Impact of Sage, Ivy Gourd and Lemongrass on Rumen Fermentation and Methane Production: An In Vitro Study

Globally, ruminants generally consume grasses; however, in the Indian subcontinent, especially in Bangladesh, they are commonly given a combination of grasses, inferior rice straw, and concentrates (Rahman et al., 2024a). The inclusion of rice straw or grass as primary components in diets often leads to poor nutrient utilization and contribute to greater environmental pollution (Khonkhaeng et al., 2021). This pollution is primarily caused by methane (CH₄), which is the second most abundant greenhouse gas produced in the rumen by methanogenic archaea (Vargas-Ortiz et al., 2022). A cow daily emits around 132–414 g of enteric CH₄, with a potential for global warming which is 28 times greater than CO2 over a century (Lee et al., 2017). Additionally, CH₄ production leads to a 10% energy loss from feed, reducing nutrient efficiency and causing a loss of 16–26 g/kg of feed consumed (Hristov et al., 2013).

To mitigate CH₄ emissions and improve nutrient utilization, researchers are investigating phytogenic supplements (PSs) as natural and sustainable solutions (Kholif et al., 2024). Sage (Salvia officinalis), a PS, exhibits varying effects on methanogenesis, ruminal fermentation, and nutrient degradation, depending on various doses like 0.5, 1.0, 1.5, and 2.0% dry matter (DM) of diet (Kholif et al., 2024). Among these doses, supplementation at 1% DM with basal diet enhance ruminal fermentation, nutrient degradability, and reduces CH₄ emissions, likely due to its phytochemicals, such as tannins, camphor, saponins, rosmarinic acid, and carnosol (Kholif et al., 2024).

Similarly, ivy gourd (Coccinia grandis) and lemongrass (Cymbopogon citratus), as promising PSs, have been used in egg production in laying hens (Rahman et al., 2021) and functional milk production in cows (Rahman et al., 2024b), respectively, due to their rich content of flavonoids, phenolics, tannins, and saponins. Tannins and saponins present in ivy gourd and lemongrass may reduce protozoa, methanogens, and fungal species by forming sterol complexes in their cell membranes, potentially decreasing enteric CH₄ emissions (Bodas et al., 2012; Goel and Makkar, 2012). However, to our knowledge, the anti-methanogenic potential of ivy gourd as a PS remains unexplored. On the contrary, it was reported that lemongrass, when included at 1% along with mangosteen peel (1%) or separately at 2% of the substrate level, significantly improves total gas production, nutrient degradability, and propionic acid production, while reducing CH₄ concentration and methanogens (Prachumchai et al., 2024; Totakul et al., 2024). Additionally, daily supplementation of lemongrass as a PS (100 g/cattle) has been reported to improve nutrient digestibility, milk yield, antioxidant status, and concentrations of desirable fatty acids (Rahman et al., 2024b). It also stimulates microbial protein synthesis and enhances the populations of cellulolytic and amylolytic bacteria in beef cattle (Wanapat et al., 2008).

In addition, the composition of basal diets along with PSs namely sage, ivy gourd, and lemongrass—particularly their crude protein levels and fibre fractions—are closely associated with CH₄ emissions (Bashar et al., 2024). When these PSs, particularly sage and lemongrass, are included at low levels (typically 1% of DM), their effectiveness is mainly attributed to secondary metabolites rather than primary nutrients (Kholif et al., 2024; Prachumchai et al., 2024). Moreover, the chemical composition of these PSs is influenced by various factors such as plant variety, region, soil type, and cultivation practices, which may influence rumen fermentation patterns and CH₄ production (Yalew et al., 2020). Among various varieties, lemongrass (Cymbopogon flexuosus) exhibited variable fibre fractions, with cellulose, hemicellulose, and lignin contents approximately 39.5%, 22.6%, and 28.5%, respectively (Madhu et al., 2017). However, to the best of our knowledge, the nutrient composition—particularly the fibre fractions and the anti-methanogenic properties of these PSs have not been studied in this subcontinent. Consequently, this study (in vitro) was designed to evaluate the composition of shrub (sage) and herbs (ivy gourd and lemongrass) and their effects on rumen organic matter degradability, microbial protein synthesis, digestible energy, metabolizable energy, short-chain fatty acids, net gas, and CH₄ production.

The experiment was under taken in the Department of Animal Nutrition at Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh. Phytogenic supplements (PSs), including sage (Salvia officinalis), ivy gourd (Coccinia grandis), and lemongrass (Cymbopogon citratus DC. staf.), were cultivated and harvested at 65 days post-planting (Rahman et al., 2021; Rahman et al., 2024b) from the Shahjalal Animal Nutrition Field Laboratory (SANFL) at BAU. The harvested PSs were oven-dried at 60°C and subsequently ground using a locally manufactured grinder equipped with a 1 mm sieve. Then, each PS was placed in a zipper-sealed bag and stored in a desiccator with silica gel at room temperature to maintain its stability and prevent moisture absorption.

Green roughage namely Napier grass (Pennisetum purpureum) and dried rice straw were collected from the SANFL, BAU. Concentrate items, including corn, mustard oil cake, soybean meal, and wheat bran, were procured from the local market. These ingredients were oven-dried at 60°C and ground to a particle size of 1 mm using a locally made grinder. Then, the concentrate mixture was prepared on a dry matter (DM) basis by combining corn, mustard oil cake, soybean meal, wheat bran, di-calcium phosphate, and salt at 15.72, 9.60, 4.96, 8.00, 0.50, and 1.00%, respectively. The basal diet was formulated considering the roughages and concentrate mixture ratio at 60: 40 and designed as the control diet without PS (Table 1). The phytogenic diets (PDs) included the basal diet supplemented with 1% DM of sage, ivy gourd, or lemongrass and considered as sage, ivy gourd, or lemongrass diet, respectively. The control diet and three PDs were arranged in a completely randomized design.

The proximate components namely DM, ash, crude protein (CP), crude fibre (CF), and ether extract (EE) of the basal diet, Napier grass, rice straw, concentrate mixture, and PSs including sage, ivy gourd, and lemongrass powder were determined in accordance with the AOAC, (2005). The fibre components of these samples, including neutral detergent fibre (NDF), acid detergent fibre (ADF), and acid detergent lignin (ADL), were determined following the procedures described by Goering and Van Soest, (1970). Then, hemicellulose was calculated by subtracting the ADF from the NDF; whereas cellulose was quantified by subtracting the ADL from the ADF. Besides, non-structural carbohydrate (NSC) were calculated using the following formula, NSC (%) = 100 – % of (CP + ash + NDF + EE). The chemical composition of the basal diet, Napier grass, rice straw, and concentrate mixture are presented in Table 1.

Table 1: Ingredients and nutrient composition of the basal diet and nutrient composition of grass, straw, and concentrate mixture.

The rearing of cannulated beef cattle and the rumen liquor collection system were approved by the Animal Welfare and Experimentation Ethics Committee (AWEEC/BAU/2025 (2) /15(b)). The in vitro study was conducted in two phases. In the first phase, rumen liquor was collected from two Holstein crossbred beef cattle (390 ± 30 kg) at a local slaughterhouse using a pre-warmed conical flask filled with CO₂. The flask was placed in a thermo flask with hot water (39°C) to maintain temperature during collection and transportation. The rumen liquor was then homogenized and filtered through a two-layer cheesecloth into a CO₂-filled warm flask. In the second phase, rumen liquor was collected from one Holstein crossbred cattle (350 kg) and one cannulated Indigenous Zebu beef cattle (198.5 kg) following the same procedure. The cannulated animal, housed at SANFL, BAU, was maintained on a 52: 48 roughage-to-concentrate diet. Cattle was supplied a daily ration of around 12 kg German grass and 2.20 kg of a concentrate mixture (66.4% corn, 16.3% wheat bran, 15.4% mustard oil cake, 0.9% di-calcium phosphate, and 1.0% salt), with ad libitum water access. To accurately represent diet digestibility across both genetic types and avoiding variability from slaughterhouse sampling, rumen liquor was collected from a cannulated Indigenous Zebu cattle under controlled conditions and from slaughterhouse-sourced Holstein crossbred cattle.

For both phases, in vitro gas production was measured described by Menke et al. (1979). For each phase, after measuring the basal diet (200 ± 10 mg) without PSs (control diet) or with PSs at 1.0% of DM (PDs: sage, ivy gourd, or lemongrass), fourteen piston syringes (2 per diet, 2 blanks, 2 hay and 2 concentrate standards) fitted with piston and long needle attached to silicon rubber tube was incubated at 39°C from the afternoon. The following day, one part of filtered liquor was mixed with two parts of an incubation medium saturated with CO₂ and stirred using a magnetic stirrer in a 39°C water bath to prepare the liquor-incubation medium. Subsequently, 30 mL of the rumen liquor-incubation medium was dispensed into each pre-warmed (39°C) piston syringe using an automatic pipette. After eliminating gas bubbles, the plastic clip on the silicon tube was secured, and the initial piston position was recorded. The syringes were then placed in an incubation apparatus set to rotate at a rate of one rotation per minute for 24 hours. The first reading was taken after 8 hours, followed by gas elimination, and the second reading was recorded at 24 hours.

Considering both hours reading of the gas for each diet, blank, hay, and concentrate standards, the net gas production (mL per 200 mg of DM) was determined for each diet (Menke et al., 1979). Then, using net gas production, CP, and EE of each diet, digestible energy (DE), metabolizable energy (ME), and organic matter (OM) degradability were calculated according to Menke et al. (1979). Short-chain fatty acids (SCFAs) were estimated using the formula of Njidda (2010), while microbial protein (MP) synthesis was calculated as 19.3 g microbial nitrogen per kg OM degradability, following Khattab et al. (2016).

After 24 hours reading, the silicon tube was inserted into the nozzle of a syringe containing 2 mL of 10 M NaOH solution. After securing the setup, the tube’s clip was loosened, allowing gas to flow from the piston syringe into the NaOH solution. The displacement of NaOH in syringe indicated CH₄ volume, as CO₂ dissolves in the solution (Fievez et al., 2005). CH₄ generation per mL of NaOH was calculated based on the total gas volume injected and the corresponding NaOH displacement. The CH₄ percentage in the total gas was determined from the CH₄-to-CO₂ ratio, while CH₄ production per mg of NDF per hour was calculated using the NDF content of the diets. Immediately after gas collection, rumen fluid pH was measured using a pH meter (Hanna, China).

All samples were analyzed in triplicate. Differences among the control diet and PDs were assessed using one-way ANOVA in SPSS 22. Tukey’s post-hoc test was applied to compare means among diets, with significance declared at P≤0.05 and tendency toward significance at P≤0.10. Then, data were presented as mean ± standard deviation.

Table 2: Nutrient profiles of phytogenic supplements used in the in vitro study.

% DM: Percentage dry matter; #Reference value: Rahman et al., 2021; Rahman et al., 2024b; TPC: Total phenolic contents; GAE: Gallic acid equivalent; TFC: Total flavonoid contents; QE: Quercetin equivalent.

Lemongrass showed higher DM (25.91%) content, followed by ivy gourd (14.21%) and sage (13.76%; Table 2) on fresh basis. On a DM basis, OM was higher in lemongrass, followed by sage and ivy gourd. CP content was 14.25% in ivy gourd and 6.78% in sage, with lemongrass showed the lowest (5.04%) value. Lemongrass also had the highest CF (25.11%), followed by ivy gourd (20.08%) and sage (18.79%). Then, EE (6.60%) and ash (14.48%) contents were highest in ivy gourd among three PSs. Lemongrass had higher level of nitrogen free extract (NFE), NDF, and ADF, followed by sage and ivy gourd, while NSC was highest in ivy gourd (28.74%) and lowest in lemongrass (13.07%).

The cellulose content was highest in lemongrass (30.93%), followed closely by ivy gourd (22.34%), while sage had the lowest cellulose content (17.50%; Figure 1). Besides, the hemicellulose content was highest in lemongrass compared to ivy gourd and sage. The lignin content was highest in lemongrass (8.54%), followed by ivy gourd (3.88%) and sage (3.04%).

pH of rumen fluid exhibited no significant variation across all phytogenic diets (PDs; Table 3). The PDs (sage, ivy gourd, or lemongrass) showed a tendency for higher OM degradability, DE, ME, SCFAs, and MP synthesis compared to the control diet (P=0.063). However, there was no significant differences among the PDs for these parameters (P>0.05). Compared to the control diet, the PDs demonstrated a 7–8% increase in OM degradability, a 7% enhancement in MP synthesis, an 8% improvement in DE, a 10–11% increase in ME, and a 14–17% higher production of SCFAs.

Compared to the control diet, the PDs namely sage, ivy gourd, or lemongrass showed a propensity of 17–18% higher net gas production in 24 hours or each hour (P=0.06), while there was no variation among all PDs (P>0.05; Table 4). The sage-based PD significantly reduced CH₄ production over 24 hours (Table 4) and on an hourly basis (Figure 2) compared to the control and ivy gourd diet (P<0.05), and no significant difference was observed between the lemongrass and sage diets (P>0.05). Additionally, CH₄ as a percentage of net gas production was 16–30% lower in PDs than in the control, with the lowest value recorded for the sage diet (P<0.01). Similarly, CH₄ production per mg of NDF was decreased by 16–30% in all PDs compared to the control (P<0.01), with sage exhibiting the greatest reduction (Figure 3).

Table 3: Effects of phytogenic supplements on in vitro rumen fermentation kinetics.

DE: Digestible energy; MJ/kg DM: Mega joule per kilogram dry matter; ME: Metabolizable energy; OMD: Organic matter digestibility; %: Percentage; SCFAs: Short-chain fatty acids; mmoL: Milli moles; MP: Microbial protein; g/kg DM: Gram per kilogram dry matter.

Table 4: Effects of phytogenic supplements on in vitro gas production.

Sage shrub was reported to contain 82.1% OM, 5.6% CP, 9.4% EE, 36.5% NDF, 17.7% ADF, and 30.6% NSC in Egypt (Kholif et al., 2024). However, the present study displayed different findings, with higher OM and CP contents but lower EE and ash values compared to previous finding. These variations are likely due to differences in geographical location, climatic conditions, and cultivation practices between Bangladesh and Ethiopia. Bangladesh is characterized by fertile alluvial soils and a humid monsoon climate, whereas Ethiopia features diverse volcanic soils and a climate ranging from arid to temperate. These environmental factors significantly contribute to nutritional differences in shrub (Yalew et al., 2020). On the contrary, the chemical composition of lemongrass in the present study nearly aligns with previous finding (Rahman et al., 2022), particularly in terms of DM, OM, CF, EE, NFE, ADF, and NSC. This similarity can be attributed to the fact that lemongrass in both studies was cultivated under identical regional conditions, on the same land, and following the same cultivation practices (Yalew et al., 2020). However, higher NDF and ADL levels were observed in the current study, which could be attributed to delayed harvesting, leading to increased lignification. Furthermore, the nutritional profile of ivy gourd analyzed in this study exhibited notable differences from the findings of Rahman et al. (2021), who assessed only the proximate composition of the leaves. The variations in DM, CF, EE, and NFE contents-except for CP and ash-may be due to differences in fibre fractions, particularly cellulose, hemicellulose, and lignin, which are present in variable concentrations across different plant parts. In addition, data on the cellulose, hemicellulose, and lignin content of sage and ivy gourd specific to this subcontinent were not available. However, their composition may vary depending on land type, cultivation practices, and harvesting processes. Besides, the total phenolic and flavonoid contents in these PSs are relatively high, as reported by Rahman et al. (2021, 2024b).

These primary nutrients and secondary metabolites especially phenolic and flavonoids, significantly influence rumen fermentation kinetics, nutrient utilization, gas production, and particularly CH₄ emissions in both in vivo and in vitro studies (Martínez-Fernández et al., 2013). However, these effects are dose-dependent like more than 1% sage supplements reduce the nutrient degradability (Benchaar et al., 2008). Consistent with the present findings, supplementation of 1% sage (Kholif et al., 2024), or 1% lemongrass combined with mangosteen peel (1%; Prachumchai et al., 2024), or 2% individually (Totakul et al., 2024), improved gas production, nutrient degradability, and rumen fermentation while reducing CH₄ emissions. These current effects may be attributed to the enhancement of beneficial rumen microbes by secondary metabolites, contributing to improved OM degradability, energy yield (DE and ME), MP synthesis, SCFAs, and overall fermentation efficiency (Wanapat et al., 2008; Cieslak et al., 2013). These PSs enhance the activity and population of cellulolytic and amylolytic bacteria (Wanapat et al., 2008; Prachumchai et al., 2024), thereby improving feed degradability which may lead to increased OM degradability, energy yield (DE and ME), SCFAs, and net gas production. In addition, these PSs contain phenolic, flavonoids, tannin, saponin, and terpenoids which are responsible for inhibiting hyper-ammonia-producing bacteria, thus conserving nitrogen for microbial growth and improve the MP synthesis in the current study (Patra and Yu, 2015; Bodas et al., 2012; Oskoueian et al., 2013).

Additionally, the secondary metabolites present in sage (1,8-cineole, thujone, camphor, and other terpenoids) or lemongrass (citral, limonene, and myrcene) were proven to reduce protozoal populations and shift rumen fermentation patterns, leading to increased propionate production (Kholif et al., 2024; Wanapat et al., 2008). This greater availability of propionate helps to utilize hydrogen, which would otherwise be used to produce CH₄, thereby contributing to a reduction in CH₄ production by 18% with sage and 5% with lemongrass in the current study. Previous studies indicated that supplementation with 1% sage (Kholif et al., 2024) and 0.50–0.75 mg/L sage essential oil (Kahvand and Malecky, 2018) has been shown to reduce CH₄ production by approximately 10.34% and 9–13%, respectively. Likewise, the inclusion of 1–2% lemongrass individually or 1% lemongrass with mangosteen peel resulted in a 2–5% and 21.0% reduction in CH₄ production under in vitro conditions (Totakul et al., 2024; Prachumchai et al., 2024) which aligns with the current study. Although the present study observed a relatively modest (5%) CH₄ reduction, this outcome is significant given the practical advantages of lemongrass, such as its lower cost, higher biomass yield, and greater DM content compared to sage and ivy gourd (Rahman et al., 2024b). Additionally, low-dose inclusion of lemongrass has been reported to improve dairy cow productivity, immunity, and milk desirable fatty acids, and zinc content (Rahman et al., 2024b, 2024c). In contrast to the findings of the present study, Glorio Patrucco et al. (2025) reported that lemongrass supplementation at 0.07% did not significantly reduce in vitro CH₄ production, which may be attributed to the relatively low doses of lemongrass essential oil used (Benchaar et al., 2008). Supplementation with either sage or lemongrass at a 1% inclusion level has been shown to reduce the abundance of methanogens (Kholif et al., 2024; Prachumchai et al., 2024). This effect is primarily attributed to key secondary metabolites: citral in lemongrass (Fidriyanto et al., 2021), and thujone and 1,8-cineole in sage (Patra and Yu, 2015), which are known to disrupt the integrity of methanogenic cell membranes, thereby contributing to decreased ruminal CH₄ production in the existing study. Moreover, additional in vivo studies are necessary to validate their applicability and confirm their effectiveness under practical feeding conditions. Besides, in the current study, ivy gourd supplementation significantly improved percentage of CH₄ of net gas production but directly failed to mitigate ruminal CH₄ production. This outcome may be attributed to suboptimal concentrations (on DM basis) of anti-methanogenic secondary metabolites, such as tannins (1.2–3.5%) and saponins (0.8–2.2%), present in ivy gourd (Benchaar et al., 2008). Notably, previous study (Goel and Makkar, 2012) has reported that dietary inclusion of tannins at 2.5% and saponins at 0.75% can exert significant anti-methanogenic effects in ruminants.

Lemongrass, sage, and ivy gourd showed distinct nutrient profiles, with lemongrass rich in dry matter and organic matter (OM), sage high in nitrogen-free extract and low in fibre, and ivy gourd highest in ash and ether extract. In an in vitro study, supplementation of phytogenic diets (PDs) with sage, ivy gourd, or lemongrass at 1% inclusion level exhibited a suggestive trend (P=0.063) toward enhancing OM degradability, microbial protein synthesis, feed energy values, short-chain fatty acids, and net gas production compared to the control diet. Among these PDs, sage demonstrated a notable reduction in CH₄ production, whereas lemongrass achieved a modest reduction, which hold practical significance when considering cost-effectiveness and better performance in cattle. The percentage of CH₄ relative to net gas volume was significantly reduced across all PDs, with the sage diet exhibiting the most substantial mitigation effect.

The authors gratefully acknowledge the financial support provided by the Ministry of Science and Technology (MoST), Government of the People’s Republic of Bangladesh, under the Research and Development (R and D) program (Project ID: 2023/07/MoST (R and D)). The authors also extend their sincere appreciation to the Department of Animal Nutrition, Bangladesh Agricultural University, and Bangladesh Agricultural University Research System for their technical support and valuable contributions throughout the research.

This study provides a comprehensive analysis of the fibre composition including cellulose, hemicellulose, and lignin content of sage, ivy gourd, and lemongrass cultivated in Bangladesh. Notably, it is the first to investigate the methane mitigation potential of ivy gourd as a phytogenic supplement using an in vitro rumen fermentation system. In contrast, the inclusion of either sage or lemongrass at the same inclusion level with the basal diet resulted in significant improvements in rumen fermentation kinetics, along with notable reductions in methane production.

Md. Aliar Rahman: Conceptualization, Methodology, Data Collection and Analysis, Writing Original Draft and Editing.

Rakhi Chowdhury: Supervision, Editing and Review.

The authors have declared no conflict of interest.