EFFECT OF DIFFERENT FEED PHOSPHATES ON BLOOD PLASMA MINERAL PROFILE IN LAYING HENS

Zbigniew Dobrzański¹, Sebastian Opaliński², Krystyna Hoffmann³, Fabiola Bubel⁴, Krystyna Pogoda-Sewerniak^2
1 Department of Environment Hygiene and Animal Welfare, The Faculty of Biology and Animal Science, Wrocław University of Environmental and Life Sciences, Poland
² Department of Environmental Hygiene and Animal Welfare, Wrocław University of Environmental and Life Sciences, Poland
³ Institute of Inorganic Technology and Mineral Fertilizers, Wroclaw University of Technology, Poland
⁴ Poltegor-Institute, Institute of Opencast Mining, Wroclaw, Poland

ABSTRACT

Three types of feed phosphates: monocalcium (MCP), dicalcium (DCP), and calcium-sodium (CSP), were used in Lohmann Brown laying hens feeding. The content of macro- (Ca, P, Mg, Na) and microelements (Cu, Fe, Zn) in blood serum of birds was determined at a peak and in final phase of laying period. Dicalcium phosphates (DCP) significantly increased mean content of phosphorus when compared to CSP, calcium in relation to MCP, and iron when compared to both phosphates (MCP and CSP). Monocalcium phosphates (MCP) in turn, significantly increased concentration of copper when compared to DCP. Generally, more macroelements (except sodium) and microelements were observed in blood hens in the final phase of laying period, when compared to the first phase of laying period (peak).

Key words: hen, phosphate, blood, mineral profile.

INTRODUCTION

The variation of mineral concentration in blood of laying poultry is an important issue due to intense skeleton development (young birds) or egg formation. While an eggshell contains mainly calcium compounds, eggs content is also rich in large amounts of micro- and macroelements, including phosphoric compounds [27, 35]. Mineral substances play a significant role in a development of immunity, health status and laying rate, influence eggs quality, mainly eggshell strength [14, 33]. Thus, the concentration of main chemical elements in blood serum is a good index of mineral supplementation in poultry feeding. The elements that have been analyzed quiet often are Ca, P, Mg, K, Na, Cu, Mn, Fe, Se and Zn. Mineral premixes, pasture chalk and feed phosphates are the main source of these elements. Some of the elements are significant in laying hens feeding, due to large requirements of birds on calcium and phosphorus, which may be supplied in a specified ratio and chemical form.

Mono-, di-, tricalcium, calcium-sodium, calcium-sodium-magnesium and ammonium phosphates are available on feed market. Apparent digestibility of phosphorus in these phosphates is very often differentiated from 65 to 90% and depends mainly on production technology applied and raw material quality [6, 11, 29]. Currently, deficiency of phosphates are observed on feed market, and they are mainly imported from non-European countries, and often their chemical composition and suitability for poultry feeding is not fully understood. There is lack of the studies concerning an influence of various feed phosphates on biochemical indices of poultry blood, including mineral composition of blood serum, and the reference values for mineral compounds in laying hens blood have still not been established.

The aim of the study was to determine an influence of three different feed phosphates on macroelements and microelements content in blood serum of laying hens.

MATERIAL AND METHODS

Animals and feeding
The experiment was conducted with the use of 60 laying hens (Lohmann Brown) divided onto three groups, 20 birds in each (5 birds in one cage). Hens were housed in a three-tier battery system with full technological equipment (MBD Company) in a vivarium of Department of Environment Hygiene and Animal Welfare, Wroclaw University of Environmental and Life Sciences.

The standard diet containing 17.1% of crude protein, 2758 kcal/kg metabolic energy, 3.41% of crude fat, 2.72% of crude fibre, 0.35% of NaCl, 3.52% of Ca, 0.55% of total phosphorus and 0.34% of available phosphorus was used during the feeding investigation which lasted 50 weeks. The detailed recipe and chemical composition of the standard diet was presented in a separate publication [17]. The three types of phosphates were used:

• MCP - monocalcium phosphate Ca (H₂PO₄)₂ - group A
• DCP - dicalcium phosphates Ca HPO₄ x2H₂O - group B
• CSP - calcium - sodium phosphates Na₂Ca₅ (PO₄) - group C

The phosphates from group A (MCP) and C (CSP) were imported products (from Finland and Russia) generally available on the market, while DCP (group B) is a new phosphate, elaborated on the basis of phosphoric acid [13]. The technology was made within the framework of the research and development project No. R-0504203 and was applied for patent protection (No P-369805). Physico-chemical analysis of phosphates with respect to crude phosphorus, solubility of P in citric acid, content of Ca, Na, Mg and Cu, Fe, Zn were conducted in the Laboratory of Institute of Inorganic Technology and Mineral Fertilizers of Wroclaw University of Technology, according to standards methods [1].

Percentage contribution of phosphates, as well as pasture chalk, was in accordance with requirements of Poultry Nutrition Standards [25]. The contribution of phosphorus from phosphates was similar in all the groups (A - C). Mineral phosphorus (phosphates) was added in a form of supplementary feed mixtures [17].

Chemical blood serum analyses
In order to determine an influence of various chemical forms of phosphorus in laying hens feeding, the analyses of the content of macro- (Ca, P, Mg, Na) and microelements (Cu, Fe, Zn) in blood serum of birds were conducted. Blood for analyses was collected twice, from a wing vein (vena brachialis) from 12 hens in each group to plastic test tubes of a volume of 1.5 ml in the following periods:

• at peak of production period (laying yield about 90%) - phase I
• at the end of production period (laying yield about 55-50%) - phase II.

The analyses of mineral components were conducted in Biochemical Laboratory of the Department of Environmental Hygiene and Animal Welfare, Wroclaw University of Environmental and Life Sciences, using Petra 400 biochemical analyzer of Horiba HBX Company. The following parameters were determined in the samples: inorganic phosphorus (P) using UV method with phosphomolybdate, calcium (Ca) using photometric method with ortho-cresolphthalein complexone, magnesium (Ma) using photometric method with xylidyl blue. Iron (Fe) was analysed using photometric test with pherene. The analyses were conducted with ready to use reagent kits of HORIBA ABX Company. The content of sodium (Na), potassium (K) and chlorine (Cl) was determined using the set of ion selective electrodes against reference electrode in ISE module aimed at measurement of electrolytes concentration integrated with Pentra 400 analyser. The analyses of copper (Cu) were conducted using colorimetric test, while those of zinc (Zn) with colorimetric test with deproteinization. To determine these parameters, the reagent kits of RANDOX Company were used.

The results were worked out statistically; mean values (x) and standard deviations (SD) were calculated. Statistical significance of differences was assessed using Duncan's test (Statgraphics ver. 5.0 computer software).

RESULTS AND DISCUSSION

Chemical composition of phosphates
Chemical composition of phosphates used is presented in Table 1. The content of phosphorus was the highest in MCP, while those of calcium in CSP. Generally, these two macroelements and basal microelements were within the ranges noted by other authors [8, 24, 29]. It was demonstrated in another detailed study [13] that the phosphates analysed do not contain excessive amounts of heavy and toxic metals (As, Cd, F, Hg, Pb).

Table 1. Results of chemical analyses of three different feed phosphates (in 1 kg of d.m.)

Specification	Monocalcium phosphate	Dicalcium phosphate	Calcium - sodium phosphate
Chemical formula	(MCP) Ca (H2PO4)2	(DCP) Ca HPO4 x2H2O	(CSP) Na2Ca5 (PO4)
Crude phosphorus (P) content, g Solubility of P in 2% citric acid , % Content of:     Calcium (Ca), g     Sodium (Na), g     Magnesium (Mg), mass %     Iron (Fe), mg     Zinc (Zn), mg     Copper (Cu), mg	227 99   177 7.6 0.33 825 27 22	185 98   250 4,7 0.46 905 14 10	180 98   310 49,0 0.31 2680 36 26

Mineral profile of serum blood
The content of macroelements in hen's blood serum is presented in Table 2 and on Figures 1-4.

Table 2. The content of macroelements in hen's blood serum in the first (I) and second (II) phase of production (mmol/l)

Group	Phosphate	Production phase	P	Ca	Mg	Na
A	MCP	I	1.84a	7.14	1.45	145.77
		II	1.76b	7.24b	1.42	146.55
B	DCP	I	1.72	6.37	1.38	146.17
		II	2.16a	9.13a	1.65	147.04
C	CSP	I	1.39a	6.58	1.40	145.39
		II	1.75b	8.13	1.58	146.20

a-b p< 0.05 (between the groups at the same production phase)

Figure 1. Mean concentration of phosphorus (P) in hen's blood serum (mmol/l)

Figure 2. Mean concentration of calcium (Ca) in hen's blood serum (mmol/l)

Figure 3. Mean concentration of magnesium (Mg) in hen's blood serum (mmol/l)

Figure 4. Mean concentration of sodium (Na) in hen's blood serum (mmol/l)

Phosphorus. The concentration of inorganic phosphorus (P) in blood serum at phase I of laying period was in the range of 1.39 mmol/l (group C) - 1.84 mmol/l (group A), and the differences between these groups were significant statistically. At phase II of laying period in turn, the value of that parameter in blood serum of laying hens was in the range of 1.75 mmol/l (group C) - 2.16 mmol/l (group B), and statistically significant differences were observed between group B, and groups A and C. Mean contents of phosphorus (together at phase I and II of laying period) differed slightly between the groups (Fig. 1), and significant differences were noted between group B and C. Generally, phosphorus from dicalcium phosphate (DCP) was available in a better manner, thus its amount in hens blood was higher.

The range of P concentration value in blood serum of laying hens noted during the experiment corresponds to values of this parameter given in the literature. For example, in hens in a period of 18-25 week of life it amounts to 1.2-3.1 mmol/l [26]. Suchý et al. [32] demonstrated the content of phosphorus in blood serum of laying hens in the range of 1.25-1.90 mmol/l. In hens of Warren line at 18th week of life, the mean value of that parameter was 3.2 mg/dl (1.03 mmol/l), while in Golden-Commet laying hens (Hubbard) it was 7.5 mg/dl (2.26 mmol/l) [3]. In turkeys and female broiler chickens at 5th week of life these values were 6.63 mg/dl (2.14 mmol/l) and 4.42 mg/dl (1.43 mmol/l), respectively [18, 19]. Similarly to others gallinaceous poultry mean serum P concentration of 36 weeks of age Japanese quails was on the level of 6.9 mg/dl (2.23 mmol/l) [22].

Calcium. Concentration of calcium (Ca) in blood serum at phase I of laying period was in the range of 6.37 mmol/l (group B) - 7.14 mmol/l (group A). No statistically significant differences between the groups were demonstrated. At phase II of laying period in turn, the value of that parameter in blood serum of laying hens was in the range of 7.24 mmol/l (group A) - 9.13 mmol/l (group B), and differences between the groups were significant statistically (p < 0.05). Mean concentration of that element (phase I and II of laying period) between the groups were however different (Fig. 2), but statistically significant differences were only noted between group B and C. Generally, calcium from dicalcium phosphate (DCP) was available in a better manner, thus its amount in blood was higher.

The range of Ca concentration value in blood serum of laying hens noted during the experiment corresponds to values of this parameter given in the literature. For example, Cerolini et al. [3], observed the calcium content on the level of 12.01-33.18 mg/dl (3.00-8.28 mmol/l) in laying hens aged 18-67 weeks. Kurtoglu et al. [23] observed relatively low range of that element, i.e. 9.9-13.76 mg/dl (2.48-3.44 mmol/l) in Brown Nick hens. Mean serum Ca concentration of 36 weeks of age Japanese quails was on the level of 16.2 mg/dl (4.05 mmol/l) [22]. In turn, in turkeys and female broiler chickens at 5th week of life these values were on average 9.88 mg/dl (2.46 mmol/l) and 6.09 mg/dl (1.52 mmol/l), respectively [18,19].

Magnesium. Concentration of magnesium (Mg) in blood serum in both phases of laying period was on a stable level irrespectively on the kind of phosphate used and in was from 1.38 to 1.65 mmol/l. Mean concentration of that element (phase I and II of laying period) were however different between the groups (Fig. 3), but these differences were not significant statistically. Thus, the phosphates applied did not influence the level of such important macroelement in blood serum of birds.

The range of concentrations obtained is similar to values given in the literature. For example, Capcarova et al. [2] give mean concentration of Mg in blood serum of Isa Brown laying hens on an average level of 1.74 mmol/l. In 60 weeks old Hyline Brown laying hens the level of that element in blood serum was 3.26 mEq/l (1.63 mmol/l) [37]. In turn, the concentration of that element in serum of 42 weeks of age broilers was on the level of 0.71 mmol/l [21].

Sodium. Concentration of sodium (Na) in blood serum in both phases of laying period was, like in the case of magnesium, on a stable level irrespectively on the kind of phosphate used. Mean values of sodium concentration noted during laying period were 145.77-147.04 mmol/l. When comparing mean concentrations of that element (phase I and II of laying period together) between the groups, almost no differences were noted (Fig. 4). Thus, the phosphates applied did not influence the level of this such important macroelement in blood serum of birds.

The data available in the worldwide literature point that Na concentration in blood serum of birds is quite stable. Garalevičienė [9] observed the values of that parameter in the range of 118.6 -150.7 mmol/l in Hisex Brown laying hens between 45 and 51 week of life. In turn, the study conducted on Ross Brown laying hens demonstrated the content of sodium in serum on an average level of 153 mmol/l [30]. Gezen et al. [10] in turn, in Lohmann Brown laying hens aged 47-56 weeks, observed sodium concentration in blood serum in the range of 149.54-158.77 mmol/l. The concentrations of that element was within the range of 136.4-150.1 mmol/l for Hybro-PG broilers, between 21 and 35 days of age [31].

The content of microelements in hen's blood is presented in Table 3 and on Figure 5.

Table 3. The content of microelements in hen's blood serum in the first (I) and second (II) phase of production (µmol/l)

Group	Phosphate	Production phase	Zn	Fe	Cu
A	MCP	I	40.64	46.97	2.96a
		II	38.36b	37.76	3.90
B	DCP	I	37.34	44.33	2.24
		II	52.57a	57.08	3.02
C	CSP	I	39.18	40.34	1.78b
		II	44.30	44.19	3.45

a-b p< 0.05 (between the groups at the same production phase)

Figure 5. Mean concentration of zinc (Zn), iron (Fe) and copper (Cu) in hen's blood serum (µmol/l)

Zinc. Concentration of zinc (Zn) in blood serum at phase I of laying period was in the range of 37.34 mmol/l - 40.64 μmol/l. At phase II of laying period in turn, the values of that parameter were slightly different, on a higher level of 37.76 μmol/l (group A) - 57.08 μmol/l (group B), and the differences observed were significant statistically. When comparing mean concentrations of that element (phase I and II of laying period together), no significant differences between the groups were noted. Thus, the phosphates applied did generally not influence the level of such important element in blood serum of birds.

The results obtained in the experiment are similar to those presented in literature data. Pavlík et al. [26] noted in the case of laying hens between 22 and 75 week of life, concentration of Zn in blood serum in the range of 52-80 μmol/l. The authors of that study noted an increase in Zn concentration in blood serum of laying hens from 22 to 47 week of life, and next a decrease up to 75 week of life. Suchý et al. [32] in turn, noted a lack of relationship of this element content in blood on the phase of laying period and laying yield level. In turn, Truchliński et al. [34] in female turkeys between 9 and 16 week of life, noted zinc content on the level of 1.38-2.02 mg/l (21.67-31.71 μmol/l).

Iron. Concentration of iron (Fe) in blood serum at phase I of laying period was in the range of 40.34-46.97 μmol/l. In turn, at second phase of laying period, the values of that parameter were slightly different, in the range of 37.76 μmol/l (group A) - 57.08 μmol/l (group B), and differences observed were significant statistically. When comparing mean concentrations of that element (phase I and II of laying period together) significant differences were noted between group B and the other ones. Thus, dicalcium phosphate (DCP) would have influenced an increase in Fe concentration in hen's blood; however the mechanism of that phenomenon is difficult to explain.

The range of Fe concentration in blood serum of laying hens noted during the experiment is generally lower when compared to those given in the literature. For example, Planas [28] gives iron concentration in serum of laying hens on a level of 466 μg/100 ml (83.46 μmol/l), and for turkey females 127-701 μg/dl (22.74-125.55 μmol/l). In turn, Króliczewska et al. [20], for 40 weeks old laying hens of ISA SHAVER line, determined average value on a level of 110.8 μmol/l, while in broilers (21-42 days of age), the values of this parameter were within the range of 18.63-24.8 μmol/l [21].

Copper. The concentration of copper (Cu) in blood serum at phase I of laying period was in the range of 1.78 μmol/l (group C) - 2.96 μmol/l (group A), and the differences between these groups were significant statistically. At the second phase of laying period in turn, the value of that parameter in blood serum of laying hens was on a stable level of 3.02-3.90 mg/l. When comparing mean concentrations of that element (phase I and II of laying period together), significant differences were noted between group A and B. Thus, monocalcium phosphate (MCP) would have influenced an increase in Cu concentration in hen's blood; however the concentration of that element was similar in a diet of all the birds.

The obtained values of Cu concentration in blood serum of laying hens are lower when compared to those given in the literature. Planas [28] gives the content of that element in blood serum of hens on an average level of 85 μg/100 ml (13.34 μmol/l). Similar values are given by Pavlík et al. [26] for laying hens housed in three different maintenance systems. The authors of that study noted an increase in copper concentration from 22 to 47 week of life, and next its decrease. Also Filizciler et al. [7] noted an increasing copper level in blood serum of hens from the beginning of laying period (3.56-4.86 μmol/l). Truchliński et al. [34] in female turkeys, between 9 and 16 weeks of age, observed copper content in the range of 1.26-1.52 mg/l (1.60-1.93 mmol/l).

The results of the investigation are difficult to compare, since no papers concerning the similar subject were found in the available literature. However, an influence of different environmental factors and feed additives on mineral profile of hen's blood is quite well documented. Pelicia et al. [27] observed increased Ca concentration in blood of semi-heavy layers while increased level of this element was supplemented into a diet. Such correlation was not determined in case of dietary P. An addition of microbiological phytase has a main influence on phosphorus concentration in poultry blood [5]. However, other authors did not obtain any clear relationship in that issue [16]. The type of calcium source and its level in hen's diet significantly influence the content of that macroelement basic for hens [15]. It was demonstrated, that humic chalk (raw material associated with brown coal deposits) significantly influences Ca and P concentration in laying hens blood serum when compared to the control group (pasture chalk) [33]. Magnesium level in turn (and Ca) in laying hens blood serum depends on Mg supply in feed, and these values decrease in a period of an intense shell formation (peak of laying period) [36]. Another authors [23] demonstrated an influence of sodium and potassium compounds on Mg concentration in hen's blood serum. Sodium concentration is stable in blood, and possible fluctuations of that important electrolyte concentration are connected to its supply with feed mixture. Gyenis et al. [12] in turn, observed a tendency of Na increase with an increase of body dry matter content. Concentration of microelements in hen's blood is quite changeable, and depends mainly on feed mixture quality, and chemical form of additives. Kurtoglu et al. [23] using sodium bicarbonate, potassium chloride and sodium chloride supplementation demonstrated their significant influence on the concentration of inorganic P, Cl, Mg, Na and K in blood serum. No influence on Ca level was noted. In turn, Dobrzański et al. [4] did not observe an influence of organic forms of Mn and Fe on the concentration in blood of hens, while such an influence was noted in the case of Cu with quite stable Zn level. An increase in Cu concentration in poultry blood is observed when EDTA and microbiological phytase is added to feed of low phosphorus availability [5].

It was stated, that phosphates application had some influence on the content of macro- and microelements in blood of laying hens. Dicalcium phosphates (DCP) significantly increased mean content of phosphorus when compared to CSP, calcium in relation to MCP, iron when compared to both phosphates. In turn, Monocalcium phosphates (MCP) significantly increased concentration of copper when compared to DCP. Generally, more macroelements (except sodium) and microelements were observed in hen's blood on the final period, when compared to the first phase of laying period.

Acknowledgements

The present study was conducted within the framework of the statutory research No Z4/S/2011 (Fac. Biol. Anim. Sci. – WUELS, Wrocław) and No S10095/I-26/W3 (Fac. Chem. – WUT, Wrocław).

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Accepted for print: 21.12.2011

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Zbigniew Dobrzański
Department of Environment Hygiene and Animal Welfare, The Faculty of Biology and Animal Science, Wrocław University of Environmental and Life Sciences, Poland
J. Chełmońskiego 38 C
51-630 Wrocław
Poland
Phone: +48 71 320 5865
email: zbigniew.dobrzanski@up.wroc.pl

Sebastian Opaliński
Department of Environmental Hygiene and Animal Welfare,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 38 C, 51-630 Wrocław, Poland
Phone: (+48 71) 32 05 865

Krystyna Hoffmann
Institute of Inorganic Technology and Mineral Fertilizers, Wroclaw University of Technology, Poland
Smoluchowskiego 25, 50-372 Wrocław, Poland
email: krystyna.hoffmann@pwr.wroc.pl

Fabiola Bubel
Poltegor-Institute, Institute of Opencast Mining, Wroclaw, Poland
Parkowa 25,
51-616 Wroclaw
email: fabiola.bubel@igo.wroc.pl

Krystyna Pogoda-Sewerniak
Department of Environmental Hygiene and Animal Welfare,
Wrocław University of Environmental and Life Sciences, Poland
Chełmonskiego 38 C, 51-630 Wrocław, Poland
Phone: (+48 71) 348 41 42

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Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.

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