Effective vitrification and warming of porcine embryos using a pH-stable, chemically defined medium

Abstract

The use of pH-stable media would simplify embryo vitrification and the warming of porcine embryos and might facilitate the application of embryo transfer in practice. In this work, we investigated whether a pH-stable basal medium constituted of Tyrode's lactate medium, polyvinyl alcohol, and HEPES for buffering was suitable for porcine embryo vitrification warming in place of the conventional gas-equilibrated media. A high percentage (>90%) of embryos survived vitrification and warming in this medium, achieving in vitro survival rates similar to embryos vitrified-warmed using the conventional protocol and their fresh counterparts. The pH-stable medium did not affect the in vivo developmental competence of the vitrified-warmed embryos. A farrowing rate of 71.4% (5/7) with 10.4 ± 3.1 piglets born was obtained for the embryos vitrified and warmed in this medium and transferred to selected recipients. This medium will enable the use of simple, safe and standardized protocols for the vitrification and warming of porcine embryos for optimal embryo survival and quality when applied under field conditions. This study opens new possibilities for the widespread use of embryo transfer in pigs.

Figure 1

pH values of basal (A), warming (B) and vitrification media (V1: (C) and V2: (D)) prepared using TCM or TL-PVA (TL) as the basal medium. Measurements of pH values were performed 0, 5, 10, 20, 30, 40, 50 and 60 min after the vitrification and warming plate preparation. Asterisks indicate significant differences between both media for each time point (*P < 0.05; **P < 0.01). A hash mark (#) indicates that from this time onwards the pH values significantly (P < 0.05) differed from the initial pH.

Figure 2. Total cell numbers in blastocysts derived from fresh (white bars) and vitrified morula and blastocysts (grey bars) after 48 and 72 h of in vitro culture, respectively.

The embryos were vitrified-warmed using TCM as the basic medium (TCM-TCM), vitrified using TL-PVA as basic medium and warmed with TCM (TL-TCM) or vitrified and warmed using TL-PVA (TL-TL). Data are expressed as the mean ± SD. Different letters represent differences (P < 0.05) among groups.

Figure 3. Experimental design of experiment 1 (In vitro embryo development after vitrification and warming using TL-Hepes or TCM-based media).

Figure 4. Experimental design of experiment 2 (In vivo embryo development of blastocysts vitrified-warmed using conventional TCM or TL-Hepes-based media).

Figure 5

Stereomicroscopic images of fresh and vitrified–warmed blastocysts (A–D) and morulae (A’–D’). (A,A’) Fresh blastocysts and morulae before vitrification. (B,B’) Blastocysts and morulae immediately after warming. (C,C’) Vitrified-warmed blastocysts and morulae after 24 h of in vitro culture. (D,D’) Vitrified-warmed blastocysts and morulae after 48 h of in vitro culture.