*Article* **How Stressful Is Maternity? Study about Cortisol and Dehydroepiandrosterone-Sulfate Coat and Claws Concentrations in Female Dogs from Mating to 60 Days Post-Partum**

**Jasmine Fusi 1, Tanja Peric 2, Monica Probo 1,\*, Alessio Cotticelli 2, Massimo Faustini <sup>1</sup> and Maria Cristina Veronesi <sup>1</sup>**


**Simple Summary:** Canine pregnancy and post-partum (total duration around 120 days) is a very intense period and could be a trigger for the activation of the Hypothalamic–Pituitary–Adrenal (HPA) axis in female dogs, given the higher allostatic load. To evaluate this activation, the concentrations of Cortisol (C) and Dehydroepiandrosterone-sulfate (DHEA-S), final products of the HPA axis, were detected in the coat and claws of 15 Dobermann Pinscher female dogs, with monthly collections from mating until the end of weaning. The C concentrations, both in the coat and claws, showed a significant trend of increase from mating until 60 days post-partum. In both matrices, the DHEA-S changes were not significant. Maternal parity and litter size did not play a significant influence on the concentrations of C and DHEA-S in both matrices. The results of the present study seem to depict maternity as a main activator of the HPA axis that, in turn, leads to the secretion of C. This is probably due to the increased allostatic load for the mothers, although it is not possible to discern the precise role of the multiple processes characterizing this period (uterine involution, lactation, nursing and grooming of the puppies).

**Abstract:** In dogs, the phase from mating to the end of weaning lasts about 120 days and encompasses many aspects that, interacting, contribute to increase the allostatic load. The coat and claws, useful for long-term change assessments, have the advantage of being collectable without invasiveness. In the present study, the Cortisol (C) and Dehydroepiandrosterone-sulfate (DHEA-S) concentration monthly changes in the coat and claws were studied in female dogs from mating to the end of weaning to assess Hypothalamic–Pituitary–Adrenal (HPA) axis activation during pregnancy and the post-partum period. The results from 15 Dobermann Pinscher female dogs showed a trend of increase of the coat C from mating to 60 days post-partum, with significant changes between mating and parturition-60 days post-partum (*p* < 0.01) and between the 30-day pregnancy diagnosis (PD) and 30–60 days post-partum (*p* < 0.05). The claws C trend showed significant increases between mating and 30–60 days post-partum (*p* < 0.05) and between the PD and 60 days post-partum (*p* < 0.01). DHEA-S in both matrices showed non-significant changes. The results suggest that maternity could play a pivotal role in the HPA axis activation, with a subsequent chronic secretion of C determining an increase in the allostatic load in the mothers. Neither maternal parity nor litter size played a significant role in the accumulation of C and DHEA-S in both matrices.

**Keywords:** dog; maternity; cortisol; dehydroepiandrosterone-sulfate; HPA axis

**Citation:** Fusi, J.; Peric, T.; Probo, M.; Cotticelli, A.; Faustini, M.; Veronesi, M.C. How Stressful Is Maternity? Study about Cortisol and Dehydroepiandrosterone-Sulfate Coat and Claws Concentrations in Female Dogs from Mating to 60 Days Post-Partum. *Animals* **2021**, *11*, 1632. https://doi.org/10.3390/ani11061632

Academic Editors: Angelo Gazzano and Asahi Ogi

Received: 27 April 2021 Accepted: 28 May 2021 Published: 31 May 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

In contrast to horse and cow newborns, newborn dog puppies are considered altricial and strictly dependent on the mother to survive until the end of weaning [1]. Canine pregnancies last about 63 ± 1 days after ovulation [2,3], and, at parturition, the phase of lactation, nursing and puppies grooming begins. In breeding facilities, the weaning of puppies starts at about 30 days after parturition, ending at 60 days post-partum. In Italy, puppies are usually sold to the new owners not earlier than 60 days of age and usually between 60 and 65 days of age. Therefore, the time elapsing between mating of the female dog and the end of weaning lasts about 120 days: altogether, this phase is complex and encompasses reproductive, metabolic, emotional and behavioral processes, often interacting among them, in turn increasing the allostatic load of the animal. This long-term period deserves scientific interest, as it represents a challenging and possibly very stressful time for the female dog [1,4]. Although many studies have focused on the specific phase of pregnancy, parturition and post-partum, to the authors' knowledge, the whole period from mating until the end of weaning has only rarely been investigated in dogs [5]. Nonetheless, like what happens in horse husbandry, the selection of breeding female dogs is not based on the actual reproductive aptitude but, more often, on their show performances and genetic value. Due to the important role of maternal aptitude in puppies rearing, a more accurate selection of breeding female dogs could ameliorate the quality of dog breeding [6], contributing to limit the still too-high percentages of perinatal mortality [7] and conveying more ethical dog breeding. Still, today, it is not uncommon to observe disturbed behavior in periparturient female dogs, possible aggressiveness towards the newborns or even cannibalism, often accompanied by insufficient grooming or nursing of the puppies, thus impairing the successful outcome of litters. Among the diverse causes underlying these troubles, environmental factors, genetics, parity and number of the newborns in a litter were suggested [1,4,6]. Other than that, the role of the Hypothalamic–Pituitary–Adrenal axis (HPA axis) in shaping maternal behavior was presumed in sheep [8,9]. A study on adrenalectomized rats reported that, when the primiparae were injected with high doses of corticosterone, they showed higher levels of maternal care (notably, licking behavior) than the other primiparous rats who received lower doses of corticosterone [9,10]. Additionally, in humans, the HPA axis was reported to exert a role in mother–infant bonding [11]. In gorillas, a correlation between maternal behavior, stress and urinary Cortisol (C) concentrations was found; higher C concentrations were found in stressed mothers, whose infants were often less-cared-for [12]. In dogs, a similar association was presumed; mothers experienced peaks of salivary C after being temporarily separated from their litters, with higher peaks in mothers who displayed the most the typical maternal care behaviors [13]. It is thus evident that the role of stress is of utmost importance when evaluating data about the experience of maternity, both in humans and animals.

Lactation, especially when associated with nursing and grooming, is a challenging physiologic phase that requires a great expenditure of energy and could represent a source of stress in relation to the litter size [14] and to parental efforts. The increased metabolic load and stress are associated with activation of the HPA axis, leading to an increase of circulating C [14]. In a recent study on female cats, reference [14] reported that the highest concentrations of C in the blood were found 4 weeks after parturition, in association with the peak of lactation, suggesting a major effect of lactation and kitten nursing on HPA axis activation. Those authors concluded that measuring the circulating C concentrations could be helpful in deepening the understanding of the reaction of queens through the physiological stresses occurring during lactation.

Other than C, dehydroepiandrosterone (DHEA) and its sulfated form, dehydroepiandrosterone sulfate DHEA-S, are also final products of the activation of the HPA axis. Among multiple actions, DHEA is notably reported to be a neuroactive steroid with antidepressant actions, associated with some types of behaviors like sex recognition and aggressiveness [15–18]. In the literature, some results about the concentrations of circulating DHEA and DHEA-S during pregnancy exist in women [19], cows [20] and, more recently, in killer whales [21]. However, the results are conflicting: in women, a decline of circulating DHEA and DHEA-S during pregnancy was reported; in killer whales, a rise of DHEA-S from early and during mid and late pregnancy in comparison to pre-fertilization and post-partum was detected; lastly, a significant increase of circulating DHEA throughout pregnancy was detected in heifers and cows [19–21].

However, the HPA axis could not be considered as a "closed system", and its activity is modulated by factors such as oxytocin, able to exert an inhibitory action on the HPA axis. A recent study reported that the salivary oxytocin concentrations in female dogs were negatively correlated with excessive sniffing/poking behavior, considered as a possible sign of maternal distress associated with lactation [22]. Moreover, an allostatic theory of oxytocin was reported. According to this theory, the oxytocin system should be able to support the refinement of a physiologic setting to cope with adaptation and survival [23].

Despite most of the studies referring to concentrations of C and DHEA/DHEA-S in the blood or less invasive matrices such as urines, feces and saliva, all those matrices depict only the concentration of a given time point, and their use is therefore not suitable when long-lasting physiological processes have to be investigated because of the need to repeat multiple samplings. To overcome these issues, in the last years, new matrices, like the hair/coat and nails/claws, have been proposed for long-term hormonal studies in humans and animals [24–28] thanks to their characteristic of providing retrospective information about long-term hormone accumulation. Thus, the coat and claws could be suitable matrices for the study of hormonal changes occurring in the long-lasting phase between mating of the female dog to puppies weaning.

Therefore, the aim of the present study was to investigate the possible activation of the maternal HPA axis during the dynamic and challenging phase lasting from mating of the female dog to puppies weaning through the assessment of C and DHEA-S concentrations in the maternal coat and claws monthly collected.

#### **2. Materials and Methods**

#### *2.1. Ethics*

Although coat and claws sampling is a noninvasive procedure, this trial was carried out in accordance with EU Directive 2010/63/EU, and it was approved by the Ethical Committee of the University of Milan (OPBA\_33\_2021). Written informed consent was signed by the owners, giving permission to submit each female dog to elective C-section, to collect coat and claws samples and to allow the record of clinical data for research purposes.

#### *2.2. Animals*

Sixteen purebred Doberman Pinscher, 2nd to 3rd parity female dogs, aged 3–6 years old (mean ± SD: 4.1 ± 1.06), belonging to a single breeder were enrolled in the study. All the female dogs were found to be healthy by a clinical examination and by routine health tests performed by a veterinarian and submitted to the common vaccination and parasite prophylaxes, fed with commercial food and housed in a single kennel with indoor and outdoor spaces. The animals were always handled and managed by the same operator. All the female dogs showed a history of previous normal pregnancies, post-partum and lactation phases. In all of them, an elective cesarean section (ELCS) was performed at the previous whelping, because of the large litter sizes and the consequent risk for dystocia. Of the 16 mated female dogs enrolled in the present study, one was diagnosed as not pregnant at 30 days after ovulation and therefore removed from the study.

#### *2.3. Estrus and Mating*

From the onset of proestrus, all the female dogs were monitored through serial vaginal smears every 48–72 h and through the assay of plasma progesterone concentrations performed every 48 h from the beginning of the signs of cytological estrus. Based on these parameters, all the female dogs were submitted to a single mating, performed with

a male of proven fertility, 48 h after the estimated ovulation, when progesterone plasma concentrations ranged between 4 and 10 ng/ml [29]. Each female dog was mated with a different healthy male of proven fertility. At the time of mating, the BCS of each female dog was assessed on a scale of 5.

#### *2.4. Pregnancy, Caesarean Section, Newborn Puppies and Post-Partum Management*

At 30 days after the estimated ovulation, pregnancy was checked by ultrasonographic examination, and the pregnant female dogs were submitted to measurement of the inner chorionic cavity (ICC) for the first calculation of the parturition date [30], while the nonpregnant female dogs were excluded from the study.

At 45 days after ovulation, a second ultrasonographic evaluation was performed to assess the normal course of pregnancy, the correct development and wellbeing of the fetuses and to measure the biparietal (BP) diameter for an additional calculation of the parturition date [30].

Given the reported higher risk for dystocia when the litter size counts more than 9 puppies [31], and because all the enrolled Dobermann Pinscher female dogs carried pregnancies with more than 9 fetuses, they were all scheduled for ELCS at term of pregnancy. The day for performing ELCS was scheduled on the basis of the concordance of several parameters, as previously reported [32–36]: date of ovulation, based on the measurement of plasma progesterone concentrations, ICC and BP. Moreover, during the last days of pregnancy, before the expected parturition date, the female dogs were checked daily to assess their clinical conditions and for ultrasonographic evaluations of the fetal wellbeing. Other than this, plasma progesterone concentrations were assessed to identify the pre-parturient decrease [37]. The ELCSs were performed only when plasma progesterone concentrations were <2 ng/ml.

For all the ELCSs, the same anesthetic and surgical protocols, aimed to minimize the possible negative effects on newborn viability and mothers' wellbeing, were performed, as *previously* reported [32,35,36].

Two expert neonatologists took care of the newborns as soon as they were extracted from the uterus, providing professional assistance. Within 5 minutes after birth, newborn viability was assessed by an Apgar score, and the puppies were classified as viable when the Apgar score was ≥7 [38]. The newborns were also evaluated for the absence of gross physical malformations or defects.

According to the viability classification, all the subjects were submitted to routine neonatal care or different degrees of neonatal assistance, as previously reported [38]. Birthweight, gender and litter size were also recorded.

Mothers and litters were discharged when female dogs were awake and showed normal behavior toward the puppies and after having verified the presence of normal mammary secretions.

A daily follow-up to update the general conditions was provided by the breeder from the day after parturition. Maternal and litter clinical data were daily recorded, together with the puppies' bodyweight until 60 days after parturition. In addition, at 1 and 2 weeks, and at 30 and 60 days after parturition, clinical examinations were scheduled. At 60 days after parturition, maternal BCS was reassessed.

#### *2.5. Samples Collection*

Coat samples were collected by using a razor (TN2300 Nomad, Rowenta® spa, Milan, Italy), allowing to shave an area of about 5 cm2 from the dorsal surface of the right forearm until the level of the skin (coat samples). The tips of the claws were collected from all the digits of both forearms with a claw clipper (C135, Candure® Services LTD, Leicester, UK) and stored (claws samples). Coat and claws samples were placed in separate paper envelopes, labeled with a univocal code and stored at room temperature until the analysis.

The first sample collection was performed the day of mating. At the time of the pregnancy diagnosis, the second coat and claws sampling was performed, collecting only the regrown area.

At parturition, the third sample collection was performed. This collection did not always respect the 30 days of interval previously scheduled for the other samplings. Given that parturition occurs at about 60–62 days after ovulation, indeed, the interval of time between the second sampling and the one at parturition could have been 30 + a few days.

In all the cases, before anesthesia induction, the regrown coat and claws were collected and stored as reported above.

At 30 and 60 days post-partum, the regrown coat and claws were further collected and stored as reported above.

At every sampling time, after the collection, the razor and the claw clipper were disinfected with a 70% alcohol solution [39].

#### *2.6. Hormone Analysis*

Coat strands and claws were washed in 3-mL isopropanol to ensure the removal of any steroids on their surface. Coat and claws steroids were extracted with methanol and measured by radioimmunoassay (RIA). The concentrations of Cortisol and Dehydroepiandrosterone sulphate (DHEA-S) were measured using a solid-phase microtiter RIA. In brief, a 96-well microtiter plate (OptiPlate; PerkinElmer Life Sciences Inc., Zaventem, Belgium) was coated with goat anti-rabbit γ-globulin serum diluted 1:1.000 in 0.15-mM sodium acetate buffer (pH 9) and incubated overnight at 4 ◦C. The plate was then washed twice with RIA buffer (pH 7.5) and incubated overnight at 4 ◦C with 200 μL of the antibody serum diluted 1:20,000 for Cortisol and1:800 for DHEA-S. The cross-reactivities of the anti-Cortisol antibody with other steroids were as follows: Cortisol 100%, cortisone 4.3%, corticosterone 2.8%, 11-deoxycorticosterone 0.7%, 17-hydroxyprogesterone 0.6%, dexamethasone 0.1%, progesterone, 17-hydroxypregnenolone, DHEA-S, androsterone sulphate and pregnenolone < 0.01%. The cross-reactivities of the anti-DHEA-S antibody with other steroids were as follows: DHEA-S, 100%; androstenedione, 0.2%; DHEA, <0.01%; androsterone, <0.01% and testosterone, <0.01%. After washing the plate with RIA buffer, the standards (5–200 pg/well), the quality control extract, the test extracts and the tracer (hydrocortisone {Cortisol [1,2,6,7-3H (N)]-}, DHEA-S [1,2,6,7-3H (N)] were added, and the plate was incubated overnight at 4 ◦C. The bound hormone was separated from the free hormone by decanting and washing the wells in RIA buffer. After the addition of 200 μL of scintillation cocktail, the plate was counted on a β-counter (Top-Count; PerkinElmer Life Sciences Inc.).

The intra- and inter-assay coefficients of variation were 3.7 and 10.1% and 3.2 and 11.8% for Cortisol and DHEA-S, respectively. The sensitivities of the assays were 1.23 pg/well and 0.54 pg/well for Cortisol and DHEA-S, respectively.

#### *2.7. Statistical Analysis*

A Shapiro–Wilk test was used to verify the normal distribution of data, followed by an ANOVA and post-hoc test, to assess the possible effects of the sampling time, litter size, maternal age and parity on the C and DHEA-S concentrations in the coat and claw samples. Statistical significance was set for *p* < 0.05 (JASP®, ver. 9 for Windows platform). The Pearson correlation test was used to assess the possible correlations between the two matrices for each hormone.

#### **3. Results**

#### *3.1. Clinical Findings*

The 15 pregnant female dogs (BCS at mating, mean ± SD: 3.0 ± 0.00) showed normal courses of pregnancy and, because all of them carried more than nine fetuses, were submitted to the ELCS at 60–63 (mean ± SD: 61.2 ± 1.09) days after ovulation, providing a total of 15 litters with a total of 163 puppies (three stillborn). The litter sizes ranged

between 10 and 13, with a mean ± SD of 10.9 ± 1.13. Seventy-five males and 85 females, mean ± SD birthweight: 427.7 ± 87.82 g, mean ± SD Apgar score: 8.9 ± 0.66, were enrolled. Consequently, all the 15 female dogs were followed for the whole period elapsing from mating until the end of weaning at 60 days post-partum, when the BCS was 2.5 ± 0.24 (mean ± SD).

#### *3.2. Coat and Claws C and DHEA-S Concentrations*

The concentrations (mean ± SD) of C and DHEA-S in the coat and claws from mating to 60 days post-partum in the 15 female dogs enrolled in the study are reported in Figures 1 and 2, respectively.

**Figure 1.** Concentrations (mean ± SD) of C in the coats and claws from mating to 60 days postpartum in the 15 female dogs. A,B *p* < 0.05; a,b *p* < 0.01 denote within-row significance. \* *p* < 0.05 denotes within-row significance.

The concentrations of C in the coats showed a trend of increase from mating to 60 days post-partum, with significant changes between mating and parturition-60 days post-partum and between the pregnancy diagnosis and 30–60 days post-partum. In the claws, the trend of C was very similar to the one observed in the coats, with significant increases between mating and 30–60 days post-partum and between pregnancy diagnosis and 60 days post-partum.

The Pearson correlation test showed a significant positive correlation (r = 0.277; *p* < 0.05) between the coat and claw C concentrations.

**Figure 2.** Concentrations (mean ± SD) of DHEA-S in the coats and claws from mating to 60 days post-partum in the 15 female dogs.

The concentrations of DHEA-S in both the coats and claws showed the same trend of increase between mating and all the subsequent sampling times but without significant changes.

The statistical analysis showed the absence of significant effects of the maternal age and litter size on the C and DHEA-S concentrations in the coats and claws, and no significant correlations of DHEA-S between the two matrices was found.

#### **4. Discussion**

To the authors' knowledge, this was the first study reporting the concentrations of C and DHEA-S- in the coat and claws of female dogs from mating to 60 days postpartum, providing new information about long-term hormonal changes during gestation and the post-partum period in dogs, a topic that needs to be further investigated also in humans [40].

Some studies previously demonstrated the usefulness of a single coat sample for the study of C [25] and DHEA-S [41] concentrations in dogs. However, the present study offered the first evidence that, using the shave–re-shave method [27,42–44] after the first sampling, it is possible to collect only the regrown matrices, providing an optimal tool to investigate the long-term hormonal variations in dogs, as previously reported for other species [45–47]. However, in valuable dogs, the owners often refuse to shave the hair, even in a small area, limiting the use of the coat as a matrix of study in many instances. A couple of recent studies [26,28] showed the usefulness of the claws as an alternative to the coat for the same long-term studies in newborn puppies. For these reasons, in the present study, both the coat and the claws were collected with the shave—re-shave and clip—re-clip method for the longitudinal analysis of C and DHEA-S long-term changes.

The 30-day collection interval was designed to address several targets. Firstly, it reduced, at the minimum, the number of samplings to restrict the possible disturbances for the pregnant/lactating female dogs. Secondly, this timing fits with the most important milestones of the reproductive process management in female dogs. Thirdly, it allowed the regrowth of a suitable amount of sample for the analysis. Fourthly, it was reasonable for identifying and studying long-term hormonal changes. About the area of the body chosen for the coat collection, the right forearm was used because it is shaved for blood collection and other clinical purposes.

About the enrollment criteria of the female dogs, in the present study, only one breed was selected to avoid possible genetic differences in HPA axis activation [48]. However, even within the same breed, some individual peculiarities in HPA axis activation could be presumed. Moreover, to avoid possible differences in the managerial, nutritional and environmental factors that could influence HPA axis activation, all the female dogs belonged to the same breeder and were managed by the same operator. Other than this, selecting a single breed and the consequent homogeneous litter size, together with similar parity, allowed an additional reduction of the possible variables in HPA axis activation. The choice of enrolling only female dogs of second and third parity was based on the hypothesized possible influence of the maternal experience in HPA axis activation, as reported for maternal behavior [6]. The exclusive inclusion of female dogs scheduled for ELCS, in addition, concurred to reduce the other possible variables related to the effects of different types of delivery on HPA axis activation, as previously observed [49].

About C, the trend of increase from mating to 60 days post-partum observed in both the coat and claws suggested a gradual activation of the maternal HPA axis during the delicate phase of pregnancy and post-partum in female dogs. The finding of the highest concentration of C at 60 days post-partum is intriguing and could be related to the complex interaction of multiple factors, such as uterine involution, lactation and, also, nursing and grooming of the puppies, therefore addressing not only metabolic and physiologic phenomena but, also, emotional and behavioral changes for female dogs. All these factors, in turn, play a role in the increase of the allostatic load for female dogs. Although it is not possible to discern the single contribution of each one of these processes on the activation of the HPA axis, it is possible to suppose that lactation and care of the puppies could promote a continuous stimulation on the HPA axis, leading to a chronic secretion of C, as evidenced by its long-term, retrospective accumulation in the coat and claws. In the present study, all the female dogs displayed normal maternal behavior in the present and previous pregnancies, so that the observed HPA axis activation could be supposed to reflect a "normal" process related to maternity in dogs. However, it is not possible to exclude that the energy expenditure needed for all of the processes above seen could stimulate C secretion in order to mobilize the depots, as supposed in reference [14], given the higher concentrations reported at the peak of lactation.

The statistical analysis did not show an effect of maternal parity on C coat and claw concentrations. This could be due to very limited differences in the parity of the enrolled female dogs. However, in the bottlenose dolphin, the profiles of C concentrations during pregnancy were not affected by age or parity [50], and the same results were reported for killer whales [21]. In a recent study, the authors of reference [14] reported that the maternal experience did not represent an influencing factor on C concentrations in the blood of queens at 4 weeks after parturition, while it was influenced by the size of the litter (higher in queens with up to three kittens than in queens with more than three kittens). In the present study, the litter size did not influence the C concentrations in the coats and claws. It should be noted that, in the enrolled female dogs, no extreme litter sizes were observed, and the litter sizes were rather homogeneous, with the number of puppies per litter ranging between 10 and 13. Moreover, about the possible effect played by the puppies on the maternal HPA axis activation, in the present study, excluding the three stillborn, the puppies were all healthy and viable at birth and for all the subsequent periods of observation, showing normal growth and development in the first 60 days of age.

Therefore, taking all these considerations together, the results of the present study should be considered representative of the "normal" conditions of both mothers and litters. Hence, it could be interesting to verify the possible differences of long-term maternal HPA axis activation according to breed, first parity, extreme litters, pathological situations of mothers and/or puppies, etc.

Beside the C concentrations in the coats and claws, it was unforeseen to observe no significant changes in the concentrations of DHEA-S in both matrices. Even if conflicting results are reported in women, cows and killer whales, the role of DHEA as a neuroactive steroid is known to be involved in behavioral changes [15–18], in stress responses [15–17] and in the stimulation of lobuloalveolar mammary gland development in rats [51]. Like C, the concentrations of DHEA-S in the coat and claws were not influenced by the maternal parity or litter size.

In a study by Fusi and colleagues [26], both the C and DHEA(S) concentrations measured in the claws collected from puppies since birth to 60 days of age were found to be higher in claws collected at birth than in the subsequent sampling times, with a trend of decreasing. Therefore, because no differences related to DHEA-S claw concentrations were found in the present study in female dogs, it could be supposed that DHEA-S is mainly synthesized by the fetus/newborn. This hypothesis could also be reinforced by the extremely different concentrations found in the present study in the claws of female dogs compared to the study in reference [26] in the claws of puppies when the same time from parturition to 60 days post-partum was concerned. In fact, in the study from reference [26], the DHEA(S) claw concentrations were about 50-fold higher in puppies at parturition, about 20-fold higher at 30 days post-partum and about 15-fold higher at 60 days post-partum, as compared to the female dogs investigated in the present study.

Additionally, about C, higher concentrations were found in the puppies of the same study when compared to the concentrations found in the female dogs of the present one, especially at parturition (about six-fold).

These comparisons seem to suggest that, in dogs, the fetal HPA axis activation plays an important role, especially around the time of parturition.

The results of the present study offer new insights in the role of maternity on the allostatic load of female dogs. This topic is of maximum importance, given the influence of the allostatic load on the health of an organism, as reported in humans [52], and suggests that, although it is a physiologic event, the periods of pregnancy and, especially, postpartum should be considered as possible stressors for female dogs.

#### **5. Conclusions**

In conclusion, the higher concentration of C in the coat and claws found at 60 days post-partum than those found at mating seems to suggest that a complex interplay among the physiologic, emotional and behavioral events in the post-partum period occurs in female dogs. Maternity seems to play a pivotal role in the HPA axis activation in female dogs, with a subsequent chronic secretion of C and long-term accumulation in the coat and claws. No significant differences were found in the DHEA-S concentrations in the different sampling times, and neither maternal parity nor litter size played a significant role in the accumulation of C and DHEA-S in both matrices.

**Author Contributions:** Conceptualization, M.C.V. and J.F.; methodology, T.P. and M.P.; software, M.F.; validation, A.C. and T.P.; formal analysis, M.F.; investigation, J.F. and M.P.; data curation, M.C.V. and J.F.; writing—original draft preparation, M.P. and J.F.; writing—review and editing, M.C.V. and J.F. and supervision, M.C.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Università degli Studi di Milano (UNIMI), grant number LINEA2\_CVERO\_2019\_AA.

**Institutional Review Board Statement:** This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Università degli Studi di Milano-Organismo Preposto al Benessere degli Animali (OPBA) (protocol code OPBA\_33\_2021).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data are available upon request from the authors.

**Acknowledgments:** The authors are grateful to the breeder Chiara Campazzo for her support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


#### *Article* **Blood Biomarker Profile Alterations in Newborn Canines: Effect of the Mother***-* **s Weight**

**Brenda Reyes-Sotelo 1, Daniel Mota-Rojas 2,\*, Patricia Mora-Medina 3, Asahi Ogi 4, Chiara Mariti 4, Adriana Olmos-Hernández 5, Julio Martínez-Burnes 6, Ismael Hernández-Ávalos 3, Jose Sánchez-Millán <sup>3</sup> and Angelo Gazzano <sup>4</sup>**


**Simple Summary:** Morphological variability in canines is associated with the mother's size and weight, which likely affects the birth weight of the puppies and their metabolic status. Identifying physio-metabolic alterations in the blood from the umbilical vein to evaluate the concentration of gases, glucose, lactate, calcium, hematocrit levels, and blood pH of newborn puppies will make it possible to determine the risk of complications due to intrauterine asphyxia. The objective of this study is to evaluate the effect of the mother's weight on the weight of liveborn and stillborn puppies during spontaneous births and the neonates' blood physiological alterations during the first minute of life. The above allowed us to identify the physio-metabolic maladjustments that newborn puppies suffer from and to determine the risk of asphyxia according to the weight category of the mothers. Results suggest that if the weight of the bitch is >16.1 kg in eutocic births, there is a higher risk of intrapartum physiological alterations and death. The results of this study allowed us to identify that the weight of dams before birth determines the weight of the puppies at birth, though there is a wide range in birth weights due to the ample morphological variability characteristics of this species.

**Abstract:** This study aims to determine the effect of the weight of bitches on liveborn and stillbirth puppies from eutocic births, and physiological blood alterations during the first minute postpartum. A total of 52 female dogs were evaluated and distributed in four categories: C1 (4.0–8.0 kg, n = 19), C2 (8.1–16.0 kg, n = 16), C3 (16.1–32.0 kg, n = 11), and C4 (32.1–35.8 kg, n = 6). The dams produced 225 liveborn puppies and 47 were classified as stillbirth type II. Blood samples were taken from the umbilical vein to evaluate the concentration of gases, glucose, lactate, calcium, hematocrit levels, and blood pH. The liveborn puppies in C2, C3, and C4 had more evident physiological alterations (hypercapnia, acidosis) than those in C1 (*p* < 0.05). These signs indicate a process of transitory asphyxiation. The stillborn pups in all four categories had higher weights than their liveborn littermates. C3 and C4 had the highest mean weights (419.86 and 433.79 g, respectively) and mortality rates (C3 = 20.58%, C4 = 24.58%). Results suggest that if the weight of the bitch is >16.1 kg in eutocic births, there is a higher risk of intrapartum physiological alterations and death. The results of this study allowed us to identify that the weight of dams before birth determines the weight of the puppies at birth.

**Keywords:** animal perinatology; asphyxia; physiological blood profile; puppy welfare; stillbirth

**Citation:** Reyes-Sotelo, B.; Mota-Rojas, D.; Mora-Medina, P.; Ogi, A.; Mariti, C.; Olmos-Hernández, A.; Martínez-Burnes, J.; Hernández-Ávalos, I.; Sánchez-Millán, J.; Gazzano, A. Blood Biomarker Profile Alterations in Newborn Canines: Effect of the Mother s Weight. *Animals* **2021**, *11*, 2307. https://doi.org/10.3390/ani11082307

Academic Editor: Michael Lindinger

Received: 29 June 2021 Accepted: 28 July 2021 Published: 5 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Mortality in dogs during the neonatal period has been estimated to reach 40% [1]. Deaths may occur in the uterus, during expulsion, immediately postpartum, or during the first weeks of life [2–4], but the highest number of stillbirths occurs during birth [5] and the first 7 days of life [6]. Approximately 60% of these deaths are associated with intrapartum asphyxiation [7] caused by dystocic deliveries [5,6,8]. Asphyxia during the birthing process also negatively impacts the newborns' adaptation to extrauterine life [9] by limiting their viability and vitality [6,10–13]. A high neurologic morbidity increases the risk of neonatal mortality [14]. The birthing process is the most critical phase for newborns [15] because the transition from fetus to neonate involves physiological, biochemical, and anatomical changes accompanied by flows of hormones that trigger the respiratory function, vascular changes, and the activation of energy metabolism [16,17]; additionally, the maternal behavior is critical for the parturition to take place in favorable conditions for the newborn puppy [18–21]. Studies of dogs have reported that a certain level of transitory asphyxiation occurs during delivery. Though this is normal, it produces hypercapnia and transitory acidosis in puppies [22,23]. If these conditions persist, they will alter gas exchange [24], delay the onset of respiration, and generate metabolic acidosis in newborns [25]. The challenges of the birthing process, together with these risk factors can determine the proportion of the liveborn (LP) vs. stillbirth (SB) puppies and the viability of the former [26–28]. Morphological variability in canines is associated with the mother's size and weight [23], for these likely affect the birth weight of the puppies [26,28–30] and their metabolic status. In both veterinary and human perinatology, analyzing blood gases and metabolites has emerged as an important tool for evaluating newborns [13,31], but reports on dogs are scarce. Studying physiological indicators provides crucial information and allows researchers to estimate variations in oxygenation levels, metabolic profiles, and the acid–base balance [32] that help determine the level of fetal hypoxia suffered during birth. Gasometry allows the monitoring of the respiratory function by measuring the concentration of certain gases (pO2, O2 saturation (SaO2), pCO2) and blood pH [11,12,33–36] and the evaluation of the acid-base balance—to estimate the newborns' metabolic status [13,33,37,38]. Variations in metabolite levels, including lactate, play an important role in metabolic acidosis [39,40] associated with hypoxic events [1,41], high blood glucose levels [36], and a general compensatory metabolism marked by excess base and bicarbonate in the blood [25]. Identifying physio-metabolic alterations in the blood of newborn puppies will make it possible to determine the risk of complications due to intrauterine asphyxia. However, evidence on hypoxia in canines, its effects, and its relations to the mother's weight as a risk factor is scant or has not been fully evaluated. Thus, the objective of this study was to evaluate the effect of the mother's weight on the weight of the LP and SB puppies during spontaneous births and the neonates' blood physio-metabolic alterations during the first minute of life. The above allowed us to identify the physiological maladjustments that newborn puppies suffer and determine the risk of asphyxia according to the weight category of the mothers.

#### **2. Materials and Methods**

#### *2.1. Infrastructure*

A network of 10 veterinary clinics in Mexico City was organized to recruit pregnant dogs. Prenatal control was performed from day 25 of pregnancy to 24 h postpartum.

#### *2.2. Study Population*

A total of 52 young multiparous bitches (2–4 births) were recruited. The inclusion criteria were: (i) clinically healthy dogs; (ii) valid vaccination/deworming record; (iii) fed a commercial formula; (iv) no history of reproductive problems; (v) radiographic and ultrasonographic evaluations to show they were apt for natural births. The exclusion criteria were: (i) primiparous females; (ii) bitches with a history of dystocia or pyometra; (iii)

previous type I SB puppies; (iv) malformed fetuses; (v) use of birth inducers or accelerators; (vi) bitches with body condition 8 or 9 (obese) as per the WSAVA scale [42]; (vii) extremely aggressive behavior. Brachycephalic and large breeds were excluded due to their reported high incidence of dystocia [3]. The 52 pregnant females were classified in 4 categories according to their weight recorded before labor (day 60 ± 2), as follows: C1 (4.0–8.0 kg, n = 19); C2 (8.1–16.0 kg, n = 16); C3 (16.1–32.0 kg, n = 11); C4 (32.1–35.8 kg, n = 6). The body weight ranges respected the general guidelines for breed size established by the Federation Cynologique Internationale (FCI) [43].

#### *2.3. Clinical History*

The clinical history of the dogs was compiled, including age, weight, alimentation, preventive medicine status, and a description of the environment where they lived. All information was recorded in the Q.vet® Ed. Professional 2016 database for veterinary clinics.

#### *2.4. Diagnoses of Pregnancy*

Diagnoses were confirmed between days 24 and 28 post-service for each dam. Fetal structures and cardiac activity were detected in the gestational sacs using a LOGIQ 400 MD ultrasound machine (General Electric, Yokohama, Japan) equipped with a 3.5 MHz convex transducer to establish probable due dates. Monitoring of fetal maturation and vitality was performed on days 40–50 of gestation. The fetal structure was defined completely to permit the early identification of pyometra cases, type I SB puppies, and malformations. X-rays of the dams' abdomens were taken on day 45 of pregnancy once bone calcification of the fetuses was achieved to discard early stages of maternal–fetal dystocia and evidence of cephalopelvic disproportion, conditions that would make caesarean sections necessary [29]; thus, excluding the dam from the study. On day 60 of pregnancy, the females were checked by ultrasound to corroborate cardiac rhythms and fetal biparietal diameters. Monitoring of births was performed with a model S80Vet Sino-Hero® vital sign monitor to evaluate the mothers' physiological parameters. Clinical signs observed in the peripartum interval included anorexia, anxiety, and nesting behaviors.

#### *2.5. Puppies*

The number of LP and SB was recorded by category.


### 2.5.1. Blood Physio-Metabolic Profiles

#### Blood Sampling

A trained veterinarian took blood samples immediately after birth in less than 10 s. An assistant held the puppy in a supine position and exposed the abdominal region; the pup's umbilical cord was carefully grasped to insert the needle (26G) of a tuberculin syringe impregnated with lithium heparin to avoid coagulation and alterations of blood values and immediately obtain 0.3 mL samples of venous blood. All samples were processed by a GEM Premier® critical blood variable analyzer (Instrumentation Laboratory Diagnostics USA/Italy) to obtain values for the metabolite's glucose (mg/dL) and lactate (mg/dL), blood gases pCO2 (mmHg) and pO2 (mmHg), the acid–base balance pH, HCO3 − (mmol/L), EB (mEq/L), Ca2+ (mmol/L), and hematocrit (Htc %). All profiles were tested for each LP and Type II SB pup.

#### 2.5.2. Birth Weight

The weight of each LP and Type II SB puppy was recorded using a digital scale (Salter Weight Tronix Ltd., West Bromwich, UK) after drawing the blood samples.

#### *2.6. Statistical Analyses*

Descriptive statistics were obtained for the variables tested following the Origin Version® 9 statistical package procedure. Normality tests were performed for all dependent variables to determine differences among the 4 categories (the dam's weight was the independent variable) regarding the parameters of the blood physio-metabolic profile: pH, pO2 (mmHg), pCO2 (mmHg), glucose (mg/dL), Ca2+ (mmol/L), lactate (mg/dL), hematocrit (Htc %), HCO3 − (mmol/L), and EB (mEq/L), as well as the weight of the LP pups (dependent variable). An analysis of variance (ANOVA) was performed with a contrast of means using a Tukey test (*p* < 0.05).

Values were considered as significant at *p* < 0.05.

$$Metabolites\_{ijk} = \mu + T\_{\bar{i}} + \text{CN}\_{\bar{i}} + P\_{\bar{i}} \left( T\_{\bar{i}} \text{CN}\_{\bar{i}} \, P\_{\bar{i}} \right) + \epsilon\_{ijk}$$

where:

*Metabolites* = pH, pCO2, pO2, glucose, Ca2+, lactate, hematocrit, HCO3 −, EB; *μ* = general mean; *Ti* = fixed effect; *CNi* = 1,2,3,4; for the case of SB Type II; *Pi* = birth weight; *e* = error.

The same statistical model was used to analyze the SB Type II neonates.

#### *2.7. Ethical Note*

The studies were performed with privately owned dogs. Each owner gave her/his informed consent before data were gathered. During the study, all dogs were treated following the directives and guidelines of Mexico's Official Norm NOM-062-ZOO-1999 on technical specifications for the production, care, and use of laboratory animals and those related to the field of applied etiological studies [44]. The experimental protocol (code CAMCA.32.18) was approved by the Committee of the Master's Program in Agricultural Sciences of the Universidad Autónoma Metropolitana-Xochimilco, Mexico City.

#### **3. Results**

#### *3.1. Weight*

Significant statistical differences were observed (see Table 1, mean and standard error of the birth weights of neonates) between the weight of the LP puppies in categories one (189.85 ± 16.50 g; *p* = 0.01), two (266.84 ± 16.23 g; *p* = 0.01), and three (374.57 ± 48.18 g; *p* = 0.0001), with those in C1 having lower weights than those in C2 and C3 (76.99–184.72 g). There were no significant differences in the weight of the LP pups in C3 (374.57 ± 48.18 g) and C4 (381.02 ± 20.24) (*p* = 0.70).

Table 1 presents data on the weight of the Type II SB pups. The pups in C1 (219.11 ± 23.05 g; *p* = 0.01) weighed less than those in C2 (297.08 ± 17.62 g; *p* = 0.001), C3 (419.86 ± 4.57 g; *p* = 0.001), and C4 (433.75 ± 12.98 g; *p* = 0.001), but there were no significant differences between the weight of the SB pups in C3 (419.86 ± 4.57 g) and C4 (433.75 ± 12.98 g) (*p* = 0.40).

#### The lowest number of SB occurred in C1 with nine (12.5%), compared to C2, C3, and C4, which had 14.45–24.48%.

**Table 1.** Mean and standard error of the birth weights of the liveborn (LP) puppies and the Type II stillbirth (SB) during the first minute of life, grouped according to the category of the mother.


*<sup>a</sup>*,*b*,*<sup>c</sup>* letters indicate differences in the categories of the mother (weight). Variance analysis (*p* < 0.05); Tukey test for independent samples (*p* < 0.05). SEM—standard error of the mean. Weight of the mothers according to category: C1 (4.00–8.00 kg), C2 (8.10–16.00 kg), C3 (16.10–32.00 kg), C4 (32.10–35.8 kg).

#### *3.2. Blood Physiometabolic Profiles*

The effect of the mother's weight on the blood physio-metabolic profiles of LP and SB are shown in Tables 2 and 3, respectively. In general, the weight category of the dam was related to metabolic changes in some of their puppies' critical blood variables.

**Table 2.** Mean and standard error of the blood physio-metabolic profile of the LP puppies grouped according to the category of the weight of the mother.


a,b,c Letters indicate differences in the category of the mother (weight). Variance analysis (*p* < 0.05); Tukey test for independent samples (*p* < 0.05). SEM—standard error of the mean. LP—number of LP in the samples. Blood samples taken in a maximum of 10 s post-birth. Weight of the mothers according to category: C1 (4.0–8.0 kg), C2 (8.10–16.0 kg), C3 (16.10–32.0 kg), C4 (32.10–35.8 kg).


**Table 3.** Mean and standard error of the blood physio-metabolic profile of the Type II SB grouped according to the category of weight of the mother.

a,b Letters indicate differences in the category of the mother (weight). Variance analysis (*p* < 0.05); Tukey test for independent samples (*p* < 0.05). SEM—standard error of the mean. LP—number of LP in the samples. SB—Stillbirth. Blood samples taken in a maximum of 10 s post-birth. Weight of the mothers according to category: C1 (4.0–8.0 kg), C2 (8.10–16.0 kg), C3 (16.10–32.0 kg), C4 (32.10–35.8 kg).

#### 3.2.1. Energy Metabolism

In terms of the level of blood lactate (see Table 2), there was a significant increase of 9.26–14.33% between the LP in C1 (4.80 ± 0.23 mg/dL, *p* = 0.001) and those in C2 (6.28 ± 0.17 mg/dL, *p* = 0.001), C3 (7.05 ± 0.27 mg/dL, *p* = 0.01), and C4 (7.27 ± 0.30 mg/dL, *p* = 0.01). The LP in C4 had the largest increase in blood lactate concentrations compared to C1 and C2 (*p* < 0.05), though there was no significant difference in lactate values between C2 and C3 (6.28 ± 0.17 mg/dL and 7.05 ± 0.27 mg/dL, respectively) (*p* = 0.07) (Table 2).

The Type II SB pups in C4 (13.08 ± 0.41 mg/dL) showed a larger increase in lactate than C1 (11.44 ± 0.52) (*p* = 0.02), but there were no significant differences between C2 and C3 (12.58 ± 0.31 and 12.5 ± 0.22, respectively) (*p* = 0.99) (Table 3).

In terms of blood glucose levels for the LP, no significant differences among the categories were found (*p* > 0.05) (Table 2). In contrast, the Type II SB pups in C1 (51.11 ± 3.18 mg/dL) had higher glucose levels than those in C3 (38.78 ± 3.97) (*p* = 0.04). There were no significant differences between C2 and C4 (42.58 ± 2.19 mg/dL and 41.91 ± 2.09 mg/dL, respectively) (*p* = 0.99) (Table 3).

#### 3.2.2. Calcium and Hematocrit

Regarding the metabolite Ca2+, a statistically significant decrease was found in the values of the LP in C1 (1.43 ± 0.01 mmol/L) compared to C2 (1.55 ± 0.01 mmol/L, *p* = 0.001), C3 (1.62 ± 0.01 mmol/L, *p* = 0.01), and C4 (1.59 ± 0.02 mmol/L, *p* = 0.01), but no significant differences in blood Ca2+ concentrations were seen among C2, C3, and C4 (*p* > 0.05) (Table 2). For the Type II SB, Ca2+ levels showed no significant differences among the categories (*p* > 0.05) (Table 3).

Hematocrit values showed that the LP from the C2 (48.65 ± 0.41, 8.5%; *p* = 0.001), C3 (50.43 ± 0.39, 12.46%; *p* = 0.01), and C4 (49.82 ± 0.55, 11.1%; *p* = 0.01) dams all had higher percentages of hematocrit than those in C1 (44.84 ± 0.55) (*p* < 0.05). However, no significant differences were seen among the LP in C2, C3, and C4 (*p* > 0.05) (Table 3). Regarding the Type II SB, there were no significant differences among the categories for this value (*p* > 0.05) (Table 3).

#### 3.2.3. Acid–Base Balance

Observations showed that blood pH had a lower imbalance in the LP from the C1 dams (7.38 ± 0.01; *p* < 0.05), compared to C3 (7.29 ± 0.01; *p* = 0.01) and C4 (7.31 ± 0.02; *p* = 0.04). While pH clearly decreased, there were no significant differences in pH values among categories C2, C3, and C4 (*p* < 0.05) (Table 2). Similarly, for the Type II SB, there were no significant differences among the categories in terms of pH (*p* > 0.05) (Table 3).

Regarding pO2, results showed that the LP from three groups—C2 (15.47 ± 0.36 mmHg, *p* = 0.01), C3 (14.48 ± 0.40 mmHg, *p* = 0.001), and C4 (15.18 ± 0.47 mmHg, *p* = 0.01)—had significant decreases compared to C1 (17.09 ± 0.44 mmHg) (*p* < 0.05), but there were no significant differences in the pO2 values among C2, C3, and C4 (*p* > 0.05) (Table 3). For blood pO2 values, we found that the Type II SB in C4 (5.75 ± 0.89 mmHg) had a significant decrease compared to C1 (9 ± 0.91 mmHg) (*p* = 0.02), but the pO2 values between C2 and C3 did not differ significantly (6.66 ± 0.63 mmHg, *p* = 0.15, and 6.21 ± 0.48 mmHg, *p* = 0.053, respectively) (Table 3).

Regarding pCO2, a statistically significant increase was observed in the LP in C2 (54.42 ± 1.35 mmHg, *p* = 0.01), C3 (55.98 ± 1.63 mmHg, *p* = 0.001), and C4 (54.78 ± 1.87 mmHg, *p* = 0.01), compared to C1 (47.69 ± 1.01 mmHg). In contrast, no significant differences were found in the pCO2 values of C2, C3, and C4 (*p* > 0.05) (Table 2). Upon observing the blood pCO2 values in the Type II SB, a significant increase was found in groups C2 (93.08 ± 2.42 mmHg, *p* = 0.01), C3 (91.78 ± 2.28 mmHg, *p* = 0.01), and C4 (94.66 ± 1.9 mmHg, *p* = 0.01) compared to C1 (1.66 ± 2.08 mmHg), but no significant differences were found in the pCO2 values from groups C2, C3, and C4 (*p* > 0.05) (Table 3).

A significant decrease in HCO3 <sup>−</sup> levels was seen in the LP in C2 (20.44 ± 0.17 mmol/L, *p* = 0.001), C3 (19.96 ± 0.18 mmol/L, *p* = 0.01), and C4 (19.86 ± 0.21 mmol/L, *p* = 0.001) compared to C1 (21.94 ± 0.26 mmol/L) (*p* < 0.05), but upon comparing the metabolite values for C2, C3, and C4, no significant differences were found (*p* > 0.05) (Table 2). Regarding the SB, no differences were observed among the categories (*p* > 0.05) (Table 3).

Observations showed that EB increased significantly in the LP in C4 (−8.82 ± 0.43 mEq/L) compared to the values for C1 (−4.77 ± 0.38 mEq/L, *p* = 0.01), C2 (−5.96 ± 0.36 mEq/L, *p* = 0.001), and C3 (−7.03 ± 0.45 mEq/L, *p* = 0.01). A similar result emerged when we compared the values for C1 and C3 (*p* = 0.001), though no significant differences were found between C2 and C3 (*p* = 0.11) (Table 2). Regarding the Type II SB, no significant differences were found among the categories (*p* > 0.05) (Table 3).

#### **4. Discussion**

*4.1. Weight*

It was proposed that the weight of puppies at birth can be influenced by diverse factors, as occurs in other mammals. These include the duration of pregnancy [27,45,46], restrictions on intrauterine growth [45,47,48], the mother's nutritional status [27], breed [49], and the weight and size of the placenta [50]. However, the results of our study suggest that the mother's weight before giving birth exerts an effect on the weight of the newborn puppies since this varied significantly among the four categories tested. A broad weight range was observed that might be attributable to this species' extensive morphological variability characteristic [51]. The recorded weights of the 272 puppies born and classified according to the weight of the dam (based on FCI guidelines, [43]) showed a mean from 204.48 to 407.39 g (157–453 g), with a mean variation of 77.46 to 202.91 g. We believe that the weight of the neonates reflected the mother's weight because our study did not consider breed as a variable. A study by Vassalo et al. [6] observed a similar effect, the mothers' body weight influenced the weight of puppies born by eutocic births and cesarean section. While it is true that the dam's nutritional status can affect the weight of the puppies—as occurs in humans [52]—this variable was not controlled in this study.

One important finding involves the weight of the SB, as this was always higher than that of the LP in all four cases (C1–C4). These differences mean 29.16–52.77 g, but categories C3 and C4 had both the highest mean weights (419.86 and 433.79 g, respectively) and the highest mortality rates (C3 = 20.58%, C4 = 24.58%). On one side, and such as other species, including humans, swine, and bovines, low birth weight is considered an important risk factor for neonatal mortality [34,53,54]. Reports on dogs affirm that low birth weight is strongly related to mortality. In this regard, Groppetti et al. [55] and Mila et al. [56] pointed out that there is a twelve-fold higher mortality risk for the lightest puppies than those with normal weight. Though low birth weight has been deemed a disadvantageous condition for neonatal survival [3,55,57,58] and has been associated with a higher risk of fetal death, our study did not reveal signs of this because the heaviest puppies presented a higher risk of fetal death than those with the lowest weight (12.5 vs. 24.58% mortality). On the other side, previous results indicate that higher birth weights reduce postnatal mortality but increase the rate of intrapartum mortality due to the difficulties of birth caused by cephalopelvic disproportion and prolonged labor that can cause hypoxia or death [28,59,60]. The mortality rate in this study was 17.27%, counting only the pups that died intrapartum. While it is true that the cause of perinatal mortality in dogs is multifactorial, the mother's weight must be considered a risk factor due to its impact on the weight of type II SB puppies, physiological alterations, and the acid–base imbalance present in puppies born in natural births.

#### *4.2. Physiometabolic Profiles*

Fetuses commonly suffer intermittent periods of light hypoxia due to uterine contractions and the mechanical pressure inherent to the birth process [61]. Vassalo et al. [6] affirmed that a state of fetal hypoxia during the perinatal period is common in newborn puppies. We measured the physiological and metabolic changes that LP experienced during eutocic births, including increases in blood levels of pCO2 and lactate of 17.38% and 51.45%, respectively, and decreases in blood pH of 1.22%, and levels of pO2 (15.27%) and bicarbonate HCO3 − (9.48%), with high EB (45.91%), all due to hypercapnia (an indicator of respiratory acidosis) as the main factor. The most evident alterations occurred in the LP groups with the heaviest dams (C2, C3, C4). These alterations in gases and blood metabolites indicated respiratory and metabolic acidosis (mixed acidosis) resulting from intermittent asphyxia in utero during natural birth. Compensatory alkalosis began as a response to the respiratory and metabolic acidosis in the LP in all groups (C1–C4) due to hypoxia. This explains why pH did not decrease drastically [62,63]. Hypoxia-induced stress increases circulating epinephrine that breaks down muscular glycogen; thus, increasing lactate concentrations [64–66]. The above slows metabolism and triggers delayed anaerobiosis, a mechanism known as a tolerance to fetal hypoxia [67].

Regarding the metabolite glucose, no significant differences were observed in the LP during the first minute of life in any of the four categories, since measurements were in a range of 94.92–103.91 mg/dL. Mila et al. [17] reported a mean plasma glucose concentration of 97 mg/dL between 10 min and 8 h postpartum. The blood glucose level of puppies in the first 24 h postpartum was established in a range of 88–133 mg/dL [68,69]. Adequate energy reserves are extremely important for neonatal survival and resistance to adverse climatic conditions [70]. However, during the first hours of life, a decrease in glucose concentrations may be seen in diverse species because the glucose supply is interrupted abruptly during birth. This decrease is associated with the rapid exhaustion of hepatic glycogen. Hypoglycemia increases blood glucagon, cortisol, and catecholamine levels, leading to gluconeogenesis, lipolysis, glycogenolysis, and the consumption of ketone bodies. Ingesting colostrum post-birth increases and maintains glucose levels. In humans, for example, values below 50 mg/dL have been observed, though these may increase to 81 mg/dL during daytime [71,72]. A similar situation has been seen in foals [73]. On another point, an increase in blood glucose in newborn piglets can be considered an accurate indicator of neonatal distress because it shows their incapacity to regulate, or

compensate for, the physiological processes during birth [13,37,74]. Mota-Rojas et al. [75] mention that high glucose concentrations in piglets are a sign of a short episode of asphyxia compared to those that manage to maintain their energy reserves. Therefore, lower glucose levels are associated with a more extended period of asphyxia and higher a consumption of energy reserves. It is important to mention that a prolonged or intermittent asphyxia in utero during birth does not necessarily lead to intrapartum stillbirth. However, these conditions can weaken newborns and reduce their capacity to adapt to extrauterine life, as documented in piglet studies [36]. The events that occur during an acute process of asphyxiation—such as metabolic acidosis and hypoxia—impact the welfare of newborns and their postnatal development.

Birth weight was reported as a risk factor for intrapartum hypoxia because newborns with a low birth weight are more likely to suffer oxygen restriction and the secondary effects of hypoxemia [76,77]. Present findings, however, indicate otherwise. The blood samples collected from the umbilical cords for the gas and metabolic analyses of the intrapartum SB indicated that the fetuses showed signs of severe metabolic acidosis moments before birth due to the low pH (range: 6.79–6.88), increased pCO2 levels up to double those registered in the LP (94.66 vs. 47.69 mmHg), lactate values as many as four times higher than in the LP (13.08 vs. 4.80 mg/dL), EB in a range of −14.26 to −15.33, and a decrease in pO2 (6.22%) and bicarbonate HCO3 − levels (6.97%). We also observed that the Type II SB showed a significant decrease in plasma glucose concentrations in every group (C1–C4), with a range of 38.78–51.11 mg/dL. This result coincides with the values < 40 mg/dL reported by Lawler [78], related to hypoglycemia, which indicate a depletion of the newborns' energy reserves, as a by-product of a previous hypoxic process [7,78] accompanied by a delay in eliminating excess liquid from the lungs, a decrease in uterine blood circulation, deficient gas exchange, and an alteration of energy metabolism [16]. We, therefore, infer that prolonged uterine contractions without fetal expulsion, caused by the prolonged birth process characteristic of this species, trigger hypoxia by increasing anaerobic glycogenolysis and the development of metabolic acidosis, as has been seen in piglets [75,79,80], humans [81], foals [82], and domesticated animals, including buffaloes [83,84]. These are conditions that reduce vitality and increase mortality [85].

Fetal asphyxia, defined as a condition of hypoxemia with hypercapnia and acidosis, caused the death of the pups at birth in this study. Fetal asphyxia caused the death of 17.27% of the puppies in this study, but the highest percentages of intrapartum deaths were seen in C3 and C4, which together represented 55.31% of the mortality of the pups from natural births.

#### Acid–Base Balance

In LP, a pH below the reference values was observed (7.35–7.45) in C2, C3, and C4, but this parameter on its own does not permit measuring the accumulated exposure to hypoxia because it is expressed logarithmically [39]. It does, however, establish the existence of acidosis in newborns and reflects fetal hypoxic stress that occurred during birth. Hence, the relation among pH, bicarbonate, and pCO2 indicates a process of metabolic acidosis [86]. In addition, the concentration of excess base (EB) must be determined, as this is an indicator of a linear tendency that determines the accumulation of acidosis after being adjusted for variations in pCO2 [87].

The intense, constant uterine contractions necessary for fetal expulsion can compress the umbilical cord, drastically decreasing both placental and umbilical blood circulation [88]. The decrease in pO2 and the increase in pCO2 combined to lower the pH, but bicarbonate in the plasma mitigated this imbalance. As a result, the high concentration of bicarbonate generated a mixed acidosis that exacerbated the increase in ionized calcium and the decrease in protein-bonded calcium [89], producing hypocalcemia at birth. Andres et al. [25] indicate that delayed breathing and metabolic acidosis are related to mortality at birth.

During pregnancy, fetuses depend on their mother for gas exchange and correct oxygenation through the placenta. This exchange is determined by the size of the fetuses, blood

gas concentrations in the mother, and the latter's capacity for transfer and transport. Thus, modifications to any of these parameters can generate a state of hypoxia and subsequent disruptions of the acid–base balance, such as metabolic acidosis [61]. It is believed that the fetus' capacity to withstand birth stress and welfare depends on both its condition at birth and the birthing process itself (duration, number of contractions, fetus thermoregulation, physiological, and metabolic changes. In addition, the newborn has to make several adjustments to adapt to extrauterine life, such as maintaining normoglycemia, thermoregulation, etc.) [18,90–95].

#### **5. Conclusions**

The results of this study allowed us to identify that the weight of dams before birth determines the weight of the puppies at birth, though there is a wide range in birth weights due to the ample morphological variability characteristic of this species. We observed that the puppies born from bitches in categories three and four had the highest birth weights (397.21 and 407.39 g, respectively), with a difference greater than 100 g from the pups in C1 and C2. The LP puppies in category 1—the lightest ones (189.85 g)—had fewer physio-metabolic alterations during the first minute of life; while the litters that weighed 16.1–35.8 kg showed more physio-metabolic alterations, which impacted their adaption to extrauterine life and caused respiratory and metabolic acidosis and hypocalcemia, all of which contributed to the total mortality of 17% as a consequence of intrapartum hypoxia.

Furthermore, all the Type II SB were heavier (C1 > 29.26 g; C2 > 30.24 g; C3 > 45.29 g; C4 > 52.73 g) than the LP born in the same group. The above suggests that puppies with higher weights experience greater difficulty during expulsion through the birth channel, which increases the risk of suffering an acute process of intrauterine asphyxia that affects their chances of survival. The highest mortality rates for the heavier categories (C3 and C4) were 20.58% and 24.48%, respectively. Therefore, this study established the mother s weight (16.1–35.8 kg) as a risk factor for fetal asphyxia in eutocic births and, consequently, a high percentage of intrapartum mortality. New studies will allow the integrated evaluation of other variables as risk factors related to in utero asphyxia in eutocic births and their influence on postpartum survival.

**Author Contributions:** Conceptualization, D.M.-R. and A.G.; investigation, B.R.-S., D.M.-R., P.M.-M., A.O.-H., I.H.-Á., J.M.-B. and J.S.-M.; writing—original draft preparation, D.M.-R., B.R.-S., A.O., C.M., A.G. and J.M.-B.; writing—review and editing, D.M.-R., A.O.-H., B.R.-S., J.M.-B., P.M.-M., J.S.-M., I.H.-Á. and B.R.-S.; project administration, D.M.-R., A.O., C.M. and A.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and the experimental protocol was approved by the Committee of the Master's Program in Agricultural Sciences (protocol code CAMCA.32.18) of the Universidad Autónoma Metropolitana-Xochimilco campus, Mexico City.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

#### **Abbreviations/Nomenclature**


#### **References**

