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Animals : an open access journal from MDPI2021; 11(5); doi: 10.3390/ani11051325

Epigenetic Changes in Equine Embryos after Short-Term Storage at Different Temperatures.

Abstract: In embryos subjected to assisted reproductive techniques, epigenetic modifications may occur that can influence embryonic development and the establishment of pregnancy. In horses, the storage temperature during transport of fresh embryos before transfer is a major concern. The aim of this study was, therefore, to determine the effects of two storage temperatures (5 °C and 20 °C) on equine embryos, collected at day seven after ovulation and stored for 24 h, on: (i) morphological development; (ii) expression of candidate genes associated with embryo growth and development, maternal recognition of pregnancy, methylation and apoptosis, and (iii) gene-specific and global DNA methylation. Embryos (n = 80) were collected on day seven or day eight after ovulation and assigned to four groups: day seven control (E7F, fresh); day seven, stored for 24 h at 5 °C (E5C); day seven, stored for 24 h at 20 °C (E20C) and day eight control (E8F, fresh 24h time control). The embryos and the storage medium (EquiHold, holding medium, Minitube, Tiefenbach, Germany) from all treatment groups were analyzed for (i) medium temperature, pH, and lipid peroxidation (malondialdehyde; MDA) and (ii) embryo morphology, mRNA expression and DNA methylation (immunohistochemistry and gene-specific DNA methylation). The size of embryos stored at 5 °C was larger (p < 0.01), whereas embryos stored at 20 °C were smaller (p < 0.05) after 24 h. There were no changes in pH and MDA accumulation irrespective of the group. The mRNA expression of specific genes related to growth and development (POU5F1, SOX2, NANOG), maternal recognition of pregnancy (CYP19A1, PTGES2), DNA methylation (DNMT1, DNMT3A, DNMT3B) and apoptosis (BAX) in the E5C and E20C were either up or downregulated (p < 0.05) when compared to controls (E7F and E8F). The immune expression of 5mC and 5hmC was similar among treatment groups. Percentage of methylation in the CpG islands was lower in the specific genes ESR1, NANOG and DNMT1 (p < 0.001) in E20C embryos when compared to E8F (advanced embryo stage). Therefore, our study demonstrates for the first time the gene-specific and global DNA methylation status of fresh equine embryos collected on days seven and eight after ovulation. Although our results suggest some beneficial effects of storage at 20 °C in comparison to 5 °C, the short-term storage, regardless of temperature, modified gene expression and methylation of genes involved in embryo development and may compromise embryo viability and development after transfer.
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Publication Date: 2021-05-06 PubMed ID: 34066466PubMed Central: PMC8148113DOI: 10.3390/ani11051325Google Scholar: Lookup
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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.

This research investigates how the storage temperature of horse embryos influences their development and the success of pregnancy. The study particularly focuses on the effect of short-term storage at two different temperature (5 °C and 20 °C) on the embryos’ development, gene expression, and DNA methylation.

Objective and Methodology

  • The researchers aimed to determine the consequences of storing equine embryos at either 5 °C or 20 °C for 24 hours.
  • The evaluation was based on certain factors: their morphological development, the expression of genes associated with embryo growth and development, recognition of pregnancy, DNA methylation, and cellular death (apoptosis).
  • 80 embryos were collected on the seventh or eighth day after ovulation and divided into four groups for comparative analysis. Two groups were controls, kept fresh without storage, while the remaining two were stored at the indicated temperatures.

Findings and Analysis

  • Embryos stored at 5 °C had increased size, while those stored at 20 °C were smaller after 24 hours.
  • No changes were observed in the acidity (pH value) and lipid peroxidation (MDA level, a parameter of oxidative stress) of the storage medium, which were consistent across all groups.
  • There were noticeable alterations in the mRNA expression of specific genes linked to growth and development, pregnancy recognition, DNA methylation and apoptosis in embryos that were stored, compared to fresh ones. These modifications varied from upregulation to downregulation.
  • The global DNA methylation – modification of DNA that usually represses gene transcription – appeared the same among all groups. However, the methylation percentage at certain specific genes decreased in embryos stored at 20 °C compared to the advanced-stage embryos (Day 8 fresh).

Conclusion

  • The research revealed, for the first time, information on gene-specific and global DNA methylation status of fresh equine embryos collected on day seven and eight post-ovulation.
  • The results suggest some benefits of storing embryos at 20 °C compared to 5 °C. However, short-term storage at any temperature affected gene expression and methylation related to embryo development and might reduce the success rate of embryo transfer and subsequent development.

Cite This Article

APA
Gastal GDA, Scarlet D, Melchert M, Ertl R, Aurich C. (2021). Epigenetic Changes in Equine Embryos after Short-Term Storage at Different Temperatures. Animals (Basel), 11(5). https://doi.org/10.3390/ani11051325

Publication

Animals : an open access journal from MDPI
ISSN: 2076-2615
NlmUniqueID: 101635614
Country: Switzerland
Language: English
Volume: 11
Issue: 5

Researcher Affiliations

Gastal, Gustavo D A
  • Center for Artificial Insemination and Embryo Transfer, Department for Small Animals and Horses, University of Veterinary Medicine Vienna, 1210 Vienna, Austria.
  • Instituto Nacional de Investigación Agropecuaria INIA, Estación Experimental La Estanzuela, Ruta 50 km 11, 39173 Colonia, Uruguay.
Scarlet, Dragos
  • Division of Obstetrics, Gynecology and Andrology, Department for Small Animals and Horses, University of Veterinary Medicine Vienna, 1210 Vienna, Austria.
Melchert, Maria
  • Center for Artificial Insemination and Embryo Transfer, Department for Small Animals and Horses, University of Veterinary Medicine Vienna, 1210 Vienna, Austria.
Ertl, Reinhard
  • Vetcore Facility, University of Veterinary Medicine Vienna, 1210 Vienna, Austria.
Aurich, Christine
  • Center for Artificial Insemination and Embryo Transfer, Department for Small Animals and Horses, University of Veterinary Medicine Vienna, 1210 Vienna, Austria.

Conflict of Interest Statement

The authors declare no conflict of interest.

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