General and Comparative Endocrinology 170 (2011) 650–656

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General and Comparative Endocrinology

journal homepage: www.elsevier.com/locate/ygcen

Relaxin and progesterone during pregnancy and the post-partum period

in association with live and stillborn calves in bottlenose dolphins
(Tursiops truncatus) q
Don R. Bergfelt a,⇑, Bernard G. Steinetz b, Salamia Lasano b, Kristi L. West c, Michelle Campbell d,
Gregg P. Adams e
a
US Environmental Protection Agency, Washington, DC 20460, USA
b
New York University School of Medicine, Tuxedo, NY 10987, USA
c
Hawaii Pacific University, Kaneohe, HI 96744, USA
d
Dolphin Quest and Quest Global Management, Waikoloa, HI 96738, USA
e
University of Saskatchewan, Saskatoon, SK, Canada S7N 5B4

a r t i c l e i n f o a b s t r a c t

Article history: The objectives of this study were to validate a relaxin and progesterone RIA for use in bottlenose dol-
Received 1 September 2010 phins, and quantify and characterize both hormones in extracts of placental tissue and serum collected
Revised 1 December 2010 during pregnancy and the post-partum period, and compare the results between dolphins with live
Accepted 5 December 2010
and stillborn calves. In Experiment 1, validation of a heterologous relaxin and progesterone RIA involved
Available online 13 December 2010
specific displacement of antibody-bound radiolabeled human relaxin or progesterone in response to
increasing volumes of pooled pregnant dolphin serum and amounts of respective hormone standards
Keywords:
added to a fixed volume of serum. The displacement curves were considered parallel and additive relative
Bottlenose dolphins
Pregnancy
to respective standard curves. In Experiment 2, immunoreactive relaxin and progesterone were detected
Progesterone in placental extracts and, in corresponding serum samples, concentrations of both hormones were higher
Relaxin during the pre-partum than post-partum periods. Circulatory concentrations of progesterone decreased
Stillbirths (P < 0.05) from relatively high concentrations during early and mid-pregnancy to intermediate concen-
trations by late pregnancy (month effect, P < 0.0001) in dolphins with live births, whereas, in dolphins
with stillbirths, the decrease in progesterone began earlier (month-by-birth status interaction,
P < 0.007); mean concentrations were lower at mid- (37%, P < 0.06) and late (25%) pregnancy. Temporally,
relaxin increased (P < 0.05) progressively from relatively low concentrations during early pregnancy to
high concentrations during late pregnancy (month effect, P < 0.0001) and was not different between birth
statuses (birth status effect, P = 0.76; month-by-birth status interaction, P = 0.17). Even though the inter-
action did not reach significance, mean relaxin concentrations were 42%, 29%, and 34% lower at early,
mid-, and late pregnancy, respectively, in dolphins with stillbirths than in those with live births. In con-
clusion, the pregnancy-specific increase in serum concentrations of relaxin and lower concentrations of
both relaxin and progesterone in association with stillbirths suggest the potential for relaxin to be used
diagnostically to determine pregnancy status, and one or both hormones to be used to assess placental
function, and, perhaps, fetal well-being in bottlenose dolphins and other cetaceans.
Published by Elsevier Inc.

1. Introduction conservation and research to increase our basic understanding of

Bottlenose dolphins (Tursiops truncatus) are the most common
species of dolphin found in aquariums and oceanariums world-
wide. Dolphins born in aquatic facilities and those obtained
from the wild are a valuable resource for education on species
q
The views expressed in this article are those of the authors and do not
necessarily reflect the views or policies of the USEPA or other institutions
represented by the authors.
⇑ Corresponding author. Fax: +1 202 564 8483.
E-mail address: bergfelt.don@epa.gov (D.R. Bergfelt).
less well known and threatened species of cetaceans (i.e., dolphins,
porpoises, and whales), and for improving dolphin husbandry and
reproductive management practices. Management of an effective
and efficient breeding program is dependent, in part, on the
capability to distinguish between pregnant and pseudopregnant
dolphins for an accurate diagnosis of pregnancy. Pseudopregnancy
in dolphins is not thoroughly understood, but it appears that the
lifespan of the corpus luteum (CL) and period of elevated concen-
trations of progesterone can occasionally be prolonged (e.g.,
5–6 months) in nonpregnant (nonbred and bred) dolphins
[29,46], well beyond the expected length of the luteal phase

0016-6480/$ - see front matter Published by Elsevier Inc.

doi:10.1016/j.ygcen.2010.12.002
 D.R. Bergfelt et al. / General and Comparative Endocrinology 170 (2011) 650–656
(16–23 days) [27]. Although an increase in serum concentrations of
progesterone in successive samples may be highly predictive of
pregnancy in dolphins (review, [30]), the presence and viability
of an embryo/fetus is still questionable until it is confirmed with
ultrasonic imaging [45]. In regard to the latter, many dolphin facil-
ities do not have funding or experience to support the use of ultra-
sonography. Thus, an alternative method to progesterone assay
and ultrasonographic imaging is needed for wider application
and acceptance among aquariums and oceanariums for distin-
guishing between pregnancy and pseudopregnancy in dolphins
and diagnosing the presence of a viable embryo/fetus.
Relaxin is a 6-kDa polypeptide related to the insulin family of
hormones and growth factors (reviews, [1,32]). In laboratory and
domestic species (reviews, [1,11,22,25,33]), the greatest sources
of relaxin production during pregnancy are the embryo/feto-pla-
cental unit and CL. Although relaxin has a major role in softening
the cervix and pelvic ligaments to facilitate parturition, it also
has an effect on the uterus to maintain myometrial quiescence,
especially as the quiescent effect of progesterone declines prior
to parturition [25,33].
Conservation of the amino acid sequence for relaxin among spe-
cies is relatively low (<76%) [33]; hence, it has been challenging to
develop and validate immunological methods to detect and char-
acterize relaxin in many domestic and nondomestic mammalian
species. Nonetheless, homologous and heterologous radioimmuno-
assays (RIA) involving canine, equine, and porcine relaxin antisera
have been used successfully to quantify immunoreactive relaxin in
plasma, serum, and urine from wild species such as leopards [7],
rhinoceroses, elephants [20,36], coyotes [5], spotted hyenas [40],
and Northern fur seals [2]. Relaxin has also been quantified in ex-
tracts of placental tissue from the spotted hyena [40] and several
species of seals [2]. Although relaxin has not been detected in dol-
phins, it has been detected in extracts of luteal tissue from a preg-
nant Antarctic minke and Bryde’s whale using a porcine relaxin RIA
with sheep anti-porcine relaxin [31]. In veterinary medicine, a
homologous canine relaxin RIA is used to diagnose pregnancy in
dogs [38], which have relatively high circulatory concentrations
of relaxin during pregnancy compared to low concentrations dur-
ing pseudopregnancy (review, [15]). Thus, considering the diagnos-
tic value of a relaxin assay in dogs, detection of relaxin in whales,
and pregnancy-specific changes in circulatory concentrations of re-
laxin in some terrestrial and marine mammals, the proposition
that relaxin may serve as a hormonal marker for diagnosing preg-
nancy in wild species (reviews, [36,39]), perhaps can be extended
to include dolphins.
Stillbirth typically refers to a fetus that is born dead [44]; how-
ever, the term has been more broadly defined for use in domestic
animal production to include spontaneous death of a fetus or neo-
nate near (i.e., late-term abortion), during, or within 24–48 h after
parturition [23]. Available information on the frequency and basis
for stillbirths in bottlenose dolphins is limited to a couple survey
reports involving multiple aquariums and oceanariums that com-
piled data from 1990 to 2009 on reproduction and survivability
[16,41]. In one report [16], 8.8% stillbirths (dead at parturition)
were recorded from a total of 113 births. In a more recent report
[41], 5.2% stillbirths (dead at parturition or within 24 h after partu-
rition) were recorded from a total of 249 births. Comparatively,
stillbirths ranging from 5% to 9% in captive bottlenose dolphins
are similar to 4–10% reported in dairy cattle [4] and 2–7% in thor-
oughbred horses [42]. Parity, dystocia, and placentitis have been
indicated as major contributing factors to stillbirths in cattle and
horses [4,42]. In horses, more than 60% of stillbirths have been
associated with placental insufficiency involving placentitis
[9,14]. Considering the placenta is a major source of relaxin in
horses (review, [10]), a preliminary study was conducted in mares
to compare serum relaxin concentrations in pregnancies with and
651
without periparturient complications (e.g., dystocia, placentitis)
[28]. In 12 pregnancies with complications, serum relaxin concen-
trations were significantly reduced compared to 10 pregnancies
without complications. Moreover, in all pregnancies in which re-
laxin concentrations were low, foals were born hypoxic and dys-
mature. Thus, it was proposed [28] that relaxin has the potential
to serve as a biomarker for evaluating placental function and fetal
well-being in mares.
The objectives of this study were to validate a relaxin and pro-
gesterone RIA for use in bottlenose dolphins (Experiment 1), and
quantify and characterize both hormones in extracts of placental
tissue and serum collected during pregnancy and the post-partum
period, and compare the results between dolphins with live and
stillborn calves (Experiment 2).
2. Materials and methods
2.1. Dolphins and dolphin management
A total of 16 female bottlenose dolphins involving 33 pregnan-
cies were represented in the present study. Eleven of the 16 dol-
phins were obtained from the wild during the 1970s through
1988, and five were born in aquatic facilities. Experiment 1 in-
volved three dolphins and five pregnancies with live births. Exper-
iment 2 involved 11 dolphins and 18 pregnancies with live births,
and nine dolphins and 10 pregnancies with stillbirths.
At the time of blood sampling, dolphins ranged in age from 7 to
34 years and were housed at one of five different facilities: (1) Dol-
phin Quest Hawaii (Waikoloa, HI, USA), (2) Dolphin Quest Oahu
(Oahu, HI, USA), (3) Dolphin Quest Bermuda (Dockyard, Bermuda),
(4) Dolphin Quest French Polynesia (Moorea, French, Polynesia),
and (5) Indianapolis Zoo (Indianapolis, IN, USA). Each of the Dol-
phin Quest oceanariums contained natural seawater in lagoon set-
tings ranging in size from 0.5 to 2 million gal. The Indianapolis Zoo
aquariums contained a combined total of 2.3 million gal of filtered
saltwater. At all facilities, pregnant and post-partum females were
housed with their respective calves and other adult and juvenile
male and female bottlenose dolphins. Dolphins were housed and
fed in compliance with the US Animal Welfare Act and by the Stan-
dards and Guidelines of the Alliance of Marine Mammal Parks and
Aquariums.
Based on fetal and neonatal survivability information from Dol-
phin Quest [41] and the Indianapolis Zoo, and use of the term in
domestic animal production [23], stillbirths were defined herein as
those pregnancies in which the fetus or neonate died before (i.e.,
late-term abortion), during, or within 24–48 h after parturition.
2.2. Collection of blood and placentas
The present study involved a retrospective analysis of archived
serum samples and placentas from bottlenose dolphins that were
collected during routine animal husbandry practices as part of a
proactive health and breeding program at the Dolphin Quest facil-
ities and Indianapolis Zoo. Practices and procedures were done in
compliance with US Department of Agriculture-Approved Program
of Veterinary Care.
Blood samples were collected from pregnant and post-partum
dolphins between 1993 and 2006. Preconditioned behavior was
used for the voluntary collection of blood samples which consisted
of signaling the dolphins to move into a dorsal or ventral-up posi-
tion with tail flukes placed on the lap of a trainer while sitting
dockside. A 21-gauge winged infusion set or butterfly needle at-
tached to a 12-ml syringe was used to draw blood from the arterial
venous network of the tail. Blood from each dolphin was placed in
a 10-ml serum separator tube and centrifuged within 45 min of
652 D.R. Bergfelt et al. / General and Comparative Endocrinology 170 (2011) 650–656
collection. Serum was transferred to cryo-vials, labeled with dol-
phin identification and date, and frozen at 20 °C. Serum samples
were initially stored on-site at the Dolphin Quest facilities or Indi-
anapolis Zoo. Later, some of the samples from Dolphin Quest and
all samples from the Indianapolis Zoo were transferred and stored
( 20 °C) at Hawaii Pacific University (Kaneohe, HI, USA).
A total of 47 serum samples were available to establish a pool of
pregnant dolphin serum for validation of the relaxin and progester-
one RIA in Experiment 1 and 114 samples were available for quan-
tifying and characterizing respective hormone concentrations
during pregnancy and the post-partum period in Experiment 2.
Five full-term placentas were collected at Dolphin Quest Hawaii
between 2003 and 2007 and were available for sampling, process-
ing, and assay of tissue extracts for relaxin and progesterone. The
placentas were collected from the birthing pool within 8 h after
delivery of live calves, frozen intact at 20 °C, and initially stored
at Dolphin Quest Hawaii. Placentas were later transferred and
stored ( 20 °C) at Hawaii Pacific University.
Serum samples from Dolphin Quest and serum and placentas
from Hawaii Pacific University were shipped frozen with dry ice
to the New York University School of Medicine (Tuxedo, NY,
USA) and stored at 20 °C until assay.
2.3. Experiment 1
Development of a relaxin RIA for dolphins was based on a ca-
nine relaxin RIA [37] that has been modified and validated for ser-
um and extracts of placental tissue from Northern fur seals [2] and
a porcine relaxin RIA used to assay extracts of luteal tissue from
pregnant whales [31]. In the present study, 125I-labeled synthetic
canine relaxin was used initially in combination with pooled preg-
nant dolphin serum and one of three relaxin antisera: (1) rabbit
polyclonal porcine relaxin antiserum (R6) which reacts specifically
with the receptor binding domain of all relaxins sequenced [21],
(2) rabbit polyclonal canine relaxin antiserum (No. 79888) which
is highly reactive with relaxin in canids and felids [7,37], and (3)
rabbit polyclonal canine relaxin antiserum (No. 80037) [37]. Rabbit
anti-porcine relaxin R6 gave the greatest immunoreactive response
(specific binding, 10–12%) compared to the rabbit anti-canine
relaxins. Moreover, when rabbit anti-porcine relaxin was used
with 125I-labeled H2 human relaxin, specific binding reached
approximately 50%. Hence, the relaxin RIA used in the present
study consisted of H2 human relaxin as 125I-labeled ligand
(30,000 cpm/tube), rabbit anti-porcine relaxin R6 as the primary
antibody (working dilution, 1:40,000), and synthetic canine relaxin
as reference standard (0.2–50 ng/tube).
Adaptation of a progesterone RIA for dolphins involved a com-
mercial kit purchased from ICN Biomedicals, Inc. (ImmuChem™
Double Antibody Progesterone 125I RIA Kit; Costa Mesa, CA, USA).
The only modification to the instructions provided by the manufac-
turer was a 75% reduction in volume of kit reagents to adjust to the
different volumes of pooled dolphin serum used in Experiment 1
and limited volumes of serum and extracts of placental tissue from
individual dolphins in Experiment 2.
Validation of the relaxin and progesterone RIA involved assay of
pooled dolphin serum in duplicate with results reported as the
average specific binding. For the relaxin assay, parallelism was
tested by comparing specific binding of increasing volumes (25,
50, and 100 ll) of serum to the profile of the canine relaxin stan-
dard curve. Additivity was tested by comparing the specific bind-
ing of increasing amounts (0, 0.8, and 1.6 ng) of canine relaxin in
50 ll of serum to corresponding canine relaxin standards in assay
buffer. For the progesterone assay, parallelism was tested by com-
paring specific binding of increasing volumes (5, 10, 20, and 40 ll)
of serum to the profile of the progesterone standard curve. Additiv-
ity was tested by comparing the specific binding of increasing
amounts (0, 0.5, 2.0, and 5.0 ng) of progesterone in 25 ll of serum
to corresponding progesterone standards in progesterone-free
serum.
2.4. Experiment 2
The relaxin and progesterone RIA validated in Experiment 1
were used to quantify and characterize respective hormones in ser-
um and extracts of placental tissue from individual dolphins in
Experiment 2.
For quantifying placental relaxin, tissue samples from each of
the five placentas were extracted in a manner similar to that de-
scribed for the spotted hyena placenta [40]. Briefly, a 250 g sample
of tissue from each placenta was cut from a site considered vascu-
larized and well-preserved (darkest red), homogenized in cold
phosphate buffered saline (PBS), centrifuged to remove tissue frag-
ments and fat, and decanted. Relaxin is uniquely soluble in acidi-
fied acetone and was isolated from each supernatant using a
highly efficient acid–acetone extraction process as originally de-
scribed [8]. Briefly, each supernatant was mixed with water, con-
centrated HCl, and acetone (70%, v/v) at 4 °C for approximately
24 h. Thereafter, each mixture was filtered, precipitated with cold
acetone (90%, v/v) for an additional 24 h, decanted, and dried. Each
of the relaxin-containing residues were reconstituted in assay buf-
fer and set aside until assay.
For quantifying placental progesterone, tissue samples from
each of the five placentas were extracted in a manner similar to
that described for the gray seal [12]. Briefly, a 5 g sample of placen-
tal tissue was cut near the same site as the tissue cut for assay of
relaxin, homogenized in 100 ml of 100% ethanol, centrifuged, and
decanted. Thereafter, each supernatant was dried under a stream
of nitrogen gas, reconstituted in 1 ml of petroleum ether, and dried
again under a stream of nitrogen gas. Each of the progesterone-
containing residues were reconstituted in assay buffer and set
aside until assay.
The capability and efficiency of extracting progesterone from
each of the dolphin placentas was examined by taking a 10 ml ali-
quot of each homogenate and adding approximately 30,000 cpms
of [3H]progesterone. After the extraction process with ethanol
and drying as described in the previous paragraph, the residues
were reconstituted in 200 ll of petroleum ether, dried again, and
counted.
Serum and placental extracts were assayed using a volume of
100 ll for relaxin and 25 ll for progesterone. For the assay of pro-
gesterone, the 25 ll of extract was diluted 1:15 in assay buffer so
concentrations could be estimated from the linear portion of the
standard curve. Samples were assayed in duplicate with results re-
ported as the average concentrations relative to respective stan-
dard curves. Minimal detectable hormone concentrations or
assay sensitivity at 90% specific binding were 15.1 ng/ml for relax-
in and 0.11 ng/ml for progesterone. Intra- and inter-assay coeffi-
cients of variation (CV) were, respectively, 8.8% and 18.9% (n = 5
assays) for relaxin and 14.6% and 8.4% (n = 3 assays) for
progesterone.
2.5. Data management and statistical analysis
Serum concentrations of relaxin and progesterone for each of
the 28 pregnancies (18 live births and 10 stillbirths) in Experiment
2 were normalized to the month of parturition (Month 0), trun-
cated according to the duration of pregnancy in bottlenose dol-
phins [6], and examined for 12 months before (Months 11 to 0)
and 4 months after (Months 1–4) parturition. Infrequent and
inconsistent collection of samples within and among pregnancies
made it difficult to statistically model the repeated nature of the
data on a month-to-month basis. Months were, therefore, grouped
 D.R. Bergfelt et al. / General and Comparative Endocrinology 170 (2011) 650–656 653

into 4-month periods; early (Months 11 to 8), mid- (Months 7
to 4), and late (Months 3 to 0) pregnancy and the post-partum
period (Months 1–4). To further facilitate the use of the statistical
model, heterogeneity in the number of observations within each 4-
month period was standardized by averaging respective hormone
concentrations within each period so that there was a single mean
value of relaxin and progesterone during early, mid-, and late preg-
nancy and the post-partum period for each of the 28 pregnancies.
Prior to statistical analyses, the data were checked for extreme
values using Dixon’s outlier test [17], tested for homogeneity using
Shapiro–Wilk’s test, and transformed by natural log or ranks if nec-
essary. Data were examined by analysis of variance for repeated-
measures using PROC MIXED procedure in SAS (version 9.2; Statis-
tical Analysis System Institute, Inc., Cary, NC, USA) to determine
the main effects of month (each 4-month period) and birth status
(live vs stillbirth) and interaction. If a significant (P 6 0.05) main
effect or interaction was detected, mean differences among the
4-month periods and between birth statuses within a 4-month
period were determined using least significant difference (LSD).
3. Results
3.1. Experiment 1
The validation process and results of tests for parallelism and
additivity are shown in Table 1 for the relaxin and progesterone
RIA.
3.2. Experiment 2
Serum concentrations of progesterone and relaxin during preg-
nancy and the post-partum period in dolphins with live and still-
born calves along with results of statistical analyses are shown in
Fig. 1. Progesterone decreased (P < 0.05) from relatively high con-
centrations during early and mid-pregnancy to intermediate con-
centrations by late pregnancy and low or undetectable
concentrations during the post-partum period. In dolphins with
stillbirths, progesterone decreased earlier (month-by-birth status
interaction, P < 0.007) such that mean concentrations were 37.4%
(P < 0.06) and 25.4% lower at mid- and late pregnancy, respectively.
Temporally, relaxin increased (P < 0.05) progressively from rela-
tively low concentrations during early pregnancy to high concen-
trations during late pregnancy before decreasing (P < 0.05) to low
or undetectable concentrations during the post-partum period
(Fig. 1). Although the relative pattern of change in relaxin between
birth statuses did not reach significance (month-by-birth status
interaction, P = 0.17), mean concentrations were 42.5%, 28.8%,
and 34.3% lower at early, mid-, and late pregnancy, respectively,
in dolphins with stillbirths compared to those with live births.
Relaxin and progesterone were detected in extracts of placental
tissue from five dolphins as shown in Table 2 and examined in rela-
tion to serum concentrations of respective hormones averaged
over the pre- and post-partum periods in four of the same dolphins
(A–D). Corresponding serum samples for Dolphin E were not avail-
able. Notably, the recovery of [3H]progesterone from each of the
placental tissue extracts was 25%, 40%, 51%, 60%, and 80%
(mean ± SD, 51 ± 21%).
4. Discussion
The collection and archiving of serum samples from pregnant
and post-partum bottlenose dolphins over multiple months and
years provided a reasonable distribution and number of observa-
tions to characterize and examine the temporal relationship be-
tween circulatory concentrations of relaxin and progesterone in
association with live and stillborn calves. Although the effect of
long-term cryopreservation on serum concentrations of relaxin
and progesterone from dolphins is unknown, it is known that re-
laxin is a highly stable protein that can withstand exposure to ex-
treme cold, heat, and long periods at ambient temperature [8,35].
Correspondingly, progesterone is a relatively stable steroid as indi-
cated by a weak or moderate negative correlation between sample
storage time (as long as 22 years at 25 °C) and serum concentra-
tions [13]. In the present study, storage conditions (i.e., locations
and temperatures) and shipping of samples were comparable
within experiments; therefore, any alteration (i.e., decline) in spe-
cific binding or serum concentrations of relaxin or progesterone
due to prolonged storage or transport would presumably be pro-
portional among samples.

Table 1
The assay validation process involved specific displacement of antibody-bound radiolabeled human relaxin and progesterone in response to increasing amounts of pooled serum
from pregnant dolphins (parallelism) and canine relaxin (cRlx) or progesterone (P4) standards in a fixed volume of serum (additivity). Volume and dose–response curves
decreased linearly and were parallel with respective standard curves for each assay (Experiment 1).

Relaxin assay Parallelism Additivity

Standard cRlx (ng) Specific binding (%) Dolphin serum (ll) Specific binding (%) Standard cRlx (ng) + Dolphin serum (ll) Specific binding (%)
0 100 25 92 0 + 50 82
0.2 78 50 82 0.8 + 50 67
0.4 76 100 62 1.6 + 50 45
0.8 59
1.6 56
3.2 52
6.3 38
12.5 34
25.0 27
50.0 21
Progesterone assay Parallelism Additivity
Standard P4 (ng) Specific binding (%) Dolphin serum (ll) Specific binding (%) Standard P4 (ng) + Dolphin serum (ll) Specific binding (%)
0 100 5 62 0 + 25 100
0.2 97 10 55 0.5 + 25 98
0.5 88 20 42 2.0 + 25 82
2.0 69 40 35 5.0 + 25 63
5.0 48
10.0 34
25.0 21
50.0 14
654 D.R. Bergfelt et al. / General and Comparative Endocrinology 170 (2011) 650–656

16 Progesterone In Experiment 1, it was first determined that radiolabeled H2

Month: P<0.0001 human relaxin with rabbit anti-porcine relaxin R6 gave the great-
14 Birth status: P=0.33 est immunoreactive response (i.e., specific binding) compared to
Interaction: P<0.007
w (4) a (7) combinations of radiolabeled canine relaxin and corresponding
12 antisera. Second, for both the relaxin and progesterone RIA,
Live births increasing volumes of pooled serum from pregnant dolphins and
10 a (9) * amounts of respective hormone standards added to a fixed volume
of serum specifically displaced radiolabeled hormones from corre-
8 b
x sponding antibodies in a decreasing and linear manner. The dis-
(7) (8)
placement curves were considered parallel and additive relative
6 to respective standard curves. Considering that anti-porcine relax-
Stillbirths y
(4) in was also used to detect relaxin in extracts of luteal tissue from
4 pregnant whales [31], the heterologous relaxin RIA validated here-
in for dolphins may be applicable for detecting and quantifying re-
Concentrations (ng/ml)

2 laxin in other cetaceans.

In Experiment 2, relaxin and progesterone were detected in
(4) z c (11)
0 multiple, full-term placentas collected after parturition. Appar-
ently, placental progesterone has been detected previously in a tis-
120
sue sample collected at sea belonging to a humpback whale [34]
Relaxin but this is the first report of detecting both progesterone and relax-
100 in in tissue samples collected from bottlenose dolphins. Compara-
(8)
Month: P<0.0001 tively, placental production of progesterone and relaxin has been
Birth status: P=0.76 reported in laboratory species (e.g., rat, rabbit) [32], domestic spe-
80 Interaction: P=0.17 cies (e.g., sheep, horse) [10,43], and wild species (e.g., hyena, seals)
[2,40]. In the present study, detection of placental relaxin and rel-
(4)
atively high serum concentrations during the pre-partum period
60 (7)
followed by low concentrations in sequential samples collected
from the same dolphins during the post-partum period is indica-
(8)
40 tive of the pregnancy-specific nature of relaxin. Thus, assay of re-
(10) laxin has potential as an alternative hormonal approach for
distinguishing between pregnancy and pseudopregnancy in
20 (5) (5)
(12) dolphins.
Serum concentrations of progesterone were relatively high dur-
a b c a
0 ing early and mid-pregnancy and, thereafter, significantly de-
-11 to -8 -7 to -4 -3 to 0 1 to 4
creased to intermediate concentrations during late pregnancy.
Temporally, serum concentrations of relaxin progressively and sig-
Months from parturition nificantly increased during early to late pregnancy. In regard to
progesterone, relatively high concentrations during early and
Fig. 1. Mean (±SEM) serum concentrations of progesterone and immunoreactive mid-pregnancy in the present study are supported by previous
relaxin during early, mid-, and late pregnancy and the post-partum period (Month
0 = parturition) are shown with statistical analyses (Experiment 2). Progesterone
studies that have collected blood samples for diagnosing preg-
concentrations with different superscripts for abclive births (solid line) and nancy in captive bottlenose dolphins [29,30]. Additional support
wxyz
stillbirths (dashed line) among months differ (P < 0.05) and concentrations for the pattern of circulatory concentrations of progesterone dur-
between birth statuses for ⁄Months 7 to 4 tended to differ (P < 0.06). Relaxin ing pregnancy is provided by a couple case studies involving longi-
concentrations with different superscripts for abcbirth statuses combined among
tudinal collection of samples in captive cetaceans. The results from
months differ (P < 0.05). Values in parentheses represent number of observations.
assay of monthly or bi-monthly sera from two killer whales [18]
and monthly feces from one bottlenose dolphin [3] indicated con-
centrations of progesterone and progestogens, respectively, were
Table 2 relatively high during the first half of pregnancy and low during
Detection of relaxin and progesterone in extracts of placental tissue and serum in the the second half.
same bottlenose dolphins (A–D) averaged over the pre- and post-partum periods. In contrast to progesterone, the progressive increase in circula-
(Month 0 = parturition) in association with live births (Experiment 2).
tory concentrations of relaxin during pregnancy has not been
Dolphin Placental tissue extract (ng/kg) Pre- and post-partum serum previously documented in bottlenose dolphins or any cetacean.
(ng/ml) Considering the lack of information in cetaceans to support the
Months 11 to 0 Months 1–4 relaxin results reported herein, comparative results have been
Immunoreactive relaxin summarized from other mammalian species. Generally, among
A 1024 36.6 (5) 14.0 (2) laboratory species (e.g., rats, rabbits) [32], domestic species (e.g.,
B 1215 45.1 (7) 16.2 (1) pigs, sheep) [11,25], and wild species (e.g., elephants, rhinoceros,
C 2580 48.9 (4) 14.0 (3)
leopards) [36,39], circulatory concentrations of relaxin are low,
D 1546 123.7 (3) 16.5 (1)
E 1380 – – intermediate, or high from early to mid-pregnancy with a gradual
or marked surge-like increase during late pregnancy, prior to
Progesterone
A 57,000 7.2 (5) 0.12 (2) parturition. Regardless of the diversity in the pattern of circulatory
B 44,800 8.1 (7) 0.13 (1) concentrations of relaxin among species, the rise in relaxin preced-
C 18,100 9.8 (4) 0.22 (2) ing parturition appears to be relatively consistent; moreover, it
D 12,400 8.3 (3) 0.13 (1) occurs concomitantly with a decline in progesterone. In controlled
E 5300 – –
studies that have examined the role of relaxin in relation to proges-
Values in parentheses represent number of observations. terone in laboratory (e.g., rats, rabbits) [32] and domestic (e.g., pig,
 D.R. Bergfelt et al. / General and Comparative Endocrinology 170 (2011) 650–656
sheep) [25] species, it has been proposed that the decrease in pro-
gesterone prior to parturition represents a necessary withdrawal of
the ‘‘progesterone block’’ that has maintained the gravid uterus in
a quiescent state. Correspondingly, the increase in relaxin repre-
sents a mechanism to maintain myometrial quiescence and protect
against premature contractions and delivery. Despite the lack of
information on relaxin in bottlenose dolphins, the combined re-
sults from previous studies in pregnant dolphins, whales, and other
laboratory, domestic, and wild species provides support for the
temporal changes in progesterone and relaxin during pregnancy
in the present study. Future studies involving frequent and consis-
tent (e.g., bi-monthly, weekly) collection of urine or feces as less
invasive approaches to acquire hormonal data may provide an
opportunity to more thoroughly characterize the pattern of circu-
latory concentrations of progesterone and relaxin throughout
pregnancy in bottlenose dolphins.
The source of systemic concentrations of progesterone and re-
laxin during pregnancy in dolphins may be placental as reported
herein as well as luteal since the CL in pregnant whales apparently
produces both progesterone [26] and relaxin [31]. Reportedly the
structural lifespan of the CL is maintained until parturition in bot-
tlenose dolphins as in many cetaceans (review, [24]); however, it is
not known if the CL is functionally required throughout pregnancy
in dolphins. In laboratory (e.g., rat) and domestic (e.g., pig) species
in which the CL is required until the end of pregnancy, the luteal
gland is the major source of progesterone and relaxin [19]. In dol-
phins, it remains to be clarified if the major source of progesterone
and relaxin throughout pregnancy is placental, luteal, or a combi-
nation of both.
In the present study, stillbirths were associated with a progres-
sive and significant decrease in serum concentrations of progester-
one that began earlier compared to live births; mean
concentrations were lower at mid- (37%) and late (25%) pregnancy.
Although the relative pattern of change in serum concentrations of
relaxin between birth statuses did not reach significance, mean
concentrations were lower during early (42%), mid- (29%), and late
(34%) pregnancy in association with stillbirths. Apart from the lack
of information on relaxin in bottlenose dolphins, apparently the
only documented case of relating a premature decline in progester-
one to stillbirths involved the intermittent collection of blood sam-
ples from captive bottlenose dolphins over approximately
9 months of pregnancy [6]. Compared to relatively high monthly
mean concentrations of progesterone in dolphins with live births,
monthly concentrations were consistently lower in a dolphin with
a stillbirth; moreover, a progressive decrease began approximately
7 months before parturition. Comparatively, in an elephant, plasma
concentrations of both progesterone and relaxin declined to pre-
pregnancy concentrations approximately 5 week before delivery
of a stillborn calf [20]. In horses, a majority of stillbirths have been
linked with placentitis [9,14] which has been associated with re-
duced production of placental relaxin [28] and hypoxic and dysma-
ture foals at birth. In the present study, placentas from dolphins
with stillbirths were apparently not collected, but it was observed
that a few stillborn calves failed to thrive at or shortly after partu-
rition as a result of poor birth condition or immaturity [41]. Future
studies are required to clarify if the reduced concentrations of pro-
gesterone and relaxin in association with stillbirths are a reflection
of placental and/or luteal dysfunction and if these hormones alone
or in combination can be used diagnostically for assessing fetal
health in dolphins.
In conclusion, the successful validation of a heterologous relax-
in and progesterone RIA for use in bottlenose dolphins led to the
detection of immunoreactive relaxin and progesterone in placental
extracts and serum samples and characterization of the temporal
relationships between these hormones during pregnancy and the
post-partum period in dolphins with live and stillborn calves.
655
The pregnancy-specific increase in serum concentrations of relaxin
and lower concentrations of both relaxin and progesterone in asso-
ciation with stillbirths suggest the potential for relaxin to be used
diagnostically to determine pregnancy status, and one or both hor-
mones to be used to assess placental function, and, perhaps, fetal
well-being in dolphins. The nature of this study and novelty of
the results illustrate the value of archived material for providing
important fundamental information about reproductive biology
of bottlenose dolphins that may be applied to enhance reproduc-
tive management practices of captive and wild populations of dol-
phins and other cetaceans.
Acknowledgments
This study was supported by grants from the National Marine
Mammal Laboratory and NYU NIEHS Center (ES00260). Dolphin
serum samples and placentas were provided by Dolphin Quest
and the Indianapolis Zoo. The authors thank Drs. Jay Sweeney,
Rae Stone, Gregg Levine, and the Dolphin Quest training staff
who manage the Dolphin Quest Reproduction Program. In addition,
the authors thank Drs. Todd Robeck for providing supplementary
reference material on reproduction in cetaceans and Mohd Beg
for assistance with statistical analyses.
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