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Reproduction in domestic animals = Zuchthygiene2025; 60(7); e70092; doi: 10.1111/rda.70092

Direct Warming of Vitrified In Vivo Equine Embryos.

Abstract: Vitrified in vitro-produced embryos can be successfully warmed in isotonic media at room temperature (RT; 22°C). However, this protocol has not been reported for in vivo embryos, which are more challenging to vitrify and warm. Study objectives were to see if vitrified in vivo embryos warmed in RT isotonic medium gave equivalent pregnancy rates to stepwise serial dilution warming, and if embryo size influenced the results. One hundred and seventeen embryos were divided into groups by size (G1:≤ 300 μm, n = 59; G2:> 300-400 μm, n = 33; G3:> 400-500 μm, n = 24) and vitrified using a commercial vitrification kit. Embryos were placed in an equilibration solution for 10 (embryos ≤ 300 μm) or 15 min (embryos > 300 μm) before being moved to a vitrification solution (≤ 90 s) and loaded onto a Cryolock prior to plunging into LN2. Warming was undertaken by placing the tip of the uncapped Cryolock into RT isotonic medium (n = 77; comprised of G1:n = 45; G2:n = 22; G3:n = 10) or by stepwise serial dilution (n = 40) with the initial 1 M sucrose solution at 42°C (G1:n = 14; G2:n = 12; G3:n = 14). Warmed embryos were transferred to Day 6 recipient mares and pregnancy rates compared between warming protocols. Ignoring embryo size, there was no difference in Day 14 pregnancy rates for vitrified embryos warmed in RT isotonic medium versus stepwise serial dilution (70.1%, 54/77 vs. 82.5%, 33/40, respectively). No statistical difference existed in pregnancy rates between warming protocols for G1 or G2 embryos (p = 1.0, p = 0.439, respectively), but for G3 embryos, the stepwise protocol results in significantly more pregnancies (p < 0.001). The largest embryo successfully warmed in RT isotonic medium was 390 μm. Whereas for the stepwise protocol, the largest embryo was 480 μm. Direct warming in RT isotonic medium is a suitable protocol for warming vitrified embryos ≤ 390 μm, although the decline in pregnancies at the upper limit of G2 would suggest that clinically this methodology is suitable for embryos ≤ 360 μm.
© 2025 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.
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Publication Date: 2025-07-02 PubMed ID: 40598845DOI: 10.1111/rda.70092Google 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.

The research article explores the viability of using room temperature isotonic medium for the warming of vitrified in vivo equine embryos. It particularly assesses the impact of embryo size on the success of these two methods: direct warming in room temperature medium and a traditional stepwise dilution process.

Objective of the Research

  • The main aim of the study was to determine if vitrified in vivo embryos warmed in room temperature (22°C) isotonic medium can produce pregnancy rates equivalent to those seen with the traditional stepwise serial dilution warming process.

Research Methodology

  • The study involved 117 embryos which were grouped by size into three categories: less than or equal to 300 μm (G1), between 300-400 μm (G2), and more than 400-500 μm (G3). The embryos were vitrified using chemicals from a commercial kit.
  • Before being dunked into liquid nitrogen (LN2) they were first placed into an equilibration solution — for a duration of 10 minutes for embryos up to 300 μm or 15 minutes for embryos above this size. Then they were moved to a vitrification solution for 90 seconds or less.
  • The rewarming of the embryos was done either by direct immersion of the uncapped Cryolock (used to hold embryos during cryopreservation) in room temperature isotonic medium or by a series of serial dilutions with an initial 1M sucrose solution at 42°C (the traditional stepwise method).
  • The warmed embryos were then transferred to day 6 recipient mares and the pregnancy rates determined after day 14.

Findings of the Research

  • Overall, the findings showed no significant difference in pregnancy rates between the two warming methods. However, the effectiveness varied with the size of the embryo. The direct warming method was found to be suitable for warming smaller vitrified embryos up to 390 μm.
  • For embryos larger than 400 μm (G3), the stepwise protocol resulted in significantly more pregnancies.
  • While the direct warming procedure seemingly could accommodate embryos up to 390 μm, the researchers suggested that, based on a noted decline in pregnancies at the upper limit of size group G2, the method may actually be clinically applicable for embryos up to 360 μm in size.

Cite This Article

APA
Couto G, Grippo A, Ismer A, Hoogewijs M, Pedro B, Vasconcelos L, Santos G, Wilsher S. (2025). Direct Warming of Vitrified In Vivo Equine Embryos. Reprod Domest Anim, 60(7), e70092. https://doi.org/10.1111/rda.70092

Publication

Reproduction in domestic animals = Zuchthygiene
ISSN: 1439-0531
NlmUniqueID: 9015668
Country: Germany
Language: English
Volume: 60
Issue: 7
Pages: e70092

Researcher Affiliations

Couto, Guilherme
  • Al Wathba Stables, Abu Dhabi, UAE.
Grippo, Agustina
  • Sharjah Equine Hospital, Sharjah, UAE.
Ismer, Ann
  • Sharjah Equine Hospital, Sharjah, UAE.
Hoogewijs, Maarten
  • Sharjah Equine Hospital, Sharjah, UAE.
Pedro, Bussade
  • Sharjah Equine Hospital, Sharjah, UAE.
Vasconcelos, Lucas
  • Al Wathba Stables, Abu Dhabi, UAE.
Santos, Gabriel
  • Al Wathba Stables, Abu Dhabi, UAE.
Wilsher, Sandra
  • Sharjah Equine Hospital, Sharjah, UAE.
  • The Paul Mellon Laboratory, Suffolk, UK.

MeSH Terms

  • Animals
  • Vitrification
  • Horses / embryology
  • Female
  • Cryopreservation / veterinary
  • Cryopreservation / methods
  • Pregnancy
  • Pregnancy Rate
  • Embryo Culture Techniques / veterinary
  • Embryo Culture Techniques / methods
  • Embryo Transfer / veterinary
  • Embryo, Mammalian / physiology

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