HAEMATOLOGICAL AND SERUM BIOCHEMICAL REFERENCE VALUES EXTRAPOLATED FROM REHABILITATED STRANDED SPINNER DOLPHINS (Stenella longirostris)

Abstract

The spinner dolphin (Stenella longirostris) is one of the smallest odontocetes that commonly strands in the Philippines. Despite its apparent abundance and cosmopolitan distribution, there is no published data on the haematological and serum chemical reference values for this species. This limitation greatly affects the ability of veterinarians and marine mammal rehabilitators to make informed decisions in the diagnosis and treatment of diseases, and the formulation of action plans when individuals of this species strand alive. This study used blood samples from two subadult female spinner dolphins that were successfully rehabilitated after stranding in 2014 to establish haematological and serum chemistry reference values for the species. The overall resulting values of the blood parameters recorded such as the WBC (5.01-10.45 10 3 /μL), RBC (4.23-6.18 10 6 /μL), and PCV (42-50%) generally demonstrated narrow ranges and were close to the published reference values for other similar conspeci c odontocetes. Although there were only two individuals of the species used in this study, the data gathered serves as a valuable reference tool for future cases of spinner dolphin strandings in the Philippines.

Full text

HAEMATOLOGICAL AND SERUM BIOCHEMICAL REFERENCE VALUES
EXTRAPOLATED FROM REHABILITATED STRANDED SPINNER
DOLPHINS (Stenella longirostris)
Leo Jonathan A. Suarez, DVM1,3*, Jonalyn I. Paz, MT1, Jean T. Balquin, MT1
and Lemnuel V. Aragones, PhD2,3
1Ocean Adventure, Camayan Wharf, West Ilanin Forest Area, Subic Bay Freeport Zone, 2222,
Philippines; 2Institute of Environmental Science & Meteorology, University of the
Philippines, Diliman, Quezon City, 1101, Philippines; 3The Philippine Marine Mammal
Stranding Network Inc, IESM, UP Diliman, Quezon City, 1101, Philippines
ORIGINAL ARTICLE
ABSTRACT
The spinner dolphin (Stenella longirostris) is one of the smallest odontocetes
that commonly strands in the Philippines. Despite its apparent abundance and
cosmopolitan distribution, there is no published data on the haematological and
serum chemical reference values for this species. This limitation greatly affects
the ability of veterinarians and marine mammal rehabilitators to make informed
decisions in the diagnosis and treatment of diseases, and the formulation of action
plans when individuals of this species strand alive. This study used blood samples
from two subadult female spinner dolphins that were successfully rehabilitated after
stranding in 2014 to establish haematological and serum chemistry reference values
for the species. The overall resulting values of the blood parameters recorded such
as the WBC (5.01-10.45 103/μL), RBC (4.23 – 6.18 106/μL), and PCV (42 – 50%) generally
demonstrated narrow ranges and were close to the published reference values for
other similar conspeci c odontocetes. Although there were only two individuals of
the species used in this study, the data gathered serves as a valuable reference tool
for future cases of spinner dolphin strandings in the Philippines.
Key words: haematology, blood values, reference range, serum chemistry, spinner dolphin
Philipp. J. Vet. Med., 56(2): 114-121, 2019
*FOR CORRESPONDENCE:
(email: leo_sj@yahoo.com)
mammal that strands in the country (Aragones
et al., 2017). Illness, disease, trauma or simply
getting lost are among a variety of reasons
that may cause a cetacean to strand. One of
the objectives of a stranding response team
for a live stranded cetacean is to rehabilitate
the animal (Gulland et al., 2001; Geraci and
Lounsbury, 2005; Laule et al., 2013). Gathering
objective information on the health status of a
stranded cetacean is important in determining
the possible cause of the stranding and the best
course of action to take towards rehabilitation
INTRODUCTION
The spinner dolphin Stenella longirostris
is one of the most common cetaceans in the
world that occurs throughout the tropics
and subtropics (Braulik and Reeves, 2018;
Reeves et al., 2002). It appears to be the most
abundant and widely distributed cetacean
species in the Philippines (Dolar et al., 2006),
as well as the most common species of marine
114

(Schwacke et al., 2009). Blood is the most
important diagnostic sample needed to assess,
diagnose, and medically manage a sick animal
(Manire et al., 2018; Flores et al., 2013).
However, haematological reference values
are necessary for the useful interpretation
of laboratory results and assessment of the
health of the animal (Schwacke et al., 2009;
Klaassen, 1999). These values usually vary
among species, between sexes, age groups, and
environmental conditions (Manire et al., 2018;
Norman SA et al., 2013; Kasamatsu et al.,
2012; Bossart et al., 2001). Unfortunately, only
a few comprehensive haematological studies
have been done on marine mammals. The
lack of haematological reference values for the
different species of cetaceans greatly affects the
ability of veterinarians and marine mammal
rehabilitators to make informed decisions in
the diagnosis, treatment, and plan of action
for live dolphin stranders (Sharp et al., 2014).
This challenge is especially important in the
Philippines because majority (65%) of cetaceans
that stranded in the country from 2005 to 2016
were alive (Aragones et al., 2017). As far as the
authors’ knowledge and research is concerned,
there is no published data on the reference
blood values for spinner dolphins despite the
species’ apparent abundance and cosmopolitan
distribution. The objective of this paper is to
establish preliminary haematological and
serum chemistry reference values for spinner
dolphins that may be used for future stranding
cases of the species in the Philippines, as well
as in other parts of the world.
MATERIALS AND METHODS
Blood samples from two stranded subadult
female spinner dolphins that were successfully
rehabilitated were used in this study. These
animals stranded in February and March
2014, respectively, in the west coast of Luzon,
Philippines. Based on the auditory brain
response test conducted in April 2014, both
individuals suffered from varying degrees of
hearing loss and were deemed un t for release.
These dolphins were provided with long-term
human care in a sea pen complex of a marine
theme park in Subic Bay Freeport Zone,
Philippines.
Since the two dolphins were already
deemed unreleaseable (see above), both were
trained basic husbandry behaviors, including
blood collection. By October 2014, both dolphins
were already trained to voluntarily present
their tail  uke for venipuncture. Venipunctures
were performed on the ventral aspect of the
tail  uke using 23G x ¾ inch Luer Lock blood
collection set (BD Vacutainer®). Blood was
collected directly into three different vacutainer
tubes containing sodium citrate, serum
separator gel, and ethylenediaminetetraacetic
acid (EDTA), respectively. Complete blood
counts (CBC) were performed manually using
the Unopette system (BMP Leukocheck®) for
white blood cell (WBC) counts, and Hayem’s
diluting reagent for red blood cell (RBC) counts.
WBC differential counts were determined by
microscopic examinations of Wrights-Giemsa-
stained smears. Blood samples in haematocrit
tubes were spun at 11,000rpm for  ve minutes
to determine the packed cell volume (PCV).
Haemoglobin was measured using a Sysmex
XF2000. Mean corpuscular volume (MCV),
mean corpuscular haemoglobin (MCH), and
mean corpuscular haemoglobin concentration
(MCHC) were calculated using Microsoft Excel
365.
Erythrocyte sedimentation rate (ESR) was
determined using Westergren ESR system.
The serum chemistry and electrolytes were
identi ed using blood serum analyser IDEXX
VetTest® and VetLyte®, respectively, except
for  brinogen, which was tested in another
laboratory using Sysmex CS1600.
Although the two dolphins appeared
healthy and apparently normal after four
weeks in the rehabilitation facility, they
continued receiving medications for another
sixteen months, as several of their blood
parameters were outside the normal ranges
when compared to the haematological
reference values extrapolated from a single
male adult pantropical spotted dolphin. Blood
test results from routine health screening,
follow-ups, and during episodes of apparent
illness between September 2015 and April
2018 were examined. For this paper, only
blood test results from samples collected when
the animals were apparently healthy and not
BLOOD VALUES FROM REHABILITATED STRANDED SPINNER DOLPHINS 115

SUAREZ et al.
receiving medications, and with sera that
appeared normal on visual inspection were
considered. A total of 44 (26,18) blood test
results were included in this investigation.
For some samples, only speci c tests were
requested, so not all parameters were
determined for these. Descriptive statistics
(mean, standard deviation, median, and range)
were used to calculate and summarize the data
using Microsoft Excel 365.
RESULTS AND DISCUSSION
The haematological and serum biochemical
pro les in this study generally showed narrow
ranges (Tables 1 and 2). The means of most of
the blood parameters were equal or were very
close to the median values of the pooled samples.
The results were compared with several
published reference values for rehabilitated
stranded and free-range delphinids, including
rough-toothed dolphin (Manire et al., 2018),
pantropical spotted dolphin (St. Aubin et al.,
2011), Atlantic bottlenose dolphin (Goldstein et
al., 2006), common dolphin, common bottlenose
dolphin (Bossart et al., 2001) and Fraser’s
dolphin (Rhinehart et al., 1995) (Tables 3 and
4).
The total WBC count for the spinner
dolphins in this study overlapped with the
reported reference ranges for Fraser’s dolphin
(4.2-7.2 x103/µL), rough-toothed dolphin (6.1-
6.7 X103/µL), pantropical spotted dolphin (7.6-
Table 1. Haematological values extrapolated from the two stranded spinner dolphins.
116

12.6 x103/µL), Atlantic bottlenose dolphin (7.73-
12.89 x103/µL), and common bottlenose dolphin
(5.6-12.4 x103/µL). Among the  ve species, the
WBC counts for the Fraser’s dolphin and rough-
toothed dolphin had the lowest and narrowest
reference ranges. Samples from the rough-
toothed dolphins were taken from healthy
individuals under human care, and most of
which were collected under a trained voluntary
procedure. The samples from the Fraser’s
dolphins, on the other hand, were taken from
a pod that was trapped in a water inlet. The
trapped Fraser’s dolphins were either  oating,
actively milling, or moving around the water
(Rhinehart et al., 1995). In contrast, the samples
from the pantropical spotted dolphin, Atlantic
bottlenose dolphin, and common bottlenose
dolphins were taken from wild animals
that were captured for health assessment,
including blood collection. Thus, it is possible
that the higher ranges noted for these three
species were in uenced by the effects of stress
after having been chased by boats, captured,
and restrained. On the other hand, the blood
sampling conditions for the rough-toothed
dolphin and Fraser’s dolphin (St. Aubin et al.,
2011), as well as the spinner dolphin in this
present investigation were less stressful. The
absolute differential WBC count in this study
is also generally similar and overlapped with
published values for most of the other species.
However, the lymphocyte count ranges are
lower in the Fraser’s dolphin (387-897/µL),
common dolphin (380-850/µL), and rough-
Table 2. Serum biochemical values extrapolated from the two stranded spinner dolphins.
BLOOD VALUES FROM REHABILITATED STRANDED SPINNER DOLPHINS 117

SUAREZ et al.
toothed dolphin (953-1135/µL). Eosinophil
counts are lower in the Fraser’s dolphin (0-239/
µL) and rough-toothed dolphin (408-712/µL),
and higher in the Atlantic bottlenose dolphin
(2080-4620/µL). Monocyte counts were higher
in the pantropical spotted dolphin and rough-
toothed dolphin in comparison to the spinner
dolphin. The RBC, PCV, and haemoglobin
ranges for the spinner dolphin were similar
or fall within the ranges for the pantropical
spotted and common dolphins.
The serum chemistry pro les in this study
demonstrate some similarities to the published
reference values for the different species.
Several parameters were similar or overlapped,
particularly the liver parameters aspartate
transferase (AST) and alkaline phosphatase
(AP). Alanine transferase (ALT) level was
higher in the spinner dolphin compared to most
of the other species but overlapped with that of
pantropical spotted dolphin (86.7-167.3 U/L)
and Fraser’s dolphin (46-156 U/L). Gamma-
glutamyltransferase (GGT) level was higher
in the spinner dolphins than the other species,
while that of total bilirubin overlapped with
reference ranges of most of the other species,
except for the Atlantic bottlenose dolphin
(0.06-0.12 mg/dL) and Fraser’s dolphin (1.1-1.7
mg/dL).
The level of globulin was also the same
or overlapped with the reference ranges of the
different species except that of the common
dolphin, which was lower at 1.8-3.0 g/dL. The
serum albumin level of the spinner dolphin
was low compared to most of the other species,
but it was within the reference range of that of
the Fraser’s dolphin (3.1-3.5 g/dL) and common
bottlenose dolphin (2.9-3.7 g/dL). Similarly,
the level of total protein was the same or
overlapped with all of the other species, except
Table 3. Published haematological values of select species of dolphins compared to the spinner
dolphin covered by this study.
118

while the level of calcium overlaps with those of
all the other species except the rough-toothed
dolphin (8.8-9.0 mEq/L).
Erythrocyte sedimentation rate, which has
been useful in determining the presence and
degree of in ammation (Bossart, et al., 2001)
and is often used as a prognostic indicator
in dolphins (Manire et al., 2018; Bossart et
al., 2001), was only determined in the rough-
toothed dolphin study. It was higher (4.9-7.1)
in the rough-toothed dolphin compared to the
spinner dolphin. Reference ranges for plasma
 brinogen levels, which has been associated
with the ESR and has almost completely
replaced ESR as a laboratory diagnostic test
in domestic animals and humans (Bossart et
al., 2001), were only available for the Atlantic
bottlenose dolphin and pantropical spotted
dolphins. The  brinogen level in the spinner
dolphin overlapped with that of the Atlantic
for that of the rough-toothed dolphin, which
was higher (7.5-7.7 g/dL).
Reference ranges for kidney function
parameters in this study overlapped with
that for the common dolphin and common
bottlenose dolphin for both blood urea nitrogen
(BUN) and creatinine. Values for lactate
dehydrogenase (LDH) and serum electrolytes
such as sodium (Na) and chloride (Cl) were
higher in the spinner dolphins, compared to
all other species. It must be noted, however,
that the serum chemistry machine of the
laboratory used in this study yielded higher
measurements for these three parameters
when compared to those from duplicate
samples sent to two other laboratories. The
potassium level overlapped with the reference
ranges for common bottlenose dolphin (3.2-4.4
mEq/L), Fraser’s dolphin (2.8-3.6 mEq/L), and
pantropical spotted dolphin (3.3-4.7 mEq/L),
Table 4. Published serum biochemical values of select species of dolphins compared to the spinner
dolphin covered by this study.
BLOOD VALUES FROM REHABILITATED STRANDED SPINNER DOLPHINS 119

SUAREZ et al.
bottlenose dolphin (48.92-227.36 mg/dL) and
was lower than that of the pantropical spotted
dolphin (249.8-488.8 mg/dL).
To further investigate the plausibility
of the extrapolated blood values, parameters
that were outside the normal limits during
the rehabilitation were compared to the  rst
twenty blood results of the two dolphins taken
within the  rst 11 and 13 weeks of their
rehabilitation, respectively. The two dolphins
had in ammation (elevated WBC count and
ESR) likely from liver insult (elevated ALT and
AST), muscle injury (elevated CK), and possibly
other soft tissue injuries (elevated LDH). They
were dehydrated (elevated PCV) and had
electrolyte imbalances. Their AP, which has
been used as a prognostic indicator for some
species of dolphins (Bossart et al. 2001), were
low. These were the same interpretation of their
blood results when compared to the reference
values of a pantropical spotted dolphin.
Fluctuations in these parameters moved
towards the normal values and plateaued,
particularly ESR, PCV, AST, Na, and Cl.
Conventionally, haematology and serum
chemistry parameters can vary with sex, age,
season, and physiological state (Manire et al.,
2018; Kasamatsu et al., 2012, St. Aubin et
al., 2011, Goldstein et al., 2006). Reference
ranges derived from animals in the wild are
known to differ from their counterparts under
human care, due to physiological adaptations
in captivity, and the stress and physiological
effects of handling for those that were caught
from the wild (Sharp et al., 2014; St. Aubin et
al., 2011; Bounous et al., 2000). Nevertheless,
some studies showed similar reference ranges
for both wild and captive dolphins (Kasamatsu
et al., 2012; Goldstein et al., 2006).
Reference ranges are normally generated
from representative samples of a species or
population in ample numbers (Manire et al.,
2018; Schwacke et al., 2009). The samples for
the reference ranges in this study came from
only two individuals and is not statistically
representative of the species or population.
However, this is currently the only available
reference value for this species and will still
serve as a valuable tool for veterinarians and
marine mammal rehabilitators for future
stranding events of spinner dolphins in the
Philippines. This is critical as the spinner
dolphin is the most common strander in the
country as per the Philippine Marine Mammal
Stranding Database (Aragones et al. 2017).
ACKNOWLEDGEMENT
This study would not have been possible if
the dolphins used in this study were not rescued
and rehabilitated. The following institutions
contributed to the efforts in saving “Valentina”
and “Scarlet”: the Philippine Marine Mammal
Stranding Network (PMMSN), Ocean
Adventure, Bureau of Fisheries and Aquatic
Resources Regional Of ces I and III, the
Provincial Veterinary Of ce and Provincial
Agriculture Of ce of Ilocos Norte. Many thanks
to Dr. Emilia Lastica for her helpful insights
in writing this manuscript. Many thanks to
the trainers and animal care team of Ocean
Adventure for helping us collect the blood
samples and ensuring the welfare of these two
dolphins during and after their rehabilitation.
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