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1.
Reprod Domest Anim ; 55(3): 351-363, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31903647

RESUMO

Oocyte maturation plays a vitally important role in the reproduction of pigs. However, the roles of messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) in the developmental process of porcine oocyte maturation are still largely unclear. In this study, a transcriptome analysis of germinal vesicle (GV) and metaphase II (MII) of oocytes from Chinese Duroc pigs was performed. A total of 1,753,030 and 2,486 differentially expressed (DE) mRNAs, 22,811 and 9,868 DE lncRNAs were identified between GV and MII stages, respectively. Furthermore, functional enrichment analysis showed that the common DE mRNAs and DE lncRNAs during the process of maturation were mainly involved in biological process and cellular components. Our study provides new insights of the expression changes of mRNAs and lncRNAs during GV and MII stages, which might contribute to the maturation of oocytes. These results greatly improve our understanding of the molecular mechanisms regulating the maturation of oocytes in pigs.


Assuntos
Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Animais , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos/métodos , Metáfase/fisiologia , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Sus scrofa
2.
Arq. bras. med. vet. zootec. (Online) ; 72(1): 25-32, Jan.-Feb. 2020. tab, graf
Artigo em Português | LILACS, VETINDEX | ID: biblio-1088924

RESUMO

A fim de avaliar o efeito de diferentes doses da rbST sobre a dinâmica folicular, a produção e a maturação in vitro de oócitos, 20 vacas Sindi, divididas em três grupos, receberam um dispositivo de progesterona intravaginal, estradiol e PGF2α, além de 2mL de solução salina (grupo controle), 250 (grupo rbST 250) ou 500mg de rbST (grupo rbST 500). Cinco dias depois, realizou-se a ovum pick up, e os complexos cumulus-oócitos (CCOs) recuperados foram selecionados, classificados e maturados in vitro. Os dados de contagem foram comparados pelo procedimento glht (General Linear Hypothesis Test), e os dados em porcentagem foram submetidos ao qui-quadrado, no programa estatístico R, onde as diferenças foram consideradas significativas quando P<0,05. Não houve diferença (P>0,05) entre os grupos quanto à quantidade de folículos e à taxa de maturação. Os grupos rbST 250 e rbST 500 foram significativamente superiores (P<0,05) ao grupo controle em relação ao número de folículos grandes (0,42±0,20 vs. 0). O grupo rbST 500 apresentou maior (P<0,05) porcentagem de oócitos viáveis (91,52%) do que os grupos controle (67,85%) e rbST 250 (53,33%). A rbST aumenta o número de folículos grandes, e 500mg de rbST aumentam a porcentagem de oócitos viáveis em vacas Sindi.(AU)


In order to evaluate the effect of different doses of rbST on the follicular dynamics, production, and in vitro maturation of oocytes, 20 Sindhi cows were divided into three groups, receiving an intravaginal progesterone device, estradiol and PGF2α, and 2mL of solution saline (Control Group), 250 (rbST 250 Group) or 500mg rbST (rbST 500 Group). Five days later, the ovum pick up was performed, and the cumulus-oocyte (CCO) complexes recovered were selected, classified, and matured in vitro. The counting data were compared by the glht (General Linear Hypothesis Test) procedure, and the percentage data were submitted to Qui- square, in the statistical program R, where differences were considered significant when P< 0.05. There was no difference (P> 0.05) between the groups regarding follicle quantity and maturation rate. The rbST 250 and rbST 500 groups were significantly higher (P< 0.05) than the Control group in relation to the number of large follicles (0.42±0.20 versus 0). The rbST 500 group presented higher (P< 0.05) percentage of viable oocytes (91.52%) than the Control (67.85%) and rbST 250 (53.33%) groups. rbST increases the number of large follicles and 500mg rbST increases the percentage of viable oocytes in Sindhi cows.(AU)


Assuntos
Animais , Feminino , Bovinos , Oócitos , Indução da Ovulação/veterinária , Hormônio do Crescimento/administração & dosagem , Técnicas de Maturação in Vitro de Oócitos/veterinária
3.
Theriogenology ; 141: 35-40, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31518726

RESUMO

Oocyte in vitro maturation (IVM) plays a pivotal role in in vitro embryo production. However, the efficiency of IVM is still low and needs to be further improved. In the present study, we evaluated the beneficial effects of mogroside V, an extract derived from Siraitia grosvenorii, on oocyte IVM. Porcine cumulus-oocyte complexes were cultured in IVM medium supplemented or not supplemented with mogroside V for 40 h. We found that mogroside V supplementation increased the percentage of oocyte first polar body extrusion and improved subsequent blastocyst formation after parthenogenetic activation. Furthermore, mogroside V reduced the levels of reactive oxygen species (ROS) and increased the mRNA expression of oxidative stress-related genes (SOD, CAT and SIRT1). Moreover, mogroside V supplementation enhanced the mitochondrial content, mtDNA copy number, mitochondrial membrane potential (ΔΨm), ATP generation, and the relative mRNA expression of mitochondria-related genes (PGC-1α and TFAM). In summary, our findings demonstrate that mogroside V supplementation reduces intracellular ROS levels and enhances mitochondrial function to promote porcine oocyte IVM.


Assuntos
Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Suínos/fisiologia , Triterpenos/farmacologia , Animais , DNA Mitocondrial , Fertilização In Vitro/veterinária , Mitocôndrias/fisiologia , Oócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
4.
Theriogenology ; 141: 54-61, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31518729

RESUMO

The concentration of fatty acids in follicular fluid reflect the physical condition of donors, and palmitic acid (PA) is a major component of follicular fluid. The present study examined the effect of PA on in vitro oocyte growth and investigated the molecular backgrounds of the PA induced-low quality oocytes. Oocyte-granulosa cell complexes (OGCs) were collected from early antral follicles of gilts. The OGCs were cultured for 14 days in a medium containing 0.5 mM PA or vehicle (BSA). PA was found to reduce granulosa cell (GCs) proliferation (0.73 fold) and viability (93.9% vs. 85.8%) and increase lipid content in oocytes and GCs. Oocytes developed in the presence of PA had low developmental ability to the blastocyst stage. In addition, PA affected developmental and epigenetic markers of histone modifications in oocytes; levels of H4K12 acetylation and H3K9 demethylation. PA affected cellular proliferation, apoptosis and endoplasmic reticulum stress markers along with reducing the phosphor-AKT/AKT levels and increasing the expression levels of caspase-3 and CHOP in GCs. Incubation of OGCs with PA increased ceramide content in the GC, and addition of ceramide to the culture medium inhibited GC proliferation. In conclusion, it is suggested that high PA content in the medium reduces viability and proliferation through ceramide accumulation, and PA impaires the developmental ability of oocytes grown in vitro. In addition, high-fat conditions induce changes in the histone modifications of oocytes grown in vitro.


Assuntos
Células da Granulosa/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Ácido Palmítico/toxicidade , Suínos , Animais , Apoptose , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Retículo Endoplasmático , Feminino
5.
Theriogenology ; 141: 91-97, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31521883

RESUMO

Autophagy is a critical process in early mammalian embryogenesis. Mammalian target of rapamycin (mTOR) inhibitors are major regulators of autophagy. However, mTOR plays a vital role in major signaling pathways controlling cell growth and metabolism; thus, more secure autophagy activation methods should be considered. The present study investigated the effects of supplementary trehalose, a novel mTOR-independent autophagy enhancer, on oocyte maturation and embryonic development after parthenogenetic activation (PA). Trehalose treatment during in vitro maturation (IVM) did not affect the nuclear maturation rates of oocytes. Oocytes treated with 25 mM trehalose during IVM had a significantly higher (P < 0.05) blastocyst formation rate (64.2%) after PA compared to that in control oocytes (52.0%). Blastocyst quality was also improved in the trehalose-treated group. The total cell numbers for blastocyst formation and expanded blastocyst formation were significantly increased in the trehalose-treated group (52.2% and 27.7%, respectively) compared to those in the control group (36.9% and 11.0%, respectively). Trehalose treatment led to the increased expression of LC3, an autophagy marker, in metaphase II oocytes and 4-cell stage embryos. Gene expression analysis revealed that the expression of several autophagy related genes (LAMP2, pATG5, and LC3) increased, while the Bax/Bcl2 ratio and pro-apoptotic Bak transcript levels were decreased in the trehalose-treated group. In conclusion, these results indicate that treatment with trehalose during IVM improved the developmental potential of porcine embryos by down-regulation of pro-apoptotic genes and up-regulation of autophagy-related genes and marker. Trehalose may be useful for the large-scale production of high-quality porcine blastocysts in vitro.


Assuntos
Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Partenogênese , Suínos , Trealose/farmacologia , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/metabolismo , Relação Dose-Resposta a Droga , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Oócitos/fisiologia , Trealose/administração & dosagem
6.
Theriogenology ; 141: 120-127, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31536861

RESUMO

Mastitis has deleterious effects on ovarian function and reproductive performance. We studied the association between plasma or follicular fluid (FF) obtained from endotoxin-induced mastitic cows, and oocyte developmental competence. Lactating Holstein cows were synchronized using the Ovsynch protocol. On Day 6 of the synchronized cycle, an additional PGF2α dose was administered, and either Escherichia coli endotoxin (LPS, 10 µg; n = 3 cows) or saline (n = 3 cows) was administered to one udder quarter per cow, 36 h later. Milk samples were collected and rectal temperatures recorded. Cows treated with LPS showed a typical transient increase in body temperature (40.3 °C ±â€¯0.4), whereas cows treated with saline maintained normal body temperature (38.9 °C ±â€¯0.04). A higher (P < 0.05) somatic cell count was recorded for cows treated with LPS. Plasma samples were collected and FF was aspirated from the preovulatory follicles by transvaginal ultrasound probe, 6 h after LPS administration. Radioimmunoassay was performed on plasma samples to determine estradiol and cortisol concentrations. Either FF or plasma was further used as maturation medium. In the first experiment, oocytes were matured in TCM-199 (Control) or in FF aspirated from cows treated with saline (FF-Saline) or LPS (FF-LPS). Cleavage rate to the 2- to 4-cell stage embryo did not differ among groups. However, the proportion of developed blastocysts on Day 7 postfertilization in the FF-LPS group tended to be lower for that in FF-Saline and was lower (P < 0.05) than that in the Control groups (10.6 vs. 22.4 and 24.4%, respectively). In the second experiment, oocytes were matured in TCM-199 (Control), or in plasma obtained from cows treated with saline (Plasma-Saline) or LPS (Plasma-LPS). Similar to the FF findings, cleavage rate did not differ among groups; however, the proportion of developing blastocysts tended to be lower in the Plasma-LPS group than in the Plasma-Saline group and was lower (P < 0.05) from that in the Control group (11.0 vs. 25.5 and 34.7%, respectively). The proportion of apoptotic cells per blastocyst, determined by TUNEL assay, did not differ among the experimental groups. The findings shed light on the mechanism by which mastitis induces a disruption in oocyte developmental competence. Further studies are required to clarify whether the negative effect on oocyte developmental competence is a result of LPS, by itself, or due to elevation of secondary inflammatory agents.


Assuntos
Bovinos , Líquido Folicular/química , Lipopolissacarídeos/toxicidade , Mastite Bovina/induzido quimicamente , Oócitos/efeitos dos fármacos , Plasma , Animais , Meios de Cultura , Fragmentação do DNA , Feminino , Técnicas de Maturação in Vitro de Oócitos/veterinária
7.
Theriogenology ; 141: 134-141, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31541782

RESUMO

The effectiveness of the use of natriuretic peptide C (NPPC) in the blocking of meiosis has already been proven in several species. However, there are no reports on the use of NPPC in the activation of metabolic processes in embryos. Whereas modulations of cAMP concentrations alter the lipid metabolism of bovine oocytes, the present study aims to evaluate the effect of NPPC on the development, lipid content and transcript levels of genes related to lipid metabolism of IVP bovine embryos. For this purpose, ovaries were obtained from a slaughterhouse, and oocytes were fertilized in vitro (D0). From D5 of in vitro culture, embryos were treated with 100 nM NPPC (NPPC group) or with no NPPC (Control group) and evaluated in terms of Blastocyst (D7) and hatching rates (D10). For the assessment of the cytoplasmatic lipid amounts, blastocysts were stained with Sudan Black B dye. The embryonic lipid profile was investigated by electrospray ionization desorption-mass spectrometry (DESI-MS). The abundance of nine transcripts related to lipid metabolism were assessed using the Biomark HD system. For statistical analysis, blastocyst and hatching rates, lipid content by the Sudan Black B and variation of gene expression between groups were compared by Student t-test. For lipid profile analysis, principal component analysis (PCA) and fold-change were performed. The embryo lipid content was similar between NPPC (881 ±â€¯3.7) and Control (883 ±â€¯5.2) groups (p > 0.05). However, cholesteryl esters and TAGs were downregulated by NPPC at multiple levels according to the DESI-MS profiles. Of the analyzed genes, ELOVL6 and SREBF1 showed an up-regulation in the control group (p < 0.05), while CPT2 was observed to be up-regulated in the NPPC-treated embryos. There was no significant difference in the blastocyst production rate between NPPC (44.4%) and Control (42.4%), however the hatching rate at D10 was higher (p < 0.05) in the NPPC group (69.77%) when compared to the Control group (48.33%). These findings demonstrate that NPPC alters the mRNA expression of genes related to lipid metabolism and that it exerts a positive effect on the hatching rates of IVP Bos taurus indicus embryos.


Assuntos
Bovinos/embriologia , Meios de Cultura/química , Técnicas de Cultura Embrionária/veterinária , Peptídeo Natriurético Tipo C/farmacologia , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/fisiologia , Bovinos/genética , Feminino , Fertilização In Vitro/veterinária , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/química , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
8.
Theriogenology ; 141: 146-152, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31541784

RESUMO

Differences in reproductive physiology between cattle breeds may help to explain distinct responses to assisted reproductive techniques and to define breed-specific protocols with improved efficiency. Germinal vesicle (GV) stage oocytes are characterized by increasing levels of chromatin compaction enclosed within the nucleus (graded from GV0 to GV3), associated with different developmental competence. The first objective of this study was to characterize chromatin configuration of GV stage oocytes recovered by OPU at random days of the estrous cycle from Nelore (Bos indicus) and Holstein (Bos taurus) cows. In Nelore 90% of the oocytes presented advanced stages of chromatin compaction associated with higher developmental competence (GV2 and GV3), while in Holstein, only 65% of the oocytes were at these stages. Then, aiming to obtain a more homogeneous population of oocytes in Holstein, we tested two synchronization protocols combining aspiration of all visible follicles at a random day (day 0), two IM injections of FSH 12 h apart on day 2, and OPU on day 4 (OPU/D4) or 5 (OPU/D5). The protocol OPU/D4 provided around 45% of the oocytes with low chromatin compaction (GV1), while the protocol OPU/D5 provided 70% of the oocytes at GV2 and 20% at GV3. Finally, we assessed the effects of a culture system known to prevent meiotic resumption on chromatin configuration of the GV2 enriched oocyte population obtained with the protocol OPU/D5. After 9 h of culture most oocytes transited from GV2 to GV3, with 90% of the oocytes at GV3 stage. This study demonstrates differences between Nelore and Holstein cows regarding patterns of chromatin configuration that may account for their different performance in IVM/IVF. In addition, it provides novel references for the design of protocols aiming to regulate oocyte quality before IVM for the optimization of IVF outcomes.


Assuntos
Bovinos/genética , Bovinos/fisiologia , Montagem e Desmontagem da Cromatina/fisiologia , Oócitos/fisiologia , Animais , Técnicas de Cultura Embrionária/veterinária , Ciclo Estral , Hormônio Foliculoestimulante , Técnicas de Maturação in Vitro de Oócitos/veterinária , Meiose , Óvulo , Doadores de Tecidos , Coleta de Tecidos e Órgãos
9.
Theriogenology ; 142: 207-215, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31614287

RESUMO

One of the major challenges of artificial reproductive technologies is to develop new methods for producing greater numbers of embryos. An oocyte fosters the ability to develop into an embryo before oocyte meiotic resumption. The aim of the present study was to assess the effect of adenosine (ADO), a purine nucleoside found in follicular fluid, on the inhibition of oocyte meiotic resumption and the production of blastocysts. The results showed the efficacy of ADO to inhibit oocyte meiotic resumption. The use of ADO (3 mM) during a pre-in vitro maturation (pre-IVM) culture period of 6 h resulted in a significant increase (p < 0.05) of blastocysts compared to control conditions with no pre-IVM culture period. No effect on the percentage of cleavage was observed. The effect of adenosine on blastocyst yield was time- and concentration-dependent with an optimum effect at 3 mM for 6 h. Supplementing the ADO pre-IVM culture medium with estradiol, follicle-stimulating hormone, progesterone, epidermal growth factor, insulin-like growth factor-2 or reelin did not improve the blastocyst yield. Transcriptional analyses of ADO-treated cumulus cells revealed that NRP1, RELN, MAN1A1, THRA and GATM were up-regulated. Finally, bioinformatic analysis identified mitochondrial function as the top canonical pathway affected by ADO. This opens up new opportunities for further investigations.


Assuntos
Adenosina/farmacologia , Bovinos , Técnicas de Cultura de Células/métodos , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Animais , Bovinos/embriologia , Bovinos/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária/métodos , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/genética , Feminino , Fertilização In Vitro/métodos , Fertilização In Vitro/veterinária , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Análise em Microsséries , Oócitos/citologia , Oócitos/fisiologia
10.
Theriogenology ; 142: 296-302, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31708194

RESUMO

Environmental stresses, such as heat stress (HS), have been shown to have diverse effects on the developmental competence of oocytes. The aim of this study was to determine the effect of exogenous conjugated linoleic acid (CLA) supplementation in maturation medium on bovine oocyte maturation and developmental competence under HS condition. Accordingly, cumulus-oocyte complexes (COCs) were cultured at 41 °C and 38.5 °C for the first and second 12 h of maturation in the presence of 0 (PC), 50 (CLA50-HS) and 100 (CLA100-HS) µM CLA. Also, a group of COCs were cultured at 38.5 °C for 24 h of maturation without CLA supplementation as negative control (NC). Nuclear maturation, level of intracellular glutathione (GSH), reactive oxygen species (ROS) content, cleavage and blastocyst rates as well as relative expression of BAX, and BCL2 genes in blastocysts were investigated. Our finding for the PC and NC groups revealed that HS decreased the percentage of MII oocytes, cleavage and blastocyst rates (P < 0.05). Moreover, HS lead to an increase in ROS levels and relative expression of BAX gene, decreased the intracellular content of GSH and relative expression of BCL2 gene (P < 0.05). However, the cleavage and blastocyst rates tended to increase in the CLA-supplemented groups compared to PC group (p < 0.10). Also, ROS and GSH levels in the matured oocytes decreased and increased in the CLA50-HS group compared to the PC group (P < 0.05), respectively. The ratio of expression levels of BAX to BCL2 genes was not different between the PC and CLA50-HS groups (P > 0.05). These findings suggest that HS has undesirable effects on the maturation competence of bovine oocyte and subsequent embryo development while administration of CLA can ameliorate some of adverse effects of HS.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Transtornos de Estresse por Calor/patologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Ácidos Linoleicos Conjugados/farmacologia , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Animais , Bovinos , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/farmacologia , Feminino , Fertilização In Vitro , Glutationa/metabolismo , Transtornos de Estresse por Calor/metabolismo , Transtornos de Estresse por Calor/veterinária , Resposta ao Choque Térmico/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/patologia , Oócitos/fisiologia , Espécies Reativas de Oxigênio/metabolismo
11.
Theriogenology ; 142: 320-327, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31711691

RESUMO

To optimize the protocols for assisted reproductive techniques (ARTs) in collared peccary (Pecari tajacu Linnaeus, 1758), we evaluated various conditions for oocyte in vitro maturation (IVM) and chemical activation. Initially, we assessed the IVM rates, cumulus-oocyte complex (COC) quality, and oocyte morphometry in the absence or presence of epidermal growth factor (EGF). There was no difference between the COCs matured in absence or presence of EGF for the expansion of cumulus cells (97.6% ±â€¯1.2 vs. 100% ±â€¯0.0), presence of first polar body (65.9% ±â€¯1.2 vs. 70.5% ±â€¯1.8), nuclear status in second metaphase (62.5% ±â€¯11.6 vs. 68.4% ±â€¯4.9), cytoplasmic maturation (100.0% ±â€¯0.7 vs. 75.0% ±â€¯0.7), reactive oxygen species levels (0.5 ±â€¯0.2 vs. 0.3 ±â€¯0.1), and mitochondrial membrane potential (1.1 ±â€¯0.2 vs. 1.1 ± 0.1). However, the zona pellucida thickness of matured COCs was reduced in the presence of EGF. Thus, the EGF group was used for further experiments. The oocytes were artificially activated with ionomycin and four secondary activator combinations [6-dimethylaminopurine (6D), 6D and cytochalasin B (6D + CB), cycloheximide (CHX), and CHX and CB (CHX + CB)]. The effect of immature COCs based on cumulus cell layers and cytoplasm homogeneity (GI and GII or GIII COCs) on embryonic development and quality was evaluated. There was no difference in the cleavage rates among the groups of secondary activators. The cleavage rates of embryos derived from GI/GII and GIII COCs were greater than 72.2% and 25.0%, respectively. Moreover, treatment with CHX showed a reduction in the cleavage rate of embryos derived from GIII COCs when compared to the cleavage rate of embryos derived from GI/GII COCs (P < 0.05). Nevertheless, higher rates of blastocyst/total GI and GII COCs were observed in the 6D group (27.6% ± 0.3) compared to CHX group (6.9% ± 0.3). Additionally, only 6D treatment resulted in the production of embryos derived from GIII COCs (25.0% ± 0.2). The percentage of the ICM/total cell ratio was also greater in blastocysts derived from 6D (42.5% ± 19.0), 6D + CB (37.9% ± 21.9), and CHX + CB (43.8% ± 19.6) groups when compared to CHX (3.6% ± 0.1) group. Thus, the combination of ionomycin and 6D could produce collared peccary embryos by activation of both GI/GII COCs and GIII COCs. These optimized IVM conditions using EGF and chemical activation using ionomycin and 6D in collared peccaries form the first steps for establishing ARTs to conserve this species.


Assuntos
Adenina/análogos & derivados , Artiodáctilos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Ionomicina/farmacologia , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Adenina/farmacologia , Animais , Artiodáctilos/embriologia , Células Cultivadas , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Transferência Nuclear/veterinária , Oócitos/citologia , Oócitos/fisiologia , Oogênese/efeitos dos fármacos , Oogênese/fisiologia , Partenogênese/fisiologia
12.
Anim Reprod Sci ; 211: 106220, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31785645

RESUMO

The knowledge about the biological events that regulate lipid metabolism in oocytes and embryos in buffalo is scarce. Lipogenesis, lipolysis, transport and oxidation of fatty acids (FAs) occur in gametes and embryonic cells of all mammalian species, as an intrinsic component of energy metabolism. In oocytes and cumulus cells, degradation of lipids is responsible for the production of ATP that is essential for the metabolic processes that lead to oocyte maturation in in vivo and in vitro culture conditions. Similarly, throughout embryo development, blastomeres have the capacity to use exogenous and/or endogenous lipid reserves to serve as an energy source necessary for early embryonic development. In addition, supplementation of culture media with L-carnitine to promote lipid metabolism during in vitro oocyte maturation and early embryonic development leads to an improved embryo quality. The limited scientific evidence available in buffalo indicates there is relatively greater oocyte lipid content as compared with many other species that undergoes a dynamic distribution during folliculogenesis and follicle maturation and that has a positive effect on oocyte maturation and embryo development when there is L-carnitine supplementation of the media. Advances in the understanding of the biological peculiarities of lipid metabolism, and the consequences of its alteration on the quality of buffalo gametes and embryos, therefore, are necessary to design specific culture media and laboratory procedures as a strategy to increase in vitro-derived embryo production rates.


Assuntos
Búfalos/fisiologia , Técnicas de Cultura Embrionária/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Metabolismo dos Lipídeos/fisiologia , Oócitos/fisiologia , Animais , Búfalos/embriologia
13.
Reprod Domest Anim ; 54 Suppl 4: 78-81, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31625232

RESUMO

The acquisition of equine oocyte developmental capacity is ensured by the follicular environment, such as granulosa cells, which could reflect the meiotic development potential of immature oocytes. This study evaluated the relationship between DNA fragmentation of granulosa cells, using the chromatin dispersion test, and equine oocyte meiotic development after in vitro maturation. Granulosa cells and cumulus-oocytes complexes (n = 50) were recovered from slaughterhouse-derived ovaries. Oocytes were in vitro matured, stained and evaluated under fluorescence microscopy. Maturation rates were classified into outstanding, medium and poor levels of maturation using 25th and 75th percentiles as thresholds. For DNA assessment, each sample was processed with the Ovoselect® kit (Halotech DNA). High, low and total DNA fragmentation percentages were compared among levels of maturation rates by ANOVA, followed by Duncan test. Results were expressed as mean ± SE. Total and high DNA fragmentation rates of granulosa cells were significantly higher (p < 0.05) in follicles whose oocytes had reached outstanding maturation level than those originating from follicles whose oocytes had reached poor maturation level. In conclusion, the DNA fragmentation analysis of equine granulosa cells can be a valuable test to identify equine oocytes showing the best meiotic competence after in vitro maturation.


Assuntos
Fragmentação do DNA , Células da Granulosa/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Animais , Feminino , Cavalos , Meiose/fisiologia
14.
Reprod Domest Anim ; 54 Suppl 4: 82-85, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31625234

RESUMO

The aim of this study was to induce the cold-inducible RNA-binding protein (CIRBP) expression on cumulus-oocyte complexes (COCs) through exposure to a sub-lethal cold shock and determine the effects of hypothermic temperatures during the in vitro maturation of bovine oocytes. Nuclear maturation, cortical granule redistribution and identification of cold-inducible RNA-binding protein (CIRBP) were assessed after 24 hr of in vitro maturation of control (38.5°C) and cold-stressed oocytes (33.5°C). The presence of CIRBP was assessed by Western blot in COCs or denuded oocytes and their respective cumulus cells. Based on the odds ratio, cold-stressed oocytes presented higher abnormal cytoplasmic distribution of cortical granules and nuclear maturation than the control group. Although CIRBP was detected in both control and cold-stressed groups, cold-stressed COCs had 2.17 times more expression of CIRBP than control COCs. However, when denuded oocytes and cumulus cells were assessed separately, CIRBP only was detected in cumulus cells in both groups. In conclusion, cold shock induced CIRBP expression, but it negatively affected nuclear maturation and cortical granule distribution of bovine oocytes. Moreover, the expression of CIRBP was only identified in cumulus cells but not in oocytes.


Assuntos
Resposta ao Choque Frio/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Proteínas de Ligação a RNA/metabolismo , Animais , Bovinos , Núcleo Celular/fisiologia , Células do Cúmulo/metabolismo , Grânulos Citoplasmáticos , Feminino , Oócitos/citologia , Oócitos/metabolismo
15.
Reprod Domest Anim ; 54 Suppl 4: 72-77, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31625244

RESUMO

The coenzyme Q10 (CoQ10) is a potent antioxidant with critical protection role against cell oxidative stress, caused by the mitochondrial dysfunction. This study evaluated the effects of CoQ10 supplementation to in vitro maturation (IVM) or embryo culture media on the maturation, fertilization and subsequent embryonic development of pig oocytes and embryos. Maturation (Experiment 1) or embryo culture (Experiment 2) media were supplemented with 0 (control), 10, 25, 50 and 100 µM CoQ10. The addition of 10-50 µM CoQ10 to the IVM medium did not affect the percentage of MII oocytes nor the fertilization or the parameters of subsequent embryonic development. Exogenous CoQ10 in the culture medium neither did affect the development to the 2-4-cell stage nor rates of blastocyst formation. Moreover, the highest concentration of CoQ10 (100 µM) in the maturation medium negatively affected blastocyst rates. In conclusion, exogenous CoQ10 supplementation of maturation or embryo culture media failed to improve the outcomes of our in vitro embryo production system and its use as an exogenous antioxidant should not be encouraged.


Assuntos
Técnicas de Cultura Embrionária/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Ubiquinona/análogos & derivados , Animais , Antioxidantes/farmacologia , Meios de Cultura/química , Técnicas de Cultura Embrionária/métodos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilização In Vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/métodos , Masculino , Oócitos/efeitos dos fármacos , Suínos , Ubiquinona/efeitos adversos , Ubiquinona/farmacologia
16.
Arq. bras. med. vet. zootec. (Online) ; 71(5): 1735-1740, set.-out. 2019. tab
Artigo em Português | LILACS, VETINDEX | ID: biblio-1038668

RESUMO

The aim of this study was to describe population and morphological characteristics of preantral follicles of not pregnant cows, pregnant cows and fetus. Ten ovaries of non-pregnant Nelore cows, eighteen ovaries of pregnant cows and eighteen ovaries of fetus were used. For pregnant cows, six ovaries from each third (initial, middle and final) were evaluated, acquired from a slaughterhouse. For fetus, the same methodology and proportion of ovaries were used. Ovaries were washed, fixed and embedded in paraffin. They were then sectioned in longitudinal sections and stained by the Hematoxylin-Eosin method. Preantral follicles were classified according to morphology (primordial, primary and secondary) and degree of viability (intact and in initial, moderate and marked atresia). Descriptive and statistical analyzes were performed through KS300 image analysis program and Tukey's test. A greater proportion of primordial follicles were found in all categories. Secondary follicles were not observed in ovaries of fetus and cows in the initial third of pregnancy. All the ovary dimensions were higher in non-pregnant cows and in the final third of cows' pregnancy, and lower in final third of pregnancy fetus. It was concluded that follicle isolation was effective in describing population and morphological characteristics of preantral follicles of cows and fetus.(AU)


Assuntos
Animais , Feminino , Bovinos , Oócitos/crescimento & desenvolvimento , Bovinos/embriologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Fertilização In Vitro/métodos
17.
Zygote ; 27(5): 321-328, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31412962

RESUMO

Around 60-80% of oocytes maturated in vivo reached competence, while the proportion of maturation in vitro is rarely higher than 40%. In this sense, butafosfan has been used in vivo to improve metabolic condition of postpartum cows, and can represent an alternative to increase reproductive efficiency in cows. The aim of this study was to evaluate the addition of increasing doses of butafosfan during oocyte maturation in vitro on the initial embryo development in cattle. In total, 1400 cumulus-oocyte complexes (COCs) were distributed in four groups and maturated according to supplementation with increasing concentrations of butafosfan (0 mg/ml, 0.05 mg/ml, 0.1 mg/ml and 0.2 mg/ml). Then, 20 oocytes per group were collected to evaluate nuclear maturation and gene expression on cumulus cells and oocytes and the remaining oocytes were inseminated and cultured until day 7, when blastocysts were collected for gene expression analysis. A dose-dependent effect of butafosfan was observed, with decrease of cleavage rate and embryo development with higher doses. No difference between groups was observed in maturation rate and expression of genes related to oocyte quality. Our results suggest that butafosfan is prejudicial for oocytes, compromising cleavage and embryo development.


Assuntos
Blastocisto/fisiologia , Butilaminas/farmacologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Ácidos Fosfínicos/farmacologia , Animais , Butilaminas/administração & dosagem , Bovinos , Relação Dose-Resposta a Droga , Feminino , Fertilização In Vitro , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/fisiologia , Ácidos Fosfínicos/administração & dosagem
18.
Theriogenology ; 140: 44-51, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31437668

RESUMO

Lysosome, an important organelle in eukaryotes, can sequester macromolecules submitted by the endocytosis and autophagy pathways for degradation and recycling. Massive macromolecular turnover is also vital to the growth and development of mammalian oocytes. However, the functional role of lysosomes in the meiotic maturation of mammalian oocytes remains largely unexplored. Here, by treating in vitro matured porcine cumulus-oocyte complexes (COCs) with chloroquine (CQ), a lysosome inhibitor, we showed that regardless of CQ concentration, lysosomal inhibition affected neither the extrusion of the first polar body (PB1), nor the ROS levels. However, CQ treatment dramatically decreased the rates of oocytes with normal chromosome alignment and cytoskeleton organization (P < 0.05), but boosted the rates of oocytes with apoptosis (P < 0.05). Subsequently, after pathenogenetic activation or in vitro fertilization, the death or fragmentation rates of oocytes treated by CQ (both 35 µM and 45 µM) were significantly higher (P < 0.05), whereas the rates of embryo cleavage, embryos developed to blastocysts, and average blastomere number per blastocyst, were all significantly lower (P < 0.05), respectively. Furthermore, CQ (35 µM) treatment activated the autophagy pathway by elevating the LC3 II/I ratio. Taken together, lysosomes could affect porcine oocyte maturation and subsequent developmental capacity partially through the chromosome organization/cytoskeleton assembly and autophagy/apoptosis pathways.


Assuntos
Lisossomos/fisiologia , Oócitos/crescimento & desenvolvimento , Suínos/embriologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Cloroquina/farmacologia , Cromossomos/metabolismo , Cromossomos/ultraestrutura , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Lisossomos/efeitos dos fármacos , Meiose/efeitos dos fármacos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
19.
Reprod Domest Anim ; 54(10): 1357-1365, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31368591

RESUMO

This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus-oocyte complexes were exposed to heat shock (41.0°C) during the first 12 hr of in vitro maturation (IVM), followed by 12 hr at 38.5°C. Control group consisted of in vitro maturation for 24 hr at 38.5°C. Oocytes were in vitro-fertilized or activated with ionomycin and cultured in vitro for 192 hr post-in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p < .01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48 hpia. Heat shock had a negative impact (p < .01) on cleavage of IVF embryos, whereas no (p > .05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192 hpia decreased in both IVF and Part embryos derived from heat-shocked oocytes. Heat shock increased (p < .05) the apoptotic index in Part blastocysts, but no effect (p > .05) was found in IVF counterparts. Heat shock also down-regulated the expression of AQP3 (p < .01) and up-regulated the expression of HSP70.1 (p < .01) in Part blastocysts, whereas it down-regulated the expression of ATP1A1 (p < .05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat-shocked oocytes.


Assuntos
Blastocisto/fisiologia , Bovinos/embriologia , Resposta ao Choque Térmico/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Partenogênese/fisiologia , Animais , Células do Cúmulo , Desenvolvimento Embrionário , Feminino , Fertilização In Vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/métodos , Masculino , Oócitos
20.
Reprod Domest Anim ; 54(11): 1443-1448, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31381183

RESUMO

To improve the efficiency of somatic cell nuclear transfer (SCNT) in sheep, we investigated the effects of recipient oocyte source, number of transferred embryos and season on the pregnancy and live lamb rates for sheep somatic cell nuclear transfer embryos. Follicle-stimulating hormone (FSH)-stimulated ovaries produced significantly more oocytes both in total and of suitable quality for maturation culture than those without FSH treatment (from slaughterhouse). However, their in vitro maturation rates were similar. Embryos were reconstructed using adult fibroblast cells into enucleated MII oocytes. The pregnancy and term rates were significantly higher in the FSH-stimulated group than in the slaughterhouse one. Oocytes from FSH-stimulated ovaries were enucleated as recipient cytoplasm for nuclear transfer in the following experiments. The transfer of 7-9 and 11-13 embryos produced significantly higher pregnancy rates than that of six embryos. However, the former groups exhibited similar live lamb rates. FSH-stimulated ovaries produced significantly more oocytes in November and December (winter) than in May to July (summer), but the associated maturation rate did not increase. Pregnancy and term rates were significantly higher when transfer occurred in winter than in summer. In conclusion, FSH treatment produced significant benefit regarding the number and quality of collected oocytes and also for the pregnancy and live lamb rates for reconstructed embryos. However, the transfer of an appropriate number of embryos (7-13) and at an appropriate season (winter) increased pregnancy and term rates.


Assuntos
Transferência Embrionária/veterinária , Técnicas de Transferência Nuclear/veterinária , Estações do Ano , Carneiro Doméstico/fisiologia , Animais , Feminino , Hormônio Foliculoestimulante/farmacologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos , Ovário/efeitos dos fármacos , Gravidez , Taxa de Gravidez
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