120 SUCCESSFUL PRODUCTION OF PIGLETS DERIVED FROM VITRIFIED MORULAE AND EARLY BLASTOCYSTS WITHOUT DIRECT EXPOSURE TO LIQUID NITROGEN

Abstract

Recently piglets have been produced from morulae and early blastocysts vitrified by using methods such as open pulled straw and microdroplet. However, a problem of pollution, such as contamination of embryos with pathological microorganisms from the liquid nitrogen (LN), to the embryos is possible, because embryos must be exposed directly to LN in these methods. Therefore, we designed a new device in which the embryo is vitrified on the stainless steel chip inserted into a 0.25-mL straw without direct contact with LN. To serve as a stainless steel chip, a part of the body of a 19 G needle was cut to reveal its inner surface as the loading site for embryos, and 2 to 15 embryos in a small amount of a vitrification solution were placed on the inner surface of the needle. Then the needle with the embryos was inserted into the 0.25-mL straw which was pre-cooled in LN. This study was conducted to determine the efficiency of vitrification using the new device for porcine embryos at early developmental stages. In Experiment 1, embryos at the compact morula to early blastocyst stages were collected from 7 gilts. The embryos were equilibrated in 20 mM HEPES-buffered PZM (Yoshioka et al. 2003 Biol. Reprod. 69, 2092–2099) (H-PZM) supplemented with 1.8 M ethylene glycol (EG), and then in 1.8 M EG, 0.3 M sucrose, and 2% (w/v) bovine serum albumin (BSA) for 5 min. After equilibration, the embryos were vitrified using H-PZM supplemented with 0.6 M sucrose, 2% (w/v) BSA, and 4 M, 5 M, 6 M, or 7 M EG (ESB). The vitrified embryos were thawed, diluted by directly plunging the needle of the device into H-PZM supplemented with 1.8 M EG and 0.6 M sucrose for 5 min at 38°C, and subsequently cultured in PZM supplemented with 10% fetal bovine serum for 48 h; fresh embryos were cultured as a control group. In Experiment 2, embryos were vitrified using 5 M EG ESB, and then 56 vitrified embryos (collected from 4 gilts) were transferred to 4 recipient gilts (12 to 15 embryos per recipient). In Experiment 1, the survival rates of vitrified embryos after 48 h of culture in 4 M, 5 M, 6 M, 7 M, and control groups were 72.7, 86.4, 90.0, 87.0, and 100% (n = 20 to 23), respectively. The 4 M group showed a significantly lower survival rate than the control group (P < 0.05; chi-square test). The expanding and hatching rates of the vitrified embryos in these groups after 48 h of culture were 68.2, 77.3, 70.0, 65.2, and 95.0%, respectively. The 7 M group had a significantly lower expanding and hatching rate than the control group (P < 0.05). In Experiment 2, 3 recipients showed estrus at 19, 29, and 44 days after their previous estrus. One recipient became pregnant and 6 normal live piglets were born. These results indicated that porcine embryos at the compact morula to early blastocyst stages can survive after vitrification using our new device, which can prevent embryos from direct exposure to LN, and that piglets can be obtained from vitrified embryos without fear of pollution from LN.