209 VIABILITY OF PORCINE EMBRYOS VITRIFIED BY THE METAL MESH VITRIFICATION METHOD AFTER SURGICAL OR NONSURGICAL TRANSFER

Abstract

The aim of the present study was to evaluate viability of porcine embryos vitrified by the metal mesh vitrification (MMV) method after surgical or nonsurgical transfer. Prepubertal gilts were treated with eCG and hCG (= Day 0), and then inseminated artificially. Expanding blastocysts that were about 200 µm in diameter were collected on Day 7. The embryos were equilibrated in 7.5% ethylene glycol (EG) + 7.5% DMSO + 20% fetal bovine serum (FBS) in PBS at 37°C for 5 min, and then transferred into 15% EG + 15% DMSO + 0.6 M trehalose + 20% FBS in PBS for 1 min. Embryos in groups of 5 were transferred in a minimum volume of the vitrification solution (less than 1 µL) onto stainless steel mesh (75 µm screen size, 1.5 mm in width by 10 mm in length, with a 3-mm right-angle bend), and then plunged into liquid nitrogen. The mesh was stored in a 1.8-mL cryotube submerged in liquid nitrogen. Warming and dilution were performed by moving the mesh from liquid nitrogen into 0.5 M trehalose + 20% FBS in PBS at 37°C for 5 min. Embryos were rinsed twice in NCSU37 + 10% FBS (mNCSU37) for 5 min. After being vitrified, embryos in groups of 20 per recipient were suspended in modified NCSU37 medium and then transferred into gilts either by surgical transfer (5 gilts) or by nonsurgical transfer (6 sows). For surgical transfer, embryos suspended in 0.1 mL of medium were transferred into the uterine horn at 15 cm above the uterine body, which was about 35 cm from the external uterine orifice. For nonsurgical transfer, an intrauterine catheter made from polyethylene tubing (1.2 m long, 3.0 mm outer diameter, 0.5 mm inner diameter) was used. A spiral guide inserted through the vagina into the cervix was used to guide the catheter into one uterine horn. The catheter was moved through the cervix and along the uterine horn. Then, embryos suspended in 1 mL of medium were transferred. Pregnancy was assessed by ultrasonography at 30 days post-estrus. With surgical transfer, 4 of 5 recipients became pregnant, and 3 gilts farrowed a total of 21 (10, 8, 3) live piglets; the fourth gilt aborted one fetus on Day 34. With nonsurgical transfer, 3 of 6 sows became pregnant. The present study demonstrates that vitrified porcine embryos can develop after both surgical and nonsurgical transfer to recipients. As a practical measure, nonsurgical transfer of vitrified porcine blastocysts may be used instead of surgical transfer.