**7. Laboratory method for semen quality assessment**

#### **7.1 Sperm concentration and progressive motility**

Sperm concentration will be assessed by direct cell count using a Bürker haemocytometer (Boeco, Humburg, Germany) (Beardon and Fuquay, 1997). The visual progressive motility of both fresh and FT sperm is evaluated at 38C under a phase contrast microscope at 200x and 400x magnification. The motility is assessed by the same person throughout the experiment.

#### **7.2 Computer-assisted sperm analysis (CASA)**

The motility patterns of diluted FT semen are assessed using the CASA system (Halminton Thorne Biosciences IVOS, Version 12 TOX IVOS, Beverly, USA). Each FT thawed semen samples is diluted with pre-warmed Modena extender (37°C) to obtain a final concentration of 50x106 spermatozoa/ml. A 5 µl of diluted semen is pipetted into the chamber and allowed the 1 min before analysis fore sample distribution and pre-warming (Iguer-Ouada and Verstegen, 2001). After the first assessment (T0), the diluted semen is evaluated after incubation at 37 C for 30 min (T30) and 60 min (T60). The camera will recognize the position of the sperm heads in successive frames. Spermatozoa heads are marked with a different color to enable the observer and the analyzer to differentiate between the different motility patterns. Each semen sample is measured twice, 3 fields are evaluated and counted at least 1000 cells per analysis. Motility patterns including (1) Curvilinear velocity (VCL, µm/s), the average velocity measured in the progression line along the whole track of cell path; (2) Average pathway velocity (VAP, m/s) , the average velocity of the smoothed cell path; (3) Straight line velocity (VSL), the average velocity measured in a straight line from the beginning to the end of the track (m/s); (4) The amplitude of the lateral head displacement (ALH), the mean width of the head oscillation as the sperm cells swim (m); (5) The beat cross-frequency (BCF, Hz), frequency of the sperm head crossing the average path in either direction; (6) The straightness (STR, %) = average value of the ratio VSL/VAP; (7) The Linearity (LIN, %) = average value of the ratio VCL/VAP.
