Generation of live piglets from cryopreserved oocytes for the first time using a defined system for in vitro embryo production

Abstract

We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42 °C during warming prevented temperature drops in a medium below 34.0 °C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38 °C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF) of the surviving oocytes after warming at 38 °C and 42 °C was not different but lower (P<0.05) than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively). However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF) from IVM medium (POM) did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group) resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized in vitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.

Figure 1. Dynamics of blastocyst formation during in vitro culture in PZM-5 medium of embryos derived by IVF from control and vitrified oocytes warmed at different temperatures.

Numbers of total blastocyst in each group are given in parentheses. Percentages (± SEM) with different superscripts (a, b) at the same time point differ significantly (P<0.05) by one-way ANOVA followed by Tukey’s multiple comparison test.

Figure 2. Changes of medium temperature during consecutive insertion of 6 vitrified microdrops (denoted with arrows) in a common 35 mm petri dish containing 2.5 ml of warming medium.

A) a typical pattern of temperature changes on a 38°C warmplate; B) a typical pattern of temperature changes on a 42°C warmplate; C) mean ± SEM values of maximum and minimum temperatures recorded on 38°C and 42°C warmplates. Values with different superscripts (a, b) between treatment groups differ significantly (P<0.05) by student’s t-test.

Figure 3. The first piglets obtained by the transfer of IVP blastocysts obtained from vitrified oocytes.

The photograph was taken 5 days after delivery.