The Open Veterinary Science Journal, 2008, 2, 7-10 7
1874-3188/08 2008 Bentham Science Publishers Ltd.
Sperm DNA Fragmentation in Rams Vaccinated with Miloxan
J. Gosálvez*,1, J. M. Vázquez2, M. Enciso1, J.L. Fernández3, A. Gosálbez1, J. R. Bridle4 and
1Departamento de Biología, Unidad de Genética, C/Darwin nº 2, Universidad Autónoma de Madrid, Madrid 28049,
2Ovigen, Granja Florencia, Zamora 49800, Spain
3Sección de Genética y Unidad de Investigación, Complejo Hospitalario Juan Canalejo, As Xubias, Coruña, Spain
4School of Biological Sciences, University of Bristol, BS8 1UD, UK
Abstract: Sperm DNA fragmentation was analysed in 113 semen samples obtained from different rams over a period of
one year. Semen samples were collected from: unvaccinated rams between January and June (Control group 1; CG1);
vaccinated rams at least 70 days after vaccination between October and December (Control group 2; CG2); vaccinated
rams 20 days after vaccination (Vaccinated group 1; VG1); and vaccinated rams 40 days after vaccination (Vaccinated
group 2; VG2). Results show Miloxan, the vaccine of interest in this study, increased the percentage of sperm cells with
fragmented DNA by 10-fold on average (from 6.5±7.9 to 63.4±24.2). However, the negative impact of vaccination on
sperm DNA fragmentation appeared to reversible; near normal levels of sperm DNA fragmentation had been restored 40
days after vaccination (21.7±10.6). On the basis of these data, the use of semen samples from vaccinated animals should
be avoided until at least one month after vaccination.
of numerous studies because loss of fertility has been shown
to be highly correlated with a high frequency of nuclei con-
taining damaged DNA in human  and in other mammals
[2, 3]. However, based on human studies, the relationship
between a high frequency of sperm containing fragmented
DNA and the capacity of the male to generate fertilization is
controversial . The origin of DNA fragmentation is
mostly unknown, although apoptosis, oxidative stress, or
persistence of DNA breaks produced during the chromatin
protamination process in spermiogenesis, could be direct
causes. It is also known that some external effectors such as
exposure to environmental pollutants, smoking, or genetic
characteristics such as a massive reduction of telomeric
DNA sequences, may trigger sperm DNA fragmentation [5,
6]. The assessment of SDF in organisms used for reproduc-
tive purposes is therefore of interest, especially when the
individuals concerned have encountered a stress episode.
This is because the assessment of classical parameters of
sperm quality does not fully correlate with the sperm DNA
quality [7, 8]. In particular, SDF should be periodically as-
sessed along the reproductive life of those animals used re-
peatedly to retrieve semen samples for assisted reproduction,
in order to avoid using damaged sperm for fertilization.
Sperm DNA Fragmentation (SDF) has been the subject
controlling the organism’s normal development with a clear
detrimental effect on individual fitness. In terms of reproduc-
tion, it is known that infectious agents can affect embryonic
Infectious agents may interfere with genetic programs
*Address correspondence to this author at the Departamento de Biología,
Unidad de Genética, C/Darwin nº 2, Universidad Autónoma de Madrid,
Madrid 28049, Spain; E-mail: firstname.lastname@example.org
survival, ovulation rates and fertilization [9,10]. From a
similar viewpoint, immunological infertility in males has
been associated with presence of auto- or iso-antibodies and
their effect on sperm proteins. These antibodies can prevent
the accessibility of spermatozoal proteins at various stages
during the fertilization process and therefore induce infertil-
ity. A number of sperm-specific proteins are therefore being
considered as candidates for the development of vaccines for
reducing fertility [11,12].
germs or toxins, when injected to living animals they may
produce side-effects, generally through the activation of the
immune system. Vaccination may affect the various standard
seminal parameters such as sperm motility, concentration
and production rate of abnormal forms . The aim of the
present investigation was to analyse if subcutaneous vaccina-
tion of rams with Miloxan (Clostridium perfringens type C,
D and C. oedematiens type B) had any effect on the percent-
age of sperm cells with fragmented DNA.
Although vaccines are composed of weakened, dead
MATERIAL AND METHODS
Semen Samples, Collection and Preparation
individuals from four main different races of ram (Castel-
lana, Assaf, Lacaune and Churra) collected along one year
were included in the analysis. All animals belong to the same
Genetic Centre (Ovigen, Zamora, Spain). The animals were
vaccinated with Miloxan (Clostridium perfringens type C, D
and Clostridium oedematiens type B; Merial, Lyon, France).
Two ml were subcutaneously administered to each animal.
Sperm samples included in the analysis were collected at
different periods throughout the year and classified into four
groups: Control Group 1 (CG1): sperm samples obtained
from January to June from unvaccinated animals. Vaccina-
One hundred and thirteen semen samples of different
8 The Open Veterinary Science Journal, 2008, Volume 2 Gosálvez et al.
tion Group 1 (VG1): sperm samples obtained in July 20 days
post-vaccination. Vaccination Group 2 (VG2): sperm sam-
ples obtained in August 40 days post-vaccination. Control
Group 2 (CG2): sperm samples obtained in October to De-
cember at least 70 days post-vaccination. Unfortunately,
during the experiment, it was only possible to assign semen
samples to particular animals in 35% of cases. Samples
within each pre-established groups were therefore considered
to be independent clusters of individuals.
ficial vagina. Immediately after collection, the semen was
diluted to a final concentration of 43x108 spermatozoa/ml
with a commercial egg yolk-based extender (Triladylt; Mini-
tube Canada, Woodstock, Canada) and frozen using 0.2 ml
straws. Only sperm samples considered of sufficiently high
quality for standard parameters, including volume, concen-
tration, motility, acrosomes and positive endosmosis, are
currently frozen at the Ovigen Center. All animals were aged
2 to 4 years, healthy, and controlled for feeding, housing,
and photoperiod conditions. These stocks are currently being
maintained for breeding purposes.
All semen samples were collected with the use of an arti-
Sperm DNA Fragmentation Analysis
mented DNA, i.e. DNA Fragmentation Index (SDFi), the
Sperm-Halomax® kit (Halotech-DNA SL, Madrid, Spain)
was used. This methodology is a variant on the SCD (Sperm
Chromatin Dispersion) test and has been used to assess
sperm DNA fragmentation in mouse , pig [14, 15] and
bull . For each experiment, frozen straws were thawed
and different aliquots were prepared to a final concentration
of 10-15x106 spermatozoa/ml. Twenty five microliters of
semen diluted to 10-15x106 spermatozoa per millilitre were
added to a vial containing low melting point agarose and
mixed. Pre-treated slides were placed onto a metallic plate
cooled to 4ºC. A drop of the agarose-sperm mix was then
spread onto the treated face of the slide and covered with a
glass coverslip for 5 minutes at 4ºC. The coverslip was
smoothly removed, and the slide was horizontally placed in
10 ml of the lysing solution provided in the kit, to remove
membranes and proteins. Finally, slides were washed for 5
min, dehydrated in sequential 70, 90 and 100% ethanol
baths. The sperm were then stained for fluorescence micros-
copy with DAPI in Vectashield Mounting Medium H-1000
(Vector, USA) followed by 2, 7 - dibrom - 4 - hydroxyl -
mercuryfluorescein di-sodium salt (DMF; Sigma, USA). The
dual emission fluorochrome combination used in the present
experiment, allowed simultaneous visualization of DNA
(blue) and proteins (green) respectively using a dual band
pass fluorescence filter block or, alternatively, a single emis-
sion could also be observed using a single band pass fluores-
cence filter block.
To determine the percentage of sperm cells with frag-
counting using the Sperm Class Analyzer DNA module (Mi-
croptic SL, Barcelona, Spain). The SCA software discrimi-
nated between spermatozoa that showed a small halo of
spreading of chromatin loops (sperm containing unfrag-
mented DNA) and large or medium halo of diffused chroma-
tin fragments (sperm containing fragmented DNA). This
software was coupled to a Leica DMLA (Leica SA, Ger-
many) motorized fluorescence microscope, controlled with
Fluorescence microscopy was used for automatic sperm
Leica based software, for automatic scanning and image
digitalization of each slide. Magnification lenses 20x were
used for facility of cell counting. A minimum of 800 sper-
matozoa per sample were counted. The SDFi (percentage of
sperm cells with fragmented DNA) was then calculated. Sta-
tistical analysis was performed using Minitab version 14 for
RESULTS AND DISCUSSION
alization showed basically two different spermatozoa mor-
phologies. Those nuclei that remained compact or displayed
very small halos of chromatin spreading corresponded to
spermatozoa harbouring unfragmented DNA (Fig. 1a,c). By
contrast, those sperm nuclei which exhibited medium or
large peripheral halos of diffusion of chromatin spots (Fig.
1b,d), contained fragmented DNA .
Sperm samples processed for DNA fragmentation visu-
Fig. (1). Visualization of the sperm DNA fragmentation in ram
under fluorescence-microscopy. (a) Sperm sample showing a low
SDFi. (b) Sperm sample of an individual after 20 days of vaccina-
tion where most of the sperm cells exhibit a large dispersion of the
chromatin. (c) Selected nuclei from a sperm cell containing
unfragmented DNA. (d) Selected nuclei from a sperm cell contain-
ing fragmented DNA. In all cases, blues colour corresponds to
DNA, whereas protein fluoresces in green and is mainly labelling
the flagelum and sperm core after the SCD test.
June), was from individuals presenting nearly 0 % of sperm
DNA fragmentation, to others exhibiting about 41% % of
SDFi,. The mean value of SDFi or this period was 6.4+7.9.
The animals studied between October and December (CG2)
showed a similar distribution of SDFi values (5.7+4.5) and
significant differences between both periods of analysis were
not obtained based on arc-sine transformed data (one-way
ANOVA: F = 0.16; p = 0.7 for 1 and 68 df). The condition of
SDF 20 and 40 days post vaccination was very different
(Fig. 2). SDFi post vaccination (VG1) significantly increased
with a mean value of 63.4+24.2 (one-way ANOVA, com-
pared to CG1 and CG2 combined: F = 484.9; p<0.0001 for 1
and 101 df).
The observed range of SDFi in the CG1 (January to
Sperm DNA Fragmentation in Rams Vaccinated with Miloxan The Open Veterinary Science Journal, 2008, Volume 2 9
Fig. (2). Descriptive statistics (box-and-whisker plots) of sperm
DNA fragmentation index in the four established groups (CG: Con-
trol Group; VG: Vaccinated group). See text for details.
with respect to the VG1 score (one-way ANOVA: F = 37.6;
p<0.0001 for 1 and 41 df), but was still significantly in-
creased, with a 3.6 fold increase in mean compared to the
two outside controls (one-way ANOVA: F = 36.8; p<0.0001
for 1 and 78 df). The mean SDFi value (21.7+10.6) for VG2
was also still very high compared with the standard values.
Forty days post vaccination (VG2) the SDFi decreased
1) vaccination can temporarily increase sperm DNA frag-
mentation in ram and 2) sperm DNA fragmentation levels
can be restored to normal levels after being elevated follow-
ing an acute stress event, such as vaccination. Practically, the
sudden large increase in the percentage of sperm cells with
fragmented DNA after vaccination is another reason to avoid
using sperm samples retrieved from animals for artificial
reproduction until at least, one month or forty days following
Two main conclusions may be obtained from this work
by infectious agents as well as by vaccination episodes
against those same agents . In boar  and in ram ,
the percentage of forward movement and normal spermato-
zoal morphology and motility were significantly reduced in
vaccinated animals. In the male germ line, only spermio-
genesis is affected. Meiosis is also likely to be affected by
the collateral effects of vaccination, as suggested by the in-
creased occurrence of univalents involving autosomes, as
well as sex chromosomes, in a group of mice which were
antirabic vaccinated, in comparison with control stocks .
In this situation, vaccines could be considered as clastogenic
effectors. In the case analyzed here, the time of recovery to a
normal rate of sperm DNA fragmentation seems to be related
to duration of spemiogenesis, more than to possible muta-
tions or alterations occurring during meiosis. Duration of
spermiogenesis varies in different species but is quite con-
stant within each species. Thus, in ram the spermiogenesis
encompasses around 18 days, while in bull and stallion it is
about 21 days [20, 21]. The timing here for sperm to reach
the maximum levels of sperm DNA fragmentation, coupled
with the quick recovery of sperm quality, seems to indicate
that spermiogenesis rather than spermatogenesis was af-
It is known that germ line cells are significantly affected
fected by vaccination. If meiotic cells or gonial cells were
affected the time to recover normal cells would be longer
than observed here. In the case analysed here from 20 to 30
days was the delay before SDF started to decrease. This sug-
gests that histone/protamine replacement and assembly dur-
ing sperm maturation were affected. Interestingly, when the
effect of foot-and-mouth disease vaccination on the various
seminal attributes of buffalo bulls was studied, it was ob-
served that vaccination also produces adverse effects on the
standard seminal parameters, up to one month after vaccina-
tion . This duration for semen quality recovery is quite
similar to that reported here.
fully understood. Probably the type of vaccine and genetic
background of the animal is determinant of the immune reac-
tion of the individual. Inoculation of live Brucella ovis into
the epididymus of rams generated intense epididymal le-
sions. In addition, an increased numbers of neutrophils,
macrophages and lymphocytes were detected, being indica-
tive of induced inflammatory response. Unfortunately, such
effects on spermatozoa were not determined in this experi-
ment . However, it is known that leucocytospermia pro-
duces oxidative stress that may result in DNA damage [23,
24]. Additionally, vaccination may trigger elevated tempera-
ture, either locally through the inflammation process, or gen-
erally, inducing fever in some cases. In fact, concentration,
motility and morphology may be adversely affected by fever,
especially during the period of spermiogenesis . Also
sperm chromatin structure seems to be affected by an in-
crease of temperature, causing extensive DNA strand breaks,
altering protein synthesis  and decreasing the protamine
disulfide bonding . Interestingly, in bulls, scrotal mild-
thermal stress caused sperm morphological abnormalities
after 11 days after insulation . They increased during the
following days, decreasing near 40 days after scrotal insula-
The effect of vaccinations on semen parameters is not
larly on sperm DNA integrity probably consists of many
factors and effectors, such as the genetic background, and the
capacity to respond to oxidative stress or temperature varia-
tions, that finally determines a response for each individual.
Substantial interactions between these factors could be an
explanation for the high levels of variance in response for
sperm DNA fragmentation values observed for animals post-
The effects of vaccination on sperm quality and particu-
lous technical assistance. This work has been supported by
grants from the MEC Petri 480PTR1995-0907-OP, BFU
2007-66340/BFI and CGL2005-02898/BOS.
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Received: November 12, 2007
Revised: December 10, 2007 Accepted: January 01, 2008