The influence of the obstetrical condition on canine neonatal pulmonary functional

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Original Study

Journal of Veterinary Emergency and Critical Care 00(0) 2015, pp 1–6 doi: 10.1111/vec.12368

The influence of the obstetrical condition on canine neonatal pulmonary functional competence ´ Liege Garcia Silva, DVM, PhD; Guilherme Vannucchi Portari, PhD; Cristina F´atima Lucio, DVM, PhD; Jaqueline Aguiar Rodrigues, DVM, MSc; Gisele Lima Veiga, DVM, PhD and Camila Infantosi Vannucchi, DVM, PhD

Abstract

Objective – To compare the influence of the obstetrical condition on neonatal respiratory performance, to estimate surfactant synthesis through lecithin and sphingomyelin ratio (L/S) in amniotic fluid of pregnant bitches and correlate the L/S with the respiratory condition during the first hour of life. Design – Prospective randomized study. Setting – University veterinary teaching hospital and private referral centers. Animals – In accordance to the condition at birth, puppies from 25 healthy bitches aged 2–6 years were allocated into: Eutocia Group – EUT (n = 19 neonates and 14 bitches); Dystocia Group – DYS (n = 8 neonates and 5 bitches) and Elective Cesarean Section Group – CS (n = 12 neonates and 11 bitches). Interventions – Amniotic fluid was drawn from amniotic sac and lecithin (L) and sphingomyelin (S) were measured by high performance liquid chromatography to obtain the L/S ratio. Neonatal physical examination was performed at 1, 5, and 60 minutes after delivery, and included the assessment of respiratory rate (RR) and respiration effort (RE). Measurements and Main Results – CS group had significantly lower amniotic L/S ratio when compared to EUT and DYS. There were no significant differences between vaginal groups (EUT versus DYS) in respect to L/S ratios. RE of eutocic neonates improved promptly, while RE of DYS and CS groups improved only 1 hour following birth. Moreover, amniotic L/S ratio positively correlated with RR after 60 minutes. Conclusions – We identified a possible influence of the delivery method on the final surfactant maturation process. Thus, neonates born by elective CS prior to the onset of expulsive uterine contractions should have their respiratory parameters carefully monitored. Additionally, we propose that assessment of respiratory parameters 1 hour following birth can serve as a practical means to indirectly estimate pulmonary maturation (ie, surfactant synthesis) in puppies. (J Vet Emerg Crit Care 2015; 00(0): 1–6) doi: 10.1111/vec.12368 Keywords: cesarean section, lecithin, lung, sphingomyelin, surfactant

Introduction The development of the fetal respiratory system is a complex and dynamic process during growth and lung maturation.1 This process begins early in pregnancy and extends into adulthood.2 Any negative influence on this ´ From the Department of Animal Reproduction (Silva, Lucio, Rodrigues, Veiga, and Vannucchi), School of Veterinary Medicine and Animal Science, University of S˜ao Paulo, S˜ao Paulo, Brazil; and the Department of Nutrition (Portari), Federal University of Triˆangulo Mineiro, Uberaba, Brazil.

Abbreviations

CS DYS EUT HPLC L/S RDS RE RR

cesarean section Dystocia group Eutocia group high performance liquid chromatography lecithin and sphingomyelin ratio respiratory distress syndrome respiratory effort respiratory rate

Supported by FAPESP Grants: 06/52766-3 and 06/59315–7. The authors declare no conflicts of interest. Address correspondence and reprint requests to Dr. Camila Vannucchi, Rua Prof. Orlando Marques de Paiva, 87, Cidade Universit´aria, S˜ao Paulo, SP, 05508-270, Brazil. Email: [email protected] Submitted January 23, 2014; Accepted July 23, 2015.  C Veterinary Emergency and Critical Care Society 2015

developmental process can impair normal pulmonary gas exchange and ultimately threaten the neonate’s life. 1

L. G. Silva et al.

Several studies utilizing different animal species have shown that the absence of a compressive force in the fetal thoracic area while passing through the birth canal reduces the breathing reflex in neonates born by cesarean section (CS).3,4 In fact, babies delivered by elective CS lack the rapid clearance of fetal lung fluid promoted by the physiological hormonal milieu at the onset of spontaneous labor.5 Additionally, infants delivered by elective CS experience transient respiratory distress due to ineffective fluid reabsorption by the lungs; this is not observed in babies born vaginally.3 In dogs, respiratory depression caused by the administration of anesthetic agents during CS contributes to the accumulation of interstitial lung fluid; however, the same phenomenon is not observed in eutocic vaginal deliveries.4 Additionally, puppies born by vaginal delivery display stronger signs of vitality (eg, respiratory and heart rate, muscle tone, irritability reflex, and normal mucous color) compared to newborns delivered via emergency or elective CS.4 Consequently, the delivery method may play an important role in a newborn’s ability to successfully respire during the immediate neonatal period. Therefore, our hypothesis is that the conditions during birth have a direct influence on canine neonatal pulmonary maturation and lung fluid clearance. Surfactant synthesis and secretion are essential for proper postnatal lung adaptation. The main function of a surfactant is to reduce the cohesion properties of water molecules, which decreases the surface tension within the alveoli and prevents atelectasis, mainly during expiration.6–8 Canine surfactant is composed of 90–95% phospholipids, 5–10% proteins, and a low percentage of carbohydrates.9 The main phospholipids are lecithin (L) and sphingomyelin (S), which vary in concentration during gestation. During late pregnancy, concentration of L begins to increase significantly, while the S concentration remains relatively unchanged. The ratio of both phospholipids (L/S ratio) is indicative of proper fetal lung function and development, that is the higher L/S ratios indicate more mature fetal lungs (ie, ample surfactant production). As the lungs develop, a significant amount of surfactant from the fetal lungs accumulates within the amniotic fluid.10 The phospholipid concentration in the amniotic fluid is correlated with the concentration of surfactant isolated from pharyngeal and tracheal aspirates of newborns.11 Thus, amniotic fluid may be a suitable material for the study of the fetal surfactant system and may help predict respiratory disorders during the neonatal period.12,13 The amniotic L/S ratio has previously been established in other animal species. For example, healthy, vaginally born calves present amniotic L/S values at birth of 2.6 ± 0.1, while calves with respiratory distress syndrome (RDS) showed significantly lower values (1.5 ± 0.1).13 Foals that reach full 2

term with no signs of dysmaturity were noted to have an amniotic L/S ratio of 2.7 ± 0.9.10. The absence, deficiency, or inactivation of surfactant causes severe respiratory disorders such as RDS in infants, which is one of the most frequent causes of neonatal underdevelopment.14 In otherwise unassisted babies with RDS, a 25% mortality rate has been reported, and 25% of live babies develop bronchopulmonary dysplasia.8 The evaluation of respiratory performance immediately following birth may be a reliable parameter that may indicate the necessity for instituting neonatal critical care. However, in domestic animals, the relationship between the clinical respiratory status and appropriate lung surfactant synthesis remains undetermined. Consequently, a practical method for estimating the stage of lung maturity in canine neonates is lacking. The development of a diagnostic procedure for puppies at risk for RDS may enable timely provision of specific neonatal care, with the aim of diminishing the high morbidity and mortality rate observed in canine neonates with RDS.15 The aim of this study was to compare the influence of delivery method (vaginal delivery [eutocia and dystocia] or elective cesarean section) with neonatal respiratory performance in the first hour of life. We further aimed to evaluate the L/S ratio in the amniotic fluid of parturient bitches and to establish whether there was a relationship between the L/S and neonatal respiratory performance.

Materials and Methods The present study complied with the ethical requirements for the use of animals in experiments, and was approved by the Bioethics Committee of the Faculty of Veterinary Medicine, University of S˜ao Paulo. Bitches were privately owned and all owners were aware of and consented to the experimental design and procedures in the study. Animals and experimental groups For the purpose of this study, we analyzed a total of 39 newborn puppies of different gender and of 4 breeds (English Bulldog, Chow Chow, Golden Retriever, and Labrador Retriever, equally distributed in each experimental group). According to the condition at birth, puppies born from 25 healthy bitches aged 2–6 years were allocated into: Eutocia Group – EUT (n = 19 neonates from 14 bitches); Dystocia Group – DYS (n = 8 neonates from 5 bitches); and Elective Cesarean Section Group – CS (n = 12 neonates from 11 bitches). As an inclusion criterion, only 1 or 2 puppies were randomly selected from each dam to compose the same experimental group. Regardless of the obstetrical condition, no drugs were used to artificially enhance fetal maturation.  C Veterinary Emergency and Critical Care Society 2015, doi: 10.1111/vec.12368

Study of canine neonatal pulmonary competence

Figure 1: Chromatogram illustrating the retention time of lecithin and sphingomyelin in the amniotic fluid of whelping bitches.

Clinical procedures and sample collection and processing Dystocia was diagnosed whenever the interval between deliveries exceeded 2 hours and was corrected with obstetric manipulation and oxytocina and calcium administration for uterine inertia. Oxytocin was infused slowly to bitches via a catheter placed into the cephalic vein; 100 mL of 0.9% physiological saline, containing 5 IU oxytocin. Calcium gluconateb (16 mg/kg) was also administered intravenously. Elective CS was performed at term following the onset of first stage of labor. Clinical signs of the first stage of labor were vaginal elimination of the mucus plug and behavioral alterations (eg, isolation, restlessness, and lack of appetite). The CS was performed only after bitches’ rectal temperature reached 37.5°C or lower. Bitches in the CS group were anesthetized according to a standardized protocol consisting of sedation with intramuscular acepromazinec (0.02 mg/kg), tramadold (2 mg/kg), or morphinee (0.3 mg/kg). Anesthesia was induced with slow intravenous administration of propofolf (1 mg/kg) and epidural blockage in the lumbosacral intervertebral space using a combination of lidocaine chlorideg (2 mg/kg), bupivacaine chlorideh (0.5 mg/kg), and fentanyli (3 ␮g/kg). Amniotic fluid was collected directly from the amniotic sac soon after its protrusion through the birth canal during the second stage of labor in EUT and DYS groups; or during hysterotomy in CS group (before placental detachment). Amniotic fluid was collected with the use of a 10 mL syringe and a sterile needle and stored in plastic tubes at –20°C until analysis of the L/S. The amniotic fluid contaminated with meconium or blood was discarded. Immediately following birth, the nasal surface of each puppy was cleaned with soft towels and the amniotic fluid removed with a manual nasal aspirator.j Subsequently, pulmonary auscultation of each puppy was performed at 1, 5, and 60 minutes after delivery. For the purpose of this study, the respiratory rate (RR) and

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respiration effort (RE) was determined for all puppies. For evaluation of the RE, the overall respiratory pattern was assessed via thoracic auscultation, determination of the regularity of inspiratory movements, presence of intercostal retractions, sternal retraction or beat of the nostrils. The RE was given a score of 0 if an irregular frequency and rhythm was noted, or any of the referred respiratory alterations was present. A score of 1 required a regular frequency and rhythm and absence of any respiratory alterations. RE results were reported in relation to the percentage of puppies displaying normal RE (ie, score 1). Analysis of lecithin and sphingomyelin concentrations in the amniotic fluid was carried out by high performance liquid chromatography (HPLC)k with ultraviolet detection on 200 nm. Lipids were extracted from amniotic fluid by a previously described method.16 The HPLC separation was done on Luna C18 column (5 ␮m, 250 mm x 4.6 mm i.d., Phenomenex) with mobile phase of acetonitrile/chloroform/water (65:21:14, v:v:v) delivered at 2 mL/min isocratically. The peaks were recorded on CR8A Chromatopac integrator and identified by comparison with standards of lecithin and sphingomyelin derived in the same manner. The quantification was performed using the areas of external standards. Thus, L/S ratio was calculated by dividing lecithin concentration by spingomyelin concentration.

Statistical analyses Values were compared using a repeated measures ANOVA. The effect of obstetrical condition groups (EUT versus DYS versus CS) was evaluated using parametric and nonparametric tests, according to data distribution and variance homogeneity of each variable. The Tukey Range test was used for multiple comparisons. Results are reported as untransformed means ± SEM. Spearman correlation test was used to determine the correlation between RR, RE, and L/S ratio. All values were considered significant at P < 0.05.

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L. G. Silva et al.

Table 1: Mean and SEM of the respiratory rate at 0, 5, and 60 minutes after birth in the eutocia (EUT), dystocia (DYS), and cesarean section (CS) groups

EUT (n = 19) DYS (n = 08) CS (n = 12)

1 min

5 min

60 min

35 ± 2.2B 33 ± 4.1 28 ± 1.9

40 ± 0.8AB 34 ± 2.0 36 ± 3.5

43 ± 9.9Aa 35 ± 2.2ab 32 ± 4.1b

Indicates a significant difference at the same time point (P < 0.05). A,B Indicates a significant difference in the same group (P < 0.05). a,b

Table 2: Percentage (%) of puppies presenting a RE score of 1 (regular respiratory effort) at 0, 5, and 60 minutes after birth in the eutocia (EUT), dystocia (DYS), and cesarean section (CS) groups

1 min EUT (n = 19) DYS (n = 08) CS (n = 12) A,B

B

26.32 12.5B 33.33B

5 min

60 min

A

77.78 37.5AB 58.33AB

94.12A 87.5A 90.0A

Indicates a significant difference in the same group (P < 0.05).

Results A total of 39 samples of amniotic fluid were analyzed for L/E ratio, one from each neonate. The quantitative analysis of amniotic lecithin and sphingomyelin through the HPLC method was easy to perform. A major component eluted at 2.8 minutes corresponding to the retention time of lecithin and a minor component eluted at 3.9 minutes corresponding to the sphingomyelin peak (Figure 1). Puppies born from CS had significantly lower amniotic L/S ratio (3.62 ± 0.44) in comparison to both EUT (7.29 ± 0.81) and DYS (8.54 ± 1.23). Also, no significant differences were observed between vaginal births (EUT and DYS groups). CS neonates had significantly lower RR values at 60 minutes after birth, when compared to the EUT group (Table 1). RE improved satisfactorily within all groups (Table 2). Regardless of the experimental group, up to 85% of all newborns presented regular RE at 60 minutes of life, with regular inspiratory movements and absence of intercostal retractions, sternal retraction or beat of the nostrils (Table 2). During lung auscultation, vesicular respiratory pattern predominated with discrete areas of inspiratory crepitation and rare stridor in caudal lobes. Considering the moment of evaluation, no significant differences on RE values were verified amongst groups (Table 2). However, the EUT group had an increase in RE at 5 minutes in comparison to RE measurements at birth (Table 2). Additionally, DYS and CS groups showed an increase in the percentage of puppies with RE score 1 only at 60 minutes of life (Table 2). Therefore, postnatal respiratory adaptation occurred earlier in the EUT 4

group compared to other groups. In regards to the correlation analyses, amniotic L/S ratio correlated positively (r = 0.35; P = 0.03) with the respiratory rate (RR) at 60 minutes after birth.

Discussion In the present study, the L/S ratio in the CS group was significantly lower than that in both vaginal groups (EUT and DYS), indicating a possible influence of the delivery method on final surfactant maturation in dogs. In late pregnancy, fetal cortisol secretion stimulates surfactant production, activates hepatic, and gastric enzymes, and promotes overall final fetal maturation.11 During labor, surfactant secretion is stimulated while lung fluid synthesis is decreased and absorption is increased.13 Such events are mediated by an increase in fetal catecholamines in response to fetal membrane rupture and the onset of the second stage of labor (expulsive uterine contractions).17 In fact, term babies born through elective CS are reported to have lower catecholamine concentrations and increased frequency of respiratory alterations.17 Therefore, performing a cesarean section prior to the onset of uterine contractions likely prevents the rise in fetal catecholamine concentrations and, ultimately, blunts the final stimulus for lung maturation. It is interesting to note that our measurements of amniotic L/S in eutocic puppies (7.29 ± 3.55) were higher than those in otherwise healthy calves (2.6 ± 0.1) and foals (2.7 ± 0.9).10,13 Therefore, we can infer that there are likely important species differences in the process of surfactant synthesis in the peri-natal period. In other words, fetal pulmonary maturation varies amongst ruminants, horses and dogs, representing a possible evolutionary feature. In the final stage of fetal lung development (the alveolar stage) is achieved in ruminants and horses during gestation, while in dogs, lung development continues during the postnatal period, when there is significant increase in the number of alveoli.18,19 Hence, it is possible that the high amniotic L/S ratio verified in the present study can be explained by a compensatory mechanism for the ensuing lung development in dogs and thereby ensuring neonatal survival in the extra-utero environment. Amniotic L/S ratios from all neonates positively correlated with RR only at 60 minutes after delivery. Puppies born under adverse obstetrical conditions slowly recover from the metabolic imbalance and the respiratory components of acidosis are not corrected in the first hour of life, regardless of the obstetric condition.20 Surfactant biotransformation can be affected by body temperature, blood pH, and perfusion, and it is impaired by hypovolemia, hypoxemia, and acidosis.8 Hypoxia severely affects the function of type II pneumocytes, can alter the  C Veterinary Emergency and Critical Care Society 2015, doi: 10.1111/vec.12368

Study of canine neonatal pulmonary competence

local synthesis of surfactant and can ultimately compromise the ability of the lungs to exchange gases.21 The respiratory effort of the EUT group improved promptly, with up to 75% of neonates producing regular RE at 5 minutes after birth. Conversely, in the DYS and CS groups, proper respiratory function occurred at the one hour evaluation point. Regarding the obstetrical condition, we have previously shown in dogs that dystocia compromises neonatal vitality (ie, Apgar score) as a consequence of persistent metabolic acidosis.22 Therefore, in the DYS group, we can infer that respiratory alterations were due to prolonged exposure to uterine contractions, which may lead to intrapartum hypoxia, metabolic acidosis and, consequently, impaired surfactant synthesis and pulmonary clearance. Puppies born by CS present with bradycardia and respiratory depression.23 Elective CS is associated with an increase in respiratory disorders even in full-term human neonates.24 In the CS group, the absence of compressive stimulation from the vaginal canal combined with the overall depression caused by anesthesia may both be responsible for the instability and greater accumulation of fluid in the lung interstitium (ie, pulmonary clearance). In light of our analysis of the correlation between the amniotic L/S ratio and RR at 60 minutes after birth, we propose that the RR of puppies should be assessed after 1 hour of whelping. This assessment may serve as a practical method to indirectly estimate pulmonary capacity to synthesize surfactant and ultimately achieve full lung maturation. Additionally, the amniotic L/S ratio can be used as a surrogate marker of respiratory performance within the first hour after birth in canine neonates, regardless of the obstetric condition. Consequently, newborns with low L/S ratios could experience some compromise in respiratory function and require intensive care during the immediate neonatal period. The development of a rapid and practical method for determining L/S ratios is a future goal. In conclusion, the obstetrical condition can influence neonatal respiratory performance during the first hour of life. Puppies born by elective CS prior to the onset of expulsive uterine contractions had significantly lower amniotic L/S ratios compared to those delivered by vaginal birth, which indicates incomplete pulmonary maturation. Therefore, the respiratory performance of puppies born by CS should be carefully monitored, and a more vigilant clinical follow-up should be performed to guarantee satisfactory neonatal health. Additionally, the correlation between amniotic L/S ratios and RR at 60 minutes after delivery suggests that assessment of the RR of puppies may be a practical method for indirectly estimating pulmonary capacity to synthesize surfactant.  C Veterinary Emergency and Critical Care Society 2015, doi: 10.1111/vec.12368

Footnotes a b c d e f g h i j k

Orastina, Merck Animal Health, Summit, NJ. ´ Merck Animal Health, Summit, NJ. Glucafos, Acepran 0,2%, Vetnil, Louveira, SP, Brazil. Tramal, Pfizer, S˜ao Paulo, SP, Brazil. Dimorf, Crist´alia, S˜ao Paulo, SP, Brazil. Propofol, Biosintetica, S˜ao Paulo, SP, Brazil. Lidojet 2%, Uni˜ao Qu´ımica, Embu-Guac¸u, SP, Brazil. Bupivaca´ına 0,5%, Crist´alia, S˜ao Paulo, SP, Brazil. Fentanil, Janssem-Cilag Farmaceutica N.V., S˜ao Jos´e dos Campos, SP, Brazil. Nasal Aspirator, Lillo do Brasil Ltda., Campo Grande, RJ, Brazil. Ultimate 3000 HPLC, Dionex Brasil, S˜ao Paulo, SP, Brazil.

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