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REP. INT. WHAL. COMMN (SPECIAL ISSUE 6) 1 06 459

Hormonal Evidence of Spontaneous Ovulation in Captive Dolphins, Tursiops truncatus and Delphinus delphis

VICKY LEE KIRBY

San Diego Zoological Society, P.O. Box 551, San Diego, CA 92112

SAM H. RIDGWAY

Naval Ocean Systems Center , Code 5103, San Diego, CA

ABSTRACT

Baseline plasma levels of progesterone and total immunoreactive estrogens were determined for four captive female bonlenose dolphins. Turslops IruncalUS. and two captive female common dolphins. Deiphlnus de/phis . Female bonlenose dolphins were kept as pairs with or without males and bied biweekly for one year periods. Female common doiphins were kept as a paIr without a male and bled biweekly for a two year period.

Baseline progesterone levels less than I ng/mJ were indicative of . resting' or anestrous females. One common dolphin and three bottlenose dolphins did not exhibit ovulations. Episodic fluctuations of progesterone from baseline to 22.1 ng/ml were observed in two bonlenose dolphins kept with and without a male and from baseline to 15 .5 ng/mJ in one common dolphin. These cycles of progesterone were considered to be indicative of ovulation. No episodic fluctuations of estrogens were observed in the bottlenose dolphins and two were observed in the one common dolphin which ovulated. Estrogen levels on a biweeldy sampling schedule did not appear to be useful.

It was concluded for captive females of T. InDICalUS and D. de/phis that (l) they can be anestrous for at least a one-year period, (2) they can be polyestrous with an observed maximum of three cycles/year for T. lruncalus and seven cyclesiyear for D. de/phis. (3) the females of T. (runcalus had spring cycles whereas no concIusion about D. de/phis seasonaliry could be made. and (4) these two species can exhibit spontaneous ovulations.

Since reproductive events are hormonally mediated, monitoring hormonal levels in animals has proven to be effective in elucidating ovulatory cycles. Gonadal function can be inferred from the gonadal steroid levels in females, since folliculogenesis can be monitored by changes in levels of estrogens (and estrus if it occurs) and episodic fluctuations in progesterone reflect luteal function, i.e. corpus luteum formation as a result of evulation (Loskutoff, Ott and Lasley, 1983). Immuno­logical techniques have been used to monitor gonadal steroid hormones in a wide variety of domestic animals, such as the goat, cow, sheep. pig, horse (Cole and Cupps, 1977; Hafez, 1974; Hogarth, 1981; McDonald, 1975), in some exotic animals such as the alpaca, elephant, rhino, okapi, primate (Fernandez-Baca, Hansel and Novoa, 1979; Hodges, Czekala and Lasley, 1979; Kassam and Lasley, 1980; Loskutoff. Ott and Lasley, 1982; Ramsey, Lasley and Stabenfeld t, 1980). and in humans (Ross, Cargille, Lipsett, Rayford, Marshall, Scrott and Rod­bard. 1970; Tepperman. 1974; Vande Wiele, Bogwnil, Dyrenfurth, Ferin, Jewelewicz, Warren, Rizkailah and Mikail, 1970).

In the past, gonadal steroid studies on whales have consisted of gross measures of bioactivi ty which consisted of injecting whale ovarian extracts into mice or rabbits and measuring progestational effects on the uterus (Callow, Laurie and Parkes. 1935; Bomskov and Unger, 1938; Jacobsen, 1941). Prelog and Meister (1949) identified progesterone in the corpora lutea of whales, and Kristofferson. Lunaas and Velie (1961) iden tified 20 beta and 20 alpha-hydroxypregnene-4-ene-3-one in Balaenoptera physalus ovaries. With the advent of specific and sensitive radioimmunoassays (RIA), studies have been conducted on long-term captive dolphins. Sawyer-

Steffan, Kirby and Gilmartin (1983) and Sa'W),er-Steffan and Kirby (1980) reponed that hormonal studies in Tursiops truncatus were feasible for monitoring ovula­tions and diagnosing pregnancies. Kirby, Cornell, Schroeder and Andrews (In prep.) reponed on tbe comparisons of progesterone levels in six species of captive odontocetes and concluded -that plasma. pro­gesterone levels could be used to study pregnancies and ovulations in odontocetes.

Several studies of delphinid steroid hormones have indicated the occurrence of infertile cycles or spontaneous ovulations. Sawyer-Steffan et al. (1983) and Sawyer­Steffan and Kirby (1980) speculated that T. truncatus is capable of infenile cycles, but no conclusions about spontaneous vs induced ovulation could be made since males were present with the females. Kirby et al. (In prep.) found infertile cycles and spontaneous ovulations in females of T. truncacus, Orcinus orca, Stenella lon­girostris, Globicephala macrorhynchus, Delphinapterus leucus, and Delphinus delphis kept with or without males .

The purpose of this study was to synthesize information from longitudinal hormonal studies on captive females of T. truncatus and Delphinus delphis, and progesterone baseline data for females of these species kept with and without access to male dolphins.

METHODS

Subjects and sample collection

Two pairs of adult T. truncatus females and one pair of adult D. delphis females were each maintained in separate circular fiberglassed-redwood tanks (lOx 2 m). One pair of bottlenose dolphins (A and B) were bled biweekly for two years (1978 and 1980), but only had access to a

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460 KIRBY & RlDGWA Y: SPO,,"'T ANEOUS OVULATION IN CAPTIVE DOLPHINS

Table I

Animal Age (years) Length (em) Year Aged

T ursiops trlUlcalus

A 13 231 1980 B 19 242 1980 C 20 227 1980 D 16 261 1980

De/phinus de/phis

E 8t-9 178 1981 F 16 180 1981

mature adult T. IruncalUS male during 1978. The other pair of bottlenose d.olphins (C and D) were bled bi­weekJy for one year (1978) and were v.ith a mature adult male of T. lruncalus for the entire year. The females of D. deLphis (E and F) were bled biweekly for two years (1979 and 1980) v.ithout access to any males. Blood samples were obtained by draining the tanks to strand the animals. The dolphins were placed on foam rubber pads and kept moist during the bleeding procedure. Blood was taken from the dorsal or the ventral aspect of the tail fluke (Ridgway, 1965). Plasma was stored in glass vials at -4 °C until analyzed.

At least one year prior to the beginning of these studies teeth were extracted from the bottlenose dolphins

. (Ridgway, Green and Sweeney, 1975), and the teeth were used to age the animals by the method of Hui (1980). Teeth were extracted from the common dolphins after the study and aged by the method of Hohn (1980). Lengths were standard, from the tip of the lower jaw to notch in the tail fluke (Norris, 1961; see Table I).

Radioimmunoassays (RIA)

Plasma was assayed for progesterone and total immuno­reactive estrogens as described by Sawyer-Steffan and Kirby (1980) using their hexane-modified techniques of Anderson, Hopper, Lasley and Yen. (\976) for progesterone. Co-chromatography validation of celite chromatography for progesterone in T I run Cal us was reported by Sawyer-Steffan and Kirby (1980) . Co­chromatography verification of celite separation of D. de/phis progesterone was performed by running hexane extracted plasma (10: 1 v/v) on (;. 2: 1 (v/v) celite: propylene glycol column, collecting seven 0.5 ml isooctane fractions. Each fraction was counted for radioactivity and RIA for progesterone immunoactivity. The interassay coefficient of variation (CV) for progesterone was 11.3 /~ (n = 13) and for total immunoreactive estrogens was 6.2 ~~ (n = 6). Sensitivity of the progesterone assay was as low as 30 pg/ml and for estrogen was as low as 3 pg/ml.

Plasma samples with progesterone levels less than

I ng/ml were evaluated as baseline levels. Samples with progesterone greater than 3 ng/ml were evaluated as indicative of ovulation. Pregnancy was diagnosed in those cases in which progesterone levels were elevated above 3 ng/m! over an extended period of time (Sawyer-Steffan el ai., 1983; Sawyer-Steffan and Kirby , 1980). Values between I and 3 ng/ml were considered indetenninate. unless subsequent sampies indicated that the proges­terone levels were continuing to rise.

RESULTS

The mean baseline level of plasma progesterone (P) for the four females of T lruncarus was 0.18±0.04 ng/ml (n = 133 samples). Progesterone levels ranged from less than 1 ng/ml to 22.1 ng/ml in animals where elevated P

E 12 .... '" 10 c

UJ 8 z 0

6 c:: l.U ~ 4 r.tl UJ Cl 2 0 a: Q.. 0

Z <: -,

24

22

20

E 18 .... a> 16 c

UJ 14 z 0 a: 12 UJ ~ r.tl 10 UJ Cl

8 0 a: Q.. 6

4

2

0 z ...: ..,

FEMALE Tursiops A MALE PRESENT

Ie::: ' 1...J

~ ~ MONTHS

1978

'~' c.:> o

FEMALE Turlliops 8 MALE PRESENT

\ \

c: ...J

~ ;;

\ \ \ \ , \ I \ I \ I \ I 'v

'..-c.:> o

MONTHS 1978

z <: ..,

z · < ..,

FEMALE Tursiops A NO MALE

, c: -' Q.. ::. <: .., MONTHS

1980

~ c.:> o

FEMALE Tursiops 8 NO MALE

'a: ' ~ Q.. ::. < -, MONTHS

1980

Fig. 1. Plasma progesterone levels in two captive females of Tursiops lruncalus (' A' and . B ') maintained together with a mature T. tnmcalus male (1978) and Wltbout access to a mature male (1980).

Table 2

Baseline plasma progesterone (P) and total immunoreactive estrogens (E,) in captive female Tursiops lruncarus and Delphinus de/phis

No male present Mature male present

Species n xP ± CI ng/lnl n xE, :: CI pgiml n xP:::CI ng}ml n x E, :: CI pg/ml

Tursiops lrunCQIUS 36 0.t5±0.03 43 31 ::3 97 0.21 =005 lOt 23 = 2 Delphinus delphis 73 0.48 :to.08 45 33±5

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REP. INT. WHAL. COMMN (SPECIAL ISSUE 6) 461

E 12 "-Cl 10 L::

~ 8 o c:: 6 UJ

l;; 4 UJ

g 2 c::

FEMALE Delphinus E

Cl. 0 f-Z.,.....r=-ra:T"i',:f:, ...JT==P, "f'l-=:rT. =:'~Z'i"1 ""i'~a::r:;::, :':;':":...J:;=r~~,........,.-< Cl. ~ U < Cl. ~

16

E 14 "-en 12 L::

, < , 0 , < ,

1979 MONTHS 1980

~ 10 FEMALE Delphinus F

o c:: 8 UJ l-en 6 w g 4 c: Cl. 2

z < , a:

Cl. <

1979

l­U o

z < ,

MONTHS

a: Cl. <

1980

Fig. 2. PI;;;ma progesterone levels in two captive females of Delphinus de/phis (' E' and' F') maintained together for two years witbout access to a male.

was not maintained (i .e. animals were not pregnant). The maximum number of cycles observed during the study was three (Table 2) . The mean total immunoreactive estrogen (Et) levels were 23 ± 2 pg!ml when a male was present and 31 ± 3 pg/ml when no male was present. No episodic fluctuations of Et were observed. Of the 94 D. delphis samples evaluated, the mean P baseline level was 0.48 ± 0.08 ng/ml with episodic fluctuations ranging from less than I ng/ml to 15.5 ng/ml. The maximum number of cycles during the study was ten (Female F , Table 1) . The mean Et level for 94 samples was 33 ± 5 pg/mJ with two periodic fluctuations observed of 79 pg!ml (April , 1980) and 80 pg!ml (July. 1980) for Female F, although plasma was not available for Female F from May and June for estrogen analysis.

Comparison of plasma from T. lruncacus females A and B in the presence of males to plasma levels for A and B without ;nales is shown in Fig. I. From the figure it can be seen that (I) female A was anestrous for 1978, when a male was present, while female B ovulated tWO times and was impregnated on the second cycle, (2) each female ovulated when no male was present, (3) each female cycled at approximately the same time of the year (spring/summer) , and (4) the profiles of the cycles with and without maies were similar. Female A had episodic P fluctuatlOns May through July 1980 with no male present, with a maximum of three sequential cycles. Female B ovulated twice and became pregnant in 1978 and ovulated again in 1980 when no male was present. The second pair of bottlenose dolphins (C and D) kept

with a mature male (1978) were anestrous for the one year period and are not shown, but had a profile similar to that of T. lruncatus A, 1978, in Fig. I.

Episodic fluctuations were observed forP in one of the common dolphins (F) while the other female (E) was anestrous for the two year period . Female F exhibited three periods of P elevation in 1979 and seven in 1980 (Fig. 2).

DISCUSSION

The primary objective of this study was to determine whether episodic fluctuations of progesterone and estrogens occur in captive females of T. (runCalUS and D. delphis that do not have access to males. This would be consistent with the hypothesis that these two species are spontaneous ovulators. It was disappointing that observable estrogen cycles were not always associated with progesterone cycles, but this may have been an artifact of the sampling time schedule in that the follicular phase may be of short duration relative to a two-week sampling schedule. Obviously, macroscopic analysis of the ovaries is necessary to confinn the relationship between episodic fluctuations of progesterone and ovulation. Still, it is generally agreed that in mammals, where captive studies have been performed, episodic progesterone levels are associated with functional corpora lutea from ovulations. Considering that (1) progesterone is produced by only three endocrine glands (corpus luteum, placenta, adrenals), (2) while adrenal progesterone may be reproductively significant in the rat (Feder, 1981) and in the armadillo (Nakakura, Czekala, Lasley and Benirscbke, 1982), it is 'not significant to the luteal phase in higher mammals such as humans (Tiez, 1976), and (3) adrenal progesterone has never been reported to be released in episodic surges similar to luteal or placental excretion patterns, the relationship between episodic progesterone surges and ovulations is reasonable .

Sawyer-Steffan and Kirby (1980) and Sawyer-Steffan el al. (1983) reported baseline P levels in captive females of T. Iruncacus as less than I ng!ml with a mean of 0.3 ng/mI. In this study, the mean baseline level for T. lruncalUS was 0.2 ng/ml and 0.5 ng!ml for D. delphis. Progesterone levels less than I ng/ml are generally considered indicative of lack of luteal activity in other mammals (Hendricks and Meyer, 1977). Kirby el al. (In prep.) and Wells (1984) observed similar levels in four other species of odoDtocetes, wi th mean P levels of 0.2 to 0.4 ng/ml for Slenella longiroslris, Orcinus orca, Lagenorhynchus obliquidens. and Globicephala macrorhyn­chus. Episodic P fluctuations from baseline to 22 ng/ml were reported for ovulations in T. lruncalus (Sawyer­Steffan el al., 1983) which are similar to observed levels in this study in which P levels fluctuated from less than I ng/ml to 22.1 ng/ml. The D . delphis females exhibited similar progesterone profiles of ovulation from baseline levels to 15.5 ng!ml. Each species exhibited multiple cycles, ..... i th T. rruncalus ranging from none to possibly three cycles in a year , and D . delphis ranging from none to seven cycles within one year. In both species, females were observed to be anestrous for a one-year period, and the D. delphis female E was anestrous for two years . The T. lruncacus female A was anestrous for one year, as was

462 KIRBY & RlDGW A Y : SPONTANEOUS OVULA TlON IN CAPTIVE DOLPHINS

the second pair of bottlenose dolphins (C and D) for the same year. T. lruncalUS females A and B (Fig. J) exhibited cycles during April through july. D . delphis female F exhibited no specific seasonality, with cycles occurring during spring, summer, and winter of one year, and during spring through late fall of the next year . Occasionally three to five weeks passed between samples, so that one apparent cycle may have actually been two cycles.

Sergeant, Caldwell and Caldwell (1973) suggested that T. lruncatus females reach sexual maturity at an average length of about 235 cm. Animal C was captured in 1971 at a length of 227 em, produced a calf in 1973, and has remained at 227 cm (through 1982), considerably under the length at which females generally became sexually mature. The smallest sexually mature females mentioned by Sergeant el al . (1973) were also 227 ern long. It is possible that Animal A was not yet sexually mature in 1978 even though she was II years of age. In 1974 she was 226 ern in length, in 1980 231 em and in 1982 245 cm. Another possibility is that length at attainment of sexual maturity varies among populations (perrin and Reilly, 1984).

Ovulations may be spontaneous in other cetacean species. Some mysticete females have been found to cycle more than once in a season and to cycle when males were not in season (Slijper, 1966). In the same paper it was mentioned that Globicephala macrorhychus and possibly Pseudorca crassidens may experience one to three ovulations either simultaneously or in close temporal association as detennined histologically. In some odonto­cetes (Lagenorhynchus, Globicephala) ovulations may be spontaneous, as evidenced by large numbers of corpora relative to body length (Harrison. 1969; Harrison, Boice and Brownell, 1972; Sergeant, 1962). Yet, it has been proposed that other species of odontocetes are reflex ovulators as evidenced by low numbers of corpora relative to body length and by active corpora lutea usually associated only with pregnancy, under the assumptions that corpora persist throughout the lifespan of the female and that all corpora albicantia are of past pregnancies. Harrison and McBrearty (1977) and Harrison and Ridgway (1971) did not find an active corpus luteum without pregnancy in T. lruncalUS. Perrin, Coe and Zweifel (1976) reported only 5,4 % of mature females of Slenella allenuala had a corpus luteum without pregnancy. For S. longiroSlris, only 2.8% of the mature females had a corpus luteum without an associated fetus (perrin. Holts and Miller. 1977). Harrison el al. (1972) reponed that in the Slenella species they examined, the corpora of pregnancy persisted and that these species are induced ovulators as evidenced by the lack of ovulation in captivity. However, it should be pointed out that the corpora observed in the ovaries of a long-tenn captive female were assumed to be the result of pregnancies before capture, rather than the result of spontaneous ovulations in captivity . They also observed that in D . delphis not every corpus could possibly be attributed to past pregnancies, hence some corpora may have been the result of lutealized follicles or accessory corpora lutea. It seems likely that they could also have been attributed to spontaneous ovulations. However, there have been questions about the basic assumptions which underly the theory of reflex ovulations in

cetaceans. Benirschke, Johnson and Benirschke (1980) reponed that histologically the corpus luteum of a non-fertilized spontaneous ovulation could not be distinguished in S. allenuala from a corpus luteum of early pregnancy and that the possibility of spontaneous ovulatlon should be examined. Harrison and Ridgway (1971) reponed that T. lTUncalus females are induced ovulators because of the relationship between length of the female and number of corpora. However, they also reponed relatively shOft sexually mature females with three or more corpora. Without histological examination of the uterus, it would not be possible to determine if these sexually mature females had ever been pregnant. Harrison and McBrearty (1977) speculated that T. lrun­calUS females are induced ovulators because a female kept with an immature male had corpora in her ovaries, although they mentioned that this female could have spontaneously ovulated. Harrison el al. (1972) speculated that T. lruncalUS females are induced ovulators, although they observed females with corpora counts that were greater than the probable number of pregnancies for animals of that size class. As can be seen above, the only evidence for induced ovulation has been the low number of corpora found in captive animals and the fact that most corpora lutea could be associated with pregnancy. The possibility that corpora of infertile cycles do not persist as long as do the corpora of pregnancy is critical to any interpretation of ovarian scars. Harrison and Ridgway (\ 971) concluded that the number of corpora albicantia does not increase relative to female body length. The argument that these two corpora cannot be distinguished does not preclude that they may persist for different lengths of time.

Considering that (I) in captivity female dolphins might have an ovulation rate that is lower than that in wild dolphins and (2) for where it is known in wild marine mammals, the proportion of ovulations that result in pregnancy is relatively ltigh (LeplOnycholes weddelli 78 % , Stirling, 1971 ; Callorhinus ursinus 80 ~~, Chapman, 1961; Phoca groenlandica 84 %, Sergeant, 1966), it would be impossible to state unequivocally that T. lruncalUS or D. delphis are induced ovulators. It seems likely that a number of delphinid species may be spontaneous ovulators, in light of anum ber of recent hormone studies of captive cetaceans that report episodic fluctuations of progesterone and/or estrogens (Kirby el al., in prep; Sawyer-Steffan and Kirby, 1980; Sawyer-Steffan el al., 1983; and Wells, ! 984), and a number of ovarian studies (Benirschke el al., 1980; Collet and Harrison, 1981; Marsh and Kasuya, 1984). Although hormonal evidence of spontaneous ovulation does not eliminate the possibility that dolphins are actually reflex ovulators in the wild, It does suggest that this question should be reviewed in a new perspective with respect to the literature on terrestrial mammals and with respect to what ovarian scars actually represent.

ACKNOWLEDGMENTS

The authors wish to express their appreciation to Ms Aleta Hohn for tbe ageing of some of the teeth , to Dr Candace Fenner and her technical staff for the collection of blood samples and to Dr Kurt Benirschke for the histology of the ovarian tissues.

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REP. INT. WHAL. COMMN (SPECIAL ISSUE 6) 463

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Note added in proof

Gross and histological examinations were made on the formalin-fixed ovaries from T. truncalUS Band D . de/phis F. In T. truncalus B, gross examination yielded a corpora count oftive yellow scars in the left ovary and nine yellow scars and one obvious corpus albicans (CA) in the right ovary. Histological examination revealed that there were 13 to 15 old CA in addition to the obvious CA. Exact corpora counts are difficult in serially step-sectioned tissue because a CA on one slide may overlap with the edge of a second CA on the next slide. Hence. this female had a total corpora count of approximately 14 small old CA and one larger CA. She was known to have a

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464 KIRBY & RlDGWA Y SPOl'iT ANEOUS OVULATION IN CAPTIVE DOLPHINS

reproducllve history of at least five ovulations and only one calf ia captivity.

In ~he D. de/phis F, gross examination yielded a corpora count of one possible degenerated corpus luteum (CL) associaled with a cystic follide in the right ovary and 8-10 yellow scars in the left ovary. Histological examination of the ovanes yielded a total corpora count of 1-2 degenerated CL and 11-13 CA. Three of these CA

were so old as to be very small and devoid of pigment and would probably have not been seen in gross examination. The relatively recent luteal tissue associated with the cystic follicle in the right ovary was not induded in the tOlal corpora count. These ovarian data suggest that gross corpora counts are not indicative of past pregnancies and that yellow scars are old corpora albicantia in these two species.