Effect of vitrification of in vitro matured prepubertal goat oocytes on embryo development after parthenogenic activation and intracytoplasmic sperm injection

Irene Menéndez-Blanco, Sandra Soto-Heras, María G. Catalá, Anna Rita Piras, Dolors Izquierdo, María Teresa Paramio (Corresponding author)

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1 Citation (Scopus)

Abstract

This work studies the effect of vitrification of in vitro matured (IVM) prepubertal goat oocytes on: 1) oocyte damage assessed by reactive oxygen species (ROS) level and apoptosis and 2) embryo development after Intracytoplasmic sperm injection (ICSI) and Parthenogenic Activation (PA). Oocytes were IVM in supplemented TCM-199 for 22-24 h. Control group oocytes matured during 24 h were directly used for the analysis after IVM. Vitrified/warmed IVM-oocytes were vitrified after 22 h of IVM in 15% ethylene glycol (EG), 15% dimethyl sulfoxide (Me2SO) and 0.5 M sucrose and after subjected to warming procedure. Oocyte ROS level was measured by staining denuded IVM-oocytes with 10 mu M 2'7' dichlorodihydrofluorescein diacetate. Apoptosis was analyzed by Annexin V (AV) Apoptosis Detection kit and Propidium iodide (PI) signal and oocytes were classified as: Live (AV(-) PI-), early apoptotic (AV(+) PI-), dead non-apoptotic (AV(-) PI+) and necrotic (AV(+) PI+). Developmental competence of vitrified/warmed oocytes was assessed by PA (5 min in 5 mu M Ionomycin plus 4 h in 2 mM 6-Dimethylaminopurine), and by ICSI fertilization. Presumptive zygotes were in vitro cultured for 8 days in commercial media BO-IVC. Vitrified/warmed oocytes showed higher ROS levels (P <0.0001), lower live oocytes (44 vs. 66%; P: 0.0025) and higher dead non-apoptotic oocytes (33 vs. 13% P: 0.023) compared to control. No differences were found on normal zygote formation (2 PN) (32 vs. 25%) or blastocyst development (0 vs. 4%) after ICSI fertilization. However, after PA, significant differences were found in cleavage rate (59 vs. 78%; P <0.0343) and blastocyst formation (1 vs. 25%; P <0.0001). In conclusion, vitrification reduced oocyte competence by increasing dead oocytes and ROS levels.

Original languageAmerican English
Pages (from-to)56-61
Number of pages6
JournalCryobiology
Volume93
DOIs
Publication statusPublished - 1 Apr 2020

Keywords

  • Goat
  • IVM-Oocytes
  • Prepubertal
  • Vitrification
  • APOPTOSIS
  • FERTILIZATION
  • OVINE OOCYTES
  • SHEEP
  • CRYOPRESERVATION
  • COMPETENCE
  • FUTURE
  • MATURATION
  • IMPROVES

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