- Email: [email protected]
Morphometric analysis of fetal development of Cavia porcellus (Linnaeus, 1758) by ultrasonography—Pilot study
Theriogenology 81 (2014) 896–900
Contents lists available at ScienceDirect
Theriogenology journal homepage: www.theriojournal.com
Morphometric analysis of fetal development of Cavia porcellus (Linnaeus, 1758) by ultrasonographydPilot study Juliana Santos a, *, Erika Fonseca b, Juliano van Melis c, Maria Angélica Miglino a a
University of São Paulo, São Paulo, Sao Paulo, Brazil Federal University of Tocantins, Araguaína, Tocantins, Brazil c Institute of Biology, Unicamp, Campinas, Sao Paulo, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2013 Received in revised form 9 December 2013 Accepted 3 January 2014
Measurements on the growth process and placental development of the embryo and fetuses of Cavia porcellus were carried out using ultrasonography. Embryo, fetus, and placenta were monitored from Day 15 after mating day to the end of gestation. Based on linear and quadratic regressions, the following morphometric analysis showed a good indicator of the gestational age: placental diameter, biparietal diameter, renal length, and crown rump. The embryonic cardiac beat was first detected at an average of 22.5 days. The placental diameter showed constant increase from beginning of gestation then remained to term and presented a quadratic correlation with gestational age (r2 ¼ 0.89). Mean placental diameter at the end of pregnancy was 3.5 0.23 cm. By Day 30, it was possible to measure biparietal diameter, which followed a linear pattern of increase up to the end of gestation (r2 ¼ 0.95). Mean biparietal diameter in the end of pregnancy was 1.94 0.03 cm. Kidneys were firstly observed on Day 35 as hyperechoic structures without the distinction of medullar and cortical layers, thus the regression model equation between kidney length and gestational age presents a quadratic relationship (r2 ¼ 0.7). The crown rump presented a simple linear growth, starting from 15 days of gestation, displaying a high correlation with the gestational age (r2 ¼ 0.9). The offspring were born after an average gestation of 61.3 days. In this study, we conclude that biparietal diameter, placental diameter, and crown rump are adequate predictive parameters of gestational age in guinea pigs because they present high correlation index. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Ultrasound Guinea pig Pregnancy Fetal age Embryo age
1. Introduction Guinea pigs (Cavia porcellus [Linnaeus, 1758]) have long been recognized as representatives of laboratory animals [1]. The first experimental use of this species reports to Lavoisier, in 1790, in investigations of the effects of heating [2]. Thereafter, these animals have been largely used as experimental models in several areas of biology [3–5]. In particular, guinea pigs have been used to study diverse aspects of gestation of mammals [6,7]. The relatively long gestational period, which allows for surgical access to * Corresponding author. Tel.: þ55 11 30917690; fax: þ55 11 3091 7805. E-mail address: [email protected] (J. Santos). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.01.004
fetuses followed by an adequate time for postsurgical monitoring, and the structural similarity of guinea pig and human placenta are among the characteristics making this species suitable models for gestational and prenatal studies [8]. Ultrasonography has been largely employed in many fields of medicine, and its usefulness has been recognized in a variety of clinical situations [9,10]. It has been shown to be a noninvasive and reliable method for pregnancy diagnosis and for monitoring fetal development [5,11]. The procedure does not offer risks to the conceptus once used under previously tested and recommended levels and intensity [10]. The establishment of metric parameters for gestational age and fetal development using ultrasonography may have
J. Santos et al. / Theriogenology 81 (2014) 896–900
important applications, because it would allow for the establishment of relationships between the chronology of fetal organs and structure development [12]. Measurements as crown rump (distance between the head and caudal poles), diameter of gestational sac, biparietal diameter of the head, femur and kidney lengths, and head and abdominal circumferences are morphometric analyses commonly used in rabbits [13], rats [10], mice [9], large animals, and humans [11]. In guinea pigs, ultrasonographic examinations have been used to measure the biparietal diameter of fetal head [8], to evaluate the morphology and blood flow of the placenta [7,14], and to estimate the number of the offspring between Days 25 and 40 of pregnancy [6]. Early diagnosis of pregnancy and a precise knowledge concerning the gestational age may be important information in a variety of studies using pregnant females. Depending on the animal species, monitoring of mating day is not always possible, and these uncertainties may compromise experimental planning and execution. Because of scarce information on the fetal development of guinea pigs, the aim of this study was to evaluate the relationship between gestational age and fetal development and morphometric measurements of fetal structures using ultrasound in pregnant guinea pigs between Day 15 of gestation and the proximity of the parturition, and then elaborate regression models that estimate gestational age. 2. Material and methods All experimental procedures were performed under veterinary supervision, according to recommendations by the National Council for Control of Animal Experimentation from Brazil, under license number 1999/2010 from the University of São Paulo Ethics Committee on Animal Experimentation. Five female guinea pigs privately owned, 3- to 4-month old, were donated to University of São Paulo, and used for this study. These animals were housed in cages, one guinea pig per cage, at 21 C, on a 12:12 hour light-to-dark cycle. Diet and water were consumed ad libitum. In this study, Day 0 of gestation was assumed to be the day of mating. Pregnant females were first examined at Day 15 of gestation, then at 5-day intervals up to Day 30. Afterward, ultrasonographic examinations were performed every 3 days until the parturition. The same operator performed all studies, without anesthesia or sedation. The animals usually stayed calm during the whole examination (about 20 minutes). The abdomen was shaved, and the guinea pig was fixed in dorsal recumbency, and a water soluble gel Aquasonic 100 (Parker) was applied over the surface of the abdomen. Three consecutive measurements were undertaken from each embryonic, fetal structures, and extra fetal structures, as follows: placental diameter (PD), biparietal fetal diameter (BFP), fetal kidney length (FKL), thoracic diameter, fetal abdominal diameter (FAD) and crown rump. All examinations used the color Doppler function to detect blood flow and pulsed Doppler to determine the fetal cardiac rate. The angle of insonation between the Doppler stream and vascular segment direction was less than 60 ,
897
the high-pass filter was set at 313 Hz, and the gain was set at 21. Abdominal diameter was measured when two echogenic lines were visible at both abdominal sides. Biparietal diameter measures were taken from the highest distance between the two cranial limits. Ultrasonographic examinations were performed using the model (GE Logiq 3, Wauwatosa, WI, USA) equipped with a convex transducer of 3 to 7 MHz and a linear transducer of 5 to 12 MHz. Statistical analyses were done by Akaike information criterion (AIC). We selected between quadratic or simple linear regression models using AIC.
3. Results Fetuses were born between Days 59 and 64. Fetuses from two females presented peristalsis at Day 55, and were born at Days 61 and 64, respectively. Fetuses in which no peristalsis was detected were born at Day 59. Raw data are showed in Table 1. On Day 15 of gestation, the embryos were identified in the uterine cavity as a hyperechoic structure displaying cardiac pulse at the Color Doppler in the gestational sac, which appeared as circular anechoic structures. One of the females had four gestational sac, and the others had only two. The crown rump presented a simple linear growth (AIC ¼ 210.427), displaying a high correlation with gestational age (r2 ¼ 0.9173). Crown-rump measurements were performed between Days 15 and 35, after that, images of the full fetuses could not be taken because of their dimensions. The beginning of skull ossification was observed on Day 30, allowing measurement of the biparietal diameter. This parameter followed a linear pattern of growth up to the end of gestation. Regression models adjusted between biparietal diameter and gestational age presented high determination coefficients, mainly for quadratic models (r2 ¼ 0.955) (Table 2). The placenta was characterized as hyperechoic structure at the first stages of gestation, with the echogenicity changing as the gestation proceeded. For example, between Days 35 and 40, they were characterized as hypoechoic structures intertwined by anechoic areas. The PD increased in the first stages of gestation, remaining fairly unaltered in later gestational stages. The PD showed a positive linear relationship with gestation age (Table 2). The embryonic cardiac beat was first detected at Day 20 of gestation. During the gestation, the heart rates ranged from 147.3 to 259.3 bpm (median ¼ 213.2 bpm). However, at the end of the gestation, between Days 59 and 65, the mean of fetal heartbeat was 205 bpm. Table 2 shows that the heart frequency shows a quadratic relationship with gestational age, reaching a peak at Days 45 to 55, but has a poor explanation power (r2 ¼ 0.56). Thoracic and abdominal diameters were well characterized only in fetuses beyond Day 35 of gestation. Table 2 presents the equation models for the relationship of thoracic and abdominal diameters with gestational age.
898
J. Santos et al. / Theriogenology 81 (2014) 896–900
Table 1 Measures of guinea fetuses after 15 days of gestation using ultrasonography. Gestational staging (d) 15 20 25 30 35 40 45 50 52 54 56 59 61 62 65
Biparietal diameter (cm)
Cardiac frequency (bpm)
1.3 1.6 1.9 2.2 2.5 2.7 2.8 2.9 3.1 3.2 3.3 3.4
163 183 199 211 219 223 223 222 220 217 212 208 206 198
Abdominal diameter (cm)
1.2 1.6 1.9 2.1 2.1 2.2 2.2 2.3 2.3 2.3 2.3
Also, by Day 35, the difference in echogenicity of the lungs (hyperechoic) and liver (hypoechoic) became evident. Kidneys were first observed at the Day 35 of gestational age. At first, the kidney image appeared hyperechoic without the distinction of medullar and cortical layers (Fig. 1), which became evident by Day 45. The regression model equation between kidney length and gestational age presented a quadratic relationship (Table 2). The beginning of intestine image was evidenced by Day 45, which appeared as hyperechoic lines in the caudal abdominal region. At Day 50, the layers of the intestine were visualized. Peristaltic movements were only observed in fetuses from animals two and three.
4. Discussion To the best of our knowledge, there are few studies on prenatal growth and development exposure to ultrasound in guinea pig at different gestational ages. The number of days from mating day to birth was 61.3 days, within the limits of the species (59–72 days) [15]. We highlight that biparietal diameter, crown rump, and PD presented the highest power of explanation to determine the gestational age. Our results showed that biparietal diameter provides a good predictor of gestational age in guinea pigs, supporting previous studies, which show the importance of biparietal diameter for clinical information concerning to gestational age and to encephalic development as well [8,16]. According to Turner and Trudinger [8] and Silva, et al. [17], biparietal diameter is the parameter which best predicts the gestational age, different from what was observed by Mu, et al. [16], who reports the measurements of fetal length and abdominal diameter as the most adequate measures to estimate fetal age in mice. Turner and Trudinger [8] registered biparietal diameter in guinea pig fetuses from Day 27, demonstrating a linear growth during most of the gestational period, followed by a plateau at the final stages. In the present study, the regression models adjusted between biparietal diameter and gestational age presented high determination coefficients.
Thoracic diameter (cm)
Placental diameter (cm)
Renal length (cm)
1.1 1.2 1.4 1.6 1.6 1.7 1.8 1.9 1.9 2.0 2.1
0.9 1.3 1.6 1.9 2.2 2.5 2.7 3.0 3.0 3.1 3.2 3.3 3.4 3.4 3.5
0.4 0.7 0.9 1.0 1.1 1.1 1.1 1.1 1.1 1.1 1.0
Crown-rump measurements could be first obtained at Day 15, and after Day 35, it was not possible to obtain because of the fetal dimensions. However, crown rump presented a high correlation with gestational age, which was similar to those reported by other studies in guinea pigs [18,19] and mice [20]. Placental diameter presented constant increase from the beginning of the gestation then remained to term, similar to that observed by Mu, et al., in mice [16]. Placental diameter presented a high correlation with the gestational age, thus, a good indicator of the gestational age in guinea pigs. Placenta was characterized as a hyperechoic structure at the initial stages, and the echogenicity was changing during the gestation. These changes in placenta echogenicity could be explained by the development of placenta lobules. The anechoic areas showed after Day 35 could correspond to a maturational phase of placental development in which
Table 2 Prediction equation based on our final models using as predecessor gestational time. Morphometric Predicted equation variables based on gestational age (GA) Biparietal diameter (DBP) Cardiac frequency (CF) Abdominal diameter (AD) Thoracic diameter (TD) Placental diameter (PD) Renal lenght (RL)
Coefficient of Akaike determination information (r2) criterion (AIC)
DBP¼0.4435183 + 0.0602337 (GA) 0.0003576 (GA)2 CF¼40.773 + 7.7351 (GA) 0.08175 (GA)2
0.9546
78.627
0.56
260.1309
AD ¼3.45912 + 0.186389 (GA) 0.001513 (GA)2 TD ¼0.054312 + 0.03253 (GA)
0.7962
PD ¼0.2655692 + 0.0857825 (GA) 0.0004205 (GA)2 RL¼3.641996 + 0.1691221 (GA) 0.001507 (GA)2
0.8935
0.7653
0.7093
6.989889
17.305
12.91243
20.3215
J. Santos et al. / Theriogenology 81 (2014) 896–900
899
Fig. 1. Ultrasonographic image showing: (A) disc-shaped placenta (arrows); (B) guinea embryo with a distinguishable head (arrows); (C) use of the flow Doppler to measure cardiac rhythm; (D) guinea embryo at 45 days with longitudinal section of the kidney (arrows).
vascularity increases. It is a characteristic of hemocorial placenta of rodents [7,21]. Before our study, the measurement of the kidney length had never been correlated with gestational age in the guinea pig. In this study, the differentiation between the renal cortex and medulla was an early observation when comparing with other species like cats, which have the same time of gestation of guinea pigs, and the differentiation between the renal cortex and medulla is only possible after Day 50 [22]. The average heart rate of guinea pig fetuses at the end of the gestation detected by ultrasonography in the present study is below the value reported by Kihlstrom [23], who measured heart rate performing a catheter in one of the umbilical arteries of guinea pig. The reasons for this difference are unclear. The visualization of difference in echogenicity between the lungs (hyperechoic) and liver (hypoechoic) became evident in this study almost at the same time as it has been identified in mole rats [24]. The examination for pregnancy diagnosis and determination of early pregnancy was easy to perform, and did not require the use of anesthesia, which favored the animal welfare. Likewise, the accuracy of ultrasonography was higher than that by abdominal distention; usually, it is visually evident on Day 40 after mating in mole rats, for example [25].
Our study shows the suitability of abdominal ultrasonography for early pregnancy diagnosis in guinea pig, with an efficiency of 100% at 15 days after mating day. At Day 15 of gestation, embryonic cardiac pulse was first detected, but heartbeat could be detected only at Day 20. Inaba and Mori [6] detected heartbeat in guinea pig fetuses at Day 34. In rodents with similar time of gestation, detection of heartbeat as indicator of fetal viability was only possible after Day 24 [24]. These differences may reflect the new technologies present in recent ultrasound machines, increasing sensitivity and image quality. In this study, we have obtained useful new data for monitoring fetal development in the guinea pig using ultrasonography. Measurements of PD, biparietal diameter, renal length, and crown rump were conclusive for the establishment of the gestational age. The next step should be evaluating a developmental profile of fetal guinea pig cardiac morphology and function to detect fetal stress. 4.1. Conclusions This study provides baseline information for the followup of fetal growth in guinea pig using ultrasonography. Biparietal diameter and PD showed the best predictive parameters of gestational age, and it seems that
900
J. Santos et al. / Theriogenology 81 (2014) 896–900
ultrasonography is a useful tool for the prediction of the parturition day in this species. Acknowledgments The authors gratefully acknowledge the Spécialité Veterinary Diagnostics for providing ultrasound machine. References [1] Harkness J, Wargner J. Biologia e clínica de coelhos e roedores. São Paulo: Roca; 1993. [2] Andrade A, Pinto SC, Oliveira RS. Animais de laboratório: criação e experimentação. Rio de Janeiro: Fiocruz; 2006. [3] Creese R, Head SD, Jenkinson DF. The role of the sodium pump during prolonged end-plate currents in guinea-pig diaphragm. J Physiol 1987;384:377–403. [4] Karol MH. Animal models of occupational asthma. Eur Respir J 1994; 7:555–68. [5] van Kan CM, de Vries JIP, Lüchinger AB, Mulder EJH, Taverne MAM. Ontogeny of fetal movements in the guinea pig. Physiol Behav 2009; 98:338–44. [6] Inaba T, Mori J. Use of echography in guinea pig for pregnancy diagnosis. The Jpn J vet Sci 1986;48:615–8. [7] Mess A. The guinea pig placenta: model of placental growth dynamics. Placenta 2007;28:812–5. [8] Turner AJ, Trudinger BJ. Ultrasound measurement of biparietal diameter and umbilical artery blood flow in the normal fetal guinea pig. Comp Med 2000;50:379–84. [9] Russo M, Meomartino L, Greco A, Catone G, Cocchia N, Tortora G, et al. Pregnancy detection in mice using ultrasound. Vet Rec 2007; 160:446–7. [10] Ypsilantis P, Deftereos S, Prassopoulos P, Simopoulos C. Ultrasonographic diagnosis of pregnancy in rats. J Am Assoc Lab Anim Sci 2009;48:734–9. [11] Pallares P, Gonzalez-bulnes A. Use of ultrasound imaging for early diagnosis of pregnancy and determination of litter size in the mouse. Lab Anim 2009;43:91–5.
[12] Almeida AH, Sterman FA, Miglino MA. Mensuração ultra-sonográfica das dimensões da placenta em gestações normais em cadelas da raça Boxer e sua relação com a idade gestacional. Braz J Vet Res Anim Sci 2003;40:136–40. [13] Chavatte-Palmer P, Laigre P, Simonoff E, Chesné P, ChallahJacques M, Renard JP. In utero characterisation of fetal growth by ultrasound scanning in the rabbit. Theriogenology 2008;69:859–69. [14] Verkestea CM, Slangena BFM, Daemenb M, van Straatenc H, Kohnend G, Kaufmann P, et al. The extent of trophoblast invasion in the preplacental vasculature of the guinea-pig. Placenta 1998;19: 49–54. [15] Rood JP, Weir J. Reproduction in female wild guinea pigs. J Reprod Fertil 1970;23:393–409. [16] Mu J, Slevin JC, Qu D, McCormick S, Adamson SL. In vivo quantification of embryonic and placental growth during gestation in mice using micro-ultrasound. Reprod Biol Endocrinol 2008;6:34. [17] Silva MRC, Sterman FA, Almeida AH. Mensuração ultra-sonográfica das dimensões do crânio fetal em gestações normais em cadelas da raça boxer e sua relação com a idade gestacional. Braz J Vet Res Anim Sci 2007;44:25–9. [18] Draper RL. The prenatal growth of guinea pig. Anat Rec 1970;18. [19] Kaufmann P, Davidoff M. The guinea-pig placenta. Adv Anat Embryol Cell Biol 1977;53:5–91. [20] Brown SD, Zurakowski D, Rodriguez DP, Dunning PS, Hurley RJ, Taylor GA. Ultrasound diagnosis of mouse pregnancy and gestational staging. Comp Med 2006;56:262–71. [21] Coan PM, Ferguson-Smith AC, Burton GJ. Developmental dynamics of the definitive mouse placenta assessed by stereology. Biol Reprod 2004;70:1806–13. [22] Zambelli D, Caneppele B, Bassi S, Paladini C. Ultrasound aspects of fetal and extrafetal structures in pregnant cats. J Feline Med Surg 2002;4:95–106. [23] Kihlstrom I. Blood pressure in the fetal guinea-pig. Acta Physiol Scand 1981;113:51–2. [24] Roellig K, Drews B, Goeritz F, Hildebrandt TB. The long gestation of the small naked mole-rat (Heterocephalus glaber Rüppell, 1842) studied with ultrasound biomicroscopy and 3D-ultrasonography. PLoS ONE 2011;6:e17744. [25] Jarvis JUM. Reproduction of naked mole rats. In: Sherman PW, Jarvis JUM, Alexander RD, editors. The biology of the naked mole rat. Princeton, New Jersey: Princeton University Press; 1991.
Contents lists available at ScienceDirect
Theriogenology journal homepage: www.theriojournal.com
Morphometric analysis of fetal development of Cavia porcellus (Linnaeus, 1758) by ultrasonographydPilot study Juliana Santos a, *, Erika Fonseca b, Juliano van Melis c, Maria Angélica Miglino a a
University of São Paulo, São Paulo, Sao Paulo, Brazil Federal University of Tocantins, Araguaína, Tocantins, Brazil c Institute of Biology, Unicamp, Campinas, Sao Paulo, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2013 Received in revised form 9 December 2013 Accepted 3 January 2014
Measurements on the growth process and placental development of the embryo and fetuses of Cavia porcellus were carried out using ultrasonography. Embryo, fetus, and placenta were monitored from Day 15 after mating day to the end of gestation. Based on linear and quadratic regressions, the following morphometric analysis showed a good indicator of the gestational age: placental diameter, biparietal diameter, renal length, and crown rump. The embryonic cardiac beat was first detected at an average of 22.5 days. The placental diameter showed constant increase from beginning of gestation then remained to term and presented a quadratic correlation with gestational age (r2 ¼ 0.89). Mean placental diameter at the end of pregnancy was 3.5 0.23 cm. By Day 30, it was possible to measure biparietal diameter, which followed a linear pattern of increase up to the end of gestation (r2 ¼ 0.95). Mean biparietal diameter in the end of pregnancy was 1.94 0.03 cm. Kidneys were firstly observed on Day 35 as hyperechoic structures without the distinction of medullar and cortical layers, thus the regression model equation between kidney length and gestational age presents a quadratic relationship (r2 ¼ 0.7). The crown rump presented a simple linear growth, starting from 15 days of gestation, displaying a high correlation with the gestational age (r2 ¼ 0.9). The offspring were born after an average gestation of 61.3 days. In this study, we conclude that biparietal diameter, placental diameter, and crown rump are adequate predictive parameters of gestational age in guinea pigs because they present high correlation index. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Ultrasound Guinea pig Pregnancy Fetal age Embryo age
1. Introduction Guinea pigs (Cavia porcellus [Linnaeus, 1758]) have long been recognized as representatives of laboratory animals [1]. The first experimental use of this species reports to Lavoisier, in 1790, in investigations of the effects of heating [2]. Thereafter, these animals have been largely used as experimental models in several areas of biology [3–5]. In particular, guinea pigs have been used to study diverse aspects of gestation of mammals [6,7]. The relatively long gestational period, which allows for surgical access to * Corresponding author. Tel.: þ55 11 30917690; fax: þ55 11 3091 7805. E-mail address: [email protected] (J. Santos). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.01.004
fetuses followed by an adequate time for postsurgical monitoring, and the structural similarity of guinea pig and human placenta are among the characteristics making this species suitable models for gestational and prenatal studies [8]. Ultrasonography has been largely employed in many fields of medicine, and its usefulness has been recognized in a variety of clinical situations [9,10]. It has been shown to be a noninvasive and reliable method for pregnancy diagnosis and for monitoring fetal development [5,11]. The procedure does not offer risks to the conceptus once used under previously tested and recommended levels and intensity [10]. The establishment of metric parameters for gestational age and fetal development using ultrasonography may have
J. Santos et al. / Theriogenology 81 (2014) 896–900
important applications, because it would allow for the establishment of relationships between the chronology of fetal organs and structure development [12]. Measurements as crown rump (distance between the head and caudal poles), diameter of gestational sac, biparietal diameter of the head, femur and kidney lengths, and head and abdominal circumferences are morphometric analyses commonly used in rabbits [13], rats [10], mice [9], large animals, and humans [11]. In guinea pigs, ultrasonographic examinations have been used to measure the biparietal diameter of fetal head [8], to evaluate the morphology and blood flow of the placenta [7,14], and to estimate the number of the offspring between Days 25 and 40 of pregnancy [6]. Early diagnosis of pregnancy and a precise knowledge concerning the gestational age may be important information in a variety of studies using pregnant females. Depending on the animal species, monitoring of mating day is not always possible, and these uncertainties may compromise experimental planning and execution. Because of scarce information on the fetal development of guinea pigs, the aim of this study was to evaluate the relationship between gestational age and fetal development and morphometric measurements of fetal structures using ultrasound in pregnant guinea pigs between Day 15 of gestation and the proximity of the parturition, and then elaborate regression models that estimate gestational age. 2. Material and methods All experimental procedures were performed under veterinary supervision, according to recommendations by the National Council for Control of Animal Experimentation from Brazil, under license number 1999/2010 from the University of São Paulo Ethics Committee on Animal Experimentation. Five female guinea pigs privately owned, 3- to 4-month old, were donated to University of São Paulo, and used for this study. These animals were housed in cages, one guinea pig per cage, at 21 C, on a 12:12 hour light-to-dark cycle. Diet and water were consumed ad libitum. In this study, Day 0 of gestation was assumed to be the day of mating. Pregnant females were first examined at Day 15 of gestation, then at 5-day intervals up to Day 30. Afterward, ultrasonographic examinations were performed every 3 days until the parturition. The same operator performed all studies, without anesthesia or sedation. The animals usually stayed calm during the whole examination (about 20 minutes). The abdomen was shaved, and the guinea pig was fixed in dorsal recumbency, and a water soluble gel Aquasonic 100 (Parker) was applied over the surface of the abdomen. Three consecutive measurements were undertaken from each embryonic, fetal structures, and extra fetal structures, as follows: placental diameter (PD), biparietal fetal diameter (BFP), fetal kidney length (FKL), thoracic diameter, fetal abdominal diameter (FAD) and crown rump. All examinations used the color Doppler function to detect blood flow and pulsed Doppler to determine the fetal cardiac rate. The angle of insonation between the Doppler stream and vascular segment direction was less than 60 ,
897
the high-pass filter was set at 313 Hz, and the gain was set at 21. Abdominal diameter was measured when two echogenic lines were visible at both abdominal sides. Biparietal diameter measures were taken from the highest distance between the two cranial limits. Ultrasonographic examinations were performed using the model (GE Logiq 3, Wauwatosa, WI, USA) equipped with a convex transducer of 3 to 7 MHz and a linear transducer of 5 to 12 MHz. Statistical analyses were done by Akaike information criterion (AIC). We selected between quadratic or simple linear regression models using AIC.
3. Results Fetuses were born between Days 59 and 64. Fetuses from two females presented peristalsis at Day 55, and were born at Days 61 and 64, respectively. Fetuses in which no peristalsis was detected were born at Day 59. Raw data are showed in Table 1. On Day 15 of gestation, the embryos were identified in the uterine cavity as a hyperechoic structure displaying cardiac pulse at the Color Doppler in the gestational sac, which appeared as circular anechoic structures. One of the females had four gestational sac, and the others had only two. The crown rump presented a simple linear growth (AIC ¼ 210.427), displaying a high correlation with gestational age (r2 ¼ 0.9173). Crown-rump measurements were performed between Days 15 and 35, after that, images of the full fetuses could not be taken because of their dimensions. The beginning of skull ossification was observed on Day 30, allowing measurement of the biparietal diameter. This parameter followed a linear pattern of growth up to the end of gestation. Regression models adjusted between biparietal diameter and gestational age presented high determination coefficients, mainly for quadratic models (r2 ¼ 0.955) (Table 2). The placenta was characterized as hyperechoic structure at the first stages of gestation, with the echogenicity changing as the gestation proceeded. For example, between Days 35 and 40, they were characterized as hypoechoic structures intertwined by anechoic areas. The PD increased in the first stages of gestation, remaining fairly unaltered in later gestational stages. The PD showed a positive linear relationship with gestation age (Table 2). The embryonic cardiac beat was first detected at Day 20 of gestation. During the gestation, the heart rates ranged from 147.3 to 259.3 bpm (median ¼ 213.2 bpm). However, at the end of the gestation, between Days 59 and 65, the mean of fetal heartbeat was 205 bpm. Table 2 shows that the heart frequency shows a quadratic relationship with gestational age, reaching a peak at Days 45 to 55, but has a poor explanation power (r2 ¼ 0.56). Thoracic and abdominal diameters were well characterized only in fetuses beyond Day 35 of gestation. Table 2 presents the equation models for the relationship of thoracic and abdominal diameters with gestational age.
898
J. Santos et al. / Theriogenology 81 (2014) 896–900
Table 1 Measures of guinea fetuses after 15 days of gestation using ultrasonography. Gestational staging (d) 15 20 25 30 35 40 45 50 52 54 56 59 61 62 65
Biparietal diameter (cm)
Cardiac frequency (bpm)
1.3 1.6 1.9 2.2 2.5 2.7 2.8 2.9 3.1 3.2 3.3 3.4
163 183 199 211 219 223 223 222 220 217 212 208 206 198
Abdominal diameter (cm)
1.2 1.6 1.9 2.1 2.1 2.2 2.2 2.3 2.3 2.3 2.3
Also, by Day 35, the difference in echogenicity of the lungs (hyperechoic) and liver (hypoechoic) became evident. Kidneys were first observed at the Day 35 of gestational age. At first, the kidney image appeared hyperechoic without the distinction of medullar and cortical layers (Fig. 1), which became evident by Day 45. The regression model equation between kidney length and gestational age presented a quadratic relationship (Table 2). The beginning of intestine image was evidenced by Day 45, which appeared as hyperechoic lines in the caudal abdominal region. At Day 50, the layers of the intestine were visualized. Peristaltic movements were only observed in fetuses from animals two and three.
4. Discussion To the best of our knowledge, there are few studies on prenatal growth and development exposure to ultrasound in guinea pig at different gestational ages. The number of days from mating day to birth was 61.3 days, within the limits of the species (59–72 days) [15]. We highlight that biparietal diameter, crown rump, and PD presented the highest power of explanation to determine the gestational age. Our results showed that biparietal diameter provides a good predictor of gestational age in guinea pigs, supporting previous studies, which show the importance of biparietal diameter for clinical information concerning to gestational age and to encephalic development as well [8,16]. According to Turner and Trudinger [8] and Silva, et al. [17], biparietal diameter is the parameter which best predicts the gestational age, different from what was observed by Mu, et al. [16], who reports the measurements of fetal length and abdominal diameter as the most adequate measures to estimate fetal age in mice. Turner and Trudinger [8] registered biparietal diameter in guinea pig fetuses from Day 27, demonstrating a linear growth during most of the gestational period, followed by a plateau at the final stages. In the present study, the regression models adjusted between biparietal diameter and gestational age presented high determination coefficients.
Thoracic diameter (cm)
Placental diameter (cm)
Renal length (cm)
1.1 1.2 1.4 1.6 1.6 1.7 1.8 1.9 1.9 2.0 2.1
0.9 1.3 1.6 1.9 2.2 2.5 2.7 3.0 3.0 3.1 3.2 3.3 3.4 3.4 3.5
0.4 0.7 0.9 1.0 1.1 1.1 1.1 1.1 1.1 1.1 1.0
Crown-rump measurements could be first obtained at Day 15, and after Day 35, it was not possible to obtain because of the fetal dimensions. However, crown rump presented a high correlation with gestational age, which was similar to those reported by other studies in guinea pigs [18,19] and mice [20]. Placental diameter presented constant increase from the beginning of the gestation then remained to term, similar to that observed by Mu, et al., in mice [16]. Placental diameter presented a high correlation with the gestational age, thus, a good indicator of the gestational age in guinea pigs. Placenta was characterized as a hyperechoic structure at the initial stages, and the echogenicity was changing during the gestation. These changes in placenta echogenicity could be explained by the development of placenta lobules. The anechoic areas showed after Day 35 could correspond to a maturational phase of placental development in which
Table 2 Prediction equation based on our final models using as predecessor gestational time. Morphometric Predicted equation variables based on gestational age (GA) Biparietal diameter (DBP) Cardiac frequency (CF) Abdominal diameter (AD) Thoracic diameter (TD) Placental diameter (PD) Renal lenght (RL)
Coefficient of Akaike determination information (r2) criterion (AIC)
DBP¼0.4435183 + 0.0602337 (GA) 0.0003576 (GA)2 CF¼40.773 + 7.7351 (GA) 0.08175 (GA)2
0.9546
78.627
0.56
260.1309
AD ¼3.45912 + 0.186389 (GA) 0.001513 (GA)2 TD ¼0.054312 + 0.03253 (GA)
0.7962
PD ¼0.2655692 + 0.0857825 (GA) 0.0004205 (GA)2 RL¼3.641996 + 0.1691221 (GA) 0.001507 (GA)2
0.8935
0.7653
0.7093
6.989889
17.305
12.91243
20.3215
J. Santos et al. / Theriogenology 81 (2014) 896–900
899
Fig. 1. Ultrasonographic image showing: (A) disc-shaped placenta (arrows); (B) guinea embryo with a distinguishable head (arrows); (C) use of the flow Doppler to measure cardiac rhythm; (D) guinea embryo at 45 days with longitudinal section of the kidney (arrows).
vascularity increases. It is a characteristic of hemocorial placenta of rodents [7,21]. Before our study, the measurement of the kidney length had never been correlated with gestational age in the guinea pig. In this study, the differentiation between the renal cortex and medulla was an early observation when comparing with other species like cats, which have the same time of gestation of guinea pigs, and the differentiation between the renal cortex and medulla is only possible after Day 50 [22]. The average heart rate of guinea pig fetuses at the end of the gestation detected by ultrasonography in the present study is below the value reported by Kihlstrom [23], who measured heart rate performing a catheter in one of the umbilical arteries of guinea pig. The reasons for this difference are unclear. The visualization of difference in echogenicity between the lungs (hyperechoic) and liver (hypoechoic) became evident in this study almost at the same time as it has been identified in mole rats [24]. The examination for pregnancy diagnosis and determination of early pregnancy was easy to perform, and did not require the use of anesthesia, which favored the animal welfare. Likewise, the accuracy of ultrasonography was higher than that by abdominal distention; usually, it is visually evident on Day 40 after mating in mole rats, for example [25].
Our study shows the suitability of abdominal ultrasonography for early pregnancy diagnosis in guinea pig, with an efficiency of 100% at 15 days after mating day. At Day 15 of gestation, embryonic cardiac pulse was first detected, but heartbeat could be detected only at Day 20. Inaba and Mori [6] detected heartbeat in guinea pig fetuses at Day 34. In rodents with similar time of gestation, detection of heartbeat as indicator of fetal viability was only possible after Day 24 [24]. These differences may reflect the new technologies present in recent ultrasound machines, increasing sensitivity and image quality. In this study, we have obtained useful new data for monitoring fetal development in the guinea pig using ultrasonography. Measurements of PD, biparietal diameter, renal length, and crown rump were conclusive for the establishment of the gestational age. The next step should be evaluating a developmental profile of fetal guinea pig cardiac morphology and function to detect fetal stress. 4.1. Conclusions This study provides baseline information for the followup of fetal growth in guinea pig using ultrasonography. Biparietal diameter and PD showed the best predictive parameters of gestational age, and it seems that
900
J. Santos et al. / Theriogenology 81 (2014) 896–900
ultrasonography is a useful tool for the prediction of the parturition day in this species. Acknowledgments The authors gratefully acknowledge the Spécialité Veterinary Diagnostics for providing ultrasound machine. References [1] Harkness J, Wargner J. Biologia e clínica de coelhos e roedores. São Paulo: Roca; 1993. [2] Andrade A, Pinto SC, Oliveira RS. Animais de laboratório: criação e experimentação. Rio de Janeiro: Fiocruz; 2006. [3] Creese R, Head SD, Jenkinson DF. The role of the sodium pump during prolonged end-plate currents in guinea-pig diaphragm. J Physiol 1987;384:377–403. [4] Karol MH. Animal models of occupational asthma. Eur Respir J 1994; 7:555–68. [5] van Kan CM, de Vries JIP, Lüchinger AB, Mulder EJH, Taverne MAM. Ontogeny of fetal movements in the guinea pig. Physiol Behav 2009; 98:338–44. [6] Inaba T, Mori J. Use of echography in guinea pig for pregnancy diagnosis. The Jpn J vet Sci 1986;48:615–8. [7] Mess A. The guinea pig placenta: model of placental growth dynamics. Placenta 2007;28:812–5. [8] Turner AJ, Trudinger BJ. Ultrasound measurement of biparietal diameter and umbilical artery blood flow in the normal fetal guinea pig. Comp Med 2000;50:379–84. [9] Russo M, Meomartino L, Greco A, Catone G, Cocchia N, Tortora G, et al. Pregnancy detection in mice using ultrasound. Vet Rec 2007; 160:446–7. [10] Ypsilantis P, Deftereos S, Prassopoulos P, Simopoulos C. Ultrasonographic diagnosis of pregnancy in rats. J Am Assoc Lab Anim Sci 2009;48:734–9. [11] Pallares P, Gonzalez-bulnes A. Use of ultrasound imaging for early diagnosis of pregnancy and determination of litter size in the mouse. Lab Anim 2009;43:91–5.
[12] Almeida AH, Sterman FA, Miglino MA. Mensuração ultra-sonográfica das dimensões da placenta em gestações normais em cadelas da raça Boxer e sua relação com a idade gestacional. Braz J Vet Res Anim Sci 2003;40:136–40. [13] Chavatte-Palmer P, Laigre P, Simonoff E, Chesné P, ChallahJacques M, Renard JP. In utero characterisation of fetal growth by ultrasound scanning in the rabbit. Theriogenology 2008;69:859–69. [14] Verkestea CM, Slangena BFM, Daemenb M, van Straatenc H, Kohnend G, Kaufmann P, et al. The extent of trophoblast invasion in the preplacental vasculature of the guinea-pig. Placenta 1998;19: 49–54. [15] Rood JP, Weir J. Reproduction in female wild guinea pigs. J Reprod Fertil 1970;23:393–409. [16] Mu J, Slevin JC, Qu D, McCormick S, Adamson SL. In vivo quantification of embryonic and placental growth during gestation in mice using micro-ultrasound. Reprod Biol Endocrinol 2008;6:34. [17] Silva MRC, Sterman FA, Almeida AH. Mensuração ultra-sonográfica das dimensões do crânio fetal em gestações normais em cadelas da raça boxer e sua relação com a idade gestacional. Braz J Vet Res Anim Sci 2007;44:25–9. [18] Draper RL. The prenatal growth of guinea pig. Anat Rec 1970;18. [19] Kaufmann P, Davidoff M. The guinea-pig placenta. Adv Anat Embryol Cell Biol 1977;53:5–91. [20] Brown SD, Zurakowski D, Rodriguez DP, Dunning PS, Hurley RJ, Taylor GA. Ultrasound diagnosis of mouse pregnancy and gestational staging. Comp Med 2006;56:262–71. [21] Coan PM, Ferguson-Smith AC, Burton GJ. Developmental dynamics of the definitive mouse placenta assessed by stereology. Biol Reprod 2004;70:1806–13. [22] Zambelli D, Caneppele B, Bassi S, Paladini C. Ultrasound aspects of fetal and extrafetal structures in pregnant cats. J Feline Med Surg 2002;4:95–106. [23] Kihlstrom I. Blood pressure in the fetal guinea-pig. Acta Physiol Scand 1981;113:51–2. [24] Roellig K, Drews B, Goeritz F, Hildebrandt TB. The long gestation of the small naked mole-rat (Heterocephalus glaber Rüppell, 1842) studied with ultrasound biomicroscopy and 3D-ultrasonography. PLoS ONE 2011;6:e17744. [25] Jarvis JUM. Reproduction of naked mole rats. In: Sherman PW, Jarvis JUM, Alexander RD, editors. The biology of the naked mole rat. Princeton, New Jersey: Princeton University Press; 1991.