Compo Biochem. Physiol. Vol. 100A, No.3, pp. 649-651, 1991Printed in Great Britain

0300-9629/91 $3.00 + 0.00© 1991 Pergamon Press pic

ACUTE STRESS SUPPRESSES PLASMA ESTRADIOLLEVELS IN FEMALE ALLIGATORS

(ALLIGATOR MISSISSIPPIENSIS)

RUTHM. ELSEY,VALENTINEA. LANCE,* TEDJOANENand LARRYMcNEASELouisiana Department of Wildlife and Fisheries, Rockefeller Wildlife Refuge, Route I, Box 20-B, GrandChenier, LA 70643, U.S.A. and *Center for Reproduction of Endangered Species, Zoological Society of

San Diego, P.O. Box 551, San Diego, CA 92112, U.S.A. Telephone: (619) 557-3944

(Received 29 January 1991)

Abstract-I. Five adult, female alligators (Alligator mississippiensisi were captured at night during thebreeding season, and a blood sample taken within 5 min of capture.

2. The alligators were physically restrained (tied to boards) and additional blood samples taken at 4,8, 12, 16, 22, 28, 38, and 48 hr after capture. After the last blood sample was collected the animals werereleased.

3. Plasma estradiol-17/J and corticosterone were measured by radioimmunoassay. Estradiol declinedsignificantly from initial values by 22 hr post capture, but remained unchanged for 48 hr.

4. Plasma corticosterone rose from a mean of 0.8 ng/rnl at capture to 12.6ng/ml after 4 hr.Corticosterone continued to rise up to 16hr then declined after 22 hr. From 28 until 48 hr corticosteroneagain increased significantly.

5. These results demonstrate that acute stress in female alligators' causes significant suppression ofplasma estradiol and a biphasic pattern of corticosterone secretion.

INTRODUCTION

It is well known that environmental stress, crowd-ing, or social subordination can result in reproduc-tive failure in many animal species including reptiles(see reviews by Stephens, 1980; Moberg, 1985;Greenberg and Wingfield, 1987). Until recently, verylittle was known about the effects of stress in reptiles,but several recent studies have shown that reptilesrespond to various stressors in a similar manner toother vertebrates (Lance, 1990). Crowding stress injuvenile alligators maintained in controlled environ-mental chambers resulted in chronically elevatedplasma corticosterone in those held at the highestdensities. Those held at the lowest density grewsignificantly faster and had significantly lower plasmacorticosterone than those at the higher densities(Elsey et al., 1990a). Adult alligators maintained forcaptive breeding experiments also showed chronicallyelevated plasma corticosterone and reduced egg pro-duction associated with increased stocking density(Elsey et al., 1990b). Similarly, juvenile alligators heldunder osmotically stressful conditions had chroni-cally elevated plasma corticosterone (Lauren, 1985).

In a previous report we documented the effect ofacute stress (restraint) on adult, wild-caught, malealligators (Lance and Elsey, 1986). Plasma corti-costerone rose dramatically and plasma testosteronedeclined to non-detectable levels within 24 hr ofcapture. Likewise, in the male painted turtle, Chry-semys picta, Licht et al. (1985) showed that plasmatestosterone declined precipitously within 24 hr ofcapture. Curiously, in male snapping turtles, Chely-dra serpentina, plasma testosterone rose during thefirst 24 hr after capture and then declined rapidly

(Mahmoud et al., 1989). Plasma androgens in malelizards, Tilqua rugosa, appeared to be unaffected bycaptivity (Bourne et al., 1986).

Only two studies on the effect of the stress ofcapture in female reptiles have been published. Inthe turtle, Chelydra serpentina, both progesteroneand estradiol increased dramatically 24 hr aftercapture in gravid females, but in non-gravid femalesonly progesterone showed a significant increase by24 hr. Both hormones returned to initial values,but did not decline significantly from baseline duringone week of captivity. Plasma corticosterone wasnot measured (Mahmoud et al., 1989). In the lizard,Lacerta vivipara, Dauphin- Villemant and Xavier(1987) showed that both corticosterone and aldos-terone increased significantly after capture. Con-sidering the contradictory results cited above weinvestigated the effect of acute stress on plasmaestradiol and corticosterone in another reptilianspecies, reproductively active, adult female alligators.

MATERIALSANDMETHODS

During May, when plasma estradiol levels are high(Lance, 1989), five adult female alligators were captured atnight on the Rockefeller Wildlife Refuge by snare as de-scribed in Chabreck (1963). Using a heparinized syringe a15ml blood sample was taken within 5-10 min of capturefrom the supravertebral branch of the internal jugular(Olson et al., 1975). The blood was stored on ice in aportable cooler until all five alligators had been caught. Thealligators were then taken back to the field laboratory wherethey were tied securely to boards. Each animal wasmeasured to the nearest em (total length) and additionalblood samples taken at 4, 8, 12, 16, 22, 28, 38, and 48 hrfrom the time of capture. The animals were released in good

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condition after the last sample was taken. Red blood cellswere separated from plasma in a clinical desk-top centrifugeimmediately after the sample was drawn. The plasma wasrapidly frozen on dry ice, and then stored at - 20°C untilassayed. Corticosterone and estradiol were measured byradioimmunoassay in duplicate aliquots of plasma usinghighly specific antisera as previously described (Lance andCallard, 1978; Lance and Lauren, 1984). The data wereana1ysed using a repeated measure ANOV A followed byDuncan's multiple range test.

RESULTS

The five female alligators ranged from 187 to222 cm total length. Female alligators are sexuallymature at a length of approximately 180 em (Joanenand McNease, 1989). Estimated weights from pub-lished growth curves (Chabreck and Joanen, 1979)were 35 to 40 kg. One of the large females (216 em)had very low estradiol levels and was probablyquiescent. Only about 50% of adult females repro-duce each year in Louisiana (Joanen and McNease,1980; Lance, 1989). The other four animals had initialestradiol levels ranging from 240 to 735 pg/ml, Theresults are presented in the upper panel of Fig. 1. Twoof the animals showed an initial rise in estradiolfollowed by a gradual decline, and two of the animalsshowed a steady decrease from the initial values. Themean values at 22, 28, 38, and 48 hr were significantlylower (P < 0.05) than the initial value. The meanplasma estradiol value after 48 hr of restraint was334 pg/ml, Plasma corticosterone at the first samplingranged from 0.07 to 1.86 ng/ml, The results arepresented in the lower panel of Fig. 1. Corticosteronerose dramatically in all of the alligators by 4 hr(P < 0.001) and remained elevated for the first16 hr. At 22 hr, however, mean corticosterone levels

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Fig. 1. Plasma estradiol-I 7.B (upper panel) and plasmacorticosterone (lower panel) in female alligators under re-straint. Data from four alligators was used for the estradiolgraph and data from five alligators for the corticosteronegraph. Each point represents the mean and the bars about

the point signify the standard error of the mean.

dropped markedly, and were not significantly differ-ent from the initial value. From 28 hr until the endof the experiment mean plasma corticosterone againincreased. At each of these last three points the meanswere significantly different from the initial sample(P < 0.05).

DISCUSSION

Our results demonstrate that in female alligatorsacute stress causes a significant decline in plasmaestradiol. Two of the animals, however, showed aninitial increase in plasma estradiol followed by adecline. These results are somewhat similar to thoseseen in the turtle, Chelydra serpentina, where plasmaestradiol rose 24 hr after capture and then fell backto initial levels (Mahmoud et al., 1989). In the gravidturtle, however, plasma estradiol rose by an order ofmagnitude (ca 30 pg/ml to more than 300 pg/ml), Thesource of this transient surge in estradiol secretion inresponse to stress is not known. Embryonic turtleadrenal tissue has been shown to synthesize andsecrete estadiol (White and Thomas, 1990), but it isnot known if adult turtle adrenal is capable ofproducing estradiol. The mean rise in estradiol in thealligators was not significant, and by 22 hr all animalshad levels significantly lower than the initial levels.While the drop in plasma estradiol was significant inthe female alligators, it was far less dramatic than thedrop in plasma testosterone seen in male alligatorssubjected to the stress of restraint. In males, plasmatestosterone was undetectable in the plasma by 28 hrof restraint (Lance and Elsey, 1986), whereas infemales plasma estradiol remained over 300 pg/mlafter 48 hr of restraint. During the reproductiveseason plasma testosterone in male alligators rangesfrom 10 to over 100 ng/ml, whereas plasma estradiolin female alligators only ranges from 300 to1000 pg/ml (Lance, 1989). The reason for this sus-tained estrogen titer may be due to the presence ofthe sex steroid binding protein (SSBP) in alligatorblood (Ho et al., 1987). Concentrations of SSBP(30-140 nM) always exceed those of estradiol(0-20 nM), whereas concentrations of testosteronein breeding males greatly exceed those of SSPB.Furthermore, the binding affinity of alligator SSBPfor estradiol is about five times that of testosterone(Ho et al., 1987). This suggests that even though thestress of restraint effectively shuts down sex steroidsecretion from the overy, there is sufficient bindingcapacity in the blood to maintain circulating estradiolconcentrations at physiological levels during the 48 hrof the experiment.

The biphasic pattern of corticosterone secretion infemale alligators under restraint is remarkable in thatit is identical to that seen in the male. In malealligators plasma corticosterone rose dramaticallyfrom 4 to 12 hr post capture, dropped at 22 hr, thencontinued to rise up to 41 hr (Lance and Elsey, 1986).This unusual pattern is difficult to explain. There areno comparable data for other species, and there areno data available on circadian rhythms of corti-costerone secretion in adult wild alligators. The nor-mal circadian pattern of corticosterone secretion inyoung captive alligators (Lance and Lauren, 1984)does not fit the pattern seen in this experiment.

Acute stress and estradiol levels in alligators

The results of this experiment showing the effectsof acute stress on corticosterone and estradiol levels,and the data of Elsey et al. (l990b) documenting anassociation of reproductive failure with chronicallyelevated plasma corticosterone levels in alligators,indicate that both acute and prolonged stress canhave deleterious effects on reproduction in thisspecies.

Aeknowledgements- The authors would like to thank DaveRichard for help in catching the alligators. Financial sup-port was provided in part by the Louisiana Department ofWildlife and Fisheries.

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