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Nanocrystalline cerium dioxide is able to protect living cells from oxidative stress under the influence of various stress factors, in particular under the one of low temperatures. This study investigates the phase-structural transformations in aqueous solutions containing CeO 2 nanoparticles (NPs) and their impact on the cryopreservation process. Differential scanning calorimetry and thermomechanical analysis were used to analyse the phase transitions in aqueous suspensions of CeO 2 NPs and aqueous solutions of the cryoprotectant dimethyl sulfoxide (Me 2 SO) with CeO 2 NPs. Various concentrations of CeO 2 NPs were tested to observe their effects on the crystallization and melting behaviours. The addition of CeO 2 NPs significantly altered the temperatures and enthalpies of melting and crystallization in water. Low concentrations of CeO 2 NPs promoted crystallization, while higher concentrations inhibited it, reducing supercooling and recrystallization during thawing. In Me 2 SO solutions, CeO 2 NPs raised the glass transition temperature and affected the recrystallization process, with higher concentrations leading to more pronounced vitrification and reduced recrystallization. We also investigated the regularities of the effect of CeO 2 NPs on phase transitions in combined cryoprotective media with Ham's F12, fetal bovine serum and Me 2 SO, which can be used in future to design the cryopreservation protocols. In the complex media, CeO 2 NPs decreased the metastability and altered eutectic crystallization patterns, indicating potential cryoprotective effects. In conclusion, CeO 2 NPs modulate the thermophysical properties of cryoprotective solutions, enhancing vitrification and reducing recrystallization, which could improve cryopreservation efficiency. Optimizing NP concentrations is crucial for practical applications in cryopreservation., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the potential protective mechanisms of aconite polysaccharide (FZPS-1) during cryopreservation, with a particular emphasis on morphological changes in autophagy in rat abdominal aorta., Methods: Thirty-six male SD rats were divided into the control group, the cryopreserved model group, and the FZPS-1 intervention group treated with different concentrations of FZPS-1. The structural changes of the abdominal aortic wall were assessed via Masson staining, while cytolysosomes were identified using transmission electron microscopy (TEM). The expression of Beclin-1, LC3-II, and P62 was detected by immunohistochemistry (IHC) and western blot separately. Bcl-2 and Bax mRNA expression was measured by RT-qPCR., Results: Compared with the control group, the abdominal aortic wall in the model group was severely damaged. Contrarily, FZPS-1 10 mg/mL and 20 mg/mL groups had relatively normal structure of the blood vessel wall, higher cytolysosome counts, and increased Beclin-1 and LC3-II expression compared with the model group (all P<0.05); P62 expression also increased in the FZPS-1 20 mg/mL group (P<0.05). Compared with the control group, the mRNA expression of Bcl-2 in the cryopreservation model group was reduced (P<0.05), while Bax was increased (P<0.05). Compared with the cryopreservation model group, the mRNA expression of Bcl-2 was upregulated, while Bax was downregulated in the FPS 10 mg/L group (P<0.05)., Conclusion: During liquid nitrogen cryopreservation, autophagy is inhibited in the rat abdominal aorta, and the blood vessel wall structure is damaged. FZPS-1, as a cryoprotectant, can enhance autophagy and mitigate blood vessel wall damage in the rat abdominal aorta., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Frozen dough technology has been widely used in the food industry at home and abroad due to its advantages of extending shelf life, preventing aging, and facilitating refrigeration and transportation. However, during the transportation and storage process of frozen dough, the growth and recrystallization of ice crystals caused by temperature fluctuations can lead to a deterioration in the quality of the dough, resulting in poor sensory characteristics of the final product and decreased consumption, which limits the large-scale application of frozen dough. In response to this issue, antifreeze proteins (AFPs) could be used as a beneficial additive to frozen dough that can combine with ice crystals, modify the ice crystal morphology, reduce the freezing point of water, and inhibit the recrystallization of ice crystals. Because of its special structure and function, it can well alleviate the quality deterioration problem caused by ice crystal recrystallization during frozen storage of dough, especially the plant-derived AFPs, which have a prominent effect on inhibiting ice crystal recrystallization. In this review, we introduce the characteristics and mechanisms of action of plant-derived AFPs. Furthermore, the application of plant-derived AFPs in frozen dough are also discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

This study investigated whether the origin of sperm (epididymal vs. ejaculate) affects the cryopreservation efficiency in agouti (Dasyprocta leporina). Five sexually mature agoutis underwent electroejaculation, resulting in obtaining four semen samples. After 15 days, the same animals were euthanized, and through retrograde flushing, sperm samples were obtained from the epididymis tails. In both collection methods, samples were evaluated for sperm parameters (sperm concentration, motility, vigor, membrane integrity, osmotic response, and morphology). Then, samples were diluted in ACP 109c, added with 20% egg yolk, and a final concentration of 6% glycerol. Finally, the samples were packaged in 0.25 mL straws and frozen in liquid nitrogen. After one week, samples were thawed and evaluated in the same way as fresh samples, with the addition of membrane integrity analysis using fluorescent probes (C-FDA/PI) and computerized analysis (CASA). Immediately after obtaining the sperm, samples obtained directly from the epididymis presented higher values (P ≤ 0.05) than those obtained by electroejaculation concerning the parameters of volume, sperm concentration, and total number of sperm (1,398.25 ± 206.0 x10 6 and 184.5 ± 78.0 x10 6 sperm). On the other hand, in the classical evaluation of the other sperm parameters and the computerized analysis (CASA) after thawing, such as total motility, no statistical differences were observed between sperm from both origins (ejaculate: 16.7 ± 8.2% and epididymal: 24.8 ± 12.0%, P > 0.05). This demonstrates the possibility of direct application of the cryopreservation protocol for agouti (D. leporina) sperm obtained via the epididymis or ejaculate., Competing Interests: Declaration of competing interest The authors state that there are no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background Aims: Intracranial (IC) locoregional delivery of chimeric antigen receptor (CAR) T cells presents an attractive delivery method to central nervous system tumors. Although IC delivery is actively being employed in early-phase clinical studies, no thaw/wash methods have been published to remove the neurotoxic cryoprotectant dimethyl sulfoxide (DMSO) from CAR T-cell products before IC administration. Thus, the aim of this study was to develop and validate a simple thaw/wash procedure., Methods: We developed a thaw/wash procedure that consist of product thaw at 37°C, equilibration for 5 min in 1 volume of preservative-free normal saline (PFNS), dilution with an additional 8 volumes of PFNS, removal of DMSO through a washing step, resuspension in 2.0 mL of PFNS and storage in a syringe at 20-25°C. Final formulated products (FPs) were assessed for quality and safety attributes and stability over 3 h from the completion of the thaw. Stability parameters included CAR T-cell viability, transgene surface expression and cytolytic activity., Results: The developed procedure reduced the calculated % of DMSO to less than 0.025%. FP cell viability and recovery (versus pre-cryopreservation) were within acceptable specifications (mean viability: 85.3%, range: 83%-88%; total nucleated cell recovery mean: 76.5%, range: 65.4%-82.5%). Other prespecified quality assurance/quality control parameters including appearance/ integrity, sterility and endotoxin level (≤1.0 EU/mL), were also met by all FPs (n = 3). Three hours' post thaw/wash stability was confirmed. All products maintained cell viability greater than 70% (mean, 80.0%; range, 79%-81%), with no significant change in transgene expression or cytolytic activity of B7-H3-CAR T cells compared with thawed not diluted/washed control CAR T cells., Conclusions: We have developed a simple thaw/wash procedure to prepare B7-H3-CAR T cells for their locoregional delivery to the neural axis. While we focus here on CAR T cells, the methods could be readily adapted to other cryopreserved immune effector cell products., Competing Interests: Conflict of interest SG and CD are co-inventors on patent applications in the fields of cell or gene therapy for cancer. SG is a consultant of TESSA Therapeutics, a member of the Data and Safety Monitoring Board (DSMB) of Immatics, on the Scientific Advisory Board of Be Biopharma and has received honoraria from Tidal, Catamaran Bio and Sanofi within the last 2 years., (Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Information on prolonging the storage duration of cold semen with acceptable fertility in roosters is limited. This study aimed to determine the efficiency of solid storage with the addition of various concentrations of serine to the Thai native rooster (Pradu Hang Dum) semen extender on semen quality and fertility potential during storage at 5°C for up to 120 h. Pooled semen was diluted with a base extender and a gelatin extender containing 0, 2, 4, and 6 mM serine, then stored at 5°C for 120 h. In Experiment 1, the semen quality and malondialdehyde (MDA) concentrations were assessed at 0, 24, 72, and 120 h after storage. In Experiment 2, fertility potential in terms of fertility and hatchability rates was determined using the most effective solid-storage semen from Experiment 1. Sperm quality decreased with increasing storage time (P < 0.05). The lowest semen quality was observed in the control group since T24 of storage compared with the other groups (P < 0.05). Progressive motility, viability, and mitochondrial function were higher (P < 0.05) in the extender supplemented with gelatin and serine groups than those in the gelatin alone group at T72 and T120. In the extender supplemented with gelatin and serine groups, the highest semen quality was observed in the gelatin with 4 mM serine groups. The differences among extenders supplemented with serine were insignificant (P > 0.05), and the lowest MDA was observed in the gelatin with 4 mM serine groups. The fertility and hatchability rates in gelatin with 4 mM serine at T24 were comparable to those in fresh semen (83.87 and 86.12% vs. 86.66 and 88.3%; P > 0.05). Those of T72 were significantly better than those of the control at the same hour of storage (64.08 and 71.61% vs. 52.38 and 64.48%), while those of T120 were not different among groups. In summary, a semen extender as a solid medium supplemented with 4 mM serine successfully preserved the rooster semen for a long duration up to 72 h of storage time., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Potatoes are consumed by millions of people and are the survival food in several countries. Cultivated varieties of potato (Solanum tubersosum L.) are results of selection and crossing of many wild species. Only 8-13% of wild potato species used for food are preserved by either in situ or ex situ methods. The U.S. National Potato Germplasm Collection maintains over 5900 accessions, of which 75% are crop wild relatives (CWR). The objective of the study was to investigate regrowth of cryogenically stored clonal propagules (shoot tips) of selected CWR accessions maintained in the collection. Sixty-nine accessions from 30 Solanum species and six accessions that are not yet assigned to a species were cryopreserved by a droplet vitrification method at the NLGRP. The post cryopreservation regrowth varied from 40 to 100% (average 68%) but was not significantly different between the tested accessions. Regrowth of six accessions tested after 10 years of cryogenic storage was between 35 and 90% (average 66%) and was significantly different from their initial regrowth (average 87%); the largest viability loss was in S. condolleanum; but for the other five accessions the regrowth was between 45 and 90% (average 72%) and suggested at least 10 years of successful storage in LN was possible. Twelve potato wild species cryopreserved in this study were reported in literature as important for developing cultivated varieties for changed weather conditions., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Transplantation has substituted dysfunctional organs with healthy organs from donors to significantly lower morbidity and mortality associated with end-stage organ disease. Since the advent of transplantation, the promise of functional replacement has attracted an exponential mismatch between organ supply and demand. Theoretical proposals to counter the increasing needs have either been to create a source through genetic engineering of porcine donors for xenotransplantation (with more potent immunosuppression protocols) or recreate one's organ in a pig using interspecies blastocyst complementation for exogenic organ transplantation (without immunosuppression). Another promising avenue has been organ banking through cryopreservation for transplantation. Although ice free preservation and acceptable early function following rewarming is critical for success in transplantation, the immunological response that predominantly defines short- and long-term graft survival has failed to captivate attention to date. It is well sorted that thermal and metabolic stress incurred at 4 °C during recovery and reperfusion of organs for clinical transplantation has varying impact on graft survival. Considering the magnitude of cellular imbalance and injury at sub-zero/ultralow temperatures in addition to the chemical toxicity of cryoprotective agents (CPA), it is essential to assess and address the immunological response associated following transplantation to maximize the success of cryopreservation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In the present work we modulated two stress factors salinity and temperature, whose ranges have been previously determined by bioassays using six pre-treatments (18 °C-29.5‰; 18 °C-35‰; 18 °C-39‰ and 20 °C-29.5‰; 20 °C-35‰; 20 °C-39‰), in order to obtain a successful cryopreservation protocol for pluteus larvae of the sea urchin P. lividus (Lamarck 1816). Toxicity tests were performed with different cryoprotectants in a range of 0.5-3 M. Best results pointed out to METH and Me 2 SO as those more suitable for cryopreservation. First an exploratory cryopreservation experiment with Me 2 SO supplemented with 0.04 M trehalose (TRE) was tested following the protocol for cryopreservation of embryos (8-h blastula) of Bellas and Paredes, 2011, which did not give satisfactory results. A cryopreservation experiment was performed with both cryoprotectants supplemented with 0.04 M trehalose on 4-arm pluteus larvae (48 h-old) developed in these pre-treatment conditions, followed by a simpler and shorter protocol with a cooling rate of 1 °C/min to -35 °C, achieving for the first time the successful cryopreservation of P. lividus larvae. When larvae were incubated in low salinity or low temperature pre-treatments, they showed delayed larval development and abnormalities. In contrast, pretreatments with high temperature and salinity showed good results. Dimethyl sulfoxide with trehalose proved to be the only effective cryoprotectant for successful cryopreservation of P. lividus larvae. The success of dimethyl sulfoxide is consistent with that described for other cases in previous literature, where dimethyl sulfoxide, although not the least toxic compound, gave the best cryopreservation result., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Zebrafish is an important animal model, thousands lines have been developed, thus having a great need for their preservation. However, the cryopreservation of fish oocytes is still limited and needs improvement. The sodium alginate hydrogel, in addition to providing support for the cells, has been shown to be a potential cryoprotectant. Therefore, the aim of this study was to evaluate the sodium alginate hydrogel encapsulation technique efficiency during zebrafish ovarian tissue vitrification. The encapsulation methodology was standardized in the first experiment. In Experiment 2, we evaluated four vitrified groups: standard protocol without encapsulation (VS); encapsulated with cryoprotectants (VS1-A); encapsulated with half the cryoprotectants concentration (VS2-A); encapsulated without cryoprotectants (VA). VS treatment (54.6 ± 12.3%; 23.7 ± 9.9%; 12.6 ± 5.0%) did not differ from the VS1-A and VA showed a lower membrane integrity percentage (1.2 ± 1.4%; 0.3 ± 0.6%; 0.5 ± 1.5%). Mitochondrial activity was significantly greater in non-encapsulated treatment (VS) when compared to the encapsulated treatments. VS1-A and VS obtained the lowest lipid peroxidation (39.4 ± 4.4 and 40.5 ± 3.3 nmol MDA/mg respectively) in which VS was not significantly different from the VS2-A treatment (63.6 ± 3.1 nmol MDA/mg), unlike, VA obtained the highest lipid peroxidation level (124.7 ± 7.9 nmol MDA/mg). The results obtained in this study demonstrate that the sodium alginate hydrogel encapsulation technique did not have a cryoprotective action, but maintained the membrane integrity when used the standard concentration of cryoprotectants. However, halving the cryoprotectant concentration of fragments encapsulated in alginate hydrogel did not cause an increase in lipid peroxidation. In addition, it provided support and prevented the oocytes from loosening from the tissue during the vitrification process, being an interesting alternative for later in vitro maturation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In contrast to the livestock industry, sperm cryopreservation has not yet been successfully established in the poultry industry. This is because poultry sperm cells have a unique shape and membrane fluidity, differing from those of livestock sperm. The objective of this review is to discuss the cellular and molecular characteristics of rooster spermatozoa as a cause for their generally low freezability. Furthermore, here, we discuss novel developments in the field of semen extenders, cryoprotectants, and freezing processes, all with the purpose of increasing the potential of rooster sperm cryopreservation. Currently, it is very important to improve cryopreservation of rooster sperm on a global scale for the protection of gene resources due to the incidence of epidemics such as avian influenza., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: In this study we investigated hydrogen bonding interactions in hydrated and frozen solutions of different cryoprotective agents (CPAs) including dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, and trehalose. We also investigated the effect of CPAs on ice crystal growth during storage and correlated this with storage stability of liposomes., Methods: FTIR spectroscopy was used to study hydrogen bonding interactions in CPA solutions in H 2 O and D 2 O, and their thermal response was analyzed using van 't Hoff analysis. The effect of CPAs on ice crystal growth during storage was investigated by microscopy and correlated with storage stability of liposomes encapsulated with a fluorescent dye., Results: Principal component analyses demonstrated that different CPAs can be recognized based on the shape of the OD band region only. Chemically similar molecules such as glycerol and ethylene glycol closely group together in a principal component score plot, whereas trehalose and DMSO appear as condensed separated clusters. The OH/OD band of CPA solutions exhibits an overall shift to higher wavenumbers with increasing temperature and changed fractions of weak and strong hydrogen interactions. CPAs diminish ice crystal formation in frozen samples during storage and minimize liposome leakage during freezing but cannot prevent leakage during frozen storage., Conclusions: CPAs can be distinguished from one another based on the hydrogen bonding network that is formed in solution. DMSO-water mixtures behave anomalous compared to other CPAs that have OH groups. CPAs modulate ice crystal formation during frozen storage but cannot prevent liposome leakage during frozen storage., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Successful cryopreservation requires the addition of cryoprotective agents (CPAs). The addition of permeating CPAs, such as glycerol, is associated with some risk to the cells and tissues. These risks are both related to the CPA themselves (CPA toxicity) and to the volume response of the cell (osmotic damage). To minimize the potential for damage during cryopreservation, mathematical models are often employed to understand the interactions between protocols and cell volume responses. In the literature, this volume response is usually captured using ideal and dilute approximations of chemical potential and osmolality, an approach that has been called into question for cells in high concentrations of CPAs. To address this, the relevance of non-ideal and non-dilute models has been explored in a number of cell types in the presence of permeating CPAs. However, it has not been explored in erythrocytes, which have a cytosolic hemoglobin content of more than 20% by volume and are cryopreserved in 40% glycerol. Because hemoglobin has been suggested to be a highly non-ideal solute, if the non-ideal and non-dilute transport model is relevant to any cells, it should be relevant to erythrocytes. Here we investigate the use, and accuracy, of both the dilute and non-dilute models in predicting cell volume changes during CPA equilibration in erythrocytes, and demonstrate that using published values for the non-ideal and non-dilute model, applied to erythrocytes, leads to model predictions inconsistent with experimental data, whereas dilute approximations align well with experimental data., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cryopreservation of gametes has revolutionized both animal agriculture and human reproductive medicine. Although many new technologies have tremendously improved the cryopreservation of oocytes and embryos, osmotic stress encountered during the equilibration process can cause their loss of function. Rational cryoprotective agent (CPA) equilibration strategies can be used to minimize this stress but require trained personnel to monitor the process in individual oocytes or embryos or require the use of suboptimal average transport parameter values in mathematically guided protocols. To enable individually optimized equilibration of CPAs in individual cells, here we establish experimental and computational techniques to track the osmotic behavior of individual bovine oocytes and embryos during CPA equilibration in real time. We designed a microfluidic device to provide a controlled flow of CPA and modified standard image analysis techniques to estimate real-time cell volume changes. In particular, we used a level-set method to define a boundary within a contour plot which could automate the image analysis process. A colour based level set algorithm coupled with contour smoothing not only provided the best fit but also reduced the segmentation time to well under a second per image. The accuracy of the automated method was comparable to human segmented images for both oocytes and embryos. This technology should enable both rapid evaluation of key biophysical parameters in oocytes and embryos undergoing CPA equilibration and the development of real-time feedback-control of CPA equilibration, enabling individual oocyte- and embryo-specific optimal protocols., (Copyright © 2022 Elsevier Inc. All rights reserved.)

This study was conducted to investigate the effect of semen extenders enriched with glutathione (GSH) on in vitro quality parameters and fertility of post-thawed turkey. In experiment 1, pools of semen diluted in glucose-based extender containing 0.5, 1, and 2 mM of GSH were cryopreserved. During the next step, a different variable such as motility and motion parameters, plasma membrane integrity (PMI) and functionality (PMF), DNA integrity, lipid peroxidation (MDA), total antioxidant capacity (TAC), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity were determined in the post-thawed samples. In the second experiment, artificial insemination was used to evaluate the fertility and hatchability performances of the post-thawed semen. The results of the first experiment showed that the extenders supplemented with 2, 1 and 0.5 mM of GSH had higher levels (p ≤ 0.05) of motility and motion parameters, PMI, PMF, TAC, CAT and SOD activity and lower abnormal morphology, DNA damage, and lipid peroxidation respectively in comparison to the control group (only extender with semen). Notably, the second experiment showed a higher rate of fertility (p ≤ 0.05) in 2 mM of GSH compared to the control group. It can be concluded that adding 2, 1 and 0.5 mM of glutathione leads to an improvement in the survival of the post-thawed turkey, while 2 mM of GSH can increase the fertility strength of the turkey sperm; hence it can be used to improve fertility and hatchability performance., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest to declare. Ethical Considerations the study was approved by Animal Ethics Committee in Urmia University, Urmia, Iran (IR-UU-AEC-3/1191/AD) and conducted under the regulations of this committee., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background Aims: Cryopreserved cellular products, as parts of hematopoietic progenitor cell (HPC) transplants, mononuclear cell reinjections for donor lymphocyte infusion or extracorporeal photopheresis, can be washed before being reinjected into the patient or infused directly, depending on local practices. The aim of washing is to reduce the incidence and severity of adverse reactions (ARs) due to the dimethyl sulfoxide (DMSO) used as a cryoprotective agent and other factors, such as dead cell debris. At the authors' cell therapy laboratory (CTL) in Poitiers, France, as in 76% of Etablissement Français du Sang (EFS) CTLs, all cryopreserved products undergo thawing in a water bath followed by washing with the COBE 2991. As this device will soon cease to be available, an alternative process needs to be assessed., Methods: The authors compared two closed systems: the authors' semi-automatic system using the traditional centrifugation method (COBE 2991) and an automated device using spinning membrane filtration (Lovo). A total of 72 HPC bags available for research were used. The authors first performed a paired comparison, processing one or two HPC bags washed by each device. A second study was carried out to compare two different washing solutions generally used by EFS CTLs along with variable storage conditions. Finally, the authors studied the efficiency of the Lovo with three or four thawed bags. The main parameters studied were viable CD34+ cell recovery and viability, CD3+ cell recovery, stability up to 6 h after washing, DMSO elimination and center feasibility., Results: The Lovo device showed better CD34+ cell recovery compared with the COBE 2991 while maintaining CD34+ viability and stability over 6 h. Moreover, Lovo efficiency seemed to be independent of the number of thawed bags processed and washing solution used in the authors' study. CD3+ cell recovery met the authors' internal specifications (cell recovery >50%), with similar results seen when processing with either the COBE 2991 or Lovo. Additionally, on average, 97% of DMSO was removed after washing with Lovo, minimizing the risk of ARs. The storage conditions post-processing indicated preferred storage conditions of 7 ± 3°C. Finally, if processing time seemed shorter using COBE 2991 for one bag washed, the Lovo device required only one staff member regardless of the number of HPC bags processed., Conclusions: The Lovo device seems to provide an opportunity to standardize HPC processing, ensuring patient safety, with, on average, 97% of DMSO removed, while improving recovery of cells of interest and maintaining viability over time in case of delayed transplant. The Lovo device consequently seems to be a serious alternative to the COBE 2991., Competing Interests: Declaration of Competing Interest MM and SB are employees of Fresenius Kabi. Fresenius Kabi provided the disposables to support the studies described in this article., (Copyright © 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

This study aimed to evaluate the cryopreservation effects on the semen of oncilla (Leopardus guttulus, n = 5, 15 ejaculates) and ocelot (Leopardus pardalis, n = 5, 17 ejaculates) and compare two extenders (commercial and non-commercial extender). An andrological exam was conducted (testicle measurements and penis evaluation), including semen collection by electroejaculation. After collection, the semen was assessed to volume, color, pH, sperm motility, vigor, sperm number in the ejaculate, viability, membrane integrity, and sperm morphology. Samples were centrifuged (300 g for 10 min) and pellet diluted in two extenders (TRIS/glucose/egg yolk and BotuCRIO®), packed into 0.25 mL French straws (20 × 10 6 spermatozoa/mL), equilibrated at 5 °C for 1 h (<0.5 °C/min), freezing in nitrogen vapor for 20 min. Thawing was achieved at 46 °C for 15 min. Thawed samples were evaluated to the same characteristics and ultrastructural analysis. There is no difference for extenders, but in ocelot the spermatozoa maintained higher quality after thawing. Major defects were increased in thawed samples, especially acrosome injuries, in both species. Semen contamination by urine was remarkable to oncilla (53% of the ejaculates) which can have reduced sperm cryoresistance of this species. Ultrastructural analysis endorsed morphological analysis under light microscopy and identified cells with acrosome vesiculation. In conclusion, the spermatozoa of ocelot were more cryoresistent and the extender commercial and non-commercial were suitable for their cryopreservation. Other extenders should be investigated for oncilla., Competing Interests: Declaration of competing interest We have no conflict of interest to declare., (Copyright © 2021. Published by Elsevier Inc.)

The aim of cell preservation technologies is to slow down damaging reactions by lowering the storage temperature. Upon dilution in a stabilizing extender, stallion sperm can be stored at refrigerator temperatures for several days. Cryopreservation allows storage for decades, but freezing and thawing cause damage and viability losses. It is assumed that by storing cells at subzero temperatures in a non-frozen supercooled state, the damaging effects of ice formation can be avoided. In this study, we have investigated if stallion sperm can be stored at -10°C in the absence of ice, and compared viability during supercooled storage with that during storage at 5°C. We found that addition of 2% Ficoll-400 to buffered saline and covering with mineral oil depressed the sample freezing point and inhibited surface-catalyzed nucleation. This allowed storage in a supercooled state at -10°C for up to 7 days. Supplementing specimens with sperm, however, increased the incidence of sample freezing. Nonetheless, with 50×10 6 sperm mL -1 , about 40% of the samples turned out to be non-frozen. Adding 100 mM sucrose was found to preserve sperm membrane intactness during supercooled storage, although this resulted in lower percentages as found with refrigerated storage. Sperm motility appeared to be lost during supercooled storage but could be partly restored by substituting buffered saline with a milk-based extender as base medium. Percentages of membrane intact sperm, however, were found to be lower. Supercooled storage holds promise for semen preservation, but further optimization of the storage solution is required to preserve sperm motility., (Copyright © 2021. Published by Elsevier Inc.)

Studies on the cryopreservation of ovarian tissue usually compare slow freezing versus vitrification and aim to optimize protocols, evaluate combinations or concentrations of cryoprotectant agents (CPAs), exposure time, and the addition of synthetic polymers. This systematic review aimed to identify the different CPAs used for the vitrification of human or primate ovarian tissue and to compare their results in terms of follicular survival and functional preservation. We searched Pubmed and EMBASE for randomized clinical trials or cohort studies comparing CPAs for human and/or primate ovarian vitrification. The highest rate of morphologically normal follicles after cryopreservation was 98% and was obtained with a combination of 27% ethylene glycol (EG) plus 27% glycerol, in addition to non-permeable synthetic polymers. The use of dimethyl sulfoxide (DMSO) in relatively low concentrations combined with EG and other CPAs yielded more than 90% of intact follicles after vitrification. The methods and outcomes varied largely among studies, making it difficult to combine their results. While there is no definite answer to what is the best combination of CPAs for vitrification of human ovarian tissue, the data reviewed here suggest that current vitrification techniques are able to preserve the integrity of most follicles., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Circumventing ice formation is critical to successful cryopreservation by vitrification of large organs. While ice formation during the cooling part of the cryogenic protocol is dictated by the evolving thermal conditions, ice formation during the rewarming part of the cryogenic protocol is also dependent on the history of cooling and storage conditions. Furthermore, while the exothermic effect of ice crystallization during cooling tends to adversely slow down the desired high cooling rates to ensure ice-free preservation, the same effect under some conditions tends to assist acceleration of rewarming during recovery of the specimen from cryogenic storage when limited crystallization does occur. The current study proposes a computational framework to study the thermal effects of crystallization during recovery from cryogenic storage, using a semi-empirical approach to account for the relationship between latent heat effects and the rewarming rate. This study adds another layer of computational capabilities to a recent study investigating similar effects during cooling. Results of this study demonstrate that the thermal effects of crystallization on the local cooling and rewarming rates cannot be neglected. It further explains how crystallization during rewarming helps in increasing the rewarming rate and, thereby, affects rewarming-phase crystallization. Counterintuitively, this study suggests that the fastest possible rewarming rate at the outer surface of the domain in an inwards rewarming problem is not always advantageous, while the proposed computational tool is essential to find an intermediate optimal rate., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Vitrification is the most common method of cryopreservation of gametes in fertility clinics due to its improved survival rates compared to slow freezing techniques. For the Open Cryotop® vitrification device, the number of oocytes, or embryos, mounted onto a single device can vary. In this work, a mathematical model is developed for the cooling of oocytes and embryos (samples). The model is solved computationally, to investigate whether varying the number of samples mounted onto the Open Cryotop® affects the cooling rates, and consequently the survival rates, of vitrified samples. Several realistic spatial arrangements of samples are examined, determining their temperature over time. In this way we quantify the effect of spatial arrangement on the cooling rate. Our results indicate that neither the spatial arrangement nor the number of mounted samples has a large effect on cooling rates, so long as the volume of the cryoprotectant remains minimal. The time taken for cooling is found to be on the order of half a second, or less, regardless of the spatial arrangement or number of mounted samples. Hence, rapid cooling can be achieved for any number or arrangement of samples, as long as device manufacturer guidelines are adhered to., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Data about cryoprotectant-free cryopreservation of human ICSI spermatozoa are limited. The aim of this investigation was to compare two technologies for cryopreservation of spermatozoa from men with oligoasthenoteratozoospermia: standard conventional freezing with 5% glycerol (freezing in glycerol) and cryoprotectant-free freezing with 5% high-molecular-weight (360 kDa) polyvinylpyrrolidone (PVP) (PVP-freezing). Capillaries with spermatozoa were cooled in vapor and then plunginged into liquid nitrogen. Head-, midpiece- and tail-abnormality of spermatozoa, mitochondrial membrane potential (MMP) and DNA fragmentation rates after cryopreservation were evaluated. After warming of spermatozoa, fertilization of oocytes (ICSI) was performed. It was detected the lower rate of morphological abnormalities of PVP-frozen spermatozoa in comparison with cells frozen with glycerol (34.6 ± 4.1% vs. 20.7 ± 4.7%, respectively) (P < 0.05). Quality of cells with high MMP after warming in spermatozoa frozen with glycerol was lower than in PVP-frozen spermatozoa (34.7 ± 4.2 vs. 54.5 ± 4.2%, respectively) (P < 0.05). It was established that the DNA fragmentation rate in PVP-frozen spermatozoa was significantly lower in comparison with spermatozoa frozen with glycerol (23.1 ± 2.5% vs. 38.8 ± 3.0%, respectively) (P < 0.05). After fertilization (ICSI) of oocytes, it was established that cleavage and blastulation rates were higher in oocytes after fertilization with PVP-frozen spermatozoa than with spermatozoa frozen with glycerol. Fertilization-, development to 8-blastomeres-, and blastocyst-rates were for PVP-frozen and spermatozoa frozen with glycerol, respectively: 94.4 ± 7.8 vs. 82.2 ± 6.2% (P > 0.1 with tendency to increasing), 90.0 ± 4.6 vs. 69.5 ± 5.1% (P < 0.05), and 45.4 ± 4.1% vs. 30.9 ± 3.3% (P < 0.05). It was concluded that permeable cryoprotectant-free freezing with 5% high-molecular-weight (360 kDa) polyvinylpyrrolidone can be applied successfully for cryopreservation of human oligoasthenoteratozoospremic spermatozoa., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Assisted reproductive technologies provide important tools for wildlife conservation but have rarely been developed for reptiles. A critical step in developing cryopreservation protocols is establishing optimal cooling rates for cell survival. The two-factor hypothesis explaining cryoinjury to cells originates from an inverted 'U' shape of recovery curves generated in many cell types thawed after cryopreservation, due to cell recovery declining at cooling rates either side of a single optimum. We generated such a curve for the yellow-spotted monitor lizard Varanus panoptes, the first for any reptile. We cryopreserved sperm using two cooling devices (LN 2 dry shipper; LN 2 bath vapour) and two sperm-holding vessels (Cassou sperm straws; Nunc CryoTubes) to generate four different cooling-rate curves during freezing. Sperm motility and viability (47.3% and 76.5% respectively) were highest when frozen in straws suspended in a LN 2 bath at an intermediate cooling rate of 73  ° C/min between 0 and -50 °C, whereas sperm frozen in straws suspended in a dry shipper at the fastest cooling rate (231  ° C/min between 0 and -50 °C) produced the lowest recovery (10.4% and 36.4% motility and viability, respectively). Sperm frozen in cryotubes at the lowest cooling rates in either LN 2 bath vapour or dry shipper produced intermediate recovery. The shape of the optimal cooling curve conformed to the two-factor hypothesis of cryoinjury, the first such evidence in reptile sperm. This in turn led to the identification of simple cryopreservation setups (LN 2 vapour with straws and cryotubes; dry shipper with cryotubes but not straws) suitable for cryopreserving lizard sperm in the field., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Antifreeze proteins (AFPs) are synthesized by diverse non-mammalian species, allowing them to survive in severely cold environments. Since the 1990s, the scientific literature reports their use for low-temperature preservation of germplasm. The aim of this systematic review was to compile available scientific evidence regarding the use of AFP for low-temperature preservation of several reproductive specimens. Internet databases were consulted using the terms: "antifreeze protein" OR "AFP" OR "antifreeze glycoprotein" OR "AFGP" OR "ice-binding protein" OR "IBP" OR "thermal hysteresis protein" AND "cryopreservation". From 56 articles, 87 experiments testing AFPs in low-temperature preservation of gametes, embryos or reproductive tissues/cells were fully analyzed and outcomes were annotated. A positive outcome was considered as a statistically significant improvement on any parameter evaluated after low-temperature preservation with AFP, whereas a negative outcome included worsening of any evaluated parameter, in comparison to untreated groups or groups treated with a lower concentration of AFP. The findings indicated that research on the use of AFP as a cryoprotectant for reproductive specimens has increased markedly over the past decade. Some experiments reported both positive and negative results, which depended, on AFP concentration in the preservation media. Variation in the outcomes associated with species was also observed. Among the 66 experiments conducted in mammals, 77.3% resulted in positive, and 28.8% in negative outcomes after the use of AFP. In fishes, positive and negative outcomes were observed in 71.4% and 33.3% of 21 experiments, respectively. Most positive outcomes included preserving cell post-warming survival. The beneficial effect of AFP supports its use in cryobiological approaches used in human and veterinary medicines and animal protein industry. Moreover, combination of different AFP types, or AFP with antioxidants, or even the use of AFP-biosimilar, comprise some promising approaches to be further explored in cryopreservation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The aim of this study was to examine the effects of long-term dietary supplementation of young Nellore bulls with rumen-protected polyunsaturated fatty acids (PUFAs) and of the inclusion of catalase in the semen extender on semen quality, in vitro sperm fertilizing ability, and intracytoplasmic lipid content in the resulting embryos. Twelve Nellore bulls were supplemented with rumen-protected PUFAs or with a basal diet from 14 to 24 months of age. The semen was collected at the end of supplementation. For cryopreservation, the ejaculate was divided into two equal volumes and catalase was added to the extender in one of the fractions. Thus, the experimental design consisted of a 2 × 2 factorial scheme with two diets (control and PUFA) and two extenders (Cat+ and Cat-). Total motility and the percentage of rapid cells in fresh semen were negatively affected by dietary supplementation with PUFAs (P < 0.05), but these effects did not persist after freezing. The frozen/thawed semen of animals fed PUFAs exhibited an increase in the percentages of damaged plasma and acrosomal membranes, as well as an increase in the proportion of lipids ions at m/z 578 and m/z 757 detected by MALDI-MS. Nevertheless, there was no effect of the treatments on in vitro embryo development. However, embryos derived from bulls supplemented with PUFAs exhibited higher lipid accumulation compared to control (P < 0.05). In conclusion, PUFA supplementation promoted worsening of semen quality without affecting the in vitro sperm fertilizing ability; however, the paternal diet affected the intracytoplasmic lipid content in the resulting embryos., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mathematical modeling of surface deformation during cryopreservation by vitrification is presented in this study. The specific problem under consideration is of a cryoprotective agent (CPA) solution vitrifying in a vial, following previously obtained cryomacroscopy observations. A multiphysics solution is proposed in this study, combining coupled effects associated with heat transfer, fluid mechanics, and solid mechanics. Consistent with previous investigations, this study demonstrates that surface deformation is the result of material flow, which is the combined outcome of temperature gradients developed during the inward cooling process, the tendency of the material to change its volume with temperature, and the exponential increase in material viscosity with the decreasing temperature. During this process, the behavior of the CPA changes from liquid to a solid-like amorphous material, where the arrested flow in the vitrified state results in mechanical stresses. Results of this study show a good qualitative agreement of surface deformation with previously obtained experimental data, and support prior investigations to explain fracture tendencies propagating from the deformed surface. Results of this study also highlight the effect of heat convection in the CPA at the early stage of cooling., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The family Orchidaceae, with over 25,000 species, includes five subfamilies and nearly 700 genera. Loss of plants in the wild has resulted from clearing of forests and excessive collection for various purposes. Moreover, the requirement of symbiotic association during seed germination under natural conditions adds a certain level of difficulty in retaining the orchid resources in the wild. Cryopreservation is an important arena in conservation science due to its potential of storing genetic resources without altering the genetic makeup. Cryopreserved orchids are a very small percentage of the species, and are also not representative of most genera. Finding effective protocols for the various explant types is of prime importance in conserving orchid diversity. Seed is the most commonly stored and directly useful explant, and direct plunging in liquid nitrogen or PVS2 vitrification appear to be suitable for most tested species. The myriad of other species should be screened as they become available, with special emphasis on seed maturity and moisture content. Studies of protocorms and protocorm-like bodies mostly employ desiccation, PVS2 vitrification or encapsulation-dehydration. Pollinia are generally stored successfully following desiccation or slow cooling. There are too few examples of shoot tip cryopreservation to make a determination, however vitrification techniques are likely the most useful for a range of genera. A systematic and coordinated effort is needed to screen all available species in as many taxa as possible, initially with seed, protocorms and pollinia. It is a charge to the orchid research community to organize this effort and fill in the required data for the large number of untested taxa. In addition, providing stored samples to established orchid cryo collections would greatly increase preservation of these endangered treasures., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The present study focuses on the effect of different levels of ergothioneine and thawing temperature on rooster semen cryopreservation. Semen was diluted in Lake extender containing ergothioneine at 5, 10, 15, and 20 µM and cryopreserved. Two thawing temperatures (37°C for 30 s and 60°C for 5 s) were consequently examined. Sperm motility parameter, membrane integrity, abnormal morphology, viability, apoptotic status, mitochondria activity, and lipid peroxidation were determined after freeze-thaw process. Ergothioneine levels of 5 and 10 µM led to higher (P < 0.05) total motility (66.58 ± 1.44 and 72.11±1.44, respectively) and average path velocity (VAP) (34.54 ± 0.89, 37.28 ± 0.89, respectively). Higher (P < 0.05) significant membrane integrity and mitochondria activity after freeze-thawing were observed in the groups supplemented with 10 µM ergothioneine (68.62 ± 1.24 and 69.12 ± 1.26, respectively). Also, 5 and 10 µM of ergothioneine led to the lowest significant percentage of apoptotic and dead sperm. The total motility and progressive motility resulted in significantly (P < 0.05) higher amount when sperm were thawed with 60°C (60.58 ± 0.91 and 24.76 ± 0.53, respectively) compared to thawed sperm in 37°C. The membrane integrity, viability and mitochondria activity led to significantly (P < 0.05) higher when sperm were thawed with 60°C (58.2 ± 0.78, 63.21 ± 0.80 and 56.85 ± 0.79, respectively). It could be concluded the addition of 5 and 10 µM ergothioneine in the semen extender and thawing temperature at 60˚C in 5 s can be an efficient strategy to preserve rooster cryopreserved semen quality., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

As a magical oligosaccharide, trehalose has been revealed to enhance the post-thaw quality of stock semen. However, information regarding the cryoprotective mechanism of trehalose during cryopreservation has not yet been determined. This study was designed to observe the effects of trehalose on the proteome of ram frozen spermatozoa by applying the isobaric tag for relative and absolute quantification (iTRAQ) strategy combined with parallel reaction monitoring (PRM). A total of 1269 proteins were identified. Among them, there were 21 differentially expressed proteins (DEPs), with 9 up-regulated proteins and 11 down-regulated proteins in spermatozoa frozen with trehalose. These DEPs were primarily located in nucleus, cytoplasm, and extracellular region. The Gene Ontology (GO) enrichment analysis demonstrated the involvement of the DEPs in signal transduction, ion binding, oxidoreductase activity, response to stress, and catabolic processes. Based on the STRING analysis, tight functional correlations were observed between 6-phosphogluconate dehydrogenase, fructose-bisphosphate aldolase A isoform 1, 14-3-3 protein epsilon, tyrosine-protein kinase Fer, and beta-hexosaminidase subunit alpha precursor. Furthermore, 10 DEPs were verified using PRM, confirming the accuracy of the iTRAQ data acquired in this study. In conclusion, trehalose can modify the protein profile of ram spermatozoa during cryopreservation, which may be associated with its cryoprotective effects. Additionally, trehalose may function on frozen spermatozoa through antioxidation, involvement in glycolysis, and increment of spermatozoa tolerance to various stresses., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This study investigated the effects of l-arginine and l-lysine on the water holding capacity, shear force, color, and protein denaturation of frozen porcine Longissimus lumborum. Four batches were prepared, each corresponding to samples of an experimental treatment: without a cryoprotective solution, injecting a 0.3% sodium tripolyphosphate and 0.5% NaCl solution, a 0.5% l-arginine solution, or a 0.5% l-lysine solution. The results showed that both l-arginine and l-lysine decreased thawing loss, cooking loss, shear force, L ⁎ values, b ⁎ values, and surface hydrophobicity, but they increased pH values, a ⁎ values, percentages of peak areas for T 21 relaxation times, and Ca 2+ -ATPase activity. Additionally, both histological and transmission electron microscopy images showed that l-lysine, and especially l-arginine could inhibit the formation of gaps between fiber bundles, alleviate the disruption of intracellular spaces, and maintain the structural integrity of sarcomeres. Overall, the results showed that both l-arginine and l-lysine hindered the structural damage of muscle fibers during freezing and protected myofibrillar proteins from denaturation, ultimately contributing to superior quality attributes., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cryopreservation of coral sperm requires reliable, travel-ready, inexpensive hardware. To this end, we developed and tested a robust, second-generation, conduction-based cryovial cooling rack assembled from 3D-printed and commercially available parts. Cooling rates from -10 to -80 °C were found to be repeatable at -22.9 ± 1.9 (rate ± SD) °C/min for 1-mL samples and -35.4 ± 3.3 °C/min for 0.5-mL samples. This represents an improvement on the variability of cooling rates in an older design, which was found to be -31.8 ± 7.1 °C/min for 1-mL samples. Design files and a manual were produced to encourage widespread use and the development of derivative designs., (Copyright © 2021 Smithsonian Institution. Published by Elsevier Inc. All rights reserved.)

The aim is to optimize the dimethylacetamide (DMA) straw freezing technology of Black silkies rooster semen through the handy patent equipment, screening the formula of freezing basic extender and optimizing the DMA addition method, and then by comparing the fertility of DMA straw frozen semen with the pellet frozen semen. After the DMA straw freezing technology is optimized, it is extended to the Youxian Partridge drake semen. The result showed that the frozen sperm motility of Lake and Ravie (LR) group is 64%, the fertility 49.57% and the hatchability 91.52%, all of which are superior to those of FEB, Beltsville Poultry Semen Extender (BPSE) and Lake (P < 0.05). The sperm motility of adding DMA stock solution is 59%, which is superior to adding DMA directly into diluted semen (P > 0.05). The fertility and hatchability of DMA straw group are 77.61% and 92.30%, respectively, and it is significantly higher than those in the pellet group (P < 0.01; P < 0.05). The fresh drake sperm motility of induction collection method is 71%, the massage collection method 61% and the frozen drake sperm motility of induction 33% while the massage 19%. The fertility of frozen drake semen group is 85.93%, while that of the fresh semen group is 88.17%. The frozen drake semen fertility of the highest batch is 93.8%. In conclusion, the world's advanced fertility of frozen semen can be obtained both in the chicken and drake through the optimized DMA straw freezing technology and the method of screening freeze-resistant individuals., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cryopreservation is required to preserve the native properties of tissue for prolonged periods of time. In this study, we evaluate the impact that 4 different cryopreservation protocols have on porcine urethral tissue, to identify a protocol that best preserves the native properties of the tissue. The cryopreservation protocols include storage in cryoprotective agents at -20 °C and -80 °C with a slow, gradual, and fast reduction in temperature. To evaluate the effects of cryopreservation, the tissue is mechanically characterised in uniaxial tension and the mechanical properties, failure mechanics, and tissue dimensions are compared fresh and following cryopreservation. The mechanical response of the tissue is altered following cryopreservation, yet the elastic modulus from the high stress, linear region of the Cauchy stress - stretch curves is unaffected by the freezing process. To further investigate the change in mechanical response following cryopreservation, the stretch at different tensile stress values was evaluated, which revealed that storage at -20 °C is the only protocol that does not significantly alter the mechanical properties of the tissue compared to the fresh samples. Conversely, the ultimate tensile strength and the stretch at failure were relatively unaffected by the freezing process, regardless of the cryopreservation protocol. However, there were alterations to the tissue dimensions following cryopreservation that were significantly different from the fresh samples for the tissue stored at -80 °C. Therefore, any study intent on preserving the mechanical, failure, and geometric properties of urethral tissue during cryopreservation should do so by freezing samples at -20 °C, as storage at -80 °C is shown here to significantly alter the tissue properties., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The aim of this study was to evaluate the effects of L-proline on the extender quality of frozen and post-thawed jackass semen. Jackass (n = 6) semen samples were collected and cryopreserved in gradient concentrations (0-80 mM) of L-proline in extenders; post-thawed semen samples were cultured in L-proline medium for 10 hours at 37°C. For cryopreservation experiment I, the motile parameters, mitochondrial membrane potential (MMP), and plasma membrane, acrosome, and chromatin structure integrities of post-thawed semen were assessed. For culture experiment II, additional ROS contents were analyzed after incubation. For the fertility trial, jennies (n = 135) were divided into group I (30 mM L-proline in cryopreservation extender), group II (40 mM L-proline in culture medium), and the control. Pregnancy was diagnosed using an ultrasound scanner 30 days after ovulation. The results of experiment I showed that, motile parameters and acrosome and chromatin structure integrities of groups I and 40 mM were significantly higher than the control (P < .05). MMP of group I was significantly higher than the control and 40 mM groups (P < .05). In experiment II, after 4 hours of incubation, motile parameters, MMP, and DNA integrity in group II were significantly higher than the control (P < .05). Additionally, 40 and 80 mM L-proline in culture medium significantly reduced ROS accumulation after 4 and 10 hours of incubation (P < .05). Pregnancy rates of the control and groups I and II were 28.85%, 40%, and 36.84%, respectively. In conclusion, the extenders containing 30 to 40 mM L-proline improved both qualities of frozen and post-thawed semen, and it will be a beneficial agent for donkey frozen spermatozoa or post-thawed semen storage., (Copyright © 2021. Published by Elsevier Inc.)