285 BIRTH OF PIGLETS AFTER NON-SURGICAL TRANSFER OF PORCINE EMBRYOS CULTURED IN PZM-4 WITH ALTERED CONCENTRATIONS OF AMINO ACIDS

We previously developed an in vitro production (IVP) system for porcine embryos and obtained piglets after surgical transfer of blastocysts cultured in Porcine Zygote Medium (PZM)-4. However, the developmental competence of pig IVP embryos to the blastocyst stage is still low and further improvement of IVC medium is needed. In the present study, we evaluated the effects of the addition of glutamine (Gln), hypotaurine (HT), taurine (Tau), BME-essential (EA) and MEM-nonessential (NA) amino acids solutions to PZM-4, and the replacement of polyvinyl alcohol (PVA) with BSA on embryo development to blastocysts. Moreover, the developmental competence of IVP blastocysts after nonsurgical embryo transfer (NS-ET), using a flexible catheter (FC) for deep intrauterine insemination, was investigated. Porcine COC from prepubertal gilts were matured and fertilized in vitro, using frozen-thawed ejaculated boar semen. Presumptive zygotes were cultured in PZM-4, as a basal culture medium, until Day 5 after IVF. Data from six replicates were analyzed by ANOVA. Addition of 0.25 to 4 mM Gln to PZM-4 (containing 5 mM HT) significantly increased the percentage of embryos that developed to blastocysts (15 to 31%), with addition of 2 mM Gln significantly increasing the total cell numbers in blastocysts (43 ± 17 cells) compared with no addition (3% and 20 ± 4 cells, respectively). Addition of 1.25 to 10 mM HT to HT-free PZM-4 supplemented with 2 mM Gln (named PZM-5) significantly increased the percentage of embryos that developed to blastocysts (22 to 28%) compared with control (no HT;; 4%). In the culture with HT-free PZM-5, addition of 5 mM Tau significantly increased blastocyst yield (17%) compared with control (4%). However, Tau addition in the presence of 5 mM HT had no effect on development to the blastocyst stage. In combinations of EA and NA added to PZM-5, a single dose of EA significantly increased the percentage of embryos that developed to blastocysts (27%) compared with no dose (19%) or with a double dose of EA (20%), while a double dose of NA significantly increased the total cell numbers in blastocysts (43 ± 16 cells) compared with no NA (37 ± 6 cells). Replacement of PVA with BSA in PZM-5 had no effect on embryo development to the blastocyst stage. Crossbred sows were used as recipients for NS-ET, and had their estrous cycle synchronized by a described previously method (Yoshioka et al., 2002 Biol. Reprod. 66, 112–119). Five days after hCG injection, a FC was introduced via the cervix into the uterine horn of recipients without sedation. Day-5 blastocysts cultured in PZM-5 were then transferred together with 5 ml of TALP-Hepes (45 to 50 blastocysts/recipient). Of 6 recipients, one sow became pregnant and farrowed 7 piglets. Our results indicate that the addition of amino acids to PZM-4 can improve porcine embryo development to the blastocyst stage, and that blastocysts cultured in a chemically defined medium, PZM-5, can develop to full-term following NS-ET.