Calcium Levels and Limestone Granulometries in the Diet of Light Layers in Their Second Production Cycle

ABSTRACT

Calcium is essential for bone development and eggshell formation in laying hen nutrition. It is therefore important to align the nutritional levels of calcium and the granulometry of limestone. The aim herein was to evaluate the effect of different calcium levels and two limestone granulometries on the performance, egg quality, digestive organ characteristics, and bone quality of layers in their second production cycle. A total of 324 Lohmann LSL Lite layers at 113 weeks of age were distributed in a completely randomized design with a 3 x 2 factorial scheme, with 3 calcium levels and 2 limestone granulometries, totalling 6 treatments with 6 replicates of 9 birds each. Performance, egg quality, digestive organ biometry, and bone characteristics were evaluated. The data were submitted to analysis of variance, and the means were compared by Tukey’s test. In addition, a canonical discriminant analysis (CDA) was carried out. There was no interaction between the factors for any of the variables. However, there was an effect of granulometry on egg weight, and of calcium levels on relative liver weight. No differences were observed in the CDA. It is concluded that coarse-grained limestone improves egg weight, and a 4.3% calcium level is recommended.

Keywords:
Calcium sources; micromineral; old hens; shell

INTRODUCTION

Minerals are involved in various metabolic pathways, acting in energy production, muscle contraction, reproduction, bone growth and development, and production. Their functions range from maintaining life to regulating the productivity of animals (Vieira et al., 2012). Calcium is the most abundant mineral in animals’ bodies and is mainly found in the skeleton (99%), which acts as a major calcium deposit (Bertechini, 2014).

In addition to bone development, calcium and phosphorus are the main factors responsible for shell formation, being related to poultry performance factors such as laying rate, egg weight, and longevity. It is worth noting that the slightest variation in the content of these minerals can influence these parameters, so they are kept within a narrow variation range by the animals’ bodies (Figueiredo Júnior et al., 2018). Furthermore, Ca deficiency in poultry can lead to delayed growth, bone fragility and poor shell quality (Bueno et al., 2019).

The eggshell formation process usually takes place at night, when the birds reduce their food intake. This situation restricts calcium intake in the gastrointestinal tract, stimulating partial calcium removal from the bone matrix, which can cause damage to the birds’ skeletal system (Bueno et al., 2016). It is therefore argued that the use of coarse limestone increases the retention time in the gizzard until the calcium is released to form the eggshell (Wang et al., 2014), thus allowing it to be released slowly and made available for shell formation when the birds are not feeding.

Factors such as the calcium level in the diet and the limestone granulometry are important, as they interfere with calcium absorption (Souza et al., 2017). The most commonly used calcium source in poultry feed formulations is calcitic limestone. This comes from limestone rocks and is abundant in nature. It contains 38% calcium and its bioavailability is close to 90%. However, it needs to go through a simple grinding process to be used in poultry diets. This process results in a specific granulometry, which can be fine or coarse (Silva, 2017).

According to Silva (2017), changes in limestone granulometry and its use, as well as different calcium percentages according to the age of the birds, contribute to a greater serum calcium circulation, which causes a reduction in the mobilization of bone calcium for shell deposition, and minimizes possible damages to the eggs. In other words, it is believed that a greater limestone granulometry can prolong the time this particle remains in the gizzard, so that it is slowly and gradually made available, without the need to remove calcium from the bones (Wang et al., 2014).

The aim of this study was to evaluate the effect of different dietary calcium levels and two limestone particle sizes on the production performance, egg and bone quality, and digestive organ biometry of light layers in their second production cycle.

MATERIALS AND METHODS

Before starting the study, the project was submitted to and approved by the Ethics Committee on the Use of Animals (CEUA), under protocol number 001.06.021.UVA.504.03.

The experiment was conducted in the laying sector of the Experimental Farm of the Vale do Acaraú State University, located in the municipality of Sobral, Ceará, Brazil (3° 36’’ S, 40° 18’’ W and 56 m above sea level). The region’s climate is characterized as Bsh (B- Dry; s- Semi-arid; h- Low latitude and altitude) according to the Köppen Climate Classification. The field research lasted four consecutive 28-day periods, covering the months of June to October.

The birds were housed in galvanized wire cages 90 cm long x 45 cm high x 45 cm wide, with three 30 cm subdivisions and a stocking density of 450 cm² bird-1. The cages had a trough-type feeder and a nipple-type drinker and were arranged in a pyramidal system. The barn where the birds were raised was manually built of masonry, with a concrete floor, a ceramic roof, and a wire screen, being positioned in an east-west orientation. A 16-hour light program was followed (12 hours of natural light + 4 hours of artificial light).

A total of 324 Lohmann LSL Lite layers were used, 113 weeks old, weighing 1.693 ± 0.092 kg, and with an average egg production of 68.6 ± 7.66%, without forced molting. As recommended by Sakomura and Rostagno (2016), the birds were selected according to body weight and egg production rate to obtain uniform experimental plots. A completely randomized experimental design (CRD) was adopted in a three x two factorial scheme, with three calcium levels (4.3%, 4.4%, 4.5%) and two limestone granulometries (fine, with a mean geometric diameter (MGD) of 0.222 mm; and coarse, with a MGD of 3.332 mm), totaling six treatments, with six replicates of nine birds each.

The diets were isoenergetic and isonutritive (Table 1), formulated according to the nutritional recommendations suggested in the strain’s manual (Lohmann Lsl Lite, 2016). The nutritional composition of the dietary ingredients used the values recommended by Rostagno et al., (2017).

Table 1
Percentage and calculated nutritional composition of the experimental diet.

Ingrediente	Fine limestone			Coarse limestone
	4.3%	4.4%	4.5%	4.3%	4.4%	4.5%
Corn grain	62.00	61.44	60.88	62.00	61.44	60.88
Soybean meal (45%)	21.54	21.64	21.75	21.54	21.64	21.75
Fine limestone	9.60	9.88	10.14	-	-	-
Coarse limestone	-	-	-	9.60	9.88	10.14
Meat meal	4.64	4.64	4.64	4.64	4.64	4.64
Vitamin-mineral supplement*	0.400	0.400	0.400	0.400	0.400	0.400
Common salt	0.32	0.310	0.310	0.32	0.310	0.310
Soybean oil	1.42	1.61	1.80	1.42	1.61	1.80
DL-Methionine	0.080	0.080	0.080	0.080	0.080	0.080
Metabolic Energy (Cal/kg)	2840	2840	2840	2840	2840	2840
Crude Protein (%)	17.00	17.00	17.00	17.00	17.00	17.00
Calcium (%)	4.3	4.4	4.5	4.3	4.4	4.5
Available phosphorus (%)	0.37	0.37	0.37	0.37	0.37	0.37
Sodium (%)	0.17	0.17	0.17	0.17	0.17	0.17
Digestible Met + Cis (%)	0.555	0.554	0.553	0.555	0.554	0.553
Digestible methionine (%)	0.312	0.312	0.312	0.312	0.312	0.312
Digestible lysine (%)	0.756	0.758	0.759	0.756	0.758	0.759
Digestible threonine (%)	0.552	0.552	0.552	0.552	0.552	0.552
Digestible tryptophan (%)	0.156	0.156	0.156	0.156	0.156	0.156

• Vitamin-mineral supplement* 0.4%: Guarantee levels (Composition per Kg of product) - iron (min) 10.00 g/kg; copper (min) 2,500.00 mg/kg; manganese (min) 20.00 g/kg; zinc (min) 20.00 g/kg; iodine (min) 208. 00 mg/kg; selenium (min) 75.00 mg/kg; vitamin A (min) 2.000.000.00 IU/kg vitamin D3 (min) 625.000.00 IU/kg; vitamin E (min) 3.000.000.00 IU/kg; vitamin K3 (min) 395.00 mg/kg; vitamin B1 (min) 250. 00 mg/kg; vitamin B2 (min) 1,000.00 mg/kg; niacin (min) 5,025.00 mg/kg; pantothenic acid (min) 1,805.00 mg/kg; vitamin B6 (min) 250.00 mg/kg; folic acid (min) 74.00 mg/kg; biotin (min) 5. 00 mg/kg; vitamin B12 (min) 2,400.00 mcg/kg; choline (min) 125.00 g/kg; methionine (min) 125.00 g/kg; xylanase (min) 3,000,000.00 U/kg; FTU/kg; enramycin (min) 1,250.00 mg/kg.

Performance parameters were subsequently asses-sed. Egg production was recorded daily until the end of each 28-day period, and the following performance variables were calculated: feed consumption (g/bird/day), egg production (%), egg weight (g), egg mass (g/bird/day), conversion per egg mass (kg/ kg), and conversion per dozen eggs (kg/dz).

The following egg quality parameters were assessed at the end of each 28-day period: albumen, yolk and shell percentage, shell thickness (mm), and egg specific gravity (g/cm3). Four eggs were selected per repetition for these measurements, two of which were used to measure specific gravity using the saline flotation method (Bezerra et al., 2015), and the other two for the other quality analyses.

The eggs were broken manually and their compo-nents separated and weighed using a high-precision scale. The percentage of each egg component was obtained by dividing the weight of the component by the weight of the egg, then multiplying the result by 100. The shells were dried without forced ventilation for 24 hours, then they were weighed, and the thickness was measured in three regions (equatorial and extremities) using a digital caliper. The average shell thickness (mm) was calculated from these values.

At the end of the experiment, 30 birds were euthanized (five per treatment) using the universal cervical dislocation method (according to CONCEA Normative Resolution Nº. 37/2018). The proventriculus, gizzard, liver, pancreas and intestines were removed to perform biometry of the digestive organs. The gizzard and intestines were properly cleaned and emptied. Afterwards, all the organs were weighed on a 0.01 g precision scale. The length of the intestines was also measured using a tape measure. All the weights obtained were used to calculate the relative weight of each organ.

The limbs (right and left) of the euthanized birds were removed and frozen until boning. The right tibias were used to analyze the following bone characteristics: weight (g), length (mm), mineral matter (g/kg) and Seedor Index (mg/mm). Weights were obtained on an electronic scale with a precision of 0.01g, and bone length was measured using a digital caliper. The bones were placed in disposable containers with the corresponding identification and sent for pre-drying in a forced ventilation oven at 65°C for 72 hours to determine the mineral content, which was performed at the UVA Animal Nutrition Laboratory (LANUT). The tibias were crushed using a mortar and pestle and returned to the containers for final drying in the oven at 105°C for 16 hours (Silva & Queiroz, 2002).

After drying, 2 g of the sample was weighed (in triplicate) into duly identified crucibles. The crucibles were then put into a muffle furnace at 600°C for 4 hours, and the weight of the ash was subsequently measured. Bone density was assessed using the Seedor index by dividing the weight of the ash (mg) by the length (mm) of the assessed bone (Seedor et al., 1991).

The left tibias were used for the strength (kgf/cm²) and bone deformity (mm) analyses, and were conducted at the Soil Mechanics Laboratory of the Federal University of Ceará (UFC). The right tibias were placed in a horizontal position and a compressive force was applied to the center of each one using a mechanical press. The maximum amount of force applied to the bone until it broke was measured using a digital extensometer and recorded as the breaking strength (kgf/cm²). The deformity (mm) was determined using an analog extensometer until the bone broke.

The data were submitted to analysis of variance (ANOVA) considering the factorial model, which included the effects of particle size and calcium levels, as well as the interaction between these factors. The Tukey’s test at 5% probability was used to compare the means. In addition, Canonical Discriminant Analysis (CDA) was performed to discriminate the main variables that differentiate the treatments and determine which variables have discriminatory power. The general model of the CDA is described in the following equation:

$Zn=\alpha +\beta 1X1+\beta 2X2+\mathrm{...}+\beta nXn$

In which: Zn is the independent variable (treatments), α is the intercept, Xi are the exploratory variables, and βi are the discriminant coefficients for each exploratory variable.

RESULTS

There was no interaction (p>0.05) between the factors (calcium levels and limestone granulometry) for the variables of performance, egg quality, digestive organ biometry and bone quality (Tables 2, 3, 4 and 5). However, there was an individual effect of the factors on egg weight and liver percentage.

Table 2
Feed intake, egg production, egg weight, egg mass, conversion per egg mass (CM) and conversion per dozen eggs (CDZ) of layers in their second productive cycle fed diets containing different dietary calcium levels and two limestone granulometries.

Variables
Factors	Intake (g/bird/day)	Production (%)	Egg weight (g)	Egg mass (g/bird/day)	CM (kg/kg)	CDZ (kg/dz)
Calcium levels
4.3 %	90.57	58.85	65.14	39.19	2.375	1.853
4.4%	91.15	59.67	66.58	39.91	2.278	1.786
4.5%	93.48	63.97	65.77	40.28	2.318	1.775
Granulometry
Fine	90.33	59.71	65.02B	38.57	2.376	1.829
Coarse	93.14	61.97	66.64A	40.94	2.275	1.781
CV1 (%)	4.91	8.26	2.95	8.83	6.98	6.70
Mean	91.74	60.76	65.83	39.80	2.326	1.806
ANOVA2			p-value
Calcium levels	0.3273	0.0839	0.2715	0.8018	0.4439	0.3233
Granulometry	0.1003	0.2464	0.0321	0.0943	0.1119	0.2970
C x G3	0.1946	0.1001	0.4605	0.1403	0.1562	0.1722

• ¹
  CV - Coefficient of variation; - 2ANOVA - Analysis of variance; 3CxG- Calcium levels x granulometry; A, B- Means followed by different capital letters in the same column differ by Tukey’s test at 5% probability.

Table 3
Percentage of albumen, yolk and shell, shell thickness and specific gravity of eggs from layers in their second productive cycle fed diets containing different dietary calcium levels and two limestone granulometries.

Variables
Factors	Albumen (%)	Yolk (%)	Shell (%)	ST1 (%)	SG2 (g/cm3)
Calcium levels
4.3 %	60.59	28.45	8.51	0.331	1.081
4.4%	61.47	28.26	8.74	0.341	1.079
4.5%	61.04	27.87	8.49	0.334	1.080
Granulometry
Fine	61.35	28.32	8.67	0.335	1.081
Coarse	60.71	28.07	8.49	0.336	1.079
CV3 (%)	2.30	3.98	3.95	4.94	0.21
Mean	61.03	28.19	8.58	0.336	1.080
ANOVA4			p-value
Calcium levels	0.3860	0.5040	0.2037	0.4175	0.4084
Granulometry	0.2255	0.5527	0.1527	0.9314	0.0767
C x G5	0.8194	0.3246	0.2247	0.1716	0.1008

• ¹
  Shell thickness; ²Specific gravity; ³Coefficient of variation; ⁴Analysis of variance; ⁵CxG - Calcium levels x Granulometry.

Table 4
Relative weight of digestive organs, and length of intestines of layers in their second productive cycle fed diets containing different dietary calcium levels and two limestone granulometries.

Variables
Factors	Prov.3 (%)	Gizzard (%)	Liver (%)	Pancreas (%)	Intestines (%)	Int. Length4 (cm)
Calcium levels
4.3 %	0.37	1.15	2.98A	0.18	2.75	149.50
4.4%	0.39	1.28	2.51B	0.21	2.80	150.90
4.5%	0.36	1.15	2.77AB	0.20	2.57	152.10
Granulometry
Fine	0.39	1.23	2.87	0.22A	2.84	151.73
Coarse	0.36	1.16	2.64	0.19B	2.57	149.93
CV1 (%)	12.74	10.48	13.11	13.94	13.36	6.96
Mean	0.37	1.19	2.75	0.20	2.71	150.83
ANOVA²				p-value
Calcium levels	0.3884	0.0579	0.0239	0.0786	0.3715	0.8586
Granulometry	0.1083	0.1056	0.0923	0.0243	0.0577	0.6432
C x G5	0.1865	0.0518	0.2516	0.1578	0.8843	0.9269

• ¹
  CV - Coefficient of variation; ²ANOVA - Analysis of variance; ³Prov - Proventricle; ⁴Int. Length - Intestine length; ⁵C x G - Calcium levels x granulometry; A, B - Means followed by different capital letters in the same column differ by Tukey’s 5% probability test.

Table 5
Seedor index (SI), strength, deformity and mineral matter (MM) of the tibias of layers in their second productive cycle fed diets containing different dietary calcium levels and two limestone granulometries.

Variable
Factors	SI (mg/mm)	Strength (kgf/cm2)	Deformity (mm)	MM (%)
Calcium levels
4.3 %	36.58	9.75	3.26	54.31
4.4%	33.64	8.70	3.14	51.89
4.5%	33.04	9.54	3.31	51.79
Granulometry
Fine	34.08	9.46	3.16	52.91
Coarse	34.76	9.27	3.32	52.41
CV1 (%)	11.66	16.75	17.69	6.90
Mean	34.42	9.37	3.24	52.66
ANOVA²	p-value
Calcium levels	0.1307	0.3564	0.7849	0.2346
Granulometry	0.6438	0.7488	0.4675	0.7111
C x G3	0.5805	0.8766	0.1852	0.8643

• ¹
  CV - Coefficient of variation; ²ANOVA - Analysis of variance; ³C X G - Calcium levels x Granulometry

Egg weight (Table 2) was lower (p<0.05) in the treatments containing fine-grained limestone and higher in those containing coarse-grained limestone. There was a higher liver percentage (p<0.05) in the treatments containing 4.3% calcium, and a lower percentage in the treatments with 4.4% calcium (Table 4). The treatments with 4.5% calcium did not differ from the others. The pancreas percentage was influenced by the limestone granulometry, showing a higher percentage when fine-grained limestone was used in the diets. There was no effect of calcium levels or limestone granulometry for all the other variables.

The summary of the canonical discriminant analysis is shown in Table 6. No studied indicator showed discriminatory power (p>0.05) to differentiate the groups according to supplementation with different calcium levels and limestone granulometries.

Table 6
Summary of the canonical discriminant analysis according to the indicators under study for the treatments with different calcium levels and two limestone granulometries.

Indicators	CCC 1	Treatments2 (%)						Variance
								(%)		(p-value)
		1	2	3	4	5	6	F1	F2	F1	F2
Performance	50	60	60	20	80	60	20	68.7	17.6	0.438	0.836
Egg characteristics	53.3	40	60	40	20	80	80	37.7	35	0.29	0.321
Guts	56.7	60	60	60	80	40	40	48.2	28.2	0.054	0.243
Bone characteristics	30	20	60	40	20	40	0	47.5	37.2	0.804	0.8

• ¹
  Total percentage of correctly classified cases: CCC = correctly classified cases; ²Percentage of correctly classified cases according to cluster; ³Statistical test: canonical functions (F1 and F2) with p<0.05 from the Wilks’ Lambda test were considered significant.

DISCUSSION

Table 2 shows that only the weight of the eggs was influenced by the limestone granulometry, whereby the treatments containing coarse-grained limestone had higher egg weights (66.64g) than those with fine-grained limestone (65.02g) (p<0.05).

The Lohmann LSL Lite Breed Manual (Lohmann LSL Lite, 2016) described performance values for birds up to 95 weeks of age. It is known that birds suffer certain physiological limitations with advancing age, such as a natural decline in laying, a reduction in the ability to digest and absorb nutrients, and a reduction in the bone mobilization of calcium, which results in worsening egg quality (Oliveira & Oliveira, 2013).

Despite the birds’ advanced age and the fact that they had not undergone forced molting, the values found in this study for egg production, feed consumption, egg weight, egg mass and feed conversion were close to those described in the manual for birds at 95 weeks. Thus, it can be deduced that the calcium levels and particle sizes used in the birds’ feed were adequate to sustain good values in the performance variables studied. It should also be noted (Table 2) that the calcium levels did not influence the performance variables, so it can be inferred that there was no additional effect from increasing the level of this mineral. Therefore, the lowest calcium level (4.3%) can be used without compromising the productivity of layers in their second laying cycle.

It is also worth noting that the eggs from all the treatments remained within the marketing standards, being classified in the category of extra type eggs (58 g to 67.99 g) and jumbo eggs (minimum 68 g), according to SDA Ordinance No. 747 dated February 6, 2023 (Brasil, 2023). This classification is in line with the Lite line’s manual, which recommends percentages of extra and jumbo eggs at 95 weeks of 47.3% and 7.2%, respectively (Lohmann LSL Lite, 2016). One of the factors that influences consumer choice the most in eggs is the size, with larger eggs being preferred (Marques, 2022). Extra and jumbo eggs are therefore more highly valued on the market, making them more profitable for producers.

The shell plays an important biological role in the formation of the embryonic chamber; but from a commercial point of view, it is a crucial encasing that must be resistant enough to prevent contamination and damage during transportation from the farm to the consumer (Gherardi & Vieira, 2018). Shell quality is an essential parameter, acting as a physical barrier against impact, microorganisms and dirt, as well as facilitating water and gas exchange through its pores (Cordeiro et al., 2017). These considerations are crucial to preserve the integrity of eggs and consumer safety.

There is a natural decline in performance as birds get older. At this stage, the eggs tend to become larger, but their shells are less thick. The decrease in shell quality associated with age is the result of a decrease in intestinal calcium absorption and bone calcium mobilization (Molnár et al., 2018). According to Figueiredo Júnior et al., (2013), young birds have a calcium absorption capacity of 60%, while older birds are closer to 40%. These factors, combined with any deficiency in the birds’ calcium supply or metabolism, result in more fragile eggshells, causing damage to the production system (Bueno et al., 2016).

According to Zhang et al., (2017), shell quality is influenced by calcium levels, feeding time and the particle size of calcium sources. As shown in Table 3, the egg quality indicators were not influenced by the treatments; in other words, dietary Ca levels and limestone particle sizes did not influence shell quality. Therefore, considering that an insufficient supply of calcium reduces egg quality, the results show that the layers were able to assimilate the necessary amount for adequate eggshell formation, even though they were at the end of the production cycle.

It was hoped to obtain better shell quality parameters with the use of coarse-grained limestone, but this was not observed. These results are in conflict with the theory that the larger particle size and lower solubility would lead to a longer retention time in the gizzard with the gradual release of calcium, thus acting as a reserve during the night when the birds are not feeding, which would help the shell formation process (Sousa et al., 2017).

Bearing in mind that the specific gravity of eggs is one of the parameters used to assess shell quality (Araújo & Albino, 2011), and that values equal to or greater than 1080 g/cm³ indicate good quality eggs (Roque & Soares, 1994), the values found in this study (Table 3) indicate that these eggs had good shell quality.

When studying the effect of the probiotic Bacillus subtilis on the performance and physiological parameters of young birds at 36 weeks of age, Sobczak & Kozłowski (2015) observed the following values for the percentage of albumen, yolk, shell and shell thickness: 66.43%; 23.78%; 9.79% and 0.355 mm, respectively. Thus, even though the birds in this study were older (113 weeks) and were not subjected to forced molting, they showed values close (61.03%, 28.19%, 8.58%, 0.336 mm) to those described by Sobczak & Kozłowski (2015), showing that they maintained the desired egg quality standards.

Contrary to what was observed in this study, Swiatkiewicz et al., (2015) studied the effect of the particle size of calcium sources and different calcium concentrations on performance and shell quality, observing that replacing fine limestone particles with coarse ones increased (p<0.05) the shell percentage and thickness, the specific density of the eggs, and the breaking strength of the eggs in older hens (69 weeks of age). However, calcium levels did not influence performance parameters, as they did in the present study.

The morphology and physiology of poultry’s gastrointestinal systems can be influenced by the type of diet provided. Therefore, biometric analysis of the digestive organs enables identifying changes resulting from the nutritional management to which birds were subjected (Farias et al., 2019).

Changes in the digestive organs, especially the proventriculus, gizzard and intestines, were expected when different particle sizes were used, as they directly participate in the digestion process and adapt quickly to the bromatological and structural conditions of the diet (Svihus, 2014). However, these specific organs were not influenced by the treatments.

Despite the increase observed in liver and pancreas weights (table 4), these values are within acceptable limits, considering that, as well as the birds being older, their productive performance was not affected. It can therefore be inferred that the diets fed to the birds did not cause metabolic disturbances and can be used safely.

The main function of calcium during the rearing and growing phases is forming the bone structure, while it is an essential component for shell formation during the laying phase. According to Mello (2015), approximately 70% of the Ca used for eggshell formation comes from the diet and 30% from the bones, due to the absence of Ca storage in the uterus. From this point of view, the bones act as large calcium deposits, which are made available to the animal whenever necessary.

Therefore, insufficient dietary calcium supply can negatively affect bone strength (Bain et al., 2016). Older birds normally tend to show a reduction in the absorption and retention efficiency of this mineral, which can affect egg quality and production (Cordeiro et al., 2017). As shown in Table 5, calcium levels and limestone granulometries did not influence the bone characteristics of the layers, indicating that regardless of the calcium level or limestone granulometry used, the birds were able to assimilate the calcium necessary to maintain bone integrity.

The combined analysis of the data using canonical discriminant analysis (Table 6) showed similarities between the evaluated variables (performance, egg characteristics, viscera and bone characteristics). Thus, it can be said that the multivariate analysis confirmed the results obtained by the univariate analysis, whereby the limestone granulometry and the calcium levels taken together did not influence the treatments.

Although the egg and viscera weights differed in the univariate analysis, two factors have to be considered: (I) multivariate analysis uses matrix algebra, unlike a simpler analysis such as univariate; and (II) all the variables in the multivariate analysis were analyzed together, which is why they differed from the univariate analysis (Angelin et al., 2023).

CONCLUSIONS

The use of coarse-grained limestone is recommended as it improves egg weight, and the dietary level of 4.3% calcium is recommended in the diet for layers in their second production cycle.

ACKNOWLEDGMENTS

To God, Tecnavic, the Planalto farm, Vale do Acaraú State University, and Funcap for the research grant.

REFERENCES

• Alvares CA, Stape JL, Sentelhas PC, Moraes JLG, Sparovek G. Koppen's climate classification map for Brazil. Meteorologische Zeitschrift 22(6):711-28;2023. https://doi.org/10.1127/0941948/2013/0507
  » https://doi.org/10.1127/0941948/2013/0507
• Angelim AM, Leite SCB, Farias MRS, et al. Alternative additives associated in the feeding of laying hens:performance, biometrics, bone traits, and economic evaluation-an unsupervised machine learning approach. Tropical Animal Health and Production 2023;55(2):74. https://doi.org/10.1007/s11250-023-03495-6
  » https://doi.org/10.1007/s11250-023-03495-6
• Araújo WAG, Albino LFT. Incubação commercial. Viçosa: Transworld Research Network; 2011.
• Bain MM, Nys Y, Dunn IC. Increasing persistency in lay and stabilising egg quality in longer laying cycles. What are the challenges?. British Poultry Science 2016;57(3):330-8. https://doi.org/10.1080/00071668.2016.1161727
  » https://doi.org/10.1080/00071668.2016.1161727
• Bertechini, AG. Exigências de minerais para aves. In: Sakomura NK, et al. Nutrição de não ruminantes. Jaboticabal: FUNEP; 2014. p.375-88. ISBN: 978-857805132-7
• Bezerra RM, Costa FGP, Givisiez PEN, et al. Glutamic acid supplementation on low protein diets for laying hens. Acta Scientiarum Animal Science 2015;37(2):129-34. https://doi.org/10.4025/actascianimsci.v37i2.25911
  » https://doi.org/10.4025/actascianimsci.v37i2.25911
• Brasil. Ministério da Agricultura, Pecuária e Abastecimento. Portaria SDA nº 747, de 06 DE fevereiro de 2023. Brasília, DF. Disponível em: https://www.in.gov.br/en/web/dou/-/portaria-sda-n-747-de-6-de-fevereiro-de-2023-462821629
  » https://www.in.gov.br/en/web/dou/-/portaria-sda-n-747-de-6-de-fevereiro-de-2023-462821629
• Bueno ICS, Gomide CA, Tenório AI, et al. Metabolismo de minerais em animais: cálcio [monografia] São Paulo (SP): Universidade de São Paulo; 2019. [cited 2023 nov 10]. Available from: https://edisciplinas.usp.br/pluginfile.php/4663535/mod_resource/content/4/Monografias/Grupo01-C%C3%A1lcio.pdf
• Bueno IJM, Surek D, Rocha C, et al. Effects of different limestone particle sizes in the diet of broiler breeders post molting on their performance, egg quality, incubation results, and pre-starter performance of their progeny. Poultry Science 2016;95(4):860-6. https://doi.org/10.3382/ps/pev438
  » https://doi.org/10.3382/ps/pev438
• CONCEA - Conselho Nacional de Controle de Experimentação Animal. Normativas do CONCEA. Diretrizes da Prática de Eutanásia. Resolução Normativa n.37, 2018 [cited 2023 Nov 02]. Available from: https://antigo.mctic.gov.br/mctic/opencms/legislacao/outros_atos/resolucoes/Resolucao_CONCEA_n_37_de_15022018.html
  » https://antigo.mctic.gov.br/mctic/opencms/legislacao/outros_atos/resolucoes/Resolucao_CONCEA_n_37_de_15022018.html
• Cordeiro CN, Bastos-Leite SC, Vasconcelos FC, et al. Chelated minerals and limestone particle sizes on performance and bone quality of brown-egg layers. Brazilian Journal of Poultry Science 2017;19(5):35-42. https://doi.org/10.3382/ps/pev438
  » https://doi.org/10.3382/ps/pev438
• Farias MRSD, Bastos-Leite SC, Moura CP, et al. Organic minerals with different chemical characteristics in diets for Hy Line White laying hens: performance, biometry of digestive organs, and bone quality. Revista Brasileira de Zootecnia 2019;48:3-8. https://doi.org/10.1590/rbz4820170329
  » https://doi.org/10.1590/rbz4820170329
• Figueiredo Júnior JP, Costa FGP, Givisiez PEN, et al. Transportadores de cálcio e fósforo em aves de postura. Revista Campo Digital 2018;13(1):70-81.
• Figueiredo Júnior JP, Costa FGP, Ludke JV, et al. Exigência de cálcio e fósforo para poedeiras leves em todas as fases de criação e ciclos de produção. Revista Eletrônica Nutritime 2013;10(4):2583-626.
• Gherardi SRM, Vieira RP. Fatores que afetam a qualidade da casca do ovo: revisão de literatura. Revista Eletrônica Nutritime 2018;15(3):8172-81.
• Lohmann Lsl Lite. Manual da linhagem: poedeiras comerciais Lohmann LSL Lite. 2016 [cited 2023 oct 15]. Available from: https://www.hyline.com/userdocs/pages/36_COM_POR.pdf
  » https://www.hyline.com/userdocs/pages/36_COM_POR.pdf
• Marques KC, Abreu KS, Mendonça MO. Fraudes em produtos avícolas. Revista Eletrônica Nutritime 2022;19(3):9082-93.
• Mello JF. Influência dos níveis de cálcio e fósforo na dieta de matrizes de codornas japonesas, no desempenho produtivo e no desenvolvimento ósseo embrionário da progênie [dissertação]. Maringá (PR): Universidade Estadual de Maringá; 2015 [cited 2023 Oct 15]. Available from: https://pesquisa.bvsalud.org/portal/resource/pt/vtt-200672 (acessed on:15 oct 2023).
• Molnár A, Maertens L, Ampe B, et al. Effect of different Split-feeding treatments on performance, egg quality and bone quality of individually housed aged laying hens. Poultry Science 2018;97(1):88-101. https://doi.org/10.3382/ps/pex255
  » https://doi.org/10.3382/ps/pex255
• Oliveira BL, Oliveira DD. Qualidade e tecnologia de ovos. 3rd ed. Lavras: UFLA; 2013.
• Roque L, Soares MC. Effects of eggshell quality and broiler breeder age on hatchability. Poultry Science 1994;73(12):1838-45. https://doi.org/10.3382/ps.0731838
  » https://doi.org/10.3382/ps.0731838
• Rostagno HS, Albino LFT, Hannas MI, et al. Tabelas brasileiras para aves e suínos: composição de alimentos e exigências nutricionais. 4th ed. Viçosa: UFV; 2017.
• Sakomura NK, Rostagno HS. Métodos de pesquisa em nutrição em monogástricos 2nd ed. Jaboticabal: FUNEP: 2016.
• Seedor JG, Quarraccio HH, Thompson DD. The biophosphonate alendronate (MK-217) inhibits bone loss due to ovariectomy in rats. Boné and Mineral Resources 1991;6(1):339-346. https://doi.org/10.1002/jbmr.5650060405
  » https://doi.org/10.1002/jbmr.5650060405
• Silva DJ, Queiroz AC. Análises de alimentos: métodos químicos e biológicos, 3rd ed. Viçosa: UFV; 2002.
• Silva, AN. Fontes e granulometria do calcário nas dietas inicial e crescimento de frangos de corte [dissertação]. Areia (PB): Universidade Federal da Paraíba; 2017 [cited 2023 Aug 12]. Available from: https://repositorio.ufpb.br/jspui/handle/123456789/20960
• Sobczak A, Kozlowski,K. The effect of a probiotic preparation containing Bacillus subtilis ATCC PTA-6737 on egg production and physiological parameters of laying hens. Annals of Animal Science 2015;15(3):711-23. https://doi.org/10.1515/aoas-2015-0040
  » https://doi.org/10.1515/aoas-2015-0040
• Sousa AM, Bastos-Leite SC, Abreu CG, et al. Chelated minerals and two limestone particle sizes on production of layers in the second laying cycle. Revista Brasileira de Saúde e Produção Animal 2017;18(1):103-12. https://doi.org/10.1590/S1519-99402017000100010
  » https://doi.org/10.1590/S1519-99402017000100010
• Souza CS, Barreto SLT, Vieites FM, et al. Cálcio e fósforo na nutrição de codornas japonesas em Postura. Science And Animal Health 2017;5(3):260-81. https://doi.org/10.15210/sah.v5i3.9166
  » https://doi.org/10.15210/sah.v5i3.9166
• Svihus B. Function of the digestive system. Journal of Applied Poultry Research 2014;23(2):306-14. https://doi.org/10.3382/japr.2014-00937
  » https://doi.org/10.3382/japr.2014-00937
• Swiatkiewicz S, Arczewska-Wlosek A, Krawczyk J, et al. Effects on performance and eggshell quality of particle size of calcium sources in laying hens diets with different Ca concentrations. Archives Animal Breeding 2015;58(2):301-7. https://doi.org/10.5194/aab-58-301-2015
  » https://doi.org/10.5194/aab-58-301-2015
• Vieira DVG, Barreto SLTD, Valeriano MH, et al. Exigências de cálcio e de fósforo disponível para codornas japonesas de 26 a 38 semanas de idade. Revista Brasileira de Saúde e Produção Animal 2012;13(1):204-13.
• Wang S, Chen W, Zhang HX, et al. Influence of particle size and calcium source on production performance, egg quality, and bone parameters in laying ducks. Poultry Science 2014;93(10):2560-6. https://doi.org/10.3382/ps.2014-03962
  » https://doi.org/10.3382/ps.2014-03962
• Zhang B, Caldas JV, Coon CN. Effect of dietary calcium intake and limestone solubility on egg shell quality and bone parameters for aged laying hens. International Journal of Poultry Science 2017;16(4):132-8. https://doi.org/10.3923/ijps.2017.132.138
  » https://doi.org/10.3923/ijps.2017.132.138

• Funding

  The authors have received funds from the FUNCAP funding agency.

• Data availability statement

  The data set generated during this study is available from the corresponding author upon reasonable request.

Edited by

Section editor:

Tatiana Carlesso dos Santos

Data availability

The data set generated during this study is available from the corresponding author upon reasonable request.

Publication Dates

• Publication in this collection
  04 Nov 2024
• Date of issue
  2024

History

• Received
  09 Feb 2024
• Accepted
  19 July 2024