Abstract:
The aim of this study was to determine whether flow cytometric evaluation of combined
merocyanine 540 and Yo-Pro 1 (M540-YP) staining would identify viable dog sperm that
had undergone membrane stabilization known to be associated with capacitation in other
species, and whether such destabilization is detected earlier than when using the tyrosine
phosphorylation and ethidium homodimer (TP-EH) stain combination with epifluorescence
microscopy. Semen from nine dogs was collected and incubated in parallel
in bicarbonate-free modified Tyrode’s medium ( BIC), medium containing 15 mM
bicarbonate (þBIC), dog prostatic fluid, and in PBS. Aliquots for staining were removed at
various time points during incubation of up to 6 hours. Staining with M540-YP allowed the
classification of dog sperm as viable without destabilized membranes, viable with destabilized
membranes, nonviable without destabilized membranes, or nonviable with
destabilized membranes. The percentage of viable sperm detected using EH (83.5 1.37%;
mean SEM) was higher than when using YP (66.7 1.37%: P < 0.05; n ¼ 54 semen
samples). On the other hand, M540-YP identified a higher percentage of viable sperm with
destabilized membranes than TP-EH (75 1.76% vs. 35 1.70%: P < 0.05; n ¼ 54 semen
samples). Staining with M540-YP indicated a rapid increase in the percentage of viable
sperm with destabilized membranes, reaching a maximum during the first 30 minutes of
incubation in þBIC. For all other treatments (i.e., BIC, prostatic fluid, and PBS), the peak
in the percentage of viable sperm with destabilized membranes was reached as much as
90 to 210 minutes later than incubation in þBIC. The lowest percentage of viable sperm
showing signs of capacitation was recorded during incubation in PBS. We conclude that YP
identifies sperm committed to cell death earlier than EH, and that the M540-YP stain
combination identifies membrane destabilization known to be associated with capacitation
in other species earlier than the TP-EH stain combination.
