In vitro maturation of equine oocytes followed by two vitrification protocols and subjected to either intracytoplasmic sperm injection (ICSI) or parthenogenic activation

Nowak Agnieszka; Kochan Joanna; Witarski Wojciech; Okólski Adam

doi:10.1016/j.theriogenology.2020.12.022

In vitro maturation of equine oocytes followed by two vitrification protocols and subjected to either intracytoplasmic sperm injection (ICSI) or parthenogenic activation

Theriogenology. 2021 Mar 1:162:42-48. doi: 10.1016/j.theriogenology.2020.12.022. Epub 2021 Jan 4.

Authors

Nowak Agnieszka¹, Kochan Joanna², Witarski Wojciech³, Okólski Adam⁴

Affiliations

¹ University of Agriculture in Krakow, Department of Animal Reproduction, Anatomy and Genomics, Al. Mickiewicza 24/28, 30-059, Krakow, Poland. Electronic address: nowak.a.a@gmail.com.
² University of Agriculture in Krakow, Department of Animal Reproduction, Anatomy and Genomics, Al. Mickiewicza 24/28, 30-059, Krakow, Poland.
³ National Research Institute of Animal Production, Department of Animal Molecular Biology, Ul. Krakowska 1, 32-083, Balice Near Krakow, Poland.
⁴ University of Agriculture in Krakow, University Centre of Veterinary Medicine UJ-UR, Al. Mickiewicza 24/28, 30-059, Krakow, Poland.

PMID: 33444915
DOI: 10.1016/j.theriogenology.2020.12.022

Abstract

The aim of this study was determine the viability and developmental competence of equine oocytes after IVM and vitrification using the Rapid-I method, as part of an effort to develop an effective equine oocyte vitrification protocol. Equine oocytes were collected by scraping ovarian follicles of slaughtered mares. A total of 1052 ovaries were used in this study, from which 3135 oocytes were obtained. Of the 2853 oocytes retrieved, 2557 underwent in vitro maturation for approximately 36 h. After in vitro culture, 1202 oocytes (47%) had a first polar body. To evaluate the toxicity of the solutions (Experiment I), oocytes were exposed to vitrification media without cryopreservation. Of all the experimental groups evaluated, the best results were obtained for IVM oocytes exposed to EquiproVitKit media (IVM + TOX EquiVitKit), with a viability rate of 69.5%. In the Experiment II, oocytes, either freshly collected from the ovary or after in vitro maturation (IVM), were vitrified using either the EquiPro VitKit or an in-house medium containing 18% Ficoll, 40% ethylene glycol and 0.3 M sucrose. Oocytes were stained with fluorescein diacetate and ethidium bromide to evaluate viability. In vitro matured oocytes vitrified using EquiproVitKit media (IVM + VIT EquiVitKit) had a cryosurvival rate of 63%. In the last part of the study (Experiment III), vitrified IVM oocytes were activated by 7.5 μM ionomycin in TCM-199 for 5 min TCM 199 (5 min) combined with 2 mM 6-DMAP in TCM-99 with 10% FBS (4.5 h) or in vitro fertilized using ICSI. Development of potential embryos after activation in TCM-199 medium, showed a cleavage rate was 10.2%, compared to 22.5% of oocytes cultured in G1/G2 medium. ICSI of vitrified IVM oocytes resulted in 20% embryo development to the 16-cell stage, compared to 33.3% in the control. The vitrification of oocytes after IVM by Rapid-I method is a good way to preserve genetic material in horses.

Keywords: Embryo development; Equine; ICSI; Oocyte maturation; Parthenogenetic activation; Vitrification.

MeSH terms

Animals
Cryopreservation / veterinary
Female
Horses
In Vitro Oocyte Maturation Techniques / veterinary
Oocytes
Parthenogenesis
Sperm Injections, Intracytoplasmic* / veterinary
Vitrification*