Even though research is going on, both at Százhalombatta and at Szarvas, for improving the percentage of successful induced spawning of carps and other fish, much remains to be achieved in this regard. The percentage of success of induced breeding is highly variable and at times is hardly 50 percent. Failures are correlated, partly with the potency of the donor pituitary, unsuitable weather conditions or poor condition of the female fish - of these, potency of the donor pituitary appears to be of considerable importance. It is, therefore, important to undertake investigations to know the most effective donor/age/season/sex/sexual stage for obtaining pituitary for hypophysation.
Attempts are being made to replace fish pituitary for hypophysation; however, the results so far obtained with mammalian pituitary hormones have been very conflicting. Results with HCG, Progesteron, and the chemical LH-RH, appear promising and these need further trials and investigations.
At present, very little is known about the eco-physiological aspects of the female fish, either breeding naturally or by hypophysation. In this regard, a very important observation has been made by the consultant, i.e., that these fish gain weight after getting pituitary injections. This may be due to hydration and also the degree of hydration may indicate/predict the success of hypophysation. This needs further elaboration.
Investigation for accelerating the sexual development/increase in number of the sexual cycles per year needs to be undertaken so as to increase the egg production potential of these cultivated fish and also to utilize the hatchery facilities available, which are presently used only for 4–5 months of the year. These investigations will lead to a substantial increase in fish seed production in Hungary. Since there is a vast scope for eel culture in Hungary and, having no natural eel resources, it is important to initiate research for the development of the hypophysation technique for breeding eels.
Heavy mortality of fry is encountered mostly at the stage when they start feeding on food organisms, partly because of lack of proper fish food organisms or because of predators and disease. Research efforts need to be int sified to solve this aspect of fish seed production.
The above are some of the major problems relating to fish seed production in Hungary.
While formulating the research programmes the consultant had detailed discussions in this regard with Director of the Institute Dr. F. Müller, Project Manager Mr. Imre Csávás, Director of Research Dr. J. Oláh, Scientific Adviser Professor E. Woynarovich, and with other scientists in charge of different units, such as breeding (both at Százhalombatta and Szarvas), nutrition, genetics, catfish culture, eel culture and instrumentation, which helped considerably to prepare this report. Availability of the staff and equipment for such programmes has also been considered and assessed.
The pituitary extract of male and female of different species when chromatographed showed three molecular species (three peaks) irrespective of their stages of sexual development, the second peak/fraction of which contained fish pituitary gonadotropin. The second fraction obtained from the pituitary extract of free-spawners (fish that breed in captivity) and non-spawners (fish that do not spawn in captivity), irrespective of their stages of sexual development, is effective in inducing spawning in ripe non-spawners, which indicates that qualitatively the gonadotropin does not differ between free-spawner and non-spawner, nor their sexes, nor their stages of sexual development. But, while working on the effects of carp pituitary fractions on vitellogenesis, ovarian maintenance and ovulation in hypophysectomized catfish Heteropneustes fossilis, it has been found that the gonadotropic content of the pituitary gland of the free-spawner is considerably more than that of the pituitary gland of non-spawner. Therefore, it is necessary to find out the level of gonadotropin according to species/age/sex/sexual stage and season to find out the most suitable donor species/age/sex/sexual stage and season to take out the pituitary for hypophysation. For this, the extract of the individual pituitary gland obtained according to species/age/sex/sexual stage/season may be run through the sephadex g-100 column (420 × 9 mm) and eluted with O.IM NaCl solution and the eluted fractions subsequently passed through the spectrophotometer at 253.7 nm to isolate the fraction showing the second peak. The fraction may be lyophilized and direct quantitative measurement may be made. The fraction may also be dialyzed to get rid of NaCl before quantitative measurement is done. Quantitative estimate can also be made by other methods.
The fraction showing the second peak containing pituitary gonadotropin from free-spawner and non-spawner may be analysed for amino-acid composition and amino-acid sequence may be established and compared.
Presently available mammalian gonadotropins FSH/LH give very conflicting results in induced spawning. However, it will be interesting to compare the amino-acid composition/sequence of the fish gonadotropin with that of the mammalian FSH and LH to find out if a similar chain is present in mammals. If so, the particular chain may be utilized for substituting the fish pituitary gonadotropin. This research needs much more experienced staff and equipment and thus this may be taken up in collaboration with other institutions where such facilities and staff are available.
The pituitary extract fraction showing the second peak containing gonadotropin may be used for acceleration of sexual development and maturation. The whole crude pituitary extract should not be used.
The milt necessary for fertilization of European catfish eggs is obtained by siphoning it with a special syphon, so as to avoid contamination with urine, or by dissecting the males. In any case, an adequate amount of milt is difficult to obtain. It is, therefore, suggested that the males may be given carp gonadotropin 0.5 mg equivalent of pituitary gland/kg four times at weekly intervals along with 1 ml spleen extract (Solcosplen solco GmbH, Basel)/kg of body weight during the spawning season. A final dose of carp gonadotropin of 4 mg equivalent of pituitary gland/kg of body weight should be injected at the time of spawning. The does of the treatments may be adjusted in subsequent trials as per the results obtained.
Considering the vast scope of eel culture in the country and lack of natural resources of elvers, it is important for Hungary to concentrate its research effort for the acceleration and maturation of sex of eels so as to eventually induce them to breed in captivity, otherwise Hungary will have to continue to import elvers from other countries forever. Hence, the following research project has been formulated.
The silver migrating eels to be kept in fresh water, having a temperature of 22–23°C with oxygen about 8 ppm.
The males may be injected with 0.2 ml spleen extract (Solcosplen solco GmbH, Basel)/100 g of body weight, along with carp pituitary gonadotropin fraction equivalent of 300 μg of pituitary gland/100 g of body weight, at fortnightly intervals for 3–4 months. Adequate control should be maintained and the enlargement of eye diameter, distinctness of lateral line, enlargement of pectoral fin and the gonadosomatic ratio should be investigated. Sexually mature males will show testicular lobes fully developed and tightly filled with sperm, with the gonadosomatic ratio in the range of 3.5 to 4 percent.
The females may be injected with carp pituitary gonadotropin fraction equivalent of 450 μg of pituitary gland/100 g of body weight at fortnightly intervals for 4–6 months. Adequate control should be maintained and the enlargement of eye diameter, distinctness of lateral line, enlargement of pectoral fin and the gonadosomatic ratio should be investigated. The oocytes size in fully mature fish should be 1–1.4 mm in diameter, whereas the gonadosomatic ratio should be in the range of 30–40 percent. For ovulating the females, two injections of pituitary gonadotropin fraction equivalent of 4.5 mg/kg (for each injection) may be given. The second injection may be given at an interval of 12 hours along with 200 μg/kg of synthetic LH-RH (luteinizing hormone - releasing hormone). The doses of the injections may be adjusted in subsequent trials according to the results obtained.
It is fairly well established that the gonadal hydration is a prerequisite for spawning of the carps. The total water content of the ovary, the appearance of fluid in the spaces between the follicular cells, the loosening of the adhesion between the follicle cells and the eggs and the increase in diameter of egg, suggest that these morphological changes accompanying ovulation are preceded by a remarkable absorption of water into the ovary and ovarian egg. Whether the conditions prevailing in ponds during spawning season do or do not favour such absorption of water needs immediate investigation, especially because these carps do mature in ponds but do not spawn there, where perhaps the water is not that much diluted with rain or flood as that of the river where they normally breed. Further, the males do spermiate in ponds but the females do not ovulate.
The sperms have no mechanical barrier to water movement and it is not yet known if any mechanism exists to oppose osmotic change. But the chorion of the egg in plaice, cod and herring are freely permeable, offer no protection against osmotic withdrawal of water in high salinities, but mechanically oppose the entry into the cytoplasm of the egg of more than a certain amount of water in low salinities. Also it is reported in certain fishes that the removal of the chorion resulted in the rapid swelling and bursting of the eggs. In herring, the semen was found to be isometic with the blood, whereas the eggs were hyposmotic. Thus, unlike the sperm, the eggs have a mechanism to regulate the entry of ion/water to the eggs with the help of chorion. Perhaps this may explain why the males spermiate in the pond where the females are unable to ovulate. However, no information is available on the osmotic mechanism of these carps during natural breeding or induced breeding. Therefore, it becomes all the more important to undertake such investigation. It is suggested that the following investigations may be carried out:
Seasonal determination of conductivity/salinity of water of river where common carp wild breeding occurs. Conductivity/salinity of water before the wild spawning and during spawning should be compared.
Determination and comparison of freezing point of blood plasma, ovarian fluid, and egg fluid (see para. 5.1.4.1) of carps spawning in nature and by hypophysation. Also comparison of these parameters may be made between successful induced bred females and unsuccessful ones.
Determination and comparison of freezing point of blood plasma and seminal fluid of males inhabiting rivers and ponds.
Relation of gondatropin with the permeability of ovarian wall.
On the basis of the information obtained, manipulation may be made with hypophysation to treat the fish with the following and then to put them in water of comparatively very much lower conductivity of water than pond water:
Adequate control should be maintained. The doses of the above treatments and the degree of conductivity of waters may be adjusted according to the results obtained on the hydration of the fish, increase in the size of the egg, the migration of the nucleus toward micropyle and successful stripping of viable eggs.
Work is already progressing on the larval rearing of these cultivable fishes as mentioned in para. 3.5. Also nutritional requirements for these larvae are under investigation for which all the facilities are available. It is important to utilize mass culture techniques already known for the zooplankton for feeding them to the successive larval stages of these cultivable fishes. Simultaneously, investigations may also be made to produce in the pond itself a controlled sequential development of algal bloom and other food organism for the larvae. The staff and research facilities are excellent to undertake such studies at the Institute.
As mentioned in para. 3.4, investigations are being carried out at Százhalombatta. However, Szarvas has a very competent team of workers and necessary equipment for such studies and there is an urgent need to study the influence of nutrition on maturation and for formulation and manipulation of feeding to advance or delay maturation.
