Successful vitrification of pronuclear-stage pig embryos with a novel cryoprotective agent, carboxylated ε-poly-L-lysine

PLoS One. 2017 Apr 27;12(4):e0176711. doi: 10.1371/journal.pone.0176711. eCollection 2017.


Vitrification is a powerful tool for the efficient production of offspring derived from cryopreserved oocytes or embryos in mammalian species including domestic animals. Genome editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas)9 are now available even for domestic species, suggesting that the vitrification of embryos at the pronuclear stage (PN) will be more important because they could provide genomic host cells to be targeted by TALENs or CRISPR/Cas9. Although we reported the successful production of piglets derived from vitrified PN embryos by a solid-surface vitrification method with glutathione supplementation, further improvements are required. The cryoprotective agent (CPA) carboxylated ε-poly-L-lysine (COOH-PLL) was introduced in 2009. COOH-PLL reduces the physical and physiological damage caused by cryopreservation in mammalian stem cells and the vitrification of mouse oocytes and embryos. Those results suggested that vitrification of COOH-PLL may help improve the developmental ability of pig embryos vitrified at the PN stage. However, it remains unclear whether COOH-PLL is available as a CPA for the vitrification of embryos in domestic species. In this study, we evaluated COOH-PLL as a CPA with ethylene glycol (EG) and Cryotop as a device for the vitrification of PN pig embryos. Exposure to vitrification solution supplemented with COOH-PLL up to 30% did not decrease developmental ability to the 2-cell stage and the blastocyst stage. After warming, most of the vitrified embryos survived regardless of the concentration of COOH-PLL (76.0 ± 11.8% to 91.8 ± 4.6%). However, the vitrified embryos without COOH-PLL showed a lower development rate up to the blastocyst stage (1.3 ± 1.0%) compared to the fresh embryos (28.4 ± 5.0%) (p<0.05). In contrast, supplementation of 20% (w/v) COOH-PLL in the vitrification solution dramatically improved the developmental ability to blastocysts of the vitrified embryos (19.4 ± 4.6%) compared to those without COOH-PLL (p<0.05). After the transfer of embryos vitrified with 30% (v/v) EG and 20% (w/v) COOH-PLL, we successfully obtained 15 piglets from 8 recipients. Taken together, our present findings demonstrate for the first time that COOH-PLL is an effective CPA for embryo vitrification in the pig. COOH-PLL is a promising CPA for further improvements in the vitrification of oocytes and embryos in mammalian species.

MeSH terms

  • Animals
  • Blastocyst / drug effects
  • Blastocyst / physiology
  • Cryopreservation / methods
  • Cryopreservation / veterinary*
  • Cryoprotective Agents / adverse effects
  • Cryoprotective Agents / pharmacology*
  • Embryo Culture Techniques / veterinary
  • Embryo Transfer / veterinary
  • Embryonic Development*
  • Female
  • Polylysine / adverse effects
  • Polylysine / chemistry
  • Polylysine / pharmacology*
  • Pregnancy
  • Pregnancy Outcome / veterinary
  • Swine / embryology*
  • Swine / genetics
  • Vitrification


  • Cryoprotective Agents
  • Polylysine

Grant support

This work was supported in part by Grants-in-Aid for Scientific Research from JSPS KAKENHI Grant Number JP25242050 (SH.H. and K.M.), JP25660215 (N.K.) and JP15H04584 (J.I.). This research was partially supported by a research project grant awarded by the Azabu University Research Services Division to J.I. and N.K.. This work was also supported by Collaborative Research Project organized by the Interuniversity Bio-Backup Project (IBBP) to K.M..