Short communication

In vitro survival and lipid peroxidation status of rabbit spermatozoa

after both chilled and frozen storage in lycopene enriched extenders
M.P. Rosato
a,n
, M. Di Iorio
a
, A. Manchisi
a
, M. Gambacorta
a
, G. Petrosino
a
, G. Centoducati
b
,
M.P. Santacroce
b
, N. Iaffaldano
a
a
Department of Animal, Plant and Environmental Sciences, University of Molise, via De Sanctis snc, 86100 Campobasso, Italy
b
Department of Public Health and Animal Sciences, University of Bari, Str. Prv. Casamassima, km 3, Valenzano, 70010 Bari, Italy
a r t i c l e i n f o
Article history:
Received 27 October 2011
Received in revised form
16 February 2012
Accepted 13 March 2012
Keywords:
Rabbit
Spermatozoa
Lycopene
Lipid peroxidation
Refrigeration
Cryopreservation
a b s t r a c t
The effects of lycopene-enriched extenders on motility, viability, osmotic resistance,
DNA integrity and lipid peroxidation of rabbit sperm were examined after both chilled
and frozen storage. Five pools of semen diluted in extenders containing 0, 0.05 or
0.1 mg/mL of lycopene were refrigerated at 5 1C for 48 h or cryopreserved. Sperm
quality was generally compromised after storage, especially post-freezing, however
lycopene limited the amount of sperm lipid peroxidation after chilling and freezing. In
chilled sperm, the highest dose of lycopene was provided to maintain the viability,
acrosome and DNA intactness similar to that of fresh semen and contained the
reduction of sperm motility, whereas in cryopreserved semen lycopene protected the
DNA integrity of sperm, even if not in a dose-dependent way. So lycopene diminished
sperm lipid peroxidation during refrigeration and cryopreservation, prolonging the
survival of rabbit sperm after liquid storage, but it had a limited effect on sperm
cryosurvival.
& 2012 Elsevier B.V. All rights reserved.
1. Introduction
Rabbit breeding for meat production is almost exclu-
sively dependent on articial insemination (AI) programs,
thus it would greatly benet if semen could be stored after
collection and subsequently used for AI without affecting
fertility. Unfortunately the ability of rabbit sperm to survive
in vitro after chilled (Rosato and Iaffaldano, 2011; Iaffaldano
et al., 2010) or frozen storage (Moce and Vicente, 2009) is
limited. This is in part due to lipid peroxidation caused by a
supra-physiological level of reactive oxygen species (ROS),
which affects sperm lipids, proteins, nucleic acids and
sugars (Bansal and Bilaspuri, 2011). The majority of ROS
are continuously neutralized by antioxidants contained in
rabbit semen itself (Mourvaki et al., 2010), but endogen-
ous antioxidants are insufcient to counteract the lipid
peroxidation that occurs during sperm storage (Castellini
et al., 2000). Several attempts have been made with
dietary supra-nutritional antioxidants supplementation
aimed to enhance rabbit sperm quality (Gliozzi et al.,
2009; Yousef et al., 2003; Castellini et al., 2002) or its
survival during refrigeration (Castellini et al., 2000), but
reports evaluating the efcacy of a direct addition of
antioxidants in the rabbit seminal extenders are lacking.
Lycopene is the most abundant carotenoid in tomatoes
and red fruits and it is considered the most efcient
antioxidant of the carotenoids (Di Mascio et al., 1989).
In reproductive biology it has attracted attention for its
role in protecting sperm from oxidative damage and
signaling male fertility (Mangiagalli et al., 2010; Zini
et al., 2010; Uysal and Bucak, 2007; Gupta and Kumar,
2002). However, the effects of lycopene-supplemented
extenders on the survivability of sperm cells after either
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/livsci
Livestock Science
1871-1413/$ - see front matter & 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.livsci.2012.03.006
n
Corresponding author. Tel.: 39 874 404707; fax: 39 874 404855.
E-mail addresses: mariapina.rosato@unimol.it,
mapirosato@hotmail.com (M.P. Rosato).
Livestock Science 146 (2012) 199202
chilled or frozen storage have not yet been explored in the
rabbit. The present study was designed to assess the
effects of lycopene supplementation of semen extenders
on sperm quality and lipid peroxidation in refrigerated
and cryopreserved rabbit semen.
2. Materials and methods
Two ejaculates per male were collected with an
articial vagina from twelve mature rabbit bucks (Centro
Genetica Martini line, Italy; average age: 14 months)
weekly trained for semen collection in a private breeding.
Bucks were housed in at deck cages with 16 h light/d
and were fed a commercial standard diet. Only ejaculates
presenting white opalescent appearance and a volume
higher than 0.5 mL were utilized and pooled (46 ejacu-
lates/pool; 5 pools in total). Each pool was divided into
seven 500 mL aliquots that were used to prepare one
of the seven treatments as follows: the rst aliquot was
left undiluted; three aliquots were diluted vefold in a
Tris-citrate-glucose (TCG) basic extender (Rosato and
Iaffaldano, 2011) containing 0, 0.05 or 0.1 mg/mL of lycopene
(Sigma L9879) from a stock solution (Srinivasan et al., 2007);
the remaining three aliquots were diluted 1:1 in a freezing
medium (TCG basic extender and 3.5 M of DMSO and 0.1 M
of sucrose, as the cryoprotectants, to reach a nal concentra-
tion of 1.75 M DMSO and 0.05 M sucrose in diluted semen)
containing 0, 0.05 or 0.1 mg/mL of lycopene. The undiluted
semen was immediately tested as fresh semen. The semen
samples diluted in TCG basic extender were refrigerated in
an incubator at 5 1C for 48 h. Semen samples diluted in the
freezing extender were cryopreserved according to Moce
et al. (2005): in brief they were packaged in 0.25 mL plastic
straws, cooled to 5 1C within 45 min, frozen 5 cm above the
liquid nitrogen level (130 1C) for 10 min and stored in a
liquid nitrogen tank (196 1C). The straws were thawed
two weeks later into a water bath at 50 1C for 12 s. Lipid
peroxidation, motility, viability, osmotic-resistance, acro-
some and DNA integrity were evaluated in fresh, chilled
and frozen/thawed semen, in duplicate. Sperm lipid
peroxidation was measured as malonaldehyde (MDA)
concentration, as described by Kadirvel et al. (2009)
and determined from the molar absorption coefcient
of MDA at 535 nm (1.5610
5
mol/L cm
1
). Mass and
forward motility, sperm viability, the functional compe-
tence of the sperm membrane (osmotic-resistance) and
acrosome integrity were evaluated according to Rosato
and Iaffaldano (2011). DNA integrity was evaluated with
the Acridine orange test described by Gandini et al.
(2006). Data were analyzed using ANOVA and Duncan
comparison test (SPSS 15.0 for Windows, 2006; SPSS,
Chicago, Ill). Pearsons correlations between MDA produc-
tion and sperm quality parameters were also tested.
Signicant differences were set at Po0.05.
3. Results
The semen quality and lipid peroxidation status
recorded in the freshly collected, chilled and frozen semen
samples processed in the absence or presence of different
amounts of lycopene in the extender are provided in
Table 1. MDA values no different from those of fresh
semen samples were found in semen chilled with both
doses of lycopene. Higher concentrations of MDA were
found in frozen sperm compared to those of both fresh
and liquid stored samples (Po0.05), with signicant
higher values of MDA in semen frozen without lycopene
with respect to those frozen with lycopene. In chilled sperm
mass and forward motility were signicantly reduced by
storage (Po0.05), but they were higher (Po0.05) in semen
extended with the highest dose of lycopene compared to
that stored in semen chilled without lycopene. No differ-
ences were found between viability, sperm osmotic-resis-
tance and DNA integrity percentages of fresh semen and
those of semen chilled stored with 0.1 mg/mL lycopene.
Frozen storage reduced signicantly motility, viability,
osmotic-resistance and acrosome integrity (Po0.05) inde-
pendently by treatment, with the exception of sperm DNA
integrity, where the semen enriched with 0.05 mg/mL
lycopene showed a DNA integrity not different from that
stored in fresh spermatozoa. In Fig. 1 it is possible to observe
a signicant inverse correlation between MDA levels and all
the sperm quality parameters (Po0.05), but DNA integrity
(P0.19).
4. Discussion
Our ndings showed that even if the quality of rabbit
semen was generally reduced and lipid peroxidation
Table 1
Sperm quality and MDA data (mean7SE) recorded in pooled samples of rabbit semen freshly collected and after chilled or frozen storage in extenders
lacking or containing different amounts (mg/ml extender) of lycopene (n5).
Semen treatment Sperm variable
MDA
nmol/10
8
sperm
Mass
motility (%)
Progressive
motility (%)
Viability (%) Osmotic
resistance (%)
Acrosome
integrity (%)
DNA
integrity (%)
Fresh undiluted 1.3270.03
d
9171.31
a
6871.01
a
8571.78
a
6871.56
a
9271.72
a
98.870.28
a
Chilled in lycopene 0 1.8170.10
c
5275.83
c
2873.74
bc
7075.52
b
5273.08
b
8571.60
b
97.170.22
c
Chilled in lycopene 0.05 1.6770.08
cd
6478.12
bc
3676.01
b
6874.86
b
5773.16
ab
8872.42
ab
98.270.23
ab
Chilled in lycopene 0.1 1.5770.04
cd
6874.89
b
3873.84
b
7773.65
ab
5574.10
b
9071.41
ab
98.170.33
ab
Frozen in lycopene 0 2.8170.22
a
2771.58
d
2171.22
c
3174.68
c
3277.03
c
5172.63
c
97.870.34
b
Frozen in lycopene 0.05 2.2370.17
b
3374.63
d
2474.30
c
3475.16
c
3475.11
c
5472.37
c
98.370.04
ab
Frozen in lycopene 0.1 2.2570.15
b
3673.20
d
2472.45
c
4175.37
c
3573.61
c
4971.31
c
97.970.27
b
adDifferent superscript letters within the same column indicate a signicant difference (Po0.05).
M.P. Rosato et al. / Livestock Science 146 (2012) 199202 200
increased by both chilled and frozen storage, lycopene
addition to extenders effectively reduced the amount of
lipid peroxidation in sperm after storage. This protective
effect improved the overall quality of liquid stored sper-
matozoa, whereas in cryopreserved semen it was limited
in protecting the DNA integrity of sperm, even if not in a
dose-dependent way. The lack of strategies to effectively
maintain the lifespan of rabbit spermatozoa during refrig-
eration or cryopreservation has been a major deterrent to
the widespread use of stored semen in AI programs. The
reduced lifespan of stored rabbit sperm has been
hypothesized to be related to the phospholipid composi-
tion and polyunsaturated components of the plasma
and organelle membranes, inuencing their uidity
and susceptibility to oxidation (Moce and Vicente, 2009;
Castellini et al., 2000). This hypothesis is conrmed by
this investigation, since we found a negative correlation
between the level of lipid peroxidation and all the sperm
qualitative characteristics of rabbit sperm cells, but DNA
integrity. This is the evidence that lipid peroxidation is
Fig. 1. Scatter plots showing Pearsons correlation coefcient (r) between MDA production and sperm quality parameters.
M.P. Rosato et al. / Livestock Science 146 (2012) 199202 201
correlated to sperm quality deterioration during storage,
thus it is necessary for an adequate antioxidant protection
of sperm. For this reason the MDA concentration was
limited during both chilled and frozen storage in a dose-
dependent way by the lycopene supplementation, mean-
ing that lycopene effectively acts as a potent antioxidant
in rabbit sperm cells, even if these antioxidant properties
were more evident after chilled storage than cryopreser-
vation. The protective role of lycopene on sperm has yet
been recognized: an oral lycopene provision is reported to
improve the initial quality of human (Gupta and Kumar,
2002) and chicken sperm (Mangiagalli et al., 2010),
whereas a lycopene supplementation to semen extenders
is reported to improve the cryosurvival of ram semen
(Uysal and Bucak, 2007) and to protect the DNA integrity
of human sperm following incubation (Zini et al., 2010). It
has been proposed that lycopene exerts an anti-oxidative
role in cells by donating its electrons to oxygen free
radicals thus quenching and neutralizing them before
they can damage cells (Di Mascio et al., 1989). In previous
studies on rabbit it has been also shown a benecial effect of
other antioxidants as vitamin E, C, selenium (Yousef et al.,
2003; Castellini et al., 2002) and all of them improved the
quality and oxidative stability of fresh rabbit spermatozoa,
whereas both vitamin E and C improved sperm chilled
storage (Castellini et al., 2000), even if the potential of these
antioxidants was assessed only following dietary adminis-
tration. An interesting result of our research was that the
integrity of rabbit sperm DNA appeared compromised during
both chilled and frozen storage, but we found no correlation
between MDA production and DNA damage, despite detect-
ing a reduction in the chromatin damage in lycopene
enriched sperm. Our ndings are consistent with those of a
study on human sperm, in which oxidative stress was linked
to DNA damage, but no correlations between nuclear altera-
tions and MDA levels were detected (Zribi et al., 2011).
Another relevant point stressed in this study was that the
level of MDA was higher in frozen than in chilled sperm, thus
rabbit sperm are highly susceptible to lipid peroxidation
during freezingthawing process. However, with the excep-
tion of an improved chromosome stability in sperm frozen
with a medium containing 0.5 mg/mL lycopene, there was a
lack of evidence of a benecial effect of lycopene addition in
counteracting the cryopreservation-induced damages. These
results suggest that even if lipid peroxidation occurs in rabbit
sperm during freezing, the increased levels of lipid peroxida-
tion are probably not the primary cause of sperm damage
during cryopreservation.
5. Conclusion
Lycopene improved the overall quality of chilled stored
sperm and minimized the loss in DNA integrity of frozen
sperm. The positive effects of lycopene addition to exten-
ders seem to be closely involved with the inhibition of
sperm lipid peroxidation after both chilled storage and
cryopreservation. So lycopene seems suitable for supple-
mentation of rabbit sperm storage solutions, even if
further work is needed to conrm the in vivo benets of
lycopene-enriched extenders.
Conict of Interest Statement
The authors have no conicts of interest to declare.
Acknowledgments
The authors thank Innocenzo Gentile, Maria
Adamkovicova and Luca Romagnoli for their assistance.
References
Bansal, A.K., Bilaspuri, G.S., 2011. Impacts of oxidative stress and
antioxidants on semen functions. Vet. Med. Int. (2011, Article ID
686137).
Castellini, C., Lattaioli, P., Bernardini, M., Dal Bosco, A., 2000. Effect of
dietary alpha-tocopheryl acetate and ascorbic acid on rabbit semen
stored at 5 degrees C. Theriogenology 54, 523533.
Castellini, C., Lattaioli, P., Bosco, A.D., Beghelli, D., 2002. Effect of
supranutritional level of dietary alpha-tocopheryl acetate and sele-
nium on rabbit semen. Theriogenology 58, 17231732.
Di Mascio, P., Kaiser, S., Sies, H., 1989. Lycopene as the most efcient
biological carotenoid singlet oxygen quencher. Arch. Biochem. Bio-
phys. 274, 532538.
Gandini, L., Lombardo, F., Lenzi, A., Span o, M., Dondero, F., 2006.
Cryopreservation and sperm DNA integrity. Cell Tissue Banking 7,
9198.
Gliozzi, T.M., Zaniboni, L., Maldjian, A., Luzi, F., Maertens, L., Cerolini, S.,
2009. Quality and lipid composition of spermatozoa in rabbits fed
DHA and vitamin E rich diets. Theriogenology 71, 910919.
Gupta, N.P., Kumar, R., 2002. Lycopene therapy in idiopathic male
infertilitya preliminary report. Int. Urol. Nephrol. 34, 369372.
Iaffaldano, N., Rosato, M.P., Paventi, G., Pizzuto, R., Gambacorta, M.,
Manchisi, A., Passarella, S., 2010. The irradiation of rabbit sperm cells
with HeNe laser prevents their in vitro liquid storage dependent
damage. Anim. Reprod. Sci. 119, 123129.
Kadirvel, G., Kumar, S., Kumaresan, A., 2009. Lipid peroxidation, mito-
chondrial membrane potential and DNA integrity of spermatozoa in
relation to intracellular reactive oxygen species in liquid and frozen
thawed buffalo semen. Anim. Reprod. Sci. 114, 125134.
Mangiagalli, M.G., Martino, P.A., Smajlovic, T., Guidobono Cavalchini, L.,
Marelli, S.P., 2010. Effect of lycopene on semen quality, fertility and
native immunity of broiler breeder. Br. Poult. Sci. 51, 152157.
Moce , E., Lavara, R., Vicente, J.S., 2005. Inuence of the donor male on the
fertility of frozenthawed rabbit sperm after articial insemination
of females of different genotypes. Reprod. Domest. Anim. 40,
516521.
Moce , E., Vicente, J.S., 2009. Rabbit sperm cryopreservation: a review.
Anim. Reprod. Sci. 110, 124.
Mourvaki, E., Cardinali, R., Dal Bosco, A., Castellini, C., 2010. In vitro
antioxidant activity of the prostatic secretory granules in rabbit
semen after exposure to organic peroxides. Reprod. Biol. Endocrinol.
2010 (8), 16. http://dx.doi.org/10.1186/1477-7827-8-16.
Rosato, M.P., Iaffaldano, N., 2011. Effect of chilling temperature on the
long-term survival of rabbit spermatozoa held either in a Tris-based
or a jellied extender. Reprod. Domest. Anim. 46, 301308.
Srinivasan, M., Sudheer, A.R., Pillai, K.R., Kumar, P.R., Sudhakaran, P.R.,
Menon, V.P., 2007. Lycopene as a natural protector against gamma-
radiation induced DNA damage, lipid peroxidation and antioxidant
status in primary culture of isolated rat hepatocytes in vitro.
Biochim. Biophys. Acta 1770, 659665.
Uysal, O., Bucak, M.N., 2007. Effect of oxidized glutathione, bovine serum
albumin, cysteine and lycopene on the quality of frozen thawed ram
semen. Acta Vet. Brno 76, 383390.
Yousef, M.I., Abdallah, G.A., Kamel, K.I., 2003. Effect of ascorbic acid and
vitamin E supplementation on semen quality and biochemical
parameters of male rabbits. Anim. Reprod. Sci. 76, 99111.
Zini, A., San Gabriel, M., Libman, J., 2010. Lycopene supplementation
in vitro can protect human sperm deoxyribonucleic acid from
oxidative damage. Fertil. Steril. 94, 10331036.
Zribi, N., Chakroun, N.F., Elleuch, H., Abdallah, F.B., Ben Hamida, A.S.,
Gargouri, J., Fakhfakh, F., Keskes, L.A., 2011. Sperm DNA fragmenta-
tion and oxidation are independent of malondialdheyde. Reprod. Biol.
Endocrinol. 2011 (9), 47. http://dx.doi.org/10.1186/1477-7827-9-47.
M.P. Rosato et al. / Livestock Science 146 (2012) 199202 202