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.

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

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.

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.