This study was conducted at the experimental base of the Ningxia Feed Engineering Technology Research Center and was approved by the Animal Ethics Committee of Ningxia University (approval No. NXU-H-2023-093).

The D. officinale powder used in this research was sourced from the dried stems of Huoshan D. officinale, cultivated in a traditional Chinese medicine base located in Taishang Village, Qingshui Town, Mentougou District, Beijing. The preparation process involved initially crushing the dried stem segments with a crusher, followed by sieving through a 40-mesh standard sieve to obtain finely powdered material for experimentation. This powder has been verified to conform to the quality standards outlined in the Pharmacopeia of the People’s Republic of China (2022 edition) for D. officinale.

Thirty-two healthy adult female poodles, aged 1–2 years with similar body weights (5.20 ± 0.26 kg), were randomly divided into four groups, each consisting of eight poodles. The groups were designated as follows: Group A (control group, 0% D. officinale), Group B (0.5% D. officinale), Group C (1% D. officinale), and Group D (2% D. officinale). The study was conducted over a 45-day period, comprising a 15-day pre-test phase and a 30-day main test phase.

The diet was formulated based on the Nutrition Requirements for Dogs and Cats (20) to fulfill the daily metabolic energy requirements of adult pet dogs, and the ingredients presented in Table 1.

The experimental poodles were individually housed in cages (1.25 m × 1.85 m). Prior to the trial, the kennels and cages underwent thorough cleaning and disinfection following standard protocols. The poodles were vaccinated and dewormed according to established procedures. The amount of food provided was calculated based on the daily maintenance metabolizable energy requirement (ME (kcal/d) = 140 × BW^0.75) for young adult test dogs, which ME and BW, respectively, represent metabolizable energy and body weight. The poodles were fed twice daily at 08:00 and 17:00, with free access to water. A comprehensive observational approach was employed to monitor the diet intake, water consumption, incidence of diarrhea, mortality and excretion throughout the poodles feeding trial.

Fecal samples were collected using the total feces collection method in the last 7 days of the experiment. Fresh fecal sample were cleaned of hair and debris, weighed and placed in sample bags. Subsequently, the samples were mixed with 10% sulfuric acid and stored at −20 °C. Upon completion of fecal sample collection, the 7 days samples were combined and homogenized, then desiccated to a consistent weight at 105 °C. The desiccated samples were pulverized through a 40-mesh sieve and stored in sample bags for nutrient analysis.

On the last day of the experiment, a blood sample was obtained from the anterior arm vein, Approximately 8 mL of blood into a vacuum blood collection tube without anticoagulants. The tube was then left at room temperature for about 3.5 h, before being centrifuged at 3,000 rpm for 10 min at 4 °C to isolate the serum. The resulting supernatant was then transferred to 1.5 mL centrifuge tubes and stored at −80 °C for subsequent analysis of serum biochemical and antioxidant parameters. Furthermore, blood samples were collected from four randomly selected poodles in each group, using blood collection tubes containing ethylene diamine tetraacetic acid (EDTA) as an anticoagulant. These samples were allowed to rest for 30 min at room temperature before undergoing the same processing procedure as the serum sample for blood metabolomics analysis.

Nutrient digestibility data, blood biochemical indices, and antioxidant parameters were initially organized using Excel 2019, followed by analysis through one-way analysis of variance (ANOVA) in SPSS version 24.0. Duncan’s multiple range test was employed for post-hoc comparisons, with differences deemed statistically significant at p < 0.05. The data are presented as mean ± standard deviation.

The correlation analysis between plasma metabolites and blood biochemical indices as well as antioxidant indices was conducted using Spearman correlation analysis.

As shown in Table 2, the inclusion of D. officinale in the diet did not lead to significant changes in the digestibility of dry matter, crude protein, and ether extract. However, it had a considerable impact on the digestibility of ash (from 64.90 to 71.39%), Ca (from 40.40 to 51.03%), and P (from 45.97 to 56.99%), although these differences were not significant (p > 0.05), it indicated that D. officinale did not interfere with the absorption of dietary nutrients in poodles.

As shown in Table 3, incorporation of D. officinale into the diet significantly increased the levels of TP and GLB in the blood (p < 0.05). The trends TP and GLB are consistent, following the addition of D. officinale, the levels of both components are significantly elevated compared to the control group (p < 0.05). The peak TP and GLB levels were noted at the 0.5% supplementation level, reaching 69.72 g/L and 34.67 g/L, respectively. No significant effects were observed for the other indicators (p > 0.05).

According to Table 4, the inclusion of D. officinale in the diet significantly influenced the activities of serum IgM, CAT, GSH-Px and T-AOC in poodles (p < 0.05). The highest IgM levels were recorded at 0.5 and 1% D. officinale supplementation, reaching 1.02 g/L and 1.04 g/L, respectively. The highest CAT levels were recorded at 0.5 and 1% supplementation, reaching 1.94 U/mL and 1.95 U/mL, respectively. However, a significant decreased in CAT content was observed with the addition of D. officinale at 2%. The peak GSH-Px activity was noted at 2% supplementation, achieving 1630.56 U/mL, while the highest T-AOC was found at 1% supplementation, reaching 7.12 U/mL. No significant effect on serum MDA content was detected (p > 0.05). These findings indicate that D. officinale enhances the immune system and antioxidant capacity in poodles.

Apparent nutrient digestibility directly reflects dietary utilization efficiency in canines. Contemporary pharmacological studies have revealed that Dendrobium has significant gastrointestinal regulatory functions, which can enhance gastric emptying capacity, improved intestinal peristalsis, and promoted digestive-excretory processes (21). Investigations using murine models further demonstrate that Dendrobium polysaccharides induce notable intestinal modifications: they stimulate the production of secretory immunoglobulin A (sIgA), increase the villus height/crypt depth ratios in the small intestine mucosa, and expand the surfaces available for nutrient absorption (22). However, this study did not find any significant affecte of D. officinale on the digestibility of nutrients in poodles, which may be attributed to species differences.

Serum biochemical indices in animal organisms serve as crucial biomarkers that reflect both physiological and metabolic status, while also providing insights into immune competence (23). Immunoglobulins, produced by plasma cells differentiated from B lymphocytes following antigen stimulation, generating as specific antibodies capable of targeted antigen binding. Notably, IgM the largest molecular weight antibody class, is predominantly synthesized and secreted by splenic and lymph node plasma cells (24). Structurally, total serum proteins include two principal fractions: albumin and globulin (25). Serum albumin plays a crucial role in maintaining osmotic pressure, ensuring the proper distribution of nutrients, and both organic and inorganic ions, while facilitating detoxification processes (26). Serum globulins are primarily composed of complement proteins and immunoglobulins, which facilitate complement activation, antigen binding, and immune regulation (27). Emerging pharmacological evidence demonstrates the immunomodulatory potential of Dendrobium phytoconstituents. Chen et al. (9) and Fan et al. (11) systematically validated that bioactive compounds derived from Dendrobium species significantly enhance immune responses through multiple pathways. Murine experimental models have revealed three distinct immunostimulatory mechanisms of Dendrobium: (1) activation of splenic lymphocyte differentiation/proliferation, (2) augmentation of peritoneal macrophage phagocytic activity, and (3) elevation of serum immunoglobulin levels to strengthen humoral immunity (16, 17). This immunoenhancement profile has been cross-species validated in avian models, with D. officinale extracts demonstrating comparable immune-boosting efficacy in poultry through analogous mechanisms (28).

The findings of this study revealed that dietary supplementation with D. officinale significantly elevated serum levels of IgM, total protein, and globulin in canines. These results further demonstrate that D. officinale enhances the host’s capacity to resist exogenous stimuli and pathogens, thereby improving immune function. This immunomodulatory effect may be attributed to the polysaccharides in D. officinale, which regulate immune responses through multi-target mechanisms, including promoting immune cell proliferation and differentiation, activating the complement system, and modulating cytokine release (15). Notably, high-dose Dendrobium supplementation (2%) exhibited immunosuppressive effects, indicating a non-linear positive correlation between Dendrobium concentration and immune competence, characterized by a distinct threshold characteristic. This phenomenon may be attributed to a dose-dependent biphasic regulatory mechanism. Previous studies have reported that certain phytochemical constituents (flavonoids and alkaloids) at elevated concentrations may induce oxidative stress or suppress immunocyte proliferation, consequently compromising immunoenhancing efficacy (29). In the present study, optimal immunomodulatory efficacy was observed at D. officinale supplementation levels of 0.5 to 1%.

The antioxidant system serves as a critical defense mechanism for organisms, enabling the scavenging of excess reactive oxygen species (ROS) and mitigating oxidative damage (30). CAT, a terminal oxidase, specifically catalyzes the decomposition of hydrogen peroxide (H₂O₂) into water and molecular oxygen, thereby reducing the cytotoxicity associated with H₂O₂ accumulation (31). As a pivotal antioxidant enzyme, GSH-Px primarily neutralizes hydrogen peroxide and lipid hydroperoxides through redox reactions (32). T-AOC quantitatively reflects the systemic balance between oxidative stress and antioxidant defenses within biological systems (33). MDA is a key byproduct of membrane lipid peroxidation, serving as a biomarker for evaluating free radical-mediated cellular injury, with its concentration positively correlating with the severity of oxidative damage (34). Research has demonstrated that polysaccharides derived from D. officinale exhibit antioxidant properties through the modulation of the antioxidant enzyme system (35). Qi et al. (36) revealed that Dendrobium candidum polysaccharides demonstrate protective effects against aortic oxidative stress in atherosclerotic mice by enhancing serum SOD and GSH-P_X, while concurrently reducing MDA concentrations. Liang et al. (13) found that both the juice of D. officinale and its polysaccharide components effectively elevated serum glutathione peroxidase activity and total antioxidant capacity in murine models, while also exhibiting anti-aging properties. This study demonstrated that incorporating D. officinale into dog food significantly enhances serum CAT and GSH-Px levels, as well as total antioxidant capacity, consistent with previous research findings. These observed effects are likely attributable to the bioactive constituents of D. officinale, particularly its rich profile of natural antioxidants including flavonoids and phenolic compounds (37). These phytochemicals have been extensively documented as potent antioxidants, effective free radical scavengers, and anti-aging agents through multiple mechanistic pathways (37, 38). Furthermore, accumulating evidence indicated that D. officinale polysaccharides upregulate the expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant gene (10, 12). This transcriptional activation subsequently enhances the expression of Glutamate-cysteine ligase catalytic subunit (GCLC) and Glutamate-cysteine ligase modifier subunit (GCLM), which are key enzymes in glutathione biosynthesis. The resultant elevation in antioxidant enzyme levels ultimately enhances systemic antioxidant capacity in biological systems (10, 12).

Metabolomics data are characterized by their high-dimensional and large-scale nature. By integrating univariate and multivariate statistical analyses and examining data from multiple perspectives based on its characteristics, differential metabolites can be accurately identified (39). This approach holds unique advantages and plays a crucial role in animal nutrition research (40). Wang (18). found that the polysaccharides in D. officinale can alleviate subacute alcoholic liver injury in mice by modulating glucose metabolism, amino acid metabolism, and lipid metabolism pathways. The present study revealed that D. officinale significantly affects the serum concentrations of ten metabolites in poodles, including Fumaric Acid, 8-isoprostane F2a, L-lactic acid, Prostaglandin E2, Thromboxane B2, 1,7-Dimethylxanthine, 5-Acetylamino-6-amino-3-methyluracil, 5-Hydroxyindole-3-Acetic Acid, Hydroxyphenylacetic acid, and Theophylline in plasma (p < 0.05). As intermediate metabolites or substrates, these compounds participate in various metabolic pathways, primarily enriched in arachidonic acid metabolism, caffeine metabolism, pyruvate metabolism, and amino acid metabolism.

The arachidonic acid metabolic pathway produces various eicosanoids that positively influence the body’s antioxidant capacity and immune response (41, 42). Prostaglandin E2, a member of the eicosanoid family, binds to the Prostaglandin E Receptor 2, thereby regulateing the antioxidant signaling pathway in macrophages. This interaction reduces free radical production and enhances the antioxidant capacity of macrophages, leading to anti-inflammatory effects (41, 42). Additionally, 8-iso Prostaglandin F2α, a prostaglandin derivative formed through free radical-catalyzed non-enzymatic peroxidation of arachidonic acid, exhibits a positive correlation with inflammatory factors levels in the body, serving as a marker for oxidative stress. Elevated levels of 8-iso Prostaglandin F2α indicate increased oxidative stress and a corresponding decline in immune function (43). In this study, the incorporation of D. officinale into poodle food led to a significant increase in plasma levels of Prostaglandin E2 and a notable decrease in 8-iso Prostaglandin F2α. Spearman correlation analysis further indicated that Prostaglandin E2 was positively correlated with antioxidant capacity, whereas 8-iso Prostaglandin F2α exhibited a negative correlation with the immune marker IgM. These findings suggest that D. officinale modulates arachidonic acid metabolism, reduces oxidative stress, and consequently enhances both antioxidant capacity and immune function. Liu et al. (44) employed metabolomics techniques to investigate the effects of D. officinale, demonstrating its capability to regulate arachidonic acid metabolism and restore lipid metabolic balance within the body.

The biotransformation of caffeine involves complex hepatic metabolic processes mediated by multiple enzyme systems, predominantly yielding various purine derivatives and uric acid metabolites (45). Specifically, 1,7-dimethylxanthine and theophylline as xanthine derivatives and end-products of purine metabolism, are ubiquitously distributed in human biological fluids (45). As the primary degradation product of caffeine through N-demethylation, theophylline undergoes further catabolism to 3-methylxanthine or 1-methylxanthine. This methylxanthine exhibits pharmacological activities including smooth muscle relaxation and bronchodilation, with emerging evidence supporting its anti-inflammatory and immunomodulatory properties (46). In the present study, dietary supplementation with D. officinale at concentrations of 0.5 to1% demonstrated no significant alteration in systemic theophylline levels. However, a 2% supplementation dose resulted in a marked reduction of serum theophylline concentrations in poodles (p < 0.05), suggesting potential adverse effects on methylxanthine homeostasis at higher dosage thresholds.

L-lactate, the principal metabolic product of the pyruvate pathway, serves as a critical energy shuttle in mammalian systems. In addition to its biosynthetic roles, it functions as a signaling molecule that regulates hormone release and the activity of various enzymes in the body (47). Studies have indicated that a moderate increase in circulating L-lactic acid levels can improve metabolic health (48). Tauffenberger et al. (49) demonstrated that L-lactate induces controlled generation of reactive oxygen species (ROS), which activates cellular defense systems, thereby providing protection against oxidative damage. The present study found that supplementation with D. officinale at a concentration of 1% significantly increased serum L-lactate levels in poodles, contributing to the enhancement of the body’s antioxidant capacity.

P-hydroxyphenylacetic acid, a principal metabolite in tyrosine catabolism, serves as the biosynthetic precursor for p-hydroxyphenylpyruvate. This intermediate undergoes enzymatic conversion via homogentisate to ultimately yield fumarate in the tricarboxylic acid (TCA) cycle (50). Evidence indicates that endogenous fumaric acid reduce the oxidative capacity of the body by binding to glutathione (51). Cheng et al. (52) elucidated that fumarate exerts immunomodulatory effects by directly inactivating the tyrosine-protein kinase LYN, subsequently suppressing B-cell activation and functional responses. In this study, D. officinale supplementation significantly reduced serum concentrations of both Phydroxyphenyl Acetic Acid and fumaric acid in poodles. Correlation analysis further revealed a negative correlation between fumaric acid and CAT levels in the body. This suggests that the observed enhancement in antioxidant capacity may be mechanistically linked to fumarate homeostasis modulation through Dendrobium intervention.