Relationship between non-return rate and chromatin
condensation of deep frozen bull spermatozoa
Ninoska Madrid-Burya, Jose´ Fe´lix Pe´rez-Gutie´rrezb,
Sonia Pe´rez-Garneloa, Pedro Moreiraa,
Bele´n Pintado Sanjuanbenitoa, Alfonso Gutie´rrez-Ada´na,*,
Julio de la Fuente Martı´neza
aDepartamento de Reproduccio´n Animal, INIA, Ctra dela Corun˜a Km 5,9, Madrid 28040, Spain
bDepartamento de Medicina y Cirugı´a Animal, Facultad de Veterinaria,
Universidad Complutense de Madrid, Madrid 28040, Spain
Received 16 May 2004; received in revised form 27 July 2004; accepted 2 November 2004
Abstract
This study analyzes the relationship between chromatin condensation and field fertility, expressed as
90 days non-return rate (NRR), of bulls actively used by AI studs. Frozen–thawed semen from five bulls
(six ejaculates per bull, three straws per ejaculate), that showed a non-return rate between 60 and 80%,
were analyzed to assess sperm chromatin condensation and stability. The chromatin condensation was
determined by flow cytometry using propidium iodide as fluorochrome, and the chromatin stability was
evaluated by inducing its decondensation with SDS and EDTA. Coefficient of variation among
replicates was less than 7% and 5% for chromatin condensation and stability, respectively. No
correlation was present between chromatin condensation and NRR. However, significant correlation
was found between chromatin stability and NRR. Chromatin stability was higher (P < 0.05) in those
bulls that showed higher fertility. The results obtained in this study conclude that assessment of stability
could be a valuable tool for routine evaluation and identification of ejaculates with high levels of sperm
chromatin abnormalities and to detect animals of higher reproductive potential.
# 2004 Elsevier Inc. All rights reserved.
Keywords: Chromatin stability; Spermatozoa; Field fertility; Bull
www.journals.elsevierhealth.com/periodicals/the
Theriogenology 64 (2005) 232–241
* Corresponding author. Tel.: +34 913473768; fax: +34 913474014.
E-mail address: agutierr@inia.es (A. Gutie´rrez-Ada´n).
0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2004.11.017

1. Introduction
The use of frozen semen for improving the bovine population genetics is accepted and
utilized worldwide. However, in order to enhance production potential, cattle artificial
insemination industry is focusing on the development of accurate methods capable of
predicting field fertility when using frozen semen. Conventional tests of semen quality
although widely used, are considered to be inconsistent predictors of reproductive
efficiency [1,2]. Reproductive efficiency of bulls is usually measured by non-return rate
(NRR), which is defined as the percentage of cows that were inseminated and were not
reinsenseminated within a specified numbers of days. Non-return rate is the result of
conception [3]. Conception at insemination depends among other factors, on the ability of
sperm chromatin to decondense and to form the male pronucleus [4]. Therefore, it is
important to establish the state of sperm chromatin in a given sample, in relation to its
fertilizing potential, especially in those males where other functional tests and semen
parameters do not explain the differences in field fertility [5–7].
Nuclear compactness is the result of DNA packing by protamines, which act in sperm
cells as histones do in somatic cells [8–10]. The nature and degree of interactions among
protamines, which include non-covalent bonds, disulfide bonds and interactions of
sulphydryl groups through zinc ions, is essential to maintain a particular physical state of
chromatin [9–11]. Excessive interactions among protamines can lead to a highly stable
chromatin, difficult to decondense, which could delay paternal nuclear formation and
induce early embryonic death. On the other hand, sperm chromatin condensation is
necessary for DNA transient inactivation and protection from environmental factors [9].
The physical state of chromatin has to provide packing and protection to DNA, but also the
ability to decondense upon fertilization. Previous studies in human and porcine species
have shown that freezing and thawing induce important changes in the sperm chromatin
resulting in a greater compactness of the sperm nuclei [12–16]. Thus, chromatin
condensation and stability may be critical factors to consider when using frozen semen.
Several assays have been developed to assess sperm chromatin condensation, including
those that utilize toluidine and aniline blue or those based on the uptake of intercalating
compounds, such as ethidium bromide, acridine orange or propidium iodide (PI) [17–20].
In order to test the stability of the chromatin structure, different methods, that can reveal
different aspects of the susceptibility of chromatin to destabilisation, have been developed.
Among these, the sperm chromatin assay (SCSA) developed by Evenson et al. [21] has
been widely used, and has been applied to evaluate the relationship between fertility and
the chromatin structure in several species, including bulls [13,31,37]. This method is based
on the destabilisation of chromatin under acid conditions, and makes use of the
metachromatic properties of acridine orange. Chromatin structural stability has been also
evaluated in human [17,23] and porcine [14] by treating spermatozoa with chelating
agents, such as EDTA, which chelates protamines zinc, and anionic detergents, such as
SDS, which disrupts non-covalent bonds [17,22]. However, this assay has never been tested
on bovine spermatozoa.
The aim of this study was to evaluate deep frozen bull spermatozoa chromatin
condensation and stability, by chromatin perturbation with EDTA and SDS, and its
relationship to NRR.
N. Madrid-Bury et al. / Theriogenology 64 (2005) 232–241 233