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Effects of Phytate and Phytase on the Performance and Immune Function of Broilers Fed Nutritionally Marginal Diets

N. Liu^*, Y. J. Ru, A. J. Cowieson, F. D. Li^*,1 and X. CH. Cheng

^* Faculty of Animal Science and Technology, Gansu Agricultural University, Lanzhou, China, 730070; Danisco Animal Nutrition, Science Park III, Singapore, 117525; Danisco Animal Nutrition, Marlborough, Wiltshire, SN8 1XN, United Kingdom; and College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, China, 471003

¹ Corresponding author: lifd{at}gsau.edu.cn

	ABSTRACT

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The effects of phytate and phytase on broiler performance and cellular and humoral immunity was assessed by using 504 Cobb 500 female broilers fed nutritionally marginal diets. At 1 d of age, the chicks were randomly allocated to 6 treatments in a 2 x 3 factorial arrangement including 2 levels of phytate P (0.22 or 0.44%) and 3 dose rates of an Escherichia coli-derived phytase [0, 500, or 1,000 phytase units (FTU)/kg of feed]. Both low- and high-phytate diets were corn and soy based with the same nutritional specifications (AME 2,900 kcal/kg, CP 21.00%, Ca 0.78%, and nonphytate P0.28%), differing only in the concentration of phytate P. The immune status of birds was measured at 14, 21, and 28 d of age. The results revealed that phytase improved feed intake, BW, and feed conversion ratio (P < 0.05), whereas high dietary phytate depressed bird performance (P < 0.05). There was no interaction between phytate and phytase on bird performance. Feeding phytase increased the percentages of erythrocyte rosette-forming cells and erythrocyte-antibody complement cells by 3.03 and 1.83% at d 21, and by 2.76 and 2.20% at d 28, respectively (P < 0.05). The percentages of CD4⁺CD8⁺ T lymphocyte subsets were also increased by phytase (P < 0.05), without affecting the ratio of CD4⁺ and CD8⁺. The levels of intestinal secretory IgA were improved with phytase at d 14, 21, and 28 (P < 0.05). Antibodies against Newcastle disease virus vaccine were enhanced at d 21 and 28 in the high-phytate diets with phytase addition (P < 0.05). Increasing the phytase dose to 1,000 FTU/kg did not improve immune function further than 500 FTU/kg. The results suggest that application of phytase in nutritionally marginal diets could enhance lymphocyte numbers and the seral and mucosal antibodies of 1- to 28-d-old broilers, suggesting that both phytate and phytase may have a role in gastrointestinal health and immune competence.

Key Words: phytate • phytase • immunity • broiler

	INTRODUCTION

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The extent to which dietary phytate influences performance and nutrient utilization of intensively farmed livestock has received considerable attention in the scientific literature in recent years. Initially, phytate was considered to be a nutritional diluent, exerting its effects via a reduction in the solubility, and thus availability, of P and, to a lesser extent, Ca (Simons et al., 1990). Recent evidence suggests that the ingestion of phytate can have a substantial effect on digestive physiology, modifying secretion and absorption dynamics (Cowieson et al., 2004, 2006b; Ravindran et al., 2006; Cowieson and Ravindran, 2007b; Liu et al., 2007). Ultimately, the mode of action of phytase is to hydrolyze phosphate groups from the phytate molecule, yielding lower molecular weight myoinositol phosphates, inositol, and inorganic P in the gastrointestinal (GI) tract (Greiner et al., 2000). However, the extraphosphoric effects of such dephosphorylation of phytate are not well elucidated and warrant further study because endogenous secretion, the GI tract microflora, and the immune status of the host may be expected to be involved. Therefore, it is of interest to understand the effect of phytase on the health and immune status of broilers. It is speculated that the degradation products of the action of phytase on phytate may regulate immunocyte activity (Vucenik and Shamsuddin, 2006; Bozsik et al., 2007), and this may be particularly true for broilers fed diets that contain a high concentration of phytate (Cowieson and Adeola, 2005). The literature reviewed by Kidd (2004) has clearly indicated that subnutrition impairs the bird’s ability to mount an effective response in natural and acquired immunity. Phytase can partially ameliorate the adverse effects of phytate in the GI tract of broilers (Cowieson et al., 2004, 2006a) and may improve mucosal immunity by enhancing nutrient uptake for the intestinal immune cells and improving mucin integrity (Cowieson and Ravindran, 2007a). Although Kettunen and Rautonen (2005) reported that exogenous enzymes increased the levels of IgA in digesta, the literature in this area is limited.

The current study was designed to assess the effects of phytate and phytase on the immune function of broilers during the first 28 d of growth by measuring the numbers of peripheral blood lymphocytes and subpopulations, the levels of specific antibodies against Newcastle disease virus (NDV) vaccine, and intestinal mucosal secretory IgA (SIgA) when broilers were fed nutritionally marginal diets.

	MATERIALS AND METHODS

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Treatments
The experiment was conducted as a 2 x 3 factorial with 2 levels of phytate P (0.22 or 0.44%) and 3 dose rates [0, 500, or 1,000 phytase units (FTU)/kg of feed] of an Escherichia coli-derived phytase (Phyzyme XP, Danisco Animal Nutrition, Wiltshire, UK). Both low- and high-phytate diets were corn and soy based with the same nutritional specifications [AME 2,900 kcal/kg, CP 21.00%, Ca 0.78%, and nonphytate P (NPP) 0.28%], differing only in the concentration of dietary phytate P (Table 1). During the experiment, no antibiotics were offered to broilers via either feed or water. Rice bran and corn germ meal were used to regulate dietary phytate.

Sampling
At 14, 21, and 28 d of age, following 6 h of fasting, 12 birds with average BW from each treatment (2 birds per replicate) were selected and weighed. Blood was obtained from each chicken by cardiac puncture and directly aliquoted into 2-mL sterile vials. One aliquot used liquid sodium heparin (1,000 USP units/mL) as an anticoagulant to obtain the whole blood for lymphocyte tests, and another was allowed to clot for 4 h for serum preparation. After centrifugation for 10 min at 3,000 x g, the serum was aliquoted (100 µL) into 1-mL vials and stored at –20°C for measurements. Birds were then killed by cervical dislocation, and the entire small intestine was excised and flushed with 0 to 4°C saline (7.5 g/L of NaCl). The lumen of the jejunum was immediately cut longitudinally to expose the brush border cells. The mucosa were gently scraped off with a glass microscope slide, diluted at 1:1 (g:mL) with calcium and magnesium-free PBS (CMF-PBS, pH 7.4), homogenized for 20 strokes, and centrifuged for 10 min at 8,000 x g. The supernatant was then separated and stored at –20°C until measurement of mucosal antibody levels.

Measurements
Lymphocytes.
Peripheral blood lymphocytes were quantified by using the method of erythrocyte-rosette formation tests described by Brain et al. (1970). Blood samples were diluted with an equal volume of CMF-PBS. The diluted blood was slowly added to the same volume of a lymphocyte separation solution. After centrifugation at 1,000 x g for 20 min, a white layer of mononuclear cells at the plasma-Ficoll interface was collected and washed twice with CMF-PBS. The mononuclear cells were diluted with CMF-PBS and counted to 5 x 10⁶/mL.

To measure erythrocyte rosette-forming cells (ERFC), the diluted lymphocytes (100 µL) were incubated with a suspension of 20% bovine serum in CMF-PBS (100 µL) and 2% sheep red blood cell solution (100 µL) at 37°C for 15 min, and then centrifuged for 5 min at 100 x g. The cell pellets were harvested and stored at 4°C for 4 h. The cells were differentiated by smear examination (Wang, 1981). Erythrocyte rosette-forming cells around which a cluster (rosette) of cells consisting of more than 3 sheep erythrocytes was formed were counted. The procedure of measuring erythrocyte-antibody complement cells (EAC) was the same as that for ERFC described above, following the incubation of diluted lymphocytes with a sensitized erythrocyte-antibody complement suspension (100 µL) containing 4% sheep red blood cells, 1:4,000 hemolysin, and 20% mouse serum.

For the assay of T lymphocyte subsets, blood mononuclear cells were isolated as described above and adjusted to a concentration of 1 x 10⁷/mL with CMF-PBS (pH 7.4). For each sample, 3 tubes containing the following combinations of monoclonal antibodies purchased from Southern Biotechnology Associates (Birmingham, AL) were set up: 10 µL of CD4-phycoerythrin (no. 2304925; amount: 10⁶ cells/µg) and 10 µL of CD3-fluorescein isothiocyanate (no. 2301061; amount: 10⁶ cells/µg); 10 µL of CD8a-phycoerythrin (no. 2308578; amount: 10⁶ cells/µg) and 10 µL of CD3-fluorescein isothiocyanate (amount: 10⁶ cells/µg); and 10 µL of IgG₁-fluorescein isothiocyanate (no. 2608595; amount: 10⁶ cells/µL) and 10 µL of IgG₁-phycoerythrin (no. 2608807; amount: 10⁶ cells/µL).

After adding 50 µL of mononuclear cell suspension to each tube, the mixture was incubated at 4°C for 20 min in the dark. Then, 500 µL of CMF-PBS solution was dispensed into each tube, and the suspension was gently mixed and left for 10 min at room temperature out of direct light. After 10 min of centrifugation at 800 x g, the cells were resuspended with 500 µL of CMF-PBS solution. The percentages of CD3⁺CD4⁺ and CD3⁺CD8⁺ lymphocytes in the mononuclear cell suspension were determined by flow cytometry (Model epicsxl, Beckman Coulter Inc., Miami, FL).

Antibodies.
Anti-Newcastle disease virus serum hem-agglutination inhibition antibody titers were measured by the micromethod (Zheng, 1984) with NDV antigen NDV antigen (4 hemagglutination units) and 0.5% rooster erythrocyte suspension. The geometric mean titer was expressed as reciprocal log2 values for the highest dilution that displayed hemagglutination inhibition.

The levels of mucosal SIgA were determined according to the method described by Schuijffel et al. (2005) with a chicken IgA ELISA quantitation kit (no. E30-103, Behyl Laboratories Inc., Montgomery, TX) with goat anti-chicken IgA-horseradish peroxidase conjugate and 1% BSA. The absorbance was read at a wavelength of 450 nm by a microplate instrument (Model Elx-808, Bio-Tek Instruments Inc., Winooski, VT).

Statistical Analysis
The experiment was a 2 x 3 factorial arrangement, with dietary phytate P and phytase dose rate being the main factors. A general linear model was used to assess the effects of dietary phytate and phytase and their interaction with SAS software (SAS Institute, 2002). All data presented as percentages were transformed to their arc-sine square root before statistical analysis, and the non-transformed data are presented in the tables. Differences were considered significant at P < 0.05. When a significant F-test was detected, means were separated by using the least significant difference test.

	RESULTS

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The chickens were healthy throughout the experiment, with a mortality of less than 2% that was unrelated to dietary treatment. No bacterial or viral disease infection was detected. Supplementation with phytase improved the feed intake, BW, and feed conversion ratio of birds (P < 0.05; Table 2), but no differences were found between phytase at 500 and 1,000 FTU/kg of feed. Birds fed the diet with a high phytate concentration had a lower BW and poorer feed conversion ratio than those fed the low-phytate diet (P < 0.05). No interaction between phytate and phytase was detected on performance parameters.

	DISCUSSION

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The effects of both phytate and phytase on immunological parameters are interesting. Studies in humans have demonstrated that lower molecular weight inositol phosphate esters are important in regulating vital cellular functions, such as ion channels and protein trafficking (Shears, 1996; Larsson et al., 1997), oocyte maturation (Ji et al., 1989), and cellular differentiation (Berridge and Irvine, 1989; Menniti et al., 1993), and may be involved in strengthening of the immune system by enhancing immunocyte activity and inhibiting pathological calcification (Shamsuddin, 2002; Vucenik and Shamsuddin, 2003; Shamsuddin and Vucenik, 2005). To our knowledge, there are no reports on the physiological benefits of low molecular weight inositol phosphate esters for broilers, although dietary manipulation can influence inositol phosphate ester concentration in biological fluids, which at least raises the possibility of nutritional intervention strategies (Grases et al., 2001). In the current study, phytase addition significantly increased the percentages of ERFC and EAC, indicating that the proliferations of T cells and B cells were induced by factors that may result from the substantial concentrations of lower inositol phosphates created by dietary phytase. ya et al. (2000) reported that phytase addition to diets with a low P concentration enhanced the bursa weight of 21-d-old Hubbard broilers. Because the bursa is the source organ for B cells, the development of the bursa may induce the proliferation of B cells. Thus, the growth-promoting effect of phytase may be expressed via both nutrient release and a physiological regulation mechanism.

The percentages of CD4⁺ and CD8⁺ T cells were enhanced by phytase addition in the current study, indicating potential for more activity of immunocytes. It has been proposed that CD4⁺ T cells may induce and enhance the immune response by secreting cytokines, whereas CD8⁺ T cells mediate cytotoxic killing of target cells (Summerfield et al., 1996). Cytokines can "reprogram" metabolism to ensure an adequate supply of nutrients for the proliferation of lymphocyte and macrophage populations, and for antibody production (Spurlock, 1997). Kettunen and Rautonen (2005) reported that the use of a combination of enzymes and betaine enhanced immune maturation by increasing CD4⁺ T cells in ileal tissue. With a similar mode of action, it is possible that phytase improves the percentages of CD4⁺ and CD8⁺ T cells. The observations herein put forth the possibility that part of the nutritional improvements associated with phytase may be mediated via improvements in immunocyte activity. Additionally, CD8⁺ cytotoxic T lymphocytes may regulate the lytic activity via a perforin pathway, as found in mice and humans (Groscurth and Filgueira, 1998).

Antibodies are important biological agents prevalent in the healthy immune repertoire, and they participate in the maintenance of immune homeostasis by exposure to environmental stimulation (Coutinho et al., 1995; Bayry et al., 2005). It has been shown that low levels of humoral antibody may be related to disease susceptibility (Parmentier et al., 2004). Serum hemagglutination inhibition antibody is a valid indicator because it is directly effective against NDV in the humoral immunity of chickens (Roy et al., 1999; Maas et al., 2003). In the current study, anti-NDV antibodies were improved in some treatments by phytase addition, indicating that dietary factors may affect specific immune responses. There is some precedent for these effects in the literature, because Gao et al. (2004) reported that supplementation of diets with nonstarch polysaccharide-degrading enzyme preparations significantly increased the anti-NDV titers of chicks.

The investigation of innate mucosal humoral immunity in this study showed that the levels of SIgA were increased by phytase addition. The mucosal epithelium is a potential effector tissue of integrated host responses, producing SIgA to protect GI-associated port of entry into the body. The degradation products of phytate by phytase may regulate immune activity of these cells (Vucenik and Shamsuddin, 2006; Bozsik et al., 2007). Kettunen and Rautonen (2005) reported that the use of xylanase, amylase, and protease or a combination of the enzymes and betaine enhanced nutrient uptake by intestinal cells and concluded that the concentration of IgA in the digesta contributed to improvements in immune competence. In the current study, birds fed on the high-phytate diets returned a lower concentration of SIgA in jejunal mucosa, which may partially result from dilution with hypersecreted mucin, as stimulated by phytate. The mechanism by which phytate influences mucin integrity is thought to be related to the highly (pH dependent) reactive nature of dietary phytate. When feed is exposed to the low pH conditions in the proximal gut, phytate is solubilized and can react electrostatically with basic amino acid residues in dietary protein. These phytate-protein complexes are variably refractory to digestion by pepsin and solubilization with HCl (Vaintrub and Bulmaga, 1991), leading to a downstream increase in the secretion of mucin by GI epithelium. Therefore, the fact that phytase addition ameliorates the excess secretion of mucin (Cowieson et al., 2004, 2006a) may contribute to maintaining a "normal" gut ecology that enhances host immunity by stimulating the immunological defense mechanisms at the mucosal and systemic level, perhaps by reducing the concentration of saprogenic compounds.

A comprehension of the effect of phytase and phytate on endogenous protein production is not only scientifically enlightening, but also assists in explaining why published phytase matrix values for amino acids and energy vary so significantly. For example, the improvements in the ileal digestibility of Thr, Ser, and Cys with phytase are superior to that of Met, perhaps because endogenous digestive and immunological proteins are essentially devoid of Met. This is partly confirmed by Cowieson and Ravindran (2007b) in that amino acid compositional changes in endogenous protein in response to the ingestion of phytate are highly correlated with the phytase-induced improvement in ileal amino acid digestibility and also the amino acid profile of mucin. Further, synthesis and loss of endogenous proteins carry a substantial net energetic cost as well as having a direct effect on the digestible energy value of the diet. It has been proposed that the effect of phytate on endogenous protein loss may negatively influence digestible energy by as much as 0.1 MJ/kg of DM intake for every 1 g/kg of dietary phytate P (Cowieson and Ravindran, 2007a), without including the effects on net energy associated with protein synthesis and turnover. Therefore, a substantial part of the extra-phosphoric effects of exogenous phytase may be mediated via beneficial changes in secretory physiology and improvements in the immunological repertoire.

In conclusion, phytate is a ubiquitous and potent anti-nutrient in monogastric diets and exerts a range of physiological, nutritional, and immunological consequences on the host. Compensatory mechanisms are in place to allow normal digestive processes to continue, but these carry a substantial nutritional cost to the animal in terms of energy, and amino acid and mineral requirements associated with synthesis, absorption, catabolism, and autolysis. An understanding of the antinutritive effects of phytate is an important first step in developing improved microbial phytases and in maximizing the potential of currently available phytase technology. The interaction between phytate and phytase and digestive physiology, cellular and humoral immunity, and microbiology warrants further investigation, particularly the role of phytase in neonate nutrition and during disease challenge.

	ACKNOWLEDGMENTS

 
The authors are grateful to the staff of Animal Immunology Laboratory of Henan University of Science and Technology (Luoyang, China) for providing the experimental facilities and carrying out most of the practical work for this experiment.

Received for publication December 12, 2007. Accepted for publication February 21, 2008.

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