Effect of phytase and limestone particle size on mineral digestibility, performance, eggshell quality, and bone mineralization in laying hens

Abstract

The effect of microbial phytase and limestone particle size (LmPS) was assessed in Lohmann Tradition laying hens from 31 to 35 wk of age. Seventy-two hens were used in a completely randomized trial according to a 2 × 2 factorial arrangement with 2 levels of phytase/basal available P (aP); 0 FTU/kg with 0.30% aP or 300 FTU/kg with 0.15% aP, and 2 limestone particle sizes; fine particles (FL, <0.5 mm) or a mix (MIX) of 75% coarse limestone (CL, 2–4 mm) and 25% FL. Diets contained equivalent levels of Ca (3.5%), phytic P (PP; 0.18%), and aP (0.30%) considering the P equivalency of phytase. Thus, dietary treatments were FL0 and MIX0 without phytase, and FL300 and MIX300 with 300 FTU/kg phytase. Performance were recorded daily and eggshell quality (eggshell weight proportion, weight, thickness, and breaking strength) was measured weekly. At the end of the trial, bone parameters (tibia breaking strength, elasticity, and ash) and the apparent precaecal digestibility (APCD) of P and Ca were determined. No differences were observed between treatments in feed intake, FCR and bone parameters. Addition of MIX increased the eggshell proportion, weight and thickness in groups receiving no phytase (+6.5, +6.9, and +4.5%, respectively) while no effect was observed in groups receiving phytase (Phytase × LmPS, P < 0.05). In hens receiving FL, the APCD of P was lower in diets supplemented with phytase (−14 percentage points; Phytase × LmPS, P < 0.001). A higher phytate disappearance was observed in hens fed diets with phytase in combination with MIX (Phytase × LmPS, P = 0.005). Phytase and MIX together increased the APCD of Ca by 7.3 percentage points (Phytase × LmPS, P < 0.001). In conclusion, addition of CL could limit the formation of Ca-phytate complex thus improving the response of the birds to phytase compared to FL.

INTRODUCTION

Around 60 to 70% of seed phosphorus (P) is complexed to myo-inositol to form phytic acid, which drastically reduces its bioavailability to the animal (Abbasi et al., 2019). Thus, phosphates are added to the diet to meet the daily requirements of poultry. The use of phytase enhances the bioavailability of P from phytic acid, and reduces P excretion (Selle and Ravindran, 2007). The positive effects of phytase on digestive performance, especially on precaecal digestibility of P are well known in broilers (Bournazel et al., 2018; Babatunde and Adeola, 2020; Ravindran et al., 2020). However, less studies measuring P digestibility at the precaecal level have been performed in laying hens (van der Klis et al., 1997; Javadi et al., 2021).

Another nutritional concern in laying hens is the high requirement for Ca occurring during the night to produce the eggshell. The diet provides approximately 60% of the Ca required (Nys, 2017), however, because of a desynchronization between dietary Ca availability during the light period and the high Ca requirement to produce the eggshell during the dark period (Nys and Leroy, 2018), additional Ca is mobilized from medullary bone (Kim et al., 2012). To reduce the gap between the supply and the demand for Ca, coarse limestone (CL) is commonly added to the diet (Molnár et al., 2018). Compared to fine limestone (FL), CL is retained for longer in the gizzard (Rao et al., 1992). The acidic content of the gizzard and a longer retention time of CL in this organ allow a slower solubilization of Ca and provide a longer diffusion of available Ca in the duodenum during the night, reducing bone Ca mobilization (Fleming, 2008). Moreover, CL has been shown to increase eggshell quality, without influencing production performance parameters (Hervo et al., 2022).

Phytic acid has the capacity to chelate some divalent cations to form phytic acid salts: phytate (Maenz et al., 1999; Angel et al., 2002). Due to the high Ca concentration in laying hen's feed, phytic acid can easily bind Ca to form unavailable complexes. This process depends on pH (Grynspan and Cheryan, 1983) and Ca-to-phytic acid ratio (Wise, 1983). In broilers, FL shows a higher capacity than CL to form insoluble complexes with phytate, thus reducing P digestibility, growth performance and bone mineralization (Manangi and Coon, 2007; Bradbury et al., 2016; Majeed et al., 2020). However, no data are available in laying hens. The objective of the current study was to assess the effect of microbial phytase, limestone particle size and their interaction on mineral digestibility in laying hens. The hypothesis was that FL could form insoluble complexes with phytic acid thus reducing phytase efficiency and P digestibility.

MATERIALS AND METHODS

The experiment was conducted under the guidelines of the French Ministry of Agriculture for Animal Research at the Experimental Poultry Unit of Tours, INRAE, Nouzilly, France (PEAT INRAE 1295, doi.org/10.15454/1.5572326250887292E12). The experimental design was approved by the Regional Ethics Committee on Animal Experimentation (Tours, France) and the French Ministry of Higher Education and Research (Paris, France; authorization: APAFIS #32000-202 1061008462021 v3).

Animal Housing and Management

Seventy-two laying hens (Lohmann Tradition, Lohmann, Saint-Fulgent, France) were housed in groups of 3 hens per cage from 18 to 25 wk of age. During this period, animals were fed according to breeder recommendations (Lohmann Breeders, 2021) to cover all nutritional requirements, that is, 3.4% Ca and 0.34% available P (aP). At 26 wk of age, hens were transferred to individual cages for a 5-wk adaptation period. This was followed by a 4-wk experimental period from 31 to 35 wk of age. Temperature was maintained between 18 and 20°C with 16 h of light per day (from 6 am to 10 pm). On each day of the 4-wk experimental period, eggs were collected and weighed to obtain egg production, egg weight and to calculate egg mass. Cumulative weekly refusals were weighed to determine the average daily feed intake (ADFI) and feed conversion ratio (FCR). Animals were weighed at the beginning and end of the experiment.

Experimental Treatments and Diets

Diets contained corn and wheat and were offered as mash form. Experimental diets were formulated to meet all nutrient requirements, with 3.5% Ca and 0.34% aP, according to breeder recommendations (Lohmann Breeders, 2021) (Table 1). At 31 wk of age, 72 laying hens were assigned to 1 of the 4 experimental treatments, with 18 replicates per treatment. A 2 × 2 factorial arrangement was used with 2 limestone particle sizes (LmPS), 100% fine limestone (FL) or a mix (MIX) of FL (25%) and coarse limestone (CL, 75%), and 2 levels of phytase, 0 FTU/kg or 300 FTU/kg. The mean diameter was below 0.5 mm for FL and between 2 and 4 mm for CL. Since the origin of limestone is known to influence the solubilization of Ca in the digestive tract of poultry (Kim et al., 2019), the same source of CL was used and CL particles were ground to obtain FL. Microbial phytase (a novel biosynthetic bacterial 6-phytase produced by Trichoderma reesei, Adisseo France SAS, Commentry, France) was added to 2 diets at 300 FTU/kg to provide 0.15% aP (only a matrix value for aP was considered). To summarize, FL0 contained fine limestone without phytase; MIX0 contained coarse and fine limestone without phytase; FL300 contained fine limestone with 300 FTU/kg phytase; and MIX300 contained coarse and fine limestone with 300 FTU/kg phytase. Feed and water were provided ad libitum except for the last day of the experiment. For the last 10 d of the experiment, hens received a diet supplemented with titanium dioxide (Ti) at 5.0 g/kg to determine the apparent precaecal digestibility (APCD) of P and Ca.

Table 1.

Ingredients, calculated, and analyzed nutrient levels, and phytase activities of experimental diets.¹

	0 FTU		300 FTU
Ingredients (g/kg)	FL	MIX	FL	MIX
Corn	532	532	537	537
Wheat	100	100	100	100
Soybean meal (46% CP)	240	240	240	240
Soybean oil	20	20	18.4	18.4
Corn gluten meal	5.3	5.3	5.3	5.3
Fine limestone2	83.7	21	87.5	22
Coarse limestone3	0	62.7	0	65.5
Monocalcium phosphate4	10.3	10.3	3.1	3.1
Phytase5	0	0	0.0091	0.0091
NaCl	3	3	3	3
Layer premix6	4	4	4	4
DL-Methionine	1.7	1.7	1.7	1.7
Nutrient composition (%)7
Calculated values8
DM	89	89	89	89
ME (kcal/kg)	2,749	2,749	2,751	2,751
CP	17.1	17.1	17.1	17.1
Dig. Lys	0,73	0,73	0,73	0,73
Dig. Met	0,25	0,25	0,25	0,25
Ca	3.50	3.50	3.50	3.50
tP	0.56	0.56	0.39	0.39
NPP	0.34	0.34	0.18	0.18
PP	0.22	0.22	0.16	0.16
aP	0.30	0.30	0.30	0.30
Analyzed values
DM	89.7	89.7	89.9	89.9
CP	17.3	17.8	17.8	17.7
Ca	3.52	3.68	3.58	3.49
tP	0.55	0.52	0.38	0.39
PP	0.20	0.16	0.19	0.19
Phytase activity (FTU/kg)	85	45	373	293

¹FL, fine limestone (<0.5 mm); MIX, mix of 25% fine limestone and 75% coarse limestone (2–4 mm); 0, diet without phytase and 0.34% NPP; 300, diet with 300 FTU/kg phytase and 0.18% NPP

²Fine limestone was <0.5 mm (geometric mean diameter = 0.21 ± 0.17 mm) and was obtained by grinding coarse limestone particles.

³Coarse limestone was between 2 and 4 mm (geometric mean diameter = 2.76 ± 0.13 mm). Calcium content in limestone was 38% Ca, according to producer.

⁴Monocalcium phosphate contained 17.0% Ca, and 22.7% P, according to producer.

⁵Phytase was a novel biosynthetic bacterial 6-phytase produced by Trichoderma reesei (Rovabio PhyPlus, Adisseo France SAS, Commentry, France).

⁶Layer premix contains the following, expressed per kilogram: vitamin A = 2,500,000 UI; vitamin D3 = 860,000 UI; vitamin E = 20,000 UI; vitamin K3 = 1,000 mg; vitamin B1 = 1,000 mg; vitamin B6 = 1,600 mg; pantothenic acid = 5,000 mg; vitamin B6 = 1,400 mg; vitamin B12 = 5.2 mg; vitamin PP = 20,000 mg; folic acid = 600 mg; biotin = 60 mg; Cu = 3,200 mg; Fe = 10,000 mg; Zn = 14,000 mg; Mn = 16,000 mg; I = 400 mg; Se = 40 mg.

⁷DM, dry matter; ME, metabolizable energy; CP, crude protein; tP, total P; NPP, nonphytate P; aP, available P; PP, phytic P.

⁸Calculated values of nutrients based on INRA-AFZ tables (2017).

Sample Collection and Analysis

Limestone Particle Size Distribution. The particle size distribution of limestone was determined by sifting the limestone through a series of sieves. Sieve sizes were according to the US standard mesh chart as follows: #100 (150 µm), #50 (300 µm), #30 (600 µm), #12 (1,700 µm), #10 (2,000 µm), #8 (2,360 µm), and #6 (3,350 µm). The geometric mean diameter (mm) and the geometric standard deviation (mm) were calculated for CL, FL, and mixed limestones, according to the method described by Wilcox et al. (1970). Limestone contained 38% Ca according to the manufacturer.

Eggshell Quality

Once per week, eggs were collected to evaluate eggshell thickness and breaking strength using a DET 6000 (Nabel Co., Kyoto, Japan). Shells were then cleaned and dried at 110°C for 2 h. Dried eggshells were weighed, and the eggshell proportion (%) was calculated.

Feed and Digesta Analysis

At 35 wk of age, all birds were euthanized by injection of sodium pentobarbital at the wing-vein. To synchronize hens, they were fasted from 10 pm the previous evening to the time of laying on the day of sampling. From the moment of laying (t lay = t0), hens were fed and euthanized 4 h later. As eggshell formation had not started, hens were considered to be in a steady state. The digesta of the distal half of the ileum were collected. Digesta were frozen at −20°C, freeze-dried for 72 h, and stored in order to determine the APCD of P and Ca and the PP disappearance. Diets and digesta of the distal ileum were analyzed to determine the Ca, tP, and Ti concentration using inductively coupled plasma optical emission spectrometry (ICP OES Thermo Scientific iCAP 7200, Courtaboeuf, France; method 990.08; AOAC International, 2006). Phytate concentration was determined in diets and digesta using a kit based on the modification of the ammonium molybdate method (Phytic Acid Assay Kit; NEOGEN Europe Ltd., Auchincruive, Scotland). Diets were analyzed for phytase activity (Adisseo France SAS, Commentry, France) and for dry matter and crude protein content by near-infrared spectroscopy, with a Büchi NIRFlex N-500 spectrometer (Büchi SARL, Vellibon-sur-Yvette, France).

Bone Parameters

The right tibia was collected for measurement of breaking strength (N) and elasticity (mm) with the 3-point destructive bending test using an Instron compression machine (Model 4411, Instron Corp., Canton, MA) loaded with a 200-N load cell. A 6-cm distance between the 2 fixed points supporting the tibia and a crosshead speed of 5.0 mm/min were held constant throughout each measurement. Tibia ash content was determined on dried defatted bones according to the method described by the Association of Official Analytical Chemists (AOAC, 1990). Briefly, soft tissues were gently removed before bones were dried at 100°C for 24 h. Tibias were defatted by immersion in a diethyl-ether solution for 24 h before ashing at 600°C overnight.

Calculations and Statistical Analysis

The apparent precaecal digestibility of P and Ca was determined using Ti as an indigestible marker. The same method was applied to determine the phytic P (PP) disappearance at the ileum level. The apparent precaecal digestibility and the PP disappearance were calculated as follows:

$$\text{APCD}(\%)=\left[1-\left(\frac{\mathrm{T}{\mathrm{i}}_{\text{feed}}}{\mathrm{T}{\mathrm{i}}_{\text{digesta}}}\times \frac{\text{minera}{\mathrm{l}}_{\text{digesta}}}{\text{minera}{\mathrm{l}}_{\text{feed}}}\right)\right]\times 100$$

where Ti_digesta and mineral_digesta are the Ti and mineral content (P, Ca, or PP; g/kg) in the freeze-dried digesta, respectively, and Ti_feed and mineral_feed are the Ti and mineral content (P, Ca, or PP; g/kg) in the feed, respectively.

A randomized block design was used (with a total of 18 blocks of 4 cages) with a 2 × 2 factorial arrangement: 2 limestone particle size and 2 levels of phytase resulting in 4 treatments, each with 18 replicates. Statistical analysis was performed using R open-source software version 3.02 (R Foundation for Statistical Computing, Vienna, Austria). All data were tested for normality according to a Shapiro test. Analysis of variance was conducted to determine the effect of phytase, limestone particle size, and their interaction. For each dependent variable, the experimental unit was the individual. The block effect of the experimental design, representing the position of the cage into the building and considering the potential variability due to the environment, was considered as a fixed effect. For egg weight and bone parameters body weight was also introduced as a covariable. Tukey's range test was used for multiple means comparisons when P < 0.05. As egg production did not follow normality, a nonparametric Kruskal-Wallis test was performed to determine the effect of phytase, limestone particle size, and their interaction. A Dunn test with a Bonferroni correction for the P value was used for multiple means comparisons. Statistical significance was considered when P < 0.05, and a trend was reported when 0.05 ≤ P < 0.10.

Results

Particle Size Distribution and Phytase Activity

As expected, fine limestone was characterized by a main proportion of particles smaller than or equal to 0.30 mm (71%) and coarse limestone showed a high proportion of particles larger than 2.36 mm (64%; Table 2). Thus, the geometric mean diameter of FL and CL was 0.21 ± 0.17 and 2.76 ± 0.13 mm, respectively. It is noteworthy that in the MIX, due to the combination of 75% CL and 25% FL, the geometric mean diameter of limestone was 1.36 ± 0.32 mm. Phytase activity in adequate P level diets was 85 and 45 FTU/kg for FL0 and MIX0, respectively. The analyzed phytase activity of FL300 and MIX300 was 373 and 293 FTU/kg, respectively (Table 1).

Table 2.

Proportion of particle size distribution for fine limestone, coarse limestone, and mixed limestone (75% coarse and 25% fine).

Sieve diameter (mm)	FL1	CL2	MIX3
>3.35	0	29.5	22.3
>2.36–3.35	0	34.5	23.9
>2.00–2.36	0	22.6	18.2
>1.70–2.00	0	13.2	10.2
>0.60–1.70	2.3	0.2	0.8
>0.30–0.60	26.3	0	5.8
>0.15–0.30	30.8	0	7.9
<0.10	40.6	0	10.9
Geometric mean diameter (mm)	0.21	2.76	1.36
Geometric standard deviation (mm)	0.17	0.13	0.32

¹FL, fine limestone.

²CL, coarse limestone.

³MIX, mixed limestone particles with 75% CL and 25% FL.

Production Performance

There was no mortality during the experimental period. No significant effect was observed on ADFI, body weight, or egg production (Table 3). Diets with FL increased FCR by 3.2% compared with MIX (P < 0.05). Phytase increased egg weight by 3.7% in diets containing FL but had no effect in MIX diets (Phytase × LmPS, P < 0.05).

Table 3.

Effect of phytase and limestone particle size on average daily feed intake, feed conversion ratio, egg production, egg weight, and egg mass.¹

Treatment4	ADFI2 (g/d)	FCR3	Final body weight (g)	Egg production (%)	Egg weight (g)
FL0	101.9 ± 1.05	1.64 ± 0.019	1,785 ± 49.6	94.1 ± 8.6	62.8 ± 0.46b
MIX0	101.8 ± 0.91	1.57 ± 0.021	1,796 ± 43.4	95.4 ± 7.5	64.0 ± 0.55ab
FL300	103.8 ± 0.88	1.60 ± 0.022	1,780 ± 39.3	96.1 ± 7.3	65.1 ± 0.54a
MIX300	101.3 ± 1.09	1.57 ± 0.021	1,882 ± 57.7	93.6 ± 8.9	63.9 ± 0.41ab
Phytase
0	101.8 ± 0.69	1.60 ± 0.015	1,790 ± 32.6	94.7 ± 8.1	63.4 ± 0.36b
300	101.9 ± 0.70	1.58 ± 0.015	1,835 ± 36.7	94.8 ± 8.4	64.5 ± 0.34a
LmPS5
FL	102.8 ± 0.69	1.62 ± 0.015a	1,782 ± 31.9	95.1 ± 8.0	63.9 ± 036
MIX	101.2 ± 0.71	1.57 ± 0.015b	1,840 ± 36.6	94.5 ± 8.5	63.9 ± 0.34
Source of variation	P value
Phytase	0.976	0.332	0.356	0.276	0.015
LmPS	0.179	0.023	0.235	0.151	0.888
Phytase × LmPS	0.275	0.077	0.392	0.231	0.021

¹Mean values of 18 replicates of 1 hen once per week during 4 wk for each treatment ± standard error of the mean (SEM).

²ADFI, average daily feed intake.

³FCR, feed conversion ratio.

⁴FL, fine limestone (<0.5 mm); MIX, mix of 25% fine limestone and 75% coarse limestone (2–4 mm); 0, diet without phytase and 0.34% NPP; 300, diet with 300 FTU/kg phytase and 0.18% NPP.

⁵LmPS, limestone particle size.

^a–bMeans within a column with different superscripts differ significantly (P < 0.05), based on Tukey's HSD means separation. Except for egg production, where different superscripts differ significantly (P < 0.05), based on Dunn test (with a Bonferroni correction for the P value) means separation.

Eggshell Quality and Bone Mineralization

The addition MIX increased the eggshell proportion (P < 0.001), weight (P = 0.005) and thickness (P = 0.003) in diets without phytase (respectively, 6.5, 6.9, and 4.5%; Phytase × LmPS; Table 4). Phytase incorporation increased eggshell breaking strength in a lower extent in hens fed MIX than in hens fed FL (Phytase × LmPS, P = 0.002): +3.2 and +6.5%, respectively. No significant effect was observed on the tibia elasticity, ash weight, ash relative content or breaking strength (Table 5). However, tibia breaking strength of hens fed with phytase tended to be lower compared with hens fed without phytase (P = 0.082).

Table 4.

Effect of phytase and limestone particle size on eggshell proportion, eggshell weight, eggshell thickness, and eggshell breaking strength.¹

Treatment2	Eggshell proportion (%)	Eggshell weight (g)	Eggshell thickness (mm)	Eggshell breaking strength (kg)
FL0	9.18 ± 0.086b	5.72 ± 0.055b	0.357 ± 0.0030b	4.72 ± 0.099b
MIX0	9.78 ± 0.088a	6.12 ± 0.073a	0.373 ± 0.0033a	5.07 ± 0.100ab
FL300	9.47 ± 0.087bc	6.13 ± 0.057a	0.366 ± 0.0029ab	5.03 ± 0.105ab
MIX300	9.54 ± 0.064ab	6.09 ± 0.045a	0.365 ± 0.0021ab	5.23 ± 0.077a
Phytase
0	9.50 ± 0.067	5.92 ± 0.050b	0.366 ± 0.0024	4.89 ± 0.072b
300	9.49 ± 0.051	6.10 ± 0.036a	0.366 ± 0.0017	5.15 ± 0.063a
LmPS3
FL	9.34 ± 0.063b	5.94 ± 0.044b	0.362 ± 0.0021b	4.89 ± 0.074b
MIX	9.67 ± 0.053a	6.11 ± 0.039a	0.369 ± 0.0018a	5.17 ± 0.061a
Source of variation	P value
Phytase	0.960	<0.001	0.884	0.005
LmPS	<0.001	0.002	0.025	0.006
Phytase × LmPS	<0.001	0.005	0.003	0.002

¹Mean values of 18 replicates of 1 hen once per week during 4 wk for each treatment ± standard error of the mean (SEM).

²FL, fine limestone (<0.5 mm); MIX, mix of 25% fine limestone and 75% coarse limestone (2–4 mm); 0, diet without phytase and 0.34% NPP; 300, diet with 300 FTU/kg phytase and 0.18% NPP.

³LmPS, limestone particle size.

^a–bMeans within a column with different superscripts differ significantly (P < 0.05), based on Tukey's HSD means separation.

Table 5.

Effect of phytase and limestone particle size on tibia breaking strength, tibia elasticity, tibia ash, and tibia ash weight.¹

Treatment2	Tibia breaking strength (N)	Elasticity (mm)	Tibia ash (%)	Ash weight (g)
FL0	94.1 ± 3.53	1.73 ± 0.066	49.8 ± 0.49	2.95 ± 0.073
MIX0	91.8 ± 3.02	1.73 ± 0.057	49.6 ± 0.54	2.77 ± 0.052
FL300	88.1 ± 2.78	1.74 ± 0.066	49.2 ± 0.66	2.77 ± 0.063
MIX300	87.9 ± 2.76	1.61 ± 0.030	49.4 ± 0.41	2.82 ± 0.063
Phytase
0	92.7 ± 2.63	1.73 ± 0.043	49.7 ± 0.35	2.87 ± 0.046
300	88.0 ± 1.92	1.68 ± 0.039	49.3 ± 0.38	2.79 ± 0.044
LmPS3
FL	90.8 ± 2.51	1.73 ± 0.046	49.5 ± 0.41	2.86 ± 0.050
MIX	90.1 ± 2.06	1.67 ± 0.034	49.5 ± 0.33	2.79 ± 0.041
Source of variation	P value
Phytase	0.082	0.354	0.113	0.207
LmPS	0.785	0.241	0.443	0.201
Phytase × LmPS	0.339	0.300	0.197	0.277

¹Mean values of 18 replicates of 1 hen for each treatment ± standard error of the mean (SEM).

²FL, fine limestone (<0.5 mm); MIX, mix of 25% fine limestone and 75% coarse limestone (2–4 mm); 0, diet without phytase and 0.34% NPP; 300, diet with 300 FTU/kg phytase and 0.18% NPP.

³LmPS, limestone particle size.

Apparent Precaecal Digestibility

As expected, laying hens receiving exogenous phytase ingested significantly less total P than others (−25%, P < 0.001; Table 6). No effect of dietary treatments was observed on Ca intake. Incorporation of MIX increased the APCD of Ca in a greater extent in hens fed phytase compared to hens fed without phytase: +9.5 and 7.0%, respectively (Phytase × LmPS, P = 0.002; Table 6). The P APCD of hens was decreased with phytase addition with a higher magnitude in hens receiving FL than MIX diets (−28 and −9%; Phytase × LmPS, P < 0.001; Table 6). The incorporation of microbial phytase increased PP disappearance by 30 compared with 10 percentage points in hens fed MIX and FL diets, respectively (Phytase × LmPS, P = 0.005; Table 6).

Table 6.

Effect of phytase and limestone particle size on P and Ca intake, apparent precaecal digestibility of P and Ca, and digestible P and Ca of feed.

	Mineral intake (g/d)		APCD (%)3		PP disappearance (%)4
Treatment5	Ca2	P1	Ca	P
FL0	3.71 ± 0.09	0.55 ± 0.012	75.6 ± 1.89b	48.6 ± 2.32a	49.58 ± 9.47b
MIX0	3.87 ± 0.13	0.53 ± 0.021	80.9 ± 2.21ab	45.6 ± 2.93a	50.57 ± 5.12b
FL300	3.71 ± 0.09	0.41 ± 0.010	75.9 ± 1.87b	34.9 ± 2.62b	59.72 ± 8.93b
MIX300	3.71 ± 0.10	0.41 ± 0.013	82. 9 ± 2.19a	41.5 ± 2.84ab	80.64 ± 8.37a
Phytase
0	3.78 ± 0.066	0.55 ± 0.012a	78.2 ± 1.56	47.3 ± 1.86a	50.04 ± 5.53b
300	3.71 ± 0.082	0.41 ± 0.008b	78.9 ± 1.62	39.1 ± 1.98b	71.14 ± 6.38a
LmPS6
FL	3.71 ± 0.079	0.47 ± 0.016	74.5 ± 1.32b	41.4 ± 2.09	53.98 ± 6.55
MIX	3.78 ± 0.070	0.53 ± 0.017	80.3 ± 1.54a	43.7 ± 2.05	66.26 ± 5.85
Source of variation			P value
Phytase	0.317	<0.001	0.735	<0.001	0.004
LmPS	0.342	0.945	<0.001	0.341	0.085
Phytase × LmPS	0.329	0.491	0.002	<0.001	0.005

¹Mean values of 17, 17, 16, and 14 replicates of 1 hen for treatment FL0, MIX0, FL300, and MIX300, respectively ± standard error of the mean (SEM).

²Mean values of 14, 15, 17, and 14 replicates of 1 hen for treatment FL0, MIX0, FL300, and MIX300, respectively ± standard error of the mean (SEM).

³APCD, apparent precaecal digestibility.

⁴Mean values of 7, 6, 5, and 6 replicates of 1 hen for the treatment FL0, MIX0, FL300, and MIX300, respectively ± standard error of the mean (SEM).

⁵FL, fine limestone (<0.5 mm); MIX, mix of 25% fine limestone and 75% coarse limestone (2–4 mm); 0, diet without phytase and 0.34% NPP; 300, diet with 300 FTU/kg phytase and 0.18% NPP.

⁶LmPS, limestone particle size.

^a–bMeans within a column with different superscripts differ significantly (P < 0.05) based on Tukey's HSD means separation.

DISCUSSION

Only a few studies have been performed regarding the effect of phytase on the APCD of P and Ca in laying hens. Moreover, there is no information on the effect of limestone particle size together with phytase on these parameters in laying hens; only data in broilers are available. As a result, part of this discussion is based on data issued from broiler trials. Phytase incorporation is known to increase APCD of P in the laying hen (van der Klis et al., 1997; Keshavarz, 2000). For instance, Bello and Korver (2019) showed that 32-wk-old hens fed 0.18% NPP with 300 FTU/kg exhibited an 18% higher APCD of P than hens fed with 0.38% NPP without phytase. Additionally, Pongmanee et al. (2020) showed no differences in APCD of P between 37-wk-old laying hens fed with 0.35% PNP without phytase or with 0.16% NPP with 300 FTU/kg, in accordance with the present results. The discrepancies between authors concerning phytase effect may be related to age of the laying hen (Bello and Korver, 2019) or to the length of the experimental period (Javadi et al., 2021). In the current experiment, it is noteworthy than hens fed FL exhibited a lower APCD of P in the diet formulated with phytase compared to other groups (P < 0.001; Table 6). Calcium can form insoluble complexes with phytate when the pH is above 5.0 (Nolan and Duffin, 1987), and this pH condition is found in the crop of the laying hen (Denbow, 2015; Classen et al., 2016). These complexes limit mineral solubilization, which in turn limits the capacity of phytase to liberate P from phytate (Selle et al., 2009). Consequently, the formation of Ca–phytate complex could reduce the APCD of P (Tamim et al., 2004). Results of the current experiment support this hypothesis: the positive impact of microbial phytase on PP disappearance was more important when using the mix of CL and FL compared to FL alone. The formation of the Ca–phytate complex is greatly influenced by limestone particle size in broilers: Manangi and Coon (2007) have shown that FL facilitates the formation of these complexes. In an in vitro assay, at pH = 6.5 and after 30 min, FL (0.03 mm) reduced PP degradation by phytase by 15% compared to CL (1.31 mm). Fine limestone is highly soluble compared to CL and rapidly provides a high quantity of soluble Ca in the gizzard (Kim et al., 2019). This soluble Ca can easily bind phytate and also decrease the APCD of P and Ca (Li et al., 2021). For example, the same authors found that, in broilers, the APCD of P was decreased from 87 to 61% in diets supplemented with phytase when limestone particle size decreased from 0.80 mm to 0.15 mm. To reinforce the significance of limestone particle size in Ca–phytate complex formation, an increase in dietary Ca in broilers did not affect the APCD of P when limestone was supplied as CL but decreases it by 16% with FL (Kim et al., 2018). Phytase inclusion allows to reduce the quantity of nonabsorbed P at the precaecal level (by 16% in the current study) decreasing in turn the P excretion (Lim et al., 2003; Abudabos, 2011). For instance, Martinez Rojas et al. (2018) showed that the use of phytase in a low P diet allows a reduction of the total amount of P present in the excreta by approximately 24% compared to a control diet receiving an adequate P level.

The level of APCD of Ca observed in this study was high (around 79%). This could be partly explained by the experimental design: laying hens were fasted from the evening (10 pm) until they laid the next day (8:30 am in average) to favor feed ingestion and ensure the presence of content in the ileal compartment. After laying, Ca and P are required to finalize medullary bone replenishment (Etches, 1987). The Ca deposition into medullary bone occurs mainly during the first 6 h after laying (Hurwitz, 1964; Clunies et al., 1993). Fasting may have induced hypocalcaemia and enhanced intestinal Ca absorption by the 1,25-OH₂-D₃ pathway (van der Klis et al., 1990; Bar, 2008) in order to meet the mineral demand. To reinforce this hypothesis, observed APCD of P was quite high when comparing to data of Keshavarz (2000) with values of 29 and 33% in hens fed 0.30% NPP without or with 300 FTU/kg, respectively. Thus, it cannot be excluded that Ca and P absorption has been increased in the current experiment due to physiological regulations. CL increased the APCD of Ca in a greater extent in hens fed diets with phytase. By favoring P absorption in phytase supplemented diets, CL decreases the propensity of Ca to complex phosphate (Hurwitz and Bar, 1965) and phytate (Selle et al., 2009) in the small intestine. Consequently, Ca would be more available for absorption.

In laying hens, P is an essential mineral involved in many functions such as bone development, energy metabolism, cell membrane structure and egg formation (Suttle, 2010). As mentioned earlier, during eggshell formation, bone resorption is stimulated to provide sufficient Ca (Dacke et al., 1993). Through this process, P is also released and excreted in the urine (Nys and Leroy, 2018). Eggshell production consequently increases the P requirement more than predicted by the small amount of P in the egg (∼120 mg). In commercial feeds, microbial phytase is commonly used to save phosphate and limit P excretion (Humer et al., 2015). In the current study, 300 FTU/kg of phytase were added, saving 7.2 g of phosphate per kg of feed with a replacement of 1.5 g aP without affecting feed intake or growth performance. These results corroborate those of Pongmanee et al. (2020), who reported an equivalency of 1.9 g aP in terms of ADFI and FCR for 300 FTU of phytase per kg in a diet containing 1.9 g aP in 37-wk-old laying hens.

It is worth noting that hens were individualized during the experimental period which was relatively short compared to practical conditions. So, the current results are probably not completely representative of commercial flocks. Egg production was maintained in the current experiment. A low dietary nonphytic P (NPP) level is known to decrease egg production (Teng et al., 2020). In their meta-analysis, Ahmadi and Rodehutscord (2012) showed that the effect of phytase on egg production is greater with low levels of NPP. Based on their results, 300 FTU of microbial phytase per kg feed containing 1.7 g NPP/kg did not significantly decrease the production performance of laying hens compared to a diet formulated with 3.4 g NPP/kg. The current study showed that phytase inclusion increased egg weight in the presence of FL, allowing to reach the same egg weight than MIX300 (Table 3). Concerning the effect of phytase on egg weight, phytase mainly acts on P utilization, which is not a major constituent of the egg as stated before. Meanwhile, the amino acids constituting proteins represent 11% of the egg white (Nys and Guyot, 2011). Phytic acid can form insoluble complexes with proteins (Kies et al., 2006; Selle et al., 2012), affecting amino acid availability. Phytase, by hydrolyzing phytate, can release amino acids and thus increase their digestibility in broilers (Rutherfurd et al., 2002). In accordance with our results, Skrivan et al. (2018) observed that inclusion of phytase at 300 FTU/kg increased egg weight by 2%, in 36-wk-old laying hens receiving 1.8 g NPP/kg. However, in the MIX diets, no effect of phytase was observed on egg weight. In the current study, CL was 2.76 mm and Scott et al. (1971) specified that particles greater than 1.0 mm were specifically retained in the gizzard. Coarse limestone has been shown to increase the gizzard grinding capacity (Roche and Martinoli, 1974) and decrease the gizzard pH (Singh et al., 2014). It is thus possible that CL improved amino acid digestibility and consequently increased egg weight compared to FL. By this mechanism, CL may hide the positive effect of phytase on egg weight.

Coarse limestone also improved eggshell weight and thickness in diets without phytase by providing solubilized Ca for an extended period compared to FL (Rao and Roland, 1990). A recent meta-analysis already described the positive effect of CL on eggshell thickness (Hervo et al., 2022): addition of CL (1.36 mm) increased eggshell thickness by 1.5% compared to FL (0.21 mm). Phytase addition and MIX together increased eggshell breaking strength. Bello et al. (2020) observed that phytase addition (600 FTU/kg) in 60-wk-old laying hen during 10 wk increased the eggshell breaking strength. Eggshell breaking strength is a synthetic indicator resulting from Ca and proteins deposition and arrangement. Indeed, proteins of the eggshell are known to play a major role in the ultrastructure (Gautron and Nys, 2007).

No effect of dietary treatment was observed on bone characteristics. Phosphorus is a major constituent of bone together with Ca in the form of hydroxyapatite (Whitehead, 2004). Even if the mineral P level was low, the extra P provided by phytase was sufficient to reach equivalent levels of bone ash content and mechanical properties compared to control diets (Table 5). Hens receiving phytase used P more efficiently for bone metabolism, as illustrated by the tibia ash weight to tP intake ratio: 5.3, 5.5, 6.6, and 7.1 for FL0, MIX0, FL300, and MIX300, respectively (Phytase: P < 0.001; data not shown). No effect of the MIX was observed on bone strength in the current study. However, a reduction of bone resorption was previously observed using CL (Guinotte and Nys, 1991; Gloux et al., 2020). Indeed, by diffusing soluble Ca into the intestine longer during the eggshell calcification period, CL limits bone mobilization. Because the structural bone content declines with age and leads to bone weakness (Whitehead, 2004), the effect of CL would be more pronounced in aged laying hens. In this way, de Witt et al. (2009) did not observe an effect of CL (2.9 mm) on bone strength before 66 wk of age. Thus, in the current study, the absence of an effect may have been due to the young age of the birds.

To conclude, this study demonstrates that using CL as a Ca source compared with FL improves the digestive utilization of P and Ca in phytase formulated diets maintaining performance and bone characteristics in a short-term. Further studies are needed to elucidate the underlying mechanisms in a dynamic way and the overall P excretion. Then, mineral requirements can be refined and nutritional strategies based on precision feeding implemented (i.e., “chrono-nutrition”).