Was performed a longitudinal prospective study, with 19 normal Golden Retrievers pregnant bitches in the period of April to August of 2010. This study was approved by the Research Ethics Committee of the Federal University of São Paulo (UNIFESP) under the number of 1859/09. The inclusion criteria were bitches with ages between 1 to 6
years old and docile behavior. The exclusion criteria were systemic pathologies like diabetes, arterial hypertension, fever; polycystic renal disease or another renal affection visualized during the realization of ultrasonography scans, beyond of renal volumes with low quality of its edges.
The ultrasonography scans were performed in the Golden Trip Kennel in the city of Embú das Artes, São Paulo, SP, Brazil. In this kennel, the dogs stay in big pickets separated in three groups by sex and age: puppies up to 6
months, puppies of 6
months to 1
year and adulthood. The pregnant bitches remain in individual bays. The maternity is located in an isolated house away of these places, were the matrix remains with the puppies until 45
days. With 45
days occurs the immunization and the puppies are directed to the pickets of the puppies up to 6
months. After the heat detection in the bitch, natural mating is performed three times on alternate days. The day of the first mating is considered the first gestational day. The animals were fed with ration twice a day and water ad libitum
The ultrasound scans were performed by two veterinarians (DSM and RFFM), utilizing an equipment SonoAce 8000Live (Samsung Medison, Seoul, Korea) equipped with a volumetric convex transducer (2 – 5
MHz). Both examiners presented one year of experience in collecting volumes in dogs by 3D ultrasonography. For the calculations of the renal volume, we used the 2D ultrasonography by the ellipsoid technique (volume
length x width x depth x 0,523) and the 3D ultrasonography by VOCAL with 30° (6 delimitation planes) and 12° (15 delimitation planes).
The first ultrasonography assessment, denominated “day 0” corresponded to non-pregnant bitches, i.e.
those animals evaluated before of the natural mating or that has mated but it wasn’t possible to identify the pregnancy. Other assessments were made every 7–14
days; which totalized 1 to 3 assessments per bitch. All animals were accompanied and grouped in three moments of time (day 0
non-pregnant bitches; days 1st
of pregnancy and days 21st
of pregnancy). The following variables were evaluated with regard to renal volume of the bitches: 1) right side, ellipsoid technique, day 0; 2) right side, ellipsoid technique, 1st
days; 3) right side, ellipsoid technique, 21th
days; 4) right side, VOCAL 30, day 0; 5) right side, VOCAL 30, 1st
days; 6) right side, VOCAL 30, 21th
days; 7) right side, VOCAL 12, day 0; 8) right side, VOCAL 12, 1st
days; 9) right side, VOCAL 12, 1st
days; 10) left side, 2D, day 0; 11) left side, 2D, 1st
days; 12) left side, 2D, 21th
days; 13) left side, VOCAL 30, day 0; 14) left side, VOCAL 30, 1st
days; 15) left side, VOCAL 30, 21th
days; 16) left side, VOCAL 12, day 0; 17) left side, VOCAL 12, 1st
days; 18) left side, VOCAL 12, 21th
To perform the ultrasound scans, the bitches were trichotomized ventrally from the sternum to the pubic portions and to the flanks and should be fasting for at least 6 hours. It was applied acoustic gel over the abdominal skin. The bitch was placed in right lateral decubitus for the left kidney evaluation and in left lateral decubitus for evaluation of contralateral kidney. Initially, for the renal volume acquisition, renal scan was performed in real time by 2D ultrasonography mode, until the complete longitudinal view of the kidney. The ideal longitudinal images were those that allowed the maximum measurement between the cranial and caudal poles with the renal pelvis visualization. A symmetrical form in “c” was formed by the renal sinus, pelvic diverticulum and renal ridge. Aiming the best contrast between the kidney and other abdominal organs, we used a predefined preset with frequency of 5
MHz, dynamic range of 75, harmonic mode and total gain of the device close to 100%. For the volumetric scanning, the 3D box was adjusted in order to embrace only the kidney in a fixed angle of 70° and fast scanning velocity in order to minimize the presence of transmissions artifacts resulting of the organs movimentation and of the animal respiratory frequency. We collected at least three volumes of each kidney by both examiners using the average of the best. The volumetric analysis was performed offline using the 1.03 version of the SonoView Pro software (Samsung Medison, Seoul, Korea).
For each one of the 18 variables, the first examiner (DSM) performed two measurements with an interval of seven days, while the second examiner (RFFM) performed an third measure, “blinded” of the first examiner results. For each renal volume using the three techniques (2D, VOCAL 30° and VOCAL 12°) were performed three measurements which was considered the average of these for the statistical analysis. For the 2D ultrasonography calculation, it was used the multiplanar mode with automatic calculation by the ellipsoid formula in which the length was obtained in the longitudinal plane (A) and the width and depth in the axial plane (B) (Figure a). For the 3D ultrasonography calculation, realized fifteen days after the 2D ultrasonography assessment, we used the VOCAL method. Plan A was selected as reference being rotated around of axis “z” so that the kidney was in lengthwise position. After the selection of the key VOCAL, the calipers were placed in the upper and lower poles of the organ. It was firstly selected the rotation angle of 30° and manual delimitation mode. In this manner the renal area was manually delimited on its external surface in the six sequential planes, and the equipment provided the image reconstructed three-dimensionally as well as its volume in cm3
(Figure b). The same protocol was followed for the contralateral kidney. Was also analyzed the VOCAL method with an rotation angle of 12°, following the same steps previously described, 7
days after the previous phase.
Figure 1 Calculation of renal volume in pregnant bitches: a) calculation by two-dimensional method (ellipsoid): in plane A is measured length and in plan B the width and depth, which is automatically calculated the volume by the software, b) calculation by three-dimensional (more ...)
All the data were collected in a specific protocol, later being transferred to an Excel 2007 spreadsheet (Microsoft Corp., Redmond, WA, USA). The statistical analysis was performed with the Statistical Package for the Social Science (SPSS) software version 17.0 for windows (SPSS Inc, Chicago, IL, USA). The statistical tests used were: ANOVA in the comparison of the renal volume (1st
measurement of the first examiner – DSM) regarding side, technique, moment of time besides the comparisons when necessary by the Turkey’s method; Shapiro-Wilk test to verify if the renal volume distribution (1st
measurement of the first examiner – DSM) regarding side, technique, moment of time is statistically normal; Pearson’s correlation coefficient (r) in order to evaluate the correlation between age (years) and the animals renal volumes at day 0, number of previous pregnancies and number of fetuses and difference of renal volume (21st
days minus 1st
day) of the animals; the intraclass correlation coefficient (ICC) to evaluate the inter- and intraobserver reproducibility. It was used the significance level (p)
0.05 in all statistical analysis.