Clinical and Biochemical Profile of Obstructive Urolithiasis in Sheep

RESEARCH ARTICLE Pub. 1515
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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 animals 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 concentrate 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.
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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 effective 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 moments 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 biochemical 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.
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Samples of at least 10 mL of urine were collected 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 performed 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 moment 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 urethral 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.
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Table 3. Mean values and standard deviations of the blood biochemical profile of male Santa Ines sheep (n = 14) sheep submitted to calculogenic diets without supplementation (G1) and supplemented with vitamin C (G2) at times (M0 to M16) of the analyzes and P value.
Profile Group Experimental Times
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 aciduria (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.
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T.A. Maciel, I.A. Ramos, R.J. Silva, et al. 2017. Clinical and Biochemical Profile…

Clinical and Biochemical Profile of Obstructive Urolithiasis in Sheep

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