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Home / Equine Research Bank / Equine Vet J / Successful vitrification of manually punctured equine embryo...
Equine veterinary journal2021; 53(6); 1227-1233; doi: 10.1111/evj.13400

Successful vitrification of manually punctured equine embryos.

Abstract: Successful vitrification of equine expanded blastocysts requires collapse of the blastocoele cavity using a micromanipulator-mounted biopsy pipette on an inverted microscope. Such equipment is expensive and requires user skill. Objective: To develop a manual method of blastocoele collapse prior to vitrification using commercial products. Methods: In vivo experiment. Methods: Seventy-nine Day 7 or 8 embryos were measured and graded. Twenty were vitrified following micromanipulator-assisted puncture and aspiration before being used to validate commercial human vitrification and warming kits containing, respectively, 2-step concentrations of DMSO and ethylene glycol (7.5%-15% v:v) and decreasing concentrations of sucrose. After warming, embryos were transferred to recipient mares. Once validated, the commercial kits were used to vitrify and warm a further 39 embryos which were punctured manually using a microneedle, 2 (5%) were damaged during puncture and excluded; 20 more embryos were vitrified without puncture. Embryos were grouped as follows: non-punctured ≤ 300µm (n = 10) and >300 to ≤560 µm (n = 10), punctured small (>300 to ≤560 µm; n = 17) and large (>560 µm; n = 10) and exposed to the equilibration solution (ES) in the kit for 6min. An additional group of punctured large embryos was exposed to ES for 8min (n = 10). For the initial warming step, embryos were exposed for 1min to the thawing solution at 42°C, before being moved to a dilution solution at room temperature. Results: Vitrified, manually punctured embryos ≤560 µm exposed to ES for 6min resulted in a pregnancy rate of 82% (14/17). Unpunctured embryos ≤300 µm gave an 80% (8/10) pregnancy rate. Larger unpunctured embryos, punctured embryos >560 µm and embryos exposed to ES for 8min gave significantly reduced pregnancy rates. Conclusions: Limited group sizes. Conclusions: High pregnancy rates can be achieved by manually puncturing ≤560 µm equine embryos prior to their vitrification and subsequent warming in commercial media.
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Publication Date: 2021-01-11 PubMed ID: 33326638DOI: 10.1111/evj.13400Google 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 presents a study conducted to develop and test a manual method of collapse of the blastocoele cavity in equine embryos for successful vitrification, using commonly available products, rather than relying on expensive equipment.

Objective and Methodology

  • The primary objective of this research was to devise a manual method that could collapse the blastocoele cavity within equine embryos before proceeding with vitrification.
  • This goal was pursued to bypass the need to use costly equipment and techniques that require specialist skills, thereby democratizing the process.
  • The researchers conducted a series of in vivo experiments where they tested and graded a total of 79 equine embryos that were 7 or 8 days old. Subsequently, 20 of these embryos were vitrified through micromanipulator-assisted puncture and aspiration.
  • Following this, commercial human vitrification and warming kits were used, which contained 2-step concentrations of DMSO and ethylene glycol (7.5%-15% v:v) and decreasing concentrations of sucrose.
  • Once the commercial kits were validated, they were used on an additional 39 embryos. These embryos were manually punctured using a microneedle. However, 2 embryos (5%) got damaged during the process and had to be discarded. Further, 20 more embryos were vitrified without puncture.

Results and Conclusion

  • Researchers observed that manually punctured embryos of ≤560 µm size that were exposed to an equilibration solution (ES) for 6 minutes had an 82% (14/17) pregnancy success rate after vitrification process.
  • Embryos of ≤300 µm size that were not punctured also exhibited an 80% (8/10) pregnancy success rate.
  • The study highlighted that larger unpunctured embryos, embryos larger than 560 µm that were punctured, and embryos exposed to ES for an extended period, yielded significantly lower pregnancy rates.
  • In conclusion, the research indicated that high pregnancy rates are achievable by manually puncturing equine embryos of ≤560 µm sizes before their vitrification and subsequent warming in commercial media. The result offers promise for making the process cost-effective and accessible to a broader range of users.

Cite This Article

APA
Wilsher S, Rigali F, Kovacsy S, Allen WT. (2021). Successful vitrification of manually punctured equine embryos. Equine Vet J, 53(6), 1227-1233. https://doi.org/10.1111/evj.13400

Publication

Equine veterinary journal
ISSN: 2042-3306
NlmUniqueID: 0173320
Country: United States
Language: English
Volume: 53
Issue: 6
Pages: 1227-1233

Researcher Affiliations

Wilsher, Sandra
  • Sharjah Equine Hospital, Sharjah, UAE.
Rigali, Florencia
  • Sharjah Equine Hospital, Sharjah, UAE.
Kovacsy, Sofia
  • Sharjah Equine Hospital, Sharjah, UAE.
Allen, Wr Twink
  • Sharjah Equine Hospital, Sharjah, UAE.

MeSH Terms

  • Animals
  • Blastocyst
  • Cryopreservation / veterinary
  • Culture Media
  • Female
  • Horses
  • Pregnancy
  • Pregnancy Rate
  • Vitrification

References

This article includes 22 references
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Citations

This article has been cited 2 times.
  1. Meuffels-Barkas J, Wilsher S, Allen WRT, Ververs C, Lueders I. Comparative reproduction of the female horse, elephant and rhinoceros: implications for advancing Assisted Reproductive Technologies (ART). Reprod Fertil 2023 Jul 1;4(3).
    doi: 10.1530/RAF-23-0020pubmed: 37439577google scholar: lookup
  2. Benammar A, Derisoud E, Vialard F, Palmer E, Ayoubi JM, Poulain M, Chavatte-Palmer P. The Mare: A Pertinent Model for Human Assisted Reproductive Technologies?. Animals (Basel) 2021 Aug 4;11(8).
    doi: 10.3390/ani11082304pubmed: 34438761google scholar: lookup
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