### NIRS Prediction Equations (Calibration and External Validation)
#### 3.3.2 Major Constituents  
NIRS prediction equations for the main fatty acids (C16:0, C18:0, C18:1 n-9, C18:2 n-6, and C18:3 n-3) of IMF were developed using the full spectra range both in the intact and minced presentation modes (Table 4), except for C18:2 n-6 in minced meat, in which models were developed in a specific wavelength range, 1176-1333/1638-1835 nm, where the model showed the best accuracy (Table 5). Regarding the test used to perform the calibration models, in the intact presentation mode (Table 4), Test 1 was selected for all parameters, with the exception of C18:1 n-9, where the best results were obtained in Test 3 (calibration test composed of 40% of samples and external validation test with 60%). Different tests were selected in the minced presentation mode. For C16:0, C18:1 n-9, and all fatty acid groups (SFA, MUFA, and PUFA), the best calibration was obtained using Test 1, whilst C18:2 n-6 and C18:3 n-3 were attained with Test 2 (calibration test composed of 60% of samples and external validation test with 40%). Test 3 was used for C18:0. As previously observed in the total lipids parameter, in general, the mean values of 1-VR were lower in the intact presentation mode, reaching the lowest values for C18:0 (<0.20) and the maximum values for C18:1 n-9 (0.41) (Table 4). According to the 1-VR statistic, calibration models for C16:0, C18:1 n-9, and C18:2 n-6 could only be used to discriminate samples with high and low analytical value in the intact presentation mode (Table 4). For the other fatty acids considered (C18:0 and C18:3 n-3), the low 1-VR value makes it impossible to use the model, even for classification as high and low values in intact muscles. The lower predictive ability in intact samples has been previously described [14; 15]. Contrarily, medium-high values of 1-VR were observed in the minced presentation mode, with values ranging from 0.50 to 0.83. More specifically, the equations developed for C18:2 n-6 and C18:3 n-3 exceeded the values of 0.7 for this statistic, being 0.83 and 0.73, respectively. Henceforth, the quantitative prediction of these fatty acids in the minced samples of LTL muscle could be possible. Regarding the RMSECV statistic, lower values were observed in the minced presentation mode compared to the intact mode (Tables 4 and 5), C18:2 n-6 and C18:3 n-3 being the best performing models, since they showed an RMSECV of 0.87 and 0.06 in minced samples (Table 5), as compared to an average of 6.37 and 0.32 g/100 g FAMEs, respectively (Table 3). Regarding RPDev and RRev statistics in the latest parameters, although satisfactory values were observed in the minced presentation mode for C18:3 n-3, with 1.90 and 10.63, respectively, the situation was not the same for C18:2 n-6, where lower values of both statistics were observed (Table 5). For the rest of the fatty acids considered in the minced presentation mode, values of RPDevranged from 1.37 (C18:0) to 1.67 (C18:1 n-9), whilst RRev values ranged from 6.04 (C18:0) to 8.86 (C16:0). Therefore, with the exception of C18:3 n-3, the predictive ability of the models should be improved to overcome the limits of reliability in terms of RPD and RER [31; 32]. Regarding the scientific literature, previous studies reported determination coefficients greater than 0.7 for C16:0, C18:0, C18:1 n-9, C18:2 n-6, and C18:3 n-3 in calibration models developed in LTL muscle [28] and subcutaneous backfat [35], although considering only one breed (Iberian pig). Additionally, [17] obtained higher determination coefficients for C16:0 and C18:0 in different sausagesfrom Iberian breed pigs, although reported lower values for C18:1 n-9, C18:2 n-6, and C18:3 n-3 than those observed in the present study. Nevertheless, to the best of our knowledge, there are no studies concerning the development of quantitative NIRS calibration models in the rest of the breeds considered in this study or for different breeds considered together.