5.1.1 Fractionation of Fish Pituitary Extract
In order to initiate the research programme suggested in Section 4, fractionation of the fish pituitary extract had been undertaken by the consultant. Acetone-dried pituitary glands were homogenized with an all-glass Potter-Elvehjem tissue homogenizer in O.IM NaCl eluant. The suspension was then centrifuged in a Tanetzki T32 C type centrifuge at 4 000 rev/min for 30 minutes. The supernatant was directly applied to a sephadex G-100 column 330 × 9 mm and equilibrated with O.IM NaCl at room temperature at about 7 ml/h. The fractions were collected every 10 minutes and were passed through Zeiss VSU 2-P type spectrophotometer at 253.7 nm to determine their absorbance. Figure 1 shows the peaks of the eluted fractions. The fractions constituting the second peak contain the gonadotropin.
Mr. I. Csengeri and Dr. Szabó are now fully familiar with the method of fractionating the fish pituitary extract as above, which would help the Institute staff to undertake the research programmes as indicated in Section 4.
5.1.2 Acceleration of Sexual Development
Migrating silver eels were caught in Lake Balaton and have been brought to the Institute to initiate the investigation as suggested in para 4.6
5.1.3 Gonadotropin and Hydration and Spawning of the Fish
In order to know whether gonadal hydration achieved with the pituitary extract injection was because of a shift of water/iron from some part of the body/tissue to the gonad or because the whole fish got hydrated and consequently the gonad, the consultant undertook the following investigation. The spawners of common carp and Chinese carps such as silver carp, bighead carp and grass carp were weighed during hypophysation whenever there was opportunity for doing so. The weight was taken after the fishes were anaesthetized with MS 222 and were tagged individually. Table 1 shows the gain and loss of weight during different stages of hypophysation and the result of stripping. Because of certain difficulties, systematic investigation could not be done; however, the results warrant further investigation in this direction as suggested in para 4.7.
The fish, on capture from the pond, lose weight in the hatchery because of stress, starvation and ejecting faecal matter. But soon after getting the pituitary extract injection they start gaining weight (Table 1, Lot No.1). The fishes of Lot No.1 were brought from the pond and kept for two days in the hatchery and then they were injected. Normally the fishes brought from the pond get the first pituitary injection immediately, which is followed by the second one after 12 hours and after 10 to 12 h after the seond injection the fishes are stripped (para. 3.2). All the fishes showed loss of weight at the time of the first injection but all gained, except one retained the same weight at the time of the second injection. At the time of stripping, this fish, however, showed a gain in weight but another one did not gain or lose weight during this period; these two fishes and one, though showing gain in weight, did not ovulate. The other four, which showed a gain in weight at the time of the second injection and at stripping, ovulated most successfully.
The fish of Lots No. 2 and 3 were injected with pituitary extract as soon as they were brought from the ponds to the hatchery. The weight of the fish of Lot No.2 was taken at the time of the first injection, the second injection and at stripping. Of nine fish, five showed loss of weight at the time of the second injection and, except for two of them, all showed further loss at the time of stripping. The one which gained during this time did not ovulate nor did the other four which lost/retained their weight, whereas the other four which gained at the second injection and also at stripping ovulated most successfully.
The weight of the fish of Lot No. 3 was taken only at the second injection and at stripping. It was seen that those fish which lost weight at stripping did not ovulate like those which lost weight in Lot No.2. Of seven fish which gained, five ovulated most successfully and two did not ovulate at all. Thus, it is clear that, on capture, the spawners lose weight and continue to do so, while the exogenous pituitary material induces increase in weight and the sum total of gain and loss indicates that those still indicating loss at the second injection/stripping do not ovulate but, on the other hand, it is not necessary that those which gain will definitely ovulate. However, the chances of success of hypophysation increase more in those which gain more. The increase in weight in a few hours after the pituitary injection appears to be because of total hydration of the spawner, the degree of which may vary depending on the internal environment of the fish/its stage of sexual development. But it is likely that this induced hydration may be influenced by the outside environment, particularly by the electrolytic level in the water in which fish lives.
A review paper on the induced spawning of commonly cultivated fishes is being prepared for publication in collaboration with Professor E. Woynarovich, Scientific Advisor of the Institute.
5.1.4 Hatchery Management
5.1.4.1 Measurement of Weight during Hypophysation
The female spawners of carps may be weighed without anaesthetizing on arrival at the hatchery before the pituitary injection with the help of a sensitive spring balance. Those fish which show loss of weight before the second injection may not be injected and sent back to the pond and those that show maximum increase in weight should be injected and kept for stripping. This practice may be initiated now and improved upon on further experience so as to have still better results of induced breeding. Also like common carp, other Chinese carps may be kept inside the hatchery in the concrete tanks during hypophysation, as has been shown during this assignment.
5.1.4.2 Selection of Eggs for Incubation
As has been pointed out in para 2.5, there was no reliable method of assessing the suitability/quality of eggs for incubation; however, the colour, elasticity or transparency of the eggs are used for appraisal of their quality. Relations between these criteria and egg quality are by no means clear. On the other hand the different constituents of the egg should have different specific gravity which may differ according to the quality of the egg. To investigate this, the consultant homogenized and centrifuged a sample of common carp ovulated unfertilized eggs. It was found that homogenization for three minutes and centrifuging for forty minutes at the speed of 4 300 rev/min was adequate to get a centrifugal pattern of homogenized eggs. Other eggs of the different samples were fertilized with live male gametes and incubated in the hatchery. The conditions and procedure for incubation were identical in all the samples.
The centrifugal pattern of the common carp egg showed four successive zones: the top zone of transparent fluid; the second one of cytoplasm, blue in colour; the third of yolk, yellow in colour, and the fourth of egg shell at the bottom. The eggs giving a fertility rate ranging 90–100 percent showed the above pattern. The volume of all the zones appeared more or less equal. The position of the cytoplasmic zone and the transparency of the fluid zone were the most important index in predicting the suitability/quality of eggs. So long as the cytoplasmic zone was on the yolk or even mixed with the yolk, the fertility rate was in the range of 70–90 percent. But, when the cytoplasmic zone started rising up in the fluid, the fertility rate decreased. The fertility rate was nil when the cytoplasmic zone was mixed with transparent fluid, which turned either turbid or less transparent. A chart of the centrifugal pattern of eggs of common carp has been made and termed as the Sinha Chart for selection of eggs for incubation (Figure 2). This will be very helpful in deciding the suitability of eggs for incubation.
Similarly, the eggs of grass carp, bighead carp and silver carp after homogenization for three minutes and centrifuging at 5 000 rev/min for 30 minutes showed the same four zones. Zones were in the same order in grass carp as those of common carp; but they were in the different order in silver carp and bighead carp. The first zone was of cytoplasm, blue in colour, the second of transparent fluid, the third of yolk, yellow in colour, and the fourth of egg shell at the bottom. The eggs having the above pattern showed a fertility rate in the range of 85–95 percent. But those with a fertility rate of 50–75 percent showed the sinking and mixing up of the cytoplasmic zone into the fluid. However, when the cytoplasmic zone was totally mixed with the fluid, the eggs had nil fertility.
A scientific paper on the selection of the eggs of these carps for incubation is being prepared for publication.
