Effect of maturation stage at cryopreservation on post-thaw cytoskeleton quality and fertilizability of equine oocytes.
Abstract: Oocyte cryopreservation is a potentially valuable technique for salvaging the germ-line when a valuable mare dies, but facilities for in vitro embryo production or oocyte transfer are not immediately available. This study examined the influence of maturation stage and freezing technique on the cryopreservability of equine oocytes. Cumulus oocyte complexes were frozen at the immature stage (GV) or after maturation in vitro for 30 hr (MII), using either conventional slow freezing (CF) or open pulled straw vitrification (OPS); cryoprotectant-exposed and untreated nonfrozen oocytes served as controls. After thawing, GV oocytes were matured in vitro, and MII oocytes were incubated for 0 or 6 hr, before staining to examine meiotic spindle quality by confocal microscopy. To assess fertilizability, CF MII oocytes were subjected to intracytoplasmic sperm injection (ICSI) and cultured in vitro. At 12, 24, and 48 hr after ICSI, injected oocytes were fixed to examine their progression through fertilization. Both maturation stage and freezing technique affected oocyte survival. The meiosis resumption rate was higher for OPS than CF for GV oocytes (28% vs. 1.2%; P < 0.05), but still much lower than for controls (66%). Cryopreserving oocytes at either stage induced meiotic spindle disruption (37%-67% normal spindles vs. 99% in controls; P < 0.05). Among frozen oocytes, however, spindle quality was best for oocytes frozen by CF at the MII stage and incubated for 6 hr post-thaw (67% normal); since this combination of cryopreservation/IVM yielded the highest proportion of oocytes reaching MII with a normal spindle (35% compared to <20% for other groups), it was used when examining the effects of cryopreservation on fertilizability. In this respect, the rate of normal fertilization for CF MII oocytes after ICSI was much lower than for controls (total oocyte activation rate, 26% vs. 56%; cleavage rate at 48 hr, 8% vs. 42%: P < 0.05). Thus, although IVM followed by CF yields a respectable percentage of normal-looking MII oocytes (35%), their ability to support fertilization is severely compromised.
Mol. Reprod. Dev. (c) 2006 Wiley-Liss, Inc.
Publication Date: 2006-02-16 PubMed ID: 16477611DOI: 10.1002/mrd.20432Google Scholar: Lookup
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- Comparative Study
- Journal Article
Summary
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This study examines the effect of maturation stage and freezing technique on the ability to successfully freeze and thaw horse egg cells (oocytes). It shows that the process of freezing and thawing oocytes can lead to problems in supporting fertilization, even if they appear normal.
Introduction
- The researchers of this study aimed to analyze the impacts of maturation stage and freezing methods on the preservation of horse oocytes.
- Oocyte cryopreservation is seen as a valuable technique, especially for preserving the value of a mare when it dies and facilities are unavailable for immediate embryo production or oocyte transfer.
Methodology
- Oocytes were frozen either at the immature stage or after a period of maturation in a lab for 30 hours.
- Two freezing techniques were used – slow conventional freezing and open pulled straw vitrification.
- After thawing, the oocytes were either matured in the lab, or incubated for different periods before staining to examine their quality.
- To assess the ability for fertilization, mature oocytes that underwent slow freezing were injected with sperm and then cultured in the lab.
- Oocytes were examined at 12-, 24-, and 48-hour intervals after injection to monitor their progression through fertilization.
Findings
- Both the freezing method and stage of maturation impacted survival of the oocytes.
- Germinal vesicle (GV) oocytes (immature oocytes) that underwent vitrification had higher rates of initiating meiosis compared to those underwent slow freezing, though the percentage was still significantly lower than the control group (non-frozen oocytes).
- Upon cryopreserving the oocytes at either stage, a significant number exhibited signs of meiotic spindle disruption, compared to the control group.
- Mature oocytes that were frozen via conventional freezing and then incubated for 6 hours after thawing had better spindle quality.
- The highest number of oocytes reaching Metaphase II with a normal spindle was found in this group, making it the best combination for examining the effect of cryopreservation on fertilizability.
- However, the rate of normal fertilization for mature oocytes frozen using conventional freezing and injected with sperm was substantially less than that of the control group.
Conclusion
- This study concluded that in-vitro maturation followed by conventional freezing yields a significant amount of mature oocytes that look normal.
- However, their potential to support fertilization is greatly impaired.
- This highlights the problematic aspect of oocyte preservation especially when it comes to maintaining the ability for fertilization post-thaw.
Cite This Article
APA
Tharasanit T, Colenbrander B, Stout TA.
(2006).
Effect of maturation stage at cryopreservation on post-thaw cytoskeleton quality and fertilizability of equine oocytes.
Mol Reprod Dev, 73(5), 627-637.
https://doi.org/10.1002/mrd.20432 Publication
Researcher Affiliations
- Faculty of Veterinary Medicine, Department of Equine Sciences, Utrecht University, Yalelaan, Utrecht, The Netherlands.
MeSH Terms
- Animals
- Cell Survival / physiology
- Cryopreservation
- Cytoskeleton / metabolism
- Female
- Horses
- Meiosis / physiology
- Oocytes / cytology
- Oocytes / metabolism
- Sperm Injections, Intracytoplasmic
- Spindle Apparatus / metabolism
Citations
This article has been cited 10 times.- Maclellan LJ, Albertini DF, Stokes JE, Carnevale EM. Use of confocal microscopy and intracytoplasmic sperm injection (ICSI) to assess viability of equine oocytes from young and old mares after vitrification. J Assist Reprod Genet 2023 Nov;40(11):2565-2576.
- Angel-Velez D, Meese T, Hedia M, Fernandez-Montoro A, De Coster T, Pascottini OB, Van Nieuwerburgh F, Govaere J, Van Soom A, Pavani K, Smits K. Transcriptomics Reveal Molecular Differences in Equine Oocytes Vitrified before and after In Vitro Maturation. Int J Mol Sci 2023 Apr 7;24(8).
- Angel-Velez D, De Coster T, Azari-Dolatabad N, Fernandez-Montoro A, Benedetti C, Bogado Pascottini O, Woelders H, Van Soom A, Smits K. New Alternative Mixtures of Cryoprotectants for Equine Immature Oocyte Vitrification. Animals (Basel) 2021 Oct 28;11(11).
- Tharasanit T, Thuwanut P. Oocyte Cryopreservation in Domestic Animals and Humans: Principles, Techniques and Updated Outcomes. Animals (Basel) 2021 Oct 13;11(10).
- Colombo M, Morselli MG, Tavares MR, Apparicio M, Luvoni GC. Developmental Competence of Domestic Cat Vitrified Oocytes in 3D Enriched Culture Conditions. Animals (Basel) 2019 Jun 7;9(6).
- Moawad AR, Choi I, Zhu J, El-Wishy ABA, Amarnath D, Chen W, Campbell KHS. Caffeine and oocyte vitrification: Sheep as an animal model. Int J Vet Sci Med 2018;6(Suppl):S41-S48.
- Fathi M, Moawad AR, Badr MR. Production of blastocysts following in vitro maturation and fertilization of dromedary camel oocytes vitrified at the germinal vesicle stage. PLoS One 2018;13(3):e0194602.
- Setti AS, Figueira Rde C, Braga DP, Ferreira RC, Iaconelli A Jr, Borges E Jr. Oocyte morphology does not affect post-warming survival rate in an egg-cryobanking donation program. J Assist Reprod Genet 2011 Dec;28(12):1177-81.
- Prentice JR, Anzar M. Cryopreservation of Mammalian oocyte for conservation of animal genetics. Vet Med Int 2010 Sep 21;2011.
- Khosravi-Farsani S, Sobhani A, Amidi F, Mahmoudi R. Mouse oocyte vitrification: the effects of two methods on maturing germinal vesicle breakdown oocytes. J Assist Reprod Genet 2010 May;27(5):233-8.
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