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

Abstract

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.