Acta Scientiae Veterinariae, 2014. 42: 1172. RESEARCH ARTICLE Pub. 1172 ISSN 1679-9216 1 Received: 28 September 2013 Accepted: 4 January 2014 Published: 14 January 2014 Setor de Obstetrícia Veterinária, Departamento de Medicina Veterinária Preventiva e Reprodução Animal, Faculdade de Ciências Agrárias e Veterinárias - UNESP, Jaboticabal, São Paulo, Brazil. CORRESPONDENCE: M.A.R. Feliciano [marcusfeliciano@yahoo.com.br - Tel.: +55 (16) 32092626]. FCAV/ UNESP, Via de Acesso Prof. Dr. Paulo Donato Castellani, Bairro Distrito Industrial, s/n. CEP 14.884-900 Jaboticabal, SP, Brazil. Triplex Doppler Ultrassonography in Prenatal of Pregnant Bitches Marcus Antonio Rossi Feliciano, Anelise Carvalho Nepomuceno, Diogo José Cardilli, Leandro Nassar Coutinho, Maria Emília Franco de Oliveira, Murillo Darapé Kirnew, Vívian Tavares de Almeida & Wilter Ricardo Russiano Vicente ABSTRACT Background: The Dopplerfl uxometry is a diagnostic technique that provides anatomical information and real-time dynamic tissue evaluation by determining the presence and direction of blood fl ow in a vessel and their hemodynamic features. Considering the importance of Doppler to pregnant women and the recent studies of vascular indices in pregnant animals and the application of news technologies for Doppler ultrasonography as spectral and power techonology, the objective of this study was to determine the absolute values of the vascular indices from the uterine, umbilical and fetal aorta arteries in pregnant bitches, using new techniques of doppler ultrassonography. Materials, Methods & Results: Twenty six multiparous bitches from the same kennel, weighting 5 - 25 kg, aged 4 - 6 years old, were selected to the current study. Doppler ultrasonography was performed to assess the maximum and minimum velocity, resistance and pulsatility indices of the uteroplacental, umbilical and fetal abdominal aorta arteries (5th, 6th, 7th and 8th gestational week). The mean pregnancy period in twenty six bitches was 60 days and the mean number of pup- pies per pregnancy was four. Twenty fi ve animals presented healthy pregnancies and one presented hydrallantois at the 7th and 8th gestational week. During Doppler exams in healthy pregnant bitches, there were progressive increase in PSV (P < 5 %) to the uteroplacental, fetal aorta and umbical arteries, constant values in the uteroplacental artery EDV (P > 5 %) but increased EDV (P < 5 %) in umbilical and fetal aorta and decreased PI and RI in the vessels evaluated (P < 5 %). For the bicthe with hydrallantois was observed EDV = 3 cm / s, PI and RI of 1.82 to 1.46; PSV was 11 cm/s, EDV = 2.1 c/ms, PI = 1.46 and RI = 0.76 at the 7th and 8th week of pregnancy, respectively, different values from those obtained in healthy bitches. Discussion: In agreement with these authors and complementing the research carried with unpublished data for veterinary medicine and power technology (as the real values of PSV and EDV obtained between the 5th and 8th gestational week), the present study suggests that this method is useful for diagnosis of pregnancy pathologies, during different stages of pregnancy in bitches. It was verifi ed by the results obtained that, in pregnant healthy bitches, the indices (PSV and EDV) of uteroplacental, umbilical and fetal aorta arteries increased progressively with the evolution of pregnancy and PI and RI decrease progressively. The progressive development of the fetal circulation coupled with the appearance of a diastolic peak velocity in these arteries, there is a drop the values of RI and PI decrease in the umbilical artery and RI decrease in the aorta artery. The increase of PSV and EDV values demonstrates the necessity of the body to intensify the maternal-fetal circulation, with consequent nutrient infusion and tissue development. In general, the physiological behavior of blood fl ow for the vascular indices observed is due to the development of gestational structures and maturation of placental and fetal circulation. Regarding pathological pregnancy. The vascular indices were different during the fi nal stage of the gestation process. These fi ndings reaffi rm the idea that, in abnormal bitches pregnancies, there is a reduction in blood fl ow and higher resistance in the umbilical placental microcirculation. Thus, it was concluded that the maternal-fetal Doppler and power technology is a useful technique for prenatal diagnosis of pathological pregnancies in bitches. Keywords: canine, pregnancy, vascular indices, Doppler. 2 M.A.R. Feliciano, A.C. Nepomuceno, D.J. Cardilli, et al. 2013. Triplex Doppler Ultrassonography in Prenatal of Pregnant Bitches. Acta Scientiae Veterinariae. 42: 1172. INTRODUCTION In pregnant women, the Doppler allows evalua- tion of the maternal utero-placental circulation (uterine artery), (umbilical arteries) and fetal available, using a non-invasive diagnosis in cases of placental insuffi ciency and fetal assessment of hemodynamic changes that occur in response to defi cit of oxygen to the fetus [10]. The Duplex Doppler for the study of pregnant bitches can help to detect canine fetal abnormalities, correlating its importance in medicine [5,12] and eva- luating the vascular indices of the maternal fetal blood vessels: peak systolic (PSV) and end-diastolic velocity (EDV), vascular resistance (RI) and pusaltivity index (PI). The spectral Doppler was used in veterinary to evaluate six animals and the values of PSV, EDV, PI and RI were determined in percentage values during pregnancy [1,11]. Although these studies have con- tributed to important advances in the evaluation of pregnant bitches, they were limited to partial search and relative vascular indices of the main vessels related to the gestational process. Considering the importance of Doppler techni- ques for prenatal and obstetric care of pregnant women and the recent studies of vascular indices of pregnant animals in veterinary medicine and the application of new technologies for Doppler ultrasonography as spec- tral and power technology, the objective of this study was to determine the absolute values of the vascular indices from the uterine, umbilical and fetal aorta ar- teries in pregnant bitches using these new techniques of doppler ultrassonography. MATERIALS AND METHODS Twenty six healthy multiparous Shi-tzu, En- glish Bulldog, French Bulldog and Pug bitches from the same kennel, weighing 5 - 25 kg, aged 4 - 6 years old, were selected for the current study by physical and obstetric examinations. The kennel owner was trained to detect the fi rst signs of proestrus, in order to determinate the ideal time for matting. The confi rmation of this period was carried out by observing the signs of estrus (i.e., acceptance of copulation by the female) and by vaginal cytology. The female in estrus and the male were put together during three days for mating or artifi cial insemination was performed. In case of natural mating, the acceptance to the male was inspected and assured. Pregnancy diagnosis was carried out following two weeks of the mating or insemination, according to methodology employed in other trial [7]. Ultrasound (US) was performed with a 7.5 and 12.0 MHz linear transducer using MyLabTM30 VET machine1. The US examinations were carried out by a single examinator. During the 5th, 6th, 7th and 8th gestational week, pulsed Doppler ultrasonography was performed to assess the PSV, end-diastolic velocity (EDV), resistan- ce indice (RI = [PSV − EDV] / PSV) and pulsatility index (PI = [PSV - EDV] / mean velocity) [8] of the uteroplacental and umbilicals and abdominal aorta fetal arteries (Figure 1). For vascular indices, Doppler ultrasonogra- phy was performed to determine vessel volume using the uniform insonation method [9]. Power Doppler ultrasonography was performed to increase the sensi- tivity in detecting the blood fl ow of the vessels and to transform the examination from angle-independent to insonation or incident angle [6]. Data were previously tested for normality of residuals and homogeneity of variances (F test). Statistical analysis The PROC MEANS-SAS™ and the GraphPad Prisma 4™ softwares were used. Signifi cance level was 5% for all tests. Raw and transformed averages were evaluated using analysis of variance and, additionally, the t-test for paired samples was applied and Tukey’s multiple comparison test between the weeks was em- ployed. Signifi cance level was expressed as a P-value. RESULTS The mean pregnancy period in twenty six bitches was 60 days and the mean number of puppies per pregnancy was four. Twenty fi ve animals presented normal gestation and there was no harm to neonates during the pregnancy ultrasound monitoring. A Pug bitch had sonographic findings compatible with hydrallantois at the 7th gestational week. This animal was monitored by ultrasound during pregnancy until indicated time for the cesarean. By Doppler ultrasonography evaluating the vascular indices of the uteroplacental artery of phy- siological pregnancy bitches presented a progressive increase for PSV (P < 0.001), constant values for EDV and decrease of PI and RI, between the 5th and 8th ges- tational weeks. The mean values of uterine artery in bitches are shown in Table 1. 3 M.A.R. Feliciano, A.C. Nepomuceno, D.J. Cardilli, et al. 2013. Triplex Doppler Ultrassonography in Prenatal of Pregnant Bitches. Acta Scientiae Veterinariae. 42: 1172. For the vascular indices of the umbilical artery of healthy bitches, there were progressive increase for PSV (P = 0.0048) and EDV (P < 0.001) and decrease for PI (P < 0.001) and RI (P < 0.001) during gestational development. The mean values of umbilical artery in bitches are sown in the Table 2. Regarding to abdominal aorta of the fetuses were showed progressive increase for PSV (P = 0.0093) and EDV (P < 0.001) and decrease for RI (P < 0.001) and PI (P < 0.001), between 5th and 8th pregnancy weeks for healthy animals. The mean values of umbilical artery in bitches are shown in Table 3. By comparing the values obtained in bitches with normal pregnancies and pathological pregnancy, it was possible to detect changes in the behavior of vascular indices of the uteroplacental artery, during late pregnancy of the bitches. It were observed EDV= 3 cm/s, PI = 1.82 and RI = 1.46 at the 7th gestational week and PSV = 11 cm/s, EDV = 2.1 cm/s, PI = 1.46 and RI = 0.76 at the 8th week of pregnancy in the bitche with hydrallantois, different values from those obtained in healthy bitches. Table 1. Mean values of the vascular indices of uterine artery in pregnant bitches. Index Weeks of pregnancy P (5%) 5th 6th 7th 8th PSV (cm/s) 15.1 ± 3.9 16.5 ± 4.1 18.9 ± 4.4 22.4 ± 6.6 < 0.05* EDV (cm/s) 7.2 ± 3.0 8.1 ± 3.1 8.9 ± 3.4 9.6 ± 4.7 0.1 PI 1.0 ± 0.3 0.8 ± 0.2 0.7 ± 0.1 0.7 ± 0.2 < 0.05* RI 0.6 ± 0.1 0.5 ± 0.1 0.5 ± 0.1 0.5 ± 0.1 < 0.05* The t-test for paired samples, 5% signifi cance level. PSV: Peak sistolic velocity; EDV: End diastolic velocity; RI: vascular resistance; PI: pulsatility index. *Signifi cant if P < 0.05. Table 2. Mean values of the vascular indices of umbilical artery in pregnant bitches. Index Weeks of pregnancy P (5%) 5th 6th 7th 8th PSV (cm/s) 14.3 ± 3.1 16.5 ± 4.4 18.0 ± 4.0 19.3 ± 6.3 < 0.05* EDV (cm/s) 2.1 ± 0.6 3.5 ± 1.6 4.1 ± 1.0 6.2 ± 2.3 < 0.05* PI 2.1 ± 0.2 2.0 ± 0.2 1.8 ± 0.4 1.6 ± 0.4 < 0.05* RI 0.8 ± 0.1 0.8 ± 0.1 0.8 ± 0.1 0.8 ± 0.1 < 0.05* The t-test for paired samples, 5% signifi cance level. PSV: Peak sistolic velocity; EDV: End diastolic velocity; PI: pulsatility index; RI: vascular resistance. *Signifi cant if P < 0.05. Figure 1. Spectral and Power Doppler ultrasound images in pregnant bitches. Note in (A), (B) and (C) Doppler assessments to determinate of vascular indices in the umbilical, uteroplacental and fetal aorta arteries. 4 M.A.R. Feliciano, A.C. Nepomuceno, D.J. Cardilli, et al. 2013. Triplex Doppler Ultrassonography in Prenatal of Pregnant Bitches. Acta Scientiae Veterinariae. 42: 1172. Table 3. Mean values of the vascular indices of abdominal aorta fetal in pregnant bitches. Index Weeks of pregnancy P (5%) 5th 6th 7th 8th PSV (cm/s) 13.4 ± 4.2 15.1 ± 3.1 15.5 ± 3.4 17.9 ± 4.6 < 0.05* EDV (cm/s) 1.9 ± 0.6 2.9 ± 0.9 3.8 ± 1.1 4.4 ± 1.1 < 0.05* PI 2.0 ± 0.2 1.9 ± 0.2 1.8 ± 0.2 1.8 ± 0.1 < 0.05* RI 0.8 ± 0.1 0.8 ± 0.1 0.8 ± 0.1 0.8 ± 0.1 < 0.05* The t-test for paired samples, 5% signifi cance level. PSV: Peak sistolic velocity; EDV: End diastolic velocity; PI: Pulsatility index; RI: Vascular resistance. *Signifi cant if P < 0.05. DISCUSSION Studies using the Triplex Doppler [1,11] and using Duplex Doppler [5,12] have demonstrated the importance of these techniques for pregnancy moni- toring in bitches. In agreement with these authors and complementing the research carried with unpublished data for veterinary medicine and power technology (as the real values of PSV and EDV obtained between the 5th and 8th gestational week), the present study suggests that this method is useful for diagnosis of pregnancy pathologies, during different stages of pregnancy in bitches. In pregnant healthy bitches the indices (PSV and EDV) of uteroplacental, umbilical and fetal aorta arteries increased progressively with the evolution of pregnancy acording with the literature [1], likewise the PI and RI decrease progressively [11]. In early pregnancy, the simultaneous presence of high values of RI and PI and the absence of diastolic fl ow in the aorta and umbilical arteries characterize a blood fl ow of high resistence. The progressive development of the fetal circulation coupled with the appearance of a diastolic peak velocity in these arteries, the values of RI and PI decrease in the umbilical artery and RI decrease in the aorta artery [13]. The reduction of RI and PI of maternal fetal blood fl ow is secondary to the perfusion increase of the vessels and fetal size increase [11]. The increase of PSV and EDV values demonstrates the necessity of the body to intensify the maternal-fetal circulation, with conse- quent nutrient infusion and tissue development [12]. In other words, there is an increasing demand to meet the vital organs (brain and heart), organs of the abdominal cavity and the placental bed [4]. In general, the physiological behavior of blood fl ow observed is due to the development of gestational structures and maturation of placental and fetal circula- tion [3,12]. Regarding to pathological pregnancy, the values of vascular indices were different at the fi nal stage of gestation process. These fi ndings reaffi rm the idea that, in abnormal bitches pregnancies, there is a reduction in blood fl ow and higher resistance in the umbilical placental microcirculation [2]. It is expected, because of the inadequate trophoblastic invasion and the evi- dent changes in the parameters of the mode Doppler for uteroplacental, umbilical and fetal arteries refl ect on image fi ndings such as the increase in placental re- sistance (abnormal dopplerfl uxometry of vessels) and in the fetal hemodynamic response (decrease in tissue oxygen supply) [5]. CONCLUSION It was concluded that the maternal-fetal Dop- pler (associated with power technology) is a useful technique for the diagnosis of pathological pregnancies and hemodynamic changes in the main vessels involved in pregnancy of bitches. SOURCE AND MANUFACTURER 1MyLabTM30 VET - ESAOTE, Genoa, Italy. Acknowledgements. The authors would like to thank FAPESP for the fi nancial support (process 2010/16913-7) to the cur- rent research and for post-doctor scholarship support (process 2011/06011-9). Ethical approval. The trial was conducted following approval (process no 017314/10) by the Animal Welfare and Ethics Committee of the School of Agrarian and Veterinary Sciences of the São Paulo State University (FCAV/UNESP - Jaboticabal). Declaration of interest. The authors report no confl icts of interest. The authors alone are responsible for the content and writing of the paper. 5 M.A.R. Feliciano, A.C. Nepomuceno, D.J. Cardilli, et al. 2013. Triplex Doppler Ultrassonography in Prenatal of Pregnant Bitches. Acta Scientiae Veterinariae. 42: 1172. www.ufrgs.br/actavet 1172 REFERENCES 1 Blanco P.G., Arias D. & Gobello C. 2008. Doppler ultrasound in canine pregnancy. Journal Ultrasound Medicine. 27:1745-50. 2 Blanco P.G., Arias D., Rube A., Barrena J.P., Corrada Y. & Gobello C. 2009. 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