61 APPLICATION OF AN OPEN DEVICE TO VITRIFY EQUINE IN VITRO-PRODUCED EMBRYOS

Abstract

Recently, we reported a >50% normal pregnancy rate for vitrified/warmed equine expanded blastocysts after blastocoele collapse via micromanipulation and vitrification in fine-diameter (250-µm) pipettes (Choi et al. 2011 Theriogenology 76, 143). This vitrification system has also yielded high pregnancy rates after transfer of blastocysts produced by intracytoplasmic sperm injection and embryo culture (IVP embryos) in our clinical program (unpublished data). However, the pipettes are difficult to load and must be manipulated to expel the embryo after warming. To improve this technique, we modified the pipettes by cutting their tip lengthwise and flaming the surface smooth, and we investigated the use of these open devices (designated “Sujo”) for vitrification of equine IVP embryos. Embryos were held in 1.5 M ethylene glycol in DMEM/F-12 + 20% FBS for 5 min; then moved to 7 M ethylene glycol and 0.6 M galactose; and within 1 min loaded on a Sujo with a minimum amount of medium, plunged into liquid N₂, and inserted into a 5-mL cryovial or 0.5-mL straw before being stored in liquid N₂. Embryos were warmed by placing the Sujo tip in 0.3 M sucrose in base medium (Dulbecco’s phosphate buffered saline with 0.1% glucose, 36 mg L^–1 of pyruvate, and 0.4% BSA). Embryos were left in this medium for 1 min and then moved to 0.15 M and then 0 M sucrose in base medium for 5 min each. In Experiment 1, we examined whether embryos stayed on the Sujo device. A total of 23 cleaved embryos were vitrified individually on Sujos; all of them were successfully recovered after warming. In Experiment 2, 18 IVP blastocysts were vitrified on Sujos (1 to 3 per Sujo), then warmed and cultured in 500-µL DMEM/F-12 + 20% FBS in an atmosphere of 5.5% CO₂, 5% O₂, and 89.5% N₂ at 38.2°C for 3 days. All 18 embryos grew in vitro. The average percentage increase in diameter (mean ± SEM) for embryos vitrified at 1 per Sujo was 73% (from 167 ± 4 to 289 ± 16 µm, 11 embryos); 2 per Sujo, 98% (from 187 ± 27 to 387 ± 106 µm, 4 embryos); and 3 per Sujo, 96% (from 176 ± 4 to 342 ± 51 µm, 3 embryos). There was no significant difference in percentage growth among treatments (one-way ANOVA; SigmaPlot 11.0). In Experiment 3, 11 IVP embryos were vitrified after loading singly on Cryolocks^®, then warmed and cultured as above. One embryo did not grow; the remaining 10 embryos grew an average of 73% (from 170 ± 3 to 294 ± 31 µm). In a preliminary study, 2 in vivo-recovered blastocysts (277 and 411 µm) were vitrified singly on Sujos and then warmed and cultured in vitro for 24 h under the above conditions increased in diameter to 594 and 729 µm, respectively. In conclusion, an open device can be used effectively for vitrification of equine IVP embryos. Further studies are needed to determine the efficiency of this system for vitrification of expanded equine blastocysts and the pregnancy rate of these embryos after transfer.

Supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts.