Impact of Cysteine as a Semen Additive on Mitigating the  Decline in Sperm Quality due to Summer Stress in Riverine  Buffaloes (Bubalus bubalis)

J Arunpandian; Neeraj Srivastava; Gyanendra Singh; Brijesh Kumar; Amala Jackson; Pradeep Chandra; Subrato Kumar Ghosh; Sanjay Kumar Singh; Meiraj Haider Khan

doi:10.48165/ijar.2023.44.01.7

Authors

J Arunpandian ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243122
Neeraj Srivastava ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243122
Gyanendra Singh ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243122
Brijesh Kumar ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243122
Amala Jackson ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243122
Pradeep Chandra ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243122
Subrato Kumar Ghosh ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243122
Sanjay Kumar Singh ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243122
Meiraj Haider Khan ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243122

DOI:

https://doi.org/10.48165/ijar.2023.44.01.7

Keywords:

Buffalo, Cysteine, Spermatozoa, Free radicals, ROS, Summer

Abstract

The seasonal variations such as temperature, rain and humid environment greatly influences reproduction, and semen quality particularly in buffalo bulls, either positively or negatively. The heat stress coupled with freezing and thawing processes generates reactive oxygen species (ROS), which further reduce post-thaw spermatozoa quality. Application of cysteine as a semen additive have been reported to alleviate such damaging effect in canine, buck, bull, human, boar, and rooster. Therefore, objective of this study was to investigate ameliorating effect of cysteine on summer stress on buffalo spermatozoa. For the experiment, 24 ejaculates with individual progressive motility >70, volume >1mL and sperm concentration 1000 million/mL from four Murrah buffalo bulls were collected and divided into four groups as Control (Group-I), and Group-II, III, and IV (4, 8, and 12 mmol cysteine, respectively) containing 80 million spermatozoa per mL. Semen was filled (French mini straw), sealed and equilibrated at 40⸰C and frozen into LN2. The group containing L-cysteine at 4 mmol concentration had higher significant (p<0.05) difference in post thaw motility, viability, plasma membrane integrity and acrosome integrity, TAC, DNA integrity and lower significant (p<0.05) difference in spermatozoa abnormality, LPO and intracellular ROS. Moreover, cysteine concentration at 8 mmol has no beneficial effect compared to the Control, whereas Group-IV had greater adverse effect. This may be due to its toxicity at this concentration. It is evident from the study that the cysteine molecules at 4 mmol concentration produced more pronounced improvement in the freezability potential of buffalo bull spermatozoa.

References

Ahirwar, M.K., Kataktalware, M.A., Prasad, K., Pal, R.P., Barman, D., Thul, M. and Rawat, N. (2018). Effect of non-genetic factors on semen quality in bulls: A review. J. Entom. Zoo. Studies, 6: 38-45.

Ahmad, M.Z., Chatagnon, G., Amirat Briand, L., Moussa, M., Tainturier, D., Anton, M. and Fieni, F. (2008). Use of gluta mine and low-density lipoproteins isolated from egg yolk to improve buck semen freezing. Reprod. Domest. Anim., 43:429-436.

Aitken, R. J., Buckingham, D. and Harris, D. (1993). Use of a xanthine oxidase oxidant generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa. J. Reprod. Fertil., 97: 441-450.

Andreea, A. and Stela, Z. (2010). Role of antioxidant additives in the protection of the cryopreserved semen against free radicals. Romanian Biotech. Let., 15: 33-41.

Atessahin, A., Bucak, M.N., Tuncer, P.B. and Kýzýl, M. (2008). Effects of anti-oxidant additives on microscopic and oxida tive parameters of Angora goat semen following the freeze– thawing process. Small Rum. Res., 77: 38-44.

Badr, M.R., Azab, A.M.S. and Rawash, Z.M. (2014). Effect of trehalose, cysteine and hypotaurine on buffalo bull sperm freezability, ultrastructure changes and fertilizing poten tials. Assiut. Vet. Med. J., 60: 38-45.

Bathgate, R. (2011). Antioxidant mechanisms and their benefit on post thaw boar sperm quality. Reprod. Domest. Anim., 46: 23-25.

Beheshti, R., Asadi, A., Eshratkhah, B., GhaleKandi, J.G. and Ghorban, A. (2011). The effect of cysteine on post-thawed

buffalo bull (Bubalus bubalis) sperm parameters. Adv. Environ. Biol., 5: 1260-1263.

Benzie, I.F. and Strain, J.J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of ‘antioxidant power’: the FRAP assay. Analytical biochem., 239: 70-76.

Bilodeau, J.F., Chatterjee, S., Sirard, M.A. and Gagnon, C. (2000). Levels of antioxidant defences are decreased in bovine sper matozoa after a cycle of freezing and thawing. Mol. Reprod. Develop, 55: 282-288.

Bisla, A., Rautela, R., Yadav, V., Singh, P., A Ngou, A.A., Kumar, A., Katiyar, R., Ghosh, S.K., Kumar, A., Bag, S., Kumar, B. and Srivastava, N. (2020). Nano-purification of raw semen minimizes oxidative stress with improvement in post-thaw quality of buffalo spermatozoa. Andrologia, e13709.

Bucak, M.N. and Uysal, O. (2008). The role of antioxidants in freezing of Saanen goat semen. Indian Vet. J., 85: 148-150. Bucak, M.N., Ateþþahin, A., Varýþlý, Ö., Yüce, A., Tekin, N. and Akçay, A. (2007). The influence of trehalose, taurine, cys teamine and hyaluronan on ram semen: microscopic and oxidative stress parameters after freeze–thawing process. Theriogenology, 67: 1060-1067.

Carter, W.O., Narayanan, P.K. and Robinson, J.P. (1994). Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J. Leukoc. Biol., 55: 253–258.

Chatterjee, S., de Lamirande, E. and Gagnon, C. (2001). Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione. Mol. Reprod. Develop., 60: 498-506.

Ciftci, H., Verit, A., Savas, M., Yeni, E. and Erel, O. (2009). Effects of N-acetylcysteine on semen parameters and oxidative/ antioxidant status. Urology, 74: 73-76.

Çoyan, K., Baþpýnar, N., Bucak, M.N. and Akalýn, P.P. (2011). Effects of cysteine and ergothioneine on post thawed Merino ram sperm and biochemical parameters. Cryobiology, 63: 1-6.

Din, O., Mustapha, A.R., Amin, B.Y., Rahul, K., Abhishek, K., Ghosh, S.K., Prasad, J.K., Ajay, K., Praveen, S., Bag, S. and Megha, P. (2018). Comparative efficacy of enrichment of spermatozoa using swim up vis-à-vis nano-technique in buffalo bull. Rum. Sci. 7: 73-76.

El-Sheshtawy, R.I., El-Sisy, G.A. and El-Nattat, W.S. (2008). Use of selected amino acids to improve buffalo bull semen cryo preservation. Global Vet., 2: 146-150.

Eskiocak, S., Gozen, A.S., Yapar, S.B., Tavas, F., Kilic, A.S. and Eskiocak, M. (2005). Glutathione and free sulphydryl con tent of seminal plasma in healthy medical students during and after exam stress. Hum. Reprod., 20: 2595-2600.

Esteves, S.C., Sharma, R.K., Thomas Jr, A.J. and Agarwal, A. (2007). Evaluation of acrosomal status and sperm viability

in fresh and cryopreserved specimens by the use of fluores cent peanut agglutinin lectin in conjunction with hypo-os motic swelling test. Int. Braz. J. urol., 33: 364-376.

Funahashi, H. and Sano, T. (2005). Select antioxidants improve the function of extended boar semen stored at 10ºC. Theriogenology, 63: 1605-1616.

Garner, D.L., and Hafez, E.S.E. (1993). Spermatozoa and sem inal plasma. In: Hafez, E.S.E.(Ed.), Reproduction in Farm Animals. Lea & Febier, Philadelphia. 167–182.

Gaurav, K., Goel, R., Shukla, M. and Pandey, M. (2012). Glutamine: a novel approach to chemotherapy-induced toxicity. Indian J. Med. Paediatr. Oncol., 33: 13.

Jerome A. and Srivastava N. (2012) Prostaglandins vis-a-vis bovine embryonic mortality: a review. Asian Pac. J. Reprod., 1(3): 238-246.

Jeyendran, R.S., Van der Ven, H.H., Perez-Pelaez, M., Crabo, B.G. and Zaneveld, L.J.D. (1984). Development of an assay to assess the functional integrity of the human sperm mem brane and its relationship to other semen characteristics. Reproduction, 70: 219-228

Kadirvel, G., Kumar, S. and Kumaresan, A. (2009). Lipid per oxidation, mitochondrial membrane potential and DNA integrity of spermatozoa in relation to intracellular reactive oxygen species in liquid and frozen-thawed buffalo semen. Anim. Reprod. Sci., 114: 125-134.

Kaeoket, K., Chanapiwat, P., Tummaruk, P. and Techakumphu, M. (2010). Supplemental effect of varying L-cysteine con centrations on the quality of cryopreserved boar semen. Asian J Androl., 12: 760.

Kasahara, E., Sato, E.F., Miyoshi, M., Konaka, R., Hiramoto, K., Sasaki, J., Tokuda, M., Nakano, Y. and Inoue, M. (2002). Role of oxidative stress in germ cell apoptosis induced by di(2-ethylhexyl) phthalate. Biochem. J., 365: 849–856

Koppers, A.J., Garg, M.L. and Aitken, R.J. (2010). Stimulation of mitochondrial reactive oxygen species production by unes terified, unsaturated fatty acids in defective human sperma tozoa. Free Rad. Biol. Med., 48: 112-119.

Krishnan, G., Bagath, M., Pragna, P., Vidya, M.K., Aleena, J., Archana, P.R., Sejian, V. and Bhatta, R. (2017). Mitigation of the heat stress impact in livestock reproduction. Theriogenology, 8: 8-9.

Kumar, A., Ghosh, S.K., Rahul, K., Rupali, R., Bisla, A., Ngou, A.A., Pande, M., Srivastava, N. and Bhure, S. (2021). Effect of Mito-Tempo Incorporated Semen Extender on Physico Morphological Attributes and Functional Membrane Integrity of Frozen Thawed Buffalo Spermatozoa. Cryoletters, 42(2): 111-119

Mata-Campuzano, M., Álvarez-Rodríguez, M., Del Olmo, E., Fernández-Santos, M.R., Garde, J.J. and Martínez-Pastor,

F. (2012). Quality, oxidative markers and DNA damage (DNA) fragmentation of red deer thawed spermatozoa after incubation at 37 C in presence of several antioxidants. Theriogenology, 78: 1005-1019.

Medeiros, C.M.O., Forell, F., Oliveira, A.T.D. and Rodrigues, JL. (2002). Current status of sperm cryopreservation: why isn’t it better? Theriogenology, 57: 327-344.

Mehmood, A., Anwar, M. and Naqvi, S.S. (2009). Motility, acro some integrity, membrane integrity and oocyte cleavage rate of sperm separated by swim-up or Percoll gradient method from frozen–thawed buffalo semen. Anim. Reprod. Sci., 111: 141-148.

Memon, A.A., Wahid, H., Rosnina, Y., Gohb, Y.M., Ebrahimi, M.B., Nadiac, F.M. and Audrey, G. (2011). Effect of hypotaurine and cysteine on sperm cytological parameters of cooled and post-thaw Boer goat semen. Elixir Int. J., 38: 4100-4104.

Michael, A.J., Alexopoulos, C., Pontiki, E.A., HadjipavlouLitina, D.J., Saratsis, P., Ververidis, H.N. and Boscos, C.M., (2010). Effect of N acetyl L cysteine supplementation in semen extenders on semen quality and reactive oxygen species of chilled canine spermatozoa. Reprod. Domest. Anim., 45: 201-207.

Mustapha, A.R. (2017). Studies on the partial deoxygenation of the extender on freezability of crossbred bull spermatozoa. PhD Thesis submitted to Deemed University ICAR-Indian Veterinary Research Institute, Izatnagar, India.

Nair, S.J., Brar, A.S., Ahuja, C.S., Sangha, S.P.S. 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.

Ngou, A. A., Ghosh, S. K., Prasad, J. K., Katiyar, R., Kumar, A.,Rautela, R., Bisla, A., Srivastava, N., Kumar, A. (2020) Exploring the role of E coli derived enzyme, Oxyrase, as an oxygen scavanger to improve the cryotolerance of sperma

tozoa of Sahiwal bull. Cryobiology, 97:85-92.

Sadeghi, M.R., Hodjat, M., Lakpour, N., Arefi, S., Amirjannati, N., Modarresi, T., Jadda, and Akhondi, M.M. (2009). Effects of sperm chromatin integrity on fertilization rate and embryo quality following intracytoplasmic sperm injection. Avicenna J. Med. Biotech., 1: 173.

Sanocka, D. and Kurpisz, M. (2004). Reactive oxygen species and sperm cells. Reprod. Biol. Endo., 2: 1-7.

Sarýözkan, S., Bucak, M.N., Tuncer, P.B., Ulutaş, P.A. and Bilgen, A. (2009). The influence of cysteine and taurine on micro scopic–oxidative stress parameters and fertilizing ability of bull semen following cryopreservation. Cryobiology, 58: 134-138.

Srivastava N., Srivastava S.K., Ghosh S.K., Jerome A., Das G.K., and Mehrotra S. (2013). Sequestration of PDC-109 protein by specific antibodies and egg yolk cryoprotects bull sper matozoa. Reprod. Dom. Anim., 48(5): 724-731.

Suleiman, S.A., Ali, M.E., Zaki, Z.M.S., El Malik, E.M.A. and Nasr, M.A. 1996. Lipid peroxidation and human sperm motility: protective role of vitamin E. J. Androl., 17: 530- 537.

Swanson, W. and Bearden, H. J. (1951). An Eosin-Nigrosin stain for differentiating live and dead bovine spermatozoa. J. Anim. Sci., 10: 981-987.

Szcesniak-Fabianczyk, B., Bochenek, M., Smorag, Z. and Ryszka, F. (2003). Effect of antioxidants added to boar semen extender on the semen survival time and sperm chromatin structure. Reprod. Biol., 3: 81-87.

Thuwanut, P., Chatdarong, K., Bergqvist, A.S., Söderquist, L., Thiangtum, K., Tongthainan, D. and Axnér, E. (2011). The effects of antioxidants on semen traits and in vitro fertilizing ability of sperm from the flat-headed cat (Prionailurusplaniceps). Theriogenology, 76: 115-125.

Topraggaleh, T.R., Shahverdi, A., Rastegarnia, A., Ebrahimi, B., Shafiepour, V., Sharbatoghli, M., Esmaeili, V. and Janzamin, E. (2014). Effect of cysteine and glutamine added to extender on post thaw sperm functional parameters of buf

falo bull. Andrologia, 46: 777-783.

Trinchero, G.D., Affranchino, M.A., Schang, L.M. and Beconi, M.T. (1990). Antioxidant effect of bovine spermatozoa on lipid peroxidation. Com. Biol., 8: 339-350.

Tripodi, L., Tripodi, A., Mammi, C., Pulle, C. and Cremonesi, F. (2003). Pharmacological action and therapeutic effects of glutathione on hypokinetic spermatozoa for enzymatic-de pendent pathologies and correlated genetic aspects. Clinical Expt. Obs. Gyn., 30: 130-136.

Tuncer, P.B., Bucak, M.N., Büyükleblebici, S., Sarýözkan, S., Yeni, D., Eken, A., Akalýn, P.P., Kinet, H., Avdatek, F., Fidan, A.F. and Gündoðan, M. (2010). The effect of cysteine and glu tathione on sperm and oxidative stress parameters of post thawed bull semen. Cryobiology, 61: 303-307.

Uysal, O. and Bucak, M.N. (2007). Effects of oxidized gluta thione, bovine serum albumin, cysteine and lycopene on the quality of frozen-thawed ram semen. Acta Veterinaria Brno., 76: 383-390.

Watson, P.F. (1975). The interaction of egg yolk and ram sper matozoa studied with a fluorescent probe. Reproduction, 42: 105-111

Woelders, H., Matthijs, A. and Engel, B. (1997). Effects of treha lose and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing. Cryobiology, 35: 93-105.