General and Comparative Endocrinology 177 (2012) 238–245

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General and Comparative Endocrinology

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Evidence of melatonin secretion in cetaceans: Plasma concentration and extrapineal

HIOMT-like presence in the bottlenose dolphin Tursiops truncatus
Mattìa Panin, Gianfranco Gabai, Cristina Ballarin, Antonella Peruffo, Bruno Cozzi ⇑
Department of Comparative Biomedicine and Food Science, University of Padova, Legnaro, PD, Italy

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

Article history: The pineal gland is generally believed to be absent in cetaceans, although few and subsequently uncon-
Received 14 September 2011 firmed reports described the organ in some species. The recent description of a complete and photographed
Revised 11 April 2012 pineal body in a bottlenose dolphin (Tursiops truncatus) prompted us to examine a series of 29 brains of the
Accepted 15 April 2012
same species, but no gland was found. We then decided to investigate if the main product of the gland, mel-
Available online 23 April 2012
atonin, was nevertheless produced and present in the plasma of this species. We collected plasma and
serum samples from a series of captive bottlenose dolphins for a period of 7 months spanning from winter
Keywords:
to summer and we determined the indoleamine concentration by radio-immunoassay (RIA). The results
Pineal gland
Melatonin
demonstrated for the first time a quantitative assessment of melatonin production in the blood of a ceta-
HIOMT cean. Melatonin levels were comparable to those of terrestrial mammals (5.15–27.74 pg/ml daylight con-
Bottlenose dolphin centration), with indications of both seasonal and daily variation although the presence of a circadian
Retina rhythm remains uncertain. Immunohistochemical analyses using as a marker hydroxyindole-O-methyl-
transferase (HIOMT, the key enzyme involved in the biosynthesis of the hormone), suggested extrapineal
melatonin production by the retina, the Harderian gland and the gut. The enzyme was unequivocally local-
ized in all the three tissues, and, specifically, ganglion cells in the retina showed a very strong HIOMT-
immunoreactivity. Our results suggest that further research might reveal unexplored aspects of melatonin
production in cetaceans and deserves special attention and further efforts.
Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction
The presence of a distinct pineal gland (epiphysis cerebri) in ceta-
ceans has been long discussed with no definite conclusions. No pine-
al gland was reported in the Amazon river dolphin (Inia geoffrensis)
[17], in the Pacific white-sided dolphin (Lagenorhynchus obliquidens)
and the spinner dolphin (Stenella longirostris) [3], in the short-
beaked common dolphin (Delphinus delphis) [37], in the dwarf
sperm whale (Kogia sima) [39], and in the fetal narwhal (Monodon
monoceros) [20]. A ‘‘rudimentary’’ pineal gland was observed by Fuse
[14] in the finless porpoise (Neophocaena phocaenoides), while the
presence of a well-formed gland was described at least in some spec-
imens of beluga (Delphinapterus leucas) and bottlenose whale
(Hyperoodon ampullatus) [25], sperm whale (Physeter macrocepha-
lus) [38], and harbor porpoise (Phocoena phocoena) [4]. However,
the presence of a pineal gland is apparently not constant among all
the individuals of a given species. A pineal gland was found in six
humpback whales (Megaptera novaeangliae) [15], whereas both
⇑ Corresponding author. Address: Department of Comparative Biomedicine and
Food Science, University of Padova, viale dell’Università 16, 35020 Legnaro, PD,
Italy. Fax: +39 049 8272796.
E-mail address: bruno.cozzi@unipd.it (B. Cozzi).
Breathnach [6] and Pilleri [44] did not find any in the same species.
In the sei whale (Balaenoptera borealis) Pilleri [45] described a pineal
gland ‘‘intimately bound with choroid plexus under the splenium of
the corpus callosum’’, but Arvy [3] reported it to be absent. Duffield
et al. [12] were able to find a pineal body only in one out of 11 spec-
imens of bowhead whale (Balaena mysticetus) harvested by Alaskan
Eskimos. In an adult blue whale (Balaenoptera musculus) no pineal
gland was found in a study [43], but the presence of the organ was
reported in a fetal specimen by others [21]. The common bottlenose
dolphin, Tursiops truncatus, a widely studied cetacean, represents an
emblematic case. Neither McFarland et al. [34] nor Ridgway [50]
were able to detect a pineal body in several brains of this species.
On the contrary, Morgane and Jacobs [36] observed a pineal gland
‘‘present up to the adult stage’’, and an unmistakable image of a large
pineal gland in a pregnant female has been published recently [28].
Variations of the size in the gland during pregnancy were described
in bats [18], squirrels [5] and humans [65], but the gland does not ap-
pear or disappear completely.
The unpredictability of the presence of a pineal organ in ceta-
ceans has never been fully explained. From the evolutionary point
of view, a specific organ should be present (or absent) in a given
species even if rudimentary, but its appearance only in random
individuals is enigmatic. Furthermore, the pineal gland is well

0016-6480/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ygcen.2012.04.012
 M. Panin et al. / General and Comparative Endocrinology 177 (2012) 238–245 239

developed in other non-cetacean marine mammals, specially veterinary medical health controls and because of this the experi-
Phocidae [9] and Otariidae [35], in which it reaches a remarkably mental design was slightly different in the two facilities, due to the
large size with high melatonin plasma levels in newborns [54]. different temporal availability of the veterinary staffs and the
Prompted by the puzzling data present in the literature we necessity to coordinate and integrate the sampling effort with rou-
examined a series of brains of T. truncatus by gross dissection to tine activities.
evaluate systematically the possible presence of a pineal gland. Gi- The animals that we sampled from have been living in the same
ven that no report is present in the literature about the secretion of facility from birth or at least for several years. They could not be
melatonin in this species, we also studied for the first time its plas- constrained into artificial light:dark cycles, due to the unfeasibility
ma concentration in captive individuals. Finally, we investigated of such procedure in their quarters. Complete darkness in the pools
extrapineal melatonin production in three tissues indicated in after sunset is impossible to achieve, since there is always a mini-
the literature as the most probable sites for other mammals: the mal nocturnal artificial lighting. Although the dolphin pools are
retina [58], the Harderian gland [41], and the gastrointestinal tract seldom directly illuminated, lights streams in from adjacent sec-
[24]. To this effect we chose the enzyme hydroxyindole-O-methyl- tors of the parks in which the dolphinaria are located. Light inten-
transferase (HIOMT) as a marker, due to its terminal position in the sity was not monitored, but we relied upon sunrise/sunset time
biosynthetic pathway of the hormone, and its high specificity for gathered from the U.S. Navy on-line service (http://aa.usno.navy.-
melatonin-producing tissues [2]. mil/).
Water is generally heated in the dolphin pool during winter
2. Materials and methods time. Temperature range therefore varies from approx. 18 °C (cold-
est winter peak) to 27 °C (warmest summer peak).
2.1. Brain samples Dolphins are usually fed at fixed hours during the day, plus
unscheduled meal sections in which feeding is a way to positively
The Mediterranean Marine Mammal Tissue Bank (MMMTB, enforce required behaviors. Cooperation during the sampling ses-
http://www.mammiferimarini.sperivet.unipd.it/eng/index.php), sions is routinely accompanied by feeding rewards. Furthermore,
established in 2002 and hosted by the Department of Experimental while blood was withdrawn from the vessels of the fluke of one
Veterinary Science of the University of Padova, contains over 2500 animal, the others were kept at distance by rewarding them with
samples of cetacean organs, including a collection of more than fish. When blood is sampled, the animal stands still at the surface
forty formalin-fixed brains, 29 of which belong to bottlenose dol- and floats only with the pectoral fins. Blood sampling from the
phins. Twenty-nine of these latter brains were cut into serial coro- flukes cannot be performed in the dark or with minimal red light.
nal sections 1 cm thick; one additional brain was cut into sagittal A permanent catheter cannot be inserted, because of the continu-
sections 1 cm thick. All sections were examined for the presence ous high mobility of the flukes. Here we stress that these are the
of a pineal gland in the epithalamus. Structures possibly identified normal operational setting for routine medical samplings.
as pineal glands were to be removed, washed in phosphate buffer For the assessment of seasonal variations of melatonin concen-
saline (PBS) pH 7.4 overnight, immersed in a cryoprotective solu- tration, one sample was scheduled to be drawn at approx 10:00
tion (30% sucrose in PBS), frozen and then cut into serial cryostat during the first week of each month (December 2008 to June
sections (8–10 lm thick). 2009, group A; February to August 2009, group B, respectively,
A few bovine pineal glands were removed at the slaughter- see Table 2). For the assessment of daily variations, sampling took
house, fixed in 10% buffered formalin overnight, processed for place at approx 10:00 and 17:30, for three subsequent days during
cryostat sectioning and used as a positive control for immunohis- the second week of December and June in dolphinarium A, and for
tochemistry (see below). two subsequent days during the first week of February and August
in dolphinarium B, respectively (see Table 3).
2.2. Specimens and sampling design for melatonin quantification Each blood sample was drawn from the ventral surface of the
tail flukes into vacuum EDTA tubes, while trained dolphins offered
A total of twelve captive bottlenose dolphins of known age, voluntarily their flukes. The plasma was then separated after cen-
maintained into two distinct dolphinaria, A (latitude 41°370 N; trifugation for 20 min at 2800g (3500 rpm) and stored at 20 °C.
n = 4) and B (latitude 37°070 N; n = 8) were involved in the present
study (Table 1). Bottlenose dolphins held in captivity are con-
stantly monitored under a strict ethical code enforced by European Table 2
laws (see below). Therefore sampling was scheduled to overlap Melatonin concentration (in pg/ml) in blood samples used for seasonal variation
assessment.
Table 1 A group
List of specimens with identification name, sex and age (in years).
DEC JAN FEB MAR APR MAY JUN
Sex Age RM1 12,90 10,57 11,54 9,64 7,13 7,95 12,67
RM2 13,25 11,64 11,93 12,91 11,78 13,03 13,35
A group
RM3 18,04 17,57 20,57 23,01 18,11 20,86 19,20
RM1 M 17
RM4 20,37 24,33 22,63 25,35 18,38 21,50 16,99
RM2 M 11
RM3 M 10 B Group
RM4 F 7
FEB MAR APR MAY JUN JUL AUG
B group PT1 16,36 21,65 21,36 22,31 14,76 10,94 27,56
PT1 F 27 PT2 14,21 9,59 14,72 14,16 7,60 12,04 15,24
PT2 M 49 PT3 11,55 17,65 14,58 11,58 8,73 10,26 25,43
PT3 M 14 PT4 12,26 12,72 14,77 15,11 8,25 9,77 24,53
PT4 F 31 PT5 6,02 11,38 14,73 13,88 5,15 6,89 19,35
PT5 F 41 PT6 15,13 21,71 19,47 16,03 11,35 27,74 /
PT6 F 13 PT7 / 16,53 15,84 / 7,61 10,77 /
PT7 F 13 PT8 / / / 19,48 11,03 23,04 20,70
PT8 F 10
/, Not determined; DEC, December; JAN, January; FEB, February; MAR, March; APR,
F, female; M, male. April; JUN, June; JUL, July; AUG, August.
240 M. Panin et al. / General and Comparative Endocrinology 177 (2012) 238–245

Table 3
Melatonin concentration (in pg/ml) in blood samples used for daily variation assessment.

A group
December June
I Day II Day III Day I Day II Day III Day
a.m. p.m. a.m. p.m. a.m. p.m. a.m. p.m. a.m. p.m. a.m. p.m.
RM1 / / 9,96 14,38 11,39 15,37 10,93 12,23 17,80 / 9,27 22,94
RM2 11,23 17,48 12,09 17,05 10,94 15,11 9,62 18,15 15,7 / 14,73 14,69
RM3 18,41 24,59 18,33 19,80 19,08 21,95 21,15 27,22 19,83 / 16,61 16,92
RM4 23,62 / 19,1 16,04 22,17 23,17 15,68 / 16,57 / 18,71 29,69
B group
February August
I Day II Day I Day II Day
a.m. p.m. a.m. p.m. a.m. p.m. a.m. p.m.
PT1 16,80 34,59 15,92 24,57 24,70 / 30,42 42,90
PT2 15,41 16,71 13,02 49,18 18,80 / 11,68 /
PT3 8,34 29,28 14,77 28,95 25,88 26,34 24,98 38,74
PT4 14,50 18,16 10,01 / 26,66 24,67 22,41 33,16
PT5 / / / / 21,36 19,71 17,33 24,43
PT6 / / / / / / / /
PT7 / / / / / / / /
PT8 / / / / / / / /

/, Not determined; a.m., before noon; p.m., after noon; DEC, December; JAN, January; FEB, February; MAR, March; APR, April; JUN, June; JUL, July; AUG, August.

2.3. Ethics of experimentation
All the blood samples from bottlenose dolphins were collected
using routine husbandry training and were obtained from unre-
strained animals, following the EU directive 1999/22/CE concerning
the keeping of wild animals in zoos (http://eur-lex.europa.eu), and
the EAAM standard for facilities housing bottlenose dolphins
(http://www.eaam.org/housing_standards/).
The bovine pineals were sampled from animals treated according
to the European Communities Council directive concerning animal
welfare during the commercial slaughtering process (86/609/EEC).
2.4. Immunohistochemistry
Formalin-fixed retinas, Harderian glands, gut (Bank IDs #54,
#89, #186, #192) and epithalamus (ID #107) samples of adult bot-
tlenose dolphins stored in the MMMTB were either embedded in
paraffin, or frozen. Both cryostat (10 lm) or paraffin embedded
sections (4 lm) were incubated for 30 min at room temperature
(RT) in 3% H2O2 in absolute methanol. After saturation with 10%
normal goat serum (NGS) for 20 min, sections were incubated
overnight at 4 °C with a validated polyclonal anti-human HIOMT
primary antibody raised in rabbit (courtesy of Dr. Fukuda, Univer-
sity of Tokyo), at a final dilution of 1:2000. The sections were then
incubated with a biotinylated anti-rabbit IgG secondary antibody
(Vector Laboratories), at a final dilution of 1:200 for 1 h at RT, then
with avidin–biotin complex (Vector Laboratories) for another hour.
Finally 3,30 -diaminobenzidine (DAB) (Vector Laboratories) was
used as a chromogen for 2–5 min, then sections were counter-
stained with haematoxylin.
For details of the specificity validation procedure of the affinity-
purified anti-HIOMT antibody please refer to [13]. As a positive
control, a few pineal glands were sampled from adult bovines ob-
tained from a commercial abattoir and treated like the dolphin
samples. Negative controls included skin samples in parallel with
the other tissues, omission of the primary antibody.
2.5. Melatonin assay
Melatonin concentrations in bottlenose dolphin plasma samples
(200 ll) were measured in duplicate by a commercial RIA kit
(RE29301, IBL International) following the manufacturer’s instruc-
tions. As the kit was originally designed for the direct quantification
of melatonin in human plasma, it was validated for bottlenose
dolphin plasma by performing parallelism and recovery tests. Paral-
lelism was assessed by calculating the regression curve between the
observed hormone concentrations and the reciprocal of the dilution
factors in bottlenose dolphin plasma samples serially diluted up to
1:8. The assay showed a good degree of parallelism, as demon-
strated by the values of the intercept of the regression curves that
were not significantly different from 0 (P > 0.05), and the regression
coefficients (R2) were always greater than 0.98. Recovery was as-
sessed in bottlenose dolphin plasma samples spiked with known
amounts of melatonin (expected melatonin concentrations ranged
between 10 and 130 pg/ml). In the concentration interval consid-
ered, recovery ranged between 90% and 117% indicating an overall
good correspondence between expected and observed hormone
concentration values, although a slight overestimation in the higher
values was observed. The sensitivity of the assay expressed as 90% of
hormone bound (B/Bo) was 3 pg/ml. The calculated intra-assay CV
ranged between 3.2% and 5.8%, and inter-assay CV was 10.8%.
2.6. Statistical analyses
Sample size is always troublesome when working with captive
marine mammals, and the use of a General Linear Model (GLM)
considering all the variables resulted unfeasible. Thus, we opted
for separate analyses of plasma melatonin concentration as fol-
lows. One-way ANOVA with Tukey’s post-hoc multiple compari-
sons was used to assess significant differences in the trend of
mean values of each month, separately for the two locations.
Two different General Linear Models (GLM), with Tukey’s post
hoc test, were employed as follows: the first, to compare mean val-
ues between overlapping months (i.e. from February to June), with
‘‘month’’ factor nested within ‘‘location’’ factor; the second, to
compare mean morning and afternoon values for each specimen,
then pooling them by month in a second analysis, with ‘‘specimen
name’’ factor nested within ‘‘period of the day’’ factor, in turn
nested within ‘‘location’’ factor. All the statistical analyses were
performed using the software Statistica (StatSoft Inc., Tulsa, OK)
and setting the significance level for P < 0.05.
 M. Panin et al. / General and Comparative Endocrinology 177 (2012) 238–245
3. Results
3.1. Gross dissection of formalin-fixed brains
Gross dissection of the series of thirty brains (29 coronal and 1
sagittal), including careful removal and inspection of the meninges,
revealed a small epithalamic body resembling a pineal gland only
in one specimen (ID #107). The individual was an adult male bot-
tlenose dolphin delivered to our facility for post-mortem evalua-
tion. A brownish-red corpuscle was clearly visible dorsal to the
thalamic enlargement, with two lateral structures recalling the
striae habenularis thalami (sin.: striae medullaris thalami), which
normally connect the pineal body to the habenular areas
(Fig. 1A). No discernible pineal recess of the third ventricle was
present. Observation at the microscope failed to reveal a parenchy-
mal pineal-like tissue, but showed instead a tangle of blood vessels
and cuboidal epithelial tissue (Fig. 1C). These elements suggested
that the structure had no glandular function, but was composed
of a densely packed assemblage of the choroid vessels connected
to the habenular walls.
3.2. Plasma melatonin in captive dolphins
Some failures in the sampling effort occurred due to unstable
cooperation by some of the animals or to insufficient blood volume
obtained after withdrawals, so the total number of samples was
less than expected (see Tables 2 and 3 for details).
The results of melatonin quantification are presented in Tables
2 and 3. Melatonin concentration of the monthly samples in the A
group ranged between 7 pg/ml (RM1, April) and 25 pg/ml (RM4,
March), and between 5 pg/ml (PT5, June) and 28 pg/ml (PT6, July)
in the B group. The values followed distinct patterns for each spec-
imen, with very little fluctuations in group A, unlike in group B dol-
phins. Fig. 2 shows the trend of the mean values of each month for
both locations. For the A specimens no significant variation among
months was observed (ANOVA; F = 0.202, P = 0.972). For the B ones
a significant variation was found (ANOVA; F = 5.585, P = 0.000), and
specifically August showed a higher value than February (Tukey’s
post hoc, P = 0.010), June (P = 0.000) and July (P = 0.025). However,
comparison of the means of the overlapping months between the
two locations (i.e. from February to June), did not result significant
(GLM, F = 1.646; P = 0.179).
Looking at the daily variation of melatonin levels (Fig. 3), in all
the individuals the values were always lower in the morning than
in the afternoon, both in winter and in summer months, with an
increase ranging between 101.8% and 377.7% in 27 out of 31 com-
parisons. Due to the paucity of samples for daily variation assess-
ment, the statistical analyses were unfortunately limited. As a
consequence, considering the mean values of subsequent sam-
plings, none of the individuals showed a significant variation be-
tween morning and afternoon concentrations, nor in A group
(GLM; Tukey’s post hoc, P = 0.163) or in B group (P = 0.052).
However, pooling the values of all the B group specimens, a highly
significant difference was found between morning and afternoon
Fig. 1. Epithalamus of bottlenose dolphin ID #107. (A) Coronal section showing a pineal-like body (rectangle) between the thalamic masses and under the corpus callosum.
Two ridges of tissue (arrowheads) flanking the corpuscle recalled the striae medullaris thalami. (B and C) Hematoxylin-eosin staining (2 and 10 magnification, respectively)
of the corpuscle, revealing a tangle of capillaries (asterisks) and cuboidal epithelium (arrowheads), typical of the choroid plexus. cc = corpus callosum, lv = lateral ventricle,
t = thalamus. Scale bar = (A) 1 cm; (B) 500 lm; (C) 50 lm.
241
Fig. 2. Trend of the mean daytime melatonin concentrations (± S.E.M.) of each
month for both locations. The A group dolphins show no significant variations
among months. In the B group, values of February, June and July were all
significantly different than those of August. Lowercase letters indicate statistically
different values (P < 0.05).
levels in February (GLM; Tukey’s post hoc, P = 0.004), but not in
August (P = 0191). On the contrary, no significant difference was
found for the A group specimens either in December (P = 0.627)
or in June (P = 0.166) (Fig. 3).
3.3. Immunohistochemistry
The epithalamic choroid-like structure, sampled from ID #107
and described above, showed no HIOMT-immunoreactive-like
(-ir-like) elements (Fig. 4A).
In all the dolphins, the investigated extrapineal tissues showed
a marked positive staining, indicating the synthesis of HIOMT. The
freshness of the samples was variable among dolphin tissues, a fac-
tor linked to postmortem interval prior to sampling. The bovine
pineal glands showed a widespread staining of cells (= pinealo-
cytes) in the parenchyma (Fig. 4B and C).
3.3.1. Retina
In the retina, the positivity was limited to individual cells lo-
cated in the ganglion cell layer (GL) and to the nerve fiber layer
(NFL) (Fig. 4E and F). However, in some regions of the same sec-
tions, the outer plexiform layer (OPL) showed an intense staining
as well (Fig. 4G). Positive cells in the GL occurred at regular inter-
vals along the circumference of the layer. The staining of their cyto-
plasm was always very intense, displaying large dark granules. The
OPL and the NFL showed less intense and more uniform positivity.
3.3.2. Harderian gland
In the Harderian glands, HIOMT-ir-like cells were localized only
in some of the lobules, accounting for a very small part of the or-
gan. A variable number of positive secretory acini was present
within each of these latter lobules (Fig. 4H). The staining was
localized uniformly in the cytoplasm of most cells (Fig. 4I), but in
242 M. Panin et al. / General and Comparative Endocrinology 177 (2012) 238–245
Fig. 3. Mean melatonin concentrations (± S.E.M.) of subsequent samplings for daily
variation assessment. No significant variation between morning (empty bars) and
afternoon values (grey bars) was found for the A group, whereas a significant
difference was found in February for the B group. Lowercase letters indicate
statistically different values (P < 0.05).
specimen ID #89 some secretory cells showed a concentration of
positive response towards the lumen of the acinus (Fig. 4J).
3.3.3. Gut
The absorptive epithelium was recognizable as small rows of
enterocytes still held together by tight junctions. A strong positiv-
ity for HIOMT was found restricted to the apical surface of these
cells, seemingly in the brush border (Fig. 4K). The precise architec-
ture of the organ in the sections was sometimes altered, due to the
detachment of the Lieberkühn’s crypts and superficial mucosa pos-
sibly due to autolytic processes or pre-agonic waste, and debris
filled the lumen. A weaker staining was sometimes found in the
surroundings of absorptive cells, possibly related to non-specific
staining of debris or local dye accumulation (Fig. 4L).
4. Discussion
Our study failed to identify a pineal gland in a series of 29 bot-
tlenose dolphin brains, thus confirming the majority of the reports
and the general opinion that a distinct epiphysis cerebri is not pres-
ent in adult cetaceans. An imperfect dissection of the brain, with
excision of meninges or vascular structures closely bound to the
gland, may lead to ripping and avulsion of a possibly tiny glandular
structure. Furthermore, sectioning of the brain in thick serial
coronal slices might hide the epiphysis at gross examination, espe-
cially if the gland is constituted by a small deep pineal followed by
an elongated stalk and a superficial part, like in rabbits and several
rodents [60]. However, the cetacean brain has been studied in de-
tail and occasional mistakes might not be repeated so frequently in
the literature. In our study careful removal of the meninges after
extraction of the brain and accurate dissection revealed no hint
of the pineal, except in individual #107 in which a cluster of cho-
roid vessels superficially (but not microscopically) resembled the
epiphysis cerebri. We note that unless a further histological investi-
gation confirms the glandular structure of a pineal-shaped organ, a
cluster of dark compact vessels may lead to a wrong identification.
Yet in at least one former occasion, referred to six humpback
whales, a pineal gland was identified and confirmed by histological
analysis of celloidin sections [15]. An incomplete development of
the fetal anlage might result in pineal tissue distribution in islets
or sheets of pinealocytes, instead of a distinct gland, like in ele-
phants [19]. Imaging of whole brain represents a useful means to
investigate the presence of a pineal gland, because of the calcium
and magnesium salt concretions (brain sand, corpora arenacea) that
can be visualized as bright spots by MRI or CT [64]. If such concre-
tions would occur also in cetaceans, pineal glands might be more
easily detectable, especially in older specimens. However, studies
involving MRI analysis of cetacean brains, performed in the bottle-
nose [30], common [39] and spinner dolphins [29], showed no
pineal gland in the dorsal thalamus. It is worth noting that a dis-
tinct pineal gland seems to be absent also in the dugong (Sirenia)
[48], and in terrestrial mammals like the elephant [19] and the
nine-banded armadillo [42]. In the bovine, HIOMT-ir pinealocytes
have been localized in the habenular and posterior commissures,
which delimit the gland rostrally and caudally, respectively [52].
Future studies with HIOMT immunohistochemistry may address
these and other neighbouring regions also in the dolphin dorsal
thalamus in search of alternative subcallosal loci of melatonin
production.
In spite of the lack of a pineal gland in our brain samples, here
we report for the first time, to the best of our knowledge, quanti-
tative data on the presence of melatonin in the blood of a cetacean
species. Overall, daytime levels varied between 5 and 28 pg/ml.
These values are consistent with what observed in rats (26 pg/
ml) and domestic pigs (10–30 pg/ml) [1], bovines (12 pg/ml) [51]
and humans (2–10 pg/ml) [49]. The annual fluctuations of melato-
nin levels were not significant for the A group, whereas the B group
in August showed significantly higher values than in February, June
and July. However, the annual profiles of both groups have exten-
sive gaps, so we do not know if also the A group would show a rise
in melatonin after June. The peak in August for the B group is rather
unexpected, since throughout the year the daytime melatonin con-
centration does not change significantly in most mammals, even in
seasonal breeders. Seasonal variations are rather described as dif-
ferences in amplitude and duration of night-time peak levels that
follow the changing length of the photoperiod. This shift of the dai-
ly rhythm is perceived in a species-specific manner as a signal for
the onset of reproductive season. Bottlenose dolphins show a con-
siderable flexibility in the reproductive seasonality, as estimated
by sighting and stranding records of calves, with diffuse peaks over
spring and summer months [57]. Hormonal variation assessment
[46] revealed that the ovulatory cycle may span 21–42 days, but
it may occur at irregular intervals. A female might be seasonally
polyestrous, then anestrous for 1–2 years and then polyestrous
again. Most males are thought to produce sperm across the season,
thus spanning the ovulating periods of all the females in a given so-
cial group. For additional details on the seasonality of reproduction
and pregnancy in cetaceans, see the review by Pomeroy et al. [46].
A relationship between melatonin concentration and reproduc-
tive stage was not established in our study, so future works
 M. Panin et al. / General and Comparative Endocrinology 177 (2012) 238–245 243

Fig. 4. Hydroxy-indole-O-methyl-transferase (HIOMT) immunohistochemistry. (A) Pineal-like body in the epithalamus of dolphin #107 showing no HIOMT-immunore-
activity of the cuboidal epithelium (arrows); a small capillary is visible (asterisk). (B) HIOMT-positive parenchyma in the bovine pineal gland. (C) Detail of rectangle in panel
(B); note that some pinealocytes are positively stained (arrows) while others are not (arrowheads). (D) Corresponding region of panel (C) with omission of the primary
antibody, showing a clearly negative staining. (E) Retina of bottlenose dolphin ID #186. Note the strong HIOMT-positive ganglion cells (arrows, rectangle) occurring at roughly
regular intervals. (F) Detail of rectangle in panel (E). Note the coarse granular appearance of positive cytoplasm of ganglion cells (arrows). (G) Retina of bottlenose dolphin ID
#192. Note the intense HIOMT-immunoreactivity of the outer plexiform layer (opl) similar to that of a ganglion cell (arrow). (H) Harderian gland of bottlenose dolphin ID #89
showing lobules with either positive (arrows, rectangle) or negative (arrowheads) secretory cells. (I) Detail of rectangle in panel (H). In this lobule all the acini (ac) are HIOMT-
positive, with uniform staining of the cytoplasm. (J) Harderian gland of bottlenose dolphin ID # 54. Note that reactivity is concentrated towards the lumen (asterisks) of the
secretory acini (ac). This section was not counterstained with haematoxylin, but nuclei are clearly visible (n). (K) Intestine of dolphin ID #192. The lumen (ln) is filled by cells
of the absorptive epithelium or of the intestinal glands that detached after autolytic processes. (L) Detail of rectangle in panel (K). Note the strong reactivity of the apical
cytoplasm (or possibly the brush border) of the enterocytes still held together in rows (arrows); a weaker positivity is shown by other cells shredded by the intestinal glands
or possibly by local dye accumulation in debris (asterisks). Abbreviations: a = artifact, ac = acinus, dm = detached mucosa, ch = choroid, cp = posterior commissure,
d = secretory duct, gl = ganglion cell layer, inl = inner nuclear layer, ipl = inner plexiform layer, is = interlobular septum, ln = lumen, mn = meninges, mu = mucosa, n = nucleus,
nf = nerve fiber layer, onl = outer nuclear layer, opl = outer plexiform layer, os = outer segment of photoreceptors, p = pineal parenchyma, rpe = retinal pigmented epithelium,
sm = submucosa, so = subcommissural organ, t = thalamus, v = ventricular space. Scale bar = (B, E, H, K) 500 lm; (A, C, D, F, G, I, J, L) = 50 lm.

comparing melatonin and sexual hormones production in this spe-
cies might shed light on this aspect. Night-time values for our spec-
imens were unfortunately unavailable, because of the technical
difficulties of performing blood sampling at night in a dolphinarium
and the need to overlap sampling with routinely programmed med-
ical venipuncture. Blood collection would be potentially harmful for
the animals and hard to carry out without a light source. For these
reasons, in the afternoon blood was taken as late as possible at the
same hour, and the corresponding melatonin values were consid-
ered as dark phase ones. Unfortunately, very few blood samples
were drawn after the sunset, and this obviously has led to a very
small significance of differences between morning and afternoon
levels, which ranged between 12 and 49 pg/ml. These values are
far below the average nocturnal peak levels observed for example
in humans (around 100 pg/ml) [22], or Weddel seals (131 pg/ml)
[16], even though some species apparently do not reach very high
levels, like cows (55 pg/ml) [51] and pigs (23 pg/ml) [1]. The signif-
icant difference between pooled morning and afternoon values in
February (for the B group) is not fully explainable, but represents
an indication that a daily variation in bottlenose dolphins may exist.
A much higher number of specimens and samples and a better sam-
pling schedule are needed to properly inquire the pattern of circa-
dian and circannual melatonin production in this species.
Since we obtained evidence of circulating plasma melatonin but
failed to identify a pineal gland in the bottlenose dolphin, we used
HIOMT immunohistochemistry to look for an alternative source of
blood melatonin. We chose to indicate reactive cells as ‘‘HIOMT-ir-
like’’ because the dolphin protein has not been cloned and purified
yet, for making affinity tests. However, cross-reactivity for tens of
proteins and enzymes between human and dolphin tissues has
been proven by a few studies [8], [26].
Our results indicate that the bottlenose dolphin retina contains
the enzyme HIOMT and therefore might be able to produce mela-
tonin. We observed a specific population of HIOMT-ir-like cells in
the GL, and their regular distribution suggests that positive ele-
ments may belong to a specific type of ganglion cells among those
recognized in the mammalian retina [31]. A marked staining was
present also in the NFL and sometimes in the OPL, whereas no po-
sitive response was shown in the outer nuclear layer (ONL). The
ONL, composed of photoreceptors nuclei, was demonstrated to be
melatonin-ir or HIOMT-ir in rodents [59], [63], bovines and hu-
mans [62]. However, the studies by Wiechmann and co-workers
were corrected by the same group [61] stating that HIOMT-immu-
nopositivity was due to cross-reactivity with recoverin. In the in-
ner nuclear layer (INL) only one report described a population of
HIOMT-ir cells in rodents [59], whereas there are no accounts for
244 M. Panin et al. / General and Comparative Endocrinology 177 (2012) 238–245
HIOMT-immunoreactivity in other retinal layers in mammals. It is
generally recognized that melatonin is synthesised in the photore-
ceptors and plays a key role in the regulation of normal physiology
of the retinal pigmented epithelium (RPE), through paracrine
secretion [40,23]. The bottlenose dolphin retina seems to be a un-
ique case, as photoreceptors apparently do not synthesize HIOMT.
Since the mammalian retina does not possess endocrine properties,
melatonin produced by ganglion cells may act in a paracrine fash-
ion, possibly modulating neurotransmission [10,11].
In our Harderian glands, we observed only a very small number
of HIOMT-ir-like cells, in few secretory acini of some lobules. This
paucity is coherent with what observed in rodents [59], minks [33],
and baboons [32]. In all these latter reports, however, both melato-
nin and the precursor N-acetyl-serotonin (NAS) were used as
markers. To the best of our knowledge in the literature there are
no accounts for immunodetection of HIOMT in the Harderian
gland. The small number of HIOMT-ir-like cells may be due to
scarce synthetic activity in the organ or to shifts in the circadian
production. Vivien-Roels et al. [59] described a more intense
immunofluorescence in glands of rodents killed at night than of
those killed during the daytime, and in fact Harderian gland mela-
tonin production is rhythmic [41].
With respect to the gut, several reports described melatonin-
immunoreactivity in enteroendocrine or enterochromaffine (EC)
cells of rats and humans [47,7,27]. However, melatonin presence
might be the result of uptake from the blood and local accumula-
tion, as demonstrated in the rat [7]. HIOMT, being a cytoplasmic
enzyme, is a more reliable indicator of the local synthesis of the
hormone. We did not detect any HIOMT-ir-like EC cells in intestine
sections, and the strong positivity observed in the brush border
suggests that staining in the enterocytes might reside in their api-
cal cytoplasm. Melatonin has potent antioxidant properties [56],
helpful to protect epithelia from free-radical damage in several or-
gans such as the Harderian gland [41], the gall bladder [55] and the
duodenum [53]. Future studies are needed to investigate the pos-
sibility of intestinal production of melatonin by enterocytes in
the dolphin.
In conclusion, our data clearly demonstrate for the first time
that melatonin is present in the blood of T. truncatus, despite the
apparent lack of a pineal gland. Our data on plasma levels of the in-
dole do not allow any conclusion on the presence of daily and sea-
sonal variations, which need to be studied more in depth in future
works. The extrapineal origin of melatonin in this species deserves
special attention, because of its very different characteristics with
respect to what is reported in the literature for most mammals.
Acknowledgments
We would like to thank Dr. Pedro Lavia for the generous help he
gave us in this study by allowing us full cooperation from his
skilled medical staff, including – among the others – Dr. Elio Vicen-
te, Dr. Ana Salbany, and Dr. Elisabetta Mantratzi. We also thank Dr.
Elena Guglielmi for her suggestions, technical opinions and in-
sights. This work is based on the technical skills and ability of Dr.
Maristella Giurisato and Mr. Giovanni Caporale, of the Dept. of
Comparative Biomedicine and Food Science of the University of
Padova: we thank them for their cooperation. We finally thank
Dr. Takahiro Fukuda for the use of his HIOMT antibody.
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