Effects of sucrose and glycerol on vitrification of buffalo oocytes

M. A. Hossain, Shahrina Akter, M. F. H. Miraz, M. Nuronnabi Islam, Jannatul Bari, M. Hasanur Alam, M. Bodruzzaman Sarker, M. E. Kabir, A. Khatun, M. A. Hashem, M. Moniruzzaman


Background: Vitrification, ultra-rapid cooling can be used to cryopreserve oocytes for embryo technology. The objective of this study was to evaluate the effects of sucrose and glycerol on vitrification of buffalo oocytes.

Methods: Cumulus-oocyte complexes (COCs) were aspirated from slaughtered buffalo ovaries. In experiment 1, the vitrification solution was supplemented with either 0, 0.25 or 0.5 M sucrose. In experiment 2, the vitrification solution was supplemented with either 0, 5 or 10 M glycerol together with 0.5 M sucrose. COCs were exposed into equilibration solution and vitrification solution for 5 min and 1 min, respectively. Then the oocytes were submerged into liquid nitrogen for 10 min using cryotops. The oocytes were thawed, diluted and washed in washing solution. Vitrified oocytes were cultured for maturation at 38.5°C for 24 hrs at 5% CO2. Then oocytes were fixed in acetic acid and ethanol and stained with aceto-orcein to examine the meiotic stages.

Results: In experiment 1, a significantly higher number of morphologically normal oocytes and cumulus cell expansion were found in 0.5 M sucrose group than others. In addition, a proportion of oocytes resumed meiosis but none of those developed to the metaphase II (MII) stage. In experiment 2, a significantly higher number of oocytes showed cumulus cell expansion as well as higher morphologically normal oocytes in 5 M and 10 M glycerol than in 0 M (control) group. In addition, 18% oocytes matured to MII stage in 5 M glycerol group.

Conclusions: Buffalo oocytes can be vitrified with a combination of sucrose and glycerol to maintain its developmental potential.


Buffalo, Glycerol, In vitro maturation, Oocytes, Sucrose, Vitrification

Full Text:



Warriach DM, Mcgill RD, Bush WPC, Chohan KR. A review of recent developments in buffalo reproduction. Asian-Australas J Anim Sci. 2015;28(3):451-5.

Gasparrini B. In vitro embryo production in buffalo species: state of the art. Theriogenology. 2002;57(1):237-56.

Kumar A, Solanki VS, Jindal SK, Tripathi VN, Jain GC. Oocyte retrieval and histological studies of follicular population in buffalo ovaries. Anim Reprod Sci. 1997;47(3):189-95.

Fahy GM, MacFarlane DR, Angell CA, Meryman HT. Vitrification as an approach to cryopreservation. Cryobiology. 1984;21(4):407-26.

Parnpai R, Liang Y, Ketudat-Cairns M, Somfai T, Nagai T. Vitrification of buffalo oocytes and embryos. Theriogenology. 2016;86(1):214-20.

Shaw JM, Kuleshova LL, MacFarlane DR, Trounson AO. Vitrification properties of solutions of ethylene glycol in saline containing PVP, Ficoll, or dextran. Cryobiology. 1997;35(3):219-29.

Wani NA, Maurya SN, Misra AK, Saxena VB, Lakhchaura BD. Effect of cryoprotectants and their concentration on in vitro development of vitrified-warmed immature oocytes in buffalo (Bubalus bulalis). Theriogenology. 2004;61(5):831-42.

Vieira AD, Mezzalira A, Barbieri DP, Lehmkuhl RC, Rubin MI, Vajta G. Calves born after open pulled straw vitrification of immature bovine oocytes. Cryobiology. 2002;45(1):91-4.

Kuleshova LL, MacFarlane DR, Trounson AO, Shaw JM. Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes. Cryobiology. 1999;38(2):119-30.

Leibo SP. Water permeability and its activation energy of fertilized and unfertilized mouse ova. J Membr Biol. 1980;53:179-88.

Mazur P. Equilibrium, quasi-equilibrium, and nonequilibrium freezing of mammalian embryos. Cell Biophys. 1990;17:53-92.

Kasai M, Komi JH, Takakamo A, Tsudera H, Sakurai T, Machida T. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J Reprod Fertil. 1990;89(1):91-7.

Hochi S, Terao T, Kamei M, Kato M, Hirabayashi M, Hirao M. Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedure. Theriogenology. 2004;61(2-3):267-75.

Schiewe MC, Rall WF, Stuart LD, Wildt DE. Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos. Theriogenology. 1991;36(2):279-93.

Oberstein N, O'Donovan MK, Bruemmer JE, Seidel GE, Carnevale EM, Squire EL. Cryopreservation of equine embryos by open pulled straw, cryoloop, or conventional slow cooling methods. Theriogenology. 2001;55(2):607-13.

Delval A, Ectors FJ, Touati K, Beckers JF, Ectors F. Vitrification of bovine embryos produced in vitro: survival, hatching and pregnancy rates. Theriogenology. 1996;45(1):178.

Hufana-Duran D, Pedro PB, Venturina HV, Hufana RD, Salazar AL, Duran PG, et al. Post-warming hatching and birth of live calves following transfer of in vitro-derived vitrified water buffalo (Bubalus bubalis) embryos. Theriogenology. 2004;61(7-8):1429-39.

Mukaida T, Nakamura S, Tomiyama T, Wada S, Kasai M, Takahashi K. Successful birth after transfer of vitrified human blastocysts with use of a cryoloop container less technique. Fertil Steril. 2001;76(3):618-20.

Nakagata N. High survival rate of unfertilized mouse oocytes after vitrification. J Reprod Fertil. 1989;87(2):479-83.

Hamano S, Koikeda A, Kuwayama M, Nagai T. Full-term development of in vitro matured, vitrified and fertilized bovine oocytes. Theriogenology. 1992;38(6):1085-90.

Dhali A, Manik RS, Das SK, Singla SK, Palta P. Vitrification of buffalo (Bubalus bubalis) oocytes. Theriogenology. 2000;53(6):1295-303.

Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: the cryotop method. Theriogenology. 2007;67(1):73-80.

Wright DL, Eroglu A, Toner M, Toth TL. Use of sugars in cryopreserving human oocytes. Reprod Biomed Online. 2004;9(2):179-86.

Huang J, Li Q, Zhao R, Li W, Han Z, Chen X, et al. Effect of sugars on maturation rate of vitrified-thawed immature porcine oocytes. Anim Reprod Sci. 2008;106(1-2):25-35.

El-Shalofy AS, Moawad AR, Darwish GM, Ismail ST, Badawy ABA, Badr MR. Effect of different vitrification solutions and cryodevices on viability and subsequent development of buffalo oocytes vitrified at the germinal vesicle (GV) stage. Cryobiology. 2017;74:86-92.

Gupta MK, Uhm SJ, Lee HT. Cryopreservation of immature and in vitro matured porcine oocytes by solid surface vitrification. Theriogenology. 2007;67(2):238-48.

Moniruzzaman M, Bao RM, Taketsuru H, Miyano T. Development of vitrified porcine primordial follicles in xenografts. Theriogenology. 2009;72(2):280-8.

Bao RM, Yamasaka E, Moniruzzaman M, Hamawaki A, Yoshikawa M, Miyano T. Development of vitrified bovine secondary and primordial follicles in xenografts. Theriogenology. 2010;74(5):817-27.

Maruska DV, Leibfried ML, First NL. Role of calcium and the calcium-calmodulin complex in resumption of meiosis, cumulus expansion, viability and hyaluronidase sensitivity of bovine cumulus-oocyte complexes. Biol Reprod. 1984;31(1):1-6.

Islam MN, Alam MH, Khatun A, Akter I, Modak AK, Hashem MA, et al. Effects of stem cell factor on in vitro growth of buffalo oocytes. Theriogenology. 2020;142:114-9.

Rajan R, Matsumura K. Development and application of cryoprotectants. Adv Exp Med Biol. 2018;1081:339-54.

Yamada C, Caetano HV, Simões R, Nicacio AC, Feitosa WB, Assumpção ME, et al. Immature bovine oocyte cryopreservation: comparison of different associations with ethylene glycol, glycerol and dimethylsulfoxide. Anim Reprod Sci. 2007;99(3-4):384-8.

Hurtt AE, Landim-Alvarenga F, Seidel GE, Squires EL. Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws. Theriogenology. 2000;54(1):119-28.

El-Shahat KH, Hammam AH. Effect of different types of cryoprotectants on developmental capacity of vitrified-thawed immature buffalo oocytes. Anim Reprod. 2014;11(4):543-8.

Kim JY, Kinoshita M, Ohnishi M, Fukui Y. Lipid and fatty acid analysis of fresh and frozen-thawed immature and in vitro matured bovine oocytes. Reproduction. 2001;122(1):131-8.

Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod. 1996;54(5):1059-69.

Purohit GN, Meena H, Solanki K. Effects of vitrification on immature and in vitro matured, denuded and cumulus compact goat oocytes and their subsequent fertilization. J Reprod Infertil. 2012;13(1):53-9.

Hammam M, El-Shahat H. Vitrification of immature and mature buffalo oocytes in glycerol solution by a simple method. Proceedings of the XII ISAH Congress on Animal Hygiene. Poland: Warsaw Agricultural University; 2005: 252-5.

Kuwayama M. Evidence-based embryo cryopreservation. J Mam Ova Res. 2005;22:193-7.

Morató R, Izquierdo D, Paramio MT, Mogas T. Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development. Cryobiology. 2008;57(2):137-41.

Succu S, Leoni GG, Bebbere D, Berlinguer F, Mossa F, Bogliolo L, et al. Vitrification devices affect structural and molecular status of in vitro matured ovine oocytes. Mol Reprod Dev. 2007;74(10):1337-44.

Liebermann J, Tucker MJ, Graham JR, Han T, Davis A, Levy MJ. Blastocyst development after vitrification of multipronuclear zygotes using the Flexipet denuding pipette. Reprod Biomed Online. 2002;4(2):146-50.

Vanderhyden BC, Caron PJ, Buccione R, Eppig JJ. Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation. Dev Biol. 1990;140(2):307-17.

Buccione R, Vanderhyden BC, Caron PJ, Eppig JJ. FSH-induced expansion of the mouse cumulus oophorus in vitro is dependent upon a specific factor (s) secreted by the oocyte. Dev Biol. 1990;138(1):16-25.

Elvin JA, Clark AT, Wang P, Wolfman NM, Matzuk MM. Paracrine actions of growth differentiation factor-9 in the mammalian ovary. Mol Endocrinol. 1999;13(6):1035-48.