t i l e Effect of Dietary Supplementation of Astragaloside IV on Growth performance, Inflammatory, and Antioxidant Status of Holstein Male Calves

Holstein male calves were selected to study the effects of supplementing diets with Astragaloside IV (ASIV) on growth performance, inflammatory, and antioxidant functions. The calves were assigned to four treatment groups with six calves each in a completely randomized design. The calvels were fed diets in which different amounts of ASIV were provided (0, 15, 30, or 60 mg/d per calf). The experimental period consisted of 7 days of adaptation followed by 120 days of data collection. Calves were fed 6 L/ day of milk replacer from 7–60 days, weaned at 60 days, and offered water, starter, and Chinese wildrye ad libitum for the whole trial period. The total dry matter intake of calves were similar among treatments, whereas the final body weight at 120 days ( P = 0.084) and average daily gain ( P = 0.025) increased with increased ASIV. There were no significant differences among treatments on body measurement indexes. Serum blood urea nitrogen levels decreased ( P = 0.028) and glucose levels increased ( P = 0.029) with increasing ASIV. The average concentrations of CAT ( P = 0.0005), GSH-Px ( P < 0.0001), and T-SOD ( P = 0.013) increased, and MDA ( P = 0.006) decreased with increased feeding amount of ASIV. Furthermore, concentrations of IL-6, IL-8, and TNF-α were not affected by treatments ( P > 0.05). In conclusion, ASIV improved the growth performance and antioxidant function of calves in a concentration-dependent manner. To further study the mechanisms underlying the action of ASIV to improve antioxidant functionality and immune level in vitro experiments should be explored.


INTRODUCTION
T he survival rate of calves is one of the main factors limiting the development of the cattle industry, and healthier calves are required to increase productivity. When the rumen and digestive tract of calves are not fully developed, they are susceptible to external environmental O n l i n e F i r s t A r t i c l e 2007). Therefore, it is important to find safe and effective antioxidant drugs to slow down the occurrence of disease in calves. Chinese herbal medicines or plant extracts are characterized by low toxicity and are affective. Astragalus membranaceus, a commonly used Chinese medicinal plant, has been shown to have pharmacological benefits (Qi et al., 2014;Li et al., 2017a). Astragaloside IV (ASIV) is the main active ingredient of Astragalus saponins, and its anti-inflammatory, anti-oxidative, and immune-regulatory activities have been previously demonstrated (Ren et al., 2013;Li et al., 2017b). Many previous studies have shown that ASIV plays an important role in the clinical treatment of atherosclerosis, cardiac fibrosis, diabetes, liver damage, and other diseases (Dai et al., 2017;He et al., 2017;Li et al., 2018;Sun et al., 2018). However, there have been few studies that have investigated the effects of supplemented ASIV on animals, especially calves.Therefore, our study investigated the effects of ASIV supplementation on the performance, plasma biochemistry, antioxidant functions, and immune indexes of calves.

Astragaloside IV
The astragaloside IV (ASIV) extract used in this study was purchased from a commercial pharmacy with a purity of 500 g/kg dry matter (Nanjing Spring and Autumn Biological Engineering Co. Ltd, Nanjing, China). The extract was prepared by extracting from the roots of the leguminous plant Astragalus and purified with high performance liquid chromatography.

Animals, diets, and experimental design
The experiment was conducted under experimental license from the Institutional Animal Care and Use Committee (IACUC20060101, 1 Jan 2006) of the Shandong Academy of Agricultural Sciences.
The experiment was conducted at the Shandong Qingye Pasture Co., Ltd from July 2018 to November 2018. We selected 24 Holstein male calves (7 days after birth) with an initial body weight of 40.79 ± 2.77 kg (mean ± SD). They were housed in individual pens and provided free access to water by bucket. Calves were randomly allocated to four groups of six calves (n = 6).
Four treatment groups received different doses of ASIV mixed into the diet (0, 15, 30, and 60 mg/d per calf). The ASIV powder was mixed with 50 ml water, and feeding was done by mixing it in milk replacer liquid or through oral syringe. The experiments consisted of two periods (i) 0-60 days when calves were fed milk replacer (Beijing Precision Animal Nutrition Research Center, Beijing, China), the milk replacer was prepared fresh in 1:7 power to water (lukewarm) ratio and fed twice per day (at 8:00 and 16:00) for a total of 6 L per cattle, and Chinese wildrye and starter (Shandong Jiurui Agricultural Group Co., Ltd, Shandong, China) ad libitum. After 60 days, calves were weaned, (ii) and 60-120 days when calves were only fed Chinese wildrye and starter ad libitum. The chemical composition of milk replacer powder, Chinese wildrye, and starter are listed in Table I.

Growth performance traits
Throughout the experiment, the amount of diet offered and refused was recorded daily to calculate dry matter intake (DMI). Body weight was continuously measured before the morning feeding in the last 3 days of each period. Average daily gain (ADG) of the calves were calculated between feeding period intervals. The feed conversion rate (FCR) was calculated by the ratio of DMI: ADG.

Body measurement indices
Body measurement indexes were measured and recorded after body weighing, and included whither height (WH), body length (BL), and heart girth (HG); they were measured in an unforced position. To study the relative growth of calves, the body length index (BLI), heart girth index (HGI), and somatic index (SI) were calculated with the following equations (Mavule et al., 2013;Liu et al., 2018): BLI=BL/WH, HGI=HG/WH, and SI=HG/BL.

Serum sampling and analysis
Approximately 10 ml of blood was collected from the jugular vein prior to the morning feeding on the last day of each period. Blood was collected in 10-ml centrifuge O n l i n e

Statistical analysis
All data were analyzed using the PROC MIXED procedure of SAS (SAS Inst. Inc., Cary, NC, 2002) based on the following statistical model: Y ijk = μ+ T i + P j + (T × P) ij + A k + e ijk, where μ = overall mean; T i = fixed effect of ASIV treatments (i = 1 to 4); P j = fixed effect of the period within two feeding periods (j = 1 to 2); (T x P) ij = fixed effect of interaction between treatment and period; A k = random effect of animals (k = 1 to 6), and e ijk represents the random residual error. P <0.05 indicated a significant difference. A tendency towards a difference was also considered for 0.05 ≤ P <0.10. Polynomial analysis was conducted to determine the quadratic or linear response to the increasing ASIV dosage in the diet. The means of each trait were compared by Turkey multiple comparisons if a significant treatment effect was found.

Growth performance
The results of growth performance are shown in Table II. The BW of calves at 60 days was not affected by the different diets (P >0.05), whereas the BW at 120 days linearly increased with increased ASIV (P= 0.084). The DMI was not affected by the different diets during the two periods and on average (P >0.05). Except for during the first 60 days, the ADG linearly increased with increased ASIV during the 60-120-day period and on average (P <0.05). The FCR was no affected during the two periods, but significance declined on average (P =0.014).

Body measurement indices
The results of body measurement indexes of calves are shown in Table III. The amount of ASIV had no effect on the BLI of calves from 0-60 day (P >0.05) and on average, and had tendency effects from 60-120 days (P = 0.061). The HGI of calves fed 30 mg/d of ASIV was higher than that of other diets (P = 0.051). However, there was no difference among treatments from 60-120 days and on average (P >0.05). The SI was not affected by different diets during the two periods and on average (P >0.05).

Serum analysis
The results of serum biochemical index of calves are shown in Table IV. The HDL, LDL, ALT, AST, and TCH content of calves were not affected by different diets during the two periods and on average (P >0.05). The TP content linearly increased and BUN content linearly decreased with increased ASIV from 0-60 days (P <0.05). GLU content linearly increased from 0-120 days (P <0.05). Moreover, the TP content from 0-60 days and GLU content on average were higher, and BUN and TG content from 0-60 days was lower for calves fed 30 mg/d of ASIV than that of the other diets (P <0.05).
The results of serum antioxidant index of calves are shown in Table V. From 0-60 days, CAT and GSH-Px content linearly increased (P <0.05), and T-SOD content tended to linearly increase (P= 0.098), and MDA content tended to linearly decrease (P= 0.076) with increased ASIV. From 60-120 days, CAT and T-SOD content linearly increased (P <0.05), and MDA content linearly decreased (P = 0.035) with increased ASIV. On average, the serum antioxidant index of calves had similar varying tendencies to those from 0-60 days. Moreover, the TCA content was not affected by the different diets during the two periods or on average (P >0.05).
The results of serum immune index of calves are shown in Table VI. The IL-6, IL-8, and TNF-α content were not affected by the different diets during the two periods and on average (P >0.05).

DISCUSSION
In the present study, supplemented ASIV had no effect on DMI, whereas the result showed a significant increase in the final body weight and ADG on average. Although some studies have reported that Astragalus polysaccharides could improve ADG and tended to increase the food intake of weaned pigs; however, the FCR did not improve (Yuan et al., 2006). Ying (2010) also observed that basal diet supplementation with Astragalus polysaccharides could increase body weight improving broiler growth performance. However, there are few studies on ASIV in ruminants at present, and body weight is an important indicator for evaluating growth and development (Heinrichs et al., 1992). In our results, the increase of calves weight is closely related to the addition of ASIV into the diet to improve the intestinal environment of calves and promote the absorption of nutrients.
The body measurement indexes can reflect the relative body shape regardless of growth and nutritional status of calves. We found that ASIV supplementation did not affect body length parameters, including BLI, HGI, and SI. This result indicated that the body size ratio was appropriate and well developed.

O n l i n e F i r s t A r t i c l e
Effect of Astragaloside IV on Growth of Holstein Calves

O n l i n e F i r s t A r t i c l e
Effect of Astragaloside IV on Growth of Holstein Calves carrier of nutrients and content changes reflect dietary protein levels. Therefore, decreased TP content indicates that the nutrient content of the protein was insufficient (Bartlett et al., 2006). In this experiment, the TP content was significantly higher than that of the control group at 60 days, and it also showed a tendency to increase at 120 days. This result indicated that ASIV may promote protein absorption, reduce protein decomposition, and consequently affect calf body weight, which is consistent with the results pertaining to growth performance. In ruminants, BUN is mainly metabolized by the liver, part of which is excreted from the body, and part of which is returned to the intestine for further anabolism for use in the body (Lapierre and Lobley, 2001). Therefore, BUN is an important indicator reflecting nitrogen metabolism, amino acid balance, and energy nitrogen balance (Abe et al., 1997). The levels of BUN were significantly decreased during the feeding trails. Thus, it was beneficial to amino acid balance and nitrogen utilization and improved the utilization efficiency of protein in the body. This also increased the body weight of calves. Both TG and TCH are neutral fats, which are important indicators of blood lipid levels and reflect the body's lipid metabolism. HDL and LDL are two lipoproteins in serum. LDL can transport TCH, while HDL is an anti-atherosclerotic lipoprotein that can transport cholesterol in surrounding tissues.
In the present study, there was no significant effect of the ASIV treatments on TG, TCH, HDL, or LDL. This result indicated that ASIV did not cause abnormal lipid metabolism in calves and had no adverse effects on the health of the body. GLU is a biochemical indicator of energy metabolism, which can reflect energy levels (Stanley et al., 2002). Our results suggested that GLU was not affected up to 60 days but increased from 60-120 days, which might have been induced by rumen and digestive tract development. Furthermore, ASIV supplementation promoted digestion and metabolism of carbohydrates, which was conducive to maintain energy balance. AST and ALT are important indicators reflecting heart and liver function, respectively. When liver tissue was damaged, serum transaminase activity would increase . The ASIV had no influence on AST and ALT, indicating that ASIV supplementation did not affect the heart and liver of calves. In healthy animals, the production and elimination of free radicals are balanced. When they become unbalanced, substances in the cells are excessively oxidized, which leads to oxidative stress (Sohal and Allen, 1990). GSH-Px can eliminate lipid peroxidation products. T-SOD and CAT are the main antioxidant enzymes in organisms and have strong abilities for scavenging free radicals. TCA is an important part of the antioxidant system, and can measure the total antioxidant capacity of the body. MDA is the end product of lipid peroxidation, which can reflect the oxidative stress state. Our results indicated that ASIV had a significant increases in the GSH-Px, T-SOD, CAT levels, and marked decreases in MDA. Although the effect of TCA was not significant, it generally increased. Our results were consistent with prvious reports. This might account of ASIV prevents animal damage by increasing the levels of antioxidant enzymes.
Other studies have also demonstrated the improvement of antioxidant status by supplementation with Astragalus polysaccharides in the diet of rats, lambs, and broilers (Yan et al., 2010;Zhong et al., 2012;Shengjun, 2018). The enhanced antioxidant enzyme activity and antioxidant status is perhaps due to bioactive compounds in ASIV, and those compounds possess different biological and pharmaceutical activities, such as antioxidant and free radical scavenging functions (Hao et al., 2018;Wang and Guo, 2019). Therefore, our results showed that ASIV could protect against oxidative stress by enhancing the body's antioxidant capacity.
Cytokines are produced by a variety of cells and have polypeptide molecules that regulate cell functions. They can participate in immune response and regulation and contribute to the prevention, diagnosis, and treatment of diseases. They are also typical inflammatory mediators of IL-6, secreted by T cells, macrophages, and smooth muscle cells. IL-6 stimulates inflammation and autoimmune processes against a range of diseases, such as atherosclerosis and diabetes. It can act as a proinflammatory cytokine and an anti-inflammatory cytokine by inhibiting IL-1 and TNF-α (Chen et al., 2019). IL-8 attracts and activates neutrophils, which are in contact with neutrophils and undergo morphological changes and release active substances, leading to local inflammatory reactions. TNF-α is secreted by macrophages and participates in the body's immune response as a proinflammatory cytokine (Pedersen and Bruunsgaard, 2003). The result of this experiment showed that ASIV had no significant effect on IL-6, IL-8, or TNF-α, indicating that ASIV supplementation did not cause an inflammatory response in calves. Meanwhile, ASIV can protect the health of the animal by inhibiting the damage of adverse external factors.

CONCLUSIONS
Based on the obtained results, ASIV supplementation in calves inhibited inflammatory response, and evidently improved the growth performance, increased the activity of serum antioxidant enzymes, and decreased the MDA content. To sum up, ASIV can enhance the O n l i n e

F i r s t A r t i c l e
Y. Wang et al. inflammatory and antioxidant capacity of calves. Based on the requirement for improved growth performance, it is recommended to feed Holstein male calves 30 mg/day.