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.

Porcine embryo production following in vitro fertilization and intracytoplasmic sperm injection from vitrified immature oocytes matured with a granulosa cell co-culture system

“…Therefore we suggest for GV oocyte vitrification to perform it with denuded oocytes using a granulosa cell co-culture system during maturation. In addition, these results clearly indicate that even if live offspring has been obtained from vitrified immature oocytes [45] the procedure for vitrification and embryo production could be improved by the use of the co-culture system and the Cryolock method in order to increase live offspring.…”

“…The vitrification of denuded oocytes and the employment of a granulosa cell co-culture during maturation can improve survival and maturation rates [5]. Recently, it was also reported the first successful piglet production from cryopreserved immature oocytes [45]. Despite this, percentages of embryo survival and development are considerably low.…”

“…The oocyte developmental stage is considered a critical factor for its survival and developmental competence after cryopreservation [40]. In comparison with other mammals, porcine oocytes have shown to be more difficult to cryopreserve in the germinal vesicle stage (GV) [26,32,45]. This fact has been attributed to their high lipid content [34], which decreases in the following developmental stages.…”

New Trends in Assisted Reproduction Techniques: Cryopreservation, In vitro Fertilization, Intracytoplasmic Sperm Injection and Physiological Intracytoplasmic Sperm Injection

“…This contributes to their low ability to survive vitrification. Despite this, it has been reported the first generation of live piglets from cryopreserved immature oocytes [29]. Even that mature oocytes show high viability rates and recovery, their developmental competence is decreased.…”

Effects of two combinations of cryoprotectants on the in vitro developmental capacity of vitrified immature porcine oocytes

“…In addition, vitrification at this stage may avoid spindle damage [10], which is one of the most common alterations observed in vitrified MII oocytes [12]. Although live offspring have been obtained from vitrified immature oocytes [13], the total yield of blastocyst stage embryos remains very low.…”