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.
Highlights
Oocyte cryopreservation has a strategic importance for the gene baking of female resources serving the preservation of genetic diversity and for the distribution of genetic lines
The percentages of fertilized and cultured oocytes that developed to the blastocyst stage in the vitrified groups warmed at 38uC or 42uC were significantly lower compared with the control group on day 5 (1.9%, 2.2% and 12.9%, respectively), day 6 (3.9%, 4.6% and 16.9%, respectively) and day 7 (4.6%, 5.2% and 17.9%, respectively) (Table 2)
When the total blastocyst yield was calculated as the percentage of day 7 blastocysts from the original number of oocytes subjected to vitrification, the vitrified group warmed at 42uC showed a significantly higher blastocyst yield compared with the vitrified group warmed at 38uC (14/615 = 2.760.3% and 19/ 534 = 4.460.4%, respectively)
Summary
Oocyte cryopreservation has a strategic importance for the gene baking of female resources serving the preservation of genetic diversity and for the distribution of genetic lines. Vitrification of matured porcine oocytes has been characterized by high survival rates; production rates and total yields of blastocyst stage embryos by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) have remained very low [2] Despite of their high survival rates, porcine oocytes vitrified at the MII stage often show spindle abnormalities, the accumulation of reactive oxygen species and parthenogenetic activation caused by the cooling/warming process, which greatly reduce their fertilization and developmental competence [3,4]. For this reason, production of live offspring from cryopreserved porcine oocytes has not been reported to date
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