NH<sub>3</sub>, ammonia; NIR, near infrared reflectance; OM, organic matter; OSS, oral-stomach sampling; P, potassium; PG, permanent grassland; RC, red clover; RF, rumen fluid; RF<sub>mix</sub>, rumen fluid sampled in different sites of the rumen; RF<sub>ret</sub>, rumen fluid sampled in the reticulum; RF<sub>tub</sub>, rumen fluid sampled using tubing OSS; S, sainfoin; TMR, total mixed ration; TMRA, acidogenic total mixed ration; TMRF, total mixed ration rich in fibers; VFA, volatile fatty acids; WC, white clover.

* Corresponding author at: INRAE, Université Clermont Auvergne, Vetagro Sup, UMRH, Saint-Genès-Champanelle, 63122, France.

E-mail address: vincent.niderkorn@inrae.fr (V. Niderkorn).

<footnote><sup>a</sup> INRAE, Université Clermont Auvergne, Vetagro Sup, UMRH, Saint-Genès-Champanelle 63122, France</footnote>

<footnote><sup>b</sup> Centre Mondial de l'Innovation Roullier, Saint-Malo 35400, France</footnote>

<footnote><sup>c</sup> INRAE, UMR PEGASE, Institut Agro, Saint-Gilles 35590, France</footnote>

<footnote><sup>d</sup> Department of Animal Nutrition and Feed Technology, Faculty of Animal Husbandry, Universitas Padjadjaran, Jatinangor, Sumedang, 45363, Indonesia</footnote>
# 1. Introduction

In vitro rumen fermentation techniques are important research tools mainly to characterize the rumen digestion of forages, diets and alternative feeding resources under controlled conditions and in different situations (Menke et al., 1979). While biochemical, enzymatic or spectrometric methods exist to predict the feeding value of forages and feeds through their chemical composition and degradability in lab conditions, the in vitro rumen fermentation methods enable precise evaluation of the activity of the rumen microbial ecosystem during the digestion and the evaluation of fermentation end-products, especially gases, volatile fatty acids (VFA) and ammonia ($NH_3$) (Getachew et al., 2005). An important purpose of the studies using the in vitro techniques is to evaluate strategies allowing more efficient rumen fermentation, reduction of enteric methane ($CH_4$) production and excessive protein degradation in the rumen (Rymer et al., 2005), mainly by testing rumen fermentation manipulators such as additives or bioactive plant extracts (Makkar et al., 2005). Another interest in using these techniques is to better understand the specific needs of microbial populations, particularly in minerals and buffers to maintain a functionally optimal rumen environment (Boudon et al., 2019).

The standard method to collect rumen fluid (RF) serving as fresh inoculum for in vitro rumen fermentation assays is through the use of ruminally cannulated animals, which allows the collection of crude rumen digesta just before the inoculation of fermenters with a minimal loss of ecosystem integrity (Mould et al., 2005). However, the use of such animals is less and less accepted in some countries, and there is a need or obligation to use alternative sampling methods. Among these methods, oral-stomach sampling (OSS) to collect the RF by the esophagus and use it fresh appears to be a method respecting at the best the integrity of the microbial ecosystem in comparison to conservation methods such as freezing due to sensitivity of some microbial populations to cell lysis (Martinez-Fernandez et al., 2019). The OSS is a minimally invasive technique for collecting ruminal contents, although it may cause some temporary discomfort to the animal during the procedure. While the method may not target a specific ruminal compartment, it can serve as a suitable source of inoculum for batch fermenters. Additionally, OSS reduces the need for permanent cannulation, supporting ethical considerations by aligning with the 3Rs (Replace, Reduce, Refine). The OSS method also allows the use of experimental animals with controlled feeding and avoids the use of slaughtered animals as a source of ruminal inoculum, which may be problematic because the microbial community is compromised by the common practice of withholding feed for long prior to slaughter.

However, the execution of OSS can be source of variability in RF characteristics due to the dilution by the saliva when the tube is introduced in the esophagus, affecting pH (Duffield et al., 2004, Boudon et al., 2025) and microbial communities (Henderson et al., 2013). Another possible source of variability in RF is the sampling site in the rumen (Geishauser and Gitzel, 1996) as the rumen content is usually composed with three phases: the liquid phase, the fiber mass, and the gaseous phase which are located in a gradient from the ventral sac to the dorsal sac and from the caudal sac to the cranial sac. The OSS also presents challenges in obtaining a representative sample of suspended feed particles, which harbor the majority of ruminal bacteria (McAllister et al., 1994). All these factors are susceptible to affect the characteristics of the inoculum and subsequently to create some biases on in vitro rumen fermentation parameters.

While numerous studies have been conducted to compare the sampling methods of RF to assess its characteristics (pH, fermentation end-products, microbial communities) with different results (e.g. Shen et al., 2012, de Assis Lage et al., 2020), few studies have compared the different sampling methods on in vitro rumen fermentation parameters, and the number of substrates tested was rather low (Van Gastelen et al., 2019). Thus, to improve the knowledge on the feasibility of OSS method as an alternative to the use of cannulated animals, there is a need to test the fermentation of a large range of contrasted substrates (forages and diets).

The objective of this study was to compare the *in vitro* rumen fermentation parameters of forages and total mixed rations (TMRs) with different chemical compositions using inocula obtained by OSS and from two sampling sites in the rumen of cannulated dairy cows.

# 2. Material and methods

The experimental part of this study was conducted at INRAE experimental farm of Méjusseuma (IE PL, 35650 Le Rheu, France; https://doi.org/10.15454/yk9q-pf68) between 2020 January 4th and 2020 Mars 26th using the experimental plan described by Boudon et al. (2025) to test different RF sampling methods when investigating the dynamics of ruminal composition during the inclusion of an acidogenic diet in lactating dairy cows. The *in vitro* rumen fermentations were conducted at the Centre Mondial de l'Innovation (Groupe Roullier, Saint-Malo, France). The experimental design and the experimental procedures were approved by a Regional Ethic Committee and the French Ministry of Research under number APAFiS #26894-2020081715322100_v2.

## 2.1. Substrates for in vitro incubations

A total of 12 freeze-dried 1-mm substrates (6 forages and 6 diets) ground using a hammer mill equipped with a 1 mm screen (BJL-8500, JL Boisson, Lusignan, France), was used as a representative set of temperate forage species and TMRs. The forages were: 2 grasses, namely cocksfoot (C, *Dactylis glomerata*) and meadow fescue (MF, *Festuca pratensis*); 2 non-tanniferous legumes, namely white clover (WC, *Trifolium repens*) and red clover (RC, *Trifolium pratense*); 1 tanniferous legume, namely sainfoin (S, *Onobrychis vicifolia*) and 1 sample of permanent grassland (PG) harvested at first stage of vegetation. The chemical composition of forage substrates is shown in Table S1. The TMR were: 3 TMR rich in fibers containing corn silage, hay and concentrate (on a dry matter (DM) basis, 50:30:20) either not supplemented (TMRF) or supplemented with Ca (TMRF+Ca) or supplemented with P (TMRF+P); 3 acidogenic TMR containing corn silage, soyabean meal and concentrate (33:17:50) either not buffered (TMRA) or buffered with bicarbonate (TMRA+Bic) or buffered with magnesium oxide (TMRA+MgO). The ingredients and chemical composition of TMR substrates are shown in Table S2.
## 2.2. Experimental design

A total of 9 runs of fermentation over 3 months (3 feeding periods of 1 month, 3 runs/period) were carried out. At each run, 4 substrates (2 forages and 2 diets) and a blank without substrate were incubated using RF collected in 3 different ways (see Section 2.3) from 4 cows. The donor cows were considered as the biological replicates, and thus served as the source of replication in the statistical model. In this way, all substrates were tested in each feeding period, alternating the distribution of substrates between runs within each period. Thus, 60 fermenters were used at each run, and each combination substrate-type of RF was tested 12 times (3 feeding periods × 4 cows) throughout the whole experiment.

## 2.3. Animals, collection and preparation of buffered RF

The experimental plan was detailed by Boudon et al. (2025). Briefly, four early lactation dairy cows (4.9 ± 0.8 years old, 712 ± 35 kg bodyweight) equipped with rumen cannula were used as donors of RF over 3 months. The animals were fed a diet based on corn silage, soya meal, dehydrated alfalfa, concentrate, minerals and vitamins, and had free access to water. To create a variability in RF characteristics, the experiment was divided in three successive one-month-feeding periods with a control diet (periods 1 and 3) and an acidogenic diet (period 2). The diets were distributed daily for 60 % at 9h00 and for 40 % at 17h00.

The day of incubations (occurring between d19 and d26 of each feeding period), before morning feeding, 500 ml of RF of each cow were collected i) in the reticulum (RF<sub>ret</sub>), ii) in several parts of the rumen, i.e reticulum and ventral sac (RF<sub>mix</sub>), and iii) using OSS (RF<sub>tub</sub>) after eliminating at least the first 200 ml collected to reduce the contamination by saliva when the probe passes through the oesophagus. The selection of sampling sites was based on the following considerations: the standard method for obtaining inoculum involves collecting a representative sample from the entire reticulorumen (mix = reference). However, there is a potential risk that inoculum obtained via OSS may not be fully representative of the whole rumen content. This risk arises from the possibility that the tube does not reach sufficient depth, resulting in sampling from the dorsal part of the rumen. The dorsal region, like the reticulum, tends to be more contaminated with saliva (Shen et al., 2012). To assess the impact of this potential bias on gas test outcomes, we also included in our comparison a sample collected from the bottom of the reticulum, which is significantly more reproducible and easier to standardize than sampling from the dorsal rumen.

The OSS system consisted in a semi-flexible 300-cm hose (20-mm diameter) made in Polyvinyl Chloride (PVC, RS Pro, UK), a manual sampling pump (Ammerlaan Import, Longnes, France) and a collecting flask (1 l Duran borosilicate glass bottle) placed between the tube and the pump (Popova et al., 2022). The inserted length was 200 cm with a stainless-steel strainer at the tip comprising 24 holes (10-mm diameter). The tube and the procedure were designed to reach the ventral sac of the rumen. At each sampling, 500 ml were taken after discarding the initial 200 ml.

Each type of RF from each cow was transferred in a separate thermos flask (n = 12), placed in a box at temperature regulated at 39 °C, transported rapidly to the laboratory (1 h transportation), and filtered through 3 layers of surgical-grade gauze (Sylamed, Paris, France). The fermentative characteristics of the different types of RF at sampling are shown in Table 1. The obtained RF were mixed 1:2 (v/v) in an artificial saliva as described by (Menke and Steingass, 1988), and used as inoculum for the *in vitro* incubations.

## 2.4. *in vitro* incubation procedure, measurements and sampling

Each substrate (0.5 g) was incubated anaerobically with 60 ml of buffered RF in 100 ml fermenters that had been flushed with N₂ to eliminate the residual oxygen, using an automated ANKOM gas production system (Ankom RF; Ankom Technology Corp., Macedon, NY) at 39 °C for 24 h. At each run, for each type of RF and each repetition (cow), a blank containing the corresponding buffered RF without substrate was included. The gas production (GP) in fermenters was determined continuously using a pressure sensor as described by Vargas-Bello-Pérez et al. (2022). The absolute pressure was recorded every 5 min to calculate cumulative pressure and converted to volume using the ideal gas law. The gas produced during the whole incubation time was collected in bags (Supel-Inert Multi-Layer Foil, 0.6 L Screw Cap Valve (SCV), Supelco, Darmstadt, Germany) for analysis of CH₄ and CO₂. At the end of the incubation period (24 h), the fermenters were transferred into the ice to stop the fermentation. The whole content of the fermenter was transferred into two 50 ml Falcon® centrifugation tubes and centrifuged at a relative centrifugal force (RCF) of 2800 g for 15 min. The supernatant was sampled using conservative solutions as described by Niderkorn et al. (2011) and stored at -20 °C for VFA and NH₃ analyses. The sampling of supernatant was done in exactly the same conditions on blanks at t = 0 at each run of fermentation and for each cow (n = 12 per run). The residue obtained after centrifugation were freeze dried (Lyovac GT2, SRK Systemtechnik GmbH, Riedstadt, Germany) and used for determination of DM and fiber (NDF and ADF) analysis.

## 2.5. Chemical analyses

Forage and TMR substrates, and rumen digesta were analyzed for DM by oven-drying at 103 °C for 48 h. Organic matter (OM) in forage substrates was analyzed by ashing at 550 °C for 6 h in a muffle furnace. The NDF and ADF contents, both expressed exclusive of residual ash, in substrates and residues were estimated using near infrared reflectance (NIR) spectroscopy at the Centre Mondial de l'Innovation (Saint-Malo, France) using a Brucker Tango (Bruker Optik GmbH, Ettlingen, Germany). Total crude protein (CP) was determined according to the AOAC (1995) Kjeldhal method 976.06. The differences between DM, NDF and ADF in plant material before the fermentation and in residue after 24 h of fermentation were used to calculate *in vitro* DM, NDF and ADF degradability (IVDMD, IVNDFD and IVADFD), respectively.
<table><caption>**Table 1**

Fermentative characteristics (pH, volatile fatty acids (VFA) and ammonia (NH₃) concentration) of the different types of rumen fluid (RF).</caption><thead><tr><th rowspan="2"></th><th colspan="3">Period 1</th><th colspan="3">Period 2</th><th colspan="3">Period 3</th></tr><tr><th>RF<sub>ret</sub></th><th>RF<sub>mix</sub></th><th>RF<sub>tub</sub></th><th>RF<sub>ret</sub></th><th>RF<sub>mix</sub></th><th>RF<sub>tub</sub></th><th>RF<sub>ret</sub></th><th>RF<sub>mix</sub></th><th>RF<sub>tub</sub></th></tr></thead><tbody><tr><td>pH</td><td>6.77 ± 0.12</td><td>6.75 ± 0.07</td><td>7.04 ± 0.13</td><td>6.98 ± 0.07</td><td>6.97 ± 0.07</td><td>7.36 ± 0.20</td><td>6.94 ± 0.05</td><td>6.93 ± 0.05</td><td>7.32 ± 0.13</td></tr><tr><td>VFA (mmol/L)</td><td>89 ± 5</td><td>108 ± 22</td><td>75 ± 7</td><td>80 ± 7</td><td>87 ± 8</td><td>62 ± 16</td><td>99 ± 6</td><td>97 ± 11</td><td>78 ± 11</td></tr><tr><td>NH<sub>3</sub> (mmol/L)</td><td>3.10 ± 1.49</td><td>3.46 ± 1.66</td><td>3.00 ± 1.14</td><td>1.33 ± 1.88</td><td>2.56 ± 1.99</td><td>2.79 ± 1.40</td><td>2.99 ± 1.10</td><td>3.22 ± 1.23</td><td>3.00 ± 0.93</td></tr></tbody></table>

RF<sub>ret</sub>, rumen fluid from the reticulum; RF<sub>mix</sub>, rumen fluid from reticulum and ventral sac; RF<sub>tub</sub>, rumen fluid obtained by oral-stomach sampling;
The total and individual VFA in the supernatant were analyzed by gas chromatography (PerkingElmer Clarus 580CPG), according to Jouany (1982). For this analysis, isovalerate specifically included 3-methylbutyrate and 2-methylbutyrate. Ammonia was measured using the Berthelot reaction (Weatherburn, 1967). The composition of fermentation gases ($CH_4$, $CO_2$) was determined by micro-GC-MS

<figure><img src="image_5.png" /><figcaption>Fig. 1. *in vitro* rumen fermentation parameters of forages and total mixed rations (TMRs) according to the type of rumen fluid (RF) (n = 3). For each type of substrates, the substrate × RF interaction effect and the RF effect are shown. The error bars represent the standard error.</figcaption></figure>
(Agilent 1260, Agilent Technologies, Ltd., Santa Clara, CA, USA) in the laboratory OSU (Rennes, France).

## 2.6. Statistical analysis

Linear mixed effect models were applied using the package (lmerTest) within R (R Core Team, 2021) v 4.1.1, in which the cow and the feeding period were random factors while the substrate, the type of RF and their interaction were considered as fixed effects (Table 3). A similar analysis was conducted by considering forages and TMRs separately (Fig. 1), and an analysis by contrasts was done to compare i) TMRF, TMRF+Ca and TMRF+P, and ii) TMRA, TMRA+MgO and TMRA+MgO (Table S4). Another mixed model was applied to analyze specifically the effect of the feeding period and interactions with substrate and the type of RF, in which just the cow was used as random factor (Table S5). Post-hoc pair-wise comparisons were conducted with the packages (emmeans) and (multcomp) using Tukey tests. For all the models, differences of GP-6h, GP-24h, % CH₄, IVDMD, IVNDFD, IVADFD, total and proportions of individual VFA, and NH₃ were declared significant at P < 0.05.

# 3. Results

## 3.1. Interaction effects between substrate and type of RF

There was no interaction effect between substrate and type of RF (Table 3). Considering specifically forages and TMRs, no interaction effect between substrate and type of RF was observed (Fig. 1).

## 3.2. Effect of substrates

Clear and highly significant effects of the substrate were observed on all fermentation parameters (P < 0.001; Table 3 and S3). A wide range of variation was found across substrates for GP, CH₄ proportion, degradability, and fermentation end-products. TMRA and WC generally showed the highest values for GP and *in vitro* degradability, while TMRF+MgO, PG, and S were among the lowest. The CH₄ proportion was highest for TMRAs and supplemented TMRFs, and lowest for forage substrates. Total VFA production was maximal for TMRA and MF, and minimal for supplemented TMRFs, PG, S and C. Substrate type also strongly influenced VFA profiles, with forage-based substrates yielding more acetate and TMRAs yielding more propionate and butyrate. The effects of mineral supplementation (Ca, P, MgO, Bic) on fermentation traits were variable and substrate-dependent (Table S4).

## 3.3. Effect of type of RF

The GP-6h and GP24h were higher for RFₘix than for RFᵣet (Tables 2 and 3). The proportion of CH₄ in fermentation gas was lower for RFᵣet than for the other types of RF. The proportion of acetate and the ratio A/P were lower for RFᵣet than for the other types of RF. The proportion of propionate was lower for RFᵣub than for RFₘix, which was lower than for RFᵣet. The proportions of total iso-VFA and iso-valerate were lower and the proportion of isobutyrate was higher for RFᵣub than for the other types of RF. NH₃ was higher for RFᵣub

<caption>**Table 2**</caption>

*in vitro* rumen fermentation parameters measured according to the type of rumen fluid (RF) (n = 3).

<table><thead><tr><th></th><th>RF<sub>ret</sub></th><th>RF<sub>mix</sub></th><th>RF<sub>tub</sub></th><th>SE</th></tr></thead><tbody><tr><td>GP-6h, ml/g DM</td><td>60.6<sup>a</sup></td><td>65.0<sup>b</sup></td><td>61.7<sup>ab</sup></td><td>1.26</td></tr><tr><td>GP-24h, ml/g DM</td><td>129<sup>a</sup></td><td>136<sup>b</sup></td><td>132<sup>ab</sup></td><td>1.8</td></tr><tr><td>CH<sub>4</sub>, %</td><td>1.94<sup>a</sup></td><td>2.83<sup>b</sup></td><td>2.99<sup>b</sup></td><td>0.138</td></tr><tr><td>IVDMD, %</td><td>59.2</td><td>59.4</td><td>57.7</td><td>0.76</td></tr><tr><td>IVNDFD</td><td>69.4</td><td>69.0</td><td>68.3</td><td>0.59</td></tr><tr><td>IVADFD</td><td>68.1</td><td>67.7</td><td>68.9</td><td>1.09</td></tr><tr><td>Total VFA, mmol/g DM</td><td>6.87</td><td>6.82</td><td>7.00</td><td>0.113</td></tr><tr><td colspan="5"><em>VFA molar proportions, mol/100 mol</em></td></tr><tr><td>Acetate</td><td>57.7<sup>a</sup></td><td>58.4<sup>b</sup></td><td>58.3<sup>b</sup></td><td>2.67</td></tr><tr><td>Propionate</td><td>26.3<sup>c</sup></td><td>25.7<sup>b</sup></td><td>25.0<sup>a</sup></td><td>2.28</td></tr><tr><td>Butyrate</td><td>10.0</td><td>9.9</td><td>10.3</td><td>0.46</td></tr><tr><td>Valerate</td><td>2.22<sup>ab</sup></td><td>2.15<sup>a</sup></td><td>2.29<sup>b</sup></td><td>0.681</td></tr><tr><td>Caproate</td><td>0.45</td><td>0.44</td><td>0.46</td><td>0.045</td></tr><tr><td>Isobutyrate</td><td>1.03<sup>a</sup></td><td>1.00<sup>a</sup></td><td>1.14<sup>b</sup></td><td>0.104</td></tr><tr><td>Isovalerate</td><td>1.41<sup>a</sup></td><td>1.43<sup>a</sup></td><td>1.65<sup>b</sup></td><td>0.115</td></tr><tr><td>Total iso-VFA</td><td>2.44<sup>a</sup></td><td>2.43<sup>a</sup></td><td>2.78<sup>b</sup></td><td>0.203</td></tr><tr><td>Ratio A/P, mol/mol</td><td>2.26<sup>a</sup></td><td>2.34<sup>b</sup></td><td>2.41<sup>b</sup></td><td>0.039</td></tr><tr><td>NH<sub>3</sub>, mmol/g DM</td><td>0.342<sup>a</sup></td><td>0.399<sup>ab</sup></td><td>0.447<sup>b</sup></td><td>0.0362</td></tr></tbody></table>

RFᵣet, rumen fluid from the reticulum; RFₘix, rumen fluid from reticulum and ventral sac; RFᵣub, rumen fluid obtained by oral-stomach sampling; GP, gas production; IVDMD, *in vitro* dry matter degradability; IVNDFD, *in vitro* neutral detergent fibre degradability; IVADFD, *in vitro* acid detergent fibre degradability; VFA, volatile fatty acids; Ratio A/P, ratio acetate/propionate.

Data shown are mean values calculated from the *in vitro* runs carried out with 12 plant substrates during three feeding periods. <sup>a-c</sup>Means within a row with different subscripts differ (P < 0.05).
than for RF<sub>ret</sub> (RF<sub>mix</sub> intermediary).

Considering specifically the different types of substrates (forages and TMRs), the type of RF affected all the parameters except for IVNDFD, IVADFD, and the proportions of butyrate and caproate for forages, and IVDMD, IVNDFD, IVADFD, NH<sub>3</sub>, total VFA and the proportions of butyrate and caproate for TMRs (Fig. 1).

### 3.4. Effect of feeding period and interactions

Very clear effects of feeding period were observed on all the parameters (P < 0.001) except for IVDMD (Table 3 and S5). The GP-6h was higher in feeding period 2 than in the other feedings (period 2 > period 3 > period 1), while values for GP-24h and the proportion of CH<sub>4</sub> in the gas were lower in feeding period 2 than in the other feeding periods (for GP-24h, period 3 > period 1 > period 2). The IVNDFD and IVADFD were higher in feeding period 2 than in the other feeding periods (for IVADFD, period 2 > period 3 > period 1). The total VFA production was lower in feeding period 3 than in the other feeding periods. The proportions of VFA were all affected by the feeding period. In particular, the ratio A/P was higher in feeding period 1 than in the other feeding periods (period 1 > period 3 > period 2), and the proportion of total iso-VFA was higher in period 2 compared to other periods (period 2 > period 3 > period 1). There was significant interaction effect between substrate and feeding period on all the parameters, except for CH<sub>4</sub> proportion and IVDMD (Table 3). Significant interaction effects between feeding period and type of RF were observed on iso-VFA, minor VFA and the ratio A/P.

## 4. Discussion

### 4.1. Validation of the set of substrates with contrasted rumen fermentation profiles

Overall, the observed values were in the range of those generally observed for the parameters from *in vitro* rumen fermentation of a variety of temperate forages and TMRs (Niderkorn et al., 2011, Li et al., 2021). As expected, we obtained a large range of rumen fermentation profiles allowing us to test in good conditions the impact of the type of RF used as inoculum for the *in vitro* assays. The clear variability in acetate:propionate ratio for forage substrates (higher values for legumes than for grasses and PG) and TMR substrates (higher values for the TMRFs than for the TMRAs) reflects contrasted carbohydrate metabolism pathways. This was consistent with numerous previous studies reporting higher acetate production from fibrous diets and increased propionate from starch-rich substrates, highlighting the influence of carbohydrate type on VFA profiles (Cantalapiedra-Hijar et al., 2009; Russell and Rychlik, 2001). The variability in rumen fermentation profiles was also reinforced by the strong effect of feeding periods differing in terms of diet offered to the cows (control vs. acidogenic diets). In particular, we observed a clear decrease in the CH<sub>4</sub> proportion in the gas and in the ratio A/P when RF was sampled during the period during which cows were fed with an acidogenic diet. These results are consistent with those observed by Kim et al. (2018) reporting changes in the rumen microbiome, CH<sub>4</sub> concentration, and methanogen diversity and abundance in cattle fed diets at different concentrate levels. In our study, the presence of interactions between substrate and feeding period underscores the importance of using standardized diets to feed the animals from which the RF is collected for the evaluation of *in vitro* rumen fermentation parameters of different substrates. The addition of minerals in TMRF created variability, even if it has to be noted that the overall reduction of GP, VFA production and degradability values when adding Ca or P contrasted

<table><caption>**Table 3**
Effects of substrate, type of rumen fluid, feeding period and their interactions on *in vitro* rumen fermentation parameters.</caption><thead><tr><th rowspan="2"></th><th colspan="6">P-values</th></tr><tr><th>Effect substrate</th><th>Effect type of RF</th><th>Effect substrate × type of RF</th><th>Effect period</th><th>Effect substrate × period</th><th>Effect type of RF × period</th></tr></thead><tbody><tr><td>GP-6h</td><td>&lt; 0.001</td><td>0.017</td><td>0.563</td><td>&lt; 0.001</td><td>0.001</td><td>0.659</td></tr><tr><td>GP-24h</td><td>&lt; 0.001</td><td>0.007</td><td>0.528</td><td>&lt; 0.001</td><td>0.023</td><td>0.981</td></tr><tr><td>CH<sub>4</sub>, %</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.318</td><td>&lt; 0.001</td><td>0.400</td><td>0.367</td></tr><tr><td>IVDMD</td><td>&lt; 0.001</td><td>0.216</td><td>0.650</td><td>0.978</td><td>0.234</td><td>0.801</td></tr><tr><td>IVNDFD</td><td>&lt; 0.001</td><td>0.411</td><td>0.785</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.558</td></tr><tr><td>IVADFD</td><td>&lt; 0.001</td><td>0.749</td><td>0.661</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.757</td></tr><tr><td>Total VFA</td><td>&lt; 0.001</td><td>0.359</td><td>0.309</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.115</td></tr><tr><td colspan="7"><em>VFA molar proportions</em></td></tr><tr><td>Acetate</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.562</td><td>&lt; 0.001</td><td>0.001</td><td>0.121</td></tr><tr><td>Propionate</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.993</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.440</td></tr><tr><td>Butyrate</td><td>&lt; 0.001</td><td>0.048</td><td>1.000</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.709</td></tr><tr><td>Valerate</td><td>&lt; 0.001</td><td>0.020</td><td>0.981</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>&lt; 0.001</td></tr><tr><td>Caproate</td><td>&lt; 0.001</td><td>0.137</td><td>0.967</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>&lt; 0.001</td></tr><tr><td>Isobutyrate</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.913</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.013</td></tr><tr><td>Isovalerate</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.994</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.004</td></tr><tr><td>Total iso-VFA</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.989</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.008</td></tr><tr><td>Ratio A/P</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.861</td><td>&lt; 0.001</td><td>0.007</td><td>0.044</td></tr><tr><td>NH<sub>3</sub></td><td>&lt; 0.001</td><td>0.028</td><td>0.939</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.186</td></tr></tbody></table>

RF, rumen fluid; GP, gas production; IVDMD, in vitro dry matter degradability; IVNDFD, in vitro neutral detergent fibre degradability; IVADFD, in vitro acid detergent fibre degradability; VFA, volatile fatty acids; Ratio A/P, ratio acetate/propionate.
with previous observations (e.g Gülşen et al., 2006). Similar effects were observed when adding buffers in TMRA.

## 4.2. Overall effect of RF sampling method

The characteristics of RF are potentially affected by the site of sampling in the rumen and the sampling method (rumen canula vs. OSS) (Shen et al., 2012). In our study, a main effect of the type of RF sampling was observed on GP at 6 h and 24 h which was higher using RF<sub>mix</sub> than for RF<sub>ret</sub>. But besides, there was a limited effect of the type of RF on the overall fermentability of substrates as shown by the similar values for DM and fiber degradabilities and total VFA production. In particular, it seems that the well-described dilution of RF by saliva when sampled using OSS (Duffield et al., 2004) does not impact negatively the intensity of the *in vitro* rumen fermentation compared to the standard method (RF<sub>mix</sub>). This result supports the beneficial effect of the methodology consisting to eliminate the first 200 ml collected to reduce the contamination by saliva when using OSS, even differences still remain on the pH and VFA of the inoculum (Boudon et al., 2025). Regarding CH<sub>4</sub> expressed in proportion of the total gas produced, it is interesting to observe that the results from RF collected by OSS (RF<sub>tub</sub>) were similar to those from inoculum collected in different points of the rumen (RF<sub>mix</sub>, standard method). This last result is comforted by similar results on the acetate proportion and acetate:propionate ratio which were shown to be highly correlated with CH<sub>4</sub> production (Russell, 1998). Indeed, acetate production results in the generation of reducing equivalents, whereas propionate production involves their consumption. Consequently, a decrease in the acetate:propionate ratio reflects a shift in electron balance that limits hydrogen availability for methanogenesis, thereby reducing CH<sub>4</sub> production (Wolin, 1960). The CH<sub>4</sub> production is a very important criteria to evaluate *in vitro* the potential of mitigation of some resources, feed additives and plant extracts, or to investigate the mode of action of rumen manipulators. So, according to our results the sampling of fresh RF using OSS remains adequate to evaluate accurately CH<sub>4</sub> production comparatively to the standard method. By contrast, using three substrates, some differences on CH<sub>4</sub> production and proportion between OSS and cannulation have been reported by Van Gastelen et al. (2019). This could be due to technical differences in sampling by both OSS and through canula as it was shown that the exact location of sampling in the rumen can change the values of rumen parameters (Shen et al., 2012). Our results also indicate this kind of difference as shown by the differences in rumen fermentation parameters between RF<sub>ret</sub> and RF<sub>mix</sub>. This variability highlights i) the need of a well standardized methodology when practicing OSS, and ii) the confirmation that the values obtained by OSS have to be considered as relative differences rather than in absolute terms (Boudon et al., 2025).

Another effect of the type of RF sampling was on NH<sub>3</sub> for which a difference was observed between fermentations done using RF<sub>tub</sub> and RF<sub>ret</sub>. NH<sub>3</sub> is also an important parameter as final end-product from microbial breakdown of protein, and consequently an indicator of efficiency of rumen protein utilization and urinary nitrogen losses. Similarly, to results on CH<sub>4</sub>, no significant difference was observed on NH<sub>3</sub> between fermentations done using RF<sub>tub</sub> and the standard method (RF<sub>mix</sub>). However, it has to be noted that the difference observed on iso-VFA (isobutyrate and isovalerate) between these two sampling methods may indicate a slight increase in protein breakdown with RF<sub>tub</sub> as these end-products are known to be related to deamination of branched-chain amino acids such as valine and leucine (Andries et al., 1987). A possible explanation could be related to the sampling of different ruminal phases, as the liquid phase may harbor a higher proportion of proteolytic and amino acid-fermenting microbes, whereas the solid phase is generally dominated by fibrolytic bacteria attached to feed particles (Kung and Hession, 1995).

Despite identical diets in periods 1 and 3, fermentation parameters did not fully return to baseline, suggesting a persistent effect of the acidotic challenge. This may reflect an incomplete recovery of the rumen microbial community, which is known to require several weeks to stabilize after dietary disturbances (Snelling et al., 2019).

## 4.3. Absence of interaction effect between substrate and RF sampling method

In this study, the interaction effect between substrate and the type of RF sampling was the key element to be taken into account in order to provide a recommendation on the suitability of OSS for the collection of fresh RF used as inoculum in *in vitro* rumen fermentation assays. The absence of such interaction using a large range of substrates on all the parameters indicates that OSS is a suitable alternative to rumen cannulation, at least to classify substrates or treatments. These results are only partly consistent with those reported by Van Gastelen et al. (2019) that showed few substrate × inoculum interactions. The absence of such interactions in our study may suggest that discarding the first 200 ml of rumen fluid collected by OSS helps reduce potential biases, which could result from differences in physico-chemical characteristics (Duffield et al., 2004) and microbial composition (Henderson et al., 2013). However, this interpretation should be made with caution, as no direct comparison with saliva-contaminated samples was performed. Overall, these results reasonably support that this method used with caution and a standardized procedure may be suitable in the context of the obligation to find alternative to the use of cannulated animals in research.

# 5. Conclusions

Overall, our results obtained on the rumen fermentation of a large range of forage and TMR substrates showed that OSS can be an acceptable alternative to the RF sampling using cannulated animals. In particular, we observed limited differences between OSS and the standard method, and no interaction between substrate and type of RF, highlighting the possibility to use OSS to provide inoculum for *in vitro* batch systems and to measure relative differences among substrates or treatments. In order to keep OSS as a research tool for ruminant scientists on the long term, it would be useful to continue the efforts undertaken to improve the standardization of the OSS method and its refinement to decrease discomfort for the animal, in parallel to the exploration of other potential alternatives such as the conservation of inocula.
CRediT authorship contribution statement

**Angelique Torrent:** Resources, Methodology, Formal analysis. **Sandra Point:** Writing – review & editing, Validation, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Vincent Niderkorn:** Writing – original draft, Data curation, Conceptualization. **Anne Boudon:** Writing – review & editing, Supervision, Investigation, Funding acquisition, Conceptualization. **Mathieu Silberberg:** Methodology, Investigation, Funding acquisition, Conceptualization. **Pierre Nozière:** Writing – review & editing, Project administration, Funding acquisition.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgements

The authors acknowledge the financial support provided by INRAE (department Animal Physiology and Livestock Systems) through the projects EvalRumPH and Alterfi. This research was also supported by the companies ADISSEO, CARGILL-PROVIMI, CCPA-DELTAVIT, CMI-ROUILLER, MG2MIX, and LALLEMAND. The authors also acknowledge the CMI Roullier (France) for laboratory facilities, and staff involved in rumen fermentation assays and lab analysis.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.anifeedsci.2025.116416.

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