Elsevier

Theriogenology

Volume 65, Issue 6, 1 April 2006, Pages 1200-1214
Theriogenology

In vitro assessment of a direct transfer vitrification procedure for bovine embryos

https://doi.org/10.1016/j.theriogenology.2005.07.015Get rights and content

Abstract

We developed a simple vitrification technique for bovine embryos that could permit direct transfer. Embryos were produced in-vitro by standard procedures. The base medium for cryopreservation was a chemically defined medium similar to SOF + 25 mM Hepes and 0.25% fatty acid free bovine serum albumin (FAF-BSA) (HCDM2). In experiment 1, embryos were first exposed to 3.5 M ethylene glycol (V1) for 1, 2 or 3 min at room temperature (20–24 °C), and then moved to 7 M ethylene glycol (V2) at 4 or 20–24 °C and loaded in 0.25-mL straws. After 45 s in 7 M ethylene glycol, straws were placed in liquid nitrogen. Embryos that were loaded at 20–24 °C had higher survival rates than those loaded at 4 °C (P < 0.05). Exposure for 1 min was best for morulae, while 3 min was best for blastocysts. In experiment 2, blastocysts were handled at 24 °C and exposed to two concentrations of ethylene glycol in V1 (3.5 or 5 M) followed by V2 as in experiment 1, two warming temperatures (20 or 37 °C) and two post-warming holding times until culture (5 or 15 min). Exposure to 5 M ethylene glycol and warming at 37 °C was the optimal combination of procedures, and embryos survived well after 15 min in straws if warmed at 37 °C. In experiment 3, ethylene glycol concentration (3, 4 or 5 M) and exposure time (0.5 or 1 min) during two-step addition of cryoprotectant were studied for bovine morulae. In experiment 4, morulae were exposed to V2 for 30 or 45 s in HCDM2 or Vigro holding medium and then held in 22–24 °C air or 37 °C water post-warming. Experiment 5 was like experiment 4 except blastocysts were used. Overall survival rates of blastocysts in experiment 5 averaged 80% of non-vitrified controls after 48 h culture. The survival rates with in vitro-produced morulae in experiments 1, 3 and 4 were unacceptable. Vitrification solutions based on Vigro tended to result in higher survival than HCDM2 for blastocysts, but not morulae. In experiment 6, the survival rate in vitro of in vivo-produced morulae and blastocysts after two-step vitrification was nearly 100%. Our vitrification technique was very effective for in vitro produced blastocysts, but not for in vitro-produced morulae.

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.

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Both authors contributed equally to this research.

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