The Influence of Pre-Mortem Conditions on Pale, Soft and Exudative (PSE) and Dark, Firm and Dry (DFD) Pork Meat

Abstract The aim of this study was to determine the effect of gender, stocking density in the transport vehicle, lairage time and season on the incidence of skin lesions on pig carcasses and PSE and DFD meat. Skin lesions on carcasses (480, in total) were assessed according to the Welfare Quality® protocol (2009). The pH and temperature measurements were performed 45 minutes after slaughter. The group of carcasses with skin lesions score 2 had significantly higher incidence of PSE and DFD meat compared to the groups of carcasses with skin lesions score 0 and 1. With regard to gender, there were no differences in meat quality parameters, as well as for the incidence of skin lesions and PSE and DFD meat. The results showed that a space allowance lower than 0.3 m2/100 kg and higher than 0.5 m2/100 kg pig had a detrimental effect to animal welfare and meat quality. Lairage time affected meat quality parameters, incidence of skin lesions score and PSE and DFD meat, where after long lairaging (> 17 h) mean pH45 and t45 values became significantly higher, as well as the incidence of skin lesions and DFD meat. After short lairaging (< 1 h) mean pH45 value became significantly lower, while mean t45 value and the incidence of PSE meat became significantly higher. A significantly higher number of skin lesions on the carcass were observed in winter compared to all other seasons. High temperatures during summer and low temperatures during winter had a negative influence on meat quality parameters and incidence of PSE and DFD meat.


INTRODUCTION
Skin lesions on pig carcasses represent a severe welfare problem and have a negative impact on pork quality and carcass grading. It has been reported that skin lesions can be a very useful predictor of pork quality [1]. Nielsen et al. [2] found that the primary infl uence of skin lesions is on meat quality rather than animal welfare. Skin lesions can cause severe economic losses to the meat industry [1]. Furthermore, skin lesions refl ect the quality of the animal's physical and social environment. Consequently, the number of skin lesions on the carcasses could provide a valuable database, regarding the management of the animals in the farm of origin, transport or in the lairage pens [3]. Guàrdia et al. [1] reported the association between carcass skin lesions and the risk of obtaining PSE and DFD meat.
Pale, soft and exudative (PSE) and dark, fi rm and dry (DFD) meat are the two main defects with regard to meat quality in pigs [4]. The fi rst occurs after exposing animals to acute stress just prior to slaughter, when the pigs still have suffi cient reserves of muscle glycogen to cause a rapid increase in lactic acid content early post-mortem. This brings the muscle pH to values below 6.0 prior to the fi rst hour after slaughter [5]. On the other hand, high pH values appear when animals suffer chronic stress and the muscle glycogen is used up rapidly during the pre-slaughter period and after slaughter, there is little lactic acid production which results in DFD meat [6].
Several practices, prior to slaughter, are associated with the occurrence of skin lesions on pig carcass, PSE and DFD meat, such as, facility design, handling during loading or unloading of pigs on or from the transport vehicle, fi ghting between pigs after mixing unfamiliar animals, the fl oor type in the transport vehicle, the stocking density and time of transportation and lairaging, the duration of total fasting time before slaughter and season [5,7,8,4,9]. Guàrdia et al. [10] found that high stocking density during transport represents a risk for occurring of PSE pork meat. As opposed to this, lowering the stocking density from 0.37 to 0.50 m 2 per 100 kg pig during transport increases the incidence of DFD pork and skin lesions [5]. Based on the previously published scientifi c literature [11,12], lairaging shorter than one hour poses a risk for developing of PSE pork, conversely lairaging longer than three hours increases the incidence of DFD pork and skin lesions on the carcasses. Season, i.e. environmental temperatures may have a negative infl uence on welfare of pigs and pork quality, where heat stress results in a higher incidence of PSE pork, while cold stress leading to higher incidence of DFD pork and skin damages [13].
The aim of this study was to determine the effect of gender, stocking density in the transport vehicle, lairage time and season on the incidence of skin lesions on pig carcasses and PSE and DFD meat.

MATERIALS AND METHODS
The study was carried out during autumn of 2014 (October and November), winter (February and March), spring (April and May) and summer (July and September) of 2015 on 30 fatteners per sampling day (480 in total, 295 barrows and 185 gilts, crossbreeds Yorkshire x Landrace, with average live weight of approximately 120 kg and 6 months old). All pigs were fattened on the same farm of origin under identical conditions. After loading, the pigs were transported for about 1-2 hours to the slaughter facility, at a mean speed of 60 km/h. The same transport vehicle and the same driver, but different stocking densities (ranged from 0.27 to 0.53 m 2 /100 kg pig) were used. Depending on the dinamics of slaughter, after unloading, pigs (n=300) were held in a lairage less than one hour -short lairaging (on average 44.60±8.24 minutes) or more than 17 hours (n=180) -long lairaging (20.00±2.37 hours), when stayed overnight and slaughtered in the morning. During lairaging stocking density in the lairage pen was 0.65 m 2 per pig. The ambient temperature during transport and lairaging varied: in autumn from 2 to 11 °C (on average 6.14±2.91°C), in winter from 1 to 3 °C (on average 2±1 °C), in spring from 16 to 20 °C (on average 16.67±3.06 °C) and in summer from 25 to 33 °C (on average 29.22±4.04 °C). The pigs were slaughtered at the same slaughterhouse with a throughput of 13,000 pigs a year and a slaughter rate of approximately 35 pigs per day. Head-only electrical stunning was used with a pair of stunning tongs. In the fi rst 15 seconds after stunning, the animals were shackled by one hind leg and exsanguinated. Following bleeding, the carcasses were processed using conventional industry practice.

Skin lesions score
Skin lesions on the left side of the pig's carcasses were visually assessed 45 minutes postmortem according to the Welfare Quality® protocol [14]. The carcass was divided into fi ve sections: 1) ears; 2) front (from the head to the back of the shoulder); 3) middle (from the back of the shoulder to the hind-quarters); 4) hindquarters and 5) legs (from the accessory digit upwards). Each part of the carcass was scored using a three-point scale: 0) no visible skin damage, only one lesion greater than 2 cm or lesions smaller than 1 cm; 1) between two and 10 lesions greater than 2 cm and 2) any wound penetrated into muscle tissue, or more than 10 lesions greater than 2 cm. The scoring of the fi ve parts of the carcass was combined in one scoring as follows: 0) all body parts with a score of zero; 1) at least one body part with a score of one; and 2) at least one body part with a score of two.

Meat quality
The pH (pH 45 ) and temperature (t 45 ) measurements were performed 45 minutes after slaughter on carcasses using the pH-meter "Testo 205" (Testo AG, Lenzkirch, Germany). Both measurements were done on the Longissimus dorsi muscle, pars lumbalis (central area of the loin). The arithmetic mean of the two consecutive pH/ temperature values measured at the same point was taken as the fi nal result. The carcasses showing pH 45 values lower than 6.0 were classifi ed as PSE meat, whilst the carcasses showing pH 45 values higher than 6.4 were classifi ed as DFD meat [4]. The carcasses with pH 45 between 6.0 and 6.4 were classifi ed as normal pork quality.

Statistical analysis
Statistical analysis of the results was conducted using software GraphPad Prism version 6.00 for Windows (GraphPad Software, San Diego California USA, www. graphpad.com). Pig carcasses were classifi ed in two groups according to the intensity of carcass skin lesions to determine the effect of skin lesions, gender, stocking density in the transport vehicle, lairage time and season on meat quality parameters (pH 45 and t 45 ) and the incidence of PSE and DFD meat: low skin lesions score group -group of carcasses with the score 0 and 1 and high skin lesions score group -group of carcasses with the score 2. The pH and temperature values of two groups of carcasses were presented by descriptive statistical parameters (mean value and standard error). Student t-test was used to examine the differences in the pH and temperature values between the two groups. Two-way ANOVA with Tukey's multiple comparison test was performed to test the effect of gender (gilts and barrows), stocking density (high -< 0.3 m 2 /100 kg pig, medium -0.3 -0.5 m 2 /100 kg pig and low -> 0.5 m 2 /100 kg pig), lairage time (long > 17 h and short < 1 h) and season (autumn, winter, spring and summer) in relation to the skin lesions score and their interaction on meat quality parameters. According to gender (gilts and barrows) and intensity of carcass skin lesions (high and low skin lesions score) pigs were distributed in 2 x 2 factorial design. Pigs were assigned to one of the six treatments arranged in a 3 x 2 factorial design according to stocking densities (high, medium and low) and intensity of carcass skin lesions (high and low skin lesions score). Furthermore, according to lairage time (long and short) and intensity of carcass skin lesions (high and low skin lesions score) pigs were distributed in a 2 x 2 factorial design. In addition, pigs were assigned to one of the eight groups arranged in a 4 x 2 factorial design according to season (autumn, winter, spring and summer) and intensity of carcass skin lesions (high and low skin lesions score). The differences among genders, stocking densities, lairage times and seasons in relation to the skin lesions score were determined by Chi-square test. The same test was used to detect the differences among genders, stocking densities, lairage times and seasons in incidence of meat quality classes in relation to the skin lesions score. In all cases, signifi cance was fi xed at level P<0.05.
Classifi cation of examined pig carcasses based on gender, stocking density in the transport vehicle, lairage time and season in relation to the skin lesions score is given in Table 1. No signifi cant difference was found between the barrows and gilts for the percentage of carcasses with skin lesions score 1 and 2 (P>0.05). The group of pigs transported in low stocking density had signifi cantly higher incidence of skin lesions compared to the groups of pigs transported at medium and high stocking density (P<0.05). Skin lesions score was signifi cantly higher in the group of pigs after long lairaging (P<0.05) compared to the group of pigs after short lairaging. The highest skin lesions score was found in the group of pigs slaughtered during winter (P<0.05), while the lowest skin lesions score was observed in the group of pigs slaughtered during spring (P<0.05).   The incidences of PSE and DFD meat in relation to the carcass skin lesions are shown in Figure 2. Pig carcasses with skin lesions score 2 had a signifi cantly higher percentage of PSE and DFD meat compared to pig carcasses with skin lesions score 1 and 0 (P<0.05). The mean values of pH 45 and t 45 between the two groups of carcasses divided according to intensity of skin lesions are given in Table 2. The group of the carcasses with high skin lesions score had signifi cantly higher mean t 45 value compared to the group of the carcasses with low skin lesions score (P<0.05). In contrast, no signifi cant difference was found among two groups for the mean pH 45 value (P>0.05). Low skin lesions score -skin lesions score 0 and 1, High skin lesions score -skin lesions score 2; a -within a row the same letters indicate a signifi cant difference between groups at P < 0.05.
The effect of skin lesions score and gender and their interaction on meat quality parameters and the incidence of meat quality classes are presented in Table 3. The pH 45 values, t 45 values and the incidence of PSE and DFD meat were signifi cantly infl uenced by the skin lesions score (P<0.05). Gender had no signifi cant infl uence on meat quality parameters (P>0.05). No signifi cant interaction effect between skin lesions score and gender was found for meat quality parameters (P>0.05).
In Table 4 are represented the effects of skin lesions score and stocking density and their interaction on meat quality parameters and the incidence of meat quality classes. Skin lesions had no signifi cant infl uence on the pH 45 value, whilst signifi cant infl uence of skin lesions was found on the t 45 value. The pH 45 and t 45 values were signifi cantly infl uenced by stocking density (P<0.05). A signifi cant interaction effect between skin lesions score and stocking density was found for meat quality parameters, as well as for the incidence of PSE and DFD meat (P<0.05). Low skin lesions score -skin lesions score 0 and 1, High skin lesions score -skin lesions score 2; SLC -skin lesions score factor, G -gender factor, SLC x G -interaction between skin lesions score and gender factor; * -P<0.05; ns -no signifi cance (P>0.05); a,b,c,d -within a row the same letters indicate a signifi cant difference between groups at P<0.05. Low skin lesions score -skin lesions score 0 and 1, High skin lesions score -skin lesions score 2; Stocking density: High -< 0.3m 2 /100 kg pig; Medium -0.3 -0.5 m 2 /100 kg pig; Low -> 0.5 m 2 /100 kg pig. SLC -skin lesions score factor, SD -stocking density factor, SLC x SD -interaction between skin lesions score and stocking density factor; * -P<0.05; ns -no signifi cance (P>0.05); a,b,c,d,e,f,g,h,iwithin a row the same letters indicate a signifi cant difference between groups at P<0.05.
The effect of skin lesions score and lairage time and their interaction on meat quality parameters and the incidence of meat quality classes are shown in Table 5. The pH 45 and t 45 values were signifi cantly infl uenced by the skin lesions score (P<0.05). Lairage time had a signifi cant infl uence on pH 45 value, while no signifi cant infl uence of this factor was found on t 45 value. A signifi cant interaction effect between skin lesions score and lairage time was found for meat quality parameters and incidence of PSE and DFD meat (P<0.05).    The effect of skin lesions score and season and their interaction on meat quality parameters and the incidence of meat quality classes are given in Table 6. The pH 45 value was not signifi cantly infl uenced by the skin lesions score (P>0.05). Contrarily, skin lesions score had a signifi cant infl uence on the t 45 value (P<0.05). Season had a signifi cant infl uence on pH 45 and t 45 values. A signifi cant interaction effect between skin lesions score and season was found for the pH 45 value and the incidence of PSE and DFD meat (P<0.05), but not for the t 45 value (P>0.05).

DISCUSSION
The percentage of pig carcasses with different skin lesions score (score 0 -39.38%, score 1 -28.96%, score 2 -31.66%), found in this study, is comparable to Dalmau et al. [3] results, who examined skin lesions according to the Welfare Quality® protocol [14] in 10 Spanish pi g slaughterhouses based on a four point scale. Their study showed that the percentage of pig carcasses with different skin lesions scores varied from 58.3% to 85% for score 1, from 5% to 30% for score 2 and from 1.7% to 10% for score 3. Guàrdia et al. [1] assessed skin lesions on pig carcasses based on a fi ve point scale. They reported that 72.4% of examined pig carcasses were scored as seriously damaged, 16.6% as slightly damaged and only 11% carcasses were found to be without any evidence of damage.
In this study, pig carcasses with skin lesions score 2 had signifi cantly higher percentage of PSE (30.92%) and DFD (28.95%) meat compared to pig carcasses with skin lesions score 1 (PSE -9.35%, DFD -10.07%) and 0 (PSE -4.23%, DFD -5.29%) (P<0.05) ( Figure 2). This indicates that the carcasses with higher skin lesions score are more prone to PSE and DFD meat. It has been found that the risk of obtaining PSE meat is doubled in carcasses with skin lesions as compared to carcasses without skin lesions (43.7% vs. 28.8%) [1]. The presence of skin lesions on the carcass was also associated with an almost four times higher risk of DFD pork (11.70% vs. 3.30%) [1]. Data showed that carcasses being scored as very slight or without skin lesions (score 0) and slightly damaged (score 1) belong to the same group, because no difference was found among these two classes for the risk of PSE and DFD meat [1]. This was also confi rmed in the present study, where no signifi cant difference was detected among carcasses with skin lesions score 1 and 0 (PSE -9.35% vs. 4.23%, DFD -10.07% vs. 5.29%) (P>0.05) (Figure 2).
The group of the carcasses with high skin lesions score had signifi cantly higher mean t 45 value compared to the group of carcasses with low skin lesions score (P<0.05) ( Table 2). Contrary to the above-mentioned fi ndings of meat quality classes according to the skin lesions score, the group of pig carcasses with low skin lesions score had lower mean pH 45 compared to the group of pig carcasses with high skin lesions score, but not signifi cantly (P>0.05) ( Table 2). Pre-slaughter procedures, imposed during transportation and lairaging at the slaughterhouse, are the most important factors infl uencing the pH of the carcass [15,16]. Therefore, if the severity of skin damages arises from aggressive activity at an early time, higher pH values and consequently increased incidence of DFD might be expected with increasing severity of skin lesions [1]. On the other hand, if fi ghting occurs just prior to slaughter, then skin lesions cause the rapid decrease of the pH value, which results to an increased occurrence of PSE meat [1].
In the present trial, similarly to the fi ndings of D'Eath et al. [17], signifi cant differences were not found between barrows and gilts for the percentage of carcasses with high score of skin lesions (34.92 vs. 26.48%, P>0.05) ( Table 1). In addition, signifi cant differences were not found between barrows and gilts for meat quality parameters (P>0.05), as well as for the incidence of PSE and DFD meat (P>0.05) ( Table 3). The same was observed by Alonso et al. [18], who did not fi nd any differences among barrows and gilts for meat quality parameters. This can be explained by the fact that boars are more aggressive than castrated male pigs or gilts and this behavior in entire males causes a high level of fi ghts among them, which lead to higher skin lesions score [4]. As well as skin damage, the carcasses from boars also tended to show a higher incidence of PSE and DFD meats, due to stress and physical activity associated with fi ghting [4].
As observed in this study, the group of pigs transported at low stocking density (42.66%) had a signifi cantly higher incidence of skin lesions compared to the groups of pigs transported at medium and high stocking density (17.50% and 31.91%, respectively) (P<0.05) ( Table 1). In addition, the interactive effects of excessive space allowance during transportation (>0.5 m 2 /100 kg) and higher skin lesions score signifi cantly increased mean pH 45 (P<0.05), mean t 45 (P<0.05) and the incidence of DFD meat (P<0.05) ( Table 4). The negative impact of excessive space allowance during transportation to slaughterhouse could be explained by the fact that pigs can be thrown around and get struck and bruised as a result of unexpected movements of the transport vehicle [19]. Likewise, if pigs can move around, they may be involved in fi ghts and this can cause muscular fatigue and glycogen breakdown which contributes to the increased incidence of DFD meat [5]. Contrarily, the interactive effects of low space allowance during transportation (<0.3 m 2 /100 kg pig) and higher skin lesions score signifi cantly decreased mean pH 45 (P<0.05) and signifi cantly increased mean t 45 (P<0.05), as well as the incidence of PSE meat (P<0.05) ( Table 4). The pigs transported in high stocking density have reduced space allowance which leads them to fi ght with each other for resting place, resulting in a higher incidence of skin lesions, and, therefore, produce lower meat quality [1]. The results obtained in our study, showed the negative effect of insuffi cient (< 0.3 m 2 /100 kg pig) and excessive (> 0.5 m 2 /100 kg) space allowance on the pH 45 and t 45 values, and, consequently, on meat quality. When the space availability was between 0.3 m 2 /100 kg pig and 0.5 m 2 /100 kg, the results indicated signifi cantly lower risks of production of PSE and DFD meat. This suggests that the pigs transported in the high and low stocking density were under greater stress and, therefore, produce inferior meat quality. The EC Directive on the protection of animals during transportation emphasizes that each pig must at least be able to stand or lie down in its natural position [10]. This could be achieved with a space allowance of around 235 kg/m 2 or 0.425 m 2 per 100 kg pig (as recommended by the EC Directive 95/29/EC and the Council Regulation (EC) No.1/ 2005 on the protection of animals during transport), as a suitable compromise among transport economy, meat quality and animal welfare [10,5].
The data observed in this study showed that incidence of skin lesions increases with longer lairage time, with the risk of skin blemish occurrence being almost twofold higher at 17 h compared to 1 h lairaging (40.00% vs. 26.67%) ( Table 1), which is in line with previous studies [20,11,12,1]. Furthermore, we observed that the interactive effects of increased lairage time (>17 h) and higher skin lesions score signifi cantly increased mean pH 45 (P<0.05), mean t 45 (P<0.05) and the incidence of DFD meat (P<0.05) ( Table 5). A long lairaging improves the colour of the pork, reduces the incidence of PSE meat and allows the pigs to rest, but at the same time stimulates aggression and fi ghting between animals and as a consequence, increases skin lesions and the risk of DFD pork [11,12,21]. It was also found that the interactive effects of decreased lairage time (<1 h) and higher skin lesions score signifi cantly decreased mean pH 45 (P<0.05), signifi cantly increased mean t 45 (P<0.05) and the incidence of PSE meat (P<0.05) ( Table 5). The main cause for the occurrence of PSE meat is acute stress just before slaughter [22]. It is generally accepted that the most stressful moments, prior to slaughter, are transport and lairaging [23]. It has been found that a short transportation and slaughter of pigs after a short stay in lairage (15-60 minutes), does not allow pigs to recover from loading stress and to acclimate to transport stress [24]. Slaughter of pigs, immediately after unloading or after a short stay in lairage (15-60 minutes), is not recommended, because animals are exhausted and agitated. This results in an increase in muscle temperature (+1°C) and lactic acid just prior to slaughter, which contributes to the increased incidence of PSE meat [25,12,26]. The main purpose of lairage is to allow pigs to recover from the stress caused by transportation and unloading to the slaughterhouse, and it is expected to improve the meat quality [1]. From an animal welfare and meat quality perspective, it is considered that the optimal lairage time is 2-4 h [20,24].
The risk of incidence of skin lesions, according to the season, was almost twofold higher in the winter (51.11%) than in autumn and summer (30.48% and 33.34%, respectively) (P<0.05), i.e. fourfold higher than in spring (13.33%) (P<0.05) ( Table 1). This is in concordance with Gosálvez et al. [27] results, who found higher skin lesions score in the winter, which led them to conclusion that low temperatures, acting as a grouping stimulator, increase fi ghting behavior. According to the results obtained in the present study, the interactive effects of low temperatures during winter and higher skin lesions score signifi cantly increased mean pH 45 (P<0.05) and the incidence of DFD meat (P<0.05) ( Table 6). Over colder months, pigs tend to huddle in order to reduce the heat loss and to create a microclimate that increases the surrounding temperature at the expense of energetic reserves in the muscle, which contributes to the increased incidence of DFD meat [5]. About 80% of the transports during winter, were carried out at temperatures lower than 15 °C, which is not inside the neutral thermal range for pigs (15-25 °C) [13]. Opposite to this, the interactive effects of high temperatures during summer and higher skin lesions score signifi cantly decreased mean pH 45 (P<0.05) and signifi cantly increased the incidence of PSE meat (P<0.05) ( Table  6). Guàrdia et al. [10] found almost twofold higher risk of the PSE condition in summer than in winter (6.5% vs. 3.4%). Their study confi rmed that pigs are very sensitive to high temperatures, because they have ineffective sweat glands that affect an animal's ability to dissipate body heat after being exposed to hot weather conditions [10].

CONCLUSION
The group of carcasses with skin lesions score 2 had a signifi cantly higher incidence of PSE and DFD meat compared to the groups of carcasses with skin lesions score 0 and 1. With regard to gender, there were no differences in meat quality parameters, as well as for the incidence of skin lesions and PSE and DFD meat. The results showed that a space allowance lower than 0.3 m 2 /100 kg pig and higher than 0.5 m 2 /100 kg had detrimental effect to animal welfare (signifi cantly higher skin lesions score) and meat quality (signifi cantly lower pH 45 value and signifi cantly higher incidence of PSE meat for stocking density < 0.3 m 2 /100 kg pig, while signifi cantly higher pH 45 value and incidence of DFD meat for stocking density > 0.5 m 2 /100 kg). Lairage time affected meat quality parameters and the incidence of skin lesions score and PSE and DFD meat, where after long lairaging (> 17 h) mean pH 45 and t 45 values became signifi cantly higher, as well as the incidence of skin lesions and DFD meat. On the other hand, after short lairaging (< 1 h) mean pH 45 value became signifi cantly lower, while mean t 45 value and the incidence of PSE meat became signifi cantly higher. A signifi cantly higher number of skin lesions on the carcass were observed in winter compared to the all other seasons. In addition, high and low temperatures had a negative infl uence on meat quality parameters and the incidence of PSE and DFD meat.
or revising it critically for important intellectual content. All authors have read and approved the fi nal manuscript.

Declaration of confl icting interests
The author(s) declared no potential confl icts of interest with respect to the research, authorship, and/or publication of this article.