This study was approved by the Animal Care and Use Committee of Rajamangala University of Technology Isan, Thailand (approval no. 01-66-006). According to Thailand’s National Research Council’s Ethics of Animal Experimentation, approval was required to collect rumen fluid from animals for this study’s main objective, which comprised laboratory examination of ruminant feeds.

Fresh Azolla (Azolla microphylla) was collected at 15 days of age and fresh turmeric rhizomes were harvested from Roi-et Province, Thailand. Fresh Azolla and turmeric were dried at 60°C, and then ground to pass through a 1-mm screen using a Cyclotech Mill (Hoganas, Sweden). The dietary treatments followed a 2 × 4 × 2 Factorial arrangement in a completely randomized design (CRD), with three replicates per treatment including blank triplicates (medium only). The first factor was two ratios of R:C at 60:40 and 40:60, the second factor was four supplementation levels of Azolla (Azolla microphylla) powder (AMP) at 0%, 3%, 6%, and 9% dry matter (DM) substrate, and the third factor was two supplementation levels of turmeric (Curcuma longa) powder (TUP) at 0 and 2% DM substrate. Details of the nutritive values of concentrate, roughage source, AMP, and TUP of the experiment are shown in Table 1. Roughage, concentrate, AMP, and TUP were dried at 60°C and passed through a screen (1 mm) to determine the chemical analysis of DM, organic matter, and CP using standard methods [15]. Following Van Soest et al [16], measurements were taken to determine the fiber content, especially neutral detergent fiber (NDF) and acid detergent fiber (ADF). Using the Folin-Ciocalteu reagent and measuring absorbance at 765 nm, the TUP was evaluated for total phenolic compounds [17], total flavonoid compounds [18], and tannin content [19].

This experiment utilized rumen fluid as a source of fermentation from two Thai native beef cattle (2 years old) with an average live weight of 250±10 kg. Before the morning feeding, a combined volume of approximately 1,000 mL of rumen fluid was obtained from each animal. All the beef cattle were fed with rice straw ad libitum and concentrate mixture at 0.5% of body weight at 14% CP and 75% TDN. The in vitro fermentation technique followed Menke et al [20]. A suction pump was used to collect 1,000 mL of rumen fluid from each specimen. A feed sample from the experiment was weighed and put into 50 mL bottles of the total mixed substrate (200 mg). Before mixing with substrates in the treatments, the rumen fluid was mixed and maintained in an anaerobic condition. Bottles with the mixtures of substrate treatment were subjected to CO₂ flushing and pre-warmed in a water bath at 39°C. The bottles were sealed with a rubber and aluminum cap and placed in an incubator at 39°C. The rumen-fluid mixture was added (40 mL) to the bottles and incubated at 39°C, as described in detail by Blümmel and Orskov [21].

During incubation, gas production of all the treatment samples was recorded at 1, 2, 4, 6, 8, 12, 24, 48, 72, and 96 h. The cumulative gas produced during fermentation was fitted to the model of Ørskov and McDonald [22]. After inoculation, the pH of the ruminal fluid was determined at 4, 8, and 12 h, with gas production recorded at each time point. The rumen fluid samples were then divided into two parts. The first portion of 20 mL rumen fluid was collected in a plastic bottle, added with 2 mL of 1 mol L⁻¹ sulfuric acid (H₂SO₄) and centrifuged at 16,000×g for 15 min. The supernatant was removed and stored at −20°C for later NH₃-N analysis using the micro-Kjeldahl method [15] and volatile fatty acid (VFA) analysis according to Samuel et al [23]. The second portion was subjected to a total direct counting approach of the protozoal population [24]. After incubation, the in vitro DM degradability (g/kg) was determined at 12 and 24 h according to Van Soest and Robertson [25]. CH₄ production was measured using a gas chromatograph (GC) machine (GC2014; Shimadzu Co., Ltd., Kyoto, Japan); a volume of 0.3 mL of the gas was kept in a 10 mL bottle, followed by injecting the gas using a GC. Based on the equation: methane production = (peak area/the slope of the standard methane graph)/gas volume (mL).

All experimental data were analyzed by the general linear model by using the procedure of SAS software [26] as a 2×4×2 factorial arrangement in a CRD. The statistical model included R:C ratio, AMP levels, TUP levels, R:C ratio×AMP level interactions, R:C ratio×TUP level interactions, AMP level×TUP level interactions, and R:C ratio×AMP level×TUP level interactions. Differences among statistical treatment parameters were taken as significant at p<0.05 and p<0.01.

Concentrate, rice straw, AMP, and TUP chemical compositions are listed in Table 1. The crude protein of the concentrate and rice straw were 14.2 and 2.2%. The AMP had 5.6% DM, 7.2% ash, 23.6% CP, 64.4% NDF, 54.4% ADF, 2.0% total phenolics, 3.6% total flavonoids, and 1.8% tannins. Moreover, TUP contained 88.3% DM, 6.4% ash, 4.5% CP, 40.4% NDF, 26.1% ADF, 8.3% total phenolics, 18.0% total flavonoids, and 16.6% tannins, respectively. In the other hands, analyzed the chemical composition of CP in Azolla microphylla and found it to be at a concentration of 31.3% [7]. However, Hasan and Chakrabarti [4] stated that the CP content of Azolla was typically between 19.0% and 30.0% under circumstances for optimal growth.

The gas production kinetics and in vitro true DM degradability are presented in Table 2. Gas production kinetics included gas production from the insoluble fraction (b), gas production rate constant for the insoluble fraction (c), the potential extent of gas production (a + b), and cumulative gas production at 96 h were affected by R:C and numerically increased with AMP and TUP supplementation (p<0.05) but no interactions were observed. Gas production kinetics increased with increasing ratios of concentrate and AMP supplementation, whereas TUP supplementation reduced gas production. In vitro DM degradability at both 12 and 24 h after incubation remarkably increased (p<0.05) with R:C ratio, AMP, and TUP supplementation, with the lowest found in the non-supplementation group. However, no interaction was found (p>0.05).

The degradabilities improved with an increase in the ratio of concentrate and increased with the addition of AMP supplementation. The fermentation process that occurs with the feed substrates can have a positive effect on the total amount of rumen gas produced. A substantial relationship has been found between the variety of roughages utilized and the concentration of carbohydrates present in feeds and rations, with the potential to greatly influence the production of gases. Previous research by Viennasay et al [27] found that feeds and plants containing phytonutrients such as CT and saponins had antibacterial capabilities against a variety of microorganisms including rumen bacteria, protozoa, and fungi. Nevertheless, Phesatcha et al [28] stated that gas production kinetics were improved by Mitragyna leaf powder, possibly because it enhanced the microbial population (particularly bacteria population) and increased substrate breakdown, thereby enhancing gas production kinetics. However, feed digestibility was unaffected by or enhanced by the addition of herbal extracts containing tannins and saponins. Similarly, no significant difference (p>0.05) was recorded in the digestibility of nutrients with the addition of 10 and 20 g/kg turmeric DM [29].

Results in Table 3 show the impacts of factors on pH, ammonia-nitrogen concentration, and protozoal population. The ruminal pH was not affected (p>0.05) by R:C, AMP, and TUP supplementation, while the rumen ammonia-nitrogen concentration reduced (p<0.05) with increasing levels of R:C, AMP, and TUP supplementation. No interaction was found between R:C, AMP, and TUP while the protozoal population decreased (p<0.05) with R:C ratio, AMP, and TUP supplementation but with no interactive results. Our results showed that the pH of the rumen was within the typical range of 6.6 to 6.8 which is considered normal for the ecology of the rumen as well as for the effectiveness of the fermentation process. Similarly, Van Soest [30] concluded that the most suitable pH range for microbial activity was between 6.2 and 7.2. AMP and TUP supplementation decreased the ammonia-nitrogen concentration. The protein-CT binding interaction may be responsible for the drop in ruminal NH₃-N concentration observed when supplemented with TUP. Supplementation with Azolla improved the fermentation process in the rumen by supplying the microorganisms with more nutrients, which increased their capacity to degrade feed. Ammonia production from feed protein breakdown due to reduced deamination caused the rumen pH to drop significantly after adding 1% DM curcuma powder [31], while AMP and TUP supplementation decreased NH₃-N content. The tannin-protein complex played a role in the proteolysis process, which inhibited the breakdown of proteins and reduced the concentration of NH₃-N. Similarly, Wanapat et al [32] and Patra and Saxena [33] who demonstrated that CT possessed dietary benefits from the production of a tannin-protein complex which reduced the availability of ruminal breakdown feed protein, and the production of NH₃-N. The relationship between protein and tannin in temulawak showed potential for decreasing NH₃-N concentration [34]. According to Sulistyowati et al [35] suggested that the active compounds found in curcuma, which have been shown to enhance the reduction of ammonia, contributed to a decrease in the number of deaminator bacteria. The reduction in ammonia nitrogen was attributed to inhibition of substrate protein degradation by microorganisms. The decreasing effect that turmeric has on ammonia nitrogen could be advantageous in terms of enhancing the use of dietary proteins in the rumen. Furthermore, the protozoal population also greatly decreased. Methanogens that are adherent on the surface of protozoa are negatively impacted by the presence of tannins and flavonoids, and this has an adverse effect on the population of protozoa. A correlation was found between the phytochemicals in turmeric, which have been associated with antibacterial properties, and the reduced effect that turmeric had on the population of microorganisms. Furthermore, Tyagi et al [14] reported that curcumin possessed powerful antibacterial effects against both gram-positive and gram-negative bacteria, while tannins, flavonoids, saponins, and oxalates have all been shown to have antibacterial properties [36,37]. Hence, the observed fermentation and microbial population decreases were directly associated with the effect of curcumin and other plant secondary metabolites contained in turmeric. The secondary metabolites found in plants possess a wide variety of antibacterial actions. These include disruption of the cell membrane, inhibition of enzymes, deprivation of substrates, and prevention of bacterial colonization [38]. Similarly, Phesatcha et al [28] showed that supplementation of Mitragyna leaves resulted in a decrease in the population of ruminal methanogens as well as the production of methane. Protozoa host methanogen archaea and also feed on the bacteria responsible for feed fermentation. Reducing protozoa can therefore improve the digestibility of DM, organic matter, and VFA, which are then turned into energy in the livers of ruminants.

Table 4 presents the details of total VFAs (C₂, C₃, C₄, and the C₂:C₃ ratio) and rumen CH₄ production. The R:C ratio increased with the addition of AMP and TUP, and the levels of propionate (C₃) and total VFA also increased (p<0.05), while acetic acid (C₂) and the ratio of C₂ to C₃ decreased (p<0.05). The addition of TUP resulted in a decrease in the production of VFA, while methane production was reduced by increasing the R:C ratio and TUP supplementation (p<0.05) at all times of incubation. However, no interaction was found between the R:C ratio, AMP, and TUP regarding VFA or methane production. In this study, supplementation with AMP and TUP resulted in a significant improvement in the production of propionate, as well as total VFA production. Accordingly, Ahmed et al [39] reported that the utilization of plant-based bioactive supplements resulted in a significant enhancement of rumen fermentation, particularly regarding the production of total VFA and propionate under in vitro conditions. The microbes used the hydrogen created during rumen fermentation to increase propionate production, which in turn improved total VFA production [3]. For ruminants, VFA are the primary source of energy for minerals and the liver is responsible for converting VFA into glucose [40]. When the value of VFA is high, this suggests that the process of turning feed into energy in the rumen continues to function correctly. Accordingly, Sulistyowati et al [35] reported that the most effective utilization of VFAs occurred when the dose of curcuma powder was 0.25% of DM. This resulted in a reduction in protozoa that ingest fiber-digesting bacteria and an increase in VFA values. The availability of phytonutrients including flavonoids and tannins also contributed to the decrease in CH₄ production induced by AMP and TUP supplementation. Specifically, as the supplemental level increased, CH₄ production decreased. Furthermore, Jayanegara et al [41] reported that tannins in proportions of more than 5% are not advised because they suppress the digestibility and performance of livestock. Including a tannin level of 2% to 5% in the diet had the most effective impact in decreasing methane gas production. By contrast, Al-Hadeethi et al [29] reported that in vitro studies had no significant impact on rumen methane production when turmeric was added at 10, 20, and 30 g/kg DM in the diet. Essentially, accordingly to Hristov et al [42] reported that the inclusion of plant-based feeds containing phytonutrients showed the potential to decrease the protozoal population and methane production, while simultaneously enhancing the concentration of VFAs in the rumen. In addition, Beyzi [43] stated that essential oils from plants effect the ecology of the rumen by influencing the protozoa and bacterial cell membranes, as well as reducing hydrogen content and inhibiting methanogenesis. Thus, the essential oils present in turmeric powder may contribute to the decrease in methane production.