Research Article

Evaluation of Fermentation Quality and In vitro Nutrient Digestibility of Oil Palm Fronds-Based Complete Silage with Various Additive Treatments

Budi Santoso*, Evi Warintan Saragih, Bambang Tjahyono Hariadi

Department of Animal Science, Faculty of Animal Science, University of Papua, Manokwari, West Papua, Indonesia.

Received | March 15, 2025; Accepted | April 04, 2025; Published | May 20, 2025

*Correspondence | Budi Santoso, Department of Animal Science, Faculty of Animal Science, University of Papua, Manokwari, West Papua, Indonesia; Email: [email protected]

Citation | Santoso B, Saragih EW, Hariadi BT (2025). Evaluation of fermentation quality and In vitro nutrient digestibility of oil palm fronds-based complete silage with various additive treatments. Adv. Anim. Vet. Sci. 13(6): 1303-1313.

DOI | https://dx.doi.org/10.17582/journal.aavs/2025/13.6.1303.1313

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

The quantity, quality, and consistent supply of feed are crucial factors affecting the productivity of ruminants. Generally, the availability of quality forage fluctuates throughout the year, particularly during the dry season, making some farmers rely on agricultural, plantation, or food industry by-products as feed for ruminants. These by-products are used to reduce feed costs and environmental pollution in most developing countries (Rusli et al., 2021). However, agricultural and plantation by-products contain high crude fiber and low palatability, which is a limiting factor for their use as ruminant feed.

Mixing agricultural or plantation by-products with materials of high-quality feed ingredients and additives is a method to improve low-quality feed based on waste, followed by anaerobic fermentation to produce complete silage. Previous studies by Lee et al. (2010) reported that beef cattle farmers and feedlot industries have shifted from the conventional feeding system where forage and concentrate are provided separately to a complete feed system. Beauchemin et al. (2002) stated that conventional feeding methods led cattle to prefer concentrated feed, increasing the risk of acidosis. Additionally, the application of complete feed in beef cattle industry is more economically advantageous than the conventional feeding system, saving time and labor (Baba et al., 2011).

Oil palm fronds are by-products of oil palm plantations, which is relatively abundant but has not been optimally used as feed for ruminants. According to Santoso et al. (2019), oil palm fronds contain 22.5% DM (dry matter), 82.7% OM (organic matter), 4.38% CP (crude protein), 79.2% NDF (Neutral Detergent Fiber), 63.4% ADF (Acid Detergent Fiber), 15.8% hemicellulose, and 3960 kcal/kg gross energy. Based on the nutrient content, oil palm fronds are classified as low-quality feed ingredients due to a minimum level of CP content and very high crude fiber fraction.

Over the decades, previous research has explored and developed various silage additives in order to improve the quality of forage preservation. In general, based on Muck et al. (2017) silage additives are classified into 4 groups based on their effect on silage preservation: (1) fermentation stimulants, (2) fermentation inhibitors, (3) aerobic spoilage inhibitors, as well as (4) nutrients and absorbents. Fibrolytic enzymes such as cellulase and xylanase are frequently applied in silage production to degrade cell wall carbohydrates into soluble sugars, enhance lactic acid production, and improve nutrient degradation (Guo et al., 2014). Elghandour et al. (2015) stated that cellulase and xylanase are enzymes that specifically break the β-1,4 bonds of cellulose and hemicellulose (xylan) to release soluble sugars. In a prior investigation, Santoso et al. (2024) reported that addition of cellulase enzyme with a dose of 6 ml/kg in complete silage containing 40 and 50% oil palm fronds was more effective in reducing crude fiber fractions such as NDF and ADF compared to a dose of 4 ml/kg. In addition, this treatment produces better silage fermentation quality as indicated by high lactic acid concentration and high Fleig Point and low ammonia concentrations.

Urea+Ca(OH)2 combination can significantly increase crude fiber digestibility, especially when applied to high crude fiber or low quality feed materials (Polyorach and Wanapat (2015). Chanjula et al. (2021) reported that the combination treatment of urea-Ca(OH)2 with a dose of 2.5% in oil palm frond silage was the most appropriate treatment because it has increased dry matter digestibility, total VFA, and nitrogen balance in Thai Native-Anglo Nubian Goats. The addition of 5% urea to rice straw can increase digestibility by breaking down carbohydrate structures more easily and enhancing protein content. In alkaline conditions, the bonds between lignin, hemicellulose, and cellulose can dissolve, resulting in structural fibers to swell (Wanapat et al., 2009). Applying Ca(OH)2 on corn crop residues effectively increases feed intake, cow milk production, also the digestibility of DM, OM, NDF, and starch (Cook et al., 2015). Despite the potential benefits, no information on the application of cellulase, xylanase, urea, and Ca(OH)2 in complete silage containing 40% oil palm fronds. Hence, this analysis evaluates the effect of adding additives of cellulase and xylanase enzymes, urea, and Ca(OH)2 to complete silage based on oil palm fronds on chemical composition, fermentation characteristics, and in vitro nutrient digestibility.

MATERIALS AND METHODS

Study Location and Material

This study was carried out at the Animal Science Faculty, Papua University, Manokwari, Indonesia. Oil palm fronds were obtained from plantations and king grass cultivated from areas in the Prafi District, Manokwari Regency. The study site was at approximately 133°48’E and 00°53’S with an altitude of about 128 m above sea level. Furthermore, cassava and tofu wastes were sourced from food industries in the Prafi and Manokwari Districts, Manokwari Regency. The cellulase and xylanase enzymes used in this study were commercial products (Novozymes). Fresh oil palm fronds were sliced into 1.5 - 2 cm pieces using a chopper, while King grass was cut to a size of 1.5 - 2 cm lengths with scissors. Tofu wastes and tapioca were oven-dried at 60°C for at least 72 h to achieve a constant weight and ground using a grinder equipped with a 1 mm sieve.

Study Design and Silage Treatments

This analysis applied a completely randomized design, consisting of six treatments (Table 1) with three replicates. The six complete silage treatments included A: 40% Oil palm fronds, 30% king grass, 10% cassava waste, 10% tofu waste, 7% molasses, and 3% Lactic acid bacteria inoculant, B: silage A + 6 ml cellulase/kg, C: silage A + 6 ml xylanase/kg, D: silage A + 3% urea, E: silage A + 3% Ca(OH)2, F: silage A + 1.5% urea + 1.5% Ca(OH)2. During the experiment, the initial concentration of LAB inoculated in silage was 3.1 × 106 cfu/ml of fresh material. The feed ingredients of each treatment were thoroughly mixed, packed into 25 × 40 cm plastic silos with a capacity of 1 kg. The silage material was compacted to expel residual oxygen and sealed. All silages were placed at 28-30°C and opened after 30 days of fermentation. Adding enzymes (silages B and C), as well as alkaline compounds (silages D, E, and F), significantly decreased (P<0.01) the NDF and hemicellulose contents compared to silage A (control).

 

Table 1: Composition of feed ingredients (%) of complete silage based on oil palm fronds.

Ingredients	Complete silages
	A	B	C	D	E	F
Oil palm fronds (%)	40	40	40	40	40	40
King grass (%)	30	30	30	30	30	30
Tofu waste (%)	10	10	10	10	10	10
Cassava (%)	10	10	10	10	10	10
Molasses (%)	7	7	7	7	7	7
Lactic acid bacteria inoculant (%)	3	3	3	3	3	3
Cellulase (ml/kg)	0	6	0	0	0	0
Xylanase (ml/kg)	0	0	6	0	0	0
Urea (%)	0	0	0	3	0	1.5
Ca(OH)2 (%)	0	0	0	0	3	1.5

 

OPF: oil palm fronds.

 

Preparation and Analysis of Silage Fermentation Quality

The fermentation duration took 30 days and then fresh silage samples were removed from the plastic silo, oven-dried at 60°C for 48 h, ground using a grinder equipped with a 1 mm sieve, and used for analysis. The DM, ash, and CP composition was analyzed using AOAC (2005) procedures. Furthermore, the Van Soest et al. (1991) procedure with modifications was used to analyze the composition of NDF and ADF.

Silage extract preparation was carried out using the procedure of Bureenok et al. (2006). Initially, two grams of fresh silage samples were placed in a plastic bottle, mixed with 70 ml of distilled water, and placed in a chiller at 4°C for 24 h. The sample was homogenized employing a shaker for 15 min and filtered through 4 cheesecloth layers. The obtained filtrate was applied to analyze pH value, lactic acid concentration, NH3-N (ammonia nitrogen), and VFA (Volatile Fatty Acids). This was followed by measuring pH value with a digital pH meter (Hanna Hi 9025, Hanna Instruments Italia Srl, Villafrance Padovana, Italy). Lactic acid and N-NH3 concentrations were measured using modified technique by Barker and Summerson (1941) as well as Chaney and Marbach (1962).

Individual VFA concentrations were divided and measured applying gas chromatography (Varian CP-9002 GC, 1500×3 mm i.d., column temperature of 130°C, detector temperature of 220°C, and nitrogen pressure at 1.25 kg/cm2; Shimadzu Co., Japan). The assessment of silage fermentation quality applied Fleig Point, which was measured with the Ozturk et al. (2006) formula: Fleig Point = 220 + (2 × DM% - 15) - (40 × pH) where the values between 85 and 100 indicate excellent quality, while 60-80, 55-60, 25-40, and < 20 represent good, moderate, poor, and very poor, respectively.

Inoculant Preparation of Lactic Acid Bacteria

The preparation of LAB inoculant in this study followed the Bureenok et al. (2006) modified methods as applied by Santoso et al. (2024). Initially, 200 g of fresh elephant grass was mixed with 1000 ml of distilled water and blended for four minutes. The grass mixture was filtered through two cheesecloth layers and 600 ml of filtrate was mixed with 18 g of glucose. The mixture was then anaerobically incubated at 30° C in 48 h. After incubation, the filtrate was used as the LAB inoculant to make complete silage. The LAB population was counted and added to the silage material by inoculating on MRS media at 35° C for three days.

Measurement of In vitro Fermentation Characteristics

Evaluation of rumen fermentation characteristics in complete feed silage samples was performed through a modified in vitro gas production method proposed by Menke and Steingass (1988), according to Santoso et al. (2024). The procedure followed was oven-drying silage samples weighed at 300 ± 5 mg and put into a 100 ml syringe (Model Fortune, Häberle Labortechnik, Germany), with each treatment having three replicates. Rumen fluid was found from 2 fistulated Ongole crossbred cattle before morning feeding using a suction pump. The sample obtained was filtered through four cheesecloth layers and put in a pre-warmed thermos (40°C). A 30 ± 1 ml mixture of rumen fluid and buffer was added to each syringe, flushed with CO2 gas, and sealed with a rubber stopper. Syringes were incubated at 39°C water bath for 48 h, with manual shaking every eight hours. At the end of incubation, 10 ml of sample was taken and the pH value was measured applying digital pH meter (Hanna, Hi 8520, Ronchi di Villafranca, Italy). Subsequently, 0.2 ml of the sub-sample was transferred to a 1.5 ml microcentrifuge tube with 1 ml of 25 g/100 ml (w/v) metaphosphoric acid solution and centrifuged at 9000 × g in 10 min for VFA analysis. NH3-N concentration was analyzed using a 2 ml sub-sample mixed with 2 ml of 20 g/L (w/v) NaCl solution.

Measurements of In vitro Nutrient Digestibility

The evaluation of DM and NDF digestibility was performed through a modification of the two-stage method of Tilley and Terry (1963) as applied by Santoso et al. (2024). Initially, 250 mg dry sample was weighed and put in a 100 ml glass tube, with each treatment having three replicates. Rumen fluid was found from 2 fistulated Ongole crossbred cattle in the morning before feeding using a suction pump, filtered through four cheesecloth layers, and placed in a pre-warmed thermos. The glass tubes containing 25 ml of rumen fluid-buffer solution mixture at 1:4 (v/v) ratio, flushed with CO2 gas, and sealed with a rubber stopper. The glass tube was incubated at 39°C water bath for 48 h, followed by hydrolysis using pepsin-HCl solution for 48 h. At the end of incubation, the residue was filtered through pre-weighed Gooch crucibles then dried at 105°C for 24 h. The DM and NDF percentage weight loss was expressed as IVDMD (in vitro dry matter digestibility) and IVNDFD (in vitro NDF digestibility).

Statistical Analysis

Data on chemical composition, fermentation characteristics, and in vitro nutrient digestibility of silage fermented for 30 days were studied applying ANOVA with the GLM procedure of SAS version 9.1 (SAS Institute, 2002, Cary, NC, USA). Duncan’s Multiple Range Test is applied to test differences between treatments when the P value <0.05.

RESULTS AND DISCUSSION

Characteristics of Fermented Grass Extract

The LAB inoculant used in this study was derived from king grass extract incubated at 30°C for 48 h. After incubation, the pH value decreased from 5.78 to 4.17, suggesting that the LAB population increased during the fermentation and produced more lactic acid. A significant increase in the LAB population was observed over 300-fold after 48 h of fermentation. The addition of glucose to the grass extract serves to ensure the growth of LAB. Furthermore, during incubation, LAB will convert glucose into lactic acid through the homofermentative pathway. The decreasing trend of pH due to increasing the LAB population after 48 h of incubation was in line with previous analysis reported by Santoso et al. (2020, 2024). The LAB inoculant population in this analysis was similar to the LAB population in the study of Astuti et al. (2017) which was 4 × 106 cfu/ml. However, the value was lower than the LAB population in timothy and orchard grass extracts which were 1.5 × 1010 cfu/ml and 2.0 × 109 cfu/ml, respectively (Masuko et al., 2002). The king grass extract characteristics after 48 h incubation at 30°C are shown in Table 2.

 

Table 2: Characteristics of king grass extract after fermentation for 48 hours.

	Grass extract
	Before incubation	After incubation
pH	5.78	4.17
LAB (× 106cfu/ml)	1.1	3.3

 

Physical Quality and Chemical Composition of Complete Silages

In general, all six oil palm fronds-based complete silages had an intact/unbroken texture and were not crushed, clumped, or slimy. All six silages had a light brown color, without rotting parts. There was slight mold growth (<2%) on the silo plastic obtained from residual oxygen during compaction. Febrina et al. (2022) reported that the physical quality assessment of oil palm fronds silage with poultry manure and urea additives showed light to dark brown coloration, intact texture, non-clumping and crumbly, acidic smell, and slight mold growth. Silage B and C have a stronger acidic smell than silage A. In comparison, silage F had no noticeable acidic smell, while D and E had a slightly acidic odor. The strong acidic smell in B and C was suspected to come from degradation of NDF and ADF fractions by cellulase and xylanase enzymes, causing an increase in water-soluble carbohydrates (WSC) available to be converted to lactic acid and OM. Furthermore, adding molasses to all silages created a more pleasant aroma, improving the feed palatability for livestock.

 

Table 3: Chemical composition (%) of oil palm fronds-based complete silage with various additive treatments.

	OPF	Complete silages						SEM	P
		A	B	C	D	E	F
Dry matter	34.6	38.8b	36.9b	37.2b	39.3b	42.6a	38.6b	0.95	*
Organic matter	93.8	93.7a	93.6a	93.3a	94.0a	86.7c	89.1b	0.35	**
Crude protein	4.4	8.9c	10.0bc	9.7bc	19.9a	7.9d	10.6b	0.32	**
NDF	87.3	72.5a	65.3b	66.4b	66.4b	65.0b	65.0b	0.56	**
ADF	67.3	48.9	43.6	44.5	44.6	45.0	47.6	1.25	NS
ADL	9.8	3.5	2.9	3.1	3.2	3.2	3.7	0.30	NS
Hemicellulose	20.0	24.9a	21.7b	21.9b	21.8b	20.0bc	17.5c	1.60	**
Cellulose	57.5	45.4	40.7	41.4	41.4	41.8	43.9	1.22	NS

 

OPF: oil palm fronds; SEM: standard error of the mean; A: complete silage without additives; B: complete silage A + cellulase; C: complete silage A + xylanase; D: complete silage A + urea; E: complete silage A + Ca(OH)2; F: complete silage A + urea + Ca(OH)2; NS: not significant (P>0.05); * (P<0.05); ** (P<0.01).

 

The DM, OM, CP, and crude fiber fractions of the six silages are listed in Table 3. The DM value of complete silage exceeded 30%, consistent with the recommendation of Chamberlain and Wilkinson (1996). In comparison, DM in silage E with Ca(OH)2 treatment was higher than others. The OM content of silage A - D was relatively similar at 93.6 %, while E and F treated with the addition of Ca(OH)2 had a lower value due to the combustion of Ca(OH)2 into the ash. Silage D with urea treatment had the highest CP content, followed by F which was treated with urea + Ca(OH)2 combination. Generally, urea is a non-protein nitrogen (NPN) compounds with a relatively high nitrogen content of 46%. The protein content in all six silages was above 7%, which was the minimum level required to support rumen microbial activity. Minson and Milford (1966) stated that when the nitrogen concentration in feed fell below the minimum threshold, it would cause a decrease in nutrient digestibility due to low rumen microbial activity.

The addition of enzymes (silages B and C), as well as alkaline compounds (silages D, E, and F), significantly reduced (P<0.01) the NDF content than silage A (control) by 9.9%, 8.4%, 8.4%, 10.3%, and 10.3%, respectively. Similarly, hemicellulose content was significantly lower (P<0.01) in silages B, C, D, E, and F with enzyme treatment and alkaline compounds compared to silage A. Santoso et al. (2024) reported that NDF content in complete silage containing 40% oil palm fronds with 6 ml/kg cellulase enzyme treatment was significantly lower (P<0.01) than at 3 ml/kg treatment. Furthermore, it was concluded that the application of cellulase enzyme at 6 ml/kg was effective in reducing the crude fiber fraction (NDF, ADF, and ADL), thus enhancing in vitro nutrient digestibility. He et al. (2018) and Ma et al. (2023) also stated that cellulase enzyme treatment on Neolamarckia cadamba leaf silage and mixed with amaranth silage and rice straw improved fermentation quality which is characterized by a decrease in NDF and ADF content. Li et al. (2019) explained that the pattern of decreasing NDF and ADF during silage fermentation was caused by the process of enzymolysis and acid solubilization processes targeting on NDF, ADF, and ADL.

A reduction in NDF content in silage treated with alkaline compounds in this study aligns with finding reported by Chanjula et al. (2021), which shows that adding 5% Ca(OH)2 and combining 2.5% urea - 2.5% Ca(OH)2 to oil palm fronds silage reduced NDF content by 18.1% and 9.6%, respectively. According to, Gunun et al. (2016), the use of a combination of 2% urea + 2% Ca(OH)2 on sugarcane bagasse was able to reduce NDF, ADF, and ADL contents by 9.1%, 13.0%, and 33.0%, respectively. The breakdown of the lignin structure in high fiber feed, accompanied by increased cellulose and hemicellulose availability, is essential for ruminant feed. The addition of alkaline compounds such as urea + Ca(OH)2 to lignocellulosic materials causes swelling, raising the internal surface area as well as reducing the polymerization and crystallinity degree, thus causing breaking down of lignin (Casperson et al., 2018).

Reductions in NDF and hemicellulose content due to enzyme and alkali treatments can significantly impact the physical properties of silage, particularly its compaction, porosity, and storage stability. Lower fiber content typically results in a finer, more degradable material that can be compacted more easily. Besides that, lower fiber content might accelerate microbial fermentation, leading to a faster pH drop and better preservation.

Fermentation Characteristics of Complete Silage

The fermentation quality of oil palm fronds-based complete silage after 30 days of fermentation with enzyme treatment and alkaline compounds is shown in Table 4. Silage fermentation quality is determined by the effect of pH, lactic acid concentration, VFA composition, NH3-N/total N, and other factors. In the good quality silage, the population of lactic acid bacteria must be dominant to increase the fermentation process and enhance the quality (Cai et al. (2003).

 

Table 4: Fermentation characteristics of oil palm fronds-based silage with various additive treatments.

	Complete Silages						SEM P
	A	B	C	D	E	F
pH	4.03c	3.87cd	3.65d	3.85 cd	4.81b	5.34a	0.07	**
NH3-N (g/kg total N)	51.5b	45.9b	45.2b	165.2a	44.8b	154.1a	4.42	**
Lactic acid (g/kg DM)	78.5e	82.6cd	84.6 cb	79.3ed	87.0b	92.3a	1.07	**
Acetic acid (g/kg DM)	25.9a	12.2b	14.7b	14.8b	16.8b	16.6b	4.06	*
Propionic acid (g/kg DM)	7.4a	5.5b	4.6b	4.9b	10.8a	8.1a	1.75	*
Butyric acid (g/kg DM)	3.4a	1.7bc	1.1c	1.5c	1.8bc	2.1b	0.25	*
Total VFA (g/kg DM)	36.6a	19.4c	20.4c	21.2c	29.2b	26.8b	3.07	*
Fleig Point	106.4b	109ab	118.5a	114.6ab	83.0c	53.6d	3.03	**

 

SEM: standard error of the mean; A: complete silage without additives; B: complete silage A + cellulase; C: complete silage A + xylanase; D: complete silage A + urea; E: complete silage A + Ca(OH)2; F: complete silage A + urea + Ca(OH)2; NS: not significant (P>0.05); * (P < 0.05); ** (P < 0.01).

 

The pH value is a key indicator of silage quality, determining the anaerobic fermentation success, and the high or low production of organic acids, particularly lactic acid (Oliveira et al., 2017). pH values of silage A, B, C, and D varied from 3.65 - 4.03, which was within the range of ideal values (4.0 - 4.5) stated by Chamberlain and Wilkinson (1996). Furthermore, xylanase enzyme treatment (silage C) significantly reduced (P<0.01) the pH value than the control. According to Del Valle et al. (2019) and Mu et al. (2023), xylanase treatment of sugarcane tops silage and a mixture of agricultural waste and alfalfa decreased the pH value after 60 and 30 days of fermentation. Cellulase and xylanase enzymes were also found to hydrolyze β-1,4-glycosidic bonds in major plant polysaccharides such as cellulose, and xylan, thereby increasing the availability of water-soluble carbohydrates (WSC) for silage fermentation by LAB. Meanwhile, combining urea + Ca(OH)2 (silage E) significantly increased (P<0.01) the pH value than control silage. The relatively high increase in pH value in silage E with the urea + Ca(OH)2 combination was associated with higher NH3-N concentration. This can be explained by the addition of urea to silage, which will rapidly decompose the protein content of plants into ammonia by plant and microbial enzymes as well as urease activity in the initial phase of ensilage when the pH is neutral.

NH3-N concentration is an index that serves the proteolysis level in silage which can assess the silage fermentation quality. Therefore, high NH3-N concentration indicates extensive protein degradation into NH3, thereby the fermentation quality of the silage is considered poor. The NH3-N concentration in urea-treated silage (silage D) and urea-Ca(OH)₂-treated silage (silage F) were significantly higher (P<0.01) than others. High concentrations of NH3-N in silage D and F can cause negative impacts on ruminants, including reducing dry matter intake. The findings show the NH3-N concentration in silage B, C, and E varied from 44 to 45 g/kg total N which consistent with the ideal concentration (<50 g/kg total N) as recommended by Chamberlain and Wilkinson (1996). The low concentration of NH3-N in cellulase and xylanase enzyme treatments is in line with previous reports by Mu et al. (2023). These results show that adding cellulase, xylanase, and Ca(OH)2 enzymes effectively reduced the concentration of NH3-N compared to silage control. The addition of these additives will be able to maintain a low pH, thereby suppressing the activity of clostridia, which is responsible for protein degradation of silage.

In fermentation process, early stage is indicated by rapid development of LAB and fermenting of the WSC to produce lactate, leading to low pH value below 4.0 (Pholsen et al., 2016). When added with cellulase (silage B), xylanase (silage C), Ca(OH)2 (silage D), and urea+Ca(OH)2 (silage E) enzymes, (P<0.01) lactic acid concentration increased in comparison with the control. Furthermore, lactic acid concentration in the four treatments was 5.2%, 7.8%, 10.8%, and 17.6%, respectively. A study by Santoso et al. (2024) found that cellulase enzyme treatment at doses of 4 and 6 ml/kg significantly enhanced lactic acid concentration of complete silage with 40% and 50% oil palm fronds. According to Bai et al. (2023), adding cellulase could give more substrate derived from lignocellulose degradation, which could further be used by LAB to produce high lactic acid. In all treatments, the lactic acid concentration remained within the normal range of 80-120 g/kg DM (Chamberlain and Wilkinson, 1996).

VFA includes acetic, propionic, butyric, and other organic acids formed through secondary fermentation that could break down into ammonia, thereby producing acetic and butyric acid. Generally, high VFA concentrations indicate inefficient silage fermentation (Chamberlain and Wilkinson, 1996). The results of this study demonstrated the concentrations of acetic acid, propionic acid, butyric acid, and total VFA in silage B, C, D E, and F were lower (P<0.05) than the control. The decreasing trend of propionic and butyric acid concentrations was due to cellulase and xylanase-treated silage, which is similar with the analysis of Mu et al. (2023). Meanwhile, Wagali et al. (2023) reported that the addition of 0.4% urea in corn silage tended to improve (P>0.05) the concentration of acetic, propionic, butyric acid, and total VFA than control silage. The ratio of total VFA to acid in silage treated with enzyme and alkali additive in this analysis indicated no significant difference (P>0.05) with the control silage. In comparison, the ratio values of total VFA to acid obtained in silage B and C fell within the normal range recommended by Lima et al. (2011), suggesting that ideal silage ratio was below 0.2.

Adding Ca(OH)2 and Ca(OH)2 + urea combination led to a significantly (P<0.01) lower Fleig Point compared to other silages. This indicated that the addition of additives caused low silage quality, based on pH value, and DM content. Fleig Point is one of the parameters applied to examine silage fermentation quality based on pH value and DM. Therefore, a higher Fleig Point value indicates better silage fermentation quality. In this study, the enzyme xylanase treatment (silage C) produced the highest Fleig Point (P<0.01) compared to other treatments. According to the classification criteria proposed by Ozturk et al. (2006), silages A, B, C, and D were categorized as high-quality silage, while E and F had medium quality. The Fleig Point value in this analysis was higher than those of Santoso et al. (2024), ranging from 88 - 93.6.

In vitro Rumen Fermentation Parameters and Gas Production

NH3-N concentration, pH value, VFA, gas production, and CH4 emissions of oil palm fronds-based complete silage after 48 h incubation can be seen in Table 5. The pH of rumen fluid is one of the indicators reflecting the fermentation process in the rumen, performing an essential function in supporting the growth of microbes. The enzyme treatment and alkaline compounds did not affect significantly (P>0.05) the rumen fluid pH. The enzyme treatment and alkaline compounds did not significantly affect (P>0.05) the rumen fluid pH. The pH values observed in this study ranged 6.83 - 6.91, which falls within the normal pH range of 6.5 - 7.0 needed for fiber digestion in the rumen (Hamchara et al., 2018). Moreover, if the rumen pH is below 6.0, it can inhibit the growth of cellulolytic bacteria. According to Dijkstra et al. (2012), the low rumen pH values can be caused by a reduction in fiber degradation and a decrease in the acetate/propionate ratio.

 

Table 5: In vitro rumen fermentation characteristics of oil palm fronds-based complete silage with various additive treatments.

	Complete silages						SEM	P
	A	B	C	D	E	F
pH	6.85	6.87	6.89	6.91	6.83	6.83	0.03	NS
NH3-N (mg/100 ml)	18.3d	16.7d	32.7b	37.8a	26.6c	34.1b	0.60	**
Acetate (mM)	28.0c	33.5a	33.9a	34.5a	32.9a	31.4b	1.19	*
Propionate (mM)	8.2b	10.7a	9.6b	8.1b	9.2b	9.8a	0.50	*
Butyrate (mM)	5.7c	7.1a	6.8b	6.8b	6.0c	7.2a	0.30	*
Total VFA (mM)	41.9b	51.3a	50.3a	49.4a	48.2a	48.4a	1.66	**
Protein Microbe (mg/100 ml)	11.9c	12.8c	30.98a	17.2b	16.0b	16.9b	0.74	**
CH4 (ml/g DM)	11.0c	13.3a	13.7a	9.9d	12.3b	12.5ab	0.43	*

 

Description: A: complete silage without additives; B: complete silage A + cellulase; C: complete silage A + xylanase; D: complete silage A + urea; E: complete silage A + Ca(OH)2; F: complete silage A + urea + Ca(OH)2; NS: not significant (P>0.05); * (P<0.05); ** (P<0.01).

 

The NH3-N concentration in complete silage C, D, E, and F was significantly higher (P<0.01) than in silage A and B. Silage treated with urea (silage D) and a combination of urea-Ca(OH)2 produced the highest concentration compared to other silage treatments. This result appropriates to Chanjula et al. (2021) reported that the goats fed with oil palm fronds silage with 5% urea treatment exhibited the highest NH₃-N concentration in the rumen, followed by the urea-Ca(OH)₂ combination and Ca(OH)₂ treatment. NH3-N serves as a primary nitrogen for microbial protein synthesis as rumen bacteria majority utilize NH3-N as their nitrogen source. The availability of NH3-N is crucial for optimal microbial growth (Wanapat et al., 2011). The N-NH3 concentration in this analysis varied from 18.3 to 37.7 mg/dl which falls within the effective range of 15 - 30 mg/dl NH3-N for enhancing rumen fermentation, microbial proliferation, and feed intake in ruminants (Wanapat and Pimpa, 1999).

VFA from microbial fermentation in the rumen serve as a primary ruminant energy nutrition (Fimbres et al., 2002). The VFA composition in the rumen is based on the substrate type, fermentation pattern, microbial population, and rumen environment (Bannink et al., 2008). According to Meale et al. (2012), in vitro DM degradation is strongly positively correlated with total VFA production. Complete silage treated with enzymes and alkaline compounds showed significantly higher (P<0.05) acetic, propionic, butyric acids, and total VFA than the control silage. In comparison with silage A, total VFA in B, C, D, E, and F increased by 22.4, 20.0, 17.9, 15.0 and 15.5%, respectively. The increase in the individual and total VFA concentration in oil palm fronds-based complete silage with cellulase enzyme treatment is appropriate to Santoso et al. (2024). Similarly, Mu et al. (2023) stated that adding cellulase and xylanase enzymes enhanced the molar VFA proportion and caused high gas production, lowering pH due to increased DM degradation.

Chanjula et al. (2021) reported that the application of urea, Ca(OH)2, and urea+Ca(OH)2 combinations in oil palm fronds silage increased the total VFA concentration four hours after feeding. Van Soest (1994) stated that the concentration of acetic acid was from the degradation and fermentation of cell walls. Meanwhile, McCollum et al. (1985) showed that propionic acid concentration in the rumen was related to the soluble carbohydrate fermentation. The VFA concentration in the analysis ranged 41.9-51.3 mM, which was lower than the optimal rumen at 70-150 mM, as suggested by McDonald et al. (2012). Compared to other studies, the variation in results was due to the in vitro methods, thereby the population of microorganisms in the fermenter tubes over prolonged incubation period was difficult to maintain.

Gas production can indicate feed degradation in the rumen. The enzyme treatment and alkaline compounds in oil palm fronds-based complete silage had significant effect (P<0.05) on CH4 production. CH4 production in silage B, C, E and F was lower (P<0.05) compared to silage A (control). The average decrease in CH4 production in the four treatments compared to silage A was 16.3%. Furthermore, the propionic acid production in ruminants competed with methanogenesis in hydrogen utilization. This suggested that increased propionic acid formation was associated with a decrease in CH4 production, leading to a lower acetic acid to propionic acid ratio in the rumen (Weimer, 1996). Increasing the concentration of propionate in silage with enzyme and alkaline compound treatments tend to be followed by a decrease in CH4 production.

In vitro Nutrient Digestibility

The in vitro digestibility measurements reflect the level of substrate digestibility by microorganisms under artificial environment conditions, that simulate the rumen environment conditions in a test tube. The DM and NDF digestibility coefficients of oil palm fronds-based complete silage are presented in Table 6.

 

Table 6: In vitro nutrient digestibility (%) of oil palm fronds-based complete silage with various additive treatments.

	Complete Silages						SEM	P
	A	B	C	D	E	F
IVDMD	44.15d	50.00b	48.22bc	46.37c	53.03a	45.71d	0.96	*
IVNDFD	43.49b	49.38a	45.89b	45.31b	52.14a	44.29b	0.90	**

 

IVDMD: in vitro dry matter digestibility; IVNDFD: in vitro neutral detergent fiber digestibility; A: complete silage without additives; B: complete silage A + cellulase; C: complete silage A + xylanase; D: complete silage A + urea; E: complete silage A + Ca(OH)2; F: complete silage A + urea + Ca(OH)2. NS: not significant (P>0.05); * (P<0.05); ** (P<0.01).

 

Enzyme, urea and Ca(OH)2 treatments in oil palm fronds-based complete silage (B, C, D and E) significantly increased (P<0.05) IVDMD compared to silage A. In this study, the values were lower than what Santoso et al. (2024) found, where 57.7% IVDMD was for silage with 40% oil palm fronds and 6 ml/kg cellulase enzyme. Therefore, to meet the nutrient requirements of ruminants, this can be done by supplementing other feed ingredients with high energy and protein. Another method that can be done is providing supplements such as urea, minerals, or buffers to increase rumen fermentation. The difference might be because of the fiber concentration, like NDF and ADF in silage. Zhang et al. (2020) stated that what was in the feed affected dry matter digestibility more than the additives used. However, Pino et al. (2018) revealed the rumen digestibility depended on things like NDF concentration, population of microorganisms, and type of feed. Ebrahimi et al. (2014) found that if 2 g/kg cellulase enzyme was added, the IVDMD in oil palm fronds silage increased by 6.9% (P<0.01), showing better improvement than the control silage.

The IVDNDF value in complete silage treated with Ca(OH)2 (silage E) and cellulase enzyme (silage B) was higher (P<0.01) than in other complete silages. Wang et al. (2019) reported that cellulase and xylanase enzymes were effective in breaking down crude fiber bonds, thereby increasing the digestibility of crude fiber in agricultural waste-based feeds. Similarly, according to Chanjula et al. (2021), treatment with 5% of Ca(OH)2 alongside 2.5% Ca(OH)2 and urea in complete silage with 40% oil palm fronds significantly increased the DM, OM, and NDF digestibility in goats. These results are appropriate to Santoso et al. (2020) who stated that there is a strong correlation on increasing cellulase dosage and the enhancement of in vitro digestibility of DM, OM, and NDF.

There are environmental benefits and challenges on utilizing oil palm fronds as a livestock feed source. Incorporating oil palm fronds into animal feed system contributes to sustainable waste management that reduce environmental pollution (Dahlan, 2000). Furthermore, Boafo et al. (2020) stated that utilizing oil palm fronds that are generated as part of existing oil palm plantation does not require additional agricultural land, reducing land use for feed crop expansion.

CONCLUSIONS AND RECOMMENDATIONS

In conclusion, the treatment of enzymes and alkali compounds in complete silage based on oil palm fronds reduced the crude fiber fraction such as NDF and hemicellulose, followed by an increase in fermentation quality. The results showed that xylanase enzyme treatment yielded the highest fermentation quality, evidenced by the lowest pH value, NH3-N concentration, propionic acid, and butyric acid, alongside the highest Fleig point in complete silage based on oil palm fronds. These findings support the use of xylanase in oil palm frond silage for ruminant feed.

ACKNOWLEDGMENTS

This study was funded by the Ministry of Education, Culture, Research, and Technology for providing financial support (No. 076/E5/PG.02.00.PL/2024). The authors expressed their gratitude for valuable support in sample preparation from assistants M.F. Rumakey and D.N. Renyaan.

NOVELTY STATEMENTS

These findings indicated that xylanase application at a dose of 6 ml/kg was a highly effective strategy to improve the quality of oil palm fronds-based silage, offering a practical and sustainable solution to upgrade fibrous agricultural by-products into valuable animal feed.

AUTHOR’S CONTRIBUTIONS

Conceptualization, Budi Santoso; methodology, Budi Santoso, Evi Warintan Saragih, and Bambang Tjahyono Hariadi; writing-original draft preparation, Budi Santoso and Evi Warintan Saragih; writing-review and editing, Budi Santoso and Evi Warintan Saragih. All authors have read and agreed to the manuscript published version.

Conflict of Interest

The authors have no conflicts of interest to disclose.

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