106 THE WARMING PROCEDURE: A FIRST STEP FOR IMPROVING THE NONSURGICAL DEEP INTRAUTERINE TRANSFER OF SOPS-VITRIFIED PORCINE EMBRYOS

Abstract

We previously reported successful nonsurgical deep intrauterine embryo transfer (ET) of fresh in vivo–derived porcine embryos. However, several trials from our laboratory demonstrated that when this procedure was used in combination with vitrified/warmed (VW) embryos, its efficiency was very low. Recently, we have shown that the one-step warming method in syringe, which was used in the earlier trials, compromises the in vitro embryo viability. The aim of this study was to confirm the negative effect of the direct warming in syringe and to evaluate the effect of alternative warming procedures on the in vivo development of VW embryos after nonsurgical ET. In Experiment 1, morulae and early blastocysts were collected on Days 5 to 6 (Day 0: onset of oestrus) and assigned to one of the following groups: 1) syringe group: vitrified embryos (n = 88) were warmed by the one-step method directly in a 1-mL syringe containing 300 μL of warming medium, which was connected to the ET catheter and then transferred to recipients (n = 6); 2) dish group: vitrified embryos (n = 194) were warmed with one-step warming method in a Petri dish containing 1 mL of warming medium, loaded into a Tom Cat catheter and transferred to recipients (n = 13); and 3) control group: fresh embryos (n = 129) were loaded in a 1-mL syringe in 100 μL of transfer medium and transferred to 9 recipients. An average of 15 embryos were transferred to each recipient on Day 4 or 5. Embryos were surgically recovered 24 h after ET. Data were analysed by ANOVA. The embryo recovery rate was similar among groups (range: 70.7 ± 4.8% to 77.2 ± 6.5%). The embryo survival (ES) and the hatching rate (HR) from the control group (94.0 ± 2.1% and 33.4 ± 7.6%, respectively) were higher (P ≤ 0.05) than those from the dish group (80.4 ± 4.6% and 14.5 ± 4.1%, respectively). All embryos from the syringe group were degenerated. Some viable recovered embryos (n = 135) were cultured for 48 h to evaluate their subsequent in vitro development. No differences were observed in ES between the control and the dish group (100.0 ± 0.0% vs 98.9 ± 1.0%). The HR in the control group (71.5 ± 2.1%) was higher (P ≤ 0.01) than that of the dish group (42.7 ± 6.1%). In experiment 2 we evaluated the reproductive performance of naturally cycling recipients after nonsurgical ET of vitrified embryos warmed with the one-step method in a Petri dish. An average of 35 VW morulae and blastocysts were transferred to each recipient (n = 10) on Days 4 to 6. Four recipients returned to oestrus at Days 21 to 22. The remaining 6 recipients were diagnosed pregnant by ultrasonography on Day 26. At Days 50 to 55, 5 recipients remained pregnant. In conclusion, the one-step in syringe warming method for vitrified porcine embryos had a completely adverse effect on the vivo viability, whereas nonsurgical ET of embryos warmed in a Petri dish allowed us to obtain promising reproductive performance.

Supported by MICINN (AGL2009-12091) and SENECA (GERM 04543/07).