SHORT COMMUNICATION Selectivity of porcine zona pellucida to bind spermatozoa with normal chromatin structure I. A. Tsakmakidis 1 , A. G. Lymberopoulos 2 & T. A. A. Khalifa 2 1 Clinic of Farm Animals, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece; 2 NAGREF, Veterinary Research Institute, Thessaloniki, Ionia, Greece Introduction Sperm-zona pellucida (ZP) interaction plays a crucial role in the fertilisation process. Moreover, ZP is an important selective barrier against spermatozoa with abnormal morphology (Kaskar et al., 1994). Liu & Baker (2007) reported that spermatozoa with single stranded or dena- tured DNA bind less or do not bind at all to the human ZP. Furthermore, DNA damaged spermatozoa can fertilise the oocyte, produce early-stage embryo, but fail to pro- duce a successful term pregnancy (Ahmadi & Ng, 1999). High incidence of nuclear chromatin instability in semen is associated with reduced breeding efficiency of boars (Evenson et al., 1994). On the basis of the aforemen- tioned considerations and taking into account that in the current literature, there is no report of the capacity of boar spermatozoa with normal or abnormal DNA to bind to homologous ZP, the aim of the present study was to investigate the selective ability of swine ZP for sperma- tozoa with normal nuclear chromatin, using hemi- zona assay (HZA). HZA, which uses bisected oocytes, has been introduced as a reliable test to evaluate the fertilising ability of boar spermatozoa (Fazeli et al., 1995). Materials and methods Four boars of proven fertility (oestrus return <10%, far- rowing rate >85%, live piglets born/sow >11) were selected and used in this study. Ten ejaculates with a sperm concentration of >0.1 · 10 9 spermatozoa ml )1 (SpermaCue photometer, Minitu ¨b, Tiefenbach, Germany), a progressive motility of >70% (subjective estimation by two individual experienced persons) and morphological abnormalities of <15% (eosin–nigrosin stain) were extended to 30 · 10 6 spermatozoa ml )1 and used in HZA application. Each one of the four boars provided 3, 3, 2 and 2 ejaculates respectively. Ovaries were obtained from gilts at a local abattoir. Cumulus–oocyte complexes were aspirated from follicles (2–6 mm) and vortexed for 5 min in sodium citrate buffer 2.96% to denude oocytes from cumulus cells. Semen samples were centrifuged at 400 g for 10 min and the sperm pellet was resuspended in SP-TALP medium at a concentration of 10 6 spermatozoa ml )1 . Dif- ferent aliquots of each semen sample were used in the pre- and post-incubation evaluation of chromatin integ- rity as well as for the HZA. Keywords hemizona—Swine zona—sperm chromatin— boar semen—sperm zona interaction Correspondence Dr Ioannis A. Tsakmakidis, DVM, PhD, Lecturer, Clinic of Farm Animals, Department of Clinical Studies, School of Veterinary Medicine, Aristotle University of Thessaloniki, 11 St. Voutyra str., PC 54627, Thessaloniki, Greece. Tel./Fax: +302310994467; E-mail: iat@vet.auth.gr Accepted: March 22, 2010 doi: 10.1111/j.1439-0272.2010.01074.x Summary This study aimed to investigate the selective ability of swine zona pellucida (ZP) to bind spermatozoa with normal nuclear chromatin. Ten ejaculates of four boars were used, while hemizona assay was applied for evaluation of bind- ing capability. The results of this study showed that swine ZP has the ability to select spermatozoa with normal chromatin structure for sperm-zona binding process. 358 ª 2011 Blackwell Verlag GmbH Æ Andrologia 43, 358–360