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Lordosis induction in juvenile red sea bream, Pagrus major, by high swimming activity
Aquaculture 212 (2002) 149 – 158 www.elsevier.com/locate/aqua-online
Lordosis induction in juvenile red sea bream, Pagrus major, by high swimming activity Minoru Kihara a,*, Shigeru Ogata b,1, Noriaki Kawano b, Itsuo Kubota b, Ryoichi Yamaguchi b a
Central Research Institute, Maruha Corporation, Wadai 16-2, Tsukuba, 300-4295, Japan Aquaculturing Operations Department, Maruha Corporation, Nagahama 3-11-3, Chuo, Fukuoka 810-0072, Japan
b
Received 16 December 2000; received in revised form 1 November 2001; accepted 4 November 2001
Abstract Lordosis is correlated with absence or malfunction of the swimbladder. However, swimbladder abnormalities do not completely explain the occurrence of lordosis. We examined whether muscle activities from vigorous tail beat induced by removal of the caudal fin could induce lordotic malformation in juvenile red sea bream. We also attempted to determine the minimal current velocity for inducing lordosis in relation to total length (TL). For this purpose, we employed relative current velocity in terms of ‘‘move of water in times of total body length per second (TL s 1)’’. We compared effects of different water current (2 vs. 4 TL s 1) for 10 days on bone malformation using juvenile of 25-mm TL. Exposure to 4 TL s 1 current velocity, but not 2 TL s 1, induced lordosis. Lordotic juvenile red sea bream had a cuneiform centrum mainly at the 15th vertebra. Lordosis was observed in fish with normal swimbladders as well. Thus, swimbladder abnormalities alone cannot completely explain the occurrence of lordosis. We removed part or all of the caudal fins of fish of 40mm TL and exposed these fish to 2 TL s 1 current for 10 days. Control fish did not show deformations in the vertebrae. Fish with complete removal of the caudal fin swam with vigorous tail beats. However, fish with complete, upper or lower caudal fin removal (36%, 29% or 7%, respectively) displayed lordosis even at the current velocity of 2 TL s 1. These findings suggested that the muscle activities from excess beat of the tail against excessive current, but not the mechanical action of the caudal fin, induced lordosis. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Lordosis; Current velocity; Muscle activity; Vertebra; Red sea bream
* Corresponding author. Present address: Utsunomiya Plant, Maruha Corporation, Kiyohara Industrial Area 8-1, Utsunomiya 321-3297, Japan. Tel.: +81-28-667-0801; fax: +81-28-667-0736. E-mail address: [email protected] (M. Kihara). 1 Present address: Taikei Gyogyo, Uomachi 3-13-7, Ishinomaki 986-0022, Japan.
0044-8486/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 1 ) 0 0 8 7 1 - 7
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1. Introduction Red sea bream Pagrus major is a marine fish of great economic importance in Japan (Foscarini, 1988). The species is reared in net pens and the juveniles are produced artificially. Thus, seed production of this fish plays an important role in the farming of red sea bream in Japan. However, serious skeletal malformations such as brachyospondylie and lordosis appear frequently in hatchery-produced juveniles (Takashima, 1978). These abnormalities are often associated with poor growth and low survival of the seed. Lordosis is correlated with absence or malfunction of the swimbladder (Takashima et al., 1980; Kitajima et al., 1981). However, swimbladder abnormalities do not completely explain the occurrence of lordosis. Divanach et al. (1997) indicated that similar malformations occurred in juvenile sea bass Dicentrarchus labrax L. despite the presence of a functional swimbladder, and related lordosis to the high current velocity ( > 10 cm s 1). Tail beat frequency increases with swimming speed (Bainbridge, 1958; Hunter and Zweifel, 1971). Thus, these findings suggest that swimming dynamics, i.e., muscle activities by water current, may induce lordosis. The role of current velocity on lordosis induction was already explored (Divanach et al., 1997; Backiel et al., 1984). However, we do not know yet how the high current velocity induced lordosis in these studies. Observing the fish, we hypothesized that abnormally high muscle activity, especially in the caudal part of the body, might be responsible for the induction of lordosis by high current velocity. Accordingly, our aim in this study was to examine whether muscle activities from the vigorous tail beat induced by removal of caudal fin, the main means of propulsion, induced lordotic malformation. For this purpose, we also conducted a preliminary study to find out the minimal current velocity to induce lordosis in juvenile-size fish.
2. Materials and methods 2.1. Fish husbandry Spontaneously spawned eggs were obtained from red sea bream broodstock reared in an indoor 20 m3 concrete square tank in our hatchery (28j26VN:129j32VE, Amami Island, Japan). Eggs were incubated, hatched and reared using generally established techniques for red sea bream hatcheries in Japan (Yamaguchi, 1978). Larvae were maintained in a tank of 75 m3 with flowing UV-sterilized seawater at 23.0 F 1.0 jC and fed-enriched rotifers Brachionus plicatilis from 4 days after hatching (DAH) to 38 DAH and enriched brine shrimp Artemia nauplius from 20 DAH on. A formula diet (Love Larvae, Hayashikane Sangyo, Shimonoseki, Japan) was also fed from 20 DAH on. To remove oil film on water surface, we used oil skimmer consisted of vinylchloride pipes equipped with an air blow system 5 DAH. Experiments were conducted in November 1997. The experiment was conducted strictly following the guideline for animal experiments of Central Research Institute of MARUHA. The guideline adheres to governmental legislation in Japan.
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2.2. Experiment 1: lordosis induction by water current Three hundred fish aged 47, 51 or 52 DAH were randomly selected and kept in a 500-l transparent cylindroconical tank. Total length (TL) of selected fish varied from 25.5 to 50.4 mm. UV-sterilized seawater at 22.0 F 1.0 jC was supplied to the tank horizontally through a vinyl-chloride pipe (13-mm I.D.) to induce a rotary water current. Horizontal water current velocity was between 10 cm s 1 (bottom wall side) and 14 cm s 1 (top wall side). Water current velocity in the tank was measured using an electromagnetic current meter (ACM100-D, Alec Electronics, Osaka). Fish were adapted to an 11 h light:13 h dark cycle (lights on from 07:00). We fed a commercial formula diet (Love Larvae) to fish using an automatic feeder (Food Timer, Seiko, Tokyo), every hour from 07:00 to 17:00. Each feeding consisted of 4.35 g per tank. After 7 days, 90 fish were randomly collected, anesthetized with 0.2 ml l 1 of 2phenoxyethanol (Wako Pure Chemical Industries, Osaka) and measured for total length with slide calipers. Then, the fish were fixed with 10% (v/v) formalin in neutral phosphate buffer solution (pH 7.4, Wako Pure Chemical Industries) for bone observation. The difference of total length measured as a straight line or as a curve was at most 1.7% (data not shown). Therefore, we measured the total length as a straight-line method. 2.3. Experiment 2: effect of current velocity Approximately 500 juvenile red sea bream aged 35 DAH were held in a 1000-l cylindroconical polycarbonate tank (stock tank). Seawater was supplied vertically to avoid horizontal rotary water current. We fed a commercial diet (Love Larvae) to fish using an automatic feeder, seven times a day (08:00, 09:00, 10:00, 11:00, 13:00, 15:00 and 17:00). Each feeding consisted of 0.2 g per tank. We randomly selected 125 fish of 25-mm TL from the stock tank at the age of 40 DAH, and randomly divided them into five groups of 25. We examined the effect of current pattern (chronic vs. intermittent) and current speed (5 vs. 10 cm s 1) on bone malformation in these fish. The current velocity of 5 or 10 cm s 1 in the present study corresponded to 2 or 4 TL s 1, respectively. Fish were treated in one of the three 10-l transparent cylinder tanks to create different water flow (Fig. 1). Tank A was for the zero rotary current group (control group, Fig. 1A). Tank A was equipped with an inverted glass bowl (10 cm in diameter) under the water supply-nozzle. The bowl dispersed the downward water current and kept the rotating current velocity less than 0.1 cm s 1. The volume of water supplied to tank A was 4 TL s 1. Tank B was equipped with a flexible nozzle of 7-mm I.D. The nozzle was bent at 45j to create chronic rotary current of 2 or 4 TL s 1 (Fig. 1B). Tank C was equipped with both an inverted glass bowl and a flexible-nozzle (Fig. 1C). Water was supplied alternately either from the top of the tank or from the nozzle for intermittent current groups (2 or 4 TL s 1). The rotary water current was applied between 08:00 and 17:00 in these groups.
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Fig. 1. A, B and C: Schematic drawing of the experimental tanks (top, side view; bottom, top view). (a) Tube for the air inlet equipped with stone (c); (b) water supply tube; (d) circular transparent tank for 10 l; (e) glass bowl (10 cm in diameter); (f) vinyl-chloride pipe (13-mm I.D.) for water outlet; (g) a vinyl-chloride pipe (25-mm I.D.); (h) a commercial flexible-straw (7-mm I.D.) bent at 45j; (i) valves.
The volume of water supplied to tank A, B or C was kept constant during the experiment. Water current velocity in the tanks was measured at approximately 2 cm high from the bottom and wall-side of tanks. We fed a commercial diet (Love Larvae) by a hand as same pattern and same amount of the above stock rearing for 10 days. Fish were collected after 10 days of exposure to one of the water current stated above. They were anesthetized, killed and fixed for bone observation as in Experiment 1. 2.4. Experiment 3: effect of caudal fin removal Sixty fish of 40-mm TL were selected from the stock tank at 60 DAH and randomly divided into four groups of 15. We adopted whole and partial caudal fin removal to assess the contribution of propulsion to lordosis. Our hypothesis is whether muscle activities from the vigorous tail beat induced by removal of caudal fin, the main means of propulsion, induce lordotic malformation without increasing the current velocity. We cut dorsal one-half, ventral one-half or whole caudal fin under anesthesia using scissors sterilized with 70% (v/v) ethanol (Fig. 2). Another group of fish (control group) were left with their caudal fin intact. All fish were placed in a cylindroconical 50-l transparent tank after the removal of fins and treated with 2.5-g antibiotics (Nifurasuchinsan, Kyowa Hakko Kogyo, Tokyo) for 2 h, during which the seawater supply was temporarily shut off. All fish were exposed to a chronic rotary water current at 8 cm s 1 (2 TL s 1) for 10 days and received a formula diet (Love Larvae) at 08:00, 09:00, 10:00, 11:00, 13:00, 15:00 and 17:00 (4.35 g per tank per feeding).
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Fig. 2. The region of caudal fin removal. Caudal fins were cut using scissors sterilized with 70% (v/v) ethanol solution under anesthesia (0.2 ml l 1 of 2-phenoxyethanol).
Caudal fins of fish that were once removed one-half or whole caudal fin were reremoved on the same part on the fifth day of the experiment to prevent regeneration. After 10 days, all fish were collected, anesthetized, killed and treated for bone observation as in Experiments 1 and 2. 2.5. Bone observation We adopted the bone staining method using alizarin red S (Okiyama, 1979) with minor modifications. Both sides of the skin, including scales, were removed from fixed fish with forceps. Then, we washed the fish with tap water for a day to remove formalin. Fish were cleared in 4% (w/v) potassium hydroxide solution with gentle shaking for 3– 7 days at room temperature. We immersed cleared fish in alizarin red solution2 and were shaken for a day. When bone was stained red, the solution was changed to 4% potassium hydroxide solution including 20% (v/v) glycerin. The fish in this solution were shaken for several days. This solution was further changed to a graded series of glycerin dissolved in 4% potassium hydroxide solutions (40%, 55%, 70%, 90% and 100% (v/v)) over several days. Bone and swimbladder of thus prepared fish were observed under a stereomicroscope (SMZ-1B, Nikon, Tokyo). We counted the number of vertebra in a caudad direction in these fish. 2.6. Statistical analysis Data were expressed as number of fish or as frequency (%). We analyzed differences between groups using chi-square analysis (Zar, 1984). For Experiment 1, we arbitrarily divided 90 sampled fish into two groups bordering on the TL of 36.0 mm, and compared the number of lordotic fish between those two groups (Fig. 5). For Experiment 2, we compared number of lordotic fish using the following 2 2 tables; bone morphology (lordosis or normal) current velocity (2 or 4 TL s 1) at either chronic or intermittent current conditions, or bone morphology current condition (chronic or intermittent) at 4 TL s 1. 2 alizarin red S, 0.5 g; acetic acid, 5 ml; glycerin, 10 ml; 1% (w/v) chloral hydrate solution, 60 ml (Wako Pure Chemical Industries).
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Fig. 3. Lordotic vertebra in side viewing. Arrow indicates cuneiform centrum (15th vertebra in this fish).
Fig. 4. Frequency distribution of the number of lordotic vertebrae. n = 90.
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Fig. 5. Size distribution of the sampled fish and lordotic fish (n = 90). More than half the fish had lordotic centrums, with more lordosis in fish less than 36.0-mm body size ( P < 0.01), arbitrarily bordering on the TL of 36.0 mm, when compared the number of lordotic fish on fish size.
For Experiment 3, we also compared number of lordotic fish using the following 2 4 table; bone morphology (lordosis or normal) caudal fin treatments (no removal, complete, upper tail or lower tail). The differences were considered statistically significant when probability was less than 0.05.
3. Results 3.1. Lordotic fish and size distribution All fish beat the tail vigorously during the experiment. Lordotic centrums were cuneiform when viewed from the side (Fig. 3). Lordosis was observed between the
Table 1 Frequency (%) of lordotic fish exposed to rotary current Current velocity 2 TL s Chronic Intermittent
a
0 0a
1
(n) (25) (25)
4 TL s b
25 21b
1
(n) (24) (24)
Fish exposed to less than 0.1 cm s 1 current (control group) had no lordotic vertebra. Number of observations are in parentheses. In both 4 TL s 1 groups, one fish died during the experiment. a,b : The different superscripts indicate statistically significant differences at P < 0.01 by chi-square analysis.
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Table 2 Effect of caudal fin removal on the appearance of lordosis Removal of the caudal fin
(n)
Lordosis frequency (%)
No removal (control) Complete Upper tail Lower tail
(14) (14) (14) (15)
0a 36b 29b 7a
Water current was loaded at 8 cm s 1 (2 TL s 1) for 10 days. The number of observations are in parentheses. One fish died during experiment in each group: ‘‘No removal’’, ‘‘Complete’’ and ‘‘Upper tail’’. a,b : The different superscripts indicate statistically significant differences at P < 0.01 by chi-square analysis.
12th and 18th vertebrae. Highest frequency of lordotic centrum was observed at the 15th vertebra (40%, Fig. 4). The total length of the sampled fish was in the range from 27.0 to 51.6 mm. More than half of the fish had lordotic centrums, with more lordosis in fish less than 36.0-mm body size ( P < 0.01, Fig. 5). All experimental fish had morphologically normal swimbladders. No other serious skeletal deformities except lordosis were seen. 3.2. Effect of current velocity Lordosis was observed only in fish exposed to 4 TL s 1 current velocity (21 –25%). Chronic and intermittence current did not affect the frequency of lordosis (Table 1). Fish exposed to 2 TL s 1 current velocity did not exhibit a cuneiform centrum. All fish had morphologically normal swimbladders. 3.3. Effect of caudal fin removal Fish with their caudal fin completely removed swam with vigorous tail beat, however, they tended to be swept away by the water current more eagerly than fish with their entire or a part of caudal fin retained. Control fish showed no vertebral deformations. However, lordosis was seen in all fish with their caudal fin partly or entirely removed (Table 2). Lordosis frequency was significantly higher in fish with their entire (36%) or upper half of (29%) of caudal fin removed than in fish with their lower caudal fin removed (7%) or entire caudal fin retained (0%) ( P < 0.01).
4. Discussion In this study, lordosis appeared mostly at the 15th vertebra. This agrees with our field observations on our hatchery products in 1996 (data not shown). On the other hand, sea bream, Sparus auratus, without swimbladder (Chatain, 1994) had the lordosis mainly at 10th caudad-counted vertebra. Therefore, we may categorize the lordosis of these fish into two groups. The vertebra of prehemal position is affected when the fish lack buoyancy. On
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the other hand, the vertebra of hemal position is affected when they have normal swimbladder, however, they are obliged to swim vigorously. Although the position of affected vertebra was slightly different from the present experiments, high current conditions induced lordosis in D. labrax having functional swimbladder as indicated by Divanach et al. (1997). Furthermore, although positions of malformed vertebrae have not been described, carp Cyprinus carpio reared in strong-water stream showed a deformation as what appeared in our present experiment (Backiel et al., 1984). Thus, it is likely that the overloading current, i.e., abnormally high muscular activity, induces lordosis in fish with widely different body shape and with different proportion of the bone versus muscle. Lordosis did not appear in fish of 25- and 40-mm TL exposed to 2 TL s 1 current velocity. This result suggests that water current slower than 2 TL s 1 will not induce lordosis in juvenile red sea bream of more than 25 mm TL, agreeing with the results in D. labrax (Divanach et al., 1997). On the other hand, lordosis appeared in fish of 40-mm TL when their caudal fin was removed even at the current velocity of 2 TL s 1. Therefore, the caudal fin should play an important role in lordosis induction. As the caudal fin of carangiform fish is the main origin of propulsion (Webb, 1997), fish without the caudal fin beat the tail more vigorously to make up for the reduction in propulsive force of caudal fin . The interaction between the removal of caudal fin and the effect of current velocity suggests that muscle activity, and not the mechanical action of the caudal fin itself, is responsible for lordosis. Thus, muscle activity from the excess beat of the tail against the current should induce lordosis in spite of morphologically normal swimbladder. It is interesting that the malformation of vertebrae (12 –18th vertebrae) was observed at the region of fulcrum for tail moving. Chatain (1994) suggested that the vertebrae affected in lordotic fish were mainly the ones where muscle pressure was the highest during swimming. Histological changes in C. carpio with lordosis induced by rearing in strong water current (Backiel et al., 1984) had hypertrophic and differentiated muscular fibers in lateral muscle fibers, which might result in abnormal tensions on the bone. Thus, histological abnormalities of muscle fibers and probably of tendons that occurred in lordotic fish may indicate that this type of lordosis does not occur in the earlier stage such as larval stage, when the formation of vertebrae and myotomal muscle is not yet completed in quality. The structure of vertebral column at approximately 9-mm TL and that of myotomal muscle at 20-mm TL do not differ from those at 68.4- and 103.8-mm TL in P. major (Matsuoka, 1987). Accordingly, possibilities of lordosis formation might be increased along with the growth stage in early juveniles. We tested the effect of intermittent current on the occurrence of lordosis in Experiment 2. We also observed that red sea bream rested at the bottom of tank at night when water current was less than 0.1 cm s 1. Accordingly, we did not apply water current to the intermittent group during the dark period (from 17:00 to 08:00, 15 h). However, 9 h of forced swimming per day, cumulatively 90 h per 10 days, induced lordosis. This irreversible damage to vertebrae suggests that the effect of current, i.e., overloaded muscle activity, is cumulative. Therefore, we suggest that the rest in the middle of swimming could not prevent lordosis. In conclusion, high current velocity and excess muscle activity caused by caudal fin removal induced lordosis in the present study. These findings suggest that muscle activities
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from the excess beat of the tail against the increased current should induce lordosis possibly by giving too much stress on the vertebrae. In intensive hatchery ponds, rapid water exchange is required for uniform dispersion of feed and oxygen, and dilution of wastes. Therefore, a strong horizontal rotary current is commonly used in such ponds, especially in circular ponds. Our findings suggest that the velocity of such rotary current should be carefully regulated to avoid lordosis in early-stage red sea bream raising.
Acknowledgements The authors thank our hatchery staff, C. Hirata, S. Yamada, T. Shigee, Y. Higo, M. Migita, H. Nakamura, H. Urui and M. Mamori-Ogata for their excellent technical assistance, and T. Shinohara of Amami Fish Farm for his valuable constructive comments. We wish to thank Dr. Brian J. Harvey of World Fisheries Trust and Prof. Takashi Sakata of Ishinomaki Senshu University for suggesting improvements to the manuscript.
References Backiel, T., Kokurewicz, B., Ogorzalek, A., 1984. High incidence of skeletal anomalies in carp, Cyprinus carpio, reared in cages in flowing water. Aquaculture 43, 369 – 380. Bainbridge, R., 1958. The speed of swimming of fish as related to size and to the frequency and amplitude of the tail beat. J. Exp. Biol. 35, 109 – 133. Chatain, B., 1994. Abnormal swimbladder development and lordosis in sea bass (Dicentrarchus labrax) and sea bream (Sparus auratus). Aquaculture 119, 371 – 379. Divanach, V., Papandroulakis, N., Anastasiadis, P., Koumoundouros, G., Kentouri, M., 1997. Effect of water currents on the development of skeletal deformities in sea bass (Dicentrarchus labrax L.) with functional swimbladder during postlarval and nursery phase. Aquaculture 156, 145 – 155. Foscarini, R., 1988. A review: intensive farming procedure for red sea bream (Pagrus major) in Japan. Aquaculture 72, 191 – 246. Hunter, J., Zweifel, J., 1971. Swimming speed, tail beat frequency, tail beat amplitude, and size in jack mackerel, Trachurus symmetricus, and other fishes. Fish. Bull. 69, 253 – 266. Kitajima, C., Tsukashima, Y., Fujita, S., Watanabe, T., Yone, Y., 1981. Relationship between uninflated swim bladders and lordotic deformity in hatchery-reared red seabream Pagrus major. Bull. Jpn. Soc. Sci. Fish. 47, 1289 – 1294 (in Japanese with English abstract). Matsuoka, M., 1987. Development of the skeletal tissues and skeletal muscles in the red sea bream. Bull. Seikai Reg. Fish. Res. Lab. 65, 1 – 113. Okiyama, M., 1979. Manuals for the larval fish taxonomy (2), general aspects of larval characters that aid identification. Aquabiology 2, 53 – 59 (in Japanese with English abstract). Takashima, F., 1978. Vertebral malformation in hatchery-reared red seabream, Chrysophrys major. Bull. Jpn. Soc. Sci. Fish. 44, 435 – 443 (in Japanese with English abstract). Takashima, F., Arai, Y., Nomura, M., 1980. Abnormal development of the swimbladder in hatchery-reared red sea-bream, Chrysophrys major. J. Tokyo Univ. Fish. 67, 67 – 73 (in Japanese with English abstract). Webb, P., 1997. Swimming. In: Evans, D. (Ed.), The Physiology of Fishes. CRC Press, New York, pp. 3 – 24. Yamaguchi, M., 1978. Tai Youshoku no Kiso to Jissai. Koseisha Koseikaku, Tokyo (in Japanese). Zar, J., 1984. Biostatistical Analysis, 2nd ed. Prentice Hall, New Jersey, pp. 61 – 78.
Lordosis induction in juvenile red sea bream, Pagrus major, by high swimming activity Minoru Kihara a,*, Shigeru Ogata b,1, Noriaki Kawano b, Itsuo Kubota b, Ryoichi Yamaguchi b a
Central Research Institute, Maruha Corporation, Wadai 16-2, Tsukuba, 300-4295, Japan Aquaculturing Operations Department, Maruha Corporation, Nagahama 3-11-3, Chuo, Fukuoka 810-0072, Japan
b
Received 16 December 2000; received in revised form 1 November 2001; accepted 4 November 2001
Abstract Lordosis is correlated with absence or malfunction of the swimbladder. However, swimbladder abnormalities do not completely explain the occurrence of lordosis. We examined whether muscle activities from vigorous tail beat induced by removal of the caudal fin could induce lordotic malformation in juvenile red sea bream. We also attempted to determine the minimal current velocity for inducing lordosis in relation to total length (TL). For this purpose, we employed relative current velocity in terms of ‘‘move of water in times of total body length per second (TL s 1)’’. We compared effects of different water current (2 vs. 4 TL s 1) for 10 days on bone malformation using juvenile of 25-mm TL. Exposure to 4 TL s 1 current velocity, but not 2 TL s 1, induced lordosis. Lordotic juvenile red sea bream had a cuneiform centrum mainly at the 15th vertebra. Lordosis was observed in fish with normal swimbladders as well. Thus, swimbladder abnormalities alone cannot completely explain the occurrence of lordosis. We removed part or all of the caudal fins of fish of 40mm TL and exposed these fish to 2 TL s 1 current for 10 days. Control fish did not show deformations in the vertebrae. Fish with complete removal of the caudal fin swam with vigorous tail beats. However, fish with complete, upper or lower caudal fin removal (36%, 29% or 7%, respectively) displayed lordosis even at the current velocity of 2 TL s 1. These findings suggested that the muscle activities from excess beat of the tail against excessive current, but not the mechanical action of the caudal fin, induced lordosis. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Lordosis; Current velocity; Muscle activity; Vertebra; Red sea bream
* Corresponding author. Present address: Utsunomiya Plant, Maruha Corporation, Kiyohara Industrial Area 8-1, Utsunomiya 321-3297, Japan. Tel.: +81-28-667-0801; fax: +81-28-667-0736. E-mail address: [email protected] (M. Kihara). 1 Present address: Taikei Gyogyo, Uomachi 3-13-7, Ishinomaki 986-0022, Japan.
0044-8486/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 1 ) 0 0 8 7 1 - 7
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M. Kihara et al. / Aquaculture 212 (2002) 149–158
1. Introduction Red sea bream Pagrus major is a marine fish of great economic importance in Japan (Foscarini, 1988). The species is reared in net pens and the juveniles are produced artificially. Thus, seed production of this fish plays an important role in the farming of red sea bream in Japan. However, serious skeletal malformations such as brachyospondylie and lordosis appear frequently in hatchery-produced juveniles (Takashima, 1978). These abnormalities are often associated with poor growth and low survival of the seed. Lordosis is correlated with absence or malfunction of the swimbladder (Takashima et al., 1980; Kitajima et al., 1981). However, swimbladder abnormalities do not completely explain the occurrence of lordosis. Divanach et al. (1997) indicated that similar malformations occurred in juvenile sea bass Dicentrarchus labrax L. despite the presence of a functional swimbladder, and related lordosis to the high current velocity ( > 10 cm s 1). Tail beat frequency increases with swimming speed (Bainbridge, 1958; Hunter and Zweifel, 1971). Thus, these findings suggest that swimming dynamics, i.e., muscle activities by water current, may induce lordosis. The role of current velocity on lordosis induction was already explored (Divanach et al., 1997; Backiel et al., 1984). However, we do not know yet how the high current velocity induced lordosis in these studies. Observing the fish, we hypothesized that abnormally high muscle activity, especially in the caudal part of the body, might be responsible for the induction of lordosis by high current velocity. Accordingly, our aim in this study was to examine whether muscle activities from the vigorous tail beat induced by removal of caudal fin, the main means of propulsion, induced lordotic malformation. For this purpose, we also conducted a preliminary study to find out the minimal current velocity to induce lordosis in juvenile-size fish.
2. Materials and methods 2.1. Fish husbandry Spontaneously spawned eggs were obtained from red sea bream broodstock reared in an indoor 20 m3 concrete square tank in our hatchery (28j26VN:129j32VE, Amami Island, Japan). Eggs were incubated, hatched and reared using generally established techniques for red sea bream hatcheries in Japan (Yamaguchi, 1978). Larvae were maintained in a tank of 75 m3 with flowing UV-sterilized seawater at 23.0 F 1.0 jC and fed-enriched rotifers Brachionus plicatilis from 4 days after hatching (DAH) to 38 DAH and enriched brine shrimp Artemia nauplius from 20 DAH on. A formula diet (Love Larvae, Hayashikane Sangyo, Shimonoseki, Japan) was also fed from 20 DAH on. To remove oil film on water surface, we used oil skimmer consisted of vinylchloride pipes equipped with an air blow system 5 DAH. Experiments were conducted in November 1997. The experiment was conducted strictly following the guideline for animal experiments of Central Research Institute of MARUHA. The guideline adheres to governmental legislation in Japan.
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2.2. Experiment 1: lordosis induction by water current Three hundred fish aged 47, 51 or 52 DAH were randomly selected and kept in a 500-l transparent cylindroconical tank. Total length (TL) of selected fish varied from 25.5 to 50.4 mm. UV-sterilized seawater at 22.0 F 1.0 jC was supplied to the tank horizontally through a vinyl-chloride pipe (13-mm I.D.) to induce a rotary water current. Horizontal water current velocity was between 10 cm s 1 (bottom wall side) and 14 cm s 1 (top wall side). Water current velocity in the tank was measured using an electromagnetic current meter (ACM100-D, Alec Electronics, Osaka). Fish were adapted to an 11 h light:13 h dark cycle (lights on from 07:00). We fed a commercial formula diet (Love Larvae) to fish using an automatic feeder (Food Timer, Seiko, Tokyo), every hour from 07:00 to 17:00. Each feeding consisted of 4.35 g per tank. After 7 days, 90 fish were randomly collected, anesthetized with 0.2 ml l 1 of 2phenoxyethanol (Wako Pure Chemical Industries, Osaka) and measured for total length with slide calipers. Then, the fish were fixed with 10% (v/v) formalin in neutral phosphate buffer solution (pH 7.4, Wako Pure Chemical Industries) for bone observation. The difference of total length measured as a straight line or as a curve was at most 1.7% (data not shown). Therefore, we measured the total length as a straight-line method. 2.3. Experiment 2: effect of current velocity Approximately 500 juvenile red sea bream aged 35 DAH were held in a 1000-l cylindroconical polycarbonate tank (stock tank). Seawater was supplied vertically to avoid horizontal rotary water current. We fed a commercial diet (Love Larvae) to fish using an automatic feeder, seven times a day (08:00, 09:00, 10:00, 11:00, 13:00, 15:00 and 17:00). Each feeding consisted of 0.2 g per tank. We randomly selected 125 fish of 25-mm TL from the stock tank at the age of 40 DAH, and randomly divided them into five groups of 25. We examined the effect of current pattern (chronic vs. intermittent) and current speed (5 vs. 10 cm s 1) on bone malformation in these fish. The current velocity of 5 or 10 cm s 1 in the present study corresponded to 2 or 4 TL s 1, respectively. Fish were treated in one of the three 10-l transparent cylinder tanks to create different water flow (Fig. 1). Tank A was for the zero rotary current group (control group, Fig. 1A). Tank A was equipped with an inverted glass bowl (10 cm in diameter) under the water supply-nozzle. The bowl dispersed the downward water current and kept the rotating current velocity less than 0.1 cm s 1. The volume of water supplied to tank A was 4 TL s 1. Tank B was equipped with a flexible nozzle of 7-mm I.D. The nozzle was bent at 45j to create chronic rotary current of 2 or 4 TL s 1 (Fig. 1B). Tank C was equipped with both an inverted glass bowl and a flexible-nozzle (Fig. 1C). Water was supplied alternately either from the top of the tank or from the nozzle for intermittent current groups (2 or 4 TL s 1). The rotary water current was applied between 08:00 and 17:00 in these groups.
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Fig. 1. A, B and C: Schematic drawing of the experimental tanks (top, side view; bottom, top view). (a) Tube for the air inlet equipped with stone (c); (b) water supply tube; (d) circular transparent tank for 10 l; (e) glass bowl (10 cm in diameter); (f) vinyl-chloride pipe (13-mm I.D.) for water outlet; (g) a vinyl-chloride pipe (25-mm I.D.); (h) a commercial flexible-straw (7-mm I.D.) bent at 45j; (i) valves.
The volume of water supplied to tank A, B or C was kept constant during the experiment. Water current velocity in the tanks was measured at approximately 2 cm high from the bottom and wall-side of tanks. We fed a commercial diet (Love Larvae) by a hand as same pattern and same amount of the above stock rearing for 10 days. Fish were collected after 10 days of exposure to one of the water current stated above. They were anesthetized, killed and fixed for bone observation as in Experiment 1. 2.4. Experiment 3: effect of caudal fin removal Sixty fish of 40-mm TL were selected from the stock tank at 60 DAH and randomly divided into four groups of 15. We adopted whole and partial caudal fin removal to assess the contribution of propulsion to lordosis. Our hypothesis is whether muscle activities from the vigorous tail beat induced by removal of caudal fin, the main means of propulsion, induce lordotic malformation without increasing the current velocity. We cut dorsal one-half, ventral one-half or whole caudal fin under anesthesia using scissors sterilized with 70% (v/v) ethanol (Fig. 2). Another group of fish (control group) were left with their caudal fin intact. All fish were placed in a cylindroconical 50-l transparent tank after the removal of fins and treated with 2.5-g antibiotics (Nifurasuchinsan, Kyowa Hakko Kogyo, Tokyo) for 2 h, during which the seawater supply was temporarily shut off. All fish were exposed to a chronic rotary water current at 8 cm s 1 (2 TL s 1) for 10 days and received a formula diet (Love Larvae) at 08:00, 09:00, 10:00, 11:00, 13:00, 15:00 and 17:00 (4.35 g per tank per feeding).
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Fig. 2. The region of caudal fin removal. Caudal fins were cut using scissors sterilized with 70% (v/v) ethanol solution under anesthesia (0.2 ml l 1 of 2-phenoxyethanol).
Caudal fins of fish that were once removed one-half or whole caudal fin were reremoved on the same part on the fifth day of the experiment to prevent regeneration. After 10 days, all fish were collected, anesthetized, killed and treated for bone observation as in Experiments 1 and 2. 2.5. Bone observation We adopted the bone staining method using alizarin red S (Okiyama, 1979) with minor modifications. Both sides of the skin, including scales, were removed from fixed fish with forceps. Then, we washed the fish with tap water for a day to remove formalin. Fish were cleared in 4% (w/v) potassium hydroxide solution with gentle shaking for 3– 7 days at room temperature. We immersed cleared fish in alizarin red solution2 and were shaken for a day. When bone was stained red, the solution was changed to 4% potassium hydroxide solution including 20% (v/v) glycerin. The fish in this solution were shaken for several days. This solution was further changed to a graded series of glycerin dissolved in 4% potassium hydroxide solutions (40%, 55%, 70%, 90% and 100% (v/v)) over several days. Bone and swimbladder of thus prepared fish were observed under a stereomicroscope (SMZ-1B, Nikon, Tokyo). We counted the number of vertebra in a caudad direction in these fish. 2.6. Statistical analysis Data were expressed as number of fish or as frequency (%). We analyzed differences between groups using chi-square analysis (Zar, 1984). For Experiment 1, we arbitrarily divided 90 sampled fish into two groups bordering on the TL of 36.0 mm, and compared the number of lordotic fish between those two groups (Fig. 5). For Experiment 2, we compared number of lordotic fish using the following 2 2 tables; bone morphology (lordosis or normal) current velocity (2 or 4 TL s 1) at either chronic or intermittent current conditions, or bone morphology current condition (chronic or intermittent) at 4 TL s 1. 2 alizarin red S, 0.5 g; acetic acid, 5 ml; glycerin, 10 ml; 1% (w/v) chloral hydrate solution, 60 ml (Wako Pure Chemical Industries).
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Fig. 3. Lordotic vertebra in side viewing. Arrow indicates cuneiform centrum (15th vertebra in this fish).
Fig. 4. Frequency distribution of the number of lordotic vertebrae. n = 90.
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Fig. 5. Size distribution of the sampled fish and lordotic fish (n = 90). More than half the fish had lordotic centrums, with more lordosis in fish less than 36.0-mm body size ( P < 0.01), arbitrarily bordering on the TL of 36.0 mm, when compared the number of lordotic fish on fish size.
For Experiment 3, we also compared number of lordotic fish using the following 2 4 table; bone morphology (lordosis or normal) caudal fin treatments (no removal, complete, upper tail or lower tail). The differences were considered statistically significant when probability was less than 0.05.
3. Results 3.1. Lordotic fish and size distribution All fish beat the tail vigorously during the experiment. Lordotic centrums were cuneiform when viewed from the side (Fig. 3). Lordosis was observed between the
Table 1 Frequency (%) of lordotic fish exposed to rotary current Current velocity 2 TL s Chronic Intermittent
a
0 0a
1
(n) (25) (25)
4 TL s b
25 21b
1
(n) (24) (24)
Fish exposed to less than 0.1 cm s 1 current (control group) had no lordotic vertebra. Number of observations are in parentheses. In both 4 TL s 1 groups, one fish died during the experiment. a,b : The different superscripts indicate statistically significant differences at P < 0.01 by chi-square analysis.
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Table 2 Effect of caudal fin removal on the appearance of lordosis Removal of the caudal fin
(n)
Lordosis frequency (%)
No removal (control) Complete Upper tail Lower tail
(14) (14) (14) (15)
0a 36b 29b 7a
Water current was loaded at 8 cm s 1 (2 TL s 1) for 10 days. The number of observations are in parentheses. One fish died during experiment in each group: ‘‘No removal’’, ‘‘Complete’’ and ‘‘Upper tail’’. a,b : The different superscripts indicate statistically significant differences at P < 0.01 by chi-square analysis.
12th and 18th vertebrae. Highest frequency of lordotic centrum was observed at the 15th vertebra (40%, Fig. 4). The total length of the sampled fish was in the range from 27.0 to 51.6 mm. More than half of the fish had lordotic centrums, with more lordosis in fish less than 36.0-mm body size ( P < 0.01, Fig. 5). All experimental fish had morphologically normal swimbladders. No other serious skeletal deformities except lordosis were seen. 3.2. Effect of current velocity Lordosis was observed only in fish exposed to 4 TL s 1 current velocity (21 –25%). Chronic and intermittence current did not affect the frequency of lordosis (Table 1). Fish exposed to 2 TL s 1 current velocity did not exhibit a cuneiform centrum. All fish had morphologically normal swimbladders. 3.3. Effect of caudal fin removal Fish with their caudal fin completely removed swam with vigorous tail beat, however, they tended to be swept away by the water current more eagerly than fish with their entire or a part of caudal fin retained. Control fish showed no vertebral deformations. However, lordosis was seen in all fish with their caudal fin partly or entirely removed (Table 2). Lordosis frequency was significantly higher in fish with their entire (36%) or upper half of (29%) of caudal fin removed than in fish with their lower caudal fin removed (7%) or entire caudal fin retained (0%) ( P < 0.01).
4. Discussion In this study, lordosis appeared mostly at the 15th vertebra. This agrees with our field observations on our hatchery products in 1996 (data not shown). On the other hand, sea bream, Sparus auratus, without swimbladder (Chatain, 1994) had the lordosis mainly at 10th caudad-counted vertebra. Therefore, we may categorize the lordosis of these fish into two groups. The vertebra of prehemal position is affected when the fish lack buoyancy. On
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the other hand, the vertebra of hemal position is affected when they have normal swimbladder, however, they are obliged to swim vigorously. Although the position of affected vertebra was slightly different from the present experiments, high current conditions induced lordosis in D. labrax having functional swimbladder as indicated by Divanach et al. (1997). Furthermore, although positions of malformed vertebrae have not been described, carp Cyprinus carpio reared in strong-water stream showed a deformation as what appeared in our present experiment (Backiel et al., 1984). Thus, it is likely that the overloading current, i.e., abnormally high muscular activity, induces lordosis in fish with widely different body shape and with different proportion of the bone versus muscle. Lordosis did not appear in fish of 25- and 40-mm TL exposed to 2 TL s 1 current velocity. This result suggests that water current slower than 2 TL s 1 will not induce lordosis in juvenile red sea bream of more than 25 mm TL, agreeing with the results in D. labrax (Divanach et al., 1997). On the other hand, lordosis appeared in fish of 40-mm TL when their caudal fin was removed even at the current velocity of 2 TL s 1. Therefore, the caudal fin should play an important role in lordosis induction. As the caudal fin of carangiform fish is the main origin of propulsion (Webb, 1997), fish without the caudal fin beat the tail more vigorously to make up for the reduction in propulsive force of caudal fin . The interaction between the removal of caudal fin and the effect of current velocity suggests that muscle activity, and not the mechanical action of the caudal fin itself, is responsible for lordosis. Thus, muscle activity from the excess beat of the tail against the current should induce lordosis in spite of morphologically normal swimbladder. It is interesting that the malformation of vertebrae (12 –18th vertebrae) was observed at the region of fulcrum for tail moving. Chatain (1994) suggested that the vertebrae affected in lordotic fish were mainly the ones where muscle pressure was the highest during swimming. Histological changes in C. carpio with lordosis induced by rearing in strong water current (Backiel et al., 1984) had hypertrophic and differentiated muscular fibers in lateral muscle fibers, which might result in abnormal tensions on the bone. Thus, histological abnormalities of muscle fibers and probably of tendons that occurred in lordotic fish may indicate that this type of lordosis does not occur in the earlier stage such as larval stage, when the formation of vertebrae and myotomal muscle is not yet completed in quality. The structure of vertebral column at approximately 9-mm TL and that of myotomal muscle at 20-mm TL do not differ from those at 68.4- and 103.8-mm TL in P. major (Matsuoka, 1987). Accordingly, possibilities of lordosis formation might be increased along with the growth stage in early juveniles. We tested the effect of intermittent current on the occurrence of lordosis in Experiment 2. We also observed that red sea bream rested at the bottom of tank at night when water current was less than 0.1 cm s 1. Accordingly, we did not apply water current to the intermittent group during the dark period (from 17:00 to 08:00, 15 h). However, 9 h of forced swimming per day, cumulatively 90 h per 10 days, induced lordosis. This irreversible damage to vertebrae suggests that the effect of current, i.e., overloaded muscle activity, is cumulative. Therefore, we suggest that the rest in the middle of swimming could not prevent lordosis. In conclusion, high current velocity and excess muscle activity caused by caudal fin removal induced lordosis in the present study. These findings suggest that muscle activities
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from the excess beat of the tail against the increased current should induce lordosis possibly by giving too much stress on the vertebrae. In intensive hatchery ponds, rapid water exchange is required for uniform dispersion of feed and oxygen, and dilution of wastes. Therefore, a strong horizontal rotary current is commonly used in such ponds, especially in circular ponds. Our findings suggest that the velocity of such rotary current should be carefully regulated to avoid lordosis in early-stage red sea bream raising.
Acknowledgements The authors thank our hatchery staff, C. Hirata, S. Yamada, T. Shigee, Y. Higo, M. Migita, H. Nakamura, H. Urui and M. Mamori-Ogata for their excellent technical assistance, and T. Shinohara of Amami Fish Farm for his valuable constructive comments. We wish to thank Dr. Brian J. Harvey of World Fisheries Trust and Prof. Takashi Sakata of Ishinomaki Senshu University for suggesting improvements to the manuscript.
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