Production and quality of eggs obtained from wolffish (Anarhichas lupus L.) reared in captivity

Aquaculture ELSEVIER

Aquaculture 122 (1994) 295-312

Production and quality of eggs obtained from wolffish (Anarhichas lupus L. ) reared in captivity D.A. Pavlov”,

E. Moksnessby*

“Chair oflchthyoiogy, Department ofBiology, Moscow State University, 119899 Moscow, Russian Federation bInstitute of Marine Research, Fbdevigen Marine Research Station, N-481 7 His, Norway

(Accepted 16 December 1993)

Abstract Maturation of females and ovulation of eggs of common wolffish, Anarhichas lupus L., were followed during the spawning season from 10 October 1992 to 28 July 1993. The broodstock was reared from larvae in the laboratory. Most females matured from the middle of December to the middle of March at water temperatures of 5.0-8.O”C. A total of 153 577 eggs from 47 females were stripped and artificially inseminated. Of these eggs, 70.6% were fertilized, but only 41.0% showed normal cleavage. Nine females had high proportions (more than 80%) of normally cleaved eggs. Fertilized but abnormally cleaved eggs were observed in almost all females, with an average proportion of 30%. There was no difference in quality of eggs obtained by internal and external artificial insemination or from females that demonstrated spawning behaviour and females without it. Survival of eggs from different females correlated to the proportion of normally cleaved eggs. The causes of poor egg quality are discussed as well as related morphology and size-frequency distribution of eggs.

1. Introduction

To choose new species of fish for aquaculture, it would be useful to take into account the type of ontogeny. Species that have early ontogeny described by Balon ( 1985 ) as “direct” are obviously attractive for artificial breeding, because something close to a juvenile organism hatches from the egg. Species with direct early ontogeny possess comparatively lower fecundity and larger egg size, but survival of eggs and young in nature is usually very high. Among marine fish, species of the family Anarhichadidae have high-quality *Corresponding author. Tel. +47-37010580;

Fax +47-37010515;

Internet: [email protected].

0044-8486/94/$07.00 0 1994 Elsevier Science B.V. AU rights reserved XSDZOO44-8486(93)0321-Y

296

D.A. Pavlov, E. Moksness /Aquaculture 122 (1994) 295-312

flesh and direct early ontogeny; they were suggested (Tilseth, 1990 ) as potential objects for marine cold-water aquaculture. The eggs of these fish are similar in size to those of salmon eggs and their fecundity is close to that of salmon of similar size. But they differ from salmon in that the hatched larvae, more than 20 mm long, have almost adult morphology and begin to feed just after hatching. Investigations of the common wolffish, Anarhichas lupus L., have shown that the life cycle of this species could be reproduced in captivity. The larvae and juvenile fish are easy to keep in the laboratory on live food and dry pellets, their survival may reach lOO%, and their growth rate is high (Ring0 et al., 1987; Moksness et al., 1989; Moksness, 1990, 1994; Stefanussen et al., 1993). According to estimations of Moksness ( 1994)) under optimal conditions common wolffish and spotted wolffish (A. minor Olafsen) can reach total wet weights of 2.5 kg and 5 kg, respectively, 2 years from hatching. Wolffish usually do not spawn in captivity; only two reports of natural spawning of common wolffish kept for several years in an aquarium were found (Ring0 et al., 1987; Ringer and Lorentsen, 1987). Fertilized eggs stuck together in the egg-mass and were protected by the male. First results of experiments with reproduction of wolffish (A. lupus) (Pavlov and Novikov, 1986) demonstrated the possibility of artificial fertilization and incubation of eggs in the laboratory. The motility of spermatozoa in undiluted semen was described in this study. Subsequent studies on the fertility of eggs obtained from wild-caught fish kept in the laboratory before final maturation (Pavlov et al., 1992) demonstrated a wide range in the proportion of normally cleaved eggs (from 0 to 89%) obtained from different females. Fertility was correlated with the temperature during final maturation of oocytes. Peculiarities of cortical reaction were described in this paper, and it was shown that the eggs were activated by spermatozoa. Later, the spawning behaviour in captive wolffish was described (Johannesen et al., 1993). Courtship was observed to start 4-5 months prior to spawning. Approximately 30-50 h before releasing of eggs, the females turned onto their side. After this “side-lying phase” lasting 12-24 h, a “labour phase” of high-intensity movement interrupted by short periods of rest was observed. At the end of the latter phase which lasted 3-6 h, the oviduct had opened. Males usually were passive during courtship, but copulation between a pair of spawners was observed twice. It was observed immediately after the labour phase and the eggs were released into the water on both occasions 8 and 15 h after copulation. The period from copulation to releasing of eggs was called the “resting phase”. The possibility of internal fertilization in wolffish has been suggested. According to Pavlov and Radzikhovskaya ( 199 1)) the gonads of males are small and the average volume of ejaculate is about 1 ml. Sperm density is much lower than in other teleost fishes with external fertilization. Estimation of the number of spermatozoa required for fertilization of eggs from one female showed that high fertility could be reached only by internal fertilization, where sperm would not be lost in the outer medium (Pavlov, 1993 ) . In the experiments with artificial insemination of eggs, it was shown (Pavlov, 1993, 1994) that the eggs could be fertilized in marine water and in ovarian fluid, but that the period of sperm motility was much

D.A. Pavlov,E. Moksness /Aquaculture 122 (1994) 295-312

297

longer in ovarian fluid, reaching several hours. The eggs could be fertilized internally by introducing milt through the oviduct of the female or externally by mixing the eggs and ovarian fluid with milt. In both cases a comparatively long period (several hours) of contact between eggs and sperm was required to attain a high fertilization rate. The eggs inseminated in ovarian fluid should be released into water before the beginning of cleavage, or after 10 h at a temperature of 11 “C and after 27 h at a temperature of 3.5 “C. Eggs kept in ovarian fluid for longer periods had abnormal cleavage and died. The latter observations corresponded to spawning behaviour and confirmed internal fertilization in wolffish. Most previous experiments with artificial insemination of wolffish eggs were unsuccessful due to unsuitable methods, and after the methods were improved and spawners were kept under optimal conditions, large proportions of normally cleaved eggs were obtained from most females (Pavlov, 1993, 1994). Previously, eggs for artificial insemination had been obtained only from wildcaught fish. However, propagation of wolffish from broodstock kept in captivity for a long time would be important for marine fish farming. A broodstock of common wolffish, consisting of fish reared from larvae on dry pellets, has been kept in the laboratory at the Institute of Marine Research (IMR), Flodevigen Marine Research Station. In 1992-l 993, a large group of these fish matured. The objective of the present study was to obtain developing eggs and young from this captive broodstock of wolffish. We studied the quality of eggs, determined on the basis of fertility, morphology, and survival. 2. Materials and methods Origin and maintenance of spawners The fish used in this study were reared from larvae, hatched from one egg-mass which was caught near the Faeroe Islands in 1987 and from larvae collected in the Barents Sea off Northern Norway in 1986, 1987, and 1988 (Moksness et al., 1989; Moksness, 1994). The fry were grown at the IMR, Flodevigen Marine Research Station. From a size of approximately 20 g, all the fish were fed commercial dry pellets (Elite Plus, produced by T. Skretting A/S, Norway). The annual temperature fluctuated between 4.7 and 13.7”C. The oxygen content was above 7 mg 02*1-’ in the outlet water of all tanks and the salinity fluctuated between 32.0 and 34.7%. The fish were exposed to a 16 h light and 8 h dark cycle. For at least 1 year before final maturation, which took place in 1992 and 1993, the fish were kept in green-walled tanks of 2-3 m3 capacity at stocking density averaging 14 fishes*m3. The total number of fish with an average weight of 1 kg was 280. Among them about 127 and 83 were tentatively identified as females and males, respectively. The females usually had larger abdomens and the males longer urogenital papillae edged or surrounded by a pinkish or red area. Beginning in early October 1992, the most mature females, those with swollen abdomens, were kept in grey-walled tanks of 1.5 x 1.5 x 0.37 m (560-680 litres) at densities of l-6 fish per tank. The water exchange rate averaged 10 lamin-‘.

298

D.A. Pavlov, E. Moksness /Aquaculture 122 (1994) 295-312

The photoperiod was 16 h light and 8 h dark with low light intensity; complete darkness was maintained after 9 January 1993. The water temperature gradually decreased from 13.8 “C at the beginning of October to 4.7 “C in the middle of April. From then until the end of May, it averaged 6.0 ’ C and increased to 10.3 a C at the end of July. The females were fed dry pellets, but they had stopped feeding approximately 1 month before final maturation. To determine the influence of the presence of males on the maturation and quality of eggs, the females were kept together with one male in each tank from 9 January to 16 February. After ovulation, the eggs of these females were stripped. As a cortical reaction did not occur, the absence of copulation and natural insemination was determined, and the eggs were inseminated artificially.

Insemination and incubation of eggs As previously reported (Pavlov and Novikov, 1986; Pavlov and Radzikhovskaya, 199 1; Johannessen et al., 1993 ), females in tanks usually release ovulated unfertilized eggs into water and males do not take part in the spawning. Beginning in early October 1992, the dates when females released eggs were recorded. Experiments with artificial insemination of eggs were conducted from 12 November until 28 May. The more mature females were observed 2-3 times a day. Ovulation was identified by the opening of the oviduct. The time of ovulation for 10 of 57 females was missed and these females released unfertilized eggs into the water. Thus a total of 47 females were used for artificial insemination. According to the previous data (Pavlov, 1993 ) , for successful fertilization there should be more than 0.2 x 1O6spermatozoa per egg. Sperm was stripped from l15 males to obtain enough for insemination of one female. The number of stripped males depended on the quantity and quality of sperm. Quality, including sperm density and motility, was evaluated for each male under the microscope. Nonmotile sperm and sperm with very low density were not used for insemination. At the beginning of the breeding season, the quantity and quality of sperm were high and sperm from one to three males was usually used for insemination of eggs from one female. Towards the end of this season these values for sperm decreased, probably due to repeated stripping of the same males, and up to 15 males were used for insemination. Sperm obtained from several males was mixed and diluted with modified Ringer solution (Cobb et al., 1973) at ratios of 1:2-l : 4. The Ringer solution was used for better subsequent mixing of eggs and sperm. The volume of the sperm solution used for insemination was measured and sperm density was counted in a haemocytometer. To take internal fertilization in wolffish into account, two methods of artificial insemination were used. In the process of internal insemination, from 10 to 25 ml of sperm solution was injected by a syringe through the genital opening of the female in the middle part of the ovary. Inseminated females were kept in the tanks and stripped after 4-6 h. This time was used, because after 6-8 h females usually released eggs into the water and the eggs stuck together. External insemination was conducted according to the method described by Pavlov ( 1993.1994). The sperm solution was placed in a cylindrical vessel. The eggs stripped from a

D.A. Pavlov, E. Moksness /Aquaculture 122 (1994) 295-312

299

female were then added and mixed with the sperm by means of up to 20 inversions of the vessel the first hour and fewer after that, at a temperature of 4-7’ C. The period of contact between eggs and sperm was 4-6 h. As shown earlier (Pavlov, 1993), such a period was enough to reach a fertilization rate close to lOO%, when Ringer solution was used for dilution of sperm. To compare egg fertility from the two methods of insemination, we compared data for 6 females inseminated internally from 25 November to 25 December and for 15 females inseminated externally from 26 December to 25 January. Most females were alive after stripping; only four fish had died. Some eggs were retained in the body cavity after stripping. Therefore, the fecundity, as determined by the number of stripped eggs, was slightly lower than the absolute fecundity of the female. The fecundity was determined by assuming that 100 ml of eggs included an average of 738 eggs. The number of spermatozoa per egg (N) was calculated by: N=Dx

V/F

where D = density of sperm solution (no. *ml- ’ ), V= volume of sperm solution (ml) and F= number of stripped eggs. As has been reported (Pavlov and Radzikhovskaya, 199 1) , the eggs of wolftish lose their capacity to stick together about 6 h after they are released into water. To prevent contact between eggs, inseminated eggs from each female were distributed on the bottom of vessels during that time. Alternatively the eggs were placed in milk mixed with sea water at a ratio of 1: 3. The eggs were washed from the milk after 6 h. The eggs from each female were then placed in upwelling incubators: cylindrical plexiglass vessels with a layer of gravel on the bottom to distribute water flow. The incubated eggs were continually mixed with water. To prevent bacterial infection, the eggs were repeatedly treated with glutaraldehyde at a concentration of 600 ppm for 5 min every third to fifth day of incubation (Pavlov and Moksness, 1993 ). The temperature was 10.0 t 0.1 ‘C during the first 43 days of incubation, 7.8 + 0.1 “C!during the subsequent 58 days, and 6.7 I?0.1 “C after that. Prior to hatching, the egg membranes became more transparent. To obtain a large number of embryos in a short time, the eggs were hatched after they were placed in nets by means of light hand pressure. Similar pressure seems to take place in Anarhichadidae in nature, when the male presses the egg-mass to provoke hatching of embryos (Marliave, 1987). After hatching, the larvae were placed in 270-litre tanks at an average stocking density of 1.5 fishml-‘. They were fed natural zooplankton during the first 2-3 weeks and then dry pellets. The average water temperature during their first month growth was 7.5 ‘C. Egg quality

The quality of eggs after stripping was evaluated using a binocular microscope placed vertically according to the method described by Chernyaev ( 198 1). The morphology of live eggs and especially the presence of cortical alveoli in the surface layer of yolk were recorded. The fertility of eggs was estimated by examining at least 150 eggs through a binocular microscope 2 days after insemination. The eggs were classified according to the scheme described earlier (Pavlov et al., 1992).

300

D.A. Pavlov,E. Moksness /Aquaculture 122 (1994) 295-312

The number of eggs with normal cleavage, abnormal cleavage, uncleaved blastodiscs and incomplete cortical reaction (unactivated) was counted. As there were few atretic eggs, eggs with follicular layers, or coagulated yolks, their number was not correlated with fertilization success, and they were excluded from analysis. As was shown before (Pavlov et al., 1992)) cleavage of blastodiscs was possible only after contact of the eggs with spermatozoa. Therefore, the fertilized eggs were identified as the eggs with cleavage. Cleavage might be normal or abnormal. Difference in egg quality between groups of females inseminated internally and externally and females with spawning behaviour and without it was determined using Student’s t-test. The diameter of eggs was measured under a binocular microscope in 10 females 2 days after insemination; 100 eggs from each female were used. Due to insignificant swelling of eggs and formation of a narrow perivitelline space in the upper parts of the eggs (Pavlov, 1986), their diameter (measured looking at the eggs from the upper side) was the same as just after stripping. To determine the survival rate, dead whitish eggs were removed from the incubators by siphon and counted. The survival rate at the beginning of eye pigmentation, before hatching, and at the stage of start-feeding was determined as the percentage of live eggs or larvae from the total initial number of eggs. The hatchability was determined as the percentage of normal larvae from the total number of live eggs 2 weeks before hatching.

3. Results Production andfertility of eggs Maturation of females and ovulation of eggs were observed during most of 10 months, from 10 October 1992 to 28 July 1993 (Fig. 1). A total of 65 females, or about 40% of all females from the broodstock, matured during this period. The water temperature, 13.8 ’ C at the beginning of the season of maturation, was de9

.I 4

L

6

13

7

12

6

6

11

e

5

10 9

2 a m

4

6

3 2 1 0

7

F m

6

)-

5 40424446465052 1992

1

3

5

7

Week

9

1113151719212325272931

number

4

1993

Fig. 1. Number of females spawned and the temperature during the season of maturation. Dashed line shows the beginning of experiments with insemination of eggs. The stars indicates presence of females with proportion of normally cleaved eggs more than 80%.

D.A. Pavlov,E. Mokmess /Aquaculture 122 (1994) 295-312

301

n = 47 g

20

I E .s B

10

2 E z’ 0 0

1

3

2 Fecundity

Fig. 2. Fecundity-frequency

“o

2’0

5

4

distribution,

3’0

6

7

(thousands/fish)

4’0

5.0

Fertilization

6.0 rate

data for 47 females.

7-o

e-0

9-o 100

60

90

(%)

16

0

0

10

20

30

40

Normal

Fig. 3. Frequency distribution mally cleaved eggs (B ) .

50

60

cleavage

70

100

(%)

of females according to fertilization

rate (A) and proportion

of nor-

creased to 4.7”C in March, and then increased to 10.3”C to the end of this season, The last female was stripped at 28 July. Her oviduct was open and the eggs were released easily, but, probably due to high temperature, they were inside of follicular layers and were not used for insemination. Most females matured from the middle of December to the middle of March at water temperatures between 5.0 and 8.O”C. The number of eggs ranged from 849 to 6863, averaging 3268 +220 eggs per fish. The fecundity-frequency distribution (Fig. 2 ) showed three modes.

302

D.A. Pavlov, E. Moksness /Aquaculture

Table 1 The fertility of eggs (%) after insemination Female, ID

1 1 2 2 3 3

No. of Total amount males of eggs

2 1 1 2 3 3

500 423 1255 723 2214 100

with sperm from different malesa

Quantity of sperm (X 106.egg-‘)

2.1 2.0 2.1 3.6 0.7 2.2

122 (1994) 295-312

Unactivated

Activated Normal cleavage

Abnormal cleavage

Uncleaved

75.8 53.7 97.1 100.0 1.6 1.9

12.6 10.9 0.8 0 71.2 86.8

9.4 16.5 0.8 0 26.6 11.3

2.2 18.9 1.5 0 0 0

aAnalysis of variance using the arc-sinus transformed values of normal and abnormal cleavage (Sokal and Rohlf, 1969, pp. 386-387) showed significant difference (PC 0.02) between free females.

As is known (Templeman, 1986)) there is a strong correlation between fecundity and body size in common wolffish, and these modes are probably connected with the presence of three age groups of females from the broodstock. We have not found any correlation between date of ovulation and fecundity, i.e. body size (r2=0.032; P=O.38). A total of 153 577 eggs were stripped from 47 females. Among them, 41 .O% of eggs had normal cleavage and 29.6% of eggs had abnormal cleavage of blastodiscs. As cleavage of eggs was possible only after contact of eggs with spermatozoa (Pavlov et al., 1992)) 70.6% of eggs were fertilized. The number of females with fertilization rates exceeding 70% was 32, or 68.1% (Fig. 3A). However, the number of females with high proportions of normally cleaved eggs wasmuch lower (Fig. 3B). The females with high fertilization rates were observed during most of the whole breeding season (Fig. 1) . Abnormally cleaved eggs were found in almost all females averaging 30.0 -+4.3% of all eggs. In 4 females, 80-100% of eggs were abnormal. There was no correlation between fecundity (i.e. size of females) and percentage of normally cleaved eggs ( r2 = 0.0 12; P= 0.47 ) . The proportions of normally cleaved eggs after internal and external insemination were 60.12 14.5% ( y1= 6 ) and 5 1.7 ? 7.8% (n = 15 ) ) respectively. The difference was not significant (P=O.59). The eggs from the first 3 stripped females were at the beginning of resorption and the fertilization rate was very low. During the period from 22 November to 1 February, 24 females or 5 1% of the females were stripped and a comparatively high fertility of eggs was observed with an average number of normally cleaved eggs of 57.0 2 6.2%. The following period of breeding, until 15 February, during which 7 females were stripped, showed low fertility and more than 50% of eggs had uncleaved blastodiscs. During this time, the males were kept together with the females. Therefore, the presence of males did not increase fertility of eggs. The comparatively high fertility of eggs obtained from 4 females with an average number of normally cleaved eggs of 68.3 2 8.7% was observed after exclusion of males from the tanks. From the beginning of March to 28 May, 8 females were

D.A. Pavlov, E. Mohmess /~Aquaculture 122 (1994) 295-312

303

ymm,

Fig. 4. Morphology of eggs just after stripping from one female. (A), Normal egg. (B) Cortical reaction and formation of blastodisc took place inside the female’s body. (C) Constriction of yolk due to damage of yolk membrane. (D) Resorbing egg. (E) Resorbing egg covered by follicular layer. bl = blastodisc; mc= micropyle; od= oil droplets; ps = perivitelline space. Natural orientation of eggs with view from a side is given, using the microscope installed vertically (Chemyaev, 198 1).

304

D.A. Pavlov, E. Mokmess /Aquaculture 122 (1994) 295-312

1

Imm

,

Fig. 5. Normal (A) and abnormal (B,C,D,E) cleavage of blastodiscs at the morula stage.

stripped and their fertility was very different with the number of normally cleaved eggs ranging from 0 to 86.3%. As was reported earlier (Pavlov, 1993, 1994), the proportion of unactivated

D.A. Pavlov,E. Mokmess /Aquaculture 122 (1994) 295-312

305

80 , EO-

A

40200

,...,.,

60-

B

3.4

3.8

4.2

4.6

Egg diameter

5.0

5.4

!

8

(mm)

Fig. 6. Size-frequency distribution of eggs from 4 females. Fertilization rate and proportion of normally cleaved eggs are following: (A) 91.7% and 88.0%, (B) 84.1°h and 0%, (C) 74.8% and 67.7%, (D) 88.5% and 32.8%.

eggs depended mostly on the number of spermatozoa per egg. This number in our experiments fluctuated from 0.4 x lo6 to 2.2 x 106, averaging 0.8 + 0.1 x 106. There was no correlation between these two values (r*=O.O34; P= 0.21). Thus the number and concentration of spermatozoa were sufficient for successful fertilization. The results of experiments with insemination of eggs from the same female by sperm from different males (Table 1) indicated that, if the number of spermatozoa was sufficient, the fertility of the eggs depended mainly on the quality of eggs, not on the quality of sperm. Johannessen et al. ( 1993) reported a “resting phase” of female spawning behaviour 8-15 h before release of eggs into water. During this phase, the female

D.A. Pavlov, E. Moksness /Aquaculture 122 (1994) 295-312

306

40

50

70

60

Normal cleavage

80

100

90

(%)

Fig. 7. The relationship between normal cleavage in the eggs (x) and the survival rate to the eyed stage (JJ). Data from 6 females. y= -57.926+ 1.4952x; ?=0.957. Table 2 Survival of eggs and larvae from females with different proportions Survival (%) Female, Total no. Normal ID of eggs cleavage (O/o) To eyed stage Before hatching 1 2 3

6790 4428 4713

67.7 78.0 88.0

‘Hatchability was determined weeks before hatching.

36.6 52.6 72.5

32.7 43.1 71.2

of normally cleaved eggs Hatchability

(o/o)”

To start-feeding 13.4 24.7 49.2

41.0 57.3 69.1

as the percentage of normal larvae from the total number of live eggs 2

spent most of the time lying on her side with open oviduct. In our study such behaviour was observed in 30% of females. The proportions of normally cleaved eggs from females with and without, spawning behaviour were 42.2? 10.8% (n= 14) and 37.2 + 6.3% (y1= 33), respectively. The difference was not significant (P=O.67).

Morphology and diameter of eggs The quality of eggs and their potential for fertilization could be evaluated just after stripping. In females showing high fertility of eggs, all of the eggs had the same morphology. Most of the oil droplets occurred in the upper part of the yolk. The cortical alveoli, 5-9 pm in diameter, were visible in the cytoplasmic layer of egg (Pavlov et al., 1992 ). In females with low fertility, some of the stripped eggs appeared abnormal. This is illustrated in Fig. 4, using the ovulated eggs from one female. The eggs potentially suitable for fertilization had no perivitelline space (Fig. 4A), and the cortical alveoli were present. In some ovulated eggs, the blastodiscs formed and the perivitelline spaces were visible (Fig. 4B). This meant that the cortical reaction took place inside the body of the female; fertilization was then impossible. In several ovulated eggs, the yolk membranes were damaged and the perivitelline spaces were too large (Fig. 4C). In such eggs, the yolks flowed out through the yolk membranes and the perivitelline spaces disappeared a few

D.A. Pavlov, E. Moksness /Aquaculture 122 (1994) 295-312

307

hours after stripping. Some eggs were at the beginning of resorption. They were less transparent and contained dark structures (Fig. 4D). Several stripped eggs, further along toward resorption, were covered by follicular layers (Fig. 4E). Among the stripped eggs of the female, the proportion of eggs of types A, B and C+D+E (Fig. 4) were 26.3, 57.5, and 16.2% (n= 179), respectively. Thus the number of eggs with the capacity to be fertilized was less then 26.3%. After insemination, normally cleaved eggs were absent. There were several possibilities for the fate of eggs after insemination with morphology which seemed to be normal (Fig. 4A). The egg might not meet the spermatozoa; in this case, the cortical reaction did not reach its end. The perivitelline space appeared, but the blastodisc did not form. The egg might be activated inside the female’s body or activated by spermatozoa without real joining of gametes. In this case, an uncleaved blastodisc formed. After joining of gametes, a blastodisc formed and cleavage began. The cleavage might be normal or abnormal Initial normal cleavage was symmetrical, whereas abnormal cleavages were often incompleted and irregular. Examples of different types of initial cleavage have been described (Pavlov et al., 1992) for White Sea wolffish. The type of cleavage was easy to recognize at the morula stage. In normal eggs all cells were distinguished clearly (Fig. 5A). The size of cells at the centre of the blastodisc was lower than that at the periphery, as is usual for cleavage of wolffish (Pavlov, 1986). In poor-quality eggs cleavage was incomplete, the borders of some cells were not visible and free spaces formed in blastodiscs (Fig. SB,C,D,E). Abnormally cleaved eggs usually died before the gastrulation step. The eggs from all females had a similar light-yellow colour. Mean egg diameter from different females ranged from 4.7 to 5.1 mm. The size-frequency distribution of eggs might reflect their quality. Normal distribution and a narrow size range (ca. 0.6 mm) (Fig. 6A) usually correlated to a high proportion ( > 80%) of normally cleaved eggs. However, in some cases normal distribution was accompanied by a large number of eggs with uncleaved blastodiscs. The presence of oocytes of smaller size and a wide size range (more than 0.8 mm) (Fig. 6B,C,D) probably was caused by abnormal maturation. In some cases, despite a high fertilization rate, a large proportion of eggs with such a size-frequency distribution cleaved abnormally. Survival during incubation and exogenous feeding Survival of eggs until the beginning of eye pigmentation correlated with the proportion of normally cleaved eggs ( r2 = 0.96) (Fig. 7). The survival of eggs from eyed stage to the stage about 2 weeks before hatching was high (Table 2). However, many embryos died just before hatching and the hatchability was low, averaging 56%. Most larvae were hatched by age 125 days from insemination. They had almost completely resorbed yolk sacks and began feeding just after hatching. The survival rate of fry from different females to 1 month from hatching varied from 9 1.1 to 100.0%. Their average wet weight was 0.28 tr 0.01 g (n=150) at this time.

308

D.A. Pavlov, E. Moksness /Aquaculture 122 (1994) 295-312

4. Discussion The results show the possibility of artificial breeding of wolffish raised in captivity. The maturation and ovulation of eggs of wolffish was observed during most of 10 months. The spawning season of wild wolffish is much shorter: in most regions of the Northwest Atlantic this species breeds in September-October (Jonsson, 1982; Templeman, 1986)) while spawning may occur between November and January (Bigelow and Schroeder, 1953). Peak spawning in southern Norway occurs, as in our study, during December, January and February (Moksness, unpubl. data). The main environmental factor which apparently determined time of maturation was the photoperiod, and the very protracted period of egg maturation, observed in captivity, probably was caused by the constant light cycle. It is well known (see reviews by Bromage et al., 1990, 1992), that timing of spawning in teleost fishes can be modified by photoperiodic change. As in our study, rainbow trout maintained under a constant photoperiod had a protracted spawning time (Duston and Bromage, 1991). Observation of the peak spawning in wolffish broodstock confirms the presence of an endogenous rhythm in the control of reproduction. Such rhythms have been described (Carillo et al., 1989; Bromage et al., 1990, 1992; Duston and Bromage, 199 1) in marine and freshwater fish species. Temperature seems to have a lesser effect on the timing of wolffrsh spawning. About 28% of females released ovulated eggs at a temperature above 10 ’ C, which was apparently too high for normal maturation. The lower effect of temperature, compared to photoperiod, on the timing of spawning was found for salmonids (Bromage et al., 1992). It is important that eggs with high proportions of normal cleavage were obtained during the entire breeding season. These results suggest that it may be possible to obtain eggs and larvae all-yearround as industry requires by manipulating the photoperiod. Our results show that the quality of sperm was good in most cases, but the quality of eggs from a large number of females was not. Observed abnormalities in the morphology of stripped eggs and eggs after insemination may have been caused by the following processes. The appearance of opaque whitish eggs and eggs with coagulated yolk means the beginning of resorption. Fertility of eggs obtained from such females was usually low. The release of eggs covered by follicular layers together with ovulated eggs apparently suggests an abnormality in the process of maturation. The presence of eggs with uncleaved blastodiscs after insemination might be caused by two factors: ( 1) Due to low spermatozoon activity, eggs were activated, but spermatozoon did not join to the female’s pronucleus. This was observed (Pavlov, 1993) in White Sea wolffish after sperm had been kept more than 20 h before insemination in the refrigerator. (2 ) The cortical reaction had begun inside the female’s body: part of the cortical alveoli had broken down and the space between micropyle and yolk surface had formed. As is well known (Ginsburg, 1968)) under these conditions the joining of gametes is impossible. This condition, called “postovulational overrippening”, has been described in many fish species kept under artificial conditions (Detlaf et al., 198 1; Korovina, 1986; Makeeva et al., 1987). Observation of eggs lacking cortical al-

D.A. Pavlov, E. Mohness /Aquaculture 122 (1994) 295-312

309

veoli and, even more, with already-formed blastodiscs just after stripping clearly suggest postovulational overrippening in some females. Special attention should be paid to analysis of the appearance of fertilized, but abnormally cleaving eggs. The proportion of such eggs after artificial insemination of wild-caught fish was usually no more than 3% (Pavlov, 1993 ) , whereas it was very high in our experiments. Large numbers of abnormally cleaving eggs obtained from a female were usually accompanied by an abnormal size-frequency distribution and a wide range of egg diameters. It is known (Kjorsvik et al,, 1990), that variation in egg diameter is very important for determining egg quality in fishes. As was reported earlier (Dzerzhinskiy and Pavlov, 1992), oocyte growth in wolffish synchronized during final maturation. In wild-caught White Sea wolffish females kept for l-2 months in the laboratory, the range of oocyte diameter was 1.3-2.0 mm at Stage IV of maturation and no more than 0.8 mm at Stage V. According to our data, this range for ovulated eggs from many females exceeded 1.0 mm and reached 1.8 mm. Therefore, it could be supposed that a portion of oocytes from some females did not reach final maturation, in particular the metaphase II of meiosis. In some cases immature eggs were obtained after stripping from females of White Sea wolffish (Pavlov et al., 1992). In these eggs the germinal vesicles broke down, but the oil droplets were still distributed in various parts of the eggs, and sometime the eggs were not completely free from the follicular layers. As is known (Detlaf, 1977)) immature eggs cannot to be fertilized or may cleave abnormally. The mean diameter of ovulated eggs obtained from different females (4.7-5.1 mm) was less than that of eggs (5.2-5.8 mm) reported for a wild population of White Sea wolffish (Dzerzhinskiy and Pavlov, 1992). Therefore the diameter of eggs from our fish might be lower than normal due to abnormalities in maturation. A reduction in egg diameter and poor quality of eggs with a fertilization rate on average of less than 50% were observed (Papst and Hopky, 1984) in broodstock of Artic charr (Salvelinus alpinus L.) kept in captivity during several years, as in our case, at comparatively high temperatures. Egg quality at the beginning of the breeding season was low. This was probably caused by high temperature (more than 10°C) during the final stages of maturation. Low fertility of eggs and resorption of oocytes in most females were observed, (Pavlov et al., 1992) for White Sea wolffish kept under similar temperatures. The temperature during subsequent periods of the breeding season was mostly 5-7°C. According to previous data (Pavlov et al., 1992; Pavlov, 1993, 1994)) this temperature was close to optimal for normal egg maturation. The low fertility of eggs from females matured in the first half of February might be related to the abnormal breeding behaviour of males which were kept together with these females for at least 3 weeks. The absence of normal male behavioural ritual leading to copulation was observed in captivity (Pavlov and Radzikhovskaya, 1991; Johannessen et al., 1993); this might provoke a delay in the opening of oviducts in females and overrippening of eggs in our experiments. This assumption was confirmed by the large proportion of eggs with uncleaved blastodiscs obtained from females kept together with males. On the whole, the low quality of eggs was caused by processes of resorption and

310

D.A. Pavlov, E. Moksness /Aquaculture

122 (1994) 295-312

desynchronization of maturation and ovulation. This has been described in several species breed in captivity (Detlaf, 1977; Detlaf et al., 198 1; Korovina, 1986; Makeeva et al., 1987). Failure in final maturation might be connected with unfavourable aspects of artificial environments. In addition to the temperature regimen and the influence of the presence of males, the composition of feed might have had a negative effect on the maturation of oocytes. As was shown (Kjorsvik et al., 1990; Lie and Mangor-Jensen, 1993), quality and composition of feed are important factors for obtaining high-quality, viable eggs in fishes. The eggs from all females were light yellow in our study, while the colour of eggs from wild females is usually more intense. Survival of inseminated eggs depends mainly on their quality. In particular, at proportions of normally cleaved eggs of 100 and 60%, it could be expected that 92 and 32%, respectively, would survive to the eyed stage. Therefore, there is no reason to incubate eggs with low proportions of normal eggs. Large number of the embryos died by l-2 weeks before hatching, probably due to high temperature. The temperature in natural spawning grounds of wolffish near the end of development decreases to about 2.5 “C in northern Norway (Falk-Petersen et al., 1990) and to - 1“C in the White Sea (Pavlov and Novikov, 1993). A hatchability close to 100% was observed after incubation of eggs of White Sea wolffish at temperatures between 0 and 1‘C before hatching (Pavlov, unpubl. data). According to published data (Ringo et al., 1987) and the present results, the survival rate of larvae and fry was very high if they were fed live organisms for at least the first 2 weeks after hatching. The main problem to be solved in the near future is the improvement of egg quality by means of keeping females in conditions favourable to normal final maturation, in particular at optimal temperature and feeding.

Acknowledgements

We would like to thank the Norwegian Research Council, Department NFFR, for financial support. The technical assistance of Inger Henriksen, Vetle Madsen, and Alexandra Pavlova is highly appreciated.

References Balon, E.K. (Editor), 1985. Early Life Histories of Fishes: New Developmental Ecological and Evolutionary Perspectives. Dr. W. Junk Publs., Dordrecht, 280 pp. Bigelow, H.B. and Schroeder, W.C., 1953. The wolf fishes. Family Anarhichadidae. Fish. Bull., 53: 502-508. Bromage, N., Duston, J., Randall, C., Brook, A., Thrush, M., Carillo, M. and Zanuy, S., 1990. Photoperiodic control of teleost reproduction. In: A. Epple, C. Scanes and M. Stetson (Editors), Progress in Comparative Endocrinology. Wiley-Liss, New York, pp. 620-626. Bromage, N., Jones, J., Randall, C., Thrush, M., Davies, B., Springate, J., Duston, J. and Barker, G.,

D.A. Pavlov,E. Moksness /Aquaculture 122 (1994) 295-312

311

1992. Broodstock management, fecundity, egg quality and the timing of egg production in the rainbow trout (Oncorhynchus mykiss). Aquaculture, 100: 141-166. Carillo, M., Bromage, N., Zanuy, S., Serrano, R. and Prat, F., 1989. The effect of modifications in photoperiod on spawning time, ovarian development and egg quality in the sea bass (Dicentrarthus ZabraxL.). Aquaculture, 81: 351-365. Chernyaev, Zh.A., 198 1. A method of lateral microscopic examination using the vertical chamber for live investigation on the development of fish eggs. In: Studies on Reproduction and Development of Fishes (Methodological Handbook). Nauka Press, Moscow, pp. 2 16-22 1 (in Russian). Cobb, J.L.S., Fox, NC. and Santer, R.M., 1973. A specific ringer solution for the plaice (Pleuronectes platessa L.). J. Fish Biol., 5: 587-591. Detlaf, T.A., 1977. Development of organization of matured egg in amphibia and fish at the final stages of oogenesis during maturation of the oocyte. In: Modern Problems of Oogenesis. Nauka Press, Moscow, pp. 99-104 (in Russian). Detlaf, T.A., Ginsburg, A.S. and Shmalgausen, O.I., 1981. Development of Sturgeons. Nat&a Press, Moscow, 224 pp. (in Russian). Duston, J. and Bromage, N., 1991. Circannual rhythms of gonadal maturation in female rainbow trout (Oncorhynchus mykiss). J. Biol. Rhythms, 6: 49-53. Dzerzhinskiy, ICE and Pavlov, D.A., 1992. Gametogenesis in the White Sea wolffish Anarhichas lupus marisalbi. J. Ichthyol., 32(6): 87-99. Falk-Petersen, I.-B., Haug, T. and Moksness, E., 1990. Observations on the occurrence, size and feeding of pelagic larvae of the common wolffish (Anarhichas lupus) in Western Finmark, Northern Norway. J. Cons. Int. Explor. Mer, 46 (2): 148-154. Ginsburg, A.S., 1968. Fertilization in Fish and the Problem of Polyspermi. Nauka Press, Moscow, 359 pp. (in Russian). Johannessen, T., Gjesaeter, J. andMoksness, E., 1993. Reproduction, spawning behaviour and captive breeding of the common wolf&h Anarhichas lupus L. Aquaculture, 115: 41-5 1. Jonsson, G., 1982. Contribution to the biology of catfish (Anarhichas lupus) at Iceland. Rit Fiskideildar, 6: 2-26. Kjersvik, E., Mangor-Jensen, A. and Holmetjord, I., 1990. Egg quality in fishes. Adv. Mar. Biol., 26: 71-113. Korovina, V.M., 1986. Effect of “overrippening” of eggs on the embryogenesis of teleost fishes. Rep. Zool. Inst. Acad. Sci. Sov. Un., 154: 115-123 (in Russian). Lie, 0. and Mangor-Jensen, A., 1993. Importance of broodstock nutrition for optimal production in aquaculture. In: Proceedings, Fish Farming Technology, pp. 3 5-4 1. Makeeva, A.P., Emel’yanova, N.G. and Verigin, B.V., 1987. About quality of eggs produced by Far East vegetative-feeding fishes Hypophthalmichthys molitrix, Aristichthys nobilis, Ctenopharyngodon idella in the conditions offarming reproduction. Vopr. Ikhtiol., 27( 5): 809-822 (in Russian). Marliave, J.B., 1987. The life history and captive reproduction of the wolfeel Anarrhichthys ocellatus at the Vancouver Public Aquarium. Int. Zoo Yb., 26: 70-8 1. Moksness, E., 1990. Weaning of wild-caught common wolffish (Anarhichas lupus) larvae. Aquaculture, 91: 77-85. Moksness, E., 1994. Growth rates of the common wolf&h, Anarhichas lupus L., and spotted wolffish, A. minor Olafsen, in captivity. Aquacult. Fish. Manage., 25 (in press). Moksness, E., Gjosaeter, J., Reinert, A. and Fjallstein, I.S., 1989. Start-feeding and on-growing of wolffish (Anarhichas lupus) in the laboratory. Aquaculture, 77: 221-228. Papst, M.H. and Hopky, G.E., 1984. Development of an Arctic charr (Salvelinus alpinus L.) brood stock. Aquaculture, 43: 323-33 1. Pavlov, D.A., 1986. Developing the biotechnology culturing White Sea wolffish, Anarhichas lupus marisalbi. II. Ecomorphological peculiarities of early ontogeny. J. Ichthyol., 26 (6) : 156- 169. Pavlov, D.A., 1993. Fertilization in wolf&h Anarhichas lupus: external or internal? Vopr. Ikhtiol., 33(5): 664-670 (in Russian). Pavlov, D.A., 1994. Maturation and artificial fertilization of the eggs of captive common wolffish (Anarhichas lupus L.) from the White Sea. Aquacult. Fish. Manage., in press.

312

D.A. Pavlov,E. Moksness /Aquaculture 122 (1994) 295-312

Pavlov, D.A. and Moksness, E., 1993. Bacterial destruction of the egg shell of common wolflish during incubation. Aquacult. Int., 1: 178-186. Pavlov, D.A. and Novikov, G.G., 1986. On the development of biotechnology for rearing of White Sea wolffish, Anarhichas lupus marisalbi. I. Experience on obtaining mature sex products, incubation of eggs and rearing of the young fish. J. Ichthyol., 26 (4): 95-106. Pavlov, D.A. and Novikov, G.G., 1993. Life history and peculiarities of common wolffish (Anarhichas lupus) in the White Sea. ICES J. Mar. Sci., 50: 271-277. Pavlov, D.A. and Radzikhovskaya, E.K., 1991. Reproductive biology peculiarities of the White Sea wolf&h, (Anarhichas lupus marisalbi) (based on experimental data). J. Ichthyol., 3 l(7): 52-62. Pavlov, D.A., Dzerzhinskiy, K.F. and Radzikhovskaya, E.K., 1992. Assessing the quality of roe from White Sea wolffish (Anarhichas lupus marisalbi), obtained under experimental conditions. J. Ichthyol., 32( 1): 88-104. Ringo, E. and Lorentsen, H., 1987. Brood protection of wolffish (Anarhichas lupus L.) eggs. Aquaculture, 65: 239-24 1. Ringo, E., Olsen, R.E. and Bee, B., 1987. Initial feeding of wolflish (Anarhichas lupus L.) fry. Aquaculture, 62: 33-43. Sokal, R.R. and Rohlf, F.J. (Editors), 1969. Biometry. W.H. Freeman and Company, San Francisco, 776 pp. Stefanussen, D., Lie, O., Moksness, E. and Ugland, K.I., 1993. Growth ofjuvenile common wolffish (Anarhichas lupus) fed practical fish feeds. Aquaculture, 114: 103-l 11. Templeman, W., 1986. Some biological aspects of Atlantic wolf&h (Anarhichas lupus) in the northwest Atlantic. J. Northwest Atl. Fish. Sci., 7 ( 1): 57-65. Tilseth, S.,‘1990. New species for cold-water farming. Aquaculture, 85: 235-245.