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British Poultry Science

ISSN: 0007-1668 (Print) 1466-1799 (Online) Journal homepage: http://www.tandfonline.com/loi/cbps20

Evaluation of cockerel sperm viability and motilityby a novel enzyme based cell viability assay

H. L. Lin, R. B. Liaw, Y. H. Chen, T. C. Kang, D. Y. Lin, L. R. Chen & M. C. Wu

To cite this article: H. L. Lin, R. B. Liaw, Y. H. Chen, T. C. Kang, D. Y. Lin, L. R. Chen & M. C. Wu(2018): Evaluation of cockerel sperm viability and motility by a novel enzyme based cell viabilityassay, British Poultry Science, DOI: 10.1080/00071668.2018.1426832

To link to this article: https://doi.org/10.1080/00071668.2018.1426832

Accepted author version posted online: 22Jan 2018.

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Research Institute, Council of Agriculture, Executive Yuan, 112 Farm Rd., 71246

Tainan, Taiwan. E-mail: mcwu@mail.tlri.gov.tw

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Abstract. 1. The results of spermatozoa assessment by the WST-8 (2-[2-methoxy-

4-nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium,

monosodium salt) assay, flow cytometry (FC) or computer-assisted spermatozoa

analysis (CASA) were compared.

2. Different live/killed ratios of cockerel semen were serially diluted to 120, 60,

and 30 × 106 cells/ml, and each sample was analysed by (1) WST-8 assay at 0, 10,

20, 30, 40, 50, 60 min, (2) viability with flow cytometry, and (3) motility with

CASA.

3. The WST-8 reduction rate was closely correlated with spermatozoa viability

and motility. The optimal semen concentration for the WST-8 assay was 120 ×

106 cells/ml, and the standard curves for spermatozoa viability and motility

predictions respectively were yviability60 = 162.8x + 104.96 (R² = 0.9594) after 60

min of incubation and ymotility40 = 225.09x + 96.299 (R² = 0.8475) after 40 min of

incubation.

4. It was conclused that the WST-8 assay is useful for the practical evaluation of

cockerel spermatozoa viability and motility. Compared to FC and CASA, the

WST-8 assay does not require expensive and complex instrumentation in the lab.

Furthermore, one well of the WST-8 reaction can be used to predict spermatozoa

viability and motility at the same time which all lead it to be efficient and

economical for semen quality assessment.

Keywords: CASA, flow cytometry, formazan, MTT, reduction rate, standard

curve

INTRODUCTION

It is necessary to evaluate the quality of semen samples before artificial

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insemination (Graham and Moce, 2005; Petrunkina et al., 2007; Dhurvey et al.,

2012; Sikka and Hellstrom, 2016). The spermatozoa mass motility test is fast and

inexpensive, but is subjective and the evaluation results can differ from one

analyst to another. Computer-assisted spermatozoa analysis (CASA) technology

was introduced in the late 1980s (Amann and Waberski, 2014; Mortimer et al.,

2015). With CASA, spermatozoal images are captured by a microscope camera,

using the defined software to determine spermatozoa variables, such as motility

and morphology (King et al., 2000; Lu et al., 2014; Maroto-Morales et al., 2016;

Santiago-Moreno et al., 2016). Flow cytometry (FC) can detect spermatozoa

labelled by various specific fluorescent probes and rapidly calculates the relevant

numbers of spermatozoa for assessing different functional characteristics (for

example, plasma and acrosomal integrity, mitochondrial function and DNA

status) (Christensen et al., 2004; Cordelli et al., 2005; Graham and Moce, 2005;

Moce and Graham, 2008; Partyka et al., 2010). Both CASA and FC can provide

accurate, reliable, objective and efficient analysis for spermatozoa quality;

however, they require expensive and complex instrumentation in the laboratory.

The MTT (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide)

reduction assay is used to determine mitochondrial dehydrogenase activities in

living cells, which is applied extensively in many studies to assess viability in

different cells (Holder et al., 2012), including spermatozoa (HARA et al., 1995;

Hazary et al., 2001; Nasr-Esfahani et al., 2002; Aziz et al., 2005; Aziz, 2006;

Byun et al., 2008). MTT is reduced by NADH to form water-insoluble purple

formazan, which has a needle-shaped crystal and accumulates in cells (Berridge et

al., 1996; Berridge et al., 2005). It requires an organic solvent to solubilise purple

formazan before measuring the absorbance. Needle-shaped crystals and organic

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solvents both damage cells (Tominaga et al., 1999). The WST-8 assay is another

enzyme-based method for determining cell viability (Ishiyama et al., 1997; Holder

et al., 2012). However, WST-8 (2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-

5-[2,4-disulfophenyl]-2H-tetrazolium, monosodium salt) formazan is water-

soluble and results in less damage and less toxicity to viable cells.

The WST-8 assay is a fast, simple and reliable method to evaluate cell

viability, however, to our knowledge there is no report describing its application

for semen quality evaluation. The purpose of the present study was to establish the

value of the WST-8 assay for cockerel spermatozoa quality assessment.

MATERIALS AND METHODS

Animals and semen collection

All experiments were carried out in accordance with the legislation governing the

ethical treatments of animals and approved by the LRI IACUC (No 106-38).

Forty adult Taiwan Native chicken L7 and L12 cockerels of 57 to 63

weeks of age were used in this study. L7 and L12 chickens are two inbred lines of

Taiwan Native chickens, selected for meat production at the Livestock Research

Institute, Tainan, Taiwan for 20 years. Cockerels were divided into 4 groups (10

cockerels each) and semen was collected routinely by abdominal massage

(Burrows and Quinn, 1937) once a week. Semen from 10 cockerels of each group

was pooled and the spermatozoa concentration was determined immediately by a

photometer (ACCUREAD, IMV, L’Aigle, France).

Experimental design

In order to obtain the standard curve of the relationship between the WST-8

reduction rate, spermatozoa viability and motility, the frozen-killed procedure was

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applied as described (Aziz et al., 2005; Byun et al., 2008). Fresh semen samples

of 4 cockerel groups were 1:1 diluted with PBS (Lonza, Basel, Switzerland) and

divided into viable and killed portions. The viable portion was kept at room

temperature, and the killed portion were plunged into liquid nitrogen and thawed

at 37°C for two cycles. Different live/killed semen were prepared by mixing

aliquots of viable and killed spermatozoa ratios of 0/10, 2/8, 4/6, 6/4, 8/2, and

10/0 (v/v), respectively. The prepared semen samples were serial diluted to 120,

60, and 30 × 106 cells/ml, and each sample were analysed by (1) WST-8 assay at

0, 10, 20, 30, 40, 50, 60 min (2) viability with flow cytometry and (3) motility

with CASA.

WST-8 assay

Cell Counting Kit-8 (CCK-8®) (Bimake, Houston, USA) was adopted to perform

WST-8 assay according to the manufacturer's instructions. CCK-8® is a redox

indicator that takes the highly water soluble tetrazolium salt WST-8 to produce a

water-soluble formazan dye upon the reduction by cellular dehydrogenases, which

is directly proportional to the number of living cells. For each sample, three

repeats of 100 μl semen were prepared in wells of the 96-well microplate (Greiner

Bio-One, Kremsmünster, Austria). Then 10 μl CCK-8® solution was added

directly to each well and examined by spectrophotometry (BioTek, Vermont,

USA) to record the absorbance at a wavelength of 450 nm after incubation at

37°C for 10, 20, 30, 40, 50, and 60 min, respectively.

Flow cytometry analysis

Guava® easyCyte microcapillary flow cytometry (Guava Technologies Inc.,

Hayward, CA, USA; distributed by IMV Technologies) with EasyKit 1 Viability

and Concentration (ref. 024708, IMV Technologies) was used to determine

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cockerel spermatozoa viability according to the manufacturer's instructions. The

kit contains SyBr14 and PI dyes with differential permeability to viable

(membrane intact) and nonviable (membrane damaged) cells. Each aliquot of

57000 spermatozoa was placed into the well of EasyKit 1 Viability and

Concentration, incubated at 37°C for 10 min, and then passed through the

easyCyte. A total of 5000 events were analysed for each sample, and results were

expressed as a percentage of viable spermatozoa (Sellem et al., 2015).

CASA analysis

Spermatozoa motility was assessed with a CEROS IITM CASA system (Hamilton

Thorne Inc., Beverly, MA, USA) with settings of 30 frames to avoid spermatozoa

track overlapping, minimum contrast = 50, non-motile head size = 7 pix, and non-

motile intensity = 95 (Moce et al., 2010). Spermatozoa concentration was

adjusted to 30 × 106 cells/ml, and a 2.6 μl aliquot was placed into a four-chamber

Leja counting slide (Leja Products B.V., Nieuw-Vennep, the Netherlands).

Motility was evaluated with a minimum 200 spermatozoa per sample, and results

were expressed in percentage of motile spermatozoa.

Statistical analysis

The CORR procedure of SASTM package v 9.2 (SAS Institute Inc., Cary, NC,

USA) was applied to calculate the Pearson correlation coefficient between WST-8

reduction rate and viability/motility of cockerel semen. P < 0.001 was considered

as extremely significant, and P < 0.01 was considered as highly significant.

Hereafter, data were analysed by the REG procedure of SASTM to result in the

standard curves for prediction of spermatozoa viability and motility.

RESULTS

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Analysis of different ratios of live/killed semen samples

The viability and motility of semen samples are presented in the Table 1. The

viability/motility of 0/10, 2/8, 4/6, 6/4, 8/2 and 10/0 live/killed samples

respectively were 0.02%/1.18%, 18.8%/20.6%, 30.4%44.4/%, 49.1%/59.5%,

63.9%/66.0%, and 78.2%/70.6%. The results demonstrate that the semen samples

prepared for the standard curves exactly reflected the different live/killed ratios.

Table 1 near here

Correlation between WST-8 reduction rate and viability/motility

Pearson correlation coefficients between the WST-8 reduction rate and

viability/motility are shown in Table 2. In all groups, the correlation increased

gradually with semen concentration and incubation period. When semen

concentration increased to 120 × 106 cells/ml, the WST-8 reduction rate and

viability are very strongly correlated (r ≧ 0.933, P < 0.001) at 10, 20, 30, 40, 50,

and 60 min; the WST-8 reduction rate is also very strongly correlated (r ≧ 0.916,

P < 0.001) with motility at 30, 40, 50, and 60 min.

Table 2 near here

Effects of WST-8 reduction rate, viability and motility

Based on the result of the correlation coefficient analysis, the strongly correlated

groups were followed with regression analysis and the three most appropriate

standard curves for prediction of spermatozoa viability and motility were obtained.

For spermatozoa viability prediction, the three standard curves were: yviability40 =

237.86x + 95.425 (R² = 0.9357), yviability50 = 192.05x + 100.02 (R² = 0.9497) and

yviability60 = 162.8x + 104.96 (R² = 0.9594) at 40, 50, and 60 min (Figure 1),

respectively. The optimal incubation period for viability prediction was 60 min,

but 40 and 50 min are also acceptable. For spermatozoa motility prediction, three

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standard curves at 40, 50, and 60 min were ymoyility40 = 225.09x + 96.299 (R² =

0.8475), ymoyility50 = 180.17x + 100.16 (R² = 0.8454) and ymoyility60 = 151.74x +

104.4 (R² = 0.8431), respectively (Figure 2). The optimal incubation for motility

prediction was 40 min.

Figure 1 and 2 near here

DISCUSSION

The WST-8 assay has been widely used to determine the viability of various types

of cells (Ishiyama et al., 1997; Holder et al., 2012). However, this study is the

first report which indicates that this method could be applied to cockerel semen

evaluation for both spermatozoa viability and motility. WST-8 is a water-soluble

tetrazolium dye, and there are several commercial ready-to-use solution kits

available. It offers a simple, rapid, reliable, quantitative and sensitive

measurement of cell viability for use in practical applications. Tominaga et al.

(1999) tested WST-8 with microplates on 5 different cell lines of HeLa (human

cervical cancer), RC (rabbit cornea), L929 (mouse connective tissue), Balb3T3

(mouse embryo) and HL60 (human acute promyelonic Leukemia) cells and

compared the sensitivity with results obtained with other tetrazolium dyes.

Compared to an MTT assay, WST-8 did not form needle-shaped crystals, so the

absorbance could be measured directly without solubilisation by an organic

solvent. Therefore, the WST-8 assay might be more efficient and reliable than the

MTT assay.

Several studies reveal that in vitro analysis of spermatozoa viability and

motility are correlated with fertilising potential in vivo (Donoghue, 1999; King et

al., 2000; Blesbois et al., 2008; Hossain et al., 2011; Farah et al., 2013;

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Petrunkina and Harrison, 2013). It is therefore important to measure spermatozoa

viability and motility before artificial insemination in order to maximise fertility.

Eosin-nigrosin staining has been extensively used to evaluate spermatozoa

viability for decades, but there are reports that double fluorescence SyBr14 and PI

staining is more effective than the eosin-nigrosin test for the assessment of

cockerel spermatozoa viability (Chalah and Brillard, 1998; Chalah et al., 1999).

SyBr14/PI double staining is a spermatozoa membrane assay. When PI penetrates

membrane-damaged spermatozoa it displaces Sybr14 fluorescence. Thus, live

spermatozoa fluoresce green and dead spermatozoa are red. The spermatozoa

membrane is important for many functions such as the ability to maintain cell

homeostasis, exercise motility and interact with the female genital tract. In this

study, different ratios of live/killed cockerel semen samples were prepared and

double staining with SyBr14 and PI was used to determine spermatozoa viability.

Each semen sample was therefore compared in different live/killed ratios between

SyBr14/PI viability and WST-8 reduction rate to obtain the prediction curves for

cockerel spermatozoa viability. Thus, the viability prediction curves attained in

this study are more accurate than the prediction from eosin-nigrosin staining

viability.

Byun et al. (2008) used the MTT assay to evaluate boar spermatozoa

viability and the resultant correlation coefficient between MTT at 1 h and 4 h of

incubation and spermatozoa viability from eosin-nigrosin staining was 0.9493 and

0.9564, respectively. Another study (Aziz et al., 2005) also reported a correlation

between MTT reduction assay at 1 h and 4 h of incubation with equine

spermatozoa viability of r = 0.954 and r = 0.977, respectively. Compared to their

findings, this study indicates that the incubation period could be shortened to 50

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min by increasing the semen concentration to 120 × 106 cells/ml to get a similar

result, which means that increasing semen concentration could improve assay

efficiency. However, this dissimilarity could also be due to different sensitivities

between the MTT and WST-8 assays, or the different dehydrogenase activity

between boar, stallion and cockerel spermatozoa.

In conclusion, the WST-8 assay needs only a plate reading

spectrophotometer instead of expensive and complex FC or CASA. Moreover,

many samples can be tested at the same time. Above all, one well of WST-8

reaction can be used to predict spermatozoa viability and motility at the same time.

All these advantages make the WST-8 assay a simple and practical tool for

evaluating cockerel spermatozoa quality in a simply-equipped laboratory or a

breeding farm.

ACKNOWLEDGEMENTS

This study was performed with the financial support of the Council of Agriculture,

Executive Yuan (1062101011003-020501L1). The authors would like to thank Mr

Ming-Chuan Chien, Mr Yung-Yao Cheng and Ms Wen-Tsen Chen of Animal

Industry Division at LRI for assistance with animal management and semen

collection.

REFERENCES

AMANN, R.P. & WABERSKI, D. (2014) Computer-assisted spermatozoa

analysis (CASA): capabilities and potential developments. Theriogenology,

81: 5-17.

Accep

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12

AZIZ, D.M. (2006) Assessment of bovine spermatozoa viability by MTT

reduction assay. Animal Reproduction Science, 92: 1-8.

AZIZ, D.M., AHLSWEDE, L. & ENBERGS, H. (2005) Application of MTT

reduction assay to evaluate equine spermatozoa viability. Theriogenology, 64:

1350-1356.

BERRIDGE, M.V., HERST, P.M. & TAN, A.S. (2005) Tetrazolium dyes as tools

in cell biology: New insights into their cellular reduction. Biotechnology

Annual Review, 11: 127-152.

BERRIDGE, M.V., TAN, A.S., MCCOY, K.D. & WANG, R. (1996) The

biochemical and cellular basis of cell proliferation assays that use tetrazolium

salts. Biochemica, 4: 14-19.

BLESBOIS, E., GRASSEAU, I., SEIGNEURIN, F., MIGNON-GRASTEAU, S.,

SAINT JALME, M. & MIALON-RICHARD, M. (2008) Predictors of

success of semen cryopreservation in chickens. Theriogenology, 69: 252-261.

BURROWS, W. & QUINN, J. (1937) The collection of spermatozoaatozoa from

the domestic fowl and turkey. Poultry Science, 16: 19-24.

BYUN, J., CHOO, S., KIM, H., KIM, Y., HWANG, Y. & KIM, D. (2008)

Evaluation of boar spermatozoa viability by MTT reduction assay in

Beltsville thawing solution extender. Asian-Australasian Journal of Animal

Sciences, 21: 494-498.

CHALAH, T. & BRILLARD, J. (1998) Comparison of assessment of fowl

spermatozoa viability by eosin-nigrosin and dual fluorescence (SYBR-14/PI).

Theriogenology, 50: 487-493.

CHALAH, T., SEIGNEURIN, F., BLESBOIS, E. & BRILLARD, J. (1999) In

vitro comparison of fowl spermatozoa viability in ejaculates frozen by three

Accep

ted M

anus

cript

13

different techniques and relationship with subsequent fertility in vivo.

Cryobiology, 39: 185-191.

CHRISTENSEN, P., STENVANG, J.P. & GODFREY, W.L. (2004) A flow

cytometric method for rapid determination of spermatozoa concentration and

viability in mammalian and avian semen. Journal of Andrology, 25: 255-264.

CORDELLI, E., ELEUTERI, P., LETER, G., RESCIA, M. & SPANÒ, M. (2005)

Flow cytometry applications in the evaluation of spermatozoa quality: semen

analysis, spermatozoa function and DNA integrity. Contraception, 72: 273-

279.

DHURVEY, M., GUPTA, V., NEMA, S., PATIDAR, A., SHIVHARE, M.,

SINGH, N. & SHAKYA, V. (2012) Modern semen evaluation techniques in

domestic animals: A review. DHR International Journal of Biomedical and

Life Sciences, 3: 62-83.

DONOGHUE, A.M. (1999) Prospective approaches to avoid flock fertility

problems: predictive assessment of spermatozoa function traits in poultry.

Poultry Science, 78: 437-443.

FARAH, O.I., CUILING, L., JIAOJIAO, W. & HUIPING, Z. (2013) Use of

fluorescent dyes for readily recognizing spermatozoa damage. Journal of

Reproduction & Infertility, 14: 120-125.

GRAHAM, J.K. & MOCÉ, E. (2005) Fertility evaluation of frozen/thawed semen.

Theriogenology, 64: 492-504.

HARA, H., TAKAI, R., TANAKA, N., MIZOGUCHI, K., TSUNO, T.,

IGARASHI, S.I. & USAMI, M. (1995) Simple methods for objective

assessment of spermatozoa viability and motility with MTT assay and

Accep

ted M

anus

cript

14

spermatozoa quality analyzer (SQA) in rats. Congenital Anomalies, 35: 477-

480.

HAZARY, R.C., CHAUDHURI, D. & WISHART, G.J. (2001) Application of an

MTT reduction assay for assessing spermatozoa quality and predicting

fertilising ability of domestic fowl semen. British Poultry Science, 42: 115-

117.

HOLDER, A.L., GOTH-GOLDSTEIN, R., LUCAS, D. & KOSHLAND, C.P.

(2012) Particle-induced artifacts in the MTT and LDH viability assays.

Chemical Research in Toxicology, 25: 1885-1892.

HOSSAIN, M.S., JOHANNISSON, A., WALLGREN, M., NAGY, S.,

SIQUEIRA, A.P. & RODRIGUEZ-MARTINEZ, H. (2011) Flow cytometry

for the assessment of animal spermatozoa integrity and functionality: state of

the art. Asian Journal of Andrology, 13: 406-419.

ISHIYAMA, M., MIYAZONO, Y., SASAMOTO, K., OHKURA, Y. & UENO,

K. (1997) A highly water-soluble disulfonated tetrazolium salt as a

chromogenic indicator for NADH as well as cell viability. Talanta, 44: 1299-

1305.

KING, L.M., HOLSBERGER, D.R. & DONOGHUE, A.M. (2000) Correlation of

CASA velocity and linearity parameters with spermatozoa mobility

phenotype in turkeys. Journal of Andrology, 21: 65-71.

LU, J.C., HUANG, Y.F. & LÜ, N.Q. (2014) Computer-aided spermatozoa

analysis: past, present and future. Andrologia, 46: 329-338.

MAROTO-MORALES, A., GARCÍA-ÁLVAREZ, O., RAMÓN, M.,

MARTÍNEZ-PASTOR, F., FERNANDEZ-SANTOS, M.R., SOLER, A.J. &

Accep

ted M

anus

cript

15

GARDE, J.J. (2016) Current status and potential of morphometric

spermatozoa analysis. Asian Journal of Andrology, 18: 863-870.

MOCÉ, E. & GRAHAM, J.K. (2008) In vitro evaluation of spermatozoa quality.

Animal Reproduction Science, 105: 104-118.

MOCÉ, E., GRASSEAU, I. & BLESBOIS, E. (2010) Cryoprotectant and

freezing-process alter the ability of chicken spermatozoa to acrosome react.

Animal Reproduction Science, 122: 359-366.

MORTIMER, S.T., VAN DER HORST, G. & MORTIMER, D. (2015) The future

of computer-aided spermatozoa analysis. Asian Journal of Andrology, 17:

545-553.

NASR-ESFAHANI, M.H., ABOUTORABI, R., ESFANDIARI, E. & MARDANI,

M. (2002) Spermatozoa MTT viability assay: a new method for evaluation of

human spermatozoa viability. Journal of Assisted Reproduction and Genetics,

19: 477-482.

PARTYKA, A., NIŻAŃSKI, W. & ŁUKASZEWICZ, E. (2010) Evaluation of

fresh and frozen-thawed fowl semen by flow cytometry. Theriogenology, 74:

1019-1027.

PETRUNKINA, A. & HARRISON, R. (2013) Fluorescence technologies for

evaluating male gamete (Dys) function. Reproduction in Domestic Animals,

48: 11-24.

PETRUNKINA, A.M., WABERSKI, D., GUNZEL-APEL, A.R. & TŌPFER-

PETERSEN, E. (2007) Determinants of spermatozoa quality and fertility in

domestic species. Reproduction, 134: 3-17.

SANTIAGO-MORENO, J., ESTESO, M.C., VILLAVERDE-MORCILLO, S.,

TOLEDANO-DÍAZ, A., CASTAÑO, C., VELÁZQUEZ, R., LÓPEZ-

Accep

ted M

anus

cript

16

SEBASTIAN, A., GOYA, A.L. & MARTINEZ, J.G. (2016) Recent advances

in bird spermatozoa morphometric analysis and its role in male gamete

characterization and reproduction technologies. Asian Journal of Andrology,

18: 882-888.

SELLEM, E., BROEKHUIJSE, M.L., CHEVRIER, L., CAMUGLI, S.,

SCHMITT, E., SCHIBLER, L. & KOENEN, E.P. (2015) Use of

combinations of in vitro quality assessments to predict fertility of bovine

semen. Theriogenology, 84: 1447-1454.

SIKKA, S.C. & HELLSTROM, W.J. (2016) Current updates on laboratory

techniques for the diagnosis of male reproductive failure. Asian Journal of

Andrology, 18: 392-401.

TOMINAGA, H., ISHIYAMA, M., OHSETO, F., SASAMOTO, K.,

HAMAMOTO, T., SUZUKI, K. & WATANABE, M. (1999) A water-soluble

tetrazolium salt useful for colorimetric cell viability assay. Analytical

Communications, 36: 47-50.

FIGURE LEGENDS

Figure 1. Linear regression between WST-8 reduction rate (ΔOD) and viability in

40, 50 and 60 min.

Figure 2. Linear regression between WST-8 reduction rate (ΔOD) and motility at

40, 50 and 60 min.

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Figure 1.

yviability40 = 237.86x + 95.425

R² = 0.9357

0

20

40

60

80

100

-0.5 -0.4 -0.3 -0.2 -0.1 0.0

Viability (%)

Δ OD (nm)

yviability50 = 192.05x + 100.02

R² = 0.9497

0

20

40

60

80

100

-0.8 -0.6 -0.4 -0.2 0.0

Viability (%)

Δ OD (nm)

yviability60 = 162.8x + 104.96

R² = 0.9594

0

20

40

60

80

100

-0.8 -0.6 -0.4 -0.2 0.0

Viability (%)

Δ OD (nm)

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Figure 2

ymotility40 = 225.09x + 96.299

R² = 0.8475

0

20

40

60

80

100

-0.5 -0.4 -0.3 -0.2 -0.1 0.0

Motility (%)

Δ OD (nm)

ymotility50 = 180.17x + 100.16

R² = 0.8454

0

20

40

60

80

100

-0.8 -0.6 -0.4 -0.2 0.0

Motility (%)

Δ OD (nm)

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Table 1. The viability and motility of cockerel semen samples in different

live/killed ratios

Live/killed ratio Viability (%) Motility (%)

0/10 0.02 ± 0.01a 1.18 ± 0.54a

2/8 18.78 ± 5.15b 20.58 ± 1.19b

4/6 30.08 ± 4.02c 44.35 ± 6.40c

6/4 49.11 ± 5.84d 59.45 ± 7.75d

8/2 63.90 ± 3.95e 66.95 ± 7.71e

10/0 78.15 ± 3.27f 70.63 ± 7.53f

Values are means ± SEM from 24 samples in 4 different experiments. a-f Different superscript letters within a column indicate statistically significant differences between different live/killed sperm ratios (P < 0.01).

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Table 2. Pearson correlation coefficients between WST-8 reduction rate and

viability/motility in different incubation periods

Correlation coefficients

Semen concentration (106 cells/ml)

Incubation period (min) 120 60 30

Viability

10 0.933*** 0.597** 0.214

20 0.933*** 0.735*** 0.621**

30 0.958*** 0.824*** 0.676***

40 0.967*** 0.860*** 0.742***

50 0.975*** 0.895*** 0.800***

60 0.979*** 0.920*** 0.838***

Motility

10 0.881*** 0.633*** 0.285

20 0.896*** 0.798*** 0.698***

30 0.916*** 0.838*** 0.695***

40 0.921*** 0.878*** 0.753***

50 0.919*** 0.900*** 0.805***

60 0.918*** 0.912*** 0.839***

WST-8 reduction rate=ΔO.D. at 450 nm. Data collected from 72 samples in 4 different experiments. ** Correlation differs significantly from zero at P < 0.01. *** Correlation differs significantly from zero at P < 0.001.