Cryobiology最新文献

{"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}

{"title":"Effect on viability and yield on Pleurotus pulmonarius and Ganoderma lucidum after 3 years of cryopreservation without cryoprotectants","authors":"N.S. Ramírez ,&nbsp;D. Lining ,&nbsp;E. Albertó ,&nbsp;G. Pose","doi":"10.1016/j.cryobiol.2025.105204","DOIUrl":"10.1016/j.cryobiol.2025.105204","url":null,"abstract":"<div><div>Due to their importance in various fields such biotechnology, medicine, food science, preserving microorganisms effectively is essential. This study investigated cryopreservation of the edible fungus <em>Pleurotus pulmonarius</em> and the medicinal mushroom <em>Ganoderma lucidum</em>, for three years at −80 °C in sorghum grains without cryoprotectants. We compared viability, growth, and yields with strains maintained on agar media at 5 °C. Our findings show that the grain technique in sorghum requires neither cryoprotectants nor a thawing bath, achieving 100 % recovery within 24 h. Importantly, there were no significant differences in yield (BE) between cryopreserved and sub-cultured strains. These findings demonstrate the effectiveness of the grain technique with no cryoprotectants for long-term preservation of filamentous edible and medicinal fungi.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"118 ","pages":"Article 105204"},"PeriodicalIF":2.3,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143064150","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":"Indian cryogenebank conserving diverse plant genetic resources for the last three decades: Achievements and way forward","authors":"S.K. Malik,&nbsp;Sangita Bansal,&nbsp;Era Vaidya Malhotra,&nbsp;Anju Mahendru Singh,&nbsp;G.P. Singh","doi":"10.1016/j.cryobiol.2025.105205","DOIUrl":"10.1016/j.cryobiol.2025.105205","url":null,"abstract":"<div><div>Ex situ conservation of plant genetic resources (PGR) plays a crucial role in sustainable growth and development, as highlighted by the Global Strategy for Plant Conservation (GSPC). Seed genebanks, a key component of ex situ conservation, have been instrumental in preserving plant diversity. However, challenges arise with the conservation of non-orthodox (recalcitrant and intermediate) seeds and vegetative tissues, which are not amenable to storage in traditional genebanks at temperatures of −20 °C. Cryopreservation, the storage of biological materials at ultra-low temperatures in liquid nitrogen, has emerged as a viable solution for conserving such non-orthodox seeds, pollen, and dormant buds. This review presents insights into the National Cryogenebank Facility at ICAR-NBPGR, India, a pioneer in developing cryopreservation techniques and cryobanking of PGR. Established in 1987, the facility focuses on conserving difficult-to-conserve species of various agri-horticultural crops, including recalcitrant and intermediate species. With a capacity to hold a quarter of a million samples, the facility employs species-specific protocols to conserve rare, threatened, and endangered plant species, wild and weedy crop relatives, and genetic stocks. Over the past 3 decades, cryopreservation protocols have been developed at this facility using a diverse range of explants, including seeds, excised embryos, embryonic axes, pollen grains, and dormant buds. Successful cryopreservation protocols have been developed for temperate and tropical plant species important for horticultural, plantation, agro-forestry, and industrial use. Priority is given to conserving indigenous crop species and capturing the genetic diversity of indigenous tropical and temperate major and minor fruits. Additionally, the facility has successfully conserved pollen grains and dormant buds of tropical and temperate fruit crops, ensuring their viability and survival over extended periods of cryostorage. Furthermore, the cryobank regularly retests cryostored germplasm to assess viability and regrowth, with promising results indicating retention of seed viability even after 25–30 years of cryostorage. This highlights the potential of cryobanking as a long-term solution for conserving plant genetic resources. The National Cryogenebank Facility at ICAR-NBPGR exemplifies advancements in cryopreservation techniques applicable to plant genetic resource conservation, contributing significantly to national, regional and global efforts towards ex situ conservation of difficult-to-store plant species and overall sustainable agricultural development.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"118 ","pages":"Article 105205"},"PeriodicalIF":2.3,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143051915","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":"Withdrawn : Application of a single \"Universal warming protocol\" for vitrified donor oocytes: A multicenter study.","authors":"Paloma Troncoso-Perez, Cristina Gonzalez-Navas, Maria Elisabetta Coccia, Rossella Fucci, Patrizia Falcone, Francesco Bertocci, Rita Picone, Daniel Fernando Sosa-Rosales, Nuria López-Perez, Enrique Criado-Scholz, Miguel Angel Vilches-Ferron","doi":"10.1016/j.cryobiol.2025.105206","DOIUrl":"10.1016/j.cryobiol.2025.105206","url":null,"abstract":"<p><p>Author wanted to withdraw the paper as they are not interested in having it published in the journal Cryobiology. Approved by EIC and Publisher. As S5 was not published in SD, so we have proceeded with expiring the article from the production system</p>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":" ","pages":"105206"},"PeriodicalIF":2.3,"publicationDate":"2025-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143074155","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":"Epididymal bull sperm selection by Percoll® density-gradient centrifugation prior to conventional or ultra-rapid freezing enhances post-thaw sperm quality","authors":"Mauricio Duma ,&nbsp;Diego A. Galarza ,&nbsp;Kelly Delgado ,&nbsp;Angie Morocho ,&nbsp;Guido Bermúdez ,&nbsp;Manuel E. Soria ,&nbsp;María S. Méndez ,&nbsp;Esteban Muñoz-León ,&nbsp;Fernando P. Perea","doi":"10.1016/j.cryobiol.2025.105200","DOIUrl":"10.1016/j.cryobiol.2025.105200","url":null,"abstract":"<div><div>This study evaluated the effectiveness of Percoll® density gradient centrifugation (Percoll-DGC) for selecting bull epididymal sperm prior to conventional slow (CS) or ultra-rapid (UR) freezing and its effects on sperm quality. Fifteen pooled samples from 30 epididymides (2 different samples/pool) of 15 bulls were split into two aliquots assigned to either CS or UR freezing. Samples were either selected using Percoll-DGC (40/80 %) or left non-selected (control), resulting in four pre-freezing treatments: Percoll-CS, Control-CS, Percoll-UR, and Control-UR. The CS freezing used 5 % glycerol, exposing sperm straws to liquid nitrogen (LN₂) vapors, while UR freezing used 100 mM sucrose with direct submersion of 30 μL samples into LN₂. Overall, sperm quality was higher in CS treatments than in UR treatments. Pre-freezing, Percoll-CS improved straight-line velocity (VSL), linearity (LIN), and beat-cross frequency (BCF) compared to Control-CS (P &lt; 0.05). Similarly, Percoll-UR treatment enhanced progressive motility (PSM), velocities, straightness (STR), amplitude of lateral head displacement (ALH), and BCF compared to Control-UR (P &lt; 0.05). Post-thaw, Percoll-CS demonstrated higher kinematic parameters, viability, and acrosome integrity compared to Control-CS (P &lt; 0.05). Meanwhile, Percoll-UR improved viability and acrosome integrity relative to Control-UR (P &lt; 0.05). Notably, both Percoll-UR and Control-UR resulted in lower DNA fragmentation compared to Percoll-CS. In conclusion, Percoll-DGC selection prior to CS freezing significantly improved post-thaw sperm quality, including kinematics, viability, and acrosome integrity. For UR freezing, Percoll-DGC primarily enhanced post-thaw viability and acrosome integrity.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"118 ","pages":"Article 105200"},"PeriodicalIF":2.3,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143001543","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}