Resumo
Piau porcine blastocysts were submitted to MALDI-TOF to identify the main phospholipids (PL). After that, in vivo blastocysts (D6) were vitrified (n=52), non-vitrified were used as control (n=42). After warming, blastocysts were in vitro cultured to assess re-expansion and hatching at 24 and 48 hours. Finally, at 48 hours, hatched blastocysts were submitted to RT-qPCR searching for BCL2A1, BAK, BAX and CASP3 genes. For MALDI-TOF, the ion intensity was expressed in arbitrary units. Blastocyst development was compared by Qui-square (P< 0.05). Among the most representative PL was the phosphatidylcholine [PC (32:0) + H]+; [PC (34:1) + H]+ and [PC (36:4) + H]+. Beyond the PL, MALDI revealed some triglycerides (TG), including PPL (50:2) + Na+, PPO (50:1) + Na+, PLO (52:3) + Na+ and POO (52:2) + Na. Re-expansion did not differ (P> 0.05) between fresh or vitrified blastocysts at 24 (33.3%; 32.7%) or 48 hours (2.4%; 13.5%). Hatching rates were higher (P< 0.05) for fresh compared to vitrified at 24 (66.7%; 15.4%) and 48 hours (97.6%; 36.0%). BAX was overexpressed (P< 0.05) after vitrification. In conclusion, Piau blastocysts can be cryopreserved by Cryotop. This study also demonstrated that the apoptotic pathway may be responsible for the low efficiency of porcine embryo cryopreservation.(AU)
Blastocistos de suínos foram submetidos ao MALDI-TOF para se identificarem os principais fosfolipídios (PL). Depois, parte destes embriões (D6) foram vitrificados (n=52), ou permaneceram frescos (grupo controle, n=42). Após o aquecimento, os blastocistos foram cultivados in vitro para se avaliar a reexpansão e a eclosão (BE) às 24 e 48 horas. Finalmente, às 48 horas, os BE foram submetidos ao RT-qPCR em busca dos genes BCL2A1, BAK, BAX e CASP3. No MALDI-TOF, a intensidade do íon foi expressa em unidades arbitrárias. O desenvolvimento embrionário foi comparado por qui-quadrado (P<0,05). Entre os PL mais representativos estavam as fosfatidilcolinas [PC (32: 0) + H] +; [PC (34: 1) + H] + e [PC (36: 4) + H] +. Além do PL, o MALDI revelou alguns triglicerídeos (TG), incluindo PPL (50: 2) + Na +, PPO (50: 1) + Na +, PLO (52: 3) + Na + e POO (52: 2) + Na. A reexpansão não diferiu (P>0,05) entre blastocistos frescos ou vitrificados às 24 (33,3%, 32,7%) e 48 horas (2,4%, 13,5%). As taxas de eclosão foram maiores (P<0,05) para o grupo fresco comparado ao vitrificado às 24 (66,7% x 15,4%) e 48 horas (97,6% x 36,0%). O BAX estava mais expresso (P<0,05) após a vitrificação. Concluindo, os blastocistos Piau podem ser criopreservados por Cryotop. Este estudo também demonstrou que a via apoptótica pode ser responsável pela baixa eficiência da criopreservação de embriões suínos.(AU)
Assuntos
Animais , Fosfolipídeos/análise , Criopreservação/veterinária , Sus scrofa/embriologia , Desenvolvimento EmbrionárioResumo
Piau porcine blastocysts were submitted to MALDI-TOF to identify the main phospholipids (PL). After that, in vivo blastocysts (D6) were vitrified (n=52), non-vitrified were used as control (n=42). After warming, blastocysts were in vitro cultured to assess re-expansion and hatching at 24 and 48 hours. Finally, at 48 hours, hatched blastocysts were submitted to RT-qPCR searching for BCL2A1, BAK, BAX and CASP3 genes. For MALDI-TOF, the ion intensity was expressed in arbitrary units. Blastocyst development was compared by Qui-square (P< 0.05). Among the most representative PL was the phosphatidylcholine [PC (32:0) + H]+; [PC (34:1) + H]+ and [PC (36:4) + H]+. Beyond the PL, MALDI revealed some triglycerides (TG), including PPL (50:2) + Na+, PPO (50:1) + Na+, PLO (52:3) + Na+ and POO (52:2) + Na. Re-expansion did not differ (P> 0.05) between fresh or vitrified blastocysts at 24 (33.3%; 32.7%) or 48 hours (2.4%; 13.5%). Hatching rates were higher (P< 0.05) for fresh compared to vitrified at 24 (66.7%; 15.4%) and 48 hours (97.6%; 36.0%). BAX was overexpressed (P< 0.05) after vitrification. In conclusion, Piau blastocysts can be cryopreserved by Cryotop. This study also demonstrated that the apoptotic pathway may be responsible for the low efficiency of porcine embryo cryopreservation.(AU)
Blastocistos de suínos foram submetidos ao MALDI-TOF para se identificarem os principais fosfolipídios (PL). Depois, parte destes embriões (D6) foram vitrificados (n=52), ou permaneceram frescos (grupo controle, n=42). Após o aquecimento, os blastocistos foram cultivados in vitro para se avaliar a reexpansão e a eclosão (BE) às 24 e 48 horas. Finalmente, às 48 horas, os BE foram submetidos ao RT-qPCR em busca dos genes BCL2A1, BAK, BAX e CASP3. No MALDI-TOF, a intensidade do íon foi expressa em unidades arbitrárias. O desenvolvimento embrionário foi comparado por qui-quadrado (P<0,05). Entre os PL mais representativos estavam as fosfatidilcolinas [PC (32: 0) + H] +; [PC (34: 1) + H] + e [PC (36: 4) + H] +. Além do PL, o MALDI revelou alguns triglicerídeos (TG), incluindo PPL (50: 2) + Na +, PPO (50: 1) + Na +, PLO (52: 3) + Na + e POO (52: 2) + Na. A reexpansão não diferiu (P>0,05) entre blastocistos frescos ou vitrificados às 24 (33,3%, 32,7%) e 48 horas (2,4%, 13,5%). As taxas de eclosão foram maiores (P<0,05) para o grupo fresco comparado ao vitrificado às 24 (66,7% x 15,4%) e 48 horas (97,6% x 36,0%). O BAX estava mais expresso (P<0,05) após a vitrificação. Concluindo, os blastocistos Piau podem ser criopreservados por Cryotop. Este estudo também demonstrou que a via apoptótica pode ser responsável pela baixa eficiência da criopreservação de embriões suínos.(AU)
Assuntos
Animais , Fosfolipídeos/análise , Criopreservação/veterinária , Sus scrofa/embriologia , Desenvolvimento EmbrionárioResumo
The impressive increase in the use of assisted reproductive technologies (ARTs), especially in cattle, during the last few years in Brazil is well known worldwide. In 2015, there were over 13.7 million artificial inseminations (AI), of which, about 77% were carried out using fixed-time AI (FTAI). This technology has helped to substantially improve reproductive efficiency in beef and dairy cattle. In relation to embryo transfer, production of in vivo derived (IVD) embryos remained relatively stable, with average production of 30-40,000 embryos per year, whereas in vitro production (IVP) of embryos had a substantial increase, from about 12,500 IVP embryos in 2000 to more than 300,000 IVP embryos after 2010. The increasing availability and use of sex-sorted sperm was one of the factors responsible for a recent shift from the predominance of IVP embryos from beef breeds to dairy breeds in Brazil. Moreover, there was also an increase from 13% in 2014 to 29% in 2015 in the percentage of vitrified/frozen embryos. Moreover, the successful use of protocols for fixed-time ET (FTET) due to their high efficiency and ease of implementation, has facilitated the dissemination of ET programs all over Brazil. However, there is room for improvement, since there are several reports of high pregnancy loss and high peripartum loss, when IVP embryos are used. The production of healthy cattle by somatic cell nuclear transfer has also increased in the last few years in Brazil, but despite substantial progress in reducing postnatal losses, no drastic increase in cloning efficiency up to parturition has occurred.
Assuntos
Feminino , Animais , Bovinos , Bovinos/embriologia , Desenvolvimento Embrionário , Superovulação , Técnicas In Vitro , Técnicas In Vitro/veterinária , Técnicas de Reprodução Assistida , Inseminação Artificial/veterináriaResumo
The impressive increase in the use of assisted reproductive technologies (ARTs), especially in cattle, during the last few years in Brazil is well known worldwide. In 2015, there were over 13.7 million artificial inseminations (AI), of which, about 77% were carried out using fixed-time AI (FTAI). This technology has helped to substantially improve reproductive efficiency in beef and dairy cattle. In relation to embryo transfer, production of in vivo derived (IVD) embryos remained relatively stable, with average production of 30-40,000 embryos per year, whereas in vitro production (IVP) of embryos had a substantial increase, from about 12,500 IVP embryos in 2000 to more than 300,000 IVP embryos after 2010. The increasing availability and use of sex-sorted sperm was one of the factors responsible for a recent shift from the predominance of IVP embryos from beef breeds to dairy breeds in Brazil. Moreover, there was also an increase from 13% in 2014 to 29% in 2015 in the percentage of vitrified/frozen embryos. Moreover, the successful use of protocols for fixed-time ET (FTET) due to their high efficiency and ease of implementation, has facilitated the dissemination of ET programs all over Brazil. However, there is room for improvement, since there are several reports of high pregnancy loss and high peripartum loss, when IVP embryos are used. The production of healthy cattle by somatic cell nuclear transfer has also increased in the last few years in Brazil, but despite substantial progress in reducing postnatal losses, no drastic increase in cloning efficiency up to parturition has occurred.(AU)
Assuntos
Animais , Feminino , Bovinos , Bovinos/embriologia , Técnicas In Vitro , Técnicas In Vitro/veterinária , Superovulação , Desenvolvimento Embrionário , Técnicas de Reprodução Assistida , Inseminação Artificial/veterináriaResumo
Over the years, many techniques for in vitro evaluation of sperm have been developed. Those assessments allow to perform structural, functional and molecular evaluations of the sperm cell. A combination of laboratory tests used simultaneously can provide more accurate information on sperm function and quality because sperm have multiple compartments with different functions. Many of those analyses have been used to assess the effect of sexing by flow cytometry on sperm cellular and molecular levels such as DNA methylation pattern, sperm shape, sperm morphology and capacity to remain viable after thawing. Considering that sexed sperm are submitted to a variety of adverse conditions during sorting, evaluation and identification of the possible damages caused by the sexing process are needed. It is expected that those information will help to develop procedures to improve results when sexed sperm is used. This review is focused on the recent results using structural, functional and molecular tests to evaluate sperm viability after sexing by flow cytometry.
Assuntos
Masculino , Animais , Bovinos , Citometria de Fluxo/veterinária , Processos de Determinação Sexual , Sêmen/fisiologia , Análise do Sêmen/veterináriaResumo
Over the years, many techniques for in vitro evaluation of sperm have been developed. Those assessments allow to perform structural, functional and molecular evaluations of the sperm cell. A combination of laboratory tests used simultaneously can provide more accurate information on sperm function and quality because sperm have multiple compartments with different functions. Many of those analyses have been used to assess the effect of sexing by flow cytometry on sperm cellular and molecular levels such as DNA methylation pattern, sperm shape, sperm morphology and capacity to remain viable after thawing. Considering that sexed sperm are submitted to a variety of adverse conditions during sorting, evaluation and identification of the possible damages caused by the sexing process are needed. It is expected that those information will help to develop procedures to improve results when sexed sperm is used. This review is focused on the recent results using structural, functional and molecular tests to evaluate sperm viability after sexing by flow cytometry. (AU)
Assuntos
Animais , Masculino , Bovinos , Processos de Determinação Sexual , Sêmen/fisiologia , Citometria de Fluxo/veterinária , Análise do Sêmen/veterináriaResumo
A constatação da presença de RNAs em espermatozoides trouxe diversos questionamentos a respeito dacontribuição dessa célula para o desenvolvimento embrionário. O gameta masculino, que até então eraconsiderado apenas um transportador do genoma paterno, começou a ser estudado na busca de se descobrir seessa célula tão especializada possuiria outros atributos. Ainda existem muitas dúvidas quanto às funções dosRNAs presentes nos espermatozoides. Acredita-se que essas moléculas possam ter alguma função no início dodesenvolvimento embrionário, ou ainda que possam indicar a qualidade da espermatogênese e estar envolvidascom a fertilidade masculina, no entanto mais estudos são necessários para defini-las.(AU)
The findings of spermatozoa RNAs brought several questions regarding potential contributions of thiscell to embryo development. The male gamete, considered until recently as a simple transporter of male genome,began to be studied to discover if such a specialized cell would also have other functions. There are still manydoubts related to the functions of RNAs found on spermatozoa, it is believed that these molecules may have somerole in early embryonic development, or may indicate the quality of spermatogenesis and be related to malefertility, but further studies are necessary to better define them.(AU)
Assuntos
Animais , Masculino , Espermatozoides/fisiologia , RNA/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologiaResumo
A constatação da presença de RNAs em espermatozoides trouxe diversos questionamentos a respeito dacontribuição dessa célula para o desenvolvimento embrionário. O gameta masculino, que até então eraconsiderado apenas um transportador do genoma paterno, começou a ser estudado na busca de se descobrir seessa célula tão especializada possuiria outros atributos. Ainda existem muitas dúvidas quanto às funções dosRNAs presentes nos espermatozoides. Acredita-se que essas moléculas possam ter alguma função no início dodesenvolvimento embrionário, ou ainda que possam indicar a qualidade da espermatogênese e estar envolvidascom a fertilidade masculina, no entanto mais estudos são necessários para defini-las.
The findings of spermatozoa RNAs brought several questions regarding potential contributions of thiscell to embryo development. The male gamete, considered until recently as a simple transporter of male genome,began to be studied to discover if such a specialized cell would also have other functions. There are still manydoubts related to the functions of RNAs found on spermatozoa, it is believed that these molecules may have somerole in early embryonic development, or may indicate the quality of spermatogenesis and be related to malefertility, but further studies are necessary to better define them.
Assuntos
Masculino , Animais , RNA , Desenvolvimento Embrionário/fisiologia , Desenvolvimento Embrionário/genética , Espermatozoides/fisiologiaResumo
A reprogramação epigenética se refere a modificações programadas do genoma, que ocorrem nos períodos da gametogênese e embriogênese e que regulam a atividade dos genes sem alteração da sequência primária de DNA, sendo herdáveis ao longo das divisões celulares. Entender como ocorrem esses mecanismos proporcionará maior compreensão acerca das modificações necessárias para a melhoria do sistema de cultivo in vitro, viabilizando ainda mais as biotécnicas de reprodução assistida, em especial a produção in vitro de embriões (AU)
The epigenetic reprogramming refers to programmed genome modifications that occur during gametogenesis and embryogenesis and regulate gene activity without altering the primary sequence of DNA, heritable during cell divisions. Understanding how these mechanisms occur, would give us a clearer indication of the which modifications are needed to improve the in vitro systems in, enabling further assisted reproductive biotechnologies, in particular, in vitro embryo production. (AU)
Assuntos
Animais , Bovinos/genética , Gametogênese/genética , Desenvolvimento Embrionário/genética , Pesquisas com EmbriõesResumo
A reprogramação epigenética se refere a modificações programadas do genoma, que ocorrem nos períodos da gametogênese e embriogênese e que regulam a atividade dos genes sem alteração da sequência primária de DNA, sendo herdáveis ao longo das divisões celulares. Entender como ocorrem esses mecanismos proporcionará maior compreensão acerca das modificações necessárias para a melhoria do sistema de cultivo in vitro, viabilizando ainda mais as biotécnicas de reprodução assistida, em especial a produção in vitro de embriões
The epigenetic reprogramming refers to programmed genome modifications that occur during gametogenesis and embryogenesis and regulate gene activity without altering the primary sequence of DNA, heritable during cell divisions. Understanding how these mechanisms occur, would give us a clearer indication of the which modifications are needed to improve the in vitro systems in, enabling further assisted reproductive biotechnologies, in particular, in vitro embryo production.
Assuntos
Animais , Bovinos/genética , Gametogênese/genética , Desenvolvimento Embrionário/genética , Pesquisas com EmbriõesResumo
Vários estudos indicam que embriões produzidos in vitro (PIV) são menos resistentes à criopreservação do que os in vivo. Isso se deve às diferenças existentes entre eles causadas, principalmente, pelo cultivo in vitro. Essa revisão visa sumarizar os principais fatores que afetam a criopreservação de embriões PIV e abordar asdiferentes estratégias que podem ser utilizadas para melhorar a qualidade e aumentar a criotolerância desses embriões. (AU)
Numerous studies indicate that in vitro-produced (IVP) bovine embryos are less resistant to cryopreservation when compared to those produced in vivo. The lower cryotolerance of those embryos are probably caused by the in vitro culture. This review aims to summarize the main factors that affect cryopreservation of IVP embryos and to describe different strategies for modifying those embryos to increasetheir quality and cryosurvival. (AU)
Assuntos
Animais , Bovinos , Criopreservação , Embrião de Mamíferos/embriologia , Vitrificação , Criação de Embriões para PesquisaResumo
Vários estudos indicam que embriões produzidos in vitro (PIV) são menos resistentes à criopreservação do que os in vivo. Isso se deve às diferenças existentes entre eles causadas, principalmente, pelo cultivo in vitro. Essa revisão visa sumarizar os principais fatores que afetam a criopreservação de embriões PIV e abordar asdiferentes estratégias que podem ser utilizadas para melhorar a qualidade e aumentar a criotolerância desses embriões.
Numerous studies indicate that in vitro-produced (IVP) bovine embryos are less resistant to cryopreservation when compared to those produced in vivo. The lower cryotolerance of those embryos are probably caused by the in vitro culture. This review aims to summarize the main factors that affect cryopreservation of IVP embryos and to describe different strategies for modifying those embryos to increasetheir quality and cryosurvival.