Ultrafast cooling reduced oxidative stress in vitrified bovine oocytes

Cryopreservation of gametes has been a revolutionary tool in assisted reproductive technology field. Traditionally, cryopreservation was performed by slowcooling (0.2 - 10°C/minute) method which induces ice formation in extracellular space and dehydration of intracellular space. Recently, fastcooling or vitrification-based approaches replaced slow cooling in human and equine gamete cryopreservation, with higher cryosurvival rates and superior clinical outcomes. Obstacles for cells to survive fastcooling include intracellular ice formation and exposure to high concentrations of cryoprotectants (CPAs) required to achieve vitrification. Increasing cooling rates should reduce ice formation, and ice formation during warming can be reduced by reducing volume of media around cell. Cryotop system is industry leader, achieving cooling rates of -3,000°C per minute. NANUQ™ Hyperquenching Cryocooler, designed to maximize cooling rate for protein crystallography, combines automated fast (< 2 minutes) plunging with removal of cold gas layer above liquid nitrogen, leading to ultra-fast cooling rates of ~ 600,000°C per minute. We hypothesized that ultrafast cooling rates would reduce oxidative stress from cryodamage of vitrified/warmed bovine oocytes. Bovine oocytes were collected from abattoirderived ovaries, matured in vitro, partially denuded of cumulus cells, then assigned to 1 of 4 groups:

1) negative control group which is not exposed to CPA and not cryocooled, 2) CPA control group which is exposed to CPA but not cooled, 3) Cryotop group with CPA exposure and cryocooling on Cryotops according to manufacturer protocols, and 4) NANUQ group with CPA exposure and cryocooling on microloops using NANUQ hyperquenching cooler. Oocytes were warmed, completely denuded and cultured for 1 hour in holding medium. Effect of treatment on oocyte oxidative stress was determined by fluorescent signal measured from cells after coincubation with green fluorescent general oxidative stress indicator CM-H2DCFDA. Images were transformed in ImageJ to derive corrected total cell fluorescence (CTCF) values, and these values were compared using ANOVA with Tukey multiple comparison on RStudio. A total of 281 oocytes were included. There were no differences (p > 0.05) in CTCF values between negative (n = 77) and vehicle control (n = 93) groups. CTCF values for cryocooled Cryotop (n = 65) and NANUQ (n = 46) groups were higher (p < 0.001) than control groups. For cryocooled groups, microloops cooled using NANUQ gave a lower (p < 0.001) CTCF value compared to Cryotop group. Experiment was repeated and in vitro fertilization was performed, but vitrified groups had poor cleavage rates and no blastocysts were produced, whereas negative and vehicle control groups had similar (p > 0.05) acceptable embryo development rates. NANUQ™ reduced postthaw oxidative stress of vitrified bovine oocytes compared to an industry standard vitrification device. Exposure to CPA did not have a significant effect on development.

Keywords: Automation, vitrification, in vitro fertilization, bovine

Acknowledgement Supported by Center for Advanced Technology, Cornell University

This manuscript was originally published in the journal Clinical Theriogenology Vol 12(3) Sept 2020.  Clinical Theriogenology is the official journal of the Society for Theriogenology (SFT) and the American College of Theriogenologists (ACT).  This content has been reproduced on the IVIS website with the explicit permission of the SFT/ACT.