Improving cryosurvival of in vitro produced bovine oocytes and embryos

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Cryopreservation of in vitro produced bovine embryos allow the maximum benefit from the genetic elite cows. However, pregnancy rates from cryopreserved embryos are still 15-20 % lower than that of their fresh counterparts. The slow rate freezing method allows the direct transfer of embryos without the need of time-consuming serial thawing to remove the high concentration of cryoprotective agents. The limiting factor of this procedure is the lower post-thaw developmental competence of embryos cooled by this method compared to vitrified embryos. To address this limitation, we supplemented in vitro produced bovine embryos with rho-associated coiled-coil containing kinase (ROCK) inhibitor (Y-27632). This treatment has been previously proved to improve the cryosurvival of vitrified IVP bovine embryos. Our results showed that in slow-cooling, exposure of embryos to ROCK inhibitor pre- and post-freeze resulted in the best post-thaw outcomes. Embryos exposed to ROCK inhibitor had similar outcomes as unfrozen control embryos and post-thaw exposure to ROCK inhibitor yielded greater benefits than pre-freezing exposure only. Overall, these results suggested that ROCK inhibitors may be beneficial for post-thaw embryo survival. For embryo vitrification, the cooling rate is a major determinant of intra-cellular ice crystals formation which are a major cause of cryodamage. Therefore, we conducted a study to characterize and evaluate the behavior of intra-cellular ice crystals during freezing and at thawing using synchrotron X-ray and X-ray diffraction. We used bovine MII oocytes vitrified using high and low cooling rates and vitrified with cryoprotective agent at concentrations ranging from 30 to 100% with 10% increments. Three types of mounts with different thermal mass including Micro-loops LD 100, thick Cryotop and thin Cryotop were used. Oocyte plunging in liquid nitrogen was conducted using an automated cooling machine (NANUQ). We observed that ice grain sizes remained consistently small when higher concentrations of cryoprotective agent were used. However, when lower CPA concentrations and slower rates of cooling and warming were used, larger ice grains were observed. The thermal response times of oocytes were significantly longer on Cryotop than for Micro-Loops LD. By employing well-optimized cooling protocols, such as a rapid cooling rate exceeding 600,000oC/min using Micro-Loop LD, oocytes vitrified effectively. This process allowed for reliable vitrification without any observable ice diffraction, or at most, faint and scattered cubic diffraction patterns. Successful vitrification was achieved using CPA concentrations as low as 6% DMSO, 6% EG, and 0.2 M sucrose, resulting in the formation of only small ice grains during warming. To test the efficacy of vitrification using the optimized cooling conditions tested in the previous study, we conducted a study to compare conditions deemed optimal in our previous study to the conventional Cryotop method. In vitro produced bovine embryos were assigned to one of three groups: NANUQ (ultra-fast cooling rate), Cryotop (fast cooling rate), and Control group (treated with the CPA as the other two groups but without freezing). Embryos were thawed and cultured for 48 hours in a time-lapse plate for monitoring post-thaw survival parameters. Sets of embryos from each group that hatched during 48 hours were used apoptotic cells staining whereas another set of embryos that hatched during the first 24 hours were used for RNA extraction and sequencing. Results showed that ultra-fast cooling improved cryosurvival of in vitro produced bovine embryos based on hatching rate and median time to re-expansion and hatching.

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115 pages


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bovine; cryopreservation; embryos; ice crystals; oocytes; synchrotron x-ray


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Union Local


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Cheong, Soon Hon

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Giordano, Julio
Duan, Jingyue
Selvaraj, Vimal

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Animal Science

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M.S., Animal Science

Degree Level

Master of Science

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Government Document




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dissertation or thesis

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