ISSN: 2047-2919 ANDROLOGY

ORIGINAL ARTICLE

Correspondence:
Marina R. S. Fortes, The University of Queensland, Sperm protamine deficiency
St. Lucia, Qld 4072, Australia.
E-mail: m.fortes@uq.edu.au correlates with sperm DNA damage
in Bos indicus bulls
Keywords:
bovine, chromomycin A3, DNA fragmentation,
1
sperm protamines, spermatozoa M. R. S. Fortes, 2N. Satake, 3D. H. Corbet, 4N. J. Corbet, 4B. M. Burns,
1
S. S. Moore and 2G. B. Boe-Hansen
Received: 18-Nov-2013
1
Revised: 23-Jan-2014 Queensland Alliance for Agriculture and Food Innovation, Centre for Animal Science, The University
of Queensland, Saint Lucia, 2School of Veterinary Science, The University of Queensland, Gatton,
Accepted: 24-Jan-2014 3
Queensland Department of Agriculture, Fisheries and Forestry, Rockhampton, Queensland, and
4
Queensland Alliance for Agriculture and Food Innovation, Centre for Animal Science, The University
doi: 10.1111/j.2047-2927.2014.00196.x of Queensland, Rockhampton, Queensland, Australia

SUMMARY
The primary purpose of spermatozoa is to deliver the paternal DNA to the oocyte at fertilization. During the complex events of fer-
tilization, if the spermatozoon penetrating the oocyte contains compromised or damaged sperm chromatin, the subsequent progres-
sion of embryogenesis and foetal development may be affected. Variation in sperm DNA damage and protamine content in
ejaculated spermatozoa was reported in the cattle, with potential consequences to bull fertility. Protamines are sperm-specific
nuclear proteins that are essential to packaging of the condensed paternal genome in spermatozoa. Sperm DNA damage is thought
to be repaired during the process of protamination. This study investigates the potential correlation between sperm protamine con-
tent, sperm DNA damage and the subsequent relationships between sperm chromatin and commonly measured reproductive phe-
notypes. Bos indicus sperm samples (n = 133) were assessed by two flow cytometric methods: the sperm chromatin structure assay
(SCSA) and an optimized sperm protamine deficiency assay (SPDA). To verify the SPDA assay for bovine sperm protamine content,
samples collected from testis, caput and cauda epididymidis were analyzed. As expected, mature spermatozoa in the cauda epidi-
dymidis had higher protamine content when compared with sperm samples from testis and caput epididymidis (p < 0.01). The DNA
fragmentation index (DFI), determined by SCSA, was positively correlated (r = 0.33  0.08, p < 0.05) with the percentage of sperma-
tozoa that showed low protamine content using SPDA. Also, DFI was negatively correlated (r = 0.21  0.09, p < 0.05) with the per-
centage of spermatozoa with high protamine content. Larger scrotal circumference contributes to higher sperm protamine content
and lower content of sperm DNA damage (p < 0.05). In conclusion, sperm protamine content and sperm DNA damage are closely
associated. Protamine deficiency is likely to be one of the contributing factors to DNA instability and damage, which can affect bull
fertility.

INTRODUCTION environment stressors, genetics, compromised spermiogenesis,

The main purpose of spermatozoa is the delivery of intact
DNA that contributes genetic information to successful fertiliza-
tion and subsequently the individual. If sperm DNA is damaged,
embryogenesis may be compromised and this is suggested as
one of the causes of male subfertility in a number of species
including: human (Larson et al., 2000; Larson-Cook et al., 2003),
bovine (Waterhouse et al., 2006; D’Occhio et al., 2013) and por-
cine (Didion et al., 2009). Sperm DNA damage evaluated with
the sperm chromatin structure assay (SCSA) was associated with
poor pregnancy outcomes and miscarriages in humans (Evenson
et al., 1999; Evenson, 2013). Sperm DNA damage higher than
20%, assessed by SCSA was reported to be associated with smal-
ler litter size in pigs (Boe-Hansen et al., 2008). Sperm DNA dam-
age is likely to have a multi-factorial cause, including
or inappropriate sperm chromatin structure, for a review see
D’Occhio et al. (2007) and Gonzalez-Marin et al. (2012). Recent
evidence also suggests a link between abnormal sperm prot-
amine content and sperm DNA damage (Simon et al., 2011). As
sperm DNA damage contributes to failure in embryonic devel-
opment further investigation of its causes is necessary.
During spermiogenesis, the mammalian spermatids undergo
critical changes in the cytoarchitecture of its chromatin, which
enables mature sperm chromatin to be highly organized and
condensed. Sperm chromatin is the most compacted configura-
tion of any eukaryotic cell DNA (Risley et al., 1986; Ward & Cof-
fey, 1991). Essential to this compact configuration, protamines
are sperm-specific nuclear proteins required for sperm DNA
packaging and structural stability, which have an arginine-rich

370 Andrology, 2014, 2, 370–378 © 2014 American Society of Andrology and European Academy of Andrology
SPERM CHROMATIN EVALUATION IN CATTLE
core and cysteine residues (Krawetz & Dixon, 1988; Fuentes-
Mascorro et al., 2000; Toshimori, 2003; Miller et al., 2010). The
disulphide cross-linkage of the cysteine residues enables a high
organizational order of sperm chromatin compaction (Kosower
et al., 1992) and the rigidity and stability of this nuclear structure
protects the paternal genome to be transported and delivered to
the oocyte (Brewer et al., 2002). There are two main variants of
protamines: protamine 1 (P1) which is the predominant variant
in eutherian mammals and protamine 2 (P2) which is only
observed in the spermatozoa of some species. The ratio of these
variants differs considerably from species to species from 100%
P1 variant in bulls, boars, rabbits and domestic cats spermatozoa
(no P2) to close to 80% P2 variants in some primate species (Cor-
zett et al., 2002). In humans, P1 : P2 ratio is approximately 55 to
45% of protamine content (Corzett et al., 2002). This P1 : P2
ratio in spermatozoa influences male fertility in humans (Silves-
troni et al., 1976; Nanassy et al., 2011; Simon et al., 2011). Sperm
protamine content and variants in other species deserve further
investigation to determine its role and relevance to sperm DNA
damage and male fertility.
Variation between bulls in terms of sperm protamine content
has been described by fluorescent microscopy (Simoes et al.,
2009). Decreased protamine content was also observed in bulls
exposed to a testicular heat insult in the form of scrotal insula-
tion (Rahman et al., 2011). Recently, a flow cytometric assay for
sperm protamine content was developed in humans and showed
a correlation between sperm protamine deficiency and sperm
DNA fragmentation, which was assessed with the sperm chro-
matin dispersion test (Tavalaee et al., 2009, 2010; Fathi et al.,
2011). A comparative assessment of sperm protamine content
and sperm DNA damage by flow cytometry would further eluci-
date the relationship between sperm protamine deficiency and
sperm DNA damage, with implications for fertilization outcome.
Understanding the link between sperm protamine content and
sperm DNA damage would contribute new knowledge to under-
stand sperm chromatin structure and susceptibility to sperm
DNA damage.
Methods for assessment of protamine content could be further
developed. Previous microscopic analysis and flow cytometric
analysis of protamine using chromomycin A3 (CMA3) staining
have only used unstained and stained contrast using visible
emission spectra to identify sperm protamine content (Evenson
et al., 1986; Simoes et al., 2009; Tavalaee et al., 2009, 2010; Fathi
et al., 2011). However, CMA3 has a shift in its absorption spec-
trum from its monomeric to dimeric and further to polymerized
molecular forms, which is dependent on pH and concentration
of CMA3 in aqueous solutions. This absorption spectrum shift
occurs in the ultraviolet and visible spectra (Hayasaka & Inoue,
1969). In this study, we have assessed whether the utilization of
both ultraviolet and visible spectra of CMA3 will enable further
information on sperm protamine content and the condensed
chromatin.
The objective of this study was to investigate whether a novel
method describing sperm protamine content, the sperm prot-
amine deficiency assay (SPDA) and sperm DNA damage (using
the SCSA) correlate in post-pubertal Bos indicus Brahman
bulls. Furthermore, correlations between SCSA, SPDA, scrotal
circumference (SC) and traditional semen parameters (i.e.
sperm morphology, concentration and motility) were also
described.
© 2014 American Society of Andrology and European Academy of Andrology
ANDROLOGY
MATERIALS AND METHODS
Two experiments are described below. For Experiment 1, the
sampling of sperm samples from testis, caput and cauda epidid-
ymis was opportunistic and shared using fresh bovine abattoir
material sourced for teaching purposes, approved by the Univer-
sity of Queensland Ethics Committee SVS/118/11/T (NF). For
Experiment 2, data and samples used were obtained from the
existing database and sperm bank established by the Co-opera-
tive Research Centre for Beef Genetics Technologies (Beef CRC)
under ethics approval granted by the J M Rendel Laboratory Ani-
mal Experimental Ethics Committee (CSIRO, Queensland)
TBC107 and RH225-06.
Unless stated otherwise, all reagents and chemicals were pur-
chased from Sigma Aldrich Co. LLC (Camp Hill, NSW, Australia).
Samples for experiment 1: bovine testicular samples
Male reproductive tract material (n = 12) were used for valida-
tion of the SPDA. From each reproductive tract, cells were
extracted from (i) testicular tissue, (ii) caput epididymidis, and
(iii) cauda epididymidis, to identify changes in CMA3 binding
that correspond to perceived level of protamine content and
minor groove accessibility of spermatogenic cells from sper-
matogenesis to mature spermatozoa. Approximately 2 9 2 cm
pieces of testicular tissue and of caput epididymidis tissue were
dissected. The tissues were macerated with 1.5 mL of cold phos-
phate buffered saline (PBS) and subsequently homogenized in a
Potter’s homogenizer. The cell suspension was then passed
through a 100 lm cell strainer to produce a crude preparation of
testicular cells. The suspension was assessed under the micro-
scope to ensure presence of spermatogenic cells. The suspension
from the caput was concentrated by centrifugation at 500 g for
5 min and resuspended into 500 lL of PBS. The cauda epidi-
dymidis was excised and flushed with 2–5 mL of PBS to obtain a
highly concentrated epididymal sperm sample. Aliquots of these
samples were snap-frozen and stored at 80 °C until flow cyto-
metric analysis.
Samples for experiment 2: animals and bull breeding
soundness evaluation
Each bull underwent a bull breeding soundness evaluation
(BBSE) based on the procedure reported by Fordyce et al.
(2006). Scrotal circumference (SC) was measured with a stan-
dard measuring tape in centimeters at approximately 6, 12, 18
and 24 months of age. Semen was collected using electroeja-
culation procedure and samples were assessed immediately
post-collection for semen volume (Vol; mL) and subjective
measures of mass activity (Mass; 5-point scale of ‘1’ no swirl
to ‘5’ fast distinct swirl with continuous dark waves) and pro-
gressive forward motility (Mot; estimated percentage in 5%
increments) based on standard protocols (Fordyce et al.,
2006).
One semen aliquot of 20–120 lL was diluted into a 0.2% glut-
eraldehyde in PBS solution for sperm morphology evaluation. A
second aliquot of 500–1000 lL raw semen was snap frozen in
liquid nitrogen for later analysis of sperm concentration, sperm
chromatin integrity and sperm protamine content. The mor-
phology of 100 spermatozoa was determined by examining a
thin cover-slip preparation of semen using phase contrast
microscopy with a differential interference contrast objective
(magnification at 91000). Spermatozoa were classified
Andrology, 2014, 2, 370–378 371
M. R. S. Fortes et al.
individually, and then allocated into categories: percentage of
normal sperm (PNS); percentage of abnormal heads (PAH); per-
centage with total droplets (PTD); and percentage with proximal
droplets (PPD). All sperm morphology assessments were con-
ducted by the same sperm morphologist accredited by the Aus-
tralian Cattle Veterinarians. Details on morphology assessment
and the experimental design of BBSE performed by the Beef CRC
were reported previously (Burns et al., 2013).
Flow cytometric analysis 1: sperm protamine deficiency assay
Sperm protamine deficiency was assessed using CMA3 from
Streptomyces, which was a fluorochrome that competed for
binding sites at the minor groove of the DNA strand (Evenson
et al., 1986; Bianchi et al., 1996; Simoes et al., 2009). The method
optimized in this study was based on a flow cytometric approach
previously described in humans (Tavalaee et al., 2010; Fathi
et al., 2011) with modifications detailed below.
Samples were thawed at 37 °C for 3 min and sperm concen-
tration of each sample was determined using the Improved Neu-
bauer chamber. Thawed sperm cell suspensions and raw
electroejaculated semen samples were diluted to approximately
50 9 106 sperm/mL in Dulbecco’s PBS (DPBS, Ca2+ and Mg2+
free), and subsequently washed by centrifugation at 500 g for
5 min. The pellet was then resuspended into DPBS, split into
two aliquots and incubated for 15 min at 37 °C in the treat-
ments: (i) no treatment and (ii) positive control treated with
5 mM dithiothreitol (DTT). The samples were washed twice by
centrifugation at 500 g for 5 min and resuspended into DPBS. A
subsequent wash was conducted by centrifugation at 500 g for
5 min. The samples were then resuspended in 0.25 mg/mL
CMA3 in McIlvaine’s buffer (17 mM citric acid, 164 mM
Na2HPO4 and 10 mM MgCl2
6H2O; pH 7.0) and incubated at
room temperature for 1 h in the dark. Finally, the samples were
washed three times by centrifugation at 500 g for 5 min and the
concentration was adjusted to a concentration of 5–
10 9 106 sperm/mL for flow cytometric analysis.
Flow cytometric analysis of samples was conducted on the
Beckman Coulter GalliosTM flow cytometer (Beckman Coulter
Inc., Miami, FL, USA) using two excitation solid state lasers, the
violet laser (405 nm) and the blue laser (488 nm), and fluores-
cence was detected on FL9 (450BP50 filter), FL10 (550BP40 filter)
and FL2 (575BP30 filter) respectively, at the low flow rate
(approximately 1.5 lL/min). Kaluza® analysis software (version
1.1, Beckman Coulter Inc.) was used to analyze the flow cyto-
metric list mode data before statistical analysis (see Experiment
1 and statistical analysis sections for details).
Flow cytometric analysis 2: sperm chromatin structure assay
The same samples thawed for SPDA were used for SCSA. The
SCSA was conducted according to the protocol described by
Evenson & Jost (2001). In short, SCSA is based on the metachro-
matic properties of acridine orange to assess chromatin stabil-
ity. Acridine orange fluoresces green when combined with
double stranded (intact) DNA and it fluoresces red when com-
bined with single stranded (damaged) DNA. Semen samples
were diluted with TNE buffer (0.15 M NaCl, 0.01 M Tris HCl,
1 mм EDTA) to obtain a sperm concentration between 6 and
10 9 106 sperm/mL. Sample analysis was carried out on the
Beckman Coulter GalliosTM flow cytometer (Beckman Coulter
Inc.) as the SPDA assay above. The blue excitation laser
372 Andrology, 2014, 2, 370–378
ANDROLOGY
(488 nm) was used to excite the fluorophore, acridine orange
and fluorescence was detected on FL1 (525BP40 filter), FL3
(620BP30 filter) and FL4 (675BP20 filter) respectively, at the low
flow rate (approximately 1.5 lL/min). After every six test sam-
ples, a reference sample was thawed and analyzed to ensure
stability of the instrument. The reference sample was an aliquot
from the same ejaculate, which was used throughout the exper-
iment. Analyses of list mode data were performed using Kaluza
(Beckman Coulter Inc.). The percentage of sperm with abnor-
mally high DNA stainability (HDS) and the proportions of
sperm with DNA fragmentation, collectively termed the DNA
fragmentation index (DFI) were determined for each sample.
Two DFI values were determined using the FL3 fluorescence
(DFI FL3) and the FL4 fluorescence (DFI FL4) for detecting
sperm with DNA damage. Likewise, HDS was detected on the
two fluorescences: HDS FL3 and HDS FL4. This use of the Gal-
lios cytometer and software, adding an FL4 reading to the stan-
dard SCSA was developed by our group previously (Fortes
et al., 2012a).
Experiment 1: verification of SPDA
In this study, we have assessed whether the utilization of the
two absorption spectrum of CMA3 will enable further informa-
tion on the availability of the DNA minor grooves on the GC-rich
sequences of the chromatin for CMA3 binding. Accessibility of
sperm DNA minor grooves was relative to sperm protamine con-
tent and sperm chromatin condensation (Bianchi et al., 1996).
To verify the SPDA, testicular cells, including somatic and sper-
matogenic cells, from three sections of bovine testis were
extracted to represent three stages of the histone-to-protamine
replacement and chromatin condensation processes. Somatic
and spermatogenic cells extracted from the testicular paren-
chyma were representative of the standard eukaryotic cell and
immature spermatogenic cells with minimal initiation of this
replacement and condensation processes and thus maximal
CMA3 access to the DNA minor grooves. Spermatogenic cells in
the caput epididymidis were used to represent cells in varying
stages of the histone-to-protamine replacement process and
thus varying states of chromatin condensation. Accordingly,
spermatogenic cells in the caput epididymidis were expected to
present varying levels of DNA minor grooves access and there-
fore varying CMA3 binding. Finally, spermatozoa from the cauda
epididymidis were used to represent mature spermatozoa, which
have completed the chromatin condensation and histone-to-
protamine replacement processes. Spermatozoa from the cauda
epididymidis are expected to be comparable in protamine con-
tent and chromatin condensation to spermatozoa in the ejacu-
lated samples. Therefore, spermatozoa from the cauda
epididymidis are expected to have minimal access to the DNA
minor grooves for CMA3 binding.
Mature bovine testis (n = 12) were collected, sectioned and
spermatogenic cells were extracted for SPDA assessment as
described above. Three subpopulations of cells were identified
in these sperm samples and categorized as: (i) low or no
CMA3 binding (LCB), minor groove binding sites are unavail-
able because of the completion of chromatin condensation
and packaging by the argenine-rich protamines; (ii) medium
CMA3 binding (MCB), histone-to-protamine replacement and
chromatin condensation processes are in transitional stages,
transition proteins may be present (transitional chromatin)
© 2014 American Society of Andrology and European Academy of Andrology
SPERM CHROMATIN EVALUATION IN CATTLE ANDROLOGY
and some CMA3 staining occurs; and (iii) high CMA3 binding
Experiments 1 and 2: statistical analyses
(HCB), where minor groove binding sites of the GC-rich chro-
Statistical analyses were performed using Statistica 7.0 (Stat-
matin are freely available for binding by CMA3. Additional
Soft Inc., Tulsa, OK, USA). Semen parameter values were
information and figures related to flow cytometric data analy-
expressed as mean  standard error of the mean (SEM). For
sis for this assay are provided as supporting information (Sup-
Experiment 1, variations in testicular protamine content
plementary 1).
between samples were compared using multiple comparisons
by the Kruskal–Wallis test. For Experiment 2, Pearson’s correla-
Experiment 2: comparison between SCSA and SPDA
tion coefficients were calculated to assess pairwise comparisons
Semen samples of 133 bulls from the Beef CRC database were
of all semen parameters and BBSE measures. Correlations were
assayed using the two flow cytometric methods: SCSA and SPDA.
deemed significant when the absolute value was higher than
Samples used in this experiment were selected on the basis of
twice the SEM (p < 0.05).
having complete BBSE data, including sperm morphology
parameters collected at approximately 24 months of age (rang-
ing from 21 to 25 months) and having an aliquot available for RESULTS
SCSA and SPDA, preserved at 80 °C from the same ejaculate
Experiment 1
collected at approximately 24 months of age.
Significant differences in CMA3 binding were observed from
The groups of cells identified in the ejaculated samples using
spermatogenic cells of each testicular section (Fig. 1, Table 1).
SPDA are also termed HPC, MPC and LPC. However, it is impor-
An example from spermatogenic samples from an individual tes-
tant to note that the meaning of protamine content here is dif-
tis and epididymis collection is shown as scatter plots (Fig. 1,
ferent from that described for Experiment 1. In ejaculated
inset a–d) that illustrate the different flow cytometric profiles,
samples, mature spermatozoa should be completely protaminat-
FL2 vs. FL10, for: (i) testicular parenchyma cells (Fig. 1a), (ii) ca-
ed. Therefore, any proportion of spermatozoa with increased
put epididymidis spermatogenic cells (Fig. 1b) and (iii) cauda
CMA3 binding is abnormal and sometimes termed as ‘protamine
epididymidis spermatozoa (Fig. 1c). Changes in CMA3 binding
deficient’ or ‘deprotaminated’ in recent literature (Simoes et al.,
were observed in all three testicular sections (n = 12: Fig. 1
2009; Ferraz et al., 2012). The terms LCB, MCB and HCB have
graph). Proportions of CMA3 binding to DNA from each testicu-
been used throughout this manuscript to facilitate readership
lar section are summarized in Table 1. The percentage of HCB
and reflect the degree of CMA3 binding observed with no specu-
was significantly higher in the cells of the testicular parenchyma
lation of its cause.

Figure 1 Sperm samples were extracted from bull testis, caput and cauda epididymidis (n = 12) for validation of the sperm protamine deficiency assay
(SPDA). Identified sperm populations were (i) Low CMA3 binding (LCB, %), (ii) Medium CMA3 binding (MCB, %); and (iii) High CMA3 binding (HCB, %).
The positive control (black bar) represents the percentage of cysteine double bond disrupted (%) cells; CMA3 positive population in pooled samples after
treatment with dithiothreitol (DTT). Inset a–d illustrates the different flow cytometric profiles, FL2 vs. FL10, for testicular parenchyma spermatogenic cells
(a), caput epididymidis spermatogenic cells (b), cauda epididymidis spermatozoa (c) and a DTT positive control sample (d). A detailed description of the
flow cytometric method of analysis is found in Supplementary 1.
(a) (b) (c) (d)

© 2014 American Society of Andrology and European Academy of Andrology Andrology, 2014, 2, 370–378 373
M. R. S. Fortes et al. ANDROLOGY
Table 1 Protamine (values indicated as average  standard error, %) of Experiment 2
sperm cells of different testicular sections assessed by sperm protamine defi- A brief description and abbreviations included in the BBSE
ciency assay (SPDA)a
measurements and semen parameters evaluated, as well as the
Testicular section n SPDA mean, standard deviation, minimum and maximum values were
provided for comparison with previous studies (Table 2). Pear-
HCB (%) MCB (%) LCB (%)
son’s pair-wise correlations estimated for comparisons between
Testicular parenchyma 12 85.35  2.69A
3.56  0.33A
11.09  2.74A SCSA, SPDA and traditional BBSE measurements are presented
Caput epididymidis 12 50.55  7.78A 33.67  5.13B 15.77  4.09A in Table 3.
Cauda epididymidis 12 2.55  0.77B 4.80  0.55A 92.64  1.18B
The results showed that both DFI measurements (DFI FL3 and
Dithiothreitol 12 98.04  0.73 0.14  0.06 1.82  0.72
positive control DFI FL4) estimated with SCSA were positively correlated with
the percentage of HCB spermatozoa observed with SPDA
a
Sperm cells were classified in three categories according to SPDA: (i) high CMA3
(r = 0.33  0.08, for FL3 and r = 0.36  0.08, for FL4). Also, both
binding (HCB), which corresponds to native chromatin; (ii) medium CMA3 bind-
ing (MCB), which corresponds to transitional chromatin; and (iii) low CMA3 DFI were negatively correlated with the percentage of LCB,
binding (LCB), which corresponds to histone replacement completed. Different which represented normal sperm protamine content according
superscript letters (A,B) represent significant differences between protamine of to SPDA (r = 0.21  0.09, for FL3 and r = 0.26  0.08 for
the sperm samples of different testicular sections.
FL4). These correlations suggested an association between lower
sperm protamine content and sperm DNA damage.
Correlations between DFI and traditional semen parameters
and caput epididymidis in comparison to spermatozoa from the were also observed. Both DFI FL3 and FL4 were negatively corre-
cauda epididymidis (z = 5.73, p < 0.0001 and z = 3.66, p < 0.001, lated with PNS (p < 0.05). Further, DFI measurements were pos-
respectively). The percentage of MCB was significantly higher in itively correlated with PAH. Correlations between DFI FL4 and
spermatogenic cells from the caput epididymidis in comparison Mass or Mot were significant and negative, whereas DFI FL3 cor-
to cells from the testicular parenchyma or spermatozoa from the relations with these traits were in the same direction but not
cauda epididymidis (z = 4.20, p < 0.0001 and z = 3.76, p < 0.001, significant (Table 3).
respectively). The percentage of LCB was significantly higher in As there was a small variation in the age of bulls at the time of
spermatozoa from the cauda epididymidis in comparison to semen collection (ranging from 21 to 25 months of age), it was
cells from the testicular parenchyma or caput epididymidis possible to evaluate the effect of age on sperm protamine con-
(z = 4.96, p < 0.0001 and z = 4.51, p < 0.0001, respectively). tent and sperm DNA damage (Table 3). Age of bull at the time of
Finally, no significant differences on CMA3 binding were identi- semen collection (Age at BBSE) was positively correlated with
fied between different cell samples from testicular sections when LCB and negatively correlated with HCB. These correlations
disulphide bridges of the DNA were disrupted by DTT (positive indicated a possible effect of age in the protamine content
control). In summary, accessibility of the DNA minor grooves assessed by SPDA. No effect of age was observed for SCSA
reduced significantly (CMA3 binding decreased) as spermato- measurements.
genic cells matured, histones were replaced by protamines and Possible effects of scrotal circumference on SCSA and SPDA
chromatin condensation increased in the trajectory from the tes- results were also evaluated (Table 3). At approximately 18 and
ticular parenchyma to the cauda epididymidis. 24 months of age, SC was negatively correlated (p < 0.05) to

Table 2 Descriptive analysis including mean, stan-

Traits Description Mean SD Max Min dard deviations (SD), maximum (Max) and minimum
(Min) values for bull breeding soundness evaluation
SC Scrotal circumference
measurements and semen parameters of post-puber-
SC6 Scrotal circumference at 6 months, cm 17.53 1.68 20.50 13.00
SC12 Scrotal circumference at 12 months, cm 21.72 2.47 28.50 16.50 tal Brahman bulls (n = 133)
SC18 Scrotal circumference at 18 months, cm 27.28 2.79 33.50 19.50
SC24 Scrotal circumference at 24 months, cm 30.89 2.69 36.50 24.00
BBSE at 24 m Bull breeding soundness evaluation at 24 months
Vol Volume of ejaculate, mL 7.12 2.31 14.00 2.00
Mass Mass activity, visual score 1–5 2.91 0.87 4.00 0.50
Mot Progressive motility, % 74.71 19.73 98.00 10.00
Conc Concentration of sample, sperm/mL 9106 235.73 266.25 1085.00 4.60
PNS Percentage of morphologically normal sperm, % 74.28 20.54 98.00 7.00
PAH Percentage of sperm with abnormal head, % 8.21 11.29 84.26 0.00
PTD Percentage of total droplets, % 8.22 13.96 81.00 0.00
PPD Percentage of proximal droplets, % 6.11 13.25 80.00 0.00
Age at BBSE Age at semen collection for BBSE, months 23.82 0.79 25.27 21.50
SPDA Sperm protamine deficiency assay
LCB Sperm with high protamine content, % 85.44 16.08 99.05 9.69
MCB Sperm with medium protamine content, % 11.53 14.03 90.08 0.53
HCB Sperm with low protamine content, % 3.03 5.67 32.53 0.08
SCSA Sperm chromatin structure assay
DFI FL3 DNA fragmentation index on FL3 3.80 2.72 12.45 0.33
DFI FL4 DNA fragmentation index on FL4 5.37 5.19 27.73 0.71
HDS FL3 High DNA stainability on FL3 13.40 10.15 43.64 1.32
HDS FL4 High DNA stainability on FL4 14.60 10.70 46.14 1.61

374 Andrology, 2014, 2, 370–378 © 2014 American Society of Andrology and European Academy of Andrology
SPERM CHROMATIN EVALUATION IN CATTLE ANDROLOGY
Table 3 Correlations  standard errors estimated for male reproductive traits: sperm chromatin structure assay (SCSA) and sperm protamine deficiency
assay (SPDA) of post-pubertal Brahman bulls (n = 133)a

Traits SCSA SPDA

DFI FL3 DFI FL4 HDS FL3 HDS FL4 LCB MCB HCB

SC6 (cm) 0.14  0.09 0.10  0.09 0.15  0.09 0.16  0.09 0.02  0.09 0.06  0.09 0.10  0.09
SC12 (cm) 0.04  0.09 0.18  0.09 0.06  0.09 0.06  0.09 0.04  0.09 0.02  0.09 0.17  0.09
SC18 (cm) 0.17  0.09 0.25  0.08 0.13  0.09 0.13  0.09 0.04  0.09 0.03  0.09 0.18  0.09
SC24 (cm) 0.16  0.09 0.19  0.09 0.17  0.09 0.17  0.09 0.05  0.09 0.15  0.09 0.24  0.08
Vol (mL) 0.07  0.09 0.05  0.09 0.10  0.09 0.10  0.09 0.08  0.09 0.10  0.09 0.03  0.09
Mass (score 1–5) 0.11  0.09 0.21  0.09 0.21  0.09 0.22  0.09 0.11  0.09 0.05  0.09 0.18  0.09
Mot (%) 0.15  0.09 0.22  0.09 0.03  0.09 0.03  0.09 0.12  0.09 0.14  0.09 0.01  0.09
Con (106 sperm/mL) 0.08  0.09 0.01  0.09 0.06  0.09 0.06  0.09 0.01  0.09 0.12  0.09 0.26  0.08
PNS (%) 0.21  0.09 0.31  0.08 0.04  0.09 0.04  0.09 0.09  0.09 0.07  0.09 0.08  0.09
PAH (%) 0.19  0.09 0.24  0.08 0.13  0.09 0.13  0.09 0.08  0.09 0.06  0.09 0.09  0.09
PTD (%) 0.05  0.09 0.10  0.09 0.07  0.09 0.07  0.09 0.07  0.09 0.06  0.09 0.05  0.09
PPD (%) 0.07  0.09 0.13  0.09 0.04  0.09 0.05  0.09 0.07  0.09 0.05  0.09 0.06  0.09
Age at BBSE 0.12  0.09 0.07  0.09 0.14  0.09 0.15  0.09 0.20  0.09 0.12  0.09 0.26  0.09
DFI FL3 (%) 0.88  0.04 0.20  0.09 0.20  0.09 0.21  0.09 0.11  0.09 0.33  0.08
DFI FL4 (%) 0.06  0.09 0.07  0.09 0.26  0.08 0.15  0.09 0.36  0.08
HDS FL3 (%) 1.00  0.00 0.08  0.09 0.08  0.09 0.02  0.09
HDS FL4 (%) 0.07  0.09 0.08  0.09 0.01  0.09
LCB (%) 0.94  0.03 0.50  0.08
MCB (%) 0.18  0.09

Traits’ abbreviations defined in Table 1. Bold numbers indicate significant correlations (p < 0.05).
a

HBC and DFI FL4. The correlations between SC18 and SC24 with
DFI FL3 were not significant (p > 0.05) but were also negative
(Table 3). The correlations between SC and semen parameters
(Mot Mass, Conc, PAH, PTD and PPD) observed were also out-
lined in Table 3.
DISCUSSION
This study has assessed sperm chromatin to establish the cor-
relation between sperm protamine content and sperm DNA
damage in B. indicus bulls. The reorganization of sperm chro-
matin is initiated during the post-meiotic phase of spermatocy-
togenesis (Sassone-Corsi, 2002). The replacement of histone
proteins to testis specific histone variants occurs during sper-
matogenesis (Churikov et al., 2004). Next, during spermiogene-
sis, histones are lost, replaced by transitional proteins and then
ultimately replaced by protamines (Lee & Cho, 1999; Kierszen-
baum, 2001; Zhao et al., 2004). The complete replacement of
histones to protamines and final compaction of chromatin is
observed in mature spermatozoa in the cauda epididymis and
therefore in the ejaculated spermatozoa. The sperm protamines
are required to maintain highly condensed sperm DNA packag-
ing and provide structural stability (Krawetz & Dixon, 1988;
Toshimori, 2003; Miller et al., 2010). The rigidity and stability of
the condensed chromatin structure in mature spermatozoa pro-
tects the paternal genome that is transported and delivered to
the oocyte (Brewer et al., 2002). As protamines are essential to
sperm chromatin structure, reduced sperm protamine content
may contribute to the susceptibility of sperm DNA damage and
there is some evidence of this reported in humans (Simon et al.,
2011). Results presented here support and extend this hypothesis
to the bovine species.
An optimized assay for bovine spermatozoa was required to
study the relationship between sperm protamine content and
sperm DNA damage. Protamines bind to the minor groove of the
GC-rich sequence of DNA (Balhorn, 1982). The fluorochrome
CMA3 forms an ion co-ordinated dimer, which binds to this
minor groove of the GC-rich DNA sequence mediated by a single
divalent metal ion, such as Mg2+ (Evenson et al., 1986; Bianchi
et al., 1996; Hou et al., 2004; Simoes et al., 2009). In mature
spermatozoa, where DNA is compacted and stabilized by
protamines, the GC-rich minor grooves are masked by the
argenine-rich sequence of the protamines, and therefore they
are inaccessible to CMA3 binding. Because CMA3 is unable to
bind when protamines are present, this staining is used as an
indicator for protamine deficiency (Tavalaee et al., 2010; Fathi
et al., 2011). Protamine deficiency is traditionally assessed by
fluorescence microscopy or flow cytometry by the presence or
absence of the emission spectrum of CMA3 observed with visible
light excitation. However, CMA3 fluorochrome has the ability to
fluoresce across the ultraviolet spectra as well as the visible light
spectra, which are concentration dependent (Hayasaka & Inoue,
1969). This study utilizes the dual spectral properties of the
CMA3 and the SPDA was optimized by the additional UV excita-
tion and absorption to explore the properties of CMA3 binding
for the assessment of sperm protamine content in more detail
(Experiment 1, Supplementary 1). To verify this method of analy-
sis by flow cytometry, cell suspensions from three sections of the
testis were extracted to represent three perceived levels of DNA
minor groove accessibility, which are related to three stages of
histone to protamine replacement and chromatin condensation
processes. This new method allowed the observation of higher
variations in protamine content than those previously reported
by microscopy in bulls (Simoes et al., 2009). Using SPDA, it was
observed that cells from testicular parenchyma and caput epidi-
dymidis had higher levels of CMA binding in comparison to
spermatozoa from cauda epididymis samples. According to the
progression of spermiogenesis, we expected cells from testicular
parenchyma to have the highest level of CMA3 binding, because
this sample included somatic cells and spermatogenic cells with
chromatin associated to histones and low levels of condensation.
Therefore, cells from testicular parenchyma were expected to
have the highest accessibility to the DNA minor grooves and

© 2014 American Society of Andrology and European Academy of Andrology Andrology, 2014, 2, 370–378 375
M. R. S. Fortes et al.
lowest protamine content as reported. Conversely, in matured
spermatozoa of the cauda epididymidis, CMA3 binding was
expected to be at its lowest, because chromatin condensation,
protamination and packaging processes were completed. The
CMA3 staining in SPDA was indicative of the lowest protamine
content in cells from testicular parenchyma (maximal staining,
85.35% HCB) and of the highest protamine content in spermato-
zoa from cauda epididymis (minimal staining, 92.64% LCB).
Considering this validation provided by Experiment 1, SPDA
may be a useful tool for studying sperm protamine content and
its relationship with sperm DNA damage in bulls.
Single stranded breaks in sperm DNA are detected as red acri-
dine orange fluorescence in SCSA (Evenson & Jost, 2001). These
breaks (i.e. sperm DNA damage) are generally caused by endo-
nucleases during spermiogenesis and are largely repaired during
the protamination process by the transition nuclear proteins (Ki-
erszenbaum, 2001). Hence, there was an expectation that sperm
DNA damage, which was irreparable at the final stages of pro-
tamination and sperm chromatin condensation, would probably
have some association to protamine content.
The relationship between sperm protamine content and
sperm DNA damage was assessed in Experiment 2. The correla-
tions observed between DFI measurements (SCSA results) and
HPC as well as LPC percentages (SPDA results) established a link
between varying sperm protamine content and varying DFI. The
correlations between sperm protamine content and DFI were
significant, but of medium magnitude (absolute r values ranging
from 21% to 36%). Therefore, our results indicate that low prot-
amine content may be one of multiple factors contributing to
sperm DNA instability and consequent damage in B. indicus
bulls. The implication is that low sperm protamine content may
affect sperm DNA integrity and cause or aggravate male
subfertility.
Heat stress is known to affect sperm DNA integrity, sperm
protamine content and the incidence of abnormal spermatozoa
in the ejaculate. Sperm protamine deficiency and altered sperm
chromatin condensation were observed in bulls exposed to tes-
ticular heat insult (Rahman et al., 2011). Further, heat stressed
spermatozoa can affect DNA methylation reprograming in early
embryonic development and reduce fertilization rates in bulls
(Rahman et al., 2013). Altered methylation patterns have been
observed in humans with subfertility and sperm protamine defi-
ciency (Nanassy et al., 2011). Therefore, sperm protamine defi-
ciency, sperm DNA damage and abnormal methylation patterns
are likely to be interlinked causes of male subfertility that can be
aggravated by heat stress. It is possible to suggest that bulls
would be an interesting model for future research on heat stress
as a cause of sperm protamine deficiency, sperm DNA damage
and abnormal methylation patterns.
Sperm DNA damage and sperm protamine content were cor-
related with some traditional semen quality parameters and
could add information to BBSE. Among BBSE measurements,
PNS has previously been described as the best predictor of field
fertility in bulls (Holroyd et al., 2002). This study confirms previ-
ously reported significant correlations between PNS and DFI
(Fortes et al., 2012a; D’Occhio et al., 2013), although here the
correlation was lower possibly because of breed differences or a
sample effect. In the above mentioned studies, samples were
from tropically adapted bulls, including Tropical Composites
and crossbred bulls, whereas in the present study only Brahman
376 Andrology, 2014, 2, 370–378
ANDROLOGY
bulls were investigated. Sperm DNA damage was also correlated
with mass activity and progressive motility in Brahman bulls,
similarly to previous results reported by D’Occhio et al. (2013).
In Bos taurus bulls, SCSA has been a valuable measure for evalu-
ating sperm quality in relation to fertility, assessed as nun return
rates after artificial insemination (Waterhouse et al., 2006). Cur-
rently, no studies have investigated the correlation between
sperm protamine content and field fertility in bulls. Our results
would suggest that sperm protamine content contributes to field
fertility, in light of its correlations with DFI, mass activity and
sperm concentration. The value of SPDA as a predictor of bull
fertility would need to be confirmed in field trials.
Measurements of SC are commonly used in genetic selection
programs in both the beef and dairy cattle industries, with the
aim to improve bull fertility. Correlations between SC and sev-
eral semen parameters (Mot Mass, Conc, PAH, PTD and PPD)
have been observed in bulls of the Beef CRC population, as dis-
cussed earlier (Corbet et al., 2009, 2011, 2013; Burns et al., 2013).
These observations embody some of the motivation for the use
of SC as an indicator trait associated with bull fertility. Geneti-
cally, SC is correlated with male and female reproductive traits,
including age at puberty of female relatives (Vargas et al., 1998;
Martinez-Velazquez et al., 2003; Fortes et al., 2012b). Larger SC
measurements in young bulls (up to 24 months) serve as an
indicator of sexual development and maturity, which could
explain the negative correlation between SC and sperm prot-
amine content (i.e. HCB percentages). The correlations between
SC and HCB may also contribute to the correlation between SC
and DFI FL4, given the link between DFI and HCB established in
these samples. In simple terms, it seems that larger SC at a
young age contributes to higher sperm protamine content and
lower degree of sperm DNA damage. These comparisons
between SPDA, SCSA and SC may serve as an additional reason
for the use of SC as an indicator trait of bull fertility.
In summary, the optimized SPDA allowed the observation of a
correlation between sperm protamine content and sperm DNA
damage in B. indicus Brahman bulls. This correlation should be
further investigated. Future research should consider investigat-
ing sperm protamine content and sperm DNA damage in the
context of interlinked male fertility factors, not only traditional
semen parameters but also heat stress and sperm methylation
patterns.
ACKNOWLEDGEMENTS
The authors acknowledge that this research used resources
from the Cooperative Research Centre for Beef Genetic Technol-
ogies (Beef CRC). In particular, we acknowledge Dr Richard Hol-
royd for leading the experiments that created the Beef CRC male
fertility dataset and Ms Bronwyn Venus for the sperm morphol-
ogy assessment. We also acknowledge Ms Avril Aspland, who
assisted with flow cytometry analysis. Financial support for flow
cytometry was provided by a start-up grant awarded by the Uni-
versity of Queensland to Dr Marina R. S. Fortes and by Meat and
Livestock Australia (B.NBP.0787).
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of
this article:
Supplementary 1: Flow cytometric data processing details for the sperm
protamine defiffiency assay.
© 2014 American Society of Andrology and European Academy of Andrology