Medycyna Weterynaryjna - Summary Med. Weter. 69 (8), 492-498, 2013

Praca oryginalna Original paper

Abbreviations used in the work: A – cross-sectional aready, BDI – bone density index, dy – yielding deformation, bl – bone length, E – Young’s modulus, GWK – thickness of cortical layer, Ix – second moment of inertia of the cross section in relation to the horizontal axis, L – spacing of supports in bending test, bw – bone weight; MRWT – mean relative wall thickness, PK – cortical surface, Wc – yielding load, Wf – maximum force moment, Wf/A – bending point resistance, Wf/mc – maximum force moment towards body weight, Wf/mk – maximum force moment towards bone weight, WK – cortical index, WPK – cortical surface index, Wy – yielding load, Wy/dy – load-to-deformation ratio, Wy/mc – yielding load towards body weight,Wy/mk – yielding load towards bone weight.

Microelements play a significant role in the correct development of bones, particularly in fast growing chickens; therefore, the assimilability of such elements can affect the growth and strength of bones (8). A bone is a storage site for growth factors necessary for its growth, repair, and maintenance. The growth factors, known as insulin-like growth factors (IGFs), trans-forming growth factor beta (TGFâ), fibroblast growth factors (FGFs), and platelet-derived growth factor (PDGFs), act synergistically to induce new bone formation (17).

Effect of copper glycine chelate

on the quality of the tibia bones in broiler chickens

MA£GORZATA KWIECIEÑ

Institute of Animal Nutrition and Bromatology, Faculty of Biology and Animal Breeding, University of Life Sciences, Akademicka 13, 20-950 Lublin, Poland

Kwiecieñ M.

Effect of copper glycine chelate on the quality of the tibia bones in broiler chickens Summary

The analyses aimed at determining the effect of administering copper in the form of glycine chelates on physical, chemical, morphometric and strength parameters of the tibia bone in broiler chickens.

300 one-day-old Ross 308 chicks were divided into 6 groups each in 5 repetitions of 10 chicks. The feed mixtures were supplemented with Cu in inorganic form (sulphate) and organic form (glycinate chelate), covering 100%, 50% or 25% of the total requirement of the component recommended for Ross 308 broiler chicks. The basic feed mixtures contained: starter 6.1 mg·kg–1 _{Cu, grower 6.21 mg·kg}–1 _{Cu and finisher 5.91}

mg·kg–1 _{Cu. After the chickens were sacrificed, the tibiae bone were isolated, weighed, measured and frozen}

for further mechanical analysis. An Instron Universal Testing Machine (Model 3369) was used to determine bone ultimate strength and maximum elastic strength. The geometric properties bones (cross-section area, second moment of interia, mean relative wall thickness) and cortical indexes (cortical layer, cortical index, cortical surface, cortical surface index) of were estimated on the basis of measuring the external and internal horizontal and vertical axes in the cross section of the bones at the site of fracture. Degreased and dried to constant weight was bone mineralization in a muffle furnace and mineral content was determined.

On the 42nd _{day higher, statistically (P £ 0.01), confirmed bone weight per 100 g of body weight was}

recorded in chickens administered a supplement of Cu-Gly. amounting to 100%, 50% and 25%, as well as S-Cu amounting to 50% and 25% of the demand, compared with chickens given the recommended dose of Cu in the form of its sulphate. The levels of both organic and inorganic Cu compounds had a significant effect on the geometrical parameters of the bones, i.e. Ix and A. The lowest values of the analyzed properties were observed with the use of Cu-S amounting to 100%. The highest values of dy and Wf/A were recorded in the group of chickens receiving an addition of Cu in its organic form, covering the demand in 50%. Supplementa-tion with Cu in its organic form resulted also in a significantly higher index of bone density. Lowering the amount of Cu to 25% of the demand in the form of a chelate resulted in a significant (P £ 0.05) increase in Fe concentration, in reference to the recommended doses of Cu-Gly. and Cu-S at the level of 2 mg·kg–1_,

respectively, by 14% and 11%.

The results of the research permitted us to claim that Cu-Gly. may be used as an alternative for sulphates. Administering 4 or even 2 mg·kg–1 _{in the form of glycine compounds did not lead to lower bone quality, and}

it even increased the value of certain parameters.

The use of adequate mineral components in animal feed manufacturing is a vital issue regarding the pro-per development and growth rate in broiler chickens. Changing nutritional demand in growing chickens accompanied by our pursuit of reduction in environ-mental pollution resulting from administering higher portions of the components require constant updating of the birds’ nutritional requirements (1). The high rate of chickens’ growth and their welfare, as well as improved nutritional value and dietary qualities of poultry products, are the main indicators for modifying the demand for nutritional components. It is possible to achieve the required aims due to balancing the diet more accurately and improving the access to their components.

In recent years studies have been performed on the utility of chelates of selected minerals and amino acids, including copper (6, 27). Copper deficiency leads to impaired durability and strength of bone collagen. The incorrect arrangement of polypeptide chains of collagen in the bone system results in impaired bone density and development disorders, fractures and numerous deformations. Femur fractures are often related to a decrease in the amount of Cu in serum and bones, which may result from a reduced level of Cu in the blood. The activity of copper on the concentration of calcium in the blood may occur in the liver through inhibition of 25-hydroxylase, which catalyses the for-mation of hydroxycholecalciferol responsible for Ca absorption in the upper section of the small intestine, on the manner of active transportation. 1.25-(OH)₂D₃ models the synthesis of the protein responsible for binding Ca2+ _{ions in the cells of the mucous membrane}

in the intestine. Reduced concentration of Ca may also be related to the dynamics of the development of the skeletal system in growing birds, which requires high absorption of calcium from the blood, and copper ions favor the process of bone mineralization. The adequate consumption of Cu is important for acquiring the optimal bone weight of the long bones (24), while balanced consumption of Zn and Cu is significant for the development of bone mass in the trabecular bone (23). Moreover, as revealed in the studies by Abdallah et al. (1), administering chicks with the organic form of minerals, including copper, increases their weight and the content of crude ash in their tibia bones. It has been proved that mineral components from organic compounds (chelates) are absorbed much better than from mineral compounds (3), making it possible to administer a lower amount of a mineral compound, which additionally reduces the cost of chicken breeding. Instead of methionine or lysine, new-generation chelates contain glycine, an amino acid of lower molecular mass. Since in the near future such chelates may become introduced on a mass scale into feed mixtures for broiler chickens it is necessary to analyze the process of bone tissue mineralization, including limb bones,

which is an important indicator of performance and the quality of nutritional procedures, as well as a measure of the organism’s health status (7, 22). Although better availability of the elements from the chelate of a metal ion with an amino acid, mainly lysine and methionine, has been sufficiently confirmed, the amino acid chelates of the new generation used in the present studies have been the subject of few studies so far (9, 10, 14, 16), especially in the context of their effect on the quality of chickens’ bones; hence, it was justified to deal with this issue.

The aim of the present study was to determine the effect of administering copper in the form of glycine chelates on physical, chemical, morphometric and strength parameters of the tibia bone in broiler chickens.

Material and methods

Animals and diets. All procedures used during the research were approved by the Local Ethics Committee for Animal Testing at the University of Life Sciences in Lublin. 300 one-day-old Ross 308 sexed male chicks were divided into 6 groups of 10 chicks each in 5 repetitions. The respective birds were weighed at the beginning of the experiment, and each of them was identified with a wing tag on day 1 of rearing. Ross 308 chicks were reared in cages in a controlled temperature and humidity environment. Throughout the term of the experiment electric lighting was used to ensure lighting for 24 hours to 10 days of age, after 16 h light. In the first week the chicks were kept at a tempe-rature of 33°C. Every week the tempetempe-rature was reduced by 2°C and finally 24°C was reached.

The experimental diets were fed from one to 42 d of age, including the starter – S (1-21 d), grower – G (22-35 d) and finisher – F (36-42 d) phases. From 1 to 42 d of age, all the chickens were provided with water and feed ad libitum. The feed mixtures were supplemented with Cu in inorganic form (sulphate) and organic form (glycinate chelate), covering 100%, 50% or 25% of the total requirement of the component recommended for Ross 308 broiler chicks. The basic feed mixtures contained: starter 6.1 mg·kg–1 _Cu,

grower 6.21 mg·kg–1 _{Cu and finisher 5.91 mg·kg}–1 _Cu.

In the experiment 8, 4 or 2 mg·kg–1 _{of copper were added}

to the premix (containing no Cu) in groups I, II and III as copper sulphate (Cu-S), and in groups IV, V and VI as copper glycinate chelate (Cu-Gly.). The demand for mineral components in feed mixtures was based on the recommen-dations issued by the Ross Company for broiler chickens of the Ross 308 line, amounting to 8 mg·kg–1 _{Cu, without}

considering its content in the components of feed mix-tures. Following these recommendations, the content of Cu should be the same at each breeding stage, which was taken into account in the experiment. The composition and calculated nutritive value of basic feed mixtures are presented in Table 1.

Bone measurement. On the last day of rearing, the birds were weighed and 10 birds from each group with body weight closest to the average weight in the group were selected for dissection. Bone samples were mechanically cleared of soft tissue and their length was measured with

an electronic slide caliper (accuracy up to 0.001 mm), every time in an identical measuring position. Next, bone circumference was measured at ½ of bone length and the samples were packed into labeled foil bags and frozen (at –25°C) until analyses.

The mechanical properties of bones such as the maximum elastic (Wy) and ultimate (Wf) strengths were determined in INSTRON 3369 apparatus (Instron, Canton, MA, USA) as described previously (12, 13, 21).

In addition, Young’s modulus was calculated according to the formula:

Wc · L E =12dyðIx

(where: E – Young’s modulus; Wc – yielding load; L – spacing of supports in bending test; dy – yielding deforma-tion; Ix – second moment of interia) and bone density index was determined (5):

BDI = bw/bl

(where: BDI – bone density index; bw – bone weight; bl – bone length).

The geometric properties and cortical indexes of bones were estimated on the basis of horizontal and vertical dia-meter measurements of the mid-diaphyseal cross section of bone. The cross-section area (A), the second moment of interia (Ix), and the mean relative wall thickness (MRWT) were determined as described previously (12, 13, 26). The thickness of cortical layer (GWK), the cortical index (WK), the cortical surface (PK) and the cortical surface index (WPK) were determined as described previously (12, 13, 21).

After evaluating the strength and structural properties, the tibial were defatted, dried to constant weight and finally mineralised in a muffle furnace at 600°C (2). The content of mineral components (Ca, Mg, Cu, Fe, Zn) in bones was determined by atomic absorption spectrometry using a Unicam 939/959 apparatus, and general P (20) with a Helios á-Unicam apparatus, using molybdenum and vana-dium as the reagent (NH₄VO₃, (NH₄)₆Mo₇O₂₄·H₂O, H₂O).

Statistical analysis. The results obtained were statisti-cally analyzed (SEM, SD and effects of diet) by ANOVA and Duncan’s one-way range test, using Statistica v.6.0 soft-ware (25). P values < 0.05 and < 0.01 were considered significant. An asterisk indicates a statistically significant difference between the control and experimental groups.

Results and discussion

Physical parameters of bone. The bones in broiler chickens after completing the breeding period were properly developed, without any traces of fractures, cracks or other damage. The strength of the bone depends on its weight and length. Enriching the feed

) % ( s t n e i d e r g n I S(1-21d) G(22-35d) F(36-42d) e zi a M 24.441 40.001 40.001 t a e h W 42.991 27.841 28.841 % 6 4 l a e m n a e b y o S 25.001 24.971 22.871 li o n a e b y o S 2.50 3.69 3.98 e t a h p s o h p m u i c l a c o n o M 0.90 0.90 0.81 e n o t s e m i L 1.40 1.13 1.09 e t a n o b r a c i b m u i d o S 0.08 0.08 0.08 l C a N 0.29 0.25 0.26 x i m e r p l a r e n i m -n i m a ti V a_0.50a a_0.50b a_0.50c * e t a rt n e c n o c n i e t o r p -t a F 1.00 – 1.00 % 9 9 e n i n o i h t e m -L D 0.30 0.23 0.23 l C H e n i s y l-L 0.42 0.28 0.27 % 9 9 e n i n o e r h t-L 0.18 0.13 0.07 : s n i a t n o c s e r u t x i m d e e f g k 1 e_{Energy,MJ 12.70 13.10 13.20} d_{Crudeprotein,% 20.20 18.20 18.10} d_{Crude ifbre,% 13.06 12.99 12.99} d_{Crudefa,t% 14.66 16.08 16.43} e_{Lysine,% 11.29 11.13 11.09} e_{Met+Cys,% 10.93 10.83 10.81} e_{TotalCa,% 10.88 10.78 10.75} e_{TotalP,% 10.66 10.65 10.63} e_{AvaliableP,% 10.42 10.41 10.39} e_{TotalCa/avaliableP 12.12 11.90 11.92} d_Cu,mg** x x x 8mgS-Cu 14.81 14.52 13.98 x x x 4mgS-Cu 10.52 10.38 19.82 x x x 2mgS-Cu 18.51 18.47 17.89 x x x 8mgCu-Gly. 13.91 14.01 13.15 x x x 4mgCu-Gly. 10.01 10.11 19.23 x x x 2mgCu-Gly. 18.01 18.05 17.98 d_{Fe,mg 40.31 39.82 38.61} d_{Zn,mg 99.71 98.50 98.52}

Tab. 1. Ingredients of the diets

Explanations: a _{composition of S premix/kg: Mn 20 000 mg,} J 200 mg, Fe 8000 mg, Zn 20 000 mg, Se 30 mg, Cu 3200 mg, vit. A 3 000 000 j.m., vit. D3 1 000 000 j.m., vit. E 15 000 mg, vit. K3 800 mg, vit. B1 600 mg, vit. B2 1600 mg, vit. B6 1000 mg, vit. B12 3200 µg, biotin 40 mg, folic acid 400 mg, nicotic acid 12 000 mg, pantothenic acid 3600 mg, choline 360 000 mg; b _{composition of G premix/kg: Mn 20 000 mg, J 200 mg, Fe 8000} mg, Zn 20 000 mg, Se 30 mg, Cu 3200 mg, vit. A 2 400 000 j.m., vit. D3 1 000 000 j.m., vit. E 10 000 mg, vit. K3 600 mg, vit. B1 400 mg, vit. B2 1200 mg, vit. B6 800 mg, vit. B12 3200 µg, biotin 40 mg, folic acid 350 mg, nicotic acid 12 000 mg, pantothenic acid 3600 mg, choline 320 000 mg;

c _{composition of F premix/kg: Mn 20 000 mg, J 200 mg, Fe 8000} mg, Zn 20 000 mg, Se 30 mg, Cu 3200 mg, vit. A 2 400 000 j.m., vit. D3 1 000 000 j.m., vit. E 10 000 mg, vit. K3 400 mg, vit. B1

400 mg, vit. B2 1000 mg, vit. B6 600 mg, vit. B12 2200 µg, biotin 10 mg, folic acid 300 mg, nicotic acid 7000 mg, pantothenic acid 3600 mg, choline 320 000 mg;

d _{values analysed;} e _{values calculated;}

** in experiment Cu was added to the premix in an amount of 8, 4 or 2 mg·kg–1 _{in the form of Cu-sulfate or in the form of} Cu-glycine chelate in an amount of 8, 4 or 2 mg·kg–1

mixture for the chickens of the Ross 308 line with a premix containing chelated Cu significantly increased the weight and length of their tibia bones (Tab. 2).

Similarly, studies by Kwiecieñ (14) revealed the highest weight and length of the femur bone when feed mixture was supplemented with copper in its organic form at 25% of the demand. On day 42, a higher, statistically (P £ 0.01), confir-med bone weight per 100 g of body mass was recorded in chickens administered an addition of Cu-Gly. amounting to 100%, 50% and 25%, and of S-Cu at 50% and 25%, compared with chickens fed with the recommended amount of Cu in the form of a sulphate. Thus, it seems that the use of Cu supplement in the form of Cu-Gly. at lower levels is sufficient and it conditions the adequate development of chickens’ tibia bone.

The literature dealing with the issue presents few studies discussing the effect of using varied forms of Cu on the quality of bones in broiler chickens. Studies by Banks et al. (4) revealed that chickens obtaining Cu-lysine had the highest weight of their tibia bone, compared with birds fed an addition of copper citrate or sulphate. Different results were obtained by Kwiecieñ et al. (15). An addition of Cu-and-lysine bioplex resulted in reduced weight of the bones, both fresh and air-dried ones, by 14.1% and 8%, respectively.

Morphometric parameters of bone. Geometric and cortical features of the bone are factors which condition the strength of the bone tissue. The levels of organic and inorganic Cu compounds had an influence on geometrical parameters of the bone, i.e. Ix and A (Tab. 3). The lowest values of the analyzed features were noted with the use of Cu-S at the level of 100%. The value of the cortical parameters in the bone in particular groups was similar (Tab. 4), which proves that the levels of organic and inorganic sources applied did not significantly affect the analyzed bone parameters. In the studies performed by Kwiecieñ (14), replacing the inorganic form of Cu with its organic equivalents at the same amount did not have any significant effect on the morphometric parameters of the tibia bone in chickens. Thus, it may be sug-gested that administering Cu in both its organic and inorganic forms results in the adequate increase in the volume and cross-section area, without excessive increase in the mass of the tissue. On the other hand, Ferket et al. (11) observed definitely thicker areas of the cortical bone in the tibia in turkeys fed organic forms of microelements (e.g. Zn, Cu, Mn and Se). Moreover, groups with an addition of elements in the organic form revealed lower incidence of defects such as varus

Tab. 2. Physical parameters of chicken tibia bones (–x ± SD)

Explanations: A, B, C, D _{– values in columns with different denoted letters differ} significantly at P £ 0.01; NS – not significant; * – P £ 0.01; SEM – standard error of the means

l a t n e m ir e p x E s r o t c a f t h g i e w e n o B _Length ) m m ( Pe(mirmme)ter ) g ( (g/100g) S -u C 8mg 21.6A_{±1.22 0.88}A_{±0.05 112.6}B_{±1.92 29.8±1,04} S -u C 4mg 22.8AB_{±1.43 1.03}B_{±0.07 109.0}A_{±1.20 28.8±1.28} S -u C 2mg 23.1AB_{±1.08 1.00}B_{±0.06 110.4}A_{±1.30 29.3±1.49} . y l G -u C 8mg 24.3BC_{±1.40 0.98}B_{±0.04 114.6}BC_{±1.06 31.5±3.07} . y l G -u C 4mg 25.7C_{±1.46 1.05}B_{±0.11 115.0}CD_{±1.85 30.4±3.20} . y l G -u C 2mg 26.2C_{±1.48 1.05}B_{±0.05 117.0}D_{±1.69 29.9±2.36} P <0.00 <0.00 <0.000 0.227 M E S 0.303 0.013 0.454 0.331 s t e i d f o s t c e ff E * * * NS

Tab. 3. Geometric features of the tibia bone in chickens (–x ± SD)

Explanations: A, B _{– values in columns with different denoted} letters differ significantly at P £ 0.01; NS – not significant; * – P £ 0.01; SEM – standard error of the means

l a t n e m ir e p x E s r o t c a f (mImx4_{) (m}A_m2₎ MRWT S -u C 8mg 104.68A_{±23.64 23.2}A_{±1.53 0.41±0.03} S -u C 4mg 151.33B_{±29.08 25.5}AB_{±1.81 0.36±0.07} S -u C 2mg 161.89B_{±23.85 27.3}B_{±3.18 0.40±0.07} . y l G -u C 8mg 146.42B_{±18.31 26.1}AB_{±2.62 0.43±0.11} . y l G -u C 4mg 135.86B_{±14.97 23.6}AB_{±2.16 0.38±0.07} . y l G -u C 2mg 150.59B_{±22.86 27.4}B_{±3.89 0.42±0.08} P <0.000 <0.006 0.431 M E S 4.073 0.436 0.011 s t e i d f o s t c e ff E * * NS

Explanations: NS – not significant

Tab. 4. Cortical indexes of the tibia bone in chickens (–x ± SD) l a t n e m ir e p x E s r o t c a f (GmWmK) (mPmK2₎ W_(%K₎ W_(%PK₎ S -u C 8mg 2.63±0.35 38.19±4.25 7.93±0.49 74.14±8.97 S -u C 4mg 2.44±0.56 38.78±7.45 8.51±0.36 85.14±6.81 S -u C 2mg 2.50±0.46 39.38±5.72 8.46±0.34 84.00±6.70 . y l G -u C 8mg 2.63±0.35 39.81±4.65 8.23±0.53 79.66±9.91 . y l G -u C 4mg 2.25±0.53 33.88±6.38 8.01±0.34 76.13±6.41 . y l G -u C 2mg 2.75±0.53 42.31±8.53 8.37±0.64 81.98±11.23 P 0.377 0.203 0.077 0.084 M E S 0.069 0.941 0.071 1.302 s t e i d f o s t c e ff E NS NS NS NS

in the 17th _{week or staggering in the 12}th_{, 15}th _{and 17}th

weeks.

The literature of the subject does not include works discussing the influence of the forms and levels of Cu on geometrical and cortical parameters of the femur bone in broiler chickens. Hence, our analyses have both a cognitive and practical contribution.

The parameters of bone strength. The studies revealed a statistically significant influence of the level of Cu on the majority of strength parameters in the chickens’ tibia (Tab. 5). An addition of Cu-Gly. at the amount limited to 50% and 25% of the demand resulted in a significant (P £ 0.01) increase in the maximum strength of bone elasticity (Wy) in the chickens’ tibia, compared with the groups receiving an addition of Cu in the form of a sulphate at 50% and 25%. The highest value of Wf/A was recorded in the group of chickens administered an addition of Cu in the organic form, covering the demand in 50%. The addition of Cu in its organic form also led to a

signifi-cant increase in bone density. As reported by Monte-agudo et al. (19), the higher the value of the index, the bones are more dense. The reason for the increased index may be accounted for by a significant increase in the weight of the tibia. On the other hand, in the studies performed by Kwiecieñ (14), the replacement of the inorganic form of Cu with its organic equiva-lents at the same levels significantly affected (P £ 0.05) the maximum elastic force. An addition of Cu-Gly. chelate at the amount recommended or reduced to 50% of the demand resulted in a significant (P £ 0.05) increase in the Wy value of the chickens’ tibia, com-pared to the groups receiving an addition of Cu in the form of a sulphate, irrespective of the level of Cu. In other studies performed by Kwiecieñ et al. (15) Cu-and-lysine chelate reduced the absolute strength of the tibia in chickens’ by 20.05%, as compared with bone strength in the control group.

The value of the examined strength parameters of the tibia in chickens and of the physical properties of the bone material, determining its strength (i.e. Wy/mc and Wf/mk) was similar, which sug-gests lack of influence resulting from Cu levels applied (Tab. 6). On the other hand, significantly higher values of Wy/mk were noted when Cu was added to the mixtures in the form of Cu-Gly. at the level of 25% and 50% of the demand, and in case of Wf/mc when Cu was applied in its organic form at 25%. The lowest values of the studied parameters were observed when the recommended amount of Cu was added in the form of Cu-Gly. Conversely, in the studies by Kwiecieñ (14), no significant

Tab. 5. Strength parameters of the chicken tibia bone (–x ± SD)

Explanations: A, B, C _{– values in columns with different denoted letters differ significantly at P £ 0.01; NS – not significant; * – P £ 0.01;} SEM – standard error of the means

l a t n e m ir e p x E s r o t c a f N•Wmym mdmy N•Wmfm N•mWmy•/dmym–1 _N•mW_m•/fA_mm–2 _N•mE–2 _mgB_•mDI_m–1 S -u C 8mg 148.4ABC_{±2.92 1.40}A_{±0.04 236.4}AB_{±15.25 103.6±4.61 10.23}AB_{±0.67 1.56}AB_{±0.21 191.6}A_±10.54 S -u C 4mg 143.5A_{±5.66 1.40}A_{±0.05 242.0}AB_{±18.01 102.8±4.36 9.52}AB_{±0.98 1.39}AB_{±0.28 208.7}B_±11.41 S -u C 2mg 146.1AB_{±3.11 1.41}AB_{±0.04 229.5}A_{±18,06 103.4±3.39 8.48}A_{±1.07 1.21}A_{±0.19 209.1}B_±7.97 . y l G -u C 8mg 152.2BC_{±4.71 1.44}AB_{±0.07 262.3}AB_{±27.03 106.1±6.09 10.14}AB_{±1.41 1.66}B_{±0.35 211.8}B_±12.20 . y l G -u C 4mg 153.2C_{±4.98 1.48}B_{±0.02 260.2}AB_{±35.84 103.8±4.32 11.15}B_{±2.08 1.69}B_{±0.38 223.8}B_±13.00 . y l G -u C 2mg 153.6C_{±6.85 1.46}AB_{±0.06 271.9}B_{±40.34 105.4±5.56 10.08}AB_{±1.85 1.50}AB_{±0.21 224.1}B_±13.80 P <0.000 0.006 0.022 0.734 0.019 0.012 <0.000 M E S 0.866 0.008 4.361 0.677 0.228 0.045 2.254 s t e i d f o s t c e ff E * * * NS * * *

Tab. 6. Yielding load (Wy) and maximum force moment (Wf) towards body weight (1 kg) and bone weight (–x ± SD)

Explanations: a, b _{– values in columns with different denoted letters differ significantly at} P £ 0.05; A, B _{– values in columns with different denoted letters differ significantly at P £ 0.01;} NS – not significant; * – P £ 0.01; ** – P £ 0.05; SEM – standard error of the means

l a t n e m ir e p x E s r o t c a f (N•Wmym/m•kg–1_{) (N•mm}W_•1y/₀m₀₀c_•kg–1_{) (N•}W_mm/fm_•k_g–1_{) (N•mm}W_•1/f₀m₀c_0•kg–1₎ S -u C 8mg 6.74A_{±0.59 59.2±5.95 11.0±0.83 96.4}a_±7.2 S -u C 4mg 6.33AB_{±0.44 65.1±2.94 10.7±1.36 109.8}ab_±8.8 S -u C 2mg 6.34AB_{±0.27 63.4±2.26 9.9±0.69 99.5}ab_±7.2 . y l G -u C 8mg 6.29AB_{±0.38 61.6±5.61 10.8±1.05 105.8}ab_±11.1 . y l G -u C 4mg 5.97B_{±0.39 62.3±4.04 10.2±1.77 105.5}ab_±12.1 . y l G -u C 2mg 5.88B_{±0.40 61.9±4.26 10.4±1.32 108.8}b_±10.7 P 0.003 0.180 0.522 0.055 M E S 0.071 0.652 0.175 1.499 s t e i d f o s t c e ff E * NS NS **

influence of the level of Cu on the values of Wy and Wf was recorded in reference to chickens’ body weight and the weight of their tibia bones.

On the 42nd _{day of their lives, chickens in individual}

experimental groups did not reveal significant dif-ferences in the content of crude ash, Zn or Cu (Tab. 7). Although the relationship between the share of crude ash and Zn was not statistically confirmed, their higher content was observed in the groups in which the diet included the organic source of Cu. Similarly, Yan and Waldroup (28) studying chickens’ bones and Mikulski et al. (17) studying turkeys’ tibia did not observe significant differences in the concentration of ash, depending on the source of the component. On the other hand, Abdallah et al. (1) observed a signifi-cantly higher percentage of the component in the form of the peptid chelate. In the studies performed by Banks et al. (4) chickens administered Cu-lysine revealed the highest content of Cu in ash, compared with chickens fed a supplement of copper citrate or sulphate. In the studies carried out by Wang et al. (27) a significant increase in the content of Cu in bone ash was recor-ded, both at the starter and grower stage, resulting from enriching the feed with Cu sulphate or organic Cu in the form of MINTERX® _{preparation, at 500 mg·kg}–1_.

The studies suggest that a reduction in Cu sup-plementation to 25% of the demand in the form of a chelate led to a significant (P £ 0.05) increase in Fe concentration, as compared with the recommended doses of Cu-Gly. and Cu-S at the level of 2 mg·kg–1_,

respectively, by 14% and 11%. In the study by Kwie-cieñ (14), the highest share of Fe in chickens’ femur was recorded with the addition of Gly-Cu at the level of 25% of the demand. At the same time, no statisti-cally significant differences were noted in the content of crude ash, Ca, P, Mg, Zn or Cu. In our own studies the highest concentration of Ca and P in chickens’ tibia (P £ 0.05) was observed with an addition of the

recommended dose of Cu in the form of Cu-Gly., while the lowest share of Mg was noted in enriching the feed with 4 mg·kg–1 _{of Cu-S. Studies by Kwiecieñ et al.}

(15) revealed that an addition of Cu-and-lysine bio-plex did not significantly vary the content of crude ash in the tibia, although it slightly reduced the share of Ca and P and slightly increased the content of Mg. On the other hand, it did not affect the content of Cu.

Conclusions

The results of the studies performed on growing broiler chickens from the day of their hatching until the 42nd _{day of their lives and fed feed mixtures}

supplemented with Cu in its inorganic (sulphates) and organic (glycine chelates) forms at three levels (100%, 50% and 25%) permit us to claim that Cu-Gly. may be used as an alternative for sulphates. The administra-tion of 4 or even 2 mg·kg–1 _{of Cu in the form of glycine}

compounds did not result in worse bone quality, and it even increased the value of certain parameters.

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