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CryoLetters 43 (4), 189-199 (2022)
© CryoLetters, editor@cryoletters.org
doi.org/10.54680/fr22410110112

PERSPECTIVE:
A STATE OF THE ART REVIEW OF ISOCHORIC CRYOPRESERVATION AND CRYOPROTECTANTS

George-Andrei Beşchea¹*⁺, Ştefan-Ioan Câmpean¹⁺,
Maria-Bianca Tăbăcaru¹, Beatrice-Georgiana Vuţoiu¹,
Alexandru Şerban¹ and Gabriel Năstase²

¹Transilvania University of Braşov, Faculty of Civil Engineering, Department of Building Services, Braşov, Romania.
²University Politehnica of Bucharest, Faculty of Mechanical Engineering and Mechatornics, Thermotechnics, engines, thermal and refrigeration equipment Department, Bucharest, Romania.
*Corresponding author’s E-mail: george.beschea@unitbv.ro
⁺Co-First authors

Abstract

There is a developing enthusiasm for discovering new methods, cryoprotectants, systems and devices for cells, tissues, and organ preservation in medicine, in sub-zero temperature conditions and a growing interest in developing more efficient and economical methods for long-term preservation of food in a frozen state. Most of the preservation protocols currently used in medicine and food preservation involve the use of atmospheric pressure, and temperatures lower than normal body temperature in medicine, or lower than room temperature in the food industry. In this state of the art review, we analyzed the results of a new preservation method that uses an isochoric system. We aimed to offer a clear overview of the potential of this new technology. Firstly, to study the origins of isochoric preservation, we searched using the WoS Database. A search with the world “isochoric” returned 488 results. A more specific search of the term “isochoric freezing” returned 94 results. From these searches, we selected the 12 most relevant articles and discuss them here in detail. We present an overall characterization and criticism of the current use and potential of this new preservation method that can be used in the medicine and food industry. The main findings indicate encouraging results for the tested biological matter, including for the preservation of food products (e.g. cherries, spinach, potatoes), biological organisms (e.g. Caenorhabditis elegans, Escherichia coli, Listeria, Salmonella typhimurium), organs (e.g. rat hearts), tissues (e.g., tilapia fish filets) or cells (e.g., mammalian cells, pancreatic cells). Accordingly, we conclude that the isochoric system holds huge potential as a new technique in the field of preservation.

Keywords: cryopreservation; cryoprotectants; historical events; isochoric systems.

Download the paper: A STATE OF THE ART REVIEW OF ISOCHORIC CRYOPRESERVATION AND CRYOPROTECTANTS (PDF)

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CryoLetters 43 (4), 200-205 (2022)
© CryoLetters, editor@cryoletters.org
doi.org/10.54680/fr22410110312

POTENTIAL APPLICATION OF CRYOGENIC FREEZER ON TURBOT SPERM CRYOPRESERVATION

İlhan Aydin¹, Atife Tuba Beken², Rafet Çağrı Öztïrk³,
Hamza Polat² and Yahya Terzi³*

¹General Directorate of Agricultural Research and Policies-TAGEM, Ankara.
²Central Fisheries Research Institute, Trabzon;
³Department of Fisheries Technology Engineering, Faculty of Marine Sciences, Karadeniz Technical University, Trabzon, Turkey.
*Corresponding author’s E-mail: yhyterzi@gmail.com

Abstract

BACKGROUND:Cryopreservation is a common practice to preserve fish sperm for prolonged periods. OBJECTIVE: To examine the effect of different freezing protocols on sperm characteristics, fertilization, and hatching rate of turbot. MATERIALS AND METHODS: Milt was obtained from ten 8-year-old turbot (54.3 ± 1.7 cm in length and 3,106 ± 283 g in weight) at the peak of spawning season. Six batches of milts with >90% motility was pooled and diluted to 1:3 by adding dimethyl sulfoxide (DMSO, 10%) as cryoprotectant. Then straws filled with semen were subjected to three freezing protocols (cooling rates). Sperm characteristics were assessed using sperm class analyzer before and after cryopreservation. Cryopreserved and fresh sperm were used for artificial fertilization to assess fertilization and hatching rates. RESULTS: Cryopreservation protocol has significantly deleterious effects on total motility, progressive motility, curvilinear velocity, straight line velocity, average path velocity, linearity index, straightness index, oscillation index, and amplitude of lateral head displacement of sperm. However, the beat frequency of cryopreserved sperm was found to be similar to control sperm. The fertilization rate of sperm subjected to three freezing protocols were similar, varying between 65.3 % and 75.6 %, and the hatching rates varied from 51.2% to 70.7%. CONCLUSION: The results show the potential application of cryopreservation in fish hatcheries.

Keywords: cryopreservation; sperm motility; sperm quality; turbot.

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CryoLetters 43 (4), 206-221 (2022)
© CryoLetters, editor@cryoletters.org
doi.org/10.54680/fr22410110512

PROTEOMIC PROFILE OF VITRIFIED IN VITRO-PRODUCED BOVINE EMBRYOS (Bos taurus indicus)

Joane Isis Travassos Vieira¹*, José Carlos Ferreira-Silva¹,
Fabiana Aparecida Cavalcante Silva², Elton Pedro Nunes Pena³,
Lucas Carvalho Freitas³, Maiana Silva Chaves¹,
João Gabriel Viana Grázia¹, Lucas Carvalho Pereira¹,
Renan Henrique dos Santos Fagundes¹, Tercilio Calsa Junior³,
José Pompeu dos Santos Filho¹, Vicente José Figueiredo Freitas⁴
and Marcos Antonio Lemos Oliveira¹

¹Laboratory of Reproductive Biotechniques, Department of Veterinary Medicine, Federal Rural University of Pernambuco, Brazil
²Laboratory of Phytosanitary diagnosis, Northeast Strategic Technologies Center (CETENE), Brazil
³Laboratory of Plant Genomics and Proteomics, Department of Genetics, Federal University of Pernambuco, Brazil
⁴Laboratory of Physiology and Control of Reproduction, Department of Veterinary Medicine, Ceará State University, Brazil.
* Corresponding author’s E-mail: ferreirasilva.jc@gmail.com

Abstract

BACKGROUND: The proteomic profile of cryopreserved in vitro produced bovine embryos is little known but can provide insights on the successful application of cryo procedures in support of animal breeding. OBJECTIVE: To identify embryonic proteins and biomarkers related to improved cryotolerance of vitrified in vitro produced bovine embryos. MATERIALS AND METHODS: Proteins were isolated from embryo pools (n = 25 embryos per replicate) and analyzed using the nanoLC - MS/MS system. Further, the UniProtKB database (Uniprot – http://www.uniprot.org/) was used for protein identification. Proteins were classified based on their molecular mass, isoelectric point, and enzymatic activity. Post-translational modification predictions and functional gene ontology analysis were performed as well. Finally, a protein-protein interaction network was created to shed light on the embryo interactome. RESULTS: Based on the MS/MS approach, 66 proteins were identified from vitrified Bos taurus embryos. The retrieved proteins were presumably annotated, which allowed a description of the qualitative and functional aspects of the embryo proteome after the vitrification process. CONCLUSION: These findings allowed us to conclude that in vitro-produced vitrified embryos expressed proteins that underlie biological processes related to reproduction, stress and lipid metabolic process, which are essential to maintain embryo viability.

Keywords: blastocyst; cryopreservation; LC/MS/MS; proteome; reproduction.

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CryoLetters 43 (4), 222-226 (2022)
© CryoLetters, editor@cryoletters.org
doi.org/10.54680/fr22410110712

USE OF BIOLOGICAL AND SYNTHETIC POLYMERS FOR HUMAN SPERMATOZOA CRYOPRESERVATION

Maryna Petrushko^1,2 and Taisiia Yurchuk¹*

¹Institute for Problems of Cryobiology and Cryomedicine (IPC&C) of the National Academy of Sciences of Ukraine, 23, Pereyaslavska Str., Kharkiv 61016, Ukraine.
²ART-Clinic of Reproductive Medicine, 38B, Gagarina av., Kharkiv 61000, Ukraine.
*Corresponding author’s E-mail: taisiya.yur@gmail.com

Abstract

BACKGROUND: Spermatozoa cryopreservation is an integral part of the assisted reproductive technologies for treatment of infertility. It is also used to preserve the reproductive potential of men. However, using a standard freezing method with glycerol leads to a decrease in morphological and functional characteristics of spermatozoa in the case of oligoasthenoteratozoospermia (OAT). Therefore, it is relevant to develop effective methods of cryopreservation for such sperm. The use of various biopolymers can stabilize the membrane and bind excess water, which forms ice crystals in the medium that causes cell damage when temperature decreases. OBJECTIVE: To study the effectiveness of using cryoprotectant mixtures based on biological and synthetic polymers [serum albumin, polyvinylpyrrolidone (PVP) and insulin] for the cryopreservation of human spermatozoa with OAT. MATERIALS AND METHODS: Human spermatozoa with OAT were cryopreserved using different cryoprotectant media containing 10% glycerol or 10% PVP, 20% albumin and 1 μg/mL human insulin. The viability, motility and mitochondrial membrane potential of spermatozoa were assessed after rewarming. RESULTS: A cryoprotectant solution containing 10% PVP, 20% human serum albumin and 1 μg/mL insulin enabled a similar level (%) of viable gametes compared with the standard method using glycerol, while the number of motile cells was significantly lower (p < 0.008). The membrane mitochondrial potential did not differ significantly from fresh sperm. CONCLUSION: The data obtained in this study show the effectiveness of a biopolymer mixture containing PVP, serum albumin and insulin for the cryopreservation of human OAT spermatozoa

Keywords: cryopreservation; insulin; oligoasthenoteratozoospermia; polyvinylpyrrolidone; serum albumin; spermatozoa.

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CryoLetters 43 (4), 227-236 (2022)
© CryoLetters, editor@cryoletters.org
doi.org/10.54680/fr22410110212

TIME DEPENDENT IMPACT OF REACTIVE OXIDANTS ON SEMINAL ATTRIBUTES, MITOCHONDRIAL MEMBRANE POTENTIAL, LIPID PEROXIDATION AND CAPACITATION-LIKE CHANGES OF KARAN-FRIES (KF) BULLS DURING CRYOPRESERVATION

Nitish. A. Kulkarni¹*, A. K. Roy¹, Sujata Pandita¹, C. G. Shashank¹ and H. S. Chethan²

¹Animal Physiology Division, ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, India.
²Animal Reproduction Gynaecology and Obstetrics Division, ICAR-NDRI, Karnal, India.
Corresponding author’s E-mail: kulkarni.nitish97@gmail.com

Abstract

BACKGROUND: Cryopreservation of semen is a valuable technique; however, it is also known to be detrimental to the structure of spermatozoa and fertility due to cryo-injury and subsequent generation of reactive oxidants. OBJECTIVE: To determine the time-dependent impact of reactive oxidants on seminal attributes, mitochondrial membrane potential (MMP), lipid peroxidation status (LPO) and early capacitation like changes. MATERIALS AND METHODS: Semen samples were collected by artificial vagina technique from six Karan-Fries (KF) bulls and subsequently examined at 0 h (before cryopreservation) and at 24 hours, 15 days and 2-months of storage for various seminal attributes, MMP (Δψm), and early capacitation-like changes. Simultaneously, LPO (TBARS) was determined in fresh and post-thaw seminal plasma. RESULTS: A sharp decrease (P<0.01) in semen quality was observed only after 24 h of cryopreservation except for viability and acrosomal integrity. Sperm viability and acrosome integrity reduced significantly up to 2 months of cryopreservation. The lipid peroxidation status was found to be lower in fresh seminal plasma (2.63±0.22 vs. 3.51±0.34 units/mL) as compared to post-thaw. Furthermore, the active Δψm of fresh semen showed a significant (P<0.01) decrease after 24 hours (77.92±0.387 vs. 54.52±0.28%) of cryopreservation, while there was a non-significant decrease in active MMP after 15 d and 2-months (53.68±0.138 and 52.76±0.16%). Moreover, significant (P<0.01) early capacitation-like changes were found in post-thaw spermatozoa (25.72±0.12 vs. 9.1±0.19%) as compared to fresh ejaculate. CONCLUSION: Spermatozoa incur the majority of damages during the early phase of cryopreservation, however the damage associated by different stressors cannot be neglected

Keywords: capacitation; cryopreservation; KF bulls; mitochondrial membrane potential; reactive oxygen species.

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CryoLetters 43 (4), 237-245 (2022)
© CryoLetters, editor@cryoletters.org
doi.org/10.54680/fr22410110412

THE ADAPTATION TO FREEZING TOLERANCE OF HYDRATED LETTUCE SEEDS: EFFECTS OF REGIONAL CLIMATE AND OF SEED CHARACTERISTICS

Yingying Han¹, Ganesh K Jaganathan¹, Jingwen Zhou¹, Shiwei Wei² and Baolin Liu¹*

¹Institute of Biothermal Science and Technology, School of Health Science and Engineering, University of Shanghai for Science and Technology, 516 Jungong Road, Shanghai, China. ²Shanghai Agrobiological Gene Center, 2901 Beidi Road, Shanghai, China.
*Corresponding author’s E-mail: blliuk@163.com

Abstract

BACKGROUND: With global warming, soil seed banks at high altitudes face dual challenges, excessive water absorption and thinner snow cover that increase underground temperature. A better understanding of freezing tolerance of hydrated seeds provides insights for conservation in natural soil seed banks. OBJECTIVE: To understand the adaptation mechanisms of seed freezing tolerance under various climates, in relation to cooling rate and seed size. MATERIALS AND METHODS: Twelve ecotypes of lettuce (Lactuca sativa) seeds were collected from different geographical locations around the world. Seeds were fully hydrated and tested for their freezing tolerance using programmed cooling methods. RESULTS: The size of seeds from different climate regions varied, and was correlated with the freezing tolerance of the hydrated seeds (P<0.05). Larger seeds showed poorer freezing tolerance. The local climates of maternal plants were also well correlated to seed freezing tolerance (P<0.05), especially under slow cooling conditions. The seeds collected in regions with high spring rainfall exhibited greater freezing tolerance. CONCLUSION: Freezing tolerance of hydrated seeds is affected by the climate of maternal plants and by seed size. Our data revealed the existence of an adaptation mechanism of freezing tolerance among various ecotypes of lettuce seeds

Keywords: climate; freezing tolerance; hydrated seeds; Lactuca sativa; seed size.

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CryoLetters 43 (4), 246-254 (2022)
© CryoLetters, editor@cryoletters.org
doi.org/10.54680/fr22410110612

EXTENDERS AND CRYOSOLUTIONS FOR GRUMATÃ (Prochilodus vimboides) SPERM PRESERVATION

Alexmiliano V. Oliveira¹*, Eduardo A. T. Lanna², Naiara C. Motta³, Guilherme Souza⁴, Genaina A. Souza⁵, Jerusa M. Oliveira⁶,
Thiago A. Freato⁷ and Felipe M. Santos⁶

¹Agricultural Research Company of Minas Gerais, EPAMIG, Viçosa, MG, Brazil.
²Department of Animal Science, Federal University of Viçosa, MG, Brazil.
³Department of Animal Science, Federal University of Lavras, Lavras, MG, Brazil.
⁴Non-governmental Organization, Piabanha Project, Itaocara, RJ, Brazil.
⁵Department of Forest Engineering, Federal University of Viçosa, MG, Brazil.
⁶Department of Animal Biology, Federal University of Viçosa, MG, Brazil.
⁷Meteorological and Climate Research Center Applied to Agriculture, State University of Campinas, Campinas, SP, Brazil.
*Corresponding author’s E-mail: alexmiliano@epamig.br

Abstract

BACKGROUND: Prochilodus vimboides populations are being reduced in rivers due to changes in their habitat, overfishing, urbanization, and pollution. OBJECTIVE: To evaluate the effect of sperm extender solutions for short-term storage and cryosolutions for freezing sperm of Prochilodus vimboides. MATERIALS AND METHODS: For short-term storage, the sperm was diluted in 0.9% NaCl, 1.2% NaCl, 5% glucose, 5% BTS®, or 6% MIII®. Sperm motility was evaluated after 0, 24, 48, and 72 h of short-term storage at 4-6°C. For cryopreservation, sperm samples were diluted in the same extenders and factorially combined with three cryoprotectants (dimethylsulfoxide, methyl glycol, and ethylene glycol). After thawing, sperm motility and oxidative stress parameters were evaluated. RESULTS: Dilution of samples in BTS® preserved sperm motility >40% for up to 48 h. Samples cryopreserved in 5% glucose and methylglycol presented higher sperm motility, lower catalase, and lipid peroxidation activities. CONCLUSION: Prochilodus vimboides sperm can be cooled for up to 48 h in an extender solution of 5% BTS® and cryopreserved in 5% glucose and methyl glycol.

Keywords: characiformes; cryopreservation; oxidative stress; short-term storage

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