Journal of Trace Elements in Medicine and Biology 42 (2017) 2529

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Journal of Trace Elements in Medicine and Biology

journal homepage: www.elsevier.com/locate/jtemb

Veterinary medicine

Effects of dietary chromium supplementation on muscle and bone

mineral interaction in broiler chicken
Abdullah A. Saeed a , Mansur A. Sandhu b, , Muhammad S. Khilji a , Muhammad S. Yousaf a ,
Habib U. Rehman a , Zafar I. Tanvir c , Tanveer Ahmad d
a
Department of Physiology, Faculty of Bio-Sciences University of Veterinary and Animal Sciences, Lahore, Pakistan
b
Department of Biomedical Sciences, Faculty of Veterinary and Animal Sciences, PMAS-Arid Agriculture University, Rawalpindi, Pakistan
c
Drug Residual Laboratory, National Veterinary Laboratory, Islamabad, Pakistan
d
Department of Livestock Production and Management, Faculty of Veterinary and Animal Sciences, PMAS-Arid Agriculture University, Rawalpindi, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: The study was conducted to ascertain the effects of dietary chromium chloride (CrCl3 6H2 O) supplemen-
Received 21 November 2016 tation on mineral interaction in blood serum, leg muscles and bones of broilers at 35th day of age. For this
Received in revised form 6 February 2017 purpose, ninety male broiler chicks were divided into three groups. One served as control (group I) while,
Accepted 9 March 2017
the other two groups were supplemented with CrCl3 (group II-12.5 mg/Kg feed; group III25 mg/Kg feed)
from 12 to 28 days of age. In serum, Cr concentration remained non-signicant however, Zn, and K con-
Keywords:
centrations decreased (P < 0.05) with both levels of Cr-supplementation. Furthermore, in muscles Cr, Cu,
Broiler
Ca and Na levels remained non-signicant but concentrations of Zn and K decreased (P < 0.05) with feed
Bone
Chromium
Cr enrichment. Chromium had a substantial effect on femur and bula Zn retention with 25 mg/Kg feed
Mineral interaction supplementation while, Cr deposition decreased (P < 0.05) in bula. Femur Ca (P < 0.002), Na (P < 0.001)
Muscle and K (P < 0.05) retention was inversely proportional to both Cr concentrations in feed. In tibia, Cu and
Na concentration decreased (P < 0.002) with high dietary Cr supplementation. Fibular Ca and Na con-
centrations remained signicantly (P < 0.001) lower in Cr supplemented groups. Bone robusticity index
was non-signicant but ash to weight ratio of femur, tibia and bula decreased (P < 0.05) in group III.
Chromium supplementation has a major effect on serum or muscle Zn and K deposition while bone
mineral interaction shows a major thrust on Zn, Ca and Na levels.
2017 Elsevier GmbH. All rights reserved.

1. Introduction
Trace elements play an important role in human and ani-
mal body homeostasis. Trivalent chromium (Cr) is a biologically
active form and mostly present in food along with other trace ele-
ments. Inorganic trivalent Cr is less toxic and this may be due to
its restricted transport across cell membrane [1]. An increase in
poultry meat quality and decrease in meat fat contents has been
reported with Cr supplementation [2,3]. It is known to affect the
metabolism of carbohydrates, lipids, and proteins [46]. Chromium
supplementation in poultry [7] and pig feed showed a boost in feed
conversion ratio [8]. One of the foremost in vivo functions of Cr
is to potentiate insulin function through glucose tolerance factor
(GTF) [9] and the activation of enzymes to stabilize different cellu-
Corresponding author at: Institt fr Veterinr Physiologie, Freie Universitt
Berlin, Germany.
E-mail address: mansur.sandhu@fu-berlin.de (M.A. Sandhu).
lar proteins and nucleic acids [10]. After feeding, a small quantity
of Cr is absorbed (ranging between 0.4 to 2%) [10] and about 90%
of this absorbed Cr in extracellular compartments is bound with
-globulin and transferrin [11,12]. In contrast to carbohydrates,
proteins and fats, the consumption of micronutrients is lower in
both human and animals. In goats, Cr deciency leads to lower Cu
in kidneys, impaired glucose tolerance leading to hyperinsuline-
mia and increased serum proteins [13]. In this complex mineral
interaction, Cr is known to interact with Zn, Cu [14] and the stimu-
latory factor for mineral mobilization is environmental stress [15].
One of the most important areas of body mineral deposition is
bones but this was given less attention in Cr supplemented mineral
homeostasis. To the best of our knowledge, there is no report on the
importance of trace mineral interactions in different body compart-
ments with Cr supplementation. Therefore, the current study was
undertaken to estimate the effect of dietary Cr supplementation on
mineral concentrations in blood serum, leg muscles and bones.

http://dx.doi.org/10.1016/j.jtemb.2017.03.007
0946-672X/ 2017 Elsevier GmbH. All rights reserved.
26 A.A. Saeed et al. / Journal of Trace Elements in Medicine and Biology 42 (2017) 2529

Table 1 2.3. Bone density and mineralization

Ingredients and chemical analyses of broiler chicken basal diet.

Ingredients in% Grower Ration The right femur, tibia and bula of each bird (the same birds
Soybean meal (44%CP) 32 used for blood serum analysis) were freed from intact muscles and
Corn 60 submerged in deionized boiling water for 15 min. After cooling,
Soybean oil 02 the boiled esh was removed carefully and the proximities were
Fish meal 02 untied from the cartilages and patella. All three bones were kept
Calcium carbonates 1.3
at 70 C in a hot air oven for 6 h to dry. The length of femur, tibia
Di-calcium phosphate 1.3
Salt 0.4 and bula were determined and each bone was marked approxi-
DL-Meth 0.12 mately at center for determination of outer diameters using a digital
Lys-HCl 0.04 Vernier Caliper. The robusticity index was calculated by using the
Vitamin premix* 0.25
following formula [17]:
Mineral premix** 0.25
bone diaphyseal circumference
The major composition of *Vitamin premix was: B12 15 mg; B6 3 g; Thiamine 1.8 g; Bone robusticity = 100
niacin 30 g; folic acid 1 g; vitamin K 2 g; D3 2000,000IU; A 9.000.000IU; E 18 g, bone maximum length
riboavin 6.6 g. This whole composition of vitamin premix was in 2.5Kg of broiler
grower feed. Along with that **mineral premix in 2.5Kg feed was as following: Cu To nd out the bone ash to weight index, the bones were com-
10 g; Fe 50 g; Zn 100 g; Mn 100 g; Se 200 mg and Iodine 1 g. pletely dried in a hot air oven at 105 C (Memmart, Germany) for
12 h by placing them in china crucible. Afterwards, the bones were
turned to ash in a mufe furnace (Thermo Scientic, USA) at 600 C
for 6 h. The ash to weight ratio was determined by weighing ash rel-
ative to dry weight of bones. The obtained ash from each bone was
2. Materials and methods
preserved separately in dry plastic zip bags at 4 C for wet digestion
and mineral estimation.
2.1. Procurement of birds and preparation of experimental diet

2.4. Tissue digestion and spectrophotometry

A total of 90one-day-old male broiler chicks (Cobb-500) were
procured and kept in an experimental shed at PMAS-Arid Agricul-
The samples of muscles, bones, feed (1.0 g) and serum (1.0 mL)
ture University, Rawalpindi. The birds were kept together for the
were digested in 10 mL of nitric acid (62%; Merck, Darmstadt,
rst 7 days and afterwards randomly divided into three experimen-
Germany) on a temperature controlled heating plate [18]. When
tal groups (n = 30). Within each group, three replicates of ten birds
the volume of nitric acid in asks was reduced to approximately
were kept on oor housing system with one-inch bedding. Repli-
around 2 mL, a quantity of 5 mL perchloricacid (70% AnalaR, USA)
cates were separated by stainless steel mesh and an 8 h dark period
was added and heated to clear up the solution till the remaining
was provided. Feed and water utensils were installed as per require-
volume was about 1 mL. Afterwards, the digested samples were
ment and provided with ad libitum basal broiler ration according
ltered with Whatman number 1 lter paper and diluted with
to National Research Council [16] recommendations. Temperature
deionized water to a volume of 10 mL. For the measurement of Cu,
during brooding was maintained by an electric brooder and in the
Ca and Zn contents, the atomic absorption spectrometry (Thermo
whole experiment environmental temperature was at 25 3 C.
Scientic, USA) was conducted. However, for the Cr determination,
All the birds were kept on a basal diet during the acclimatization
graphite furnace atomic absorption spectrometry was performed
period. For the experiment, two different levels of chromium chlo-
as described by Krzysik et al. [19]. For the determinations of Na and
ride (CrCl3 6H2 O) were added to basal broiler grower diet as given
K, the samples were run through a ame photometer (Sherwood
in Table 1. The rst treatment consisted of basal diet (Group I); with
M300, USA). The standard calibrations of the instrument for min-
the addition of CrCl3 at the dose rate of 12.5 mg/Kg feed (Group II)
eral assays were conducted with serial concentrations of chloride
and 25 mg/Kg feed (Group III) respectively. The treated feeds were
salts prepared of each element.
started with the culmination of the 12th day of brooding age and
For results accuracy, the samples were immediately preserved
the supplementation was withdrawn one week before sampling on
by refrigeration to prevent decomposition. All the samples were run
28th day of age. All the experimental procedures and maintenance
in duplicate and averages of these duplicates were calculated for
of the broiler was according to the animal welfare guidelines and
data analysis. The glassware was presoaked in HNO3 (6 M) for one
by the approval of the University Synopsis Review Committee of
day, boiled and thoroughly rinsed with deionized water to remove
the University of Veterinary and Animal Sciences, Lahore.
any debris and impurities. Before using, all the glassware was kept
dry in hot air oven. From all calculations, blanks were subtracted
before data analysis.
2.2. Blood and tissue sampling
2.5. Statistical analysis
At 35 days of age, 3 mL of blood was collected from the wing
vein of three birds from each replicate and was kept in sterilized All data was analyzed through one-way analysis of variance
glass vacuum containers at 4 C for 2 h. The collected blood samples (ANOVA) using SigmaPlot, version 11.0 software package. The
were centrifuged at 800 g for 5 min in a temperature-controlled group differences were analyzed by the Holm-Sidak method. Dif-
centrifuge (4 C; Thermo Scientic Bench top Centrifuge) and the ferences were considered signicant when the F-test was P < 0.05.
serum was stored at 20 C in polyethylene tubes for further min-
eral analysis. After blood sampling, the birds were sacriced by 3. Results
decapitation and the right leg was excised at the pelvic girdle.
All the muscles present on the femur and tibia were removed, 3.1. Feed and serum minerals
minced with disposable scalpel blades, and kept at 20 C. The
bones (femur, tibia and bula) were collected, boiled, cleaned and The feed Cr was measured in basal and experimental feed after
kept frozen in separate polythene zip lock bags at 20 C until supplementation with two different concentrations of CrCl3 6H2 O
analysis. (12.5 and 25 mg/Kg feed). As expected, the concentration of Cr
 A.A. Saeed et al. / Journal of Trace Elements in Medicine and Biology 42 (2017) 2529 27

Table 2 4. Discussion
The effects of chromium supplementation on broiler blood serum and leg muscle
mineral (mEq/l) concentrations at 5 weeks of age.
In the studies of mineral nutrition, the most important part is
Serum Muscles interaction of minerals in different body compartments. However,
C 12.5 25 SEM Sig C 12.5 25 SEM Sig this interaction is diverse due to the use and deposition of min-
erals in different physiological processes [20,21]. Cr is one of the
Cr 01.1 01.2 01.2 0.02 NS 02.1 02.3 02.1 0.06 NS
Zn 0.08a 0.03b 0.02b 0.01 ** 01.2a 0.63b 0.33c 0.08 *** essential micronutrient required by avian and mammalian species
Cu 0.07 0.06 0.07 0.01 NS 0.17 0.18 0.19 0.03 NS to maintain physiological integrity. Also, Cr is an active module
Ca 83.5 77.0 86.5 5.5 NS 146.0 149.0 153.5 6.5 NS of Glucose Tolerance Factor (GTF) which has an active physiolog-
Na 110.4 95.7 117.4 11.3 NS 92.2 107.8 110.4 7.4 NS ical role in empowering insulins action [9]. Therefore, Cr has an
K 14.6a 5.6b 9.9b 1.5 * 188.2a 145.9b 144.1b 8.7 *
important role in maintaining the blood glucose concentration by
C-control; 12.5 and 25 are concentrations of CrCl3 H2 O (mg/Kg feed); SEM = standard muscle, adipocytes and liver glucose uptake [22]. In our study, a
error of mean; Sig = statistical signicance.
non-signicant difference in serum Cr was observed. This may be
NS = P > 0.05; * = P < 0.05; ** = P < 0.002; *** = P < 0.001.
ac
Means with different superscripts within the same row differ signicantly. due to the decreased absorption of Cr, formation of Cr oxides with
the natural chelating compounds of feed in the intestine [23], a
suboptimal amount of niacin in the feed [24] or one week Cr with-
was lower in basal/control feed (40.03 1.88; P < 0.05), signicantly drawal period. However, the decrease in Cu and Zn concentrations
higher (54.20 1.66; P < 0.05) with the addition of 12.5 mg/Kg feed maybe due to the fact that Cr, along with Cu, Zn and Fe compete
Cr and remained at the uppermost level (65.03 7.58; P < 0.05) with transferrin binding sites in blood and with this competition
when feed was supplemented with 25 mg/Kg feed Cr. In blood some mineral levels decrease. This decrease of serum Zn can also
serum, the concentration of Cr was non-signicant when compared be correlated with the increase in bone Zn deposition, because in
to the control group while, Zn concentrations were momen- serum, Cr competes with Zn [12] while binding with proteins. Due
tously reduced (P < 0.002) in both Cr supplemented groups when to the availability of free Zn in serum, it may have the tendency to
compared to control. Within Cr-supplemented groups, serum Zn deposit in the bones. Contrary to our results, a group of researchers
concentration was inversely proportional to the concentration of reported elevated K levels in sows blood because of Cr supplemen-
feed Cr (Table 2) however; blood serum levels of Cu, Ca and Na tation [25] and this may be due to differences in animal species.
remained unaffected by Cr feeding. Serum K was negatively affected Pechova et al. [26] also reported no impact of Cr supplementation
by feed Cr supplementation (Table 2). When compared to the con- on circulating Cr and other minerals. Stress can also interfere with
trol group, serum K remained (P < 0.05) lower in both treatment normal metabolism of trace minerals, and usually increases the
groups. concentrations of serum Zn [25]. The animals in thermo-neutral
zone showed a negative correlation with Cr supplementation and
concentrations of serum Zn, Fe, Cu which resulted in decreased lev-
3.2. Muscle mineral concentrations
els [27,28]. In our experiment, we used whole thigh and leg muscles
because we wanted to see the generalized deposition of minerals
In muscle samples Cr, Cu, Ca and Na concentrations were non-
rather than the use of one single muscle. Feed Cr supplementa-
signicant in both treatment groups either fed 12.5 mg/Kg or
tion did not affect muscle Cr accumulation. The absorption of Cr is
25 mg/Kg feed Cr as given in Table 2. However, Zn concentration
inversely related to the concentration of Cr present in the alimen-
reduced (P < 0.001) signicantly in both treatment groups com-
tary tract and an increase in dietary Cr can reduce its absorption
pared to control group but within treatment groups, the decrease
[12]. In turkey leg muscle, Anderson et al. [29] reported no increase
was even more pronounced with the increment of high Cr feed.
in the muscle Cr concentration even with extremely high dietary
There was a signicant (P < 0.05) decline in muscle K concentration
Cr concentrations (200 g/g of diet). On the other hand, Gler and
compared to control (Table 2), although K concentration remained
Eeta [30] reported no effect of Cr supplementation in rabbit mus-
unaffected with different levels of Cr supplementation.
cle mineral concentrations. In our experiment there was no effect of
Cr supplementation on muscle Cu, Ca and Na concentrations. Simi-
3.3. Bone mineral concentrations larly, other researchers found no effect of Cr treatment on liver and
serum Cu, Zn levels [14,31]. We found decreased retention of Zn in
The supplementation of Cr did not bring around any signicant leg muscles of birds supplemented with dietary Cr. These results
effect on the robusticity index of femur, tibia and bula while, ash contradict with Sahin et al. [14] but are in line with the ndings
to weight ratio was signicantly lower (P < 0.05) in 25 mg/Kg Cr of Tufft and Nockels [32], this may be due to different sources of
fed group (Table 3). The pattern of decreased ash to weight ratio Cr and the duration of Cr supplementation. Furthermore, muscle
remained similar in all three bones from lower to higher Cr fortied K concentration decreased by feed Cr supplementation. Both Na
groups. In femur and tibia, Cr retention remained non-signicant and K are important for maintaining cell homeostasis, osmotic bal-
but bular Cr concentration decreased signicantly (P < 0.001) with ance and are key ions for sarcolemma depolarization. The resultant
the increase of Cr in feed as shown in Table 3. The absorption of Zn hypokalemia may result in decreased muscle function [33] and can
in femur and bula was higher (P < 0.002) in Group III birds, but in be a potential reason for decreased movement in broiler chickens
the case of tibia stayed in a non-signicant range. The presence of along with increased muscle mass. The induction of hypokalemia
Cu decreased signicantly (P < 0.002) in tibia of group III birds when also antagonizes the effects of Ca ions [34], however, we did not
compared to group I, and remained statistically irrelevant in femur notice any gross pathological symptoms and this may be due to the
and bula. In the femur and bula of Cr supplemented birds, Ca continuous supply of K+ through feed/water.
deposition concentration was signicantly (P < 0.001) lower than in The mechanism of bone Cr interaction with other minerals is
group I, whereas in tibia the Ca level remained non-signicant. The not yet comprehended [35]. In this study, we are reporting the
concentration of Na in femur (P < 0.001), tibia (P < 0.002) and bula disposition of minerals in different bones of chicken after Cr sup-
(P < 0.001) was signicantly higher in control group as compared plementation, which has not yet been reported or ascertained from
to both Cr fortied groups. Femur K level decreased (P < 0.05) after review of literature. In our experiment there is no increase in serum
25 mg/Kg feed Cr supplementation while, remained non-signicant Cr hence the deposition of Cr also remained lower in femur, tibia
in tibia and bula. and bula. This decrease in Cr concentration is due to decreased
28 A.A. Saeed et al. / Journal of Trace Elements in Medicine and Biology 42 (2017) 2529

Table 3
The impact of dietary chromium chloride on leg bones robusticity index, ash/weight ratio and bones mineral analysis (mEq/l) of broiler.

Femur Tibia Fibula

C 12.5 25 SEM Sig C 12.5 25 SEM Sig C 12.5 25 SEM Sig

Robusticity Index 34.46 36.56 34.53 1.19 NS 41.19 41.54 43.17 1.06 NS 61.63 71.65 77.26 7.92 NS
Ash/weight ratio 0.28a 0.24a 0.19b 0.05 * 0.29a 0.22ab 0.21b 0.04 * 0.34a 0.25a 0.17b 0.08 ***
Cr 2.12 2.04 1.81 0.09 NS 1.66 2.08 1.11 0.20 NS 1.89a 1.79a 1.24b 0.05 ***
Zn 6.85b 6.31b 7.20a 0.16 * 6.58 6.77 7.11 0.13 NS 3.35b 2.23b 4.82a 0.22 **
Cu 0.14 0.13 0.14 0.01 NS 0.33a 0.14ab 0.11b 0.18 ** 0.21 0.11 0.17 0.04 NS
Ca 5661a 5237b 5009b 74.2 ** 5352 5110 5147 58.3 NS 2526a 1192c 1946b 95.8 ***
Na 578.9a 288.1b 280.3b 16.79 *** 702.1a 295.7b 350.8b 57.1 ** 221.2a 79.81b 98.09b 7.85 ***
K 126.1b 126.7b 192.4a 11.36 * 154.8 127.6 155.8 20.08 NS 69.27 54.07 95.95 7.40 NS

C-control; 12.5 and 25 are concentrations of CrCl3 H2 O (mg/Kg feed); SEM = standard error of mean; Sig = statistical signicance.
NS = P > 0.05; * = P < 0.05; ** = P < 0.002; *** = P < 0.001.
ac
Treatment groups within one bone analysis are different if they do not share a common letter.

absorption of Cr [12], binding with transferrin [36] or more usage Acknowledgements

in GTF like molecules for enhancing insulin function. On the other
hand, the Zn depositions in femur and bula of group III birds were
signicantly higher than group I and group II. These results are cor-
roborated by Sahin and Sahin [27], who reported the retention of
Zn after chromium picolinate supplementation. The supplementa-
tion of Cr in broilers decreased the deposition of Cu in tibia due
to lesser tendency of Cu to deposit in bones [37,38]. Some sort of
Zn-Cu antagonism may also be responsible for less deposition of
Cu. Sirirat et al. [31] reported no change in Cu and Ca by increased
Cr level in diet. The decrease in bula and femur Ca concentra-
tions in our results shows a negative effect of Cr supplementation.
The removal of Ca in bone tissue is deleterious but in avian species
bula is a rudimentary structure and this maybe the major cause
of reduced mineral deposition. The ossication and mineralization
starts with the maturation of the chondrocytes at diaphysis and
then expands toward epiphyses. The distal end of bula epiphysis
has no Parathyroid hormone-related protein, which is obligatory to
grow epiphysis [39] and may impair minerals deposition. Similarly,
Na concentration in all three bones was decreased with Cr sup-
plementation when compared to control. On the contrary, higher
level of Cr supplementation enhanced the deposition of K in femur.
Interestingly, 25 mg Cr/Kg feed supplementation resulted in high K
deposition. Almost similar but non-signicant effect of Cr supple-
mentation on Na and K was observed [40] in bulls. The increase in
femur K concentration can be correlated with the decrease in serum
K concentration after 25 mg/Kg feed Cr group as also reported in
sows [26]. A decrease in bone Ca may be due to decreased activity
of ALP in blood [41], and this has a tendency to increase the bone
breaking strength and decrease mineralization. The bone robus-
ticity index is an important measurement for the determination
of bone strength [42]. The decline in robusticity index is a clear
explanation of stronger bone and if the bone weight/bone length
index is higher the bone is more dense [43]. The decrease in ash
contents in our experiment shows a decrease in mineralization of
bones with the increase in Cr supplementation. This may be due to
the interaction of Cr with Zn, Na, K and to some extent with Ca for
its mineralization; hence the decrease in ash/weight ratio can be
negatively correlated with bone-breaking strength.
In conclusion, we can state that Cr-supplementation has a
potent effect on serum, muscle and bone mineral interaction espe-
cially among Zn, Ca, Na and K but this interaction is different in
different body compartments. The decrease in bone weight may be
due to less deposition of Ca and Na in the bones.
Funding
The presented research was not carried out under any specic
grant from the commercial, public or non-prot funding agencies.
We authors are thankful to Mrs. Madonna Shriver for making
English language corrections in the manuscript.
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