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ANIMAL NUTRITION |
* College of Animal Resource Science, Kangwon National University, Chunchon-200-701, Republic of Korea;
and
College of Animal Husbandry, Konkuk University, Seoul, 143-701, Republic of Korea
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: pig • immunity • ß-glucan • antibiotic • growth • digestibility
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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). Glucans with ß –1,3 and ß –1,6 glycosidic linkages (ß-glucan) are major structural components of yeast and fungal cell walls (Jorgensen and Robertsen, 1995). ß-Glucan is known to possess antitumor (Ohno et al., 1987) and antimicrobial activities (Hetland et al., 2000) by enhancing the host immune function. It has beneficial effects on weaned pig growth (Mowat, 1987; Stokes et al., 1987) because it elicits specific immune reactions and increases the nonspecific immunity and tolerance to oral antigens as an immunomodulator. Supplementing nursery pig (18 ± 2 d age) diets with 0.025% ß-glucan increased growth performance but also increased the susceptibility to Streptococcus suis infection (Dritz et al., 1995). These authors suggested that a complex interaction exists between growth performance and disease susceptibility in pigs fed ß-glucan.
Schoenherr et al. (1994) reported that ß-glucan (Macrogard-S) supplementation improved growth performance and feed efficiency in nursery pigs. Immunopotentiation effected by binding of a (1 
3)-ß-glucan molecule or particle probably includes activation of cytotoxic macrophages, helper T-cells and natural killer cells promotion of T-cell differentiation, and activation of the alternative complement pathway (Bohn and BeMiller, 1995). These studies were conducted to evaluate the effects of ß-glucan on performance, immune response, and digestibility of nutrients in weanling pigs in comparison with antibiotics.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Two hundred ten weaned, castrated male pigs (Duroc x Landrace x Yorkshire; 6.38 ± 0.92 kg of BW) were allotted by weight to 5 treatments with 3 replicates comprised of 14 pigs per replicate pen (6.38 ± 0.92 kg). Pigs were housed in partially slotted and solid concrete floor pens, with a self-feeder and nipple waterer to allow ad libitum access to feed and water. The ß-glucan was fed at 0, 0.01, 0.02, 0.03, or 0.04%. The composition of phase-I (2 wk) and phase-II (3 to 5 wk) diets is presented in Table 1. Corn was replaced by ß-glucan on an equal percent basis. The experiment was conducted for 5 wk, during which the BW and ADFI were recorded for each phase. The source of ß-glucan was Saccharomyces cerevisiae from IS 2(KCTC 0959BP), IS 9(KCTC 0960BP), IB 54(KCTC 0961BP), and IB 56 (KCTC 0962BP) strains. This product is named Glucagen (Enbiotec Company, Seoul, Korea).
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). All pigs in all pens were fed diets mixed with chromic oxide on d 28, and fecal grab samples were collected from all pigs on d 32 to 35 during phase II. The analyzed composition of the diets used for the digestibility trial is presented in Table 2. Because the feces were collected over a 3-d period, they were pooled, mixed, and a representative sample was collected from each pen. For chemical analysis, the representative fecal samples were dried in a forced-air drying oven (Model FC-610; Advantec, Toyo Seisakusho Co. Ltd., Tokyo) at 60°C for 72 h and ground with a 1-mm mesh disc mill (Model BM-D-100; Disc Mill, McCoy Corporation, San Marcos, TX).
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One hundred forty-four weanling, castrated male pigs (Duroc x Landrace x Yorkshire; 6.18 ± 1.31 kg of BW) were allotted by weight to 4 dietary treatments for 8 wk. Each treatment had 3 replicates with 12 pigs per pen. The 4 treatment diets contained: no ß-glucan or antibiotics (control, T1); 0.02% ß-glucan (T2); antibiotic (T3); and 0.02% ß-glucan and antibiotics (T4). The antibiotics fed during phase I (0 to 2 wk) were 0.15% apramycin (Apralan; KBNP Inc., Gunpocity, Kyungki-Do, Korea) and 0.10% Carbadox (Mecadox, Seoul Vet. Pharm., Umsong, Chungcheongbuk-Do, Korea). The antibiotics fed during phase II (3 to 8 wk) were 0.10% chlortetracycline (CTC, Yuhan Corporation, Anyang-City, Kyungki-Do, Korea) and 0.10% Carbadox. The performance study was conducted for 5 wk, but the immune response was studied until 8 wk. The composition of the control diet (T1) during phases I and II was the same as in Exp. 1, but the treatment diets (T2 to T4) were adjusted by replacing corn on an equal percent basis (Table 1
). The source of ß-glucan and the facilities and management were the same as in Exp.1. The BW and ADFI were recorded at the end of phase I and at 5 wk during phase II. A digestibility trial was also conducted on 28 d using chromic oxide (0.25%), as an indicator as in Exp. 1.
An additional 3 pens of 2 pigs each per treatment were assigned and penned separately for antibody titer studies. They were vaccinated (i.m.) after weaning with atrophic rhinitis vaccine (Pfizer Co., Seoul, Korea) at 9 d with 1 mL and 14 d with 2 mL. For antibody quantification by using an ELISA (Kit No. JBPA1, Jeil-Bio Co., Seoul, Korea), blood was then drawn from the jugular vein at 15, 24, and 46 d postvaccination using a 22-gauge sterile needle into a 10 mL syringe and then transferred to a BD Vacutainer (Becton Dickinson, Franklin Lakes, NJ) without anticoagulant. The blood was centrifuged at 1,500 x g for 10 min, and serum was collected and stored for further analysis.
During the study, the effects of ß-glucan on porcine lymphocyte populations were measured using flow cytometry, with fluorescence-activated cell sorter Calibur and CellQuest programs (Becton Dickinson, Franklin Lakes, NJ) and monoclonal antibodies specifically reactive with porcine major histocompatibility complex (MHC) class II, cluster of differentiation antigens 2, 4, and 8 (CD2, CD4, and CD8, respectively), B-cells, NonT/NonB cells (N), and granulocytes (G). The blood was drawn from 6 randomly selected pigs per group (2 per replicate) at 4 and 8 wk after weaning, and the leukocyte population was measured using flow cytometry. Blood was drawn from the jugular vein by using a 22-gauge needle into a 10-mL syringe and then transferred to a BD Vacutainer (Becton Dickinson) containing sodium heparin as an anticoagulant and stored at 4°C until analysis.
Chemical Analyses
Chemical analyses of the experimental diets were carried out following the AOAC (1990) methods. The DM content of feed and fecal samples was measured by using a hot air oven (FC-610; Advantec, Toyo Seisakusho Co. Ltd, Tokyo, Japan). The GE was determined in a bomb calorimeter (Model 1241; Parr Instrument Co., Moline, IL). The chromium concentration of diets and fecal samples was determined by an automated spectrophotometer (Model V-550; Jasco Co., Tokyo, Japan) according to the procedure of Fenton and Fenton (1979). In short, samples of feed (5 g) or feces (1 to 2 g) were ashed in a muffle furnace at 450°C overnight. After cooling, 15 mL of a digestion mixture (10 g of sodium molybdate dihydrate dissolved in 500 mL of a 150:150:200 mixture of distilled water:concentrated sulfuric acid:70% perchloric acid) was added to each sample and heated on a hot plate (surface temperature up to 300°C) until a yellowish or reddish color developed. The samples were heated for an additional 10 to 15 min, removed from the hot plate and allowed to cool. The digests were then quantitatively transferred to 200-mL volumetric flasks with distilled water and made to volume. Approximately 10 mL of the diluted digest was poured into a centrifuge tube, capped, and centrifuged (VS-550; Vision Scientific Co. Ltd., Seoul) for 5 min at 700 x g. The OD was measured in a cuvette vs. distilled water at 440 nm with an automated spectrophotometer (Model V-550; Jasco Co., Tokyo). Standard curves were prepared by using a stock solution of pure Cr2O3 (100mg/100 mL), diluted to several working standards of 5, 10, 20, 40, 60, or 80 mg/100 mL, and carrying them through each method. The optical density was plotted against milligrams of Cr2O3. The digestibility was then calculated using the following formula:
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in which
The Ca and P contents in the feed and feces were measured according to AOAC (1984; method 7.099b).
Lymphocyte Subpopulation Assay. Three milliliters of blood were mixed with 3 mL of acid citrate dextrose (ACD) solution (25 mM citric acid, 51.7 mM sodium acetate, 81.6 mM D-glucose). The blood solution was then centrifuged at 1,500 x g for 10 min, and white blood cells (WBC) were collected with a sterile Pasteur pipette and placed on the surface of Hypaque Ficoll (Histopaque 1.803; Sigma Chemical Co.) and centrifuged at 500 x g for 30 min. The lymphocytes were obtained from the interface between Ficoll and plasma, and cell suspensions were washed 3 times and resuspended in a Ca- and Mg-free phosphate buffered saline (11.3 mM sodium phosphate, 3.8 mM potassium phosphate, 125 mM sodium chloride, 100 units of penicillin/mL, and 100 µg of streptomycin/mL).
Cells were then incubated with a panel of monoclonal antibodies (mAb), obtained from Seoul National University, Korea, specific for various swine leukocyte differentiation antigen markers. The panel of mAb included PT85A (MHC class II), H42A (IgG2a), and mAb specific for porcine CD2, CD4, CD8, and B and N cells. Lymphocytes were subtyped by the flow cytometry Cell-Quest program (Davis et al., 1990).
Fifteen microliters of each mAb and 1 x 107 lymphocytes were mixed in a well of a V-bottomed 96-well microplate (Stockwell Scientifics, Scottsdale, AZ), and the mixture of lymphocytes and mAb were incubated at 4°C for 30 min. The cells were washed 3 times with the cold washing buffer [450 mL of PBS, 50 mL of ACD, 5 mL of 20% sodium azide, 10 mL of 
-globulin-free horse serum (Gibco-BRL, Grand Island, NY), 20 mL of 250 mM EDTA, and 1 mL 0.5% of phenol red], and the supernatant was discarded.
The cell suspension was incubated with 100 µL of 0.02 mg/mL of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG + IgM antibody (Caltag Lab, Burlingame, CA) at 4°C for 30 min. The cells were then washed 3 times with the second washing buffer [450 mL of PBS, 50 mL of ACD, 5 mL of 20% sodium azide, 20 mL of 250 mM EDTA, 1 mL of 0.5% phenol red] and fixed with 200 µL of 20% PBS-formalin (20 mL of 38% formalin, 980 mL of PBS). To analyze the lymphocyte subpopulation, cells were counted and analyzed with the fluorescence-activated cell sorter Calibur and Cell-Quest programs.
Antibodies were measured in the previously collected blood using an ELISA kit. Formalin-inactivated Pasteurella multocida whole cells were coated in 96-well V-bottomed microplate. Sera were diluted 2-fold serially and incubated for 2 h. After washing 3 times with washing buffer, anti-pig IgG peroxidase conjugate (Sigma Chemical Co.) was added to each well. The O-phenylenediamine was used as the substrate, and absorbance was measured at 490 nm with an ELISA reader (Microplate autoreader; BioTek Instruments, Winooski, VT).
Statistical Analysis
Statistical analysis was performed using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC). Pens were the experimental unit for all analysis. Experiment was a randomized complete block design and appropriate linear and quadratic components of the treatment effects were determined. Experiment 2 was also a randomized complete block design, and the differences between means were tested by least significance difference. The data for the antibody study (Exp. 2) was analyzed using 1-way analysis of variance with pen as an experimental unit. An alpha level of 0.05 was used to determine statistical significance, and a level of 0.10 was considered a trend.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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). There was a trend for a linear increase (P = 0.086, P = 0.068) for ADG from 0 to 0.04% inclusions during phase II and in the overall study, respectively. No effect (P > 0.10) was found on ADFI or G:F in Phase II (3 to 5 wk) or in overall study (0 to 35 d).
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). Except for Ca (P = 0.055), no quadratic effects were observed because of dietary ß-glucan supplementation.
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). During 3 to 5 wk in phase II, there was a trend (P < 0.10) for ADG to increase with the addition of antibiotics and ß-glucan compared with animals fed the control diet though their ADG did not differ compared with pigs fed diets supplemented solely with ß-glucan (T2) or antibiotics alone (T3). The overall (0 to 35 d) ADG was greater (P < 0.05) for the pigs fed the combination diet (T4) than for ß-glucan or control fed pigs and was similar for pigs fed only the antibiotics (T3).
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). Except GE, pigs fed the antibiotic diet (T3) had similar nutrient digestibilities to pigs fed the combination diet (T4).
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). At 46 d, antibody titers to Pasteurella multocida were greater in control pigs than those fed the antibiotic (T3) and combination diet (T4).
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). At 46 d, pigs fed antibiotic and ß-glucan combination diet (T4) showed greater (P < 0.05) titers than ß-glucan (T2) and antibiotic (T3) supplemented diets.
At 4 wk, only MHC-II lymphocytes were greater (P < 0.10) in ß-glucan supplemented diets than pigs fed the other treatments (Table 8). At 8 wk, the CD4 cells were greater (P < 0.05) in pigs fed ß-glucan supplemented diets (T2), and the CD2 cells were greater (P < 0.05) in antibiotic added diets (T3) when compared with pigs fed the other diets (Table 8).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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. Dritz et al. (1995) reported improved ADFI when pigs were fed 0.025% ß-glucan for 28 d, but we saw no improvement in ADFI. It is common worldwide to add pharmacological Zn and acidifiers in weanling pig commercial diets. These enhancers were used both in basal (control) and treatment diets. We wanted to see if there were any additional effects of ß-glucan over and above these performance enhancers.
Ko et al. (2000) reported that antibiotic supplementation to growing pigs improved weight gain, feed efficiency, and total gain when compared with antibiotic-fed pigs, which is similar to some of our observations. The effect of antibiotic supplementation on growth performance in pigs varies with age, health status, and environment (Chesson, 1994), and perhaps that is why we did not see a consistent antibiotic effect. Even ß-glucan alone also failed to show any effect on performance at 0.02% that was contradictory to our findings in Exp. 1.
Few reports are available on the effect of ß-glucan on nutrient digestibility. No effect was seen on digestibilities of DM, GE, CP, ash, or P with ß-glucan supplementation in growing (Ko et al., 2000) or finishing pigs by (Bae et al., 1999). The linear increase in nutrient digestibilities in our studies did not culminate in a linear increase in weight gains in the ß-glucan supplemented diets; only a trend was seen.
In Exp. 2, antibiotics had a more positive effect on nutrient digestibilities than ß-glucan alone. However, Yoo et al. (1985) and Min (1992) did not find any effect on nutrient digestibilities due to antibiotic supplementation with growing-finishing pigs. The increase in nutrient digestibilities might have improved the weight gains of pigs fed the combination diet (T4) when compared with nonsupplemented diet.
Pasteurella multocida has 5 capsular serotypes A, B, D, E, and F, but serotypes A and D are more prevalent in Korean swine farms, so we studied antibody response against serotypes A and D only. There was a significant reduction in the antibody response to the 2 Pasteurella antigens in the treatment groups compared with control. High animal-to-animal variation in responding to vaccine may have prevented treatment differences. Hiss and Sauerwein (2003) reported that ß-glucan supplementation did not show any effect on the immune response, e.g., serum haptoglobin and antibody response to porcine reproductive and respiratory syndrome vaccination. Reduced haptoglobin concentrations and increased mortality rates after Streptococcus suis challenge in ß-glucan fed pigs (Dritz et al., 1995) have also been reported.
The subset of porcine lymphocyte populations MHC-II (4 wk), and CD4 and CD8 (8 wk), showed greater lymphocytes in ß-glucan supplemented diets as compared with pigs fed the other diets. Either ß-glucan or antibiotics or both at 4 and 8 wk did not influence the B, N, and G cells. The CD4 cells showed lowest numerical values of all the cells measured during 4 wk. Previously, Suzuki et al. (1989) showed that the proliferative responses of spleen cells from ß-glucan administered mice T-cell and B-cell mitogens were greater than those from normal mice. Oral administration of ß-glucan also enhanced the activities of natural killer cells and peritoneal macrophages. In addition, ß-glucan stimulated cytotoxic T-lymphocytes, B-cells, and macrophages in mice (Cross et al., 2001). Our results indicate marginal benefits of ß-glucan supplements on immune parameters in pigs.
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IMPLICATIONS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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1 Corresponding author: bjchae{at}kangwon.ac.kr
Received for publication January 21, 2005. Accepted for publication January 3, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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3)-ß-D-glucans as biological response modifiers: A review of structure-functional activity relationships. Carbohydrate Polymers 28:3–14. 3) beta-D-glucan, SSG, obtained from Sclerotinia sclerotiorum IFO 9395. J. Pharmacobiodyn. 10:478–486.[Medline]This article has been cited by other articles:
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  Z. Jin, Y. X. Yang, J. Y. Choi, P. L. Shinde, S. Y. Yoon, T.-W. Hahn, H. T. Lim, Y. Park, K.-S. Hahm, J. W. Joo, et al. Potato (Solanum tuberosum L. cv. Gogu valley) protein as a novel antimicrobial agent in weanling pigs J Anim Sci, July 1, 2008; 86(7): 1562 - 1572. [Abstract] [Full Text] [PDF]
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