ORIGINAL RESEARCH
published: 22 April 2021
doi: 10.3389/fvets.2021.667318

Clinical Findings in Healthy Dogs Fed
With Diets Characterized by Different
Carbohydrates Sources
Manuela Gizzarelli, Serena Calabrò, Alessandro Vastolo*, Giuseppe Molinaro,
Ines Balestrino and Monica Isabella Cutrignelli
Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy

Edited by:
Luciano Trevizan,
Federal University of Rio Grande doSul, Brazil
Reviewed by:
Sónia Félix Lucena,
University of Evora, Portugal
Nikola Puvača,University Business Academy in Novi
Sad, Serbia*Correspondence:
Alessandro Vastolo
alessandro.vastolo@unina.it
Specialty section:
This article was submitted toAnimal Nutrition and Metabolism,a section of the journal
Frontiers in Veterinary Science
Received: 12 February 2021
Accepted: 26 March 2021
Published: 22 April 2021
Citation:
Gizzarelli M, Calabrò S, Vastolo A,
Molinaro G, Balestrino I andCutrignelli MI (2021) Clinical Findings
in Healthy Dogs Fed With Diets
Characterized by Different
Carbohydrates Sources.
Front. Vet. Sci. 8:667318.
doi: 10.3389/fvets.2021.667318

In recent years, pet owners have become more interested in the ingredients, andquality of pet-food, and several studies have demonstrated that feed management
could affect healthy status. Recently, some authors indicated that commercial diets
formulated without cereals, or using unconventional protein, and starch sources, cancause a reduction in taurine levels in both whole blood, and plasma. Nevertheless, thespecific mechanism by means of which nutritional factors determine this reduction is
not completely clear. Thirty neutered half-breed dogs were recruited at a kennel in theprovince of Naples (Italy) to investigate the influence of carbohydrates sources, anddietary density of nutrients on healthy status of dogs in terms of blood count, andbiochemical parameters. The dogs were housed in the kennel and divided into three
distinct groups. Three iso-energy, and iso-nitrogen commercial kibble diets (named GF1,
GF2, and CB) with different protein, and carbohydrates contents, and carbohydrates
sources were chosen for the trial. The chemical composition and amino acid profile ofeach of the three tested diets were analyzed. Moreover, blood samples of each dog
were collected to evaluate the hematological and biochemical profiles. The taurine level
was determined both on plasma and whole blood. The effect of the diets was analyzed
statistically, and all tested diets were compared to the control one. There were significant
differences between the three tested diets as regards their chemical composition. The
concentrations of all amino acids seem to reflect protein content diets. The hematological
profile resulted within the ranges considered physiological for the canine species forall subjects. Compared to the control diet, the three tested diets showed significant
differences in blood count for MCHC and platelets. The biochemical profile showedsignificant differences between the diets, particularly their AST, fructosamine, lipase, andtriglycerides values. The diets did not affect the blood and plasma taurine levels. They
resulted in higher than optimal reserve levels. Preliminary results showed that the sourcesof carbohydrates and use of balanced diets affected only some biochemical parametersand did not alter the levels of taurine in healthy adult dogs.
Keywords: blood count, biochemical profile, taurine, pea, spelt, oats, pumpkin, amino acids

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INTRODUCTION

MATERIALS AND METHODS
Animals and Diets

In all developed countries, the companion animal population
has gradually increased over the last 20 years, particularly
in great urban centers (1). In Italy, about 39% of the
population live with a cat and/or a dog (2). As a result, petowners have been giving more attention to animal welfare
and have become increasingly interested in the characteristics
and production processes of commercial diets (3). The pet
industry has produced several diets with particular ingredientsor nutrients, suggested by specific claims, which could indicate
beneficial effects, for example, grain-free diets which were
formulated using tubers, and legumes as starch sources. Eachcarbohydrate source has a unique nutritional composition that
could affect both the production process and use of nutrients(4). For instance, legume grains (pea and lentil) are richin soluble dietary fiber (SDF, mean value: 26.9% of total
dietary fiber, TDF), and proteins (CP, mean value: 23.87%
DM) compared to cereals grains (e.g., corn and rice) (CP:10.72% DM, SDF: 10.00% TDF) (5–7). Moreover, the aminoacid profile of different protein sources varied significantly,
legume protein is less rich in essential amino acids (e.g.,taurine, L-carnitine) compared to protein sources of animal
origin (6, 8, 9).
In 2018, the Food and Drug Administration (10) publisheda report on the possible link between grain-free diets and dilated
cardiomyopathy related to taurine deficiency. Cardiomyopathy ischaracterized by a dilation of the left ventricle or both ventricles,
in association with impairment of ventricular contractions (11)and it causes cardiac dysfunction (12). Moreover, some studies
have indicated a correlation between taurine reduction due to theadministration of grain-free diets and dilated cardiomyopathy indogs (13–15).
Nevertheless, the results are controversial in the literature. A
recent study conducted (16) on 86 Golden Retrievers observed
significantly higher taurine values in whole blood in dogs fed
a diet containing cereal compared to dogs fed grain-free diets.
Significant differences in plasma taurine levels, however, were
not observed.
On the contrary, Donadelli et al. (17) demonstrated
significant increases in plasma taurine and whole blood
taurine levels in Golden Retrievers fed grain-free dietsfor 26 weeks. Similarly, Pezzali et al. (18) found thatgrain-free diets had no effect on taurine levels. A
recent review (19) highlighted some limits as far as
current literature is concerned as regards identifying
the specific nutritional causes of taurine deficiency and,
consequently, dilatative myocardiopathy development in
the dog.
The purpose of this study was to evaluate if the administrationof three diets (two grain-free: GF1, and GF2 vs. one cereal-based:
CB), over a medium-term period (5 weeks), formulated withdifferent carbohydrate sources and amounts could affect blood
profile, and biochemical parameters with particular regard totaurine levels in healthy dogs. We hypothesized that different
carbohydrates sources could influence the healthy status ofadult dogs.

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The nutritional double-blind trial was performed at a private
kennel located in the province of Naples (Italy). At the
beginning of the trial, a veterinarian clinically examined
50 adult dogs and performed, hematological, biochemical,
and parasitological tests (20) to exclude subjects with signsof pathologies. Subsequently, 30 adults, neutered, halfbreed dogs (age 4 ± 1.20 years, weight 20.79 ± 6.38 kg,
BCS 3.96 ± 0.95 on five point scale) were recruited.
Each dog was housed in an individual box of 8 m2 (2× 4) consisting of a closed rest portion (2 × 2), andan open common walking area for five adjacent boxes.
Before the beginning of the study, and during the firstdays of each adaptation period, all dogs were submitted
to copromicroscopic analysis for intestinal nematodes(Trichuris, Toxocara, Toxascaris, and Ancylostomidae),cardiopulmonary nematodes (Angiostrongylus and Capillaria),
Cestode (Dipylidium and other Taeniidae), and Protozoa(Giardia and Cystoisospora). If they were found to bepositive, they were immediately treated with specificdeworming drugs.
After enrollment, the dogs were divided into three distinctgroups (blue, red, and black), homogeneous for sex, age,weight, and BCS. For the experimentation, three commercial
dry diets (Farmina-pet food, Nola, Italy) named GF1, GF2, andCB, respectively, were chosen and administered alternately toexperimental groups following a Latin square scheme (3 diets× 3 groups); each experimental period had a total duration of
50 days (15 days of adaptation and 35 days of trial). Each diet
was administered in a ratio of 110 kcal/kg0.75 of metabolizableenergy (EM) (6).
The diets were characterized by similar energy densities (3,995± 4.73 kcal/kg), formulated mainly with the same protein source(chicken), but consisting of different carbohydrates sources(cereal grain vs. legume or tubers).
The ingredients of each diet were the following:• Diet CTR: rice, beet pulp, poultry, and turkey meals, fat andoil, and minerals;• Diet GF1: boneless chicken, dehydrated chicken protein, sweet
potato, chicken fat, dried eggs, herring, dehydrated herring
protein, fish oil (from herring), pea fiber, and dried carrot;• Diet GF2: boneless chicken, dehydrated chicken protein,pea starch, chicken fat, dried pumpkin, dried eggs, herring,
dehydrated herring protein, fish oil (from herring), pea fiber,and dried carrot;• Diet CB: boneless chicken, dehydrated chicken protein,spelt, oats, chicken fat, dried eggs, herring, dehydrated
herring protein, dried beet pulp, fish oil (from herring), anddried carrot.

Diets Chemical Composition and Amino
Acid Profile
An aliquot of 500 g for each diet was analyzed by means ofnear-infrared spectroscopy (NIRS DS 2005F, FOSS, Hilleroed,2

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Grain-Free Diets and Healthy Status

FIGURE 1 | Study timeline.

(TP); Albumin (Alb); Alkaline phosphatase (AP); Glutamic
Pyruvic Transaminase (GPT); Alanine Transaminase (ALT);
Gamma-glutamyl transferase (GGT); Aspartate Transferase(AST); Glutamate dehydrogenase (GLDH); Fructosamine (Fr);Glucose (GLU); α-amylase; Lipase (LP); Sodium (Na); Potassium(K); Calcium (Ca); Chloride (Cl); Phosphorus (P); Magnesium(Mg); Cholesterol (Col); Triglycerides (Tri); Creatinine (Crea);
BUN, Creatine kinase (CK).
At the end of each phase from five dogs per group blood were
sampled to determine taurine on whole blood; after samplingblood was collected in a tube containing lithium heparin.
Subsequently, it was gently mixed and stored at a temperatureof −80◦ C up to the shipment on dry ice to the Amino Acid
Laboratory (University of California, Davis, CA, USA). Whole
blood taurine concentrations were determined using a Beckman
6,300 amino acid analyzer (Beckman Industries, Palo Alto,
CA, USA).
Taurine in plasma was determined by reversed phase
chromatography hyphenated to a triple quadrupole mass
spectrometer (LC-MS/MS Sciex API4000QTRAP plus Agilent
HPLC and CTC PAL autosampler, Santa Clara, CA, USA) (26).

Denmark) to determine its chemical composition (21, 22). Total
dietary fiber, and proportion soluble, and insoluble fractions were
determined according to (23, 24). The diets were analyzed also to
determine its amino acid profile using high-performance liquid
chromatography (HPLC, Agilent Technologies 1290, California,
CA, United States) according to Spitze et al. (25).

Clinical Examination, Weight Checks, andSampling
At the beginning of each experimental period, the dogs were
subjected to physical examination, weighed, and their body
condition score (BCS) was evaluated. At recruitment and the endof each nutritional phases (Figure 1), fasted dogs were clinicallyevaluated and ∼10 mL of blood was collected in three tubes:• two with EDTA, one for determination of the blood count, andone for the dosage of plasmatic taurine;• one for whole blood with separator gel from which to obtainthe serum for determination of the biochemical profile.
The whole blood samples intended for the evaluation of the bloodcount were refrigerated, and quickly transported to the clinicalanalysis laboratory of the Department of Veterinary Medicine
and Animal Production of the Federico II University of Naples.
Each blood sample was analyzed using an impedance device tocarry out an instrumental count (HeCo 5 Vet C, Real Time
Diagnostic Systems; San Giovanni a Valdarno, Italy) after slow
and constant mixing for 20 min.
At the kennel, to obtain the serum, the gel separator tubes wereleft at room temperature for about 15 min until the clot formed,
then the samples were centrifuged for 10 min at a speed of 1,500
× g. The serum was stored at −80◦ C and subsequently sent
on dry ice to a reference laboratory (Kornwestheim, Germany)
where the following parameters were determined using a
Beckman biochemical analyzer (Beckman Coulter AU5400;
Olympus America, Melville, NY, USA): Globulin; Total protein

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Statistical Analysis
The effects of the diets were analyzed by one-way analysis ofvariance (ANOVA). Tukey’s HSD test was used when significant
differences were observed.
To compare each diet with the control one, the Dunnett
test was used, which allows multiple comparisons. All statisticalanalyses were performed using the software JMP 14 (SASInstitute, NC, USA).

RESULTS
Table 1 shows the chemical composition of the diets usedduring the trial. All statistical analyses were referred comparing
the diets. GF1 showed the highest value in protein, and3

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TABLE 1 | Diets chemical composition (g/kg as is) and energy content (ME kcal/kg as is).
Nutritional characteristics

CTR*

GF1

GF2

CB

RMSE
19.1

Crude Protein

240

363A

316B

317B

Total fat

100

185

193

194

51.5

Crude Fiber

38.0

23.0

23.7

24.7

9.41

Ash

99.0

65.0a

60.0b

60.3b

20.3

Metabolizable Energy*

3,200

3,999

3,990

3,997

140

*Chemical composition of control diet as reported in the label; GF1, grain-free diet 1; GF2, grain-free diet 2; CB, cereal based diet; along the row the capital letters indicate differences
for P < 0.01 and P < 0.001; lowercase indicated differences for P < 0.05. RMSE, root mean square error. The statistical comparison was performed between GF1, GF2, and CB diets.
*ME, calculated according to the predictive equation indicated by NRC 2006 (6).

TABLE 2 | Amino acids profile of diets used during the trial.
GF1

GF2

CB

GF1

GF2

% as isCB

FEDIAF*g/1,000 kcal ME

Protein

36.3

31.6

31.7

97.19

85.25

84.91

45.00

Alanine

2.19

1.99

1.90

5.86

5.37

5.09

NA

Arginine

2.11

1.88

1.80

5.65

5.07

4.82

1.30

Aspartic acids

2.83

2.80

2.26

7.58

7.55

6.05

NA

Glutamic acids

4.29

4.17

4.54

11.5

11.3

12.2

NA

Glycine

3.24

2.67

2.86

8.67

7.20

7.66

NA

Histidine

0.76

0.72

0.68

2.03

1.94

1.82

0.58

Hydroxyproline

1.01

0.71

0.95

2.70

1.92

2.54

NA

Isoleucine

1.13

1.12

0.97

3.03

3.02

2.60

1.15

Leucine

2.32

2.20

2.07

6.21

5.94

5.54

2.05

Lysine

2.08

2.11

1.65

5.57

5.69

4.42

0.25

Phenylalanine

1.30

1.22

1.18

3.48

3.29

3.16

1.35

Proline

2.20

1.77

2.21

5.89

4.78

5.92

NA

Serine

1.43

1.32

1.29

3.83

3.56

3.46

NA

Threonine

1.27

1.22

1.08

3.40

3.29

2.89

1.30

Tyrosine

0.87

0.84

0.77

2.33

2.27

2.06

NA

Valine

1.54

1.48

1.35

4.12

3.99

3.62

1.48

Cystine

0.42

0.37

0.43

1.12

1.00

1.15

NA

Methionine

1.10

1.00

0.93

2.95

2.70

2.49

1.00

Tryptophane

0.33

0.33

0.32

0.88

0.89

0.86

0.43

Taurine

0.22

0.23

0.18

0.59

0.62

0.48

NA

*Nutritional values and FEDIAF (2020) (27) recommendation based on a metabolizable energy requirement of 110 kcal ME/kg0.75; GF1, grain-free diet 1; GF2, grain-free diet 2; CB,
cereal based diet; NA, not applicable.

acid, glycine, alanine, arginine, aspartic acid, and lysine. The
concentrations of all amino acids seem to reflect protein
content of the diets.
The results of the blood count are reported in Table 3.
All recorded values fall into the relative physiologicalrange for canine species. The analysis of variance did
not show differences between the diets. Nevertheless, theDunnett test indicated significant (P < 0.01) differences
between CTR diet and each tested one for MCHC, which
resulted in 1 g/dL lower in CTR than GF1, GF2, and CB.
Moreover, platelet values resulted significantly (P < 0.05)
higher for CTR compared to that of CB diet (328 vs. 287
K/uL, respectively).

ash (P < 0.01 and P < 0.05, respectively). The energy
nutrients (protein, carbohydrates, and lipids) of GF1, GF2,
and CB diets were equally proportioned. Moreover, the three
diets tested resulted in higher protein and energy levels thanthe CTR one, which showed higher carbohydrates content.
Total dietary fiber (TDF) content of GF1 was significantly(P < 0.05) lower than CB one (data not reported 76.78
vs. 83.73 vs. 94.30 g/kg as is, for GF1, GF2, and CB,
respectively). Moreover, all diets showed higher incidence ofsoluble fiber than insoluble fiber (60.65; 58.61; and 63.15%
TDF, respectively).
Table 2 shows the amino acid concentrations of the three
diets. In all cases, the most present amino acids were glutamic

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TABLE 3 | Blood profile of dogs in function of administered diet.
Diets
Items

CTR vs.

Units

CTR

GF1

GF2

CB

RMSE

GF1

GF2

CB

RBC

M/uL

6.88

7.00

6.95

6.76

0.64

NS

NS

NS

WBC

K/uL

12.8

11.4

12.6

13.00

2.42

NS

NS

NS

Hgb

g/dL

16.4

15.8

16.3

16.2

1.77

NS

NS

NS

Hct

%

48.1

47.9

47.3

47.3

4.57

NS

NS

NS

MCV

fL

69.2

68.4

68.0

68.7

2.80

NS

NS

NS

MCH

Pg

23.8

24.0

23.9

24.1

1.16

NS

NS

NS

MCHC

g/dL

34.4

35.1

35.1

35.1

0.70

**

**

**

Plt

K/uL

328

298

306

287

42.0

NS

NS

*

CTR, control; GF1, grain-free diet 1; GF2, grain-free diet 2; CB, cereal based diet; RBC, red blood cells; WBC, white blood cells; Hgb, hemoglobin; Hct, hematocrit; MCV, medium
corpuscular volume; MCH, medium corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; Plt, platelets; *, **, P < 0.05 and P < 0.01, respectively; NS, notsignificant; RMSE, root mean square error.

TABLE 4 | Biochemical profile of dogs in function of diets.
Diets
Items

Units

CTR

GF1

CTR vs.
GF2

CB

RMSE

GF1

GF2

CB

Gl

g/L

37.6

35.8

34.7

36.0

5.47

NS

NS

NS

PT

g/L

66.9

67.1

64.9

65.2

6.25

NS

NS

NS

Alb

g/L

29.4

28.9

30.3

29.6

1.62

NS

NS

NS

AP

U/L

38.2

30.5

31.3

30.7

11.8

NS

NS

NS

Crea

µmol/L

80.7

72.6

74.2

76.0

12.1

NS

NS

NS

BUN

mmol/L

6.81

5.74

6.11

6.57

1.46

NS

NS

NS

CK

U/L

113

106

103

102

27.0

NS

NS

NS

ALT

U/L

39.3

39.1

38.5

39.3

8.44

NS

NS

NS

GGT

U/L

3.11

3.04

2.96

3.19

0.97

NS

NS

NS

AST

U/L

40.0A

33.9AB

30.5B

31.2B

7.61

*

***

*

GLDH

U/L

3.05

3.08

3.12

3.42

1.29

NS

NS

NS

Fr

µmol/L

204A

187B

186B

188B

9.93

***

***

***

Glu

mmol/L

5.03

4.79

4.94

4.95

0.64

NS

NS

NS

α-amylase

U/L

781

763

759

797

109

NS

NS

NS

LP

U/L

133A

67B

65B

69B

15.9

***

***

***

Chol

mmol/L

4.48

4.93

4.78

4.99

0.86

NS

NS

NS

Tri

mmol/L

0.88A

0.59B

0.58B

0.56B

0.17

***

***

***

CTR, control diet; GF1, grain-free diet 1; GF2, grain-free diet 2; CB, cereal based diet; Gl, Globulin; PT, Total protein; Alb, Albumin: AP, Alkaline phosphatase; Crea, Creatinine; CK,Creatine kinase; ALT, Alanine Transaminase; GGT, Gamma-glutamyl transferase; AST, Aspartate Transferase; GLDH, Glutamate dehydrogenase; Fr, Fructosamine; Glu, Glucose; LP,
Lipase; chol, Cholesterol; Tri, Triglyceride along the row the capital letters indicate differences to P < 0.01 and P < 0.001; NS, not significant, *, ***: p < 0.05, p < 0.001, respectively;
RMSE, root mean square error.

phosphorus, and magnesium which were always significantly
higher in CTR than in GF1, GF2, and CB diets.
In Table 6, whole blood and plasma taurine levels are shown.
In both cases, taurine levels were not significantly affected by theadministered diet, even if the CTR diet showed the lowest valueof both parameters.

Table 4 shows biochemical values. The nutritional treatment
affected only a few parameters. The analyses of varianceevidenced that the dogs showed significantly higher (P < 0.01)
values as regards AST, fructosamine, lipase, and triglycerides
when fed CTR diets. The Dunnett test evidenced the differences
between CTR diets vs. each tested diet more clearly, in particular
for triglycerides which were about double when CTR diet
was administered.
Table 5 shows the mineral profile. All parameters are within
the physiological ranges for healthy dogs. With both statisticalanalyses, significant differences were observed for potassium,

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DISCUSSION
All diets showed nutritional characteristics able to satisfy thenutritional requirement of adult dogs located in a kennel

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Grain-Free Diets and Healthy Status

TABLE 5 | Mineral profile (mmol/L) of dogs in function of diets and comparison of each diet to control one.
Diets
Items

CTR

GF1

CTR vs.
GF2

CB

RMSE

GF1

GF2

CB

Na

147

147

146

146

2.40

NS

NS

NS

K

4.92A

4.55B

4.41B

4.59B

0.40

*

***

*

Ca

2.43

2.45

2.44

2.44

0.14

NS

NS

NS

Cl

111

111

110

111

2.71

NS

NS

NS

P

1.33A

1.37A

1.24AB

1.18B

0.18

NS

NS

*

Mg

0.90A

0.81B

0.80B

0.82B

0.08

***

***

**

CTR, control diet; GF1, grain-free diet 1; GF2, grain-free diet 2; CB, cereal based diet; Na, Sodium; K, Potassium; Ca, Calcium; Cl, Chloride; P, Phosphorus; Mg, Magnesium; along therows the capital letters indicate differences to p < 0.01 and p < 0.001; NS, not significant, *, **, ***, p < 0.05, p < 0.01, p < 0.001, respectively; RMSE, root mean square error.

TABLE 6 | Blood (n = 60) and plasma (n = 120) taurine levels of dogs in function of diets.
Diets
Taurine

Units

CTR

GF1

CTR vs.
GF2

CB

RMSE

GF1

GF2

CB

Whole bloodµmol/l

288

316

316

318

51.3

NS

NS

NS

Plasmaµmol/l

101

128

127

125

32.8

NS

NS

NS

CTR, control diet; GF1, grain-free diet 1; GF2, grain-free diet 2; CB, cereal based diet; NS, not significant; RMSE, root mean square error.

differences that resulted from our trial. However, it is interesting
to note that in the study by Anturaniemi et al. (31), higher
erythrocyte counts, and hemoglobin levels occurred in dogs fed
with a high protein diet when as compared to those fed on thelowest protein diet. In the present study, the increased values of
MCHC in dogs fed with diet which was richer in proteins thanthe control may corroborate the same trend, also consideringthat dietary protein may play a role in maintaining appropriate
red blood cell indices (32). Clearly, further study is needed tobetter determine which dietary factor is responsible.
Some pre-analytical and analytical bias cannot be excluded,
even if points sampling procedures and processing werestandardized at all times (33, 34). Moreover, no specifictrend for platelets in dogs or diets during the trial wasobserved. Furthermore, platelets numbers could vary in different
physiological conditions, above all due to the presence of plateletclumps, often caused by sampling procedures and collection (35).
In addition, the biochemical profile also falls into thephysiological range for canine species. The differences related tofructosamine, lipase, and triglycerides obtained by the Dunnett
test seem to indicate a dietary effect on carbohydrates and lipid
metabolism. In particular, the redaction of these parameters
observed when the dogs were fed GF1, GF2, and CB diets could
be related to the different proportions of carbohydrates, lipids
and proteins in these diets compared to CTR. The sources ofcarbohydrates used in the formulation of the control diet showeda higher content of nitrogen free extractives than the others(NFE: 42.3 vs. 30.83; 34.83, and 34.60% as is, respectively, for
the CTR, GF1, GF2, and CB diets), while the fat and protein
contents are significantly lower in the control diet than thoseused for the trial. The reduction of AST observed with all diets
than the control could be indicative of lower hepatic stress.

(6). In particular, the amount of crude protein, total fat,and metabolizable energy fall into the levels recommended by
FEDIAF 2020 (27). During the experimental period, we did not
find refusals probably due to the high palatability of all diets.
Considering that no significant differences were observed asregards live weight, and body condition score (final live weight
21.59 ± 5.70 kg; BCS 3.97 ± 0.81 on a five-point scale) we
can assert that the amount of feed administered was correctly
calculated in all groups (28).
The amount of essential amino acids (g/1,000 kcal) in
the three tested diets were about double the minimum levels
recommended by FEDIAF (27) for an adult dog which
has metabolized energy requirement equal to 110 kcal/kg0.75 .
All experimental diets are able to fully satisfy even the
aforementioned nutritional requirements for a dog with a lower
energy necessity (95 kcal/kg0.75 ), also considering an apparent
availability of 70% (29).
There were no significant changes observed in thehematological profiles within the diets. All dogs showed theblood count values within the ranges considered physiological.
The only exceptions were recorded for two factors, MCHC(CTR vs. GF1, GF2, and CB, P < 0.01) and platelets (CTR vs.
CB, P < 0.05). Actually, from our study, whether these findings
are coincidental or just a trend cannot be established, nor itis possible to give a definitive explanation. It is essential tounderline that, even if there were significant differences, values
for MCHC and platelets were both within ranges. Considering
the absence of clinical signs or clinicopathological alterations andthe negativity as regards the mainly canine vector-borne diseases,
it seems likely that the results were due to any pathological cause.
There is very little data available concerning the influence of
diet on hematological parameters (30, 31) that could clarify the

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Grain-Free Diets and Healthy Status(45) observed that the whole blood taurine concentration was
lower in dogs fed whole grain rice, rice bran, or barley. Freidet al. (46) in a retrospective study on dogs affected by dilatative
myopathies, observed that dogs fed a non-traditional diet (grainfree contained novel ingredients such as peas or lentils as themain component) showed status improvement after their dietwas changed.
Donadelli et al. (17) did not observe a reduction in plasmaamino acids and taurine status when Labrador Retrievers werefed with a commercial grain-free diet after 26 weeks.
In our study, conducted on healthy dogs, increased taurine
levels in whole blood and plasma were observed after 5 weeksof the administration of three diets. The control diet (usedbefore the trial) could be defined as a traditional diet (grain
inclusive with rice and beet pulp). While the three diets tested
showed particular nutritional characteristics, and carbohydrates
ingredients: GF1 (grain-free with sweet potato, pea fiber, driedcarrot); GF2 (grain-free with pea starch, dried pumpkin, pea
fiber, dried carrot); and CB (grain inclusive with spelt, oats,
dried beet pulp, dried carrot). It seems possible to affirm thatthe relative proportions of the nutrients in the diets rather
than the use of novel ingredients could affect taurine level.
Indeed, all tested diets are characterized by the use of highquality protein sources (dehydrated and fresh chicken, herring,and eggs), and high levels of protein inclusion allow protein andamino acids requirements to be satisfied and, consequently, thetaurine status.
Moreover, the relative lower root means square error of
taurine in whole blood compared to plasma one confirms theprevious observation (45) that taurine has greater stability inwhole blood.

Aspartate aminotransferase is an enzyme that is found mainly inthe liver and heart and, in lower concentrations, in the kidneys
and muscles and low levels of AST are indicative of good health,while when the liver or muscle cells are damaged, the enzyme
is released into the blood in higher quantities. Although elevatedserum levels of AST could be considered a sign of a hepatic injury
or disease, concomitant with other variations of hematologicalparameters (e.g., lower ALT values) and other clinical signs (36).
In our case, clinical signs of hepatic injury were not observed, andall parameters could be considered physiological. Nevertheless,higher metabolic activity in the liver could be indicated by thehigher AST values registered with CTR, and GF1 diets (37).
Another important aspect may have also concerned thecarbohydrate sources used. While the CTR diet was formulatedwith rice (source of starch) and beet pulp, which mainly provides
insoluble fiber, in the other three diets carbohydrates sources
such as pea starch, spelt and oats, which are characterized by
low glycemic index, and carrot, squash and pea fiber as sourcesof dietary fiber were used (38, 39). These ingredients guarantee
a greater intake of soluble dietary fiber, able to modulate
the post-prandial glycemic response. Moreover, these different
dietary components guarantee the maintenance of the balanceof saprophytic bacterial populations of the large intestine as
it is fermented here, thereby producing short-chain fatty acids(40), in particular butyrate, which is considered the main energy
source for erythrocytes and colonocytes. Dietary fiber has been
indicated as a nutritional factor able to modify lipid absorption
reducing, directly and indirectly, bile acid reabsorption (41). The
decreasing of triglycerides observed with diets GF1, GF2, and CB
could be due to limited absorption of triglycerides in the small
intestine (41, 42). The reduced lipid absorption was confirmed by
the significant reduction of pancreatic lipase production. Indeed,
Stock-Damge’ et al. (43), administering a diet supplemented
with 5 g/d of wheat bran for 4 weeks, observed significantly
higher (P < 0.05) pancreatic secretion and lower (P < 0.05)
lipase concentration.
The significantly higher serum concentration of potassium,
phosphorus, and magnesium registered when the dogs fed
control diet could be related to the higher concentration ofphosphorus in this diet (12 vs. 8 mg/kg). On the other hand,
the higher value of phosphorus level in serum of dog fed
GF1 diet could be related to the higher bioavailability of thiselement that mainly derived from animal sources in this diet.
Moreover, CTR and GF1 diets were characterized by the higher
Ca:P ratio (1.50; 1.25; 1.12 and 1.12, CTR; GF1; GF2; CB diet,respectively) (6).
Although no statistically significant differences were observed,
there was an increased value in both parameters compared to theinitial values of 288.27 and 101 nmol/l, recorded with the control
diet. Another interesting aspect is that in all cases the taurine
levels were higher than the optimal reserve levels indicatedby FEDIAF (27) (>40 nmol/l in plasma and >200 nmol/l
in whole blood) and by University researchers California (44)(>70 nmol/ml in plasma and >250 nmol/ml in whole blood),
regardless of the dilatative cardiomyopathy risk in adult dogs.
The literature concerning the effect of diet on taurine
concentration in blood and serum is controversial. Delaney et al.

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CONCLUSION
Our preliminary results showed that only a few hematologicalparameters were affected when balanced diets were administeredto healthy dogs. The sources of carbohydrates (starch and dietaryfiber) and the appropriate equilibrium between energy nutrients(e.g., protein, fat, and starch) could modify the indicators oflipid, and carbohydrate metabolism (AST, fructosamine, lipase,triglycerides) and improve liver function.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will bemade available by the authors, without undue reservation.

ETHICS STATEMENT
All the procedures used in the study have been approvedby the Ethics Committee for the care and use of animals of
the University of Naples Federico II in accordance with local
and national regulations and guidelines (Legislative Decree 26
of 04/03/2014).

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Grain-Free Diets and Healthy Status

AUTHOR CONTRIBUTIONSFUNDING

MC: conceptualization. AV, GM, and IB: formal analysis.
IB and AV: methodology and data curation. SC and
AV: statistical analysis. MG and GM: clinical visitation.
MG and AV: writing—original draft. SC and MC:
writing—review and editing. MC and MG: supervision.
All authors contributed to the article and approved thesubmitted version.

This trial was partially supported by Farmina Pet Food (Nola,
Italia) and by DMVPA founding.

ACKNOWLEDGMENTS
The authors would like to thank Dog Kennel Service s.r.l. (Nola,
Italy) for the support and hospitality during the trial.

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Conflict of Interest: The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interest.
Copyright © 2021 Gizzarelli, Calabrò, Vastolo, Molinaro, Balestrino and Cutrignelli.
This is an open-access article distributed under the terms of the Creative Commons
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