The effects of cysteine and L-carnitine on the DNA integrity of post-thaw sperm of frozen buck semen

Document Type : Full paper (Original article)


Department of Theriogenology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, 41522, Egypt


Background: The addition of antioxidants to semen extenders is fundamental to limit oxidative changes resulting from cryopreservation. Aims: This experiment was designed to clarify the effects of cysteine and L-carnitine (LC) on post-thaw sperm criteria. Methods: Semen samples were collected once weekly from five mature Zaraibi bucks for 10 successive weeks. Fresh semen samples were evaluated for basic semen characteristics, and the accepted samples were pooled and divided into seven aliquots: control (Tris egg yolk extender), cysteine (2.5, 5 and 10 mM) and LC (2.5, 5 and 7.5 mM). Aliquots were diluted and subjected to cryopreservation procedures. After thawing, sperm criteria were evaluated regarding motility, viability, plasma membrane (hypo-osmotic swelling test, HOST) and acrosome integrity, total antioxidant capacity (TAC), level of malondialdehyde (MDA), and DNA comet assay. Results: Addition of both 10 mM cysteine and 7.5 mM LC significantly increased post-thaw sperm motility, viability, HOST, and acrosome integrity. Comet assay revealed significant decreases in DNA damage with best results at 2.5 mM cysteine and 7.5 mM LC. Besides, 10 mM cysteine and 7.5 mM LC exhibited significant diminish in MDA levels. Cysteine was positively correlated with sperm viability, acrosome integrity, and comet, but negatively correlated with MDA and tail length. Positive correlations were declared between LC and progressive motility, viability, HOST and TAC, while negative correlations were found with MDA, comet, tail DNA, tail moment, and olive tail moment. Conclusion: Supplementing cysteine and LC to frozen buck semen improved sperm criteria and decreased DNA damage, possibly due to their effectiveness in the suppression of MDA formation.


Abd-Allah, M; El Kelawy, H; Ibrahim, H; El-Gaafary, N and El-Ela, A (2021). Effect of treatment with L-carnitine, co-enzyme q10 and zinc-methionine as stimulants on semen quality and some blood parameters of Damascus goat bucks during summer season of Egypt. J. Product. Dev., 26: 671-688.
Adekunle, E; Iyanda, O; Majekodunmi, B; Akosile, A; Ojo, S; Odeyemi, A; Logunleko, M; Oderinwale, O; Sorongbe, T and Daramola, J (2022). Preservative effects of cysteine on sperm viability of West African dwarf goat bucks chilled at 4oC. Niger. J. Anim. Prod., 49: 49-59.
Alahmar, AT (2019). Role of oxidative stress in male infertility: An updated review. J. Hum. Reprod. Sci., 12: 4-18.
Alcay, S; Gokce, E; Toker, MB; Onder, NT; Ustuner, B; Uzabacı, E; Gul, Z and Cavus, S (2016). Freeze-dried egg yolk based extenders containing various antioxidants improve post-thawing quality and incubation resilience of goat spermatozoa. Cryobiology. 72: 269-273.
Alomar, M (2021). Effects of oxidized glutathione, cysteine and taurine supplementations on motility charateristics of different goat spermatozoa types. Iran. J. Vet. Sci. Technol., 13: 84-92.
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. Biotechnol. Lett., 15: 26-32.
Atessahin, A; Bucak, MN; Tuncer, PB and Kızıl, M (2008). Effects of anti-oxidant additives on microscopic and oxidative parameters of Angora goat semen following the freeze-thawing process. Small Rumin. Res., 77: 38-44.
Balamurugan, B; Ghosh, S; Lone, S; Prasad, J; Das, G; Katiyar, R; Mustapha, AR; Kumar, A and Verma, M (2018). Partial deoxygenation of extender improves sperm quality, reduces lipid peroxidation and reactive oxygen species during cryopreservation of buffalo (Bubalus bubalis) semen. Anim. Reprod. Sci., 189: 60-68.
Bucak, MN; Ateşşahin, A and Yüce, A (2008). Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze-thawing process. Small Rumin. Res., 75: 128-134.
Bucak, MN; Tuncer, PB; Sarıözkan, S; Başpınar, N; Taşpınar, M; Çoyan, K; Bilgili, A; Akalın, PP; Büyükleblebici, S and Aydos, S (2010). Effects of antioxidants on post-thawed bovine sperm and oxidative stress parameters: antioxidants protect DNA integrity against cryodamage. Cryobiology. 61: 248-253.
Chowdhury, S; Das, S; Gupta, T; Sana, D and Bose, S (2014). Evaluation of frozen semen by acrosomal integrity and sperm concentration-two vital quality parameters of male fertility in bovines. ERMR., 4: 101-107.
Cirit, Ü; Bağış, H; Demir, K; Agca, C; Pabuccuoğlu, S; Varışlı, Ö; Clifford-Rathert, C and Agca, Y (2013). Comparison of cryoprotective effects of iodixanol, trehalose and cysteamine on ram semen. Anim. Reprod. Sci., 139: 38-44.
Çoyan, K; Başpınar, N; Bucak, MN and Akalın, PP (2011). Effects of cysteine and ergothioneine on post-thawed Merino ram sperm and biochemical parameters. Cryobiology. 63: 1-6.
da Silva Maia, M; Bicudo, SD; Sicherle, CC; Rodello, L and Gallego, ICS (2010). Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen cryopreserved in extenders with antioxidants. Anim. Reprod. Sci., 122: 118-123.
de Souza, CV; Brandão, FZ; Santos, JDR; Alfradique, VAP; dos Santos, VMB; da Cruz Morais, MC; Rangel, PSC; da Silva, AA and Souza-Fabjan, JMG (2019). Effect of different concentrations of l-carnitine in extender for semen cryopreservation in sheep. Cryobiology. 89: 104-108.
Elsayed, DH; El-Shamy, AA; Abdelrazek, HM and El-Badry, D (2019). Effect of genistein on semen quality, antioxidant capacity, caspase-3 expression and DNA integrity in cryopreserved ram spermatozoa. Small Rumin. Res., 177: 50-55.
Evans, G and Maxwell, WC (1987). Salamons’ artificial insemination of sheep and goats. 2nd Edn., Sydney: Butterworths.
Gadea, J; Gumbao, D; Gómez-Giménez, B and Gardón, JC (2013). Supplementation of the thawing medium with reduced glutathione improves function of frozen-thawed goat spermatozoa. Reprod. Biol., 13: 24-33.
Gülçin, İ (2006). Antioxidant and antiradical activities of L-carnitine. Life Sci., 78: 803-811.
Iqbal, S; Riaz, A; Andrabi, S; Shahzad, Q; Durrani, A and Ahmad, N (2016). l-Cysteine improves antioxidant enzyme activity, post-thaw quality and fertility of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa. Andrologia. 48: 943-949.
Jeulin, C and Lewin, LM (1996). Role of free L-carnitine and acetyl-L-carnitine in post-gonadal maturation of mammalian spermatozoa. Hum. Reprod. Update. 2: 87-102.
Jeyendran, R; Van der Ven, H; Perez-Pelaez, M; Crabo, B and Zaneveld, L (1984). Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. Reproduction. 70: 219-228.
Jia, B; Memon, S; Liang, J; Lv, C; Hong, Q; Wu, G and Quan, G (2021). Trehalose modifies the protein profile of ram spermatozoa during cryopreservation. Theriogenology. 171: 21-29.
Koracevic, D; Koracevic, G; Djordjevic, V; Andrejevic, S and Cosic, V (2001). Method for the measurement of antioxidant activity in human fluids. J. Clin. Pathol., 54: 356-361.
Küçük, N; Aksoy, M; Uçan, U; Ahmad, E; Naseer, Z; Ceylan, A and Serin, I (2014). Comparison of two different cryopreservation protocols for freezing goat semen. Cryobiology. 68: 327-331.
Longobardi, V; Salzano, A; Campanile, G; Marrone, R; Palumbo, F; Vitiello, M; Zullo, G and Gasparrini, B (2017). Carnitine supplementation decreases capacitation-like changes of frozen-thawed buffalo spermatozoa. Theriogenology. 88: 236-243.
Lv, C; Larbi, A; Wu, G; Hong, Q and Quan, G (2019). Improving the quality of cryopreserved goat semen with a commercial bull extender supplemented with resveratrol. Anim. Reprod. Sci., 208: 106127-106137.
Memon, AA; Wahid, H; Rosnina, Y; Goh, Y; Ebrahimi, M and Nadia, F (2012). Effect of antioxidants on post thaw microscopic, oxidative stress parameter and fertility of Boer goat spermatozoa in Tris egg yolk glycerol extender. Anim. Reprod. Sci., 136: 55-60.
Mia, M; Khandoker, M; Husain, S; Faruque, M; Notter, D and Apu, A (2013). Genetic and phenotypic parameters for semen characteristics and their relationship with scrotal circumference in Black Bengal bucks. Iran. J. Appl. Anim. Sci., 3: 709-717.
Naijian, H; Kohram, H; Shahneh, AZ and Sharafi, M (2013). Effects of various concentrations of BSA on microscopic and oxidative parameters of Mahabadi goat semen following the freeze-thaw process. Small Rumin. Res., 113: 371-375.
NRC (2006). Nutrient requirements of small ruminants: sheep, goats, cervids, and new world camelids. 1st Edn., Washington D.C., China Legal Publishing House. PP: 271-300.
Ohkawa, H; Ohishi, N and Yagi, K (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem., 95: 351-358.
Peris, SI; Bilodeau, JF; Dufour, M and Bailey, JL (2007). Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm. Mol. Reprod. Dev., 74: 878-892.
Peris-Frau, P; Martín-Maestro, A; Iniesta-Cuerda, M; Sánchez-Ajofrín, I; Cesari, A; Garde, JJ; Villar, M and Soler, AJ (2020). Cryopreservation of ram sperm alters the dynamic changes associated with in vitro capacitation. Theriogenology. 145: 100-108.
Pradieé, J; Cardoso, T; Silva, E; Gonçalves, A; Gastal, G; Rosa, C; Mondadori, R; Pegoraro, L; Vieira, A and Lucia Jr, T (2016). Effect of β-mercaptoetanol and cysteine on post-thawing quality and oxidative activity of ram sperm and on the viability of vitrified sheep embryos. Arq. Bras. Med. Vet. Zootec., 68: 1309-1315.
Rostami, B; Ebrahimi, D; Sadeghipanah, H; Masoumi, R and Shahir, MH (2020). Effects of supplementation of tris-egg yolk extender with different sugars and antioxidants on freezability of ram semen. Cryobiology. 92: 62-66.
Sánchez-Rubio, F; Soria-Meneses, PJ; Jurado-Campos, A; Bartolome-Garcia, J; Gomez-Rubio, V; Soler, AJ;
Arroyo-Jimenez, M; Santander-Ortega, M; Plaza-Oliver, M and Lozano, M
(2020). Nanotechnology in reproduction: Vitamin E nanoemulsions for reducing oxidative stress in sperm cells. Radic. Biol. Med., 160: 47-56.
Sarıözkan, S; Bucak, MN; Tuncer, PB; Büyükleblebici, S and Cantürk, F (2014). Influence of various antioxidants added to TCM-199 on post-thaw bovine sperm parameters, DNA integrity and fertilizing ability. Cryobiology. 68: 129-133.
Setyawan, EE; Cooper, TG; Widiasih, DA; Junaidi, A and Yeung, CH (2009). Effects of cryoprotectant treatments on bovine sperm function and osmolyte content. Asian J. Androl., 11: 571-581.
Shafiei, M; Forouzanfar, M; Hosseini, SM and Esfahani, MHN (2015). The effect of superoxide dismutase mimetic and catalase on the quality of postthawed goat semen. Theriogenology. 83: 1321-1327.
Shahzad, Q; Mehmood, MU; Khan, H; ul Husna, A; Qadeer, S; Azam, A; Naseer, Z; Ahmad, E; Safdar, M and Ahmad, M (2016). Royal jelly supplementation in semen extender enhances post-thaw quality and fertility of Nili-Ravi buffalo bull sperm. Anim. Reprod. Sci., 167: 83-88.
Sharafi, M; Zhandi, M and Sharif, AA (2015). Supplementation of soybean lecithin-based semen extender by antioxidants: complementary flowcytometric study on post-thawed ram spermatozoa. Cell Tissue Bank., 16: 261-269.
Shaygannia, E; Ghandehari-Alavijeh, R; Tavalaee, M and Nasr-Esfahani, MH (2020). The protective effects of alpha lipoic acid on human sperm function during freezing-thawing. Cryoletters. 41: 344-350.
Tabarez, A; García, W and Palomo, MJ (2017). Effect of the type of egg yolk, removal of seminal plasma and donor age on buck sperm cryopreservation. Small Rumin. Res., 149: 91-98.
Toker, MB; Alcay, S; Gokce, E and Ustuner, B (2016). Cryopreservation of ram semen with antioxidant supplemented soybean lecithin-based extenders and impacts on incubation resilience. Cryobiology. 72: 205-209.
Vasquez, J; Florentini, E; Camargo, L; Gonzales, J and Valdivia, M (2013). Hypoosmotic swelling test in epididymal ram (Ovis aries) spermatozoa. Livest. Sci., 157: 618-622.
Watson, P (1975). Use of a Giemsa stain to detect changes in acrosomes of frozen ram spermatozoa. Vet. Rec., 97: 12-15.
Zhu, Z; Zeng, Y and Zeng, W (2022). Cysteine improves boar sperm quality via glutathione biosynthesis during the liquid storage. Anim. Biosci., 35: 166-176.