The key field scientific observation and test station of Changbai Mountain Wildlife Resources, owned by the Ministry of Agriculture, was chosen as the feeding trial site. Fifty healthy male raccoon dogs of about 120 ± 5 days old were randomly selected and divided into five groups (group PRA0, PRA1, PRA2, PRA4, PRA8). Racoon dog litters were split up between groups to eliminate the influence of genetic factors. There were 10 replicates in each group and one in each replicate. There was no significant difference in weight among the groups (P > 0.05).

All animals were weighed prior to the experiment and randomly distributed to five experimental groups employing the completely randomized design (CRD), and subjected to diets with PRA extract levels of 0 g/kg (control group, PRA0), 1 g/kg (PRA1), 2 g/kg (PRA2), 4 g/kg (PRA4), and 8 g/kg (PRA8). According to the optimal concentration of PRA extract to alleviate various diseases in rats in previous studies, and the body weight and the daily feed intake of raccoon dog in this study, the conversion factor of mg/kg to mg/m², the appropriate addition level converted into diet might be 1.0–3.2 g/kg. Therefore, the doses of 1, 2, and 4 g/kg were selected. In order to observe whether the excessive dose has a negative impact on raccoon dogs, a dose of 8 g/kg was also selected. The PRA extract was purchased from Nanjing Dasf biotechnology co., Ltd. The PRA extract was determined by HPLC, according to the peak area ratio of sample and standard substance, the content of paeoniflorin in PRA extract was 11.86%, and the content of albiflorin was 3.92%. Referring to NRC (1982) (11) and related literature (12, 13) about the nutrient requirements of raccoon dogs during the breeding period, the experimental diets were prepared with extruded corn, soybean cypress, extruded soybean, rice bran cypress, DDGS, fish meal, meat and bone meal, chicken meal, hemoglobin powder, glucose, and soybean oil as raw materials. The composition and nutrition level of the diets were shown in Table 1. The pre-feeding period lasted for 7 days and the trial lasted for 40 days.

Before the study, the raccoon dogs were immunized routinely. In the study, raccoon dogs were raised in a single cage. They were fed by specially assigned personnel once at 07:30 and 14:30 every day. They had free access to food and water. The breeding houses were kept clean and dry. The health status of raccoon dogs was observed and recorded every day.

The digestion and metabolism trails were carried out from September 19, 2020, to September 21, 2020, for 3 days. Using the total fecal collection method, the feces collected in 3 days were mixed evenly, sprayed with a small amount of 10% sulfuric acid solution, dried at 65°C to constant weight, ground through a 40-mesh sieve, and preserved. Before urine collection, 10% sulfuric acid 20 mL was added to the collection bucket to fix nitrogen. The 3-day urine was evenly mixed and filtered and stored at −20°C.

On the 36th day after the beginning of the feeding experiment, each group of raccoon dogs had blood taken in the morning (before feeding). Blood samples were collected with a 5 ml procoagulant tube. The samples were quickly transferred to the laboratory, and centrifuged for 10 min by 3,500 r/min. The separated serum was packed in a 1.5 mL Eppendorf tube and stored at −80°C. Raccoon dogs were euthanized and the small intestinal tissue was collected and stored in liquid nitrogen immediately, then transferred to −80°C for further testing.

After the beginning of the trial period, weight on the first day was recorded as the initial weight, and weight at the end of the experiment was recorded as the final weight, the average daily gain (ADG) of each raccoon dog was calculated. The daily given feed and leftover feed of each raccoon dog was recorded so that the average daily feed intake (ADFI) and feed-gain weight ratio (Feed/Gain, F/G) were calculated. The content of dry matter, crude protein, crude fat, and crude ash in basic diet was measured, along with feces (14). Additionally, the protein content in urine was measured.

The calculation method was as follows:

Serum lactate dehydrogenase (LDH), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), and Alkaline phosphatase (ALP) were determined by automatic biochemical analyzer Selectra E (the Netherlands). Serum superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity, malondialdehyde (MDA) content, IgA, IgG, and IgM were determined by Nanjing Jiancheng kit. Serum D-Lactate (D-lac), Diamine oxidase (DAO), jejunal mucosal interleukin-18 (IL-18), and Tumor necrosis factorα (TNF-α) were detected by double-antibody sandwich enzyme-linked immunosorbent assay (microplate reader, BioTek, Vermont, USA).

The small intestine tissue soaked in neutral formaldehyde was washed with running water for more than 2 h and dehydrated with gradient alcohol. The fixed tissue was embedded in paraffin and sectioned into 5-μm thickness. For hematoxylin and ecosin (H&E) staining, the slices were dewaxed and stained with hematoxylin (Solaibao, China) for 5 min. and stained with eosin (Sangon Biotech, China) for 3 min. The staining was observed under an optic microscope (Olympus, Japan).

The data were processed by Excel 2016, and a one-way analysis of variance (one-way ANOVA) was carried out by SAS 9.4 software GLM program. The results were expressed as “mean ± standard deviation.” Duncan's method was used for multiple comparisons. P < 0.01 means extremely significant difference, P < 0.05 means significant difference, and P > 0.05 means that the difference was not significant.

The effects of dietary PRA extract levels on the growth performance of raccoon dogs were shown in Table 2. There were no significant differences in final weight, ADG, and F/G among treatments (P > 0.05), indicating a minimal effect of PRA extract on the growth of raccoon dogs. In contrast, PRA extract level had a significant effect on ADFI (P < 0.01). Raccoon dogs in the PRA1 groups and PRA2 groups had higher ADFI than those in other groups (P < 0.01).

The effects of PRA extract supplementation on nutrient digestibility are shown in Table 3. Dietary PRA extract supplementation decreased the digestibility of DM, and CP (P < 0.01). Raccoon dogs in the PRA8 groups had higher dry matter excretion than those in t other groups (P < 0.01). Besides, the dry matter digestibility and protein digestibility of group PRA8 were significantly lower than those of other groups (P < 0.01). The level of dietary PRA extract had no significant effect on the fat digestibility of raccoon dogs (P > 0.05).

As shown in Table 4, there was a very significant difference among the nitrogen intake groups of raccoon dogs (P < 0.01). It was significantly higher in group PRA2 than in group PRA0 and PRA1, and nitrogen intake in groups PRA0 and PRA1 was significantly higher than that in groups PRA4 and PRA8 (P < 0.01). There was a very significant difference among fecal nitrogen groups (P < 0.01) and group PRA8 was significantly higher than that of other groups (P < 0.01). There was a significant difference among nitrogen deposition groups (P < 0.05), that in group PRA1 and PRA2 was significantly higher than that in group PRA8 (P < 0.05). There was no significant difference in urinary nitrogen, NPU, and PBV between the five groups (P > 0.05).

As shown in Table 8, different dietary PRA extract levels had no significant effect on the activities of D-Lac and DAO in the serum of raccoon dogs (P > 0.05). Different dietary levels of PRA had no significant effect on the content of IL-18 in the small intestinal mucosa of the raccoon dogs (P > 0.05) but had a significant effect on the content of TNF-α in the small intestinal mucosa of raccoon dog (P < 0.05). The content of TNF-α in group PRA1 and group PRA2 was significantly higher than that in group PRA0.

As shown in Figure 1A, there was less enteritis cell infiltration in the small intestine in each group. As shown in Figure 1B, there was no significant difference in villus height and crypt depth among the groups (P > 0.05). Compared with the group PRA0, the ratio of villus height to crypt depth in group PRA1and group PRA2 increased significantly (P < 0.05).

At the end of the experiment, there was no significant difference in the final weight, average daily gain, and feed-to-weight ratio among different groups, which indicated that different dietary PRA extract levels had no effect on the growth performance of raccoon dogs. In recent years, the research of PRA extract was mainly focused on its pharmacological effects, such as analgesia, anti-inflammation, antibacterial, anti-oxidation, anti-cancantiliver fibrosis, anti-autoimmune diseases, anti-cardiovascular diseases, anti-cerebrovascular diseases, and anti-neurodegenerative diseases (5, 7, 9, 15). PRA extract is mostly in the form of compatibility with other traditional Chinese medicine to form a compound when it was added to the feed. The research has shown that the addition of Chinese herbal medicine residue composed of (Radix astragali, Radix angelicae sinensis, Radix rehmanniae preparata, Paeoniae raidix alba, and etc.) could promote the growth and development of piglets to a certain extent, especially fermented Chinese herbal medicine residue (16). Furthermore, the compound prescription of Chinese herbal medicine containing PRA was also studied in sows. Dietary supplementation with the herb residues (from Radix astragali, Radix angelicae sinensis, Radix rehmanniae preparata, Raidix paeoniae alba, and etc.), during the perinatal period improved the reproductive performance in sows (17). CZKJKL (composed of saikosaponins, poly-saccharide of Atractylis macroceohala, TGP) can efficiently relieve weanling stress in piglets (18).

In this experiment, PRA extract was added to the diet, not compound Chinese herbal medicine, which did not play a role in promoting the growth of raccoon dogs. However, with the increasing of the content of PRA extract, the average daily feed intake of the raccoon dogs increased at first and then decreased, indicating that PRA extract had a certain effect on the palatability of diet, but the palatability of diet will decrease when the content of PRA extract was too high.

The nutrient digestibility of raccoon dogs can affect the growth of animals. The main reasons that the addition of Chinese herbal medicine to feed can improve the absorption of nutrients in animals can be listed as follows: 1. Chinese herbal medicine is rich in nutrients, such as amino acids, minerals, vitamins, etc., which can make the diet composition better and more balanced, so as to improve the feed conversion efficiency (19). 2. Chinese herbal medicine has a local stimulating effect, which can promote intestinal peristalsis and improve the digestion and utilization of diet (20). 3. Chinese herbal medicine contains digestive enzymes, such as lipase, amylase, sugar invertase, and proteolytic enzyme, which can effectively promote animal digestion of diet (21). 4. Chinese herbal medicine can adjust the structure of intestinal flora, improve the intestinal environment of animals, promote the reproduction of beneficial bacteria, prevent intestinal villus atrophy, and promote the absorption of nutrients by intestinal mucosa (22–27). In this study, the addition of PRA extract did not promote the digestion of nutrients compared to group PRA0 (0 g/kg PRA extract). The reason may be that the PRA extract plays a role in relieving inflammation and improving immunity, so it failed to promote the absorption of nutrients in the intestine. However, when the supplementary amount was 8 g/kg, the PRA extract did not have a negative effect on the growth of raccoon dogs, which may be due to the animals themselves being in a healthy state.

The feed intake of animals determines the amount of nitrogen intake. The protein was mainly absorbed in the small intestine. Nitrogen metabolism reflects the utilization rate of protein by raccoon dogs. Net protein utilization and biological value of protein were used to measure the degree of protein utilization in feed and the protein requirement of animals. The research showed that the addition of Chinese herbal medicine (30% pine needles, 20% mugwort, 40% garlic, and 10% Astragalus mongholicus as dry matter) to the diet could decrease fecal and urinary N contents and increased N retention of Mongolian lambs (28). Moderate dietary CTHM (Fructus Ligustri Lucidi seed, Radix Astragali root, Radix Codonopsis root) supplementation (300–500 mg/kg DM) has been reported to increase the non-ammonia N flux in the small intestine and absorption of essential amino acids in growing sheep (21). In this experiment, PRA extract supplemented with 1 and 2 g/kg did not significantly improve the nitrogen deposition of raccoon dogs, which was consistent with the results of their growth performance. This may be because the extract of PRA extract has no effect on the absorption and utilization of amino acids. However, when the amount of PRA extract was added to 8 g/kg, the nitrogen deposition of raccoon dogs decreased, and there was no significant difference in net protein utilization and protein biological value groups, which may be due to a decrease in nitrogen intake and an increase in nitrogen excretion in the high dose group. The average daily gain of raccoon dogs in the 8 g/kg PRA extract group was not significantly lower than that in other groups, however, it was still suggested that it was not suitable to add a high dose of PRA to the diet for a long time.

The intestinal barrier function can be indirectly reflected by detecting intestinal permeability (40). The level of D-lac in peripheral blood can reflect the changes in intestinal mucosal damage and permeability (41). The level and activity of DAO in peripheral blood can reflect the injury and repair of the intestinal mucosal epithelium (42). Research has shown that Curcumin, a Chinese herbal medicine, can reduce the levels of D-lactic acid and DAO in the blood of rats with enteritis induced by methotrexate (MTX) (43). Treatment with glycyrrhizic acid significantly reduced D-lac but did not inhibit DAO activity in MTX-induced enteritis (44). In this experiment, with the PRA extract, there was no significant change in the levels of D-lac and DAO in peripheral blood, because the raccoon dogs were in a healthy physiological state and their intestinal mucosa was not damaged. IL-18 is mainly produced by activated macrophages and epithelial cells in the intestine. It plays an important role in the process of immune injury (45–47). TNF-α can participate in the intestinal mucosal inflammatory response and play an important role in promoting the necrosis and exfoliation of intestinal mucosal epithelial cells (48). TNF-α can also increase the permeability of local intestinal mucosa by reducing the TJs protein in the tight junction between cells (49). Paeoniflorin is one of the effective components of PRA extract. Some studies have shown that paeoniflorin inhibited the levels of TNF-α both in sepsis model rats in vivo and RAW264.7 cells in vitro (50). Furthermore, paeoniflorin inhibited atherosclerotic inflammatory cytokines IL-1β, IL-6, and TNF-α via the blockade of the TLR-4-mediated NF-κB signaling path-way (51). Other results indicate that paeoniflorin protects mice against lethal LPS challenge, at least in part, through inhibiting TNF-α and IL-1β production and accelerating IL-10 expression (52). In this experiment, different dietary levels of PRA extract had no significant effect on the level of IL-18 in the small intestine of the raccoon dog, but the added level of 1–2 g/kg PRA extract had a significant effect on the level of TNF-α, indicating that the PRA extract has a certain regulatory effect on the intestinal immune stress response of raccoon dog.

The villus length, crypt depth, etc. of the small intestine are important indexes to measure the digestion and absorption function of the small intestine. As the length of the villi increases, the number of absorbing cells on the villi increases, and the contact area between the small intestine and nutrients is larger, which is more conducive to nutrient absorption. crypt depth of the Small intestinal primarily reflects the rate of epithelial cell update rate. The ratio of villus length to crypt depth may comprehensively reflect the functional status of the small intestine. The increase of the ratio indicates that the digestion and absorption capacity of the small intestine is enhanced. Plant extracts have a certain effect on improving the intestinal histomorphology of animals. Research (53) has shown that resveratrol increased the ratio of villus height to crypt depth, increased the number of goblet cells, and reduced the histopathological damage of jejunum of ducks caused by acute heat stress. Dietary supplementation with Ampelopsis grossedentata extract facilitated nutrient adsorption in hens via intestinal histology changes for the reason that the villus height in duodenum and villus height to crypt depth ratio in duodenum and jejunum of A. grossedentata extract group was significantly higher than that of basal diet group (54). Piglets from Forsythia suspensa extract-fed sows had higher villus height in the ileum, and villus height to crypt depth ratio in jejunum and crypt depth in ileum compared with those from sows fed a control diet (55). In this study, from the villus height, crypt depth, and their ratio in the small intestine of Ussuri raccoon dog, there was no significant difference between the diet supplemented with groups PRA extract and the group control, though there was still a trend of enhanced digestion and absorption capacity of the small intestine. This might be related to the different physiological stages and health status of animals. It can only explain the preventive effect of PRA extract to a certain extent, the follow-up observations based on IBD raccoon dog models might be further helpful in clarifying the effects of PRA extracts.