RESUMO
Dietary rumen-protected polyunsaturated fatty acids (PUFAs) rich in linoleic acid (LA) may affect embryo yield, and LA can modulate the molecular mechanisms of lipid uptake in bovine blastocysts produced in vitro. In embryos, membrane lipids, such as phosphatidylcholines (PCs) and sphingomyelins (SMs), affect cryopreservation success. The aim of the present study was to evaluate embryonic developmental rates after the IVF of oocytes retrieved from Nellore heifers fed for approximately 90 days with rumen-protected PUFAs rich in LA. In addition, we evaluated embryo cryotolerance and the membrane structure lipid composition using matrix-assisted laser desorption ionisation mass spectrometry of fresh and vitrified embryos. Embryo development to the blastocyst stage (mean 43.2%) and embryo survival after vitrification and warming (mean 79.3%) were unaffected by diet. The relative abundance of one lipid species (PC ether (PCe; 38:2, which means that this lipid has 38 carbon atoms and 2 double bonds in the fatty acyl residues) was increased after PUFAs supplementation. However, 10 ions were affected by cryopreservation; ions consistent with PC 32:0, PC 34:1, SM 24:1, PC 40:6 or PC 42:9, PC plasmalogen (PCp) 44:10 or PC 42:7, triacylglycerol (TAG) 54:9 and a not assigned ion (m/z 833.2) were lower in blastocysts that survived to the cryopreservation process compared with fresh blastocysts, whereas the abundance of the ions PC 36:3 or PC 34:0, PCe 38:2 or PC 36:6 and PC 36:5 or PCe 38:1 were increased after cryopreservation. Thus, the results demonstrate that the mass spectrometry profiles of PC, SM and TAG species differ significantly in bovine blastocysts upon cryopreservation. Because the lipid ion abundances of fresh and vitrified-warmed embryos were distinct, they can be used as potential markers of post-cryopreservation embryonic survival.
Assuntos
Criopreservação/veterinária , Gorduras Insaturadas na Dieta/administração & dosagem , Ectogênese , Embrião de Mamíferos/metabolismo , Fenômenos Fisiológicos da Nutrição Materna , Lipídeos de Membrana/metabolismo , Oocistos/metabolismo , Animais , Animais Endogâmicos , Blastocisto , Brasil , Bovinos , Estudos Cross-Over , Embrião de Mamíferos/citologia , Feminino , Fertilização in vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Ácido Linoleico/administração & dosagem , Ácido Linoleico/metabolismo , Masculino , Lipídeos de Membrana/química , Oocistos/citologia , Oocistos/isolamento & purificação , Recuperação de Oócitos/veterinária , Plasmalogênios/química , Plasmalogênios/metabolismo , Preservação do Sêmen/veterinária , VitrificaçãoRESUMO
UNLABELLED: We examined whether culturing embryos with linoleic acid (LA) in semi-defined medium reduces lipid accumulation and improves cryosurvival after vitrification. Embryos were cultured with LA (100 µM) and a semi-defined medium was used during in vitro culture (IVC), in which the fetal calf serum was substituted by bovine serum albumin (BSA). There was a reduction (P < 0.05) in the embryonic development rate ( CONTROL: 25.8% versus LA: 18.5%), but the proposed system was effective in promoting the decrease (P = 0.0130) in the intracellular lipid content ( CONTROL: 27.3 ± 0.7 versus LA: 24.6 ± 0.7 arbitrary fluorescence units of embryos stained with the fluorescent dye Nile Red), consequently increasing (P = 0.0490) the embryo survival after 24h of culture post-warming ( CONTROL: 50.0% versus LA: 71.7%). The results question the criteria used to evaluate the efficiency of an in vitro production system specifically with relation to the maximum number of blastocysts produced and suggest that might be more appropriate to improve the desired characteristics of embryos generated in accordance with the specific purpose of in vitro embryo production, commercial or scientific. In conclusion, supplying LA to serum-free culture medium was found to adversely affect the rates of embryo development to the blastocyst stage, but significantly reduced embryo lipid accumulation and improved cryopreservation survival.
Assuntos
Blastocisto/efeitos dos fármacos , Criopreservação/métodos , Citoplasma/química , Ácido Linoleico/farmacologia , Lipídeos/análise , Adaptação Fisiológica/efeitos dos fármacos , Animais , Blastocisto/fisiologia , Bovinos , Meios de Cultura/química , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária , Feminino , Fertilização/fisiologia , Congelamento , Masculino , Soroalbumina Bovina/farmacologia , Espermatozoides/fisiologia , Fatores de TempoRESUMO
This study examined the effects of antioxidant supplementation and O2 tension on embryo development, cryotolerance and intracellular reactive oxygen species (ROS) levels. The antioxidant supplementation consisted of 0.6 mM cysteine (CYST); 0.6 mM cysteine + 100 µM cysteamine (C+C); 100 IU catalase (CAT) or 100 µM ß-mercaptoethanol (ß-ME) for 3 or 7 days of in vitro culture (IVC). Two O2 tensions (20% O2 [5% CO2 in air] or 7% O2, 5% CO2 and 88% N2 [gaseous mixture]) were examined. After 7 days of antioxidant supplementation, the blastocyst frequencies were adversely affected (P < 0.05) by CYST (11.2%) and C+C (1.44%), as well as by low O2 tension (17.2% and 11.11% for 20% and 7% O2, respectively) compared with the control (26.6%). The blastocyst re-expansion rates were not affected (P > 0.05) by the treatments (range, 66-100%). After 3 days of antioxidant supplementation, the blastocyst frequencies were not affected (P > 0.05) by any of the antioxidants (range, 43.6-48.5%), but they were reduced by low O2 tension (P < 0.05) (52.1% and 38.4% for 20% and 7% O2, respectively). The intracellular ROS levels, demonstrated as arbitrary fluorescence units, were not affected (P > 0.05) by antioxidant treatment (range, 0.78 to 0.95) or by O2 tension (0.86 and 0.88 for 20% and 7% O2, respectively). The re-expansion rates were not affected (P > 0.05) by any of the treatments (range, 63.6-93.3%). In conclusion, intracellular antioxidant supplementation and low O2 tension throughout the entire IVC period were deleterious to embryo development. However, antioxidant supplementation up to day 3 of IVC did not affect the blastocyst frequencies or intracellular ROS levels.
Assuntos
Antioxidantes/farmacologia , Blastocisto/fisiologia , Técnicas de Cultura Embrionária/métodos , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Dióxido de Carbono/farmacologia , Catalase/farmacologia , Bovinos , Criopreservação , Meios de Cultura/farmacologia , Cisteamina/farmacologia , Cisteína/farmacologia , Feminino , Masculino , Mercaptoetanol/farmacologia , Nitrogênio/farmacologia , Espécies Reativas de Oxigênio/metabolismo , VitrificaçãoRESUMO
The effects of intracellular (cysteine and ß-mercaptoethanol) and extracellular (catalase) antioxidant supplementation at different times during in vitro production (IVM and/or in vitro culture (IVC)) on bovine embryo development, intracellular reactive oxygen species (ROS) levels, apoptosis and re-expansion rates after a vitrification-thawing process were examined. Blastocyst frequencies were not affected by either antioxidant supplementation (40.5%-56.4%) or the timing of supplementation (41.7%-55.4%) compared with control (48.7%; P>0.05). Similarly, antioxidants and the moment of supplementation did not affect (P>0.05) the total number of blastomeres (86.2-90.5 and 84.4-90.5, respectively) compared with control (85.7). However, the percentage of apoptotic cells was reduced (P<0.05) in groups supplemented during IVM (1.7%), IVC (2.0%) or both (1.8%) compared with control (4.3%). Intracellular ROS levels measured in Day 7 blastocysts were reduced (P<0.05) in all groups (0.60-0.78), with the exception of the group supplemented with ß-mercaptoethanol during IVC (0.88), which did not differ (P>0.05) from that in the control group (1.00). Re-expansion rates were not affected (P>0.05) by the treatments (50.0%-93.0%). In conclusion, antioxidant supplementation during IVM and/or IVC reduces intracellular ROS and the rate of apoptosis; however, supplementation does not increase embryonic development and survival after vitrification.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Bovinos , Fertilização in vitro , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/fisiologia , Blastocisto/citologia , Bovinos/embriologia , Bovinos/metabolismo , Células Cultivadas , Criopreservação , Técnicas de Cultura Embrionária/métodos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilização in vitro/métodos , Técnicas de Maturação in Vitro de Oócitos/métodos , Espaço Intracelular/metabolismo , Masculino , Vitrificação/efeitos dos fármacosRESUMO
Recent advances in the field of animal assisted reproduction have allowed faster dissemination of animals with superior genetics. In cattle, the development of some biotechnologies such as the in vitro production (IVP) of embryos optimized the use of oocytes, which represents a great improvement in embryo production world-wide. Therefore, females with high net merit can be multiplied more efficiently to maximize the number of their offspring. The use of ultrasound-guided transvaginal follicle aspiration combined with IVP allowed wide commercial transfer of IVP-embryos with satisfactory pregnancy results. Yet, there are a number of unanswered areas including the level of repeatability and predictability in IVP systems. Thus, the aim of this study was to evaluate the repeatability of some variables related to IVP of bovine embryos from Nellore breed. The study was conducted from 2011 to 2012 using 42 Nellore cows housed at the Centro APTA Bovinos de Corte located in Sertãozinho-SP, Brazil. Donors were kept under the same management throughout the entire experimental period. All animals underwent a total of 25 transvaginal ultrasound-guided follicular aspiration sections. Oocytes were retrieved and handled similarly by Sexing Technologies using semen from 11 sires. Repeatability was evaluated in the following variables: total number of oocytes, embryos and viable oocytes, viable o
O artigo não possui resumo em português.
RESUMO
Several factors can influence the recovery of the amount of oocytes for in vitro embryo production (IVEP), including the age of the donor, the time and the frequency at which the animal is collected and climate station year. Another factor is the individual variation of each animal in the number of oocytes and embryos. Thus, the aim of this work was to evaluate the effect of the number of aspirations and the influence of the months of the year in the amount of total and viable oocytes in Nellore cows. The date was collected from January 2011 to December 2012. Non-lactating, cycling Nellore cows (n = 42) of high genetic value were used as oocyte donors. The procedures were carried out in a farm in southeast Brazil (21816S/48582W), located in humid tropical climate. All the donors were maintained on pasture consisting of guinea grass (Panicum maximum and Urochloa brizantha), getting protein mineral supplement energy and water ad libitum daily. The analyzes were performed by proc GLIMMIX of SAS 9.3 (SAS Inst., Inc., Cary, NC). The amount of total oocytes (P=0.16) and viable (P=0.11) did not influenced by the number of aspirations. However, the months of the year have an effect on the amount of total and viable (P 0.0001) oocytes. Probably the production of oocytes did not suffer influences the number of aspirations performed mainly because the intervals between each OPU
O artigo não possui resumo em português.