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Effect of Green Tea Extract (Camellia sinensis) on Fertility Indicators of Post-Thawed Bull Spermatozoa.

Byline: Hamayun Khan, Momen Khan, Muhammad Subhan Qureshi, Shakoor Ahmad, Ali Gohar, Hameed Ullah, FarmanUllah, Arab Hussain, Pershotam Khatri, Said Sajjad Ali Shah, Hamid Rehman and Azmatullah Khan

Abstract

Green tea extract is plant-dervied natural antioxidant. The objective of this study was to explore the antioxidative effect of green tea extract on the cryopreserved bull semen. Experiments were carried out on Achai breeding bulls of similar age at the Semen Production Unit, Harichand, District Charsadda, Khyber Pakhtunkhwa. Collection of semen was done using artificial vagina (42degC) for three weeks. Semen was extended in egg yolk extender at various inclusion levels (0.0%, 0.25%, 0.5%, 0.75 and 1.0%) of green tea extract. Testing was consummated for quality characteristics of post-thawed spermatozoa such as motility, viability and plasma membrane integrity. The data was statistically analyzed with one-way analysis of variance using SPSS. The highest significant (P<0.05) response was obtained in extenders containing 0.75% green tea extract for all studied parameters.

Based on the findings of the current study, it can be concluded that supplementation of green tea extract in semen extender showed significant response on the post-thawed spermatozoa motility, viability and membrane integrity of Achai bull.

Key words

Green tea extract, Post thaw, Achai bull semen.

INTRODUCTION

Semen cryopreservation is an important technique, widely used in ruminate breeding progamme to increase animal yields for fullfilling the rising demand of meat, milk and work in developing countries. Freeze-thawing procedure of sperm is performed characteristically for artificial insemination (Bucak et al., 2008). During cryopreservation, semen is exposed to cold shock and atmospheric oxygen, this raise up the level of lipid peroxidation (Bucak et al., 2011). Experiments have shown that oxidative stress produced in these procedures due to high reactive oxygen species (ROS) are associated with low quality of seminal material and death of sperm cells with abnormal morphology, thus resulting in a predominant impairment for profitable semen cryopreservation (Janice et al., 2000).

It is well known that natural antioxidants of bovine semen are not enough to safeguard the sperm integrity against oxidative stress during the process of cryopreservation (Sreejith et al., 2006; Nichi et al.,2006). Additionally, during freeze-thawing cycles, the intensity of naturally occurring antioxidants in bovine semen get diminished (Bilodeau et al., 2000). Lower fertility and conception rate have been reported in different animal studies with frozen-thawed semen (Maxwell et al., 1993; Bhosrekar et al., 2001). To cope up with these harmful effects, supplementation of antioxidant may be a feasible approach to improve cryopreservation tech-niques (Anghel et al., 2010) for successful artificial insemination.

Recently plant-derived antioxidants have been getting major focus due to their lower cytotoxicity and are considered to be better than synthetic antioxidants (Gupta and Sharma, 2006; Nagulendran et al., 2007; Sen et al., 2010; Ibrahim et al., 2014). Additionally, green tea (Camila sensis) holds greater antioxidant protection than the ordinary polyphenols in antioxidant vitamins such as C and E (Tedeschi et al., 2004), also its antioxidative activity is stronger than butylated hydroxyanisole, butylated hydroxytoluene and DL-alpha-tocopherol. Toxicity generated with tea polyphenols is lesser than butylated hydroxyanisole, butylated hydroxytoluene and dl-alpha-tocopherol (Chen and Wan, 1994). It is well documented that Green tea has valuable effects on health due to abundant amount of polyphenols that are the major water-soluble components of green tea infusions.

Green tea polyphenols include epigallocatechin-gallate (EGCG), epicatechin-gallate (ECG), epicatechin (EC), and epigallocatechin (EGC) (Wittayarat et al., 2013). In addition to strong antioxidant and antioxidative characteristics, green tea polyphenols (GTP) are potent scavengers of ROS superoxide, hydrogen peroxide, hydroxyl radicals, and nitric oxide produced. In the recent past, it has been confirmed that EGCG prevents spontaneous mutations and chromosomal damage induced by ROS in somatic cells (Roy et al., 2003).

Plant-derived extracts are excellent sources of the natural antioxidant. Several studies documented that the plant-derived antioxidant or herbs are associated with improvement of spermatozoa in freezing extender. Antioxidative protection property of green tea extract on the quality of canine, avian and mouse semen have been confirmed (Wittayarat et al., 2013; Al-Daraji, 2011; Abshenas et al., 2011). On the other hand, comprehensive assessment of the green tea extract in the semen extenders for successful ruminant breeding plan is not well documented. Though recently one study has been reported in Sahiwal bull assessing green tea extract effect at low concentration (0.25% and 0.5%) in semen extender.

Therefore, current study was an attempt to clarify the effect of different level of green tea extract (0.25, 0.5, 0.75 and 1.0) on post-thawed spermatozoa functional indicators in Achai breeding bulls; a dairy and light drought indigenous cattle breed of Khyber Pakhtunkhwa that have the potential for continued existence under a mountainous and sub-mountainous environment (Khan et al., 2008).

MATERIALS AND METHODS

Experimental design

Standard procedure as previously described was used for the semen collection through artificial vagina (42degC) from four mature Achai bulls (Andrabi et al., 2008; Gohar et al., 2014). Experimental animals were nurtured in clean and hygienic environment maintained at Semen Production Unit Harichand, Distt Charsadda, Khyber Pakhtunkhwa, Pakistan. Ejaculates were collected twice a week for three weeks successively. Immediately after collection, the ejaculates were transferred to laboratory and kept in water bath at 37oC. The semen samples were subjected to gross (volume, color) and microscopic (percentage motility) evaluation. Semen ejaculates having motility above 65% were included in this study.

Preparation of green tea extract

Standard protocol was utilized to prepare green tea extract as previously described (Chan et al., 2007; Ali et al., 2014). Briefly, green tea leaves were powdered in a laboratory blender. Four grams of powdered tea leaves were mixed with 200 ml of methanol, kept for 18 h at room temperature followed by centrifugation at 6200 rpm for 20 min. Supernatant was taken and filtered through filter paper. Extract was kept frozen until use.

Preparation of semen extender

Semen samples showing normal characteristics of motility above 65% were pooled to eliminate bull effect and to have adequate semen. Semen samples were pooled and extended in egg yolk citrate extender (Khan and Ijaz, 2007). Briefly, Tris HCl 24.20 g, citric acid 13.4 g, fructose 10g, glycerol 70ml, egg yolk 200 ml, streptomycin 1 g, benzyl penicillin 500,000 IU, distil water up to 1000ml, at 37degC within 10 min after collection. The concentration of spermatozoa was adjusted to 80 x 106 sperms per ml.

Table I.- Inclusion level of green teat extract in semen extender.

Sr.###Green tea###Extract###Total volume (ml)

No.###extract (%age)###volume (l)###(extract+extender)

1###0.00###0.00###10

2###0.25###25###10

3###0.5###50###10

4###0.75###75###10

5###1.00###100###10

Inclusion level of green tea extracts in extended semen

The composition of the green tea extract in semen extender at different concentration has been indicated in Table I. To evaluate the effect of green tea extracts, different inclusion levels of green tea extract (0.0%, 0.25%, 0.5%, 0.75% and 1%) were added to individual test tubes. Tubes containing green tea extract were incubated at 37degC for 30 min to allow methanol to evaporate followed by the addition of the extended semen to tubes. The tubes were incubated at 37degC for 5 min to allow uptake of green tea extracts by spermatozoa. Semen samples were transferred from 37degC to 4degC for 2 h for equilibration. Extended semen was filled into 0.5 ml straws in cold cabinet unit and was frozen first at -120degC by keeping in nitrogen vapors for 7 min, 4 cm above liquid nitrogen. Semen straws were then frozen at -196degC by dipping in liquid nitrogen.

Post-thawed semen evaluation

At the time of analysis, one straw of semen from each treatment was thawed separately at 37degC for 30s to perform the semen quality parameters. Five semen straws were evaluated for each treatment.

Spermatozoa motility

For the assessment of motility, one straw of semen was thawed in water bath at 37degC for 30 seconds and then semen straw was emptied in a test tube kept in water bath. A drop (10l) of semen was placed on prewarmed (37degC) glass slide and covered with cover slip. Percentage motility was assessed at X40 under phase-contrast microscope (Olympus BX51 TF, Japan) attached with closed circuit camera following the standard procedure (Ijaz et al., 2009).

Spermatozoa viability

Eosin-nigrosin stain was used to evaluate the viability (live/dead %) of the frozen-thawed semen. Briefly described as a small drop of frozen-thawed semen was placed on a pre-warmed slide and mixed with a relatively larger drop of the supravital stain [1% (w/v) eosin B, 5% (w/v) nigrosin dissolved in 3% tri-sodium citrate dehydrate solution] to prepare a thin and uniform smear. After air-drying, the smear was observed under a phase-contrast microscope at 100x as previously described (Mahmood and Ijaz, 2006). Two hundred spermatozoa were counted for unstained heads of spermatozoa (live) and/or stained/partial stained heads of spermatozoa (dead).

Plasma membrane integrity

Hypo-osmotic swelling test (HOS) as previously described was used to evaluate plasma membrane integrity (Gohar et al., 2014). Briefly, a hypo-osmotic solution (190 mOsm/L) was prepared by dissolving 0.735 g of tri-sodium citrate dihydrate and 1.351 g D (-) fructose in 100 mL of de-ionized distilled water. The test was performed by first mixing 500 L of hypo-osmotic solution with 50 L of each frozen-thawed semen sample and incubated for 45 minutes at 37degC. After incubation, the sample was mixed gently and examined under a phase-contrast microscope (40x). Two hundred spermatozoa per slide were counted, and the percentage of spermatozoa exhibiting tail curling (intact membrane) was determined.

Statistical analysis

Statistical analysis was conducted using the Statistical Package for Social Science (SPSS for Windows version 12, SPSS Inc., Chicago, IL, USA). Data was presented as mean S.E. Data was analyzed using one-way analysis of variance (ANOVA). Differences were considered significant at P < 0.05.

RESULTS

Post-thaw sperm functional assays of different level of green tea extract in semen egg yolk extender are expressed in Table II. Data were expressed as percentage of mean with standard error of the respective fertility indicator including sperm motility, viability and plasma membrane integrity of the post-thaw spermatozoa in Achai bull's semen with various concentration of green tea extract. The current findings demonstrated positive response of the green tea extract on post-thawed quality indicators. For motility of post-thawed spermatozoa, significant response of green tea extract in semen extender was attained at 0.75% (P<0.05) inclusion level in comparison with the other treated groups at 0.5%and 0.25% including the control (0.0%). Likewise, the current result indicated that post-thawed viability of sperms were statistically maximum (P<0.05) at 0.75% inclusion level of green tea extract compared to the other treated groups at 0.25% and 0.50% and 0.0% (the control).

Table II.- Appraisal of green tea extracts inclusion in semen extender on post-thawed fertility indicators of Achai bull spermatozoa.

Fertility indicator of post-thaw spermatoza###Concentration of green tea extract in semen extender

###0.00%###0.25%###0.50%###0.75%###1.00%

Molitiy SE###34.310.33a###36.212.5a###43.330.88b###52.640.66c 48.130.04d

Viability SE###49.501.2a###51.661.1a###57.521.4b###64.322.4c###59.211.4d

Plasma membrane integrity SE###38.562.7a###40.322.0a###46.310.57b###53.340.88c 49.331.6d

Furthermore, result obtained in the current study further demonstrated that the post-thawed percentage of spermatozoa with intact plasma membranes was higher (P<0.05) at 0.75% green tea extract inclusion level in comparation with at 0.25% and 0.50% together with the control (0.0%). On the other hand, the significant decrease in the response of green tea extract toward the post thaw fertility indicator were also observed at 1% when compared with 0.75 % inclusion level. Thus current study elucidated the enhancement of the fertility indicators of the post-thaw spermatozoa at 0.75% inclusion level of green tea extract in semen extender.

DISCUSSION

Nowadays usage of frozen semen in ruminants has become a routine technique with widespread relevance in countries depending on artificial insemination to increase animal productivity. Semen proficiency is the gauge of successful insemination in ruminant breeding plans. Measurement of sperm functional indicators i.e. sperm motility, viability and plasma membrane integrity is imperative for the appraisal of potential fertilizing capacity of the cryopreserved processed semen (Huynh et al., 2000; Rodriguez-Martinez, 2003, 2006). Consequently, the high motility of post-thaw spermatozoa in conjunction with its entire structural integrity of diverse components is recognized as a decisive factor in assessment of semen characteristic for the fertility potential in A.I. Enterprise.

On the other hand, production of reactive oxygen species in cryopreservation has been associated with deterioration of sperm motility, plasma membrane integrity, viability of bull semen (Bailey et al., 2000; Bilodeau et al., 2001; Chatterjee and Gagnon, 2001; Ansari et al., 2010, 2011, 2012). Hence, various studies attempted to explore the best method of semen preservation with different extenders for successful artificial insemination in ruminant breeding plan. In recent time, green tea has become a central focus of extensive research investigation in the field of reproductive biology for exploration of its potential effects on mammalian reproduction (Wittayarat et al., 2013; Al-Daraji, 2011; Abshenas et al., 2011). We believe to be pioneers in elucidating the beneficial effect of green tea extract on the post-thaw spermatozoa functional characteristics of Achai bull.

Current findings indicated that addition of green tea extract to the semen extender enhanced the sperm functional factors such motility, viability and plasma membrane integrity which also confirmed recently reported antioxidative protection of the green tea extract in Sahiwal bull spermatozoa (Ali et al., 2014). Additionally, some studies have reported positive effect of green tea extract for semen preservation in various mammalian species. Findings of the current study were in line with the observations as reported in several mammalian species including canine, rabbit and avian species (Wittayarat et al., 2013; Jirina and Anton, 2013; Al-Daraji, 2011). In the canine species, it was demonstrated that addition of green tea extract to tris-egg yolk extender enhanced the motility and viability of chilled canine sperm.

In case of avian study, it was clarified that green tea had dominant antioxidant effect against lipid peroxidation that was naturally occurring during cryopreservation of avian semen, thus improved the avian semen quality. Based on these studies, together with current study, it was evident that low concentration of green tea exerted excellent effect in semen extender to enhance post thaw quality parameter. In our study, at 0.75 % concentration of green tea extract in semen extender, the optimal response toward spermatozoa motility, viability and plasma semen extender was observed. Likewise, in Sahiwal bull, the maximium response of green tea extract was evident at 0.5% concentration in semen extender. Recently enhancement of sperm motility has been elucidated in rabbit with low concentration of green tea extract ([?]0.75 mg/L) (Jirina and Anton, 2013).

Therefore, it is construed from these studies including the current finding that high fraction of motile and viable spermatozoa in processed semen samples might indicated that at low concentration, green tea extract have potentially prevented the damages usually occurring during the process of cryopreservation by ROS production. Thus, ascertained characteristically the potential antioxidative role of green tea extract against the oxidative stress produced by ROS during cryopreservation. On the other hand, various studies carried in field of reproductive biology has demonstrated that higher doses of antioxidants supplementation have been associated with detrimental effect whereas at physiological point the addition of such antioxidants are commonly not dangerous (Bouayed and Bohn, 2010).

In recent study also, the response of green tea extract on fertility indicator was not more significantly improved at higher concentration (1.0%) that has been remarkably supported by the another recent study carried out in rabbit where interestingly decreased sperm motility was observed with increased concentration of green tea extract in semen extender (Jirina and Anton, 2013). Hence elucidation of dose dependent response of green tea extract in the semen extender has been evident from these studies. However, further studies in more high concentration of green tea extract in the semen extender would clarify the dose dependent nature of the green tea in semen extender biology.

As an established fact, the green tea extract contain ample amount of catechin polyphenols having the antioxidant characteristics and are strongly associated with fight against oxidative stress (Chyu et al., 2004). Considering the current findings as well as earlier studies, it seems reasonable that the positive effects of green tea extract on semen quality may be ascribed to catechin polyphenols content on reduction of the oxidative stress that naturally occurred during cryopreservation of semen,despite the difference in experimental animal model. Though the functional mechanism through which green tea polyphenol is involved in the inhibition of oxidative stress during semen preservation is not clear and remains to be elucidated. However, it was demonstrated that polyphenols might bind to components of the sperm membrane and would have prevented the lipid membrane oxidation induced by free radicals (Hyon, 2004).

Additionally, the other potential factors might be attributed to the association of green tea polyphenols with inhibition of egg yolk oxidation in semen extender (Ponglohapan et al., 2004). In most animal studies including ours, egg yolk extenders were widely used for the preservation of semen (Wittayarat et al., 2013; Quintero-Moreno et al., 2004).

CONCLUSION

The current study demonstrated the significant response of the green tea extract on the fertility factors such as viability, motility and plasma membrane integrity in cryopreserved Achai breeding bull semen. The findings generated from the current study could be used in the field of animal reproduction to further elucidate antioxidative role of green tea extract (Camellia sinensis) on the cryopreserved semen in the other breeds of cattle and buffalo in different region of the world under diverse environmental conditions.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the technical staff of Semen Production Unit of the Govet Cattlle and Breeding Farm Harichand, Charrsada, Pakistan.

Statement of conflict of interest

Authors have declared no conflict of interest.

REFERENCES

Abshenas, J., Babaei, H., Zare, M.H., Allahbakhshi, A. and Sharififar, F., 2012. The effects of green tea (Camellia sinensis) extract on mouse semen quality after scrotal heat stress. Vet. Res. For., 2: 242-247.

Al-Daraji, H.J., 2011. Effect of diluent supplementation with different levels of green tea on roosters' semen quality during in vitro storage. Int. J. Pl. Anim. environ. Sci., 3: 51-56.

Ali, H., Riaz, A., Ghafoor, A., Javeed, A., Ashraf, M. and Satter, A., 2014. Antioxidative protection by Strawberry and green tea extracts during cryopreservation of Sahiwal bull semen. Pak. J. Life Soc. Sci., 12: 97-100.

Andrabi, S.M.H., Ansari, M.S., Ullah, N. and Afzal, M., 2008. Effect of non-enzymatic antioxidants in extender on post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa. Pak. Vet. J., 28: 159-162.

Anghel, A., Zamfirescu, S., Dragomir, C., Nadolu, D., Elena, S. and Florica, B., 2010. The effects of antioxidants on the cytological parameters of cryopreserved buck semen. Rom. Biotech. Lett., 15: 26-32.

Ansari, M.S., Rakha B.A., Ullah, N., Andrabi, S.M.H., Iqbal, S., Khalid, M. and Akhter, S., 2010. Effect of exogenous glutathione in extender on the freezability of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa. Anim. Sci. Pap. Rep., 28: 235-244.

Ansari, M.S., Rakha, B.A., Andrabi, S.M.H., Ullah, N., Iqbal, R., Holt, W.V. and Akhter, S., 2012. Glutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa. Reprod. Biol., 12: 271-276. https://doi.org/10.1016/j.repbio.2012.10.001

Ansari, M.S., Rakha, B.A., Ullah, N., Andrabi, S.M.H. and Akhter, S., 2011. Glutathione addition in tris-egg yolk extender improves the quality of cooled buffalo (Bubalus bubalis) bull semen. Pakistan J. Zool., 43: 46-55.

Bailey, J.L., Bilodeau, J.F. and Cormier, N., 2000. Semen cryopreservation in domestic animals: A damaging and capacitating phenomenon: Minireview. J. Androl., 21: 1-7.

Bhosrekar, M.R., Rane, R.S. and Mazokari, R.C., 2001. Fertility of murrah buffalo bulls in Bhilawadi area of Sangli district. In: Proceedings of the XVII Annual Convention and National Seminar on Fertility Management of Farm Animals Under Adverse Agro-climatic Conditions, Jodhpur, India, 6-8 October., pp. 18.

Bilodeau, J.F., Blanchette, S., Gagnon, C. and Sirard, M.A., 2001. Thiols prevent H2O2-mediated loss of sperm motility in cryopreserved bull semen. Theriogenology, 56: 275-286. https://doi.org/10.1016/S0093-691X(01)00562-3

Bilodeau, J.F., Chatterjee, S., Sirard, M.A. and Gagnon, C., 2000. Level of antioxidant defense are decreased in bovine spermatozoa after a cycle of freezing and thawing. Mol. Reprod. Dev., 55: 282-288. https://doi.org/10.1002/(SICI)1098-2795(200003)55:33.0.CO;2-7

Bouayed, J. and Bohn, T., 2010. Exogenous antioxidants-double-edged swords in cellular redox state. Oxid. Med. cell. Long., 4: 228-237.

Bucak, M.N., Atessahin, A. and Yce, A., 2008. Effect of antioxidants and oxidative stress parameters on ram semen after the freeze-thawing process. Small Rumin. Res., 75: 128-134. https://doi.org/10.1016/j.smallrumres.2007.09.002

Bucak, M.N., Baspinar, N., Tuncer, P.B., oyan, K., Sanzakan, S., Akalin, P.P., Bykleblebici, S. and Kkgnay, S., 2011. Effect of curcumin and dithioerythritol on frozen-thawed bovine semen. Andrologia, 44(Suppl. 1): 102-109. https://doi.org/10.1111/j.1439-0272.2010.01146.x

Chen, W.J. and Wan, SQ., 1994. Research progress on polyphenols of tea. Nat. Prod. Res. Develop., 6: 74-80.

Chan, E.W.C., Lim, Y.Y. and Chew, Y.L., 2007. Antioxidative activity of Camellia sinensis and tea from lowland population in Malaysia. Fd. Chem., 102: 1214-1222. https://doi.org/10.1016/j.foodchem.2006.07.009

Chatterjee, S. and Gagnon, C., 2001. Production of reactive oxygen species by spermatozoa undergoing cooling, freezing and thawing. Mol. Reprod. Develop., 59: 451-458. https://doi.org/10.1002/mrd.1052

Chyu, K.Y., Babbidge, S.M., Zhao, X., Dandillaya, R., Rietveld, A.G., Yano, J., Dimayuga, P., Cercek, B. and Shah, P.K., 2004. Differential effects of green-tea derived catechin on developing versus established atherosclerosis in apolipoprotein E-null mice. Circulation, 109: 2448-2453. https://doi.org/10.1161/01.CIR.0000128034.70732.C2

Gohar, A., Khan, H., Yousaf, M.S., Ahmad, J., Ali, Q., Khan, M., Khan, D., Hayat, Y., Ali, F., Ahmad, I., Saleem, M. and Ullah, F., 2014. Assessment of alpha lipoic acid inclusion in semen extender on cryopreservation of Nili-Ravi buffalo bull spermatozoa. Life Sci. J., 11: 45-50.

Gupta, V.K. and Sharma, S.K., 2006. Plants as natural antioxidant. Nat. Prod. Radiance, 5: 326-334.

Huynh, P.N., Hikim, A.P., Wang, C., Stefonovic, K., Lue, Y.H., Leung, A., Atienza, V., Baravarian, S., Reutrakul, V. and Swerdloff, R.S., 2000. Long-term effects of triptolide on spermatogenesis, epididymal sperm function, and fertility in male rats. J. Androl., 21: 689-699.

Hyon, S.H., 2004. A non-frozen living tissue bank for allotransplantation using green tea polyphenols. Yon med. J., 45: 1025-1034.

Ibrahim, A.H.B., Ahmed, R.H. and Ahmed, M.R., 2014. Antioxidant effect of green tea leaves extract on in vitro production of sheep embryos. Pakistan J. Zool., 46: 167-175.

Ijaz, A., Hussain, A., Aleem, M., Yousaf, M.S. and Rehman, H., 2009. Butylated hydroxytoluene inclusion in semen extender improves the post-thawed semen quality of Nili-Ravi buffalo. Theriogenology, 71: 1326-1329. https://doi.org/10.1016/j.theriogenology.2008.12.023

Janice, L., Bilodeau, J. and Cormier, N., 2000 . Semen cryopreservation in domestic animals: A damaging and capacitation phenomenon. J. Androl., 21: 1-7.

Jirina, K. and Anton, K., 2013. Effect of green tea extract on motility parameters of rabbit sperm. J. Microbiol. Biotech. Fd. Sci., 2: 1-13. http://www.jmbfs.org/section_animal_physiology/?issue_id=2422andarticle_id=10

Khan, M.I.R. and Ijaz, A., 2007. Assessing undiluted, diluted and frozen-thawed Nili-Ravi buffalo bull sperm by using standard semen assays. Ital. J. Anim. Sci., 6: 784-787. https://doi.org/10.4081/ijas.2007.s2.784

Khan, M.S., Rehman, Z.U., Khan, M.A. and Ahmad, S., 2008. Genetic resources and diversity in Pakistani cattle. Pak. Vet. J., 28: 95-102.

Mahmood, S.A. and Ijaz, A., 2006. Effect of cold shock on frozen-thawed spermatozoa of buffalo and cow bulls. Proc. 5th Asi. Buff. Cong. Naning, China, 18-22 April., pp. 701-708.

Maxwell, W.M.C., Evans, G., Rhodes, S.L., Hillard, M.A. and Bindon, B.M., 1993. Fertility of superovulated ewes after intrauterine or oviducal insemination with low numbers of fresh or frozen-thawed spermatozoa. Reprod. Fertil. Develop., 5: 57-63. https://doi.org/10.1071/RD9930057

Nagulendran, K., Velavan, S., Mahesh, R. and Begum, V.H., 2007. In vitro antioxidant activity and total polyphenolic content of Cyperus rotundus rhizomes. e-J. Chem., 4: 440-449. https://doi.org/10.1155/2007/903496

Nichi, M., Bols, P.E.J., Zge, R.M., Barnabe, V.H., Goovaerts, I.G.F., Barnabe, R.C. and Cortada, C.N.M., 2006. Seasonal variation in semen quality in Bos indicus and Bos taurus bulls raised under tropical conditions. Theriogenology, 66: 822-828. https://doi.org/10.1016/j.theriogenology.2006.01.056

Ponglohapan, S., Essen-Gustavsson, B. and Linde, F.C., 2004. Influence of glucose and fructose in the extender during long-term storage of chilled canine semen. Theriogenology, 62: 1498-1517. https://doi.org/10.1016/j.theriogenology.2004.02.014

Quintero-Moreno, A., Rigau, T. and Rodriguez-Gil, J.E., 2004. Regression analyses and motile sperm subpopulation structure study as improving tools in boar semen quality analysis. Theriogenology, 61: 673-690. https://doi.org/10.1016/S0093-691X(03)00248-6

Rodriguez-Martinez, H., 2003. Laboratory semen assessment and prediction of fertility: still utopia? Reprod. Domest. Anim., 38: 312-318. https://doi.org/10.1046/j.1439-0531.2003.00436.x

Rodriguez-Martinez, H., 2006. Can we increase the estimative value of semen assessment? Reprod. Domest. Anim., 41: 2-10. https://doi.org/10.1111/j.1439-0531.2006.00764.x

Roy, M., Chakrabarty, S., Sinha, D., Bhattacharya, R.K. and Siddiqi, M., 2003. Anticlastogenic, antigenotoxic and apoptotic activity of epigallocatechin gallate: a green tea polyphenol. Mutat. Res., 523: 33-41. https://doi.org/10.1016/S0027-5107(02)00319-6

Sen, S., Chakraborty, R., Sridhar, C., Reddy, Y.S.R. and De, B., 2010. Free radicals, antioxidants, diseases and phytomedicines: current status and future prospect. Int. J. pharm. Sci. Rev. Res., 3: 91-100.

Sreejith, N., Brar, A.S., Ahuja, C.S., Sangha, S.P. and Chaudhary, K.C., 2006. A comparative study on lipid peroxidation, activities of antioxidant enzymes and viability of cattle and buffalo bull spermatozoa during storage at refrigeration temperature. Anim. Reprod. Sci., 96: 21-29. https://doi.org/10.1016/j.anireprosci.2005.11.002

Tedeschi, E., Menegazzi, M., Yao, Y., Suzuki, H., Forstermann, U. and Kleinert, H., 2004. Green tea inhibits human inducible nitric-oxide synthase expression by down-regulating signal transducer and activator of transcription-1 {alpha} activation. Mol. Pharmacol., 65: 111-120. https://doi.org/10.1124/mol.65.1.111

Wittayarat, M., Ito, A., Kimura, T., Namula, Z., Luu, V.V., Do, L.T.K., Sato, Y., Taniguchi, M. and Otoi, T., 2013. Effects of green tea polyphenol on the quality of canine semen after long-term storage at 5degC. Reprod. Biol., 13: 251-254. https://doi.org/10.1016/j.repbio.2013.07.006
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Author:Khan, Hamayun; Khan, Momen; Qureshi, Muhammad Subhan; Ahmad, Shakoor; Gohar, Ali; Ullah, Hameed; Ull
Publication:Pakistan Journal of Zoology
Article Type:Report
Date:Aug 31, 2017
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