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The relationship of maternal characteristics and circulating
progesterone concentrations with reproductive outcome in the
bottlenose dolphin (Tursiops truncatus) after artificial
insemination, with and without ovulation induction, and
natural breeding
J.K. O’Brien
a,b,
*, T.R. Robeck
a
a
SeaWorld and Busch Gardens Reproductive Research Center, SeaWorld Parks and Entertainment, 2595 Ingraham Street, San Diego,
California 92109, USA
b
Faculty of Veterinary Science, University of Sydney, NSW 2006, Australia
Received 6 October 2011; received in revised form 20 December 2011; accepted 11 February 2012
Abstract
Bottlenose dolphins (Tursiops truncatus) undergoing natural breeding and artificial insemination (AI) were examined to characterize
serum progesterone concentrations and determine relationships among age, parity, and reproductive outcome. Progesterone profiles of
five cycle types (n ⫽119 total cycles from 54 animals) were characterized as follows: (i) conception and production of a live term calf
(conceptive-term, n ⫽73); (ii) conception and abortion after Day 60 (conceptive-abortion, n ⫽12); (iii) unknown conception status with
prolonged, elevated progesterone and absence of a fetus (conceptive-unknown, n ⫽14); (iv) conception failure with normal luteal phase
progesterone concentrations (non-conceptive, n ⫽14, AI cycles only); and (v) conception failure with progesterone insufficiency
occuring after spontaneous ovulation or owing to premature ovulation induction using GnRH (non-conceptive-PI, n ⫽6, AI cycles only).
By Day 21 post-insemination (PI), progesterone concentrations were similar (P ⬎0.05) among conceptive-term, conceptive-abortion
and conceptive-unknown, and higher (P ⬍0.05) for conceptive-term than non-conceptive and non-conceptive-PI cycles. Progesterone
concentrations of known conceptive cycles peaked by Week 7 PI (P ⬍0.05) and remained elevated for the remainder of pregnancy
(Weeks 8 up to 54, ⱖ5 days pre-partum). During midpregnancy (Days 121–240), conceptive-term cycles had higher (P ⬎0.05)
progesterone concentrations than conceptive-abortion and unknown conception status cycles. Parity was not associated with reproduc-
tive outcome based on cycle type (P ⬎0.05). Age of females in conceptive-unknown (26.5 ⫾10.1 yrs) and conceptive-abortion (22.1 ⫾9.4
yrs) groups was higher (P ⬍0.05) than in conceptive-term (15.7 ⫾7.2 yrs). The conceptive-unknown cycle type possibly represents
undetected early embryonic loss occurring before Day 60 PI. Length of gestation using known conception dates was 376.1 ⫾11.0 days
and the range of this parameter (355–395 days) has implications for peri-parturient management procedures for the species.
© 2012 Elsevier Inc. All rights reserved.
Keywords: Cetacean; Embryonic loss; Gestation; Luteal insufficiency; Senescence; Seasonality
1. Introduction
Initial studies on the reproductive endocrinology of
the female bottlenose dolphin (Tursiops truncatus) ex-
amined concentrations of steroids, including progester-
* Corresponding author. Tel.: ⫹1 619 225 3176; fax: ⫹1 619 225
3178.
E-mail address: justine.obrien@seaworld.com (J.K. O’Brien).
Available online at www.sciencedirect.com
Theriogenology 78 (2012) 469 – 482
www.theriojournal.com
0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2012.02.011
Author's personal copy
one, in a small number of ovariectomized, non-preg-
nant and pregnant females [1]. Circulating progesterone
concentrations diagnostic of pregnancy in the bottle-
nose dolphin were later characterized as values exceed-
ing 3 ng/mL that were maintained at this concentration
for more than 6 to 8 wk [2]. Monthly progesterone
profiles have since been described in the parturient
bottlenose dolphin after serial sampling of captive an-
imals, but several discrepancies exist. For example, a
biphasic pattern of serum progesterone during preg-
nancy was reported by Cornell, et al. [3], but not by
Kirby [4]. Further, although a similar biphasic proges-
terone profile was reported by Schroeder and Keller [5],
the timing of the progesterone decline before the sec-
ond increase differed from that described by Cornell,
et al. [3].
Progesterone profiles published to date comprise
only the last ⬃11 mo of gestation, as known conception
dates based on ovulation timing were not available
[3–5]. Consequently, there remains a dearth of infor-
mation on progesterone profiles in early pregnancy
(Weeks 1– 8) for this species. In other mammalian
species, the first 4 to 8 wk of pregnancy represent the
period where embryonic loss is most likely to occur
(ruminants [6,7], horses [8], humans [9]). Because
pregnancy confirmation by transabdominal ultrasono-
graphic detection of a dolphin conceptus is most reli-
ably performed after Day 50 of gestation using known
conception dates (TR Robeck, unpublished), knowl-
edge of normal progesterone profiles in early pregnancy
would be useful to determine if they differ from those
of cycles with known conception failure or presumptive
embryonic loss.
Available evidence indicates that the primary source
of progesterone during pregnancy in the bottlenose dol-
phin is the CL [10]. In agreement with the well docu-
mented role of progesterone as a mammalian pregnancy
support hormone [11,12], sustained periods of abnor-
mally low concentrations of progesterone in bottlenose
dolphin pregnancies result in abnormal fetal develop-
ment [10]. Whereas the normal hormone milieu in the
parturient dolphin is yet to be thoroughly characterized,
documentation of progesterone concentrations through-
out normal pregnancy would assist with the clinical
management of pregnancy abnormalities, such as pro-
gesterone insufficiency. Monitoring pregnancies with
known conception dates through the advent of artificial
insemination (AI) will also allow determination of the
true gestation length range for this species, which has
implications for peri-parturient management practices.
The overall goal of this research was to characterize
circulating progesterone profiles during pregnancy, us-
ing known conceptions dates, to provide baseline data
for gestational health assessments for the species. Spe-
cific objectives were to examine females undergoing
natural breeding and AI (with and without ovulation
induction in the latter) to: (i) characterize weekly pro-
gesterone concentrations during conceptive and non-
conceptive cycles; (ii) examine the relationship of pro-
gesterone concentration during early pregnancy with
reproductive success; (iii) examine the relationship of
maternal age and parity at conception with progester-
one concentrations and reproductive success; (iv) char-
acterize the gestation length for the species using
known conception dates; and (v) examine seasonal ef-
fects on conception timing in naturally mated females.
2. Materials and methods
2.1. Ethics of experimentation
All samples were collected as part of routine hus-
bandry procedures for the bottlenose dolphin. All pro-
cedures described within were reviewed and approved
by each institution’s Animal Care and Use Committee,
and were performed in accordance with the Animal
Welfare Act for the care of Marine Mammals.
2.2. Animals
Animals (n ⫽54 total) were located at Dolphin
Quest, Hawaii and Bemuda (n ⫽8), Dolphin Adven-
tures, Mexico (n ⫽5), Genoa Aquarium, Italy (n ⫽2),
Harderwijk Aquarium, the Netherlands (n ⫽1), Mundo
Marino, Argentina (n ⫽2), the Navy Marine Mammal
Program, San Diego (n ⫽1), SeaWorld Orlando (n ⫽
10), SeaWorld San Antonio (n ⫽7), SeaWorld San
Diego (n ⫽15), and Zoomarine, Portugal (n ⫽2). All
female bottlenose dolphins were ⬎6 yrs of age and
weighed at least 180 kg. Animals were housed in pools
containing ⱖ850 m
3
of natural processed salt water or
were housed in numerous connected ocean pools hold-
ing 300 to 600 m
3
of salt water (ambient temperatures
14 –28 °C). Dolphins were fed, at approximately 4 to
5% of their body weight per day, a diet of frozen-
thawed whole fish (herring, Clupea harengus, capelin,
Mallotus villosus, and Columbia River smelt, Thaleich-
thys pacificus).
2.3. Progesterone concentrations
Blood samples (n ⫽979) were collected on a vol-
untary basis from vessels in the ventral aspect of the tail
470 J.K. O’Brien and T.R. Robeck / Theriogenology 78 (2012) 469 – 482
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fluke using a 21 g winged blood collection set. Blood
collections were also performed with restrained animals
at the time of AI or when voluntary collections were not
possible (⬃10% of collections). The blood was allowed
to clot in a serum separator tube for 20 min and cen-
trifuged at ⬃1000 g for 15 to 30 min. Serum was
removed and stored at ⫺80 °C until analysis. Samples
were collected at 7 to 10 days intervals from Day 0 (day
of estimated natural breeding or observed ovulation
after AI) until Day 28, and on a bimonthly to monthly
basis thereafter until either parturition or conception
failure/fetal abortion was confirmed.
Serum progesterone concentrations were determined
at commercial laboratories using a chemiluminescence
immunoassay (CIA, Siemens Healthcare Diagnostics,
Inc., Deerfield, IL, USA), or were determined at Sea-
World San Diego using an immunoenzymometric assay
(IEA; TOSOH BioScience NV, Tessenderlo, Belgium).
Both assays have been previously validated for use in
dolphins by (i) demonstrating parallelism between the
standard curve and a series of pooled serum sample
dilutions (r⫽0.96), and (ii) obtaining a high recovery
rate (ⱖ87%) of exogenous progesterone after its addi-
tion to a pooled dolphin serum sample (range, 5-20
ng/tube). Both CIA and IEA values were highly corre-
lated in validation trials (R⫽0.95, P ⬍0.05, n ⫽31).
Sensitivity of all assays was 0.1 ng/mL, and intra- and
interassay variation was ⬍10%. Routine internal qual-
ity control checks were performed by the commercial
and in-house laboratories, and sample reanalysis was
performed whenever an outcome outside of the ex-
pected range for the respective stage of the reproductive
cycle was observed.
2.4. Artificial insemination and ovulation induction
Inseminations were performed using chilled or fro-
zen-thawed sex-sorted and non-sexed spermatozoa
(ⱖ50 ⫻10
6
progressively motile spermatozoa). De-
tailed AI methods in the bottlenose dolphin have been
presented elsewhere [13–16]. Briefly, females were
synchronized using altrenogest (0.044 mg/kg for a min-
imum of 20 days, Regu-mate, Intervet, Inc., Millsboro,
DE, USA) and ovulated from 16 to 31 days after the
end of altrenogest treatment (n ⫽54 total cycles).
Timing of AI was determined using urinary LH moni-
toring (whereby inseminations occurred 26 –30 h fol-
lowing initiation of the LH surge, or 17–18 h following
the LH surge peak) and ovulation timing was confirmed
by ultrasound. Using this regimen, females were insem-
inated no earlier than 12 h before ovulation and no later
than an estimated 2 h after ovulation, with conceptions
occurring at both ends of this range (T. R. Robeck, J. K.
O’Brien, K. J. Steinman, G. A. Montano, unpublished
observations).
A subset of females (n ⫽12 cycles) were induced to
ovulate using a GnRH analogue, whereas all other
females (n ⫽42 cycles) ovulated spontaneously. Ani-
mals undergoing ovulation induction were adminis-
tered Cystorelin (Merial, Duluth, GA, USA; 3 ⫻250
g iv every 1.5–2 h) once a growing follicle reached a
size indicative of imminent ovulation (ⱖ1.5 cm) and
urinary estrogens displayed presumptive peak values
(ⱖ2.1 ng/mg Cr) [13]. Animals were then inseminated
26 to 27 h after the first Cystorelin injection.
One to 2 h before each AI procedure, females were
premedicated with Valium (Diazepam, Abbott Lab,
Chicago, IL, USA; 0.1– 0.2 mg/kg). Intrauterine insem-
ination was performed while the animal was in lateral
recumbency using a flexible endoscope (6 –12 mm in
diameter and 190 –300 cm long; Olympus, America,
Melville, NY, USA) preloaded witha5to7-Fr custom
made catheter, 250 to 350 cm long; Smiths Medical
PM, Inc., Waukesha, WI, USA).
2.5. Ultrasonography for monitoring ovarian activity
and pregnancy diagnosis
Transabdominal ultrasonography was used to mon-
itor ovarian activity and ovulation during AI procedures
[13] and confirm pregnancy [17] by detection of an
embryonic vesicle or fetus, in conjunction with thick-
ening of the endometrium. Ultrasonographic examina-
tions were performed with either a GE Logiqbook (GE
LogiqGE Medical Systems, Milwaukee, WI, USA) us-
ing a 3.5 MHz transducer (wide footprint convex linear
transducer) or an Aloka 900 machine (Aloka, Walling-
ford, CT, USA). Animals with progesterone concentra-
tions remaining above 5 ng/mL from Day 28 post-AI
were considered pregnant; ultrasound examinations
were performed weekly to biweekly thereafter until
further confirmation of pregnancy by the presence of an
embryonic vesicle and thereafter a viable fetus. Once a
fetus was detected, ultrasound examinations were per-
formed monthly or bimonthly at the discretion of each
institution.
2.6. Age and reproductive history
For animals in this study, institutional records for
non-North American animals, and records documented
in the bottlenose dolphin studbook for North American
animals [18] were used to collate data on age and parity
at the time of conception for examining the influence of
these parameters with reproductive outcome (n ⫽113
471J.K. O’Brien and T.R. Robeck / Theriogenology 78 (2012) 469 – 482
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cycles in 53 animals, non-conceptive cycles with ab-
normal follicular luteinization were not included).
2.7. Gestation length
Calculation of gestation length in artificially insem-
inated females was based on the interval between the
day of ovulation (Day 0) and parturition (n ⫽17 preg-
nancies in 16 animals). Gestation length was deter-
mined in naturally bred animals (n ⫽56 pregnancies in
24 animals) with Day 0 denoted as the day of final
breeding activity and further confirmed by the subse-
quent progressive increase in serum progesterone con-
centrations. For females where breeding activity was
not recorded, Day 0 was designated as the midpoint
between the last sample with baseline progesterone
concentration and the first sample with increased pro-
gesterone concentration (if the interval exceeded 10
days, data from that female were not included).
2.8. Influence of season on the timing of natural
conception
The influence of season on the timing of natural
conception was also examined in North American-
based females (n ⫽31, held at SeaWorld San Antonio,
SeaWorld San Diego and SeaWorld Orlando) using
data for term pregnancies (n ⫽56) that were monitored
in the current study, and for that of previous term
pregnancies (n ⫽55) exhibited by the same animals.
For the 55 cycles where the date of conception was
unknown, the month of conception was determined by
subtracting 376 days (i.e., the average gestation length
determined in this study) from the date of parturition.
Females in this dataset had access to a proven male(s)
year-round except when they were temporarily trans-
ferred to holding pools for medical procedures (for 7
days or less).
2.9. Data and statistical analyses
Characterization of progesterone concentrations dur-
ing conceptive and non-conceptive cycles after natural
breeding and AI, and examination of the relationship
between progesterone concentrations during pregnancy
with reproductive outcome was conducted using sam-
ples from 119 cycles and 54 animals (Table 1). Proges-
terone data were compared at weekly intervals for all
cycles types during the first 4 wk after insemination
(Days 1–7, Days 8 –14, Days 15–21, Days 22–28) but
after Day 28, weekly comparisons were only made for
conceptive-term cycles as there were insufficient data
in remaining cycles for appropriate statistical analysis.
Progesterone data were also examined at 4-wk intervals
(i.e., data pooled across Weeks 1– 4, Weeks 4 – 8, etc.)
during pregnancy for conceptive-term, conceptive-
abortion and cycles of unknown conception status. Pro-
gesterone data were also divided into three periods for
comparative purposes across cycle types: early preg-
Table 1
Cycle classification and number of bottlenose dolphins, cycles and samples included in the progesterone (P
4
) characterization study.
Cycle type Cycle type description No. animals
per cycle type
No. cycles
(NAT, AI)
No. serum
samples
analyzed
Conceptive - term Conceptive cycle: production of a term,
live calf
49 73 (56 NAT, 17
AI)
657
Conceptive - abortion Conceptive cycle: abortion occurring after
Day 60
11 12 (4 NAT, 8 AI) 95
Unknown conception status –prolonged,
elevated progesterone without
visualization of a fetus
Conceptive (embryonic loss occurring
before Day 60 and prolonged
maintenance of the CL [retained CL])
or non-conceptive with a retained CL
10 14 (5 NAT, 9 AI) 147
Non-conceptive Non-conceptive cycle and normal
follicular luteinization (serum P
4
ⱖ1.5
ng/mL by Day 7 post-insemination)
12 14 (14 AI) 55
Non-conceptive with progesterone
insufficiency
Non-conceptive cycle: abnormal follicular
luteinization occurring after
spontaneous ovulation (n ⫽2) or
premature ovulation induction with
GnRH (n ⫽4). Serum P
4
ⱕ1.5 ng/mL
by Day 7 post-insemination)
5 6 (6 AI) 25
Day 0 ⫽day of estimated natural breeding or observed ovulation after AI. Overall, a total of 54 animals were included in the study.
AI, artificial insemination (n ⫽54 total cycles); NAT, natural breeding (n ⫽65 total cycles).
472 J.K. O’Brien and T.R. Robeck / Theriogenology 78 (2012) 469 – 482
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nancy (Days 0 –120), midpregnancy (Days 121–240),
and late pregnancy (Day 241 to parturition). Finally,
examination of the relationships of age and parity at
conception with progesterone concentrations during
normal pregnancies included data from 73 cycles and
37 animals.
Statistical analyses were performed using SigmaStat
(version 3.5; SSPS, San Rafael, CA, USA). Progester-
one data were checked for normality (Kolmogorov–
Smirnov test) and accordingly analyzed using ANOVA,
repeated measures ANOVA and repeated measures ANOVA
on ranks. All pairwise multiple-comparison procedures
between means were conducted using either Tukey or
Dunn’s tests. Pearson product moment correlation was
used to examine the relatedness between age and parity.
Proportion data for the seasonal incidence of concep-
tion were analyzed by
2
analysis. For all analyses, P ⬍
0.05 was considered significant. Unless noted, data are
presented as the mean ⫾SD.
3. Results
Examination of 119 cycles based on progesterone
profiles and ultrasound examinations revealed five cat-
egories of cycles occurring after insemination (Table
1): (i) 61.3% (73/119) were conceptive and resulted in
a live term calf; (ii) 10.1% (12/119) resulted in abor-
tion; (iii) 11.8% (14/119) were of unknown conception
status, and included prolonged luteal activity/elevated
progesterone; (iv) 11.8% (14/119) were non-conceptive
with normal follicular luteinization; and (v) 5.0% (6/
119) were non-conceptive with abnormal follicular lu-
teinization. Overall, 71.4% (85/119) of monitored cy-
cles resulted in a documented conception, and 85.9% of
those conceptions (73/85) resulted in a live, term calf.
3.1. Characterization of progesterone concentrations
during conceptive and non-conceptive cycles after
natural breeding and AI
3.1.1. Relationship of progesterone concentration
with reproductive outcome
There were no differences in progesterone concen-
trations among conceptive and non-conceptive cycles
(with normal or abnormal follicular luteinization) dur-
ing the first 14 days following insemination (P ⬎0.05).
By Day 21, progesterone concentrations were similar
(P ⬎0.05) among conceptive cycles (with term calves
or aborted fetuses) and cycles of unknown conceptive
status. Although progesterone concentrations by Day
21 were higher (P ⬍0.05) for conceptive-term cycles
than non-conceptive cycles, concentrations of cycles
resulting in fetal abortion or of unknown conception
status were similar (P ⬎0.05) to that of non-conceptive
cycles. By Day 28, progesterone concentrations of all
conceptive cycle types and cycles of unknown concep-
tive status were similar (P ⬎0.05), and higher than that
of non-conceptive cycles (P ⬎0.05). By Week 5 (Days
29 –35) and Week 6 (Days 36 – 42), there continued to
be no difference in progesterone concentration among
conceptive cycle types and cycles of unknown concep-
tion status (P ⬎0.05, data not shown).
When progesterone data were grouped into 4-wk
increments (Fig. 1), there were no differences (P ⬎0.05)
in concentrations among conceptive-term, conceptive-
abortion and unknown conception status cycle types. Due
to the low number of samples for conceptive-abortion
cycles from Week 17 onwards, Weeks 17 to 36 were
grouped together for comparison; during this period, con-
centrations were lower (P ⬍0.05) for cycles resulting in
fetal abortion (11.8 ⫾7.4 ng/mL, n ⫽10) than concep-
tive-term cycles (21.6 ⫾9.7 ng/mL, n ⫽204).
Comparisons of progesterone data after it was di-
vided into three gestational phases demonstrated no
significant differences among conceptive-term, concep-
tive-abortion and unknown conception status cycle
types in early and late pregnancy (Table 2). During
midpregnancy, conceptive-term cycles had higher (P ⬎
0.05) concentrations than conceptive-abortion and un-
known conception status cycles.
3.1.2. Progesterone profiles of conceptive-term cycles
During the first 4 wk after insemination, progester-
one concentrations underwent a progressive increase
(P ⬍0.05; Table 3), reached peak values at Week 7
(P ⬍0.05) and thereafter remained elevated throughout
gestation (Fig. 2). When comparisons were made with
pooled weekly data (combined across four weekly in-
tervals), progesterone concentrations (Fig. 1) under-
went an increase (P ⬍0.05) from baseline by Week 4
post-insemination, increased (P ⬍0.05) further by
Week 8, and remained elevated at similar concentra-
tions for the remaining months of gestation, except for the
last 4 wk (Weeks 53-56) when concentrations declined
slightly but not significantly. Blood samples were col-
lected more than 1 wk before parturition for all but two
cycles (where blood was collected at 5 and 7 days before
parturition) thereby preventing examination of potential
changes occurring pre-partum. Samples collected from
four females at Day 1 post-partum displayed baseline
progesterone concentrations (1.2 ⫾0.4 ng/mL).
When progesterone data were divided into three
phases, concentrations during early pregnancy were
lower (P ⬍0.05) than midpregnancy and similar (P ⬎
473J.K. O’Brien and T.R. Robeck / Theriogenology 78 (2012) 469 – 482
Author's personal copy
0.05) to late pregnancy (Table 2). Progesterone concen-
trations within each of the three phases of pregnancy
were similar for naturally bred and AI groups (P ⬎
0.05, data not shown).
3.1.3. Progesterone profiles of conceptive-abortion
cycles
Progesterone concentrations (Fig. 1) increased (P ⬍
0.05) between the intervals of Weeks 1 to 4 and Weeks
5 to 8, and remained elevated at similar concentrations
for the remaining weeks while a viable fetus was pres-
ent. Of the 12 cycles analyzed in this group, fetal loss
had occurred in 9 cases (9/12, 75.0%) by Weeks 33 to
36, and occurred on average for all cases by Weeks 21
to 24 (153.8 ⫾75.5 days; range: Day 69 –265). Fetal
loss coincided with progesterone decline to baseline
(ⱕ1.5 ng/mL) in all females but one where progester-
Fig. 1. Pooled weekly serum progesterone profiles of conceptive cycles (Conceptive-term, Conceptive-abortion) and of cycles of unknown
conception status that represent possible early embryonic loss (Conceptive-unknown) in bottlenose dolphins. Note that data were pooled at 4-wk
intervals, and across AI and natural breeding groups. Day 0 ⫽day of ovulation (confirmed by urinary hormone monitoring and ovarian ultrasound
for females undergoing AI, or estimated from serum progesterone data, with or without breeding behavior, for naturally bred females). Data are
the mean ⫾SD.
Table 2
Serum progesterone concentrations in bottlenose dolphins from conceptive cycles and cycles of unknown conceptive status at early, mid and
late gestation following natural breeding and AI (Day 0 ⫽day of estimated natural breeding or observed ovulation after AI). Data are the
Mean ⫾SD.
Stage of gestation (days of gestation) Cycle type (no. samples) Unknown conception status – prolonged
elevated progesterone
§
Conceptive-term* Conceptive-abortion
‡
Early (0-120) 16.3 ⫾12.4
A
(333) 13.3 ⫾10.6 (78) 17.07 ⫾17.2 (120)
Mid (121–240) 22.0 ⫾9.6
aB
(176) 11.1 ⫾8.3
b
(7) 15.68 ⫾11.8
b
(18)
Late (241-parturition or baseline serum
progesterone, ⱕ1.5 ng/mL)
18.8 ⫾10.2
AC
(148) 8.0 (1)
¶
15.09 ⫾11.9 (5)
¶ Value was not included in statistical analyses.
* Conceptive-term (live, term calf produced);
‡
Conceptive-abortion (abortion occurred after Day 60);
§
Unknown conception status with
elevated progesterone (early embryonic loss before Day 60 and retained CL, or a retained CL without conception).
abc
Values without a common superscript within the same column (cycle type) are different (P ⬍0.05).
ABC
Values without a common superscript within the same row (stage of gestation) are different (P ⬍0.05).
474 J.K. O’Brien and T.R. Robeck / Theriogenology 78 (2012) 469 – 482
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one concentrations remained elevated after abortion on
Day 80. This animal was given prostaglandin F
2
␣
(25
mg im, twice daily, Lutalyse, Pfizer Animal Health,
New York, NY, USA) on Days 224, 225, and 226 to
lyse the CL that had been retained for 142 days after
fetal loss. Only progesterone values before abortion
were included in data summaries and analyses.
When data were divided into the three phases of
gestation, only one sample was available for the late
pregnancy phase (as all females but one had aborted
Fig. 2. Weekly serum progesterone profiles during normal pregnancy in the bottlenose dolphin (data pooled across AI and natural breeding
groups). Day 0 ⫽day of ovulation (confirmed by urinary hormone monitoring and ovarian ultrasound for females undergoing AI or estimated from
serum progesterone data, with or without breeding behavior, for naturally bred females). Note the decline (*P ⬍0.05) in progesterone
concentrations for non-conceptive cycles compared to conceptive cycles at Week 3 and Week 4 (inset graph). Data are the Mean ⫾SD.
Table 3
Serum progesterone concentrations in bottlenose dolphins from conceptive and non-conceptive cycle types during the first 4 wk following
natural breeding and AI (Day 0 ⫽day of estimated natural breeding or observed ovulation after AI). Values are the Mean ⫾SD.
Week post-
insemination (d)
Cycle type (no. samples) Non-conceptive
progesterone
insufficiency
¶
Conceptive-term* Conceptive-
abortion
†
Unknown conception status
(prolonged, elevated
progesterone)
‡
Non-conceptive
§
0 (0) 0.7 ⫾0.7 (17) 1.1 ⫾0.9 (3) 1.3 ⫾1.5 (9) 1.3 ⫾0.4 (6) 0.1 ⫾0.0 (2)
1 (1–7) 3.2 ⫾2.3 (36) 2.8 ⫾2.8 (9) 3.3 ⫾3.1 (9) 2.4 ⫾1.5 (10) 0.4 ⫾0.3 (6)
2 (8–14) 6.0 ⫾3.2 (26) 5.1 ⫾4.0 (10) 5.6 ⫾5.7 (9) 4.9 ⫾2.1 (11) 3.0 ⫾2.2 (6)
3 (15–21) 12.5 ⫾9.1 (26)
a
9.1 ⫾4.5 (9)
ab
10.7 ⫾7.0 (11)
ab
3.9 ⫾3.3 (13)
b
0.6 ⫾0.5 (6)
b
4 (22–28) 17.1 ⫾10.4 (30)
a
9.4 ⫾6.5 (7)
a
12.6 ⫾7.2 (11)
a
0.7 ⫾1.3 (15)
b
0.3 ⫾0.2 (5)
b
All weeks (0-28) 8.3 ⫾8.8 (135)
a
6.0 ⫾5.1 (38)
a
7.1 ⫾6.9 (49)
a
2.7 ⫾2.6 (55)
b
1.0 ⫾1.5 (25)
c
* Conceptive-term (live, term calf produced);
†
Conceptive-abortion (abortion occurred after Day 60);
‡
Unknown conception status with
elevated progesterone (early embryonic loss before Day 60 and retained CL, or a retained CL without conception);
§
Non-conceptive with
normal follicular luteinization and CL function;
¶
Non-conceptive with abnormal follicular luteinization and progesterone insufficiency.
ab
Values without a common superscript within the same row are different (P ⬍0.05).
475J.K. O’Brien and T.R. Robeck / Theriogenology 78 (2012) 469 – 482
Author's personal copy
their fetus by this stage) and was therefore not included
in statistical analyses. Progesterone concentrations
were similar (P ⬎0.05) for early and midpregnancy
(Table 2).
3.1.4. Progesterone profiles of unknown conception
status cycles with prolonged, elevated progesterone
Progesterone concentrations (Fig. 1) increased (P ⬍
0.05) between the intervals of Weeks 1 to 4 and Weeks
5 to 8, and remained elevated at similar concentrations
for the following 20 wk, after which progesterone con-
centrations fluctuated (P ⬍0.05) before declining to
baseline values (ⱕ1.5 ng/mL), on average, by 25 wk
(173.1 ⫾102.8 days; range, Days 62–282). Progester-
one concentrations were similar (P ⬎0.05) across
early, mid and late “gestation”.
3.1.5. Non-conceptive cycles
For non-conceptive cycles with presumptive normal
follicular luteinization and luteal activity (serum P
4
ⱖ1.5 ng/mL by Day 7 post-insemination), serum pro-
gesterone (Table 3,Fig. 2) reached peak concentrations
by Day 14 and declined (P ⬍0.05) to baseline values
(ⱕ1.5 ng/mL) by Day 28.
For the 12 females undergoing ovulation induction
before AI, the timing of GnRH administration was
retrospectively determined to be suboptimal in four
cycles when the induction protocol was being devel-
oped. In those cycles, the LH surge was induced pre-
maturely, and the resultant CL secreted reduced (P ⬍
0.05) concentrations of progesterone during the 28-
days monitoring period than non-conceptive cycles
where normal luteinization occurred after spontaneous
ovulation, or after induced ovulation when GnRH was
administered at the correct time (i.e., after complete
maturation of the preovulatory follicle). In the six cy-
cles where abnormal luteinization occurred after spon-
taneous ovulation (n ⫽2) or after ovulation induction
(n ⫽4), progesterone concentrations did not differ
between each week (Weeks 1– 4, P ⬎0.05).
After confirmation of conception failure, seven fe-
males underwent a second estrous cycle, with ovulation
and or breeding activity being detected at 33.1 ⫾2.4
days after the initial AI procedure.
3.2. Relationship of age and parity at conception on
progesterone concentrations and pregnancy outcome
after natural breeding and AI
Age was higher (P ⬍0.05) for females undergoing
fetal abortion and cycles of unknown conception status
than for females which produced a live, term calf (Ta-
ble 4). Of the 85 confirmed pregnancies recorded in this
study, there were 12 cases of fetal loss, thereby giving
an overall loss rate of 14.1%. For confirmed concep-
tions, age of naturally bred females was lower (P ⬍
0.05) than that of females undergoing AI (15.3 ⫾6.3
and 21.4 ⫾8.5 yrs, respectively). The significant in-
teraction of age at conception with insemination
method thereby prevented comparison of fetal loss rate
between natural breeding and AI cycles.
There was a positive correlation of parity and age of
the female at insemination for all cycle types (r
2
⫽
0.70, P ⬍0.05, n ⫽119). Parity did not differ across all
cycles types (Table 4;P⬎0.05) and was therefore not
associated with reproductive outcome after insemina-
tion. There was no influence (P ⬎0.05) of parity on
serum progesterone concentrations during term preg-
nancies; primiparous animals (n ⫽13 cycles from 13
females) had similar progesterone concentrations to
multiparous animals (n ⫽60 cycles from 33 females)
throughout different stages of pregnancy (data not
shown). Similarly, there was no association (P ⬎0.05)
of female age on serum progesterone concentrations
throughout all stages of pregnancy (data not shown).
Table 4
Age and parity at time of insemination of bottlenose dolphins undergoing conceptive and non-conceptive cycles (data pooled for natural
breeding or AI). Data are the mean ⫾SD.
Reproductive
Parameter
Cycle type (no. cycles)
Conceptive-term*
(n ⫽73)
Conceptive-abortion
‡
(n ⫽12)
Unknown conception
status-prolonged, elevated
progesterone
§
(n ⫽14)
Non-conceptive
¶
(n ⫽14)
Age (y) 15.7 ⫾7.2
a
22.1 ⫾9.4
bc
26.5 ⫾10.1
b
18.9 ⫾4.3
ac
Parity (0 onwards) 2.4 ⫾1.8 2.3 ⫾1.5 3.6 ⫾2.6 2.7 ⫾1.5
* Conceptive-term (live, term calf produced);
‡
Conceptive-abortion (abortion occurred after Day 60; Day 0 ⫽day of estimated natural breeding
or observed ovulation after AI);
§
Unknown conception status with elevated progesterone (early embryonic loss before Day 60 and retained
CL, or a retained CL without conception);
¶
Non-conceptive with normal follicular luteinization and CL function.
abc
Values without a common superscript within the same row are different (P ⬍0.05).
476 J.K. O’Brien and T.R. Robeck / Theriogenology 78 (2012) 469 – 482
Author's personal copy
3.3. Gestation length and influence of season on
conception timing
Overall gestation length for conceptive cycles where
a live, term calf was produced was 375.5 ⫾8.7 days
with a range of 355 to 399 days. Length of gestation
was not different for animals undergoing natural breed-
ing and AI (374.9 ⫾8.1 days, n ⫽56, range: 357–399
days and 376.1 ⫾11.0 days, n ⫽17, range: 355–395,
respectively).
For naturally bred animals, conceptions occurred
in every month of the year (Fig. 3) with a higher
(P ⬍0.05) proportion of females conceiving in the
summer months (March–August: 50/111, 45.0%)
than the months of spring (March–May: 28/111,
25.2%), fall (September–November: 21/111, 18.0%)
and winter (December–February: 13/111, 11.7%).
The proportion of animals conceiving was higher
(P ⬎0.05) during spring than winter, and similar (P ⬍
0.05) for winter and fall periods. Conception was
more likely to occur in the months of summer than
all other seasons (P ⬍0.05), except for SeaWorld
Orlando where a similar (P ⬎0.05) proportion of
conceptions occurred during the summer and spring
(results not shown).
4. Discussion
This study provides new information regarding pro-
gesterone profiles during pregnancy in the bottlenose
dolphin, particularly during early pregnancy under a
serial blood sampling regimen. Weekly serum proges-
terone concentrations of cycling or pregnant females
have never been reported in the bottlenose dolphin, or
any other delphinid. Circulating progesterone concen-
trations increased exponentially during the first month
of gestation, a period when the maternal recognition of
pregnancy likely occurs in this species.
Mechanisms of pregnancy recognition vary across
mammalian species, but the process typically occurs at
11 to 13 days after ovulation. If conception has oc-
curred, biochemical signals from the uterus maintain
CL function and pregnancy status, whereas in the ab-
sence of a viable conceptus, and in most species exam-
ined to data, the CL undergoes lysis under the influence
of uterine-derived prostaglandin F
2
␣
(reviewed by Rob-
erts, et al., [19]; Spencer et al. [20]). Placentae of
cetaceans are of the diffuse, epitheliochorial type [21].
Although anatomical features of placentae have been
well described for a few delphinids at macroscopic and
microscopic levels (reviewed by Miller, et al. [22]),
Fig. 3. Annual distribution of conceptions in the study population of naturally reproducing bottlenose dolphins (n ⫽31 females at three North
American facilities) for cycles resulting in a term calf (n ⫽111).
477J.K. O’Brien and T.R. Robeck / Theriogenology 78 (2012) 469 – 482
Author's personal copy
information on biochemical signals involved in luteoly-
sis and pregnancy recognition have not been reported
for delphinids nor any cetacean.
Evidence indicates that ungulates of the Artiodactyla
are more closely related to cetaceans than other taxa,
giving rise to the proposed new order of placental
mammals, Cetartiodactyla [23]. It is therefore logical to
examine pregnancy recognition processes within the
Artiodactyla in an attempt to hypothesize about such
processes in dolphins in the context of the findings on
progesterone profiles reported herein. Artiodactylans
with the same diffuse, epitheliochorial placenta as ce-
taceans include the camel and species of the suborder
Suina, including pigs and hippopotamuses [24 –26].In
pigs and camels, estrogens derived from the conceptus
are thought to be primarily responsible for eliciting
pregnancy recognition and suppressing luteolysis of the
CL by prostaglandin F
2
␣
[27,28]. Like the bottlenose
dolphin [10], the primary source of progesterone in pigs
and camels is the CL [29,30]. Examination of concen-
trations of prostaglandin F
2
␣
and its metabolites in
serial serum samples from conceptive and non-concep-
tive bottlenose dolphin cycles would be the first logical
step to examine the control of luteolysis in this species.
It would seem reasonable to hypothesize that the ma-
ternal recognition of pregnancy occurs before Day 14
after ovulation in the bottlenose dolphin, as data herein
revealed that progesterone concentrations in non-con-
ceptive cycles decreased after this time. The specific
mechanisms involved in pregnancy recognition would
require further examination of maternal hormones, as
well as in vitro studies on steroid and protein metabo-
lism by early dolphin embryos.
The occurrence of elevated progesterone concentra-
tions without evidence of a conceptus or fetus, in the
presence or absence of males, has been reported in
several ex situ delphinids, including bottlenose dol-
phins, killer whales [31,32] and false killer whales
[33,34]. In those studies, the source of progesterone
was not established; not unexpectedly, serial ultrasound
examination of ovaries and uteri in the present study
indicated that the source of progesterone in dolphins
with prolonged, elevated concentrations of the steroid
was the CL from the follicular phase ovulation. Similar
observations were reported in the Pacific white-sided
dolphin, where one female underwent two periods of
prolonged elevated progesterone after ovulation in the
presence of an anatomically functional CL [35].In
species which do not experience delayed embryonic
implantation, the occurrence of early embryonic death
after pregnancy recognition is a common cause of pro-
longed maintenance of the CL and accompanying ele-
vated progesterone concentrations [36,37]. However,
the specific incidence of this condition and the factors
causing it has not been established in cetaceans.
An interesting cetacean case by Benirschke and
Marsh [38] describes the presence of an elongated pre-
implantation embryonic vesicle (⬃14 cm in length and
widest diameter of 4 cm) comprising a viable tropho-
blast and a degenerating embryo (less than 0.5 cm
long). The specimen was collected post-mortem from a
wild ⬃28 yrs old short-finned pilot whale. Though it is
not possible to ascertain the speed of conceptus degra-
dation following embryonic death, the presence of a
histologically active CL in this female makes it reason-
able to speculate that prolonged maintenance of the CL
would have ensued. In the current study where known
conception dates were utilized, the earliest indicator of
pregnancy using ultrasound occurred at Day 44 and was
characterized by uterine horn fluid and a thickened
endometrium. If cetaceans are similar to other mamma-
lian species where most pregnancy loss occurs during
the preimplantation stages, it is probable that a number
of animals in the unknown conception status group in
the current study had conceived and undergone unde-
tected embryonic loss. This hypothesis is supported by
the observation of a small volume of clear uterine horn
fluid (ⱕ6cm
2
) in five cycles of the unknown concep-
tion status group (two natural breeding and three AI
cycles) after Day 40.
In horses, prolonged maintenance of the CL without
pregnancy detection is relatively common after mating
(up to 25% of females during a breeding season [37]),
and the condition also occurs in non-mated mares,
albeit on a less frequent basis (⬃4% [37,39]). Though
the mechanisms underlying its cause in the latter sce-
nario are poorly understood, modulation in prostaglan-
din F
2
␣
secretion, in the presence or absence of uterine
inflammation and infection, can result in a retained CL
in that species [37,39,40]. Dolphins with a retained CL
in the present study, representing 12.4% of insemina-
tions with normal follicular luteinization, were clini-
cally normal based on hematology and chemistry val-
ues. However, transient uterine inflammation cannot be
excluded and might have contributed to embryonic loss
and the ensuing persistent CL in some cases. In light of
the similar progesterone profiles among non-pregnant
animals with a retained CL and those undergoing nor-
mal pregnancy, examination of profiles of other hor-
mones, such as relaxin [41] and prostaglandin F
2
␣
is
warranted to help establish potential factors leading to
prolonged maintenance of the CL in the dolphin. Sub-
478 J.K. O’Brien and T.R. Robeck / Theriogenology 78 (2012) 469 – 482
Author's personal copy
stantial variation in the interval of elevated progester-
one, 62 to 282 days for individuals with a retained CL
in the present study supported the high probability of
different etiologies of this condition.
As expected, approximation of conception timing in
naturally bred animals can be adequately performed by
weekly progesterone analyses as described herein, and
similar progesterone profiles were displayed after nat-
ural and artificial inseminations. In cases where weekly
blood collections are not feasible, daily urinary estro-
gen and LH monitoring [13,14,35] can also be used to
retrospectively determine conception timing, but this
approach is typically more time-intensive and costly. In
contrast to studies where a decline in progesterone
secretion was reported in the first 2 to 3 mo [3] or first
3 to 4 mo of gestation [5], progesterone concentrations
in the current study underwent a progressive increase
until Week 7 and remained elevated thereafter. The
most probable reason for these discrepancies is the
relatively low numbers (⬍10) of serum samples at each
monthly time-point in the earlier studies. In the current
study, monthly values (data pooled across 4 wk) com-
prised an average of 47 samples. It was previously
suggested that primiparous dolphins might have re-
duced progesterone concentrations compared to their
multiparous counterparts [3]. In contrast, results herein
which comprised a larger animal and sample dataset
demonstrated no influence of parity on progesterone
concentrations during early, mid, or late pregnancy.
Together with standard ultrasound examinations to
assess fetal growth and development [42], clinical man-
agement of pregnant dolphins would benefit from the
inclusion of bimonthly progesterone concentration de-
termination to monitor fetal wellbeing and to allow
detection of fetal distress. As in other mammalian spe-
cies, abnormal changes in progesterone profiles may
indicate placentitis [43,44] and detection of such anom-
alies will facilitate appropriate monitoring and treat-
ment of the dam and fetus. Progestagen therapy in
bottlenose dolphins in response to abnormal progester-
one profiles (serum progesterone, ⬍5ngmL
⫺1
in two
or more consecutive samples) and retarded fetal growth
has led to normalization of fetal development based on
established growth curves for the species [10]. How-
ever, the lack of information on the endocrinology of
the pregnant dolphin, particularly just before parturi-
tion, has prevented such treatment from culminating in
the birth of a live calf [10]. In the current study, pro-
gesterone concentrations of two females sampled at 5
and 7 days pre-partum were 12.1 and 8.5 ng/mL, re-
spectively; at 1 days post-partum, progesterone from
four females was 1.2 ⫾0.4 ng/mL. Further sampling is
required to characterize peri-parturient hormone
changes and establish normal reference values to fur-
ther enhance clinical management practices for this
species.
Strong evidence for reproductive senescence exists
for many mammalian species [45,46], including del-
phinids [47]. Bottlenose dolphins can produce offspring
into their 40s [18], but the proportion of ovulatory
cycles that result in a term, live calf across different age
classes has not been examined. Features of reproduc-
tive aging in the female have been investigated most
extensively among delphinids in the short-finned pilot
whale (Globicephala macrorhychus)[48 –50]. Post-
mortem examinations of whales from fishery activities
demonstrated that the pregnancy rate declined from
65% for females aged 6 to 12 y to 24% in females aged
30 to 36 y [49]. Interestingly, the proportion of females
possessing ovarian CL but no conceptus increased for
this species after 20 yrs of age. An unpublished study in
the bottlenose dolphin by T. Katsuya (cited in Marsh
and Kasuya [49]), reported that the annual pregnancy
rate declined from 50% for 10-yr-old females to 34%
for those aged 30 to 35 y. In the present study, the
average age of females producing a live, term calf (16
yrs) was significantly lower than that of females under-
going fetal abortion (22 yrs) or of females of unknown
conception status exhibiting potential early embryonic
loss (27 yrs). Gross anatomy of the reproductive tract
and appearance of the endometrium were normal dur-
ing all AI procedures in this study. While undetected
reproductive abnormalities may have contributed to
presumptive early embryonic loss reported herein, par-
ticularly in naturally breeding females where endo-
scopic examinations were not conducted, the possibility
of oocyte aging as a factor in the reduced reproductive
fitness of older females is worth consideration and
examination in future research.
The timing of bottlenose dolphin AI is dependent on
the endogenous LH surge, which therefore leads to
some inseminations being performed at undesirable
hours relative to staff schedules. Protocols for ovulation
induction and timed insemination have been developed
for many domesticated species, without decreasing
conception rates, to synchronize insemination attempts
and thereby improve the efficiency of management pro-
cedures (e.g., Burke, et al. [51]). In the current study,
12 dolphins underwent ovulation induction using
GnRH; of those, four females displayed progesterone
concentrations indicative of abnormal follicular lutein-
ization and did not conceive. In sheep, induction of the
479J.K. O’Brien and T.R. Robeck / Theriogenology 78 (2012) 469 – 482
Author's personal copy
LH surge before adequate follicular and oocyte matu-
ration resulted in abnormal CL formation and similarly
abnormal progesterone profiles [52]. For the eight re-
maining animals undergoing subsequent ovulation in-
duction, additional follicular maturation, as assessed by
preovulatory follicle diameter and urinary estrogens,
was allowed to occur before GnRH treatment. Of those
eight, one displayed elevated progesterone for 246 days
without the detection of a fetus and four females con-
ceived, but only one conceptive cycle resulted in a live
term calf (abortion occurred at Days 69, 100, and 182
of gestation in the remaining females [all were ⬍25 y
of age], respectively). Clearly, further research is re-
quired in the bottlenose dolphin to determine the ap-
propriate timing of GnRH for use in ovulation induc-
tion protocols.
With the exclusion of cycles where abnormal follic-
ular luteinization occurred, the overall incidence of
fetal abortion in the current study was 10.6% (12/113).
Due to the aforementioned difficulties in detecting the
preimplantation dolphin embryo, it was not possible to
attribute early embryonic loss to the cause of prolonged
elevated progesterone in cycles where a fetus was not
detected (14/113, 12.4%), but as mentioned previously,
evidence suggests that the loss of the embryo may have
occurred in a number of those cycles, possibly at a
higher incidence for older females. If only AI cycles
(with normal follicular luteinization) are considered,
the overall incidence of pregnancy failure in the current
study based on embryonic loss (with the assumption
that all females in the unknown conception status group
underwent early embryonic loss) and fetal loss (17/48,
35.4%) appears high. However, if dolphins aged 25 y
and older are excluded (n ⫽9 females), rates of pre-
sumptive early embryonic loss (6/39, 15.4%), fetal loss
(2/39, 5.1%) and combined loss (8/39, 20.5%) were
comparable to rates of pregnancy loss after AI in non-
aged domesticated species [53,54].
An average gestation length of 376 days and a biolog-
ical range of 355 to 399 days were observed for naturally
bred and artificially inseminated dolphins undergoing re-
productive monitoring in the current study. The frequently
published estimate of gestation length in the bottlenose
dolphin of 12 mo clearly underestimates normal gestation
length potential in this species. The integration of this new
knowledge of normal gestation length with current partu-
rition prediction techniques (fetal growth curves [42]; rec-
tal temperature monitoring [55]) may improve the efficacy
of peri-partum management practices.
The gestation length for live term foals is also
highly variable both within and across breeds. Ex-
amination of 433 Thoroughbred normal pregnancies
from known conception timing to birth revealed a
gestation length range of 315 to 388 days, which was
influenced by month of foaling and foal sex [56]. The
exact mechanisms involved in the seasonal and fetal
effects on gestation length have not been unraveled,
but environmental and nutritional factors are hypoth-
esized to be involved [56]. In line with potential
nutritional effects, it has been suggested that a re-
duced embryo development rate during the first 2 mo
of pregnancy in the horse will lead to a prolonged
gestation length [57,58]. Research into early preg-
nancy diagnosis and embryonic development in do-
mesticated species has benefited from high resolution
provided by transrectal ultrasonography. Modifica-
tion of currently used ultrasound probes in delphin-
ids, most notably a decrease in probe size, may
facilitate the application of transrectal or transvagi-
nal ultrasonography for early detection and monitor-
ing of embryos in these species.
Analysis of parturition dates indicated that wild and
captive bottlenose dolphins exhibit seasonal trends in
reproduction which are partly influenced by the geo-
graphic origin of that animal [59]. Naturally bred dol-
phins in the present study conceived in all months of
the year, as has been reported previously [3]. Non-
significant peaks in calving occurred in the ex situ
Californian population during spring and fall months,
but an effect of season on conception/calving timing
was not possible to ascertain due to the small number of
pregnancies in that study (n ⫽36 [3]). In contrast,
conceptions that were monitored in the present study
(n ⫽111, derived from three North American facilities)
occurred significantly more often in the summer (June
through August, 45%) than in any other season. These
findings agreed with preliminary data on the seasonality
of bottlenose dolphin sperm production in males at a
Californian facility [16].
Determination of gestation length from known con-
ception dates has been possible in the bottlenose dol-
phin with the advent of reproductive monitoring and AI
technologies. The results of the present study have
practical implications for husbandry procedures of the
parturient dolphin, and increase our knowledge on fac-
tors that significantly influence reproductive outcome
after natural or artificial insemination. Evidence sug-
gests that prolonged luteal activity may be the culmi-
nation of a non-conceptive or conceptive cycle, and
additional investigation into early embryonic mortality
is required to further characterize reproductive effi-
ciency in the bottlenose dolphin.
480 J.K. O’Brien and T.R. Robeck / Theriogenology 78 (2012) 469 – 482
Author's personal copy
Acknowledgments
This research was supported by SeaWorld Parks and
Entertainment (SWP&E). Brad Andrews (SWP&E)
and Dr. Jim McBain (SWP&E) are particularly thanked
for institutional support. The authors are also grateful to
Karen Steinman, Dr. Gisele Montano, Michelle Bue-
scher and Angela Ho (SeaWorld and Busch Gardens
Reproductive Research Center) and to all animal care,
animal training, curatorial and veterinarian staff at Sea-
World San Diego, SeaWorld San Antonio and Sea-
World Orlando for assistance with this research. In
particular, we acknowledge Dr. Todd Schmitt for con-
ducting serial ultrasound examinations and blood col-
lections, Marilyn Dudley for assistance with breeding
records, and Pam Thomas and Melinda Tucker for
progesterone assay analyses. We also acknowledge all
animal care, animal training, curatorial and veterinary
staff at the following institutions for their collaborative
efforts in this research, in particular: Wayne Phillips
and Dr. Paola Smolensky of Dolphin Adventures (Mex-
ico); Dr. Jay Sweeny and Michelle Campbell of Dol-
phin Quest, Hawaii (USA) and Dolphin Quest Bemuda
(Bemuda); Drs. Claudia Gili and Maddalena Iannac-
cone of Genoa Aquarium (Italy); Dr. Niels Van Elk at
the Harderwjik Dolfinarium (the Netherlands); Dr. Ju-
lio Loureiro and Jose Mendez of Mundo Marino (Ar-
gentina); Drs. Eric Jensen and Cynthia Smith of the US
Navy Marine Mammal Program and Drs. Ana Salbany
and Luis Roque of Zoomarine (Portugal). The national
and international transport of biological samples was
conducted under the National Marine Fisheries Service
(NMFS permit numbers: 782–1694 and 116 –1691).
This is a SeaWorld Technical Contribution Number
2011– 06-T.
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