Unless otherwise indicated chemicals were obtained from Sigma Chemical Company (St. Louis, MO).

In Vitro Maturation of Oocytes

Ovaries were collected at a local abattoir and transported to the laboratory in PBS containing 75 ^g/ml potassium penicillin G, 50 ^g/ml streptomycin sulfate, and 0.1% (w/v) polyvinyl alcohol (PVA). Cumulus-oo-cyte complexes (COCs) were collected from antral follicles (3.0-6.0 mm in diameter) of ovaries by aspiration. COCs having at least three layers of compacted cumulus cells were selected and cultured in NCSU23 medium supplemented with 0.6 mM cysteine, 10 ng/ml epidermal growth factor (EGF), 10% (v/v) porcine follicular fluid, 75 ^g/ml potassium penicillin G, 50 ^g/ml streptomycin sulfate, and 10 IU/ml of eCG and hCG (Teikoku Zouki Co., Tokyo, Japan). They were cultured for 22 h with hormones and then 22 h without hormones in a humidified atmosphere of 5% CO2 and 95% air at 38.5°C.

Electric Activation of IVM Oocytes

IVM oocytes with expanded cumulus cells were treated with 1 mg/ml hyaluronidase dissolved in Tyrode lactose medium supplemented with 10 mM Hepes and 0.3% (w/v) polyvinylpyrrolidone (PVP) (Hepes-TL-PVP), then denuded of cumulus cells by gentle pipetting. Oocytes having extruded the first polar body were selected and washed twice in an activation solution consisting of 0.3 M mannitol (Nacalai Tesque Inc., Tokyo, Japan), 50 ^M CaCl2, 100 ^M MgCl2, and 0.01% PVA. Next, they were lined up between two wire electrodes (1.0 mm apart) of a fusion chamber overlaid with 0.2 ml of activation solution, and a single 150-V/mm DC pulse was applied for 100 ^sec using an electrical pulsing machine (ET-1; Fu-jihira, Tokyo, Japan). Activated oocytes were finally treated with 5 ^g/ml cytochalasin B (CB) for 3 h.

In Vitro Culture of Embryos

In vitro culture of IVP embryos except for those in experiment 3 was performed in 20-^l droplets of NCSU23 supplemented with 4 mg/ml BSA under paraffin oil in a plastic Petri dish in humidified air with 5% CO2 at 38.5°C. For culturing embryos that developed beyond the morula stage, 10% (v/v) fetal calf serum (FCS) was added to the medium.

For culturing the IVP embryos for the first 2 days in experiment 3, NCSU medium was modified to contain 0.17 mM sodium-pyruvate and 0.17 mM sodium-lactate instead of glucose. The gas atmosphere used in experiment 3 was 5% CO2, 5% O2, and 90% N2.

Removal of Cytoplasmic Lipid Droplets from Embryos (Delipation)

Removal of cytoplasmic lipid droplets was carried out by a method described previously. Briefly, to polarize lipid granules in the cytoplasm, embryos were centrifuged (12 000 X g, 23 min) at room temperature in Hepes-TL-PVP containing 7.5 ^g/ml CB using a 1.5-ml microcentrifuge tube (Treff Lab, Schweiz, Switzerland). The resultant lipid layer was removed by micromanipulation using a beveled suction pipette (diameter, 28-32 ^m) attached to micromanipulators (MO-108; Narishige, Tokyo, Japan) under an inverted microscope (TE300; Nikon, Tokyo, Japan).

Noninvasive delipation of embryos was also carried out. IVP morulae at 4 days after activation were treated with 4% trypsin (in PBS) for 3.5 min, so that the zona pellucidae were swollen due to slight digestion. Embryos were then washed twice by PBS + 10% FCS. Embryos with swollen zona were centrifuged in the presence of CB as described above to polarize the cytoplasmic lipid droplets in the perivitelline space. After centrifugation embryos were subjected to vitrification within 2 min.

Vitrification of Embryos

Cryopreservation of embryos was carried out by vitrification using the MVC method. All solutions used during vitrification and thawing were prepared with TCM-199 containing 20 mM Hepes, 4.2 mM Na-HCO3, 75 ^g/ml potassium penicillin G, and 50 ^g/ml streptomycin sulfate as the basal medium. Embryos were equilibrated with equilibration solution containing 7.5% (v/v) ethylene glycol (Nacalai Tesque), 7.5% (v/ v) dimethylsulfoxide (DMSO; Wako Pure Chemical Industries Co., Osaka, Japan), and 20% (v/v) calf serum (JRH Biosciences, Inc., Lenexa, KS) for 4 min followed by exposure to vitrification solution containing 15% ethylene glycol, 15% DMSO, 0.5 M sucrose (Nacalai Tesque), and 20% calf serum. Embryos were then loaded onto an MVC plate (Cryotop; Kitazato Supply, Tokyo, Japan) and immediately plunged into liquid nitrogen. The process from exposure of embryos to plunging was completed within 1 min. Embryos were thawed by immersing the MVC plate directly into thawing solution containing 1 M sucrose and 20% calf serum at 37°C for 1 min. Recovered embryos were transferred to a diluent solution containing 0.5 M sucrose and 20% calf serum and kept for 3 min, after which they were kept for 10 min in a washing solution containing 20% calf serum in order to remove cryoprotectant. All solutions except for the thawing solution, were used at room temperature.

Fixation and Staining of Embryos

Cryopreserved and control IVP embryos were fixed with aceto-alcohol (1:3) 8 days after activation. Embryos were stained with 1% aceto-orcein to determine cell number.

Experiment 1

This experiment was performed to examine survival of IVP porcine blastocysts after cryopreservation. As the cryotolerance of in vivo-derived porcine blastocysts is known to reach maximum at the perihatching stage, including the expanded blastocyst stage, expanded blastocysts 6 days after parthenogenetic activation were subjected to vitrification.

We also tested whether survival of IVP porcine blastocysts after vitrification can be improved by delipation, which is known to be effective in rendering in vivo-derived porcine embryos cryotolerant. IVP embryos at the morula stage were delipated at 4 days after activation and further cultured for 2 days. Delipation at the morula stage was determined by our preliminary data that delipation of embryos at the expanded blastocyst stage turned out to be technically difficult. Embryos developing to expanded blastocysts were vitrified. Vitrified embryos and control IVP embryos were cultured up to Day 8 to examine their survival and cell number.

Experiment 2

This experiment was conducted to examine the possibility of cryopre-serving IVP porcine embryos at an early cleavage stage, assuming that IVP porcine embryos such as transgenic or cloned embryos are transferred to recipients at an early developmental stage.

At 2 days after activation, IVP embryos at the 4-cell stage were deli-pated, further cultured for 15 h, and vitrified. As a control, nondelipated embryos at the 4-cell stage were also vitrified. Vitrified embryos and nonvitrified IVP embryos were cultured up to Day 8 to examine their development to the blastocyst stage.

Experiment 3

The aim of this experiment was to investigate the possibility of cryo-preserving IVP embryos without using micromanipulation for delipation.

Survival of embryos vitrified after delipation by the noninvasive method (tryp/centr-morula; Fig. 1a) was compared with other embryos, including delipated morulae, morulae centrifuged without trypsin treatment (centr-morula; Fig. 1b), and intact control morulae.

Vitrified embryos and nonvitrified IVP embryos were cultured up to Day 8 to examine their development to the blastocyst stage.

Statistical Analysis

Survival rates of embryos were compared using x2-test. The Student /-test was used to compare mean cell numbers of embryos.