MECHANICAL, CHEMICAL AND SPECTROSCOPIC ANALYSIS OF MINERALISATION RATE AS METHODS OF BONES QUALITY DETERMINATION IN BROILER CHICKENS

Dorota Jamroz, Tomasz Wertelecki, Roman Żyłka, Rafał Bodarski, Agnieszka Gajda-Janiak

ABSTRACT

Different method for bone quality assay: bone strength and elasticity, fourier transform infrared spectroscopy for estimation of mineral/organic matrix ratio and chemical composition of ash in bones of chickens, were used. Values of estimated indices are in clearly relation to kind of bones. In young chickens, the strength and elasticity parameters were significant lower in femur as in tibia. Presented results of mineral/organic matrix ratio and Ca-content in ash of bones stand in negative relation to elasticity (r² = -0.72; -0.69) and in young broiler chickens measurements of mechanical parameters are for bone quality a informative characteristic.

Key words: bone, quality, strength, elasticity.

INTRODUCTION

The increased frequency of leg disorders in the intensive rearing of chickens is the one of essential problems in broiler production [1,2,6,7,12,18,19]. The locomotion ability of chickens depends not only from good functioning muscles but also from mechanical and chemical properties of bones, especially from mineral/organic matrix ratio. This remains mainly in relation to calcium, but to phosphorus status of animals organism, too. In numerous studies was demonstrated, that the effectivity of phosphorus metabolism can be measured through phosphorus retention or bone quality parameters [8,9,10,11,14,15,17]. The source of phosphorus in feed mixtures and supplementation with microbial phytase of diets rich in domestical grains and other plant compounds containing phytate-phosphorus influenced the P-utilization and bone quality [3,5,11,13,15,20,21].

The objective of the presented investigations was to determine of bone quality of broiler chickens fed mixtures contained two kinds of phosphorus sources: mono- (MCP) or dicalcium phosphates (DCP) composed with grains: wheat, barley and maize. The mechanical, chemical and infrared spectroscopy methods of determination of bone quality parameters, ratio between organic matrix and mineral ingredients in bones, and Ca, P, Mg retention in chickens were estimated.

MATERIALS AND METHODS

Animals and environment

The experiment was performed on 100 Hubbard HI-Ye broiler chickens (♂) with average initial body weight 42.5 g, kept in battery cages. Day-old chickens were randomly divided into two groups, each comprised of 12 replicates (cages) with 4 or 5 birds. The temperature in the room was initially 29-31°C and was gradually reduced to 21°C. The lighting program was 24 hours light for the first 10 days, later 19 hours light and 5 hours darkness. The birds had free access to drinking water (nyple system).

Chickens were fed mixtures in mash form: starter in period 1-14 days and grower in days 15-32 with average protein content 219/203 g ˇ kg^-1 and energy (ME) density 12.5 and 13.1 MJ kg^-1 (2 987.5 kcal and 3 130.9 kcalˇkg^-1) respectively (Table 1). Mixtures were diversified in content of two phosphates: group I (monocalcium phosphate MCP) or group II (dicalcium phosphate DCP). Diets were fed ad libitum.

In experiment chickens were weighed in days 1, 7, 14, 21, 28 and 32; daily feed intake was in period of balance trial registered. In days 28-32 were calcium, phosphorus and magnesium balances performed. The collection period was five days, in them the both feed intake and quantity of excrements were recorded.

At day 32^nd the 20 chickens per each group were killed and from their both legs the muscles were removed, bones were cleaned, and stored for estimation of bone quality parameters (20 left legs). In bones from the each right leg of 10 chickens the chemical composition (content of ash, Ca, P, Mg) was estimated.

Analytical methods

The chemical composition of compounds of diets and excrements were determined according to standard methods (AOAC, 1990). Phosphorus content in diets and bones was analysed after previous mineralization by the ammonium vanadomolybdate method using a Specol 11 Carl Zeiss Jena spectrophotometer at a wave length of 470 nm. Calcium and magnesium in diets, bones and excrements were determined by atomic absorption spectrophotometry using ASS-3 EA-30 type apparatus Carl Zeiss Jena.

Bone strength parameters - mechanical properties

Two different analytical estimations of mechanical bone properties were used: perpendicular to the bone axis (estimation A) and parallel to the bone length axis (estimation B). The measurements were done on fresh bones that had been stored in a refrigerator at a temperature of 4-5°C. Mechanical performed measurements were taken in the Institute of Agricultural Engineering of Agricultural University, Wrocław on INSTRON instrument [11].

In the first method (A), bone deflection h was measured by a standard method, in which the force F was applied to the shaft of the bone supported at both epiphyses at 25 mm distance (Fig.1). Force F was variable up to the bone breaking point. Similarly in the second method, the elasticity coefficient α_n= F/h was calculated. After mechanical test, the bones were cut in the breaking place and the cut surface were scanned. Then the cut surface area and the surface moment of inertia were measured. On the basis of the data obtained, the Young's Modulus was calculated from the equation:

E_n = α_n · L³/ω

where:
L - supported distance (25 mm),
ω - cut surface moment of inertia.

On the basis of the data obtained at the mechanical test were also calculated: maximal loading force F_n (strength at breaking point), maximal bone deflection h_n (deflection at breaking point), maximal percent bone deflection U_n = h_n / L and the energy at the breaking point W_n = ∫Fdh (from zero deflection to breaking point).

In the second method (B), 8 mm length of the bone central shaft was taken. A force F variable up on to the value of l kN was put perpendicular to the cut surface (Fig.1), and length deformation x (=h) was measured. Hook's law concerning elasticity, where the deformation vector is proportional to the force applied, the elasticity coefficient α₁ = F/x was calculated. After mechanical test the cut surface was scanned and the cut surface area measured using an own program. On the basis of the data obtained, the Young's Modulus was calculated from the equation:

E₁ = α₁ · l/S

where:
l - length of cut (8 mm),
S - cut surface area.

Fourier transform infrared spectroscopy (FTIR)

For FTIR, fresh bones dissected from the right legs of 10 chickens per group were defatted by standard method dried for 24 h in 50°C and then pulverized to fine powder in room temperature. Next, fine powder was lyophilised in vacuum for 24 h. and mixed with salt KBr to total weight of sample 3 mg (3-5% of bones powder) and pressed to tablet form for spectrometer analyses. The infrared spectra were recorded in the FTIR Mattson IR 300 spectrometer, according to FTIR standards, where peak positions near 1 030 cm^-1 and near 1 650 cm^-1 were assigned to the phosphate stretching vibration of links P-O in PO₄ (hydroxyapatite - mineral matrix) and the amide 1 (Amide 1 – collagen specimen) stretching vibration of the bone organic matrix [4, 16].

The mineral/organic matrix ratio was calculated from the ratio of absorbency (in the vibration intensity units) of the phosphate links at 1 030 cm^-1 to the amide 1 links at 1 650 cm^-1 (Fig. 2).

Experimental data were evaluated statistically by one- or two factorial analysis of variance using SASŽ procedures. Differences between treatment means were tested according to Duncan's multiple range test (Duncan, 1955).

RESULTS AND DISCUSSION

The growth and development of chicken was in period of 21^st day of life in both group similar (Table 2).

The calcium retention in chickens (Table 3) fed diet contained MCP was higher (75.3%) in comparison with DCP-groups, was only 65% of consumed Ca (p<0.01). High P-retention (54%) was stated in DCP-group and in MCP-group was lower, about 42.7% (p<0.01). Similar, higher Mg utilization was determined in chickens from DCP- group (40.3%) however significant lower (35%) was the magnesium retention in MCP group (p<0.05).

The parameters characterized the leg bones strength and elasticity were similar in chickens from both groups (Table 4). The strength of tibia in chickens from MCP-groups was 215.6 N, for DCP - 211.1 N; the elasticity 2.29 and 2.38 x 10⁵ Nm^-1, respectively. Differences between groups in Young Modulus (Nm^-2) were small, only the deflection of tibia between chickens from both groups was significantly different.

The differences in main bone quality parameters, totally lower in femur characteristic, were not similar in groups as for tibia. The greatest differences, however not significant, were observed in femur elasticity (1.37 for MCP and 1.53 x 10⁵ Nm^-1 for DCP). Significant better Young’s Modulus (p<0.05) parameters were estimated in bones from DCP – chickens (Table 4).

The Young Modulus measured at the bone length (Estimation B) was influenced from kind of used phosphate and was lower for femur as for tibia bones (p<0.05) and significant higher in DCP – group (p<0.05). The mineral/organic matrix ratio was significant (p<0.05) lower only in chickens fed diets with DCP (Table 4).

Two-factor analyse of variance of mainly physical parameters of bones confirm the significant (p<0.05 and p<0.01) differences between tibia and femur quality. All parameters: strength, elasticity, break energy, Young Modulus were significantly (p<0.01) lower in femur. Also better quality of bones was stated in chickens fed with MCP in the diet (Table 4a).

The kind of used phosphates remained without evidently influence on chemical composition of crude ash and content of organic matter in bones (Table 5). Only very small differences in Ca, P, Mg level in ash of tibia or femur were stated.

The mineral/organic matrix ratio of femur bone has corresponded with mechanical parameters of bones like strength, deflection and energy break (estimation A). The higher mineral/organic matrix ratio pointed on higher strength of break and on the other side lower elasticity of bone.

Calculated correlations coefficients for some important indices shown, that Ca-content in bones is negatively correlated to elasticity of bones (p<0.05) and the kind of used phosphates has not clearly changed the correlations coefficients (Table 6).

In present studies the different method for bone quality assay were used. The balance of macro elements in organism of chickens, bone strength and elasticity parameters as a picture of mineralisation processes in bones, fourier transform infrared spectroscopy for estimation of mineral/organic matrix ratio and chemical composition of ash in bones were determined.

The response of young chickens on different phosphate kinds in diets was visible only in some estimated parameters, only. Better retention of phosphorus and magnesium was stated by using of DCP in diets and this results are in contrary with observations of another authors [3,17]. However the better calcium retention was observed in chickens fed diets contained MCP supplement and this result was in agreement with other publications [3,17].Values of estimated indices are in clearly relation to kind of bones. In young chickens, the strength and elasticity parameters were significant lower in femur as in tibia. Contrary to this results in layer hens strength parameters of tibia are significant lower as of femur [11] and the breaking incidences of tibia are in layer keeping an important problem. And the kind of phosphates – sources of Ca and P play a significant role in mechanical properties of bones. Non await stabile was the chemical composition of bone ash, without influence of calcium-phosph ate kind. Presented results of mineral/organic matrix ratio and Ca-content in ash of bones stand in negative relation to elasticity (r² = -0.72; -0.69). However in layers the bone ash composition is a good information of bone quality, in young broiler chickens measurements of mechanical parameters are for broiler bone quality more an informative characteristic.

CONCLUSIONS

The mineral/organic ratio and Ca-content in the ash of bones stand in negative relation to elasticity of bones. Those parameters may be a informative characteristic of bone quality.

Research project number GW105/2002 Agricultural University of Wrocław, Poland

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Roman Żyłka
Department of Biophysics
Agricultural University of Wrocław
Norwida 25/27, 50-375 Wrocław, Poland

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