1 Department of Animal Nutrition and Feed Quality, Wrocław University
of Environmental and Life Sciences
Katedra Żywienia Zwierząt i Paszoznawstwa, Uniwersytet Przyrodniczy we Wrocławiu
2 Department of Histology and Embryology, Wrocław University of Environmental
and Life Sciences
Zakład Histologii i Embriologii, Uniwersytet Przyrodniczy we Wrocławiu
3 Department of Physics and Biophysics, Wrocław University of Environmental
and Life Sciences
Katedra Fizyki i Biofizyki, Uniwersytet Przyrodniczy we Wrocławiu
Broiler chickens within 1–28 days post hatch were fed diets containing different amounts of Ca (11 or 9 g/kg) and P-available. (4.5–3.7 g/kg). the dynamics of bone growth, bone metric and mecha-nical parameters, chemical composition and histological picture were determined. the body weight noted on day 28 post hatch in treatments II (9 g Ca and 3.7 g P-avail./kg) and III (11 g Ca and 3.7 g P-avail./kg) was significantly lower than in treatment I (11 g Ca and 4.5 g P-avail./kg) and Iv (9 g Ca; 4.5 g P-avail.). the bone strength evaluated on day 7 and 28 post hatch and elasticity para-meters measured on day 28 were significantly lower in chickens of treatment Iv. the best chemical composition of whole tibia and its parts was stated only on day 28 in birds from treatment I. In younger ones, the significantly higher bone ash content was found in the same treatment. Analysis of mineral composition of tibia parts in relation to the level of Ca and P-available in diets and lack of significant differences show that it is not depending on the level of these macroelements in feed mixtures. It must be noted that the contents of Ca and P-available as 11 and 4.5 or 11 and 3.7 g/kg For citation – Do cytowania: Jamroz D., Wertelecki t.J., Kuryszko J., Żyłka r., Kaleta-Kuratewicz K., 2011. Dynamics of bones development in early growth of chickens fed diets with different amounts of calcium and phosphorus. Zesz. Nauk. UP Wroc., Biol. Hod. Zwierz., LXIII, 583: 95–121.
diet, was the most beneficial nutritional variant for bone quality and ash content in bone, however without marked changes in Ca and P content in it. Moreover, it should be stated that only one of the applied nutritional variants 9 g Ca and 4.5 g P-available in feed mixture (treatment Iv) significantly or insignificantly decreased the bone quality parameters.
response of chicken in early growth phase to the diversified Ca and P level in feed mixtures, determined on the basis of mechanical and metric properties, chemical composition and histologi-cal picture of long bone – tibia, indicates the substantial adaptative abilities and ambiguous tends in dynamics of analyzed parameters. Greater, but unclear differences in bone quality parameters among treatments, just from 28 day of chickens life became significant.
KEY WOrDS: broilers, bones, chemical composition, histological structure
iNTROdUCTiON
the optimum levels of phosphorus and calcium applied in broiler diets depends on many factors such as genotype and age of birds, dynamics of growth and physical activity, en-docrine status, kind of cereal grains applied in diets (NSP and phytates content), sources of dietary phosphorus and calcium, supplementation of diets with enzymes, especially phytase and some other (Bleux et al. 2002, Borges et al. 2003, Edwards 1988, Hemme et al. 2005, Huyghebaert 1996, Jamroz et al. 2001, 2003, tatara et al. 2009).
the European Commission and Environmental Protection Agency (2003) have re-commended the level of 0.65 to 0.78% of total phosphorus in starter mixtures for broiler chickens; in grower and finisher diets the phosphorus quantity should vary at limits of 0.65% and within 0.57 to 0.67%, respectively. the NrC (1994) and Polish recommen-dations (2005) gives the values similar to the EU data – for total P 0.64–0.68, and for P-available 0.38 to 0.45%. the recommended calcium contents varies between 0.85 and 0.97%. For different hybrids the diversified values related to the requirement for Ca and P are given. For instance the Cobb-vantress Hybrid (2002) have recommended the amount of 0.84–0.90% of calcium and 0.40–0.45% of available phosphorus during all growth phases, while the Hubbard ISSA (2002) recommends the amounts of 1.1–1.2% of Ca and 0.45% of P available. Also in the studies by Huyghebaert (1996), Huyghebaert et al. (2009) and Jamroz et al. (2001, 2004, 2007) the relatively wide range of phosphorus and calcium for broiler chickens is recommended.
the purpose of presented investigation was to evaluate the response of young chic-kens at the first four weeks post hatch, to the different Ca and P levels in the diets. As an substantial parameters of evaluation of birds reaction, the dynamics of bone development – changes in the chemical, metric and mechanical parameters of bones as well as histo-logical structure of them were analyzed. In opinion of rodehutscord (2009) these are the one of data which can explain the response of birds to the diversified mineral composition of diets. the measurements were performed during early phase of growth, when the im-portant processes that determine the correct formation of structure and strenght of bones are occurring (rath et al. 2000, Whitehead 2005).
mATERiAl ANd mEThOdS
One hundred and sixty eight one-day old Hubbard Flex male broiler chickens, with aver-age initial body weight ca. 39.5 g (±0.91 standard deviation for averaver-age values in treat-ments) were randomly assigned to four treatments, kept in battery cages. Each treatment had three replications (cages) with 14 birds per one. the room temperature was gradually reduced from initial 32°C to 20°C after first four weeks post hatch. relative humidity in-side room gradually growed up from 64% at start to 71% in the last week of experiment. the lighting programm during whole experimental period was 24 hours of light a day. the birds had free access to drinking water given via nipple system.
Chickens were fed mixtures in mash form (starter from 1st to 14th day of life and gro-wer from 15th to 28th day) containing 215/205 g of crude protein, 12.3/11.6 g lysine and 9.3/9.0 g total sulphur amino acids (tSAA) per kg of mixture, respectively. the apparent metabolizable energy (AME) value amounted ca. 12.0 MJ/kg of mixture (table 1).
Four feeding variants of the isoenergetical and isoprotein feed mixtures offered ad libitum were different in calcium and phosphorus contents: treatment I have received mixture composed according to the relatively high Hubbard recommendations (2002) with Ca – 11.0 g/kg and 4.5 g/kg of P-available. Birds of treatment II were fed mixture containing 9.0 g/kg of Ca and 3.7 g/kg of P-available. Chickens of treatment III were fed mixture containing 11.0 g Ca/kg and 3.7 g of P-available and in treatment Iv diets conta-ining amounts of Ca and P close to the european recommendations (9.0 g of Ca and 4.50 g of available phosphorus per kg of diet).
the dietary electrolyte balances (DEB) in diets, were calculated according to the mo-del given by Hooge (Borges et al. 2003) DEB = [% Na+ in diets x 434.98 +% K+ in diets x 255.74 –% Cl- in diets x 282.06] and estimated values varied between 207 mEq/kg in starter and 197 mEq/kg of grower mixture.
the feed mixtures contained the vitamin/mineral premix free of feed antibiotics and feed enzymes – carbohydrases and phytases and there no additional calcium and pho-sphorus sources were used. the coccidiostat Diclazuril was applied only.
Body weight and bone quality
the dynamics of bone mineralisation processes was estimated in chickens. On day 1, post hatch, birds were weighed individually then eight birds (n=8) from each treatment (3 or 2 birds with average body weight per replication within treatment) were randomly selected and weighed individually then the birds with treatments’ average weight were used for examinations. this procedure was repeated on day 7, 14, 21 and 28, always in the morning. Birds at the age of 7, 14, 21 and 28 days were killed by cervical dislocation and then the both legs were prepared out. the muscles were removed then the tibia bones were cleaned. In fresh bones all metric and mechanical measurements were performed (16 bones per each treatment). the bones intended for defatting and chemical analysis were wrapped in the foil and stored for 2 days in refrigerator in +4 C. Whole tibia bones were subjected to the measurements and analysis according to the procedures described by Kim et al. (2004) and tatara (2009) and Jamroz et al. (2004, 2007). Four fresh tibia bones from each treatment and for each age of chickens were randomly chosen for histo-logical evaluation, another 6 bones were taken for mechanical and metric measurements. the remaining 6 bones were subjects to chemical composition analysis.
table 1 tabela 1 Composition of the experimental feed mixtures
Skład mieszanek doświadczalnych
treatment – Grupa doświadczalna
i ii iii Iv
Starter Grower Starter Grower Starter Grower Starter Grower Components (g/kg) – Składniki mieszanek
Maize – Kukurydza 329.6 355.9 337.8 363.1 332.3 363.1 336.8 367.3 Wheat – Pszenica 286.0 295.3 286.0 295.3 286.0 290.0 286.0 290.0 Soya bean meal
śruta sojowa 310.0 280.0 310.0 280.0 310.0 280.0 310.0 280.0 Soya oil – Olej sojowy 30.0 24.0 28.0 23.0 28.0 23.0 28.0 23.0 L-Lysine – L-Lizyna 2.2 2.2 2.2 2.2 2.2 2.2 2.2 2.2 DL-Methionine DL-Metionina 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 Limestone Kreda paszowa 17.2 17.3 14.0 14.1 19.5 19.5 12.0 12.0 Ca-phosphate Fosforan wapnia 14.5 14.8 11.5 11.8 11.5 11.7 14.5 15.0 Salt – Sól 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 Premix1 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
Determined nutrients (g/kg) – Oznaczone składniki pokarmowe Crude protein Białko surowe 215.6 204.9 216.5 205.1 215.8 205.1 214.9 205.6 Lysine 12.31 11.61 12.31 11.61 12.31 11.61 12.31 11.61 tSAA2 9.21 9.02 9.21 9.02 9.21 9.02 9.21 9.02 treonine 7.60 7.20 7.60 7.20 7.60 7.20 7.60 7.20 tryptophan 2.42 2.20 2.42 2.20 2.42 2.20 2.42 2.20 AMEn (MJ/kg)3 12.04 12.03 12.06 12.05 12.04 12.04 12.07 12.04 Ca 11.01 11.02 9.04 9.01 11.03 11.03 9.02 9.04 P total P całkowity 6.97 6.97 6.30 6.31 6.29 6.27 6.99 7.04 P avail. (calcul.)4 P dostępny (obliczony) 4.49 4.51 3.71 3.70 3.70 3.71 4.50 4.56 Ca:P avail. 2.45 2.44 2.44 2.43 2.98 2.97 2.00 1.98 Mg 2.00 1.95 1.98 1.92 2.03 1.98 1.96 1.90
1 added per kilogram of diet: retinyl palmitate, 5.5 mg; cholecalciferol, 0.05 mg; DL-α-tocopheryl acetate, 20 mg; menadione, 3 mg; thiamin, 2.5 mg; riboflavin, 4.5 mg; pyridoxine, 4 mg; cyanocobalamin, 0.015 mg; nicotinic acid, 25 mg; Ca-pantothenate, 8 mg; folic acid, 1.2 mg; choline chloride, 450 mg; DL-methionine, 1.0 mg; Mn, 74 mg as MnO; Fe, 30 mg as Fe2SO4·H2O; Zn, 45 mg as ZnO; Cu, 4 mg as CuO; Co, 0.4 mg as CoSO4; iodine, 0.3 mg as KI
zawartość w 1 kg diety: palmitynian retinylu, 5.5 mg; cholekalciferol, 0.05 mg; octan DL-α-tokoferolu, 20 mg; menadion, 3 mg; tiamina, 2.5 mg; ryboflawina, 4.5 mg; pirydoksyna, 4 mg; kobalamina, 0.015 mg; kwas nikotynowy, 25 mg; pantotenian Ca, 8 mg; kwas foliowy, 1.2 mg; chlorek choliny, 450 mg; DL-metionina, 1.0 mg; Mn, 74 mg jako MnO; Fe, 30 mg jako Fe2SO4•H2O; Zn, 45 mg jako ZnO; Cu, 4 mg jako CuO; Co, 0.4 mg as CoSO4; jod, 0.3 mg jako KI;
2 tSAA – total Sulphur Amino Acids – aminokwasy siarkowe łącznie tSAA – aminokwasy siarkowe łącznie
3 calculated according to European table of Energy values for Poultry Feedstuffs (1989), 3rd Edition, WPSA and the chemical composition of the diets according to GfE Empfehlungen DLG (1999)
obliczono zgodnie z Europejskimi tabelami Wartości Energetycznej Pasz dla Drobiu (1989), Wyd. III, WPSA skład chemiczny określono zgodnie z normami GfE Empfehlungen DLG (1999)
4 calculated according to Polish recommendations for Poultry Nutrition obliczono zgodnie z polskimi zaleceniami żywienia drobiu
For the fresh bones characteristics the metric, bone mass and mechanical parame-ters, breaking strength (maximum force causing bone breaking) and deflection were determined using an INStrON 5544 (USA) apparatus. Bone deflection was measured by a standard method, in which the force (F) was applied to the shaft of bone supported on both epiphyses, at L=8 mm distance for estimations made during first two weeks of experiment and L=13 mm in measure-ments made on day 21 and 28 of chickens life. the rate of movement of head during breaking force estimation was 0.5 mm/min for bones of 1 and 2 week-old chickens and 0.8 mm/min for bones of 3 and 4 week-old birds. Force (F) was increased from zero up to the bone breaking point. On the basis of obtained data of the mechanical measure-ments the following parameters were evalu-ated: the maximum loading force Fn (force at breaking point) expressed as newtons (N), maximum bone deflection hm (deflection at breaking point, expressed in meters (m) and the maximum relative bone deflection Um = hm/L (%). During measurements the elasticity coefficient k=F/h, where F is the acting force and h is the deflection, was also calculated. It was calculated as the slope of the deflection curve (F=f(h)) at the begin-ning, straight part and breaking work (work needed to break the bone) calculated as the integral of the deflection curve from zero to the breaking point. Elasticity coefficient is
expressed as newton per meter (N/m), and breaking work is calculated in joules (J). the detailed technique of measurements was presented by Jamroz et al. (2004).
In the defatted (in ether extract in 45oC for 24 hours, according to Soxhlet method,) and dried bones the crude ash and the contents of Ca, P and Mg were determined. the se-parate, chemical analysis of these ingredients in epiphysis proximalis and diaphysis were made to localize of a weak parts of tibia in aspect of bone fractures. the cuts were made using electric saw at places as presented on Figure 1.
Histological examinations
the histological examinations were performed in order to check the influence of diets containing different amounts of Ca and P, on the structure of bone tissue at the three age-points. the samples were taken out from the epiphysial zone, metaphysial area as well as
Fig. 1. Experimental cross-section places of tibia epi- and diaphysis
rys. 1. Miejsca eksperymentalnego cięcia kości piszczelowej
Epiphysis Nasada kości
Diaphysis trzon kości
from the diaphysis of the tibia bone. those were fixed for 72 hours in the 4% aqueous, solution of formalin buffered with calcium carbonate. After this procedure, the segments were rinsed in the tap water and then decalcified in the 10% solution of EDtA within 7 days and next with the mixture of formic acid and sodium citrate for 14 days. Such pre-pared biological material was dehydrated in the alcoholic series and paraffin embedded. Material was cutted into 7 µm thick sections and stained with hematoxylin (topographic, cells nuclei) and eosin (cell’ cytoplasm) according to the Delafield’s method. the histo-logical assayes were done using light microscope Nicon Eclipse 80I. the microphotogra-phies were obtained using the Canon PS66 camera.
Balance trial – minerals retention
the balances of calcium, phosphorus, magnesium were carried out for the days 7–10 (starter period) and 252–8 (grower period) of chickens life. the feed intake was pre-cisely controlled and the excrements were quantitatively collected twice a day during four days. For above periods both quantity and chemical composition of feed and excrements were recorded. these data were obtained treating all sub-groups in each treatment as one (n=3).
Analytical methods
the chemical composition of used raw materials, complete diets and tibia bones was determined according to standard methods AOAC (2005): the nitrogen content by Kjel-dahl-method using Kjeltec 2300 Foss tecator apparatus (Sweden), crude protein by multiplying of the N-content by 6.25, crude fat by ether extraction, crude fibre by the Henneberg-Stohmann method using an Fibertec tecator (Sweden) apparatus. the miner-alisation of samples was carried out with HNO3. Phosphorus was analyzed after previous mineralization with nitric acid (HNO3) and perchloric acid (HClO4) by the ammonium vanadomolybdate method using spectrophotometer Specol 11 (Carl Zeiss, Jena) at a wave length of 470 nm; calcium and magnesium were determined by atomic absorption spec-trophotometry using AAS-3 EA-30 type apparatus (Carl Zeiss, Jena).
For the determination of the amino acids, the samples of components were hydrolysed with 6M hydrochloric acid (HCl) for 24 h at 110oC and then amino acids were separated according to the Moore (1963) and Moore and Stein method (1963). For the sulphur ami-no acids determination the feed samples were oxidized (0oC, 16 h) with formic acid plus hydrogen peroxide (H2O2) (9:1) before the HCl hydrolysis and then were separated using an Analysator AAA 400 Ingos (Prague, Czech republic). For tryptophan contents the samples after alkaline hydrolysis with lithium hydroxide (LiOH) (110oC, 16 h) and 4-di-methyloaminobenzaldehyde (DMAB) were examined colorimetrically at a wave length of 590 nm according to the Landry and Delhaye (1992). On the basis of obtained results the amino acids contents in feed mixtures was optimalized and then analyzed in complete mixtures considering that the correct amino acid contents in feed is important for regular synthesis of organic matrix – bone proteins.
the energy density in the diets was calculated on the basis of determined nutrients and according to the formula published in the European tables of Energy values of Feeds for Poultry, WPSA (1989).
Statistical analysis
All obtained separate data for chickens and data for pens as a experimental units and data for balance trial were evaluated statistically by one-factorial ANOvA, and mineral composition of epi- and diaphysis by two-factorial ANOvAusing StatSoft Statistica® software (2005). the differences for all parameters were tested according to the following statistical model:
yij = μ + ai + eij or
yijk = μ + ai + bj + (ab)ij + eijk
where yij or yijk is the variance associated with parameter a, μ is the overall mean, ai is the treatment effect, bj is the body weight, bone characteristics, age or part of tibia effect, (ab) ij is the interaction efect and eij or eijk is an error term. the individual measurements were treated as the experimental units and differences between treatment means were analyzed for significance (P<0.01or 0.05) using tukey’s test. the data are presented as average valu-es and are accompanied by standard deviation valuvalu-es (±SD). Because of a lack of significant interactions in three-factorial analysis of variance (diets, age, part of bones, Ca and P level) the presentation of these data in very complicated tables was omitted.
All procedures that were carried out with animals have been approved by the Local Ethic Commission for animal experiments.
RESUlTS
the growth rate of chickens was estimated on the basis of individual body weight mea-sured at the 7-daily intervals. Data obtained on day 7, 14, 21 and 28 post hatch were significantly different between treatments (P<0.05) (table 2). the higher body weight of chickens as compared to other treatments, was registered on day 7 (P<0.05) and insig-nificantly different on day 14 post-hatch in treatment II (mixture with 9 g Ca and 3.7 g P-available/kg); on 21 day the significantly higher body weight than in treatment I and II was observed in treatment Iv (9 g Ca and 4.5 g P-available/kg). Seven days later the high-est body weight was registered in the same treatment (Iv) but also in treatment I. On day 28 significantly lower body weight was observed in chickens fed mixture containing 3.7 g P available/kg and 11 or 9 g Ca. the feed intake, strictly controlled at balance periods of 7–10 and 25–28 days of life, in which the retention of Ca, P, Mg was determined (table 9) was similar in treatments I–III, except of higher feed intake obtained in treatment Iv (P<0.05).
In mechanical and physical parameters of tibia bones significantly lower values for strength against breaking were registered in treatment Iv but also in treatment III on day 7, and on 28 day of life – in treatment II, III and Iv (table 3). Generally, higher values of strenght were estimated on 28 day of life in treatments fed diet with higher Ca and P con-centration – I and III (P<0.01) than in other treatments. No clear tendency was observed in characteristics of tibia elasticity and in parameters of maximum deflection in relation to maximum strength. In measurements performed on day 7 post hatch only in chickens from treatment Iv, the higher elasticity value and lower strenght of bones were stated. On day 28 post hatch, highest, statistically confirmed values of elasticity of bones were registered in chickens of treatment I, II and III as compared to the treatment Iv.
table 2 tabela 2 Body weight of chickens randomly selected for bone quality estimations during experiment (g)
(mean ±SD)
Masa ciała kurcząt losowo wybranych do oznaczeń jakości kości podczas eksperymentu (g) (średnia, ±SD)
Ca and P levels in feed mixtures (g/kg)
Poziom Ca i P w mieszankach
Days post-hatch – Dzień po wykluciu
1 7 14 21 28 i 11 and 4.5 39.0 107 a 325a 596a 1114a 3.8 8.8 20.7 93 121 ii 9 and 3.7 38.9 116 b 335a 608a 1014b 2.9 12.3 26.1 108 157 iii 11 and 3.7 40.0 107 a 317ab 633ab 1026b 2.8 13.3 37.7 79 136 Iv 9 and 4.5 40.5 107 a 301b 673b 1144a 3.6 17.2 39.0 87 124 n per treatment
Liczba ptaków w grupie 42 32 24 16 8
the average values in the same column marked with superscript a,b differ significantly by P<0.05 Wartości w kolumnach oznaczone różnymi literami a, b różnią się istotnie przy P<0,05
Other mechanical and physical parameters, such as maximum bone deflection and breaking work were inconsiderably affected by both, Ca and P level in diets, only on day 28 the significantly higher deflection was stated. the deflection of tibia was greater in very young chickens.
In general, the higher calcium (11.0 g/kg) level applied in diets of chickens of treat-ment I and III, positively influenced the mechanical parameters (strength, elasticity) of tibia bone. Such effect was noted in 28 day-old birds from this treatment in comparison to others. Other bone parameters were different in treatments and there no regular tenden-cies of changes were observed.
the development of tibia bones examined from the point of view of metric features allow to state the insignificant differences among treatments in parameters measured on day 7, 14 and 21 post hatch (table 4). On day 28 of life the significantly (P<0.05) longer