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

144 T.A.A. Khalifa et al. / Animal Reproduction Science 104 (2008) 143–163
significantly influenced NCI. The variability of NCI within a frozen ejaculate affected efficiency of IVEP.
Significant negative correlations were observed between incidence of NCI and both fertilization rate and
developmental capacity of embryos after maturation of oocytes for 18 h. The significant variation in IVEP
traits due to season was independent of the effect of sperm chromatin instability.
? 2007 Elsevier B.V. All rights reserved.
Keywords: Bull semen; Chromatin stability; Embryo production
1. Introduction
Failure of fertilization and embryonic mortality, particularly after AI, have long been recog-
nized as potential sources of loss in breeding cows and numerous studies have reported on them
(Gordon, 1996). The potential sire effects on bovine embryonic development have been shown
to occur as early as the initiation of S-phase in the zygote and after expression of the embryonic
genome at the four- to eight-cell stage (Eid et al., 1994). Therefore, laboratory assessment of semen
must include testing of most sperm attributes relevant for fertilization and embryo development,
such as evaluation of sperm genomic integrity (Evenson and Wixon, 2006).
Mammalian sperm genome is composed of nuclear DNA (Gledhill, 1970), mitochondrial DNA
(Sutovsky et al., 2003) and cytoplasmic messenger RNAs (Miller, 2000). The DNA in sperm
nuclei is bound to basic nuclear proteins to form the deoxyribonucleoprotein (DNP) complex
(Livolant, 1984). The only essential component of the sperm needed to form normal embryos is a
nucleus with an intact nuclear matrix (Ward et al., 1999). A high level of sperm nuclear chromatin
instability (NCI) in semen is associated with reduced breeding efficiency of bulls (Ballachey et
al., 1988; Karabinus et al., 1990; Dobrinski et al., 1994; Anzar et al., 2002; Madrid-Bury et al.,
2005).
The presence of sperm with NCI in freshly ejaculated semen is considered an uncompensable
defect, which cannot be tolerated at levels greater than 15–20% of spermatozoa (Barth and Oko,
1989). Fresh semen from bulls classified as questionable or unsatisfactory potential breeders
contains a higher percentage (13.50%) of spermatozoa with abnormal DNA condensation than
does semen from bulls classified as satisfactory potential breeders (7.10%) (Dobrinski et al., 1994).
Freshly diluted semen of mature dairy bulls contains 0.53–8% sperm with NCI (Krzyzosiak et
al., 2000). The factors that may affect the incidence of NCI in fresh semen are individuality and
semen quality characteristics (Dobrinski et al., 1994) as well as variation between and within
ejaculates of the same individual (Duty et al., 2002). However, the effects of breed, successive
ejaculations and dilution rates on the incidence of NCI in fresh semen of beef bulls have not yet
been investigated.
Previous studies on cooled bull semen (Salisbury et al., 1961; Hanada et al., 1965) did not
show any changes in sperm DNP complex for storage up to 24 h. Moreover, the literature con-
tains conflicting results regarding the impact of cryopreservation on sperm genome. Freezing
and/or thawing of semen altered DNP complex of bull sperm (Salisbury et al., 1964). On the
contrary, overcondensation of sperm chromatin has been observed in frozen-thawed semen of
bulls (Dobrinski et al., 1994; Anzar et al., 2002). Other authors did not find any effect for
cryopreservation of bull semen on sperm chromatin stability or DNA integrity (Martin et al.,
2004).
Concerning the variation range (0–15%) of NCI in frozen semen, many factors have been
studied, such as individuality and sperm traits (Dobrinski et al., 1994; Januskauskas et al., 2003),