Assessment of stallion spermatozoa viability by flow cytometry and light microscope analysis

Viability of spermatozoa can be assessed by numerous methods, but many are slow and poorly repeatable, and subjectively assess only 100 to 200 spermatozoa per ejaculate. We collected two ejaculates from each of 4 stallions, and extended them to 50x10(6) sperm/mL in a nonfat dried milk solids glucose extender (EZ Mixin). Half the ejaculate was freeze-killed by immersing in liquid nitrogen for 10 min. Aliquots using appropriate volumes of live and freeze-killed spermatozoa provided the following ratios of live:dead spermatozoa: 100:0, 75:25, 50:50, 25:75, 0:100. We determined the viability of each aliquot by 1) motility; 2) eosin-nigrosin staining; and 3) dual fluorescent staining. For the latter, aliquots incubated with SYBR-14 and propidium iodide had live and dead spermatozoa quantitated by fluorescent microscope (2 x 100 sperm/sample) and flow cytometry (10,000 sperm/sample). We found a linear relationship between the ratio of live:dead spermatozoa and the percentage of spermatozoa counted as live (P<0.0001). For fresh spermatozoa, correlation coefficients of the known live:dead ratio were high for all methods (eosin-nigrosin, r>0.75; fluorescent microscope, r>0.76; flow cytometry, r>0.75; motility, r>0.76). To determine viability of cryopreserved equine spermatozoa, we froze 17 fresh ejaculates from 6 stallions in a glycine extender. Each sample was thawed, extended 1:1 with EZ Mixin and evaluated as above. Cryopreserved spermatozoa assessed by flow cytometry tended to be less well correlated (r<0.68) with the other methods, and estimates were significantly higher with eosin-nigrosin staining (P<0.001). This study shows that different methods may equally estimate viability of fresh equine spermatozoa. However, evaluation by flow cytometry appears to be less precise with cryopreserved spermatozoa.