In vitro assessment of a direct transfer vitrification procedure for bovine embryos
Introduction
Since the first successful cryopreservation of mammalian embryos [1], cryopreservation of bovine embryos has been widely used commercially [2], [3]. However, there are opportunities for simplifying these cryopreservation methods for production.
Cryopreservation procedures are designed to avoid intracellular ice crystal formation. The first strategy for dealing with intracellular ice, termed conventional cryopreservation throughout this paper, is based on the principle of dehydration. Cooling rates are optimized to remove water from the embryo, preventing cryoinjury from ice crystal formation, while minimizing chemical toxicity/osmotic stress from exposure to high concentrations of salts. The most commonly used procedure for cryopreserving in vivo-produced bovine embryos consists of equilibration of embryos in cryoprotectant for 5–10 min, seeding at around −6 °C, and cooling at about 0.5 °C/min to around −32 °C, followed by plunging into liquid nitrogen. Originally, embryos were conventionally cryopreserved with glycerol (1–1.5 M). Glycerol resulted in acceptable pregnancy rates [4]; however, its use necessitated a sequential dilution of cryoprotectant upon thawing. The development of direct transfer [5] and an eventual switch from glycerol to ethylene glycol, a more permeable cryoprotectant [6] resulted in simplifying the process after thawing. The second strategy for dealing with intracellular ice is called vitrification. Rall and Fahy [7] first described vitrification of mammalian embryos in 1985. This technique requires high solution viscosity, rapid cooling rates, small volumes, and the use of high concentrations of cryoprotectant solutions to bring about a physical state similar to glass. With this procedure, crystalline ice does not form, and there is no concentration of solutes during the cryopreservation process [8]. Vitrification is attractive due to the fact that it is a relatively rapid and inexpensive procedure, and it has been shown to be beneficial for embryos that have lower cryosurvival, such as in vitro-produced embryos [9]. Numerous publications comparing conventional cryopreservation and vitrification of in vivo- or in vitro-produced bovine embryos report either similar or improved survival rates after vitrification [9], [10], [11], [12], [13], [14], [15], [16], [17]. Although multiple formulations, methodologies, and containers have been described, only one vitrification protocol has been tested extensively with field data [16]. This study used glycerol-based cryopreservation media to compare conventional cryopreservation and vitrification. Although the resulting pregnancy rates for both methods (conventional cryopreservation: 45.1% vitrification: 44.5%) were nearly identical, vitrification has not been adapted for routine cryopreservation of bovine embryos.
Vitrification is an economical alternative to conventional cryopreservation that might be implemented for routine field use; however, there is a need for more practical and reliable methodology as well as large field trials for evaluation. The purpose of this study was to develop a simple, rapid, efficacious vitrification technique based on ethylene glycol to permit in-straw dilution for direct transfer of bovine embryos. In this series of experiments morulae and blastocysts were obtained in vitro (with a subset in vivo) and vitrified after different exposure times, loading temperatures, cryoprotectant concentrations, warming temperatures, and post-warming holding temperatures and times to optimize vitrification procedures.
Section snippets
IVP/IVF/IVC
Oocytes were aspirated from 3- to 8-mm follicles of abbatoir-derived ovaries derived from mature cull cows. Oocytes were matured in chemically defined medium (CDM) [18] supplemented with 0.5% fatty acid free bovine serum albumin (FAF-BSA) (Sigma Chemical Co., St. Louis, MO, USA; A6003), 15 ng/mL NIDDK-oFSH-20, 1 μg/mL USDA-LH-B-5, 0.1 μg/mL E2, 50 ng/μL epidermal growth factor (Sigma E9644) and 0.1 mM cysteamine for 23 h at 38.5 °C and 5% CO2 in air. Sperm were separated through a Sperm-Talp [19]
Experiment 1: effects of cryoprotectant exposure time in V1, and loading temperature on vitrification of morulae and blastocysts
Results for experiment 1 are presented in Table 1. Loading straws with medium at room temperature (22–24 °C) resulted in higher re-expansion rates than at 4 °C when averaged over other factors (56 vs. 46%; P < 0.05). Short exposure times in V1 resulted in improved survival for morulae, but longer exposure was better for blastocysts. The re-expansion of blastocysts in the 3 min, 24 °C group was nearly identical to non-vitrified controls (82% versus 84%); however morulae had a lower survival rate.
Discussion
Vitrification has not been widely adopted by embryo transfer practitioners for commercial use in cattle [2], [8], [16]. The present study revealed that a simple two-step addition of cryoprotectant for vitrification appears to be efficacious, indicating that techniques could potentially reduce the overall cost of cryopreservation of embryos without compromising in vitro viability. The media formulations that we used were based on previous studies of the EFS40 vitrification media [21] with some
Acknowledgements
This research was supported in part by a grant from Bioniche. JFDS was supported by a scholarship from CONACyT and INIFAP, Mexico. DJW was supported by the Edward J. Carroll memorial scholarship. Zella Brink and students at the Embryo Transfer Laboratory at Colorado State University assisted with many aspects of this work.
References (36)
The current status and future of commercial embryo transfer in cattle
Anim Reprod Sci
(2003)- et al.
Production, freezing and transfer of bovine IVF embryos and subsequent calving results
Theriogenology
(1995) One-step method for direct transfer of frozen-thawed bovine embryos
Theriogenology
(1984)- et al.
Direct transfer of frozen-thawed bovine embryos
Theriogenology
(1992) Vitrification of the oocytes and embryos of domestic animals
Anim Reprod Sci
(2000)- et al.
Post-thaw survival and pregnancy rates of in vitro produced bovine embryos after vitrification
Theriogenology
(1994) - et al.
Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide
Theriogenology
(1993) - et al.
In-straw dilution of bovine IVF-blastocysts cryopreserved by vitrification
Theriogenology
(1994) - et al.
Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification
Cryobiology
(1999) - et al.
Field trial to compare pregnancy rates of bovine embryo cryopreservation methods: vitrification and one-step dilution versus slow freezing and three-step dilution
Theriogenology
(1997)
A new paper container for the vitrification of bovine embryos
Reprod Fertil Develop
Simple and efficient methods for vitrification of mammalian embryos
Anim Reprod Sci
Sugars exert major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes
Cryobiology
Vitrification properties of solutions of ethylene glycol in saline containing PVP, Ficoll, or dextran
Cryobiology
Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration
Anim Reprod Sci
Effects of vitrification medium composition on the survival of bovine in vitro produced embryos, following in straw-dilution, in vitro and in vivo following transfer
Anim Reprod Sci
Devitrification in glass-forming aqueous solutions
Cryobiology
Effect of culture system on the yield and quality of bovine blastocysts as assessed by survival after vitrification
Theriogenology
Cited by (0)
- 1
Both authors contributed equally to this research.