Effects of tamanu crude oil as feed additive on rumen digestibility, fermentation, methane production through in vitro study

Dimas Hand Vidya Paradhipta¹, Yasmine Dhiya Ulhaq¹, Bayu Aji Saputro¹, Tribhuvana Mahachakri Guntoro¹, Fathin Alif Djatmiko¹, Chusnul Hanim¹ and Budi Leksono²

¹ Department of Animal Feed and Nutrition, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
dimas.hvp@ugm.ac.id
² Research Center for Applied Botany, National Research and Innovation Agency (BRIN), Bogor 16911, Indonesia

Abstract

This study aimed to evaluate the effects of tamanu crude oil (TCO) supplementation as a feed additive on ruminal fermentation, digestibility, methane production through an in vitro technique. TCO is rich of plant secondary metabolite and unsaturated fatty acids such as C18:1 and C18:2. A basal diet consisting of Pennisetum purpureum and commercial concentrate (7:3, dry matter basis) was supplemented with TCO at levels of 0%, 0.01%, 0.05%, 0.10% of sample weight dry matter basis. Rumen fluid was obtained from two animal donors and used for in vitro fermentation over 48 hours. In the results, TCO level at 0% and 0.01% were not different on ruminal dry matter and organic matter digestibility, also total volatile fatty acid (VFA). However, TCO level at 0.05% and 0.10% had lower values of these variables than TCO at 0% and 0.01% (p<0.05). Notably, methane production was lower across all supplementation treatments compared to none (p<0.05), with the most favorable balance between ruminal digestibility, fermentation and methane mitigation were observed at 0.01% of TCO application. The findings suggest that TCO at 0.01% may serve as a promising natural feed additive to reduce enteric methane emissions without impairing ruminal function through in vitro study.

Keywords: in vitro, methane, rumen, supplementation, tamanu crude oil

Introduction

Enteric methane emissions from ruminant livestock make a significant contribution to global greenhouse gas, which become a consideration in the development of livestock industry (Hristov et al 2018; Min et al 2022). Ruminants contribute around 17% of global enteric methane emissions (Benaouda et al 2020). Methane production is conducted by ruminal fermentation activity, which methanogenic archaea is the main contributor. This methane is primarily produced via microbial fermentation, which methanogenic archaea convert free hydrogen (H₂) and carbon dioxide (CO₂), resulting in methane formation (Ale Enriquez and Ahring, 2024). Enteric methane emissions indicate low efficiency of livestock in utilizing or absorbing feed nutrients, with up to 12% of the gross energy intake lost as methane (Ale Enriquez and Ahring, 2024). At the farmers level, the enteric methane emissions by livestock could be high because mainly the animal is fed using low quality of feed. High portion of roughage in the diet without considering balance ration, could increase hydrogen free in the rumen, which increase the methane emissions. Thus, the study to decrease the greenhouse gas in the ruminant’s sector is necessary to create green livestock farming.

Modification of ruminal fermentation is targeted to inhibit the growth of methanogenic archaea or reducing the availability of free hydrogen in the rumen. In recent years, dietary manipulation has emerged as a promising approach to mitigate ruminal methane production. The dietary manipulation could be conducted by developing feed additives or supplement, which could help to modify the rumen ecosystem to inhibit the growth of methanogenic archaea (Honan et al 2021). Moreover, the approach technology through additives is more applicable for industry. According to previous studies, plant-derived bioactive compounds, especially those containing lipids and plant secondary metabolites like phenolics and flavonoids, could modify the ruminal fermentation and results in decreased etheric methane emission, altering rumen microbial populations and fermentation pathways (Ku-Vera et al 2020; Min et al 2022). On the other side, lipid supplementation can inhibit methanogenesis in particular by reducing protozoal populations and redirecting hydrogen use away from methane synthesis (Honan et al 2021; Min et al 2022). Development of feed additives containing plant secondary metabolite or essential fatty acid needs further exploration, with consideration of local resources (Paradhipta et al 2023).

Tamanu (Calophyllum inophyllum) is grown easily in tropical area as one of an origin plant from Indonesia. Tamanu plant has a wide distributed from Sumatra to Papua Island, especially along coastal areas (Leksono, 2014). Tamanu crude oil (TCO), obtained from the seeds, is traditionally recognized for its antimicrobial, anti-inflammatory, antioxidant properties. It is rich of coumarin dan flavonoid (Van Thanh et al 2019; Zakaria et al 2014) and fatty acids consisting of 41% to 52% saturated fatty acid (SFA) and 18% to 22% unsaturated fatty acid (UFA) (Makiej et al 2024). Nowadays, Indonesia’s TCO sector is expanding rapidly, following supportive government initiatives. This occurred because TOC could be used for alternative biofuel. In fact, crude oil production from tamanu kernel is higher than other biofuel crops such as Jatropha curcas, Reutealis trisperma and is nearly comparable to oil palm, reaching up to 20 tons per hectare per year (Leksono et al 2014). The oil yield from tamanu kernel is also relatively high, ranging from 37% to 58%, which is greater than that of other biofuel feedstocks (Leksono et al 2014). Despite its medicinal and biofuel relevance, the potential use of tamanu crude oil (TCO) in ruminant nutrition remains largely underexplored. Given its unique phytochemical profile, TCO may exert modulatory effects on rumen fermentation, including methane mitigation.

Dose of additives directly influence the reduction of methane emission in the rumen (Honan et al 2021; Min et al 2022). Accordingly, this research was designed to assess the influence of TCO added at varying levels on in vitro ruminal fermentation and methane emission. The findings are expected to provide initial insights into the feasibility of using TCO as a natural feed additive for lowering enteric methane emissions from ruminants.

Materials and methods

Preparation

The TCO was obtained from National Research and Innovation Agency (BRIN). In addition,

Basal diet consisted of Pennisetum purpureum (Elephant grass) and commercial concentrate in a 7:3 ratio, following the practical condition in smallholder farmers. Diet was dried at 55^oC during 48 h for chemical analyses and ruminal incubation. TCO was applied to the diet with different levels consisted of 0%, 0.01%, 0.05%, 0.10% of dry matter (DM). Due to small concentration, TCO from each level was dissolved using diethyl ether at ratio 1:9. After applied, the basal diet was dried in room temperature for 6 h to evaporate the diethyl ether. In addition, TCO was sub-sampled around 20 g for fatty acid profile analyses using Gas Chromatography-Mass Spectrometry (GC-MS, Agilent 8890 and 5977B) with a column (DB-FastFAME, 30 m x 250 µm x 0.25 µm) according to the procedure of Christie (1998).

Chemical composition of basal diet

Basal diet, after dried at 55^oC during 48 hours, were reduced particle size to pas 1 mm screen using a Wiley mill. Concentration of DM was determined by drying 1 g of the sample at 105°C for 24 hours following AOAC (2005) method 934.01. Organic matter (OM) was analyzed by combusting the samples in a muffle furnace (Advantec KM-420, Japan) at 550°C for 5 hours. Determination of crude protein (CP) was conducted using the procedure of Kjeldahl ((AOAC, 2005); method 984.13) with a nitrogen analyzer (B-324, 412, 435, 719 S Titrino, BUCHI, Flawil, Switzerland). Ether extract (EE) was determined using the Soxhlet extraction method ((AOAC, 2005) method 920.39). Crude fiber (CF) was determined by sequential extraction using acid and alkali (method 978.10; (AOAC, 2005)).

Ruminal incubation

In order to ruminal in vitro digestibility, the rumen fluid was collected before morning feeding from two cannulated Bali cattle (Bos indicus). The cattle, averaging 300 kg in body weight, were fed same as basal diet. The basal diet was applied to the animals for 2 weeks before collecting rumen fluid. Rumen fluid was strained through double-layered cheesecloth and then combined with a buffer solution at a 1:4 ratio, following the procedure described by Tilley and Terry (1963), then maintaining anaerobic conditions by flushing with carbon dioxide (Paradhipta et al 2023). The in vitro incubation was carried out in 100 mL glass serum bottles containing 500 mg of dried sample and 40 mL of rumen buffer. Each dietary treatment was incubated using 3 replications, along with 2 blanks and 2 standards (Pangola grass). The ruminal incubation was conducted in two batches for all dietary treatments. The bottles were sealed with rubber stoppers and aluminum caps, incubated at 39°C for 48 hours.

After incubation, 5 mL of accumulated gas was collected using a syringe from incubation bottle and then transferred to vacuum tube for methane analysis. The methane analyses followed the procedure of previous studies (Paradhipta et al 2023; Fitriani et al 2024). The contents of incubation bottle were filtered using Gooch crucibles, the residue was used to determine in vitro dry matter digestibility (IVDMD) and organic matter digestibility (IVOMD). A pH meter (Ohaus AB23PH-F, China) was used to determine the pH from rumen buffer, while volatile fatty acids (VFAs) were measured via gas chromatography (GC 8A, Shimadzu Corp., Japan) (Filípek and Dvořák, 2009). Ammonia-N (NH₃-N) concentration was determined calorimetrically as described by previous study (Chaney and Marbach, 1962).

Ethical approval

The animal care and in vitro procedures were carried out in accordance with the ethical standards of the Ethics Committee of the LPPT, UGM (No. 00007/III/UN1/LPPT/EC/2024).

Data Analysis

A completely randomized design was employed for data analysis, using the PROC GLM procedure in SAS. The model structure was defined as Y_ij = µ + T_i + e_ij, where Y_ij corresponds to the response variable, µ is the grand mean, T_i the treatment effect of TCO, e_ij the random error term. Tukey’s test was used for post-hoc comparisons, with significant differences accepted at p≤0.05, tendencies indicated for values between 0.05 and 0.10.

Results and discussion

Chemical composition of basal diet

The chemical compositions of basal diet are presented in Table 1. In the present study, basal diet contained 85.92% OM, 10.12% CP, 2.78% EE, 31.76% CF. Commonly in the local farmers, the diet contained high CF concentration with low energy and CP concentrations. The unbalanced diet for ruminant, especially high CF concentration, potentially increase methane production (Hristov et al 2018; Min et al 2022). The basal diet in the present study represented the diet condition in the small holder farmers. The majority of cattle production in Indonesia, approximately 90%, is contributed by smallholder farming (Agus and Widi, 2018). Thus, the use of TCO was expected to help the reduction of methane by livestock industry in Indonesia.

Fatty acid profiles of TCO

Besides being rich sources of coumarin dan flavonoid (Van Thanh et al 2019; Zakaria et al 2014), the present study discovered that TCO contained high levels of oleate (C18:1) and linoleate (C18:2) fatty acids (Table 2). The information of fatty acid profile in TCO was in limited study. Thus, present study was conducted to investigate it. The concentration of C18:1 and C18:2 were 39.4% and 24.3%, respectively. In addition, the concentrations of palmitate (C16:0) and stearate (C18:0) in TCO were 16.0% and 16.7%, respectively. Generally, concentrations of SFA, monounsaturated fatty acid (MUFA), polyunsaturated fatty acid (PUFA) were 35.0%, 40.7% and 24.3%, respectively. Previous studies reported that the supplementation of oil-rich UFA potentially reduced a methane emission in the rumen through in vitro study (Kliem et al 2019; Ibrahim et al 2021). However, the effectiveness of oil-rich UFA in modifying rumen fermentation depends on the dose and its composition of fatty acid (Amanullah et al 2022; Kliem et al 2019; Rasmussen and Harrison, 2011).

Ruminal digestibility and fermentation

Application of TCO in different level affected the results of IVDMD and IVOMD (Figure 1). Application of TCO at 0% and 0.01% resulted in higher IVDMD (p=0.001; 57.4% and 59.9% vs. 51.4% vs. 38.1%) and IVOMD (p=0.001; 61.2% and 61.4% vs. 52.9% vs. 38.7%) than TCO at 0.05% and followed by TCO at 0.10%. The present study discovered that supplementation of TCO more than 0.01% could decrease digestibility. The concentration of plant secondary metabolite combined with UFA in 0.05% and 0.10% of TCO application might be in toxic level for rumen microbial. In the low dose, plant secondary metabolites and UFA can help to increase digestibility. But, in high dose, it can be a toxic and reduce digestibility (Bodas et al 2012; Kliem et al 2019; Ku-Vera et al 2020; Rasmussen and Harrison, 2011). Both IVDMD and IVOMD were reduced at the high inclusion level (more than 0.05%), indicating that higher levels of TCO may inhibit fibrolytic microbial populations in the rumen. These effects are likely due to the antimicrobial activity of bioactive compounds present in TCO, such as flavonoids and phenolics, which are known to suppress the growth of cellulolytic bacteria (Bodas et al 2012; Honan et al 2021; Ku-Vera et al 2020). Previous studies reported that UFA might also affect the growth of cellulolytic bacteria depending on the type and dose (Amanullah et al 2021, 2022; Ibrahim et al 2021). Application of TCO at 0.01% had a similar digestibility compared to none. In this case, the TCO at 0.01% could be a rumen modifier, which had a similar digestibility and also reduced methane (Figure 2) without presenting negative effects on digestibility and ruminal fermentation (Table 3).

Figure 1. Effects of tamanu crude oil supplementation on the in vitro dry matter digestibility and in vitro organic matter digestibility. a,b,cMeans with different superscripts differ significantly (p≤0.05)

The ruminal pH was not affected by supplementation of TCO (Table 3). Generally, rumen pH values remained within the optimal physiological range (6.0–7.2) across all treatments (Bodas et al 2012; Faniyi et al 2019). The rumen pH was affected by the concentrations of ammonia-N and total VFA during ruminal fermentation (Bodas et al 2012; Honan et al 2021; Amanullah et al 2022). In addition, concentrations of ammonia-N and total VFA tended to decrease, which was supported by the results of IVDMD and IVOMD. Application of TCO at 0.05% and 0.10% had a lower ammonia-N concentration than none (p=0.065; 14.8 and 14.0 mg/100 mL vs. 18.1 mg/100 mL), while TCO at 0% and 0.01% were not different (18.1 and 16.9 mg/100 mL). The present study indicated that supplementation TCO more than 0.05% could inhibit proteolytic activity in the rumen. The reduction in ammonia-N might indicate a shift in nitrogen metabolism, potentially due to a reduction in proteolytic activity or deamination by rumen microbes, which could be attributed to the antimicrobial properties of the secondary metabolites in TCO, such as calophyllolide and other phenolic compounds (Makiej et al 2024; Van Thanh et al 2019). Additionally, the supplementation of UFA could also affect the rumen fermentation, which caused the concentration of ammonia-N (Amanullah et al 2021, 2022; Ibrahim et al 2021).

Total VFA production showed a decreasing trend as TCO levels increased, with TCO at 0% and 0.01% had higher concentration of total VFA than TCO at 0.05% and 0.10% (p=0.071; 98.3 and 97.8 mMol vs. 82.0 and 80.5 mMol). This trend might reflect a partial inhibition of microbial fermentation by plant secondary metabolites or UFA present in TCO, which supported by previous studies (Bodas et al 2012; Ibrahim et al 2021). The present study also indicated that supplementation of TCO at 0.01% did not change the ruminal fermentation. And the supplementation more than 0.01% had possibility to be toxic for rumen microbes, especially cellulolytic. This could be identified by the decrease of total VFA production in tendency. The molar proportions of propionate and butyrate were not affected by TCO supplementation (Table 3). However, acetate tended to decrease, where TCO at 0% and 0.01% had higher acetate than TCO at 0.05% and 0.10% (p=0.074; 61.8 and 60.8 mMol vs. 48.1 and 48.0 mMol). A lower acetate concentration in the rumen is typically associated with reduced methanogenesis and more efficient energy utilization (Cañete, 2025; Faniyi et al 2019).

The present study discovered that higher level of TCO supplementation decrease the production of methane (Table 3 and Figure 2). Application of TCO at 0.10% had lowest methane production, followed by TCO at 0.01% and 0.05%, then TCO at 0% (p= 0.008; 69435.9 ppm vs. 78204.1 and 73058.3 ppm vs. 90306.1 ppm). The presence of UFA and plant secondary metabolites in TCO may modulate microbial activity in a way that favors reduced methanogenesis (Bodas et al 2012; Hristov et al 2018; Ibrahim et al 2021). Plant secondary metabolites shown potential as methane inhibitors by affecting rumen fermentation patterns and inhibiting protozoa and archaea(Bodas et al 2012; Ku-Vera et al 2020). Moreover, previous studies reported that supplementation of oil rich fatty acids of C18:2 and C18:3 could decrease the population of methanogenic archaea through in vitro study (Amanullah et al 2021, 2022). In the present study, TCO was rich of C18:2, which could help to decrease a methane production. Moreover, in some case, C18:2 fatty acid could increase the population of Fibrobacter succinogenes and Ruminococcus flavefaciens (Amanullah et al 2021). The present study reported that TCO supplementation at 0.01% was the recommended level because it could decrease methane production without had any negative effects on digestibility. This is a promising indication that TCO could be used as a natural feed additive to reduce methane emissions while maintaining rumen function.

Figure 2. Effects of tamanu crude oil supplementation on ruminal methane production

Conclusion

The present study concluded that TCO could be used as feed additive to reduce methane emission. Supplementation of TCO at 0.01% was effective to reduce methane production without presenting any negative effects on ruminal digestibility and fermentation.

Acknowledgment

The present study was supported by the RIIM LPDP Grant and BRIN, grant No 82/11.7/HK/2022.

Conflict of Interest

The authors declare no conflicts of interest.

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