Interaction between Phytase and Calcium Source, Concentration and Particle Size on Broiler Performance and Skeletal Integrity

Calcium (Ca) and phosphorus (P) are essential minerals for many biological processes and skeletal health, however, they have a complex interactive relationship. Phytate is the naturally occurring storage form of P in plants, with the main storage site of phytate-P being seeds (Tamim et al., 2004). As poultry diets are comprised mainly of seed based ingredients, there is a considerable amount of phytate-P present in the diets (Angel et al., 2002; Tamim and Angel 2003). However, phytate-P is relatively poorly available and consequently inorganic sources of P are used to meet bird requirements (Angel et al., 2002; Tamim and Angel 2004; Selle et al., 2009). This results in higher dietary total P than what is required by the bird and excess P is excreted (Tamim and Angel et al., 2003). Diets with lower total Ca levels are desirable to enhance P digestibility, feed conversion efficiency and weight gain, however, lower total Ca level may have a negative effect on broiler welfare, in particular adverse effects on skeletal and leg health (Shim et al., 2012).

Calcium particle size influences Ca metabolism and is correlated to gizzard retention time with longer retention times reportedly improving shell quality in laying hens. The porosity of the Ca source and in vivo solubility of the Ca source are also important for Ca metabolism, and skeletal health (Zhang and Coon 1997).

The ability to replace high concentrations of limestone with a HSC source at lower concentrations may be beneficial for poultry, facilitating a reduction in total dietary Ca levels, whilst retaining intestinal absorption (Walk et al., 2012). This would result in less Ca-phytate interactions and reduce excess P excretion. Both reducing the need for inorganic P to be added to the diet, in turn reducing the cost of the diet. In previous work conducted by Bradbury et al (2015) it was observed that HSC may be too soluble at high concentrations, especially when phytase is present. Therefore the purpose of this study was to repeat the original study, inclusive of particle size to establish whether a larger particle size would help alleviate the problematic effects of such a highly soluble Ca source. 

All experimental procedures were approved by The University of Sydney Animal Ethics Committee. A total of 2400 Ross 308 day old male broiler chicks were obtained from a commercial hatchery and randomly allocated across 95 deep litter floor pens (1.5 m x 1.5 m) with 25 birds per pen and six replicate pens per treatment. Birds were maintained at 31°C for the first five days and reduced 0.5°C per day until 21°C was reached. The lighting regime was 23h:1h (light:dark) for the first five days then 18h:6h (light:dark) for the remainder of the study. Dietary treatments were arranged as a 2 x 2 x 2 x 2 factorial, comprising two sources of Ca (limestone or HSC), two dietary concentrations of Ca (6.0 or 9.0 g/kg, 5.5 and 8.0 g/kg during the starter and grower periods, respectively), two Ca particle sizes (< 0.5 mm or > 0.5 mm) and two phytase inclusions (0 or 1000 FTU/kg). Birds were fed a starter diet from d1- d14 and were fed a grower diet from d15-d28. Birds had ad libitum access to feed and water. On day 27 the skeletal health of six birds from two replicate pens of each dietary treatment was evaluated using the latency to lie (LTL) procedure as outlined in Bradbury et al., 2014. Pen body weight and feed intake measurements were recorded on d1, d14 and d28 of the study. On day 28 four birds per pen were euthanized via injection of a lethal dose (1ml/2kg) of sodium pentobarbitone into the jugular vein. Terminal body weight was recorded for each bird and the left tibia and foot were removed and stored at -20°C for foot ash analysis.

All bird performance data were analysed using the full factorial procedure in JMP 8.0 (SAS Institute Inc. NC, USA). All results are expressed as treatment means with statistical significance defined as P < 0.05. If significance was determined comparison between all pairs was performed using a Tukey-Kramer HSD test. Behavioural LTL times were analysed using Cox’s proportional hazard survival analysis in GenStat (14th Edition). 

Results for the starter and grower period are shown in Table 1. During the starter period a three-way interaction was observed for Ca source*Ca concentration*particle size (P = 0.046) on feed intake. Birds that were fed limestone to supply 6.0 g/kg dietary Ca with a mean particle size of < 0.5 consumed significantly more than birds that received HSC to supply 9.0 g/kg dietary Ca with > 0.5 mm particle size. Furthermore birds that were fed limestone to supply 9.0 g/kg dietary Ca with a mean particle size > 0.5 mm consumed significantly less than birds fed limestone to supply 9.0 g/kg dietary Ca with a mean particle size < 0.5 mm. Body weight gain was influenced by a Ca concentration*phytase interaction (P < 0.0001) where phytase significantly improved body weight gain at both dietary Ca concentrations compared to birds fed diets without phytase.

Birds that were fed HSC with phytase > 0.5 mm particle size had a significantly lower FCR than birds fed HSC without phytase > 0.5 mm particle size (P < 0.05) resulting in a three-way interaction for Ca source*particle size*phytase. Additionally birds that received limestone with phytase > 0.5 mm particle size had a significantly lower FCR than birds fed limestone without phytase > 0.5 mm particle size (P < 0.05).

Grower period feed intake was influenced by multiple three-way interactions. Birds fed HSC 8.0 g/kg dietary Ca with phytase consumed more fed than birds fed HSC 8.0 g/kg dietary Ca without phytase (P < 0.05) (between Ca source*Ca concentration*phytase (P = 0.001)). Birds fed HSC with phytase > 0.5 mm particle size had the highest feed intake and consumed significantly more feed than HSC without phytase > 0.5 mm particle size (P < 0.05), HSC with phytase < 0.5 mm particle size (P < 0.05) Ca source*particle size*phytase (P = 0.001). Ca concentration*particle size*phytase (P = 0.034) influenced feed intake where birds that were fed 8.0 g/kg dietary Ca with phytase and > 0.5 mm particle size consumed significantly more feed than bird fed 8.0 g/kg dietary Ca without phytase > 0.5 mm particle size and 8.0 g/kg dietary Ca with phytase < 0.5 mm particle size.

Body weight gain at the conclusion of the grower period was not influenced by dietary treatment. Phytase inclusion significantly increased live weight gain of broilers during the grower period (P < 0.001). Feed conversion ratio was significantly influenced by the Ca source*phytase interaction with birds that were fed limestone with phytase had a significantly lower FCR compared to birds fed limestone without phytase and HSC with and without phytase (P < 0.05). 

Table 1 - Effect of dietary treatment on feed intake, body weight gain and FCR during the starter and grower period.

Birds fed higher Ca concentration diets had a higher foot ash percentage (P<0.001). Phytase supplementation significantly increased foot ash (P = 0.0003) compared to birds that did not receive phytase.

Latency to lie results were significantly correlated with body weight (P = 0.007). For every 100g increase in body weight the chance of the bird sitting increased by 19%. In a dietary treatment comparison, birds fed HSC 5.5g/kg dietary Ca without phytase > 0.5mm particle size was the only diet to have a significantly better standing time during the LTL test (P = 0.02), with the birds being 21% less likely to sit at any given point during the LTL test.

There have been few studies that have reported the use of HSC on broiler performance. Walk et al., (2012) reported that broilers that were fed 0.9% dietary Ca from limestone consumed more feed than birds that were fed 0.9% dietary Ca from HSC. These results are consistent with the starter period of this study, whereby birds that were fed limestone 6.0 g/kg dietary Ca with < 0.5 mm particle size consumed significantly more than birds fed HSC 6.0 g/kg with < 0.5 mm particle size. The results of the study also show that birds that were fed limestone 6.0 g/kg dietary Ca < 0.5 mm particle size consumed significantly more feed than birds fed HSC 6.0 g/kg dietary Ca > 0.5 mm particle size. Zhang and Coon (1997) reported that larger Ca particles have a greater retention time in the gizzard when compared to smaller Ca particles and could explain why birds fed the larger particle size consumed less fed.

In this study, body weight was determined to be the most correlated with standing times and this is in agreement with Kestin et al., (2001) and Sherlock et al., (2010). The results of the LTL show that HSC source at the lower inclusion level (5.5 g/kg) was the only diet whereby the birds were statistically less likely to sit, implying better skeletal and leg health. However, it is interesting to note that birds that performed best in the LTL test (HSC 5.5 g/kg dietary Ca without phytase > 0.5 mm particle size) does not correlate to the greatest foot ash percentage. Foot ash responded positively with higher Ca concentrations and phytase inclusion in the diet that is consistent with Walk et al., (2012) who reported main effects of Ca concentration and phytase inclusion on tibia ash.

From the available literature, studies investigating Ca particle size report conflicting results. The beneficial effects of larger Ca particle size of egg shell and bone status in layers are documented, however, more research in broilers investigating effects on mineral digestibility and Ca particle retention in the gizzard are required. The results in this paper suggest that a HSC source when used at lower concentrations and coupled with an exogenous phytase results in bird performance comparable with higher concentrations of Ca from limestone. Further research is required to determine the economic implications of using a HSC source at lower concentrations. 

ACKNOWLEDGEMENTS: This study was funded by AB Vista Feed Ingredients. The senior author is in receipt of a scholarship from the Poultry CRC. 

Angel R, Tamim NM, Applegate TJ, Dhandu AS & Ellestad LE (2002) Journal of Applied Poultry Research 11: 471-480.

Bradbury EJ, Wilkinson SJ, Cronin GM, Thomson PC, Bedford MR & Cowieson AJ (2014) Animal 8: 1071-1079.

Bradbury EJ, Wilkinson SJ, Cronin GM, Thomson PC, Walk CL & Cowieson AJ (2015) (Unpublished Data).

Cowieson AJ, Acamovic T & Bedford M (2004) British Poultry Science Abstracts 45: 101- 108.

Selle PH, Cowieson AJ & Ravindran V (2009) Livestock Science 124: 126-141.

Sherlock L, Demmers TGM, Goodship AE, McCarthy D & Wathes CM (2010) British Poultry Science 51: 22-30. S

him MY, Karnuah AB, Mitchell AD, Anthony NB, Pesti GM & Aggrey SE (2012) Poultry Science 91: 1790-1795.

Tamim NM & Angel R (2003) Journal of Agricultural and Food Chemistry 51: 4687-4693.

Tamim NM, Angel R & Christman M (2004) Poultry Science 83: 1358-1367.

Walk CL, Addo-Chidie EK, Bedford M & Adeola O (2012) Poultry Science 91: 2255-2263.

Zhang B & Coon CN (1997) Poultry Science 76: 1702-1706.