Neubauer chamber

A total of 12 ejaculates coming from different stallions (n = 12) were used. Animals were housed at the Equine Reproduction Service, Autonomous University of Barcelona (Spain), which is a European Union (EU)-approved equine semen collection center (Authorization code: ES09RS01E) that operates under strict protocols of animal welfare and health control. Since all stallions used in this study were semen donors and were housed at the Equine Reproduction Service, the local ethics committee at our University indicated that no further ethics authorization was required.
Semen samples were collected using a Hannover-type artificial vagina (Minitüb GmbH, Tiefenbach, Germany) with an in-line nylon mesh filter to separate the gel fraction. Gel-free semen ere subsequently diluted 1:5 (v:v) in a Kenney extender [29 ], previously warmed at 37 °C. Sperm concentration was assessed with a Neubauer chamber (Paul Marienfeld GmbH & Co. KG, Lauda-Köningshofen, Germany), and each sample was split into two different fractions. The first one was used to assess the quality of the fresh semen, whereas the other was divided into nine different sub-fractions that were cryopreserved in the presence or absence of different concentrations of the three AQP-inhibitors.

CFs were plated at a density of 156 cells/mm² on 35-mm plastic dishes and stimulated under the conditions indicated for each experiment. After 16 h of simulated reperfusion, cells were washed two times with PBS and treated with trypsin EDTA (0.5%) and EDTA 0.2% (1×) to detach cells, followed by administration of FBS (10%) to induce inactivation. After detachment, aliquots of 20 μl of sample plus 20 μl of trypan blue (0.5%) reagent were homogenized, and then 8 μl was transferred to a Neubauer chamber (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) to count viable cells (unstained) using optic microscopy. Experiments were performed in duplicate and repeated six times. At least 1,000 cells/ml was counted in each sample.

For the treatment with 100 ng/mL NGF or 100 ng/mL anti–NGF neutralizing antibody (RnD Systems, Minneapolis, MN, USA), 1.5 mL/well cell suspension of 6.7 × 10⁴ cells/mL were plated in 12-well plates (Falcon^TM, Durham, NC, USA) in serum-free, albumin containing medium [43 (link)] and cultured for 72 h. After that, the cells were washed with PBS and incubated with 10 µg/mL Mitomycin C (Sigma-Aldrich^®, St. Louis, MO, USA) in serum-free albumin-containing medium for 30 min at 37 °C ensuring cell cycle arrest [42 (link)]. Then the cells were washed twice with albumin-containing medium and subsequently treated with albumin-containing medium for two times 48 h supplied with 100 ng/mL recombinant human NGF [45 (link)] or with 100 ng/mL anti NGF neutralizing antibody (RnD Systems) for altogether 96 h. After completion of treatments, the cells were used for cell counting with trypan blue staining (Sigma, Darmstadt, Germany) in a Neubauer chamber (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany).