Cryobiology最新文献

{"title":"Is isochoric vitrification feasible? Cycling between modeling, experimental observations, and first principles","authors":"Yoed Rabin,&nbsp;Prem K. Solanki","doi":"10.1016/j.cryobiol.2025.105216","DOIUrl":"10.1016/j.cryobiol.2025.105216","url":null,"abstract":"<div><div>Isochoric cryopreservation is preservation of biological material in a constant-volume container, to benefit from a presumed baroprotective effect. From its inception, the premise for the isochoric approach is that pressure elevation can be achieved by permitting some ice formation in the system. The idea of integrating vitrification into an isochoric chamber followed at a later stage, where vitrification is a century old established approach of ice-free cryopreservation. At face value, the resulting term “isochoric vitrification” encompasses an internal contradiction revolving around ice formation. We have previously shared modeling results pointing to this contradiction and, hence, questioning the feasibility of the process (Cryobiology 2023, 111, 9–15). More recently, Ali et al. (Cryobiology 2024, 116, 104935) claimed to have obtained contradictory experimental evidence while stating that isochoric vitrification is feasible. The objective for this communication is to share our skepticism about this claim with the community of cryobiology.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"118 ","pages":"Article 105216"},"PeriodicalIF":2.3,"publicationDate":"2025-02-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143467291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessment of alternative freezing methods for preservation at −80 °C of radiata pine embryogenic cultures: A six-year study","authors":"K.P. Sandoval,&nbsp;A. Castander-Olarieta,&nbsp;P. Moncaleán,&nbsp;I.A. Montalbán","doi":"10.1016/j.cryobiol.2025.105217","DOIUrl":"10.1016/j.cryobiol.2025.105217","url":null,"abstract":"<div><div>Somatic embryogenesis is an essential component of breeding programs for <em>Pinus radiata</em> aimed at implementing multi-varietal forestry. Coupled with this technique, the long-term cryopreservation of embryogenic cultures is necessary to maintain the viability of the cell lines, but this entails high maintenance costs. In this research we evaluated the application of a protocol for long-term storage at −80 °C in an ultra-low freezer to preserve several radiata pine embryogenic cell lines. Also, we studied the influence of several parameters to optimize the protocol, such as the effect of dimethyl sulfoxide cryoprotectant solution, the effectiveness of alternative freezing methods, the use of post thawing treatments and the addition of sodium butyrate at maturation stage. We found that the use of dimethyl sulfoxide cryoprotectant enhanced somatic embryo production; slow cooling was the only viable method for preserving embryogenic cell lines at −80 °C and the use of sodium butyrate was not highly effective to improve maturation and germination stages. Moreover, we have regenerated embryogenic cell lines up to their conversion into plants after six years of storage. In line with these findings, the protocol to storage in an ultra-low freezer represents an economical alternative to preserve somatic embryogenic cultures of <em>Pinus radiata</em>.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"119 ","pages":"Article 105217"},"PeriodicalIF":2.3,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143482475","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Procyanidin B2 alleviates damage to mouse testicular tissue after freezing by inhibiting oxidative stress and apoptosis","authors":"Guorui Cao ,&nbsp;Chunyuan Li ,&nbsp;Jian Zhang ,&nbsp;Liwen Deng ,&nbsp;Rong Li ,&nbsp;Changlong Xu","doi":"10.1016/j.cryobiol.2025.105196","DOIUrl":"10.1016/j.cryobiol.2025.105196","url":null,"abstract":"<div><div>For infertile patients who are unable to obtain sperm or prepubertal boys who require radiotherapy, testicular tissue freezing can be used for later transplantation and is a potentially effective method of preserving male fertility. Oxidative stress caused by the freezing process is an important cause of tissue damage. Procyanidin B2 (PCB2) is a polyphenolic natural compound widely distributed in plants that is known for its anti-inflammatory, anticancer, and neuroprotective properties, and its antioxidant capabilities are particularly noteworthy. Research has indicated that PCB2 exerts a protective effect on the reproductive system. However, its specific role in mitigating testicular tissue cryoinjury and the underlying mechanisms remain unclear. This study investigated whether adding PCB2 to a cryoprotective solution of mouse testicular tissue can alleviate the cryoinjury of testicular tissue and its possible mechanism. Our findings revealed that frozen mouse testicular tissue presented decreased cell viability and induced oxidative stress. Conversely, PCB2 effectively mitigated these adverse effects. In addition, PCB2 improved the tubular structural disorganization caused by freezing and increased the expression of proteins related to the junction function of Sertoli cells. Further experiments indicated that PCB2 activated the nuclear respiratory factor 2 (Nrf2)/heme oxygenase 1 (HO-1) antioxidant signaling pathway, increased the activity of downstream antioxidant enzymes, and improved mitochondrial kinetic homeostasis. Additionally, PCB2 ameliorated apoptosis while increasing the expression levels of key enzymes involved in testosterone synthesis. In summary, these results suggest that PCB2 attenuates damage to mouse testicular tissue during freezing by inhibiting oxidative stress and apoptosis.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"118 ","pages":"Article 105196"},"PeriodicalIF":2.3,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142930567","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mitochondrial respiratory analysis of cryopreserved PBMCs isolated from human blood","authors":"C. Payne ,&nbsp;E. Louw ,&nbsp;N. Baines ,&nbsp;B. Botha ,&nbsp;C. Lombard ,&nbsp;B. Allwood ,&nbsp;G. Maarman","doi":"10.1016/j.cryobiol.2025.105212","DOIUrl":"10.1016/j.cryobiol.2025.105212","url":null,"abstract":"<div><div>Mitochondrial bioenergetics of PBMCs have been linked with several factors that contribute to a better understanding of several human diseases. Due to the complex logistics of clinical studies, samples are often cryopreserved for later analysis. Current data on whether cryopreservation negatively affects the mitochondrial function of PBMCs is discrepant. We isolated and cryopreserved peripheral blood mononuclear cells (PBMCs) from human whole blood and tested mitochondrial function using a substrate-uncoupler-inhibitor-titration protocol on the Oroboros instrument. After three months of storage in a cryopreservation medium (at −80 °C), several aspects of mitochondrial bioenergetics were measured. We demonstrate that cryopreservation did not adversely affect mitochondrial parameters (routine, leak, complex-I linked OXPHOS, cytochrome-c response, ETS capacity, the contributions of the N and S-pathways to ETS, ROX, complex-IV activity and mitochondrial coupling). Therefore, after three months of cryopreservation at −80 °C, human PBMC-mitochondria were fully coupled and functional. Therefore, clinical studies may cryopreserve PBMCs for later mitochondrial analyses.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"118 ","pages":"Article 105212"},"PeriodicalIF":2.3,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143373794","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Modeling and typical cases analyze at the cell-scale of transmembrane transport and intracellular crystallization and recrystallization during the freeze-thaw process","authors":"Pengsong Yuan ,&nbsp;Xueqiang Dong ,&nbsp;Haocheng Wang ,&nbsp;Xian Wang ,&nbsp;Maoqiong Gong","doi":"10.1016/j.cryobiol.2025.105210","DOIUrl":"10.1016/j.cryobiol.2025.105210","url":null,"abstract":"<div><div>Mechanical and solute damage caused by ice crystals during the freeze-thaw process of biological samples in cryopreservation are principal determinants of their activity. In this study, a numerical model is constructed by comprehensively considering the phenomenon of crystallization during cooling, recrystallization during rewarming, and the transmembrane transport of water and cryoprotective agent (CPA). The computational findings of the model demonstrate that higher cooling rates result in an increased volume of intracellular crystallization, with a correspondingly elevated intracellular nucleation temperature. By integrating the trend of CPA concentration variation during the cooling process, it is determined that the rates of 0.5 °C·min⁻¹ and 1 °C·min⁻¹ inflict minimal harm to mouse oocytes. During the rewarming process, the rate influences the intracellular ice volume, specifically the higher the rate of rewarming the smaller the increase in intracellular ice volume, and it is recommended that a high-power pulse be added before recrystallization to reduce the effects of recrystallization in practical applications. The pick-and-place operation of the cryopreservation vials can lead to recrystallization, and based on the calculations, it is recommended that the cryopreservation temperature should be lower than −160 °C and the operation time should be controlled within 90 s. The parameter scanning showed that a cooling rate of 0.4–1.8 °C·min⁻¹ and an initial DMSO concentration of 0.1–0.3 M are more favorable for the efficient recovery in the water bath of mouse oocytes. The model constructed in this study can provide valuable numerical guidance for practical cryopreservation protocols of biological specimens.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"118 ","pages":"Article 105210"},"PeriodicalIF":2.3,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143188585","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparison of different freezing rates on post-thaw viability, proliferation, and stemness of sheep spermatogonial stem cells","authors":"Balakrishnan Binsila ,&nbsp;Tomy A. Tomcy ,&nbsp;Balaganur Krishnappa ,&nbsp;Muhammed Sadikh ,&nbsp;Natesan Ramachandran ,&nbsp;Atul P. Kolte ,&nbsp;Sellappan Selvaraju","doi":"10.1016/j.cryobiol.2025.105203","DOIUrl":"10.1016/j.cryobiol.2025.105203","url":null,"abstract":"<div><div>The application of spermatogonial stem cells (SSC) will be more effective and feasible following the successful cryopreservation and transfer of SSCs in livestock. Like other cells, SSCs are also sensitive to cryoinjury; hence composition of the cryomedia and freezing protocols need to be optimized. The present study aims to optimize the best freezing rates by minimising the ice crystallization and dehydration effect in order to maximize the post-thaw SSCs survivability and stemness characteristics. Three different freezing protocols with varied cooling profiles, cooling profile 1 (isopropanol based freezing): 1 °C/min from 0 °C to −10 °C, 0.5 °C/min up to −40 °C, further reduced to 0.25 °C/min up to −50 °C and 0.1 °C/min to −60 °C; cooling profile 2 (using programmable freezer): 1 °C/min up to 4 °C, 0.3 °C/min up to −8 °C, and cooled at 0.5 °C/min to −50 °C, further decrease to −90 °C (8 °C/min) and cooling profile 3 (uncontrolled rapid freezing): 3.3 °C/min from 0 °C to −10 °C, 5 °C/min up to −40 °C, 2 °C/min to −50 °C and 1.2 °C/min up to −60 °C, were compared for cryopreservation efficiency. The overall viability (91.41 ± 2.00 % Vs 74.59 ± 2.34 %), stemness activity (1.34 ± 0.095 OD units Vs 0.356 ± 0.026 OD units), and proliferation rate (0.849 ± 0.019 OD units Vs 0.749 ± 0.015 OD units) of post-thaw SSC culture irrespective of the freezing regimes were significantly decreased when compared to pre-freeze SSC culture characteristics. The post-thaw viability was significantly greater in cooling profile 1 (79.64 ± 4.1 %) when compared to cooling profile 2 (69.72 ± 2.4 %) and cooling profile 3 (75.43 ± 4.8 %). Also, cooling profile 1 yielded greater (<em>p</em> &lt; 0.05) post-thaw stemness activity (0.456 ± 0.044 OD units) when compared to other methods. The study suggests that the cooling profile 1 using isopropanol based freezing can be recommended for preservation of viability and stemness characteristics of SSCs.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"118 ","pages":"Article 105203"},"PeriodicalIF":2.3,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143037420","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Species-specific optimisation of cryopreservation media for goat and buffalo adipose-derived mesenchymal stem cells","authors":"Michelle Abraham ,&nbsp;Sandeep Goel","doi":"10.1016/j.cryobiol.2025.105211","DOIUrl":"10.1016/j.cryobiol.2025.105211","url":null,"abstract":"<div><div>Adipose-derived mesenchymal stem cells (ADSCs) are promising for clinical and veterinary applications due to their ease of isolation, high yield, and multilineage differentiation potential. Effective cryopreservation is vital to ensure their availability for large-scale applications. This study evaluated cryopreservation strategies for goat (gADSCs) and buffalo (bADSCs) ADSCs, using combinations of intracellular (dimethyl sulfoxide, DMSO) and exocellular cryoprotectants, including fetal bovine serum (FBS), polyethylene glycol (PEG), trehalose, bovine serum albumin (BSA), and dextran. Post-thaw parameters such as viability, recovery, metabolic activity, clonogenicity, oxidative stress, apoptosis, and senescence were assessed. Results revealed species-specific differences in cryopreservation requirements. gADSCs were optimally preserved in a medium with 5 % DMSO, 3 % FBS, 2 % PEG, 3 % trehalose, and 2 % BSA, while bADSCs performed best in an FBS-free medium containing 5 % DMSO, 2 % PEG, 3 % trehalose, and 2 % BSA. DMSO-FBS formulations supported high recovery and metabolic activity but were associated with increased oxidative stress and apoptosis. Dextran-based cryomedia effectively preserved gADSCs but failed to maintain bADSC functionality. Biochemical composition analysis indicated significantly higher lipid content in bADSCs, likely influencing cryopreservation efficacy. These findings underscore the need for tailored cryopreservation strategies to address species-specific differences. Incorporating exocellular cryoprotectants reduced FBS dependency, minimised oxidative damage, and maintained functional attributes. This study highlights the potential for optimised, cost-effective biobanking solutions that accommodate species-specific requirements, advancing the use of ADSCs in veterinary and translational research.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"118 ","pages":"Article 105211"},"PeriodicalIF":2.3,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143373799","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inactivation of porcine epidemic diarrhea virus as a SARS-CoV-2 surrogate at sub-zero temperatures by isochoric freezing","authors":"Chenang Lyu ,&nbsp;Xinyu Liang ,&nbsp;Jing Jiang ,&nbsp;Fengqing Wang ,&nbsp;Ran An ,&nbsp;Jielin Yang ,&nbsp;Dapeng Wang","doi":"10.1016/j.cryobiol.2025.105209","DOIUrl":"10.1016/j.cryobiol.2025.105209","url":null,"abstract":"<div><div>Given the resilience of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on frozen food, there is a risk that contaminated products could serve as vectors for viral transmission. Yet, methods capable of inactivating the virus at sub-zero temperatures without compromising the food's taste and quality are scarce. The high-pressure environment that arises spontaneously during isochoric freezing has demonstrated efficacy in suppressing or inactivating harmful microorganisms, such as bacteria; however, its effectiveness against coronavirus and the mechanisms involved remain unclear. In this study, we employed Porcine epidemic diarrhea virus (PEDV) as a proxy for SARS-CoV-2 to examine the effects of isochoric freezing on PEDV infectivity and to evaluate post-treatment alterations in the integrity of viral nucleic acids and envelopes, as well as changes in antigenic properties. Our experimental findings indicate that after a 6-h isochoric freezing treatment at −20 °C and 201 MPa, the titer decreased by 1.18 log₁₀(TCID₅₀/mL). While the viral nucleic acid remained intact post-treatment, the envelope's integrity was significantly impaired, accounting for the loss of infectivity. Moreover, the antigenic properties of the virus showed a slight increase following isochoric freezing treatment. This study pioneers the exploration of isochoric freezing technology's inactivation effects on coronaviruses, including preliminary mechanisms, offering novel perspectives for managing coronavirus contamination in frozen food and highlighting the potential of isochoric freezing in vaccine inactivation processes.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"118 ","pages":"Article 105209"},"PeriodicalIF":2.3,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143188584","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Eliminating osmotic stress during cryoprotectant loading: A mathematical investigation of solute–solvent transport","authors":"Joseph R. Kangas,&nbsp;Christopher J. Hogan Jr.,&nbsp;John C. Bischof","doi":"10.1016/j.cryobiol.2025.105198","DOIUrl":"10.1016/j.cryobiol.2025.105198","url":null,"abstract":"<div><div>Osmotic stresses during cryoprotectant loading induce changes in cellular volume, leading to membrane damage or even cell death. Appropriate model-guided mitigation of these osmotic gradients during cryoprotectant loading is currently lacking, but would be highly beneficial in reducing viability loss during the loading process. To address this need, we reformulate the two-parameter formalism described by Jacobs and Stewart for cryoprotectant loading under the constraint of constant cell volume. We then derive simple, concise, analytic solutions to these equations, showing the transient extracellular permeating and nonpermeating cryoprotectant concentrations required to load a cell at constant volume, thus eliminating osmotic stresses during cryoprotectant loading. Additionally, we show analytic approximations of both ramp (linear) as well as step-wise loading and how one can use the hydraulic conductivity <span><math><msub><mrow><mi>L</mi></mrow><mrow><mi>p</mi></mrow></msub></math></span>, membrane permeability <span><math><msub><mrow><mi>P</mi></mrow><mrow><mi>s</mi></mrow></msub></math></span>, cell volume <span><math><msub><mrow><mi>V</mi></mrow><mrow><mi>o</mi></mrow></msub></math></span>, and osmotically inactive fraction to derive cryoprotectant loading protocols that minimize osmotic stress. We also present timescales for water and cryoprotectant transport which can be used to estimate loading times as well as <span><math><msub><mrow><mi>L</mi></mrow><mrow><mi>p</mi></mrow></msub></math></span> and <span><math><msub><mrow><mi>P</mi></mrow><mrow><mi>s</mi></mrow></msub></math></span>. We discuss how previous optimized loading strategies are inherently sensitive to parameter uncertainties and biological variability, increasing the likelihood of exceeding critical osmotic limits. By contrast, the proposed protocol provides a larger buffer against deviations, offering a safer and more robust solution to CPA loading. Importantly, we demonstrate that the volume-loss-free CPA loading protocols outlined in this paper occur on the same timescale as conventional and step-loading methods, suggesting that these protocols could be a safer alternative for CPA loading.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"118 ","pages":"Article 105198"},"PeriodicalIF":2.3,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143001677","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Impact of particle size of nanoliposomes on the biological response of frozen-thawed sperm in Holstein bulls","authors":"Touba Nadri ,&nbsp;Armin Towhidi ,&nbsp;Saeed Zeinoaldini ,&nbsp;Dariush Gholami ,&nbsp;Gholamhossein Riazi ,&nbsp;Felipe Martínez-Pastor","doi":"10.1016/j.cryobiol.2025.105208","DOIUrl":"10.1016/j.cryobiol.2025.105208","url":null,"abstract":"<div><div>Nanoliposomes could improve the delivering profile of protecting substances in sperm-freezing extenders. This study aimed to assess the impact of nanoliposomes with varying particle sizes and containing 2.5 mM glutathione (GSH) on the post-thaw quality of bull sperm. Ejaculates were obtained weekly from six mature Holstein bulls for six weeks using an artificial vagina. Semen was pooled and mixed with the lecithin-based extender. Semen was pooled and diluted with lecithin-based extenders containing 2.5 mM encapsulated GSH in nanoliposomes with particles in the size range of &lt;50, 50–100, or 100–200 nm and Andromed (control). Intracellular GSH was 4.8 ± 0.14 nmol/10⁸ sperm pre-freezing, decreasing by 58 % to 2.8 ± 0.14 nmol/10⁸ sperm post-thawing in control (no differences among treatments). Sperm motility, viability, membrane functionality, apoptotic status, mitochondrial activity, lipid peroxidation, and DNA fragmentation were assessed after cryopreservation. Compared to the control, the 50–100 nm group showed significantly higher viability (72.7 % ± 2.7 vs. 61.2 % ± 2.7) and plasma membrane integrity (82.0 % ± 1.6 vs. 73.3 % ± 1.6). Conversely, the 100–200 nm group showed lower acrosomal integrity (P &lt; 0.05 vs. others) and increased DNA fragmentation (P &lt; 0.05 vs. 50–100 nm and control). Supplementing the lecithin-based freezing extender with 50–100 nm nanoparticles loaded with 2.5 mM GSH could enhance bull semen cryopreservation.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"118 ","pages":"Article 105208"},"PeriodicalIF":2.3,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143074169","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}