The experimental procedures used throughout this study were approved by the Local Ethics Committee on Animal Experimentation of the University of Life Sciences of Lublin, Poland. The birds were maintained in an animal house according to the guidelines of this committee. All efforts were made to minimize the number of animals used as well as their suffering.
Animals and Experimental Design
A total of 120 1-day-old Ross 308 broiler chickens were obtained from a commercial hatchery. The birds were weighed after hatching and randomly selected to one of three dietary treatments, each group containing 40 chickens. The chickens were assigned to either a control group (the 0 Suppl group; 40 birds divided into 10 pens with 4 birds per pen), or a group fed with lowered level of organic Cu in the form of glycinate chelate (Cu-Gly) as experimental group I (the Cu25% group; 40 birds divided into 10 pens with 4 birds in each pen), or a group fed with lowered level of organic Cu in the form of glycinate chelate simultaneously enriched with phytase as experimental group II (the Cu25% + phyt group; 40 birds divided into 10 pens with 4 birds in each pen). All birds were raised in battery cages (76 × 97 × 45 cm, width × length × height) placed in an environmentally controlled room and kept under standard rearing conditions and air temperature set at the optimal level depending on age. During the first week, the chickens were kept at 33 °C, which was reduced by 2 °C weekly, until the final temperature of 24 °C. The chickens had constant access to fresh water and appropriate feed supplied ad libitum in accordance with this stage of the production cycle (Table 1). To evaluate the growth rate, the birds’ daily body weight gains were recorded. The birds were fed a diet corresponding to the periods of rearing: starter (1–21 days), grower (22–35 days), and finisher (36–42 days). The chickens received a starter diet in the form of crumble, and grower and finisher diets in the form of pellets. At the end of the experiment, 10 birds randomly selected from the control (1 bird from each pen) and experimental groups I and II (1 bird from each pen) were weighed and slaughtered by cutting the carotid arteries. Ten hours before the slaughter, the selected birds were not given feed, but only provided with unlimited access to water.
Table 1 Composition and nutritive value of the experimental diet
Immediately after slaughter, the tibiae were dissected and cleaned from the remnants of adherent tissues and their weight and length measured. Directly after the measurements, each bone was wrapped in gauze soaked in isotonic saline and frozen at −25 °C for further analyses.
Supplementation of Cu Amino Acid Chelate and Phytase
The control group (the 0 Suppl group) was fed basal diet supplemented with the premix which did not provide external Cu (0 mg kg−1). The experimental diets were formulated by supplementing a corn-wheat-soyabean meal mixture (Table 1) with lowered levels (25% of the total daily recommended amount for Ross 308 broiler, 4 mg kg−1) of Cu from Gly-Cu, with or without phytase (500 FTU kg−1). The experiment involved the use of Glystar Forte chelate (Arkop Sp. z o.o., Bukowno, Poland) containing 16% of Cu and Ronozyme® HiPhos 6-phytase (DSM Nutritional Products, Mszczonów, Poland) produced by a genetically modified strain of Aspergillus oryzae. Application of glycine chelate was in accordance with the EU Directive 1334/2003 [18].
The basal corn-wheat-soybean meal diet (Table 1) containing (by analysis) 6.1 mg kg−1 (starter), 6.21 mg kg −1 (grower), and 5.91 mg kg−1 (finisher) of Cu from plants as the feed basis was formulated to meet or exceed nutritional requirements [10]. The amount of Cu in the premix was based on nutritional recommendations for Ross 308 broilers [10, 19], i.e., 16 mg kg−1 of Cu, irrespective of its content in the components of the basal diet. According to these recommendations, the Cu content should be the same in all periods of rearing, which was taken into account in the study [10, 19].
The nutrient composition of the basal diet was analyzed using standard methods: total phosphorus, calorimetrically with a Helios Alpha UV-VIS apparatus (Spectronic Unicam, Leeds, UK) [20], phytic phosphorus, by the Frühbeck et al. method [21], and the Cu, Fe, and Ca content in feed samples determined after ashing at 550 °C using the AAS flame technique in a Unicam 939 AA Spectrometer (Shimadzu Corp., Tokyo, Japan) apparatus, according to the methods of AOAC [20].
The amino acid composition in the diet was determined by ion exchange chromatography using an INGOS AAA 400 amino acid analyzer with post-column derivatization of ninhydrin and spectrophotometric detection [20]. Cysteine and methionine (sulfur amino acids) were determined in a separate analysis as described previously [15], according to the method of AOAC [20]. Assimilable lysine was determined based on the difference between total lysine and the so-called residual lysine which did not react with DNFB (dinitrofluorobenzene) [15]. Following this reaction, the tested samples were again subjected to acid hydrolysis [22].
Serum Biochemical Analyses
Each chicken was fasted for 12 h before blood collection. The blood was collected using standard venipuncture from the brachial vein; next, after clotting at room temperature, it was centrifuged and frozen at −80 °C for further analysis. The blood serum concentrations of copper, calcium, and phosphorus were determined by a colorimetric method using a Metrolab 2300 GL unit (Metrolab SA, Argentina) and sets of biochemical reagents produced by BioMaxima (Lublin, Poland) [20].
Growth Hormone and Bone Turnover Markers
The serum concentration of chicken growth hormone, insulin-like growth factor 1 (IGF-1), osteocalcin, and leptin were determined using an enzyme-linked immunosorbent assay kit (ELISA; Uscn Life Science Inc. Wuhan, China) with minimum detectable concentrations of 0.056 ng ml−1, 7.4 pg ml−1, 0.67 pg ml−1, and 14.8 pg ml−1, respectively.
Mechanical Properties
The mechanical properties of the tibia were determined for all the groups after 3-h thawing at room temperature using the three-point bending test of bone mid-diaphysis. The mechanical properties were examined on a Zwick Z010 universal testing machine (Zwick GmbH & Co. KG, Ulm, Germany), equipped with a measuring head of operation range up to 10 kN, linked to a computer with testXpert II 3.1 software (Zwick GmbH & Co. KG, Ulm, Germany), registering the relationship between force perpendicular to the longitudinal axis of the bone and the resulting displacement. The distance between the supports was set at 40% of the total bone length. The measuring head loaded bone samples with a constant speed of 10 mm min−1 until fracture [23]. The ultimate load was determined as the force causing bone fracture and the yield load as maximal force under an elastic (reversible) deformation of the bone [24]. Moreover, on the basis of measured geometric and mechanical traits, the material properties of the mid-diaphyseal fragment of the bone were calculated. These traits describe the specific mechanical properties of the midshaft cortical tissue and are independent of the bone size and the conditions under which the strength tests were conducted. The bending moment can be described as a yield load adjusted to the bone length, and it indicates the bone elastic load capability [25]. The elastic stress reflects the elastic strength of midshaft cortical bone; the ultimate stress is equal to the maximum stress a bone can withstand in bending before fracture [25].
Geometric Parameters
The geometric properties such as the cross section area (A), the mean relative wall thickness (MRWT), and the cortical index (CI; defined as the ratio of the thickness of the cortical part to the thickness of the midshaft measured at the middle part of the bone) were estimated on the basis of the horizontal and vertical diameter measurements of the mid-diaphyseal cross section of the bone with the previously described method [26]. Moreover, as during the strength analysis the bone was loaded in the A-P plane, the second (cross-sectional) moment of inertia Ix and the radius of gyration Rg about the medial-lateral (M-L) axis were calculated [25]. The second moment of inertia Ix is not a direct bone geometric trait, but is a critical property in terms of the bone bending rigidity evaluation.
The samples of the proximal end of each bone were subjected to histology as described previously [2]. The site and size (approximately 3 mm in length of both analyzed cartilages) of the areas of interest which were measured were chosen on the basis of motoric properties of the body—the knee joint in particular—as was described previously [27]. Two methods of staining were used: the Goldner’s trichrome to assess the morphology of the growth plate and articular cartilage, and the safranin-O staining to visualize the cartilage proteoglycans [28]. Briefly, the sagittal sections through the middle of the lateral condyle of each tibia were cut strictly according to the previously described method and equipment [27]. Safranin-O staining was applied to the visual assessment of Mankin’s histological and histochemical grading system for evaluation of the articular cartilage [29, 30].
The thickness of the following zones: reserve (I), proliferation (II), hypertrophy (III), and ossification (IV) were measured at four sites along the growth plate cartilage, and an average was calculated as described previously [30]. Similarly, the thickness of the main zones of the articular cartilage, i.e., horizontal (superficial surface, I), transitional (II), and radial (III), was measured as described previously [2].
The Picrosirius red staining (PSR) was employed to assess the morphology of the articular cartilage and to evaluate the distribution of thick (mature) and thin (immature) collagen fibers in the articular cartilage [31,32,33]. The sections stained with PSR were analyzed using a Leica DM 2500 microscope (Leica Microsystems, Wetzlar, Germany) equipped with filters to provide circularly polarized illumination. Images were documented by a high-resolution digital camera (Leica Microsystems, Wetzlar, Germany).
The bone volume (BV), tissue volume (TV), relative bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), and fractal dimension (Fd) of the trabecular bone were measured as described previously [28].
Ca, P, and Ash Content in Bone
After evaluating the strength and structural properties, the bones were defatted, dried at 105 °C to a constant mass, and finally mineralized in a muffle furnace at 500 °C [2, 22]. The content of the mineral components (Ca, P, Cu) of the bones was determined by an atomic absorption spectrometry using a Unicam 939/959 apparatus [20]. The percentage of bone ash and the content of Ca, P, and Cu in the bone were calculated as part of components from the crude ash.
Statistical Analysis
All results are expressed as mean ± SD (standard deviation). The differences between the means were tested with one-way ANOVA and post hoc Tukey’s HSD test as the correction for multiple comparisons. Normal distribution of data was examined using the Shapiro-Wilk W-test, and equality of variance was tested by the Brown-Forsythe test. A P value of less than 0.05 was considered statistically significant. All statistical analyses were carried out by means of Statistica 12 software (StatSoft, Inc., Tulsa, OK, USA; http://www.statsoft.com).