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Effect of Glutathione and L-Cysteine Supplementation on the Quality of Frozen Beetal Buck Spermatozoa


Effect of Glutathione and L-Cysteine Supplementation on the Quality of Frozen

Beetal Buck Spermatozoa

Muhammad Hammad Fayyaz*, S. Murtaza Hassan Andrabi and Muhammad Shafiq Haider

Animal Reproduction and Genetics Program, Animal Sciences Institute, National Agricultural Research Centre, PARC, Islamabad 44000


This study was designed to assess the cryosurvival of beetal buck spermatozoa with the supplementation of Glutathione (GLU) and L-cysteine (CYS) in the extender. In the first experiment, semen samples from each of the five mature bucks were pooled and diluted in Tris-citric acid (TCA) extender containing Glutathione (0.5mM, 1mM), L-cysteine (5mM CYS, 10mM CYS), and control. In experiment 2, semen samples (n=5) were diluted in TCA extender containing differential doses of combined antioxidants (0.5 mM GLU+ 5mM CYS, 0.5mM + 10mM CYS, 1mM GLU + 5mM CYS, 1mM GLU + 10mM CYS and Control). At post-thawing, progressive motility, plasma membrane integrity (PMI), and livability of buck spermatozoa were higher (P< 0.05) with 1mM GLU and 10mM CYS as compared to control. In the second experiment, post-thaw progressive motility, PMI, and livability were higher (P< 0.05) with 1mM GLU + 10mM CYS and 1mM GLU + 5mM CYS compared to other doses of GLU+CYS and control. We concluded that optimized supplementation of Glutathione and L-cysteine in TCA extender ameliorated frozen-thawed quality of beetal buck spermatozoa.

Article Information

Received 22 April 2019

Revised 30 May 2020

Accepted 22 April 2021

Available online 13 December 2021

(early access)

Published 19 July 2022

Authors’ Contributions

MHF preformed the research, analyzed the data and prepared the manuscript. SMHA edited the manuscript and supervised the overall research. MSH designed the research and assisted in analyzing the data.

Key words

Buck semen, Cryopreservation, Cryo-injuries, Oxidative stress.


* Corresponding author:

0030-9923/2022/0005-2473 $ 9.00/0

Copyright 2022 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (

Artificial insemination (AI) is one of the major assisted reproductive techniques used to impregnate the female without direct involvement of the male. It offers a low cost and easy way to disseminate desired valuable genetic factors (Leboeuf et al., 2000). Breeding programs of goat majorly depend upon AI of frozen-thawed spermatozoa (Andrabi et al., 2016). Success in fertilization is the main goal of artificially incorporated cryopreserved spermatozoa in the female reproductive tract (Purdy, 2006). Although, cryopreservation process is an efficient technique to store spermatozoa for long periods but its initiated injuries are also harmful (Andrabi, 2009). Oxidative stress is one of the major injuries faced by spermatozoa during the freeze-thawing process (Fayyaz et al., 2017). Reactive oxygen species (ROS) are highly reactive molecules that are normally produced during cellular respiration (Aitken et al., 2016). The rate of ROS generation is high during the freeze-thaw process (Shah et al., 2017) which compromises fertilization potential spermatozoa by affecting its various qualities i.e. motility, acrosomal integrity, plasma membrane integrity, mitochondrial membrane potential, and DNA integrity (Baumber et al., 2000). Buck sperm also constitutes a high amount of polyunsaturated fatty acids (PUFA) (Dolatpanah et al., 2008) which are more susceptible to lipid peroxidation (Shah et al., 2017) leading to ROS generation (Alvarez and Storey, 1995). Therefore, a powerful exogenous antioxidant system should be required to avoid cryoinjuries and lipid peroxidation (Irvine, 1996). In this regard, the addition of antioxidants in cryodiluent is considered helpful to improve the frozen-thawed quality of spermatozoa (Rao et al., 2013).

Glutathione is the biological antioxidant that normally exists in semen. Moreover, exogenous supplementation of glutathione has also been reported to provide intracellular defense against oxidative stress (Salmani et al., 2013). Interestingly, cysteine is the main component of glutathione, which has also been reported to prevent lipid peroxidation in spermatozoa (Topraggaleh et al., 2014). Cysteine also consists of thiol groups, which individually act as a non-enzymatic antioxidant and easily penetrates the sperm (Çoyan et al., 2011).

There is no comprehensive data available on the combined useful effects of glutathione and L-cysteine on post-thaw sperm quality of buck. Therefore, the present


Table I.- Motility, plasma membrane integrity (PMI) and liveability of frozen thawed beetal buck spermatozoa supplemented with different concentrations of glutathione (GLU) and L-cysteine (CYS). Values are the mean±S.E.M. of five treatment groups. Means with different superscripts are significantly different (P<0.05).




Experiment 1





0.5mM GLU












10mM CYS




Experiment 2


40.50 ± 1.48A


55.45± 1.40A

0.5mM GLU + 5mM CYS

55.00 ± 1.48B


62.12± 1.55B

0.5mM GLU + 10mM CYS

55.50 ± 1.48B

58.61± 1.72B

64.50± 1.53C

1mM GLU + 5mM CYS

58.50 ± 1.40BC

63.90± 1.70C

67.50± 1.60D

1mM GLU + 10mM CYS

60.00 ± 1.40BC

65.00± 1.70C

68.30± 1.60D


study was designed to determine the optimal combination of glutathione and L-cysteine in extender, to improve the post-thaw quality of beetal buck spermatozoa.

Materials and methods

Semen from Five healthy beetal bucks each buck was collected twice a week (for 6 weeks) using an artificial vagina at 42°C. Collected samples with more than 60% motility and 1.5 × 109 sperm concentration per mL were pooled and subdivided into five aliquots. Each aliquot was diluted with tris-citric extender [Tris 3.07 g/100mL, citric acid 1.64 g/100mL, fructose 1.26 g/100mL, 1000 IU/mL streptomycin sulfate, 5 mL of glycerol (v/v); 15mL of egg yolk (v/v)] containing different concentrations of glutathione (0.5mM GLU, 1mM GLU) and L-cysteine (5mM CYS, 10mM CYS) alone and in combination viz. 0.5mM GLU+5mM CYS, 0.5mM GLU+10mM CYS, 1mM GLU+5mM CYS, 1mM GLU+10mM CYS.

For freezing, extended sperm suspensions were cooled from 37°C gradually to 4°C in 90 min and equilibrated at 4°C for 2 h in the cold cabinet and packaged in 0.54 mL French straws (50 million sperm/straw). Packaged straws were placed horizontally on racks in cold cabinet then held at liquid nitrogen vapors 4 cm higher than the level of liquid nitrogen for 10 min followed by immersing in liquid nitrogen at -196°C for storage.

For post-thaw sperm evaluation assays, motility, plasma membrane integrity (PMI), and livability were considered as criteria. Motility analysis was performed according to Gillan et al. (2008). PMI of spermatozoa was analyzed by hypo-osmotic swelling (HOS) assay as described by Correa and Zavos (1994). Post-thaw livability was assessed by eosin–nigrosine staining. A total of 200 spermatozoa were counted in different fields of the phase-contrast microscope. Spermatozoa gaining partial or complete stain were recorded non-viable, and spermatozoa with unstained heads were considered as viable.

All data were expressed as mean with the standard error of the mean (SEM) among different treatment groups. One-way analysis of variance (ANOVA) was performed for data analysis and the Tukey test as post-hoc were performed on SPSS (version 20.0). The probability level of P < 0.05 was considered significant.


Table I shows the effects of differential concentrations of GLU and CYS (combined or separate) on post-thaw progressive motility, PMI, and livability. All sperm parameters were higher (P<0.05) in 1mM GLU, 5mM CYS and 10mM CYS groups than control. The highest sperm parameters were observed in the 10mM CYS group. The highest motility was observed in 1mM GLU + 10mM CYS and 1mM GLU + 5mM CYS groups. A similar trend was seen in the case of livability and PMI, in which high concentration groups exhibited significantly higher results as compared to control and other groups.


The plethora of ROS during the freeze-thaw process affects sperm quality parameters, compromising its survival, declining its internal antioxidant defense system, and fertilization potential. Frozen sperm is more prone to lipid peroxidation as compared to fresh sperm.

This is the very first study to evaluate the combined effect of GLU and CYS on the post-thaw quality of buck spermatozoa. In this study, supplementation of two antioxidants in different combinations significantly improved the sperm quality in terms of motility, PMI, and livability. Our results are in agreement with the study conducted in bovine semen (Sattar et al., 2016). However, these findings contradicted the studies conducted in ram (Zhandi and Sharafi, 2015) and buck (Salmani et al., 2013) in which inclusion ratios of GLU were high (5mM and 10mM). This may be due to mitochondrial DNA degradation is associated with high GLU concentration (Abdi-Benemar et al., 2015). Also, it has been studied that the high levels of antioxidants negatively affect plasma membrane functionality (Salmani et al., 2013). Therefore, improving the effects of GLU on PM and other parameters may be due to its low inclusions which affected the functionality of the plasma membrane. Results of our study agreed with the findings of Zhandi et al. (2015) who reported that low concentrations of GLU in freezing extender improved the frozen-thawed sperm qualities of buck.

Cysteine is a well-known ROS scavenger. In this study addition of CYS in extender also positively affected motility, plasma membrane integrity, and livability agreeing with the studies performed in bull (Topraggaleh et al., 2014) ram (Çoyan et al., 2011), and buck (Memon et al., 2011).

However, this is the first study on the combined effect of two related antioxidants tested on frozen-thawed buck sperm quality. Our findings were consistent with the same study in ram. This discrepancy may be due to the difference in antioxidant concentrations used and semen characteristics of the two species. Our results indicated that the optimum combination of GLU and CYS in freezing extender can protect buck sperm from cryoinjuries.

In conclusion, this study exhibited that glutathione and L-cysteine at the concentration of 1mM and 10mM may be the optimum combination in extender to ameliorate cryopreservation of Beetal buck spermatozoa.

Statement of conflict of interest

The authors have declared no conflict of interests.


Abdi-Benemar, H., Jafaroghli, M., Khalili, B., Zamiri, M.J., Ezazi, H. and Shadparvar, A.A., 2015. Small Rumin. Res., 130: 166-170.

Aitken, R.J., Gibb, Z., Baker, M.A., Drevet, J. and Gharagozloo, P., 2016. Reprod. Fertil. Dev., 28: 1-10.

Alvarez, J.G. and Storey, B.T., 1995. Mol. Reprod. Dev., 42: 334-346.

Andrabi, S.H., Mehmood, A., Anwar, M. and Khan, M.F., 2016. Small Rumin. Res., 143: 61-66.

Andrabi, S., 2009. Reprod. Domestic Anim., 44: 552-569.

Baumber, J., Ball, B.A., Gravance, C.G., Medina, V. and Davies-Morel, M.C., 2000. J. Androl., 21: 895-902.

Correa, J.R. and Zavos, P.M., 1994. Theriogenology, 42: 351-360.

Çoyan, K., Başpınar, N., Bucak, M.N. and Akalın, P.P., 2011. Cryobiology, 63: 1-6.

Dolatpanah, M.B., Towhidi, A., Farshad, A., Rashidi, A. and Rezayazdi, A., 2008. J. Anim. Sci., 21: 29.

Fayyaz, M.H., Ahmad, M. and Ahmad, N., 2017. Andrologia, 49: e12652.

Gillan, L., Kroetsch, T., Maxwell, W.C. and Evans, G., 2008. Anim. Reprod. Sci., 103: 201-214.

Irvine, D.S., 1996. Rev. Reprod., 1: 6-12.

Leboeuf, B., Restall, B. and Salamon, S., 2000. Anim. Reprod. Sci., 62: 113-141.

Memon, A.A., Wahid, H., Rosnina, Y., Gohb, Y.M., Ebrahimi, M., Nadiac, F.M. and Audrey, G., 2011. Elixir Int. J., 38: 4100-4104.

Purdy, P.H., 2006. Small Rumin. Res., 63: 215-225.

Rao, T.K.S., Kumar, N., Patel, N.B., Chauhan, I. and Chaurasia, S., 2013. Vet. World, 6: 579.

Salmani, H., Nabi, M.M., Vaseghi-Dodaran, H., Rahman, M.B., Mohammadi-Sangcheshmeh, A., Shakeri, M. and Zhandi, M., 2013. Small Rumin. Res., 112: 123-127.

Sattar, A., Younas, F., Andrabi, S.M.H., Ahmad, N., Mehmood, M.U. and Durrani, A.Z., 2016. Anim. Reprod. Sci., 169: 99-135.

Shah, S., Andrabi, S. and Qureshi, I., 2017. Andrologia, 49: e12713.

Topraggaleh, T.R., Shahverdi, A., Rastegarnia, A., Ebrahimi, B., Shafiepour, V., Sharbatoghli, M. and Janzamin, E., 2014. Andrologia, 46: 777-783.

Zhandi, M., Shakeri, M., Towhidi, A., Sharafi, M. and Emamverdi, M., 2015. Protective role of glutathione in buck semen cryopreservation. Iran. J. Vet. Res., 16: 298-300.

Zhandi, M. and Sharafi, M., 2015. Cell Tissue Bank, 16: 443-448.

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