The experimental subjects comprised of 40 lambs, approximately 1 month old, with an average weight of 6.30 ± 0.10 kg. All animal experiments were carried out at the Baodi farm in Kalatale Town, Aksu City. The Experimental Animal Welfare Ethics Committee of Xinjiang Agricultural University approved the experiment (approval number: 2022016) to ensure compliance with all relevant ethical regulations for animal experiments and research.

The Glutathione assay kit, Malondialdehyde (MDA) assay kit, Catalase assay kit, Total Superoxide Dismutase assay kit, and Total Antioxidant Capacity (T-AOC) assay kit were all purchased from Nanjing Jiengcheng Biotechnology Co., Ltd. The Interleukin-2 (IL-2) test kit, Interleukin-4 (IL-4) test kit, Tumor Necrosis Factor-α (TNF-α) test kit, Immunoglobulin G (IgG) test kit, Immunoglobulin A (IgA) test kit, and Cu²⁺ colorimetric reagent is purchased from Shanghai Vanco Bio-Technology Co., Ltd. Other equipment used in the study included a Bio Tek Instruments (Icn), a Veterinary Blood Analyzer (ABAXIS Europe), a Centrifuge (TDL-40B) from Shanghai Anting Scientific Instrument Factory, and a Flame Atomic Absorption Spectrophotometer (SP-3520 AA) from Shanghai Yili Scientific Instrument Co., Ltd.

The RJP extraction involved weighing and immersing Ruoqiang jujube in anhydrous ethanol and petroleum ether for degreasing and drying. The dried sample was boiled and extracted, and the resulting solution is concentrated to a concentration of 1 g/mL. The concentrated liquid is then mixed with Sevag reagent (chloroform: n-butanol = 4:1) at a volume ratio of V_RJP: V_{Sevag reagent} = 2:5. The mixture was vigorously shaken, left to stand for 30 min, and then centrifuged for 10 min at 4000 rpm/min. The aqueous phase was collected through three repetitions. Anhydrous ethanol is then added to achieve an 80% ethanol concentration. The mixture was refrigerated at 4°C overnight, and the precipitate, washed and dried with anhydrous ethanol to obtain dry RJP material.

The preparation of RJP-Cu involved combining extracted RJP and sodium citrate in 300 mL of water. The mixture was heated in a water bath, stirred continuously and pH adjusted using a 20% NaOH solution. CuSO₄ was added slowly, stirring ceased when insoluble matter appeared, and the mixture was centrifuged at 4000 rpm for 1 min to obtain the supernatant. The liquid was collected, concentrated, cooled, and anhydrous ethanol was added. After refrigerating at 4°C overnight, the mixture was centrifuged at 4000 rpm for 1 min to precipitate. RJP-Cu was dissolved in water at a ratio of 1:10. A dialysis bag, soaked in flowing water for 24 h, concentrated the remaining liquid. After alcohol precipitation, drying, and refinement, RJP-Cu was obtained (Figure 1).

To assess the influence of sodium citrate content on Cu²⁺ concentration, the reaction time was set at 1 h, temperature at 60°C, pH at 9.0, and sodium citrate content at 0.0625 g, 0.125 g, 0.25 g, 0.5 g, and 1 g, respectively. To assess the influence of reaction temperature on Cu²⁺ concentration.

To assess the influence of reaction temperature on Cu²⁺ concentration, the reaction time fixed at 1 h, pH at 9.0, and sodium citrate content at 0.5 g, the reaction temperature was varied at 30°C, 40°C, 50°C, 60°C, and 70°C. To assess the influence of pH value on Cu²⁺ concentration, the reaction time was set at 1 h, temperature at 50°C, and sodium citrate content at 0.5 g, the pH value was adjusted to 7.0, 8.0, 9.0, 10, and 11, respectively. In each of the aforementioned experimental groups, the average value was determined based on three repetitions, and a significance analysis was conducted to identify the impact of different single factors on the RJP-Cu.

According to the results of univariate experimental, sodium citrate content (A), reaction temperature (B) and pH value (C) were selected as key variables. −1, 0 and +1 indicated low, medium and high levels respectively, and the response surface analysis of 3 factors and 3 levels was conducted. The design factor level is shown in (Table 1).

Based on the response surface experimental results, RJP-Cu was prepared under the following conditions: a reaction time of 1 h, a sodium citrate content of 2.5 g, a reaction temperature of 50°C, and a pH value of 8.0. RJP-Cu was completely dissolved in distilled water, and a 1000 Da molecular weight cutoff dialysis bag was employed for 2 days to concentrate the dialyzed RJP-Cu. RJP-Cu, with free impurities removed, was obtained after alcohol precipitation.

The polysaccharide content in RJP and RJP-Cu was determined using the phenol-sulfuric acid method (Yue F et al., 2022). The protein content in RJP and RJP-Cu was assessed using the BCA kit method (Rogatsky E, 2021). The glucuronic acid content in RJP and RJP-Cu was measured using the sulfuric acid-carbazole method (Maccari F and Volpi N, 2022). The Cu²⁺ in the samples was analyzed via flame atomic absorption spectrometry (Bakircioglu D et al., 2022).

RJP and RJP-Cu samples were prepared for SEM analysis to observe the shape and characteristics of the samples (Mobin M and Rizvi M, 2017). The infrared spectra (FT-IR) of RJP and RJP-Cu were measured using a Fourier transform infrared spectrometer. A small amount of each sample mixed with KBr particles, was ground into a powder, dried, and then pressed into thin slices. These slices were subsequently scanned and measured in the range of 400–4000 cm⁻¹ (Yi Y et al., 2020).

Forty lambs with similar organism weights were selected. After 14 days of adaptation to the feeding regimen, the lambs were divided in to 5 groups based on their organism weights, with 8 lambs in each group. These groups were as follows: the Cu sulfate group (CuSO₄, 4 mg/kg), the RJP-Cu high-dose group (RJP-Cu-H, 5.3 mg/kg), the RJP-Cu medium-dose group (RJP-Cu-M, 2.6 mg/kg), the RJP-Cu low-dose group (RJP-Cu-L, 1.3 mg/kg), and the blank control group, blank control group received no additional treatment (Figure 2).

After a total of 14 days of feeding, all groups were maintained on a basal diet for an additional 28 days. The experimental lambs were fed in groups and had free access to water. Feeding occurred once daily at 10:00 and 19:00, each feeding session lasting for 1.5 h. Any remaining feed was collected at 11:30 and 20:30 daily. The feeding mode, management practices, and environmental conditions were consistent for all groups throughout the duration of the experiment.

The feeding conditions for each group of lambs were meticulously recorded on a daily basis throughout the entire experiment. Additionally, the organism mass of each lamb was weighed once a week. Measurements for organism height, organism diagonal length, chest circumference, and tube circumference were taken at the beginning and end of the experiment. Various metrics such as average daily gain, average daily feed intake, feed-to-weight gain ratio, organism height, organism diagonal length, chest circumference, and tube circumference were calculated. Furthermore, blood samples were collected from the jugular vein of the lambs on the 28th day after feeding.

On the 28th day following the cessation of feeding, blood samples were gathered for the assessment of T-AOC, CAT, GSH-PX, MDA, and SOD. Additionally, on the 14th and 28th days after the termination of feeding, blood samples were obtained to measure the Cu²⁺ in the serum using a Cu²⁺ analysis kit and the levels of IgG, IgA, IL-2, IL-4, TNF-α were measured using a ELISA kits.

On the 28th day following the cessation of feeding all groups of lambs were administered a 15 mL normal saline rectal injection, and rectal feces were collected. The collected samples were preserved in liquid nitrogen tanks and transported to the laboratory for DNA extraction. DNA quantification was carried out using nanodrop, and following purification, PCR amplification and fluorescence quantification were performed. Subsequently, a sequencing library was prepared for computer-based sequencing. Nomi Biological Company conducted the analysis of intestinal microorganisms and indexes related to SCFAs.

SPSS 24.0 software is suitable for conducting single-factor experimental analysis to determine significant differences. Design Expert 10.0.7 software is employed for the statistical analysis of response surface experimental outcomes.

RJP and RJP-Cu exhibited prominent and extensive hydroxyl (-OH) stretching vibration absorption peaks, along with moderately strong (-CH₂-) C-H bond stretching vibration peaks in the range of 3400–3200 cm^-1 and 3000–2800 cm^-1, respectively. These peaks are characteristic of polysaccharide absorption. The similarity in their position, shape, and intensity indicates a shared carbon chain structure and monosaccharide composition. Upon Cu²⁺ chelation, RJP displayed a stretching vibration peak of -CH₂OH (3288.04–2930.58 cm^-1), while the C=O stretching vibration peak at 1619.94 cm^-1 disappeared. Furthermore, changes in C-O stretching (1407.06 cm^-1, 1041.22 cm^-1) and a shift in the C-O wave number (from 1407.06 cm^-1 to 1416.18 cm^-1) suggest a strong binding affinity of Cu²⁺ to the O in C-O, indicating a chemical interaction. The characteristic absorption peak at 1594.95 cm^-1 in RJP-Cu indicates the presence of SO₄ ^2-, implying that RJP simultaneously chelates SO₄ ^2- along with Cu²⁺, a form of physical chelation. These results affirm that Cu²⁺ forms both chemical and physical chelation bonds with RJP, resulting in the formation of RJP-Cu (Figure 5).

As indicated in Table 4, compared to the control group, there were no statistically significant differences in the counts of leukocytes, lymphocytes, monocytes, neutrophil granulocytes, red blood cells, hematocrit levels, or platelets between the RJP-Cu-H, M, and L groups (p > 0.05). This suggests a high level of safety. This indicates that all doses of RJP-Cu have a good safety profile.

The final weight, daily weight gain, and daily feed intake of lambs in the RJP-Cu-H, M, and L groups were significantly higher than the control group (p < 0.05), and all indicators were measured exhibiting a dose-dependent pattern. Furthermore, the feed-to-gain ratio in the RJP-Cu-H, M, and L groups was significantly lower than that in the control group (p < 0.05). This suggests that RJP-Cu effectively promotes lamb growth and improves feed utilization efficiency (Table 6).

There were no significant differences in the initial organism height, initial oblique organism length, initial chest circumference, and initial tube circumference were observed among all experimental groups (p > 0.05). However, the final organism height, final oblique organism length, final chest circumference, and final tube circumference in the RJP-Cu-H, M, and L groups were significantly higher than those in the control group (p < 0.05), and this increase was dose-dependent. These results suggest that RJP-Cu effectively promotes the growth and bone development of lambs (Table 7).

The serum Cu²⁺ levels in the CuSO₄ group, as well as the RJP-Cu-H, M, and L groups, were significantly higher than those in the control group on both day 14 and day 28 (p < 0.05). This indicates that the CuSO₄ group and the RJP-Cu-H, M, and L groups all demonstrated a notable and effective Cu²⁺ supplementation effect (Figure 6).

As indicated in Table 8, the T-AOC, CAT, and T-SOD levels in the RJP-Cu-H, M, and L groups were significantly higher compared to those in the control group (p < 0.05). This result suggests that RJP-Cu could effectively eliminate excess free radicals within the organism.

The serum IgA levels in lambs from the RJP-Cu-H, M, L groups, and the CuSO₄ group were significantly higher than those in the control group on both day 14 and day 28 (p < 0.05). Specifically, on day 14, the serum IgA levels in lambs from the RJP-Cu-H group were significantly higher than those in the CuSO₄ group (p < 0.05) (Figure 7A). Furthermore, it is evident that the serum IgG levels in lambs from the RJP-Cu-H, M, and CuSO₄ groups on day 14 were significantly higher compared to the control group (p < 0.05). On day 28, the serum IgG levels in lambs from the RJP-Cu-H group and the CuSO₄ group were significantly higher than those in the control group (p < 0.05). These results indicate that the RJP-Cu-H group effectively stimulates the production of IgA and IgG in lamb serum (Figure 7B).

The serum interleukin-2 (IL-2) levels in the RJP-Cu-H group and CuSO₄ group were significantly higher than those in the RJP-C-L group and control group on day 14(p < 0.05). On day 28, the serum IL-2 levels in the RJP-Cu-H, M, and L groups were significantly higher than those in the control group (p < 0.05) (Figure 8A). Additionally, IL-4 content in the RJP-Cu-H group was significantly higher than that in the RJP-Cu-M, L, CuSO₄, and control groups on day 14 (p < 0.05). On day 28, no significant differences were observed in IL-4 content among all groups (p > 0.05) (Figure 8B). TNF-α content in the CuSO₄ group was significantly higher than that in the RJP-Cu-M and L, and control groups on day 14 (p < 0.05), with no significant difference observed between the RJP-Cu-H group and the CuSO₄ group (p > 0.05). On day 28, the TNF-α content in all experimental groups was significantly higher than that in the control group (p < 0.05) (Figure 8C). These results indicate that the RJP-Cu-H group can enhance the secretion of IL-2, IL-4, and TNF-α in the serum of lambs.

The number of unique Operational Taxonomic Units (OTUs) for the control, RJP-Cu, and CuSO₄ groups were 1793, 2349, and 1845, respectively. There were 754 common intestinal flora species shared among the three groups, and 259 OTUs were common between the control and RJP-Cu groups, and 637 OTUs were common between the RJP-Cu and CuSO₄ groups. In summary, the RJP-Cu-H group exhibited an increased diversity of intestinal microflora species in lambs (Figure 9A). As shown in Figure 9B, the coverage for each group is greater than 0.995 and there is no significant difference among each groups (p > 0.05), Chao1, Pielou_e, Observed_species, Faith_pd, Simpson index and Shannon index in RJP-Cu group are significantly higher than those in the control group (p < 0.05). Both Principal Coordinate Analysis (PCoA) and Non-Metric Multidimensional Scaling (NMDS) maps revealed differences in the composition of flora among the control, RJP-Cu, and CuSO₄ groups (Figure 9C). The UPGMA cluster analysis diagram further illustrated that the control group exhibited distinct clustering, and there were noticeable differences in the composition of flora among the control, RJP-Cu, and CuSO₄ groups (Figure 9D).

The RJP-Cu group exhibited an increased abundance of Spirochaetes, Bacteroidetes, and Verrucomicrobia compared to the control group, and the abundance of beneficial bacteria Actinobacteria decreased. These results indicated that at phylum level RJP-Cu effectively enhanced the prevalence and quantity of beneficial bacteria and reducing the presence of harmful bacteria, contributing to a healthier composition of the intestinal flora (Figure 10A). At genus level the RJP-Cu group increased the abundance of beneficial bacteria Prevotella, Oscillospira, Ruminococcus, Coprococcus, and Akkermansia, and decreasing the abundance of the harmful bacteria Clostridium compare to the control and CuSO₄ groups (Figure 10B).

After confirming the regulatory impact of RJP-Cu on bacterial composition at both the phylum and genus levels, LDA EFfect Size (LEfSe) was used to further discern the specific effects of RJP-Cu on lamb intestinal bacteria. Substantial changes were observed in the predominant intestinal flora of the RJP-Cu, CuSO₄, and control group. Most of the beneficial bacteria in the RJP-Cu group have the potential to promote host intestinal health, while the control group retained a certain amounted of pathogenic bacteria. These findings highlight that RJP-Cu can increase the abundance of beneficial bacteria and reducing the prevalence of harmful bacteria, thereby promoting intestinal health (Figure 10C).

According to the log fold change (logFc) analysis result, compared to the control group, the RJP-Cu group demonstrated a significant increase in processes related to flavonoid biosynthesis (p < 0.05), clavulanic acid biosynthesis (p < 0.05), siterpenoid synthesis, and atrazine degradation (p < 0.05). Conversely, it showed a decrease in activities associated with cell apoptosis and lipopolysaccharide biosynthesis (Figure 10D).

SCFAs are vital for maintaining intestinal health. The isovaleric acid content in the RJP-Cu group was significantly higher compared to both the CuSO₄ and control groups (p < 0.05). Additionally, the levels of valerate, propionic acid, and isobutyric acid in the RJP-Cu-H group and the CuSO₄ group were significantly higher than those in the control group (p < 0.05). Moreover, the contents of valerate and isobutyric acid in the RJP-Cu group were higher than those in the CuSO₄ group, but not statistically significant (p > 0.05) (Table 9). These findings demonstrate that RJP-Cu can stimulate the synthesis of multiple SCFAs.