Ruminal methane mitigation potential of selected medicinal plants using in vitro fermentation assays

Muhammad Lawal^1,2,4, Mohd Azrul Lokman¹, Sadiku Musa Otaru², Muhd Danish Daniel Abdullah³, Fauziah Tufail Ahmad¹ and Mohd Effendy Abd Wahid¹

¹ Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, Kuala Nerus, Terengganu, Malaysia
effendy@umt.edu.my
² Small Ruminant Research Programme, National Animal Production Research Institute, Ahmadu Bello University, Shika, Nigeria
³ Institute of Climate Adaptation and Marine Biotechnology, Universiti Malaysia Terengganu, Kuala Nerus, Terengganu, Malaysia
⁴ Agricultural Education Department, Federal College of Education Katsina, Katsina, Nigeria

Abstract

Reduction of methane emissions from ruminants is a global priority due to its effect on the environment. In the current study, two experiments were conducted to assess in vitro anti-methanogenic potential of four selected medicinal plants. In the first experiment, ground leaves of Guiera senegalenesis, Leptadenia hastata, Combretum micranthum and Diospyros mespiliformis were extracted through ultra-sonication using water, absolute ethanol and 70% ethanol to produce twelve extracts. The extracts were incorporated at a 5 mg/g inclusion level in an in vitro gas production assay to evaluate their effects on rumen fermentation. In the second experiment, whole leaf powder of the plants were supplemented at 300 mg/g to determine their effect on rumen fermentation. Methane production and rumen fermentation parameters were measured in both experiments. Results of gas production parameters were analysed using two-way ANOVA. Most plant extracts significantly reduced methane production with ethanol extract of C. micranthum, water extract of D. mespiliformis and 70% ethanol extract of G. senegalensis having the highest reductions. Digestibility markedly varied among the extracts. In the second experiment, leaf powders of C. micranthum and G. senegalensis reduced methane production without reducing total volatile fatty acids production.

Keywords: Combretum micranthum, Diospyros mespiliformis, extract, Guiera senegalenesis, Leptadenia hastata, solvent

Introduction

Ruminant production, while offering numerous benefits to farmers, communities and countries at large, have been implicated to cause climate change through emission of methane which is a potent greenhouse gas. Methane is a short lived greenhouse gas and is reported to have a global warming potential twenty five folds that of carbon dioxide (Forster et al 2021). A large number of ruminant population in different parts of the world is managed under extensive grazing systems and there is need to develop enteric methane mitigation strategies appropriate for their conditions. It has been suggested that the utilization of plants containing plant secondary compounds could be an appropriate option to reduce methane emissions from ruminants in such systems due to their widespread abundance especially in the tropical part of the world. Plant secondary compounds are phytochemicals like tannins, flavonoids and saponins produced by plants for protective purposes. These compounds have been reported to reduce methane emissions from ruminants by about 16% (Arndt et al 2022).

Guiera senegalensis, Leptadenia hastata, Combretum micranthum and Diospyros mespiliformis are widely abundant medicinal plants found in the semi-arid region of Nigeria which ruminants commonly browse on. These plants have been reported to contain phytochemicals with anti-methanogenic properties. Hence, they have been selected to assess their methane reduction ability. Medicinal plants have been used in various forms to investigate their anti-methanogenic efficacy, for example whole leaves, leaf extracts, bark extracts, marc/pomace (Lambo et al 2024). The use of crude extracts especially from the leaves has been a very common practice because it provides an opportunity to evaluate the efficacy of the phytochemicals without the confounding effects of some factors associated with whole leaves such as digestibility, bioavailability and release of the phytochemicals which can affect results of the experiments. A number of studies (Jo et al 2022; Ibrahim et al 2022; Bharanidharan et al 2021) used crude extracts obtained from leaves of certain medicinal plants using organic solvents and assessed their anti-methanogenic potential. The efficacy of an extract can be affected by solvent polarity due to differences in solubility of phytochemicals. Solvents of different polarities are able to extract different compounds and can therefore have divergent anti-methangonic effect. However, the use of plant extracts especially those obtained using organic solvents for reducing enteric methane by farmers practicing extensive system of management is impracticable due to high cost of organic solvents. Additionally, the process of extraction to obtain crude extracts for use at large scale by farmers is also difficult. Nevertheless, in the current study, both forms of the medicinal plants, that is, crude extracts and leaf powder will be used to evaluate their effect on methane production. Moreover, since water is a cheap and more readily available solvent, it will be used alongside ethanol to assess its effect on methane reduction.

Materials and methods

The study involved two in vitro gas production experiments. The first experiment involved determination of effects of crude extracts of G. senegalensis, L. hastata, C. micranthum and D. mespiliformis on ruminal methane production. A subsequent experiment was carried out to validate certain observations previously made in Experiment 1. The second experiment investigated the effect of supplementation of whole leaf powder of the plants on methane production and rumen fermentation properties.

Experiment 1

In vitro gas incubation

The procedure described by Fievez et al (2005) was used for in vitro gas production incubation. Incubations were conducted in 50 ml plastic syringes (Terumo, Japan) which were graduated to 1 ml. Timothy hay (chemical composition shown in Table 1) was used as incubation substrate. Dried leaves of the four medicinal plants were extracted with 100% ethanol, 70% ethanol and water via ultra-sonication to produce twelve crude extracts. The twelve crude extracts obtained from the medicinal plants were used as treatments. There were two controls: a negative control (CON) which was untreated and a positive control chloroform (CHL). Chloroform is a strong methane inhibitor and even though it is banned due to its carcinogenic effect, it is still used as a laboratory model in in vitro experiments. Approximately 100 mg of hay was mixed with 50 mg/g of the respective plant extracts and inoculated with 10 ml of media prepared from rumen fluid and McDougal buffer. Chloroform was included at 3 µl per incubation bottle. Rumen fluid was collected from at least three different slaughtered cows at the abattoir and mixed with McDougal buffer at 1:2 ratio. Syringes were then placed at inverted position and incubated in a water bath set at 39°C for 48 hours with the syringes being shaken at least twice a day. At the end of the 48 hours incubation, the total amount of gas produced was observed using the graduation on the syringe and recorded. The amount of methane present in the gas produced was measured using carbon dioxide absorption method of Fievez et al (2005). Four milliliters of 10 molar sodium hydroxide was added into the syringes which led to absorption of carbon dioxide and the remaining volume of gas left was considered to be the amount of methane produced. Short chain fatty acids were predicted using the formula below:

In vitro dry matter digestibility

The two step digestion method of Tilley and Terry (1963) was used in the current study. In vitro digestion was conducted in 120 ml serum bottles. Approximately 200 mg of Timothy hay was weighed into Taffeta fiber bags (Despal et al 2022), placed into the bottles and treated with 50 mg/g of plant extracts. Inoculation media was prepared as previously mentioned and 20 ml of the media was dispensed into the serum bottles while flushing with carbon dioxide. The bottles were sealed with butyl stoppers and crimped with aluminum caps using a manual hand crimping tool. After sealing, the bottles were placed into an incubator (Memmert, GMBH) set at 39°C. Incubation was carried out for 48 hours with bottles being shaken at least twice a day. The bottles were also vented at 24 hours and 48 hours. At the end of the incubation, the bottles were placed on ice to stop fermentation, uncapped and 4 ml of samples were collected and stored for ammonia nitrogen determination. Pepsin-hydrochloric acid solution was prepared according to Holden (1999) and 1 ml was added to each bottle after which the bottles were placed back into the incubator for another round of 48 hours incubation. At the end of the incubation, the fiber bags were removed from the bottles and rinsed until the rinsing water became clear. The bags were compressed to squeeze out excess water and then placed into an oven set at 60°C to dry. After drying, the bags were removed from the oven and placed into a desiccator and allowed to cool. The bags were then weighed and their weights were recorded. The difference between the final weight of the bags after drying and the initial weight of the bags before digestion was calculated and expressed as percentages of the initial weight.

Ammonia nitrogen was determined using the phenol-hypochlorite method (Souza et al 2013). Samples for ammonia nitrogen determination were stabilized with 50% sulfuric acid (Broderick & Kang 1980) and stored at -20°C. To initiate the reaction, 10 µl of thawed sample was added to falcon tubes followed by addition of 1.5 ml phenol solution and 1.5 ml sodium hypochlorite solution. The mixture was incubated at 39°C for 15 minutes and the absorbance read at 630 nm. A standard calibration curve of ammonium chloride was constructed and used to determine ammonia-nitrogen concentrations of the samples.

Validation of Experiment 1 results

Following promising observations in Experiment 1 using the absolute ethanol extract of C. micranthum, a follow up experiment was conducted to validate the effect under different conditions. Due to impracticality of producing absolute ethanol extract at a large scale or for in vivo studies, and the observation that water extract of the plant increased methane production, the second experiment focused on evaluating the effect of leaf powder of the plant that has been leached of the water soluble compounds to see whether it could also greatly reduce methane production just as the absolute ethanol extract did. The follow up experiment was conducted in a different location and incubations were carried out using rumen fluid collected from slaughtered cattle at the abattoir. Incubations and measurement of methane production was conducted as described in the first experiment. The experiment included three treatments: absolute ethanol extract of C. miranthum (CME), leaf powder of C. micranthum (CMP) and leaf powder of C. micranthum that has been leached of water soluble compounds (LCM). The treatments were included at 50 mg/g for the extract and 200 mg/g for the leaf powders. Leaching of water soluble compounds from C. micranthum leaf powder was achieved through several rounds of ultra-sonicating the powder in water with subsequent decantation of the liquid extract obtained after each round until the liquid extract produced became clear. The leaf pomace was dried to constant weight in an oven at 55°C. Two controls: negative, with no treatment (CON) and positive, treated with 30 µl chloroform (CHL) were also included just as in the first experiment. Two runs of the experiment were conducted with each treatment having six replicates.

Experiment 2

The use of whole leaves compared to extracts would be an easier and more practicable approach for peasant farmers. Hence, in the current experiment dried leaves of medicinal plants that have been ground into powder form were used as experimental treatments. From Experiment 1, L. hastata showed little anti-methanogenic potential. Hence, the other three medicinal plants (G. senegalensis, C. micranthum, D. mespiliformis) that showed higher level of methane reduction were selected for this experiment. The experiment sought to determine the effect of supplementation with leaf powder on methane reduction and fermentation properties. Incubations were conducted in 120 ml serum bottles using rumen fluid from slaughtered cattle using the method described by Theodorou et al (1994). Approximately 0.5 g of Timothy hay was directly weighed into bottles and incubated with 50 ml of inoculated media prepared as described in Experiment 1. The experiment included four treatments: a control (CON) with no supplementation and leaf powders of C. micranthum (CMI), D. mespiliformis (DME), G. senegalensis (GSE) supplemented at 300 mg/g inclusion rate. Four runs of the experiment were conducted with each treatment replicated three times. Gas production was derived from pressure readings of bottles measured using Benetech GM522 (Shenzhen, China) digital pressure manometer. Methane was measured using carbon dioxide absorption method. Thirteen milliliters of gas sample was collected from bottles into 12 ml syringe through a three-way stop cock followed by addition of 2 ml of 10 molar sodium hydroxide solution. The syringe was agitated vigorously for about a minute which led to absorption of the carbon dioxide present in the gas. The remaining gas left was considered to be the amount of methane produced. At the end of incubations, bottles were immediately chilled to stop fermentation. Two milliliters samples of rumen fluids were collected into 5 ml sample tubes followed by addition of 0.5 ml 50% sulfuric acid and stored at -20°C for total volatile fatty acids and ammonia nitrogen determination. Drawell pH200E (Chongqing, China) meter was used to measure pH of the rumen fluids. Digestibility was determined using the two-step digestion method previously described in Experiment 1. Ammonia nitrogen was determined using the sodium-hypochlorite method described in Experiment 1. Total volatile fatty acids was determined using the improved Montgomery spectrophotometric method described by Aramrueang et al (2022) with little modification. Approximately, 400 µl of thawed rumen fluid sample was pipetted into 5 ml stoppered glass tubes, followed by the addition of 400 µl concentrated ethylene glycol and 100 µl sulfuric acid (90 g/l). The mixture was heated at 100°C for 10 minutes followed by rapid cooling in chilled water. Then 500 µl of hydroxylamine HCl (18 g/l), 1 ml of sodium hydroxide (75 g/l), 500 µl of hydrochloric acid (140 g/l) and 2 ml of iron (III) chloride (3.5 g/l) were sequentially added to the mixture and mixed vigorously. The absorbance of the resulting mixture was read at 513 nm using SPM23a spectrophotometer (Nanjing, China). A standard calibration curve of acetic acid was constructed and used to extrapolate the concentrations of the samples.

Statistical analysis

Data obtained from the experiments were analysed using two-way ANOVA. The twelve extracts (in Experiment 1) and three leaf powders (in Experiment 2) were used as the treatments, while the runs in each of the gas production experiments were considered as blocks and treated as fixed factors to account for variation between runs. Significance was declared at 95% confidence interval. All data were analysed in SPSS version 22.

Results

Effect of plant extracts on methane reduction, fermentation parameters and digestibility

Table 2 below shows the effect of plant extracts on rumen fermentation. There was an effect of treatment on total gas production (p<0.001). Absolute ethanol and 70% aqueous ethanol extracts of L. hastata markedly produced more total gas compared to the control. All other extracts produced less total gas compared to the control with absolute ethanol extract of C. micranthum producing the least amount of total gas similar to chloroform.

Several plant extracts produced less methane compared to the control (p<0.001). However, absolute ethanol extract of C. micranthum which was statistically similar to the positive control chloroform, produced the least amount of methane. Although not different from the control, absolute ethanol extract of L. hastata and water extract of C. micranthum produced more methane than the control. The effect of water extract relative to ethanol extracts on methane production differed among the medicinal plants. Water extract of G. senegalensis reduced methane production (18.5%) but not as much as absolute ethanol (25.0%) and 70% ethanol (28.1%) extracts. For L. hastata, water extract had a higher methane reduction (15.5%) compared to the 70% ethanol extract (4.05%), while the absolute ethanol extract increased methane production. For C. micranthum, unlike absolute ethanol extract and 70% ethanol extract which respectively reduced methane production by (87.5%) and (12.8%), water extract of the plant not only failed to reduce methane but increased its production. Water extract of D. mespiliformis had a higher methane reduction (31.1%) compared to the both absolute ethanol (16.5%) and 70% ethanol (24.7%) extracts of the plant.

Most of the plant extracts lowered the amount of short chain fatty acids produced compared to the control (p<0.001) with the exception of absolute ethanol and aqueous ethanol (70%) extracts of L. hastata as well as aqueous ethanol (70%) extract of C. micranthum. Substrates treated with absolute ethanol extract of L. hastata had the highest dry matter digestibility while those treated with aqueous ethanol (70%) of D. mespiliformis had the lowest digestibility although both were not different from the control (p>0.05). There was tendency of treatments to affect pH of incubations (p=0.053). Among all the incubations, control incubations released the least amount of ammonia nitrogen while incubations treated with absolute ethanol extract of L. hastata or water extract of C. micranthum were the ones that released the highest ammonia nitrogen.

Results of follow up experiment is given in Table 3 below. All treatments with the exception of C. micranthum leaf powder produced less total gas compared to the control (p<0.001). Chloroform and absolute ethanol extract of C. micranthum produced less methane compared to the control (p<0.001). Water leached C. micranthum leaf powder produced less methane relative to the control although they were similar. Compared to the control, all the treatments produced less SCFAs with the exception of C. micranthum leaf powder (p<0.001).

Effect of supplementation with leaf powder on fermentation and dry matter digestibility

As shown in Table 4 below, all the fermentation variables were affected by the treatments except total volatile fatty acids production which was not affected (p=0.731) and CH₄/TGP which tended (p=0.056) to be affected. Supplementation with medicinal plants affected gas production (p=0.026), leading to reduction of total gas production to various degrees. DME produced 22.0% less total gas compared to the control. Relative to the amount of substrate incubated, all the medicinal plants (p<0.001) yielded less gas (total gas production/substrate incubated – ml/g) in the following order: DME (40.0%) > GSE (32.0%) > CMI (24.0%). The three medicinal plants reduced total methane production to various extents (p=0.012) in the following order: DME (32.0%) > GSE (10.0%) > CMI (7.00%). Methane production in relation to the amount of substrate incubated and digestion were all reduced by the medicinal plants (p<0.001). Both DME and GSE (p<0.001) reduced dry matter digestibility compared to control while CMI increased it (p<0.001). Supplementation affected pH with CMI and GSE having similar pH values which were lower than the values for the control and DME which in turn were similar. There was no difference in total volatile fatty acids produced among the treatments (p=0.731) although DME had a relatively lower production than the others. However, when related to the amount of substrate incubated, supplementation of all the medicinal plants led to lower yield of total volatile fatty acids compared to the control (p=0.007). Both CMI and DME produced less ammonia nitrogen compared to the control (p<0.001) while GSE was similar to it.

Discussion

Medicinal plants which are good storehouse of phytochemicals have anti-methanogenic properties and their use have been suggested to be an appropriate strategy for reducing methane emissions in subsistence and extensive systems of ruminant production practiced in large part of the world. In the current study, the anti-methanogenic properties of extracts and whole leaves of four selected medicinal plants were assessed.

Effect of plant extracts on gas production, digestibility and rumen fermentation

Phytochemicals like tannins, saponins and flavonoids have antimicrobial properties and can affect rumen fermentation in various ways (Patra & Saxena 2009). For example, tannins are known to reduce methane but can also reduce digestibility and affect animal production (Makkar, 2003). Flavonoids are also known to reduce methane production (Oskoueian et al 2013). Most of the plant extracts used in the current study were rich in phytochemicals with the exception of extracts of L. hastata. The various extracts used in the current study had varying concentrations of phytochemicals and varying effects on rumen fermentation depending on the plant and the type of solvent used for extraction. Ethanol extract of G. senegalensis had a higher methane reduction compared to the water extract possibly due to higher quantity of flavonoids. The 70% ethanol extract which had even higher flavonoids had the highest methane reduction in the plant showing a dose dependent manner of the flavonoid in methane reduction. However, the high methane reduction of the extract was accompanied by a reduction in dry matter digestibility. Absolute ethanol extract of C. micranthum remarkably had a very high methane reduction reducing it by 87.0%. Interestingly, this reduction was not associated with reduction in dry matter digestibility implying that methane reduction by this extract could have been through direct inhibition of methanogens or through reduction of hydrogen availability. Conversely, water extract of C. micranthum increased methane production suggesting the presence of methane promoting compounds. The 70% ethanol extract of the plant minimally reduced methane production. It is possible that the extract contained both methane reducing compounds and methane promoting compounds due to the solvent’s ability to be able to extract different types of phytochemicals from the plant leave. Water extract of D. mespiliformis which had the highest amount tannins reduced methane production by 31.0% without significantly affecting dry matter digestibility. This implies that the tannins present in this plant could have reduced hydrogen availability or directly inhibited methanogens hence the observed reduction in methane without negatively affecting dry matter digestibility. The reduction in total gas production and methane by ethanol based extracts of D. mespiliformis could be due to the high levels of flavonoids which could have inhibited and directly killed some of the rumen fibrolytic microbes hence the reduced dry matter digestibility and gas production observed with addition of these extracts. Extracts of L. hastata were relatively less effective in methane reduction possibly due to the low concentration of phytochemicals recorded in the plant. Nevertheless, water extract of the plant had a modest 15.0% reduction of methane. Although L. hastata had low phenolic content, it yielded high amount of extracts suggesting the presence of soluble non fiber carbohydrates. The increased total gas production, SCFA production and digestibility observed with the plant could have been due to presence of soluble non fiber carbohydrates. (Sinz et al 2019) assessed the effects several plant extracts on rumen fermentation and reported a similar observation where extracts of buckwheat seeds and ginkgo leaves increased total gas production and short chain fatty acids. Both plant extracts turned out to have low phenolic compounds but had good amount of non fiber carbohydrates. Contrary to expectation, all the extracts used this experiment led to an increase in the amount of ammonia nitrogen produced compared to the control. Tannins which were present in most of the plant extracts are generally known to decrease ammonia nitrogen production through forming complexes with proteins and reducing deamination (Min et al 2003). A number of studies (Mbisha 2024); (Yang et al 2009) have reported increased ammonia nitrogen release with inclusion of plant extracts. Rumen bacteria utilize ammonia nitrogen as one of their main nitrogen source (Bach et al 2005). Phytochemicals in plant extracts are known to inhibit rumen bacteria (Patra & Saxena 2009). It is possible that plant extracts used, reduced bacterial activity leading to decreased utilization of ammonia nitrogen already existing in the rumen fluid thus leading to a higher ammonia nitrogen compared to the control where normal bacterial activity could have led to using up of ammonia present in the rumen fluid.

A follow up experiment for Experiment 1 was conducted at a different location to confirm the substantial reduction of methane production by absolute ethanol extract C. micranthum. Additionally, since it will be difficult for farmers to produce ethanol extracts of the plants and because water extract of the plant increased methane production, whole leaf of C. micranthum that had been leached of water soluble compounds was also tested whether it could exert the same or similar effect in methane reduction. Although while there was reduction in methane by both the extract and whole leaves, the magnitude of methane reduction observed in the first experiment was not recorded in the present experiment. This could be due to difference(s) in diets which can affect microbial composition of rumen fluids used in the different locations. Rumen fluid is greatly influenced by the type of feed given to animals and can greatly affect the microbial composition and how they respond to dietary treatments (Neves et al 2025).

Effects of supplementation with whole leaves on rumen fermentation

The reduction in gas production relative to the quantity of substrate incubated observed with supplementation of the three medicinal plants compared to the control indicates that all the three plants inhibited fermentation to some extent. Phenolic compounds like tannins and flavonoids can directly inhibit microbial activity thereby reducing digestion and consequently gas production (Patra and Saxena 2009). Hence, the presence of these compounds in the medicinal plants could have led to the reduced fermentation observed. (Akinbode et al 2023) reported a similar observation where inclusion of Cassia fistula leaf powder reduced in vitro gas production linearly with increasing levels of supplementation. However, overall gas production was similar relative to the control with supplementation of C. micranthum and G. senegalensis, while D. mespiliformis produced lower gas. This means that even though the medicinal plants reduced fermentation, overall gas production which is indicative of total fermentation, was not impaired in C. micranthum and G. senegalensis. Apart from phytochemicals, medicinal plants also contain nutrients like fiber which can serve as substrate thereby contributing to fermentation. Supplementation of the medicinal plants was substantial up to 30.0%, therefore the presence of additional fermentation substrate in the medicinal plants helped in stabilizing/making up overall fermentation despite their inhibitory effect on fermentation. The lower gas production recorded with D. mespiliformis supplementation however indicates that the plant inhibited fermentation beyond a level which additional fermentation substrate present in it could not make up for its inhibition of fermentation.

The reduction in methane production observed with supplementation of the three medicinal plants in the current experiment indicate that they do have some anti-methanogenic properties. Medicinal plants and the secondary compounds which they contain are able to reduce methane through several ways (Patra 2010). The methane reduction ability exhibited by the medicinal plants appears to be majorly through inhibition of fermentation and to a lesser extent through direct inhibition of methanogenesis. This can be explained by the ratio of methane to total gas which was reduced by 13.0% with supplementation of DME, while CMI and GSE both reduced it by 6.00%. This shows even though methane reduction was mainly through reduced fermentation, the three plants also had some direct effect on methanogenesis.

The two step digestion represents total digestion including both microbial digestion in the rumen as well as enzymatic digestion in the abomasum. While all the three plants reduced rumen digestion, GSE and DME also reduced total digestibility by approximately 9.00% and 10.0% respectively. On the other hand, CMI appeared to enhance post ruminal digestion as observed in its increased total digestibility compared to the control. Reduction of digestion by phenolic compounds can be through inhibition of microbial activity as well as through formation of complexes with nutrients and enzymes (McSweeney et al 2001). C. micranthum leaves contain about 25.0% crude protein (Zampaligré et al 2013). It is possible that some nutrients present in the plant especially protein were bound and not digested in the rumen but were freed up and digested during the pepsin-hydrochloric acid digestion thus contributing to improved overall digestibility. Additionally, flavonoids have been reported to increase dry matter digestibility (Hassan et al 2020). C. micranthum leaves are rich in flavonoids (Tine et al 2024) and the presence of these compounds could have led to the improved digestibility observed with supplementation CMI.

The pH of the rumen fluids of the various treatments were within normal range with rumen fluids of C. micranthum and G. senegalensis having a little lower pH compared to the control. Lower pH can indicate the accumulation of volatile fatty acids and the lower pH observed in the two plants can be explained by the higher amount of volatile fatty acids observed in the two plants. Ammonia is an environmental pollutant and can indirectly contribute to production of nitrous oxide (Chanu et al 2020), hence its reduction is beneficial. Both C. micranthum and D. mespiliformis reduced ammonia nitrogen release and this could have been possible through formation of protein complex by bound tannins present in the plants.

Conclusion

This study found that extraction solvents can influence plant extracts which can lead to divergent effects on in vitro rumen fermentation. While plant extracts elicited divergent effects on rumen fermentation depending on solvent of extraction, whole leaf powder of most plants reduced in vitro methane production. Whole leaf powders of D. mespilormis reduced methane production but also reduced digestibility and VFA limiting its use for methane reduction in ruminants feasible. The utilization of leaf powders of C. micranthum and G. senegalensis should be investigated further in in vivo study to evaluate their effect of methane reduction and animal productivity.

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