**2. Results**

#### *2.1. Anabolic E*ff*ect of 17*β*-Testosterone (Esters)*

All animals from both groups were weighed at regular weekly intervals during the treatment experiment, and the body weight gain (BW) in kg is shown in Tables 1 and 2. The anabolic e ffect was expressed in a graphical form of the dependence of the body weight of experimental pigs on the time interval for both treatment and control groups. Two linear regression models were used to highlight the growth trends of both groups of pigs and the statistical assessment of the anabolic e ffect of 17β-testosterone (Figure 1). The average weekly body weight gains were calculated from the detected

BW data for each group of pigs and are shown in Supplementary Materials Table S1 and Figure S1. All animals were clinically monitored during the experiment and were in good health until slaughter at the end of the experiment. The treatment experiment was performed without any problems.


**Table 1.** Body weight of the individual pigs treated with a hormone preparation containing a testosterone ester combination.

**Table 2.** Body weight of the individual pigs in the control group.


**Figure 1.** A combined diagram of two regression models for testosterone treated pigs (male and female) and the control group of pigs.

#### *2.2. Targeted Analysis of 17* β*-Testosterone in Plasma*

#### 2.2.1. Identification of Analytes

Standards of 17β-testosterone, testosterone propionate, testosterone isocaproate, testosterone decanoate, and 17β-testosterone-D2 internal standard were always identified on the basis of the retention time (see Figure 2) obtained from the chromatogram and mass accuracy (MA) parameters calculated from mass spectra for precursor and product ions of each analyte by comparing the theoretical mass *m*/*z* with the measured experimental mass *<sup>m</sup>*/*<sup>z</sup>*. The obtained results and the calculated MA values determined by the standards are presented in Supplementary Materials Table S3 and in the diagrams showing the detected experimental mass spectra of the analyte standards, always in comparison with the theoretical mass spectra (Supplementary Materials Figures S2 and S3).

**Figure 2.** Chromatogram of the analysed plasma samples fortified with standards at 10 μg L−<sup>1</sup> (ppb), RT indicates retention time, AA indicates the peak area, SN indicates signal to noise ratio, black chromatogram represents 17β-testosterone, red chromatogram represents IS 17β-testosterone-D2, green chromatogram represents 17β-testosterone propionate, blue chromatogram represents 17β-testosterone decanoate and the yellow chromatogram represents 17β-testosterone isocaproate.
