Birth of piglets from in vitro-produced, zona-intact porcine embryos vitrified in a closed system

Abstract

As the importance of swine models in biomedical research increases, it is essential to develop low-cost, high-throughput systems to cryopreserve swine germplasm for maintenance of these models. However, porcine embryos are exceedingly sensitive to low temperature and successful cryopreservation is generally limited to the use of vitrification in open systems that allow direct contact of the embryos with liquid nitrogen (LN(2)). This creates a high risk of pathogen transmission. Therefore, cryopreservation of porcine embryos in a "closed" system is of very high importance. In this study, in vitro-produced (IVP) porcine embryos were used to investigate cryosurvival and developmental potential of embryos cryopreserved in a closed system. Optimal centrifugal forces to completely disassociate intracellular lipids from blastomeres were investigated using Day-4 embryos. Cryosurvival of delipidated embryos was investigated by vitrifying the embryos immediately after centrifugation, or after development to blastocysts. In this study, centrifugation for 30 min at 13,000 g was adequate to completely delipidate the embryos; furthermore, these embryos were able to survive cryopreservation at a rate comparable to those centrifuged for only 12 min. When delipidated embryos were vitrified at the blastocyst stage, there was no difference in survival between embryos vitrified using OPS and 0.25 mL straws. Some embryos vitrified by each method developed to term. These experiments demonstrated that porcine embryos can be cryopreserved in a closed system after externalizing their intracellular lipids. This has important implications for banking swine models of human health and disease.

Fig. 1

Thermodynamic properties of the vitrification solution used in this experiment. Tg = vitrification temperature; Td = devitrification temperature; Tm = melting temperature.

Fig. 2

Delipidation of Day 4 porcine embryos by centrifugation: complete disassociation of intracellular lipids after centrifugation (a); externalized lipid droplets outside blastomeres at 48 h after centrifugation (b); embryos immediately after warming showing externalized lipid droplets (c); and vitrified blastocysts 24 h after warming (d). The scale bars in (a) and (b) represent 20 μm, and in (c) and (d) represent 50 μm.

Fig. 3

Cryosurvival of cryopreserved porcine Day 4 embryos using OPS vitrification after 48 h of culture as assessed by the formation of blastocysts. Control was non-centrifuged and non-vitrified embryos. A portion of warmed embryos were treated with 0.1% pronase to remove their zona pellucida immediately after warming (zona-free group). Values in parenthesis indicate number of embryos (three replications). ^a,bColumns without a common superscript differed (P < 0.05).

Fig. 4

In vitro survival of porcine embryos vitrified at blastocyst stage after delipidation using centrifugation at 13,000 g for 30 min at 4-8 cell stages. Number of blastocysts in each treatment group is shown in parenthesis (three replications). ^a,bWithin each category, columns without a common superscript differed (P < 0.05).