In silico-designed vitrification protocols: an approach to improve survival of in vitro produced-bovine embryos

in Reproduction
Authors:
Iris Martínez-Rodero Department of Animal Medicine and Surgery, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain

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Judith Diaz-Muñoz Department of Animal Medicine and Surgery, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain

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Adam Z Higgins School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, Oregon, USA

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Manel López Béjar Department of Animal Health and Anatomy, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain

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Teresa Mogas Department of Animal Medicine and Surgery, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain

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https://orcid.org/0000-0002-6733-1328
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Tania García-Martínez Department of Animal Medicine and Surgery, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain

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Correspondence should be addressed to T Mogas; Email: teresa.mogas@uab.cat

*(I Martínez-Rodero and J Diaz-Muñoz contributed equally to this work)

†(T Mogas and T García-Martínez share senior authorship)

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In brief

In silico predictions validated in this study demonstrate the potential for designing shorter equilibration protocols that improve post-warming re-expansion and hatching rates of D7 and D8 in vitro-produced bovine embryos. Our results benefit the livestock industry by providing a refined and reproducible approach to cryopreserving bovine embryos, which, in addition, could be useful for other mammalian species.

Abstract

The cryopreservation of in vitro-produced (IVP) embryos is vital in the cattle industry for genetic selection and crossbreeding programs. Despite its importance, there is no standardized protocol yielding pregnancy rates comparable to fresh embryos. Current approaches often neglect the osmotic tolerance responses to cryoprotectants based on temperature and time. Hereby, we propose improved vitrification methods using shorter dehydration-based protocols. Blastocysts cultured for 7 (D7) or 8 days (D8) were exposed to standard equilibration solution (ES) at 25ºC and 38.5ºC. Optimized exposure times for each temperature and their impact on post-warming re-expansion, hatching rates, cell counts, and apoptosis rate were determined. In silico predictions aligned with in vitro observations, showing original volume recovery within 8 min 30 s at 25ºC or 3 min 40 s at 38.5ºC (D7 blastocysts) and 4 min 25 s at 25ºC and 3 min 15 s at 38.5ºC (D8 blastocysts) after exposure to ES. Vitrification at 38.5ºC resulted in D7 blastocysts re-expansion and hatching rates (93.1% and 38.1%, respectively) comparable to fresh embryos (100.0% and 32.4%, respectively), outperforming the 25ºC protocol (86.2% and 24.4%, respectively; P < 0.05). No differences were observed between D7 and D8 blastocysts using the 38.5ºC protocol. Total cell number was maintained for D7 and D8 blastocysts vitrified at 38.5ºC but decreased at 25ºC (P < 0.05). Apoptosis rates increased post-warming (P < 0.05), except for D8 blastocysts vitrified at 38.5ºC, resembling fresh controls. In conclusion, based on biophysical permeability data, new ES incubation times of 3 min 40 s for D7 blastocysts and 3 min 15 s for D8 blastocysts at 38.5ºC were validated for optimizing vitrification/warming methods for bovine IVP blastocysts.

 

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  • Abdalla H, Shimoda M, Hara H, Morita H, Kuwayama M, Hirabayashi M & & Hochi S 2010 Vitrification of ICSI- and IVF-derived bovine blastocysts by minimum volume cooling procedure: effect of developmental stage and age. Theriogenology 74 10281035. (https://doi.org/10.1016/j.theriogenology.2010.04.033)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Appeltant R, Somfai T & & Kikuchi K 2018 Faster, cheaper, defined and efficient vitrification for immature porcine oocytes through modification of exposure time, macromolecule source and temperature. Cryobiology 85 8794. (https://doi.org/10.1016/j.cryobiol.2018.09.004)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Benson JD, Kearsley AJ & & Higgins AZ 2012 Mathematical optimization of procedures for cryoprotectant equilibration using a toxicity cost function. Cryobiology 64 144151. (https://doi.org/10.1016/j.cryobiol.2012.01.001)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • G & & Mapletoft R 2013 Evaluation and classification of bovine embryos. Animal Reproduction 10 344348.

  • Caamaño JN, Gómez E, Trigal B, Muñoz M, Carrocera S, Martín D & & Díez C 2015 Survival of vitrified in vitro-produced bovine embryos after a one-step warming in-straw cryoprotectant dilution procedure. Theriogenology 83 881890. (https://doi.org/10.1016/j.theriogenology.2014.11.021)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Campos-Chillòn LF, Walker DJ, De La Torre-Sanchez JF & & Seidel GE 2006 In vitro assessment of a direct transfer vitrification procedure for bovine embryos. Theriogenology 65 12001214. (https://doi.org/10.1016/j.theriogenology.2005.07.015)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Do VH, Walton S, Catt S & & Taylor-Robinson AW 2017 A comparative analysis of the efficacy of three cryopreservation protocols on the survival of in vitro-derived cattle embryos at pronuclear and blastocyst stages. Cryobiology 77 5863. (https://doi.org/10.1016/j.cryobiol.2017.05.007)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Dormand JR & & Prince PJ 1980 A family of embedded Runge-Kutta formulae. Journal of Computational and Applied Mathematics 6 1926. (https://doi.org/10.1016/0771-050X(8090013-3)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Dujíčková L, Makarevich AV, Olexiková L, Kubovičová E & & Strejček F 2021 Methodological approaches for vitrification of bovine oocytes. Zygote 29 111. (https://doi.org/10.1017/S0967199420000465)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Edashige K 2016 The movement of water and cryoprotectants across the plasma membrane of mammalian oocytes and embryos and its relevance to vitrification. Journal of Reproduction and Development 62 317321. (https://doi.org/10.1262/jrd.2016-048)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Fahy GM, Lilley TH, Linsdell H, Douglas MS & & Meryman HT 1990 Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms. Cryobiology 27 247268. (https://doi.org/10.1016/0011-2240(9090025-y)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ferré LB, Kjelland ME, Strøbech LB, Hyttel P, Mermillod P & & Ross PJ 2020 Review: recent advances in bovine in vitro embryo production: reproductive biotechnology history and methods. Animal 14 9911004. (https://doi.org/10.1017/S1751731119002775)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • García-Martínez T, Mogas T, Mullen SF, Martínez-Rodero I, Gulieva RE & & Higgins AZJC 2021 Effect of cryoprotectant concentration on bovine oocyte permeability and comparison of two membrane permeability modelling approaches. Scientific Reports 11 15387. (https://doi.org/10.1038/s41598-021-94884-0)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • García-Martínez T, Martínez-Rodero I, Roncero-Carol J, Yánez-Ortiz I, Higgins AZ & & Mogas T 2022 Impact of equilibration duration combined with temperature on the outcome of bovine oocyte vitrification. Theriogenology 184 110123. (https://doi.org/10.1016/j.theriogenology.2022.02.024)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gómez E, Carrocera S, Martín D, Pérez-Jánez JJ, Prendes J, Prendes JM, Vázquez A, Murillo A, Gimeno I & & Muñoz M 2020 Efficient one-step direct transfer to recipients of thawed bovine embryos cultured in vitro and frozen in chemically defined medium. Theriogenology 146 3947. (https://doi.org/10.1016/j.theriogenology.2020.01.056)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hagedorn M, Kleinhans FW, Artemov D & & Pilatus U 1998 Characterization of a major permeability barrier in the zebrafish Embryo1. Biology of Reproduction 59 12401250. (https://doi.org/10.1095/biolreprod59.5.1240)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Han YM, Yamashina H, Koyama N, Lee KK & & Fukui Y 1994 Effects of quality and developmental stage on the survival of IVF-derived bovine blastocysts cultured in vitro after freezing and thawing. Theriogenology 42 645654. (https://doi.org/10.1016/0093-691x(9490381-r)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Jin B, Kawai Y, Hara T, Takeda S, Seki S, Nakata Y, Matsukawa K, Koshimoto C, Kasai M & & Edashige K 2011 Pathway for the movement of water and cryoprotectants in bovine oocytes and embryos. Biology of Reproduction 85 834847. (https://doi.org/10.1095/biolreprod.110.088641)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kaidi S, Donnay I, Lambert P, Dessy F & & Massip A 2000 Osmotic behavior of in vitro produced bovine blastocysts in cryoprotectant solutions as a potential predictive test of survival. Cryobiology 41 106115. (https://doi.org/10.1006/cryo.2000.2272)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kasai M, Ito K & & Edashige KJHR 2002 Morphological appearance of the cryopreserved mouse blastocyst as a tool to identify the type of cryoinjury. Human Reproduction 17 18631874. (https://doi.org/10.1093/humrep/17.7.1863)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kleinhans FW 1998 Membrane permeability modeling: Kedem-Katchalsky vs a two-parameter formalism. Cryobiology 37 271289. (https://doi.org/10.1006/cryo.1998.2135)

  • Kuwayama M 2007 Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology 67 7380. (https://doi.org/10.1016/j.theriogenology.2006.09.014)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lagarias JC, Reeds JA, Wright MH & & Wright PE 1998 Convergence properties of the Nelder--Mead simplex method in low dimensions. SIAM Journal on Optimization 9 112147. (https://doi.org/10.1137/S1052623496303470)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Martinez-Rodero I, Garcia-Martinez T, Ordonez-Leon EA, Vendrell-Flotats M, Olegario Hidalgo C, Esmoris J, Mendibil X, Azcarate S, Lopez-Bejar M, Yeste M, et al.2021 A shorter equilibration period improves post-warming outcomes after vitrification and in straw dilution of in vitro-produced bovine embryos. Biology (Basel) 10. (https://doi.org/10.3390/biology10020142)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Martinez-Rodero I, Salas-Huetos A, Ordonez-Leon A, Hidalgo CO, Yeste M, Mercade E & & Mogas T 2022 Cryoprotectant role of exopolysaccharide ID1 in the vitrification/in-straw warming of in vitro-produced bovine embryos. In Reproduction in Domestic Animals 57(Supplement 5) 5357. (https://doi.org/10.1111/rda.14191)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Martínez-Rodero I, Salas-Huetos A, Diaz-Muñoz J, Ordóñez-León EA, García-Martínez T, Yeste M, Olegario Hidalgo C & & Mogas T 2024 Blastocoel fluid aspiration improves vitrification outcomes and produces similar sexing results of in vitro-produced cattle embryos compared to microblade biopsy. Theriogenology 218 142152. (https://doi.org/10.1016/j.theriogenology.2024.01.042)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Mazur P & & Schneider U 1986 Osmotic responses of preimplantation mouse and bovine embryos and their cryobiological implications. Cell Biophysics 8 259285. (https://doi.org/10.1007/BF02788516)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Min SH, Lee E, Son HH, Yeon JY & & Koo DB 2013 Forced collapse of the blastocoel enhances survival of cryotop vitrified bovine hatching/hatched blastocysts derived from in vitro fertilization and somatic cell nuclear transfer. Cryobiology 66 195199. (https://doi.org/10.1016/j.cryobiol.2013.01.004)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Mogas T 2018 Update on the vitrification of bovine oocytes and invitro-produced embryos. Reproduction, Fertility, and Development 31 105117. (https://doi.org/10.1071/RD18345)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Morató R, Izquierdo D, Paramio MT & & Mogas T 2010 Survival and apoptosis rates after vitrification in cryotop devices of in vitro-produced calf and cow blastocysts at different developmental stages. Reproduction, Fertility, and Development 22 11411147. (https://doi.org/10.1071/RD10013)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Morató R & & Mogas T 2014 New device for the vitrification and in-straw warming of in vitro produced bovine embryos. Cryobiology 68 288293. (https://doi.org/10.1016/j.cryobiol.2014.02.010)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Mullen SF, Li M, Li Y, Chen ZJ & & Critser JK 2008 Human oocyte vitrification: the permeability of metaphase II oocytes to water and ethylene glycol and the appliance toward vitrification. Fertility and Sterility 89 18121825. (https://doi.org/10.1016/j.fertnstert.2007.06.013)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ordonez-Leon EA, Martinez-Rodero I, Garcia-Martinez T, Lopez-Bejar M, Yeste M, Mercade E & & Mogas T 2022 Exopolysaccharide ID1 Improves Post-Warming Outcomes after Vitrification of In Vitro-Produced Bovine Embryos. International Journal of Molecular Sciences 23. (https://doi.org/10.3390/ijms23137069)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Otsuka J, Takahashi A, Nagaoka M & & Funabashi H 2002 Optimal equilibration conditions for practical vitrification of two-cell mouse embryos. Comparative Medicine 52 342346.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Paynter SJ 2005 A rational approach to oocyte cryopreservation. Reproductive Biomedicine Online 10 578586. (https://doi.org/10.1016/s1472-6483(1061664-1)

  • Paynter SJ, Fuller BJ & & Shaw RW 1999 Temperature dependence of Kedem–Katchalsky membrane transport coefficients for mature mouse oocytes in the presence of ethylene glycol. Cryobiology 39 169176. (https://doi.org/10.1006/cryo.1999.2199)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Raju R, Bryant SJ, Wilkinson BL & & Bryant G 2021 The need for novel cryoprotectants and cryopreservation protocols: insights into the importance of biophysical investigation and cell permeability. Biochimica et Biophysica Acta. General Subjects 1865 129749. (https://doi.org/10.1016/j.bbagen.2020.129749)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Rios GL, Mucci NC, Kaiser GG & & Alberio RH 2010 Effect of container, vitrification volume and warming solution on cryosurvival of in vitro-produced bovine embryos. Animal Reproduction Science 118 1924. (https://doi.org/10.1016/j.anireprosci.2009.06.015)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Rizos D, Ward F, Boland MP & & Lonergan P 2001 Effect of culture system on the yield and quality of bovine blastocysts as assessed by survival after vitrification. Theriogenology 56 116. (https://doi.org/10.1016/s0093-691x(0100538-6)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Saha S & & Suzuki T 1997 Vitrification of in vitro produced bovine embryos at different ages using one- and three-step addition of cryoprotective additives. Reproduction, Fertility, and Development 9 741746. (https://doi.org/10.1071/r97024)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sanches BV, Lunardelli PA, Tannura JH, Cardoso BL, Pereira MH, Gaitkoski D, Basso AC, Arnold DR & & Seneda MM 2016 A new direct transfer protocol for cryopreserved IVF embryos. Theriogenology 85 11471151. (https://doi.org/10.1016/j.theriogenology.2015.11.029)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Shampine LF & & Reichelt MW 1997 The MATLAB ode suite. SIAM Journal on Scientific Computing 18 122. (https://doi.org/10.1137/S1064827594276424)

  • Sudano MJ, Paschoal DM, Rascado Tda S, Magalhães LC, Crocomo LF, de Lima-Neto JF & & Landim-Alvarenga Fda C 2011 Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification. Theriogenology 75 12111220. (https://doi.org/10.1016/j.theriogenology.2010.11.033)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Vajta G, Rindom N, Peura TT, Holm P, Greve T & & Callesen H 1999 The effect of media, serum and temperature on in vitro survival of bovine blastocysts after Open Pulled Straw (OPS) vitrification. Theriogenology 52 939948. (https://doi.org/10.1016/S0093-691X(9900184-3)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Valente RS, Marsico TV & & Sudano MJ 2022 Basic and applied features in the cryopreservation progress of bovine embryos. Animal Reproduction Science 239 106970. (https://doi.org/10.1016/j.anireprosci.2022.106970)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Van Soom A, Ysebaert MT & & De Kruif A 1997 Relationship between timing of development, morula morphology, and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos. Molecular Reproduction and Development 47 4756. (https://doi.org/10.1002/(SICI)1098-2795(199705)47:1<47::AID-MRD7>3.0.CO;2-Q)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Vian AM & & Higgins AZ 2014 Membrane permeability of the human granulocyte to water, dimethyl sulfoxide, glycerol, propylene glycol and ethylene glycol. Cryobiology 68 3542. (https://doi.org/10.1016/j.cryobiol.2013.11.004)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Vieira AD, Forell F, Feltrin C & & Rodrigues JL 2007 In-straw cryoprotectant dilution of IVP bovine blastocysts vitrified in hand-pulled glass micropipettes. Animal Reproduction Science 99 377383. (https://doi.org/10.1016/j.anireprosci.2006.06.010)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Wang X, Al Naib A, Sun DW & & Lonergan P 2010 Membrane permeability characteristics of bovine oocytes and development of a step-wise cryoprotectant adding and diluting protocol. Cryobiology 61 5865. (https://doi.org/10.1016/j.cryobiol.2010.05.001)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Warner RM, Brown KS, Benson JD, Eroglu A & & Higgins AZ 2022 Multiple cryoprotectant toxicity model for vitrification solution optimization. Cryobiology 108 19. (https://doi.org/10.1016/j.cryobiol.2022.09.002)

    • PubMed
    • Search Google Scholar
    • Export Citation