Effect of the ABCA1 inhibitor probucol on post thaw characteristics of dog spermatozoa

During freezing and thawing, capacitation-like changes occur within the sperm plasma membrane, impairing fertilizing capacity. In extended fresh canine semen, addition of probucol (PRO), a cholesterol transporter antagonist specific for ABCA1 reduced capacitation-like changes. The aim of this study was to investigate effects of probucol (PRO) on post-thaw dog semen characteristics. Ejaculates from 17 stud dogs were collected. Sperm-rich fractions from dogs collected on the same day (n = 2–3) were pooled (13 pooled samples) and divided into 5 aliquots that were frozen according to the Uppsala protocol with treatment T1: untreated control, T2 addition of 250 μM PRO and T3 of 500 μM PRO before dilution with cooling medium and equilibration for 1 h (+4°C), T4 addition of 250 μM PRO and T5 of 500 μM PRO before dilution with freezing extender. Straws were stored in liquid nitrogen at −196°C until analyses. Kinematic data (IVOS II, IMV Technologies), sperm concentration (NucleoCounter SP-100, ChemoMetec) and sperm morphology (Hancock's solution) were assessed. For tyrosine phosphorylation, a semen smear was prepared on a glass slide, fixed with 4% paraformaldehyde, permeabilized with Triton-X and incubated with mouse anti-phosphotyrosine antibody (clone 4G10R; Merck Millipore, Darmstadt, Germany). Then, slides were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Breda, the Netherlands) and stained with Hoechst 33342 (Sigma,St. Louis, MO, USA) and microscopically evaluated as described by Petrunkina (3). Because not all data were normally distributed, comparisons among treatments were done by Friedman test followed by Wilcoxon test. Motility, progressive motility (P), viability, total number of morphologically abnormal sperms as well as ALH, BCF, DAP, DCL, DSL, VAP, VCL, and VSL were reduced after thawing, irrespective of treatment (p < 0.01). Motility, P, linearity (LIN), straightness (STR) and total capacitation were decreased by addition of PRO in comparison to controls (p < 0.01); but not the other kinematic characteristics and pathomorphology. Tyrosine phosphorylation demonstrated a decrease in non-capacitated sperm after freezing-thawing (p < 0.001) that was less pronounced after addition of 500 μM PRO (T3 and T5). Although addition of the cholesterol transporter antagonist probucol at 500μM decreased capacitation like changes after freezing-thawing of dog sperm, the treatment did not counteract the decrease of all other sperm characteristics investigated.

• VetImaging