THE INFLUENCE OF LIQUID CALCIUM SOURCE ON EGGSHELL QUALITY

It is estimated that the world’s population will enlarge by 25% by the middle of this century, resulting in the food production increase by at least 60%. Intensifying egg production is one of the most aff ordable solutions to provide animal protein. Among many other eff orts to improve the quality of eggs, special attention is paid to attempts to provide a better endurance and strength of eggshell, due to the fact that the production of eggs with broken, cracked or soft shells incurs signifi cant economic losses. One of the most important factors to achieve this goal is careful adjustment of calcium in the diet of laying hens, but the nutritional role of calcium is closely linked to that of phosphorus and the eff ect of vitamin D. Th e aim of the experiment was to determine whether two diff erent nutritional supplements of calcium, applied in drinking water of laying hens for two weeks have any signifi cant infl uence on the shell egg quality. Statistically very signifi cant diff erence in egg production was observed in the treatment with the product containing calcium and phosphorus. On the other hand, the product consisting of calcium and vitamin D3 had no benefi cial impact on the egg parameters. Based on the obtained results and literature data, it can be concluded that the amount and source of calcium in the diet of laying hens is a very complex and not fully solved issue. Th erefore, especially keeping in mind the duration of the experiment, additional research is needed on this subject.


INTRODUCTION
It is estimated that until 2050 the world's population will enlarge by 25%, and the demand for increase in food production will grow by at least 60% (FAOSTAT, 2013). Eggs constitute one of the most aff ordable source of animal protein available, so the number of laying fl ocks is rapidly increasing in developing countries. In Europe, the priority is to increase egg production by breed-ing for increased persistency in lay and stability in egg quality so that the laying cycle of commercial fl ocks can be extended to 90-100 weeks (Bain et al., 2016).
A number of factors have an impact on the quality of eggs, such as breed and age of laying hens, inheritance, feed, the environmental conditions or diseases. Endurance and strength of eggshells require special attention as the production of eggs with broken, cracked or soft shells incurs signifi cant economic losses. Eggshells of laying hens are highly organized mineral structures built of spherical calcite crystals deposited on the outer surface of the protein membrane, around egg white. Th e crystals of calcium carbonate are formed by crystallization from secreted supersaturated solution in the distal part of the oviducts. It takes about 20 hours for the formation of an eggshell, which indicates a large demand for a constant supply of the necessary amounts of calcium (Vitorovic et al., 2004).
During the laying period, the fi rst challenge is to adjust the energy and protein requirements to optimize egg output and to carefully control body weight. Th e crude protein concentration and amino acids in the layer diet are also important, methionine being the main limiting amino acid. Th e provision of insuffi cient dietary calcium during the rearing or laying period has an adverse eff ect on both eggshell quality and bone strength (Bain et al., 2016). Th e laying hens requirement for dietary calcium within the diet for diff erent ages is in the range from 0.9 to 1.2% during the growth period of the pullet, increasing to 2 to 2.5% just prior to the onset of lay and 3.5 to 4.5% once a lay is established (Bouvarel et al., 2011).
Shell formation takes place mainly during the night when consumption is very low. Th erefore, it is recommended to replace a portion of a fi ne pulverized calcium source in the diet with a larger particle size limestone, which takes more time to dissolve in the digestive tract, and thus presents calcium available overnight (Pavlovski et al., 2003). It is also recommended to implement the socalled night diet, when hens are stimulated to feed intake by periodic lighting.
Having all the above mentioned in mind, the aim of this study was to determine the infl uence of two nutritive supplements, as additional calcium sources administered in the drinking water on the quality of the eggshell.

MATERIALS AND METHODS
Th e experiment was conducted on 34200 Lohmann Brown laying hens, 78 weeks of age. All of them were fed with a mixture of standard ingredient structure as shown in the Table 1, and the chemical composition shown in the Table 2. Cage rearing facility consisted of three batteries, each containing 184 cages. Th e layers were thus divided into 3 experimental groups consisting of 11,400 individuals per each (R, C and L), which were of uniform performance and egg production characteristics. Every group had its own dispenser of drinking water. During 8 hours a day an experimental group R was given a product which contained calcium 5 g/l and phosphorus 144 g/l, in the dosage of 1 l per 1000 l of drinking water. At the same time experimental group L received another product containing calcium 75 g/l and vitamin D3 300 000 IU/l, in a dosage of 1 l per 1000 l of drinking water. For the control group C only pure water was provided. As diff erent additives were used, it was not the goal to make a comparison between them, but to gain, in practical farm conditions, a quick insight into their impact in relation to the control group.
Th e experiment lasted for 15 days. During that period, there were fi ve samplings of eggs, always within the same central part of the hall. Th e same number of eggs (30 eggs of A and 30 eggs of SS class) were taken from each group separately. Out of every 30 eggs, 14 were selected for weight measurements, with 2 lightest and 2 heaviest eggs discarded from statistics. Th e remaining 10 eggs were measured for the eggshell thickness using a micrometer aft er rinsing the shell in water to remove any adhering albumen. Later on, in order to determine the eggshell weight, drying at 105 °C for two hours was performed with the shell membrane intact. Th is was followed by weighing on an analytical scale. Th e percentage of eggshell breakage was also recorded as a percentage of the cracked eggs in relation to the number of whole eggs.
Statistical analysis was done using Microsoft Excel 2007, while the diff erences in measurements between the groups were tested by paired two-tailed t-test and ANOVA Two-Factor.

RESULTS AND DISCUSSION
Th e obtained results are given for each parameter separately as follows: Th e number of produced and cracked eggs (Table 3), the percentage of eggshell breakage (Table 4), Egg weight (Table 5), Eggshell weight (Table 6) and Eggshell thickness (Table 7). Th ese tables show that there were no statistically signifi cant diff erences in eggshell quality parameters between the experimental groups. Th e only parameter that diff ered signifi cantly comparing to the control group (< 0.01) was the number of eggs in the group R, which was given product containing calcium and phosphorus. Dissimilarity of all other results had no statistical importance.
Some authors, like Tunc and Cufadar (2015), who investigated infl uence of dietary large calcium sources (limestone, oyster shell and egg shell) on performance and eggshell quality parameters in laying hens observed no eff ect. However, the diet used in their experiment, containing at least 50% large calcium sources, had positive eff ect on mineral contents of tibia. Also, in earlier research of Keshavarz and Nakajima (1993) no benefi t of adding more calcium to limit the age deterioration in shell quality was determined, as well as no infl uence of such dietary treatments on egg production. Th is could be due to some other factors like breed and age of laying hens, inheritance and the environmental conditions, which could be dominant. Increasing the dietary level of Ca without benefi cial eff ects on shell quality indicate that the National Research Council (NRC, 1994) estimation of calcium (Ca) requirement of 3.25 g per hen per day is adequate for optimum shell formation.
On the other hand, experiment performed by Zhang et al. (2017), which lasted for 10 weeks, showed that aged non-molted laying hens (77 weeks) or older molted second cycle layers (94 weeks) require lower calcium solubility and higher calcium intake compared to relative younger laying hens (36 weeks old) to maximize shell quality and bone status. Th e amounts 3.94 -4.89 g of Ca intake for a hen per day from calcium carbonate source with a solubility range of 30.1-39.8% was recommended for older layers. Th erefore, according to these authors, the NRC recommendation might not be suffi cient to support the performance variables such as egg shell quality and bone status in older non-molted laying hens, or older molted second cycle laying hens. Pavlovski et al. (2003) and Vitorovic et al. (2004) similarly suggested usage of limestone of larger particle size to improve eggshell quality of laying hens, while Wang et al. (2013) had the same conclusion for ducks. Sharma et al. (2009) used liquid additional supplement source of calcium and phosphorus in their experiment and showed that overall egg and shell quality was improved in correlation with the activity of herbal constituents of the products owing to mineralization properties. Jadhav et al. (2010) also proved that supplementation of the same liquid calcium and phosphorus source was effi cacious in enhancing bioavailability of these minerals, thereby improving overall performance and bone mineralization in broilers.
During two weeks of our experiment, additional calcium from liquid source given through drinking water, especially supplement with calcium and vitamin D3 in group L, had no signifi cant impact on the examined parameters. It possibly means that the maximal exploitation of hens, in these circumstances, was achieved through adequate nutrition. In the used complete feed all nutrients were balanced and aligned with the needs of Lohmann Brown breed, while feed calcium was at the level of 3.95%. On this issue McDowell (2017) emphasized that the usage of liquid supplements to provide minerals might not provide enough calcium due to the solubility problems. Also, the duration of the experiment of only two weeks is a possible limiting factor.
Amino acids, in particular methionine and lysine (Bain et al., 2016), as well as fatty polyunsaturated linoleic acid are also important for adequate egg production. Certain improvement in the trial was observed in the treatment with the product containing calcium and phosphorus, which indicates the signifi cance of both minerals and demonstrates the importance of balancing Ca-P ratio in the diet of laying hens (Sefer and Sinovec, 2008). Th e establishment of Ca and P requirements of commercial layers is a continuous challenge for poultry nutritionists and egg producers as the needs for these two minerals seem to constantly change (Pelicia et al., 2009). Two-week experiment seems to be a short time to gain a quick insight into the impact of the applied supplements within the farm conditions and in limited material possibilities, compared to the fi ve-week trial carried out by Jadhav et al. (2010). In order to get a clearer picture and deeper understanding of obtained results, the continuity of investigations by extending the experiment, or by examining some other sources and diet regimens of laying hens are required. Also, as no signifi cant diff erences in eff ects of two additives of diff erent composition were found, this research limitation has no infl uence on fi nal conclusions, but affi rms the need to engage these complex facts in further investigations as well.

CONCLUSION
Based on the obtained results and literature data, it could be concluded that the amount and source of calcium in the diet of laying hens is a very complex and still not fully solved issue. Many factors infl uence the quality of eggshell. Taking into account signifi cant economic losses incurred by the production of eggs with broken, cracked or soft shells, as well as demand to increase food production for 60% by the middle of this century, it is obvious that all research eff orts for improvement in breeding and production of laying hens are necessary and important. Th e extension of the research is required and the topic of this paper should also be addressed.