5.0 INDUCED BREEDING OF ORNAMENTAL FISH USING VARIOUS INDUCING AGENTS

5.1 INTRODUCTION

Fish seed is a veiy important component for fish culture. The success of fish

culture depends mainly on the year round supply of quality fish seed. In nature, the

maturation of gametes and associated spawning in fish are largely influenced by variation

in environmental factors such as light, water temperature, water level of the habitat, pH,

salt concentration etc (De Vlaming 1972; Billard and Breton, 1979; Grim, et al., 1981;

Santhanam, 1990 & Ram, et al., 2001). To increase seed production considerable

attempts have been made employing different ways and means by several workers

(Alikunhi, et al., 1963, and 1965; Chaudhury, et ah, 1966). So far in the regulation of

reproduction and induced breeding many products are utilized. An excellent technique

ensure to get pure and required fish seed is elan vital (Anon, 1995; Piska, 1998). The

choice of natural hormones for this purpose depends on many criteria including species,

cost, availability and training. Techniques developed for particular species need

continuous evaluation on its efficiency and cost effectiveness of the method (Lee, et ah,

1986). The most commonly used method for stimulation of maturation in fish is the

administration of fish pituitary extract (Okoniewski & Klodzinska 1975; Kozlowski,

1994; Donaldson and Hunter 1983). However this simple method is not always

successful. In many cases, much better results in controlled reproduction are obtained

when pure or synthetic hormones are used for reproductive stimulation (Takashima, et

al, 1984; Grim, et al, 1986; Glubokov, et al, 1991; Drori, 1994; Yaron, 1995). On the

other hand extract of carp pituitary combined with other honnones, mainly with hcG, has

proved successful (Ahmad, et al., 1986; Thalathiah, et ah, 1988 and Kozlowski 1994).

The commonly adopted technique for induced spawning of carp is hypophysation

i.e., by injecting the mature carps with carp pituitary (Hypophysis) gland extract. Induced

spawning in fish employing hypophysation as a tool was first realized and developed by

the Brazilians (Von Ihering, 1937). The hypophysation involving exogenous

administration of the pituitary extract has been the main inducing practice used in quality

50

seed production programme of different fishes before and even after the availability of

ovaprim on commercial level (Ram, et al., 2001)

Induction of oocyte maturation and /or ovulation has been reported in gold fish

(Lam, et al., 1975); Plaice Pleuronecter mastessa and goby Acanthogobus flavimanus

(Aida, et al., 1978); Kyn Plecoglossus altivetes (Hirose and Shida 1974); rainbow trout

Salmo gairdneri (Crim, et al., 1983) and Coho salmon Onchorhynchiis kisntchi

(Donaldson, et al., 1981; Van den kraak, et al., 1983). On the other hand in gold fish

single or multiple injections of LHRH or its analogues at different doses and regimes

could not induce a very high rate of ovulation (Chang et al., 1984; Sokolowska, et al.,

1984) and Afiican cat fish (De Leeuw, et al., 1985).Similar result was also found in an

expeiiment using hcG for induced spawning of Tai local caip and cat fish

(Nagamvonchon 1981).

Fish pituitary extract have long been applied to induce spawning in teleosts

(Clemens and Sneed, 1962). Tang (1960) stated that treatment with fish pituitaries in

combination with human chorionic gonadotropin increases the effectiveness and better

success was achieved. Reproduction must be induced by pituitary injection (Ihering and

Azevedo, 1934, 1936: Fontenele, 1953,1959; Solano, 1973 and Da silva, et al., 1977).

Houssay (1931) of Argentina injected pituitary gland of some small sized viviparous fish

and brought about premature birth of the young ones. Von Ihering (1937) developed

proper technique for induced ovulation using fish pituitary. Experiments on induced

spawning using pituitary injections were performed in early 1950's in China on

Molopharyngodon piceus, Aristichthys nobilis, Hypophthalmicthys molitrix and

Ctenopharyngodon idella. They obtained better success gradually. In India, pituitary

hormones were successfully administered to C. catla, L. rohita, C. mrigala and spawning

were observed (Choudhuri and Alikunhil957). Recently, experiments were successfiilly

performed on various fish species by Yusuf Kama(1987); Rao, et al., (1989); Rao and

Janakiram,(1991); Duff Munshi,(1991); Goswami, (1997); Mohapatra (2000) in

C. batrachiis, Sheel and Singh(1981); Kohil and Goswami (1987) in H. fossilis and

Kurub, et al., (1993) in L. dussumeri. But this technique suffers from disadvantages

51

(i.e.) difficult to evaluate the gonadotropic potency of the pituitary gland used and

standardize its activity.

The administration of ovaprim was based on Linpe method (Peter, et al., 1988).

Ovaprim has been successfully tested on Thai carp Puntius gonionotus in Thailand

(Leelapatra 1988), in the Indian major carps (Nandeesha, et al., 1990), in the Channel cat

fish Jctalums punctatus at USA (Goudie, et al., 1992) in Walking cat fish C. batrachus of

Malaysia (Cheah and Yeo 1994) and Sand whiting Shillago ciliate in Australia

(Battaglene 1966) and C. catla in India (Srinivas, et al, 1999).

The administration of ovatide has been successfully tested in C. catla; L. rohita;

C. mrigala; C.idella; L. calbasu; P.javanicus; T.musullah; T.khudree by Thakur and

Reddy (1997); O.patio by Mukherjee and Das (2001); C.carpio by Sarma, et al, (2000)

and C.catla by Pandey, et al, (2000).

"Homeopathic preparation of Natrum muriaticum is seen as canying information

in to the body when it is taken in dose form, perhaps as biological instmctions"

(^^ww.iyghtfore.com/homeolist/00000526.htm). Generally constitutional medicines are

rarely indicated during labor, since the process of childbirth creates stresses, which

require the use of medicine for acute symptoms. Vishakan (2002) successfully spawned

the black molly and gold fish using Natrum muriaticum.

In the present study an attempt was made to induce the Puntius conchonicus &

Poecilia sphenops using various inducing agents (Pituitary extract, ovaprim, ovatide,

Natrum muriaticum & herbal preparation of Pedalium murex and Mucuna pniriens) and

evaluated the efficacy. Very limited information is available on the induced breeding of

fishes using various inducing agents and their relative perfonnance ability. So assessment

and comparative efficiency of various hormones on induced breeding of chosen

ornamental fishes is highly essential.

52

5.2 MATERIALS AND METHODS

Sexually matured ornamental fish Rosy barb (Puntiiis conchonicus -egg la)dng)

and Black molly {Poecilia sphenops - live bearing) of identical size (Length 5.5 ±

0.187cm; Weight 5.6 ± 0.225 for Rosy barb; Length 7.7 ± 0.245cm; Weight 8.8 ± 0.837g

for Black molly) were procured from the local aquarist and maintained acclimated to the

laboratory for 15 days. Filtered dechlorinated tap water was used . The experimental

tanks were cleaned and the water was replenished every alternate day. Continuous

aeration were provided and the fish were fed ad libitum with chopped firesh water mussel.

The unconsumed feed remains and faeces were removed after 1 hr of feeding to avoid

contamination of water. Uniform sized fishes with identical maturity condition were

used for the experiments.

5.2.1 Preparation of inducing agents

5.2.2 Alcoholic preparation of herbal extract

2 gm dry powder was taken in clean test tube (1:1 ratio of ethanol and dis. H2O)

was added and mixed well atid kept overnight at room temperature, during this period

also it was shaken 3 times at different intervals to maximize the leaching, following day

the mixture was centrifuged at 3500 rpm for 20 min. The supernatant was'collected, made

to constant volume (2ml) and stored in a separate clean containers. Each experimental

female fish was injected 30 \i\ I 8-9 g. size groups of black molly and 30 |J,1 / 5-6 g. size

group of Rosy barb. The preparation was injected intramuscularly below the dorsal fin

by a tuberculin syringe.

5.2.3 Pituitary gland extract

Pitutary gland was dissected out from the mature female Common carp, Cyprinus

carpio and was preserved in acetone. During the experimental work, the acetone

preserved pituitary gland was homogenized in 1ml of 0.9% Na.Cl solution and the

supernatant was injected at 20 p,l A5-6 g. in^o rosy barb and 20 |.il / 8-9 g. black molly

(Detei-mination of optimum dose as was stated in chapter III - Table 7-10).

53

5.2.4 Ovaprim

This is a combination of synthetic salmon GnRHa and dopamine inhibitor

domperidone (Glaxo company, Canada). This commercially available hormone ovaprim

was injected at 10 |J, 1 / 8-9 g. to black molly and at 10 |il / 5-6 g. to rosy barb females

(Determination of optimum dose as was stated in chapter III).

5.2.5 Ovatide

This also a combination of synthetic salmon GnRHa and Dopamine inhibitor

domperidone (Hemmo Phanna, Mumbai).. This ovatide was injected at lOfxl / 8-9 g. in to

black molly and at 10 JJ. 1 / 5-6 g. in to rosy barb (Detennination of optimum dose as was

stated in chapter III).

5.2.6 Natrum muriaticum

The homeopathic preparation, Natrum muriaticum one lakh potency, was

procured from a local homeopathy medical shop. Tliey were kept at room temperature

(28 ± 2 °C). The preparation was diluted in distilled water in the ratio of 0.5 ml: 100ml

and injected at the rate of 10|J.l / 8-9 g. to black molly fish at 10}i. / 5-6 g. to rosy barb.

5.2.7 Control Animal

With identical maturity and size a control group for each tj pe of fish were also

maintained. The female were sam injected with vehicle (1:1 ratio ethanol and dis. H2O)

of similar quantity.

5.2.8 Method of Administration

The selected inducing agents were administered intr^amuscularly by injecting the

predetermined dose in the dorsal musculature between the lateral line and the dorsal fin.

Tuberculin syringe was used for this induced breeding injection purpose. The needle was

inserted under a scale, and aimed towards anterior direction at an angle of about 30 °.

A finger tip is often pressed onto the skin just anterior to the point of insertion

when the needle is withdrawn (Plate 5a, 5b). During the injection time, fish eyes were

54

I\^ I \^ \J

Method of administration

Intramuscular injection

covered with a wet cloth, to avoid the struggling of fish and to minimize the stress. All

the set of treated fish were maintained separately in identical environmental conditions

in the laboratory. After administering the prescribed dose to the selected group of fancy

fishes, they were continuously monitored to obtain the result.

5.2.9 Statistical analysis

The effect of different inducing agents such as pituitary extract, ovaprim, ovatide,

Natrum muriaticum, herbal extract (Pedalium murex, Mucuna pniriens) and their

inducing ability through the latency period, number of eggs and young ones produced

was evaluated. One way ANOVA was employed to find out the differences among means

and multiple comparison among means were made using Duncan multiple range test

(Zarl984).

5.3 RESULT

In order to understand the possibility for reproductive modulations, various

inducing agents like pituitary extract, ovaprim, ovatide, Natrum muriaticum and herbal

extract ( Pedalium murex and Mucuna pruriens) were used on the two familiar fancy

fishes (Poecilia sphenops and Puntius conchonicus) for reproductive performance.

Among these Pedalium murex and Mucuna pruriens were found to be more effective

than other and therefore used for fiirther studies (Table 17, 18)

5.3.1 Rosy barb

The observed result on the effect of herbal extract {Pedalium murex and Mucima

pruriens) and other inducing agents are depicted in the Table (17). During the

experimental duration of 15 days for rosy barb, the control group has not spawned but the

treated {Pedalium murex and Mucuna pruriens) fish spawned within 20 and 24 hr

respectively. In the treated group the egg production was found to be higher than the

control The Pedalium murex treated fish laid 280 ± 15.811 eggs in 20 ± 1.414 hr and

Mucuna pniriens treated fish laid 254 ± 15.811 eggs in 24 ± 1.225 hr. The other inducing

agents ovaprim induced rosy barb laid only 114.2 ± 3.701 eggs in 24 ± 3.162 hrs and

ovatide treated fish laid 113.6 ± 4.159 eggs in 24 ± 2.739 hr. Pituitaiy extract induced

55

rosy barb laid 109 ±2.915 eggs in 26 ± 2.915 hr and Natrum muriaticum treated fish laid

in 116 ± 2.645 eggs in 25.4 ± 1.817 hr. The control rosy barb spawned 65 ± >15 between

20 - 30 days. Here in these experiments gestation time to spawn was low and egg

production was high in Pedalium murex and Muciina pruriens injected fishes than the

other inducing agents.

The spawned eggs of rosy barb were settled to the bottom. The fertilized egg were

round and transparent while unfertilized eggs were too round but opaque. The zygote

undei-went cleavage at room temperature and the larvae hatched out after 48 hr. The

lai-vae started feeding only after a day or two (i.e. after opening of the mouth) the larvae

feed with infusoria as starter feed and egg yolk prepared feed (strained through a cloth)

subsequently.

Significant difference (P<0.05) in the latency period and number of eggs spawned

by rosy barb was found between Pedalium murex and Miicima pruriens and other

inducing agents (Table 19 &20).

5.3.2 Black nioUy

The effect of herbal extract Pedalium murex and Mucuna pruriens over other

inducing agents are depicted in the Table (18). During the experimental duration (15

days) for black molly, the control hasn't spawned but the Pedalium murex and Mucuna

pruriens treated fishes spawned in 42& 48 hr, respectively. In the extract treated group

the larval production was found to be higher than the control. The Pedalium murex and

Mucuna pruriens treated fish given birth of 54 ± 2.449 young ones in 42 ± 1.414 hr and

51.4 ± 2.608 young ones in 48 ± 1.414hr, respectively. The other inducing agents

ovaprim induced black molly given birth of 27.6 ± 1.673 young ones in 23.6 ± 0.894 hr

and ovatide treated fish given birth of 23.4 ± 1.140 young ones in 22.8 ± 0.837 hr.

Pituitary extract induced black molly given birth of 36.4 ± 1.673 young ones in 30.4 ±

1.673 hr and the Natrum muriaticum treated fishes delivered 45.6 ± 3.286 young ones in

23 ± 0.707 hr. The control fish delivered 20 ± 4.147 young ones between 30 - 40 days.

Here also the gestation time was low and the number of young ones production was

56

high in PedaUiim murex and Mucuna pruriens than the other inducing agents. The black

molly young ones were delivered mostly in batches in all its delivery attempts

obser\'ed. The whole or batch - lajdng process extended up to 30 minutes. The freshly

bom young ones bear yolk and they settle to the bottom for a while and after one or two

minutes, started swimming. Their yolk were absorbed in 36-48 hr and the larvae start

feeding after 48hr.

Significant difference (P<0.05) in the latency period and number of young ones

spawned by Black molly was found between Pedalium murex and Muciina pruriens as

well as other inducing agents (Table 21,22).

5.4 DISCUSSION

The result of the experiments presented in this chapter elucidate the role of

alcoholic preparation of herbal extracts in the regulation of ovulation and breeding in

Rosy barb and Black molly. Present findings demonstrated that alcoholic preparation of

Pedalium murex and Mucuna pruriens are the highly effective stimulants for ovulation

in the Rosy barb and Black molly . Similar reports are also available for the homeopathic

preparation of Natrum muriaticum in Gold fish and Black molly (Vishakan 2002).

Mating and spawning are very important events in fish breeding . It has been reported in

several species viz., Anarhichus lupas, (Johannesen, et ah, 1993); Tliymallus thymallus,

(Darchambeam and Poncin, 1997); Macrozoarces americanus (Yau and Crim 1995).

These events also vary from species to species.

Oocyte maturation and ovulation were induced by many factors such as

environmental parameters (photoperiod, temperature, water level), feed etc. The

administration of homione play key role in maturation of gonads. Administration of

hormones assumes a major role in the maturation and spawning in teleosts (Tan Fennin,

1992; Drori, et al., 1994 and Bhattacharya, 1999). Various ov'ulatoiy agents are used for

inducing maturation and spawning in a variety of teleosts with considerable success

(Marimuthu, 2002 and Vishakan, 2002).

57

In the present study natural, homeopathic and synthetic hormones (pituitary

extract, natnim muriaticum, ovaprim, ovatide) were used to asses their impact on latency

period and number of eggs spawned. Pituitary extract tested black molly and rosy barb

fish, exerts the latency period of 30.4 ± 1.073 hr. and 26 ± 2.915 hr. respectively and

their production was 36.4 ± 1.673 young ones 109 ± 2.915 eggs respectively. Similarly

many reports on the administration of pituitary extract for induced breeding of fishes

were available but the latency period vary with species. Banerji (1974) reported 6.25 hr

latency period in channa piinctatus, Kohil and Goswami (1987) mentioned 22-25 hr in

H.fossilis. Inyang, et ah, (1994) stated 11-16 hr in Clarias fariepinus and Clarias

anguillaris and Haniffa, et al, (2000) observed 23-24 hr in C.striatus.

Despite the involvement of numerable variables, lack of standardized

gonadotropin preparations and variation in effectiveness of heterologous gonadotiopin

preparation , pituitary extract remain most commonly used preparation for induced

spawning in several cultured fish species (Donaldson and Hunter, 1983; Weil, et al.,

1986; Peter, et al., 1988). The gonadotropin present in the pituitary extract directly acts

on the gonads of recipient fish and induces final maturational events followed by

ovulation and spermiation exactly in accordance to its level. Normally female recipients

are injected in two split doses of pituitary extract. The first on priming injection of

gonadotropin stimulates the onset of inducing steroids. The second injection the resolving

dose induce ovulation mediated through the action of maturation inducing steroids

(Levavi-zermonsky and Yaron 1986). The induction of spawning through the LINPE

technique use of GnRH and dopamine receptor antagonist pimozide or domperidone,

stimulates the noimal ovulatory surge of gonadotropin (Lin, et al., 1987). Ram, et al,

(2001) showed pituitary extract injected fish only gro^vn yolk oocytes to the advanced

stage of Graffian vesicle breakdown (GVBD), and follicular separation after 4 hr.

Deharaj (1984) stated that the determination of dosage is arbitrary due to the

unknown potency of pituitary glands therefore and in case of ovaprim this problem is

over come. Peter, et al, (1987) stated that the potency of ovaprim is uniform, it contains

salmon gonadotropin releasing hormone (GnRHa), which is loiown to be 17 times more

58

potent than LH-RH. Ovaprim is the combination of sGnRHa and domperidone, the

antagonist. Francis, et al, (1991) reported that the administration of GnRH together

with domperidone has been successful in spawning as alternative to pituitary. Piska

(1998) reported that ovatide is an indigenous, cost effective and new hormonal

formulation for induced breeding of fishes, which is an alternative for ovaprim.

Ovaprim is the commercial name of hormonal preparation with 20 ^g of sGnRHa

and 10 mg of domperidone in one 1 ml of solution . It was used in this study for induced

breeding and seed production of commercially important ornamental fishes. Ovaprim

were tested in both black molly and rosy barb at 30|j.l/fish (i.e 30|il/7-8gm body weight

of Black molly and 30 ^1 /5-6 gm body weight of rosy barb), latency period of 32.6 ±

0.894 for black molly and 24 ± 3.162 hr for rosy barb recorded. Cheah and Lee (2000)

reported that ovulation in Neosilunis ater achieved only when ovaprim was administered

and the latency period was 17-23hr. Different synthetic GnRHa in combination with a

dopamine antagonist have been shown to induce spawning of C.gariepinus (De leeuw, et

al., 1985) and H.fossolis (Alok, et ah, 1993). The average longer latency period of

C.punctatus was 24.50 ± 0.5 hr. The latency period was longer than those of ovaprim

treated C. batrachus 12-15 hr (Cheah and Yeo, 1994), Ovaprim used in C. striatus 24-

36hr, H.fossilis (10-14.5hr), (Vijayakumar, et al., 1998), O.bimaculatus 5-6 hr, (Sridhar,

et al., 1998) and carp 10-12hr (Srinivas, et al., 1999). In contrast, the latency period of

ovaprim administered Channel cat fish was 92 ± 39 hr (Goudie et al., 1992).

Ovatide is highly active and ready to use injectable solution containing a synthetic

pepetide which is an analogue of gonadotropin releasing honnone (GnRH) with a

dopamine antagonist, ovatide treated black molly and rosy barb showed reduced latency

duration to (22-24hr). Tliakur and Reddy (1997) reported in ovatide injected

Ctenopharyngodon idella the latency period was 10.5 hrs. Reddy and Mathur(2000)

observed the latency period ranged fi-om 6.3 to 9 hr in Catla.Rohii and Mrigal fishes.

The experimental preparation of Natrum muriaticum is also a natural material.

There appears to be no experimental information regarding the role of Natrum

muriaticum in ovulation of teleosts. Natrum muriaticum is a highly effective stimulus for

59

ovulation in Black molly and Rosy barb, thus bring down the latency period to 23 ±

0.707 and 25.4 ± 1.817hr respectively Mitra and Raizada (1986) have attempted

homeopathic preparation on major carp for breeding purpose and obtained a favourable

result. Then Vishakan (2002) tested the same on gold fish and Black molly and brought

out good result.

The alcoholic preparation of herbal extract {Pedalium murex and Miicuna

pruriens) treated in some ornamental fishes black molly and rosy barb fish, bring down

the latency period to 42 ± 1.414hr; 48 ± 1.414 hr respectively and 20 ± 1.414hr, 24 ±

1.225 hr respectively. Ageel et al., (1994) have also reported that ethanolic extract of

Orchis maculate bark was found to produce a significant increase in penile erection

index, increased intromission and ejaculation firequencies, indication of enhanced sexual

arousal in male rats. Ananthakumar et al, (1994) noted that the total alkaloids firom the

seed of Mucuna pruriens were found to bring about a noteworthy increase in the

spermatozoa counts and in the weights of testis.

The observation of the experiment, regarding number of egg spawned and latency

period, confirmed that the alcoholic preparation of herbal extract {Pedalium murex and

Mucuna pruriens) was found to be the most potent ovulating agent for induced breeding

in fishes. The administration of single dose of Pedalium murex or Mucuna pruriens

extract reduces the stress in the brood fish considerably. The egg and spawn obtained are

comparatively larger in size and healthy. These herbal products are easily available,

alcoholic preparation of herbal extract techniques also simple and above all has a high

probability of spawning success.

Albeit various agents have been used successfully to induce ovulation in fishes.

Human chorionic gonadotropin and other synthesized hormones are expensive and are

not easily available to rural farmers. To overcome these problems and to encourage the

fish farmers in their breeding programmes, the use of herbal extract from specimens

which are easily available and economically viable is very much advocated. Herbal

extract is very cheap and easily obtainable in and around their locality. In the present

60

study on herbal extract {Pedaliiim miirex and Muciina pruriens) to Black molly and Rosy

barb have been recorded better perfonnance and effective than other inducing agents.

This study further reveal that different fertility agents impart reproductive ability

at different level in different species of fishes. That is any one fertility compound may not

impart uniform impact in all fish species. Therefore it is dire necessary to evaluate and

recognize species specific fertility agents and optimum dose to achieve success in farmed

fishes.

61

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Table 19. ANOVA (one-way) table showing the effect of Pedalium murex and Mucuna pruriens and other inducing agents on the latency period of Puntius conchonicus*. The means were compared using Duncan multiple range rest

Source of variance

SS DF MS F-value Sig

Between 109.500 5 21.900 4.006 0.01

Treatment

Within

Treatment 131.200 24 5.467

Total 240.700 29

** P < 0.05 at 5% level of significant Duncan**^

Treatment N Subset of alpha =05

Treatment N 1 2

PM 5 20.00 24.00

OVA 5 24.00

OVT 5 24.00

MP 5 25.40

NM 5 26.00

PE 5 .238

Sig 1.000

A, uses Inducing agents Mean sample size=5.000(Pm- Pedalium murex; OVA-Ovaprim; OVT-Ovatide;MP-Mucuna pruriens;NM-Natrum muriaticum; PE-Pituitary extract)

Table 20. ANOVA (one-way) table showing the effect of Pedalium murex and Mucuna pruriens and other inducing agents on the egg spawned by Puntius conchonicus*. The means were compared using Duncan multiple range rest

Source of SS DF MS F-value Sig variance

Between 1303372.8 5 26074.506 16.388** 0.000

Treatment

Within 38186.00 24 1571.083

Treatment

Total 240.700 29

** P < 0.01 at 1% level of significant Duncan**^

Treatment N Subset of alpha =05

Treatment N 1 2 3

PE 5 109.00

OVT 5 113.60

OVA 5 114.20

NM 5

MP 5 116.00

PM 5 214.00

Sig .803 1.000 280.00

A, uses Inducing agents Mean sample size=5.000 (PE-Pituitary extract; OVT-Ovatide OVA-Ovaprim; NM-Natrum muriaticum; MP-Mucuna pruriens; PM- Pedalium murex)

Table 21. ANOVA (one-way) table showing the effect of Pedalium murex and Mucuna pruriens and other inducing agents on the latency period of Poecilia sphenops*. The means were compared using Duncan multiple range rest

Source of variance

SS DF MS F-value Sig

Between 2969.767 5 593.953 404.968** 0.000

Treatment

Within 35.200 24 1.467

Treatment

Total 3004.967 29

** P < 0.01 at 1% level of significant

Duncan**^

Treatment N Subset of alpha =05

Treatment N 1 2 3 4

OVT 5 20.00

NM 5 22.80

OVA 5 23.00

PE 5 23.60

MP 5

PM 5 30.40 42.00 48.00

Sig 1.000 1.000 1.000

A, uses Inducing agents Mean sample size=5.000 (OVT-Ovatide; NM-Natrum muriaticum; OVA-Ovaprim; PE-Pituitary extract; MP-Mucuna pruriens; PM- Pedalium murex)

Table 22. ANOVA (one-way) table showing the effect of Pedalium murex and Mucuna pruriens and other inducing agents on the number of young ones spawned by Poecilia sphenops*. The means were compared using Duncan multiple range rest

Source of variance

SS DF MS F-value Sig

Between 3995.867 5 799.173 157.214** 0.000

Treatment

Within 122.000 24 5.083

Treatment

Total 4117.867 29

** P < 0.01 at 1% level of significant Duncan**^

Treatment N Subset of alpha =05

Treatment N 1 2 3 4 £

OVT

OVA

PE

NM

PM

MP

Sig

5

5

5

5

5

5

23.40

27.60

1.000

36.40

1.000

46.60

1.000

51.40

54.00

.081

A, uses Inducing agents Mean sample size=5.000 (OVT-Ovatide; OVA-Ovaprim; PE-Pituitary extract; NM-Natrum muriaticum; PM- Pedalium murex; MP-Mucuna pruriens)