Vitrification of immature bovine cumulus-oocyte complexes: effects of cryoprotectants, the vitrification procedure and warming time on cleavage and embryo developmentReportar como inadecuado




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Reproductive Biology and Endocrinology

, 10:73

First Online: 06 September 2012Received: 26 April 2012Accepted: 30 August 2012

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.

KeywordsCattle Cumulus-oocyte-complexes Germinal vesicle stage Cryoprotectants Ethylene glycol Dimethyl sulfoxide Vitrification Warming time In vitro maturation In vitro fertilization AbbreviationsCOCsCumulus-oocyte complexes

GVGerminal vesicle

VSVitrification solution

EGEthylene glycol

DMSODimethyl sulfoxide

CSCalf serum

TCM-199Tissue Culture Medium-199

IIFIntracellular ice formation

DPBSDulbecco’s phosphate buffered saline

MEMMinimum Essential Medium

IVMIn vitro maturation

IVFIn vitro fertilization

BOBrackett-Oliphant

IVCIn vitro culture

BSABovine serum albumin.

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Autor: Jennifer R Prentice-Biensch - Jaswant Singh - Reuben J Mapletoft - Muhammad Anzar

Fuente: https://link.springer.com/







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