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Use of confocal microscopy and intracytoplasmic sperm injection (ICSI) to assess viability of equine oocytes from young and old mares after vitrification

  • Gamete Biology
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Abstract

Background

The impact of vitrification on oocyte developmental competence as a function of donor age remains an important issue in assisted reproductive technologies (ARTs).

Methods

Equine germinal vesicle (GV) or metaphase II (M(II) oocytes were vitrified using the Cryotop® method. Spindle organization and chromosome alignment were evaluated from confocal imaging data sets of in vivo (IVO) or in vitro (IVM) matured oocytes subjected to vitrification or not. Intracytoplasmic sperm injection (ICSI) from the same groups was used to assess developmental potential.

Results

An increase in chromosome misalignment was observed in spindles from older mares when compared to those of younger mares (P < 0.05). When MII oocytes subjected to vitrification were examined following warming, there was no difference in the percentage of oocytes displaying chromosome misalignment. Next, GV oocytes, collected from the ovaries of younger and older mares, were compared between fresh IVM and IVM following vitrification and warming. For nonvitrified samples, an age difference was again noted for spindle organization and chromosome alignment, with a higher (P < 0.05) percentage of normal bipolar meiotic spindles with aligned chromosomes observed in nonvitrified oocytes from young versus older mares. Vitrification led to a reduction of spindle length (P < 0.05) for oocytes from old mares, whether vitrified at GV or MII stages, whereas this effect was not observed in oocytes from young mares except those vitrified at GV and subjected to IVM. Oocyte developmental potential after vitrification was evaluated after ICSI of vitrified and warmed MII or GV oocytes from young mares. From 25 MII oocytes, 18 oocytes were injected with sperm, and six blastocysts were produced, which, upon transfer to mares’ uteri, resulted in four pregnancies. Immature (GV) oocytes collected from live mares were also vitrified, warmed, and matured in vitro before ICSI. In this group, nonvitrified, control, and vitrified oocytes did not differ (P > 0.05) with respect to the incidence of maturation to MII, cleavage after ICSI, or blastocyst development.

Conclusion

These findings demonstrate an effect of maternal age in an equine model at the level of meiotic spindle integrity and chromosome positioning that is influenced by both the meiotic stage at which oocytes are vitrified and whether meiotic maturation occurred in vivo or in vitro.

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Acknowledgements

This study was supported by the Cecil and Irene Hylton Family Foundation. Dr. Raul Gonzalez-Castro assisted with statistical analyses. Dr. Elena Ruggeri provided measurements for experiment 1A, and Drs. Jennifer Hatzel and Fabio Amoroso aspirated immature oocytes in experiment 2B.

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Authors and Affiliations

  1. Department of Biomedical Sciences, Colorado State University, 1601 Campus Delivery, Fort Collins, Colorado, 80523, USA

    Lisa J Maclellan, Joanne E Stokes & Elaine M Carnevale

  2. Seven Creeks Equine Reproduction, Euroa, 3666, Australia

    Lisa J Maclellan

  3. Bedford Research Foundation, Bedford, MA, 01730, USA

    David F. Albertini

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  1. Lisa J Maclellan
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  2. David F. Albertini
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Correspondence to Elaine M Carnevale.

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Maclellan, L.J., Albertini, D.F., Stokes, J.E. et al. 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 40, 2565–2576 (2023). https://doi.org/10.1007/s10815-023-02935-4

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