IMMUNOLOGY, HEALTH, AND DISEASE

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1 IMMUNOLOGY, HEALTH, AND DISEASE Influence of dietary calcium level, calcium source, and phytase on bird performance and mineral digestibility during a natural necrotic enteritis episode D. M. Paiva,* C. L. Walk, and A. P. McElroy * 1 * Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061; and AB Vista Feed Ingredients, Marlborough, Wiltshire, SN8 4AN, United Kingdom ABSTRACT The objective of this study was to determine the influence of Ca source [highly soluble calcified seaweed (HSC) or limestone], phytase supplementation, and dietary levels of Ca on bird performance and mineral digestibility (Ca and P) during a necrotic enteritis (NE) episode. Cobb 500 male broilers were weighed and randomized into 8 treatment groups (9 pens/treatment; 30 birds/pen) at day of hatch. The 21-d trial was designed as a factorial, which included 2 dietary levels of Ca (0.6 and 0.9%), 2 Ca sources (limestone or HSC), and 2 levels of an Escherichia coli phytase (0 or 1,000 FTU/kg). One unit of phytase (FTU) is defined as the quantity of enzyme that releases 1 µmol of inorganic phosphorus/min from mol/l of sodium phytate at ph 5.5 at 37 C. Birds were placed on used litter from a previous flock that exhibited clinical signs of NE. Birds and feed were weighed on d 7, 14, and 21, and BW gain, feed intake, and feed conversion were calculated for each of these periods and cumulatively. Mortality was recorded daily and ph of the gizzard and duodenum were measured on d 7, 14, and 21. Ileal digesta (8 birds/pen) was collected on d 7, 14, and 21. Significance is reported at P < Birds began exhibiting clinical signs of NE on d 9, and elevated NE-associated mortality persisted until the end of the trial. Significantly higher mortality was observed when broilers were fed diets with 0.9% Ca from HSC compared with birds fed diets with 0.6% Ca, regardless of Ca source. Broilers fed 0.6% Ca diets supplemented with phytase were heavier than the other treatments regardless of Ca source. Broilers fed diets formulated with HSC had significantly higher feed conversion then broilers fed diets formulated with limestone. The gizzard of broilers fed 0.9% Ca in the diet was significantly less acidic than the gizzard of broilers fed 0.6% Ca in the diet. Broilers fed 0.6% Ca in diets supplemented with phytase showed significant improvements in P and Ca digestibility. In conclusion, higher dietary Ca (0.9% vs. 0.6%) had a negative effect on mortality associated with NE and on bird performance. Key words: calcium, high soluble calcium, phytase, necrotic enteritis 2013 Poultry Science 92 : INTRODUCTION True dietary requirements for Ca and P are of ongoing interest to poultry nutritionists. Diet supplementation with exogenous enzymes, especially phytases, represents a challenge when trying to establish the ideal dietary levels of Ca and P in the diet. The impacts of phytase supplementation on poultry performance [BW gain (BWG), feed intake (FI), and feed conversion (FC)] and mineral digestibility have been extensively reported by several authors (Ravindran et al., 1995; Selle et al., 2009; Rutherfurd et al., 2012). However, little information exists in the literature regarding the 2013 Poultry Science Association Inc. Received May 8, Accepted August 18, Corresponding author: amcelroy@vt.edu impact of phytases on intestinal health and Ca and P requirements during disease. The main purpose of phytase supplementation is to increase nutrient availability. Initially, the supplementation of diets with phytases targeted improvements in P availability and digestibility (Perney et al., 1993; Selle et al., 2009). However, research has shown that phytase supplementation also has a significant impact on the availability of other nutrients, such as proteins and amino acids, minerals, and carbohydrates (Cowieson et al., 2006, 2009). Calcium must be soluble in the intestinal lumen to be absorbed. Calcium solubility in the intestine is closely related to small intestinal ph (around 6.0) and phytate concentration. When ph is close to neutral, phytate forms mineral chelates that are highly insoluble (Sebastian et al., 1996; Tamim et al., 2004; Plumstead et al., 2008). In addition, a high ratio of dietary Ca to P reduces the digestibility and absorption of Ca and P due 3125

2 3126 PAIVA et al. to increased precipitation of Ca-P complexes (Plumstead et al., 2008; Selle et al., 2009). Selle et al. (2000) have reported that maximum insolubility of phytatemineral chelates occurs between ph 4 and 7. Calcium is one of the divalent cations with the lowest affinity for phytate. However, because Ca is the mineral added in highest concentrations in poultry diets, it has a greater impact in forming mineral-phytate chelates than other dietary minerals, making both Ca and P unavailable for absorption (Sebastian et al., 1996; Maenz et al., 1999; Tamim et al., 2004). Thus, the problem with phytate digestion is not a lack of compatible endogenous enzymes but poor substrate solubility in the small intestine. This effect is increased when high amounts of Ca are present (Tamim et al., 2004; Cowieson et al., 2011). Benefits from supplementing broiler diets with lower levels of Ca are supported by performance results. Anderson et al. (1984) reported that BWG, FC, and bone ash were all significantly reduced by increasing dietary Ca from regular industry standards (0.9%) to 1.5% in the diet. Sebastian et al. (1996) were the first to challenge NRC (1994) recommended Ca levels in diets supplemented with phytase. In their study, efficacy of supplemental phytase was significantly affected by dietary Ca levels, and the optimal growth performance and retention of P and Ca was achieved at the lowest level of dietary Ca tested (0.6%). A healthy intestine is able to digest and absorb nutrients released by phytase from the phytate molecule resulting in improvements in bird performance. However, when the gastrointestinal tract is colonized by enteric pathogens, damage to the intestinal lining impairs function of the intestinal mucosa and utilization of nutrients, and these nutrients may become available to enteric pathogens. One enteric disease of concern for the commercial poultry industry related to impaired intestinal function is necrotic enteritis (NE). Necrotic enteritis has reemerged in recent years as an important multifactorial enterotoxemia in poultry. The increasing numbers of flocks affected by NE are likely consequent to attempts to reduce the use of antimicrobials and coccidiostats in poultry feed by the industry. Clostridium perfringens is the etiological agent associated with NE. Although the pathogenesis of NE is not completely elucidated, it is believed that the toxins produced by C. perfringens are responsible for producing the lesions observed during NE. Researchers suggest that the most important toxins in the pathogenesis of NE are netb and α-toxin. NetB forms pores in the cellular membrane causing an influx of ions (Ca, Na, Cl, and so on) that eventually lead to osmotic cell lysis (Keyburn et al., 2010). Alpha-toxin is a zinc-dependent phospholipase sphingomyelinase that hydrolyzes phospholipids and promotes cellular membrane disorganization. In addition to Zn, α-toxin also depends on Ca for full activity. Calcium ions are essential for the binding of α-toxin to lipid films (Moreau et al., 1988; Petit et al., 1999; Titball et al., 1999). Therefore, there is strong evidence suggesting that Ca is involved in the pathogenesis of NE. The objective of this study was to evaluate the effects of phytase supplementation, Ca sources, and Ca levels on broiler performance, gastrointestinal ph, and mineral digestibility during naturally occurring NE. MATERIALS AND METHODS Birds and Husbandry Experimental protocols were approved by Virginia Tech Institutional Animal Care and Use Committee. Cobb 500 male broilers (n = 2,160) were acquired from a commercial hatchery, randomized, weighed by pen, and placed into floor pens relative to the different treatment groups from d 0 to 21. Each treatment group was replicated by 9 pens of 30 birds each. Birds were placed on used litter from a previous flock that had presented clinical signs of NE. Following the previous trial, litter was removed from each pen and mixed in the center of the house. Mixed litter was then redistributed to pens, and fresh pine shavings were layered on top of the mixed used litter. Feed and water were administered ad libitum throughout the study. Experimental Diets and Bird Performance Diets were formulated to meet or exceed NRC (1994) recommended nutrient levels, with the exception of Ca. In addition, in diets supplemented with phytase, available P was reduced by approximately 0.15% so excess available P would not be present in the diet. Experimental diets consisted of a factorial design with 2 total Ca levels (0.9% and 0.6%), 2 Ca sources (limestone and calcified marine seaweed), and 2 levels of phytase (0 and 1,000 FTU/kg). One unit of phytase (FTU) is defined as the quantity of enzyme that releases 1 μmol of inorganic phosphorus/min from mol/l of sodium phytate at ph 5.5 at 37 C. Calcium was supplemented in the diets as a combination of dicalcium phosphate and limestone or highly soluble calcified marine seaweed (HSC; Vistacal, AB Vista Feed Ingredients, Marlborough, UK). Limestone is constituted almost exclusively of calcite, which is the most stable polymorph of calcium carbonate. Highly soluble calcified marine seaweed is composed of 65% calcite, 23% aragonite, and 12% vaterite. Aragonite and vaterite are less stable polymorphs of calcium carbonate, meaning they are the same chemically but differ in structure. The difference in structure is what gives HSC higher solubility under the same conditions. The phytase used was an Escherichia coli 6-phytase expressed in Trichoderma reesei and contained an expected activity of 5,000 FTU/g (Quantum Blue, AB Vista Feed Ingredients, Marlborough, UK). Diets were analyzed for Ca, total P, and recovered phytase activity, and results were comparable with formulated values (Table 1). Titanium dioxide was included in the diets

3 CALCIUM LEVEL AND SOURCE AND PHYTASE 3127 Table 1. Calculated and analyzed composition and nutrient content of experimental diets 0 (FTU/kg) phytase 1,000 (FTU/kg) phytase HSC 1 Limestone HSC Limestone Item LCa 2 SCa 2 LCa SCa LCa SCa LCa SCa Ingredient (%, unless otherwise indicated) Corn Soybean meal, 48% Poultry fat Salt dl-methionine l-lysine HCl l-threonine HSC Limestone Dicalcium phosphate Sodium phosphate Mineral premix Vitamin premix Titanium dioxide Phytase Calculated composition CP Ca Total P Available P Ca:P ratio 1.3:1 2:1 1.3:1 2:1 1.3:1 2:1 1.3:1 2:1 Nutrient composition Energy (ME, kcal/kg) 3,046 3,046 3,046 3,046 3,046 3,046 3,046 3,046 Analyzed composition DM CP Ca Total P Phytase (FTU/kg) <50 <50 <50 < High solubility calcified seaweed (Vistacal, AB Vista Feed Ingredients, Marlborough, UK). 2 LCa = low Ca (0.6%); SCa = industry standard Ca (0.9%). 3 Titanium dioxide was added to diets as an indigestible marker to calculate apparent ileal digestibility. 4 Sand was supplemented in place of phytase in diets containing 0 FTU/kg of phytase to equal 100%. as an indigestible marker, and it was determined using a colorimetric method previously described by Short et al. (1996). On d 7, 14, and 21, birds and feed were weighed by pen to calculate average BWG, average FI, and FC. Mortality was recorded daily and used to adjust FI and FC according to bird days. Ca and P Digestibility On d 7 (n = 7 birds/pen), 14 (n = 6 birds/pen), and 21 (n = 6 birds/pen), randomly selected birds were euthanized by cervical dislocation, and ileal digesta (defined as Meckel s diverticulum to the ileal cecal junction) was sampled, pooled per pen, and frozen ( 80 C) until further analysis. Frozen digesta samples were freeze-dried and ground (1-mm screen) before mineral analysis. Digesta and diet samples were wet ashed using nitric and perchloric acids. Phosphorus was determined by the alkalimeter ammonium molybdate method, and color intensity was read using a spectrophotometer measuring absorbance at 410 nm (method , AOAC International, 2000). Calcium was determined using flame atomic absorption spectroscopy (method AOAC International, 2000). All samples (Ca, P, and titanium) were assayed in duplicates. Calcium and phosphorus apparent ileal digestibility (AID) was calculated using the following equation: AID = [(nutrient/tio 2 ) diet (nutrient/tio 2 ) ileum ]/ ph (nutrient/tio 2 ) diet. On d 14 and 21, 2 birds/pen of average BW were euthanized to obtain measurements of ph from the gizzard and duodenum. From each bird, ph measurements were obtained directly from the digesta contents in the lumen using a digital ph meter (Mettler-Toledo, Columbus, OH) and spear-tip electrode (Sensorex, Garden Grove, CA). The ph values from each gastrointestinal section/bird were used to obtain a mean ph value/pen for analysis. Statistical Analysis Data were analyzed as a factorial using the fit model platform in JMP 9.0 (SAS Institute Inc.,

4 3128 PAIVA et al. Cary, NC), and means were separated using Tukey s honestly significant difference test. Pen served as the experimental unit. Data were analyzed for main effects (Ca, P, and phytase) and for the interaction of main effects. Statistical significance was accepted at P < The P-values of mortality data are reported as squared arcsine transformed percentage mortality data. Performance RESULTS Birds began exhibiting clinical signs of NE on d 9, and elevated NE-associated mortality persisted until the end of this trial at d 21. No significant differences in mortality were observed from d 0 to 7 or d 0 to 14 (data not shown). However, from d 0 to 21, mortality was significantly affected by a 2-way interaction between Ca source and Ca levels (Figure 1). Mortality was significantly higher when broilers were fed 0.9% HSC diets compared with 0.6% Ca, whereas no differences in mortality occurred between birds fed 0.9 or 0.6% Ca with limestone. Calcium source did not significantly affect mortality when broilers were fed equivalent levels of Ca in the diet, and phytase had no influence on NEassociated mortality at any day measured. From d 0 to 7, a 3-way interaction (Ca level Ca source phytase) was observed for BWG (Figure 2A). Broilers fed diets supplemented with phytase gained more weight than broilers fed diets that were not supplemented with phytase regardless of Ca level or Ca source, with the exception of those with 0.6% Ca from limestone where nonphytase-supplemented broilers had BWG similar to phytase-supplemented birds. A 2-way interaction between Ca level and Ca source significantly affected FI from d 0 to 7 (Figure 2B). When broilers were fed diets formulated using HSC as the Ca source, dietary levels of Ca did not significantly affect FI. However, when limestone was used as the Ca source, broilers fed 0.6% Ca diets had higher FI than broilers fed 0.9% Ca diets. A 2-way interaction between Ca levels Figure 1. Effects of dietary Ca level and Ca source on broiler mortality (%) for d 0 to 21. HSC = highly calcified marine seaweed. Treatment means lacking a common letter (a,b) are significantly different (P < 0.05). and phytase also significantly affected FI during the first week. A significant decrease in FI was observed when broilers were fed 0.9% Ca diets without phytase, but there was no difference in FI due to phytase supplementation in broilers fed diets with 0.6% Ca (Figure 2C). From d 0 to 14 and d 0 to 21, Ca source or phytase supplementation significantly affected BWG (Table 2). Broilers fed diets formulated with limestone gained significantly more weight than broilers fed diets formulated with HSC. Additionally, broilers fed phytasesupplemented diets gained more weight than those on diets without phytase. Dietary levels of Ca did not affect BWG for the same periods. Phytase significantly increased FI from d 0 to 14 (Table 2). Feed intake was not significantly affected by any factors from d 0 to 21. From d 0 to 7, no differences in FC were observed (data not shown), but Ca source had a significant impact on FC from d 0 to 14 and d 0 to 21 (Table 2). Diets formulated with limestone as the Ca source resulted in more efficient FC compared with diets formulated with HSC as a Ca source. Ca and P Digestibility A 3-way interaction of Ca level, Ca source, and phytase significantly influenced P and Ca digestibility. Optimal P digestibility was observed when broilers were fed 0.6% Ca diets formulated with limestone and supplemented with phytase regardless of feeding period (Table 3). On d 7, P digestibility was improved by phytase supplementation in the 0.6% Ca diet with limestone as the Ca source but not with HSC as the Ca source. In contrast, with 0.9% Ca in the diets, phytase supplementation improved P digestibility with HSC as the Ca source but not limestone. The use of HSC as the Ca source in the 0.9% Ca diet not supplemented with phytase resulted in lower P digestibility compared with the 0.6% Ca, nonphytase-supplemented diet on all sampling days, but this wasn t observed with the use of limestone as the Ca source. The improvement in P digestibility by phytase supplementation in the 0.6% limestone diets was also observed on d 14 and 21, whereas it had no effect in other diets regardless of Ca source or level. Similar to P digestibility results on d 7, Ca digestibility was improved by phytase supplementation in broilers fed the 0.6% Ca diets formulated with limestone and 0.9% Ca diets formulated with HSC. However, this response was not seen on d 14 and 21. On d 14, phytase supplementation only improved Ca digestibility of 0.9% Ca diets formulated with limestone. On d 21, broilers fed 0.6% Ca diets supplemented with phytase and formulated with limestone had higher Ca digestibility than those fed 0.9% Ca diets supplemented with phytase and formulated with limestone. Generally, on d 14 and 21, 0.6% Ca diets resulted in improved Ca digestibility, regardless of Ca source or phytase supplementation.

5 CALCIUM LEVEL AND SOURCE AND PHYTASE 3129 Figure 2. Effects of (A) dietary Ca level, Ca source, and phytase on BW gain for d 0 to 7; (B) dietary Ca level and Ca source on feed intake for d 0 to 7; and (C) dietary Ca level and phytase on feed intake for d 0 to 7. HSC = highly calcified marine seaweed. Treatment means lacking a common superscript (a,b) are significantly different (P < 0.05).

6 3130 PAIVA et al. Table 2. Effects of dietary calcium levels, calcium sources, and phytase on BW gain, feed intake, and feed conversion d 0 to 14 and 0 to 21 BW gain (kg) Feed intake (kg) Feed conversion (kg:kg) Variable d 0 to 14 d 0 to 21 d 0 to 14 d 0 to 21 d 0 to 14 d 0 to 21 Ca level 0.60% Ca % Ca SEM Ca source Limestone 0.40 A 0.84 A B 1.38 B HSC 0.39 B 0.80 B A 1.46 A SEM Phytase 0 FTU/kg 0.37 B 0.79 B 0.47 B ,000 FTU/kg 0.41 A 0.85 A 0.51 A SEM P-value Ca level Ca source Phytase < A,B Means within a column and main treatment effect lacking a common superscript are significantly different (P < 0.05). ph Differences in ph measurements were only seen in the gizzard on d 21 (data not shown). The gizzard of broilers fed 0.6% Ca diets was significantly more acidic than the gizzard of broilers fed 0.9% Ca, with values of 2.64 and 2.96, respectively. DISCUSSION New regulations concerning antimicrobial use in feeds and market demand for a product raised without the aid of growth promoters has had a significant impact on the incidence of NE in commercial broiler houses. In this experiment, broilers were fed nonmedicated diets, and high mortality due to naturally occurring NE was observed from d 9 until the end of the trial (d 21). Higher mortality resulting from NE was observed when broilers were fed industry standard levels of Ca (0.9%) in the diet, particularly when diets were formulated using a highly soluble and highly available Ca source (HSC). Research to understand risk factors, pathogenesis, prevention, and control of NE has been hindered by the necessity to induce NE infection through challenge models, often with manipulations of diet and other factors. Our laboratory has repeated ability to evaluate naturally occurring NE, without dietary manipulations or oral inoculation of birds with high counts of C. perfringens, which do not reflect industry conditions. The complete mechanism by which C. perfringens leads to NE has not been completely elucidated. Clostridium perfringens is an extracellular pathogen; thus, its virulence is mostly associated with its ability to produce toxins, such as α-toxin and netb (Songer, 1996), which lead to subsequent mucosal damage along the intestinal lining (Petit et al., 1999; Titball et al., 1999; Keyburn et al., 2010). Membrane recognition by α-toxin is a complex event. The mechanism involves a Ca-mediated phospholipid recognition, which allows α-toxin to bind to the enterocyte cellular-membrane (Titball et al., 1999). The toxin netb also depends on Table 3. Effects of dietary calcium level, calcium source, and phytase supplementation on Ca and P apparent ileal digestibility (AID) on d 7, 14, and 21 Variable Ca source Phytase (FTU/kg) Phosphorus AID (%) Calcium AID (%) d 7 d 14 d 21 d 7 d 14 d 21 Ca level (%) 0.60 Limestone DE 0.63 BC 0.58 CD 0.49 BC 0.58 ABC 0.50 ABC 1, A 0.75 A 0.72 A 0.65 A 0.67 A 0.63 A HSC BC 0.69 B 0.69 AB 0.54 ABC 0.62 A 0.61 AB 1, B 0.64 BC 0.64 ABC 0.57 ABC 0.58 ABC 0.54 ABC 0.90 Limestone CDE 0.62 C 0.61 BCD 0.54 ABC 0.58 AB 0.47 ABC 1, BC 0.61 CD 0.58 CD 0.50 BC 0.49 CD 0.43 C HSC E 0.55 E 0.57 CD 0.44 C 0.45 D 0.45 C 1, BCD 0.56 DE 0.54 D 0.60 AB 0.52 BCD 0.46 BC SEM P-value Ca Ca source phytase < < < A E Means within a column lacking a common superscript are significantly different (P < 0.05).

7 Ca to cause cell lysis through the formation of pores in the enterocyte membrane, which results in an influx of ions into the cytoplasm (Keyburn et al., 2010). The influx of ions into the cell cytoplasm eventually leads to osmotic cell lysis. In addition to osmotic lysis, another mechanism involving Ca and netb activity has been suggested that would induce cell death (Kennedy et al., 2009). Kennedy et al. (2009) reported that the α-toxin (pore-forming toxin) of Clostridium septicum forms Ca-permeable pores, which increase intracellular Ca. It is believed that this Ca influx induced a cascade of events consistent with programmed necrosis because it was associated with calpain activation and release of cathepsins from lysosomes. In addition, Kennedy et al. (2009) also observed deregulation of mitochondrial activity leading to an increase in reactive oxygen species and dramatically decreasing ATP levels. The increase in mortality due to NE observed when broilers were fed 0.9% Ca compared with 0.6% Ca in this trial, in association with other research findings of Ca participation in the mode of action of α-toxin and netb, strongly suggest that dietary Ca level is an important factor in NE pathogenesis. The effect of Ca source also supports this theory. Because HSC is more soluble than limestone and can be supplemented at lower total dietary Ca levels in the presence of phytase (Walk et al., 2012), formulating diets with an industry standard level of Ca (0.9%) from HSC likely resulted in an excess of Ca in the intestinal lumen and more extensive damage to the intestinal mucosa. In the past, Ca involvement with NE was thought to be associated with an increase in gastrointestinal ph with high levels of Ca in the diet (Williams, 2005). In theory, the increase in gastrointestinal ph would favor the proliferation of C. perfringens in the intestinal lumen. However, our data did not suggest that higher mortality when broilers were fed 0.9% Ca in the diet was the result of ph changes. Changes in gastrointestinal ph were not observed until d 21, which was well after the initial phase of the NE outbreak. In addition, ph changes were restricted to the gizzard. Therefore, there was indication that Ca impact on mortality due to NE was most likely associated with toxin activity and not changes in gastrointestinal ph. Phytase supplementation improved BWG regardless of Ca source and Ca levels. Initially, when broilers were fed 0.9% Ca in the diet, phytase supplementation also increased FI, which partially explains the improvements in BWG. However, after d 14, phytase supplementation did not significantly affect FI. Improvements in BW and BWG due to phytase supplementation have been reported previously in reduced P diets (Cowieson et al., 2004, 2006; Selle et al., 2009). In this study, available P was adjusted from 0.45 to 0.30% when diets were supplemented with phytase to minimize the effects of excess P in the diets. These improvements have been associated with extraphosphoric effects of phytase that result from the release of nutrients, such as Ca, carbohydrates, and amino acids, which become available CALCIUM LEVEL AND SOURCE AND PHYTASE 3131 for digestion and absorption and ultimately result in improvements in animal performance in reduced P and Ca diets. After d 7, Ca source had a significant impact on BWG and FC. When broilers were fed diets formulated with HSC, a reduction in BWG was observed. Because there were no differences in FI due to Ca source for the same periods, an increase in FC was expected. The HSC is a more soluble Ca source than limestone (Walk et al., 2012). Therefore, when broilers are fed diets formulated with HSC, the concentration of soluble Ca in the intestinal lumen is expected to be higher than when broilers are fed diets formulated with limestone. Walk et al. (2012) reported broilers fed diets with 0.9% Ca from HSC without phytase were significantly lighter and ate less than birds fed diets with 0.9% Ca from limestone. In addition, broilers fed diets with reduced levels of Ca (0.75, 0.6, or 0.45%) from HSC plus phytase consumed feed and gained weight equivalent to broilers fed an industry standard diet (0.9% Ca) formulated with limestone (Walk et al., 2012). The lack of differences in FI, FC, and BWG in their study could be consequent to the fact that the birds were not undergoing an intestinal challenge, and Ca concentrations had no obvious impact on intestinal environment. The differences observed in broiler performance due to Ca source in this trial were likely due to the impact of Ca in the pathogenesis of NE. As previously discussed in this manuscript, high Ca concentrations in the intestinal lumen could have a negative impact on the mucosal lining during a NE episode as a result of toxin activity. Therefore, the reduction in BWG may be attributed to the reduction in ability to digest and absorb nutrients by birds fed diets formulated with HSC. This reduction in the ability to digest and absorb nutrients was likely due to more severe damage to the intestinal mucosa when broilers were fed diets formulated with HSC compared with broilers fed diets formulated with limestone. In this experiment, dietary Ca levels significantly affected P digestibility. A high ratio of dietary Ca to P reduces the digestibility and absorption of Ca and P due to increased precipitation of Ca-P complexes (Plumstead et al., 2008; Selle et al., 2009). This effect is more evident when formulating diets using HSC at standard industry levels (0.9% Ca). Because HSC is more soluble then limestone, there is more Ca in solution that binds to P and precipitates. Phosphorus digestibility was also affected by exogenous phytase supplementation. Improvements in P digestibility due to phytase supplementation result from phytate P hydrolysis, which releases bound P from the phytate molecule and improves P availability (Tamim et al., 2004; Selle et al., 2009). Optimal P digestibility was observed when feeding diets formulated with phytase and 0.6% Ca. This interaction between Ca and P has been described before and can be attributed to phytase activity and formation of mineral-phytate chelates. Tamim et al. (2004) reported that phytase activity is decreased with increasing levels of Ca. Calcium is the mineral added in

8 3132 PAIVA et al. highest concentrations in poultry diets. Therefore, Ca has a greater impact than other dietary minerals in the formation of mineral-phytate chelates (Sebastian et al., 1996; Tamim et al., 2004). The phytate molecule can carry up to 12 negative charges, thus having the potential to chelate 6 Ca atoms (Selle et al., 2009). When Ca forms mineral-phytate chelates, they precipitate, becoming unavailable for digestion and absorption. In addition, when these complexes precipitate they also become unavailable for phytase attack and further decrease P availability. Calcium digestibility was also affected by phytase supplementation. As discussed, Ca tends to form mineral-phytate chelates that precipitate. Therefore, phytase supplementation improves P and Ca digestibility by reducing the formation of mineral-phytate chelates. In addition, improvements in Ca digestibility were observed when broilers were fed 0.6% Ca in the diet. Similar to P digestibility, excess Ca decreases digestibility due to the precipitation of Ca and P. Similar to results of Walk et al. (2012), no differences in Ca digestibility were observed when comparing diets formulated with limestone or HSC, which was surprising due to the greater solubility of HSC compared with limestone. In this study, the lack of differences in Ca digestibility could be consequent to greater destruction of the intestinal mucosa (through mechanisms previously described in this manuscript) when feeding broilers diets formulated with HSC as a Ca source. Another hypothesis for the lack of differences in Ca digestibility when feeding limestone alternatively to HSC was proposed by Walk et al. (2012). These authors suggested that to observe improvements in Ca digestibility due to Ca source, further reductions in dietary Ca might be necessary. In this trial, feeding lower levels of Ca (0.6% compared with 0.9%) in the diet resulted in improvements in bird performance and mineral digestibility, as shown in previous publications (Anderson et al., 1984; Sebastian et al., 1996; Selle et al., 2009). A healthy intestine is able to digest and absorb nutrients released by exogenous phytases supplemented in the diets, resulting in improvement in bird performance. However, when the gastrointestinal tract is colonized by enteric pathogens, damage to the intestinal lining impairs function of the intestinal mucosa and consequently nutrient utilization, and unused nutrients may become available for pathogenic bacteria utilization. Evidence from this research involving a natural occurrence of NE, suggests that the presence of greater amounts of soluble Ca in the intestine with 0.9% dietary Ca compared with 0.6% Ca significantly aggravated the enteritis caused by C. perfringens. Furthermore, a more soluble Ca source favored the incidence and severity of NE. Although not specifically examined in broilers to date, research has shown that Ca influx into murine myoblast cells was dependent on pore formation by α toxin of C. septicum and extracellular Ca availability (Kennedy et al., 2009). The result of increased Ca influx was a cascade of events culminating in necrotic cell death. Collectively, results for this study support the involvement of Ca in the development and pathogenesis of a naturally occurring NE episode and indicate the need for further research to evaluate Ca requirements in growing broilers, particularly during enteric disease. REFERENCES Anderson, J. O., D. C. Dobson, and O. K. Jack Effect of particle size of the calcium source on performance of broiler chicks fed diets with different calcium and phosphorus levels. Poult. Sci. 63: AOAC International Official Methods of Analysis. 17th ed. Assoc. Off. Anal. Chem., Arlington, DC. Cowieson, A. 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