498 I. A. TSAKMAKIDIS ET AL. Copyright © 2007 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2007; 27: 498–505 DOI: 10.1002/jat JOURNAL OF APPLIED TOXICOLOGY J. Appl. Toxicol. 2007; 27: 498–505 Published online 16 March 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jat.1239 Study on the in vitro effect of zearalenone and α α α - zearalenol on boar sperm–zona pellucida interaction by hemizona assay application I. A. Tsakmakidis, 1 A. G. Lymberopoulos, 1 E. Vainas, 1 C. M. Boscos, 2 S. C. Kyriakis 3 and C. Alexopoulos 2, * 1 NAGREF, Veterinary Research Institute, Ionia, 57008 Thessaloniki, Greece 2 Clinic of Production Animals, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 11 St. Voutyra St., 54627 Thessaloniki, Greece 3 Foundation of Biochemical Research, Academy of Athens, 4 Soranou Ephesius St., 11527 Athens, Greece Received 24 November 2006; Revised 29 January 2007; Accepted 29 January 2007 ABSTRACT: The mycotoxin zearalenone (zen) impairs fertility in farm animals. The aim of the present study was to investigate the effect of zearalenone and its major metabolite ( α α α-zearalenol) on boar semen binding capacity, under in vitro conditions. Extended boar semen was exposed to three different concentrations of zen and α α α-zen (40, 60 and 80 μg ml -1 of semen) for 1 h. Afterwards, the semen was washed and incubated with homologous oocyte hemizona for 4 h. A signifi- cant decrease (P < 0.001) in the number of tightly attached spermatozoa on the hemizona was obtained at concentrations of 60 μg ml -1 and 80 μg ml -1 of zen and α α α-zen. In conclusion, zen and α α α-zen affected the sperm–zona interaction by re- ducing the ability of boar spermatozoa to bind to the zona pellucida. Copyright © 2007 John Wiley & Sons, Ltd. KEY WORDS: zearalenone; mycotoxin; boar sperm; zona binding; hemizona assay; in vitro incubation * Correspondence to: Assoc. Professor C. Alexopoulos, St. Voutyra 11, 54627 Thessaloniki, Greece. E-mail: kalex@vet.auth.gr Contract/grant sponsor: General Secretariat of Research and Technology (GSRT) of the Ministry of Development. Contract/grant sponsor: Veterin ABEE. Introduction The worldwide contamination of food and feed with mycotoxins is a signifficant problem. Zearalenone (zen) is a common mycotoxin in pig farms which is implicated in reproductive disorders with great agro-economic im- portance. Zen produced by Fusarium mold in cereals and grains has been shown to decrease the reproductive potential of boars (Diekman and Green, 1992). Previous studies showed that approximately 60% of the consumed zen was excreted in urine as α-zearalenol (α-zen) which is the predominant metabolite of zen in pigs (Kuiper- Goodman et al., 1987; Olsen, 1989; Zollner et al., 2002). High levels of zen were reported to occur naturally in animal feeds [500–600 ppm in Minnesota, USA (Christensen et al., 1972), 135 ppm in Finland (Mirocha et al., 1979), 100 ppm in Hungary (Palyusik, 1977), 64 ppm in Indiana, USA (Long et al., 1982), > 803 μg kg -1 , in Germany, 120 μg kg -1 , in Bulgaria (Bottalico and Perrone, 2002)]. Dietary supplementation of boars with zen resulted in significant decreases in testis weight, testosterone production and sperm motility (Christensen et al., 1972; Young and King, 1986). How- ever, in a previous study on the effects of the direct interaction between zen or α-zen and sperm cells, it was found that exposure of boar semen to zen or α-zen at concentrations of 125, 187.5 and 250 μM significantly induced damage to the sperm plasma membrane and reduced the percentage of acrosome reacted spermatozoa (Tsakmakidis et al., 2006). A number of tests relating to sperm factors and fertility have been developed, such as the classical spermiogram, hypoosmotic swelling test, chromatin inte- grity and sperm–oocyte interaction assays (Matas et al., 1996; Gil et al., 2005). Moreover, Saacke (1983) reported that incubation of semen at a temperature higher than 35 °C allowed resolution of any latent injury in sperma- tozoa. Sperm–zona pellucida (ZP) interaction plays a crucial role in determining sperm ability not only to fertilize the oocyte but also to sustain early embryonic development (Flesch and Gadella, 2000). Consequently, the hemizona assay (HZA) has been introduced as a reliable test to evaluate the fertilizing ability of boar spermatozoa (Fazeli et al., 1995). The objective of the present study was to investigate the in vitro effect of zen or α-zen on sperm motility, viab- ility and binding ability to zona pellucida using HZA.