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1.
PLoS One ; 15(10): e0241105, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33119667

RESUMO

The aim of our study was to examine the effects of crocin (0.5 (C0.5), 1 (C1) and 1.5 (C1.5) mM) and naringenin (50 (N50), 100 (N100) and 150 (N150) µM) in cryopreservation extender for freezing rooster semen. Sperm motility, viability, abnormalities, membrane functionality, active mitochondria, apoptosis status, lipid peroxidation (LP), GPX, SOD, TAC, the mRNA expression of pro-apoptotic (CASPASE 3) and anti-apoptotic (Bcl-2) genes, fertile eggs, hatched eggs and hatching rate were investigated following freeze-thawing. C1 and N100 resulted in higher (P < 0.05) total motility and progressive motility in comparison to the control group. The C1 and N100 groups improved viability, membrane functionality and reduced lipid peroxidation. We found higher values for active mitochondria with C1 and N100 compared to control group. The C1 and N100 groups showed lower percentages of early apoptosis when compared with control group. Also, C1 and N100 had higher TAC, compared to the control group. The mRNA expressions of BCL-2 in the C1 and N100 groups were significantly higher than that of other treatments. The expression of CASPASES 3 was significantly reduced in C1 and N100 group (P < 0.05) when compared to control group. Significantly higher percentages of fertile eggs, hatched eggs and hatching rate were observed in C1 and N100 compared to the control group. In conclusion, crocin at 1 mM and naringenin at 100 µM seem to improve the post-thawing rooster semen quality, fertility and could protect the sperm by reducing the pro-apoptotic (CASPASE 3) and increasing anti-apoptotic (Bcl-2) genes.


Assuntos
Carotenoides/farmacologia , Crioprotetores/farmacologia , Flavanonas/farmacologia , Preservação do Sêmen/veterinária , Sêmen/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Galinhas , Criopreservação/métodos , Criopreservação/veterinária , Fertilidade/efeitos dos fármacos , Masculino , Preservação do Sêmen/métodos , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/citologia
2.
Anim Sci J ; 91(1): e13451, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32926550

RESUMO

This study assessed the effects of cryoprotectant concentration during equilibration on the efficiency of bovine blastocyst vitrification and the expression of selected developmentally important genes. In vitro produced bovine blastocysts were equilibrated in either 7.5% ethylene glycol (EG) + 7.5% DMSO (Va group) or in 2% EG + 2% DMSO (Vb group) then vitrified on Cryotop® sheets in 16.5% EG + 16.5% DMSO + 0.5M sucrose. After warming, embryos were cultured for 48 hr. Re-expansion, hatching, and the numbers of total and membrane damaged cells were compared among vitrified groups and a control. There was no significant difference between the vitrified groups in survival, cell numbers and the extent of membrane damage. Vitrification increased the number of membrane-damaged cells in both groups, however, in a greater extent in the Vb group. Vitrification increased (p < .05) the expression of the HSP70 gene in Va but not in Vb embryos. The expression of IGF2R, SNRPN, HDAC1, DNMT3B, BAX, OCT4, and IFN-t genes were the same in control and vitrified groups. In conclusion, the concentration of cryoprotectants during equilibration did not affect survival rates; however, normal cell numbers could be maintained only by equilibration in 15% cryoprotectants which was associated with increased HSP70 expression.


Assuntos
Blastocisto , Sobrevivência Celular/efeitos dos fármacos , Criopreservação/métodos , Criopreservação/veterinária , Crioprotetores/farmacologia , Fertilização In Vitro/métodos , Fertilização In Vitro/veterinária , Vitrificação/efeitos dos fármacos , Animais , Bovinos , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo
3.
PLoS One ; 15(9): e0238573, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32877463

RESUMO

Sugars are commonly supplemented into vitrification solution to dehydrate cells in order to reduce the formation of fatal intracellular ice crystals. Natural honey is a mixture of 25 sugars (mainly fructose and glucose) that have different biological and pharmacological benefits. The present study was designed to determine if honey can be used as a nonpermeating cryoprotectant in vitrification of bovine oocytes. In the first experiment, denuded-MII oocytes were exposed to 0.25, 0.5, 1.0, 1.5 or 2.0 M of honey or sucrose. Natural honey and sucrose caused similar ooplasm dehydration. A significant relationship existed between time and ooplasm volume change (P < 0.05), during dehydration and rehydration phases, in both honey and sucrose solutions. In the second experiment, the immature cumulus-oocyte complexes (COCs) were vitrified in an EG/DMSO-based vitrification solution containing honey (0.5, 1 or 1.5 M) or sucrose (0.5 M) as a gold standard. The vitrified-warmed COCs were matured in vitro and evaluated for nuclear maturation. The maturation (MII) rate was greater in nonvitrified control (81%) than vitrified groups (54%, P < 0.05). In the third experiment, COCs were either remained nonvitrified (control) or vitrified in 1.0 M honey or 0.5 M sucrose, followed by IVM, IVF and IVC (for 9 days). Cleavage rate was greater in control (74%) than in vitrified groups (47%, P < 0.05), without significant difference between sugars. Blastocyst rate was 34, 13 and 3% in control, honey and sucrose groups respectively (P < 0.05). In conclusion, natural honey acted as a nonpermeating cryoprotectant in vitrification solution and improved the embryonic development in vitrified bovine COCs.


Assuntos
Crioprotetores/farmacologia , Mel , Oócitos/citologia , Vitrificação , Animais , Blastocisto/citologia , Bovinos , Desidratação , Técnicas de Maturação in Vitro de Oócitos , Oócitos/efeitos dos fármacos , Concentração Osmolar , Análise de Regressão , Soluções , Sacarose/farmacologia
4.
Ecotoxicol Environ Saf ; 204: 111005, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32738624

RESUMO

Freezing temperatures is an important stressor in the arctic regions and has a significant influence on the population dynamics and geographic distribution of terrestrial invertebrates. Toxic metals in the environment can interfere with protective cold-acclimation responses of organisms. It is therefore important to evaluate the combined effects of cold stress and environmental contaminants. Here, we aimed to investigate the effects of Hg (HgCl2) on various physiological aspects of freeze-tolerance in the earthworm (Enchytraeus albidus). We measured the levels of the cryoprotectant glucose, the glycogen content (source of glucose molecules for cryoprotection and fuel for metabolism), and changes in the composition of membrane phospholipid fatty acids (PLFA) as an indicator of lipid peroxidation. Freezing at -6 °C had no effect on survival in uncontaminated soil, however, survival of freezing in Hg contaminated soil was clearly reduced, especially at extended exposure times. Thus, the LC50 value in frozen soil decreased from 8.3 mg Hg kg-1 (when exposed for 17 days) to only 4.2 mg Hg kg-1 after 36 days' exposure indicating that combined effects of Hg and freezing became larger at prolonged exposure times. Hg caused a depletion of glycogen reserves (almost 50% at 12 mg kg-1 dry soil), but despite this effect worms were able to maintain a constant cryoprotectant level (about 0.12 mg glucose mg-1 dry weight) at all Hg concentrations. Hg had clear negative effects on the proportion of unsaturated PLFAs, which could be an indication of lipid peroxidation. Since a high proportion of unsaturated fatty acids in the membrane is important for invertebrate freeze-tolerance, our results suggest that the negative effect of Hg on freeze-tolerance in E. albidus is related to degraded membrane functionality at low temperature.


Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Congelamento , Cloreto de Mercúrio/efeitos adversos , Oligoquetos/efeitos dos fármacos , Animais , Crioprotetores/farmacologia , Relação Dose-Resposta a Droga , Ácidos Graxos/metabolismo , Glucose/farmacologia , Glicogênio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Mercúrio/efeitos adversos
5.
Poult Sci ; 99(6): 2841-2851, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32475417

RESUMO

The objective of this study was to investigate differences in mRNA expression between fresh and frozen-thawed sperm in roosters. In trial 1, gene expression profiles were measured using microarray with Affymetrix GeneChip Chicken Genome Arrays. The results showed that 2,115 genes were differentially expressed between the 2 groups. Among these genes, 2,086 were significantly downregulated and 29 were significantly upregulated in the frozen-thawed sperm group. Gene Ontology (GO) analysis showed that more than 1,000 differentially expressed genes (DEG) of all significantly regulated genes were involved in GO terms including biological processes, molecular function, and cellular component. Kyoto Encyclopedia of Genes and Genomes analysis showed that DEG were significantly (P < 0.05) enriched on ribosome, oxidative phosphorylation, proteasome, cell cycle, oocyte meiosis, and spliceosome pathways. In trial 2, ejaculated semen was collected from 18 roosters and divided into 5 recombinant HSP90 protein-supplemented groups (0.01, 0.1, 0.5, 1, or 2 µg/mL) and one control group with no recombinant HSP90 protein supplementation to evaluate the effect of recombinant HSP90 protein in the extender on post-thaw quality of rooster semen. The results showed that post-thaw sperm viability and motility was significantly improved (P < 0.05) in the extender containing 0.5 and 1 µg/mL of recombinant HSP90 protein compared with the control. Our preliminary results will provide a valuable basis for understanding the potential molecular mechanisms of cryodamage in frozen-thawed sperm and theoretical guidance to improve the fertility of frozen-thawed chicken sperm.


Assuntos
Galinhas/fisiologia , Crioprotetores/farmacologia , Perfilação da Expressão Gênica/veterinária , Preservação do Sêmen/veterinária , Espermatozoides/efeitos dos fármacos , Transcriptoma , Animais , Galinhas/genética , Congelamento , Masculino , Preservação do Sêmen/métodos
6.
Acta Vet Scand ; 62(1): 31, 2020 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-32552825

RESUMO

BACKGROUND: Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Gums have been suggested as an alternative cryoprotectant to glycerol for stallion spermatozoa. Therefore, the present experiment was designed to verify whether the effect of addition of cashew gum (CG), or nanoparticles (NP) containing CG, to the extender before cooling on sperm quality in stallion semen. Ejaculates from 6 stallions were extended and split between six treatment groups (control, a-tocopherol [TOC], CG1, CG0.5, NP1 and NP0.5), stored in cryotubes at 4 °C. RESULTS: Aliquots were analysed by computer-assisted sperm motility analysis on the day of collection, and after 24 h and 48 h of cold storage. After 48 h, the total motility with NP1 (78.53 + 6.31%) was similar to control 85.79 + 6.31% at 0 h. The same pattern was observed for progressive motility. Membrane integrity assessed by flow cytometer was similar between control, TOC and G1 at all storage times. The DNA fragmentation in the control group increased at all time points, whereas chromatin integrity was maintained after 24 h in TOC and NP0.5 compared to 0 h. There was no increase in the proportion of live spermatozoa producing hydrogen peroxide, but there was a tendency for an increased proportion of spermatozoa in the live superoxide category in CG1 after 24 h cooled storage. CONCLUSIONS: The addition of CG or CG-derived NP to extender for stallion semen was not harmful to the sperm cells.


Assuntos
Anacardium/química , Crioprotetores/farmacologia , Cavalos/fisiologia , Nanopartículas/química , Preservação do Sêmen/veterinária , Sêmen/fisiologia , Animais , Crioprotetores/química , Gengiva/química , Masculino , Preservação do Sêmen/instrumentação
7.
Indian J Ophthalmol ; 68(7): 1412-1416, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32587179

RESUMO

Purpose: Due to the COVID-19 pandemic, most of the eye banks have limited/stopped corneal collection, as this is a highly contagious disease. This has led to shortage of donor corneas worldwide. Glycerol preservation of tissue remains a viable option in this scenario. The objective is to compare fresh corneal tissue (FCT) with glycerol-preserved cornea (GPC) in emergency corneal transplantation. Methods: This was a retrospective cohort study conducted in a tertiary care centre of Uttarakhand. Medical records of the patients who underwent therapeutic penetrating keratoplasty (TPK) were reviewed. FCT group included patients who underwent TPK with fresh corneal tissue and GPC group included patients who underwent TPK with glycerol preserved cornea. The indications and outcomes of TPK in the terms of therapeutic success were analysed and compared between both the groups. Results: A total of 94 eyes of 91 patients underwent TPK from October 2011 to August 2017. FCT group included 60 eyes of 57 patients and GPC group included 34 eyes of 34 patients. The primary indication of TPK was infectious keratitis in both the groups (FCT-81.6%; GPC - 91.2%) There was no significant difference in the therapeutic success in both the groups (P = 0.741, Odds ratio- 1.59 with 95% CI- 0.39-6.44). Complications included glaucoma (FCT-21.7%; GPC- 35.2%) graft infection (FCT- 18.33% GPC- 2.9%); graft rejection (FCT-11.66%, GPC- 0%); and graft failure (FCT-88.33%, GPC-100%). Conclusion: The GPC is comparable to FCTs in therapeutic transplant and can be a useful interim procedure in saving the eyes in cases of infective keratitis in the time of crisis.


Assuntos
Betacoronavirus , Doenças da Córnea/cirurgia , Infecções por Coronavirus/epidemiologia , Emergências , Glicerol/farmacologia , Pneumonia Viral/epidemiologia , Doadores de Tecidos , Preservação de Tecido/métodos , Doenças da Córnea/complicações , Transplante de Córnea/métodos , Infecções por Coronavirus/complicações , Crioprotetores/farmacologia , Feminino , Seguimentos , Sobrevivência de Enxerto , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/complicações , Estudos Retrospectivos , Acuidade Visual
8.
PLoS One ; 15(6): e0234339, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32516324

RESUMO

Hypothermic storage of boar semen provides the possibility to omit antibiotics from semen extenders so long as sperm quality is maintained and bacterial growth prevented. The objective of this study was to determine an optimal cooling-rate frame for boar semen preserved at 5°C in an antibiotic-free extender. Semen from eight boars extended in AndroStar® Premium was cooled from 30°C to 5°C using seven different cooling rates, ranging initially from 0.01 to 0.36°C min-1 and reaching 5°C between 2 h and 24 h after dilution. Sperm motility, membrane integrity, membrane fluidity, mitochondrial membrane potential and the response to the capacitation stimulus bicarbonate remained at a high level for 144 h at 5°C when the semen was initially cooled in a cooling-rate frame ranging from 0.01 to 0.09°C min­1 in the temperature zone from 30 to 25°C, followed by 0.02 to 0.06°C min-1 to 10°C and 0.01 to 0.02°C min­1 to the final storage temperature. A cooling rate of 0.07°C min-1 in the temperature zone from 30 to 10°C led to a reduced response to bicarbonate (P < 0.01) and fast cooling to 5°C within 1 h with a cooling rate of 0.31°C min-1 resulted in lower values (P > 0.05) of all sperm parameters. In a further experiment, slow cooling with a holding time of 6 h at 22°C induced after 6 h storage a temporary increase in Escherichia coli of 0.5 × 103 to 2.4 × 103 CFU mL-1 in the sperm-free inoculated extender. Overall, the load of mesophilic bacteria in the stored semen was below 6 × 103 CFU mL-1, a level that is not regarded as critical for sperm quality. In conclusion, appropriate cooling protocols were established for the antibiotic-free storage of boar semen at 5°C, allowing the application of hypothermic preservation in research and in artificial insemination.


Assuntos
Criopreservação/métodos , Preservação do Sêmen/métodos , Manejo de Espécimes/métodos , Animais , Secreções Corporais/efeitos dos fármacos , Líquidos Corporais/efeitos dos fármacos , Crioprotetores/farmacologia , Masculino , Sêmen/efeitos dos fármacos , Sêmen/metabolismo , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/fisiologia , Sus scrofa/metabolismo , Suínos , Temperatura
9.
Poult Sci ; 99(5): 2766-2774, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32359614

RESUMO

Artificial insemination is used in almost 95% of turkey reproductive flocks and is becoming more important in chickens, particularly broiler breeders, as well as in assisted reproduction of wild birds kept in breeding centers. Diluted semen is recommended for artificial insemination. Pooled semen samples collected twice a week by dorso-abdominal massage from 2 chicken lines: laying-ISA Brown (ISA-B) and meat type-Hubbard Flex (H-F) were divided into 5 parts: neat semen and diluted in 1:2 ratio with 4 extenders: basic EK; EK + 1 µg/mL organic selenium and 8 µg/mL vitamin E; EK + 10 mg/mL of royal jelly; and EK + 0.25 g/mL of lyophilized bovine colostrum. Diluted semen samples were evaluated after 15 min and then 24 h storage at 4°C. Sperm concentration, motility, motility parameters (with Sperm Class Analyzer), and morphology were evaluated in the neat semen, whereas in diluted and stored samples, the last 3 traits were determined. In case of both lines, dilution did not affect (P > 0.05) the number of live normal cells (78.0-81.1% in ISA Brown and 73.8-68.7% in Hubbard Flex) in relation to neat semen; however, bovine colostrum addition increased (P < 0.05) the percentage of bulb head sperm (5.7 vs. 10.0% and 12.1 vs. 17.6%, for ISA and Hubbard, respectively) and decreased sperm motility (67.4 vs. 92.9% and 67.3 vs. 98.5% for ISA and Hubbard). The 24 h storage of neat semen and semen diluted with colostrum caused (P < 0.05) the unfavorable changes in all evaluated traits and both chicken lines, whereas semen dilution with remaining extenders decreased the percentage of live normal cells (by 18.8-23.4% ISA and by 20.9-25.5% Hubbard) but did not affect sperm motility (81.5-87.6% for ISA and 81.1-96.6% for Hubbard). Sperm motility and motility parameters depended both on the extender and chicken line.


Assuntos
Galinhas/fisiologia , Crioprotetores/farmacologia , Preservação do Sêmen/veterinária , Sêmen/fisiologia , Animais , Inseminação Artificial , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos
10.
Cryobiology ; 94: 49-56, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32387287

RESUMO

This study aimed to improve a sperm cryopreservation protocol for farmed Pacific abalone, Haliotis discus hannai. Dimethyl sulfoxide (Me2SO), glycerol, ethylene glycol (EG), propylene glycol (PG), and methanol were chosen as cryoprotectants (CPAs). Four different equilibration time (5, 10, 30, and 60 min), and two types of equilibration temperature (4 °C and 20 °C) were selected at the present experiment. Most equilibration temperatures with each CPA showed significant differences among different equilibration time. Post-thaw sperm motility of five CPAs showed no significant difference at two equilibration temperature. Based on these results, 8% Me2SO, 8% EG, 6% PG, 2% glycerol, and 2% methanol were chosen to determine optimal conditions for sperm cryopreservation of H. discus hannai. The highest post-thaw sperm motility (8% Me2SO: 50.6%, 8% EG: 45.6%, 2% glycerol: 44.5%, 6% PG: 28.7%, 2% methanol: 25.4%) was achieved after exposing sperm to liquid nitrogen (LN2) vapor for 10 min at 5 cm above the LN2 surface and then submerging them in LN2 for at least 2 h followed by thawing at 60 °C with seawater and recovering them at 20 °C with seawater. In this study, 8% Me2SO and 2% glycerol were chosen to check post-thaw sperm quality to estimate percentages of plasma membrane integrity (PMI), mitochondrial potential analysis (MP), and acrosome integrity (AI) using fluorescent techniques. No significant difference in PMI, MP, and AI was found between sperm cryopreserved with 8% Me2SO and those cryopreserved with 2% glycerol. The current study has demonstrated that 8% Me2SO was optimal for sperm cryopreservation for H. discus hannai with 5 min of equilibration time, 5 cm of rack height and 60 °C of thawing temperature. The present research provides more effective cryopreservation methods for H. discus hannai sperm than previous studies.


Assuntos
Criopreservação/métodos , Gastrópodes , Preservação do Sêmen/métodos , Espermatozoides , Animais , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Glicerol/farmacologia , Masculino , Metanol/farmacologia , Propilenoglicol/farmacologia , Motilidade Espermática/efeitos dos fármacos
11.
Cryobiology ; 94: 26-31, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32389624

RESUMO

In our study, a systematic development of a new large-scale sperm cryopreservation protocol was carried out in northern pike (Esox lucius). The effect of 2 sugar based (glucose and trehalose) extenders, 3 dilution ratios (1:3, 1:9 and 1:19) 2 vol straws (0.5 and 5 mL) and a 10 mL cryotube, 2 different cryopreservation methods (Polystyrene box-P. box and Controlled Rate Freezer-CRF), as well as 3 different thawing periods (3, 3.5 and 4 min) were investigated on the motility of thawed sperm. The glucose based extender showed significantly higher pMOT (1:3-18 ± 16%, 1:9-20 ± 13%, 1:19-16 ± 12%) at all dilution ratios than in the trehalose based extender (1:3-0.3 ± 1%, 1:9-1±1%, 1:19-4±2%). A similar tendency was recorded in VCL and STR at a ratio 1:3 and 1:9. No significant difference was measured in sperm movement between the P. box and CRF using the 0.5 mL straw. Similarly no significant difference was observed in all motility parameters with 10 mL cryotube frozen in CRF at a ratio 1:3-1:19. An effective and short thawing period (3 min) was experimentally specified for the 10 mL cryotube cryopreserved in the CRF. In all large-scale cryopreservation methods, high pMOT (straw CRF: 57 ± 10%, straw P. box: 50 ± 9%, cryotube CRF: 41 ± 10%), and STR were measured, and no significant difference was recorded in all motility parameters. Our results demonstrate the effectiveness of our newly developed extender and the applicability of 3 different large-scale cryopreservation methods in pike sperm. Our protocols could be new prospective candidates for future exploitation in hatchery practice.


Assuntos
Criopreservação/métodos , Esocidae , Preservação do Sêmen/métodos , Espermatozoides , Animais , Crioprotetores/farmacologia , Glucose/farmacologia , Masculino , Trealose/farmacologia
12.
Cryobiology ; 94: 40-48, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32416082

RESUMO

This study aimed to evaluate the comparative effects of Purslane aqueous extract (PAE), Purslane methanolic extract (PME) and Purslane ethanolic extract (PEE on the quality of frozen-thawed goat spermatozoa. Collected semen with motility >75% and sperm concentration >1.0 × 109 sperm/ml was pooled and divided into 10 equal aliquots and supplemented by basic extender containing 25, 50 or 100 µg/ml of Purslane aqueous extract (PAE25µg/ml, PAE50µg/ml, PAE100µg/ml, respectively), basic extender containing 25, 50 or 100 µg/ml of Purslane methanolic extract (PME25µg/ml, PME50µg/ml, PME100µg/ml, respectively), basic extender containing 25, 50 or 100 µg/ml of Purslane ethanolic extract (PEE25µg/ml, PEE50µg/ml, PEE100µg/ml, respectively). Control diluent contained no additives. For the determination of sperm quality, frozen straws were thawed and then the sperm characteristics were assessed. Results indicated that higher (P < 0.05) percentages of total motility, viability, mitochondrial activity and lower percentages of malondialdehyde (MDA) for PAE50µg/ml, PME50µg/ml and PEE50µg/ml than those of the control. In addition, PME50µg/ml resulted in the highest) P < 0.05) total motility and the lowest (P < 0.05) MDA levels compared to other treatments. Compared to the control group, PME50µg/ml resulted in higher integrity (P < 0.05) of plasma membranes and in lower amounts of apoptotic and dead spermatozoa. PME50µg/ml and PAE50µg/ml showed higher (P < 0.05) percentages of progressive motility, DNA integrity and live post-thawed spermatozoa than those of the control. No significant differences in the motility, viability, mitochondrial activity and number of live sperms were observed between PME50µg/ml and PAE50µg/ml treatments. In conclusion, the results of this study indicated that 50 µg/ml purslane extracts could be used for the cryopreservation. However, the results of methanolic extract was more beneficial compared to other extracts.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Extratos Vegetais/farmacologia , Portulaca , Preservação do Sêmen/métodos , Espermatozoides , Animais , Cabras , Masculino , Motilidade Espermática/efeitos dos fármacos , Trometamina
13.
Cryobiology ; 94: 80-88, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32437677

RESUMO

The aims of this project were to transfer hormone-induced spermiation and sperm cryopreservation protocols developed in the model salamander species, Ambystoma tigrinum, to three threatened newt species. Additionally, we tested if supplementation with trehalose or thawing at different temperatures impacts post-thaw sperm parameters. Hormone stimulation protocols were applied to male Notophthalmus meridionalis (N = 10), Neurergus kaiseri (N = 5) and Tylototriton kweichowensis (N = 6) with sperm collected periodically up to 24-28 h post-spermiation dose. Samples of adequate sperm concentration (>70%) were cryopreserved in solutions of 10% Me2SO + 1% BSA with or without a 10% trehalose cryodiluent. Frozen sperm samples were thawed at either 20 °C or 40 °C and examined for post-thaw motility parameters and abnormalities in head and tail structure. The spermiation response to exogenous hormone treatment was significantly different between newt species, with a success rate of 0% for N. kaiseri, 67% for T. kweichowensis, and 100% for N. meridionalis. Sperm concentration varied with time of collection after hormone administration in both T. kweichowensis and N. meridionalis. For N. meridionalis, structural abnormalities decreased in samples collected over the 24 h period (p < 0.0001) and a thaw temperature of 40 °C resulted in higher relative total sperm motility (p < 0.0001). This is the first study to describe the cryopreservation of sperm from two newt species and demonstrates the transferability of ART developed in a salamander to two newt species.


Assuntos
Criopreservação/métodos , Espécies em Perigo de Extinção , Salamandridae , Preservação do Sêmen/métodos , Espermatozoides , Animais , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Masculino , Soroalbumina Bovina/farmacologia , Trealose/farmacologia
14.
Cryobiology ; 94: 57-65, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32437678

RESUMO

The advantageous influence of quercetin (Q) supplementation in an extender has not yet been evaluated for rooster semen cryopreservation. This research was purposely conducted in order to assess the effect of different quercetin concentrations added into an extender on the sperm quality of the rooster subsequent to a freezing-thawing process. After the freezing-thawing process, spermatozoa quality parameters (membrane functionality, acrosome integrity, motility, viability, and abnormal morphology), endogenous enzymes (SOD, CAT, and GPx), mitochondrial activity, DNA fragmentation index, lipid peroxidation (MDA), and ROS were all evaluated. A total of 75 neat pooled ejaculates (3 ejaculates/rooster) were collected from 25 arbor acres roosters (24 wks) twice a week using abdominal massage technique, then divided into five equal aliquots and diluted with an extender containing different doses of Q (CS-Q) as follows: casein extender without Q (control only), casein extender containing 0.040 mg/mL quercetin (CS-Q 0.040), 0.020 mg/mL quercetin (CS-Q 0.020), 0.010 mg/mL quercetin (CS-Q 0.010), and 0.005 mg/mL quercetin (CS-Q 0.005). Our results depicted that adding to the extender with a 0.010 mg/mL Q enhanced (P < 0.01) sperm motility, membrane function, viability, mitochondrial activity, intact acrosome (P < 0.05), SOD (P < 0.001), CAT, and GPx (P < 0.01) compared to the control group at post-thaw. Compared to the control group and other treatment groups after the freeze-thawing process, the addition of 0.005 mg/mL Q into the extender also showed higher (P < 0.05) improvement in the quality of sperm parameters and a higher (P < 0.01) SOD and CAT but did not improve mitochondrial activity and sperm viability. In addition, there was a lower degree of DNA fragmentation index, lower (P < 0.05) lipid peroxidation and ROS in frozen-thawed sperm treated with 0.010 mg/mL and 0.005 mg/mL Q than in control and the other treatment groups. In addition, 0.020 mg/mL Q supplementation into the extender also reduced DNA fragmentation and improved GPx activity compared to the control group at post-thaw. Different concentrations of Q 0.010 and 0.005 mg/mL added to the extender reduced the percentage of abnormal spermatozoa compared to the other groups. The results of this study showed for the first time that the inclusion of an extender with a suitable quercetin concentration of 0.010 mg/mL improved the post-thawed quality of rooster semen.


Assuntos
Caseínas/farmacologia , Criopreservação/métodos , Crioprotetores/farmacologia , Quercetina/farmacologia , Preservação do Sêmen/métodos , Sêmen , Animais , Galinhas , Fragmentação do DNA , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Análise do Sêmen , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos
15.
J Appl Microbiol ; 129(3): 521-531, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32281210

RESUMO

AIM: To evaluate the bacterial composition of collared peccary semen and foreskin mucosa, and to verify the sensitivity of isolates to antimicrobials used in semen conservation and to Aloe vera gel, which is an alternative external cryoprotectant. METHODS AND RESULTS: Nine foreskin mucosa and ejaculate samples from adult animals were used. Sperm characteristics and bacterial load were evaluated in fresh semen. The preputial mucosa and semen bacterial isolates were identified and tested against five concentrations of each antimicrobial (streptomycin-penicillin and gentamicin) and A. vera gel. Corynebacterium sp. and Staphylococcus sp. were isolated in greater numbers than others in both semen (64·10 and 20·51%, respectively) and the foreskin mucosa (60·60 and 24·25%, respectively), and ranged from 0·4 to 21 × 105 colony-forming units (CFU) per ml. The average load of Corynebacterium sp. was negatively correlated (P < 0·05) with the sperm membrane integrity (r = -0·73055) and curvilinear velocity (r = -0·69048). Streptomycin-penicillin and gentamicin inhibited most micro-organisms, and A. vera showed lower antimicrobial activity. CONCLUSION: Several Gram-positive bacteria are present in semen and foreskin mucosa of collared peccary, and the benefits of using primarily penicillin-streptomycin and gentamicin antimicrobials in the bacterial control of diluted semen of these animals are strongly indicated. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides insight into the reproductive microbiota of captive male-collared peccary. This work provides a theoretical basis to assist reproductive biotechnologies for ex situ conservation of the species.


Assuntos
Artiodáctilos/microbiologia , Prepúcio do Pênis/microbiologia , Microbiota , Sêmen/microbiologia , Espermatozoides/fisiologia , Aloe , Animais , Antibacterianos/farmacologia , Artiodáctilos/fisiologia , Bactérias Aeróbias/classificação , Bactérias Aeróbias/efeitos dos fármacos , Bactérias Aeróbias/isolamento & purificação , Crioprotetores/farmacologia , Masculino , Membrana Mucosa/microbiologia , Espermatozoides/citologia
16.
Cryobiology ; 94: 107-115, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32259523

RESUMO

Chinese hamster ovary (CHO) cell lines are the most widely used in vitro cells for research and production of recombinant proteins such as rhGH, tPA, and erythropoietin. We aimed to investigate changes in protein profiles after cryopreservation using 2D-DIGE MALDI-TOF MS and network pathway analysis. The proteome changes that occur in CHO cells between freshly prepared cells and cryopreserved cells with and without Me2SO were compared to determine the key proteins and pathways altered during recovery from cryopreservation. A total of 54 proteins were identified and successfully matched to 37 peptide mass fingerprints (PMF). 14 protein spots showed an increase while 23 showed decrease abundance in the Me2SO free group compared to the control. The proteins with increased abundance included vimentin, heat shock protein 60 kDa, mitochondrial, heat shock 70 kDa protein 9, protein disulfide-isomerase A3, voltage-dependent anion-selective channel protein 2. Those with a decrease in abundance were myotubularin, glutathione peroxidase, enolase, phospho glyceromutase, chloride intracellular channel protein 1. The main canonical functional pathway affected involved the unfolded protein response, aldosterone Signaling in Epithelial Cells, 14-3-3-mediated signaling. 2D-DIGE MALDI TOF mass spectrometry and network pathway analysis revealed the differential proteome expression of FreeStyle CHO cells after cryopreservation with and without 5% Me2SOto involve pathways related to post-translational modification, protein folding and cell death and survival (score = 56, 22 focus molecules). This study revealed, for the first time to our knowledge the proteins and their regulated pathways involved in the cryoprotective action of 5% Me2SO. The use of 5% Me2SO as a cryoprotectant maintained the CHO cell proteome in the cryopreserved cells, similar to that of fresh CHO cells.


Assuntos
Criopreservação , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Proteoma/efeitos dos fármacos , Animais , Células CHO , Cricetulus , Proteoma/metabolismo , Proteômica
17.
Cryobiology ; 94: 95-99, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32304703

RESUMO

Sperm cryopreservation is an essential approach for assisted reproduction and genetic resources conservation in captive giant pandas. Cryopreservation, however, leads to a significant decrease in sperm quality and, consequently, a low fertilization rate. Therefore, it is mandatory to disclose more suitable and efficient freezing strategies for sperm cryopreservation. In the present study, we compared for the first time the performance of two commercial freeze extender (INRA96 versus TEST) freezing methods on post-thawed semen quality. Semen cryopreserved with the INRA96 showed better total motility (73.00 ± 4.84% vs 57.56 ± 3.60%, P < 0.001), membrane integrity (60.92 ± 2.27% vs 40.53 ± 2.97%, P < 0.001) and acrosome integrity (90.39 ± 2.74% vs 84.26 ± 4.27%, P < 0.05) than stored with TEST. There was no significant difference in DNA integrity after thawing between the two extenders (95.69 ± 3.60% vs 94.26 ± 4.84%). In conclusion, the INRA96 method showed to be better for giant panda sperm cryopreservation and should therefore be recommended for use in order to increase success of artificial insemination.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Preservação do Sêmen/métodos , Sêmen , Ursidae , Animais , Masculino , Análise do Sêmen , Espermatozoides/efeitos dos fármacos
18.
Cryobiology ; 94: 73-79, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32315650

RESUMO

As known for different metabolic functions, α-lipoic acid (ALA) has been tested for spermatozoa preservation of animals as well as of human, but not for fish spermatozoa. The present study determined the effects of ALA on short and long-term (cryopreservation) preservation of common carp (Cyprinus carpio) spermatozoa, for the first time. For that, spermatozoa were diluted in extenders containing 0 (control), 0.025, 0.05, 0.1, 0.5, 1, 2, 5, and 10 mM of ALA concentrations in both short-term preservation and cryopreservation. Spermatozoa motility parameters by computer-assisted semen analysis, viability, lipid peroxidation and catalase activity in spermatozoa were conducted in both 2nd and 120th hours of short-term storage and post-thaw samples. Higher percentages of total spermatozoa motility (80 ± 3) and viability (87 ± 3) were observed in 0.5 mM ALA group after 120 h of incubation. In post-thaw samples, higher percentages of these parameters were in 1 mM ALA group (74 ± 3 and 83 ± 2, respectively). Moreover, the results have shown that the addition of ALA until concentrations of 2 mM improved especially spermatozoa curvilinear velocity, maintained viability, and suppressed excessive lipid peroxidation during the preservations. In conclusion, the additions of 0.5 mM ALA for short-term preservation and 1 mM ALA for cryopreservation were the optimal concentrations, and shown the protective effects on common carp spermatozoa, when considering all measured parameters together.


Assuntos
Carpas , Criopreservação/métodos , Crioprotetores/farmacologia , Preservação do Sêmen/métodos , Espermatozoides , Ácido Tióctico/farmacologia , Animais , Catalase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Análise do Sêmen , Motilidade Espermática/efeitos dos fármacos
19.
Cryobiology ; 94: 66-72, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32339491

RESUMO

Type and concentration of cryoprotective agents (CPAs) are important factors which influence the likelihood of a successful ovarian tissue vitrification outcome. In an attempt to address this factor, the present study was conducted to evaluate the impacts of different synthetic polymers (Supercool X-1000, Supercool Z-1000 and PVP K-12) on vitrification of bovine ovarian tissue. From each ovarian pair, fragments were recovered and immediately fixed for analysis (fresh control) or submitted to vitrification, either or not followed by in vitro culture for one or five days. Vitrification was performed using the ovarian tissue cryosystem (OTC) system. The ovarian tissues were intended for histological and viability analysis [Reactive oxygen species (ROS) production and degenerate cells assay (Ethidium homodimer-1)], as well as immunolocalization of AQP3 and AQP9 were measured. The results showed that during almost all the periods after warming, in treatment groups which contain polymer (X-1000, Z-1000 and PVP), the percentage of morphologically normal follicles was the highest in the X-1000 samples. Furthermore, post-thawed X-1000 group revealed stronger labeling for AQP9 in primordial and transitional follicles, when compared with others. However, morphology after cryopreservation did not correlate with follicle viability and function where the levels of degeneration and tissue damage of PVP K-12 group were lower in comparison with X-1000 group and only in PVP K-12 group, ROS level was similar to that of the fresh control group. We believe that in addition to permeating CPAs, the addition of one (Supercool X-1000) or maybe a combination (Supercool X-1000 and PVP K-12) of non-permeating polymers could be useful to improve the outcome for vitrified bovine ovarian tissue.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Ovário , Polímeros/farmacologia , Vitrificação/efeitos dos fármacos , Animais , Bovinos , Feminino
20.
Transplant Proc ; 52(7): 2055-2058, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32299705

RESUMO

BACKGROUND: Lutropin, a luteinizing hormone (LH), is a glycoprotein hormone secreted by gonadotropic cells of the anterior pituitary gland. LH receptors are a promising therapeutic target. The aim of the study was to analyze the protective effect of LH as a component of preservation solutions on the prevention of ischemia-reperfusion injury in isolated porcine kidneys. METHODS: The study used Biolasol solution (Biochefa, Sosnowiec, Poland) modified with the addition of ascorbic acid and LH at a concentration of 0.01 µg/L, 0.1 µg/L, and 1 µg/L. The study was carried out on 40 kidneys from 20 Polish large white pigs. Kidneys were divided into 4 groups, then washed out of the blood, and cooled by complete immersion in a 500-mL preservation solution at 4°C. The total storage time for grafts in the preservation solutions was 2 hours. After this time, the kidneys were perfused. Perfundates intended for biochemical analyses were collected from the renal vein after 0 minutes and after 30 minutes of perfusion. Another perfusion was performed after 24-hour renal storage. The activity of aspartate aminotransferase and alanine aminotransferase, as well as sodium, potassium, total protein, and urea concentrations was determined. RESULTS: LH as a component of preservation solutions affects the degree of ischemia-reperfusion injury of isolated porcine kidneys. CONCLUSIONS: The results obtained suggest that the efficacy of LH in improving ischemic kidney function is dependent on the dose used. The best graft protection was obtained using the lowest concentration of LH: 0.01 µg/L.


Assuntos
Crioprotetores/farmacologia , Rim/efeitos dos fármacos , Hormônio Luteinizante/farmacologia , Soluções para Preservação de Órgãos/farmacologia , Preservação de Órgãos/métodos , Animais , Perfusão , Polônia , Suínos
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