## 3 Results and Discussion
### Multi-Elemental ICP-OES Analysis of Donkey Milk Samples  
Data were collected as described in Section 2.6. In Figure 1, a first overview of the ICP-OES profiles is given; mean concentrations element by element are reported in Table A1 in Appendix A. In the plots, the average content of Ca, Fe, K, Mg, Na, P, and Zn for freeze-thaw milk (FT-) and reconstituted (R-) milk at T\({}_{0}\) and T\({}_{1}\) are shown (bars represent the standard deviation among samples). These values are in strong agreement with the concentrations reported by [PERSON] et al. [40], [PERSON] et al. [41], and [PERSON] et al. [9] in Turkish and Italian donkey milks, respectively.  
Figure 1 shows that, regardless of sampling time, the composition of reconstituted milk appears less rich than FT-milk. Considering sampling, as far as FT-milk is concerned, the differences between T\({}_{0}\) and T\({}_{1}\) are mainly associated to the concentration of K and Fe.  
On the contrary, for R-milk, the (already lower) concentrations remain fairly constant between the two sampling times.  
The analysis has revealed that R-milk generally exhibits a lower concentration of elements compared to FT samples. This observation aligns with findings from other researchers, such as [PERSON] et al. [42], who also observed a similar aspect, and they imputed it to oxidation and the disintegration of the mineral compounds, as previously proposed by [PERSON] et al. [43] and by [PERSON] and [PERSON] [44].  
ASCA was used to evaluate whether the conservation process and the lactation period are significant effects. The model was calculated on auto-scaled data, and both variables appeared significant (\(p\) < 0.001).  
A more insightful and engaging interpretation of the data can be accomplished by examining the PCA biplot (Figure 2). The PCA model was computed by extracting four Principal Components (PCs), accounting for a total explained variance of 90%. In Figure 2, it is evident that FT- and R-milk samples tend to separate along the first principal component.  
Figure 1: Bar plot of the average concentrations of the diverse quantified elements with standard deviations. (**A**) Average concentration (\(\mu\)g/mL) of Ca, K, Mg, Na, and P in FT-milk collected at T\({}_{0}\); (**B**) Average concentration (\(\mu\)g/mL) of Ca, K, Mg, Na, and P in FT-milk collected at T\({}_{1}\); (**C**) Average concentration (\(\mu\)g/mL) of Ca, K, Mg, Na, and P in R-milk collected at T\({}_{0}\); (**D**) Average concentration (\(\mu\)g/mL) of Ca, K, Mg, Na, and P in R-milk collected at T\({}_{1}\); (**E**) Average concentration (\(\mu\)g/mL) of Fe and Zn in FT-milk collected at T\({}_{0}\); (**F**) Average concentration (\(\mu\)g/mL) of Fe and Zn in FT-milk collected at T\({}_{1}\); (**G**) Average concentration (\(\mu\)g/mL) of Fe and Zn in R-milk collected at T\({}_{0}\); (**H**) Average concentration (\(\mu\)g/mL) of Fe and Zn in R-milk collected at T\({}_{1}\).  
FT-samples (dark blue and yellow diamonds) predominantly cluster with positive PC1 scores, while the majority of reconstituted individuals (red and purple dots) exhibit negative PC1 scores.  
In agreement with what has been previously observed, for the reconstituted milk, no difference with respect to T\({}_{0}\) and T\({}_{1}\) can be appreciated. Conversely, FT-milk is prone to form two groups according to the sampling time. As expected, the biplot shows that the highest concentrations of metals are present in the FT-milk samples, in particular Fe, Mg, and Ca for T\({}_{0}\), and K, P, and Na for T\({}_{1}\). The behavior of Zn is opposite to the other elements and peculiar. This is probably due to the fact that the concentration of this metal is, on average, the same in FT- and R-milk, but two R-milk and one FT-milk samples have slightly higher concentrations of Zn.