Immediately after collection, the sperm concentration was measured using a Thoma–Zeiss counting chamber and a light microscope (Olympus CH2, Tokyo, Japan) with a 40× magnification.
An aliquot of the semen (about 0.5 mL) was centrifuged at 700× g for 15 min to obtain seminal plasma (SP) and to quantify NGF concentration by using ELISA.
An aliquot of each semen sample derived from the different collections was diluted with a modified TALP [13 (link)] to achieve a final concentration of 107 sperm/mL. The effect of storage was evaluated at different time points (0-2-4-6 h) in semen samples supplemented at time 0 with 100 ng/mL exogenous NGF (Merck, Milan, Italy), according to the dose–response curve previously described [13 (link)], and compared to vehicle samples diluted with PBS instead of NGF. The samples were evaluated for motility, viability, and necrotic/apoptotic processes as described below. Furthermore, receptor expression was also evaluated.
An aliquot of the semen (about 0.5 mL) was centrifuged at 700× g for 15 min to obtain seminal plasma (SP) and to quantify NGF concentration by using ELISA.
An aliquot of each semen sample derived from the different collections was diluted with a modified TALP [13 (link)] to achieve a final concentration of 107 sperm/mL. The effect of storage was evaluated at different time points (0-2-4-6 h) in semen samples supplemented at time 0 with 100 ng/mL exogenous NGF (Merck, Milan, Italy), according to the dose–response curve previously described [13 (link)], and compared to vehicle samples diluted with PBS instead of NGF. The samples were evaluated for motility, viability, and necrotic/apoptotic processes as described below. Furthermore, receptor expression was also evaluated.
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