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1.
Biosci Biotechnol Biochem ; 84(4): 686-694, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-31852366

RESUMEN

Budded viruses (BVs) of baculovirus such as Autographa californica nucleopolyhedrovirus (AcNPV) have recently been studied as biological nanomaterials, and methods for their longer-term storage without deterioration would be desirable. The cryopreservation of virions with a naturally occurring saccharide like trehalose as a cryoprotectant is known to be useful for maintaining the viral structure and function. In this study, we examined how useful trehalose is as protectant for BV cryopreservation during repeated freeze-thaw cycles: 1) membrane fusion between liposomes (multilamellar vesicles, MLVs) and BVs, 2) infection of insect culture cells (Sf9 cells) by RFP-expressing BVs, and 3) morphologies of these BVs were investigated by fluorescent dequenching assay, fluorescence microscopy, and transmission electron microscopy (TEM), respectively. The results suggest that the BVs deteriorate in quality with each freeze-thaw cycle, and this deterioration can be diminished with the use of trehalose to an extent similar to that seen with storage on ice.


Asunto(s)
Crioprotectores/farmacología , Congelación , Fusión de Membrana , Trehalosa/farmacología , Virión/fisiología , Animales , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Células Sf9
2.
Int J Nanomedicine ; 14: 8095-8104, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31632020

RESUMEN

Introduction: Nimbolide (Nim), a limonoid obtained from the neem tree, Azadirachta indica, has several pharmacological properties, including anticancer effects in different type of cancers. No drug-delivery system has been reported for enhancing the therapeutic application of this novel hydrophobic molecule. Methods: In the present research, poly(lactic-co-glycolic acid) (PLGA) nanoparticles of Nim (Nim-nano) were formulated by nanoprecipitation, characterized for physicochemical properties, and screened for anticancer potential in breast (MCF-7 and MDA-MB-231) and pancreatic (AsPC-1) cancer cell lines. Results: The Nim-nano had a particle size of 183.73±2.22 nm and 221.20±11.03 nm before and after lyophilization, respectively. Cryoprotectants (mannitol and sucrose) significantly inhibited growth in particle size due to lyophilization. The ζ-potential of the Nim-nano was -22.40±4.40 mV. Drug loading and encapsulation efficiency of Nim-nano were 5.25%±1.12% and 55.67%±12.42%, respectively. The Nim-nano exhibited sustained release of Nim for more than 6 days in PBS (pH 7.4) and showed two- to three-fold enhanced cytotoxicity in breast and pancreatic cancer cell lines compared with free Nim. Conclusion: The Nim-nano formulation has great potential for treatment of cancers, such as pancreatic and breast cancer. Further, the PLGA-polymer surface can be modified by conjugation with polyethylene glycol, receptor-binding ligands (eg, folic acid), and other that which may lead to targeted delivery of Nim in the treatment of cancer.


Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Limoninas/administración & dosificación , Limoninas/uso terapéutico , Nanopartículas/administración & dosificación , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias de la Mama/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Crioprotectores/farmacología , Liberación de Fármacos , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Concentración 50 Inhibidora , Limoninas/química , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Electricidad Estática
3.
Anim Reprod Sci ; 208: 106127, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31405456

RESUMEN

The purpose of the current study was to evaluate the effects of resveratrol (RSV) on the quality of frozen-thawed goat sperm. Semen samples from four bucks were divided into five aliquots and diluted with a commercial bull semen extender containing: no antioxidant (RSV-0, control), 10 µM RSV (RSV-10), 50 µM RSV (RSV-50), 100 µM RSV (RSV-100) and 250 µM RSV (RSV-250). After thawing, sperm motility, abnormal morphology, membrane and acrosome integrity, mitochondrial activity, phosphatidylserine (PS) distribution, and oxidative stress were evaluated. The results indicated that in comparison with the control, when the concentration of RSV was 10 or 50 µM, the total motility, progressive motility, membrane and acrosome integrity, and mitochondrial activity of post-thaw spermatozoa was greater (P < 0.05). Additionally, the use of extenders containing RSV-10 or RSV-50 resulted in a greater percentage of viable spermatozoa as compared to the other groups (P < 0.05). Importantly, there were more viable spermatozoa (49.61 ±â€¯0.61%) and less non-viable spermatozoa (49.16 ±â€¯1.01%) in the RSV-50 group compared to the other extenders (P < 0.05). Furthermore, the use of the extenders containing RSV-10 and -50 resulted in a reduction in ROS production in frozen-thawed spermatozoa as compared to the control (P < 0.05). There, however, was no difference among extenders for abnormal morphology and PS distribution. In conclusion, supplementation with RSV, at a concentration of 10 or 50 µM in the semen extender, can improve the post-thaw goat sperm quality, which may occur as a consequence of inhibition of ROS generation.


Asunto(s)
Criopreservación/veterinaria , Cabras , Resveratrol/farmacología , Análisis de Semen/veterinaria , Preservación de Semen/veterinaria , Semen/efectos de los fármacos , Animales , Antioxidantes/farmacología , Membrana Celular/efectos de los fármacos , Crioprotectores/farmacología , Masculino , Estrés Oxidativo/efectos de los fármacos , Semen/fisiología , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos
4.
Anim Reprod Sci ; 208: 106111, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31405475

RESUMEN

The aim of this study was to evaluate the in vitro and in vivo quality of frozen-thawed sperm obtained from the Combatiente Español avian breed, with sperm having previously been diluted in N-methylacetamide (NMA). Experimental groups were established: fresh control semen (C); semen diluted without cryoprotectant (T1); semen diluted with extender containing NMA (T2); frozen-thawed sperm (with NMA) containing 500 × 106 spermatozoa (T3); frozen-thawed sperm (with NMA) containing 250 × 106 spermatozoa (T4). In the different groups, sperm motility and viability were assessed using a computer-assisted semen analyzer and flow cytometer, respectively. To evaluate the fertilizing capacity of the sperm, the percentage of fertile eggs was determined. The fertility rate after insemination with frozen-thawed semen was poor, and the concentration of the inseminating dose did not affect fertility rate (9.4 ±â€¯2.7% and 7.0 ±â€¯2.3%, respectively). The results indicate insemination using diluted semen without CPA leads to a reduced fertility, and the addition of 9% NMA to the extender has a greater negative effect on this in vivo variable. Furthermore, inclusion of NMA in the freezing-thawing processes reduced capacity of sperm for fertilization. Sperm viability was reduced during the freezing process, and the dilution in NMA extender affected both sperm viability and motility. The results indicate rooster fertility is negatively affected by sperm dilution, NMA addition and the frozen-thawed effects. Frozen-thawed sperm from Combatiente Español roosters maintained fertilizing capacity for no more than 6 days after insemination, whereas for fresh sperm this capacity was maintained for 14 days.


Asunto(s)
Acetamidas/farmacología , Congelación , Galliformes/fisiología , Inseminación Artificial/veterinaria , Preservación de Semen/veterinaria , Animales , Criopreservación/veterinaria , Crioprotectores/farmacología , Femenino , Masculino , Análisis de Semen , Espermatozoides/efectos de los fármacos
5.
Anim Reprod Sci ; 208: 106126, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31405480

RESUMEN

The aim of this study was to compare the effect of three sugars and Equex paste in a freezing extender for donkey sperm cryopreservation. Ejaculates (n = 18) were collected from six Andalusian donkeys of proven fertility were pooled (two ejaculates per pool) and cryopreserved using a freezing extender containing three different sugars (glucose, fructose and sorbitol), with or without the addition of Equex paste. Sperm quality was assessed before and after freezing-thawing for motility, morphology, plasma membrane integrity, acrosome integrity and DNA integrity. The use of sorbitol in the freezing extender improved total and progressive sperm motility (P < 0.05) and amplitude of lateral head displacement (P < 0.01), but it reduced the values for other sperm motility variables compared with glucose (P < 0.001). The use of fructose resulted in a reduction in values for most CASA variables (P < 0.05), whereas addition of Equex paste did not have any beneficial effect on values for these variables (P > 0.05). Glucose was more effective in maintaining sperm morphology (P < 0.05), while there was no beneficial effect with the addition of Equex paste (P > 0.05). Supplementation of fructose and Equex paste in the freezing extender decreased plasma membrane integrity (P < 0.05) as compared with glucose, but there were no differences between treatments for acrosome and DNA integrity (P > 0.05), even after 24 h of incubation. The use of different sugar sources in the extender could affect the in vitro post-thaw quality of cryopreserved donkey spermatozoa, with sorbitol being an interesting alternative for improving the sperm quality. Results of the present study indicate the use of Equex paste could negatively affect post-thaw outcomes for sperm viability in this species.


Asunto(s)
Criopreservación/veterinaria , Crioprotectores/farmacología , Equidae/fisiología , Preservación de Semen/veterinaria , Espermatozoides/fisiología , Animales , ADN/química , Congelación , Masculino , Análisis de Semen , Preservación de Semen/métodos , Motilidad Espermática/fisiología
6.
Int J Nanomedicine ; 14: 4895-4909, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31456636

RESUMEN

Introduction: Insulin is given by injection, because when administered orally, it would be destroyed by enzymes in the digestive system, hence only about 0.1% reaches blood circulation. The purpose of the present study was to use pH sensitive polyelectrolyte methyl methacrylate (MMA)/itaconic acid (IA) nanogels as carriers in an attempt to improve absorption of insulin administered orally. Methods: Insulin (Ins) was incorporated into the MMA/IA nanogels (NGs) using the polyelectrolyte complexation (PEC) method to form Ins/NGs-PEC. Several parameters, including Ins:NGs ratio, pH, incubation time and stirring rate were optimized during preparation of InsNGs-PEC. The prepared formulations were characterized in terms of particle size (PS), polydispersity index (PdI), zeta potential (ZP) and percent entrapment efficiency (% EE). Results: The optimized InF12 nanogels had a PS, PdI, ZP and %EE of 190.43 nm, 0.186, -16.70 mV and 85.20%, respectively. The InF12 nanogels were lyophilized in the presence of different concentrations of trehalose as cryoprotectant. The lyophilized InF12 containing 2%w/v trahalose (InF12-Tre2 nanogels) was chosen as final formulation which had a PS, PdI, ZP and %EE of 430.50 nm, 0.588, -16.50 mv and 82.10, respectively. The in vitro release of insulin from InF12-Tre2 nanogels in the SGF and SIF were 28.71% and 96.53%, respectively. The stability study conducted at 5±3°C for 3 months showed that lnF12-Tre2 nanogels were stable. The SDS-PAGE assay indicated that the primary structure of insulin in the lnF12-Tre2 nanogels was intact. The in-vivo study in the diabetic rats following oral administration of InF12-Tre2 nanogels at a dose of 100 IU/kg body weight reduced blood glucose level significantly to 51.10% after 6 hours compared to the control groups. Conclusions: The pH sensitive MMA/IA nanogels are potential carriers for oral delivery of insulin as they enhanced the absorption of the drug.


Asunto(s)
Liofilización , Insulina/administración & dosificación , Polielectrolitos/química , Polietilenglicoles/administración & dosificación , Polietileneimina/administración & dosificación , Administración Oral , Animales , Crioprotectores/farmacología , Diabetes Mellitus Experimental/tratamiento farmacológico , Portadores de Fármacos/química , Liberación de Fármacos , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Masculino , Ratas Sprague-Dawley , Espectroscopía Infrarroja por Transformada de Fourier , Temperatura Ambiental , Factores de Tiempo
7.
Nat Commun ; 10(1): 3491, 2019 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-31375674

RESUMEN

Despite the wide applications, systematic mechanobiological investigation of 3D porous scaffolds has yet to be performed due to the lack of methodologies for decoupling the complex interplay between structural and mechanical properties. Here, we discover the regulatory effect of cryoprotectants on ice crystal growth and use this property to realize separate control of the scaffold pore size and stiffness. Fibroblasts and macrophages are sensitive to both structural and mechanical properties of the gelatin scaffolds, particularly to pore sizes. Interestingly, macrophages within smaller and softer pores exhibit pro-inflammatory phenotype, whereas anti-inflammatory phenotype is induced by larger and stiffer pores. The structure-regulated cellular mechano-responsiveness is attributed to the physical confinement caused by pores or osmotic pressure. Finally, in vivo stimulation of endogenous fibroblasts and macrophages by implanted scaffolds produce mechano-responses similar to the corresponding cells in vitro, indicating that the physical properties of scaffolds can be leveraged to modulate tissue regeneration.


Asunto(s)
Materiales Biocompatibles/química , Crioprotectores/farmacología , Porosidad/efectos de los fármacos , Andamios del Tejido/química , Cicatrización de Heridas , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Fibroblastos , Gelatina/química , Gelatina/efectos de los fármacos , Humanos , Macrófagos , Masculino , Ensayo de Materiales/métodos , Ratones , Cultivo Primario de Células , Medicina Regenerativa/métodos , Piel/lesiones , Resistencia a la Tracción
8.
J Assist Reprod Genet ; 36(8): 1713-1720, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31273587

RESUMEN

STUDY QUESTION: Does cryoprotection of spermatozoa using a vitrification protocol with improved cryoprotective agents and a novel device for large storage lead to better outcomes than conventional slow freezing? Vitrification of human sperm using sucrose and dextran-based cryoprotectant (CPA4) with a new vitrification device resulted in significantly better sperm motility and progressive motility and improved DNA integrity with lower DNA fragmentation compared with conventional slow freezing. WHAT IS KNOWN ALREADY: A major limitation to clinical implementation of vitrification is the right balance between the volume of spermatozoa suspension cryopreserved and a standardised use of CPAs for survival of spermatozoa. STUDY DESIGN, SIZE, DURATION: This was a control versus current clinical practice study using 30 fresh human semen samples to carry out the different cryoprotectant analyses followed by a further 23 semen samples to test the novel vitrification protocol. PARTICIPANTS/MATERIALS, SETTING, METHODS: All human specimens fulfilled the following criteria: > 5 million spermatozoa/mL, > 20% total motility, ≥ 1.8 mL in volume, with all participants falling within the age range of 25-45 inclusively. The concentration, progressive motility, non-progressive motility, immotility, and various morphokinetic variables including DAP, DCL, DSL, LIN, and STR were then determined using the IVOS II™ Clinical CASA system (Hamilton Thorne, Beverly, MA, USA) on the basis of the 5th Edition of WHO Laboratory Manual for the Examination and Processing of Human Semen. MAIN RESULTS AND THE ROLE OF CHANCE: Among the 6 cryopreservation methods in this study, vitrification with the funnel-shaped device using CPA4 best preserves the 13 sperm parameters evaluated by CASA system. Conventional slow freezing and vitrification with the device using seminal plasma also protects sperm quality, but the overall motilities are statistically lower in comparison with the novel vitrification approach with cryoprotective media using the device. DNA fragmentation significantly increased after cryopreservation through the method of conventional slow freezing (p = 0.07). There was no significant difference in DNA fragmentation between fresh control and vitrification (p = 1.000). LIMITATIONS, REASONS FOR CAUTION: Extensive training is required to minimise the human error in using the vitrification device to perform cryopreservation. Each operator can only handle one sample at a time with device vitrification, whereas several samples can be processed without the need for special training with conventional slow freezing. WIDER IMPLICATIONS OF THE FINDINGS: The presented study shows that a new vitrification method could improve survival sperm rate. Human sperm vitrification using our novel protocol gives higher motility and progression and lower percentage of DNA fragmentation than conventional slow freezing. Our findings indicate that this method could supersede the current clinical practice in particular for patients with oligospermia as it reduces osmotic damage, time, and cost.


Asunto(s)
Criopreservación/instrumentación , Criopreservación/métodos , Crioprotectores/farmacología , Preservación de Semen/métodos , Motilidad Espermática/fisiología , Espermatozoides/fisiología , Vitrificación/efectos de los fármacos , Humanos , Masculino , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos
9.
Food Chem ; 298: 124868, 2019 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-31260976

RESUMEN

This work aimed to evaluate the efficacy of protein hydrolysates from bighead carp gill in inhibiting protein oxidation and quality loss in surimi. Firstly, antioxidant activity of hydrolysates in vitro was assessed, then 1%, 2% hydrolysates with better antioxidant activity and 4% sucrose were added to surimi respectively and stored at -18 °C for 4 months. Peptide sequences of above hydrolysates were also identified. The results suggested that hydrolysates of neutral protease had excellent 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability (42.93%) and Fe2+-chelating activity (73.27%). Compared with surimi without treatments, surimi with hydrolysates had higher sulfhydryl and salt-soluble protein concentrations, greater Ca2+-ATPase activity, lower disulfide bonds, carbonyls and hydrophobicity, as well as better gel strength and texture (p < 0.05). This study demonstrated that gill hydrolysate had antioxidant and dose-dependent cryoprotective effects on surimi that were comparable to the effects of sucrose, representing it as an alternative to sugar cryoprotectants in surimi industry.


Asunto(s)
Antioxidantes/farmacología , Cyprinidae , Productos Pesqueros , Proteínas de Peces/farmacología , Hidrolisados de Proteína/farmacología , Animales , Antioxidantes/química , Crioprotectores/química , Crioprotectores/farmacología , Proteínas de Peces/química , Congelación , Branquias/química , Interacciones Hidrofóbicas e Hidrofílicas , Oxidación-Reducción , Péptidos/análisis , Hidrolisados de Proteína/química , Compuestos de Sulfhidrilo/química
10.
BMC Musculoskelet Disord ; 20(1): 316, 2019 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-31279341

RESUMEN

BACKGROUND: Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and meniscus regeneration. The optimum cryopreservation medium has not been determined, but dimethylsulfoxide (DMSO) should be excluded, if possible, because of its toxicity. The purposes of our study were to examine the possible benefits of higher concentrations of serum and the effectiveness of 100% serum (without DMSO) for the cryopreservation of synovial MSCs. METHODS: Human synovium was harvested from the knees of four donors with osteoarthritis during total knee arthroplasty. Synovial MSCs (8 × 105 cells) were suspended in 400 µL medium and used as a Time 0 control. The same number of synovial MSCs was also suspended in 400 µL α-MEM medium containing 10% fetal bovine serum (FBS) (5% DMSO, and 1% antibiotic), 95% FBS (and 5% DMSO), or 100% FBS (no DMSO) and cryopreserved at - 80 °C for 7 days. After thawing, the cell suspensions (1.5 µL; 3 × 103 cells) were cultured in 60 cm2 dishes for 14 days for colony formation assays. Additional 62.5 µL samples of cell suspensions (1.25 × 105 cells) were added to tubes and cultured for 21 days for chondrogenesis assays. RESULTS: Colony numbers were significantly higher in the Time 0 and 95% FBS groups than in the 10% FBS group (n = 24). Colony numbers were much lower in the 100% FBS group than in the other three groups. The cell numbers per dish reflected the colony numbers. Cartilage pellet weights were significantly heavier in the 95% FBS group than in the 10% FBS group, whereas no difference was observed between the Time 0 and the 95% FBS groups (n = 24). No cartilage pellets formed at all in the 100% FBS group. CONCLUSION: Synovial MSCs cryopreserved in 95% FBS with 5% DMSO maintained their colony formation and chondrogenic abilities to the same levels as observed in the cells before cryopreservation. Synovial MSCs cryopreserved in 100% FBS lost their colony formation and chondrogenic abilities.


Asunto(s)
Condrogénesis/efectos de los fármacos , Criopreservación/métodos , Crioprotectores/farmacología , Células Madre Mesenquimatosas , Membrana Sinovial/citología , Anciano , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Crioprotectores/química , Dimetilsulfóxido/química , Dimetilsulfóxido/farmacología , Femenino , Humanos , Articulación de la Rodilla/citología , Trasplante de Células Madre Mesenquimatosas , Osteoartritis/terapia , Suero/química
11.
Theriogenology ; 136: 36-42, 2019 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-31242456

RESUMEN

We examined the effects of different freezing extenders, cryoprotectant agents (CPA) and initial thawing temperatures for preparing doses of refrozen stallion sperm for intracytoplasmic sperm injection (ICSI). Single ejaculates, from twelve stallions, were frozen in lactose-EDTA-egg yolk extender (LE) with 5% glycerol. In experiment 1, sperm were initially thawed to 5 °C or 37 °C, before being diluted in LE or skim milk-egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF) or 5% of a combination of both (GMF). In experiment 2, frozen sperm were initially thawed to 5 °C, diluted and refrozen in SMEY containing 2, 4, 6 or 8% GLY or GMF. In Experiment 1, sperm motility was reduced after each cryopreservation cycle (P < 0.05). Extender type did not affect motility after refreezing (P > 0.05), but sperm initially thawed to 5 °C exhibited higher motility than sperm thawed to 37 °C (P < 0.05). In addition, sperm refrozen in SMEY containing MF or GMF exhibited higher motility than sperm refrozen in GLY alone (P < 0.05). In experiment 2, there was an interaction between CPA and CPA concentration (P < 0.05). Sperm refrozen with GMF had higher motility than refrozen sperm with GLY (P < 0.05), and while GLY concentration did not affect post-thaw motility (P > 0.05). Sperm refrozen with 6 or 8% GMF exhibited the highest motility (P < 0.05). In conclusion, sperm motility is best maintained when thawing and refreezing stallion sperm in low sperm concentration ICSI doses by initially thawing the sperm to 5 °C and diluting the sperm in a freezing extender with 8% GMF.


Asunto(s)
Criopreservación/veterinaria , Crioprotectores/farmacología , Caballos/fisiología , Preservación de Semen/veterinaria , Inyecciones de Esperma Intracitoplasmáticas/veterinaria , Espermatozoides/fisiología , Animales , Congelación , Glicerol/farmacología , Masculino , Leche , Motilidad Espermática
12.
Anim Reprod Sci ; 207: 107-117, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31204090

RESUMEN

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.


Asunto(s)
Bovinos , Criopreservación , Crioprotectores/farmacología , Factor de Crecimiento Nervioso/farmacología , Preservación de Semen , Semen/efectos de los fármacos , Animales , Criopreservación/métodos , Criopreservación/veterinaria , Crioprotectores/administración & dosificación , Combinación de Medicamentos , Eyaculación/fisiología , Estimulación Eléctrica , Congelación/efectos adversos , Masculino , Factor de Crecimiento Nervioso/administración & dosificación , Semen/fisiología , Análisis de Semen/veterinaria , Preservación de Semen/métodos , Preservación de Semen/veterinaria , Recuperación de la Esperma
13.
Poult Sci ; 98(11): 6071-6077, 2019 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-31180125

RESUMEN

The aim was to compare the effect of dimethylacetamide (DMA) and N-methylacetamide (NMA) concentrations on the quality and fertility of post-thaw chicken semen. Ejaculates were obtained from 30 Hi-Line White roosters and processed according to the following treatments: lake pre-freezing extender + 0.1 M trehalose (LPF-T) + 6% DMA (control treatment), LPF-T + 9% DMA, LPF-T + 6% NMA, and LPF-T + 9% NMA. Sperm quality (viability, motility, and kinetic traits) was assessed before and after cryopreservation. A total of 15 laying hens per treatment were inseminated to assess fertility and embryo viability. Sperm cryopreserved in presence of DMA had significantly better in vitro quality compared to NMA, showing the highest proportion of viable and progressive motile sperm recovered after thawing. Furthermore, proportion of progressive motile sperm and the VCL, LIN, ALH, and WOB mean values were significantly improved in semen samples frozen/thawed with 6% compared to 9% cryoprotectant concentration. However, the best cryoprotective action on sperm quality played by DMA and the lowest cryoprotectant concentration did not translate into a concomitant advantage in in vivo semen fertility that showed no differences between cryoprotectant and cryoprotectant concentration treatments. Finally, the cryoprotectant DMA and NMA showed an opposite effect on embryo viability in comparison with the effect played on in vitro semen quality, being NMA more efficient than DMA on preserving viable embryos. The present results suggest the urgency to further decrease the cryoprotectant concentration in poultry semen freezing procedures and to assess the specific toxic effect of cryoprotectant on sperm integrity, fertility, and embryo development.


Asunto(s)
Acetamidas/farmacología , Pollos/fisiología , Crioprotectores/farmacología , Fertilidad/efectos de los fármacos , Análisis de Semen/veterinaria , Semen/efectos de los fármacos , Animales , Congelación , Masculino
14.
Anim Reprod Sci ; 207: 61-72, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31208850

RESUMEN

Semen cryopreservation is a very important technique for assisted reproduction; however, the cryopreservation process is harmful because it results in a reduction in sperm motility and viability, and leads to premature signals of capacitation, resulting in lesser than desirable fertility rates after artificial insemination. A fraction of seminal plasma, enriched in proteins that contain type II fibronectin domains (FNII) can reverse molecular indicators of cryo-capacitation. The beneficial effects of these proteins, however, depend on the relative abundance in seminal plasma. To create a safe additive for improving frozen sperm functionality, in the present study there was cloning and expression of a recombinant peptide containing four FNII domains (named TrxA-FNIIx4-His6) and evaluation of its effect after addition to frozen/thawed ram sperm. The cDNA for this protein was expressed in E. coli and after denaturation and re-naturalization of the protein, toxicity and binding capacity were assessed. By fluorescent labelling assessment, there was binding of the protein to the thawed sperm. At the two doses used (0.15 and 0.3 µM), TrxA-FNIIx4-His6 had the capacity to reverse the molecular indicators of cryo-capacitation as indicated by the reduction on phosphorylated substrates of PKA. Furthermore, the supplementation with this protein resulted in a normal capacitation process as evidenced by the increase in the in vitro fertilization rate when the greatest concentration of the protein was evaluated (73.25 ±â€¯2.95; 40.13 ±â€¯11.82 for 0.3 µM and control, respectively). There was no effect of protein supplementation on sperm objective motility compared to untreated sperm. In conclusion, the use of TrxA-FNIIx4-His6 is a promising biotechnological approach for cryopreserving ram sperm and maintaining sperm viability.


Asunto(s)
Criopreservación/veterinaria , Fertilización In Vitro/veterinaria , Péptidos/farmacología , Proteínas de Plasma Seminal/farmacología , Capacitación Espermática/efectos de los fármacos , Animales , Clonación Molecular , Criopreservación/métodos , Crioprotectores/química , Crioprotectores/metabolismo , Crioprotectores/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Fertilización In Vitro/métodos , Fibronectinas/química , Fibronectinas/genética , Inseminación Artificial/métodos , Inseminación Artificial/veterinaria , Masculino , Péptidos/genética , Dominios Proteicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Análisis de Semen/veterinaria , Preservación de Semen/métodos , Preservación de Semen/veterinaria , Proteínas de Plasma Seminal/química , Proteínas de Plasma Seminal/genética , Ovinos , Capacitación Espermática/fisiología , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos
15.
J Food Sci ; 84(6): 1547-1553, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-31107547

RESUMEN

Frozen storage of lobster meat (Homarus americanus) can result in undesirable quality changes that decrease consumer acceptability of these products. Current seafood industry methods use cryoprotective agents that contain phosphates including sodium tripolyphosphates (STPP). However, recent evidence suggests that cryoprotective mixtures that combine different carbohydrates and STPP can have equal or even greater cryoprotective properties compared to using STPP alone. The objective of this study was to compare the overall consumer acceptability of lobster meat stored for 6 months in different blends of these cryoprotective solutions. One hundred and seven panelists were recruited to score the acceptability of the lobster samples using nine-point hedonic scales. A check-all-that-apply (CATA) question containing 27 literature-informed, sensory descriptors was also used to identify terms frequently used to describe lobster meat. Analysis of variance analysis, indicated a significant increase for overall liking (22.1%, P < 0.0001), liking of flavor (23.6%, P < 0.0001) and texture (15.6%, P = 0.000) scores for samples stored in a novel carbohydrate blend plus sodium chloride (NaCl) and STPP compared to the water control. Subsequent penalty analysis revealed that overall liking scores were most positively associated with the attributes tender, sweet, moist and soft. Moreover, the attributes with the highest positive mean impact were more frequently used to describe lobster samples stored in solutions containing NaCl and the novel carbohydrate blend, as well as NaCl and STPP (Lobster-3 and Lobster-5 samples, respectively). PRACTICAL APPLICATION: The positive impact on the sensory quality of this novel blend of cryoprotective compounds (carbohydrates and NaCl) is proof of concept that this mixture is comparable, if not better than preservatives currently used by the seafood industry. Given the necessary regulatory approval and industry acceptance, lobster processors may consider this novel blend as a suitable alternative to freeze lobster products for up to 6 months.


Asunto(s)
Crioprotectores/farmacología , Conservación de Alimentos/métodos , Conservantes de Alimentos/farmacología , Nephropidae/química , Mariscos/análisis , Animales , Aromatizantes/química , Conservación de Alimentos/instrumentación , Congelación , Humanos , Carne/análisis , Nephropidae/efectos de los fármacos , Polifosfatos/farmacología , Cloruro de Sodio/farmacología , Gusto
16.
Cell Tissue Bank ; 20(3): 367-378, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31139967

RESUMEN

Cryopreservation exposes sperm to physical and chemical stresses causing cell damages and impairs sperm functions. The aim of this study was to evaluate the association between motility and sperm chromatin/DNA damage before and after cryopreservation and investigate the effects of folic acid and nicotinic acid on post-thaw sperm quality. Thirty semen samples were obtained from 30 normozoospermic men, aged between 25 and 45 years old. Each sample were divided into five aliquots to form the following groups: fresh, cryopreserved with sperm-freeze only (control), with nicotinic acid (10 mM), with folic acid (50 nM), and with a combination of folic acid (50 nM) + nicotinic acid (10 mM). Sperm viability and motility in each group were assessed by eosin-nigrosine staining and computer-aided sperm analysis respectively. Sperm chromatin quality was studied by aniline blue, toluidine blue, acridine orange staining methods and sperm chromatin dispersion test. Cryopreservation led to a significant reduction in sperm quality in comparison to fresh sample groups (p < 0.05). Sperm chromatin damage was negatively correlated with the percentage of progressively motile cells. Supplementation of the cryopreservation medium with folic acid or nicotinic acid induced a significant improvement in sperm parameters and chromatin quality, compared to control groups (p < 0.05). Meanwhile, the combination of folic acid + nicotinic acid showed a significant protective effect in post thaw sperm. In conclusion, cryopreservation generated oxidative stress, inducingsperm cryodamage, reducing progressive motility and sperm quality, as an indicator of significant chromatin/DNA damage. Folic acid and nicotinic acid exhibited a potential cryoprotective effect by enhancing sperm quality.


Asunto(s)
Acrosoma/efectos de los fármacos , Cromatina/química , Criopreservación , Daño del ADN , Ácido Fólico/farmacología , Niacina/farmacología , Motilidad Espermática , Adulto , Crioprotectores/farmacología , Humanos , Masculino , Persona de Mediana Edad , Análisis de Semen/métodos , Preservación de Semen/métodos , Espermatozoides/efectos de los fármacos
17.
Reprod Domest Anim ; 54(8): 1131-1138, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31145501

RESUMEN

The current study aimed to explore the potential usefulness of liquid or lyophilized egg yolk plasma (EYP) as a substitute for low-density lipoproteins (LDL) for cryopreservation of canine spermatozoa. In the first experiment, a total of 20 ejaculates harvested from six Beagles were frozen in extenders containing 6% LDL (control) or liquid or lyophilized EYP at one of three concentrations (20%, 40% or 60%). Motility parameters were assessed 10 min after thawing using computer-assisted sperm analysis. For both liquid and lyophilized EYP, the 40% concentration yielded motility similar (p > 0.05) to that observed with the control extender. In the second experiment, 12 ejaculates collected from the same six dogs were frozen in 6% LDL (Control), 40% liquid EYP or 40% lyophilized EYP extenders. Spermatozoal membrane integrity (hypo-osmotic swelling test [HOSt] and SYBR14/propidium iodide [PI] staining), acrosome integrity (FITC-Pisum sativum agglutinin staining) and DNA integrity (acridine orange staining) characteristics were evaluated 10 min after thawing. Both liquid and lyophilized 40% EYP-based extenders successfully preserved all assessed integrity parameters as efficiently as the control. Results of this study suggest that lyophilized EYP is a viable alternative to LDL in freezing extenders for dog semen.


Asunto(s)
Criopreservación/veterinaria , Perros , Yema de Huevo/química , Lipoproteínas LDL/farmacología , Preservación de Semen/veterinaria , Espermatozoides/efectos de los fármacos , Animales , Crioprotectores/farmacología , Liofilización , Congelación , Masculino , Análisis de Semen/veterinaria , Preservación de Semen/métodos , Motilidad Espermática/efectos de los fármacos
18.
Reprod Domest Anim ; 54(8): 1069-1077, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31099063

RESUMEN

Spermatozoa are highly specialized cells, and energy metabolism plays an important role in modulating sperm viability and function. Rosiglitazone is an antidiabetic drug in the thiazolidinedione class that regulates metabolic flexibility and glucose uptake in various cell types, but its effects on boar sperm metabolism are unknown. In this study, we investigated the potential effect of rosiglitazone against time-dependent deterioration of boar spermatozoa during liquid preservation at 17°C. Freshly ejaculated semen was diluted with Beltsville Thawing Solution (BTS) containing different concentrations of rosiglitazone, and the motility, membrane and acrosome integrity of sperm were detected. Besides, we measured glucose uptake capacity, l-lactate production level, mitochondrial membrane potential, adenosine triphosphate (ATP) content and mitochondrial reactive oxygen species (mROS) production of sperm after boar semen had been incubated with or without rosiglitazone, iodoacetate (glycolysis inhibitor) and rotenone (electron transport chain inhibitor) for 5 days. The addition of rosiglitazone significantly enhanced sperm quality and had a strong protective effect on the sperm membrane and acrosome integrity during storage. BTS containing 50 µM rosiglitazone maintained the total motility of liquid-preserved sperm above 60% for 7 days. Rosiglitazone improved sperm quality by regulating energy metabolism manner of preserved sperm, protected the sperm mitochondrial membrane potential, enhanced sperm ATP production and in the meanwhile reduced mROS through enhancing glycolysis but not oxidative phosphorylation. The data suggested the practical feasibility of using rosiglitazone for improving boar spermatozoa quality during semen preservation.


Asunto(s)
Hipoglucemiantes/farmacología , Rosiglitazona/farmacología , Preservación de Semen/veterinaria , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Porcinos , Animales , Crioprotectores/farmacología , Metabolismo Energético , Masculino , Motilidad Espermática
19.
Poult Sci ; 98(9): 4161-4171, 2019 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-31065720

RESUMEN

Chicken semen conservation is an important tool for programs of genetic diversity management and of endangered breeds' conservation. However, the method still needs to be improved in order to be applied in a wide variety of environments and breeds. Our objective was to compare the effects of 2 external cryoprotectants saccharides (sucrose and raffinose) on the sperm freezability of a Thai local breed, Pradu Hang Dum, in which semen was frozen with a simple freezing method using nitrogen vapors and dimethyl formamide (DMF). Thirty-six males were selected on their motility vigor score for the experiments. In a first experiment, a large range of sucrose and raffinose doses were tested. Semen quality was evaluated after incubation at 5°C or after cryopreservation in straws in the saline Blumberger Hahnen Sperma Verdünner diluent + DMF (6% v/v) with or without sucrose/raffinose. The best targeted doses of sucrose and raffinose were then kept for experiment 2 that was focused on cryopreserved semen. In this experiment, semen quality was measured on frozen-thawed sperm: different objective motility data evaluated by computer-assisted sperm analysis (CASA), membrane integrity, acrosome integrity, mitochondria function evaluated using flow cytometry, lipid peroxide production assessed by the thiobarbituric acid test. Fertility obtained with frozen-thawed semen supplemented or not with sucrose or raffinose was also evaluated after artificial insemination of laying hens. The presence of sucrose at the osmotically inactive dose 1 mmol significantly increased the vigor motility, membrane integrity, acrosome integrity, and mitochondrial functions of frozen-thawed sperm (P < 0.05), and showed the highest levels of fertility after sperm cryopreservation (91% vs. control 86%, P < 0.001). Raffinose showed negative effects on in vitro semen quality from 1 to 100 mmol. Fertility was also negatively (P < 0.001) affected by raffinose (fertility rate 66 to 70%). We thus showed in the present study the high success of a simple chicken sperm cryopreservation method with an external cryoprotectant easily available and cheap, the sucrose, used at an osmotically inactive low concentration.


Asunto(s)
Pollos/fisiología , Crioprotectores/farmacología , Fertilización/fisiología , Rafinosa/farmacología , Preservación de Semen/veterinaria , Espermatozoides/fisiología , Sacarosa/farmacología , Animales , Femenino , Masculino , Análisis de Semen/veterinaria , Preservación de Semen/métodos
20.
Anim Reprod Sci ; 205: 126-133, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-31047761

RESUMEN

The aim of the present study was to establish a protocol for solid surface vitrification of peccary ovarian tissue by using different cryoprotectants. Ovarian pairs from five adult females were fragmented and two fragments (fresh control group) were immediately subjected to morphological evaluation using classical histology, transmission electron microscopy, and viability analysis using fluorescent probes. The remaining fragments (n = 18) were vitrified using a solid surface method with different concentrations (3 or 6 M) of ethylene glycol (EG), dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). After 2 weeks, samples were re-warmed and evaluated. A decrease in the percentage of morphologically normal preantral follicles (PFs) was verified for all the groups in comparison to the fresh control (92.0 ± 2.8%); however, if only the primordial follicles are considered, the most effective preservation (P < 0.05) was achieved with the use of EG at 3 M (74.2±7.3%) or DMSO at 6 M (75.0 ± 4.2%). Ultrastructural analysis indicated there were well-preserved PFs in all the groups evaluated, having well-defined membranes, a few vacuoles, and organelles that were uniformly distributed throughout the cytoplasm, mainly round and elongated mitochondria in close association with lipid droplets. Viability was preserved (P < 0.05) with the use of EG at 3 (97%) or 6 (97%) M, DMSO at 3 (100%), and DMF at 6 (97%) M. Solid surface vitrification, therefore, is an effective method for conservation of peccary female germplasm, especially with the use of EG at 3 M, which was highly effective for preservation of both the morphology and viability of PFs.


Asunto(s)
Artiodáctilos/fisiología , Crioprotectores/farmacología , Ovario/fisiología , Conservación de Tejido/veterinaria , Vitrificación/efectos de los fármacos , Animales , Supervivencia Celular , Femenino
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