Cryopreservation of equine embryos by open pulled straw, cryoloop, or conventional slow cooling methods.
Abstract: Cryopreservation of equine embryos with conventional slow-cooling procedures has proven challenging. An alternative approach is vitrification, which can minimize chilling injuries by increasing the rates of cooling and warming. The open pulled straw (OPS) and cryoloop have been used for very rapid cooling and warming rates. The objective of this experiment was to compare efficacy of vitrification of embryos in OPS and the cryoloop to conventional slow cool procedures using 0.25 mL straws. Grade 1 or 2 morulae and early blastocysts (< or = 300 microm in diameter) were recovered from mares on Day 6 or 7 post ovulation. Twenty-seven embryos were assigned to three cryopreservation treatments: (1) conventional slow cooling (0.5 degrees C/min) with 1.8 M ethylene glycol (EG) and 0.1 M sucrose, (4) vitrification in OPS in 16.5% EG, 16.5% DMSO and 0.5 M sucrose, or (3) vitrification with a cryoloop in 17.5% EG, 17.5% DMSO, 1 M sucrose and 0.25 microM ficoll. Embryos were evaluated for size and morphological quality (Grade 1 to 4) before freezing, after thawing, and after culture for 20 h. In addition, propidium iodide (PI) and Hoechst 33342 staining were used to assess percent live cells after culture. There were no differences (P > 0.1) in morphological grade or percent live cells among methods. Mean grades for embryos after culture were 2.9 +/- 0.2, 3.1 +/- 0.1, and 3.3 +/- 0.2 for conventional slow cooling, OPS and cryoloop methods, respectively. Embryo grade and percent live cells were correlated, r = 0.66 (P < 0.004). Thus OPS and the cryoloop were similarly effective to conventional slow-cooling procedures for cryopreserving small equine embryos.
Publication Date: 2001-03-10 PubMed ID: 11233786DOI: 10.1016/s0093-691x(01)00429-0Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research study explores the effectiveness of various methods of cryopreserving, or freezing, small horse embryos: conventional slow-cooling, open pulled straw (OPS), and cryoloop. It concludes that all three methods are similarly effective.
Objective of the Study
- The main goal of this experiment was to compare the efficacy of two vitrification methods (OPS and cryoloop) with conventional slow cooling techniques for preserving small horse embryos. Vitrification is a rapid freezing process aimed at reducing freeze-associated injuries.
Procedure of the Study
- The study used embryos which were either ‘grade 1 or 2 morulae’ and ‘early blastocysts’, not exceeding 300 microm in diameter, taken from mares on day 6 or 7 following ovulation.
- Three different methods of cryopreservation were followed for twenty-seven embryos – conventional slow cooling, vitrification in OPS, and vitrification with a cryoloop. Each method uses different concentration of cryoprotectant substances.
- The embryos were evaluated for size and morphological quality before freezing, after thawing, and after being cultured for 20 hours.
- The study used propidium iodide (PI) and Hoechst 33342 staining to calculate the percentage of live cells after culture.
Results of the Study
- The experiment revealed no significant differences in morphological grade or percentage of live cells among the three cryopreservation methods.
- However, the study did find a correlation (r = 0.66, P < 0.004) between embryo grade and percent live cells.
Conclusion of the Study
- The study concluded that the OPS and the cryoloop were equally as effective as the conventional slow-cooling procedures for cryopreserving small equine embryos.
Cite This Article
APA
Oberstein N, O'Donovan MK, Bruemmer JE, Seidel GE, Carnevale EM, Squires EL.
(2001).
Cryopreservation of equine embryos by open pulled straw, cryoloop, or conventional slow cooling methods.
Theriogenology, 55(2), 607-613.
https://doi.org/10.1016/s0093-691x(01)00429-0 Publication
Researcher Affiliations
- Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins 80523, USA.
MeSH Terms
- Animals
- Benzimidazoles / chemistry
- Cryopreservation / methods
- Cryopreservation / veterinary
- Female
- Fluorescent Dyes / chemistry
- Horses / embryology
- Horses / physiology
- Insemination, Artificial / veterinary
- Male
- Microscopy, Fluorescence / veterinary
- Pregnancy
- Propidium / chemistry
Citations
This article has been cited 2 times.- Qi B, Ji QS, Hou GH, Li L, Cao XF, Wu J. Comparison of conventional and directional freezing for the cryopreservation of human umbilical vein endothelial cells. Int J Ophthalmol 2014;7(5):768-72.
- Han X, Ma H, Jiao A, Critser JK. Investigations on the heat transport capability of a cryogenic oscillating heat pipe and its application in achieving ultra-fast cooling rates for cell vitrification cryopreservation. Cryobiology 2008 Jun;56(3):195-203.
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