In the present study, we analyzed the effects of mycotoxins and E2 on motility (including characteristics of hyperactivated motility) and acrosome integrity in stallion sperm exposed to E2 or estrogenic mycotoxins for 2 hours. In general, it is accepted that capacitation and hyperactivation are linked events in the continuum of changes that a sperm must undergo in preparation for fertilization of an oocyte, and previous reports have suggested that stallion sperm require relatively long incubations to induce signs of capacitation [34–38]. For example, McPartlin et al.  reported that a 6 hour incubation was needed to observe procaine-induced hyperactivation in stallion sperm. In the present study, a 2 hour incubation was chosen for both motility (CASA) and flow cytometric analysis because, in our experimental conditions, longer incubation times (e.g. 4 hours) significantly reduced sperm viability. Moreover in a previous study , we reported deleterious effects of mycotoxins on the chromatin structure stability of stallion sperm after 2 hours of incubation.
The results of the present study indicate that a 2 hour in vitro exposure to α-ZOL affected proportions of motile sperm (total and progressive motility) only when the toxin was present at a high concentration (0.1 mM). The effects of α-ZOL on the proportion of motile equine sperm resembled those previously reported for ZEA and α-ZOL on boar sperm [17, 18], even though different experimental conditions were used in the earlier studies. In the current study, exposure to E2 did not affect the percentages of motile sperm (total or progressive); this corresponds with the findings of Luconi et al.  for human sperm. On the other hand, at high concentrations (0.1 mM) both E2 and ZEA and its derivatives affected VAP; this suggests that, while the percentages of motile sperm are not affected by estrogenic mycotoxins, the speed at which the sperm move is consistently reduced by exposure to this family of mycotoxins. In particular, a pronounced reduction in VAP was observed after exposure to α-ZOL or ZEA. Again, the findings for the effect of E2 are in agreement with those reported by Luconi et al.  for human sperm. With respect to the effects on motility parameters, the following rank of activity was apparent: α-ZOL > ZEA > β-ZOL = E2. Moreover, at the concentrations examined, α-ZOL was the only ZEA derivative able to reduce the proportion of motile sperm in stallion semen, whereas all the tested mycotoxins influenced the velocity of sperm movement of the remaining motile cells and, in addition, there was a trend for the sperm motility parameters to change in a way suggesting hyperactivation of motility; i.e., reductions in VSL and LIN and a concomitant increase in ALH. More specifically, at the highest tested concentration (0.1 mM), ZEA inhibited VSL, α-ZOL reduced VSL and LIN and increased ALH, and β-ZOL reduced LIN. At the same concentration, E2 reduced VSL and LIN. In the only previous report  of the effect of ZEA derivatives on sperm motility parameters, β-ZOL and α-ZOL were found to stimulate VCL in boar sperm when introduced at concentrations ranging from 0.2 to 20 μM, after 5 hours of incubation. The current study is the first to examine the effects of E2 on sperm motility parameters associated with hyperactivation. The ranking of the effects on hyperactivation-related motility parameters in stallion sperm was α-ZOL > E2 > ZEA = β-ZOL.
Neither ZEA nor E2 influenced the proportion of live acrosome-reacted sperm during a 2 hour incubation. By contrast, 0.1 mM α-ZOL or β-ZOL stimulated an increase in the percentage of live acrosome-reacted sperm. In a previous study on pig sperm , ZEA and α-ZOL at higher concentrations (125 to 250 μM) reduced the proportion of live acrosome-reacted cells and concomitantly increased the proportion of deteriorated (dead) sperm. These conflicting results could relate to differences in the experimental conditions used, such as incubation time and methods of determination. We did not observe the deterioration effect and, besides the likely contribution of the relatively short incubation employed in the current study, we note the advice that detection of the acrosome status of incubated sperm should preferably be performed at the incubation temperature because temperature shock can easily induce membrane damage . In the present study, E2 did not influence the percentage of live acrosome-reacted sperm.
Conflicting data on whether, and at what molecular level, estrogens may affect the AR are still not completely resolved. The currently favored theory is that E2 inhibits the progesterone- or ATP-induced AR in both human [23, 42, 43] and bovine  sperm. However, studies in the mouse have documented a stimulatory effect of E2 on the AR [45, 46]. On the other hand, it may be significant that different incubation conditions (medium, time), staining (FITC-PNA or FITC-Pisum sativum agglutinin – PSA) and evaluation techniques (fluorescence microscope or flow cytometry) were used in the various studies. In the current study, a ranking of the magnitude of the effect on the AR of stallion sperm was Beta-zearalenol > β-ZOL > E2 = ZEA.
In the present study, a 2 hour incubations in the presence of ZEA, its derivatives or E2 were sufficient to induce changes in equine sperm motility, hyperactivated motility, and in the percentage of sperm that had acrosome-reacted. The most potent compound was α-ZOL, which modified sperm motility (total and progressive motility), sperm velocity (VAP), most of parameters related to hyperactive motility (VSL, LIN and ALH) and induced the AR to a larger extent than any of the other estrogenic compounds studied. Beta-zearalenol affected only the percentage of acrosome-reacted sperm. As a result of the contrasting effects of related chemicals, there must be suspicions that, in the stallion, the pathways that regulate capacitation and hyperactivation are separate and independent, as previously reported in other species [47–49].
Apparently, the mycotoxin ZEA influenced hyperactivation-associated parameters to a lesser extent than E2, whereas both ZOL derivatives induced premature acrosome reactions and thereby adversely affected the fertilizing capacity of stallion sperm. The α isomer of ZOL still possessed the apparently estrogenic capacity to induce hyperactivated motility, whereas the β stereo-isomer had lost this property.
Importantly, the current study showed that neither ZEA, its derivatives nor E2 affected sperm cell deterioration during the 2 hour incubation. Previous studies reported a significant decrease in the percentage of viable boar sperm after in vitro exposure to ZEA, α-ZOL and β-ZOL [17, 19]; however, this occured at higher concentrations of mycotoxin  and at longer incubation times than in the present study .