Breed-specific ecobiometry and ultrasound factors predictive of fetal maturity in healthy English Bulldog bitches subjected to elective cesarean section

The aims of the current study are to build an equation to predict gestational age (GA) and to compare ultrasound parameters indicative of delivery in healthy English Bulldog bitches subjected to elective cesarean section. Sixteen pregnant female dogs were included in the study. Inner chorionic cavity (ICCDD) and biparietal (BPD) diameters were measured at 30 and 50 days after artificial insemination, respectively, to estimate GA at embryonic and fetal stages. BPD, heart rate (HR), and intestinal peristalsis were measured at 48 h, 24 h, and 6 hours before delivery to compare fetal development. ICCD and BPD values were subjected to linear regression and parameters predictive of elective cesarean section were compared through Student’s t-test before delivery. The number of conceptuses did not influence pregnancy duration. Both ICCD and BPD were significantly correlated to GA; their formulas presented accuracy of ± 1 and ± 2 days, respectively, in comparison to that of progesterone dosage. Based on the comparative Research, Society and Development, v. 10, n. 10, e555101019091, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.19091 2 ultrasound assessment, BPD has significantly increased from 48 h to 6 h before delivery (≥3 cm), regardless of the number of pups, whereas HR has significantly decreased within 6 h before delivery (HR <200 bpm). There was not statistically significant difference in parameter “intestinal peristalsis” among measurement times. The current study is pioneer in highlighting that measuring ICCD and BPD in the formula is a useful tool to predict GA in the herein investigated breed and that fetal parameters such as BPD and HR are fetal maturity predictors.

ultrasound assessment, BPD has significantly increased from 48 h to 6 h before delivery (≥3 cm), regardless of the number of pups, whereas HR has significantly decreased within 6 h before delivery (HR <200 bpm). There was not statistically significant difference in parameter "intestinal peristalsis" among measurement times. The current study is pioneer in highlighting that measuring ICCD and BPD in the formula is a useful tool to predict GA in the herein investigated breed and that fetal parameters such as BPD and HR are fetal maturity predictors. Keywords: Canine bitches; Breed; Cesarean section; Fetuses; Gestation; Ultrasound.

Introduction
Parturition date prediction is an important factor to help planning cesarean sections, even in unknown-coverage cases.
It is known that gestation duration in domestic dogs can range from 58 to 71 days after the mating day (Concannon et al., 1989). This range may be influenced by sperm longevity in females' reproductive tract, as well as by the interval between estrus onset and ovulation, and the release of immature oocytes, whose maturation takes place within 72 hours in the reproductive tract (Concannon et al., 1989;Feldman & Nelson, 2003;Concannon, 2011).
Ecobiometry is also a useful tool to estimate the likely delivery date based on anatomical measurements taken of conceptuses (Luvoni & Grioni 2000) at different pregnancy times (Groppetti et al., 2015). However, ecobiometric data are not always applied to all canine breeds, due to great diversity in species' size and different conformational types. Recent study has applied a new approach to gestational ultrasound examination given the need of planning elective cesarean sections for brachycephalic breeds such as English Bulldog, miniature breeds such as Yorkshire and Chihuahua (O'Niell et al., 2017), among other breeds such as Boston Terrier and Scottish Terrier, who often have obstructive dystocia due to fetal-maternal disproportion (Jutkowitz, 2005;Linde-Forsberg, 2005).
Fetal maturity must be taken into consideration at the time to make decisions about elective cesarean section, since it enables identifying fetal structures as to their defined architecture (Nyland & Mattoon, 2015). Intestinal peristalsis , reduced heart rate (Lopate, 2008;Davidson & Baker, 2009;Lamm & Makloki, 2012;Gil et al., 2014;Nyland & Mattoon, 2015), as well as reduced umbilical artery resistivity index (Giannico et al., 2015) are also factors that should be taken into account at the time to make decisions about cesarean section.
The aforementioned variables must be analyzed together, rather than in separate, to enable defining the proper time for elective cesarean section . The set of information often generates uncertainties about breeds lacking detailed information. Among them, one finds English Bulldog, whose high dystocia incidence justifies the frequency of elective cesarean sections animals belonging to this species are often subjected to (Dobak et al., 2018).
The aims of the current study were to build an equation to predict gestational age at embryonic and fetal stages and to compare ultrasound parameters indicative of delivery in English Bulldog bitches subjected to elective cesarean section.

Research
The current research is an experimental study of quantitative nature (Pereira et al., 2018) aimed at evaluating ultrasound data and at building an equation predictive of gestational age in canine bitches subjected to elective cesarean section.

Experimental Animals and Procedure
Sixteen (n=16) pregnant English Bulldog bitches were evaluated in the current study. All animals were healthy, presented weight ranging from 20 to 25 kg, were in the age group 1 year and 8 months to 4 years, and belonged to private kennels in Ceará State (Northeastern Brazil). They fed on commercial granulated dog food and had access to water ad libitum.
They had known reproductive history and/or presented normal standards in clinical (physical inspection), laboratory (blood count), abdominal and pelvic ultrasound assessments. All experimental procedures were approved by the Ethics Committee on Animal Experimentation (Comitê de Ética para Uso Animal -CEUA, Ceará State University, Fortaleza, CE, Brazil), under protocol number 5999508/2015.

Two-dimensional ultrasound evaluations
Ultrasound examinations were performed for consecutive six months in private kennels located in the metropolitan region of Fortaleza City, Ceará State (Northeastern Brazil). They were carried out by the same operator with the aid of Logiq E (General Electronics, GE) 8-13 MHz multifrequency linear probe (LA523 reference). Evaluations were preferably carried out in the morning, in animals who fasted for at least eight hours. Animals were kept in supine position, without sedation. Their abdominal and pelvic regions were previously shaved and water-soluble acoustic gel was used to perform the exam.

Experimental design number 1 to predict gestational age
After confirmatory pregnancy results, canine bitches were evaluated at two different times: the first evaluation was carried out at 30 days after artificial insemination (AI) and the second evaluation, at 50 days after AI. Both assessments were performed to predict gestational age through the application of mathematical equations at embryonic and fetal stages through linear regression and ANOVA (Software R 2.15.1).
ICCD values were measured at 30 days after AI, by taking into consideration the mean of two inner chorionic cavity diameter measurements taken at 90º cross-section angles, close to the trophoblastic reaction; embryonic vesicles were spherical and presented well-defined margins. BPD values, in their turn, were assessed at 50 days after IA; they were obtained through the longitudinal section of the skull and measured by taking into consideration the parallel distance between parietal bones ( Figure 1). All measurements were taken in two different conceptuses located more cranial to the birth canal in each uterine horn, and their application to build the equations was based on the mean value of these measurements (Luvoni, 2013). Source: Authors.

Experimental design number 2 for fetal growth evaluation
Ultrasound assessments were performed at 48 h, 24 h, and 6 h before surgical intervention, based on two-dimensional findings of fetal maturity. At least five fetuses from each pregnant dog were evaluated when litter size was equal to, or larger Research, Society andDevelopment, v. 10, n. 10, e555101019091, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.19091 5 than, five fetuses. All conceptuses were measured when litter comprised less than 5 individuals. The evaluation procedure took into consideration the smallest and largest fetuses in the same pregnancy. Biparietal diameter was measured as described in experiment n. 1; it was used as parameter to assess fetal growth.
Fetal functionality parameter was evaluated through intestinal peristalsis, which was classified based on bowel movement intensity. Peristalsis was classified as absent (score 0), when bowel movement was not identified; as light (score 1), when this movement was only seen in a few segments of it at reduced intensity; as moderate (score 2), when movement was identified in multiple segments, although at reduced intensity; and as evident (score 3), when bowel movement was identified in multiple segments at evident intensity.
Heart rate was measured as parameter to assess fetal viability and stress. All fetuses presented parietal stratification and compartmentalization of cardiac chambers; this outcome has evidenced fetal maturity. All cesarean sections were performed in surgical environment by using inhalational anesthetic. Apgar score was applied to newborn puppies to prove ultrasound assessment effectiveness in indicating successful cesarean sections.

Statistical analysis
Results were expressed as mean and standard deviations. All tests were performed in R Software version 2.15.1.
Linear regression method and ANOVA were used to calculate the gestational age based on measurements taken in evaluations carried out at 30 and 50 days after artificial insemination. The number of conceptuses was assessed based on their influence on pregnancy duration. The accuracy of values that have resulted from formulas generated to estimate gestational age was compared to that of pregnancy duration values obtained through serum progesterone dosage using Pearson's test. Parameters such as fetal growth (biparietal diameter), heart rate, and intestinal peristalsis 48 h, 24 h, and 6 h before delivery were subjected to ANOVA and Student's t-test in order to find factors associated with fetal maturity; P values ≤ 0.05 were considered significant.

Ultrasound findings
Ultrasound assessment time ranged from 20 to 30 minutes: 25 minutes, on average. All canine bitches presented normal gestational course without complications during the intraoperative and postoperative periods. Two of the 16 evaluated English Bulldog bitches went into labor before surgical intervention; there was no neonatal death (n. 6 and 10). Nulliparous (02/16), primiparous (03/16), and multiparous (11/16) canine bitches, whose age ranged from 16 to 49 months, were evaluated, as shown in Table 1. If one considers the LH hormone peak as day zero, pregnancy duration ranged from 58 to 63 days (60 ±

days).
Litter size ranged from two to nine puppies; in total, 89 puppies were born during the current study. Two of them presented prenatal death due to mummification (fetuses born from dogs n. 3 and 7), and six presented birth abnormalities featured by fetal hydrops, eventration, and cleft lip (fetuses born from dogs n. 1, 4, 7, 10, 12 and 16). All fetal abnormalities were identified through ultrasound, except for cleft lip. Minimum and maximum Apgar scores were 9 and 10, respectively. *N. of days after LH peak, which was considered as day 0. Source: Authors.

Formula used to predict gestational age in English Bulldog bitches
Equations built for embryonic ecobiometry assessment at 30 days after AI and for fetal ecobiometry assessment at 50 days after AI (Table 2) were useful to predict gestational age at embryonic and fetal stage. Both ICCD and BPD were significantly and linearly correlated to gestational age. The accuracy of values obtained through these formulas was ± 1 and ± 2 days, respectively, in comparison to pregnancy duration estimated through hormonal dosage. Moreover, the number of conceptuses did not influence pregnancy duration (P>0.05).
Findings recorded in experiment n. 1 have shown that the formulas used to measure fetal structures enabled accurately predicting gestational age at embryonic and fetal stage in English Bulldog bitches.

Biparietal diameter and heart rate should be analyzed together
According to Table 3, the analysis based on fetal parameters such as biparietal diameter (BPD) and heart rate (HR) has shown significant ultrasound findings at 48 h, 24 h, and 6 h before elective cesarean section. BPD values have significantly increased from 48 h to 6 h before delivery among the three evaluated times. BPD has increased by 0.11 ± 0.03 cm between 48 h and 24 h before cesarean section, as well as by 0.14 ± 0.05 cm, between 24 h and 6 h before cesarean section. Pre-delivery BPD was ≥ 3 cm (minimum: 3.00 cm and maximum: 3.17 cm) 6 h before cesarean section, regardless of the number of puppies.
Although heart rate values did not differ between 48 h and 24 h before cesarean section, these values have significantly reduced during the last 6 h before delivery. During this period, fetal HR ranged from 165 beats per minute (bpm) to 210 bpm, most of the litter presented HR values <200 bpm. On the other hand, there was not statistically significant difference in intestinal peristalsis among scores analyzed at different prepartum times.
Results of experiment n. 2 have shown that fetal parameters such as BPD and HR should be analyzed together in order to help making decisions about the proper time to perform elective cesarean section, with emphasis on the incidence of BPD larger than 3 cm and HR <200 bpm in English Bulldog puppies, within the last 6 h before delivery. *Parameters analyzed by measuring the parallel distance between parietal bones (biparietal diameter), heart rate per minute (bpm for heart rate), and bowel movements, which were classified as absent (score 0), light (score 1), moderate (score 2) and evident (score 3). **Times when ultrasound assessment was performed before elective cesarean section; a, b, c indicates statistically significant differences between columns (evaluated times). Source: Authors.
The current study was the first to evaluate GA estimates in brachycephalic English Bulldogs. This breed is highly prone to dystocia (Jackson, 2004;Bergstrom et al., 2006;Dobak et al., 2018), due to incidence of dorsum-ventrally flattened pelvis in bitches belonging to this species, as well as to disproportionately large fetal skull (Johnston et al., 2001;Linde-Forsberg, 2005;Evans & Adams, 2010;Wydooghe et al., 2013), which ends up leading to the need of emergency (Jutkowitz, 2005) or elective cesarean sections (Dobak et al., 2018).
Although breed is reported to be determining factor for gestation duration and litter size (Okkens et al., 2001) and litter size is reported to lead to longer pregnancy duration (Eils et al., 2005;Groppetti et al., 2015;Alonge et al., 2016), the number of conceptuses did not influence pregnancy duration in the current study. Assumingly, the number of English Bulldog puppies would not influence pregnancy duration because this breed is not highly prolific. However, the canine bitches investigated in the present study were subjected to elective cesarean sections, and it was not possible estimating the influence of the number of conceptuses on gestational duration.
Delivery date or gestational age can be estimated by measuring gestational vesicle diameter or chorionic cavity diameter (Yeager et al., 1992;Luvoni & Grioni, 2000), biparietal diameter (Kutzler et al., 2003), thoracic and abdominal, body lengths (England et al., 1990), placental thickness (Maldonado et al. 2012) and diencephalon-telencephalic vesicle diameter (Beccaglia et al., 2008). However, only ICCD and BPD measurements were evaluated in the current study because they provide accurate results to estimate gestational age at embryonic and fetal stage, respectively (Kutzler et al., 2003;Luvoni & Beccaglia, 2006;Lopate, 2008;Michel et al., 2011). These parameters were sufficient to measure GA in healthy English Bulldog bitches since pregnancy duration calculated through the adopted formulas was equivalent to that of hormonal progesterone dosages.
Although the incidence of complications during parturition in English Bulldogs remains unknown, most owners and breeders often make the option for elective cesarean section due to fear of dystocic births and, consequently, to avoid litter loss. Thus, ultrasound is an important method used to assess fetal maturity parameters (Carvalho, 2014;Nyland & Mattoon, 2015). However, few parameters are reliable and breed-specific, due to the subjectivity of this assessment. Based on evaluations of fetal growth parameters (BPD), HR and intestinal peristalsis performed in the current study, it is possible emphasizing that the joint evaluation of BPD and HR is highly recommended for decision-making about the proper time to perform elective cesarean sections in English Bulldog bitches.
Intrauterine fetal growth can be monitored by measuring fetal structures (Freitas et al., 2016). BPD is an accurate measurement taken at 40 days after conception, when bone mineralization has already started (Luvoni, 2013); thus, it is useful to estimate canine GA. This is the reason why BPD was herein assessed to estimate fetal maturity. It is important understanding breed-specific BPD in the pre-delivery period to make the right decision about the time to perform cesarean sections, since its value significantly increases in the last 48-6 h before delivery due to exponential fetal growth in the final third of pregnancy. In addition, BPD measuring through ultrasound enables inferring about fetuses in the litter presenting smaller size or delayed growth. However, so far, BPD was not accurate in predicting the best time to perform cesarean section in experimental dogs belonging to giant breeds, since they present great variation in litter size (Alonge et al., 2016) -further studies should be conducted to assess the influence of litter size on specific breeds, such as English Bulldog.
Mean fetal HR is 230 bpm; it reaches approximately 214 bpm at early pregnancy, increases to approximately 238 bpm at the 40 th gestational day, and declines close to parturition (Carvalho, 2014;Nyland & Mattoon, 2015). However, normal fetal HR values or intervals before delivery in canine species remain unknown. The current findings have shown cardiac deceleration 6 h before delivery, sharp fetal HR decrease in a short time interval (at least 5 min) and return to normal frequencies as indicative of eminent delivery (Giannico et al., 2015). Since HR below 180 bpm is indicative of fetal distress (Johnson et al., 2001), values identified during the last 6 h before delivery were considered indicative of imminent delivery and, therefore, indicative of immediate cesarean delivery. Most fetuses in the current study were fully developed and presented reduced HR during this period, although their mothers did not show signs of active labor.
Finally, although intestinal peristalsis is key parameter used to assess complete canine fetal organogenesis and to evaluate fetal maturity , the current findings have shown that this parameter should not be evaluated in separate, at the time to make decisions about performing cesarean sections. Score 3 was identified in all three analyzed periods, including 48 h before delivery, and it evidenced early organogenesis. Since intestinal peristalsis evaluation is subjective, and its early visibility depends on the adopted ultrasound equipment , we do not recommend using ultrasound, in separate, to determine the functionality and maturation of this organ.

Conclusion
Chorionic cavity and biparietal diameters are accurate measurements used to calculate gestational age in healthy English Bulldog bitches. Moreover, biparietal diameter measurements in association with heart rate are important and useful parameters to help identifying fetal maturity during ultrasound examinations before delivery.