V.

BIOLOGY OF AMPHIPRION SEBAE

5.1. Introduction

The pomacentrid Amphiprion is comprised of several species of marine reef

fish. It is commonly known as clownfish or anemonefish. It is distributed in tropical

and sub-tropical seas. The anemonefish is omnivorous and it attains a standard length

of about 100 mm, depending on the species. They are popular among marine aquarists

all over the world because they are generally small and hardy. They have attractive

colours. They are highly adaptable to life in captivity and display interesting

behaviour Collingwood (1868), Alava and Gomes (1989). was the first person to note

the association between a giant sea anemone and a beautiful little fish hovering near

by on a reef in the South china sea (Neff et al., 1994).

The clownfish named for its brilliant orange and white colours, is a member of

a family Pomacentridae. Anemonefishes are obligate symbionts with large sea

anemones and are distributed mainly on coral reefs of the Indo-West Pacific Sea

(Allen, 1991; Fautin and Allen, 1992). The family Pomacentridae includes 28 species

of Amphiprion and one species of Premnas. About a dozen different species of

anemones from three different families, Stichodactylidae, Actiniidae and

Thalassinidea are associated with the clownfishes.

The anemonefishes usually live in groups consisting of a monogamous

breeding pair and a few juveniles (Fricke, 1974, Allen, 1975; 1979; Fricke and Fricke,

1977; Moyer and Nakazono, 1978; Ross, 1978 and Hattori, 1991). At least 8 of 28

species of anemonefishes are protandrous hermaphrodites that change sex from male

to female and their juveniles have ambisexual gonads (Fricke and Fricke, 1977;

Moyer and Nakazono, 1978; Fricke, 1979, 1983; Hattori, 1991; Hattori and

45
Yanagisawa, 1991). In coral reefs, coast sea anemones are usually distributed sparsely

(Allen, 1975; Ross, 1978 and Hattori, 1991).

Anemonefishes rarely move between sea anemones (Fricke, 1974; Allen,

1975; Ross, 1978 and Hattori 1991). They usually live in isolated groups (Fricke and

Fricke, 1977; Moyer and Nakazono, 1978 and Fricke, 1979). Under such

circumstances, protandry may function to secure a mate in an isolated group without

immigration of adults (Warner, 1984 and Ross, 1978). Fricke and Fricke (1977)

hypothesized as follows: 1. Due to the limited size of the individual sea anemones, not

more than two adult anemonefishes live together; 2. When a mate disappears, it is

very difficult to replace the vacated position with a fish from other sea anemones; 3.

When a female disappears, the male changes sex to a female and a juvenile soon

becomes a functional male; 4. When a male disappears, a juvenile becomes a

functional male. Thus, new pair formation is always accompanied by sex change or

sex differentiation in an isolated group.

Symbiotic relationship with anemones

Most tropical anemones live in a light rich environment because algae, which

live in tentacles, need sunlight to photosynthesize and to fix carbon, processes that

provide nutrients for the anemone (Neff et al., 1994). The powerful sting of most

anemones, derived from microscopic structures called nematocysts, drives most fishes

away, but the clownfish has an ability to acclimatize to these stings after persistent

contact. The clownfish initiates contact by touching the anemone with its tail, where it

appears to be sensitive to the initial stings, later within few hours; it develops

immunity to the stings. The physiological nature of this immunity and symbiosis has

never been fully understood.

46
 Another important feature of the symbiotic relationship is its beginning at an

early age, in fact, so early that it starts almost before they are born. Clownfish eggs

are laid in close proximity to the anemone, generally beneath the oral disc of the host

and are then tended primarily by the male.

The symbiotic relationship between living clownfish and sea anemone is

classified as mutualism, because both the partners are benefited. Mutual benefit is

evidenced by the clownfish’s fierce territorial defense of the anemones against

anemone eating fishes, such as butterfly fishes, which have been observed to nibble at

the tentacles. Other possible benefits to the anemone include the availability of the

nitrogen from clownfish excretions, as the coral reef represents a nutrient-poor

environment from the stand point of growing algae.

The clownfish may provide valuable fertilizer for the symbiotic algae within

the host anemone. Direct feeding of the anemones by their symbiotic clownfish

occurs often in the aquarium (Neff et al., 1994). It is observed that the captive

clownfish often grabs big chunks of food at feeding time and brings it back to secure

at home of their host anemone. It is felt that this is of great benefit in raising captive

anemones. Although one would think that this also occurs in the wild, it has rarely

been observed.

5.2. Maintenance of the breeding pair

The young ones of the clownfishes were collected from their natural living

place in Gulf of Mannar and reared them upto adult forms in 1 ton capacity FRP tank

i.e., Mother tank (Plate: 5.2.1) by feeding with minced beef, edible oyster, monoceros

and prawn. During their rearing period the growth rate of experimental fishes were

promising but they did not spawn. So, the matured adult pairs were collected along

47
with their anemones for our studies. The paired fishes were identified by their

territorial behavior and transferred to the breeding tank (115 lit.) (Plate: 5.2.2).

Plate. 5.2.1. Mother Aquarium

Plate. 5.2.2. Breeding Tank

48
5.3. Pathological studies and health management

Fishes tend to get injuries and infection occasionally due to fighting and

shock. Water borne diseases also affect fish health in the tank. The affected fishes can

be identified by their sluggish movement and reduced rate of feeding. As soon as an

infected fish is identified, it is removed from the tank and treated in an appropriate

way. The following are some of the common disease, which affect Amphiprion during

the process of captive rearing.

New tank syndrome

A New tank, which has been carefully setup will become unhealthy after a few

days and fish mortality will result, when it is put to use without proper maturation.

The old tanks will have nicely balanced bacterial population with the nitrogen cycle

proceeding satisfactorily and hence no build up of ammonia or nitrites will occur. In

a new tank the first thing to occur is the growth of bacterial population, which

decomposes the organic matter such as fish faeces, uneaten food etc. When sufficient

ammonia accumulates it forms the substrate for growth of Nitrosomonas, which

convert ammonia to nitrites. When nitrite accumulates Nitrobacter is formed which

converts in it to nitrates which are non-toxic unless in very huge quantities

(40-60 ppm).

Wounds or injuries

Injuries are caused by chasing and capturing of fishes in the tank. The colour

of the eyes is changed into milky white due to injury. The affected fish is treated with

the bath treatment of quarantine for 3hrs in the concentration of 2g/l. The recovery of

49
the fish by this quarantine treatment is encouraging. When the wound continues for a

long period in the fish it is better to treat it with antibiotic powder.

Tail-rot disease

Tail-rot disease is caused by the biting of small sized fishes while one chases

away the other from their host anemones. It is noticed that at the beginning of tail-rot

disease, the fin membranes disintegrate and the fin rays are also exposed. Sometimes

blood also oozes out from the affected fins. At the early stage of infection it is better

to cut down the tail edges and then dip the tail region into 1% Silver nitrate solution

for wound healing. It is then followed by the chloromycetin or ciprofloxacin dip

treatment twice a day. This treatment cures the diseased fishes within a period of eight

days.

Wasting disease

In the early stages of this disease, the fish behaves normally and a little later,

the sick fish forsakes its anemone and is found shimmy miserable, often in a corner of

the tank. It also appears to loose interest in taking food. Then the fish will die

suddenly. There will be no obvious signs of wastage or internal parasites in this type

of wasting disease. The affected fish is removed as quickly as possible to a separate

tank to prevent infection of the remaining Amphiprion. The separated fish is given a

bath treatment of quarantine for 3 hrs (2g/2l). The fish will be recovered within 3-4

days.

Pop-eye

Sometimes one or both eyes of the clownfish are affected by pop-eye disease.

If only one eye is affected, the cause is likely to be the external physical damage. In

50
this case the affected eye will be cured by itself within a week, if it is given hygienic

food in healthy environmental conditions. If the infection is on both the eyes, it is

often caused by the intake of raw seafood by the fish. If both the bulged eye is

infected by the intake of raw food, there is a possibility for the occurrence of a life

threatening fungal disease called Ichthyophonus. In this case, the infection is

symptomized by lethargic behavior of fish in addition to the bulged eyes. If only one

eye is bulged, the treatment with chloramphenicol may be effective. But if both the

eyes are bulged due to fungal disease, food soaked in a 1% solution of

phenoxyethanol controls the internal fungus.

5.4. Reproductive physiology of anemonefish

Fishes may exhibit hermaphroditism, unisexuality (parthenogenesis),

bisexuality (gonochorism), or a combination of sexualities.

Hermaphroditism

Hermaphroditism may be genetically programmed or a function of the social

surrounding and is particularly prevalent in tropical reef regions. In synchronous or

simultaneous hermaphroditism, the left gonad is the ovary and the right one is the

testis or vice versa or there may be an ovotestis. Although self-fertilization exists, it is

rare because recessive traits become present such as albinism. Synchronous

hermaphroditism is usually found in species where potential mates are sparsely

dispersed because if the energy costs are the same for asynchronous hermaphrodite as

for a male or female, it is clearly advantageous. In general, these species are

continuously mature reproductively so that a chance encountered is always fruitful.

51
 In asynchronous, consecutive, successive, or sequential hermaphrodites, the

fish start functionally as one sex and then switches to another. In protandric

hermaphrodites, the fish is first male, then female; this is more common in

anemonefishes especially in A. sebae. The theory behind this reproductive method is

that it has evolved under conditions where there is a strong female fecundity

exponential increase because of volume change and asymmetry of age-specific

fecundities between the two sexes. Protandry occurs when there is no advantage to

males accruing larger size and, presumably, the gain is considerably greater than the

cost of the change.

Egg Laying

Actually, internal fertilization is very rare among teleosts. Eggs of the vast

majority of teleost’s, especially marine fishes, are released before fertilization these

are the oviparous fishes (literally, “egg-laying” fishes). Males and females swim close

together so that the eggs are shed into a cloud of spermatozoa. Because mating and

courtship occur in many oviparous species, one important aspect is that the activity of

sperm depends on small concentrations of Ca or Mg ions, which allows sperm to

remain active in salt water for up to an hour or so as opposed to a minute or so in

freshwater. The eggs are then fertilized and develop in the environment outside the

female. An egg membrane is present, and the embryonic stage is nourished entirely by

the yolk.

52
5.5. Induced breeding of A. sebae

5.5.1. Introduction

Breeding is a kind of reproductive process, which increases the population of

an organism. Actually, the aquatic organisms are breed casually in their natural

environmental conditions. But, in captive conditions these aquatic organisms do not

breed successfully due to the lack of or less availability of suitable nutrients,

temperature, salinity, PH, photoperiod and dissolved oxygen etc. Due to the lack of

these elements in captive conditions it may lead to the delayed gonadal development

and the failure of releasing gametes (egg and sperm) of the fish in an appropriate time.

Moreover, the lack of appropriate spawning environmental conditions and the stress

caused by environmental factors are also responsible for the reproductive dysfunction

of the fish in captive condition (Pankhurst and Van Der Kraak, 1997)

Breeding is commonly of two types namely Natural breeding and Artificial

breeding. An animal which breeds in their natural habitat without an involvement of

exogenous inducing agent is called natural breeding. An animal which breed in their

natural environment or in captive conditions with an involvement of exogenous

inducing agent is called artificial breeding. The artificial breeding methods have many

breeding techniques used to breed in different species. Among them, the induced

breeding technique is more popular and profitable in aquaculture. It is also

successfully practiced in food fishes as well as in ornamental fishes.

When the fishes are reared in captive conditions, it shows some degree of

reproductive dysfunction in both male and female fishes. In that case, the females are

seriously affected than the males. The common problem of the female fish in captive

culture is to undergo the failure of the final oocyte maturation (FOM) and ovulation

53
(release of eggs), it often leads to the delayed spawning activities in female fishes. In

males the dysfunction of the reproductive process leads to the reduction of milt

quality and quantity (Zohar, 1989).

Gametogenesis

The reproductive cycle of the fish is divided into two phases. The first phase

constitutes the mitotic multiplication and growth of the gametes, where as the second

phase constitutes the preparation and release of the mature gametes. The first

reproductive phase in the female is referred to as vitellogenesis. The major event of

this phase is the production of the yolk protein precursor vitellogenin and its

accumulation into the growing oocyte in female fish (Specker and Sullivan, 1994).

The second phase of the reproductive cycle in female is referred to as the final oocyte

maturation. This stage constitutes a number of cytological, bio-chemical and nuclear

changes. These all prepare the oocyte for ovulation and fertilization (Jalabert,

et al., 1991).

In males the first phase of the reproductive cycle is referred to as

spermatogenesis, the production of flagellated immobile spermatozoa is called as

spermatogenesis (Callard, 1991). While the second phase of the male reproductive

cycle is referred to as the process of final testicular maturation which constitutes the

acquisition of motility capacity of the spermatozoa. The production of expressible

milt is called spermiation (Nagahama, 1994).

In some fishes the spermatogenesis and spermiation is temporarily separated.

The spermiation occurs after the conclusion of spermatogenesis (Malison et al.,

1994). In some other fishes the two reproductive processes of spermatogenesis and

spermiation can occur simultaneously. Actually the spermatogenesis takes place prior

54
to the spawning season of the fish. But the spermiation takes place during the

spawning season of the fish (Berlinsky et al., 1994).

Endocrine control of final oocyte maturation and spermiation

In vertebrates the reproduction is regulated by the brain. The hypothalamus

releases the Gonadotropin-releasing hormone (GnRH). This GnRH stimulates the

pituitary to release the Gonadotropin (Trudeau and Peter, 1995). All the vertebrates

(except the placental mammals) possess the two forms of GnRH (Sherwood et al.,

1994). The Teleost fish possess three forms of GnRH such as a universal form of

GnRH, cGnRH and sGnRH. Although all the three forms of GnRH are potent, they

stimulate to release the GtH (Zohar et al., 1995). Among them, the sGnRH reaches

the pituitary and stimulates them to release the GtH.

Similarly, it is now recognized that the teleost fishes have two forms of GtH

such as GtH 1, and GtH II (Schulz, 1995). Among them, the GtH II is the prominent

form during the whole reproductive cycle of the fish. The other form of GtH1 is

mostly involved in vitellognesis and spermatogenesis. Moreover, the hormonal

treatments of the fish also grouped into two categories upon where the hormones act.

The first category include the GtH preparation that act on the gonads of the fish. The

second category includes various GnRHa, which act at the level of the pituitary gland

(Mylonas et al., 1998).

The major events of the natural spawning are influenced by the environmental

factors as well as the internal factors of the fish. It differs from species to species that

trigger the hormone cascade of the fish which leads to the egg maturation and

ovulation (egg release) in females and the sperm maturation and spermiation (release

of sperms) in males.

55
A simple description of GNRH which induce the ovulation and spermiation in
fishes

In fishes, the hypothalamus part of the brain releases the gonadotrophin

releasing hormone (GnRH). Then this GnRH travels to the pituitary gland. When it

reaches the pituitary, the GnRH initiates to stimulate the pituitary gland to release the

gonadotrophin hormone (GtH). After that, the gonadotrophin hormone travels to the

ovary and testeis; it stimulates them to produce the steroids and prostaglandins

hormone. Finally, these hormones act directly on the gonads to induce the final

maturation of oocytes and release the eggs (ovulation) in females and release the

sperms or milts in males.

Material used for induced breeding in fishes

In captive condition of some fish species the hormone cascade is naturally

blocked by a compound called dopamine which inhibits the production or release of

egg or sperm under certain environmental and physiological conditions such as stress

and longer photoperiod. Such a critical situation needs to use the appropriate induced

breeding agent for releasing of eggs and sperms in fishes.

Although, the Carp pituitary act as an inducing agent in the Indian and

Chinese carp (Park et al., 1994), many of the commercially cultured fishes are

induced by the LHRHa, that has been successfully in the final oocyte maturation and

synchronized ovulation in fishes (Park et al., 1997). Similarly, the Human chronic

gonadotropin also induced the final oocyte maturation in the fishes (Peter et al.,

1988). Moreover, the different forms of GnRHa (Gonadotropin releasing hormone

agonist) are responsible for stimulating the secretion of endogenous gonadotropin

(Zohar and Mylonas, 2001). In the catfish, Heteropneustes fossilis the commercial

56
product LHRHa which contains dopamine antagonist pimozide has been successfully

used to induce the oocyte maturation and ovulation (Nayak et al., 2001). The ovaprim

and ovatide are a kind of analogue of Salmon Gonadotropin releasing hormone

(sGnRHa) with a dopamine blocker which are used for inducing fishes (Rutaisire and

Booth, 2004; Hill et al., 2005 and Sahoo et al., 2007).

An important inducing agent ovatide is a mixture of synthetic GnRHa analog

and a dopamine antagonist which are used to induce the spawning activity of Striped

Snakehead, Channa striata (Marimuthu et al., 2007). Nevertheless, the Green

Sturgeon, Acipencer medirostris was treated with GnRHa along with the dopamine

antagonist that has induced the ovulation and spermiation of the fish (Van Eenennaam

et al., 2008).

Moreover, the Striped bass, Morone saxatilis was treated with the combination

of LHRHa and HCG which actively induced the final oocyte maturation of the fish.

Nevertheless, the catfish reproductive potentials were measured by using the

homoplastic and heteroplastic pituitary hormone. The experimental results have

revealed that both the hormones are successfully used for inducing the catfish

reproduction (Nwokoye et al., 2008).

The application of GnRHa in fishes is more popular now-a-days. The mature

sea bass was treated with GnRHa loaded microsphere which induced the multiple

spawn in the European sea bass, without a negative egg quality. But in the GnRHa

injected fish was spawn only once in a stipulated period. The proper dose of implants

is necessary to induce a single or multiple spawn in the fish. In some experimental

studies among the GnRHa implants containing 0, 25, 50, 75 and 100 µg dose level the

75 µg GnRHa treated fish was better than the other implants of GnRHa treated fish

57
respectively. So, in these types of GnRHa implants or injections or microsphere

hormone application studies, fishes were helpful to develop an improved or advanced

induced spawning protocol for using the GnRHa in Spotted rose snapper (Lutjanus

guttatus) and other fish species (Ibarra-Castro and Alvarez- Lajonchere, 2009).

The Gonazon is another more effective induced spawning agent which is

commercially available in the market now. The Gonazon does not have the dopamine

antagonist but it contains the GnRH analog azagly - nafarelin. They have been found

to induce the spawning process of EU Salmonids. Now a variety of successfully

inducing agents are commercially available in the market including GnRHa that have

effectively induced about 15 fish species. The dagin has promoted the process of

induced spawning in many fish species. For instance, the Common carp pituitary and

dagin have effectively induced the common reproductive process in fishes (Yaron

et al., 2009).

The induced spawning agent ovaprim has effectively induced the breeding in

the ornamental fish aquaculture industries. It was the first animal drug used as a

spawning agent in the ornamental broodstock. The ovaprim is a two part drug

consisting of a shorter synthetic version of GnRH and a domperidone.

Actually, the ovaprim was used as a spawning aid to induce the ovulation and

spermiation. The ovaprim function is to initiate the reproductive cascade of the fish

and the domperidone is an active compound of ovaprim that helps to block the

inhibitory effects of dopamine. Therefore, the domperidone is essential for induced

spawning of the fish for which the reproductive cascade would be stopped due to the

stress that lead to the release of dopamine in the fish, so that dopamine will block the

GnRH activity in fishes (Roy et al., 2009).

58
 The characteristic of induced spawning was observed through the comparative

study on the hormonal administration of GnRHa. The GnRHa injected groups of

fishes are released well in advance than the implanted group of fishes (Basaran et al.,

2009). The hormone administration through the diet is the most advanced, compatible,

and accepted method for induced breeding in fish.

Basically the brain pituitary gonadal axis controls the reproductive mechanism

of the fish. The brain is stimulated by environmental cues like water temperature,

salinity, rainfall, photoperiod, pH, feeding etc. (Zohar et al., 2010). The ovulation and

the spermiation process are affected by the sex steroids that have been produced in the

fish. In order to control the amount of hormone, the fish has a positive and negative

feedback mechanism for hormone regulation in the fishes (Zohar et al., 2010).

Nevertheless, the hormone induction is actively involved in the sex reversal of

Xiphophorus maculates, Platyfish and Cyprinus carpio, Koi carp. In that experiment

the fish fry was fed with the food along with 17α-methyltestosterone (MTS) for sex

reversal activity of the fish. Whereas, the hatchery breeds fish do not reproduce or

breed spontaneously. The result of the experiment revealed that the 17α-methyl

testosterone was sufficient to induce the sex reversal in both fishes (Kumar and

Haniffa, 2011).

Similar work was also carried out in the fry of Siamese fighting fish,

(Betta splendens) which was treated with the oral hormonal administration through

the diet. The diet has the various concentration of 17α-methyltestosterone. This

17α-Methyltestosterone effectively induced the sex reversal of Siamese fighting fish

fry (Kipouros et al., 2011). The ovaprim, carp pituitary and human chronic

gonadotropin is an inducing aid that are treated with the wild carp and they produced

59
a good quality of milt than the ovaprim and carp pituitary treated fish. Hence, the

HCG was found to act as an inducing agent better than the ovaprim and carp pituitary

(Seifi et al., 2011).

The carp family species of Caspian Kutum, Rutilus frisii effectively induced

the maturation and ovulation with the spawning aid LHRH-A2 and chlorpromazine.

At the same time, the same type of fish were treated with LHRH-A2 alone, the less

significant results were obtained than the LHRH A2 and chlorpromazine treated fish

(Ahmadnezhad et al., 2012). Moreover, the GnRHa was used to produce the sex

steroid hormones in river catfish. Similarly the testosterone, 11-Ketotestosterone, 17β-

estradiol were found to act as a sex steroid hormone which induced the reproductive

activity of the river catfish (Adebiyi et al., 2013).

Before conducting the induced breeding experiments, the dose effect of

gonadotropin releasing hormone analogue (GnRH-A) on ovulation by GnRH-a

injection in A. sebae was studied. During this experiment, the sexually matured

females (each 3 pairs) were injected with 25, 50 and 75 µg of GnRH-A/kg body

weight. As a result, all the GnRH-a treated test fishes were ovulated within 12 days,

where as no ovulatory response occurred in control fishes. The optimum dose for

better results (such as fecundity rate, hatching rate and less spawning interval days)

were observed in the fish treated with 50 µg of GnRH-A/kg body weight. That is why

we have used the dose of 50 µg GnRHa/Kg body weight in our induced breeding

studies.

In the present study the male and female A. sebae were treated with 50 µg

GnRH loaded polyanhydride microsphere. Based on the final results of this

experiment it was found that the male and female fish A. sebae expressed their full or

60
maximum reproductive potentials when treated with 50 µg GnRHa loaded

polyanhydride microsphere.

5.5.2. Materials and Method

A. sebae were collected from Gulf of Mannar. They were fed with mussels,

polychaete worms, edible oysters and clams. They were acclimatized to laboratory

conditions (pH 7.8-8.2, salinity 32-38 ppt, and temperature 27-32oC, light duration

12 h/day) for a period of 60 days.

Microsphere preparation: To prepare GnRHa containing microspheres, 22.6 µg of

GnRHa (82.3% active ingredient) were dissolved in 100 µl of distilled water

containing 25 µg of gelatin. This solution was kept at 65oC with an addition of 1ml of

methylene chloride containing 452.4 µg of p (FAD-SA) of molecular weight 43 kDa.

The mixture was agitated and the coarse emulsion was cooled on ice. It was slowly

allowed to reach room temperature, after evaporation of the MeCl2, the microspheres

were filtered through a 250 µg sieve in order to remove any amorphous material.

The microspheres were then weighed and recovery was estimated by the ratio

of the weight of collected microspheres to the combined weight of the polymer,

gelatin and GnRHa used. The microspheres were stored at -20oC under nitrogen

atmosphere (Mylonas et al., 1995).

The amount of GnRHa entrapped in the microspheres was estimated indirectly

by measuring the amount of GnRHa in all solutions used for microsphere preparation

and rinsing of the microspheres. It was found that 1.6 mg microsphere/kg of body

weight containing 50 µg GnRHa/kg of body weight. The shape and size of the

microspheres were evaluated using a compound microscope at 40X magnification.

61
Ovulation and spermiation: Studies on in vitro release and degradation and in vivo

GnRHa release were carried out by the techniques followed by Mylonas et al. (1995).

In order to evaluate the efficiency of the GnRHa loaded p (FAD – SA) microspheres

in inducing ovulation and spermiation, the experiments with captive reared

anemonefish, A. sebae were designed according to the methods suggested by Mylonas

et al. (1995).

In the first experiment six sexually matured females (15±1g) were

injected with GnRHa loaded microspheres at a dose of 1.6 mg/kg body weight (50 µg

GnRHa/kg of body weight). As a control, a group of six females were injected with

microsphere vehicle only. In order to assess the progress of ovulation at various times

(multiples of five days) after treatment the ovarian biopsy were collected from test

fishes. These test fishes were reared in rectangular glass tanks (150 l each) connected

with flow through water system under ambient temperature ranging from 25-30oC.

In the second experiment, six sexually matured males (15±1 g) were injected

with GnRHa loaded microspheres at a dose of 1.6 mg microspheres/kg body weight

(50 µg GnRHa/kg of body weight). The other group of six fishes were injected with

microsphere vehicle and this group served as the control. At every two days after

treatment, the total amount of sperm was collected from all tested males by stripping.

In the third experiment, six sexually matured females (15±1 g) and six

spermiating males (15±1 g) were maintained in aquarium tanks (150 l). The test fishes

were treated with 1.6 mg microspheres /kg body weight (50 µg GnRHa/kg body

weight). Six pairs of control fishes were also maintained for comparison. The females

were tested for ovulation for every three days after treatment. The sperm was

collected from test fishes once in a week after post-treatment by stripping.

62
5.5.3. Results

Matured female fish treated for a period of 35 days with GnRHa-loaded p

(FAD-SA) microspheres induced ovulation in 65% of the tested sexes and when the

treatment was prolonged to 50 days, 80% of the experimental fishes ovulated

(Fig.5.5.1). The ovulation started 15 days after hormonal treatment, 50% of ovulation

was achieved after 30 days of treatment and 80% ovulation was obtained after 50 days

of treatment. Total failure of ovulation in non-treated fish underlines the fact that in

captivity, the ovaries in the females do not undergo the maturation process of the

ovary (Table.5.5.1).

In the second group of experiment, the GnRHa loaded p (FAD-SA)

microspheres were effective in enhancing sperm production (Fig.5.5.2). The mean

total sperm volume of GnRHa microsphere treated fish was higher than control at

every sample time within the duration of two weeks of experimental period. Two days

after treatment, sperm production was doubled when compared to sperm in control,

but in males treated for 14 days, the sperm production was 4 times higher than that of

the control (Table.5.5.2). This increase in sperm volume was the clear indication of

higher sperm cell production.

In the third experiment, all the females and males treated with GnRHa loaded

microspheres ovulated and spermiated within fourteen days of treatment (Fig.5.5.3).

This higher enhancement of ovulation and spermiation with in a shorter period of

treatment may be attributed to the injection of GnRHa-loaded microspheres (1.6 mg

microspheres having 50 µg GnRHa /kg of body weight). The first fish spawned after 6

days of treatment and the cent percent ovulation was obtained within 21 days.

63
 At the same time, in the control fishes the ovulation commenced only after

15th day (Table.5.5.3). After two days of injection, the sperm production increased

three times more than control. After 21 days, microsphere treated fishes produced five

times more sperm than control (Table.5.5.4). The presence of both male and female

fish in one anemone may be another favourable factor for the higher inducement of

ovulation and spermiation in all the test fishes.

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Table.5.5.1. Cumulative percentage of ovulation in A. sebae treated with GnRHa
loaded p (FAD-SA) microspheres (50 µg GnRHa/1.6 mg
microsphere)

Cumulative % of ovulation
Days
Treated Control

15 10 0

20 25 0

25 40 0

30 50 0

35 65 0

40 70 0

45 75 0

50 80 0
CUMULATIVE OVULATION (%)

TIME AFTER TREATMENT (DAY)

Fig.5.5.1. Cumulative percentage of ovulation in six A. sebae. Broodstock versus

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Table.5.5.2. Variations in sperm volume (m1/g) in relation to treatment period in
GnRHa microsphere treated A. sebae males

Cumulative % of spermiation
Days
Treated Control

0 0.2 0.1

2 0.2 0.1

4 0.35 0.15

6 0.42 0.15

8 0.56 0.20

10 0.64 0.25

12 0.76 0.20

14 0.8 0.20

Fig.5.5.2. Mean total sperm from six A. sebae. Broodstock in response to treated
with GnRHa-loaded p (FAD-SA) microspheres (50 µg GnRHa/1.6 mg
microsphere)

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Table.5.5.3. Variations in cumulative percentage of ovulation in relation to
treatment period in matured A. sebae in aquarium tanks having
both male and female

Cumulative % of ovulation
Days
Treated Control

6 10 0

9 30 0

12 50 0

15 70 20

18 80 20

21 100 20

Fig.5.5.3. Cumulative percentage of ovulation in GnRHa microsphere treated

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Table.5.5.4. Variations in sperm volume (ml/kg) in relation to treatment period
in GnRHa microsphere treated A. sebae males reared in cement
tanks having both male and female in one anemone

Cumulative % of spermiation
Days
Treated Control

7 0.6 0.2

14 0.8 0.2

21 1.0 0.2

Fig.5.5.4. Mean total sperm from GnRHa microsphere treated six male A. sebae
reared in cement tanks having both C and T fish in one anemone.

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5.5.4. Discussion

The above observations corroborate the results obtained in the studies made by

Mylonas et al. (1995) in Striped bass and Atlantic salmon. In the third experiment,

treatment of both male and female fish with GnRHa-loaded microspheres induced

spermiation and ovulation in all the test fish within 21 days (Table.5.5.4). Captive-

reared marine fishes can undergo final oocyte maturation and spermatid formation

leading to optimum ovulation and spermiation without any hormonal manipulation

(Zohar, 1989). Unfortunately, females do not spawn in captivity and the eggs have to

be stripped manually and fertilized artificially (Mylonas et al., 1995). Moreover,

ovulation in many of the marine fishes is asynchronous within broodstocks and it may

prolong from 1 to 3 months for all the fish to ovulate.

During this period, the test fish has to be verified for ovulation frequently

(2 to 3 times a week). This may cause handling stress leading to very high mortalities.

GnRHa has been successfully used in the past to synchronize ovulation in various

cultured Salmonids (Zohar, 1989). A GnRHa delivery system can alleviate the need

for repetitive handling then by preventing mortality due to handling stress,

furthermore, due to the long-acting properties of p (FAD-SA) microspheres, a single

treatment can successfully induce ovulation up to 6 weeks prior to the natural onset of

ovulation (Mylonas et al., 1995).

The sperm availability is a general factor for consideration in aquaculture, and

in some situations it may be a limiting factor (Billard, 1989). In hatcheries where the

eggs are manually stripped from the females and fertilization is achieved in vitro with

an excess of sperm there is often shortage of sperms. Hence, relatively large amounts

of sperms are collected from a selected group of males every time a female is found to

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have ovulated. Sperm collection may occur many times over the course of a week,

and often over the course of a day, and as a result the males can run out of sperm

(Fig.5.5.4). Hormonal manipulations can be used to increase sperm production but the

effects are short-lives, or require multiple treatments ranging from once weekly to

once daily (Weil and Crim 1983; Saad and Billard, 1987 and Garcia, 1993). Hence,

the GnRHa delivery system can be better used to increase sperm production many

times and also for a prolonged period of time.

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