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Injection and stripping of spawners
51. INJECTION AND STRIPPING OF SPAWNERS
The advanced technique of the artificial propagation of common carp involves a hormonal treatment prior to hand stripping of the mature spawners to obtain sperm and eggs.
Note 1: How much dried hypophysis do you require?
| Females | First injection: | 0.3 mg/kg |
| Second injection: | 3.5 mg/kg | |
| Males | One injection: | 2.0 mg/kg |
Example: You are planning to treat 34 females (average weight 4 kg) and 17 males (average weight 2.5 kg). You will require the following quantities of dried hypophysis:
(a) for the first injection of the females: 34 fish x 4 kg x
0.3 mg/kg = 40.8 mg
(b) for the second injection of the females: 34 fish x 4 kg x 3.5
mg/kg = 476 mg
(c) for the injection of the males: 17 fish x 2.5 kg x 2 mg/kg =
85 mg
It is good practice to add 10% to these calculated quantities of dried hypophysis to compensate for the losses occurring during the injections. If you know the average weight of one dried hypophysis (about 2.5-3 mg), you may then easily calculate how many hypophysis glands are necessary in each case.
Note 2: How to prepare the 0.65% salt solution?
Dissolve 6.5 g of common kitchen salt in 1 litre of distilled water. You may also use boiled and filtered water.
Note 3: How much salt solution do you require?
| Females | First injection: | 1 ml/fish |
| Second injection: | 1.5 ml/fish | |
| Males | One injection: | 1.5 ml/fish |
Example: For the above example (Note 1), you will require the following volumes of salt solution:
(a) for the first injection of the females: 34 fish x 1 ml =34
ml
(b) for the second injection of the females: 34 fish x 1.5 ml =
51 ml
(c) for the injection of the males: 17 fish x 1.5 ml = 26 ml
It is good practice to add about 10% to these calculated volumes of salt solution to compensate for later losses.
Note 4: The 100 litre anaesthetic solution may be used several times for a full day to save on the high cost of MS 222.
Note 5: Which anaesthetic should be used?
Three chemicals may be used as fish anaesthetics:
(a) MS 222 or tricaine methane-sulphonate (Sandoz, Basel, Switzerland) is a very mild and safe anaesthetic from which fish easily recover. Fish may be kept in it for about 30 minutes at 20-25°C. But it is now thought to be a possible carcinogenic in man; it is rather expensive and must be used sparingly.
(b) Quinaldine or 2-4 methylquinolin is a toxic chemical which should be handled with great care and stored safely. During the treatment of the fish, they should be carefully watched and transferred quickly to well oxygenated water if necessary.
(c) Phenoxyethanol is milder and less effective than MS 222 but is far cheaper.
Note 6: How should anaesthetics be used?
It is advisable to determine the correct dose of anaesthetic to be used by preliminary tests. For common carp for example the following doses are used in Hungary for 24°C water:
MS 222 1:10000 or 10 g dissolved in 100 l
water
Quinaldine 1:40000 or 2.5 ml dissolved in 100 l
water
Phenoxyethanol 1: 5 000 or 200 ml dissolved in
100 l water
These injections replace the natural release in the bloodstream of such hormones by the hypophysis, at the command of the hypothalamus.
As seen earlier, only two basic environmental factors have to be at optimum level: water temperature and dissolved oxygen content.
As far as possible, it is better to use batches of spawners similar m size to simplify calculations. The spawners are weighed in a netting bag, using weighing scales.
54. The gonadotropin hormones to be injected in the breeders are extracted from dried hypophyses.
Fresh hypophyses are collected, for example, from live sexually mature fish prior to their marketing and then prepared for preservation as further described in the last section of this manual.
(1) The necessary number of hypophyses is calculated on the basis of the dose of hormones to be injected in the breeders brought into the hatchery, see Note 1. (2) The dried glands are carefully ground into a fine powder in a mortar. (3) The resulting fine powder is then well mixed with a certain volume of a 0.65% salt solution which extracts the gonadotropin hormones from the gland tissue, see Notes 2 and 3. (4) It is the supernatant fluid which is used for the injections.
(1) Early in the morning, selected mature females are brought in from the storage ponds. They are put into a small mobile tank. At about 8 a.m. they are slightly anaesthetized (2) and receive the first injection of hypophysis extract (3). They are then stored in a large tank with a good water supply (4).
At about 8 p.m., they are anaesthetized again (5), their genital opening is sutured (6) and they receive the second injection of hypophysis extract (7). They are stored in the large tank (8) and a male fish is generally added (9) as an indicator of ovulation. The latter occurs about 10 hours later, at which point the females are anaesthetized (10) and stripped of their eggs (11). More detail on this procedure is given in the following pictures.
(2) They are anaesthetized, using for example MS 222, see
Notes 4, 5 and 6.
(3) From now on the anaesthetized breeders should be watched
carefully to see if their opercules keep moving. The life of the
fish is in danger when this movement stops. In this event the
breeder should be immediately removed from the anaesthetic
solution and put into well-aerated fresh water.
(4) The anaesthetized fish can be handled very easily.
This is the preparatory injection of gonadotropin hormones which will result in the initial development of the dormant eggs: their nucleus will migrate near to the micropyle and their first hydration will take place.
The type of injection varies according to the presence or absence of scales on the body.
(A) For mirror carps, the injection is intramuscular. It is generally given in the muscles below the tip of the dorsal fin, at a 45' angle. To avoid losing some of the injected solution, a finger should be kept on the punctured skin which should be slowly massaged (1, 2).
(B) For scaly carps, the injection is intraperitoneal. It is generally given into the body cavity from behind the base of the abdominal fin.
58. After being injected, the females are placed in a large tank.
A plentiful supply of well-aerated water should be provided. The water temperature should be kept constant, if possible within the optimum spawning temperature range of 22°C-24°C. To reduce stress, the females should be kept in a quiet environment and left undisturbed for 10-12 hours.
The genital opening is closed to prevent egg losses through wild spawning.
A needle and strong cotton thread are used as shown in steps 1 to 4. Nylon thread should be avoided because it cuts into the skin too easily as the fish swim.
This is the decisive injection of gonadotropin hormones which should cause the eggs to ripen fully. According to the carp strain used the type of injection varies as described above for the first injection.
As the length of the ovulation period is closely related to the water temperature, the latter should be measured and noted regularly throughout the night.
(1) As the end of the ovulation period approaches, the females
with fully ripe eggs start looking for a place to spawn, moving
along the sides of the tank.
(2) If an indicator male has been added to the tank, it will then
closely follow ripe females as for spawning.
62. The length of the ovulation period is closely related to the water temperature.
The sum of the hourly water temperatures should reach 240°C-260°C. By measuring the water temperature in the tank every hour, it is possible to predict the time when the females will reach full maturity relatively well, especially if the water temperature varies.
If, on the contrary, the water temperature remains nearly constant, the graphic method may be used. According to the actual water temperature (°C) prevailing during ovulation (1), the number of hours necessary (2) may be read on the horizontal scale. For example, at 24°C ovulation takes place a little more than 11 hours after the first injection. As the exact length of the ovulation period varies slightly from one female to the other, the result will be only a close approximation.
After stripping, the females are gently replaced in the tank where the well-oxygenated water supply will help them to recover from narcotization (2).
A similar crowding procedure is also used to remove the females from the tank and to transport them back to the broodstock pond.
They are then prepared for stripping.
(1) First, the suture of the genital opening is cut and the
cotton string carefully removed.
(2) The free genital opening is immediately closed with the thumb
of the left hand to prevent egg loss.
(3) The ventral part of the body is then carefully dried with
a clean towel.
65. Small anaesthetized females weighing 4 kg or less are stripped by one man.
The fish is held firmly with a towel by the tail and pressed under the right arm. The right hand gently massages the lower flanks of the female, from front to back, releasing the ripe eggs from the ovaries into a plastic bowl. The eggs should run along the side of this bowl, as they are sensitive to shocks.
Remember that no water should reach the eggs at this stage: the bowl, the fish and the hands should be carefully dried.
It is also necessary to make sure that the female is stripped empty of ripe eggs. A good female breeder may provide up to 20% of its own live weight in ripe eggs. A 5-kg female for example produces about 1 kg of them, containing 700 000 to 1 000 000 eggs.
The head is held down in a towel by one person. A second person holds the tail part of the body with one hand while stripping the eggs into a plastic bowl with the other hand.
Very large females may provide several kilograms of eggs and, in such cases each kilogram of eggs is collected into a separate bowl. Water should be avoided and the female stripped empty.
67. This is a summary of the treatment applied in the hatchery to the male spawners to obtain sperm.
In the morning the required number of mature males is brought in the hatchery from the storage ponds (1), which is normally half the number of ripening females.
They are temporarily stored in a separate large tank with a good water supply (2).
After the females have received their second injection the treatment of the males starts by placing them in an anaesthetic solution (3), see above. One injection of 1.5 ml of hypophysis extract is given to each fish, which corresponds to a dose of 2 mg dry hypophysis per kilogram live weight (4).
As for the females, the type of injection varies also according to the carp strain. One of these injected males is placed with the ripening females to act as an ovulation indicator (5). The other males are kept in a large storage tank with a good water supply (6).
At least 6 hours after the hormone injection, the males are ready. They are anaesthetized (7) and the milt is collected (8) while the females are stripped.
68. The milt containing the spermatozoids should also be kept dry as it is collected.
The anaesthetized male is placed on its back on a table
preferably on a piece of foam rubber. Its ventral surface is
carefully dried with a clean towel. If a
slight pressure along the flanks of the belly releases
milt, it is collected in a dry small glass. If the milt is less
abundant, it is better to use a milt collector,
sucking the milt from the genital opening.
A 4-5 kg male spawner provides an average of 20 ml milt.
