Effect of Variations in the Gas Outlet Location on an In Vitro Rumen Simulation Technique (RUSITEC^®) System

Abstract

The rumen simulation technique (RUSITEC^®) is a known model for research in rumen microbiology and fermentation. However, our research group observed inconsistencies in gas production across trials. This study investigated the effects of different gas outlet locations on digestibility, ruminal fermentation, gas production, and microbial protein synthesis. Fifteen fermenters tested three different gas outlet locations within the RUSITEC^® equipment: (1) gas outlet directly on the effluent vessel for output liquid (EV); (2) gas outlet directly on fermenter cap (F); and (3) gas outlet on both effluent vessel and fermenter cap (EVF). Data were analyzed using a completely randomized design in SAS (v. 9.4) with the MIXED procedure, and significance was set at p < 0.10. Results showed that altering the gas outlet location did not affect nutrient digestibility (p > 0.10), microbial protein synthesis (p > 0.10), and volatile fatty acid (VFA) production when expressed on a molar basis (p > 0.10). However, total gas production (p = 0.108) was higher in the EVF group and ammonia nitrogen produced in the fermenter was higher in group F (p = 0.081). Furthermore, methane (CH₄) production was underestimated when the gas outlet location was in just one of the locations when compared to the EVF group (p = 0.006). VFA proportion was also affected, with lower acetate (p = 0.005) and higher butyrate (p = 0.014) for group EV. These results indicate that the location of the gas outlet is an important methodological factor affecting fermentation measurements in the RUSITEC system, with outlets positioned in both the effluent and fermenter vessels enhancing gas recovery.

1. Introduction

The rumen simulation technique (RUSITEC^®) is a semicontinuous in vitro model originally developed by Czerkawski and Breckenridge [1] that has since become a well-established tool for investigating ruminal fermentation under controlled laboratory conditions. By maintaining mixed ruminal microbial populations in a semi-continuous culture environment, RUSITEC^® supports sustained fermentation over multiple days, enabling researchers to continuously monitor digestion dynamics, nutrient utilization, and end-product formation [1]. One of the principal strengths of in vitro methodologies such as RUSITEC^® lies in their capacity to generate high-resolution data with reduced costs, faster turnaround, and enhanced environmental control when compared to traditional in vivo trials [2].

In ruminant nutrition research, the reliability of any in vitro system depends largely on its ability to faithfully replicate the complex physicochemical and microbial environment of the natural rumen. Key parameters such as incubation temperature, pH stability, redox potential, dilution rate, and digesta turnover must be carefully maintained to preserve microbial diversity and fermentation pathways that resemble those occurring in vivo [2]. When successful, these systems enable mechanistic studies of nutrient breakdown, methane (CH₄) production, and microbial protein synthesis without the ethical and logistical constraints associated with cannulated animals or whole-animal metabolism studies [3].

A key advantage of the RUSITEC^® system is its ability to quantify both the volume and composition of gases produced during fermentation, including CH₄ and carbon dioxide (CO₂), which are critical indicators of ruminal fermentation efficiency and environmental impact [4]. Precise gas measurement is especially important given the increasing emphasis on CH₄ mitigation in ruminant systems, as enteric CH₄ represents a significant contributor to agricultural greenhouse gas emissions [5]. Accurate quantification of gas output allows for comparisons across dietary treatments, feed additives, and microbial modulators, making RUSITEC^® a valuable platform for screening mitigation strategies [4].

A study conducted by Adebayo Arowolo et al. [6] indicates that fermentation gases in RUSITEC^® are initially dissolved in the liquid phase and then evolve to the gaseous headspace, and that saturation factors reflect the rate at which dissolved gas moves into the gas phase.

These findings underline the importance of understanding gas partitioning and sampling methodologies within semi-continuous culture systems, particularly when comparing gas output between treatments or experimental configurations. Despite growing use of RUSITEC^® for gas measurements, relatively little attention has been paid to the implications of gas outlet location on the capture and quantification of fermentation gases.

In our laboratory, ongoing investigations have identified potential factors affecting the accuracy and consistency of gas outflow measurements, prompting a reconsideration of the standard gas collection methodology. Specifically, the possibility was raised that gas produced in the liquid phase or within areas of the vessel not directly connected to a single outlet may escape detection when using a single sampling point. Dual gas collection ports (one at the top of the fermenter and an additional outlet positioned lower on the vessel) could potentially capture gases that otherwise remain dissolved or are released outside of the primary sampling line, improving the recovery of total fermentation gases. Improved gas capture would be particularly consequential for CH₄ measurements, which are essential for evaluating dietary mitigation strategies and understanding fermentation energetics [7].

Based on these considerations, we hypothesize that the implementation of dual gas outlets in a RUSITEC^® system would enhance the recovery of fermentation gases, yielding greater measured CH₄ output without disrupting core ruminal fermentation parameters or nutrient digestibility. The primary objective of the present study was to evaluate the effect of different gas sampling points in a RUSITEC^® system on digestibility, ruminal fermentation characteristics, gas production dynamics, and microbial protein synthesis. By elucidating the influence of gas outlet configuration on fermentation measurements, this work aims to refine methodological approaches in in vitro rumen modeling and improve the accuracy of fermentation gas quantification used in ruminant nutrition research.

2. Material and Methods

The current experiment was carried out at Washington State University (Pullman, WA, USA), in accordance with the animal care guidelines approved by the Institutional Animal Care and Use Committee (WSU/IACUC; ASAF #6608 approved on 1 July 2021).

2.1. Experimental Design and Treatments

The RUSITEC^® (adapted from Czerkawski and Breckenridge [1]; Pullman, WA, USA) system used in this study was custom designed and constructed at the Washington State University (WSU) Department of Animal Science Farm Shop, incorporating modifications to accommodate specific experimental requirements relative to the original design described by Czerkawski and Breckenridge [1]. In WSU’s configuration, the gas outlet is positioned at the top of the fermenter cap, along with dedicated ports for the artificial saliva inlet and liquid effluent outlet. This configuration aims to maintain anaerobic conditions while providing discrete sampling points for effluent, saliva, and fermentation gases, allowing for detailed temporal monitoring of fermentation dynamics.

The experiment was carried out using a total of 15 fermenters, which were evenly distributed across treatments, with five units allocated per treatment. A completely randomized experimental design was implemented, comprising three treatments that differed according to the location of gas output within the system. Detailed descriptions of the RUSITEC^® system setup, including its configuration, operational procedures, and daily management routines, are provided by Castro et al. [8].

The study extended over a 10-day period, consisting of an initial 7-day adaptation phase to allow for the stabilization of fermentation conditions, followed by a 3-day sampling phase (days 8 through 10). The experimental treatments were defined based on variations in the gas outlet position within the fermenters, as described below:

• Effluent vessel (EV): gas outlet directly on the effluent vessel for output liquid.
• Fermenter (F): gas outlet directly on fermenter cap.
• Effluent vessel + Fermenter (EVF): gas outlets on both effluent vessel and fermenter cap.

Throughout the trial, fermenters were supplied with a total mixed ration (TMR;

Table 1. Chemical composition of the ingredients, (% of DM unless otherwise noted of the total mixed ratio diet).

Ingredient Composition	% DM
Corn silage	24.20
Alfalfa hay	12.52
Triticale silage	12.27
Corn ground fine	18.78
Soybean meal, 48%	7.19
Urea	1.06
Barley	8.35
Pea screenings	0.00
Rice hulls	14.49
Mineral mixture 1	1.14
Chemical Composition
Dry matter (DM)	91.87
Crude protein (CP)	13.37
Non-fibrous carbohydrate (NFC) 2	39.57
Neutral detergent fiber (NDF)	36.14
Ether-extract (EE)	2.90 *
Ash	8.02
Starch	37.02

¹ Contained per kilogram of the supplement: 0.0079 g of vitamin A 30 S, 0.0021 g of vitamin D 30 S, 1.1263 g of vitamin E Lutavin 50, 103.03 g of ammonium sulfate, 209.54 g of limestone, 264.91 g of dicalcium phosphate, 421.30 g of salt (iodized, 0.01%), 0.0159 g of cobalt carbonate and 0.0562 g of sodium selenite. ² NFC = 100 − (CP + NDF + EE + Ash), NRC [9]. * Estimated value.

). Feedstuffs were ground through a 4-mm screen using a Wiley mill (Model 4, Philadelphia, PA, USA) and placed in polyester bags for incubation.

2.2. Experimental Apparatuses and Incubations

Each fermenter was hermetically sealed with an airtight cap, functioning as a closed thermodynamic system to ensure anaerobic conditions and the complete capture of gas and liquid outputs. The lid was equipped with three ports: an inlet for continuous infusion of artificial saliva, a gas outlet connected to the fermenter headspace, and a liquid effluent outlet. For 24-h gas collection, the gas outlet was connected to 5-L polypropylene sampling bags (Tedlar bags; Environmental Samply Supply Inc., Oakland, CA, USA), allowing for quantitative recovery of fermentation gases while maintaining system integrity. The liquid effluent outlet was connected via Tygon tubing (3/8″ ID × 1/2″ OD; VWR Scientific^®, model 1370 GM, Radnor, PA, USA) to sealed 2-L plastic collection flasks maintained in an ice bath to immediately halt microbial activity and prevent post-collection fermentation or volatilization of fermentation products. These collection flasks were also equipped with gas outlets connected to sampling bags to ensure proper pressure equilibration and prevent gas loss. Effluent volume and weight were recorded daily to quantify liquid outflow. In accordance with the experimental design, gas outlets not required for specific treatments were securely sealed to maintain strict closed-system conditions and prevent gas leakage or atmospheric contamination.

At the beginning of the trial, each fermenter was inoculated with 2200 mL of ruminal fluid obtained from two cannulated Angus cows (759.77 ± 64.15 kg), following Ribeiro et al. [10], fed a chopped hay diet (55% alfalfa, 25% straw, 10% barley hay, and 10% water). Rumen fluid was collected 3 h after morning feeding, filtered through four layers of cheesecloth (grade 40), and transported to the laboratory in pre-warmed insulated containers. Additionally, 400 g of solid digesta (200 g per cow) was collected as initial inoculum. In the laboratory, ruminal fluid was homogenized, filtered again, maintained under continuous O₂-free N gas (N₂) flushing to ensure anaerobic status, and simultaneously inoculated into all fermenters. A 500 mL subsample was frozen at −20 °C as a baseline reference.

At the start of the experiment (day 1), each fermenter was inoculated with rumen fluid, after which a sample of solid ruminal digesta (20 g, wet weight) was introduced in a pre-labeled polyester bag. In addition, a separate polyester bag containing the experimental diet (20 g, wet weight) was prepared and placed into each vessel. To establish anaerobic conditions that closely resemble the rumen environment, the fermenters were flushed with nitrogen (N₂) gas prior to sealing, thereby ensuring the removal of oxygen and the maintenance of strictly anoxic conditions.

Following an initial 24 h incubation period, the bag containing rumen solid digesta was removed and replaced with a new bag containing fresh diet. From this point forward, each fermenter consistently contained two bags representing different incubation durations. The feeding protocol consisted of opening the fermenters once daily at 1300 h to remove the bag that had been incubated for 48 h and replace it with a newly prepared feed bag. During each bag exchange, fermenters were again flushed with N₂ gas to minimize oxygen contamination and preserve anaerobic conditions throughout the experiment.

Artificial saliva was prepared fresh on a daily basis according to the formulation described by McDougall [11], with a target pH of 8.2 to support optimal buffering capacity. This buffer solution was continuously infused into each fermenter at a constant dilution rate of 1.11 mL/min, corresponding to a total daily infusion volume of approximately 1600 mL.

2.3. Measurements

The true dry matter (DM) disappearance after 48 h incubation was determined from days 8 to 10. Diet bags were removed from each fermenter, gently squeezed to remove excess liquid, rinsed until clear, dried at 55 °C for 72 h in a forced-air oven (VWR Scientific^®, 158 Model 1370 GM, Radnor, PA, USA), and further dried at 105 °C for 2 h. The difference between the initial and residual weights was used to calculate DM disappearance.

Gas bags were closed before opening the fermenter or effluent collection. Daily gas production was measured with a flowmeter (Omega Engineering Inc., Stamford, CT, USA) connected to a vacuum pump and manometer, with pressure differentials recorded after stabilization. Gas samples (30 mL) were collected from bags with a syringe, capped, and stored for later CH₄ determination.

Fermenter fluid pH was measured daily (days 1–10) during bag exchanges with a pH meter (HI9813-5, Hanna Instruments, Smithfield, RI, USA). Effluent volume and weight were determined with a graduated cylinder and scale. For volatile fatty acid (VFA) analysis, 8 mL fermenter and effluent samples were preserved with 2 mL of 25% (w/w) metaphosphoric acid [12]. Concurrently, 10 mL aliquots were preserved with 100 μL of H₂SO₄ (50% v/v) for ammonia nitrogen (NH₃-N) determination. Samples were centrifuged at 1000× g for 15 min at 4 °C (Eppendorf, 5804 R, Hamburg, Germany), filtered, transferred into screw-cap vials, and stored at −20 °C until analysis. VFA and NH₃-N concentrations (mmol/L) were multiplied by daily effluent output (L/d) to calculate production rates (mmol/d). Methane concentration and microbial protein synthesis were determined as described in Castro et al. [8].

2.4. Chemical Analyses

Residual feed samples recovered from the fermenters were initially dried in a forced-air oven (VWR Scientific®, model 1370 GM, Radnor, PA, USA) at 55 °C for 72 h to remove moisture while preserving sample integrity. Following drying, residues collected during the sampling period (days 8–10) were pooled within the fermenter to generate composite samples representative of each experimental unit. These composites were subsequently ground to pass through a 1-mm screen using a Wiley mill (Model 4, Arthur H. Thomas Co., Philadelphia, PA, USA) to ensure homogeneity prior to chemical analyses.

The chemical composition of the samples was determined according to standard procedures outlined by AOAC [13]. Dry matter content was measured using method 930.15, whereas ash concentration was determined by combustion following method 942.05. Total nitrogen content was quantified using method 990.03 via combustion analysis with a protein/nitrogen analyzer (LECO^® FP-528, St. Joseph, MI, USA), allowing for the precise estimation of crude protein. Neutral detergent fiber (NDF) concentrations were determined according to the method of Van Soest et al. [14], incorporating the use of heat-stable amylase and correction for residual ash to improve accuracy.

For the liquid effluent fraction, volatile fatty acid (VFA) concentrations were measured using gas chromatography as described by Wang et al. [15], ensuring the separation and quantification of individual fatty acids. Ammonia nitrogen concentrations were analyzed following the colorimetric procedure outlined by Broderick and Kang [16], providing an estimate of nitrogen availability within the fermentation system.

2.5. Statistical Analysis

Only average data from the last three days were considered. The experiment followed a completely randomized design with three treatments, using the fermenter as the experimental unit. The data were tested for normality and subjected to analysis of variance with a 10% significance level. All analyses were performed using the MIXED procedure of SAS software version 9.4 (SAS Inst., Cary, NC, USA), according to the model:

Yijkl = μ + Ti + εijkl

(1)

where μ = overall mean; Ti = fixed effect of treatment i; and εijkl = random error with mean zero and variance of σ², the variance between measurements across fermenters.

3. Results

Gas outlet location did not affect the ruminal fluid pH within the fermenters (p = 0.795) or NH₃–N concentration in the effluent (p = 0.869). However, the NH₃–N concentration within the fermenter was influenced by the gas outlet location (p = 0.081), with greater concentrations observed when gas was sampled at the effluent vessel (EV; 11.53 mmol) or both the fermenter and effluent vessel (EVF; 11.82 mmol) compared with sampling exclusively at the fermenter headspace (F; 8.59 mmol). Gas outlet location did not affect DM disappearance or overall nutrient digestibility (

Table 2. Effect of variations in the gas outlet location in a RUSITEC system on the fermentation parameters, in vitro digestibility, gas production, and volatile fatty acid profile.

Item	Gas Outlet Location			SEM 1	p-Value
	Effluent Vessel	Fermenter	Effluent Outlet + Fermenter
Fermentation parameter
pH	6.96	6.99	6.96	0.032	0.795
NH3 Fermenter 2, mmol	11.53 a	8.59 b	11.82 a	1.013	0.081
NH3 Outflow 3, mmol	14.63	15.02	14.21	1.082	0.869
In vitro digestibility, %
Dry matter	56.50	56.61	51.50	2.630	0.326
Crude protein	74.98	70.76	70.19	3.519	0.590
NDF	32.42	29.59	34.28	6.108	0.862
Starch	96.28	96.98	95.68	0.568	0.301
Gas production
Total, L/d	0.27 b	0.29 b	0.56 a	0.076	0.030
Methane, mg/d	10.20 c	27.91 b	42.88 a	5.882	0.006
Volatile fatty acid production, mmol/d
Acetate	1160.44	1162.51	1166.49	187.93	0.999
Propionate	443.22	413.94	393.99	69.56	0.898
Butyrate	259.04	219.29	159.15	56.46	0.475
Total	1862.70	1794.73	1723.64	306.04	0.950
Volatile fatty acid production, %
Acetate	64.67 b	70.71 a	72.63 a	1.408	0.005
Propionate	24.07	23.43	23.15	0.619	0.575
Butyrate	11.25 a	5.87 b	4.21 b	1.479	0.014

¹ SEM = Standard error of the mean. ² NH₃ Fermenter, mmol = Ammonia nitrogen in the fermenter. ³ NH₃ Outflow, mmol = Ammonia nitrogen in the outflow. ^a,b,c Different superscript letters within a row indicate significant differences between groups (p < 0.05).

).

Methane production was significantly influenced by gas outlet location (p = 0.006), with the highest CH₄ output observed in EVF (42.88 mg/d), intermediate values in F (27.91 mg/d), and the lowest values in EV (10.20 mg/d). Similarly, total gas production was greater in EVF (0.52 L/d) compared with F (0.27 L/d) and EV (0.29 L/d), which did not differ from each other (Table 1).

Total VFA production, expressed in molar concentration and absolute output, was not affected by gas outlet location. However, the VFA profile was altered, as the acetate proportion was greater in F (72.63%) and EVF (70.71%) compared with EV (64.67%; p = 0.005). Conversely, butyrate proportion was greater in EV (11.25%) compared with EVF (4.21%) and F (5.87%; p = 0.014) (Table 2). Nitrogen metabolism parameters were not affected by the gas outlet location (

Table 3. Effect of variations in the gas outlet location in a RUSITEC system on microbial protein synthesis, nitrogen digestion, utilization efficiency, and nitrogen flow dynamics.

Item	Gas Outlet Location			SEM 1	p-Value
	Effluent Vessel	Fermenter	Effluent Vessel + Fermenter
NH3 N, g/d 2	0.08	0.06	0.09	0.014	0.329
NAN, g/d 3	0.29	0.30	0.26	0.021	0.295
Bacterial N, g/g 4	0.21	0.21	0.21	0.020	0.989
NANMN, g/d 5	0.08	0.09	0.06	0.012	0.224
N dig	0.77	0.74	0.71	0.027	0.310
N use efficiency 6	0.56	0.57	0.61	0.050	0.761
G Bac/OM dig, g/d 7	28.41	21.47	26.11	3.212	0.331
RUP flow 8	0.26	0.25	0.23	0.182	0.561
N flow 9	0.47	0.46	0.44	0.013	0.311
RDP flow 10	0.23	0.24	0.26	0.018	0.562

¹ SEM = Standard error of the mean. ² NH₃-N, g/d = Ammonia nitrogen. ³ NAN, g/d = Non-ammonia nitrogen. ⁴ Bacterial N, g/g = Bacterial nitrogen. ⁵ NANMN, g/d = Non-ammonia non-microbial nitrogen. ⁶ N use efficiency = Nitrogen use efficiency. ⁷ g Bac/OM dig, g/d = Grams of bacteria per organic matter digested. ⁸ RUP flow = Rumen undegradable protein flow. ⁹ N flow = Nitrogen flow. ¹⁰ RDP flow = Rumen degradable protein flow.

).

4. Discussion

The present study was designed to evaluate how variations in gas outlet location within a modified RUSITEC^® system influence measured fermentation variables, with particular emphasis on distinguishing true biological responses from methodological artifacts. Because the RUSITEC system operates as a closed, semi-continuous fermentation environment, the accuracy of measured outputs, especially gaseous products, is inherently dependent on system configuration. Therefore, interpretation of treatment effects requires careful consideration of how modifications to the system, such as gas outlet positioning, affect the recovery and quantification of fermentation end products rather than the fermentation process itself.

Across treatments, gas outlet location did not affect core indicators of fermentation extent, including ruminal pH, nutrient digestibility (DM, CP, NDF, and starch), total VFA production, and most nitrogen metabolism variables. These results demonstrate that the microbial ecosystem within the fermenters remained stable regardless of outlet configuration, and that substrate degradation and fermentation pathways proceeded similarly across treatments. The absence of differences in digestibility is particularly important, as it indicates that microbial attachment, enzymatic activity, and feed degradation kinetics were not disrupted by the modified gas outlet configurations. This aligns with previous studies showing that RUSITEC systems provide a controlled and reproducible environment for maintaining microbial fermentation over extended incubation periods [3,17,18]. Importantly, these findings confirm that the modified RUSITEC system used in this study preserved its biological functionality despite structural adjustments, supporting its suitability for fermentation studies.

In contrast to the stability observed in fermentation-related variables, gas production measurements were highly sensitive to outlet configuration. Total gas production and CH₄ output were both significantly greater when gas was collected simultaneously from the fermenter and effluent vessel (EVF), whereas single-outlet configurations (EV or F) resulted in consistently lower measured values. Because digestibility, VFA production, and microbial protein synthesis were unaffected, these differences cannot be attributed to changes in microbial activity or substrate fermentation. Instead, they indicate that gas outlet configuration directly affects the efficiency of gas recovery within the system, leading to the underestimation of total gas and CH₄ production when only a single collection point is used.

These findings can be explained by the dynamics of gas partitioning in RUSITEC systems. Fermentation gases are not exclusively present in the headspace but are initially dissolved in the liquid phase and gradually released depending on saturation, pressure, and mixing conditions [6]. As liquid flows from the fermenter to the effluent vessel, changes in physicochemical conditions can promote the release of dissolved gases, which may then accumulate in the effluent compartment. If gas is collected only from the fermenter headspace or only from the effluent vessel, a fraction of total gas production is likely not captured. This mechanism is consistent with previous descriptions of gas behavior in rumen simulation systems and in vitro fermentation techniques [19,20,21]. Therefore, the dual-outlet configuration likely improved gas recovery by capturing both headspace gas and gas released during liquid transfer, resulting in a more complete measurement of total gas production.

Beyond gas recovery efficiency, the results of this study also highlight the importance of considering physicochemical gradients within the RUSITEC system when interpreting fermentation outputs. In semi-continuous systems, localized differences in pressure, gas solubility, and fluid flow can create microenvironments that influence the partitioning of gases between phases. As described by Adebayo Arowolo et al. [6], dissolved gases may accumulate in the liquid phase until saturation thresholds are reached, after which they are released depending on mixing intensity and system configuration. In the present study, the presence of a gas outlet exclusively at the fermenter or effluent vessel likely limited the ability to capture these transient gas fluxes, particularly during periods of liquid transfer. The dual-outlet configuration, in contrast, may have reduced localized pressure gradients and facilitated more continuous gas release, improving the overall recovery of fermentation gases. This interpretation is consistent with earlier work indicating that gas measurement in in vitro systems is highly sensitive to system design and gas evacuation dynamics [19,20,21].

The implications of incomplete gas recovery are particularly relevant for CH₄ quantification. Methane production was substantially underestimated in single-outlet configurations compared with the dual-outlet system, highlighting the sensitivity of CH₄ measurements to methodological factors. Given the increasing use of RUSITEC systems for evaluating CH₄ mitigation strategies [4,5], inaccurate gas recovery could lead to erroneous conclusions regarding the efficacy of dietary interventions. Similar concerns have been raised in other in vitro systems, where methodological differences in gas collection can significantly affect the measured CH₄ output [22,23]. Thus, the present results reinforce the need for careful standardization of gas collection procedures in modified RUSITEC systems to ensure comparability across studies.

Although the total VFA production was not affected by outlet configuration, changes in VFA proportions were observed, particularly for acetate and butyrate. The effluent-only configuration resulted in lower acetate and higher butyrate proportions compared with the other treatments. However, these shifts occurred in the absence of differences in total VFA production or digestibility, suggesting that they are unlikely to reflect biologically meaningful changes in fermentation pathways. Instead, they may be linked to the incomplete recovery of fermentation gases, particularly CH₄, which is closely associated with hydrogen balance and acetate formation. Methanogenesis serves as a major hydrogen sink in the rumen, and reductions in measured CH₄ are often associated with shifts in VFA profiles [21,24]. In the present study, the correspondence between lower CH₄ recovery and reduced acetate proportion in the EV treatment supports the interpretation that these differences are methodological rather than biological in nature. In conventional RUSITEC systems, VFA production and profiles are generally stable and primarily driven by diet composition rather than system configuration. Previous studies have reported acetate as the predominant VFA, followed by propionate and butyrate, with relatively consistent proportions across experiments [4,19]. The absence of differences in total VFA concentration in the present study is therefore consistent with these reports and further supports the conclusion that fermentation pathways were not biologically altered by outlet configuration.

Nitrogen metabolism variables were largely unaffected by gas outlet location, further supporting the conclusion that microbial activity was stable across treatments. The only observed difference was a lower ammonia-N concentration within the fermenter for the fermenter-only configuration. However, this effect was not accompanied by changes in effluent ammonia-N, microbial protein synthesis, or nitrogen flow variables. Because ammonia concentration in RUSITEC systems is primarily governed by the balance between protein degradation and microbial uptake [25,26], the lack of consistent responses across nitrogen-related variables suggests that this isolated difference is unlikely to represent a true shift in nitrogen metabolism. Instead, it may reflect minor variations in gas–liquid equilibrium, mixing patterns, or system dynamics associated with outlet configuration [19,27]. Therefore, this result should be interpreted cautiously and considered of limited biological relevance. Microbial protein synthesis in RUSITEC systems is generally considered a robust indicator of microbial activity and is often reported to be relatively stable across treatments when nitrogen supply is adequate [25,26]. The lack of differences observed in the present study is therefore consistent with previous findings using unmodified RUSITEC systems and further supports the conclusion that microbial growth and nitrogen utilization were not affected by gas outlet configuration.

These findings also have important implications for experimental repeatability and comparability across laboratories. Differences in RUSITEC configuration, particularly regarding gas outlet positioning and collection strategies, may contribute to variability in reported CH₄ production even when diets and substrates are similar. This may partially explain inconsistencies observed across studies evaluating CH₄ mitigation strategies using in vitro systems [4,5]. Without standardized approaches to gas collection, methodological differences may confound treatment effects, limiting the ability to draw robust conclusions or compare results across experiments. The present results therefore reinforce previous recommendations emphasizing the need for methodological standardization in RUSITEC systems [3], particularly for studies focused on gas emissions and environmental outcomes.

In addition, the improved gas recovery observed with the dual-outlet configuration suggests that some previous RUSITEC studies may have underestimated absolute CH₄ production when using single-outlet systems. While relative treatment comparisons may still be valid within a given experimental setup, absolute values of CH₄ production should be interpreted cautiously when gas recovery efficiency is unknown or incomplete. This is particularly relevant when RUSITEC data are used to parameterize models, validate mitigation technologies, or extrapolate in vitro findings to in vivo conditions. Ensuring complete gas capture is therefore not only a methodological consideration but also a critical factor for the broader application and interpretation of RUSITEC-derived data.

From a broader perspective, this study contributes to the ongoing effort to standardize RUSITEC methodologies and improve the reliability of in vitro fermentation measurements. While the RUSITEC system has been widely used and validated for studying ruminal fermentation [1,2], variations in system design and operation can introduce methodological biases that affect specific outputs, particularly gas-related measurements. The present results highlight that even relatively small modifications, such as gas outlet positioning, can have substantial effects on measured variables. Therefore, greater attention should be given to system configuration and the reporting of methodological details to enhance reproducibility and comparability across studies.

In summary, this expanded evaluation of gas outlet location demonstrates that accurate gas recovery in RUSITEC systems depends on capturing gases from both the fermenter headspace and the effluent vessel. Failure to do so may result in systematic underestimation of CH₄ production and potential misinterpretation of fermentation outcomes. By adopting improved gas collection strategies, such as dual-outlet configurations, researchers can enhance the precision of gas measurements while preserving the biological integrity of the system.

5. Conclusions

This study demonstrates that the location of the gas outlet in the RUSITEC system is a critical methodological factor that can substantially influence the measurement of fermentation gases. Our results indicate that single-outlet configurations may underestimate CH₄ production and slightly alter the profile of volatile fatty acids, particularly reducing the proportion of acetate. Importantly, variations in gas outlet position did not affect nutrient digestibility or microbial protein synthesis, suggesting that the overall microbial activity and feed degradation processes remained stable regardless of gas collection strategy. These findings highlight the need for careful consideration of gas sampling points when designing and interpreting RUSITEC experiments, as methodological choices can directly affect the quantification of gases, particularly CH₄, which is a key indicator of fermentation efficiency and an important environmental parameter. By implementing dual or strategically positioned gas outlets, researchers can more accurately capture fermentation gases that might otherwise remain dissolved in the liquid phase or escape detection, thereby improving the precision and reliability of in vitro fermentation measurements.