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CryoLetters 43 (6), 303-315 (2022)
© CryoLetters, editor@cryoletters.org
doi.org/10.54680/fr22610110112

PERSPECTIVE:
A GUIDE TO SUCCESSFUL mL TO L SCALE VITRIFICATION AND REWARMING

Lakshya Gangwar¹, Shaunak S. Phatak¹, Michael Etheridge¹ and John C. Bischof*^1,2

¹Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN, 55455 USA
²Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, 55455 US
*Corresponding author’s E-mail: bischof@umn.edu

Abstract

Cryopreservation by vitrification to achieve an “ice free” glassy state is an effective technique for preserving biomaterials including cells, tissues, and potentially even whole organs. The major challenges in cooling to and rewarming from a vitrified state remain ice crystallization and cracking/fracture. Ice crystallization can be inhibited by the use of cryoprotective agents (CPAs), though the inhibition further depends upon the rates achieved during cooling and rewarming. The minimal rate required to prevent any ice crystallization or recrystallization/devitrification in a given CPA is called the critical cooling rate (CCR) or critical warming rate (CWR), respectively. On the other hand, physical cracking is mainly related to thermomechanical stresses, which can be avoided by maintaining temperature differences below a critical threshold. In this simplified analysis, we calculate ΔT as the largest temperature difference occurring in a system during cooling or rewarming in the brittle/glassy phase. This ΔT is then used in a simple “thermal shock equation” to estimate thermal stress within the material to decide if the material is above the yield strength and to evaluate the potential for fracture failure. In this review we aimed to understand the limits of success and failure at different length scales for cryopreservation by vitrification, due to both ice crystallization and cracking. Here we use thermal modeling to help us understand the magnitude and trajectory of these challenges as we scale the biomaterial volume for a given CPA from the milliliter to liter scale. First, we solved the governing heat transfer equations in a cylindrical geometry for three common vitrification cocktails (i.e., VS55, DP6, and M22) to estimate the cooling and warming rates during convective cooling and warming and nanowarming (volumetric heating). Second, we estimated the temperature difference (ΔT) and compared it to a tolerable threshold (ΔTmax) based on a simplified “thermal shock” equation for the same cooling and rewarming conditions. We found, not surprisingly, that M22 achieves vitrification more easily during convective cooling and rewarming for all volumes compared to VS55 or DP6 due to its considerably lower CCR and CWR. Further, convective rewarming (boundary rewarming) leads to larger temperature differences and smaller rates compared to nanowarming (volumetric rewarming) for all CPAs with increasing failure at larger volumes. We conclude that as more and larger systems are vitrified and rewarmed with standard CPA cocktails, this work can serve as a practical guide to successful implementation based on the characteristic length (volume/surface area) of the system and the specific conditions of cooling and warming.

Keywords: critical cooling rate; critical warming rate; temperature difference; vitrification.

Download the paper: A GUIDE TO SUCCESSFUL mL TO L SCALE VITRIFICATION AND REWARMING (PDF)

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CryoLetters 43 (6), 316-321 (2022)
© CryoLetters, editor@cryoletters.org
doi.org/10.54680/fr22610110312

THE USE OF PECTINS AS PART OF A CRYOPROTECTIVE SOLUTION FOR LONG-TERM STORAGE OF HUMAN PLATELET CONCENTRATES

M.I. Sergushkina¹*, A.N. Khudyakov¹, O.O. Zaitseva¹,
T.V. Polezhaeva¹, O. N. Solomina¹, K. A. Vetoshkin²
and M. A. Butolina²*

¹Institute of Physiology of Komi Scientific Centre of the Ural Branch of the Russian Academy of Sciences, FRC Komi SC UB RAS, Syktyvkar, Russian Federation
²The Federal State-Financed Scientific Institution Kirov Research Institute of Hematology and Blood Transfusion under the Federal Medical Biological Agency, Kirov, Russian Federation
*Corresponding author’s E-mail: mara.kovalkova@mail.ru

Abstract

BACKGROUND: Pectins have unique properties and great potential to become an indispensable component of cryoprotective environment for platelet freezing. OBJECTIVE: To investigate the possibility of including pectins (apple pectin AU-701, tanacetan) into the composition of a cryoprotective solution for platelets during low-temperature storage. MATERIALS AND METHODS: Samples of platelet concentrates (PC) were frozen under the protection of complex solutions and stored in an electric freezer at -80°C for 1 and 6 months. RESULT: The study showed that of the basic cryoprotectants, the best effect in the preservation of PC was with dimethylacetamide (DMAC). The use of pectins as an additive to the base solution of DMAC statistically improves the preservation of PC after exposure to low temperatures (-80°C) for 30 and 180 days. CONCLUSIONS: We conclude that DMAC is more promising as a basis for the development of a new combined cryoprotectant for PC freezing. Moreover, the chemical structure of pectin determines the level of its cryoprotective action in relation to the preservation of PC.

Keywords: cryopreservation; pectins; platelet concentrate.

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CryoLetters 43 (6), 322-327 (2022)
© CryoLetters, editor@cryoletters.org
doi.org/10.54680/fr22610110512

ANTIBIOGRAM OF MICROORGANISMS ISOLATED FROM FRESH AND FROZEN SEMEN OF CROSSBRED FRIESWAL BULLS

Naimi Chand, Megha Pande*, Shrikant Tyagi, Ajayvir S Sirohi,
Sumit Mahajan, Suresh Kumar, Sarika and Ankur Sharma

Division of Cattle Physiology and Reproduction, ICAR-Central Institute for Research on Cattle, Meerut Cantt-250001, Uttar Pradesh, India.
*Corresponding author’s E-mail: drncmudgal75@rediffmail.com

Abstract

BACKGROUND: The bacterial contaminants in the semen are a major concern for most of the semen production laboratories because they adversely affect the semen quality. During sperm cryopreservation, the inclusion of antimicrobials in extenders may help to minimize bacterial growth. However, due to bacterial resistance to commonly used antimicrobials, they cannot fully assure microbiological safety to the frozen semen. OBJECTIVE: To estimate the microbial load and antibiogram of microorganisms isolated from the fresh and frozen bull semen. MATERIALS AND METHODS: The bacterial load was estimated in fresh and frozen semen samples of crossbred Frieswal bulls by the pour plate method. Microorganisms were identified as Gram positive and Gram negative by Gram staining. The representative bacterial colonies were streaked onto different specific media which were further confirmed by biochemical tests. Bacterial isolates were subjected to in vitro antibiotic sensitivity test. RESULTS: The average microbial load of fresh and frozen semen samples was found to be 8397.4±524.31 cfu/mL and 680.87±105.41cfu/mL, respectively. Microorganisms belonging to Staphylococcus aureus, Staphylococcus epidermidis, Proteus, Klebsiella, Bacillus cereus, Bacillus subtilis, Actinomyces, E. coli, Rhodococcus, Neisseria and Micrococcus were identified in the semen samples. The antibiotic sensitivity testing of the bacterial isolates revealed that benzyl penicillin was found to be the least effective against the isolated organisms while gentamicin and spectinomycin were found to be most effective among the antibiotics used. Lincomycin, tylosin and streptomycin showed moderate efficacy against the bacterial isolates. CONCLUSION: Gentamicin, tylosin, lincomycin, and spectinomycin (GTLS) antibiotic combination is more effective against bacterial isolates and may be added to semen extender to better control bacterial load and semen quality.

Keywords: antibiogram; bacteria; bull semen; sensitivity

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CryoLetters 43 (6), 328-333 (2022)
© CryoLetters, editor@cryoletters.org
doi.org/10.54680/fr22610110712

EVALUATION OF VACUUM INFILTRATION VITRIFICATION AND CRYO-MESH CRYOPRESERVATION TECHNIQUES WITH Arabidopsis thaliana SHOOT TIPS

Milana Lukić^1,2, Bryn Funnekotter^1,2, Michael J. Considine^3,4,5,
Eric Bunn^1,2,3, and Ricardo L. Mancera^1*

¹Curtin Medical School, Curtin University, GPO Box U1985, Perth WA 6845, Australia.
²Kings Park Science, Department of Biodiversity, Conservation and Attractions, Kings Park WA 6005, Australia.
³School of Molecular Sciences, The University of Western Australia, Perth WA 6009, Australia.
⁴The UWA Institute of Agriculture, The University of Western Australia, Perth WA 6009, Australia.
⁵Department of Primary Industries and Regional Development, Perth WA 6000, Australia
*Corresponding author’s E-mail: R.Mancera@curtin.edu.au

Abstract

BACKGROUND: Novel cryo-techniques are continuously being developed that may better improve cryogenic survival in plants, with the aim of reducing exposure times to otherwise toxic cryoprotective agents whilst maximising regeneration rates. OBJECTIVE: This study used cryo-mesh and vacuum infiltration vitrification with two vitrification solutions (PVS2 and PVS3) to develop an optimised cryopreservation protocol for Arabidopsis thaliana. MATERIALS AND METHODS: Shoot tips from 10-day old seedlings of wild type A. thaliana were cryopreserved using either vacuum infiltration vitrification or the cryo-mesh technique. Shoot tips were treated for up to 60 min in increments of 10 min with PVS2 and PVS3, and for an additional 180 and 300 min incubation for cryo-mesh prior to exposure to liquid nitrogen. RESULTS: Both methods resulted in very high regeneration rates, but which decreased after longer exposure to the vitrification solutions. The highest regeneration rate for vacuum-infiltration vitrification was attained after only 30 min incubation in PVS2 (92.5%) and 50 min incubation in PVS3 (93.55%). In the case of cryo-mesh the highest regeneration was observed after 180 min incubation in either PVS2 (100%) or PVS3 (92.2%). CONCLUSION: Vacuum-infiltration vitrification is more effective than cryo-mesh by reducing exposure times to cryoprotective solutions whilst achieving very high regeneration rates of shoot tips of A. thaliana.

Keywords: Arabidopsis thaliana; cryo-mesh; plant cryopreservation; PVS2; PVS3; vacuum-infiltration vitrification.

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CryoLetters 43 (6), 334-340 (2022)
© CryoLetters, editor@cryoletters.org
doi.org/10.54680/fr22610110212

CAN HONEY IMPROVE THE QUALITY OF CRYOPRESERVED CROSS BRED RAM SEMEN ADDED TO TRIS EGG YOLK EXTENDER?

Arjuma Khatun^1*, M.R. Fazili², A.A. Malik¹, M. Naikoo¹,
A.R. Choudhury³, Syed Shah⁴, F.A. Lone¹, S. Qureshi⁵
and I. Hussain⁵

¹Division of Animal Reproduction, Gynaecology and Obstetrics;
²Mountain Livestock Research Institute;
³Division of Veterinary Anatomy and Histology;
⁴Frozen Semen Station;
⁵Division of Veterinary Microbiology and Immunology,
Faculty of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir, University of Agricultural Sciences and Technology of Kashmir, Shuhama, Alusteng, Srinagar – 190006, J & K, India.
Corresponding author’s E-mail: khatunarjuma786@gmail.com

Abstract

BACKGROUND: Honey can improve the quality of cryopreserved ram semen because of its multinutrient and cryoprotective nature added to standard tris egg yolk extender. OBJECTIVE: Different concentrations of honey were added to the standard tris egg yolk extender to improve the post-thaw quality of crossbred ram semen. METHOD: Thirty six (36) ejaculates from eight healthy cross bred rams were pooled and divided into four aliquots. Standard tris egg yolk extender without any alteration acted as Control (C) and was supplemented with different concentrations of honey, viz. T1 (honey 1.5%), T2 (2.5%), and T3 (3.5%). RESULTS: The percent (mean±S.E.M) sperm motility at pre-freeze was significantly (P<0.05) higher in Group T2 and at post-thaw in Group T3 in comparison to T1 and C treatment groups. The percent (mean±S.E.M) HOST reacted spermatozoa at post-thaw was significantly (P<0.05) higher in Group C and at pre-freeze the value was significantly (P<0.05) higher in the same treatment group than Group T1. The mean MDA level (mean±S.E.M) at post thaw was significantly (P<0.05) lower in Group T3 than the treatment groups C and Group T1. CONCLUSION: From this study it is concluded that the addition of 3.5% honey to the standard tris egg yolk extender provides better protection to ram semen than the addition of 1.5% honey (i.e., Control).

Keywords: cryopreservation; honey; IVF; ram; semen; tris egg yolk.

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CryoLetters 43 (6), 341-348 (2022)
© CryoLetters, editor@cryoletters.org
doi.org/10.54680/fr22610110412

CRYOPRESERVATION OF SEEDS OF THE HIGHLY VALUED TROPICAL TIMBER SPECIES Swietenia mahagoni

Ysmel Entensa¹, Abel González-Morales¹, Claudia Linares¹,
José Gerardo Vázquez¹, Marcos Edel Martínez-Montero¹,
Byron E. Zevallos-Bravo², Elliosha Hajari³, Monika Höfer⁴,
Ariel Villalobos-Olivera¹ and José Carlos Lorenzo¹*

¹Laboratory for Plant Breeding and Conservation of Genetic Resources, Bioplant Centre, University of Ciego de Ávila, Ciego de Ávila, 69450, Cuba.
²Universidad Estatal del Sur de Manabí (UNESUM), Ecuador.
³Plant Improvement; Agricultural Research Council-Tropical and Subtropical Crops; Private Bag X11208, Nelspruit, 1200, South Africa.
⁴Julius Kühn Institute, Institute for Breeding Research on Fruit Crops, Pillnitzer Platz 3a, 01326 Dresden, Germany.
*Corresponding author’s E-mail: jclorenzo@bioplantas.cu

Abstract

BACKGROUND: Swietenia mahagoni wood is one of the most valuable in world trade and, as a result, natural populations have been decimated due to unsustainable harvesting. The decline in natural population levels is being exacerbated by climate change. In order to ensure the preservation of valuable genotypes, there is an urgent need to develop strategies to conserve the genetic diversity present within this species. At present, cryopreservation is the most viable option for the long-term storage of plant germplasm, particularly for long-lived species which are challenging to maintain in the field. OBJECTIVE: To cryopreserve intact seeds of S. mahagoni, with the dual goal of retaining the biosynthetic capacity of plants, which is critical since this species is highly valued for medicinal purposes. MATERIALS AND METHODS: Seeds at a moisture content of 6% were immersed in liquid nitrogen (LN) before warming and recovery. Plantlet establishment and growth were assessed over a period of 70 days and anthraquinone synthesis was determined in roots, stems and leaves. RESULTS: The results showed an initial lag in the germination rate of cryopreserved seeds compared with control seeds; however, this difference disappeared over time. The lag in seedling emergence observed in cryostored seeds was also evident in the plant characteristics measured following 30 days of culture when all plant parameters measured were significantly higher in plants produced from control than cryostored seeds. However, after 70 days of growth, these differences were no longer apparent. Anthraquinone levels were also initially lower (at 30 days) in plants regenerated from cryopreserved seeds than those from control seeds, however, this difference was substantially reduced by 70 days thereby indicating the ability of these plants to accumulate secondary metabolites, albeit at a reduced rate, during the early stages of development. CONCLUSION: In S. mahagoni, the delay in anthraquinone production in plants regenerated from cryostored seeds during the early stages of development may have occurred as a consequence of the preferential allocation of resources towards the initiation of recovery processes in response to the stresses imposed by cryopreservation. Once the stresses were overcome and plant growth resumed, resources could be directed to secondary processes such as anthraquinone synthesis.

Keywords: anthraquinones; cryopreservation; ex situ conservation; genetic resources; Swietenia mahagoni.

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CryoLetters 43 (6), 349-356 (2022)
© CryoLetters, editor@cryoletters.org
doi.org/10.54680/fr22610110612

THE EFFECT OF DIFFERENT PRESERVATION MEDIA AND TEMPERATURES ON SPERM QUALITY AND DNA INTEGRITY IN MOUSE CAUDA SPERMATOZOA

Cengiz Yildiz, Nurdan COŞKUN ÇETİN*, İlker YAVAŞ,
Oğuz Kaan YALÇIN, Firdevs YILMAZ and Fikret KARACA

Hatay Mustafa Kemal University, Faculty of Veterinary Medicine, Department of Reproduction and Artificial Insemination, Hatay-TURKEY
*Corresponding author’s E-mail: nurdancsknctn@gmail.com

Abstract

BACKGROUND: Mouse sperm can be stored for long or short-time periods. Nevertheless long-term storage leds to significantly reduced sperm quality and fertility because of cryodamage. Thus, in the storage of semen in mice, it is necessary to focus on media and temperatures that gives good results in short-term storage. OBJECTIVE: To determine favorable media for short-term storage of mice spermatozoa by evaluating progressive motility, viability, membrane function integrity, acrosome integrity and fragmented DNA rates at various storage temperatures. MATERIALS AND METHODS: Mouse spermatozoa were collected from epididymides of mature CD1 males and samples were stored at 24°C and 4°C for 60 h. RESULTS: Motility, viability and membrane function of mice spermatozoa were greatest when stored in KSOM media. Motility and viability were not different when stored at refrigerator or room temperature in KSOM compared to HTF or PBS mediums for 48 h, but were after 60 h. There wasn’t any significant variation in terms of acrosome integrity in different preservation conditions. Fragmented DNA rates were similar in fresh sperm with KSOM and HTF media, while there was higher damage in PBS medium at 60 h. Overall, sperm parameters were affected significantly by the time of storage and type of preservation medium, and PBS extender was not suitable for mice spermatozoa at room and refrigerated temperatures as it caused the lowest progressive motility, viability, membrane function integrity and the highest DNA damage. CONCLUSION: Mice spermatozoa stored in KSOM retained the best sperm quality parameters both 24°C and 4°C for the first 48 h.

Keywords: cooling; DNA integrity; KSOM; mouse; room temperature; sperm quality.

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CryoLetters 43 (6), 357-367 (2022)
© CryoLetters, editor@cryoletters.org
doi.org/10.54680/fr22610110812

EFFECTS OF CELL CONCENTRATION DURING CRYOPRESERVATION ON THE POST-THAW QUALITY OF SANTA INÊS RAM SPERM

Matheus Batista de OLIVEIRA¹, Julio Constantino Jerí MOLINA¹, Rebeca Santos da SILVA¹, Alexandre Floriani RAMOS²,
Phillip Hamilton PURDY³ and Hymerson Costa AZEVEDO⁴

¹Universidade Federal de Sergipe (Federal University of Sergipe), Av. Marechal Rondon, São Cristóvão, SE, 490100-000, Brazil.
²Empresa Brasileira de Pesquisa Agropecuária — EMBRAPA (Brazilian Agricultural Research Corporation) — Embrapa Recursos Genéticos e Biotecnologia (Embrapa Genetic Resources and Biotechnology), Av. W5 Norte, Brasília, DF, 70770-917, Brazil.
³United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Animal Germplasm Program, 1111 S. Mason St., Fort Collins, CO, 80521-4500, USA.
⁴Empresa Brasileira de Pesquisa Agropecuária — EMBRAPA (Brazilian Agricultural Research Corporation) — Embrapa Tabuleiros Costeiros (Embrapa Coastal Tablelands), 3250, Av. Beira Mar, Aracaju, SE, 49025-040, Brazil.
*Corresponding author’s E-mail: hymerson.azevedo@embrapa.br

Abstract

BACKGROUND: Non-surgical artificial insemination techniques for sheep may benefit from larger numbers of sperm in the insemination dose because the ewe’s cervix is convoluted and often cannot be traversed with an insemination gun resulting in deposition of the sperm at the os cervix. OBJECTIVE: To compare a range of sperm concentrations when cryopreserving semen from Santa Inês rams and determine the effects of this on post-thaw quality. MATERIALS AND METHODS: One ejaculate from each ram (n = 10) was diluted to four sperm concentrations to obtain the following groups: G-400, G-800, G-1200, and G-1600 x 106 sperm/mL. The semen samples were packaged in 0.25 mL straws, cooled to 5°C, cryopreserved in liquid nitrogen vapor, thawed in a water bath (40°C/20 s), and were analyzed for computerized kinetics, capacitation and acrosome integrity, and plasma membrane integrity of sperm. RESULTS: The G-400 treatment resulted in samples with the highest linearity and progressive motion (P < 0.05) and had significantly greater plasma membrane integrity, and lower capacitation and acrosome reaction rates compared to G-1600 (P < 0.05). Overall, use of the G-400 treatment resulted in better kinetics, less plasma membrane damage and less early capacitation. However, despite reducing the ejaculate yield and increasing the costs of the semen freezing process, the G-800 and G-1200 treatments make a greater absolute number of sperm with good kinetics, plasma membrane integrity and capacitation status available. CONCLUSION: Ram sperm concentration impacts cryopreservation, and higher concentrations may be advantageous if a single artificial insemination protocol is desirable.

Keywords: acrosome reaction; capacitation; cryopreservation; computer-assisted sperm analysis; motion kinetics; plasma membrane integrity.

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