Eapen Jacob, 2013. Studies on the captive breeding and reproductive biology of two indigenous ornamental fishes of the Western Ghats. Ph. D Thesis, Mahatma Gandhi University, Kottayam, India CHAPTER 3 CAPTIVE BREEDING TECHNIQUES FOR PRISTOLEPIS MARGINATA 3.1 INTRODUCTION Manipulation of fish reproduction is advantageous for the development of hatchery management. Stocking of natural waters with captive bred seeds is an important and in some cases very successful tool for conservation of endangered species. Captive breeding is one of the major steps in the conservation programme of a species. Although the merits of supplemental breeding are debated (Snyder et al., 1996, 1997; Hutchins et al., 1997), this approach has become a common management technique for many fish species (eg., Brown et al., 2000; Hedrick et al., 2000). Captive breeding initiatives should not merely produce larger numbers of individuals of a threatened species, they should also aim to maintain a high proportion of the gene 165
diversity that is present in the wild population (Leus, 2011). It does not mean mere the production of large number of individual; it demands lots of parameters to be taken into consideration. For naturally outbreeding species, high levels of inbreeding often have negative effects on life history traits related to reproduction and survival (Frankham et al., 2002). In addition, the smaller the captive population stays, the faster gene diversity will be lost through genetic drift and inbreeding. Pristolepis marginata is an indigenous frehshwater fish of the Western Ghats and belonged to the family nandidae. It is a very compatible fish in the aquarium except for its breeding time. It readily takes any food, both artificial and natural. Breeding in captive conditions is one of the desirable qualities for an ideal ornamental fish. Hence attempts were made to breed this fish in captive conditions. Anna Mercy et al., (2003) has made studies on the captive breeding and breeding behavior of this species. In this chapter different methods to raise the common catopra, Pristolepis marginata, in captivity has been explained. Different methods include (1) natural breeding by simulating the natural habitat of this fish species and (2) hormonal techniques. 3.2 MATERIALS AND METHODS 3.2.1 Collection and maintenance of brooders The main objective of this experiment was to test whether it is possible to make Common Catopra spawn in captive condition and to study the different reproductive behavioural features. The specimens collected directly from its natural habitat as well as F1 individuals which produced in the hatchery were used for captive breeding experiments. Fishes from natural habitats were collected from Bharathapuzha river 166
(about 150 km away from the hatchery site) and were brought to the hatchery in the College of Fisheries. Collections were mainly made by means of cast net operations. Caste nets of comparatively smaller mesh size of about 5 mm were made use of. The broodstock was raised on a mixed diet of artificial pelleted feed, live feeds like moina, mosquito larvae, blood worm and egg yolk. A weekly water exchange at a rate of 2/3 was ensured. The maturity condition of the brooders were assessed based on the macroscopic characters such as body size, bulginess of belly and different behavioral features it shown had been taken as signal of readiness to spawn. As the specimens became sexually mature they were separated sex-wise and kept in separate tanks of the same dimensions mentioned above. The experiments were conducted in the laboratory at the College of Fisheries, Cochin. 3.2.2 Sexual dimorphism For the sake of captive breeding experiments the mature specimens were segregated sex-wise. Different macroscopic and visual features were made use of for determining the sex of the individual. They include 7. Special features in vent region 8. Bulginess of the stomach 9. Special behavioural patterns 167
3.2.3 Captive Breeding Experiments The biological studies revealed that P. marginata is a fish showing strong parental care mechanisms, which including territoriality, nest building, courting aggression etc. the biological studies also showed that the fish is a lithophyl; a clear indication that the presence of particular sized pebbles is a critical factor in the spawning of P. marginata. The presence of the pebbles, along with other factors, stimulated the fish to start reproductive activities. The factors played as stimulants to initiate reproductive activities in a sexually mature male P. marginata are, 1. Presence of proper breeding substratum 2. Presence of a ripe female 3. Clear oxygenated water 4. Absence of intruders into the vicinity of nesting area The breeding experiments were designed based on the above factors and based on the previous studies on fishes showing similar habits (Pal et al., 2003; Das and Zamal, 2000). It became evident that this fish showed similar behavior shown by a number of cichlid species. In the present study two types of breeding techniques were successfully applied. 1) Natural mode of reproduction by simulating the factors available in the natural habitat of the fish, and 2) Application of hormonal method based on Linpe (Peter et al.,1990) method. Here the hormones were orally administered along with feed. 168
3.2.4 Natural mode of reproduction Breeding tank set up for Natural breeding Males and females are released together in a spawning pond where external cues are manipulated, (e.g. temperature, water exchange, water quality, photoperiod and presence of the nest), to stimulate the fish into spawning naturally. The spawners may be left to incubate their eggs, or else they may be separated, depending on their brooding behaviour. This technique is also called uncontrolled breeding or semi-controlled breeding. A breeding tank was set up simulating the factors available in the natural habitat of the fish. The dimensions of the aquarium was 3 ft X 2 ft X 2 ft. the tank was made up of glass sheets of thickness 8 mm. The floor of the tank was provided with a bed of pebbles of 3 inches thickness. The size of the pebbles varied from 0.5 cm to 2.00 cm. The pebbles were washed in prior to avoid sand and soil particles. Studies showed that during nest making the male fish arrange the pebbles in a particular way, mainly based on the size range, to make depression like structure made of larger pebbles. Smaller ones were kept at the periphery. All the movements of the stones were done by the male fish and its thick lips were the sole instruments all the activities. It is probable to develop special structures to strengthen the lips of the male fish by the onset of breeding activities which is evident a number of fishes showing parental care. Canister filter was fitted in the tank instead of under gravel filter, which may interfere the nest making activities. Clean filtered freshwater of ph 7.0 to 7.5 was used. A single pair of sexually 169
mature male female fishes were introduced in each trial. The male and female fishes for breeding was selected based on the following features. Active males were selected by stripping the abdominal region of fish. Fully ripe males showed traces of milt as white material on applying pressure to abdomen. It also selected based on the basis of its behavioural features. Sexually active female started nest building and exhibition of aggressive behavior even in the absence of ripe female fish. Ripe females were selected based on its morphological specialties. It includes bulginess of belly and special features in the cloacal region. In a ripe female of P. marginata, the cloacal region was found to be reddish, soft and little risen structures from the surroundings. Soon after introduction of breeding pair into the breeding tank, the male started its nest building activities, along with territorial and aggressive behaviours. It became a clear indication that the conditions in the breeding tank and the pair are right in all means. After spawning, eggs were collected from the nest in a random sampling method to find out the average number of eggs per spawning. 3.2.5 Hormonal Application Hormones in breeding experiments were administered in different ways (Haniffa and Sridhar,2002). It includes 3. Intra-muscular injections 4. Through oral administration along with feed 5. Subcutaneous transplants. 170
In the present study, in P. marginata, hormonal application through oral ways found effective. In this experiment breeding tank was set up as explained above. Male and female fishes for the hormonal study were selected and kept separately. The fishes were fed with mosquito larvae, which were kept in a mixture of ovaprim and water at ratio 3:1 for 45 minutes. This method was adopted from Burton (1998), where it was practiced in Artemia. Ovaprim is a hormone complex used for fish breeding, manufactured by Syndel laboratories, Canada, on the basis of Linpe method (Peter et al., 1990). Each fish was fed with approximately fifty numbers of hormone treated mosquito larvae, and released into the breeding tank pairwise. Its activity changes, intensity of behavioural patterns and latency period were studied. In the present case time taken by the fish to spawn successfully after co-habitating with male is termed Latency period. All the spawning activities were observed visually, recorded the incidence of different behavior patterns and videographed the activities futire studies and analysis. 3.3 RESULT 3.3.1 Sexual dimorphism There was no clear cut sexual dimorphism in P. marginata. Even though no morphological features to distinguish the sexes were found, some characters were useful in identifying sexes. This included, size, bulged stomach, characteristic features of genital area and certain behavioural attributes. Sexually mature males started nest building and territorial aggression when it had reached gonadal maturity. That was regarded as a good sign to select sexually active males for captive breeding experiments. Males at this stage produced traces of white milky milt at the time of stripping on the 171
stomach. Females showed well distinguished bulged stomach at its ripe stage. In addition to this, the genital area of a ripe female was found to be reddish, swollen and soft. (Plate 3.2 Figs 1 &2) Courtship in P. marginata, prolonged for more than 48 hrs. Spawning occurred as a culmination to all the courtship activities. Courtship and reproductive patterns are detailed in chapter 4. Spawning occurred inside the depression of the pebble nest. The spawning activity lasted for about 2 hours and the release of eggs was done in batches. Each batch which consisted of 25-30 eggs was released after short courtship. Typical short courtship lasted for 2 minutes. The ovulated female was led by the male towards the depression in the centre part of the pebble nest. Some special behavioral patterns are exhibited at this stage. It included bumping, lateral display, rubbing, and tail quivering. After two minutes of intensive behavioural display both the fishes stood motionless for a while, with their genital organs at a closer position. A batch of eggs was then released. After the release of the eggs, male and female became separated. The female moved away from the site. The male was then found to be repairing the pebble nest. After an interval of 5 minutes the female again came to the periphery of the nest and a similar process is repeated. This activity is repeated for a number of times until all the ovulated eggs are released. Each of this two minutes procedure is considered as a unit. So the complete spawning is divided into a number of units. The time interval between initial units was shorter and it increased as time passed. Towards the end, the time interval reached close to 15 minutes. Each unit of spawning activity was different in itself. This is explained in the chapter 4. 172
The released eggs were found attached to the pebbles. The eggs were slightly adhesive in nature. Average diameter of the fertilized egg was found to be 2.6 ± 0.3 mm in diameter. A major portion of the eggs went inside the pebble nest fitting themselves among the voids of the pebbles. After spawning, the female left the place and she took no role in any of the activities there after. The male constantly guarded the nest site, against the intruders, both conspecifics and heterospecifics. It went on repairing the nest continuously. Finally the nest was transformed into a heap of stones. Male continued its guarding by hovering over the nest site and periodically fanning it. It was continued for three to four days till the larva were hatched out. Soon after spawning a sample of eggs were removed using droppers for developmental biology studies. 3.3.2 Natural Breeding simulating natural habitat An experimental tank was prepared simulating the natural habitat of the fish. Perfectly mature and conditioned male and female fishes were introduced into it. Soon after introduction the fishes did not show any indication of breeding behavior. After half an hour the fishes started its special breeding behavioral signs like chasing, nubbing etc. The longest latency period observed was 74 hours and a shortest of 66 hours. The average latency period was found to be 70.4±2.9 hours. 62 % of pairs reacted positively to the natural method of captive breeding. The average no of eggs per spawning in this method is found out as 278.6. The average survival rate after one month was found to be 34.6±5.3 %. The survival rate was found at the range 29-42 %. 173
3.3.3 Hormonal method of captive breeding Fishes treated with hormone gave better results than the non-treated ones. The average latency period has fallen down to 46.4±1.5 hours. A maximum latency period of 48 hrs and minimum of 44 hours were recorded. The positive response of breeding pairs also showed improvement. It increased to 73 %. The mean number of eggs spawned by each pair ws calculated as 256.5±52.4 and the mean survival rate was found to be 28.25±3.8. The details of the captive breeding via different induction methods have been represented in plate 3.1. 3.4 DISCUSSION There are records of captive breeding of nandids namely Nandus nandus (Sarkar, 2009; Pal et al.,2003) and Badis badis (Barlow,1964) available. Parental care of the eggs and hatchlings was observed in P. marginata. It agrees with general behaviour of the fishes belonging to family nandidae. P. marginata, also showed very strong parental care and great resemblance towards cichlid breeding behavior as reported by Annett (1999), Barlow (2000) and Keenleyside (1991) in cichlids. P. marginata exhibited all the aspects of guarders (Perrone, 1979 and Balon 1984). Breeding behaviour or courtship behaviour is a very important act in fish breeding. It varies from the simple swimming of the breeders along the side of each other to the elaborate act of nest building and instances of male competition inherent in group spawning. The absence of positive breeding behaviour from any one of the breeders resulted in termination of breeding (Munshi and Hughes, 1992). In the case of P. marginata, the withdrawal from the activities of spawning by the female parent could 174
be marked as the end of spawning, or in other words complete release of the ovulated eggs from the ovary. The biological studies revealed that the male individual has a larger size than the female of the same age. In P.arginata the male fish has the sole responsibility of all the parental care mechanisms. The larger size of the male justifies this sort of behavior. In all the activities including nest building the only weapon the male used was its thick lips. Comparatively large sized eggs and low fecundity are also connected with the guarding nature of the species. The structure of the nests made by the fish is quite interesting. During the nest building it avoided smaller sized pebbles and sand particles. The remaining larger ones are arranged in a special manner to make lots of interspaces among them. The eggs are directly released into the nest. The down falling eggs directly got into these voids. The embryonic developments, hatching, and early growth are taking place inside the voids only. The young ones emerged out as free swimming larvae after a period of 4-5 days. Till then, the male fish guarded the nest site by repairing the damages, fanning for oxygenation and defending from the enemies. The nest of P. marginata can be explained as a network of underground tunnels which facilitated free life of the larvae and young ones inside there. The captive studies have shown that maximum production of the young ones can be achieved by removing the stones, by which the pebble nest is made of, soon after the complete hatching of the eggs within the nest. The male parent also has to be removed. By providing proper larval rearing measures higher survival rate can be achieved than in 175
the natural means. It is evident that, however, higher larval mortalities are happening inside the tunnel system of the pebble nest most probably of the lack of enough quantity of food material. Similar results were obtained in the case of angel fish (Pterophyllum scalare) under captive conditions in commercial farms (Personal Observation.) It is revealed that oral administration of ovaprim through the mediation of mosquito larvae has given better performance. The length of latency period has fallen to 46.4 than that of the 70.4 in natural mode of reproduction. Percentage of pairs positively responded to breeding experiment was increased to 73 % from 62 %. But a lowering trend was noticed in the case of survival rate. It fallen down to 28.25 % from 34.6 %. P. marginata showed male parental care. Sargent (1995) have explained about the male parental care and cannibalism in fishes. But in P. marginata no filial cannibalism was evident as in many related species. Fishes that have parental care exhibit monogamy at the time of spawning (Perrone, 1978). Monogamy is a common event in the family cichlidae. Monogamy in cichlid fishes is usually considered to be the inevitable outcome of essential biparental care; species that care for large clutches directly on the substrate are biparental and monogamous, while those that utilise safer breeding sites are uniparental and polygamous (Keenleyside 1991). Same situation is evident in P. marginata. The present study has practical implications on the context of biodiversity conservation and large scale hatchery production of the species. This may help the species to strengthen in its natural habitats at the same time hatchery production may 176
help to boost the ornamental fish industry. It can fulfil specific tasks that should be an integral part of the overall conservation action plan for a species (Leus, 2011). 177