**8. Lipid composition of sperm plasma membrane**

The lipid compositions of the plasma membrane of the mammalian spermatozoa are markedly different from those of somatic cells. In general, the sperm plasma membrane contains approximately 70% phospholipids, 25% neutral lipids, and 5 % glycolipids (Flesch and Gadella, 2000). All lipid components located in the sperm membranes responsible for the fluidity of membrane lipid bilayers, regulation of sperm maturation, spermatogenesis, capacitation, acrosome reaction and membrane fusion (Parks and Hammerstedt, 1985; Martinez and Morros, 1996; Sanocka and Kurpisz, 2004).

Sperm plasma membrane are made up of a phospholipids bilayer, with the major phospholipids were choline phosphoglycerides (CP), ethanolamine phosphoglycerides (EP) and sphingomyelin which their proportions differed between species. These phospholipids contain a high proportion of long chain, polyunsaturated docosapentanoyl (22:5) and

from inner to outer leaflet of cell plasma membrane which is the hallmark of apoptosis during the degradation phase. Following manufacturer instructions, sperm cells are washed twice with PBS (pH 7.4) by centrifugation at 400 g for 5 min. Sperm pellet are resuspended with HEPES buffer (10mM HEPES/NaOH, pH7.4, 150mM NaCl, 5mM KCl, 1mM MgCl2, 1.8mM CaCl2, 2x106 sperm/ml). One hundred l of sperm suspension are mixed well with 5 l annexinV-FITC conjugate and 3 l of Propidium iodide (PI;20g/ml) and incubated for 15 min at room temperature in the dark. Two hundred spermatozoa are assessed under fluorescent microscope at 400x magnification. The apoptotic sperm cells will fluorescence green while necrotic sperm cells fluorescence red. Alternatively, flow cytometry analysis can

Fig. 5. FCS/SCC two-dimensional histogram, flow cytometry analysis of frozen-thawed boar spermatozoa (supplemented with L-cysteine) stained with Annexin-V/PI: Q3 represent

The lipid compositions of the plasma membrane of the mammalian spermatozoa are markedly different from those of somatic cells. In general, the sperm plasma membrane contains approximately 70% phospholipids, 25% neutral lipids, and 5 % glycolipids (Flesch and Gadella, 2000). All lipid components located in the sperm membranes responsible for the fluidity of membrane lipid bilayers, regulation of sperm maturation, spermatogenesis, capacitation, acrosome reaction and membrane fusion (Parks and Hammerstedt, 1985;

Sperm plasma membrane are made up of a phospholipids bilayer, with the major phospholipids were choline phosphoglycerides (CP), ethanolamine phosphoglycerides (EP) and sphingomyelin which their proportions differed between species. These phospholipids contain a high proportion of long chain, polyunsaturated docosapentanoyl (22:5) and

the viable spermatozoa with intact plasma membrane, while Q2 represent dead

also be used instead of fluorescent microscope (Fig. 5).

spermatozoa with non-intact plasma membrane

**8. Lipid composition of sperm plasma membrane** 

Martinez and Morros, 1996; Sanocka and Kurpisz, 2004).

docosahexanoyl (22:6) groups which both lipids represent approximately 50 to 60 % of total phospholipids in boar and bull spermatozoa (Pursel and Graham, 1967; Johnson et al., 1969; Parks and Lynch, 1992). Cholesterol was the major sterol in sperm lipids of all species. Cholesterol to phospholipid molar ratios were 0.26, 0.30, 0.36, and 0.45 for sperm plasma membrane of the boar, rooster, stallion, and bull, respectively (Parks and Lynch, 1992).Glycolipids represented less than 10% of total polar lipids for all species.

The susceptibility of spermatozoa to cold shock differ among species because of the differences of lipid composition of the sperm plasma membrane among species (Flesch and Gadella, 2000).The resistance of the mammalian spermatozoa to cold shock was high in species in which the cholesterol to phospholipids molar ratio and the phospholipids saturation is high (Darin-Bennett and White, 1977). The avian spermatozoa have a high level of cold shock resistant and have a higher level of saturated phospholipids compared to mammalian sperm (Parks and Lynch, 1992). The plasma membrane of the boar spermatozoa is characterized by a high protein, low cholesterol and high proportion of EP compared to other species (Parks and Lynch, 1992; Nikolopoulou et al., 1985). In contrast, the protein content and EP proportion of rooster sperm plasma membrane is low while the cholesterol content is intermediate (Parks and Lynch, 1992).

As mentioned above the sperm plasma membrane has a very high amounts of polyunsaturated fatty acids (PUFAs) especially docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) (Johnson et al., 1969; Parks and Lynch, 1992). It has been suggested that the proportion of unsaturated fatty acid influence the properties of sperm plasma membrane (Miller et al., 2005). High levels of long chain PUFAs, DPA and DHA, are associated with an increased membrane fluidity (Quinn, 1985). During cryopreservation, the fluidity of the plasma membrane from boar spermatozoa is significantly decreased when compared to fresh spermatozoa which tend to restrict the post-thawed sperm quality (Buhr et al., 1994). In human, sperm with a high level of membrane fluidity had a higher postthawed motility compared to sperm with a low level of membrane fluidity after cryopreservation (Giraud et al., 2000).
