Minimum Volume Vitrification of Immature Feline Oocytes

Colombo, Martina; Luvoni, G.C.

doi:10.3791/61523

Cited by 8 publications

(7 citation statements)

References 15 publications

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“…Oocytes were vitrified by the Cryotop method (26,27), as previously described for cat oocytes (28). Briefly, groups of 4-8 oocytes were equilibrated at room temperature in an equilibration solution containing 7.5% (v/v) ethylene glycol (EG) and 7.5% dimethylsulfoxide (DMSO) in Medium 199, with 20% fetal bovine serum (FBS) for 15 min.…”

Section: Vitrification and Warming Of Immature Cumulus-oocyte Complexmentioning

confidence: 99%

Inhibition of Apoptotic Pathways Improves DNA Integrity but Not Developmental Competence of Domestic Cat Immature Vitrified Oocytes

et al. 2020

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Being a model for endangered wild felids, cryopreservation protocols for domestic cat oocytes are under continuous development. Immature vitrified oocytes (VOs) are a valuable resource for fertility preservation programs, but they often degenerate after warming and their in vitro development is poor. Since the exact mechanisms are not clear, this study assessed whether vitrification might trigger two apoptotic markers (DNA fragmentation and caspase activity, Experiment I) and the effects of a chemical inhibitor (i.e., the pan-caspase inhibitor Z-VAD-FMK) on the same markers (Experiment II) and on VOs in vitro development (Experiment III). The overarching aim was to check whether apoptosis inhibition might be a strategy to improve cat oocytes cryotolerance. In Experiment I, vitrification induced DNA fragmentation and increased caspase activity in VOs incubated for 24 h after warming (DNA fragmentation: 59.38%; caspase activity: 414.6 ± 326.8) compared to a fresh control (9.68%; 199.6 ± 178.3; p = 0.02). In Experiment II, the addition of Z-VAD-FMK to vitrification-warming and incubation media decreased DNA fragmentation and caspase activity (8.82%; 243.7 ± 106.9) compared to control (untreated) VOs (69.44%; 434.5 ± 248.3; p < 0.001). In Experiment III, Z-VAD-FMK brought maturation rates of treated VOs close to those of fresh oocytes (53.13 and 65.38%, respectively, p = 0.057), but there were no differences in VOs embryo development (cleavage rates; Z-VAD-FMK-treated VOs: 34.38%; control VOs: 31.78%; p = 0.69). In summary, vitrification increased apoptotic markers in cat VOs, and while Z-VAD-FMK was able to hinder DNA damage and caspase activity, its addition was not determinant for embryo development. To make the best use of VOs, other oocyte in vitro maturation and embryo culture strategies, such as the addition of other inhibitors or their prolonged use, should be investigated.

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Section: Vitrification and Warming Of Immature Cumulus-oocyte Complexmentioning

confidence: 99%

Inhibition of Apoptotic Pathways Improves DNA Integrity but Not Developmental Competence of Domestic Cat Immature Vitrified Oocytes

et al. 2020

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“…Concerning the ultra-rapid freezing systems, pellets and straws were chosen for their ease of application. Other devices usually employed for oocyte vitrification [Cryotops, ( 54 )] or denudation [stripper tips, ( 55 )] have been tested for spermatozoa vitrification ( 16 , 56 ), as well as carrier systems specifically designed for this purpose, such as the SpermVD ( 57 ) or others ( 58 ). In this study, pellets and straws were basically comparable, likely because the volume of ultra-rapid frozen spermatozoa was the same in both the systems and the presence of the straw did not have a significant influence on cooling rates.…”

Section: Discussionmentioning

confidence: 99%

Ultra-Rapid Freezing Preserves Morphofunctional Integrity and Fertilizing Ability of Epididymal Cat Spermatozoa

et al. 2022

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Vitrification and ultra-rapid freezing, which are more commonly used for oocytes and embryos, have recently been applied to spermatozoa in an attempt to make semen cryopreservation in field conditions easier compared to conventional freezing. It is well-known that in case of unexpected death of rare and wild animals, preserving epididymal spermatozoa from isolated testicles represents a great chance of salvaging male germplasm for future use in assisted reproductive technologies. The aim of this study was to evaluate the morphofunctional integrity of cat epididymal spermatozoa ultra-rapid frozen in pellets or straws with two different extenders [E1 (Tris buffer with 20% egg yolk and 0.25 M sucrose) or E2 (Ham's F10 with 1% bovine serum albumin and 0.4 M sucrose)] and to test whether spermatozoa preserved by the best combination were able to fertilize oocytes and produce embryos in vitro by intracytoplasmic sperm injection (ICSI) of in vitro matured cat oocytes. The results showed that E1 and E2 in straw or pellet were comparable (at warming, about 30% normal morphology, 45% intact membranes, and 20% intact acrosomes), except for post-warming motility that was better maintained along time by E1 pellet (21.7 ± 7.4% at warming and 3.6 ± 2.9% after 6 h). Such spermatozoa could fertilize conspecific oocytes and support embryonic development (cleavage 35.5%) as well as frozen control spermatozoa (cleavage 54.29%, p = 0.22). In conclusion, cat epididymal spermatozoa better maintained their morphofunctional features after ultra-rapid freezing with E1 and could successfully produce embryos in vitro after ICSI. This underscores their usefulness as cryobanked material for fertility and biodiversity preservation purposes.

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“…In this view, two protocols are presented that promote oocyte ultra-rapid freezing, also known as vitrification, in feline and ovine oocytes 3 , 4 . While Succu and colleagues highlight the importance of postwarming culture conditions and timing for the restoration of oocyte developmental potential in juvenile donors 4 that might have implications also in prepubertal human patients, Colombo and Luvoni focus their attention on the development of field-friendly protocols, mainly aiming at safeguarding wildlife biodiversity 3 .…”

Section: Discussionmentioning

confidence: 99%