Flow cytometric evaluation of mitochondrial activity and membrane integrity in fresh and cryopreserved rainbow trout (Oncorhynchus mykiss) spermatozoa

There are still several obstacles to effective cryopreservation of trout spermatozoa as seen by the large variability of fertility rates obtained with frozen-thawed spermatozoa. We investigated the applicability of flow cytometry to estimate the quality of frozen-thawed trout spermatozoa. Flow cytometric analysis was performed on fresh and cryopreserved pools of sperm after fluorescent staining with rhodamine 123 to quantify mitochondrial function and propidium iodide to assess plasma membrane integrity. Several cryoprotectants that penetrate the membrane were tested: Me2SO, dimethylacetamide, glycerol, methanol, and propylene glycol at various concentrations in the freezing medium (0, 5, 10, and 15%). ATP content was evaluated by bioluminescence before and after freezing. The percentage of fresh spermatozoa with a functional mitochondrion and an intact membrane was 99%. After cryopreservation, the percentage of spermatozoa with damaged membrane varied between 55 and 90%, while only a few cells (between 0.5 and 17%) displayed both an undamaged membrane and an intact mitochondrion. The plasma membrane was better protected with 10% Me2SO, while methanol and propylene glycol showed toxic effects. The best cryoprotectant for the mitochondrion was Me2SO at a concentration of 5 or 10%. These results are in agreement with fertility rates generally observed. Frozen-thawed spermatozoa lost between 50 and 90% of their initial ATP content, and the origin of this loss is discussed. (C) 1997 Academic Press.