Clinical and Biochemical Profile of Obstructive ... 1515.pdfBlood count, total plasma protein (PPT), and plasma fibrinogen (PF) were performed in two mo-ments of the experiment, the

Acta Scientiae Veterinariae, 2017. 45: 1515.

RESEARCH ARTICLE Pub. 1515

ISSN 1679-9216

1

Received: 24 July 2017 Accepted: 25 November 2017 Published: 19 December 2017

*Article based on a Thesis submitted by the senior author in partial fulfillment of requirements for the Master’s Degree (Programa de Pós-graduação em Sanidade e Reprodução de Ruminantes), Universidade Federal Rural de Pernambuco (UFRPE), Garanhuns, PE, Brazil. Unidade Acadêmica de Medici-na Veterinária (UAMV), Universidade Federal de Campina Grande (UFCG), Campina Grande, PB, Brazil. 2Programa de Pós-graduação em Medicina Veterinária, Universidade Estadual Paulista (UNESP), Jaboticabal, SP, Brazil. 3Setor de Clínica e Cirurgia de Grandes Animais, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil. 4Departamento de Medicina Veterinária, UFRPE, Recife, PE. 5Instituto Federal de Alagoas (IFAL), Santana do Ipanema, AL, Brazil. 6Departamento de Morfologia e Fisiologia Animal, UNESP, Jaboticabal. 7Clínica de Bovinos de Garanhuns, UFRPE, Garanhuns, PE. 8Laboratório de Anatomia e Patologia Animal, Unidade Acadêmica de Garanhuns (UAG), UFRPE, Garanhuns. CORRESPONDENCE: D. Oliveira [[email protected] - Tel.: +55 (87) 3764-5500]. Laboratório de Anatomia e Patologia Animal, Unidade Acadêmica de Garanhuns (UAG) - UFRPE. Av. Bom Pastor s/n. CEP 55292-270 Garanhuns, PE, Brazil.

Clinical and Biochemical Profile of Obstructive Urolithiasis in Sheep*

Thiago Arcoverde Maciel1, Inalda Angélica Ramos2, Rafael José da Silva3, Pierre Castro Soares4, Cleyton Charles Dantas Carvalho4, Rinaldo José de Souto Maior Júnior5, Lizandra Amoroso6,

Silvana Martinez Baraldi Artoni6, José Augusto Bastos Afonso7 & Daniela Oliveira8

ABSTRACT

Background: Urolithiasis is a metabolic disease of complex and multifactorial etiology, characterized by uroliths formation in the urinary system. It becomes clinically important when obstruction occurs, with little chance of reversal of the situation, and prevention is the best option for maintaining the integrity of the reproductive capacity. For this, the aim of this research was to know the clinical and biochemical profile of urolithiasis and predict the evolution of the disease in Santa Ines sheep breed fed with calculogenic diet, as well as to evaluate the prevention potential of vitamin C.Materials, Methods & Results: In this study, 14 healthy male Santa Ines sheep, aged approximately 90 days, were divided into two groups (G1- without vitamin C and G2 - with vitamin C, 1 g/dayly/orally) fed on calculogenic diet for 120 days. The animals were examined weekly, including observation of clinical signs, and blood and urine analysis were performed. Final leukogram revealed leukocytosis by neutrophilia in G2. There was aciduria in 57.14% (G1) and 71.43% (G2) and the presence of blood cells, bacteria and sperm to the urinary sediment. There were hyperproteinemia and proteinuria, but no significant elevation in serum albumin and globulin, urea and creatinine concentrations. Urine urea remained high. Serum uric acid was higher in M15 and M16, although inferior to those obtained in urine. No hyperglycemia was reported and urine concentrations remained stable over time, with a peak observed at M16. Serum AST presented a significant increase in M12 and M13, but returned to normal just after. Urine revealed significant turbidity and density changes for G1 and G2. Haematuria was observed in G1 and G2; however, changes in color and odor were evidenced only in G2. It was verified that, although most of the ani-mals presented aciduria, 42.86% of G1 and 28.57% of G2 presented alkaline urinary pH. Crystals found in both groups were amorphous phosphate, calcium carbonate, triple phosphate and calcium phosphate. Discussion: High-grain diet becomes a risk factor to the development of the disease, even face to high water consumption. The confinement condition, associated to the hyperprotein diet and high phosphorus levels characterized by the large supply of con-centrate to which the animals were submitted, are implicated as risk factors for the disease development. Urinary acidification is cited as an efficient alternative for the prevention of formation of struvite calculous and calcium phosphate, happened often in sheep species, so vitamin C was used. Clinical characteristics of urolithiasis are related to the location/degree of obstruction and time elapsed from obstruction to clinical care, and may begin abruptly with anorexia, colic, depression, strangulation with oliguria, hematuria or anuria. Intensity and manifestation of clinical signs of urolithiasis is dependent on the occurrence and degree of obstruction as a result of pain and consequent metabolic disorders. The design of the biochemical profile has been shown to be effective in elucidating the behavior of renal function biomarkers, such as urea and creatinine over of the process of calculus formation and installation of the disease. Presence of crystals, flaking cells and other elements such as cylinders can cause an increase in urinary density. Although the urinary pH of herbivores is naturally alkaline, it varies according to the animal’s diet. Though the perceptual of the urinary acidification superior on G2, it did not prove to be effective in preventing the development of obstructive urolithiasis, which occurred in 28.57% (2/7) of the animals that received vitamin C. Thus, other concentrations should be tested, with possibility of success and favorable conditions (economic and palatability) than the current acidifier.

Keywords: sheep disease, metabolism, urinary tract, acidification, urine.

2

T.A. Maciel, I.A. Ramos, R.J. Silva, et al. 2017. Clinical and Biochemical Profile of Obstructive Urolithiasis in Sheep. Acta Scientiae Veterinariae. 45: 1515.

INTRODUCTION

Urolithiasis is the most important disease that affects the urinary tract of ruminants, responsible for causing serious economic losses, such as the prema-ture exit of animals for breeding, treatment expenses and death of the affected animals [15,33]. It becomes clinically important when uroliths causes obstruction and then inflammation, hydronephrosis and uremia [2,20,24,38,42].

After the beginning of clinical signs, there is little chance of reversion and if surgical treatment is required most animals become unsuitable for repro-duction [17]. Thus, aiming at the reproductive integrity of the animal the best results are obtained with the prevention of the disease. Thus, the biochemical profile of the animals must be known and the correction of all possible factors must be done. Among prevention methods, urinary acidification is cited as an efficient alternative for small ruminants because of their com-mon alkaline urinary pH [38].

Ammonium chloride has been used in the diet of several species, for providing urinary acidification [12,32]. However, a disadvantage of this use is the potentiation of urolith formation after its interruption [17]. Vitamin C has been used as a potential acidifying of urine in human patients with urinary infections [44], but in ovine species 120 mg of vitamin C was not effec-tive in maintaining urinary acidification [16]. Therefo-re, the present research aimed to evaluate the clinical and biochemical profile of urolithiasis and predict the evolution of the disease in Santa Ines sheep fed with calculogenic diet, as well as the prevention potential of urinary acidification of higher dose of vitamin C.

MATERIALS AND METHODS

Animals

Fourteen healthy, male (uncastrated) sheep of the Santa Inês breed were used, approximately 90 days old and with a mean weight of 23.53 ± 4.45 kg. Before the beginning of the experiment, the parasitological examination of feces, control of endoparasites with sulfaquinoxaline (Sulfaquinoxalina)1 and oral sodium closantel (Diantel 10%)2 and vaccination against clos-tridiosis (Poli-Star)3 were performed.

Random distribution in two experimental groups was followed: Group 1 (without vitamin C, n = 7) and Group 2 (with vitamin C supplementation,

1 g/animal/day, n = 7), dose determined on the basis of acidification at a dose of 3 to 4 mg/kg/day [3,17]. Animals went through a 15 - day adaptation period where they received a balanced diet to better adapt the ruminal microbiota as well as the animals to the facilities and experimental management.

Animals were confined in individual masonry stalls measured 2.0 m x 1.0 m for small ruminants from the Academic Unit of Garanhuns (UAG), Federal Rural University of Pernambuco State (UFRPE), Per-nambuco, Brazil, where they were placed under equal conditions of temperature, humidity and luminosity throughout the experimental period.

For food and water composition it was utilized the protocol suggested by Maciel et al. [31].

Clinical and biochemical analysis

Blood count, total plasma protein (PPT), and plasma fibrinogen (PF) were performed in two mo-ments of the experiment, the first one was performed at experimental time zero (M0) to ensure the sanitation of the animals and the second immediately after finding the obstruction or final period of the experiment (M16).

The animals were rigorously examined follo-wing a technique described and evaluated for bioche-mical and urinalysis profiles at seven day intervals (M0, M1, M2, ..., M16 - 119 days of confinement) [14]. All blood and urine harvests were carried out si-multaneously before feeding, in the morning, between 6 and 8 h, with the sheep restraint on manually using a halter, and in a standing position to minimize stress. Hemogram was performed according to the previou-sly described methodology [23], after venipuncture collection of 4 mL of blood, using vacuum tubes with EDTA-sodium and 25x8 mm needle (BD Vacutainer)4. For determination of glucose concentrations (Glicose PAP Liquiform Kit)5, aliquots of 4 mL of blood were collected in vacuum tubes with sodium fluoride/EDTA. Aliquots of 8 mL of blood were collected in vacuum tubes without anticoagulant, to obtain serum for the determination of enzyme activity, aspartate aminotransferase (AST, AST\GOT Liquiform5 kit) and gamma-glutamyltransferase (GGT, Gama GT Liqui-form5 kit), early biomarkers of acute renal failure (urea, Uréia UV Liquiform5 kit; and creatinine, Creatinina K5 kit), and total serum protein (Proteínas Totais5 kit)/ albumin (Albumina5 kit) and uric acid (Ácido Úrico Liquiform5 kit) dosages. Blood serum was stored in 1 mL aliquots6, and frozen at -20°C.

3


Samples of at least 10 mL of urine were col-lected from each animal of the two groups by natural or forced urination, and respiration was interrupted by occlusion of the nostrils for 10 to 20 s [19] until the appearance of the first signs of obstructive urolithiasis. Urine samples were collected in sterile flasks (standard collector, 50 mL) and immediately sent to the Garanhuns Cattle Clinic (Clinica de Bovinos de Garanhuns / UFR-PE) and fractionated for urinalysis [51] and storage in 1 mL aliquots in a freezer (-20°C) after centrifugation. Again, glucose, GGT, urea, creatinine, albumin, uric acid and total protein (Sensiprot5) were evaluated.

Serum and urine biochemical analyzes were carried out using kits in an automatic biochemical analyzer (Labmax 240)5 of the Laboratory of Clinical Analysis of the UFRPE Veterinary Hospital (HOVET / UFRPE) using as calibrator Calibra H5 and as control Qualitrol 2H5.

Statistical analysis

The statistical analysis of the data was per-formed as a computational aid of the SAS program7 (Version 9.2) by PROC GLM, where the variables were submitted to analysis of variance and test of interaction between the group and observed moment. When significance was observed in the interaction, the analysis was performed to observe the effect of the mo-ment in each group and the effect of the group at each moment [13]. Tukey test was performed considering a 5% probability.

RESULTS

The mean total feed intake was 4.6% of the live weight/day and at the end of the experiment the animals weighed 37.19 ± 6.88 kg on average, with a mean weight gain of 13.66 kg/LW/animal. The mean water consumption and feed intake for G1 and G2 were 2,545 mL and 2,228 mL and 1,112 g and 1,059 g, being 778 g and 741 g of concentrate/animal/day, respectively (Table 1).

There was no significant change in body temperature in the days preceding urolithiasis, and in clinical signs. A decrease in ruminal motility, eno-phthalmos and slightly congestive conjunctivae were present in all affected animals, a slight increase in heart and respiratory rates was also observed (124 ± 19 beats/min and 60 ± 35 motions/min, respectively).

Although bladder rupture was not observed in this study, there was rupture of the penile urethra in

only one animal, which presented anuria, edema and tenderness to palpation of the ventroabdominal region (scrotal region up to xiphoid cartilage) and edema (Figure 1), as well as vocalization, bruxism, and di-gging movements. During necropsy, urethral rupture and intense edema were evident throughout the penile area (Figure 2).

Clinical signs that characterized urolithiasis were: apathy or restlessness, strangury, contraction and abdominal sprains with exposure and licking of the penis, postural changes (arching of the back, abduction of the pelvic limbs and raised tail), areas of necrosis and firm to palpation with congestive glans and ure-thral process, as well as discomfort and sensitivity to examination of the urogenital tract (Figure 3). At the beginning of the clinical signs it was observed that the appetite was not affected, but was reduced or absent after the period of 24 h.

Mean values of the CBC variables can be vi-sualized in Table 2. The CBC analysis did not reveal significant changes. There was no change in the red series. However, the final leukogram revealed an inver-sion in the number of segmented neutrophils, although only in G2 at the end there was leukocytosis (12,729 ± 9,104) by neutrophilia (8,681 ± 9,431).

Mean values of the serum and urinary bio-chemistry in the different groups and experimental moments are shown in Tables 3 and 4. There was a significant difference in concentrations between groups for both serum and urinary protein, with higher values for group 2, revealing proteinuria in 42.85% (G1) and 100% (G2) of the animals.

There was no significant elevation in serum concentrations of albumin and globulin, as well as observed for urinary concentrations and for serum urea and creatinine concentrations.

Urine urea concentrations did not vary over time, as there was no difference between groups, but remained high.

Serum uric acid had higher values in M15 and M16 (1.13 and 0.12 mg/dL), which were elevated in comparison to baseline (0.02 mg/dL). Nevertheless, their concentrations were inferior to those obtained in the urine.

No hyperglycemia was reported and urine concentrations remained stable over time, with a peak observed at M16.

4


Tabl

e 1.

Mea

n va

lues

and

sta

ndar

d de

viat

ions

of d

aily

wat

er a

nd fe

ed in

take

of m

ale

Sant

a In

es s

heep

(n =

14)

sub

mitt

ed to

cal

culo

geni

c di

ets

with

out s

uppl

emen

tatio

n (G

1) a

nd s

uppl

emen

ted

with

vita

min

C (

G2)

at t

he ti

mes

(M

0 to

M16

) of

the

anal

yzes

.

Var

iabl

eG

roup

Exp

erim

enta

l Tim

es

M0

M1

M2

M3

M4

M5

M6

M7

G1

172.

86 ±

122

.30

927.

14 ±

452

.46

1,94

8.57

± 6

45.2

52,

277.

14 ±

633

.90

2,47

7.14

± 6

33.2

92,

407.

14 ±

863

.09

3,23

7.14

± 1

,037

.91

2,77

0.00

± 5

08.0

0

Wat

er (m

L/D

day)

G2

228.

57 ±

211

.77

1,09

5.71

± 4

61.3

31,

567.

14 ±

463

.56

1,62

7.14

± 3

16.6

92,

338.

57 ±

74.

932,

257.

14 ±

436

.95

3,27

4.29

± 6

26.8

71,

820.

00 ±

961

.77

GA

200.

7i1,

011.

4h1,

757.

9fgh

1,95

2.1ef

g2,

407.

9cdfe

g2,

332.

1defg

3,25

5.7ab

cd2,

200.

0defg

G1

492.

86 ±

18.

9046

5.71

± 7

1.15

538.

57 ±

19.

5256

7.86

± 1

46.2

51,

115.

00 ±

200

.17

1,15

2.57

± 1

77.5

61,

687.

86 ±

618

.37

1,59

6.67

± 5

59.8

5

Food

(g/d

ay)

G2

495.

71 ±

11.

3448

4.29

± 2

1.49

540.

00 ±

26.

4666

5.00

± 0

1,08

7.86

± 1

57.8

794

2.14

± 3

22.5

71,

600.

00 ±

411

.56

1,38

9.29

± 4

57.4

9

GA

494.

3f47

5.0f

539.

3f61

6.4f

1,10

1.4cd

e1,

047.

4de1,

643.

9ab1,

485.

0abc

Dif

fere

nt l

ower

case

let

ters

on

the

sam

e lin

e re

pres

ent

sign

ifica

nt d

iffe

renc

e be

twee

n m

omen

ts (

P <

0.0

5);

Dif

fere

nt c

apita

l le

tters

in

the

sam

e co

lum

n re

pres

ent

a si

gnifi

cant

dif

fere

nce

betw

een

the

grou

ps a

t ea

ch

mom

ent (

P <

0.0

5); * G

A=

Gen

eral

ave

rage

s.

Tabl

e 1.

Mea

n va

lues

and

sta

ndar

d de

viat

ions

of

daily

wat

er a

nd f

eed

inta

ke o

f m

ale

Sant

a In

es s

heep

(n

= 1

4) s

ubm

itted

to

calc

ulog

enic

die

ts w

ithou

t su

pple

men

tatio

n (G

1) a

nd s

uppl

emen

ted

with

vita

min

C

(G2)

at t

he ti

mes

(M

0 to

M16

) of

the

anal

yzes

.

Var

iabl

eG

roup

M8

M9

M10

M11

M12

M13

M14

M15

M16

GA

*

G1

3,41

0.00

± 1

,083

.77

3,31

1.67

± 1

,616

.11

1,59

1.67

± 3

52.8

974,

026.

67 ±

891

.60

3,15

2.00

± 1

,102

.69

3,49

3.33

± 1

,368

.45

2,63

6.00

± 1

,425

.69

4,29

7.50

± 1

,099

.47

2,66

7.50

± 8

75.9

52,

544.

7A

Wat

er (m

L/D

day)

G2

3,10

3.33

± 5

03.0

22,

492.

00 ±

899

.68

1,25

0.00

± 5

78.5

23,

805.

00 ±

820

.10

3,24

3.33

± 9

66.4

33,

733.

33 ±

1,4

15.3

12,

639.

17 ±

790

.02

2,33

0.00

± 6

67.7

62,

232.

00 ±

896

.84

2,22

8.3B

GA

3,25

6.7ab

cd2,

939.

1abcd

e1,

420.

8gh3,

938.

0a3,

201.

8abcd

3,61

3.3ab

2,63

7.7bc

def

3,45

4.3ab

c2,

425.

6cdfe

g

G1

1,58

9.17

± 4

64.4

81,

429.

17 ±

509

.57

1,89

0.83

± 4

64.6

41,

704.

17 ±

362

.88

1,24

9.17

± 3

76.0

172

5.00

± 2

35.3

566

5.83

± 4

38.5

51,

298.

75 ±

455

.91

1,06

6.25

± 3

46.1

81,

111.

46

Food

(g/d

ay)

G2

1,59

3.33

± 2

71.8

61,

315.

83 ±

265

.56

1,65

2.50

± 3

58.5

21,

565.

83 ±

487

.51

1,21

3.33

± 4

92.8

976

4.17

± 2

45.0

869

0.83

± 4

99.3

41,

169.

00 ±

538

.72

1,07

5.00

± 4

55.0

81,

059.

49

GA

1,59

1.3ab

1,37

2.5ab

cd1,

771.

7a1,

635.

0ab1,

231.

3bcd

744.

6ef67

8.3f

1,22

6.7bc

d1,

071.

1de

Dif

fere

nt lo

wer

case

lette

rs o

n th

e sa

me

line

repr

esen

t sig

nific

ant d

iffe

renc

e be

twee

n m

omen

ts (

P <

0.0

5); D

iffe

rent

cap

ital l

ette

rs in

the

sam

e co

lum

n re

pres

ent a

sig

nific

ant d

iffe

renc

e be

twee

n th

e gr

oups

at e

ach

mom

ent (

P <

0.0

5); * G

A=

Gen

eral

ave

rage

s.

(con

tinue

s...)

(...c

ontin

uatio

n)

5


Tabl

e 2.

Mea

n va

lues

and

sta

ndar

d de

viat

ions

of t

he h

emog

ram

of m

ale

Sant

a In

es s

heep

(n =

14)

sub

mitt

ed to

cal

culo

geni

c di

ets

with

out

supp

lem

enta

tion

(G1)

and

sup

plem

ente

d w

ith v

itam

in C

(G

2) a

t the

initi

al ti

me

(M0)

and

at t

he e

nd o

f th

e ex

peri

men

t (M

F).

Para

met

erM

0M

FR

efer

ence

val

ues*

G1

G2

G1

G2

Ery

thro

cyte

s (x

106

mL

)13

.24

± 1

.27

11.5

9 ±

2.2

710

.65

± 1

.13

9.83

± 1

.19

9 -

15

Hem

oglo

bin

(g/d

L)

13.9

8 ±

0.7

513

.51

± 1

.63

11.9

5 ±

1.4

711

.48

± 1

.65

9 -

15

Hem

atoc

rit (

%)

36 ±

435

± 4

31 ±

330

± 3

27 -

45

MC

V (

fL)

27.3

8 ±

3.4

030

.78

± 2

.96

29.1

4 ±

2.7

427

.22

± 1

.36

28 -

40

MC

HC

(%

)38

.26

± 2

.77

38.5

3 ±

2.9

438

.56

± 2

.74

37.6

1 ±

031

- 3

4

TPP

(g/

dL)

5.94

± 0

.62

6.33

± 0

.74

6.96

± 0

.26

6.61

± 0

.42

6 -

7.5

PF (

mg/

dL)

243

± 7

930

0 ±

141

371

± 1

5034

3 ±

230

100

- 50

0

Leu

kocy

tes

(/μL

)8,

915

± 4

,021

9,19

3 ±

1,3

429,

300

± 2

,670

12,7

29 ±

9,1

044

- 12

Segm

ente

d (/

μL)

4,76

1 ±

1,1

143,

437

± 7

864,

999

± 2

,295

8,68

1 ±

943

170

0 -

6,00

0

Ban

d (/

μL)

00

00

Rar

e

Lym

phoc

ytes

(/μ

L)

5,85

9 ±

745

5,59

6 ±

925

4,07

2 ±

901

3,72

0 ±

981

2,00

0 -

9,00

0

Mon

ocyt

es (

/μL

)11

2 ±

325

2 ±

217

129

± 8

026

7 ±

180

0 -

750

Bas

ophi

ls (

/μL

)11

0 ±

080

± 1

110

3 ±

190

± 3

70

- 30

0

Eos

inop

hils

(/μ

L)

012

7 ±

47

625

± 0

106

± 3

70

- 10

00*S

ourc

e: J

.W. K

ram

er [2

9]. M

CV

= M

ean

Cel

l Vol

ume.

MC

HC

= M

ean

Cel

l Hem

oglo

bin

Con

cent

ratio

n. T

PP=

Tot

al P

lasm

a Pr

otei

n. P

F= P

lasm

a Fi

brin

ogen

.

6


Tabl

e 3.

Mea

n va

lues

and

sta

ndar

d de

viat

ions

of

the

bloo

d bi

oche

mic

al p

rofil

e of

mal

e Sa

nta

Ines

she

ep (

n =

14)

she

ep s

ubm

itted

to

calc

ulog

enic

die

ts w

ithou

t su

pple

men

tatio

n (G

1) a

nd

supp

lem

ente

d w

ith v

itam

in C

(G

2) a

t tim

es (

M0

to M

16)

of th

e an

alyz

es a

nd P

val

ue.

Profi

leG

roup

Exp

erim

enta

l Tim

es

M0

M1

M2

M3

M4

M5

M6

M7

M8

M9

M10

Tota

l pro

tein

(g

/dL

) (6

.0-

7.9g

/dL

)

G1

7.45

± 0

.81

7.62

± 0

.49

7.81

± 0

.54

7.98

± 0

.38

7.51

± 0

.44

7.68

± 0

.45

8.34

± 0

.48

1,05

9.49

7.74

± 0

.33

7.92

± 0

.45

7.84

± 0

.45

G2

8.02

± 0

.98

8.13

± 0

.61

7.96

± 0

.61

8.67

± 0

.71

7.67

± 0

.66

8.18

± 0

.67

8.78

± 0

.49

8.66

± 0

.52

8.01

± 0

.50

8.11

± 0

.69

8.58

± 0

.67

GA

7.74

de7.

88cd

e7.

88cd

e8.

33ab

cd7.

59e

7.93

cde

8.56

abc

8.59

abc

7.87

cde

8.01

bcde

8.21

bcde

Alb

umin

(g/

dL)

(2.4

-3g/

dL)

G1

3.09

± 0

.24

3.03

± 0

.22

2.76

± 0

.27

2.80

± 0

.21

2.63

± 0

.22

2.70

± 0

.25

2.70

± 0

.29

2.94

± 0

.24

2.7

± 0

.20

2.90

± 0

.33

2.85

± 0

.23

G2

3.21

± 0

.29

3.10

± 0

.15

2.78

± 0

.15

3.06

± 0

.15

2.67

± 0

.02

2.85

± 0

.14

2.85

± 0

.20

3.01

± 0

.25

2.85

± 0

.19

2.87

± 0

.30

2.95

± 0

.22

GA

3.15

a3.

06ab

2.77

bc2.

93ab

c2.

65c

2.77

bc2.

80bc

2.98

abc

2.81

bc2.

88ab

c2.

90ab

c

Glo

bulin

(g/

dL)

(3.5

-5.

7g/d

L)

G1

4.36

± 0

.59

4.60

± 0

.37

5.04

± 0

.365

5.18

± 0

.41

4.89

± 0

.34

4.98

± 0

.30

5.64

± 0

.22

5.59

± 0

.32

4.98

± 0

.30

5.03

± 0

.25

4.99

± 0

.37

G2

4.81

± 0

.78

5.03

± 0

.55

5.18

± 0

.56

5.61

± 0

.60

5.00

± 0

.56

5.33

± 0

.59

5.92

± 0

.42

5.64

± 0

.45

5.16

± 0

.53

5.24

± 0

.63

5.63

± 0

.73

GA

4.59

e4.

82de

5.11

bcde

5.40

abcd

4.95

cde

5.15

bcde

5.80

a5.

62ab

5.06

bcde

5.13

bcde

5.31

abcd

Alb

/Glo

b (0

.42-

0.76

)

G1

0.42

± 0

.01

0.01

± 0

.01

0.01

± 0

.01

0.35

± 0

.02

0.35

± 0

.02

0.35

± 0

.02

0.32

± 0

.02

0.35

± 0

.02

0.36

± 0

.02

0.36

± 0

.03

0.37

± 0

.03

G2

0.40

± 0

.03

0.38

± 0

.03

0.35

± 0

.02

0.35

± 0

.02

0.35

± 0

.02

0.34

9 ±

0.0

20.

32 ±

0.0

20.

35 ±

0.0

30.

36 ±

0.0

30.

35 ±

0.0

40.

35 ±

0.0

4

GA

0.41

a0.

39a

0.35

b0.

35b

0.35

b0.

35b

0.32

b0.

35b

0.36

b0.

36b

0.36

b

Ure

a (m

g/dL

) (1

7.12

-42

.8m

g/dL

)

G1

39.8

7 ±

4.3

7abc

39.5

9 ±

7.3

5Aab

c23

.34

± 7

.65 e

25.4

7 ±

5.9

9de45

.14

± 7

.02B

a25

.03

± 4

.92de

31.1

6 ±

5.0

7bcde

26.6

5 ±

10.

57de

30.0

7 ±

9.3

9bcde

29.5

3 ±

5.4

0bcde

37.6

0 ±

6.2

5Aab

cd

G2

46.9

6 ±

8.4

7b31

.70

± 3

.90B

cde

20.2

8 ±

5.8

5e28

.34

± 5

.24cd

e60

.09

± 9

.92A

a30

.29

± 9

.84cd

e25

.28

± 7

.65de

30.5

5 ±

10.

46cd

e27

.61

± 5

.46c

de32

.85

± 7

.99cd

e27

.99

± 7

.31B

cde

GA

43.4

2b35

.64cd

e21

.81f

26.9

1ef52

.62a

27.6

6ef28

.22de

f28

.60de

f28

.94de

f31

.06de

32.7

9cde

Cre

atin

ine

(mg/

dL)

(1.2

-1.

9mg/

dL)

G1

0.99

± 0

.12

0.89

± 0

.12

0.82

± 0

.09

0.81

± 0

.09

1.24

± 0

.16

1.20

± 0

.10

0.97

± 0

.08

1.09

± 0

.13

0.99

± 0

.13

1.00

± 0

.13

1.11

± 0

.16

G2

1.04

± 0

.10

1.04

± 0

.10

0.83

± 0

.08

0.97

± 0

.09

1.15

± 0

.09

1.21

± 0

.07

1.08

± 0

.07

1.20

± 0

.18

1.07

± 0

.06

1.11

± 0

.06

1.14

± 0

.07

GA

1.02

cd0.

96de

0.82

f0.

89ef

1.20

ab1.

21ab

1.02

cd1.

15bc

1.03

cd1.

05cd

1.12

bc

Uri

c A

cid

(mg/

dL)

G1

0.01

± 0

.01

0.01

± 0

.01

0.01

± 0

.02

0.04

± 0

.03

0.09

± 0

.04

0.07

± 0

.04

0.08

± 0

.02

0.14

± 0

.10

0.07

± 0

.04

0.08

± 0

.05

0.08

± 0

.02

G2

0.02

± 0

.02

0.03

± 0

.02

0.02

± 0

.04

0.05

± 0

.03

0.09

± 0

.05

0.07

± 0

.03

0.08

± 0

.02

0.10

± 0

.03

0.08

± 0

.02

0.06

± 0

.02

0.06

± 0

.02

GA

0.02

c0.

02c

0.02

c0.

04bc

0.09

ab0.

07ab

c0.

08ab

c0.

12a

0.07

abc

0.07

abc

0.07

abc

Glu

cose

(m

g/dL

) (5

0-80

mg/

dL)

G1

67.6

6 ±

7.7

169

.60

± 3

.97

71.0

2 ±

4.3

177

.36

± 4

.75

59.1

8 ±

4.2

973

.59

± 4

.19

65.3

7 ±

5.0

877

.22

± 5

.48

72.1

2 ±

4.0

967

.05

± 6

.61

60.6

7 ±

5.2

9

G2

61.6

4 ±

10.

7759

.88

± 9

.82

73.9

8 ±

4.7

462

.31

± 2

1.60

64.8

3 ±

8.0

263

.72

± 3

.08

60.6

7 ±

10.

0673

.39

± 13

.37

75.0

3 ±

4.2

365

.64

± 5

.40

58.7

1 ±

6.0

3

GA

64.6

5bcde

64.7

4bcde

72.5

0abc

69.2

6abcd

62.0

1cdef

69.2

0abcd

63.0

2bcde

f75

.30a

73.4

6ab66

.40ab

cde

59.6

9def

AST

(U

/L)

(60-

280U

/L)

G1

150.

20 ±

26.

5815

2.84

± 3

8.36

135.

59 ±

36.

9013

4.49

± 1

8.78

139.

58 ±

13.

6214

4.81

± 2

1.47

147.

19 ±

27.

6817

0.69

± 1

0.03

161.

03 ±

27.

9419

4.57

± 5

6.40

219.

66 ±

64.

43

G2

149.

81 ±

38.

3815

1.05

± 5

1.22

116.

34 ±

18.

0514

4.29

± 2

2.41

116.

66 ±

23.

7515

1.25

± 3

1.53

142.

61 ±

30.

4015

6.17

± 2

6.88

149.

59 ±

26.

1218

2.34

± 3

3.56

194.

01 ±

18.

64

GA

150.

01cd

151.

95cd

125.

97d

139.

39d

128.

12d

148.

03cd

144.

90cd

163.

43cd

155.

75cd

188.

92cd

206.

83cd

GG

T (

U/L

) (2

0-52

U/L

)

G1

42.7

2 ±

10.

8348

.35

± 1

7.60

65.0

2 ±

20.

3673

.98

± 2

0.15

75.5

3 ±

28.

4270

.07

± 2

6.55

73.6

8 ±

22.

3679

.73

± 18

.43

69.7

6 ±

17.3

368

.96

± 17

.47

69.7

0 ±

19.2

7

G2

37.8

1 ±

11.

5041

.80

± 1

0.76

59.8

8 ±

13.

4768

.45

± 1

1.71

61.9

2 ±

8.2

768

.19

± 1

1.01

66.8

3 ±

17.

8177

.57

± 12

.15

69.6

2 ±

13.4

867

.73

± 12

.90

69.7

5 ±

15.2

8

GA

40.2

6d45

.08d

62.4

5cd71

.21cd

68.7

2cd69

.13cd

70.2

6cd78

.65c

69.6

9cd68

.39cd

69.7

2cd

Dif

fere

nt l

ower

case

let

ters

on

the

sam

e lin

e re

pres

ent

sign

ifica

nt d

iffe

renc

e be

twee

n m

omen

ts (

P <

0.0

5);

Dif

fere

nt c

apita

l le

tters

in

the

sam

e co

lum

n re

pres

ent

a si

gnifi

cant

dif

fere

nce

betw

een

the

grou

ps a

t ea

ch

mom

ent (

P <

0.0

5); *

GA

= G

ener

al a

vera

ges.

(con

tinue

s...)

7


Table 3. Mean values and standard deviations of the blood biochemical profile of male Santa Ines sheep (n = 14) sheep submitted to cal-culogenic diets without supplementation (G1) and supplemented with vitamin C (G2) at times (M0 to M16) of the analyzes and P value.

Profile GroupExperimental Times

M11 M12 M13 M14 M15 M16 GA* P

Total protein (g/dL) (6.0-7.9g/

dL)

G1 8.11 ± 0.35 8.16 ± 0.45 9.01 ± 0.58 8.68 ± 0.36 8.66 ± 0.53 8.45 ± 0.63 8.06B

G2 8.62 ± 0.46 8.91 ± 0.46 8.98 ± 0.54 8.65 ± 0.67 8.44 ± 0.46 8.30 ± 0.75 8.38A

GA 8.36abcd 8.54abc 8.99a 8.66ab 8.56abc 8.38abcd <0.0001

Albumin (g/dL) (2.4-3g/dL)

G1 2.93 ± 0.27 2.90 ± 0.32 3.14 ± 0.43 3.06 ± 0.36 3.06 ± 0.27 2.90 ± 0.29 2.89

G2 2.88 ± 0.31 2.97 ± 0.24 3.04 ± 0.24 2.93 ± 0.18 2.73 ± 0.15 2.70 ± 0.25 2.91

GA 2.91abc 2.93abc 3.09ab 3.00ab 2.91abc 2.80bc <0.0001

Globulin (g/dL) (3.5-5.7g/dL)

G1 5.17 ± 0.17 5.26 ± 0.26 5.87 ± 0.50 5.62 ± 0.30 5.60 ± 0.73 5.55 ± 0.55 5.18B

G2 5.74 ± 0.47 5.95 ± 0.56 5.94 ± 0.54 5.72 ± 0.53 5.71 ± 0.32 5.60 ± 0.51 5.47A

GA 5.46abc 5.60ab 5.90a 5.67ab 5.65ab 5.58ab <0.0001

Alb/Glob (0.42-0.76)

G1 0.36 ± 0.02 0.36 ± 0.03 0.35 ± 0.04 0.35 ± 0.03 0.36 ± 0.05 0.34 ± 0.03 0.36A

G2 0.34 ± 0.04 0.34 ± 0.04 0.34 ± 0.03 0.34 ± 0.02 0.32 ± 0.004 0.32 ± 0.01 0.35B

GA 0.35b 0.35b 0.34b 0.35b 0.34b 0.33b <0.0001

Urea (mg/dL) (17.12-

42.8mg/dL)

G1 30.51 ± 4.42bcde 23.73 ± 6.36Be 34.98 ± 5.75abcde 27.71 ± 6.89cde 41.54 ± 7.35ab 30.92 ± 7.92bcde 31.82

G2 31.14 ± 6.18cde 34.70 ± 4.76Acd 39.56 ± 4.43bc 29.09 ± 4.58cde 38.32 ± 5.51bcd 31.25 ± 10.75cde 33.33

GA 30.82de 29.21def 37.27bcd 28.40def 39.93bc 31.09de <0.0001

Creatinine (mg/dL) (1.2-1.9mg/

dL)

G1 1.05 ± 0.12 1.16 ± 0.17 1.34 ± 0.23 1.31 ± 0.16 1.30 ± 0.13 1.26 ± 0.18 1.08B

G2 1.10 ± 0.08 1.13 ± 0.07 1.33 ± 0.20 1.28 ± 0.13 1.29 ± 0.11 1.33 ± 0.06 1.13A

GA 1.08bcd 1.14bc 1.33a 1.29a 1.30a 1.30a <0.0001

Uric Acid (mg/dL)

G1 0.08 ± 0.03 0.09 ± 0.03 0.07 ± 0.03 0.09 ± 0.06 0.18 ± 0.18 0.12 ± 0.07 0.08

G2 0.05 ± 0.03 0.08 ± 0.02 0.08 ± 0.03 0.08 ± 0.03 0.06 ± 0.03 0.11 ± 0.12 0.06

GA 0.07abc 0.09ab 0.08abc 0.08ab 0.13a 0.12a <0.0001

Glucose (mg/dL) (50-80mg/

dL)

G1 59.37 ± 6.20 62.28 ± 5.537 57.68 ± 16.70 52.84 ± 5.43 59.69 ± 13.86 64.77 ± 10.47 65.90A

G2 67.54 ± 6.02 61.23 ± 2.30 53.39 ± 4.74 53.99 ± 4.74 56.24 ± 5.82 62.49 ± 7.37 63.43B

GA 63.45bcdef 61.76cdef 55.53ef 53.41f 58. 00def 63.79bcdef <0.0001

AST (U/L) (60-280U/L)

G1 218.60 ± 53.16 273.87 ± 95.21 405.14 ± 174.59 94.79 ± 75.78 98.13 ± 98.61 26.97 ± 6.55 174.00

G2 249.91 ± 75.58 382.13 ± 201.67 442.05 ± 186.63 180.82 ± 46.27 197.75 ± 48.36 175.79 ± 70.36 189.43

GA 234.25c 328.00b 423.59a 133.02d 155.06cd 119.98d <0.0001

GGT (U/L) (20-52U/L)

G1 68.72 ± 21.77 71.59 ± 25.19 83.50 ± 28.12 92.65 ± 28.55 152.31 ± 43.36 153.08 ± 35.17 78.11

G2 5.22 ± 16.31 83.90 ± 20.13 100.06 ± 35.80 116.00 ± 61.88 138.54 ± 61.48 119.96 ± 36.76 75.59

GA 71.97cd 77.74c 91.78bc 104.33b 146.05a 136.52a <0.0001

Different lowercase letters on the same line represent significant difference between moments (P < 0.05); Different capital letters in the same column represent a significant difference between the groups at each moment (P < 0.05); *GA= General averages.

(...continuation)

The serum activity of AST remained within normal values, but presented a significant increase in M12 and M13 (328 and 423.59 U/L), however, the concentrations returned to normal in the subsequent moments.

The results of the physical-chemical analysis and urinary sedimentation are shown in Tables 5 and 6, respectively. Physical analysis of urine revealed significant turbidity and density changes for G1 and G2. Haematuria (28.57% and 57.14%) was observed in

G1 and G2, however, changes in color and odor were evidenced only in G2.

It was verified that, although the numerical majority of the animals presented a tableau of acidu-ria (64.29%), being 57.14% (G1) and 71.43% (G2), a great percentage of animals 42.86% of the G1 and 28, 57% of G2 presented alkaline urinary pH. The predo-minance of crystals for both groups was amorphous phosphate, as well as those of calcium carbonate, triple phosphate and calcium phosphate.

8


Table 4. Mean values and standard deviations of the urinary biochemical profile of male Santa Ines sheep (n = 14) submitted to calculogenic diets without supplementation (G1) and supplemented with vitamin C (G2) at different times (M0 to M16) of the analyzes and values of P.

Vari-able

GroupExperimental Times

M0 M1 M2 M3 M4 M5 M6

Total protein (g/dL)

G1 0.79 ± 0.89 0.48 ± 0.38 1.11 ± 0.92 1.91 ± 0.37 1.12 ± 0.44 3.08 ± 1.42 3.49 ± 0.70

G2 1.00 ± 0.83 1.37 ± 0.96 1.97 ± 0.82 2.40 ± 1.05 2.83 ± 2.11 4.49 ± 2.68 4.49 ± 2.75

MG 0.90c 1.04bc 1.58abc 2.16abc 1.97abc 3.78a 3.99a

Albu-min (g/

dL)

G1 0.05 ± 0.02 0.03 ± 0.01 0.50 ± 0.81 0.09 ± 0.04 0.03 ± 0.02 0.08 ± 0.02 0.11 ± 0.05

G2 0.05 ± 0.03 0.07 ± 0.02 0.09 ± 0.03 0.08 ± 0.06 0.04 ± 0.01 0.08 ± 0.03 0.10 ± 0.03

MG 0.05b 0.05b 0.30a 0.09b 0.03b 0.08b 0.11b

Urea (mg/dL)

G1 2,385.95 ± 2,161.25 7,313.47 ± 16,821.09 1,242.96 ± 390.34 1,794.70 ± 550.36 2,588.90 ± 916.95 2,456.76 ± 984.94 3,077.49 ± 915.27

G2 3,370.37 ± 1,468.24 2,444.30 ± 1,558.91 1,716.70 ± 1,058.70 2,257.90 ± 606.65 4,519.72 ± 1,309.21 2,821.20 ± 794.23 2,519.72 ± 822.53

MG 2878 4879 1480 2026 3554 2639 2820

Cre-atinine (mg/dL)

G1 75.69 ± 82.01 69.21 ± 59.19 130.33 ± 82.54 117.82 ± 24.60 82.04 ± 25.47 159.01 ± 53.23 158.03 ± 23.34

G2 122.13 ± 57.18 154.99 ± 95.46 153.93 ± 60.28 158.34 ± 52.91 101.32 ± 27.92 185.99 ± 112.63 186.79 ± 67.85

MG 98.91c 108.80bc 143.04abc 138.08abc 91.68c 172.50abc 171.30abc

Uric Acid (mg/dL)

G1 11.35 ± 6.96 11.55 ± 4.37 13.75 ± 8.55 19.00 ± 4.19 22.00 ± 10.94 29.63 ± 12.20 36.67 ± 10.36

G2 18.57 ± 10.38 25.78 ± 9.70 17.97 ± 2.85 25.39 ± 9.18 20.44 ± 5.58 34.08 ± 21.24 37.14 ± 7.28

MG 14.96f 18.12def 15.69ef 22.19cdef 21.22cdef 31.86abc 36.89ab

Glu-cose (mg/dL)

G1 2.59 ± 1.66 2.03 ± 0.51 2.93 ± 0.76 3.72 ± 0.60 3.26 ± 0.42 4.14 ± 1.03 5.55 ± 1.42

G2 3.33 ± 1.28 3.68 ± 0.889 3.60 ± 1.70 4.04 ± 1.98 3.09 ± 0.85 4.67 ± 1.42 4.59 ± 1.12

MG 2.96bc 2.85bc 3.27bc 3.88abc 3.17bc 4.40ab 5.07a

GGT (U/L)

G1 3.17 ± 2.16 2.64 ± 1.91B 10.50 ± 19.35 6.54 ± 3.85 1.53 ± 1.12 7.19 ± 5.29 4.64 ± 5.13

G2 3.66 ± 1.55ab 2.97 ± 1.11abA 7.14 ± 3.60ab 5.40 ± 3.51ab 2.54 ± 2.77ab 3.97 ± 3.04ab 7.48 ± 4.72ab

MG 3.42ab 2.81ab 8.82ab 5.97ab 2.04ab 5.58ab 6.06ab

Different lowercase letters on the same line represent significant difference between moments (P < 0.05). Different capital letters in the same column represent a significant difference between the groups at each moment (P < 0.05). GA = General averages.


Vari-able

GroupExperimental Times

M7 M8 M9 M10 M11 M12 M13


G1 2.89 ± 1.35 2.96 ± 1.48 3.19 ± 1.38 2.82 ± 1.71 2.97 ± 1.73 2.16 ± 0.82 1.5 ± 0.98

G2 5.67 ± 4.23 4.52 ± 1.14 4.70 ± 1.65 4.02 ± 1.61 4.29 ± 1.73 3.68 ± 1.13 3.10 ± 2.04

MG 4.28a 3.68ab 3.89a 3.42abc 3.63ab 2.92abc 2.17abc

Albu-min (g/

dL)

G1 0.16 ± 0.08 0.10 ± 0.03 0.08 ± 0.03 0.09 ± 0.03 0.16 ± 0.01 0.11 ± 0.06 0.12 ± 0.08

G2 0.19 ± 0.07 0.15 ± 0.05 0.10 ± 0.03 0.09 ± 0.05 0.19 ± 0.07 0.16 ± 0.07 0.16 ± 0.10

MG 0.17ab 0.12ab 0.09b 0.09b 0.18ab 0.14ab 0.14ab

Urea (mg/dL)

G1 1,838.56 ± 712.65 2,478.22 ± 648.99 5,044.34 ± 2,567.42 2,413.53 ± 1,419.37 2,657.29 ± 985.446 1,541.54 ± 618.62 1,701.17 ± 757.31

G2 2,255.96 ± 1,645.50 3,290.20 ± 987.53 4,007.22 ± 1,197.03 3,245.08 ± 721.46 3,307.48 ± 1,569.48 3,420.31 ± 582.04 2,941.31 ± 1,135.16

MG 2047 2853 4566 2829 2982 2481 2321

Cre-atinine (mg/dL)

G1 138.98 ± 65.76 138.37 ± 49.74 197.87 ± 129.22 114.55 ± 68.72 145.51 ± 56.10 130.21 ± 66.42 88.30 ± 25.17

G2 149.09 ± 79.49 201.40 ± 46.91 228.86 ± 52.43 235.00 ± 35.46 156.41 ± 51.85 172.32 ± 28.40 135.29 ± 35.61

MG 144.03abc 167.46abc 212.17a 174.77abc 150.96abc 151.27abc 111.79bc

Uric Acid (mg/dL)

G1 29.95 ± 12.45 26.88 ± 11.89 42.46 ± 18.40 24.79 ± 16.03 21.82 ± 12.96 21.22 ± 5.80 12.69 ± 2.90

G2 23.77 ± 10.08 33.30 ± 8.92 35.12 ± 16.03 31.71 ± 5.06 24.75 ± 4.41 20.34 ± 5.01 14.89 ± 1.76

MG 26.86bcdef 29.84abcd 39.07a 28.56abcde 23.28cdef 20.74cdef 13.79f

(...continuation)

(continues...)

(continues...)

9


Glu-cose (mg/dL)

G1 3.23 ± 1.46 2.90 ± 0.84 3.20 ± 0.47 3.00 ± 1.00 4.25 ± 1.61 3.04 ± 1.17 3.03 ± 0.59

G2 3.04 ± 0.66 3.84 ± 0.66 3.96 ± 1.61 4.53 ± 0.64 4.00 ± 1.11 3.72 ± 1.16 3.67 ± 1.13

MG 3.13bc 3.33bc 3.55bc 3.74abc 4.12abc 3.38bc 3.35bc

G1 8.80 ± 4.04 5.03 ± 1.93 2.87 ± 1.94 15.44 ± 18.93 3.32 ± 8.01 3.68 ± 5.63 5.51 ± 10.01

GGT (U/L)

G2 13.98 ± 9.39a 6.61 ± 3.44ab 3.10 ± 4.77ab 3.43 ± 5.74a 14.17 ± 28.78ab 5.35 ± 8.35ab 10.03 ± 16.88ab

MG 11.39a 5.76ab 2.97ab 9.43ab 8.75ab 4.52ab 7.77ab


(...continuation)

(...continuation)Table 4. Mean values and standard deviations of the urinary biochemical profile of male Santa Ines sheep (n = 14) submitted to calculogenic diets without supplementation (G1) and supplemented with vitamin C (G2) at different times (M0 to M16) of the analyzes and values of P.

Variable GroupExperimental Times

GA PM14 M15 M16


G1 3.53 ± 3.50 5.48 ± 5.29 4.69 ± 4.10 2.58B

G2 5.13 ± 4.58 2.86 ± 2.06 4.07 ± 2.50 3.52A

MG 4.33a 4.17a 4.38a <0.0001

Albumin (g/dL)

G1 0.07 ± 0.05 0.13 ± 0.08 0.10 ± 0.09 0.12

G2 0.10 ± 0.03 0.10 ± 0.08 0.07 ± 0.03 0.11

MG 0.08b 0.12ab 0.08b <0.0001

Urea (mg/dL)

G1 2,224.82 ± 781.20 3,366.29 ± 934.93 2,854.66 ± 363.32 2,780.8

G2 2,391.50 ± 1,456.55 3,061.87 ± 939.45 2,987.43 ± 2,196.03 2,959.7

MG 2308 3214 2921 0.0867

Creatinine (mg/dL)

G1 134.55 ± 54.38 209.93 ± 65.25 227.60 ± 72.59 134.01B

G2 0.10 ± 0.03 181.52 ± 72.37 214.99 ± 91.06 168.21A

MG 144.41abc 195.72ab 221.29a <0.0001

Uric Acid (mg/dL)

G1 15.11 ± 3.22 22.72 ± 13.05 22.44 ± 6.28 22.74B

G2 18.68 ± 5.39 18.15 ± 4.60 26.11 ± 13.77 25.16A

MG 16.74def 20.44cdef 24.28cdef <0.0001

Glucose (mg/dL)

G1 2.34 ± 1.10 4.30 ± 1.84 5.33 ± 1.50 3.42B

G2 3.14 ± 1.53 3.43 ± 1.01 4.94 ± 1.84 3.83A

MG 2.74c 3.87abc 5.14a <0.0001

G1 0 ± 0B 0.10 ± 0.21 1.45 ± 3.20 4.96

GGT (U/L) G2 13.46 ± 14.44bA 14.39 ± 17.28b 0.26 ± 0.58 ab 6.85

MG 6.73ab 7.25ab 0.85b 0.0030


(continues...)


10


Figure 1. Santa Inês ovine with obstructive urolithiasis caused by cal-culogenic diet. Animal with abdominal and preputial edema to clinical examination.

Figure 2. Santa Inês ovine with obstructive urolithiasis caused by cal-culogenic diet. Sheep with abdominal and preputial edema at necropsy.

Figure 3. Santa Inês ovine with obstructive urolithiasis caused by calculo-genic diet. Glans congestion and necrosis of the urethral process.

Table 5. Physical-chemical findings of urinalysis of male Santa Ines sheep (n = 14) submitted to calculogenic diets without supplementation (G1) and supplemented with vitamin C (G2), at the end of the experiment.

Parameter Finding% of samples

G1 G2

ColorPale yellow 100 71.43

Amber/Dark amber 0 28.57

SmellSui Generis 100 85.71

Altered 0 14.29

Aspect

Clear 42.86 28.57

Slightly Cloudy 14.28 14.29

Cloudy 42.86 57.14

Glucose Normal 100 100

Protein

Absent 28.57 0

Traces 57.14 42.86

30 - 100 mg/dL 14.29 42.86

> 100 mg/dL * 14.28

BloodAbsent 71.43 42.86

Present 28.57 57.14

pH5 - 6.5 57.14 71.43

>6.5 42.86 28.57

Density

<1,015 0 14.28

1,015 - 1,040 85.71 42.86

> 1,040 14.29 42.86

11


DISCUSSION

High-grain diet becomes a risk factor to the development of the disease, even face to high water consumption [38,41,54]. This was confirmed because 35.71% (5/14) of the animals developed the disease, 42.86% (3/7) of G1 and 28.57% (2/7) of G2. The confinement condition, associated to the hyperprotein diet and high phosphorus levels characterized by the large supply of concentrate to which the animals were submitted, are implicated as risk factors for the disease development [3,42,46,50].

No significant changes in body temperature was previously described [35], and reported a preva-lence of 87.1% of the animals in their study with body temperature below 39.5°C, with only 12.9% with hi-gher values. Rectal temperature at clinical examination may be increased, normal or below normal [1].

Observed clinical signals were also reported [34,42]. Urinary stasis results in pain and the release of prostaglandins, adrenaline and noradrenaline that act directly in the gastric center, leading to a colic syn-drome with decreased ruminal motility and anorexia [36,38], as well as tachycardia and tachypnea [52]. Mucosal congestion may be a result of uremia, urinary stasis, dehydration or development of endotoxemia, originating from infections in the urinary tract and rupture of the urethra or bladder [11,22].

The same signs of obstruction were observed in 27.4% of the animals studied by Morais [35]. In cases of urethral necrosis and rupture of the bladder there is extravasation of urine into the connective tissue of the abdominal wall and foreskin or abdominal cavity causing edema, tissue irritation, necrosis, urinary rea-bsorption and uremia [22,26,42]. These signals were not observed by Ferreira [16].

The symptomatology observed in the present study is in agreement with the reports of other authors [35,42,45]. The clinical picture of animals with uroli-thiasis is related to the location/degree of obstruction and time elapsed from obstruction to clinical care [6], and may begin abruptly with anorexia, colic, depression, strangulation with oliguria, hematuria or anuria [7,52].

Reduced or absent appetite in 53.22% of the animals was seen at CBG/UFRPE [35]. The rumen fullness also presented different degrees, varying from moderately empty to full. Timpanism was observed in only one of the animals. Highlighting should be given to the depraved appetite (gnawing stakes and bay walls) observed in some animals presented. Hydatidosis was present in all animals, but in varying degrees (from mild to severe). These data revealed that in this experiment the presence of appetite and water consumption are directly related to the time that has elapsed since the installation of the clinical signs, being suppressed as the level of pain increased, and leading to depraved appetite.

Table 6. Findings of urinary sedimentation of male Santa Ines sheep (n = 14) with 60 to 90 days of age submitted to calculogenic diets without supplementation (G1) and supplemented with vitamin C (G2).

Finding% of samples

G1 G2

Erytrocytes 28.57 28.57

Squamous Urethral Epithelium 100 71.43

Cells Squamous Transitional Epithelium 0 14.29

Leucocytes 85.71 71.43

Bacterias 0 28.57

Spermatozoa 42.86 28.57

Crystals

Amorphous Phosphate 71.43 42.86

Calcium carbonate 28.57 0

Triple Phosphate 28.57 0

Calcium phosphate 14.29 0

12


The CBC analysis did not reveal significant changes, such as hyperfibrinogenemia [35]. During the acute inflammatory process, interleukins and tissue ne-crosis factor are released, raising plasma fibrin concen-tration for several days, with peak between the fifth and seventh days [29]. The results of the hemogram after the first signs show the fibrinogen concentrations obtained, as there was no time to trigger the whole process [29]. There was leukocytosis by neutrophilia [37] typical in cases of acute renal infection, cystitis and urethritis.

The analysis of the total protein revealed, from the M9 values higher than the established standard for the species, 6.0-7.9 g/dL [25,30]. Expressive increase was evidenced from M11 (> 8.36 g/dL), remaining high up to M16. As a consequence of this behavior, there was a progressive increase in urinary concentrations, which were more expressive from the M5 and the peak of 4.38 g/dL in M16. Botelho et al. [4] found an average of 1.84 g/dL for cattle with urolithiasis.

Proteinuria usually occurs in animals fed with high protein content, a fact observed in this study, since the diet was composed of more than 20% crude protein (CP), which reflected in the high serum protein concen-trations and in urinary excretion [4]. Other factors to be considered in proteinuria are: inflammation in the uro-genital tract, hemoglobinuria and hematuria [18,19,21].

There is usually no presence of protein in the urine, however, those with low molecular weight may pass through the glomerular filtration barrier [48]. It is a protein with molecular weight of 69,000 from 0.2 to 0.3% of its blood albumin concentration may appear in the glomerular filtrate and consequently in the urine [39].

In contrast to other studies [21,33,53], there was no increase in serum urea and creatinine concen-trations. It demonstrates that the time factor (manifes-tation of clinical signs at the time of the test) is deter-minant for the expression of these biomarkers. Thus, in acute conditions, time may not be sufficient for the installation of post-renal azotemia, due to urinary stasis and consequent reabsorption of these compounds [9] or for renal filtration rate deficiency resulting in increased concentrations of creatinine [10,27,28].

Urea concentrations are constantly influenced by diet, serum concentration and excretion increase with increasing dietary protein content [5]. In diets with low crude protein levels, most of the urea is recycled and very little is lost in the urine [40], explaining the

observed values. Urine creatinine showed small oscilla-tions along the time, and significantly higher values were recorded from M5, with a peak at M16 (221.29 mg/dL). Botelho et al. [4] obtained concentrations of 60.9 mg/dL for the bovine species with urolithiasis.

No hyperglycemia was reported [35,47], probably associated with stress conditions of affected animals. Urine concentrations remained stable over time, with a peak observed at M16. Glucose undergoes glomerular filtration becoming part of the ultrafiltrate, but is completely reabsorbed by the proximal tubular pathway to the tubular and peritubular fluid [35,48].

The enzymatic serum activity of GGT was elevated since M2, with a peak in M15 (146.05 U/L), corroborating other studies [1,35], but with higher concentrations than those reported by the authors. Most affected animals had body scoring between 4 and 5 consistent with a probable liver steatosis, justifying the activity of GGT. However, urinary concentrations showed great oscillation throughout the moments.

Presence of crystals, flaking cells and other elements such as cylinders may characterize turbidity [49]. This may explain the observed turbidity, since the percentage of erythrocytes and leukocytes found in both groups was expressive, as well as the desqua-mation cells of the urethral epithelium in G1 (100%) and G2 (71.43%), and of the transitional epithelium in G2 (14.29%) and bacteria. When urine collection is performed by natural urination, scaly cells can be found in addition to red blood cells, leukocytes and other elements such as cylinders, mucus, bacteria and crystals [19,48]. Other components such as sperm can also be found.

The presence of red blood cells may be due to lesions in the genitourinary tract, including the foreskin or penis, a fact justified by lesions caused by uroliths at obstruction sites [19,48].

The urinary density of the small ruminants va-ries from 1015 to 1045 [7,8,25,39,43]. Refractometry revealed, therefore, hyperestenuria in G1 of 14.29% and of 42.86% in G2, already hyposostenuria was manifested in only 14.28% of G2. Presence of crystals, flaking cells and other elements such as cylinders can cause an increase in urinary density [49].

For the animals of the present study vitamin C was not effective in acidifying and maintaining urine pH, even under the high concentration used (1 g/ani-mal/day). In contrast, doses of only 3 to 4 mg/kg/day to

13


obtain satisfactory results in acidifying sheep urine [3]. However, the use of this concentration was tested by Ferreira et al. [17], who administered 120 mg/animal/day, but these showed that vitamin C was not effective in maintaining urinary acidification. It suggests that the particularities of the ruminants’ digestive tract influence the use, metabolism and excretion of this element, or that other concentrations need to be tested.

Although the urinary pH of herbivores is naturally alkaline, it varies according to the animal’s diet [7,19,43]. Thus, acidosis can occur as a result of metabolic or respiratory acidosis [35,48]. This fact justifies the situation of acidosis in both groups, as a result of the diet rich in concentrate to which the animals were submitted, and not only in the one that received vitamin C.

CONCLUSIONS

Diet rich in grains and the imbalance between Ca and P minerals, especially when their ideal propor-tions (2:1) are not observed, represent the great risk for the formation of uroliths and, consequently, the development of obstructive urolithiasis, as these factors trigger metabolic imbalances such as elevation in plas-ma protein concentrations and consequent excretion, which may not only cause lesions at the glomerular level but also saturate the urine, creating the ideal medium for precipitation of crystals and formation of the nuclei originators of uroliths.

Although water restriction is cited as a risk factor in the development of urolithiasis, it has been observed that under confinement conditions with ex-cessive supply of concentrate, even under high water

intake, animals can develop disease. This fact was observed in the G1 that presented the highest mean water intake, and, nevertheless, a higher percentage of affected animals.

The intensity and manifestation of clinical signs of urolithiasis is dependent on the occurrence and degree of obstruction as a result of pain and consequent metabolic disorders. The design of the biochemical profile has been shown to be effective in elucidating the behavior of renal function biomarkers such as urea and creatinine over of the process of calculus formation and installation of the disease. It was evidenced that the high concentrations of urea and creatinine commonly observed by the authors in their reports suggest delay in the time elapsed from the installation of clinical signs and the demand for care, not necessarily renal damage.

MANUFACTURERS

1Vansil Saúde Animal. Descalvado, SP, Brazil.2Hipra Saúde Animal. Porto Alegre, RS, Brazil.3Vallé S.A. São Paulo, SP, Brazil.4Becton Dickinson Indústrias Cirúrgicas. Curitiba, PR, Brazil.5Labtest Diagnóstica S.A. Lagoa Santa, MG, Brazil.6Eppendorf do Brasil Ltda. São Paulo, SP, Brazil.7SAS Institute Inc. Cary, NC, USA.

Acknowledgements. T.A. Maciel received MSc scholarship from FACEPE/IBPG.

Ethical approval. All methodology adopted for the develop-ment of the present research was submitted and approved by the Ethics Committee on the Use of Animals (CEUA) of the Federal Rural University of Pernambuco (UFRPE), Recife-PE, under protocol number 13726/2012-97.

Declaration of interest. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of paper.

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