Abstract
BackgroundThe present studies evaluated the effects of cryoprotectants, the vitrification procedure and time in the warming solution containing sucrose on cleavage and embryo development of immature (GV stage) bovine cumulus-oocyte complexes (COCs).MethodsTwo experiments were conducted. In Experiment 1, COCs (n = 420) were randomly assigned to four groups: 1) Control group: no treatment; 2) VS1 group: COCs were exposed to vitrification solution 1 (VS1) containing 7.5% ethylene glycol [EG] + 7.5% dimethyl sulfoxide [DMSO] + 20% calf serum [CS] in TCM-199 at 37 C for 5 min; 3) VS1 + VS2 group: COCs were exposed to VS1 for 5 min followed by VS2 (15% EG + 15% DMSO + 17.1% sucrose + 20% CS) at 37 C for 45–60 sec; and 4) Vitrified group: COCs were exposed to VS1 and VS2, loaded on cryotops, vitrified in liquid nitrogen and then warmed in TCM-199 + 17.1% sucrose + 20% CS at 37 C for 1 min. In Experiment 2, COCs (n = 581) were assigned to the same groups, but those in VS1, VS1 + VS2 and Vitrified groups were sub-divided and exposed to the warming solution for either 1 or 5 min. After treatment and/or warming, all COCs in both experiments underwent in vitro maturation, in vitro fertilization and in vitro culture.ResultsCleavage and blastocyst rates did not differ among Control, VS1 and VS1 + VS2 groups in either experiment. In Experiment 2, there was no effect of time in the warming solution.However, both cleavage and blastocyst rates were lower (P < 0.001) in the Vitrified group than in the Control, VS1 and VS1 + VS2 groups (40.9 and 1.6% vs 92.2 and 34.4%, 79.4 and 25.2%, and 80.2 and 20.8%, respectively in Experiment 1, and 25.0 and 1.7% vs 75.3 and 27.2%, 67.9 and 19.5%, and 62.7 and 22.5%, respectively in Experiment 2).ConclusionsThe permeating cryoprotectants (EG and DMSO) present in VS1 and VS2 solutions and the time in the warming solution containing sucrose had no adverse effects on cleavage and blastocyst rates of immature bovine COCs. However, cleavage rate and early embryo development were reduced following the vitrification and warming.
Highlights
The present studies evaluated the effects of cryoprotectants, the vitrification procedure and time in the warming solution containing sucrose on cleavage and embryo development of immature (GV stage) bovine cumulus-oocyte complexes (COCs)
Chemicals and supplies Dulbecco’s phosphate buffered saline (DPBS), newborn calf serum (CS), Tissue Culture Medium-199 (TCM-199) and Minimum Essential Medium (MEM) non-essential amino acids were purchased from Invitrogen Inc. (Burlington, ON, Canada)
The immature COCs at the germinal vesicle (GV) stage were aspirated from follicles (3–8 mm in diameter) using an 18-gauge needle attached to a 5 ml syringe containing approximately 1.0 ml of DPBS supplemented with 5% CS (v/v)
Summary
The present studies evaluated the effects of cryoprotectants, the vitrification procedure and time in the warming solution containing sucrose on cleavage and embryo development of immature (GV stage) bovine cumulus-oocyte complexes (COCs). Slow freezing and vitrification are two common methods of cryopreservation for mammalian oocytes and embryos. Slow (controlled) freezing, cells dehydrate because of increased salt concentrations in the extracellular compartment resulting in a reduced likelihood of intracellular ice formation, but this results in increased intracellular salt concentrations referred to as the “solution effect” which can cause cell damage [6]. Vitrification exposes cells to relatively high concentrations of cryoprotectants and ultra-rapid cooling [7]. Vitrification is used to avoid chilling injury and ice crystal formation in the cryopreservation of tissue, embryos and oocytes [8,9,10]. Vitrification does not require a sophisticated and expensive programmable cell freezer, and is a fairly quick procedure, it requires skill and experience
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