Abstract
PurposeTo define the effect of sperm agglutination, associated with incubation under capacitating conditions, on accuracy of membrane assessment via flow cytometry and to develop methods to mitigate that effect.MethodsSperm motility was measured by CASA. Sperm were stained with PI-PSA or a novel method, LD-PSA, using fixable live/dead stain and cell dissociation treatment, before flow-cytometric analysis. Using LD-PSA, acrosome reaction and plasma membrane status were determined in equine sperm treated with 10 μm A23187 for 10 min, followed by 0, 1, or 2 h incubation in capacitating conditions.ResultsUsing PI-PSA, measured membrane integrity (MI; live sperm) was dramatically lower than was total motility (TMOT), indicating spurious results (“zombie sperm”). Sperm aggregates were largely of motile sperm. Loss of motility after A23187 treatment was associated with disaggregation and increased MI. On disaggregation using LD-PSA, MI rose, and MI then corresponded with TMOT. In equine sperm incubated after A23187 treatment, as the percentage of live acrosome-reacted sperm increased, TMOT decreased to near 0.ConclusionFlow cytometry assesses only individualized sperm; thus, agglutination of viable sperm alters recorded membrane integrity. As viable sperm become immotile, they individualize; therefore, factors that decrease motility, such as A23187, result in increased measured MI. Disaggregation before assessment allows more accurate determination of sperm membrane status; in this case we documented a mismatch between motility and live acrosome-reacted equine sperm that may relate to the poor repeatability of A23187 treatment for equine IVF. These findings are of profound value to future studies on sperm capacitation.
Highlights
Conventional in vitro fertilization (IVF) is not successful in the horse [1]
Total motility (TMOT) was significantly decreased (P < 0.05) at 0 h when sperm were exposed to concentrations of A23187 ≥ 5 μM in N medium, and at 0.5 h when sperm were exposed to ≥ 5 μM A23187 in P (BSAcontaining) medium (Fig. 1a)
In this report, using a novel method for membrane analysis (LD-PSA), we present new information on the physiology of equine sperm exposed to the calcium ionophore A23187
Summary
Current work toward understanding the basis of equine IVF has been focused on factors affecting sperm capacitation in this species [2]. While certain media components (notably, calcium, bovine serum albumin (BSA), and bicarbonate) are known to promote capacitation in other species [7, 8], the effect of these components on capacitation of equine sperm is unclear. Albumin is a cholesterol acceptor which promotes cholesterol efflux from the sperm plasma membrane – an important mechanism in capacitation – in other species including humans, pigs, and mice [9,10,11]. Presence of BSA in the medium is not associated with a reduction in membrane cholesterol levels in equine sperm [12]
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