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(176) EFFECTS OF CRYOPRESERVATION AND COOLING ON PLASMA MEMBRANE LIPID DISORDER AND INTEGRITY IN BOAR SPERMATOZOA. Guthrie, Howard1, Welch, Glenn1, Cooper, Bruce1, 1 Germplasm and Gamete Physiology Laboratory, ARS, USDA, Beltsville, MD ABSTRACT- This study was conducted to test the hypothesis that freezing and thawing of boar spermatozoa caused changes in plasma membrane lipid fluidity or disorder resembling those of bicarbonate (BIC)-induced in vitro capacitation. Semen from each of ten ejaculates were subjected four treatments: incubation at ambient temperature for 1) 0.2 hr or 2) 7 hr, 3) processing to 5°C in freezing extender, and 4) freezing in 0.5 mL straws by the static liquid N2 vapor method. Plasma membrane lipid disorder was determined using fluorescence flow cytometry for merocyanine (MC) binding (in the absence and presence of 15 mM BIC) and for Annexin V (AV) binding. Percent motile sperm (MOT), plasma membrane integrity (PMI), and percent sperm with normal apical ridges, respectively, were reduced from 81, 93, and 97%, at 0.2 hr to 54, 82, and 82% in sperm cooled to 5°C; and further reduced to 25, 33, and 56% after thawing (P < 0.05). In the absence of BIC only 1.4% of viable sperm bound MC and treatment had no significant effect. The ability of BIC to increase the number of sperm binding MC decreased at 7 hr (15%) and after cooling to 5°C (6%) compared to 0.2 hr (32%). BIC did not significantly increase MC binding after thawing. Sperm binding of AV was low in viable cells (2%) and treatment had no significant effect. Binding of AV increased in the non-viable sperm population only after cooling to 5°C (7%) or after cryopreservation (47%). The absence of evidence for cooling and cryopreservation effects on sperm plasma membrane lipid disorder does not support the hypothesis that cryopreservation causes changes in the plasma membrane that resemble BIC-induced in vitro capacitation in viable boar sperm. KEY WORDS: merocyanine binding, annexin V binding, sperm motility, plasma membrane integrity |
