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Pilot Study
Tyrosine supplementation and hair coat pigmentation in puppies with black
coats –A pilot study
Adrian Watson
1
*, Eric Servet
1
, Marta Hervera
2
and Vincent C. Biourge
1
1
Royal Canin, Research Center, Aimargues, France
2
Nutrition & Endocrinology Unit, ONIRIS National College of Veterinary Medicine, Food Science and Engineering Nantes-Atlantic, 44307
Nantes Cedex 3, France
Summary
The appearance of a red hue to the hair in black coated cats and dogs has previously been reported as a “red hair syndrome”.
Such changes in hair colour are related to an alteration in the proportions of two types of pigments produced by melanocytes;
black eumelanin and brown pheomelanin. In black cats, it has been demonstrated that higher levels of phenylalanine + tyrosine
(Phe+Tyr) than those recommended for growth are required to support eumelanin synthesis. The purpose of this study was to
evaluate if a similar observation could be made in dogs. Twelve black coated puppies (Black Labrador retrievers and
Newfoundlands) were divided into 3 groups of 4 and fed 3 diets with increasing concentrations of Phe+Tyr (A: 4 g/Mcal;
B: 5.8 g/Mcal; C: 7 g/Mcal) for a period of 6 months. Quantification of plasma amino acids (Phe, Tyr, Cys) and spectroco-
lourimetry of hair samples from the Labrador retrievers (as the a* dimension of CIE Lab system) were performed at the begin-
ning, during and at the end of the study. There was a significant negative linear relationship between plasma Tyr levels and a*
values of hair in Labrador dogs on diets A and B, suggesting that a diet with total Phe+Tyr content of 6 g/Mcal (higher than
the growth recommended allowance) was necessary to ensure an optimal black coat colour in these puppies and that levels up
to 7 g/Mcal can lead to a more intense black coat colour. Moreover, similar to what was found in kittens, plasma levels of Tyr
up to 54 μmol/l did not guarantee an optimal black colour coat and led to the “reddish hair”appearance in black coated
puppies.
Keywords: dog: nutrition: black coat: red coat syndrome: tyrosine: phenylalanine: melanin: melanocytes
Introduction
Melanocytes are the cells responsible for generating hair
colour. They reside within the follicle and synthesise
two types of melanin pigments, black eumelanin and
reddish-brown pheomelanin. A study by Ito (1993)
showed that, in animals, the relative proportions of
these two pigments molecules in the hair are determined
by tyrosinase activity in melanocytes. Low tyrosinase
activity results in higher proportions of pheomelanin syn-
thesis, while higher activity dictates elevated eumelanin
production. Tyrosinase activity is known to be stimulated
by tyrosine (Tyr) concentration resulting in a greater syn-
thesis of eumelanin, as shown by Slominski in 1989.In
addition, Morris et al. (2002) illustrated, using a cat
model, that the tyrosine requirement can be derived dir-
ectly from the diet or by the hydroxylation of the essen-
tial amino acid phenylalanine (Phe).
Coat reddening in cats and dogs is a phenomenon
which is capricious and poorly characterised. Yu et al.
(2001), and subsequently Morris et al. (2002), demon-
strated that if black cats were provided with diets contain-
ing reduced levels of Tyr and Phe, red coats were
observed, and also that these could be reverted to
black when cats were returned to their original commer-
cial pet food diet. Anderson et al. (2002) subsequently
confirmed that eumelanin production in black kittens
*Corresponding author: adrian.watson@effem.com
Journal of Applied Animal Nutrition, Vol. 3; e10; page 1 of 4 doi:10.1017/jan.2015.8
© Cambridge University Press and Journal of Applied Animal Nutrition Ltd. 2015
Journal of Applied Animal Nutrition
could be compromised by a lack of Tyr and Phe in the
diet. It was estimated that the amount of these 2
amino acids required to produce a black coat was twice
as high as that required for normal growth.
The National Research Council (NRC, 2006) recom-
mend dietary levels of Tyr and Phe combined for grow-
ing dogs are 3.25 g/MCal and 2.50 g/Mcal before and
after 3 months of age, respectively. However, no data
has been published evaluating the dietary Tyr and Phe
requirements in growing dogs with respect to coat colour.
The aim of this pilot study, therefore, was to investigate
how dietary levels of Tyr can affect coat colour of grow-
ing black dogs.
Materials and Methods
Animals
The study involved 12 puppies; six female Newfoundlands
(NFL), 3 male and 3 female black Labrador retrievers. At
the beginning of the study the NFL were 3 months old and
had an average weight of 13.1 ± 0.9 kg (Mean, +/−SD).
The male LR were on average 2.5 months old and weighed
8.0 ± 1.0 kg; the females were on average 3.5 months old
and also weighed 8.0 ± 0.9 kg. All of the puppies were
healthy and had homogeneous black coats. Before entering
the study, all dogs were fed with the same commercial diet
for growth of large breed puppies (Royal Canin, Maxi
Junior, Aimargues, France). For the period of the
study, all puppies were housed in the same well-ventilated,
temperature controlled indoor facility, with daily exercise
regimes.
Diets
The dogs were provided with one of three experimental
diets which varied in their Tyr and Phe content (labelled
Diets A, B and C; Table 1). Diet B was a standard Royal
Canin Maxi Junior (Aimargues, France). Diets were
nutritionally complete and balanced for growth (accord-
ing NRC recommended allowances, NRC, 2006). Initial
energy requirements were calculated based on NRC
(2006). Table 1 shows proximal analyses, total Tyr, Cys
and Phe content, as well as ingredient lists for the three
diets. Other dietary amino acid contents were in excess
of NRC recommended RA for growing puppies.
Experimental design
The 12 dogs were divided into 3 groups balanced with
respect to breed and sex, whereby each contained 2
NFL, 1 male LR and 1 female LR. Each group was main-
tained on one of the three experimental diets chosen at
random and fed twice per day. The amount of diet con-
sumed by each dog was recorded daily and all dogs were
weighed weekly. Food intake was regulated such that
maximum weight gain was 0.7 kg/week for LR and
1.2 kg/week for NFL.
A heparinised blood sample was collected from each
dog 5 hours after feeding at time zero, 3 and 6 months.
Tubes were centrifuged for 15 minutes at 2000 rpm and
the plasma transferred into separate tubes. At time zero,
4 months and 6 months an 8 × 8 cm square of hair was
shaved on the right lateral area of the abdomen of all
Labrador retrievers for spectrophotometry (each time at
the same location).
Table 1. Composition of 3 diets provided to dogs in order to evaluate the effect of tyrosine intake on coat colour.
Diet A B C
g/Mcal
Moisture 13.4 18.0 18.0
Crude Protein 89.5 92.7 94.2
Crude Fat 37.3 36.2 36.2
Total Dietary Fibre 16.3 17.7 17.7
Tyrosine total 1.5 2.4 3.8
Tyrosine free 0 0 1.6
Phenylalanine total 2.5 3.4 3.2
Phenylalanine free 0.2 0.1 0.5
Phe + Tyr total 4.0 5.8 7.0
Cystine total 0.2 0.4 0.6
Metabolisable Energy
(Mcal/kg)*
4.1 4.0 4.0
Feed materials (in
descending order
by weight)
Corn flour, pork gelatin, beef greaves, corn,
poultry fat, beet pulp, pork fat, poultry liver
hydrolysates, mineral mix, soy oil, vitamin
mix, fish oil, d-l methionine, L-Lysine-HCl,
L-Tryptophan
Beef greaves, corn flour, corn, beet pulp,
pork fat, poultry liver hydrolysates,
poultry fat, mineral mix, soy oil, vitamin
mix, fish oil
Diet C was the same that diet
B but 0.7% of crystalline
tyrosine was added during
coating
*Calculated based on Total Dietary Fiber (NRC, 2006)
Journal of Applied Animal Nutrition
Adrian Watson et al.2
Analytical methods
Plasma was mixed with an equal volume of 0.28 mol/l
sulfosalicylic acid. The resulting precipitate was removed
by centrifugation at 16,000 g. Lithium hydroxide was
added to the supernatant to adjust pH to 2.2. Plasma
amino acid concentrations were determined by Ion
Exchange Column Chromatography (7300 Beckman
Amino Acid Analyser, Beckman Instruments, Palo
Alto, CA) on a 0.4 × 10 cm column packed with spheric-
al cation exchange resin. An equivalent of 20 µl of
plasma was injected onto the analysis column.
For the quantitative assessment of hair colour, hair
samples were analysed with a spectrocolourimeter
(Spectro-guide
®
45/0, gloss, BYK Gardner, Brant
Industries, Germany) according to the CIE Lab colour
measurement system (Hunter Associates Laboratory,
Inc. Reston, VA, USA). Colour is determined as a
numeric value of a* (red to green axis) and b* (yellow
to blue axis). Lightness is determined as L* on a numer-
ical scale (from black = 0 to white = 100).
Statistics
Data were analysed using GLMs through the MIXED
procedure of SAS v9.1.3 (SAS Institute Inc., Cary, NC,
USA). Differences were considered significant at p ≤0.05.
Ethical Statement
The experimental protocol used in this study adhered to
European Union Guidelines (Official Journal of the European
Communities L 358, 18/12/1986). The study was also
approved by the Royal Canin Ethics Committee (RC90).
Results and Discussion
During the study puppies gained appropriate weight and
growth/development occurred as expected for breed
norms (data not shown). There was no difference in weight
gain between dietary groups. Average daily food intakes
(+/−SD) for NFL and LR were 593± 86 and 452 ± 60
g /dog /day respectively. No differences were observed
in the food intake between diet groups (p > 0.05).
Levels of Tyr, Phe and Cys in the blood of the puppies
on each diet are summarized in Table 2. There was no dif-
ference in the levels of each amino acid between the NFL
and LR within diet groups (p > 0.05). There were no sig-
nificant differences in plasma Cys and Phe between diet
groups. No changes were observed in the plasma Phe or
Cys between the different sampling points (p > 0.05).
Plasma Tyr concentrations at 3 and 6 months were signifi-
cantly higher in the diet C group than the diet B and A
groups (p < 0.05). In addition, dogs fed diet B had a
higher Tyr concentration than those fed diet A (p < 0.05).
Spectrophotometric hair analysis showed no differ-
ences between groups with respect to L* and b* dimen-
sions at any of the three sampling points. There was no
difference in a* values between groups at the start of the
study and those of the Labrador puppies fed Diet C did
not change during the study. However, the a* values of
Labrador puppies fed Diets A and B had increased
Table 2. Plasma levels of tyrosine (Tyr) and cystine (Cys) of dogs consuming 3 diets with increasing Phe+Tyr content (A: 4 g/Mcal; B: 5.8 g/Mcal;
C: 7 g/Mcal) after time zero, 3 and 6 months of diet consumption. Averages are shown (± standard error) for 4 puppies.
Month Tyr Cys Phe
AB CABCABC
Initial 59.0 ± 6.38
x
71.5 ± 8.09
x
66.9 ± 6.16
x
34.9 ± 3.09 39.8 ± 2.39 41.3 ± 4.98 70.0 ± 5.10 74.2 ± 2.00 80.8 ± 1.70
335.1 ± 1.96
ay
54.0 ± 1.33
by
79.1 ± 4.12
cxy
46.5 ± 2.35 41.3 ± 0.40 48.6 ± 1.95 71.1 ± 2.40 78.9 ± 3.20 74.9 ± 3.20
637.7 ± 2.84
ay
53.8 ± 1.56
by
86.9 ± 5.20
cy
48.2 ± 1.47 36.8 ± 3.09 42.0 ± 3.50 74.1 ± 3.40 81.8 ± 9.70 77.3 ± 4.30
abc
different superscripts indicate significant differences of the values within a row, among groups (p <0.05);
xy
different superscripts indicate significant differences of the values
within a column, over time (p < 0.05).
Figure 1. Changes in the a* dimension of CIE Lab index over time; three diets
with increasing Phe+Tyr (A: 4 g/Mcal; B: 5.8 g/Mcal; C: 7 g/Mcal) assessed at
time zero, 4 and 6 months of diet consumption (n = 2/diet group). Letters over
the bars denote significant differences (p < 0.05) within the same diet group
over time; *indicate significant differences among diet groups at the same sam-
pling point (p > 0.05).
Journal of Applied Animal Nutrition
Canine hair pigmentation and nutrition 3
significantly after 4 months and did not change thereafter
(p < 0.05; Figure 1).
There was evidence of an association between plasma
Tyr concentration and the a* dimension in the Labrador
puppies. In particular, a* decreased significantly in associ-
ation with the increased plasma Tyr seen for the group B
dogs vs group A dogs. The decrease in a* was greater
for the dogs in group C, although there was no significance
between groups B and C. The CIE Lab method was used
by Busch-Kschiewan et al. (2004) in a study on white dogs
and permitted corroboration of what was determined visu-
ally. In humans, spectrophotometric measures have been
described as an alternative to chemical methods of asses-
sing hair eumelanin and pheomelanin variation. Shekar
et al. (2008) showed that dimension a* of CIE Lab spectro-
colourimetic index is a good approximation of pheomela-
nin concentration in hair, as it measures a continuum of the
red-green spectra. A lower a* value therefore reflects a
lower hair pheomelanin concentration, manifesting as a
less reddened hair shaft.
Plasma Tyr levels were negatively correlated with a*
values in the dogs fed diets A and B (Figure 2) suggesting
a relationship between higher plasma Tyr levels and higher
eumelanin contents in the hair of black coat puppies. The
results agree with those found in kittens by Anderson et al.
(2002), where a positive correlation between plasma Tyr
levels and pyrrole-2,3,5-tricarboxylic acid was reported,
with the latter used as an indicator of eumelanin concen-
tration in the hair of cats. Raising plasma Tyr concentra-
tion from 40 to 54 μmol/L effected the most significant
reduction in the a* dimension (Figure 2), which is similar
to previous data from cats. Increased plasma Tyr levels in
the puppies in group C did not result in a further increase
in a* values over and above group B, possibly reflecting
that a point exists beyond which increasing dietary tyro-
sine has no effect, or could indeed be an artefact of our
relatively small sample sizes.
Conclusion
In summary, young black dogs maintained on a diet of
4 g/Mcal Phe+Tyr (diet A) showed reduced levels of
black pigmentation, even though they maintained the
normal growth trajectory and appeared healthy. Black
pigmentation was restored by increasing the Tyr content
of the diet. These initial results suggest that the nutrition-
al requirements for growth are insufficient to also meet
the demands of regular melanin synthesis, as highlighted
previously by Anderson et al. (2002) for cats. Normally,
growth is the most nutritionally demanding life stage.
Thus, a greater need for Phe+Tyr for other physiological
processes, such as hair pigmentation, may have to be
considered. Further studies to understand this relation-
ship better would be merited.
Declaration of interest
Eric Servet, Adrian Watson and Vincent Biourge are all
employees of Royal Canin SAS, Aimargues, France.
References
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dietary phenylalanine or tyrosine for melanin deposition in hair than
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Figure 2. Relationship between plasma tyrosine (Tyr) levels and a* dimension
spectrocolourimetric values for the hair coat of puppies consuming 3 different
diets (A: 4 g/Mcal; B: 5.8 g/Mcal; C: 7 g/Mcal). Values of animals fed A and B
(solid line; a* = 7.6-0.09 × Tyr (μmol/l); r
2
= 0.98) and B and C diets showed a
closer relationship (dashed line; a* = 3.9-0.02 × Tyr (μmol/l); r
2
= 0.91). Each
value is the mean of two animals.
Journal of Applied Animal Nutrition
Adrian Watson et al.4