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Keywords:

  • blow;
  • whale;
  • testosterone;
  • progesterone

Abstract

  1. Top of page
  2. Methods
  3. Results
  4. Discussion
  5. Acknowledgments
  6. Literature Cited

The feasibility of using analysis of hormone content of whale blow samples to assess reproductive function is addressed. A suitable collection method and analytical technique using liquid chromatography-mass spectrometry (LC-MS) has been developed. Blow samples were collected opportunistically from free-ranging humpback whales (Megaptera novaeangliae) (n= 35) and North Atlantic right whales (Eubalaena glacialis) (n= 18) using a 13-m carbon fiber pole with a collection device. Samples were analyzed for the presence of testosterone and progesterone using a 55% isocratic gradient with LC-MS. Testosterone was detected in four humpback whale samples and eight northern right whale samples. Progesterone was detected in seven humpback whale samples and eight northern right whale samples. This is the first documented use of lung mucosa to determine the presence of reproductive hormones in any free-swimming cetacean and may provide a novel non-invasive technique to quantify the hormonal condition of free-swimming animals that spend brief periods of time at the water's surface.

Non-invasive techniques to assess reproductive and adrenal function in terrestrial species are well established (Matsumoto et al. 1999, Czekala and Sicotte 2000, Romero et al. 2000, Czekala and Robbins 2001, Larter and Nagy 2001, Graham et al. 2002, Stoinski et al. 2002, Hagey and Czekala 2003, Cross et al. 2004, Dloniak et al. 2004). Urine and fecal sampling have been used to determine hormone concentrations in a number of species including gorillas, timber wolves, feral horses, and deer. Measuring endocrine function using saliva is frequently used with humans (Connor et al. 1982, Riad-Fahmy et al. 1982, Sannikka et al. 1983, Ellison et al. 1986), in particular children (Granger et al. 1999; Shirtcliff et al. 2000, 2002) and more recently with wildlife (Lutz et al. 2000, Iwata et al. 2003, Hogg et al. 2005). These different non-invasive sampling methods can be easily adapted to a variety of terrestrial species yet collecting non-invasive samples from free-ranging marine mammals offers challenges.

In more recent years, a number of different studies have used non-invasive sampling to describe endocrine function in marine mammals. Initial studies with captive animals used saliva in monk seals, Monachus schauinslandi (Pietraszek and Atkinson 1994, Theodorou and Atkinson 1998), false killer whales, Pseudorca crassidens (Atkinson et al. 1999), and bottlenose dolphins, Tursiops truncatus (Hogg et al. 2005); urine in killer whales, Orcinus orca (Walker et al. 1988, Robeck et al. 1993); and milk in bottlenose dolphins, Tursiops sp. (West et al. 2000) to determine both reproductive and adrenal hormones. Non-invasive sampling (feces or urine) in free-ranging pinnipeds is slowly becoming more commonplace with basal hormone concentrations existing for the harbor seal, Phoca vitulina richardii (Gulland et al. 1999), Steller sea lion, Eumetopias jubatus (Mashburn and Atkinson 2004), and Weddell seal, Leptonychotes weddellii (Constable et al. 2006). Seals spend a portion of their life on shore, making fecal and urinary sampling possible. However, for the larger and completely aquatic species, such as cetaceans, collecting samples proves more difficult.

The endocrine function of the great whales is little understood. Recently fecal sampling has been used to determine both reproductive and glucocorticoid hormone concentrations in the North Atlantic right whale (Eubalaena glacialis) (Rolland et al. 2005, Hunt et al. 2006). The North Atlantic right whale is breeding at such low rates there are grave concerns for their recovery (Donovan et al. 2001, Kraus et al. 2005). This low rate may be driven by breeding dysfunction (Reeves et al. 2001) but enormous logistic difficulties prevent the systematic collection of samples from free-swimming breeding whales (Gales et al. 2005), which is needed to investigate their hormonal profiles. Fecal sampling has been shown to be a powerful tool in the analysis of northern right whales hormonal profiles. Yet, this sampling technique is only viable in regions where the whales are feeding and defecating. The use of dogs to assist with sample location and collection has greatly improved sample sizes in this species (Rolland et al. 2006). For other baleen whales, particularly those in the Southern Hemisphere, fecal sampling can be problematic due to the logistical difficulties of obtaining samples in the feeding grounds of baleen whales. Many baleen whales in the Southern Hemisphere feed near the Antarctic (Mackintosh 1943, Chittleborough 1956, Dawbin 1956, Chittleborough 1965) and repeated fecal sampling may not be viable.

In light of these issues, an alternative non-invasive sampling technique for the repeated and systematic collection of samples from baleen whales for hormonal profiles is needed. This study was designed to determine the feasibility of using exhaled air (blow) samples from baleen whales to determine reproductive state. To our knowledge using lung mucosa to determine reproductive state has not been used in any species, although the analysis of rat nasal and lung mucosa has shown the presence of progesterone, testosterone, and estradiol (Brittebo 1982, Brittebo and Rafter 1984, Brittebo 1985).

The objectives of this pilot study were to: (1) determine a suitable collection substrate and method for obtaining blow samples from free-swimming whales; (2) develop suitable extraction and analytical techniques for hormone determination in blow samples; and (3) determine the presence of testosterone or progesterone in two species of baleen whale, the humpback whale, Megaptera novaeangliae, and the North Atlantic right whale.

Methods

  1. Top of page
  2. Methods
  3. Results
  4. Discussion
  5. Acknowledgments
  6. Literature Cited

Sample Collection

Blow samples were collected opportunistically from humpback whales in October 2003 and October 2004 and from northern right whales in August 2005. The blow samples were collected using either cotton gauze (2003) or inert nylon stocking (2004 and 2005) secured over a 5-in. (12.7 cm) bamboo ring at the end of a 13-m carbon fiber pole bracket-mounted to the bow of a 5-m aluminum vessel (Moore et al. 2001). Blow samples were collected simultaneously with deployment of suction-cup tags (Dtags) held on the opposite side of the cantilevered pole (see Miller et al. 2004 for details of tag deployment). The collection device was extended into the cloud of blow as the whale surfaced. The quality of the blow sample was a subjective scoring determined by the amount of blow that was observed to pass over the blow collection device during sampling. Samples were rated as 3 if the end of the pole was within 5 m of the whale's blowholes when it surfaced. Sample was rated as 2 if there were high winds during collection or if the sample was collected when the pole approached within 5–10 m of the whale. Any sample collection that was further than 10 m from the whale was rated as the poorest quality, 1.

The collection material was changed from cotton gauze to nylon stocking as scan analysis of the 2003 samples showed interference from some of the cotton gauze. Millipore net and nylon stocking were tested as alternative collection materials. These were tested for interference and their ability to absorb liquid. Nylon stocking that had been cleaned by sonication for 15 min in 100% acetonitrile and then for a further 15 min with Milli-Q water; changing the water every 5 min was deemed the most suitable collection material.

During October 2003 (n= 9) and October 2004 (n= 26) blow samples were collected from humpback whales in breeding condition at 26°28'S, 153°05'E (Peregian Beach, Australia) as they migrated south to their feeding grounds. The sex of the individuals was determined through behavioral observations (Table 1). That is, animals with a calf present were classed as females, singing whales were classed as males, while all other animals were classed as unknown. Of the eight unknown individuals in 2003, two were escorts to female-calf pairs. Of the four unknown individuals in 2004, one was an escort to a female-calf pair. It is believed that these escort animals may be males (Glockner-Ferrari and Ferrari 1990). In August 2005 (n= 18) blow samples were collected from North Atlantic right whales in their feeding grounds at 44°35'N, 66°28'W (Bay of Fundy, Canada). The sex of the individuals was determined through identification of known individuals using the New England Aquarium Northern Right Whale Catalog (Hamilton and Martin 1999) and one unknown individual was identified as a female due to the presence of a calf (Table 1).

Table 1.  Blow samples collected from humpback whales in 2003 (Mn03) and 2004 (Mn04) and from northern right whales in 2005 (Eg05). Sex in humpback whales was determined through behavioral observations, singing whales were classed as males and animals with a calf present were classed as females. Sex in northern right whales was determined through known individual identifications (*) or were classed as female if a calf was present. Pod composition codes are: F = female, C = calf, E = escort, and S = single animal. Quality of the sample: 1 = poor, 2 = good, and 3 = excellent. Those animals with testosterone and/or progesterone present in their samples are shown.
Animal IDSexPod compositionQuality of sampleTestosteroneProgesterone
Mn03–01UF/C/2E1  
Mn03–03US2  
Mn03–04US3  
Mn03–05UF/C/E2  
Mn03–06US1  
Mn03–07US3X 
Mn03–09FF/C1  
Mn03–10U (escort)F/C/E3X 
Mn03–11UF/C/E1  
Mn04–01FF/C3 X
Mn04–02FF/C3  
Mn04–03FF/C/E3  
Mn04–04FF/C3  
Mn04–05FF/C/E3  
Mn04–06FF/C3 X
Mn04–07FF/C/E3 X
Mn04–08FF/C3  
Mn04–09FF/C/E2 X
Mn04–10FF/C2  
Mn04–11FF/C2 X
Mn04–12FF/C2 X
Mn04–13UF/C/E2X 
Mn04–14FF/C2  
Mn04–15FF/C/E2  
Mn04–16UF/C/E2X 
Mn04–17FF/C2  
Mn04–18UF/C/E2  
Mn04–19FF/C/E2  
Mn04–20U (escort)F/C/E2 X
Mn04–21FF/C2  
Mn04–22FF/C2  
Mn04–23FF/C2  
Mn04–24FF/C/E1  
Mn04–25FF/C1  
Mn04–26FF/C1  
Eg05–01F*S3  
Eg05–02US3  
Eg05–03US3 X
Eg05–04US2X 
Eg05–05F*S2  
Eg05–06F*F/C/E3  
Eg05–07FF/C3X 
Ego5–08F*S3  
Eg05–09F*S3 X
Eg05–10FF/C3  
Eg05–11U2 animals3X 
Eg05–12F*S3 X
Eg05–13US2 X
Eg05–14US3X 
Eg05–15F*S3XX
Eg05–16US3 X
Eg05–17F*S3XX
Eg05–18US3XX

To prevent degradation of the samples 5 mL of inhibitor mix (100 mM MnCl2/ 100 μg/mL amoxicillin/potassium clavulanate) was added after collection and samples were stored on ice until return to shore. Samples were then stored (with the cotton gauze or nylon stocking) at −20°C for 3–4 wk in the field and then at −80°C until assay at the lab. Seawater samples were also collected at the time of blow collection to ensure that any results were from the blow samples and not seawater contamination.

Hormone Extraction

Blow sample volumes were small (50 μL) and so liquid chromatography-mass spectrometry (LC-MS) was validated for testosterone and progesterone in captive bottlenose dolphin blow (Tursiops truncatus) (Hogg et al. 2005). Whale blow samples were extracted using “Envi-Chrom P” solid phase extraction (SPE) cartridges (Sigma-Aldrich, Sydney, NSW, Australia). The SPE procedure was as follows: (1) samples were centrifuged at 3,000 rpm (1,500 g) for 15 min to remove all sample and inhibitor from the stocking before being loaded onto the SPE cartridge; (2) SPE cartridges were preconditioned using 20 mL acetonitrile (ACN) and then 5 mL Milli-Q water (higher than the manufacturer's recommendations, to completely remove interfering extraneous material) (Vickers et al. 2001); (3) samples were loaded onto the cartridges at 5 mL/min; (4) cartridges were then washed with 7.5 mL of Milli-Q water to remove salts and the eluant discarded; (5) elution of hormones was carried out with 5 mL 100% ACN and eluant dried under nitrogen; (6) samples were reconstituted in 60 μL 60% ACN (to prevent hormone precipitation) for LC-MS analysis.

Hormone Analysis

Hormone analysis was conducted using an LC-10AD liquid chromatograph connected to a LCMS-2010 (Shimadzu, Japan). Chromatographic separation was achieved using an Alltech Macrosphere 300 Å, C8, 5 μm, 150 mmHg 2.1 mm HPLC column (Alltech Associates Ltd., Sydney, Australia). Two forms of LC-MS analyses were conducted, a gradient scan for mass-to-charge ratios (m/z) from 100 to 900 in both positive and negative modes; and SIM mode analysis for testosterone and progesterone. A 5-μL injection was used for each analysis. The MS instrumental parameters for all hormones were: detector gain, 2 kV; capillary voltage, 4.5 kV; drying gas was nitrogen at 4.5 L/min; drying gas temperature, 250°C; nebulizer pressure, 6.89 MPa; ionization source at 200°C.

Gradient Scan Analysis

The LC conditions for the gradient scan were: 10%–90% mobile phase B for 0– 30 min, 90% mobile phase B for 5 min, 90%–10% mobile phase B for 5 min, and 10 min at 10% mobile phase B to re-equilibrate the column. Two scan runs were conducted: (1) mobile phase A = 0.5% acetic acid, mobile phase B = 0.5% acetic acid, 90% ACN; (2) mobile phase A = 0.5% formic acid, mobile phase B = 0.5% formic acid, 90% ACN. Gradient scan analyses were conducted on the collection material with and without the inhibitors added.

SIM Analysis

LC conditions for the SIM analysis were: testosterone = 55% isocratic mobile phase B (mobile phase A = 0.5% acetic acid; B = 0.5% acetic acid, 90% ACN) and progesterone = 50% isocratic mobile phase B (where A = 0.5% formic acid; B = 0.5% formic acid, 90% ACN). The MS SIM analysis for testosterone used an m/z 289.20 [M + H]+ with zero variability of detector range. A second m/z 330.25 [M + H + CH2CN]+ was the acetylated adduct of testosterone and was used to further confirm the retention time (RT) (Bressolle et al. 1996). The acetylated adduct of 330.25 was present in both the biological samples and the testosterone standards with a ratio of approximately 30% compared to the m/z 289.2 peak. Data acquisition for testosterone was 10 min. The MS SIM for progesterone used an m/z 315.20 [M + H]+ and a second m/z 356.25 [M + H + CH2CN]+, the acetylated adduct of progesterone, was used to confirm the RT. The progesterone acetylated adduct was present in both the biological samples and the progesterone standards with a ratio of approximately 70% compared to the m/z 315.20 peak. Data acquisition for progesterone was 12 min. Deuterated progesterone and deuterated testosterone (Cambridge Isotope Laboratories, Andover, MA) were used as internal standards (Kawakami and Montone 2002). The m/z of the internal standards was 324.20 [M + H]+ and 291.15 [M + H]+ for progesterone and testosterone, respectively.

Assay Precision and Accuracy

The RT for testosterone was 5.0 min and for progesterone was 6.0 min. Extraction recovery for testosterone was 83.3% ± 6.8% (50 ng/mL) and 85.8% ± 4.6% (1 ng/mL); and for progesterone was 87.7% ± 5.2% (50 ng/mL) and 98.7% ± 0.2% (0.5 ng/mL). Intra-assay variability for testosterone was 6.2% ± 0.04% and for progesterone was 2.8% ± 0.04%. Inter-assay variability for testosterone was 7.3% ± 0.02% and for progesterone was 4.4% ± 0.04%. (All results are given as mean ± SD).

Chromatogram Analysis

The gradient analyses chromatograms of the seawater samples and the collection material were compared to the gradient analyses chromatograms of the blow samples. Any m/z values that were from the seawater samples and collection material were removed from the whale blow analysis so that all remaining m/z values could be attributed to the whale blow. The SIM chromatograms were assessed for testosterone and progesterone peaks. The presence of testosterone and progesterone was determined if the peak correlated to the appropriate RT and was greater than a 3: 1 signal to noise ratio.

Results

  1. Top of page
  2. Methods
  3. Results
  4. Discussion
  5. Acknowledgments
  6. Literature Cited

Gradient Scan Analysis

Scan analysis of the cotton gauze showed a number of different m/z values that may cause interference when used as a collection substrate (Fig. 1A). Scan analysis of the cleaned nylon stocking showed little to no interference with blow samples (Fig. 1B). Millipore net showed little interference with blow samples but did not absorb liquid efficiently. The samples collected in 2003 were excluded from the gradient scan analysis due to a high level of background noise caused by the cotton gauze.

image

Figure 1. (A) A 10%–90% gradient scan of cotton gauze. (B) A 10%–90% gradient scan of cleaned nylon stocking.

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SIM Analysis

All humpback whale samples collected in 2003 were analyzed for the presence or absence of testosterone and progesterone. Within the sample collection improvement only quality 2 and 3 samples collected in 2004 were analyzed. Only 16 of the 18 northern right whale samples collected were analyzed. Testosterone was identified in 22% (2 of 9) and 8% (2 of 24) of the humpback whale blow samples collected in 2003 and 2004, respectively (Table 1, Fig. 2A). Three of the humpback whale blow samples with testosterone present were collected from whales in female-calf-escort pods. No testosterone was found in any of the samples from known female humpback whales. The fourth humpback whale sample with testosterone present was collected from an adult of unknown sex. In northern right whale blow samples, testosterone was identified in 50% (8 of 16) of the samples (Fig. 2B). Progesterone was not identified in any humpback whale blow samples collected in 2003. However, in 2004, female humpback whales were targeted for sampling and progesterone was identified in 35% (7 of 24) of blow samples (Fig. 2C). Progesterone was identified in 50% (8 of 16) of the northern right whale blow samples; four of these samples came from known female whales (Fig. 2D). Three of the northern right whale samples had both progesterone and testosterone present, two of which were from known females (Table 1).

image

Figure 2. (A) Mass spectral chromatogram showing presence of testosterone in humpback whale blow (Mn03–07) collected in the 2003 season. (B) Mass spectral chromatogram showing presence of testosterone in northern right whale blow (Eg05–18) collected in the 2005 season. (C) Mass spectral chromatogram showing presence of progesterone in humpback whale blow (Mn04–07) collected in the 2004 season. (D) Mass spectral chromatogram showing presence of progesterone in northern right whale blow (Eg05–16) collected in the 2005 season.

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Discussion

  1. Top of page
  2. Methods
  3. Results
  4. Discussion
  5. Acknowledgments
  6. Literature Cited

This method is the first documented use of lung mucosa to determine the presence of reproductive hormones in baleen whales and provides a unique opportunity to collect samples from free-swimming animals that spend brief periods of time at the water's surface. The ability to detect measurable levels of testosterone and progesterone in the blow of baleen whales is probably due to their diving adaptations. Whales breathe less frequently and tend to exchange a larger percentage of their total lung volume with each breath than do terrestrial mammals (Ridgway et al. 1969). The heavy vascularization of their lungs (reviewed in Pabst et al. 1999) likely allows different compounds to diffuse across the wall from the blood stream and into the organic lung mucosa (Dellman and Eurell 1998), similar to the process where compounds move from the blood stream into human saliva (Vining and McGinley 1987). Therefore, their blow should not be viewed simply as air and water, but as a matrix of organic material. Due to the volume of air exhaled and the presence of lung mucosa in the blow, we have shown that it is feasible to use blow sampling for hormonal analysis in baleen whales.

Analysis of different collection materials showed that cotton gauze was unsuitable for whale blow collection. Although Millipore net was inert, it was unsuitable as it did not absorb enough liquid. There were concerns that if the pole bounced whilst at sea that any blow on the net would be dislodged. Nylon stocking absorbed liquid easily but interfering compounds were noted if the stocking was not cleaned. Sonication with 100% acetonitrile proved an effective cleaning method. Caution should be used when choosing collection materials for hormonal analysis as different materials may cause interference affecting hormonal results.

A preliminary analysis of unknown m/z values shows that testosterone and progesterone precursors and metabolites also may be present in the whale blow samples. Whether these compounds are from the bloodstream or whether they have been produced through metabolism in the lungs is undetermined. By using more sensitive technologies like MS-MS it may be possible to better understand which form of the reproductive hormones are present in whale blow. With further analytical development and enhanced collection techniques, whale blow has the potential to provide answers to a range of physiological questions.

In humpback whales, it is believed that escorts of female-calf pods are typically males waiting for the opportunity to mate (Glockner-Ferrari and Ferrari 1990). The presence of testosterone in these blow samples supports this theory. It is unknown at this time if blow sampling can be used to determine sex of an individual. Yet, it may be possible to use a testosterone/estradiol ratio (Gross et al. 1995) to determine sex of a humpback whale from a blow sample in the future.

The presence of testosterone in blow samples from two females is not surprising as there is no significant difference in fecal testosterone concentration between pregnant female and male northern right whales (Rolland et al. 2005). In addition, female testosterone excretion has been documented in dogs (Pineda 1989), Ridley sea turtles (Rostal et al. 1998), and alligators (Lance 1987).

All female humpback whale blow samples collected in 2004 were from known lactating females (due to the presence of a calf). Humpback whales nurse their young until they return to the breeding area the following season and may experience post-partum ovulation during their migration to the feeding grounds (Chittleborough 1958). The presence of progesterone in seven samples, and not in the other fourteen, may be attributed to females being at different stages of the estrus cycle, the suppression of progesterone as a result of lactation, or to the quality of the blow sample. Of the eight northern right whale blow samples that had progesterone present, four were known females and four were unknown individuals.

A goal of this pilot study was to show that there is sufficient material in the blow of free-swimming whales to be able to find and measure steroid hormones. A suitable collection and analysis method for whale blow samples has been presented here. The LC-MS method employed in this study has shown the presence of testosterone and progesterone in whale blow samples. Currently further analysis of samples is being conducted to develop a more sensitive method and determine a suitable endogenous substance to account for dilution, similar to how cortisol concentrations in urine samples are expressed as a ratio of creatinine (Jones et al. 1990). An endogenous substance that accounts for dilution will allow for the determination of hormone concentrations from whale blow using tandem mass spectrometry. It is essential when collecting blow samples for hormone analysis that the collection material is inert; the sample is collected as close to the whale as possible and that a broad spectrum antibiotic is added to the samples to ensure that there is no degradation of the sample during storage (Hogg et al. 2005). Although weather is a factor that is beyond control, collecting samples when there is little to no wind is advisable.

Future collection of blow samples from individuals of known sex or in conjunction with genetic sampling will determine the practicality of sex determination from blow sampling. It is difficult at this time to ascertain the biological reasons behind the testosterone and progesterone results found here due to the small sample size of this preliminary study and the little to no information on the hormonal cycling of northern right and humpback whales. Development of a more sensitive analysis and collection techniques will enable blow sampling to be used to assess reproductive function in baleen whales in both their breeding areas and feeding grounds. Because blow samples can be relatively easily collected using a tag-deployment system, future work may provide material useful to study whale genetics, and other hormones to study stress, diving physiology, and fertility. With time this powerful tool will allow us to better understand the endocrine cycles of baleen whales and may make it possible to assess the reproductive dysfunction in declining or threatened populations.

Acknowledgments

  1. Top of page
  2. Methods
  3. Results
  4. Discussion
  5. Acknowledgments
  6. Literature Cited

We would like to thank the Pain Management Research Institute, Northern Clinical School, Sydney, Australia, for the use of their LC-MS. Thanks must also go to the team of the Humpback Acoustic Research Collaboration (HARC), in particular Michael Noad and Doug Cato, for their support of the humpback whale field seasons. Thank-you also to Nicoletta Biassoni, Jim Partan, and Alessandro Bocconcelli for their assistance with blow sample collection in Australia and Canada. This work was funded by the KEST Foundation and the Australian Marine Mammal Research Centre, Sydney, Australia. Humpback whale samples were collected under the following permits: Queensland EPA WISP01331503, DEH Australia E2002/00030, ZPB Australia ethics ZPB 3d/04/03, University of Queensland ethics ZOO/ENT/216/03/USNR/DSTO (2003), ZOO/ENT/239/04/USNR/DSTO (2004). Northern right whale samples were collected under the following permits: University of Florida Ethics OE1149, ZPB Australia ethics 7c/06/05, DFO Canada MAR-SA-2005–003, and DEH Australia 2005/65888.

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  5. Acknowledgments
  6. Literature Cited
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