Effect of the levels of calcium and particle size of limestone on laying hens 1

This experiment was conducted to evaluate the effect of levels of calcium (Ca) and particle size of limestone (LS) for laying hens. It was used 216 Dekalb White laying hens at 25 to 49 weeks of age in a completely randomized design in a 3 × 2 factorial arrangement, composed of three levels of calcium (3.92, 4.02 and 4.12%) and two particle sizes of limestone (thin 0.60 mm; and thick 1.00 mm), resulting in 6 treatments with six replicates of six birds. There was no significant effect of levels of calcium and limestone particle size neither of the interaction calcium × particle size for feed intake, egg weight, specific gravity, shell thickness, shell percentage, Haugh unit, calcium and phosphorus digestibility and percentage of digestive tract. Production, mass, conversion by mass and conversion by egg dozen had their best results with 4.12% of calcium in the diet. Percentage of gizzard and feed intake observed at 3:00 p.m. improved as limestone granulometry improved from 0.60 to 1.00 mm. There was an interaction effect among calcium level and limestone granulometry for breaking strength of tibia, bone density, length of the small intestine and feed intake at 6:00 a.m.; 9:00 a.m., 12:00 p.m., 6:00 p.m. and 9:00 p.m., respectively. It is recommended the addition of 4.12% of calcium and limestone in the thick granulometry (1.00 mm) for a better performance of commercial laying hens. More studies are needed to elucidate the mechanisms involved in the alteration of nutritional behavior of laying hens in response to changes in the levels of calcium and in the granulometry of limestone in the rations.


Introduction
The great genetic development of laying hens in recent years has changed important physiological aspects in these birds, requiring further research to equate them (Geraldo, 2006).In this context, the development of body and digestive tract of these birds have been object of concern, with problems related to uniformity of flocks and digestibility of nutrients, affecting their productivity.
Calcium is one of the main elements for maintenance and egg production of laying hens (Narváez et al., 1997) and it has been studied to improve the quality of the eggshell and bone system of modern laying hens, arousing the attention of nutritionists for studies on calcium sources and its physical and chemical characteristics.The calcium requirements by laying hens are considered an ongoing challenge for nutritionists and poultry producers (Roland & Gordon, 1996).The reasons for this fall on the continuous genetic improvement of commercial strains, in addition to the differences in particle size of limestone, which directly influence the availability of this mineral and poultry ability to adjust feed intake to meet their daily needs.Calcium absorption depends on several factors, including the mineral availability, calcium and phosphorus serum levels, vitamin D3, parathyroid hormone, gastrointestinal pH, fiber and fat contents and mineral granulomety in the diet (Stringhini, 2004).
The reason for the larger grain size of the calcium source for laying hens is related to improved availability of dietary calcium in the evening period, concomitantly with the eggshell formation in the shell gland.According to Leeson & Summers (1997), limestone with large particles are longer retained in the gizzard, and by being dissolved more slowly, the chicken has a strategic reserve of calcium to be used at the time the egg is being formed in the shell gland.
This study was conducted to evaluate the effects of calcium levels and particle size of limestone on the growth performance and feeding behavior of light laying hens in the stage of egg production.

Material and Methods
Experiment 1 was conducted at the Laboratório de Pesquisa em Nutrição de Aves of the Centro de Ciências Humanas, Sociais e Agrárias (CCHSA), campus III of the Universidade Federal da Paraíba, located in the city of Bananeiras -PB, micro-region of the Paraiba marsh.The experiment was constituted of six periods of 28 days.It was used 216 Dekalb White hens at 25 weeks of age.Poultry were selected by weight and, after two weeks of feeding on a single diet they were again selected based on the eggs production, so that poultry with extreme means of production were discarded whereas the others were weighed in a group of six and distributed in cages.Each cage had nipple drinker for every four hens housed and a trough-type feeder at the front of the cage.
In parallel with the first experiment, it was performed a digestibility trial (experiment 2) by using the method of total excreta collection.A group of 72 poultry were weighed and housed in a battery of cages equipped with trays to allow the collection of total excreta.The birds were fed on the 12 experimental diets for 10 days (five days for adaptation and five of excreta collection).
Experiments 1 and 2 were conducted in a randomized design, in a 3 × 2 factorial arrangement, with three levels of calcium (3.92, 4.02 and 4.12%) and two limestone particle sizes (thin = 0.60 mm and thick = 1.00 mm), which resulted in six combinations, evaluated with six repetitions of six birds.It was offered 120 g of feed/bird/day with water ad libitum throughout the experimental period.The experimental diets were formulated based on corn and soybean meal, according to the dietary recommendations of Rostagno et al. (2005) for light commercial layers, except calcium, which was supplemented by the addition of limestone to replace the inert (washed sand).
A diet with adequate calcium level (Rostagno et al., 2005), 4.02%, was included as positive control and two diets (isoproteic and isocaloric) containing low (3.92%) and high (4.12 %) calcium content, were formulated to meet or to exceed the nutritional requirements of calcium for poultry (Table 1).The particle size of limestone was obtained by analysis in sieves shaker, according to the standards of the supplier, considering as fine granulometry 30 to 50% of limestone retained on the 0.60 mm sieve and coarse 50 to 80% of limestone retained on the 1.00 mm sieve.Regardless of particle size (fine or coarse), the limestone was used to guarantee a minimum level of 38% calcium.
Dead poultry and food scraps were considered to adjust the consumption, egg production and food conventions per mass and per dozen eggs.
The average temperatures registered in the experiment were 21.6 ºC (minimal temperature) and 32.0 °C (maximal termperature).Minimal and maximal relative humidity were 39 and 85% respectively, and they were recorded by using a digital thermo-hygrometer at 9:00 a.m. and 5:00 p.m.The lowest temperatures were recorded at night and the highest in the day when feed intake is increased.
The food scraps removed from the trough feeder and buckets were weighed at the end of each period.Egg production was daily recorded; the average egg weight and specific gravity of eggshells were obtained in the last five days of each experimental period.
The variables studied in experiment 1 were feed intake, egg production, egg weight, egg mass, conversion per mass and per dozen eggs, specific gravity, eggshell, eggshell percentage, eggshell thickness and Haugh unit.
The specific gravity of eggshell was determined by floating eggs method in 17 salt solutions, ranging in density from 0.0025 unit, starting from 1.060 to 1.100 (Butcher & Miles, 2003).
To determine shell thickness, measurements were taken at three points in the equatorial region of the egg, with a Mitutoyo micrometer.At the end of each period, three eggs per plot were analyzed, weighed on a 0.01 g precision digital scale and broken on a glass flat surface to obtain the height of the albumen, by using an altimeter (Ames).Haugh unit values were calculated according to the formula presented by Card & Nesheim (1966).At the end of experiment 1, two poultry per plot were sacrificed after fasting for two hours to assess the gizzard weight.It was taken the digestive tract weight from the proventriculus to the rectum, including the cecum and pancreas, and liver was removed.Results were expressed as body weight percentage (Choi et al., 1986).
The small intestine length (duodenum to ileum) was measured by using a tape measure, and intestine extended on a flat surface for measurement.The relative length of the small intestine was expressed in cm/kg body weight (Choi et al., 1986).
The left tibia of each poultry was removed and used in the analysis of shear strength with three point bending.The analysis was performed on an Instron (kgf) press of the Departamento de Engenharia Civil of the Universidade Federal de Campina Grande, Paraíba.
For bone densitometry, radiograph images of the right tibia were obtained.After extraction of soft tissues and ligaments, bones were radiographed together with the density reference in an X-ray Siemens Tridoros 812E machine with a capacity of 38 kilovolt (KV), four milliamps and 10 seconds.As a densitometric reference in radiographs, it was used an aluminum ladder (6063, ABNT) of 12 steps (0.5 mm thick for the first step) ranging from 0.5 to 0.5 mm until the 10 th step, the 11 th with 6.0 mm thick and the 12 th with 8.0 mm thick, each step with 5 × 25 mm 2 area.
After being X-rayed, the images were scanned in an HP scanner (Scanjet 4C -Brazil), with transparency adapter for HP Scanjet 4C, and stored in a microcomputer.In densitometric analysis, it was used the Image-Pro Plus (Media Cybernetics -USA) software, which recaptured the radiographed images, being made initially the density calibration and bone density measurement in millimeters of equivalent aluminum (mmAl).
The program calibration was carried out by selecting specific density regions in the aluminum ladder.The values obtained led to a standard curve which was correlated to the values found on bone scans of interest.The reading of bone density was performed at three points (proximal epiphysis, diaphysis and distal epiphysis) of the right tibia of light laying hens at 49 weeks of age.
During the experiment 1, it was conducted an evaluation of the feeding behavior at the end of the second, fourth and sixth periods, respectively.In this evaluation, feed intake was controlled, being provided 150 g feed/poultry.The feed offers occurred at 5:00 a.m. and weights for measuring consumption at 6:00 a.m.; 9:00 a.m.; 12 p.m.; 3:00 p.m., 6:00 p.m. and 9:00 p.m., respectively.
In experiment 2, excreta samples were taken twice daily, in the morning and in the afternoon, weighed and a 10% aliquot was stored in a freezer at -10°C until laboratory analysis.The contents of total calcium in the diets and excreta were analyzed by spectrophotometer.Subsequently, -11,000 mg; Niacin -25,000 mg; folic acid -400 mg; pantothenic acid -10,000 mg; Se -300 mg. 2 Composition per kg of product: MN -150,000 mg; zinc -100,000 mg; iron -100,000 mg; copper -16,000 mg; iodine -1,500 mg.
Table 1 -Composition of experimental diets mineral digestibility was calculated (calcium and phosphorus).The test was conducted in a completely randomized design with 12 combinations, each one with three replicates of two birds.Statistical analysis was performed by using the program SAEG 5.0, developed by the Universidade Federal de Viçosa -UFV (1983).After the analysis of variance, when interactions were significant, the effects of each level of a major factor were studied within the other.In the absence of significant interaction, main effects were assessed by SNK test (P<0.05).

Results and Discussion
There was no significant effect (P>0.05) in calcium levels and particle size of limestone, or the interaction between these two factors on feed intake (Table 2).This result corroborates studies in literature which point out that there was no influence of particle size of limestone in feed consumption (Oliveira et al., 2002, Geraldo et al., 2004, Couto et al., 2008).
Egg production has improved (P<0.01) with 4.12% calcium in the diet.However, there was no significant effect (P>0.05) of limestone particle size, which contradicts, in part, the results described by Geraldo et al. (2006), who found a positive residual effect of fine granulometry in phases of rearing and re-rearing of hens on the average egg production at the production stage.There was no effect (P>0.05) of calcium levels and particle size of limestone on the egg weight.Geraldo et al. (2006), studying calcium levels and particle sizes of limestone for hens and their effects on yield and quality of eggs, also found no difference in egg weight among groups, then the calcium level and particle size of limestone used did not affect the egg weight.
Regardless of limestone granulometry, the best results for egg mass were observed with 4.12% calcium provided all day.This result exceeds the level of 4.02% calcium recommended by Rostagno et al. (2005) for light laying hens.The best conversions per mass and per dozen eggs (P<0.01) were observed with 4.12% calcium in the diet (Table 3).There was no effect of limestone particle size on these variables.Ito et al. (2006)    dietary calcium and limestone particle size on laying hens performance and noticed no significant effect (P>0.05) of limestone particle size on the feed conversion per mass and per dozen eggs.The specific gravity was not affected (P>0.05) by the main factors (calcium level and limestone particle size) or by their interaction.This result contradicts that obtained by Ito et al. (2006), who observed eggshell specific gravity reduction obtained by providing diets containing fine granulometry compared to the diets with limestone of coarse and mixed granulometry.
There was no significant effect (P>0.05) of calcium levels and particle size of limestone, or their interaction on the shell thickness, shell percentage and Haugh unit (Table 4).
A result opposite to that observed in this study was described by Pavloviski et al. (2003), who evaluated the replacement of powdered limestone (fine granulometry) for 60 or 80% granular limestone (coarse granulometery) and noted improvement in the eggshell quality (greater thickness and shell weight, higher breaking strength and lower shell deformation); however, there is evidence in the literature that there is no influence of limestone particle size in the eggshell percentage (Jardim Filho et al., 2005, Gerard et al., 2006, Ito et al., 2006), indicating that the hens were able to assimilate well the limestone offered, preserving the shell quality.For Haugh unit, Ito et al. (2006) found no significant effect (P>0.05) of the calcium fractionation or limestone particle size, as observed in this result.There was no significant effect (P>0.05) of calcium levels and granulometry or their interaction, on the digestibility of calcium and phosphorus (Table 5).
This was probably caused by the small spacing among the levels of calcium studied.Pelica et al. ( 2009) commented that animals fed diets deficient in this mineral had increased their absorption rates whereas high dietary levels caused reduction.These same authors noted that the increased level of dietary calcium from 3 to 4.5% increases the calcium losses in the feces of laying hens.
According to Roland (1986), larger particles from the source, inadequate levels and exposure to factors that reduce the use of calcium improves the availability of this mineral in chickens and help to explain the lack of effect of  experimental diets on the calcium digestibility.Furthermore, the use of levels very close to calcium requirements for poultry did not come to induce calcium deficiency and excess.There was no significant effect (P>0.05) of calcium levels on the tibia bone resistance to fracture.However, the coarse texture of the limestone improved (P<0.05) resistance.
The interaction calcium × granulometry improved (P<0.05) the tibia bone resistance to breakage when using 4.12% calcium associated with coarse limestone (Table 6).
Results of Fleming et al. (1998) found better bone resistance when added in the form of limestone gravel to feed.However, Jardim Filho ( 2005), working on different sources and textures of limestone in the diet of laying hens, observed no effect on bone resistance in hens until 49 weeks of age.For Saunders-Blades et al. ( 2009), diets containing coarse particles of limestone can improve the welfare of laying hens by reducing cases of osteoporosis and preventing the suffering of poultry with bones breaking at the end of the production cycle.
Bone density was not affected (P>0.05) by calcium levels or limestone granulometry, which did not occur (P<0.01) when 4.12% calcium and coarse limestone were combined (Table 6).Paz et al. (2008), evaluating bone density in laying hens, found differences among levels tested for the tibia density and stated that bones of poultry fed 3.8% calcium had higher densities than those fed on 1.8% calcium.This proves that the elevated levels of calcium in the diet increases bone density.The best result of the interaction (calcium level and limestone particle size) on bone density coincided with the best result of bone resistance to breakage, which occurred at 4.12% calcium associated with coarse limestone indicating a positive and narrow correlation between results of these two variables.No significant effects (P>0.05) of calcium levels and limestone particle size or their interaction (calcium × size) were observed on the digestive tract proportion to the body weight of laying hens during production stage (Table 7).The result is consistent with the findings of Geraldo et al. (2006), who noted no significant effect of calcium and limestone texture on the digestive tract percentage of laying hens.
There was no significant effect (P>0.05) of the interaction between calcium levels and limestone particle size on the gizzards percentage.The low (3.92%) and high (4.12%)calcium levels promoted the best results (P<0.01) for gizzards percentage than the control (4.02%).
The use of coarse limestone in the diet (P<0.01)caused a higher percentage of gizzards than the fine limestone granulometry.This result may be related to the greater  Table 7 -Morphometric measurements of the digestive tract, gizzard and small intestine length in hens fed diets with three levels of calcium retention of limestone gravels in the gizzard.A similar effect of coarse ground limestone on the increase of gizzard proportion was noted by several authors, who attributed these results to larger amounts of this mineral source retained in the gizzard of poultry (Rao & Roland, 1992, Zhang & Coon, 1997), improving calcium intake.There was no significant effect (P>0.05) of calcium levels or limestone particle size on the small intestine length.The interaction calcium levels × limestone particle size had no effect (P<0.01) on this variable when it was used 4.12% calcium associated with coarse limestone (1.00 mm; Table 6), and this indicates that the limestone fine texture affects the intestinal length.Geraldo et al. (2006) found no significant effect of calcium levels or limestone particle size on the small intestine length.However, the use of limestone with coarse texture result in a larger quantity of this ingredient retained in the gut of birds (Bertechini, 2006), which explains the longer length of this part of the intestine of birds.
The assessment of feeding behavior showed some intriguing results, starting with the significant effect on poultry consumption from 5 to 6 a.m.(P<0.01), when birds fed diets containing 3.92% calcium had peak of feed intake at this time, much higher than that of birds fed other calcium levels (4.02 and 4.12%).The limestone particle size affected (P<0.01)feed intake until 6:a.m. in the morning (Table 8).The combination of calcium levels and limestone particle size also influenced (P<0.01) until 6 a.m., when 3.92% calcium associated with limestone fine particle size resulted in higher consumption (Table 9).
From 6:00 a.m. to 9:00 a.m.(Table 8), feed intake began to stabilize among calcium levels and it was significantly higher (P<0.01) at 4.12%.The limestone particle size did not affect (P> 0.05) consumption from 6:00 a.m. to 9:00 a.m., however, the interaction calcium levels × limestone particle size had effect (P<0.05) at this period of day inasmuch as 4.12% calcium regardless of limestone particle size, resulted in higher feed intake (Table 9).
Feed intake from 9:00 a.m. to 12:00 p.m. was higher (P<0.05) in birds fed the diet with 3.92% calcium, but this result did not differ from that obtained with 4.12% calcium in the diet.Feeding behavior at this time of day was inversely proportional to that at 9:00 a.m., when the lowest intakes   were observed for poultry fed on the diet with 3.92 and 4.02% calcium.At 12:00 p.m., there was no significant effect (P<0.05) of limestone of particle size on feed intake, whose highest (P<0.01)value was obtained with the fine grains.The combination of calcium levels and limestone particle size also affected (P<0.01)feed intake from 9:00 a.m. to 12:00 p.m.; the highest values were obtained with 4.12% calcium associated with fine particle size (Table 9).
From 12:00 p.m. to 3:00 p.m., feed intake was not affected (P>0.05) by calcium or by the interaction calcium levels × limestone particle size, indicating balance among calcium levels.The coarse limestone influenced (P<0.01)feed intake more than fine granulometry.This increased consumption of diets with limestone with a 1 mm limestone at the hottest time of a day (from 12:00 p.m. to 3:00 p.m.) was very important because it certainly led to greater retention of limestone gravels in the gizzard, increasing the input of calcium in the afternoon and in the evening (Rao & Roland, 1992;Zhang & Coon, 1997), when the formation of the eggshell is initiated and completed, respectively.
The consumption of diet with 4.02% calcium was the highest in the period from 3:00 p.m. to 6:00 p.m. (P<0.01),but it did not differ (P>0.05)statistically from that obtained at 4.12 % calcium.At this time, limestone particle size as an isolate factor did not affect feed intake; however, the interaction calcium levels × limestone particle size improved (P<0.01) feed intake in poultry fed the diet with 4.02% calcium and coarse limestone.
From 6:00 p.m. to 9:00 p.m., there was a reduction in feed intake for the day: the highest consumption (P<0.01) at this time was recorded in birds fed the diet with 4.12% calcium.The limestone particle size as an isolate factor did not affect feed intake, but the interaction calcium levels × limestone particle size improved (P<0.01) consumption when birds were fed diet with 4.12% calcium and coarse limestone.
The highest feed consumption measured throughout the day via feeding behavior of laying hens occurred between 9:00 a.m. and 12:00 p.m., a result which contradicts those obtained by Xin et al. (2002) that indicate the increase in food consumption at around 3:00 p.m.

Conclusions
The addition of 4.12% calcium together with the supply of coarse limestone (1.00 mm) improves the performance of laying hens without affecting the quality of bones and eggs.More studies are needed to elucidate the mechanisms involved in changing the eating behavior of hens in response to changes in calcium levels and limestone particle size in the diet.
studied the fractionation of Means in columns followed by different letters are different (P<0.01) by SNK test.

Table 2 -
Feed intake, egg production, egg weight and egg mass in laying hens fed diets with three levels of calcium Means in columns followed by different letters are different (P<0.01) by SNK test.

Table 3 -
Feed conversion by egg mass, feed conversion per dozen eggs and egg specific gravity of hens fed diets containing three levels of calcium

Table 4 -
Thickness, eggshell percentage and egg Haugh unit of laying hens fed diets with three levels of calcium Means in columns followed by different letters are different (P<0.01) by SNK test.*Significant effect by SNK test (P<0.05).**Significant effect (P<0.01) by SNK test.

Table 5 -
Mineral digestibility of experimental diets, breaking strength and bone density of tibia in laying hens fed diets with three levels of calcium Means followed by at least one capital letter in the same column and tiny line do not differ at 5% probability by SNK test.CV = Coefficient of variation.

Table 6 -
Effect of interaction between calcium levels and particle size of lime on the breaking strength, bone density of the tibia and the length of the small intestine of laying hens Means in columns followed by different letters are different (P<0.01) by SNK test.PV = Weight.

Table 9 -
Effect of the interaction levels of calcium × particle size of limestone in the feed intake of laying hens measured at 6:00 a.

Table 8 -
Feed intake as a function of calcium level and limestone particle size for laying hens