| Two different combinations of fluorophores were used to assess the functional capabilities of three spermatozoal organelles, identify specific cellular phenotypes and to estimate spermatozoal viability, morphology and potential fertility. The integrity of spermatozoal plasma membranes were assessed using dihydroethidium (HED), carboxy dimethyl fluorescein diacetate (CDMFDA) or propidium iodide (PI). The integrity of acrosomal membranes also were assessed using fluorescein isothiocyanate-labeled Pisum sativum agglutinin (PSA). The mitochondrial function of sperm cells were quantified using rhodamine 123 (R123). Protocol 1 consisted of PI, R123 and CDMFDA; and protocol 2 consisted of HED, R123 and PSA. Cryopreserved samples from 8 bulls were analyzed. Samples were thawed and then incubated with either staining combination for 1.5 hr prior to flow cytometric analysis after 1.5 and 4 hr. Incubation-induced changes in mitochondrial function and integrity of plasma and acrosomal membranes were simultaneously assessed using protocol 1 and 2. Similarly, the percentage of viable, moribund, dead and severely degenerated spermatozoa in a sample were identified based on the function of specific organelles. These cellular phenotypes were associated with the percentage of spermatozoa in the sample with abnormal heads, intact acrosomes at 0 hr, normal morphology, motility at 4 hr and vacuoles/craters. Additionally, these same cellular phenotypes at both incubation times were associated with both ejaculate and cumulative nonreturn rates. In conclusion, these particular combinations of fluorophores can be used to assess changes in spermatozoal organelle function, identify specific cellular phenotypes and provide quantifiable information reflective of spermatozoal viability, morphology and fertilizing potential. |
