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Feasibility of replacing antibiotic feed promoters with the Chinese traditional herbal medicine Bazhen in weaned piglets
Livestock Production Science 107 (2007) 97 – 102 www.elsevier.com/locate/livsci
Feasibility of replacing antibiotic feed promoters with the Chinese traditional herbal medicine Bazhen in weaned piglets T.F. Lien ⁎, Y.M. Horng, C.P. Wu Department of Animal Science, National Chiayi University, Chiayi, Taiwan, ROC Received 14 October 2005; received in revised form 6 September 2006; accepted 11 September 2006
Abstract This study investigated the feasibility of replacing antibiotic as growth promoter for weaned piglets with the Chinese traditional herbal medicine Bazhen. Thirty-six weaned piglets (average initial body weight 8.92 ± 1.18 kg) were used in this study. Pigs were blocked according to weight, sex and litter origin, and then randomly assigned to control (basal diet), antibiotics (100 ppm chlortetracycline and 100 ppm oxytetracycline) and Bazhen (1%) groups, with three pens per group. Piglets had free access to water and feed during this 8-week study period. Experimental results indicated that average daily gain of the Bazhen group was higher than that of the control group, but lower than that of the antibiotics group. However, feed intake was reduced while feed to gain ratio was improved in the Bazhen group compared to the control and antibiotics groups. The Bazhen group showed higher neutrophil activity and IgG levels than the control group. The increase in white blood cell counts of the Bazhen group when challenged with Escherichia coli lipopolysaccharide was higher than that in the control and antibiotics groups. Moreover, IL-6 and TNF-α (tumor necrosis factor-α) levels were higher in the Bazhen group than in the antibiotics group. Red blood cells and γglobulin levels were higher in the Bazhen group than in the antibiotics group and in the control group, respectively. On the other hand, the antibiotics group displayed the highest amount of SRBC (sheep red blood cell) antibody titers, and differed significantly from the control group. Some immune responses such as white and red blood cells, IL-6 and TNF-α levels were greater at weaning in the piglets of the Bazhen supplemented group than in those of the antibiotics group. Thus, Bazhen could be a potential substitute for antibiotics (chlorotetracycline and oxytetracycline) in weaned piglets feed. © 2006 Elsevier B.V. All rights reserved. Keywords: Bazhen; Antibiotics; Immune response; Piglets
1. Introduction Antibiotics are generally added to animal diets to maintain animal health and improve growth performance. However, their use for therapeutic and growth improvement purposes at least in Europe is not allowed. Therefore, new immuno-potentiators with high efficiency and low toxicity are required. ⁎ Corresponding author. Tel.:+886 5 2717536. E-mail address: [email protected] (T.F. Lien). 1871-1413/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2006.09.008
Classical Chinese pharmacopoeia describes numerous herbal formulations that are used to treat a wide variety of diseases and conditions. Many Chinese herbal medicines have been reported to boost immune system efficiency (Hu, 1997; Liu et al., 1998; Kong et al., 2004) and such medicines offer a great potential for practical application. Bazhen is rich in flavonoids and polyphenols. Recent reports on Bazhen demonstrated that it could stimulate immuno-competence (Pang and Zheng, 2001; Wang and Chen, 2000), and it was used for hundreds of years in China. A previous study on chickens also showed that
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T.F. Lien et al. / Livestock Production Science 107 (2007) 97–102
Bazhen can elevate γ-globulin levels and antioxidant function (Lien et al., unpublished). We thus posited that Bazhen may be beneficial for pig immune response. This study, therefore, investigated the effects of supplemental Bazhen on growth performance and immune response in piglets, and assessed its feasibility as a substitute for supplemental antibiotics.
were provided ad libitum throughout the 8-week experiment. The experimental animals were reared following the guidelines in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999). Pigs were vaccinated during the 1st and 3rd weeks of the experiment for classical swine fever, and foot-and-mouth disease during the 2nd week of the experiment.
2. Materials and methods 2.3. Methods and measurements 2.1. Preparation of Bazhen powder Bazhen, a traditional Chinese prescription medicine, is composed of eight herbal medicines. Bazhen is commonly used to adjust qi (a form of “vital energy” that is important to general health according to Chinese belief) and induce hematopoiesis to remedy poor appetite, extreme tiredness, and dizziness. The following eight Chinese herbal medicines were purchased from a traditional Chinese drug market in Taiwan: Atractylodis Rhizoma (Atractylodes ovata A.P. De Candolle); Codonopsitis Radix (Codonopsis pilosula Nannfeldt); Hoelen (Poria cocos Wolf); Glycyrrhizae Radix (Glycyrrihiza uralensis Fischer et DC.); Angelicae Sinensis Radix (Angelica sinensis Diels); Ligustici Chuanxiong Rhizoma (Ligusticum chuanxiong Hortorum); Paeoniae Raix Rubra (Paeonia albiflora Pallas var. trichocarpa Bunge); and Rehmanniae Radix et Rhizoma ( Rehmannia glutinosa Liboschitz). Each herb was pulverized into a fine powder and passed through an 80-mesh sieve. Equal amounts of all eight Chinese herbal powders were mixed together as feeding additive. Polyphenol and flavonoid components of Bazhen were then analyzed. Polyphenol level was determined using the procedure proposed by Singleton and Rossi (1965). Flavonoid level was examined following the method described by Jia et al. (1999). 2.2. Animal treatment Thirty-six crossbred (Landrace × Duroc) weaned pigs (4 weeks old, average initial body weight 8.92± 1.18 kg) were blocked according to weight, sex and litter origin, and then randomly allotted to three dietary groups. Each group comprised three pens (4 pigs/per pen): control fed a basal diet (Table 1), formulated according to NRC (1998), antibiotics (supplemented with 100 ppm chlortetracycline and 100 ppm oxytetracycline), and Bazhen (basal diet supplemented with 1% Bazhen). Piglets received 2 successive feeds that were switched at 10 kg body weight. Feed and water
Mean daily body weight gain, feed intake and feed to gain ratio were determined for each pen. Escherichia coli lipopolysaccharide (LPS), an endotoxin, was applied as a stress-inducing agent at a dosage of 100 μg/kg BW injected i.m. at the 5th week of the experiment. Blood was sampled at 0 h and 24 h after LPS injection. White blood cell counts were measured using a microcell counter (Sysmex F-800, Japan). Red blood cells, mean corpuscular volume
Table 1 Composition of basal diets, g/kg (as feed basis) Ingredients
Piglet weight 8–10 kg
10–35 kg
Yellow corn meal Steamed corn meal Full fat soybean meal Fish meal Skim milk powder Whey Fat Limestone, pulverized Calcium phosphate, dibasic Vitamin premix a Mineral premix b Iodine salt Total
– 325 390.3 50 30 150 32.4 5.2 4.1 5 5 3 1000
475.8 – 325.6 50 – 100 24.7 4.7 6.2 5 5 3 1000
Calculated value Crude protein (%) Metabolizable energy (MJ/kg) Lysine (%) Methionine + cystine (%) Calcium (%) Total phosphorus (%)
23.70 13.66 1.52 0.77 0.81 0.65
20.90 13.66 1.24 0.66 0.70 0.60
a Vitamin premix supplied per kilogram of diet: vitamin A, 8000 IU; vitamin D3, 1200 IU; vitamin E, 40 IU; vitamin K3, 4 mg; vitamin B1, 0.6 mg; vitamin B2, 8 mg; vitamin B12, 40 μg; niacin, 80 mg; pantothenic acid, 34 mg; biotin, 50 μg; folic acid, 750 μg. b Mineral premix supplied per kilogram of diet: Fe (FeSO4·7H2O, 20.09% Fe), 140 mg; Cu (CuSO4·5H2O, 25.45% Cu), 17 mg; Mn (MnSO4·H2O, 32.49% Mn), 33 mg; Zn (ZnSO4, 80.35% Zn), 75 mg; Se (NaSeO3, 45.56% Se), 0.17 mg; Co (CoSO4·H2O, 32% Co), 0.7 mg.
T.F. Lien et al. / Livestock Production Science 107 (2007) 97–102 Table 2 Growth performance of weaned piglets1 Items Average daily feed intake (kg/day) Average daily gain (kg) Feed/gain ratio
Control
Bazhen a
Antibiotics b
0.78 ± 0.03
0.67 ± 0.01
0.77 ± 0.02 a
0.402 ± 0.005c
0.421 ± 0.004b
0.448 ± 0.011a
1.95 ± 0.03a
1.60 ± 0.02 c
1.71 ± 0.02b
1
Means ± S.E. (n = 3). Means in the same row with different superscripts are significantly different (P b 0.05). Antibiotics: supplemented with 100 ppm chlortetracycline and 100 ppm oxytetracycline.
a,b
99
against sheep red blood cells (SRBC), 2 mL 2.5% of SRBC isolated from black-belley sheep was injected twice at weeks 5 and 6. Blood samples were taken at week 7. Antibody titers against SRBC were measured in duplicate using the hemagglutination procedure presented by Van Heugten and Spears (1997) to investigate humoral immune response. To examine the mitogen of phytohemagglutinin (PHA) skin swelling response, 1 mL of a solution containing 150 μg/mL PHAwere injected s.c. at week 7 and the swollen ear skin thickness was measured using a micrometer 24 h after injection, as described by Kegley and Spears (1995). 2.4. Statistical analysis
(MCV) and mean corpuscular hemoglobin concentration (MCHC) were measured with a microcell counter at the end of the experiment. Serum IL-6 (interleukin6) and TNF-α (tumor necrosis factor-α) levels after LPS injection were measured with an ELISA kit (R&D System Inc., USA). Neutrophil activity was examined at the end of the experiment employing the method described by Morrow-Tesch and Anderson (1994). Briefly, 100 μL fresh blood and 100 μL nitroblue tetrazolium (NBT) were added to each well and incubated at 37 °C for 10 min, after that samples were smeared over a slide and stained with May Grunwald and Giemsa. Finally, the cells were viewed with a microscope to count intracellular formazan deposits. Serum immunoglobulin G (IgG) levels were determined in triplicate at the end of the experiment using the ELISA procedure described by Leslie and Frank (1989). Rabbit anti-porcine IgG (Sigma Chemico Co., USA) diluted 1:1000 with coating buffer was used as the primary antibody, while rabbit anti-porcine IgG conjugated with horseradish peroxidase diluted 1:1000 with PBS buffer was used as the secondary antibody. Purified porcine IgG was adopted as the standard. Total globulin and γ-globulin levels were measured using electrophoresis. The percentage of each gel band was measured with a densitometer (Helena Co., 8JF00105, USA). Classical swine fever antibody titers were determined with an ELISA kit (JENO, Korea). To examine the antigen of ovalbumin response, 1 mL of a solution containing 0.5 mg ovalbumin in a mixture of 0.5 mL Freund's incomplete adjuvant was injected i.m. at weeks 5 and 6. Blood samples were taken after 2 weeks. Ovalbumin antibody titers were measured in duplicate by an ELISA scheme with rabbit anti-ovalbumin IgG conjugated horseradish peroxidase diluted 1:1000 with potassium phosphate buffer (Fitzgerald, Inc., USA) as described by Van Heugten and Spears (1997). To measure antibody titers
The statistical analysis software (Version 9.1.3, SAS, 1998) was applied to analyze the variance between groups, and the significance was determined with Duncan's multiple range test (SAS, 1998) according to the following model, Y ¼ l þ Ti þ Pj þ eijk where Y is the dependent variable, μ represents the mean, T is the treatment effect, P is the pen effect and e is the random residual error term. 3. Results The Bazhen components were analyzed and high levels of polyphenol (85.03 mg/g) and flavonoid (18.32 mg/g) were found. Table 2 lists the effects of Bazhen supplementation on growth performance of weaned piglets. The average daily gain of the Bazhen group was higher than that of the control group (P b 0.05), but lower than that of the antibiotics group (P b 0.05). However, the Bazhen group Table 3 Immune responses in weaned piglets after LPS challenge at the 5th week of the experiment1 Items
Control
Bazhen c
Increased white blood cell 7.46 ± 1.08 (×103/μL) Interleukin-6 (pg/mL) 1285 ± 18a,b Tumor necrosis factor-α 1036 ± 63a,b (pg/mL) 1
Antibiotics a
11.09 ± 0.81
9.08 ± 0.11b
1318 ± 64a 1180 ± 63a
1114 ± 15 b 1028 ± 99 b
Means ± S.E. (n = 3). Means in the same row with different superscripts are significantly different (P b 0.05). LPS: Escherichia coli lipopolysaccharide. 100 μg LPS/kg BW was injected i.m. at the 5th week of the experiment. Blood was sampled at 0 and 24 h after LPS injection.
a, b
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displayed reduced feed intake and improved feed to gain ratio compared to the control and antibiotics groups (P b 0.05). One pig in the control group and one in the Bazhen group died, the death in the Bazhen group being caused by LPS injection. Table 3 shows the effects of Bazhen supplementation on the immune responses of weaned piglets after LPS challenge at the 5th week of experiment. The change in white blood cell counts after 24 h for LPS challenged pigs in the Bazhen group was the greatest, followed by that in the antibiotics group, and differed significantly from that in the control group (P b 0.05). Cytokine IL-6 and TNF-γ levels in the Bazhen group were higher than those in the antibiotics group. Table 4 lists the effects of Bazhen supplementation on the immune responses of weaned piglets at the 8th week of experiment. IgG level and neutrophil activity in the Bazhen and antibiotics groups were higher than those in the control group (P b 0.05). SRBC antibody titers in the antibiotics group were greater than those in the control group (P b 0.05). Other parameters such as classical swine fever antibody titers, antibodies against ovalbumin and PHA skin challenge showed no difference between the groups (P N 0.05). Table 5 lists the effects of Bazhen supplementation on serum traits of weaned piglets. Analytical results revealed that the Bazhen group had a higher red blood cell level than the antibiotics group, and their γ-globulin level exceeded that of the control group. Other parameters including mean corpuscular volume, mean corpuscular hemoglobin concentrations, albumin and Table 4 Immune responses of weaned piglets at the 8th week of the experiment1 Items
Control
Bazhen
Antibiotics
Immunoglobulin G 141.98 ± 0.51b 176.33 ± 0.51a 174.92 ± 0.29 a (mg/dL) Neutrophil activity 13.44 ± 1.02b 20.44 ± 0.31a 18.75 ± 1.31a (%) Antibody titer of 8.25 ± 0.25 8.58 ± 0.42 8.75 ± 0.25 classical swine fever (log2) SRBC antibody titer 3.25 ± 0.01a,b 3.29 ± 0.08 a 3.17 ± 0.07b (log2) Ovalbumin antibody 20.90 ± 1.17 22.61 ± 0.78 23.42 ± 0.50 titer (mg/dL) PHA skin swelling 5.09 ± 0.41 4.38 ± 0.61 4.46 ± 0.81 test (mm) 1
Means ± S.E. (n = 3). Means in the same row with different superscripts are significantly different (P b 0.05). SRBC: sheep red blood cells. PHA: phytohemagglutinin.
a, b
Table 5 Serum traits of weaned piglets at the 8th week of the experiment1 Item
Control 6
Red blood cells (×10 / μL) MCV (fL) MCHC (g/dL) Albumin (%) Total globulin (%) Total globulin/albumin (%) γ-Globulin (%)
Bazhen a,b
Antibiotics a
6.22 ± 0.06b
6.28 ± 0.22
6.69 ± 0.28
52.41 ± 0.77 31.25 ± 0.65 44.82 ± 4.08 55.18 ± 4.08 1.23 ± 0.33
54.76 ± 1.55 30.52 ± 0.53 40.67 ± 4.43 59.32 ± 4.43 1.46 ± 0.28
17.93 ± 1.63b
21.49 ± 1.03a 18.52 ± 1.99a,b
56.28 ± 2.33 31.20 ± 0.68 41.61 ± 0.84 58.39 ± 0.84 1.41 ± 0.05
1
Means ± S.E. (n = 3). Means in the same row with different superscripts are significantly different (P b 0.05). MCV: mean corpuscular volume. MCHC: mean corpuscular hemoglobin concentration.
a, b
total globulin did not differ significantly among the three groups (P N 0.05). 4. Discussion The Bazhen pigs displayed a reduced feed intake, and their average daily gain was lower than that of the antibiotics group. But their average daily gain was greater than that of the control group, and consequently, their feed to gain ratio was improved. Recent reports have verified that Bazhen can strengthen the immune system. Pang and Zheng (2001) indicated that a diet supplemented with Bazhen stimulated immune ability in old mice. Ju et al. (2001) also demonstrated that the proliferation ability of T and B cells of mice with damaged bone marrow was restored to normal levels by feeding a diet supplemented with Bazhen. Additionally, Wang and Chen (2000) reported that a diet supplemented with Bazhen improved the activity of spleen lymphocytes and the secretion of cytokine IL-2 in humans. Moreover, Ju and Tang (2000) demonstrated that supplementing mice diet with Bazhen stimulated bone marrow cell proliferation and restored cytokines IL-1, IL-2 and IL-3 to normal levels. The results of this study are in agreement with those of previous studies. Thus, Bazhen may replace antibiotics of chlortetracycline and oxytetracycline in disease prevention supplementation in the present experimental conditions. The lipopolysaccharide of bacterial cell wall component is a stress-inducing agent. Because LPS can induce inflammatory responses that mediate aspects of the acute phase response, LPS injection results in white blood cell proliferation and TNF-α and IL-6 release (Panesar et al., 1999; Ghezzi et al., 2000; Peck et al.,
T.F. Lien et al. / Livestock Production Science 107 (2007) 97–102
2004). White blood cell level was higher in the Bazhen group than in the control and antibiotics groups after LPS injection. Pigs in the Bazhen group also had greater TNF-α and IL-6 levels than in the antibiotics group, indicating that their immune system had an increasing response ability. Interleukin-6, produced mainly by monocytes, is a multiple functional cytokine involved in many aspects of systemic inflammatory response (Akira et al., 1990). It stimulates B cell and macrophage proliferation and differentiation, and promotes antibody (e.g. IgG) production (Urashima et al., 1996). Neutrophil activity and IgG levels were highest in the Bazhen group, which may be possibly due to the high IL-6 level. Neutrophils are critical cells in humoral and innate immunity and play vital roles in phagocytosis and bacteria killing. They account for the majority (about 40–75%) of leukocytes, and can rapidly respond to chemokines released by inflammatory regions when induced by LPS challenge. Furthermore, neutrophils have high a phagocytosis capacity to kill bacteria (Burg and Pillinger, 2001). Measuring neutrophil activity also indicates the characteristics of its phagocytosis. Thus, increased activity means that neutrophils have strong phagocytosis ability (Morrow-Tesch and Anderson, 1994). In this study, γ-globulin level was also increased in the Bazhen group; γ-globulin primarily contains immunoglobulin, and thus, the immunoglobulin G level in the Bazhen group was also elevated. This study indicated that red blood cell counts in the Bazhen group were higher than in the control and antibiotics groups. Bazhen has also been used to remedy symptom of anemia; Ju et al. (2001) indicated that Bazhen enhanced hematopoiesis in γ-ray damaged mice. The addition of Bazhen to piglet diet had no effect on their specific antibody response: the SRBC antibody titer was higher in the antibiotics group than in the control group, but not in the Bazhen group, and classical swine fever and ovalbumin antibody titers did not differ between groups. Some immune response such as white and red blood cells, IL-6, and TNF-α levels were greater at weanling in the Bazhen supplemented group than in the antibiotics group. Bazhen could then be a potential substitute for antibiotics (chlortetracycline and oxytetracycline) in piglet diet. However, these results need to be confirmed on a greater number of pigs, and the dose– effect relationship needs to be further investigated. 5. Conclusion Bazhen supplementation in the diet of weaned piglets induced higher average daily gain than in the control
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group, though gains remained lower than in the antibiotics group. However, feed intake was reduced with Bazhen supplementation and feed to gain ratio was improved. Bazhen supplementation stimulated the following immune response parameters than control group: IgG, γ-globulin levels, neutrophil activity, and white blood cell counts following challenge with LPS. The immune response results of white and red blood cells, IL-6 and TNF-α levels in Bazhen group were greater than those obtained with antibiotic supplementation. This investigation therefore suggests that the addition of Bazhen in weaned piglet diets may be a potential substitute for antibiotic (chlortetracycline and oxytetracycline) supplementation. Acknowledgment The authors would like to thank the Council of Agriculture Executive Yuan of the Republic of China, Taiwan, for financially supporting this research. References Akira, S., Hirano, T., Taga, T., Kishimoto, T., 1990. Biology of multifunctional cytokines: IL-6 and related molecules (IL-1 and TNF). FASEB J. 4, 2860–2867. Burg, N.D., Pillinger, M.H., 2001. The neutrophil: function and regulation in innate and humoral immunity. Clin. Immunol. 99, 7–17. FASS, 1999. Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Federation of Animal Science Societies. Ghezzi, P., Sacco, S., Agnello, D., Marullo, A., Caselli, G., Bertini, R., 2000. LPS induces IL-6 in the brain and in serum largely through TNF production. Cytokine 12, 205–1210. Hu, Y.L., 1997. Progress in the study of immunopharmacology of Chinese herbal medicine. Chin. J. Immunol. 3, 96–98. Jia, Z., Tang, M., Wu, J., 1999. The determination of flavonoid content in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64, 555–559. Ju, H.I., Tang, I.I., 2000. Effect of Bazhen decoction on the bone marrow cells and related cell factors of mice exposed to Co γ-ray. J. Chin. Immunol. 16, 43–48. Ju, H.I., Wu, H., Tang, I.I., 2001. Effects of Bazhen tang on the immune and hematopoiesis of mice that damaged by Co γ-ray. J. Beijing Chin. Med. Univ. 24, 40–45. Kegley, E.B., Spears, J.W., 1995. Immune response, glucose metabolism, and performance of stressed feeder calves fed inorganic or organic chromium. J. Anim. Sci. 73, 2721–2726. Kong, X., Hu, Y., Rui, R., Wang, D., Li, X., 2004. Effects of Chinese herbal medicinal ingredients on peripheral lymphocyte proliferation and serum antibody titer after vaccination in chicken. Intern. Immunopharm. 4, 975–982. Leslie, H., Frank, C.H., 1989. Practical Immunology, Third ed., p. 23. Liu, J.X., Wang, C.Y., Bai, Y.M., Fang, Y.P., 1998. Influence of Astragalus and other nine natural medicines on Newscastle disease antibody titers (ND-HI), coefficient of the immune organs and gains of chicks. Chin. J. Vet. Sci. Technol. 12, 30–32.
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Morrow-Tesch, J., Anderson, G., 1994. Immunological and hematological characterizations of the wasting pig syndrome. J. Anim. Sci. 72, 976–983. National Research Council, 1998. Nutrient requirements of swine. National Academy Press, Washington D.C. Panesar, N., Tolman, K., Mazuski, J.E., 1999. Endotoxin stimulates hepatocyte interleukin-6 production. J. Surg. Res. 85, 251–258. Pang, H.P., Zheng, X., 2001. Study of Bazhen tang on cellular immunology. Chin. Mod. Apply Pharmacol. 18, 279–281. Peck, O.M., Williams, D.L., Breuel, K.F., Kalbfleisch, J.H., Fan, H., Tempel, G.E., Teti, G., Ook, J.A., 2004. Differential regulation of cytokine and chemokine production in lipopolysaccharide-induced tolerance and priming. Cytokine 26, 202–208.
Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 144–158. Statistical Analysis System Institute, 1998. SAS/STAT User's guide, Version 6.06, 4th ed. Cary, NC. Urashima, M., Chauhan, D., Hatziyanni, M., Ogata, A., Hollenbaugh, D., Aruffo, A., Anderson, K.C., 1996. CD40 ligand triggers interleukin-6 mediated B cell differentiation. Leuk. Res. 20, 507–515. Van Heugten, E., Spears, J.W., 1997. Immune response and growth of stressed weaning pigs fed diets supplemented with organic or inorganic forms of chromium. J. Anim. Sci. 75, 409–416. Wang, B.I., Chen, I.C., 2000. Bazhen tang stimulated animal immune function. Chin. Med. Study 13, 20–23.
Feasibility of replacing antibiotic feed promoters with the Chinese traditional herbal medicine Bazhen in weaned piglets T.F. Lien ⁎, Y.M. Horng, C.P. Wu Department of Animal Science, National Chiayi University, Chiayi, Taiwan, ROC Received 14 October 2005; received in revised form 6 September 2006; accepted 11 September 2006
Abstract This study investigated the feasibility of replacing antibiotic as growth promoter for weaned piglets with the Chinese traditional herbal medicine Bazhen. Thirty-six weaned piglets (average initial body weight 8.92 ± 1.18 kg) were used in this study. Pigs were blocked according to weight, sex and litter origin, and then randomly assigned to control (basal diet), antibiotics (100 ppm chlortetracycline and 100 ppm oxytetracycline) and Bazhen (1%) groups, with three pens per group. Piglets had free access to water and feed during this 8-week study period. Experimental results indicated that average daily gain of the Bazhen group was higher than that of the control group, but lower than that of the antibiotics group. However, feed intake was reduced while feed to gain ratio was improved in the Bazhen group compared to the control and antibiotics groups. The Bazhen group showed higher neutrophil activity and IgG levels than the control group. The increase in white blood cell counts of the Bazhen group when challenged with Escherichia coli lipopolysaccharide was higher than that in the control and antibiotics groups. Moreover, IL-6 and TNF-α (tumor necrosis factor-α) levels were higher in the Bazhen group than in the antibiotics group. Red blood cells and γglobulin levels were higher in the Bazhen group than in the antibiotics group and in the control group, respectively. On the other hand, the antibiotics group displayed the highest amount of SRBC (sheep red blood cell) antibody titers, and differed significantly from the control group. Some immune responses such as white and red blood cells, IL-6 and TNF-α levels were greater at weaning in the piglets of the Bazhen supplemented group than in those of the antibiotics group. Thus, Bazhen could be a potential substitute for antibiotics (chlorotetracycline and oxytetracycline) in weaned piglets feed. © 2006 Elsevier B.V. All rights reserved. Keywords: Bazhen; Antibiotics; Immune response; Piglets
1. Introduction Antibiotics are generally added to animal diets to maintain animal health and improve growth performance. However, their use for therapeutic and growth improvement purposes at least in Europe is not allowed. Therefore, new immuno-potentiators with high efficiency and low toxicity are required. ⁎ Corresponding author. Tel.:+886 5 2717536. E-mail address: [email protected] (T.F. Lien). 1871-1413/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2006.09.008
Classical Chinese pharmacopoeia describes numerous herbal formulations that are used to treat a wide variety of diseases and conditions. Many Chinese herbal medicines have been reported to boost immune system efficiency (Hu, 1997; Liu et al., 1998; Kong et al., 2004) and such medicines offer a great potential for practical application. Bazhen is rich in flavonoids and polyphenols. Recent reports on Bazhen demonstrated that it could stimulate immuno-competence (Pang and Zheng, 2001; Wang and Chen, 2000), and it was used for hundreds of years in China. A previous study on chickens also showed that
98
T.F. Lien et al. / Livestock Production Science 107 (2007) 97–102
Bazhen can elevate γ-globulin levels and antioxidant function (Lien et al., unpublished). We thus posited that Bazhen may be beneficial for pig immune response. This study, therefore, investigated the effects of supplemental Bazhen on growth performance and immune response in piglets, and assessed its feasibility as a substitute for supplemental antibiotics.
were provided ad libitum throughout the 8-week experiment. The experimental animals were reared following the guidelines in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999). Pigs were vaccinated during the 1st and 3rd weeks of the experiment for classical swine fever, and foot-and-mouth disease during the 2nd week of the experiment.
2. Materials and methods 2.3. Methods and measurements 2.1. Preparation of Bazhen powder Bazhen, a traditional Chinese prescription medicine, is composed of eight herbal medicines. Bazhen is commonly used to adjust qi (a form of “vital energy” that is important to general health according to Chinese belief) and induce hematopoiesis to remedy poor appetite, extreme tiredness, and dizziness. The following eight Chinese herbal medicines were purchased from a traditional Chinese drug market in Taiwan: Atractylodis Rhizoma (Atractylodes ovata A.P. De Candolle); Codonopsitis Radix (Codonopsis pilosula Nannfeldt); Hoelen (Poria cocos Wolf); Glycyrrhizae Radix (Glycyrrihiza uralensis Fischer et DC.); Angelicae Sinensis Radix (Angelica sinensis Diels); Ligustici Chuanxiong Rhizoma (Ligusticum chuanxiong Hortorum); Paeoniae Raix Rubra (Paeonia albiflora Pallas var. trichocarpa Bunge); and Rehmanniae Radix et Rhizoma ( Rehmannia glutinosa Liboschitz). Each herb was pulverized into a fine powder and passed through an 80-mesh sieve. Equal amounts of all eight Chinese herbal powders were mixed together as feeding additive. Polyphenol and flavonoid components of Bazhen were then analyzed. Polyphenol level was determined using the procedure proposed by Singleton and Rossi (1965). Flavonoid level was examined following the method described by Jia et al. (1999). 2.2. Animal treatment Thirty-six crossbred (Landrace × Duroc) weaned pigs (4 weeks old, average initial body weight 8.92± 1.18 kg) were blocked according to weight, sex and litter origin, and then randomly allotted to three dietary groups. Each group comprised three pens (4 pigs/per pen): control fed a basal diet (Table 1), formulated according to NRC (1998), antibiotics (supplemented with 100 ppm chlortetracycline and 100 ppm oxytetracycline), and Bazhen (basal diet supplemented with 1% Bazhen). Piglets received 2 successive feeds that were switched at 10 kg body weight. Feed and water
Mean daily body weight gain, feed intake and feed to gain ratio were determined for each pen. Escherichia coli lipopolysaccharide (LPS), an endotoxin, was applied as a stress-inducing agent at a dosage of 100 μg/kg BW injected i.m. at the 5th week of the experiment. Blood was sampled at 0 h and 24 h after LPS injection. White blood cell counts were measured using a microcell counter (Sysmex F-800, Japan). Red blood cells, mean corpuscular volume
Table 1 Composition of basal diets, g/kg (as feed basis) Ingredients
Piglet weight 8–10 kg
10–35 kg
Yellow corn meal Steamed corn meal Full fat soybean meal Fish meal Skim milk powder Whey Fat Limestone, pulverized Calcium phosphate, dibasic Vitamin premix a Mineral premix b Iodine salt Total
– 325 390.3 50 30 150 32.4 5.2 4.1 5 5 3 1000
475.8 – 325.6 50 – 100 24.7 4.7 6.2 5 5 3 1000
Calculated value Crude protein (%) Metabolizable energy (MJ/kg) Lysine (%) Methionine + cystine (%) Calcium (%) Total phosphorus (%)
23.70 13.66 1.52 0.77 0.81 0.65
20.90 13.66 1.24 0.66 0.70 0.60
a Vitamin premix supplied per kilogram of diet: vitamin A, 8000 IU; vitamin D3, 1200 IU; vitamin E, 40 IU; vitamin K3, 4 mg; vitamin B1, 0.6 mg; vitamin B2, 8 mg; vitamin B12, 40 μg; niacin, 80 mg; pantothenic acid, 34 mg; biotin, 50 μg; folic acid, 750 μg. b Mineral premix supplied per kilogram of diet: Fe (FeSO4·7H2O, 20.09% Fe), 140 mg; Cu (CuSO4·5H2O, 25.45% Cu), 17 mg; Mn (MnSO4·H2O, 32.49% Mn), 33 mg; Zn (ZnSO4, 80.35% Zn), 75 mg; Se (NaSeO3, 45.56% Se), 0.17 mg; Co (CoSO4·H2O, 32% Co), 0.7 mg.
T.F. Lien et al. / Livestock Production Science 107 (2007) 97–102 Table 2 Growth performance of weaned piglets1 Items Average daily feed intake (kg/day) Average daily gain (kg) Feed/gain ratio
Control
Bazhen a
Antibiotics b
0.78 ± 0.03
0.67 ± 0.01
0.77 ± 0.02 a
0.402 ± 0.005c
0.421 ± 0.004b
0.448 ± 0.011a
1.95 ± 0.03a
1.60 ± 0.02 c
1.71 ± 0.02b
1
Means ± S.E. (n = 3). Means in the same row with different superscripts are significantly different (P b 0.05). Antibiotics: supplemented with 100 ppm chlortetracycline and 100 ppm oxytetracycline.
a,b
99
against sheep red blood cells (SRBC), 2 mL 2.5% of SRBC isolated from black-belley sheep was injected twice at weeks 5 and 6. Blood samples were taken at week 7. Antibody titers against SRBC were measured in duplicate using the hemagglutination procedure presented by Van Heugten and Spears (1997) to investigate humoral immune response. To examine the mitogen of phytohemagglutinin (PHA) skin swelling response, 1 mL of a solution containing 150 μg/mL PHAwere injected s.c. at week 7 and the swollen ear skin thickness was measured using a micrometer 24 h after injection, as described by Kegley and Spears (1995). 2.4. Statistical analysis
(MCV) and mean corpuscular hemoglobin concentration (MCHC) were measured with a microcell counter at the end of the experiment. Serum IL-6 (interleukin6) and TNF-α (tumor necrosis factor-α) levels after LPS injection were measured with an ELISA kit (R&D System Inc., USA). Neutrophil activity was examined at the end of the experiment employing the method described by Morrow-Tesch and Anderson (1994). Briefly, 100 μL fresh blood and 100 μL nitroblue tetrazolium (NBT) were added to each well and incubated at 37 °C for 10 min, after that samples were smeared over a slide and stained with May Grunwald and Giemsa. Finally, the cells were viewed with a microscope to count intracellular formazan deposits. Serum immunoglobulin G (IgG) levels were determined in triplicate at the end of the experiment using the ELISA procedure described by Leslie and Frank (1989). Rabbit anti-porcine IgG (Sigma Chemico Co., USA) diluted 1:1000 with coating buffer was used as the primary antibody, while rabbit anti-porcine IgG conjugated with horseradish peroxidase diluted 1:1000 with PBS buffer was used as the secondary antibody. Purified porcine IgG was adopted as the standard. Total globulin and γ-globulin levels were measured using electrophoresis. The percentage of each gel band was measured with a densitometer (Helena Co., 8JF00105, USA). Classical swine fever antibody titers were determined with an ELISA kit (JENO, Korea). To examine the antigen of ovalbumin response, 1 mL of a solution containing 0.5 mg ovalbumin in a mixture of 0.5 mL Freund's incomplete adjuvant was injected i.m. at weeks 5 and 6. Blood samples were taken after 2 weeks. Ovalbumin antibody titers were measured in duplicate by an ELISA scheme with rabbit anti-ovalbumin IgG conjugated horseradish peroxidase diluted 1:1000 with potassium phosphate buffer (Fitzgerald, Inc., USA) as described by Van Heugten and Spears (1997). To measure antibody titers
The statistical analysis software (Version 9.1.3, SAS, 1998) was applied to analyze the variance between groups, and the significance was determined with Duncan's multiple range test (SAS, 1998) according to the following model, Y ¼ l þ Ti þ Pj þ eijk where Y is the dependent variable, μ represents the mean, T is the treatment effect, P is the pen effect and e is the random residual error term. 3. Results The Bazhen components were analyzed and high levels of polyphenol (85.03 mg/g) and flavonoid (18.32 mg/g) were found. Table 2 lists the effects of Bazhen supplementation on growth performance of weaned piglets. The average daily gain of the Bazhen group was higher than that of the control group (P b 0.05), but lower than that of the antibiotics group (P b 0.05). However, the Bazhen group Table 3 Immune responses in weaned piglets after LPS challenge at the 5th week of the experiment1 Items
Control
Bazhen c
Increased white blood cell 7.46 ± 1.08 (×103/μL) Interleukin-6 (pg/mL) 1285 ± 18a,b Tumor necrosis factor-α 1036 ± 63a,b (pg/mL) 1
Antibiotics a
11.09 ± 0.81
9.08 ± 0.11b
1318 ± 64a 1180 ± 63a
1114 ± 15 b 1028 ± 99 b
Means ± S.E. (n = 3). Means in the same row with different superscripts are significantly different (P b 0.05). LPS: Escherichia coli lipopolysaccharide. 100 μg LPS/kg BW was injected i.m. at the 5th week of the experiment. Blood was sampled at 0 and 24 h after LPS injection.
a, b
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displayed reduced feed intake and improved feed to gain ratio compared to the control and antibiotics groups (P b 0.05). One pig in the control group and one in the Bazhen group died, the death in the Bazhen group being caused by LPS injection. Table 3 shows the effects of Bazhen supplementation on the immune responses of weaned piglets after LPS challenge at the 5th week of experiment. The change in white blood cell counts after 24 h for LPS challenged pigs in the Bazhen group was the greatest, followed by that in the antibiotics group, and differed significantly from that in the control group (P b 0.05). Cytokine IL-6 and TNF-γ levels in the Bazhen group were higher than those in the antibiotics group. Table 4 lists the effects of Bazhen supplementation on the immune responses of weaned piglets at the 8th week of experiment. IgG level and neutrophil activity in the Bazhen and antibiotics groups were higher than those in the control group (P b 0.05). SRBC antibody titers in the antibiotics group were greater than those in the control group (P b 0.05). Other parameters such as classical swine fever antibody titers, antibodies against ovalbumin and PHA skin challenge showed no difference between the groups (P N 0.05). Table 5 lists the effects of Bazhen supplementation on serum traits of weaned piglets. Analytical results revealed that the Bazhen group had a higher red blood cell level than the antibiotics group, and their γ-globulin level exceeded that of the control group. Other parameters including mean corpuscular volume, mean corpuscular hemoglobin concentrations, albumin and Table 4 Immune responses of weaned piglets at the 8th week of the experiment1 Items
Control
Bazhen
Antibiotics
Immunoglobulin G 141.98 ± 0.51b 176.33 ± 0.51a 174.92 ± 0.29 a (mg/dL) Neutrophil activity 13.44 ± 1.02b 20.44 ± 0.31a 18.75 ± 1.31a (%) Antibody titer of 8.25 ± 0.25 8.58 ± 0.42 8.75 ± 0.25 classical swine fever (log2) SRBC antibody titer 3.25 ± 0.01a,b 3.29 ± 0.08 a 3.17 ± 0.07b (log2) Ovalbumin antibody 20.90 ± 1.17 22.61 ± 0.78 23.42 ± 0.50 titer (mg/dL) PHA skin swelling 5.09 ± 0.41 4.38 ± 0.61 4.46 ± 0.81 test (mm) 1
Means ± S.E. (n = 3). Means in the same row with different superscripts are significantly different (P b 0.05). SRBC: sheep red blood cells. PHA: phytohemagglutinin.
a, b
Table 5 Serum traits of weaned piglets at the 8th week of the experiment1 Item
Control 6
Red blood cells (×10 / μL) MCV (fL) MCHC (g/dL) Albumin (%) Total globulin (%) Total globulin/albumin (%) γ-Globulin (%)
Bazhen a,b
Antibiotics a
6.22 ± 0.06b
6.28 ± 0.22
6.69 ± 0.28
52.41 ± 0.77 31.25 ± 0.65 44.82 ± 4.08 55.18 ± 4.08 1.23 ± 0.33
54.76 ± 1.55 30.52 ± 0.53 40.67 ± 4.43 59.32 ± 4.43 1.46 ± 0.28
17.93 ± 1.63b
21.49 ± 1.03a 18.52 ± 1.99a,b
56.28 ± 2.33 31.20 ± 0.68 41.61 ± 0.84 58.39 ± 0.84 1.41 ± 0.05
1
Means ± S.E. (n = 3). Means in the same row with different superscripts are significantly different (P b 0.05). MCV: mean corpuscular volume. MCHC: mean corpuscular hemoglobin concentration.
a, b
total globulin did not differ significantly among the three groups (P N 0.05). 4. Discussion The Bazhen pigs displayed a reduced feed intake, and their average daily gain was lower than that of the antibiotics group. But their average daily gain was greater than that of the control group, and consequently, their feed to gain ratio was improved. Recent reports have verified that Bazhen can strengthen the immune system. Pang and Zheng (2001) indicated that a diet supplemented with Bazhen stimulated immune ability in old mice. Ju et al. (2001) also demonstrated that the proliferation ability of T and B cells of mice with damaged bone marrow was restored to normal levels by feeding a diet supplemented with Bazhen. Additionally, Wang and Chen (2000) reported that a diet supplemented with Bazhen improved the activity of spleen lymphocytes and the secretion of cytokine IL-2 in humans. Moreover, Ju and Tang (2000) demonstrated that supplementing mice diet with Bazhen stimulated bone marrow cell proliferation and restored cytokines IL-1, IL-2 and IL-3 to normal levels. The results of this study are in agreement with those of previous studies. Thus, Bazhen may replace antibiotics of chlortetracycline and oxytetracycline in disease prevention supplementation in the present experimental conditions. The lipopolysaccharide of bacterial cell wall component is a stress-inducing agent. Because LPS can induce inflammatory responses that mediate aspects of the acute phase response, LPS injection results in white blood cell proliferation and TNF-α and IL-6 release (Panesar et al., 1999; Ghezzi et al., 2000; Peck et al.,
T.F. Lien et al. / Livestock Production Science 107 (2007) 97–102
2004). White blood cell level was higher in the Bazhen group than in the control and antibiotics groups after LPS injection. Pigs in the Bazhen group also had greater TNF-α and IL-6 levels than in the antibiotics group, indicating that their immune system had an increasing response ability. Interleukin-6, produced mainly by monocytes, is a multiple functional cytokine involved in many aspects of systemic inflammatory response (Akira et al., 1990). It stimulates B cell and macrophage proliferation and differentiation, and promotes antibody (e.g. IgG) production (Urashima et al., 1996). Neutrophil activity and IgG levels were highest in the Bazhen group, which may be possibly due to the high IL-6 level. Neutrophils are critical cells in humoral and innate immunity and play vital roles in phagocytosis and bacteria killing. They account for the majority (about 40–75%) of leukocytes, and can rapidly respond to chemokines released by inflammatory regions when induced by LPS challenge. Furthermore, neutrophils have high a phagocytosis capacity to kill bacteria (Burg and Pillinger, 2001). Measuring neutrophil activity also indicates the characteristics of its phagocytosis. Thus, increased activity means that neutrophils have strong phagocytosis ability (Morrow-Tesch and Anderson, 1994). In this study, γ-globulin level was also increased in the Bazhen group; γ-globulin primarily contains immunoglobulin, and thus, the immunoglobulin G level in the Bazhen group was also elevated. This study indicated that red blood cell counts in the Bazhen group were higher than in the control and antibiotics groups. Bazhen has also been used to remedy symptom of anemia; Ju et al. (2001) indicated that Bazhen enhanced hematopoiesis in γ-ray damaged mice. The addition of Bazhen to piglet diet had no effect on their specific antibody response: the SRBC antibody titer was higher in the antibiotics group than in the control group, but not in the Bazhen group, and classical swine fever and ovalbumin antibody titers did not differ between groups. Some immune response such as white and red blood cells, IL-6, and TNF-α levels were greater at weanling in the Bazhen supplemented group than in the antibiotics group. Bazhen could then be a potential substitute for antibiotics (chlortetracycline and oxytetracycline) in piglet diet. However, these results need to be confirmed on a greater number of pigs, and the dose– effect relationship needs to be further investigated. 5. Conclusion Bazhen supplementation in the diet of weaned piglets induced higher average daily gain than in the control
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group, though gains remained lower than in the antibiotics group. However, feed intake was reduced with Bazhen supplementation and feed to gain ratio was improved. Bazhen supplementation stimulated the following immune response parameters than control group: IgG, γ-globulin levels, neutrophil activity, and white blood cell counts following challenge with LPS. The immune response results of white and red blood cells, IL-6 and TNF-α levels in Bazhen group were greater than those obtained with antibiotic supplementation. This investigation therefore suggests that the addition of Bazhen in weaned piglet diets may be a potential substitute for antibiotic (chlortetracycline and oxytetracycline) supplementation. Acknowledgment The authors would like to thank the Council of Agriculture Executive Yuan of the Republic of China, Taiwan, for financially supporting this research. References Akira, S., Hirano, T., Taga, T., Kishimoto, T., 1990. Biology of multifunctional cytokines: IL-6 and related molecules (IL-1 and TNF). FASEB J. 4, 2860–2867. Burg, N.D., Pillinger, M.H., 2001. The neutrophil: function and regulation in innate and humoral immunity. Clin. Immunol. 99, 7–17. FASS, 1999. Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Federation of Animal Science Societies. Ghezzi, P., Sacco, S., Agnello, D., Marullo, A., Caselli, G., Bertini, R., 2000. LPS induces IL-6 in the brain and in serum largely through TNF production. Cytokine 12, 205–1210. Hu, Y.L., 1997. Progress in the study of immunopharmacology of Chinese herbal medicine. Chin. J. Immunol. 3, 96–98. Jia, Z., Tang, M., Wu, J., 1999. The determination of flavonoid content in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64, 555–559. Ju, H.I., Tang, I.I., 2000. Effect of Bazhen decoction on the bone marrow cells and related cell factors of mice exposed to Co γ-ray. J. Chin. Immunol. 16, 43–48. Ju, H.I., Wu, H., Tang, I.I., 2001. Effects of Bazhen tang on the immune and hematopoiesis of mice that damaged by Co γ-ray. J. Beijing Chin. Med. Univ. 24, 40–45. Kegley, E.B., Spears, J.W., 1995. Immune response, glucose metabolism, and performance of stressed feeder calves fed inorganic or organic chromium. J. Anim. Sci. 73, 2721–2726. Kong, X., Hu, Y., Rui, R., Wang, D., Li, X., 2004. Effects of Chinese herbal medicinal ingredients on peripheral lymphocyte proliferation and serum antibody titer after vaccination in chicken. Intern. Immunopharm. 4, 975–982. Leslie, H., Frank, C.H., 1989. Practical Immunology, Third ed., p. 23. Liu, J.X., Wang, C.Y., Bai, Y.M., Fang, Y.P., 1998. Influence of Astragalus and other nine natural medicines on Newscastle disease antibody titers (ND-HI), coefficient of the immune organs and gains of chicks. Chin. J. Vet. Sci. Technol. 12, 30–32.
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T.F. Lien et al. / Livestock Production Science 107 (2007) 97–102
Morrow-Tesch, J., Anderson, G., 1994. Immunological and hematological characterizations of the wasting pig syndrome. J. Anim. Sci. 72, 976–983. National Research Council, 1998. Nutrient requirements of swine. National Academy Press, Washington D.C. Panesar, N., Tolman, K., Mazuski, J.E., 1999. Endotoxin stimulates hepatocyte interleukin-6 production. J. Surg. Res. 85, 251–258. Pang, H.P., Zheng, X., 2001. Study of Bazhen tang on cellular immunology. Chin. Mod. Apply Pharmacol. 18, 279–281. Peck, O.M., Williams, D.L., Breuel, K.F., Kalbfleisch, J.H., Fan, H., Tempel, G.E., Teti, G., Ook, J.A., 2004. Differential regulation of cytokine and chemokine production in lipopolysaccharide-induced tolerance and priming. Cytokine 26, 202–208.
Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 144–158. Statistical Analysis System Institute, 1998. SAS/STAT User's guide, Version 6.06, 4th ed. Cary, NC. Urashima, M., Chauhan, D., Hatziyanni, M., Ogata, A., Hollenbaugh, D., Aruffo, A., Anderson, K.C., 1996. CD40 ligand triggers interleukin-6 mediated B cell differentiation. Leuk. Res. 20, 507–515. Van Heugten, E., Spears, J.W., 1997. Immune response and growth of stressed weaning pigs fed diets supplemented with organic or inorganic forms of chromium. J. Anim. Sci. 75, 409–416. Wang, B.I., Chen, I.C., 2000. Bazhen tang stimulated animal immune function. Chin. Med. Study 13, 20–23.