*3.2. Blood Parameters*

Table 2 shows the effects of sampling time, genetic type and their interaction on blood parameters of Apulo-Calabrese and crossbreeds.

Sampling time statistically influenced (*p* < 0.05) all blood parameters, except ALP and sodium whilst the effect of genetic type was significant (*p* < 0.05) for glucose, albumin/globulin ratio, urea, creatinine, AST and potassium. Of particular interest was the interaction between the genetic type and the sampling time since it lays emphasis on the possibility that variation of plasma components between basal and exsanguination is influenced by the genetic type. This interaction was statistically significant (*p* < 0.05) for lactate, albumin/globulin, urea, creatinine, AST, ALT, ALP, sodium, and potassium.

At exsanguination, significantly higher levels of lactate (*p* < 0.05) were found in both genetic types. Higher levels of lactate in the blood have been associated with physical stress [33]. The highest value of lactate in this study was found in Apulo-Calabrese which showed a lower concentration of basal lactate and were driven with less difficulty (minor duration) during loading, as shown in Table 1. According to Broom et al. [34], different breeds cope differently to the handling and transport procedures which could explain the higher levels of lactate found in Apulo-Calabrese in this experiment. Other welfare indicators of stress such as CK and cortisol did not differ between the two genetic types, which is in agreemen<sup>t</sup> with the results found by Lebret et al. [17] in the French local Basque and Large White pigs. The similar levels of basal cortisol found in Apulo-Calabrese and crossbreeds in this experiment contrasted with the result by Li et al. [16] who found higher levels of plasma cortisol in Erhualian with respect to Pietrain. Plasma glucose did not differ between both genetic types at exsanguination. However, slightly lower levels of glucose were found in Apulo-Calabrese compared with crossbreeds.

Significantly lower levels (*p* < 0.05) of albumin/globulin were found at exsanguination in Apulo-Calabrese than the crossbreeds, the values obtained were however within the normal physiological range for pigs [35]. With the exception of some globulins, plasma proteins are produced in the liver and are indicators of colloid osmotic pressure of the blood. Lower values of the concentrations may be due to a lack of dietary protein or hepatic damage [35], whilst higher values have been associated with dehydration due to the length of the journey [4].

Higher levels of urea and creatinine in the blood have been associated with food deprivation stress and an increase in physical activity as a result of the transport procedures [36]. In this experiment, significantly lower levels of creatinine (*p* < 0.05) were found in Apulo Calabrese at the basal condition when compared with the crossbreeds whereas serum urea did not differ between the two genetic types. At exsanguination Apulo-Calabrese showed significantly higher levels of urea and lower levels of creatinine when compared with crossbreeds. The values of urea and creatinine obtained in this study were within the normal physiological range for pigs and were in agreemen<sup>t</sup> with the results found by Dikic et al. [37] in local Turopolje breed and their crossbreeds [Turopolje × (CHypor × Swedish Landrace)].

**Table 2.** Effects of sampling time (T), genetic type (GT), and their interaction (T × GT) on least square means (L.S.M) and standard error of means (S.E.M.) of blood parameters of Apulo-Calabrese and crossbreed pigs on farm (baseline) and at exsanguination.


Means on the same row with different superscript letters (a, b, **c**) indicate significant effects (*p* < 0.05) of the interaction between sampling time (T) and genetic type (GT).

AST, ALT, and ALP are chemical indicators of tissue function: elevated levels of these enzymes occur when liver and pancreas are damaged. Significantly higher levels (*p* < 0.05) of AST were found in Apulo-Calabrese at exsanguination when compared with crossbreeds. It is interesting to note that the concentration of AST increased slightly in Apulo-Calabrese from T0 to T1, unlike in the crossbreeds which demonstrated a remarkable increase at slaughter compared with the values obtained at the basal level. According to Pugliese and Sirtori [21] local breeds are reared mostly in an extensive system where pigs forage for food in their surroundings. The elevated levels of AST found in Apulo-Calabrese at the basal condition could be a marker of an overworking hepatic metabolism due to their feeding with formula rations given to conventional fast-growing breeds. Nevertheless, the value obtained was within the range of values found in healthy pigs [38].

Despite the similar levels of ALT found in both genetic types of pigs at exsanguination, there was an increase in this parameter within both genetic types from T0 to T1.

The levels of sodium and potassium found in Apulo-Calabrese at exsanguination were higher when compared with crossbreeds and with values obtained at the basal condition. According to Mota-Rojas et al. [39] transport and slaughter can cause an increase in the concentrations of sodium

and potassium, respectively. The values reported in this study were, however, within the normal physiological range for pigs [35].

The results of the PCA performed on the changes in blood concentrations between T1 and T0 are reported in Figure 1, where the score and the loadings plots are shown. Two samples were not included in this analysis since they appeared to be outliers in orthogonal distance plot. Multivariate analysis generated three Principal Components (PCs: PC1, PC2, and PC3) explained 30%, 14%, and 10% of the total variance, respectively. The two components that explained the most differences between the two genetic types were PC2 and PC3, since plotting these two components samples displayed to be clustered (Figure 1a). The score plot (Figure 1a) presents the graphical projection of the samples into a two-dimensional space with PC2 (t2 in Figure 1a) values as the *x* axis and PC3 (t3 in Figure 1a) as the *y* axis. The red and blue ellipses represent the Mahalanobis distances for the crossbreed and Apulo-Calabrese pigs, respectively, while the black ellipse showed the average area of Mahalanobis distances for the complete population. Figure 1b graphically displays PCA loadings, numerically presented in Table S2. In Figure 1b the variables weighting the most in each PC are displayed: variables which have little contribution to a direction (PC) have almost zero weight (like urea for PC3), while the ones that contribute the most in the definition of the PCs show higher or lower weights (like glucose for PC3). Therefore, the blood parameters that contribute the most in the explanation of the differences among crossbreed and Apulo-Calabrese pigs are grouped together at the opposite quartiles. The results obtained from the PCA were consistent with those observed in the mixed model. Interestingly, the variables that weighted most in PC2 were urea (0.431), AST (−0.406), alb/glob (−0.350), lactate (0.331), and ALP (−0.327) (Table S2), which were the blood parameters that were influenced by the time × genetic type interaction in univariate results. PC3 resulted to be mainly related to glucose (−0.587), which was influenced by the genetic type in the univariate analysis.

**Figure 1.** Results of principal components analysis (PCA) on the variations of blood parameters between T1 and T0: (**a**) score plots for principal component 2 (t2) and principal component 3 (t3) of Apulo-Calabrese (blue) and crossbreeds (red) samples; (**b**) loadings plot with the weights of variables included in principal component 2 (p2) and principal component 3 (p3).
