Live Piglets Derived from In Vitro-Produced Zygotes Vitrified at the Pronuclear Stage1

Abstract

We report the successful cryopreservation of in vitro-produced porcine zygotes. Follicular oocytes from prepubertal gilts were matured (IVM), fertilized (IVF), and cultured (IVC) in vitro. At 10 or 23 h after IVF, the oocytes were centrifuged to visualize pronuclei. Zygotes with two or three pronuclei were used for solid surface vitrification (SSV). Survival of vitrified-warmed zygotes was determined by their morphology. To assess their developmental competence, vitrified (SSV), cryoprotectant-treated (CPA), and untreated (control) zygotes were subjected to IVC for 6 days. Survival and developmental competence did not differ between control and CPA zygotes. The proportion of live zygotes after SSV and warming (93.4%) was similar to that in the controls (100%). Cleavage and blastocyst formation rates of SSV zygotes after vitrification (71.7% and 15.8%, respectively) were significantly lower than those of controls (86.3% and 24.5%, respectively; ANOVA P<0.05). Blastocyst cell numbers of SSV and control embryos were similar (41.2+/-3.4 and 41.6+/-3.3, respectively). There was no difference in developmental ability between zygotes cryopreserved at an early (10 h after IVF) or late (23 h after IVF) pronuclear stage. Storage in liquid nitrogen had no effect on the in vitro developmental competence of vitrified zygotes beyond the reduction induced by the vitrification itself. When the embryo culture medium was supplemented with 1 muM glutathione, the rate of development of cryopreserved zygotes to the blastocyst stage did not differ significantly from that of control glutathione-treated zygotes (18.6% and 22.1%, respectively). To test their ability to develop to term, vitrified zygotes were transferred to five recipients, resulting in three pregnancies and the production of a total of 17 piglets. These data demonstrate that IVM-IVF porcine zygotes can be cryopreserved at the pronuclear stage effectively without micromanipulation-derived delipation, preserving their full developmental competence to term.

Changes in mitochondrial function in porcine vitrified MII-stage oocytes and their impacts on apoptosis and developmental ability

“…Porcine oocytes are particularly sensitive to low temperature. It was confirmed that their survival was essentially lost after exposure to a temperature of 15°C or below for a short period [2,30,35]. High lipid content in porcine oocytes was considered to be one of the main reasons for their sensitivity to low-temperature [23].…”

Factors Affecting Cryopreservation of Porcine Oocytes

“…The largeness of mammalian oocytes [9] and high intracellular lipid content specific to porcine species [10] are the most frequently referred factors which make porcine oocytes extremely sensitive to low temperatures. Nevertheless, since survival and development of vitrified porcine zygotes (having the same size and lipid content as oocytes) is significantly higher than those of vitrified mature or immature oocytes [11,12], ultra-structural characteristics specific to those unfertilized oocytes should also be considered as potential reasons for their low cryotolerance. In pigs, traditional freezing could be successfully applied for semen [5] and even for in vivo produced embryos [13][14][15]; however it does not seem to work well for oocytes [16].…”

In-straw Cryoprotectant Dilution for Bovine Embryos Vitrified Using Cryotop

“…For example, decreasing the sample volume to <1 μl and using carrier systems of minimum capacity increase the heat conduction of the sample, which can then be cooled very quickly. Various techniques have been reported in studies and have been applied with varying degrees of success in several mammalian species [13][14][15], including humans [16]. However, these methods have not been widely used for bovine ET on farms because of the necessity of sequential dilution of cryoprotectants upon warming of vitrified embryos in a laboratory setting.…”