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

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.

Keywords: Confocal microscopy; Cytoskeleton; Equine; ICSI; Oocyte; Vitrification.