Additive effects of Momordica charantia powder in ruminant diets on in vitro gas production, fermentation parameters and methane emission

Risikat Mojisola Akinbode¹, Abibat Folashade Rahman¹, Ronke Yemisi Aderinboye¹, Azeez Olanrewaju Yusuf² and Kafayat Omowumi Adebayo¹

¹ Department of Animal Nutrition, Federal University of Agriculture, Abeokuta (FUNAAB), P.M.B. 2240, Abeokuta, Nigeria
akinboderm@funaab.edu.ng
² Department of Animal Production and Health, FUNAAB. Abeokuta, Nigeria

Abstract

This study evaluated the additive effects of Momordica charantia powder (MCP) on in vitro gas production and fermentation parameters of ruminant diets. A basal diet of Megathyrsus maximus and concentrate (ratio 6:4) containing six levels (0, 10, 20, 30, 40 and 50 mg/g DM) of Momordica charantia powder was incubated for 48 hours. Data on total gas, methane production, degradability, and fermentation parameters were analyzed using one-way ANOVA. Results showed a significant (p<0.05) reduction in total gas production with 30–50 mg/g MCP inclusion. Methane volume and proportion decreased significantly (p<0.05) at 20 mg/g MCP and above. Inclusion of 30 mg/g MCP and above reduced the in vitro metabolizable energy, organic matter digestibility and short chain fatty acid concentration. However, MCP inclusion had no effect on the pH, ammonia nitrogen, or total volatile fatty acids concentration. In addition, 40 and 50 mg/g MCP in the diet reduced bacterial populations, while 20 to 50 mg/g MCP decreased the fungal and protozoal populations. In conclusion, inclusion of 20 mg/g MCP in ruminant diets can effectively reduce methane production without adverse effects on fermentation parameters or the organic matter digestibility of the diets.

Key words: bioactive compounds, microbial population, Momordica charantia, Phytogenic additive, nutrient degradability

Introduction

Ruminant production is under immense pressure due to emission of greenhouse gas which is a major contributing factor to decrease in animal performance and environmental damage (Beauchemin et al 2020). About 30 % of all anthropogenic methane (5.9 × 10⁹ metric tons CO₂ equivalent) is released from enteric methane emission, primarily from feed fermentation in the rumen (CCAC 2019). These emissions lead to disruption of nitrogen (N) and phosphorus cycles, indirect impacts on the ecosystem, loss of energy and organic matter which undermine the efficiency and productivity of ruminants (Benchaar and Greathead 2011). Numerous scientific researches have been conducted to lessen enteric methane production and improve performance in ruminants through the use of certain antibiotic growth promoters (Makkar et al 2007). However, health concerns about the antimicrobial resistance and residual effects associated with the use of these antibiotics has necessitated the recent increase in the search for natural and safe alternative to antibiotics such as phytobiotic additives.

Phytobiotics are wide range of natural substances obtained from herbs, spices and other plants. Many medicinal plants and their extracts are rich source of phytobiotics such as tannins, saponin, flavonoids and alkaloids. These phytochemicals have been reported to manipulate rumen microbial populations, suppress methanogenic archaea or protozoa, shift fermentation towards propionate production and enhance nutrient digestibility (Li et al 2024). A previous study showed a strong potential of a medicinal plant ( Cassia fistula) to reduce methane (Akinbode et al 2023). However, the effectiveness of plant bioactive compounds is often plant-specific, dose-dependent and influenced by diet composition (Honan et al 2021). Momordica charantiais one of such medicinal plants that contains several bioactive compounds such as polysaccharides, terpenoids, saponins, polypeptides, flavonoids, alkaloids and sterols (Jia et al 2017). It grows well in many tropical and subtropical regions where it had been used in many traditional and folk medicine (Polito et al 2016) for the treatment of variety of ailments. Despite the abundant bioactive compounds in Momordica charantia, the reported effects of the activity of this plant in manipulating rumen fermentation is limited. Hence, this study aims to evaluate the additive effect of Momordica charantia powder at varying levels in the diet of ruminants on in vitro gas production and fermentation parameters.

Materials and methods

Location of the study and experimental diets

The experiment was conducted at the Laboratory of Animal Nutrition Department, Federal University of Agriculture, Abeokuta (7°13’28” N; 3°25’2” E) Ogun State, Nigeria.

Momordica charantia (whole plant) were harvested within the University premises. The plant was cleaned and air-dried till it becomes brittle. The air-dried samples were milled into powdered form using an electric stainless-steel grinder. The powdered material was stored in an airtight container for subsequent use. Momordica charantia powder was included in the experimental diet (Megathyrsus maximus and concentrate diet in ratio 6:4) at varying levels of 0, 10, 20, 30, 40 and 50 mg/g as shown in Table 1.

In vitro gas production study

The in vitro gas production procedure of Menke and Steingass (1988) was followed. Rumen fluid was collected from a cow immediately post-slaughter at a reputable abattoir in Abeokuta, Ogun State, Nigeria. The rumen fluid collected was strained through four-layered cheeses cloth under a continuous flushing of carbon dioxide (CO₂) in the laboratory. The rumen fluid and buffer solution mixed in ratio 1:2 served as the inoculum for the incubation process.

Two hundred milligram (200 mg) each of diet containing varying levels of 0, 10, 20, 30, 40 and 50 mg/g Momordica charantia were weighed into 100 ml capacity glass syringes with fitted silicon tube along with 30 mL inoculum. Each treatment was replicated ten (10) times in a Completely Randomized Design. Samples were incubated at 39 °C for a period of 48 hours and volume of gas produced in each syringe was read at three hours interval. Three blanks were included in the run to correct for gas production not arising from feed degradation.

Determination of methane gas from substrate incubated

Volume of methane gas produced from the fermentation of substrates incubated was determined by dispensing 4 ml of 10N Sodium hydroxide (NaOH) into three syringes per treatment at the end of 48 hours incubation period. The NaOH introduced absorbed the Carbon-dioxide produced during the fermentation while the remaining volume of gas was recorded as methane following the procedure of Fievez et al (2005).

In vitro dry matter digestibility

The fermentation residue was oven dried at 105 °C overnight and the dry matter digestibility (DMD) was calculated thus:

In vitro organic matter digestibility, Metabolizable energy and short chain fatty acid of substrate

The in vitro organic matter digestibility, metabolizable energy and shortchain fatty acids of the diets were estimated using gas produced at 24 hours incubation period as follows:

ME (MJ/kg) = 2.20 + 0.136GV + 0.057CP + 0.0029(CF)²(Menke and Steingass 1988)

OMD (%) = 14.88 + 0.889GV + 0.45CP + 0.651A (Menke and Steingass 1988)

SCFA (mmol/g DM) = 0.0239GV – 0.0601 (Getachew et al 2002)

where GV = gas volume at 24 h of incubation period; CP = crude protein of the diet (g/kg DM), CF = crude fibre (g/kg DM) and A = Ash content of the diet (g/kg DM).

Chemical analysis

The dry matter content of Momordica charantia,andthe experimental diet ( Megathyrsus maximus and concentrate in ratio 60:40) were determined by drying samples to a constant weight at 65 °C for 48-hour according to AOAC (2005), method 934.01. Crude protein concentration was determined following the Kjeldahl procedure (AOAC 2005), method 984.13. Ether extract content was determined with the use of Soxhlet extraction (AOAC 2005), method 920.39. Neutral and acid detergent fibre were determined according to Van Soest et al (1991). The phytochemical compositions of Momordica charantia was also analyzed; tannins and saponin contents were determined according to the method of de Lima et al (2011); Obadoni and Ochuko (2001) respectively. Flavonoids and total phenol contents were quantified following standard methods (Nasseri et al 2019; Popoviciu et al 2020, respectively) while alkaloids and glycoside contents were determined according to the procedure of Harborne (1973); Onwuka (2005) respectively.

Rumen fermentation parameters determination

Immediately after the 48-hour incubation period, the pH of the incubation fluid was measured using a portable pH meter (PH- 200 model). Ammonia-nitrogen (NH₃-N) concentration of fluid was determined using the steam distillation procedure of a Kjeldahl system (Ogubai and Sereke, 1997); where a 10 ml of NaOH and incubation fluid each was distilled and the ammonia collected through the distillation process was titrated against 0.1 N HCl. Then, the ammonia-nitrogen concentration in the fluid was calculated. Total volatile fatty acids concentration was determined with the use of Markham apparatus as described by Barnett and Reid (1956).

The microbial population of the incubation fluid was also determined using a serial dilution technique according to Adams and Moss (2007). The diluted sample (1 ml) was inoculated on Nutrient Agar and incubated at 37⁰C for 24 hours after which enumeration of bacterial colony was carried out as described by Prescott et al (2005). Fungi counts were done by inoculating 1 ml of diluted sample on Potato Dextrose Agar, incubated at 37⁰C for 5 days and Fungi colonies were enumerated. While the protozoan count was determined using Neubauer hemocytometer (Gürelli and Ito, 2014)

Statistical analysis

Data obtained from this study were subjected to statistical analysis using General Linear Model (GLM) procedures of SAS (2014) in a completely randomized design. Where significant differences were observed, treatment means were separated using Duncan Multiple Range Test. Significant differences were tested at 5 % probability level.

The experimental model used was defined as:

 Y_ij= µ+T_i+ Ɛ_ij ,Y

Where,Y_ij is the observation, μ is the population mean, T_i is the effect of treatments, Ɛ_ij is the residual error.

Results and discussion

Chemical composition (%) of basal diet used for the study

The chemical composition of basal diet used in this study is presented in Table 2. The diet contained 62.0% dry matter, 14.2% crude protein, 10.5% ash, 2.7% ether extract, 59.8% NDF and 30.8% ADF. This composition indicates a feed that can support maintenance and growth in goats. However, the relatively high NDF may limit digestibility, lower energy density, shift volatile fatty acids production patterns towards higher acetate production and hence, increase methane production (Li et al 2025). This form the main reason phytogenic additive is being employed in ruminant feeding to reduce methane production.

Phytochemical composition of Momordica charantia

Table 3 presents the phytochemical composition of Momordica charantia. The plant contained tannins (179 mg/100g), total phenol (153 mg/100g), flavonoids (1567 mg/100g), glycoside (0.99 mg/100g), alkaloids (6.95 %) and saponins (15.5 %). This corroborates the findings of Gayathry and John (2022). The secondary metabolites are responsible for the variety of activities such as anti-inflammatory, antioxidants, anthelmintic, as well as antimicrobial activity against pathogenic organisms while promoting the proliferation and growth of beneficial organisms in the gut (Sharma et al 2022). Ability of many secondary metabolites to modify rumen fermentation and suppress methanogenesis have been reported (Singh et al 2020; Ahmed at al 2024).

In vitro gas production and post-incubation parameters of substrate

The additive effects of Momordica charantia powder (MCP) on in vitro gas volume and post incubation parameters is presented in Table 4. The volume of gas produced at 24 and 48 h of incubation periods decreased significantly (p<0.05) at 30 to 50 mg/g MCP. Similar result was observed by Kang et al (2016), when high level of Momordica charantia saponin (0.60 mg/mL) was included in a diet. Notably, highly purified Momordica charantia saponin was used by these authors which may be the reason for the similar effect obtained at 30 mg/g MCP since whole plant of Momordica charantia was used in this study. Decreased gas production had been reported in diet containing Cassia fistula leaf powder (Akinbode et al 2023) and cinnamon powder (Aderinboye et al2020); which are also rich source of bioactive compounds. However, Faniyi et al (2021) and Antonius et al (2023) reported increased gas production when diets were supplemented by some herbal plants. Differences in the concentration and structure of the metabolites in the herbal plants and their levels of inclusion in the diet might be responsible for the variation in the observation made in various studies compared.

As observed in this study, inclusion of varying levels of MCP in the diets reduced methane production. Plant metabolites have been demonstrated to modulate rumen fermentation (Singh et al 2020). For instance, saponins are amphipathic glycosides which can have effects on microbial activity to impact fermentation depending on the dose. Tannins can also interact with rumen microbes and their enzymes, impacting digestion of protein and fibre, and methane production. However, ensuring the appropriate dosage of bioactive compounds in animal diets is essential to prevent adverse effects on digestibility and productivity. As noticed in this study, inclusion of 20 mg/g MCP in the diet caused a significant reduction in methane production without affecting the dry matter and organic matter digestibility, metabolizable energy and short chain fatty acids of the diet while higher levels reduced methane but adversely affected the digestibility and the feed value (Table 4). This implies that adding 20 mg/g MCP in the diet of ruminants may improve performance without negative impact on the environment due to methane emission.

In vitro fermentation parameters of diets containing Momordica charantia powder

The pH, total volatile fatty acids and ammonia-nitrogen concentration of the incubation fluid was unaffected (p>0.05) with the inclusion of MCP in the diet while the microbial population was altered (p<0.05) (Table 5). This aligns with the previous findings that some plant bioactive compounds may modulate rumen microbial population without significantly altering the core fermentation parameters (Patra and Saxena, 2010). The lack of effect on fermentation parameters suggest that fermentation activity was not impaired by the inclusion of MCP in the diets and that the basal fermentative capacity of the rumen microbiota was maintained. In addition, bacteria population was unaffected (p> 0.05) with inclusion of up to 30 mg/g MCP in the diets. This suggests that addition of 30 mg/g MCP in the diet has no detrimental effect on cellulolysis and cellulolytic bacterial growth. The reduction in bacterial population at higher inclusion levels of MCP (40 and 50 mg/g) and suppression of fungi and protozoa at 20 mg/g and above indicates that MCP contains potent antimicrobial compounds which made bacteria strived well at lower inclusion levels. Momordicin, charantin, saponin and flavonoids which are present in Momordica charantia have been investigated to exhibit antimicrobial properties (Grover and Yadav, 2004). Well-documented is the fact that protozoa and fungi in the rumen are sensitive to plant secondary metabolites, especially saponins which have defaunation effects (Holtshausen et al 2009). The suppression of protozoa has been linked with methane reduction which was also evident in this study as inclusion of 20 mg/g MCP resulted to approximately 31% methane reduction. These findings suggest that inclusion of Momordica charantia at appropriate levels can selectively suppress some microbes such as protozoa which are known to contribute to methane emission and protein degradation in the rumen. The use of plant secondary metabolites to selectively reduce rumen microbial population without affecting the overall rumen fermentation have been documented (Patra and Saxena, 2010).

Conclusion

Inclusion of 20 mg/g Momordica charantia powder in ruminant diets reduced total gas protozoa and methane production without adversely affecting diet digestibility. The selective antimicrobial effects of Momordica charantia powder highlight its potential as a natural feed additive, supporting sustainable livestock production and reducing reliance on antibiotics. Thus, Momordica charantia as a phytogenic feed additive can positively influence rumen fermentation by mitigating greenhouse gas emissions while maintaining feed value.

Conflict of Interest

The authors declared no conflict of interest

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