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Poult Sci 2007. 86:1955-1959
© 2007 Poultry Science Association
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METABOLISM AND NUTRITION

Effect of Polysavone (Alfalfa Extract) on Abdominal Fat Deposition and Immunity in Broiler Chickens

X. F. Dong*, W. W. Gao{dagger}, J. M. Tong*,1, H. Q. Jia*, R. N. Sa* and Q. Zhang*

* State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China 100094; and {dagger} Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China 100094

1 Corresponding author: tjm606{at}263.net


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Two hundred 1-day-old male commercial Arbor Acres broiler birds were randomly distributed to a control group and a polysavone group (5 replicates of 20 birds each) to investigate the influence of polysavone, a natural extract from alfalfa, on abdominal fat deposition and immunity in broiler chickens. Birds in the control group were supplied with a basal diet, and 0.06% polysavone was added to the basal diet of birds in the polysavone group. Body weight and feed consumption for each replicate were recorded weekly. At 3, 4, 5, and 6 wk of age, 4 birds from each replicate were randomly selected for blood and organ sampling. Polysavone had no significant effect on feed intake, BW, or feed:gain ratio in the experimental period, and it decreased the abdominal fat weights at 5 and 6 wk of age. Polysavone improved (P <0.05) the relative thymus and spleen weights at 6 wk of age and the bursa weights at 4 and 5 wk of age compared with the control group. At 4 and 6 wk of age, the proliferation of T and B lymphocytes in the polysavone group was significantly greater (P <0.05) than that in the control group. When birds were 4 and 5 wk of age, polysavone resulted in a significant increase (P <0.05) in serum anti-Newcastle disease virus hemagglutination inhibition antibody titer. These results showed that polysavone may decrease abdominal fat deposition and enhance immunity without an adverse effect on the performance of broiler chickens.

Key Words: alfalfa • extract • fat • immunity • broiler


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
With the future ban on the use of antibiotics as growth promoters in animal feed and increased concerns over food safety, environment contamination, and general health risks, the search for growth-promoting and immune system-strengthening alternatives is necessary. Concurrently, animal products with a high fat content, which is a risk factor for cardiovascular diseases, are not welcomed by consumers. At present, plant extracts are being paid great attention to as feed additives by virtue of their advantage of being natural.

Polysavone is a natural extract of alfalfa (Medicago sativa L.) and contains polysaccharides (18.63%), triterpenoid saponins (5.58%), and flavonoids (5.89%). Plant polysaccharides definitely possess an immunomodulating effect in many ways, and they regulate the balance of the neuroendocrine immune network (Nie and Zhang, 1999; Chen et al., 2002; Guo et al., 2004; Kong et al., 2006). The immune-enhancing function of alfalfa polysaccharides has been studied in broiler chickens and swine in China (Zhao et al., 1993, 2005; Jiang and Yu, 2005). Saponins are compounds found in a number of plants. Previous studies had suggested that alfalfa saponins may prevent hypercholesterolemia, reduce egg production, and depress growth in mammals and birds (Heywang and Bird, 1954; Anderson, 1957; Heywang et al., 1959; Malinow et al., 1977, 1979, 1980, 1981; Whitehead et al., 1981). Recently, Ilsley et al. (2005) reported that quillaja saponins may potentiate an immune response in the weaned piglet but have a detrimental effect on the utilization of feed. Flavonoids isolated from plants are used in the treatment of certain physiological disorders in humans, and some flavonoids exhibit unusual hormonal activities as estrogens when fed to livestock. Plant flavonoids are likely to be exploited as animal hormones and as antimicrobial, antiinflammatory, and antitumor compounds in the future (Dakora, 1995).

Studies in our laboratory have shown that polysavone may depress abdominal fat deposition and be beneficial to the growth of broiler chickens (Tong et al., 2004). In the current study, the influence of polysavone on abdominal fat deposition and the immunity of broiler chickens was investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Polysavone and Its Composition
Polysavone is a solid compound that is obtained from alfalfa (Medicago sativa L.) by hot water extraction. Fresh alfalfa at full bloom was harvested and air-dried for 2 to 3 d to a 15% moisture content. The dried alfalfa was boiled in hot water at 100°C for 2 h. The contents were then filtered through 2 layers of gauze, and the extract was spray-dried. The extract consisted of polysaccharides (18.63%), saponins (5.58%), flavonoids (5.89%), CP (21.58%), moisture (9.87%), ash (18.64%), and unknown compounds (19.81%). Repetitious examination showed that the proportion of components in polysavone was constant within a minute range.

Selected Dose of Polysavone, and Experimental Birds and Treatment
All procedures were approved by the Beijing Administration Office of Laboratory Animals. In a preliminary experiment, we observed the effect of gradients of polysavone (0.01, 0.03, and 0.05%) on the serum anti-New-castle disease virus (NDV) antibody titer of broiler chickens, and found that at a dose of 0.05%, polysavone significantly improved the antibody titer, whereas at other concentrations, polysavone failed to increase the antibody titer of broiler chickens. In addition, in a study by Tong et al. (2004) of gradients of polysavone (0.03, 0.06, and 0.09%), 0.06% polysavone had the optimal effect of inhibiting abdominal fat deposition and promoting performance. Accordingly, in the current study, 0.06% polysavone was selected.

Two hundred 1-d-old male Arbor Acre broiler birds (Beijing Huadu Broiler Co., Beijing, China) were randomly allocated to 2 groups of 100 birds each, with 5 replicates of 20 birds each. Birds in the control group were supplied with a basal diet, and those in the polysavone group received an experimental diet supplemented with 0.06% polysavone at the expense of maize, based on the control diet; other factors were the same as those in the control group. The basal maize-soybean diet (Table 1Go) was formulated to meet the NRC (1994) nutrient requirements. All birds were placed in wire cages in a temperature-controlled house. The ambient temperature was gradually decreased from 32°C on d 0 to 25°C on d 21 and then kept constant. Continuous lighting was provided throughout the trial. Feed and water were provided for ad libitum consumption. Fresh diets were prepared and supplied each day. Body weight and feed consumption per replicate were determined weekly. All birds were inoculated with the Newcastle disease IV strain vaccine (Qian Yuan Hao Biological Co., Ltd., Beijing, China) on d 7 and with the infectious bursal disease intermediate vaccine (Qian Yuan Hao Biological Co., Ltd.) on d 21 by intranasal and intraocular administration.


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  		Table 1. Composition of the basal diets in the experiment
 
Sampling Time and Procedures
At 3, 4, 5, and 6 wk of age, 4 birds per replicate were randomly selected and weighed after feed deprivation for 12 h, and fresh blood was obtained by cardiac puncture into heparinized blood collection tubes for the lymphocyte proliferation assay and into nonheparinized tubes for the NDV hemagglutination inhibition (HI) assay. Immediately after blood sampling, the birds were killed by cervical dislocation. The carcasses were then opened and the abdominal fat pad, thymus, spleen, and bursa were removed and weighed. Relative organ weight was calculated as [organ weight (g)/BW (kg)].

Leukocyte Preparation
Fresh heparinized blood from 2 birds in each replicate at 4 and 6 wk of age was separated by Percoll gradient (Amersham Pharmacia Biotech, Uppsala, Sweden) centrifugation and washed 3 times with Hanks’ balanced salt solution to obtain peripheral blood mononuclear cells. Peripheral blood mononuclear cells were counted, and cell viability was determined by the trypan blue exclusion method. The cells were suspended and adjusted to 1 x107 cells/mL in RPMI 1640 medium (Sigma-Aldrich, Inc., St. Louis, MO) containing 100 U/mL of penicillin, 100 µg/mL of streptomycin, and 2 mM L-Gln (complete media), and then supplemented with 10% heat-inactivated fetal bovine serum (Qian Yuan Hao Biological Co., Ltd.).

Lymphocyte Blastogenesis Assay
A peripheral blood mononuclear cell suspension (1 x107 cells/mL) was tested for the blastogenic response to concanavalin A (ConA, Sigma-Aldrich Inc., St. Louis, MO) and lipopolysaccharide (LPS, from Escherichia coli, Sigma-Aldrich Inc.). A 100-µL quantity of cell suspension and ConA (or LPS) was added to a 96-well microtiter plate to provide a final concentration of 25 µg of ConA (or LPS)/mL. No cell suspension was added to control wells. After cultures were incubated for 48 h in a incubator at 37°C and 5% CO2, 10 µL of 3-(4,5-dimethylthiazolyl)-2,5-diphenyl tetrazolium bromide (5 mg/mL, Sigma-Aldrich, Inc.) was added to each well, and the plates were incubated for 4 h. A 90-µL quantity of sodium dodecanesulfonate (20%) was added to each well to stop the reaction. The plates were placed at room temperature for 24 h, and the results are reported as optical density at 570 nm with an automated microplate analyzer (318MC, Sanco Instrument Co. Ltd., Shanghai, China).

Assay of Serum HI Antibody
Newcastle disease virus HI antibody quantification was done by using the hemagglutination and HI procedures. The nonheparinized blood samples (1.5 mL/ chicken) were placed at 37°C for 2 h and then centrifuged at 829 xg/min for 30 min. Serum samples were collected and frozen at –20°C for assays. Briefly, HI tests were carried out by using serial 2-fold dilutions of serum and 4 hemagglutination units of the NDV antigen (Qian Yuan Hao Biological Co.). Serum dilutions ranged from 1:2 to 1:2,048. The geometric mean titer was expressed as reciprocal log2 values for the highest dilution that displayed HI.

Statistical Analysis
Data were analyzed by SPSS 11.00 software for Windows (SPSS Inc., Chicago, IL). The differences between groups were determined by 1-way ANOVA. Duncan’s multiple-range tests were performed. The level of statistical significance was set at P <0.05. Data were expressed as mean ±SD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Polysavone on Abdominal Fat Deposition and the Performance of Broiler Chickens
The effects of polysavone on abdominal fat deposition and the performance of broiler chickens are summarized in Table 2Go. Birds in the polysavone group had significantly lower (P <0.05) relative abdominal fat weights compared with the control birds at wk 5 and 6. Polysavone had no significant (P >0.05) effect on feed intake, BW, and feed:gain ratio from 0 to 3 wk of age and at 4 to 6 wk of age. In general, there were no significant differences in feed intake (91.6 vs. 91.2), BW (2.006 vs. 2.036), and feed:gain ratio (2.03 vs. 2.00) between the 2 groups.


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  		Table 2. Effects of polysavone on the performance of broiler chickens
 
Effects of Polysavone on Relative Immune Organ Weights of Broiler Chickens
The effects of polysavone on relative immune organ weights are shown in Table 3Go. Polysavone improved the relative thymus and spleen weights (P <0.05) compared with those of the control group at 6 wk of age. At 4 and 5 wk of age, relative bursa weights in the polysavone group were significantly higher (P <0.05) than those of the control group.


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  		Table 3. Effects of polysavone on immune organ weights of broiler chickens (n = 20)
 
Effects of Polysavone on Lymphocyte Proliferation and Serum HI Antibody Titer of Broiler Chickens
The effects of polysavone on lymphocyte proliferation and the serum HI antibody titer of broiler chickens are presented in Tables 4Go and 5Go. At 4 and 6 wk of age, the proliferation of T and B lymphocytes in the polysavone group was significantly greater (P <0.05) than that of the control group. Polysavone resulted in a significant increase (P <0.05) in serum HI antibody titer at 4 and 5 wk of age.


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  		Table 4. Effects of polysavone on lymphocyte proliferation of broiler chickens (n = 10)
 

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  		Table 5. Effects of polysavone on anti-Newcastle disease virus antibody titer (log2) of broiler chickens (n = 20)
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Polysavone reduced abdominal fat deposition and improved the immune response (both humoral immunity and cell-mediated immunity) of broiler chickens in the current study. Plant polysaccharides (Nie and Zhang, 1999; Chen et al., 2002; Guo et al., 2004; Kong et al., 2006), saponins (Ilsley et al., 2005), and flavonoids (Dakora, 1995), respectively, possess the function of immune enhancement, which has been found in alfalfa polysaccharides (Zhao et al., 1993, 2005; Jiang and Yu, 2005). Polysavone, a compound containing these 3 active substances, exhibited obvious activity in improving immunity, perhaps as a result of synergism among its active ingredients or the simple combination of the functions of each component. The immune system participates in lipid metabolism by the cytokines, and the incretion system regulates lipogenesis or lipolysis by the hormones; for example, interleukin-1 plays an important role in lipid metabolism by regulating insulin levels under physiological conditions (Matsuki et al., 2003). Therefore, the reduction of abdominal fat deposition caused by polysavone should be related to enhancement of the immune response.

In the current study, polysavone had no adverse effects on the performance of broiler chickens, which was consistent with the study of Tong et al. (2004). Previous studies have suggested that alfalfa saponins have adverse effects on the performance of birds (Heywang and Bird, 1954; Anderson, 1957; Heywang et al., 1959; Whitehead et al., 1981) and the adverse effects of saponins have been attributed to depressed feed consumption because of the bitter taste (Cheeke et al., 1983; Milgate and Roberts, 1995). Jenkins and Atwal (1994) found that dietary triterpenoid saponins had adverse effects on the growth and feed consumption of chicks when fed at a level of 0.30% (gypsophila saponins) or higher (quillaja saponins), whereas steroidal saponins (sarsaponins) had no effect up to a level of 0.90%. Therefore, it was concluded that the effects of saponins may depend on the level in the diets and the type of saponin.

Diets containing alfalfa saponins at levels of greater than 0.10% (Anderson, 1957) and 0.15% (Heywang and Bird, 1954) had adverse effects on the performance of broiler chickens. In the current study, the content of saponins in the diet was only 0.003% and may not have been enough to cause bitterness, thereby reducing feed intake and performance, in spite of triterpenoid saponins being present in polysavone. Previously, Heywang et al. (1959) reported a lack of effect of alfalfa meal on performance, which was also found by Kocaoglu et al. (2004), and this may have been due to the less adverse effect of saponins in alfalfa meal compared with extracted alfalfa saponins. Similarly, the saponins in polysavone are different from extracted alfalfa saponins, which may be another cause of the lack of an adverse effect of polysavone on performance.


   ACKNOWLEDGMENTS
 
This research was financially supported by a grant (2004CB117507) from the National Basic Research Program (also called the 973 Program), Ministry of Science and Technology, People’s Republic of China. The authors also thank all the teachers and students who offered us help in this study.

Received for publication January 19, 2007. Accepted for publication May 19, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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Dakora, F. D. 1995. Plant flavonoids: Biological molecules for useful exploitation. J. Plant Physiol. 22:87–99.

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Kocaoglu, G. B., K. M. Iscan, F. Uyanik, M. Eren, and A. A. Can. 2004. Effect of alfalfa meal in diets of laying quails on performance, egg quality and some serum parameters. Arch. Anim. Nutr. 58:255–263.[ISI][Medline]

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Malinow, M. R., P. McLaughlin, L. Papworth, C. Stafford, G. O. Kohler, A. L. Livingstone, and P. R. Cheeke. 1977. Effects of alfalfa saponins on intestinal cholesterol absorption in rats. Am. J. Clin. Nutr. 30:2061–2067.[Abstract/Free Full Text]

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