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Effect of phosphorylated mannans and pharmacological additions of zinc oxide on growth and immunocompetence of weanling pigs

M. E. Davis^*, D. C. Brown^*, C. V. Maxwell^*,1, Z. B. Johnson^*, E. B. Kegley^* and R. A. Dvorak

^* Department of Animal Science, University of Arkansas, Fayetteville 72701 and and Alltech, Nicholasville, KY 40356

Abstract

Three experiments were conducted to evaluate the efficacy of phosphorylated mannans (MAN) and pharmacological levels of ZnO on performance and immunity when added to nursery pig diets. Pigs (216 in each experiment), averaging 19 d of age and 6.2, 4.6, and 5.6 kg of BW in Exp. 1, 2, and 3, respectively, were blocked by BW in each experiment, and penned in groups of six. A lymphocyte blastogenesis assay was performed in each experiment to measure in vitro lymphocyte proliferation response. In Exp. 1, diets were arranged as a 2 x 2 factorial with two levels of Zn (200 and 2,500 ppm) and two levels of MAN (0 and 0.3% from d 0 to 10, and 0 and 0.2% from d 10 to 38). Zinc oxide increased (P < 0.05) ADG, ADFI, and G:F from d 0 to 10, and ADG and ADFI from d 10 to 24. In Exp. 2, diets were arranged as a 2 x 3 factorial with two levels of Zn (200 and 2,500 ppm) and three levels of MAN (0, 0.2, and 0.3%). Pigs fed 2,500 ppm Zn from d 0 to 10 had greater (P < 0.05) ADG, ADFI, and G:F than pigs fed 200 ppm Zn. From d 10 to 24, ADG was similar when pigs were fed 200 ppm Zn, regardless of MAN supplementation; however, ADG increased (P < 0.05) when 0.2% MAN was added to diets containing 2,500 ppm Zn (MAN x Zn interaction, P < 0.05). In Exp. 3, diets were arranged as a 2 x 3 factorial with two levels of MAN (0 and 0.3%) and three levels of Zn (200, 500, and 2,500 ppm). Zinc was maintained at 200 ppm from d 21 to 35, so only two dietary treatments (0 and 0.3% MAN) were fed during this period. Average daily gain was greater (P < 0.05) from d 7 to 21 when pigs were fed 2,500 ppm Zn compared with pigs fed 200 or 500 ppm Zn. The addition of MAN improved (P < 0.05) G:F from d 7 to 21 and d 0 to 35. Lymphocyte proliferation of unstimulated cells and phytohemagglutinin-stimulated cells was decreased (P < 0.05) in cells isolated from pigs fed MAN compared with cells isolated from pigs fed diets without MAN. Lymphocyte proliferation of pokeweed mitogen-stimulated cells isolated from pigs fed MAN was less (P < 0.05) than for pigs fed diets devoid of MAN when diets contained 200 ppm Zn; however, MAN had no effect on lymphocyte proliferation when the diet contained 500 or 2,500 ppm Zn (MAN x Zn interaction, P < 0.05). Although the magnitude of response to MAN was not equivalent to that of pharmacological concentrations of Zn, MAN may improve growth response when pharmacological Zn levels are restricted.

Key Words: Growth • Lymphocyte Transformation • Mannans • Swine • Zinc •

Introduction

Pharmacological concentrations of Zn are commonly added to nursery pig diets during the early weaning period because of the improved growth response often elicited. Additionally, high levels of dietary Zn have been implicated in the prevention of diarrhea after weaning (Holm and Poulsen, 1996). One mechanism by which Zn may improve performance in young pigs is by stabilizing the microbial flora in the intestinal tract at a time when the enteric environment is disrupted by the transition from a milk diet to a solid diet (Katouli et al., 1999). The European Union has recently limited Zn in swine diets to 500 ppm. However, there has been no reported benefit to supplementing ZnO at levels less than 1,000 ppm (Dove and Ewan, 1990; Kornegay et al., 1993).

Phosphorylated mannans (MAN) derived from the cell wall of the yeast Saccharomyces cerevisiae may provide an alternative to feeding pharmacological levels of ZnO. Previous research investigating the benefits of adding MAN to livestock diets has reported improved growth response in poultry (Kumprecht et al., 1997) and pigs (Davis et al., 2002). Like pharmacological additions of ZnO, MAN influences the microbial population in the intestinal tract by preventing bacteria from attaching to mannose residues on intestinal epithelial cell surfaces and thereby colonizing the intestinal tract (Oyofo et al., 1989a,b; Spring et al., 2000). However, MAN may also alter the immune response due to the presence of mannose receptors on many cells of the immune system (Djeraba and Quere, 2000).

The objectives of this study were 1) to assess the efficacy of MAN and pharmacological additions of ZnO for improving growth and efficiency in weanling pigs, 2) to investigate the potential of MAN to act synergistically with 500 ppm Zn to improve growth and efficiency, and 3) to determine whether the additives act to modulate T- and B-lymphocyte proliferative responses of the weaned pig.

Materials and Methods

Animals and Housing
In each experiment, 216 weanling barrows (Hampshire x Duroc sires mated to Yorkshire x Landrace females), averaging 19 d of age and 6.2, 4.6, and 5.6 kg of initial BW in Exp. 1, 2, and 3, respectively, were obtained from Tyson Foods, Inc. (The Pork Group, Inc., Rogers, AR), from a single source and transported to the University of Arkansas off-site nursery facility. Pigs were divided into weight groups (blocks) in each experiment and further subdivided into pens with six pigs per pen. Pigs in each experiment had ad libitum access to feed and water and were housed in an environmentally controlled nursery. Each pen measured 1.63 x 1.19 m and had two nipple waterers, a five-hole feeder, and Maxima nursery flooring (Agra Flooring Int. Ltd., Calgary, Alberta, Canada). For the first week of the trial, the ambient temperature was maintained at 29°C and decreased 0.5°C each week of the experiment.

Diets
Basal diets (Table 1) fed in each of the three experiments were supplemented with ZnO (Zinc Nacional, S. A., Monterrey, Mexico; 72% Zn) and/or MAN (Bio-Mos, Alltech, Nicholasville, KY) to provide the dietary treatments. Substitutions were made to the basal diets at the expense of corn. Diets were formulated to contain 200 ppm Zn and 1.50% total Lys during Phase 1, 1.35% total Lys during Phase 2, and 1.20% total Lys during Phase 3. Dietary nutrients met or exceeded the recommended values according to NRC (1998). Calculated Zn concentrations for each basal diet were made using published values according to NRC (1998) for each feed ingredient and the ZnO content of the trace mineral premix. Pharmacological levels of Zn were obtained by supplementation with ZnO. The amount of ZnO required to supply approximately 500 and 2,500 ppm Zn was calculated to be 0.05% to provide 300 ppm Zn and 0.32% to provide 2,300 ppm Zn, respectively, in addition to the 200 ppm Zn in the basal diet.

Experiment 2.
Six dietary treatments were arranged as a 2 x 3 factorial and consisted of two levels of Zn (200 and 2,500 ppm) and three levels of MAN (0, 0.2, and 0.3%). Dietary treatments were fed throughout Phases 1 (d 0 to 10), 2 (d 10 to 24), and 3 (d 24 to 38) of the experiment. Treatment diets were formulated by supplementing the basal diets with 0.32% ZnO, 0.2% MAN, and 0.3% MAN. Diets were randomly assigned to pens within each weight block such that there were six pens representing each dietary treatment.

Experiment 3.
Six dietary treatments were arranged as a 2 x 3 factorial and consisted of three levels of Zn (200, 500, and 2,500 ppm) and two levels of MAN (0 and 0.3%). Phosphorylated mannans were added at 0.3% in this experiment due to the performance response observed in Exp. 2. Dietary treatments were fed throughout Phase 1 (d 0 to 7) and Phase 2 (d 7 to 21). The addition of MAN remained the same during Phase 3 (d 21 to 35); however, Zn was maintained at 200 ppm during Phase 3 in all diets, resulting in only two dietary treatments (0% and 0.3% MAN) fed during Phase 3. Treatment diets were formulated by supplementing the basal diets with 0.05 and 0.32% ZnO during Phases 1 and 2, and 0.3% MAN in each of the three phases. Diets were randomly assigned to pens within each weight block such that there were six pens representing each dietary treatment.

Data Collection
In each experiment, individual pig BW and feed disappearance from each pen were determined at the initiation and termination of Phase 1, and weekly during Phases 2 and 3. Average daily gain, ADFI, and G:F were calculated on a per-pen basis for each phase.

Blood samples were obtained on d 10, 11, 14, and 15 during Exp. 1; d 24, 25, 28, and 29 during Exp. 2; and d 21, 22, 26, and 27 during Exp. 3. On each of the four sampling days within each experiment, one pig was randomly selected from 18 pens for sampling. On the first sampling day, 18 pens were selected such that each treatment was represented on each day, whereas the remaining 18 pens were sampled on the second sampling day. This procedure was repeated for the last two sampling days within each experiment. The first two days were considered to be one replication, and the last two days were considered to be the second replication, such that each pen was sampled in each replication within each experiment.

Lymphocyte Blastogenesis Assay.
In vitro lymphocyte proliferation response was measured using a lymphocyte blastogenesis assay from methods adapted from Blecha et al. (1983). A 15-mL blood sample was obtained in heparinized tubes via vena cava puncture from two randomly selected pigs in each pen, for a total of 72 pigs sampled (18 pigs per treatment in Exp. 1, 12 pigs per treatment in Exp. 2 and 3). Whole blood was mixed in a 1:1 ratio with 0.15 M PBS (pH 7.4). Peripheral blood mononuclear cells were isolated by gradient centrifugation using Ficoll gradient (Histopaque 1077, density = 1.077g/mL; Sigma Chemical Co., St. Louis, MO). Any remaining erythrocytes were lysed by adding 1 mL of sterile water to the isolated pellet for 20 s. Cells were resuspended in RPMI medium at a concentration of 2 x 10⁶ cells/mL and plated in triplicate in 96-well, round-bottom plates in 100-µL aliquots. The T and B lymphocyte proliferation was stimulated using phytohemagglutinin (PHA, Sigma) and pokeweed mitogen (PWM, Sigma), respectively. One to two weeks before each experimental assay, a preliminary test was conducted to titrate various concentrations of the mitogens to determine optimal concentrations before each experiment. Tested mitogen concentrations ranged from 10 to 80 µg/mL for PHA, and 5 to 50 µg/mL for PWM. The concentration that gave the maximum response (based on the arithmetic mean) was used. Mitogens were administered at a concentration of 30 and 10 µg/mL (Exp. 1), 30 and 20 µg/mL (Exp. 2), and 50 and 25 µg/mL (Exp. 3), respectively, for PHA and PWM, to stimulate lymphocyte proliferation. Incubation, labeling with tritiated thymidine (specific activity = 6.7 Ci/mmol; ICN Pharmaceuticals, Inc., Irvine, CA), and cell harvesting followed procedures outlined by van Heugten and Spears (1997). Cells were incubated for 48 h at 37°C and 5% CO₂. Following the 48-h incubation, tritiated thymidine (1 µCi/well in 50 µL of RPMI) was added to each well and the cultures were incubated for an additional 18 h. Cells were harvested on glass fiber mats and the radioactivity was measured as counts per minute on a liquid scintillation analyzer (TRI-CARB 2200CA, Packard Instrument Co., Downers Grove, IL).

Statistical Analysis
In each experiment, ANOVA was performed using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC), with pen as the experimental unit. For performance data, the model included the effects of weight block, Zn, MAN, and the Zn x MAN interaction. For blood analysis data, the model included the effects of replication, Zn, MAN, and the Zn x MAN interaction. The sampling day x treatment interaction was evaluated for the blood analysis data and was not statistically significant (P > 0.20). Therefore, the first two and last two sampling days were combined for two replications. When a significant interaction was observed, treatment means were separated using the PDIFF option of the LSMEANS statement in PROC GLM. Main effect means were evaluated when the interaction was not significant.

Results

There was no growth response to MAN addition to diets during Exp. 1 (Table 2). Supplementation of ZnO at pharmacological levels in diets fed to pigs from d 0 to 10 after weaning increased (P < 0.05) ADG, ADFI, and G:F, and ADG and G:F in the overall experiment (d 0 to 38) compared with pigs fed diets containing 200 ppm Zn. From d 10 to 24, ADG and ADFI increased (P < 0.05) when pigs were fed diets containing 2,500 ppm Zn compared with those fed 200 ppm. Proliferation response of lymphocytes was not different among dietary treatments fed in this experiment.

View larger version (14K): [in this window] [in a new window]   Figure 1. Effect of phosphorylated mannans and zinc oxide added to nursery pig diets on ADG from d 10 to 24 after weaning in Exp. 2 (mannan x zinc interaction, P < 0.05). Error bars indicate standard error and values with different letters indicate differences at P < 0.05.

View larger version (14K): [in this window] [in a new window]   Figure 2. Effect of dietary inclusion of zinc oxide and phosphorylated mannans (MAN) on the proliferation of pig lymphocytes in response to in vitro administration of pokeweed mitogen in Exp. 3 (mannan x zinc interaction, P < 0.05). Error bars indicate standard error, and values with different letters indicate differences at P < 0.05.

The pharmacological addition of ZnO in these experiments consistently improved gain, feed intake, and efficiency during the early postweaning period, which is consistent with previous research extensively reviewed by Cromwell (2001). Additionally, supplementing ZnO to provide 500 ppm Zn in weanling pig diets did not result in any growth benefit over supplementation of Zn to provide the pigs’ dietary requirement. This concurs with previous research, which reported no improvement in growth when supplementing ZnO at levels less than 1,000 ppm (Dove and Ewan, 1990; Kornegay et al., 1993).

Although the growth-promoting benefits observed from the supplementation of pharmacological concentrations of Zn are consistent across many experiments, the effects from supplementing MAN in swine diets have not been as extensively investigated. A series of four experiments were conducted by LeMieux et al. (2001) evaluating MAN and ZnO supplementation in nursery pig diets. Similar to the results of our experiments, response to supplemental MAN was variable across the four experiments. Generally speaking, although there were effects of MAN on ADG and G:F regardless of Zn concentration in the diet, responses to MAN also occurred in synergy with pharmacological ZnO supplementation. However, this synergy was not evident when MAN was added to diets containing 500 ppm Zn. The variation in growth response to MAN supplementation suggests that there may be several factors influencing the weanling pig’s response to MAN supplementation, such as environmental conditions, herd health status, disease challenge, or other dietary components.

To determine whether pharmacological Zn concentrations or MAN supplementation modulated immune function of weanling pigs, the proliferation response of lymphocytes isolated from pigs in each experiment was evaluated in vitro. In the third experiment, the lower lymphocyte proliferation observed in unstimulated cells and cells administered PHA in MAN-fed pigs indicated that lymphocytes of the MAN-fed pigs are in a more suppressed state than those in pigs fed diets devoid of MAN. The Zn x MAN interaction observed with PWM stimulation reinforces this idea of suppressed lymphocyte stimulation, such that lymphocyte proliferation was lower with MAN supplementation when the diet contained 200 ppm Zn, whereas additional Zn supplementation seemed to mask this effect. There is evidence that the addition of high concentrations of Zn increases the proliferation response of lymphocytes. Additional Zn added to broiler breeder diets resulted in an increased cellular immune response in their offspring (Kidd et al., 1993), whereas the addition of excess Zn in turkey diets directly resulted in an increase in cellular immunity (Ferket and Qureshi, 1992).

Phytohemmaglutinin stimulates the nonspecific proliferation of primarily T cells, whereas PWM stimulates the nonspecific proliferation of T and B cells (Roitt et al., 1985). The observations in this study indicate that MAN acts to suppress the proliferative lymphocyte response both in an unstimulated resting state, as well as when activated with mitogens. These findings are supported by observations of the effect of mannose oligosaccharides on lymphocyte function in vitro (Muchmore et al., 1990; Podzorski et al., 1990). Stimulation of the immune response is energetically costly and results in metabolic alterations that redistribute resources away from growth (Spurlock, 1997). The depression in lymphocyte responsiveness due to MAN supplementation may be a mechanism by which growth performance is improved in weanling pigs.

Implications

Although responses were inconsistent, phosphorylated mannans may have potential to improve growth performance in the presence of pharmacological levels of ZnO and modulate lymphocyte proliferation when supplemented in nursery pig diets. The addition of phosphorylated mannans with 500 ppm dietary zinc did not result in a synergistic response above either added singly. Although the magnitude of response to phosphorylated mannan supplementation was not equivalent to that of pharmacological concentrations of Zn, phosphorylated mannans may result in an improved growth response in situations in which zinc supplementation is restricted.

¹ Correspondence: B-128 Animal Science (phone: 501-575-2111; fax: 501-575-7294; e-mail: cmaxwell{at}uark.edu).

Received for publication April 8, 2003. Accepted for publication September 25, 2003.

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