Open pulled straw (OPS) vitrification: A new way to reduce cryoinjuries of bovine ova and embryos

TLDR

Cryopreservation efficiency varies widely across species, developmental stages, and origin, largely due to intracellular lipid content, microtubule organization, and cryoprotectant penetration governed by volume‑to‑surface ratio. This study reports vitrification of in vitro produced bovine embryos at multiple developmental stages using the Open Pulled Straw (OPS) method. OPS achieves ultra‑rapid cooling and warming (>20,000 °C/min) with brief (<30 s) exposure to concentrated cryoprotectants, thereby reducing chilling, toxic, and osmotic damage. Embryos vitrified from Day 3–7 reached blastocyst rates comparable to controls, Day 1–2 embryos showed only modest survival loss, 81 % of Day 8 hatched blastocysts survived, 25 % of 184 vitrified oocytes developed into blastocysts, and pregnancies were achieved after transfer at both oocyte and blastocyst stages, demonstrating OPS’s potential to advance reproductive cryobiology and biotechnology.

Abstract

Although cryopreservation of certain mammalian embryos is now a routine procedure, considerable differences of efficiency exist depending on stage, species and origin (in vivo or in vitro produced). Factors that are suspected to cause most of these differences are the amount of the intracellular lipid droplets and the different microtubular structure leading to chilling injury as well as the volume/surface ratio influencing the penetration of cryoprotectants. A new approach, the Open Pulled Straw (OPS) method, which renders very high cooling and warming rates (over 20,000 degrees C/min) and short contact with concentrated cryoprotective additives (less than 30 sec over -180 degrees C) offers a possibility to circumvent chilling injury and to decrease toxic and osmotic damage. In this paper we report the vitrification by the OPS method of in vitro produced bovine embryos at various stages of development. Embryos cryopreserved from Day 3 to Day 7 (Day 0 = day of fertilization) exhibited development into blastocysts at rates equivalent to those of control embryos; even those cryopreserved on Day 1 or 2 exhibited only somewhat reduced survival. Eighty-one percent of Day 8 hatched blastocysts also survived the procedure. The method was also successfully used for bovine oocytes; of 184 vitrified oocytes, 25% developed into blastocysts after fertilization and culture for 7 days. Pregnancies were achieved following transfer after vitrification at both the oocyte and blastocyst stage. The OPS vitrification offers a new way to solve basic problems of reproductive cryobiology and may have practical impact on animal biotechnology and human assisted reproduction.