2.5.2. Superoxide Dismutase (SOD) Activity Assay

The Superoxide Dismutase Activity kit (Thermo Fisher, Waltham, MA, USA), a colorimetric assay, was used for detection and quantification of superoxide dismutase activity in mice plasma preparations. Plasma samples were incubated for 20 min at room temperature after the addition of the sample and substrate and chromogenic detection reagent. The optical densities were recorded at 450 nm.

#### 2.5.3. Reduced Glutathione (GSH) Detection and Quantification Assay

Glutathione Colorimetric Detection Kit (Thermo Fisher), a colorimetric assay, was used for detection and quantification of reduced glutathione (GSH) levels in plasma preparations. Detection reagen<sup>t</sup> and reaction mixture were added to samples and after 20 min of incubation at room temperature the optical densities were recorded at 405 nm.

#### 2.5.4. Total Reactive Oxygen Species (ROS) Assay

Total Reactive Oxygen Species (ROS) Assay Kit 520 nm (Thermo Fisher) was used to analyze the total ROS levels in mice plasma preparations. 10 μl of each plasma sample were added to 100 μL of 1× ROS Assay Stain. After for 60 min of incubation at 37 ◦C and 5% CO2, signals were analyzed using a fluorescent microplate reader o ff the 488 nm (blue laser) in the FITC channel.

## 2.5.5. Detection of telomerase by ELISA Assay

Quantitative determination of mouse telomerase concentrations was performed in plasma preparations using a colorimetric sandwich-ELISA assay, Mouse TE(telomerase) ELISA Kit (Elabsciences ®, Houston, TX, USA. The optical density (OD) was measured at 450 ± 2 nm.

#### *2.6. Bone Marrow Cells Recovery from Mice*

Immediately after the sacrifice of CTR, ET-FPP ® and LT-FPP ® mice, bone marrow was obtained from both tibias and femurs of the hind legs of mice [51–54]. Bone marrow was then placed in physiological solution (NaCl) and disrupted with the blunt end of a 5-mL syringe plunger. Bone marrow cells were isolated using a Falcon ® 100 μm cell strainer (Corning, Corning, NY, USA), obtaining a uniform single-cell suspension from bone marrow. The single-cell suspensions were washed twice in PBS and immediately processed for following analysis.

#### *2.7. Ovarian Germ Cells Recovery from Mice*

Immediately after the sacrifice of CTR, ET-FPP ® and LT-FPP ® mice, ovaries were dissected [51–53,55], placed in physiological solution (NaCl) with 1% of trypsin and 0.1 μM of EDTA, separated from the remaining reproductive system with a cutter and disrupted with the blunt end of a 5-mL syringe plunger. Ovarian germ cells were isolated using a Falcon ® 100 μm cell strainer (Corning), connective tissue and debris were allowed to settle, obtaining a uniform single-cell suspension from ovarian tissue. The single-cell suspensions were washed twice in PBS and immediately processed for following analysis.

#### *2.8. Detection of Telomeres by PNA Kit*/*FITC for Flow Cytometry*

Detection of telomeres was performed in bone marrow cells and in ovarian germ cells of CTR, ET-FPP ® and LT-FPP ® mice obtained immediately after the sacrifice. To this purpose a Telomere PNA Kit/FITC for Flow Cytometry (Dako-Agilent, Santa Clara, CA, USA) was used. The kit allows detection of telomeres in nucleated haematopoietic cells using a fluorescence in situ hybridization and a fluorescein-conjugated peptide nucleic acid (PNA) probe. Results were evaluated by flow cytometry using a light source with excitation at 488 nm.
