Equine ovarian tissue viability after cryopreservation and in vitro culture.
Abstract: Ovarian tissue cryopreservation allows the preservation of the female fertility potential for an undetermined period. The objectives of this study were to compare the efficiency of cryoprotective agents (CPAs; dimethyl sulfoxide, DMSO; ethylene glycol, EG; and propylene glycol, PROH) using slow-freezing and vitrification methods, and evaluate the viability of cryopreserved equine ovarian tissue after 7 days of culture. Fresh and cryopreserved ovarian fragments were evaluated for preantral follicle morphology, stromal cell density, EGFR, Ki-67, Bax, and Bcl-2 protein expression, and DNA fragmentation. Vitrification with EG had the highest rate of morphologically normal preantral follicles, while DMSO had the lowest (76.1 ± 6.1% and 40.9 ± 14.8%, respectively; P < 0.05). In slow-freezing, despite that DMSO had the highest percentage of morphologically normal follicles (77.7 ± 5.8%), no difference among the CPAs was observed. Fluorescence intensity of EGFR and Ki-67 was greater when vitrification with EG was used. Regardless of the cryopreservation treatment, DMSO had the highest (P 0.05) among treatments after thawing. After in vitro culture, the percentage of normal follicles was similar (P > 0.05) between slow-freezing and vitrification methods; however, vitrification had greater (P < 0.05) stromal cell density than slow-freezing. In summary, equine ovarian tissue was successfully cryopreserved, increasing the viability of the cells in the ovarian tissue after thawing when using DMSO and EG for slow-freezing and vitrification methods, respectively. Therefore, these results are relevant for fertility preservation programs.
Copyright © 2017 Elsevier Inc. All rights reserved.
Publication Date: 2017-04-23 PubMed ID: 28583597DOI: 10.1016/j.theriogenology.2017.04.029Google Scholar: Lookup
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- Journal Article
Summary
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This study explores the efficiency of various cryoprotective agents in the preservation of equine ovarian tissue. It was found that dimethyl sulfoxide (DMSO) and ethylene glycol (EG) were effective for slow-freezing and vitrification methods respectively, offering potential advancements for fertility preservation programs.
Research Methodology
- The team conducted the study with an aim to explore the efficacy of different cryoprotective agents (CPAs), including dimethyl sulfoxide (DMSO), ethylene glycol (EG), and propylene glycol (PROH) in the slow-freezing and vitrification (rapid cooling) techniques.
- The evaluation of the viability of cryopreserved equine ovarian tissue after 7 days of culture was also part of the study.
- Criteria of evaluation included preantral follicle morphology, stromal cell density, and the expression of several proteins (EGFR, Ki-67, Bax, and Bcl-2).
- DNA fragmentation in the follicles was also noted as a measure of cellular damage.
Key Findings
- Vitrification with ethylene glycol resulted in the highest rate of preantral follicles maintaining their normal morphology.
- Slow freezing with dimethyl sulfoxide also resulted in a high percentage of morphologically normal follicles.
- The expression of certain proteins, EGFR and Ki-67, was noted to be higher when vitrification with EG was undertaken.
- Despite the cryopreservation method used, DMSO resulted in the highest Bax/Bcl-2 ratio, which indicates a higher level of apoptosis, or programmed cell death.
- DNA fragmentation, a sign of cellular damage, was observed to be similar across all treatments after thawing.
- Post in vitro culture, the percentage of normal follicles was similar between slow-freezing and vitrification methods, but vitrification was noted to have more stromal cell density than the slow-freezing technique.
Conclusions and Implications
- The study concluded that equine ovarian tissue could be effectively cryopreserved, and the viability of the cells in the ovarian tissue post-thawing increased when using DMSO for slow-freezing and EG for vitrification methods.
- These findings are particularly significant for fertility preservation programs, offering new potential for improved preservation of female fertility potential.
Cite This Article
APA
Gastal GDA, Aguiar FLN, Alves BG, Alves KA, de Tarso SGS, Ishak GM, Cavinder CA, Feugang JM, Gastal EL.
(2017).
Equine ovarian tissue viability after cryopreservation and in vitro culture.
Theriogenology, 97, 139-147.
https://doi.org/10.1016/j.theriogenology.2017.04.029 Publication
Researcher Affiliations
- Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, IL, USA.
- Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, IL, USA.
- Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, IL, USA.
- Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, IL, USA.
- Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, IL, USA.
- Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, IL, USA.
- Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS, USA.
- Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS, USA.
- Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, IL, USA. Electronic address: egastal@siu.edu.
MeSH Terms
- Animals
- Cryopreservation / veterinary
- Cryoprotective Agents / pharmacology
- Female
- Fertility Preservation
- Freezing
- Horses / physiology
- Ovary / drug effects
- Ovary / physiology
- Tissue Culture Techniques / veterinary
- Tissue Preservation / veterinary
- Tissue Survival
- Vitrification
Citations
This article has been cited 6 times.- Nikiforov D, Russo V, Nardinocchi D, Bernabò N, Mattioli M, Barboni B. Innovative multi-protectoral approach increases survival rate after vitrification of ovarian tissue and isolated follicles with improved results in comparison with conventional method. J Ovarian Res 2018 Aug 7;11(1):65.
- Ishak GM, Bashir ST, Dutra GA, Gastal GDA, Gastal MO, Cavinder CA, Feugang JM, Gastal EL. In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels. Reprod Biol Endocrinol 2018 Jul 28;16(1):71.
- Borges AA, Lira GPO, Nascimento LE, Queiroz Neta LB, Santos MVO, Oliveira MF, Silva AR, Pereira AF. Influence of Cryopreservation Solution on the In Vitro Culture of Skin Tissues Derived from Collared Peccary (Pecari tajacu Linnaeus, 1758). Biopreserv Biobank 2018 Apr;16(2):77-81.
- Martins SD, Oliveira MAF, Azevedo VAN, Costa FDC, Silva IGMD, de Morais SM, Báo SN, Silva JRV, Ceccatto VM, Araújo VR. Punica granatum L. Modulates Antioxidant Activity in Vitrified Bovine Ovarian Tissue. Int J Mol Sci 2026 Jan 16;27(2).
- Kong Q, Stavrev D, Rahimi G, Todorov P, Pei C, Isachenko E, Salama M, Skala C, Isachenko V. Dynamic in vitro 3D culture of cryopreserved human ovarian tissue: transcriptomic analysis by RNA sequencing. J Ovarian Res 2026 Jan 16;19(1):55.
- Wang T, Zhang Z, Qu C, Shi Y, Chen S, Li W, Li M. Loss of FUT8 impairs embryonic development by reducing cAMP production in granulosa cells. J Assist Reprod Genet 2025 Jul;42(7):2437-2448.
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