Fillet shelf-life of Atlantic halibut Hippoglossus hippoglossus L. fed elevated levels of α-tocopheryl acetate
Abstract
Fish fillet quality may be influenced by the antioxidant level in preslaughter diet. Thus, the effects of dietary α-tocopheryl acetate supplementation and feeding time on the flesh quality of farmed Atlantic halibut Hippoglossus hippoglossus L. were investigated. Halibut of mean initial weight of 312 ± 12.3 g were divided into two groups and fed commercial diets, supplemented with different levels of α-tocopheryl acetate at the dietary inclusion levels of 189 and 613 mg kg−1 diet. Fish were sampled after 6,9,12 and 24 weeks. Over the experimental period, they reached a final mean weight of 1320 ± 108.4 g. Tissue α-tocopherol of fillet and liver was significantly affected by the levels of α-tocopheryl acetate given with the diets (P < 0.001). In storage on ice, fillets of fish fed the diets high in α-tocopheryl acetate exhibited significantly lower (P < 0.001) levels of lipid oxidation. The colour of fillets in all groups deteriorated slightly, but diet did not affect this process. Halibut fed the supplemented diets for longer periods were better protected against lipid oxidation (P < 0.001) and colour deterioration (P < 0.01) than those fed for shorter periods. However, after 9 days of storage, lipid oxidation levels were still extremely low [< 0.6 µg malondialdehyde (MDA) g−1 fillet], even in fillets of fish fed the low α-tocopheryl acetate diet for a short period preslaughter. Different slaughtering methods tested at the end of the trial showed that percussive stunning can delay the onset of rigor mortis by 8–12 h compared with bleeding of the fish. These results suggest that halibut fillets have enhanced shelf-life stability even at low doses of dietary α-tocopheryl acetate, and that other factors in the antioxidant defence mechanisms of the species might play a major role in the prevention of lipid oxidation.
Introduction
Atlantic halibut Hippoglossus hippoglossus L. is an important food fish of ‘excellent flavour’ (Wheeler 1978). Falling catches, good growth potential in captivity and a high market value have made this species a prime candidate for aquaculture in the last two decades (Shields, Gara & Gillespie 1999). However, even with further expansion of the European halibut aquaculture industry, very little research has focused on the quality characteristics of this valuable species.
Diet is a preslaughter factor that can have a major effect on the quality of fish. Through modifying the fish feed, the quality of the final product can be greatly enhanced. For example, the supplementation of feeds with dietary α-tocopheryl acetate, a stable ester of the lipid-soluble antioxidant α-tocopherol (vitamin E), has been shown to reduce lipid oxidation in fish (Frigg, Prabucki & Ruhdel 1990; Baker & Davies 1997). This is of great importance as fish are considered very nutritious because of their high content of polyunsaturated fatty acids (PUFAs), which are highly susceptible to oxidation. Lipid oxidation is of major concern in fresh, high-value seafood products because it results in the loss of texture, colour, flavour and highly nutritious PUFAs (Frigg et al. 1990; Waagbø, Sandnes, Torrissen, Sandvin & Lie 1993). This is especially important in reared Atlantic halibut, as these fish often have a higher fillet fat content than their wild counterparts. However, improved product quality (i.e. protection against lipid oxidation and thus rancidity) has been achieved by feeding α-tocopheryl acetate above recommended requirement levels. This has been demonstrated in sea bass Dicentrarchus labrax L. (Gatta, Pirini, Testi, Vignola & Monetti 2000; Pirini, Gatta, Testi, Trigari & Monetti 2000) in refrigerated storage and also in turbot Scophthalmus maximus L. under retail conditions on ice (Ruff, FitzGerald, Cross, Hamre & Kerry 2002a).
The first post-slaughter occurrence that has a major impact on the flesh quality of a fish is rigor mortis. This is influenced by the handling conditions before slaughter and the degree of stress experienced by the fish during the slaughter process (Sikorski, Kolakowska & Sun Pan 1990). An animal that struggles at slaughter goes into rigor very quickly because of a rapid decrease in ATP and pH post mortem, caused by vigorous movement (i.e. greater muscle activity). Low muscle activity at slaughter can be achieved by either anaesthetizing the fish (Clarke 1999; Robb, Kestin & Warriss 2000; Akse & Midling 2001) or applying slaughter methods that leave the fish insensible instantaneously, i.e. spiking of the brain (Boyd, Wilson, Jerrett & Hall 1984; Korhonen, Lanier & Giesbrecht 1990), destroying the spinal chord (Mochizuki & Sato 1994), a swift and accurate blow to the head (Kestin, Wotton & Adams 1995; Berg, Erikson & Nordtvedt 1997) or percussive stunning (Ruff, FitzGerald, Cross, Teurtrie & Kerry 2002b). These slaughtering techniques, if performed correctly, result in a slower onset of rigor mortis and a less intense rigor (Robb 2001). As degradative processes only set in with the resolution of rigor (Sikorski et al. 1990), delaying the time period before rigor as well as a long-lasting rigor ensures that the fish remain fresh for longer.
With very little information available on the quality characteristics of Atlantic halibut, it is important to concentrate research efforts on factors that affect the final quality of the product. Therefore, the aim of the present study was to investigate the effect of dietary α-tocopheryl acetate and a novel slaughtering method on the susceptibility of fresh fillets, in ice storage, to lipid oxidation and colour deterioration, and on the development of rigor mortis in reared Atlantic halibut.
Materials and methods
Diets
Halibut diets were provided by TROUW Aquaculture (Renfrew, UK). The diets were commercially available halibut feeds, formulated with high-quality fishmeal and oil. The two diets were enriched (pre-extrusion) with 189 and 613 mg α-tocopheryl acetate kg−1 diet respectively. The proximate composition of the diets were as follows: crude protein, 48.3 ± 0.46%; crude lipid, 26.8 ± 0.63%; moisture, 2.3 ± 0.32%; ash, 10.7 ± 0.15. The α-tocopheryl acetate used was Roche Rovimix E50 SD (Roche Products, UK), which is α-tocopheryl acetate combined with a diluent providing 500 g kg−1 feed.
Fish
The experiment was carried out in a marine pump-ashore unit (Bantry Bay, south-west Ireland). Halibut were obtained from Mannin Seafarms Ltd (Isle of Man, UK) and had an initial mean weight of 312 ± 12.3 g. The fish were held in two flat-bottomed, square tanks (2 m2, 2000 L) at a stocking density of 45 fish per tank, with a flow rate of 20 L min−1. They were exposed to a natural light cycle and ambient Irish sea water temperatures (7–15 °C over the experimental period). The water quality was monitored on a regular basis. Ammonia, nitrate and nitrite never exceeded the lowest detectable levels of 0.6, 10 and 0.15 mg L−1 respectively. The sea water had a salinity of 35 g L−1 and a pH of 8.0–8.3. Oxygen levels ranged between 80% and 90%. During the experimental period, the fish were fed twice daily to satiation.
Sampling of fish
Six fish were sampled at the start of the experiment. After 6, 9, 12 and 24 weeks of feeding the experimental diets, six fish from each treatment were sampled. The fish were bled (gill and caudal vein cut) in ice water (60% flaky ice/40% sea water, −0.6 ± 0.04 °C), gutted and filleted. Four fillets per fish were obtained. Tissue samples were vacuum packed and stored at −20 °C until analysis for proximate composition (fillet samples) and α-tocopherol content (fillet samples, liver, heart and kidney). Whole fillets were stored on and covered with ice in a commercial display cabinet under fluorescent illumination (similar to market conditions) for up to 9 days after slaughter. On days 0, 2, 4, 7 and 9 after slaughter, fillet pH and colour were measured, and samples for the determination of lipid oxidation were removed from each fillet. Furthermore, after 24 weeks of feeding, an additional 10 fish (2 × 5) from each dietary treatment were sampled applying two different slaughtering methods. These were: (A) bleeding in ice water as used throughout the experiment; and (B) percussive stunning followed by bleeding in ice water as described by Ruff et al. (2001). The time course of rigor mortis over 84 h after slaughter was measured using pH, rigor index (Bito, Yamada, Mikumo & Amano 1983) and compression force (Sørensen, Tobiassen, Joensen, Midling & Akse 2001). Measuring points and times were as described by Ruff et al. (2002b).
Analytical procedures
Proximate composition analysis was performed using standard methods (AOAC International 1999). The proximate composition of the fish was reported on a wet weight basis. Moisture was analysed using a microwave moisture analyser CEM AVC-80 (CEM Corporation, Matthews, NC, USA), crude fat using a CEM FAS-9001 automated solvent extractor (CEM Corporation), crude protein using a Tecator 2020 digester (Tecator Inc., Herndon, VA, USA) and a Kjeltech system 1026 distillation unit (Tecator) and ash using a Nabertherm oven (Nabertherm, Lilienthal, Germany). Samples from each of the four fillets per fish (taken from the cranial end of the fillet) were combined and analysed in duplicate.
The samples were analysed for their α-tocopherol content by means of tissue saponification (Buttriss & Diplock 1984) followed by the extraction procedure of Sheehy, Morrissey & Flynn (1994). Samples were quantified by high-performance liquid chromatography (HPLC) using a Waters model S10 pump, a Waters 717 autosampler, a Machery-Nagel Nucleosil 5 C18 (250 × 0.4 mm) reverse-phase column and a Waters model 486 UV-visible wavelength detector (Millipore Corporation, Milford, MA, USA) set at 292 nm. The mobile phase used was methanol:water (97:3) at a flow rate of 2 mL min−1. Data were recorded and evaluated using the Millipore Millenium 2010 chromatography management system. Samples from each of the four fillets per fish [taken at approximately one-third (7–8 cm) of the fillet length from the caudal end of the fillet] were analysed and treated as replicates. Each organ was analysed in duplicate.
Lipid oxidation was measured by means of a distillation-colorimetric technique, the 2-thiobarbituric acid method (Ke, Ackman, Linke & Nash 1977). Absorbance was read at 538 nm using a Milton Roy Spectronic 20D+ spectrophotometer (Milton Roy, formerly Bausch & Lomb, Rochester, NY, USA). Levels of thiobarbituric acid-reactive substances (TBARS levels) were expressed as malondialdehyde (MDA) equivalents (mg kg−1 tissue). Samples from each of the four fillets per fish (taken from the cranial end of the fillet) were combined and analysed in duplicate.
Fillet pH was measured using a WTW pH meter 320/Set-1 using a Mettler Toledo type LoT406-M6-DXK-S7/25 probe (Wissenschaftliche-Technische Werkstätten, Weilheim, Germany). One measurement from each of the four fillets per fish was taken at the thickest part (≈ 6 cm from the cranial end) of the fillet, and these readings were treated as replicates.
Surface colour measurements (Hunter L* a* b* values) on fillets were recorded using a Minolta Chromameter CR-300 (Minolta Camera Co., Osaka, Japan). The colour variables calculated by the chromameter were Hunter L* a* b* (Hunt 1977) where L* describes lightness (+L* = white, –L* = black), a* red–green chromaticity (+a* = red, –a* = green) and b* yellow–blue chromaticity (+b* = yellow, –b* = blue). The colour of each of the four fillets per fish was measured in triplicate (along the length of the fillet), and values were combined to one mean value per fish for each of the three colour variables measured. From Hunter a* and b* values, the angle of hue and chroma were calculated using the following equations:
The hue angle describes what is generally known as the colour. The chroma defines the purity or degree of saturation of a colour (the relative absence of white or grey in a colour).
Statistical analysis
All data were analysed using SPSS 10.0 (SPSS Inc., Chicago, IL, USA) and tested for normality and homogeneity of variance before being subjected to analysis. Percentage data were subjected to arcsine transformation before analysis. The effects of dietary treatment and feeding duration on tissue α-tocopherol, fish wet weight and fillet proximate composition were analysed by two-way analysis of variance (anova). When significant (P < 0.05) effects were found, groups were compared using Tukey's test with a significance level of P < 0.01. To establish the effect of feeding duration only (including the initial sample) on fillet proximate composition, one-way anova was carried out, followed by Tukey's test with P < 0.05. For the evaluation of proximate composition, fish wet weight was used as a covariate. A repeated measure three-way anova was carried out for the parameters fillet MDA, pH, Hunter L*, hue angle and chroma. When neither the three-way interaction between the within-subjects factor (storage day) and the between-subjects factors (dietary treatment and feeding duration) nor the two-way interaction between the between subjects factors was significant (P > 0.05), the model was reduced, leaving these interaction terms out. Significant (P < 0.05) main effects and significant (P < 0.01) interactions with the within-subject factor allowed for post hoc tests (Tukey's and Student's t-test) to establish overall differences (P < 0.01) and differences on each individual storage day (P < 0.01) respectively. Regression analyses (best fit) were carried out to determine the relationships between parameters. Peak rigor (rigor index, compression force) as well as initial and ultimate pH data were analysed for slaughter and diet effects by two-way anova with P < 0.05.
Results
No difference in growth was observed between groups. After 5 months of feeding, the fish had reached a mean weight of 1320 ± 108.4 g. The average fillet yield was 47 ± 0.4%.
Effects of diet
Diet significantly (P < 0.05) affected the fat content of fillets of halibut fed for 12 and 24 weeks (Table 1). However, the comparison of means was not significant (P > 0.01).
| Week | Diet | Moisture (%) | Fat (%) | Protein (%) | Ash*(%) |
|---|---|---|---|---|---|
| Initial | 75.7 ± 0.66a | 2.1 ± 0.50c | 20.6 ± 0.27a | 1.7 ± 0.12a | |
| 12 weeks | 189 | 71.1 ± 0.53b | 6.7 ± 0.49b | 19.3 ± 0.10b | 1.4 ± 0.13ab |
| 613 | 70.4 ± 0.98 | 8.8 ± 1.16 | 19.2 ± 0.23 | 1.4 ± 0.12 | |
| 24 weeks | 189 | 69.2 ± 1.00c | 9.3 ± 0.70a | 18.2 ± 0.48c | 1.4 ± 0.05b |
| 613 | 66.8 ± 1.08 | 12.2 ± 1.45 | 17.4 ± 0.31 | 1.2 ± 0.14 |
- Effects of feeding duration (one-way anova followed by Tukey's test).
- abc Different superscripts within one column indicate significant (P < 0.05) differences between weeks.
- * Differences in ash content between weeks were not significant (P > 0.05) when fish wet weight was used as a covariate.
Tissue α-tocopherol levels (initial mean values of 9.6 ± 1.31, 1144.5 ± 83.88, 652.6 ± 88.00 and 136.3 ± 14.58 µg g−1 in fillets, liver, heart and kidney respectively) are presented in Table 2. Diet significantly (P < 0.001) affected vitamin E incorporation in fillet and liver in a dose-dependent manner, with fish fed the diet high in α-tocopheryl acetate incorporating more than those receiving low levels of α-tocopheryl acetate. Atlantic halibut from all dietary treatments incorporated α-tocopherol in the following order: liver and heart > kidney > muscle.
| Weeks of feeding the experimental diets | Between subjects effects | Main effects* | |||||
|---|---|---|---|---|---|---|---|
| Tissue | Diet | 6 | 9 | 12 | 24 | ||
| Fillet | 189 | 9.7 ± 0.89 | 6.2 ± 0.54 | 10.2 ± 1.12 | 11.2 ± 2.38 | Diet P < 0.001 | 613 > 189 (P < 0.001) |
| 613 | 11.6 ± 0.94 | 17.1 ± 2.62 | 11.3 ± 1.77 | 15.9 ± 0.53 | Week P = 0.214 Diet × week P = 0.005 | ||
| Liver | 189 | 253.3 ± 29.79 | 97.4 ± 7.69 | 204.9 ± 17.72 | 199.1 ± 14.23 | Diet P < 0.001 | 613 > 189 (P < 0.001) |
| 613 | 694.1 ± 71.14 | 992.4 ± 67.26 | 250.2 ± 33.64 | 645.8 ± 44.10 | Week P = 0.004 Diet × week P < 0.001 | 6 > 12 (P = 0.001) | |
| Heart† | 189 | 594.1 ± 78.66 | 181.1 ± 18.49 | 301.1 ± 35.77 | – | Diet P = 0.012 | NS |
| 613 | 510.9 ± 58.21 | 629.7 ± 100.27 | 345.1 ± 39.42 | 634.9 ± 90.20 | Week P = 0.002 Diet × week P = 0.001 | 6 > 12 (P = 0.002) | |
| Kidney† | 189 | 93.2 ± 11.19 | 29.6 ± 4.85 | 90.0 ± 11.70 | – | Diet P < 0.001 | NS |
| 613 | 95.8 ± 13.67 | 76.7 ± 18.92 | 68.1 ± 4.37 | 123.0 ± 12.70 | Week P = 0.124 Diet × week P < 0.001 | ||
- * NS, not significant (P > 0.01).
- † For heart and kidney, week 24 was not included in the analysis because of an incomplete data set.
Dietary treatment significantly affected lipid oxidation (Fig. 1, Tables 3 and 4). Overall, MDA levels were significantly higher (P < 0.001) in fish fed the diet low in α-tocopheryl acetate. The same effect was observed on storage days 2, 4 and 7. Diet had no effect on fillet pH and colour or the time course of rigor mortis.
Effect of diet on malondialdehyde (MDA µg g−1) of fillets of H. hippoglossus displayed on ice for 9 days. Each point represents the mean ± SEM of four feeding times combined (open triangles,189; filled diamonds, 613 diet codes).
| Full factorial model Parameter | Reduced modelParameter | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Between subjects | MDA | pH | Hunter L* | Hue | Chroma | Between subjects | MDA | Hue | Chroma |
| Diet | P < 0.001 | P = 0.997 | P = 0.585 | P = 0.116 | P = 0.559 | Diet | P < 0.001 | P = 0.269 | P = 0.675 |
| Week | P < 0.001 | P = 0.709 | P < 0.001 | P = 0.077 | P = 0.404 | Week | P < 0.001 | P = 0.124 | P = 0.433 |
| Diet* week | P = 0.620 | P = 0.649 | P = 0.239 | P = 0.163 | P = 0.433 | ||||
| Within subjects | Within subjects | ||||||||
| Day | P < 0.001 | P < 0.001 | P < 0.001 | P < 0.001 | P < 0.001 | Day | P < 0.001 | P < 0.001 | P < 0.001 |
| Day* diet | P = 0.001 | P = 0.046 | P = 0.073 | P = 0.903 | P = 0.120 | Day* diet | P < 0.001 | P = 0.915 | P = 0.343 |
| Day* week | P < 0.001 | P < 0.001 | P = 0.003 | P < 0.001 | P = 0.010 | Day* week | P < 0.001 | P < 0.001 | P = 0.007 |
| Day* diet* week | P = 0.476 | P < 0.001 | P = 0.012 | P = 0.431 | P = 0.440 | ||||
- Italic P-values indicate neither a significant three-way interaction of between-subject factors and within-subject factor nor a two-way interaction between the between-subject factors. Thus, the full factorial model was reduced, leaving these interaction terms out.
- Bold P-values indicate significant (P < 0.05) between-subjects effects (main effects) and significant (P < 0.01) within-subjects effects (diet and week effects on individual days) in the relevant model for each parameter.
| Parameter | Main effects | Effects on individual storage days | ||||||
|---|---|---|---|---|---|---|---|---|
| Diet | Week | 0 | 2 | 4 | 7 | 9 | ||
| MDA | 189 > 613 P < 0.001 | 6 > 24 P < 0.001 | Diet Week | NS†P < 0.01 | P < 0.001 P < 0.01 | P < 0.001 P ≤ 0.001 | P = 0.001 P = 0.001 | NS NS |
| pH | No effect | No effect‡ | Week | NS | P < 0.01 | NS | P < 0.01 | NS |
| Hunter L* | No effect | 6 > 12 P = 0.002 | Week | NS | P < 0.01 | P < 0.001 | P < 0.01 | P < 0.01 |
| 6 > 24 P = 0.001 | ||||||||
| 9 > 12 P < 0.001 | ||||||||
| 9 > 24 P < 0.001 | ||||||||
| Hue angle | No effect | No effect‡ | Week | P < 0.01 | NS | NS | NS | P < 0.01 |
| Chroma | No effect | No effect‡ | Week | P < 0.001 | NS | NS | NS | NS |
- † NS, not significant (P > 0.01).
- ‡No main effects, but within-subject effects (see Table 1) indicate significant (P < 0.01) interactions with day and therefore post hoc tests were performed within each day.
Effects of feeding duration
The fillet proximate composition change with prolonged feeding of the diets (Table 1). The moisture, protein and ash content decreased significantly (P < 0.05) from the start of the experiment to the end, whereas the fat content increased significantly (P < 0.05). However, when fish wet weight was used as a covariate, the change in fillet ash content was not significant (P > 0.05).
Feeding duration significantly (P < 0.01) affected α-tocopherol incorporation (Table 2). In liver and heart, tissue α-tocopherol levels dropped significantly (P < 0.01) from 6 to 12 weeks of feeding.
Feeding duration had an effect on lipid oxidation, in that 24 weeks resulted in significantly lower MDA (P < 0.001) levels than 6 weeks of feeding (Fig. 2a, Tables 3 and 4). The results obtained on individual storage days were variable however, 24 weeks of feeding always resulted in the lowest MDA values.
Effect of feeding duration on(a) malondialdehyde (MDA µg g−1),(b) pH,(c) Hunter L*,(d) hue (H°ab) and(e) chroma (C*ab) of fillets of H. hippoglossus displayed on ice for 9 days. Each point represents the mean ± SEM of the two diets combined (filled circles, 6; open circles, 9; filled squares, 12; and open squares, 24 weeks of feeding).
The fillet pH (Fig. 2b) was affected by feeding duration on individual storage days (Tables 3 and 4). On day 2, the pH was significantly higher (P < 0.01) in fillets from fish fed for 6 weeks than in fish fed for 12 weeks, whereas on day 7, this significant difference (P < 0.01) was reversed. However, the pH remained relatively stable (6.2–6.4) over the storage time, and no trends indicated overall group differences.
The surface colour of fillets from all groups deteriorated over storage time (Fig. 2c–e). Hunter L* values increased as fillets got paler (Fig. 2c). Feeding the diets for 6 and 9 weeks resulted in significantly higher (P < 0.01) Hunter L* values than feeding them for 12 or 24 weeks (Tables 3 and 4). This result was also reflected on individual storage days 2–9. The angle of hue decreased over storage time (Fig. 2d). No main effects were found (Tables 3 and 4) but, on day 0 of cold storage, fillets of fish fed the diets for 12 weeks had a significantly higher hue angle (P < 0.01) than those fed for 9 weeks. However, on day 9 of storage, 24 weeks of feeding resulted in a significantly higher hue angle (P < 0.01) than any shorter feeding duration. This phenomenon had already started to develop as a trend by storage day 2. No main effects were found for chroma (Fig. 2e, Tables 3 and 4) but, on day 0 of storage, chroma was significantly lower (P < 0.001) for fillets of fish fed for 24 weeks than for fish fed for shorter periods. For the remaining storage period, the chroma of all groups started to increase from day 4 onwards with a trend towards lower values at 24 weeks of feeding. Both hue angle and chroma were correlated to Hunter b* in a polynomial model of the third order (r2 = 0.99 and r2 = 0.98 respectively), but no strong correlation with Hunter a* could be established (Fig. 3a and b). This demonstrated that, in halibut fillets, yellow chromaticity was the major factor in terms of colour changes. The models showed that Hunter b* values increased (fillets appeared to be more yellow) with decreasing hue and increasing chroma (colour became more intense). It was also demonstrated that lipid oxidation was related to Hunter b* in a polynomial model of the third order with r2 = 0.86 (Fig. 3c).
Relationship between(a) hue (H°ab),(b) chroma (C*ab) and(c) malondialdehyde (MDA µg g−1) and Hunter a* (grey circles)/Hunter b* (black squares) of fillets of H. hippoglossus. Combined means of all diets, all feeding durations and 0–9 days of display on ice. All relationships with Hunter b* follow a polynomial model of the third order (y = ax3 + bx2 + cx + d).
Slaughter effect
The assessment of rigor mortis showed no significant differences (P > 0.05) in initial and ultimate pH between the two slaughtering methods investigated (Fig. 4). The rigor index data showed that bled fish were in full rigor at the time of the first measurement (4 h after slaughter), whereas fish subjected to percussion reached full rigor 12 h after slaughter. Compression force measurements showed a similar trend with fish subjected to percussion in full rigor 16 h after slaughter. Rigor was fully resolved 36 h after slaughter. The comparison of peak compression force points (4 h and 16 h for fish bled and killed by percussive stunning respectively) revealed that bled fish were significantly stiffer (P < 0.05) than halibut killed by percussion.
Effect of slaughtering method on the time course of (a) pH, (b) rigor index (%) and (c) compression force (g) of whole H. hippoglossus subjected to two different slaughtering methods. Each point represents the mean ± SEM of the two diets combined (open squares, bleeding in ice water; filled squares, percussive stunning, followed by bleeding in ice water).
Discussion
Effect of dietary α-tocopheryl acetate and feeding duration on tissue composition
In the present study, dietary α-tocopheryl acetate supplementation had no effect on halibut fillet proximate composition, which is in agreement with studies on sea bass and turbot (Gatta et al. 2000; Ruff et al. 2002a). Elevated levels of α-tocopheryl acetate in the diet resulted in increased tissue α-tocopherol concentrations in fillet and liver, but not in heart and kidney. A dose-dependent incorporation of α-tocopherol was found in kidney and heart (as well as in fillet and liver) of Atlantic salmon Salmo salar L. and turbot (Hamre & Lie 1995; Ruff et al. 2002a). These authors suggested that liver has the ability to store excess α-tocopherol. However, they also reported much lower vitamin E levels in heart tissue than found in halibut. This suggests that, in Atlantic halibut, heart as well as liver might be a major storage site for the vitamin. That Atlantic halibut might generally have the ability to store large amounts of α-tocopherol was reflected in the very high concentrations of α-tocopherol incorporated into tissues, even in fish fed the low α-tocopheryl acetate diet. Fillet α-tocopherol concentrations detected were similar to those found in comparable studies with Atlantic salmon and sea bass (Stéphan & Lamour 1993; Bjerkeng, Hamre, Hatlen & Wathne 1999; Gatta et al. 2000). Nevertheless, lower fillet α-tocopherol was found in Atlantic salmon and turbot (Onibi, Scaife, Fletcher & Houlihan 1996; Ruff et al. 2002b; Ruff et al. 2002a). As discussed previously by Ruff et al. (2002a), the incorporation of α-tocopherol is most likely determined by various factors, such as fish species and fat content, size and age as well as rearing conditions, which would account for the disparate results reported by various authors.
Prolonged feeding of the experimental diets resulted in increased fat and decreased moisture and protein content in the fillet. The fat content appeared to increase with fish size, as also observed in Atlantic halibut by Nortvedt & Tuene (1998). The decrease in moisture resulted from the increase in fat, a well-understood reciprocal relationship (Shearer 2001). The higher fat content of fish fed for longer periods also suggests less oxidative stability of the fillets of these fish. This was, however, not the case; the levels of lipid oxidation were in fact lower with prolonged feeding of the experimental diets (see below).
Feeding duration affected α-tocopherol incorporation insofar as, in liver and heart, a 12-week feeding period resulted in lower α-tocopherol concentrations than only 6 weeks of feeding. However, tissue α-tocopherol concentrations fluctuated considerably over time, as also observed by Watanabe, Takeuchi, Wada & Uehara (1981) in muscle and liver of rainbow trout Oncorhynchus mykiss (Walbaum). Fillet α-tocopherol had reached saturation after 6 weeks of feeding.
Effect of dietary α-tocopheryl acetate and feeding duration on lipid oxidation and colour deterioration
Lipid oxidation was affected by diet, as the fillet α-tocopherol levels reflected. Although lower dietary α-tocopheryl acetate resulted in a higher degree of lipid oxidation, MDA values were extremely low (< 0.6 mg kg−1 fillet), even after 9 days of storage. This contrasts with MDA concentrations of 2–4 mg kg−1 fillet after 9 days of refrigerated storage, when 70–170 mg of α-tocopheryl acetate kg−1 diet were fed to Atlantic salmon and turbot (Onibi et al. 1996; Ruff et al. 2002a, b). These low levels of lipid oxidation in the halibut are quite surprising, considering the relatively high fat content of the fillets.
Lipid oxidation was positively affected by longer feeding times with 24 weeks resulting in particularly decreased levels of MDA. This finding is in agreement with those of Onibi et al. (1996), who found better protection by α-tocopherol against lipid oxidation when Atlantic salmon were fed for 11 as opposed to 7 weeks. This time effect was also observed for colour deterioration, especially for Hunter L* values, but also for hue angle and chroma. The improved fillet preservation with prolonged feeding cannot be explained by fillet α-tocopherol levels, as these did not increase over the experimental period. However, the fish may have adjusted better to the diets after a longer time and achieved a greater balance in their overall antioxidant systems.
The connection between Hunter b* values and lipid oxidation
Although colour development of the fillets was not affected by dietary treatment, lipid oxidation was directly linked to colour, especially to Hunter b* values. With an increase in MDA values, fillets became more yellow. This is in agreement with studies on tilapia (species name not given) and turbot (Lee, Yang, Lin & Chow 1998; Ruff et al. 2002a, b). Undeland, Hall & Lingnert (1999) linked an increase in Hunter b* values to an increase in the formation of yellow pigment. It has been suggested that the formation of yellow pigment resulted from polymerization of fluorescent Schiff's bases, which derive from a reaction of carbonyls (i.e. MDA) with amino compounds (i.e. phospholipids, free amino acids, peptides and proteins) (Fujimoto 1970; Pokorny, El-Zeany & Janicek 1974; Gardner 1979; Erickson 1993).
This hypothesis was supported by the present study with Hunter b* values relating to MDA in a polynomial model. This might be evidence of some MDA in halibut being metabolized to yellow pigment and, therefore, not detected as the aldehyde. However, fillets of vitamin E-supplemented turbot showed a greater increase in MDA than the halibut, even though the same relationship between MDA and Hunter b* (polynomial model with r2 = 0.95) was present (Ruff et al. 2002b). This same relationship in turbot and halibut refutes the possible explanation of lower levels of MDA in halibut resulting from enhanced formation of fluorescent products and subsequently yellow pigment. Even if the polymerization of fluorescent products had only happened to a certain extent, amino compound-bound MDA would still have been detected by the TBARS method used in the present study (method involves heat and hydrolysis with HCl).
The low levels of lipid oxidation in Atlantic halibut compared with other fish species
The hypothesis that low levels of MDA could be caused by the binding of MDA not only to amino compounds, but also to nucleotides also seems unlikely, as this should still result in a deterioration of sensory parameters, which normally occurs with the formation of the secondary oxidation products such as MDA (Milo & Grosh 1993). Although sensory analysis was not carried out in the present study, it has been reported that storage of Atlantic halibut for 21 days on ice had no negative effect on sensory evaluation. In fact, a more ‘pronounced’ and positive taste was found (Dr R Nortvedt, Institute of Nutrition, Directorate of Fisheries, Bergen, Norway, pers. comm.). Moreover, almost no deterioration of chemical, microbiological and sensory parameters were found until after 21 days of ice storage of Atlantic halibut (Akse & Midling 2001). Other studies on the shelf-life of Atlantic halibut in our own laboratory did not show any significant increase in bacterial load over 10 days of cold storage (unpubl. data). Such an increase could possibly have inhibited lipid oxidation as suggested by Aubourg, Sotelo & Gallardo (1997).
Consequently, it seems very likely that, in halibut, lipid oxidation is inhibited before the formation of quality-deteriorating secondary oxidation products. It is thus quite possible that dietary α-tocopheryl acetate levels as low as 189 mg kg−1 diet and muscle α-tocopherol concentrations of 6–10 mg kg−1 tissue are sufficient to protect fillets from lipid oxidation and the formation of carbonyls in this species. However, this was not the case in another flatfish species, turbot (Ruff et al. 2002a, b).
Although dietary α-tocopheryl acetate had an effect on lipid oxidation in fillets of Atlantic halibut in the present study, the strong hyperbolic relationship between the two parameters, as reported for turbot (Ruff et al. 2002a, b) and rainbow trout (Frigg et al. 1990), could not be observed. Therefore, antioxidant defence mechanisms other than α-tocopherol seem to be very important in this species.
Antioxidant mechanisms other than α-tocopherol
Both Atlantic halibut and turbot belong to the same order (Pleuronectiformes, flatfishes), but halibut are known to be much more active fish than turbot (halibut are often regarded as flatfish with an almost pelagic behaviour). Thus, these fish would have a more active metabolism, higher protein turnover and be exposed to a higher oxidative load as a result of this difference in life style. Therefore, halibut may have adapted to the greater oxidative stress loads through modulation of their chemical and biochemical make-up, which would provide greater protection from oxidative stress. Supporting this theory are the findings of Hoare (2000), who reported higher haematocrit and better resistance to disease challenge in farmed Atlantic halibut than in farmed turbot. A higher protein turnover in a more active animal might promote the reaction of hydroperoxides (primary oxidation products) with amino acids, protein and nucleotides, which would lead to reduced production of MDA (Gardner 1979; Frankel, Neff & Brooks 1987). It was also demonstrated that a fluorescence was formed from the interaction of DNA and hydroperoxides (Frankel et al. 1987), which could possibly account for the fillet colour changes observed in the present study. By suggesting that halibut might be exposed to a higher oxidative load than turbot, one must consider that the former might be lower in oxidation-promoting trace elements (e.g. iron) and higher in those promoting the antioxidant defence system (e.g. selenium).
Another hypothesis might be that other oxidation inhibitors, such as enzymes (e.g. superoxide dismutase, catalase and glutathione peroxidases), lipid and water-soluble small-molecule antioxidants (e.g. glutathione, ubiquinol and ascorbate), are present in higher concentrations in halibut. The enzymes are mainly preventive antioxidants, removing active oxygen species, whereas the other antioxidants (including α-tocopherol) act as chain-breaking antioxidants. Some of the antioxidants also interact and regenerate each other. The loss of antioxidants as a result of oxidative stress as well as antioxidant-regenerating reactions should be in a certain order, determined by their reduction potential (Buettner 1993). Owing to this so-called ‘pecking order’, all antioxidants mentioned should be lost before oxidation of fatty acids. However, this ‘pecking order’ should merely be taken as a guideline, as it is only valid under standard thermodynamic conditions (Buettner 1993). Undeland et al. (1999) showed that the antioxidant loss in whole herring Clupea harengus L. fillets was α-tocopherol > ascorbic acid > glutathione peroxidase, which demonstrates that the ‘pecking order’ is not an absolute. However, the antioxidant loss in mackerel Scomber scombrus L. white muscle was shown to be ascorbic acid = total glutathione > α-tocopherol > ubiquinone 10 (Petillo, Hultin, Krzynowek & Autio 1998). These two studies demonstrate that the antioxidant loss seems to be species dependent. The actual antioxidant concentrations seem to be quite important in the pathways of lipid oxidation, especially for antioxidants with low reduction potentials. As the muscle α-tocopherol concentrations in the halibut from the present study seemed almost too low to prevent the accumulation of MDA to the observed extent, one must consider the possibility that higher concentrations of certain antioxidants (other than α-tocopherol) or other compounds are present and, therefore, effectively prevent lipid oxidation in this species over a long time. However, further research needs to be undertaken to investigate and quantify this hypothesis.
The effect of slaughtering method on the time course of rigor mortis
Slaughtering method significantly affected the time course of rigor mortis in halibut. No slaughter-related differences in initial and ultimate pH were detected, which was probably because of the high variation observed in the data. However, bled fish had reached full rigor in the first 4 h after slaughter, whereas this was the case in halibut subjected to percussion 12–16 h after slaughter. This is in agreement with the findings of Akse & Midling (2001), who reported a slight delay in rigor onset when Atlantic halibut were killed by a blow to the head instead of by CO2 anaesthetization. The delay in the onset of rigor in halibut subjected to percussion can be explained by the fact that these fish showed no muscle activity at slaughter, whereas bled fish struggled for a considerable time period before they died. Less muscle activity at slaughter results in higher energy reserves and a slower onset of rigor (Robb 2001). Not only did bled fish go into rigor faster than those killed by percussion, they were also stiffer at the time point of full rigor. This was also observed in turbot and Atlantic salmon, which were stiffer when bled than when anaesthetized or pin-bolted (Clarke 1999; Ottera, Roth & Torrissen 2001). In a similar study on turbot, a much more pronounced delay in the onset of rigor mortis in fish subjected to percussive stunning was observed (full rigor was reached 30–48 h after slaughter); even bled fish did not reach full rigor before ≈ 20 h after slaughter (Ruff et al. 2002b). Again, the difference between the two species might result from the higher activity of halibut, which would lead to much lower energy reserves and, therefore, a more rapid onset of rigor.
In conclusion, we can confirm the findings of Akse & Midling (2001) that ‘farmed Atlantic halibut is likely to be accepted in the market as fresh fish at least one week longer than other white fish species’. Dietary α-tocopheryl acetate concentrations as low as 189 mg kg−1 diet are sufficient to reduce lipid oxidation in ice-stored fillets of Atlantic halibut, even after just 6 weeks of feeding the vitamin in a finishing diet. However, other possible mechanisms involved in preventing lipid oxidation in this species need to be investigated further. Moreover, the onset of rigor mortis can be delayed in Atlantic halibut by 8–12 h if fish are killed by percussive stunning instead of bleeding.
Acknowledgments
This project was funded by the European Union (FAIR-GT 97-4467). We gratefully acknowledge the support of TROUW Aquaculture who supplied the trial diets. We would also like to thank Dave Evans for skilful fish husbandry, Anna-Marie Callaghan and Gwenola Teurtrie for technical assistance, and Dr Kathleen O'Sullivan for advice on statistical evaluation of the data.
