The experiment took place at the Tropical Feed Resources Research and Development Center (TROFREC), which is part of the Department of Animal Science in the Faculty of Agriculture at Khon Kaen University. The university is in Khon Kaen Province, Thailand, at the precise coordinates of 16°26′48.16″N and 102◦49′58.8″E. The experiment was conducted during the winter season, specifically from December 2021 to March 2022, with an average temperature falling between 28°C and 31°C. The research station of the institute was the location where the experimental animals were housed, as it was specifically designed for research purposes. The rumination behavior and digestibility inside the facility were unaffected by the exterior environment. The cattle were accommodated in four enclosures, each measuring 3×5 square meters, and supplied with cement water containers. The animals had comparable age and body weight (BW) before the commencement of the study. The animals were measured, and their initial BW was documented. Subsequently, they were dewormed using Ivomec F from Kos Introtech, and given an injection of vitamin AD3E from Phenix, Anitech Total Solution. Before the commencement of the research, the experimental cattle had a training period of no less than 2 weeks in separate enclosures, allowing the animals to acclimate effectively to the facilities. All experimental cattle remained in good health throughout the study. Following this acclimation period, the animals were subjected to dietary treatments designed to evaluate the effects of WBTP substitution.

The Faculty of Agriculture, Department of Animal Science, at Khon Kaen University (KKU) in Khon Kaen, Thailand, was the site of the research investigation. The WBT was cultivated and collected at the Faculty of Agriculture, Department of Agronomy, KKU. To achieve a moisture content of less than 10%, the freshly harvested WBT were cut into pieces measuring 3–5 cm and sun-dried for 72 hours. Before being used as a substrate, each WBT was subjected to milling via a 1-mm screen. Subsequently, the WBT powder with a moisture content of 30% was formed into pellets using the procedure described by Seankamsorn et al [14]. The prepared pellets were sun-dried for approximately 72 hours to achieve a moisture content of less than 10%, ensuring optimal storage stability and feed quality.

A quartet of Thai Indigenous beef cattle were used, each with an average BW of 310±20.0 kg. A 4×4 Latin square design was implemented to allocate the cattle. Dietary regimens included four levels of WBTP replacing corn meal, which was initially included at 10% in the control concentrate diet. The corresponding WBTP inclusion levels in the concentrate were 0%, 3.3%, 6.6%, and 10% for the 0%, 33%, 66%, and 100% replacement treatments, respectively. The concentrated diets used in the experiment were produced based on the nutritional needs of Thai native beef cattle, as specified by the recommendation of the Working Committee of Thai Feeding Standard for Ruminants. Each animal was administered a concentrated diet that was equivalent to 1% of their BW two times per day, at 8:00 a.m. and 4:00 p.m. This was followed by the unrestricted feeding of rice straw. Table 1 presents the chemical compositions of the dietary concentrate combination treatment, rice straw, corn meal, and WBTP. The concentrate diets were created using local feed ingredients that had comparable CP levels, ranging from 14.87% to 15.71% CP, and similar gross energy content, ranging from 4.06 to 4.02 megacalories/kg DM, in each treatment. Each session of the research lasted 21 days, and it was conducted over four consecutive periods. The initial 14-day period was intended to facilitate the subjects’ adaptation to the diet provided. The subsequent 7-day period was dedicated to the collection of samples using the total collection technique in specialized enclosures that were specifically designed to measure the digestibility of fecal matter. The quantity of food ingested and rejected was documented daily. At the commencement and conclusion of each interval, black-and-white images were recorded.

The cattle were relocated to the metabolism enclosures to investigate digestibility after the trial concluded. Consecutive samples of concentrated diet, WBTP, rice straw, and leftover food were obtained and preserved at a temperature of −20°C until further investigation. Fecal samples were collected, measured, documented, and combined by the animals during the final seven days of each session. Finally, the samples were maintained at a temperature of −20°C. After a 7-day collection period, defrosted samples from each animal were pooled and analyzed. The portions were subsequently thoroughly mingled, and smaller portions of the feed that was consumed and the feed that was not consumed were taken. Feed and fecal samples were dried in a forced-air oven at 60°C for 72 hours. After drying, they were ground using a Cyclotec Mill (Tecator) to achieve a particle size capable of passing through a 1 mm screen.

The standard method of the Association of Official Analytical Chemists [15] was used to analyze the chemical compositions of feed and feces. This included the constituents of DM (no. 967.03), organic matter (OM), and ash (no. 492.05), as well as ether extract (EE, no. 920.39). A nitrogen analyzer (Leco FP828 Nitrogen Analyzer; LECO) was employed to evaluate the CP content. The following procedure was used to compute NFC: According to Holtshausen [16]:

The neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents of the samples were determined by simmering them in NDF and ADF solutions. Additionally, the analysis was conducted in accordance with the methodology established by Van Soest et al [17], and α-amylase was added. The gross energy (GE) content of experimental diets was determined by employing an adiabatic calorimeter bomb (AC500, LECO). The procedure of Keawpila et al [18] was used to determine the estimated metabolizable energy (ME) content of experimental diets.

Hematological specimens were obtained from the vein located in the jugular on the final day of each phase at two specific time points: immediately before the morning meal and 4 hours after the beginning of nourishment. Concurrently with the collection of rumen fluid, hematological samples have been taken from the jugular vein., with an estimated volume of 10 mL. Each tube contained 12 mg of ethylenediaminetetraacetic acid to prevent coagulation. To separate the plasma, it was centrifuged at a force that was 500 times the acceleration due to gravity for 10 minutes. The material was thereafter maintained at −20°C until analysis of blood urea nitrogen (BUN) concentrations was conducted using the L-type Wako UN diagnostic device manufactured in Tokyo, Japan. The diacetyl monoxime method was subsequently employed to implement an automated colorimetric technique. Using a gastric tube connected to a vacuum compressor, ruminal fluid samples (100 mL) were collected on the final day of the feeding study, both before and four hours after feeding. The early part of the rumen fluid was eliminated to prevent saliva contamination. The pH of the ruminal fluid was swiftly measured using a HANNA Instruments HI 8424 microcomputer from Singapore. The fluid samples were segregated into two separate parts after the pH measurements. The initial 45 mL of fluid samples were transferred to a 60 mL plastic container and combined with 9 mL of 1 M H₂SO₄. This study aimed to ascertain the amounts of volatile fatty acids (VFA), namely acetic acids (C2), propionic acids (C3), and butyric acids (C4). The second sample of ruminal fluid, measuring one milliliter and containing nine milliliters of 10% formaldehyde in formalin, was designated for assessing bacterial and protozoal populations. The ruminal fluid samples were centrifuged for 15 minutes at a force of 16,000×g after collection. The supernatant, the liquid layer above the debris, was then tested to quantify the VFA present. VFA concentration was determined using gas chromatography (Nexis GC-2030; Shimadzu) with a flame ionization detector and a capillary column (DB-WAX 30 m, 0.25 mm, 0.25 μm; Agilent Technologies). The bacterial and protozoal populations were quantified using hemacytometers (Tiefe depth 0.1 mm and 0.0025 mm²; ISO LAB Laborgerate) and a microscope, under the technique outlined by Galyean et al [19]. The calculation of apparent digestibility and gross energy intake (GEI) was done according to the report of Suriyapha et al [5] following:

Where: GEI = gross energy intake, GE = gross energy, ME = metabolizable energy, MEI = metabolizable energy intake, MEI:GEI = energy efficiency (MEI to GEI ratio).

The SAS Proc GLM procedure was employed to evaluate the data as a 4×4 Latin square design, as indicated by the following equation [20]:

Where Y_ijk, is the observation from animal j, receiving diet i, in period k; μ is the overall mean, M_i is the effect of the level of WBTP (i = 1, 2, 3, 4), Aj is the effect of animal (j = 1, 2, 3, 4); P_k is the effect of the period (k = 1, 2, 3, 4); and ɛijk is the residual effect. To identify any significant differences among the treatments, Tukey’s Multiple Comparison Test was employed. Orthogonal polynomials were utilized to statistically compare the treatment trends.

Ruminal VFA concentrations and proportions, along with BUN levels, were analyzed using a repeated-measures mixed model that included baseline values (0 h pre-feeding) as covariates for the initial values at time 0 h before feeding using the Proc Mixed procedure of SAS [20] as shown in the model.

Where Y_ijkm is the observation for a specific variable, μ is the overall mean, P_i is the random effect of period, G_k is the random effect of cow, tm is the random effect of sampling time (0 h pre- and 4 h post-feeding), and T_j is the fixed effect of treatment. The residual random term is denoted by ɛ. The trend effect of WBTP substitution levels was investigated using orthogonal polynomials.

Table 2 illustrates the impact of substituting corn meal in the diet with WBTP on feed intake. Feed intake across all WBTP replacement levels showed no statistically significant differences (p>0.05), indicating the potential of WBTP as a viable substitute for corn meal without compromising intake.

The effects of substituting corn meal with WBTP on nutrient intake, energy utilization, and digestibility are presented in Table 3. The intake of OM, DM, EE, NDF, ADF, and NFE was not affected by replacing corn meal with WBTP (p>0.05). Substituting WBTP for corn meal did not significantly alter GEI, MEI, or their ratio (p>0.05). Furthermore, the digestibility of DM, OM, CP, EE, NDF, ADF, and NFE did not significantly differ between the various WBTP replacement levels (p>0.05).

The effects of substituting corn meal with WBTP on ruminal pH, BUN, and microbial population are presented in Table 4. Ruminal pH ranged from 6.73 to 7.04. BUN values, ranging from 5.50 to 8.75 mg/dL, fell within the typical range for tropical ruminants. In the present study, at 0 hours post-feeding, protozoal populations increased significantly with 100% WBTP substitution.

Table 5 presents the effects of replacing corn meal with WBTP on total VFA concentrations and VFA profiles in the rumen of Thai-native beef cattle. No significant differences were found in total VFA concentrations ranging from 84.56 to 101.87 mM (p>0.05). Similarly, the proportions of C2, C3, and C4 remained unchanged (p>0.05), despite the incorporation of WBTP into the concentrate as a replacement for corn meal.

The findings of this study suggest that the total DM intake (DMI) of Thai-native cattle fed a rice straw-based diet is within the typical range of 2.39% to 2.69% of BW [5,7]. Furthermore, replacing corn meal in the concentrate diet with WBTP did not negatively impact total DMI and nutrient intake compared to the control group. Pelleting, which involves heat treatment, can disrupt cellular structures, alter starch–protein interactions, and induce starch gelatinization, thereby enhancing the palatability of WBTP in terms of taste, odor, and aroma [6,21]. Additionally, the pelleting process, which improves nutrient availability in WBTP, may have contributed to the comparable digestibility observed between the 100% WBTP and control groups. Moreover, substituting corn meal with WBTP in the concentrate diet can balance feed utilization by improving the sensory qualities of the feed [21]. Building on the feed intake analysis, the following section explores the impact of WBTP substitution on nutrient utilization and energy balance. With feed intake remaining unaffected, the study further evaluates nutrient digestibility and energy utilization under WBTP-based diets.

Digestibility of DM, OM, CP, EE, NDF, and ADF was unaffected by WBTP substitution at levels up to 100%, potentially improving overall health and productivity. Starch gelatinization during processing increases the surface area for microbial activity, which may maintain digestibility at levels similar to corn meal [22]. The rate of carbohydrate fermentation is influenced by several factors, including monosaccharide type and arrangement, molecular size, and feed physical structure [23].

Replacing corn meal with WBTP sustained energy intake levels in Thai-native beef cattle, demonstrating its efficacy in meeting the energy requirements of ruminants without adverse effects. The recommended metabolizable energy for maintenance (MEm) in Thai-native cattle is 0.12 Mcal/kg BW^0.75/day. In this experiment, the MEI varied between 0.20 and 0.22 Mcal/kg BW^0.75/day, indicating a favorable energy balance sufficient to sustain growth. Replacing corn meal with WBTP maintained both GEI and MEI, likely due to the consistent DMI, which ensured sufficient overall energy intake for growth. In contrast, Unnawong et al [7] reported that feeding Thai-native beef cattle a concentrated diet containing modified WBTP increased energy intake, possibly due to the appetite-stimulating effects of modified starch. In the present study, the MEI/GEI efficiency (0.60–0.63) was consistent with values reported by Kongphitee et al [24], who observed an MEI/GEI ratio of 0.56–0.70 when using cassava pulp in the diets of Thai-native beef cattle. Therefore, the energy requirements of Thai-native beef cattle can be met by substituting WBTP for corn meal in the concentrate diet. While WBTP substitution showed no adverse effects on energy utilization, long-term studies are needed to assess potential impacts on growth performance and production efficiency.

The ruminal pH is within the ideal range for microbial activity (pH 6.20 to 7.00) [25]. This pH range is crucial for effective feed degradation and rumen health. Agarwal et al [26] also noted that ruminal microbes are most active within a pH range of 6.0 to 7.0, depending on rumen conditions. The variation in ruminal pH at 0 hours post-feeding may be due to inconsistent feeding behavior under ad libitum conditions [27,28]. Although ruminal pH fluctuations were within the optimal range, the slight variability observed immediately post-feeding suggests that WBTP may influence the buffering capacity of ruminal contents, possibly due to changes in starch composition and fermentation dynamics. On the other hand, the stable ruminal pH observed after four hours of feeding and throughout the day suggests that the rumen microbial population adapted to the diet over time [4,29]. Even at WBTP inclusion levels as high as 100%, ruminal fermentation and pH remained stable, demonstrating resilient ruminal metabolism and consistent microbial activity [25,26].

BUN concentrations in Thai-native cattle typically range from 6.30 to 25.50 mg/dL [5,7]. In this study, BUN levels were unaffected by WBTP substitution, likely due to the similar CP and nitrogen sources in the feed. Unnawong et al [7] also reported no significant changes in BUN and ruminal NH₃-N levels when using steamed WBT in ruminant feed, supporting the comparable BUN levels observed here.

Before feeding, rumen protozoal populations may fluctuate due to factors such as circadian rhythms, nutrient availability, and microbial interactions. Protozoa feed on bacteria and starch, and in the absence of fresh feed, they adjust their population based on the availability of fermentable substrates [19]. Moreover, Dennis [30] found that rumen protozoa populations increased with higher energy and nitrogen levels, such as those in diets containing pelleted mixtures. Pelleted feed enhances the fermentation environment, supporting protozoal growth. Galyean [19] suggested that the availability of glucose, a key nutrient for protozoa, may be influenced by the physical properties of pellets. However, protozoal populations did not change after four hours of feeding, likely indicating that the protozoal community had adapted to the diet during the 21-day feeding period.

The VFA profiles and total VFA concentrations remained stable four hours post-feeding, likely due to the similar nutrient composition of WBTP and corn meal [4], resulting in comparable ruminal fermentation dynamics [31]. The consistent VFA concentrations across treatments highlight the ability of WBTP to maintain stable fermentation patterns. Although not statistically significant, the slight increase in propionate at 66% WBTP substitution may suggest improved glucogenic potential, meriting further investigation. Srichompoo et al [4] also found that substituting WBTP for corn meal did not alter VFA profiles during in vitro fermentation after four hours of incubation. The heat produced during pelleting or steaming may have modified WBTP’s amylopectin, creating branched, gelatinized structures [7]. These changes produce denser starch granules, often referred to as resistant starch, which reduces starch solubility in the rumen [21] Unnawong et al [7] demonstrated that increasing the level of steamed WBT up to 30% in the diet resulted in similar ruminal VFA concentrations and profiles compared to the control group.