Effect of substrate dispersion method and particle size on in vitro digestibility, gas production kinetics and composition, and fermentation characteristics in different feed types

Our objective was to evaluate the influence of substrate dispersion method and particle size on in vitro digestibility, gas production kinetics and composition, and fermentation characteristics of different feed types. Alfalfa hay, tall fescue hay, ground corn, soybean meal, and a total mixed ration (TMR) were used as substrates. Treatments were designed according to a 2 × 2 × 5 factorial arrangement: two substrate dispersion methods (loose substrate and in filter bags), two particle sizes (1 mm and 2 mm), and five feeds. Filter bags decreased (P ≤ 0.001) digestibility and gas production compared with loose samples. Filter bags decreased digestibility and total gas production to a greater extent for forages, whereas the effects on concentrate feeds were less pronounced. Using filter bags decreased (P ≤ 0.015) both methane production and methane concentration in headspace gas across all feeds. Substrates incubated in filter bags showed a lower molar proportion of acetate and a greater molar proportion of propionate than loose substrates (P< 0.001). In general, energy-rich feeds incubated using filter bags had a decreased (P ≤ 0.036) acetate-to-propionate ratio, while substrate dispersion method did not influence (P ≥ 0.16) the VFA profile of protein-rich feeds. Incubating substrates in filter bags alters in vitro digestibility, gas production, and fermentation characteristics regardless of particle size. However, the magnitude of responses is greater for forage-based feeds than concentrate feeds.

1 Introduction

In vitro gas production systems are widely employed for evaluating diets and feed additives globally (Dijkstra et al., 2005; Yáñez-Ruiz et al., 2016). However, there are many variations in the application of the method, including differences in inoculum source, medium composition, pressure measurement systems, substrate dispersion method inside the bottles, substrate mass, and processing methods (Rymer et al., 2005). Differences in in vitro gas production system methodologies may affect measurements of digestibility and gas production and potentially compromise the ability to accurately and precisely compare feeds and diets both within and across laboratories.

Most studies with in vitro gas production systems have reported incubating substrates directly in bottles (Schofield et al., 1994; Huhtanen et al., 2008; Christodoulou et al., 2025). However, there are alternative methodologies for substrate inclusion that have incubated the substrates inside filter bags (Ankom Technology, Macedon, NY, USA), rather than directly into the bottles (Eun and Beauchemin, 2007; Yang et al., 2014; Trotta et al., 2024). The main appeal of using filter bags lies in their operational simplicity, and in the elimination of the need for quantitative transfer of residues after incubation to measure digestibility (Krizsan et al., 2013). In addition, some authors (Castro-Montoya and Dickhoefer, 2019; Camacho et al., 2023; Ajayi et al., 2025) have employed filter bags to evaluate digestion characteristics of individual feeds and investigate potential associative effects among feeds.

There are limited studies available comparing the incubation of substrates in bottles versus filter bags and these often yield mixed results (Krizsan et al., 2013; He et al., 2016; Sampaio et al., 2024). Adhesion of loose substrates to the surface of the flask decreased in vitro dry matter (DM) digestibility and total gas production (He et al., 2016; Sampaio et al., 2024). Conversely, potential residue losses during the filtration process of loose substrates could lead to artificially inflated digestibility estimates. In contrast, there is concern that incubating substrates within filter bags could decrease the surface area available for microbial contact and potentially alter the diffusion of fermentation end-products out of the filter bag. Accumulation of fermentation end-products within the filter bag microenvironment impaired microbial activity and degradation within the bag (Marinucci et al., 1992; Valente et al., 2011b). Indeed, several studies (Ramin et al., 2013; Schlau et al., 2021; García et al., 2024) have shown that the use of filter bags decreased in vitro DM digestibility, methane production, and total gas output. However, the effect of filter bags appears to depend on the type of substrate used. For instance, some studies (Krizsan et al., 2013; Castro-Montoya and Dickhoefer, 2019) report that the use of filter bags decreases digestibility and gas production to a greater extent in forages than in concentrate feeds, while others (Sampaio et al., 2024) have shown the opposite trend, with a greater decrease observed in concentrate feeds. Therefore, the overall impact of using filter bags in in vitro gas production systems remains uncertain, particularly across different feed types.

An additional consideration in in vitro incubation systems for feed evaluation is the substrate particle size. Substrate particle size of feeds being evaluated, whether in situ or in vitro, should be large enough to resemble particle size in in vivo conditions, but small enough to ensure proper microbial access, minimize sampling bias, and reduce experimental variation (Lowman et al., 2002; Nocek, 1988). Currently, it is recommended that the samples should be ground to pass through 1-mm screen to measure in vitro digestibility and gas production characteristics (Yáñez-Ruiz et al., 2016; Camacho et al., 2022). However, some studies (Earing et al., 2010; Mesgaran et al., 2010; Altman et al., 2022) using in vitro gas production systems have employed a 2-mm particle size. There are a few studies evaluating the impact of changing particle size on in vitro digestibility and gas production characteristics. Larger particle sizes generally reduce in vitro digestibility and gas production, although this effect may be feed-dependent (Lowman et al., 2002; Damiran et al., 2008; He et al., 2016). As microbial access to substrates within the filter bags may be restricted, and larger particles have a reduced specific surface area, the use of filter bags could have a greater impact on digestibility and gas production in substrates processed at larger particle sizes.

To our knowledge, no studies have investigated the combined effects of substrate dispersion method and particle size on in vitro digestibility and gas production kinetics. We hypothesized that incubating samples in filter bags reduces in vitro digestibility, as well as the extent and rate of gas production and composition, with this effect being greater for processed feeds with larger particle sizes. Our objective was to evaluate whether the method of substrate dispersion and particle size affects in vitro digestibility, gas production kinetics and composition, and fermentation characteristics in different feed types.

2 Materials and methods

All animal care and handling procedures were approved by the Animal Care and Use Committee of the University of Kentucky (protocol 2023-4296).

2.1 Substrates, processing, and chemical composition

Alfalfa hay, tall fescue hay, ground corn, soybean meal, and a total mixed ration (TMR; Table 1) were used as substrates. Substrates were chosen because of their common use in ruminant diets and contrasting chemical compositions. All substrates were ground using a knife mill fitted with a 2-mm screen sieve (Model 3 Wiley Mill, Thomas Scientific, Swedesboro, NJ, USA). Subsequently, half of each substrate was ground through the same mill using a 1-mm screen sieve.

Table 1

Table 1. Chemical composition of basal diet and feed samples.

Chemical composition of the basal diet and substrates are presented in Table 1. Dry matter concentration was analyzed by oven-drying for 24 h at 105°C. Total nitrogen (N) concentration was analyzed by combustion using a CN628 Carbon/Nitrogen Determinator (Leco Corporation, St. Joseph, MI; AOAC, 1995; method 990.03). Crude protein concentration was calculated by multiplying N concentration × 6.25. Crude fat concentration was analyzed by high-temperature solvent extraction with diethyl ether using an ANKOM X15 Extractor (ANOKM Technology Method 2, Macedon, NY, USA). Total starch concentration was quantified using an enzymatic-colorimetric method (AOAC, 2023; method 2014.10), with the colorimetric step substituted by amperometric detection using a YIS-2950D-1 Biochemistry Analyzer (YSI Life Sciences, Yellow Springs, OH). Neutral detergent fiber (aNDF) concentration was quantified using an ANKOM200 Fiber Analyzer (ANOKM Technology, Macedon, NY, USA) with heat-stable α-amylase and omitting sodium sulfite.

2.2 Treatments, experimental design, and in vitro incubation

Treatments were structured in a 2 × 2 × 5 factorial arrangement, as follows: two substrate dispersion methods inside the bottles (loose substrates and into filter bags; 25 µm pore size, ~44 cm² surface area; Cat. #F57; Ankom Technology, Macedon, NY, USA), two particle sizes (1 mm and 2 mm), and five feeds (alfalfa hay, tall fescue hay, ground corn, soybean meal, and TMR). The experiment was replicated over four daily incubations, with two bottles per treatment in each incubation.

Approximately 500 mg of each feed was weighed directly into 250-mL incubation bottles (Cat. #7056; Ankom Technology, Macedon, NY, USA) or weighed into acetone-cleaned, labeled, filter bags. Filter bags were heat-sealed and secured inside the bottles using silicone-coated, steel-lined cable ties to ensure they remained fully submerged throughout the incubation period. On the morning of incubation, 2 mL of deionized water was added to each bottle to wet the substrate and promote uniform dispersion of the inoculum throughout the substrate.

Two ruminally-cannulated Angus × Holstein crossbred steers (body weight: 522 ± 11 kg) were used as inoculum donors. Steers were fed daily at 0700 h with a corn-silage-based diet formulated to exceed requirements for ruminally degradable protein, metabolizable protein, vitamins, and minerals (Table 1; NASEM, 2016). Approximately 4 h after feeding, ruminal contents (both liquid and solid digesta) were collected from the solid-liquid interface of the rumen mat of each steer’s rumen mat and pooled. The pooled ruminal contents were immediately placed into insulated bottles (YETI Rambler Gallon Jug, Yeti Holdings, Inc., Austin, TX) and transported to the laboratory.

Upon arrival, ruminal contents were blended under a CO2 headspace for 30 seconds and then strained through four layers of cheesecloth. A mixture of buffer solution, macro- and micro-mineral solutions, and reducing solution was prepared as described by Goering and Van Soest (1970). The combined solution was maintained at 39°C on a hot plate equipped with a temperature probe, with continuous agitation using a stir bar and constant infusion with CO2. After 30 minutes, rumen fluid was added into the buffered solution and mixed thoroughly at a buffer:rumen fluid ratio of 4.5:1. Subsequently, 100 mL of the prepared medium was dispensed into each incubation bottle using a FlexiPump Pro dispensing pump (Interscience International, St. Nom, France).

Bottles were placed in a 39°C water bath to equilibrate. The valves on the bottle caps were simultaneously opened to release any accumulated pressure. Then, valves were connected to an automated pressure transducer system (Memograph M RSG45 Data Manager; Endress+Hauser, Greenwood, IN) described previously by Seeforth and Trotta (2025), with cumulative gas pressure readings recorded every 5 minutes over a 72-h incubation period.

2.3 Sampling and measurements

At the end of the 72-h incubation period, bottles were placed in an ice bath to halt fermentation. After 15 minutes, a 10-mL gas sample was collected from each bottle using a syringe. The gas sample was collected into an empty vacuum evacuated test tube (Vacutainer^®, Becton, Dickinson and Company, East Rutherford, NJ, USA) using a line connected to the vent valve on the bottle cap. Gas samples were analyzed for methane concentration by gas chromatography with flame ionization detection (HP 7890A, Agilent Technologies, Santa Clara, CA, USA) as described by Trotta et al. (2023). Following gas sampling, bottles were opened, and the pH of the incubation medium was immediately measured using a pH meter (HM-21P, DKK TOA Electronics Ltd., Tokyo, Japan). A 1-mL aliquot of the fluid was then transferred into 1.5 mL centrifuge tubes containing 0.1 mL of 85 mM 2-ethyl butyrate (internal standard) and 0.1 mL of 50% metaphosphoric acid for volatile fatty acids (VFA) and ammonia analysis. Samples were frozen at -20°C for subsequent analysis.

The VFA were analyzed by gas chromatography with flame ionization detection (8890 GC System; Agilent Technologies Inc., Santa Clara, CA, USA) as described by Trotta et al. (2023). Ammonia concentration was quantified using the glutamate dehydrogenase method (Kun and Kearney, 1974), adapted for use in a multimode plate reader (BioTek Synergy HTX; Agilent Technologies Inc., Santa Clara, CA, USA) as described by Trotta et al. (2024).

For loose incubation, the contents of each bottle were carefully transferred to acetone-rinsed, labeled, and pre-weighed F57 filter bags. Samples were filtered using a vacuum pump and then heat-sealed. All filter bags, including the ones initially used for substrate incubations, were washed thoroughly with tap water until the rinse water ran clear, and sequentially oven-dried at 55°C and 105°C for 24 h. After drying, filter bags were then placed in a desiccator and weighed to estimate the apparent undigested DM residue. Subsequently, filter bags were processed in neutral detergent solution with heat-stable α-amylase (17,400 liquefon units/g; Spezyme Fred; Genencor International, Inc., Palo Alto, CA) using an ANKOM200 Fiber Analyzer (Ankom Technology Corp., Macedon, NY, USA). Filter bags were washed three times with hot distilled water, submerged in acetone for 3 min, and sequentially oven-dried at 55°C and 105°C for 24 h, placed in a desiccator and weighed to estimate the true undigested DM residue.

2.4 Calculations

The apparent in vitro DM digestibility (apparent IVDMD, g/kg), true in vitro DM digestibility (true IVDMD, g/kg), and in vitro NDF digestibility (IVNDFD, g/kg) were calculated using the following equations (Equations 1–3):

$\begin{array}{ll}\text{Apparent} \text{IVDMD} =\frac{\text{DMi}-\text{DMr}}{\text{DMi}} \times 1000& (1)\end{array}$

$\begin{array}{ll}\text{True} \text{IVDMD} =\frac{\text{DMi}-\text{NDFr}}{\text{DMi}} \times 1000& (2)\end{array}$

$\begin{array}{ll}\text{IVNDFD} =\frac{\text{NDFi}-\text{NDFr}}{\text{NDFi}} \times 1000& (3)\end{array}$

where DMi is the mass of DM incubated; DMr is the mass of the apparently undigested DM residue; NDFi is the mass of NDF incubated; NDFr is the mass of the undigested NDF residue.

Assuming ideal gas behavior, constant temperature, and a closed system, total gas production (GP, in mL) was calculated from the vessel pressure corrected to current atmospheric pressure into standard atmospheric pressure (101.33 kPa) using the following equation (Equation 4):

$\begin{array}{ll}\text{GP} = \frac{\text{ΔP} \times {\text{V}}_{\text{headspace}}}{{\text{P}}_{\text{STP}}} \times 1000 & (4)\end{array}$

where ΔP is the net pressure change in kPa (corrected for atmospheric pressure), V_headspace is the headspace volume (L), and P_STP is the standardized pressure (kPa). The volume of the headspace was estimated for each bottle by subtracting the volume of the contents (inoculum, substrate, filter bag, and clip) from the total volume of the bottle. The volume of the contents was estimated from the difference between the bottle weight at filling and the empty bottle. The total volume of each bottle was determined by subtracting the weight of the empty bottle from that of the water-filled bottle. Methane production was obtained by multiplying the total gas production by the methane concentration in the headspace gas.

The cumulative gas production curve was adjusted individually for each bottle using a first-order exponential model using the PROC NLIN procedure in SAS (version 9.4; SAS Institute Inc., Cary, NC, USA), as follows (Equation 5):

$\begin{array}{ll}\text{G}(\text{t}) = \text{G} \times [1-{\text{e}}^{(-\text{k} \times \text{time})}]& (5)\end{array}$

where G(t) is the cumulative gas production (mL) at time t, G is the asymptotic gas production (mL), and k is the fractional rate of gas production (h^-1).

2.5 Statistical analysis

All analyses were performed using GLIMMIX procedure of SAS (version 9.4; SAS Institute Inc., Cary, NC). Data were analyzed according to a completely randomized design including the fixed effects of substrate dispersion method, particle size, feed, and their interactions. Observations from two bottles in each treatment were averaged for each day of incubation, and the averaged bottle was considered the experimental unit (n = 4). When a significant two-way interaction between method or particle size and feed was detected, simple effects of method or particle size were evaluated within each feed level using an F-test. Least squares means and their standard errors were computed for each method and feed combination. Significance was declared at P< 0.05 and tendency was considered at 0.05< P< 0.10.

3 Results

3.1 In vitro digestibility and gas production characteristics

There was no interaction (P ≥ 0.70) between substrate dispersion method and particle size on in vitro digestibility (Table 2). Incubating samples using filter bags decreased (P ≤ 0.001) all in vitro digestibility characteristics compared with loose incubation. However, we observed an interaction (P ≤ 0.003) between dispersion method and feed type for all in vitro digestibility characteristics. Overall, filter bags decreased (P ≤ 0.022) true IVDMD in forages but had no effect (P ≥ 0.32) on concentrate feeds (Figure 1B). Similarly, filter bag incubation reduced (P ≤ 0.013) both apparent IVDMD and IVNDFD in TMR and tall fescue hay (Figures 1A, C). In contrast, filter bags did not influence (P > 0.11) apparent IVDMD but decreased (P< 0.013) IVNDFD in ground corn. For soybean meal, using filter bags did not affect (P > 0.15) apparent IVDMD but increased IVNDF (P< 0.002). In addition, filter bag incubation tended to decrease (P ≥ 0.087) both apparent IVDMD and IVNDFD in alfalfa hay.

Table 2

Table 2. Effects of substrate dispersion method (M), particle size (PS), and feed (F) on in vitro digestibility and gas production and composition.

Figure 1

Figure 1. Effects of substrate method dispersion on apparent in vitro dry matter digestibility [apparent IVDMD, (A)], true in vitro dry matter digestibility [true IVDMD, (B)], in vitro neutral detergent fiber digestibility [IVNDFD, (C)], and gas production at 72 h [GP72h, (D)], according to the type of feed. [Loose, substrates incubated loose in bottles; Filter bag, substrates incubated using filter bags].

True IVDMD tended to be greater (P = 0.097) for substrates ground to 1 mm compared with substrates ground to 2 mm (Table 2). Likewise, we observed an interaction (P< 0.012) between particle size and feed type on true IVDMD. Tall fescue substrates ground to 2 mm had decreased (P< 0.001) true IVDMD compared with those ground to 1 mm (Figure 2). No effects (P ≥ 0.35) of particle size were detected for the other feeds. Particle size did not influence (P ≥ 0.16) apparent IVDMD or IVDNDF (Table 2).

Figure 2

Figure 2. Effects of particle size on true in vitro dry matter digestibility (true IVDMD) according to the type of feed. [1-mm, substrates processed through 1-mm sieve; 2-mm, substrates processed through 2-mm sieve].

No effect of particle size (P ≥ 0.17) or interaction effect between method and particle size (P ≥ 0.30) was found on gas production characteristics (Table 2). Filter bag incubation decreased (P ≤ 0.001) total gas and methane production, whether expressed as mL/g incubated DM or mL/g digested DM. Similarly, methane concentration in headspace gas decreased (P< 0.001) when substrates were incubated in filter bags compared with loose substrates. Nevertheless, the effect of method dispersion on gas production characteristics depended (P ≤ 0.028) on feed type. Using filter bags decreased (P ≤ 0.013) total gas production at 72 h across all feeds, except for soybean meal, which was unaffected (P > 0.22; Figure 1D). Likewise, filter bag incubation decreased (P ≤ 0.001) asymptotic gas production in TMR and tall fescue, tended to decrease it in alfalfa hay (P = 0.059), but had no effect in ground corn or soybean meal (P ≥ 0.15; Figure 3A). When expressed as mL/g digested substrate, filter bags decreased (P ≤ 0.001) asymptotic gas production in TMR and tall fescue but did not influence (P ≥ 0.15) asymptotic gas production in alfalfa hay, ground corn, and soybean meal (Figure 4A). Using filter bags decreased (P ≤ 0.015) both methane production and the concentration of methane in the headspace across all feeds, even though the magnitude was greater for TMR, tall fescue hay, and ground corn (Figures 3C, D, 4B).

Figure 3

Figure 3. Effects of substrate method dispersion on maximum gas production [Maximum GP, (A)], gas production rate [GP rate, (B)], methane concentration (C), and methane production (D) according to the type of feed. [Loose, substrates incubated loose in bottles; Filter bag, substrates incubated using filter bags].

Figure 4

Figure 4. Effects of substrate method dispersion on maximum gas production [Maximum GP, (A)] and methane production (B) according to the type of feed. [Loose, substrates incubated loose in bottles; Filter bag, substrates incubated using filter bags].

Incubating substrates using filter bags tended to increase (P = 0.098) gas production rate (Table 2). We observed an interaction (P< 0.002) between substrate dispersion method and feed type on gas production rate. Filter bag incubation increased (P< 0.003) gas production rate in tall fescue hay and tended to increase gas production rate in TMR (P = 0.056) and alfalfa hay (P = 0.097; Figure 3B). Conversely, filter bag incubation decreased (P< 0.034) gas production rate in ground corn and had no effect (P > 0.45) on soybean meal.

3.2 Fermentation characteristics

There were no interactions (P ≥ 0.74) between method and particle size for pH or ammonia concentration (Table 3). Media pH was greater (P< 0.022) for substrates incubated using filter bags, whereas it was decreased (P< 0.036) with 1 mm particle size. On average, final pH ranged from 6.93 to 6.97, showing that this effect was of low relevance or magnitude. Substrates ground to 1 mm tended to have a greater (P = 0.064) ammonia concentration than those ground to 2 mm. Nevertheless, the effect of particle size on ammonia concentration tended (P = 0.073) to depend on feed type. Specifically, processing samples to 2 mm decreased (P< 0.036) and tended to decrease (P = 0.094) ammonia concentrations in tall fescue hay and alfalfa hay, respectively. In contrast, particle size did not affect (P ≥ 0.11) ammonia concentration for the remaining feeds. Furthermore, filter bag incubation tended to decrease (P = 0.068) ammonia concentration compared with loose incubation.

Table 3

Table 3. Effects of substrate dispersion method (M), particle size (PS), and feed (F) on in vitro fermentation characteristics.

There was no interaction (P > 0.73) between method and particle size on total VFA concentration (Table 3). Substrates incubated in filter bags had a lesser molar proportion of acetate and a greater molar proportion of propionate than those incubated in bottles (P ≤ 0.001). As a result, filter bag incubation decreased (P< 0.001) acetate-to-propionate ratio. However, the effects of substrate dispersion method on acetate and propionate proportions, and acetate-to-propionate ratio varied (P ≤ 0.001) by feed type. The use of filter bags decreased both (P ≤ 0.036) acetate and the acetate-to-propionate ratio in tall fescue hay, TMR, and ground corn, but had no effect (P ≥ 0.16) on alfalfa hay and soybean meal (Figures 5A, C). Likewise, the molar proportion of propionate was increased (P ≤ 0.001) in TMR and ground corn incubated in filter bags but remained unchanged (P ≥ 0.14) in alfalfa hay, tall fescue hay, and soybean meal (Figure 5B).

Figure 5

Figure 5. Effects of substrate method dispersion on molar proportions of acetate (A), propionate (B), valerate (C), and acetate-to-propionate ratio (D) according to the type of feed [Loose, substrates incubated loose in bottles; Filter bag, substrates incubated using filter bags].

Filter bag incubation tended to increase (P = 0.099) butyrate molar proportion. On average, filter bag incubation increased (P< 0.001) molar proportion of valerate and tended to decrease (P = 0.088) the molar proportion of isobutyrate compared with loose incubation (Table 3). However, the effect of method on isobutyrate molar proportion tended to depend (P = 0.083) on feed type. The isobutyrate molar proportion was less (P< 0.004) in ground corn samples incubated in filter bags, while it was not affected (P ≥ 0.18) for the other feeds. Similarly, the effect of method on valerate molar proportion depended on particle size (P = 0.071) and feed type (P< 0.015). Filter bag incubation increased (P< 0.001) the molar proportion of valerate in samples ground to 2 mm, but had no effect (P > 0.17) on samples ground to 1 mm. Furthermore, filter bags tended to increase (P = 0.059) and increased (P<0.001) valerate molar proportions in alfalfa hay and ground corn, respectively, while filter bags did not influence valerate in the other feeds (Figure 5D). No effects (P ≥ 0.12) of particle size or interaction with feed type (P ≥ 0.81) were observed on VFA molar proportions.

4 Discussion

In this study, we aimed to investigate whether the substrate dispersion method and substrate particle size affected in vitro digestibility, gas production kinetics and composition, and fermentation characteristics across different substrate types. Contrary to our hypothesis, the overall effects of substrate dispersion methods were independent of substrate particle size. In fact, the effect of particle size was limited on in vitro digestibility and gas production. The effects of substrate particle size on in vitro digestibility and gas production are inconsistent across studies (Wilman and Adesogan, 2000; Lowman et al., 2002; Bossen et al., 2008; Damiran et al., 2008; Tagawa et al., 2017). Such variability may be attributed to differences in incubation time and substrate type. The lack of a particle size effect observed in the present study may be explained by the long incubation period (72 h). Under long-term incubation, the smaller surface area of substrates ground to 2 mm may have been compensated by the extended time available for microbial digestion. Indeed, previous studies (Tagawa et al., 2017) have reported that particle size affects DM digestibility at early incubation times but not after 24 h of incubation. Similarly, increasing particle size in long-term incubation (48 h to 96 h) did not affect true DM or NDF digestibility (Wilman and Adesogan, 2000; Bossen et al., 2008). Moreover, some authors (Lowman et al., 2002) have reported that increasing particle size decreases total gas production in forages but not in concentrate feeds. In our study, we observed a trend toward decreased true IVDMD in tall fescue hay ground to 2 mm, although gas production was unaffected. Additionally, tall fescue hay processed to 2 mm showed lower ammonia concentrations in the fermentation medium compared with substrates ground to 1 mm, which may indicate reduced substrate degradability. Although these differences were small, the results suggest that grinding substrates to 2 mm may reduce in vitro digestibility in slowly digestible feeds.

Overall, the effects of substrate dispersion method on digestibility and total gas varied by feed type. For forages, filter bag incubation decreased apparent IVDMD, true IVDMD, and IVNDFD by 6.43%, 5.09%, and 11.8%, respectively. Among concentrate feeds, ground corn incubated in filter bags showed reductions of 2.55%, 0.81%, and 11.4% in apparent IVDMD, true IVDMD, and IVNDFD, respectively, whereas soybean meal showed increases of 2.22%, 0.93%, and 9.35%, respectively. Total gas production decreased by 17.1% in forages and 6.75% in concentrates when incubated in filter bags. Therefore, changing the method of substrate dispersion impacted digestibility and gas production to a greater extent in forages, while the effects on concentrate feeds, although present, were relatively minor. Similar results have been reported by other authors (Krizsan et al., 2013; Schlau et al., 2021; García et al., 2024) when comparing substrates incubated in filter bags versus dispersed freely in the medium. In addition, the greater digestibility of soybean meal incubated in filter bags was unexpected, particularly because it was not accompanied by greater gas production. In fact, gas production was decreased for soybean meal incubated in filter bags.

Several factors may explain the differences in in vitro digestibility and total gas production between substrate dispersion methods (substrates in filter bags vs. loose substrates). Feed particles can aggregate in filter bags as they become hydrated. This may impair the flow of incubation medium and the associated microbes through the sample, thereby limiting microbial access to the substrate. A similar pattern has been suggested when filter bags are used in pressurized systems for estimating fiber content in feedstuffs, and has been proposed as one of the reasons for the greater fiber values obtained with filter bags compared with conventional loose refluxing systems (Barbosa et al., 2015).

Filter bags used in the current study had an average pore size of 25 µm (#F57, Ankom Technology). Based on size, bacteria should be able to readily pass through the pores in the bag. Hence, no compromise in microbial degradation would be expected due to bacterial exclusion. However, the filter bags used in this study are composed of non-woven fabric, which results in an irregular pore structure (Valente et al., 2011a). Previous in situ incubation studies (Meyer and Mackie, 1986) reported a decrease in bacterial counts inside filter bags as pore size decreased, although this effect was more strongly associated with the proportion of pore surface area than with pore size itself. Furthermore, this pore size is smaller than the average width of most rumen ciliate protozoa genera (Williams and Coleman, 1992). Early studies (Lindberg et al., 1984) using in situ incubation reported a linear increase of total protozoa inside the bags as pore size increased from 10 µm to 36 µm. Likewise, increasing bag pore size from 40 µm to 100 µm enhanced protozoal counts as well as dry matter and fiber digestibility of substrates incubated in a semi-continuous culture system (Carro et al., 1995). Protozoa are known to play a key role in the ruminal degradation of organic matter (Williams and Coleman, 1992). Several studies have shown that partial or total defaunation decreases fiber digestibility (Newbold et al., 2015; Li et al., 2018; Dai and Faciola, 2019). Hence, it is possible that the 25 µm pore size and the irregular pore structure of the filter bags used may have restricted the entry of some protozoa into the bags, which could have contributed to the decrease in dry matter and fiber digestibility in the current study. This may also help explain why the effects of filter bags were more pronounced for forages than for concentrates, given the key role of protozoa in fiber degradation.

The use of filter bags in in situ or in vitro incubation assays assumes that the environment inside the bag is identical or at least similar to that outside the bag. However, a body of evidence suggests that this assumption may not be entirely valid (Meyer and Mackie, 1986; Lindberg et al., 1984; Marinucci et al., 1992; Valente et al., 2011b). Some authors (Úden et al., 1974; Marinucci et al., 1992) have reported limited fluid exchange between the filter bag and the surrounding medium. This limitation has been attributed to the small pore size of the bags and, more importantly, to the lack of pressure against filter bags or physical agitation especially in in vitro systems (Marinucci et al., 1992). Such factors may be critical for dislodging particles or facilitating fluid movement through the filter material (Marinucci et al., 1992). As a consequence, gas accumulation and a decline in pH within the bags have been observed, impairing microbial degradation, particularly by fiber-degrading microorganisms (Úden et al., 1974; Nocek et al., 1979; Marinucci et al., 1992). Unfortunately, we did not measure pH and VFA concentrations inside the bags, and the buffered media pH exhibited minimal variation. Nonetheless, we observed that using filter bags shifted the fermentation profile toward increased propionate compared with loose incubation, particularly for the TMR and corn, and to a lesser extent for tall fescue hay. Similarly, Sampaio et al. (2024) reported changes in the VFA profile towards propionate when substrates were incubated in filter bags.

Two main reasons may explain this shift in the fermentation profile toward propionate. First, even though we cannot conclude from this study, it is possible that the microenvironment inside the bags (e.g., VFA and gas accumulation) might have favored the activity of bacteria that degrade non-fibrous carbohydrates (e.g., amylolytic bacteria) while impairing fibrolytic bacteria, thereby altering the fermentation pattern toward propionate. Secondly, the increase in propionate proportion could be a direct effect of a potential decrease in protozoal entry into the bags. Indeed, it has been observed that decreasing ruminal protozoa shifts the fermentation pattern from acetate toward propionate both in vivo (Li et al., 2018; Dai and Faciola, 2019) and in vitro (Spanghero et al., 2022). This shift has been attributed to impaired fiber digestibility in the absence of protozoa (Newbold et al., 2015; Dai and Faciola, 2019). Additionally, protozoa compete with amylolytic bacteria for dietary starch, which is mostly fermented into acetate by protozoa (Williams and Coleman, 1992). Hence, a potential decrease in protozoa within the bags may have favored starch utilization by amylolytic bacteria, resulting in increased propionate proportion. This likely accounts for the more pronounced increase in propionate observed for TMR and corn incubated in filter bags, given their high starch contents.

In the current study, one of the most notable effects of using filter bags was the decrease in methane production. Methane production decreased by 34.4% for forages and 26.2% for concentrate feeds when incubated in filter bags. Similar findings have been reported in previous studies (Ramin et al., 2013; Sampaio et al., 2024) comparing substrates incubated in filter bags versus directly in bottles. For instance, Sampaio et al. (2024) observed that methane production decreased by 47.2% in a high-forage diet and 68.8% in a high-concentrate diet with filter bag incubation. Likewise, Ramin et al. (2013) reported an average decrease of 8% in methane production across feeds, although the magnitude of the filter bag’s effect varied considerably depending on the type of feed. At first glance, the decrease in methane production can be attributed, at least partially, to the decreased amount of digested organic matter in samples incubated using filter bags. Indeed, there is a well-established positive linear relationship between the amount of digested organic matter and methane production in the rumen (Dai et al., 2022). To account for differences in digestibility, we expressed methane production relative to the amount of substrate digested. Even after this correction, substrates incubated in filter bags had decreased methane production compared with substrates incubated in bottles, particularly for TMR, tall fescue, and corn. In addition, methane concentration in the headspace gas was reduced by 24.8% for forages and 20.6% for concentrate feeds under filter bag incubation. These results appear to suggest that the use of filter bags altered ruminal fermentation and the fermentation end-products, as previously discussed.

There could be multiple explanations for greater propionate proportions observed with filter bag incubations. First, the shift in the fermentation profile from acetate to propionate can result in decreased metabolic hydrogen (H2) availability for methanogens (Janssen, 2010), which could partially account for the decreases in methane production observed in samples incubated in filter bags. Second, the potential restriction of ciliate protozoa influx into the filter bags may have further contributed to the decrease in methane production. Rumen protozoa play a key role in methanogenesis, primarily due to their capacity to generate H₂ in hydrogenosomes and to harbor both epi- and endosymbiotic methanogens, protecting them from oxygen toxicity (Fenchel and Finlay, 2006). Indeed, one of the most consistent effects of decreasing protozoa in the rumen is a concomitant decrease in methane production (Newbold et al., 2015; Li et al., 2018; Dai and Faciola, 2019; Dai et al., 2022; Spanghero et al., 2022). This effect has been attributed, at least in part, to decreased fiber degradation in the absence of rumen protozoa (Dai and Faciola, 2019). However, even when accounting for known methane-related variables (e.g., feed intake, fiber digestibility), Dai et al. (2022) reported that a decrease in total protozoa counts, especially isotrichids, was associated with lower methane output. Together, these findings appear to suggest that the use of filter bags alters the fermentation pattern, ultimately impairing methane production.

Also, we observed that samples incubated in filter bags tended to exhibit greater gas production rates compared with those incubated directly in bottles. At first glance, this finding appears to contradict our previous argument that filter bags affect microbial degradation processes. However, a closer examination of the gas production rates and the estimated gas production curves suggests a different interpretation. In general, filter bags increased the gas production rate in forages but either decreased or did not influence the gas production rate in concentrate feeds. A noteworthy observation relates to the asymptotes of the fitted gas production curves (Figure 6). Gas production from forages incubated in filter bags tended to plateau much earlier, whereas gas production from the same feeds incubated in bottles continued to increase and had not reached a plateau even after a 72 h-incubation. Asymptotic gas production and gas production rate were negatively correlated (data not shown). Thus, the greater gas production rates observed for forages in filter bags likely reflect a modeling artifact resulting from the lower asymptotic gas production. In fact, the rapid stabilization in gas production, coupled with the overall lower gas production, suggests an environment inside the filter bags that is not conducive to sustained microbial degradation. This interpretation is further supported by the gas production patterns observed for concentrate feeds. As gas production reached a plateau at similar time points and showed a minimal difference in asymptotic gas production between incubation methods, the gas production rate was either lower (e.g., for corn) or unaffected (e.g., for soybean meal) in samples incubated in filter bags.

Figure 6

Figure 6. Cumulative gas production over a 72-h incubation period according to the substrate method dispersion and feed type [(A) Alfalfa hay, (B) TMR, (C) Tall fescue hay, (D) Ground corn, (E) Soybean meal; Loose, substrates incubated loose in bottles; Filter bag, substrates incubated using filter bags].

5 Conclusions

Incubating samples within filter bags alters in vitro digestibility, gas production, and fermentation characteristics regardless of particle size. These effects are more pronounced in forage-based feeds than concentrate feeds. Future research should focus on elucidating the compositional and functional differences in microbial communities residing inside the filter bags compared with those in the surrounding incubation medium and the impact of restricting protozoa access on feedstuff characterization.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement

The animal study was approved by University of Kentucky IACUC Committee. The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

LS: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. EM: Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. RT: Conceptualization, Project administration, Supervision, Writing – review & editing. ED: Writing – review & editing. DH: Conceptualization, Methodology, Project administration, Writing – review & editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This work is funded by Hatch Capacity Grant Project no. KY007088 from the USDA National In-stitute of Food and Agriculture and Project 201807121511 from USDA/ARS and the University of Kentucky Agricultural Experiment Station.

Acknowledgments

The authors thank the staff of the University of Kentucky C. Oran Little Research Center for their assistance with rumen fluid collection and animal management. The authors thank Winston Lin and Hugo Hamilton of the University of Kentucky Ruminant Nutrition Laboratory for their assistance with laboratory analyses. The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support provided to the first author during his PhD program.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The authors RT, DH declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

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