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1.
Sensors (Basel) ; 21(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34451003

RESUMO

The mechanical properties of biological cells, especially the elastic modulus and viscosity of cells, have been identified to reflect cell viability and cell states. The existing measuring techniques need additional equipment or operation condition. This paper presents a cell's viscoelasticity measurement method based on the spheroidization process of non-spherical shaped cell. The viscoelasticity of porcine fetal fibroblast was measured. Firstly, we introduced the process of recording the spheroidization process of porcine fetal fibroblast. Secondly, we built the viscoelastic model for simulating a cell's spheroidization process. Then, we simulated the spheroidization process of porcine fetal fibroblast and got the simulated spheroidization process. By identifying the parameters in the viscoelastic model, we got the elasticity (500 Pa) and viscosity (10 Pa·s) of porcine fetal fibroblast. The results showed that the magnitude of the elasticity and viscosity were in agreement with those measured by traditional method. To verify the accuracy of the proposed method, we imitated the spheroidization process with silicone oil, a kind of viscous and uniform liquid with determined viscosity. We did the silicone oil's spheroidization experiment and simulated this process. The simulation results also fitted the experimental results well.


Assuntos
Técnicas de Imagem por Elasticidade , Animais , Simulação por Computador , Módulo de Elasticidade , Elasticidade , Suínos , Viscosidade
2.
IEEE Trans Biomed Eng ; PP2020 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-33156778

RESUMO

OBJECTIVE: The invisibility of domestic oocyte nucleus in bright field currently forces operators to blindly aspirate nucleus out in oocyte enucleation, usually causing large cytoplasm losses and poor developmental competences of cloned embryos. Although fluorescent labeling of nucleus allows for nucleus localization, the involved photobleaching problems and barriers to the execution of enucleation process limit its online-application in oocyte enucleation. This paper reports a novel label-free oocyte enucleation method for precise removal of the nucleus with less cytoplasm loss. METHODS: The relative positions between the injection pipette and nucleus for complete removal of nucleus with less cytoplasm loss were determined through a finite element modeling of nucleus aspiration. To position injection pipette to the above positions relative to nucleus, the appropriate oocyte orientation and trajectory of injection pipette inside oocyte were derived according to the offline-calibrated 3-D distribution of nucleus and the simulated dynamic drift of nucleus that occurs as injection pipette is maneuvered inside oocyte. Finally, a robotic label-free precise enucleation procedure was established. RESULTS: The experimental results on more than 1000 porcine oocytes proved that this system is capable of reducing cytoplasm loss by 60% at the same level of enucleation success rate and almost doubling the cleavage rate of clone embryos in comparison to blind aspiration method. CONCLUSIONS: The results prove that our method significantly improves the developmental competence of cloned embryos in comparison to manual enucleation method. SIGNIFICANCE: Our method is expected to improve the extremely low success rate of animal cloning in the future.

3.
PeerJ ; 8: e9913, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33083108

RESUMO

The MPF and MAPK genes play crucial roles during oocyte maturation processes. However, the pattern of MPF and MAPK gene expression induced by melatonin (MT) and its correlation to oocyte maturation quality during the process of porcine oocyte maturation in vitro remains unexplored. To unravel it, in this study, we cultured the porcine oocytes in maturation medium supplemented with 0, 10-6, 10-9, and 10-12 mol/L melatonin. Later, we analyzed the MPF and MAPK gene expression levels by RT-PCR and determined the maturation index (survival and maturation rate of oocytes). The GSH content in the single oocyte, and cytoplasmic mitochondrial maturation distribution after porcine oocyte maturation in vitro was also evaluated. We also assessed the effects of these changes on parthenogenetic embryonic developmental potential. The oocytes cultured with 10-9mol/L melatonin concentration showed higher oocyte maturation rate, and MPF and MAPK genes expression levels along with better mitochondrial distribution than the 0, 10-6, and 10-12 mol/L melatonin concentrations (p < 0.05). No significant difference was observed in the survival rates when the oocytes were cultured with different melatonin concentrations. The expression of the MPF gene in the oocytes cultured with 10-6 mol/L melatonin was higher than with 10-12 and 0 mol/L melatonin, and the expression of the MAPK gene in 10-6 and 10-12 group was higher than the control (p < 0.05). As far as the embryonic developmental potential is concerned, the cleavage and blastocyst rate of oocytes cultured with 10-6 and 10-9 mol/L melatonin was significantly higher than the 10-12 mol/L melatonin and control. In conclusion, 10-9-10-6 mol/L melatonin significantly induced the MPF and MAPK gene expression; besides, it could also be correlated with GSH content of single oocyte, mitochondrial maturation distribution, and the first polar body expulsion. These changes were also found to be associated with parthenogenetic embryo developmental potential in vitro.

4.
Animals (Basel) ; 10(2)2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-32012669

RESUMO

Melatonin treatment can improve quality and in vitro development of porcine oocytes, but the mechanism of improving quality and developmental competence is not fully understood. In this study, porcine cumulus-oocyte complexes were cultured in TCM199 medium with non-treated (control), 10-5 M luzindole (melatonin receptor antagonist), 10-5 M melatonin, and melatonin + luzindole during in vitro maturation, and parthenogenetically activated (PA) embryos were treated with nothing (control), or 10-5 M melatonin. Cumulus oophorus expansion, oocyte survival rate, first polar body extrusion rate, mitochondrial distribution, and intracellular levels of reactive oxygen species (ROS) and glutathione of oocytes, and cleavage rate and blastocyst rate of the PA embryos were assessed. In addition, expression of growth differentiation factor 9 (GDF9), tumor protein p53 (P53), BCL2 associated X protein (BAX), catalase (CAT), and bone morphogenetic protein 15 (BMP15) were analyzed by real-time quantitative PCR. The results revealed that melatonin treatment not only improved the first polar body extrusion rate and cumulus expansion of oocytes via melatonin receptors, but also enhanced the rates of cleavage and blastocyst formation of PA embryos. Additionally, melatonin treatment significantly increased intraooplasmic level of glutathione independently of melatonin receptors. Furthermore, melatonin supplementation not only significantly enhanced mitochondrial distribution and relative abundances of BMP15 and CAT mRNA, but also decreased intracellular level of ROS and relative abundances of P53 and BAX mRNA of the oocytes. In conclusion, melatonin enhanced the quality and in vitro development of porcine oocytes, which may be related to antioxidant and anti-apoptotic mechanisms.

5.
RNA Biol ; 16(10): 1494-1503, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31276432

RESUMO

Mammalian fertility is reduced during heat exposure in the summer, but is regained as temperatures decrease in the autumn again. However, the mechanism underlying the phenomenon remains unknown. We investigated heat stress tolerance of germ cells and their surrounding somatic cells, and discovered that microRNA ssc-ca-1 was upregulated after heat stress in cultured porcine granulosa cells (GCs), but not in serum-starved GCs. Ssc-ca-1 inhibited heat shock protein 70 (Hsp70) expression through its 3'- and 5'-UTRs. Although Hsp70 mRNA transcription was induced in GCs by in vivo exposure to heat in the summer, ssc-ca-1 inhibited Hsp70 expression. In ovarian cultures, heat stress-induced Hsp70 expression in primordial but not in growing follicles; ssc-ca-1 expression did not change in primordial follicles, but increased in growing follicles. Consistently, ssc-ca-1 was present in testicular cells and exhibited the same function as in ovarian cells. It modulated the different Hsp70 expression between spermatogonial stem cells and Sertoli cells after scrotal heat stress. This mechanism is of relevance to mammalian fertility in parts of the world dominated by heat stress associated with global climate change.


Assuntos
Células Germinativas/metabolismo , Resposta ao Choque Térmico/genética , MicroRNAs/genética , Termotolerância/genética , Animais , Apoptose/genética , Biomarcadores , Feminino , Regulação da Expressão Gênica , Células da Granulosa/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Masculino , Folículo Ovariano , Células de Sertoli/metabolismo , Suínos
6.
Micromachines (Basel) ; 10(5)2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31137867

RESUMO

As there are significant variations of cell elasticity among individual cells, measuring the elasticity of batch cells is required for obtaining statistical results of cell elasticity. At present, the micropipette aspiration (MA) technique is the most widely used cell elasticity measurement method. Due to a lack of effective cell storage and delivery methods, the existing manual and robotic MA methods are only capable of measuring a single cell at a time, making the MA of batch cells low efficiency. To address this problem, we developed a robotic MA system capable of storing multiple cells with a feeder micropipette (FM), picking up cells one-by-one to measure their elasticity with a measurement micropipette (MM). This system involved the following key techniques: Maximum permissible tilt angle of MM and FM determination, automated cell adhesion detection and cell adhesion break, and automated cell aspiration. The experimental results demonstrated that our system was able to continuously measure more than 20 cells with a manipulation speed quadrupled in comparison to existing methods. With the batch cell measurement ability, cell elasticity of pig ovum cultured in different environmental conditions was measured to find optimized culturing protocols for oocyte maturation.

7.
Vet Microbiol ; 216: 176-182, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29519513

RESUMO

NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV) causing clinical disease outbreaks has been recently reported in China. The recombination occurring among PRRSV strains could lead to the emergence of novel and more virulent viruses. In our previous study, a novel recombinant type 2 PRRSV (TJnh1501) between NADC30-like and modified-live virus (MLV)-like derived from the Chinese highly pathogenic PRRSV was shown to have higher pathogenicity than NADC30-like PRRSV. It remains unknown whether the emergence of the novel recombinant PRRSV strain can lead to variable protection efficacy of the MLV vaccines. In this paper, two typical commercial MLV vaccines were used to evaluate their efficacy to block TJnh1501 infection and onset of clinical symptoms. Our results showed that both MLV vaccines could shorten the period of fever and reduce viral loads in sera, but were not able to reduce the clinical signs and lung lesions indicating that the two commercial MLV vaccines provide limited cross-protection efficacy against the novel recombinant type 2 PRRSV infection. This study gives valuable suggestions for the use of MLV vaccines to control PRRSV infection in the field.


Assuntos
Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Recombinação Genética , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Proteção Cruzada , Filogenia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Suínos , Potência de Vacina , Vacinas Atenuadas/administração & dosagem , Carga Viral , Virulência
8.
J Lab Autom ; 20(4): 471-80, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25911396

RESUMO

This article presents a simple weighing method for spherical cells to avoid the high cost of correlated devices in traditional cell-weighing methods. In this method, the constant falling speeds of the spherical objects in liquid are derived to estimate their masses online. Using this method, the detected density of one type of microbead is highly in accordance with the known value. This method is proved to be capable of detecting tiny variations of the cell mass (at least within 1% of the cell mass). Finally, the proposed method is applied in nuclear transplantation operations, and, for the first time, the proper amount of the removed cytoplasm in porcine enucleation is estimated. The proposed method is able to weigh cells with a success rate of 92% at an average speed of 22 s/cell, and it can be performed on traditional microoperation systems, which makes it easily applicable in biological applications.


Assuntos
Técnicas Citológicas/instrumentação , Técnicas Citológicas/métodos , Oócitos/citologia , Oócitos/fisiologia , Algoritmos , Animais , Citoplasma/fisiologia , Desenho de Equipamento , Micromanipulação , Microesferas , Modelos Biológicos , Técnicas de Transferência Nuclear , Robótica/instrumentação , Robótica/métodos , Suínos
9.
Theriogenology ; 80(1): 50-7, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23623352

RESUMO

An ovine fetal fibroblast cell line highly expressing TLR4 was established by inserting TLR4 into a reconstructive p3S-LoxP plasmid. Transgenic sheep overexpressing TLR4 were produced by transferring TLR4-transfected fetal fibroblasts into metaphase (M)II-stage enucleated oocytes (using SCNT). Because reconstructed embryos derived from MII-stage enucleated oocytes matured in vivo using a delayed-activated method had a higher pregnancy rate (18.52%) than that from MII-stage enucleated oocytes matured in vitro, the former procedure was used. Nine TLR4-transgenic live births were confirmed using polymerase chain reaction and Southern blot analysis. Increased expression of TLR4 at mRNA and protein levels in ear tissues of transgenic lambs were verified using reverse transcription polymerase chain reaction and immunohistochemistry, respectively. More toll-like receptor 4 protein was expressed by peripheral blood monocytes and/or macrophages collected from 3-month-old TLR4-transgenic than nontransgenic lambs at 0, 1, and 4 hours after lipopolysaccharide stimulation. Furthermore, interferon-γ and tumor necrosis factor α secreted by monocytes and/or macrophages of TLR4-transgenic lambs were significantly higher at 1 hour. Therefore, lipopolysaccharide-induced inflammatory responses from monocytes and/or macrophages occurred sooner in TLR4-transgenic lambs, consistent with an enhanced host immune response. In conclusion, transgenic sheep overexpressing TLR4 are a primary model to investigate the role of transgenic animals in disease resistance and have potential for breeding sheep with disease resistance.


Assuntos
Animais Geneticamente Modificados/fisiologia , Resistência à Doença/fisiologia , Expressão Gênica , Ovinos/fisiologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/fisiologia , Animais , Cruzamento , Clonagem de Organismos/métodos , Clonagem de Organismos/veterinária , Feminino , Feto/citologia , Fibroblastos/ultraestrutura , Vetores Genéticos , Lipopolissacarídeos/administração & dosagem , Macrófagos/imunologia , Monócitos/imunologia , Oócitos/ultraestrutura , Gravidez , RNA Mensageiro/análise , Receptor 4 Toll-Like/análise
10.
Rev Sci Instrum ; 84(12): 123703, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24387437

RESUMO

This paper presents a novel micropipette aspiration (MA) method based on a common pneumatic micro-injection system. This method is the first to quantify the influence of capillary effect on aspiration pressure using a balance pressure model, and in return, uses the capillary effect to quantify the aspiration pressure. Subsequently, the seal between the cell and the micropipette is detected to judge and exclude the ineffective MA attempts. The rationality of the balance pressure model is validated by the designed micropipette-filling experiments. Through applied to elasticity-determination of the cells with different sizes, the feasibility and versatility of this MA method are proved. With abilities to quantify aspiration pressures and detect the seam between the cell and the micropipette, our method is expected to advance the application of the commercial pneumatic injector in the MA of cells. Moreover, with the quantified volume of the liquid entering into the micropipette during MA process, our method also has a potential applicability to the study of the permeability of the cell membrane in the future.


Assuntos
Pressão , Sucção/instrumentação , Animais , Módulo de Elasticidade , Estudos de Viabilidade , Modelos Teóricos , Células-Tronco Neurais/citologia , Ratos
11.
BMC Vet Res ; 8: 196, 2012 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-23082910

RESUMO

BACKGROUND: Toll-like receptor 2 (TLR2) is important to host recognition of invading gram-positive microbes. In goats, these microbes can cause serious mastitis, anthrax, tetanus, and other problems. Transgenic goats constitutively over-expressing TLR2 in many tissues serve as a suitable model for the study of the role of TLR2 over-expression in bacterial clearance. RESULTS: Capra hircus TLR2 over-expression vector (p3S-LoxP-TLR2) was used to generate transgenic goats by egg microinjection. The integration efficiency was 8.57%. Real-time PCR and immunohistochemical results confirmed that the goats over-expressing the TLR2 gene (Tg) expressed more TLR2 than wild-type goats (WT). Monocyte-macrophages from the bloodstreams of transgenic goats were stimulated with synthetic bacterial lipoprotein (Pam3CSK4) and by the promotion of interleukin-6 (IL-6) and IL-10 expression in vitro. The oxidative damage was significantly reduced, and lysozyme (LZM) secretion was found to be up-regulated. Ear tissue samples from transgenic goats that had been stimulated with Pam3CSK4 via hypodermic injection showed that transgenic individuals can undergo the inflammation response very quickly. CONCLUSIONS: Over-expression of TLR2 was found to decrease radical damage to host cells through low-level production of NO and MDA and to promote the clearance of invasive bacteria by up-regulating lysozyme secretion and filtration of inflammatory cells to the infected site.


Assuntos
Doenças das Cabras/metabolismo , Muramidase/metabolismo , Estresse Oxidativo/fisiologia , Receptor 2 Toll-Like/biossíntese , Animais , Animais Geneticamente Modificados , Feminino , Doenças das Cabras/genética , Doenças das Cabras/imunologia , Cabras , Histocitoquímica/veterinária , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Malondialdeído/metabolismo , Muramidase/imunologia , Óxido Nítrico/metabolismo , RNA/química , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Regulação para Cima
12.
Theriogenology ; 76(7): 1207-14, 2011 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-21820723

RESUMO

Dromedary camel oocytes have the ability to spontaneous parthenogenetic activation and development in vivo and in vitro. The present study was conducted to investigate changes in mitochondrial distribution, adenosine triphosphate (ATP), and glutathione (GSH) contents and [Ca(2+)] oscillation during in vitro maturation and spontaneous parthenogentic activation of dromedary camel oocytes. Dromedary camel cumulus-oocyte complexes (COCs) were matured in TCM199 medium supplemented with 10% FCS + 10 µg/mL FSH + 10 IU hCG + 10 IU eCG + 10 ng/mL EGF and 50 µg/mL gentamycine. Maturation was performed at 38.5 °C under 5% CO(2) in humidified air for 40 h. After maturation and removal of cumulus cells, oocytes were classified into: immature cultured (Group 1); metaphase II (M II, Group 2); and spontaneously parthenogenetically activated (with 2 polar bodies, Group 3); cleaved embryos (Group 4); and immature oocytes served as a control (Group 5). Cytoplasmic mitochondrial distribution, ATP-GSH contents, calcium [Ca(2+)] oscillation were determined. Results indicated that M II and spontaneously parthenogenetically activated oocytes represent 37.53% and 32.67% of the cultured oocytes, respectively, and 3.3% cleaved and developed to 2-16-cell stage embryos. Mitochondrial distribution, ATP-GSH contents and [Ca(2+)] oscillation were significantly (P < 0.01) differ between immature and matured dromedary camel oocytes. Mitochondrial distribution showed clustering form in matured oocytes without polar body. High polarized mitochondrial distribution (HPM) was detected in M II and spontaneously parthenogenetically activated oocytes, and the intensity of MitoTracker Red was higher in spontaneously parthenogenetically activated than M II. ATP-GSH contents and the duration of [Ca(2+)] oscillation were significantly (P < 0.01) higher in spontaneously parthenogenetically activated than M II oocytes or that matured without polar body. In conclusion, the higher incidence of spontaneously parthenogenetically activated in vitro matured dromedary camel oocytes could be attributed to the high polarized mitochondrial distribution associated with significantly higher ATP-GSH contents and duration of [Ca(2+)] oscillation.


Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Camelus , Glutationa/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterinária , Mitocôndrias/metabolismo , Oócitos/metabolismo , Animais , Polaridade Celular , Feminino , Mitocôndrias/ultraestrutura , Oócitos/ultraestrutura
13.
Theriogenology ; 75(4): 638-46, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21074839

RESUMO

Deterioration in the quality of mammalian mature oocytes during metaphase-II (M-II) arrest is called "oocyte aging". Although histone acetylation may affect the progression of aging in murine oocytes, the mechanism is unknown. The objective was to determine the role of ooplasmic reactive oxygen species (ROS) in acetylation of histone H4 at lysine 12 (acH4K12) in porcine aged oocytes in vitro. Based on immunostaining with a specific antibody, acetylation of H4K12 in porcine oocytes increased during in vitro aging, which coincided with changing patterns of ooplasmic ROS content. Furthermore, both hydrogen peroxide (H(2)O(2)), and the mitochondrial membrane potential disrupter, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which can moderately elevate oocyte ROS content, significantly increased acetylation levels of H4K12 in porcine oocytes. It was noteworthy that acetylation in the CCCP group was decreased when ROS was counteracted by cysteine, a common antioxidant. In addition, the intracellular mRNA abundance of acetyltransferase gene HAT1 in aged and H(2)O(2) treated oocytes was higher than in M-II phase oocytes, suggesting that HAT1 was involved in this reaction. After parthenogenetic activation, a lower proportion of oocytes developed to the blastocyst stage after CCCP or H(2)O(2) treatment when compared with M-II phase oocytes (20 and 0% for CCCP and H(2)O(2) groups, respectively, versus 42% for the M-II group, P < 0.05). In conclusion, elevated levels of H4K12 acetylation were attributed to increased ooplasmic ROS content during porcine oocyte aging in vitro.


Assuntos
Histonas/metabolismo , Oócitos/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Suínos/genética , Acetilação/efeitos dos fármacos , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Técnicas de Cultura de Células , Cisteína/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Epigenômica , Feminino , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Peróxido de Hidrogênio/farmacologia , Oócitos/efeitos dos fármacos
14.
Reprod Fertil Dev ; 21(2): 323-32, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19210923

RESUMO

The present study aims to investigate major changes in porcine oocytes during ageing in vitro. After the oocytes were cultured for 44, 56, 68 and 80 h, changes to porcine oocytes in ultrastructure, mitochondrial distribution, glutathione (GSH) and ATP content, Ca(2+) release patterns and developmental competence after electro-activation were observed. Mitochondria were evenly distributed in oocytes at 44 h, aggregated in clusters or in peripheral cytoplasm at 68 h and dimly dispersed throughout ooplasm at 80 h. Mitochondrial shape during ageing was also observed by transmission electron microscopy (TEM) at the same time intervals. Most mitochondria were spherical at 44 h, and became elongated when the culture time was extended to 68 h and 80 h. Moreover, mitochondrial clustering became increasingly loose from 56 h. Lipid droplets in oocytes appeared prominent and electron-dense at 44 h, but electron density was lost at 56 h. Lipid droplets were solidified as of 68 h. There was an age-dependent decrease in ATP content per oocyte. Glutathione content per oocyte decreased significantly and remained lower after 56 h. Amplitudes of [Ca(2+)] rise decreased dramatically following 56 h, and the time required for [Ca(2+)] to plateau became shorter after electro-activation with prolonged culture time. Cleavage and blastocyst rates of aged oocytes progressively decreased, while the fragmentation rate gradually increased after electro-activation. It is concluded that abnormal changes in mitochondria, lipid droplets, Ca(2+) release after electro-activation, and ATP and GSH content in oocytes during ageing may result in poor developmental competence of parthenotes.


Assuntos
Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio , Senescência Celular , Glutationa/metabolismo , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Oócitos/metabolismo , Animais , Células Cultivadas , Estimulação Elétrica , Feminino , Mitocôndrias/ultraestrutura , Oócitos/ultraestrutura , Partenogênese , Suínos , Fatores de Tempo
15.
Anim Reprod Sci ; 114(1-3): 279-88, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19008058

RESUMO

The present study was to investigate effects of synthetic oviductal fluid (SOF) and Charles Rosenkrans medium (CR1) culture systems on developmental competence and cell apoptosis of ovine in vitro fertilization (IVF) embryos. Ovine presumptive IVF zygotes were cultured in the following six media: (1) SOF supplemented with amino acids (SOFaa) and 8 mg/ml bovine serum albumin (BSA) for 9 days (SOFaaBSA); (2) SOFaa supplemented with 10% fetal bovine serum (FBS) for 9 days (SOFaaFBS); (3) SOFaaBSA for first 3 days and then SOFaaFBS for later 6 days (SOFaaBSA-FBS); (4) CR1 supplemented with amino acids (CR1aa) and 8 mg/ml BSA for 9 days (CR1aaBSA); (5) CR1aa supplemented with 10% FBS for 9 days (CR1aaFBS); (6) CR1aaBSA for first 3 days and then CR1aaFBS for later 6 days (CR1aaBSA-FBS). The rates of blastocyst and hatched blastocyst in group 1, group 3 and group 6 were not different (P>0.05), but were greater than in other three groups (P<0.05). In SOF and CR1 cultural system, SOFaaBSA and CR1aaBSA-FBS provided the highest blastocyst rates respectively. Both numbers of total cell and trophectoderm (TE) in expanded or hatched blastocyst from SOFaaBSA were significantly higher than CR1aaBSA-FBS (P<0.05). However, the inner cell mass (ICM) cell number and ratio of ICM to TE cell in expanded or hatched blastocysts were not different between two groups (P>0.05). The apoptotic signals were firstly observed at 8-cell stage in two groups and became stronger and stronger with the development of embryos. Rates of embryos with apoptotic signals in group 6 at morula or blastocyst were greater than in group 1 (P<0.05). The apoptotic nuclei numbers of morula or blastocyst in group 6 were also significantly higher than group 1 (P<0.05). It is concluded that CR1aaBSA-FBS can support in vitro development of ovine IVF embryos, but SOFaaBSA is more suitable.


Assuntos
Apoptose/efeitos dos fármacos , Meios de Cultura/química , Técnicas de Cultura Embrionária/veterinária , Fertilização In Vitro/veterinária , Ovinos/embriologia , Zigoto/crescimento & desenvolvimento , Animais , Contagem de Células , Feminino , Coloração e Rotulagem
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