105vitrification of Bovine Embryos Using the CLV Method

Abstract

Vitrification has become the preferred method for cryopreserving in vitro-produced bovine embryos (IVP). Here we introduce a technique for vitrification developed at CryoLogic (the CLV Method), in conjunction with a study comparing the post-thaw viability of IVP embryos frozen by the widely used open pulled straw method (OPS—Vajta et al., 1997 Cryo-Letters 18, 191) and the new CLV Method. Vitrification on thin metal surfaces has been explored and demonstrated previously (Le Gal & Massip 1999, Cryobiology 38, 290), and Dinnyes presented a Solid Surface Vitrification (SSV) (Dinnyes et al., 2000, Biol. Reprod. 63, 513). The CLV Method utilizes vitrification on the surface of a solid metal block. This surface has been custom shaped and treated to enhance vitreous formation. The method also includes handling, storage and thawing protocols designed to avoid damage from crystallization of the unstable glass. Briefly, the block is precooled in LN2 to −196°C. Up to 10 embryos are collected into a droplet of medium (3μL), on the end of a fibre carrier attached to a handle. The droplet is presented to the vitrification surface, where it is converted into a glass bead by cooling rates comparable to that of plunging into solid/liquid phase nitrogen (−210°C) (Arav et al., 2001 Theriogenology 55, 313). The glass bead, fibre and handle are transferred quickly into a half-sealed, precooled straw, and the handle seals the open end. A bead is thawed very rapidly by removing the handle, fibre and bead from the straw and transferring the bead into washing medium. COCs collected from bovine ovaries obtained from abattoirs were matured, fertilized and cultured for 6 days (Fry et al., 2003 Theriogenology 59, 446). Embryos reaching the blastocyst or expanding blastocyst stage of development were graded (Grades 1, 2, and 3), equilibrated for 5min in HEPES-199 medium with 20% FCS (HFm), placed in HFm with 10% EG, and 10% DMSO (VS1) for 2min, and then transferred to HFm with 20% EG, 20% DMSO (VS2) for 30s (Vajta). Between 5 and 10 IVP embryos were processed and collected for vitrification, either in an OPS plunged into LN2, or in a 3μL droplet vitrified by the CLV Method. The two sets of specimens were stored in LN2, and later thawed. Both OPS tips and beads were thawed in 0.5mL of HFm with 0.2M sucrose at 39°C. Embryos were maintained at 39°C, examined after 5min for contraction, and again after 6 h for re-expansion. They were then transferred to culture medium, incubated and examined at 24 and 48h to assess hatching. As shown in Table 1, the CLV method appears to be satisfactory for maintaining membrane integrity (expansion) and developmental potential (hatching) for even poorer grade embryos, that might be more sensitive to the stresses of cryopreservation. Table 1 Re-expansion and hatching rates of graded thawed bovine embryos vitrified by OPS or CLV methods

Cell Counts and Survival to Vitrification of Bovine In Vitro Produced Blastocysts Subjected to Sublethal High Hydrostatic Pressure

“…However, comparisons are also technically difficult to make because of the differences in terms of culture systems or embryo stage at vitrification. In bovine, Lindemans et al. (2004) compared in vitro survival rates of bovine blastocysts vitrified by Open Pulled Straw (OPS) or CVM, and no differences were found in terms of re‐expansion or hatching rates between the two systems, and their results were in both cases comparable to those here presented.…”

Solid‐Surface Vitrification and In‐Straw Dilution After Warming of In Vitro‐Produced Bovine Embryos

“…Briefly, a 0.6 μL droplet of the vitrification solution containing an embryo was placed in a hook attached to a straw plug (called Fyberplugs™; Cryologic, Mulgrave, Australia) using a pipette and immediately exposed to the solid metal surface by touching the drop containing the embryo to the metal surface cooled at −196°C (Lindemans et al. ). Then the Fyberplugs were inserted into short plastic straw that were also cooled to −196°C by contact with metal block as described by Lindemans et al.…”

“…Then the Fyberplugs were inserted into short plastic straw that were also cooled to −196°C by contact with metal block as described by Lindemans et al. (). After at least 1 week storage in LN 2 tanks, vitrified embryos were warmed by firstly twisting off the Fyberplugs from the short plastic straws inside LN 2, to release pressure before removal from the short plastic straws, and immediate plunging of the drop containing the embryo in 0.25 m sucrose solution that was pre‐warmed at 39°C.…”

“…With this method, it is possible to use vitrification solutions with lower cryoprotectant concentrations, using higher cooling rates (>20 000°C/min) and minimum standard volumes (0.6 μL) (Lindemans et al. ; Peachey et al. ; Dinnyes and Nedambale ).…”

Practical applications of new research information in the practice of bovine embryo transfer

“…A good example is the changes that were made in the 1970s from surgical to non-surgical embryo collections and transfers that revolutionised the ET industry (Baker and Jillella 1978;Rowe et al 1980). Other new technologies are constantly being applied to several areas within the cattle industry, including (but not limited to) embryo production (Lonergan 2007), stem cell research (Oback 2009), in vivo-and in vitro-produced embryo cryopreservation Vajta and Kuwayama 2006), vitrification (Lindemans et al 2004;Horvath and Seidel 2006), embryo lipolysis (Tominaga et al 2000), ultrasound-guided follicular aspirations for in vitro embryo production (IVP; Callesen et al 1987;van der Schans et al 1991;Looney et al 1994) and the use of sexed spermatozoa in embryo production (Garner and Seidel 2008). However, these technologies raise the following questions: (1) which new advancements can benefit my practice; and/or (2) is the product or procedure regulated and, if so, what are these regulations?…”