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Submitted to: Meeting Abstract
Publication Type: Abstract Only Publication Acceptance Date: 7/28/2013 Publication Date: 7/28/2013 Citation: Purdy, P.H., Spiller, S.F., Blackburn, H.D. 2013. Rooster sperm plasma membrane protein and phospholipid organization and reorganization attributed to cooling and cryopreservation. Meeting Abstract. Annual Meeting of the Society for Cryobiology, Bethesda, MD, July 28-31, 2013. Interpretive Summary: Technical Abstract: Cholesterol to phospholipid ratio is used as a representation for membrane fluidity, and predictor of cryopreservation success but results are not consistent across species and ignore the impact of membrane proteins. Therefore, this research explored the modulation of membrane fluidity and protein organization of rooster sperm during cooling and cryopreservation using the fluorescent stains Merocyanine 540 (phospholipid order) and Rhodamine 640 (protein order). Rooster semen samples (3 roosters and breeds per pool; 4 pools total) were diluted in Lakes Low Temperature (LLT) and Sasaki diluent and cooled to 5 °C over 30 minutes (Step 1). The samples were then diluted with the penetrating cryoprotectants (CPA) glycerol (LLT) or methylacetamide (Sasaki), equilibrated for 30 minutes (Step 2), loaded into straws and cryopreserved. The cryopreservation and thawing (Step 3) was performed using the optimized procedures for each diluent. Computer automated semen analysis (CASA) was used to analyze motility characteristics and flow cytometry was used to analyze plasma membrane phospholipid, protein organization and plasma membrane integrity (PMI) at each step. Percent values from CASA and flow cytometry fluorescent medians (FM) were transformed using arcsine and Box-Cox, respectively. The GLM ANOVA was used to detect differences in motility characteristics and membrane organization and included the effects of cryopreservation step and rooster semen pool. In the total motility analyses cryopreservation diluent/CPA was a significant (P < 0.05) source of variation at Step 2 (83 and 63% motile sperm for LLT and Sasaki diluents, respectively) and at Steps 2 and 3 in the progressive motility analyses (24 and 15% for LLT; 63 and 5% for Sasaki at Steps 2 and 3, respectively). Differences in PMI were detected only following cryopreservation (Step 3; 56 and 46% for LLT and Sasaki, respectively; P < 0.05). Phospholipid organization was impacted by the diluent at step 1 (0.75 and 0.42 FM for LLT and Sasaki, respectively; P < 0.05) and the LLT samples had a significant change in phospholipid organization at step 2 (0.38 FM) relative to step 1 (0.75 FM; P < 0.05) indicating a less fluid membrane due to glycerol addition. Sasaki samples had a non-significant increase (greater membrane fluidity) in FM at step 2 (0.60 FM) relative to step 1 (0.42 FM) due to methylacetamide addition. Following cryopreservation the phospholipid organization of LLT (0.79 FM) and Sasaki (0.84 FM) samples were similar. A significant decline in detectable protein from steps 1 to 3 (2.3 and 1.13 FM, respectively; P < 0.05) was observed for LLT although step 2 (1.7 FM) was not different from the other steps. The detectable protein in Sasaki samples had a non-significant decrease from step 1 (1.9 FM) to step 2 (0.98 FM) but increased from step 2 to 3 (1.46 FM); a reorganization status similar to step 1. Our results demonstrate that during cryopreservation the diluent and CPA influence membrane phospholipid and protein organization and manifest as differences in motilities and PMI. Following additional investigation, we believe this assay may be used to monitor and predict the post-thaw quality of sperm samples. |
