Fisheries Science 62(5), 796-799 (1996)

Influence on Post-mortem Rigor of Fish Body and Muscular ATP Consumption by the Destruction of Spinal Cord in Several Fishes

Masashi Ando, Akira Banno, Michio Haitani, Hideaki Hirai, Takayuki Nakagawa, and Yasuo Makinodan

Department of Fisheries, Faculty of Agriculture, Kinki University, Nakamachi, Nara 631, Japan

(Received December 12, 1995)

Influence of destruction of spinal cord on the post-mortem rigor and muscular ATP consumption was examined in yellowtail, red sea bream, and plaice. In case of yellowtail and red sea bream, the sam ple whose spinal cord was destroyed (tested sample) showed 6-12 hours delay in reaching full-rigor com pared with the control sample. On the other hand, in case of plaice, the tested sample attained full-rigor faster than the control, and the control sample did not reach to full-rigor during 24h storage. Rate of ATP consumption was slower in the tested than the control in yellowtail and red sea bream. In case of plaice, the ATP content was almost the same in both of the tested and the control, and was much in the control for 15-24h storage. Then, it was concluded that destruction of the spinal cord delayed ATP consumption and process of post-mortem rigor except for the case of plaice, and such a specialty of plaice would be caused by the inferiority of autonomic nerve development. Key words: post-mortem rigor, ATP, spinal cord, nerve, fish

In the killing of a live cultured fish, punch at the brain Nara, Japan). They were punched at the brain and their or a cut of the spinal cord is done generally. In some cases, spinal cord cut (control sample). Tested samples were pre in addition to the cut of the spinal cord, a wire is pushed in pared as follows. In the fish whose spinal cord was cut, a to destroy the spinal cord completely. It is thought that the wire (2mm in diameter, 30cm in length) was pushed in to freshness of fish is maintained for longer period by this destroy the spinal cord (tested sample). The samples were method, but there are few studies which are concerned the stored at 5•Ž for 24 h. Three individuals were examined in influence on the freshness by such a method. Nakayama et each fish. al. *1 has reported the delay effect on the post-mortem rigor and ATP consumption which was evaluated by ATP/IMP Measurement of the Rigor Index ratio by the destruction of spinal cord about red sea Post-mortem rigor was evaluated according to the bream. ATP consumption has been reported as the cause method of Bito et al. 19)Briefly, fish were put on a horizon of post-mortem rigor in frigate mackerel,1) pacific mack tal table protruding the half of the body length from the erel,2-4) red sea bream,2,3,5,6) carp,3,7-10) yellowtail,11-14) edge of the table. The distance from the horizontal line to flathead,12) Japanese striped knifejaw,12.14) sardine,4,15) the base of the tail (L) was measured at selected time inter plaice16,17) and horse mackerel.18) In comparison of slaugh vals after death, and the rigor index was calculated by ap ter methods, death by struggling is the fastest in ATP ex plying these values to the following equation. haustion and onset of post-mortem rigor in yellowtail13) and horse mackerel.18) Iwamoto et al.5) showed that proc ess of post-mortem rigor was influenced by the storage tem perature, and the rigor proceeded slower at 10•Ž than 0•Ž storage because ATP consumption rate was slower at

10•Ž. According to these reports, the decrease of ATP Determination of A TP Content content is the major factor of post-mortem rigor progress. Amount of ATP content was determined according to The purpose of the present study is to examine the the method of Yokoyama et al.20) One gram of muscle was influence of destruction of the spinal cord on the rigor mor cut from the dorsal muscle of the fish body which was used tis process and ATP consumption rate in several fishes. for measurement of rigor index. The muscle was homogenized with 5ml of 10% perchloric acid (PCA). Materials and Methods The homogenate was centrifuged at 3,000•~g for 15min, and the residue was washed twice with 5% PCA. The su Samples pernatants were combined and neutralized with 10 and 1N Yellowtail Seriola quinqueradiata (50-59cm, 2.3-3.3 KOH solution. The neutralized solution was centrifuged at kg), red sea bream Pagrus major (27-39cm, 714-1240g), 3,000•~g for 15 min, and the precipitate was washed with and plaice Paralichthys olivaceus (33-38cm, 386-560g) 10ml of neutralized 5% PCA-KOH solution and cen were obtained alive from a fish store (Uoshin suisan Co., trifuged. The supernatants were combined and diluted to

*1 Proceedings of Annual Meeting of the Japanese Society of Scientific Fisheries , 1995, Tokyo, p. 294. Post-Mortem Rigor of Fish 797

25ml with distilled water. The PCA extract was filtered through a 0.45ƒÊm membrane, and 10ƒÊ1 of the filtrate was analyzed by a CAPCELLPAK 5C18-AR (4.6•~150mm, Shiseido, Japan) which was equilibrated with a mixture of 20mm citric acid, 20mm acetic acid, and 40mm triethyla mine (pH 4.8). Flow rate was 1ml/min and the column temperature was held at 40•Ž. The eluate was monitored by UV absorption at 260nm.

Results and Discussion

Process of Post-mortem Rigor The results of rigor index are shown in Figs. 1-3. In case of yellowtail and red sea bream, the control sample body started to be stiff within 3h storage, and was in maximum rigor in 6-9h after death. On the other hand, a tested sam ple showed slower process of rigor than the control. In the Fig. 3. Change of rigor index of plaice during storage at 5•Ž. tested sample of yellowtail, post-mortem rigor started at 6 The symbols are the same of Fig. 1. h and progressed rapidly till 9h storage. Maximum rigor was observed at 15h after death. In the tested sample of red sea bream, post-mortem rigor progressed rapidly in 3 rigor was attained at 24 h after death. According to these 6 h, but gradually in 6-21 h storage. After that, maximum results, process of rigor mortis was delayed significantly by the destruction of the spinal cord in yellowtail and red sea bream. By the destruction of the spinal cord, the fish body stopped struggling promptly. This effect would be due to the destruction of the autonomic nerve. In case of the con trol sample, autonomic nerve would function even after the death of fish for a short time, so the muscle moves at random till autonomic nerve stops their function. Then, ATP consumption rate of the control in the early period of storage would be faster than the tested, and it might be the cause of the earlier onset of post-mortem rigor. In case of plaice, however, the opposite result was ob tained (Fig. 3). The onset of rigor mortis was earlier for 3 hours in the tested than the control. And the tested sample maintained the higher level of rigor than the control espe cially in 18-24h storage. In case of plaice, body struggling was not observed with or without destruction of the spinal cord. Plaice lives on the bottom of sea and it swims less than other fishes. So, it would be speculated that autonom Fig. 1. Change of rigor index of yellowtail during storage at 5•Ž. ic nervous system which controls muscle exercise in plaice •œ: the sample whose spinal cord was cut and destroyed by a wire. doesn't develop so well as pelagic fishes which swim con •›: the sample which was killed by cutting the spinal cord. stantly. Then, instead of stopping the function of auto nomic nervous system, some stimulation would be given to plaice muscle by the destruction of the spinal cord, and the post-mortem rigor progressed earlier.

Change of ATP Contents The results of ATP content determination are shown in Figs. 4-6. In case of yellowtail (Fig. 4), ATP content was almost the same between the tested and the control during 3 h storage. In 6-15 h storage, ATP content was more in the tested. In case of red sea bream (Fig. 5), ATP content was decreased rapidly till 6 h storage in the control. In the tested, ATP content was decreased slower than the control during storage for 15h. As the case of plaice (Fig. 6), ATP content was almost the same between the control and the tested during 15h storage, and the control sample main tained a higher level of ATP content than the tested in 18 24h storage. These difference in ATP contents between the tested and the control would have a relationship to the F ig. 2. Change of rigor index of red sea bream during storage at 5•Ž. destruction of autonomic nervous system as described The symbols are the same of Fig. 1. 798 Ando et al.

Fig. 4. Change of muscular ATP contents of yellowtail during storage Fig. 6. Change of muscular ATP contents of plaice during storage at at 5•Ž.•¡ 5•Ž. : the sample whose spinal cord was cut and destroyed by push The symbols are the same of Fig. 4. ing the wire. • : the sample which was killed by cutting the spinal cord. The process of rigor was different in each fish specie, and this would be due to the difference in body shape. In case of yellowtail, its body is spindle-shaped, and influence of forming myosin-actin cross-bridge would be reflected to rigor index easily. On the other hand, in the cases of red sea bream and plaice, their body shape is flat in compared to that of yellowtail. So, when ATP is consumed and my ofibrils decrease their extensibility, it would be hard to be reflected in rigor index. In the early period of storage, the decrease rate of ATP content was the fastest in red sea bream. The difference of ATP consumption rate would be due to the degree of strug gling after death compared with other fishes. In addition to this, difference of myofibrillar ATPase activity exists among fish species. Myofibrillar ATPase activity of fish has been reported as to plaice,17,24) red sea bream,24) carp,) and sardine,25) and the activity of sardine is higher than Fig. 5. Change of muscular ATP contents of red sea bream during that of plaice and red sea bream. Such a difference of ATP storage at 5•Ž. The symbols are the same of Fig. 4. degrading activity would be the cause of the variation in the ATP consumption rate during storage.

above. Influence of Muscle Cutting on Post-mortem Rigor It was shown in some cases that the change of ATP con At the beginning of this study, we measured the rigor in tents did not correspond to the progress of rigor mortis. In dex in the sample whose body was not accepted for any the yellowtail, rigor mortis progressed rapidly in 3-9 h damage for excision of a piece of muscle to determine the storage; but ATP content decreased slightly during the ATP content. Because we thought that the cutting of mus same period. In the tested red sea bream, the rigor was cle gave damage to the fish body, and rigor index might be progressed gradually in 6-21 h, but the content of ATP lower than the actual. However, damaged sample showed decreased rapidly in 9-15 h storage. These cases show that higher index and faster process of rigor than the intact sam ATP content does not always correspond with post mor ple (Fig. 7). This result suggests that cutting of muscle tem rigor. might cause ATP exhaustion, and promote post-mortem Post-mortem rigor occurs with the lost of muscle exten rigor. Then, to discuss about the exact relationship be sibility after the consumption of ATP .21) However, Bito et tween the rigor index and ATP contents, an experiment al.14) reported that ATP content was not always coincided would be needed as follows; several samples which are with the rigor process of several fish species. Strength of killed at the same time are provided, and ATP contents are muscle contraction would be different among fish species, determined when each sample shows a specific rigor index. and it might be the cause of the variation in the relation be According to this method, the relationship between the tween the rigor state and ATP content. In case of tilapia, ATP contents and rigor index of intact fish body would be Toyohara and Shimizu22) reported that post-mortem rigor clarified. proceeded faster in 0•Ž than 10•Ž storage. But muscle con Muscle cell is connected with a neural end and it accepts, traction progressed faster in l0•Ž.23) These reports suggest a neural pulse at the cell membrane.26) Ca2+ is released that muscle contraction does not always correspond to the from sarcoplasmic reticulum by the pulse, and muscle con rigor mortis process. tracts by the elevation of Ca2+ concentration. The central Post-Mortem Rigor of Fish 799

Fig. 7. Influence of muscle excision on the progress of post-mortem rigor in red sea bream. (A) the sample whose muscle was not excised during storage. (B) the sample whose muscle was excised partially for the determination of ATP content. The symbols are the same of Fig. 1. nervous system exists in the spinal cord and it links with peripheral nerves. By nature, the pulse from the central nervous system causes the muscle contraction. As the case of the present study, destruction of the central nervous sys tem might stop the pulse to the muscle fiber, and stop the release of Ca2+ from sarcoplasmic retiiculum, and resultantly, muscuscusccmusclecontraction was also stopped complete ly. To study about the detail mechanism, histological ob servations on the connecting site of neural end and distri bution of Ca2+ would be needed.

References

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