FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Cryobiology / Effect of dehydration prior to cryopreservation of large equ...
Cryobiology2009; 59(1); 36-41; doi: 10.1016/j.cryobiol.2009.04.003

Effect of dehydration prior to cryopreservation of large equine embryos.

Abstract: Cryopreservation of equine embryos>300microm in diameter results in low survival rates using protocols that work well for smaller equine embryos. These experiments tested the potential benefit of incorporating a dehydration step prior to standard cryopreservation procedures. Forty-six, day 7-8, grade 1, equine embryos 300-1350microm in diameter were subjected to one of the following treatments: (A) 2 min in 0.6M galactose, 10min in 1.5M glycerol, slow freeze (n=21); (B) 10min in 1.5M glycerol, slow freeze (n=15); (C) 2min in 0.6M galactose, 10min in 1.5M glycerol, followed by exposure to thaw solutions, then culture medium (n=5); (D) transferred directly to culture medium (n=5). Frozen embryos were thawed and subjected to a three-step cryoprotectant removal. Five embryos from each treatment were evaluated morphologically after 24 and 48h culture (1=excellent, 5=degenerate/dead). All treatments had at least 4/5 embryos with a quality score >or=3 at these time points except treatment B (2/5 at 24h, 1/5 at 48h). Subsequent embryos from treatment A (n=16) or B (n=10) were matched in sets of two for size and treatment, thawed, and immediately transferred in pairs to 13 recipients. Only two recipient mares were pregnant; one received two 400microm embryos from treatment A, and the other one 400 and one 415microm embryo from treatment B. There was no advantage of incorporating a 2min dehydration step into the cryopreservation protocol for large equine embryos.
Get Full Text
Publication Date: 2009-04-16 PubMed ID: 19375416PubMed Central: PMC2706293DOI: 10.1016/j.cryobiol.2009.04.003Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article
  • Research Support
  • N.I.H.
  • Extramural
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research paper analyses the effects of a dehydration step before cryopreservation on large equine embryos. The results suggest that inclusion of a 2min dehydration step does not provide any significant benefits to the survival rates of such embryos post cryopreservation.

Methodology and Experimental Groups

  • The researchers worked with 46 grade 1 equine embryos ranging in size from 300-1350 micrometers, all at day 7-8 of development.
  • These embryos were subjected to one of four treatments which varied in terms of exposure to galactose, glycerol, a thaw solution, a slow freeze process, and direct transfer to a culture medium.
  • Embryos were divided and subjected to treatments A (21 embryos), B (15 embryos), C(5 embryos), and D (5 embryos).

Post Treatment Evaluations

  • The embryos were slow frozen and then thawed.
  • The embryos underwent a three-step cryoprotectant removal process.
  • Each group had 5 embryos evaluated morphologically after 24 and 48 hours of culture.
  • The evaluation used a score from 1 (excellent) to 5 (degenerate or dead).
  • All treatments had most of the embryos score at least 3 except for group B which had fewer.

Embryo Transfer

  • Remaining embryos from groups A and B were matched in pairs for size and treatment.
  • These embryos were thawed and immediately transferred into pairs to 13 recipient mares.
  • Out of the 13 recipient mares, only two were pregnant – one that received two 400 micrometer embryos from group A, and the other received one 400 and one 415 micrometer embryo from group B.

Conclusions

  • The researchers concluded that the inclusion of a 2min dehydration step in the cryopreservation protocol for large equine embryos does not confer any significant advantages.
  • Therefore, it seems that such a step might not be necessary for the successful cryopreservation of larger embryos.

Cite This Article

APA
Barfield JP, McCue PM, Squires EL, Seidel GE. (2009). Effect of dehydration prior to cryopreservation of large equine embryos. Cryobiology, 59(1), 36-41. https://doi.org/10.1016/j.cryobiol.2009.04.003

Publication

Cryobiology
ISSN: 1090-2392
NlmUniqueID: 0006252
Country: Netherlands
Language: English
Volume: 59
Issue: 1
Pages: 36-41

Researcher Affiliations

Barfield, J P
  • Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO 80523-1683, USA. jbarfiel@colostate.edu
McCue, P M
    Squires, E L
      Seidel, G E

        MeSH Terms

        • Animals
        • Cryopreservation / methods
        • Cryoprotective Agents / pharmacology
        • Culture Media
        • Embryo Culture Techniques
        • Embryo Transfer / methods
        • Female
        • Fertilization in Vitro / methods
        • Freezing
        • Glycerol / chemistry
        • Horses
        • Pregnancy
        • Pregnancy, Animal
        • Time Factors
        • Water

        Grant Funding

        • T32 HD007031 / NICHD NIH HHS
        • HD07031 / NICHD NIH HHS

        References

        This article includes 25 references
        1. Bass LD, Denniston DJ, Maclellan LJ, McCue PM, Seidel GE Jr, Squires EL. Methanol as a cryoprotectant for equine embryos.. Theriogenology 2004;62:1153–1159.
          pubmed: 15289054
        2. De La Torre-Sanchez F, Preis K, Seidel GE Jr. Metabolic regulation of in vitro-produced bovine embryos I. Effects of metabolic regulators at different glucose concentrations with embryos produced by semen from different bulls.. Reprod Fertil Dev 2006;18:585–596.
          pubmed: 16836965
        3. Hinrichs K, Choi YH, Walckenaer BE, Varner DD, Hartman DL. In vitro-produced equine embryos: production of foals after transfer, assessment by differential staining and effect of medium calcium concentrations during culture.. Theriogenology 2007;68:521–529.
          pubmed: 17586036
        4. Hiraoka K, Hiraoka K, Kinutani M, Kinutani K. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts.. Hum Reprod 2004;19:2884–2888.
          pubmed: 15347597
        5. Hochi S. Cryopreservation of follicular oocytes and preimplantation embryos in cattle and horses.. J Reprod Dev 2003;49:13–21.
          pubmed: 14967945
        6. Hochi S, Maruyama K, Oguri N. Direct transfer of equine blastocysts frozen-thawed in the presence of ethylene glycol and sucrose.. Theriogenology 1996;46:1217–1224.
          pubmed: 16727984
        7. Lawitts JA, Biggers JD. Joint effects of sodium chloride, glutamine, and glucose in mouse preimplantation embryo culture media.. Mol Reprod Dev 1992;31:189–194.
          pubmed: 1554503
        8. Leibo SP. A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos.. Theriogenology 1984;21:767–790.
          pubmed: 16725925
        9. Lin L, Du Y, Krager PM, Li J, Bolund L, Yang H, Zhang X, Kuwayama M, Vajta G. Induced blastocoele collapse improves survival rates of porcine blastocysts after vitrification.. Reprod Fert Dev 2008;20:121.
        10. Liu Z, Foote RH. Sodium chloride, osmolyte, and osmolarity effects on blastocyst formation in bovine embryos produced by in vitro fertilization (IVF) and cultured in simple serum-free media.. J Assist Reprod Genet 1996;13:562–568.
          pubmed: 8844313
        11. Maclellan LJ, Bass LD, McCue PM, Squires EL. Effect of cooling large and small equine embryos prior to cryopreservation on pregnancy rates after transfer.. Theriogenology 2003;59:306.
        12. Maclellan LJ, Carnevale EM, Coutinho da Silva MA, McCue PM, Seidel GE Jr, Squires EL. Cryopreservation of small and large equine embryos pre-treated with cytochalasin-B and/or trypsin.. Theriogenology 2002;58:717–720.
        13. McKinnon AO, Squires EL. Morphological assessment of the equine embryo.. J Am Vet Med Assoc 1988;192:401–406.
          pubmed: 3281922
        14. McWilliams RB, Gibbons WE, Leibo SP. Osmotic and physiological responses of mouse zygotes and human oocytes to mono- and disaccharides.. Hum Reprod 1995;10:1163–1171.
          pubmed: 7657759
        15. Mukaida T, Oka C, Goto T, Takahashi K. Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts.. Hum Reprod 2006;21:3246–3252.
          pubmed: 16936299
        16. O’Donovan MK. Cryopreservation and metabolic assessment of equine embryos, Master of Science Thesis.. Colorado State University; Fort Collins, CO: 2000.
        17. Pfaff RT. Effects of cryoprotectants on viability of equine embryos, Master of Science Thesis.. Colorado State University; Fort Collins, CO: 1995.
        18. Scherzer J, Fayrer-Hosken RA, Ray L, Hurley DJ, Heusner GL. Advancements in large animal embryo transfer and related biotechnologies.. Reprod Dom Anim 2008;43:371–376.
          pubmed: 18226021
        19. Slade NP, Takeda T, Squires EL, Elsden RP, Seidel GE Jr. A new procedure for the cryopreservation of equine embryos.. Theriogenology 1985;24:45–48.
          pubmed: 16726058
        20. Squires EL, Seidel GE Jr. Collection and transfer of equine embryos, Animal Reproduction and Biotechnology Laboratory.. Vol. 8. Colorado State University; Bulletin: 1995.
        21. Stachecki JJ, Cohen J, Willadsen SM. Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium.. Cryobiology 1998;37:346–354.
          pubmed: 9917351
        22. Stachecki JJ, Cohen J, Willadsen SM. Detrimental effects of sodium during mouse oocyte cryopreservation.. Biol Reprod 1998;59:395–400.
          pubmed: 9687313
        23. Tharasanit T, Colenbrander B, Stout TAE. Effect of cryopreservation on the cellular integrity of equine embryos.. Reproduction 2005;129:789–798.
          pubmed: 15923394
        24. Weon-Young S, Yoon S-H, Yoon H-J, Lee S-M, Lim J-H. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele.. Hum Reprod 2003;18:137–139.
          pubmed: 12525454
        25. Young CA, Squires EL, Seidel GE Jr, Kato H, McCue PM. Cryopreservation procedures for day 7–8 equine embryos.. Equine Vet J 1997;25(Suppl):98–102.
          pubmed: 9593539
        Subscribe to our newsletter
        Join 99,344 horse owners like you who receive our email updates on horse health and nutrition.