animals

Article
Umbilical Cord Blood Gas Parameters and Apgar Scoring in
Assessment of New-Born Dogs Delivered by Cesarean Section
Agnieszka Antończyk 1 , Małgorzata Ochota 2, * and Wojciech Niżański 2

1 Wrocław University of Environmental and Life Sciences, Department and Clinic of Surgery,
50-366 Wrocław, Poland; [email protected]
2 Wrocław University of Environmental and Life Sciences, Department of Reproduction and Clinic of Farm
Animals, 50-366 Wrocław, Poland; [email protected]
* Correspondence: [email protected]

Simple Summary: The article presents the results of the clinical evaluation and umbilical cord blood
analysis obtained from newborn pups delivered by the elective Caesarean section. In human medicine
both the umbilical cord blood and Apgar score were proved to provide valuable information on
neonatal status. In veterinary medicine very few reports concerning the relation between the clinical
neonatal assessment (Apgar score) and umbilical cord blood parameters exist. All puppies show mild
respiratory acidemia regardless the Apgar scores result. The lactates were not elevated whereas all
the puppies with low Apgar sores had higher glucose and mortality rates. Only pups with low initial
Apgar scores were at risk of death within first 24 h. Adaptation to the extra-uterine life is crucial
and any practical improvement in neonatal diagnostics and care would be beneficial for newborn
puppy survival.






Abstract: The article presents the results of the clinical evaluation (Apgar scores, AS) and umbilical
Citation: Antończyk, A.; Ochota, M.; cord blood gas analysis (UCBGA) obtained from clamped umbilical cords of newborn pups delivered
Niżański, W. Umbilical Cord Blood
by the elective Caesarean section. The study was planned as a controlled clinical study, the newborns
Gas Parameters and Apgar Scoring in
were allocated into one of the groups, I—critical neonates (severe distress, AS ≤ 3), II—weak
Assessment of New-Born Dogs
neonates (moderate distress, AS 4–6) and III—healthy neonates (no distress, AS ≥ 7). The following
Delivered by Cesarean Section.
parameters were evaluated: pH (pH units), carbon dioxide partial pressure (pCO2 ; mmHg), oxygen
Animals 2021, 11, 685. https://doi.
org/10.3390/ani11030685
partial pressure (pO2 ; mmHg), actual bicarbonate (cHCO3 − ; mmol/L), total carbon dioxide (cTCO2 ;
mmol/L), base excess of extracellular fluid (BE(ecf); mmol/L), base excess of blood (BE(b); mmol/L),
Academic Editors: Monica De Los oxygen saturation (csO2 ; %), lactate (Lac; mg/dL), hematocrit (Hct; %PCV), hemoglobin (cHgb;
Reyes and Nucharin Songsasen g/dL), glucose (Glu; mg/dL), ions (Na, K, Ca, Cl). The majority of puppies had low AS at birth (AS
4–6 in 38.1% and AS ≤ 3 in 57.1% of the neonates), but most of them (85.7%) improved by the 20th
Received: 20 January 2021 min. reaching AS of 7 and more. Moreover, puppies with lower AS (≤ 3) were at higher risk of death
Accepted: 26 February 2021 within the first 24h (20.8% did not survive). The positive correlation was found between Apgar score
Published: 4 March 2021 measured at 0 min and pH (r = 0.46, p = 0.01), and between Apgar score (at 0 min) and base excess
in whole blood measured [BE(b)] r = 0.36, p = 0.03). Whereas, a negative correlation was detected
Publisher’s Note: MDPI stays neutral
between Apgar score at 0 and 5th min and glycemia (r = −0.42, p = 0.05, r = −0.34, p = 0.02 respectively.
with regard to jurisdictional claims in
Overall, the puppies with higher glucose levels had lower Apgar scores and were at higher risk of
published maps and institutional affil-
death. Furthermore, in our study, the newborn puppies had mild acidemia with elevated pCO2
iations.
levels and the HCO3 at the lower range of normal limits, suggesting the mixed component in the
acidemic state. Adaptation to extra-uterine life is crucial and any practical improvement in neonatal
diagnostics and care would be beneficial for newborn puppy survival.

Copyright: © 2021 by the authors.

Keywords: dog; neonate; Apgar score; umbilical cord blood; Cesarean section
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://
1. Introduction
creativecommons.org/licenses/by/ The transition from fetus to neonate is a crucial time of physiological adaptation.
4.0/). In fetal life, the oxygen is delivered through placental circulation, carbon dioxide and

Animals 2021, 11, 685. https://doi.org/10.3390/ani11030685 https://www.mdpi.com/journal/animals

Animals 2021, 11, 685 2 of 12

other fetal waste products go back to the mother’s circulation same route. In most cases,
birth asphyxia and postnatal related problems originate from the interruption of uterine,
placental or umbilical circulation. The transition from fetal to neonatal life is a crucial
moment marked by profound changes to the physiology and biochemistry of all organs
of the body. If improval fails, these changes would result in fatal consequences and
most often death of a neonate [1,2]. The natural sequence of events in normal delivery
has been mastered over the ages to heighten the chances of a newborn to survive. The
medical approach, apart from bringing new challenges, also offers the opportunity to
closely supervise and control the neonatal state at birth. Proper diagnosis and relevant
clinical care would play an important role in increasing puppy survival [3]. Currently, in
veterinary medicine, it is essential to better understand the pattern of changes which occur
in a neonate at the moment of birth and its influence on the newborn homeostasis and
performance [4,5].
The observed mortality rate in puppies is still high, around 20% [6–9]. Sadly, in veteri-
nary medicine, weaker puppies often die underdiagnosed, due to clinical and therapeutic
restrictions. The particularities of canine newborns, their size and physiology make it very
difficult to develop or adopt standardized measures for neonatal diagnostics, assistance
and direct therapy [10,11] contributing to inaccurate diagnosis and probably explaining
the high mortality level [9]. The early and accurate detection of factors contributing to
newborn puppies’ fatalities could improve neonatal outcomes [3,12].
The standard care in human neonatal assessment includes Apgar scoring (AS) and
detailed clinical examination [13,14]. However, in neonatal intensive care units, the umbili-
cal cord blood gases evaluation is also performed and considered essential [15]. Similar to
human medicine for some years now the most advanced veterinary hospitals use modified
Apgar scores to assess newborn puppies and kittens [16]. Similarly to human medicine,
it quickly summarizes the newborn state and helps to decide what level of medical or
emergency care is needed. In veterinary medicine, where usually more than one puppy
or kitten is delivered, it has also another benefit—to help the staff to quickly determine
the state of each puppy in the litter and set priorities which one needs urgent or sec-
ondary attention [3]. A few papers have been published on Apgar score use in veterinary
medicine [3,9,12]. The systematic approach in newborn puppies evaluation proved to be
effective in the timely detection of weak puppies with low Apgar scores that need urgent
attention. It is very useful for short-term prognosis and also correlates with the future
survival rate [8]. In addition to the current newborn status, the evaluation of umbilical
cord blood gases analysis (UCBGA) proved to be an objective measure for the evaluation
of fetal oxygenation status and metabolic acidosis at the moment of delivery [9,15]. The
UCBGA has been shown to be more reliable than the Apgar scoring system in providing
objective evidence of birth asphyxia and is now widely recommended especially for high-
risk deliveries [17] in human medicine. The most important measurements are the pH,
partial pressure of carbon dioxide (pCO2 ) and base deficit (BE), which help to determine
whether the acidosis is metabolic or respiratory. Apart from these, other parameters can be
assessed like ions, glucose, lactate or PCV, all providing valuable information on prenatal
uterine condition, placental perfusion or impaired fetal nutrient supply [18]. Currently,
not many publications are available concerning UCBGA in dogs, most probably due to
different umbilical cord morphology in dogs than in humans and significantly lower birth
weight and thus limited vessel size in puppies. Silva et al. studied only venous gases [9],
whereas Mila et al. evaluated blood glucose, lactate and β-hydroxybutyrate concentra-
tion [7], and Groppetti et al. correlated neonatal umbilical cord blood lactate concentration
with neonatal viability [12]. All the publications aimed at finding a relationship between
studied parameters and postnatal puppy vitality and survival. Unfortunately, limited data,
different time schedules, and the variety of vessels used to obtain the blood make it very
difficult to compare the presented results. Since the veterinary neonatology is still lacking
thorough evaluation of umbilical cord blood parameters and its correlation with neonatal
health and performance, the main objective of this study was to describe the umbilical cord
Animals 2021, 11, 685 3 of 12

blood parameters indicating the character and level of biochemical and physical changes
that occur at the time of the transition from fetal to neonatal state in newborn puppies
delivered through Cesarean section.

2. Materials and Methods

2.1. Animal Selection
The 18 pregnant bitches of different breeds, including English Bulldog, German
Shepherd, Boston Terrier, French Bulldog, Yorkshire Terrier, Welsh Corgi Pembroke, Great
Dane, Shar-pei, Bull Terrier, Labrador, Shetland Sheepdog, were enrolled in the study. The
mean bodyweight of the females was 28.11 kg (3.5–77 kg) and the mean age was 3.83 (1.5–7).
The decision on elective Cesarean section was made by the vet responsible for the surgical
procedure, based solely on clinical indications only and independently of this research.
The patient qualification, time schedule and the veterinary procedures were performed
under the Good Clinical Practice (GCP) guidelines. The Cesarean section was scheduled
based on progesterone measurements during heat and LH peak determination and was
performed on 63–64 days after LH peak, depending on the clinical evaluation of the dam,
presence of milk and US foetal monitoring (heart rate).

2.2. Anesthetic Protocol

All females underwent standard anesthetic protocol used at our Clinic for elective
Cesarean sections. After admission the bitch was clinically assessed, weighted, and an intra-
venous catheter was placed, the preoxygenation was started and a single dose of 0.2 mg/kg
s.c. meloxicam was administered (Metacam® 5 mg/mL, Boehringer Ingelheim, Warsaw,
Poland). Anesthesia was induced with intravenous bolus administration of propofol at
4–6 mg/kg to effect (Propofol-Lipuro® , 10 mg/mL B. Braun Melsungen AG, Germany),
then the bitch was intubated and the anesthesia was maintained with isoflurane in oxygen
(1.5–2.5%, IsoVet® , Piramal Healthcare, Morphet, United Kingdom ® ). Intravenous fluids
(crystalloids) were given in each case at the rate of 5 mL/kg/h. After removal of the last
puppy opioids: morphine 0.3 mg/kg, (Morphini Sulfas® , WZF Polfa Warsaw, Poland),
or buprenorphine 0.02 mg/kg (Bupaq Multidose® Richter Pharma AG, Wels, Austria), or
methadone (Comfortan® , Eurovet Animal Health BV, Bladel, Holand) 0.3 mg/kg were
given to provide further analgesia [19].

2.3. Surgical Procedure and Umbilical Cord Sampling

A standard ventral midline approach was performed, the skin, subcutaneous tissue
and abdominal wall were incised to enter the abdominal cavity and expose the uterine body
and horns. Then, the edges of the abdominal incision were protected with moist swabs.
Thereafter, the uterine incision was performed at a ventral surface of the uterine body
and the closest neonate was delivered immediately. Directly after the puppy extraction
from the uterus the fetal membranes were torn and removed from the muzzle and the
umbilical cord was double clamped before placenta detachment. First, forceps were placed
as close to the placenta as possible, and the second forceps about 1 cm from the newborn’s
abdominal wall. The clamped vessel was then positioned between clamps to allow blood
collection. At least 100 mcl of mixed umbilical cord blood was collected into a heparinized
syringe with 25G needle, and taken for further analysis. The placenta and the remaining
fetal membranes were removed using gentle traction. The same procedure was performed
for all the fetuses, then the uterus was carefully checked for remaining fetal membranes,
placentas or debris and sutured with a single continuous pattern (Monosyn® 3/0, B. Braun
Aesculap Chifa Sp. z o.o., Nowy Tomyśl, Poland). The abdominal cavity was lavaged
with warmed saline before closing. The midline was sutured with a single continuous
pattern and synthetic absorbable material, similarly, the subcutaneous tissue was sutured
using absorbable material and continuous pattern, then the skin was routinely closed with
absorbable material and intradermal pattern.
Animals 2021, 11, 685 4 of 12

2.4. Neonatal Care and Resuscitation

Depending on the size of the litter, 2–3 staff members with similar experience and skills
gave first aid and, if necessary, performed resuscitation. Immediately after placental de-
tachment, each puppy was placed on a heated blanket, the heart rate (HR), respiration rate
(RR), mucous membrane colour, muscle tone and reflex irritability were briskly evaluated.
Then, depending on the initial state, neonatal care or resuscitation was initiated. Neonatal
care began with removing free fluid from the upper airways using the syringe bulb and
clamping of the umbilical cord. Thereafter breathing was stimulated by a vigorous rubbing
with a towel and the puppy was checked for obvious physical defects. The responding
pups, i.e., vocalizing and breathing spontaneously, were moved into a veterinary incubator
(Rcom MX-B90N, Autoelex Co., Ltd. Gyeongsangnam-do, Korea). The non-improving
pups underwent neonatal resuscitation. If the heartbeat was present, the oxygen (constant
flow or mask) was supplied and gentle stimulation of the abdominal area was started.
The GV26 acupuncture point was stimulated and vigorous massage was continued. In
puppies with asystole cardiac compression was performed at a rate of 120 per minute
and epinephrine (0.01 mg/kg starting dose and continued if necessary at 0.1 mg/kg) was
administered in the jugular vein. If there were no improvement after 20 min of intensive
care it was stopped and the puppy was considered dead. All puppies were evaluated at
5th and 20th min using Apgar scoring and then a full clinical examination was performed
around the moment of discharge to make sure all neonates can be reunited with the dam
and sent home. Each owner was contacted by phone 48 h and 7 days after Cesarean section
to confirm the survival/death of the puppies.

2.5. APGAR Scoring (AS)

The Apgar scoring was always done by the same person, using a modified Apgar
scoring system adapted to be used in bitches [16]. The first evaluation was performed
just after delivery, before first aid or neonatal care was instituted (0 min) and at the
following times: 5th, 20th. The final evaluation was carried out around the time of
discharging, i.e., 60th–90th min after birth. The AS reference range for the heart rate (HR)
was: >220 bpm (2 points), 180–220 bpm (1 point) to <180 (0 points). Similarly, for the
respiration rate (RR): >15 breaths per minute (2 points), 6–15 (1 point) to <6 (0 points).
The reflex irritability detected after a gentle compression of a tip of a paw was evaluated
depending on the degree of reaction: crying and quick leg retraction (2 points), weak
leg retraction and no or just weak vocalization (1 point), and no leg retraction and no
vocalization (0 points). Spontaneous movement of a newborn was rated as: 2—strong
movement, 1—weak movement, and 0—absent. The color of mucous membranes was
assessed as dark pink (2 points), light pink (1 point) and pale to cyanotic (0 points).
All points together provided the final Apgar score: 7–10 no distress, healthy newborn;
4–6 moderate distress, weak newborn; and 0–3 severe distress, critical newborn [16].

2.6. Umbilical Cord Blood Gas Analysis (UCBGA)

The blood samples for UCBGA were collected in heparinized syringes and analyzed
immediately after sampling (POC: Point-Of-Care testing) using EPOC VET handheld
analyzer (Siemens Healthineers, Erlangen, Germany). The following parameters were
evaluated: pH (pH units), carbon dioxide partial pressure (pCO2 ; mmHg), oxygen partial
pressure (pO2 ; mmHg), actual bicarbonate (cHCO3 -; mmol/L), total carbon dioxide (cTCO2 ;
mmol/L), base excess of extracellular fluid (BE(ecf); mmol/L), base excess of blood (BE(b);
mmol/L), oxygen saturation (csO2 ; %), lactate (Lac; mg/dL), hematocrit (Hct; %PCV),
hemoglobin (cHgb; g/dL), glucose (Glu; mg/dL), ions (Na, K, Ca, Cl). The blood gas
analysis was normalized by puppies’ body temperature, estimated as 37 ◦ C.

2.7. Study Design

The study was planned as a controlled clinical study, the newborns were allocated
into one of the groups, I—critical neonates (severe distress in AS evaluation), II—weak
Animals 2021, 11, 685 5 of 12

neonates (moderate distress in AS evaluation) and III—healthy neonates (no distress

in AS evaluation), depending on the initial Apgar points obtained immediately after
delivery (0 min). Neonates classified as critical received no more than 3 Apgar points in
the initial examination, weak neonates received 4–6 points, whereas healthy received 7
and more Apgar points. Thereafter each puppy was re-examined using AS at 5th and
20th min. Finally, before discharging (around 60–90 min after delivery) each puppy was
evaluated again.
The initial (0 min) AS allowed to identify the weakest puppies which would need
the most intensive care and to see whether this initial Apgar score would correlate with
UCBGA. The following AS at 5 min was carried out to determine if the neonatal state
improved after the neonatal care or resuscitation provided and to select puppies that need
further attention and those which can be placed in the incubator. The following evaluation
was performed at 20th min to check the status of all pups and to decide on stopping the
resuscitation in non-responding critical neonates [1]. The last AS was carried out before
discharging to make sure all neonates can be reunited with the dam and discharged home.
Each owner was contacted by phone 48 h and 7 days after Cesarean section to confirm the
survival/death of the puppies.

2.8. Statistical Analysis

Puppies were divided based on the initial Apgar score results into groups of I—
critical neonates, II—weak neonates and III—healthy neonates. Statistical analysis included
standard descriptive statistics and normality testing using the Kolmogorov–Smirnov test
with the Lilliefors correction. One way repeated measures analysis of variance was used to
compare Apgar scores within different time points. The results of UCBGA were compared
among groups using one-way analysis of variance. In the case where the third group
was too small, a t-test was used to compare two sets of data. The correlation between
UCBGA and the Apgar scores results were evaluated with Spearman’s correlation test.
The significance level was set to 0.05 in all the abovementioned tests. Calculations were
performed using Statistica 13 (StatSoft, Kraków, Poland).

3. Results
Eighty-five puppies were born in 18 Cesarean sections. One to six puppies were
delivered in each Cesarean section. The collection of umbilical cord blood was successful
in 42 of them (1 to 5 puppies per dam), and these pups were enrolled in the study, however,
in the remaining 43, the amount of the collected blood was not enough to perform analysis.
Statistical analysis revealed differences between Apgar scores obtained at different
time points (p < 0.00001) (Table 1). At 0 min of birth, only 4.8% of the neonates (n = 2) had an
Apgar score of 7 or above, which was the cut-off value for considering the puppy as normal.
In total, 38.1% (n = 16) and 57.1% (n = 24) of the neonates had Apgar scores of 4–6 and of 3
or below, respectively. At 5 min of birth, 31.0% (n = 13) pups were considered normal with
an Apgar score of 7 or above. Furthermore, 52.4% (n = 22) and 16.7% (n = 7) had Apgar
score of 4–6 and 3 or below, which classified the pup as weak or critical, respectively. At
20 min of birth, the vast majority of newborns (85.7%, n = 36) were scored at 7 or higher.
Four newborns (9.5%) were classified as weak (4–6 AS), however, critical pups were also
found (4.8%, n = 2). Two newborns did not survive and had an Apgar score of 0 at 20 min.

Table 1. Summary of Apgar Score (AS) at 0, 5th and 20th min of birth (n = 42).

AS 0 Min AS 5th Min AS 20th Min

Mean ± sd 2.7 ± 2 * 5.3 ± 2.3 * 7.9 ± 2.3 *
Min–max 0–8 0–9 0–10
* indicates significant difference between time points (p < 0.00001).

The initial Apgar score (AS) assessment was indicative of further neonatal perfor-
mance. All puppies classified with Apgar score ≥7 were in good condition in the following
Animals 2021, 11, 685 6 of 12

assessments, receiving similar or higher Apgar scores. Similarly, in the group of puppies
classified as weak (AS 4–6 at 0 min), all improved enough to be classified as normal (AS ≥ 7)
at 20th min despite the fact that some of them 43.75 % (n = 7) still remained weak (AS 4–6)
at 5th min. However, in the group of critical neonates at 0 min, only 8.3% (n = 2) improved
enough to receive AS ≥ 7 in the 5th min, but in the 20th min, as many as 75 % (n = 18)
improved significantly and were classified with AS ≥ 7. The remaining 58.33 % (n = 14)
received only AS of 4–6 at 5th min, however, only 16.67 % (n = 4) of them still remained
weak and received AS 4–6 at 20th min. Unfortunately, the 33.33 % (n = 8) of pups were still
critical at 5th min and two (8.33%) of them died before the next assessment in 20th min
(Figure 1), while three out of the four puppies assessed as weak at 20 min died within the
first 24 h (12.5%). The remaining 37 pups were healthy and were developing normally in
the final check on the 7th day after birth.

Figure 1. The following results of Apgar score assessments in puppies classified as critical (AS ≤ 3), weak (AS 4–6) and
normal (AS ≥ 7) immediately after birth (0 min). red rectangles—newborns with Apgar score < 4; yellow rectangles—
newborns with Apgar score 4–6; green rectangles—newborns with Apgar score > 6.

The mean results of umbilical cord blood gas analysis including all the investigated
parameters are presented in Table 2. The average pH was 7.2, which in adult dogs indicates
mild acidemia, however, there is no reference range for newborn puppies. Additionally, all
the investigated puppies had higher mean pCO2 comparing to reference values in adults
(35–45 mmHg). The mean glucose level was also quite high (72.64 mg/dL) and correlated
negatively with the AS evaluation, while the mean lactates were within the normal adult
dog range (2.45 mmol/L) (Table 2).
The retrospective analysis of umbilical cord blood gas analysis (UCBGA) parameters
in puppies classified as critical, weak, or healthy at different time points of the study (0, 5th,
20th min) was also performed (Table 3). Spearman’s correlation test showed the presence
of a positive correlation between Apgar score measured at 0 min and pH (r = 0.46, p = 0.01)
and between Apgar score (at 0 min) and base excess in whole blood measured [BE(b)]
r = 0.36, p = 0.03). Whereas, a negative correlation was detected between Apgar score at
Animals 2021, 11, 685 7 of 12

0 min and glycemia (r = −0.42, p = 0.05) as well as Apgar score measured at 5 min of birth
(r = −0.34, p = 0.02). No statistically significant correlation was found between any of the
other blood gas parameters examined.

Table 2. Mean values of umbilical cord blood gas parameters measured at birth (0 min, n = 42), mean ± SD.

pH pCO2 pO2 HCO3 − Na+ K+ Ca++ Cl− Glu Lac cHgb BE(b) BE(ecf)
Hct %
Units mm Hg mm Hg mmol/L mmol/L mmol/L mmol/L mmol/L mg/dL mmol/L g/dL mmol/L mmol/L
mean 7.2 ± 58.6 ± 19.7 ± 22.8 ± 132.7 3.5 ± 0.6 ± 109.5 72.6 ± 2.5 ± 39.3 ± 13.6 ± −6.0 ± −5.2 ±
SD 0.1 12.7 13 3.4 ± 6.1 0.8 0.4 ± 2.8 24.7 1.5 11.5 3.6 3.6 3.8
min 6.9 32.1 5 16.2 117. 2.1 0.3 105 18 0.9 10 3.9 −16.6 −14.6
max 7.3 84.5 69.3 29.7 144.0 5.8 1.7 119 131 9.3 62 21 −1.1 1

Table 3. The comparison between the chosen parameters of umbilical cord blood gas analysis
(UCBGA) collected at birth (0 min) in puppies classified with Apgar score (AS) as critical, weak or
healthy at different time points of the study (0, 5th, 20th min).

Apgar Score Parameters 0 Min 5th Min 20th Min

n = 24 n=7 n=2
pH units 7.2 ± 0.1 * 7.2 ± 0.1 7.1 ± 0.01 A
pCO2 mmHg 60.2 ± 12.4 60 ± 13.2 69.7 ± 14.7
CRITICAL
BE(b) mmol/L −7.1 ± 4 * −7.8 ± 3.3 −9.2 ± 2.9 A
AS < 4
BE(ecf) mmol/L −6.2 ± 4.2 * −6.5 ± 3.4 −7.1 ± 4.5 A
Lac mmol/L 2.6 ± 1.7 2.3 ± 1.2 3.9 ± 0.8 A
Glu mg/dL 79.3 ± 21.8 95.3 ± 2.8 * 90 ± 33.9 A
n = 16 n = 22 n=4
pH units 7.2 ± 0.1 * 7.2 ± 0.1 7.2 ± 0.1
pCO2 mmHg 57.6 ± 13.2 59.8 ± 13 54 ± 9.2
WEAK
BE(b) mmol/L −4.5 ± 2.3 * −5.9 ± 4.1 −7.2 ± 4.8
AS 4–6
BE(ecf) mmol/L −3.7 ± 3 * −5 ± 1 −6.8 ± 4.8
Lac mmol/L 2.1 ± 1 2.4 ± 1 2.1 ± 0.1
Glu mg/dL 67.9 ± 24.4 72.4 ± 15.3 * 72 ± 20
n=2 n = 13 n = 36
pH units 7.3 ± 0.1 A 7.2 ± 0.1 7.2 ± 0.1
pCO2 mmHg 47.3 ± 10.1 A 56.5 ± 13 58.5 ± 12.8
NORMAL
BE(b) mmol/L −5.6 ± 0.1 A 5.3 ± 3 5.8 ± 3.4
AS > 6
BE(ecf) mmol/L −5.6 ± 0.6 A −4.7 ± 3.8 −4.9 ± 3.8
Lac mmol/L 2.8 ± 0.8 A 2.7 ± 2.1 2.4 ± 1.5
Glu mg/dL 30.5 ± 3.5 A 64.4 ± 29.2 * 71.8 ± 25
* Indicates significant differences between values in a column (p < 0.05); A Indicates values excluded from
statistical analysis due to the low number of cases (n = 2).

4. Discussion
Identification of newborn puppies at high risk of developing adverse outcomes after
delivery is the main goal in advancing veterinary neonatology. The presented research
intended to give a chance for a better understanding of basic chemical and physical changes
which occur in a neonate just after birth and to establish the most apparent clinical and
biochemical findings influencing the early neonatal life. In human medicine, both the
umbilical cord blood and Apgar score were proved to provide valuable information on the
neonatal status. To the best of the authors’ knowledge, this is the first report presenting
the full umbilical cord blood gas analysis in newborn puppies together with the clinical
evaluation of their health state.
In dogs, same as in human medicine, the initial newborn puppy assessment should
include Apgar scoring, however, based on the obtained results and the available literature,
the Apgar score obtained just after birth (0 min) seems to be less predictive for further
puppy outcome in comparison to the next evaluation carried out few minutes after birth.
It has been noted in human babies that the transition time may vary in normal infants,
Animals 2021, 11, 685 8 of 12

so the low initial Apgar score is often due to a transient low oxygen saturation, which
resolves spontaneously when normal respiration is established [20]. Hence, in humans,
the Apgar score is routinely measured at 1 min and at 5 min after birth [21]. In this study,
most of the newborns who scored as critical (n = 24) immediately after birth, improved
and developed satisfactory condition in the following evaluations (75% of puppies had
AS ≥ 7 in 20th min after birth). Our Apgar score results at 0 min were much lower than
those reported by Vilar et al. [22], however, it might be due to the different surgical and
anesthetic protocols used or the exact time of the initial Apgar assessment, which in our
study was performed prior to any medical attention provided for a newborn. The exact
time of cord clamping could also influence the neonatal state as reported by Pereira et al.
who described a higher vitality in puppies in which the umbilical cord was preserved intact
for a least 3 min after birth to allow the residual blood to flow to the puppies [23]. On the
other hand, Davidson [1] suggested that neonates delivered via Cesarean section tend not
to initiate respiration spontaneously, which may affect their initial Apgar score records.
Considering the above, the vitality scoring performed immediately after birth seems to be
useful in predicting short-term abnormalities, but it does not provide information on the
long-term outcome, and the way of puppy handling might play a role in the subsequent
neonatal performance.
In dogs, some authors measured the Apgar score for the first time at 5 min after birth,
which same as in our investigations, corresponded much better with the further puppy
performance [3,9,22,24,25]. Nevertheless, the authors of this paper agree with Batista
et al. [3] that the initial (0 min) Apgar assessment should still be performed, mostly to
help the veterinary staff in triage and evaluating the amount of care a pup would need.
Contrary to human medicine, where typically only one infant is born, veterinary personnel
usually deals with several puppies. Therefore, early and accurate identification of the
neonate’s condition is essential to define which newborn requires immediate assistance
and to provide an adequate level of clinical care necessary to improve the viability and
survival of each puppy [3]. The suitable first aid provided for a newborn might be crucial
for its further performance and survival. It is worth noting that 11.9% (n = 5) of pups did
not survive the first 24 h, despite neonatal critical care provided, indicating some predictive
value of the initial Apgar scoring.
The detailed comparison of Apgar score results obtained here and reported in the
literature is difficult due to different time schedules of Apgar evaluation, mode of delivery
and the potential influence of medications given to the dams. Some authors measured
Apgar score only once [3,12,16], whereas others twice at 0 and 60th min [22], or three times
at 0, 5th and 60th min [9] or at 5th, 15th and 60th min after delivery [24]. The adopted time
schedule of Apgar score recording in our study aimed firstly (AS at 0 min) at recognizing
the weakest newborns and indicating whether a puppy requires medical intervention. The
second examination (AS at 5 min) helped to determine whether a neonate improved after
the neonatal care or resuscitation provided. The following scoring (AS at 20 min) confirmed
the status of neonates and allowed the decision to stop resuscitation in non-responding
pups. All newborns underwent a full clinical examination before discharging to ensure all
puppies are well enough to go home. In our clinical settings such time schedule turned
out to be the most efficient for the veterinary staff involved in neonatal care and decision
making process.
In human babies, the pH and BE possess the high prognostic value of an adverse
neonatal outcome as they reflect respiratory status and acid–base imbalances [15,26]. When
linked to the current newborn status it would help to determine the underlying cause of the
blood gas changes. The same as other authors [25,27], we found the newborns to be mildly
acidemic with a mean pH value of 7.2, while Lucio et al. [27] reported for vaginally born
puppies the pH at 7.1 and Vassalo et al. [25], similar to our results, at 7.2 for both eutocia and
Cesarean section. The acid production in newborns is buffered to maintain the pH within
limits. However, if the delivery of oxygenated blood through the placenta is disrupted,
hypoxia occurs and fetal metabolism shifts to the anaerobic pathway with the production
Animals 2021, 11, 685 9 of 12

of lactates. With high lactic acid production, the buffer system becomes insufficient leading
to neonatal acidosis, low pH and poor Apgar scores [28,29]. We observed lower pH values
in puppies classified as critical at birth (7.18 vs. 7.27 in puppies receiving 7 or more points
in Apgar scoring at birth). The lower pH was positively correlated with BE increase and
a trend towards higher pCO2 levels. However, the puppies examined in our research
had low mean concentrations of lactates (2.45 mmol/L), bicarbonates on the lower border
(22.78 mmol/L), decreased BE(b) (−6.04 mmol/L) and moderate increased partial pressure
of CO2 (58.59 mmHg) which suggest both, respiratory and metabolic component as the
reason of the pH changes. This is in agreement with other papers where authors reported
mixed acidosis being the cause of poor neonatal performance [25,27]. Moreover, in our
study puppies receiving high Apgar scores at birth (AS ≥ 7) had also lower pCO2 and
higher pH compared to the rest of the investigated pups. Unfortunately, we had only
two such puppies, which did not allow for the statistical analysis, but their results still
suggest the respiratory component in the observed neonatal acidemia. Same as mentioned
before, the discrepancies between our results and those reported by other authors may
be due to different vessels used for obtaining the blood sample. We collected the mixed
(venous–arterial) umbilical cord blood, whereas Lucio et al. and Vassalo et al. [25,27]
collected venous blood from the jugular vein to evaluate neonatal acidemia.
Recently, several studies have examined the efficiency of the combination of Apgar
scoring system with umbilical cord blood gas assessment (UCBGA), lactate measurement
or acid–base analysis, proving to be a useful diagnostic tool especially in the intensive
care units [15,26]. Articles published in human medicine showed that the measurement
of lactate concentration in umbilical blood is a good predictive factor for neonatal out-
come [30–33]. Further, the relationship between the type of delivery and the lactate content
was reported, with the highest lactate level in babies delivered instrumentally and the low-
est after Cesarean section [34]. Our results demonstrated the low mean value of umbilical
lactate concentration (2.45 mmol/L). This result is consistent with the study of Groppetti
et al. [12] who showed the lowest lactate levels in puppies born by elective Cesarean section
(3.9 mmol/L) compared to the emergency procedures (6.3 mmol/L) or vaginal parturition
(6.8 mmol/L). While, Castagnetti et al. [35] reported for vaginal delivery the initial lactate
at the level of 6.7 mmol/L in surviving newborns, and 10 mmol/L in those that died within
the first day of life. Hyperlactatemia would develop in the late stage of fetal hypoxia, when
the lack of oxygen leads to anaerobic metabolism and the production of lactic acid [33].
Thus the mode of delivery and the uterine activity during labor, may contribute to the
switching into the anaerobic condition and therefore affect the level of lactates. Moreover,
Groppetti et al. [12] sampled the umbilical cord up to 5 min after delivery, whereas Castag-
netti et al. [35] obtained the blood sample by puncture of puppy’s finger pad. The time of
sampling in the first report and another vessel used for blood collection may explain the
differences in the presented results. Groppetti et al. [12] also found a relationship between
lactate concentrations in healthy and unhealthy neonates, and proposed the cut-off value
for lactate at 5 mmol/l, to distinguish between healthy and distressed pups. However, we
did not observe such a correlation between lactate levels in newborns classified as critical
and weak. Unfortunately, we were not able to include into this comparison the results
obtained in pups classified with Apgar score over 7 at birth, due to the low number of cases
(only two puppies), however their lactate levels were 2.24 and 3.38 mmol/L respectively.
Interestingly, the two critical puppies which died within first 20 min after delivery had
higher initial lactate levels (4.42 and 3.3 mmol/L, respectively).
We also noted differences in the glucose levels between puppies classified in different
time points (0 and 5 min) as critical (AS ≤ 3), weak (AS 4–6) and normal (AS ≥ 7). Puppies
classified based on Apgar score as still being critical at 5 min, had significantly higher
levels of glucose in comparison to the weak and normal neonates. Critical pups from 0 and
5th min showed also a negative correlation between AS and glucose levels, which may
indicate its impact on the newborn clinical state. The available literature does not provide
sufficient information on the relationship between glucose concentration in umbilical blood
Animals 2021, 11, 685 10 of 12

and neonatal survival in dogs. Castagnetti et al. [35] observed a tendency to higher glucose
levels in non-surviving comparing to surviving neonates, which is in agreement with our
results. On the contrary, other studies showed that the low glucose levels were related
to poor newborn puppy performance. Vessalo et al. [25] found that newborn puppies
with glucose levels below 40 mg/dL had usually low Apgar scores and poor reflexes
(suckling, rooting and righting reflex). Our results showed the opposite relationship:
pups receiving 7 and more Apgar points in the initial evaluation had lower mean glucose
concentration (30.5 ± 3.53 mg/dL). In humans, similarly to our results, Khan et al. [36]
showed that babies with fetal distress (identified by the meconium-stained fetal fluid
and fetal bradycardia) had higher glucose levels in the umbilical cord blood. Since, the
glucose concentration decreases with the impaired utero-placental perfusion or increased
fetal consumption [18], the mode of delivery in our study (elective Cesarean section)
vs. eutocia and dystocia resolved via Cesarean section in Vassalo et al. [25] research
may explain the presented discrepancies. These findings were also confirmed by Lucio
et al. (2021) showing differences in maternal and neonatal glucose levels depending
on delivery mode [37]. Authors found hyperglycemia in puppies from C-section and
fetal dystocia groups whereas puppies from eutocia and maternal dystocia remained
normoglycemic. One of the proposed theories suggested hyperglycemia is the response
to peripartum stress (increasing catecholamine, hypoxia, etc.). This explanation may
support our findings that newborns with low Apgar scores had higher glucose. On the
other hand, Lucio et al. [37] and Vanspranghels et.al [30] suggested that glucose level
reflects rather maternal physiology than fetal status and did not appear to be useful for
predicting neonatal morbidity. Moreover, the abovementioned concern regarding the vessel
used for blood sampling could have also contributed to the differences in the presented
results. Considering the above, further studies are necessary to explain these changes in
the reported results.
Although puppies born via elective Cesarean section may initially present asphyxia
and obtain low Apgar scores, in most of the cases, their state improves substantially
within the next few minutes when neonatal care is provided and the proper respiration
is established. In the light of the presented results, the umbilical cord blood collection
was feasible, and the cord vessels provided sufficient blood for further analysis. UCBG
analysis contributed to obtaining new and important data regarding newborn metabolic
states at the transition time. If further investigated, the umbilical cord blood may allow
us to set the reference values for the evaluated parameters and present association with
medium and long-term neonatal outcomes. The improvement of neonatal assessment
protocol should elucidate all factors and circumstances affecting the neonatal state, provide
practical knowledge for practicing veterinarians, and hopefully decrease the mortality rates
in puppies.

5. Conclusions
The detailed evaluation of a newborn state is crucial in the overall understanding of
neonatal physiology. In our study, the newborn puppies had mild acidemia with elevated
pCO2 levels and the HCO3 at the lower range of normal limits suggesting the mixed
component in the acidemic state. However, the lack of reference values in the available
literature makes it very difficult to interpret the obtained data. Contrary to what was
expected, the lower Apgar score results were, the higher glucose levels were found in
newborns, and higher mortality rates were observed. Only pups with the lowest initial
Apgar scores were at risk of death within the first 24 h.
It is necessary to increase the knowledge and understanding of clinical and metabolic
changes in newborn puppies, since the puppy mortality rate is still high in veterinary
medicine. Adaptation to the extra-uterine life is crucial and any practical improvement in
neonatal diagnostics and care would be beneficial for newborn puppy survival.
Animals 2021, 11, 685 11 of 12

Author Contributions: A.A.—conceptualisation, data collection, data evaluation and statistical

analysis, M.O.—conceptualisation, data collection and writing of the manuscript, W.N.—supervising
the study, reviewing and editing the paper. All authors have read and agreed to the published version
of the manuscript.
Funding: This study was co-funded by Wroclaw University of Environmental and Life Sciences,
project no B030/0026/20. The publication costs were co-financed under the Leading Research Groups
support project from the subsidy increased for the period 2020–2025 in the amount of 2% of the
subsidy referred to Art. 387 (3) of the Law of 20 July 2018 on Higher Education andScience, obtained
in 2019.
Institutional Review Board Statement: The study was approved by the II Local Ethics Committee
for Animal Experiments of the University of Environmental and Life Sciences in Wroclaw (No.
047/2020).
Data Availability Statement: The data that support the findings of this study are available from the
corresponding author [MO], upon reasonable request.
Conflicts of Interest: No conflict of interest to be declared.

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