EVALUATION OF DIFFERENT STUNNING METHODS ON ASPECTS OF ANIMAL WELFARE AND MEAT QUALITY OF MATRINXÃ (BRYCON CEPHALUS)

Abstract

Three methods of stunning fish (electronarcosis, CO2 narcosis, and thermal shock) were compared to study their influence on welfare and meat quality of matrinxä (Brycon cephalus). Parameters such as water quality and the time to reach clinical indicators of unconsciousness were observed. Rigor mortis index and muscular shrinkage were evaluated 3 and 5 h after stunning and at 1, 4, 7, 12 and 18 days of storage. None of the methods extended shelf-life; however, electronarcosis provided faster clinical indicators of unconsciousness and did not cause loss of meat quality. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

Three methods of stunning fish (electronarcosis, CO2 narcosis, and thermal shock) were compared to study their influence on welfare and meat quality of matrinxä (Brycon cephalus). Parameters such as water quality and the time to reach clinical indicators of unconsciousness were observed. Rigor mortis index and muscular shrinkage were evaluated 3 and 5 h after stunning and at 1, 4, 7, 12 and 18 days of storage. None of the methods extended shelf-life; however, electronarcosis provided faster clinical indicators of unconsciousness and did not cause loss of meat quality.

- Keywords: CO2 narcosis, electronarcosis, shelf-life, thermal shock, unconsciousness -

(ProQuest: ... denotes formulae omitted.)

INTRODUCTION

Because fish is the most internationally traded food commodity, quality control and safety, including meat quality and ethical aspects of animal welfare, are fundamental in aquaculture production (FAO, 2009; LAMBOOIJ etaL, 2006b). Thus, one of the most critical production stages to maintain fish quality is the stunning process.

Techniques for fish slaughter have been the subject of numerous studies regarding quality control, efficiency and safety procedures (CONTE, 2004). Several studies have also aimed to minimise the time required to establish death and implicitly reduce stress and pain (LAMBOOIJ et cd., 2002; ACERETE et cd., 2009). Slaughter is usually a two-step process. First, the animal is stunned to induce insensitivity to pain, and death is induced by various methods, including bleeding or oxygen deprivation (OLIVEIRA and GALHARDO, 2007). These two phases may occur together, but when they occur separately, the stunning time should be minimised to avoid recovery of consciousness before death (LINES et cd., 2003). When correctly applied, the stunning methods should cause little stress, improve the physical properties of meat, reduce muscular energy exhaustion, generate less lactic acid, maintain muscle pH balance, delay rigor mortis and, consequently, produce better fish quality (CONTE, 2004).

A typical stunning method in the fish processing industry is thermal shock (ASHLEY, 2007), in which water temperature is decreased to approximately 1°C. This low temperature decreases metabolic rate and oxygen consumption, causing the fish to become immobilised until death (RIBAS et cd., 2007). The effectiveness of this method depends on the difference between culture and stunning water temperature. Thus, thermal shock is more effective when applied to tropical fish species (ACERETE eta I, 2004) and is most often used in warm climate regions owing to ease of application and positive results on meat quality (ROBB and KESTIN, 2002b; SCHERER et cd., 2005; LAMBOOIJ et cd., 2006b; BAGNI et cd., 2007).

Some studies demonstrate that electronarcosis can damage fish quality when not conducted properly (ROBB and ROTH, 2003; LINES et cd., 2003; POLI et al., 2005b). Nevertheless, it has been demonstrated that species such as Atlantic salmon (Salmo salar) (KIESSLING étal., 2004), rainbow trout (Oncorhynchus mykiss) (AZAM et cd., 1990), eel [Anguila anguila) (MORZEL and VAN DE VIS, 2003) and common carp [Cyprinus carpió) (LAMBOOIJ et al., 2006a) show good positive results on fish quality because of the low stress level provided by this method.

C02 narcosis is mostly used for salmon and trout. This method consists of placing fish inside a box containing water saturated with C02, which produces H2C03 in equilibrium with HC03 and H+. This procedure decreases the blood pH and, hence, induces a toxic effect in the animals' brain (POLI et cd., 2005b). Sea bass stunned by C02 narcosis show high energy reserve in muscle and low plasma cortisol levels (POLI et cd., 2005a). This method is efficient and quick for narcotising a large number of fish and is useful for several species of all sizes, but it is highly stimulating to the fish during the first few minutes. The use of a controlled C02 delivery system or inert gases to promote anoxia in a more humane way is needed to prevent fish suffering (POLI et al, 2005). Therefore, studies concerning different stunning methods may contribute to achieve greater fish shelf-life and assure animal welfare.

Matrinxä [Brycon cephalus) is a freshwater native fish species from the Upper Amazon basin in Peru, Bolivia and Brazil. This species has been considered a promising candidate for Brazilian aquaculture because of its rapid growth rate, good adaptation to captivity and increasing economic importance among farmed fishes in Brazil (ROMAGOSA et cd., 1999; GOMES et cd., 2000). Although this fish has been the subject of several studies in Brazil, few researchers have studied the biochemical, physical and sensory changes during refrigerated storage (ALMEIDA et cd., 2006). Despite the fact that stunning methods are a widely studied subject worldwide, the effects of stunning stress on the physiological responses and meat quality of native freshwater fish species are unknown in Brazil. Thus, we aimed to compare the efficacy of the three stunning methods (electronarcosis, thermal shock and C02 narcosis) in evaluating parameters such as behaviour and meat quality of matrinxä (B. cephalus) stored in ice.

MATERIAL AND METHODS

Ten month old matrinxä specimens (n = 90; weight, 535.10± 121.36 g) were purchased from a local breeding farm. Fish underwent a 24-h fasting period in outdoor tanks. Then, the animals were captured with a net and immediately transferred to plastic boxes (120 L) inside the laboratory. All animals in the treatment (n = 30) were stunned at the same time. As the procedures were the same for all animals, they were subjected to the same stress level. The slaughter methods were performed on the same day to avoid the influence of weather.

For electronarcosis stunning, salinity of the tank water was adjusted to achieve a specific conductance of 700 p,S. Two aluminium plates (65x35 cm) were placed parallel to each other inside a box with a distance of 49 cm between them to create an electric field. One of the plates was covered with a polyethylene screen to avoid short circuit by fish contact. An electrode connected to a device capable of discharging 220 V was attached to each plate. Fish were simulta- neously subjected to an electric current of 7.3 A and 155 V for 3 min.

In C02 narcosis, fish (n = 30) were placed in a slaughter box and C02 was diffused into the water (23.2°C) for 30 min until apparent death. For thermal shock, fish (n = 30) were immersed in a slaughter box containing water and ice (1:1) for 14 min until apparent death. This is the usual stunning procedure for freshwater farmed fish in the Brazilian aquaculture industry.

Water quality parameters (pH, temperature, salinity, specific conductance and dissolved oxygen) were monitored with a multi-parameter probe (Horiba multi-parameters, template U-10) during all procedures. Fish behaviour was also carefully monitored from the beginning of the stunning procedures until apparent death. All stunning treatments should make fish insensitive to pain; therefore, some behaviours were used as indicators of brain function in the absence of brain activity measuring equipment, as suggested by KESTIN et al. (2002). These included no vestibulo-ocular reflex (VOR), opercular (no breathing) or swimming movements, upside down posture and the absence of a reflex when the lateral line was stimulated with a pin. After stunning, fish were kept cold in isothermal boxes containing ice and housed in a cold chamber at 4°C for 18 days. The parameters evaluated were as follows:

a) Rigor mortis index (RI), which was measured during each sampling period with three replicates per treatment. This parameter was measured initially every 30 min after stunning to monitor its onset (RI = 100%) and later at 1, 4, 7, 12 and 18 days of storage to verify resolution. RI was determined using the equation described by BITO et al., 1983.

...

where RI is the rigor index, D0 is the initial distance between the table surface and the caudal fin base and D is the final distance between the table surface and the caudal fin base.

b) Fillet length shrinkage, which was measured using three right fish fillets per treatment with the aid of a digital calliper (Starrett, 799A series) at 0, 3 and 5 h after death and at 1, 4, 7, 12 and 18 days of storage. Fish fillets were stored under refrigeration at 4°C at time 0. Fillet shrinkage was measured in the samples at each sampling time. The percentage of variation in fillet length was determined using the following equation:

...

where L is the final fillet length and L0 is the initial fillet length.

c) Volatile base nitrogen (VBN), which was determined in three left fish fillets per treatment immediately after confirming death (time 0) and at 1, 4, 7, 12 and 18 days of storage according to HOWGATE (1976).

d) Instrumental colour analysis, which was performed in three left fish fillets per treatment at 1, 4, 7, 12 and 18 days of storage using a portable colourimeter (Miniscan XE, Hunterlab). The instrument was calibrated with standard black and white before each reading. A D65 light source, 10° viewing angle and 33-mm diameter area were used for the analysis. Colour measurements were expressed according to the CIELab colour scale, where L* denotes lightness, a* denotes red to green variation and b* denotes yellow to blue variation.

e) Instrumental texture, which was measured in three left fish fillets per treatment at 1,4, 7, 12 and 18 days of storage with a texturometer (TA-XT2Í, Stable Micro Systems) previously calibrated with a 5 kg standard weight. Fillets were compressed perpendicularly with the aid of an aluminium probe (SMS P/20) and measured at 40% compression and a 2 mm/s speed with a platform distance of 20 mm in three different locations.

f) Sensory evaluation, which was performed by five trained panellists with three fish per treatment at 1, 4, 7, 12 and 18 days of storage, according to the European Union official scheme (European Union Regulation, EEC 103/76 modified by regulation 2406/96) that determines four levels of fish freshness.

g) Statistical analysis: data were subjected to analysis of variance (ANOVA) at a 5% significance level. Tukey's test was applied to assess differences among stunning methods and regression analyses were performed where appropriate. All analyses were performed with the statistical program SAS version 9.1.3 (SAS/STAT®, 2002).

RESULTS AND DISCUSSION

Water quality parameters

As parameters such as water quality may have varied during stunning causing stress and consequently compromising meat quality, these parameters were monitored and are described in Table 1. As anticipated, in electronarcosis, parameters such as pH, dissolved oxygen and temperature remained stable during application of the electrical current. Salinity increased because NaCl was added to improve specific conductance of the water. pH decreased after adding C02 due to the production of carbonic acid; however, dissolved oxygen levels did not decrease. Other parameters such as specific conductance, temperature and salinity remained stable. Water temperature was low during the thermal shock stunning method. Dissolved oxygen was higher in the low temperature water (PROENÇA and BITTENCOURT, 1994). pH, conductance and salinity values also remained within expected ranges.

Time required to reach clinical indicators of unconsciousness

Clinical indicators of unconsciousness were observed when fish remained upside down without opercular or muscular movement or a VOR (KESTIN etal, 2002). Considering that these data were not statistically analysed, as each stunning method was simultaneously performed on thirty fish to simulate the fish industry environment, unconsciousness seemed to differ among treatments (Table 2).

Stunning by electronarcosis resulted in clinical indicators of fish unconsciousness almost instantaneously and apparently faster than those of the other treatments. It is possible to use electronarcosis to instantaneously stun and kill eels by using an electric current for longer periods (ROBB et cd., 2002), similar to what we used on matrinxä. Other studies have also reported that stunning fish in water with an electric current is a suitable method (LINES et al, 2003) because it promotes rapid clinical indicators of unconsciousness.

In thermal-shock-stunning gradual loss of consciousness was observed. Although the fish were stunned in groups (n = 30), all fish presented simultaneous clinical signs of unconsciousness. Time to achieve unconsciousness using thermal shock stunning seemed shorter than in electronarcosis, but longer than that in C02 narcosis (Table 2), as observed for turbot [Scophthalmus máximos) (MORZEL et cd., 2003).

In C02 narcosis stunning, the time required to reach clinical indicators of unconsciousness was apparently longer than that in the other treatments. Similar studies in other species have reported shorter times to reach loss of consciousness (ROBB and KESTIN, 2002a; ROBB et al, 2002a; KESTIN, 2003; ROTH et cd., 2006). These variations may be due to metabolic and physiological differences among species or the manner in which the stunning was executed (if fish are exposed to saturated water or if the gas is diffused into water afterwards). As observed in our experiment, C02 narcosis stimulates fish, which is obvious by their quick and violent reactions such as repeated swimming movements, attempts to escape and abnormal activity after stunning (POLI et cd., 2005b).

Rigor mortis

The time required for the onset of full rigor mortis did not differ (P > 0.05) among stunning methods. Two hours were required for the onset of rigor mortis, which remained for 96 h (4 days) of storage. These data can vary greatly among species, stunning methods and preservation temperatures after death, as reported by RUFF et cd. (2002) in their study of S. máximos (20-36 h) and by PARISI et cd. (2002) in their study of sea bass stunned by thermal shock and stored for 11 days at 4°C.

The onset of rigor mortis occurs within 2 h when tilapia are slaughtered at 0°C and stored in ice (CURRAN et cd., 1986), as observed in the present study.

Muscular fillet shrinkage

Similar behaviours were observed within treatments for the muscular fillet shrinkage evaluations, and a significant (P < 0.05) increase was observed 12 h after storage. Shrinkage rate tended to stabilise after this time and was higher in fillets from fish stunned by C02 narcosis (COa: 10.28%; thermal shock: 9.47%; electronarcosis: 8.52%; Fig. 1). In cod (Gados morhoa) fillets, (MISIMI et cd., 2008), the maximum period for tissue shrinkage ( 11 h) is similar for fish stressed and not stressed by the stunning method (bleeding); however, the shrinkage rate (11%) is higher in fillets that originated from stressed fish, as observed in our study with matrinxä.

At the time of death, fish muscles are relaxed, soft and have an elastic structure. Gradually, the muscles become harder, changes their geometric form and decrease in size (ROBB et cd., 2000a). Fillet shrinkage rate depends on the species (EINEN et cd., 2002) and the time of filleting. Fish muscles tend to have greater shrinkage if filleted before rigor mortis because the muscles are separated from the bone structure, which normally prevents such retraction (LAMBOOIJ et cd., 2010).

VBN

Stunning by electronarcosis resulted in higher levels of VBN during the storage period (19.02 mg 100 g"1) than those of the other methods (C02: 17.76 mg 100 g-1; thermal shock: 17.89 mg 100 g'1), probably because of the presence of blood. However, it is important that fish should not be consumed after 18 days of storage regardless of treatment according to sensory evaluation and once they have been categorised as class B (European Union Regulation, EEC 103/76 modified by regulation 2406/96). VBN levels during the 18 day storage period were below the maximum allowed by the Brazilian Control Department for Products of Animal Origin (RIISPOA), which is 30 mg 100 g"1. In another study with matrinxä stored in ice for 16 days after stunning by thermal shock, VBN levels were 19 mg 100 g-1 and fillets were considered appropriate for consumption according to the sensory evaluation table proposed by the Torry Research Station, Aberdeen (LESSI et cd., 2004).

Total VBN content in fish stored in ice is low during the first storage phase and tends to increase rapidly only when fish quality deteriorates (HUSS, 1997). This was not observed in the present study because VBN levels were stable throughout the entire storage period even when fish were not appropriate for human consumption. HUSS (1997) reported that VBN values should not be used to estimate the first changes in fish but to evaluate the degree of deterioration during the last steps. However, this has been confirmed mainly with regard to sea fish species (LESSI et cd., 2004). Therefore, on the ba- sis of the three stunning methods used in this study, VBN was not an effective tool to evaluate matrinxä under cold storage.

Sensory analysis

No differences were observed among the tested methods with regard to sensory aspects. The evaluated methods could not extend fish shelf life as the fish stored in ice were classified as 'extra' up to day 4 (96 h), 'fresh' (category A) up to day 18 (432 h) and 'rancid' (category B) after this period (Fig. 2).

A study by LESSI et al (2004) on matrinxä stunned in ice and water and subsequently refrigerated showed that fish maintained good condition until day 26, and fish quality was classified as optimal during the first 13 days. The sensory evaluation was based on the Torry Research Station table, which considers culture conditions, fish habitat and weight at slaughter.

Colour evaluation (L*, a* and b*)

The stunning methods did not alter the intensity of yellow colour (b*) of the fillets. This parameter increased constantly throughout time in the three treatments and could be explained by a single equation, b* = 10.4684 + 0.004209x. The lightness (L*) of the fillets increased (P < 0.05) significantly during storage period for C02 narcosis and thermal shock treatment. L* values for both methods presented little but constant increase until day 18 of storage. In contrast, fillets from fish stunned by electronarcosis had consistent lightness (L*) throughout storage (average, 53.9 ± 1.58).

Red colour intensity (a*) did not differ during the storage period, but the intensity was higher (P < 0.05) in fish stunned by electronarcosis (4.84 ± 0.6) than in those stunned by C02 narcosis (0.58 ± 0.6) and thermal shock (0.80 ± 0.6). The highest red intensity was observed due to haemorrhaging caused by the electric current applied during electronarcosis. Higher a* and lower L* values were observed in turbot stunned by electronarcosis (MORZEL et al., 2003). Stunning by electricity is a fast and efficient method to render the fish insensitive and induce unconsciousness (ROBB and ROTH, 2003; LAMBOOIJ et al., 2004). However, injuries, such as vertebra fractures and arterial disruption, caused by this kind of stunning can promote blood stains and haemorrhaging in the meat (ROTH, 2003), compromising product quality. Thus, further studies investigating different electric shock variables (voltage, amperage, etc.) are necessary to improve L* and a* parameters of fillets.

Instrumental texture

Texture was measured with a texturometer to evaluate if the tested methods could cause alterations in muscle texture; however, no differences in fillet resistance were observed when compression strength was applied, regardless of the kind of treatment. In contrast, compression strength diminished during cold storage (Fig. 3). During cold storage, fish suffer degradation in muscle structure owing to protein dénaturation and enzyme actions, causing fish muscle to become less rigid, particularly in the first 24 h after slaughter (TOYOHARA and SHIMIZU, 1988; OKA et al., 1990; MOCHIZUKI and SATO, 1996). ROTH et al. (2007) did not observe differences in fillet resistance after comparing different stunning methods (brain percussion, bleeding and electronarcosis) and meat texture in turbot.

In summary, the European Union regulations were not effective for this species because some evaluation parameters, such as gills and abdominal cavity odour, only occur in typical sea fish. Therefore, developing a specific quality index method (QIM) for matrinxä or utilising an existent scheme for species with similar habitats and feeding behaviour is recommended. Further investigations concerning voltage variations as well as the stunning time and its consequences on fish quality are suggested with the purpose of minimising or eliminating possible damage caused by haemorrhages associated with electric shock.

CONCLUSIONS

The three stunning methods tested were effective in maintaining meat quality when applied to matrinxä. Stunning by electronarcosis seemed slightly deficient when considering colour because of the hemorrhages caused by this method suggests the need for further research. However, the haemorrhaging did not compromise other quality parameters. Electronarcosis is considered to be the most efficient method for matrinxä welfare because it seemed to clinically indicate unconsciousness faster than the other treatments.

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