# Daily changes in blood serum levels of 17beta-estradiol and 11-ketotestosterone in the mature carp cyprinus caprio L.

K. Beniarz, M. Sokolovska, Alexis Fostier, P. Epler

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K. Beniarz, M. Sokolovska, Alexis Fostier, P. Epler. Daily changes in blood serum levels of 17beta-estradiol and 11-ketotestosterone in the mature carp cyprinus caprio L.. Chronobiologia, 1986, 13, pp.23-27. hal-02728619

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DAILY CHANGES IN BLOOD SERUM LEVELS OF 17 β-ESTRADIOL AND 11-KETOTESTOSTERONE IN THE MATURE CARP *CYPRINUS CARPIO* L.

KRZYSZTOF BIENIARZ *, MIROSLAWA SOKOLOWSKA *, ALEXIS FOSTIER **, PIOTR EPLER *

*Department of Ichthyobiology and Fisheries, Academy of Agriculture, Kraków-Mydlniki, Poland* 
*Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, Rennes, France**

Very few studies deal with the analysis of daily changes in sex steroid levels in fish<sup>12,14</sup>.

In carp, 17β-estradiol has been found in blood as well as in ovarian tissue by gas chromatography⁹ and identified as a compound derived from pregnenolone in incubations of ovaries³, while 11-ketotestosterone has been identified as a metabolite of pregnenolone in incubation of the testes of another Cyprinid, the goldfish¹¹.

A short-term rhythmicity of various physiological and biochemical processes has been observed, among others, in Cyprinids<sup>5,13,19</sup>. Daily changes in blood gonadotropin (GTH) levels have been recorded in goldfish<sup>2,8,20</sup> and in carp during spawning<sup>10</sup>. Such changes have been also observed for cortisol in goldfish<sup>4,15,16,18</sup> and for β-estradiol in common carps<sup>14</sup>.

The aim of this investigation was to define daily changes in the blood serum 17 β-estradiol levels of mature females and 11-ketotestosterone levels of mature male carp. Mention should be made that the results obtained, except of cognitive value, can be useful for artificial reproduction of carp, a fish which is of great economic value in many countries in the world.

MATERIALS AND METHODS

Investigations were carried out in the Department of Ichthyobiology and Fisheries, Academy of Agriculture in Kraków, during the first week of June 1979. A total of 21 mature common carp females and 24 mature males aged

<footnote>Key-words: Carp; Circadian; Estradiol; Fish; Testosterone.</footnote>

<footnote>This work was supported by a grant (Resort Project R.III. 14) from the Jagiellonian University, Kraków, Poland.</footnote>

<footnote>Received February 14, 1984.</footnote>

<footnote>Chronobiologia 13, 23, 1986.</footnote>
4 years, weighing about 3 kg were used. Females and males had completed vitellogenesis and spermatogenesis respectively, however, they did not give gametes spontaneously. Injections of pituitary extracts (hypophysation) were necessary to induce ova or sperm release. Fish were kept under natural light conditions (LD = 16:8); water temperature was about 19° C. Females and males were divided into 2 groups respectively. Each group was sampled every 4h during 24h, beginning at 08⁰⁰ (group 1) and 10⁰⁰ (group 2). Thanks to such a group division each fish was sampled every 4h within a sex group. In this way a relatively high frequency of blood sampling during 24h was ensured with a low number of blood sampling from the same fish. One group of females consisted of 10 and the second of 11 fish. Each group of males consisted of 12 fish. Samples of blood serum after deep freezing were sent to the Laboratoire de Physiologie des Poissons, Rennes (France) to be tested for 17 β-estradiol (blood serum from females) and 11-ketotestosterone (for males) using radioimmunoassay. The 17 β-estradiol assay⁷ included a chromatographic step which increased the specificity of the measurement while in 11-ketotestosterone assay⁷ a specific antibody was used having a low cross reactivity with testosterone (1.1%). Data obtained in this work were statistically analyzed using cosinor analysis¹.

## RESULTS

17 β-estradiol levels changed during 24h in group 1 between an average of 0.11 to 0.45 ng/ml of blood serum and in group 2 between 0.21 to 0.58 ng/ml.

11-ketotestosterone levels changed during 24h in group 1 between an average of 0.23 to 2.09 ng/ml of blood serum and in group 2 between 0.88 to 3.00 ng/ml.

In all samples: 1. the lowest level of estradiol was below 0.10 and the highest one was 4.01 ng/ml (detection limit 0.10 ng/ml); 2. the lowest level of 11-ketotestosterone was below 0.08 ng/ml and the highest one 5.14 ng/ml of blood serum.

The circadian rhythmicity of estradiol is statistically proved (error ellipse omits the center of the circle) (fig. 1). Confidence intervals are very large which means that individual variability is very high.

The highest levels of estradiol (0.46-0.58 ng/ml) were observed between 09⁰⁰ and 11⁰⁰, amplitude ($A^±$) = 1.1080, acrophase ($Φ$) = -6.8036h.

The circadian rhythm of 11-ketotestosterone is very well marked and has been statistically proved; error ellipses in fact omit the center of cosinor circle being rather far from the center of the circle. The highest levels of 11-ketotestosterone (2.09-3.00 ng/ml) were observed between 10⁰⁰ and 12⁰⁰ (fig. 1), ($A^±$) = 1.4007, ($Φ$) = 11.032h.

## DISCUSSION

Up to this moment nobody has investigated changes during 24h in 17 β-estradiol and 11-ketotestosterone in carp, except MUSIALEK et al.¹⁴. They found the highest levels of estrogen in the blood serum of carp all year round at 12⁰⁰ which is also in agreement with the data from this experiment. The highest levels of cortisol in goldfish found by PETER and HONTELA¹⁵ and PETER
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<table><caption>Fig. 1 - Circadian rhythm of estradiol in the blood of female carps and of 11-ketotestosterone in the blood of male carps.</caption><thead><tr><th rowspan="2">key to ellipses</th><th rowspan="2">n</th><th rowspan="2">rhythm detection, p</th><th colspan="3">mesor, M</th><th colspan="3">amplitude, A</th><th rowspan="2">acrophase, Φ<br>0.95 confidence<br>area in hours</th></tr><tr><th>M ±</th><th>SE</th><th>CL</th><th>A ±</th><th>SE</th><th>CL</th></tr></thead><tbody><tr><td>E ♀</td><td>21</td><td>0.05</td><td>5.8563</td><td>1.5312</td><td>3.0325</td><td>1.1080</td><td>0.349</td><td>0.6917</td><td>-6.8036<br>-4.4989 +4.3708</td></tr><tr><td>K ♂</td><td>24</td><td>0.05</td><td>0.3356</td><td>0.093</td><td>0.184</td><td>1.0407</td><td>0.098</td><td>0.181</td><td>11.032<br>-0.546 +0.437</td></tr></tbody></table>

E = estradiol; K = 11-ketotestosterone; p < 0.05.

et al.¹⁶ were more or less in the same part of the day as estradiol and 11-ketotestosterone in this experiment with carp.

In HONTELA and PETER'S⁸ study, a large peak of serum GTH was detected at 12⁰⁰ when vitellogenic females of goldfish were exposed to a LD = 16:8 and 21° C regime. At a later stage, in mature females, BRETON et al.² detected a GTH peak at 11⁰⁰, while STACEY et al.²⁰ could only detect a GTH surge in ovulatory females and it occurred during the dark phase. In the mature common carp a small increase in blood plasma levels of 17 β-estradiol observed in the morning between 09⁰⁰ and 11⁰⁰, is rather related to a GTH increase occurring during the last part of the dark phase or early in the morning since, at this stage, the release of endogenous hormone, induced by luteinizing hormone-releasing hormone (LH-RH) or an intraperitoneal injection
of pituitary extracts stimulates estradiol secretion 5 to 7h after the GTH increase²¹.

Fewer data are available for males than for females. BRETON et al.² show a small increase of plasma GTH levels at 11⁰⁰, but not statistically significant in spermiating goldfish. Such a 11-ketotestosterone increase, observed between 10⁰⁰ and 12⁰⁰ in male carp, could be related to the GTH increase in the morning.

Besides, changes in the responsiveness of the goldfish ovary have been shown according to the time of GTH injection¹⁷. In this study oocyte development has been recorded. However, if these changes are related to variations in the availability of the ovarian receptors for GTH, we can expect that the steroidogenic activity will fluctuate during the day, even without drastic changes in GTH secretion. Authors did not state a circadian rhythmicity but only changes during 24h.

In this paper the most suitable method from those applied in chronobiology was used to find if the changes stated during 24h were of circadian rhythm type. On the basis of the data found in this work it is possible to conclude that in carp steroid hormones in blood plasma change during 24h reaching the highest levels in the first or middle parts of light day and that some of them (17 β-estradiol and 11-ketotestosterone) are of circadian rhythm nature.

## ACKNOWLEDGEMENTS

The authors wish to thank Prof. Józef Surowiak for critically reviewing this manuscript and Sylvian Marlot for technical assistance.

## SUMMARY

Daily changes in blood serum levels of 17 β-estradiol and 11-ketotestosterone in the mature carp *Cyprinus carpio* were investigated. Fish were kept under natural light conditions (LD = 16:8) water temperature about 19° C. Females and males were divided into 2 groups respectively. Each group was sampled every 4h during 24h beginning at 08⁰⁰ (group 1) and 10⁰⁰ (group 2).

The 17 β-estradiol assay⁷ included a chromatographic step which increased the specificity of the measurement while in 11-ketotestosterone assay⁶ a specific antibody was used, having a low cross reactivity with testosterone (1.1%). Data obtained in this work were statistically analyzed using cosinor circle with error ellipses¹.

The highest levels of estradiol (0.46-0.58 ng/ml) were observed between 09⁰⁰ and 11⁰⁰ and of 11-ketotestosterone (2.09-3.00 ng/ml) between 10⁰⁰ and 12⁰⁰.

It was proved statistically that changes in the estradiol and 11-ketotestosterone levels during 24h are of circadian rhythm type. The problem of hormonal changes during 24h, connected with sexual maturation of fish, is discussed.

## REFERENCES

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Requests for reprints should be addressed to:

KRZYSZTOF BIENIARZ
Department of Ichthyobiology and Fisheries
Academy of Agriculture
ul. Ambrosowa 6
30-149 Kraków-Mydlniki, Poland