Effects of cysteamine during in vitro maturation on viability and meiotic competence of vitrified buffalo oocytes

Document Type : Full paper (Original article)


1 Department of Animal Reproduction & Artificial Insemination (AI), National Research Center, Dokki, Giza, Egypt

2 Department of Theriogenology, Faculty of Veterinary Medicine, Benha University, Kaliobia, Egypt


The aims of the present study were to assess the effects of cysteamine as an anti-oxidant on the rate of in vitro maturation (IVM) of buffalo oocytes (experiment 1), and their viability and nuclear status following vitrification (experiment 2). Immature oocytes with compact cumulus cells obtained from the ovaries of slaughtered animals were harvested and then cultured in the maturation medium with no cysteamine (control) or 50 μM cysteamine (treated). Oocytes were vitrified in vitrification solution 1 (VS1): 1.5 M ethylene glycol (EG) + 1.5 M dimethyl sulfoxide (DMSO) for 45 s (step one). After this initial exposure, oocytes were transferred to VS2: 3 M EG + 3 M DMSO in a holding medium for 25 s (step two). After warming, oocytes were evaluated morphologically and then cultured for a further 2 h in the cysteamine-supplemented or control maturation media. The oocytes were evaluated morphologically, stained with trypan blue for viability evaluation. The maturation rate of oocytes was higher (P<0.05) for IVM media with cysteamine compared with controls. There was no significant difference in morphology, survivability and maturation rate between the two
vitrification groups (cysteamine-treated and untreated groups) but the morphology, survivability and percentages of metaphase-II oocytes in both groups of vitrified oocytes were lower compared with their respective controls. In conclusion, the addition of cysteamine to the maturation medium improved nuclear maturation of buffalo oocytes but had no positive effect on their cryoresistance during vitrification.


Ali, AA; Bilodeau, JF and Sirard, MA (2003). Antioxidant requirements for bovine oocytes varies during in vitro maturation, fertilization and development. Theriogenology. 59: 939-949.
Balasubramanian, S and Rho, GJ (2007). Effect of cysteamine supplementation of in vitro matured bovine oocytes on chilling sensitivity and development of embryos. Anim. Reprod. Sci., 98: 282-292.
Bedaiwy, MA; Falcone, T; Mohamed, MS; Aleem, AA; Sharma, RK; Worley, SE; Thornton, J and Agarwal, A (2004). Differential growth of human embryos in vitro: role of reactive oxygen species. Fertil. Steril., 82: 593-600.
De Matos, DG; Gasparrini, B; Pasqualini, SR and Thompson, JG (2002). Effect of glutathione synthesis stimulation during in vitro maturation of ovine oocytes on embryo development and intracellular peroxide content. Theriogenology. 5: 1443-1451.
Elamaran, G; Singh, KP; Sing, MK; Singla, SK; Chauhan, MS; Manik, RS and Palta, PO (2012). Oxygen concentra-tion and cysteamine supplementation during in vitro production of buffalo (Bubalus bubalis) embryos affect mRNA expression of BCL-2, BCL-XL, MCL-1, BAX and BID. Reprod. Dom. Anim., 47: 1027-1036.
El-Naby, Al HH; Mahmoud, KGhM; Ahmed, YF; Abouel-Roos, MEA and Abdel-Ghaffar, AE (2013). Effect of season of the year and ovarian structures on oocytes recovery rate, quality and meiotic competence in Egyptian buffaloes. Glob. Vet., 10: 408-412.
El-Sokary, MMM; Kandiel, MMM, Mahmoud, KGhM; Abouel-Roos, MEA and Sosa, GAM (2013). Evaluation of viability and nuclear status in vitrified mature buffalo oocytes. Glob. Vet., 10: 297-302.
Gasparrini, B (2002). In vitro embryo production in buffalo species: state of the art. Theriogenology. 57:  237-256.
Gasparrini, B; Boccia, L; Marchandise, J; Palo, RD; Georgr, F; Donay, I and Zicarelli, L (2006). Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development. Theriogenology. 65: 275-287.
Gasparrini, B; Neglia, G; Palo, RD; Campanile, G and Zicarelli, L (2000). Effect of cysteamine during in vitro maturation on buffalo embryo development. Theriogenology. 54: 1537-1542.
Gasparrini, B; Sayond, H; Neglia, G; De Matos, DG; Donny, I and Zicarelli, L (2003). Glutathion synthesis during in vitro maturation of buffalo (Bubalus bubalis) oocytes: effects of cysteamine on embryo development. Theriogenology. 60: 943-952.
Grupen, CG; Nagashima, H and Nottle, MB (1995). Cysteamine enhances in vitro development of porcine oocytes matured and fertilized in vitro. Biol. Reprod., 53: 173-178.
Kelly, J; Kleemann, D; Kuwayama, M and Walker, S (2005). Effect of cysteamine on survival of bovine and ovine oocytes vitrified using the minimum volume cooling (MVC) cryotop method. Reprod. Fertil. Dev., 18: 158 (abst.).
Kitagawa, Y; Suzuki, K; Yoneda, A and Watanabe, T (2004). Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos. Theriogenology. 62: 1186-1197.
Kobayashi, M; Lee, ES and Fukui, Y (2006). Cysteamine or beta-mercaptoethanol added to a defined maturation medium improves blastocyst formation of porcine oocytes after intracytoplasmic sperm injection. Theriogenology. 65: 1191-1199.
Ledda, S; Leoni, G; Bogliolo, L and Naitana, S (2001). Oocyte cryopreservation and ovarian tissue banking. Theriogenology. 55: 1359-1371.
Liu, RH; Sun, QY; Li, YH; Jiao, LH and Wang, WH (2003). Effects of cooling on meiotic spindle structure and chromosome alignment within in vitro matured porcine oocytes. Mol. Reprod. Dev., 65: 212-218.
Mahmoud, KGhM; Al-Shimaa, Al HH; Ahmed, YF; Eldebaky, HAA; Abou El-Roos, MEA and Abd-El Ghaffar, AE (2014). Effect of cumulus cells and meiotic stages on oocytes survivability and meiotic competence in vitrified buffalo oocytes. Pakistan J. Zool., 46: 1185-1192.
Mahmoud, KGhM and El-Sokary, MMM (2013). Im-provement of the efficacy of buffalo oocytes vitrification. Glob. Vet., 11: 420-431.
Mahmoud, KGhM; El-Sokary, MMM; Scholkamy, TH; Abou El-Roos, MEA; Sosa, GAMand Nawito, M (2013). Effect of cryodevice type and cryoprotectant concentration on vitrified buffalo oocytes at MII stage. Anim. Reprod., 10: 689-696.
Mahmoud, KGhM; Mohamed, YMA; Amer, MA; Noshy, MM and Nawito, M (2010c). Aneuploidy in in-vitro matured buffalo oocytes with or without cumulus cells. Nat. Sci., 8: 46-51.
Mahmoud, KGhM; Scholkamy, TH; Ahmed, YF; Seidel, JrGE and Nawito, MF (2010a). Effect of different combinations of cryoprotectants on in vitro maturation of immature buffalo (Bubalus bubalis) oocytes vitrified by straw and open pulled straw methods. Reprod. Dom. Anim., 45: 565-571.
Mahmoud, KGhM; Scholkamy, TH; Seidel, JrGE and Elsisy, GA (2010b). Influence of meiotic stages on the survival and developmental ability of vitrified-warmed buffalo oocytes. GJBB., 5: 220-225.
Mahmoud, KGhM and Seidel, JrGE (2010). Viability and DNA fragmentation assessment of bovine embryos vitrified by different methods. Researcher. 2: 14-20.
Mukherjee, A; Kumar, D; Singh, KB; Chauhan, MS; Singla, SK; Palta, P and Manik, RS (2010). Assessment of DNA damage during in vitro development of buffalo (Bubalus bubalis) embryos: effect of cysteamine. Reprod. Dom. Anim., 45: 1118-1121.
Oyamada, T and Fukui, Y (2004). Oxygen tension and medium supplements for in vitro maturation of bovine oocytes cultured individually in a chemically defined medium. J. Reprod. Dev., 50: 107-117.
Roushandeh, AM; Mooghahi, MHN; Pasbakhsh, P; Abddvahabi, A; Akbari, M, Shokrgozarand, Aand Sobhani, A (2006). Effect of cysteamine on the rate of in vitro maturation of oocytes in two media. Acta Med. Iran., 44: 167-171.
Roushandeh, AM and Roudkenar, MH (2009). The influence of meiotic spindle configuration by cysteamine during in vitro maturation of mouse oocytes. Iran. Biomed. J., 13: 73-78.
Sadeesh, EM; Shah, F; Balhara, AK; Thirumaran, SMK; Yadav, S and Yadav, PS (2014). Effect of growth factor
and antioxidant on in vitro maturation of oocytes and cleavage rates of in vitro produced Indian buffalo (Bubalus bubalis) embryos. Vet. Arhiv., 84: 459-474.
Scholkamy, TH; Samah, FD and Mahmoud, KGhM (2015). Effect of vitrification by straw and cryotop on DNA integrity using comet assay with reference to brilliant cresyl blue exposure in buffalo oocytes. Alexandria J. Vet. Sci., 46: 114-120.
Singhal, S; Prasad, S; Singh, B; Prasad, JK and Gupta, HP (2009). Effect of including growth factors and antioxidants in maturation medium used for in vitro culture of buffalo oocytes recovered in vivo. Anim. Reprod. Sci., 113: 44-50.
Somfai, T; Ozawa, M; Noguchi, J; Kaneko, H; Karja, NWK; Farhudin, M; Dinnyes, A; Nagai, T and Kikuchi, K (2007). Developmental competence of in vitro-fertilized porcine oocytes after in vitro maturation and solid surface vitrification: effect of cryopreservation on oocyte antioxidative system and cell cycle stage. Cryobiology. 55: 115-126.
Subramaniam, A; Devarajan, KP and Mohanan, M (1990). Incidence and avoidance of zona fracture in cryopreserved bovine ova and embryos. N. Z. Vet. J., 38 : 156-157.
Takahashi, M; Nagai, T; Hamano, S; Kuwayama, M; Okamura, N and Okano, A (1993). Effect of thiol com-pounds on in vitro development and intra cellular glutathione content of bovine embryos. Biol. Reprod., 49: 228-232.
Tarkowski, AK (1966). An air-drying method for chromosome preparations from mouse eggs. Cytogenetics. 5: 394-400.
Vajta, G (2000). Vitrification of the oocytes and embryos of domestic animals. Anim. Reprod. Sci., 60-61: 357-364.
Yadav, RC; Sharma, A; Garg, N and Purohit, GN (2008). Survival of vitrified water buffalo cumulus-oocytes-complexes and their subsequent development in vitro. Bulg. J. Vet. Med., 11: 55-64.