Effect of anti-freeze protein (AFP) on the cooling and freezing of equine embryos as measured by DAPI-staining.
Abstract: Equine embryos recovered on Day 6 after ovulation were cooled to +4 degrees C, or frozen with AFP alone or together with glycerol. Twenty embryos (140-200 microm in diameter) were randomly assigned to 6 treatment groups. In the first 3 groups, the embryos were cooled from room temperature to +4 degrees C at a rate of 3 degrees C/min and warmed again at a rate of 32 degrees C/min in a programmable freezer. In the second 3 groups, the embryos were frozen using a standard protocol, stored in liquid nitrogen for 5-7 days and then thawed in a 37 degrees C waterbath. After cooling/warming or freezing/thawing all the embryos were stained with DAPI. The percentage of dead cell area was significantly lower in the cooling groups than in the freezing groups and no significant differences were apparent between the cryoprotectants used in the study.
Publication Date: 1998-05-21 PubMed ID: 9593536DOI: 10.1111/j.2042-3306.1997.tb05108.xGoogle Scholar: Lookup
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- Comparative Study
- Journal Article
Summary
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The research article studies the efficacy of anti-freeze protein (AFP) in the cooling and freezing of horse embryos, detailing the processes used and the results obtained, which showed AFP alone, or together with glycerol, could significantly reduce cell death during cooling and freezing.
Methodology
- The research team collected equine embryos on the sixth day after ovulation.
- These embryos, measuring between 140-200 microm in diameter, were cooled to +4 degrees Celsius. This cooling was done with the anti-freeze protein (AFP) alone or in conjunction with glycerol.
- A total of twenty embryos were arbitrarily divided into six different treatment groups.
- For the first three groups, the embryos underwent cooling from room temperature to +4 degrees Celsius. This cooling process had a rate of 3 degrees Celsius per minute. Warming also took place, clocking in at a rate of 32 degrees Celsius per minute. A programmable freezer facilitated this technique.
- The remaining three groups had embryos that were frozen using a customary protocol. These were then stored in liquid nitrogen for a period spanning five to seven days. The embryos were then thawed in a 37 degrees Celsius water bath.
- After the embryos were cooled and heated again or frozen and defrosted later, they were all stained with DAPI (a nucleic acid stain).
Findings
- The research yielded notable results relating to the impact of cooling and freezing on equine embryos.
- The percentage of dead cell area was considerably reduced in the cooling groups in comparison to the freezing ones.
- Moreover, it was discovered that there were no discernible differences between the cryoprotectants used in the study.
- From this, it can be inferred that using the anti-freeze protein (AFP) either alone or alongside glycerol can play a crucial role in minimizing cell death during the freezing and cooling of equine embryos.
Cite This Article
APA
Lagneaux D, Huhtinen M, Koskinen E, Palmer E.
(1998).
Effect of anti-freeze protein (AFP) on the cooling and freezing of equine embryos as measured by DAPI-staining.
Equine Vet J Suppl(25), 85-87.
https://doi.org/10.1111/j.2042-3306.1997.tb05108.x Publication
Researcher Affiliations
- Haras Nationaux-INRA Equine Reproduction, Nouzilly, France.
MeSH Terms
- Animals
- Antifreeze Proteins
- Cohort Studies
- Cryopreservation / methods
- Cryopreservation / veterinary
- Cryoprotective Agents / pharmacology
- Embryo, Mammalian / chemistry
- Embryo, Mammalian / drug effects
- Embryo, Mammalian / physiology
- Female
- Fluorescent Dyes / chemistry
- Freezing
- Glycoproteins / pharmacology
- Horses / embryology
- Indoles / chemistry
- Random Allocation
Citations
This article has been cited 7 times.- Choi HW, Jang H. Application of Nanoparticles and Melatonin for Cryopreservation of Gametes and Embryos.. Curr Issues Mol Biol 2022 Sep 5;44(9):4028-4044.
- Ordóñez-León EA, Martínez-Rodero I, García-Martínez T, López-Béjar M, Yeste M, Mercade E, Mogas T. Exopolysaccharide ID1 Improves Post-Warming Outcomes after Vitrification of In Vitro-Produced Bovine Embryos.. Int J Mol Sci 2022 Jun 25;23(13).
- Robles V, Valcarce DG, Riesco MF. The Use of Antifreeze Proteins in the Cryopreservation of Gametes and Embryos.. Biomolecules 2019 May 9;9(5).
- Voets IK. From ice-binding proteins to bio-inspired antifreeze materials.. Soft Matter 2017 Jul 19;13(28):4808-4823.
- Kim HJ, Lee JH, Hur YB, Lee CW, Park SH, Koo BW. Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant.. Mar Drugs 2017 Jan 27;15(2).
- Bang JK, Lee JH, Murugan RN, Lee SG, Do H, Koh HY, Shim HE, Kim HC, Kim HJ. Antifreeze peptides and glycopeptides, and their derivatives: potential uses in biotechnology.. Mar Drugs 2013 Jun 10;11(6):2013-41.
- Brockbank KG, Campbell LH, Greene ED, Brockbank MC, Duman JG. Lessons from nature for preservation of mammalian cells, tissues, and organs.. In Vitro Cell Dev Biol Anim 2011 Mar;47(3):210-7.
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