One of the promising biological approaches is bioaugmentation with homoacetogenic bacteria (homoacetogens), which compete with methanogens for H₂ in the rumen, thereby reducing CH₄ emissions (Ungerfeld, 2020).

During ruminal fermentation, H₂ and CO₂ serve as the primary substrates for methanogens; methanogenesis acts as the main H₂ sink, keeping dissolved H₂ levels low (1–10 Pa), which is essential for maintaining efficient fermentation pathways (Kohn and Boston, 2000; Mackie et al., 2023; Fregulia et al., 2024).

Homoacetogens convert H₂ and CO₂ into acetate via the Wood–Ljungdahl pathway, offering an alternative electron sink to methanogenesis (Danielsson et al., 2012). However, the effectiveness of this approach depends on several factors, including rumen pH, substrate availability, and the ability of homoacetogens to establish and outcompete methanogens in the complex rumen ecosystem (Gagen et al., 2010).

According to Karekar et al. (2022) homoacetogens exhibit a versatile metabolism that is suitable for diverse substrates and can act as a carbon sink by converting CO₂ into bioproducts, potentially improving efficiency by diverting H₂ away from methanogenesis. However, their competitive advantage in mature rumen systems appears limited, as methanogens overwhelmingly dominate H₂ utilization and suppress homoacetogenic activity. Experimental approaches that integrate methanogenesis inhibition—such as the use of 2-bromoethanesulfonic acid (BES)—with microbial bioaugmentation strategies have demonstrated promising potential for mitigating enteric CH₄ production. For instance, in the study by Murali et al. (2021) BES treatment increased headspace H₂ and reduced acetate; subsequent bioaugmentation with Acetitomaculum ruminis and Acetobacterium woodii restored acetate levels by 45% and 70%, respectively. Similarly, Stefanini Lopes and Ahring (2023) demonstrated that combining a kangaroo-derived homoacetogenic consortium with almond-shell biochar improved acetic acid production in vitro, albeit temporarily, highlighting transient benefits and the need for stabilization strategies.

Although homoacetogenesis is energetically less favorable than methanogenesis (Conrad, 2023) its competitiveness can be enhanced through strategies such as supplementing substrates like glucose, glycerol, and xylose, along with H₂ and CO₂, to leverage its mixotrophic advantages (Tsapekos et al., 2022). To enhance the viability of homoacetogenesis, strategies such as co-supplementation with acetogenesis stimulants (e.g., fumarate, malate, or nitrate) and optimizing feeding regimens have been explored (Morgavi et al., 2010). Additionally, genetic screening of ruminant microbiomes has identified novel homoacetogenic strains with greater resilience to rumen conditions, offering potential for further development (Henderson et al., 2015).

Additional measures include the introduction of acetogenesis stimulants, such as yeast cultures, maintaining a lower ruminal pH, and identifying novel acetogen strains capable of thriving at low H₂ thresholds and increasing their densities in the rumen (Yang et al., 2015).

Propionate-producing bacteria, along with nitrate- and nitrite-reducing, and sulfate-reducing bacteria, have thermodynamic advantages over methanogens in utilizing H₂ as an electron donor (Lan and Yang, 2019). However, their low abundance or the absence of necessary substrates in the rumen limits their activity (Choudhury et al., 2022). Enhancing the propionate-producing pathway can be achieved by supplementing animals with propionate precursors such as fumarate and malate or introducing functionally complementary propionate-producing bacterial consortia as additives (Jeong et al., 2024). Given the low natural concentrations of nitrate and sulfate in the rumen, using these compounds as additives could stimulate the growth of nitrate- and sulfate-reducing bacteria. However, toxic by-products such as nitrite and hydrogen sulfide (H₂S) must be carefully managed (Latham et al., 2016). Strategies to mitigate toxicity risks include combining sulfate-reducing bacteria (SRB) with nitrate-reducing, sulfur-oxidizing bacteria or employing SRB strains capable of utilizing H₂S or nitrite (Greene et al., 2003).

Exploring microbes that compete with methanogens and redirect H₂ away from methanogenesis presents a promising strategy for reducing CH₄ emissions in the rumen (Lan and Yang, 2019). Despite its potential, bioaugmentation with homoacetogenic bacteria faces challenges, including the need for long-term microbial stability in the rumen and variations in host responses across different animal species. Large-scale field trials are necessary to evaluate the long-term feasibility and effectiveness of this approach under commercial farming conditions (Wallace, 2004). Future research should focus on strain selection, microbial adaptation strategies, and possible synergies with other methane mitigation technologies to improve implementation (Martin et al., 2010).

Bacteriophages (phages), traditionally applied in phage therapy to treat bacterial infections such as enteric diseases, sepsis, and chronic infections (Lin et al., 2017), are gaining attention for broader roles, including food preservation, microbiome modulation, and even environmental applications like climate change mitigation (Elois et al., 2023). Recently, phage therapy has been proposed as a novel strategy to target methanogenic archaea in the rumen to reduce enteric CH₄ production (Lobo and Faciola, 2021). By selectively lysing methanogens, phages may suppress methane formation without significantly disturbing other rumen microbial populations (Morkhade et al., 2020).

The conceptual appeal of phage-based CH₄ mitigation lies in its specificity, ecological safety, and potential to bypass some of the limitations associated with chemical inhibitors or vaccines. However, this strategy remains in its infancy, and several critical challenges must be addressed.

To date, only a limited number of studies have investigated the isolation and characterization of archaeal phages that target rumen methanogens. For example, Ouwerkerk et al. (2011) initiated the development of a phage library specifically targeting the dominant methanogenic archaea in Australian livestock systems. However, experimental evidence on the in vivo efficacy of such phages remains limited. The effectiveness of archaeaphage therapy mainly relies on the ability to identify highly specific phages that can infect predominant methanogenic species—such as Methanobrevibacter ruminantium and Methanobacterium spp.—without disrupting beneficial rumen microbial functions (Lobo and Faciola, 2021). Despite their potential, the identification of archaeal phages remains limited, underscoring a substantial knowledge gap in our understanding of phage-host interactions within methanogenic communities. Among fully sequenced microbial genomes, six archaeal phages have been described, including Methanobacterium phage psi M1, Methanobacterium phage psi M2 (a variant of M1), and Methanobacterium phage psi M100, all of which belong to the Siphoviridae phage family. These phages demonstrate the capacity to infect key rumen methanogens such as Methanobacterium spp., a dominant archaeal genus in the rumen. Moreover, members of the Siphoviridae family have shown infectivity toward Methanobacterium, Methanobrevibacter, and Methanococcus species (Mcallister and Newbold, 2008). Leahy et al. (2013) presented the complete genome sequence of the rumen methanogen Methanobrevibacter ruminantium M1, offering critical insights into its metabolic and cellular pathways. A prophage identified in M. ruminantium encodes 69 phage-related proteins, including the lytic enzyme PeiR from prophage φMru, which shows potential as a biocontrol agent against ruminal methanogens. A novel approach was proposed, utilizing viral enzyme-loaded nanoparticles that effectively lyse not only the original methanogen host strain but also a diverse range of ruminal methanogen species in pure in vitro cultures, resulting in significant CH₄ reductions of up to 97% (Altermann et al., 2018). However, this broad-spectrum activity raises concerns about potential disruption to the natural rumen microbial ecosystem.

Rumen phage populations are highly diverse and individualized, with concentrations ranging from 10⁷ to 10⁹ particles per milliliter (Swain et al., 1996). This high diversity, coupled with host-specific microbial interactions, raises concerns about the stability, persistence, and consistent efficacy of introduced phages within the rumen ecosystem. To date, no study has comprehensively identified the phage taxa present in the rumen and their specific archaeal hosts, nor has it assessed their interactions with the methanogen community at a large scale. These knowledge gaps underscore a critical barrier to the development of phage-based CH₄ mitigation strategies in ruminants, highlighting the need for advanced metagenomic and host-linkage studies to inform future applications.

One proposed strategy to mitigate CH₄ emissions is the development of vaccines targeting methanogenic archaea in the rumen. These vaccines aim to elicit an immune response that reduces methanogen populations, thereby lowering methane production without adversely affecting essential microbial communities in the rumen (Wedlock et al., 2013). Developing an effective methane-reducing vaccine requires identifying immunogenic proteins unique to methanogens to ensure a robust immune response while maintaining overall gut health (Baca-González et al., 2020). Research indicates that vaccines targeting key methanogen species can significantly alter rumen archaeal populations, leading to a measurable reduction in methane emissions (Williams et al., 2009). However, long-term efficacy remains a critical challenge, as the rumen microbiome is highly dynamic and capable of adapting to immune pressures over time (Wedlock et al., 2010).

In vivo (Wright et al., 2004; Zhang et al., 2015), and in vitro (Cook et al., 2008) studies evaluating antimethanogenic vaccines have reported variable and often time-dependent effects on enteric CH₄ production. Notably, the lack of a consistent reduction in CH₄ emissions—despite increased methanogen-specific antibody titers and observed shifts in archaeal community composition—suggests that vaccine formulations may lack broad-spectrum efficacy against the diverse rumen methanogen populations (Williams et al., 2009). Moreover, population-level differences in immune responses across species and breeds introduce high inter-animal variability, complicating the predictability and scalability of vaccine interventions (Buddle et al., 2011). One of the major limitations in the development of antimethanogenic vaccines is the challenge of identifying antigens that are both conserved and immunogenic across the diverse array of methanogenic archaea present in the rumen. Methanogens exhibit high variability in surface structures and protein epitopes (Reeve, 1992), which complicates the formulation of a broadly protective vaccine. In addition, variation in host immune response—driven by genetic background, physiological status, and rumen microbiota composition—leads to inconsistent antibody production and limited uniformity in microbial suppression. Some animals exhibit high antibody titers with negligible impact on archaeal populations or methane output, while others respond poorly to vaccination protocols. These issues have been reported in both dairy and sheep trials and represent key barriers to reliable implementation (Wedlock et al., 2013; Subharat et al., 2016). Another source of variation is animal age, as it is well-known that young animals are more susceptible to infectious diseases than adults (Watson et al., 1994). Moreover, the durability of the immune response and the potential for microbial adaptation or vaccine escape remain unresolved. Further research is needed to identify robust antigen targets and optimize delivery systems that can consistently elicit long-term methane mitigation across diverse ruminant populations.

Despite these constraints, vaccination remains a promising and potentially cost-effective approach for mitigating methane emissions. It offers practical advantages, particularly for grazing systems with limited access to feed additives. However, successful implementation will require optimized antigen discovery, improved delivery systems (e.g., oral or slow-release formulations), and robust field trials to assess long-term impacts on CH₄ emissions, animal performance, and microbial ecology (Baca-González et al., 2020).

The advantages and challenges of biological strategies for reducing methane emissions from ruminants are presented through a SWOT analysis, which is presented in Table 3.

Algal biomass is increasingly positioned as a sustainable, circular feed ingredient with the potential to lower the carbon footprint of ruminant production. Beyond serving as a high-quality nutrient source, specific macro- and microalgal taxa contain bioactive compounds that modulate rumen microbiology and hydrogen sinks, thereby holding high potential for enteric CH₄ mitigation. Recent reviews highlight both the promise and the practical constraints (supply, processing costs, and standardization) associated with scaling algae for livestock systems (De Bhowmick and Hayes, 2023; Wanapat et al., 2024).

The summary report of the literature analysis on the effects of supplementing ruminant diets with probiotic bacteria is presented in Table 4.

Among seaweeds, red macroalgae of the genus Asparagopsis remain the most potent enteric CH₄ mitigation option in vivo. Multiple trials in beef cattle have demonstrated substantial reductions when A. taxiformis is included at low dietary levels, with reported decreases often exceeding 50% and, in some cases, approaching 80%, depending on the diet composition and inclusion rate. The primary mechanism involves the inhibition of the methyl-coenzyme M reductase (MCR) pathway by halogenated methane analogs—especially bromoform (CHBr₃)—which suppresses the terminal step of methanogenesis (Thorsteinsson et al., 2023; Kelly et al., 2025).

Efficacy varies with species, dose, basal diet, and type of supplement used in the study (freeze-dried biomass vs. stabilized actives) (Alvarez-Hess et al., 2024). In a finishing-diet research study, a proprietary bromoform-containing algae product (“Alga 1.0”) fed at 69 or 103 g/d reduced methane yield by 39% and 64%, respectively, without affecting digestibility but decreasing DMI by ~10–13%. These data underscore the trade-off between mitigation and intake that may emerge at higher effective doses (Colin et al., 2024).

Safety and residue outcomes are an active area of research. Transfer of CHBr₃ to milk and urine has been detected under certain conditions in dairy cows fed Asparagopsis. However, tissue accumulation was not observed, and excretion appeared transient in that study. Additionally, some trials with Asparagopsis armata at 0.5–1.0% of dietary OM in dairy cows reduced CH₄ yield but also lowered DMI, highlighting the need for careful dosing and monitoring of animal performance and product quality (including iodine/halogen load) (Muizelaar et al., 2021). Similarly, in dairy cows, supplementation with A. taxiformis at 0.3% of dietary OM reduced enteric CH₄ emissions by ~30% during the first 8 weeks, with no sustained effect from week 9 to 12. This inclusion level also led to reductions in DMI (~7%) and ECM (~2%), shifts in VFA profiles (↓acetate; ↑propionate, butyrate, valerate), and elevated concentrations of bromine and iodine in milk (5-fold and 9-fold higher than controls, respectively), highlighting the need for long-term evaluation of efficacy, safety, and product integrity (Angellotti et al., 2025).

By contrast, brown and green seaweeds generally lack halomethanes at higher levels; their antimethanogenic potential is less consistent and often modest. For example, bromoform-free brown/green species included at 10 g/kg diet DM did not reduce CH₄ in RUSITEC tests, whereas metabolomics indicate these taxa contain phenolics (e.g., phlorotannins) and other sulfated compounds that could influence fermentation. Species, season, and geography contribute to pronounced chemical variability (Nørskov et al., 2021; Roskam et al., 2022).

Several microalgae and cyanobacteria have shown methane-mitigating potential—though, to date, none match Asparagopsis in vivo. In vitro study comparing Chlorella vulgaris, Tetraselmis spp., and Nannochloropsis oceanica found the lowest CH₄ yield with N. oceanica at 10% of incubated DM, likely linked to its high n-3 PUFA content (Meehan et al., 2021). Likewise, Dunaliella salina, when used as an additive with maize forages, lowered biogas/CH₄ kinetics without compromising fermentation characteristics (Elghandour et al., 2023).

In vivo findings are mixed and context-dependent. Some studies report that Chlorella can increase methanogenic archaea and protozoa in goats, whereas others (including associative feeding strategies with low-level Chlorella) suggest potential to improve fermentation while decreasing CH₄ (Tsiplakou et al., 2017; Kholif et al., 2023). Cyanobacteria Spirulina (Arthrospira spp.) is widely used as a protein/antioxidant supplement. Across small-ruminant studies, Spirulina supplementation has been shown to modulate the rumen microbiome, but it yields inconsistent methane responses. In lactating goats, ≈1% of diet DM—especially when combined with live yeast—lowered Methanobrevibacter prevalence and predicted CH₄, though effects were small-scale (Emara Rabee et al., 2025). In ewes, graded doses of methanogen inhibitors shifted community structure without reducing total methanogens, and Methanobrevibacter tended to increase at the highest inclusion rate (Christodoulou et al., 2023). In lambs, ~3% (fresh-weight basis) of the altered microbiota did not produce consistent enteric CH₄ outcomes (Wang et al., 2024b). Collectively, Spirulina may influence archaeal ecology at low inclusion rates, yet robust, controlled trials are needed to clarify its effects on CH₄ emissions.

Microalgal feed supplements appear to modulate rumen fermentation and H₂ disposal pathways (e.g., favoring propionate or microbial lipid sinks); however, the magnitude of CH₄ suppression is typically lower than that achieved with Asparagopsis.

Collectively, the literature supports algae as a diverse toolbox for enteric methane abatement. Asparagopsis (via bromoform) delivers the most considerable and reproducible reductions—especially in high-concentrate systems—while brown/green macroalgae and microalgae offer nutritional value and modest, formulation-dependent CH₄ mitigation. Critical research gaps include: (1) scalable, cost-efficient cultivation and processing for consistent bioactive content; (2) long-term animal health and product-quality surveillance (residues, iodine/halogens); (3) delivery formats that sustain efficacy without depressing intake; and (4) robust performance data in pasture-based and dairy systems.

Biochar (BH) has garnered increasing interest as a potential CH₄ mitigation agent in ruminant nutrition due to its high surface area, porosity, and adsorptive capacity, which may modulate rumen fermentation and microbial dynamics. Proposed mechanisms include altering microbial habitats, reducing hydrogen availability for methanogenesis, and promoting the proliferation of alternative hydrogen-utilizing microbes (Leng et al., 2013; Saenab et al., 2018). However, evidence for its effectiveness remains inconsistent across studies (Winders et al., 2019; Sperber et al., 2022).

In a recent two-phase study in beef cattle, supplementation with tailored (“fit-for-purpose”) biochars yielded modest reductions in CH₄ emissions (8.8–12.9%) under controlled pen conditions. Still, no effect was observed under grazing systems, highlighting a disconnect between controlled trials and practical field application (Martinez-Fernandez et al., 2024). Similarly, in dairy cattle, a Latin square trial revealed that neither biochar nor biochar–urea blends affected CH₄ emissions or productive performance (Terler et al., 2023), while supplementation at 1% DM in lactating Holsteins also yielded no benefits (Dittmann et al., 2024). A study in lambs found no favorable effects on CH₄ production or growth, both in vitro and in vivo (Lind et al., 2024). Additionally, mineral-enriched biochar failed to elicit any changes in CH₄ or rumen fermentation in Holstein steers (Ni et al., 2024). By contrast, an in vivo study in ewes reported improved feed efficiency and reduced CH₄ emissions with biochar supplementation (Burezq and Khalil, 2025), indicating that host species, diet type, and biochar formulation may all influence response. This inconsistency likely stems from differences in pyrolysis conditions, feedstock type, particle size, and chemical composition of the biochar used. Smaller particle sizes and acidic pH have been associated with greater CH₄ mitigation (Zhou et al., 2017; Osman et al., 2022), while the presence of phenolic compounds may exert additional antimicrobial effects. A recent quantitative review confirmed the modest average efficacy of biochar across studies but emphasized the substantial heterogeneity and lack of dose–response consistency, calling for standardization in biochar production and application protocols (Pepeta et al., 2024).

Overall, while biochar shows mechanistic potential as a CH₄ mitigation tool, primarily through indirect modulation of ruminal hydrogen metabolism, current in vivo evidence does not yet support its broad implementation in commercial livestock systems. Future work should focus on defining optimal biochar types, inclusion levels, and diet contexts, as well as the possible synergistic effects with other mitigation agents.

Garlic (Allium sativum) and its organosulfur compounds—such as allicin, diallyl sulfide, diallyl disulfide, and allyl mercaptan – have attracted attention as natural feed additives for mitigating enteric CH₄ emissions in ruminants. These compounds exhibit antimicrobial activity against methanogenic archaea and rumen protozoa and have been shown to alter fermentation profiles by promoting propionate production, thereby redirecting H₂ away from methanogenesis (Shang et al., 2019; Sari et al., 2022).

However, the efficacy of garlic-based interventions appears highly variable. It is influenced by multiple factors, including the specific compound used, its concentration and stability, the delivery matrix (e.g., oil, extract, powder), and interactions with the basal diet (Kamel et al., 2008; Sari et al., 2022).

Recent in vivo evidence supports the methane-reducing potential of garlic-derived products under controlled and grazing conditions. In a respiration chamber study with mid-lactation dairy cows, supplementation with a garlic–citrus extract over 18 days reduced CH₄ production (−10.3%), intensity (−11.7%), and tended to lower CH₄ yield (−9.7%) without affecting dry matter intake or milk yield. Propionate concentrations increased, while Methanobrevibacter abundance declined. Similarly, under grazing conditions, daily supplementation of 33 g/cow of GCE over 12 weeks improved DMI and ECM yield. This led to an 8.39% reduction in milk GHG intensity, as determined by a life cycle assessment, although CH₄ was not directly quantified in the study (Khurana et al., 2024).

Meta-analyses and recent reviews have emphasized the heterogeneity in response to garlic supplementation, highlighting formulation sensitivity as a key factor influencing efficacy (Shang et al., 2019; Sari et al., 2022a, Ding et al., 2023; Martin and Chaudhry, 2024). Several studies demonstrated that garlic products provide a range of biological benefits to ruminants (Ogbuewu et al., 2019; Yang et al., 2021). While garlic-based products offer a promising natural approach to CH₄ mitigation, especially at practical inclusion levels that do not compromise intake or animal performance, their persistence and repeatability under commercial conditions remain uncertain.

In summary, garlic and its bioactive constituents have demonstrated potential for mitigating CH₄ through both direct inhibition of methanogens and fermentation shifts that favor propionate production. However, the success of such strategies depends heavily on compound selection, dosing, delivery method, and dietary context. Long-term, multi-period in vivo studies are needed to confirm sustained efficacy, evaluate adaptation, and guide the development of commercially viable formulations.

Tannins—classified as condensed (CT) or hydrolyzable (HT) based on their chemical structure—are among the most widely studied plant secondary compounds for enteric CH₄ mitigation in ruminants. Their antimethanogenic effects are attributed to multiple mechanisms, including suppression of protozoa and associated methanogens, shifts in VFAs production (typically characterized by reduced acetate and increased propionate), and complexation with dietary proteins and carbohydrates, which can reduce H₂ availability for methanogenesis (Patra and Saxena, 2011; Goel and Makkar, 2012). The extent of mitigation depends heavily on the type of tannin, the botanical source, the inclusion rate, and the adaptation period.

A comprehensive meta-analysis by Jayanegara et al. (2012) covering both in vitro and in vivo data confirmed an apparent, dose-dependent reduction in CH₄ emissions, particularly with CT sources. More recently, a systematic review by Cardoso-Gutierrez et al. (2021) focused on tropical forages and reported consistent CH₄ suppression across multiple studies. However, the magnitude of reduction was highly variable and linked to the specific plant species and dosage employed. Goel and Makkar (2012) highlighted that CT mitigates CH₄ primarily via indirect mechanisms, such as reducing fiber digestion and thus limiting H₂ availability. In contrast, HT appear to exert more direct antimethanogenic effects by inhibiting the growth and activity of methanogens and hydrogen-producing microbes. Animal-level studies further demonstrate the complex and dose-dependent impacts of tannin supplementation on CH₄ mitigation and animal productivity. In dairy goats, stepwise inclusion of quebracho-derived condensed tannins (CT; 0–6% of diet DM) elicited non-linear responses, with milk yield peaking at approximately 4% CT, beyond which diet digestibility declined and effects on methane emissions became inconsistent (Battelli et al., 2024). Similarly, dietary inclusion of hydrolyzable tannins (HT) has been associated with improvements in milk yield and udder health, further supporting their utility in dairy systems (Ali et al., 2017). In an earlier in vivo study, Beauchemin et al. (2007a) reported a 14% reduction in CH₄ emissions following dietary supplementation with Quebracho tannin extract, accompanied by a shift in VFA production toward propionate, a competitive H₂ sink. Comparable results were observed by Grainger et al. (2009) who supplemented condensed tannins from Lotus pedunculatus and reported up to 29% CH₄ reduction without adverse effects on dry matter intake or animal productivity.

In vitro investigations support the potential of forage-derived tannins. For example, purified CT extracts from Hedysarum coronarium (sulla) and Lotus corniculatus (big trefoil) decreased CH₄ production by up to ~15% at inclusion rates of 30 g/kg DM. However, gas production and fermentation efficiency were negatively affected at the highest levels (Verma et al., 2023). These findings underscore the importance of optimizing tannin inclusion levels to mitigate undesirable effects on rumen fermentation and animal productivity.

In summary, tannins represent a viable strategy for mitigating enteric CH₄ emissions in ruminants, particularly when their use is aligned with dietary context and production objectives. Low-to-moderate inclusion levels (<3–4% of diet DM) have been shown to reduce CH₄ output without adversely affecting animal performance; however, higher doses may impair nutrient digestibility and feed efficiency. Effective formulation requires careful consideration of tannin type (condensed vs. hydrolyzable), bioactivity, and interactions with the basal diet to ensure sustained mitigation and production efficiency.

In addition, key knowledge gaps remain regarding the mechanisms by which tannins reduce methanogenesis, including their effects on nutrient utilization, direct inhibition of methanogens, suppression of protozoa, and modulation of hydrogen sinks within the rumen environment. Addressing these uncertainties through targeted in vivo research will be essential to optimizing tannin-based strategies for practical application.

Saponins—diverse glycosides abundant in legumes and tropical plants—are recognized for their antiprotozoal and antimicrobial properties (Patra and Saxena, 2009; Goel and Makkar, 2012). By suppressing rumen protozoa—key partners of methanogenic archaea—saponins diminish hydrogen transfer to methanogens, thereby reducing CH₄ formation. They also act directly against methanogens, shifting fermentation toward propionate production —a competitive hydrogen sink (Hristov et al., 2013; Pen et al., 2006; Patra and Saxena, 2009; Firkins and Mitchell, 2023). Commercial saponin sources such as Yucca schidigera and Quillaja saponaria are well-characterized: QS contains ~10% triterpenoid saponins across 20+ structures, while YS offers ~4.4% steroidal saponins spanning 28 variants (Kholif, 2023). Other promising sources include Sapindus saponaria, which exhibits potent antiprotozoal activity (Hu et al., 2018), and fenugreek (Trigonella foenum-graecum), notable for its high saponin content (~4.63 g per 10 g) and potential antimethanogenic action (Singh and Garg, 2006; Visuvanathan et al., 2022).

In vitro, S. saponaria fruit extracts (100 mg/g) significantly decreased CH₄ without impairing fermentation. At the same time, inclusion of its seed pericarp reduced protozoa and improved weight gain in sheep, though CH₄ was not measured (Navas-Camacho et al., 2001; Hess et al., 2003). Fenugreek extracts also inhibited total gas and CH₄ production and shifted VFAs toward propionate in vitro (Dey, 2015; Niu et al., 2021), while improving nitrogen utilization without affecting intake or digestibility (Wina et al., 2005).

Although saponins exhibit considerable potential to reduce enteric methane emissions across a range of inclusion levels, thereby supporting environmentally sustainable ruminant nutrition (Ridla et al., 2021). Evidence suggests that their effects may not be consistently sustained over time. Several long-term in vitro studies have indicated that the methane-suppressing effects of certain saponin extracts on rumen microbial fermentation may be transient rather than permanent (Wang et al., 1998; Cardozo et al., 2004). This attenuation may be partly explained by microbial adaptation, as rumen microbes can adjust to repeated exposure to bioactive compounds such as saponins (Makkar and Becker, 1997; Wallace et al., 2002).

However, in vivo responses to saponin supplementation remain inconsistent. For instance, supplementation of whole-plant Yucca schidigera or Quillaja saponaria at 10 g/kg DM failed to reduce CH₄ emissions in lactating dairy cows (Holtshausen et al., 2009), while lower-dose inclusion in sheep yielded only numerical reductions (Pen et al., 2007). Similarly, in dairy goats, supplementation with fenugreek seeds at 0.1 kg/d had no significant impact on milk yield or health status (El-Tarabany et al., 2018; Akbağ et al., 2022). By contrast, substantial CH₄ reductions of 28%, 35.8%, and 47.9% were observed in sheep supplemented with tea seed saponins at 5, 10, and 20 g/kg DM, respectively (Zhang et al., 2021), highlighting the role of the botanical source and dose in determining efficacy. Beyond ruminant systems, low-level inclusion of fenugreek (0.04%) has demonstrated benefits in aquaculture species—improving growth, antioxidant capacity, and immune function (Yu et al., 2019; Abdel-Wareth et al., 2021; Yang et al., 2022; Paneru et al., 2022), indicating the broader applicability of saponins across animal production systems. A recent meta-analysis encompassing 66 in vivo treatments (up to 40 g/kg DM) revealed no adverse effects on feed intake; however, the effects on productivity and fermentation were highly variable and dependent on the plant source, animal species, and dietary context (Yanza et al., 2024).

These findings underscore the need for additional long-term, species-specific studies to better understand the persistence of saponin-induced CH₄ mitigation and to refine supplementation strategies for practical livestock systems.

The summary report of the analysis of literature data on the effects of supplementation of ruminant diets with garlic, tannins, or saponins is shown in Table 5.

Essential oils (EOs) are plant-derived volatile compounds with antimicrobial properties that have been explored as natural feed additives to mitigate enteric CH₄ emissions in ruminants. Their effects are attributed to the modulation of rumen microbial communities, the inhibition of methanogens and protozoa, and alterations in fermentation profiles (Castillejos et al., 2005; Calsamiglia et al., 2007; Patra and Yu, 2012). Compounds such as thymol, eugenol, carvacrol, cinnamaldehyde, and flavonoids (e.g., naringin, hesperidin) have demonstrated methane-reducing potential in both in vitro and in vivo systems (Busquet et al., 2005a; Patra and Yu, 2015; Yu et al., 2024).

In vitro studies report CH₄ reductions ranging from 10% to 91%, depending on EO type, dose, and microbial sensitivity (Busquet et al., 2006; Cobellis et al., 2016). For example, garlic oil constituents—diallyl disulfide and allyl mercaptan—reduced CH₄ production by up to 74% in batch cultures (Busquet et al., 2005a), high-carvacrol oregano oil reduced methane by 22% at 1000 mg/L, although with concurrent suppression of VFA production and feed digestion (Benchaar and Hassanat, 2024).

Similarly, citrus flavonoids (naringin and hesperidin, each at 10 g/kg DM) or citrus flavonoid extract (20 g/kg DM) significantly reduced CH₄ and ammonia concentrations in vitro, alongside declines in archaea Methanobrevibacter spp. and protozoa Isotricha spp. populations (Yu et al., 2024). The authors suggest that flavonoids may possess synergistic effects in mitigating ruminal CH₄ and have the potential to enhance N utilization. Using the rumen simulation technique (RUSITEC), Soliva et al. (2011) reported a 91% reduction in daily CH₄ emissions, accompanied by a decrease in protozoal counts and an increase in total bacterial populations, highlighting the strong methane-mitigating potential of the garlic oil under controlled in vitro conditions. In another in vitro study, five essential oils—clove, eucalyptus, garlic, oregano, and peppermint – reduced CH₄ production by 34.4%, 17.6%, 42.3%, 87.0%, and 25.7%, respectively, at 1.0 g/L, with oregano oil showing the most significant CH₄ inhibition (Patra and Yu, 2012).

In vivo, results have been inconsistent. Agolin^® Ruminant (a commercial EOs blend) reduced CH₄ emissions by 8.8%, improved milk yield by 4.1%, and enhanced feed efficiency by 4.4% in lactating dairy cows (Belanche et al., 2020). A carbon footprint modelling study confirmed a 6% reduction in GHG emissions across several feeding strategies (Becker et al., 2023). However, Benchaar and Hassanat (2025) found no effect of the same blend (1 g/day) on lactational performance or CH₄ output in dairy cows. Castro-Montoya et al. (2015) reported a 15% CH₄ reduction after 6 weeks of supplementation with 0.2 g/d of Agolin^® Ruminant in dairy cows. Interestingly, no significant changes were seen in beef heifers supplemented with the same dose.

Conversely, several studies have reported inconsistent or non-significant effects of essential oil supplementation on CH₄ mitigation and animal performance. For example, an EOs blend of cresols, thymol, limonene, vanillin, eugenol, and salicylates (1.2 g/day) did not confer any measurable benefits in mid-lactation Holstein dairy cows in terms of CH₄ mitigation, lactational performance, or rumen fermentation parameters (Joch et al., 2019). Likewise, Jiménez-Ocampo et al. (2021) demonstrated CH₄ reductions with 1.5 g/kg DMI of naringin and chitosan in in vivo trials. However, in situ tests using the same doses (1.5–3.0 g/kg DMI) showed no significant changes in CH₄ or nutrient use. Supplementation with eucalyptus and anise oils at 0.5 g/animal/day in sheep had no significant effect on methane production (Wang et al., 2018). An in vitro experiment using rumen inoculum from Daragh ewes demonstrated that sage, pine, and clove EOs at 300–900 mg/L led to dose-dependent CH₄ suppression and improved the ruminal fatty acid profile (Bokharaeian et al., 2023).

These contrasting findings underscore the complexity of host–additive interactions and suggest that the delivery method, dosage, and microbial adaptation may have a significant influence on experimental results.

Recommended effective doses for CH₄ mitigation typically range from 20 to 1000 mg/L in vitro and 500 to 1000 mg/day in vivo. However, high doses may impair fibre digestion and reduce feed intake (Cobellis et al., 2016; Joch et al., 2019). Long-term exposure to EOs may induce microbial adaptation, reducing their effectiveness over time.

Thus, EOs supplementation should be approached with caution—strategies such as encapsulation, rotational use, or combination with other phytochemicals are recommended to sustain efficacy while minimizing adverse effects (Benchaar and Greathead, 2011; Patra and Yu, 2015).

Ezema (2013) described probiotics as live, non-pathogenic, and non-toxic microorganisms that, when administered in appropriate amounts, confer beneficial effects on the host animal. Their mechanism of action includes improving feed digestibility, enhancing beneficial microbial populations, competing with methanogens for substrates (e.g., hydrogen), and modulating ruminal fermentation pathways (Uyeno et al., 2015). In ruminant nutrition, commonly used probiotics—also referred to as direct-fed microbials—include yeast species such as Saccharomyces cerevisiae, as well as bacterial genera including Bacillus, Bifidobacterium, Enterococcus, Lactobacillus, Propionibacterium, Megasphaera elsdenii, and Prevotella bryantii (Seo et al., 2010).

The summary report of the literature analysis on the effects of supplementing ruminant diets with probiotic bacteria is presented in Table 6.

Bacterial probiotics have been shown to improve rumen function, enhance dry matter intake, feed efficiency, and weight gain in ruminants (Elghandour et al., 2015). They may also inhibit pathogenic microbes, modulate gut microbiota, and stimulate the immune system via bacteriocin production (Khan et al., 2016). Additionally, their supplementation has been associated with increased milk yield, fat-corrected milk, and milk fat content (Elghandour et al., 2015; Khan et al., 2016).

Studies of Bacillus subtilis supplementation in cattle have reported improvements in digestibility, performance, milk production, reductions in somatic cell counts, reductions in CH₄ emissions, and stimulation of proteolytic and amylolytic bacterial growth (Sun et al., 2013; Jia et al., 2022). The inclusion of B. subtilis under in vitro conditions has demonstrated potential for reducing ruminal methane production when supplemented in mid-lactation dairy cow diets, suggesting its promise as a methane mitigation additive (Sarmikasoglou et al., 2024). In young Holstein calves, dietary supplementation with a probiotic mixture (L. plantarum, Pediococcus acidilactici, Pediococcus pentosaceus, and B. subtilis) has been shown to enhance health status and decrease the need for medicinal treatments (Wang et al., 2022).

M. elsdenii, a lactic acid-utilizing bacterium, has also been investigated for its probiotic potential. Its capacity to metabolize lactate into VFAs such as butyrate and propionate supports pH stability and reduces lactate accumulation, which can limit methanogenic activity (Carberry et al., 2012; Cabral and Weimer, 2024). A recent meta−analysis by Susanto et al. (2023) integrating 32 studies (136 data points) found that M. elsdenii inclusion significantly reduced CH₄ emissions (p < 0.05), while simultaneously improving fermentation profiles (e.g., increased propionate, butyrate, isobutyrate, valerate; decreased lactic acid and acetate proportion) and enhancing livestock performance (e.g., average daily gain, body condition score, carcass traits).

Yeast-based probiotics have emerged as a potential strategy for mitigating enteric CH₄ emissions in ruminants. Although supplementation with live yeast, particularly Saccharomyces cerevisiae, is known to stimulate cellulolytic bacterial populations, potentially increasing H₂ production—a key substrate for methanogenesis—it may also simultaneously enhance the proliferation of alternative H₂-utilizing microorganisms. This dual microbial modulation may lead to a net reduction in CH₄ production by diverting metabolic H₂ flux away from methanogens and toward competing fermentation pathways, such as propionate or acetogenesis. Such mechanisms suggest that yeast probiotics could play a supportive role in reducing CH₄ emissions while improving overall rumen function and fermentation efficiency (Newbold and Rode, 2006; Chaucheyras-Durand et al., 2008; Newbold et al., 1996; Fonty and Chaucheyras-Durand, 2006). In several in vitro studies, the addition of S. cerevisiae has been shown to decrease CH₄ production (Bayat et al., 2015; Kamal et al., 2025).

While direct anti-methanogenic effects of yeast are less pronounced, their supportive role in maintaining rumen health and competitive microbial dynamics can indirectly contribute to CH₄ mitigation. Additionally, S. cerevisiae can improve feed intake, nutrient digestibility, rumen ecology, and growth performance (Khalouei et al., 2020; Phesatcha et al., 2021), and milk production in dairy cows (Majdoub-Mathlouthi et al., 2009; Moallem et al., 2009; Maamouri et al., 2014; Bayat et al., 2015; Rossow et al., 2018; Perdomo et al., 2020; Cattaneo et al., 2023). It can also reduce oxidative stress and improve dairy cattle performance under heat-stress conditions (Perdomo et al., 2020; Benedetti et al., 2024). Despite promising results, the application of probiotics in ruminants for CH₄ mitigation remains limited compared to chemical inhibitors or feed formulation strategies. In addition, the effectiveness of probiotics is often inconsistent due to variations in strain specificity, dosage, delivery method, dietary context, and host microbiome composition. Long-term, large-scale in vivo studies under commercial conditions are necessary to validate their efficacy in CH₄ reduction and assess potential interactions with other mitigation strategies.

Nonetheless, probiotics—particularly when used in synergistic combinations or in conjunction with complementary additives—represent a sustainable and biologically integrated strategy for mitigating methane. In addition to their environmental benefits, probiotics contribute to enhanced rumen health, improved nutrient utilization, and increased overall animal productivity.