Non-surgical deep intrauterine transfer of superfine open pulled straw (SOPS)-vitrified porcine embryos: evaluation of critical steps of the procedure.
Previous trials achieved extremely poor results when using the one-step warming method in a syringe in combination with non-surgical deep intrauterine transfer (NET) of superfine open pulled straw (SOPS)-vitrified embryos. This study aimed to assess the effect of the warming procedure on the in vitro and in vivo development of SOPS-vitrified embryos. The effect of the passage of the vitrified-warmed (VW) embryos through the NET catheter was also evaluated. Groups of 4 to 6 morulae and blastocysts, collected from weaned sows, were SOPS-vitrified in 1 μL of vitrification medium, warmed by the one-step warming method in a dish or in a 1-mL syringe and cultured in vitro for 48 h to evaluate the embryo survival (ES) and hatching rates (HR). Warming in syringe had a deleterious effect (P < 0.05) on the in vitro ES (60.5 ± 10.4%) and HR (39.6 ± 9.5%) of VW embryos in comparison with embryos warmed in a dish (85.4 ± 10.6% and 69.0 ± 8.4%, respectively). This decreased embryonic development was due to the increased time required between the removal of the straws from the liquid nitrogen and the contact of the embryos with the warming medium when the warming was performed in a syringe in comparison with that for the warming in a dish. After verifying that the passage of VW embryos through the NET catheter does not have a damaging effect on their further in vitro development, the negative effect of warming in a syringe was also confirmed after NET. Fifteen fresh and SOPS-vitrified embryos warmed in a syringe or in a dish were transferred to each recipient (n = 28) and recovered 24 h later to assess their developmental progression. All embryos from the syringe group were found to have degenerated at recovery. The in vivo ES and HR from the dish group (80.4 ± 3.4% and 14.2 ± 7.2%, respectively) were lower (P < 0.05) than those from the fresh group (94.0 ± 4.1% and 36.8 ± 7.8%, respectively). Combining the warming in a dish and the NET procedure, 35 VW embryos were transferred to each of 10 gilts. Five recipients farrowed an average of 10.4 ± 0.9 piglets. In conclusion, the method of one-step warming in a syringe has a negative effect on the in vitro and in vivo viability of SOPS-vitrified porcine embryos. In addition, NET of SOPS-vitrified embryos warmed by the one-step method in a dish showed promising reproductive performance of recipients. However, despite the great potential of this technology, further developments are required for large-scale commercial applications.
Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Regional Campus of International Excellence, University of Murcia, E-30071 Murcia, Spain.
J
J
Gomis
C
C
Cuello
J
J
Sanchez-Osorio
M A
MA
Gil
I
I
Parrilla
M A
MA
Angel
C
C
Maside
D
D
del Olmo
J M
JM
Vazquez
J
J
Roca
E A
EA
Martinez
D000818:Animals; D001755:Blastocyst / Q000502:physiology; D015925:Cryopreservation / Q000295:instrumentation / Q000379:methods / Q000662:veterinary*; D046149:Embryo Culture Techniques / Q000662:veterinary; D004624:Embryo Transfer / Q000379:methods / Q000662:veterinary*; D047108:Embryonic Development; D005260:Female; D006358:Hot Temperature; D007315:Insemination, Artificial / Q000662:veterinary; D009028:Morula / Q000502:physiology; D011247:Pregnancy; D034421:Sus scrofa / Q000196:embryology*; D020858:Tissue and Organ Harvesting / Q000662:veterinary