Chapter 21
Vitrification of Porcine Oocytes and Zygotes in Microdrops
on a Solid Metal Surface or Liquid Nitrogen
Tamas Somfai and Kazuhiro Kikuchi
Abstract
Oocyte cryopreservation is a potent approach to keep female germplasm safe from epidemic diseases. In the
last decade, we developed simple, cheap, and robust vitrification protocols which enable quick cryopreservation of immature porcine oocytes and zygotes in large numbers. In this chapter, we describe vitrification
procedures for porcine oocytes and zygotes where they are vitrified in 1–2 μL aliquots of a defined (proteinfree) vitrification medium and dropped either on a metal surface pre-cooled from the bottom with liquid
nitrogen (solid surface vitrification) or directly into liquid nitrogen. Vitrified microdrops can be stored in
cryo-vials in liquid nitrogen. Low concentrations of permeating cryoprotectants during equilibration and
proper temperatures during equilibration and warming are crucial for achieving high survival rates. The
device used for cooling does not seem to affect system efficacy as vitrification of oocytes or zygotes either on
Cryotop
® sheets or in microdrops were equally effective.
Key words Immature oocyte, In vitro embryo production, Porcine, Vitrification, Zygote
1 Introduction
Oocyte cryopreservation is a potent approach to keep female germplasm safe from epidemic diseases, and it enables the flexible use of
oocytes in time and space. Porcine oocytes are very sensitive to low
temperatures due to their high lipid content [1], and probably for
this reason, they do not survive traditional slow freezing [2]. Several
different vitrification protocols have been applied on porcine
oocytes with relatively high survival rates; however, subsequent
embryo development was greatly compromised [3]. Earlier results
suggested that porcine oocytes tend to survive vitrification procedures at higher rates after in vitro maturation compared with those
vitrified before maturation [4]. However, the subsequent developmental competence of oocytes vitrified at the immature stage was
remarkably better than that of those vitrified after maturation
[4]. For this reason, we developed a vitrification protocol specifically for immature porcine oocytes by the modification of the
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_21, © Springer Science+Business Media, LLC, part of Springer Nature 2021
455
Vitrification of Porcine Oocytes and Zygotes in Microdrops
on a Solid Metal Surface or Liquid Nitrogen
Tamas Somfai and Kazuhiro Kikuchi
Abstract
Oocyte cryopreservation is a potent approach to keep female germplasm safe from epidemic diseases. In the
last decade, we developed simple, cheap, and robust vitrification protocols which enable quick cryopreservation of immature porcine oocytes and zygotes in large numbers. In this chapter, we describe vitrification
procedures for porcine oocytes and zygotes where they are vitrified in 1–2 μL aliquots of a defined (proteinfree) vitrification medium and dropped either on a metal surface pre-cooled from the bottom with liquid
nitrogen (solid surface vitrification) or directly into liquid nitrogen. Vitrified microdrops can be stored in
cryo-vials in liquid nitrogen. Low concentrations of permeating cryoprotectants during equilibration and
proper temperatures during equilibration and warming are crucial for achieving high survival rates. The
device used for cooling does not seem to affect system efficacy as vitrification of oocytes or zygotes either on
Cryotop
® sheets or in microdrops were equally effective.
Key words Immature oocyte, In vitro embryo production, Porcine, Vitrification, Zygote
1 Introduction
Oocyte cryopreservation is a potent approach to keep female germplasm safe from epidemic diseases, and it enables the flexible use of
oocytes in time and space. Porcine oocytes are very sensitive to low
temperatures due to their high lipid content [1], and probably for
this reason, they do not survive traditional slow freezing [2]. Several
different vitrification protocols have been applied on porcine
oocytes with relatively high survival rates; however, subsequent
embryo development was greatly compromised [3]. Earlier results
suggested that porcine oocytes tend to survive vitrification procedures at higher rates after in vitro maturation compared with those
vitrified before maturation [4]. However, the subsequent developmental competence of oocytes vitrified at the immature stage was
remarkably better than that of those vitrified after maturation
[4]. For this reason, we developed a vitrification protocol specifically for immature porcine oocytes by the modification of the
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_21, © Springer Science+Business Media, LLC, part of Springer Nature 2021
455