Methane mitigation potential of herbal mixture supplementation in total mixed rations: Effects on in vitro fermentation characteristics, rumen protozoa, and methanogenic archaea

Introduction

Livestock production plays a vital role in global food security by providing milk, meat, fiber, and livelihood opportunities to millions of people worldwide. Among livestock species, ruminants contribute substantially to the agricultural economy through their ability to convert fibrous feed resources into valuable animal products. However, enteric methane production during ruminal fermentation has emerged as a major environmental concern due to its contribution to greenhouse gas emissions and climate change. Methane possesses a global warming potential approximately 28 times greater than carbon dioxide over a 100-year period and accounts for a significant proportion of greenhouse gases emitted from the livestock sector. Methane production in the rumen is a natural consequence of anaerobic fermentation, where methanogenic archaea utilize hydrogen and carbon dioxide generated during microbial degradation of feed. Although methanogenesis serves as a hydrogen sink that maintains rumen fermentation, it represents an energy loss of approximately 2-12% of gross dietary energy consumed by the animal. Therefore, reducing enteric methane emissions not only contributes to environmental sustainability but also improves feed energy utilization and animal productivity. Several methane mitigation strategies have been investigated, including dietary manipulation, chemical inhibitors, ionophores, probiotics, vaccines, and plant-derived bioactive compounds. Among these approaches, phytogenic feed additives have gained considerable attention because of their natural origin, consumer acceptability, and minimal risk of chemical residues in animal products. Herbs and their bioactive constituents such as tannins, saponins, flavonoids, essential oils, and phenolic compounds have been reported to suppress methanogenic archaea, reduce ruminal protozoal populations, and alter fermentation pathways, thereby decreasing methane production. The use of herbal mixtures rather than individual herbs may provide synergistic effects due to the complementary actions of diverse phytochemicals. Previous studies have demonstrated that certain herbal combinations can effectively reduce methane production while maintaining normal rumen fermentation and nutrient digestibility. However, the efficacy of herbal mixtures depends on their composition, inclusion level, and interaction with rumen microbial populations. Therefore, identifying optimum combinations and dose levels is essential before recommending their practical application in ruminant feeding systems. In vitro gas production techniques provide a rapid, economical, and reliable method for screening feed additives for their methane mitigation potential. Furthermore, advances in molecular biology, particularly quantitative polymerase chain reaction (qPCR), have enabled precise quantification of specific rumen microbial populations involved in methanogenesis and fiber degradation, thereby improving the understanding of mechanisms underlying methane reduction. The present study was undertaken to evaluate the methane mitigation potential of selected herbal mixture combinations incorporated into total mixed rations using in vitro gas production techniques. The study further aimed to identify the optimum inclusion level of the most promising herbal mixture and to investigate its effects on rumen fermentation characteristics, protozoal population, microbial biomass production, and the abundance of methanogenic archaea and fibrolytic bacteria using qPCR analysis. The findings are expected to contribute toward the development of sustainable and environmentally friendly feeding strategies for reducing enteric methane emissions from ruminants without compromising rumen function and feed utilization.

Materials and Methods

Experimental design

The present study was conducted to evaluate the methane mitigation potential of selected herbal mixture combinations incorporated into total mixed rations (TMR) using the in vitro gas production technique. Herbal mixture combinations that exhibited superior methane reduction potential in previous in vitro screening studies were selected for further evaluation in TMR. The investigation was carried out in three successive experiments. The effects of these treatments on total gas production, methane production, percentage methane in total gas, in vitro true dry matter digestibility (IVTDMD), methane production per 100 mg truly digested substrate (TDS), pH, ammonia nitrogen, total volatile fatty acids (TVFA), microbial biomass production, and total protozoal count were evaluated.

Experiment 2: Validation of the most effective herbal mixture combination

Based on the results of Experiment IV, the herbal mixture combination supplemented at 60 mg (0.05%) level, which exhibited the lowest methane production per 100 mg truly digested substrate without adversely affecting digestibility, was selected for further validation. The selected herbal mixture was evaluated at three inclusion levels:

• 60 mg (0.04%) • 60 mg (0.05%) • 60 mg (0.06%) along with an unsupplemented control.

The effects on total gas production, methane production, percentage methane in total gas, IVTDMD, methane production per 100 mg TDS, pH, ammonia nitrogen, TVFA concentration, microbial biomass production, and total protozoal count were studied.

Experiment 3: Quantification of rumen methanogens and fibrolytic bacteria by qPCR

Based on the findings of Experiment V, rumen microbial populations were quantified in the following treatments:

• Control • Herbal mixture 60 mg (0.04%) • Herbal mixture 60 mg (0.05%) • Herbal mixture 60 mg (0.06%) Quantitative polymerase chain reaction (qPCR) was employed to determine the abundance of selected methanogenic archaea and fibrolytic bacteria.

• Preparation of total mixed ration and herbal mixture supplementation The basal substrate consisted of a total mixed ration formulated according to the nutrient requirements of ruminants. Feed ingredients were dried, ground through a 1mm sieve, and thoroughly mixed to obtain a homogeneous substrate.The selected herbal mixture combinations were incorporated into the total mixed ration at the respective dose levels prior to incubation. Herbal supplementation levels were expressed as percentages of substrate dry matter.

• Collection of rumen inoculum

Rumen liquor was collected before the morning feeding from healthy adult donor ruminants maintained on a standard feeding regimen. The rumen contents were strained through four layers of muslin cloth under continuous flushing with carbon dioxide to maintain anaerobic conditions. The filtered rumen liquor was transferred immediately to pre-warmed insulated flasks and transported to the laboratory for use as inoculum.

In vitro gas production technique

The in vitro gas production study was conducted following the procedure of Menke and Steingass (1988) [17] . Approximately 500 mg of substrate was weighed into calibrated fermentation syringes. The incubation medium consisted of buffered mineral solution and rumen liquor mixed under continuous carbon dioxide flushing to maintain anaerobic conditions. The substrate and corresponding herbal treatments were incubated with the inoculum-buffer mixture at 39 °C for 24 h. Blank incubations containing inoculum and buffer without substrate were maintained simultaneously for correction of gas production values. At the end of incubation, total gas production was recorded. Methane concentration in the gas produced was determined using gas analytical procedures, and methane production was expressed as millilitres per incubation and as methane production per 100 mg truly digested substrate.

• Determination of in vitro true dry matter digestibility Following incubation, fermentation residues were subjected to neutral detergent extraction for estimation of truly undigested substrate. In vitro true dry matter digestibility (IVTDMD) was calculated as: IVTDMD (%) = [(Initial substrate DM -Residual DM) / Initial substrate DM] × 100 Methane production per 100 mg truly digested substrate was calculated using methane production values and digestibility measurements.

Measurement of fermentation characteristics ph

The pH of fermentation liquor was measured immediately after incubation using a calibrated digital pH meter.

• Ammonia nitrogen Ammonia nitrogen concentration was estimated using standard analytical procedures and expressed as mg/dl.

• Total volatile fatty acids Total volatile fatty acid concentration was determined by steam distillation and expressed as mM/l.

• Microbial biomass production Microbial biomass production was estimated from substrate degradation and gas production data and expressed as mg per incubation.

• Total protozoal count Total protozoa were enumerated microscopically using a haemocytometer after fixation of rumen fluid samples. Protozoal counts were expressed as ×10⁵ cells/ml.

• Quantification of rumen microbial population by quantitative PCR Genomic DNA was extracted from fermentation samples using a commercial DNA extraction kit according to the manufacturer's instructions. DNA concentration and purity were determined spectrophotometrically. Quantitative PCR was performed using species-specific primers targeting:

Amplification reactions were carried out using SYBR Green chemistry in a real-time PCR system. Standard curves were generated using known copy numbers of target genes. Microbial abundance was expressed as log10 copies of target gene per millilitre of rumen fluid.

• Statistical analysis

The experiment was conducted in a Completely Randomized Design (CRD) with six replicates per treatment. Data were subjected to one-way analysis of variance (ANOVA) using appropriate statistical procedures. Treatment means were compared using Duncan's Multiple Range Test (DMRT). Differences among means were considered significant at p<0.05 and highly significant at p<0.01 where applicable. Results were expressed as mean ± standard error (SE).

Result and discussion

Effect of herbal mixture combinations on in vitro gas production, digestibility and methane production

The effects of herbal mixture combinations on in vitro gas production characteristics are presented in Table 1. Total gas production ranged from 27.16 to 41.00 ml among the treatments. The control group recorded the highest (p<0.05) total gas production, whereas supplementation of herbal mixture at 60 mg (0.05%) resulted in the lowest gas production. The reduction in gas production may be attributed to the presence of phytochemicals such as tannins, saponins and essential oils that modulate rumen microbial activity and fermentation pathways. Methane production was significantly (p<0.05) reduced in all herbal mixture supplemented groups compared with the control. The greatest reduction was observed with supplementation of herbal mixture at 60 mg (0.05%), which decreased methane production from 9.16 ml in the control to 5.16 ml, representing a reduction of approximately 43.7%. Similarly, methane production expressed per 100 mg truly digested substrate was significantly reduced in herbal mixture supplemented groups. The reduction in methane production could be attributed to suppression of methanogenic archaea and rumen protozoa by bioactive compounds present in the herbal mixture. Saponins are known to reduce protozoal populations, thereby indirectly decreasing associated methanogens, while tannins inhibit hydrogen-producing microorganisms and methanogenic activity. The percentage methane in total gas production was also significantly reduced by herbal supplementation. The lowest value was observed in the 80 mg (0.025%) treatment (18.19%) compared with the control (22.34%). These findings indicate that herbal supplementation effectively altered rumen fermentation towards reduced methanogenesis without adversely affecting nutrient utilization. In vitro true dry matter digestibility was not significantly affected by herbal supplementation, suggesting that the methane mitigation effect was achieved without compromising feed degradability. Similar observations have been reported by Patra and Saxena (2010), who concluded that plant secondary metabolites can suppress methanogenesis while maintaining normal rumen fermentation. This bar chart in figure 1 illustrates methane production (mL CH₄ g⁻¹ VS) across different experimental treatments. The control group exhibited the highest methane production (138.6 mL CH₄ g⁻¹ VS), whereas methane production progressively decreased with increasing levels of BBS supplementation. Treatments T1 (10% BBS), T2 (20% BBS), T3 (30% BBS), and T4 (40% BBS) recorded methane production values of 114.2, 92.7, 71.4-, and 54.1-mL CH₄ g⁻¹ VS, respectively. Error bars represent the variability among replicates. The results indicate a dose-dependent reduction in methane production with increasing BBS inclusion levels. The reduction in methane production from 138.6 mL CH₄ g⁻¹ VS in the control to 54.1 mL CH₄ g⁻¹ VS in T4 suggests that increasing BBS inclusion effectively suppresses methane generation during fermentation. This effect may be attributed to bioactive compounds present in BBS that alter rumen microbial activity and methanogenic pathways."

Effect of BBS supplementation on methane production

Effect of herbal mixture combinations on rumen fermentation characteristics and microbial population

The effects of herbal mixture supplementation on rumen fermentation parameters are presented in Table 2. The pH values ranged from 6.64 to 6.76 and did not differ significantly among treatments, indicating that supplementation of herbal mixtures did not disturb the normal rumen environment. The observed pH values were within the optimum range required for cellulolytic bacterial activity. Ammonia nitrogen concentration was significantly (p<0.05) reduced in most herbal mixture supplemented groups compared with the control. The reduction in ammonia nitrogen concentration may be attributed to decreased deamination of amino acids and improved utilization of nitrogen by rumen microorganisms. Similar reductions in ruminal ammonia concentration following supplementation of phytogenic additives have been reported previously. Total volatile fatty acid concentration did not differ significantly among treatments, indicating that overall fermentative activity remained unaffected. Maintenance of VFA production together with reduced methane emission suggests improved fermentation efficiency and diversion of metabolic hydrogen away from methanogenesis.

Validation of the optimum herbal mixture inclusion level

Table 3: Effect of potential herbal mixture combinations in total mixed rations studied in vitro on total gas (ml), methane (ml), percentage of methane on total gas, true dry matter digestibility and methane (ml) per 100 mg truly digested substrate (Mean ± SE)

Formula

Percentage Change=(Treatment-Control) /Control×100

Effect of optimum herbal mixture on rumen fermentation characteristics

Herbal mixture supplementation significantly influenced microbial biomass production and protozoal population. Microbial biomass production was significantly higher (p<0.05) in most herbal mixture supplemented groups compared with the control. The highest microbial biomass was observed in the 60 mg (0.05%) treatment. The increase in microbial biomass indicates improved efficiency of microbial protein synthesis and utilization of fermentation substrates. Enhanced microbial growth may contribute to improved nutrient availability for the host animal.Total protozoal counts were significantly lower (p<0.05) in all herbal mixture supplemented treatments than in the control group. The reduction in protozoal population is likely associated with the antiprotozoal activity of saponins and other phytochemicals present in the herbal mixture. Since rumen protozoa maintain symbiotic associations with methanogenic archaea, suppression of protozoal populations may partly explain the reduction in methane production observed in the present study. Relationship between methane production and total protozoal count in herbal mixture-supplemented total mixed rations Figure 2 illustrates the association between methane production and total protozoal count in herbal mixturesupplemented TMR. A strong positive correlation (r = 0.894, p<0.01) was observed, indicating that methane production increased with increasing protozoal population. Scatter plot showing the relationship between methane production (ml/24 h) and total protozoal count (×10⁵ cells/ml) in control and herbal mixture-supplemented total mixed rations (TMR). Each point represents the mean value of six observations. The regression analysis demonstrated a strong positive correlation between methane production and protozoal population (r = 0.894, p<0.01; R² = 0.799), indicating that treatments with lower protozoal counts produced less methane. The results suggest that suppression of rumen protozoa by herbal supplementation may contribute to methane mitigation through disruption of the symbiotic association between protozoa and methanogenic archaea. Approximately 79.9% (R² = 0.799) of the variation in methane production could be explained by differences in protozoal abundance. The control treatment exhibited the highest protozoal count and methane production, whereas herbal mixture supplementation, particularly at 60 mg (0.05%), markedly reduced both parameters. The reduction in protozoal population may be attributed to the presence of phytochemicals such as saponins, tannins, flavonoids, and essential oils in the herbal mixture. These compounds possess antiprotozoal activity and can alter rumen microbial ecology. Rumen protozoa are known to maintain close symbiotic associations with methanogenic archaea by supplying hydrogen generated during feed fermentation. Therefore, a reduction in protozoal numbers decreases the availability of hydrogen for methanogenesis, resulting in lower methane production. The strong correlation observed in the present study supports the hypothesis that suppression of protozoal populations is one of the major mechanisms through which the herbal mixture mitigates enteric methane production. The findings are further supported by the qPCR results, which demonstrated a significant reduction in the abundance of whole methanogens, Methanobacteriales, Methanosarcinales, and Methanomicrobiales in herbal mixture-supplemented treatments. Thus, the decrease in methane production appears to be associated with both reduced protozoal populations and direct inhibition of methanogenic archaea.

Effect of herbal mixture supplementation on rumen methanogenic archaea and fibrolytic bacteria

Quantitative PCR analysis demonstrated that herbal mixture supplementation significantly reduced the abundance of whole methanogens, Methanobacteriales and Methanosarcinales compared with the control. Whole methanogen abundance decreased from 9.11 log10 copies/ml in the control to 6.36 log10 copies/ml in the 60 mg (0.05%) treatment. Similarly, Methanobacteriales and Methanosarcinales populations were markedly reduced following herbal supplementation. These findings provide molecular evidence supporting the reduction in methane production observed during in vitro fermentation. The abundance of fibrolytic bacteria such as Fibrobacter succinogenes and Ruminococcus albus was reduced in some treatments, whereas Ruminococcus flavefaciens remained unaffected. Despite these changes, IVTDMD was maintained, indicating that fibre degradation was not adversely affected. The reduction in methanogenic archaea may be attributed to the antimicrobial effects of phytochemicals present in the herbal mixture. The close association between decreased methanogen abundance, lower protozoal population and reduced methane production suggests that suppression of rumen methanogenesis was the primary mechanism responsible for methane mitigation in the present study. Figure 3 represents the Effect of herbal mixture supplementation on the abundance of rumen methanogenic archaea quantified by qPCR. Values represent mean ± SE (n = 6). Herbal mixture supplementation at 0.05% and 0.06% significantly reduced the abundance of whole methanogens, Methanobacteriales, and Methanosarcinales compared with the control treatment. Different superscripts indicate significant differences among treatments (p<0.05; p<0.01).

Synergistic phytochemical action

Reduced methane production, decreased protozoa and methanogens, improved microbial efficiency

Practical implications

The findings of the present study demonstrate that supplementation of the selected herbal mixture at 60 mg (0.05%) of dietary dry matter can serve as an effective natural strategy for mitigating enteric methane emissions from ruminants. The observed reduction in methane production, coupled with decreased populations of rumen protozoa and methanogenic archaea, indicates that the herbal mixture has the potential to improve rumen fermentation efficiency while lowering greenhouse gas emissions. Unlike synthetic methane inhibitors, herbal feed additives are generally regarded as environmentally friendly, biodegradable, and residue-free, making them more acceptable to consumers and compatible with sustainable livestock production systems.

Limitations of the study

The present investigation provides valuable insights into the methane mitigation potential of herbal mixture supplementation in total mixed rations; however, certain limitations should be acknowledged while interpreting the findings. Firstly, the study was conducted under in vitro conditions using the gas production technique. Although in vitro systems provide a rapid, controlled, and cost-effective approach for evaluating feed additives, they may not fully replicate the complex physiological and microbial interactions occurring within the rumen of live animals. Secondly, the long-term adaptation of rumen microbial populations to continuous herbal supplementation was not evaluated. Microbial communities may undergo adaptive changes over extended periods, which could influence the persistence and magnitude of methane mitigation effects.

Thirdly, the present study assessed the efficacy of the herbal mixture as a composite formulation, and the individual contributions of specific herbal ingredients and their bioactive phytochemicals were not separately quantified. Therefore, the relative roles of individual components in methane suppression remain to be elucidated. Fourthly, animal performance parameters such as feed intake, nutrient utilization efficiency, body weight gain, milk yield, milk composition, reproductive performance, and animal health indicators were not assessed because the study was limited to in vitro experimentation. Finally, although significant reductions in archaea and protozoal populations were observed, comprehensive characterization of the rumen microbiome using advanced molecular approaches such as metagenomics, metatranscriptomics, or metabolomics was beyond the scope of the present investigation. Therefore, further in vivo studies incorporating animal performance measurements, long-term microbial assessments, and comprehensive rumen microbiome analyses are necessary to validate the practical applicability of the herbal mixture under commercial livestock production systems.

Future Perspectives

Although the present study demonstrated the effectiveness of herbal mixture supplementation in reducing methane production under in vitro conditions, further investigations are necessary before its practical application in ruminant feeding systems. Future research should focus on validating these findings under field conditions and elucidating the long-term impacts of herbal supplementation on animal performance and rumen ecology.

In vivo Validation Studies

The methane mitigation potential of the identified optimum herbal mixture supplementation level (60 mg at 0.05%) should be evaluated in live animals through controlled feeding trials to confirm its efficacy under practical production conditions.

Evaluation of Animal Performance

Future studies should assess the effects of herbal supplementation on productive parameters such as feed intake, nutrient utilization, growth performance, milk yield, milk composition, carcass characteristics, and overall animal health to ensure that methane mitigation is achieved without compromising productivity.

Direct Measurement of Methane Emissions

Methane reduction should be quantified using advanced in vivo measurement techniques such as respiration chambers, the sulphur hexafluoride (SF₆) tracer technique, or emerging sensor-based technologies to obtain accurate estimates of enteric methane emissions.

Long-Term Rumen Microbial Adaptation

Extended-duration studies are required to investigate whether rumen microbial populations adapt to continuous herbal supplementation and whether the methane mitigation effect remains stable over time.

Mechanistic Studies on Rumen Microbiome

High-throughput molecular approaches such as metagenomics, metatranscriptomics, and metabolomics may be employed to better understand the interactions between herbal phytochemicals, rumen microorganisms, and fermentation pathways involved in methane suppression.

Optimization of Herbal Formulations

Further research should explore different combinations and concentrations of individual herbal ingredients to identify the most effective formulations and determine potential synergistic effects among bioactive compounds.

Economic Feasibility and Farm-Level Adoption

Comprehensive cost-benefit analyses should be conducted to evaluate the economic viability of herbal supplementation under commercial farming conditions, including the availability, processing costs, and scalability of herbal ingredients.

Environmental Sustainability Assessment

Future studies should quantify the contribution of herbal supplementation toward reducing the carbon footprint of livestock production systems and assess its role in achieving climate-smart and sustainable animal agriculture.

Overall, the promising methane mitigation effects observed in the present investigation warrant further in vivo validation and large-scale field studies to establish herbal mixture supplementation as a practical, environmentally friendly, and economically viable strategy for reducing enteric methane emissions from ruminants.

Conclusion

The present study demonstrated that supplementation of selected herbal mixture combinations in Total Mixed Rations (TMR) effectively mitigated enteric methane production under in vitro conditions without adversely affecting rumen fermentation or feed digestibility. Among the various treatments evaluated, the herbal mixture supplemented at 60 mg (0.05%) emerged as the most effective inclusion level. This treatment significantly reduced methane production from 9.16 ml in the control to 5.16 ml, representing a 43.67% reduction, while methane production per 100 mg truly digested substrate decreased from 5.52 to 3.23 ml, corresponding to a 41.49% reduction. The reduction in methane production was associated with a substantial decline in rumen protozoal population. Total protozoal counts decreased from 7.21 × 10⁵ cells/ml in the control to 4.26 × 10⁵ cells/ml in the 60 mg (0.05%) treatment, representing a 40.92% reduction. Furthermore, qPCR analysis confirmed that methane mitigation was accompanied by a marked suppression of methanogenic archaea. Whole methanogen abundance decreased from 9.11 to 6.36 log₁₀ copies/ml, while significant reductions were also observed in Methanobacteriales (32.90%), Methanosarcinales (40.16%), and Methanomicrobiales (32.50%) populations. These findings indicate that the herbal mixture exerted its methane-reducing effect through both direct inhibition of methanogens and indirect suppression of protozoa-associated methanogenesis. Importantly, supplementation of the herbal mixture did not adversely affect in vitro true dry matter digestibility (IVTDMD), ruminal pH, total volatile fatty acid production, or microbial biomass synthesis, indicating that normal rumen fermentation and feed utilization were maintained. The enhanced microbial biomass observed in supplemented treatments further suggests improved efficiency of rumen microbial activity. Overall, the results demonstrate that the herbal mixture supplemented at 60 mg (0.05%) possesses considerable potential as a natural phytogenic feed additive for reducing enteric methane emissions from ruminants. The combined reductions in methane production, protozoal population, and methanogenic archaeal abundance, together with the maintenance of digestibility and fermentation characteristics, highlight its suitability for sustainable livestock production. However, since the present investigation was conducted under in vitro conditions, future in vivo studies are warranted to validate the efficacy, safety, long-term effects, animal performance responses, and economic feasibility of this herbal mixture under practical feeding conditions before field-level recommendations can be made.

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