freezing medium
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2022 ◽  
Vol 18 (1) ◽  
Author(s):  
Ruixue Zhang ◽  
Hemeng Dong ◽  
Pengpeng Zhao ◽  
Chunmei Shang ◽  
Hang Qi ◽  
...  

Abstract Background Semen cryopreservation has become an essential tool for conservation efforts of the giant panda (Ailuropoda melanoleuca); however, it is severely detrimental to sperm quality. Evidence has shown that antioxidants have the potential to reverse cryopreservation-induced damage in sperm. The purpose of this study was to screen effective antioxidants that could retain sperm quality during cryopreservation and to determine the optimal dose. Seven antioxidant groups, including resveratrol (RSV = 50 μM, RSV = 100 μM, RSV = 150 μM), lycium barbarum polysaccharide (LBP = 2 mg/mL, LBP = 4 mg/mL), laminaria japonica polysaccharides (LJP = 1 mg/mL) or combination (LBP = 2 mg/mL, LJP = 1 mg/mL and RSV = 100 μM) were assessed. Results RSV, LBP, LJP, or a combination of RSV, LBP, and LJP added to the freezing medium significantly improved sperm progressive motility, plasma membrane integrity, acrosome integrity, and mitochondrial activity during the cryopreservation process. Furthermore, the activities of glutathione peroxidase and superoxide dismutase were also improved. The levels of reactive oxygen species and malondialdehyde in semen were notably reduced. Hyaluronidase activity and acrosin activity were significantly increased in LBP-treated sperm. However, sperm total motility and DNA integrity were not significantly different between the groups. Conclusions RSV (50 μM) or LBP (2 mg/mL) are the best candidate antioxidants for inclusion in the freezing medium to improve the quality of giant panda spermatozoa during semen cryopreservation.


Author(s):  
Daniel Kaiser ◽  
Natalie Maureen Otto ◽  
Oliver McCallion ◽  
Henrike Hoffmann ◽  
Ghazaleh Zarrinrad ◽  
...  

Cell therapies have significant therapeutic potential in diverse fields including regenerative medicine, transplantation tolerance, and autoimmunity. Within these fields, regulatory T cells (Treg) have been deployed to ameliorate aberrant immune responses with great success. However, translation of the cryopreservation strategies employed for other cell therapy products, such as effector T cell therapies, to Treg therapies has been challenging. The lack of an optimized cryopreservation strategy for Treg products presents a substantial obstacle to their broader application, particularly as administration of fresh cells limits the window available for sterility and functional assessment. In this study, we aimed to develop an optimized cryopreservation strategy for our CD4+CD25+Foxp3+ Treg clinical product. We investigate the effect of synthetic or organic cryoprotectants including different concentrations of DMSO on Treg recovery, viability, phenotype, cytokine production, suppressive capacity, and in vivo survival following GMP-compliant manufacture. We additionally assess the effect of adding the extracellular cryoprotectant polyethylene glycol (PEG), or priming cellular expression of heat shock proteins as strategies to improve viability. We find that cryopreservation in serum-free freezing medium supplemented with 10% human serum albumin and 5% DMSO facilitates improved Treg recovery and functionality and supports a reduced DMSO concentration in Treg cryopreservation protocols. This strategy may be easily incorporated into clinical manufacture protocols for future studies.


2021 ◽  
Vol 8 (12) ◽  
pp. 302
Author(s):  
Iván Yánez-Ortiz ◽  
Jaime Catalán ◽  
Ariadna Delgado-Bermúdez ◽  
Augusto Carluccio ◽  
Jordi Miró ◽  
...  

In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed prior to cryopreservation, thereby exposing sperm cells to further oxidative stress. The exogenous addition of antioxidants to the freezing medium can reduce the detrimental effects caused by ROS generation. Therefore, the aim of this study was to evaluate how the addition of different reduced glutathione (GSH) concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) to fresh sperm affect their cryotolerance. Total and progressive motility, kinematic parameters and motile sperm subpopulations were significantly (p < 0.05) different from the control in treatments containing 8 mM and 10 mM GSH, but not at lower concentrations. Plasma and acrosome membrane integrity, mitochondrial membrane potential (MMP) and intracellular superoxide levels (O2−) were not affected (p > 0.05) by any GSH concentration. Interestingly, however, the addition of 8 mM or 10 mM GSH reduced (p < 0.05) the percentages of viable sperm with high overall ROS levels compared to the control. In conclusion, frozen-thawed donkey sperm are able to tolerate high GSH concentrations, which differs from what has been observed in other species. This antioxidant capacity suggests that ROS could be important during post-AI and that the impact of using exogenous antioxidants like GSH to improve the sperm resilience to freeze-thawing is limited in this species.


Cryobiology ◽  
2021 ◽  
Vol 103 ◽  
pp. 186-187
Author(s):  
Reyon Dcunha ◽  
Ananda Hanumappa ◽  
Sneha Guruprasad Kalthur ◽  
Sadhana P. Mutalik ◽  
Srinivas Mutalik ◽  
...  

Author(s):  
V. Sapanidou ◽  
S. Lavrentiadou ◽  
M. Errico ◽  
I. Panagiotidis ◽  
I. Efraimidis ◽  
...  

Author(s):  
da Costa Silva RJ ◽  
◽  
da Silva MHM ◽  
Valadão L ◽  
da Silva FM ◽  
...  

Boar semen cryopreservation has a high potential in the swine industry, allowing the large-scale use of genetically superior animals, improving efficiency, product quality, helping to reduce the risk of disease spread and gathering needs from the market. From a genetic point of view, semen freezing is desirable for genetic diversification, favouring a more efficient reproduction as well as the constitution of germplasm banks, including for repopulation in case of disease outbreak. However, freezing this semen for long periods for practical use is limited by the reduced viability and fertilization potential caused to sperm during the cryopreservation process and consequently low conception rates and smaller litters after artificial insemination. In part, the decrease in the fertilizing power of frozen spermatozoa may be associated with oxidative damage due to excessive formation of Reactive Oxygen Species (ROS), osmotic stress and cell damage due to ice formation during cryopreservation. To suppress the damage caused by ROS, the present study was conducted to determine the impact of supplementation with three antioxidants, these being ascorbic acid, a-tocopherol and reduced glutathione, evaluating the parameters of semen quality, viability, total and progressive motility, vigour and agglutination rate after thawing. For this purpose, semen was collected from five boars, each being collected three times, at weekly intervals, always at the same time. Immediately after harvesting, the macroscopic (colour, appearance, and volume) and microscopic evaluation of the semen (mass motility, concentration, progressive individual motility, spermatic vigour and spermatic morphology) were evaluated. Subsequently, the semen was placed at 15°C for two hours and centrifuged at 800 x g for 10 minutes also at 15°C, removing the supernatant. For the freezing medium, a base medium consisting of a commercial MR-A extender, supplemented with 3% v/v glycerol, 10% v/v egg yolk and 0.20% w/v Sodium Dodecyl Sulfate (SDS) was used. The nine treatments used in the study were, respectively, ascorbic acid at concentrations of 100, 200 and 400μL, a-Tocopherol at concentrations of 200, 400 and 800μM and reduced Glutathione at concentrations of 2.5, 5 and 10 mg/l and numbered as T1 to T9, respectively. In the control group, semen was frozen in a medium without adding any antioxidant. The semen belonging to the different treatments and to the control was placed in 0.25ml insemination French straws and incubated at 6°C for two hours. The subsequent freezing was carried out in nitrogen vapours (-120°C) for ten minutes and immersed in liquid nitrogen after this period. After 7 days, the semen was thawed in a water bath at 37°C for 20 seconds, the straws dried on paper, placed on a microscope slide heated to 37°C and evaluated according to the parameters described above. Regarding the comparison between the different treatments, it was observed that the sperm viability obtained in the treatments with ascorbic acid as well as glutathione reduced, was not statistically different from the control group. Higher values of ascorbic acid and reduced glutathione reduced sperm viability after thawing. As for the use of a-tocopherol at a concentration of 400μM, the best results of the entire study were obtained, with sperm viability of 31.52% (±1.50). Regarding sperm motility and agglutination rate, a-tocopherol also showed the best results at the concentration of 200μM, in which the mean sperm motility was 2.57 ± 0.15 and 2.07 ± 0.15, respectively. The results of the present study allow us to infer that the addition of 200μM or 400μM of a-tocopherol to the swine semen-freezing medium has a positive effect on sperm viability parameters after thawing.


Author(s):  
Martina Contino ◽  
Elena Maria Scalisi ◽  
Roberta Pecoraro ◽  
Chiara Failla ◽  
Sara Ignoto ◽  
...  

Opuntia sp. contain antioxidant phytochemicals resistant to ROS damage, whose excess negatively affect fertilization. We investigate the activity of fruit extracts of O. dillenii and O. ficus indica (cv red and yellow) on sperm quality and cryopreservation. In the first experiment, we exposed the samples to extracts (50 &micro;l) for 1 hour to then evaluate semen parameters (vitality, motility, acrosome reaction, DNA fragmentation and oxidative stress). The results showed a significant increase in the motility (86%&plusmn;0.19 for OFI cv yellow, 82%&plusmn;0.15 for OFI cv red and 90%&plusmn;0.08 for O. dillenii) compared to the control (80%&plusmn;0.17). Moreover, we noted a reduction of DNA fragmentation on treated (3%&plusmn;0.03 in OFI cv yellow, 7%&plusmn;0.09 in OFI cv red and 5%&plusmn;0.07 in O. dillenii) than the control (40%&plusmn;0.14). Furthermore, the oxidative stress was reduced after exposure to solutions (3.15mV in the control and 2.94mV in the treated). In the second experiment, 50 &micro;l of solutions were added to the Freezing medium. After thawing, we observed an improvement in vitality and the number of intact acrosomes. Our results suggest that Opuntia sp. fruit extracts improve sperm quality, both before and after cryopreservation, optimizing the potential of fertilization of sperm cells.


2021 ◽  
Author(s):  
Ruixue Zhang ◽  
Hemeng Dong ◽  
Pengpeng Zhao ◽  
Chunmei Shang ◽  
Hang Qi ◽  
...  

Abstract Background: Semen cryopreservation has become an essential tool for the reproduction and long-term preservation of giant pandas (Ailuropoda melanoleuca). However, cryopreservation is severely detrimental to sperm quality, including motility, plasma membrane, acrosome integrity, and mitochondrial activity. It is necessary to screen a effective antioxidants for improvement of sperm quality. Eight groups of antioxidants were added to the freezing medium.Results:The results showed that lycium barbarum polysaccharide (LBP), Laminaria japonica polysaccharides (LJP), or goujresveratrol (RSV) added to the freezing medium significantly improved sperm motility, acrosome integrity, and mitochondrial activity during the cryopreservation process. Furthermore, the activities of glutathione and superoxide dismutase were also improved. However, the levels of reactive oxygen species and malondialdehyde in semen were notably reduced. Regarding fertility, acrosin activity was significantly increased in LBP-treated sperm. Unfortunately, sperm viability, DNA fragmentation, and hyaluronidase activity were not significantly different between the above groups. Conclusions: LBP (2.0 mg/mL) or RSV (50 μM) are the best candidate antioxidants for inclusion in the freezing medium for improving the quality of panda spermatozoa, during semen cryopreservation.


2021 ◽  
Vol 42 (5) ◽  
pp. 2959-2978
Author(s):  
Renata Vieira do Nascimento ◽  
◽  
Vanessa Alves Pereira ◽  
Priscila Silva Almeida-Monteiro ◽  
Yara Silvino Sales ◽  
...  

The study aimed to evaluate the in vitro antioxidant action of glycosaminoglycans (GAGs) from the skin of Oreochromis niloticus, and to determine their ideal concentration to supplement the sperm freezing medium of Prochilodus brevis. In experiment 1, the in vitro antioxidant properties of GAGs were verified through the analysis of DPPH, chelating ferrous ability, and total antioxidant capacity. In experiment 2, milt pools were formed, which were frozen in solution supplemented or not with different GAGs concentrations: 0 (control), 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg mL-1 (total of 10 treatments). The samples were evaluated for membrane integrity, DNA integrity, sperm morphology, and sperm kinetics. The results of experiment 1 showed that the GAGs exhibited, with the increase of the concentration, significant antioxidant action, for all the evaluated tests, mainly in the chelating ferrous ability. In experiment 2, it was observed that the increase of GAGs concentration decreased kinetic parameters (P < 0.05), however, the control and 0.5 mg mL-1 GAGs concentration showed similar results. For the other parameters (membrane integrity, DNA integrity, and sperm morphology), there was no decrease in results with the increase of GAGs concentration. In conclusion, GAGs extracted from O. niloticus skin have antioxidant action, and the concentration of 0.5 mg mL-1 was the most adequate to supplement the P. brevis sperm-freezing medium.


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