Mar Biol DOI 10.1007/s00227-011-1676-9
S H O R T CO M MU N I C A T I O N
Use of blubber levels of progesterone to determine pregnancy in free-ranging live cetaceans Sergi Pérez · Ángel García-López · Renaud De Stephanis · Joan Giménez · Susana García-Tiscar · Philippe Verborgh · Juan Miguel Mancera · Gonzalo Martínez-Rodriguez
Received: 19 October 2010 / Accepted: 14 March 2011 © Springer-Verlag 2011
Abstract We tested the possibility of measuring progesterone levels in bubbler samples collected from free-ranging live bottlenose dolphins (n = 11) and long-Wnned pilot whales (n = 2) as a tool to evaluate the pregnancy status of individuals. Samples were collected during January 2004 and September–October 2005 in the Strait of Gibraltar and Gulf of Cadiz and used for sex-determination by genetic methods (skin samples) and for quantiWcation of progesterone levels by enzyme immunoassay. Photo-identiWcation tracking of females after taking the biopsy was used to observe the presence of newborns and, in this way, to determine if the female was pregnant at the time of sampling.
Communicated by U. Sommer. S. Pérez (&) · J. Giménez · P. Verborgh CIRCE (Conservation, Information and Research on Cetaceans), C/Cabeza de Manzaneda 3, Algeciras-Pelayo, 11390 Cádiz, Spain e-mail:
[email protected] R. De Stephanis Department of Conservation Biology, Estación Biológica de Doñana, CSIC, C/Americo Vespucio, s/n, 41092 Isla de la Cartuja, Sevilla, Spain S. García-Tiscar Department of Ecology, Universidad Autónoma de Madrid, Madrid, Spain Á. García-López · G. Martínez-Rodriguez Instituto de Ciencias Marinas de Andalucía, Consejo Superior de Investigaciones CientíWcas, Avenida República Saharaui nº 2, 11510 Puerto Real, Cádiz, Spain J. M. Mancera Departamento de Biología, Facultad de Ciencias del Mar y Ambientales, Polígono Universitario Río San Pedro, 11510 Puerto Real, Cádiz, Spain
Mean progesterone levels from pregnant bottlenose dolphins (n = 2) were around 9 times higher than those from non-pregnant females (n = 9), with no overlap between concentration ranges demonstrating that this method could constitute an eVective tool for determining pregnancy in wild populations of bottlenose dolphins and other cetacean species.
Introduction Proper conservation management of wild cetacean populations requires a great variety of Weld information. Among other parameters, information about reproductive status, including pregnancy rates, is of great value as an index for future recruitment into the population (e.g., reproductive success) allowing for instance more accurate estimations of the viability of wild populations in the near future (e.g., using numeric models such as population viability analysis). Despite this importance, current knowledge about reproductive biology of wild cetacean populations is very scarce (Mansour et al. 2002) and, in many cases, such limited information constraints to a great extent the decisionmaking process for future conservative actions (Smit 2003). DiVerent methods, including ultrasonography examinations and blood, feces, milk, and urine samplings, have been applied to determine pregnancy in captive, stranded, and by-catched cetaceans (Walker et al. 1988; Kjeld et al. 1992; West et al. 2000; Brook et al. 2004; Westgate and Read 2007; Biancani et al. 2009). On the contrary, very few alternative methods have been reported for this purpose in wild populations. Among them, Perryman and Lynn (2002) used allometric measurements taken from aerial photographs of gray whales (Eschrichtius robustus), and Rolland et al. (2005) showed that progestin concentrations in fecal
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material could diVerentiate pregnant from non-pregnant right whales (Eubalaena glacialis). Though the latter method appears to be eVective, in most Weld situations, fecal collection is impractical or often requires special detection systems to increase collection eYciency (Rolland et al. 2006). Recently, Kellar et al. (2006) reported the measurement of progesterone levels in blubber samples collected from stranding animals showing 16-fold higher values in pregnant compared with non-pregnant shortbeaked common dolphins (Delphinus delphis), Northern right whale dolphins (Lissodelphis borealis), and PaciWc white-sided dolphins (Lagenorhynchus obliquidens). Here, we tested the possibility of applying this methodology to free-ranging populations of cetaceans. For this purpose, bottlenose dolphins (Tursiops truncatus) and long-Wnned pilot whales (Globicephala melas) inhabiting the Strait of Gibraltar and the Gulf of Cadiz were selected as test populations. Such area gives shelter to important populations of cetaceans, including diVerent species of dolphins and whales, which conservation management is becoming challenging due to the constantly increasing maritime traYc through the area and the high levels of human activity in the environment (de Stephanis et al. 2008a; Verborgh et al. 2009).
Materials and methods Bottlenose dolphins and long-Wnned pilot whales skin and blubber samples were collected from the research vessel Elsa during January 2004 and September–October 2005 in the frame of a project focused on the study of the social structure and diet of both species in the Strait of Gibraltar and Gulf of Cadiz (5°30⬘W, 35°58⬘N). As previous studies in other cetacean species have showed that blubber steroid concentrations may change with anatomical location (Kellar et al. 2006, 2009), we collected all the biopsies from the mid-lateral region, near the dorsal Wn of the animals, in order to reduce variability. Biopsy sampling, which was performed following the methods described by de Stephanis et al. (2008b), complied with the current Spanish and European laws and was made under the authorization of the Spanish Ministry of Environment. A total of 28 samples (23 from bottlenose dolphins and 5 from long-Wnned pilot whales) were sexed by genetic analysis of the skin biopsies as described previously (de Stephanis et al. 2008b, c). Only 13 samples belonging to females, 11 of bottlenose dolphins and 2 of pilot whales, were selected for progesterone measurements in blubber biopsies. The females sampled, which belong to a population of 250 bottlenose dolphins and 213 long-Wnned pilot whales (Jiménez Torres 2009; Verborgh et al. 2009), were therefore tracked along the following year through photo-identiWcation surveys, and when the
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presence of neonates was recorded, the animal was considered to be pregnant at the time the biopsy was carried out. Gestation period for bottlenose dolphins has been estimated to be around 12 months (Kasuya 1995), while for longWnned pilot whales this period was reported to range between 10 and 16 months (Rothery et al. 1995). Among the 11 bottlenose dolphin females studied, 2 were found to be pregnant, while the 2 pilot whale females sampled were pregnant as well. For progesterone measurements, cross-sectional subsamples of biopsies (ca. 50 mg) were homogenized in liquid nitrogen using a mortar and pestle and extracted exactly as described by Kellar et al. (2006). Final steroid extracts were reconstituted in 1 ml EIA buVer and progesterone concentrations were then quantiWed in 50 l duplicate samples by enzyme immunoassay using a commercially available kit and following the manufacturer instructions (Progesterone EIA Kit #582601#; Cayman Chemical Company). Progesterone concentrations of standard curve used in the assay ranged between 2.5 and 0.0006 ng/ml, while the detection range of the assay following the dilutions described was 1,000–1.3 ng/g of bubbler. Intra-assay coeYcient of variation was 2.2% (all the samples were assayed in one plate and then, an inter-assay variation was not applicable) while extraction eYciency (determined by cold spiking of Wve bottlenose dolphin samples) was 119%. Main cross-reactivity (>1%; given by the supplier) for the antibody used was detected with pregnenolone (61%), 17-estradiol (7.2%), and 5-pregnan-3-ol-20-one (6.7%).
Results Blubber progesterone levels obtained in the present study were comparable between bottlenose dolphins and longWnned pilot whales (Table 1). Mean progesterone levels from pregnant bottlenose dolphins were around 9 times higher than those from non-pregnant females, with no overlap between concentration ranges; there was a 2.5-fold diVerence between the lowest level measured in pregnant bottlenose dolphins and the highest level recorded in nonpregnant females. Relatively high blubber concentrations of progesterone were measured in the two samples analyzed from pregnant pilot whale females, although samples from non-pregnant individuals were not available for comparative purposes.
Discussion The results presented in the current study provide evidences supporting the concept that the techniques developed by Mansour et al. (2002) and Kellar et al. (2006) for assessing
Mar Biol Table 1 Progesterone concentration in the blubber of pregnant, non-pregnant, and immature females of six cetacean species Status
T. truncatus
G. melas
D. delphisa
L. borealisa
L. obliquidensa
B. acutorostatab
Pregnant Average
54.82 § 22.86
45.28 § 28.40
261 § 29
312 § 44
161
132 § 22
Minimum
31.96
16.89
132
196
–
22.8
Maximum
77.7
73.68
415
402
–
454
n
2
2
18
5
1
22 Not available
Non-pregnant/mature Average
6.16 § 3.62
Minimum
1.65
Not available
13.7 § 1.8
15.0 § 7.5
12.1 § 8.4
6.75
2.11
3.75
Maximum
12.7
33.3
34.7
20.5
n
9
19
6
2
Inmature Average
n
16.5 § 2.7
14.2 § 2.30
18.1 § 9.1
1.95 § 0.32
Minimum
Not available
Not available
0.92
0.98
0.11
1.36
Maximum
48.2
33.1
34.4
3.43
36
18
4
6
The progesterone concentrations are reported as ng/g of blubber extracted. Average values are displayed with standard error a From Kellar et al. (2006) b From Mansour et al. (2002)
pregnancy in stranded cetaceans by the determination of progesterone amounts in bubbler samples can be successfully applied to free-ranging live bottlenose dolphins and long-Wnned pilot whales, as the collection of a small bubbler biopsy from the animal was enough to quantify reliably progesterone levels. Despite the fact that these measurements require the use of invasive techniques for biopsies collection, our observations indicate that samplings cause minor short-term disturbances to both target and non-target individuals, and the wound healing seems to be fast, with no evident infection or excessive inXammation at the biopsy site (Giménez et al. 2011). While considerable diVerences in the absolute progesterone levels measured in bubbler samples have been reported among several cetacean species (see Table 1), which could reXect either species-speciWc diVerences or/and diVerences in the reproductive status of the sampled animals, a common feature for all of them is that progesterone amounts were much higher in pregnant females than in non-pregnant individuals. For bottlenose dolphin, we found a 2.5-fold diVerence between the lowest level in a pregnant female and the highest level in a non-pregnant individual, values well comparable with the results obtained for other delphinids (4-fold) and minke whales (6.5-fold) (Mansour et al. 2002; Kellar et al. 2006). DiVerences in progesterone concentrations of milk and blood samples have been also probed to be highly eVective for diVerentiating pregnant from non-pregnant individuals in several cetaceans species maintained in captivity, including bottlenose dolphins (Sawyer-Stefan et al. 1983; West et al. 2000), although the
collection of such type of samples from wild populations is impracticable. In these cases, the measurement of progesterone concentrations in blubber biopsies as demonstrated in the present study could constitute an eVective and economic alternative for determining pregnancy in bottlenose dolphins. However, in order to establish the speciWc values, ranges and time of the year to determine pregnancy with the highest reliability in these species, further studies analyzing bubbler progesterone levels along the year and during diVerent phases of pregnancy are required, as hormone levels are known to Xuctuate with season and reproductive stage of the individuals (Kellar et al. 2009; Bergfelt et al. 2011). In this regard, long-term photo-identiWcation data are required to identify successfully mother-calf associations. The current report represents the Wrst contribution addressing the study of the reproductive biology of wild cetacean populations of the Strait of Gibraltar and, therefore, establishes a basis for further studies focused on this important aspect. Such studies would complement the already existing information about abundance, distribution, feeding ecology, social structure, and genetics of cetacean populations in this area (de Stephanis et al. 2008a, b, c; Guinet et al. 2008; Verborgh et al. 2009; García-Tíscar 2010) and thus, they may help in the design of proper conservation management programs of marine mammals populations to face the challenges of the near future, such as the constantly increasing maritime traYc between Spanish and Moroccan harbors or the increasing whale watching activities in the Strait of Gibraltar.
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Mar Biol Acknowledgments Our study was made possible due to the Wnancial support received during several cetacean campaigns in the Iberian Peninsula. We thank the Foundation Loro Parque, the Foundation Biodiversidad and CEPSA. This work would not have been possible without the help of diVerent volunteers and researchers, especially to R. Esteban, N. Seller Fernandez, Z. Munilla, Y. Yaget, C. Corbella, C. Jiménez, D. Alarcon, M. Carbó, S. Villar, and N. Pérez.
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