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. 2020 Jan 29;99(3):1502–1514. doi: 10.1016/j.psj.2019.11.009

Limestone particle size, calcium and phosphorus levels, and phytase effects on live performance and nutrients digestibility of broilers

S Majeed 1, R Qudsieh 1,2, FW Edens 1, J Brake 1,1
PMCID: PMC7587709  PMID: 32111318

Abstract

Limestone particle size (PS) affects its solubility and thus can influence broiler performance by altering the rate of calcium (Ca) release into the gastrointestinal tract. The objective of this research was to determine, using 2 × 2 × 2 factorial arrangement, the influence of PS (fine and coarse) and supplemented phytase (0 and 1,000 FYT/kg) in diets formulated with 2 Ca and Pi levels (positive control [PC]; negative control [NC]) on live performance, bone ash, and apparent ileal nutrients digestibility (AID). Starter PC: 0.9 Ca and 0.45 Pi; NC: 0.72 Ca and 0.03 Pi. Grower PC: 0.76 Ca and 0.38 Pi; NC: 0.58 Ca and 0.23 Pi. The 8 diets were assigned randomly to a total of 1,512 birds, with 21 birds per pen and 9 pens per treatment. The main effects of PS and Ca and Pi levels had no influence on feed intake (FI), body weight gain (BWG), or feed conversion ratio. Adding phytase improved BWG by 8 g and 50 g and FI by 25 g and 56 g at 0–14 D (P ≤ 0.05) and 0–35 D (P ≤ 0.05), respectively. Interaction between Ca and Pi levels and phytase improved BWG and FI for 0–14 D (P ≤ 0.05) and BWG during 15–28 D (P ≤ 0.05) for PC without phytase and for PC and NC with phytase when compared with NC without phytase. Birds fed PC without phytase, or either PC or NC with phytase were about 96 g heavier than NC without phytase. Birds fed either PC or NC diet with coarse limestone or PC with fine limestone gained approximately 14 g more (P ≤ 0.05) than birds fed NC with fine limestone for BWG at 0–14 D (P ≤ 0.05). Phytase increased tibia bone ash (14 D) by 1% (P ≤ 0.05). AID of Ca and Pi at 14 D was improved (P ≤ 0.05) by 66% when phytase was added to coarse limestone. Results indicate that phytase improved broiler performance without being affected by PS. Furthermore, phytase had greater influence on coarse limestone than on fine limestone for bone ash and AID. Ca and Pi levels were the most influential factors in determining bone ash although phytase inclusion could lead to an improvement during early days.

Key words: limestone, particle size, broiler, phytase, performance

Introduction

Calcium (Ca) is the most abundant mineral in the body (Applegate and Angel, 2008) and performs critical functions in bone mineralization, blood clotting, intracellular signaling, and muscle contractions (Suttle, 2010). Being important as it is, excess Ca in the diet can lead to undesirable effects. Excess dietary Ca can interfere with the availability of other minerals such as zinc, magnesium, and manganese (NRC, 1994), and Ca can chelate lipids making them unavailable, decreasing the energy profile of feed (Edwards et al., 1960). Ca has the capacity to bind with inorganic phosphorus (Pi) and form Ca-phytate complex in the gut which interferes with Pi availability (Hurwitz and Bar, 1971). In poultry diets, more often than not, Ca is oversupplemented because of its being used as a vehicle in various premixes (Kim et al., 2018), which is not taken into account when formulating diets. Limestone is the primary source of Ca in poultry diets; it is readily available and inexpensive (Blount, 2013). One can argue that with accelerated growth of broilers over the years, the Ca requirement must have increased because of a stronger skeleton being required to support greater body mass. Contrary to that supposition, the factual situation is that over the years, Ca requirements have dropped (Driver et al., 2005, Ziaei et al., 2008 Singh et al., 2013) compared with NRC requirements of 1994. The Cobb broiler performance and nutrient supplement guide (Cobb, 2018) and Ross broiler nutrition specification guide (Ross, 2014) both recommend less Ca than the NRC requirements of 1994. This points to modern fast-growing, high-yielding broilers being able to more efficiently use Ca than smaller, slower growing broilers of the past.

Phytase initially became commercially available because of legislation designed to limit Pi pollution of the environment in the Netherlands (Selle and Ravindran, 2007) as it had been demonstrated that phytase inclusion reduced Pi excretion (Simons et al., 1990, Paik, 2003). Additional trials have shown phytase to increase amino acid availability (Selle et al., 2000), feed energy profile (Ravindran et al., 1999a, Ravindran et al., 2000, Ravindran et al., 2001, Selle et al., 1999, Selle et al., 2001, Selle et al., 2003), and feed intake (FI) (Onyango et al., 2005, Bahadoran et al., 2011), which led to its widespread use in broiler feed. It has been well established by now that phytase improves broiler performance, especially in cases of low Pi content in the diets (Nelson et al., 1971, Broz et al., 1994, Cowieson and Adeola, 2005), but more work needs to be done on phytase interaction with various ingredients in the feed. Excess Ca in the diet has been known to bind phytate, resulting in a Ca-phytate complex, which is refractory to hydrolysis by phytase (Wise, 1983), thus negatively affecting phytase activity.

Various studies in layers have attributed Ca retention as a function of limestone particle size (PS) solubility (Roland, 1986, Cheng and Coon, 1990, Zhang and Coon, 1997a). Eusebio-Balcazar et al. (2018) demonstrated that coarse PS improved bone integrity of layer hens, which indicated a positive effect of coarse limestone inclusion instead of fine limestone. Compared to layers, fewer studies have been conducted in broilers in terms of PS, although in broilers, PS does affect Ca solubility and availability (Anwar et al., 2016, Kim et al., 2018). It is thought that compared with fine, coarse limestone will be retained longer in the ventriculus (gizzard). Longer retention will increase total dissolution and increase Ca availability associated with slower Ca release, which might be beneficial as there will be less interference with gut pH and Pi digestibility.

In the poultry industry, particularly in broilers, PS has not been given much consideration. The purpose of this experiment was to observe whether PS, its interaction with different Ca and Pi levels, and phytase inclusion will have an impact on live performance, digestibility, and bone ash of broilers.

Material and methods

All animal work conformed to the Guide for Care and Use of Agricultural Animals in Research and Teaching (FASS, 2010) and North Carolina State University approved Institutional Animal Care and Use Committee protocol (IACUC #16-157). A total of 1,512 male birds of Cobb 500 were placed randomly into 72 pens with 21 birds per pen. Initial litter brooding temperature was set at 35°C and was reduced gradually to 27°C by 15 D of age and was held at 27°C until 21 D of age. From 22 D of age until the end of experiment, temperature was maintained at approximately 24°C. When chicks were placed into the floor pens, there was one supplemental drinker and 3 supplemental feed trays available in each pen in addition to 2 tube feeders and one bell drinker. Supplemental drinkers and 2 supplemental feeders were removed after 7 D while the remaining feeder tray was removed after 14 D. Fluorescent-type bulbs provided light during the experiment and were lit for 23 h during the first week, reduced to 20 h at 14 D and to 18 h from 15–21 D. After 22 D, only natural light was provided. Temperature and mortality were checked twice daily.

Experimental Diets

A total of 8 experimental diets were tested in a 2 × 2 × 2 factorial treatment design (Table 1). Factors tested were limestone PS (fine and coarse), phytase (0 and 1,000 FYT/kg), and Ca and Pi levels (positive control [PC] and negative control [NC]). There was a total of 72 pens with 9 replicate pens and 21 birds per pen for each factorial dietary treatment. Birds were fed crumbled starter (Table 2) from 0–16 D and pelleted grower (Table 3) from 17–35 D ad libitum. All diets had titanium dioxide included at the rate of 0.5 g/kg of diet as an inert marker for calculation of apparent ileal digestibility (AID). Limestone (Huber engineered material, Quincy, IL) was obtained in 2 PS: fine limestone (Hubercarb Q200) PS was 190 μm, and coarse limestone (Hubercarb 12–40) PS was 900 μm. The PC had Ca of 0.9 and Pi of 0.45 in starter and Ca of 0.76 and Pi of 0.38 in grower. The NC had Ca of 0.72 and Pi of 0.3 in starter and Ca of 0.58 and Pi of 0.23 in grower. The supplemented phytase enzyme was Ronozyme Hiphos 2,500 GT (Koninklijke DSM N.V., Heerlen, Netherland) and was included at the rate of 1,000 FYT/kg. Feed samples were collected during feed manufacturing. Phytase activity measured in the mash feed was on average around 600 FYT/kg and in pelleted feed around 300 FYT/kg. Phytase was added either on top of the PC or with an assigned matrix value of 0.18% for Ca and 0.15% for Pi when added to NC. Pelleting temperature for both starter and grower was 180°C. PS was measured for limestone and for each treatment at both phases using Ro-TaP sieve shaker following the method ANSI/ASAE S319.3 (ASAE, 2008). All diets had a geometric average PS (Dgw) of 600 μm for both starter and grower. PS range for fine limestone was as follows with percentage in brackets: 297 μm (23%), 212 μm (26%), 150 μm (18%), 103 (15%), 73 μm (9%), and 53 μm (4%). PS range for the coarse limestone was as follows: 1,191 μm (36%), 841 μm (25%), 594 μm (17%), 420 μm (12%), and 297 μm (3%).

Table 1.

Experimental diets design.

Limestone particle size Calculated Ca:AvP1
 Phytase2
Starter Grower
Fine 0.90:0.45 0.76:0.38 1,000
0.90:0.45 0.76:0.38 0
0.72:0.30 0.58:0.23 1,000
0.72:0.30 0.58:0.23 0
Coarse 0.90:0.45 0.76:0.38 1,000
0.90:0.45 0.76:0.38 0
0.72:0.30 0.58:0.23 1,000
0.72:0.30 0.58:0.23 0
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1

The Ca and available phosphorus (AvP) levels used in this design are referred to as positive control (PC, 0.9:0.45 and 0.76:0.38) and negative control (NC, 0.72:0.3 and 0.58:0.23).

2

Phytase (RONOZYME HiPhos) was expected to provide 1,000 FYT/kg feed and was assigned a matrix value of 0.18% for Ca and 0.15% for AvP.

Table 2.

Experimental diet composition and nutrient content (starter).

 Treatment
Limestone particle size
 Fine
 Coarse
Ca and AvP level1
 PC
 NC
 PC
 NC
Phytase 1,000 0 1,000 0 1,000 0 1,000 0
Ingredient (%)
 Corn 55.66 55.66 55.66 55.66 55.66 55.66 55.66 55.66
 Soybean meal 48% 36.59 36.59 36.59 36.59 36.59 36.59 36.59 36.59
 Poultry by-product meal 1.29 1.29 1.29 1.29 1.29 1.29 1.29 1.29
 Poultry fat 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50
 Deflouridated phosphorus 1.55 1.55 0.68 0.68 1.55 1.55 0.68 0.68
 Limestone fine 0.49 0.49 0.70 0.70 0.00 0.00 0.00 0.00
 Limestone coarse 0.00 0.00 0.00 0.00 0.49 0.49 0.70 0.70
 Sodium chloride 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50
 DL-Methionine 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23
 Choline chloride, 60% 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20
 Mineral premix 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20
 Vitamin premix2 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
 L-lysine 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09
 Selenium premix3 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
 Coban4 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
 L-Threonine 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
 Titanium 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50
 Ronozyme HiPhos®5 0.04 0.00 0.04 0.00 0.04 0.00 0.04 0.00
 Sand 0.00 0.04 0.66 0.70 0.00 0.04 0.66 0.70
Calculated nutrient content (%)
 Metabolizable energy (kcal/kg) 2,958 2,958 2,958 2,958 2,958 2,958 2,958 2,958
 Crude protein 22.50 22.50 22.50 22.50 22.50 22.50 22.50 22.50
 Calcium 0.90 0.90 0.72 0.72 0.90 0.90 0.72 0.72
 Total phosphorus 0.67 0.67 0.52 0.52 0.67 0.67 0.52 0.52
 Available phosphorus 0.45 0.45 0.30 0.30 0.45 0.45 0.30 0.30
 Total lysine 1.30 1.30 1.30 1.30 1.30 1.30 1.30 1.30
 Total methionine 0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.58
Analyzed nutrient content (%), as fed
 Crude fat 4.01 3.99 3.87 3.95 3.91 4.29 4.10 3.86
 Crude protein 21.24 21.34 21.68 20.91 22.31 21.13 21.89 21.18
 Crude fiber 3.30 3.40 3.60 3.40 3.50 3.30 3.40 3.40
 Ash 5.13 4.84 4.62 4.89 5.44 5.59 5.27 5.12
 Calcium 0.77 0.80 0.65 0.59 0.86 0.95 0.79 0.67
 Total phosphorus 0.63 0.62 0.52 0.49 0.66 0.69 0.58 0.52
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1

The Ca and available phosphorus (AvP) levels used in this design are referred to as positive control (PC, 0.9:0.45) and negative control (NC, 0.72:0.3).

2

Vitamin premix supplied the following per kg of diet: 6,614 IU vitamin A, 1,984 IU vitamin D3, 33 IU vitamin E, 0.02 mg vitamin B12, 0.13 mg biotin, 1.98 mg menadione (K3), 1.98 mg thiamine, 6.6 mg riboflavin, 11 mg d-pantothenic acid, 3.97 mg vitamin B6, 55 mg niacin, and 1.1 mg folic acid.

3

Selenium premix provided 0.2 mg Se (as Na2SeO3).

4

Coban supplied monensin sodium at 90 mg/kg of feed.

5

Phytase (RONOZYME HiPhos) was expected to provide 1,000 FYT/kg feed and was assigned a matrix value of 0.18% for Ca and 0.15% for AvP.

Table 3.

Experimental diet composition and nutrient content (grower).

 Treatment
Limestone particle size
 Fine
 Coarse
Ca and AvP level1
 PC
 NC
 PC
 NC
Phytase 1,000 0 1,000 0 1,000 0 1,000 0
Ingredient (%)
 Corn 65.45 65.45 65.45 65.45 65.45 65.45 65.45 65.45
 Soybean meal 27.69 27.69 27.69 27.69 27.69 27.69 27.69 27.69
 Poultry by-product meal 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
 Poultry fat 1.58 1.58 1.58 1.58 1.58 1.58 1.58 1.58
 Deflouridated phosphorus 1.13 1.13 0.26 0.26 1.13 1.13 0.26 0.26
 Limestone fine 0.41 0.41 0.60 0.60 0.00 0.00 0.00 0.00
 Limestone coarse 0.00 0.00 0.00 0.00 0.41 0.41 0.60 0.60
 Sodium chloride 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30
 DL-Methionine 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19
 Choline chloride, 60% 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20
 Mineral premix 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20
 Vitamin premix2 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
 L-lysine 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15
 Selenium premix3 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
 Coban4 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
 L-Threonine 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
 Titanium 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50
 Ronozyme HiPhos®5 0.04 0.00 0.04 0.00 0.04 0.00 0.04 0.00
 Sand 0.00 0.04 0.68 0.72 0.00 0.04 0.68 0.72
Calculated nutrient content (%)
 Metabolizable energy (kcal/kg) 3,025 3,025 3,025 3,025 3,025 3,025 3,025 3,025
 Crude protein 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00
 Calcium 0.76 0.76 0.58 0.58 0.76 0.76 0.58 0.58
 Total phosphorus 0.58 0.58 0.43 0.43 0.58 0.58 0.43 0.43
 Available phosphorus 0.38 0.38 0.23 0.23 0.38 0.38 0.23 0.23
 Total lysine 1.15 1.15 1.15 1.15 1.15 1.15 1.15 1.15
 Total methionine 0.51 0.51 0.51 0.51 0.51 0.51 0.51 0.51
Analyzed nutrient content (%), as fed
 Crude fat 4.01 4.02 4.22 4.10 4.21 4.21 3.92 3.84
 Crude protein 21.04 20.84 21.00 20.39 20.22 19.88 22.25 20.85
 Crude fiber 3.10 3.00 3.60 3.90 3.30 3.40 3.80 8.00
 Ash 4.62 4.85 5.14 5.01 4.92 4.88 5.15 5.05
 Calcium 0.77 0.83 0.59 0.52 0.82 0.83 0.64 0.61
 Total phosphorus 0.67 0.68 0.54 0.48 0.65 0.67 0.53 0.51
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1

The Ca and available phosphorus (AvP) levels used in this design are referred to as positive control (PC, 0.76:0.38) and negative control (NC, 0.58:0.23).

2

Vitamin premix supplied the following per kg of diet: 6,614 IU vitamin A, 1,984 IU vitamin D3, 33 IU vitamin E, 0.02 mg vitamin B12, 0.13 mg biotin, 1.98 mg menadione (K3), 1.98 mg thiamine, 6.6 mg riboflavin, 11 mg d-pantothenic acid, 3.97 mg vitamin B6, 55 mg niacin, and 1.1 mg folic acid.

3

Selenium premix provided 0.2 mg Se (as Na2SeO3).

4

Coban supplied monensin sodium at 90 mg/kg of feed.

5

Phytase (RONOZYME HiPhos) was expected to provide 1,000 FYT/kg feed and was assigned a matrix value of 0.18% for Ca and 0.15% for AvP.

The Ca solubility was calculated for both coarse and fine limestone by solubilizing weighed samples of coarse and fine limestone in 0.1 N HCl at 42°C for 10 min with agitation and recovering quantitatively the undissolved residue. The difference between the weighed sample and insoluble residue was calculated to be the solubility and was expressed as a percentage solubility (Zhang and Coon, 1997b). Solubility was 72.8 and 53.0% for fine and coarse limestone, respectively. Calcium concentration in both fine and coarse limestone was also determined; the concentration was 39.5% for fine and 38.9% for coarse.

Data Collection

Chicks were weighed in groups and individually on 1, 14, 28, and 35 D of age with feed weigh-back on the same days to determine live performance based on body weight (BW), body weight gain (BWG), FI, and feed conversion ratio (FCR). FCR and FI were calculated from feed weigh-back and group BW while individual weights were used to calculate coefficients of variation (CV).

Ileal content and left tibia were collected from 3 birds/pen at 15 D and 2 birds/pen at 36 D. The ileum, distal to Meckel's diverticulum and about 1 inch proximal to the ileocecal junction, was separated from bird's intestinal tract, and its content was expressed into marked plastic tubes, placed on ice, and frozen to be used for AID. The left tibia was sampled by separating it from the femur at the knee joint and ankle joint removing it from the foot. The tibia was cleaned by removing all the muscles and tendons. Afterward, it was placed in a marked plastic bag and frozen to be used for bone ash analysis.

Tibia Bone Ash Analysis

Defatted tibia bones were dried at 105°C in an oven for 24 h, then were placed in furnace at 600°C for 24 h, and the residues weighed and expressed in % (Hall et al., 2003).

Digestibility Analysis

Ileal digesta and feed samples were freeze-dried at −50°C and ground. Digestibility analysis of both digesta and feed samples for amino acids was performed following the procedure of AOAC Official method 982.30 (AOAC, 2006a), and that for Ca and Pi was conducted using AOAC official method 968.08 (AOAC, 2006b). Titanium content in ileal digesta and feed samples was determined according to Myers et al. (2004).

Ileal digestibility was calculated according to the procedure described by Dilger and Adeola (2006).

Data Analysis

Data were analyzed using the General Linear Model procedure of SAS (Statistical Analysis System, 2017). Means were separated using Tukey's test (Statistical Analysis System, 2017). Significance among main effects, their interactions, and means was based on P ≤ 0.05 or less. The pen was considered to be the experimental unit.

Results and discussion

The PS of starter and grower feed were similar, being around 600 μm. This is important as different PS of feed can influence the bird's performance as demonstrated by Amerah et al. (2008). PS uniformity was also high among treatments ensuring birds in the trial got similar feed PS. There was no 3-way interaction between the 3 main effects investigated, so only main effects and 2-way interactions between any 2 out of the 3 main effects are discussed. Individual BW data were analyzed for CV calculation at day 0, 14, 28, and 35. All CVs for BW were less than 10%, indicating that birds were uniform.

Live Performance

Live performance results are shown in Table 4 (FI and BWG) and Table 5 (FCR and mortality). The main effects of PS and Ca and Pi levels did not affect FI, BWG, or FCR. Mortality was largely unaffected by the factors with no significant interactions, except for 35 D and 15–28 D in which NC had higher mortality than PC. Overall mortality in the entire trial was less than 2%. In an earlier study (McNaughton et al., 1974), fine PS improved BW of broilers compared with coarse limestone although the coarse PS was 2,360–3,350 μm which is greater than what was used for this study (900 μm). Similarly, Managi and Coon (2007) observed that the best weight gain and FI were with fine PS (137–299 μm) compared with coarse (>500 μm) or very fine (28 μm) PS. Bradbury et al. (2018) came to a conclusion similar to the outcomes of this study, which showed that PS had no significant influence on either BWG or FCR. Findings reported in literature with regard to limestone PS effects are variable depending on the PS investigated.

Table 4.

Feed Intake (FI) of male broilers as affected by limestone particle size (PS), phytase enzyme, Ca and Pi levels, and their interactions.

PS1 Phytase2 Ca and P3 Age period (D)
FI (g/bird)
 BWG (g/bird)
0–14 15–28 29–35 0–28 0–35 0–14 15–28 29–35 0–28 0–35
Main effects
Fine 544 1,962 1,424 2,506 3,930 398 1,311 930 1,707 2,637
Coarse 544 1,942 1,423 2,487 3,909 398 1,305 928 1,707 2,635
P value 0.90 0.30 0.86 0.38 0.42 0.99 0.58 0.89 0.10 0.91
SE 4.2 13.3 6.9 15.2 18.0 3.0 7.7 12.0 9.9 14.6
0 532B 1,937 1,423 2,469b 3,892b 392B 1,297b 925 1,686B 2,611b
1,000 557A 1,966 1,424 2,523a 3,948a 404A 1,319a 932 1,728A 2,661a
P value 0.00 0.13 0.86 0.01 0.03 0.01 0.05 0.67 0.00 0.02
SE 4.2 13.3 6.9 15.2 18.0 3.0 7.7 12.0 9.9 14.6
PC 547 1,964 1,420 2,510 3,930 401 1,310 938 1,715 2,652
NC 543 1,939 1,427 2,482 3,910 395 1,307 920 1,700 2,619
P value 0.51 0.21 0.45 0.20 0.43 0.19 0.75 0.30 0.29 0.12
SE 4.2 13.3 6.9 15.2 18.0 3.0 7.7 12.0 9.9 14.6
Interactions
Fine 0 533 1,952 1,423 2,484 3,908 394 1,305 928 1,694 2,622
Coarse 0 531 1,922 1,422 2,453 3,875 390 1,290 923 1,678 2,600
Fine 1,000 556 1,971 1,425 2,527 3,952 402 1,318 932 1,720 2,652
Coarse 1,000 559 1,961 1,423 2,520 3,943 406 1,321 933 1,736 2,669
P value 0.72 0.60 0.99 0.58 0.64 0.33 0.40 0.90 0.24 0.36
SE 6.0 18.8 9.8 21.5 25.4 4.3 10.9 17.0 14.0 20.6
0 NC 522C 1,912 1,429 2,434 3,863 382B 1,281B 912 1,661b 2,572b
0 PC 542B 1,962 1,416 2,504 3,920 402A 1,314A 939 1,771a 2,650a
1,000 NC 563A 1,967 1,425 2,531 3,956 408A 1,333A 928 1,739a 2,667a
1,000 PC 551A 1,967 1,423 2,516 3,940 400A 1,306A,B 937 1,718a 2,655a
P value 0.01 0.18 0.56 0.06 0.16 0.00 0.01 0.59 0.01 0.03
SE 6.0 18.8 9.8 21.5 25.4 4.3 10.9 17.0 14.0 20.6
Fine NC 539 1,931 1,428 2,470a 3,898 387C 1,289B 931 1,675C 2,607
Fine PC 549 1,992 1,421 2,542b 3,962 409A 1,334A 929 1,739A 2,668
Coarse NC 546 1,949 1,426 2,495a 3,921 403A,B 1,324A,B 908 1,724A,B 2,632
Coarse PC 544 1,935 1,419 2,479a 3,897 393A,B 1,286B 947 1,690B,C 2,637
P value 0.29 0.05 0.99 0.05 0.09 0.00 0.00 0.23 0.00 0.18
SE 6.0 18.8 9.8 21.5 25.4 4.3 10.9 17.0 14.0 20.6
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Means within each column and treatment effect that don't possess any lowercase superscript letter in common differ significantly (P ≤ 0.05).

Means within each column and treatment effect that don't possess any uppercase superscript letter in common differ significantly (P ≤ 0.01).

Abbreviations: BWG, body weight gain; SE, standard error of mean.

1

Limestone particle size: fine (190 μm) and coarse (900 μm).

2

Phytase: had a dose of 1,000 FYT/kg and a matrix value of 0.18% for Ca and 0.15% for Pi.

3

The Ca and Pi had 2 levels, NC (negative control) had Ca 0.72 and P 0.3 in starter and Ca 0.53 and P 0.23 in grower while PC (positive control) had Ca 0.9 and P 0.45 in starter and Ca 0.76 and P 0.38 in grower.

Table 5.

Feed conversion ratio (FCR) and mortality of male broilers as affected by limestone particle size (PS), phytase enzyme, Ca and Pi levels, and their interactions.

PS1 Phytase2 Ca and P3 Age period (D)
FCR (g:g)
 Mortality (%)
0–14 15–28 29–35 0–28 0–35 0–14 15–28 29–35 0–35
Main effects
Fine 1.371 1.497 1.539 1.468 1.491 0.14 0.17b 0.67 0.94
Coarse 1.373 1.498 1.543 1.457 1.485 0.14 1.00a 0.50 1.61
P value 0.89 0.98 0.89 0.22 0.54 1.00 0.05 0.70 0.35
SE 0.01 0.01 0.02 0.01 0.01 0.14 0.29 0.31 0.51
0 1.361 1.500 1.542 1.465 1.491 0.15 0.83 0.504 1.45
1,000 1.383 1.495 1.539 1.461 1.485 0.13 0.34 0.662 1.10
P value 0.17 0.69 0.91 0.67 0.56 0.94 0.24 0.72 0.63
SE 0.01 0.01 0.02 0.01 0.01 0.14 0.29 0.31 0.51
PC 1.366 1.500 1.52 1.464 1.482 0.14 0.17B 0.33 0.64B
NC 1.378 1.494 1.56 1.461 1.494 0.14 1.00A 0.83 1.91A
P value 0.45 0.64 0.15 0.73 0.27 1.00 0.01 0.26 0.01
SE 0.01 0.01 0.02 0.01 0.01 0.14 0.29 0.31 0.51
Interactions
Fine 0 1.355 1.498 1.536 1.466 1.491 0.02 0.32 0.68 0.96
Coarse 0 1.367 1.501 1.549 1.463 1.491 0.23 1.33 0.33 1.94
Fine 1,000 1.387 1.496 1.542 1.470 1.491 0.26 0.01 0.66 0.92
Coarse 1,000 1.380 1.493 1.537 1.452 1.478 0.00 0.67 0.67 1.28
P value 0.58 0.15 0.78 0.41 0.51 0.18 0.67 0.69 0.67
SE 0.02 0.01 0.03 0.01 0.01 0.12 0.41 0.44 0.41
0 NC 1.372 1.506 1.574 1.466 1.503 0.29 1.32 0.68 2.23
0 PC 1.350 1.494 1.511 1.464 1.479 0.00 0.33 0.33 0.66
1,000 NC 1.385 1.483 1.549 1.457 1.485 0.02 0.66 0.99 1.60
1,000 PC 1.382 1.507 1.529 1.465 1.485 0.28 0.00 0.33 0.61
P value 0.58 0.15 0.46 0.53 0.25 0.13 0.70 0.72 0.70
SE 0.02 0.01 0.03 0.01 0.01 0.20 0.41 0.44 0.41
Fine NC 1.396a 1.501 1.539 1.475 1.496 0.00 0.33 0.67 0.94
Fine PC 1.346b 1.493 1.538 1.462 1.486 0.28 0.00 0.67 0.94
Coarse NC 1.360a,b 1.487 1.584 1.450 1.491 0.28 1.67 1.00 2.89
Coarse PC 1.386a,b 1.507 1.502 1.467 1.478 0.00 0.33 0.00 0.33
P value 0.02 0.27 0.16 0.07 0.87 0.16 0.23 0.26 0.08
SE 0.02 0.01 0.03 0.01 0.01 0.20 0.41 0.44 0.71
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Means within each column and treatment effect that don't possess any lowercase superscript letter in common differ significantly (P ≤ 0.05).

Means within each column and treatment effect that don't possess any uppercase superscript letter in common differ significantly (P ≤ 0.01).

Abbreviation: SE, standard error of mean.

1

Limestone particle size: fine (190 μm) and coarse (900 μm).

2

Phytase: had a dose of 1,000 FYT/kg and a matrix value of 0.18% for Ca and 0.15% for Pi.

3

The Ca and Pi had 2 levels, NC (negative control) had Ca 0.72 and P 0.3 in starter and Ca 0.53 and P 0.23 in grower while PC (positive control) had Ca 0.9 and P 0.45 in starter and Ca 0.76 and P 0.38 in grower.

The phytase main effect was significant for all age periods for BWG except at 29–35 D and for FI at 0–14 D, 0–28 D, and 0–35 D, but it was not significant for FCR. Earlier investigators have shown that phytase improved live performance or was associated with a similar performance in response to adequate Ca and Pi when it was supplemented in a diet with inadequate Ca and Pi (Singh et al., 2003, Shaw et al., 2011, Ceylan et al., 2012). The BWG and FI show that phytase was most effective for the first 14 D, and as the bird aged, the phytase effect was not significant. This was in agreement with the study by Olukosi et al. (2007) who concluded that young chicks have a problem retaining Ca and Pi when given a corn-soy diet. This can be relieved by phytase supplementation. One reason why young birds have a lower Ca and Pi bioavailability might be associated with lower production of endogenous phytase. As birds age, the endogenous phytase activity increases (Morgan et al., 2015) leading to a reduced effect of dietary phytase on live performance.

Interaction between PS and phytase had no significant impact on live performance parameters even though it has been shown that in the ventriculus, coarse limestone results in more Pi being liberated from phytate (Joardar, 2019). Kim et al. (2018) came to the conclusion that phytase, when supplemented with coarse limestone, could better negate harmful effects of Ca on Pi digestibility than fine limestone. Managi and Coon (2007) also demonstrated in vitro that coarse limestone improved phytase activity, but in a live trial, coarse limestone (>519 μm) had a negative effect on BWG. In the present study, improved phytase activity was noted with coarse limestone, but it did not influence live performance. This response could be due to the fact that a higher activity of phytase is required to facilitate an advantage associated with the use of coarse limestone. Supplementing optimum dietary Ca and Pi levels with increasing phytase activity, which releases additional Ca and Pi, did not have a significant effect on live performance.

There was an interaction effect between phytase and Ca and Pi levels on live performance. The NC without phytase had lower FI (0–14 D) and BWG (0–14, 15–28, 0–28, and 0–35 D) than PC, NC+phytase, and PC+phytase. Adding phytase to a PC diet did not improve performance compared with PC without phytase, which showed that the phytase effect is prevalent when broilers are fed a Ca- and Pi-deficient diet. This was supported by dos Santos et al. (2013). Low Pi in diets is associated with decreased BWG and FI (Denbow et al., 1995, Potter et al., 1995). The addition of phytase to NC likely increased phytate degradation causing greater release of phytate-bound Ca and Pi, which led to mineral levels similar to PC, eliciting a live performance similar to PC. The interaction between Ca and Pi levels and PS was significant for BWG (0–14 D, 15–28 D, and 0–28 D). The NC with coarse PS improved live performance compared with the NC with fine PS, which might be associated with the longer ventricular retention of the coarse limestone as well as slower release of Ca (Anwar et al., 2016). Longer retention time in the ventriculus would indicate slower Ca release with sustained Ca availability reducing the potential for Ca interference with Pi digestibility, thereby assuring the likelihood of optimal Ca and Pi for growth and development.

Bone Ash

Bone ash results (Table 6) for the PS were not significant for either 14 D or 35 D. This observation was in agreement with results from a recent investigation in which inclusion of dietary limestone of various PS (200 μm, 1,000 μm, 2,000 μm, and 3,000 μm) did not influence tibial bone ash content in 82-week postmolting broiler breeders (Bueno et al., 2016). Neither Joardar (2019) nor Managi and Coon (2007) found a PS effect on tibial ash. Similarly, Bradbury et al. (2018), who studied broiler skeletal integrity, were not able to demonstrate an effect of PS on foot bone ash content.

Table 6.

Tibia bone ash, Ca, and Pi apparent ileal digestibility (AID) of male broilers as affected by limestone particle size (PS), phytase enzyme, Ca and Pi levels, and their interactions.

PS1 Phytase2 Ca and P3 Age (D)
Bone ash (%)
 Ca AID (%)
 Pi AID (%)
14 35 14 35 14 35
Main effects
Fine 50.0 52.5 65.1A 54.8a 63.9A 56.5A
Coarse 50.3 52.6 56.1B 48.4b 53.4B 52.1B
P value 0.39 0.78 0.00 0.01 0.00 0.00
SE 0.15 0.19 1.25 1.25 1.09 0.96
0 49.6B 52.3 56.7B 52.5 55.8B 51.2B
1,000 50.7A 52.7 64.4A 50.7 61.5A 57.5A
P value 0.00 0.11 0.00 0.28 0.00 0.00
SE 0.15 0.19 1.25 1.17 1.09 0.96
PC 50.1A 52.8a 52.4B 49.9b 56.7b 48.4B
NC 49.4B 52.2b 68.7A 53.3a 60.6a 60.2A
P value 0.00 0.02 0.00 0.05 0.01 0.00
SE 0.15 0.19 1.25 1.17 1.09 0.96
Interactions
Fine 0 49.7 52.4 69.7A 55.3 63.5A 53.7
Coarse 0 49.6 52.2 43.8C 49.6 48.1B 48.6
Fine 1,000 50.5 52.5 60.5B 54.2 64.3A 59.4
Coarse 1,000 50.9 52.9 68.3A 47.2 58.7A 55.6
P value 0.29 0.27 0.00 0.70 0.00 0.64
SE 0.21 0.27 1.76 1.66 1.55 1.36
0 NC 48.4C 52.0 66.3 54.7 57.9 57.0
0 PC 50.9A 52.6 47.1 50.3 53.6 45.3
1,000 NC 50.3B 52.4 71.1 51.8 63.3 63.4
1,000 PC 51.1A 53.1 57.7 49.6 59.7 51.5
P value 0.00 0.78 0.10 0.50 0.82 0.93
SE 0.21 0.27 1.76 1.66 1.55 1.36
Fine NC 49.3 51.9 71.8 54.5a 66.6 61.3
Fine PC 50.9 53.1 58.3 55.0a 61.2 51.8
Coarse NC 49.4 52.5 65.6 52.0a 54.6 59.2
Coarse PC 51.1 52.6 46.5 44.8b 52.1 45.0
P value 0.66 0.05 0.11 0.02 0.36 0.09
SE 0.21 0.27 1.76 1.66 1.55 1.36
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Means within each column and treatment effect that don't possess any lowercase superscript letter in common differ significantly (P ≤ 0.05).

Means within each column and treatment effect that don't possess any uppercase superscript letter in common differ significantly (P ≤ 0.01).

Abbreviation: SE, standard error of mean.

1

Limestone particle size: fine (190 μm) and coarse (900 μm).

2

Phytase: had a dose of 1,000 FYT/kg and a matrix value of 0.18% for Ca and 0.15% for Pi.

3

The Ca and Pi had 2 levels, NC (negative control) had Ca 0.72 and P 0.3 in starter and Ca 0.53 and P 0.23 in grower while PC (positive control) had Ca 0.9 and P 0.45 in starter and Ca 0.76 and P 0.38 in grower.

In this experiment, phytase improved tibial bone ash at 14 D, but the phytase effect was lost at 35 D. Numerous studies have shown phytase improving bone ash content at 14 or 21 D (Qian et al., 1996, Johnston and Southern, 2000, Li et al., 2015). It is generally accepted that dietary phytase results in improved bone ash content due to greater availability of Ca and Pi through liberation of these minerals from phytate (Selle and Ravindran, 2007). Dietary phytase is most effective during the first 2–3 wk in chicks, but the phytase effect diminishes as birds age, which would account for lack of phytase influence at 35 D in this study (Olukosi et al., 2007). The high Ca and Pi levels in the PC treatment improved bone ash compared with the low Ca and Pi in the NC treatment at 14 D and 35 D in this study. These observations were expected as Ca and Pi are major constituents of bone, and increasing their levels would result in greater availability, leading to a greater ash content of tibial bones. Driver et al. (2005) have reported similar results from experiments in which diets with higher Ca and Pi levels resulted in greater tibial bone ash percentage than diets with low Ca and Pi levels in 16-day-old broilers. Venäläinen et al. (2006) observed that Ca and Pi influenced tibial bone ash percentage through 35 D of age when the starter diet had Ca of 0.9% and Pi of 0.45% compared with starter diets with Ca of 0.8% and Pi of 0.40%. Other studies also have reported greater tibial bone ash content with increasing dietary Ca and Pi levels (Nelson et al., 1990, Onyango et al., 2003).

The interaction between PS and phytase inclusion, as well as the interaction between PS and Ca and Pi levels, did not affect bone ash percentage. However, at 14 D, the interaction between phytase and Ca and Pi levels was highly significant (P = 0.0005), with the NC+no phytase treatment having the lowest bone ash percentage compared with the PC treatment, regardless of phytase inclusion. The inclusion of phytase in the NC treatment slightly improved bone ash percentage; however, the improvement did not reach the bone ash percentage of the PC treatment. By 35 D of age, loss of significance of interactions between phytase and Ca and Pi levels was found. These observations suggest that dietary Ca and Pi levels are as important concerning bone mineralization as supplemental phytase in NC diets during the early starter feed phase. Similarly, Gautier et al. (2018) found that broilers fed diets with nonphytate phosphorus (NPP) at 0.53% had a higher bone ash percentage than broilers fed diets with NPP at 0.45%. The addition of phytase to the 0.45% NPP diet improved bone ash percentage but to a lower percentage than the 0.53% NPP diet without phytase. However, the addition of phytase to the PC diet (0.53% NPP) further improved bone ash (Gautier et al., 2018); the same response was not found in the present study.

Nutrient Digestibility

The AID of Ca, Pi, and amino acids was calculated (Table 6, Table 7, Table 8). The PS had a significant effect on Ca and Pi digestibility, with fine PS (190 μm) exhibiting better digestibility of both Ca and Pi at 14 and 35 D than coarse PS (900 μm).

Table 7.

Amino acid AID of male broilers as affected by limestone particle size (PS), phytase enzyme, Ca and Pi levels, and their interaction at 14 D of age.

PS1 Phytase2 Ca and P3 (%)
Lysine Arginine Histidine Leucine Isoleucine Methionine Glycine Proline Threonine Cysteine
Main effects
Fine 80.1a 84.1a 77.4A 77.9a 77.6A 88.1a 71.2A 75.3a 70.5A 61.0A
Coarse 75.5b 80.8b 72.8B 73.8b 73.0B 84.6b 67.6B 71.7b 64.3B 52.9B
P value 0.04 0.02 0.01 0.02 0.01 0.01 0.01 0.02 0.00 0.00
SE 1.33 1.00 1.16 1.19 1.19 0.95 1.22 1.06 1.37 1.62
0 75.8b 80.8b 73.6 74.3b 73.6a 85.5 67.5b 72.6 66.0 55.2
1,000 79.8a 84.1a 76.6 77.5a 77.1b 87.1 71.3a 74.4 68.9 58.8
P value 0.04 0.02 0.08 0.05 0.04 0.265 0.03 0.22 0.15 0.12
SE 1.33 1.00 1.16 1.19 1.19 0.95 1.22 1.06 1.37 1.62
PC 77.0 82.2 74.9 75.2 75.0 84.9 69.4 73.4 66.6 56.1
NC 78.5 82.7 75.3 76.5 75.6 87.7 69.3 73.6 68.3 57.9
P value 0.42 0.70 0.83 0.46 0.70 0.08 0.96 0.91 0.38 0.43
SE 1.33 1.00 1.16 1.19 1.19 0.95 1.22 1.06 1.19 1.62
Interactions
Fine 0 80.3a 83.9A 78.0A 78.3a 77.9a 88.6 71.8A 76.5A 72.0a 62.4a
Coarse 0 71.3b 77.8B 69.3B 70.2b 69.2b 82.5 63.2B 68.6B 60.0b 48.0b
Fine 1,000 79.9a 84.2A 76.8A 77.5a 77.4a 87.5 70.6A 74.1A 69.0a 57.8a
Coarse 1,000 79.6a 83.9A 76.4A 77.4a 76.7a 86.7 72.0A 74.8A 68.8a 59.7a
P value 0.02 0.00 0.01 0.01 0.02 0.06 0.01 0.01 0.01 0.02
SE 1.88 1.42 1.64 1.68 1.69 1.35 1.73 1.50 1.68 1.69
0 NC 77.1 81.3 74.0 75.1 74.1 77.1 67.7 72.6 67.3 55.7
0 PC 74.5 80.3 73.3 73.4 73.0 74.5 67.4 72.5 64.7 54.6
1,000 NC 79.9 84.1 76.6 77.9 77.1 79.9 71.1 74.5 69.2 60.0
1,000 PC 79.6 84.0 76.5 77.1 77.0 79.6 71.5 74.3 68.5 57.5
P value 0.55 0.75 0.87 0.79 0.79 0.55 0.83 0.98 0.63 0.74
SE 1.88 1.42 1.64 1.68 1.69 1.88 1.73 1.50 1.94 2.30
Fine NC 81.5 84.7 78.2 78.8 78.5 81.5 71.8 76.3 72.3 64.1
Fine PC 78.7 83.4 76.7 77.1 76.8 78.7 70.5 74.3 68.8 58.0
Coarse NC 75.6 80.7 72.4 74.2 72.8 75.6 66.8 70.8 64.3 51.7
Coarse PC 75.3 80.9 73.2 73.4 73.2 75.3 68.4 72.5 64.4 54.1
P value 0.51 0.59 0.48 0.78 0.79 0.51 0.41 0.23 0.37 0.07
SE 1.88 1.42 1.64 1.68 1.69 1.88 1.73 1.64 1.94 1.69
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Means within each column and treatment effect that don't possess any lowercase superscript letter in common differ significantly (P ≤ 0.05).

Means within each column and treatment effect that don't possess any uppercase superscript letter in common differ significantly (P ≤ 0.01).

Abbreviations: AID, apparent ileal digestibility; SE, standard error of mean.

1

Limestone particle size: fine (190 μm) and coarse (900 μm).

2

Phytase: had a dose of 1,000 FYT/kg and a matrix value of 0.18% for Ca and 0.15% for Pi.

3

The Ca and Pi had 2 levels, NC (negative control) had Ca 0.72 and P 0.3 in starter and Ca 0.53 and P 0.23 in grower while PC (positive control) had Ca 0.9 and P 0.45 in starter and Ca 0.76 and P 0.38 in grower.

Table 8.

Amino acid AID of male broilers as affected by limestone particle size (PS), phytase enzyme, Ca and Pi levels, and their interaction at 35 D of age.

PS1 Phytase2 Ca and P3 (%)
Lysine Arginine Histidine Leucine Isoleucine Methionine Glycine Proline Threonine Cysteine
Main effects
Fine 84.3A 88.7A 83.0a 82.8a 82.5A 83.2A 76.7A 79.8A 75.8A 80.6A
Coarse 86.2B 87.9B 82.1b 81.9b 81.4B 80.9B 75.4B 77.2B 74.1B 77.5B
P value 0.00 0.00 0.04 0.02 0.01 0.00 0.00 0.00 0.00 0.00
SE 0.25 0.21 0.28 0.28 0.28 0.42 0.36 0.35 0.34 0.52
0 84.8 87.9 82.2 82.0 81.5 81.4b 75.3b 78.0 74.6 78.5b
1,000 85.7 88.7 82.6 82.7 82.4 82.7a 76.8a 78.9 75.3 79.8a
P value 0.15 0.08 0.60 0.09 0.06 0.05 0.05 0.26 0.17 0.05
SE 0.25 0.21 0.28 0.28 0.28 0.42 0.36 0.35 0.34 0.52
PC 85.6 88.4 83.2a 82.6 82.5 82.5A 76.9 79.3a 76.1a 79.6A
NC 85.0 88.0 81.6b 81.8 81.6 81.6B 75.5 77.7b 73.8b 78.7B
P value 0.28 0.29 0.01 0.18 0.13 0.01 0.07 0.04 0.02 0.00
SE 0.25 0.21 0.28 0.28 0.28 0.42 0.36 0.35 0.34 0.52
Interactions
Fine 0 85.5 88.7A 82.7 82.3 82.2 82.6 77.5 78.6 75.6 79.7
Coarse 0 84.2 87.1B 81.7 81.7 80.8 80.3 73.4 77.5 73.6 77.3
Fine 1,000 86.0 88.7A 83.2 83.2 82.8 83.8 78.0 79.4 75.9 81.5
Coarse 1,000 85.4 88.7A 82.5 82.2 82.0 81.5 76.2 79.0 74.6 79.7
P value 0.74 0.00 0.70 0.61 0.39 0.97 0.14 0.51 0.46 0.53
SE 0.36 0.31 0.40 0.40 0.40 0.72 0.51 0.51 0.49 0.79
0 NC 84.2 87.3 80.9b 80.8b 80.5B 80.3 73.5B 76.2B 73.1 77.0b
0 PC 85.4 88.4 83.5a 83.2a 83.1A 82.6 77.2A 79.9A 76.1 80.0a
1,000 NC 85.4 88.6 82.4a,b 82.5a 83.1A 82.9 76.5A 78.7A 74.4 80.3a
1,000 PC 86.0 88.7 83.4a 82.9a 82.2A,B 82.5 77.1A 79.7A 76.1 79.3a
P value 0.45 0.13 0.05 0.01 0.01 0.06 0.00 0.01 0.20 0.02
SE 0.36 0.31 0.40 0.40 0.40 0.72 0.51 0.50 0.49 0.79
Fine NC 85.3a,b 87.5B 80.9C 80.9B 80.6B 81.7B 74.0B 76.5B 73.1B 78.9B
Fine PC 87.2a 89.8A 85.0A 84.7A 84.3A 84.7A 79.4A 81.5A 78.4A 82.3A
Coarse NC 84.7b 88.4B 82.4B 82.4B 81.9B 80.3B 75.9B 78.3B 74.5B 78.5B
Coarse PC 83.9b 87.3B 81.8B,C 81.5B 81.0B 80.3B 74.9B 78.1B 73.8B 77.0B
P value 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
SE 0.36 0.31 0.40 0.40 0.40 0.72 0.51 0.50 0.49 0.79
Open in a new tab

Means within each column and treatment effect that don't possess any lowercase superscript letter in common differ significantly (P ≤ 0.05).

Means within each column and treatment effect that don't possess any uppercase superscript letter in common differ significantly (P ≤ 0.01).

Abbreviations: AID, apparent ileal digestibility; SE, standard error of mean.

1

Limestone particle size: fine (190 μm) and coarse (900 μm).

2

Phytase: had a dose of 1,000 FYT/kg and a matrix value of 0.18% for Ca and 0.15% for Pi.

3

The Ca and Pi had 2 levels, NC (negative control) had Ca 0.72 and P 0.3 in starter and Ca 0.53 and P 0.23 in grower while PC (positive control) had Ca 0.9 and P 0.45 in starter and Ca 0.76 and P 0.38 in grower.

Kim et al. (2018) concluded that larger PS had better Ca and Pi digestibility at 28 D, when they defined small PS as 75 μm and large PS as 402 μm. Bradbury et al. (2018) considered fine PS to be <0.5 mm and coarse to be >0.5 mm, and they observed no significant effect of PS on either Ca or Pi digestibility. Anwar et al. (2016), on the other hand, concluded that coarse PS resulted in better true and apparent Ca digestibility with fine PS being <500 μm and coarse PS ranging between 1,000 and 2,000 μm. Guinote and Nys (1991) reported that fine PS (<150 μm) supported better Ca retention than medium PS (600–1,180 μm) and coarse PS (>1,180 μm).

It is accepted that different PSs vary in solubility, and it is well known that in vitro fine PS is more soluble than coarse PS (de Witt et al., 2006, Managi and Coon, 2007, Kim et al., 2018). In the ventriculus, it is thought that coarse limestone particles are retained longer as Roland (1986) concluded that PS > 900 μm will be retained longer in the ventriculus of layers, and Zhang and Coon (1997a) also came to the same conclusion. The longer retention time of coarse limestone particles in the ventriculus appears to sustain longer Ca availability to the bird than fine PS (de Witt et al., 2006). The greater and sustained Ca release from coarse limestone particles is at a slower rate than that from fine limestone particles because of differences in solubility. The differences in the solubility of coarse and fine limestone particles will influence Pi and Ca digestibility, with fine PS causing greater variability in Ca and Pi digestibility. One hypothesis to explain the reduction of AID in birds fed coarse PS is that an elevated dietary Ca level will downregulate the Ca transporter in the small intestine, thereby reducing Ca uptake (Li et al., 2012), while additional Ca can bind to Pi precipitating the insoluble calcium phosphate, which passes through the intestinal tract undigested (Heaney and Nordin, 2002).

Inclusion of phytase enzyme improved both Ca and Pi digestibility at 14 D while only Pi digestibility was affected at 35 D. Phytase degrades phytate in poultry diets, thus improves Ca and Pi availability, which can then result in better live performance (Bougouin et al., 2014, Bradbury et al., 2016, Scholey et al., 2017). Ca and Pi levels also affect Ca and Pi digestibility, where higher inclusion levels in the diet resulted in decreased AID and lower levels improved AID at 14 D and 35 D of age. These responses were likely due to a stoichiometric mixture of Ca and Pi, which regulates their intestinal uptake. Phosphate is absorbed in the small intestine by passive diffusion via paracellular phosphate transport and active transport via sodium-dependent phosphate cotransporters (Sabbagh et al., 2011). Calcium transport is mediated primarily via the calcitriol-dependent Calbindin-D28k in the chick intestine. The Ca and Pi transporters are regulated according to chymal Ca and Pi content, with high concentrations causing low transporter activity and low concentrations causing high transporter activity (Li et al., 2012, Adedokun and Adeola, 2013). No significant interaction for Ca and Pi AID was observed at 35 D, but there was a significant interaction between PS and phytase enzyme at 14 D for both Ca (P = 0.0001) and Pi (P = 0.0025).

Along with Ca and Pi digestibility, the AID of 22 amino acids (lysine, methionine, cysteine, threonine, arginine, histidine, taurine, hydroxyproline, aspartic acid, serine, glutamic acid, proline, lanthionine, glycine, alanine, valine, leucine, isoleucine, tyrosine, phenylalanine, hydroxylysine, and ornithine) was determined for 14 D (Table 7) and 35 D (Table 8); results are shown for only 10 out of the 22 amino acids tested (lysine, methionine, cysteine, threonine, arginine, histidine, proline, glycine, leucine, and isoleucine). Similar to Ca and Pi, PS had a strong effect on amino acid digestibility, with fine PS improving digestibility of all the amino acids for both 14 D and 35 D. The reason for this divergence in amino acid digestibility with fine PS vs. coarse PS is not yet understood.

Phytase inclusion improved the AID of 10 (arginine, lysine, isoleucine, glycine, leucine, glutamic acid, serine, aspartic acid, hydroxyproline, and phenylalanine) out of 22 amino acids analyzed at 14 D and 5 (hydroxyproline, methionine, cysteine, glycine, and ornithine) of 22 amino acids at 35 D; not all listed amino acids are shown in tables. These observations show that as birds age, the phytase inclusion effect dissipates significantly. The phytate molecule appears to form a protein-phytate complex, which interferes with protease activity that can degrade the complex proteins (Selle et al., 2000). Phytate has been reported to increase endogenous amino acid flow which can negatively impact amino acid availability (Ravindran et al., 1999b, Cowieson et al., 2004). Thus, phytase improved AID of certain amino acids by degrading phytate. Furthermore, phytase has an effect on the sodium-dependent transporters, which may also contribute to the observed effects (Truong et al., 2015). The various Ca and Pi ratios did not significantly affect AID of amino acids at 14 D, but Ca and Pi ratios at high levels did positively influence the AID for 6 (hydroxyproline, threonine, cysteine, methionine, ornithine, and histidine) of 21 amino acids at 35 D.

At 14 D, the interaction between PS and phytase was significant; other interactions were not significant. The majority of the amino acids tested showed coarse PS without phytase as the only treatment that was different and had a low AID. Inclusion of phytase in coarse PS treatments resulted in significant improvement in amino acid AID, making it similar to fine PS treatment either with or without phytase. This indicated that phytase enzyme had a stronger influence with coarse limestone PS, which was also observed by Kim et al. (2018) who noted a negative effect of dietary Ca on Pi AID which could be fixed when a larger PS of limestone (400 μm) was used in the presence of phytase, however the effect of fine PS could not be reversed with phytase inclusion. At 35 D, the majority of the amino acids' (hydroxyproline, glutamic acid, proline, glycine, alanine, cysteine, valine, isoleucine, leucine, tyrosine, phenylalanine, hydroxylysine, ornithine, and histidine) AIDs were influenced by the interaction between Ca and Pi levels and phytase. There was also a significant interaction between PS and Ca and Pi levels. Fine PS+PC treatments had greater amino acid AIDs than other treatments.

The results of this experiment demonstrated that phytase inclusion improved live performance (FI and BWG), bone ash (14 D), and nutrient digestibility (except Ca at 35 D), but it is most effective at younger ages; however, adding phytase to diets formulated with sufficient amount of Ca and Pi did not add any benefits. Phytase also improved Pi AID at both 14 D and 35 D, indicating its effectiveness in decreasing Pi content in excreta. On the other hand, PS of 190 μm and 900 μm did not alter tibia bone ash or live performance, although it is possible that NC treatment with coarse PS until 28 D can lead to a live performance similar to the PC treatment with fine limestone PS. The fine PS improved Ca and Pi digestibility, but this did not improve live performance compared with coarse PS. This indicates that transporters in the digestive tract play a huge role in intestinal uptake and that they can be upregulated or downregulated because of FI and nutrient concentration, which can also influence digestibility. Ca and Pi levels were the most influential factors in determining bone ash, although phytase inclusion can lead to an improvement during early days. Results of tibia bone ash and digestibility analyses point to the fact that phytase is more effective when used with NC treatments and with coarse PS.

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