Bull Vet Inst Pulawy 49, 117-120, 2005
EFFECT OF ALPHA-KETOGLUTARATE GIVEN
TO PREGNANT SOWS ON THE DEVELOPMENT
OF THE HUMERUS AND FEMUR IN NEWBORNS
EWA ŚLIWA, SYLWESTER KOWALIK, MARCIN R. TATARA, WITOLD KRUPSKI1,
PIOTR MAJCHER2, IWONA ŁUSZCZEWSKA-SIERAKOWSKA3,
STEFAN G. PIERZYNOWSKI4 AND TADEUSZ STUDZIŃSKI
Department of Animal Physiology, 3Department of Animal Anatomy
Faculty of Veterinary Medicine,
Agricultural University of Lublin, 20-033 Lublin, Poland
1
II Department of Radiology,
2
Department of Orthopaedy, Traumatology and Rehabilitation,
Medical University of Lublin, 20-950 Lublin, Poland
4
Department of Cell and Organism Biology,
Lund University, SE-223 62 Lund, Sweden
e-mail: [email protected]
Received for publication August 19, 2004.
Abstract
Alpha-ketoglutarate (AKG) was given to pregnant
sows every day starting from the 91st d of pregnancy to
farrowing. At the same time control pregnant sows were treated
in the same way with physiological saline (PhS). Newborn
piglets from the experimental (AKG) and control (PhS) mothers
were euthanised in 1 h after their birth, and the femora and
humeri were isolated and frozen at –25oC until further analyses.
Using quantitative computed tomography (QCT) method,
volumetric bone density of the cortical and trabecular bone
compartments were estimated. Moreover, geometrical and
mechanical parameters of the bones and the level of osteocalcin
and alkaline phosphatase activity in serum were determined. It
was found that the administration of AKG to pregnant sows
during 24 d before parturition decreased bone density in their
newborns.
Key words: sow, pregnancy, piglets, alphaketoglutarate, bone formation.
The maintenance of the structural and
functional homeostasis of bones, that is their formation
and metabolism is tightly regulated. Many different
environmental and nutritional factors and genes
determine bone turnover and the mineralization process.
Bone growth and formation are rapid during
development in prenatal and neonatal time in humans
and in pigs (7). At present time there is more and more
new information about the role of alpha-ketoglutarate
(AKG) in skeletal development and growth progress in
pigs in postnatal life and in post-hatching turkeys as well
(4, 5, 8). Further studies show a fundamental role of
AKG given per os in the growth of the whole body and
bones in postnatal life (4-6, 8). These studies indicated
that mammalian gastrointestinal tract acts as an
important system influencing the development and
process of the mineralization of the skeleton during
postnatal life. AKG has an important role in general
growth and maintenance of metabolic functions of bones
(5, 6). The compound given as a dietary supplement is a
source of glutamine and increases mechanical and
geometrical properties of bones (8). AKG is also the
main source of energy for cells of the gastrointestinal
tract, induces the proliferation of intestinal cells and is
an important factor for microbial flora present in the
healthy gut (4-6). Interactions between bacteria and
animal host are important for the maintenance of the gut
barrier in normal function and activity, which is very
important during post-weaning period. AKG
administered enterally is directly utilized not only
through the bacterial processes but also through
intestinal cells and finally is converted into glutamine
(4-6). AKG is a precursor of glutamine, synthesized in
human skeletal muscles. It makes about 50% of the free
amino acid pool in bloodstream and its rapid decrease is
associated with injury or stress conditions. Glutamine
improves protein synthesis in the skeletal muscle, and is
an energy source for immune cells as well (4-6, 10).
There are still no investigations on the influence of AKG
given as a dietary supplement to pregnant sows on the
development of the skeletal system during the 3rd
trimester of gestation. The purpose of this study was to
identify the effect of dietary supplementation of
pregnant sows with AKG on bone formation in their
newborns.
118
Material and Methods
Experimental
design
and
sampling
procedure. The experiment was carried out on 6 sows
of Large Polish White breed and on 24 their newborns.
The sows were housed under standard rearing conditions
(temperature and humidity) with free access to fresh
water and were fed standard commercial diet for
pregnant sows. Sows were fed at 7.00 a.m. and 3.00 p.m.
The experimental procedure was conducted from the 91st
d of pregnancy to the parturition. The time of parturition
was calculated from the date of the fertilization. The
sows of experimental group were treated per os with
AKG in the doses of 0.4 g/kg b.w. every day, whereas
those of the control group were given in the same
manner physiological saline (PhS) in doses of 300 ml
per sow. The newborns were divided into 2 groups, an
experimental (AKG) and a control one (PhS). Four
newborn piglets were taken randomly from every litter
of every experimental and control sows. The weight of
the individual newborns was measured just after their
birth. In 1 h after the birth the animals from both groups
were euthanised with lethal doses of pentobarbital
sodium (Morbital; Biowet Puławy, Poland) introduced
intravenously and their left and right femora and humeri
were isolated. The bones were stored at –25oC until they
were analysed and tested at the temperature about 20oC.
Analysis of the geometrical and mechanical
parameters. The geometrical parameters such as cross
sectional area (A), second moment of inertia (Ix) and
mean relative wall thickness (MRWT) were examined
through the measurement of the horizontal and vertical
diameter in the middle shaft of the femur and humerus
according to Ferretti’s et al. method (2, 3). Mechanical
properties of the bones were estimated using three-point
bending test in INSTRON 4302 apparatus coupled with
a computer, registering relationship between force acting
perpendicularly to the length of bone (2, 3). The results
of this mechanical analysis were presented graphically
and the maximum elastic strength (Wy) and the ultimate
strength were estimated (2, 3).
Volumetric bone mineral density. Volumetric
bone mineral density of the humerus and femur were
measured with quantitative computed tomography
(QCT) method and SOMATOM AR. T - SIEMENS
apparatus supplied with VR 3 software. Bone density
was measured for the cortical compartment of bone
using 2 mm thick cross sectional QCT scans in the
middle of diaphysis of (C1) and 1 cm from this scan
towards distal part of the bones (C2). Bone density was
measured for the trabecular compartment of bone using
the same method in the distal part of the bones. Cortical
and trabecular bone density were calculated by
automatic computation.
Blood sample analysis. The blood samples
were collected from the subclavian vein, just after the
birth from not-suckling piglets. The samples were
centrifuged immediately and the serum was stored at –
25oC until further analysis. The osteocalcin
concentration was determined using Human Osteocalcin
ELISA kit (Diagnostic System Laboratories Inc., Texas,
USA). The serum total alkaline phosphatase (AP)
activity was assessed using photocolorimetric methods
and the length of the wave was 405 nm.
Statistical analysis. All data are presented as a
mean ± standard error (±SE). Statistical analyses were
performed using STATISTICA 5.0 software. The
Student’s t-test was used to determine statistical
significance level of differences in variables between the
investigated groups. The level of statistic significance
was set at P≤0.05 for all comparisons.
Results
The body weight of newborns was higher in
experimental group (1716 g ±74) than in control one
(1324 g ±85). Both bones showed a tendency for higher
values of the length and weight in newborns, whose
mothers were treated with AKG (Tables 1 and 2).
Similarly Weight/Length Index reached in AKG group
higher value than in control one although the differences
were not statistically significant as well (Tables 1 and
2). Geometrical parameters of the femur and humerus,
including the cross sectional area and the second
moment of inertia were significantly higher in newborns
from AKG group than in control one (Tables 1 and 2).
The MRWT of the bones showed a tendency for higher
values in the control group than in the AKG one.
However, the differences were not statistically
significant (Tables 1 and 2). Prenatal administration of
AKG lowered the mean values of the maximum elastic
strength and ultimate strength of both bones in newborns
in comparison with the controls. The differences were
statistically significant (Tables 1 and 2). The bone
density of trabecular part of the femur was significantly
higher in the control than in AKG group. QCT of
trabecular part of the femur reached the value 0.804
g/cm3 (±0.04) in the control and 0.703 g/cm3 (±0.02) in
the AKG group. The value of density for trabecular part
of the humerus was significantly higher in the control
group (1.080 g/cm3 ±0.04) when compared with the
AKG group (0.858 g/cm3 ±0.01). The density of cortical
part of the femur in C1 was statistically higher in the
control (1.693 g/cm3 ±0.04) in comparison with the
AKG group (1.362 g/cm3 ±0.06). The density of cortical
part of the humerus in C1 was statistically higher in the
control (1.735 g/cm3 ±0.05) in comparison with the
AKG group (1.498 g/cm3 ±0.02). The density of cortical
part of the femur in C2 was significantly higher in the
control (1.673 g/cm3 ±0.01) in comparison with the
AKG group (1.225 g/cm3 ±0.08). The density of cortical
part of the humerus in C2 was statistically higher in the
control (1.862 g/cm3 ±0.02) in comparison with the
AKG group (1.517 g/cm3 ±0.02). The density of both
compartments of the bones were lower when newborns
were under influence of AKG. The level of osteocalcin
in serum was significantly higher in the control group
(12.75 ng/ml ±2.67) in comparison with the AKG group
(9.83 ng/ml ±0.57). The activity of total serum alkaline
phosphatase was significantly higher in the AKG group
(2356.6 IU/l ±35) in comparison with the control
(1745.66 IU/l ±70).
119
Table 1
Characteristics of femur properties in piglets born by sows treated
with AKG or PhS and examined an hour after farrowing
Group
PhS
AKG
Number of examined bones
24
24
Length (cm)
5.06 (±0.08)
5.13 (±0.06)
Weight (g)
6.76 (±0.41)
7.38 (±0.37)
Cross-sectional area (mm2)
31.3 (±1.18)
21.83 (±0.9)*
Second moment of inertia (mm4)
139.1 (±8.12)
78.31 (±5.79)*
Mean relative wall thickness
1.00 (±0.07)
0.66 (±0.05)*
Weight/Length Index (WLI)
1.33 (±0.06)
1.43 (±0.05)
Ultimate strength (N)
466.5 (± 38.3)
208.5 (±26.5)*
Maximum elastic strength (N)
356.18 (±25.6)
99.16 (±2.7)*
* P ≤0.05
Table 2
Characteristics of humerus properties in piglets born by sows treated
with AKG or PhS and examined an hour after farrowing
Group
PhS
AKG
Number of examined bones
24
24
Length (cm)
5.19 (±0.08)
5.18 (±0.06)
Weight (g)
7.65 (±0.36)
8.13(±0.32)
Cross-sectional area (mm2)
33.55 (±1.94)
23.91 (±2.01)*
Second moment of inertia (mm4)
131.2 (±12.5)
80.42 (±9.04)*
Mean relative wall thickness
0.83 (±0.03)
0.65 (±0.66)*
Weight/Length Index (WLI)
1.46 (±0.06)
1.58 (±0.06)
Ultimate strength (N)
544.3 (±44.5)
233.7 (±23.3)*
Maximum elastic strength (N)
338.0 (±32.3)
151.66 (±20.2)*
* P≤0.05
120
Discussion
Exogenous AKG may be converted to
glutamate and glutamine in intestinal cells and serves as
a precursor of glutamine in Krebs cycle (4-6). Glutamine
is a conditionally essential amino acid and main energy
source for all types of cells in the organism. It is an
important factor in stress and in growth during neonatal
life particularly during weaning and after this time when
the animals diet and bacterial flora are changed (4-6).
Glutamine and glutamate are chemically similar.
Glutamate is a neuromediator in the central nervous
system. Bone metabolic processes are regulated by
glutamate receptors where they serve as a signaling
system between bone cells. The intercellular connexion
in bones is very similar to synapses in the central
nervous system (1, 10). In this model, alphaketoglutarate was given via the digestive tract to
pregnant sows during the last 3 weeks of pregnancy
when the growth of the whole body, including the
skeletal system, is very rapid. Despite numerous studies
performed in growing animals, it is still unknown what
is the influence of prenatal administration of AKG on
skeletal development in mammals. These studies are
novel and for the first time present results indicating that
maternal AKG administration markedly influences
mineral density and mechanical and geometrical
properties of foetal bones in swine. Considering the
results obtained, prenatal administration of AKG
increased the length and weight of the examined bones
but lowered their all mechanical and geometrical
parameters. This effect on the processes of bone
mineralization is opposite to the effects observed during
development in postnatal period. Our earlier studies
showed that the enteral administration of AKG to pigs
and turkeys in postnatal life increased all the mechanical
and geometrical parameters of the examined bones and
mineralization process as well. It seems to indicate that
there is an axis connecting the gut and bones. The
digestive tract plays the important role in the
maintenance of density and mass of bones (4-6, 8). The
presence of this axis gives the possibility of the
influence in different way on the bone processes. As a
proof that AKG does not inhibit the whole foetal
development of piglets, there were higher body weight
values obtained after its administration, when compared
to the control piglets. Furthermore, the activity of total
alkaline phosphatase was higher in the experimental
group. The decline in serum osteocalcin level was
caused probably by a decrease in the processes of
skeletal system mineralization. It shows that AKG plays
an important role in the development and function of
many tissues and organs in the growing piglets but it is
still unknown why AKG presents an adverse effect in
prenatal and postnatal life (4-6, 10). AKG given
enterally to pregnant sows is probably metabolized in
about 90% through intestinal cells and bacteria and is
used as a “fuel”. It stimulates processes in the digestive
tract and influences directly the whole metabolism of
treated animal. What part of this renewed fuel crosses
the placenta in bloodstream and what form is available
for foetal cells is still unknown. In conclusion, further
studies are needed to explain this mechanism of
different action of AKG on the development of the
skeletal system in foetus than during its postnatal life.
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