Spermatogonia cryopreservation is a method to preserve valuable genomes from both maternal and paternal origin. The damage associated with the application of this technology on post-thaw cell quality is important to assess, including at the epigenetic level. This study aimed to assess post-thawed spermatogonia quality by evaluating alterations in plasma membrane integrity, DNA integrity (fragmentation and apoptosis), lipid peroxidation (malondialdehyde levels) and epigenetic modifications (DNA methylation profile). We observed that plasma membrane integrity (fresh 78.98% ± 5.66; cryopreserved 62.81% ± 3.25; P = 0.003) and DNA integrity (fresh 32.95% ± 2.28; cryopreserved 37.28% ± 1.87; P = 0.0026) were affected by cryopreservation, while no difference in lipid peroxidation was observed (fresh 1.13% ± 0.45; cryopreserved 0.91% ± 0.96; P = 0.701). While global levels of DNA methylation were unaffected by cryopreservation (fresh 82.80% ± 0.47; cryopreserved 83.32% ± 0.81; P = 0.745), some differentially methylated cytosines (DMC) were observed in cryopreserved versus fresh spermatogonia (156 DMC). This study showed that spermatogonia cryopreserved according to our protocol provides a good supply of undamaged cells for several applications. The significance of the few detected DMCs deserves further attention since it may affect gamete differentiation and epigenetic profile.
{"title":"Cryopreservation did not affect spermatogonia global methylation profile in Senegalese sole (Solea senegalensis).","authors":"Almeida M Mafalda, Cabrita Elsa, Laizé Vincent, Brionne Aurélien, Labbé Catherine, Fatsini Elvira","doi":"10.1016/j.cryobiol.2024.105162","DOIUrl":"https://doi.org/10.1016/j.cryobiol.2024.105162","url":null,"abstract":"<p><p>Spermatogonia cryopreservation is a method to preserve valuable genomes from both maternal and paternal origin. The damage associated with the application of this technology on post-thaw cell quality is important to assess, including at the epigenetic level. This study aimed to assess post-thawed spermatogonia quality by evaluating alterations in plasma membrane integrity, DNA integrity (fragmentation and apoptosis), lipid peroxidation (malondialdehyde levels) and epigenetic modifications (DNA methylation profile). We observed that plasma membrane integrity (fresh 78.98% ± 5.66; cryopreserved 62.81% ± 3.25; P = 0.003) and DNA integrity (fresh 32.95% ± 2.28; cryopreserved 37.28% ± 1.87; P = 0.0026) were affected by cryopreservation, while no difference in lipid peroxidation was observed (fresh 1.13% ± 0.45; cryopreserved 0.91% ± 0.96; P = 0.701). While global levels of DNA methylation were unaffected by cryopreservation (fresh 82.80% ± 0.47; cryopreserved 83.32% ± 0.81; P = 0.745), some differentially methylated cytosines (DMC) were observed in cryopreserved versus fresh spermatogonia (156 DMC). This study showed that spermatogonia cryopreserved according to our protocol provides a good supply of undamaged cells for several applications. The significance of the few detected DMCs deserves further attention since it may affect gamete differentiation and epigenetic profile.</p>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":" ","pages":"105162"},"PeriodicalIF":2.3,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142616413","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}
Pub Date : 2024-11-04DOI: 10.1016/j.cryobiol.2024.105155
Tom Brookshaw , Barry Fuller , Eloy Erro , Tamnia Islam , Sweta Chandel , Elizaveta Zotova , Clare Selden
For the efficient delivery of a cell therapy a treatment must be provided rapidly, at clinical scale, contain a sufficient active cellular component (biomass), and adhere to a multitude of regulatory requirements. Cryopreservation permits many of these demands to be met more readily. Here we present the cryopreservation and recovery of large volume (2.5L) alginate encapsulated liver cell spheroids (AELS), suitable for use with a novel bioartificial liver device (HepatiCan™) for the treatment of those suffering from acute liver failure (ALF), in regulatory approved cryobags and a cryopreservation process optimised for large volumes. By first assessing the thermal profiles of large scale cryobags with a thermal mimic, the feasibility of cryopreserving a full patient dose simultaneously (3x cryobags containing 833 ml biomass each) was investigated, allowing for small and subsequently large-scale testing of cellular functional recoveries. Work presented here demonstrates that optimised reproducible cooling and warming profiles could be achieved with these large volumes, leading to high biomass recoveries at full clinical scale. The recovered AELS also had high regeneration potential, achieving full pre-freeze viable cell densities within 3 days, indicating that the cell therapy could be delivered rapidly to patients with ALF. This study has presented the feasibility for rapid delivery of large volume cell therapies, whilst further research into improved speed of post-thaw recovery is warranted.
{"title":"Cryobiological aspects of upscaling cryopreservation for encapsulated liver cell therapies","authors":"Tom Brookshaw , Barry Fuller , Eloy Erro , Tamnia Islam , Sweta Chandel , Elizaveta Zotova , Clare Selden","doi":"10.1016/j.cryobiol.2024.105155","DOIUrl":"10.1016/j.cryobiol.2024.105155","url":null,"abstract":"<div><div>For the efficient delivery of a cell therapy a treatment must be provided rapidly, at clinical scale, contain a sufficient active cellular component (biomass), and adhere to a multitude of regulatory requirements. Cryopreservation permits many of these demands to be met more readily. Here we present the cryopreservation and recovery of large volume (2.5L) alginate encapsulated liver cell spheroids (AELS), suitable for use with a novel bioartificial liver device (HepatiCan™) for the treatment of those suffering from acute liver failure (ALF), in regulatory approved cryobags and a cryopreservation process optimised for large volumes. By first assessing the thermal profiles of large scale cryobags with a thermal mimic, the feasibility of cryopreserving a full patient dose simultaneously (3x cryobags containing 833 ml biomass each) was investigated, allowing for small and subsequently large-scale testing of cellular functional recoveries. Work presented here demonstrates that optimised reproducible cooling and warming profiles could be achieved with these large volumes, leading to high biomass recoveries at full clinical scale. The recovered AELS also had high regeneration potential, achieving full pre-freeze viable cell densities within 3 days, indicating that the cell therapy could be delivered rapidly to patients with ALF. This study has presented the feasibility for rapid delivery of large volume cell therapies, whilst further research into improved speed of post-thaw recovery is warranted.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"117 ","pages":"Article 105155"},"PeriodicalIF":2.3,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142496674","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}
Pub Date : 2024-11-02DOI: 10.1016/j.cryobiol.2024.105160
Kieran T. Smith , Kanav Khosla , Guebum Han , Tim Humphrey , Nicholas Phelps , John Bischof
Cryopreservation of aquatic embryos or larvae is needed to help safeguard genetics from important wild and captive species, increase aquaculture output, and meet the global demand for protein. To this end, the development of a cryopreservation protocol for nauplius larvae of the commercially important aquaculture species Litopenaeus vannamei, or Pacific White Shrimp, was pursued. Toxicity screening was performed using multiple cryoprotective agents (CPA), and a multi-constituent CPA cocktail was developed to achieve reliable vitrification of shrimp larvae encapsulated in 1.0-μL droplets containing gold nanoparticles. Vitrification and ultra-rapid laser warming were used to cryopreserve and revive nauplius-V stage larvae. Laser warming parameters were optimized to protect the pigmented eye spot from laser-induced ablation, and ice recrystallization inhibitors (IRIs) were tested to induce long-term survival. Approximately 54 % of revived larvae resumed active swimming, but all failed to molt to the zoea-I stage of development or live beyond 15 h post warming.
{"title":"Revival of cryopreserved larvae from the important aquaculture species Pacific White Shrimp (Litopenaeus vannamei) using vitrification and ultra-rapid laser warming","authors":"Kieran T. Smith , Kanav Khosla , Guebum Han , Tim Humphrey , Nicholas Phelps , John Bischof","doi":"10.1016/j.cryobiol.2024.105160","DOIUrl":"10.1016/j.cryobiol.2024.105160","url":null,"abstract":"<div><div>Cryopreservation of aquatic embryos or larvae is needed to help safeguard genetics from important wild and captive species, increase aquaculture output, and meet the global demand for protein. To this end, the development of a cryopreservation protocol for nauplius larvae of the commercially important aquaculture species <em>Litopenaeus vannamei</em>, or Pacific White Shrimp, was pursued. Toxicity screening was performed using multiple cryoprotective agents (CPA), and a multi-constituent CPA cocktail was developed to achieve reliable vitrification of shrimp larvae encapsulated in 1.0-μL droplets containing gold nanoparticles. Vitrification and ultra-rapid laser warming were used to cryopreserve and revive nauplius-V stage larvae. Laser warming parameters were optimized to protect the pigmented eye spot from laser-induced ablation, and ice recrystallization inhibitors (IRIs) were tested to induce long-term survival. Approximately 54 % of revived larvae resumed active swimming, but all failed to molt to the zoea-I stage of development or live beyond 15 h post warming.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"117 ","pages":"Article 105160"},"PeriodicalIF":2.3,"publicationDate":"2024-11-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564155","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}
Pub Date : 2024-10-31DOI: 10.1016/j.cryobiol.2024.105156
Alisson R.S. Silva , Jacqueline B. Copetti , André C. Monteiro , Marcelo Gotardo , Jeferson D. de Oliveira , Mario H. Macagnan , Elaine M. Cardoso , Karolyn Ogliari
One of the biggest challenges in studying vitrification protocols for small volumes of biological materials, especially the microdroplet vitrification protocol, is measuring the solidification rate, requiring equipment with a high level of technology, making it practically impossible to measure the degree of crystallization. An alternative is using mathematical models applied in computer simulations (CFD), helping to improve and develop new vitrification protocols. This study investigates the vitrification process utilizing the microdroplet method through experimental and numerical analysis. Droplets of mineralized water are deposited onto a copper substrate, temperature data is collected, and images of the process are taken with a high-speed camera. Numerical simulations are performed using ANSYS Fluent® software to analyze temperature and solidification behavior. Droplet contact angle measurements are also conducted to determine boundary conditions for numerical simulations. Mesh refinement is conducted using the Grid Convergence Index method, ensuring accuracy in computational results. The simulations employ a solidification model, considering phase enthalpy and thermal properties of the droplet, environment, and substrate. Results show good agreement between numerical and experimental data regarding solidification dynamics and temperature profiles. Furthermore, the study examines the influence of cooling surface geometry on the vitrification process. The contact area between the droplet and the surface increases by machining a cavity on the copper substrate, leading to enhanced cooling rates and reduced stabilization time. This research provides insights into optimizing vitrification processes, contributing to advancements in cryopreservation and material science applications.
{"title":"Numerical and experimental investigation of a droplet vitrification process: A thermofluidic analysis for cryopreservation","authors":"Alisson R.S. Silva , Jacqueline B. Copetti , André C. Monteiro , Marcelo Gotardo , Jeferson D. de Oliveira , Mario H. Macagnan , Elaine M. Cardoso , Karolyn Ogliari","doi":"10.1016/j.cryobiol.2024.105156","DOIUrl":"10.1016/j.cryobiol.2024.105156","url":null,"abstract":"<div><div>One of the biggest challenges in studying vitrification protocols for small volumes of biological materials, especially the microdroplet vitrification protocol, is measuring the solidification rate, requiring equipment with a high level of technology, making it practically impossible to measure the degree of crystallization. An alternative is using mathematical models applied in computer simulations (CFD), helping to improve and develop new vitrification protocols. This study investigates the vitrification process utilizing the microdroplet method through experimental and numerical analysis. Droplets of mineralized water are deposited onto a copper substrate, temperature data is collected, and images of the process are taken with a high-speed camera. Numerical simulations are performed using ANSYS Fluent® software to analyze temperature and solidification behavior. Droplet contact angle measurements are also conducted to determine boundary conditions for numerical simulations. Mesh refinement is conducted using the Grid Convergence Index method, ensuring accuracy in computational results. The simulations employ a solidification model, considering phase enthalpy and thermal properties of the droplet, environment, and substrate. Results show good agreement between numerical and experimental data regarding solidification dynamics and temperature profiles. Furthermore, the study examines the influence of cooling surface geometry on the vitrification process. The contact area between the droplet and the surface increases by machining a cavity on the copper substrate, leading to enhanced cooling rates and reduced stabilization time. This research provides insights into optimizing vitrification processes, contributing to advancements in cryopreservation and material science applications.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"117 ","pages":"Article 105156"},"PeriodicalIF":2.3,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142560718","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}
Pub Date : 2024-10-31DOI: 10.1016/j.cryobiol.2024.105157
Hao Cheng , Jun Mei , Jing Xie
Temperature abuse occurs frequently during transportation and frozen storage, which affects the quality of frozen aquatic products. Recrystallization generated by temperature abuse leads to irreversible damage to the muscle tissue and microstructure, and exacerbates undesirable oxidation reactions, thus reducing the quality of frozen aquatic products. In this study, a modeling system of temperature abuse alternating between −24 °C and −7 °C was established to evaluate the effect of temperature abuse on the stability of frozen large yellow croaker. The results revealed that temperature abuse caused water migration with the extension of storage time, as well as poorer texture, color, and freshness. Furthermore, the structure of myofibrillar protein (MP) was severely damaged, with a gradual decrease in total sulfhydryl groups and Ca2+-ATPase activity, a loosening of the secondary structure, and a disruption of the protein conformation. The confocal laser scanning microscopy (CLSM) analysis also found that temperature abuse exacerbated protein aggregation. Therefore, temperature abuse during transportation and frozen storage could affect the stability of large yellow croaker negatively, and it mainly originated from the growth of ice crystals and the effect of recrystallization. The study was supposed to provide new insights into the improvement of frozen aquatic products quality.
{"title":"Stability of large yellow croaker (Pseudosciaena crocea) as affected by temperature abuse during frozen storage: Quality attributes, myofibril characteristics, and microstructure","authors":"Hao Cheng , Jun Mei , Jing Xie","doi":"10.1016/j.cryobiol.2024.105157","DOIUrl":"10.1016/j.cryobiol.2024.105157","url":null,"abstract":"<div><div>Temperature abuse occurs frequently during transportation and frozen storage, which affects the quality of frozen aquatic products. Recrystallization generated by temperature abuse leads to irreversible damage to the muscle tissue and microstructure, and exacerbates undesirable oxidation reactions, thus reducing the quality of frozen aquatic products. In this study, a modeling system of temperature abuse alternating between −24 °C and −7 °C was established to evaluate the effect of temperature abuse on the stability of frozen large yellow croaker. The results revealed that temperature abuse caused water migration with the extension of storage time, as well as poorer texture, color, and freshness. Furthermore, the structure of myofibrillar protein (MP) was severely damaged, with a gradual decrease in total sulfhydryl groups and Ca<sup>2+</sup>-ATPase activity, a loosening of the secondary structure, and a disruption of the protein conformation. The confocal laser scanning microscopy (CLSM) analysis also found that temperature abuse exacerbated protein aggregation. Therefore, temperature abuse during transportation and frozen storage could affect the stability of large yellow croaker negatively, and it mainly originated from the growth of ice crystals and the effect of recrystallization. The study was supposed to provide new insights into the improvement of frozen aquatic products quality.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"117 ","pages":"Article 105157"},"PeriodicalIF":2.3,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142544274","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}
Pub Date : 2024-10-28DOI: 10.1016/j.cryobiol.2024.104980
Hussain Ahmed , Muhammad Umar Ijaz , Mehreen Riaz , Farhad Ullah , Haney Samir , Muhammad Shuaib , Ayman A. Swelum
This comprehensive study was carried out to investigate the effects of taxifolin in the freezing medium on post-thaw semen quality, and fertility potential of buffalo bull spermatozoa. Taxifolin was also evaluated for radical scavenging activity through DPPH (2, 2-diphenyl-1-picrylhydrazyl), and Nitric oxide (NO) inhibition (%) during in vitro conditions. Collected semen samples from four buffalo bulls were initially evaluated (consistency, volume, motility, and concentrations); the accepted samples were pooled, and diluted in extenders containing different doses of taxifolin (0 μM [control], 2 μM, 5 μM, 10 μM, and 20 μM). Diluted semen was gradually cooled (2 h, 4 °C), equilibrated (4 h, 4 °C), and then frozen in liquid nitrogen (−196 °C). Data analysis revealed that taxifolin supplementation (10 μM) displayed the highest DPPH-scavenging ability, and NO inhibition compared to the control (% DPPH, 67.31 vs. 13.50; and % NO, 78.25 vs. 21.25) respectively. Moreover, taxifolin supplementation (10 μM) significantly (P < 0.05) improved progressive motility (%), average path velocity (μm/sec), straight-line velocity (μm/sec), and seminal plasma glutathione peroxidase (μM), and superoxide dismutase (U/mL) of buffalo bull spermatozoa than control. Sperm plasma membrane integrity, mitochondrial transmembrane potential, acrosome integrity (%) and seminal plasma adenosine triphosphate (nmol/million), and total antioxidant capacity (μM/L) were enhanced significantly with taxifolin (10 and 20 μM) supplementing group. Lastly, taxifolin supplementation significantly improved the fertility rate (%, 69.09 vs. 40.38) compared to the control. Further studies to assess mechanisms by which taxifolin improves semen quality and fertility of buffalo spermatozoa are warranted.
这项综合研究旨在探讨冷冻培养基中的紫杉叶素对水牛精子解冻后精液质量和生育潜力的影响。研究还通过 DPPH（2, 2-二苯基-1-苦基肼）和一氧化氮（NO）在体外条件下的抑制率来评估 Taxifolin 的自由基清除活性。对从四头水牛身上采集的精液样本进行初步评估（一致性、体积、运动性和浓度）；将接受的样本集中起来，并在含有不同剂量（0 μM[对照组]、2 μM、5 μM、10 μM 和 20 μM）taxifolin 的扩展剂中稀释。稀释后的精液逐渐冷却（2 小时，4°C），平衡（4 小时，4°C），然后冷冻在液氮中（-196°C）。数据分析结果表明，与对照组相比，补充紫杉叶素（10 μM）的 DPPH 清除能力和 NO 抑制能力最高（DPPH 抑制率分别为 67.31% vs. 13.50；NO 抑制率分别为 78.25% vs. 21.25）。此外，与对照组相比，补充紫杉叶素（10 μM）能显著（P < 0.05）提高水牛精子的渐进运动率（%）、平均路径速度（μm/秒）、直线速度（μm/秒）、精浆谷胱甘肽过氧化物酶（μM）和超氧化物歧化酶（U/mL）。水牛精子的精浆膜完整性、线粒体跨膜电位、顶体完整性（%）、精浆三磷酸腺苷（nmol/million）和总抗氧化能力（μM/L）在补充紫杉叶素（10 和 20 μM）组有显著提高。最后，与对照组相比，补充 taxifolin 能显著提高受精率（%，69.09 vs. 40.38）。有必要开展进一步研究，以评估 taxifolin 改善水牛精液质量和生育能力的机制。
{"title":"Taxifolin: A flavonoid in the freezing medium augments post-thaw semen quality and in vivo fertility potential of buffalo bull spermatozoa","authors":"Hussain Ahmed , Muhammad Umar Ijaz , Mehreen Riaz , Farhad Ullah , Haney Samir , Muhammad Shuaib , Ayman A. Swelum","doi":"10.1016/j.cryobiol.2024.104980","DOIUrl":"10.1016/j.cryobiol.2024.104980","url":null,"abstract":"<div><div>This comprehensive study was carried out to investigate the effects of taxifolin in the freezing medium on post-thaw semen quality, and fertility potential of buffalo bull spermatozoa. Taxifolin was also evaluated for radical scavenging activity through DPPH (2, 2-diphenyl-1-picrylhydrazyl), and Nitric oxide (NO) inhibition (%) during <em>in vitro</em> conditions. Collected semen samples from four buffalo bulls were initially evaluated (consistency, volume, motility, and concentrations); the accepted samples were pooled, and diluted in extenders containing different doses of taxifolin (0 μM [control], 2 μM, 5 μM, 10 μM, and 20 μM). Diluted semen was gradually cooled (2 h, 4 °C), equilibrated (4 h, 4 °C), and then frozen in liquid nitrogen (−196 °C). Data analysis revealed that taxifolin supplementation (10 μM) displayed the highest DPPH-scavenging ability, and NO inhibition compared to the control (% DPPH, 67.31 vs. 13.50; and % NO, 78.25 vs. 21.25) respectively. Moreover, taxifolin supplementation (10 μM) significantly (P < 0.05) improved progressive motility (%), average path velocity (μm/sec), straight-line velocity (μm/sec), and seminal plasma glutathione peroxidase (μM), and superoxide dismutase (U/mL) of buffalo bull spermatozoa than control. Sperm plasma membrane integrity, mitochondrial transmembrane potential, acrosome integrity (%) and seminal plasma adenosine triphosphate (nmol/million), and total antioxidant capacity (μM/L) were enhanced significantly with taxifolin (10 and 20 μM) supplementing group. Lastly, taxifolin supplementation significantly improved the fertility rate (%, 69.09 vs. 40.38) compared to the control. Further studies to assess mechanisms by which taxifolin improves semen quality and fertility of buffalo spermatozoa are warranted.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"117 ","pages":"Article 104980"},"PeriodicalIF":2.3,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459934","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}
Pub Date : 2024-10-25DOI: 10.1016/j.cryobiol.2024.104982
Cumali Kaya , Burcu Esin , Melih Akar , Cansu Can , Mesut Çevik
The presented study covers testicular tissue and epididymal spermatozoa cryopreservation processes in bulls and aims to investigate the effects of these applications on spermatological parameters, cell viability in testicular tissue, and the expression of the PARP-1 gene, a DNA repair enzyme. Testes of 20 bulls over 2 years old, slaughtered in a slaughterhouse, were used in the study. After spermatological evaluations, the semen obtained from the cauda epididymis was frozen in liquid nitrogen vapor according to the straw method and stored in liquid nitrogen (−196 °C). Testicular tissue pieces obtained from the testicles were frozen by the slow freezing method in cryotubes in diluents containing Dimethylsulfoxide (DMSO) and Ethylene Glycol (EG) cryoprotectants and stored in liquid nitrogen (−196 °C). The total motility (TM) (85.89 ± 12.83 %), progressive motility (PM) (54.02 ± 15.77 %), and kinematic parameter values of fresh sperm were significantly higher compared to the TM (57.62 ± 13.13 %), PM (29.60 ± 10.76 %), and kinematic parameter values after thawing (P < 0.05). Significant decreases in plasma membrane integrity (PMI) and viability and an increase in chromatin condensation and morphological disorders in the head, middle part, and tail regions were observed in post-thaw semen samples (P < 0.05). When the effects of DMSO and EG on cell viability after thaw in frozen testicular tissue were evaluated, it was observed that the cell viability values of testicular tissues frozen with EG (45.70 ± 10.00) were statistically significantly lower than those frozen with DMSO (51.20 ± 7.70) (P < 0.05). When the effects of both cryoprotectants on gene expression in tissue and semen samples were examined, it was determined that gene expression increased on average 0.19 ± 0.27 times in the tissue samples in the DMSO group compared to fresh tissue samples and 0.17 ± 0.19 times in the tissue samples in the EG group. It was determined that gene expression levels increased by an average of 1.20 ± 1.08 times in post-thaw epididymal spermatozoa samples compared to fresh semen samples. The results show that cryopreservation can activate cellular repair mechanisms by stimulating PARP-1 gene expression and affect gene expression by activating specific pathways in tissues and cells.
{"title":"Investigation of the efficacy of different cryoprotectants in the freezing of testicular tissue and epididymal sperm: Spermatological parameters, tissue viability and PARP-1 gene expression","authors":"Cumali Kaya , Burcu Esin , Melih Akar , Cansu Can , Mesut Çevik","doi":"10.1016/j.cryobiol.2024.104982","DOIUrl":"10.1016/j.cryobiol.2024.104982","url":null,"abstract":"<div><div>The presented study covers testicular tissue and epididymal spermatozoa cryopreservation processes in bulls and aims to investigate the effects of these applications on spermatological parameters, cell viability in testicular tissue, and the expression of the PARP-1 gene, a DNA repair enzyme. Testes of 20 bulls over 2 years old, slaughtered in a slaughterhouse, were used in the study. After spermatological evaluations, the semen obtained from the cauda epididymis was frozen in liquid nitrogen vapor according to the straw method and stored in liquid nitrogen (−196 °C). Testicular tissue pieces obtained from the testicles were frozen by the slow freezing method in cryotubes in diluents containing Dimethylsulfoxide (DMSO) and Ethylene Glycol (EG) cryoprotectants and stored in liquid nitrogen (−196 °C). The total motility (TM) (85.89 ± 12.83 %), progressive motility (PM) (54.02 ± 15.77 %), and kinematic parameter values of fresh sperm were significantly higher compared to the TM (57.62 ± 13.13 %), PM (29.60 ± 10.76 %), and kinematic parameter values after thawing (P < 0.05). Significant decreases in plasma membrane integrity (PMI) and viability and an increase in chromatin condensation and morphological disorders in the head, middle part, and tail regions were observed in post-thaw semen samples (P < 0.05). When the effects of DMSO and EG on cell viability after thaw in frozen testicular tissue were evaluated, it was observed that the cell viability values of testicular tissues frozen with EG (45.70 ± 10.00) were statistically significantly lower than those frozen with DMSO (51.20 ± 7.70) (P < 0.05). When the effects of both cryoprotectants on gene expression in tissue and semen samples were examined, it was determined that gene expression increased on average 0.19 ± 0.27 times in the tissue samples in the DMSO group compared to fresh tissue samples and 0.17 ± 0.19 times in the tissue samples in the EG group. It was determined that gene expression levels increased by an average of 1.20 ± 1.08 times in post-thaw epididymal spermatozoa samples compared to fresh semen samples. The results show that cryopreservation can activate cellular repair mechanisms by stimulating PARP-1 gene expression and affect gene expression by activating specific pathways in tissues and cells.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"117 ","pages":"Article 104982"},"PeriodicalIF":2.3,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459933","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}
Pub Date : 2024-10-23DOI: 10.1016/j.cryobiol.2024.104979
Jia-Yu Wu , Huan-Yu Kang , Yong Guo , Xi-Hui Sheng , Xiang-Guo Wang , Kai Xing , Long-Fei Xiao , Xue-Ze Lv , Cheng Long , Xiao-Long Qi
Cryopreservation causes higher reactive oxygen species (ROS) concentrations, leading to oxidative stress and lipid peroxidation damaging sperm, and using antioxidants can improve semen quality after freeze-thaw. Natural astaxanthin (ASTA) can be inserted into cell membranes and its antioxidant properties are stronger than other antioxidants. We aimed to investigate the effects of ASTA supplementation in the Beltsville Poultry Semen Extender (BPSE) on post-thaw rooster semen quality and to explore the potential mechanism of rooster semen quality change. The qualifying semen ejaculates collected from 30 adult male Jinghong No. 1 laying hen breeder roosters (65 wk old) were pooled, divided into four aliquots, and diluted with BPSE having different levels of ASTA (0, 0.5, 1, or 2 μg/mL). Treated semen was cryopreserved and kept in liquid nitrogen. The entire experiment was replicated three times independently. Sperm viability, motility, curvilinear velocity, amplitude of lateral head displacement, straightness, plasma membrane integrity, and acrosome integrity were observed to be highest (P < 0.05) with 1 μg/mL ASTA at freeze-thawing. Higher (P < 0.05) antioxidant enzyme (CAT-like, SOD) activities and free radical (·OH, O2.-) scavenging ability, less ROS and malondialdehyde (MDA) concentrations were recorded with the addition of appropriate concentrations of ASTA compared to control. In addition, the levels of mitochondrial membrane potential (MMP), adenosine triphosphate (ATP), and lactate dehydrogenase (LDH) in the 1 μg/mL ASTA group improved compared to the control group, and decreased the amount of AIF protein level but increased the Bcl-2 protein level (P < 0:05). Collectively, these results demonstrate that adding ASTA in the BPSE promoted rooster freeze-thaw sperm quality, which may be related to reducing ROS levels, protecting the antioxidant defense system, preventing lipid peroxidation, improving mitochondrial structural and functional integrity, and inhibiting sperm apoptosis.
{"title":"Effect of natural astaxanthin on sperm quality and mitochondrial function of breeder rooster semen cryopreservation","authors":"Jia-Yu Wu , Huan-Yu Kang , Yong Guo , Xi-Hui Sheng , Xiang-Guo Wang , Kai Xing , Long-Fei Xiao , Xue-Ze Lv , Cheng Long , Xiao-Long Qi","doi":"10.1016/j.cryobiol.2024.104979","DOIUrl":"10.1016/j.cryobiol.2024.104979","url":null,"abstract":"<div><div>Cryopreservation causes higher reactive oxygen species (ROS) concentrations, leading to oxidative stress and lipid peroxidation damaging sperm, and using antioxidants can improve semen quality after freeze-thaw. Natural astaxanthin (ASTA) can be inserted into cell membranes and its antioxidant properties are stronger than other antioxidants. We aimed to investigate the effects of ASTA supplementation in the Beltsville Poultry Semen Extender (BPSE) on post-thaw rooster semen quality and to explore the potential mechanism of rooster semen quality change. The qualifying semen ejaculates collected from 30 adult male Jinghong No. 1 laying hen breeder roosters (65 wk old) were pooled, divided into four aliquots, and diluted with BPSE having different levels of ASTA (0, 0.5, 1, or 2 μg/mL). Treated semen was cryopreserved and kept in liquid nitrogen. The entire experiment was replicated three times independently. Sperm viability, motility, curvilinear velocity, amplitude of lateral head displacement, straightness, plasma membrane integrity, and acrosome integrity were observed to be highest (P < 0.05) with 1 μg/mL ASTA at freeze-thawing. Higher (P < 0.05) antioxidant enzyme (CAT-like, SOD) activities and free radical (·OH, O2<sup>.-</sup>) scavenging ability, less ROS and malondialdehyde (MDA) concentrations were recorded with the addition of appropriate concentrations of ASTA compared to control. In addition, the levels of mitochondrial membrane potential (MMP), adenosine triphosphate (ATP), and lactate dehydrogenase (LDH) in the 1 μg/mL ASTA group improved compared to the control group, and decreased the amount of AIF protein level but increased the Bcl-2 protein level (P < 0:05). Collectively, these results demonstrate that adding ASTA in the BPSE promoted rooster freeze-thaw sperm quality, which may be related to reducing ROS levels, protecting the antioxidant defense system, preventing lipid peroxidation, improving mitochondrial structural and functional integrity, and inhibiting sperm apoptosis.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"117 ","pages":"Article 104979"},"PeriodicalIF":2.3,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142460032","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}
Male fertility preservation is still challenged by cell damage induced during sperm cryopreservation and impaired sperm structure and function. Sperm ultra-rapid freezing, despite a higher protective effect compared to conventional freezing method, is still associated with suboptimal sperm cryosurvival and needs to be modified to increase its efficiency in sperm protection. Sperm freezing media supplemented with antioxidants can improve sperm parameters following freezing-warming process. In this study, we aimed to investigate the effect of employing ultra-rapid freezing and myo-inositol on sperm cryosurvival. Thirty semen samples with normal sperm parameters were collected and each one was divided into four portions to cryopreserve by conventional freezing, ultra-rapid freezing, conventional freezing + myo-inositol 2 mg/ml, and ultra-rapid freezing + myo-inositol 2 mg/ml. Sperm samples warmed after at least 24 h of freezing and sperm cryosurvival were analyzed by evaluation of sperm motility, viability, morphology and DNA fragmentation index (DFI). Freezing method had a significant influence on post-thaw sperm DFI and morphology (p < 0.05) and the interaction between freezing method and antioxidant supplementation significantly affected sperm morphology (p < 0.05). The highest percentage of sperm normal morphology and minimal DFI was achieved using ultra-rapid freezing supplemented by myo-inositol antioxidant compared to other groups (P < 0.05). The highest sperm DNA damage after freezing-warming was observed following the conventional freezing method. In conclusion, sperm freezing method was identified as factor strongly influencing sperm DFI and morphology after thawing/warming. Sperm samples can be rapidly frozen using the modified freezing media supplemented by myo-inositol without impacting sperm DNA and morphology.
{"title":"The effect of myo-inositol antioxidant activity on human sperm parameters and DNA damage in ultra-rapid and conventional freezing methods","authors":"Parastoo Salehi , Nadia Sheibak , Fatemehsadat Amjadi , Reza Nejatbakhsh , Zahra Zandieh","doi":"10.1016/j.cryobiol.2024.104978","DOIUrl":"10.1016/j.cryobiol.2024.104978","url":null,"abstract":"<div><div>Male fertility preservation is still challenged by cell damage induced during sperm cryopreservation and impaired sperm structure and function. Sperm ultra-rapid freezing, despite a higher protective effect compared to conventional freezing method, is still associated with suboptimal sperm cryosurvival and needs to be modified to increase its efficiency in sperm protection. Sperm freezing media supplemented with antioxidants can improve sperm parameters following freezing-warming process. In this study, we aimed to investigate the effect of employing ultra-rapid freezing and myo-inositol on sperm cryosurvival. Thirty semen samples with normal sperm parameters were collected and each one was divided into four portions to cryopreserve by conventional freezing, ultra-rapid freezing, conventional freezing + myo-inositol 2 mg/ml, and ultra-rapid freezing + myo-inositol 2 mg/ml. Sperm samples warmed after at least 24 h of freezing and sperm cryosurvival were analyzed by evaluation of sperm motility, viability, morphology and DNA fragmentation index (DFI). Freezing method had a significant influence on post-thaw sperm DFI and morphology (p < 0.05) and the interaction between freezing method and antioxidant supplementation significantly affected sperm morphology (p < 0.05). The highest percentage of sperm normal morphology and minimal DFI was achieved using ultra-rapid freezing supplemented by myo-inositol antioxidant compared to other groups (P < 0.05). The highest sperm DNA damage after freezing-warming was observed following the conventional freezing method. In conclusion, sperm freezing method was identified as factor strongly influencing sperm DFI and morphology after thawing/warming. Sperm samples can be rapidly frozen using the modified freezing media supplemented by myo-inositol without impacting sperm DNA and morphology.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"117 ","pages":"Article 104978"},"PeriodicalIF":2.3,"publicationDate":"2024-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142399682","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}
Pub Date : 2024-10-18DOI: 10.1016/j.cryobiol.2024.104981
Clara Slade Oliveira , Viviane Luzia da Silva Feuchard , Carolina Romano Capobiango Quintão , Leticia Zoccolaro Oliveira , Naiara Zoccal Saraiva
Given the significant variation in lipid levels among bovine embryos, our study was designed to associate lipid content to oxidative stress in individual embryos undergoing vitrification, and to assess how this and other morphological parameters impacts cryosurvival. Linear and logistic regression were performed to understand the influence of the variables in the cryosurvival. T-test or Kruskal Wallis were employed to compare means. Vitrified embryos revealed a positive correlation between lipid content and oxidative stress post-warming both 2 h (p = 0.025, n = 64) and 48 h (p < 0.001, n = 122) after warming. Lipid levels explained (p < 0.001) up to 51 % (multiple R-squared) of oxidative stress variability. Compared to fresh embryos, a negative influence (p = 0.01) of vitrification-warming procedures was detected in lipid levels. Vitrified embryos exhibited lower (p < 0.001, n = 90) mean lipid content compared to fresh counterparts 48 h post-warming, and similar (p = 0.24) oxidative stress levels. No impact of lipid content or oxidative stress levels was detected on hatchability or embryo quality 48 h post-warming (n = 99). Expansion just after (0 h) and 2 h after warming resulted in a higher chance of hatching (p = 0.015 and p = 0.008, OR 1.30 and 1.58), and a positive association was observed between expansion at 0 h (p = 0.002) and embryo area (p = 0.047) with cell number. In conclusion, a decrease in lipid levels was found following vitrification-warming procedure and an individual association between lipids and oxidative stress is present in vitrified embryos. Lipids or oxidative stress levels was not linked to survivability of vitrified embryos 48 h following warming. Expansion at 0 h indicates a better chance for hatching and higher cell numbers in vitrified embryos.
{"title":"Impact of lipid content on oxygen reactive species and viability predictors in vitrified bovine embryos","authors":"Clara Slade Oliveira , Viviane Luzia da Silva Feuchard , Carolina Romano Capobiango Quintão , Leticia Zoccolaro Oliveira , Naiara Zoccal Saraiva","doi":"10.1016/j.cryobiol.2024.104981","DOIUrl":"10.1016/j.cryobiol.2024.104981","url":null,"abstract":"<div><div>Given the significant variation in lipid levels among bovine embryos, our study was designed to associate lipid content to oxidative stress in individual embryos undergoing vitrification, and to assess how this and other morphological parameters impacts cryosurvival. Linear and logistic regression were performed to understand the influence of the variables in the cryosurvival. T-test or Kruskal Wallis were employed to compare means. Vitrified embryos revealed a positive correlation between lipid content and oxidative stress post-warming both 2 h (p = 0.025, n = 64) and 48 h (p < 0.001, n = 122) after warming. Lipid levels explained (p < 0.001) up to 51 % (multiple R-squared) of oxidative stress variability. Compared to fresh embryos, a negative influence (p = 0.01) of vitrification-warming procedures was detected in lipid levels. Vitrified embryos exhibited lower (p < 0.001, n = 90) mean lipid content compared to fresh counterparts 48 h post-warming, and similar (p = 0.24) oxidative stress levels. No impact of lipid content or oxidative stress levels was detected on hatchability or embryo quality 48 h post-warming (n = 99). Expansion just after (0 h) and 2 h after warming resulted in a higher chance of hatching (p = 0.015 and p = 0.008, OR 1.30 and 1.58), and a positive association was observed between expansion at 0 h (p = 0.002) and embryo area (p = 0.047) with cell number. In conclusion, a decrease in lipid levels was found following vitrification-warming procedure and an individual association between lipids and oxidative stress is present in vitrified embryos. Lipids or oxidative stress levels was not linked to survivability of vitrified embryos 48 h following warming. Expansion at 0 h indicates a better chance for hatching and higher cell numbers in vitrified embryos.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"117 ","pages":"Article 104981"},"PeriodicalIF":2.3,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459935","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}
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