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1.
Theriogenology ; 179: 245-253, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34922135

RESUMO

Glycerol is widely used as a cryoprotectant to protect the sperm from freezing damage during cryopreservation. However, glycerol at a high concentration has toxic effects on the sperm. Therefore, we explored the effects of partially replacing glycerol with cholesterol-loaded cyclodextrin (CLC) in a cryoprotectant on protamine deficiency, in vitro capacitation, and fertilization ability of freeze-thawed Yanbian Yellow cattle sperm. We used fresh semen, control (6% glycerol), and four treatment-I, II, III, and IV (3% glycerol + 0, 0.75, 1.5, and 3 mg/mL CLC, respectively)-groups. Computer-assisted semen analysis; JC-1, CMA3, and FluoZin-3-AM staining; flow cytometry; and IVF were conducted. Replacing a portion of glycerol with 1.5 mg/mL CLC significantly improved sperm motility, viability, plasma membrane integrity, acrosome integrity, and membrane lipid disorders, mitochondrial membrane potential (MMP), capacitation, and fertilization ability (P < 0.05) compared with the control. Additionally, in group I and III, the protamine deficiency were significantly lower (P < 0.05) than in the control group. It was found that 6% glycerol has a higher degree of damage to sperm DNA integrity than 3% glycerol. Overall, this study revealed that partial replacement of glycerol with CLC can be used as a novel cryoprotection method to reduce the toxicity of glycerol and improve the quality of thawed Yanbian Yellow cattle sperm.


Assuntos
Ciclodextrinas , Preservação do Sêmen , Animais , Bovinos , Colesterol , Criopreservação/veterinária , Crioprotetores/farmacologia , Ciclodextrinas/farmacologia , Fertilização , Congelamento , Glicerol , Masculino , Protaminas/farmacologia , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides
2.
Theriogenology ; 178: 1-7, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34735977

RESUMO

The aim of the present study was to establish a vitrification protocol for ovine preantral follicles, which can retain viability after thawing and to evaluate the impact of different vitrification treatments on apoptosis and development-related gene expression. Preantral follicles were isolated from cortical slices of ovaries by the mechanical method of isolation. The isolated preantral follicles (200-300 µm) were randomly assigned into four groups. Group1 - Control Fresh preantral follicles (256 follicles); Group 2- Vitrification treatment A (259 follicles) (Vitrification solution 1 (VS1) - Fetal bovine serum (FBS)10%, Ethylene glycol (EG):1.8 M, Dimethyl sulfoxide (DMSO): 1.4 M, Sucrose-0.3 M for 4 min; VS2- FBS10%, EG:4.5 M, DMSO: 3.5 M, Sucrose:0.3 M for 45 s), Group 3 - Vitr. treatment B (235 follicles) (VS1-FBS 20%, EG:1.3 M, DMSO1.05 M for 15 min, VS2- FBS 20%, EG:2.7 M, DMSO:2.1 M for 5 min) and Group 4-Vitrification treatment C (248 follicles) (VS1-Glycerol(Gly):1.2 M for 3 min, VS2- Gly:1.2 M, EG:3.6 M for 3 min, VS3- Gly3M, EG: 4.5 M for 1 min). Preantral follicles were placed in corresponding vitrification treatments and later plunged immediately into liquid nitrogen (-196 °C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue dye exclusion method as well as for gene expression. The results showed that the low concentration of cryoprotectants (vitrification treatment B) negatively affected the viability of preantral follicles in comparison with control follicles. There was no significant difference in the viability rates among the Control (87%), Treatment A (79%) and Treatment C (75%). The percentage of viable preantral follicles (73%) derived from Treatment B was significantly decreased (P<0.05%) in comparison to that of control. The expression of apoptotic gene BAK was higher in the vitrification treatment B group. Expressions of the other apoptosis-related genes i.e. Bcl2L1, BAD, BAX, Caspase 3, and Annexin showed no significant difference among the groups. The expression pattern of development competence genes GDF-9 and BMP-15 were higher (P < 0.05) in vitrification treatment A and C, respectively. Expression of NOBOX gene was significantly increased in preantral follicles with Vitrification treatment B compared to the control group. We conclude that both the Vitrification treatment A and Treatment C were the efficient vitrification treatment methods for the vitrification of ovine preantral follicles.


Assuntos
Criopreservação , Vitrificação , Animais , Ensaios Clínicos Veterinários como Assunto , Criopreservação/veterinária , Crioprotetores/farmacologia , Etilenoglicol/farmacologia , Feminino , Expressão Gênica , Folículo Ovariano , Ovinos
3.
Theriogenology ; 177: 29-33, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34656834

RESUMO

This study investigated the effects of supplementing Lake extender with cysteamine (CYS) on rooster semen quality in cold storage and it's fertility performance. Semen samples were diluted with Lake extender supplemented with different concentrations of CYS (0, 1, 2, 4 and 8 mM) and were cooled and stored at 5 °C for a period of 46 h. Motility, membrane functionality, viability, lipid peroxidation, and mitochondria membrane potential were evaluated at 0, 23 and 46 h of storage. Fertility was assessed at 23 h of storage. Although at the beginning time (0 h), parameters were not affected, 1 mM of CYS improved (P ≤ 0.05) total motility, progressive motility and mitochondria membrane potential during 23 and 46 h storage. Moreover, 1 and 2 mM CYS improved (P ≤ 0.05) membrane functionality and viability compared to other groups. Lipid peroxidation was lower (P ≤ 0.05) in samples diluted with 1 and 2 mM CYS compared to the others. Artificial insemination with 23-hrs cooled-stored semen produced the higher (P ≤ 0.05) fertility rate in groups received 1 and 2 mM CYS compared to the control group. In conclusion, addition of 1 and 2 mM CYS to the extender could be helpful to protect rooster semen against structural and functional damages of cooling storage process.


Assuntos
Preservação do Sêmen , Sêmen , Animais , Galinhas , Crioprotetores/farmacologia , Cisteamina/farmacologia , Fertilidade , Masculino , Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides
4.
Theriogenology ; 177: 127-132, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34700069

RESUMO

ATP-binding cassette (ABC) transporters perform multiple functions in reproductive tissues. During ovarian tissue vitrification, the plasma membrane has important functions in the influx or efflux of water, and substances such as cryoprotectants and channel proteins that are required in this process. Thus, the present study aimed to verify the relative abundance of mRNA transcript of ABC transporters ABCB1, ABCG2, and MRP2 after vitrification and in vitro culture (IVC) of ovine ovarian tissue. For this study, the ovarian cortex fragments were proportioned into four groups as fresh control, vitrified control, fresh culture, and vitrified culture groups. After vitrification and in vitro culture, the ovarian tissue was evaluated using morphological procedures. Further, relative abundance of ABCB1, ABCG2, and MRP2 transporter mRNA transcripts in the ovarian cortex subjected to aforementioned treatment conditions were evaluated using qPCR. Our results showed a negative association between degenerated follicles and mRNA transcript abundances of ABCB1 and ABCG2. In addition, the percentage of growing follicles in the ovine ovarian cortex after vitrification was similar to that of the fresh control tissue without in vitro culture. The in vitro culture of fresh and vitrified tissue however, showed a significant decrease in the percentage of growing follicles. To the best of our knowledge, we believe that our data for the first time has studied the relative abundances of ABCB1 and ABCG2 mRNA transcripts in the ovine ovarian cortex. In addition, alterations of these protein channels may be indicative of a deleterious effect of osmotic stress on follicular survival during vitrification. Furthermore, these effects were detectable only after the IVC of the ovarian tissues. Nonetheless, further studies are required to investigate the functions of ABC transporters in ovine folliculogenesis, especially after in vitro culture of ovarian tissue.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Vitrificação , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Criopreservação/veterinária , Crioprotetores/farmacologia , Regulação para Baixo , Feminino , Ovinos
5.
Trop Anim Health Prod ; 53(6): 552, 2021 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-34812951

RESUMO

The present experiment was carried out with the objectives to study the effects of antioxidants superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GSH) on cryopreservation of Black Bengal buck semen. Semen ejaculates (n = 60) were collected from eight bucks by artificial vagina method and diluted with Tris citrate egg yolk glycerol extender. To study the effect of antioxidants, SOD was added @ 0, 100, and 150 IU/ml; CAT was added @ 0, 200, and 400 IU/ml while GSH was added @ 0, 1, and 2 mM of diluted semen. Semen samples were equilibrated and vapor frozen in liquid nitrogen. Semen samples were evaluated after 48 h of storage for post thaw in vitro characters such as motility, viability, functional membrane integrity, and acrosome integrity. Semen extenders supplemented with SOD @ 100 and 150 IU/ml and GSH @ 1 and 2 mM had a higher (p < 0.01) number of motile cells, viable cells, HOST reacted cells, and acrosome intact cells than their respective controls. Further, semen extenders added with catalase @ 200 and 400 IU/ml had more (p < 0.05) number of viable, HOST reacted cells and significantly higher (p < 0.01) acrosome intact sperm cells than its control group. It can be concluded that supplementation of antioxidants SOD, GSH, and CAT had a beneficial effect on cryopreservation of Black Bengal buck semen.


Assuntos
Preservação do Sêmen , Animais , Catalase , Criopreservação/veterinária , Crioprotetores/farmacologia , Suplementos Nutricionais , Feminino , Glutationa Redutase , Masculino , Sêmen , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides , Superóxido Dismutase
6.
BMC Cancer ; 21(1): 1076, 2021 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-34600510

RESUMO

BACKGROUND: Approximately 20% of MM patients harbor glucocorticoid (GC) resistance and are not responsive to therapeutic effect. Chaperoneheat-shock proteins Hsp90 is needed for ligand docking, The imbalance of Hsp90/GRα (glucocorticoid receptor α) may be an important cause of GC resistance. Recent studies have indicated that EPA could repress cancer cell growth by regulating critical influential factors in progression of cancer, consisting of resistance to drugs, chemosensitivity. The aim of the present study was to test the cytotoxic effects of EPA alone or EPA + Dexamethasone in dexamethasone-resistant MM cell (MM.1R) and investigate whether DHA can induce apoptosis and reverse acquired glucocorticoid resistance in dexamethasone-resistant MM cell (MM.1R). METHODS: Cell Counting Kit-8 (CCK-8) was used to detect the proliferation of MM.1R cells after treating with EPA alone and EPA combined with DEX. Mitochondrial membrane potential was measured by flow cytometry and GRα and Hsp90 protein expression were assessed by western blot analysis. RESULTS: EPA alone was able to inhibit cell proliferation as evidenced by CCK-8 assay and the tumor growth was remarkably suppressed by EPA + Dexamethasone, Cell apoptosis after EPA treatment was obviously observed by Flow cytometry analysis of the mitochondrial membrane potential. Analysis of Hsp90 and GRα proteins in MM.1R cells incubated with EPA revealed down-regulation of Hsp90 and up-regulation of GRα. Accordingly, the Hsp90/GRα ratio was significantly decreased with the increase of EPA concentration. CONCLUSIONS: EPA might be used as a new effective treatment for reversal of glucocorticoid-resistance in multiple myeloma.


Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ácido Eicosapentaenoico/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Receptores de Glucocorticoides/metabolismo , Apoptose/efeitos dos fármacos , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Crioprotetores/farmacologia , Dexametasona/farmacologia , Dimetil Sulfóxido/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Glucocorticoides/farmacologia , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Sincalida/farmacologia
7.
Int J Mol Sci ; 22(19)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34638994

RESUMO

The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the "solution effect damage" during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.


Assuntos
Aloenxertos/diagnóstico por imagem , Criopreservação/métodos , Veia Femoral/diagnóstico por imagem , Corantes Fluorescentes , Congelamento , Imagem Óptica/métodos , Veia Safena/diagnóstico por imagem , Aloenxertos/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Veia Femoral/efeitos dos fármacos , Humanos , Microscopia Confocal/métodos , Veia Safena/efeitos dos fármacos , Doadores de Tecidos , Enxerto Vascular/métodos
8.
An Acad Bras Cienc ; 93(4): e20190314, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34705929

RESUMO

Due to the reduction of the jaguar population, the formation of somatic cell cryobanks represents an interesting tool for its conservation. Nevertheless, the success of these cryobanks depends on the cryoprotectants used in cryopreservation. We evaluated the effects of the intracellular cryoprotectants (10% dimethyl sulfoxide, DMSO; 10% ethylene glycol, EG) in the absence or presence of an extracellular cryoprotectant (0.2 M sucrose, SUC) on the morphology, confluence, viability, and metabolism of somatic cells derived from five jaguars belonging to Brazilian zoos. The morphology was presented in a descriptive manner, while the confluence, viability and metabolic activity were presented as means and compared using statistical tests. Non-cryopreserved cells were used as control and compared to frozen/thawed cells using cryoprotectants. No difference was observed for the morphology and confluence among non-cryopreserved and cryopreserved cells, regardless of the cryoprotectants. Only cryopreserved cells in EG (45.8%±12.9) had a reduction in their viability when compared to non-cryopreserved cells (97.8%±1.1). Only cryopreserved cells in DMSO with SUC (76.0%±2.7) or absence of SUC (77.0%±3.7) maintained their metabolic activity after thawing, when compared to non-cryopreserved cells (100.0%±6.7). Therefore, combinations of DMSO in the absence and presence of SUC were efficient in the cryopreservation of somatic cells of jaguars.


Assuntos
Produtos Biológicos , Panthera , Animais , Criopreservação , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia
9.
Andrologia ; 53(11): e14224, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34496074

RESUMO

This study evaluated the effects of freezing extenders, cryoprotectants and their concentrations, presence of Orvus Es Paste and freezing rates for cryopreserving dromedary camel sperm. Semen (five males; 2 ejaculates/male) was frozen in one of the following extenders (Green Buffer® or INRA96® ), cryoprotectants (3 and 6% glycerol or ethylene glycol), with or without Orvus Es Paste and freezing at two different heights (1 and 4 cm) above liquid nitrogen. Sperm motility recovery parameters were evaluated post-thaw (0 and 1 hr), vitality and acrosome integrity (0 hr). Green Buffer showed higher total motility recoveries (p < .001). Higher percentage of cryoprotectant improved both total and progressive motility at 0 hr (p < .001; p = .003) and 1 hr (p < .001; p = .005). Acrosome integrity at thawing increased in the presence of ethylene glycol (p < .001) and Orvus Es Paste (p = .001). Kinematics were affected by extender, cryoprotectant concentration and Orvus Es Paste at 0 and 1 hr, and type of cryoprotectant only influenced them at 0h. Our findings showed strong interactions among type of cryoprotectant and concentration, and extender and Orvus Es Paste. Generally, combining Green Buffer, 3%-6% ethylene glycol or 6% glycerol without Orvus Es Paste, regardless of the freezing rates, yielded the highest post-thaw parameters for camel sperm.


Assuntos
Camelus , Preservação do Sêmen , Animais , Criopreservação , Crioprotetores/farmacologia , Suplementos Nutricionais , Congelamento , Masculino , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides
10.
ACS Biomater Sci Eng ; 7(10): 4757-4762, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34587440

RESUMO

Sorbitol, mannitol, xylitol, and erythritol, four readily available sugar alcohols with poor or no membrane permeability, are converted into their corresponding dipropionates by acylating their primary hydroxyl groups. With enhanced membrane permeability, these diesters are expected to permeate the cell membranes and, upon their hydrolysis, release the corresponding sugar alcohols inside the cells. NIH-3T3 cells incubated with these diesters before being frozen at -80 °C exhibited considerably higher total recovery over those incubated with the free sugar alcohols or media only. Among the four diesters, those of sorbitol, especially mannitol, showed cryoprotective effects comparable to that shown by 5% DMSO. This work has demonstrated the feasibility of converting readily available, naturally occurring compounds into membrane-permeable derivatives that serve as water-soluble, nontoxic alternatives to DMSO.


Assuntos
Álcoois Açúcares , Água , Animais , Criopreservação , Crioprotetores/farmacologia , Camundongos , Sorbitol
11.
Theriogenology ; 176: 18-25, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34564013

RESUMO

The objective of this study was to evaluate extender supplemented with melatonin and freezing curves on the antioxidant enzyme activity, peroxidation lipid and sperm characteristics of cryopreserved Brycon orbignyanus milt. Males (n = 16) and females (n = 5) were hormonally induced with two doses (0.5 mg and 5.0 mg kg-1) of carp pituitary extract, and their gametes were collected by light abdominal massage. The fresh milt was diluted at a ratio of 1:4 (milt:extender) in the following solutions: (Control) 10% methyl glycol (MG) + 5% Beltsville thawing solution; (M1) Control + 1 mM melatonin; and (M2) Control + 2 mM melatonin. The freezing curves were C1 (automated freezer) and C2 (dry shipper for 24 h). After each curve was recorded, the straws were transferred to a liquid nitrogen container until the analyses were performed. The samples were thawed in a water bath (60 °C, 8 s) and evaluated using the Sperm Class Analyzer software for the parameters total motility, progressive motility, curvilinear velocity, straight line velocity, mean displacement velocity, straightness, and linearity. The following were also measured: motility time, vitality, morphology, oxidative stress (lipid peroxidation, activity of catalase and superoxide dismutase, quantification of nitric oxide), and fertilization and hatching rates. The data were analyzed within R by one-way analysis of variance and Tukey's test for comparison of means (p < 0.05). A significant difference (p < 0.05) was observed between the solutions in vitality, morphology, motility, and fertilization rate, the solutions with melatonin having the best values. Total motility, progressive motility, and motility time were significantly different. Among oxidative stress markers, only lipid peroxidation and superoxide dismutase activity showed an effect of the curve × solution interaction (p < 0.05), the solutions with melatonin yielding the lowest values. The fertilization and hatching rates were also higher under the melatonin treatments, regardless of the curve. Melatonin 2 mM and slow curve are indicated for the cryopreservation of fish species sperm as it led to the slowest detrimental spermatozoa effects and better fertilization and hatching rates.


Assuntos
Caraciformes , Melatonina , Preservação do Sêmen , Animais , Antioxidantes , Criopreservação/veterinária , Crioprotetores/farmacologia , Feminino , Congelamento , Peroxidação de Lipídeos , Lipídeos , Masculino , Melatonina/farmacologia , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides
12.
Andrologia ; 53(10): e14199, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34392549

RESUMO

Post-thawing cryoinjuries in boar spermatozoa due to oxidative stress may be reduced by adding nanoencapsulated antioxidants to freezing extenders. This study evaluated post-thawing kinetics, structural and biochemical functions of boar spermatozoa frozen with extenders including resveratrol and vitamin E loaded into polymeric nanocapsules. Resveratrol was added at 0 (control), 5, 10, 20, 40 and 80 µg/ml, whereas Vitamin E was added at 0 (control), 50, 100, 200 and 400 µg/ml. Both antioxidants were tested in free and nanoencapsulated presentations. In contact with empty nanocapsules, some sperm kinetics parameters were impaired compared to the control (p < .05), whereas lipoperoxidation declined (p < .05). With inclusion of 40 µg/ml nanoencapsulated resveratrol, some sperm kinetics parameters were improved (p < .01), but sperm motility, structural and biochemical functions did not differ from the control (p > .05). No improvement in sperm quality occurred with inclusion of vitamin E, although sperm kinetics with 400 µg/ml nanoencapsulated vitamin E was reduced compared to the control (p < .01). Inclusion of 40 µg/ml nanoencapsulated resveratrol benefitted boar sperm kinetics after thawing, but no improvement resulted from inclusion of vitamin E.


Assuntos
Preservação do Sêmen , Animais , Antioxidantes/farmacologia , Criopreservação , Crioprotetores/farmacologia , Congelamento , Masculino , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides , Suínos
13.
Biomed Res Int ; 2021: 1340281, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34336999

RESUMO

The purpose of this study was to develop an efficient vitrification system for cryopreservation of dog skin tissues as a source of stable autologous stem cells. In this study, we performed vitrification using four different cryoprotectants, namely, ethylene glycol (EG), dimethyl-sulfoxide (Me2SO), EG plus Me2SO, and EG plus Me2SO plus sucrose, and analyzed the behaviors of cells established from warmed tissues. Tissues vitrified with 15% EG, 15% Me2SO, and 0.5 M sucrose had a normal histological appearance and the highest cell viability after cell isolation, and thus, this cocktail of cryoprotectants was used in subsequent experiments. We evaluated proliferation and apoptosis of cells derived from fresh and vitrified tissues. These cells had a normal spindle-like morphology after homogenization through subculture. Dog dermal skin stem cells (dDSSCs) derived from fresh and vitrified tissues had similar proliferation capacities, and similar percentages of these cells were positive for mesenchymal stem cell markers at passage 3. The percentage of apoptotic cell did not differ between dDSSCs derived from fresh and vitrified tissues. Real-time PCR analysis revealed that dDSSCs at passage 3 derived from fresh and vitrified tissues had similar expression levels of pluripotency (OCT4, SOX2, and NANOG), proapoptotic (BAX), and antiapoptotic (BCL2 and BIRC5) genes. Both types of dDSSCs successfully differentiated into the mesenchymal lineage (adipocytes and osteocytes) under specific conditions, and their differentiation potentials did not significantly differ. Furthermore, the mitochondrial membrane potential of dDSSCs derived from vitrified tissues was comparable with that of dDSSCs derived from fresh tissues. We conclude that vitrification of dog skin tissues using cocktail solution in combination of 15% EG, 15% Me2SO, and 0.5 M sucrose allows efficient banking of these tissues for regenerative stem cell therapy and conservation of genetic resources.


Assuntos
Células-Tronco Mesenquimais/citologia , Pele/citologia , Vitrificação , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Derme/citologia , Cães , Feminino , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo
14.
Cells ; 10(7)2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34359838

RESUMO

The open carrier system (OC) is used for vitrification due to its high efficiency in preserving female fertility, but concerns remain that it bears possible risks of cross-contamination. Closed carrier systems (CC) could be an alternative to the OC to increase safety. However, the viability and developmental competence of vitrified/warmed (VW) oocytes using the CC were significantly lower than with OC. We aimed to improve the efficiency of the CC. Metaphase II oocytes were collected from mice after superovulation and subjected to in vitro fertilization after vitrification/warming. Increasing the cooling/warming rate and exposure time to cryoprotectants as key parameters for the CC effectively improved the survival rate and developmental competence of VW oocytes. When all the conditions that improved the outcomes were applied to the conventional CC, hereafter named the modified vitrification/warming procedure using CC (mVW-CC), the viability and developmental competence of VW oocytes were significantly improved as compared to those of VW oocytes in the CC. Furthermore, mVW-CC increased the spindle normality of VW oocytes, as well as the cell number of blastocysts developed from VW oocytes. Collectively, our mVW-CC optimized for mouse oocytes can be utilized for humans without concerns regarding possible cross-contamination during vitrification in the future.


Assuntos
Blastocisto/citologia , Criopreservação/métodos , Fertilização In Vitro/métodos , Oócitos/citologia , Vitrificação , Animais , Biomarcadores/metabolismo , Blastocisto/metabolismo , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Caderinas/genética , Caderinas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Feminino , Expressão Gênica , Masculino , Metáfase , Camundongos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Espermatozoides/fisiologia , Sacarose/farmacologia
15.
Cells ; 10(7)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34360005

RESUMO

Adipose tissue senescence is implicated as a major player in obesity- and ageing-related disorders. There is a growing body of research studying relevant mechanisms in age-related diseases, as well as the use of adipose-derived stem cells in regenerative medicine. The cell banking of tissue by utilising cryopreservation would allow for much greater flexibility of use. Dimethyl sulfoxide (DMSO) is the most commonly used cryopreservative agent but is toxic to cells. Trehalose is a sugar synthesised by lower organisms to withstand extreme cold and drought that has been trialled as a cryopreservative agent. To examine the efficacy of trehalose in the cryopreservation of human adipose tissue, we conducted a systematic review of studies that used trehalose for the cryopreservation of human adipose tissues and adipose-derived stem cells. Thirteen articles, including fourteen studies, were included in the final review. All seven studies that examined DMSO and trehalose showed that they could be combined effectively to cryopreserve adipocytes. Although studies that compared nonpermeable trehalose with DMSO found trehalose to be inferior, studies that devised methods to deliver nonpermeable trehalose into the cell found it comparable to DMSO. Trehalose is only comparable to DMSO when methods are devised to introduce it into the cell. There is some evidence to support using trehalose instead of using no cryopreservative agent.


Assuntos
Adipócitos/efeitos dos fármacos , Tecido Adiposo/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Células-Tronco/efeitos dos fármacos , Trealose/farmacologia , Adipócitos/citologia , Adipócitos/metabolismo , Adipócitos/transplante , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Tecido Adiposo/transplante , Diferenciação Celular , Crioprotetores/metabolismo , Dimetil Sulfóxido/metabolismo , Humanos , Lipectomia/métodos , Permeabilidade , Medicina Regenerativa/métodos , Células-Tronco/citologia , Células-Tronco/metabolismo , Trealose/metabolismo
16.
Reprod Domest Anim ; 56(10): 1274-1278, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34418185

RESUMO

Artificial insemination (AI) with cryopreserved semen is an important tool to preserve endangered species, including European donkey breeds. Sperm vitrification is an alternative method to conventional freezing using high cooling rates and non-permeable cryoprotectant agents (CPAs). In donkeys, sperm vitrification was firstly developed in spheres by directly dropping the sperm (30 µl) into the liquid nitrogen. The vitrification media contained a combination of sucrose and bovine serum albumin as non-permeable CPAs and resulted in better sperm parameters after warming than extenders containing glycerol. Thereafter, sperm vitrification was optimized using an aseptic protocol, which consists of volumes up to 160 µl vitrified at 300 million sperm/ml using 0.25-ml straws with outer covers, obtaining similar sperm parameters as conventional freezing for total motility (52.7 ± 15.6% versus. 58.2 ± 16.1%), progressive motility (44.3 ± 15.0% versus. 44.7 ± 18.2%) and plasma membrane integrity (49.2 ± 11.2% versus. 55.4 ± 9.0%), respectively. In order to vitrify larger volumes of sperm, a procedure using 0.5-ml straws was evaluated; however, this methodology failed when compared to conventional freezing or other vitrification protocols, obtaining poor sperm quality after warming. Recently, a new methodology was developed for warming 0.25-ml straws in a water bath and after AI using the vitrified sperm, the uterine inflammatory response solved faster, and pregnancy rates were greater (22%) than frozen semen (10%) but not statistically different. In conclusion, all these findings confirm that sperm vitrification can be performed in donkeys as an alternative to conventional freezing for AI in jennies.


Assuntos
Criopreservação/veterinária , Equidae/fisiologia , Vitrificação , Animais , Criopreservação/métodos , Crioprotetores/farmacologia , Feminino , Congelamento , Inseminação Artificial/veterinária , Masculino , Gravidez
17.
Theriogenology ; 173: 279-294, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34411905

RESUMO

In situations where an excessive generation of reactive oxygen species overwhelms antioxidant capacity, a harmful effect on sperm function is exerted. Antioxidants are molecules capable of minimizing this detrimental effect, which is important in pig sperm due to the high content of polyunsaturated fatty acids in their plasma membrane. The present systematic review aims at evaluating whether supplementing semen extenders (for liquid storage at 17 °C) or freezing and/or thawing media (for cryopreservation) with antioxidants influences sperm quality and functionality parameters, and in vitro/in vivo fertility outcomes. We defined inclusion and exclusion criteria in a PICOS table according to PRISMA guidelines, and conducted a literature search through MEDLINE-PubMed in November 2020. After systematic selection, 75 studies were included: 47 focused on cryopreservation and 28 on liquid storage at 17 °C. More than 70% of the studies included in this review showed that adding semen extenders for liquid storage and/or freezing/thawing media for cryopreservation with antioxidants enhances sperm quality and functionality parameters. In addition, this supplementation improves in vivo/in vitro fertility outcomes, supporting the hypothesis that the beneficial effect observed upon sperm quality has a positive impact on reproduction outcomes.


Assuntos
Antioxidantes , Preservação do Sêmen , Animais , Criopreservação/veterinária , Crioprotetores/farmacologia , Congelamento , Masculino , Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides , Suínos
18.
Andrologia ; 53(10): e14202, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34363239

RESUMO

In the present study, the effect of cholesterol-loaded cyclodextrin (CLC) on the quality of low sperm doses at post-thaw was evaluated. Twenty four ejaculates (6 from each bull) were collected and split into eight aliquots. First four aliquots were diluted up to 20-, 15-, 10- and 5-million sperm/0.25 ml, and remaining four were treated with CLC at the rate of 1 mg/120 million spermatozoa, followed by dilution up to 20-, 15-, 10- and 5-million sperm/0.25 ml. The diluted semen was equilibrated, cryopreserved and evaluated post-thaw. The averages of total motility, progressive motility, average path velocity, straight linear velocity, membrane intact spermatozoa and noncapacitated spermatozoa were higher (p < .05) in CLC-treated sperm doses compared to control ones. However, the moribund spermatozoa, capacitated spermatozoa and acrosome-reacted spermatozoa were reduced (p < .05) in CLC-treated spermatozoa compared to control. The curvilinear velocity and linearity did not differ (p > .05) between control and CLC-treated sperm doses. In conclusion, treatment of spermatozoa with CLC at the rate of 1 mg/120 million spermatozoon attenuates the dilution effect and improves the quality of bovine low sperm insemination doses during cryopreservation; hence it could be a favourable cryoprotectant for preserving bovine semen at higher dilutions.


Assuntos
Ciclodextrinas , Preservação do Sêmen , Animais , Bovinos , Colesterol , Criopreservação , Crioprotetores/farmacologia , Ciclodextrinas/farmacologia , Inseminação , Masculino , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides
19.
Poult Sci ; 100(9): 101290, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34311322

RESUMO

Avian spermatozoa are highly susceptible to reactive oxygen species (ROS) produced during the cryopreservation. The aim of the current study was to investigate the antioxidant effects of resveratrol (RSV) during rooster semen cryopreservation. Changes in expression of AMP-activated protein kinase as a possible mechanism behind the beneficial effects of resveratrol were also evaluated. Semen samples were collected from ten Ross broiler breeders (52-wk) using abdominal massage, then divided into 4 equal aliquots and cryopreserved in Beltsville extender that contained different concentrations (0 µM, 0.01µM, 0.1µM, and 1µM) of RSV. higher percentage (P < 0.05) of total motility and membrane integrity was observed in RSV-0.1 compared to the other frozen groups. Moreover, higher percentage of sperm mitochondrial activity was observed in the RSV-0.01 and RSV-0.1 compared to the frozen control (P < 0.05). The lowest percentage of apoptotic like changes was found in the RSV-0.1 in comparison to the other groups (P < 0.05). RSV-0.01 and RSV-1 groups produced the lowest levels of H2O2 and O2- compared to the other frozen groups, respectively. Malondialdehyde (MDA) concentration, velocity average path (VAP), and linearity (LIN) were not affected by different concentrations of RSV (P > 0.05). We observed a dose-dependent increase in AMP-activated protein kinase expression in groups exposed to RSV. Thus, RSV-1 increased AMP-activated protein kinase phosphorylation but had no positive effects on post thaw sperm parameters. Our findings suggest that RSV-0.1 improve thawed sperm functions, and these effects might be mediated through activation of AMP-activated protein kinase.


Assuntos
Preservação do Sêmen , Sêmen , Animais , Galinhas , Criopreservação/veterinária , Crioprotetores/farmacologia , Peróxido de Hidrogênio , Masculino , Resveratrol/farmacologia , Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides
20.
Sci Rep ; 11(1): 15428, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326357

RESUMO

Cryopreservation is the only reliable method for long-term storage of biological material that guarantees genetic stability. This technique can be extremely useful for the conservation of endangered species and restock natural populations for declining species. Many factors have negatively affected the populations of high economical value shellfish in Spain and, as a result, many are declining or threatened nowadays. This study was focused on early-life stages of Venerupis corrugata, Ruditapes decussatus and Ruditapes philippinarum to develop successful protocols to enhance the conservation effort and sustainable shellfishery resources. Firstly, common cryoprotecting agents (CPAs) were tested to select the suitable permeable CPA attending to toxicity. Cryopreservation success using different combinations of CPA solutions, increasing equilibrium times and larval stages was evaluated attending to survival and shell growth at 2 days post-thawing. Older clam development stages were more tolerant to CPA toxicity, being ethylene-glycol (EG) and Propylene-glycol (PG) the least toxic CPAs. CPA solution containing EG yielded the highest post-thawing survival rate and the increase of equilibration time was not beneficial for clam larvae. Cryopreservation of trochophores yielded around 50% survivorship, whereas over 80% of cryopreserved D-larvae were able to recover after thawing.


Assuntos
Bivalves , Conservação dos Recursos Naturais/métodos , Criopreservação/métodos , Espécies em Perigo de Extinção , Pesqueiros , Larva , Frutos do Mar , Animais , Bivalves/efeitos dos fármacos , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Glicerol/farmacologia , Larva/efeitos dos fármacos , Propilenoglicol/farmacologia , Espanha
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