Astragalus membranaceus root supplementation improves average daily gain, rumen TVFA production and immunity and antioxidant factors of Tibetan sheep

Background: The use of antibiotics as supplements in animal feed is restricted due to possible health hazards associated with them. Consequently, there is increasing interest in exploiting natural products as antibiotics with no detrimental side effects. In this study, we examined the effect of Astragalus membranaceus root (AMT) supplementation on dry matter intake, growth performance, rumen fermentation and immunity of Tibetan sheep. Materials and methods: Twenty-four male Tibetan sheep (31 ± 1.4 kg; 9 months old) were assigned randomly to one of four dietary treatments with different levels of AMT: 0 g/kg, 20 g/kg, 50 g/kg and 80 g/kg dry matter (A 0 , A 2 , A 5 and A 8 , respectively) in addition to their basal diets. A 0 acted as a control group and measurements were recorded over a 56-d feeding period. Results: Sheep fed with AMT had a higher average daily gain (ADG) and a lower feed:gain ratio (F:G) than controls ( P < 0.001). Rumen concentrations of NH 3 -N ( P < 0.001), total volatile fatty acids (TVFA) ( P = 0.028), acetate ( P = 0.017) and propionate ( P = 0.031) in A 5 and A 8 were higher than in A 0 . The addition of AMT in the feed significantly increased serum antioxidant and immunity factors of the sheep and increased the concentrations of serum interleukin, immunoglobulin and tumour necrosis factor-α (TNF-α) ( P = 0.010). Conclusions: We concluded that AMT can be used as a feed additive to improve growth performance and rumen fermentation and enhance the immunity of Tibetan sheep. Some responses exhibited a dose-dependent response, whereas other did not exhibit a pattern, with an increase in AMT. The addition of 50 g/kg and 80 g/kg AMT of total DMI showed the most promising results.

a control group and measurements were recorded over a 56-d feeding period.
Results: Sheep fed with AMT had a higher average daily gain (ADG) and a lower feed:gain ratio (F:G) than controls ( P < 0.001). Rumen concentrations of NH 3 -N ( P < 0.001), total volatile fatty acids (TVFA) ( P = 0.028), acetate ( P = 0.017) and propionate ( P = 0.031) in A 5 and A 8 were higher than in A 0 . The addition of AMT in the feed significantly increased serum antioxidant and immunity factors of the sheep and increased the concentrations of serum interleukin, immunoglobulin and tumour necrosis factor-α (TNF-α) ( P = 0.010).
Conclusions: We concluded that AMT can be used as a feed additive to improve growth performance and rumen fermentation and enhance the immunity of Tibetan sheep. Some responses exhibited a dose-dependent response, whereas other did not exhibit a pattern, with an increase in AMT. The addition of 50 g/kg and 80 g/kg AMT of total DMI showed the most promising results.

Background
The high-altitude Qinghai-Tibetan Plateau (QTP) is characterized by low air temperature, low air oxygen content, a short growing season and sparse vegetation of poor quality during the winter [1].
As one of the major livestock species on the QTP, numbering over 50 million head, Tibetan sheep are well adapted to the harsh environment and provide meat and income for most nomadic and seminomadic peoples in these regions [2].
Feeding antibiotics to livestock to increase production and improve livelihoods is a common practice in many countries [3]. However, the use of antibiotics as supplements in animal feed is often restricted due to residues and resistant strains of bacteria, and the rising awareness of hazards associated with antibiotics [4]. Consequently, there is increasing interest to exploit natural products that have no public health hazards.
Many Chinese herbs, with low toxicity, have been used as feed additives for livestock to improve growth performance and/or reduce mortality [5]. For example, Angelica sinensis (root), Epimedium brevicornu (full plant), Schisandra chinensis (fruit) and Acanthopanax senticosus (root) can enhance immune functions, Isatis indigotica (root), Rheum palmatum (stem and root) can reduce pathogenic microorganisms, and Citrus reticulata (peel) and Crataegus pinnatifida (fruit) can increase animal appetite and improve feed conversion rate [6].
Astragalus membranaceus, a legume, is widely distributed throughout the temperate regions of the world [7]. The dried root of Astragalus membranaceus (AMT) contains polysaccharides, saponins and other biological active substances, which have been used in Chinese traditional medicine for nearly 2000 years as a booster of the immune system [8,9]. Pharmacological studies have shown that AMT possesses immuno-stimulant, tonic, hepato-protective, diuretic, antidiabetic, analgesic and sedative properties [10,11]. In addition, it has been reported that AMT and its extracts can improve growth performance and immunity of chickens and pigs [12]. However, little is known on the effects of AMT on ruminants. The aim of this study is to fill this gap by determining the effects of dietary AMT supplementation on growth performance, rumen fermentation and immunity and antioxidant factors in Tibetan sheep. Dried roots of Astragalus membranaceus were purchased from the Gansu Hebo Chinese Herbal Medicine (Long) Science and Technology Co., Ltd, China. The roots were washed, dried, ground and passed through a 1 mm sieve. The contents of AMT, determined by the ultraviolet branch photometer method, included 125.8 g/kg astragalus polysacharin, 0.12 g/kg calycosin-7-glucoside and 0.87 g/kg astragaloside IV. The main bioactive ingredients in AMT conforms to Pharmacopopoeia of the People's Republic of China [13].

Experimental design
Twenty-four 9-month-old male Tibetan sheep (31 ± 1.4 kg), with similar genetic backgrounds, were used in a completely randomized design. Each sheep was penned individually (each pen = 2.5 × 3.5 m) and had free access to fresh water. The sheep were offered 450 g concentrate (Menyuan Yongxing Ecological Agriculture and Animal Husbandry Development Co. LTD, Haibei, China) and ad libitum oat hay daily, at 07:00 and 17:00. The compositions of the feed are presented in Table 1. After a 2-week adaptation period, the sheep were assigned randomly to one of four treatments (n = 6 per treatment) that differed in the level of AMT: control -0 g/kg (A 0 ), 20 g/kg (A 2 ), 50 g/kg (A 5 ) and 80 g/kg (A 8 ) dry matter intake (DMI) for 56 days. The AMT, mixed with 10 g concentrate, was fed separately to each sheep at 07:00, and the amount fed each sheep was based on DM intake from pretrial feed intakes, when the sheep consumed approximately 800 g DM oat hay along with the 450 g concentrate, and the amount of AMT was then adjusted weekly.  [15].
Jugular vein blood samples were collected into vacuum tubes before the morning feeding on days 14,

Statistical analysis
Results are presented as means ± SEM. Two-week values (time) of dry matter intake, TVFAs, and immune factors for each sheep were analyzed by repeated measures ANOVA using the mixed procedure of SAS (Version 9.1; SAS Institute) with day as the independent variable. A Tukey's test was used to separate means where a treatment x period interaction was significant.
Orthogonal contrasts were used to evaluate differences due to level of AMT and test the linear, quadratic, and cubic effect of an increasing proportion of AMT. The results are presented as least square means. Correlation analysis of variables was done using bivariate correlation analysis (SPSS correlation analysis). Statistical significance was accepted at P < 0.05.

Results
Effect of AMT on dry matter intake and average daily gain Body weight (BW) did not differ among groups (P = 0.973) at the beginning of the study (day 0), but was higher (P < 0.001) in sheep supplemented with AMT on day 56 than in controls ( Table 2). The addition of AMT increased the average DMI of sheep (P = 0.001). All the spplemented concentrate was consumed by the sheep in each treatment, therefore the differences in total DMI reflected mainly the differences in oat hay intake. The groups consuming AMT all had higher ADG than controls (P < 0.001) and there was a linear increase (P < 0.001) with an increase in AMT. The ADG of groups A 8 , A 2 and A 5 were 79%, 21% and 25%, higher, respectively, than group A 0 . The highest feed:gain (F:G) ratio occurred in the A 0 group, and the lowest ratio in the A 8 group (P < 0.001), and there was a linear decrease (P < 0.001) with an increase in AMT. Table 2 The effect of Astragalus membranaceus root (AMT) on body weight (BW), average daily gain (ADG) and feed:gain (F:G) ratio and dry matter intake (DMI) of Tibetan sheep a-c, means within a row followed by different lower-case letters differ significantly (P < 0.05). 1BW = body weight; DMI = dry matter intake; ADG = average daily gain; F:G ratio = feed to gain ratio. Effect of AMT on rumen pH and volatile fatty acids Rumen pH decreased (P = 0.016) on days 28 and 56 in the AMT fed groups when compared to controls (Table 3) and was lower on day 56 than on days 14 and 28 in all groups (P = 0.003). Rumen acetate concentration was higher (P = 0.017) in sheep consuming AMT than in controls on days 28 and 56 and acetate was also higher on day 56 than day 14 in all four treatment groups (P = 0.022).
Generally, AMT increased rumen propionate concentration (P = 0.031) and there was a linear increase (P < 0.001) with an increase in AMT. There was no consistent trend in rumen butyrate concentration in relation to the level of AMT (P = 0.059). The concentration of TVFA in the rumen was higher in the AMT fed sheep than controls (P = 0.028), was higher on day 56 than days 14 and 28 (P = 0.041) and increased linearly (P < 0.007) with an increase in AMT. The A:P ratio was lower in A 2 , A 5 and A 8 groups than in A 0 on day 56 (P < 0.001), with the lowest value in the A 8 group. The addition of AMT increased (P < 0.001) rumen NH 3 -N concentration on days 28 and 56, the NH 3 -N concentration increased significantly (P < 0.001) with days and the treatment x day interaction was significant (P = 0.041) ( Table 3). Table 3 The effect of AMT on rumen pH and VFA on days 14, 28 and 56 in a-c, means within a row followed by different lower-case letters differ significantly (P < 0.05). A-C, means within a column followed by different capital letters differ significantly (P < 0.05). 1TVFA = total volatile fatty acids; A/P = Acetate/Propionate; NH 3 -N = ammonia-N.

Effect of AMT on serum immune factors
The serum concentrations of IgA (P = 0.016) and IgG (P = 0.045) were higher in A 5 and A 8 than in A 0 and A 2 on day 56 (Table 4). In addition, serum concentrations of IgA (P = 0.004), IgG (P = 0.006) and IgM (P = 0.020) in the AMT fed lambs were higher on day 56 than on 14. The interaction between  a-c, means within a row followed by different lower-case letters differ significantly (P < 0.05). A-C, means within a column followed by different capital letters differ significantly (P < 0.05). 1Ig = immunoglobulin; IL = interleukin; sCD14 = soluble CD14; TNF-α = tumour necrosis factor-α.  a-c, means within a row followed by different lower-case letters differ significantly (P < 0.05). A-C, means within a column followed by different capital letters differ significantly (P < 0.05). 1INS = insulin; GH = growth hormone; T-AOC = total antioxidant capacity; SOD = superoxide dismutase.  [18], who reported no change in DMI, ADG and feed conversion in Ujumqin male lambs receiving AMT at 50 g/kg DMI for 30 days. The increase in DMI in the present study did not increase linearly with an increase in AMT, but increased only at 80 g/kg AMT, while ADG and feed conversion improved linearly with an increase in AMT intake. These different patterns illustrated the importance of testing different levels of AMT on the responses of sheep. Our results, however, were consistent with reports in monogastrics in which ADG and feed conversion in early weaned piglets [12] and ADG and DMI in chickens [19] increased with AMT. In these studies, the improvements were attributed mainly to astragalus polysaccharide, which ranged between 2.4 and 9.6 mg/kg AMI in the three groups of the present study. It was reported that astragalus polysaccharide accelerates muscle growth and increases carcass weight through the expressions of related genes and proteins [20]. In addition, an in vitro study with rumen fluid showed that supplementary AMT can improve feed digestibility in steers [3].

Effect of AMT supplementation on rumen fermentation
Optimal fibrolytic bacteria growth was found to be at a pH between 6.2 and 7.2 [21] and its activity was inhibited at a pH below 6.0 [22]. Although the pH of the rumen fluid decreased with the addition of AMT, the pH of all groups fell within the optimal range for sheep. The pH was negatively correlated with the concentration of TVFA, as was reported previously for ruminants [23].
The TVFA produced in the rumen provides about 75 percent of the ruminants' energy [24]. In the current study, the concentration of rumen TVFA and propionic and acetic acids increased with AMT supplementation, indicating that AMT enhanced rumen fermentation. The increase in propionic acid was linear with an increase in AMT, but acetic acid increased with time but did not show a pattern of increase with an increase in AMT and as a result, the A:P ratio was reduced with AMT supplement.  [26]. The increase of rumen NH 3 -N concentration with the addition of AMT in this study was also reported in a previous report by Zhong et al. (2012) [18], which indicated that AMT can play a role in promoting dietary protein degradation and microbial protein synthesis.
Effect of AMT supplementation on immune factors in the serum A number of herbs are immunologically active, functioning as excellent immuno-modulating agents.
Astragalus polysaccharides and saponins stimulate macrophages and B cell activation [28], promote antibody formation, activate complement and increase T lymphocyte proliferation [29], while astragaloside increases T, B lymphocyte proliferation and promotes antibody production [30].
Bioactive fractions, isolated from AMT, were found to be the most potent with respect to its mitogenicity on murine splenocytes [8]. The addition of 50 and 80 g/kg DMI AMT in this study showed the strongest effect in increasing serum IgA, IgG and IgM concentrations, especially toward the latter stages of the study; IgA, IgG and IgM are important indicators of humoral immunity. Immunoglobulin is an antibody-active animal protein secreted by plasma cells and plays an important role in both specific and non-specific immunity [31,32]. Immune enhancement was also observed in lambs with a supplementation of 50 g/kg DMI AMT [18].
Interleukin (IL) plays an important role in transmitting information, activating and regulating immune cells and mediating T and B cell activation, proliferation and differentiation. In the present study, the concentration of serum interleukin (IL-2, IL-4, IL-10) generally increased with an increase in the level of AMT. The increase in IL-4 was linear with an increase in AMT, whereas a definite pattern of increase was not detected for IL-2 and IL-10. A study by Hou et al. (2015) showed that AMT increased the percentage of IL-4-expressing T helper (Th) cells in the blood and prevented the decline in IL-6 mRNA expressions in spleen of mice [33]. The cytokine IL-2 is produced by activated T cells, and stimulates both the innate and acquired immune responses, including the release of secondary cytokines such as TNF, IL-1 and IL-6 [34]. IL-4 and IL-10 enhances the B-cell response and alternative activation of macrophages to promote tissue repair [35]; whereas, IL-6 functions as a differentiation factor on B cells and an activation factor on T cells [36]. Kurashige et al. (1999) observed that AMT protects against the reduction of IL-2 production in mice lymphocytes treated with a carcinogen [37].
Astragalus polysaccharides have important immuno-stimulating effects by promoting IL-2 production and splenocyte proliferation in vitro [38], while saponins have been shown to have high IL-2 inducing activity [34].
Serum TNF-α, which plays an important role in antigen enhancing and inflammatory regulation of macrophages [11], increased in concentration linearly with an increase in AMT consumed. TNF-α is induced mainly by activated peritoneal macrophages, which are important cells of the innate immune response that can eliminate invading microorganisms through phagocytosis [18,39]. In vitro studies reported an increased production of TNF-α by peritoneal macrophages of C3H/HeN mice when cells were cultured with an aqueous extract of AMT [40]. Bedir et al. (2000) [41] found that astragaloside enhanced mRNA expression of IL-1β and TNF-α, while Wei et al. (2016) [42] reported that astragalus polysaccharide increased gene expression and production of NO, TNF-α, and IL-6 in RAW264.7 cells.
Consequently, the current results demonstrated that adding AMT to Tibetan sheep diet can booster the immune system as it increases serum immunoglobulins and regulates the secretion of a number of cytokines.

Effect of AMT on serum hormones and antioxidant properties
In the present study, the addition of 80 g/kg DMI AMT increased serum GH concentration in the sheep, as was also observed in broilers offered AMT [43]. However, this response was not found in lambs fed AMT at 50 g/kg DMI [18]. Growth hormone (GH) promotes metabolism and growth by regulating the secretion of insulin-like growth factor to stimulate the entry of amino acids into cells and to accelerate the synthesis of DNA and RNA, thereby promoting the synthesis of proteins [44].
Oxidative stress is believed to be a primary factor contributing to animal diseases [45]. Astragalus polysaccharides have been shown to enhance antioxidant status and to scavenge free radicals [46] and at a dietary concentration of 5 g/kg DMI enhanced serum antioxidant enzymatic activity and improved antioxidant status of broilers [47]. In addition, flavonoids and saponins extracted from AMT possessed antioxidative activities [48]. The main parameters for assessing oxidative status in animals are T-AOC and T-SOD [49]. In this study, the addition of 80 g/kg DM AMT increased T-AOC concentration on day 56. In other studies, the addition of 50 g/kg AMT to lamb diet [18] and 10 g/kg AMT to broiler diet [50] increased liver T-AOC concentration. The addition of AMT did not affect SOD in the present study but there was an increase in concentration with time for every treatment.

Conclusions
This study demonstrated that supplementary AMT increased DMI and improved ADG and feed conversion rates in Tibetan sheep. AMT had beneficial effects on rumen fermentation and improved the immunity and antioxidant activities of Tibetan sheep. Some responses were dose dependent and increased with an increase in AMT, while other responses increased without a pattern. Based on the present study, we concluded that best results for Tibetan sheep could be obtained at 50 g/kg to 80 g/kg DMI of AMT.