Effective vitrification and warming of porcine embryos using a pH-stable, chemically defined medium

Abstract

The use of pH-stable media would simplify embryo vitrification and the warming of porcine embryos and might facilitate the application of embryo transfer in practice. In this work, we investigated whether a pH-stable basal medium constituted of Tyrode’s lactate medium, polyvinyl alcohol, and HEPES for buffering was suitable for porcine embryo vitrification warming in place of the conventional gas-equilibrated media. A high percentage (>90%) of embryos survived vitrification and warming in this medium, achieving in vitro survival rates similar to embryos vitrified-warmed using the conventional protocol and their fresh counterparts. The pH-stable medium did not affect the in vivo developmental competence of the vitrified-warmed embryos. A farrowing rate of 71.4% (5/7) with 10.4 ± 3.1 piglets born was obtained for the embryos vitrified and warmed in this medium and transferred to selected recipients. This medium will enable the use of simple, safe and standardized protocols for the vitrification and warming of porcine embryos for optimal embryo survival and quality when applied under field conditions. This study opens new possibilities for the widespread use of embryo transfer in pigs.

Achievements and future perspectives of embryo transfer technology in pigs

“…Tyrode's lactate (TL)–HEPES–PVA medium (TL‐PVA), which is used routinely for the collection and transfer of embryos, was tested. There was no difference in either post‐warming in vitro or in vivo development between the TL‐PVA medium and the traditional TCM‐199 medium (Cuello et al., ). Therefore, at present, a defined, pH‐stable (without CO2 gassing) medium for the collection, handling, vitrification, warming and transfer of porcine embryos is available, which is ideal for ET programmes under field conditions.…”

Vitrification of in Vivo-derived Porcine Morulae and Blastocysts Alters Metabolic and Stress Response Pathway Alterations

“…Vitri cation and warming were performed according to a previously described protocol [16] in 4-well tissue culture plates (Nunc A/C, Roskilde, Denmark). The basic medium (BM) for vitri cation and warming was TL-HEPES-PVA and all media were held at 39 °C.…”

“…Pig embryos have been challenging to cryopreserve due to their high amount of cytoplasmic lipids, which results in extreme chilling sensitivity [6], but signi cant advances have been made for this technique, thanks to the development of vitri cation protocols (reviewed in [5,[7][8][9][10]). The implementation of vitri cation protocols, particularly the open pulled straw (OPS) system [11] using super ne OPS (SOPS; [12] straws, has provided excellent viability in vitro reaching > 80% with vitri ed morulae and > 90%) in blastocysts [1][2][3]; further, encouraging farrowing rates (range: 38.9-75%) have been achieved after nonsurgical [13][14][15] and surgical [15,16] transfer of vitri ed morulae and blastocysts. However, although the published reports are few, pregnancy we have observed a higher pregnancy loss for vitri ed embryos (10-20%) than that of fresh embryos (< 2.5%) [17].…”

Developments of reproductive management and biotechnology in the pig

“…The challenges with AI regarding export of porcine genetics have been discussed earlier. Although sensitive to chilling and highly susceptible to intracellular ice formation, recent progress in oocyte and embryo cryopreservation is promising (Saragusty and Arav, 2011;Cuello et al, 2016;Nohalez et al, 2018). Porcine embryos have the potential to substantially accelerate genetic gain in pig populations and to facilitate international transport of genetics, while decreasing the carbon footprint due to reduced live animal transportation.…”

“…et al, 1989;Kobayashi et al, 1989;Brüssow and Rátky 1996;Besenfelder et al, 1997 Ovum pick-up (OPU) in donor sows OPU device for sows, OPU technique optimization, skill acquisition on live animals Brüssow et al, 1997;Antosik et al, 2007;Ikoma et al, 2014 II. Gametes: In vitro In vitro maturation (IVM), fertilization (IVF) and culture Funahashi and Day 1993; Laurincik et al,, 1994;Brüssow et al, 2000;Romar et al, 2012;Gil et al, 2017;Soriano-Úbeda et al, 2017 Oocytes/ embryos: In vitro Cryopreservation/ vitrification of embryos/ oocytes Berthelot et al, 2000;Cuello et al, 2016;Nohalez et al, 2018 III. Recipient sow synchronization Hormonal synchronization protocol Wilson et al, 1998;Martynenko et al, 2004;Brüssow et al, 2018 ET on recipient sows ET into the cranial portion of the corpus uteri Webel et al,, 1970;Galvin et al,, 1994;Hazeleger and Kemp 1994;Li et al, 1996;Yonemura et al, 1996;Rátky et al, 2001;Martinez et al, 2004;Martinez et al, 2016 533 The selection of recipient gilts or sows will have a major impact on ET results (Brüssow et al, 2018).…”