Research Note
Effect of daily lithium chloride administration on bone mass and strength
in growing broiler chickens,1
B. M. Harvey,∗ 2 M. Eschbach,† E. A. Glynn,∗ S. Kotha,† 3 M. Darre,∗ D. J. Adams,§ R. Ramanathan,∗ 4
R. Mancini,∗ and K. E. Govoni∗ 5
∗
Department of Animal Science, University of Connecticut, Storrs, CT; † Department of Mechanical Engineering,
University of Connecticut, Storrs, CT; and § Orthopaedic Surgery, University of Connecticut Health Center,
Farmington, CT
ABSTRACT The objective was to determine the effects of oral lithium chloride supplementation on bone
strength and mass in broiler chickens. Ninety-six broilers were assigned to 1 of 2 treatment groups (lithium
chloride or control; n = 48/treatment). Beginning at 1
or 3 wk of age, chickens were administered lithium chloride (20 mg/kg body weight) or water daily by oral gavage. At 6 wk of age, chickens were euthanized and bone
and muscle samples were collected. A 24 h lithium chloride (20 mg/kg body weight) challenge determined that
serum lithium chloride increased within 2 h and cleared
the system within 24 h, demonstrating the effective
delivery of lithium chloride. Treatment did not influence body weight (P ≥ 0.20) or feed intake (P ≥ 0.81),
demonstrating that lithium chloride did not negatively
affect broiler growth. To determine bone strength, 3point bending was performed on the femora and tibiae obtained from control and lithium chloride-treated
birds in the 1 wk group. Lithium chloride-treated birds
had a 22% reduction in stiffness compared with control in the femora (P = 0.02) without a corresponding
reduction in elastic modulus. No differences were observed in yield or ultimate load and in the corresponding calculations of stresses (P ≥ 0.26). The toughness
of tibiae was not altered in lithium chloride compared
with control (P = 0.11). Bone length and micro-CT
imaging were performed on the tibiae of control and
lithium chloride groups. No differences (P ≥ 0.52) in
bone length, cortical or trabecular bone volume, trabecular thickness, number, or spacing were observed.
Lithium chloride treatment did not affect pectoralis
muscle color or lipid oxidation (P > 0.05). In conclusion, lithium chloride treatment in broilers did not negatively affect growth or meat quality. A reduction in
bone stiffness of the femur with lithium chloride treatment was observed, however unlike the mouse model,
the dosages of lithium chloride used in the current study
did not result in anabolic effects on broiler long bones.
Key words: bone, broiler, lithium chloride, meat quality
2015 Poultry Science 94:296–301
http://dx.doi.org/10.3382/ps/peu079
INTRODUCTION
Skeletal problems affect several agricultural species
and are responsible for increased costs of production
as well as negatively impacting the welfare of the animals. Skeletal disease costs the broiler industry up to
$120 million per year (Sullivan, 1994; Fleming, 2008).
It is estimated that 13 to 41% of layers suffer from
fractures related to osteoporosis and ultimately result
in death (Webster, 2004). These skeletal disorders are
a serious concern for both efficiency of production and
the welfare of birds. Therefore, identification of novel
methods to improve bone quality and strength can be
used to increase efficiency in poultry production, as well
as improve animal welfare.
Skeletal development is a complex and tightly
regulated process. In growing animals, adequate bone
formation is dependent on several processes including
cartilage and bone matrix formation. Chondrocytes, osteoblasts and osteoclasts are key cell types involved
in forming new bone and providing proper structure
of the skeleton. During this complex process, several
C 2015 Poultry Science Association Inc.
Received May 17, 2014.
Accepted October 9, 2014.
1
This work was supported by the Storrs Agricultural Experiment Station (KEG) and Summer Undergraduate Research Fund Grant from
the University of Connecticut (BMH and KEG).
2
Current Address: Department of Biochemistry & Cell Biology, Stony
Brook University, Stony Brook, NY.
3
Current Address: Department of Biomedical Engineering, Rensselaer
Polytechnic Institute, Troy, NY.
4
Current Address: Department of Animal Science, Oklahoma State
University, Stillwater, OK.
5
Corresponding author: [email protected]
296
RESEARCH NOTE
factors including hormones, growth factors, and signaling pathways are involved in regulating these cell types.
One key signaling pathway that has received attention
is the Wnt/β -catenin signaling pathway (Canalis, 2010;
Tamura et al., 2010). The Wnt/β -catenin pathway is
critical in bone formation and can affect the ability
of osteoblasts to create bone matrices (Boyce et al.,
2005; Piters et al., 2008). In the Wnt/β -catenin signaling pathway, Wnt glycoproteins bind with lipoprotein receptor-related protein (LRP5) or LRP6 and
a 7-transmembrane serpentine Frizzled (Fz) receptor,
which causes accumulation of hypophosphorylated β catenin in the cytosol of the cell (Piters et al., 2008).
The β -catenin is then translocated into the nucleus
where it reacts with a lymphoid-enhancer binding factor and a T-cell specific transcription factor, resulting
in transcription of target genes whose products stimulate bone growth (Piters et al., 2008). The amount of
β -catenin accumulation in the cytosol is controlled by
glycogen synthase kinase-3β (GSK-3β ), which phosphorylates β -catenin and essentially tags it for degradation (Clement-Lacroix et al., 2005; Piters et al., 2008).
Lithium (Li) is an inhibitor of the activity of
GSK-3β , thereby increasing the number of β -catenin
molecules in the cell, and, thus, increases transcription
in Wnt target genes (Hedgepeth et al., 1997; ClementLacroix et al., 2005). Lithium chloride (LiCl) has anabolic effects on bone (Clement-Lacroix et al., 2005;
Vestergaard, 2008; Loiselle et al., 2013; Satija et al.,
2013). Specifically, in mice, oral LiCl supplementation
resulted in a 35% increase in trabecular bone volume to
total bone volume fraction (BV/TV) as well as a 130%
increase in mineral apposition rate via the Wnt/β catenin pathway (Clement-Lacroix et al., 2005). In addition, Li administered to humans as a psychotropic
drug has been shown to reduce the risk of bone fractures (Vestergaard, 2008). These studies indicate that
LiCl supplementation has a positive impact on bone
quality and strength. Based on the previous findings
that LiCl treatment alters the Wnt signaling pathway
to improve bone quality and strength, we hypothesized
that LiCl supplementation would improve bone mass,
architecture, and bone strength in growing broilers. To
test our hypothesis, we supplemented growing broilers
with LiCl beginning at 1 wk and 3 wk of age and determined growth, bone mass, bone strength and muscle
quality at 6 wk of age.
MATERIALS AND METHODS
Animals
Animal protocols were approved by the University of
Connecticut Institutional Animal Care and Use Committee. Ninety-six 1 d male broiler chicks (Cobb500)
were obtained from Burr Farm (Danielson, CT). At 5 d
of age, birds were organized into LiCl or control (CON)
groups, and then further organized into subgroups with
treatment starting at 1 or 3 wk of age. Overall, birds
297
were separated into 16 pens (6 birds/pen) with 4 pens
per group at each time point (1 wk or 3 wk). Birds were
housed on littered floors (pine shavings) at a density of
16 square feet per bird with 16L:8D. Birds were fed a
commercial broiler starter crumble (Blue Seal; ME =
1,430 kcal; CP = 20 to 21%; methionine = 0.45%; lysine = 1.26%; calcium = 1.0%; phosphorus = 0.73%)
upon arrival and transitioned to a commercial broiler
grower/finisher crumble (Blue Seal; ME = 1,425 kcal;
CP = 18 to 19%; methionine = 0.38%; lysine = 1.03%;
calcium = 1.0%; total P = 0.66%) at 1.5 to 2 wk of age.
Birds were provided water ad libitum throughout the
experiment. Body weights and feed intake were measured every 3 to 4 d for all treatment groups. Feed was
provided ad libitum for the duration of the experiment.
LiCl Treatment
Beginning at 1 or 3 wk of age, LiCl birds were administered 1 mL of a LiCl solution (20 mg/kg of body
weight (BW) dissolved in water) while CON birds were
administered 1 mL of water daily by oral gavage. Body
weight was measured twice per wk and used to prepare daily LiCl concentrations. The concentration of
LiCl was chosen based on previous studies in mice and
poultry in which the dose was not lethal or toxic to
the animals, but a treatment effect was observed (Scott
et al., 1973; Clement-Lacroix et al., 2005).
Sample Collection
At 6 wk of age, 16 birds from each group (1 wk LiCl, 1
wk CON, 3 wk LiCl, and 3 wk CON) were euthanized by
CO2 inhalation and decapitation. Following euthanasia and decapitation, muscle, femora, and tibiae (left
and right legs) were collected. Tibiae and femora were
stripped of muscle tissue and stored in 70% ethanol
solution at 4◦ C for strength and μCT analysis. The
right leg was used for μCT analysis for 1 and 3 wk
samples. The left leg was used for 3-point bending for
1 wk samples. The breast muscle was removed, placed
on polystyrene trays (Cryovac foam tray, 21 × 14.6 ×
2.54 cm3 , Cryovac Food Packaging Inc., Duncan, SC),
overwrapped with oxygen-permeable film (E-Z Wrap
Crystal Clear Polyvinyl Chloride Wrapping Film, Koch
Supplies, Kansas City, MO) and stored at 4◦ C for 7 d
until analysis was performed.
Li Assay
To test the effectiveness of the oral administration of
the LiCl dose, 9 birds were administered a single oral
dose of LiCl (20 mg/kg BW) and 6 birds were administered water (CON) at 5 wk of age. These were extra
birds that arrived with the experimental animals and
were kept in similar conditions, but not used for the experiment. At 2, 8, and 24 h after treatment, birds were
euthanized by CO2 inhalation and decapitation. Following euthanasia and decapitation, blood was collected
298
HARVEY ET AL.
into a tube immediately following decapitation, serum
harvested as previously described (Govoni et al., 2003)
and stored at −20◦ C until analysis was performed. A
Lithium Enzymatic Assay Kit (Diazyme Laboratories,
Poway, CA) was used to determine serum concentrations of Li according to the manufacturer’s protocol.
Strength Testing
Chicken femora and tibiae from the right legs (n =
5 to 6 per treatment) were subjected to a 3-point bend
test to failure to obtain differences in mechanical properties (using the Tinius Olsen testing device) between
the 2 treatments (CON vs. LiCl). Based on the limited
differences observed in the μCT analysis, only the 1 wk
samples were analyzed since this group had the longest
length of treatment. The 3-point bend tests were performed with a span of 50 mm and a displacement rate of
5 mm/min. Samples were kept wet throughout the entire experiment. The displacements were measured using the Tinius Olsen, which had a resolution of ±0.01%.
The loads were measured using a force transducer with
a maximum capacity of 1,000 N and a resolution of
± 0.5% accuracy. The data were recorded using National Instrument’s LabView and the load displacement
curves were analyzed using Mathworks’ MATLAB software to determine ultimate load (N), stiffness (N/mm),
0.002 yield load (N), and energy (N·mm).
The bones were sectioned, after testing, close to failure, to obtain their area moment of inertia values.
This allowed the data to be normalized into stress
and strain. Photographs were taken of the cross sections and analyzed using The MathWorks MATLAB.
Strain was calculated from the recorded deflection using
Equation 1. Stress was calculated from the recorded
load using Equation 2. Flexural modulus was calculated
from the recorded stiffness using Equation 3.
6Dd
, Strain Equation
L2
F LD
, Stress Equation
Equation 2. σ =
8I
SL3
, Flexural Modulus Equation
Equation 3. E =
48I
Equation 1. ε =
In these equations, deflection is d, vertical span (distance from the application of force on the top of the
bone to the bottom of the chicken bone directly below it) of the testing specimen is D, the testing span is
L, the load is F, the stiffness is S, and the moment of
inertia is I.
Micro-Computed Tomography Imaging
(μCT)
Bone length and bone morphometry were measured
in the tibiae of the left legs (n = 6 per treatment)
using cone beam micro-focus X-ray computed tomography (VivaCT40, Scanco Medical AG, Brüttisellen,
Switzerland). Scanning was performed at 55 kV and
145 mA, collecting 1,000 projections per rotation at 300
msec integration time. Three-dimensional images were
reconstructed using standard convolution and backprojection algorithms with Shepp and Logan filtering, and rendered within a 35.8 mm field of view at
a discrete density of 23,324 voxels/mm3 (isometric 36
mm voxels). Segmentation of mineralized tissue from
marrow and soft tissue was performed in conjunction
with a constrained Gaussian filter to reduce noise, applying hydroxyapatite-equivalent density thresholds of
140 and 280 mg/cm3 for trabecular and cortical bone,
respectively (bone tissue density is ∼1,000 mg/cm3 ).
Trabecular morphometry was measured within a 3.5
mm longitudinal span (100 serial virtual sections)
of the proximal metaphysis. Standard algorithms describing three-dimensional trabecular morphometry
were applied to obtain direct measures of trabecular
architecture and mineral density, including bone volume fraction (BV/TV), trabecular thickness, spacing,
and trabecular number. Cortical morphometry was
quantified through a 28 mm span (800 serial virtual
sections) at mid-diaphysis to obtain measures of bone
volume and mineral density.
Color Measurements
The surface color of chicken breasts was measured
through the overwrap film using a HunterLab MiniScan XE Plus Spectrophotometer (45/0 LAV, 2.54-cmdiameter aperture, Illuminant A, 10◦ standard observer;
Hunter Associates Laboratory, Inc., Reston, VA). Three
scans from each breast muscle were obtained and averaged for statistical analysis. The CIE L∗ , a∗ , and b∗
values were used to estimate muscle darkening, redness,
and yellowness, respectively.
Lipid Oxidation
Thiobarbituric acid reactive substances (TBARS)
were measured as an indicator of lipid oxidation according to Witte et al., 1970. Five grams of breast muscle
were blended with 25 mL of trichloracetic acid solution
(20%) and 20 mL distilled water. The mixture was homogenized using a Waring table-top blender (Dynamics
Corp. of America, New Hartford, CT) for 1 min and filtered through Whatman (#1) filter paper. One mL of
filtrate was mixed with 1 mL of TBA solution (20 mM)
and incubated at 25◦ C for 20 h. After incubation, absorbance was measured using a Shimadzu UV-2101 PC
spectrophotometer (Shimadzu Inc., Columbia, MD) at
532 nm against a blank consisting of 2 mL acid/water
mix (TCA/water 1:1 vol/vol) and 2 mL TBA solution.
RESEARCH NOTE
299
Statistical Analysis
Birds were randomly assigned to treatment (LiCl or
CON) and age at the start of treatment (1 wk and
3 wk). Fixed effects tested were treatment and age.
Body weight and feed intake data were analyzed using mixed model procedure (PROC MIXED). For feed
intake, the pen was used as the experimental unit (n =
4/group). For BW, animal was used as the experimental
unit (n = 24/group) with pen included in the random
statement. Strength testing data were analyzed using
the Student t test. All other variables were analyzed
using ANOVA (PROC ANOVA) with SAS Version 9.3
software. One pen of LiCl treated birds in the 1 wk
group was removed due to improper dosing during the
experiment. A significant difference was determined at
P ≤ 0.05.
RESULTS
Lithium Chloride
In a subset of broilers, a single oral administration
of LiCl successfully increased circulating concentrations
of LiCl 334% in the blood within 2 h compared with
CON (P = 0.02). Therefore, our dose was effective in
elevating LiCl in the blood. However, by 8 and 24 h
following the single dose, serum concentrations of LiCl
returned to those of CON bird (data not shown).
Feed Intake and Body Weight
Daily administration of LiCl did not affect feed intake
between CON and LiCl groups beginning treatment at
1 and 3 wk of age (P ≥ 0.81; Figure 1A). In addition,
we did not observe an effect of LiCl treatment on BW
in the 1 or 3 wk groups (P ≥ 0.20; Figure 1B).
Bone Strength and Mass
Consistent with BW, femur and tibia bone lengths
were not different between CON and LiCl broilers (P
≥ 0.63; Table 1). Femur stiffness was reduced by 22% in
birds given LiCl (P = 0.02), but no difference was observed in tibia (P = 0.25). There was no effect of LiCl
treatment on yield load and ultimate load in tibia or
femur bones (P ≥ 0.26). The energy to fracture (area
under the load-deformation curve) of the LiCl tibia was
reduced by 32.6% over CON (P = 0.01), but no differences were observed in femurs (P = 0.44). Material parameters, such as elastic modulus, yield and ultimate
stress were not different between groups in both the
tibiae and the femora (P ≥ 0.27). Toughness (as determined by energy) of the tibiae treated with LiCl
tended to be 34% lower in comparison with CON (P =
0.11). Micro-CT analysis showed that LiCl treatment
did not alter the tibial BV/TV in the metaphysis or diaphysis (P ≥ 0.52) or the trabecular thickness, spacing
Figure 1. Lithium chloride (LiCl) treatment did not affect feed
intake (A) or BW (B) in growing broilers. Broilers were given oral
LiCl (20 mg per kg BW) supplement daily beginning at 1 or 3 wk of
age (6 birds per pen; 4 pens per treatment). Data are presented as
mean. Feed intake SEM = 0.0168 (Control-1 week), 0.0168 (Control-3
week), 0.0168 (LiCl-1 week), 0.0195 (LiCl-3 week). Body weight SEM
= 0.0164 (Control-1 week), 0.0164 (Control-3 week), 0.0165 (LiCl-1
week), 0.0190 (LiCl-3 week).
and number in the metaphysis region (P ≥ 0.56; data
not shown).
Meat Quality
Lithium Cl did not influence darkening (L), redness
(a) or yellowness (b) of the breast meat (P ≥ 0.17; data
not shown). In addition, lipid oxidation was not altered
by daily LiCl treatment (P ≥ 0.75; data not shown).
300
HARVEY ET AL.
Table 1. Three-point bending analysis of femur and tibia bones in broilers supplemented with lithium chloride (LiCl) at 1 wk of age.
Femur1
Variable
Length (mm)
Stiffness (N/mm)
Energy (N mm)
Yield Stress (MPa)
Ultimate Stress (MPa)
Modulus (GPa)
Energy (MJ/m3 )
Yield Load (N)
Ultimate Stress (N)
Tibia
Control
LiCl
Control
LiCl
85.9 ± 1.0
171.6 ± 7.1
437.4 ± 38.6
45.4 ± 2.5
54.7 ± 2.6
1.10 ± 0.08
2.06 ± 0.91
245.6 ± 12.8
301.3 ± 10.4
86.7 ± 1.1
133.3 ± 12.3∗
472.5 ± 20.6
55.7 ± 12.1
65.1 ± 15.7
1.19 ± 0.34
2.85 ± 0.82
254.9 ± 19.2
291.0 ± 15.8
120.0 ± 2.4
253.4 ± 22.4
614.4 ± 47.1
42.1 ± 2.6
58.4 ± 4.5
1.99 ± 0.16
2.38 ± 0.36
250.3 ± 24.6
343.4 ± 28.8
118.8 ± 1.7
203.3 ± 34.2
414.0 ± 47.8∗
54.5 ± 10.3
68.8 ± 7.6
2.05 ± 0.30
1.57 ± 0.28†
295.6 ± 28.7
357.1 ± 28.4
Data are presented as mean ± SE.
indicates a significant difference between LiCl (n = 5 to 6 per treatment) and Control
(n = 6 per treatment) at P ≤ 0.05.
†
indicates P = 0.11.
1
∗
DISCUSSION
Skeletal disorders have been a concern in the poultry
industry for many years. In broilers, the rapid growth
rate and increased muscle production make these birds
susceptible to leg disorders leading to lameness, poor
health, and reduced growth rates (Fleming, 2008). Although proper management, breeding, and nutrition
can reduce bone-related issues, osteoporosis and legdisorders are still a concern in the layer and broiler
industries, respectively (Whitehead and Fleming, 2000;
Fleming, 2008). Therefore, identification of key targets
for improving bone quality or treating skeletal disorders
is necessary.
Lithium chloride is used in treatment of humans
with manic-depression states and increases bone mineral density in patients treated with LiCl (Cohen
et al., 1998). Additionally, LiCl improves bone formation and mechanical properties in rodent models
(Loiselle et al., 2013) and stimulates osteoblastogenesis
in vitro (Satija et al., 2013) through the Wnt-β -catenin
pathway. Therefore, this supplement has the potential
to be used to improve bone formation and quality in
poultry. Although the acceptance of LiCl use in commercial production is far reaching, this model has the
potential to identify novel mechanisms to improve bone
development in poultry.
Similar to previous reports (Scott et al., 1973), daily
administration of LiCl (20 mg/kg BW) was not toxic
or lethal to birds during the 6 wk period. However,
in a time course analysis over 24 h, LiCl was cleared
from the blood within 8 h. This finding suggests that
either more frequent administration of LiCl or a larger
dose is needed to keep LiCl concentrations sustained
in broilers. Additionally, although the current dose was
chosen to prevent lethality or side effects as previously
reported (Scott et al., 1973), it was less than previously
used in mice (Clement-Lacroix et al., 2005). Therefore,
additional studies with increased doses are warranted.
Importantly, the dose of LiCl in the current study did
not affect feed intake or BW in the growing broilers.
These findings are important to demonstrate that, if
LiCl was used to improve bone formation and quality,
it would not have a negative impact on production or
feed costs. Additionally, LiCl treatment did not affect
muscle quality as determined by color and lipid oxidation analysis. This suggests that LiCl can be used as
a supplement to target bone health without having a
negative impact on meat quality. In the event that LiCl
was used in a production setting, further studies are
needed to determine if LiCl is detected in the muscle.
However, based on the rapid clearance from the blood,
it is unlikely that there will be accumulation of LiCl in
the muscle.
Bone strength is an important indicator of the ability of the bone to carry weight and withstand force.
Adequate bone strength is important in broilers, which
grow rapidly and produce large amounts of muscle. A
common method to determine bone strength is the use
of 3-point bending of whole long bones. In the current
study, treatment with LiCl reduced bending stiffness in
the femurs of broilers, but did not affect the load applied during 3-point bending strength tests. Reduced
bending stiffness could suggest a potential positive effect by reduced risk of fracture; however, growing birds
need to be evaluated at later time points. Less energy
was needed to bend the tibiae of LiCl broilers as compared with controls implying a reduction in mineralization. However, this is one point in time, so it is unclear
if these differences could be due to a delay in mineralization in the LiCl group. Importantly, these findings
demonstrate that LiCl supplementation appears to affect femur stiffness in 6 wk old broilers after 5 wk of
LiCl treatment. Further studies with samples taken at
multiple time points during growth, including in ovo
treatment, are needed to determine if the reduced bone
stiffness at this time point would have positive or negative outcome on bone strength and quality over time.
Previous reports using a similar dose of LiCl improved bone formation and mass in mice (ClementLacroix et al., 2005); therefore, it was surprising that
LiCl did not affect bone volume or trabecular thickness
RESEARCH NOTE
and number in the current study. The lack of effect of
LiCl in the current study could be due to many factors including the dose and/or frequency of LiCl treatment, that these were healthy, fast growing birds from
a flock with limited bone abnormalities (abnormalities
were not observed during necropsy in the current experiment), the number of animals, or that the previous
studies evaluating the effects of LiCl on bone were conducted in human and rodent models.
Overall, LiCl, a known stimulator of the Wnt-β catenin signaling pathway, is not lethal to broilers and
does not negatively impact growth, feed intake, or muscle quality using the current dose reported. Although
the current dose and treatment duration did not impact
bone volume and trabecular number or thickness as previously observed in mice, it did have an apparent effect
on bone stiffness in femur and energy in 3-point bending of tibiae. It is not clear at this time if these effects
have positive or negative impacts on bone integrity. Future studies using multiple time points, a greater dose
of LiCl similar to rodent models, a bone development
impaired model and a laying hen model are warranted
to evaluate the potential of LiCl to improve bone mass
and quality in poultry.
ACKNOWLEDGMENTS
We thank the University of Connecticut Poultry
Farm for assistance with care of the birds and David
Schreiber and Karen More for technical assistance.
REFERENCES
Boyce, B. F., L. Xing, and D. Chen. 2005. Osteoprotegerin, the bone
protector, is a surprising target for beta-catenin signaling. Cell.
Metab. 2:344–345.
301
Canalis, E. 2010. Update in new anabolic therapies for osteoporosis.
J. Clin. Endocrinol. Metab. 95:1496–1504.
Clement-Lacroix, P., M. Ai, F. Morvan, S. Roman-Roman, B.
Vayssiere, C. Belleville, K. Estrera, M. L. Warman, R. Baron, and
G. Rawadi. 2005. Lrp5-independent activation of Wnt signaling
by lithium chloride increases bone formation and bone mass in
mice. Proc. Natl. Acad. Sci. U. S. A. 102:17406–17411.
Cohen, O., T. Rais, E. Lepkifker, and I. Vered. 1998. Lithium carbonate therapy is not a risk factor for osteoporosis. Horm. Metab.
Res. 30:594–597.
Fleming, R. H. 2008. Nutritional factors affecting poultry bone
health. Proc. Nutr. Soc. 67:177–183.
Govoni, K. E., T. A. Hoagland, and S. A. Zinn. 2003. The ontogeny
of the somatotropic axis in male and female Hereford calves from
birth to one year of age. J. Anim. Sci. 81:2811–2817.
Hedgepeth, C. M., L. J. Conrad, J. Zhang, H. C. Huang, V. M. Lee,
and P. S. Klein. 1997. Activation of the Wnt signaling pathway:
a molecular mechanism for lithium action. Dev. Biol. 185:82–91.
Loiselle, A. E., S. A. Lloyd, E. M. Paul, G. S. Lewis, and H. J. Donahue. 2013. Inhibition of GSK-3beta rescues the impairments in
bone formation and mechanical properties associated with fracture healing in osteoblast selective connexin 43 deficient mice.
PLoS One 8:e81399.
Piters, E., E. Boudin, and W. Van Hul. 2008. Wnt signaling: a win
for bone. Arch. Biochem. Biophys. 473:112–116.
Satija, N. K., D. Sharma, F. Afrin, R. P. Tripathi, and G. Gangenahalli. 2013. High throughput transcriptome profiling of lithium
stimulated human mesenchymal stem cells reveals priming towards osteoblastic lineage. PLoS One 8:e55769.
Scott, J. T., T. M. Ferguson, J. W. Bradley, and C. R. Creger. 1973.
The effect of low levels of lithium chloride in the diet of the laying
hen. Poult. Sci. 52:2336–2337.
Sullivan, T. W. 1994. Skeletal problems in poultry: estimated annual
cost and descriptions. Poult. Sci. 73:879–882.
Tamura, M., E. Nemoto, M. M. Sato, A. Nakashima, and H. Shimauchi. 2010. Role of the Wnt signaling pathway in bone and
tooth. Front. Biosci. (Elite Ed) 2:1405–1413.
Vestergaard, P. 2008. Skeletal effects of central nervous system active
drugs: anxiolytics, sedatives, antidepressants, lithium and neuroleptics. Curr. Drug Saf. 3:185–189.
Webster, A. B. 2004. Welfare implications of avian osteoporosis.
Poult. Sci. 83:184–192.
Whitehead, C. C., and R. H. Fleming. 2000. Osteoporosis in cage
layers. Poult. Sci. 79:1033–1041.
Witte, V. C., G. F. Krause, and M. E. Baily. 1970. A new extraction
method for determiniy 2- thiobarbiturie acid values of pork and
beef during storage. J. Food Sci. 35:582–585.