• ~!q

Judd, H. L. and S. H. Ridgway (1977) Twenty-four hour patterns of circulating androgens art:dcortisol in male dolphins. In: Ridgway, S.H. and K. Benirschke, (Eds) Br eeding dolphins. present status. suggestions for the future. National Technical Information Service, U.S. Dept. of commerce, pp. 308.

TWENTY-FOUR HOUR PATTERNS OF CIRCULATING

ANDROGENS AND CORTISOL IN MALE DOLPHINS

BY

Howard L. Judd, M.D. and SamH. Ridgway D.V.M., Ph.D.

FROM

The Department of Reproductive Medicine School of Medicine University of California, San Diego

AND

The Unde~sea Sciences Department Naval Undersea Center San Diego, California 92132

Supported by gran~s from the American Cancer Institute (#75 5017) and the Rockefeller Foundation (R-75029)

Some controversy exists concerning whether there is an annual rut or

period of increased sexual activity in male cetaceans (Asdell, 1964; Slijper,

1962; Ridgway and Green, 1967; Harrison et al., 1972). There is also a lack

of knowledge concerning the criteria for determining sexual maturity in male

dolphins. Some serum testosterone levels from immature Tursiops truncatus

and one mature animal of the same species were presented by Harrison and

Ridgway (1971). Several captive T. truncatus males under eleven years of age

had testosterone levels similar to those of human males before puberty. One

mature male was sampled at about monthly intervals for a 24 month period,

March 1969 through February 1971. Plasma levels in this animal were much

higher like those in other mature mammals. The highest levels were found

in the fall of the first year (September, October), and the following spring

(April and May). During the subsequent fall the levels did not rise but did

increase the next January and February.

A potential problem exists when animals are sampled at infrequent intervals

(monthly) to determine possible seasonal variation of circulating testosterone

levels. The concentration of this androgen is not steady but may fluctuate

significantly over short periods of time. These ultradian (less than one day)

fluctuations of testosterone may be profound in some animals (bovine)

(Katongole, et al. 1971) while they are of less magnitude in other mammals (man)

(Naftolin, et al. 1973). In addition, testosterone has been shown to have a

diurnal rhythm (highest at night and in the morning) as shown by most studies

in humans (Judd, et al. 1973) as well as monkeys (Goodman, et al. 1974). If

profound ultradian and or diurnal fluctuation of testosterone exists in dolphins,

monthly sampling would be useless in the investigation of a possible annual

rhythm. In addition, if there is a great daily variation, several samples would

need to be taken at various times during the day to make meaningful correlations

of serum testosterone levels for the purpose of sexual maturity determination.

To examine the possibility of ultradian and or diurnal fluctuations of

testosterone in dolphins we sampled 2 male dolphins at intervals of twenty

minutes throughout a twenty four hour period.

MATERIALS AND METHODS

Two male dolphins were available for these studies. Maui was a 19 year

old Atl antic bottlenose and Ivan was a 7 year old Pacific bottlenose. Maui

had been in captivity for over 13 years and Ivan for about five years. The

age of both animals had been determined by extracting a tooth and sectioning

it (Sergeant et al., 1973; Ridgway et al., 1975).

The animals were removed from the water and placed in transport slings.

Each was laid on a foam rubber pad and an intravenous catheter was put into

the brachiocephalic vein through a needle inserted at the midli ne on a plane

intersecting the anterior insertion of the flipper. After the catheter was

inserted the animals were picked up in the sling and placed in a transport

container where they rested on foam rubber that had been cut out so that the

flippers could hang downward. The container was then filled with water up to

the animal IS eye level. All parts of the animal that were out of water were

sprayed continuously to keep them moist and cool. The catheter was long

enough so that the tip, which was built to take the hub of a syringe, could be

placed on the animal IS back. Blood samples were collected at the start of

the experiment at 8 a.m. one morning and every 20 minutes thereafter until

the next morning at 8 a.m. After each blood sample was drawn, heparinized

saline was used to flush the catheter. Before the next sample was obtained,

three ml of fluid was aspirated from the tubing to insure the next blood sample

would not be diluted with the heparinized saline solution. Blood samples were

centrifuged immediately after they were drawn and stored in a freezer.

The procedure we employed to maintain the animals in the transport container

has been used many times without noticalbe ill effect on dolphins (Ridgway, 1972).

,.

Although both of these animals had been transported previously in transport

slings this was their first exposure to this particular experimental situation.

Each showed some signs of restlessness during the experimental period but

both were fine and took food when placed back in the water after the experiment.

Serum testosterone (T), androstenedione (~), dehydroepiandrosterone

(DHEA), and cortisol were measured on each sample by previously described

radioimmunoassay procedures (Judd, et al. 1973, Judd and Yen, 1973a, Hopper

and Ye n, 1975).

RESULTS

Fig. 1 shows the levels of the three androgens and cortisol in Maui.

At the beginning of sampling the plasma testosterone level was 5206 pg/ml.

There was a steady decline in the circulating level of this hormone for the

remainder of the study with the levels reading a nadir of 1345 pg/ml at the

end of sampling. Minimal, if any, pulsatile activity was observed. Andros­

tenedione levels were also highest at the beginning of sampling (1536 pg/ml),

and showed a similar pattern of decline through the entire study reaching a nadir

of 263 pg/ml. Again, minimal if any, pulsatile activity was observed. Dehydro­

epiand~osterone levels were at or below the level of detectability in the

majority of samples, with the highest levels being observed after 2200 hours.

Cortisol levels were below 50 ng/ml until 1800 hours. Periodic fluctuations

of t his honnone were observed duri ng the rema i nder of the study with the

highest level being 160 ng/ml. In general, the fluctuations of cortisol did

not coincide with those seen for dehydroepiandrosterone.

Fig. 2 shows the concentrations of the same hormones found in Ivan.

Testosterone levels were considerably lower in this dolphin with the highest

level being 842 pg/ml. Testosterone levels remained low for the entire study

and showed minimal pulsatile activity. Androstenedione levels were also lower

in I van with the highest concentration of 850 pg/ml being observed at the

beginning of sampling. During the first 4 hours of sampling there was a

gradual fall of androstenedione to the 400 pg/ml range where it remained for

the balance of the study. Minimal, if any, pulsatile activity was observed .

For dehydroepiandrosterone the levels were higher than in Maui and ranged between

642 and 2391 pg/ml. Some pulsatile fluctuations were observed particularly

after 1800 hours. Cortisol levels were at or below 50 ng/ml until 1800 hours.

After 1800 hours there was a continual rise in the concentrat ion of this

hormone reaching its highest level of 380 ng/ml at the end of the study.

Except for one pulsatile burst, minimal pulsatile activity wa s observed .

DISCUSSION

The purpose of this study was to determine if ultradian and or diurnal

fluctuations of circulating testosterone are present in male dolphins. Maui

was 19 years of age and was presumed to be sexually mature although he had

not bred. His initial testosterone level of 5206 pg/ml was similar to the

levels reported earlier for a dolphin known to have impregnanted a female

(Harrison and Ridgway, 1971). It was Qlso within the range reported earlier for

other mature mammals. Ivan was 7 years of age and was possibly sexually mature.

However, his testosterone levels were within the range reported for sexually

immature dolphins (Harrison and Ridgway, 1971). In both dolp hins, ultradian

and diurnal fluctuations of testosterone were not seen. However, a steady

decline of testosterone was observed in Maui. This raises the question if the

stress of the study may have affected the testicular secretion of testosterone.

In humans, the stress associated with non-gonadal surgery has been shown to have

an effect on testicular testosterone secretion (Aono, et al. 1972). If the

stress associated with this study affected testicular androgen secretion it may

have blocked any ultradian or diurnal fluctuations of this androgen in addition

to causing a fall in the cir~ulating level.

In both animals there was a fall in the concentration of androstenedione.

This androgen has been shown to be the secretory product of the testis as well

as the adrenals (Dorfman and Shipley, 1956). The fall observed in both animals

may also reflect decreased testicular secretion.

Some pulsatile activity was observed for cortisol, particularly at night.

In addition, cortisol levels were higher at night than during the day. Thus,

the diurnal rhythm of cortisol appears to be present in dolphins.

In summary, ultradian and diurnal fluctuations of circulating testos­

terone were not found in two male dolphins sampled every 20 minutes for 24

hours. The steady decrease of testosterone in one of the animals suggested

that the test situation may have been too stressful to obtain physiological

data. We plan to develop a test procedure that is less stressful and repeat

the experiment. The animals will be exposed to the test situation several

times so that they are adapted and therefore under less stress before the study

is repeated.

-b-

ACKNOWLEDGMENTS

We thank Ms. G. Laughlin for her excellent technical assistance in

collecting the samples and doing the hormonal analyses. Dr. J.C. Sweeney

catheterized the animals using a technique he developed and Rob Hormann,

Tom Harris, Bob Wood and Gene Orr remained overnight to care for the

animals during the experiment.

,. -7-

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K. Koshiyama, and K. Matsumoto. Influence of major surgical stress on plasma

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2. Asdel, S.A. Patterns of Mammalian Reproduction. 2nd ed. London, Constable

(1964) .

3. Dorfman, R.I. and R.A. Shipley. "Androgen". John Wiley and Sons, Inc.

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4. Goodman, R.L., J. Hotchiss, F.J. Karsch, and E. Knob; 1 . Diurnal variations

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6. Harrison, R.J. and S.H. Ridgway . Gonadal activity in bottlenose dolphins

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menstrual cycle. J. Clin. Endocrin. Metab. 36: 475-481 (1973a).

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' -

11. Naftolin, F., H.L. Judd, and S.S.C. Yen. Pulsatile patterns of gonadotropins

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LEGEND

Fig. 1 24 hour patterns of circulating testosterone, androstenedione,

dehydroepiandrosterone (DHEA), and cortisol in a 19 year old

Atlantic bottlenose dolphin.

Fig. 2 24 hour patterns of circulating androgens and cortisol in a

7 year old Pacific bottlenose dolphin.

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