The acidic probe LysoSensor is not useful for acrosome evaluation of cryopreserved ram spermatozoa

To try new acrosomal probes for assessing ram spermatozoa, we compared the LysoSensor probe, which labels acidic organelles, with the frequently used peanut agglutinin acrosomal probe (PNA-PE; phycoerythrin as fluorescent moiety). The previous microscopic observations showed a lack of relationship of LysoSensor with acrosomal status. Semen was obtained from five rams and frozen in four pools. Each pool was analysed carrying out a triple staining propidium ioide/PNA-PE/LysoSensor Green DND-189 to test acrosome labelling, and a double staining SYBR-14/PI, to assess sperm viability. Stained samples were analysed by flow cytometry. All measurements were replicated. Data were processed using agreement and repeatability tests. LysoSensor labelling did not agree with PNA (mean of differences: 30.8%; coefficient of agreement: 22.6%), confirming microscopic observations. Nevertheless, when LysoSensor was compared with SYBR-14/PI, the agreement was high (mean of differences: -0.05%; coefficient of agreement: 5.07%). Repeatability of both methods was high and similar. LysoSensor did not seem to specifically stain the acrosome, but it may accumulate in the cytoplasm and label viable spermatozoa. Therefore, LysoSensor might not be used as an acrosomal probe in ram spermatozoa, but it could be used in other kind of studies, taking advantage of its pH sensitivity.