The experiment received official review and approval from the Institutional Animal Care and Use Committee of Khon Kaen University, in accordance with the Ethics of Animal Experimentation established by the National Research Council of Thailand (Record No. IACUC-KKU-110/66 and Reference No. 0201.2.11/73).

The product of phytonutrient pellets, MARABAC, was followed by Wanapat et al [7]. The bioactive compounds from MARABAC extract were prepared from 10 g of MARABAC pellet powder mixed with 100 mL of deionized water and extracted by using the microwave extraction method in an optimal condition of microwave power at 100 W for 10 to 15 min (until the temperature reached 60°C) according to the procedure of Phupaboon et al [8].

The microencapsulated MARABAC (mMARABAC) was formulated at a 1:1 ratio between the MARABAC extract solution and the wall material containing 2% of chitosan media dissolved in a 1% acetic acid (C2) solution as an ionic gelation technique. After being stirred for overnight, the homogeneous media was processed through the spray-drying technique (B-191 mini spray dryer; Bǚchi, Flawil, Switzerland) at optimal conditions, and the dried powders of mMARABAC were collected, hermetically sealed, and then stored at a temperature of −20°C until use in in vitro experiments according to the procedure used by Phupaboon et al [8], Nouri [10] and Kurek and Pratap-Singh [11].

The measurement of the total polyphenolic content (TPC), total flavonoid content (TFC), and antioxidant capability of extracts containing bioactive compounds was performed utilizing a 96-well microplate assay. The biochemical assay mode of an EnSight multimode plate reader (PerkinElmer Inc., Waltham, MA, USA) and Kaleido Data Acquisition software were utilized in this study, following the approach defined by Phupaboon et al [8]. The assay employed label-free applications. The antioxidant activity was assessed using three methods: the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method [12], the 2, 2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid; ABTS) radical scavenging activity [13] and the ferric reducing antioxidant power (FRAP) method [14]. The analyses were conducted in triplicate, and the outcomes were reported as a percentage of radical scavenging inhibition and millimoles of Trolox equivalents per gram of dry matter (mmol TROE/g DM).

The roughage and concentrate samples were taken, combined into composite samples, and further processed by chopping and grinding for the purpose of conducting chemical analysis. The analysis of all samples included determining DM (ID 967.03) and ash (ID 492.05) content using the standard AOAC procedures [15]. The Nitrogen Analyzer (Leco FP828; LECO Corporation, Saint Joseph, MI, USA) was to determine the amount of nitrogen (N) components and evaluate the crude protein (CP) content. Van Soest et al [16] outlined a standard method for determining the content of neutral detergent fiber (NDF) and acid detergent fiber (ADF).

Rumen fluid was collected from a sample of four male 4-year-old Thai native beef cattle with an average weight of 200±50 kg. The cattle were housed in separate pens and provided with a daily ration of concentrate that constituted at 0.5% of body weight. Additionally, animal s were given ad libitum access to rice straw at 7:00 a.m. and 4:00 p.m. for a period of seven days prior to the collection of rumen fluid. On the final day of the seven-day period, collected 1,000 mL of rumen fluid from each animal before morning feeding. The rumen fluid was filtered through four layers of cheesecloth into pre-warmed thermos flasks, mixed the rumen fluid from each collected animal, and then transported to the laboratory. Table 1 presents a description of the ingredients and chemical composition of the concentrate and rice straw.

The design of the experiment was a completely randomized design (CRD). Four levels of mMARABAC supplementation at 0, 5, 10, and 15 mg/500 mg on DM basis. The dietary substrate had a 60:40 roughage to concentrate ratio, and the roughage source was rice straw. The rice straw and concentrate were ground to a particle size that could pass through a 1 mm sieve using a blender before being utilized for in vitro gas production testing. The gas production experiment was performed with three replicates for each treatment, along with an additional three replicates for the control treatment. In total, 15 serum bottles were created (3 replicates×4 treatments+3 replicates of blank). Measurement of rumen fermentation parameters, specifically NH₃-N, VFAs, and microbial diversity, was carried out at two sampling time points: 12 hours and 24 hours of incubation. A total of 20 bottles were arranged for this experiment (2 replicates×4 treatments×2 sampling times+2 replicates of blank). An additional twenty bottles were made to investigate the IVDMD at two different sampling times, namely 12 hours and 24 hours of incubation. This was achieved by conducting two replicates for each of the four treatments, as well as including two replicates of a blank sample. A quantity of 500 mg of substrate was weighed and transferred into 50 mL serum bottles. Subsequently, mMARABAC was added in different levels ranging from 0, 5, 10, and 15 mg based on a dry matter basis. Following the addition of substrates, the bottles received a CO₂ flushing procedure and were subsequently injected with 40 mL of artificial saliva. The artificial saliva was made in accordance with the methodology outlined by Menke and Steingass [17]. Following the injection, the serum bottles were sealed with an aluminum lid and subjected to incubation at a temperature of 39°C until the time of sampling.

A sequence of incubation durations involving 1, 2, 4, 6, 8, 12, 18, 48, 72, and 96 hours was conducted to assess gas production. The gas generated during each incubation period was utilized to investigate the accumulation of gas by using the Ørskov and McDonald [18] equation (1) model.

In the given context, the variable “a” represents the gas production derived from the fraction that is immediately soluble, measured in milliliters (mL). Similarly, the variable “b” represents the gas production derived from the fraction that is insoluble, also measured inmL. The variable “c” denotes the gas production rate constant for the insoluble fraction, measured in mL per hour (mL/h). The variable “t” represents the duration of incubation time, measured in hours. The sum of “a” and “b” represents the potential extent of gas production, measured in mL. Lastly, the variable “Y” denotes the amount of gas produced at specific time points “t”, measured in mL. The serum bottles were taken out of the incubator after 12 and 24 hours of incubation, and their pH levels were promptly tested using a HANNA pH meter manufactured by HANNA instruments in Romania. Subsequently, the fermentation liquid received filtration using a four-layered cheesecloth, resulting in its division into two separate phases. The liquid phase was utilized for the analysis of NH₃-N and VFAs concentrations, while the solid part was designated for the examination of the microbial population. The first part of the samples underwent centrifugation for 15 minutes at 16,000×g, and then the supernatant was collected and kept at −20°C until analysis. The concentration of NH₃-N was determined utilizing the micro-Kjeldahl method as described in the study conducted in AOAC [19]. Additionally, the VFAs concentration, which includes C2, propionic acid (C3), and butyric acid (C4), was measured using gas chromatography (GC) with a GC-2014 (Shimadzu, Kyoto, Japan) according to So [20]. The subsequent set of samples was appropriately managed and maintained in a refrigerated environment in preparation for DNA analysis according to Koike and Kobayashi [21]. After 12 and 24 hours of incubation in the IVDMD study, serum bottles were used to collect gases for the study of CH₄ production. A volume of 10 mL of gas was obtained by means of a 10 mL syringe. This gas was subsequently introduced into serum bottles with a capacity of 25 mL, which were sealed using a combination of rubber lids and aluminum caps. The bottles were then securely covered with parafilm. The measurement of CH₄ production was conducted using GC (specifically, the GC-17A System; Shimadzu). The gas chromatograph was equipped with a thermal conductivity detector and a Shin carbon column with dimensions of 3 m×3 mm. The activated charcoal used in the column had a mesh size of 60/80 and was sourced from Kyoto, Japan. The methodology employed for CH₄ measurement followed the work of Sittijunda et al [22].

Following a 12-hour and 24-hour incubation period, the serum bottles were taken out of the incubator and subsequently subjected to freezing for preservation until further examination. The IVDMD was computed following the methodology outlined in the study conducted by Menke and Steingass [17]. In a concise manner, the serum vials were extracted from the freezer and subsequently thawed at room temperature. Afterwards, the fermentation liquid samples were filtered using Gooch crucibles with a porosity of 40 mm, which were pre-weighed before the filtration process. The filtered Gooch crucibles were then subjected to oven-drying at a temperature of 100°C for a duration of 24 hours. Following the drying process, the Gooch crucibles were weighed, and the residual DM was determined. In order to determine IVDMD, the DM values of the residue were subtracted from the DM of the blank residue.

The data were analyzed using the generalized linear model technique of SAS [23] in a CRD. The data were subjected to analysis using the specified model.

The variable Yij represents the observed data, m denotes the average value, Mi represents the impact of the distinct mMARABAC levels (with i ranging from 1 to 4), and 1ij represents the residual effect. The findings are reported in the form of mean values, accompanied by the standard error of the means. The determination of differences between treatment means was conducted using Tukey’s test. Statistically significant differences were accepted when the p-value was less than 0.05 (p<0.05). Orthogonal polynomials were employed to analyze the trends of mMARABAC-level responses.

Table 1 displays the chemical composition of the concentrate, rice straw, and mMARABAC. The concentrate and rice straw contained 14.6% and 2.4% CP, respectively. The mMARABAC contained 3.02 TPC g GAE/kg DM, 0.16 TFC g QUE/kg DM, 76.6% DPPH radical inhibition, 79.7% ABTS radical inhibition, and 22.34 FRAP g TROE/kg DM).

The gas production kinetics are shown in Table 2. Supplementation of mMARABAC significantly affected the insoluble fraction of gas production (b), rate of gas production value (c), and potential extent of gas (|a|+b) value (p<0.05), while the soluble fraction of gas production (a) was not influenced by the treatments. Furthermore, the mMARABAC increased the cumulative gas at 96 h after incubation (p<0.05) when the mMARABAC was supplemented with 5 mg of the substrate in comparison to 0 mg of mMARABAC. However, adding mMARABAC to the diet had no effect on the IVDMD (p> 0.05).

The effects of mMARABAC supplementation on pH, NH₃-N concentration and CH₄ production are presented in Table 3. mMARABAC supplementation had no effect on ruminal pH and NH₃-N (p>0.05). Nevertheless, the level of mMARABAC supplementation in the diet, which ranged from 5 to 15 mg of the substrate, resulted in a decrease in CH₄ production at 24 hours (p<0.05).

The effectiveness of mMARABAC supplementation on total VFAs, C2, C3, C4, C2:C3 ratio are presented in Table 4. The concentration of total VFAs, C2, C3, C4, and the C2:C3 ratio were not influenced by mMARABAC supplementation (p> 0.05).

Table 5 shows the effects of mMARABAC supplementation on microbial diversity. The effect of mMARABAC supplementation on Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens, Butyrivibrio fibrisolvens and Megasphaera elsdenii differed between treatments (p>0.05). While, the mMARABAC supplement did not demonstrate a statistically significant effect on methanobacterial (p<0.05).