Colección, vitrificación y transferencia post-calentamiento de embriones equinos producidos in vivo: una revisión de literatura


Palabras clave:

soluciones liquidas, colección de embriones, vitrificación, calentamiento, transferencia embrionaria, yeguas, burras


Embriones equinos pequeños, iguales o menores a trescientos micrometros de diámetro (≤ 300 µmØ) son obtenidos antes del día siete post ovulación (PO). Embriones grandes, mayores a trescientos micrometros de diámetro (>300 µmØ) son recuperados durante o después del día siete PO. No existen experimentos que prueben hasta que punto los medios de lavado y de colección embrionaria influencian la sobrevivencia post vitrificación. Mas conocimiento es necesario acerca de las mezclas de criopreservadores; penetrantes o no penetrantes y el efecto que ejercen sobre el índice de sobrevivencia embrionaria post vitrificación y el índice de gestación (ISEPVIG). El tipo de porta embrión, el tamaño del embrión y el volumen de la solución vitrificante varían entre embriones pequeños y grandes. Un índice de transferencia de temperatura (ITT) elevado optimiza en ISEPVIG. El tipo de soluciones de mantenimiento y transferencia utilizadas durante la post-vitrificación es un área escasamente explorada. El propósito de este estudio es proveer información para ayudar en la adaptación de protocolos de vitrificación dependiendo en el tamaño del embrión. Un objetivo adicional es facilitar el acceso a información acerca de soluciones empleadas antes y durante la vitrificación; así como las soluciones usadas durante las etapas de calentamiento y transferencia embrionaria.





ALLEN WR, Kydd J, Boyle MS, Antczak DF. 1985. Between‐species transfer of horse and donkey embryos: A valuable research tool. Equine Veterinary Journal. 17(S3):53-62.

ALVARENGA MA, Alvarenga FC, Meira C. 1993. Modifications in the technique used to recover equine embryos. Equine Veterinary Journal. 25(S15):111-112.

BARFIELD JP, Sanchez R, Squires EL, Seidel GE. 2008. 60 Vitrification and conventional cryopreservation of equine embryos. Reproduction, Fertility and Development. 21(1):130-130.

BARFIELD JP, McCue PM, Squires EL, Seidel Jr GE. 2009. Effect of dehydration prior to cryopreservation of large equine embryos. Cryobiology. 59(1):36-41.

BATTUT I, Colchen S, Fieni F, Tainturier D, Bruyas JF. 1997. Success rates when attempting to nonsurgically collect equine embryos at 144, 156 or 168 hours after ovulation. Equine Veterinary Journal. 29(S25):60-62.

BETTERIDGE KJ, Eaglesome MD, Mitchell D, Flood PF, Beriault R. 1982. Development of horse embryos up to twenty two days after ovulation: observations on fresh specimens. Journal of Anatomy. 135(Pt1):191-209.

BOTTREL M, Fortes T, Ortiz I, Hidalgo M, Dorado J. 2017. Short communication: Establishment and maintenance of donkey-in-mule pregnancy after embryo transfer in a non-cycling mule treated with oestradiol benzoate and long-acting progesterone. Spanish Journal of Agricultural Research. 15(4):1-4.

BOTTREL M, Ortiz I, Pereira B, Díaz-Jiménez M, Hidalgo M, Consuegra C, Morató R, Mogas T, Dorado J. 2019. Cryopreservation of donkey embryos by the cryotop method: Effect of developmental stage, embryo quality, diameter and age of embryos. Theriogenology. 125:242-248.

BOTTREL M, Mogas T, Pereira B, Ortiz I, Díaz-Jiménez M, Consuegra C, Hidalgo M, Morató R, Dorado J. 2020a. The cryoprotective effect of Ficoll 70 on the post-warming survival and quality of Cryotop-vitrified donkey embryos. Theriogenology. 148:180-185.

BOTTREL M, Hidalgo M, Mogas T, Pereira B, Ortiz I, Díaz-Jiménez M, Consuegra C, Morató R, Dorado J. 2020b. One-step warming does not affect the in vitro viability and cryosurvival of cryotop-vitrified donkey embryos. Theriogenology. 152:47-52.

CAMILLO F, Vannozzi I, Rota A, Di Luzio B, Romagnoli S, Aria G, Allen WR. 2003. Successful non‐surgical transfer of horse embryos to mule recipients. Reproduction in domestic animals. 38(5):380-385.

CAMILLO F, Panzani D, Scollo C, Rota A, Crisci A, Vannozzi I, Balbo S. 2010. Embryo recovery rate and recipients' pregnancy rate after nonsurgical embryo transfer in donkeys. Theriogenology. 73(7):959-965.

CAMPOS-CHILLON LF, Suh TK, Barcelo-Fimbres M, Seidel Jr GE, Carnevale EM. 2009. Vitrification of early-stage bovine and equine embryos. Theriogenology. 71(2):349-354.

CARNEVALE EM, Ramirez RJ, Squires EL, Alvarenga MA, Vanderwall DK, McCue PM. 2000. Factors affecting pregnancy rates and early embryonic death after equine embryo transfer. Theriogenology. 54(6):965-979.

CHOI YH, Gustafson-Seabury A, Velez IC, Hartman DL, Bliss S, Riera FL, Roldán JE, Chowdhary B, Hinrichs K. 2010. Viability of equine embryos after puncture of the capsule and biopsy for preimplantation genetic diagnosis. Reproduction. 140(6):893-902.

CHOI YH, Velez IC, Riera FL, Roldán JE, Hartman DL, Bliss SB, Blanchard TL, Hayden SS, Hinrichs K. 2011. Successful cryopreservation of expanded equine blastocysts. Theriogenology. 76(1):143-152.

DERBALA MK, Abdou MS. 2017. Transfer of Large Equine Embryos in Arabian Mares. ARC Journal of Animal and Veterinary Sciences. 3(4):19-24.

DIAZ F, Bondiolli K, Paccamonti D, Gentry GT. 2016. Cryopreservation of Day 8 equine embryos after blastocyst micromanipulation and vitrification. Theriogenology. 85(5):894-903.

DORADO J, Bottrel M, Ortiz I, Díaz-Jiménez M, Pereira B, Consuegra C, Carrasco JJ, Gómez-Arrones V, Domingo A, Hidalgo M. 2020. Factors Affecting Embryo Recovery Rate, Quality, and Diameter in Andalusian Donkey Jennies. Animals. 10(11):1967.

ELDRIDGE-PANUSKA WD, di Brienza VC, Seidel Jr GE, Squires EL, Carnevale EM. 2005. Establishment of pregnancies after serial dilution or direct transfer by vitrified equine embryos. Theriogenology. 63(5):1308-1319.

FANELLI D, Panzani D, Rota A, Tesi M, Camillo F, Bollwein H, Herrera C. 2020. Cryopreservation of donkey embryos: Comparison of embryo survival rate after in vitro culture between conventional freezing and vitrification. Theriogenology. 154:11-16.

FERRIS RA, McCue PM, Trundell DA, Morrissey JK, Barfield JP. 2016. Vitrification of large equine embryos following manual or micromanipulator-assisted blastocoele collapse. Journal of Equine Veterinary Science. 100(41):64-65.

FREEMAN DA, Weber JA, Geary RT, Woods GL. 1991. Time of embryo transport through the mare oviduct. Theriogenology. 36(5):823-830.

GUIGNOT F, Reigner F, Perreau C, Tartarin P, Babilliot JM, Bed'hom B, Vidament M, Mermillod P, Duchamp G. 2015. Preimplantation genetic diagnosis in Welsh pony embryos after biopsy and cryopreservation. Journal of Animal Science. 93(11):5222-5231.

GUIGNOT F, Blard T, Barrière P, Gasgogne T, Gaude Y, Yvon JM, Mermillod P, Reigner F. 2016. Easy, quick and cheap technique to cryopreserve Welsh B pony blastocyst. Journal of Equine Veterinary Science. 100(41):53.

HENDRIKS WK, Stout TAE. 2010. Slow freezing is less damaging to equine embryos than is vitrification. Animal Reproduction Science. 121:279-280.

HENDRIKS WK, Roelen BAJ, Colenbrander B, Stout TAE. 2015. Cellular damage suffered by equine embryos after exposure to cryoprotectants or cryopreservation by slow‐freezing or vitrification. Equine veterinary journal. 47(6):701-707.

HERRERA C. 2021. Vitrification of Equine In Vivo-Derived Embryos After Blastocoel Aspiration. In: Wolkers WF, Oldenhof H. (eds) Cryopreservation and Freeze-Drying Protocols. Methods in Molecular Biology, vol 2180. Humana, New York, NY.

HINRICHS K. 1990. Work in progress: A simple technique that may improve the rate of embryo recovery on uterine flushing in mares. Theriogenology. 33(5):937-942.

HINRICHS K, Choi YH. 2012. Equine embryo biopsy, genetic testing, and cryopreservation. Journal of Equine Veterinary Science. 32(7):390-396.

HIRAOKA K, Kitamura S. 2015. Clinical efficiency of Piezo-ICSI using micropipettes with a wall thickness of 0.625 μm. Journal of assisted reproduction and genetics. 32(12):1827-1833.

HOCHI S, Fujimoto T, Oguri N. 1995. Large equine blastocysts are damaged by vitrification procedures. Reproduction, fertility and development. 7(1):113-117.

HUDSON J, McCue PM, Carnevale EM, Welch S, Squires EL. 2006. The effects of cooling and vitrification of embryos from mares treated with equine follicle-stimulating hormone on pregnancy rates after nonsurgical transfer. Journal of Equine Veterinary Science. 26(2):51-54.

IULIANO MF, Squires EL, Cook VM. 1985. Effect of age of equine embryos and method of transfer on pregnancy rate. Journal of Animal Science. 60(1):258-263.

KINGMA SG, Thibault ME, Betteridge KJ, Schlaf M, Gartley CJ, Chenier TS. 2011. Permeability of the equine embryonic capsule to ethylene glycol and glycerol in vitro. Theriogenology. 76(8):1540-1551.

LANDIM E ALVARENGA FC, Alvarenga MA, Meira C. 1993. Transmission electron microscopy of equine embryos cryopreserved by different methods. Equine Veterinary Journal. 25(S15):67-70.

LEGRAND E, Krawiecki JM, Tainturier D, Corniere P, Delajarraud H, Bruyas JF. 2001. Does the embryonic capsule impede the freezing of equine embryos? EQUINE EMBRYO TRANSFER. 62.

MACLELLAN LJ, Carnevale EM, Coutinho da Silva MA, McCue PM, Seidel Jr GE, Squires EL. 2002. Cryopreservation of small and large equine embryos pre-treated with cytochalasin-B and/or trypsin. Theriogenology. 58(2-4):717-720.

MCCUE PM, Ferris RA, Lindholm AR, DeLuca CA. 2010. Embryo recovery procedures and collection success: results of 492 embryo-flush attempts. Proceedings of the annual convention of the American Association of Equine Practitioners. December (56):318-321.

MCKINNON AO, Squires EL. 1988. Equine embryo transfer. Veterinary Clinics of North America. Equine Practice. 4(2):305-333.

MEADOWS S, Lisa H, Welsh C. 2000. Factors affecting embryo recovery, embryo development and pregnancy rate in a commercial embryo transfer programme. Proceedings of the 1st European Equine Gamete Group, Havemeyer Foundation Monograph Series. 1:61-2.

MOUSSA M, Bersinger I, Doligez P, Guignot F, Duchamp G, Vidament M, Mermillod P, Bruyas JF. 2005. In vitro comparisons of two cryopreservation techniques for equine embryos: Slow-cooling and open pulled straw (OPS) vitrification. Theriogenology. 64(7):1619-1632.

OBERSTEIN N, O'donovan MK, Bruemmer JE, Seidel Jr GE, Carnevale EM, Squires EL. 2001. Cryopreservation of equine embryos by open pulled straw, cryoloop, or conventional slow cooling methods. Theriogenology. 55(2):607-613.

OGURI N, Tsutsumi Y. 1974. Non-surgical egg transfer in mares. Reproduction. 41(2): 313-320.

OTTMANN L, Reigner F, Allamellou JM, Magistrini M, Guignot F. 2018. Preimplantation Genetic Diagnosis in Donkey Embryos After Biopsy. Journal of Equine Veterinary Science. 66:195.

PANZANI D, Rota A, Vannozzi I, Kindahl H, Govoni N, Camillo F. 2006. Cervical catheterization is not responsible for the low pregnancy rate following transcervical embryo transfer in donkeys. Animal Reproduction Science. 94(1-4):370-373.

PANZANI D, Rota A, Crisci A, Kindahl H, Govoni N, Camillo F. 2012a. Embryo quality and transcervical technique are not the limiting factors in donkey embryo transfer outcome. Theriogenology. 77(3):563-569.

PANZANI D, Rota A, Romano C, Pratelli G, Sabatini C, Camillo F. 2012b. Birth of the first donkey foals after transfer of vitrified embryos. Journal of Equine Veterinary Science. 32(7):419.

PANZANI D, Vannozzi I, Bocci C, Rota A, Tesi M, Camillo F. 2016. In vitro evaluation by DAPI staining of fresh, cooled and vitrified donkey embryos. Journal of Equine Veterinary Science. 41:71-71.

PEÑA-ALFARO CE, Barros LO, Carneiro GF, Gastal MO, Gastal EL. 2014. Embryo transfer in Pega donkeys (Equus asinus) in Brazil. Journal of Equine Veterinary Science. 34(1):185.

PÉREZ-MARÍN CC, Vizuete G, Galisteo JJ. 2017. Embryo recovery results in Hispano-Arabe horse and Spanish donkey breeds. Livestock Science. 206:76-81.

PÉREZ‐MARÍN CC, Vizuete G, Vazquez‐Martinez R, Galisteo JJ. 2018. Comparison of different cryopreservation methods for horse and donkey embryos. Equine Veterinary Journal. 50(3):398-404.

ROSER JF, Etcharren MV, Miragaya MH, Mutto A, Colgin M, Losinno L, Ross PJ. 2020. Superovulation, embryo recovery, and pregnancy rates from seasonally anovulatory donor mares treated with recombinant equine FSH (reFSH). Theriogenology. 142:291-295.

SÁNCHEZ R, Blanco M, Weiss J, Rosati I, Herrera C, Bollwein H, Burger D, Sieme H. 2017. Influence of embryonic size and manipulation on pregnancy rates of mares after transfer of cryopreserved equine embryos. Journal of Equine Veterinary Science. 49:54-59.

SCHERZER J, Davis C, Hurley DJ. 2011. Laser‐assisted vitrification of large equine embryos. Reproduction in Domestic Animals. 46(6):1104-1106.

SCOTT BR, Carwell DB, Hill RA, Bondioli KR, Godke RA, Gentry GT. 2012. Evaluation of capsule permeability in the equine blastocyst. Journal of Equine Veterinary Science. 32(12):795-798.

SILVA PC, Oliveira JP, Dutra GA, Paiva SO, Caram DF, Junqueira RG, Jacob JC. 2018. Taxa de recuperação e características morfológicas de embriões muares (Equus caballus x Equus asinus). Pesquisa Veterinaria Brasileira. 38(7):1453-1457.

SQUIRES EL, García RH, Ginther OJ. 1985. Factors affecting success of equine embryo transfer. Equine Veterinary Journal. 17(S3):92-95.

STOUT TAE, Meadows S, Allen WR. 2005. Stage-specific formation of the equine blastocyst capsule is instrumental to hatching and to embryonic survival in vivo. Animal reproduction science. 87(3-4):269-281.

URÍAS-CASTRO C, Boeta AM. 2020. Post vitrification pregnancy rate of in vivo produced embryos derived from equids. Review. Revista Mexicana de Ciencias Pecuarias. 11(4):1142-1149.

VENDRAMINI OM, Bruyas JF, Fieni F, Battut I, Tainturier D. 1997. Embryo transfer in Poitou donkeys, preliminary results. Theriogenology. 47(1):409.

WILSHER S, Rigali F, Couto G, Camargo S, Allen WR. 2019. Vitrification of equine expanded blastocysts following puncture with or without aspiration of the blastocoele fluid. Equine Veterinary Journal. 51(4):500-505.

WILSHER S, Rigali F, Kovacsy S, Allen WR. 2021. Successful vitrification of manually punctured equine embryos. Equine Veterinary Journal. 53(6):1227-1233.





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