Concentration of elements in the hair of growing and adult dogs Concentration of elements in the hair of growing and adult dogs

Abstract

The aim of the study was to investigate element concentrations in the hair of growing and adult dogs. Overall, 39 dogs were recruited in a kennel, split in 4 groups: G1, 10 puppies of small size breeds; G2, 6 puppies of medium size breed; G3, 11 adult dogs of medium size breeds; G4, 12 adult dogs of toy breeds. The dogs of each group were fed 4 different complete diets, according to the requirements. Hairs were sampled at the beginning of the study (D0) and after 60 days (D60). Elements were analysed in triplicate using ICP-OES. In the hair of the adult dogs of the G3 group the concentration of Al (p < .01) was lower and in the G2 group the concentration of Zn was higher (p < .01) than in the other groups. Cu and Mn concentrations in the hair were higher (p < .01 and p < .05, respectively) in puppies fed G1 and G2 diets in comparison to adult dogs fed G3 and G43 diets. Hair concentrations of K, Li and Na were higher at D60 in comparison with D0 (p < .01). Correlations between concentrations in the hair of Al and Fe, Al and Mg and Fe and Mg were highly significant (p < .01). Present data do not support the use of element concentrations in the hair to assess their nutritional supply but, likely, they can be used in relation to physiological status of the animals. Further studies are required to investigate the factors affecting element concentrations in the hair of dogs.

• Highlights
• Concentrations of Cu, Mn and Zn in the hair differed between growing and adult dogs;
• Al concentration in hair was highly correlated with Fe and Mg;
• Several factors other than nutritional supply regulate element deposition in the hair

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Italian Journal of Animal Science
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Concentration of elements in the hair of growing
and adult dogs
Sandy Sgorlon, Alessandro Mattiello, Linda Ronutti, Misa Sandri & Bruno
Stefanon
To cite this article: Sandy Sgorlon, Alessandro Mattiello, Linda Ronutti, Misa Sandri & Bruno
Stefanon (2019) Concentration of elements in the hair of growing and adult dogs, Italian Journal of
Animal Science, 18:1, 1126-1134, DOI: 10.1080/1828051X.2019.1621687
To link to this article: https://doi.org/10.1080/1828051X.2019.1621687
© 2019 The Author(s). Published by Informa
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Published online: 04 Jun 2019.
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PAPER
Concentration of elements in the hair of growing and adult dogs
Sandy Sgorlon , Alessandro Mattiello, Linda Ronutti, Misa Sandri and Bruno Stefanon
Dipartimento di Scienze Agrarie, Alimentari, Ambientali e Animali, University of Udine, Udine, Italy
ABSTRACT
The aim of the study was to investigate element concentrations in the hair of growing and adult
dogs. Overall, 39 dogs were recruited in a kennel, split in 4 groups: G1, 10 puppies of small size
breeds; G2, 6 puppies of medium size breed; G3, 11 adult dogs of medium size breeds; G4, 12
adult dogs of toy breeds. The dogs of each group were fed 4 different complete diets, accord-
ing to the requirements. Hairs were sampled at the beginning of the study (D0) and after 60
days (D60). Elements were analysed in triplicate using ICP-OES. In the hair of the adult dogs of
the G3 group the concentration of Al (p<.01) was lower and in the G2 group the concentration
of Zn was higher (p<.01) than in the other groups. Cu and Mn concentrations in the hair were
higher (p<.01 and p<.05, respectively) in puppies fed G1 and G2 diets in comparison to adult
dogs fed G3 and G43 diets. Hair concentrations of K, Li and Na were higher at D60 in compari-
son with D0 (p<.01). Correlations between concentrations in the hair of Al and Fe, Al and Mg
and Fe and Mg were highly significant (p<.01). Present data do not support the use of element
concentrations in the hair to assess their nutritional supply but, likely, they can be used in rela-
tion to physiological status of the animals. Further studies are required to investigate the factors
affecting element concentrations in the hair of dogs.
HIGHLIGHTS
Concentrations of Cu, Mn and Zn in the hair differed between growing and adult dogs;
Al concentration in hair was highly correlated with Fe and Mg;
Several factors other than nutritional supply regulate element deposition in the hair
ARTICLE HISTORY
Received 27 March 2019
Revised 13 May 2019
Accepted 17 May 2019
KEYWORDS
Elements; dog; hair; diet
Introduction
The element status of an organism can be investi-
gated in various biological fluids and tissues, as blood,
urine and teguments (Długaszek 2019), and in some
studies, hair has been considered a matrix to assess
retrospectively element concentrations in relation to
the nutritional status of an individual (W
ojciak et al.
2004). According to these authors, the hair closer to
the scalp is highly related to the nutrient intakes of
minerals in the previous 6–8 weeks, even though
the chemical form of element supplementations has
also to be taken into consideration (Kuhlman and
Rompala 1998).
The biological mechanisms involved in element
deposition in hair are very complex, depending upon
the concentration in the blood (Długaszek 2019),
even though the breed of the dogs can in some cir-
cumstances be another factor to be considered
(Davies et al. 2017). Nonetheless, some physiological
conditions can influence the utilisation of some ele-
ments, as was reported in dogs (So et al. 2016)and
humans (Song et al. 2007; Suliburska 2011). For these
reasons, other authors did not considered hair a reli-
able matrix to assess dietary intakes of elements in
animals (Combs 1987) and humans (Cho and Yang
2018;Długaszek 2019).
The analysis of elements can also be named as
ionome, this term referring to the amount of mineral
and trace elements found in an organism (Salt et al.
2008). This concept was recently applied to dogs
(So et al. 2016), thanks to the recent availability of
high-throughput screening (HTS) technologies of
inductively coupled plasma (ICP) spectroscopy, often
integrated with mass spectrometry. Nevertheless, the
definition of reference values of essential macro
and micro nutrient and toxic elements can form an
CONTACT Prof. Bruno Stefanon bruno.stefanon@uniud.it Dipartimento di Scienze Agrarie, Alimentari, Ambientali e Animali, University of Udine,
via delle Scienze 208, Udine 33100, Italy
Supplemental data for this article can be accessed here.
ß2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ITALIAN JOURNAL OF ANIMAL SCIENCE
2019, VOL. 18, NO. 1, 1126–1134
https://doi.org/10.1080/1828051X.2019.1621687

interesting integrating diagnostic tool to investigate,
at least in part, the healthy condition of an individual
(Klevay et al. 2004;Daviesetal.2017; Skalnaya et al.
2018; Stepanova et al. 2018). In fact, the advantages
of using hair matrix is that it is easy to collect and, in
the case of pets, can be sampled directly from the
owner. Moreover, hair analysis allow to trace back the
exposure to polluted environment or to high inges-
tion of toxic elements, as Pb, Cd, Ni, Cr and Hg
(Nadal et al. 2005; Xie et al. 2017; Stepanova et al.
2018). However, in the case of dogs, there is still a
paucity of data about the element concentrations in
hair and on the factors affecting them (So et al. 2016;
Davies et al. 2017), which makes all these aspects
interesting to unravel.
The aim of this study was to gain additional infor-
mation on element concentrations in hair of growing
and adult dogs housed in a common environment
and under controlled feeding regimes in a defined
window time. In the study, attention was paid espe-
cially for macro and micro nutrients, considered the
important role they have in the regulation of physio-
logical processes.
Material and methods
Animal, diets and experimental design
For the study, 39 dogs were recruited in the same
kennel. The inclusion criteria were a healthy condition,
as confirmed by clinical examinations, free from exter-
nal and internal parasites, and without drugs adminis-
tration in the last 4 months. All protocols, procedures
and the care of the animals complied to Italian legisla-
tion on animal care (DL n.116, 27/1/1992) and the
study was approved by the ethical committee of the
University of Udine (OBPA University of Udine,
Protocol #2/2017). At the end of the study, dogs
returned to the diet regularly fed in the kennel.
According to kennel availability, the 39 dogs were
allocated to 4 groups: G1 group, 10 puppies of small
size breeds; G2 group, 6 puppies of medium size
breed; G3 group, 11 adult dogs of medium size
breeds; G4 group, 12 adult dogs of toy breeds. Dogs
were housed alone in box with a free access to an
external area and free access to tap water from muni-
cipality. Details of the dogs enrolled for the study are
reported in Supplementary Table S1. The adult dogs
(G3 and G4 groups) were resident in the kennel from
at least 6 months and received the commercial kibbles
K3 and K4, respectively, since at least 2 months from
the beginning of the study. The puppies (G1 and g2
groups) were born in the kennel and weaned with
commercial kibbles K1 and K2, respectively. These
foods had chemical and nutritive compositions appro-
priated for the age and the size of the dogs and the
daily amount of kibble offered to the dogs followed
the doses indicated in the commercial labels.
At the beginning of the study (D0), a sample of
hair was collected with a scissor from the medial part
of the left leg, taking care to cut as closer to the skin
as possible. The area was chosen since it is not in con-
tact with leash or collar and not easy visible. The
same area was sampled with the same procedure after
60 days (D60). The D0 hairs were sampled on 3rd of
June and the D60 on 2nd of August. Just after the col-
lections, the samples were stocked in a paper enve-
lope and stored until element analysis.
Chemical and element analysis of the kibbles and
hair samples
Before the beginning of the study, a sample of each
of the 4 kibbles was collected and analysed for dry
matter, crude protein, crude fibre, lipids and ash using
the methods prescribed by AOAC (2005).
For element analysis, about 1000 g of kibbles were
grinded into fine powder with a blender and dried in
an oven at 105 C for 24 h and then left to equilibrate
in a desiccator for 30 min before weighting. For hair
about 500 mg of sample was cleaned by soaking for
2 h in a 2:1 mixture of methanol and chloroform, fol-
lowed by washing twice in MilliQ water (O’Connell
et al. 1999). This procedure was applied to remove the
hair samples from environmental contaminants before
the element analysis. Samples were then dried to con-
stant weight at 105 C for 24 h and left to equilibrate
in a desiccator for 30 min before weighting. After thor-
oughly mix, a sample of 200 mg of each kibble and
hair was mineralised at 180 C for 10 min with a micro-
wave system Mars 5 (CEM, MARS Xpress, Matthews,
NC, USA) in a Teflon digestion tube containing 1 ml of
H
2
O
2
at 30% (v/v) (Sigma Aldrich, Milan, Italy) and 9 ml
of HNO
3
at 65% (v/v) (PanReac AppliChem VWR, Milan,
Italy), according to US Environmental Protection
Agency (USEPA) procedure number 3052 (USEPA
1996). After mineralisation, the digestion tubes were
cooled to room temperature and the liquid samples
were filtered (0.45 lm PTFE), diluted (1:20) and ana-
lysed for the element contents using an inductively
coupled plasma optical emission spectroscopy ICP-OES
(Vista MPX, Varian Inc., Palo Alto, CA, USA) with detec-
tion limit <1 ppm.
The accuracy of the analytical procedure adopted
for ICP-OES analysis was checked by running standard
ITALIAN JOURNAL OF ANIMAL SCIENCE 1127

solutions of elements of 0.005, 0.05, 0.5, 5.0 and
50 ppm every 20 samples. Yttrium was used as the
internal standard. Quality control for ICP-OES was per-
formed using reagent blank samples, and triplicate
readings for each sample. Certified standard reference
material (tomato leaves 1573a from the National
Institute of Standards and Technology, Gaithersburg,
MD, USA) was treated as the samples (n¼3). The
recovery of the elements in the standard material was
on average 97% of the certified values with an RSD
average of 1.3%. Method detection limits (lg/l) in ICP-
OES were: 4.0 for Al, 1.0 for Ag, 0.7 for Ba, 0.3 for Ca,
0.5 for Cd, 1.2 for Co, 1.0 for Cr, 1.5 for Cu, 0.9 for Fe,
4.0 for K, 1.0 for Li, 0.1 for Mg, 0.13 for Mn,1.5 for Na,
2.1 for Ni, 25.0 for P, 8.0 for Pb, 0.1 for Se and 0.8
for Zn.
Cortisol determination
For the extraction of cortisol from the hair, 150 mg of
sample were weighted from each sample and placed
into 15 ml glass vial (Accorsi et al. 2008). Samples
were washed three times with 2.5 ml of isopropanol
(2-propanol 99.5%, Sigma Aldrich, Milan, Italy) and
3 min of vortex. Isopropanol was discarded after each
wash and after the final wash hair samples were
placed on a plastic disk and let dry for 48 h at room
temperature. Dried hair samples were trimmed with a
blade and 50 mg of trimmed hair were weighted and
placed into a 15 ml glass centrifuge tube with 5 ml of
methanol. Samples were incubated in water bath at
45 C for 18 h under moderate shaking. At the end of
incubation, tubes were centrifuged at 5000 gfor
10 min and 2 ml of supernatant was transferred to a
1.5 ml Eppendorf tube and centrifuged in a spin-vac-
uum (Centrifugal System, RC 10.10, Jouan, Cologno
Monzese, Italy) at 40 C until completed evaporation
of methanol. Dried samples were then reconstituted
with 0.6 ml of PBS, with 0.1% bovine serum albumin
(Sigma Aldrich, Milan, Italy) and frozen until analysis.
Cortisol concentration was measured according to the
RIA procedure described by Sgorlon et al. (2015). All
samples were assayed in duplicate, the sensitivity of
the assay was 3.125 pg/well and the intra-assay and
inter-assay coefficients of variation in high and low
cortisol reference samples were 5.9% and 9.1% and
13.5% and 15.1%, respectively.
Statistical analysis
Data were imputed on a spreadsheet and analysed
with the SPSS software (SPSS 2018). Before statistical
analysis, normality of distribution was checked for
each element with the nonparametric Kolmogorov-
Smirnoff test. Data of elements were not normally dis-
tributed and a natural logarithmic transformation was
then applied. The Kolmogorov–Smirnoff test run on
logarithmic data indicated a Gaussian distribution. The
natural logarithmic concentrations of the elements in
the hair were analysed using a mixed model proced-
ure, with fixed factors for group (4 levels; G1, G2, G3
and G4), time of sampling (2 levels, D0 and D60), sex
(2 levels, male and female), group and time of sam-
pling (G x D) and group and sex (G x S) interactions.
For each element, the covariate for element intake
was applied and dogs, repeated within time, were
considered as random factor.
The rejection of null hypothesis was considered for
p<.05, with the least square differences method and
correction for multiple testing with FDR. Pearson cor-
relations among elements were calculated. Cortisol
concentration of hair was analysed using the ANOVA
model, with group (4 levels; G1, G2, G3 and G4) as
fixed factor.
Results
The crude protein and crude fibre contents of the com-
plete diets (Table 1) differed between puppies (G1 and
G2) and adult dogs (G3 and G4), whilst the oil and fat
contents were lower for the G3 group in comparison to
Table 1. Proximate analysis and concentrations of elements
of the kibbles administered to the dog during the study.
Item Unit K1 K2 K3 K4
Crude Protein % as fed 30.2 31.7 23.6 26.6
Oil and fat “18.3 17.6 9.5 18.3
Crude Fibre “2.9 3.2 4.6 4.2
Ash “6.2 7.0 8.7 6.6
Metabolisable energy kcal 4008 3930 3399 3943
Ag lg/g b.d.l. b.d.l. b.d.l. b.d.l.
Al “198.6 247.7 16.8 35.6
Ba “5.1 6.5 4.9 3.5
Ca “5988.2 7723.3 10129.9 8460.2
Cd “b.d.l. b.d.l. b.d.l. b.d.l.
Co “b.d.l. b.d.l. b.d.l. b.d.l.
Cr “1.2 b.d.l. b.d.l. 0.3
Cu “9.6 9.3 9.0 10.3
Fe “90.2 118.2 92.3 94.5
K“3996.6 4211.9 3571.8 4256.2
Li “0.2 0.3 0.3 0.4
Mg “628.0 503.6 786.2 687.2
Mn “60.5 45.1 35.2 42.8
Na “1679.5 2488.2 2217.0 2015.2
Ni “0.7 b.d.l. b.d.l. b.d.l.
P“5407.7 6599.2 10506.4 7231.5
Pb “b.d.l. 0.8 b.d.l. b.d.l.
Sr “11.9 14.0 17.7 20.5
Zn “131.1 132.3 84.7 140.4
G1: Growing dogs of small size, fed K1 diet; G2: Growing dogs of medium
size, fed K2 diet; G3: Adult dogs of medium size, fed K3 diet; G4: Adult
dogs of small size, fed K4 diet; b.d.l.: below detection limit.
1128 S. SGORLON ET AL.

the other groups. The element concentrations varied
widely between diets, but not relevant differences
between puppies and adult kibbles were observed. Only
Al content was consistently higher in the puppy diets.
The concentrations of Ag, Cd, Co, Cr, Ni and Pb ele-
ments were below the detection limits of the ICP-OES
instrument for all or for some of the kibbles.
According to the labelling of the 4 kibbles, the
form of mineral additives supplemented in the diets
was the same for Ca (calcium carbonate and calcium
phosphate), Cu (copper sulphate pentahydrate), Fe
(iron carbonate), Mn (manganese oxide), Na (sodium
chloride) P (calcium phosphate) and Zn (zinc oxide).
The mean intake of kibbles in the 4 groups (Figure 1)
was significantly higher (p<.01) for the adult dogs of
medium size (G3) in comparison to the others groups.
The lowest kibbles intakes was recorded for the pup-
pies of small size (G1) and adult of small size (G4).
The mean and standard deviation of the concentra-
tions of the element in the hair samples before natural
logarithm transformation between the 4 groups at D0
and D60 is reported in the Table 2. The hair concen-
trations of Ag, Cd, Co, Cr, Ni and Pd were lower than
the detection limits for most of the hairs samples of
the dogs collected either at D0 or D60 and no statis-
tical analysis was performed. Statistical analysis with
mixed model of the natural logarithmic transformed
data of element concentrations revealed significant
differences between diets for Al, Cu, Mn and Zn (Table
3and Figure 2). In particular, the adult dogs of the G3
group showed the lowest hair concentrations of Al, Cu
(p<.01), Mn and Zn (p<.05), while the hairs of
0
100
200
300
400
500
G1 G2 G3 G4
g/d
CBAC
Figure 1. Average daily intake of kibbles during the study. A,
B and C denote means significantly different for p<.01. G1:
Growing dogs of small size, fed K1 diet; G2: Growing dogs of
medium size, fed K2 diet; G3: Adult dogs of medium size, fed
K3 diet; G4: Adult dogs of small size, fed K4 diet. For K1, K2,
K3 and K4 diets, see Table 1.
Table 2. Mean concentrations and number of samples below the detection limits of the elements in the hair sampled from dog
fed commercial diets at the beginning of the study (D0) and after 60 days (D60).
Element Unit
D0 D60
SEMG1 G2 G3 G4 G1 G2 G3 G4
Ag lg/g 2.9 2.8 4.8 11.4 10.6 11.8 6.8 20.7 3.5
Al lg/g 554.5 510.2 326.0 613.2 645.9 711.4 297.8 937.5 58.6
Ba lg/g 2.2 3.2 2.1 2.3 2.0 3.5 1.2 3.2 0.2
Ca lg/g 1306.3 1297.5 1430.6 1342.1 1582.9 1482.4 1252.5 1715.5 117.8
Cd lg/g 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0
Co lg/g 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Cr lg/g 0.4 0.9 0.2 0.3 1.0 0.7 0.7 0.9 0.1
Cu lg/g 11.3 10.0 8.7 9.6 11.8 10.6 8.9 10.2 0.2
Fe lg/g 518.4 355.8 324.8 451.1 541.9 448.7 236.9 663.2 42.5
Klg/g 179.3 200.0 115.3 243.0 537.0 338.6 383.2 479.1 29.2
Li lg/g 0.2 0.1 0.2 0.2 0.7 0.7 0.4 1.0 0.1
Mg lg/g 532.1 340.0 311.8 486.6 436.1 290.2 242.7 479.2 30.5
Mn lg/g 9.9 12.4 6.4 8.0 12.5 10.2 5.6 9.7 0.7
Na lg/g 293.0 375.8 302.3 414.8 1202.1 734.3 1530.2 1116.1 75.1
Ni lg/g 0.1 0.0 0.3 0.2 0.9 0.8 0.0 1.1 0.1
Plg/g 326.4 267.4 296.5 300.4 336.0 292.4 273.6 296.6 8.2
Pb lg/g 0.2 0.1 0.1 0.1 0.1 0.0 0.0 0.0 0.0
Sr lg/g 2.4 2.9 2.8 2.2 2.6 3.9 5.1 4.3 0.4
Zn lg/g 229.5 234.8 181.4 217.7 245.1 235.7 194.1 217.0 3.8
Number of Samples lower than b.d.l.
Ag No <b.d.l. 6 4 9 10 7 4 8 10
Cd No <b.d.l. 8 5 7 11 9 5 11 10
Co No <b.d.l. 9 6 11 12 9 6 11 12
Cr No <b.d.l. 34644216
Ni No <b.d.l. 7 6 7 10 4 2 11 4
Pb No <b.d.l. 9 5 10 10 9 6 11 12
G1: Growing dogs of small size; G2: Growing dogs of medium size; G3: Adult dogs of medium size; G4: Adult dogs of small size; b.d.l.: below detec-
tion limit.
ITALIAN JOURNAL OF ANIMAL SCIENCE 1129

puppies fed K1 and K2 diets (G1 and G2 groups) had
a significant higher content of Cu and Mn. The G2
group showed a significantly higher concentration of
Zn in comparison to G3 and G4 groups.
Time of sampling (Figure 3) caused a significant
increase of K, Li and Na (p<.01) concentrations in the
hair at D60 in comparison to D0. Cortisol concentra-
tion in hairs was measured at the end of the study
(D60) and represented the deposition of the hormone
during the time window of 60 days of regrowth. No
significant differences were observed between the 4
dietary groups (Figure 4).
Table 3. Results of the statistical analysis and estimated mean concentrations of the elements in the hair sampled from dog fed
commercial diets at the beginning of the study (D0) and after 60 days (D60). Since data were not normally distributed, a natural
logarithmic transformation was applied.
Main Effects and Interactions (pvalues)
CovariateElement Unit G1 G2 G3 G4 D0 D60 SEM Diet Time Sex G TGS
Al lg/g 6.510 6.560 5.470 6.220 6.120 6.260 0.110 .009 NS NS NS NS 0.731
Ba lg/g 0.340 1.540 0.900 0.160 0.850 0.620 0.090 NS NS NS NS NS 0.141
Ca lg/g 6.930 7.120 7.370 6.910 7.090 7.080 0.080 NS NS NS NS NS 0.439
Cu lg/g 2.370 2.360 2.280 2.220 2.280 2.220 0.020 .009 .027 NS NS NS 0.056
Fe lg/g 6.260 5.840 5.300 6.140 5.860 5.910 0.080 NS NS NS NS NS 0.803
Klg/g 5.280 5.840 5.900 5.200 5.160 5.930 0.110 NS .000 NS NS NS 0.001
Li lg/g 0.990 1.380 1.530 1.010 1.270 1.260 0.090 NS .000 NS .043 NS 0.831
Mg lg/g 5.770 5.530 6.240 5.670 5.950 5.660 0.080 NS .022 NS NS NS 0.076
Mn lg/g 2.360 2.400 1.820 1.710 2.130 2.020 0.090 .022 NS NS NS NS 0.371
Na lg/g 6.230 6.120 6.440 6.230 5.680 6,860 0.120 NS .000 NS .022 NS 0.968
Plg/g 5.780 5.610 5.670 5.660 5.680 5.670 0.030 NS NS NS NS NS 0.821
Sr lg/g 0.460 0.990 1.280 0.730 0.760 0.970 0.090 NS NS NS NS NS 0.432
Zn lg/g 5.370 5.560 5.320 5.300 5.370 5.400 0.020 .000 NS NS NS NS 0.003
G1: Growing dogs of small size; G2: Growing dogs of medium size; G3: Adult dogs of medium size; G4: Adult dogs of small size; G T¼Interaction
Group and Time of Sampling; G S¼Interaction Group and Sex.
0
2
3
5
6
8
9
G1 G2 G3 G4
ln(µg/g)ln(µg/g)
ln(µg/g)ln(µg/g)
Al
A A B A
0
1
2
3
G1 G2 G3 G4
Cu
A A B B
0
1
2
3
G1 G2 G3 G4
Mn
aabb
2
3
4
5
6
G1 G2 G3 G4
Zn
AB A B B
Figure 2. Effect of the diets on the mean concentrations of Al, Cu, Mn and Zn in the hair of dogs and reported as natural loga-
rithm. These elements significantly differed between groups of dogs (Table 3) and the data reported are the average concentra-
tions of the hair sampled at the beginning of the study (D0) and after 60 days (D60). Within each diagram, a, b denote means
which significantly differ for p<.05 and A, B denote means which significantly differed for p<.01. G1: Growing dogs of small
size, fed K1 diet; G2: Growing dogs of medium size, fed K2 diet; G3: Adult dogs of medium size, fed K3 diet; G4: Adult dogs of
small size, fed K4 diet. For K1, K2, K3 and K4 diets, see Table 1.
1130 S. SGORLON ET AL.

The correlations between concentrations in the hair
of Al and Fe, Al and Mg and Fe and Mg were highly
significant (p<.01), with coefficients equal to 0.922,
0.715 and 0.978, respectively (Figure 5).
Discussion
From a nutritional point of view, some elements are
considered essential for vertebrates and they fulfil
several roles, as structural, physiological, catalytic and
regulatory. From a nutritional point of view, elements
are often classified as macroelements, meaning that
the inclusion in the diet is in the order of g/kg (Ca, Cl,
K, Mg, Na, P, S) and microelements, which are
included in mg/kg (Cr, Cu, F, Fe, I, Mn, Si, Se, Zn).
However, other elements are involved in biological
processes, and their inadequate supply or excess of
intake can lead to imbalances and toxicity.
Available data on the concentrations of elements in
the hair of dogs are very limited, but the identification
of threshold values of elements –if any –in the hair
of healthy dogs could be of interest as a complemen-
tary diagnostic tools. Davies et al. (2017) analysed the
element concentrations in the hair of Labrador and
other breeds of dogs. The animals were healthy or
affected from medial coronoid process disease and
the authors found significant variations for some ele-
ments as a function of disease and breed. The concen-
trations measured by Davies et al. (2017) differed from
the data reported in the present study for Al, Fe, K
and Mn (Al: from 13 to 50 mg/kg; Fe: from 17 to
59 mg/kg; K: 3 to 89 mg/kg; Mn: 0.4 to 2.9 mg/kg);
instead, for other elements, the concentrations were
similar (Table 2). So et al. (2016) published the iono-
mic profile of canine hair pre and post lipopolysac-
charide (LPS) induced stress. Before the treatment, the
concentrations in the hair of Al was close to that
reported by Davies et al. (2017), but for Fe, K and Mn
data differed from those reported in present study
and from those of Davies et al. (2017). Other differen-
ces between the 2 studies and the present data were
shown for Cu, Zn, Mg. The reasons of the observed
discrepancy are not easy to explain, and the lack of
systematic data on the element profile of hair in dogs
0
100
200
300
400
500
600
06D0D
µg/gµg/gµg/g
K
0.0
0.3
0.6
0.9
1.2
06D0D
Li
0
300
600
900
1200
1500
06D0D
Na
Figure 3. Effect of the time of sampling on the concentration
of K, and Li and Na in the hair of dogs at the beginning (D0)
and after 60 days (D60) of the study. These elements signifi-
cantly differed between time of sampling for the groups G1, G2
G3 and G4 (Table 3). G1: Growing dogs of small size, fed K1
diet; G2: Growing dogs of medium size, fed K2 diet; G3: Adult
dogs of medium size, fed K3 diet; G4: Adult dogs of small size,
fed K4 diet. For K1, K2, K3 and K4 diets, see Table 1.
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
G1 G2 G3 G4
ng/g
Figure 4. Estimated means of cortisol concentrations in the
hair samples regrowth after 60 days from the beginning of
the study. G1: Growing dogs of small size, fed K1 diet; G2:
Growing dogs of medium size, fed K2 diet; G3: Adult dogs of
medium size, fed K3 diet; G4: Adult dogs of small size, fed K4
diet. For K1, K2, K3 and K4 diets, see Table 1.
ITALIAN JOURNAL OF ANIMAL SCIENCE 1131

in relation to the diet, breed and environment do not
allow to explain these differences.
For Cu and Mn, significant differences were
observed between young (G1 and G2) and adult (G3
and G4) dogs (Figure 2), but for Al the lowest concen-
tration was measured in G3 dogs and for Zn the high-
est concentrations were detected in G2 dogs. The
additives of the 4 kibbles had the same form and, but
the bioavailability of elements depends from several
factors, other than their chemical form (Kuhlman and
Rompala 1998). Once absorbed from the gut, the
incorporation of elements in the hair follicles depends
upon the metabolic activity and serum concentrations
(Suliburska et al. 2011; So et al. 2016;Długaszek 2019).
It must be considered that Cu, Mn and Zn are
enzymes cofactors and components of redox enzymes
(Cu: superoxide dismutase, cytochrome oxidase; Mn:
arginase, glycosyltranferase; Zn: superoxide dismutase),
metalloproteins (Cu: ceruloplasmin; Zn: metallothio-
nein) and hormones (Zn: insulin, growth hormone,
sexual hormones). Thus, the observed variations
are presumably related to the variable physiological
involvements of these elements, especially when com-
paring growing with adult animals.
Moreover, positive and negative interactions among
elements and other nutrients of the diet (Długaszek
2019), age (Suliburska 2011) and healthy conditions
(Suliburska et al. 2011; So et al. 2016) have been
reported to affect element concentrations in the hair.
For instance, dietary intake of Ca, P and Fe affects the
uptake of other elements in the hair (Długaszek 2019).
The content of Ca, Mg, Fe and Zn in the diets of
women correlated inversely with Cu level in the hair
(Suliburska 2011). Pathological conditions can modify
element concentrations in the hair both in humans
(Cho and Yang 2018; Skalnaya et al. 2018) and dogs
(So et al. 2016; Davies et al. 2017). Nonetheless, contact
of the hair with the secretions of sebaceous, apocrine
and eccrine glands can contribute to the elemental
profile and, probably, for this reason significant correla-
tions between concentrations in hair and elemental
intakes are usually low (Combs 1987). Environmental
pollution too has been reported to affect element con-
centrations in the hair (Combs 1987; Nouioui et al.
2018; Stepanova et al. 2018;Długaszek 2019).
The high difference of Li, K and Na concentrations
(p<.01) measured in the hairs sampled at D0 in com-
parison to D60 (Figure 3) would indicate that weather
is another factor playing a role in the regulation of
element concentrations in hairs. The D0 samples were
collected at the beginning of June and the D60 at the
beginning of August, and the hair concentrations of
these cations could have traced the variation of depo-
sitions from the mild to the hot period of the year. In
2017, the weather was particularly dump and hot in
June and July in North-East of Italy, as can be con-
firmed from the climate records. So et al. (2016)
reported a relevant and significant increase of Na and
K in hair after LPS treatment and the authors consid-
ered this as an effect of the enhancement of adrenal
aldosterone in the serum of the dogs after immune
stress. A similar mechanism can be claimed for the
dogs exposed to high temperature. It is noteworthy
0
300
600
900
1200
1500
0 300 600 900 1200 1500
0
300
600
900
1200
1500
0 300 600 900 1200 1500
0
300
600
900
1200
1500
0 300 600 900 1200 1500
Fe,
µ
gkg
Mg,
µ
gkg
Fe,
µ
g/g
Al,
µ
g/g
Al,
µ
g/g
Mg, µg/g
Figure 5. Significant correlations (p<.01; n¼78) between
the concentrations of Aluminium and Iron (Al vs Fe; r¼0.922),
Aluminium and Magnesium (Al vs Mg; r¼0.715) and
Magnesium and Iron (Fe vs Mg; r¼0.978) in the hair of the 4
groups of dogs fed different diets. Correlations were calculated
for the hairs sampled at the beginning of the study and after
60 days of regrowth (n¼78).
1132 S. SGORLON ET AL.

that also the study of Davies et al. (2017) a significant
increase of some elements in seek dogs was observed.
The dogs enrolled in the present study were clinic-
ally healthy and during the 60 days of the trial no
signs of diseases were reported. To assess if dogs were
exposed to environmental stressors, hair cortisol con-
centrations in the hair samples collected at D60 was
also measured (Figure 4). Cortisol is considered an
endocrine biomarkers of environmental stress (Sandri
et al. 2015; So et al. 2016; Colussi et al. 2018) and its
concentration in the hair has been used to assess
medium term exposure to unfavourable conditions
(Accorsi et al. 2008; Bryan et al. 2013). The concentra-
tion of hair cortisol did not differ between groups and
were within the normal values reported from the
authors, suggesting that dogs did not have any par-
ticular challenge during the 60 days of the study.
Interestingly, high (p<.01) coefficients of correl-
ation were found for Al, Fe and Mg (Figure 5). Fe
metabolism is highly regulated at intestinal, cellular
and renal levels and that this element participates to
several activity, including oxygen transportation at
blood and muscular levels. In the research of Davies
et al. (2017), Fe concentration in the hair was not
related to breed or disease conditions and in the
study of So et al. (2016), LPS treatment had very small
effect on Fe in the hair. Fe binds to transferrin, enter-
ing the cellular through specific transferrin receptors
(P
erez et al. 2001), but also a non-transferrin bound
iron system of transport has been reported (P
erez
et al. 2005). Al and Fe have physical and chemical
characteristics in common, as ionic radius, charge
density, chelation by particular compounds (Ward
et al. 2001), and a competition with abiogenic Al
3þ
ions in protein binding sites has been reported
(Dudev et al. 2018;Długaszek 2019). This can explain
the strict correlation between Al and Fe reported in
Figure 5. If the presence of Al was related to the iron-
aluminium alloy used in feed manufacturing is not
possible to state, but the G1 and G2 diets (with high
concentrations of Al) were manufactured by the same
company, and the G3 and G4 by another industry
(with low concentrations of Al). These aspects
deserves further investigations, since Al accumulation
in the body was considered responsible for the etiopa-
thogenesys of erythropoietic (Nesse and Garbosa
2001) and nervous (Zatta et al. 2003) systems related
diseases. For Mg, quantitative relationship with Fe in
body fluids and red cells (Długaszek 2019) and hair
(Długaszek et al. 2014) have been reported, but for
these elements other factors, as breed, probably inter-
acted with the deposition in the hair.
Conclusions
The study was performed to evaluate the concentra-
tions of elements in the hair of growing and adult
dogs fed diets differing for composition and elemental
supply. To assess the mechanisms of element uptake
and transfer from blood to tissues and hair, deeper
investigations are required, as blood and biopsy sam-
ples, but bioethical issues limit the accessibility of
these types of specimen. Instead, hair is a very simply,
inexpensive and not invasive matrix which can offer
the opportunity to retrospectively investigate the clin-
ical conditions of dogs.
The data gathered from this study can implement
the information on the concentrations of elements in
the hair, offering preliminary data for further investiga-
tions the factors regulating elemental concentrations
in the hair of dogs.
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
Sandy Sgorlon http://orcid.org/0000-0001-9247-6441
Bruno Stefanon http://orcid.org/0000-0002-7414-5830
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