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Equine veterinary journal2025; doi: 10.1111/evj.14539

A comparison of the efficacy of three commercial human embryo vitrification kits for cryopreservation of in vivo produced equine embryos.

Abstract: Different cryoprotectants can influence the ability of embryos to successfully survive vitrification and subsequent warming before transfer. Objective: To compare pregnancy rates for embryos ≤500 μm vitrified, without puncture or aspiration of the blastocoele cavity, with one of three commercial human embryo vitrification kits containing the same penetrating cryoprotectants (DMSO and EG) but varying in their non-penetrating cryoprotectants (NPCPAs; sucrose, trehalose, dextran serum supplement [DSS], and hydroxypropyl cellulose [HPC]). Methods: In vivo experiments. Methods: Embryos (n = 108) were vitrified using either a Kitazato (NPCPAs = trehalose, hydroxypropyl cellulose), Vit Kit Freeze (NPCPAs = sucrose, DSS), or Vit Kit Freeze NX (NPCPAs = trehalose, DSS) vitrification kit by exposing each embryo to kit-specific equilibration solution for 15 min, before the vitrification solution for ≤90 sec including loading onto a Cryolock device, which was capped and plunged into LN2. All embryos were warmed in the same media by placing the Cryolock tip into 1 mL of 1.0 M sucrose in a HEPES-based medium (1 min), followed by 0.5 M sucrose (4 min) and then commercial Holding medium (4 min) before transfer to a Day 6 recipient mare. For each kit, embryos were divided by size into three groups (G1 ≤ 300 μm; G2 > 300-400 μm; G3 > 400-500 μm; n = 8-14/group/kit). Results: Pregnancy rates were equivalent for the Kitazato, Vit Kit Freeze, and Vit Kit Freeze NX kits for embryos in G1 (12/14 [85.7%] vs. 8/11 [72.7%] vs. 7/8 [87.5%], respectively, p = 0.63) and for G2 (10/12 [83.3%] vs. 10/11 [90.9%] vs. 9/11 [81.8%], respectively, p = 0.81). For G3 embryos, pregnancy rates were higher for the Kitazato versus either of the other kits (10/14 [71.4%] vs. 3/12 [21.4%] vs. 2/14 [14.3%], respectively, p = 0.003). Conclusions: Limited numbers. Conclusions: Different non-penetrating/extracellular cryoprotectants can influence the success of vitrifying equine embryos 400-500 μm. The combination of trehalose and hydroxypropyl cellulose appears to be beneficial in this respect. Unassigned: Verschiedene Kryoprotektionsmittel können die Fähigkeit der Embryonen beeinflussen, die Vitrifikation und die anschließende Erwärmung vor dem Transfer erfolgreich zu überstehen. Unassigned: Vergleich der Schwangerschaftsraten von Embryonen, die ≤500 μm vitrifiziert wurden, ohne Punktion oder Aspiration der Blastozelenhöhle, mit einem von drei kommerziellen Kits zur Vitrifikation menschlicher Embryonen, die dieselben durchdringenden Kryoprotektoren (DMSO und EG), aber unterschiedliche nicht durchdringende Kryoprotektoren (NP‐CPAs; Saccharose, Trehalose, Dextran Serum Supplement [DSS], Hydroxypropyl Cellulose [HPC]) enthalten. Unassigned: In vivo Studie. Methods: Embryonen (n = 108) wurden entweder mit einem Kitazato (NPCPAs = Trehalose, Hydroxypropylcellulose), Vit Kit Freeze (NPCPAs = Saccharose, DSS) oder Vit Kit Freeze NX (NPCPAs = Trehalose, DSS) vitrifiziert, indem jeder Embryo 15 Minuten lang einer kit‐spezifischen Äquilibrierungslösung ausgesetzt wurde, bevor er für ≤90 Sekunden in eine Cryolock‐Vorrichtung geladen wurde, die verschlossen und in LN2 getaucht wurde. Alle Embryonen wurden in denselben Medien erwärmt, indem die Cryolock‐Spitze in 1 mL 1,0 M Saccharose in einem HEPES‐basierten Medium (1 Minute), gefolgt von 0,5 M Saccharose (4 Minuten) und anschließendem handelsüblichen Holding‐Medium (4 Minuten) vor dem Transfer zu einer Empfängerstute an Tag 6 gelegt wurde. Für jedes Kit wurden die Embryonen nach Größe in drei Gruppen eingeteilt (G1 ≤3 00 μm; G2 > 300–400 μm; G3 > 400–500 μm; n = 8–14/Gruppe/Kit). Unassigned: Die Schwangerschaftsraten waren für die Kits Kitazato, Vit Kit Freeze und Vit Kit Freeze NX für Embryonen in G1 (12/14 [85,7%] vs. 8/11 [72,7%] vs. 7/8 [87,5%], jeweils, p = 0,63) und für G2 (10/12 [83,3%] vs. 10/11 [90,9%] vs. 9/11 [81,8%], jeweils, p = 0,81) gleich. Bei G3‐Embryonen waren die Schwangerschaftsraten bei Kitazato höher als bei den anderen Kits (10/14 [71,4%] vs. 3/12 [21,4%] vs. 2/14 [14,3%], p = 0,003). Unassigned: Begrenzte Anzahl. Unassigned: Verschiedene nichtpenetrierende/extrazelluläre Kryoprotektoren können den Erfolg der Vitrifizierung von 400–500 μm Embryonen beeinflussen. Die Kombination von Trehalose und Hydroxypropylcellulose scheint in dieser Hinsicht von Vorteil zu sein.
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Publication Date: 2025-06-26 PubMed ID: 40574307DOI: 10.1111/evj.14539Google 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 study compares the effectiveness of three commercial human embryo vitrification kits in preserving equine embryos for future use. The research found that the kind of non-penetrating compounds in the kit can significantly affect the success of embryo vitrification, particularly for larger embryos.

Objective of the Research

  • The aim of the study was to compare the rates of successful pregnancies resulting from in vivo equine embryos cryopreserved using three commercial human embryo vitrification kits. Each of these kits contained the same penetrating cryoprotectants (Dimethyl Sulfoxide [DMSO] and Ethylene Glycol [EG]) but varied in their non-penetrating cryoprotectants (NPCPAs; sucrose, trehalose, dextran serum supplement [DSS], and hydroxypropyl cellulose [HPC]).

Research Methods

  • For the experiment, 108 embryos were vitrified using one of the three kits: Kitazato (trehalose, hydroxypropyl cellulose as NPCPAs), Vit Kit Freeze (sucrose, DSS as NPCPAs), or Vit Kit Freeze NX (trehalose, DSS as NPCPAs).
  • Each embryo exposed to a kit-specific equilibration solution for 15 minutes, followed by the vitrification solution for less than or equal to 90 seconds, resulting in the loading onto a Cryolock device and plunged into liquid nitrogen (LN2).
  • All embryos were then warmed using the same method in a specified medium, followed by a transfer to a Day 6 recipient mare.
  • The embryos were categorized into three groups based on their size to check for variations in success rates due to size.

Results of the Study

  • It was observed that the pregnancy rates were similar for embryos in group 1 (up to 300 μm in size) and group 2 (size 300-400 μm) for all three kits.
  • However, for group 3 embryos (size 400-500 μm), the success rates were higher when the Kitazato kit, with trehalose and hydroxypropyl cellulose as NPCPAs, was used.

Conclusions

  • The outcomes of the study show that the type of non-penetrating/extracellular cryoprotectants significantly influences the success rate of vitrification, particularly for larger equine embryos between 400-500 μm in size.
  • The combination of trehalose and hydroxypropyl cellulose as NPCPAs was found to be particularly beneficial for this size range of embryos.

Cite This Article

APA
Wilsher S, Ismer A, Grippo A, Hoogewijs M, Bussade P, Kovacsy S. (2025). A comparison of the efficacy of three commercial human embryo vitrification kits for cryopreservation of in vivo produced equine embryos. Equine Vet J. https://doi.org/10.1111/evj.14539

Publication

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

Researcher Affiliations

Wilsher, Sandra
  • Sharjah Equine Hospital, Sharjah, UAE.
Ismer, Ann
  • Sharjah Equine Hospital, Sharjah, UAE.
Grippo, Agustina
  • Sharjah Equine Hospital, Sharjah, UAE.
Hoogewijs, Maarten
  • Sharjah Equine Hospital, Sharjah, UAE.
Bussade, Pedro
  • Sharjah Equine Hospital, Sharjah, UAE.
Kovacsy, Sofia
  • Sharjah Equine Hospital, Sharjah, UAE.

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Citations

This article has been cited 1 times.
  1. Jung SE, Moon Y, Lim M, Jeong H, Seo H. Glucose-Enhanced Cryopreservation of hCAR-T Cells: Improved Recovery and Reduced Apoptosis. Biomol Ther (Seoul) 2025 Nov 1;33(6):963-974.
    doi: 10.4062/biomolther.2025.144pubmed: 41084238google scholar: lookup
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