**5. Animal Welfare Aspects**

As previously reported, slaughterhouses are becoming bigger and the distance between farms and slaughterhouses is, in some cases, very large.

European law sets out several compulsory requirements on the transfer of poultry to slaughterhouses: density in the crates; allowing drinking and feeding if more than 12 h are needed to reach the abattoir; limits to faecal matter falling from animals in upper layers to the underlying crates; and temperature and ventilation in the trucks during transport. These rules mainly aimed at fast-growing broilers produced on intensive farms. However, very little data are available with respect to transport of birds from free-range systems [11]. It is expected that the more active animals used in PPP systems will respond to this transport stress differently to fast-growing chickens.

Poultry transport to the slaughterhouse is one of the critical factors affecting animal welfare, quality and meat hygiene. These different stressing situations can reduce bird welfare and increases the risk of body injuries (broken wings/legs and overall distress) and mortality [36]. Chickens are caught and placed in crates to reach the slaughterhouse and during the transport they have no feed and water, are exposed to environmental changes (i.e., movement, noise, vibration), subject to even extreme conditions of temperature and humidity, forced to counteract the track movement [37].

Many studies have focused on the effects of transport stress on different blood traits [38,39]. Zhang et al. [40] reported that transportation of broilers caused decrease in glycogen in breast and thigh muscles. In addition, transport stress is associated with enhanced skeletal muscle energy metabolism, causing mitochondrial superoxide production, acceleration of lipid peroxidation and the induction of cellular damage [41]. In chickens, stress and kinetic activity before slaughter are also involved in pH variations during the early stages of rigor [42], whereas the final pH of meat mainly depends on the glycogen content at the time of slaughter [43].

Thus, time spent in transit to the slaughterhouse is a major concern in terms of welfare and meat quality. The effect of transport duration on animal welfare and the resulting meat quality in broilers have been well researched but data on the interaction between genetic strain and transport duration are sparse. It has been reported that the effect of stress could be different in fast- and slow-growing poultry strains [44].

Fast-growing strains tend to produce meat with a slower pH decline, higher ultimate pH and, consequently, greater water-holding capacity [45]. On the other hand, Berri et al. (2007) [46] reported that slow-growing strains suffer more from the lag-phase between catching and slaughter due to their high kinetic activity (i.e., wing flapping) during transport and slaughter. Accordingly, when broilers are subjected to stressful conditions, 'have been well researched could be different from that in standard broilers.

Our previous results [47] sugges<sup>t</sup> that a 4-h journey to the slaughterhouse, compared to immediate slaughter in a MPPU, negatively affects some animal welfare traits (tonic immobility, creatine kinase, heterophil/lymphocyte ratio, lysozymes, reactive oxygen species, glucose and haptoglobin) in free-range chickens. The slow-growing chickens showed the highest susceptibility to stress, even with a greater antioxidant defence due to their foraging behaviour. Accordingly, a less stressful slaughtering procedure should be developed for all chicken strains with shorter resting times in the farm, transport and animal storage at the slaughterhouse. This is particularly important for PPP in order to sustain the high welfare standard achieved during life and to maintain meat quality.

#### **6. Qualitative and Sanitary Implications**

The introduction of a MPPU could have an impact on the quality and hygiene/safety traits of the meat based on three main paradigms: reduction of pre-slaughter stress, transport procedures and proper implementation of the slaughter process (i.e., well-managed small-scale facilities, small number of animals of the same flock slaughtered per day).

Currently, it is understood that that the reduction of pre-slaughtering stress, especially catching, crating and transport, could affect meat traits. The increased level of epinephrine and glucocorticoids in animals exposed to *ante-mortem* stresses can affect *post-mortem* metabolism and, therefore, meat quality [48]. Pre-slaughtering stress, in particular due to transport, may increase muscle glycogenolysis resulting in glycogen decrease in both breast and thigh muscle [40]. Furthermore, acceleration of lipid peroxidation and induction of muscular cellular damage have been reported after stressful transport, associated with enhanced skeletal muscle energy metabolism and mitochondrial superoxide production [41].

Despite there is not a general consensus, these stressful events could therefore affect conversion of muscle to meat and the related protein functionality, following a reduced consumer acceptability and processing functionality of the meat caused by the changes in the water holding capacity, colour, tenderness, texture and shelf-life of meat and derived products [49]. Thigh meat have been reported to be affected more than breast meat by this phenomenon [50].

As previously reported, studies on the effects of pre-slaughtering practices on meat quality have mainly been conducted in fast-growing broilers, where muscle abnormalities (PSE—Pale, Soft and Exudative and DFD—Dark Firm and Dry condition) were also recorded but when slow-growing strains were considered, they seemed more subjected to stress than fast-growing genotypes due to high kinetic activity during catching, transport and wing-flapping during slaughter [43]. Castellini et al. [51] evaluated the effect of transport duration (0 h vs. 4 h) and chicken genotype (fast- vs. slow-growing strains) reared under free-range conditions. They observed that transport affected the fatty acid profile of breast and drumstick muscle, with a decrease in polyunsaturated fatty acids and antioxidant content (α-tocotrienol, α, δ-tocopherol and carotenoids) and an increase in TBARS (Thiobarbituric acid reactive substances) in breast meat (Figure 4). The decrease in γ-tocopherol, retinol and TBARS was more relevant in birds that were more active, probably due to the higher kinetic activity and the higher peroxidability of their meat. Furthermore, in this study, the breast muscles from 4 h-transported chickens showed significantly less lightness, and also meat tenderness (shear forcevalue) was affected by genotype and transport: meat from slow-growing birds was tougher, whereas after transport, in both genotypes, higher tenderness was observed. Nevertheless, neither PSE nor DFD were recorded.

**Figure 4.** Variation (% with respect to no transport) of antioxidants (α-tocotrienol, α-, γ-, δ-tocopherol, retinol and carotenoids) and TBARS in fast- and slow-growing chicken strains after 4 h of transport (modified by [51]).

A PPP system together with a MPPU, when slow-growing strains are used and reduction in the number of chickens to be caught and slaughtered, combined with the absence of transport limits the time spent struggling in crates and, therefore, improves/preserves meat quality.

From a hygiene point of view, there is a large consensus that pre-slaughter stress increases the spread of infectious diseases [37]. The stress that birds experience during pre-slaughter procedures can enhance colonisation by *Campylobacter* spp. [52] and its spread throughout the flock [53].

Previous thinning of the flocks was considered as a major risk factor for contamination of chicken carcasses with *Campylobacter* spp. at the slaughterhouse and catching of the birds for crating further increases *Campylobacter* spp. contamination [54].

In addition, transport vehicles and crates can be considered to be a source of *Campylobacter* contamination [55]. *Campylobacter* from the processing plant can survive on crates for a period sufficient to contaminate the majority of farms in the catchment (it survives for at least 3 h after sanitisation) [56] and poses a contamination risk for uninfected birds belonging to other unrelated flocks [57,58]. Reduction in the time that animals spend in the crates and limiting slaughter to a small number of animals per day that could be caught without prolonged struggling, as well as the absence of transport, could improve the hygiene level of the carcasses.

With regard to *Salmonella* spp., environmental stress could weaken the immune response of birds, with an increase in number of pathogens on the crates [59]. For this reason, reduction in the handling procedures and the absence of transport, as with a MPPU, could strongly influence the prevalence of pathogens at the slaughterhouse. GMP (Good Manufacturing Practices) or guidelines on operator behaviour during pre-slaughter steps could be useful for informing producers about correct handling and crating procedures (with regards to timing, animal density and welfare) to be adopted in MPPU.

Nonetheless, it is reported that the older age of the animals at slaughter, generally adopted in PPP, increased the contamination of caeca by *Campylobacter* spp. [60,61]. Furthermore, when the prevalence of infected animals in the flock is high (i.e., slow-growing genotype with a relatively longer period of rearing), no reduction in *Campylobacter* spp., even without transport, were observed [47].

The same consideration was not so for *Salmonella* spp., as different authors reported no shedding animals and no positive carcasses in PPP systems and MPPUs, respectively [47,62].

With regard to the slaughter practices in a MPPU, all the procedures are carried out on a manual basis instead of using industrial-scale, automated commercial processing lines [62]. Furthermore, differences in the structures and equipment adopted, as well as in the procedure implemented may strongly affect the hygiene level of the carcasses. For example, in Europe the decontamination strategies could not be used and the limited space available in the truck reduce the possibility of using water-bath chilling with chlorinated water.

Reports on the effect of a MPPU on sanitary traits in poultry meat are scarce [62,63]. It seems likely that the slaughter of a single homogeneous batch of chickens from the same flock during one-day operations could reduce the cross-contamination reported when animals come from different batches and flocks to the same slaughterhouse [54] and, therefore, a daily slaughter rotation of the flocks with a properly cleaned and disinfected MPPU is strongly suggested [64].

Other specific aspects on the possible contamination route inside a MPPU are dependent on structure and equipment. Scalding, defeathering, evisceration and chilling are considered to be the major routes of contamination by both *Salmonella* and *Campylobacter* spp. [65] and have to be carefully considered during HACCP implementation in a MPPU.

In particular, due to the limited space inside a MPPU, chilling could be carried out in two steps (pre-chilling and chilling) which could be performed within the MPPU and in the farm, respectively [66]. This could have the advantage of allowing the MPPU to be cleaned and disinfected immediately after slaughter, while chilling and storage of carcasses are performed in the farm.

The use of an air chiller could be more practical for a MPPU, even if counter-flow water-chilling and decontamination strategies, when allowed by national legislation, could be more effective in reducing carcass contamination [62,65]. High carcass density in the chiller could also be avoided to allow proper chilling of the meat and reduce cross-contamination between carcasses [54,67].

In the USA, a technical survey on *Salmonella* and *Campylobacter* showed that *Campylobacter* prevalence was significantly higher in MPPUs and this was partly due to wastewater and compost. In view of this, the processing of waste should be improved for optimum control of human pathogens.

In Europe, animal by-products must be disposed of as quickly as possible to avoid contamination of the meat for human consumption (Regulation EC 1069/2009), thus providing proper protection of the environment from food-borne pathogens.

The prototype of MPPU shown here was provided by a detailed HACCP manual, with a risk assessment based on hazard probability and severity at each step of the process, validated during the first three months of slaughter and after one year of activity. One of the operators of the MPPU should be responsible for the HACCP plan, including SSOPs. Cleaning and disinfection of the truck and equipment and assessment of the risk of carcass contamination due to scalding, defeathering and evisceration steps (GMPs) and carcass chilling, as the real CCP (Critical Control Point) able to prevent the growth of pathogens, also have to be taken into consideration.

The absence of *Salmonella* on the carcasses, as well as counts of *Campylobacter* spp., following the criteria lay down by EC Regulation 2073/2005, could be adopted in a MPPU as evidence of the hygiene level, as already performed in conventional industrial slaughterhouses. A reliable carcass sampling could be planned, according to EC Regulation 2073/2005, with 50 samples which should be derived from 10 consecutive sampling sessions

Place and day of slaughtering must be provided to the veterinarian officer to permit Official controls of MPPU.
