Oil palm (Elaeis guineensis) is a major agricultural crop widely cultivated in tropical regions. Global production of palm oil byproducts are more than 80 million tons annually (Gulbake et al., 2026), both from plantations such as oil palm fronds (OPF) and from palm oil processing, which produces byproducts like palm kernel meal (PKM) and palm oil decanter meal (PODM). These oil palm byproducts (POB) have considerable potential as ruminant feed due to their availability and nutritional properties (Afdal et al., 2025). However, the three types of POB exhibit distinct characteristics and chemical compositions. Oil palm fronds are the most abundant byproduct in plantations, yet they have nutritional limitations in meeting the energy and nutrient requirements of ruminants. In contrast, PKM and PODM are produced in smaller quantities but have higher nutritional value per unit biomass, providing essential nutrients such as minerals (P, Ca, Mg, Mn, Fe, Cu and Zn) (Morrison et al., 2022)  and protein, as well as energy to support animal growth and productivity (Ginting et al., 2018).
       
Several studies have reported that each of these byproducts has limitations when used as a sole feed ingredient. Providing OPF at up to 50% in rations has been shown to reduce body weight gain by 28.63% compared to the control diet in goats (Ebrahimi et al., 2013). Hasanuddin et al., (2025) stated that PODM, a by-product from palm oil plant could be utilized as animal feed ingredient. Darlis et al., (2023) reported that feeding 60% PODM to goats causes color changes in the digestive tract. Devika et al., (2021) state that PODM can used 20% in concentrate mixture without adverse effect on nutrient utilization in lamb. Kumar et al., (2025) recommended using PKM, an unconventional feed ration for livestock. Pam kernel kernel meal (PKM) contains high levels of copper (Cu), exceeding the concentration found in palm kernel cake (PKC) (21-29 ppm), which is considered excessive for most ruminants. The recommended inclusion level of PKM in rations ranges from 20% to 50%.
       
To maximize the utilization of these byproducts while meeting the nutrient and energy requirements of ruminants, it is necessary to formulate a balanced combination of OPF, PODM and PKM. The availability of energy in rations for ruminants and microbial growth in the rumen can be assessed through digestibility and fermentation activity. The primary fermentation end-products in the rumen include volatile fatty acids (VFA) mainly acetate, propionate and butyrate as well as carbon dioxide, methane and ammonia. However, there is currently no information on the effects of POB inclusion levels as a forage substitute on digestibility and rumen fermentation. Therefore, the objective of this study is to evaluate the effect of POB combinations as a forage substitute on digestibility and rumen fermentation, including VFA concentration, ammonia production and methane gas emissions, through an in vitro study.
       
The research was carried out in Jambi, Indonesia in 2024. The experimental feed ingredients consisted of a combination of forage and POB, with the following composition: OPF (35%), PODM (40%) and PKM (25%), the chemical composition of OPF, PODM and PKM are presented in Table 1. The treatments were as follows: T0 = 90% kumpai forage + 10% rice bran (RB), T1 = 60% kumpai forage + 30% POB + 10% RB, T2 = 30% kumpai forage + 60% POB + 10% RB and T3 = 90% POB + 10% RB. Each ration was dried in an oven at 60°C for 24 hours, ground through a 2 mm sieve and stored until use. The chemical composition of  experimental rations were  presented in Table 2.




       
The in vitro procedure was conducted following the method described by Tilley and Terry (1963). Rumen fluid was filtered and mixed with a buffer solution at a 4:1 ratio. Fifty milliliters of this mixture were transferred into fermentation vessels, sealed with rubber stoppers and incubated at 39°C for 72 hours. After incubation, samples were collected for the analysis of digestibility, volatile fatty acids (VFA) including acetate, propionate, butyrate and total VFA ammonia concentration and methane gas production. Digestibility was calculated by comparing the difference in the amount of nutrients before and after incubation divide with the amount of nutrients before incubation. VFA concentrations were determined using gas chromatography (Shimadzu GC-14A), ammonia concentration was measured using the microdiffusion Conway technique (Masterson, 2013), while methane gas production was measured according to Afzalani et al., (2022).
       
The effect of forage substitution with POB in the ration on VFA concentration, ammonia and methane gas production was analyzed using one-way analysis of variance (ANOVA), followed by Duncan’s range test, using the SAS software (SAS Institute Inc., Railg, North Carolina, 2002).
       
The digestibility, molar proportions of acetate, propionate and butyrate, as well as total VFA concentration, ammonia and methane gas production, in diets containing different levels of POB are presented in Table 3.


       
The results showed that the digestibility of DM, OM and CP in feed containing 60% POB was lower (P<0,05) compared to feed without POB. Whereas, there was no difference in digestibility between 60% and 90% POB in the ration. The difference in molar proportion of acetate and propionate was not significant (P>0,05) among treatments. However, the molar proportion of butyrate, total VFA concentration, ammonia and methane gas production were significantly affected by the treatments. The molar proportion of butyrate increased significantly (P<0.05) with higher levels of POB in the ration. At 90% POB, the proportion of butyrate was significantly higher (P<0.05) than at 0%, 30% and 60% POB. However, there was no significant difference between the 0% and 30% POB levels. The total VFA concentration at 60% POB was significantly lower than in the control diet (0% POB). However, the differences in total VFA concentrations among the 0%, 30% and 90% POB levels were not significant.
       
The decrease in digestibility with increasing POB in the ration might be due to the chemical structure differences between forage and palm waste, especially OPF. Oil Palm Frond contains higher lignin compared to grass. Stypinski et al., (2024) stated that high lignin content in the ration can reduce digestibility and energy utilization.
       
The molar proportions of acetate, propionate and butyrate are influenced by feed composition, nutrient availability and the presence of rumen microbes (Sirisan et al., 2022). In this study, since the chemical composition of the feed was relatively similar across treatments, the molar proportions of acetate and propionate did not differ significantly. However, the higher butyrate proportion at 90% POB may be attributed to variations in microbial populations responsible for feed fermentation. Sheng-Wei et al., (2025), demonstrated that rumen microflora at the genus level are significantly correlated with ruminal fermentation parameters. Specifically, Ruminococcus was positively correlated with butyrate concentration. Therefore, the higher molar proportion of butyrate in this study may be associated with a higher population of Ruminococcus in rumen fluid.
       
The VFAs are key metabolic products of microbial fermentation. Microbial species with greater digestive abilities, particularly for certain nutrients, tend to produce higher amounts of VFAs. The present study showed that total VFA production at 60% POB was lower than that in the control diet (93 mM). According to McDonald et al., (2002), that the concentration of VFA for rumen microbial growth is 70-150 mM. In this study, even the lowest VFA concentration remained sufficient to support microbial growth. Differences in VFA concentrations were influenced by nutritional composition, particularly carbohydrate content. Wijayanti et al., (2012), reported that VFA production declines as crude fiber content increases. Crude fiber components in feed include cellulose, hemicellulose, lignin, starch and water-soluble carbohydrates. Cellulose and starch are degraded into pyruvic acid, which is then fermented by rumen microbes to produce VFAs.
       
Although the protein content in the feed was the same, the resulting ammonia concentrations differed. The highest ammonia concentration was observed at 90% POB. Even though the digestibility is lower than without POB. Ammonia concentration is related to protein digestibility and nitrogen utilization by ammonia (Turren et al., 2026). The high ammonia concentration  in feed containing 90% POB might be caused by nitrogen from protein digestion not being efficiently used by microbes, so it accumulates as ammonia. Similar to butyrate concentrations, there was a tendency for ammonia to increase as the proportion of POB in the feed increased. Jiao et al., (2022), reported that butyrate-producing microbes can enhance the activity of proteolytic microbes, leading to increased protein fermentation and higher ammonia production.
       
The results showed that methane gas concentration decreased when forage was substituted with POB up to 60% in the ration. This might be caused by the fat content in the feed. At 60% POB, the fat content is 25.18%, which is higher than the control feed. The high fat content in the feed can reduce the availability of hydrogen for forming methane gas because the hydrogen is used for the biohydrogenation process (Yang et al., 2024). Besides that, palm kernel meal and palm decanter contain phenolic compounds that can inhibit the activity of methanogen bacteria. Huang et al., (2023) showed that phenolic compounds can act as hydrogen acceptors, reducing hydrogen availability for methanogens. According to Lan et al., (2019), Hidrogen produced during fermentation serves as the primary substrate for methanogenic microbes in methane formation. The low methane concentration observed in this study may also be influenced by ammonia, as higher ammonia concentrations have been associated with reduced methane production, it means that the concentration of ammonia and butyrate are negative correlated with methane gas production as shown in Fig 1. Shi et al., (2024), reported that elevated ammonia concentrations can inhibit methane gas production.

It can be concluded that the use of palm oil byproduct combinations up to a 60 percent level in the ration can reduce methane gas production and the solubility of protein into ammonia, sufficiently supporting microbial growth.

The authors would like to thank to Rector University of Jambi. which was financed this research grand.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.