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New Challenges in Cryopreservation

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A special issue of Animals (ISSN 2076-2615). This special issue belongs to the section "Animal Reproduction".

Deadline for manuscript submissions: closed (31 January 2022) | Viewed by 30853

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Special Issue Editors

Dr. Daniela Bebbere

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Guest Editor

Department of Veterinary Medicine, University of Sassari, via Vienna 2, 07100 Sassari, Italy
Interests: developmental biology; ovine and bovine reproduction; gene expression; epigenetics; oocytes; embryos; assisted reproductive technologies; cryopreservation

Dr. Sara Succu

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Guest Editor

Department of Veterinary Medicine, University of Sassari, Via Vienna 2, 07100 Sassari, Italy
Interests: ruminant reproduction; oocyte quality; embryo development; cryopreservation (oocytes, semen, embryos); in vitro culture; follicular dynamic; fertility

Special Issue Information

Dear colleague,

Cryopreservation is a fundamental procedure to maintain the structure and function of cells and tissues when stored at low temperatures for long periods. Despite being developed in the early 1900s, it maintains an essential role in the conservation of living material in different fields, including health, biodiversity conservation, and biotechnologies.

The field has evolved significantly since that time, and extensive research has resulted in the development of new approaches to create an acceptable balance betweenpositive and negative effects of cryopreservation. Efforts were mainly addressed to limit the damages caused by exposure to low temperatures and to reduce thestill high costs, increasing its applicability. Most recent technologies have opened new opportunities to optimize protocols and understand cell biological response to cryopreservation.

Aim of this special issue is to publish high-quality research papers as well as review articles addressing new or alternative approaches in the field of cryopreservation of cells and tissues.

Original, high quality contributions that are not yet published or under review by other journals are sought.

Potential topics include, but are not limited, to the following:

Novel cryopreservation technologies, including protocols, devices or equipment.
Recent developments in the use of new cryoprotective molecules.
Molecular and cellular mechanisms involved in the response to cryopreservation.
Effects of cryopreservation on the epigenome of cells.
Novel strategies in cryopreservation applied to reproduction (gametes, embryos and gonads).
Use of nanotechnologies in cryopreservation.

Dr. Daniela Bebbere
Dr. Sara Succu
Guest Editors

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Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2400 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

Cryopreservation methods
Cryoprotectants
Cryodevices
Gametes
Embryos
Gonads
Somatic cells
Biodiversity conservation
Cryopreservation equipment

Published Papers (11 papers)

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Editorial

Jump to: Research, Review, Other

3 pages, 193 KiB

Open AccessEditorial

New Challenges in Cryopreservation: A Reproductive Perspective

by Daniela Bebbere and Sara Succu

Animals 2022, 12(13), 1598; https://doi.org/10.3390/ani12131598 - 21 Jun 2022

Cited by 1 | Viewed by 1225

Abstract

Cryopreservation is a fundamental procedure to preserve the structure and function of cells and tissues by storing them at low temperatures for long periods [...] Full article

(This article belongs to the Special Issue New Challenges in Cryopreservation)

Research

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14 pages, 1761 KiB

Open AccessArticle

Freezing Protocol Optimization for Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm under Field Conditions

by Daniela Alejandra Medina-Chávez, Ana Josefa Soler, Alicia Martín-Maestro, Silvia Villaverde, Irene Sánchez-Ajofrín, Patricia Peris-Frau, Enrique del Olmo, Alfonso Bisbal, Olga García-Álvarez, María del Rocío Fernández-Santos and José Julián Garde

Animals 2022, 12(7), 869; https://doi.org/10.3390/ani12070869 - 30 Mar 2022

Cited by 5 | Viewed by 1797

Abstract

Creating germplasm banks of wild species, such as the Iberian red Deer (Cervus elaphus hispanicus) can be challenging. One of the main difficulties is the obtention and cryopreservation of good-quality reproductive cells when the spermatozoa are obtained from epididymides after death. To avoid a loss of seminal quality during transport, developing alternative methods for cooling and freezing sperm samples under field conditions is necessary. The objective of this study was to evaluate the effects of different durations of equilibrium and different techniques of cooling and freezing on Iberian red deer epididymal sperm quality after thawing to optimize the processing conditions in this species. Three experiments were carried out: (I) evaluation of refrigeration in straws or tubes of 15 mL; (II) study of equilibration period (0, 30, 60, or 120 min); and (III) comparison of four freezing techniques (liquid nitrogen vapor in a tank (C), liquid nitrogen vapor in a polystyrene box (B), dry ice (DY), and placing straws on a solid metallic plate floating on the surface of liquid nitrogen (MP)). For all experiments, sperm motility and kinematic parameters, acrosomal integrity, sperm viability, mitochondrial membrane potential, and DNA integrity were evaluated after thawing. All statistical analyses were performed by GLM-ANOVA analysis. Samples refrigerated in straws showed higher values (p ≤ 0.05) for mitochondrial activity and lower values (p ≤ 0.05) for apoptotic cells. Moreover, the acrosome integrity showed significant differences (p ≤ 0.05) between 0 and 120 min, but not between 30 and 60 min, of equilibration. Finally, no significant differences were found between freezing in liquid nitrogen vapors in a tank or in a box, although there was a low quality after thawing when the samples were cryopreserved in dry ice or by placing straws on a solid metallic plate floating on the surface of liquid nitrogen. In conclusion, under field conditions, it would be possible to refrigerate the sperm samples by storing them in straws with a 120 min equilibration period and freezing them in liquid nitrogen vapors in a tank or box. Full article

(This article belongs to the Special Issue New Challenges in Cryopreservation)

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16 pages, 2327 KiB

Open AccessArticle

The Role of Aquaporin 7 in the Movement of Water and Cryoprotectants in Bovine In Vitro Matured Oocytes

by Tania García-Martínez, Iris Martínez-Rodero, Joan Roncero-Carol, Meritxell Vendrell-Flotats, Jaume Gardela, Alfonso Gutiérrez-Adán, Priscila Ramos-Ibeas, Adam Z. Higgins and Teresa Mogas

Animals 2022, 12(4), 530; https://doi.org/10.3390/ani12040530 - 21 Feb 2022

Cited by 1 | Viewed by 2416

Abstract

Aquaglyceroporins are known as channel proteins, and are able to transport water and small neutral solutes. In this study, we evaluate the effect of exposure of in vitro matured bovine oocytes to hyperosmotic solutions containing ethylene glycol (EG), dimethyl sulfoxide (Me₂SO) or sucrose on the expression levels of AQP3, AQP7 and AQP9. Moreover, we studied whether artificial protein expression of AQP7 in bovine oocytes increases their permeability to water and cryoprotectants. Exposure to hyperosmotic solutions stimulated AQP3 and AQP7 but not AQP9 expression. Oocytes exposed to hyperosmotic Me₂SO solution exhibited upregulated AQP3 expression, while AQP7 expression was upregulated by EG hyperosmotic exposure. Microinjection of oocytes at the germinal vesicle stage with enhanced green fluorescent protein (EGFP) or EGFP+AQP7 cRNAs resulted in the expression of the corresponding proteins in ≈86% of the metaphase-II stage oocytes. AQP7 facilitated water diffusion when bovine MII oocytes were in presence of Me₂SO solution but not EG or sucrose solution. However, the overexpression of this aquaporin did not increase membrane permeability to Me₂SO or EG. In summary, cryoprotectant-induced increase of AQP3 and AQP7 expression could be one of the mechanisms underlying oocyte tolerance to hyperosmotic stress. Water diffusion appears to be improved when AQP7 overexpressed oocytes are exposed to Me₂SO, shortening the time required for oocytes to achieve osmotic balance with cryoprotectant solutions. Full article

(This article belongs to the Special Issue New Challenges in Cryopreservation)

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14 pages, 15762 KiB

Open AccessArticle

Molecular and Histological Evaluation of Sheep Ovarian Tissue Subjected to Lyophilization

by Daniela Bebbere, Amir Arav, Stefano Mario Nieddu, Giovanni Pietro Burrai, Sara Succu, Pasquale Patrizio and Sergio Ledda

Animals 2021, 11(12), 3407; https://doi.org/10.3390/ani11123407 - 29 Nov 2021

Cited by 6 | Viewed by 2022

Abstract

Cryopreservation is routinely used to preserve cells and tissues; however, long time storage brings many inconveniences including the use of liquid nitrogen. Freeze-drying could enable higher shelf-life stability at ambient temperatures and facilitate transport and storage. Currently, the possibility to freeze-dry reproductive tissues maintaining vitality and functions is still under optimization. Here, we lyophilized sheep ovarian tissue with a novel device named Darya and a new vitrification and drying protocol and assessed effects on tissue integrity and gene expression. The evaluation was performed immediately after lyophilization (Lio), after rehydration (LR0h) or after two hours of in vitro culture (IVC; LR2h). The tissue survived lyophilization procedures and maintained its general structure, including intact follicles at different stages of development, however morphological and cytoplasmic modifications were noticed. Lyophilization, rehydration and further IVC increasingly affected RNA integrity and caused progressive morphological alterations. Nevertheless, analysis of a panel of eight genes showed tissue survival and reaction to the different procedures by regulation of specific gene expression. Results show that sheep ovarian tissue can tolerate the applied vitrification and drying protocol and constitute a valid basis for further improvements of the procedures, with the ultimate goal of optimizing tissue viability after rehydration. Full article

(This article belongs to the Special Issue New Challenges in Cryopreservation)

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13 pages, 327 KiB

Open AccessArticle

Effects of Inositol Supplementation in Sperm Extender on the Quality of Cryopreserved Mesopotamian Catfish (Silurus triostegus, H. 1843) Sperm

by Zafer Doğu, Erdinç Şahinöz, Faruk Aral, İsmail Koyuncu and Özgür Yüksekdağ

Animals 2021, 11(11), 3029; https://doi.org/10.3390/ani11113029 - 21 Oct 2021

Cited by 5 | Viewed by 1857

Abstract

In this study, the effects of supplemented inositol on sperm extenders were examined on the spermatozoa motility rate and duration, total antioxidant and oxidant status, apoptotic spermatozoa and DNA damage, during the sperm post-thaw process of Mesopotamian Catfish (Silurus triostegus, H. 1843). The semen was frozen in diluents containing different inositol concentrations (5, 10, 20 and 40 mg). Increasing levels of inositol linearly improved the spermatozoa motility rate and duration significantly (p < 0.05). MDA and TOS were linearly decreased, however, TAS and GSH linearly increased (p < 0.05). The increasing inositol levels resulted in a linear and quadratic decrease in DNA damage in the comet assay, 8-hydroxydeoxyguanosine and the determined percentage of apoptotic spermatozoa (p < 0.05). These results suggest that there are many positive effects of the use of supplemental inositol on enhancing sperm cryopreservation efficiency in Silurus triostegus. Full article

(This article belongs to the Special Issue New Challenges in Cryopreservation)

9 pages, 847 KiB

Open AccessArticle

Cryoprotective Effects of Ergothioneine and Isoespintanol on Canine Semen

by Alexandra Usuga, Irene Tejera, Jorge Gómez, Oliver Restrepo, Benjamín Rojano and Giovanni Restrepo

Animals 2021, 11(10), 2757; https://doi.org/10.3390/ani11102757 - 22 Sep 2021

Cited by 12 | Viewed by 2312

Abstract

Sperm undergo oxidative stress due to excessive production of reactive oxygen species (ROS) during cryopreservation. Some unconventional natural antioxidants can reduce ROS-induced changes in cryopreserved canine sperm. This study aimed to identify the cryoprotective effects of ergothioneine and isoespintanol on the quality of thawed canine semen. Twelve ejaculates from six dogs were cryopreserved in a tris-yolk extender without (control) or with 50 (E50), 100 (E100), or 150 (E150) µM ergothioneine or 20 (I20), 40 (I40), or 60 (I60) µM isoespintanol. We evaluated the motility and kinetics of thawed sperm using computerized analysis; determined morphology by eosin-nigrosin staining; functional membrane integrity using hypoosmotic tests, and structural membrane and acrosome integrity; mitochondrial membrane potential by fluorescence microscopy; and ROS production by spectrophotometry. Data were statistically analyzed using mixed models and Tukey tests. E100 increased total (60.6% vs. 49.6%) and progressive (26.4% vs. 20.1%) motility, straight line velocity (41.3 vs. 35.9 µm/s), and rapid sperm (17.6% vs. 12.3%) compared with controls. However, E150 reduced the numbers of hyperactive sperm. E100, I40, and I60 reduced the abnormal morphology and ROS production, and all concentrations of both antioxidants increased acrosomal integrity. We concluded that ergothioneine and isoespintanol reduce deleterious sperm alterations and oxidative stress in thawed canine semen. Full article

(This article belongs to the Special Issue New Challenges in Cryopreservation)

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9 pages, 694 KiB

Open AccessArticle

Effects of Saccharides Supplementation in the Extender of Cryopreserved Rooster (Gallus domesticus) Semen on the Fertility of Frozen/Thawed Spermatozoa

by Olga Stanishevskaya, Yulia Silyukova, Nikolai Pleshanov, Anton Kurochkin, Elena Fedorova, Zoya Fedorova, Oksana Perinek, Anna Prituzhalova and Inessa Meftakh

Animals 2021, 11(1), 189; https://doi.org/10.3390/ani11010189 - 14 Jan 2021

Cited by 10 | Viewed by 2118

Abstract

The aim of this study was to create balanced media for the cryopreservation of rooster semen in pellets to maintain the functional state of the sperm after thawing. Fructose was replaced by trehalose in experimental media in proportions of 10% (LCM-T10) and 20% (LCM-T20), while LCM was used as a control. After artificial insemination of the hens, the eggs were incubated (n = 400). To determine the functional safety of spermatozoa in the genital tract of hens after 5, 10, and 15 days from the last insemination, we used a method for assessing the interaction of sperm with the perivitelline membrane. Significantly higher rates of egg fertilization (82–86%) were obtained when using LCM-T10 and LCM-T20 compared to control (79%, p < 0.05). Egg fertility on the 5th day from the last insemination with the LCM-T20 diluent reached 100% versus 86% in the control; on the 10th day, the fertility rates were 55% versus 20%, respectively. The best results for fertility duration were obtained by freezing spermatozoa with LCM-T20 medium. The numbers of interaction points of spermatozoa with the perivitelline membrane were as follows: on the 5th day from the last insemination with LCM-T20—461.5 ± 11.5 holes/cm² (LCM-control—13.7 ± 2.7 holes/cm²), p < 0.01; on the 10th day with LCM-T20—319.3 ± 12.9 holes/cm² (LCM-control—14.9 ± 3.5 holes/cm²); and on the 15th day with LCM-T20—345.2 ± 11.1 holes/cm² (LCM-control—0 holes/cm²). In conclusion, the use of trehalose in LCM diluent medium can increase the fertility of frozen/thawed sperm and the duration of their fertility in the genital tract of hens. Full article

(This article belongs to the Special Issue New Challenges in Cryopreservation)

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13 pages, 2809 KiB

Open AccessArticle

The Effect of Adding Different Levels of Curcumin and Its Nanoparticles to Extender on Post-Thaw Quality of Cryopreserved Rabbit Sperm

by Sameh A. Abdelnour, Mahmoud A. E. Hassan, Amer K. Mohammed, Ahmad R. Alhimaidi, Naif Al-Gabri, Khalid O. Al-Khaldi and Ayman A. Swelum

Animals 2020, 10(9), 1508; https://doi.org/10.3390/ani10091508 - 26 Aug 2020

Cited by 46 | Viewed by 4949

Abstract

The cryopreservation process adversely affects sperm function and quality traits, causing some changes at biochemical and structural levels, due to mechanical, thermal, osmotic, and oxidative damage. Supplementation with curcumin nanoparticles could prevent and even revert this effect and could enhance the post/thawed sperm quality in the rabbit. The study amid to explore the effect of curcumin (CU) and curcumin nanoparticles (CUNPs) supplementation in semen extender on post/thawed rabbit sperm quality. Twelve fertile, healthy rabbit bucks were included, and the ejaculates were collected using artificial vaginas. Rabbit pooled semen was cryopreserved in tris-yolk fructose (TYF) extender without any supplement (control group) or extender supplemented with CU at levels of 0.5, 1 or 1.5 µg/mL (CU0.5, CU1.0, and CU1.5, respectively) or CUNPs at levels of 0.5, 1, 1.5 (CUNPs0.5, CUNPs1.0, and CUNPs1.5, respectively) and was packed in straws (0.25 mL) and stored in liquid nitrogen (−196 °C). Results revealed that CUNPs1.5 had a positive influence (p < 0.05) on post-thawing sperm progressive motility, viability, and membrane integrity as compared with the other groups. Percentages of dead sperm, abnormalities, early apoptotic, apoptotic, and necrotic sperm cells reduced (p < 0.05) in CUNPs1.5 as compared to other treatments. Using 1.5 µg/mL of CUNPs significantly improved total antioxidant capacity (TAC), GPx, while MDA and POC reduced (p < 0.05) in CU1.5 in comparison with other groups. SOD values were enhanced (p < 0.05) in CUNPs1.0 and CUNPs1.5 in relation with other treatments. Conclusively, the addition of curcumin and its nanoparticles to the extender can improve the post-thawed quality of rabbit sperm via redox signaling and reduce the apoptosis process. Full article

(This article belongs to the Special Issue New Challenges in Cryopreservation)

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16 pages, 1664 KiB

Open AccessFeature PaperArticle

Long-Term Phenotypic and Proteomic Changes Following Vitrified Embryo Transfer in the Rabbit Model

by Ximo Garcia-Dominguez, Francisco Marco-Jiménez, David S. Peñaranda and José Salvador Vicente

Animals 2020, 10(6), 1043; https://doi.org/10.3390/ani10061043 - 17 Jun 2020

Cited by 11 | Viewed by 3455

Abstract

Nowadays, assisted reproductive technologies (ARTs) are considered valuable contributors to our past, but a future without their use is inconceivable. However, in recent years, several studies have evidenced a potential impact of ART on long-term development in mammal species. To date, the long-term follow-up data are still limited. So far, studies have mainly focused on in vitro fertilization or in vitro culture, with information from gametes/embryos cryopreservation field being practically missing. Herein, we report an approach to determine whether a vitrified embryo transfer procedure would have long-term consequences on the offspring. Using the rabbit as a model, we compared animals derived from vitrified-transferred embryos versus those naturally conceived, studying the growth performance, plus the weight throughout life, and the internal organs/tissues phenotype. The healthy status was assessed over the hematological and biochemical parameters in peripheral blood. Additionally, a comparative proteomic analysis was conducted in the liver tissue to investigate molecular cues related to vitrified embryo transfer in an adult tissue. After vitrified embryo transfer, birth weight was increased, and the growth performance was diminished in a sex-specific manner. In addition, vitrified-transferred animals showed significantly lower body, liver and heart weights in adulthood. Molecular analyses revealed that vitrified embryo transfer triggers reprogramming of the liver proteome. Functional analysis of the differentially expressed proteins showed changes in relation to oxidative phosphorylation and dysregulations in the zinc and lipid metabolism, which has been reported as possible causes of a disturbed growth pattern. Therefore, we conclude that vitrified embryo transfer is not a neutral procedure, and it incurs long-term effects in the offspring both at phenotypic and molecular levels. These results described a striking example of the developmental plasticity exhibited by the mammalian embryo. Full article

(This article belongs to the Special Issue New Challenges in Cryopreservation)

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Review

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17 pages, 368 KiB

Open AccessReview

Oocyte Cryopreservation in Domestic Animals and Humans: Principles, Techniques and Updated Outcomes

by Theerawat Tharasanit and Paweena Thuwanut

Animals 2021, 11(10), 2949; https://doi.org/10.3390/ani11102949 - 13 Oct 2021

Cited by 14 | Viewed by 4468

Abstract

Oocyte cryopreservation plays important roles in basic research and the application of models for genetic preservation and in clinical situations. This technology provides long-term storage of gametes for genetic banking and subsequent use with other assisted reproductive technologies. Until recently, oocytes have remained the most difficult cell type to freeze, as the oocytes per se are large with limited surface area to cytoplasm ratio. They are also highly sensitive to damage during cryopreservation, and therefore the success rate of oocyte cryopreservation is generally poor when compared to noncryopreserved oocytes. Although advancement in oocyte cryopreservation has progressed rapidly for decades, the improvement of cryosurvival and clinical outcomes is still required. This review focuses on the principles, techniques, outcomes and prospects of oocyte cryopreservation in domestic animals and humans. Full article

(This article belongs to the Special Issue New Challenges in Cryopreservation)

Other

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7 pages, 385 KiB

Open AccessBrief Report

Long-Term Effects Following Fresh/Vitrified Embryo Transfer Are Transmitted by Paternal Germline in a Large Size Rabbit Cohort

by Ximo Garcia-Dominguez, José Salvador Vicente, María P. Viudes-de-Castro and Francisco Marco-Jiménez

Animals 2020, 10(8), 1272; https://doi.org/10.3390/ani10081272 - 25 Jul 2020

Cited by 5 | Viewed by 1885

Abstract

The concept of developmental programming suggests that the early life environment influences offspring phenotype in later life, whose effects may also be manifested in further generations. Valuable pieces of evidence come from the fields applying assisted reproductive technologies (ARTs), which deprive embryos of their optimal maternal environment and were thus associated with subsequent developmental deviations. Recently, we demonstrated that the in vitro manipulations during a vitrified embryo transfer procedure incurs a cumulative and transgenerational decline in the growth performance of the resulting offspring. Here, we provide a longitudinal study to investigate whether previous developmental deviations could be indistinctly paternally or maternally transmitted using crossbred mattings. Our findings revealed that early embryo manipulations through fresh and vitrified embryo transfer incurred paternally transmissible effects over the growth pattern and adult body weight, which seemed not inheritable via the female germline. Similar inheritable effects were observed after fresh and vitrified embryo transfer, suggesting that disturbing optimal embryo development through in vitro manipulations was the principal trigger of transmissible effects, rather than embryo cryopreservation per se. Full article

(This article belongs to the Special Issue New Challenges in Cryopreservation)

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