Meat Science 98 (2014) 247–254 Contents lists available at ScienceDirect Meat Science journal homepage: www.elsevier.com/locate/meatsci Effect of electrical and mechanical stunning on bleeding, instrumental properties and sensory meat quality in rabbits R. Lafuente a, M. López b,⁎ a b Gobierno de Aragón, Departamento de Sanidad, Bienestar Social y Familia, Vía Universitas 36, 50071 Zaragoza, Spain Universidad de Zaragoza, Departamento de Producción Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Miguel Servet 177, 50013 Zaragoza, Spain a r t i c l e i n f o Article history: Received 15 November 2013 Received in revised form 25 April 2014 Accepted 30 May 2014 Available online 8 June 2014 Keywords: Stunning Neck dislocation Bleeding Colour pH Organoleptic a b s t r a c t Different voltage and frequency (T-1 = 49 V, 250 Hz; T-2 = 130 V, 172 Hz; T-3 = 22 V, 833 Hz) combinations of electrical stunning and cervical dislocation (T-4) were studied in 101 commercial rabbits in an industrial abattoir. Electrical stunning accelerated the early muscular acidification, providing lower pH-45 and pH-2 h values on Longissimus dorsi and Biceps femoris and higher pH-24 h on Biceps femoris than cervical dislocation (P b 0.02). Furthermore, meat from rabbits stunned with electrical methods showed more redness (a* with mean values 1.17–1.30 vs. 0.66, P b 0.02), although this cannot be associated to low exsanguination levels because electrical methods tend to produce even higher bleeding percentage than mechanical stunning (P = 0.063). Haematin content in muscle, water-holding capacity and cooking losses were similar in all treatments. Shear force did not change because of the stunning methods, but the members of experienced panel found the meat coming from electrical stunning T-1 (with intermediate voltages and frequencies) tougher and less juicy than the meat obtained with other electrical applications or with cervical dislocation (P b 0.05). © 2014 Elsevier Ltd. All rights reserved. 1. Introduction The implementation of the European legislation on Animal Protection at the moment of slaughter has led the EU rabbit sector to replace pre-slaughter mechanical stunning methods (such as the commonly used cervical dislocation), with procedures that guarantee in commercial facilities a sufficient degree of unconsciousness and lack of sensitivity to avoid or minimize the reactions of fear, anxiety, pain and stress until the death of the animal. In most cases, both in the EU and in other countries, electrical methods have been chosen for rabbits, with very few abattoirs using gas, others using pneumatically powered stunning guns (OMAFRA, 2011) and some experiments with captive bolt apparatus (SchüttAbraham, Knauer-Kraetzl, & Wormuth, 1992) which have had little practical repercussion. The introduction of electrical stunners, with good results from the point of view of the welfare of the animal (Anil, Raj, & McKinstry, 1996, 1998, 2000; María, López, Lafuente, & Mocé, 2001) and improving the work in the abattoir in terms of comfort and risk, gave rise to initial suspicions from the part of specialized workers, who reported that the carcasses obtained after electric stunning showed more pinkness than those obtained by traditional procedures, which were very pale. This made the sector regard post-electrical stunning bleeding as deficient and, also, question the conservation time of the carcasses and the quality of the meat. These ideas were not new, since previous references on ⁎ Corresponding author. Tel.: +34 976 762324; fax: +34 976 761612. E-mail address: [email protected] (M. López). http://dx.doi.org/10.1016/j.meatsci.2014.05.031 0309-1740/© 2014 Elsevier Ltd. All rights reserved. electrical stunning used on pigs discouraged its use on fattened pigs due to the damage caused to the carcasses and the reduction of the period of conservation of the meat because of its high blood-holding level (Ducksbury & Anthony, 1929). There are also some old results on preslaughter electro-narcosis used in rabbits, as it was studied by Croft (1952), although the methodology was limited and not enough to provoke insensibility periods which were acceptable for the species. Today the electrical possibilities to stun rabbits in commercial practice are varied and they use indiscriminate combinations of high and low voltages, together with high and low frequencies and, in turn, with different intensities and application times. This situation, as well as the negative opinion previously mentioned, led us to develop a work protocol designed to provide information about some electrical combination which would successfully induce an adequate degree of insensibility during the required time and also which would not affect the parameters of the carcass or the quality of the meat. Results on the degree and pattern of desensitization and recovery of rabbits in the conditions of this study were previously published (López, Lafuente and María, 1998; María et al., 2001). Few studies have been carried out on pre-slaughter stunning in rabbits since then (López et al., 2008; Rota Nodari, Lavazza, & Candotti, 2009; Xiong, Zhu, Zhao, Shi, & Tang, 2006) or the results are hardly applicable in commercial practice (Apata, Eniolorunda, Amao, & Okubanjo, 2012). The present study shows the results on rabbit meat quality obtained by applying three electrical combinations of pre-slaughter stunning with a commercial abattoir machine, and compares them with that achieved with the use of cervical dislocation, an unauthorized practice in EU slaughterhouses, which is nevertheless 248 R. Lafuente, M. López / Meat Science 98 (2014) 247–254 widely applied in smaller processing in many countries (McNitt & Swanson, 2005). 2. Materials and methods 2.1. Animals, experimental design and slaughter traits Terminal rabbits from a three way crossbreeding scheme, from the same commercial farm and similar conditions on the farm and during transport to the slaughterhouse were used. The transport was carried out in a truck. Animals were housed in 8 floors of cages towers (12 rabbits/cage), with a duration of 3.5–4 h of transport and resting for about 2 h pre-slaughter. The choice of each rabbit was at random, but they were within the commercial range of 1.8–2.0 kg. Sex was not considered due to its little effect on meat quality in rabbits (Carrilho, Campo, Olleta, Beltrán, & López, 2009; Cavani et al., 2000; Trocino, Xiccato, Queaque, & Sartori, 2003), nor age (62–65 days), but position on the tower cage was considered (Liste et al., 2009), selecting rabbits from all cages in the tower for each treatment. The experiment was conducted following the Spanish and European animal welfare regulations for animal husbandry, transport and slaughter. A total of 101 rabbits were used. Rabbits were divided into 4 groups and stunned using one of the following methods: T-1, voltage and power frequency usually used at this slaughterhouse (49 V and 250 Hz) (n = 24); T-2, high voltage (130 V) and low frequency (172 Hz) (n = 24); T-3, low voltage (22 V) and high frequency (833 Hz) (n = 27); T-4, traditional cervical dislocation (CD) (n = 26). Rabbits received the electric shock in the frontal sinus (Fossa temporalis) through a V-shape jagged electrode, which provided a good grip. The slaughterer was the worker usually responsible for the operation in the abattoir. The contact time of the electrodes was always under 2 s. The combination of extreme electric values (high voltage + high frequency, low voltage + low frequency) was ignored because its effects on the degree of insensibility and the pattern of recovery in rabbits were similar to some of the electric combinations mentioned (López et al., 1998; María et al., 2001) and, also, because these combinations are not often used in commercial slaughterhouses. Every rabbit was weighed before stunning and slaughtered 10 s after stunning by exsanguination from the main blood vessels on both sides of the neck, oesophagus and trachea. Ninety seconds after slaughter, the rabbit was weighed again: the difference in weight accounted for the blood loss. For the calculations, we used the absolute values of blood weight as well as the percentage in relation to the weight of the live animal. Immediately after the completion of dressing, which happened 10– 13 min after stunning and 2–3 min after evisceration, the carcasses were aired for 6 min (normal time in this slaughterhouse) and then placed in standard storing crates and protected by polyethylene sheets; they were later kept in cold storage rooms at a temperature of 0–4 °C. The pH of the Longissimus dorsi (LD) and Biceps femoris (BF) from the left side was determined in the storage room firstly 45 min after and then 2 h after the dressing process (CRISON, pHmeter model 507; Crison Instruments, Alella, Barcelona, Spain). The measurements were taken with a penetration electrode in the LD at the level of the 5th–6th lumbar vertebra and in the middle of the BF muscle, the sites recommended by the World Rabbit Scientific Association (Blasco, Ouhayoun, & Masoero, 1992). 2.2. Instrumental and sensory determinations After 24 h of cooling, the carcasses were evaluated in the laboratory of the University of Zaragoza, where the pH-24 (pHu; according to Hulot & Ouhayoun, 1999) was obtained and then the muscles of the dorsalproximal region of the hind limbs (Biceps femoris, Semitendinosus, Semimembranosus, Abductor cruris, Gluteus and Tensor fasciae latae) (HL muscles) were dissected, wrapped in aluminium foil and kept at a temperature of 4 °C for a later chemical colour analysis and to determine the water-holding capacity (WHC). The Longissimus dorsi muscle from both sides was also dissected dividing it into two portions, cranial and caudal. The cranial ones were used to determine the colour on the area of the cut, as detailed below. They were later vacuum-packaged in a polyethylene bag and used to study the cooking losses (CL) and the muscle texture. The caudal sides were vacuum-packaged in a polyethylene bag and reserved for the sensory analysis. A Minolta CM 2002 (Minolta Inc., Osaka, Japan) spectrophotometer was used for measuring physical colour in the LD muscle within the CIELAB system (CIE, 1976) with a D65 illuminant and a 10º observer, with an aperture size of 2.54 cm, following the methodology of reference given by Honikel (1998) for meat products. A chemical analysis of the total haem pigments from a minced sample of the HL muscles was carried out to determine the parts per million of haematin per gram of muscle using the method described by Hornsey (1956), with spectrophotometer readings (HITACHI U-1100; Hitachi Ltd., Chiyoda, Tokio, Japan) of optical density at wavelengths 640λ and 512λ (Alberti et al., 2005). All measurements were carried out in duplicate. Water holding capacity was measured using the modified Grau and Hamm (1953) technique as described by Carrilho et al. (2009), to determine the percentage of expelled juice in a 5 g minced sample of the HL muscles under a weight of 2250 g for 5 min. Water holding capacity was calculated as the difference between the initial and final weights of the samples over the initial weight (percentage of expelled juice). Two samples were used from each rabbit. Both cranial portions of the LD were kept in polyethylene bags and water-bathed (FLANGE, GFL Series D3006; GFL Gesellschaft für Labortechnik mbH, Burgwedel, Germany) at 75–80 °C for 20–25 min to assess the loss of weight after cooking. Cooking loss was expressed as a percentage and was calculated as the difference between the initial and final weights over initial weight of both portions (CL %). Texture was later analysed on these samples after cutting them as prisms (2 cm length × 1 cm2 base) using a scalpel and with the muscle fibres parallel to longitudinal axis. On these samples we studied the maximum shear force required to break the sample perpendicularly to the muscle fibres. A 4301 series INSTRON (Instron Corp. Barcelona, Spain) machine equipped with a Warner–Bratzler (WB) device was used. The sensory analysis was carried out 72 h after sacrifice. A trained 11-member taste panel (ISO 8586) evaluated the samples in red-lit cabins. Four sessions with 12 samples in each session were required to obtain the data. In every session, a comparative multi-sample test with three samples each time in a balanced incomplete-block design was used to detect differences in sensory features among the four treatments. The order of presentation was changed between panellists and sessions to avoid first order and carry-over effects (Macfie, Bratchell, Greenhoff, & Vallis, 1989). Both caudal parts of Longissimus dorsi were used. They were cooked unseasoned on a SAMMIC P8D-2 (Sammic, Azkoitia, Gipuzkoa, Spain) double plate grill at 200 °C, inside aluminium paper, until the internal temperature reached 65 °C (probe JENWAY 2000; Jenway, Staffordshire, United Kingdom). Each sample was cut in 11 pieces, which were served at random to the taste panel on a hot dish. Bottled water and bread were given to the panel at the beginning of the session and in between dishes. All the panellists tasted pieces from all treatments, and giving a total of 132 assessments per treatment. The parameters were tenderness, juiciness, flavour intensity and overall appraisal in a non-structured scale of 100 points (where 0 stood for very tough, no juiciness, no flavour intensity and low overall appraisal, and 100 stood for maximum tenderness, very juicy, high flavour intensity and high overall appraisal). 2.3. Statistical analysis Data were processed using the analysis of variance option of the GLM procedure of the SPSS Statistics (19.0) with the stunning treatment as a fixed effect. For the sensory test, session was also included in the R. Lafuente, M. López / Meat Science 98 (2014) 247–254 249 Table 1 Body weight pre-stunning (LW), body weight post-bleeding and blood loss in the four stunning treatments (mean ± SD). Variable Stunning treatment Body weight pre-stunning, g Body weight post-bleeding, g Blood loss, g Blood loss, LW % P-value T-1 (49 V–250 Hz) T-2 (130 V–172 Hz) T-3 (22 V–833 Hz) T-4 (CD) 1998.89 ± 13.63 1932.59 ± 13.19 66.30 ± 11.49 3.32 ± 0.53 2001.85 ± 17.35 1932.96 ± 16.98 68.89 ± 8.92 3.45 ± 0.42 1994.00 ± 13.73 1926.40 ± 13.45 67.60 ± 13.00 3.39 ± 0.63 2043.85 ± 14.21 1979.62 ± 13.90 64.23 ± 13.02 3.14 ± 0.58 model after analysing the consensus of the panellists for each descriptor. Because the pH was determined in both LD and BF muscles, the effect of stunning treatment, the effect of the muscle and their interaction on pH values were studied. When F-tests were significant, means were compared by Duncan's multiple range test with signification level of P ≤ 0.05. The probability value less than 0.10 was discussed as a trend. Likewise, a Pearson correlation analysis was performed to evaluate the relationship between some physicochemical (bleeding with colour variables; pHu and L* with colour variables, WHC, CL, WB shear force) or sensory variables (tenderness, juiciness, flavour intensity and overall appraisal). When r was significant, for predictive purposes the residual coefficient of variation (RCV) (Berg & Butterfield, 1976) was calculated by simple linear regression analysis. 3. Results The results on bleeding are shown in Table 1. Although there were no significant differences between the stunning methods (P N 0.05), cervical dislocation would tend to cause lower bleeding percentage because Duncan (P = 0.063) separated means of T1 (3.32ab ± 0.53), T2 (3.45a ± 0.42), T3 (3.39ab ± 0.63) and T4 (3.14b ± 0.58). Regarding pH, there was no significant interaction S × M (P N 0.636) but the effect of muscle factor was significant (P b 0.001) in pH-45, pH-2 and pHu. In consequence, pH values 45 min, 2 h and 24 h post mortem are shown in Table 2 separately for LD and BF. pH-45 values for any electrical combination were lower than those obtained through mechanical stunning both in LD and BF (P b 0,01). pH-2 h was also significant lower after electrical stunning in both muscles. Twenty-four hours after slaughter the effect of the treatment on the pH was only significant in the Biceps femoris but with the opposite result, that is to say, the final pH levels after electrical stunning were higher than those obtained through mechanical stunning (P b 0,05). Only pHu of T-2 was not significantly different from T-4. Results of chemical and physical determination of meat colour are shown in Table 3. The effect of stunning method on colour chemical parameters was not significant. In turn, the correlation between haematin content and bleeding level in absolute or relative values was not significant in either treatment. Considering all data together as those of a single group (T1–4), the amount of haematin (λ = 640) was associated to bleeding expressed in absolute values, and this correlation was positive .600 .523 .518 .195 (r = 0.250, P b 0.05). The correlation was not significant when bleeding was considered in percentage vs. haematin λ640, or in haematin λ512 vs. absolute or relative bleeding. On the other hand, the level of bleeding was not related to colour parameters CIE L*a*b* (Table 3), which showed mean values 48.03 ± 2.86 in L* (lightness) and 2.16 ± 1.25 in b* (yellowness). There was no effect of stunning method on these values. The a* (redness) values ranged from 1.24 in meat obtained by electrical stunning to 0.66 in meat obtained by mechanical stunning (P b 0.05). The percentage of expelled juice per minced sample from HL muscles was 16.25 ± 3.43%, with no statistical effect of stunning method (Table 3). In turn, the percentage of cooking loss in the LD portion water-bathed was 14.73 ± 3.17% on average. The shear force values did not change with the stunning method. Sensory analysis showed difference on tenderness (P ≤ 0.05), but the highest and lowest levels of tenderness were obtained within the electrical methods, while T-4 meat was not significantly different from the other groups. Thus, T-1 provided the least tender meat, and this meat was also the least juicy (P ≤ 0.05) (Table 4). The effect of stunning method on flavour intensity and overall appraisal (panel mean marks 55.30 ± 15.73 and 48.17 ± 15.72 respectively) was not significant. As we can see in Table 5, sensory tenderness and juiciness presented a significant positive correlation (r = 0.400, P b 0.01). There was no correlation between flavour and tenderness or juiciness, but the overall appraisal was linked to each of the sensory characteristics that were assessed: it increased with higher levels of tenderness, juiciness and flavour (P b 0.01). In relation to the physico-chemical traits, within each electric stunning groups the pHu of LD or BF muscles showed a significant and negative correlation with L*, WHC and CL, and not significant or with high residual coefficients of variation with most of the other features (Table 6). Within mechanical group there was no relation between pHu and cooking losses, but was significant and positive with haematin content (λ = 640). After the combination of the four groups (T1–4) we observed the usual relations between pH values and the instrumental properties of meat: an increase of pH-24 is accompanied by a higher WHC and lower cooking losses, higher redness and lower lightness and yellowness, as well as a greater shear force and less haematin, except in group T-4, as just indicated, what is less frequent. In general BF showed a slightly lower RCV than LD; therefore, it could be a best Table 2 Effect of stunning on pH 45 min, pH 2 h and pHu values in Longissimus dorsi and Biceps femoris muscles (mean ± SD). Variable Stunning treatment P-value T-1 (49 V–250 Hz) T-2 (130 V–172 Hz) T-3 (22 V–833 Hz) T-4 (CD) Longissimus dorsi pH 45 min pH 2 h pH 24 h 6.79b ± 0.26 6.56b ± 0.23 6.00 ± 0.17 6.84b ± 0.24 6.56b ± 0.22 5.99 ± 0.20 6.84b ± 0.31 6.61b ± 0.26 5.98 ± 0.26 7.06a ± 0.22 6.74a ± 0.18 5.91 ± 0.12 .001 .015 .275 Biceps femoris pH 45 min pH 2 h pH 24 h 6.54b ± 0.29 6.47b ± 0.23 6.18b ± 0.18 6.62b ± 0.31 6.40b ± 0.19 6.12ab ± 0.21 6.57b ± 0.29 6.46b ± 0.24 6.16b ± 0.25 6.82a ± 0.25 6.68a ± 0.24 6.01a ± 0.17 .002 .000 .016 a, b: different letters within row mean differences among treatments, P b 0.05. 250 R. Lafuente, M. López / Meat Science 98 (2014) 247–254 Table 3 Effect of stunning on instrumental characteristics of rabbit meat: chemical (haematin content) and physical (L*a*b*) colour, water holding capacity (WHC), cooking losses and texture (shear force) (mean ± SD). Variable Haematin (ppm/g) 512λ 640λ L* a* b* WHC (%) Cooking losses (%) Shear force (kg/cm2) Stunning treatment P-value T-1 (49 V–250 Hz) T-2 (130 V–172 Hz) T-3 (22 V–833 Hz) T-4 (CD) 27.11 ± 10.58 28.66 ± 22.94 47.54 ± 2.87 1.25a ± 0.65 1.87 ± 1.39 15.27 ± 3.99 14.41 ± 3.18 2.59 ± 0.67 28.72 ± 11.46 26.76 ± 17.48 47.67 ± 3.41 1.30a ± 0.69 2.35 ± 1.15 16.67 ± 3.09 14.55 ± 3.16 2.48 ± 0.62 28.67 ± 13.64 27.75 ± 16.06 47.90 ± 2.81 1.17a ± 0.91 2.04 ± 1.20 16.53 ± 3.76 14.61 ± 3.43 2.46 ± 0.59 31.29 ± 15.57 37.42 ± 24.93 48.95 ± 2.22 0.66b ± 0.70 2.37 ± 1.25 16.50 ± 2.79 15.26 ± 3.04 2.46 ± 0.74 .734 .241 .277 .011 .421 .457 .810 .905 a, b: different letters within row mean differences among treatments, P b 0.05. predictor of physicochemical properties. Also L* was significantly correlated with some instrumental features, improving estimation of some variables with respect to BF. 4. Discussion In the process of slaughter, the stunning method should ideally lead to the highest bleeding efficiency after throat cutting to obtain better quality meat. In the production chain, badly bled carcasses are related to a more intense colour and a less attractive aspect, and vice versa. To the best of our knowledge, only one experiment has been performed to study the effect of stunning on blood loss in rabbits (López et al., 2008). Newly weaned rabbits that weighed 1 kg were slaughtered after electrical stunning and compared to others slaughtered by the halal rite without previous stunning. The group electrically stunned tended to show lower bleeding levels than the halal group. Moreover, the percentage of blood expelled was higher than the present experiment (4.06% electrical stunning, 4.64% halal, P = 0.075) may be due to different calculation procedure, to different bleeding times or to different maturity of the animals (Kotula & Helbacka, 1966a; Swatland, 2006), whereby a comparison is quite difficult. In general, our results for the levels of blood expelled are near the 3.6% referred to by Ouhayoun (1986) for 10–11 week old commercial rabbits with a weight of 2.3 kg. According to Gregory (1998), the weight of blood in an animal is usually about 8% of its live weight and, normally, only about half that blood is voided during slaughter. When bleeding is delayed after stunning, the volume of blood could decrease (Omojola, 2007). In relation to the effect of stunning procedure on bleeding and comparing to other species of similar size, Kotula and Helbacka (1966a, 1966b) studied the kosher method in chickens, which was efficient for a bleeding period of 300 s when compared to other methods such as brain perforation, electricity, CO2 or pneumatically powered captive bolt stunner. According to the authors, it leads to high blood retention in the offal, something that happens when animals are not stunned before slaughter. Kuenzel and Ingling (1977) also studied chicken that had not previously been stunned: when jugular veins and carotid arteries are cut on one side with partial damage to the backbone, they show spasmodic muscle movements which result in lower bleeding levels. Even if the captive bolt stunner only produces brain disruption, with no brain penetration, the bleeding levels during 120 s are lower than in the case of electrical stunning (Göskoy et al., 1999). As refers to electrical methods, Kuenzel and Ingling (1977) note that cardiac fibrillation occurs when applying voltages of 95 V and AC (alternating current) in chicken, which could be the reason for poor bleeding. The same authors found wounds in heart tissues in chickens after 130 V AC had been applied and pointed out that, if heartbeat is kept active during bleeding, bleeding efficiency is higher. Göskoy et al. (1999) reach the same conclusion when, after 120 s of bleeding, the efficiency was significantly higher in chickens that did not fibrillate after electrical stunning. On the other hand, Schütt-Abraham, Wormouth, and Fessel (1983) compared the bleeding level in chickens that survived electrical stunning with those that were electrocuted and only found significant differences during the first 90 s (2.48% vs. 2.08% in live weight, respectively). Ninety seconds later the differences were no longer significant (2.98% vs. 2.78%). According to the authors, this could mean that the effect produced by the stunning method disappears if time is given when bleeding poultry. In this context and according to the results of Papinaho and Fletcher (1995), a bleeding period of 150 s would be enough to eliminate the effect of the stunning method on chicken bleeding. The authors did not obtain significant differences when they compared unstunned chickens (0 mA) to chickens stunned at different currents (50, 100, 150 and 200 mA). However, Contreras and Beraquet (2001) showed that 360 s is not enough to eliminate the effect of stunning. These authors found that, when compared to chickens stunned by applying 20 and 40 V, unstunned chickens showed lower blood losses. Therefore, two factors should be taken into account in after-slaughter bleeding: the stunning method used and the bleeding time. According to Kotula and Helbacka (1966a) and Schütt-Abraham et al. (1983), for the same bleeding times, the stunning method affects the blood expelled in the initial post mortem moments. Our study suggests that mechanical methods might produce poorer bleeding efficiency than electrical stunning, something similar to what happened when animals were slaughtered without stunning (Contreras & Beraquet, 2001; Kuenzel & Ingling, 1977). Or maybe it could be that the stunning method causes some differences in the speed of blood loss after slaughter, faster bleeding with electrical stunning and slower bleeding in the case of mechanical stunning. In our experiment the 90 s bleeding time accounts for the time needed for the animals to go along the chain, from the initial Table 4 Effect of stunning on sensory traits of the rabbit meat (mean ± SD). Variable Tenderness Juiciness Flavour intensity Overall appraisal Stunning treatment P-value T-1 (49 V–250 Hz) T-2 (130 V–172 Hz) T-3 (22 V–833 Hz) T-4 (CD) 45.96a ± 23.12 43.18a ± 19.44 55.45 ± 17.81 48.60 ± 15.59 53.72b ± 20.82 48.15b ± 20.31 55.05 ± 16.02 49.63 ± 14.60 51.91b ± 23.79 48.53b ± 18.07 53.65 ± 14.24 46.69 ± 16.83 51.13ab ± 20.60 49.14b ± 19.90 57.05 ± 14.62 47.80 ± 15.79 a, b: different letters within row mean differences among treatments, P b 0.05. .030 .050 .374 .482 R. Lafuente, M. López / Meat Science 98 (2014) 247–254 Table 5 Pearson's correlation coefficients (r) between traits of the sensory analysis. Variable Tenderness Juiciness Flavour intensity Overall appraisal Tenderness Juiciness Flavour intensity Overall appraisal 1 .400⁎⁎ 1 .005 .060 1 .279⁎⁎ .287⁎⁎ .213⁎⁎ 1 ⁎⁎ = P ≤ 0.01. slaughter to the cutting machine of the distal part of the front limbs. So, unfortunately, we could not determine if rabbits bled after this moment, as the experiment took place in a normalised abattoir. We cannot, therefore, make any other inferences since, if weighed again, the amount of blood expelled might have been the same for both methods, or different. However, three factors suggest correct bleeding of the rabbits with any of the stunning methods: no effect of stunning on the haematin content in the hind limb muscles, few correlations between colour parameters and bleeding, and the amount of blood eliminated. This discards the misconception that electrical stunning is connected to poor bleeding. In fact, very little blood is retained in the meat during slaughter, and most of the remaining blood is in the offal and major blood vessels (Gregory, 1998). Warris (1977) and Warris and Rhodes (1977) indicated that fresh meat contains very little residual blood and that haemoglobin increases redness in meat only in cases of poor bleeding. The only significant correlation in absolute measurements between haematin (λ = 640) and bleeding was positive (r = 0,250, P b 0.05), which would indicate that rabbits with higher levels of pigments in their meat show higher blood losses, which is difficult to explain. When colour is determined in the CIE L* a* b* space, a* values could indicate that the common belief might have a scientific base after all, since mechanical stunning goes along with low redness. However, in the literature we find contradictory results. In the experiment by Dal Bosco, Castellini, and Bernardini (1997) electrical and mechanical stunning had no effect on the level of redness in the muscle Longissimus dorsi of rabbit. The same happened in chicken breast after electrical stunning vs. captive bolt stunning (Hillebrand, Lambooy, & Veerkamp, 1996), although electrical stunning vs. beheading shows different a* values in this species (McNeal, Fletcher, & Buhr, 2003). In this last case, values for electrical stunning are lower than values for beheading. Finally, the effect of electrical stunning and halal was not significant on the values of redness in rabbit meat, but, subjectively, the colour of the carcass immediately after dressing was redder with electrical stunning and paler in halal sacrifice (López et al., 2008). It is important to take into account the high variability in most colour variables, both in our 251 case and in the above mentioned studies, which might have made it impossible to find significant effect of the stunning methods in these parameters. It is possible to establish the differences in the post-mortem acidification process: faster with electrical stunning and initially slow but constant and more intense after 24 h with mechanical stunning (at least on muscle Biceps femoris). The differences in initial acidification have been reported in rabbits (Civera, Julini, Quaglino, & Ferrero, 1989; Dal Bosco et al., 1997; Ouhayoun, 1988). Hulot and Ouhayoun (1999), in an extensive review, reported that the stress of electro anaesthesia accelerates muscular acidification, although it does not modify ultimate pH in meat. López et al. (2008) found that ultimate pH was, in fact, modified because of stunning, both in the Longissimus dorsi and in the Biceps femoris, and in the present study the effect stays in the pH-24 of the Bicep femoris. These differences between electrical and mechanical methods can be due to the increase in lactate in the blood produced by the strong muscle contractions caused by electrical stunning (Sybesma & Groen, 1970). Nevertheless, Lee, Hargus, Webb, Rickansrud, and Hagberg (1979) and Papinaho and Fletcher (1995, 1996) in their studies on chicken breast, obtained initial pH values and also values during the first 4 h which were higher in electrically stunned animals than in unstunned animals. The first authors also reported slower glycolysis (measured as an increase of the levels of lactate in the muscle) and, consequently, higher pH levels in stunned chickens. On the other hand, Overstreet, Marple, Huffman, and Nachreiner (1975) obtained lower pH values in electrically stunned pigs than in unstunned pigs, corresponding to low lactate levels in carcasses that came from unstunned animals. Later, Velarde, Gispert, Faucitano, Manteca, and Diestre (2000) confirmed that electrical stimulation of the nervous system in pigs accelerates the degradation of muscular glycogen into lactate, which results in a rapid pH fall in some muscles. The present study obtained the same results in rabbits, and the data suggest that the ATP activity which determines the changes in pH fall (Monin, 1988) could be more intense in electrically stunned rabbits and, consequently, the glycogen reserves in the muscles were used up more quickly in these groups. As a result, after electrical stunning the pH-24 was higher than after mechanical stunning, at least in the muscle Biceps femoris. Taking into account the nature of the muscles, the contractile and metabolic features affect acidification regardless of the stunning method used. The muscles studied here have been classified as oxy glycolitic metabolism by Ouhayoun and Delmas (1983), as the muscle LD has a high amount of type IIB fibres of glycolitic activity and variable proportions of type I and type IIA fibres, whereas the muscle BF only has these last types of fibres and consequently shows a higher oxidative trend. In such a way, the BF showed more acid pH levels during the first 2 h of cooling (Ouhayoun & Delmas, 1988), as well as a Table 6 Pearson's correlation coefficients (r) and residual coefficient of variation (%, in brackets) between pHu Longissimus dorsi, pHu Biceps femoris and L* with physicochemical parameters. Group and variable L* a* b* Haematin (ppm/g) 512λ 640λ T-1 −.641*** (4.64) −.709*** (4.26) 1 −.625*** (5.58) −.842*** (3.86) 1 −.810*** (3.43) −.891*** (2.66) 1 −.503** (3.93) −.816*** (2.62) 1 −.676*** (4.39) −.816*** (3.44) 1 .210 .123 −.357* (48.71) .232 .316 −.509* (45.51) .445** (69.76) .453** (69.48) −.443** (69.86) .215 .029 −.399* (97.81) .294*** (68.75) .343*** (67.57) −.457*** (63.98) −.132 −.226 .299 −.340 −.431* (44.19) .440* (43.97) −.335* (55.34) −.344* (55.17) .363* (54.73) −.123 −.302 .514** (45.13) −.255** (55.92) −.343*** (54.33) .398*** (53.06) −.353* (36.49) −.234 −.018 −.122 −.007 −.053 −.363* (44.35) −.375* (44.12) .230 −.074 .313 −.078 −.249** (43.18) −.132 .053 −.110 −.088 −.067 −.239 −.058 .013 −.379* (53.57) −.281 .247 .026 .423* (60.37) −.239 −.218* (67.00) −.073 .030 T-2 T-3 T-4 T1–4 pHu LD pHu BF L* pHu LD pHu BF L* pHu LD pHu BF L* pHu LD pHu BF L* pHu LD pHu BF L* * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001. WHC (%) CL (%) Shear force (kg/cm2) −.561** (21.63) −.511** (22.46) .301 −.666*** (13.82) −.663*** (13.88) .814*** (10.76) −.604*** (18.15) −.681*** (16.67) .450** (20.33) −.464** (14.98) −.473** (14.90) .467** (14.96) −.569*** (17.38) −.578*** (17.24) .495*** (18.37) −.648*** (16.83) −.572** (18.12) .428* (19.97) −.490* (18.96) −.619** (17.09) .679*** (15.98) −.633*** (18.17) −.755*** (15.39) .569** (19.30) −.132 −.120 .223 −.519*** (18.41) −.556*** (17.89) .501*** (18.63) −.321 −.166 −.037 .177 .497* (21.59) −.546** (20.85) .314 .336 −.531** (20.30) .250 .455* (26.85) −.157 .105 .296** (24.80) −.327*** (24.54) 252 R. Lafuente, M. López / Meat Science 98 (2014) 247–254 quicker acidification and a higher pH 24 h. In relation to Hudson's (2012) observations about pH decline rate related to mitochondrial content and meat quality, the red BF showed lower overall changes between initial and final values than the white LD. The high glycolitic potential of the LD accounts for the low ultimate pH (Delmas & Ouhayoun, 1990; Ouhayoun & Dalle Zotte, 1993; Ouhayoun & Delmas, 1988). Our pHu values of BF and LD confirm those obtained by other authors for rabbits raised in the standard conditions of commercial farms (Carrilho et al., 2009; Pla, Hernández, & Blasco, 1996; Trocino et al., 2003) or in other housing conditions (Combes, Moussa, Gondret, Doutreloux, & Remignon, 2005; Dalle Zotte et al., 2009; Xiccato et al., 2013). Some experiences in alternative housing with very high availability to perform movements by a low density (Volek et al., 2012) or physical stimuli induced by endurance training (Gondret, Hernández, Rémignon, & Combes, 2009) showed a decreased frequency of type IIB and increased type I and type IIA in BF muscle of trained rabbits compared with sedentary rabbits. Trained rabbits had a similar pHu to the sedentary rabbits but, according to Gondret et al. (2009), the former ones improved their oxidative energy metabolism and had a greater proportion of succinate dehydrogenase-positive myofibers rich in mitochondria and myoglobine, and, accordingly, a greater redness in BF. Also regardless of the stunning method, the pHu obtained is relatively high in this experiment, with mean values of 5.97 for the LD and 6.01–6.18 for BF. These pH values close to 6 are not infrequent in rabbit meat but they are high if compared to the results obtained in other studies (Dalle Zotte, Rizzi, & Riovanto, 2008; Hernández, Ariño, Grimal, & Blasco, 2006; Pla, Zomeño, & Hernández, 2008). These values could be caused firstly by long distance transportation, as the rabbits used in the experiment were carried in a lorry for at least 3 h and had to wait over 2 h in the abattoir, which could cause stress in animals and high ultimate pH in meat (Dal Bosco et al., 1997; Ouhayoun, 1988). Secondly, the rabbits had to rest before slaughter (this has not been properly estimated but, in any case, it was over 2 h) which, according to a study on chicken by Khan and Nakamura (1970), might reduce the rate of glycolysis after sacrifice and result in high pH 24 h. Finally, in our study the time between the end of the dressing process and cooling was about 15 min, during which the carcasses were at a temperature of 21 °C; they were later placed in a cold storage room at 0–4 °C (65% humidity), so the carcass cooling started soon after slaughter. This could bring about a halt in the consumption of the energy reserves of the muscle (Ouhayoun, Daudin, & Raynal, 1990) as low temperatures act on myosin ATPasa and actomyosin ATPasa activity (Ouhayoun, Delmas, Monin, & Roubiscoul, 1990) and post mortem changes in the muscle are slower with low temperatures (−1 °C) (Honikel, Roncalés, & Hamm, 1983). The WHC was low according to previous studies in which slaughter was carried out after electrical stunning (13–14%, Carrilho et al., 2009), or electrical and halal (13.7 and 12.8%; López et al., 2008) and unlike after cervical dislocation, when it was higher (18–19%; Lite, 1989). Evidently, even when using the same technique in the lab, the differences cannot be attributed only to the stunning method, as, for instance, the muscles were also different: Biceps femoris in the first two studies, Longissimus dorsi in Lite (1989) and in the group Biceps femoris, Semitendinosus, Semimembranosus, Abductor cruris, Gluteus and Tensor fasciae latae in our experiment. Ouhayoun (1988) reports that WHC in the muscle Abductor cruris (an oxidative muscle) in rabbits improves if electrical stunning is applied instead of mechanical stunning, whereas in the Longissimus dorsi the effect is not relevant. In the present study, the effect of stunning was not significant in the WHC of the HL muscles, nor in the cooking losses in the LD nor in the shear force texture parameter on the LD, which confirms Dal Bosco et al. (1997) when they studied the effect of electrical and mechanical stunning on these quality parameters of rabbit meat. In chickens, Papinaho and Fletcher (1995) obtain less cooking losses when they were electrically stunned, if compared with no stunning, but the differences are small according to the authors. Texture results in chickens are contradictory. In a first experiment on poultry with an Allo-Kramer blade, Papinaho and Fletcher (1995) reported higher toughness with high pre-slaughter electric intensities than with low intensities or no stunning. Nevertheless, the same authors in 1996 and McNeal and Fletcher (2003) could not find differences between the several electrical systems and no stunning. The Allo-Kramer cutting values in turkey breast were not affected by the stunning methods studied by Goodwin, Mickelberry, and Stadelman (1961), based on Nembutal, brain removing, electrical blade, CO2, reserpine or no treatment. Also with Allo-Kramer, Lee et al. (1979) obtained tougher meat in broilers after pre-cooling tank when chickens were electrically stunned rather than when they were sacrificed unstunned. After being stored at 2 °C for 24 h, the former were tenderer. Contreras and Beraquet (2001) obtained similar results using Warner–Bratzler shears in broilers. According to the first authors, this would mean that electrical stunning causes tenderness in chicken meat after some storing time (4 h), which we could not observe in our experiment. Our WB shear force values support those found in other studies (Carrilho et al., 2009; Hernández & Dalle Zotte, 2010) As sensory analysis is concerned, and considering that the panellists did not find the effect of electrical vs. mechanical stunning significant, in our study we observed that sensory tenderness and juiciness were positively correlated, as it is often observed in young rabbits (Hernández, Pla, Oliver, & Blasco, 2000; Jehl & Juin, 1999), although this relation is not so clear in older rabbits (Gondret, Juin, Mourot, & Bonneau, 1998). Both variables were scored differently between treatments, being T-1 the least valued for both qualities. There is no reason to think of any meat deterioration in this group that could account for the lower score: the pre-slaughter treatment was similar to that in other groups and this group did not stand out when considering their desensitization pattern compared to other combinations of frequency and intensity (María et al., 2001). We should point out that, although there was no effect of the stunning procedure on WHC or on shear force values, this T-1 group showed high pHu, which was significantly correlated to these traits in the total T1–4. Therefore, we can infer that some differences which were not detected in the instrumental evaluation might appear when compared sample to sample in the sensory analysis. These results of our study are especially important, as treatment T-1 is the one applied in this commercial abattoir on a regular basis. According to the above said, this electric combination must be changed if we want to offer better meat quality to consumers. Also our results indicate that as the meat from electric stunning has a high pHu level, it is prone to have more conservation problems, although, as a positive thing, the rabbit meat is quickly commercialized and it is consumed before 24–48 h post mortem. Moreover, low values in tenderness and juiciness do not affect the panellists' global appraisal, but, if both sensory features improved, the overall appraisal would also be better due to the positive correlation between them. Finally, working procedures in abattoirs should be revised to correct anything necessary in order to obtain the best possible sensory qualities of the product for the market. In our case both T-2 and T-3 treatments were similar from a welfare point of view (María et al., 2001), but pHu in T-2 is near to the lower pH values of this experiment; therefore, it could be a more adequate treatment. More studies would be necessary to take decisions about the best electrical combinations in the abattoirs and, even, to know which stunning method is the best to obtain a high meat quality. Simple protocols designed to compare the routine procedure with respect to some alternative methods, taking into account a taste panel and using pHu of LD or, better, of BF or L* as moderate predictors of the physicochemical traits, could be useful to improve meat quality. 5. Conclusions This study has shown that electrical stunning accelerates the early muscular acidification, can provide high pHu values on some muscles of rabbits and can be linked to higher redness, but not to low exsanguination levels. Extreme voltage and frequency could be preferable to R. Lafuente, M. López / Meat Science 98 (2014) 247–254 medium voltage and frequency levels with the aim of improving sensory qualities, such as tenderness and juiciness. 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