Effect of Polyvinylpyrrolidone on Vitrification of Buffalo (Bubalus bubalis) Oocytes


  • Jannatul Bari Department of Animal Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
  • M.N. Islam Department of Animal Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
  • Md Hasanur Alam Department of Animal Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
  • A. Khatun Department of Animal Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
  • M.A. Hashem Department of Animal Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
  • M. Moniruzzaman Department of Animal Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh




Buffalo, cryopreservation, in vitro maturation, oocytes, PVP.


Vitrification, a method of rapid cooling, is an alternate cryopreservation method of oocytes and embryos. The present study was aimed to examine the effect of polyvinylpyrrolidone (PVP) on vitrification of buffalo oocytes. Cumulus oocyte complexes (COCs) with fully grown oocytes (120-130 µm in diameter) were aspirated from slaughtered buffalo ovaries for vitrification. COCs were treated with equilibration solution at room temperature for 5 min and then transferred to a vitrification solution for 1 min. Then the COCs were submerged into liquid nitrogen (-196̊C) for a while using cryotops. The COCs were thawed, diluted, and washed in a washing solution for 5 min, respectively. Vitrified oocytes were incubated for in vitro maturation (IVM) at 38.5̊C under an atmosphere of 5% CO2 in the air for 24 hrs. Cumulus cells surrounding the oocytes were removed mechanically, oocytes were fixed in acetic acid and ethanol, and stained with aceto-orcein to examine the meiotic stages of oocytes. The numbers of morphologically normal oocytes after vitrification were higher in 5% PVP than 0 and 10% PVP groups. A proportion of oocytes treated with 5% PVP reached the metaphase II (MII) stage while none of the oocytes from 0% and 10% PVP groupsdeveloped beyond anaphase I and metaphase I (MI) stages, respectively. These results show that PVP can be used as a cryoprotectant for the vitrification of buffalo oocytes.


Rahman SR, Islam MN, Harun-ur-Rashid M, Sarker NR, Siddiki MS, Islam MZ, Islam MA. Buffalo milk yield, quality, and marketing in different agro-climatic districts of Bangladesh. J Buff Sci 2019; 8(3): 62-67. https://doi.org/10.6000/1927-520X.2019.08.03.2 DOI: https://doi.org/10.6000/1927-520X.2019.08.03.2

Vencato J, Badon T, Bedin S, Cogo P, Simonato M, Stelletta C. Biochemical and fatty acids composition of water buffalo (Bubalus bubalis) follicular fluid. J Buff Sci 2014; 3(3): 82-88. https://doi.org/10.6000/1927-520X.2014.03.03.3 DOI: https://doi.org/10.6000/1927-520X.2014.03.03.3

Totey SM, Pawshe CH, Singh GP. In vitro maturation and fertilization of buffalo oocytes (Bubalus bubalis) effects of media, hormones and sera. Theriogenology 1993; 39: 1153-1171. https://doi.org/10.1016/0093-691X(93)90014-V DOI: https://doi.org/10.1016/0093-691X(93)90014-V

Gasparrini B. In vitro embryo production in buffalo species: state of the art. Theriogenology 2002; 57: 237-256. https://doi.org/10.1016/S0093-691X(01)00669-0 DOI: https://doi.org/10.1016/S0093-691X(01)00669-0

Berdugo-Gutiérrez JA, Echeverri JJ, Tarazona AM, López-Herrera A. Differences of the fertility fotential between buffaloes (Bubalus bubalis) and cattle (Bos indicus): the role of antimullerian hormone (AMH). J Buff Sci 2017; 6: 74-80. https://doi.org/10.6000/1927-520X.2017.06.03.2 DOI: https://doi.org/10.6000/1927-520X.2017.06.03.2

Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at -196 ̊C by vitrification. Nature 1985; 313: 573-575. https://doi.org/10.1038/313573a0 DOI: https://doi.org/10.1038/313573a0

Abdel‐Ghani MA, El‐Sherry TM, Abdelhafeez HH. Effect of growth differentiation factor‐9 (GDF‐9) on the progression of buffalo follicles in vitrified-warmed ovarian tissues. Reprod Dom Anim 2016; 51(5): 795-803. https://doi.org/10.1111/rda.12753 DOI: https://doi.org/10.1111/rda.12753

Hufana-Duran D, Pedro PB, Venturina HV, Hufana RD, Salazar AL, Duran PG, Cruz LC. 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): 1429-1439. https://doi.org/10.1016/j.theriogenology.2003.08.011 DOI: https://doi.org/10.1016/j.theriogenology.2003.08.011

BonDurant RH, Drost M, Zambrano-Varon J, Campanile G, Gasparrini B, Zicarelli L. Importation of in vitro-produced Bubalus bubalis embryos from Italy into the United States: A case report. Theriogenology 2007; 68(3): 454-460. https://doi.org/10.1016/j.theriogenology.2007.05.049 DOI: https://doi.org/10.1016/j.theriogenology.2007.05.049

Mahesh YU, Gibence HR, Shivaji S, Rao BS. Effect of different cryo-devices on in vitro maturation and development of vitrified-warmed immature buffalo oocytes. Cryobiology 2017; 75: 106-116. https://doi.org/10.1016/j.cryobiol.2017.01.004 DOI: https://doi.org/10.1016/j.cryobiol.2017.01.004

Parnpai R, Liang Y, Ketudat-Cairns M, Somfai T, Nagai T. Vitrification of buffalo oocytes and embryos. Theriogenology 2016; 86: 214-220. https://doi.org/10.1016/j.theriogenology.2016.04.034 DOI: https://doi.org/10.1016/j.theriogenology.2016.04.034

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: 219-229. https://doi.org/10.1006/cryo.1997.2043 DOI: https://doi.org/10.1006/cryo.1997.2043

Purohit GN, Meena V, Solanki K. Morphological survival and subsequent in vitro maturation of denuded and cumulus compact bubaline oocytes cryopreserved by ultra rapid cooling. J Buff Sci 2012; 1(1): 78-83. https://doi.org/10.6000/1927-520X.2012.01.01.14 DOI: https://doi.org/10.6000/1927-520X.2012.01.01.14

Nakagata N. High survival rate of unfertilized mouse oocytes after vitrification. J Reprod Fertil 1989; 87: 479-483. https://doi.org/10.1530/jrf.0.0870479 DOI: https://doi.org/10.1530/jrf.0.0870479

Marques CC, Santos-Silva C, Rodrigues C, Matos JE, Moura T, Baptista MC, Horta AE, Bessa RJ, Alves SP, Soveral G, Pereira RM. Bovine oocyte membrane permeability and cryosurvival: Effects of different cryoprotectants and calcium in the vitrification media. Cryobiology 2018; 81: 4-11. https://doi.org/10.1016/j.cryobiol.2018.03.003 DOI: https://doi.org/10.1016/j.cryobiol.2018.03.003

El-Shalofy AS, Moawad AR, Darwish GM, Ismail ST, Badawy AB, 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. https://doi.org/10.1016/j.cryobiol.2016.11.010 DOI: https://doi.org/10.1016/j.cryobiol.2016.11.010

Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: The Cryotop method. Theriogenology 2007; 67: 73-80. https://doi.org/10.1016/j.theriogenology.2006.09.014 DOI: https://doi.org/10.1016/j.theriogenology.2006.09.014

Wright DL, Eroglu A, Toner M, Toth TL. Use of sugars in cryopreserving human oocytes. Reprod Biomed Online 2004; 9: 179-186. https://doi.org/10.1016/S1472-6483(10)62127-X DOI: https://doi.org/10.1016/S1472-6483(10)62127-X

Huang J, Li Q, Zhao R, Li W, Han Z, Chen X, Xiao B, Wu S, Jiang Z, Hu J, Liu L. Effect of sugars on maturation rate of vitrified-thawed immature porcine oocytes. Anim Reprod Sci 2008; 106: 25-35. https://doi.org/10.1016/j.anireprosci.2007.03.023 DOI: https://doi.org/10.1016/j.anireprosci.2007.03.023

Mukaida T. Blastocyst cryopreservation: ultrarapid vitrification using cryoloop technique. Reprod Biomed Online 2002; 6: 221-225. https://doi.org/10.1016/S1472-6483(10)61713-0 DOI: https://doi.org/10.1016/S1472-6483(10)61713-0

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: 831-842. https://doi.org/10.1016/j.theriogenology.2003.06.002 DOI: https://doi.org/10.1016/j.theriogenology.2003.06.002

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: 91-94. https://doi.org/10.1016/S0011-2240(02)00109-8 DOI: https://doi.org/10.1016/S0011-2240(02)00109-8

Vincent C, Pickering SJ, Johnson MH, Quick J. Dimethyl sulfoxide affects the organization of microfilaments in the mouse oocyte. Mol Reprod Dev 1990; 26: 227-235. https://doi.org/10.1002/mrd.1080260306 DOI: https://doi.org/10.1002/mrd.1080260306

Haaf F, Sanner A, Straub F. Polymers of N-vinylpyrrolidone: synthesis, characterization and uses. Polymer Journal. 1985; 17(1): 143. https://doi.org/10.1295/polymj.17.143 DOI: https://doi.org/10.1295/polymj.17.143

Steirteghem AC, Nagy Joris H, Liu J, Staessen C, Smitz J, Wisanto A, Devroey P. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1993; 8: 1061-1066. https://doi.org/10.1093/oxfordjournals.humrep.a138192 DOI: https://doi.org/10.1093/oxfordjournals.humrep.a138192

Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biol Reprod 1995; 52: 709-720. https://doi.org/10.1095/biolreprod52.4.709 DOI: https://doi.org/10.1095/biolreprod52.4.709

Hamano K, Li X, Funauchi K, Furudate M, Minato Y. Gender preselection in cattle with intracytoplasmically injected, flow cytometrically sorted sperm heads. Biol Reprod 1999; 60: 1194-1197. https://doi.org/10.1095/biolreprod60.5.1194 DOI: https://doi.org/10.1095/biolreprod60.5.1194

Catt SL, Catt JW, Gomez MC, Maxwell WM, Evans G. Birth of a male lamb derived from an in vitro matured oocyte fertilised by intracytoplasmic injection of a single presumptive male sperm. Vet Rec 1996; 139: 494-495. https://doi.org/10.1136/vr.139.20.494 DOI: https://doi.org/10.1136/vr.139.20.494

Cochran R, Meintjes M, Reggio B, Hylan D, Carter J, Pinto C, Paccamonti D, Godke RA. Live foals produced from sperm‐injected oocytes derived from pregnant mares. J Equine Vet Sci 1998; 18: 736-740. https://doi.org/10.1016/S0737-0806(98)80504-2 DOI: https://doi.org/10.1016/S0737-0806(98)80504-2

Martin MJ. Development of in vivo matured porcine oocytes following intracytoplasmic sperm injection. Biol Reprod 2000; 63: 109-112. https://doi.org/10.1095/biolreprod63.1.109 DOI: https://doi.org/10.1095/biolreprod63.1.109

Hirao Y, Itoh T, Shimizu M, Iga K, Aoyagi K, Kobayashi M, Kacchi M, Hoshi H, Takenouchi N. In vitro growth and development of bovine oocyte‐granulosa cell complexes on the flat substratum: effects of high polyvinylpyrrolidone concentration in culture medium. Biol Reprod 2004; 70: 83-91. https://doi.org/10.1095/biolreprod.103.021238 DOI: https://doi.org/10.1095/biolreprod.103.021238

Chung JT, Tosca L, Huang TH, Xu L, Niwa K, Chian RC. Effect of polyvinylpyrrolidone on bovine oocyte maturation in vitro and subsequent fertilization and embryonic development. Reprod Biomed Online 2007; 15: 198-207. https://doi.org/10.1016/S1472-6483(10)60709-2 DOI: https://doi.org/10.1016/S1472-6483(10)60709-2

Yang BC, Im GS, Chang WK, Lee YK, Oh SJ, Jin DI, Im KS, Lee CK. Survival and in vitro development of immature bovine oocytes cryopreserved by vitrification. Asian-australas J Anim Sci 2002; 16(1): 23–28. https://doi.org/10.5713/ajas.2003.23 DOI: https://doi.org/10.5713/ajas.2003.23

Titterington JL, Robinson J, Killick SR, Hay DM. Fertilization and early embryology: Synthetic and biological macromolecules: protection of mouse embryos during cryopreservation by vitrification. Hum Reprod 1995; 10: 649-653. https://doi.org/10.1093/oxfordjournals.humrep.a136004 DOI: https://doi.org/10.1093/oxfordjournals.humrep.a136004

Wang Y, Okitsu O, Zhao XM, Sun Y, Di W, Chian RC. The effect of minimal concentration of ethylene glycol (EG) combined with polyvinylpyrrolidone (PVP) on mouse oocyte survival and subsequent embryonic development following vitrification. J Assist Reprod Genet 2013; 31: 55-63. https://doi.org/10.1007/s10815-013-0136-5 DOI: https://doi.org/10.1007/s10815-013-0136-5

Gupta MK, Uhm SJ, Lee HT. Cryopreservation of immature and in vitro matured porcine oocytes by solid surface vitrification. Theriogenology 2007; 67(2): 238-248. https://doi.org/10.1016/j.theriogenology.2006.07.015 DOI: https://doi.org/10.1016/j.theriogenology.2006.07.015

Moniruzzaman M, Bao RM, Taketsuru H, Miyano T. Development of vitrified porcine primordial follicles in xenografts. Theriogenology 2009; 72: 280-288. https://doi.org/10.1016/j.theriogenology.2009.01.024 DOI: https://doi.org/10.1016/j.theriogenology.2009.01.024

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: 817-827. https://doi.org/10.1016/j.theriogenology.2010.04.006 DOI: https://doi.org/10.1016/j.theriogenology.2010.04.006

Islam MN, Md Hasanur Alam, Khatun A, Akter I, Modak AK, Hashem MA, Moniruzzaman M. Effects of stem cell factor on in vitro growth of buffalo oocytes. Theriogenology 2020; 142: 114-119. https://doi.org/10.1016/j.theriogenology.2019.09.044 DOI: https://doi.org/10.1016/j.theriogenology.2019.09.044

Maruska DV, Lorraine Leibfried M, 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. https://doi.org/10.1095/biolreprod31.1.1 DOI: https://doi.org/10.1095/biolreprod31.1.1

Motlik J, Johnsen KHH, Fulka J. Breakdown of the germinal vesicle in bovine oocytes cultivated in vitro. J Exp Zool 1978; 205: 377-83. https://doi.org/10.1002/jez.1402050306 DOI: https://doi.org/10.1002/jez.1402050306

Kuleshova LL, Shaw JM, Trounson AO. Studies on replacing most of the penetrating cryoprotectant by polymers for embryo cryopreservation. Cryobiology 2001; 43: 21-31. https://doi.org/10.1006/cryo.2001.2335 DOI: https://doi.org/10.1006/cryo.2001.2335

Suzuki T, Boediono A, Takagi M, Saha S, Sumantri C. Fertilization and development of frozen-thawed germinal vesicle bovine oocytes by a one-step dilution method in vitro. Cryobiology 1996; 33: 515-524. https://doi.org/10.1006/cryo.1996.0055 DOI: https://doi.org/10.1006/cryo.1996.0055

Checura CM, Seidel GE Jr. Effect of macromolecules in solutions for vitrification of mature bovine oocytes. Theriogenology 2007; 67: 919-930. https://doi.org/10.1016/j.theriogenology.2006.09.044 DOI: https://doi.org/10.1016/j.theriogenology.2006.09.044

Kim C, Yong H, Lee G, Cho J. Effect of the polyvinylpyrrolidone concentration of cryoprotectant on mouse embryo development and production of pups: 7.5% of PVP is beneficial for in vitro and in vivo development of frozen-thawed mouse embryos. J Reprod Dev 2008; 54: 250-253. https://doi.org/10.1262/jrd.19185 DOI: https://doi.org/10.1262/jrd.19185

Fahy GM, Lilley TH, Linsdell H, Douglas MS, Meryman HT. Cryoprotectant toxicity and cryoprotectant toxicity reduction in search of molecular mechanisms. Cryobiology 1990; 27: 247-268. https://doi.org/10.1016/0011-2240(90)90025-Y DOI: https://doi.org/10.1016/0011-2240(90)90025-Y

Maro B, Howlett SK, Houliston E. Cytoskeletal dynamics in the mouse egg. J Cell Sci Suppl 1986; 5: 343-359. https://doi.org/10.1242/jcs.1986.Supplement_5.22 DOI: https://doi.org/10.1242/jcs.1986.Supplement_5.22

Johnson MH, Pickering SJ. The effect of dimethyl sulfoxyde on the microtubular system of the mouse oocyte. Development 1987; 100: 313-324. DOI: https://doi.org/10.1242/dev.100.2.313

Magistrini M, Szöllösi D. Effects of cold and of isopropyl-N-phenylcarbamate on the second meiotic spindle of mouse oocytes. Eur J Cell Biol 1980; 22: 699-707.

Pickering SJ, Johnson MH. The influence of cooling on the organization of the meiotic spindle of the mouse oocyte. Hum Reprod 1987; 2: 207-216. https://doi.org/10.1093/oxfordjournals.humrep.a136516 DOI: https://doi.org/10.1093/oxfordjournals.humrep.a136516

Lee James C, and Serge N Timasheff. In vitro reconstitution of calf brain microtubules: effects of solution variables. Biochemistry 1977; 16: 1754-1764. https://doi.org/10.1021/bi00627a037 DOI: https://doi.org/10.1021/bi00627a037

Algaier Joseph and Richard H. Himes. The effects of dimethyl sulfoxide on the kinetics of tubulin assembly. Biochim Biophys Acta 1988; 954: 235-243. https://doi.org/10.1016/0167-4838(88)90078-7 DOI: https://doi.org/10.1016/0167-4838(88)90078-7

Dumoulin JC, Bergers-Janssen JM, Pieters MH, Enginsu ME, Geraedts JP, Evers JL. The protective effects of polymers in the cryopreservation of human and mouse zonae pellucidae and embryos. Fertil Steril 1994; 62: 793-798. https://doi.org/10.1016/S0015-0282(16)57006-X

Liang YY, Srirattana K, Phermthai T, Somfai T, Nagai T, Parnpai R. Effects of vitrification cryoprotectant treatment and cooling method on the viability and development of buffalo oocytes after intracytoplasmic sperm injection. Cryobiology 2012; 65(2): 151-156. https://doi.org/10.1016/j.cryobiol.2012.04.006 DOI: https://doi.org/10.1016/j.cryobiol.2012.04.006

Whittingham DG. Survival of mouse embryos after freezing and thawing. Nature 1971; 233(5315): 125-126. https://doi.org/10.1038/233125a0 DOI: https://doi.org/10.1038/233125a0

Dumoulin JC, Bergers-Janssen JM, Pieters MH, Enginsu ME, Geraedts JP, Evers JL. The protective effects of polymers in the cryopreservation of human and mouse zonae pellucidae and embryos. Fertil Steril 1994; 62(4): 793-798. https://doi.org/10.1016/S0015-0282(16)57006-X DOI: https://doi.org/10.1016/S0015-0282(16)57006-X

Friedler S, Giudice LC, Lamb EJ. Cryopreservation of embryos and ova. Fertil Steril 1988; 49: 743-64. https://doi.org/10.1016/S0015-0282(16)59879-3 DOI: https://doi.org/10.1016/S0015-0282(16)59879-3




How to Cite

Bari, J. ., Islam, M. ., Alam, M. H. ., Khatun, . A. ., Hashem, M. ., & Moniruzzaman, M. . (2020). Effect of Polyvinylpyrrolidone on Vitrification of Buffalo (Bubalus bubalis) Oocytes. Journal of Buffalo Science, 9, 152–158. https://doi.org/10.6000/1927-520X.2020.09.16