CURCUMIN AND L CYSTEINE SUPPLEMENTATION IN SEMEN EXTENDER MITIGATE CRYOPRESERVATION-INDUCED CRYODAMAGE IN BARBARI BUCK SEMEN SEMEN

Dr. Rajesh Kumar; Jayanth MN; Bhoopendra Singh; Sushant Srivastava; Jaswant   Singh; 6.	Manoj Kumar  Verma

doi:10.56093/ijans.v96i3.176097

Authors

Dr. Rajesh Kumar 9554188943
Jayanth MN
Bhoopendra Singh Professor, Department of Veterinary Gynaecology & obstetrics College of Veterinary Science & Animal Husbandry Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya, UP-224229, India, Postal Code: 224229
Sushant Srivastava Professor & Head, Department of Veterinary Gynaecology & obstetrics College of Veterinary Science & Animal Husbandry Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya, UP-224229, India, Postal Code: 224229
Jaswant Singh Professor and Head, Department of Animal Genetics and Breeding, College of Veterinary Science & Animal Husbandry Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya, UP-224229, India, Postal Code: 224229
6. Manoj Kumar Verma Associate Professor, Department of Animal Genetics and Breeding, College of Veterinary Science & Animal Husbandry Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya, UP-224229, India, Postal Code: 224229

https://doi.org/10.56093/ijans.v96i3.176097

Keywords:

Barbari Buck, Cryodamage, Cryopreservation, Curcumin, L Cysteine, Semen

Abstract

Artificial insemination with frozen semen has made a significant contribution to the widespread propagation of sperm, limiting the spread of sexually transmitted diseases. The study aimed to evaluate effect of curcumin (1.0 μg/mL and 2.0 μg/mL ) and L cysteine (2 mM/L and 5 mM/L) supplementation in semen extender mitigate cryopreservation-induced cryodamage in barbari buck semen semen. The present experiment was carried out on four Barbari bucks, reared at Deep Frozen Semen Lab, College of Veterinary Sciences & Animal Husbandry, ANDUAT, Kumarganj, Ayodhya, Uttar Pradesh. Twenty-four ejaculates (six from each buck) were collected, processed, frozen, and evaluated for various seminal markers. In conlusion, the post thaw seminal attributes were significantly higher except sperm abnormality in all treatments groups than those of control. Furthermore, enzyme leakage were significantly lower in treatment group than those of control one. In conclusion, the supplementation of 2µg/ml Curcumin seems to be beneficial in mitigating cryopreservation-induced cryodamage.

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Author Biographies

Bhoopendra Singh, Professor, Department of Veterinary Gynaecology & obstetrics College of Veterinary Science & Animal Husbandry Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya, UP-224229, India, Postal Code: 224229

Department of Veterinary Gynaecology & obstetrics

Professor
Sushant Srivastava, Professor & Head, Department of Veterinary Gynaecology & obstetrics College of Veterinary Science & Animal Husbandry Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya, UP-224229, India, Postal Code: 224229

Professor & Head

Department of Veterinary Gynaecology & obstetrics
Jaswant Singh, Professor and Head, Department of Animal Genetics and Breeding, College of Veterinary Science & Animal Husbandry Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya, UP-224229, India, Postal Code: 224229

Department of Animal Genetics and Breeding

Professor and Head
6. Manoj Kumar Verma, Associate Professor, Department of Animal Genetics and Breeding, College of Veterinary Science & Animal Husbandry Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya, UP-224229, India, Postal Code: 224229

Associate Professor

Department of Animal Genetics and Breeding

References

Abah KO, Fontbonne A, Partyka A, Nizanski W. 2023. Effect of male age on semen quality in domestic animals: potential for advanced functional and translational research?. Veterinary Research Communications 47(3):1125-37.

Abdelnour SA, Hassan MA, El‐Ratel IT, Essawi WM, El‐Raghi AA, Lu Y, Sheiha AM. 2022. Effect of addition of l‐carnitine to cryopreservation extender on rabbit post‐thaw semen parameters, antioxidant capacity, mitochondrial function, apoptosis and ultrastructure changes. Reproduction in domestic animals 57(8):902-11.

Abdelnour SA, Hassan MA, Mohammed AK, Alhimaidi AR, Al-Gabri N, Al-Khaldi KO, Swelum AA. 2020. The effect of adding different levels of curcumin and its nanoparticles to extender on post-thaw quality of cryopreserved rabbit sperm. Animals 10(9):1508.

Adel O, El‐Sherbiny HR, M. Shahat A, Ismail ST.2024. N‐Acetylcysteine Supplementation Improves Testicular Haemodynamics, Testosterone Levels, Seminal Antioxidant Capacity and Semen Quality in Heat‐Stressed Goat Bucks. Reproduction in Domestic Animals 59(8):e14709.

Ahmed-Farid OA, Nasr M, Ahmed RF, Bakeer RM. 2017. Beneficial effects of curcumin nano-emulsion on spermatogenesis and reproductive performance in male rats under protein deficient diet model: enhancement of sperm motility, conservancy of testicular tissue integrity, cell energy and seminal plasma amino acids content. Journal of Biomedical Science 24(1):66.

Alam K, Srivastava S, Jaiswal A, Kumar R, Verma AK. 2023a. Mitigation of cryopreservation-induced changes in Murrah buffalo (Bubalus bubalis) spermatozoa using L-cysteine hydrochloride. The Indian Journal of Veterinary Science and Biotechnology 19(6):104-8.

Alam K, Srivastava S, Sakhahari DS, Kumar R, Verma AK, Verma SK. 2023b. L-Cysteine Ameliorates Ultra-low Temperature Preservation Induced Cryodamage of Murrah Buffalo Spermatozoa. The Indian Journal of Veterinary Sciences and Biotechnology 19(6): 127-129.

Alam K, Srivastava S, Singh B, Kumar R, Kumar R, Sakhahari DS. 2024a. Cryopreservation of bovine semen using extract of Cinnamomum zeylanicum. Cryoletters 45(3):168-76.

Alam K, Srivastava S, Verma SK, Verma AK, Saurabh, Kumar R, Kumar R, Jaiswal A, Alam A, Rana T. 2024b. The Effect of Different Concentrations of Melatonin on the Quality of Frozen and Thawed Semen of Jamnapari Goat. Reproduction in Domestic Animals 59(11):e14743.

Alam K, Srivastava S, Verma SK, Verma AK, Singh B, Kumar R, Jaiswal A, Verma RK, Alam A, Singh J, Singh RR. 2025. Methionine supplementation improves freezability of Jamnapari Buck semen. Journal of Environmental Biology 46(3):329-35.

An Z, Xun W, Zhou H, Hou G, Shi L. 2025. Effectiveness of curcumin on oxidative stress in goat semen: explorations regarding semen quality, sperm apoptosis, ultrastructure, and markers of oxidative stress. Antioxidants 14(10):1242.

Anand M, Yadav S, Dhariya R, Vaswani S, Chaudharya D. 2023. Effect of time interval between the semen collection and its dilution on seminal attributes during semen cryopreservation in goats. Asian Journal of Research in Animal and Veterinary Sciences 11(2):114-8.

Andrabi SMH. 2009. Factors affecting the quality of cryopreserved buffalo (Bubalus bubalis) bull spermatozoa. Reproduction in domestic animals 44(3): 552-569.

Anghel A, Zamfirescu S, Coprean D, Sogorescu E. 2009. The effects of cystein, bovine serum albumin and vitamin e on the calitative parameters of frozen-thawed ram semen. Annals of the Romanian Society for Cell Biology 14(2): 97-103.

Anghel A, Zamfirescu S, Dragomir C, Nadolu D, Elena S, Florica B. 2010. The effects of antioxidants on the cytological parameters of cryopreserved buck semen. Romanian biotechnological letters 15(3):26-32.

Ansari MS, Rakha BA, Akhter S. 2011. Effect of L-cysteine in extender on post-thaw quality of Sahiwal bull semen. Animal Science Papers & Reports. 1;29(3).

Arivazhagan M, Kesavan M, Rai B, Balamurugan P, Arulkumar S. 2020. Effect of area specific mineral mixture and antioxidants supplementation on semen evaluation of Barbari bucks. Journal of . Entomolology and Zoology Studies 8(4):720-3.

Arunpandian J, Srivastava N, Singh G, Kumar B, Jackson A, Chandra P, Ghosh SK, Singh SK, Khan MH. 2023. Impact of Cysteine as a Semen Additive on Mitigating the Decline in Sperm Quality due to Summer Stress in Riverine Buffaloes (Bubalus bubalis). Indian Journal of Animal Reproduction 2023 Jun 1(1).44(1): 32-40.

Ashutosh K, Srivastava S, Kumar R, Kumar P. 2025. Effect of bradykinin on freezability of abattoir derived murrah bull epididymal spermatozoa. Buffalo Bulletin 44(4):399-408.

Barbas JP, Baptista MC, Carolino N, Simões J, Margatho G, Pimenta J, Claudino F, Ferreira FC, Grilo F, Pereira RM. 2024. Effect of breed and season in buck semen cryopreservation: The Portuguese animal germplasm bank. Veterinary Sciences 11(7):326.

Behboodi HR, Samadi F, Riasi A, Najafi M, Ansari M, Ebadi M. 2024. A comparative study between curcumin and curcumin nanoparticles on reproductive performance and antioxidant system of aged roosters. Poultry Science 103(10):104030.

Bilodeau JF, Blanchette S, Gagnon C, Sirard MA. 2001. Thiols prevent H2O2-mediated loss of sperm motility in cryopreserved bull semen. Theriogenology 56(2):275-86.

Brito LF, Silva AE, Barbosa RT, Unanian MM, Kastelic JP. 2003. Effects of scrotal insulation on sperm production, semen quality, and testicular echotexture in Bos indicus and Bos indicus× Bos taurus bulls. Animal reproduction science 79(1-2):1-5.

Bucak MN and Tekin N. 2007. Protective effect of taurine, glutathione and trehalose on the liquid storage of ram semen. Small Ruminant Research. 73(1-3):103-8.

Bucak MN, Ateşşahin A, Varışlı Ö, Yüce A, Tekin N, Akçay A. 2007. The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen: microscopic and oxidative stress parameters after freeze–thawing process. Theriogenology 67(5):1060-7.

Bucak MN, Ateşşahin A, Yüce A. 2008. Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze–thawing process. Small ruminant research 75(2-3):128-34.

Bucak MN, Başpınar N, Tuncer PB, Coyan K, Sarıözkan S, Akalın PP, Büyükleblebici S, Küçükgünay S. 2012. Effects of curcumin and dithioerythritol on frozen‐thawed bovine semen. Andrologia 44:102-9.

Bucak MN, Sarıözkan S, Tuncer PB, Sakin F, Ateşşahin A, Kulaksız R, Çevik M. 2010. The effect of antioxidants on post-thawed Angora goat (Capra hircus ancryrensis) sperm parameters, lipid peroxidation and antioxidant activities. Small ruminant research 89(1):24-30.

Comhaire FH, Christophe AB, Zalata AA, Dhooge WS, Mahmoud AM, Depuydt CE. 2000. The effects of combined conventional treatment, oral antioxidants and essential fatty acids on sperm biology in subfertile men. Prostaglandins, Leukotrienes and Essential Fatty Acids (PLEFA). 2000 Sep 1;63(3):159-65.

Dattilo M, Giuseppe DA, Ettore C, Ménézo Y. 2016. Improvement of gamete quality by stimulating and feeding the endogenous antioxidant system: mechanisms, clinical results, insights on gene-environment interactions and the role of diet. Journal of assisted reproduction and genetics 33(12):1633-48.

Dhara S, Thakur S, Anwar SM, Sharma M, Houzha R, Sharma RK, Gupta HP. 2022. Correlation of certain biochemical constituents of seminal plasma with semen characteristics in pantja buck. Indian Journal of Animal Research 1(1):1-6.

Elsheshtawy R. 2020. Assessment of cattle bull semen preservability using tris extender enriched with turmeric extract. Egyptian Journal of Veterinary Sciences 51(3):357-62.

Fonseca JF, Torres CA, Maffili VV, Borges AM, Santos AD, Rodrigues MT, Oliveira RF. 2018. The hypoosmotic swelling test in fresh goat spermatozoa. Animal Reproduction (AR) 2(2):139-44.

Gallo A, Menezo Y, Dale B, Coppola G, Dattilo M, Tosti E, Boni R. 2018. Metabolic enhancers supporting 1-carbon cycle affect sperm functionality: an in vitro comparative study. Scientific Reports 8(1):11769.

Gangwar C, Kharche SD, Ranjan R, Kumar S, Goel AK, Jindal SK, Agarwal SK. 2015. Effect of vitamin C supplementation on freezability of Barbari buck semen. Small Ruminant Research 129:104-7.

Gangwar, C., Ranjan, R., Kumar, S., Kharche, S. D., Goel, A. K., Ramachandran, N., & Jindal, S. K. 2014. Use of chelating agent for optimum post thaw quality of buck semen. The Indian Journal of Animal Sciences 84(8): 839–841. https://doi.org/10.56093/ijans.v84i8.43215

Gupta S, Kumar A, Mahajan A, Sharma P, Sachan V, Aggrawal J, Yadav S, Saxena A, Kumar Swain D. 2022. Curcumin in a tris‐based semen extender improves cryosurvival of Hariana bull spermatozoa. Andrologia 54(1):e14255.

Hayanti SY, Sholikin MM, Jayanegara A, Ulum MF, da Costa MA, Fitriawaty F, Surya S, Hadiatry MC, Asmarasari SA, Handiwirawan E, Anggraeny YN. 2022. Effect of supplementing L-cysteine and its group analogs on frozen semen quality of bulls: A meta-analysis. Veterinary World 15(11):2517.

Iqbal S, Riaz A, Andrabi SM, Shahzad Q, Durrani AZ, Ahmad N. 2016. L‐Cysteine improves antioxidant enzyme activity, post‐thaw quality and fertility of Nili‐Ravi buffalo (Bubalus bubalis) bull spermatozoa. Andrologia 48(9):943-9.

Ismail AA, Abdel-Khalek AK, Khalil WA, Yousif AI, Saadeldin IM, Abomughaid MM, El-Harairy MA. 2020. Effects of mint, thyme, and curcumin extract nanoformulations on the sperm quality, apoptosis, chromatin decondensation, enzyme activity, and oxidative status of cryopreserved goat semen. Cryobiology 97:144-52.

Jannatifar R, Parivar K, Roodbari NH, Nasr-Esfahani MH. 2019. Effects of N-acetyl-cysteine supplementation on sperm quality, chromatin integrity and level of oxidative stress in infertile men. Reproductive Biology and Endocrinology. 2019 Feb 16;17(1):24.

Joe B, Lokesh BR. 1994. Role of capsaicin, curcumin and dietary n—3 fatty acids in lowering the generation of reactive oxygen species in rat peritoneal macrophages. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1224(2):255-63.

Kadirvel G, Kumar S, Kumaresan A, Kathiravan P. 2009. Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol level, membrane fluidity and intracellular calcium. Animal Reproduction Science. 2009 Dec 1;116(3-4):244-53.

Kafi A, Khalid M, Herath T, Kershaw C. 2024. Cysteine supplementation pre-freeze and post-thaw improves integrity and reduces oxidative stress in cryopreserved ram spermatozoa. Cryobiology 114:104854.

Khalil WA, Hassan MA, El-Harairy MA, Abdelnour SA. 2023. Supplementation of thymoquinone nanoparticles to semen extender boosts cryotolerance and fertilizing ability of buffalo bull spermatozoa. Animals 13(18):2973.

Kinkar D, Sarkar D, Debbarma A, Rahman M, Swain S, Karunakaran M. 2020. Review on recent advancement in semen additives for improving cryopreservation of bull semen. J. Entomol. 8:1493-505.

Koohestanidehaghi Y, Torkamanpari M, Shirmohamadi Z, Lorian K, Vatankhah M. 2021. The effect of cysteine and glutamine on human sperm functional parameters during vitrification. Andrologia 53(1):e13870.

Kulaksiz R, Ari Uç, Yildiz S, Lehimcioğlu Nc, Öztürkler Y. 2020. Seasonal variations in testicular measurements, fresh sperm quality and post-thaw sperm motility in gurcu goat bucks: Gurcu goats, freezability, seasonal variation, semen characteristics, testes. Slovak Journal of Animal Science 53(04):161-7.

Kumar N, Rai B, Bhat SA, Kharche SD, Gangwar C, Jindal SK, Chandra S. 2016. Effect of management system and season on semen freezability in Jakhrana bucks. Veterinary World 9(2):199.

Kumar Patel, G., Haque, N., Madhavatar, M., Kumar Chaudhari, A., Kumar Patel, D., Bhalakiya, N., Jamnesha, N., Patel, P. and Kumar, R., 2017. Artificial insemination: A tool to improve livestock productivity. Journal of Pharmacognosy and Phytochemistry, 6(6S), 307-313.

Kumar S, Kumar A, Singh AK, Honparkhe M, Singh P, Malhotra P. 2018. Improvement in post-thaw semen quality by minimizing the lipid peroxidation following herbal treatment in sub fertile buffalo bulls. The Pharma Innovation 1: 7(5, Part D):240.

Kumar V, Srivastava S, Alam K, Kumar R, Kumar R, Kumar P. 2023. Effect of Amla (Phyllanthus emblica) Extract on Fresh and Cryopreserved Seminal Attributes of Jamnapari Buck. The Indian Journal of Veterinary Sciences and Biotechnology 19(6):96.

Kumar V, Srivastava S, Alam K, Kumar R, Kumar S and Kumar P. 2024. Moringa oleifera leaves extract enhances fresh and cryopreserved seminal attribute of Jamnapari buck. The Haryana Veterinarian 63(1): 65-68.

Kutty CI and Mathew S. 2000. Seasonal variations in physical attributes of semen in Malabari and cross-bred bucks of Kerala. Indian Journal of Animal Reproduction 21: 22-26.

Leboeuf B, Restall B, Salamon S. 2000. Production and storage of goat semen for artificial insemination. Animal reproduction science 18: 62(1-3):113-41.

Mathuria N, Verma RJ. 2008. Curcumin ameliorates aflatoxin-induced toxicity in mice spermatozoa. Fertility and sterility 90(3):775-80.

Memon AA, Wahid H, Rosnina Y, Gohb YM, Ebrahimi MB, Nadiac FM, Audrey G. 2011. Effect of hypotaurine and cysteine on sperm cytological parameters of cooled and post-thaw Boer goat semen. Elixir Int. J. 38:4100-4.

Metwaly AM, El-Siefy EM, El-Halawany RS, EL-Behnsawy BE. 2016. Effect of cysteine supplementation on freezability of Baladie goat spermatozoa. Journal of Sustainable Agricultural Sciences 42(4):120-30.

Michael AJ, Alexopoulos C, Pontiki EA, Hadjipavlou-Litina DJ, Saratsis P, Ververidis HN, Boscos CM. Effect of antioxidant supplementation in semen extenders on semen quality and reactive oxygen species of chilled canine spermatozoa. Animal reproduction science 112(1-2):119-35.

Mishra B, Alam MG, Khandokar MA, Mazumder S, Munsi MN. 2010. Qualities of goat semen in Tris-Citrate-Glucose extender containing glutathione. Bangladesh Veterinarian 27(2):46-55.

Naz RK. 2011. Can curcumin provide an ideal contraceptive? Molecular Reproduction and Development 78(2): 116-123.

Naz RK. 2014. The effect of curcumin on intracellular pH (pHi), membrane hyperpolarization and sperm motility. Journal of Reproduction & Infertility 15(2): 62.

Omasanya OK, Hassan JO, Oloye AA, Oyewusi IK, Oni OO, Olurode SA, Oloruntuga OO, Adeusi AA, Bassahwa AP, Adetomiwa AS, Mustapha L. 2021. Effects of ejaculation frequency on semen characteristics and serum testosterone concentration in Red Sokoto bucks. Nigerian Journal of Animal Production 48(6):34-45.

Omur AD and Çoyan K. 2016. Protective effects of the antioxidants curcumin, ellagic acid and methionine on motility, mitochondrial transmembrane potential, plasma membrane and acrosome integrity in freeze-thawed Merino ram sperm. Veterinární medicína 61(1): 10-16.

P Kumar, S K Maurya, R Kumar, R Kumar, Rishikant, R Diwakar, K D Singh. 2025. Comparative effects of melatonin and melatonin nanoparticle for cryopreservation of Barbari Buck semen in the summer season. Indian Journal of Animal Sciences 95(9): ***–***

Panwar S, Singh AK, Kumar A, Honparkhe M, Ratta NS, Singh P. 2024. Effect of cysteine supplementation in semen extender on freezability and fertility of Murrah buffalo semen. The Indian Journal of Animal Sciences 94(6):545-552.

Peruma P, Selvaraju S, Barik AK, Mohanty DN, Das S, Mishra PC. 2011. Role of reduced glutathione in improving post-thawed frozen seminal characters of poor freezable Jersey crossbred bull semen. Indian Journal of Animal Sciences 81(8):807.

Perumal P, Sunder J, De AK, Bhattacharya D, Nahak AK, Vikram R, Chakurkar EB. 2023. Seasonal stress on semen quality profiles, seminal biochemical and oxidative stress attributes in endangered Teressa goat of Andaman and Nicobar Islands. Asian Pacific Journal of Reproduction 12(6):288-98.

Ranjan R, Goel AK, Kharche SD, Ramachandran N, Gangwar C, Jindal SK. 2014. Comparison of normal and dual staining techniques for evaluating viability and acrosome status in frozen thawed buck spermatozoa. Indian Journal of Small Ruminants (The) 20(2):50-3.

Ranjini A. 1998. Effect of processing and freezing procedures on the acrosome morphology of buck spermatozoa (Doctoral dissertation, Department of Animal Reproduction, College of Veterinary and Animal Sciences, Mannuthy).

Rushworth GF, Megson IL. 2014. Existing and potential therapeutic uses for N-acetylcysteine: the need for conversion to intracellular glutathione for antioxidant benefits. Pharmacology & therapeutics. 141(2):150-9.

Sarıözkan S, Bucak MN, Tuncer PB, Ulutaş PA, Bilgen A. 2009. The influence of cysteine and taurine on microscopic–oxidative stress parameters and fertilizing ability of bull semen following cryopreservation. Cryobiology 58(2):134-8.

Saurabh, Srivastava S, Singh B, Alam K, Kumar R, Kumar R, and Dubey M K. 2021a. Effects of ascorbic acid incorporation on Cytomorphological attributes on Cryopreservability (-1960 C) of Murrah bull Semen. The Pharma Innovation Journal 10(7): 1563-1567.

Saurabh, Srivastava S, Singh B, Alam K, Kumar R, Kumar R, Sharma P. 2021b. Testicular Biometry, Body-weight, Seminal Attributes and their Correlations in Murrah Bulls. The Indian Journal of Veterinary Science and Biotechnology 17(4): 26-30.

Shah SA, Andrabi SM, Qureshi IZ. 2017. Freezability of water buffalo bull (Bubalus bubalis) spermatozoa is improved with the addition of curcumin (diferuoyl methane) in semen extender. Andrologia 49(8):e12713.

Shamsuddin M, Amiri Y, Bhuiyan MM. 2000. Characteristics of buck semen with regard to ejaculate numbers, collection intervals, diluents and preservation periods. Reproduction in Domestic Animals 35(2):53-7.

Sharma A and Sood P. 2021. Seminal quality attributes and their interrelationships in Gaddi and Chegu bucks. Indian Journal of Small Ruminants (The) 27(1):50-5.

Sharma A, Sood P. 2019. Cryopreservation and fertility of frozen thawed Chegu goat semen. Indian Journal of Animal Research 53(11):1414-9.

Sharma OP. 1976. Antioxidant activity of curcumin and related compounds. Biochemical pharmacology 25(15):1811-2.

Sharma S, Sharma NK, Kataria N, Sinha NK. 2013. Leakage of phosphatases and fertility of buck semen cryopreserved under different freezing modes. International Journal of Applied Sciences and Biotechnology 1(4):202-7.

Shiny M. 2011. Freezability of buck spermatozoa treated with chelating agent for fibronectin type II proteins (Doctoral dissertation, COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY, THRISSUR).

Sinha MP, Sinha AK, Singh BK, Prasad RL. 1996.The effect of glutathione on the motility, enzyme leakage and fertility of frozen goat semen. Animal reproduction science 41(3-4):237-43.

Soleimanzadeh A and Saberivand A. 2013. Effect of curcumin on rat sperm morphology after the freeze-thawing process. Veterinary Research Forum 4(3): 185-189.

Stradaioli G, Noro T, Sylla L, Monaci M. 2007. Decrease in glutathione (GSH) content in bovine sperm after cryopreservation: comparison between two extenders. Theriogenology 67(7):1249-55.

Szcześniak-Fabiańczyk B, Bochenek M, Smorag Z, Ryszka F. 2003. Effect of antioxidants added to boar semen extender on the semen survival time and sperm chromatin structure. Reprod Biol. 3(1):81-7.

Tudu KC, Sarkar D, Mandal A, Mondal M, Das SK, Rai S, Behera R, Bhakat C, Karunakaran M. 2023. Effect of L-cysteine supplementation on cryopreservation of Black Bengal Buck Semen. Indian Journal of Animal Research 57(10):1285-8.

Tuli RK, Holtz W. 1994. Effect of glycerolization procedure and removal of seminal plasma on post-thaw survival and GOT-release from Boer goat spermatozoa. Theriogenology. 1994 Jan 1;42(3):547-55.

Uysal O and Bucak M. 2007. Effects of oxidized glutathione, bovine serum albumin, cysteine and lycopene on the quality of frozen-thawed ram semen. Acta Veterinaria Brno. 76(3): 383-390.

Verma AK, Kumar R, Srivastava S, Alam K, Kumar R, Sakhahari DS, Verma SK. 2023. Methionine as a semen additive to improve Murrah buffalo (Bubalus bubalis) semen cryopreservation. The Indian Journal of Veterinary Science and Biotechnology 19:39-43.

Wang F, Liu S, Shen Y, Zhuang R, Xi J, Fang H, Pan X, Sun J, Cai Z. 2014. Protective effects of N acetylcysteine on cisplatin induced oxidative stress and DNA damage in HepG2 cells. Experimental and therapeutic medicine 8(6):1939-45.

Watson P F. 2000. The causes of reduced fertility with cryopreserved semen. Animal reproduction science 60: 481-492.

Wurlina W, Hariadi MU, Safitri E, Susilowati S, Meles DK. 2020. The effect of crude guava leaf tannins on motility, viability, and intact plasma membrane of stored spermatozoa of Etawa crossbred goats. Veterinary world 13(3):530.

Xu B, Bai X, Zhang J, Li B, Zhang Y, Su R, Wang R, Wang Z, Lv Q, Zhang J, Li J. 2023. Metabolomic analysis of seminal plasma to identify goat semen freezability markers. Frontiers in Veterinary Science 10:1132373.

Zafarullah M, Li WQ, Sylvester J, Ahmad M. Molecular mechanisms of N-acetylcysteine actions. Cellular and Molecular Life Sciences CMLS 60(1):6-20.

Zhang L, Diao RY, Duan YG, Yi TH, Cai ZM. 2017. In vitro antioxidant effect of curcumin on human sperm quality in leucocytospermia. Andrologia 49(10):e12760.

Zhu Z, Ren Z, Fan X, Pan Y, Lv S, Pan C, Lei A, Zeng W. 2017. Cysteine protects rabbit spermatozoa against reactive oxygen species-induced damages. PLoS One 12(7):e0181110.

Zuge RM, Bertolla RP, Nichi TB, Cortada CN, Bols PE, Barnabe VH. 2008. Correlation between bovine sperm membrane integrity and mitochondrial activity in Bos taurus bulls. In16th International Congress on Animal Reproduction, Budapest, Hungary. 13(17):July 2008 (Vol. 13, No. 17, p. 2008).