Submitted to: Theriogenology
Publication Type: Proceedings
Publication Acceptance Date: September 27, 2004
Publication Date: December 8, 2004
Citation: Guthrie, H.D., Welch, G.R. 2004. Impact of extender and cryopreservation on membrane and morphological integrity in boar sperm. Theriogenology. 63:396-410. Interpretive Summary: Because cryopreservation causes changes in the lipid and protein organization and content in the plasma membrane of sperm, this study was conducted to determine if biochemical markers for increased phospholipid scrambling in the plasma membrane could be used to evaluate or predict sperm tolerance to cryopreservation procedures. Boar semen was divided into four parts and analyzed 1) fresh, 2) undiluted and held at room temperature, 3) processed for freezing at room temperature, and 4) freeze-thawed. Plasma membrane phospholipid scrambling was determined using fluorescence flow cytometry for merocyanine (MC) binding and for Annexin V (AV) binding. In viable sperm neither processing at room temperature or freeze-thawing had any effect on plasma membrane binding of MC and AV compared to fresh semen. The use of MC and AV were not found to be suitable markers for sperm evaluation or to predict sperm tolerance to cryopreservation procedures.
Technical Abstract: This study was conducted to determine if semen processing and cryopreservation of boar spermatozoa caused changes in plasma membrane lipid disorder resembling those of bicarbonate (15 mM)-induced in vitro capacitation and apoptosis. Sperm from ten ejaculates were analyzed 1) upon entry to the lab (fresh), 2) held nonextended at 25 C, 3) thawed after freezing, and 4) processed in cryopreservation media at 25 C without freezing. Plasma membrane phospholipid scrambling was investigated by incubation of sperm with merocyanine and FITC-annexin-V (AV), and the shift from low to high fluorescence binding was determined by flow cytometry. Compared to no bicarbonate, bicarbonate increased (P < 0.05) the percentage of viable sperm with high merocyanine fluorescence in fresh (0.7 to 36.3%) or nonextended semen (1.6 to 18.3%), but not in processed (2.1 to 7.7%) or thawed semen (4 to 5.6%). In the absence of bicarbonate the percentage of sperm with high merocyanine fluorescence was increased (P < 0.05) nonextended, processed, and thawed compared to fresh semen. The incidence of apoptosis (percentage of viable sperm with high AV fluorescence) was 1.5 % in fresh and 1.8% in nonextended semen and was increased (P< 0.05) in processed and thawed semen (5.6 and 4.5%, respectively). In the plasma membrane the effects of processing and cryopreservation of semen are not equivalent to the effects of bicarbonate-induced capacitation in boar sperm.