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ANIMAL NUTRITION |
National Feed Engineering Technology Research Center, State Key Laboratory on Animal Nutrition, China Agricultural University, Beijing, China
| Abstract |
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at 3 and 4.5 h and increased plasma interleukin-10 from 3 to 7.5 h after LPS challenge. The ß-glucan treatments had no effect on growth hormone. In conclusion, ß-glucan can selectively influence performance and partially offer benefits on somatotropic axis and immune function in weaned piglets challenged with LPS.
Key Words: beta-glucan performance immunity lipopolysaccharide piglet
| INTRODUCTION |
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Beta-glucans extracted from the cell walls of bakers yeast (Saccharomyces cerevisiae) are being referred to as biological response modifiers because of their ability to potentiate the immune system (Miura et al., 1996
). The stimulatory effects of ß-glucan on specific and non-specific immune responses have been demonstrated in mammals (Williams et al., 1989
; Suzuki et al., 1990
). In pigs, the results of studies are inconsistent. Hiss and Sauerwein (2003)
reported that feeding diets with 150 and 300 ppm of ß-glucan had marginal benefits on performance and no effect on immune responses. In contrast, other researchers (Schoenherr et al., 1994
; Dritz et al., 1995
) reported that ß-glucan had beneficial effects on pig performance. To our knowledge, there are no reports in the literature to suggest the exact mechanism by which ß-glucan exerts its activities in pigs. Therefore, this study was conducted to evaluate the effects of dietary supplementation with ß-glucan on performance and to explore the potential mechanism by which ß-glucan exerts its effects by determining the immunological and somatotropic response in pigs challenged with lipopolysaccharide (LPS).
| MATERIALS AND METHODS |
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The ß-glucan used in this study was extracted from bakers yeast (Saccharomyces cerevisiae) according to the method published by Hunter et al. (2002)
, with some modifications. Briefly, 500 g of dry bakers yeast was added to 3 L of NaOH (1 mol/L) and mixed well. The material was heated to 115°C at a pressure of 0.6 kg/cm2 (8.5 pounds/inch2) for 45 min and then allowed to settle for 3 h. The sediment was isolated by centrifugation at 2,000 x g for 15 min and then resuspended in distilled water. After washing 3 times with distilled water, 2.5 L of 3% HCl was added to the sediment, and the mixture was heated at 85°C for 3 h. The sediment was harvested by centrifugation (2,000 x g for 15 min) and washed 3 times with distilled water. The solid material was washed 3 times with 200 mL of 3% H2O2 at 20°C for 3 h. The pellet was then washed 2 times in 100% acetone. The harvested solid material was lyophilized to dryness. Chemical analysis of the final product indicated 4.19% protein (Lowry et al., 1951
), 1.26% lipids (AOAC, 1990
), and 86.1% ß-glucan (Dubois et al., 1956
).
Animals and Experimental Design
Our protocols were approved by the Institutional Animal Care and Use Committee of China Agricultural University. Landrace x Large White, crossbred pigs were selected. Before weaning, pigs had access to creep feed with no antibiotic but with 250 ppm of Cu as Cu-SO4·5H2O. The same basal diet was used in all trials (Table 1
). The diets were offered in meal form and formulated to meet or exceed the requirements for all nutrients (NRC, 1998
).
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To determine the effects of graded levels of dietary ß-glucan on pig performance, 100 weaned pigs (8.65 ± 0.42 kg of BW and 28 ± 2 d of age) were randomly allotted to 1 of 5 treatments for a 35-d trial. Each group was randomly assigned to a diet containing 0, 25, 50, 100, or 200 ppm of ß-glucan. Each treatment was replicated using 5 pens with 4 pigs (2 males and 2 females) per pen.
Throughout the 5-wk feeding trial, pigs were housed in 2.0 x 2.0-m nursery pens equipped with woven wire flooring. The temperature in the room was controlled (25 to 27°C). Each pen contained a self-feeder and nipple waterer to provide ad libitum access to feed and water. Pigs were weighed and feed disappearance was measured weekly to determine ADG, ADFI, and G:F for each pen.
Experiment 2
Eighty weaned pigs (8.23 ± 0.56 kg of BW and 28 ± 2 d of age) were allotted to 1 of 2 treatments for a 35-d trial. Each group was randomly assigned to a diet containing 0 or 50 ppm of ß-glucan. Each treatment was replicated using 10 pens (5 pens of females and 5 pens of males) with 4 pigs per pen. The experimental procedures were identical to Exp. 1. Feed waste was collected daily, and the BW of pigs was measured weekly for calculation of ADG, ADFI, and G:F for each pen.
On d 14 and 28 of the trial, 1 pig per pen was selected randomly, and heparin-anticoagulated blood samples were collected from the anterior vena cava of each pig after fasting for 8 h (from 2200 to 0600) for assessment of the lymphocyte proliferation index.
Experiment 3
To determine the influence of ß-glucan on the immune and somatotropic responses of weaned pigs to a LPS (E. coli serotype O55: B5, Sigma Chemical Inc., St. Louis, MO) challenge, 24 weaned barrows (8.89 ± 0.20 kg of BW and 28 d of age) were randomly allotted to 1 of 4 treatments, each replicated with 6 pigs. The 4 treatments were arranged in a 2 x 2 factorial, with the main effects of LPS challenge (saline vs. LPS) and dietary addition of ß-glucan (0 vs. 50 ppm). Pigs were housed individually in metabolic cages (1.2 x 0.6 m) in an environmentally controlled room (25 to 27°C) in which a constant, 24-h light schedule was maintained. The pigs were allowed free access to feed and water.
On d 29, venous catheters were fitted nonsurgically on the right external jugular vein of each pig and equipped with remote sampling devices that allowed for frequent blood sampling without disturbing the experimental animals (Carroll et al., 1999
). Catheter patency was maintained by flushing twice daily with heparinized saline. On d 31, half of the pigs in each dietary treatment were randomly assigned to receive 25 µg of LPS/kg of BW or an equivalent amount of saline. The LPS was dissolved in a saline solution such that 0.1 mL of solution/kg of BW would achieve the desired dosage. The injections of LPS were given intraperitoneally in the lower abdominal region.
At 1800 of d 30, feed was removed, and at 0730 the next morning, catheters were flushed and blood samples were taken from each pig. At 0800, pigs were weighed and the LPS treatments were initiated. Blood samples were taken at 0, 1.5, 3, 4.5, 6, and 7.5 h after LPS injection, and centrifuged (2,000 x g for 20 min) to separate the plasma. Plasma was stored at 20°C until analysis. The plasma concentrations of IL-6, tumor necrosis factor-
(TNF-
), IL-10, IGF-I, cortisol, and GH were determined.
Lymphocyte Proliferation Assays
Lymphocyte proliferation was measured using a colorimetric assay, with 3-(4,5-dimethlthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT, M-2128, Sigma Chemical Inc.) in cultures of purified peripheral blood mononuclear cells according to the method of Mosmann (1983)
. Briefly, blood was centrifuged (3,000 x g) at room temperature for 10 min in 5 mL of histopaque (density 1.077; Sigma-Aldrich, Dorset, UK) in 10-mL centrifuge tubes. The separated cells (approximately 1 mL) were then transferred into another 10-mL centrifuge tube containing 4 mL of Roswell Park Memorial Institute medium 1640 (RPMI; Gibco Life Technologies, New York, NY) and mixed well.
After centrifugation at 2,000 x g for 10 min at room temperature, the supernatant was discarded, and the pellet was resuspended in 4 mL of RPMI. After repeating this process 2 additional times, pelleted cells were resuspended in 4 mL of RPMI supplemented with penicillin G (100 U/mL; Merck KGaA, Darmstadt, Germany), streptomycin (100 µg/mL; Sigma Chemical Inc.), and 10% heat-inactivated newborn calf serum (HyClone Laboratories Inc., Logan, UT), counted, and plated into polypropylene 96-well culture dishes (Costar Corp., Cambridge, MA) at 1 to 3 million cells/ mL, with a total culture volume of 200 µL.
Lymphocyte mitogen phytohemagglutinin (PHA; Sigma Chemical Inc.) or concanavalin A (ConA; Type IV, C-2010, Sigma Chemical Inc.) was added at a final concentration of 16 mg/mL of culture medium, and then the plates were incubated at 37°C in an incubator in an atmosphere of 5% CO2 (Heraeus, Hongkong, China) for 66 h. Subsequently, 10 µL of MTT solution [5 mg of MTT/mL in phosphate-buffered saline (0.07 M, pH 7.6)] was added to each well, and the plates were incubated at 37°C for 6 h. After incubation, 100 µL of 10% sodium dodecyl sulfate (Sigma Chemical, Inc.) in 0.04 M HCl was added to lyse the cells and solubilize the MTT crystals. The plates were read at 570 nm using an automated microplate reader (Model 550, BioRAD Laboratories Inc., Hercules, CA). Lymphocyte proliferation was expressed as a proliferation index, which was calculated as the absorbance of wells incubated with PHA or ConA divided by the absorbance of wells incubated without PHA or ConA.
Assessment of Cytokines and Hormones
Plasma IL-6 was measured using a commercially available swine ELISA kit (BioSource International Inc., Camarillo, CA). Plasma TNF-
and IL-10 were also measured using commercially available ELISA kits (R&D Systems Inc., Minneapolis, MN). The sensitivity of the assays (minimum detectable concentration) was 15 pg/mL for IL-6, 3.7 pg/mL for TNF-
, and 10 pg/mL for IL-10, with a within-assay CV of less than 10% for all 3 assays. The assays were analyzed colorimetrically using a plate reader.
Plasma IGF-I, GH, and cortisol concentrations were determined using commercially available swine ELISA kits (Biocode S. A., Liege, Belgium; Beijing North Institute of Biological Technology, Beijing, China; and Beijing Beimian Dongya Institute of Biological Technology, Beijing, China, respectively). The sensitivity (minimum detectable concentration) was 5 ng/mL for IGF-I, 0.1 ng/mL for GH, and 1 ng/mL for cortisol, with a within-assay CV of <5% for cortisol and <10% for the other 2 assays. All assays were performed in duplicate.
Statistical Analysis
All data were processed using SAS (SAS Inst. Inc., Cary, NC). In Exp. 1, dose effects of dietary ß-glucan on pig performance were evaluated by polynomial contrasts. In Exp. 2, performance was analyzed using a factorial procedure. This model consisted of dietary treatment, sex, and their interaction. The lymphocyte proliferation index was analyzed by ANOVA with time-repeated measurements (Gill and Hafs, 1971
). The statistical model for lymphocyte proliferation index consisted of dietary treatment, time, sex, and all possible interactions. In Exp. 3, plasma cytokine and hormone data were analyzed as a factorial arrangement using repeated measures. The statistical model consisted of dietary treatment, endotoxin challenge, time, and all possible interactions. Pen was used as experimental unit for the performance data in Exp. 1 and 2, whereas individual pig was used as the experimental unit in the lymphocyte proliferation index, and individually penned pigs were used as the experimental unit for Exp. 3. Differences with probabilities of P < 0.05 were considered significant, whereas P = 0.10 was considered a trend.
| RESULTS |
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Performance data are presented in Table 2
. The ADG of pigs during d 0 to 14 tended to respond to ß-glucan addition in a quadratic fashion (P = 0.08). There was a quadratic response to the addition of ß-glucan from 0 to 200 ppm, with the greatest numerical ADG observed in the pigs fed 50 ppm of ß-glucan, during d 14 to 28 (P = 0.041) and d 0 to 28 (P = 0.031). No effect of ß-glucan on G:F and ADFI was observed.
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As expected, a positive effect of ß-glucan was observed for ADG (Table 3
). Pigs supplemented with 50 ppm of ß-glucan had greater ADG on d 0 to 14 (P = 0.10), d 14 to 28 (P = 0.05), and d 0 to 28 (P = 0.035). In addition, ADFI of pigs fed diets supplemented with ß-glucan increased d 0 to 28 (P = 0.025) and d 28 to 35 (P = 0.015). The G:F in each period was not affected by dietary ß-glucan supplementation. The increase in ADG may be related to an increase in ADFI in pigs that consumed the diet supplemented with 50 ppm of ß-glucan compared with pigs that received the control diet. No effect of sex or ß-glucan x sex interaction on ADG, ADFI, or G:F was observed.
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Plasma IL-6 concentrations in pigs with an LPS injection were increased (P < 0.001) at all time points after injection (Table 5
). Pigs that received diets supplemented with ß-glucan had lower IL-6 plasma concentrations at 1.5 (P = 0.043), 3 (P = 0.019), and 4.5 h (P = 0.011) after injection compared with nonsupplemented pigs.
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concentrations in pigs injected with LPS were increased (P < 0.001) at 1.5, 3, and 4.5 h after injection. At 6 h after challenge, a tendency was observed for pigs injected with LPS to have greater (P = 0.057) TNF-
concentrations. Pigs that received diets supplemented with ß-glucan had lower plasma TNF-
concentrations at 3 (P = 0.043) and 4.5 h (P = 0.041) after injection than nonsupplemented pigs. Pigs fed a diet supplemented with ß-glucan tended to have lower TNF-
concentrations in plasma at 1.5 (P = 0.09) and 6 h (P = 0.085) after challenge compared with nonsupplemented pigs. There was a tendency for ß-glucan x LPS interactions for plasma TNF-
concentrations at 3 (P = 0.090) and 4.5 h (P = 0.085) after challenge. Plasma IL-10 concentrations increased at 3 (P = 0.046), 4.5 (P < 0.001), and 6 h (P = 0.001) after an LPS injection. Pigs fed a diet supplemented with ß-glucan had greater plasma IL-10 concentrations at 3 (P = 0.045), 4.5 (P = 0.022), 6 (P = 0.041), and 7.5 h (P = 0.029) after LPS injection compared with nonsupplemented pigs.
Plasma IGF-I concentrations decreased at 1.5 (P = 0.012), 3 (P < 0.001), 4.5 (P = 0.002), 6 (P < 0.001), and 7.5 h (P = 0.003) after an LPS challenge (Table 6
). Pigs that consumed ß-glucan-supplemented diets tended to have greater plasma IGF-I at 3 (P = 0.068), 4.5 (P = 0.082), 6 (P = 0.093), and 7.5 h (P = 0.096) postchallenge compared with nonsupplemented pigs.
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No effects of dietary ß-glucan supplementation, LPS injection, or both on plasma GH concentrations were observed.
| DISCUSSION |
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The optimal concentration of ß-glucan to improve growth in our study was much less than previously reported. A possible explanation for this divergence is the difference in purity, molecular weight, conformation, and extraction methods. Insufficient removal of protein from ß-glucan isolated from yeast cells could cause a reduction in ß-glucan efficacy. Protein can bind the active terminal residues of ß-glucan, thereby preventing ß-glucan from interacting with its receptors (our unpublished data). In addition, studies on structure-function relationships of ß-glucan using murine systems have revealed that molecular weight, conformation, and degree of branching affect the binding of ß-glucan with its receptors and therefore influence its activities (Ohno et al., 1986a
,b
, 1992
).
The exact mechanism through which ß-glucan supplementation improves pig growth performance is unclear. To the best of our knowledge, no studies have been conducted to explore the potential of ß-glucan to modulate the production of cytokines, especially antiinflammatory cytokines. In Exp. 3, diets supplemented with 50 ppm of ß-glucan partially suppressed increases in plasma concentrations of TNF-
and IL-6 and enhanced the increase in plasma concentrations of IL-10 brought about by an LPS challenge. It is well known that IL-6 and TNF-
are proinflammatory cytokines that not only modulate immunity but also can directly regulate nutrient metabolism and cause detrimental effects on animal performance (Spurlock, 1997
). The antiinflammatory cytokine IL-10 (Enk and Katz, 1992
) can inhibit T-cell proliferation, development, and function (Fuirebtubi et al., 1989
) as well as the secretion of Th1- and Th2-type cytokines (Fuirebtubi et al., 1989
; De Waal Malegyt et al., 1991
). Furthermore, IL-10 can also suppress the activity of the signal transduction of nuclear transcription factor
B, which is a major transcription factor of proinflammatory cytokines (Clarke et al., 1998
; Schottelius et al., 1999
). If feeding ß-glucan promotes secretion of antiinflammatory cytokines, such as IL-10, and decreases secretion of proinflammatory cytokines, such as TNF-
and IL-6, then less activation of the immune system during the feeding period would be achieved, which could result in increased growth performance. This hypothesis is partially supported by data of Exp. 2 and 3. In Exp. 2, pigs fed diets supplemented with 50 ppm of ß-glucan tended to have attenuated lymphocyte proliferation on d 14, indicating that cellular immunity may be suppressed. In Exp. 3, supplementing pigs with 50 ppm of ß-glucan resulted in a partially suppressed increase in TNF-
and IL-6 production and enhanced increase in IL-10 production after an LPS challenge. This may help to explain the increased growth performance observed in Exp.1 and 2 and in other studies.
Proinflammatory cytokines, such as IL-6 and TNF-
, are not only primarily associated with immune system, but also have the potential to alter many aspects of neuroendocrine function, including the somatotropic axis (Mandrup-Poulsen et al., 1995
; Maule and Vanderkooi, 1999
). In the current study, the proinflammatory cytokine responses of pigs to an LPS challenge were altered by ß-glucan supplementation. Therefore, we further investigated the influence of ß-glucan on somatotropic response in LPS-challenged pigs. In the current study, a tendency of mitigating the reduction of plasma IGF-I was observed in pigs supplemented with ß-glucan after an LPS challenge. Hasselgren (1993)
proposed that the reductions of plasma IGF-I may indicate the repartition of nutrients away from normal growth and toward immune response. Other studies also indicated that plasma IGF-I concentration is associated with pig performance (Prickett et al., 1992
; Hathaway et al., 1993
; Hathaway et al., 1996
). In our study, although not significantly different, the tendency of mitigating the reduction of plasma IGF-I may be an indication that ß-glucan supplementation alleviates the alterations in somatotropic axis after an LPS challenge, which might have been associated with the improved performance in pigs supplemented with ß-glucan compared with control pigs.
Additionally, one of the most interesting observations from our study was that pigs supplemented with 100 and 200 ppm of ß-glucan had lower ADG than pigs supplemented with 50 ppm of ß-glucan. Poutsiaka et al. (1993)
observed that stimulation of human macrophages, in vitro, with ß-glucan resulted in increased synthesis of IL-1 receptor antagonist, whereas proinflammatory IL-1ß was not increased unless very high ß-glucan doses were applied. Increased concentrations of IL-1 have been associated with decreased feed intake and growth performance (Klasing et al., 1987
; Blecha et al., 1995
). If feeding ß-glucan alters the balance of IL-1 and IL-1 receptor antagonists such that IL-1 is preferentially secreted, immune system activation will increase, potentially resulting in decreased growth performance. Additionally, Hoffman et al. (1993)
demonstrated that ß-glucan at selective doses can either induce or suppress the release of TNF-
from mononuclear phagocytes. Therefore, it is possible that diets supplemented with 100 and 200 ppm of ß-glucan in the current study promoted the secretion of proinflammatory cytokines such as IL-1, IL-6, and TNF-
instead of decreasing secretion. This could explain the decreased growth performance for pigs fed diets supplemented with 100 and 200 ppm of ß-glucan compared with pigs fed diets supplemented with 50 ppm of ß-glucan.
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| Footnotes |
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2 Corresponding author: defali{at}public2.bta.net.cn
Received for publication October 7, 2004. Accepted for publication April 5, 2006.
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