Effects of yeast and dried kratom leaves (Mitragyna speciosa [Korth] Havil.) supplementation on digestibility, rumen fermentation, blood metabolites and nitrogen balance in goats

Animal care

The study was conducted at the Prince of Songkla University Laboratory of Animal Nutrition and Experimental Farm. The Institutional Animal Care and Use Committee of Prince of Songkla University gave its approval and authorization to all protocols and procedures (approval code: AG012/2022).

Preparing dried kratom leaves and yeast

The green vein type fresh kratom leaves of M. speciosa Korth (Rubiaceae) were gathered in Tambon Namphu, Ban Na San District, Surat Thani Province, Thailand in October 2020, where the herbarium vouchers (PSU No. 012821) have been stored. The Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Songkhla, Thailand has verified the authenticity of the plant material. PSU No. 10/2563 was authorized by the Thai Ministry of Agriculture to solely use plant materials for research [9] and approved the method of keeping kratom leaves safe before feeding them to animals. Crabtree-negative yeasts, Candida tropicalis KKU20 (C. tropicalis KKU20; CBS 94^T (U45749) obtained from the Department of Animal Science, Faculty of Agriculture, Khon Kaen University (Khon Kaen, Thailand) and had a content of ≥1.15×10¹³ cfu/g) [10].

Animals, design, treatments, and management

Four male Thai Native×Anglo Nubian crossbred goats were obtained from Experimental Goat Farm of the Prince of Songkla University, in Hat Yai City, Songkhla Province, Thailand, 90110. These goats aged 8±1 months with BW of 20±0.13 kg were randomly assigned to a 4×4 Latin square experiment with a 2×2 factorial arrangement of treatments and assigned to the effects of 2 levels of yeast (Y) (Y, 0 and 0.5g/kg dry matter [DM]) and 2 levels of DKTL (DKTL, 0 and 4.44 g/kg DM). The animals were as follows: T1 = control group containing 0Y+0DKTL, T2 = 0Y+4.44DKTL, T3 = 0.5Y+0DKTL, and T4 = 0.5Y+4.44DKTL. The diets were created to achieve a daily gain of 100 g in accordance with NRC [11] requirements.

All goats were kept individually in a ventilated metabolism crate (0.115×0.95 m) in well-ventilated sheds where water and mineral salt were always available. These goats were fed twice a day at 0700 am and 1700 pm and had free access to water. Vaccinations and other preventative precautions were carried out before the trial’s start in accordance with the Institutional Animal Care and Use Committee of the Prince of Songkla University. The experiment was carried out over four 21-day intervals, with the first 14 days dedicated to adjusting to and measuring feed consumption. Samples of feed, urine, and excrement from the previous 7 days were collected for chemical analysis. All animals were given their own total mixed rations (TMR) consisting of 30% Pangola grass hay (as roughage sources) and 70% concentrated diet. The ingredients and nutritional composition of TMR and Pangola grass hay (PGH) are shown in Table 1.

Table 1

Ingredients and nutrition of total mixed ration (TMR) as the basic diet and pangola grass hay (PGH) fed to the goats

Feed and fecal sampling procedures

Daily records of the amount of feed provided and the number of samples were kept throughout the experiment. Using the entire collection method, samples of feces, feed, and refusals were taken from each individual goat at the conclusion of each period. Using AOAC [12] method, DM, ash, ether extract (EE), and crude protein (CP) were assessed. According to Van Soest et al [13] acid detergent fiber (ADF) and neutral detergent fiber (NDF) were identified. By employing the techniques outlined by Jamil et al [14], alkaloids were identified and extracted from the plant. The modified vanillin-HCL technique was used to assess the DKTL samples for CT and SP [15]. A measurement was made with an atomic absorption spectrophotometer. The chemical composition of the DKTL is shown in Table 2.

Table 2

Chemical composition and nutritive values of dried kratom leaves (DKTL) used in the experimental diets (on a dry matter basis) for goats

Urine sampling method

To keep the final pH below three and prevent nitrogen (N) extinction, whole urine was collected on the same days as feces in a plastic container treated with 10% H₂SO₄. Using the AOAC [16] total N measurement method, urine samples were collected at roughly 100 mL of total volume, frozen, and pooled at the conclusion of each session. The number of microbial purines absorbed (x mmol/d) corresponding to the purine derivatives excreted (Y mmol/d) was calculated according to Chen et al [17] as follows:

Where Y is the excretion of purine derivatives (mmol/d); x is the microbial purines absorbed (mmol/d); BW is the body weight. Microbial N supplied to the small intestine was calculated from microbial purine absorbed (x) according to the equation of Chen and Gomes [18]:

Rumen fluid sampling procedures

On the final day of the data collection period, rumen fluid samples were taken at 0- and 4-hours following feeding. Each time, rumen fluid was collected from the center of the rumen using a stomach tube connected to a vacuum pump. The pH and temperature of the rumen fluid were measured right away using a portable temperature and pH meter (HANNA Instruments HI 98153 microcomputer pH meter, Kallang Avenue, Singapore). The rumen fluid samples were then filtered through four layers of cheesecloth. To stop the microbial activity fermentation process, 45 mL of rumen fluid were collected and kept in a plastic bottle with 5 mL of sulfuric acid solution (1 M). The mixture was centrifuged for 15 minutes at 16,000×g (Table Top Centrifuge PLC-02, Enfield, CT, USA). The supernatant was analyzed for ammonia nitrogen (NH₃-N) using a Kjeltech Auto 1030 Analyzer (Foss, Hilleroed, Denmark), and volatile fatty acids (VFAs) were assessed using high-pressure liquid chromatography (HPLC; ETL Testing Laboratories, Inc., Cortland, NY, USA) [19]. According to the Moss et al [20] equation, ruminal CH₄ can be estimated using VFA proportions as follows: CH₄ production = 0.45 (acetate)× 0.275 (propionate)+0.4 (butyrate).

Another sample about 20 mL of ruminal fluid was collected to analyze for bacteria, protozoa, and zoospores and one mL was added to a formalin solution. In this procedure 1 mL of ruminal fluid from 20 mL was kept in 9 mL of 10% formaldehyde. Samples were kept chilled after dilution. For protozoa counts, a Sedgewick-Rafter chamber (S.I Scientific Supplies Co., Ltd., Bangkok, Thailand) was used with a cover slip. Finally, to find the average per square, 25 large squares were counted randomly and divided the number of protozoa counted by 25. For bacterial and fungi counts, a Petroff-Hausser chamber (Xinxiang Vic Science & Education Co., Ltd., Henan, China) was used [21] and to enumerate the bacteria, protozoa, and fungi according to Galyean’s [22] procedures using a microscope (Olympus BX51TRF, No. 2B04492, Olympus optical Co. Ltd., Tokyo, Japan).

Blood sampling analysis

Blood samples (about 10 mL) were taken from the jugular vein at 0 and 4 h post feeding in tubes containing 12 mg of ethylenediaminetetraacetic acid on the last day of the data collection period. The plasma was kept at −20°C until it was analyzed blood urea nitrogen (BUN).

Statistical analyses

All data were conducted using the general linear model (GLM) procedure of SAS (SAS Inst. Inc., Carry, NC, USA). The model used was: Model, Y_ijk = μ+A_i+P_j+Y_k+K_l+YK_kl+E_ijkl, where Y_ijkl = nutrient intake or rumen fermentation values; μ = overall mean; A_i = effect of animal; P_j = effect of period; Y_k = effect of level of Y; K_l = effect of level of DKTL; YK_kl = effect of interaction of level of Y and DKTL; E_ijkl = error of the term. Treatment means were statistically compared using Duncan’s multiple range test [23] to identify differences between means. Significant differences were declared if p<0.05.

All data were statistically analyzed in a 2×2 factorial arrangement in a 4×4 Latin square design by analysis of variance using GLM procedure of SAS (SAS Inst. Inc., USA). The results are presented as mean values and standard error of the means. The statistical model included supplementation of Y, supplementation of DKTL and interactions between Y and DKTL supplementation.

Chemical composition

The feed ingredients and chemical compositions composed of basal diet (TMR), PGH, and DKTL are summarized in Table 1 and 2. The TMR, PGH, and DKTL contained 162, 59, and 21 g/kg CP on DM basis, respectively. Additionally, DKTL consisted of 91.5% DM, 87.0% OM, 13.5% ash, 19.8% NDF and 16.8% ADF. Moreover, secondary plant metabolites, such as alkaloids, particularly mitragynine, total phenolics, total CT, total SP content, and total flavonoids content in DKTL were 41, 41, 83, 52, and 112 g/kg DM, respectively. Macro minerals were Ca, 8 g; P, 2 g; K, 15 g; Mg, 3 g; S, 13 g; and 0.1 g Na, respectively. These variations may be a result of different materials, growing locations, and plant factory processing. However, the chemical content of Kratom leaves may vary depending on factors such as the environment in which the plant growth, season, light intensity, and weather conditions, etc. [24,25].

Feed intake and nutrient digestibility in goats

There were no Y×DKTL interactions (p>0.05) with respect to feed intake, nutrient intake, apparent digestibility, digestible nutrient intake, and estimated energy intake (Tables 3, 4). However, the goats fed T4 (0.5Y+4.44DKTL) had the greatest total DMI. Candida tropicalis can improve fibrous material digestion, antioxidant function, and rumen microbial activity [26]. Furthermore, Candida tropicalis on biomass hydrolysate with ammonium sulfate as a nitrogen source [27] increased yield performance by boosting DM uptake [28]. Habeeb [29] found that yeast cell wall products increased the palatability of feed. 5′-nucleotide and glutamate can affect the amount eaten and the cell wall concentration was effective in stimulating microbial activity. Yeast fermentation with raw materials and then feeding to ruminants often results in increasing the fermentation efficiency in the rumen, digestion and number of microorganisms that allow animals to obtain more protein microorganisms [30]. Nutrients intake of organic matter (OM), CP, EE, NDF and ADF were not significantly different (p>0.05). Similarly, Chanjula et al [7] studied DKTL in goat and found no difference in dietary intake in terms of OMI, CPI, NDFI and ADFI. Dias et al [31] investigated the effect of yeast supplementation in low starch and high starch levels in the diet on rumen fermentation and digestibility in dairy cows. It was found that yeast supplementation affected total protein digestibility in cows fed the diet. In addition, García et al [32] studied the effects of yeast and monensin supplementation in sheep and found no effect on DM digestibility.

Table 3

Effects of feeding combinations of yeast and dried kratom leaves (Mitrangyna speciosa (Korth) Havi.) supplementation on feed intake and nutrient intake in goats

Table 4

Effects of feeding combinations of yeast (Y) and dried kratom leaves (Mitrangyna speciosa (Korth) Havi.) (DKTL) supplementation on apparent digestibility and digestible nutrient intake of nutrients in goats

The phytochemical or anti-nutritional chemicals such as mitragynine, CT, SP, and phenolic acids contained in the kratom leaves affected the total feed intake and digestibility. Kratom leaf supplementation increased by more than 4.44 g/h/d similar to the study of Sultana et al [33]; Su and Chen [34] reported that the CT and SP content was content in dried moringa leaves reduces nutrient digestibility. Sanguisorba officinalis supplementation 100 mg was found to decrease nutrient digestibility [35]. Mitragynine binds to many receptors such as opioid, serotonin and adrenergic receptors, etc., resulting in pharmacological properties such as reducing acid secretion, intestinal peristalsis, appetite and anti-inflammatory, etc. Especially high levels of DKTL supplementation may reduce intestinal peristalsis. However, the exact mechanism is still unknown which requires further detailed study.

Ruminal fermentation and blood urea nitrogen

The rumen fermentation is presented in Table 5. The temperature of the rumen did not differ between the groups (p>0.05). The ruminal pH parameters changed when supplemented with yeast; the value remained stable between 6.70 and 6.81. Yeast cells affected the ruminal pH compared to control. In all cases the pH values remained above 6.5, the physiological range of suitable for the microorganisms fermentation (cellulolytic bacteria) and protein digestion [36].

Table 5

Effects of feeding combinations of yeast (Y) and dried kratom leaves (Mitrangyna speciosa (Korth) Havil.) (DKTL) supplementation on ruminal fermentation in goats

Ruminal NH₃-N concentration was not affected by yeast and DKLT supplements, which is consistent with other studies [7]. Our findings demonstrated that DKTL feeding produced improved rumen fermentation. Targeted additions of CT and SP-containing feedstuffs may improve rumen efficiency by maintaining a higher pH and promoting microbial protein production [37]. Supplementation with DKTL led to reduced levels of NH₃-N and BUN when compared to the control group. The CT and SP in DKTL, which may form protein complexes can efficiently increase bypass protein.

DKTL was added to the diet, and this resulted in a decrease in BUN concentrations at 0 hours after feeding. To determine whether there was a connection between rumen NH₃-N and CP intake, blood BUN was also measured. All the crucial indications of rumen stability and function are the BUN levels [38]. BUN levels were a 16.28 to 20.57 mg/dL range. However, Anantasook et al [39] and Patra and Yu [40] found that CT produced a protein-tannin complex that limited the availability of ruminal breakdown dietary protein and decreased NH₃-N production, however, due to the high level of supplementation the results were inconsistent with the present study. Furthermore, tannin binding made more protein unavailable for bacterial conversion to NH₃-N. There is a lot of proof that DKTL affects the rumen’s microbial fermentation [41–43]. Moreover, reducing NH₃-N levels in the rumen resulted in decreased BUN values.

Volatile fatty acid and methane production

The VFA profile and methane production are shown in Table 6. The total VFA showed no interaction between the DKTL level or yeast (p>0.05). Total VFA increased at 4h after feeding when yeast was supplemented (p<0.05). The concentration of C2 decreased upon an interaction in the DKTL and yeast, due to the increased ratio of C3 synthesis. Furthermore, concentration of C3 increased when goat was fed DKTL and yeast (p<0.05). The mechanisms producing a higher VFAs concentration in yeast-fed animals appeared to be associated with an increased activity of the anaerobic microflora. By increasing rumen propionic, reducing protozoa, and consequently lowering methane production, feed containing CT and SP significantly improved rumen fermentation [44,45]. The anticipated shift from CH₄ to H₂, which is advantageous for the host’s energy supply, caused the VFA profile to go from C2 to C3 [46].

Table 6

Effects of feeding combinations of yeast (Y) and dried kratom leaves (Mitrangyna speciosa (Korth) Havil.) (DKTL) supplementation on volatile fatty acid profiles in goats

Methane production at 0 and 4 h after feeding with the Y level of 0.5 and DKTL 4.44 g/kg DM decreased when compared to other groups. The average was between 20.33–29.42 and 21.63–29.06 g/d. Rumen CH₄ production was strongly related to microbial fermentation. Tannin can reduce CH₄ emission by affecting rumen H+ exchange capacity and can also affect all the end fermentation characteristics. Reduction in CH₄ can be achieved indirectly by decreasing protozoal abundance [47]. Moreover, CH₄ production was consequently decreased by 0.5Y+4.44DKTL supplementation. This could be the result of the suppression of rumen protozoal number and methanogens. Similarly, in a study by Phesatcha et al [46], it was discovered that supplementing with Mitragyna speciosa Korth leaf pellets can influence ruminal fermentation by reducing C2 concentration and mitigating CH₄ production, consequently leading to an increase in C3 concentration.

N metabolism and utilization

There were no interaction effects on N balance and microbial protein between Y and DKTL fed to the animals (p>0.05), except N efficiency and urine litres per day (p<0.05) (Table 7). The N efficiency was the highest (59.29%) when feeding DKTL at 4.44% without Y (Figure 1). Supplementation Y and with various DKTL levels affected the percentage of N intake fecal and N intake absorbed. Increased levels of Y supplementation at 0.5% with high levels of DKTL at 4.44% in goat diet decreased the fecal N intake to 23.07% and increased the absorbed N intake to 76.93%. Y and DKTL showed an interaction on N. The most important metric for assessing ruminants’ protein nutrient status is efficiency. Similarly, Viennasay et al [48] observed that increasing the digestibility of CP led to a rise in retained nitrogen. Increased CP and CT consumption increased protein transport from the rumen to the small intestine [49]. Current study found that the nitrogen efficiency was positive in accordance with the total feed intake, probably due to the amount of digestibility and the nutrients intake.

Table 7

Effects of feeding combinations of yeast (Y) and dried kratom leaves (Mitrangyna speciosa (Korth) Havi.) (DKTL) supplementation on N balance and microbial protein in goats

Figure 1

Effects of feeding combinations of Yeast (Y) and dried kratom leaves (Mitrangyna speciosa (Korth) Havi.) (DKTL) supplementation on N efficiency. ^a,b Means with different superscripts are significantly different.

Ruminal microorganism population

Feeding Y with DKTL changed purine derivative (allatoin, purine derivative [PD] excretion and PD absorption) and microbial protein synthesis which was higher with Y supplementation at 0.5% of Y (p<0.05). The bacterial population was between (1.53 – 2.11×10⁹ cell/mL) and the fungus (0.96 – 1.37×10⁶ cell/mL), respectively (Table 8).

Table 8

Effects of feeding combinations of yeast (Y) and dried kratom leaves (Mitrangyna speciosa (Korth) Havi.) (DKTL) supplementation on the microorganism count in the rumen fluid of goats

Goats fed Y with DKTL also showed no effect on bacterial and fungal populations at various feeding times and mean values. However, the protozoal population decreased when feeding DKTL to animals after 0 and 4 h morning post feeding. The reduction in protozoa populations caused by DKTL supplementation (p<0.01) may be explained by the CT and SP in the DKTL. CT and SP binds portions of the cell membrane of protozoa and methanogens [50]. It has a considerable impact on methanogenic archaea in the rumen [46]. It has the capacity to prevent the transfer of hydrogen while also limiting the development of methanogens.

With high concentrate-based diets, one of the most frequently observed effects of live yeast supplementation is an increase in the number and activity of the bacterial population in the rumen, which appears to improve the rumen’s capacity to metabolize lactic acid and control ruminal pH [51]. To increase production levels in domesticated ruminants, diets rich in cereal grains are frequently offered.