Gestational ultrasonography and Dopplerfluxometry in capuchin monkeys (Sapajus apella) zoometric

Theriogenology 108 (2018) 63e73

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Gestational ultrasonography and Dopplerfluxometry in capuchin monkeys (Sapajus apella) zoometric ~o a, c, K.G. Oliveira a, d, I.S. Sodre  a, S.F.S. Domingues a, b, c S.A. Miranda a, b, *, D.L. Lea , Castanhal, PA, CEP 68740-970, Brazil Laboratory of Amazon Animal Biotechnology and Medicine, Federal University of Para , Castanhal, PA, CEP 68740-970, Brazil Animal Sciences Post-Graduation Program, Federal University of Para c Animal Health and Production in Amazon Post-Graduation Program, Federal Rural University of the Amazon, Bel em, PA, CEP 66.077-830, Brazil d National Primate Center, Ananindeua, PA, CEP 66645-000, Brazil a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 January 2017 Received in revised form 7 November 2017 Accepted 20 November 2017 Available online 26 November 2017

The objectives of the current study were as follows: 1) to evaluate blood flow in the uterine (UA) and umbilical (Uma) arteries in pregnant capuchin monkeys by measuring the resistive index (RI) and pulsatility index (PI); 2) to determine the presence or absence of the early diastolic notch and diastolic flow in the UA and Uma flow waveforms, respectively; 3) to perform conceptus echobiometry for fetal growth assessment during pregnancy; 4) to describe the moment that the fetal organs were initially observed; and 5) to determine when the diagnosis of fetal gender is possible. Seven healthy, sexually mature female Sapajus apella were examined in Weeks -20 to -1 before whelping (whelping Week 0). Triplex Doppler was used to assess the blood flow and fetal heart rate, and B-mode ultrasonography was used to assess the fetal organs and conceptus measurements, including the gestational sac latero-lateral longitudinal (LLL) and latero-lateral transversal (LLT), the crown rump length (CRL), biparietal diameter (BPD), occipito-frontal diameter (OFD), head circumference (HC), abdominal circumference (AC), femur length (FL) and fetal organ. All the pregnancies ended with a normal whelping and the birth of a live newborn. Prior to whelping, all conceptus dimensions increased significantly, whereas the RI and PI of both the UA and Uma decreased significantly. For the UA, the RI and PI were (mean ± SEM) 0.835 ± 0.017 and 2.157 ± 0.129, 0.808 ± 0.008 and 1.920 ± 0.041, and 0.761 ± 0.006 and 1.759 ± 0.036 on periods -3, -2 and -1, respectively. For the Uma, the RI and PI were 0.97 ± 0.01 and 2.50 ± 0.02 at Week -17 and were 0.64 ± 0.02 and 0.98 ± 0.04 at Week -1, respectively. The complete disappearance of the early diastolic notch in the UA, and the complete appearance of diastolic flow in the Uma occurred on Week -1 and Week -11, respectively. Linear regression analyses regarding the relationship of the weeks before whelping (WBW) with the conceptus parameters were as follows: CRL ¼ 16.93 þ 0.93 WBW and FL ¼ 3.62 þ 0.22 WBW (R2 ¼ 0.98, P < 0.0001); AC ¼ 13.46 þ 0.67 WBW (R2 ¼ 0.96, P < 0.0001); BPD ¼ 4.46 þ 0.20 WBW (R2 ¼ 0.95, P < 0.0001); LLL ¼ 11.46 þ 0.45 WBW, LLT ¼ 9.52 þ 0.38 WBW and HC ¼ 16.68 þ 0.80 WBW (R2 ¼ 0.92, P < 0.0001); Uma RI ¼ 0.64e0.02 WBW and PI ¼ 0.79e0.11 WBW (R2 ¼ 0.90, P < 0.0001); and OFD ¼ 5.84 þ 0.27 WBW (R2 ¼ 0.86, P < 0.0001). The authors concluded that for fetal echobiometry UA and Uma perfusion were important endpoints to assess fetal vitality in the capuchin monkey. Moreover, it was possible to describe the moment when the organs were visualized and perform sexing. © 2017 Elsevier Inc. All rights reserved.

Keywords: Blood flow Fetal development Fetal sexing Organogenesis Ultrasonography

1. Introduction Gynecologic and obstetric ultrasonography is a widely used

* Corresponding author. Parque Mangal das Garças, Passagem Carneiro da Rocha, m, Para , Brazil. Cidade Velha, 66020-160, Bele E-mail address: [email protected] (S.A. Miranda). https://doi.org/10.1016/j.theriogenology.2017.11.023 0093-691X/© 2017 Elsevier Inc. All rights reserved.

method to evaluate reproductive health in nonhuman primates (NHPs) kept in captivity. Ultrasonography has been used to study gestation in Macaca mulatta [1,2], Macaca fascicularis [3,4], Callithrix jacchus [5e8], Saguinus fuscicollis [9], Cebus apella (Sapajus apella) [10], Cercopithecus aethiops [11], Aotus azarai infulatus [12,13], Pan troglodytes, Gorilla gorilla gorilla, Mandrillus sphinx, Erythrocebus patas [14], Papio anubis [15], Aotus nancymaae [16] and Chlorocebus sabaeus [17]. With an increased number of studies on NHP

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pregnancy, e.g., zika virus studies, it is crucial to properly describe normal and abnormal fetal growth and development [10] and to evaluate the efficacy of assisted reproductive technologies in these species. The neotropical primate Sapajus apella (capuchin monkey) is a valuable model for biomedical research [18e22] and for the development of reproductive biotechnologies for other endangered NHPs from the genus Sapajus [22]. However, there is a paucity in studies regarding fetal development in S. apella. Moreover, no information is available describing when fetal organs are formed in NHPs. Usually, only a description of the heart appearance and heartbeat are reported in neotropical [10,16,23] and old world monkeys [3,4,14], and the fetal gender was observed only in Macaca fascicularis [3]. Associations between fetal echobiometry and gestational age in capuchin monkeys have been studied in the context of predicting the whelping time. Furthermore, the well-being of the capuchin fetus has been evaluated based only on the fetal heartbeat [10]. In humans, relationships among fetal size, well-being, and characteristics of blood flow in the uterine artery (UA) [24e27] and in the umbilical artery (Uma) [28e31] have been reported to evaluate fetal viability by two-dimensional B-mode ultrasonography, augmented with color Doppler and spectral Doppler (triplex Doppler). Triplex Doppler has been used clinically for obstetrics and gynecology not only in women [32e34] but also in mice [35], capuchin monkeys [36], bitches [37] and cats [38]. It is described in women [39], bitches [37], queens [38], and mice [35] that the appearance of the Uma diastolic flow is responsible for the decreased RI and PI in the Uma. Moreover, in women [40] and other animal species [37,38,41], the early diastolic notch is normally present in the UA wave flow and disappears concurrent with the significant decreases in the RI and PI of the UA. Among the Doppler indices, the PI and the RI are commonly used in obstetric applications and do not depend on the insonation angle [42]; thus, they may even be used to compare studies done by different researchers because it is not necessary to standardize the insonation angle. Therefore, the use of triplex Doppler to assess heart beat and blood flow characteristics in the UA and Uma may be important for assessing fetal vitality in the capuchin monkey. We suggest that changes in these arteries, as the gestational age advances, may reflect increased perfusion in these arteries, which closely may follow increases in fetal dimensions. Moreover, the use of B-mode ultrasonography to obtain parameters, such as the description of fetal organs and fetal echobiometry, may also be important for assessing fetal vitality, to estimate the gestational age and the date of parturition in capuchin monkeys, as well as diagnosis the fetal gender. Considering the absence of reports regarding fetal development in S. apella, the objectives of the current study were as follows: 1) to evaluate blood flow in the UA and Uma in pregnant capuchin monkeys by measuring the resistive index (RI) and pulsatility index (PI); 2) to determine the presence or absence of the early diastolic notch and diastolic flow in the UA and Uma flow waveforms, respectively; 3) to perform conceptus echobiometry for fetal growth assessment during pregnancy; 4) to describe the moment that the fetal organs were initially observed; and 5) to assess when the diagnosis of fetal gender is possible.

Research (no. 055/2009/CEPAN/IEC/SVS/MS). Seven healthy (n ¼ 7), sexually mature female Sapajus apella, aged 10e15 years, provided by the National Primate Center (CENP), had gestation monitored by ultrasonography. These females previously had one or two uncomplicated pregnancies (four and three females, respectively) and whelpings and had live, healthy newborns. The animals were group housed in cages that were 3.85  2.30  2.55 m (length, width and height, respectively) with one male and three females under a natural photoperiod, i.e., under 12 h of light and 12 h of dark, at CENP, Ananindeua, Brazil (1220 5700 S, 48 220 5200 W). The climate was humid tropical, with an average annual temperature of 28  C. The diet consisted of fresh fruit, milk and commercial pellet chow (MEGAZOO P18, Betim, MG, Brazil) and water was available ad libitum. Vitamins, minerals and eggs were supplied once a week. 2.2. Ultrasonographic examination Inside the cages, the animals were caught with a primate capture net and were then subjected to a “handheld” restraint to collect the vaginal swab samples. The vaginal swab samples were collected daily in the morning to detect the onset of menses (defined as day 1 of the menstrual cycle) and the follicular growth phase [36]. After two weeks, ultrasound examinations were done to diagnosis pregnancy. Thereafter, ultrasonographic examinations were done only on the pregnant females at Weeks -20, -17, -16, -15, -13, -12, -11, -8 and -1 (whelping Week 0). The week of the examination relative to whelping was calculated in retrospect. After physical restraint with a primate capture net and a “handheld” restraint, ultrasound examinations were done under a chemical restraint (in intramuscular) treatment with the association of tiletamine hydrochloride and zolazepam (dose 4.4 mg/kg; Zoletil® 50, Virbac, Sao Paulo, SP, Brazil). The ultrasounds were conducted with an HDI 4000 PHILIPS

2. Materials and methods 2.1. Animals This research adhered to the American Society of Primatologists principles for the ethical treatment of primates. The experimental protocol was approved by the Ethical Committee in Animal

Fig. 1. Ultrasonography in the capuchin monkey female placed in dorsal recumbency after chemical restraint.

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scanner (Philips Medical Systems, Bothel, WA, USA) equipped with multi-frequency microconvex (5e8 MHz) and linear-array (5e12 MHz) transducers. For these examinations, the females were placed in dorsal recumbency (Fig. 1). To ensure acoustic coupling between the transducer and the skin, the abdomen was shaved (with a blade razor). and a coupling gel was applied (Aquasonic, Parker Laboratories Inc., Fairfield, NJ, USA). Each examination lasted ~1 h under chemical restraint reinforced one time. Initially, the uterus was located with B-mode ultrasonography using the anechoic urinary bladder as a landmark. When the gestational sac was observed, the latero-lateral longitudinal (LLL; Fig. 2A) and latero-lateral transversal (LLT; Fig. 2B) diameters were measured in the longitudinal and transversal section of the uterine body, respectively. When the fetus was detected ultrasonographically, fetal echobiometry was performed to assess fetal development. The fetal measurements included the crown rump length (CRL; Fig. 3A) on the longitudinal section of the fetus, which was obtained from the top of the cranium to the base of the tail, the biparietal diameter (BPD), occipito-frontal diameter (OFD), the head circumference (HC) on the transverse section of the fetal head, which was obtained at the level of the thalami (Fig. 3B), the abdominal circumference (AC; Fig. 3C) on the transverse section of the fetal abdomen, which was obtained at the level of the stomach, and the femur length (FL; Fig. 3D) obtained at the distance from the outer edges of each metaphysic. All the conceptus echobiometry end points were recorded to the nearest centimeter. Using color Doppler, the right and left UAs (along each side of the uterine body) were identified. The Uma was identified with color flow mapping in the umbilical cord near the placental surface. Once the UA and Uma were located and identified, flow waveforms were measured with spectral Doppler. The caliper was positioned approximately in the center of the UA (Fig. 4A and B) and Uma (Fig. 4C, D and E) to record the waveforms and calculate RI and PI. Correct identification of arterial flow was achieved when the spectral Doppler graph pattern had at least three consecutive systolic peaks with similar velocities and amplitudes. For each recording, the RI [43] and PI [44] for each waveform were calculated automatically by the software of the ultrasound scanner, as follows:

RI ¼

peak systolic velocity  end diastolic velocity peak systolic velocity

PI ¼

65

peak systolic velocity  end diastolic velocity mean velocity

For evaluating the UA blood flow, the gestation was divided into three periods before whelping, as follows: -3 (Weeks -20 to -17); -2 (Weeks -16 to -9) and -1 (Weeks -8 to -1). The presence or absence of the early diastolic notch in all the UA waveforms (Fig. 4A and B) and the moment that the Uma systolic and diastolic flow arose (Fig. 4C, D and 4E) during the analysis gestational period were observed and noted. The moment that the fetal organs were seen (Fig. 5) and the fetal gender diagnosis (Fig. 6) were both reported. The fetal heart rate (FHR) was measured using the spectral Doppler mode on a long-axis view of the heart. 2.3. Statistical analyses Linear regression was used to generate equations describing the relationship between the days before whelping with conceptus echobiometry, using the RI and PI of the Uma. In addition, ANOVA was used to determine the effect of the weeks before whelping on the RI and PI of the Uma. Repeated measures ANOVA was performed to analyze the effect of the weeks before whelping on the RI and PI of the UA. Differences between the means were determined with Fisher's protected least significant difference post hoc test. For the RI and PI, a Student's t-test was used to compare the means between the left and right UA (for each examination). The mean ± SD was used to determine the FHR. There were recorded the week that the absence of the early diastolic notch in the UA waveforms were noted and the moment that the Uma diastolic flow rise was observed, as well as the week that the fetal organs were seen. All the analyses were done with StatView software (SAS Institute Inc., Cary, NC, USA), and P < 0.05 was considered significant. 3. Results All the pregnancies resulted in a normal, uncomplicated whelping (one live newborn per female). Therefore, the chemical restraining protocol was safe and provided good sedation. The latero-lateral longitudinal (LLL; Fig. 7A) and latero-lateral transversal (LLT; Fig. 7B) diameters of the gestational sac were measured on Weeks -20 to -15 and -20 to -13, respectively. The LLL and LLT diameters of the gestational sac were best fit with a linear function model (Table 1). Linear regression equations for the relationships between the pre-whelping interval and the LLL and LLT diameters

B

A LLL

DVI

LLT

LLI

Fig. 2. B-mode ultrasonography of a gestational sac on Week -19 before whelping (whelping Week 0) in the capuchin monkey. (A) Latero-lateral longitudinal (LLL) and (B) Laterolateral transversal (LLT) diameters of gestational sac measured in the longitudinal and transversal section of the uterine body, respectively.

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Fig. 3. B-mode echobiometry of the capuchin monkey fetus. (A) Longitudinal section of the fetus crown rump length (CRL), which was obtained from the top of the cranium to the base of the tail, on Week -16 (whelping Week 0). (B) Transverse section of the fetal head, which was obtained at the level of the thalami, showing the biparietal diameter (BPD), occipito-frontal diameter (OFD) and head circumference (HC) on Week -12. (C) Transverse section of the fetal abdomen, which was obtained at the level of the stomach (S), showing abdominal circumference (AC) on Week -8. (D) Measurement of the femur length (FL), obtained at the distance from the outer edges of each metaphysic on Week -8.

of the gestational sac are shown (Table 1). For each equation, both the intercept and slope were significant. The LLL and LLT diameters increased as the gestational age advanced (Fig. 8). The fetus was detected ultrasonographically at Week -17; thereafter, the CRL was measured from Weeks -17 to -11 (Fig. 7C). The BPD, OFD, HC and AC were assessed from Weeks -16 to -1 (Fig. 7D), and the FL was measured from Weeks -13 to -1 (Fig. 7E). The CRL, BPD, OFD, HC, AC and FL were best fit with a linear function model (Table 2). For each linear regression of weeks before whelping and various fetal measures, both the intercept and slope were significant (Table 2). The CRL, BPD, OFD, HC, AC, and FL increased as the gestational age advanced (Fig. 9). In early pregnancy, the gestational sac diameters were important parameters to evaluate conceptus growth (R2 ¼ 0.92). The visualization of the gestational sac was useful to detect early pregnancy and estimate the gestational age prior to embryo visualization [10]. From Week -17, the embryo was seen, and the CRL became a more accurate parameter in assessing gestational age (R2 ¼ 0.98). The LLT diameter and CRL were measured for more time, from Weeks -20 to -13 and Weeks -17 to -11, respectively. As gestation proceeded, the FL (R2 ¼ 0.98), from Weeks -13 to -1, and the AC (R2 ¼ 0.96) and BPD (R2 ¼ 0.95), from Weeks -12 to -1, became important endpoints to evaluate fetal development. The blood perfusion values of the UA (Fig. 7F) and Uma (Fig. 7G) were evaluated from Weeks -20 to -1 (Periods -3 to -1) and from Weeks -17 to -1 before whelping, respectively. There was no significant difference between the right and left UAs for the RI and PI on any week of the pregnancy. The mean RI and PI of the UA and

Uma both decreased as the gestational age advanced (Fig. 10). There was an effect (P < 0.05) of interval before whelping on the RI and PI of the UA and Uma (Fig. 10). During the pregnancy, increased flow in the UA occurred during two periods for the RI and during three periods for the PI, as represented by significant decreases in both the RI and PI in the UA (Fig. 10A). The early diastolic notch of the UA was apparent from the start of the study to period -1 (Weeks -8 to -1) before whelping (Fig. 4A), when it disappeared (Figs. 4B and 10A). The resistive and pulsatility index of the Uma were best fit with a linear function model (Table 3). For each linear regression of weeks before whelping and the resistive and pulsatility index of the Uma, both the intercept and slope were significant (Table 3). The blood perfusion in the Uma increased as the gestational age advanced and occurred during four periods for the RI and during three periods for the PI, as represented by decreases in the RI and PI in the Uma (Fig. 10B). In the Uma, systolic flow was detected from Week -17 before whelping (Fig. 4C), whereas the initial diastolic flow (Figs. 4D and 10B) and complete diastolic flow (Figs. 4E and 10B) were detected at Week -12 and Week -11, respectively. The moment that the Uma diastolic flow raised matched the decrease of the RI and PI (Fig. 10B). The fetal heart and stomach were observed ultrasonographically at Week -17 (Fig. 7H); the urinary bladder at Week -16 (Fig. 7I); and the lung, liver, kidney, bowel and fetal sex identification, including two males and five females, were identified at Week -12 (Fig. 7J), matching with the appearance of the diastolic flow and the decreased Uma RI and PI. The mean FHR on Weeks -17 to -1 was

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Fig. 4. Triplex Doppler of the uterine (UA) and umbilical (Uma) arteries in pregnant capuchin monkey. (A) UA on Week -7 (Period -1; whelping Week 0) with an early diastolic notch (EDN). (B) UA on Week -1 (Period -1) without EDN (arrow). (C) Uma on Week -17 with sistolic flow (arrow) (D). Uma on Week -12 with systolic flow (white arrow) and initial diastolic flow (red arrow). (E) Uma on Week -4 with sistolic (white arrow) and diastolic flow (red arrow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

194 ± 12.4. 4. Discussion The decreased uterine artery RI and PI (Fig. 10A) as the gestational age advanced in capuchin monkeys reflected increased blood perfusion in these arteries, which closely followed increases in fetal dimensions (Fig. 9), providing support for the hypothesis that changes in the uterine blood supply may be related to the increased nutritional demands of the growing fetus. Moreover, the pronounced decrease in UA blood flow resistance may be attributed to the concurrent invasion of the trophoblast into the endometrium during placentation and vascular dilatation, because new world monkeys also have discoid and hemochorial placentas [45], as well as what occurs in women [24,40,46]. The placental shape describes the pattern that makes contact with the uterine wall, where the nutrient exchange occurs, i.e., for the discoid placenta, the placenta attaches to the uterine wall as one disc that shares an umbilical cord. However, the maternalefetal interface describes the type of barrier that separates the maternal and fetal tissue, i.e., for the hemochorial placenta, both the maternal epithelial and endothelial cells are degraded, leaving only the trophoblast cells in direct contact with the maternal blood [45]. Similar to what was found in capuchin monkeys (Fig. 4A), the early diastolic notch is normally present in the UA wave flow in women [40] and other animal species [33,41], representing a decrease in the blood flow shortly after systole [47]. However, it disappeared entirely at the end of gestation (Fig. 4B), concurrent

with significant decreases in the RI and PI of the UA (Fig. 10A), which reflected the increased blood perfusion in these arteries. It was noteworthy that the RI and PI of the UA could be monitored throughout much of the pregnancy, whereas the range during which the fetal measurements could be assessed was more limited. The initial and complete appearance of the Uma diastolic flow that reflected the increased blood perfusion in these arteries and the progressive development of the fetal/placental circulation were responsible for the decreased RI and PI (Fig. 9B), consistent with similar events in women [39], bitches [37], queens [38], and mice [35] shortly after the end of organogenesis (Week -12). The emergence of Uma diastolic flow may be caused by changes associated with the maturation of the placenta and/or fetal cardiovascular development [35], because the discoid placenta attaches to the uterine wall as one disc that shares an umbilical cord and reflects the increased nutrient exchange for the growing fetus [45]. Therefore, using the RI and PI to evaluate the UA and Uma blood flow in the pregnant capuchin monkeys were effective, practical and rapid parameters that could be obtained in a short period of time. Among the Doppler indices, the PI and the RI are commonly used in obstetric applications and do not depend of the insonation angle [42], and thus, they may even be used to compare studies done by different researchers, because it is not necessary to standardize the insonation angle. Moreover, Joern et al. (1997) [48] reported that the qualitative Doppler parameters (RI and PI) are superior to the quantitative parameters. The RI and PI were widely used in gestational ultrasonography in women [31,39] dogs [37], queens [38], mice [35], and mares [41], and they are used to study

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Fig. 5. B-mode ultrasonography of the organogenesis in the capuchin monkey fetus on Week -8 before whelping. (A) heart (arrow); (B) stomach (S), urinary bladder (B) and kidney (arrow); (C) lung (LU), liver (LI) and bowel (B).

reproductive physiology in women [49] and capuchin monkeys [36]. In women, abnormal placentation and trophoblastic invasion may result in abnormal waveform patterns of the UA [47,50] and Uma [51], which manifest as elevated RI and PI. However, although the importance of evaluating the UA and Uma in pregnant women was already reported [50], some earlier

studies with nonhuman primates [1,7,10,13e16] performed only the conceptus echobiometry with B-mode, and the FHR was assessed with M-mode ultrasonography [16]. This study was the first to describe many of these variables in nonhuman primates. As gestation proceeds, different biometric parameters became important endpoints to evaluate fetal development. The

Fig. 6. Fetal sex identification in the capuchin monkey by B-mode ultrasonography on Week -12 before whelping. (A) Male and (B) female. The arrows show the fetal sex.

S.A. Miranda et al. / Theriogenology 108 (2018) 63e73

-17

-20

A

-15

-16

-13

-8

-11

-12

69

-1

Week before whelping

Latero-lateral longitudinal

B

Latero-lateral transversal

C

Crown rump lenght

D

Biparietal

Occipito-frontal

Head circumference

E

Abdomen

Femur

F

Uterine artery

G

Umbilical artery

H

Heart

Stomach

I

Urinary bladder

J

Lung

Liver

Kidney

Bowel

Sex

Fig. 7. Pre-whelping interval and end points assessed during ultrasonographic examinations in pregnant capuchin monkey: (A) Latero-lateral longitudinal diameter and (B) Laterolateral transversal diameter of gestational sac; (C) Crown rump length; (D) Biparietal diameter, occipito-frontal diameter, head circumference and abdominal circumference; (E) Femur length; (F) Resistive index, pulsatility index of uterine arteries and the presence or absence of the early diastolic notch; (G) Resistive index, pulsatility index, systolic flow (B) initial diastolic flow ( ) and complete diastolic flow (C) of umbilical artery; (H) Fetal heart and stomach; (I) Urinary bladder; (J) Lung, liver, kidney, bowel and fetal sex identification.

Table 1 Linear regression analyses regarding the relationship between the weeks before whelping (WBW) and the gestational sac diameters in pregnant capuchin monkeys. Gestational sac diameter

Regression equation

Probability

LLL ¼ 11.46 þ 0.45 WBW LLT ¼ 9.52 þ 0.38 WBW

Latero-lateral longitudinal Latero-lateral transversal

R2

Intercept

Slope

0.92 0.92

<0.0001 <0.0001

<0.0001 <0.0001

R2 ¼ coefficient of determination.

Gestational sac diameters 5.0

A

5.0

Latero-lateral transversal (cm)

Latero-lateral longitudinal (cm)

5.5

4.5 4.0 3.5 3.0 2.5 2.0 -21

B

4.5 4.0 3.5 3.0 2.5 2.0

-20

-19 -18 -17 Week before whelping

-16

-15

-14

-21

-20

-19

-18 -17 -16 -15 Week before whelping

-14

-13

Fig. 8. Linear regression analyses regarding the relationship of weeks before whelping with (A) the latero-lateral longitudinal and (B) latero-lateral transversal diameters of gestational sac in pregnant capuchin monkeys.

combination of various biometric parameters improves the prediction of gestational age [50] and has been used in humans to help distinguish symmetric from asymmetric intrauterine growth restriction [52]. The values found in this study for the CRL, BPD, FL, and the gestational period were important for echobiometry, which corroborates with what was described by Corradini et al. [10] in Sapajus apella. However, Corradini et al. [10] measured only the GS (mean of cephalocaudal and ventrodorsal measurements), embryo/ fetal greatest length, BPD, thorax height and FL and did not assess the OFD, HC and AC. In Papio anubis [15] the CRL, BPD, HC,

transverse cerebellar diameter, AC, and FL were measured, and in Macaca mulatta [1], the gestational sac, CRL, BPD, HC, head area, AC, abdominal area and FL were measured to assess fetal growth. The same measurements obtained in Macaca mulatta and the OFD were made in Aotus azarai infulatus [13], while in Aotus nancymaae, only BPD was measured [16]. In this study, the relationships between conceptus echobiometry and the weeks before whelping were best fit with a linear function model. In a similar manner, the linear model was selected as optimal for gestational sac, CRL, BPD and FL in Sapajus apella [10]

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Table 2 Linear regression analyses regarding the relationship between the weeks before whelping (WBW) and the various fetal measures in pregnant capuchin monkeys. Fetal echobiometry

Regression equation

Probability

CRL ¼ 16.93 þ 0.93 WBW BPD ¼ 4.46 þ 0.20 WBW OFD ¼ 5.84 þ 0.27 WBW HC ¼ 16.68 þ 0.80 WBW AC ¼ 13.46 þ 0.67 WBW FL ¼ 3.62 þ 0.22 WBW

Crown rump length Biparietal diameter Occipito-frontal diameter Head circumference Abdominal circumference Femur length

R2

Intercept

Slope

0.98 0.95 0.86 0.92 0.96 0.98

<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

R2 ¼ coefficient of determination.

Fetal ecobiometry 4.5

A

7

4.0

6

3.5

5

3.0 BPD

CRL

8

4

2.5

3

2.0

2

1.5

1

1.0

0

0.5 -18

6

-17

-16

-15 -14 -13 -12 Week before whelping

-11

-10

-18 16

C

-14

-12 -10 -8 -6 Week before whelping

-4

-2

0

-16

-14

-12 -10 -8 -6 Week before whelping

-4

-2

0

D

12 HC

4 OFD

-16

14

5

3

10 8

2

6

1

4 2

0 -18

14

-16

-14

-12 -10 -8 -6 Week before whelping

-4

-2

0

-18

3.5

E

12

F

3.0

10

2.5

8

FL

AC

B

2.0

6 1.5

4

1.0

2

0.5

0 -18

-16

-14

-12 -10 -8 -6 Week before whelping

-4

-2

0

-14

-12

-8 -6 -10 Week before whelping

-4

-2

0

Fig. 9. Linear regression analyses regarding the relationship of weeks before whelping with (A) crown rump length (CRL), (B) biparietal diameter (BPD), (C) occipito-frontal diameter (OFD), (D) head circumference (HC), (E) abdominal circumference (AC) and (F) femur length (FL) in pregnant capuchin monkeys.

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Uterine arteries

A 0.86

2.2 Pulsatility index

0.84 Resistive index

a

2.3

a

a

0.82 0.80 0.78

2.1 2.0

b

1.9

b c

1.8

0.76 0.74

-2 Period before whelping

-3

1.7

-1

-3

-2 Period before whelping

-1

Umbilical artery B 1.00

a

a

a

a

a

3.25 3.00

0.95

0.85

b

0.80

c

c

0.75

c

c c

0.70

2.50

a a

a

a a

2.25 2.00

b

1.75

b

1.50

c

c c

1.25

d

0.65 0.60

Pulsatility index

b

0.90 Resistive index

2.75

c

c

1.00 -17 -16 -15 -13 -12 -11 -9

-7

-5

0.75 -4

-3

-2

-1

-17 -16 -15 -13 -12 -11 -9

-7

-5

-4

-3

-2

c

-1

Week before whelping

Week before whelping

Fig. 10. (A) Means (±SEM) (C) resistive index (A) pulsatility index of the uterine arteries on periods -3 (weeks -20 to -17), -2 (weeks -16 to -9) and -1 (weeks -8 to -1) before whelping in pregnant capuchin monkeys. The arrow indicates the period that the early diastolic notch disappears. (B) Means (±SEM) (C) resistive index (A) pulsatility index of the umbilical artery in pregnant capuchin monkeys. The arrows indicate the means of days that the umbilical ( ) systolic flow appears ( ), initial diastolic flow ( ) and complete diastolic flow appear. a-d Within a line, values without a common letter differ (P < 0.05).

Table 3 Linear regression analyses regarding the relationship between the weeks before whelping (WBW) and the resistive and pulsatility index of the umbilical artery in capuchin monkeys. Blood vessel

Umbilical artery

Index

Resistive Pulsatility

Regression equation

Probability

RI ¼ 0.64e0.02 WBW PI ¼ 0.79e0.11 WBW

R2

Intercept

Slope

0.90 0.90

<0.0001 <0.0001

<0.0001 <0.0001

R2 ¼ coefficient of determination.

and for gestational sac, CRL and FL in Macaca mulatta [1]. In contrast, the linear-quadratic model for AC (R2 ¼ 97.6) and the linear cubic model for BPD (R2 ¼ 99.4%) and HC (R2 ¼ 99.3%) were the best representative functions in Macaca mulatta. However, the linear model for BPD and HC were high, too, at 95.3% and 95.8%, respectively [1]. There was a significant effect of the interval before whelping on these parameters, which was concomitant with decreases in the RI and PI in the UA and Uma. Therefore, we inferred that assessing conceptus echobiometry and changes in flow characteristics in the

UA and Uma during pregnancy were extremely important for evaluating fetal development and well-being. In women, for example, an undersized AC measurement is a sensitive marker for intrauterine growth restriction and may reflect a reduction in the size of the liver or other intra-abdominal organs [53]. Furthermore, it is described, also in women, that BPD and HC reflect the head size, which in turn reflects brain growth [53]. Microcephaly, for example, is typically diagnosed when the HC is below the mean for the gestational age [54]. In Callithrix jacchus, the BPD was the best parameter to relate with the gestational age, showing that the

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S.A. Miranda et al. / Theriogenology 108 (2018) 63e73

diagnosis of a small BPD for gestational age results in fetal death seven days after birth [7]. The fetal heart, stomach, urinary bladder, lung, liver, kidney, bowel and fetal sex were ultrasonographically observed to match with the appearance of diastolic flow and a decrease in the Uma RI and PI. Fetal development progresses rapidly from Week -17, allowing the ultrasonographic recognition of organogenesis, with all organs visible at Week -12. Nevertheless, research in NHPs describes only the appearance of the heart, FHR and biometry [4,10,14,16]. This study is the first to describe those fetal organs in nonhuman primates. At Week -12, it was also possible to perform the fetal sex determination, which was not described in neotropical primates. In 1988, Tarantal & Hendrickx [3] identified the sex of the fetus in Macaca fascicularis by ultrasonography as early as gestational day 70e75. In small ruminants, the diagnosis of fetal gender by ultrasonography may contribute with the reproductive biotechnologies after an artificial insemination with sexed semen [55,56], transfer of sexed embryos [57] or embryos produced in vitro [58]. The FHR did not show great variation during pregnancy (194 ± 12.4 bpm) compared to findings reported by Corradini et al. [10], where a decrease in heart rate with advancing pregnancy was observed, providing even a linear equation. However, Corradini et al. [10] used ketamine hydrochloride (10 mg/kg) as the chemical restraint of the females, which was different from the anesthetic used in this study protocol (the association of tiletamine hydrochloride and zolazepam; 4.4 mg/kg), which may have influenced this variation. Moreover, in this study, the fetal heart rate was within the mean of the physiological parameters (165 a 225 bpm) described for the genus Cebus [59]. In Aotus azarae infulatus, there was no statistically significant difference in the heart rate values between the tiletamine and zolazepam protocol (doses 1.11e8.33 mg/kg; mean 4.72 mg/kg) and the physical restraint protocol [60]. Thus, in this study, we used the dose recommended by the manufacturer, which was the mean dose used in other studies. The development of well-being assessment methods is augmented human obstetrics, resulting in the early diagnosis of abnormal pregnancies or fetal distress, facilitating early intervention. Determining Doppler predictors of normal obstetric outcomes in capuchin monkeys could be helpful in future studies of abnormal gestation in NHPs. Thus, triplex Doppler can be used with frequency to monitor pregnancies in S. apella to evaluate the efficacy of assisted reproduction programs and biomedical research. 5. Conclusion The equations obtained in this work, relating the weeks before the birth with the parameters measured during pregnancy (LLL, DVL, LLT, BPD, OFD, HC, AC and FL) and the Uma RI and PI allowed us to determine the gestational age to assess the anatomical fetal growth. Moreover, it was possible to describe the moment when the organs were visualized and to perform sexing. The changes in the UA and Uma RI and PI; the disappearance of EDN in the UA flow wave; and the appearance of the diastolic component of the Uma flow wave occurred simultaneously with fetal growth, demonstrating also that the evaluation of blood flow in these arteries is an important parameter to assess fetal vitality in Sapajus apella. Thereby, the ultrasound allows for studying the physiology of pregnancy in the species S. apella, establishing reference parameters that can be used to monitor normal and risk pregnancies. Conflicts of interest The authors declare that there is no potential conflict of interest

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