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# Rhythms of reproduction in teleost fish

Roland Billard, Bernard Breton

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Roland Billard, Bernard Breton. Rhythms of reproduction in teleost fish. Rhythmic activity of fishes, Academic Press, INC., 1978, 0-12-690650-5. hal-01600956

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# RHYTHMS OF REPRODUCTION IN TELEOST FISH

R. BILLARD and B. BRETON

I.N.R.A., Laboratoire de Physiologie des Poissons
78350 JOUY EN JOSAS, France

## Introduction

Reproduction is a cyclic phenomenon which occurs periodically in fish, sometimes once every two or three years but more usually, once or several times a year. But if rhythm is defined as a succession of reproductive cycles, rhythmicity can hardly be an innate characteristic, since this cyclic reproductive activity is usually considered to result from adaptation to a changing environment mediated through the endocrine system. Fish live in a large variety of biotopes leading to a great diversity of reproductive patterns. This paper describes some types of reproductive cycle and their associated hormonal changes.

The reproductive cycle is divided into two parts, gametogenesis and spawning.

Gametogenesis is the formation of highly specialised gametes (oocytes or spermatozoa) from simple germ cells (spermatogonia or oogonia). The duration of gametogenesis varies according to species and rearing temperature. In *Oryzias latipes*, the preleptotene-early spermatid interval is 5 days at 25°C and 12 days at 15°C (Egami and Hyodo-Taguchi, 1967). The leptotene-spermatozoa interval is 12 days in *Oryzias* and 14 days in *Poecilia* (Billard, 1968). The total duration of spermatogenesis at 25°C was estimated at 36 days in *Poecilia* (Billard, 1969). In the viviparous species *Xiphophorus*, at 24°C, a reproductive cycle of 35 days includes a gestation period of 27 days (Siciliano, 1972).

Spawning comprises the sequences of events leading to the liberation of gametes, meiosis resumption, oocyte maturation, ovulation and oviposition in females and spermiation and sperm release in males. These processes are complex and more variable in duration.

The endocrine changes during the reproductive cycle include variations in the levels of pituitary and plasma
gonadotropin (GTH*) and plasma sex steroids. Much information on fish hormones is now available but heterogeneity in the assays and the lack of international standards preclude definitive interpretation. Methodologies for GTH radioimmunoassay (RIA), GTH bio-assay and steroid analyses have been discussed respectively by Crim *et al.*, (1975), Jalabert *et al.*, (1974) and Sandor and Idler (1972).

## Circannual Rhythm of Reproduction in Fish Living in Temperate and Cold Zones

### Gametogenesis

Although gametogenesis and its endocrine correlates have been described in only a few species of teleost fish, some typical groups can be identified. Experimental variation of the environment gives some indication of the factors determining the reproductive cycle.

*Salmonids* In this group, gametogenesis occurs in summer and autumn and can be correlated with decreasing photoperiod and temperature.

In Europe, trout gametogenesis begins with vitellogenesis in females; the division of type B spermatogonia in males starts in late spring or early summer. During the summer, the gonadosomatic index (GSI) increases regularly and, normally, spermatogenesis ends in September or October and vitellogenesis in October or November. An example of a spermatogenetic cycle is given in Fig. 1. A peak of t-GTH secretion is observed at the beginning of the spermatogenetic cycle when a slight multiplication of type A spermatogonia is seen in the testis and when some type B spermatogonia appear. At the same time, high levels of estradiol 17β are present in the plasma. During intense testicular activity, leading to a sharp increase of the GSI, there is a parallel rise of t-GTH and a slight increase in the concentration of estradiol. t-GTH pituitary content is maximum at the beginning and at the end of spermatogenesis. A similar pattern of development was found in studies of pituitary gonadotropic cells by immuno-fluorescence (Fig. 2). At the beginning of the oogenetic cycle in female rainbow trout (Fig. 3), a high level of estradiol and a slight rise in t-GTH were noted. t-GTH pituitary content measured either by RIA or bioassay, plasma t-GTH, estradiol, oocyte diameter and GSI rose simultaneously.

Studies of reproductive development in Atlantic salmon (chilko variety) and brook trout have been reported by

<figure><img src="image_3.png" /><figcaption>Fig. 1. Endocrine components of the spermatogenetic cycle in rainbow trout (Breton *et al.*, unpublished), RIA for GTH and estradiol 17β (E2), importance of the germ cell is measured by the germ cell index (Billard *et al.*, 1974).</figcaption></figure>

Crim *et al.*, (1975). In salmon, plasma GTH rises slightly during vitellogenesis and attains very high levels during the spawning season. In brook trout males and females, as in rainbow trout, the rise of GSI is closely associated with a rise of t-GTH. Testosterone and 11-ketotestosterone levels rise before the spawning season in male brook trout (Sangalang and Freeman, 1974). Additional information on steroid hormones in the plasma of the Atlantic salmon is due to Schmidt and Idler (1962). They observed notable variations associated with migration and sexual maturation (see Ozon 1972 for review).

<footnote>*Footnote: c-GTH : carp gonadotropin
s-GTH : salmon gonadotropin
t-GTH : trout gonadotropin</footnote>
<figure><img src="image_4.png" /></figure>

Fig. 2. Evolution of the fluorescence intensity of gonadotropic cells in the pituitary of male rainbow trout during the reproductive cycle; details of the immunofluorescence technique in Billard *et al.*, (1971); intensity and fluorescence is estimated by arbitrary units (from Escaffre *et al.*, unpublished).

Circannual rhythms have been investigated as a preliminary to attempts to advance the time of reproduction (see De Vlaming, 1974, for review). Recent work has confirmed that photoperiod variation is the main factor involved in gonadal development in salmonids (Breton and Billard, 1977). Constant long (16L-8D) or short (8L-16D) photoperiods did not induce gonadal recrudescence; only a decreasing photoperiod (16L-8D to 8L-16D) from February 24 to June 19 induced full spermatogenesis and spermiation. The amount of plasma t-GTH and the efficiency of spermatogenesis were significantly increased at 16° showing that temperature is a factor, though not limiting, in reproduction in trout.

Other species which show a similar annual reproductive rhythm are *Salmo salar* (Jones, 1940), *Salvelinus fontinalis* (Henderson, 1962), *Plecoglossus altivelis* (Honma and Tamura, 1962), *Embiotoca jacksoni* (Lagios, 1965), *Sebastodes paucispinis* (Moser, 1967) and *Cymatogaster aggregata* ♀ (Wiebe, 1968).

*Cyprinids* Gametogenesis usually starts in autumn, continues slowly during the winter and ends in the spring.

(a) *Roach* The reproductive cycle of the male roach has been described by Escaffre and Billard (1976). In

<figure><img src="image_5.png" /></figure>

Fig. 3. Endocrine components of the oogenetic cycle in rainbow trout. Top graph: measurements of t-GTH content in the pituitary by RIA (●), and bioassay (o) (Breton *et al.*, unpublished).

autumn there is a rise of type B spermatogonia; meiosis starts in late December and is continuous until April. Spermiogenesis occurs in March, April and May and spermatotozoa are very abundant in May and June. Pituitary concentrations of GTH exhibit some variation, rises occurring in autumn and late winter and in late spring at the time of spermiation.

(b) *Carp* The pattern of the reproductive cycle in the carp is dependent on the thermal rearing regime. Some data are available on the c-GTH pituitary content in males measured by RIA (Fig. 4) and estimated by immunofluores-

<footnote>*Footnote: t-GTH : trout gonadotropin</footnote>
cence (Fig. 5). Pituitary concentration of GTH (by RIA) decreases in the winter and rises during the spring at the time of gonadal recrudescence and the spawning season.

<figure><img src="image_6.png" /></figure>

**Fig. 4.** Spermatogenesis in carp: evolution of the germ cells in the testis in spring and early summer. Variation of the pituitary content of immunoreactive c-GTH (µg/ml fresh gland) between January and July (From Weil, unpublished).

In another study (Fig. 5), spermatogenesis occured in spring and a second wave was observed in the summer. Gonadotropic cell fluorescence was very strong in spring, during the first wave, and very weak in summer and early autumn during the second spermatogenetic wave. There may have been a cyclic depletion of the pituitary but this possibility requires further study. Eleftheriou *et al*., (1968) have shown that the evolution of gonadal development is highly correlated with the level of plasma estradiol 17β and to a much lesser extent with 16-ketoestradiol. Gonadal development is associated with rising temperature,

<figure><img src="image_7.png" /></figure>

**Fig. 5.** Evolution of the GSI, spermatogenetic cycle and immunological fluorescence of GTH pituitary cells in carp *Cyprinus carpio* throughout the annual reproductive cycle (after Escaffre, unpublished).

and spawning occurs when the temperature cycle is at its maximum.

(c) **Goldfish** In goldfish, c-GTH increases with gonadal recrudescence (Fig. 6). In the males, androgens also rise prior to the spawning season but estrogen does not (Schreck and Hopwood, 1974). In females, androgens and estrogens are present during the spawning season. The amount of pituitary c-GTH, estimated by immunofluorescence decreases during gonadal recrudescence but rises later, minimum fluorescence intensity occurring in summer as for carp. Other studies have been carried out on cyclic variation in goldfish. Beach (1959) found that the surface of the pituitary basophil cells first increases with increasing oocyte diameter and decreases near maturity. The neurosecretory material in the NPO also varies; it is very low in the spring, and high in summer and autumn (Mieszkowska and Jasinski, 1973).

(d) **Tench** A peak of GTH concentration is observed in the plasma in February, at the beginning of the sperma-
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Annual variation in GSI, intensity of fluorescence of the gonadotropic pituitary cells (males) and plasma c-GTH in goldfish (Esmasse and Gillet, unpublished).</figcaption></figure>

togenetic cycle (Breton *et al.*, unpublished). It is low in April and rises subsequently as spermatogenesis progresses. It decreases slightly during the spawning season; pituitary concentration of GTH is very high in the middle of the spawning season. This experiment was carried out in Poland (52°N), where gametogenesis started in winter.

(e) *Other species* Other cyprinids e.g. *Couesius plumbeus* (Ahsan, 1966), *Abramis brama* (Shikhshabekov, 1974) and various other species such as *Hypseleotris galii* (Mackay, 1973), *Agonus cataphractus* (Le Gall, 1969), *Dicentrarchus labrax* (Barnabe, 1976) exhibit a reproductive cycle similar to that of roach and carp. Tench and other species have a different pattern of reproductive cycle, gametogenesis occurring entirely in rising photoperiod and temperature. Typical of these are, *Cymatogaster aggregata* male (Wiebe, 1968), *Pylodictus olivaris* (Turner and Summerfelt, 1971), *Paralabrax clatharus* (Smith and Young, 1966), *Notropis stramineus* (Summerfelt and Minckley, 1969), *Scomber scomber* (Bara, 1960), *Chromis chromis* (Contini and Donato, 1973), *Cynoscion regalis* (Merriner, 1975) and *Epinephelus* (Brusle and Brusle, 1975).

*Pleuronectids* In the female plaice, plasma steroids, such as cortisol, testosterone and estradiol are low in concentration in the summer, during the resting period and rise with gonadal recrudescence, (Wingfield and Grimm, 1977). Estradiol reaches a maximum and drops just before the peak spawning season, while testosterone and cortisol reach their maximum levels just at the beginning of the peak spawning period. Testosterone decreased during

this period while cortisol levels fell later. GTH pituitary content was minimal prior to and during the spawning season. Except for estradiol, males exhibit similar seasonal changes. The highest steroid level was recorded in January and February before the peak spawning season. In immature plaice, a pronounced cortisol peak is observed, indicating that seasonal changes of this steroid are independent of the gonadal cycle and may be associated with feeding activity as suggested by Campbell *et al.*, (1976) for the winter flounder. In this latter species, seasonal variations in the peripheral steroid levels are closely associated with GSI, as in plaice: testosterone levels are higher in females than in males. 11-Ketotestosterone remains low throughout the year in females and appears to be more important in the males than testosterone (Campbell, 1975; Campbell *et al.*, 1976). Additional information on reproduction in pleuronectids is given by Pitt (1966) and Lahaye (1972).

*Experimental modification of the reproductive cycle* In cyprinids, both temperature and photoperiod are important factors and may interact. In goldfish females (Fig. 7) taken in October, gonadal recrudescence is more rapid

<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Effects of various photoperiod-temperature combinations of gonadal development (GSI) in the goldfish (Gillet, unpublished).</figcaption></figure>
when fish are under decreasing temperature (17→10°C) than under constant elevated temperature (20°C). The effect of photoperiod is dependent on the temperature regime. In January, long day lengths are stimulatory especially when associated with a low temperature (10°C). In March, when oogenesis is more advanced, only long day and low temperature regimes are stimulatory. Experiments carried out on tench, under three temperature regimes, show that GSI peaks earlier and that the pattern of plasma GTH levels is different in heated ponds (Fig. 8); the number of oogenesis waves and consequently of spawnings is increased

<figure><img src="image_10.png" /><figcaption>**Fig. 8.** Annual reproductive cycle of female tench under three thermal regimes I (Natural), II (I + 3°C), and III (II + 3°C). A: GSI, B: temperature (total degree days), C: pituitary content of immunoreactive gonadotropin (c-GTH equivalent), D: plasma gonadotropin (Breton *et al.*, unpublished).</figcaption></figure>

at the higher temperatures (Breton *et al.*, unpublished). GTH pituitary content is at its maximum during the inter-

val between successive spawnings.

*Ovulation, Spermiation, Spawning*

*Spawning season* Three main spawning seasons can be identified in France, late autumn and winter (salmonids), early spring (pike, perch), and late spring and early summer (tench, roach, gudgeon).

Some species of fish, e.g. *Chromis chromis* (Contini and Donato, 1973), *F. diaphanus* (Fournier and Magnin, 1975), *Notropis longirostris* (Heins and Clemmer, 1976) reproduce in summer, but most of these are intermittent spawners. The duration of the spawning period may depend on the latitude; in the USSR for instance, the spawning season of bream varies from 58-60 days in the north to 16 days in the Volga delta (Shikhshabekov, 1974). Spawning of the sea bass occurs in winter in the Mediterranean area, in spring in Brittany and during early summer in Ireland when the temperature reaches 10-12°C. The period of running sexual products is usually longer than the breeding season, especially for the males. Sperm release begins before ovulation and can also be observed by hand, stripping long after the breeding season has ended. Running males can be observed as late as May for trout (Billard, unpublished) and November for carp (Shikhshabekov, 1974). Sperm taken at the end of the reproductive season has been shown to be of poor quality in tench (Horoszewicz, personal communication) and in sea bass (Billard *et al.*, 1977).

*Endocrine changes during ovulation and spermiation* Changes have been observed in the hormonal status of rainbow trout during the period of ovulation and spermiation (Breton and Manac'H, unpublished). Estradiol 17β decreased while t-GTH was still rising 15 days after ovulation. At the end of spermatogenesis estradiol reaches its maximum level and is followed by a peak in testosterone; a second peak of estradiol occurs at the same time as t-GTH. Spermiation starts immediately afterwards.

Data reported earlier indicate a rise of t-GTH at the time of spermiation (Fig. 1) and oocyte maturation + ovulation (Fig. 3) in salmonids. Pituitary t-GTH is also at its maximum in the final stages of the reproductive cycle in salmonids (Fig. 1) and in cyprinids. A surge of plasma c-GTH is observed at the time of ovulation in goldfish (Breton *et al.*, 1972). In males, androgens (testosterone and 11-ketotestosterone) increase just before spermiation and reach a peak, which occurs earlier for testosterone than for 11-ketotestosterone. This suggests that 11-ketotestosterone may be more closely associated with the phenomenon than testosterone. 11-Ketotestosterone also rises in the winter flounder at the time of spawning (Campbell, 1975; Campbell *et al.*, 1976).
Usually, a rise in plasma androgen and estrogen and a surge of gonadotropin occur at the time of ovulation and spermiation. Thus, hormonal requirements appear to be more important for the final stages of the cycle than for gametogenesis. This is also illustrated by the technique of hypophysation used to induce ovulation and sometimes spermiation in many species of fish (review by Pickford and Atz, 1957).

*Conclusion* The hormonal changes which induce ovulation are probably triggered by environmental factors which may be either abiotic (e.g. temperature) or biotic (e.g. social environment, vegetation). Thus spawning time may be linked to environmental change. However, Cushing (1969) has shown a relative fixity of spawning seasons for plaice, herring, cod and sockeye salmon. The link between spawning and environment may thus be more strict for freshwater than for marine fish which are less subject to rapid environmental change.

*Conclusion*

Various patterns of annual periodicity in fish reproduc-
tion can be identified in the temperate and cold zones.
These patterns are not characteristic of a family. Gameto-
genesis can start either in autumn (roach) or in late
winter (tench) in cyprinids. Most of the salmonid species
exhibit gametogenesis in summer and spawn in winter, but
*Thymallus* is a spring spawner. Sometimes the time for
sexual maturity of male and female fish does not coincide
closely; thus in the pike, ovulation in females does not
always synchronise with spermiation in males. An extreme
case is shown by the viviparous fish *Cymatogaster aggre-
gata*, in which males produce sperm in the summer which is
stored after copulation in the ovary of the female until
the end of vitellogenesis and oocyte maturation in January.

The duration of gametogenesis as well as of the breed-
ing season is longer when gametogenesis starts in autumn.
The duration of incubation should also be considered.
When reproduction occurs in winter, the hatching period
for some freshwater species (mainly salmonids) is delayed
because of the large amount of vitellus and the low tem-
perature. In northern Sweden, salmon spawn in the autumn
and 'swim up' occurs five to six months later. The same
phenomenon occurs to a lesser extent in pike which is a
late winter spawner; the eggs are bigger and incubation
(>200 degree-days) is longer than for spring spawning
fish. If the hatching and resorption periods are consid-
ered, the reproductive cycles of winter and early spring
spawners overlap and fry are released into the water when
food is available.

Photoperiod and temperature are probably the main seasonal timing agents for temperate and cold water fish.

<figure><img src="image_11.png" /><figcaption>Fig. 9. Circadian rhythms in plasma GTH concentration during a 24 h period in goldfish maintained under various environmental conditions (Gillet, unpublished).</figcaption></figure>

Field observations have not established which factor is
essential but experimental work has given us some clues.
The effects of temperature are important in cyprinids and
in other groups which are often intermittent spawners.
Gametogenesis may occur over a large temperature range
(10 to 30°C in goldfish; Gillet, personal communication)
provided that nutrition is adequate. In the natural envir-
onment, carp exhibit completion of gametogenesis at around
10-15°C and full gametogenesis may be obtained at 23°C
(Gupta, 1975). Thermal requirements are more strict for
ovulation in the carp, the minimum spawning temperature
being 17°C (Shikhshabekov, 1974). Ovulation can be induced
in goldfish at 12°C by antiestrogen implantation in the
pituitary (Billard and Peter, 1977). This suggests an
action of temperature at gonadal and at central nervous system and pituitary levels.

Photoperiod variation is probably the main environmental timing agent for some species such as salmonids and gasterosteids (De Vlaming, 1972). The presence of a photosensitive circadian rhythm, similar to that responsible for the annual rhythm of reproduction in birds (Follett, 1973), has been demonstrated in some fish which reproduce under long photoperiods, e.g. stickleback (Baggerman, 1972), *Heteropneustes fossilis* (Sundararaj and Vasal, 1976), and *Oryzias latipes* (Chan, 1976). The effect of photoperiod variation on the reproductive cycle may be mediated through an endogenous rhythm of light receptivity in some fish.

## Rhythm of Reproduction in Fish Living in Tropical and Equatorial Zones

### Tilapia

Field studies have been carried out on *Tilapia* in both tropical and equatorial zones. Although environmental factors such as photoperiod and temperature do not vary much in the latter zone there is a seasonal reproductive cycle. It is usually said that breeding activity corresponds to the onset of the rainy season.

Studies by Moreau (1970) in Malagasy showed that gametogenesis and breeding occurred in the austral summer and coincided with the highest temperature; food availability and growth rate was also maximum during this period. Observations by Kiener (1963) showed that at the same latitude, i.e. under the same photoperiod regime, the duration of the breeding season varied with the altitude and probably with temperature. Data from Hyder (1970) for *T. leucostica* in the equatorial zone are less clear. Seasonal testicular activity was restricted to the autumn and winter. In females, GSI variation was not always correlated with the percentage of breeding females, particularly in June or July. Histological features showed a resting period from July to September, as in males. This lack of gonadal activity can hardly be correlated with the known environmental factors, although Hyder suggested a possible effect of illumination and temperature. It should be noted, however, that air temperature rather than water temperature was recorded. The role of rainfall in the reproduction of *Tilapia* is a matter of controversy (see Hyder, 1970, for discussion).

### Other Species of Tropical Fish

A study by Payne (1975) of a tropical stream-dwelling cyprinid, *Barbus liberiensis* showed an annual rhythm of reproduction with a phase of gonad recrudescence (January to April) and a breeding season in May to July followed by a resting period.

Rainfall was among the environmental factors important for the initiation of the various stages of reproduction; it coincided with the beginning of the breeding season. The end of the breeding season corresponded with a low somatic condition, low water temperature and declining rainfall. Gametogenesis started at the beginning of the dry period and took place during a period of slightly increasing temperature, photoperiod and ionic concentration in the water. At the end of the period of gametogenesis, no fish showed an empty foregut. A similar annual reproductive cycle was reported for *Barbutor* (Mathur, 1962), and *Heteropneustes fossilis* (Sundararaj and Vasal, 1976).

The spawning season of Caribbean reef fishes was studied by Munro *et al.*, (1973). The majority of the species studied spawn mostly in February, March and April when water temperatures are minimum at around 28°C. The Sergeant Major, *Abudefduf saxatilis*, which is eurythermal (Graham, 1972) has two main spawning periods (March and April, and July to September). Okera (1974) indicates an annual spawning season of 3-4 months duration (August to November) in the Indian ocean for *Sardinella gibbosa* and a much longer period (at least August to February) for *S. albella*. The reproductive cycles of some viviparous fish have been studied by Mendoza (1962) who found great variations in the length of the reproductive season for 3 species living in the same lake; a short reproductive cycle with one brood was noted for *Alloophorus* and *Goodea* and a breeding season extending over a period of 8 to 9 months for *Neophorus*.

### Fish Showing Continuous Reproduction Throughout the Year

In the study of Munro *et al.*, (1973), two groups of tropical fish (Carangidae and Lutjanidae) appear to spawn throughout the year. Some other species of fish can reproduce continuously provided environmental conditions are favourable; continuous reproduction was observed in the guppy *Poecilia reticulata* kept at 27°C under a constant 12L-12D photoperiod (Billard, 1966). Production of spermatzoa continued throughout the year with some variations.

Young were born continuously during a 12 month period. Intervals between two parturitions were longer and more variable in winter than in summer, suggesting variations in the duration of the reproductive cycle. These variations may have been due to changes in food availability. Such a dependence of frequency of spawning on food availability has been demonstrated in *Gasterosteus aculeatus* by Wootton and Evans (1976). In other poecilid fish such as *Gambusia*, reproduction is virtually continuous under favourable conditions. Barney and Anson (1921) considered that the cyclicity of egg production was not temperature dependent but was governed by the size and metabolic potential of
the mother. In another viviparous fish *Brachyrhaphis episcopi*, Turner (1938) found that breeding occurred throughout the year. Many species of marine fish appear to be continuous breeders in Indian waters (Quasim, 1973). This phenomenon of continuous reproduction in fish is well known to aquaculturists; aquarium fish kept under constant environmental conditions may exhibit continuous gametogenesis provided food availability if sufficient. Slight changes such as temperature variation or water substitution, induce spawning.

## Conclusion

In tropical or equatorial zones, most fish show an annual rhythm of reproduction. The environmental factors responsible for the various phases of the cycle are not readily identified by field observations, where many parameters vary at the same time. Gametogenesis seems to be initiated by subtle environmental changes (including slight photoperiod variations), but more dramatic events such as seasonal rainfall and flooding are required to induce ovulation as shown in the carp by Khanna (1958). This seems to have adaptive value since food availability for the young is greater during the rainy season. At the limit of the temperate and tropical zones, John (1963) noted that the reproductive cycle of *Rhinichthys osculus* in the Chiricahua mountains (Arizona) is basically regulated by photoperiod but that spawning activity was enhanced in swollen streams and flash floods.

## Other Rhythms Associated with Reproduction

### Spawning Rhythm

**Circadian spawning activity** The daily rhythm of oviposition of the medaka (*Oryzias latipes*) (Robinson and Pugh, 1943) is well known. Eggs are laid just before dawn almost every day during the breeding season. Under artificial illumination (14L-10D), spawning occurs 30 minutes after light onset (Yamauchi and Yamamoto, 1973). Work by Egami and Nambu (1961) showed that stimuli other than photoperiodicity may also be important in determining the time of oviposition. Other species showing circadian spawning rhythms are known. The hermaphrodite, oviparous fish *Rivulus marmoratus* exhibits a 24 h rhythm of internal self-fertilization and oviposition (Harrington, 1963). During the reproductive season, *Menidia audens* lays eggs mainly in the morning (Hubbs, 1976).

Two species of *Trichopsis* spawn at the end of the light period (Marshall, 1967) and *Pomatomus saltatrix* shows daily spawning activity near sundown (Norcross *et al.*, 1974); this is probably connected with photoperiod. Similarly, the sardine spawns in the evening (Gamulin and Hure, 1956).

**Tidal and lunar spawning rhythms** A daily rhythm of spawning has been reported for *Pagrus ehrenbergii* which is a tropical intermittent spawner having a 4-6 month spawning period which coincides with the rains (Stepkina, 1973). Spawning activity may occur at different times during a 24 h period and is observed when more saline and colder (denser) ocean water moves tidally to the shelf.

In Japan, the puffer *Fugu niphobles*, exhibits a lunar spawning rhythm associated with the evening tide (Nozaki *et al.*, 1976). There are other examples of lunar and tidal rhythmicity in spawning and even in gonad development (see Schwassmann, 1971 for review).

May (personal communication) has shown a rhythmic spawning activity occurring once a month in a population of *Polydactylus sexfilis*, suggesting lunar influences.

*Other rhythms associated with spawning* Spawning intervals of 1.9 and 2.7 days have been shown to occur in the zebrafish, *B. rerio* (Eaton and Farley, 1974).

Migration is often associated with spawning. It is unnecessary to recall the well known rhythmic reproductive migration of many species, but some fish show subtle migratory rhythms. For instance, the hogchoker *Trinectes maculatus* spawns in brackish water and the juveniles move upstream and congregate in a low-salinity nursery area where they remain during the winter. In the spring, they return to the spawning area and in September to the nursery area. This rhythmic movement continues until the fourth year; as the fish mature the migration progressively extends towards higher salinities. Thus the fish revisit the spawning area, seasonally, even when they are immature (Dovel *et al.*, 1969).

### Circadian Rhythms

Circadian endocrine rhythms have been demonstrated in fish. Gonadotropin levels may vary during a 24 h period but this variation seems to depend on season, photoperiod and on the sex of the fish. In cyprinids, there is a peak of pituitary GTH concentration at 16.00 hours which disappears after pinealectomy (De Vlaming and Vodicnik, 1977). In goldfish, plasma c-GTH levels vary according to temperature, photoperiod and sex of the fish. In salmonids, a circadian rhythm in the pituitary content of GTH was shown by O'Connor (1972) but the specificity of the assay used and the reference to FSH are not convincing. Some oscillations in GTH levels in trout plasma throughout a 24 h sampling period are shown in Fig. 10; testosterone levels seem lower during the night but for both hormones any apparent differences are not statistically significant. In an experiment which involved four samplings per day, Schreck *et al.*, (1972, 1973) did not find major circadian variations for androgens and estrogens in males.
<figure><img src="image_12.png" /><figcaption>Fig. 10. Variation in T~4~GTH and testosterone (RIA - ng/ml) during a 24 h period in male rainbow trout (resting stage) experiment carried out in May, (Fostier *et al.*, unpublished).</figcaption></figure>

## Other Rhythms

A seasonal cycle was observed in the seminal vesicles and sperm ducts (Rastogi, 1969). Organs other than gonads show some variations in activity or weight during the annual cycle of reproduction. One of the more obvious changes is in the liver of the female. Usually, the liver weight rises during the resting period and gonadal recruitment, and is minimum at the end of the spawning season. The liver is considered as the main source of lipid for vitellogenesis (Takahashi, 1974). This annual rhythmic variation of liver weight does not always appear to be connected with a specific gonadal event. In a 18 month study of brown trout, a rise of HSI was observed in December during the first year (when trout were still immature) and during ovulation in the second year. Lipid content of the body and viscera also vary with the season and usually show a peak before the onset of vitellogenesis (Rao, 1967; Shikhshabekov, 1974; Lizenko *et al.*, 1975). Usually variation is greater in males than in females (Lapin, 1973).

Seasonal variation of feeding activity (Homans and Vladykov, 1954) and circadian rhythms of food consumption (Stepkina, 1973) have been reported. Thyroid activity is low during the reproductive and breeding season and high in the resting period for *Agonus cataphractus*, (Le Gall, 1969) but for a gobid fish, *Leucopsarion petersi*, Tamura and Honma (1973) found the highest activity in the breeding season. More accurate information has been reported for brook trout: T~3~ decreased during gametogenesis and was minimum during the spawning period (White and Henderson, 1977).

## Summary

Most teleost fish species exhibit an annual rhythm of reproduction which is correlated with some climatic events. In temperate zones, photoperiod and temperature variations are the most obvious environmental factors involved in determining the annual reproductive cycle. In tropical zones, photoperiod and temperature are also involved but additional factors such as the rainy season contribute. Annual rhythms of reproduction are also observed in fish at the equator and the timing agents are then less obvious (temperature, food availability, rainfall, sunshine, etc.).

The reproductive cycle consists of two parts, gametogenesis and spawning; these include ovulation, oviposition, spermination + sperm release and spawning behaviour.

Gametogenesis appears to depend on regular and long term changes (circannual variations of temperature or photoperiod for some species: both temperature and photoperiod, sometimes with interaction, for others). The presence of a circadian, light-sensitive phase has been demonstrated in fish suggesting that induction of gametogenesis by photoperiod change may be mediated through an endogenous daily rhythm. Endocrine studies have shown that relatively low and regularly increasing amounts of GTH are required for gametogenesis indicating a secretion type 'tonic release' compatible with regular change.

Spawning seems to require more specific stimuli, even for fish with continuous reproduction, and appears to be the most critical period of the reproductive cycle. These stimuli are sometimes a thermal shock in temperate zones (cyprinids, pike), and flooding, rainfall and probably many others in tropical and equatorial zones. Endocrine studies indicate that ovulation usually occurs after a GTH surge associated with other hormonal changes, and with variations in gonadal receptivity to gonadotropin. These are probably brought about by climatic stimuli. It is thus likely that spawning is triggered by climatic events. This may be an adaptation to the environment since hatching occurs soon after reproduction (at least for small egg breeders) and since vulnerable larvae
require adequate environmental conditions. However, this 'trigger' concept may be associated with endogenous rhythms; such rhythms exist for ovulation-oviposition in *O. latipes*, for instance. Mammalian species have evolved an endogenous cyclical mechanism for ovulation, perhaps because homeothermy and gestation and suckling processes render the young less sensitive to the immediate environment. Nevertheless, fish should not be regarded as being more primitive in their reproductive strategies than mammals, on the contrary, they have developed modes of reproduction which are adaptive, varied and often original.

### Acknowledgement

Thanks are due to our colleagues, Mrs. Escaffre and Reinaud, Drs. Fostier, Gillet, Jalabert and Weil, who provided us with personal results and criticised the manuscript. Mrs. Daifulu improved the English presentation of the manuscript. Graphs were drawn or redrawn by Miss Briand. Laboratory work was partly supported by grants from the E.E.C., E.D.F. and C.N.R.S.

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Rhythmic Activity
of Fishes

edited by

J.E.Thorpe

Academic Press
London New York San Francisco
A Subsidiary of Harcourt Brace Jovanovich Publishers

<figure><img src="image_13.png" /></figure>
Rhythmic Activity
of Fishes

Edited by

J. E. THORPE

Freshwater Fisheries Laboratory
Department of Agriculture and Fisheries
Faskally, Pitlochry
Perthshire, UK

1978

<figure><img src="image_14.png" /></figure>

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