Volume 196 - 1993 - Part 26 of 45
Transcription
Volume 196 - 1993 - Part 26 of 45
ICES mar. Sei. Symp., 196: 117-121. 1993 The measurement of muscle fatigue in walleye pollock (Theragra chalcogramma) captured by trawl Gang X u, Takafumi A rim oto, and Yoshihiro Inoue Xu, G ., A rim oto, T ., and Inoue, Y. 1993. The measurem ent of muscle fatigue in walleye pollock (Theragra chalcogramma) captured by trawl. - ICES mar. Sei. Symp., 196: 117-121. Walleye pollock ( Theragra chalcogramma) were captured by otter trawl in the fishing grounds off Hokkaido, northern Japan. In order to investigate the muscle fatigue of a fish in relation to its swimming ability during the capture process, samples of the dorsal white muscle of captured fish with a mean fork length of 43 cm were taken on board and the concentrations of lactic acid and ATP-related compounds (A D P, AM P, IMP) were later determ ined in the laboratory. Lactic acid concentration ranged from 52 to 239 mg 100 g -1 wet muscle and A T P concentration from 0.13 to 1.57 //mol g _1 wet muscle in the fish immediately after capture. Some of the fish had a higher ATP concentration of about 1 /^mol g ~ 1with a lower lactic acid concentration of about 50 mg 100 g -1 , very similar to that observed in the fish after 24 h recovery in a 500 litre holding tank. During a recovery period of 24 h, the A TP concentration did not increase, but the concentration of ATP-related compounds such as A D P and AM P increased gradually. The resuits suggest that some of the walleye pollock might not have experienced complete muscle fatigue, and might be able to swim longer inside the net, which was towed at speeds of 4.0 to 4.6 knots. Gang X u: Fisheries and Marine Institute o f M emorial University o f Newfoundland, PO Box 4920, St J o h n ’s, Newfoundland, Canada A I C 5R3; Takafum i A rim oto: Tokyo University o f Fisheries, M inato-Ku, Tokyo, 108 Japan; Yoshihiro Inoue: National Institute o f Fisheries Engineering, Kachidoki, Chuo-Ku, Tokyo, 104 Japan. Introduction Changes in carbohydrate metabolism in fish following severe muscular exercise have been well reported in relation to muscle fatigue (Beamish, 1966a, 1968; Prit chard et al., 1971; Johnston and Goldspink, 1973;Driedzic and Hochachka, 1976). In most fishes, the lateral musculature consists of two main fibre types, usually term ed red and white muscle. The red muscle is aerobi cally active by itself during swimming at sustained speeds, whereas during burst swimming the white muscle becomes active together with the red muscle. The white muscle carries out anaerobic glycolytic metabolism which results in glycogen depletion and lactic acid accumulation (Bone, 1966). The glycogen in white muscle is one of the major fuel sources supplying the energy required during swimming. In the glycolytic pathways, the formation of A TP, which serves as the immediate source of energy for muscle contraction, is the most important function. In view of the relationship between energy supply and muscle contraction, studies on the concentrations of muscle lactic acid and ATPrelated compounds may improve the understanding of muscle fatigue in fish after strenuous exercise, such as swimming inside towed fishing gears. Much of the older literature in this area has been reviewed by Driedzic and Hochachka (1978). Walleye pollock ( Theragra chalcogramma) are fre quently captured by otter trawls in the offshore fishery of northern Japan. Observations with an underw ater video camera during the capture process have shown that most of the fish were inactive, both in the net mouth and inside the net, which was towed at about 3.8 knots (Inoue et al., 1992). However, from the video record ings, it could not be determined whether the fish had been exhausted by swimming along with the trawl at the towing speed. In this study, a series of experiments were carried out to determine the level of muscle fatigue in captured walleye pollock. 117 Material and methods All research tows were made by two 124 G .R .T . type commercial trawlers RV “No. 85 Y awata-maru” during 27-28 July 1988 and RV “No. 67 Eishou-maru” during 24-25 July 1989, in the fishing ground off Kushiro on the east coast of Hokkaido, Japan. The gear was towed along depth contours ranging from 154 to 235 m. The towing speed varied between 3.8 and 4.6 knots accord ing to sea conditions. The towing duration was between 50 and 180 min, depending on the num ber of fish inside or around the net. Samples of walleye pollock were taken at random about 2 min after the net was hauled aboard at the end of each tow. In the 1988 trials, muscle samples were taken from the fish immediately after capture. In the 1989 trials, the fish were first sampled from the catch immediately after capture and then placed in an aerated 500 litre holding tank for recovery. Muscle samples were then taken from the fish after recovery periods of 0.3, 6, and 24 h (Table 1). For analysis of both lactic acid and ATP-related compounds, muscle samples (1 g) were cut from the dorsal white muscle block above the anus of freshly killed fish. The muscle pieces were then placed in a scintillation vial with 5 ml of cold (0°C) perchloric acid (PCA) solution (6%) to remove protein. A fter the muscle pieces were homogenized in ice, the samples were kept in a “Styrofoam” bag containing dry ice and brought back to Tokyo University of Fisheries for bio chemical analyses. The mixture was filtered with a 0.45 ,um syringe filter and the filtrate was centrifuged at 3000 g for 8 min. The supernatant solution was stored at —30°C until analysis. Lactic acid concentration was determined colorimetrically by the method of Barker and Summerson (1941). All readings were made at 560 nm in a spectrophotom eter (Hitachi, UV-1000). Lactic acid concentration is expressed as milligrams of lactic acid per 100 g wet muscle. A T P and its related compounds, A D P , A M P, and IMP, were determined by high-performance liquid chromatography (HPLC), measuring UV absorption at 254 nm (Suwetja et al., 1989). A 5 /A PCA extract was injected into a TSKgel O DS-80™ column (5 /jm, 4.6 X 150 mm, Tosoh) equilibrated with 3% m ethanol in 0.05M K2H P 0 4 buffer, pH 6.5 (PB). Elution was con ducted at a flow rate of 0.5 ml m in- 1 , first with a linear gradient between 3% and 15% m ethanol in PB for 30 s, then with 15% methanol in PB for 19.5 min. Identifi cation of ATP-related compounds was carried out by comparing the retention time of peaks in H PL C between the sample and standard compounds. A T P concen tration is expressed as micromoles A T P per 1 g wet muscle, as are the A D P , A M P, and IMP concentrations. All changes in lactic acid and ATP-related compounds were tested for significance using Student’s t test. Results In the 1988 trial, sampling was carried out over a total of five tows. Six to eight individuals were sampled for each tow. For the fish immediately after capture, the concen tration levels of muscle lactic acid varied between 52 and 239 mg 100 g ' 1. From a total of 38 samples, 14 samples had lactic acid concentrations that were under 100 mg 100 g ' 1. O n the other hand, muscle A T P concentrations in the fish immediately after capture were relatively low. With the exception of a few samples, most were under the level of 1/^mol g“ 1 (Fig. 1). The mean values of lactic acid and A TP concentrations for samples in each tow are given in Table 2. Most of the fish immediately after capture were characterized by a high lactic acid and low A T P concentration in their white muscles. However, some fish had a low lactic acid and high A T P concen tration. These conflicting results between individuals and tows may be associated with differences in operating conditions, or variations in fish exertion during the capture process. However, we found no conclusive evi dence to explain the relationships quantitatively. In 1989, measurem ents were carried out in fish after different recovery periods. Fish were removed from the holding tank and decapitated immediately. The mean value of lactic acid concentration for five individuals after 18 min recovery was 154.2 mg 100 g“ 1. This value Table 1. Operating conditions of the fishing boats during the sea trials collecting the samples of walleye pollock for biochemical analyses. Tow no. 1 2 3 4 5 6 7 8 118 Sample date Towing period (min) Towing depth (m) Towing speed (kt) Fishing method 27 Jul 1988 27 J u l 1988 27 Jul 1988 27 Jul 1988 27 Jul 1988 24 Jul 1989 25 Jul 1989 25 Jul 1989 130 50 180 125 125 135 85 78 163-215 205 - 2 3 5 195 190 185 205 165 154 4.6 4.0 4.2 4.5 4.1 3.8 4.5 4.3 Bottom trawl Mid-water trawl Bottom trawl Bottom trawl Bottom trawl Bottom trawl Bottom trawl Bottom trawl during the recovery process. A fter 24 h recovery, the total free adenylate pool was restored to 12.15 ,Mmol g~ 1, which was three times as much as that after 18 min recovery. The energy charge, [(ATP) +0.5(A D P)]/ [(ATP) + (A D P) + (AMP)] as defined by Atkinson (1968), was low at about 0.20 in all states of recovery (Table 3). 30% 30% n»38 n *3 8 20 20 10 10 0 0 0 100 L a c tic 200 0 300 a c id (mg/100g) 0.5 1. 0 1.5 A T P ((imol/g) Discussion Figure 1. Percent frequency distributions of concentrations of muscle lactic acid and A TP in walleye pollock immediately after capture in tows 1 to 5 (see Table 1). was higher than that for the fish immediately after capture (121.7 mg 100 g_1). A fter 6 h recovery, the lactic acid concentration had not changed significantly (P> 0.05), compared with that in the fish after 18 min recovery. By contrast, the fish after 24 h recovery showed a great reduction in lactic acid concentration (Fig. 2). This mean value was 52.6 mg 100 g - 1, very close to that in some fish with low lactic acid. A TP concentration remained low and unchanged dur ing all recovery periods (Fig. 2). In addition to lactic acid and A TP, measurements of A D P, A M P, and IMP were also carried out to observe the total change in the free adenylate pool. The concentrations of both A D P and AM P in muscle increased, whereas IMP decreased as the recovery period increased. The total free adenylate pool [(ATP) + (A D P) + (AM P)j increased gradually Many investigators have used lactic acid as a measure of muscle fatigue in fish following exercise (Beamish, 1966a). For Atlantic cod (Gadus morhua) of about 40 cm length, white muscle lactic acid in the fish after swimming at 130 cm s-1 for 30 min was higher (189 mg 100 g~*) than that in unexercised fish (66.8 mg 100 g-1 ). Despite 4 h recovery, its lactic acid level was almost unchanged and remained high (153.7 mg 100 g ~ ’). A fter 8 h recovery, the muscle lactic acid disappeared gradu ally to near unexercised levels (52.9 mg 100 g“ 1) (Beamish, 1968). With reference to lactic acid concen tration in plaice Pleuronectes platessa L. immediately after capture by otter trawl, a high level of 297-396 mg 100 g“ 1 in white muscle was observed (W ardle, 1978). That muscle lactic acid in walleye pollock immediately after capture was much higher than after 24 h recovery is in accord with the observations of Beamish (1968) and Wardle (1978). Based on absolute lactic acid values, it seems likely that most of the walleye pollock captured by otter trawl have experienced strenuous muscular exer s* 15, 10 200 ■8 c AMP IMP 3 8. £o 5 -3 E ADP ATP < o 00.3 24 0 0.3 6 24 R ec ov ery p erio d (ho u rs) Figure 2. Changes in muscle lactic acid and ATP-related compounds in walleye pollock after different recovery periods. Values shown are means for five individuals (see text). Table 2. Mean values for muscle lactic acid and ATP-related compounds of walleye pollock immediately after capture by trawls. Tow no. 1 2 3 4 5 Mean No. of fish Fork length (cm) Lactic acid (mg/100 g) ATP (Mmol/g) 6 8 8 8 8 4 1 .7 ± 4 .r 45.1 ± 4.5 40.0±4.8 41.8+6.1 43.9±5.4 82.2 ± 16.0 140.3± 19.6 156.3+37.9 104.8±36.2 115.3±52.7 0.98±0.33 0.41 ±0.11 0.52±0.12 0.41 ±0.11 0.34±0.11 42.5±5.4 121.7±43.9 0.5110.27 a Standard errors (s.e.). 119 Table 3. pollock. Comparisons of the values of total free adenylate pool and energy charge after different recovery periods in walleye Recovery period (h) Metabolite Adenylate pool (umol/g)a Energy chargeb No. of fish 0.3 6 24 5 -7 5 -7 4.74±0.63c 0.17±0.01 5.79±0.58 0.21 ±0.01 12.15±1.26 0.16±0.02 a (ATP) + (ADP) + (AMP). b f(ATP) +0.5(ADP)]/[(ATP) + (ADP) + (AMP)]. cStandard errors (s.e.). tion and become fatigued during the capture process. However, in some of the walleye pollock, only lower lactic acid levels, corresponding to that after 24 h recov ery, were detected. These fish are likely to have a different level of muscle fatigue from that in most of the fish caught by trawls. With respect to changes in nucleotides, Jones and Murray (1957) reported that in rested cod Gadus callarias, A TP concentration in muscle was 5.34 «mol g- 1 , and in exhausted cod which were caught by trawl, A TP decreased to a low level of 0.26 «mol g_1, with a striking increase in IMP. Usually the rate of utilization of ATP for muscle action is related to its rate of production through glycolytic metabolism. As the work load of the tissue exceeds its aerobic capabilities, the 5'-A M P de aminase converts A M P to IMP, and the adenylate pool is decreased, resulting in muscle A TP reduction (Driedzic and Hochachka, 1978). O ur observations in fish immediately after capture showed that A TP concen trations in white muscle were low for most of the fish. In contrast to the gradual disappearance of muscle lactic acid following recovery, muscle A T P was not restored significantly in any recovery period (P>0.05) (Fig. 2). However, the obvious increase in the total free adeny late pool after recovery might account for some of the muscular recovery from fatigue (Table 3). The changes in the free adenylate pool were very similar to the patterns between rested and exhausted fish observed by Jones and Murray (1957) and Driedzic and Hochachka (1978). On the other hand, the level of energy charge was almost unchanged and remained low during recov ery periods. This fact may indicate that these fish had not completely returned to an unexercised state even after 24 h recovery. It seems likely that slow muscular recov ery from fatigue might be associated with degree of fatigue, as well as experimental conditions including influences of engine noise and vessel vibration. In the present study, the capture of walleye pollock could have occurred at any time during the tow. It is hard to assign qualitative figures to strength of exercise and physical condition of fish (Beamish, 1966a). When fish are able to detect trawl gears visually, they have been observed to maintain station with the gear as it is towed. When fish are unable to swim to keep up with the trawl. 120 they become exhausted and drop back into the codend (Wardle, 1983). However, in the absence of vision, fish can only react to a moving net by a startle reaction when struck by the net (Glass and W ardle, 1989). Since our trawls were towed in the deep sea at about 200 m, the fish were estimated to recognize the trawl gear probably with a low acuity in this dark environment (Zhang and A rim oto, 1993). During the capture process video recordings were taken to observe the behaviour of walleye pollock at a water tem perature of 2°C, while artificial light was provided by a halogen lamp of 150 W. The video recordings showed that most of the walleye pollock inside the net did not swim actively and drifted passively towards the codend, even in the artificial light condition (Inoue et a i , 1992). For G adidae of similar size, it was found that at low tem peratures from 0 to 5°C, Atlantic cod could only maintain endurance swimming for several seconds at high speeds of above 2 m s_1 (Beamish, 1966b; He, 1991). For walleye pollock of 5053 cm length, the maximum swimming speed estimated from muscle contraction at 5°C was 2 . 1 m s -1 (Arimoto et al., 1991). H ere, if the walleye pollock swim to keep up with the trawl at towing speeds of 4.0 to 4.6 knots (2.1 to 2.4 m s~ !), the fish would be exhausted to a great extent after burst swimming for a short time. Wardle (1983) suggested that the fish in the codend are exhaus ted to varying degrees by their efforts made during the capture process. Therefore, some captured walleye pol lock with a lower lactic acid and higher A T P concen tration probably had not experienced complete muscle fatigue during the capture process and could have swum for longer. This result implies that inactive fish observed with an underwater video camera might not have swum long with the trawl at high speeds, and before becoming completely exhausted most of them already dropped into and struggled in the codend, while some were quiet in the codend. Acknow ledgem ents We are grateful to D rs Takaaki Shirai, Ken Suzuki, and Toshio H irano, professors of the D epartm ent of Food Science and Technology of Tokyo University of Fish eries, for encouragement and valuable suggestions dur ing the course of this study. We thank M r Frank Chopin for his kind inspection of the manuscript. Thanks are also due to the captains and crew of RV “No. 85 Y awatam aru” and “No. 67 Eishou-maru” for their assistance during the cruise. R eferences A rim oto, T ., Xu, G ., and Matsushita, Y. 1991. Muscle contrac tion time of captured walleye pollock Theragra chalco gramma. Bull. Jap. Soc. Sei. Fish., 57: 1225-1228. Atkinson, D. E. 1968. The energy charge of the adenylate pool as a regulatory param eter. Interaction with feedback modi fiers. Biochemistry, 7: 4030-4034. B arker, S. B., and Summerson, W. H. 1941. The colorimetric determ ination of lactic acid in biological material. J. Biol. C hem ., 138: 535-554. Beamish, F. W. H. 1966a. Muscular fatigue and mortality in haddock Melanogrammus aeglefinus caught by otter trawl. J. Fish. Res. Bd Can., 23: 1507-1521. Beamish, F. W. H. 1966b. Swimming endurance of some northern Atlantic fishes. J. Fish. Res. Bd Can., 23: 341-347. Beamish, F. W. H. 1968. Glycogen and lactic acid concen trations in Atlantic cod Gadus morhua in relation to exercise. J. Fish. Res. Bd C an., 25: 837-851. Bone, Q. 1966. On the function of the two types of myotomal muscle fibres in elasmobranch fish. J. Mar. Biol. Assoc. UK, 46: 321-349. Driedzic, W. D ., and Hochachka, P. W. 1976. Control of energy metabolism in fish white muscle. Am. J. Physiol., 230: 579-582. Driedzic, W. D., and Hochachka, P. W. 1978. Metabolism in fish during exercise. In Fish physiology, vol. 5: locomotion, pp. 503-543. Ed. by W. S. H oar and D. J. Randall, Academic Press, London. 576 pp. Glass, C. W ., and Wardle, C. S. 1989. Comparison of the reactions of fish to a trawl gear, at high and low light intensities. Fish. R es., 7: 249-268. H e, P. 1991. Swimming endurance of the Atlantic cod. Gadus morhua L., at low tem perature. Fish. Res., 12: 65-73. Inoue, Y., Matsushita, Y ., and A rim oto, T. 1992. Swimming performace of walleye pollock Theragra chalcogramma in deep/low tem perature trawl fishing ground (this volume). Johnston, I. A ., and Goldspink, G. 1973. A study of the swimming performance of the crucian carp Carassius carassius (L) in relation to the effects of exercise and recovery on biochemical changes in the myotomal muscles and liver. J. Fish Biol., 5: 249-260. Jones, D. R ., and Murray. J. 1957. Nucleotides in the skeletal muscle of codling Gadus callarias. Biochem. J., 66: 5-6. Pritchard, A. W ., H unter, J. R ., and Lasker, R. 1971. The relation between exercise and biochemical changes in red and white muscle and liver in the jack mackerel Trachurus symmetricus. Fish. Bull., 69: 379-386. Suwetja, I. K., Hori, K., and Miyazawa, K. 1989. Changes in content of ATP-related compounds, hom arine, and trig onelline in marine invertebrate during ice storage. Bull. Jap. Soc. Sei. Fish., 55: 559-566. W ardle, C. S. 1978. Non-release of lactic acid from anaerobic swimming muscle of plaice Pleuronectes platessa L.: a stress reaction. J. Exp. Biol., 7: 141-155. W ardle, C. S. 1983. Fish reactions to towed fishing gears. In Experimental biology at sea, pp. 167-195. Ed. by A. G. Macdonald and I. G. Priede. Academic Press, New York. 414 pp. Zhang, X ., and A rim oto, T. 1993. Visual physiology of walleye pollock Theragra chalcogramma in capturing process of trawl net (this volume). 121