Cryobiology最新文献

{"title":"Reducing dimethyl sulfoxide content in Jurkat cell formulations suitable for cryopreservation","authors":"Alexandra Roesch ,&nbsp;Roland Windisch ,&nbsp;Christian Wichmann ,&nbsp;Willem F. Wolkers ,&nbsp;Gideon Kersten ,&nbsp;Tim Menzen","doi":"10.1016/j.cryobiol.2025.105238","DOIUrl":"10.1016/j.cryobiol.2025.105238","url":null,"abstract":"<div><div>Cell-based medicinal products (CBMPs) are usually cryopreserved in formulations containing up to 10 % dimethyl sulfoxide (Me₂SO) at temperatures below −145 °C. Although Me₂SO effectively protects cells during the freezing process, it can be damaging to cells at ambient temperatures and lead to side effects in patients. The aim of this study was to reduce the amount of Me₂SO in cryopreservation formulations for an immortalized T cell line (Jurkat cells). A design of experiment (DoE) approach was applied for formulation development using seven different excipients, i.e., Me₂SO, trehalose, sorbitol, proline, ectoine, poloxamer 188 (P188) and poly vinyl pyrrolidone 40 (PVP). A DoE model was generated to predict optimal formulations resulting in a high post-thaw viability and a high glass transition temperature of the formulation to allow for frozen storage without the use of liquid nitrogen. Subsequently a stability study was performed with promising lead candidates over three months at storage temperatures of −145 °C, −80 °C, −40 °C. Three benchmark solutions were used, i.e., Cryostor CS10, CryoSOfree as well as 10 % Me₂SO in Roswell Park Memorial Institute Medium (RPMI). The excipient affecting the post-thaw viability of Jurkat cells the most was, as expected, Me₂SO, which led to increased viabilities at higher concentrations. Most formulations resulted in similar viabilities for cells stored at −145 °C and −80 °C, whereas samples stored at −40 °C did not survive. In general, benchmark formulations resulted in slightly higher viabilities than the tested formulations. Furthermore, cell samples stored at −80 °C were recultivated in cell culture and the viability was assessed after 24h. The cell viability after 24h was much lower compared to the cells analyzed directly post-thaw, indicating that freeze-thaw damages continue to unfold after thawing. In summary, several promising excipients and combinations thereof, e.g., trehalose and PVP, were identified for the cryopreservation of Jurkat cells with reduced concentrations of Me₂SO or Me₂SO-free cryopreservation. Additionally, storage at −80 °C is possible for the developed formulations.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"119 ","pages":"Article 105238"},"PeriodicalIF":2.3,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143760375","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":"Vitrification of porcine immature oocytes and pronuclear parthenotes delays development at the time of embryonic genome activation: A time-lapse study","authors":"Christopher G. Grupen ,&nbsp;Azelle Hawdon ,&nbsp;Yuji Hirao ,&nbsp;Kazuhiro Kikuchi ,&nbsp;Tamás Somfai","doi":"10.1016/j.cryobiol.2025.105239","DOIUrl":"10.1016/j.cryobiol.2025.105239","url":null,"abstract":"<div><div>Vitrification procedures have become indispensable for the preservation of female germplasm and the storage and transport of embryos despite the potential detrimental effects on embryo viability. The aim of this study was to examine the effects of vitrification on porcine embryo developmental kinetics by time-lapse imaging. In the first comparison, cumulus enclosed oocytes at the germinal vesicle (GV) stage were either vitrified-warmed or not vitrified (control) prior to in vitro maturation (IVM) and artificial activation (AA). In the second comparison, pronuclear (PN) parthenotes produced after IVM and AA were either vitrified-warmed or not vitrified (control). Embryo development was monitored for 6 d using the Primo Vision time-lapse system, and corresponding cohorts were assessed after incubation under standard in vitro culture (IVC) conditions. The cumulative time taken to progress to most of the developmental stages was significantly longer for embryos of the vitrified groups than for those of the control groups. Analysing the duration for each developmental interval revealed that the 4- to 5-cell and 5- to 8-cell periods were remarkably slower in the vitrified groups, compared with the control groups. The incidence of atypical blastomere divisions tended to increase following vitrification of GV oocytes. Under standard IVC conditions, blastocyst formation rates were lower for embryos of the vitrified groups than for those of the control groups. Given that the period of greatest developmental delay coincides with the major embryonic genome activation phase in porcine embryos, we propose that the vitrification of GV oocytes and PN parthenotes adversely impacts epigenetic processes.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"119 ","pages":"Article 105239"},"PeriodicalIF":2.3,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143746890","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":"Dormant bud cryopreservation of Rubus idaeus L.","authors":"Olena Bobrova ,&nbsp;Jiri Zamecnik ,&nbsp;Milos Faltus ,&nbsp;Alois Bilavcik","doi":"10.1016/j.cryobiol.2025.105240","DOIUrl":"10.1016/j.cryobiol.2025.105240","url":null,"abstract":"<div><div>Cryopreservation of dormant buds offers a reliable and cost-effective method for the long-term preservation of plant genetic resources, particularly for woody plants like raspberry (<em>Rubus idaeus</em> L.). This study aimed to develop an optimized protocol for the cryopreservation of dormant raspberry buds by investigating the role of dehydration and freezing conditions in maintaining bud viability. Dormant canes of two raspberry varieties, ‘Sanibelle' and ‘Willamette', were collected and prepared by dehydrating single-node cane segments at −4 °C until the desired water content was achieved. The study focused on assessing the impact of dehydration on bud viability and the physical state of water within the buds, using differential scanning calorimetry to analyse thermal transitions during cooling and heating. The cryopreservation process followed a two-step freezing protocol, with the first step involving slow cooling to −30 °C, followed by rapid immersion in liquid nitrogen (−196 °C). After cryopreservation, buds were thawed either slowly at +4 °C or rapidly at +38 °C, with rehydration taking place over 14 days. The study found that the most effective preservation (74–86 % of buds) occurred when the buds were dehydrated to 22–23 % humidity and had a water activity of 0.83–0.85. Rapid thawing significantly improved bud survival, especially for moderately dehydrated buds (with water content 25–35 %), while recrystallization during slow thawing was associated with lower viability. The results highlight the complexity of cryopreservation protocols, where factors such as dehydration levels, cooling and thawing rates, and bud dormancy status play crucial roles in ensuring successful long-term storage.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"119 ","pages":"Article 105240"},"PeriodicalIF":2.3,"publicationDate":"2025-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143734918","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":"Effects of growth factor supplementation on the proliferation of cryopreserved canine amniotic membrane stem cells","authors":"Matheus Ferreira de Almeida ,&nbsp;Priscilla Avelino Ferreira Pinto ,&nbsp;Lina Castelo Branco Motta ,&nbsp;Tiago Gonçalves do Santos ,&nbsp;Meline de Paula Coutinho ,&nbsp;Jennifer Jullie Pichinelli Noronha ,&nbsp;Vitória Mattos Pereira ,&nbsp;Sarah Ingrid Pinto Santos ,&nbsp;Carlos Eduardo Ambrósio","doi":"10.1016/j.cryobiol.2025.105241","DOIUrl":"10.1016/j.cryobiol.2025.105241","url":null,"abstract":"<div><div>Amniotic membrane-derived mesenchymal stem cells (AM-MSCs) possess significant proliferative and multilineage differentiation potential. However, prolonged in vitro culture and cryopreservation can negatively impact cell viability and expansion capacity. This study investigates the effects of growth factor supplementation on the proliferation of advanced-passage canine AM-MSCs (cAM-MSCs) before and after cryopreservation. cAM-MSCs were isolated from late-gestation canine fetuses, cultured in vitro, and supplemented with different growth factors: fibroblast growth factor 2 (FGF-2), fibroblast growth factor 4 (FGF-4), platelet-derived growth factor-ββ (PDGF-ββ), vascular endothelial growth factor-β (VEGF-β), and transforming growth factor-β1 (TGF-β1), with a control group receiving no supplementation. Growth curves were analyzed before and after cryopreservation, and gene expression of SCAPER (mitotic potential) and TP53 (apoptosis) was quantified using qPCR. As result, supplementation with FGF-2, FGF-4, and PDGF-ββ significantly enhanced cAM-MSC proliferation without altering the expression of proliferation-associated genes. These findings suggest that growth factor supplementation may be a viable strategy to improve cell viability in long-term culture and post-thaw recovery.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"119 ","pages":"Article 105241"},"PeriodicalIF":2.3,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143714824","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":"Cooling-rate dependence of the cryopreservation of aquaporin-overexpressing cells with a non-permeable cryoprotectant","authors":"Sumire Matsuo ,&nbsp;Kenji Yamazaki ,&nbsp;Masato Yasui ,&nbsp;Youichiro Abe ,&nbsp;Tsutomu Uchida","doi":"10.1016/j.cryobiol.2025.105237","DOIUrl":"10.1016/j.cryobiol.2025.105237","url":null,"abstract":"<div><div>Dehydration of intracellular water is an important factor in the cryopreservation of cells, but questions remain as to the appropriate amount and timing of dehydration and the detailed mechanism of the freezing process. Answering these questions will lead to improvements in cryopreservation methods that have remained unchanged for more than half a century and to an increase in the number of cell types that can be cryopreserved. Therefore, we aimed to reveal the time point when cells were dehydrated in their cooling process and how much their viabilities were improved by dehydration. We conducted cryopreservation experiments using cells with enhanced water permeability due to membrane overexpression of the water transport channel protein (AQP4). The AQP4-expressing cells or non-AQP4-expressing cells were cryopreserved under different cooling rates after addition of the membrane-permeable cryoprotectant (CPA) Me₂SO, the non-membrane-permeable CPA trehalose, or no CPA. The results showed that no cryopreservation was successful without CPAs, even in the AQP4-expressing cells with increased water permeability. At slow freezing rates below 35 °C/min, viability with Me₂SO was maintained with decreasing in the cooling rate, but with trehalose, the viability decreased. At cooling rates above 80 °C/min, the viability of AQP4-expressing cells was significantly higher than that of AQP4-non-expressing cells. These results suggest that dehydration due to the osmotic-pressure difference generated after extracellular freezing is fatal to cells.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"119 ","pages":"Article 105237"},"PeriodicalIF":2.3,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143725101","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":"Understanding osmoregulation, cryoprotectant toxicity, and cold tolerance in sea urchin eggs: Implications for cryopreservation","authors":"S. Campos,&nbsp;Uxía Rodriguez,&nbsp;J. Troncoso,&nbsp;E. Paredes","doi":"10.1016/j.cryobiol.2025.105235","DOIUrl":"10.1016/j.cryobiol.2025.105235","url":null,"abstract":"<div><div>This study investigates the challenges and potential solutions for cryopreserving sea urchin (<em>Paracentrotus lividus</em>) eggs. It focuses on understanding the thresholds and osmoregulatory mechanisms, cryoprotectant toxicity, and cold tolerance in these eggs, which are especially difficult to preserve due to high sensitivity to cryoprotectants and low-temperature injury. Key findings suggest that stepwise addition of cryoprotectants, particularly dimethyl sulfoxide (Me₂SO) combined with other agents like DMF or methanol, reduces toxicity and enhances larval survival. The study also highlights that short-term exposure to low temperatures (4 °C) minimally impacts egg viability, offering insights for optimizing cooling protocols. By examining osmolarity, salinity, and cryoprotectant interactions, the research proposes vitrification as a promising cryopreservation technique. These findings will contribute to refining current embryo cryopreservation protocols and provides information for the future vitrification of sea urchin eggs, with implications for marine conservation and aquaculture.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"119 ","pages":"Article 105235"},"PeriodicalIF":2.3,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143725118","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":"Ellagic acid maintains post-thaw goat sperm quality via protecting mitochondrial function from ROS damage","authors":"Zhendong Zhu ,&nbsp;Wenjia Li ,&nbsp;Kexin Ding ,&nbsp;Eslam M. Bastawy ,&nbsp;Ahmed Mohamed Kamel ,&nbsp;Xin Kou ,&nbsp;Lingjiang Min","doi":"10.1016/j.cryobiol.2025.105231","DOIUrl":"10.1016/j.cryobiol.2025.105231","url":null,"abstract":"<div><div>This study aimed to investigate the effects of ellagic acid (EA), an antioxidant, on goat sperm quality after freezing and thawing. Goat semen was frozen using Tris-citric acid-glucose (TCG) extender containing 0, 1.25, 2.5, 5, and 10 μM of EA. Egg yolk represented 20 % (v/v) and glycerol represented 5 % (v/v) of the extender's final concentration. Goat sperm post-thaw motility, acrosome integrity, plasma membrane integrity, mitochondrial activity, ATP content, NADH/NAD⁺ levels, and NADH-CoQ activity were evaluated. Moreover, to elucidate how EA enhanced the goat sperm characteristics, the post-thaw sperm mitochondrial reactive oxygen species (ROS) level, malondialdehyde (MDA) level, oxidative DNA damage, apoptosis, levels of NADH dehydrogenase 1 (MT-ND1) and NADH dehydrogenase 6 (MT-ND6) proteins, and the 4-hydroxynonenal (4-HNE) level were also measured after thawing. The results demonstrated that motility, plasma membrane integrity, and acrosome integrity rates were enhanced in the group treated with 5 μM of EA compared to the other concentrations (0 μM, 1.25 μM, 2.5 μM, 5, and 10 μM). Moreover, mitochondrial activity and ATP content were notably superior in the 5 μM EA group compared to all other treatment groups, along with a considerable decrease in ROS and MDA levels. The 4-HNE level and oxidative DNA damage in sperm were also reduced by EA supplementation. Additionally, it was found that EA (5 μM) significantly (p &lt; 0.05) decreased sperm apoptosis levels. Furthermore, the addition of 5 μM EA maintained the post-thaw sperm MT-ND1 and MT-ND6 levels and reduced the negative impact of ROS on MT-ND1 and MT-ND6, thereby sustaining mitochondrial function for ATP generation. These results suggest that ellagic acid supplementation could maintain goat post-thaw sperm quality by reducing ROS damage and maintaining mitochondrial function for ATP generation. Antioxidant treatments, such as ellagic acid are a useful tool for maintaining frozen-thawed sperm quality.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"119 ","pages":"Article 105231"},"PeriodicalIF":2.3,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143684447","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 uncoupling and glycolysis stimulation are beneficial for kinematics, functionality and oxidative homeostasis of cryopreserved ram sperm","authors":"Álvaro de Miranda Alves ,&nbsp;João Diego de Agostini Losano ,&nbsp;Roberta Ferreira Leite ,&nbsp;Bruno Rogério Rui ,&nbsp;Daniel de Souza Ramos Angrimani ,&nbsp;Thais Rose dos Santos Hamilton ,&nbsp;Camilla Mota Mendes ,&nbsp;Mayra Elena Ortiz D'Avila Assumpção ,&nbsp;Marcilio Nichi","doi":"10.1016/j.cryobiol.2025.105236","DOIUrl":"10.1016/j.cryobiol.2025.105236","url":null,"abstract":"<div><div>The aim of the present study was to improve bioenergetics and oxidative status of cryopreserved ram sperm by uncoupling mitochondrial activity and stimulating glycolysis. To verify a potential synergism between mitochondrial uncoupling and glycolysis stimulation, as well as to determine the optimal concentrations of the respective treatments, the study was divided into two experiments. In Experiment 1, ejaculates from eight rams were diluted with the commercial extender (Botubov®), supplemented with the mitochondrial uncoupler CCCP (0, 1, 10, and 20 μM), with or without 5 mM glucose, and then subjected to cryopreservation. After thawing, sperm function and oxidative status analyses were conducted to determine the optimal CCCP concentration, which was selected for Experiment 2. In Experiment 2, ejaculates from seven rams were diluted with the commercial extender (Botubov®) and supplemented with CCCP at doses of 0, 2.5, 5, and 10 μM, with or without 5 mM glucose. After thawing, an analysis of sperm bioenergetics was performed. Differences between treatments were assessed using ANOVA, followed by LSD mean comparison test for the combination of factors. In both experiments, total and progressive motility were higher in the CCCP 10 μM + glucose 5 mM group. This same group exhibited less susceptibility to lipid peroxidation, lower DNA fragmentation (Experiment 1), and greater mitochondrial activity (Experiment 2). Furthermore, treatments with only CCCP were deleterious to sperm. In conclusion, the use of the mitochondrial uncoupler CCCP at a dose of 10 μM combined with 5 mM glucose was promising in improving post-thaw sperm attributes.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"119 ","pages":"Article 105236"},"PeriodicalIF":2.3,"publicationDate":"2025-03-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143684446","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":"Evaluation of post-transfer fertility of vitrified goat morulae and blastocysts using an innovative protocol in micropipette tips","authors":"María Macarena Bruno-Galarraga ,&nbsp;Jimena Fernandez ,&nbsp;Agustí Noya ,&nbsp;Alejandro Gibbons ,&nbsp;Marcela Cueto","doi":"10.1016/j.cryobiol.2025.105234","DOIUrl":"10.1016/j.cryobiol.2025.105234","url":null,"abstract":"<div><div>In 2022, we developed an innovative protocol consisting of three vitrification solutions with the addition of sucrose to vitrify goat embryos in micropipette tips (S-VitriTip) and obtained promising <em>in vitro</em> results. However, to date, this protocol has not been tested <em>in vivo</em> in recipient females. The study aimed to evaluate the embryo survival and post-transfer fertility of goat morulae and blastocysts vitrified by the S-VitriTip protocol. Embryo recovery from donor goats (<em>n</em> = 23) was carried out on Days 8 and 9 after sponge removal. Immediately after recovery, morulae and blastocysts were vitrified using the S-VitriTip protocol. Briefly, all embryos were exposed to three different increasing solutions of cryoprotectants (glycerol, ethylene glycol, sucrose), loaded into micropipette tips and stored in liquid nitrogen. After warming, embryos were transferred in pairs into recipient goats (<em>n</em> = 47); morulae and blastocysts were transferred on the same day they were recovered, either Day 8 or Day 9 after sponge removal. On day 25 after embryo transfer, ultrasound diagnosis was performed. No significant differences were observed on embryo survival nor on post-transfer fertility according to embryo stage or day of embryo recovery (42 and 50 %, respectively; P &gt; 0.05). Embryo survival and post-transfer fertility of vitrified morulae of 30 % and 40 % were obtained, highlighting our study as one of the first to report efficient reproductive rates through the <em>in vivo</em> transfer of cryopreserved goat morulae, thus demonstrating the effectiveness of this innovative vitrification protocol.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"119 ","pages":"Article 105234"},"PeriodicalIF":2.3,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143673561","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":"Bacterial survival below zero: Impact of storage time on bacterial viability in bull semen","authors":"Aleksandar Cojkic ,&nbsp;Ingrid Hansson ,&nbsp;Jane M. Morrell","doi":"10.1016/j.cryobiol.2025.105233","DOIUrl":"10.1016/j.cryobiol.2025.105233","url":null,"abstract":"<div><div>Although freezing methods have been optimized for preserving sperm integrity, their effectiveness in sustaining bacterial viability is unknown. Therefore, culturing thawed semen samples might not give an accurate picture of the bacteria in the original sample. The aim of this study was to assess how cryopreservation and storage duration influence bacterial populations and the survival of distinct bacterial species. Semen samples were collected from 14 bulls, samples were diluted in equal proportions of antibiotic-free semen extender and transported to the laboratory at 6 °C overnight. Aliquots of semen were cultured within 24 h after semen collection on Plate Count Agar to calculate number of bacteria, and blood agar plates (5 % bovine blood) for identification of bacterial species. The remaining samples were diluted 1:1 in Brain Heart Infusion (BHI) broth with 30 % glycerol and stored at −80 °C. The frozen samples were thawed and cultured for quantification of bacteria as described for fresh semen, after 6 and 13 days at −80 °C. The isolated bacteria were re-cultured on blood agar, incubated for one day at 37 °C before identification by Matrix assisted laser desorption ionization-time of flight mass spectrometry. Total bacterial counts remained consistent across fresh and cryopreserved samples regardless of storage duration. A total of 31 bacterial species were identified, with 20 detected in fresh samples, 16 present after 6 days of storage, and 18 observed after 13 days. Ten species persisted across all time points, while others were unique to a specific sampling day, including nine species on day 1, two species on day 6, and five species on day 13. These findings suggest that while cryopreservation does not alter the overall bacterial load, the survival of individual species varies depending on storage conditions.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"119 ","pages":"Article 105233"},"PeriodicalIF":2.3,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143644327","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}