the effect of coenzyme q10 and α-tocopherol in skim milk–based extender for preservation of...

Accepted Manuscript

The effect of coenzyme Q10 and α-tocopherol in skim milk-based extender forpreservation of Caspian stallion semen in cool condition

Iman Yousefian, MSc Ahmad Zare-Shahneh, PhD Mahdi Zhandi, PhD

PII: S0737-0806(14)00153-1

DOI: 10.1016/j.jevs.2014.04.002

Reference: YJEVS 1713

To appear in: Journal of Equine Veterinary Science

Received Date: 21 January 2014

Revised Date: 19 April 2014

Accepted Date: 29 April 2014

Please cite this article as: Yousefian I, Zare-Shahneh A, Zhandi M, The effect of coenzyme Q10 andα-tocopherol in skim milk-based extender for preservation of Caspian stallion semen in cool condition,Journal of Equine Veterinary Science (2014), doi: 10.1016/j.jevs.2014.04.002.

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http://dx.doi.org/10.1016/j.jevs.2014.04.002

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The effect of coenzyme Q10 and α-tocopherol in skim milk-based extender for preservation of Caspian stallion semen in cool condition Iman Yousefian, MSc; Ahmad Zare-Shahneh, PhD; Mahdi Zhandi, PhD Department of Animal Science, The University of Tehran, Karaj, Iran. Keywords: α-Tocopherol, cooling, coenzyme Q, stallion, spermatozoa Corresponding Author: Dr Ahmad Zare-Shahneh,

Professor of Animal physiology Dept. of Animal Science, The University of Tehran Karaj Iran [email protected] Tel 0098 26 3224 8082 Fax 0098 26 3224 6752

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The effect of coenzyme Q10 and α-tocopherol in skim milk-based extender

forpreservation of Caspian stallion semen cooled to 5 C condition

Abstract

The purpose of this study was to determine the effects of different concentrations

of coenzyme Q10 (CoQ10) and α-tocopherol (T) along with their interaction effects

on the quality of preserved stallion semen at 5ᵒC for a period of 48 h. Semen was

collected and diluted with skim milk-based extender that was supplemented with

different antioxidants: no antioxidant (negative control, NC), 0.9% (v/v) dimethyl

sulfoxide (positive control, PC), α-tocopherol [5 (T5) or 10 (T10) Mm], CoQ10 [1

(C1) or 2 (C2) µM], 1µM CoQ10 + 5mM α-tocopherol (C1T5), 1µM CoQ10 +

10mM α-tocopherol (C1T10), 2µM CoQ10 + 5mM α-tocopherol (C2T5) and 2µM

CoQ10 + 10mM α-tocopherol (C2T10), then kept at 5ᵒC. The results showed that

C1 extender resulted in higher total motility (62.44±3.82) and plasma membrane

integrity (65.16±3.63%) compared to negative control after 48 h of storage (P <

0.05). Different concentrations of α-tocopherol had no significant effects on sperm

quality, with the exception of plasma membrane integrity, compared to NC and

PC extender (P > 0.05). Also, C1T5 extender improved total and progressive

motility, plasma membrane integrity and functionality, and decreased lipid

peroxidation compared to NC and C2T10 extenders over 48 h of storage at 5ᵒC (P

< 0.05). The C1T5 extender was similar to C1 and T5 extenders in all semen

parameters evaluated during storage time. In conclusion, between above-

mentioned extenders, C1T5 could improve stallion sperm quality during 48 h of

storage. In the present study, none of extenders had effect on sperm quality until

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24 h storage.

Key words: α-Tocopherol, cooling, coenzyme Q, stallion, spermatozoa.

1. Introduction

The elimination of seminal plasma during preparation of equine semen for long-

time storage may raise the sensitivity of sperm to oxidative stress. This is because

seminal plasma contains enzyme scavengers and antioxidants [1]. Therefore, it is

important to restore optimal levels of antioxidants in semen by adding them into

the extender [2]. Sensitivity of ejaculated semen to oxidative damage is attributed

to effects of reactive oxygen species (ROS) on the sperm membrane, especially

polyunsaturated fatty acids (PUFAs) of phospholipids [1]. While low levels of

ROS are important for normal functioning of sperm cells [3], excessive

production of ROS should be avoided, because it causes irreversible injury of

sperm membrane structure as well as a reduction in membrane integrity and

motility of spermatozoa [4]. A loss of membrane fluidity of spermatozoa

decreases sperm motility and contributes to a loss of fertility due to lipid

peroxidation (LPO) [5].

Vitamin E (α-tocopherol) can prevent lipid peroxidation by incorporation into

membranes of cell [5]. Some investigators have suggested beneficial effects of α-

tocopherol supplemented extender on equine and turkey spermatozoa [6,7].

Coenzyme Q10 is a lipophilic and non-enzymatic antioxidant that is endogenously

produced at considerably high level in cells of the testis [8]. During the

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peroxidation process, fully reduced form of CoQ10 (ubiquinol; CoQH2) acts as an

antioxidant [9] and it can directly eliminate lipid peroxyl radicals (LOOᵒ). As well

as, CoQH2 enables to regenerate α-tocopherol from the α-tocopheroxyl radical

[10]. Also, CoQ10 has an energy carrier role in mitochondria [11]. So, process of

ATP synthesis and energy generation is related to the availability and existence of

CoQ10 in the sperm cell [12]. A study reported a strong correlation between

motility and CoQH2 content in seminal fluid [13]. A significant increase in sperm

motility has been led by administration of CoQ10 in infertile men with idiopathic

asthenozoospermia [14]. Based on these data, it seems that CoQ10 has a significant

role on sperm quality.

Therefore this study was conducted to determine the effects of CoQ10, α-

tocopherol and their combinations on the quality of equine spermatozoa during 48

h of cooled-storage.

2. Materials and Methods

2.1 Materials

The antioxidants (coenzyme Q10 and α-tocopherol) used in this study were

purchased from Sigma–Aldrich chemicals (St. Louis, MO, USA), and other

chemicals were obtained from Merck (Darmstadt, Germany).

2.2. Collection and primary evaluation of semen

Semen was obtained from three mature Caspian horses individually housed at

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National Animal Breeding Center and Promotion of Animal Products, Karaj, Iran.

Age of the stallions was between 11 and 13 years. A total of 5 ejaculates per

stallion were collected during this experiment at twice a week, during the

breeding season (between July and August) using a Missouri artificial vagina.

After collection, the ejaculates were filtered through a sterile gauze to eliminate

the gel fraction. Only ejaculates with more than 70 % motile sperm were used.

2.3. Preparation of semen and extender

The basic extender used for this study Kenney extender [including glucose (4.9 g),

non-fat dried skim milk (2.4 g), Na penicillin (1.5 × 105 units), streptomycin (150

mg), and distilled water to 100 ml; 15]. All collected ejaculates (n=3) in each

replicate (r=5) were pooled to eliminate variability between stallions. The pooled

semen was extended at a 1:1 ratio with the Kenney extender (prewarmed to 37oC).

Diluted semen was loaded into 50 mL plastic tubes and centrifuged at 600 × g for

10 minute at room temperature. After centrifugation, most (≥90%) supernatant

(seminal plasma) was removed from each tube; the sperm-rich pellet was

resuspended and divided into 10 equal aliquots. Each sperm aliquot was diluted

with one of the following extenders to a final concentration of 50 × 106

spermatozoa/ml. Used extenders in this study was Kenney extender containing no

antioxidant (negative control, NC), 0.9% (v/v) dimethyl sulfoxide (positive

control, PC), α-tocopherol [5 (T5) or 10 (T10) Mm], CoQ10 [1 (C1) or 2 (C2)

µM], 1µM CoQ10 + 5mM α-tocopherol (C1T5), 1µM CoQ10 + 10mM α-

tocopherol (C1T10), 2µM CoQ10 + 5mM α-tocopherol (C2T5) and 2µM CoQ10 +

10mM α-tocopherol (C2T10).

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2.4. Cooling process

Tubes containing different extenders were transferred to a polystyrene box with

600 mL water (30°C) and placed in a refrigerator at 5°C. Temperature reached to

5°C in approximately 8 h. For doing every test, the sub-aliquots were separated

and warmed for 10 minute to 37oC.

2.5. Evaluation of warmed sperm quality

2.5.1. Motility and velocity parameters

After warming, semen was maintained at 37oC, and different parameters of

spermatozoa motility were measured using a CASA (CEROS version 12.3;

Hamilton-Thorne Bio-sciences, Beverly, MA, USA). The system parameters for

CASA were used according to Nouri et al [16]. Semen sample was placed in a

chamber and the loaded chamber placed on the warm stage of the microscope

(37◦C). Afterwards, three randomly selected microscopic fields were examined

five times each near the center and the mean of these 15 scans was calculated and

used for statistical analysis. Motility data was characterized as follows: total

motility (TM, %); progressive motility (PM, %); average path velocity (VAP,

µm/s); straight line velocity (VSL, µm/s); curvilinear velocity (VCL, µm/s);

amplitude of lateral head displacement (ALH, µm); beat cross frequency (BCF,

Hz); straightness (STR, %); linearity (LIN, %).

2.5.2. Plasma membrane functionality

The hypo-osmotic swelling test (HOST) was designed to determine functional

membrane integrity as previously described by Nie and Wenzel (2001) with

modifications [17]. Briefly, 10µl of semen was mixed with 100 µl of a hypo-

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osmotic solution [1.712 g sucrose dissolved in 50 mL of distilled water

(osmolarity: 100 mOsm/kg)] in a 1.5 ml Eppendorf tube. This mixture was

incubated at 37°C for 30 minutes. After incubation, one drop of this mixture was

placed on a pre-warmed microscope slide and mounted with a cover slip. Two

hundred cells were counted for evidence of plasma membrane swelling (tail

coiling) by phase-contrast microscope (CKX41; Olympus, Tokyo, Japan) at ×400

magnification. As intact plasma membrane functionality, percentage of

spermatozoa with tail coiling (HOST+) was recorded for each sample.

2.5.3. Plasma membrane integrity

Evaluation of plasma membrane integrity was examined by staining with Eosin-

Nigrosin [18]. Briefly, 20 µL aliquot from each of the treatment groups was

stained by 20 µL the Eosin–Nigrosin stain. Then, smears were prepared on a

warm slide and spreader the stain with a second slide. The plasma membrane

integrity of the stained samples was assessed by counting two hundred sperm

(viable and non-viable) under phase-contrast microscopy at ×400 magnification.

2.5.4. MDA concentrations

MDA concentrations, as indices of the LPO in the sperm cell, were measured

using the thiobarbituric acid reaction and according to the method of described by

Esterbauer and Cheeseman [19]. Briefly, 1 ml of the extended semen was mixed

with 1 ml of cold 20% (w/v) trichloroacetic acid (TCA ) to sediment precipitate

protein. The precipitate was pelleted by centrifuging with 960 ×g for 15 minute,

and 1 ml of the supernatant was incubated with 1 ml of 0.67% (w/v) thiobarbituric

acid in a boiling water-bath at 100◦C for 10 minute. After cooling, the absorbance

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was determined by a spectrophotometer (UV-1200 Spectrophotometer Shimadzu,

Japan) at 532 nm. All MDA concentrations were reported as nmol/ml.

2.6. Statistical analysis

The study was replicated five times. All data were analyzed using MIXED

procedure of SAS version 9.1 (SAS Institute, 2002, Cary, NC, USA). The results

were expressed in Lsmean ± standard error of the mean (SEM). All data were

checked for normal distribution by UNIVARIATE procedure and Shapiro–Wilk

test and all percentage data were normalized through ArcSin√x transformation.

3. Results

total and progressive motility (Table 1 and 2), plasma membrane integrity (Table

3) and plasma membrane functionality (Table 4) in all extenders were

significantly decreased with increasing storage time (P < 0.05). Lipid peroxidation

(Table 5) in all extenders was significantly increased with increasing storage time

(P< 0.05). There were no significant differences between extenders on all

parameters after 24 h of storage (P > 0.05).

3.1. Motility and velocity parameters

As shown in Table 1, total motility was significantly higher in C1 and C1T5

compared to NC, C2 and C2T10 extenders after 48 h of storage (P < 0.05). There

were no significant differences between T5, C1 and C1T5 extenders for total

motility during storage time (P > 0.05). Results showed that progressive motility

after 48 h of storage was significantly higher in C1T5 compared to NC, PC, T10

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and C2T10 extenders (Table 2; P < 0.05). The percentage of progressively motile

spermatozoa did not differ between T5, C1, C1T5, C1T10, C2 and C2T5

extenders during 48 h of storage (P > 0.05). Also, there were no significant

differences in sperm motion parameters between extenders after 48 h of storage (P

> 0.05; unpublished data).

3.2. Plasma membrane integrity of spermatozoa

Plasma membrane integrity of spermatozoa after 48 h of storage was significantly

higher in T5, C1 and C1T5 compared to NC and C2T10 extenders over 48 h of

storage (P < 0.05; Table 3). Also, there were no significant differences in plasma

membrane integrity between T5, C1, C1T5 and C2T5 extenders during storage

time (P > 0.05).

3.3 Plasma membrane functionality of spermatozoa

Plasma membrane functionality of spermatozoa was significantly higher in C1T5

compared to NC, PC, T10, C2 and C2T10 extenders after 48 h of storage (Table

4; P < 0.05). There were no significant differences in plasma membrane

functionality between T5, C1, C1T5, C1T10 and C2T5 extenders during storage

time (P > 0.05

3.4. MDA concentrations

As shown in Table 5, LPO was significantly lower in C1T5 compared to NC and

C2T10 treatments after 48 h of storage (P < 0.05).

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4. Discussion

In the present study, effects of different concentrations of CoQ10 and α-tocopherol

on the quality of equine spermatozoa during 48 h of cooled-storage were

investigated. Results of the present study showed that C1T5 extender can improve

sperm quality parameters (total and progressive motility, plasma membrane

integrity and functionality), as well as reduced level of MDA compared to NC and

C2T10 extenders over 48 h of storage at 5ᵒC. The effect of C1T5 extender was

similar to C1 and T5 extenders effects on all semen parameters evaluated during

storage time. Therefore, they could be also an alternative to improve semen

quality. In the present study, none of extenders had effect on sperm quality until

24 h of storage. This may be due to present of basal antioxidative activity in

extended semen during 24 h of storage at 5ᵒC [20]. It seems that extended semen

will require to addition amount of antioxidant(s) after 24 h of storage. As shown

in results, high concentration of antioxidants may act as an oxidation stimulator.

In spermatozoa, oxidative stress reduces motility, intracellular ATP levels and

viability and induces the damage to the structure of the lipid matrix during sperm

storage [21]. Like in other domestic animals, in horses, sperm plasma membranes

have a high content of PUFAs, thereby elevating their susceptibility to oxidative

[21]. Thus, it is necessary to use antioxidants at appropriate levels in extender in

order to prevent cellular damage.

There is little published information on the influence of CoQ10 on sperm quality.

Exogenous administration of CoQ10 was effective in improving sperm quality,

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especially motility in infertile men [13,22-24]. This is in agreement with results of

the present study. This improvement in sperm motility may be due to the bio-

energetic role of CoQ10 in the mitochondrial respiratory chain and ATP

production, as well as the antioxidant function of CoQ10 and α-tocopherol

[10,25,26]. The improvement of oxidative phosphorylation might affect motility

of sperm cells. It is noteworthy that the fully reduced form of CoQ10 and α-

tocopherol prevents LPO in most subcellular membranes [10,27,28]. Coenzyme

Q10 in combination with α-tocopherol also reduced MDA level during the storage

period. The α-tocopherol can be recycled by CoQ10 [10], thereby may provide

more protection against oxidation-induced stress. The addition of α-tocopherol

into the semen extender of boars [29] and bulls [30] decreased level of MDA

during preservation. Another study indicated that α-tocopherol addition to turkey

semen extender did not affect on MDA level during storage [31]. The α-

tocopherol-supplemented extender (200µg/mL) was useful to maintenance

motility of boar spermatozoa during cryopreservation [27]. Ball et al [32] reported

that α-tocopherol did not effect on motility of stallion spermatozoa. The result of

later study is similar to that in our study in which α-tocopherol failed to change

sperm motility compared to negative control during storage time. However,

results of the present study showed that α-tocopherol in combination with CoQ10

led to improved sperm motility after 48 h of storage. This may be due to an effect

of CoQ10 on mitochondrial ATP production. One the other hand, parameters of

sperm quality was reduced in C2T10 and NC extenders. α-Tocopherol and CoQ10

effects may vary with the concentration. Inappropriate levels of antioxidants may

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be toxic and act as an oxidation stimulator instead as an antioxidant [27,33,34].

Semi-quinone form (a redox state of CoQ10) has the pro-oxidant effect and act in

cytotoxic oxidants formation [28]. Therefore, at high concentrations, they have

negative effects on integrity of membranes and sperm oxosome, as well as,

metabolic activity of spermatozoa during preservation of semen by increasing in

ROS production.

Oxidative stress can induce an increase in oxidation of plasma membrane PUFAs

that in turn results disruption of plasma membrane functional integrity during the

storage time [21]. During liquid preservation of semen for 48 h, plasma membrane

functionality was protected in T5, C1, C1T5, C1T10 and C2T5 extenders

compared to other extenders. It seems that α-tocopherol can accommodate to the

sperm plasma membrane and protects that by its antioxidant property during the

storage time [29]. In this study, α-tocopherol failed to improve sperm plasma

membrane functionality compared to negative control that is agreement with

results of Upreti et al [35] and Baumber et al [36]. However, other researchers

reported a protective effect of α-tocopherol on sperm plasma membranes

[5,27,29,37,38]. This discrepancy can be attributed to differences in the

processing procedure and different species.

It is also possible that CoQ10 eliminated lipid peroxyl radicals directly, besides

regenerated the active form of α-tocopherol [39], and thereby may provide more

protection for plasma membrane. The results of this study showed that

combination of these antioxidants is more effective in protecting membranes. In

rat testes, combination of CoQ10 and α-tocopherol was more effective in the

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prevention of cadmium-induced LPO [40]. By decreasing PUFAs peroxidability,

therefore, membrane integrity effectively is maintained. The cell membrane lipids

and lipoprotein lipids exist in the circulation have been protected via inhibition of

peroxidation by ubi-quinol [11], which is in agreement with our results about

MDA level. So, membrane integrity improvement in C1T5 compared to C2T10

and negative control might be due to lower effect of ROS on sperm plasma

membranes due to the antioxidant properties of these compounds.

The structure of the lipid matrix changes caused cellular damage and sperm death

due to membrane dysfunction and alterations in cell permeability [21]. By

preventing of dis-arrangement of sperm membranes lipids, integrity of plasma

membrane is maintained and consequently survives sperm cell [41]. Also,

production of malondialdehyde and the more potent 4-hydroxynonenol

compounds by degradation of lipids within the plasma membrane is toxic to

spermatozoa [1]. So, accumulation of these compounds may lead to reduced

sperm viability. The data obtained in the present study showed that there is

significantly difference in sperm plasma membrane integrity between T5, C1,

C1T5 and C2T5 compared to NC and C2T10 during 48 h of storage. Other studies

on cattle [42], dogs [43], horses [38] and pigs [44] reported that α-tocopherol-

supplemented extender can increase viability of spermatozoa during semen

preservation, which is in agreement with our results. The antioxidants CoQ10 and

α-tocopherol can negate free radical lipids [27,39], thereby prevent accumulation

of cytotoxic aldehydes. Coenzyme Q10 regulates mitochondrial permeability of

transition pores [10]. It seems that CoQ10 is able to deactivate mitochondrial

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membrane potential depolarization, ATP fall, and caspase-9 activation induced by

cytochrome c release and DNA fragmentation in cell by preventing mitochondrial

permeability of transition pore opening [10].

5. Conclusions

The results of present study showed that CoQ10 and α-tocopherol in Kenney

extender could improve the quality of cooled-stored stallion semen. The

combination of α-tocopherol at 5mM concentration and CoQ10 at level of 1 µM

reduced MDA level and improved quality parameters of sperm during

conservation at 5oC for 48 h. However, in the present study, none of extenders had

effect on sperm quality until 24 h of storage.

Acknowledgments

We would like to thank the National Animal Breeding Center and Promotion of

Animal Products, Karaj, Iran (Dr. M. Bahraini) for providing stallions and

laboratory equipment. We are also acknowledging the financial support of

University of Tehran for this research. The authors would like to acknowledge the

financial support of Iran National Science Foundation under grant number

843171.

References

[1] Ball BA. Oxidative stress, osmotic stress and apoptosis: Impacts on sperm

function and preservation in the horse. Anim Reprod Sci. 2008;107:257-67.

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[2] Memon AA, Wahid H, Rosnina Y, Goh Y, Ebrahimi M, Nadia F, et al. Effect

of butylated hydroxytoluene on cryopreservation of Boer goat semen in Tris egg

yolk extender. Anim Reprod Sci. 2011;129:44-9.

[3] Sanocka D, Kurpisz M. Reactive oxygen species and sperm cells. Reprod Biol

Endocrinol. 2004;2:1-7.

[4] Pagl R, Aurich J, Aurich C. Reactive oxygen species and their influence on

stallion semen fertility-a review. Pferdeheilkunde. 2006;22:212-7.

[5] Zaniboni L, Cerolini S. Liquid storage of turkey semen: changes in quality

parameters, lipid composition and susceptibility to induced in vitro peroxidation

in control, n-3 fatty acids and alpha-tocopherol rich spermatozoa. Anim Reprod

Sci. 2009;112:51-65.

[6] Agueero A, Miragaya M, Mora N, Chaves M, Neil D, Beconi M. Effect of

Vitamin E Addition in Equine Sperm Preservation. Comun Biol. 1995;13:343-56.

[7] Donoghue A, Donoghue D. Effects of water-and lipid-soluble antioxidants on

turkey sperm viability, membrane integrity, and motility during liquid storage.

Poult Sci. 1997;76:1440-5.

[8] Kalen A, Appelkvist E-L, Chojnacki T, Dallner G. Nonaprenyl-4-

hydroxybenzoate transferase, an enzyme involved in ubiquinone biosynthesis, in

the endoplasmic reticulum-Golgi system of rat liver. Biol Chem. 1990;265:1158-

64.

[9] Mancini A, Conte G, Milardi D, Marinis L, Littarru G. Relationship between

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

sperm cell ubiquinone and seminal parameters in subjects with and without

varicocele. Andrologia. 1998;30:1-4.

[10] Turunen M, Olsson J, Dallner G. Metabolism and function of coenzyme Q.

Biochim Biophys Acta. 2004;1660:171-99.

[11] Littarru GP, Tiano L. Bioenergetic and antioxidant properties of coenzyme

Q10: recent developments. Mol Biotechnol. 2007;37:31-7.

[12] Lewin A, Lavon H. The effect of coenzyme Q10 on sperm motility and

function. Mol Aspects Med. 1997;18 Suppl:S213-9.

[13] Alleva R, Scararmucci A, Mantero F, Bompadre S, Leoni L, Littarru G. The

protective role of ubiquinol-10 against formation of lipid hydroperoxides in

human seminal fluid. Mol Aspects Med. 1997;18:221-8.

[14] Balercia G, Buldreghini E, Vignini A, Tiano L, Paggi F, Amoroso S, et al.

Coenzyme Q10 treatment in infertile men with idiopathic asthenozoospermia: a

placebo-controlled, double-blind randomized trial. Fertil Steril. 2009;91:1785-92.

[15] Ijaz A, Ducharme R. Effect of various extenders and taurine on survival of

stallion sperm cooled to 5 C. Theriogenology. 1995;44:1039-50.

[16] Nouri H, Towhidi A, Zhandi M, Sadeghi R. The Effects of Centrifuged Egg

Yolk Used with INRA Plus Soybean Lecithin Extender on Semen Quality to

Freeze Miniature Caspian Horse Semen. J Equine Vet Sci. 2013;33:1050-3.

[17] Nie G, Wenzel J. Adaptation of the hypoosmotic swelling test to assess

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

functional integrity of stallion spermatozoal plasma membranes. Theriogenology.

2001;55:1005-18.

[18] Zhandi M, Ghadimi V. The Effect of Glutathione-Supplemented INRA82

Extender on Miniature Caspian Stallion Sperm Quality during storage at 5° C. J

Equine Vet Sci. 2013; doi: 10.1016/j.jevs.2013.11.009.

[19] Esterbauer H, Cheeseman KH. Determination of aldehydic lipid

peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol.

1990;186:407-21.

[20] Kankofer M, Kolm G, Aurich J, Aurich C. Activity of glutathione

peroxidase, superoxide dismutase and catalase and lipid peroxidation intensity in

stallion semen during storage at 5 C. Theriogenology. 2005;63:1354-65.

[21] Baumber J, Ball BA, Gravance CG, Medina V, Davies-Morel M. The effect

of reactive oxygen species on equine sperm motility, viability, acrosomal

integrity, mitochondrial membrane potential, and membrane lipid peroxidation. J

Androl. 2000;21:895.

[22] Li W, Li K, Huang YF. Biological function of CoQ10 and its effect on the

quality of spermatozoa. Zhonghua Nan Ke Xue. 2006;12:1119-22.

[23] Safarinejad MR. Efficacy of coenzyme Q10 on semen parameters, sperm

function and reproductive hormones in infertile men. J Urol. 2009;182:237-48.

[24] Mancini A, Balercia G. Coenzyme Q10 in male infertility: Physiopathology

and therapy. Biofactors. 2011;37:374-80.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

[25] Almeida J, Ball BA. Effect of α-tocopherol and tocopherol succinate on lipid

peroxidation in equine spermatozoa. Anim Reprod Sci. 2005;87:321-37.

[26] Bentinger M, Tekle M, Dallner G. Coenzyme Q–biosynthesis and functions.

Biochem Biophys Res Commun. 2010;396:74-9.

[27] Breininger E, Beorlegui NB, O'Flaherty CM, Beconi MT. Alpha-tocopherol

improves biochemical and dynamic parameters in cryopreserved boar semen.

Theriogenology. 2005;63:2126-35.

[28] Ernster L, Dallner G. Biochemical, physiological and medical aspects of

ubiquinone function. Biochim Biophys Acta. 1995;1271:195.

[29] Cerolini S, Maldjian A, Surai P, Noble R. Viability, susceptibility to

peroxidation and fatty acid composition of boar semen during liquid storage.

Anim Reprod Sci. 2000;58:99-111.

[30] Beconi M, Affranchino M, Schang L, Beorlegui N. Influence of antioxidants

on SOD activity in bovine sperm. Biochem Int. 1991;23:545.

[31] Long J, Kramer M. Effect of vitamin E on lipid peroxidation and fertility

after artificial insemination with liquid-stored turkey semen. Poult Sci.

2003;82:1802-7.

[32] Ball B, Medina V, Gravance C, Baumber J. Effect of antioxidants on

preservation of motility, viability and acrosomal integrity of equine spermatozoa

during storage at 5 C. Theriogenology. 2001;56:577-89.

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[33] Cao G, Cutler RG. High concentrations of antioxidants may not improve

defense against oxidative stress. Arch Gerontol Geriatr. 1993;17:189-201.

[34] Nohl H, Gille L, Kozlov AV. Antioxidant-derived prooxidant formation from

ubiquinol. Free Radical Bio Med. 1998;25:666-75.

[35] Upreti G, Jensen K, Oliver J, Duganzich D, Munday R, Smith J. Motility of

ram spermatozoa during storage in a chemically-defined diluent containing

antioxidants. Anim Reprod Sci. 1997;48:269-78.

[36] Baumber J, Ball BA, Linfor JJ. Assessment of the cryopreservation of

equine spermatozoa in the presence of enzyme scavengers and antioxidants. Am J

Vet Res. 2005;66:772-9.

[37] Surai P, Kostjuk I, Wishart G, Macpherson A, Speake B, Noble R, et al.

Effect of vitamin E and selenium supplementation of cockerel diets on glutathione

peroxidase activity and lipid peroxidation susceptibility in sperm, testes, and liver.

Biol Trace Elem Res. 1998;64:119-32.

[38] Vasconcelos Franco JS, Chaveiro A, Góis A, Moreira da Silva F. Effects of

α-tocopherol and Ascorbic Acid on Equine Semen Quality after Cryopreservation.

J Equine Vet Sci. 2013;33;787-793.

[39] Bentinger M, Brismar K, Dallner G. The antioxidant role of coenzyme Q.

Mitochondrion. 2007;7:S41-S50.

[40] Ognjanović BI, Marković SD, Ðorđević NZ, Trbojević IS, Štajn AŠ, Saičić

ZS. Cadmium-induced lipid peroxidation and changes in antioxidant defense

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system in the rat testes: Protective role of coenzyme Q10 and Vitamin E. Reprod

Toxicol. 2010;29:191-7.

[41] Emamverdi M, Zhandi M, Zare Shahneh A, Sharafi M, Akbari‐Sharif A.

Optimization of Ram Semen Cryopreservation Using a Chemically Defined

Soybean Lecithin‐Based Extender. Reprod Domestic Anim. 2013; 48:899-904.

[42] Bansal AK, Bilaspuri GS. Antioxidant effect of vitamin E on motility,

viability and lipid peroxidation of cattle spermatozoa under oxidative stress. Anim

Sci Pap Rep. 2009;27:5-14.

[43] Michael A, Alexopoulos C, Pontiki E, Hadjipavlou-Litina D, Saratsis P,

Ververidis H, et al. Effect of antioxidant supplementation in semen extenders on

semen quality and reactive oxygen species of chilled canine spermatozoa. Anim

Reprod Sci. 2009;112:119-35.

[44] Satorre M, Breininger E, Beconi M. Cryopreservation with α-tocopherol and

Sephadex filtration improved the quality of boar sperm. Theriogenology.

2012;78:1548-1556.

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Table 1. The effect of different extenders on total motility (%) of spermatozoa during storage

at 5°C (Lsmean±SEM).

Treatment Time of storage at 5oC SEM

0 6 24 48

NC 87.69A 86.37A 74.17B 51.27Cc 3.82

PC 87.34A 87.19A 73.68B 51.89Cbc 3.82

T5 90.40A 87.63A 76.39B 57.67Cabc 3.82

T10 89.62A 88.90A 70.58B 53.93Cbc 3.82

C1 89.60A 87.63A 81.16AB 62.44Bab 3.82

C1T5 91.40A 87.14AB 79.96B 67.90Ca 3.82

C1T10 90.79A 86.05A 74.96B 54.48Cbc 3.82

C2 91.30A 87.89A 75.76B 51.58Cc 3.82

C2T5 90.98A 88.76A 74.80B 56.54Cbc 3.82

C2T10 89.83A 86.39A 71.98B 49.52Cc 3.82

A, B, C Different letters within the same row indicate significant difference (P < 0.05).

a, b, c Different letters within the same column indicate significant difference (P < 0.05).

NC: extender without antioxidants, PC: 0.9% (v/v) dimethyl sulfoxide, T5: extender with

5mM α-tocopherol, T10: extender with 10mM α-tocopherol, C1: extender with 1µM CoQ10,

C1T5: extender with 1µM CoQ10 + 5mM α-tocopherol, C1T10: extender with 1µM CoQ10 +

10mM α-tocopherol, C2: extender with 2µM CoQ10, C2T5; extender with 2µM CoQ10 +

5mM α-tocopherol, C2T10; extender with 2µM CoQ10 + 10mM α-tocopherol.

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Table 2. The effect of different extenders on progressive motility (%) of spermatozoa during

storage at 5ᵒC (Lsmean±SEM).

Treatment Time of storage at 5oC SEM

0 6 24 48

NC 51.36A 49.21A 38.82B 28.70Cb 3.87

PC 49.34A 46.41AB 39.20B 27.98Cb 3.87

T5 50.55A 48.76AB 41.51BC 35.47Cab 3.87

T10 52.67A 51.34A 37.12B 27.74Cb 3.87

C1 52.91A 50.04A 45.90A 34.86Bab 3.87

C1T5 51.37A 49.72A 45.31AB 39.92Ba 3.87

C1T10 52.96A 49.63A 40.56B 29.34Cab 3.87

C2 53.80A 45.76B 41.37B 30.39Cab 3.87

C2T5 52.97A 48.14AB 41.47B 29.58Cab 3.87

C2T10 50.98A 46.28A 37.82B 26.02Cb 3.87



NC: extender without antioxidants, PC: 0.9% (v/v) dimethyl sulfoxide, T5: extender with

5mM α-tocopherol, T10: extender with 10mM α-tocopherol, C1: extender with 1µM CoQ10,

C1T5: extender with 1µM CoQ10 + 5mM α-tocopherol, C1T10: extender with 1µM CoQ10 +

10mM α-tocopherol, C2: extender with 2µM CoQ10, C2T5; extender with 2µM CoQ10 +

5mM α-tocopherol, C2T10; extender with 2µM CoQ10 + 10mM α-tocopherol.

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Table 3. The effect of different extenders plasma membrane integrity (%, eosin-negrosin

staining) of spermatozoa during storage at 5ᵒC (Lsmean±SEM).

Treatment Time of storage at 5ᵒC SEM

0 6 24 48

NC 82.86A 79.43A 69.38B 54.44Cc 3.63

PC 81.12A 79.17AB 70.81B 57.12Cbc 3.63

T5 83.08A 80.83A 77.51A 65.89Bab 3.63

T10 83.66A 80.58AB 72.25B 57.70Cbc 3.63

C1 88.36A 85.00AB 76.60B 65.16Cab 3.63

C1T5 87.81A 85.34A 79.70A 70.34Ba 3.63

C1T10 87.68A 85.10AB 75.84B 58.88Cbc 3.63

C2 89.32A 85.02AB 76.50B 58.74Cbc 3.63

C2T5 89.80A 88.58AB 78.76B 60.86Cabc 3.63

C2T10 87.92A 86.22A 74.16B 53.70Cc 3.63



NC: extender without antioxidants, PC: 0.9% (v/v) dimethyl sulfoxide, T5: extender with 5mM

α-tocopherol, T10: extender with 10mM α-tocopherol, C1: extender with 1µM CoQ10, C1T5:

extender with 1µM CoQ10 + 5mM α-tocopherol, C1T10: extender with 1µM CoQ10 + 10mM α-

tocopherol, C2: extender with 2µM CoQ10, C2T5; extender with 2µM CoQ10 + 5mM α-

tocopherol, C2T10; extender with 2µM CoQ10 + 10mM α-tocopherol.

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Table 4. The effect of different extenders on plasma membrane functionality (%, positive HOS)

of spermatozoa during storage at 5ᵒC (Lsmean±SEM).


0 6 24 48

NC 71.38A 71.34A 66.26A 38.87Bbc 3.75

PC 72.76A 72.10A 66.81A 37.82Bbc 3.75

T5 73.62A 72.96A 68.88A 48.32Bab 3.75

T10 73.38A 71.66A 67.72A 40.38Bbc 3.75

C1 70.02A 69.10A 66.76A 42.94Babc 3.75

C1T5 72.62A 71.50A 67.76A 51.10Ba 3.75

C1T10 72.48A 70.30A 65.58A 43.20Bab 3.75

C2 72.82A 68.22A 65.14A 39.80Bbc 3.75

C2T5 74.70A 71.58A 67.06A 41.58Babc 3.75

C2T10 72.88A 71.90A 65.76A 37.46Bc 3.75








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Table 5. The effect of different extenders on MDA concentration (nm/ml) of semen during

storage at 5ᵒC (Lsmean±SEM).


0 6 24 48

NC 0.18C 1.84B 3.30A 4.42Aa 0.50

PC 0.19C 1.88B 3.05AB 4.33Aab 0.50

T5 0.21B 1.86A 2.77A 3.14Aab 0.50

T10 0.20C 1.77B 3.23A 4.21Aab 0.50

C1 0.18C 1.79B 3.06AB 3.97Aab 0.50

C1T5 0.23B 1.61AB 2.62A 2.94Ab 0.50

C1T10 0.19C 1.69B 3.27A 4.18Aab 0.50

C2 0.21B 1.53B 3.13A 4.32Aab 0.50

C2T5 0.20B 1.61B 3.26A 3.95Aab 0.50

C2T10 0.22C 1.89B 3.43A 4.40Aa 0.50








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