To prepare HPs, bee honeycombs were crushed, stirred, and boiled in distilled water at a ratio of 3:1 (water to honeycomb) for 30 min. After filtering the mixture through a 120-mesh gauze, the residue was subjected to two additional extraction cycles under the same conditions. Filtrates from all the extractions were pooled and concentrated under reduced pressure. Thereafter, the polysaccharides were precipitated by adding 95% ethanol for 6 h, followed by centrifugation at 4000 rpm for 10 min. Finally, the precipitate was collected and dried in an oven at 40°C to yield crude HPs.

Animal experiments were conducted at the Poultry Production Training Centre of Jiangsu Agri-Animal Husbandry Vocational College. Before the experiment, all equipment (chicken cages, waterers, and feeders) and the entire chicken house were thoroughly disinfected. After a 7-day post-disinfection period, the chicks were introduced into the facility. Notably, the temperature conditions during the experiment were as follows: days 1–3: maintained at 35°C; days 4–7: gradually reduced to 33°C; day 14: decreased to 27°C; day 21: reduced to 23°C and then stabilized at 20–21°C for the rest of the experiment. During the experimental period, the chickens had free access to feed and water. Environmental parameters were dynamically adjusted based on meteorological conditions to maintain optimal humidity (40–60%), lighting (16 h/8 h light/dark photoperiod), and ventilation rates (0.5–1.0 m/s). Health management procedures, including vaccination protocols, were as follows: Marek’s disease vaccine (CVI94 strain) administration on day 1, Newcastle disease vaccine (LaSota strain) on day 7, infectious bronchitis vaccine (H120 strain) on day 14, and infectious bursal disease vaccine (attenuated IBDV) on day 21. All immunizations were administered via intramuscular injection (0.2 mL per chick) under strict aseptic conditions. Disease monitoring was conducted daily based on clinical observations.

Male Cyan-Shank Partridge chicken chicks (n = 300; 1-day-old) with average body weight of 0.035 kg were randomly divided into five treatment groups with six replicates per group (10 chicks per replicate): Experimental Group 1 (HPs300): basal diet + 300 mg/kg HPs; Experimental Group 2 (HPs600): basal diet + 600 mg/kg HPs; Experimental Group 3 (HPs1200): basal diet + 1,200 mg/kg HPs; Negative control group (NC): basal diet alone; and Positive control group (PC): antibiotic group (basal diet + 250 mg/kg 99% chlortetracycline hydrochloride). Supplementary Table S1 shows the composition and nutritional levels of the basal diet. Notably, the experiment lasted for 60 days.

Fasting weights were measured in the morning in each replicate group at 1, 20, 40, and 60 days. Additionally, average daily gain (ADG) was calculated for each group. Feed intake and residual feed were accurately recorded to determine average daily feed intake (ADFI) and feed-to-gain ratio (F/G).

At 60 days of age, one chicken was randomly selected from each replicate, weighed, and slaughtered for performance assessment. Slaughter performance parameters examined included dressed weight, half-eviscerated weight with giblets, eviscerated weight, pectoral muscle weight, leg muscle weight, abdominal fat weight, dressed yield, percentage of half-eviscerated weight with giblets, percentage of eviscerated weight, pectoral muscle ratio, leg muscle ratio, and abdominal fat ratio. All measurements were conducted following the Chinese Agricultural Industry Standard Performance terminology and Measurements for Poultry(NY/T 823-2020).

Serum samples were collected from the chickens at the end of the experiment (60 days old) for various biochemical analyses. After fasting for 12 h with ad libitum access to water, blood samples were collected from one randomly selected chicken in each replicate (six chickens per group) via wing vein puncture (5 mL per chicken) and allowed to stand for 10min. Thereafter, the samples were centrifuged at 2500 rpm for 8 min to separate the serum, which was stored at -80°C for subsequent analysis.

Serum malonaldehyde (MDA) and glutathione peroxidase (GSH-Px) levels were measured using the TBA and colorimetric methods, respectively. Additionally, serum catalase (CAT) activity and total protein (TP) content were determined using the ammonium molybdate and Coomassie Brilliant Blue methods, respectively. Moreover, IgA, IgM, and IgG levels were determined via enzyme-linked immunosorbent assay (ELISA) using TECAN M200Pro kits (Nanjing Jiancheng Bioengineering Institute). Additionally, superoxide dismutase (SOD) and albumin (ALB) levels were measured using a Hitachi 3100 biochemical analyzer (Shanghai Coibo Biotechnology Co., Ltd).

Significant differences between groups were determined using one-way analysis of variance with SPSS 26.0, followed by Duncan’s multiple range post-hoc test. Additionally, we conducted orthogonal polynomial trend analysis for HPs dose-response relationships with SPSS. Results are expressed as mean ± standard error of the mean (SEM). Statistical significance was set at p < 0.05.

The growth performance indices ADG, ADFI, and F/G were calculated using the following formulae:

Table 1 shows the growth performance of Cyan-Shank Partridge chickens fed diets supplemented with HPs. Compared with the NC group, supplementation with HPs (HPs300, HPs600, and HPs1200) significantly increased body weight at 20 days and ADG from day 1 to 20 (p < 0.05). Even the HPs1200 group demonstrated a significant increase compared to the PC group. Additionally, ADFI was significantly higher in the HPs1200 group versus NC (p<0.05) during days 41-60, though no significant difference was observed compared to the PC group. The PC group did not exhibit significant differences in the growth performance indicators compared to the NC group in this study.

Trend analysis ( Table 2 ) of different HPs supplementation levels (NC, HPs300, HPs600, and HPs1200) across growth performance phenotypes revealed that only three indicators exhibited linear significant trends (p < 0.05): body weight at day 20, ADG from days 1-20, and ADFI during days 41-60. No significant trends were observed in other parameters or in quadratic/cubic analyses.

Table 3 shows the slaughter performance of Cyan-Shank Partridge chickens fed diets supplemented with HPs. Although there were no significant differences (p> 0.05) in most slaughter performance parameters between the groups, leg muscle ratio was significantly higher (p< 0.05) in HPs1200 and PC groups than in NC group ( Figure 1 ). Furthermore, trend analysis ( Table 4 ) revealed a significant linear relationship between leg muscle ratio and HPs supplementation dosage (p < 0.05). No significant difference was observed for all the slaughtering performance parameters between HPs groups and the PC group.

Table 5 shows the serum antioxidant indices of Cyan-Shank Partridge chickens fed diets supplemented with HPs. At 60 days of age, serum MDA levels were significantly lower in both HPs and PC groups compared to the NC group (p < 0.05, Figure 2 ). Although there were no significant differences (p> 0.05) in serum CAT and GSH-Px activities among the groups, serum SOD activity was significantly higher (p < 0.05, Figure 2 ) in groups HPs600 and HPs1200 than in NC group. There was also no significant for all the serum antioxidant indices between the HPs and PC groups.

Trend analysis of HPs dosage on serum antioxidant indices demonstrated a significant linear correlation exclusively with MDA levels (p < 0.05), whereas the linear association with SOD activity did not achieve statistical significance (p = 0.068), approaching borderline relevance ( Table 6 ).

Table 7 shows the serum immunological indices of chickens fed diets supplemented with HPs for 60 days. Although there were no significant differences (p> 0.05) in serum TP and ALB levels among the groups, serum IgA, IgM, and IgG levels were significantly higher (p< 0.05) in group HPs1200 than in NC group ( Figure 3 ). Notably, there were no significant differences (P> 0.05) in serum IgA, IgM, and IgG levels between groups HPs600 and HPs1200. Additionally, serum IgG level was significantly higher (p< 0.05) in group HPs1200 than in HPs300 and PC groups. However, no significant difference was observed between the NC and PC groups across all measured indices.

The trend analysis of HPs dosage and changes in immune indicators revealed that only IgM and IgG showed a significant linear relationship with HPs dosage. Although the linear relationship between IgA and dosage did not reach statistical significance, it approached a borderline p-value of 0.063. Notably, ALB demonstrated a significant quadratic relationship with dosage based on quadratic term testing ( Table 8 ).

Since the implementation of a nationwide ban on antibiotics use in feed production and restricted use in livestock farming in China in 2020, researchers have increasingly explored the use of polysaccharides as feed additives in broiler chickens to enhance disease resistance and improve production performance. For example, Astragalus mongholicus polysaccharides (Liu P et al., 2024), Abrus precatorius (licorice)polysaccharides (Liu Y, 2024), Medicago sativa (alfalfa) polysaccharides (Yang et al., 2017), and Taraxacum (dandelion) polysaccharides (Shan et al., 2023) have been added to feed to improve ADG, feed conversion rate, and slaughter indices. Additionally, Saccharomyces cerevisiae (yeast) polysaccharides (Wang et al., 2020) and Lentinus edodes (shiitake) polysaccharides (Mo et al., 2024) have been shown to improve ADG in broilers.

HPs, an animal-derived polysaccharide isolated from bee combs, have rarely been studied as a poultry feed additive. Our study showed that HPs supplementation in poultry feed significantly enhanced the growth rate of Cyan-Shank Partridge chickens during the early rearing phase (1–20 days) and notably increased ADFI in the later stages compared to the NC group. Even the HPs1200 group demonstrated superior ADG from days 1–20 compared to the positive control (PC) group. Trend analysis revealed significant linear correlations between HPs dosage and three indicators: body weight on day 20, ADG from days 1–20, and ADFI during days 41–60. Collectively, these findings further demonstrate a dose-dependent growth-promoting effect of HPs.

Additionally, high-dose HPs supplementation (HPs1200) and addition of antibiotics (PC) significantly increased (p< 0.05) leg muscle ratio compared with that in the NC group. And the trend analysis also revealed a significant linear relationship between leg muscle ratio and HPs supplementation dosage. Although not statistically significant, HPs supplementation tended to improve the other slaughter performance parameters. Collectively, these findings suggest that HPs supplementation in feed can effectively improve both growth and slaughter performance in Cyan-Shank Partridge chickens.

Oxidative stress is characterized by the excessive production of reactive oxygen species (ROS; superoxide anions, hydroxyl radicals, and hydrogen peroxide) and reactive nitrogen species (RNS; nitric oxide, nitrogen dioxide, and peroxynitrite), which can competitively deplete the reduced hydrogen required for cellular metabolism, leading to cellular metabolic damage (Sies et al., 2017). Under normal conditions, the balance between oxidation and antioxidant systems in animals maintains homeostasis. Disruption of this equilibrium may result in pathological outcomes such as infection, aging, and tumorigenesis (Poprac et al., 2017) indicators, such as CAT, GSH-Px, MDA, and SOD, are widely used to evaluate antioxidant capacity and overall health status in animals (Blokhina et al., 2003; Payne and Southern, 2005; Zhao and Shen, 2005). Previous studies have demonstrated the potential of polysaccharides to modulate the antioxidant system. For example, Chen et al. (2023) found that Atractylodes polysaccharide supplementation significantly reduced serum MDA levels and increased SOD and GSH-Px activities in Lingnan yellow chickens. Similarly, Yang et al. (2017) reported that Medicago sativa polysaccharide supplementation enhanced GSH-Px activity in 28-day-old broilers and SOD activity in 35-day-old broilers. Zhang et al. (2022) reported that Ziziphus jujuba Mill (jujube) polysaccharide supplementation improved CAT, SOD, GSH-Px, and total antioxidant capacity (T-AOC) activities and decreased serum MDA levels in Gallus gallus, thereby improving egg production. Similarly, Yin et al. (2018b) revealed that HPs significantly elevated serum SOD and CAT activities and reduced MDA levels in immunosuppressed mice.

In the present study, supplementation with HPs at three levels and the antibiotic-supplemented group both significantly decreased serum MDA levels in Cyan-Shank Partridge chickens compared to the NC group; furthermore, trend analysis revealed a significant linear dose-response relationship exclusively between HPs dosage and MDA levels (p < 0.05). Notably, serum SOD activity was significantly higher (p< 0.05) in groups HPs600 and HPs1200 than in NC group. Antioxidant capacity is mediated by enzymes like SOD, which neutralizes reactive oxygen species (ROS) by dismutating superoxide radicals into less harmful molecules (e.g., hydrogen peroxide and oxygen), thereby reducing oxidative stress and cellular damage. In this process, SOD acts as the “core guardian” of antioxidant defense, systematically blocking the generation of malondialdehyde (MDA)—a terminal product of lipid peroxidation—by eliminating its precursor, superoxide radicals. Consequently, we hypothesize that the addition of hydrophilic polymers (HPs) enhances SOD enzymatic activity through molecular interactions that stabilize its active conformation or promote substrate accessibility. This amplified SOD function would accelerate the clearance of oxidative indicators such as ROS and MDA, thereby progressively strengthening the organism’s antioxidant capacity through this dual-action mechanism. Although not statistically significant, other indices, such as GSH-Px and CAT activities, showed an increasing trend following HPs supplementation compared with those in the NC group. There was also no significant for all the serum antioxidant indices between the HPs and PC groups. Collectively, these findings suggest that HPs supplementation, similar to antibiotic supplementation, can enhance antioxidant capacity in Cyan-Shank Partridge chickens by mitigating oxidative stress, thereby promoting overall health.

TP is composed of ALB and immunoglobulin, both of which play multiple roles, including maintaining normal colloid osmotic pressure and acid-base balance in blood vessels, transporting various metabolites, and regulating the physiological functions of transported substances. Additionally, these proteins are closely associated with immune functions. Moreover, the three immunoglobulins in the serum, IgA, IgM, and IgG, are the primary antibodies involved in humoral immunity. Notably, serum IgA, IgM, and IgG concentrations reflect the level of humoral immune response in organisms (Wang et al., 2001). Previous studies have demonstrated the immunomodulatory effects of polysaccharides. Liu S et al. (2024) demonstrated that adding different doses of herbal compound polysaccharides to feed significantly increased serum IgG, IgA, and IgM levels in broilers. Similarly, Zhao (2021) found that supplementation with 0.5% Dioscorea polysaccharides significantly elevated serum IgA, IgM, and IgG levels in broiler chickens.

In the present study, there were no significant differences (p> 0.05) in serum TP and ALB levels among the groups. However, high-dose HPs supplementation (HPs1200) significantly increased (p< 0.05) serum IgA, IgM, and IgG levels compared with those in NC group. Notably, the IgG level was significantly higher (p < 0.05) in the HPs1200 group compared to the PC group. Trend analysis revealed that IgM and IgG exhibited significant linear relationships with HPs dosage, whereas ALB demonstrated a significant quadratic relationship with dosage based on quadratic term testing. Collectively, these findings suggest that high-dose HPs supplementation enhances B cell differentiation in plasma cells, thereby promoting immunoglobulin synthesis and secretion and improving immune responses in Cyan-Shank Partridge chickens.

Our study showed that HPs supplementation in poultry diet at 1,200 mg/kg significantly improved growth performance in Cyan-Shank Partridge chickens, as evidenced by enhanced body weight at 20 days of age, ADG during the early rearing phase (1–20 days), and ADFI during the late phase (41–60-day). Additionally, HPs supplementation significantly increased leg muscle ratios. Moreover, HPs supplementation reduced serum MDA levels and elevated SOD activity, indicating improved antioxidant capacity. Furthermore, high-dose HPs supplementation significantly upregulated serum immunoglobulin (IgA, IgM, and IgG) levels, indicating enhanced humoral immunity. Conclusively, these findings indicate that HPs supplementation at 1,200 mg/kg may improve growth performance, slaughter performance, antioxidant capacity, and immune function in Cyan-Shank Partridge chickens.