Effect of cryoprotectants on camel oocytes Vitrification

Document Type : Original Article


1 Zoology and Entomology Department, Faculty of Science, Al-Azhar University (Girls).

2 Animal and Poultry Production Division, Desert Research Center, 11753, Cairo, Egypt.


The objective of this study was to show how two cryoprotectants affect the vitrification rate in immature camel oocytes when used in a separate or a mixed form. As a result, their ability to mature after being warmed was recorded. The cumulus oocytes complexes (COCs) were divided into four groups, control; EG (30 percent ethylene glycol) in vitrification solution (VS); DMSO (30 percent dimethyl sulfoxide); MIX (15 percent EG + 15 percent DMSO) in vitrification solution (VS). COCs were impeded in equilibration solution (ES) for 2 minutes before being transferred to a vitrification solution containing 30% cryoprotecting agent (EG, DMSO, or MIX), 20% FBS, and 0.5 M sucrose in the medium in each vitrification group. Oocytes were put into the open pulled straw (OPS) and subsequently frozen for one hour in liquid nitrogen (LN2). The oocytes were then thawed and warmed for 2.5 minutes at 37°C in four warming solutions (WS) containing varying amounts of sucrose. The findings revealed that the oocyte maturation rate was high. In comparison, DMSO treatment had a better recovery rate, viability, and more negligible damage effect than EG and MIX.


Main Subjects

[1] Elsharnoby H, Kandil OM, Abu-Elnaga HA . Dromedary camel epididymal sperm characteristics at breeding and non-breeding seasons. Al-Azhar Bulletin of Science 2021 ; 32(1-C), 1-9.‏
[2] Miragaya  MH, Chaves  MG , Agüero  A. Reproductive Biotechnology in South American Camelids. Small Rumin. Res. 2006; 61: 299–310.
[3]    Mullen SF, Fahyb GM. Chronologic review of mature oocyte vitrification research in cattle, pigs, and sheep. Theriogenology.  2012; 78: 1709–1719.
[4]    Casillas F, Betancourt M, López A, Juárez-Rojas L, Retana-Márquez S. State of the ART of slow freesing and vitrification of immature, mature oocytes and embryonic stages. Int. J Curr. Res. (2016); 8(07): 34737-34744.
[5] Elsherief A, Adel M , Rashed AS, Bakry S, Mehany A. Intracytoplasmic Sperm Injection (ICSI) with fresh versus cryopreserved testicular sperm in egyptians non-obstructive azoospermic patients. Al-Azhar Bulletin of Science. 2020; 31(2-C), 11-25.‏
[6] Fernandez-Reyez F, Ducolomb Y, Romo  S, Casas E, Salazar Z et al. Viability, maturation and embryo development in vitro of immature porcine and ovine oocytes vitrified in different devices. Cryobiol. 2012; 64(3): 261-266.
[7] Hwang  IS, Hochi S . Recent progress in cryopreservation of bovine oocytes. Bio Med. Res. Int. (2014); 570647.
[8] Sprícigo  JF,   Diógenes  MN,  Leme  LO, Guimarães  AL , Muterlle CV,  Silva  BDM, et al. Effects of different maturation systems on bovine oocyte quality, plasma membrane phospholipid composition and resistance to vitrification and warming. PLoS One 2015; 10(6), e0130164.
[9] Zeron Y, Pearl  M, Borochov A, Arav  A . Kinetic and temporal factors influence chilling injury to germinal vesicle and matured bovine oocyte. Cryobiol. 1999; 38: 35–42.
[10] Succu  S, Leoni  G, Berlinguer  F, Mossa  F,  Galioto  M, Naitana S . Vitrification devices affect structural and molecular status of in vitro matured ovine oocytes. Mol. Reprod. Dev. 2007 ; 74: 1337–44.
[11] Cetin Y,  Bastan  A . Cryopreservation of immature bovine oocytes by vitrification in straws, Anim. Reprod. Sci. 2006; 92: 29–36.
[12] Cha  KY ,  Chung   HM,  Lim   JM ,  Ko  JJ,  Han  SY , Choi  DH,  Yoon  TK . Freezing immature oocytes. Mol. Cell. Endocrinol. 2000; 169: 43–47.
[13] Dinnyes  A , Dai  Y,   Jiang   S, Yang  X . High developmental rates of vitrified bovine oocytes following parthenogenetic activation, IVF, and somatic cell nuclear transfer. Biol. Reprod. 2000; 63: 513–518.
[14] Kartberg AJ, Hambiliki F, Arvidsson T, Stavreus-Evers A, Svalander P. Vitrification with DMSO protects embryo membrane integrity better than solutions without DMSO. Reprod. BioMed.  2008; Online, 17(3): 378-384. www.rbmonline.com/Article/3216 on web 3 July 2008.
 [15] Ruiz  J,  Landeo  L,  Mendoza  J,  Artica  M,  Correa  JE, Silva  M,  Miragaya  M,  Ratto  MH . Vitrification of in vitro mature alpaca oocyte: Effect of ethylene glycol concentration and time of exposure in the equilibration and vitrification solutions. Anim. Reprod. Sci. 2013; 143: 72– 78.
[16] Skidmore JA, Billah M, Allen WR. Investigation of factors affecting pregnancy rate after embryo transfer in the dromedary camel. Reprod. Fertil. Dev.  2002; 14: 109 –116.
 [17] Skidmore JA, Billah M, Loskutoff NM. Developmental competence in vitro and in vivo of cryopreserved, hatched blastocysts from the dromedary camel (Camelus dromedarius). Reprod. Fertil. Dev. 2004; 16: 605–609.
[18] Skidmore JA, Adams GP, Billah M. Synchronisation of ovarian follicular waves in the dromedary camel (Camelus dromedarius). Anim. Reprod. Sci. 2009; 114: 249-255.
[19] Nowshari  MA, Syed  AA,  Saleem  S . Offspring resulting from transfer of cryopreserved embryos in camel (Camel dromedarius). Theriogenology. 2005; 63: 2513–2522.
[20] Aller  JF, Rebuffi  GE,  Cancino  AK . Successful transfer of vitrified llama (Lama glama) embryos. Anim. Reprod. Sci.  2002; 73: 121-127.
[21] El-Badry   DA , Scholkamy   TH,   Darwish  GM .Vitrification of dromedary camel oocytes: Effect of maturation state, equilibration and vitrification time and type of cryoprotectant. J. Egypt Vet. Med. Assoc. 2015 ; 75 (1): 7-21.
[22] Al-Soudy  A,  El-Sayed  A , El-Itriby   HA,  Hussein  EH . Cryopreservation of immature and mature camel (Camelus dromedarius) oocytes by open pulled straw vitrification. Arab J. Biotech. 2016 ;19(2): 1-8.
[23] Madboly  MM,  EL-Sayed  E . Effect of cryoprotectants on the quality of dromedary camel oocytes with particular reference to its ultrastructure under vitrification technique.Egyptian J. Anim. Prod. 2017; 54(3): 245-254.

[24] Fathi  M, Moawad  AR,  Badr  MR. Developmental Potential of Dromedary Camel Oocytes Vitrified at the Germinal Vesicle Stage: Effects of Different Cryoprotectant Combinations and Cryo-Carriers. Reprod. Fert. Dev. 2017; 30(1): 164-164. https://doi.org/10.1071/RDv30n1Ab49

[25] Fathi  M,  Moawad  AR,  Badr   MR . Production of blastocysts following in vitro maturation and fertilization of dromedary camel oocytes vitrified at the germinal vesicle stage. PLoS ONE. 2018; 13(3): e0194602. https://doi.org/10.1371/ journal.pone.0194602
[26] Moawad  M,  Hussein  HA, Abd El-Ghani  M, Darwish G,  Badr  M . Effects of cryoprotectants and cryoprotectant combinations on viability and maturation rates of Camelus dromedarius oocytes vitrified at germinal vesicle stage.Reprod. Dom. Anim.  2019; 54:108–117. https://doi.org/10.1111/rda.13319
[27] Saadeldin  IM,  Moulavi  F, Swelum  AA,  Khorshid  SS, Hamid  HF,  Hosseini  SM . Vitrification of camel oocytes transiently impacts mitochondrial functions without affecting the developmental potential after intracytoplasmic sperm injection and parthenogenetic activation. Environ. Sci. Pol. Res.  2020 ; 27: 44604–44613. https://doi.org/10.1007/s11356-020-11070-x
[28] Russo  R,  Monaco  D,  Rubessa  M, El-Bahrawy  K,  El-Sayed  A, Martino  A, Beneult B,  Ciannarella F,  Dell’Aquila  M, Lacalandra  G,  Uranio  M. Confocal fluorescence assessment of bioenergy/ redox status of dromedary camel (Camelus dromedarius) oocytes before and after in vitro maturation. Reprod. Biol. Endocrinol. 2014 ; 12:16(1): 1-10.
 [29] Ashour, G.; Kamel, A. M.; El-Bahrawy, K. A.; El-Sayed, A. A. and Ghanem, N. (2021): Effect of ascorbic acid supplementation on in vitro production of camel embryos cultured under oxidative stress. J. Camelid Sci., 14 (1): 35-42.
[30] El-sayed  M,  Taha  N,  Hammam  A,  Sawiress F. Effect of antioxidant supplementation on in vitro maturation of Camelus dromedaries oocytes. Nat. Sci. 2015; 13(2):13-17.
[31] Nowshari  MA,  Wernery  U .  2003.  The effect of harvesting technique on efficiency of oocyte collection and their meiotic competence in dromedary camels. Ninteenth meeting A.E.T.E. 12–13 September, Rostock, Germany, P. 200.
[32] El-Shahat   KH,  Hammam AM . Effect of different types of cryoprotectants on developmental capacity of vitrified-thawed immature buffalo oocytes. Anim. Reprod. 2014; 11(4): 543-548.
 [33] Rao  BS,  Mahesh   YU,  Charan  KV,  Suman  K, Sekhar  N,  Shivaji  SEffect of vitrification on meiotic maturation and expression of genes in immature goat cumulus oocyte complexes. Cryobiol. 2012; 64(3); 176-184.
[34] Yoon  TK,  Kim  TJ,  Park  S E, Hong  SW,  Ko  JJ,  Chung  HM  et al . Live births after vitrification of oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil. Steril. 2003;79: 1323–1326.
[35] Gupta  PS,  Nandi  S,  Ravindranatha  BM,  Sarma  PV. Trypan blue staining to differentiate live and dead buffalo oocytes and its effect on embryo development. Buffalo J. 2002;18: 321-329.
[36] Khattab  AK ,  Abu Elnaga  NA, Shedeed  HA,  Kamel  AM,  Abd Rabu   MA,  El-Bahrawy   KA  . Antioxidant supplementation effects on in vitro maturation and fertilization of dromedary camel oocytes. J. Camelid Sci. 2020; 13: 10-21.
[37] Amer   H , Moosa  A . Effect of season and culture media on the competence of dromedary camel oocyte to mature in vitro. Global Veterinaria. 2009; 3(1): 1-8.
[38]  El-Sharawy  ME,  Almadaly  EA,  El-Domany  WB,  Essawy   MM,  Shamiah SM,  El-Shamaa   IS,  Zaghloul  HK . Ultrastructural changes in immature ovine cumulus-oocyte complexes vitrified in conventional and open pulled straws. Small Rum. Res. 2021; 199, 106367.
[39] Ali  R,  Razzaque  W, Sharma  U, Pandey  A,  Hussain  K . Survivability of caprine oocytes vitrified in conventional and open pulled straws. Turkish J. Vet. Anim. Sci.  2014; 38 (6): 707–711.
[40] El-Shalofy, A. S.; Moawadm, A. R.; Darwish, G. M.; Ismail S. T.; Badawy, A. B. and Badr, M. R. (2017): Effect of different vitrification solutions and cryodevices on viability and subsequent development of buffalo oocytes vitrified at the germinal vesicle (GV) stage. Cryobiol., 74:86-92. https://doi.org/10.1016/j.cryobiol.2016. 11.010 PMID: 27908686.
[41] Amiridis  GS, Cseh S . Assisted reproductive technologies in the reproductive management of small ruminants. Anim. Reprod. Sci., 130(3-4): 152-161.
[42]  Moawad, A. R.; Fisher, P.; Zhu, J.; Choi, I.; Polgar, Z. Dinnyes, A. et al. (2012): In vitro fertilization of ovine oocytes vitrified by solid surface vitrification at germinal vesicle stage. Cryobiol. 2012; 65(2): 139-144. https://doi. org/10.1016/j.cryobiol.2012.04.008 PMID: 22579520.
[43] Moawad  AR,  Zhu  J,  Choi  I,  Amarnath  D,  Chen  W,  Campbell  KH . Production of good-quality blastocyst embryos following IVF of ovine oocytes vitrified at the germinal vesicle stage using a cryoloop. Reprod. Fertil. Dev.  2013; 25(8):1204-1215. https://doi.org/10.1071/RD12215 PMID: 23336581.
[44] Shirazi  A,  Naderi  MM,  Hassanpour  H,  Heidari  M,  Borjian  S,  Sarvari  A  et al. The effect of ovine oocyte vitrification on expression of subset of genes involved in epigenetic modifications during oocyte maturation and early embryo development. Theriogenology. 2016; 86(9), 2136-2146.
[45] Moawad  AR,  Xu  B , Tan  SL,  Taketo  T. l-carnitine supplementation during vitrification of mouse germinal vesicle stage-oocytes and their subsequent in vitro maturation improves meiotic spindle configuration and mitochondrial distribution in metaphase II oocytes. Hum. Reprod. 2014 ; 29(10): 2256-2268. https://doi. org/10.1093/humrep/deu201 PMID: 25113843.
[46] El-Shalofy  ASM . Some trials for cryobanking of egyptian buffalo oocytes B.Sc. Thesis, Cairo University. 2016 ; pp: 86, 56.
 [47] Sharma  GT,  Loganathasamy K. Effect of meiotic stages during in vitro maturation on the survival of vitrified-warmed buffalo oocytes, Vet. Res. Commun. 2007; 31: 881–893.
[48] Gomes CM,  Silva CA,  Acevedo  N,  Baracat  E,  Serafini  P,  Smith  GD . Influence of vitrification on mouse metaphase II oocyte spindle dynamics and chromatin alignment. Fertil. Steril. 2008; 90(4):1396-1404. https://doi.org/10.1016/j.fertnstert.2007.08.025 PMID: 18359482.
[49] Yang  BC,  Im  GS,  Chang WK,  Lee  YK,  Oh  SJ,  Jin DI  etal. Survival and in vitro development of immature bovine oocytes cryopreserved by vitrification. Asian Australasian J.Anim. Sci. 2003 ; 16: 23–28. https://doi.org/10.5713/ajas.2003.23
[50] Gautam SK,  Verma V,  Singh  B,  Palta  P,  Singla  SK , Chauhan  MS ,et al  Effect of slow freezing on morphology and developmental competence of buffalo (Bubalus bubalis) immature oocytes., Anim. Reprod. Sci.  2008 ; 105: 311–318.
[51] Sharma  GT,  Dubey  PK,  Chandra  V. Morphological changes, DNA damage and developmental competence of in vitro matured, vitrified- thawed buffalo (Bubalus bubalis) oocytes: A comparative study of two cryoprotectants and two cryodevices. Cryobiol. 2010 ; 60: 315-321.
[52] Otoi  T, Yamamoto  K,  Koyama  N,  Tachikawa  S, Murakami  M,  Kikkawa  Y, et al . Cryopreservation of mature bovine oocytes following centrifugation treatment. Cryobiol.  1997;  34: 36–41.
[53] Ferreira  CR, Saraiva  SA,  Catharino  RR, Garcia  JS,  Gozzo  FC,  Sanvido GB, et al . Single embryo and oocyte lipid fingerprinting by mass spectrometry [S]. J. lipid res. 2010 ; 51(5), 1218-1227.
 [54] Kafi  M, Mesbah  F,  Nili H,  Khalil  A. Chronological and ultrastructural changes in camel (Camelus dromedarius) oocytes during in vitro maturation. Theriogenology. 2005 ;  63: 2458-2470.
[55] Kacinskis  MA, LucciLuque  CM,  Bao SN . Morphometric and ultrastructural characterization of Bosindicus pre antral follicles. Anim. Reprod. Sci. 2005 ; 47: 45-57.
[56] Mondadori  RG, Santin  TR,  Fidelis  AAG,  Porfirio EP, Bao  SN. Buffalo (Bubalus bubalis) preantral follicle population and ultrastructural characterization of antral follicle oocyte. Reprod. Dom. Anim. 2008 ; 45: 33-37.
[57] KhalifaMH . In vitro morphological and molecular assessment of buffalo (Bubalus bubalis) oocytes post vitrification. B.Sc. Thesis, Fac. Agric. Cairo Univ. Cairo, Egypt 2016 ;  p.34.
[58] Men  H,  Monson RL, Rutledge JJ . Effect of meiotic stages and maturation protocols on bovine oocyte’s resistance to cryopreservation. Theriogenology. 2002;  57, 1095–103
[59] Khalili  MA, Shahedi A, Ashourzadeh  S,  Nottola SA, Macchiarelli  G,  Palmerini  M G. Vitrification of human immature oocytes before and after in vitro maturation: a review. J. assisted reprod. gen. 2017 ; 34(11):1413-1426.
[60] Luvoni  GC .Gamete cryopreservation in the domestic cat. Theriogenology 2006 ;  66: 101–111.
[61]  Peris-Frau P,  Soler  AJ,  Iniesta-Cuerda  M,  Martín-Maestro  A,  Sánchez-AjofrínI, Medina-Chávez  DA ,et al . Sperm cryodamage in ruminants: understanding the molecular changes induced by the cryopreservation process to optimize sperm quality. Int. j. mol. Sci. 2020 ; 21(8): 2781.
[62] Le Gal  F,  Massip  A. Cryopreservation of cattle oocytes: effect of meiotic stage, cyclohexamide treatment, and vitrification procedure.Cryobiol. 1999 ; 38: 290-300.
[63] Sharma TO,  Kharche  SD,  Majumdar AC. Vitrification of in vitro matured goat oocytes and the effect on in vitro fertilization. Small Rum. Res.  2006 ; 64: 82-86.
[64] Mahmoud K,  Scholkamy  TH, Ahmed  YF , Seidel Jr  GE, Nawito MF. 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. 2010 ; 45(4), 565-571.   
[65]  Yamada C,  Caetano  IIV, Simoes  R,  Nicacio   AC,  Feitosa  WB,  Assumpcao  ME et al. Immature bovine oocytes cryopreservation: Comparison of different associations with ethylene glycol, glycerol and dimethylsulfoxide. Anim. Reprod. Sci. 2007 ; 99: 384-388.
[66] Szurek  EA,  Eroglu A. Comparison and avoidance of toxicity of penetrating cryoprotectants. PloS one. 2011 ; 6(11), e27604.
[67] Youm  HW,  Lee  JR,  Lee  J,  Jee  BC,  Suh  CS,  Kim  SH . Optimal vitrification protocol for mouse ovarian tissue cryopreservation: effect of cryoprotective agents and in vitro culture on vitrified–warmed ovarian tissue survival. Human reprod.  2014 ; 29(4), 720-730.
[68] Babaei  HI,  Nematollahi-Mahani  SN,  Kheradmand  A,  Ayen E. In vitro survival rate of bovine oocytes following vitrification in glass capillary micropipette (GCM).Iranian J Vet. Res., University of Shiraz. 2006,  7(1): 1-14.
[69] Kim  DH,  Park  HS,  Kim  SW,  Hwang  IS, Yang  BC,  Im  GS ,et al. Vitrification of immature bovine oocytes by the microdrop method. J. Reprod. Dev. 2007 ; 53: 843–851.
[70] Wani NA, Misra  AK, Maurya SN. Maturation rates of vitrified-thawed immature buffalo (Bubalus bubalis) oocytes: Effect of different types of cryoprotectants. Anim. Reprod. Sci. 2004 ; 84: 327–335.
[71] Kharche  SD,  Sharma GT,  Majumdar AC. In vitro maturation and fertilization of goat oocytes vitrified at the germinal vesicle stage. Small Rum. Res. 2005 ; 57: 81–84.
[72] Tharasanit T, Colenbrander B, Stout TA. Effect of maturation stage at cryopreservation on post-thaw cytoskeleton quality and fertilizability of equine oocytes. Mol. Reprod. Dev. 2006 73: 627–637.
[73] Apparicio M,  Ruggeri  E,  Luvoni  GC. Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification. Reprod. Domest. Anim. 2013 ; 48(2):240-244. https://doi.org/10.1111/j.1439-0531.2012.02138.x PMID: 22775765.
[74] Isachenko V,  Montag  M,  Isachenko E, Dessole  S,  Nawroth  F,  van der Ven  H. Aseptic vitrification of human germinal vesicle oocytes using dimethyl sulfoxide as a cryoprotectant. Fertil. Steril.  2006 ; 85 (3): 741-748. https://doi.org/10.1016/j.fertnstert.2005.08.047 PMID: 16500347.
[75] Shahedi A,  Hosseini  A,  Khalili  MA,  Norouzian  M,  Salehi  M, Piriaei  A,  et al. The effect of vitrification on ultrastructure of human in vitro matured germinal vesicle oocytes. Eur. J. Obstet. Gynecol. Reprod. Biol. 2013; 167(1): 69-75. https://doi.org/10.1016/j.ejogrb.2012.11.006 PMID: 23260597.
[76] Jin  B,  Kawai  Y,  Hara  T,  Takeda  S, Seki  S,  Nakata  Y, et al . Pathway for the movement of water and cryoprotectants in bovine oocytes and embryos. Biol. Reprod.  2011 ; 85: 834–847.