The increasing demand for beef, driven by population growth, urbanization, and rising incomes, has intensified livestock production systems worldwide (Pulina et al., 2021). Consequently, feed costs and competition for conventional feed resources have increased, particularly in tropical regions where feed availability and production efficiency are major concerns. Therefore, the use of locally available agro-industrial by-products as alternative feed resources has gained increasing attention for improving the sustainability and cost-efficiency of ruminant production systems (Shah et al., 2025).

Palm decanter cake (PDC) is a solid by-product generated during the crude palm oil extraction process in palm oil mills. During processing, fresh fruit bunches are sterilized and mechanically pressed to extract crude palm oil. The resulting slurry is then separated using a decanter centrifuge, and the solid fraction recovered from this process is referred to as PDC or decanter cake (Chan et al., 2021). PDC production accounts for approximately 4%–5% of the fresh fruit bunches processed, resulting in substantial quantities being generated annually in palm oil-producing countries (Husin et al., 2022). Despite its abundant availability, the utilization of PDC as a livestock feed ingredient remains relatively limited (Lim et al., 2022). Because it originates from residual mesocarp tissues and oil-bearing solids, PDC contains moderate crude protein, relatively high ether extract, and considerable fiber fractions, suggesting its potential as an alternative energy-rich feed ingredient for ruminants (Adebiyi et al., 2020); (Abdullah et al., 2025). Recent studies have further demonstrated the potential of solid decanter cake as a ruminant feed ingredient. (Santoso et al., 2025)reported that diets containing solid decanter cake at inclusion levels of 8.0% and 17.5% achieved dry matter digestibility values ranging from 62.62% to 65.71% and supported microbial fermentation when combined with readily fermentable carbohydrate sources. Based on these findings, PDC inclusion levels of 10% and 20% of dietary DM were selected as practical moderate and relatively high inclusion rates, respectively, within the complete FTMR dietary regimens evaluated in the present study. (Abdullah et al., 2025) also found that a PDC-based diet supported weight gain in goats, providing additional evidence of its potential as an alternative feed resource for ruminants. More recently, an in vitro study using ruminal inoculum from beef cattle showed that solid decanter cake maintained dry matter degradability at 57.73% and increased total volatile fatty acid and propionate production as well as microbial protein synthesis (Yulistiani et al., 2026). Nevertheless, the relatively high fiber and residual lipid contents of PDC may limit nutrient accessibility and lead to variable animal responses depending on its inclusion level, overall dietary composition, and processing method.

Fermented total mixed ration (FTMR) is widely used in ruminant production systems because of its benefits for feed preservation, palatability, and nutrient utilization. Although lactic acid bacteria (LAB) are commonly applied as silage inoculants to improve fermentation quality (Yin et al., 2023), their effects on nutrient utilization and aerobic stability may be limited, increasing interest in alternative additives such as Bacillus subtilis. This bacterium is tolerant of adverse environmental conditions and can produce lignocellulolytic enzymes that enhance fiber degradation and improve silage quality (Guo et al., 2022). In addition, B. subtilis produces antimicrobial compounds that suppress undesirable microorganisms, thereby reducing silage spoilage and improving aerobic stability (Bai et al., 2020); (Bonaldi et al., 2021). As a primarily aerobic bacterium, B. subtilis may help establish favorable anaerobic conditions through oxygen consumption, thereby supporting microbial balance (Su et al., 2020). Previous studies have further reported that B. subtilis supplementation can enhance rumen microbial activity, improve nutrient digestibility, and stabilize rumen fermentation (Meenongyai et al., 2023).

Combining PDC with B. subtilis in FTMR may provide complementary functions beyond those expected from either component alone. PDC supplies residual lipids, proteins, and structural carbohydrates, whereas B. subtilis may improve the fermentation environment by consuming oxygen, suppressing undesirable microorganisms, and producing extracellular enzymes. These microbial activities could increase the accessibility of nutrients associated with the fibrous PDC fraction. However, these proposed mechanisms were not measured directly in the present study and should be regarded as a biological rationale rather than evidence of synergistic activity.

Although PDC and B. subtilis have independently demonstrated potential as feed ingredients and microbial additives for ruminants, respectively, limited information is available on animal responses to FTMR containing both components. In particular, it remains unclear whether complete dietary regimens containing PDC and B. subtilis can improve nutrient utilization while maintaining favorable ruminal fermentation, microbial populations, and metabolic status in beef cattle under tropical production conditions.

Therefore, this study evaluated the effects of complete FTMR dietary regimens containing PDC and B. subtilis on feed intake, nutrient digestibility, ruminal fermentation characteristics, ruminal microbial populations, and serum metabolites in Angus crossbred bulls. We hypothesized that, compared with the control FTMR, the dietary regimens containing PDC and B. subtilis would increase the apparent digestibility of dry matter, organic matter, and fiber fractions. We further hypothesized that these regimens would maintain ruminal pH within the physiological range, increase volatile fatty acid production, and support favorable ruminal microbial populations without adversely affecting feed intake or serum metabolite profiles.

The relatively high CP and EE contents of PDC reflect residual protein and lipid fractions remaining after palm oil extraction, whereas the elevated NDF and ADF contents indicate its fibrous nature. The high OM concentration further suggests that PDC may provide fermentable substrates for rumen microbial activity. Inclusion of up to 20% PDC with PB in FTMR maintained nutrient balance and satisfied the nutrient requirements of beef cattle. The slight increase in dietary DM likely reflected the lower moisture content of PDC, whereas residual oil contributed to the increased EE content and potentially greater dietary energy density (Abdullah et al., 2025); (Adebiyi et al., 2020). However, dietary EE concentrations remained within the recommended range for finishing cattle (Sciences et al., 2016). Fiber fractions increased only slightly with PDC inclusion, suggesting minimal effects on rumen fill and feed intake. Although elevated dietary NDF may reduce intake because of rumen fill limitations and slower digestion (Shi et al., 2023), the changes observed in the present study were minimal. Increased ash content reflected the mineral composition of PDC without affecting overall OM concentration. Given its low inclusion level (0.05% or 0.10% of dietary DM), PB would be expected to contribute little directly to the nutrient composition of the FTMR. However, its independent effect on chemical composition could not be determined with this design.

Final pH was the only fermentation characteristic determined in the present study. Although the mean final pH of 4.22 indicated acidification of the FTMR, the concentrations of lactic, acetic, and butyric acids, ammonia-N, yeast and mold counts, and aerobic stability were not evaluated. Consequently, the fermentation quality and preservation stability of the FTMR could not be characterized comprehensively. The extent to which the fermentation process contributed to nutrient availability and subsequent animal responses also could not be determined. Therefore, the observed animal responses reflect the final fed dietary regimens rather than direct evidence of improved FTMR fermentation quality, aerobic stability, or silage fermentation kinetics. Without organic acid profiles or measurements during ensiling, the pathways and rate of acidification could not be established from final pH alone.

The numerically greater DMI observed in cattle fed 20% PDC with 0.05% PB may be related to differences in dietary physical characteristics. The greater bulk density of PDC may have increased dietary density and reduced rumen fill limitations, thereby allowing greater intake (Lee et al., 2021). Moderate PB supplementation may also have influenced ruminal fermentation and digesta kinetics, which can affect feed intake (Silva et al., 2024). Despite these numerical differences, OM and CP intakes were unaffected, indicating that PDC inclusion did not compromise overall nutrient intake. These results suggest that PDC can be included at up to 20% DM without adverse effects on feed intake, indicating acceptable palatability and dietary adaptation in FTMR systems. In contrast, the greater EE intake observed in cattle fed dietary regimens containing PDC was consistent with the higher lipid content of PDC (Adebiyi et al., 2020). Similarly, greater NDF and ADF intakes in cattle fed diets containing 10% or 20% PDC with 0.05% PB were consistent with the numerically greater DMI and may be associated with the previously reported fibrolytic activity of B. subtilis(Bontà et al., 2024).

Among the dietary regimens evaluated, the diet containing 20% PDC and 0.10% PB resulted in the greatest apparent digestibility of DM, OM, EE, NDF, and ADF. This response may reflect complementary functions of the greater amount of fibrous substrate supplied by PDC and the higher nominal dose of B. subtilis provided by PB. Although the fibrous matrix and residual lipid content of PDC may restrict nutrient accessibility, its structural carbohydrates provide substrates for microbial degradation. Potential mechanisms reported for B. subtilis include the production of extracellular enzymes such as cellulases, xylanases, and proteases, which may partially disrupt the feed matrix and facilitate the degradation of fiber and protein fractions (Rosazza et al., 2023); (Bontà et al., 2024). During ensiling, B. subtilis may also consume residual oxygen and produce antimicrobial compounds that suppress undesirable microorganisms, potentially creating conditions that favor preservation and subsequent nutrient utilization (Su et al., 2020); (Bai et al., 2020); (Bonaldi et al., 2021). These potential microbial and enzymatic processes are hypotheses drawn from previous studies and were not tested here. Therefore, the greater apparent nutrient digestibility observed in cattle fed the diet containing 20% PDC and 0.10% PB should be interpreted as a response to the complete dietary regimen.

However, enzyme activity, viable B. subtilis counts after fermentation, and fermentation quality were not measured in the present study. These mechanisms should therefore be regarded as biologically plausible explanations rather than processes demonstrated by the current results. Moreover, because the treatment structure did not independently separate the effects of PDC and PB, the greater digestibility cannot be attributed conclusively to B. subtilis or to a synergistic PDC-by-PB interaction. Instead, it should be interpreted as an animal response to the complete dietary regimen containing 20% PDC and 0.10% PB. A complete factorial study incorporating measurements of fermentation quality, viable microbial counts, and extracellular enzyme activities would be required to verify the proposed complementary mechanism.

The absence of treatment effects on rumen fermentation indicates that PDC inclusion (10%–20% DM) with PB did not alter overall fermentation, likely because of the similar fermentable substrate composition across diets. Ruminal NH3-N concentrations were also unaffected, consistent with the isonitrogenous formulation of the diets, reflecting balanced nitrogen supply and microbial utilization (Zhang et al., 2026). Although CP digestibility increased in cattle fed 10% PDC with 0.10% PB (Table 2, p<0.05), this was not accompanied by an increase in NH3-N, suggesting efficient microbial assimilation of released nitrogen. NH3-N concentrations remained within the optimal range for microbial activity (5.0–20.0 mg/dL), consistent with high-performance feeding conditions (Niazifar et al., 2024).

Natural LAB in fermented cassava pulp may interact synergistically with supplemented B. subtilis. As a facultative anaerobe, B. subtilis can scavenge residual oxygen, thereby promoting anaerobiosis and supporting LAB and obligately anaerobic rumen microorganisms (Gresse et al., 2025). The combined use of B. subtilis, LAB, and yeast has also been reported to enhance utilization of fibrous feed and modulate the rumen microbiota (Wei et al., 2025). However, the absence of treatment effects on total and individual VFA concentrations indicates that PDC combined with PB did not alter the overall ruminal fermentation profile, possibly reflecting comparable fermentable substrate availability among diets (Tanuwiria et al., 2025). Thus, no statistically detectable differences in ruminal fermentation profiles were observed among the complete dietary regimens. Synergistic or antagonistic interactions between PDC and PB could not be evaluated with this design.

Total VFA, acetate, and butyrate concentrations increased at 2 h post-feeding, consistent with enhanced postprandial ruminal fermentation and the accompanying decline in ruminal pH (Viquez-Umana et al., 2025). In contrast, propionate concentration was unaffected by sampling time, indicating that the increase in total VFA was primarily attributable to acetate and buty-rate. Although the C₂:C₃ ratio decreased after feeding, this response should be interpreted cautiously because propionate did not increase significantly. Therefore, the lower C₂:C₃ ratio alone does not confirm a shift in ruminal fermentation toward propionate production.

Dietary treatment did not affect predicted CO₂ or CH₄ production. However, the tendency for a treatment × sampling time interaction for predicted CH₄ (p = 0.06) indicated numerical variation in postprandial responses among treatments. The postprandial increases in predicted CO₂ and CH₄ were consistent with enhanced ruminal fermentation and the associated changes in VFA production (Lee et al., 2021) Because CH₄ was estimated from VFA stoichiometry rather than measured directly, these results should not be interpreted as evidence of altered enteric methane emissions.

Direct microscopic counting revealed no statistically detectable treatment differences in total bacterial, protozoal, or fungal zoospore populations. Although treatment effects were not significant, total bacterial and fungal counts declined numerically in the diets containing PDC combined with PB. This numerical decline may partly reflect the greater dietary lipid supply from PDC because dietary fatty acids can disrupt microbial membranes and reduce microbial attachment to fibrous feed particles, potentially affecting fibrolytic bacteria and anaerobic fungi (Sun et al., 2022). However, VFA concentrations were unaffected by dietary treatment, indicating that the numerical decline in microbial counts did not substantially alter the overall ruminal fermentation profile.

Sampling time affected total bacterial and fungal zoospore counts, indicating temporal variation in these broad microbial groups after feeding, consistent with previously reported temporal changes in ruminal microbial populations (Torres-Velázquez et al., 2025). Greater nutrient digestibility may occur without a corresponding increase in total microbial counts because digestibility may be associated with changes in microbial community composition or substrate accessibility rather than total microbial abundance alone. (Wang et al., 2024), for example, reported greater apparent total tract nutrient digestibility in cows fed FTMR together with changes in fecal microbial community composition. Fermentation of the FTMR may also have modified the feed matrix and increased nutrient availability before ingestion, thereby facilitating subsequent digestion without increasing total ruminal microbial counts (Bai et al., 2024). Furthermore, apparent total tract digestibility represents nutrient disappearance throughout the gastrointestinal tract and therefore cannot be explained solely by ruminal microbial abundance. The direct microscopic method used in the present study quantified only broad microbial groups and could not specifically quantify fibrolytic or amylolytic bacteria, methanogens, or the supplemented B. subtilis. It also could not characterize microbial community composition or metabolic function, which would require molecular and functional approaches (Cammack et al., 2018). Although B. subtilis has been reported to produce extracellular enzymes that may contribute to nutrient degradation (Rosazza et al., 2023); (Bontà et al., 2024), neither viable B. subtilis counts nor microbial enzyme activities were measured in the present study. Consequently, the greater nutrient digestibility observed with the complete dietary regimen containing 20% PDC and 0.10% PB cannot be mechanistically linked to changes in the ruminal microbiota based on the present data. Instead, it may reflect improved substrate accessibility or microbial functions that were not captured by total microscopic counts. Further studies using quantitative molecular methods, microbial community profiling, and direct measurements of fibrolytic and proteolytic activities are required to determine whether B. subtilis alters specific ruminal microbial populations or their metabolic functions.

Stable serum glucose concentrations likely reflected similar propionate supply among treatments, indicating comparable energy metabolism (Rathert-Williams et al., 2023). The postprandial increases in glucose and SUN represented normal metabolic responses to feeding and ruminal protein degradation (Khan et al., 2026). However, SUN concentrations remained within the normal physiological range, and the absence of treatment effects suggests similar nitrogen metabolism among diets, likely because of the isonitrogenous formulation and balanced nitrogen utilization (NASEM, 2016). Despite differences in dietary fat content, serum triglyceride concentrations did not differ among the complete dietary regimens. This likely reflects the moderate dietary fat levels and extensive ruminal lipid metabolism, resulting in similar fatty acid availability for absorption (Jiang et al., 2024); (Bionaz et al., 2020). The treatment structure should be considered when interpreting these findings. Because diets containing PDC without PB and PB without PDC were not included, the independent contributions of PDC and B. subtilis could not be distinguished. Therefore, the observed responses should be attributed to the complete dietary regimens rather than to either component independently. The use of five animals may have limited the statistical power to detect modest treatment differences. Therefore, nonsignificant results should be interpreted as an absence of statistically detectable differences rather than evidence of biological equivalence. Further studies using a complete factorial design and a larger number of animals are warranted. Among the regimens evaluated, the diet containing 20% PDC and 0.10% PB resulted in greater apparent digestibility of several nutrient fractions without statistically detectable differences in ruminal fermentation characteristics, microbial populations, or serum metabolites.

Under the conditions of this study, FTMR containing up to 20% PDC and supplemented with B. subtilis did not adversely affect feed intake, ruminal fermentation characteristics, microbial populations, or serum metabolites in Angus crossbred bulls. Among the dietary regimens evaluated, the regimen containing 20% PDC and 0.10% PB resulted in the greatest apparent digestibility of DM, OM, EE, NDF, and ADF. These findings indicate that PDC may be incorporated at up to 20% of dietary DM in B. subtilis supplemented FTMR as an alternative feed resource for beef cattle in oil palm-producing regions. However, the independent and interactive effects of PDC and B. subtilis require confirmation using a complete factorial design.

The authors declare that there are no conflicts of interest related to this article.

The authors gratefully acknowledge the financial support provided by the Agricultural Research Development Agency (Public Organization), Thailand, under Project No. 4838448. The authors also sincerely thank the Faculty of Natural Resources and Agro-Industry, Kasetsart University Chalermphrakiat Sakon Nakhon Province Campus, and the School of Integrated Science, Kasetsart University, Bangkok, for their valuable support and provision of research facilities. In addition, the authors appreciate AMCOVET Co., Ltd. for supplying the probiotics used in this study.The authors gratefully acknowledge the financial support provided by the Agricultural Research Development Agency (Public Organization), Thailand, under Project No. 4838448. The authors also sincerely thank the Faculty of Natural Resources and Agro-Industry, Kasetsart University Chalermphrakiat Sakon Nakhon Province Campus, and the School of Integrated Science, Kasetsart University, Bangkok, for their valuable support and provision of research facilities. In addition, the authors appreciate AMCOVET Co., Ltd. for supplying the probiotics used in this study.

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