**7.5 The functional integrity of the sperm plasma membrane**

The functional integrity of the sperm plasma membrane will be assessed using a short hypoosmotic swelling test (sHOST) (Perez-Llano et al., 2001). Spermatozoa are incubated, at 38 C for 30 min, with 75 mOsm/kg a hypo-osmotic solution that consist of 0.368 % (w/v) Nacitrate and 0.675 % (w/v) fructose (Merck, Germany) in distilled water. Following this incubation time, 200 µl of the semen-hypo-osmotic solution is fixed in 1000 µl of a hypoosmotic solution plus 5 % formaldehyde (Merck, Germany), for later evaluation. Two hundred spermatozoa are assessed under a phase contrast microscope at 400x magnification. The coiled tail (sHOST positive) spermatozoa found following incubation are functional intact plasma membrane.

#### **7.6 DNA damage**

DNA damage can be evaluated by Acridine orange (AO) staining or Halomax staining method (Fig. 4). The technique is modified after Thuwanut et al. (2008). Briefly, two smears from each sample were prepared on glass slide and air-dried. Each smear is fixed overnight in Carnoy's solution, freshly prepared with methanol and glacial acetic acid (3:1 v/v). The slide is removed from the fixative solution, air-dried, and then stained with 1% (100 mg/ml) AO (Sigma) in distilled water for 10 min. The AO staining solution is prepared by adding 10 ml of 1% AO in distilled water to 40 mL of 0.1 M citric acid (Merck, Darmstadt, Germany) and 2.5 ml of 0.3 M Na2HPO4.7H2O (Merck, Darmstadt, Germany) pH 2.5. The AO staining solution will be prepared daily and stored in the dark at room temperature until use. After staining, the slide is gently washed by distilled water and covered with the cover slip. One thousand spermatozoa are evaluated under the fluorescence microscope. The heads of the sperm cells with normal DNA (double-stranded) have green fluorescence, while those with damaged or single stranded DNA showed orange or red fluorescence. The results are expressed as the proportion of the damage/single stranded DNA per 1,000 counted spermatozoa.

Fig. 4. Sperm DNA damage (black circle) stained with a commercial kit (Halomax staining)
