Theriogenology xxx (2014) 1–6

Contents lists available at ScienceDirect

Theriogenology
journal homepage: www.theriojournal.com

DNA fragmentation and sperm head morphometry in cat

epididymal spermatozoa
Valentina Vernocchi a, Maria Giorgia Morselli a, Anna Lange Consiglio b,
Massimo Faustini c, Gaia Cecilia Luvoni a, *
a
Dipartimento di Scienze Veterinarie per la Salute, la Produzione Animale e la Sicurezza Alimentare, Università degli Studi di Milano,
Milano Italy
b
Ospedale Grandi Animali, Università degli Studi di Milano, Lodi, Italy
c
Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano, Milano, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a
Received 7 February 2014 putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle
Received in revised form 7 July 2014 differences in sperm head morphometry might be related to DNA status. Several tech-
Accepted 10 July 2014
niques are available to analyze sperm DNA fragmentation, but they are labor-intensive and
require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm
Keywords:
chromatin dispersion test and developed for spermatozoa of different species, but not for
Cat
cat spermatozoa, became commercially available. The first aim of the present study was to
Epididymal spermatozoa
DNA verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for
Morphometry the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose,
DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxy-
nucleotidyl transferase–mediated nick-end labeling (TUNEL) were compared. The second
aim was to investigate whether a correlation between DNA status, sperm head
morphology, and morphometry assessed by computer-assisted semen analysis exists in cat
epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-
Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable
evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be
independent from all the measured variables of sperm head morphology and morphom-
etry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to
the completion of the standard analysis of fresh or frozen semen used in assisted repro-
ductive technologies.
Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction containing fragmented DNA may be an important param-

eter of semen quality and a useful index of fertility potential.
The evaluation of DNA status is not included in the Spermatozoa with severe DNA damage remain func-
standard semen analysis, but the frequency of spermatozoa tionally intact, with normal fertilizing ability, but a high
incidence of DNA fragmentation results in a significant
decrease in pregnancy rates [1]. The exact mechanism of
Author contributions: GCL and VV contributed to design the study, sperm DNA damage has not yet been clarified, but envi-
analyze the data, and draft the article. MF performed statistical analysis.
ronmental stresses, gene mutations, chromosomal abnor-
Laboratory work was carried out by VV, MGM, and ALC. All authors have
approved the final article. malities, or oxidative damages might be involved [2].
* Corresponding author. Tel.: þ39 02 503 18147; fax: þ39 02 503 18148. Several methods have been developed to assess sperm
E-mail address: [email protected] (G.C. Luvoni). DNA fragmentation such as in situ nick translation,

0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2014.07.014
2 V. Vernocchi et al. / Theriogenology xxx (2014) 1–6

terminal deoxynucleotidyl transferase–mediated nick-end Health, Animal Science and Food Safety, University of
labeling (TUNEL), comet assay, acridine orange test, and Milan, Italy. Informed owner consent was obtained.
the sperm chromatin structure assay [3,4]. However, these Epididymides and vasa deferentia were dissected and
techniques are labor-intensive and require expensive squeezed to collect epididymal fluid in a warmed (37  C)
instrumentations. PBS without calcium and magnesium.
Recently, a kit (Sperm-Halomax) based on the sperm
chromatin dispersion (SCD) test [5] and specifically 2.2. Experimental design
developed for boar [6], bull [7], stallion [8], and dog
semen [9,10], but not for cat semen, became commercially 2.2.1. Experiment 1: Sperm-Halomax assay versus TUNEL test
available. Spermatozoa are immersed in an agarose matrix Epididymal fluid collected from 10 cats was divided into
on a slide and briefly incubated in a lysing solution to two aliquots for the evaluation of DNA status by Sperm-
remove membranes and proteins. DNA fragmentation Halomax for canine spermatozoa (Halotech DNA SL,
produces large halos, whereas sperm with low levels of Madrid, Spain) and TUNEL test (Calbiochem FragEL DNA
fragmentation show circumscribed halos. The evaluation fragmentation detection kit, fluorescent–terminal deoxy-
can be performed by fluorescence or light microscopy. nucleotidyl transferase (TdT) enzyme; EMD Millipore Bill-
This kit would allow the routine assessment of sperm erica, MA, USA).
DNA fragmentation in laboratories dealing with andro-
logical examinations and assisted reproductive technolo- 2.2.2. Experiment 2: Correlation between sperm DNA status,
gies (ARTs). head morphology, and morphometry
Among different methods for the evaluation of sperm Epididymal fluid collected from the remaining 18 cats
DNA fragmentation, TUNEL presents a good correlation was used for the evaluation of DNA status by Sperm-
with the SCD test [4,11] and has been previously used for Halomax, for the conventional sperm head morphology
cat semen [12]. evaluation, and for the sperm head morphometry by CASA.
In cats, significant advances in ART have been achieved,
thanks to the embryo production by intracytoplasmic 2.3. Sperm-Halomax assay
sperm injection (ICSI) of mature oocytes [13]. Sperm se-
lection for ICSI is based on motility and morphology pat- Sperm DNA fragmentation indexes (DFIs) were evalu-
terns, and the evaluation of the DNA status is not generally ated in semen samples diluted in PBS at the concentration
performed [14]. Because the sperm head consists almost of 5  106 to 10  106 sperm/mL and processed following
entirely of DNA, subtle differences in sperm head the manufacturer’s instructions. Briefly, 25 mL of diluted
morphometry might be related to DNA content and orga- samples were added to a vial with 50 mL of low melting
nization, as demonstrated in dogs [15] and humans [16]. To agarose and mixed. Pretreated slides were placed onto a
the authors’ knowledge, similar studies have not been metallic plate that was previously cooled at 4  C. A drop of
performed in cats. the cell suspension (5 mL) was spread onto the treated face
The first aim of the present study was to verify the of the cooled slide, covered with a glass cover slip, and
suitability of Sperm-Halomax assay, specifically developed maintained at 4  C for 5 minutes. The cover slip was
for canine semen, for the evaluation of DNA status of smoothly removed, and the layered sample was covered
epididymal cat spermatozoa. For this purpose, values of with the lysing solution provided in the kit for 5 minutes.
DNA fragmentation obtained with Sperm-Halomax and Finally, the slides were washed for 5 minutes with distilled
TUNEL were compared. The second aim was to investigate water, dehydrated in sequential 70% and 100% ethanol
whether a correlation between DNA status, sperm head baths, and stained for 35 minutes in 1:1 Wright solution
morphology, and morphometry assessed by computer- (Merck, Whitehouse Station, NJ, USA) and phosphate buffer
assisted semen analysis (CASA) exists in cat epididymal (pH 6.88, Merck). When the slides were perfectly dried,
spermatozoa. they were mounted with Eukitt and observed under bright-
These data could contribute to achieve a better diag- field microscopy with 40 magnification lens. A minimum
nosis in case of infertility due to male factors, to obtain a of 500 spermatozoa was evaluated in each sample. Intact
better evaluation of spermatozoa used in ARTs, and to sperm showed a small and compact halo, intensely colored,
refine the epididymal sperm selection criteria for ICSI. around the spermatozoa head. Spermatozoa with frag-
The use of epididymal cat spermatozoa is currently a mented DNA presented a widespread and soft halo of
subject of interest with the purpose of establishing an chromatin dispersion. Spermatozoa showing a halo of
efficient gene banking model for threatened and endan- dispersion were considered to have high DFI (percentage of
gered wild felids and contributing to the preservation of spermatozoa with fragmented DNA over the total number
genetic material from valuable males that die unexpectedly of spermatozoa counted per sample) [17].
or undergo orchiectomy for medical reasons.
2.4. TUNEL test
2. Materials and methods
Sperm DFI was evaluated using a detection kit (Calbio-
2.1. Semen collection chem FragEL DNA fragmentation detection kit, fluorescent–
TdT enzyme; EMD Millipore Billerica). The principle of
Epididymal spermatozoa were collected from 28 tom- Fluorescein-FragEL is that TdT catalyzes the addition of
cats subjected to routine castration at the Department of fluorescein-labeled and -unlabeled deoxynucleotides to the
 V. Vernocchi et al. / Theriogenology xxx (2014) 1–6 3

30 -OH ends generated by endonucleases during apoptotic 2.7. Statistical analysis

degeneration.
Sperm samples were smeared on a slide and air-dried. Results of experiment 1 (Sperm-Halomax assay vs.
Then, the smears were fixed in 4% paraformaldehyde in TUNEL test) have been evaluated by the Bland–Altman plot
PBS for 15 minutes at room temperature and washed technique [20] to assess the agreement between tests,
twice in Tris-buffered saline (TBS) for 15 minutes. The considering the TUNEL procedure as reference method.
slides were then covered with the permeabilization so- In experiment 2, to establish the reference values for the
lution (protein kinase 2 diluted 1:100 in Tris solution morphological sperm variables (area, aspect, perimeter,
10 mM) and incubated for 6 minutes in a moist chamber. dmax, dmin, radmax, radmin, radius ratio, roundness), a
The slides were washed twice in TBS solution and main- nonparametric approach (2.5–97.5 percentile of the distri-
tained in a moist chamber. In the dark, an aliquot of the bution) was followed on 2425 spermatozoa.
equilibration solution (TdT equilibration buffer diluted 1:5 Variables not determined on a single spermatozoon (i.e.,
in sterile water (Sigma Chemical Co., St. Louis, MO, USA)) DFI and head anomalies) were submitted to the calculation
was added to each slide that was incubated for 30 mi- of the 95% confidence interval as indicative reference values.
nutes in a moist chamber at room temperature. After the Aiming to evaluate the multivariate relations between
removal of the equilibration solution, an aliquot of the DFI and the morphological variables, a principal compo-
labeling solution (Fluorescein-FragEL TdT labeling reaction nent analysis (PCA) was applied: data were submitted to
mix diluted 1:20 in TdT enzyme) was added to each slide PCA after normalization and the varimax rotation. The
that was incubated for 90 minutes in a moist chamber at number of retained components was calculated when at
37  C. The slides were washed three times in TBS, and a least the 90% of the total variability was explained. More-
drop of an antifade reagent (Gel Mount; BiØmeda Cor- over, the Pearson univariate correlation between DFI and
poration, Foster City, CA, USA) was added. The slides the morphological variables was calculated (P < 0.05).
covered with a cover slip were examined under fluores-
cent microscope (Eclipse E600, Nikon Corporation, Tokyo,
3. Results
Japan) with 40 magnification lens and oil immersion. A
40 ,6-diamidino-2-phenylindole filter (330–380 nm) was
3.1. Experiment 1: Sperm-Halomax assay versus TUNEL test
used to visualize the total cell population (blue). Using a
fluorescein filter (465–495 nm), a bright green signal
Epididymal cat spermatozoa processed with an SCD test
indicated positive staining (DNA-fragmented sperm cells),
as Sperm-Halomax developed for dogs produce similar
whereas dull green or hard to visualize cells signified a
patterns than those described in dog spermatozoa [9].
nonreactive cell [18]. At least 500 spermatozoa of each
Spermatozoa with unfragmented DNA do not show or show
sample were analyzed randomly to evaluate the per-
very small halos of dispersion of DNA loops, whereas those
centage of TUNEL-positive sperm cells (DFI).
with DNA fragmentation release peripheral halos from the
central core (Fig. 1).
2.5. Conventional sperm head morphology
No differences were observed in DFI values obtained
with Sperm-Halomax and TUNEL (4.34  0.93% vs.
Undiluted samples were stained with a rapid Giemsa-
4.26  0.83%; P ¼ 0.84).
Wright stain (Diff-Quick, Merck), and in each sample, a
The Bland–Altman test was applied to evaluate the level
total of 200 spermatozoa was evaluated under light mi-
of agreement between the TUNEL test and Sperm-
croscope (Diaplan Leitz, Wetzlar, Germany) with 100
Halomax. The results showed that there was a good
magnification lens and oil immersion. Abnormal sperm
agreement between the considered tests, because all points
heads, included those that were pear-shaped, large, small,
lay within the boundaries (Fig. 2).
or amorphous, were recorded.

2.6. CASA sperm head morphometry

The same slides stained for the conventional

morphology were examined for the evaluation of the
sperm head morphometry using a light microscope
(Olympus BX51, Olympus America Inc., Melville, NY, USA)
with 100 magnification lens and oil immersion equipped
with a video camera (Scion Corporation 1394, Frederick,
MD, USA) interfaced to a computer. The software used for
image acquisition and analysis was Image-Pro Plus 5.1;
Media Cybernetics (Immagini & Computer, Bareggio, Italy).
Each sperm head was measured for different parame-
ters: area (mm2), aspect (ratio between major and minor
axes of the ellipse), perimeter (mm), maximum diameter
Fig. 1. Spermatozoa processed with Sperm-Halomax kit and stained with
(dmax, mm), minimum diameter (dmax, mm), maximum Wright solution. Those with a small halo have normal status of DNA and the
radius (radmax, mm), minimum radius (radmin, mm), radius spermatozoon with a large halo contains fragmented DNA. Bar represents
ratio, and roundness [19]. 10 mm.
4 V. Vernocchi et al. / Theriogenology xxx (2014) 1–6

4. Discussion

Present data show that Sperm-Halomax assay, specif-

ically developed for canine semen and based on SCD test,
provides a reliable evaluation of DNA fragmentation of
epididymal feline spermatozoa. Most of the differences
between the DFIs obtained with the Sperm-Halomax assay
and TUNEL test were within the 95% confidence interval
limits, suggesting that the level of agreement between the
two methods of analysis is satisfactory.
The conditions for sperm DNA fragmentation may not
be the same in different animals, mainly because protamine
residues, which form an important part of sperm chro-
Fig. 2. Bland–Altman plot for Sperm-Halomax/TUNEL DNA fragmentation matin, differ between species [21]. It has been found that
index results agreement. The line boundaries indicate the 95% CI of the canine and feline spermatozoa are characterized by only
difference between variables. Diff: difference between DFIs obtained by protamine 1 [22,23], and this could be the reason that the
TUNEL and Halomax. Mean: mean of DFIs obtained by TUNEL and Halomax.
SCD test protocol designed for dogs has resulted equally
efficient in analyzing DFI in cats.
3.2. Experiment 2: Correlation between sperm DNA status,
Cat spermatozoa processed with Sperm-Halomax pro-
conventional head morphology, and CASA morphometry
duce images of similar characteristics to those obtained in
dogs [9,10]. Discrimination of the size of the halos was easy
The calculated reference values for the morphological
to establish in cat sperm samples because the size of the
variables of the sperm head were as follows: area 7.34–
halos of DNA dispersion was large as those obtained in
15.59 mm2; aspect 1.69–2.86; perimeter 10.44–14.87 mm;
dogs.
dmax 4.09–6.19 mm; dmin 1.90–2.96 mm; radmax 2.12–
In the present work, DFI of cat epididymal spermatozoa
3.20 mm; radmin 0.90–1.44 mm; radius ratio 1.83–3.24;
ranged from 2.4% to 5.7%. These values are in agreement
and roundness 1.11–1.44.
with those reported in the literature and obtained with
The 95% confidence intervals for DFI and head anoma-
different methods [12,24].
lies evaluated with conventional analysis were 0.037% to
In humans, semen with 30% of spermatozoa with frag-
0.044% and 0.034% to 0.047%, respectively.
mented DNA is considered of low or poor quality to be used
The results for PCA analysis are reported in Table 1; the
in assisted reproduction [25]. In feline sperm samples,
first three components account for the 96.62% of the total
additional data are necessary to establish a solid threshold
variability. In particular, the morphological variables are
value of this parameter.
mainly expressed in the first two principal components
To the authors’ knowledge, this is the first time that
(PC) with high correlations. The third component is rep-
the relationship between conventional sperm head
resented by the DFI only, accounting for the 7.6% of the total
morphology, CASA morphometry, and DNA status has been
variability. Being the PC orthogonal vectors, DFI seems to be
assessed in cat spermatozoa.
independent from the other measured variables. The
With CASA system, the post-acquisition processing of
multivariate results are confirmed by the calculation of the
digitalized data offers an objective and detailed character-
Pearson correlation coefficients: none of the r coefficients
ization of several sperm morphometric parameters that
resulted significant.
cannot be detected by conventional visual evaluation. In
the present study, the analysis of more than 2400 sper-
Table 1
Principal component analysis (PCA) loadings for morphological variables
matozoa representing 18 mature tomcats would also
and DNA fragmentation index (DFI) on the first three components. contribute to the definition of normal values of morpho-
metric measurements that can be used as a background for
Attribute PC 1 (51.91%) PC 2 (37.29%) PC 3 (7.63%)
further extended studies aimed at better investigating the
Roundness L0.93238 0.34192 0.05891
phenomenon of teratozoospermia in this species.
Radius ratio L0.91686 0.37916 0.06608
Aspect L0.90646 0.39293 0.09087 Present data indicate that DFI is independent from
Radius maximum L0.88915 0.45212 0.01863 sperm head morphology and morphometry. This finding
(mm) confirms what has been demonstrated in boar [26], but it is
Diameter maximum L0.88565 0.45974 0.00636 in contrast with the general assumption that head shape is
(mm)
Head anomalies (%) L0.83809 0.14168 0.01752
mainly related to the status of sperm DNA due to the fact
Perimeter (mm) L0.74541 0.66100 0.03964 that most of the sperm head is compacted chromatin.
Diameter minimum 0.23555 0.96457 0.10689 Significant relationships among sperm morphometry and
(mm) the percentage of denatured DNA have been described in
Radius minimum 0.26324 0.95855 0.08731
dogs [15,27], bulls [28], brown bears [29], and humans [30].
(mm)
Area (mm2) 0.43677 0.89489 0.07500 In feline epididymal spermatozoa, it has been previously
DFI (%) 0.17188 0.41807 0.89131 shown that head abnormalities are strongly correlated
The loadings represent the correlation with the corresponding PC. Load-
with, and could accurately predict, sperm DNA defects
ings with values greater than 0.7 are bold typed. In brackets on the revealed by TUNEL test [12]. However, the conventional
headers: variability explained by the PC. evaluation of sperm head morphology by diff-quick
 V. Vernocchi et al. / Theriogenology xxx (2014) 1–6 5

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