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Breakdown of intramuscular connective tissue in cod (Gadus morhua L.) and spotted wolffish (Anarhichas minor O.) related to gaping
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LWT 39 (2006) 1143–1154 www.elsevier.com/locate/lwt
Breakdown of intramuscular connective tissue in cod (Gadus morhua L.) and spotted wolffish (Anarhichas minor O.) related to gaping R. Ofstada,, R.L. Olsenb, R. Taylorc, K.O. Hannessona a
Matforsk, Oslov 1, 1430 Aas, Norway Norwegian College of Fishery Science, University of Tromsoe, Norway c INRA-Theix, St Genes Champanelle, France
b
Received 15 February 2005; accepted 7 June 2005
Abstract The purpose of this work was to compare the microstructure of muscle connective tissue in cod and wolffish and to study its degradation during ice-storage to determine which structural alterations are related to the gaping phenomenon. Gaping is very often found in fillets of codfish, but not in wolffish. The results showed that detachments between myofibres occurred prior to the myotomes to myocommata detachments, which occurred more rapidly and to a larger extent in cod than in wolffish. Changes at the ultrastructural level in myocommata proper were mainly related to gaps between collagen fibres and between collagen fibres and cells in the extracellular matrix (ECM). The mean collagen fibre diameter was smaller and the collagen network in myocommata proper was denser in wolffish than in cod. After 7 days of ice-storage the collagen fibres, particular in cod, appeared sparsely packed. The results implied that the increased gaping during ice-storage might be due to degradation of proteoglycans and glycoproteins, important for the spatial organization of the collagen fibres and in anchoring cells in the ECM to the collagen network. r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Fish; Connective tissue; Gaping; Microstructure; Muscle
1. Introduction Gaping is a phenomenon in which the connective tissue fails to hold the fish fillet together resulting in gaps and tears at the myofibre–myocommata attachments and between myofibres. This results in fillets with unacceptable appearance and might also influence textural properties. Differences exist between fish species and individuals in their propensity to gape (Love, 1988). Gaping accompanied by softening of the tissue occurs as results of the general deterioration processes during storage and may be increased by rapid and strong muscle contractions during rigor (Bremner, 1999; Love, 1988; Taylor, Fjæra, & Skjervold, 2002). For a given species the rate of deterioration depends on the biological status, catch or slaughter history, and temperature during storage (Lavety, Afolabi, Corresponding author. Tel.:+4764970293; fax: +4764970333.
E-mail address: [email protected] (R. Ofstad).
& Love, 1988; Robb, Kestin, & Wariss, 2000; Sheehan, OConnor, Buckley, & FitzGerald, 1996). In comparison with the muscle of wild cod, softer texture and fillet gaping are often seen in pen raised Atlantic cod (Gadus morhua L.) (Ang & Haard, 1985; Ofstad et al., 1996; Rustad, 1992). Also farmed Atlantic salmon (Salmo salar) suffers from gaping (Lave´ty et al., 1988). In contrast to fillets from salmon and cod, spotted wolffish (Anarhichas minor O.) fillets apparently do not gape (Love, Lavety, & Steel, 1969). Fish flesh is organized in concentric muscle segments (myotomes) surrounded by intramuscular connective tissue (IMCT). Major constituents identified in the IMCT include collagen and elastic fibres, cells (fibroblasts, adipocytes, macrophages), glycoproteins and proteoglycans. The proteoglycans consist of a protein core with variable amounts of covalent attached linear polysaccharide with sulphated carbohydrate side chains, i.e. glycosaminoglycans. The myotomes are separated by the myocommata, where groups of collagen fibres runs roughly parallel to each
0023-6438/$30.00 r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2005.06.019
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other with adjacent groups set at an angle resulting in crisscross layers which provide considerable mechanical strength (Bremner, 1999). A layer of thin connective tissue (endomysium) surrounds each muscle cell (myofibre) and often endomysium connects to the perimysium, which is contiguous with the myocommata. Both collagen I and collagen V is present in the IMCT of fish; collagen I is the major type in the myocommata (Sato, Yoshinaka, Itoh, & Sato, 1989). The structural changes inducing gaping have been thoroughly reviewed by Bremner (1992, 1999). In blue grenadier (Macruronus novaezelandiae) and spotted trevalla (Seriolella punctata) gaping was related to degradation of fine collagen fibres at the myotendinous junctions connecting the myocommata and the myofibres leading to loosening of the structures in this area (Bremner, 1992; Bremner & Hallet, 1985, 1986; Hallet & Bremner, 1988). The work of Fletcher, Hallett, Jerrett, and Holland, 1997 indicated that in Chinook salmon (Oncorhynchus tshawytscha) the site for detachment when fish were electrically stimulated post mortem, might be within the muscle cell where the thin costameric filaments from the terminal Z-line join the inner surface of the sarcolemma at the adherens junction. Softening of rainbow trout (Oncorhynchus mykiss) flesh was related to weakening of the pericellular connective tissue due to fragmentation/disordering of the collagen fibre network structure, but no structural changes in myocommata were observed (Ando, Toyohara, & Sakaguchi, 1992; Ando, Toyohara, Shimizu, & Sakaguchi, 1991). In Atlantic salmon (Salmon salar) loss of myofibreto myofibre (endomysium) attachments was evident prior to any loss of myocommata integrity during cold storage (Taylor et al., 2002). Degradation of IMCT in pacific rockfish (Sebastes sp.) has been related to both increased solubilization of collagen and degradation of the glycosaminoglycans (Kim & Haard, 1992). Early post mortem decomposition of the pericellular connective tissue has also been reported for bovine meat (Hannesson, Pedersen, Ofstad, & Kolset, 2003; Nishimura, Hattori, & Takahashi, 1996). Even though gaping was described more than 30 years ago and much work on this topic has been done, the mechanisms behind are far from understood. The purpose of this work was to compare the microstructure of connective tissue in cod and wolffish and to determine the type of structural changes which were related to post mortem myofibre–myocommata detachments and gaping. 2. Materials and methods 2.1. Fish Wild caught and thereafter pen raised Atlantic cod (G. morhua L.) weighing 3–4 kg and farmed spotted wolffish (Anarchichas minor O.) weighing 2–3 kg, were used. The fish were obtained from different fish farms in Northern Norway in January and February (cod) and January and
November (wolffish). Five fish of each species were carefully netted from the pens, and killed by a blow to the head and filleted within 1 h after slaughter. Strips of muscle samples were excised from the fillet near the dorsal fin of each of the five fish within 2 h (day 0) and at days 2 and 7 post mortem. All fillets were kept in plastic bags and stored in ice. 2.2. Images Images of the fish fillets were taken with a CCD camera (Cannon digital D30) equipped with a standard 55 mm macro-objective. Tungsten light was used. 2.3. Microscopy 2.3.1. Cryo-sections Muscle samples of 4 4 3 mm3 were obtained from the white muscle beneath the dorsal fin of the cod and wolffish and frozen as previously described by Ofstad, Kidman, Myklebust, and Hermansson (1993). Sections of 5 mm thickness were cut in a cryostat (Leitz 1720 Digital Instruments GMBH, Heidelberg, Germany) and mounted on poly-L-lysin coated glass slides which were kept at 20 1C until use. After staining, the cover slips were photographed in a Leica DMLD microscope (Leica Microsystems, Nussloch GmbH, Germany) by a Spot RT Color Camera (Diagnostic Instruments Inc., Burroughs Sterling Heights, Michigan, USA). To study the presence of sulphated glycosaminoglycans, Alcian blue 8GX (Gurr Biological Stains, BDH, Pool, UK), 0.4 g in 8.20 g/l acetate buffer pH 5.8 containing either 12.20 or 81.32 g/l MgCl2 were used according to the procedures described by Tingbø, Kolset, Ofstad, Enersen, and Hannesson (2005). At 12.20 g/l MgCl2 most acidic groups stain whereas at 81.32 g/l MgCl2 only negatively charged groups of sulphated proteoglycans stain (Scott & Dorling, 1965). 2.3.2. Plastic-sections Muscle blocks of 1 1 3 mm3 were fixed in 2.5 g/100 ml formaldehyde and 1.0 g/100 ml glutaraldehyd with 0.2 g/ 100 ml glucose in Ringer’s buffer (pH 6.8) and embedded in Epon/Araldite (Serva, Heidelberg, Germany) as previously described by (Ofstad et al., 1993). For all samples, 1.5 mm thick plastic sections were cut perpendicular to the fibres, allowing examination of myofibre–endomysium, myofibre–myocommata junctions and myocommata proper. Optical microscopy observations were of sections stained in 0.1 g/100 ml toluidine blue dissolved in 1 g/100 ml aqueous sodium borate solution. A minimum of four fish of each species was examined at each sampling time. For transmission electron microscopy (TEM) thin sections (70–90 nm) were stained with uranyl acetate and lead citrate. Sections were examined and photographed using a TEM, 100 CXII (Jeol Ltd., Tokyo, Japan) at an accelerating voltage of 80 kV and instrument magnification of 1500 and 10,000. A total of eight microscopic fields
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were obtained by systematic random sampling from the myotendinous junction and myocommata proper of each section. Three fish were examined at each sampling time. In fish, structural alterations may be very focally. Thus two or more blocks from each of the three fish were investigated. The micrographs presented were chosen out of many and illustrated the most typical features and post-mortal changes. 2.3.3. Quantification Optical microscopy with a 10 objective was used to measure myofibre to myofibre detachment and myofibre to myocommata detachment. Quantification of structural changes was performed as previously described for Atlantic salmon (Taylor et al., 2002). This involved counting the numbers of myofibres attached and partly or completely detached from the neighbouring myofibre for a minimum of 200 myofibre-to-myofibre attachments. In addition, myofibre-to-myocommata detachments were determined in a similar manner by counting the number of the myofibres attached and partly or completely detached from the myocommata, for a minimum of 50 myofibre-tomyocommata attachments for each sample. Type and degree of structural changes in the myocommata were quantified on TEM images by the use of a transparent point lattice test system with spacing of 20 mm (equivalent to 5 mm on the image scale). The structures being overlaid by the points were classified, counted and given as percent of the total points of connective tissue or as percent of total detachment points on each image. These percentages thus represent the relative area of the structures being counted. The following structures were counted: collagen fibres, cells (fibroblasts, adipocytes, macrophages) and other structures (blood vessels and nerves), gaps between collagen fibres, gaps between collagen fibres and cells, gaps between sarcolemma and extracellular matrix and intracellular gaps between myofibres and sarcolemma. A minimum of 1500 points was counted on the 24 micrographs obtained from three fish of each species at each sampling time. This should give a 10% standard error, when structure counted is estimated to occupy 7% of the area in ECM (Ang & Haard, 1985; Bozzola & Russell, 1999; Ofstad et al., 1996; Rustad, 1992). In addition, collagen fibre diameters in the myofibre–myocommata junction zone and in the myocommata proper were measured using a measuring magnifier with metric scale. Hundred fibres were measured randomly on four micrographs with magnification 10,000. Three fish sampled immediately after slaughter (day 0) were analysed, and the fibre diameter was given as the mean diameter of a total of 300 collagen fibres. Subsequently, transparent lattices with spacing of 7 mm (equivalent to 250 nm on the image scale) were superimposed randomly on the same microscopic images. The number of collagen fibres in a total of 80 squares representing an area of 5 mm2 was counted for each specimen. The figures are given as the mean of 3 fish.
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2.4. Statistical analyses Analysis of variance was done with the GLM-procedure in SAS (SAS Institute Inc., Cary, NC, USA, version 8.12). Tukey’s test was used for multiple comparisons of detachments as quantified on micrographs. All significant tests were performed at the 0.05 level. 3. Results 3.1. Visual appearance Fillets of cod and wolffish stored 2 and 7 days in ice are shown in Fig. 1. After 2 days of storage some gaps and tears (arrows) could be seen in the cod fillet. The wolffish had a smooth appearance with no visible gaps and tears. After 7 days of storage, the cod fillets had clearly visible gaps and tears and showed gaping. In the wolffish fillets some minor gaps and tears were present, but no clear gaping could be seen. Although the cod were obtained in January and February from two separate fish farms, no farm specific differences in visual appearance or in structural degradations were noticed. Similar observations were made for the wolffish obtained from two farms in November and January. 3.2. Light microscopic observations Light microscopic images of cod and wolffish fillets excised immediately after death, and after 2 and 7 days storage in ice are shown in Fig. 2. At day 0 both endomysium and myocommata showed a strong and consistent staining even though some gaps could be seen. In cod both loss of myofibre–myofibre attachments (arrows) as well as loss of myofibre–myocommata attachments (arrowhead) were evident after 2 days of storage In wolffish only a few minor detachments could be seen after 2 days. At day 7 post mortem, most of the myofibres were detached in both species. In cod the myofibre–myocommata detachment appeared however to be more extensive than in the wolffish.
Fig. 1. Images of fish fillet of cod (a and b) and wolffish (c and d) stored in ice, 2 days (a and c) and 7 days (c and d) after filleting. In the cod images (a and b) gaps and tears are visible.
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Fig. 2. Cryo-sections of white muscle stained with Alcian blue with 12.20 g/l MgCl2. Cod (a–c) and wolffish (d–f) excised immediately after death (a and d) and after 2 (b and e) and 7 (c and f) days storage in ice. At 12.20 g/l MgCl2 most acidic groups, i.e. myfibres (my), endomysium (e) and myocommata (mc) stain blue. Myofibre–myofibre detachments are indicated with arrows and myofibre–myocommata detachments with arrowheads. The bars indicate 200 mm.
When tissue sections were stained with Alcian blue containing low concentration of MgCl2 (12.20 g/l), myocommata as well as endomysium and myofibres stained (Fig. 2). When increasing the concentration of MgCl2 to the high level (81.32 g/l), only the sulphated glycosaminoglycans in the connective tissue stained (both endomysium and myocommata) as shown in Fig. 3. A stronger and more consistent staining was seen in the endomysium and myocommata of wolffish than in cod at day 0. For both fish species the blue colour was weakened during storage. After 7 days of storage myocommata in cod were hardly visible, but still quite distinct in wolffish. Quantification of the detachments, which were observed in micrographs of toluidine blue stained plastic sections, is shown in Table 1. For both species a significant (***Po0:0001) loss of myofibre to myofibre adhesion
was observed within 2 days. The percentage of detachments was somewhat higher for cod than for wolffish. The myofibre to myocommata adhesion changed significantly (***Po0:0001) for cod from time 0 to day 2. For wolffish, a significant (**Po0:01) increase in the numbers of detachments was not registered until day 7. 3.3. Ultrastructural observations To determine which structures were changing at the ultrastructural level as gaping developed, the fish muscle was examined by TEM. Structural changes occurring between the myofibres are shown in Fig. 4. Since there were only minor differences between cod and wolffish, only the micrographs of cod are shown. In the 0 day sample (Fig. 4a) the myofibres were attached and the structure
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Fig. 3. Cryo-sections of white muscle stained with Alcian blue with 81.32 g/l MgCl2. Cod (a–c) and wolffish (d–f) excised immediately after death (a and d) and after 2 (b and e) and 7 (c and f) days storage in ice. At 81.32 g/l MgCl2 only the sulphated glycosaminoglycans in the connective tissue, i.e. endomysium (e) and myocommata (mc) stain blue. The bars indicate 200 mm.
Table 1 Quantification by light microscopy of myofibre–myofibre and myofibre–myocommata detachments in white muscle from cod and wolfish Myofibre–myofibre (mean7SD) Cod Day 0 Day 2 Day 7
Myofibre–myocommata (mean7SD) Wolffish
a
31.877.7 99.771.0b 99.171.8b
Cod a
4.773.2 88.178.3b 95.878.3b
Wolffish a
2.773.9 90.5713.8b 92.6714.8b
4.176.7a 14.9713.1a 77.4732.2b
A minimum of 200 myofibre-to-myofibre attachments and 50 myofibre-to-myocommata attachments were counted on four fish of each species at each sampling time. The mean is given in percent7SD of the total number counted. Figures in the same colon with different letters are significant different at *Po0:05.
appeared intact. After 2 days of storage the sarcolemma was often detached and gaps occurred in the endomysium causing separation between myofibres (Fig. 4b). Structural changes in the myocommata of cod and wolffish are shown in Fig. 5a–c and d–f, respectively. In the
0 day muscle myocommata appeared dense with closely packed collagen layers surrounding various cells in the ECM (Fig. 5a and d). After 2 days storage in ice, myocommata showed signs of disintegration. In cod (Fig. 5b) there were clearly visible gaps, whereas in wolffish
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Fig. 4. TEM micrographs showing the endomysial (e) layer of a transversally sectioned cod white muscle excised immediately after death (a) and stored 2 days in ice (b). Endomysium (e), myofibres (my), sarcolemma (sl) and collagenous fibres in the endomysium (c).
Fig. 5. TEM micrographs showing myocommata and myofibrils (mf) of a transversally sectioned white muscle of cod (a–c) and wolffish (d–f) excised immediately after death (a and d) and stored 2 (b and e) and 7 (c and f) days in ice. Gaps are indicated (*).
the myocommata appeared less dense (Fig. 5e) than in the 0 day image. In the samples taken after 7 days storage, the gaps were clearly evident in both species, but the connective tissue seemed more disintegrated in cod (Fig. 5c) than in wolffish (Fig. 5f). The percentage of
points classified as detachments increased significantly (**Po0:01) with storage in ice for both species and the percent detachment points were significant higher (***Po0:001) for cod than for wolffish both after 2 and 7 days of ice storage (Table 2).
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Table 2 Quantification on TEM images of the degree and type of structural changes in myocommata of cod and wolffish by the use of a transparent point lattice test system
Cod day 0 Cod day 2 Cod day 7 Wolffish day 0 Wolffish day 2 Wolffish day 7
Detachments (DP/TP*100)
Collagen fibre–fibre (P/DP*100)
Cell–collagen fibre (P/DP*100)
Sarcolemma–ECM (P/DP*100)
Myofibrill–sarcolemma (P/DP*100)
0.870.8a 14.577.6b 61.878.3c 0.070.0d 4.170.0e 8.472.1f
23.8741.2 57.1734.4 76.978.3 0.070.0 44.9739.2 56.4716.5
42.9751.5 38.7734.2 11.478.1 0.070.0 28.2724.5 18.3711.1
0.070.0 3.574.4 4.975.9 0.070.0 0.070.0 6.3710.9
0.070.0 0.871.3 6.473.2 0.070.0 33.3757.7 19.0723.8
The structures being overlaid by the points (P) were classified and counted and given as percent of the total points (TP) of connective tissue or as percent of total detachment points (DP) on each image. A minimum of 1500 points was counted on the 24 micrographs obtained from three fish of each species at each sampling time. The mean is given in percent7SD. Figures in the first colon with different letters are significant different at *Po0:05.
Examination at higher magnification of both fish species after 2 and 7 days of ice storage revealed that the gaps occurred at four different sites as illustrated for cod in Fig. 6. Gaps were also located at the same sites in wolffish (results not shown). In the mid-region of myocommata (myocommata proper) gaps occurred within the collagen layers between the collagen fibres (Fig. 6a) or at the collagen fibre–cell junctions (Fig. 6b). Gaps in the myomere–myocommatal junction zone were due to detachments between the sarcolemma/basement membrane and the extracellular matrix (Fig. 6c) or within the myofibre between the myofibrils and the sarcolemma (Fig. 6d). To see if there were differences between the species regarding the dominant type of gaps which developed during storage, all gaps were classified as one of those four types illustrated in Fig. 6. The percentage of point of the total detachment points thus representing the relative area of the different gaps are given in Table 2. Both in cod and wolffish, the majority of detachments were within the collagen layers between the collagen fibres. At 7 days post mortem, 75% and 55% of the points were classified as collagen fibre–fibre detachments in cod and wolffish, respectively. The 10–40% of the points were classified as detachments between cells and collagen fibres. The percent detachments between collagen fibres increased during storage, whereas the percent detachments between cells and collagen fibres tended to decrease. In cod the highest percent of this type of gaps were in the 0 day sample. The individual differences as reflected in the high standard deviation of these figures at day 0 and day 2, were however large. In general there were less detachments in the myomere–myocommatal junction zone than in myocommata proper. Less than 10% of the detachments were between sarcolemma and ECM. The percent of this type of detachment increased during storage, but no differences between the two species were observed. The percent of gaps between myofibrils and sarcolemma (intracellular) tended to increase during storage as well, but was much higher for wolffish (20–30%) than for cod (1–6%) both after 2 and 7
days of ice storage. Also for this type of gap there were large individual differences as reflected in the high standard deviations. The cause of the large individual differences regarding the amount and type of gaps in the myocommata is unknown, and should be studied. The composition of the myocommata of the two species as classified on the same TEM images is given in Table 3. The percentage of points classified as collagen and cells were similar in cod and wolffish. Collagen made up 85% of the relative area and different cells such as fibroblasts, macrophage-like cells and a few fat droplets (only in wolffish) made up 15%. Fibroblasts were the most abundant cell type in connective tissue. The main structural difference in ECM between cod and wolffish was related to the size and the density of collagen fibres in the myocommata proper (Fig. 7 and Table 3). The mean diameter of the collagen fibres regardless of position was 59 and 45 nm for cod and wolffish, respectively. This difference between the two species was significant at a level of 0.05. Fig. 7 shows micrographs of transversally sectioned collagen fibres from the myomere–myocommatal junction zone (Figs. 7a and d) and the myocommata proper (Figs. 7b and e) sampled at day 0. The first layer of collagen, closest to the myofibre, had small and densely packed fibres. In myocommata proper of wolffish the fibres were also small (Fig. 7e), whereas in cod there was both small and large fibres randomly distributed (Figs. 7b). This is also reflected in the large standard deviation of the mean diameter in cod (Table 3). Comparing muscle samples from the two different positions of cod and wolffish separately, a significant difference (*Po0:05) was found in the collagen fibre diameter in the myocommata proper between cod and wolffish, but not of those in the first layer. The collagen fibres in cod were significantly (*Po0:05) larger in the myocommata proper than in the first layer at the myomere–myocommatal junction zone, whereas in wolffish there were no differences in the collagen diameters between the two positions. In wolffish the collagen fibres in myocommata proper were significantly (*Po0:05) more densely packed than in cod (Table 3). In cod the collagen
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Fig. 6. TEM micrographs of cod showing gaps (*) in the myocommata proper between the collagen–fibres (a), at the collagen fibril–cell junction (b), in the myomere–myocommatal junction zone between the sarcolemma (sl) and the extracellular matrix (c) and intracellular between the fine myofibrils (mf) and the sarcolemma (sl) (d). Similar types of gaps were also found in wolffish (results not shown).
Table 3 Quantification on TEM images of the composition of myocommata in cod and wolffish by the use of a transparent point lattice test system Collagen (P/TP*100)
Cod Wolffish
85.978.5 84.277.4
Cells (P/TP*100)
14.178.5 15.877.4
Diameter collagen fibril (nm)
Number of collagen fibrils per mm2
Myofibre surface
Myocommata proper
Myofibre surface
Myocommata proper
33.7a74.8 36.9a75.0
83.9b733.7 52.8c78.4
195a773 249a759
75b730 187c747
The structures being overlaid by the points (P) were classified, counted and expressed as percent of the total points (TP) of connective tissue on each image. A minimum of 1500 points was counted on the 24 micrographs obtained from three fish of each species at each sampling time. The mean is given in percent7SD. Collagen fibril diameters were measured on 100 fibrils either in the first collagen layer at the myofibre surface or in the middle of myocommata proper. The number of collagen fibrils representing an area of 5 mm2 was counted in the same two locations for each specimen. The figures are given as the mean7SD of 3 fish. Figures with different letters are significant different at *Po0:05.
fibres in the myomere–myocommatal junction zone were more (*Po0:05) densely packed than in the myocommata proper, whereas in wolffish there were no differences between the two positions. After 7 days of storage in ice, the collagen fibres appeared less densely packed than at day 0. This was more pronounced in cod (Fig. 7c) than in wolffish (Fig. 7f). No obvious structural alteration or fragmentation of the collagen fibres could be seen. In the 0 day samples (Fig. 7b, e) the structures filling the spaces between the collagen
fibres appeared tiny and amorphous. In cod at day 7, these structures (arrow) were grainy and stained darker than at day 0. 4. Discussion The present study showed that gaping occurred faster and to a larger extent in cod than in wolffish during 7 days ice-storage of pre-rigor filleted flesh. It has previously been reported that cod is particularly prone to softening and
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Fig. 7. TEM micrographs of transversally sectioned collagen fibres of cod (a–c) and wolffish (d–f) at the myomere–myocommatal junction zone (a and d) and the myocommata proper (b, c, e, f) sampled at day 0 (a, d, b, e) and at day 7 (c, f). Proteoglycans are arrowed in panel c.
gaping (Ang & Haard, 1985; Ofstad et al., 1996; Rustad, 1992) while wolffish much more rarely shows this property (Love et al., 1969). The current study is the first time that the structural changes resulting in gaping has been compared in these two species and that the components related to gaping have been localized and quantified. The overall structure of the muscle connective tissue in cod and wolffish was the same as found for other species of fish (Bremner & Hallet, 1985, 1986; Fletcher et al., 1997; Hallet & Bremner, 1988; Taylor et al., 2002).
Examination of cryo-sectioned muscle samples revealed that gaping was caused by both loss of myofibre–myofibre and myofibre–myocommata adhesion. Detachments of the myofibres were evident as gaps between the myofibres and between the myofibres and myocommata in micrographs stained with Alcian blue with the lower concentration of MgCl2 (12.20 g/l). A simultaneous decrease in stainability of the connective tissue by Alcian blue with the higher concentration of MgCl2 (81.32 g/l) indicated a structural disintegration of the sulphated glycosaminoglycans in
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ECM. This was more apparent in cod than in wolffish. Whether the stronger staining of the connective tissue in the wolffish muscle than in cod with Alcian blue suggests a higher content of sulphated glycosaminoglycans or a higher degree of sulphatation is not known. Early post mortem degradation of glycosaminoglycans and proteoglycans has previously been reported in both fish (Kim & Haard, 1992; Olsson, Olsen, & Ofstad, 2003) and bovine meat (Hannesson et al., 2003; Nishimura et al., 1996). Quantification of the myofibre–myofibre and myofibre– myocommata detachments by microscopy of plastic sections revealed that an extensive loss in myofibre to myofibre adhesion had occurred in both cod and wolffish after 2 days storage in ice. Examination of the larger frozen samples indicated that the endomysia of wolffish were more stable than that of cod. Only few and small gaps were visible in the micrograph of the 2 days stored wolffish sample. This divergence may be due to different preparation procedures. Samples for fixation and plastic embedding are cut into much smaller pieces and thus exposed to more manipulation than those fixed in liquid nitrogen. Others have reported that structural changes of early post mortem disintegration of the pericellular connective tissue as observed using light microscopy could only be demonstrated after compression of the muscle (Ando et al., 1991; Nakayama, Goto, & Ooi, 1996). The more pronounced intermyofibrillar spaces in the cryo-sectioned samples of cod may indicate that after 2 days of ice-storage the endomysia of cod were weaker than those of wolffish. Some myofibre–myofibre detachments were also seen in the day 0 samples, particularly in cod, regardless of the preparation procedure used. This may also indicate that the connective tissue of cod was weaker and more vulnerable than that of wolffish. Ando et al. (1992) reported that thin collagen fibres in endomysium disintegrated within few hours. However, artefacts caused by preparation cannot be excluded. In cod attachments between myofibres and myocommata were significantly lost in the 2 days ice-stored sample, but not until the day 7 sample in wolffish. The slower change of the myofibre to myocommata than the myofibre to myofibre adhesion has also been reported for Atlantic salmon (Taylor et al., 2002). Ultrastructural examination showed that the loss of myofibre-to-myofibre adhesion was by both detachment of sarcolemma and structural disintegration of the endomysium (breaks in sarcolemma have previously been demonstrated in pen fed cod stored 2 days in ice (Ofstad et al., 1996)). Similar breakdown of the endomysial sheaths has been reported in other fish species (Ando et al., 1991, 1992; Taylor et al., 2002), avian (Liu, Nishimura, & Takahashi, 1994, 1995) and mammalian muscle (Hannesson et al., 2003; Taylor & Koohmaraie, 1998). Detachment of the sarcolemma was ascribed to breaks in the muscle cytoskeleton connection from the sarcomeres to the endomysium (Papa et al., 1997; Taylor & Koohmaraie, 1998; Taylor et al., 2002). Weakening of the pericellular connective tissue induced by disintegration of thin collagen fibrils in rainbow
trout and sardine (Sardinops melanosticta) muscle, was ascribed to degradation of type V collagen (Sato et al., 1997; Sato, Koike, Yoshinaka, Sato, & Shimizu, 1994; Sato, Ohashi, Ohtsuki, & Kawabata, 1991). Both these events may be involved in the observed myofibre–myofibre detachments in our study. Moreover, the reduced staining of sulphated glycosaminoglycans indicates that degradation of the proteoglycans contributed as well. Ultrastructural changes in myocommata were similar in cod and wolffish, but occurred faster and to a larger extent in cod than in wolffish. The structural changes could be related to at least four different mechanisms for detachments. The collagen fibre–fibre detachment which increased during storage was the most important one in both species. In general the collagen fibres appeared less densely packed after 7 days of ice storage. This supports, as suggested by Bremner (1999) and Taylor et al. (2002), that there are several mechanisms involved in the degradation, but that the gaps are usually in the connective tissue layer. In the present study a reduced staining of sulphated glycosaminoglycans was seen in the light micrographs and a more grainy appearance of the amorphous ground substance filling the spaces between the collagen fibrils, was evident in the TEM images as seen at high magnification after 7 days storage in ice. No structural disintegration or fragmentation of the collagen fibres could be seen. The solubility of collagen I, the major type in myocommata, is also previously reported not to change in rainbow trout during short time chilled storage (Sato et al., 1991, 1994, 1997). Thus degradation of myocommata proper is probably mainly caused by degradation of the proteoglycans and glycoproteins, which function is structural, binding collagen fibres together (Scott, 1996) and anchoring cells to collagen or basement membranes (Reichardt, 1999). Degradation of the proteoglycans which may be caused by several enzymes such as lysosomal enzymes, matrix metalloproteinases and aggrecanases, probably involves both degradation of the sulphated glycosaminoglycans as well as the peptide core (Hannesson et al., 2003; Kim & Haard, 1992; Nishimura et al., 1996). In the day 0 (only cod) and day 2 samples a significant amount of gaps were seen in the collagen fibril–cell junction particularly along the lines of the processes. Bremner (1999) also reported gaps in this area. He suggested that the cells represented a point of weakness. The large standard deviation of these types of gaps may thus be related to the large variation in the number of such cells between the individuals. In the TEM images rather few gaps were seen in the myomere–myocommatal junction zone, either extracellular between the basement membrane and the ECM or intracellular between myofibrils and sarcolemma. The percent of extracellular gaps increased during storage. This is in accordance with earlier results, showing that a progressive breakdown of the invaginating collagen fibres and the basal lamina occurred during storage of blue grenadier and spotted trevalla (Bremner & Hallet, 1985, 1986). Percent gaps caused by intracellular
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breakage were more significant in wolffish than in cod after ice-storage, but the individual variations were substantial. In electrical stimulated Chinook salmon it was shown that gaping was due to deterioration of the links between the thin filaments at the adherent junctions on the inner surface of the sarcolemma (Fletcher et al., 1997). We used pre-rigor filleted fish in our experiments, which reduced the mechanical stress and thus might have reduced the number of intracellular gaps. To elucidate if there were compositional differences between the two species, the components in the myocommata were quantified. The distribution of collagen and different cells in myocommata were similar in cod and wolffish, except that wolffish contained some fat droplets. The major differences between the two species were the mean collagen fibre diameter and the fibre density in the myocommata proper. The collagen fibres were on average smaller and more densely packed in wolffish than in cod. Similar differences between the two species were not seen in the myomere–myocommatal junction zone. The size of the collagen fibres (35 nm) in the myotendinous junction was as those (30–70 nm) reported for hoki (Macruronus novaezelandiae) (Hallet & Bremner, 1988). Taylor (unpublished results) found in trout a similar mean collagen fibre diameter in the middle layer of myocommata proper as reported in our work, and moreover that the fibre diameter was larger in the middle region than in the myomere–myocommatal junction zone regardless of muscle region investigated. Another difference between cod and wolffish was the fibre size distribution which was larger in cod (30–180 nm) than in wolffish (33–74 nm). In decorin knock out mice a wide range in collagen fibre diameters has been described together with fragile and reduced tensile strength of the skin (Danielson et al., 1997). Moreover, in the tough M. semitendinosus bovine muscle the mean collagen fibre diameter was larger, the fibre size were unimodale and more tightly bundled with less matrix in-between compared to the tender M. psoas major (Eggen, Pedersen, Lea, & Kolset, 2001). Wolffish is regarded as having tougher connective tissue than cod. The reason why collagen fibre diameter in fish species varies is however not known. It is possible that the presence of minor collagen types such as Type V collagen may affect the diameter of collagen fibres. Whether collagen V, or other collagen types such as FACIT collagens (fibril-associated collagens with interrupted triple helices) are involved in post mortem degradation of myocommata should be further studied. The myofibre to myofibre detachments occurred prior to the myofibre–myocommata detachments in both fish species. The myofibre to myocommata attachment was more stable in wolffish than in cod. Moreover, the myofibre to myocommata detachments were mainly caused by fractures in the extracellular matrix (ECM) and these structural changes occurred faster and to a larger extent in cod than in wolffish. According to Baily and Light (1989) large collagen fibres and densely packed fibre bundles are less vulnerable to degradation by proteases during storage of bovine meat. The more densely packed collagen fibres in
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wolffish than in cod could thus explain the slower breakdown of the connective tissue in this species. Therefore, differences in gaping propensity between cod and wolffishmost probably are related to differences in the myocommata structure, i.e. the collagen fibre diameter and packing density, and the composition of matrix between the fibres. For both species the ECM was mainly broken between the collagen fibres and at the collagen fibre–cell junctions. A possible mechanism of importance for myofibre–myocommata detachment may be degradation of proteoglycans that organize and stabilize the collagen fibres in the layered structure of myocommata proper. In addition mechanical forces acting upon these structures during rigor mortis may also contribute to enhance this type of detachment. Further studies are needed to elucidate the full degradation mechanisms of the different components and the relationship to species-dependent structural features in connective tissue of fish white muscle. Acknowledgements The Norwegian research council through Grant no. 134988/I10 supported this study. The assistance of Grethe Enersen and Per Lea (Matforsk), and Helga-Marie Bye (Inst of medical biology, University of Tromsoe) were highly appreciated. References Ando, M., Toyohara, H., & Sakaguchi, M. (1992). Post-mortem tenderization of rainbow trout muscle caused by the disintegration of collagen fibres in the pericellular connective tissue. Nippon Suisan Gakkaishi, 58(3), 567–570. Ando, M., Toyohara, H., Shimizu, Y., & Sakaguchi, M. (1991). Postmortem tenderization of rainbow trout (Oncorhyncus mykiss) muscle caused by gradual disintegration of the extracellular matrix structure. Journal of the Science of Food and Agriculture, 55, 589–597. Ang, J. F., & Haard, N. F. (1985). Chemical composition and postmortem changes in soft textured muscle from intensely feeding Atlantic cod (Gadus morhua, L.). Journal of Food Biochemistry, 9, 49–64. Baily, A. J., & Light, N. D. (1989). Connective tissue in meat and meat products. New York: Elsevier Applied Science Publisher. Bozzola, J. J., & Russell, L. D. (1999). Quantitative electron microscopy. In J. J. Bozzola, & L. D. Russell (Eds.), Electron microscopy. Principles and techniques for biologists (pp. 321–340). Sudbury, MA: Jones and Barlett Publishers. Bremner, H. A. (1992). Fish flesh structure and the role of collagen: Its post mortem aspects and implication for fish processing. In H. H. Huss, M. Jakobsen, & J. Liston (Eds.), Quality assurance in the fish industry (pp. 39–62). London: Elsevier Science Publisher. Bremner, H. A. (1999). Gaping in fish flesh. In K. Sato, M. Sakaguchi, & H. A. Bremner (Eds.), Extracellular matrix of fish and shellfish (pp. 81–94). Trivandrum, India: Research Signpost. Bremner, H. A., & Hallet, I. C. (1985). Muscle fibre–connective tissue junctions in the fish blue grenadier (Macruronus novaezelandiae). A scanning electron microscope study. Journal of Food Science, 50, 975–980. Bremner, H. A., & Hallet, I. C. (1986). Degradation in muscle fibre–connective tissue junctions in the spotted trevalla (Seriolella punctata) examined by scanning electron microscopy. Journal of the Science of Food and Agriculture, 37, 1011–1018.
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R. Ofstad et al. / LWT 39 (2006) 1143–1154
Danielson, K. G., Baribault, H., Holmes, D. F., Graham, H., Kadler, K. E., & Iozzo, R. V. (1997). Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility. Journal of Cell Biology, 136(3), 729–743. Eggen, K. H., Pedersen, M. E., Lea, P., & Kolset, S. O. (2001). Structure and solubility of collagen and glycosaminoglycans in two bovine muscles with different textural properties. Journal of Muscle Foods, 12(4), 245–261. Fletcher, G. C., Hallett, I. C., Jerrett, A. R., & Holland, A. J. (1997). Changes in the fine structure of the myocommata–muscle fibre junction related to gaping and exercised muscle from king salmon (Oncorhynchus tshawytscha). Lebensmittel-Wissenschaft undTechnologie, 246–252. Hallet, I. C., & Bremner, A. H. (1988). Fine structure of the myocommata–muscle fibre junction in hoki (Macruronus novaezelandiae). Journal of the Science of Food and Agriculture, 44, 245–261. Hannesson, K. O., Pedersen, M. E., Ofstad, R., & Kolset, S. O. (2003). Breakdown of large proteoglycans in bovine intramuscular connective tissue early postmortem. Journal of Muscle Foods, 14(4), 301–318. Kim, K., & Haard, N. F. (1992). Degradation of proteoglycans in skeletal muscle of Pacific rockfish (Sebastes sp.) during ice storage. Journal of Muscle Foods, 3, 103–121. Lave´ty, J., Afolabi, O. A., & Love, R. M. (1988). The connective tissue of fish. IX Gaping in farmed species. International Journal of Food Science and Technology., 23, 23–30. Liu, A., Nishimura, T., & Takahashi, K. (1994). Structural-changes in endomysium and perimysium during postmortem ageing of chicken semitendinosus muscle—contribution of structural weakening of intracellular connective tissue to meat tenderization. Meat Science, 38(2), 315–328. Liu, A., Nishimura, T., & Takahashi, K. (1995). Structural weakening of intramuscular connective tissue during post mortem ageing of chicken semitendinosus muscle. Meat Science, 39(1), 135–142. Love, R. M. (1988). Gaping. In R. M. Love (Ed.), The Food fishes their intrinsic variation and practical implication (pp. 161–180). London: Farrand Press. Love, R. M., Lavety, J., & Steel, P. J. (1969). The connective tissues of fish. II. Gaping in commercial species of frozen fish in relation to rigor mortis. Journal of Food Technology, 4, 39–44. Nakayama, T., Goto, E., & Ooi, A. (1996). Observation of characteristic muscle structure related to delay in red sea-bream rigor mortis by spinal cord destruction. Fisheries Science, 62(6), 977–984. Nishimura, T., Hattori, A., & Takahashi, K. (1996). Relationship between degradation of proteoglycans and weakening of the intramuscular connective tissue during post-mortem ageing of beef. Meat Science, 42, 251–260. Ofstad, R., Egelandsdal, B., Kidman, S., Myklebust, R., Olsen, R. L., & Hermansson, A. M. (1996). Liquid loss as effected by post mortem ultrastructural changes in fish muscle: Cod (Gadus morhua) and salmon (Salmo salar). Journal of the Science of Food and Agriculture, 71, 301–312. Ofstad, R., Kidman, S., Myklebust, R., & Hermansson, A. M. (1993). Liquid holding capacity and structural changes during heating of fish muscle, cod (Gadus morhua L.) and salmon (Salmo salar). Food Structure, 12, 163–174.
Olsson, G. B., Olsen, R. L., & Ofstad, R. (2003). Post-mortem structural characteristics and water-holding capacity in Atlantic halibut muscle. Lebesmittel-Wissenschaft und-Technologie, 36, 125–133. Papa, I., Taylor, R. G., Astier, C., Ventre, F., Lebart, M. C., Roustan, C., et al. (1997). Dystrophine cleavage and sarcolemma detachment are early post mortem changes on bass (Dicentrarchus labrax) white muscle. Journal of Food Science, 62(5), 917–921. Reichardt, L. F. (1999). Extracellulat matrix molecules and their receptors. In T. Kreis, & R. Vale (Eds.), Extracellular matrix, anchor and adhesion proteins (pp. 335–344). New York: Oxford University Press. Robb, D. H. F., Kestin, S. C., & Wariss, P. D. (2000). Muscle activity at slaughter: I. Changes in flesh colour and gaping in rainbow trout. Aquaculture, 182, 261–269. Rustad, T. (1992). Muscle chemistry and quality of wild and farmed cod. In H. H. Huss, M. Jakobsen, & J. Liston (Eds.), Quality assurance in the fish industry (pp. 19–27). London: Elsevier Science Publisher. Sato, K., Ando, M., Kubota, S., Origasa, K., Kawase, H., Toyohara, H., et al. (1997). Involvement of type V collagen in softening of fish muscle during short-term chilled storage. Journal of Agriculture and Food Chemistry, 45, 343–348. Sato, K., Koike, A., Yoshinaka, R., Sato, M., & Shimizu, Y. (1994). Post mortem changes in type I and V collagens in myocommatal and endomysial fractions of rainbow trout (Oncorhynchus mykiss) muscle. Journal of Aquatic Food Product Technology, 3(2), 5–11. Sato, K., Ohashi, C., Ohtsuki, K., & Kawabata, M. (1991). Type V collagen in trout (Salmo gairdneri) muscle and its solubility change during chilled storage of muscle. Journal of Agriculture and Food chemistry, 39, 1221–1225. Sato, K., Yoshinaka, R., Itoh, Y., & Sato, M. (1989). Molecular species of collagen in the intramuscular connective tissue in fish. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 92, 87–91. Scott, J. E. (1996). Proteodermatan and proteokeratan sulfate (decorin, lumican/fibromodulin) proteins are horseshoe shaped. Implications for their interactions with collagen. Biochemistry, 35(27), 8795–8799. Scott, J. E., & Dorling, J. (1965). Differential staining of acid glycosaminoglycans (mucopolysaccharides) by Alcian blue in salt solutions. Histochemie, 5(3), 221. Sheehan, E. M., OConnor, T. P., Buckley, D. J., & FitzGerald, R. (1996). Effect of dietary fat intake on the quality of raw and smoked salmon. Irish Journal of Agriculture and Food Research, 35, 37–42. Taylor, R. G., Fjæra, S. O., & Skjervold, P. O. (2002). Salmon fillet texture is determined by myofiber–myofiber and myofiber–myocommata attachment. Journal of Food Science, 67(6), 1–5. Taylor, R. G., & Koohmaraie, M. (1998). Effects of post mortem storage on the ultrastructure of the endomysium and myofibrils in normal and callipyge longissimus. Journal of Animal Science, 76, 2811–2817. Tingbø, M. G., Kolset, S. O., Ofstad, R., Enersen, G., & Hannesson, K. O. (2005). Sulfated glycosaminoglycans in the extracellular matrix of muscle tissue in Atlantic cod (Gadus morhua L.) and Spotted wolffish (Anarhichas minor O.). Comparative Biochemistry and Physiology B, 140, 349–357.
LWT 39 (2006) 1143–1154 www.elsevier.com/locate/lwt
Breakdown of intramuscular connective tissue in cod (Gadus morhua L.) and spotted wolffish (Anarhichas minor O.) related to gaping R. Ofstada,, R.L. Olsenb, R. Taylorc, K.O. Hannessona a
Matforsk, Oslov 1, 1430 Aas, Norway Norwegian College of Fishery Science, University of Tromsoe, Norway c INRA-Theix, St Genes Champanelle, France
b
Received 15 February 2005; accepted 7 June 2005
Abstract The purpose of this work was to compare the microstructure of muscle connective tissue in cod and wolffish and to study its degradation during ice-storage to determine which structural alterations are related to the gaping phenomenon. Gaping is very often found in fillets of codfish, but not in wolffish. The results showed that detachments between myofibres occurred prior to the myotomes to myocommata detachments, which occurred more rapidly and to a larger extent in cod than in wolffish. Changes at the ultrastructural level in myocommata proper were mainly related to gaps between collagen fibres and between collagen fibres and cells in the extracellular matrix (ECM). The mean collagen fibre diameter was smaller and the collagen network in myocommata proper was denser in wolffish than in cod. After 7 days of ice-storage the collagen fibres, particular in cod, appeared sparsely packed. The results implied that the increased gaping during ice-storage might be due to degradation of proteoglycans and glycoproteins, important for the spatial organization of the collagen fibres and in anchoring cells in the ECM to the collagen network. r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Fish; Connective tissue; Gaping; Microstructure; Muscle
1. Introduction Gaping is a phenomenon in which the connective tissue fails to hold the fish fillet together resulting in gaps and tears at the myofibre–myocommata attachments and between myofibres. This results in fillets with unacceptable appearance and might also influence textural properties. Differences exist between fish species and individuals in their propensity to gape (Love, 1988). Gaping accompanied by softening of the tissue occurs as results of the general deterioration processes during storage and may be increased by rapid and strong muscle contractions during rigor (Bremner, 1999; Love, 1988; Taylor, Fjæra, & Skjervold, 2002). For a given species the rate of deterioration depends on the biological status, catch or slaughter history, and temperature during storage (Lavety, Afolabi, Corresponding author. Tel.:+4764970293; fax: +4764970333.
E-mail address: [email protected] (R. Ofstad).
& Love, 1988; Robb, Kestin, & Wariss, 2000; Sheehan, OConnor, Buckley, & FitzGerald, 1996). In comparison with the muscle of wild cod, softer texture and fillet gaping are often seen in pen raised Atlantic cod (Gadus morhua L.) (Ang & Haard, 1985; Ofstad et al., 1996; Rustad, 1992). Also farmed Atlantic salmon (Salmo salar) suffers from gaping (Lave´ty et al., 1988). In contrast to fillets from salmon and cod, spotted wolffish (Anarhichas minor O.) fillets apparently do not gape (Love, Lavety, & Steel, 1969). Fish flesh is organized in concentric muscle segments (myotomes) surrounded by intramuscular connective tissue (IMCT). Major constituents identified in the IMCT include collagen and elastic fibres, cells (fibroblasts, adipocytes, macrophages), glycoproteins and proteoglycans. The proteoglycans consist of a protein core with variable amounts of covalent attached linear polysaccharide with sulphated carbohydrate side chains, i.e. glycosaminoglycans. The myotomes are separated by the myocommata, where groups of collagen fibres runs roughly parallel to each
0023-6438/$30.00 r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2005.06.019
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other with adjacent groups set at an angle resulting in crisscross layers which provide considerable mechanical strength (Bremner, 1999). A layer of thin connective tissue (endomysium) surrounds each muscle cell (myofibre) and often endomysium connects to the perimysium, which is contiguous with the myocommata. Both collagen I and collagen V is present in the IMCT of fish; collagen I is the major type in the myocommata (Sato, Yoshinaka, Itoh, & Sato, 1989). The structural changes inducing gaping have been thoroughly reviewed by Bremner (1992, 1999). In blue grenadier (Macruronus novaezelandiae) and spotted trevalla (Seriolella punctata) gaping was related to degradation of fine collagen fibres at the myotendinous junctions connecting the myocommata and the myofibres leading to loosening of the structures in this area (Bremner, 1992; Bremner & Hallet, 1985, 1986; Hallet & Bremner, 1988). The work of Fletcher, Hallett, Jerrett, and Holland, 1997 indicated that in Chinook salmon (Oncorhynchus tshawytscha) the site for detachment when fish were electrically stimulated post mortem, might be within the muscle cell where the thin costameric filaments from the terminal Z-line join the inner surface of the sarcolemma at the adherens junction. Softening of rainbow trout (Oncorhynchus mykiss) flesh was related to weakening of the pericellular connective tissue due to fragmentation/disordering of the collagen fibre network structure, but no structural changes in myocommata were observed (Ando, Toyohara, & Sakaguchi, 1992; Ando, Toyohara, Shimizu, & Sakaguchi, 1991). In Atlantic salmon (Salmon salar) loss of myofibreto myofibre (endomysium) attachments was evident prior to any loss of myocommata integrity during cold storage (Taylor et al., 2002). Degradation of IMCT in pacific rockfish (Sebastes sp.) has been related to both increased solubilization of collagen and degradation of the glycosaminoglycans (Kim & Haard, 1992). Early post mortem decomposition of the pericellular connective tissue has also been reported for bovine meat (Hannesson, Pedersen, Ofstad, & Kolset, 2003; Nishimura, Hattori, & Takahashi, 1996). Even though gaping was described more than 30 years ago and much work on this topic has been done, the mechanisms behind are far from understood. The purpose of this work was to compare the microstructure of connective tissue in cod and wolffish and to determine the type of structural changes which were related to post mortem myofibre–myocommata detachments and gaping. 2. Materials and methods 2.1. Fish Wild caught and thereafter pen raised Atlantic cod (G. morhua L.) weighing 3–4 kg and farmed spotted wolffish (Anarchichas minor O.) weighing 2–3 kg, were used. The fish were obtained from different fish farms in Northern Norway in January and February (cod) and January and
November (wolffish). Five fish of each species were carefully netted from the pens, and killed by a blow to the head and filleted within 1 h after slaughter. Strips of muscle samples were excised from the fillet near the dorsal fin of each of the five fish within 2 h (day 0) and at days 2 and 7 post mortem. All fillets were kept in plastic bags and stored in ice. 2.2. Images Images of the fish fillets were taken with a CCD camera (Cannon digital D30) equipped with a standard 55 mm macro-objective. Tungsten light was used. 2.3. Microscopy 2.3.1. Cryo-sections Muscle samples of 4 4 3 mm3 were obtained from the white muscle beneath the dorsal fin of the cod and wolffish and frozen as previously described by Ofstad, Kidman, Myklebust, and Hermansson (1993). Sections of 5 mm thickness were cut in a cryostat (Leitz 1720 Digital Instruments GMBH, Heidelberg, Germany) and mounted on poly-L-lysin coated glass slides which were kept at 20 1C until use. After staining, the cover slips were photographed in a Leica DMLD microscope (Leica Microsystems, Nussloch GmbH, Germany) by a Spot RT Color Camera (Diagnostic Instruments Inc., Burroughs Sterling Heights, Michigan, USA). To study the presence of sulphated glycosaminoglycans, Alcian blue 8GX (Gurr Biological Stains, BDH, Pool, UK), 0.4 g in 8.20 g/l acetate buffer pH 5.8 containing either 12.20 or 81.32 g/l MgCl2 were used according to the procedures described by Tingbø, Kolset, Ofstad, Enersen, and Hannesson (2005). At 12.20 g/l MgCl2 most acidic groups stain whereas at 81.32 g/l MgCl2 only negatively charged groups of sulphated proteoglycans stain (Scott & Dorling, 1965). 2.3.2. Plastic-sections Muscle blocks of 1 1 3 mm3 were fixed in 2.5 g/100 ml formaldehyde and 1.0 g/100 ml glutaraldehyd with 0.2 g/ 100 ml glucose in Ringer’s buffer (pH 6.8) and embedded in Epon/Araldite (Serva, Heidelberg, Germany) as previously described by (Ofstad et al., 1993). For all samples, 1.5 mm thick plastic sections were cut perpendicular to the fibres, allowing examination of myofibre–endomysium, myofibre–myocommata junctions and myocommata proper. Optical microscopy observations were of sections stained in 0.1 g/100 ml toluidine blue dissolved in 1 g/100 ml aqueous sodium borate solution. A minimum of four fish of each species was examined at each sampling time. For transmission electron microscopy (TEM) thin sections (70–90 nm) were stained with uranyl acetate and lead citrate. Sections were examined and photographed using a TEM, 100 CXII (Jeol Ltd., Tokyo, Japan) at an accelerating voltage of 80 kV and instrument magnification of 1500 and 10,000. A total of eight microscopic fields
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were obtained by systematic random sampling from the myotendinous junction and myocommata proper of each section. Three fish were examined at each sampling time. In fish, structural alterations may be very focally. Thus two or more blocks from each of the three fish were investigated. The micrographs presented were chosen out of many and illustrated the most typical features and post-mortal changes. 2.3.3. Quantification Optical microscopy with a 10 objective was used to measure myofibre to myofibre detachment and myofibre to myocommata detachment. Quantification of structural changes was performed as previously described for Atlantic salmon (Taylor et al., 2002). This involved counting the numbers of myofibres attached and partly or completely detached from the neighbouring myofibre for a minimum of 200 myofibre-to-myofibre attachments. In addition, myofibre-to-myocommata detachments were determined in a similar manner by counting the number of the myofibres attached and partly or completely detached from the myocommata, for a minimum of 50 myofibre-tomyocommata attachments for each sample. Type and degree of structural changes in the myocommata were quantified on TEM images by the use of a transparent point lattice test system with spacing of 20 mm (equivalent to 5 mm on the image scale). The structures being overlaid by the points were classified, counted and given as percent of the total points of connective tissue or as percent of total detachment points on each image. These percentages thus represent the relative area of the structures being counted. The following structures were counted: collagen fibres, cells (fibroblasts, adipocytes, macrophages) and other structures (blood vessels and nerves), gaps between collagen fibres, gaps between collagen fibres and cells, gaps between sarcolemma and extracellular matrix and intracellular gaps between myofibres and sarcolemma. A minimum of 1500 points was counted on the 24 micrographs obtained from three fish of each species at each sampling time. This should give a 10% standard error, when structure counted is estimated to occupy 7% of the area in ECM (Ang & Haard, 1985; Bozzola & Russell, 1999; Ofstad et al., 1996; Rustad, 1992). In addition, collagen fibre diameters in the myofibre–myocommata junction zone and in the myocommata proper were measured using a measuring magnifier with metric scale. Hundred fibres were measured randomly on four micrographs with magnification 10,000. Three fish sampled immediately after slaughter (day 0) were analysed, and the fibre diameter was given as the mean diameter of a total of 300 collagen fibres. Subsequently, transparent lattices with spacing of 7 mm (equivalent to 250 nm on the image scale) were superimposed randomly on the same microscopic images. The number of collagen fibres in a total of 80 squares representing an area of 5 mm2 was counted for each specimen. The figures are given as the mean of 3 fish.
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2.4. Statistical analyses Analysis of variance was done with the GLM-procedure in SAS (SAS Institute Inc., Cary, NC, USA, version 8.12). Tukey’s test was used for multiple comparisons of detachments as quantified on micrographs. All significant tests were performed at the 0.05 level. 3. Results 3.1. Visual appearance Fillets of cod and wolffish stored 2 and 7 days in ice are shown in Fig. 1. After 2 days of storage some gaps and tears (arrows) could be seen in the cod fillet. The wolffish had a smooth appearance with no visible gaps and tears. After 7 days of storage, the cod fillets had clearly visible gaps and tears and showed gaping. In the wolffish fillets some minor gaps and tears were present, but no clear gaping could be seen. Although the cod were obtained in January and February from two separate fish farms, no farm specific differences in visual appearance or in structural degradations were noticed. Similar observations were made for the wolffish obtained from two farms in November and January. 3.2. Light microscopic observations Light microscopic images of cod and wolffish fillets excised immediately after death, and after 2 and 7 days storage in ice are shown in Fig. 2. At day 0 both endomysium and myocommata showed a strong and consistent staining even though some gaps could be seen. In cod both loss of myofibre–myofibre attachments (arrows) as well as loss of myofibre–myocommata attachments (arrowhead) were evident after 2 days of storage In wolffish only a few minor detachments could be seen after 2 days. At day 7 post mortem, most of the myofibres were detached in both species. In cod the myofibre–myocommata detachment appeared however to be more extensive than in the wolffish.
Fig. 1. Images of fish fillet of cod (a and b) and wolffish (c and d) stored in ice, 2 days (a and c) and 7 days (c and d) after filleting. In the cod images (a and b) gaps and tears are visible.
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Fig. 2. Cryo-sections of white muscle stained with Alcian blue with 12.20 g/l MgCl2. Cod (a–c) and wolffish (d–f) excised immediately after death (a and d) and after 2 (b and e) and 7 (c and f) days storage in ice. At 12.20 g/l MgCl2 most acidic groups, i.e. myfibres (my), endomysium (e) and myocommata (mc) stain blue. Myofibre–myofibre detachments are indicated with arrows and myofibre–myocommata detachments with arrowheads. The bars indicate 200 mm.
When tissue sections were stained with Alcian blue containing low concentration of MgCl2 (12.20 g/l), myocommata as well as endomysium and myofibres stained (Fig. 2). When increasing the concentration of MgCl2 to the high level (81.32 g/l), only the sulphated glycosaminoglycans in the connective tissue stained (both endomysium and myocommata) as shown in Fig. 3. A stronger and more consistent staining was seen in the endomysium and myocommata of wolffish than in cod at day 0. For both fish species the blue colour was weakened during storage. After 7 days of storage myocommata in cod were hardly visible, but still quite distinct in wolffish. Quantification of the detachments, which were observed in micrographs of toluidine blue stained plastic sections, is shown in Table 1. For both species a significant (***Po0:0001) loss of myofibre to myofibre adhesion
was observed within 2 days. The percentage of detachments was somewhat higher for cod than for wolffish. The myofibre to myocommata adhesion changed significantly (***Po0:0001) for cod from time 0 to day 2. For wolffish, a significant (**Po0:01) increase in the numbers of detachments was not registered until day 7. 3.3. Ultrastructural observations To determine which structures were changing at the ultrastructural level as gaping developed, the fish muscle was examined by TEM. Structural changes occurring between the myofibres are shown in Fig. 4. Since there were only minor differences between cod and wolffish, only the micrographs of cod are shown. In the 0 day sample (Fig. 4a) the myofibres were attached and the structure
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Fig. 3. Cryo-sections of white muscle stained with Alcian blue with 81.32 g/l MgCl2. Cod (a–c) and wolffish (d–f) excised immediately after death (a and d) and after 2 (b and e) and 7 (c and f) days storage in ice. At 81.32 g/l MgCl2 only the sulphated glycosaminoglycans in the connective tissue, i.e. endomysium (e) and myocommata (mc) stain blue. The bars indicate 200 mm.
Table 1 Quantification by light microscopy of myofibre–myofibre and myofibre–myocommata detachments in white muscle from cod and wolfish Myofibre–myofibre (mean7SD) Cod Day 0 Day 2 Day 7
Myofibre–myocommata (mean7SD) Wolffish
a
31.877.7 99.771.0b 99.171.8b
Cod a
4.773.2 88.178.3b 95.878.3b
Wolffish a
2.773.9 90.5713.8b 92.6714.8b
4.176.7a 14.9713.1a 77.4732.2b
A minimum of 200 myofibre-to-myofibre attachments and 50 myofibre-to-myocommata attachments were counted on four fish of each species at each sampling time. The mean is given in percent7SD of the total number counted. Figures in the same colon with different letters are significant different at *Po0:05.
appeared intact. After 2 days of storage the sarcolemma was often detached and gaps occurred in the endomysium causing separation between myofibres (Fig. 4b). Structural changes in the myocommata of cod and wolffish are shown in Fig. 5a–c and d–f, respectively. In the
0 day muscle myocommata appeared dense with closely packed collagen layers surrounding various cells in the ECM (Fig. 5a and d). After 2 days storage in ice, myocommata showed signs of disintegration. In cod (Fig. 5b) there were clearly visible gaps, whereas in wolffish
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Fig. 4. TEM micrographs showing the endomysial (e) layer of a transversally sectioned cod white muscle excised immediately after death (a) and stored 2 days in ice (b). Endomysium (e), myofibres (my), sarcolemma (sl) and collagenous fibres in the endomysium (c).
Fig. 5. TEM micrographs showing myocommata and myofibrils (mf) of a transversally sectioned white muscle of cod (a–c) and wolffish (d–f) excised immediately after death (a and d) and stored 2 (b and e) and 7 (c and f) days in ice. Gaps are indicated (*).
the myocommata appeared less dense (Fig. 5e) than in the 0 day image. In the samples taken after 7 days storage, the gaps were clearly evident in both species, but the connective tissue seemed more disintegrated in cod (Fig. 5c) than in wolffish (Fig. 5f). The percentage of
points classified as detachments increased significantly (**Po0:01) with storage in ice for both species and the percent detachment points were significant higher (***Po0:001) for cod than for wolffish both after 2 and 7 days of ice storage (Table 2).
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Table 2 Quantification on TEM images of the degree and type of structural changes in myocommata of cod and wolffish by the use of a transparent point lattice test system
Cod day 0 Cod day 2 Cod day 7 Wolffish day 0 Wolffish day 2 Wolffish day 7
Detachments (DP/TP*100)
Collagen fibre–fibre (P/DP*100)
Cell–collagen fibre (P/DP*100)
Sarcolemma–ECM (P/DP*100)
Myofibrill–sarcolemma (P/DP*100)
0.870.8a 14.577.6b 61.878.3c 0.070.0d 4.170.0e 8.472.1f
23.8741.2 57.1734.4 76.978.3 0.070.0 44.9739.2 56.4716.5
42.9751.5 38.7734.2 11.478.1 0.070.0 28.2724.5 18.3711.1
0.070.0 3.574.4 4.975.9 0.070.0 0.070.0 6.3710.9
0.070.0 0.871.3 6.473.2 0.070.0 33.3757.7 19.0723.8
The structures being overlaid by the points (P) were classified and counted and given as percent of the total points (TP) of connective tissue or as percent of total detachment points (DP) on each image. A minimum of 1500 points was counted on the 24 micrographs obtained from three fish of each species at each sampling time. The mean is given in percent7SD. Figures in the first colon with different letters are significant different at *Po0:05.
Examination at higher magnification of both fish species after 2 and 7 days of ice storage revealed that the gaps occurred at four different sites as illustrated for cod in Fig. 6. Gaps were also located at the same sites in wolffish (results not shown). In the mid-region of myocommata (myocommata proper) gaps occurred within the collagen layers between the collagen fibres (Fig. 6a) or at the collagen fibre–cell junctions (Fig. 6b). Gaps in the myomere–myocommatal junction zone were due to detachments between the sarcolemma/basement membrane and the extracellular matrix (Fig. 6c) or within the myofibre between the myofibrils and the sarcolemma (Fig. 6d). To see if there were differences between the species regarding the dominant type of gaps which developed during storage, all gaps were classified as one of those four types illustrated in Fig. 6. The percentage of point of the total detachment points thus representing the relative area of the different gaps are given in Table 2. Both in cod and wolffish, the majority of detachments were within the collagen layers between the collagen fibres. At 7 days post mortem, 75% and 55% of the points were classified as collagen fibre–fibre detachments in cod and wolffish, respectively. The 10–40% of the points were classified as detachments between cells and collagen fibres. The percent detachments between collagen fibres increased during storage, whereas the percent detachments between cells and collagen fibres tended to decrease. In cod the highest percent of this type of gaps were in the 0 day sample. The individual differences as reflected in the high standard deviation of these figures at day 0 and day 2, were however large. In general there were less detachments in the myomere–myocommatal junction zone than in myocommata proper. Less than 10% of the detachments were between sarcolemma and ECM. The percent of this type of detachment increased during storage, but no differences between the two species were observed. The percent of gaps between myofibrils and sarcolemma (intracellular) tended to increase during storage as well, but was much higher for wolffish (20–30%) than for cod (1–6%) both after 2 and 7
days of ice storage. Also for this type of gap there were large individual differences as reflected in the high standard deviations. The cause of the large individual differences regarding the amount and type of gaps in the myocommata is unknown, and should be studied. The composition of the myocommata of the two species as classified on the same TEM images is given in Table 3. The percentage of points classified as collagen and cells were similar in cod and wolffish. Collagen made up 85% of the relative area and different cells such as fibroblasts, macrophage-like cells and a few fat droplets (only in wolffish) made up 15%. Fibroblasts were the most abundant cell type in connective tissue. The main structural difference in ECM between cod and wolffish was related to the size and the density of collagen fibres in the myocommata proper (Fig. 7 and Table 3). The mean diameter of the collagen fibres regardless of position was 59 and 45 nm for cod and wolffish, respectively. This difference between the two species was significant at a level of 0.05. Fig. 7 shows micrographs of transversally sectioned collagen fibres from the myomere–myocommatal junction zone (Figs. 7a and d) and the myocommata proper (Figs. 7b and e) sampled at day 0. The first layer of collagen, closest to the myofibre, had small and densely packed fibres. In myocommata proper of wolffish the fibres were also small (Fig. 7e), whereas in cod there was both small and large fibres randomly distributed (Figs. 7b). This is also reflected in the large standard deviation of the mean diameter in cod (Table 3). Comparing muscle samples from the two different positions of cod and wolffish separately, a significant difference (*Po0:05) was found in the collagen fibre diameter in the myocommata proper between cod and wolffish, but not of those in the first layer. The collagen fibres in cod were significantly (*Po0:05) larger in the myocommata proper than in the first layer at the myomere–myocommatal junction zone, whereas in wolffish there were no differences in the collagen diameters between the two positions. In wolffish the collagen fibres in myocommata proper were significantly (*Po0:05) more densely packed than in cod (Table 3). In cod the collagen
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Fig. 6. TEM micrographs of cod showing gaps (*) in the myocommata proper between the collagen–fibres (a), at the collagen fibril–cell junction (b), in the myomere–myocommatal junction zone between the sarcolemma (sl) and the extracellular matrix (c) and intracellular between the fine myofibrils (mf) and the sarcolemma (sl) (d). Similar types of gaps were also found in wolffish (results not shown).
Table 3 Quantification on TEM images of the composition of myocommata in cod and wolffish by the use of a transparent point lattice test system Collagen (P/TP*100)
Cod Wolffish
85.978.5 84.277.4
Cells (P/TP*100)
14.178.5 15.877.4
Diameter collagen fibril (nm)
Number of collagen fibrils per mm2
Myofibre surface
Myocommata proper
Myofibre surface
Myocommata proper
33.7a74.8 36.9a75.0
83.9b733.7 52.8c78.4
195a773 249a759
75b730 187c747
The structures being overlaid by the points (P) were classified, counted and expressed as percent of the total points (TP) of connective tissue on each image. A minimum of 1500 points was counted on the 24 micrographs obtained from three fish of each species at each sampling time. The mean is given in percent7SD. Collagen fibril diameters were measured on 100 fibrils either in the first collagen layer at the myofibre surface or in the middle of myocommata proper. The number of collagen fibrils representing an area of 5 mm2 was counted in the same two locations for each specimen. The figures are given as the mean7SD of 3 fish. Figures with different letters are significant different at *Po0:05.
fibres in the myomere–myocommatal junction zone were more (*Po0:05) densely packed than in the myocommata proper, whereas in wolffish there were no differences between the two positions. After 7 days of storage in ice, the collagen fibres appeared less densely packed than at day 0. This was more pronounced in cod (Fig. 7c) than in wolffish (Fig. 7f). No obvious structural alteration or fragmentation of the collagen fibres could be seen. In the 0 day samples (Fig. 7b, e) the structures filling the spaces between the collagen
fibres appeared tiny and amorphous. In cod at day 7, these structures (arrow) were grainy and stained darker than at day 0. 4. Discussion The present study showed that gaping occurred faster and to a larger extent in cod than in wolffish during 7 days ice-storage of pre-rigor filleted flesh. It has previously been reported that cod is particularly prone to softening and
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Fig. 7. TEM micrographs of transversally sectioned collagen fibres of cod (a–c) and wolffish (d–f) at the myomere–myocommatal junction zone (a and d) and the myocommata proper (b, c, e, f) sampled at day 0 (a, d, b, e) and at day 7 (c, f). Proteoglycans are arrowed in panel c.
gaping (Ang & Haard, 1985; Ofstad et al., 1996; Rustad, 1992) while wolffish much more rarely shows this property (Love et al., 1969). The current study is the first time that the structural changes resulting in gaping has been compared in these two species and that the components related to gaping have been localized and quantified. The overall structure of the muscle connective tissue in cod and wolffish was the same as found for other species of fish (Bremner & Hallet, 1985, 1986; Fletcher et al., 1997; Hallet & Bremner, 1988; Taylor et al., 2002).
Examination of cryo-sectioned muscle samples revealed that gaping was caused by both loss of myofibre–myofibre and myofibre–myocommata adhesion. Detachments of the myofibres were evident as gaps between the myofibres and between the myofibres and myocommata in micrographs stained with Alcian blue with the lower concentration of MgCl2 (12.20 g/l). A simultaneous decrease in stainability of the connective tissue by Alcian blue with the higher concentration of MgCl2 (81.32 g/l) indicated a structural disintegration of the sulphated glycosaminoglycans in
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ECM. This was more apparent in cod than in wolffish. Whether the stronger staining of the connective tissue in the wolffish muscle than in cod with Alcian blue suggests a higher content of sulphated glycosaminoglycans or a higher degree of sulphatation is not known. Early post mortem degradation of glycosaminoglycans and proteoglycans has previously been reported in both fish (Kim & Haard, 1992; Olsson, Olsen, & Ofstad, 2003) and bovine meat (Hannesson et al., 2003; Nishimura et al., 1996). Quantification of the myofibre–myofibre and myofibre– myocommata detachments by microscopy of plastic sections revealed that an extensive loss in myofibre to myofibre adhesion had occurred in both cod and wolffish after 2 days storage in ice. Examination of the larger frozen samples indicated that the endomysia of wolffish were more stable than that of cod. Only few and small gaps were visible in the micrograph of the 2 days stored wolffish sample. This divergence may be due to different preparation procedures. Samples for fixation and plastic embedding are cut into much smaller pieces and thus exposed to more manipulation than those fixed in liquid nitrogen. Others have reported that structural changes of early post mortem disintegration of the pericellular connective tissue as observed using light microscopy could only be demonstrated after compression of the muscle (Ando et al., 1991; Nakayama, Goto, & Ooi, 1996). The more pronounced intermyofibrillar spaces in the cryo-sectioned samples of cod may indicate that after 2 days of ice-storage the endomysia of cod were weaker than those of wolffish. Some myofibre–myofibre detachments were also seen in the day 0 samples, particularly in cod, regardless of the preparation procedure used. This may also indicate that the connective tissue of cod was weaker and more vulnerable than that of wolffish. Ando et al. (1992) reported that thin collagen fibres in endomysium disintegrated within few hours. However, artefacts caused by preparation cannot be excluded. In cod attachments between myofibres and myocommata were significantly lost in the 2 days ice-stored sample, but not until the day 7 sample in wolffish. The slower change of the myofibre to myocommata than the myofibre to myofibre adhesion has also been reported for Atlantic salmon (Taylor et al., 2002). Ultrastructural examination showed that the loss of myofibre-to-myofibre adhesion was by both detachment of sarcolemma and structural disintegration of the endomysium (breaks in sarcolemma have previously been demonstrated in pen fed cod stored 2 days in ice (Ofstad et al., 1996)). Similar breakdown of the endomysial sheaths has been reported in other fish species (Ando et al., 1991, 1992; Taylor et al., 2002), avian (Liu, Nishimura, & Takahashi, 1994, 1995) and mammalian muscle (Hannesson et al., 2003; Taylor & Koohmaraie, 1998). Detachment of the sarcolemma was ascribed to breaks in the muscle cytoskeleton connection from the sarcomeres to the endomysium (Papa et al., 1997; Taylor & Koohmaraie, 1998; Taylor et al., 2002). Weakening of the pericellular connective tissue induced by disintegration of thin collagen fibrils in rainbow
trout and sardine (Sardinops melanosticta) muscle, was ascribed to degradation of type V collagen (Sato et al., 1997; Sato, Koike, Yoshinaka, Sato, & Shimizu, 1994; Sato, Ohashi, Ohtsuki, & Kawabata, 1991). Both these events may be involved in the observed myofibre–myofibre detachments in our study. Moreover, the reduced staining of sulphated glycosaminoglycans indicates that degradation of the proteoglycans contributed as well. Ultrastructural changes in myocommata were similar in cod and wolffish, but occurred faster and to a larger extent in cod than in wolffish. The structural changes could be related to at least four different mechanisms for detachments. The collagen fibre–fibre detachment which increased during storage was the most important one in both species. In general the collagen fibres appeared less densely packed after 7 days of ice storage. This supports, as suggested by Bremner (1999) and Taylor et al. (2002), that there are several mechanisms involved in the degradation, but that the gaps are usually in the connective tissue layer. In the present study a reduced staining of sulphated glycosaminoglycans was seen in the light micrographs and a more grainy appearance of the amorphous ground substance filling the spaces between the collagen fibrils, was evident in the TEM images as seen at high magnification after 7 days storage in ice. No structural disintegration or fragmentation of the collagen fibres could be seen. The solubility of collagen I, the major type in myocommata, is also previously reported not to change in rainbow trout during short time chilled storage (Sato et al., 1991, 1994, 1997). Thus degradation of myocommata proper is probably mainly caused by degradation of the proteoglycans and glycoproteins, which function is structural, binding collagen fibres together (Scott, 1996) and anchoring cells to collagen or basement membranes (Reichardt, 1999). Degradation of the proteoglycans which may be caused by several enzymes such as lysosomal enzymes, matrix metalloproteinases and aggrecanases, probably involves both degradation of the sulphated glycosaminoglycans as well as the peptide core (Hannesson et al., 2003; Kim & Haard, 1992; Nishimura et al., 1996). In the day 0 (only cod) and day 2 samples a significant amount of gaps were seen in the collagen fibril–cell junction particularly along the lines of the processes. Bremner (1999) also reported gaps in this area. He suggested that the cells represented a point of weakness. The large standard deviation of these types of gaps may thus be related to the large variation in the number of such cells between the individuals. In the TEM images rather few gaps were seen in the myomere–myocommatal junction zone, either extracellular between the basement membrane and the ECM or intracellular between myofibrils and sarcolemma. The percent of extracellular gaps increased during storage. This is in accordance with earlier results, showing that a progressive breakdown of the invaginating collagen fibres and the basal lamina occurred during storage of blue grenadier and spotted trevalla (Bremner & Hallet, 1985, 1986). Percent gaps caused by intracellular
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breakage were more significant in wolffish than in cod after ice-storage, but the individual variations were substantial. In electrical stimulated Chinook salmon it was shown that gaping was due to deterioration of the links between the thin filaments at the adherent junctions on the inner surface of the sarcolemma (Fletcher et al., 1997). We used pre-rigor filleted fish in our experiments, which reduced the mechanical stress and thus might have reduced the number of intracellular gaps. To elucidate if there were compositional differences between the two species, the components in the myocommata were quantified. The distribution of collagen and different cells in myocommata were similar in cod and wolffish, except that wolffish contained some fat droplets. The major differences between the two species were the mean collagen fibre diameter and the fibre density in the myocommata proper. The collagen fibres were on average smaller and more densely packed in wolffish than in cod. Similar differences between the two species were not seen in the myomere–myocommatal junction zone. The size of the collagen fibres (35 nm) in the myotendinous junction was as those (30–70 nm) reported for hoki (Macruronus novaezelandiae) (Hallet & Bremner, 1988). Taylor (unpublished results) found in trout a similar mean collagen fibre diameter in the middle layer of myocommata proper as reported in our work, and moreover that the fibre diameter was larger in the middle region than in the myomere–myocommatal junction zone regardless of muscle region investigated. Another difference between cod and wolffish was the fibre size distribution which was larger in cod (30–180 nm) than in wolffish (33–74 nm). In decorin knock out mice a wide range in collagen fibre diameters has been described together with fragile and reduced tensile strength of the skin (Danielson et al., 1997). Moreover, in the tough M. semitendinosus bovine muscle the mean collagen fibre diameter was larger, the fibre size were unimodale and more tightly bundled with less matrix in-between compared to the tender M. psoas major (Eggen, Pedersen, Lea, & Kolset, 2001). Wolffish is regarded as having tougher connective tissue than cod. The reason why collagen fibre diameter in fish species varies is however not known. It is possible that the presence of minor collagen types such as Type V collagen may affect the diameter of collagen fibres. Whether collagen V, or other collagen types such as FACIT collagens (fibril-associated collagens with interrupted triple helices) are involved in post mortem degradation of myocommata should be further studied. The myofibre to myofibre detachments occurred prior to the myofibre–myocommata detachments in both fish species. The myofibre to myocommata attachment was more stable in wolffish than in cod. Moreover, the myofibre to myocommata detachments were mainly caused by fractures in the extracellular matrix (ECM) and these structural changes occurred faster and to a larger extent in cod than in wolffish. According to Baily and Light (1989) large collagen fibres and densely packed fibre bundles are less vulnerable to degradation by proteases during storage of bovine meat. The more densely packed collagen fibres in
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wolffish than in cod could thus explain the slower breakdown of the connective tissue in this species. Therefore, differences in gaping propensity between cod and wolffishmost probably are related to differences in the myocommata structure, i.e. the collagen fibre diameter and packing density, and the composition of matrix between the fibres. For both species the ECM was mainly broken between the collagen fibres and at the collagen fibre–cell junctions. A possible mechanism of importance for myofibre–myocommata detachment may be degradation of proteoglycans that organize and stabilize the collagen fibres in the layered structure of myocommata proper. In addition mechanical forces acting upon these structures during rigor mortis may also contribute to enhance this type of detachment. Further studies are needed to elucidate the full degradation mechanisms of the different components and the relationship to species-dependent structural features in connective tissue of fish white muscle. Acknowledgements The Norwegian research council through Grant no. 134988/I10 supported this study. The assistance of Grethe Enersen and Per Lea (Matforsk), and Helga-Marie Bye (Inst of medical biology, University of Tromsoe) were highly appreciated. References Ando, M., Toyohara, H., & Sakaguchi, M. (1992). Post-mortem tenderization of rainbow trout muscle caused by the disintegration of collagen fibres in the pericellular connective tissue. Nippon Suisan Gakkaishi, 58(3), 567–570. Ando, M., Toyohara, H., Shimizu, Y., & Sakaguchi, M. (1991). Postmortem tenderization of rainbow trout (Oncorhyncus mykiss) muscle caused by gradual disintegration of the extracellular matrix structure. Journal of the Science of Food and Agriculture, 55, 589–597. Ang, J. F., & Haard, N. F. (1985). Chemical composition and postmortem changes in soft textured muscle from intensely feeding Atlantic cod (Gadus morhua, L.). Journal of Food Biochemistry, 9, 49–64. Baily, A. J., & Light, N. D. (1989). Connective tissue in meat and meat products. New York: Elsevier Applied Science Publisher. Bozzola, J. J., & Russell, L. D. (1999). Quantitative electron microscopy. In J. J. Bozzola, & L. D. Russell (Eds.), Electron microscopy. Principles and techniques for biologists (pp. 321–340). Sudbury, MA: Jones and Barlett Publishers. Bremner, H. A. (1992). Fish flesh structure and the role of collagen: Its post mortem aspects and implication for fish processing. In H. H. Huss, M. Jakobsen, & J. Liston (Eds.), Quality assurance in the fish industry (pp. 39–62). London: Elsevier Science Publisher. Bremner, H. A. (1999). Gaping in fish flesh. In K. Sato, M. Sakaguchi, & H. A. Bremner (Eds.), Extracellular matrix of fish and shellfish (pp. 81–94). Trivandrum, India: Research Signpost. Bremner, H. A., & Hallet, I. C. (1985). Muscle fibre–connective tissue junctions in the fish blue grenadier (Macruronus novaezelandiae). A scanning electron microscope study. Journal of Food Science, 50, 975–980. Bremner, H. A., & Hallet, I. C. (1986). Degradation in muscle fibre–connective tissue junctions in the spotted trevalla (Seriolella punctata) examined by scanning electron microscopy. Journal of the Science of Food and Agriculture, 37, 1011–1018.
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Danielson, K. G., Baribault, H., Holmes, D. F., Graham, H., Kadler, K. E., & Iozzo, R. V. (1997). Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility. Journal of Cell Biology, 136(3), 729–743. Eggen, K. H., Pedersen, M. E., Lea, P., & Kolset, S. O. (2001). Structure and solubility of collagen and glycosaminoglycans in two bovine muscles with different textural properties. Journal of Muscle Foods, 12(4), 245–261. Fletcher, G. C., Hallett, I. C., Jerrett, A. R., & Holland, A. J. (1997). Changes in the fine structure of the myocommata–muscle fibre junction related to gaping and exercised muscle from king salmon (Oncorhynchus tshawytscha). Lebensmittel-Wissenschaft undTechnologie, 246–252. Hallet, I. C., & Bremner, A. H. (1988). Fine structure of the myocommata–muscle fibre junction in hoki (Macruronus novaezelandiae). Journal of the Science of Food and Agriculture, 44, 245–261. Hannesson, K. O., Pedersen, M. E., Ofstad, R., & Kolset, S. O. (2003). Breakdown of large proteoglycans in bovine intramuscular connective tissue early postmortem. Journal of Muscle Foods, 14(4), 301–318. Kim, K., & Haard, N. F. (1992). Degradation of proteoglycans in skeletal muscle of Pacific rockfish (Sebastes sp.) during ice storage. Journal of Muscle Foods, 3, 103–121. Lave´ty, J., Afolabi, O. A., & Love, R. M. (1988). The connective tissue of fish. IX Gaping in farmed species. International Journal of Food Science and Technology., 23, 23–30. Liu, A., Nishimura, T., & Takahashi, K. (1994). Structural-changes in endomysium and perimysium during postmortem ageing of chicken semitendinosus muscle—contribution of structural weakening of intracellular connective tissue to meat tenderization. Meat Science, 38(2), 315–328. Liu, A., Nishimura, T., & Takahashi, K. (1995). Structural weakening of intramuscular connective tissue during post mortem ageing of chicken semitendinosus muscle. Meat Science, 39(1), 135–142. Love, R. M. (1988). Gaping. In R. M. Love (Ed.), The Food fishes their intrinsic variation and practical implication (pp. 161–180). London: Farrand Press. Love, R. M., Lavety, J., & Steel, P. J. (1969). The connective tissues of fish. II. Gaping in commercial species of frozen fish in relation to rigor mortis. Journal of Food Technology, 4, 39–44. Nakayama, T., Goto, E., & Ooi, A. (1996). Observation of characteristic muscle structure related to delay in red sea-bream rigor mortis by spinal cord destruction. Fisheries Science, 62(6), 977–984. Nishimura, T., Hattori, A., & Takahashi, K. (1996). Relationship between degradation of proteoglycans and weakening of the intramuscular connective tissue during post-mortem ageing of beef. Meat Science, 42, 251–260. Ofstad, R., Egelandsdal, B., Kidman, S., Myklebust, R., Olsen, R. L., & Hermansson, A. M. (1996). Liquid loss as effected by post mortem ultrastructural changes in fish muscle: Cod (Gadus morhua) and salmon (Salmo salar). Journal of the Science of Food and Agriculture, 71, 301–312. Ofstad, R., Kidman, S., Myklebust, R., & Hermansson, A. M. (1993). Liquid holding capacity and structural changes during heating of fish muscle, cod (Gadus morhua L.) and salmon (Salmo salar). Food Structure, 12, 163–174.
Olsson, G. B., Olsen, R. L., & Ofstad, R. (2003). Post-mortem structural characteristics and water-holding capacity in Atlantic halibut muscle. Lebesmittel-Wissenschaft und-Technologie, 36, 125–133. Papa, I., Taylor, R. G., Astier, C., Ventre, F., Lebart, M. C., Roustan, C., et al. (1997). Dystrophine cleavage and sarcolemma detachment are early post mortem changes on bass (Dicentrarchus labrax) white muscle. Journal of Food Science, 62(5), 917–921. Reichardt, L. F. (1999). Extracellulat matrix molecules and their receptors. In T. Kreis, & R. Vale (Eds.), Extracellular matrix, anchor and adhesion proteins (pp. 335–344). New York: Oxford University Press. Robb, D. H. F., Kestin, S. C., & Wariss, P. D. (2000). Muscle activity at slaughter: I. Changes in flesh colour and gaping in rainbow trout. Aquaculture, 182, 261–269. Rustad, T. (1992). Muscle chemistry and quality of wild and farmed cod. In H. H. Huss, M. Jakobsen, & J. Liston (Eds.), Quality assurance in the fish industry (pp. 19–27). London: Elsevier Science Publisher. Sato, K., Ando, M., Kubota, S., Origasa, K., Kawase, H., Toyohara, H., et al. (1997). Involvement of type V collagen in softening of fish muscle during short-term chilled storage. Journal of Agriculture and Food Chemistry, 45, 343–348. Sato, K., Koike, A., Yoshinaka, R., Sato, M., & Shimizu, Y. (1994). Post mortem changes in type I and V collagens in myocommatal and endomysial fractions of rainbow trout (Oncorhynchus mykiss) muscle. Journal of Aquatic Food Product Technology, 3(2), 5–11. Sato, K., Ohashi, C., Ohtsuki, K., & Kawabata, M. (1991). Type V collagen in trout (Salmo gairdneri) muscle and its solubility change during chilled storage of muscle. Journal of Agriculture and Food chemistry, 39, 1221–1225. Sato, K., Yoshinaka, R., Itoh, Y., & Sato, M. (1989). Molecular species of collagen in the intramuscular connective tissue in fish. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 92, 87–91. Scott, J. E. (1996). Proteodermatan and proteokeratan sulfate (decorin, lumican/fibromodulin) proteins are horseshoe shaped. Implications for their interactions with collagen. Biochemistry, 35(27), 8795–8799. Scott, J. E., & Dorling, J. (1965). Differential staining of acid glycosaminoglycans (mucopolysaccharides) by Alcian blue in salt solutions. Histochemie, 5(3), 221. Sheehan, E. M., OConnor, T. P., Buckley, D. J., & FitzGerald, R. (1996). Effect of dietary fat intake on the quality of raw and smoked salmon. Irish Journal of Agriculture and Food Research, 35, 37–42. Taylor, R. G., Fjæra, S. O., & Skjervold, P. O. (2002). Salmon fillet texture is determined by myofiber–myofiber and myofiber–myocommata attachment. Journal of Food Science, 67(6), 1–5. Taylor, R. G., & Koohmaraie, M. (1998). Effects of post mortem storage on the ultrastructure of the endomysium and myofibrils in normal and callipyge longissimus. Journal of Animal Science, 76, 2811–2817. Tingbø, M. G., Kolset, S. O., Ofstad, R., Enersen, G., & Hannesson, K. O. (2005). Sulfated glycosaminoglycans in the extracellular matrix of muscle tissue in Atlantic cod (Gadus morhua L.) and Spotted wolffish (Anarhichas minor O.). Comparative Biochemistry and Physiology B, 140, 349–357.