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1.
Trop Anim Health Prod ; 53(1): 103, 2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33417110

RESUMO

To investigate the effect of supplementation of L-arginine (AR) on sub-fertile buffalo-bulls' ejaculates, 25 ejaculates of poor motility (40 to 55%) were collected by artificial vagina from 5 buffalo-bulls and extended with Tris-yolk extender (1:10) supplemented with different concentrations of AR (0, 3, 4, 5, and 6 mM). Semen was cooled gradually to 4 °C within 2 h and incubated at 4 °C for additional 2 h. Incubated semen samples were evaluated by computer-assisted semen analysis. Results showed that addition of 5 mM AR increased (P < 0.05) total sperm motility and rapid progressive motility percentages, while decreased (P < 0.05) non-motile sperm and static sperm percentages compared with AR-free (control) extender. Increasing the AR level to 6 mM increased (P < 0.05) the percentages of sperm progressive motility and rapid and slow progressive motilities, while decreased (P < 0.05) the non-progressive sperm motility percentages compared with AR-free extender. Supplementation of 5 mM AR improved (P < 0.05) sperm straight linear, curve linear, and average path velocities (36 ± 0.13, 20.6 ± 5.3, and 33.2 ± 8.5, respectively) in comparing with control and other AR treatments. Addition of AR (5 and 6 mM) improved (P < 0.05) the percentages of vitality (89.8 ± 1.9 and 80.0 ± 3.4, respectively), normality (44.3 ± 3.6 and 44.8 ± 1.5, respectively), and functional sperm (20.4 ± 8.6 and 21.0 ± 0.61, respectively), and decreased abnormal neck and tail percentages compared with AR-free extender. All AR levels decreased (P < 0.05) the abnormal neck and tail percentages. Addition of all AR levels had no significant (P > 0.05) effect on the activity of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase in semen extender. Supplementation of Tris-yolk extender with L-arginine (5 or 6 mM) can improve sperm motility, velocity, vitality, and functional sperm and can decrease tail and neck abnormalities of sub-fertile buffalo ejaculate after 4 h incubation at cool temperature.


Assuntos
Arginina/farmacologia , Búfalos/fisiologia , Crioprotetores/farmacologia , Ração Animal/análise , Animais , Arginina/administração & dosagem , Arginina/metabolismo , Criopreservação/veterinária , Crioprotetores/administração & dosagem , Crioprotetores/metabolismo , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Masculino , Sêmen/efeitos dos fármacos , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos
2.
AAPS PharmSciTech ; 21(3): 108, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32215761

RESUMO

The oral bioavailability of therapeutic proteins is limited by the gastrointestinal barriers. Encapsulation of labile proteins into nanoparticles is a promising strategy. In order to improve the stability of nanoparticles, lyophilisation has been used to remove water molecules from the suspension. Although various cryoprotections were employed in the preparation of lyophilised nanoparticles, the selection of cryoprotectant type and concentration in majority of the developed formulation was not justified. In this study, nanoparticles were fabricated by cationic chitosan and anionic Dz13Scr using complex coacervation. The effect of cryoprotectant types (mannitol, sorbitol, sucrose and trehalose) and their concentrations (1, 3, 5, 7, 10% w/v) on physiochemical properties of nanoparticles were measured. Cellular assays were performed to investigate the impact of selected cryoprotectant on cytotoxicity, glucose consumption, oral absorption mechanism and gastrointestinal permeability. The obtained results revealed that mannitol (7% w/v) could produce nanoparticles with small size (313.2 nm), slight positive charge and uniform size distribution. The addition of cryoprotectant could preserve the bioactivity of entrapped insulin and improve the stability of nanoparticles against mechanical stress during lyophilisation. The gastrointestinal absorption of nanoparticles is associated with both endocytic and paracellular pathways. With the use of 7% mannitol, lyophilised nanoparticles induced a significant glucose uptake in C2C12 cells. This work illustrated the importance of appropriate cryoprotectant in conservation of particle physiochemical properties, structural integrity and bioactivity. An incompatible cryoprotectant and inappropriate concentration could lead to cake collapse and formation of heterogeneous particle size populations.


Assuntos
Diabetes Mellitus/tratamento farmacológico , Insulina/química , Nanopartículas/química , Oligonucleotídeos/química , Animais , Crioprotetores/administração & dosagem , Estabilidade de Medicamentos , Liofilização/métodos , Insulina/administração & dosagem , Polímeros/química
3.
Anim Reprod Sci ; 213: 106259, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31987317

RESUMO

Egg yolk (EY) is conventionally used to reduce sperm cryodamage, however, there has not be evaluation of whether there is a dose-dependent effect with inclusion of EY in semen extender. To enhance the knowledge about the protective effect of EY during cryopreservation of dog semen, a specific study was designed to evaluate the dose-dependent protection of the EY against osmotic and cryogenic damage of dog sperm. In the first experiment, sperm stored in an extender that contained graded EY concentrations (0 %, 5 %, 10 %, and 20 %) were diluted with hypo- or hyper-osmotic solutions (final osmolality of 75, 150, 300, 500, 1000 mOsm/kg). Results from sperm kinetic, membrane integrity (MI), mitochondrial activity, and normal morphology evaluations indicated osmotic stress has especially marked effects on the kinetic capacity of spermatozoa, however, there were no direct effects on mitochondrial activity. In both hypo- and hyper-osmotic conditions, EY had a protective effect regardless of concentration. In the second experiment, semen samples were diluted in extenders at increasing EY concentrations (0 %, 5 %, 10 %, and 20 %) and cryopreserved. Effects on sperm kinetics, membrane and acrosome integrity and mitochondrial membrane potential indicated there was improved sperm viability after thawing when the EY concentration was 5 % and 10 %, and lesser viability when it was 20 %. These results indicate, for the first time, that EY reduces osmotic and cryogenic damage when used at 5 % or 10 % concentrations, and that these concentrations can be used to protect dog spermatozoa more effectively than the conventionally used concentration (20 %).


Assuntos
Crioprotetores/farmacologia , Cães/fisiologia , Gema de Ovo , Pressão Osmótica/efeitos dos fármacos , Preservação do Sêmen/veterinária , Espermatozoides/efeitos dos fármacos , Animais , Membrana Celular , Crioprotetores/administração & dosagem , Feminino , Masculino , Análise do Sêmen , Espermatozoides/fisiologia
4.
Anim Reprod Sci ; 207: 107-117, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31204090

RESUMO

Nerve growth factor-ß (NGF) is a seminal plasma protein associated with improved sperm membrane integrity and motility in mammalian species. The objective of this study was to compare post-thaw semen quality from both ejaculated and pididymal-collected bull sperm incubated with purified NGF prior to cryopreservation. Semen was obtained from Angus × Simmental crossbred bulls (n = 10) collected by electroejaculation, followed by castration and epididymal sperm collections 3 days later. Semen samples were incubated with extender having 0 ng/mL (CONT), 0.5 ng/mL (LOW), 5 ng/mL (MED), or 50 ng/mL (HIGH) of purified NGF prior to cryopreservation. Sperm motility was assessed in each sample prior to treatment and cryopreservation and at post-thaw. Flow cytometry was used for post-thaw assessment of sperm viability (SYBR-14/PI), acrosome integrity (FITC-PNA/PI), and chromatin stability (acridine orange). Values for post-thaw sperm motility and velocity variables were decreased, while linearity was increased in samples of the HIGH compared with CONT group (P < 0.01), but there were no differences in epididymal samples (P> 0.05). Samples from the HIGH group also had a lesser amplitude of lateral head displacement at 2.5 and 3 h post-thaw (P < 0.01). Post-thaw sperm viability, acrosome integrity, and DNA fragmentation index were not affected by NGF treatment in either ejaculated or epididymal sperm (P> 0.05). In conclusion, supplementation of freezing extender with NGF had minimal effects on post-thaw sperm quality in bulls. Results indicate NGF may have a function in preventing premature sperm hyperactivation in ejaculated, but not epididymal-collected spermatozoa. Fertility studies, both in vitro and in vivo, are warranted to ascertain the relevancy of these findings.


Assuntos
Bovinos , Criopreservação , Crioprotetores/farmacologia , Fator de Crescimento Neural/farmacologia , Preservação do Sêmen , Sêmen/efeitos dos fármacos , Animais , Criopreservação/métodos , Criopreservação/veterinária , Crioprotetores/administração & dosagem , Combinação de Medicamentos , Ejaculação/fisiologia , Estimulação Elétrica , Congelamento/efeitos adversos , Masculino , Fator de Crescimento Neural/administração & dosagem , Sêmen/fisiologia , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Recuperação Espermática
5.
Skin Res Technol ; 25(4): 461-468, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30623998

RESUMO

BACKGROUND: Nipple pain is the second most common reason for early weaning, exceeded only by the insufficient milk supply. Nipple fissures can bring other problems, acting also as a portal for bacteria and leading to mastitis. This work proposes the breast protector composite development using materials with tissue repair and moisturizing properties, aligned with a low-cost procedure, aiming not only to relieve pain, but also to heal the nipple fissures caused by breastfeeding. MATERIALS AND METHODS: For the dressings, production was used Natural Latex extracted from the rubber tree and glycerol. The Samples were evaluated chemically and physically by the techniques of Scanning Electron Microscopy, Fourier transform infrared spectroscopy, mechanical traction, and contact angle. The samples were also biologically evaluated by the hemolytic and cytotoxic activity assays. RESULTS: From the physical-chemical assays, the matrix with glycerol has high pore density; the natural latex and glycerol do not covalently interact, indicating that the glycerol can be released; the glycerol addition makes the matrix more elastic but fragile, and increase the wettability. From the biological assays, both materials showed no hemolytic effects; and the cytotoxicity results showed that glycerol did not present cytotoxicity in the fibroblasts, but show a dose-dependent influence in the keratinocytes. CONCLUSION: The material developed for application in breast fissures has mechanical properties similar to those found for materials for dermal applications, present high wettability and pore density. Furthermore, the material showed no cytolytic activity and the tests with skin cell cultures demonstrated the biocompatibility.


Assuntos
Bandagens/tendências , Aleitamento Materno/efeitos adversos , Mamilos/patologia , Dor/prevenção & controle , Bandagens/normas , Materiais Biocompatíveis/química , Crioprotetores/administração & dosagem , Crioprotetores/química , Feminino , Glicerol/administração & dosagem , Glicerol/química , Humanos , Látex/química , Teste de Materiais/métodos , Microscopia Eletrônica de Varredura , Mamilos/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/patologia , Espectroscopia de Infravermelho com Transformada de Fourier , Cicatrização/efeitos dos fármacos
6.
Zygote ; 27(1): 17-24, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30516456

RESUMO

SummaryThe aim of this study was to compare different concentrations of soy lecithin (LEC0.01%, LEC0.05% and LEC0.1%) with egg yolk (Control) in cooling extenders during the storage of semen at 5ºC for 5 days. Twelve dogs (n = 12) were selected, and semen was cooled and assessed after 2, 24, 48, 72, 96 or 120 h. At each time point, sperm were analyzed for kinetic patterns (using computer-assisted sperm analysis), mitochondrial activity (3'3- diaminobenzidine assay), lipid peroxidation (TBARS assay), DNA fragmentation (SCSA®) and plasma and acrosome membrane integrity (eosin/nigrosin and fast green/rose Bengal stains, respectively). The Control group (1814.4 ± 197.2) presented the highest rates of lipid peroxidation at 120 h. Conversely, progressive motility (42.8 ± 4%), linearity (45.4 ± 1%), and VAP (88 ± 3%) were higher in the Control group. In addition, there was lower mitochondrial activity in the Control group at 72 h. Therefore, our data show that lecithin used at these concentrations was not able to maintain sperm viability at as high qualities as would egg yolk. Moreover, the decrease in high mitochondrial activity and the persistence of sperm motility may indicate a compensatory mechanism in canine spermatozoa (i.e., glycolytic pathway). Furthermore, these higher lipid peroxidation indexes could indicate the necessity for future therapy using extenders and antioxidants over a long cooling time for dog sperm.


Assuntos
Gema de Ovo/química , Lecitinas/farmacologia , Preservação do Sêmen/métodos , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Animais , Crioprotetores/administração & dosagem , Crioprotetores/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Lecitinas/administração & dosagem , Masculino , Mitocôndrias/efeitos dos fármacos , Soja/química , Motilidade Espermática
7.
Cryobiology ; 85: 25-32, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30312592

RESUMO

Microinjection has proven useful for introduction of low-permeability cryoprotective agents (CPAs) into fish eggs or embryos for cryopreservation. In this work, we examined the suitable conditions for single or combined microinjection into the perivitelline space (PS) and the yolk mass (YM) of embryos of the Japanese whiting, an alternative marine fish model for embryo cryopreservation studies. The parameters examined were injection volume, CPA type and concentration, vehicle (diluent), and suitable developmental stage. Somites and tail elongation embryos tolerated single or combined injection with 2.1 and 15.6 nl in the PS and YM, respectively, whereas earlier embryonic stages tolerated only up to 8.2 nl in the YM. The injected solutions diffused rapidly throughout the PS and YM and remained contained within each compartment unless in the case of structural damage caused by injection of larger volumes. Yamamoto solution was marginally better as a vehicle for microinjection of CPAs than fish Ringer and phosphate buffer saline whereas » artificial sea water was clearly unsuitable. Ethylene glycol was well tolerated by embryos in all developmental stages whereas 1, 2-propylene glycol was suitable only for early embryonic stages. Overall, microinjection was efficient in delivering high loads of CPAs inside whiting embryos more swiftly than previously obtained for this species by immersion-based impregnation protocols. Embryos microinjected with CPAs showed a decrease in embryo nucleation temperature and an increase in chilling tolerance. CPA-microinjected embryos will provide valuable materials to optimize the remaining parameters that are critical for successful cryopreservation such as cooling and warming strategies.


Assuntos
Criopreservação/métodos , Crioprotetores/administração & dosagem , Desenvolvimento Embrionário/efeitos dos fármacos , Peixes , Microinjeções/métodos , Animais , Embrião não Mamífero/efeitos dos fármacos
8.
Br J Neurosurg ; 32(6): 657-660, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30295552

RESUMO

Background: One of the challenges faced by the modern-day NHS is workplace shortages, and experienced radiographers for intra-operative neurosurgical imaging is one such scenario. We describe our method for Percutaneous Retrogasserian Glycerol Rhizotomy (PRGR) using frameless neuronavigation which can be used effectively in such scenarios.Method: Stealth neuronavigation is used for needle placement within the foramen ovale and injection of glycerol, under sedation.Results: In our experience of ten procedures, it is accurate, safe and effective. Good results were obtained on all occasions. It can be repeated as often as necessary using the same Stealth® CT scan and reduces exposure for staff and patients, where repeated injections are required.Conclusion: This simple modification of PRGR technique is effective and safe provided the surgeon has previous experience in undertaking this procedure.


Assuntos
Crioprotetores/administração & dosagem , Glicerol/administração & dosagem , Neuronavegação/métodos , Rizotomia/métodos , Neuralgia do Trigêmeo/cirurgia , Idoso , Humanos , Injeções , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios X , Resultado do Tratamento
9.
Int J Pharm ; 552(1-2): 27-38, 2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30236648

RESUMO

This work investigates the impact of nanoparticle (NP) composition and effectiveness of cryo-/lyo-protectants in a freeze drying process, which was employed to convert liquid dispersions of polyelectrolyte complex (PEC) NPs into completely redispersible powders. PEC NPs, with and without peptide, were produced by complex coacervation. The cryo-/lyo-protectants investigated were mannitol, trehalose (TRE) and poly(ethylene glycol) (PEG). The solid state of lyophilised powders was studied by thermal analysis and X-ray diffraction. Cytotoxicity studies were done by MTS assay and flow cytometry. The presence of a cryoprotectant was essential to achieve a successful powder reconstitution. The concentration of TRE was optimised for each type of PEC NPs. Protamine- and hyaluronate-based NPs reconstituted better than chitosan- and chondroitin sulphate-based NPs, respectively. PEG polymers were found to be more effective cryoprotectants than TRE and best results were achieved using co-freeze drying of NPs with TRE and PEG. These ternary NPs/TRE/PEG samples were crystalline, with expected better storage stability. PEG polymers were well tolerated by Caco-2 cells, with the exception of linear PEG 10 kDa. This work shows that, as regards the formulation design and maximising NP loading in the dried product, optimisation of the cryoprotectant type and content is needed as it is highly dependent not only on the type of polyelectrolyte pair in the PEC, but also the polyions ratio.


Assuntos
Quitosana/química , Sulfatos de Condroitina/química , Crioprotetores/química , Ácido Hialurônico/química , Nanopartículas/química , Protaminas/química , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Quitosana/administração & dosagem , Sulfatos de Condroitina/administração & dosagem , Crioprotetores/administração & dosagem , Liofilização , Humanos , Ácido Hialurônico/administração & dosagem , Nanopartículas/administração & dosagem , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Protaminas/administração & dosagem , Trealose/administração & dosagem , Trealose/química
10.
Anim Reprod Sci ; 197: 162-169, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30150093

RESUMO

The objective of the present study was to investigate the effects of two different concentrations of dissolved oxygen (DO, 4 and 8) ppm in the extender on oxidative stress affecting plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and deoxyribonucleic acid (DNA) damage of bull spermatozoa following cryopreservation. For the experiment, nitrogen (N2) gassing of the extender for varied time intervals yielded extender with DO concentration of 4 ppm and 8 ppm (Groups II and III, respectively). For the Control (Group I) without N2 gassing, a DO concentration of 11.7 ppm was recorded. Following sample selection, ejaculates were divided into three aliquots and were extended to have 80 × 106 spermatozoa/mL of extender in the three groups. Semen samples were evaluated for reactive oxygen species (ROS), lipid peroxidation (LPO), total antioxidant capacity (TAC) and superoxide dismutase (SOD) at the fresh, pre-freeze, and post-thaw stages. Evaluation of PMI, MMP, and DNA damage were conducted on frozen-thawed samples. There were greater (P < 0.05) increase in ROS and LPO and decrease in TAC concentrations in Group I than Groups II and III. Mean values of SOD at the post-thaw stage was greater (P < 0.05) in Group II than Group I. There was a similar trend in the PMI in Groups II and III; MMP and DNA integrity in Group II was greater compared with Group I. In conclusion, results indicate there was a beneficial effect of maintaining DO concentrations at 4 rather than of 8 or 11.7 ppm in extender for sustaining post-thaw semen quality.


Assuntos
Bovinos , Criopreservação/veterinária , Crioprotetores/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Preservação do Sêmen/veterinária , Animais , Criopreservação/métodos , Masculino , Nitrogênio/farmacologia , Oxigênio/farmacologia , Sêmen , Análise do Sêmen , Preservação do Sêmen/métodos , Motilidade Espermática , Espermatozoides
11.
Prog Urol ; 28(7): 382-386, 2018 Jun.
Artigo em Francês | MEDLINE | ID: mdl-29526581

RESUMO

INTRODUCTION: The intravesical instillation of dimethyl sulfoxide (iDMSO), performed without anesthestic, is a therapeutic option for the painful bladder syndrome/interstial cystitis (PBS/IC). Some patients are against those iDMSO because of bad tolerance. Our study evaluates the tolerance and the outcome of the iDMSO under general anesthetic (GA) after the failure of the iDMSO without anesthetic. PATIENTS AND METHODS: From May 2013 to April 2016, 11 patients with a PBS, 9 women (81.8 %), have been treated by iDMSO without anesthetic, without improvement because of bad tolerance and no possibility to have a one hour contact between the bladder and the DMSO. The 11 patients were evaluated by mictional calendar and Sant O'Leary score. All the patients had a hydrodistension and a per os treatment without improvement. OUTCOMES: Six new iDMSO were performed under general anesthetic in ambulatory surgery with good tolerance for the 11 patients. The frequency and the nocturia before iDMSO without anesthetic and after iDMSO under general anesthetic were 32.2minutes [15; 60] and 6.3 per night [3; 10] and 126.9minutes [25; 240] and 3 per night [2; 6], so a variation respectively of 96.4minutes [0; 180] and of 3.75 per night [2; 6]. The symptom score and the problem index were 17.5 [13; 20] and 15.5 [13; 16] before and 13.5 [4; 20] and 12 [1; 16] after iDMSO under general anesthetic; a variation of 3.2 [0; 9] and 4 [0; 12]. CONCLUSION: The iDMSO under general anesthetic seems to improve objectively and subjectively the patients who are not improved by the instillations without anesthetic because of bad tolerance. LEVEL OF EVIDENCE: 4.


Assuntos
Anestesia Geral/métodos , Crioprotetores/administração & dosagem , Cistite Intersticial/tratamento farmacológico , Dimetil Sulfóxido/administração & dosagem , Administração Intravesical , Adulto , Crioprotetores/efeitos adversos , Dimetil Sulfóxido/efeitos adversos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
12.
Anim Reprod Sci ; 195: 345-354, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31262406

RESUMO

The effects of adding egg yolks (EY) of different avian species to the Tris glycerol extender was evaluated for post-thawing quality of buck semen. The semen samples collected from seven Aardi bucks were pooled and diluted 1:4 with the Tris glycerol diluents containing the egg yolks of chicken (C), pigeon (P), goose (G), Japanese quail (Q), duck (D), or turkey (T). The diluted semen samples were gradually cooled to 5 °C within 2 h, equilibrated at 5 °C for 2 h, and then frozen in liquid nitrogen vapor for 8 min before storing them at -196 °C. The frozen straws were thawed at 37 °C for 30 s and evaluated for sperm motility, vitality, abnormality, plasma-membrane integrity, and DNA fragmentation. The malondialdehyde (MDA) and reduced glutathione (GSH) activities were measured. The egg yolk samples of the above-mentioned avian species (n = 7/species) were analyzed for moisture, ash, protein, fatty acid (FA), and trace element contents. The use of the chicken EY (C) diluent resulted in better post-thawing buck semen quality, including the total progressive sperm motility parameters, sperm vitality, plasma membrane intactness, DNA integrity, and sperm abnormalities percentages, in comparison with the EY diluents of the other avian species. The lowest MDA and the highest GSH activities were observed in the C diluent. The chicken EY had the highest percentages of margaric and linolenic FAs and the lowest percentages of palmitoleic and myristic FAs. Moreover, the percentage of oleic FA was lower in the chicken EY than in the EYs of other species, except turkey. Additionally, the chicken EY had the significantly lowest concentration of Cu, Zn, Fe, and Mn. In conclusion, the use of chicken egg yolk extender is recommended for buck semen cryopreservation. The egg yolks of the other avian species studied cannot substitute the chicken EY as they gave poorer post-thawing semen quality. The EY composition, especially FA profile and trace elements concentration, significantly affected the post-thawing quality of buck semen.


Assuntos
Criopreservação/veterinária , Crioprotetores/química , Gema de Ovo/química , Glicerol/química , Controle de Qualidade , Preservação do Sêmen/veterinária , Espermatozoides/fisiologia , Animais , Membrana Celular , Galinhas , Columbidae , Coturnix , Criopreservação/métodos , Crioprotetores/administração & dosagem , Patos , Congelamento , Glicerol/administração & dosagem , Cabras , Masculino , Preservação do Sêmen/métodos , Motilidade Espermática
13.
Theriogenology ; 103: 83-89, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28779613

RESUMO

It was hypothesized that dimethyleacetamide (DMA) can be used as an alternate to glycerol for cryopreservation of Indian red jungle fowl semen. Four concentrations of DMA (4%, 6%, 8% and 10%) in extender were compared with previously optimized cryopreservation protocol based on 20% glycerol (control) for Indian red jungle fowl. Sperm motility, plasma membrane integrity, viability, and acrosome integrity were assessed at the stage of post-dilution, cooling, equilibration, and freeze-thawing. The whole experiment was repeated/replicated for five times independently. Sperm motility, plasma membrane integrity, viability and acrosome integrity were recorded highest (P < 0.05) at post-dilution, cooling, equilibration, and freeze-thawing in extender having 6% DMA compared to control and other experimental extenders. The highest (P < 0.05) recovery rates of all aforementioned parameters were also recorded in extender having 6% DMA; thus, 6% DMA was further compared with control (20% glycerol) for fertility after artificial insemination. Eggs were collected for five days after artificial insemination with semen cryopreserved in extender containing 6% DMA and control. The higher no. of fertilized eggs, fertility, no. of hatched eggs, hatch (%) and hatchability were recorded with semen cryopreserved in extender having 6% DMA compared to control. It is concluded that 6% DMA maintained higher post-thaw quality and fertility of Indian red jungle fowl semen and is a better replacement of glycerol.


Assuntos
Acetamidas/farmacologia , Criopreservação/veterinária , Crioprotetores/farmacologia , Espermatozoides/efeitos dos fármacos , Acetamidas/administração & dosagem , Animais , Galinhas , Crioprotetores/administração & dosagem , Relação Dose-Resposta a Droga , Fertilidade , Congelamento , Masculino , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos , Motilidade Espermática/efeitos dos fármacos
14.
Cryobiology ; 77: 19-24, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28602768

RESUMO

Cryopreservation of fish embryos requires the swift uptake of considerable amounts of cryoprotectant (CPA) but this process is hampered by the low permeability of the egg chorion. This study examined the relative efficiency of ultrasound to promote the incorporation of CPAs in two different embryonic developmental stages (somites and tail elongation) of Japanese whiting Sillago japonica and performed a preliminary cryopreservation trial using the best conditions determined during the study. Embryos tolerated ultrasound densities up to 37.5 W/cm2 well for up to 3 min but had significant mortality at 50 W/cm2. Hatching rates of somites embryos sonicated at 37.5 W/cm2 for 1-3 min in 10 and 20% Me2SO solutions were comparable (61-72%) to that of sonication in artificial seawater (65-86%) but decreased sharply at the concentration of 30% (0-55%); at similar conditions, tail elongation embryos had comparatively lower survival. Me2SO content of sonicated embryos at the somites and tail elongation stages increased significantly by 58-191% and 27-123%, respectively, compared to controls exposed to Me2SO without ultrasound. Pre-exposure to Me2SO before sonication increased the CPA uptake further by 36% without impairing survival. A preliminary cryopreservation trial after ultrasound-mediated impregnation of somites embryos with a CPA solution containing 20% PG and 10% MeOH did not yield live embryos after freeze-thawing but resulted in a significant decrease of nucleation temperature and increase of the proportion of morphologically intact embryos after freeze-thawing. These results suggest that sonication might be useful for fish embryo cryopreservation although it may require combination with other techniques to enhance CPA permeation.


Assuntos
Criopreservação/métodos , Crioprotetores/administração & dosagem , Dimetil Sulfóxido/administração & dosagem , Embrião não Mamífero , Ondas Ultrassônicas , Animais , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Peixes , Congelamento , Metanol/administração & dosagem , Metanol/farmacologia , Perciformes , Permeabilidade , Propilenoglicol/administração & dosagem , Propilenoglicol/farmacologia
15.
J Cardiovasc Pharmacol ; 69(6): 382-388, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28581447

RESUMO

Cold cardioplegia is used to induce heart arrest during cardiac surgery. However, endothelial function may be compromised after this procedure. Accordingly, interventions such as adenosine, that mimic the effects of preconditioning, may minimize endothelial injury. Herein, we investigated whether adenosine prevents cold-induced injury to the endothelium. Cultured human cardiac microvascular endothelial cells were treated with adenosine for different durations. Phosphorylation and expression of endothelial nitric oxide synthase (eNOS), p38MAPK, ERK1/2, and p70S6K6 were measured along with nitric oxide (NO) production using diaminofluorescein-2 diacetate (DAF-2DA) probe. Cold-induced injury by hypothermia to 4°C for 45 minutes to mimic conditions of cold cardioplegia during open heart surgery was induced in human cardiac microvascular endothelial cells. Under basal conditions, adenosine stimulated NO production, eNOS phosphorylation at serine 1177 from 5 minutes to 4 hours and inhibited eNOS phosphorylation at threonine 495 from 5 minutes to 6 hours, but increased phosphorylation of ERK1/2, p38MAPK, and p70S6K only after exposure for 5 minutes. Cold-induced injury inhibited NO production and the phosphorylation of the different enzymes. Importantly, adenosine prevented these effects of hypothermic injury. Our data demonstrated that adenosine prevents hypothermic injury to the endothelium by activating ERK1/2, eNOS, p70S6K, and p38MAPK signaling pathways at early time points. These findings also indicated that 5 minutes after administration of adenosine or release of adenosine is an important time window for cardioprotection during cardiac surgery.


Assuntos
Adenosina/administração & dosagem , Temperatura Baixa/efeitos adversos , Crioprotetores/administração & dosagem , Células Endoteliais/efeitos dos fármacos , Hipotermia Induzida/efeitos adversos , Lesões do Sistema Vascular/prevenção & controle , Células Cultivadas , Citoproteção , Esquema de Medicação , Células Endoteliais/enzimologia , Células Endoteliais/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Lesões do Sistema Vascular/enzimologia , Lesões do Sistema Vascular/etiologia , Lesões do Sistema Vascular/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
16.
Biomed Res Int ; 2017: 7279341, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28523277

RESUMO

The objective of this study was to investigate the effect of L-carnitine (LC), hypotaurine (HT), and taurine (T) on the quality of frozen-thawed chicken semen. Pooled semen samples were divided into seven aliquots (control, 1 mM LC, 5 mM LC, 1 mM HT, 10 mM HT, 1 mM T, and 10 mM T) and subjected to cryopreservation. Postthaw sperm motility was determined by IVOS system and sperm characteristics were assessed with fluorochromes and flow cytometry. The highest sperm motility and the highest percentage of viable sperm were in the HT1 group (P < 0.01 and P < 0.05) following cryopreservation. After thawing, we observed a higher percentage of sperm without apoptosis and membrane reorganization changes in the LC1 and T1 group when compared to the control (P < 0.05). There was a higher percentage of live sperm without lipid peroxidation (LPO) in all treatments (P < 0.01; P < 0.05), when compared to the control group. The percentage of sperm with high mitochondrial potential significantly increased with LC1, T1, and T10 (P < 0.05). Supplementation of the diluent with LC1, LC5, and T1 significantly (P < 0.05) reduced DNA susceptibility to fragmentation, compared to the control and HT1 groups. These results indicate that the addition of examined antioxidants improves the quality of cryopreserved chicken semen.


Assuntos
Carnitina/administração & dosagem , Crioprotetores/administração & dosagem , Sêmen/efeitos dos fármacos , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Taurina/análogos & derivados , Taurina/administração & dosagem , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Galinhas , Criopreservação/métodos , Suplementos Nutricionais , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Sêmen/metabolismo , Análise do Sêmen/métodos , Preservação do Sêmen/métodos , Espermatozoides/metabolismo , Superóxido Dismutase/metabolismo
17.
Andrologia ; 49(9)2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28295478

RESUMO

To investigate the effects of different concentrations of various cryoprotectants (CPs) on the cell viability as well as expression of spermatogenesis-related genes, such as CREM, Stra8 and HSP70-2 in frozen-thawed bovine calf testicular tissue, immature bovine (Qinchuan cattle) calf testicular tissue was collected and cryopreserved in the cryomedia containing different concentrations (5%, 10%, 15% and 20%) of the following three CPs: glycerol, ethylene glycol (EG) and dimethyl sulphoxide (DMSO) respectively. After 1 month cryopreservation in liquid nitrogen, cell viability was evaluated using Trypan blue exclusion under a bright-field microscope. The mRNA expression of the three genes was also evaluated using qRT-PCR. The results indicated that different concentrations of glycerol, EG and DMSO in cryomedia during cryopreservation could protect bovine calf testicular tissue in various ways to avoid freezing or cryopreservation-induced expression changes in spermatogenesis-related genes. The highest cell viability and the three spermatogenesis-related genes (CREM, Stra8 and HSP70-2) expression level came from the cryomedia containing glycerol, EG and DMSO at 10% concentration respectively (p < .05). Meanwhile, compared with the other CPs, the frozen-thawed bovine calf testicular tissue treated with 10% DMSO exhibited the highest cell viability and mRNA expression level of the spermatogenesis-related genes (CREM, Stra8 and HSP70-2).


Assuntos
Bovinos , Criopreservação , Crioprotetores/administração & dosagem , Testículo , Animais , Sobrevivência Celular , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Masculino
18.
Reprod Fertil Dev ; 29(11): 2140-2148, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28241901

RESUMO

Cryopreservation is an effective method for the long-term storage of valuable germplasm in the field of reproductive research. The present study examined the developmental capacity of post-thaw bovine blastocysts during vitrification after supplementation with antifreeze glycoprotein 8 (AFGP8). Survival and re-expansion rates in culture during the 12h after thawing were significantly higher in the AFGP8-treated than untreated group. In addition, blastocysts from the AFGP8-treated group exhibited lower rates of apoptosis. Real-time reverse transcription-polymerase chain reaction analysis showed that the expression of the Bcl-2 gene, coding for an anti-apoptotic protein, was increased significantly, whereas the expression of the pro-apoptotic gene Bax was decreased significantly in the AFGP8-treated group. The cellular proliferation rate and mitochondrial membrane potential were significantly higher in post-thaw re-expanded blastocysts from the AFGP8-treated compared with untreated group. In addition, outgrowth potential in post-thaw blastocysts in re-expanded blastocysts after vitrification was significantly increased in the AFGP8-treated compared with untreated group. Together, these results are the first to demonstrate that the addition of AFGP8 during vitrification can help protect bovine blastocysts against chill-induced injury.


Assuntos
Proteínas Anticongelantes/administração & dosagem , Blastocisto/efeitos dos fármacos , Criopreservação/veterinária , Crioprotetores/administração & dosagem , Fertilização In Vitro/veterinária , Vitrificação , Animais , Blastocisto/metabolismo , Bovinos , Criopreservação/métodos , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Feminino , Fertilização In Vitro/métodos , Potencial da Membrana Mitocondrial/efeitos dos fármacos
19.
Reprod Fertil Dev ; 29(11): 2235-2244, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28356183

RESUMO

The aim of the present study was to develop a protocol for the successful cryopreservation of Saltwater crocodile spermatozoa. Sperm cells were frozen above liquid nitrogen vapour in phosphate-buffered saline (PBS) containing either 0.3M trehalose, 0.3M raffinose or 0.3M sucrose and compared with glycerol (0.3-2.7M). Although the highest levels of mean post-thaw motility were observed following cryopreservation in 0.3M trehalose (7.6%) and 0.3M sucrose (7.3%), plasma membrane integrity (PI) was best following cryopreservation in 2.7M glycerol (52.5%). A pilot study then assessed the cytotoxicity of glycerol and sucrose prior to cryopreservation and revealed no loss of survival when spermatozoa were diluted in 0.68M glycerol or 0.2-0.3M sucrose once cryoprotectants were washed out with PBS or Biggers, Whitten and Whittingham medium containing sperm capacitation agents (BWWCAP). A final study refined the combined use of permeating (0.68 or 1.35M glycerol) and non-permeating (0.2 or 0.3M sucrose) cryoprotectants. Spermatozoa were cryopreserved in liquid nitrogen vapour at rates of approximately -21°Cmin-1 (fast freeze) or -6.0°Cmin-1 (slow freeze). Post-thaw survival was highest with a combination of 0.2M sucrose and 0.68M glycerol and when these cryoprotectants were washed out with BWWCAP, regardless of whether spermatozoa were frozen using a fast (motility 14.2±4.7%; PI 20.7±2.0%) or slow (motility 12.0±2.7%; PI 22±4%) cryopreservation rate.


Assuntos
Jacarés e Crocodilos , Criopreservação/métodos , Preservação do Sêmen/métodos , Espermatozoides , Animais , Crioprotetores/administração & dosagem , Masculino , Motilidade Espermática , Sacarose/administração & dosagem , Trealose/administração & dosagem
20.
Int Urogynecol J ; 28(7): 1085-1089, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27987022

RESUMO

INTRODUCTION AND HYPOTHESIS: For decades, intravesical dimethyl sulfoxide (DMSO) cocktail therapy has been used for the treatment of interstitial cystitis/bladder pain syndrome (IC/BPS), but little is known about its long-term efficacy. We aimed to assess the long-term efficacy of intravesical DMSO/heparin/hydrocortisone/bupivacaine therapy in patients with IC/BPS. METHODS: Patients with IC/BPS from our institutions who underwent this therapy with >2 years follow-up were surveyed with O'Leary-Sant interstitial cystitis symptom and problem index questionnaires before and after therapy. Chart reviews and telephone surveys were then conducted to determine their posttherapy course. RESULTS: Of 68 eligible women, 55 (80.0%) with a median follow-up of 60 months (range 24-142) were surveyed. Their mean age at therapy onset was 44.8 years and their mean body mass index was 26.2 kg/m2. There were statistically significant improvements in O'Leary-Sant and pain scores of 23-47% at both 6 weeks and the end of the follow-up period. At the end of the follow-up period, 19 of the 55 women (34.5%) were cured (requiring no further treatment) and 12 (21.8%) were significantly improved (requiring only ongoing oral medication). Univariate and multivariate analyses showed that DMSO treatment failure was more likely in patients with pretreatment day-time urinary frequency more than 15 episodes per day (OR 1.41), nocturia more than two episodes per night (OR 2.47), maximum bladder diary voided volume <200 ml (OR 1.39) and bladder capacity under anaesthesia <500 ml (OR 1.6). CONCLUSIONS: At a median follow-up of 60 months, intravesical DMSO cocktail therapy appeared moderately effective for the treatment of IC/BPS. Treatment failure was more frequent in patients with pretreatment symptoms of reduced bladder capacity.


Assuntos
Crioprotetores/administração & dosagem , Cistite Intersticial/tratamento farmacológico , Dimetil Sulfóxido/administração & dosagem , Administração Intravesical , Adulto , Idoso , Idoso de 80 Anos ou mais , Anti-Inflamatórios/administração & dosagem , Anticoagulantes/administração & dosagem , Quimioterapia Combinada , Feminino , Seguimentos , Heparina/administração & dosagem , Humanos , Hidrocortisona/administração & dosagem , Pessoa de Meia-Idade
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