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Effects of Chinese herbal polysaccharides on the immunity and growth performance of young broilers
Effects of Chinese Herbal Polysaccharides on the Immunity and Growth Performance of Young Broilers1 H. L. Chen,* D. F. Li,*2 B. Y. Chang,† L. M. Gong,* J. G. Dai,* and G. F. Yi‡ *Ministry of Agriculture Feed Industry Center, China Agricultural University, Beijing, China, 100094, †Feed Science Institute of Chinese Academy of Agriculture Science, Beijing, 100081; and ‡University of Missouri, Columbia, Missouri 65211 ABSTRACT Two trials were conducted to study the effects of two Chinese herbal polysaccharides [achyranthan (ACH), a low-molecular-weight polysaccharide, and astragalan (APS), a high-molecular-weight polysaccaride] on the immunity and growth performance of young broilers. Trial 1 was a 28-d growth assay, in which 7-d-old broilers (n = 240) were randomly allotted to one of three dietary treatments, with eight replicate pens per treatment and ten chickens per pen. Dietary treatments included a control corn-soy-fishmeal (Treatment 1), a diet with 200 mg/kg APS (Treatment 2), and a diet with 200 mg/kg ACH (Treatment 3). Blood samples were collected by cardiac puncture on Days 7, 14, 21, and 28 for determination of serum parameters, and chickens were killed on Day 28 to measure immune organ indexes. Trial 2 was an in vitro trial to study the effects of different concentrations of polysaccharides on broiler splenocyte functions.
In Trial 1, feeding either APS or ACH had no significant effects on growth performance of broilers relative to the control. However, compared to the control, feeding ACH significantly increased microhemagglutination inhibition (HI) antibody titers, bursa of Fabricius index, serum albumin, serum calcium, and nitric oxide (NO) concentrations at Day 28 (P ≤ 0.05). In Trial 2, both polysaccharides showed significant immunostimulating effects. They increased NO and interleukin-2 (IL-2) production of splenocytes and enhanced splenocyte proliferation in a dosedependent manner (P < 0.05). Those results indicate that the immunostimulating effects of APS are not as pronounced as those of ACH. Achyranthan showed immunostimulating effects in both the growth assay and in vitro studies. Therefore, ACH may be a Chinese herbal polysaccharide that has the potential to be used as a feed additive to improve broilers’ immunity.
(Key words: polysaccharide, immunity, growth, broiler) 2003 Poultry Science 82:364–370
err et al., 1994; Dritz et al., 1995). Chinese herbal polysaccharides have aroused great interest because of their natural origin, lack of drug residue and low side effects (see review by Tian and Feng, 1994). In clinical practice, there are two basic ways to administer polysaccharides, either by oral administration or injection. All kinds of polysaccharides can be injected with satisfying immunostimulating effects, but it is labor intensive and may cause additional stress to the animal. Oral administration is the preferred way because of its convenience, but it is argued that oral administration may decrease the immunostimulating effects of polysaccharides because of the possibility of being destroyed by intestinal enzymes (Wang and Gong, 2001). In the current study, two Chinese herbal polysaccharides, astragalan (APS) and achyranthan (ACH) were investigated. APS is extracted from the Chinese herb Astragalus membranaceus, which belongs to the Fabaceae (Le-
INTRODUCTION In poultry production, it is very important to improve immunity so as to prevent infectious diseases. A variety of factors can induce immunodeficiency, such as vaccination failure, infection by immune suppressive diseases, and abuse of antibiotics. Utilization of immunostimulants is one solution to improve the immunity of animals and to decrease their susceptibility to infectious diseases (Liu, 1999). Polysaccharides are considered natural immunostimulants that have been shown to promote the secretion of cytokines and antibodies, as well as enhance the function of natural killer cells, T and B lymphocytes (Nie and Zhang, 1999). Polysaccharides have also been shown to promote the growth performance of young pigs (Shoenh-
2003 Poultry Science Association, Inc. Received for publication February 28, 2002. Accepted for publication July 18, 2002. 1 Financial support by Natural Science Foundation of China. 2 To whom correspondence should be addressed: defali@ public2.bta.net.cn.
Abbreviation Key: ACH = achyranthan; ADG = average daily gain; AFDI = average feed daily intake; ANAE = a-naphthyl acetate esterase; APS = astragalan; ConA = concanavalin A; HI = microhemagglutination inhibition; IL = interleukin; ND = Newcastle disease; NO = nitric oxide.
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guminosae) family. Astragalus membranaceus is a frequently used herbal medicine in China to treat fatigue, sweating, poor appetite, heart disease, hepatitis, etc., due to its tonic and immunostimulating properties. APS is the main ingredient of Astragalus membranaceus. It is a large-molecular-weight polysaccharide that has been intensively studied in human medicine, but rarely utilized in animal production (Shan et al., 2000). It is usually administered by injection, and little is known about its effectiveness after oral administration. ACH is a novel low-molecularweight polysaccharide extracted from the Chinese herb Achyranthes bidentata, which has shown potent immunostimulating effects in human and rats when administered orally or by injection (Tian and Feng, 1994). To our knowledge, no research has been conducted in farm animals to investigate the efficacy of APS and ACH. Therefore, the objective of the current research was to explore the effects of APS and ACH on the growth performance and immunity of young broilers.
MATERIALS AND METHODS Trial 1: Animal Feeding Trial Experimental Animals and Materials. Two hundred forty 1-d-old Arbor Acre broilers were fed the control diet for 7 d. Subsequently, broilers were randomly allotted to three treatments with eight replicate pens per treatment and 10 chickens per pen. Treatment 1 was fed a corn-soyfishmeal control diet. Treatments 2 and 3 were fed the same diet as Treatment 1 but supplemented with 200 mg/ kg APS or 200 mg/kg ACH, respectively. All diets were formulated to meet the NRC (1994) nutrient requirements (Table 1). On d 7 of the study, broilers were i.m. vaccinated with 0.2 mL Newcastle Disease (ND) Clone-30 modified live vaccine.3 Blood samples were collected by cardiac puncture on Days 7, 14, 21, and 28. Peripheral blood T-lymphocytes were separated by density-gradient centrifugation according to the method of Chen (2000), whereas peripheral blood T lymphocyte proliferation was determined according to the method of Lin (1999a). Peripheral blood T lymphocytes counts were determined by the histochemical demonstration of a-naphthyl acetate esterase (ANAE) on blood films according to Lin (1999a), with the lymphocytes with reddish-brown reaction products regarded as being a-naphthylacetate esterase (ANAE) positive (ANAE+). Serum antibody titers were determined by means of a microhemagglutination inhibition (HI) test (Fu and Liu, 1997). Broilers were weighed and feed intakes recorded on Days 7 and 28. On Day 28, 16 chicks from each treatment were randomly selected and euthanized to collect immune organs, which included spleen, bursa of Fabricius, and thymus. Spleen index,
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Qilu Animal Health Products Factory, Shangdong, China. Bayer Corp., Tarrytown, NY. 5 Zhongsheng Biochemical Co., Ltd., Bejing, China. 6 Changzhou Xianfeng Drying Equipment Co., Ltd., China.
TABLE 1. Ingredients and chemical composition of the basal pretreatment and treatment diet1 Ingredients Corn Wheat Soybean meal Fish meal Limestone Monocalcium phosphate Salt Premix2 Soybean oil Calculated chemical analysis Crude protein Calcium Total phosphorus ME, kcal/kg
Composition (%) 58.9 2.0 30.0 4.0 1.0 1.2 0.3 1 1.5 21 0.9 0.68 3,100
1 The basal pretreatment and treatment diet was the same. Treatment diet was basal diet supplemented with 200 mg/kg astragalan or achranthan. 2 Premix provided the following per kilogram of diet: vitamin A (as vitamin A palmitate), 10,000 IU; cholecalciferol, 1,500 IU; vitamin E (as vitamin acetate), 20 IU; vitamin K, 2.2 mg; thiamine (as thiamine HCl), 1.1 mg; riboflavin, 8 mg; vitamin B12, 0.02 mg; biotin, 0.6 mg; folic acid, 0.7 mg; niacin, 50 mg; pantothenic acid, 20 mg; pyridoxine (as pyridoxine HCl), 2 mg; copper (as cupric sulfate 5H2O) 20 mg; iron (as ferrous sulfate 7H2O), 100 mg; zinc (as zinc sulfate 7H2O), 100 mg; selenium (as sodium selenite), 0.3 mg; iodine (potassium iodate), 1 mg; manganese (as manganese sulfate H2O), 120 mg.
bursa of Fabricius index, and thymus index were calculated as: immune organ weight × 1,000 BW. Concentrations of serum albumin and Ca2+, activities of alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase were assayed by an automatic biochemical analyzer (RA1000)4 using commercially available kits.5 Serum nitric oxide (NO) concentration was assayed by the method of Sun and Zhou (1997). ACH (molecular weight 1400) was provided by Shanghai Organic Chemistry Institute. Astragalus membranaceus was purchased from Jianlian Herbal Drug Store and APS extracted as described below. Extraction of APS. Extraction of APS was conducted according to the method of Liu (1989). In sum, 2 L of water was added to 1 kg Astragalus membranaceus, boiled and simmered for 2 h. The extraction was repeated, and two extracts were combined and filtered. The filtrate was concentrated to 500 mL in a vacuum desiccator (Model FZG-15)6 Changzhou Xianfeng Drying Equipment Co., Ltd.) at 70 C and 95% ethanol added so as to yield a 60% ethanol solution for polysaccharide precipitation. The resulting solution was filtered to recover a precipitate, which was then dissolved in water. The resulting solution was filtered again and concentrated to 200 mL and 95% ethanol added to yield an 80% ethanol solution, which was stored at 4 C overnight. Subsequently, the final product was vacuum-dried, and the resulting powder was APS. The molecular weight of APS was determined by the sephadex gel filtration method as described by Xiao and Zeng (2000) and was determined to be 67,600.
Trial 2: In Vitro Study
4
Proliferation of Splenocytes by Mitogen ConA Stimulation. Proliferation of splenocytes was determined us-
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ing a previously described method (Lin, 1999b). Briefly, spleens were aseptically removed from one 21-d-old Arbor acre broiler that was normally fed, minced into small pieces, and passed through a tissue sieve (200 mesh per inch) to prepare single-cell suspension in RPMI 1640.7 The suspension was centrifuged at 280 × g for 15 min, the supernatant was discarded, and the precipitate was resuspended in RPMI 1640. The suspension was then layered onto Ficoll-hypaque (density = 1.077)8 and separated by density-gradient centrifugation at 976 × g for 30 min. Splenocytes were on the top of Ficoll and in the form of a white band. The upper liquid was gently removed, then splenocytes were transferred into a new tube and washed with RPMI 1640 three times, after which splenocytes were suspended in 2 mL of RPMI 1640 complete media.7 The cells were detected by trypan blue dye exclusion. The cell density was counted and adjusted to 107 cell/mL. One hundred-microliter cell suspensions, 10 µL polysaccharide with different concentrations, and 100 µL of RPMI 1640 with 5 µg/mL of ConA [AUTH QUERY: Spell out ConA]9 were added into 96-well plate. The cells were then incubated at 37 C with 5% CO2. After a 72-h incubation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium) salts9 was added to the cell culture to make a final concentration 5 µ/mL. The cell continued to incubate for 4 h, then 100 µL acidified isopropylalcohol10 was added to the culture and vibrated for at least 10 min to fully dissolve the colored material. The absorbance of each sample was read via an automated ELISA reader11 (Sunrise, TECAN) at 570 nm. Splenocyte NO Assay. Splenocytes were prepared and incubated as described above, except that the culture time was changed to 24 h. At the end of the incubation, 50 µL supernatant was removed from each well into a new plate. Fifty µL of Greiss reagent (2% sulphanilamide, 5% H3PO4, 0.2% napthylethylenediamine) (Sigma Chemical, Co.) was added to the supernatant, mixed, and incubated at room temperature for 10 min. The amount of colored products was determined by spectrophotometry at 540 nm in an automated ELISA Reader. The amount of nitrite was estimated according to the standard curve generated, using a known concentration of sodium nitrite. Assay of IL-2 Production of Splenocytes. The assay of IL-2 production of splenocytes was conducted according to the method of Liu and Li (1999), briefly described by the following procedure. In Vitro Induction of IL-2 Splenocytes were prepared as described above, suspended in RPMI 1640 complete media, and adjusted to a cell density of 107 cell/mL. A 1-mL cell suspension and 1 mL of ConA (5) µg/mL) were added to a 24-well plate, and incubated at 37 C with 5% CO2 for 24 h. The cell culture was then transferred into
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Gibco BRL, Grand Island, NY. Tianjin Blood Research Center, Tianjin, China. 9 Sigma Chemical Co., St. Louis, MO. 10 Shanghai Chemical Factory, Shanghai, China. 11 Sunrise, Tecan, Austria. 8
FIGURE 1. Effects of herbal polysaccharides on Newcastle Disease (ND) microhemagglutination inhibition (HI) titers of broilers. HI titers are expressed as the geometric means of all HI titers (×log2). a,bMeans within each bar without a common letter are different (P < 0.05).
a tube, and centrifuged at 600 × g for 20 min, after which the supernatant was collected and stored at −20 C for determination. Preparation of Target Cells. A 1-mL cell suspension with a cell density of 107 cell/mL and 1 mL ConA (20 µg/mL) solution were added to a 24-well plate, and incubated at 37 C with 5% CO2 for 48 h. The cell culture was collected and purified by density-gradient centrifugation at 976 C g for 30 min on cushions of Ficoll-Hypaque. The precipitate was then washed and centrifuged twice with RPMI 1640. The final precipitate was suspended in RPMI 1640 with 20mg/mL α-MM (methyl-α-mannoside),9 and the cell density was adjusted to 108 cell/mL. Determination of IL-2. Fifty microliters of the target cell suspensions, 50 µL of IL-2 sample, and 50 µL of RPMI 1640 were added to a 96-well plate, and incubated at 37 C with 5% CO2 for 33 h. At the end of the incubation, Mossman’s colorimetric assay with MTT salts was used for the measurement of viable cell number (see above for splenocytes proliferation). The absorbance of each sample was read on an automated ELISA reader at 570 nm.
Statistical Analysis All data were subjected to ANOVA using the GLM procedure of SAS software (SAS Institute, 1996). The mean differences among different treatments were separated by Duncan’s multiple range tests. A level of P ≤ 0.05 was used as the criterion for statistical significance.
RESULTS Trial 1 Growth performance of broilers is shown in Table 2. Compared to the control, feeding ACH increased ADG by 8.45%; however there were no significant differences in ADG, AFDI, feed-to-gain ratio, or percentage of mortality among treatments. The broiler immune system responded differently to APS and ACH, as shown in Figure 1 and Table 3. The
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UTILIZATION OF CHINESE HERBS IN BROILERS TABLE 2. Effects of Chinese herbal polysaccharides on the growth performance of broilers (Day 28)
Initial weight, g Final weight, g ADG,2 g ADFI,3 g Feed:gain Mortality, %
1
Control
Astragalan
Achyranthan
SEM
P-value
126 871 36 73 2.06 4.16
135 891 36 71 1.98 3.08
138 947 39 79 2.04 5.27
1.28 18.53 0.51 0.81 0.03 0.18
0.71 0.22 0.12 0.23 0.82 0.24
1
Data represents the mean value for each treatment (80 birds per treatment). Average daily gain. 3 Average feed daily intake. 2
feeding of APS had no effect on ND HI titers, serum NO, bursa of Fabricius index, spleen index, or thymus index of broilers, whereas ACH significantly increased ND HI titers, bursa of Fabricius index, and serum NO concentration at d 28. The two polysaccharides had no significant effects on ANAE+ percentage or T-lymphocyte proliferation at d 21, although ACH numerically increased T-lymphocyte proliferation by 31.62%. As shown in Table 4, compared to the control, feeding ACH and APS increased serum albumin significantly at d 28 (P < 0.01). At d 28, feeding ACH increased (P < 0.05) serum Ca concentration compared with that of the control. Both polysaccharides had no effect on serum alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase activities at D 28.
Trial 2 As shown in Figure 2, compared to the control, both polysaccharides increased proliferation of splenocytes in a dose-dependent manner, with ACH having a stronger effect than APS. As shown in Figure 3, compared with the control, both polysaccharides increased NO production in a dose-dependent manner, with ACH causing a greater response than APS. Both polysaccharides increased the IL-2 production of splenocytes in a slightly different dose-dependent manner (Figure 4). For APS, IL-2 production reached a plateau at the dose of 50 µg/mL, and then decreased with increasing dosage. For ACH, IL-2 production peaked at the dose of 100 µg/mL.
DISCUSSION Growth Performance The current study indicated no effect of supplementing diets with Chinese herbal polysaccharides (APS and ACH) on the growth performance of young broilers. A body of literature has shown that β-glucan (polysaccharide mainly composed of glucose) could stimulate the growth response of fish (Onarheim, 1992), shrimp (Sung et al., 1994), and pigs (Shoenherr et al., 1994; Dritz et al., 1995). Sohn et al. (2000) proposed that the growthpromoting effects of polysaccharides were partly due to their immunostimulating effects, thus reducing bacterial and viral infection. β-glucan has been reported to decrease inflammatory cytokine production, such as interleukin-1 (IL-1), which might cause body fever and growth retardation, so β-glucan may indirectly promote growth (Dritz et al., 1995). In contrast, it has been proposed that immunostimulation may have adverse effects on growth, because more nutrients will be repartitioned to synthesize antibodies and develop immune organs, and thereby decreasing the nutrients available for growth (Hevener et al., 1999; Takahashi et al., 2000). Klasing (1998) proposed that immunostimulation would not necessarily decrease growth, because the immune system needs a relatively small amount of nutrients in relation to the nutrients needed for growth. Moreover, immunostimulation may be different from immune stresses caused by infectious diseases. In the current study, immunostimulation may have been limited to the immune system,
TABLE 3. Effects of Chinese herbal polysaccharides on immunity of broilers1 Item
Control
Astragalan
Achyranthan
SEM
P-value
ANAE+ (%)2 T-lymphocyte proliferation2 (OD570nm) Spleen index3 (g/kg) Thymus index3 (g/kg) Bursa of Fabricius index3 (g/kg) Serum NO3 (nmol/L)
38.2 0.664 1.164 4.789 2.664a 0.653a
40.3 0.681 1.370 4.691 3.235ab 0.749ab
42.5 0.874 1.167 5.047 3.420b 0.844b
4.32 0.11 0.26 0.26 0.37 0.08
0.23 0.08 0.62 0.44 0.05 0.04
Means within each row without a common superscript differ significantly (P ≤ 0.05). Data represents the mean value for each treatment. 2 a-naphthyl acetate esterase+ (ANAE+) and T-lymphocyte proliferation were determined on Day 21. 3 Spleen, thymus, and bursa of Fabricius index = immune organ weight × 1,000/BW. Those indexes and serum nitric oxide (NO) were determined on Day 28. a,b 1
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CHEN ET AL. TABLE 4. Effects of Chinese herbal polysaccharides on the serum parameters of broilers (Day 28)1 Item
Control
Astragalan
Achyranthan
SEM
P-value
Albumin (g/L) Ca (mg/100 mL) ALP2 (U/L) GOT3 (U/L) GPT4 (U/L)
12.0a 9.16a 1,131 173.8 2.67
14.6b 9.86ab 1,323 182.8 2.00
14.6b 11.62b 1,051 175.2 2.10
0.05 0.405 207.8 26.8 0.67
<0.01 0.04 0.84 0.52 0.46
Means within each row without a common superscript are different (P < 0.05). Data represents the mean value for each treatment. 2 Alkaline phosphatase. 3 Aspartate aminotransferase. 4 Alanine aminotransferase. a,b 1
and not accompanied with the whole-body reactions and the series of physiological and metabolic changes such as fever, decreased feed intake, acute phase protein synthesis, and increased nutrient catabolism. It is possible that the growth-stimulating effects of polysaccharides may be related to the dosage or supplementation level in the diets. Hayen and Dollmann (2000) reported that dietary supplementation of zymosan (a kind of yeast glucan in broilers) increased ADG by 14.3% and improved feed efficiency by 8.7%. However, at a high level of zymosan supplementation, ADG was reduced. Dritz et al. (1995) reported that the effects of glucan on growth performance of piglets was related to dosage, in which addition of 250 mg/kg glucan to the diet increased ADG and ADFI, whereas addition of 1% of glucan decreased feed intake and ADG. Therefore, the dosage employed in this study may not have been optimal to provide a growth response.
Serum Parameters and Immune Response In the present study, it was found that ACH significantly increased serum Ca2+ concentration. Bai et al. (1997) also reported that letinan (a polysaccharide extracted
FIGURE 2. Splenocyte proliferation in response to Chinese herbal polysaccharides. Splenocyte proliferation is measured by Mossman’s colorimetric method and expressed as an optical density of the value at > 570 nm. Each dilution was tested in triplicates. a–cMeans represented at each point without a common superscript are different (P < 0.05).
from mushrooms) increased plasma Ca2+ concentration. Ca2+ is an important regulator of lymphocyte function in that it is involved in the signal transduction of lymphocytes and promotes the proliferation of lymphocytes (Imboden et al., 1985). The increased Ca2+ induced by ACH may be of some significance in the immunomodulation of broilers. Both Chinese herbal polysaccharides increased serum albumin concentration, but the underlying mechanism is not quite clear yet. APS did not show any significant immunostimulating effects on broilers when administered by feed. Contrary to our findings, APS could promote mouse (Liang et al., 1995), human (Wang et al., 1989), and chick (Tang et al., 1998) T-lymphocyte proliferation. It could also stimulate broiler splenocyte IL-2 production (Liu and Li, 1999), NO (Cheng et al., 2001), and antibody production (Geng et al., 1995), as well as broiler ANAE+ percentage (Bi et al., 2000) when administered by injection. NO is reported to be a potentially critical factor in the regulation of macrophage activity and host defense (Hibbs et al., 1988). ANAE+ percentage is, in fact, the T-lymphocyte percentage, and thus an indicator of cellular immunity. But here no significant effect of two polysaccharides was found on ANAE+ percentage. In Trial 2, APS was found to have significant immunostimulating effects promoting NO, IL2 production, and splenocyte proliferation in vitro. The reason for the lack of immunostimulating effect of APS when delivered in the feed may be caused by the physical function of the gastrointestinal tract. It may have been destroyed by intestinal enzymes or, because it is a largemolecular-weight polysaccharide, it may no have been able to pass though the intestinal barrier and enter the blood circulation so as to be utilized by the immune system. In contrast to APS, ACH showed consistent immunostimulating effects in both Trial 1 and Trial 2, which is in agreement with other reports. ACH was reported to stimulate TNF, IL-2, and NO production of mouse splenocyte (Xiang et al., 1994b; Li and Li, 1999). It has also been shown to promote the proliferation of splenocytes, increase the total immunoglobulin G (IgG) and serum hemolysin content of lipopolysaccharide-injected mice (Xiang et al., 1994a). In Trial 2, APS had significant immunostimulating effects, which included increased NO, IL2 production of splenocytes and splenocyte proliferation.
UTILIZATION OF CHINESE HERBS IN BROILERS
FIGURE 3. Effects of Chinese herbal polysaccharides on nitric oxide (NO) production of splenocytes. NO production was measured using Greiss reagent and expressed as nmol/L. Each dilution was tested in triplicates. a–cMeans represented at each point without a common letter are different (P < 0.05).
To our knowledge, this was the first time that ACH was used in broiler feed and found to have significant immunostimulating effects. Based on the results of the current study, oral administration of ACH via feed delivery did not decrease its immunostimulating effects, as observed in Trial 2. The reason may be that the molecular weight of ACH was much smaller than that of APS, so it was more easily absorbed by the intestinal enterocytes and finally utilized by the immune system (Tian and Feng, 1994). ND HI titer is an indicator of specific humoral immunity. It is of great importance to improve HI titers. We demonstrated that ACH but not APS significantly increased ND HI titers, indicating an enhancement of humoral immunity. The authors thought that the mechanism may lie in two points. First, ACH could stimulate the development of bursa of Fabricius as reflected by bursa index, while bursa of Fabricius is the center of humoral immunity. Second, ACH could be easily absorbed and utilized by the immune system as discussed
FIGURE 4. Effects of herbal polysaccharides on interleukin-2 (IL-2) release from splenocytes. IL-2 production was measured by Mossman’s colorimetric method, and expressed as an optical density 570 nm (OD 570 nm). Each dilution was tested in triplicates. a,bMeans represented at each point without a common letter are different (P < 0.05).
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above. Therefore, ACH may be useful to improve broilers’ immunity against ND in case of vaccination failure. In the present study, it was found that large-molecularweight Chinese herbal polysaccharide, APS, was not as effective as the small molecular weight polysaccharide, ACH, in eliciting the immunity of broilers. In vitro, both polysaccharides increased splenocyte proliferation, NO, and IL-2 production in a quite similar, dose-dependent manner. However, oral administration of APS via feed delivery may decrease its efficacy due to intestinal enzyme digestion and its difficulty passing through the intestinal barrier and entering the general circulation. Thus, the smaller-molecular-weight Chinese herbal polysaccharide, ACH, may have more potential to be utilized as a feed additive to improve the immunity of young broilers.
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Sun, A. Y., and W. X. Zhou. 1997. Determination of Nitric oxide. Page 370 in Biomedicine of Nitric Oxide. C. S. Zhang, ed. Shanghai Medical University Press, Shanghai, China. Sung, H. H., G. H. Kou, and Y. L. Song. 1994. Vibriosis resistance induced by glucan treatment in tiger shrimp. Fish Pathol. 29:11–15. Takahashi, K., T. Mashiko, and Y. Akiba. 2000. Effect of dietary concentration of xylitol on growth in male broiler chicks during immunological stress. Poult. Sci. 79:743–747. Tang, X. M., Y. L. Hu, and B. K. Zhang. 1998. Effect of astragalan on chick lymphocyte transformation. China Vet. J. 18:269– 271. (in Chinese). Tian, G. Y., and Y. C. Feng. 1994. Progress in the research and application of polysaccharides. Chem. Prog. 6:44–48. (in Chinese). Wang, D. C., X. R. Li, and Y. Sun. 1989. Influence of astragalan on immunologic function of human periphery blood lymphocytes. China Cancer J. 11:180–183. (in Chinese). Wang, J., and X. G. Gong. 2001. Advance in the study on the antitumor activity and immunomodulating action of polysaccharides. Chin. J. Biochem. Pharm. 22:51–53. (in Chinese). Xiang, D. B., J. B. Ge, and X. Y. Li. 1994a. Achyranthan improved mouse humoral immunity. Shanghai Immunol. J. 3:134–136. (in Chinese). Xiang, D. B., C. Jiang, and X. Y. Li. 1994b. Influence of Achyranthes bidentata polysaccharides on function of T lymphocytes and natural killer cell. Chinese J. Pharmacol. Toxicol. 8:209– 212. (in Chinese). Xiao, G. W., and H. P. Zeng. 2000. Separation, purification and determination of Longkui polysaccharide. Chin. Herbal Med. 31:162–164. (in Chinese).
In Trial 1, feeding either APS or ACH had no significant effects on growth performance of broilers relative to the control. However, compared to the control, feeding ACH significantly increased microhemagglutination inhibition (HI) antibody titers, bursa of Fabricius index, serum albumin, serum calcium, and nitric oxide (NO) concentrations at Day 28 (P ≤ 0.05). In Trial 2, both polysaccharides showed significant immunostimulating effects. They increased NO and interleukin-2 (IL-2) production of splenocytes and enhanced splenocyte proliferation in a dosedependent manner (P < 0.05). Those results indicate that the immunostimulating effects of APS are not as pronounced as those of ACH. Achyranthan showed immunostimulating effects in both the growth assay and in vitro studies. Therefore, ACH may be a Chinese herbal polysaccharide that has the potential to be used as a feed additive to improve broilers’ immunity.
(Key words: polysaccharide, immunity, growth, broiler) 2003 Poultry Science 82:364–370
err et al., 1994; Dritz et al., 1995). Chinese herbal polysaccharides have aroused great interest because of their natural origin, lack of drug residue and low side effects (see review by Tian and Feng, 1994). In clinical practice, there are two basic ways to administer polysaccharides, either by oral administration or injection. All kinds of polysaccharides can be injected with satisfying immunostimulating effects, but it is labor intensive and may cause additional stress to the animal. Oral administration is the preferred way because of its convenience, but it is argued that oral administration may decrease the immunostimulating effects of polysaccharides because of the possibility of being destroyed by intestinal enzymes (Wang and Gong, 2001). In the current study, two Chinese herbal polysaccharides, astragalan (APS) and achyranthan (ACH) were investigated. APS is extracted from the Chinese herb Astragalus membranaceus, which belongs to the Fabaceae (Le-
INTRODUCTION In poultry production, it is very important to improve immunity so as to prevent infectious diseases. A variety of factors can induce immunodeficiency, such as vaccination failure, infection by immune suppressive diseases, and abuse of antibiotics. Utilization of immunostimulants is one solution to improve the immunity of animals and to decrease their susceptibility to infectious diseases (Liu, 1999). Polysaccharides are considered natural immunostimulants that have been shown to promote the secretion of cytokines and antibodies, as well as enhance the function of natural killer cells, T and B lymphocytes (Nie and Zhang, 1999). Polysaccharides have also been shown to promote the growth performance of young pigs (Shoenh-
2003 Poultry Science Association, Inc. Received for publication February 28, 2002. Accepted for publication July 18, 2002. 1 Financial support by Natural Science Foundation of China. 2 To whom correspondence should be addressed: defali@ public2.bta.net.cn.
Abbreviation Key: ACH = achyranthan; ADG = average daily gain; AFDI = average feed daily intake; ANAE = a-naphthyl acetate esterase; APS = astragalan; ConA = concanavalin A; HI = microhemagglutination inhibition; IL = interleukin; ND = Newcastle disease; NO = nitric oxide.
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guminosae) family. Astragalus membranaceus is a frequently used herbal medicine in China to treat fatigue, sweating, poor appetite, heart disease, hepatitis, etc., due to its tonic and immunostimulating properties. APS is the main ingredient of Astragalus membranaceus. It is a large-molecular-weight polysaccharide that has been intensively studied in human medicine, but rarely utilized in animal production (Shan et al., 2000). It is usually administered by injection, and little is known about its effectiveness after oral administration. ACH is a novel low-molecularweight polysaccharide extracted from the Chinese herb Achyranthes bidentata, which has shown potent immunostimulating effects in human and rats when administered orally or by injection (Tian and Feng, 1994). To our knowledge, no research has been conducted in farm animals to investigate the efficacy of APS and ACH. Therefore, the objective of the current research was to explore the effects of APS and ACH on the growth performance and immunity of young broilers.
MATERIALS AND METHODS Trial 1: Animal Feeding Trial Experimental Animals and Materials. Two hundred forty 1-d-old Arbor Acre broilers were fed the control diet for 7 d. Subsequently, broilers were randomly allotted to three treatments with eight replicate pens per treatment and 10 chickens per pen. Treatment 1 was fed a corn-soyfishmeal control diet. Treatments 2 and 3 were fed the same diet as Treatment 1 but supplemented with 200 mg/ kg APS or 200 mg/kg ACH, respectively. All diets were formulated to meet the NRC (1994) nutrient requirements (Table 1). On d 7 of the study, broilers were i.m. vaccinated with 0.2 mL Newcastle Disease (ND) Clone-30 modified live vaccine.3 Blood samples were collected by cardiac puncture on Days 7, 14, 21, and 28. Peripheral blood T-lymphocytes were separated by density-gradient centrifugation according to the method of Chen (2000), whereas peripheral blood T lymphocyte proliferation was determined according to the method of Lin (1999a). Peripheral blood T lymphocytes counts were determined by the histochemical demonstration of a-naphthyl acetate esterase (ANAE) on blood films according to Lin (1999a), with the lymphocytes with reddish-brown reaction products regarded as being a-naphthylacetate esterase (ANAE) positive (ANAE+). Serum antibody titers were determined by means of a microhemagglutination inhibition (HI) test (Fu and Liu, 1997). Broilers were weighed and feed intakes recorded on Days 7 and 28. On Day 28, 16 chicks from each treatment were randomly selected and euthanized to collect immune organs, which included spleen, bursa of Fabricius, and thymus. Spleen index,
3
Qilu Animal Health Products Factory, Shangdong, China. Bayer Corp., Tarrytown, NY. 5 Zhongsheng Biochemical Co., Ltd., Bejing, China. 6 Changzhou Xianfeng Drying Equipment Co., Ltd., China.
TABLE 1. Ingredients and chemical composition of the basal pretreatment and treatment diet1 Ingredients Corn Wheat Soybean meal Fish meal Limestone Monocalcium phosphate Salt Premix2 Soybean oil Calculated chemical analysis Crude protein Calcium Total phosphorus ME, kcal/kg
Composition (%) 58.9 2.0 30.0 4.0 1.0 1.2 0.3 1 1.5 21 0.9 0.68 3,100
1 The basal pretreatment and treatment diet was the same. Treatment diet was basal diet supplemented with 200 mg/kg astragalan or achranthan. 2 Premix provided the following per kilogram of diet: vitamin A (as vitamin A palmitate), 10,000 IU; cholecalciferol, 1,500 IU; vitamin E (as vitamin acetate), 20 IU; vitamin K, 2.2 mg; thiamine (as thiamine HCl), 1.1 mg; riboflavin, 8 mg; vitamin B12, 0.02 mg; biotin, 0.6 mg; folic acid, 0.7 mg; niacin, 50 mg; pantothenic acid, 20 mg; pyridoxine (as pyridoxine HCl), 2 mg; copper (as cupric sulfate 5H2O) 20 mg; iron (as ferrous sulfate 7H2O), 100 mg; zinc (as zinc sulfate 7H2O), 100 mg; selenium (as sodium selenite), 0.3 mg; iodine (potassium iodate), 1 mg; manganese (as manganese sulfate H2O), 120 mg.
bursa of Fabricius index, and thymus index were calculated as: immune organ weight × 1,000 BW. Concentrations of serum albumin and Ca2+, activities of alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase were assayed by an automatic biochemical analyzer (RA1000)4 using commercially available kits.5 Serum nitric oxide (NO) concentration was assayed by the method of Sun and Zhou (1997). ACH (molecular weight 1400) was provided by Shanghai Organic Chemistry Institute. Astragalus membranaceus was purchased from Jianlian Herbal Drug Store and APS extracted as described below. Extraction of APS. Extraction of APS was conducted according to the method of Liu (1989). In sum, 2 L of water was added to 1 kg Astragalus membranaceus, boiled and simmered for 2 h. The extraction was repeated, and two extracts were combined and filtered. The filtrate was concentrated to 500 mL in a vacuum desiccator (Model FZG-15)6 Changzhou Xianfeng Drying Equipment Co., Ltd.) at 70 C and 95% ethanol added so as to yield a 60% ethanol solution for polysaccharide precipitation. The resulting solution was filtered to recover a precipitate, which was then dissolved in water. The resulting solution was filtered again and concentrated to 200 mL and 95% ethanol added to yield an 80% ethanol solution, which was stored at 4 C overnight. Subsequently, the final product was vacuum-dried, and the resulting powder was APS. The molecular weight of APS was determined by the sephadex gel filtration method as described by Xiao and Zeng (2000) and was determined to be 67,600.
Trial 2: In Vitro Study
4
Proliferation of Splenocytes by Mitogen ConA Stimulation. Proliferation of splenocytes was determined us-
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ing a previously described method (Lin, 1999b). Briefly, spleens were aseptically removed from one 21-d-old Arbor acre broiler that was normally fed, minced into small pieces, and passed through a tissue sieve (200 mesh per inch) to prepare single-cell suspension in RPMI 1640.7 The suspension was centrifuged at 280 × g for 15 min, the supernatant was discarded, and the precipitate was resuspended in RPMI 1640. The suspension was then layered onto Ficoll-hypaque (density = 1.077)8 and separated by density-gradient centrifugation at 976 × g for 30 min. Splenocytes were on the top of Ficoll and in the form of a white band. The upper liquid was gently removed, then splenocytes were transferred into a new tube and washed with RPMI 1640 three times, after which splenocytes were suspended in 2 mL of RPMI 1640 complete media.7 The cells were detected by trypan blue dye exclusion. The cell density was counted and adjusted to 107 cell/mL. One hundred-microliter cell suspensions, 10 µL polysaccharide with different concentrations, and 100 µL of RPMI 1640 with 5 µg/mL of ConA [AUTH QUERY: Spell out ConA]9 were added into 96-well plate. The cells were then incubated at 37 C with 5% CO2. After a 72-h incubation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium) salts9 was added to the cell culture to make a final concentration 5 µ/mL. The cell continued to incubate for 4 h, then 100 µL acidified isopropylalcohol10 was added to the culture and vibrated for at least 10 min to fully dissolve the colored material. The absorbance of each sample was read via an automated ELISA reader11 (Sunrise, TECAN) at 570 nm. Splenocyte NO Assay. Splenocytes were prepared and incubated as described above, except that the culture time was changed to 24 h. At the end of the incubation, 50 µL supernatant was removed from each well into a new plate. Fifty µL of Greiss reagent (2% sulphanilamide, 5% H3PO4, 0.2% napthylethylenediamine) (Sigma Chemical, Co.) was added to the supernatant, mixed, and incubated at room temperature for 10 min. The amount of colored products was determined by spectrophotometry at 540 nm in an automated ELISA Reader. The amount of nitrite was estimated according to the standard curve generated, using a known concentration of sodium nitrite. Assay of IL-2 Production of Splenocytes. The assay of IL-2 production of splenocytes was conducted according to the method of Liu and Li (1999), briefly described by the following procedure. In Vitro Induction of IL-2 Splenocytes were prepared as described above, suspended in RPMI 1640 complete media, and adjusted to a cell density of 107 cell/mL. A 1-mL cell suspension and 1 mL of ConA (5) µg/mL) were added to a 24-well plate, and incubated at 37 C with 5% CO2 for 24 h. The cell culture was then transferred into
7
Gibco BRL, Grand Island, NY. Tianjin Blood Research Center, Tianjin, China. 9 Sigma Chemical Co., St. Louis, MO. 10 Shanghai Chemical Factory, Shanghai, China. 11 Sunrise, Tecan, Austria. 8
FIGURE 1. Effects of herbal polysaccharides on Newcastle Disease (ND) microhemagglutination inhibition (HI) titers of broilers. HI titers are expressed as the geometric means of all HI titers (×log2). a,bMeans within each bar without a common letter are different (P < 0.05).
a tube, and centrifuged at 600 × g for 20 min, after which the supernatant was collected and stored at −20 C for determination. Preparation of Target Cells. A 1-mL cell suspension with a cell density of 107 cell/mL and 1 mL ConA (20 µg/mL) solution were added to a 24-well plate, and incubated at 37 C with 5% CO2 for 48 h. The cell culture was collected and purified by density-gradient centrifugation at 976 C g for 30 min on cushions of Ficoll-Hypaque. The precipitate was then washed and centrifuged twice with RPMI 1640. The final precipitate was suspended in RPMI 1640 with 20mg/mL α-MM (methyl-α-mannoside),9 and the cell density was adjusted to 108 cell/mL. Determination of IL-2. Fifty microliters of the target cell suspensions, 50 µL of IL-2 sample, and 50 µL of RPMI 1640 were added to a 96-well plate, and incubated at 37 C with 5% CO2 for 33 h. At the end of the incubation, Mossman’s colorimetric assay with MTT salts was used for the measurement of viable cell number (see above for splenocytes proliferation). The absorbance of each sample was read on an automated ELISA reader at 570 nm.
Statistical Analysis All data were subjected to ANOVA using the GLM procedure of SAS software (SAS Institute, 1996). The mean differences among different treatments were separated by Duncan’s multiple range tests. A level of P ≤ 0.05 was used as the criterion for statistical significance.
RESULTS Trial 1 Growth performance of broilers is shown in Table 2. Compared to the control, feeding ACH increased ADG by 8.45%; however there were no significant differences in ADG, AFDI, feed-to-gain ratio, or percentage of mortality among treatments. The broiler immune system responded differently to APS and ACH, as shown in Figure 1 and Table 3. The
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UTILIZATION OF CHINESE HERBS IN BROILERS TABLE 2. Effects of Chinese herbal polysaccharides on the growth performance of broilers (Day 28)
Initial weight, g Final weight, g ADG,2 g ADFI,3 g Feed:gain Mortality, %
1
Control
Astragalan
Achyranthan
SEM
P-value
126 871 36 73 2.06 4.16
135 891 36 71 1.98 3.08
138 947 39 79 2.04 5.27
1.28 18.53 0.51 0.81 0.03 0.18
0.71 0.22 0.12 0.23 0.82 0.24
1
Data represents the mean value for each treatment (80 birds per treatment). Average daily gain. 3 Average feed daily intake. 2
feeding of APS had no effect on ND HI titers, serum NO, bursa of Fabricius index, spleen index, or thymus index of broilers, whereas ACH significantly increased ND HI titers, bursa of Fabricius index, and serum NO concentration at d 28. The two polysaccharides had no significant effects on ANAE+ percentage or T-lymphocyte proliferation at d 21, although ACH numerically increased T-lymphocyte proliferation by 31.62%. As shown in Table 4, compared to the control, feeding ACH and APS increased serum albumin significantly at d 28 (P < 0.01). At d 28, feeding ACH increased (P < 0.05) serum Ca concentration compared with that of the control. Both polysaccharides had no effect on serum alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase activities at D 28.
Trial 2 As shown in Figure 2, compared to the control, both polysaccharides increased proliferation of splenocytes in a dose-dependent manner, with ACH having a stronger effect than APS. As shown in Figure 3, compared with the control, both polysaccharides increased NO production in a dose-dependent manner, with ACH causing a greater response than APS. Both polysaccharides increased the IL-2 production of splenocytes in a slightly different dose-dependent manner (Figure 4). For APS, IL-2 production reached a plateau at the dose of 50 µg/mL, and then decreased with increasing dosage. For ACH, IL-2 production peaked at the dose of 100 µg/mL.
DISCUSSION Growth Performance The current study indicated no effect of supplementing diets with Chinese herbal polysaccharides (APS and ACH) on the growth performance of young broilers. A body of literature has shown that β-glucan (polysaccharide mainly composed of glucose) could stimulate the growth response of fish (Onarheim, 1992), shrimp (Sung et al., 1994), and pigs (Shoenherr et al., 1994; Dritz et al., 1995). Sohn et al. (2000) proposed that the growthpromoting effects of polysaccharides were partly due to their immunostimulating effects, thus reducing bacterial and viral infection. β-glucan has been reported to decrease inflammatory cytokine production, such as interleukin-1 (IL-1), which might cause body fever and growth retardation, so β-glucan may indirectly promote growth (Dritz et al., 1995). In contrast, it has been proposed that immunostimulation may have adverse effects on growth, because more nutrients will be repartitioned to synthesize antibodies and develop immune organs, and thereby decreasing the nutrients available for growth (Hevener et al., 1999; Takahashi et al., 2000). Klasing (1998) proposed that immunostimulation would not necessarily decrease growth, because the immune system needs a relatively small amount of nutrients in relation to the nutrients needed for growth. Moreover, immunostimulation may be different from immune stresses caused by infectious diseases. In the current study, immunostimulation may have been limited to the immune system,
TABLE 3. Effects of Chinese herbal polysaccharides on immunity of broilers1 Item
Control
Astragalan
Achyranthan
SEM
P-value
ANAE+ (%)2 T-lymphocyte proliferation2 (OD570nm) Spleen index3 (g/kg) Thymus index3 (g/kg) Bursa of Fabricius index3 (g/kg) Serum NO3 (nmol/L)
38.2 0.664 1.164 4.789 2.664a 0.653a
40.3 0.681 1.370 4.691 3.235ab 0.749ab
42.5 0.874 1.167 5.047 3.420b 0.844b
4.32 0.11 0.26 0.26 0.37 0.08
0.23 0.08 0.62 0.44 0.05 0.04
Means within each row without a common superscript differ significantly (P ≤ 0.05). Data represents the mean value for each treatment. 2 a-naphthyl acetate esterase+ (ANAE+) and T-lymphocyte proliferation were determined on Day 21. 3 Spleen, thymus, and bursa of Fabricius index = immune organ weight × 1,000/BW. Those indexes and serum nitric oxide (NO) were determined on Day 28. a,b 1
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CHEN ET AL. TABLE 4. Effects of Chinese herbal polysaccharides on the serum parameters of broilers (Day 28)1 Item
Control
Astragalan
Achyranthan
SEM
P-value
Albumin (g/L) Ca (mg/100 mL) ALP2 (U/L) GOT3 (U/L) GPT4 (U/L)
12.0a 9.16a 1,131 173.8 2.67
14.6b 9.86ab 1,323 182.8 2.00
14.6b 11.62b 1,051 175.2 2.10
0.05 0.405 207.8 26.8 0.67
<0.01 0.04 0.84 0.52 0.46
Means within each row without a common superscript are different (P < 0.05). Data represents the mean value for each treatment. 2 Alkaline phosphatase. 3 Aspartate aminotransferase. 4 Alanine aminotransferase. a,b 1
and not accompanied with the whole-body reactions and the series of physiological and metabolic changes such as fever, decreased feed intake, acute phase protein synthesis, and increased nutrient catabolism. It is possible that the growth-stimulating effects of polysaccharides may be related to the dosage or supplementation level in the diets. Hayen and Dollmann (2000) reported that dietary supplementation of zymosan (a kind of yeast glucan in broilers) increased ADG by 14.3% and improved feed efficiency by 8.7%. However, at a high level of zymosan supplementation, ADG was reduced. Dritz et al. (1995) reported that the effects of glucan on growth performance of piglets was related to dosage, in which addition of 250 mg/kg glucan to the diet increased ADG and ADFI, whereas addition of 1% of glucan decreased feed intake and ADG. Therefore, the dosage employed in this study may not have been optimal to provide a growth response.
Serum Parameters and Immune Response In the present study, it was found that ACH significantly increased serum Ca2+ concentration. Bai et al. (1997) also reported that letinan (a polysaccharide extracted
FIGURE 2. Splenocyte proliferation in response to Chinese herbal polysaccharides. Splenocyte proliferation is measured by Mossman’s colorimetric method and expressed as an optical density of the value at > 570 nm. Each dilution was tested in triplicates. a–cMeans represented at each point without a common superscript are different (P < 0.05).
from mushrooms) increased plasma Ca2+ concentration. Ca2+ is an important regulator of lymphocyte function in that it is involved in the signal transduction of lymphocytes and promotes the proliferation of lymphocytes (Imboden et al., 1985). The increased Ca2+ induced by ACH may be of some significance in the immunomodulation of broilers. Both Chinese herbal polysaccharides increased serum albumin concentration, but the underlying mechanism is not quite clear yet. APS did not show any significant immunostimulating effects on broilers when administered by feed. Contrary to our findings, APS could promote mouse (Liang et al., 1995), human (Wang et al., 1989), and chick (Tang et al., 1998) T-lymphocyte proliferation. It could also stimulate broiler splenocyte IL-2 production (Liu and Li, 1999), NO (Cheng et al., 2001), and antibody production (Geng et al., 1995), as well as broiler ANAE+ percentage (Bi et al., 2000) when administered by injection. NO is reported to be a potentially critical factor in the regulation of macrophage activity and host defense (Hibbs et al., 1988). ANAE+ percentage is, in fact, the T-lymphocyte percentage, and thus an indicator of cellular immunity. But here no significant effect of two polysaccharides was found on ANAE+ percentage. In Trial 2, APS was found to have significant immunostimulating effects promoting NO, IL2 production, and splenocyte proliferation in vitro. The reason for the lack of immunostimulating effect of APS when delivered in the feed may be caused by the physical function of the gastrointestinal tract. It may have been destroyed by intestinal enzymes or, because it is a largemolecular-weight polysaccharide, it may no have been able to pass though the intestinal barrier and enter the blood circulation so as to be utilized by the immune system. In contrast to APS, ACH showed consistent immunostimulating effects in both Trial 1 and Trial 2, which is in agreement with other reports. ACH was reported to stimulate TNF, IL-2, and NO production of mouse splenocyte (Xiang et al., 1994b; Li and Li, 1999). It has also been shown to promote the proliferation of splenocytes, increase the total immunoglobulin G (IgG) and serum hemolysin content of lipopolysaccharide-injected mice (Xiang et al., 1994a). In Trial 2, APS had significant immunostimulating effects, which included increased NO, IL2 production of splenocytes and splenocyte proliferation.
UTILIZATION OF CHINESE HERBS IN BROILERS
FIGURE 3. Effects of Chinese herbal polysaccharides on nitric oxide (NO) production of splenocytes. NO production was measured using Greiss reagent and expressed as nmol/L. Each dilution was tested in triplicates. a–cMeans represented at each point without a common letter are different (P < 0.05).
To our knowledge, this was the first time that ACH was used in broiler feed and found to have significant immunostimulating effects. Based on the results of the current study, oral administration of ACH via feed delivery did not decrease its immunostimulating effects, as observed in Trial 2. The reason may be that the molecular weight of ACH was much smaller than that of APS, so it was more easily absorbed by the intestinal enterocytes and finally utilized by the immune system (Tian and Feng, 1994). ND HI titer is an indicator of specific humoral immunity. It is of great importance to improve HI titers. We demonstrated that ACH but not APS significantly increased ND HI titers, indicating an enhancement of humoral immunity. The authors thought that the mechanism may lie in two points. First, ACH could stimulate the development of bursa of Fabricius as reflected by bursa index, while bursa of Fabricius is the center of humoral immunity. Second, ACH could be easily absorbed and utilized by the immune system as discussed
FIGURE 4. Effects of herbal polysaccharides on interleukin-2 (IL-2) release from splenocytes. IL-2 production was measured by Mossman’s colorimetric method, and expressed as an optical density 570 nm (OD 570 nm). Each dilution was tested in triplicates. a,bMeans represented at each point without a common letter are different (P < 0.05).
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above. Therefore, ACH may be useful to improve broilers’ immunity against ND in case of vaccination failure. In the present study, it was found that large-molecularweight Chinese herbal polysaccharide, APS, was not as effective as the small molecular weight polysaccharide, ACH, in eliciting the immunity of broilers. In vitro, both polysaccharides increased splenocyte proliferation, NO, and IL-2 production in a quite similar, dose-dependent manner. However, oral administration of APS via feed delivery may decrease its efficacy due to intestinal enzyme digestion and its difficulty passing through the intestinal barrier and entering the general circulation. Thus, the smaller-molecular-weight Chinese herbal polysaccharide, ACH, may have more potential to be utilized as a feed additive to improve the immunity of young broilers.
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