Biol Trace Elem Res (2018) 182:339–353

DOI 10.1007/s12011-017-1092-1

Effect of Dietary Phytase Supplementation on Bone and Hyaline

Cartilage Development of Broilers Fed with Organically
Complexed Copper in a Cu-Deficient Diet
Siemowit Muszyński 1 & Ewa Tomaszewska 2 & Małgorzata Kwiecień 3 &
Piotr Dobrowolski 4 & Agnieszka Tomczyk 2

Received: 15 April 2017 / Accepted: 28 June 2017 / Published online: 15 July 2017
# The Author(s) 2017. This article is an open access publication

Abstract Tibial mechanical, chemical, and histomorphometric analysis showed a positive effect of Cu
histomorphometrical traits were investigated for growing supplementation on real bone volume and trabecular thick-
male Ross 308 broiler chickens fed diets that had copper ness in the tibia metaphyseal trabeculae; additionally, phytase
(Cu) from organic source at a lowered level of 25% of the increased the trabeculea number. The supplementation with
daily requirement (4 mg kg−1 of a premix) with or without Cu significantly increased the total articular cartilage and
phytase. Dietary treatments were control non-copper, non- growth plate cartilage thickness; however, the changes in
phytase group (0 Suppl); 4 mg kg−1 Cu non-phytase group thickness of particular zones were dependent upon phytase
(25%Cu); and 4 mg kg−1 Cu + 500 FTU kg−1 phytase group addition. In summary, dietary Cu supplements given to grow-
(25%Cu + phyt). The results show that birds fed with the ing broilers with Cu in their diet restricted to 25% of the daily
addition of phytase exhibited improved weight gain and final requirement had a positive effect on bone metabolism, and
body weight and had increased serum IGF-1 and osteocalcin phytase supplementation additionally improved cartilage
concentrations. The serum concentration of Cu and P did not development.
differ between groups; however, Ca concentration decreased
in the 25%Cu + phyt group when compared to the 25%Cu Keywords Organic copper . Phytase . Bone
group. Added Cu increased bone Ca, P, Cu, and ash content histomorphometry . Mechanical testing . Broiler chicken
in Cu-supplemented groups, but bone weight and length in-
creased only by the addition of phytase. Bone geometry,
yield, and ultimate strengths were affected by Cu and phytase Introduction
addition. A decrease of the elastic stress and ultimate stress of
the tibia in Cu-supplemented groups was observed. The Copper (Cu) as an essential trace element has many physio-
logical functions in animals and humans, including skeletal
mineralization, erythropoiesis, leukopoiesis, connective tissue
* Siemowit Muszyński
[email protected] synthesis, myelin formation, melanin pigment synthesis, cat-
echolamine metabolism, thermal regulation, cholesterol me-
tabolism, immune function, cardiac function, and glucose
1
Department of Physics, Faculty of Production Engineering, metabolic regulation [1, 2]. It is known that Cu takes part in
University of Life Sciences in Lublin, Akademicka 13,
20-950 Lublin, Poland
bone metabolism as an essential co-factor needed for the ac-
2
tion of lysyl oxidase [3]. This Cu-dependent enzyme initiates
Department of Animal Physiology, Faculty of Veterinary Medicine,
University of Life Sciences in Lublin, Akademicka 12,
the critical process of covalent cross-linkage formation in elas-
20-950 Lublin, Poland tin and collagen in bones and other connective tissues [1, 4].
3
Institute of Animal Nutrition and Bromatology, University of Life
Inhibition of lysyl oxidase enzyme activity results in
Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland osteolathyrism and negatively affects bone and cartilage for-
4
Department of Comparative Anatomy and Anthropology, Maria
mation [5]. Furthermore, studies on Cu supplementation indi-
Curie-Skłodowska University, Akademicka 19, cate that Cu deficiency leads to bone loss (osteopenia or oste-
20-033 Lublin, Poland oporosis), demineralization, a failure of ossification of growth
340 Muszyński et al.

centers, and neuropenia [1, 2]. It is caused mainly by the anion, forms insoluble salts with Cu and other divalent cations
decreased function of osteoblasts (bone tissue-forming cells) [14]. These complexes are indigestible for poultry, and the
because the action of osteoclasts (bone tissue-resorbing cells) utilization of microelements bound in phytate form is lowered
remains unaffected [3]. All of these phenomena lead to the or impossible [14]. However, it is well known that the use of
decrease in bone mechanical strength and result in consequent phytase, an enzyme that hydrolyzes phytate complexes, in-
fractures [4, 6]. creases the availability of trace minerals [16, 17]. Studies to
Trace minerals are essential in the diets of poultry because quantify the bioavailability of dietary Cu given at marginally
they participate in the biochemical processes required for the deficient amounts from alternative sources of Cu are numer-
maintenance of normal growth and development, including ous [2, 6, 11]. However, to date, the relationship between the
bone and eggshell formation and development of the avian dietary status of Cu and the presence of phytase on bone
embryo [7]. Moreover, in poultry, there is substantial interest health and cartilage development has not been well studied,
in using Cu as a health promoter as an alternative to antibiotics including on broiler chickens.
that can produce equivalent effects on chicken performance With regards to the important role of Cu in bone metabolism
[8]. Copper is often added to poultry diets at prophylactic during development, the objective of this study was to compare
concentrations for its growth-promoting effects by influencing the effects of administrating amino acid Cu complex in grow-
the microbial populations within the gastrointestinal tract [9]. ing male Ross 308 chickens fed a Cu-poor diet, which ensured
The Cu requirement in the diets of chicken has been higher Cu bioavailability through enhanced absorption from
established to be 8 mg kg−1 [10], but a greater amount of Cu the intestine, to those fed the same diet but which were addi-
than is required has often been included in commercial broiler tionally enriched with phytase. This was assessed on the basis
diet. The reason for this is that the differences in the concen- of the mechanical, geometric, and histomorphometric parame-
trations of Cu and its bioavailability in various feed ingredi- ters of the tibia, bone Cu content, and determination of the
ents are as a result of the variations in processing procedures concentration of hormones of the somatotropic axis in serum.
and in the cultivating conditions used for feed ingredients It is hypothesized that the use of Cu in the more assailable form
[10]. Therefore, several supplemental Cu sources such as in- of glycinate chelate might improve the development of the
organic sources (copper sulfate or carbonate) and organic skeletal system in broiler chickens, even if it is administered
sources (chelated form with higher Cu bioavailability) have at the reduced level of 25% of the daily recommended dose.
been added to livestock diet, including broilers [6, 11–13]. Thus, to evaluate the possible response of broilers to organical-
Chelates are reported to have significantly higher absorption ly complexed Cu and the effect of phytase inclusion into the
rates from the intestine due to their unique chemical structure diet, the present study was conducted with a control diet devoid
as compared to inorganic salts, and it has been shown that of both phytase and mineral supplementation of Cu in a trace
supplementation with 4 mg kg−1 of Cu from organic sources mineral premix.
may be sufficient for normal broiler growth [11]. Furthermore,
organically complexed trace minerals make available alterna-
tive pathways for absorption, thus leading to a reduction in the Materials and Methods
excretion of minerals to the environment [8, 11]. Therefore, it
seems that the use of a mineral chelate allows for maintaining The experimental procedures used throughout this study were
performance while concomitantly reducing the mineral con- approved by the Local Ethics Committee on Animal
tent in manure [13, 14]. Experimentation of the University of Life Sciences of
The skeletal system consists of bones, cartilage, ligaments, Lublin, Poland. The birds were maintained in an animal house
and tendons and accounts for about 20% of the body weight of according to the guidelines of this committee. All efforts were
most vertebrates. It has been demonstrated that the use of Cu made to minimize the number of animals used as well as their
in the form of glycinate compounds at much reduced amounts suffering.
as opposed to using sulfate at its recommended dose does not
cause deterioration in the physical, mechanical, and morpho- Animals and Experimental Design
metric properties of femur in chickens [2, 14, 15]. Thus, with
regard to the important role of Cu in the development of A total of 120 1-day-old Ross 308 broiler chickens were ob-
bones, it is hypothesized that the use of Cu in the more digest- tained from a commercial hatchery. The birds were weighed
ible and assimilable form of chelate might improve the growth after hatching and randomly selected to one of three dietary
and development of the skeletal system in broiler chickens. treatments, each group containing 40 chickens. The chickens
Phytic acid is a naturally occurring organic complex found were assigned to either a control group (the 0 Suppl group; 40
in plants. About 70% of P present in the feedstuffs of plant birds divided into 10 pens with 4 birds per pen), or a group fed
origin used in the feed for poultry is in phytate form, inacces- with lowered level of organic Cu in the form of glycinate
sible to non-ruminant animals [14]. Phytic acid, as a reactive chelate (Cu-Gly) as experimental group I (the Cu25% group;
Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with... 341

40 birds divided into 10 pens with 4 birds in each pen), or a components of the basal diet. According to these recommen-
group fed with lowered level of organic Cu in the form of dations, the Cu content should be the same in all periods of
glycinate chelate simultaneously enriched with phytase as ex- rearing, which was taken into account in the study [10, 19].
perimental group II (the Cu25% + phyt group; 40 birds divid- The nutrient composition of the basal diet was analyzed
ed into 10 pens with 4 birds in each pen). All birds were raised using standard methods: total phosphorus, calorimetrically with
in battery cages (76 × 97 × 45 cm, width × length × height) a Helios Alpha UV-VIS apparatus (Spectronic Unicam, Leeds,
placed in an environmentally controlled room and kept under UK) [20], phytic phosphorus, by the Frühbeck et al. method
standard rearing conditions and air temperature set at the op- [21], and the Cu, Fe, and Ca content in feed samples deter-
timal level depending on age. During the first week, the mined after ashing at 550 °C using the AAS flame technique
chickens were kept at 33 °C, which was reduced by 2 °C in a Unicam 939 AA Spectrometer (Shimadzu Corp., Tokyo,
weekly, until the final temperature of 24 °C. The chickens Japan) apparatus, according to the methods of AOAC [20].
had constant access to fresh water and appropriate feed sup- The amino acid composition in the diet was determined by
plied ad libitum in accordance with this stage of the produc- ion exchange chromatography using an INGOS AAA 400
tion cycle (Table 1). To evaluate the growth rate, the birds’ amino acid analyzer with post-column derivatization of nin-
daily body weight gains were recorded. The birds were fed a hydrin and spectrophotometric detection [20]. Cysteine and
diet corresponding to the periods of rearing: starter (1– methionine (sulfur amino acids) were determined in a separate
21 days), grower (22–35 days), and finisher (36–42 days). analysis as described previously [15], according to the method
The chickens received a starter diet in the form of crumble, of AOAC [20]. Assimilable lysine was determined based on
and grower and finisher diets in the form of pellets. At the end the difference between total lysine and the so-called residual
of the experiment, 10 birds randomly selected from the control lysine which did not react with DNFB (dinitrofluorobenzene)
(1 bird from each pen) and experimental groups I and II (1 bird [15]. Following this reaction, the tested samples were again
from each pen) were weighed and slaughtered by cutting the subjected to acid hydrolysis [22].
carotid arteries. Ten hours before the slaughter, the selected
birds were not given feed, but only provided with unlimited Serum Biochemical Analyses
access to water.
Immediately after slaughter, the tibiae were dissected and Each chicken was fasted for 12 h before blood collection. The
cleaned from the remnants of adherent tissues and their weight blood was collected using standard venipuncture from the
and length measured. Directly after the measurements, each brachial vein; next, after clotting at room temperature, it was
bone was wrapped in gauze soaked in isotonic saline and centrifuged and frozen at −80 °C for further analysis. The
frozen at −25 °C for further analyses. blood serum concentrations of copper, calcium, and phospho-
rus were determined by a colorimetric method using a
Supplementation of Cu Amino Acid Chelate and Phytase Metrolab 2300 GL unit (Metrolab SA, Argentina) and sets
of biochemical reagents produced by BioMaxima (Lublin,
The control group (the 0 Suppl group) was fed basal diet Poland) [20].
supplemented with the premix which did not provide external
Cu (0 mg kg−1). The experimental diets were formulated by Growth Hormone and Bone Turnover Markers
supplementing a corn-wheat-soyabean meal mixture (Table 1)
with lowered levels (25% of the total daily recommended The serum concentration of chicken growth hormone, insulin-
amount for Ross 308 broiler, 4 mg kg−1) of Cu from Gly- like growth factor 1 (IGF-1), osteocalcin, and leptin were de-
Cu, with or without phytase (500 FTU kg−1). The experiment termined using an enzyme-linked immunosorbent assay kit
involved the use of Glystar Forte chelate (Arkop Sp. z o.o., (ELISA; Uscn Life Science Inc. Wuhan, China) with mini-
Bukowno, Poland) containing 16% of Cu and Ronozyme® mum detectable concentrations of 0.056 ng ml − 1 ,
HiPhos 6-phytase (DSM Nutritional Products, Mszczonów, 7.4 pg ml−1, 0.67 pg ml−1, and 14.8 pg ml−1, respectively.
Poland) produced by a genetically modified strain of
Aspergillus oryzae. Application of glycine chelate was in ac- Mechanical Properties
cordance with the EU Directive 1334/2003 [18].
The basal corn-wheat-soybean meal diet (Table 1) contain- The mechanical properties of the tibia were determined for all
ing (by analysis) 6.1 mg kg−1 (starter), 6.21 mg kg −1 (grower), the groups after 3-h thawing at room temperature using the
and 5.91 mg kg−1 (finisher) of Cu from plants as the feed basis three-point bending test of bone mid-diaphysis. The mechani-
was formulated to meet or exceed nutritional requirements cal properties were examined on a Zwick Z010 universal test-
[10]. The amount of Cu in the premix was based on nutritional ing machine (Zwick GmbH & Co. KG, Ulm, Germany),
recommendations for Ross 308 broilers [10, 19], i.e., equipped with a measuring head of operation range up to
16 mg kg −1 of Cu, irrespective of its content in the 10 kN, linked to a computer with testXpert II 3.1 software
342 Muszyński et al.

Table 1 Composition and

nutritive value of the Ingredients (%) Starter Grower Finisher
experimental diet (1–21 days) (22–35 days) (36–42 days)

Corn 24.5 40.0 40.0

Wheat 42.9 27.9 28.8
Soybean mealf 25.0 24.9 22.9
Soybean oil 2.50 3.69 3.98
Phosphate 1—Ca 0.90 0.90 0.81
Fodder chalk 1.40 1.13 1.09
Sodium bicarbonate 0.08 0.08 0.08
NaCl 0.29 0.25 0.26
Premix vitamin (no Cu) 0.50a 0.50b 0.50c
Concentrate protein—fattyg 1.00 – 1.00
DL-Methionine 99% 0.30 0.23 0.23
L-Lysine HCl 0.42 0.28 0.27
L-Threonine 99% 0.18 0.13 0.07
The nutritional value of diet
ME (MJ kg−1)e 12.7 13.1 13.2
Total protein (%)d 21.2 20.4 19.9
Crude fiber (%)d 1.64 1.59 1.73
Crude fat (%)d 4.57 5.42 5.53
Lysine, total (%)d 1.28 1.14 1.08
Met + Cys (%)d 0.92 0.81 0.82
Total Ca (%)d 0.87 0.79 0.76
Total P (%)d 0.65 0.66 0.64
Phytate P (%)d 0.32 0.32 0.32
Bioavailable P (%)e 0.42 0.41 0.39
Total Ca/P bioavailablee 2.12 1.90 1.92
Cu from plants in basal diet (mg kg−1)d 6.04 6.07 5.91
Fe (mg kg−1)d 40.31 39.82 38.61
Cu (mg kg−1)d
4 mg Cu-Gly 10.28 10.34 10.21
4 mg Cu-Gly + Phyt 11.12 10.87 11.01
a
Content of vitamins and minerals per 1 kg of starter: Mn 100 mg, I 1 mg, Fe 40 mg, Cu 16 mg, Se 0.15 mg,
vitamin A 15000 UI, vitamin D3 5000 UI, vitamin E 75 mg, vitamin K3 4 mg, vitamin B1 3 mg, vitamin B2 8 mg,
vitamin B6 5 mg, vitamin B12 0.016 mg, biotin 0.2 mg, folic acid 2 mg, nicotinic acid 60 mg, pantothenic acid
18 mg, choline 1800 mg
b
Content of vitamins and minerals per 1 kg of grower: Mn 100 mg, I 1 mg, Fe 40 mg, Cu 16 mg, Se 0.15 mg,
vitamin A 12000 UI, vitamin D3 5000 UI, vitamin E 50 mg, vitamin K3 3 mg, vitamin B1 2 mg, vitamin B2 6 mg,
vitamin B6 4 mg, vitamin B12 0.016 μg, biotin 0.2 mg, folic acid 1.75 mg, nicotinic acid 60 mg, pantothenic acid
18 mg, choline 1600 mg
c
Content of vitamins and minerals per 1 kg of finisher: Mn 100 mg, I 1 mg, Fe 40 mg, Cu 16 mg, Se 0.15 mg,
vitamin A 12000 UI, vitamin D3 5000 UI, vitamin E 50 mg, vitamin K3 2 mg, vitamin B1 2 mg, vitamin B2 5 mg,
vitamin B6 3 mg, vitamin B12 0.011 μg, biotin 0.05 mg, folic acid 1.5 mg, nicotinic acid 35 mg, pantothenic acid
18 mg, choline 1600 mg
d
Analyzed values
e
Calculated values
f
Forty-six percent total protein in dry matter
g
One kilogram concentrate protein-fatty contains 2% crude fat, 39% crude protein, 10.8 MJ ME

(Zwick GmbH & Co. KG, Ulm, Germany), registering the between the supports was set at 40% of the total bone length.
relationship between force perpendicular to the longitudinal The measuring head loaded bone samples with a constant
axis of the bone and the resulting displacement. The distance speed of 10 mm min−1 until fracture [23]. The ultimate load
Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with... 343

was determined as the force causing bone fracture and the yield distribution of thick (mature) and thin (immature) collagen
load as maximal force under an elastic (reversible) deformation fibers in the articular cartilage [31–33]. The sections stained
of the bone [24]. Moreover, on the basis of measured geometric with PSR were analyzed using a Leica DM 2500 microscope
and mechanical traits, the material properties of the mid- (Leica Microsystems, Wetzlar, Germany) equipped with fil-
diaphyseal fragment of the bone were calculated. These traits ters to provide circularly polarized illumination. Images were
describe the specific mechanical properties of the midshaft cor- documented by a high-resolution digital camera (Leica
tical tissue and are independent of the bone size and the condi- Microsystems, Wetzlar, Germany).
tions under which the strength tests were conducted. The bend- The bone volume (BV), tissue volume (TV), relative bone
ing moment can be described as a yield load adjusted to the volume (BV/TV), trabecular thickness (Tb.Th), trabecular
bone length, and it indicates the bone elastic load capability separation (Tb.Sp), trabecular number (Tb.N), and fractal di-
[25]. The elastic stress reflects the elastic strength of midshaft mension (Fd) of the trabecular bone were measured as de-
cortical bone; the ultimate stress is equal to the maximum stress scribed previously [28].
a bone can withstand in bending before fracture [25].

Geometric Parameters Ca, P, and Ash Content in Bone

The geometric properties such as the cross section area (A), the After evaluating the strength and structural properties, the
mean relative wall thickness (MRWT), and the cortical index bones were defatted, dried at 105 °C to a constant mass, and
(CI; defined as the ratio of the thickness of the cortical part to finally mineralized in a muffle furnace at 500 °C [2, 22]. The
the thickness of the midshaft measured at the middle part of the content of the mineral components (Ca, P, Cu) of the bones
bone) were estimated on the basis of the horizontal and vertical was determined by an atomic absorption spectrometry using a
diameter measurements of the mid-diaphyseal cross section of Unicam 939/959 apparatus [20]. The percentage of bone ash
the bone with the previously described method [26]. Moreover, and the content of Ca, P, and Cu in the bone were calculated as
as during the strength analysis the bone was loaded in the A-P part of components from the crude ash.
plane, the second (cross-sectional) moment of inertia Ix and the
radius of gyration Rg about the medial-lateral (M-L) axis were Statistical Analysis
calculated [25]. The second moment of inertia Ix is not a direct
bone geometric trait, but is a critical property in terms of the All results are expressed as mean ± SD (standard devia-
bone bending rigidity evaluation. tion). The differences between the means were tested with
The samples of the proximal end of each bone were sub- one-way ANOVA and post hoc Tukey’s HSD test as the
jected to histology as described previously [2]. The site and correction for multiple comparisons. Normal distribution of
size (approximately 3 mm in length of both analyzed carti- data was examined using the Shapiro-Wilk W-test, and
lages) of the areas of interest which were measured were cho- equality of variance was tested by the Brown-Forsythe
sen on the basis of motoric properties of the body—the knee test. A P value of less than 0.05 was considered statisti-
joint in particular—as was described previously [27]. Two cally significant. All statistical analyses were carried out
methods of staining were used: the Goldner’s trichrome to by means of Statistica 12 software (StatSoft, Inc., Tulsa,
assess the morphology of the growth plate and articular carti- OK, USA; http://www.statsoft.com).
lage, 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 Results
[27]. Safranin-O staining was applied to the visual assessment
of Mankin’s histological and histochemical grading system for Body Weight
evaluation of the articular cartilage [29, 30].
The thickness of the following zones: reserve (I), prolifer- The initial body weights of the control and birds treated with
ation (II), hypertrophy (III), and ossification (IV) were mea- the organic Cu form (regardless of the presence of phytase)
sured at four sites along the growth plate cartilage, and an were similar (Table 2). At the end of the experiment, chickens
average was calculated as described previously [30]. fed Cu-poor diet supplemented with the phytase (the
Similarly, the thickness of the main zones of the articular Cu25% + phyt group) weighed significantly more than the
cartilage, i.e., horizontal (superficial surface, I), transitional birds from the control group (the 0 Suppl group) and the
(II), and radial (III), was measured as described previously [2]. Cu25% group (Table 2). Daily weight gain was also signifi-
The Picrosirius red staining (PSR) was employed to assess cantly higher in the Cu25% + phyt group compared to the
the morphology of the articular cartilage and to evaluate the control and the Cu25% groups (Table 2).
344 Muszyński et al.

Table 2 The body weight (initial

and 42-day-old as final body Group Number Body weight (g) Daily weight gain (g)
weight) of control broilers and
supplemented with Cu in 25% of Initial body weight Final body weight
daily demand dependently on
phytase supplementation 0 Suppl 10 44.1 ± 1.0 2262 ± 127a 49.4 ± 4.3a
Cu25% 10 43.8 ± 1.0 2281 ± 144a 53.3 ± 3.4a
Cu25% + phyt 10 44.0 ± 0.8 2716 ± 160b 63.6 ± 3.8b
SEM 0.17 46 1.31
P value P = 0.775 P < 0.001 P < 0.001

Mean values in columns with different lowercase letters differ significantly at P < 0.05; data given are mean ± SD
(standard deviation)
SEM standard error of the mean, 0 Suppl the control group without received Cu in premix, Cu25% the group
received Cu in 25% of daily demand from Cu-Gly, Cu25% + phyt the group received Cu in 25% of daily demand
from Cu-Gly with phytase

The Content of Ca, Total P, and Cu in Blood Serum The Content of P, Ca, and Cu in Bone

The Cu serum concentration of the birds from both experi- The bone P, Ca, and bone crude ash content in the
mental groups (irrespective of the phytase supplementation) chickens supplemented with Cu in the Cu-Gly form
reached similar values as those in the control group (Fig. 1). in the Cu-poor diet, irrespective of the phytase pres-
Similarly, total P serum concentration was similar in the con- ence, was higher compared to the control values
trol chickens and birds supplemented with the Cu-Gly, regard- reached in the 0 Suppl group (Fig. 2). However, the
less of the phytase addition (Fig. 1). The Ca serum concentra- bone Ca/P ratio did not differ between groups (Fig. 2).
tion of the chickens from the Cu25% + phyt group was lower The bone Cu content in both groups supplemented
compared to the control values noted in chickens from the with Cu in 25% of daily demand was higher compared
Cu25% group (Fig. 1). However, the serum Ca/P ratio did to the control chickens which were fed the Cu-
not differ between groups (Fig. 1). deprived diet (Fig. 2).

Fig. 1 The serum concentration

of calcium (Ca), copper (Cu),
phosphorus (P), and Ca/P ratio in
42-day-old chickens treated with
Cu in organic (Cu-Gly) form
dependently on phytase
supplementation. Data given are
mean ± SD. a, b Values with
different letters differ significantly
at P < 0.05. 0 Suppl—the control
group without received Cu in
premix. Cu25%—the group
received Cu in 25% of daily
demand from Cu-Gly. Cu25% +
phyt—the group received Cu in
25% of daily demand from Cu-
Gly with phytase
Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with... 345

Fig. 2 The bone content of calcium (Ca), phosphorus (P), copper (Cu), given are mean ± SD. a, b Values with different letters differ significantly
crude ash, and bone Ca/P ratio in 42-day-old chickens treated with Cu in at P < 0.05. The description of the groups as in Fig. 1
organic (Cu-Gly) form dependently on phytase supplementation. Data

Bone Morphology, Geometry, and Mechanical Properties Bone Histomorphometry

The intake of Cu in the Cu-Gly form at the concentration of The microscopic assessment of cancellous bone in both ex-
25% of daily requirement with the addition of phytase in- perimental groups supplemented with Cu-Gly at the concen-
creased the bone weight, length, and weight/length ratio tration of 25% of the daily requirement showed a significant
compared to the other groups (Table 3). Moreover, Cu-Gly increase in the real bone volume (BV/TV), the mean (Tb.Th
administration at 25% of daily requirement significantly al- mean), and the maximal (Tb.Th max) trabecular thickness
tered all measured diameters; however, the greatest increases (Table 4). However, an increase in the mean (Tb.Sp mean)
were noted in the group additionally supplemented with and the maximal (Tb.Sp max) trabecular space in the
phytase (Table 3). As a result, the chickens fed the Cu- Cu25% group when compared to the control group was noted
poor diet irrespective of the phytase addition had an in- while the phytase addition resulted in a decrease of the Tb.Sp
creased cross section area as compared to the control mean and the Tb.Sp max (Table 4). Moreover, in the
chickens. However, the mean relative wall thickness de- Cu25% + phyt group, an increase in the trabecula number
creased in the Cu25% group, while the cortical index in- (Tb.N) was observed. The fractal dimension (Fd) of the tra-
creased in the Cu25% + phyt group compared to the control becular bone increased significantly in the Cu25% + phyt
0 Suppl group (Table 3). The change of the cross section group and decreased in the Cu25% group when compared to
dimensions of the midshaft resulted in a significant change the control group.
of the values of the midshaft volume and the cross-sectional
moment of inertia in both groups fed the Cu-poor diet (with Morphology of the Articular and Growth Plate Cartilages
the highest values in the group with phytase addition) when
compared to control birds fed the Cu-deprived diet The examined joints had no visible lesions or degener-
(Table 3). Similarly, an increase in the radius of gyration ative changes. The supplementation of Cu-Gly to the
of both experimental groups was observed. feed of growing chickens (regardless of phytase addi-
The addition of phytase in the Cu-poor diet resulted in an tion) significantly increased the total, I, and II zones
increase in the yield strength, the ultimate strength, and the of the articular cartilage, while the greatest change was
bending moment compared to the control diet (Cu-deprived) observed in the group fed with the phytase additive
and the Cu-poor diet without the phytase. However, for both (Fig. 3). Zone III became wider in the Cu25% group
experimental groups, the Cu supplementation led to a signif- and narrowed in the Cu25% + phyt group when com-
icant decrease of the elastic stress and the ultimate stress of the pared to the control group. As a result, the articular
midshaft cortical bone (Table 3). cartilage total thickness increased in both experimental
346 Muszyński et al.

Table 3 The physical,

mechanical, and geometric Item Group SEM P value
properties of tibia obtained from
42-day-old broilers in the control 0 Suppl Cu25% Cu25% + phyt
group and supplemented with Cu (n = 10) (n = 10) (n = 10)
in 25% of daily demand
dependently on phytase Bone general properties
supplementation Bone weight (g) 19.4a 20.1a 26.2b 0.9 P < 0.001
Bone length (mm) 110a 110a 115b 1 P < 0.001
Bone weight/bone length (g mm−1) 0.176a 0.182a 0.228b 0.01 P < 0.001
Bone geometrical properties
Horizontal internal diameter h (mm) 4.41a 5.08b 5.76c 0.20 P < 0.001
Horizontal external diameter H (mm) 6.83a 8.62b 11.18c 0.50 P < 0.001
Vertical internal diameter b (mm) 2.07a 4.46b 4.81b 0.33 P < 0.001
Vertical external diameter B (mm) 4.52a 7.44b 8.70c 0.49 P < 0.001
Cross section area A (mm2) 17.1a 32.6b 55.0c 4.5 P < 0.001
Mean relative wall thickness MRWT (–) 0.86a 0.71b 0.89a 0.04 P = 0.009
Cortical index CI (%) 44.3ab 40.9a 46.3b 1.1 P = 0.024
Midshaft volume (cm3) 0.76a 1.44b 2.53c 0.21 P < 0.001
Moment of inertia Ix (mm4) 30a 157b 348c 40 P < 0.001
Index of gyration Rg (mm) 1.31a 2.16b 2.45c 0.14 P < 0.001
Bone mechanical properties
Yield strength (N) 149a 158a 383b 29 P < 0.001
Ultimate strength (N) 239a 248a 549b 40 P < 0.001
Elastic stress (MPa) 133a 46b 61b 11 P < 0.001
Ultimate stress (MPa) 215a 69b 86b 18 P < 0.001
Bending moment (N·m) 1.64a 1.75a 4.41b 0.35 P < 0.001

Mean values in rows with different lowercase letters differ significantly at P < 0.05; data given are means
SEM standard error of the mean, 0 Suppl the control group without received Cu in premix, Cu25% the group
received Cu in 25% of daily demand from Cu-Gly, Cu25% + phyt the group received Cu in 25% of daily demand
from Cu-Gly with phytase

groups with the most significant increase being ob- Furthermore, the addition of phytase to the Cu-poor diet in
served in the Cu25% group supplemented with Cu with- the Cu25% + phyt group resulted in an increase of the total and
out the addition of phytase (Fig. 3). all particular zones of the growth plate cartilage as compared to

Table 4 The
histomorphometrical parameters Item Group SEM P value
of trabeculae of cancellous bone
in tibia obtained from 42-day-old 0 Suppl Cu25% Cu25% + phyt
broilers in the control group and (n = 10) (n = 10) (n = 10)
supplemented with Cu in 25% of
daily demand dependently on BV/TV (%) 16.2a 18.2b 21.5b 0.08 P < 0.001
phytase supplementation
Tb.Th mean (μm) 33.8a 55.5b 53.4b 1.69 P = 0.023
Tb.Th max (μm) 117a 150b 148b 7.45 P < 0.001
Tb.Sp mean (μm) 186a 287b 132a 13.00 P < 0.001
Tb.Sp max (μm) 597ab 696b 381a 39.70 P = 0.012
Fd (–) 1.54b 1.46a 1.62c 0.016 P < 0.001
Tb.N (mm−1) 4.75a 4.07a 6.13b 0.62 P < 0.001

Mean values in rows with different lowercase letters differ significantly at P < 0.05; data given are means
SEM standard error of the mean, BV/TV relative bone volume, Tb.Th trabecular thickness, Tb.Sp trabecular
separation, Fd fractal dimension of trabecular bone, Tb.N trabecular number, 0 Suppl the control group without
received Cu in premix, Cu25% the group received Cu in 25% of daily demand from Cu-Gly, Cu25% + phyt the
group received Cu in 25% of daily demand from Cu-Gly with phytase
Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with... 347

Fig. 3 The morphology of the articular cartilage and the growth plate of mean ± SD. a, b Values with different letters differ significantly at
tibia obtained from 42-day-old chickens treated with Cu in organic (Cu- P < 0.05. The description of the groups as in Fig. 1
Gly) form dependently on phytase supplementation. Data given are

the control group and in zone I and zone III when compared to staining pattern with safranin-O was observed around
the birds fed the Cu-poor diet without the phytase additive (Fig. chondrocytes (Fig. 4b, c). In addition, there was no evident
3). The Cu-poor diet without the phytase addition resulted in an gradient in the safranin-O staining within the control chickens
increase of the total, zone II, and zone IV thickness when com- fed the Cu-deprived diet and their articular cartilages had a
pared to the control 0 Suppl group (Fig. 3). very poor red staining pattern when compared to the other Cu-
Gly-supplemented groups (Fig. 4).
Proteoglycan Content in the Articular Cartilage

In all examined groups, the osteochondral junction was intact Distribution of Thick and Thin Collagen Fibers
and the surface of the cartilage was smooth without irregular- and Proteoglycans in the Articular Cartilage
ities according to Mankin’s semiquantitative scoring system
(Fig. 4). Proteoglycan staining with SO showed a lower pro- The structural information obtained from the analysis of
teoglycan content (displaying a weaker staining pattern) in the fibrous components in the PSR-stained section revealed
cartilage from the control group (Fig. 4a), while the chickens a difference between large (mature red-orange) and thin
treated with the Cu-Gly at the concentration of 25% of daily (immature green) collagen fibers. The supplementation
requirement demonstrated a higher, but only moderate, stain- of Cu (irrespective of phytase addition) enhanced thin
ing pattern linked with a higher content of proteoglycans. The (green) and decreased thick (red) fibers in the articular
concentration of proteoglycans in the Cu-Gly-supplemented cartilage resulting in greener radial fibers (Fig. 5).
groups, irrespective of the phytase addition, exhibited a grad- Moreover, thin collagen fibers (green) were distinctly
ual increase with the distance from the periphery of the carti- discernible in the layer located near the calcified carti-
lage to the end of zone I and loss of safranin-O staining from lage at the cartilage-bone interface (Fig. 5b, c). An op-
the beginning of zone II (Fig. 4b, c). The most intensive posite proportion was observed in the control birds.
348 Muszyński et al.

Fig. 4 The representative images

of safranin-O staining carried out
on formaldehyde-fixed sections
from the tibial articular cartilage
of 42-day-old chickens treated
with Cu in organic (Cu-Gly) form
dependently on phytase
supplementation. The cartilage
from the control group displayed
a very low proteoglycan content
(displaying weaker staining
pattern), while the chickens
treated with the Cu-Gly at the
concentration of 25% of daily
demand demonstrated higher but
not strong staining patterns linked
with a higher content of
proteoglycans. The most
intensive staining with safranin-O
was observed around
chondrocytes. Vertical section of
zone I and upper part of zone II of
the tibial articular cartilage from
the control group fed the Cu-
deficient control diet (a), the
Cu25% + phyt group (b), and the
Cu25% + phyt group (c).
Magnification ×200. The
description of the groups as in
Fig. 1

Hormonal Analysis the intensively reared animals and could adversely affect the
profitability of livestock production [34]. Additionally, to pre-
The results of the analysis of the growth hormone, leptin, and vent other health problems associated with rapid growth, the
bone turnover markers are presented in Fig. 6. It was shown broilers of the fast-growing breeds are fed restricted amounts of
that the addition of phytase to the Cu-poor diet for Ross 308 feed, especially during the rearing period [35, 36], which can
broiler increased the concentration of osteocalcin compared to lead to aggressive behavior around feeders [37]. The mecha-
the other groups, while the concentration of IGF-1 increased nisms for long bone growth are similar across most animal
only when compared to the control Suppl 0 group (Fig. 6). species, but the growth rate is the fastest in the proximal tibia
The experimental diets did not influence the serum concentra- of domestic fowl [38]. This, in combination with the general
tion of other examined hormones, namely, growth hormone weakness of the leg and other leg abnormalities in intensively
and leptin. reared poultry, can deprive the birds of locomotive freedom,
leading to suffering, discomfort, fear, and distress during rear-
ing [34]. The syndromes that cause lameness in the birds of a
Discussion flock can affect one or several musculoskeletal tissues of a
single bird or of the whole flock at the same time [39]. Weak
Adequate Cu intake in poultry production is necessary to not bones or tendons result in, e.g., dyschondroplasia,
only reach genetically optimal growth but also to maintain osteochondrosis, or bone loss. Thus, the potential beneficial
proper function of the skeletal system, which is an important effects of supplementation with trace elements in organic form
mineral source and which provides structural support for well- on bone health are being increasingly investigated [15, 28, 40].
muscled and fast-growing breeds [14, 25]. For this reason, the Such studies are necessary because supplementation with che-
overloading of long bones could result in locomotion problems lated forms of trace elements is an attractive strategy deserving
that have a negative impact on the performance and welfare of further evaluation in poultry breeding as traditional inorganic
Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with... 349

Fig. 5 The representative images

of PSR staining carried out on
formaldehyde-fixed sections from
the tibial articular cartilage of 42-
day-old chickens treated with Cu
in organic (Cu-Gly) form
dependently on phytase
supplementation. The large
collagen fibers are orange or red,
and the thick ones, including
reticular fibers, are green. Vertical
section of zone I of the tibial
articular cartilage from the control
group fed the Cu-deficient control
diet (a), the Cu25% + phyt group
(b), and the Cu25% + phyt group
(c). Magnification ×200. The
description of the groups as in
Fig. 1

mineral salts are often used at levels higher than the recom- Our study showed that phytase addition to broilers supple-
mended dosage in order to avoid trace mineral deficiency [28]. mented with 4 mg kg−1 of organic Cu (the Cu25% + phyt
Nevertheless, studies concerning how the skeletal system will group) increased the final body weight, growth rate, and per-
adapt to a Cu-poor diet are still limited and further studies are centage tibia ash as compared to the broilers whose diets were
therefore required, especially given that phytase is studied devoid of copper (the control 0 Suppl group) or those without
mainly in relation to the bioavailability of P [41]. added phytase (the Cu25% group). It was not supported by

Fig. 6 The serum concentration

of insulin-like growth factor 1
(IGF-1), growth hormone,
osteocalcin, and leptin in 42-day-
old chickens treated with Cu in
organic (Cu-Gly) form
dependently on phytase
supplementation. Data given are
mean ± SD. a, b Values with
different letters differ significantly
at P < 0.05. The description of the
groups as in Fig. 1
350 Muszyński et al.

other studies performed on broiler chickens that were fed con- control 0 Suppl group. In conclusion, it can be assumed that
trol Cu-deficient diets, where increase of body weight after Cu the supplementation of Cu-Gly, even at lowered levels, posi-
supplementation was observed at lowered levels [4, 42]. tively influenced bone metabolism resulting in more mature
Furthermore, Cu bone content increased in both Cu- bone and better mechanical strength in growing broilers. It is
supplemented groups when compared to the control 0 Suppl in agreement with results from another study performed on
group. These findings differed from those presented by Bao Cu-deficient chickens [4]. The bones of birds from the Cu-
et al. [42], where supplementation with organic Cu at 50% of deficient control group had lower tolerance to deformation
the recommended dose had no effect on the total Cu content of and were able to absorb less energy before fracture.
tibia when compared to the Cu-deficient control group. Our Moreover, they demonstrated a reduced range of plastic de-
results also did not support studies showing that dietary formation. However, even the lowest level of supplementation
phytase supplementation improved mineral availability (P, (2 ppm) was shown to be sufficient in preventing the bone
Ca, Cu, Mg, Fe) or Ca retention [43]. Our experimental diets changes seen in the Cu-deficient birds [4]. It seems that the
containing Cu at 25% of the recommended dose increased Cu, amount of Cu needed to maintain the mechanical integrity of
Ca, P, and ash content in bone irrespective of phytase addition, bone is substantially less than that required for growth [4].
while the serum Ca concentration was decreased by phytase Another study showed that after supplementing the diet with
supplementation. There are a few studies showing no effect of phytase, the tibia became stronger [41]. It is suggested that
dietary phytase supplementation on selected minerals [44, 45]. phytase supplementation results in better mineralization by
Furthermore, although serum Ca concentration decreased af- improving dietary quality through the release of other trace
ter phytase supplementation when compared to the Ca25% minerals [17]. However, in contrast to our study, diets de-
group and bone Ca and P content increased in both Cu- scribed contained higher level of phytase (600–1050
supplemented groups, the plasma and the bone Ca/P ratios FTU kg−1) and different level of phosphorus [17, 41].
remained unchanged. Probably, the dietary P and Ca concen- More detailed comparison of our findings with those from
tration, bird breed, and age impacted Ca retention after other studies is somewhat complicated, as no other studies spe-
phytase supplementation while having no effect in P retention. cifically into the effects of dietary phytase supplementation in
Similar observation was made by Chung et al. [43]. Cu-poor diets are available. Our study has shown improvement
Our broiler chickens from the Cu25% + phyt group had of the bone development in all Cu-Gly-treated broilers.
longer and heavier tibiae with an increased bone weight/bone However, the reserve (I), proliferative (II), and hypertrophic
length ratio. Different results were obtained in the other study (III) zones in the tibia’s growth plate cartilage of the Cu25% +
with the Cu-deficient chickens. Here, no difference was ob- phyt chickens were wider when compared to other birds. The
served in the body weight or in the basal morphology of tibia histological variation in birds with Cu deficiency suggested a
between chickens fed the Cu-poor diet and of those from the defective or disorganized proliferation and mineralization within
Cu-deficient control group [46]. There is also another study the analyzed zones of the growth plate. Thus, decreased bone
conducted with Cu-Gly given to growing chickens throughout mechanical resistance observed in the control 0 Suppl group
a 6-week period which showed that the reduction of dietary could be linked with a failure in the mineral deposition within
copper to 25% of the recommended level resulted in an in- the extracellular matrix of the bone. Furthermore, the chickens
crease in bone’s weight but without changes to its length [15]. fed the control 0 Suppl diet showed strongly osteoporotic can-
In our study, a significant alteration was noted in bone cellous bone. The widened reserve and hypertrophy zones ob-
geometry, which was dependent on phytase supplementation. served in the Cu25% + phyt group could be due to the increased
Our broilers from the Cu25% + phyt group had more mature number of differentiating chondrocytes that resulted in the accu-
bones with a greater cross-sectional area of the midshaft which mulation of cells within particular zones after Cu and phytase
resulted in the increase of its mechanical strength and in de- supplementation. Also, an increase in bone ash mineral content
creased stresses. There was a 129 and 121% increase in the and the trabecular number relating to the supplementation of
ultimate strength of the bones in birds supplemented with phytase and Cu-Gly were observed in the present study. Thus,
phytase (Cu25% + phyt), compared to the control Cu- it is reasonable to speculate that the association of osteocalcin
deficient (0 Suppl) or Cu-poor diet without the addition of with an increased cell proliferation and maturation in the growth
phytase (Cu25%), respectively. Significantly increased value plate of our phytase-treated chickens mediated by the increase of
in yield strength revealed an increase to the extent of elastic IGF-1 concentration could be the main cause of the improve-
deformation after phytase supplementation. However, al- ment of bone development. The results obtained in the current
though bones of birds which form the Cu25% + phyt group study also indicate that the positive effects of phytase inclusion in
were deforming and breaking under significantly higher loads, the Cu-Gly diet on growth and development were mediated by
the reduced values of yield and ultimate stresses showed that improved activity of the somatotropic axis, as bone and cartilage
tibiae from both Cu-supplemented groups were subjected to homeostasis is regulated by GH acting via IGF-1 stimulator of
lower mechanical stresses before fractures as compared to the the proliferation which was increased by 37% in the Cu25% +
Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with... 351

phyt group. Some studies suggest an important role of nutrition metabolism is directly associated with many physiological
in the regulation of serum hormones like growth hormone, or processes of biological adaptation. Moreover, the bone or-
IGF-1, one of somatomedins [47]. Nevertheless, the mechanism ganic matrix contributes to bone elasticity and changes in
for exogenous enzymes—like phytase—on hormone regulation the composition and spatial arrangement of soft bone tis-
is problematic and requires further examination. Currently, there sues may also be responsible for the improved mechanical
are a lack of studies relating to phytase supplementation and the strength of bones observed in the experimented Cu-
somatotropic axis. However, the study on dietary supplementa- supplemented groups [25].
tion with the enzyme xylanase reported an increase in the con- The present study also revealed many side effects of the Cu-
centration of blood IGF-1 in 21-day-old broilers. This indicates deficient diet, e.g., very thin articular cartilage, especially the
that enhanced digestion and absorption of nutrients caused by shortening of the superficial (I) zone. It can result in accelerated
enzyme supplementation has effects on blood hormone concen- degradation of the articular cartilage linked with the loss of its
tration [48]. elasticity causing difficulties in movement. Furthermore, it can
The non-collagenous proteins contribute to a variety of provoke irreversible deformation caused by the impact of the
functions within the bone such as matrix stabilization, cal- load during movement. Finally, the diet devoid of Cu supple-
cification, and other metabolic regulatory activities. mentation resulted in very low proteoglycan content in the
Approximately 10% of different non-collagenous proteins articular cartilage (Fig. 4). Proteoglycans also provide stability
are expressed by osteoblasts and chondrocytes, and expres- to the articular cartilage, and the degradation of proteoglycans
sion of these proteins is considered to be a specific marker entails destabilization of the collagen network.
[49, 50]. Neither the proteoglycan content nor the alter-
ation in collagenous fibers in the articular cartilage was
reported in broiler chickens after nutritional modification Conclusions
with phytase supplementation. Based on the obtained re-
sults, it can be concluded that both the presence of Cu in To the best our knowledge, this is the first study that has
the diet and the phytase inclusion significantly enhanced examined both the mechanical properties of bone as well as
the content of proteoglycans in the articular cartilage (Fig. the histomorphometry of cancellous bone and hyaline carti-
4). The phytase addition could exert a protective effect on lage in chickens fed a diet low or deficient in copper but also a
chickens’ articular cartilage in which degenerative changes diet low in copper in relation to phytase supplementation.
will occur much later when animals are fed the Cu- Irrespective of the phytase presence, the organic form of cop-
deprived or Cu-poor diet, as was shown in the morphom- per can lead to changes within the articular cartilage, as was
etry and the distribution of proteoglycans (Fig. 4). The indicated by the morphological analysis and the proteoglycan
phytase inclusion improved the morphology of the articu- content. Phytase supplementation showed beneficial effects
lar cartilage, and its beneficial effect was particularly man- on differentiation in chicken growth plate chondrocytes, there-
ifest in the widening of the superficial (I) zone. This by sustaining their proliferative state and maintaining their
modification can positively alter the load distribution in sensitivity to growth factors, such as IGF-1. Furthermore,
the joint and improve the elasticity of the articular carti- the phytase addition influenced the trabecular architecture of
lage, protecting it against the degradation in well-muscled the tibia with subsequent improvement of its growth and de-
and fast-growing broilers. Furthermore, the increase of the velopment. However, the response of the skeletal system to
thickness of the transitional (II) zone can be equally ben- phytase supplementation could differ among avian species
eficial, as it can also improve the transfer and distribution due to differences in physiology of the digestive tract and
of the load through the joint. It can have other functional gut microflora.
consequences, as the physical properties of articular carti-
lage are determined by the diversity in the components of
Acknowledgements The authors wish to thank ARKOP Sp. z o.o.
the matrix and the difference in the quantitative mutual (Bukowno, Poland) for providing the necessary organically complexed
relations of collagen fibers. The content of proteoglycans copper (GLYSTAR FORTE Cu) used for the purposes of this experiment.
plays an essential role in the destabilization of the colla- This work was financially supported by the project NN 311543540 from
gen network [49]. The proportion of thin and thick fibers the Ministry of Science and Higher Education, Poland.
should give additional structural information about the in-
fluence of phytase addition on collagen synthesis in the Compliance with Ethical Standards The experimental procedures
articular cartilage (Fig. 5). The fine (green) collagen fibers used throughout this study were approved by the Local Ethics
Committee on Animal Experimentation of the University of Life
might indicate an ongoing process of collagen synthesis
Sciences of Lublin, Poland.
occurring after Cu supplementation, the intensity of which
was phytase-dependent (Fig. 5). Collagen is a major com- Conflict of Interest The authors declare that they have no conflict of
ponent of the extracellular matrix of many tissues, and its interest.
352 Muszyński et al.

Open Access This article is distributed under the terms of the Creative 14. Świątkiewicz S, Koreleski J, Zhong DQ (2001) The bioavailability
Commons Attribution 4.0 International License (http:// of zinc from inorganic and organic sources in broiler chickens as
creativecommons.org/licenses/by/4.0/), which permits unrestricted use, affected by addition of phytase. J Anim Feed Sci 10:317–328
distribution, and reproduction in any medium, provided you give appro- 15. Kwiecień M, Winiarska-Mieczan A, Zawiślak K, Sroka S (2014)
priate credit to the original author(s) and the source, provide a link to the Effect of copper glycinate chelate on biomechanical, morphometric
Creative Commons license, and indicate if changes were made. and chemical properties of chicken femur. Ann Anim Sci 14:127–
139. doi:10.2478/aoas-2013-0085
16. Sebastian S, Touchburn SP, Chavez ER (1998) Implications of
phytic acid and supplemental microbial phytase in poultry nutrition:
a review. World Poult Sci J 54:27–47. doi:10.1079/WPS19980003
References 17. Aoyagi S, Baker DH (1995) Effect of microbial phytase and 1,25-
dihydroxycholecalciferol on dietary copper utilization in chicks.
1. Hunt CD (1998) Copper and boron as examples of dietary trace Poult Sci 74:121–126
elements important in bone development and disease. Curr Opin 18. European Commission (2003) European Communities Directive
Orthop 9:28–36 no. 1334/2003 of the European Parliament and the Council. OJ
L187:11–15
2. Tomaszewska E, Dobrowolski P, Kwiecień M, Winiarska-Mieczan
19. Aviagen (2014) Ross 308 broiler nutrition specification. Aviagen
A, Tomczyk A, Muszyński S (2017) The influence of the dietary
Incorporated Publishing, Huntsville
cu-glycine complex on the histomorphology of cancellous bone,
20. AOAC (2000) Official methods of analysis of AOAC
articular cartilage, and growth plate as well as bone mechanical
INTERNATIONAL, 17th edn. AOAC International, Gaithersburg
and geometric parameters is dose dependent. Biol Trace Elem Res
21. Frühbeck G, Alonso R, Marzo F, Santidrián S (1995) A modified
178:54–63. doi:10.1007/s12011-016-0894-x
method for the indirect quantitative analysis of phytate in food-
3. Rodríguez JP, Ríos S, González M (2002) Modulation of the pro-
stuffs. Anal Biochem 225:206–212. doi:10.1006/abio.1995.1145
liferation and differentiation of human mesenchymal stem cells by
22. Žilić SM, Božović IN, Savić S, Šobajić S (2006) Heat pro-
copper. J Cell Biochem 85:92–100. doi:10.1002/jcb.10111
cessing of soybean kernel and its effect on lysine availability
4. Opsahl W, Zeronian H, Ellison M, Lewis D, Rucker RB, Riggins
and protein solubility. Cent Eur J Biol 4:572–583. doi:10.
RS (1982) Role of copper in collagen cross-linking and its influence
2478/s11535-006-0039-x
on selected mechanical properties of chick bone and tendon. J Nutr
23. Tomaszewska E, Kwiecień M, Dobrowolski P, Klebaniuk R,
112:708–716
Muszyński S, Olcha M, Blicharski T, Grela ER (2016) Dose-
5. Hong HH, Pischon N, Santana RB, Palamakumbura AH, Chase dependent effects of probiotic supplementation on bone character-
HB, Gantz D, Guo Y, Uzel MI, Ma D, Trackman PC (2004) A role istic and mineralization in female turkeys. Anim Prod Sci. doi:10.
for lysyl oxidase regulation in the control of normal collagen depo- 1071/AN16289
sition in differetnting osteoblast cultures. J Cell Physiol 200:53–62. 24. Ferretti JL, Capozza RF, Mondelo N, Montuori E, Zanchetta JR
doi:10.1002/jcp.10476 (1993) Determination of femur structural properties by geometric
6. Tomaszewska E, Dobrowolski P, Kwiecień M (2016) Alterations in and material variables as a function of body weight in rats. Evidence
intestinal and liver histomorphology, and basal hematological and of sexual dimorphism. Bone 14:265–270
biochemical parameters in relation to different sources of dietary 25. Muszyński S, Kwiecień M, Tomaszewska E, Świetlicka I,
copper in adult rats. Ann Anim Sci. doi:10.1515/aoas-2016–0056 Dobrowolski P, Kasperek K, Jeżewska-Witkowska G (2017)
7. Yenice E, Mızrak C, Gültekin M, Atik Z, Tunca M (2015) Effects of Effect of caponization on performance and quality characteristics
organic and inorganic forms of manganese, zinc, copper, and chro- of long bones in Polbar chickens. Poult Sci 96:491–500. doi:10.
mium on bioavailability of these minerals and calcium in late-phase 3382/ps/pew301
laying hens. Biol Trace Elem Res 167:300–307. doi:10.1007/ 26. Tomaszewska E, Kwiecień M, Muszyński S, Dobrowolski P,
s12011-015-0313-8 Kasperek K, Blicharski T, Jeżewska-Witkowska G, Grela ER
8. Scott A, Vadalasetty KP, Sawosz E, Łukasiewicz M, Vadalasetty (2017) Long bone characteristics are altered by caponization and
RKP, Jaworski S, Chwalibog A (2016) Effect of copper nanoparti- breed different. Br Poult Sci. doi:10.1080/00071668.2017.1280770
cles and copper sulphate on metabolic rate and development of 27. Dobrowolski P, Tomaszewska E, Kurlak P, Pierzynowski SG
broiler embryos. Anim Feed Sci Technol 220:151–158. doi:10. (2016) Dietary 2-oxoglutarate mitigates gastrectomy–evoked struc-
1016/j.anifeedsci.2016.08.009 tural changes in cartilage of female rats. Exp Biol Med 241:14–24.
9. Banks KM, Thompson KL, Rush JK, Applegate TJ (2004) Effects doi:10.1177/1535370215595466
of copper source on phosphorus retention in broiler chicks and 28. Tomaszewska E, Dobrowolski P, Kwiecień M, Wawrzyniak A,
laying hens. Poult Sci 83:990–996 Burmańczuk N (2016) Comparison of the effect of a standard in-
10. National Research Council (1994) Nutrient requirements of poultry, clusion level of inorganic zinc to organic form at lowered level on
9th edn. National Academic Press, Washington. doi:10.17226/2114 bone development in growing male Ross broiler chickens. Ann
11. Bao YM, Choct M, Iji PA, Bruerton K (2007) Effect of organically Anim Sci 16:507–519. doi:10.1515/aoas-2015-0087
complexed copper, iron, manganese and zinc on broiler perfor- 29. Bobinac D, Spanjol J, Zoricic S, Maric I (2003) Changes in articular
mance, mineral excretion and accumulation in tissues. J Appl cartilage and subchondral bone histomorphometry in osteoarthritic
Poult Res 16:448–455. doi:10.1093/japr/16.3.448 knee joints in humans. Bone 32:284–290
12. Tomaszewska E, Dobrowolski P, Kwiecień M, Burmańczuk N, 30. Tomaszewska E, Dobrowolski P, Winiarska-Mieczan A,
Badzian B, Szymańczyk S, Kurlak P (2014) Alterations of liver Kwiecień M, Tomczyk A, Muszyński S (2017) The effect
histomorphology in relation to copper supplementation in inorganic of tannic acid on the bone tissue of adult male Wistar rats
and organic form in growing rats. Bull Vet Inst Pulawy 58:479–486. exposed to cadmium and lead. Exp Toxicol Pathol 69:131–
doi:10.2478/bvip-2014-0073 141. doi:10.1016/j.etp.2016.12.003
13. Tomaszewska E, Dobrowolski P, Kwiecień M (2016) Intestinal 31. Tomaszewska E, Dobrowolski P, Siwicki AK (2012) Maternal
alterations, basal hematology and biochemical parameters in ado- treatment with dexamethasone at minimal therapeutic doses inhibits
lescent rats fed different sources of dietary copper. Biol Trace Elem neonatal bone development in a gender-dependent manner. Livest
Res 171:185–191. doi:10.1007/s12011-015-0522-1 Sci 146:175–182. doi:10.1016/j.livsci.2012.03.008
Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with... 353

32. Suvara SK, Layton C, Bancroft JD (2013) Bancroft’s theory and 43. Chung TK, Rutherfurd SM, Thomas DV, Moughan PJ (2013)
practice of histological techniques, 7th edn. Churchill Livingstone Effect of two microbial phytases on mineral availability and reten-
Elsevier, Oxford tion and bone mineral density in low-phosphorus diets for broilers.
33. Rich L, Whittaker P (2005) Collagen and picrosirius red staining: a Br Poult Sci 54:362–373. doi:10.1080/00071668.2013.783902
polarized light assessment of fibrillar hue and spatial distribution. 44. Rutherfurd SM, Chung TK, Thomas DV, Zou ML, Moughan PJ
Braz J Morphol Sci 22:97–104 (2012) Effect of a novel phytase on growth performance, apparent
34. Mendes AS, Paixão SJ, Sikorski RR, Bonamigo DV, Morello MG, metabolizable energy, and the availability of minerals and amino
Ponzoni RAR (2016) Photogrammetry: a non-invasive and objec- acids in a low-phosphorus corn-soybean meal diet for broilers.
tive method for detecting locomotion problems in broiler chickens. Poult Sci 91:1118–1127. doi:10.3382/ps.2011-01702
Braz J Poult Sci 18:573–580. doi:10.1590/1806-9061-2015-007 45. Um JS, Lim HS, Ahn SH, PaikK IK (2000) Effects of microbial
35. van Krimpen MM, de Jong IC (2014) Impact of nutrition on welfare phytase supplementation to low-phosphorus diets on the perfor-
aspects of broiler breeder flocks. World Poult Sci J 70:139–150. mance and utilization of nutrients in broiler chickens. Asian
doi:10.1017/S0043933914000129 Australas J Anim Sci 13:824–829. doi:10.5713/ajas.2000.824
36. De Jong IC, Jones B (2006) Feed restriction and welfare in domes- 46. Riggins RS, Cartwright AG, Rucker RB (1979) Viscoelastic prop-
tic birds. In: Bels V (ed) Feeding in domestic vertebrates. CABI, erties of copper deficient chick bone. J Biomech 12:197–203
Wallingford, pp 120–135 47. Hajati H, Rezaei M, Sayyahzadeh H (2009) The effects of
37. Hocking PM, Jones EKM (2006) On-farm assessment of environ- enzyme supplementation on performance, carcass character-
mental enrichment for broiler breeders. Br Poult Sci 47(4):418–425. istics and some blood parameters of broilers fed on corn-
doi:10.1080/00071660600825074 soybean meal-wheat diets. Int J Poult Sci 8:1199–1205.
38. Kirkwood JK, Spratt DMJ, Duignan PJ, Kember NF (1989) doi:10.3923/ijps.2009.1199.1205
Patterns of cell-proliferation and growth-rate in limb bones of the
48. Gao F, Jiang Y, Zhou GH, Han ZK (2008) The effects of xylanase
domestic-fowl (Gallus-domesticus). Res Vet Sci 47:139–147
supplementation on performance, characteristics of the gastrointes-
39. Mench J (2004) Lameness. In: Weeks C, Butterworth A (eds)
tinal tract, blood parameters and gut microflora in broilers fed on
Measuring and auditing broiler welfare. CABI, Wallingford, pp
wheat-based diets. Anim Feed Sci Technol 142:173–184. doi:10.
3–17
1016/j.anifeedsci.2007.07.008
40. Tomaszewska E, Muszyński S, Dobrowolski P, Kwiecień M,
Winiarska-Mieczan A, Świetlicka I, Wawrzyniak A (2017) Effect 49. Tomaszewska E, Dobrowolski P, Bieńko M, Prost Ł, Szymańczyk
of zinc level and source (zinc oxide vs zinc glycine) on bone me- S, Zdybel A (2015) Effects of 2-oxoglutaric acid on bone mor-
chanical and geometric parameters, and histomorphology in male phometry, densitometry, mechanics, and immunohistochemistry in
Ross 308 broiler chicken. Braz J Poult Sci 19:159–170. doi:10. 9-month-old boars with prenatal dexamethasone-induced
1590/1806-9061-2016-0285 osteopenia. Connect Tissue Res 56:483–492. doi:10.3109/
41. Qian H, Veit HP, Kornegay ET, Ravindran V, Denbow DM (1996) 03008207.2015.1069822
Effects of supplemental phytase and phosphorus on histological and 50. Tomaszewska E, Dobrowolski P, Prost Ł, Hułas-Stasiak M,
other tibial bone characteristics and performances of broilers fed Muszyński S, Blicharski T (2016) The effect of supplementation
semi-purified diets. Poult Sci 75:618–626 of glutamine precursor on the growth plate, articular cartilage and
42. Bao YM, Choct M, Iji PA, Bruerton K (2010) Trace mineral inter- cancellous bone in fundectomy-induced osteopenic bone. J Vet
actions in broiler chicken diets. Br Poult Sci 51:109–117. doi:10. Med Sci 78:563–571. doi:10.1292/jvms.15-0385
1080/00071660903571904