Treatment with Zinc, D-aspartate and Coenzyme Q10 protects bull
spermatozoa against exogenous oxidative damage
R. Gualtieri1, V. Barbato1, I. Fiorentino1, S. Braun1, A. Merolla1, S. Sudhakaran1,
S. Longobardi2, R. Talevi1
1Università
degli studi di Napoli Federico II, Dept. Biology Complesso Universitario Monte S.
Angelo, Napoli, Italy.
2Merck Serono SpA, Medical Division, Roma, Italy.
Introduction
Oxidative stress manifests when reactive oxygen species (ROS) , such as hydroxyl radicals, superoxide anions and hydrogen peroxide, overwhelm the
antioxidant defence system in cells. In vivo and in vitro, high ROS levels have been proven to impair sperm quality, fertilization and embryo
developmental competence. Recently, studies have reported that Zinc, D-Asp and CoQ10, contained in the dietary supplement Genadis (Merck
Serono), have protective effects on human and bull sperm motility and DNA fragmentation in vitro in the absence of exogenous oxidative stress (Talevi
et al., 2013; Gualtieri et al., 2014). Moreover, treated bull spermatozoa improved the percentages of on time embryos at day 3 and blastocysts at day 8
and the latters were endowed with a lower percentage of DNA fragmented blastomeres (Gualtieri et al., 2014). In literature, there are many studies that
consider the effects of exogenous oxidative stress on spermatozoa, although only few of these use xanthine-xanthine oxidase system to induce this
stress, producing superoxide anions, and analyze the consequent effects of treatment with antioxidants.
Fig 1
TOTAL MOTILITY (%)
Fig 2
X-XO
TREATED
PROGRESSIVE MOTILITY
(%)
80
Aim of research
The main aim of this study was to
assess the in vitro effect of pretreatment with Zinc, D-Asp and
CoQ10 on oxidative stress induced
by
xanthine-xanthine
oxidase
system in bull spermatozoa
CONTR
60
X-XO
TREATED
60
**
40
#
20
0
1H
2H
1H
2H
3H
Fig 3
CONTR
X-XO
TREATED
30
TUNEL POSITIVE (%)
Data showed that the treatment with X-XO for 2h induced a significant decrease of total
(Fig 1) and progressive (Fig 2) motility (CONTR vs X-XO: total motility 23,75±3,8 vs
13,75±2,2, # P<0,05; progressive motility 22,75±4,2 vs 9,75±3,3, ## P<0,01) . The
decrease of total and progressive motility was significantly prevented by pre-treatment
with Zinc, D-Asp and CoQ10 (X-XO vs TREATED: total motility 13,75±2,2 vs 34,3±5,1,
progressive motility 9,75±3,3 vs 32,3±5, ** P<0,01). Analysis of sperm kinetics
showed no significant differences between samples.
TUNEL assay (Fig 3, 4) demonstrated that spermatozoa with fragmented DNA
significantly increased in X-XO compared to control (CONTR 17,7%, vs X-XO 27,4% ##
P<0,01), whereas such as increase was significantly prevented by the pre-treatment with
antioxidant (TREATED 11,2% vs X-XO 27,4%, ** P<0,01).
0
0
The study was performed on 10 frozen/thawed bull semen samples.
Each experiment included three groups: control (CONTR), treated with 0.1mM xanthine0.01 U/ml xanthine oxidase (X-XO) alone or after pre-treatment with 10µg/mL Zinc
chloride, 500µg/mL D-Asp and 40µg/mL CoQ10 (TREATED). Bull spermatozoa were pretreated with antioxidants for 1h and then treated with X-XO for 2h.
Sperm kinetics was evaluated by computer assisted semen analysis (CASA) every hour
and nuclear DNA integrity was measured by the TUNEL assay. All samples were labelled
with Tunel mixture and Hoechst 33342 10µg/mL.
Results
##
20
3H
Results
**
40
0
Materials and Methods
CONTR
80
25
##
20
**
15
10
5
0
0
3H
Fig. 4
Conclusions
Taken together, these data demonstrate that in vitro treatment of bull spermatozoa with the
xanthine-xanthine oxidase system (X-XO) determines a rapid decrease of sperm motility and a
concomitant increase of sperm DNA fragmentation that are prevented by pre-incubation with
Zinc, D-Asp and CoQ10.
Data demonstrate that antioxidants protect bull spermatozoa from exogenous oxidative stress
in vitro. The pre-treatment of sperm with Zinc, D-Asp and CoQ10 could protect them from
oxidative stress during in vitro handling. Experiments in progress on this animal model will
allow to understand the role played by sperm exogenous oxidative damage and its prevention
by Zinc, D-Asp and CoQ10 on embryo developmental competence and activation of DNA repair
pathways.
References
Talevi R, Barbato V, Fiorentino I, Braun S, Longobardi S, Gualtieri R.Protective effects of in vitro treatment with zinc, d-aspartate and coenzyme q10 on
human sperm motility, lipid peroxidation and DNA fragmentation. Reprod Biol Endocrinol. 2013;11:81.
Gualtieri R, Barbato V, Fiorentino I, Braun S, Rizos D, Longobardi S, Talevi R Treatment with zinc, d-aspartate, and coenzyme Q10 protects bull sperm
against damage and improves their ability to support embryo development. Theriogenology. 2014 ;82(4):592-8.
.