Dietary addition of Astragalus polysaccharide (APS) in dogs: palatability, blood biochemistry and immunity

ABSTRACT This study explored the palatability of dog feed in response to different levels of Astragalus polysaccharide (APS), and the impact of APS on blood biochemistry and immunity in growing Beagles. A total of three APS treatments were manufactured: a control diet (CD, 0 mg/kg APS), a low APS concentration diet (LD, 400 mg/kg) and a high APS concentration diet (HD, 800 mg/kg). Based on our findings, APS did not have an influence on the first-choice ratio and consume ratio. In feeding trial, weight gain and feed efficiency were decreased in dogs fed the HD. APS increased the white blood cell count and serum total protein in Beagles. However, red blood cell count and haematocrit values followed an opposite trend. Serum levels of antibodies against canine distemper virus and rabies virus were improved by APS addition. Furthermore, C-reactive protein level was decreased in dogs fed the HD. As it can be concluded, this study indicated that APS could be safely and beneficially used in dog foods without compromising palatability. Further research is needed to investigate the feasibility of APS in coping with obesity and study the detailed mechanisms by which dietary APS improves immunity of Beagles.


Introduction
In pet nutrition field, feed additives with health-beneficial effects have attracted considerable interest (Pawar et al. 2017;Bruni et al. 2020). Herbal polysaccharides from natural resources have great potential as functional additives because of their significant bio-activities (Yu et al. 2018). Astragalus polysaccharide (APS), a major active ingredient of the roots of Astragalus membranaceus, possesses immunomodulatory, antioxidant, antiinflammatory, anti-tumor and antiviral activities (Zheng et al. 2020). The application of APS in livestock animals has verified the benefits of dietary APS in promotion of growth, feed utilization, immunity, gut health and antioxidant status (Zahran et al. 2014;Wu 2018;Song et al. 2019;Yang et al. 2019). However, research on the function of APS in pet foods is limited, which warrants study. When introducing a new ingredient in pet foods, palatability is a crucial attribute for success. Ingredients with antioxidant properties could increase feed palatability by preventing oxidation and providing an odour that animals prefer (Chen et al. 2016). In addition, herb additives might be able to directly improve the odour of feed, resulting in an increased feed intake (Lei et al. 2018). The feed intake improving effect of dietary APS has been reported in Nile tilapia (Oreochromis niloticus) (Zahran et al. 2014) and lambs (Zhong et al. 2012). However, dogs have superior sense of smell. Whether diets containing APS would be well accepted by dogs is still unknown, a fact that awaits further investigation.
Generally, pet owners are willing to provide foods that can improve health of their animals without any safety loopholes.
The safety of dietary APS has been demonstrated in livestock animals (Zahran et al. 2014;Wu 2018;Song et al. 2019;Yang et al. 2019). Furthermore, the intravenous injection of Astragalus membranaceus extract had no negative influence on Beagles (Yu et al. 2007). However, the safety of dietary addition of APS, which might be different from that of intravenous injection, has not been verified in dogs. To assess the physiological changes of animals in response to new dietary ingredients, the haematological and serum biochemical parameters are commonly determined. According to Zhong et al. (2012), dietary APS had no negative impact on blood biochemistry of lambs. Besides, the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were not affected by dietary APS addition in weaned piglets (Yang et al. 2019). At the same time, dietary APS did not have an effect on plasma haematology of chickens that were exposed to immunological stress (Liu et al. 2015). However, an active ingredient may induce various reactions in physiological status in different animal species (Onasanya et al. 2015). Hence, it is necessary to investigate the impact of dietary addition of APS on blood biochemistry of dogs.
Among the various claims promoted for functional feed additives, immune enhancing effect stands out as being of great importance. In laboratory animals, APS has been demonstrated possessing strong immune-potentiating effects, including stimulation of T-and B-cell proliferation, promoting the production of antibodies, macrophages activation, cytokines expression regulation and induction of surface antigen expressions on lymphocytes (Zheng et al. 2020). Besides, the immune-modulating activity of APS has also been demonstrated in livestock animals (Zhong et al. 2012;Liu et al. 2015;Wu 2018). Furthermore, studies showed that the intravenous injection of APS improved the immune response in dogs, such as increasing the antibody titres against rabies (Qiu et al. 2010;Liu et al. 2012). However, no information is available concerning immune responses of dogs to APS that is provided through dietary route.
This study was carried out to assess palatability of dog feed in response to variations in APS addition, and it also examined the impacts of dietary APS on blood biochemistry and immune responses in growing Beagles. The present study aimed to evaluate the feasibility, safety and benefit of APS as a functional additive in canine foods.

Material and methods
Protocols used here were all in accordance with the guidelines issued by the Animal Care Advisory Committee of the Institute of Laboratory Animals of Sichuan Academy of Medical Sciences (AE2020009).

Experimental diets
APS (with a purity of 80%) used in this study was a commercial product purchased from Shaanxi Ciyuan Biotechnology Co., Ltd (China), which was mainly composed of glucose, mannose, arabinose, xylose, galactose, rhamnose and ribose. The composition and the analysis of the basal diet are shown in Table 1. The nutrient requirements for the basal diet were based on the Association of American Feed Control Officials (AAFCO 2004). The low APS concentration diet (LD) and high APS concentration diet (HD) were prepared by adding APS into the basal diet to obtain final APS dose at 400 and 800 mg/kg, respectively. The control diet (CD) was the basal diet. Pellets were produced and stored in vacuum bags until they were used according to Hong et al. (2020).

Palatability trial
Palatability was measured using the most common palatability assessment method: two-bowl test (Griffin 2003). Diets were paired-compared using two comparisons: CD vs LD and CD vs HD. Dogs were offered diets in separate bowls for 1 h per day (09:00) for five consecutive days (Chen et al. 2016). To ensure there would be leftovers, the amount of feed offered was 30% higher than the recommendation for the maintenance of adult dogs according to the guidelines of the NRC (2006) and de Brito et al. (2010). After 1 h, first-choice data were recorded, feed residues were weighed to calculate the consume ratio: Feeding trial Before the experimental procedures, Beagles were adapted to the experimental conditions for two weeks according to Hong et al. (2020). During the acclimation period, all Beagles were vaccinated with a combined vaccine, which included canine parvovirus (CPV), canine parainfluenza virus (CPIV) canine adenovirus (CAV) and canine distemper virus (CDV) antigens as part of standard veterinary care. After the acclimation, six Beagles (3.80 ± 0.43 kg) were randomly allocated into one of the three treatment groups (CD, LD, HD). Beagles were fed twice per day for five weeks. Feed rations were adjusted weekly according to Dobenecker et al. (2013). A rabies virus (RV) vaccine and another combined booster vaccine against CPIV, CAV, CPV and CDV were applied during the feeding trial (on day 16). All Beagles were weighed every week. Total weight gain (WG) was calculated by subtraction the initial body weight from the observed weight at the end of the feeding trial. The feed efficiency (FE) was calculated using the following formula:

Sample collection and analysis
At the end of week two and five (on day 15 and 36), blood samples were obtained. After 12 h of fasting, blood were collected into two different types of tubes (containing EDTA-K 2 or with no additive) from each dog via the jugular vein. Immediately after collection, tubes without additive were centrifuged to obtain serum.

Statistical analysis
The statistical significance of the palatability trial result was analysed by a two-tailed t-test. The results from the feeding trial were statistically evaluated by one-way analysis of variance (ANOVA) using SPSS (version 18.0: SPSS, Chicago).

Results
The palatability trial results are shown in Figures 1 and 2. In terms of first-choice ratio and consume ratio, significant differences occurred neither between the CD and the LD, nor between the CD and the HD (P > 0.05).
The body weight (BW) changes of Beagles in the feeding trial are shown in Figure 3. In the first three weeks, there was no significant difference in BW among the groups (P > 0.05). However, at the end of fifth week, BW of Beagles fed the HD was significant lower than that of the LD and CD groups (P < 0.05). As described in Figure 4, the FE was significantly lower in the HD group than in the other two groups (P < 0.05).
As presented in Table 2, after two weeks of dietary APS feeding, no significant differences were observed in haematology parameters among the three groups (P > 0.05). However, some significant differences in haematology parameters were Figure 1. The first-choice ratios of the feed containing different levels of APS and control feed. CD: control diet; LD: low APS concentration diet; HD: high APS concentration diet; bars refer to mean values; different letters above bars indicate a significant difference (P < 0.05); error bars refer to standard deviation. Figure 2. The consume ratios of the feed containing different levels of APS and control feed. CD: control diet; LD: low APS concentration diet; HD: high APS concentration diet; bars refer to mean values; different letters above bars indicate a significant difference (P < 0.05); error bars refer to standard deviation.
observed after five weeks of dietary APS administration. Dogs fed the HD had a higher level of WBC (P < 0.05). At the same time, dietary APS addition significantly increased MCH and MCHC values (P < 0.05). However, RBC and HCT values exhibited an opposite trend (P < 0.05). As shown in Table 3, the majority of the serum biochemistry parameters were not significantly influenced by APS addition (P > 0.05), with the exception of serum TP concentrations. Dietary inclusion of APS at the level of 800 mg/kg significantly increased the TP concentration (P < 0.05).
Serum antibody responses and CRP levels are shown in Table 4. The levels of antibodies against CPIV and CAV were not significantly affected by dietary APS (P > 0.05). However, following APS administration for five weeks, the serum level of CPV antibody was increased in the HD fed Beagles (P < 0.05), while the CDV antibody level was improved in both LD and HD group (P < 0.05). Moreover, Beagles fed diets containing APS had a higher level of RV antibody (P < 0.05). Beagles in the HD group had a lower serum CRP level (P < 0.05).

Discussion
Palatability is an important criterion for introducing a new functional ingredient in pet foods. This study for the first time indicates the palatability of APS-containing feed in canine species. The two-bowl test is commonly used for pet food palatability assessments (Griffin 2003). The firstchoice ratio, a key parameter in two-bowl test, shows the first feed tasted, reflecting the olfactory attractiveness (Tobie et al. 2015). On the other hand, the consume ratio compares how much of the two feeds provided simultaneously is consumed in a certain period of time (Tobie et al. 2015). In this study, both the first-choice ratio and consume ratio showed no significant differences between the groups. Thus, we can conclude that APS can be used as a functional additive in dog foods without any palatability-related drawbacks.
Safety assessment of dietary APS is another objective of this study. Functional ingredients should offer benefits to pets without any side effects. In our study, Beagles were all in a clinically healthy situation throughout the experimental period. The BW of Beagles fed LD, which was close to that observed in our previous study (Hong et al. 2020), showed no significance difference from that of dogs in the CD group. However, at the end of fifth week, the lower BW was observed in Beagles fed high levels of APS. This growth inhibitory effect of high dose APS has also been reported in large yellow croaker (Larimichthys crocea) , rats (Wang et al. 2009) and mice . Based on the correlation analysis, FE was positively correlated with BW in this study (r = +0.636, P = 0.066), implying that the poor growth may be partly due to the decreased FE caused by 800 mg/kg APS. Though the significant BW difference, the haematology and serum biochemical analysis  revealed that Beagles in the HD group were as healthy as that of the other two groups. The blood biochemical parameters are useful in clinical chemistry, for which reference ranges are provided for assessment of animal physiological status. In the present study, ALP, CK, TP, ALB, CREA and TC were out of reference ranges according to Rørtveit et al. (2015). This might be due to the age-related changes in blood biochemistry. Studies have demonstrated that the growth stage has a significant influence on blood biochemical parameters in animals (Jezek et al. 2006;Kiran et al. 2012). Meanwhile, the levels of these parameters were close to those of Beagles in other studies (Assarasakorn et al. 2006;Hong et al. 2020). These findings implied that the physiological status of the Beagles was not impaired by dietary APS with the dose up to 800 mg/kg. When comparing the    haematology parameters among the groups, there were some changes caused after five weeks of dietary APS administration. WBC, which plays an important role in immune responses, was increased in the HD fed Beagles. This might be partly due to an improved proliferation of immune cells. Studies have demonstrated that APS could enhance the lymphocytes proliferation (Fan et al. 2012). Besides, for centuries, the root of Astragalus membranaceus has been used to promote haematopoiesis in China (Zhu and Zhu 2007). However, to our surprise, after five weeks of dietary APS administration, Beagles had lower RBC compared with the CD group. Furthermore, HCT, a parameter reflecting the fractional volume of a blood sample occupied by packed RBC (Kim et al. 2012), was also decreased. This unexpected WBC increasing and RBC decreasing effect of dietary APS has also been observed in lambs (Zhong et al. 2012). However, these results are inconsistent with the findings carried out on immunosuppressive dogs, which showed that with the increasing dose of injected APS, the density of RBC increased (Qiu et al. 2010). The reason for this phenomenon is still unknown, which is worthy of further investigation. MCH and MCHC are two useful parameters in the diagnosis of anaemia, which reflect content and concentration of HGB in RBC (Buttarello 2016). In this study, both MCH and MCHC values were increased after APS administration for five weeks, indicating an improved oxygen-carrying capacity of RBC. The elevated MCH and MCHC might be correlated with a change in blood iron level. There is a positive connection between MCH, MCHC and iron status in blood (Rusia et al. 1996). APS was reported to be able to regulate the blood iron level in mice (Ren et al. 2016). However, additional investigations are warranted to determine this hypothesis in dogs. Most of serum biochemical parameters were not significantly influenced by dietary APS, except for serum TP. Dogs fed the HD had a higher TP content. TP, an indicator of health and stress, usually varies in different physiological and pathological conditions Zhu W et al. 2019). The elected serum TP content in 800 mg/kg APS group might be due to an increase in content of blood proteins like antioxidant enzymes and bactericidal peptides . The enhanced serum levels of these proteins as an effect of APS addition have been already demonstrated by previous studies (Zahran et al. 2014;Hamid et al. 2017;Zhao et al. 2018).
Because of the great importance of immunity in animal health, functional ingredients with immune-promoting properties have been attracting much attention in pet food industry. APS is a well-known immunostimulant (Zheng et al. 2020). Studies have demonstrated that APS exerted better preventive than therapeutic effects against pathogens (Ge et al. 2015), which makes APS be a great candidate in functional foods with health protective effects. For the first time, this study demonstrates that dietary administration of APS for five weeks enhanced immune responses in growing Beagles. Vaccine responses were used as markers for specific immune function in this study. We assessed the levels of antibodies against five specific vaccine antigens in the blood sample collected. CPV and CDV are two common and preventable infectious canine diseases, which are major causes of mortality in dogs (Litster et al. 2012). In this study, concentrations of antibodies against CPV and CDV were improved by dietary APS supplementation. RV, the agent of rabies, can cause a severe encephalomyelitis in carnivore populations (Wunner and Briggs 2010). The level of antibody against RV was also improved in APS treatment groups. The increased antibody levels here might be due to an enhanced activated status of B cells and macrophages. It is well known that antibodies are produced by activated B cells. The antibody production is also modulated by activated macrophages products (Dimitriu and Fauci 1978). In mice, APS was found to be able to activate B cells and macrophages (Shao et al. 2004). However, the levels of antibodies against CPIV and CAV here were not influenced by dietary APS, which suggested that dietary APS could target a specific antigen by improving corresponding antibody production. However, the mechanism of this phenomenon should be investigated in the future. In addition to the antibody responses, CRP, an acute-phase reactant synthesized by the liver in systemic response to inflammation (Kuribayashi et al. 2003), was also assessed. In this study, serum CRP levels in Beagles were all normal (0.8-18.9 µg/mL) (Kuribayashi et al. 2003;Satyaraj et al. 2013). Furthermore, dogs fed the HD for five weeks had a lower CRP level, indicating a better health status.
In conclusion, dietary inclusion of APS up to 800 mg/kg did not negatively affect palatability of dog feed. The growing Beagles could tolerate 800 mg/kg of dietary APS without adverse effects on blood biochemistry. Furthermore, dietary addition of APS influenced immune responses of Beagles by enhancing response to specific vaccines and alleviating inflammation. Considering the significant growth inhibitory effect of 800 mg/kg APS, 400 mg/kg APS addition dose might be appropriate for growing Beagles. The present work is the first study that demonstrated APS could be used as a beneficial functional additive in dog foods. This may be of great interest to those involved in pet nutrition research and the pet food industry. However, further research is needed to clarify the mechanism of the immunity improvements by dietary APS in Beagles. Besides, this study indicated that APS might be potentially used to cope with obesity in dogs, which would be worthy of investigation in the future.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Availability of data and materials
The data presented in this study are available on request from the corresponding author.