84 SUCCESSFUL CRYOPRESERVATION OF PORCINE EMBRYOS BY THE METAL MESH VITRIFICATION (MMV) METHOD

Abstract

The present study was designed to generate piglets from porcine embryos by the metal mesh vitrification (MMV) method. Prepuberal gilts were administered eCG and hCG and were artificially inseminated. Morulae and expanding blastocysts (diameter = approximately 200 μm) were collected at 144 h and 168 h after hCG injection, respectively. The metal mesh and the plastic plate (control) were used as sample containers for ultrarapid vitrification. The metal mesh (75 μm stainless steel mesh) was 1.5 mm wide and 10 mm long, and the 3 mm at the end of the mesh was bent at a right angle. Plastic plates, made from 0.25 mL plastic straws, were the same size and form as the metal mesh. Embryos were equilibrated with 7.5% ethylene glycol (EG) + 7.5% DMSO + 10% fetal bovine serum (FBS) in PBS for 5 min, followed by exposure to 15% EG + 15% DMSO + 0.6 M trehalose + 10% FBS in PBS for 1 min. Embryos were picked up on the metal mesh or loaded onto plastic plates with minimum volume of the solution, and then plunged into liquid nitrogen. Sample containers were placed in 1.8-mL cryotubes and stored in liquid nitrogen. Warming and dilution were performed by moving the container from liquid nitrogen into 0.5 M trehalose + 10% FBS in PBS at 37°C for 5 min. Embryos were rinsed twice in 4 mg/mL BSA + 10% FBS in NCSU37 (mNCSU37) for 5 min. Survival of the vitrified embryos was assessed after culture in mNCSU37 for 24 h (expanding blastocysts) or 48 h (morulae), and those that survived to fully expanded, hatching or hatched blastocysts were scored as viable. The vitrified warmed embryos were transferred surgically to recipient gilts. Experiment 1: The survival rates of expanding blastocysts vitrified by MMV or the control method were compared. The rate by MMV was significantly higher (84%; P < 0.01 by χ² test) than that of the control (53%). Experiment 2: The developmental stage (morula or expanding blastocyst) suitable for vitrification was examined. Survival of expanding blastocysts was significantly higher (84%; P < 0.05) than that of morulae (55%). Experiment 3: Expanding blastocysts were vitrified by MMV, warmed, and transferred into recipients (20 embryos per recipient). Eight of 10 recipients were found to be pregnant. Seven recipients farrowed a total of 37 live and 1 stillborn piglets. The other recipient miscarried and farrowed 4 stillborn piglets. These results showed that the viability of vitrified warmed porcine embryos was affected by the material of the sample container (Experiment 1) and also by the developmental stage (Experiment 2). Since the transferred embryos showed an excellent ability to develope into piglets (Experiment 3), the MMV method developed in the present study seems to be an effective preservation method for porcine embryos.