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1.
Anim Sci J ; 91(1): e13493, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33314533

RESUMO

Although successful fertilization is completed by only 150 sperm in the pig oviduct, more than 50,000 sperms are required to achieve a fertilization rate of more than 70% by pig in vitro fertilization (IVF). In this study, to improve the efficiency of pig IVF, the effects of hypoxic conditions and treatment with creatine and methyl-beta cyclodextrin (MßCD) on the glycolytic pathway were investigated. Under low O2 conditions, zig-zag motility was strongly induced within 30 min; however, the induction disappeared at 60 min. Although caffeine suppressed zig-zag motility under low O2 conditions, creatine induced and sustained zig-zag motility until 120 min. Additionally, pretreatment with MßCD for 15 min greatly enhanced zig-zag motility via ATP production in sperm incubated with creatine under low O2 conditions. Sperm pretreated with MßCD were used for IVF in medium containing creatine under low O2 conditions. A fertilization rate of approximately 70% was achieved with only 1.0 x 104 sperms/mL, and there were few polyspermic embryos. Therefore, our novel method was beneficial for efficient production of pig embryos in vitro. Moreover, the zig-zag motility may be a novel movement which boar capacitated sperm exhibit in the culture medium.


Assuntos
Anaerobiose/fisiologia , Creatina/farmacologia , Fertilização In Vitro/métodos , Fertilização In Vitro/veterinária , Fertilização/efeitos dos fármacos , Motilidade Espermática , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Espermatozoides/fisiologia , Suínos/fisiologia , beta-Ciclodextrinas/farmacocinética , Animais , Sinergismo Farmacológico , Ejaculação/fisiologia , Feminino , Fertilização/fisiologia , Masculino , Interações Espermatozoide-Óvulo/fisiologia , beta-Ciclodextrinas/farmacologia
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(8): e0237766, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822364

RESUMO

Semen contains epithelial cells that can be cultured in vitro. For somatic cell nuclear transfer applications, it is essential to know whether clone(s) produced from semen-derived epithelial cells (SedECs) are healthy and reproductively competent. In this study, the semen and fertility profile of a cloned bull (C1) that was produced from a SedEC were compared with its donor (D1) and with two cloned bulls (C2, C3) that were produced from commonly used skin-derived fibroblast cells (SkdFCs). We observed variations in some fresh semen parameters (ejaculated volume and mass motility), frozen-thawed sperm parameters (plasma membrane integrity, and computer-assisted semen analysis (CASA) indices), but values are within the normal expected range. There was no difference in sperm concentration of ejaculated semen and frozen-thawed semen parameters which include sperm motility, percentage of live and normal morphology sperm, and distance traveled through oestrus mucus. Following in vitro fertilization (IVF) experiments, zygotes from C1 had higher (P < 0.05) cleavage rates (81%) than C2, C3, and D1 (71%, 67%, and 75%, respectively); however, blastocyst development per cleaved embryo and quality of produced blastocysts did not differ. The conception rate of C1 was 46% (7/15) and C2 was 50% (8/15) following artificial insemination with frozen-thawed semen. Established pregnancies resulted in births of 7 and 6 progenies sired by C1 and C2, respectively, and all calves show no signs of phenotypical abnormalities. These results showed that semen from a cloned bull derived from SedECs is equivalent to semen from its donor bull and bulls cloned from SkdFCs.


Assuntos
Búfalos/fisiologia , Clonagem de Organismos/veterinária , Células Epiteliais/citologia , Sêmen/citologia , Animais , Criopreservação/veterinária , Feminino , Fertilidade , Fertilização , Fertilização In Vitro/veterinária , Inseminação Artificial/veterinária , Masculino , Análise do Sêmen/veterinária , Motilidade Espermática
4.
Anim Sci J ; 91(1): e13430, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32677174

RESUMO

Mitochondria are necessary for the transition from oocyte to embryo and for early embryonic development. Mitofusin 1 is the main mediator of mitochondrial fusion and homeostasis. We investigated Mitofusin 1 expression levels in porcine somatic cell nuclear transfer (SCNT) embryos. The rate of blastocyst formation in SCNT embryos was reduced significantly compared with that of parthenogenetic activation embryos. SCNT embryos showed significantly decreased Mitofusin 1 expression and mitochondrial membrane potential, while exhibiting increased reactive oxygen species and apoptosis. Mitochondrial functional changes were observed in the SCNT embryos and may be correlated with low levels of Mitofusin 1 to negatively affect development.


Assuntos
Apoptose/genética , Blastocisto , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Expressão Gênica/genética , Estudos de Associação Genética/veterinária , Potencial da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Partenogênese/genética , Suínos/genética , Suínos/fisiologia , Animais , Células Cultivadas , Técnicas de Cultura Embrionária/veterinária , Fertilização In Vitro/veterinária , Técnicas de Transferência Nuclear/veterinária , Espécies Reativas de Oxigênio/metabolismo
5.
Anim Sci J ; 91(1): e13401, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32524695

RESUMO

The aim of this study was to examine whether a morphological approach is efficient for selecting high-quality porcine embryos produced by in vitro fertilization (IVF) under high polyspermy conditions. Frozen-thawed Meishan epididymal spermatozoa showing moderate and high polyspermy were subjected to IVF (1 × 105  sperms/ml). Under conditions of moderate polyspermy, 4-cell embryos selected at 48 hr after IVF (single selection) and 8-cell embryos selected at 79 hr after IVF from the collected 4-cell embryos (double selection) showed high developmental competence. Likewise, 4- and 8-cell embryos produced by IVF under high polyspermy conditions also showed high competence for development to blastocysts. However, blastocysts derived from high polyspermy conditions had significantly fewer cells than those produced under moderate polyspermy conditions. Furthermore, the frequency of nuclear and chromosomal abnormalities in 4- and 8-cell embryos produced under conditions of high polyspermy was significantly (p < .05) higher in comparison to moderate polyspermy conditions. These findings suggest that although high polyspermy affects the frequency of nuclear and chromosomal anomalies in porcine IVF embryos, subsequent selection based on morphological features of 4- and 8-cell embryos even under high polyspermy conditions, could be an alternative option for selecting porcine IVF embryos with high development ability.


Assuntos
Blastocisto/citologia , Desenvolvimento Embrionário , Fertilização In Vitro/veterinária , Interações Espermatozoide-Óvulo , Espermatozoides/fisiologia , Suínos/embriologia , Suínos/fisiologia , Animais , Aberrações Cromossômicas/veterinária , Feminino , Masculino
6.
PLoS One ; 15(4): e0232536, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353075

RESUMO

Sperm migration towards an oocyte in the female reproductive tract is an important step for successful fertilization. Although several sperm-chemotactic factors have been identified in mammals, it is unclear whether these chemoattractants contribute to sperm migration towards an oocyte that is the final destination for sperm. Furthermore, chemoattractants for bovine sperm are still undiscovered even though the follicular fluid attracts sperm in cattle. Here, we demonstrated that a single bovine cumulus-oocyte complex (COC) had the ability to attract sperm, suggesting that the COC secreted sperm chemoattractants. We identified stromal cell-derived factor 1 (SDF1), which was expressed in COCs, and its receptor CXCR4 in sperm, as a candidate. Our results showed that bovine sperm preferentially migrated to the area with a high SDF1 concentration and occasionally showed turn movements by asymmetric flagellar bends during the migration. We also demonstrated that increasing the intracellular Ca2+ concentration via Ca2+ channels was related to SDF1-induced sperm chemotaxis. Finally, a CXCR4 inhibitor significantly suppressed the in vitro bovine sperm migration towards a COC. Taken together, we propose that SDF1 is a chemotactic factor for bovine sperm to regulate their migration towards an oocyte via the CXCR4 receptor.


Assuntos
Quimiocina CXCL12/metabolismo , Quimiotaxia/fisiologia , Receptores CXCR4/metabolismo , Motilidade Espermática/fisiologia , Animais , Bovinos , Células do Cúmulo/metabolismo , Feminino , Fertilização In Vitro/veterinária , Microscopia Intravital , Masculino , Oócitos/metabolismo , Espermatozoides/fisiologia
7.
Trop Anim Health Prod ; 52(5): 2749-2752, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32462440

RESUMO

The objective of the present study was to evaluate efficiency of in vitro fertilization (IVF) in Nelore, Brangus, and Girolando oocyte donors. Ovum pickup (OPU) from the donors was conducted every 15 days to assess oocyte recovery, IVF, and post-transfer pregnancy percentage. For Nelore, the mean numbers of total and viable oocytes recovered (23.5 ± 1.1 and 14.0 ± 1.0, respectively) were higher (p < 0.05) than those for Brangus (12.7 ± 1.9 and 6.6 ± 1.0, respectively) and Girolando (12.5 ± 1.4 and 6.8 ± 0.7, respectively); Brangus and Girolando did not differ from each other (p > 0.05). The percentage of blastocyst production differed (p < 0.05) between Nelore (48.4 ± 2.4%), Brangus (40.3 ± 3.6%), and Girolando (38.9 ± 2.6%), but those in Brangus and Girolando did not differ (p > 0.05). The percentage of blastocysts (transferred) that resulted in pregnancy did not differ (p > 0.05) between Nelore (45.5 ± 3.8%), Brangus (41.7 ± 4.1%), and Girolando (40.7 ± 3.7%). Of the breeds studied, Nelore donors are more efficient for IVF, but conditions of this study.


Assuntos
Fertilização In Vitro/veterinária , Doação de Oócitos/veterinária , Animais , Blastocisto , Cruzamento , Bovinos , Feminino , Fertilização In Vitro/estatística & dados numéricos , Doação de Oócitos/estatística & dados numéricos , Oócitos , Gravidez , Taxa de Gravidez
8.
J Anim Sci ; 98(4)2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32277240

RESUMO

Colony-stimulating factor 3 (CSF3), also known as granulocyte colony-stimulating factor, is used to reduce the incidence of mastitis in cattle. Here, we tested whether recombinant bovine CSF3 at 1, 10, or 100 ng/mL acts on the bovine oocyte during maturation or on the developing embryo to modify competence for development and characteristics of the resultant blastocyst. For experiment 1, oocytes were matured with or without CSF3. The resultant embryos were cultured in a serum-free medium for 7.5 d. There was no effect of CSF3 on cleavage or on development to the blastocyst stage except that 100 ng/mL reduced the percent of putative zygotes and cleaved embryos becoming blastocysts. Expression of transcripts for 93 genes in blastocysts was evaluated by RT-PCR using the Fluidigm platform. Transcript abundance was affected by one or more concentrations of CSF3 for four genes only (CYP11A1, NOTCH2, RAC1, and YAP1). For experiment 2, cumulus-oocyte complexes (COC) were fertilized with either X- or Y-sorted semen. Putative zygotes were cultured in medium containing CSF3 treatments added at the beginning of culture. There was no effect of CSF3, sex, or the interaction on the percent of putative zygotes that cleaved or on the percent of putative zygotes or cleaved embryos becoming a blastocyst. For experiment 3, CSF3 was added from day 4 to 7.5 of development. There was no effect of CSF3 on development to the blastocyst stage. Transcript abundance of 10 genes was increased by 100 ng/mL CSF3, including markers of epiblast (NANOG, SOX2), hypoblast (ALPL, FN1, KDM2B, and PDGFRA), epiblast and hypoblast (HNF4A) and trophectoderm (TJAP1). Results are indicative that concentrations of CSF3 higher than typical after therapeutic administration can reduce oocyte competence and act on the embryo to affect characteristics of the blastocyst.


Assuntos
Bovinos/embriologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Reprodução/efeitos dos fármacos , Animais , Blastocisto/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilização In Vitro/veterinária , Oócitos/efeitos dos fármacos , Proteínas Recombinantes
9.
Arq. bras. med. vet. zootec. (Online) ; 72(2): 443-451, Mar./Apr. 2020. ilus, tab
Artigo em Português | LILACS, VETINDEX | ID: biblio-1128368

RESUMO

O objetivo foi avaliar protocolos de maturação in vitro (MIV) para oócitos de cutias, seguida de fertilização in vitro (FIV) e ativação partenogenética (AP). Os oócitos imaturos (CCOs) foram obtidos por fatiamento do ovário, após OSH, e submetidos a três grupos: MAT - 16 (16 horas de maturação), MAT - 20 (20 horas de maturação) e MAT - 24 (24 horas de maturação), em incubadora de cultivo a 38,8°C, com atmosfera de 5% de CO2 e 95% de umidade relativa. A maturação foi analisada pela presença do primeiro corpúsculo polar. Em seguida, os CCOs maduros foram submetidos à FIV, com período de coincubação dos CCOs e dos espermatozoides de 15h, a 38,8ºC e 5% de CO2, e AP com ionomicina. Os grupos de MIV foram analisados utilizando-se o teste qui-quadrado e, nos experimentos de FIV e AP, foram analisadas a taxa de clivagem e a proporção de desenvolvimento embrionário. A análise estatística foi realizada utilizando-se o programa SAS. Houve diferença significativa entre os grupos de maturação, tendo os grupos MAT - 20 e MAT - 24 apresentado maior porcentagem de oócitos maturados in vitro. As taxas de clivagem e de desenvolvimento embrionário foram de 8,6% e 2,9%, respectivamente, na FIV, e de 63,6% e 15,1%, na AP. Entretanto, nos dois casos, o embrião não passou do estágio de mórula.(AU)


The objective was to evaluate IVM protocols for agouti oocytes, followed by in vitro fertilization (IVF) and parthenogenetic activation (PA). The immature oocytes (CCOs) were obtained by slicing the ovary after OSH and submitted to three groups: MAT - 16 (16 hours maturation), MAT - 20 (20 hours maturation) and MAT - (24 hours maturation), in a culture incubator at 38.8°C, with an atmosphere of 5% CO2 and 95% relative humidity. The maturation was analyzed by the presence of the first polar corpuscle. Then, mature CCOs were submitted to IVF, with co-incubation period of CCOs and spermatozoa from 15h to 38.8°C and 5% of CO2, and PA with inomycin. The IVM groups were analyzed using the chi-square test and in the FIV and PA experiment the rate of cleavage and the rate of embryonic development were analyzed. Statistical analysis was performed using the SAS program. There was a significant difference between the maturation groups, and the MAT - 20 and MAT - 24 groups showed a higher percentage of matured oocytes in vitro. The rates of cleavage and embryonic development were 8.6% and 2.9%, respectively in FIV and 63.6% and 15.1% in PA. However, in both cases the embryo did not pass beyond the morula stage.(AU)


Assuntos
Animais , Oócitos , Fertilização In Vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Dasyproctidae , Partenogênese , Ionomicina
10.
Trop Anim Health Prod ; 52(5): 2273-2281, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32144659

RESUMO

The aim of this study was to evaluate the impact of increased shadow supply in integrated crop-livestock-forest systems on in vitro embryonic development and physiological parameters related to stress response in Nellore heifers (Bos indicus). For the study, animals (n = 16) were randomly divided into two groups and kept in areas with different afforestation systems, the integrated crop-livestock-forest (ICLF) and the integrated crop-livestock (ICL) system. The microclimate of the ICLF system provided better comfort conditions than ICL. No differences of respiratory rate, rectal temperature, cortisol, T3, T4, oocyte quality, and cleavage rate between the systems were verified. A higher blastocyst rate was observed in the ICLF (p < 0.05). The results demonstrate that Nellore heifers managed in ICLF during summer in Midwest of Brazil showed higher production of in vitro embryos, without typical changes in its physiological parameters. The results observed in the present study indicate that zebu females are able to respond satisfactorily to the intense heat conditions; however, we believe that the long period to which these animals are exposed to these conditions interferes in the oocyte competence and embryo development.


Assuntos
Bovinos/fisiologia , Embrião de Mamíferos/fisiologia , Fertilização In Vitro/veterinária , Microclima , Animais , Desenvolvimento Embrionário , Feminino , Temperatura Alta
11.
Theriogenology ; 144: 122-131, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31951983

RESUMO

Genetic selection for the best suited offspring drives the dairy industry to use young genitors and assisted reproductive technologies (ART) to reduce generation intervals. However, sperm samples collected from peri-pubertal bulls have lower counts and quality compared to samples from adult bulls. Moreover, our previous study identified differentially methylated regions (DMRs) in sperms from early-, peri- and post-pubertal bulls. The aim of this study was to further investigate the impacts of paternal age on early embryos. To achieve this, we evaluated the transcriptome and the epigenome of bovine blastocysts generated from spermatozoa of bulls at 10, 12, and 16 months of age and used in vitro fertilization (IVF) of oocytes recovered from the same adult cows. A total of 259 probes were differentially expressed and 6953 probes were differentially methylated in the 10- vs 16-month and the 12- vs 16-month groups. Ingenuity Pathway Analysis (IPA) of transcriptomic data demonstrated that energy-related pathways such as oxidative phosphorylation, EIF2 signaling, and mitochondrial dysfunction were affected the most by the age of the bull. Meanwhile, IPA analysis of the epigenome revealed that protein kinase A signaling, RAR activation, and other pathways were influenced by paternal age. Overall, we showed that the bull's age mainly influenced metabolism-related pathways in blastocysts, and this could therefore impact subsequent development.


Assuntos
Envelhecimento , Blastocisto/fisiologia , Bovinos/fisiologia , Epigenoma , Fertilização In Vitro/veterinária , Transcriptoma , Animais , Regulação da Expressão Gênica no Desenvolvimento , Masculino
12.
Theriogenology ; 144: 152-157, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31951984

RESUMO

Resveratrol (Resv; 3,4,5-trihydroxy-trans-stilbene) is a phytoalexin with antioxidant activity that modulates redox homeostasis in oocytes and improves in vitro embryo production. Cold storage of cat ovaries for a period longer than 24 h alters oxidative status of oocytes after in vitro maturation and reduces their developmental competence. The aim of this study was to evaluate the effect of resveratrol supplementation to the maturation medium on embryo development of oocytes after storage of domestic cat ovaries at 4 °C for 24 h or 48 h. Cumulus-oocyte complexes (COCs) were recovered from ovaries of domestic queens and cultured in maturation medium supplemented with (+) or without (-) 5 µM resveratrol for 24 h. COCs collected from fresh ovaries were matured in vitro (IVM) in standard conditions as control. After IVM, oocytes were in vitro fertilized (IVF) and presumptive zygotes cultured for 7 days. Oocyte nuclear maturation, reactive oxygen species (ROS) and glutathione (GSH) levels as well as cleavage, blastocyst formation and blastocyst cell number were determined. There were no differences in the maturation rates of oocytes between the control and stored groups, irrespective of resveratrol supplementation. Resveratrol treatment during IVM significantly increased the level of GSH and reduced the level of ROS of oocytes recovered from ovaries stored for 48 h as compared to the non-treated group (48 h-). The rate of blastocyst formation from oocytes recovered from ovaries after 48 h storage that underwent IVM with resveratrol was higher (P < 0.05) than that of oocytes matured without resveratrol and similar to that of control oocytes. Resveratrol treatment increased (P < 0.05) cell number in blastocysts from 24 h + and 48 h + groups as compared to their respective counterparts. In conclusion, our results demonstrated that resveratrol supplementation during IVM can reverse the adverse effect of oxidative stress on oocytes, and enhances embryo development after ovary storage at 4 °C for 48 h. These results may provide a basis for improving culture conditions and extend the possibility of storage of cat ovaries for more than 24 h thus ensuring successful in vitro embryo production.


Assuntos
Oócitos/fisiologia , Ovário/efeitos dos fármacos , Resveratrol/farmacologia , Preservação de Tecido/veterinária , Animais , Antioxidantes/farmacologia , Gatos , Temperatura Baixa , Técnicas de Cultura Embrionária/veterinária , Feminino , Fertilização In Vitro/veterinária , Glutationa/metabolismo , Espécies Reativas de Oxigênio , Preservação de Tecido/métodos
13.
Anim Reprod Sci ; 213: 106279, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31987329

RESUMO

Incubation gas atmosphere affects the development of in vitro produced embryos. In this study, there was examination of effects of two different oxygen (O2) tensions (5 % and 21 %) during in vitro maturation (M5 and M21) and/or fertilization (F5 and F21) on embryo production and quality in deer and sheep. There was assessment of the percentage of embryos with cell cleavage occurring, percentage that developed to the blastocyst stage, and analysis of the relative abundance of mRNA transcript for genes important for development to the blastocyst stage. The O2 tension treatment did not affect (P > 0.05) percentage cleavage or blastocyst development in either species. In sheep, there was a greater abundance of SHC1, GPX1, TP53, BAX and NRF1 mRNA transcript (P < 0.05) in M21 F5-derived embryos. In deer, there was a greater abundance of SOD2 mRNA transcript (P < 0.05) when oocytes had been matured under relatively lesser O2, regardless of the tension used during fertilization. There was a lesser abundance of SOX2 mRNA transcript (P < 0.05) in the M5F21 compared to the other three treatment groups. The AKR1B1 mRNA transcript was in greater abundance (P < 0.05) in M21 F21 as compared to M21 F5 and M5F21 group, and there was a greater abundance PLAC8 mRNA transcript (P < 0.05) in M21 F21, as compared to all other treatment groups. In conclusion, while O2 tension had no effect on developmental rates it did affect the relative abundance of mRNA transcript of multiple genes related to important cell functions during development.


Assuntos
Cervos/embriologia , Técnicas de Cultura Embrionária/veterinária , Fertilização In Vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oxigênio/farmacologia , Ovinos/embriologia , Animais , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos
14.
Theriogenology ; 144: 45-55, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31911322

RESUMO

Capacitation-like changes affect sperm of several species, such as ram, reducing cell survival and fertilizing competence. Proteins from seminal plasma stabilize sperm plasma membranes, being an interesting focus to develop strategies for improving cryopreserved ram semen performance. To date, biotechnologies are focused to reduce damage in frozen-thawed ram spermatozoa through the addition of bioactives. Serine Protease Inhibitor Kazal-type 3 (SPINK3) is a little protein synthesized by mouse seminal vesicle and secreted to seminal plasma. While attached to the sperm, this protein binds to non-capacitated sperm and blocks calcium entry, avoiding a premature physiological capacitation and consequently, acrosome reaction. Due to these characteristics, SPINK3 has been proposed as a decapacitating factor. The aim of this work was to assess whether heterologous SPINK3 is able to protect ram sperm from the well-known cell damages produced by freezing/thawing and to understand the mechanisms by which it is acting. Sperm were supplemented with 13 µM SPINK3 before freezing in an egg yolk-based extender or after thawing and selection. Under both conditions, SPINK3 decreased intracellular calcium content (p < 0.05) and reduced the 25 kDa tyrosine phosphorylated protein demonstrating a decapacitating effect, although the addition of the protein before cryopreservation was not enough to improve other sperm parameters. However, the addition of SPINK3 post thawing was able to significantly ameliorate viability, motility, mitochondrial status and to avoid the increase of lipid peroxidation (p < 0.05). Moreover, sperm treated with SPINK3 was not only still capable to fertilize, but also improved it, as evidenced by an increase in the oocyte cleavage rate (p < 0.05) although, the embryo development and embryo quality were not affected. Our findings would contribute to develop a strategy for improving sperm quality by using decapacitating proteins. In fact, the outcomes of this work demonstrate that SPINK3 is able to reduce sperm cryo-injuries when is added after thawing, improving functionality and thus in vitro fertilization results.


Assuntos
Criopreservação/veterinária , Preservação do Sêmen/veterinária , Inibidores de Serinopeptidase do Tipo Kazal/farmacologia , Ovinos/fisiologia , Espermatozoides/fisiologia , Animais , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Fertilização In Vitro/veterinária , Regulação da Expressão Gênica , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Análise do Sêmen , Inibidores de Serinopeptidase do Tipo Kazal/genética , Inibidores de Serinopeptidase do Tipo Kazal/metabolismo , Motilidade Espermática
15.
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
16.
Theriogenology ; 141: 82-90, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31518732

RESUMO

Relative to alanine and serine amino acid levels, glutamine is highly abundant in follicular fluid, and is an important source of energy required for oocyte maturation and embryo development. Thus, glutamine is an essential component of in vitro embryo culture media. However, glutamine has poor stability and degrades spontaneously in solution to form ammonia and pyrrolidonecarboxylic acid. In the present study, we aimed to explore the effect of substituting l-glutamine with glycine-glutamine, a more stable glutamine, on development of early parthenogenetic embryos and in vitro fertilization (IVF) embryos in bovine. Results revealed that glycine-glutamine can significantly increase cleavage rate (parthenogenetic embryos:87.24% vs. 72.61%, IVF embryos:89.33% vs. 83.79%, P < 0.01), blastocyst number (parthenogenetic embryos:24.98% vs. 18.07%, IVF embryos:33.53% vs. 27.29%, P < 0.01), and blastocyst number (parthenogenetic embryos:96 vs. 76, IVF embryos:114 vs. 109, P < 0.01), reduce blastocyst apoptosis (parthenogenetic embryos:3.72% vs. 6.65%, IVF embryos:2.53% vs.6.23%, P < 0.01), alleviate embryo ammonia toxicity, and reduce the content of reactive oxygen species (ROS) compared with the l-glutamine. In addition, glycine-glutamine can alter epigenetic reprogramming by increasing the expression of HDAC1 (Histone Deacetylase 1) and decreasing the relative expression levels of H3K9 acetylation in early parthenogenetic embryos and IVF embryos. From our present study, we concluded that glycine-glutamine is an effective substitute of glutamine in modified synthetic oviduct fluid with amino acids (mSOFaa).


Assuntos
Bovinos/embriologia , Técnicas de Cultura Embrionária/veterinária , Fertilização In Vitro/veterinária , Glutamina/química , Glicina/química , Partenogênese/fisiologia , Acetilação , Amônia/metabolismo , Animais , Apoptose , Meios de Cultura , Dipeptídeos/química , Dipeptídeos/farmacologia , Glutamina/farmacologia , Glicina/farmacologia , Histonas/metabolismo , Espécies Reativas de Oxigênio
17.
Theriogenology ; 141: 113-119, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31536860

RESUMO

The objective of this research was to develop a system for piglet production by transvaginal ultrasound-guided ovum pick-up (OPU), in vitro production (IVP) of embryos and embryo transfer. First, to establish a culture system for a small number of oocytes or embryos, we evaluated the effect of different incubation volumes and culture densities on fertilizing ability and developmental competence in vitro. Porcine oocytes derived from slaughterhouse ovaries were matured, fertilized and then cultured in vitro in groups as follows: 50 oocytes in 500 µL medium for IVM, 20 oocytes in 100 µL medium for IVF and 20 embryos in 40 µL medium for IVC (Group I); 20 in 100 µL for IVM, 20 in 100 µL for IVF and 20 in 40 µL for IVC (Group II); and 10 in 100 µL for IVM, 10 in 100 µL for IVF and 10 in 40 µL for IVC (Group III). Percentages of sperm penetration, cleavage and blastocyst formation did not differ among the groups. Second, to increase the collection efficiency of porcine oocytes by transvaginal ultrasound-guided OPU, the effects of aspiration pressure on follicular oocyte collection were assessed. Oocytes were aspirated from ovaries of live sows using 80 or 100 mmHg. The recovered oocytes were divided into four categories according to the surrounding cumulus cells and quality of oocytes. The number of oocytes recovered using 100 mmHg pressure was significantly higher than with 80 mmHg pressure. However, there were no significant differences in the population of oocytes grouped by the morphological criteria, number of blastocysts per session and the total cell number in blastocysts between the two vacuum pressures. Finally, 81 oocytes obtained by OPU from five donor sows were subjected to IVP and 47 transferable embryos (9.4 ±â€¯4.0 [mean ±â€¯SD] morulae/blastocysts per session) were obtained at 5 days after IVF. When they were transferred into five recipient gilts (5-16 embryos per recipient), three of five recipients became pregnant and farrowed a total of 12 live piglets. The present results demonstrate that porcine blastocysts can be produced by OPU-IVP and develop to full term after embryo transfer.


Assuntos
Blastocisto/fisiologia , Técnicas de Cultura Embrionária/veterinária , Fertilização In Vitro/veterinária , Óvulo/fisiologia , Suínos , Ultrassonografia/veterinária , Animais , Doadores de Tecidos , Coleta de Tecidos e Órgãos
18.
Theriogenology ; 141: 16-25, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494458

RESUMO

The possible impact of natural heat stress on animal fertility is currently a major concern for breeding companies. Here, we aimed to address this concern by determining the effects of natural heat stress on the fertility of Holstein bulls located in the Netherlands. Semen samples were collected from six bulls at two locations in March 2016 (low temperature-humidity index (THI) group; maximum THI of 51.8 and 55 at their respective locations) or August (high THI group; maximum THI of 77.9 and 80.5 during meiotic and spermiogenic stages of spermatogenesis, 42 to 14 days prior to semen collection). The effect of heat stress on semen quality was assessed by sperm morphology, motility, reactive oxygen species production, lipid peroxidation, viability, and DNA fragmentation. Moreover, we evaluated the development of embryos generated in vitro by low and high THI semen, and determined inner cell mass/trophectoderm ratio, apoptotic cell ratio, and embryonic gene expression in day-8 blastocysts. An increase in cell death (propidium iodide-positive cells; P = 0.039) was observed in the high THI group (31.5%) compared to the low THI group (27.6%). Moreover, a decrease (P < 0.001) was observed in the total blastocyst rates at day 7 post-insemination (15.3 vs 20.9%) and day 8 (23.2 vs 29.6%) in the high THI compared to the low THI group, respectively. There were no differences in the relative abundance of candidate transcripts examined. In conclusion, sperm samples from dairy bulls obtained during a period with higher THI had reduced viability and led to a decrease in blastocyst development and delayed hatching, compared to semen collected during a period with low THI.


Assuntos
Bovinos/fisiologia , Técnicas de Cultura Embrionária/veterinária , Fertilização In Vitro/veterinária , Temperatura Alta , Umidade , Espermatogênese , Animais , Bovinos/embriologia , Sobrevivência Celular , Desenvolvimento Embrionário , Regulação da Expressão Gênica , Masculino , Motilidade Espermática , Espermatozoides
19.
Theriogenology ; 142: 26-33, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31574397

RESUMO

Asiatic acid is a pentacyclic triterpene enriched in the medicinal herb Centella asiatica, and it has been suggested to possess free radical scavenging and anti-apoptotic properties. The purpose of the current study was to explore the effects of asiatic acid on porcine early-stage embryonic development and the potential mechanisms for any observed effects. The results showed that 10 µM asiatic acid supplementation during the in vitro culture period dramatically improved developmental competence in porcine embryos derived from parthenogenetic activation (PA), somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF). Further analysis revealed that asiatic acid attenuated H2O2-induced intracellular reactive oxygen species (ROS) generation. Notably, asiatic acid not only enhanced intracellular GSH levels but also attenuated mitochondrial dysfunction. Gene expression analysis revealed that asiatic acid upregulated expression of the antioxidant-related gene Sod-1 and the blastocyst formation related gene Cox-2, while downregulating expression of the apoptosis-related gene Caspase-9 in SCNT blastocysts. These results suggest that asiatic acid exerts beneficial effects on early embryonic development in porcine embryos and that asiatic acid may be useful for improving the in vitro production of porcine embryos.


Assuntos
Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Fertilização In Vitro/veterinária , Técnicas de Transferência Nuclear/veterinária , Triterpenos Pentacíclicos/farmacologia , Suínos/embriologia , Animais , Meios de Cultura/química , Glutationa/metabolismo , Potencial da Membrana Mitocondrial , Partenogênese , Espécies Reativas de Oxigênio
20.
Equine Vet J ; 52(2): 258-261, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31232484

RESUMO

BACKGROUND: Monozygotic multiple pregnancy is rare in horses, but may be more common after transfer of an in vitro produced (IVP) embryo. OBJECTIVES: To determine the occurrence, incidence, characteristics and outcome of monozygotic siblings arising from in vivo and IVP equine embryos. STUDY DESIGN: Retrospective case series. METHODS: A total of 496 fresh in vivo and 410 frozen-thawed IVP blastocysts, produced by intracytoplasmic sperm injection (ICSI) of in vitro matured oocytes from Warmblood mares, were transferred into recipient mares. The likelihoods of pregnancy and multiple pregnancy were calculated, and the clinical features and outcome of any multiple pregnancy were recorded. RESULTS: The likelihood of pregnancy after transfer of a single IVP or in vivo embryo was 62% (254/410) and 83% (413/496) respectively. The incidence of multiple pregnancy was 1.6% (4/254) and 0% (0/413) for IVP and in vivo blastocysts, respectively. More specifically, three IVP blastocysts yielded twin embryo propers/fetuses, and one IVP conceptus developed three distinct embryonic bodies. Interestingly, only one embryonic vesicle was detected at all ultrasonographic examinations prior to embryo proper development. Multiple embryonic bodies only became apparent at later scans to check for an embryo proper and heartbeat, or when the recipient mare aborted. Two twin pregnancies aborted spontaneously at 3 and 9 months, respectively, while the heartbeat was lost from all three embryos in the triplet pregnancy before day 35 of gestation. Twin reduction by per rectum compression of one fetal thorax was attempted at day 50 of gestation in the fourth case; however, both fetuses were lost. MAIN LIMITATIONS: Small number of cases. CONCLUSIONS: In vitro embryo production resulted in a higher incidence of multiple monozygotic pregnancy, which could only be diagnosed after development of the embryo proper and is likely to result in pregnancy loss later in gestation if left untreated.


Assuntos
Transferência Embrionária/veterinária , Fertilização In Vitro/veterinária , Animais , Blastocisto , Feminino , Cavalos , Gravidez , Gravidez Múltipla , Estudos Retrospectivos
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