Animal Reproduction Science 104 (2008) 143–163 Factors affecting chromatin stability of bovine spermatozoa T.A.A. Khalifa a,∗ , C.A. Rekkas b , A.G. Lymberopoulos b , A. Sioga c , I. Dimitriadis d , Th. Papanikolaou d a Department of Theriogenology, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt b NAGREF, Veterinary Research Institute, Ionia, Thessaloniki, Greece c Department of Histology and Embryology, School of Medicine, Aristotle University of Thessaloniki, Greece d Department of Reproduction and Obstetrics, Veterinary Faculty, University of Thessaly, Karditsa, Greece Received 8 August 2006; received in revised form 31 January 2007; accepted 16 February 2007 Available online 28 February 2007 Abstract The structural stability of transcriptionally inert paternal chromatin is of vital importance for the fertiliza- tion process and early embryonic development. Accordingly, a series of eight experiments were conducted during a 7-month period to investigate: (1) effects of bull breed, individuality, successive ejaculations, semen quality characteristics (SQC), semen dilution rates and hypothermic storage of semen in a Tris-egg yolk extender on incidence of sperm nuclear chromatin instability (NCI), and (2) effects of the interaction between variation of NCI within a frozen ejaculate and variation of oocytes quality due to maturation time and/or season on the efficiency of in vitro embryo production (IVEP). Semen samples were collected once a week from six bulls using an AV and only ejaculates (n = 220) of >0.30 × 10 9 sperm/ml and ≥60% motility were used. NCI was measured by: (1) detection of lysine-rich histones in sperm chromatin using aniline blue staining, (2) sperm susceptibility to acid-induced nuclear DNA denaturation in situ using acridine orange test, and (3) sperm susceptibility to nuclear chromatin decondensation (NCD). Bovine oocytes (n = 695) were matured in vitro for 18 or 24 h, fertilized after sperm selection through a swim-up procedure and cultured for 72 h. The results showed that the 2nd ejaculates were superior to the 1st ones with respect to chromatin stability. Dilution of semen to 49.67 ± 8.56 × 10 6 sperm/ml (1:19) decreased resistance of sperm to NCD. Cooling of semen had no significant effect on chromatin stability. Cryopreservation of semen augmented sperm vulnerability to DNA denaturation. Improvement of SQC (semen volume, sperm motility, velocity, viability and morphological normalcy) was generally concomitant with increase of sperm resistance to NCI. While Blonde d’Aquitaine bulls had a resistance to NCD higher than Limousine bulls in fresh semen, the former showed a greater susceptibility to DNA denaturation than the latter in cooled semen. Individuality ∗ Corresponding author. Present address: c/o Christine Cooreman, 19 Gravias street, 54645 Thessaloniki, Greece. Tel.: +30 2310 869 500. E-mail address: drtarekkhalifa@in.gr (T.A.A. Khalifa). 0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.02.019