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Equine veterinary journal2024; doi: 10.1111/evj.14081

Successful vitrification of equine embryos >300 microns without puncture or aspiration.

Abstract: Equine embryos >300 μm require puncture before vitrification. Protocols that do not require pre-puncture would make vitrification easier and allow for its widespread use. Objective: To design a successful vitrification protocol for embryos >300 μm without puncture as a pre-treatment. Methods: Experimental in vivo study. Methods: Thirty-eight embryos were divided into 3 groups (G1: ≤300 μm, n = 11; G2: >300-500 μm, n = 20; G3: >500 μm, n = 7). Embryos were vitrified using a human vitrification kit. Following a 15 min exposure to equilibration solution (ES; 7.5% DMSO +7.5% ethylene glycol [EG] in a base medium [BM] of M199 HEPES-buffered medium [H199] + hydroxypropyl cellulose + gentamycin), embryos were exposed for ≤90 s to a vitrification solution (15% DMSO +15% EG + 0.5 M trelahose in BM), loaded onto a Cryolock and plunged into LN2. Warming was undertaken by plunging the Cryolock tip into 1 mL of H199 + 20% FBS + pen/strep +1 M sucrose at 42°C for 1 min. The embryos were then moved to a 0.5 M sucrose solution for 4 min, then placed in Vigro Hold for 4 min prior to transfer to a recipient. Results: Pregnancy rates were 81.8% (9/11) for G1, 80% (16/20) for G2, and 0% (0/7) for G3. The largest embryo to survive was 480 μm. Conclusions: Limited numbers and only one pregnancy was followed to term. Conclusions: Equine embryos ≤480 μm can be successfully vitrified using a protocol with a longer exposure time to the ES. This does not appear to have a negative effect on early embryonic development.
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Publication Date: 2024-03-07 PubMed ID: 38450769DOI: 10.1111/evj.14081Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research aims to create a successful vitrification process for horse embryos that are greater than 300 micrometers in size, without having to puncture them beforehand, thus making the process simpler to implement.

Research Overview

  • The study dealt with testing a novel vitrification process on horse embryos. Vitrification is a method of freezing embryos to retain their viability and it requires precise control over the process to prevent damage. Usually, horse embryos greater than 300 micrometers in size require a puncture before they can be vitrified. The objective of this study, however, was to find an efficient vitrification process that didn’t involve puncturing the embryo as a pre-treatment.
  • Three groups of different-sized embryos were used in the study, with the aim of testing the effectiveness of a new vitrification protocol. Thirty-eight embryos were divided into groups according to their sizes; less than or equal to 300 micrometers, between 300 and 500 micrometers, and greater than 500 micrometers.

    • Methods

    • The embryos were vitrified using a kit designed for human embryo vitrification. However, the process was altered to fit the requirements of this study.
    • They were initially exposed to an equilibration solution (ES), followed by a vitrification solution. The exposure times for these solutions were meticulously managed to ensure the process didn’t harm the embryos. Afterwards, these embryos were frozen using liquid nitrogen and warmed up later using a specified procedure.
    • After being warmed, the embryos underwent further treatment and were then transferred to a recipient.

    Results

    • The results displayed varying rates of success. The pregnancy rates for embryos ≤300 μm and those >300 to 500 μm were similar, at roughly 80%. However, there were no successful impregnations from the largest embryo group that are >500 μm.
    • The largest embryo that successfully survived the vitrification process was 480 micrometers.
    • The researchers concluded that the method allows successful vitrification of horse embryos smaller than or equal to 480 micrometers. This alteration to the vitrification process did not appear to harm the early development of the embryos.

Conclusions

  • While the sample size was limited and only one pregnancy was followed all the way to term, the study was able to achieve its main objective.
  • Further research might look into refining the approach for larger embryos or examining why the treatment was unsuccessful for the largest group in this study.

Cite This Article

APA
Kovacsy S, Ismer A, Funes J, Hoogewijs M, Wilsher S. (2024). Successful vitrification of equine embryos >300 microns without puncture or aspiration. Equine Vet J. https://doi.org/10.1111/evj.14081

Publication

Equine veterinary journal
ISSN: 2042-3306
NlmUniqueID: 0173320
Country: United States
Language: English

Researcher Affiliations

Kovacsy, Sofia
  • Sharjah Equine Hospital, Sharjah, UAE.
Ismer, Ann
  • Sharjah Equine Hospital, Sharjah, UAE.
Funes, Javier
  • Sharjah Equine Hospital, Sharjah, UAE.
Hoogewijs, Maarten
  • Sharjah Equine Hospital, Sharjah, UAE.
Wilsher, Sandra
  • Sharjah Equine Hospital, Sharjah, UAE.

References

This article includes 42 references
  1. Kuwayama M, Gandhi G, Kagalwala S, Ramani R. Vitrification: an overview.. .
  2. Vajta G. Vitrification in ART: past, present, and future.. Theriogenology 2020;150:276-279.
  3. Eldridge-Panuska WD, Caracciolo di Brienza V, Seidel GE Jr, Squires EL, Carnevale EM. Establishment of pregnancies after serial dilution or direct transfer by vitrified equine embryos.. Theriogenology 2005;63:1308-1319.
  4. Choi YH, Velez IC, Riera FL, Roldán JE, Hartman DL, Bliss SB. Successful cryopreservation of expanded equine blastocysts.. Theriogenology 2011;76:143-152.
  5. Diaz F, Bondiolli K, Paccamonti D, Gentry GT. Cryopreservation of Day 8 equine embryos after blastocyst micromanipulation and vitrification.. Theriogenology 2016;85:894-903.
  6. Sanchez R, Blanco M, Weiss J, Rosati I, Herrera C, Bollwein H. Influence of embryonic size and manipulation on pregnancy rates of mares after transfer of cryopreserved equine embryos.. J Equine Vet 2017;49:54-59.
  7. Wilsher S, Rigali F, Couto G, Camargo S, Allen WR. Vitrification of equine expanded blastocysts following puncture with or without aspiration of the blastocoele fluid.. Equine Vet J 2019;51:500-505.
  8. Wilsher S, Rigali F, Kovacsy S, Allen WR. Successful vitrification of manually punctured equine embryos.. Equine Vet J 2021;53:1227-1233.
  9. Battut I, Colchen S, Fieni F, Tainturier D, Bruyas JF. Success rates when attempting to non-surgically collect equine embryos at 144, 156 or 168 hours after ovulation.. Equine Vet J 1998;29(S25):60-62.
  10. Boyle MS, Sanderson MW, Skidmore JA, Allen WR. Use of serial progesterone measurements to assess cycle length, time of ovulation and timing of uterine flushes in order to recover equine morulae.. Equine Vet J 1989;21(S8):10-13.
  11. Betteridge KJ. The structure and function of the equine capsule in relation to embryo manipulation and transfer.. Equine Vet J 1989;21(S8):92-100.
  12. Oriol JG, Betteridge KJ, Clarke AJ, Sharom FJ. Mucin-like glycoproteins in the equine embryonic capsule.. Mol Reprod Dev 1993;34:255-265.
  13. Flood PF, Betteridge KJ, Diocee MS. Transmission electron microscopy of horse embryos 3-16 days after ovulation.. J Reprod Fertil 1982;S32:319-327.
  14. Budik S, Walter I, Tschulenk W, Helmreich M, Deichsel K, Pittner F. Significance of aquaporins and sodium potassium ATPase subunits for expansion of the early equine conceptus.. Reproduction 2008;135:497-508.
  15. Enders AC, Schlafke S, Kantz KC, Liu IKM. Endodermal cells of the equine yolk sac from Day 7 until formation of the definitive yolk sac placenta.. Equine Vet J 1993;25(S15):3-9.
  16. Pfaff R, Seidel GE, Squires EL, Jasko DJ. Permeability of equine blastocysts to ethylene glycol and glycerol.. Theriogenology 1993;39:284 (Abstract).
  17. Legrand E, Bencharif D, Tainturier D, Bruyas JF. Does the embryonic capsule impede the freezing of equine embryos.. 2000. p. 62-65.
  18. Gillard Kingma SE, Thibault ME, Betteridge KJ, Schlaf M, Gartley CJ, Chenier TS. Permeability of the equine embryonic capsule to ethylene glycol and glycerol.. Theriogenology 2011;76:1540-1551.
  19. McCue PM, DeLuca CA, Ferris RA, Wall JJ. How to evaluate equine embryos.. Proc Am Assoc Equine Practnrs 2009;55:252-256.
  20. Coello A, Campos P, Remohi J, Meseguer M, Cobo A. A combination of hydroxypropyl cellulose and trehalose as supplementation for vitrification of human oocytes: a retrospective cohort study.. J Assist Reprod Genet 2016;33:413-421.
  21. Wilsher S, Allen WR. An improved method for non-surgical embryo transfer in the mare.. Equine Vet Educ 2004;16:39-44.
  22. Vanderzwalmen P, Ectors F, Wirleitner B, Stecher A, Desmet D, Griendl J. Intracellular concentration of cryoprotectant during vitrification and slow-freezing cryopreservation procedures.. 2016. p. 35-45.
  23. Weiss J, Blanco M, Sanchez R, Arbouin C, Schockemöhle P. Comparison between an open and a closed vitrification system for equine forced-collapsed embryos.. J Equine Vet 2016;41:52-53 (Abstract).
  24. Landim-Alvarenga FC, Alvarenga MA, Meira C. Transmission electron microscopy of equine embryos cryopreserved by different methods.. Equine Vet J 1993;25(S15):67-70.
  25. Betteridge KJ. Equine embryology: an inventory of unanswered questions.. Theriogenology 2007;S68:9-21.
  26. Luyet B. On the amount of liquid water remaining amorphous in frozen aqueous solutions.. Biodynamica 1969;10:277-291.
  27. Luyet B, Rasmussen D. Study by differential thermal analysis of the temperatures of instability of rapidly cooled solutions of glycerol, ethylene glycol, sucrose and glucose.. Biodynamica 1968;10:167-191.
  28. Rasmussen D, Luyet B. Complimentary study of some non-equilibrium phase transitions in frozen solutions of glycerol, ethylene glycol, glucose and sucrose.. Biodynamica 1969;10:321-331.
  29. Fahy GM. Cryoprotectant toxicity neutralizers reduce freezing injury.. Cryo-Letters 1983;4:309-314.
  30. Fahy GM, MacFarlane DR, Angell CA, Meryman HT. Vitrification as an approach to cryopreservation.. Cryobiology 1984;21:407-426.
  31. Vanderzwalmen P, Connan D, Grobet L, Wirleitner B, Remy B, Vanderzwalmen S. Lower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentration of cryoprotectant solutions.. Hum Reprod 2013;28:2101-2110.
  32. Swain JE, Smith GD. Cryoprotectants.. 2010. p. 24-38.
  33. Leibo SP, Pool TB. The principal variables of cryopreservation: solutions, temperatures, and rate changes.. Fertil Steril 2011;96:69-76.
  34. Edashige K. Permeability of the plasma membrane to water and cryoprotectants in mammalian oocytes and embryos: its relevance to vitrification.. Reprod Med Biol 2017;16:36-39.
  35. Mitsuhata S, Hayashi M, Fujii Y, Motoyama H, Endo Y. Effect of equilibration time on clinical and neonatal outcomes in human blastocysts vitrification.. Reprod Med Biol 2020;19:270-276.
  36. Valdez DM Jr, Hara T, Miyamoto A, Seki S, Jin B, Kasai M. Expression of aquaporin-3 improves the permeability to water and cryoprotectants of immature oocytes in the medaka (Oryzias latipes).. Cryobiology 2006;53:160-168.
  37. Edashige K, Ohta S, Tanaka M, Kuwano T, Valdez DM Jr, Hara T. The role of aquaporin 3 in the movement of water and cryoprotectants in mouse morulae.. Biol Reprod 2007;77:365-375.
  38. Seki S, Kouya T, Hara T, Valdez DM Jr, Jin B, Kasai M. Exogenous expression of rat aquaporin-3 enhances permeability to water and cryoprotectants of immature oocytes in the zebrafish (Danio rerio).. J Reprod Dev 2007;53:597-604.
  39. McCue PM. Equine embryo transfer: clinical perspectives.. Clin Theriogenol 2017;9:369-375.
  40. McCue PM, Ferris RA, Lindholm AR, DeLuca CA. Embryo recovery procedures and collection success: results of 492 embryo-flush attempts.. Proc Am Assoc Equine Practnrs 2010;56:318-321.
  41. Panzani D, Rota A, Marmorini P, Vannozzi I, Camillo F. Retrospective study of factors affecting multiple ovulations, embryo recovery, quality, and diameter in a commercial equine embryo transfer program.. Theriogenology 2014;82:807-814.
  42. Cuervo-Arango J, Claes AN, Stout TAE. Horse embryo diameter is influenced by the embryonic age but not by the type of semen used to inseminate donor mares.. Theriogenology 2018;115:90-93.

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