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Therapeutic Effect of Huangjingzanyu Optimized Formula on Sperm Quality and Activities of Succinic Dehydrogenase and Lactate Dehydrogenase-C4 in Rat Asthenospermia Model.

Byline: Yanfei Zheng, Baoxing Liu, Ji Wang, Jianxin Chen, Zhaozhu Jiao, Zhuojun Yuan and Qi Wang

Abstract This study was aimed at investigating the effects of Huangjingzanyu optimized formula on the energy metabolism of sperm and on the activities of mitochondrial marker enzymes. Polyglycoside of Tripterygium wilfordii (GTW) was used to establish rat asthenospermia models. We investigated the mechanism of the action of the Huangjingzanyu optimized formula on mitochondrial energy metabolism by measuring sperm adenosine triphosphate (ATP) content and changes in succinic dehydrogenase (SDH) and lactate dehydrogenase-C4 (LDH-C4) activities, Compared to the model group, the sperm ATP content was significantly increased in the high- and moderate dose Huangjingzanyu optimized formula.

The SDH and LDH-C4 activities were also significantly increased after treatment with the high- and moderate dose. No significant difference was noted between the optimized formula small-dose group and the model group.

The Huangjingzanyu optimized formula can increase sperm ATP content by improving the activities of SDH and LDH-C4 and other mitochondrial marker enzymes for energy metabolism, thereby improving sperm motility.

Key words: Asthenospermia, Huangjingzanyu optimized formula, ATP, SDH, LDH-C

INTRODUCTION

Asthenospermia is a type of semen abnormality which accounts for approximately 46% of infertility (Guo and Chang, 2003). Although there are several drug available in the market, but none of these has been approved by FDA with curative effect (Crimmel et al., 2001).

Traditional Chinese medicine has specific therapeutic effects on male infertility and has been widely applied in clinical practice with convincing therapeutic efficacy (Zhang, 2012).

The Huangjingzanyu capsule is the first Chinese medicine (National New Drug Certificate: 220010103) used to treat male infertility.

The original Huangjingzanyu formula is composed of 20 herbs, whereas Professor Qi Wang developed the Huangjingzanyu optimized formula (Wu, 2012; Chen et al., 2009) using five herb extracts: Rhizoma polygonati, tuber fleeceflower root, wolfberry, red sage root, and dandelion. These compounds have been incorporated to preserve the health benefits of the original herb formula.

Previous studies have demonstrated that Huangjingzanyu capsules can improve sperm motility (Liu et al., 2006). In addition, the ATP content of sperm was significantly increased after treatment with high and moderate doses of the capsules. Further studies studies are therefore, required to investigate the physiological mechanism involved in the improved motility.

Succinic dehydrogenase (SDH) and lactate dehydrogenase-C4 (LDH-C4) are closely related to sperm production, metabolism, and energy acquisition (Li and Li, 2011; Guo et al., 1998; Duan et al., 2003; Cheema et al., 2013). SDH activity represents the physiological conditions of substrate oxidation and energy metabolism, along with mitochondrial function (Ji et al., 2009).

LDH-C4 can bind to the shuttle system and can couple with electron transport in mitochondria, thereby generating ATP by oxidative phosphorylation (Xin and Lan, 2008). LDH-C4 has a relatively high affinity for lactate, and energy can be produced through oxidative dehydrogenation of lactate. Therefore, SDH and LDH-C4 are the key enzymes for sperm energy metabolism.

Hence, sperm ATP content is directly related to motility, whereas SDH and LDH-C4 serve as marker enzymes for production of ATP in mitochondria. In order to investigate the mechanism of sperm mitochondrial energy metabolism, we have measured in this study the sperm ATP content and changes in SDH and LDH-C4 activities before and after treatment with the drug.

MATERIALS AND METHODS

Tested drugs and chemicals

Huangjingzanyu capsules of Beijing Chinese and Western Integrative Andrology Pharmaceutical Beijing, China, batch number 031105 and polyglycoside of Tripterygium wilfordii (GTW) of Zhejiang Apeloa Jiayuan Pharmaceutical Co. Ltd Dongyang, Zhejiang, China, batch number: 0309010 were used.

Three concentrations viz., 0.5, 1 and 2 g/mL of the Huangjingzanyu optimized formula were provided by Beijing China-Japan Friendship Hospital. These drug formulations were suspended in 0.5% carboxymethyl cellulose (CMC) before clinical use.

Besides that Medium 199 (M199) containing Earle's balanced salt solution and L-glutamine produced by Invitrogen, California, USA batch number: 11298800; and the chemicals for preparation of SDH incubation medium (0.1 M sodium succinate, 0.1 M PBS pH 7.4, nitro blue tetrazolium, dimethyl sulfoxide) (Ji et al., 2009) were purchased from Beijing Chemical Reagent Co. Beijing, China.

Animals

Male adult Sprague-Dawley, rats (grade II, weighing between 230-235 g) were supplied by Beijing Vital River, A Charles River Company) Certificate Number: SCXK-(Beijing) 2002-0003). The animals were housed separately and provided with necessary water and food ad libitum. The animal housing temperature was maintained at 24+-1oC, with a natural light/dark cycle.

Modeling methods

GTW tablets were crushed suspended in water and given to the rats by gavage feeding at 20 mg/kg once a day continuously for 30 days (Fang et al., 2000).

Animal grouping and drug administration

Seventy rats were adaptively fed for 1 week and then were randomly divided into 7 groups, each with 10 rats, the control group, the GTW model group, the negative control group, the Huangjingzanyu capsule group, the Huangjingzanyu optimized formula (= optimized formula) high-dose group, the moderate-dose group, and the low-dose group.

GTW at 2 mg/kg.d was administered to GTW model group by gavage feeding for 30 days. The animals were then sacrificed to measure each parameter. The control group was given 2 mL 0.5% CMC once a day for 60 days.

After the animal models were established the negative control group was administered 0.5% CMC at 2 mL/d by gavage feeding for 30 days.

Huangjingzanyu capsules were administered to Huangjingzanyu group at 465.mg/kg.d by gavage feeding for 30 days and different doses of the optimized formula were administered by gavage feeding at 12000 mg/kg.d (high dose), 6000 mg/kg.d (moderate dose) and 3000 mg/kg.d, small-dose groups for 30 days.

The animals in each group were weighed once a week for drug dose adjustment.

Sperm quality analysis method

Sperm collection and processing

Following Yu et al. (2000) with minor adjustments, we used a diffusion method to collect sperm from the tail of the epididymis. After drug administration, the animals were sacrificed.

The left tail of the epididymis was obtained and placed in 3 mL of saline at 37oC (preheated). Then, the specimen was cut into small pieces and incubated for 1 min. Next, a 50 uL sperm suspension was added into l mL of M199 medium and placed in a water bath at 37oC for 5 min.

Finally, a 13 uL suspension was added to a pre-warmed hemocytometer for sperm quality analysis.

Sperm quality analysis

The pre-treated sperm suspension to a counting board of WL-9000 sperm quality analysis system (Computer aided sperm analysis; Beijing WeiliNew Century Science and Tech. Div. Co, Beijing, China) at a test temperature of 37 C, Five fields of view were selected, and the analysis was completed within 2 minutes.

The main test parameters included sperm density (x 106/mL), sperm motility (%), sperm viability (%), straight-linear velocity (VSL, um/s), average path velocity (VAP, um/s), and curvilinear velocity (VCL, um/s).

ATP content assay

ATP CLS type II bioluminescent kit produced by Roche, Basel, Switzerland was used for ATP content assay (Xue et al., 2003). The specimens in each group were first defrosted. Then, a 0.l mL sperm suspension was added to pre-boiled 0.9 mL Tris-EDTA (pH 7.8), placed in 100oC boiling water for 2 min, and centrifuged for 1 min at 10,000 rpm/min.

Next, 100 uL of luciferase was added to 100 uL of supernatant and was then placed on ice for further testing. The standard curve of ATP was first generated using standard ATP samples. Then, each specimen was sequentially tested with a bioluminescence detection system (Turner Biosystem, USA), and ATP content was measured based on the ATP standard curves.

SDH activity assay

The methods for sperm suspension processing and the biochemical reactions were based on Ruiz-Pesini et al. (1998) with a few adjustments for improvement. The specimens in each group were washed with a rinse solution (0.1 M PBS +0.2% BSA) three times and then centrifuged for 8 min at 1500 r/min.

After the supernatant was removed, the incubation solution and the sperm suspension was mixed at a ratio of 2:1 and incubated in a water bath at 37 C for 2 h. A 10 uL sample was obtained to make a smear.

After air-drying, neutral balsam was used for slide mounting. Quantitative image analysis was then conducted on the smears, using a CMIAS multifunctional true color pathological image analysis system developed by the Air Force General Hospital and the Beijing University of Aeronautics, Beijing, China.

Each smear was analyzed at 200 x magnification over 5 randomly selected fields of view. More than 100 sperm cells were tested for integrated optical density (IOD), mean optical density (MOD), and positive area (um2). Because IOD integrates positive area and MOD, IOD was mainly used to represent SDH activity.

LDH-C4 assay

LDH-C4 activity was determined in strict accordance with the kit instructions. LDH-C4 assay kit produced by Shenzhen Huakang Biomedical Engineering Co., Ltd., Shenzhen, China was used in this study. After the specimens in each group were defrosted, substrates A and B (750 uL each) were added into the same reaction tube and pre-warmed at 37oC for 5 minutes.

Then, a 500-uL aliquot was added to 10 uL of defrosted sperm suspension. After rapid mixing, the samples were tested using a semi-automatic biochemical analyzer (Photometer 5010) (Boehringer Mannheim, Germany).

Statistical methods

All data are expressed as means +- standard deviations. The between-group comparison was conducted using a univariate analysis of variance (ANOVA) with SPSS11.0 software, with P Less than0.05 considered as statistically significant. The correlation between two variables was analyzed using a linear correlation analysis.

RESULTS

Modeling results

Compared with the control group, sperm density, motility, and viability were significantly decreased in the GTW model group. The results are shown in Table I.

Table I.-###Comparison of sperm density, motility, and

###viability between two groups ( +- s).

###Sperm###Sperm###Sperm

###Groups###N###concentration motility###live-rate

###(106/ml)###(%)###(%)

###Normal###10###11.41+-7.34###12.66+-6.15 34.85+-10.61

###Model###10###2.93+-0.97###4,42+-4.07###21.92+-8.53

Sperm velocity in the GTW model group

Compared with the control group, VSL, VAP, and VCL were significantly decreased in the GTW model group (PLess than0.05). The results are shown in Table II.

Table II.-###Comparison of sperm velocity between two###

###groups (X+- s).

Groups###n###VSL(um/s)###VAP (um/s)###VCL (um/s)###

Normal###10###15.19+-2.55###23.05+-2.69###52.20+-5.56###

Model###10###8.75+-2.6315###15.56+-3.63###38.80+-18.22

Sperm quality

The effects of the Huangjingzanyu optimized formula on sperm density, motility, and viability. Compared with the GTW model group, sperm density, motility, and viability were significantly increased in groups treated with high and moderate doses of the optimized formula (PLess than0.05). The therapeutic efficacies of those groups were similar to the Huangjingzanyu capsule group (PGreater than0.05).

The sperm density, motility, and viability were increased in the low-dose group. However, the difference was not statistically significant (PGreater than0.05). These results are shown in Table III and Figure 1.

Table III.-###Comparison of sperm density, motility, and###

###viability among groups (X+- s).###

Groups###n###Sperm###Sperm###Sperm###

###concentration###motility (%)###live-rate (%)###

###(106/ml)

Normal###10###11.41+-7.34###12.66+-6.15###34.85+-10.61###

Negative###10###3.81+-3.21###7.73+-5 .56###24.96+-7.67

Model###10###2.93+-0.97###4.42+-4.07###21.92+-8.53###

HJZY###10###8.87+-3 .11###12.82+-5.95###38.23+-23.69

Optimized###10###7.28+-3.09###10.14+-5.33###34.49+-15.34

Sperm velocity

Compared with the GTW model group, VSL, VAP, and VCL were significantly increased in the group treated with a moderate dose of the optimized

formula and the group treated with the Huangjingzanyu capsule (PLess than0.05). VCL was significantly increased in the group treated with the high dose of the optimized formula. VSL and VAP were significantly increased in the group treated with a low dose of the optimized formula. The results are shown in Table IV.

Sperm ATP content

Compared with the control group, the sperm ATP content was significantly decreased in the GTW model group; the difference was statistically significant (PLess than0.05). Compared with the GTW model group, the sperm ATP content was significantly increased in the groups treated with the Huangjingzanyu capsule and the high dose of the optimized formula (PLess than0.05).

No significant difference was noted in the group treated with the low dose of the optimized formula (PGreater than0.05). The results are shown in Table V.

Table IV.-###Comparison of sperm velocity among groups

###(X+-s).

###Groups###n###VSL(um/s)###VAP(um/s)###VCL(um/s)

###Normal###10###15.19+-2.55###23.05+-2.69###52.20+-5.56

###Negative###10###9.82+-3.67###18.15+-4.72###41.98+-8.68

###Model###10###8.75+-2.63###15.56+-3.64###38.80+-18.22

s###HJZY###10###13.54+-6.99###18.30+-6 .56###51.59+-11.25

###optimized###10###11.92+-5.35###16. 49+-7.75###50.76+-17.43

###optimized###9###14.16+-3.51###22.30+-4.17###50.67+-12.55

###optimized###9###13.22+-4. 86###20.66+-4.60###48.63+-10. 68

formula low

-dose

Table V.-###Comparison of sperm ATP content among###

###(pmol/106) sperm

###Normal###7###5.35+-1.61

###Negative###8###3.62+-2.85

###Model###10###1.59+-0.48

###HJZY###9###5.96+-4.75

###Optimized formula high-dose###9###5.59+-3.45

###Optimized formula moderate-dose###9###6.O1+-4.38

###Optimized formula low -dose###7###4.74+-4.32

Sperm SDH activity

Compared with the control group, the SDH activity was significantly decreased in the GTW model group; the difference was statistically significant (PLess than0.05).

Compared with the GTW model group, the SDH activity was significantly increased in the groups treated with high and moderate doses of the optimized formula (PLess than0.05). No significant difference was noted in the group treated with the low dose of the optimized formula (PGreater than0.05). The results are shown in Table VI.

Table VI.-###Comparison of sperm SDH activity among###

###groups (X+- s).###

Groups###n###IOD###

Normal###6###92.30+-17.74

Negative###6###50.44+-22.26###

Model###6###24.99+-10.28###

HJZY###6###97.60+-28.76###

Optimized formula high-dose###6###92.48+-37.93###

Optimized formula moderate-dose###6###118.20+-74.60

Optimized formula low -dose###6###61.61+-21.04

Compared with the control group, although the sperm LDH-C4 activity was decreased in the GTW group, the difference was not statistically significant (PGreater than0.05). Compared with the GTW model group, the sperm LDH-C4 activity was significantly increased in the groups treated with high and moderate doses of the optimized formula (PLess than0.05).

No significant difference was noted in the group treated with the low dose of the optimized formula (PGreater than0.05). The results are shown in Table

VII.

ATP content

Groups n

(pmol/106) sperm

Table VII.- Comparison of sperm LDH-C4 activity among###

###groups (X +- s).###

Groups###n###LDH-C4

###(mU/106 sperm)###

Normal###8###24.52+-10.44###

Negative###7###32.04+-26.96

Model###7###20.64+-11.81

HJZY###7###59.64+-52.24###

Optimized formula high-dose###7###61.75+-20.63###

Optimized formula moderate-dose###6###81.82+-55.41###

Optimized formula low -dose###6###41.58+-34.10

Correlation analysis between ATP and sperm SDH and LDH-C4 activities

In each group, sperm ATP content was positively correlated with sperm viability and SDH and LDH-C4 activities, respectively: sperm viability: r=0.805, PLess than0.05; SDH activity: r=0.908, PLess than0.01; LDH-C4 activity: r=0.866, PLess than0.05.

DISCUSSION

Based on the original Huangjingzanyu capsules, the Huangjingzanyu optimized formula is composed of five herbs: Rhizoma polygonati, tuber fleeceflower root, wolfberry, red sage root, and dandelion. A low dose of Rhizoma polygonati decoction can increase serum LDH activity in mice (Li and Zhao, 2010).

Tuber fleeceflower root can improve the survival rate and motility of sperm after thawing from frozen samples (Shi and Xu, 2010). Lycium barbarum polysaccharide is the major functional component in wolfberry and has a protective effect on the rat reproductive system (Huang et al., 2003a,b; sereflisan et al., 2013).

Red sage root liquid extract can effectively increase sperm motility and can improve sperm quality when sperm are processed in vitro (Zhao et al., 1998; Lu et al., 2002). In addition, the total salvianolic acid from red sage root extract can increase cellular ATP contents (Gong et al., 2013).

Dandelion can facilitate antigen engulfment among leukocytes and reticuloendothelial cells, thereby inhibiting immune responses.

In addition, dandelion has a relatively strong anti-inflammatory effect in the reproductive tract. With the interactions of different herbal components, the Huangjingzanyu optimized formula promotes the complementary effects of clearing and strengthening, which have the overall effects of "strengthening the kidneys and tonifying essence, activating blood circulation and removing blood stasis, eliminating wet and clearing dampness", thus promoting sperm production and fertilization.

Our early study showed HJZY optimized formula could restore the continuity of plasma structures of sperm heads, cervical parts and tails, render sperm heads to restore its well-stacked and homogeneous shape and correct the twisting deformity of sperm tails, thus to improve the sperm quality and enhance sperm motility capacity (Chen et al., 2008).

Sperm primarily use ATP produced from aerobic oxidation in mitochondria to maintain their motility. ATP content directly reflects mitochondrial functional status (Shi et al., 2008). SDH is mainly present in the mitochondria of germ cells in the testicles and is the key enzyme for aerobic respiration in testicular energy metabolism (Burgos et al., 1995).

Reduced SDH enzymatic activity can affect sperm energy metabolism, leading to a significant reduction in sperm motility (Xu et al., 2011). SDH can catalyze the transfer of succinate to fumarate, followed by ATP production after dehydrogenation.

Therefore, SDH activity is associated with ATP production. LDH-C4 is closely related to sperm production, metabolism, and energy acquisition. LDH-C4 can bind to the shuttle system and couple with electron transport in mitochondria, thereby aiding in the generation of ATP by oxidative phosphorylation. Therefore, mitochondrial ATP content is related to SDH and LDH-C4 activity levels.

GTW can inhibit sperm maturation. The extent of sperm mitochondrial damage is positively correlated with drug administration duration and dosage. Short-term drug administration can reduce LDH and SDH activity levels. In this study, sperm motility and viability were significantly reduced in the GTW model group compared with the control group. ATP content was also decreased in the GTW model group.

ATP content was significantly increased in the group treated with high and moderate doses of Huangjingzanyu optimized formula, suggesting that an increase in ATP content might be one reason for the improved sperm motility. And correlation analysis further demonstrated that ATP content was significantly correlated with SDH and LDH-C4 activity levels in each group, suggesting that the Huangjingzanyu optimized formula can increase ATP production by increasing the activity levels of mitochondrial marker enzymes related to energy metabolism, such as SDH and LDH-C4, thereby improving sperm motility.

The Huangjingzanyu optimized formula can increase ATP production in sperm flagella by increasing the activity levels of mitochondrial SDH and LDH-C4 in sperm, thereby playing a role in improving sperm motility.

ACKNOWLEDGEMENTS

The work is supported by the National Basic Research Program of China (973 program)under grant no.2011CB505403, and National Science Foundation of China under no.81273767.

Conflict of interest declaration.

There is no conflict of interest or otherwise.

Policy of Ethics Committee

This research has been accepted by Ethics Committee of Beijing University of Chiese Medicine.

REFERENCES

BURGOS, C., MALDONADO, C., GEREZ DE BURGOS, N.M. AND BLANCO, A., 1995. Intracellular localization of the testicular and sperm specific lactate dehydrogenase isozyme-C4 in mice. Biol. Reprod. 1,

84-92.

CHEEMA, R. S., VASHISHT, N., BHAKRI, G.. AND GANDOTRA, V. K., 2013. Molecular characterization and immunolocalization of sperm surface proteins (SPAM-1 and LDH-C4) in dog. Anim. Sci., 7: 96-106.

CHEN, B., HAN, D., LIU B.X. AND WANG, Q., 2009. Using AFM to observe sperm's ultrastructures in rats with asthenospermia before and after treatment with new Chinese Medicine Optimized Formula. Progr. Mod. Biomed., 21 : 4001-4005.

CHEN, B., WANG, Q. AND HAN, D., 2008. Study on Ultrastructural alteration of asthenospermia treated with huangjing zanyu optimized formula. Chinese J. Androl.,

5: 30-34.

CRIMMEL, A.S., CONNER, C.S. AND MONGA, M., 2001.

Withered yang: a review of traditional Chinese medical treatment of male infertility and erectile dysfunction. J. Androl., 22:173-182.

DUAN, C. AND GOLDBERG, E., 2003. Inhibition of lactate dehydrogenase C4(LDH- C4)blocks capacitation of mouse sperm in vitro. Cytogenet. Genome Res., 103:

352-359.

FANG, Q., JIANG, X.J. AND XIA, W.P., 2000. Morphological study on Kangning oral solution treatment for kidney insufficiency infertility caused by polyglycoside of Tripterygium wilfordii in rat testis. Acta Univ. Trad. Med. Sinen. Pharmacol. Shanghai, 4: 50-53.

GONG, W., XIAO, Y., ZHANG, M., WANG, Y. AND WANG, Y.,

2013. Synergistic protective effects of salvianolic acids and Panax notoginseng saponins on cardiomyocytes with hypoxia-reoxygenation injury. China J. Chin. Mater. Med., 7: 1046-1051.

GUO, J. AND CHANG, D.G., 2003. Integrated traditional Chinese and western medicine in andrology. People's Medical Publishing House, China, Beijing, pp. 245-256.

GUO, Y.J., LI, S.G. AND WANG, T.X., 1998. Enzymic

cytochemical studies of LDH-C4 in infertile spermatozoa. Chin. J. Histochem. Cytochem., 3:

443-449.

HUANG, X.L., YAN, J. AND WU, X.M, 2003a. The effects of Lycium Barbarum Polysaccharides on the damage inhibition of germ cell in mice induced by H2O2 . Food Sci., 12:116.

HUANG, X.L., YANG, M.L. AND WU, X.M., 2003b. Study on protective action of Lycium Barbarum Polysaccharides on DNA impairments of testicle cells in mice. J. Hyg. Res., 6: 599.

JI, Y. B., WU, P. AND LANG, L., 2009. Study on mitochondrial damage caused by solanine in mice testicular germ cells. Drug Eval. Res., 32:117-120.

LI, L. AND ZHAO, Y., 2010. The effect of Rhizoma polygonati

decoction on mice motility. J.Qiqihar Med. Coll., 12:

1862-1863.

LI, N. AND LI, J.Y., 2011. LDH-C: The most important testis-specific gene. Progr. Mod. Biomed., 20:

3975-3977.

LIU, B.X., WANG, Q., ZHAO, H.W. AND XIN, Z.C., 2006.

Effect of Hungjingzanyu Capsules on ultrastructure of sperm flagella in rats with asthenospermia. J. Beijing Univ. Chinese Med., 11: 765-767+793.

LU, Y., LIANG, Y.J. AND CHEN, S,Q., 2002. The use of extractant of salvia miltiorrhiza in sperm culture in vitro. Liaoning J. Tradit. Chinese Med., 7: 432-433.

RUIZ-PESINI, E., DIEZ, C., LAPEnA, A.C., PeREZ-MARTOS, A., MONTOYA, J., ALVAREZ, E., ARENAS, J. AND LOPEZ-PeREZ, M.J, 1998. Correlation of sperm motility with mitochondrial enzymatic activities. Clin. Chem., 44:1616-1620.

sEREFLIsAN, H.,CEK, s. AND sEREFLIsAN, M., 2013. The reproductive cycle of Potomida littoralis (Cuvier, 1798) (Bivalvia: Unionidae) in Lake GOlbasi, Turkey. Pakistan J. Zool., 5: 1311-1319.

SHI, B.W. AND XU, C.M., 2010. The preliminary study on protective effects of tuber fleeceflower root in human semen cryopreservation. Zhejiang J. Tradit. Chinese Med., 8: 610-612.

SHI, C., GUO, K. AND YEW, D.T., 2008. Effects of ageing and Alzheimer's disease on mitochondrial function of human platelets. Exp. Gerontol., 6: 589-594.

WU, T., 2012. Observation of therapeutic effect of Huangjing zanyu Capsule on asthenospermia. Asia-Pa. Tradit. Med., 6:73

XIN, N. AND LAN, F.H., 2008. Advances in research on sperm-specific lactate dehydrogenase-C4 [J]. J. Fuzhou Gen. Hosp., 1: 60-61+52.

XIONG, C.L., WU, M.Z. AND LIU, J.H., 2002. Human spermatology, Wu Han: Hubei Science and Technology Press, pp. 416-417.

XU, G.C., GAO, Q.Y., HAN, G.L. AND WANG, E.M., 2011.

Effect of omethoate on the testis succinic dehydrogenase (SDH) and the quality of sperm in male mice. China Anim. Husband. Vet. Med., 7: 148-150.

YU, L.C., WANG, M.Y. AND YANG, X.F., 2000. Application of computer-assisted sperm analysis (CASA) system to the detection of influence of cadmium on rat sperm motility. Chinese J. Ind. Hyg. Occup. Dis., 4: 223-225.

ZHANG, Z.S., 2012. Clinical observation on Traditional Chinese Medicine (TCM) differential treatment for male infertility caused by oligoasthenozoospermia and asthenospermia. Mod. J. Integr. Tradit. Chinese West.,

36: 4050-4052.

ZHAO, J.C., YANG, J.N. AND SUN, Y., 1998. A protective effect of salvia miltiorrhiza on genetic damage of germ cell induced by mitomycin C (MMC). Carcinog. Terat. Mutag., 1: 39-42.
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Author:Zheng, Yanfei; Liu, Baoxing; Wang, Ji; Chen, Jianxin; Jiao, Zhaozhu; Yuan, Zhuojun; Wang, Qi
Publication:Pakistan Journal of Zoology
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