Bull Vet Inst Pulawy 48, 461-465, 2004
INFLUENCE OF ALPHA-KETOGLUTARATE
ON CORTICAL BONE DENSITY, GEOMETRICAL PROPERTIES
AND MECHANICAL ENDURANCE
OF THE HUMERUS IN TURKEYS
MARCIN R. TATARA, PIOTR MAJCHER1, WITOLD KRUPSKI2,
STEFAN G. PIERZYNOWSKI3 AND TADEUSZ STUDZIŃSKI
Department of Animal Physiology, Faculty of Veterinary Medicine,
Agricultural University of Lublin, 20-033 Lublin, Poland
1
Department of Orthopaedy, Traumatology and Rehabilitation,
2
2-nd Department of Radiology,
Medical University of Lublin, 20-50 Lublin, Poland
3
Department of Cell and Organism Biology,
Lund University, SE-223 62 Lund, Sweden
e-mail: [email protected]
Received for publication April 19, 2004.
Abstract
The aim of this study was to estimate an influence of
alpha-ketoglutarate (AKG) on volumetric bone density and
geometrical and mechanical properties of the humerus in farm
turkeys. All birds were kept under standard rearing conditions
with constant access to water and appropriate feed, supplied in
accordance with the stage of production cycle. Bone samples
were collected 14 weeks after beginning of the experiment. To
the first group belonged the control turkeys. The second group of
animals consisted of turkeys that were treated with AKG from
the 22nd to 110th d of life. Using Quantitative Computed
Tomography (QCT) method, volumetric bone density of the
cortical bone in the humerus was estimated. Both the mechanical
and geometrical properties of the bone were assessed according
to Ferretti method. The obtained results showed anabolic
influence of enteral AKG administration on cortical bone
density, geometrical properties and mechanical endurance of the
humerus. Moreover, the humerus in turkey may be utilised as a
model of cortical bone for further investigations of factors
conditioning skeletal homeostasis.
Key words: turkeys, alpha-ketoglutarate,
humerus.
Bone tissue in vertebrates fulfils three main
functions such as supportive and locomotory, protective
for the central nervous system and bone marrow, and
metabolic as the main reservoir of calcium and
phosphorus in the body. The locomotory and protective
functions in vertebrates are generally provided by the
cortical part of bone tissue, whereas the trabecular bone is
characterised by relatively high rate of metabolic
processes (16). As opposite to the femur, tibia or ulna that
consist the cortical and the trabecular bone in turkey, the
humerus is built of the cortical bone only. It enables the
utilisation of this bone in turkey as an experimental model
for investigations of factors determining volumetric bone
density, geometrical properties, and mechanical
endurance of long bones, conditioned by cortical bone
only.
The advances made in the field of genetics and
nutrition during last 30 years have resulted in increased
growth rates and body weight in livestock (4, 5, 15). On
the other hand, the incidence of skeletal system growth
problems, such as rickets, avian tibial dyschondroplasia
and osteochondrosis rapidly increased as well. To
guarantee proper skeletal growth and development, the
growth plate cartilage that regulates long bone
development, have to maintain a tightly controlled
balance between cartilage synthesis and degradation as
well as chondrocytes proliferation and apoptosis (15). In
rapidly developing bones like the tibia, femur, humerus or
ulna in poultry, the growth plate cartilage determines the
rate of longitudinal growth as well as the ultimate length
and width of bones. The investigations of high rate
skeletal development in vertebrates elucidated that the
growth plate chondrocytes turnover in broiler chickens is
four times faster than in rabbits (15). Due to their
intensive growth and relatively short fattening time (about
16 weeks in Poland), turkeys are an attractive
experimental model for studies on conditions that
determine proper growth, development, and healing of the
skeletal system (20, 22). Moreover, the turkey model
offers a possibility of investigations of feeding,
physiological, pharmacological, and toxicological factors
influencing development and mineralisation of the
skeletal system (21).
Alpha-ketoglutarate (AKG) is a molecule
determining the overall rate of the citric acid cycle in the
organism. It is a nitrogen scavenger and a source of
462
glutamine that stimulates protein synthesis and inhibits
protein degradation in muscles. Moreover, AKG as a
precursor of glutamine is a metabolic fuel for the
gastrointestinal tract (9, 10). Glutamate released from
nervous fibers penetrates into bone tissue and regulates
its metabolism. It is synthesised by the reductive
amination of AKG in perivenous hepatocytes as well
(14, 17, 24, 26). Together with ascorbate, and Fe2+,
AKG steers hydroxylation of peptide-bound proline to
hydroxyproline via prolyl hydrolase, increasing the
conversion of pro-collagen to collagen and bone matrix
formation (12, 23).
To investigate the influence of alphaketoglutarate enteral administration on growth,
development, and mineralisation of cortical bone in
turkey, the volumetric bone density and geometrical and
mechanical properties of the humerus were estimated.
Material and Methods
Experimental
design
and
sampling
procedure. The study was performed on 16 HB-91 male
turkeys that were kept under standard rearing conditions
with a constant access to fresh water, appropriate feed
supplied in accordance with the stage of production
cycle, and air temperature set at the level of 28oC. The
experiment was conducted from the beginning of the 4th
week after hatching until the moment that the production
cycle was completed i.e. in the 17th week of life. On the
22nd d of life, all experimental turkeys were divided into
two weight-matched groups. The first group (Group 1)
consisted of 8 turkeys that received placebo
(physiological saline – PhS). To the second group
(Group 2) belonged 8 turkeys that were treated with
alpha-ketoglutarate (AKG) in the dosage of 0.4 g/kg
body mass per day. Both AKG and PhS were
administered every day throughout 14 weeks at the same
volumes, directly into the crop with the use of a tube.
After 14 weeks of the experiment, the turkeys were
slaughtered; left and right humeri were X-rayed, isolated
and frozen at –25oC for further analyses.
Volumetric bone density. Using Quantitative
Computed Tomography (QCT) method and SOMATOM
AR. T – SIEMENS apparatus supplied with VR 3
software, the volumetric bone density of the humerus
was determined. Bone density of the cortical bone was
measured using 2 mm thick, cross sectional, diaphyseal
QCT scans at approximately 40 and 47.5% of humerus
length along the longitudinal axis, measuring from distal
extremities of this bone.
Analysis of mechanical and geometrical
properties. Mechanical properties of right and left
humeri were estimated using three-point bending test
according to Ferretti`s et al. (7, 8) method in INSTRON
4302 apparatus linked with a computer, registering
relationship between forces perpendicular to length of
bone and resulting in displacement. Results were
presented graphically and maximum elastic strength
(Wy) and ultimate strength (Wf) were estimated. On the
basis of the measurement of horizontal and vertical
diameters of cross section of the humerus, both external
and internal diameters, cross-sectional area (A), second
moment of inertia (Ix), mean relative wall thickness
(MRWT) and cortical index (CI) were estimated (2, 7,
8).
Statistical analysis. All data are presented as
mean ± standard error (±S.E.). Statistical analyses were
performed using Statistica software (version 5.1). All
investigated parameters in both groups of turkeys were
found to be normally distributed in accordance to
Kolomogorov-Smirnov test. The Student’s t-test was
used to determine statistical significance of differences
in variables between the analysed groups. A significance
level of P≤0.05 was used for all comparisons.
Results
The body mass of turkeys at the beginning of
the 4th week was 522.6 g (±30.6) in control group and
520.7 g (±9.25) in AKG treated group, whereas at the
age of 17 weeks its value reached 8602.5 g (±304.1) and
8678 g (±395.2), respectively. No significant differences
between the investigated groups were found in body
mass both at the beginning and at the end of the
experiment (P = 0.95 and P = 0.88). Considering the
weight and length of the humerus no significant
differences between experimental and control groups
were observed as well (P = 0.15 and P = 0.18) (Table 1).
Results of volumetric bone density measurement of the
cortical bone showed its significantly higher values in
AKG treated animals. 14.93% and 12.69% increase in
volumetric bone density of cortical bone was obtained in
AKG group, analysing measuring scans placed in the 40
and 47.5% of humerus length, respectively (Fig. 1). The
administration of alpha-ketoglutarate for 14 weeks
induced higher values of all analysed geometrical
parameters and the obtained differences were
statistically significant (Table 1). The analysis of
mechanical properties of the humerus revealed
significantly higher values of the maximum elastic
strength of the bone in turkeys that received AKG, when
compared to the control group. The ultimate strength of
the humerus was significantly increased in the AKG
treated birds as well (Table 1).
Discussion
Due to different properties and metabolic rate,
the investigation on cortical bone model is
complementary to the models including both the cortical
and the trabecular bone. To evaluate an influence of
various factors determining bone development, growth,
and skeletal homeostasis in mammals the calvaria was
used as a cortical bone model in numerous studies (18,
19). Because of very intensive growth and development
of the skeletal system in poultry they are attractive for
investigations in this field. The humerus of turkey is
pneumatic and it is built-up from the cortical bone, what
makes this bone suitable for investigations on cortical
bone properties in birds.
463
Table 1
Characteristics of turkey humerus
Investigated parameter
Group 1
(control group)
Group 2
(AKG treated group)
Number of investigated bones
16
16
Weight (g)
48.2 (±2.32)
52.86 (±2.24)
Length (mm)
138.75 (±1.25)
136.37 (±1.24)
Cross-sectional area (mm2)
49.5 (±2.04)
*64.43 (±2.42)
4
Second moment of inertia (mm )
779.8 (±38.5)
*987.7 (±68.7)
Mean relative wall thickness
0.244 (±0.014)
*0.329 (±0.008)
Cortical index
19.39 (±0.97)
*24.6 (±0.47)
Maximum elastic strength (N)
626 (±26.7)
*782 (±36.9)
Ultimate strength (N)
1059 (±47.4)
*1188 (±45.1)
3
Cd [g /cm ]
* statistically significant differences (P<0.05) between the control and AKG treated group parameters.
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
*
*
Cd I
Cd I
Cd II
Control
AKG
Cd II
Fig. 1. Volumetric bone density of the cortical bone (Cd) of the humerus in placebo (control) and AKG treated turkeys. The measured scans
were placed at the 40% (Cd I) and 47.5% (Cd II) of total humerus length. * Statistically significant differences (P<0.001) between the
investigated groups.
Quantitative Computed Tomography (QCT)
method and estimation of geometrical and mechanical
properties of bones are commonly accepted methods,
making it possible to assess the quality of the skeletal
system in vertebrates. They also enable in-depth analysis
of the changes occurring during development, growth,
and bone metabolism modulation (20, 22). QCT is noninvasive in determination of the volumetric bone density
(g/cm3) and enables the assessment of skeletal system
mineralisation. In comparisons to other methods like
DEXA (Dual-energy X-ray Absorptiometry) that
determines areal bone mineral density (g/cm2) - BMD, it
guarantees the measurements without errors occurring in
the case of DEXA due to presence of surrounding soft
tissues (6, 13). Considering that even 0.1% X-ray
attenuation is detectable by QCT, the application of this
method in investigation of the skeletal system in
vertebrates enables very precise bone density
measurement (20, 25). Moreover, it enables to assess
changes in the bone density that can be difficult to state
by simple bone weight measurement. The mechanical
properties of bones are conditioned mainly by the bone
structure, their mineralisation, maturity, and geometrical
properties which include such parameters as crosssectional area, second moment of inertia, mean relative
wall thickness and cortical index. The quality of the
material and the structure of the trabecular bone, however,
play the decisive role (2, 7, 8, 20). Considering the
humerus that consists of no trabecular bone in turkey, its
mechanical endurance is provided due to geometrical
properties, cortical bone density and quality of bone tissue
only.
The obtained results prove advantageous
influence of alpha-ketoglutarate administration via
digestive tract on humerus quality in turkeys slaughtered
at the age of 17 weeks. Similar body mass in the
464
beginning of the experiment and no statistically
significant difference in the final body mass value
between the investigated groups confirm that geometrical
properties, mechanical endurance and volumetric bone
density of the humerus were not influenced by
differentiated body mass of turkeys. The increased
volumetric bone density in the mid-shaft of this bone
indicates that the bone tissue synthesis and mineralisation
was higher in the cortical bone. This anabolic effect on
bone tissue can be explained by digestive processes that
are influenced by enteral AKG, which acts as an energy
donor and as a substrate for the synthesis of amino acids,
mainly proline. Improved proline synthesis, regulated by
AKG, ascorbate, and Fe2+ increases its following
hydroxylation to hydroxyproline that is a principal amino
acid of collagen, main organic constituent of bone tissue
(12, 21, 23). Increase in the geometrical properties like
cross-sectional area, second moment of inertia, mean
relative wall thickness and cortical index may indicate
that AKG may stimulate chondrocytes proliferation on the
epiphyseal cartilage level that is responsible for
longitudinal growth and bone width (15). Similar effect of
AKG was observed in other investigations performed on
the osteotomised and denervated ulna model in turkey
(20). The higher values of MRWT and CI of the humerus
in experimental turkeys indicate that the cortical bone
thickness was increased due to 14 - week AKG
administration. Due to the higher volumetric bone density
of the cortical bone and risen geometrical properties, the
higher mechanical endurance of the humerus was
demonstrated as well. These results are in accordance
with other studies performed on rat, pig, and turkey
models (1, 11, 20, 21).
Next to the mechanism of increased proline
synthesis, the other action that may be engaged in bone
metabolism regulation by AKG is enhanced glutamate
synthesis and its utilisation as neuro-signaling by the
peripheral nervous system, which was previously reported
(3, 24).
All obtained results indicate that enteral AKG
administration in turkeys improves skeletal system quality
by anabolic effect on cortical bone formation. Due to
positive effect of dietary supplementation of AKG on
humerus properties it may be considered as an efficient
factor in prevention of skeletal disorders during fast
growth and development in turkeys and perhaps other
poultry species.
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